IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
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PrinciPal SPonSor:<br />
SPonSorS:<br />
Program Handbook<br />
HoSTED BY:<br />
SuPPorTED BY:
Welcome<br />
on behalf of the regional organising committee, i have great pleasure in welcoming you to the <strong>17th</strong><br />
international congress on nitrogen <strong>Fixation</strong> in Fremantle, Western australia. This congress, the first of<br />
which was held in 1974, provides the scientific community with the opportunity for a vital exchange of<br />
ideas on nitrogen fixation. let’s not forget that nitrogen fixation is as vital to our survival on the planet<br />
as photosynthesis!<br />
As always, the congress will feature<br />
high quality research across numerous<br />
disciplines and identify the emerging<br />
areas in the field. The strength of the<br />
international nitrogen fixation community<br />
lies in its people – our colleagues range<br />
from students and up-and-coming postdocs<br />
to the many experts in their field. If<br />
this is your first time attending this congress can I encourage you to<br />
take the opportunity to engage with as many colleagues as you can.<br />
Establishing research collaborations is what the meeting is all about.<br />
The <strong>17th</strong> <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong> also provides us<br />
with an opportunity to consider the questions– where are we now with<br />
nitrogen fixation and where are we going? On one hand we have the<br />
very exciting opportunities being provided by molecular technologies,<br />
with advances in these areas coming at an ever-increasing rate.<br />
On the other hand, many of the challenges confronting successful<br />
application of nitrogen fixation technology in agriculture remain<br />
the same e.g. developing new legumes and rhizobia for agriculture;<br />
achieving successful inoculation through improved technologies; and<br />
understanding the conundrums surrounding the life of rhizobia in soil<br />
and rhizosphere such as ineffective populations and competition.<br />
2<br />
Fremantle is the major port city of Western Australia, a state that<br />
is known for its magnificent coastline, incredible landscapes and<br />
unique wildlife. From tropical mangroves in the far north to the<br />
limestone cliffs pounded by the southern ocean, Western Australia<br />
offers a myriad of opportunities for travelers. Fremantle has been<br />
the gateway to Western Australia for well over 150 years and its<br />
multicultural history is reflected in its many cafes, restaurants,<br />
museums and other cultural sites. What better than a chance to browse<br />
the markets, or relax after the science in the cafes and restaurants?<br />
I sincerely hope that this will be a productive and memorable<br />
congress for all participants.<br />
associate Professor Graham o’Hara<br />
Director, Centre for Rhizobium Studies<br />
Murdoch University, Western Australia<br />
Congress Secretariat<br />
EEcW Pty ltd<br />
47 Hampden Road, Nedlands<br />
Phone: 08 9389 1488<br />
Fax: 08 9389 1499<br />
Emal: info@eecw.com.au
<strong>IN</strong>VENT<strong>IN</strong>G THE FUTURE <br />
<strong>IN</strong> <strong>IN</strong>OCULANTS<br />
<strong>Nodulaid</strong> N/T<br />
N/T stands for Nodulating Trigger<br />
<strong>Nodulaid</strong> N/T is unique. In addition to the nitrogen-fi xing rhizobia present in all<br />
inoculants, it also contains a Plant Growth Promoting Rhizobacteria (PGPR), Bacillus<br />
subtilis. This powerful active acts as a Nodulating Trigger. The combination of these 2<br />
ingredients adds an extra boost to the yield increases achieved by regular inoculants.<br />
Benefi ts of Inoculating with <strong>Nodulaid</strong> N/T<br />
The inclusion of the Nodulating Trigger results in more and healthier nitrogen-fi xing<br />
nodules per plant. Superior nodulation means a potential for increased nitrogen fi xation,<br />
producing greater, more vigorous plant root structure and resulting in greener, healthier<br />
early season plant foliage. This sets the crop up for a higher yield potential.<br />
<strong>Nodulaid</strong> N/T has been proven to increase crop performance in chickpeas, beans<br />
and lupins – even in paddocks that contain background or native rhizobia in the soil.<br />
<strong>Nodulaid</strong> N/T sets the crop up for optimal performance<br />
Nodulator Clay Granules<br />
Placing Nodulator Clay Granules into the soil adjacent to pulse seeds at sowing<br />
allows the roots to be inoculated as they emerge. Germinating seeds and developing<br />
root systems with easy access to higher levels of inoculant, result in better nodulation<br />
and better nitrogen fi xation.<br />
Nodulator Clay Granules are different, they provide:<br />
Dust and lump-free formulation<br />
Uniform size and smooth surface for superior fl ow<br />
High counts of live rhizobia<br />
Ideal for ‘dry seeding’<br />
Toll Free: 1800 558 399 Email: info.au@beckerunderwood.com www.beckerunderwood.com.au
The <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong> Series<br />
First 1974 Pullman, WA, USA (William E. Newton)<br />
Second 1976 Salamanca, Spain (Claudino Ridriguez-Barrueco)<br />
Third 1978 Madison, WI, USA (William H. Orme-Johnson)<br />
Fourth 1980 Canberra, Australia (Alan Gibson)<br />
Fifth 1983 Noordwijkerhout, The Netherlands (Cees Veeger)<br />
Sixth 1985 Corvallis, OR, USA (Harold J. Evans)<br />
Seventh 1988 Cologne, Germany (Hermann Bothe)<br />
Eighth 1990 Knoxville, TN, USA (Peter Gresshoff)<br />
Ninth 1992 Cancun, Mexico (Rafael Palacios)<br />
Tenth 1995 St. Petersburg, Russia (Igor Tikhonovich)<br />
Eleventh 1997 Paris, France (Claudine Elmerich)<br />
Twelfth 1999 Foz do Iguacu, Brazil (Fabio O. Pedrosa)<br />
Thirteenth 2001 Hamilton, ONT, Canada (Turlough M. Finan)<br />
Fourteenth 2004 Beijing, P.R. China (Yi Ping Wang)<br />
Fifteenth 2007 Cape Town, South Africa (Felix Dakora)<br />
Sixteenth 2009 Big Sky, MT, USA (John W. Peters)<br />
<strong>International</strong> Steering Committee<br />
(Provides continuity between congresses and decides among<br />
applicants for upcoming congresses)<br />
William Newton (USA)<br />
Felix Dakora (RSA)<br />
Ray Dixon (UK)<br />
Claudine Elmerich (France)<br />
Hauke Hennecke ( Switzerland)<br />
Fabio Pedrosa (Brazil)<br />
John Peters (USA)<br />
Igor Tikhonovich (Russia)<br />
Yi-Ping Wang (PRC)<br />
4
17 ICNF Organising Committee<br />
Graham O’Hara, Chair, Murdoch University<br />
Lambert Brau, Murdoch University<br />
Michael Dilworth, Murdoch University<br />
John Howieson, Murdoch University<br />
Wayne Reeve, Murdoch University<br />
Elizabeth Watkin, Curtin University<br />
Ron Yates, Department of Agriculture and Food, Western Australia<br />
Lynne Greenaway, Congress Secretariat, EECW<br />
Renee Bennett, Congress Secretariat, EECW<br />
5<br />
17 ICNF Program Advisory Committee<br />
Ross Ballard (Australia)<br />
Ton Bisseling (The Netherlands)<br />
Robert Boddey (Brazil)<br />
Nantakorn Boonkerd (Thailand)<br />
Roz Deaker (Australia)<br />
Matthew Denton (Australia)<br />
Allan Downie (UK)<br />
Jean-Jacque Drevon (France)<br />
Hans-Martin Fischer (Switzerland)<br />
Ken Giller (The Netherlands)<br />
Rene Guerts (the Netherlands)<br />
David Herridge (Australia)<br />
Mariangela Hungria (Brazil)<br />
Michael Hynes (Canada)<br />
Didier Leseur (France/Thailand)<br />
Kristina Lindstrom (Finland)<br />
Kiwamu Minamisawa (Japan)<br />
Giles Oldroyd (UK)<br />
Mark Peoples (Australia)<br />
Xavier Perret (Switzerland)<br />
Phillip Poole (UK)<br />
Clive Ronson (New Zealand)<br />
Luis Rubio (Spain)<br />
Janet Sprent (UK)<br />
Jens Stougaard (Denmark)<br />
Michael Udvardi (USA)<br />
Murray Unkovich (Australia)<br />
Graham Walker (USA)
KeYNoTe SPeAKeRS<br />
Allan Downie<br />
After completing a PhD, post-doc and a fellowship in microbial bioenergetics, I moved from Canberra<br />
to the John Innes (Norwich) in 1981 and started working on Rhizobium-legume interactions. My work<br />
on identifying various nodulation genes included the characterisation of a secreted nodulation protein<br />
that forms cation-selective ion channels. This led me to analyse Ca++ changes in legume root hairs in<br />
response to Nod factors in WT and nodulation-defective mutants, leading to a proposed Nod-factor<br />
activated signalling pathway. More recently I have been trying to understand how Nod-factor-induced<br />
Ca++ signalling might be coupled to initiation of rhizobial infection of root hairs.<br />
Ken Giller<br />
Ken Giller is Professor Plant Production Systems at Wageningen University. He leads a group of scientists<br />
with profound experience in systems analysis and simulation modelling scenarios of change. Ken’s<br />
research has focused on nitrogen fixation in tropical legumes and he is author of the standard text<br />
“<strong>Nitrogen</strong> <strong>Fixation</strong> in Tropical Cropping Systems” published in second edition in 2001. He has 5 books,<br />
>200 papers in peer-reviewed, international journals (H factor=37) and >100 book chapters. He leads<br />
a US$19.2M project “N2Africa – Putting nitrogen fixation to work for smallholder farmers in Africa<br />
(www.N2Africa.org)” across 8 countries in Africa funded by the Bill & Melinda Gates Foundation.<br />
Giles Oldroyd<br />
Western agricultural systems are reliant on the application of inorganic nitrogen fertilisers that greatly<br />
enhance yield. However, production and application of nitrogen fertilisers account for a significant<br />
proportion of fossil fuel usage in food production and the major global source of nitrous oxide emissions,<br />
a very potent greenhouse gas. Prof Giles Oldroyd studies the mechanisms by which some species of<br />
plants are capable of forming beneficial interactions with nitrogen fixing bacteria, that provide a natural<br />
source of nitrogen for plant growth. A long term aim of this research is to reduce agricultural reliance on<br />
nitrogen fertilisers. Giles completed his PhD in 1998 at the University of California, Berkeley, studying<br />
plant/pathogen interactions and then moved to Stanford University to complete a postdoc on legume/<br />
rhizobial interactions in the laboratory of Prof. Sharon Long. He has been an independent researcher<br />
at the John Innes Centre since 2002. From 2002 to 2007 he held a BBSRC David Philips Fellowship and<br />
since 2007 has been a tenured member of the JIC faculty. In 2008 he was made the head of the Institute<br />
Strategic Programme Grant “Plant sensing and responding to the Environment” and between 2010-2011<br />
he was the Deputy Director of the John Innes Centre. He has been recognised by a number of awards<br />
for his research: EMBO young investigator; European Research Council young investigator; Society of<br />
Experimental Biology Presidents medal; Royal Society Wolfson Research Merit award and the BBSRC<br />
David Phillips Fellowship.<br />
Phil Poole<br />
Phil Poole is originally from Perth but slipped up while on the typical Aussie European tour of Europe<br />
by staying to do post doctoral work in the biochemistry department at Oxford. From there he moved<br />
to Reading and then to the John Innes Centre in 2007. Phil works on the physiology and genetics of root<br />
nodule and rhizosphere/soil bacteria. He is particularly interested in:<br />
1) The regulation of nutrient exchange between Rhizobium and their host legumes<br />
2) Development and differentiation of the legume bacteroids<br />
3) The molecular basis of competitive success of bacteria in the plant rhizosphere<br />
6
Cottesloe Beach © Tourism Western Australia<br />
Luis M. Rubio<br />
Luis M. Rubio is Assistant Professor at the Center for Plant Genomics and Biotechnology of the Technical<br />
University of Madrid (Spain). He obtained BS and PhD in Biology from the University of Seville. He<br />
worked as posdoctoral researcher at the University of Wisconsin-Madison and as Associate Scientist at<br />
the University of California-Berkeley. His main research interest is the biochemistry and biosynthesis of<br />
nitrogenase, with a focus on the study of iron-molybdenum cofactor biosynthesis. His ongoing research<br />
also includes improvement of biological hydrogen production by altered forms of nitrogenase.<br />
Janet Sprent<br />
Janet Sprent is Emeritus Professor of Plant Biology in the University of Dundee and Honorary Research<br />
Fellow at the James Hutton Institute, Dundee. She has spent decades working on nitrogen cycling and<br />
fixation, especially by legumes, working in all continents, including Antarctica. Current collaborations are in<br />
Europe, Africa, India and Australia and focus on nodulation processes in understudied legumes. Her talk will<br />
have a global coverage of the great variety of legumes, their nodule structure and nodulating bacteria and<br />
will speculate on how legumes may have evolved over time to fill particular geographic niches<br />
Jens Stougaard<br />
Jens Stougaard´s primary research activity is on genes regulating development of nitrogen fixing root<br />
nodules and mycorrhiza formation in legumes. Currently the mechanisms of Nod-factor perception,<br />
the function of receptors involved and the downstream signal transduction cascades are in focus. The<br />
plant model system used for this research is Lotus japonicus that is also used for investigating the long<br />
range signalling integrating root nodule development into the general developmental program of the<br />
plant. Genetics, genomics and biochemical methods are used to identify and characterise components of<br />
regulatory circuits. In order to improve the genetic analysis and to establish a system for reverse genetics,<br />
a large-scale insertion population based on the germ-line specific activity of the LORE1 retroelement is<br />
being established and made available to the community.<br />
Michael Udvardi<br />
Michael Udvardi: I earned my PhD at The Australian National University where I worked on the biochemistry<br />
of transporters of the symbiosome membrane that mediate traffic of nutrients between plant cells and<br />
rhizobia in nitrogen-fixing legume nodules. During my postdoc years, I used molecular biology approaches<br />
to isolate and characterize plant genes involved in nodule metabolism. Over the years, my research<br />
interests have broadened, although work on symbiotic nitrogen fixation remains a core activity of my<br />
lab. I am now a Professor at the Samuel Roberts Noble Foundation where we are developing and applying<br />
tools for functional genomics in Medicago truncatula. Using forward and reverse-genetic approaches,<br />
we are currently identifying new genes required for nodule development, differentiation, and symbiotic<br />
nitrogen fixation.<br />
7<br />
Amberley Estate © Tourism Western Australia
VeNUe<br />
The Esplanade Hotel Fremantle is privately owned. The Hotel offers<br />
a warm, friendly and professional service in the simple, elegant and<br />
relaxed surroundings. The uniqueness of the Heritage and Colonial<br />
architecture blends with the character of Fremantle’s historical Port<br />
and Fishing Boat Harbour.<br />
The Esplanade Hotel Fremantle is the first hotel in WA to be declared<br />
carbon neutral.<br />
The Esplanade Hotel Fremantle realises that climate change and<br />
greenhouse emissions present pressing global challenges that<br />
will only be successfully resolved if the majority of individuals<br />
and businesses get serious about reducing their damaging carbon<br />
footprint. The Hotel’s carbon-neutral status has been achieved by<br />
securing 9.6ha of rainforest in Ecuador through Rainforest Rescue<br />
to offset the Hotel’s remaining carbon emissions.<br />
8<br />
Esplanade Hotel Fremantle<br />
Corner Marine Tce & Essex St<br />
Fremantle, Western Australia<br />
Tel: +61 8 9432 4539<br />
Fax +61 8 9430 453<br />
www.esplanadehotelfremantle.com.au<br />
Orion<br />
Lift<br />
SouTHErn croSS Gala BallrooM<br />
Pleiades<br />
Atrium<br />
Garden<br />
Restaurant<br />
Prince Regent<br />
Main Foyer Entrance<br />
Sirius<br />
Southern Cross Lobby<br />
King Sound<br />
inDian ocEan SuiTE<br />
Admiralty Gulf
GeNeRAl <strong>IN</strong>foRmATIoN<br />
aTM’s<br />
Automatic Teller Machine is located next to Cafe Panache at the<br />
Esplanade hotel, alternatively various bank ATM’s are located on<br />
South Terrace (cappuccino strip)<br />
Banks<br />
Most major banks are located in the Fremantle City Centre. Please<br />
check with concierge staff for specific bank locations.<br />
car rental<br />
Budget Rent a Car: 13 27 27<br />
Hertz: 13 30 39<br />
Avis: 13 63 33<br />
child care<br />
Please note that no official arrangements have been made for child<br />
care during the conference. Please check with your hotel as they may<br />
be able to assist you further with babysitting services during your stay.<br />
congress Dinner<br />
Venue: The View Restaurant<br />
Time: 19:00 – 23:00<br />
Dress: Smart Casual<br />
Map of Fremantle<br />
A map of the City of Fremantle is included in your delegate bag.<br />
Meals<br />
All tea breaks will be served in the Southern Cross Foyer amongst the<br />
displays. Lunches each day will be served in the Atrium Restaurant.<br />
Special Dietary Requirements: Please see registration desk. The<br />
variety of selection on the Atrium buffet should be sufficient to<br />
cater for all dietary needs.<br />
Messages<br />
The Registration Desk will receive all messages which can be<br />
collected from the Registration Desk.<br />
Telephone: +61 (0) 439 912 333<br />
The Congress Secretariat will accept no responsibility for undelivered<br />
messages.<br />
name Badge<br />
It would be appreciated if delegates wear their name badge at all<br />
times as this identifies them as eligible for catering and entry to<br />
congress sessions.<br />
9<br />
Parking<br />
Collie street car park is a multi story, undercover facility located on<br />
Collie Street, behind the Esplanade Hotel.<br />
Valet parking for in-house guests at the Esplanade is provided at<br />
a $24.50 per day. For non hotel guests valet parking is available at<br />
$28 per day and must be pre-booked 48 hours in advance by calling<br />
9432 4815.<br />
Privacy Statement<br />
In registering for <strong>17th</strong> <strong>International</strong> <strong>Nitrogen</strong> <strong>Fixation</strong> Congress<br />
relevant details may be incorporated into a delegate list for the<br />
benefit of all delegates, sponsors, exhibitors, EECW Pty Ltd and<br />
other parties directly related to the Congress. Should you wish for<br />
your details not to be included in the final list, please advise the<br />
registration desk.<br />
registration Desk<br />
The Registration Desk will be located in the Hotel foyer and will be<br />
open as follows:<br />
Sunday 27 November 14.00 – 17.00<br />
Monday 28 November 08.00 – 17.30<br />
Tuesday 29 November 08.00 – 17.30<br />
Wednesday 30 November 08.00 – 17.30<br />
Thursday 1 December 08.00 – 12.30<br />
Smoking Policy<br />
The Esplanade Hotel has a no smoking policy throughout the venue.<br />
Due to West Australian Government regulations this no smoking<br />
policy applies to most restaurants, bars and shopping centres<br />
throughout Western Australia.<br />
Shuttle Bus<br />
Shuttle Bus Services can be booked in advance for airport transfers<br />
to and from Fremantle. To receive this service, all pick-ups must be<br />
pre-booked at least 48 hours in advance, preferably between 8am –<br />
5pm Australian Western Standard Time Monday to Friday. To book<br />
contact Fremantle Shuttle Service:<br />
To Book:<br />
T: (08) 9457 7150 or 0437 197 240 (between 9am – 5.30pm AWST)<br />
Visitors to Fremantle<br />
Fremantle is one of the easiest cities in the world to explore by<br />
foot, bicycle or scooter. All leading attractions are located in close<br />
proximity and the ‘free’ Fremantle CAT bus completes regular<br />
circuits (every 15 minutes) around the City of Fremantle, stopping<br />
at popular places of interest and offering easy access to major<br />
attractions and venues.<br />
useful local Telephone numbers<br />
The Esplanade Hotel Fremantle: +61 8 9432 4000<br />
Quest Harbour Village Apartments: +61 8 9430 3888<br />
Rosie O’Grady’s Irish Pub: +61 8 9335 1645<br />
Swan Taxis: 13 13 30<br />
Bus & Rail Information: 13 62 13
PRoGRAm<br />
Sunday 27 November 2011<br />
14:00 - 17:00 Registration Desk Open<br />
17:30 - 19:30 Welcome Reception – Cicerello’s Fishing Boat Harbour Fremantle<br />
monday 28 November 2011<br />
08:00 - 17:00 Registration desk open<br />
09:00 - 10:30 coNfeReNce oPeN<strong>IN</strong>G ceRemoNY Chair: Graham O’Hara & Michael Dilworth, Room: Sirius<br />
10:30 - 11:00 Morning Tea<br />
convenor Welcome Graham o’Hara, Centre for Rhizobium Studies, Murdoch University, Australia<br />
Welcome to country Marie Taylor<br />
opening Address Peter roberts, Chair Western Panel, GRDC<br />
contemporary challenges for<br />
Symbiotic <strong>Nitrogen</strong> fixation<br />
John Howieson, Centre for Rhizobium Studies, Murdoch University, Australia<br />
11:00 - 12:30 PleNARY SeSSIoN Chair: Bill Newton, Room: Sirius<br />
11:00 - 11:30 allan Downie<br />
John Innes Centre, UK<br />
11:30 - 12:00 luis M. rubio<br />
Universidad Politecnica De Madrid, Spain<br />
12:00 - 12:30 Giles oldroyd<br />
John Innes Centre, UK<br />
12:30 - 13:30 Lunch – Atrium<br />
Characterisation of a legume pectate lyase required for initiation and growth<br />
of infection threads during nodule infection by rhizobia<br />
Changes in nif gene expression profiles during nitrogenase biogenesis<br />
Establishing beneficial interactions with the symbiosis signalling pathway<br />
13:30 - 15:00 PleNARY SeSSIoN Chair: Allan Downie, Room: Sirius<br />
13:30 - 14:00 Ken Giller<br />
Wageningen University, The Netherlands<br />
N2Africa – Putting <strong>Nitrogen</strong> <strong>Fixation</strong> to work for smallholder farmers in Africa<br />
14:00 - 14:30 Jens Stougaard<br />
Aarhus University, Denmark<br />
Nod factor perception, signal transduction and the role of LysM type receptors<br />
14:30 - 15:00 Kristina lindström<br />
University of Helsinki, Finland<br />
Rhizobium taxonomy and diversity – Is there anything more to learn?<br />
15:00 - 15:30 Afternoon Tea<br />
15:30 - 16:50 coNcURReNT SeSSIoN 1:<br />
coNcURReNT SeSSIoN 2:<br />
coNcURReNT SeSSIoN 3:<br />
field Applications I<br />
function & control of <strong>Nitrogen</strong>ase Taxonomy & evolution<br />
Chair David Herridge Michael Dilworth Kristina Lindström<br />
Room Sirius Pleiades Orion<br />
15:30 - 15:50 neil Ballard<br />
Bill newton<br />
nikolay Provorov<br />
Global Pasture Consultants, Australia<br />
Virginia Polytechnic Institute &<br />
All-Russia Research Institute For<br />
Application of Western Australian<br />
State University, USA<br />
Agricultural Microbiology, Russia<br />
legume and Rhizobium technologies Carbon monoxide adducts<br />
Mathematical simulation of<br />
in the developing world –<br />
of Azotobacter vinelandii<br />
evolutionary events in the<br />
a practitioner’s perspective<br />
Mo-nitrogenase<br />
N2-fixing plant-microbe symbioses<br />
15:50 - 16:10 raj Malik<br />
Stefan nordlund<br />
rene Guerts<br />
Department of Agriculture & Food<br />
Stockholm University, Sweden<br />
Wageningen University, The Netherlands<br />
Western Australia, Australia<br />
Regulation of <strong>Nitrogen</strong>ase activity in Parasponia to unravel genetic<br />
Impact of break crops on wheat Rhodospirillum rubrum – an interplay constraints underlying Rhizobium<br />
productivity in Western Australia of PII proteins, DRAT, DRAG and symbiosis<br />
cropping<br />
membrane sequestration<br />
16:10 - 16:30 Felipe Burgos<br />
Svetlana Yurgel<br />
Jelena Jalovaja<br />
Centre For Rhizobium Studies,<br />
Washington State University, USA<br />
Moscow Institute of Physics &<br />
Murdoch University, Australia<br />
GlnD and symbiosis: Control beyond<br />
Technology, Russia<br />
Use of remote sensing to assess the the nitrogen stress response?<br />
Comparative-genomic reconstruction<br />
symbiotic performance of Rhizobium<br />
of the NifA regulon evolution in<br />
leguminosarum var. vicieae strains<br />
and field pea<br />
alphaproteobacteria<br />
10
16:30 - 16:50 Felix Dakora<br />
Tshwane University of Technology,<br />
South Africa<br />
Identifying cowpea genotypes that<br />
increase nitrogen contribution,<br />
household food security, and human<br />
nutrition/health in Africa<br />
Tuesday 29 November 2011<br />
Emilio Jiménez-Vicente<br />
Universidad Politécnica de Madrid, Spain<br />
The FdxN protein is required for the<br />
biosynthesis and activity of the NifDK<br />
and NifH nitrogenase components in<br />
Azotobacter vinelandii<br />
Tadashi Yokoyama<br />
Tokyo University of Agriculture and<br />
Technology, Japan<br />
Genetic diversity of native soybean<br />
and mungbean bradyrhizobia from<br />
different topographical regions<br />
along the southern slopes of the<br />
Himalayan Mountains in Nepal<br />
08:00 - 17:30 Registration desk open<br />
09:00 - 10:30 PleNARY SeSSIoN Chair: Clive Ronson, Room: Sirius<br />
09:00 - 09:30 Wayne reeve<br />
Centre for Rhizobium Studies, Murdoch<br />
University, Australia<br />
The GEBA - Root Nodule Bacteria Community Sequencing Project<br />
09:30 - 10:00 Michael Hynes<br />
Glycerol utilization by Rhizobium leguminosarum requires an ABC<br />
University of Calgary, Canada<br />
transporter and affects competition for nodulation<br />
10:00 - 10:30 Phil Poole<br />
John Innes Centre, UK<br />
Metabolic transitions of Rhizobium from rhizosphere to bacteroid<br />
10:30 - 11:00 Morning Tea<br />
11:00 - 12:20 coNcURReNT SeSSIoN 4:<br />
coNcURReNT SeSSIoN 5:<br />
coNcURReNT SeSSIoN 6:<br />
Applications of New Technologies Bacteroids & Symbiosomes<br />
field Applications II<br />
Chair Ravi Tiwari Michael Hynes Felix Dakora<br />
Room Sirius Pleiades Orion<br />
11:00 - 11:20 Pascal Gamas<br />
Hauke Hennecke<br />
lori Phillips<br />
CNRS-<strong>IN</strong>RA, France<br />
ETH, Institute of Microbiology, Swithzerland DPI-Vic, Australia<br />
Molecular dissection of the<br />
New facets of Bradyrhizobium<br />
Comparative diversity of<br />
rhizobium-legume interaction by japonicum carbon metabolism and rhizosphere and endophytic<br />
RNA seq and laser micro-dissection copper trafficking which support microbial communities in faba<br />
approaches<br />
bacteroid function<br />
cropping systems<br />
11:20 - 11:40 Xavier Perret<br />
David Day<br />
Kanjana chansa-ngavej<br />
University of Geneva, Switzerland<br />
Flinders University, Australia<br />
Chulalongkorn University, Thailand<br />
Profiling the symbiotic responses Identification of transport<br />
Selection and field trials of<br />
of Sinorhizobium fredii strain<br />
proteins on the symbiosome<br />
soybean rhizobium biofertilizers<br />
NGR234 with RNA-seq<br />
membrane in soybean<br />
with DNA fingerprints and can be<br />
kept at room temperature<br />
11:40 - 12:00 David chiasson<br />
Youguo li<br />
nicole Seymour<br />
The University of Adelaide, Australia<br />
Huazhong Agricultural University, China DEEDI, Australia<br />
The use of heterologous expression Identification of a novel symbiotic <strong>Nitrogen</strong> fertiliser reduces<br />
systems to characterise the function gene participating in synthesis of nodulation of mungbean but gives<br />
of the soybean transcription<br />
a bacteroid-specific electron carrier no yield advantage in central<br />
factor GmSAT1<br />
menaquinone<br />
Queensland trials<br />
12:00 - 12:20 Elena Dolgikh<br />
Vanessa Melino<br />
All-Russia Research Institute for<br />
Centre for Rhizobium Studies,<br />
Agricultural Microbiology (ARRIAM), Russia Murdoch University, Australia<br />
New application of N2-fixing organisms: Inside out: an in-depth characterisation<br />
heterologous expression of rhizobial of effective (Fix<br />
glycosylatransferases involved in<br />
chitin oligosaccharides synthesis<br />
+ ), sub-optimal<br />
(Fix partial ) and ineffective (Fix- ryan Farquharson<br />
CSIRO Land And Water, Australia<br />
Symbiotic performance of a<br />
herbicide tolerant Medicago littoralis<br />
)<br />
N2-fixation in Trifolium symbiosis<br />
12:20 - 13:30 Lunch – Atrium<br />
13:30 - 15:00 PleNARY SeSSIoN Chair: Craig Atkins, Room: Sirius<br />
13:30 - 14:00 Graham o’Hara<br />
Centre for Rhizobium Studies,<br />
Murdoch University, Australia<br />
Fifteen years of revolution in legume N-fixation in southern Australia<br />
14:00 - 14:30 Michael udvardi<br />
The Samuel Roberts Noble Foundation, USA<br />
Functional genomics of symbiotic nitrogen fixation in legumes<br />
14:30 - 15:00 clive ronson<br />
University of Otago, NZ<br />
The requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis<br />
15:00 - 16:00 PoSTeR SeSSIoN 1 (Even numbers) /Afternoon Tea<br />
11
16:00 - 17:20 coNcURReNT SeSSIoN 7:<br />
field Applications III<br />
coNcURReNT SeSSIoN 8:<br />
Plant Symbiotic Genes<br />
coNcURReNT SeSSIoN 9:<br />
Native legume RNB<br />
Chair Ron Yates Michael Udvardi Elizabeth Watkin<br />
Room Sirius Pleiades Orion<br />
16:00 - 16:20 Elizabeth Drew<br />
Matthew Bellgard<br />
Hukam Singh Gelot<br />
SARDI, Australia<br />
CCG, Murdoch University, Australia<br />
Jai Narain Vyas University, India<br />
Impacts of pea genotype and soil Interpreting sub-optimal nitrogen Burkholderia phymatum isolated<br />
rhizobia on N2-fixation by field pea fixation in Lupinus angustifolius from root nodules of two species<br />
utilising the genome sequence of of Indigofera from alkaline soils of<br />
both symbiotic partners<br />
the Indian Thar desert<br />
16:20 - 16:40 Julie Grossman<br />
Brett Ferguson<br />
robert Walker<br />
North Carolina State University, USA<br />
University of Queensland, Australia<br />
Curtin University, Australia<br />
Effect of cover crop planting history Novel components involved in<br />
The phylogenetic diversity of<br />
on Rhizobium leguminosarum<br />
legume nodule development and Australian Burkholderia root<br />
ecology in organic farming systems autoregulation<br />
nodule bacteria<br />
16:40 - 17:00 amanda Bonython<br />
Michael Djordjevic<br />
Kerstin Huss-Danell<br />
SARDI, Australia<br />
Australian National University, Australia Swedish University of Agricultural Sciences,<br />
Improved nodulation of<br />
Nodule specific CLE peptides<br />
Sweden<br />
regenerating messina plants<br />
mediate crosstalk between local Nodulation and nitrogen fixation<br />
nodule development and systemic in native legumes in Scotland<br />
autoregulation of nodulation pathways and Sweden<br />
17:00 - 17:20 Keletso Mohale<br />
Senjuti Sinharoy<br />
Wen Feng chen<br />
Tshwane University of Technology<br />
Samuel Roberts Noble Foundation, USA China Agricultural University, China<br />
Symbiotic functioning and rhizobial Identification & functional<br />
Advances in rhizobial diversity<br />
biodiversity of Bambara groundnut characterization of Medicago truncatula and biogeography in China<br />
(Vigna subterranean L.Verdc) grown in transcription factor mutants with<br />
farmers’ field in South Africa<br />
impaired symbiotic nitrogen fixation<br />
Wednesday 30 November 2011<br />
08:00 - 17:30 Registration desk open<br />
09:00 - 10:30 PleNARY SeSSIoN Chair: Giles Oldroyd, Room: Sirius<br />
09:00 - 09:30 Janet Sprent<br />
Poles apart: Nodulation in native legumes from the Southern and<br />
University of Dundee, UK<br />
Northern hemispheres<br />
09:30 - 10:00 Erik limpens<br />
Wageningen University, The Netherlands<br />
Formation of a symbiotic interface in rhizobial and mycorrhizal symbioses<br />
10:00 - 10:30 anton Hartman<br />
Molecular characterization of the active diazotrophic community in<br />
Helmholtz Zentrum Munich, Germany uninoculated and inoculated field-grown sugarcane (Saccharum sp.)<br />
10:30 - 11:00 Morning Tea<br />
11:00 - 12:20 coNcURReNT SeSSIoN 10:<br />
coNcURReNT SeSSIoN 11:<br />
coNcURReNT SeSSIoN 12:<br />
ecology of RNB<br />
Nodule formation<br />
PGPR & Plant Production<br />
Chair Janet Sprent Jens Stougaard Anton Hartman<br />
Room Sirius Pleiades Orion<br />
11:00 - 11:20 Samson chimphango<br />
Shin okazaki<br />
Sharon Fox<br />
University of Cape Town, South Africa<br />
Nara Women’s University, Japan<br />
Centre for Rhizobium Studies, Murdoch<br />
Characterization of the cape floristic Hijacking the host nodulation<br />
University, Australia<br />
region rhizobia using 16S rRNA gene signaling by rhizobial type III<br />
Differential effects of Pseudomonas<br />
sequences and their distribution in secretion system<br />
fluorescens WSM3457 on enhanced<br />
different soil types<br />
Medicago nodulation<br />
11:20 - 11:40 Macarena Gerding<br />
christian Staehelin<br />
Khanok-on amprayn<br />
Universidad De Concepcion, Chile<br />
Sun Yat-Sen University, China<br />
University of Sydney Australia, Australia<br />
Mesorhizobium strains from the Characterization of type 3 effectors in Proteomics of rice-PGP<br />
South African herbaceous legume<br />
Lessertia spp. differ in their<br />
competitive ability in Australian soils<br />
the Rhizobium-legume symbiosis microorganisms’ interactions<br />
11:40 - 12:00 Julie ardley<br />
Kazuhiku Saeki<br />
Fernando Gonzales-andres<br />
Centre for Rhizobium Studies,<br />
Nara Women’s University, Japan<br />
University of Leon, Spain<br />
Murdoch University, Australia<br />
Mesorhizobium loti mutant lacking In vitro plant growth promotion<br />
The symbiosis between Listia spp. superoxide dismutase displays properties of endophitic bacteria<br />
and Methylobacterium and Microvirga Lotus-host accession-dependent isolated from banana (Musa sp.)<br />
rhizobia: specificity in epidermally<br />
infected legumes<br />
nitrogen fixation capacities<br />
roots in Dominican Republic<br />
12
Thursday 1 December 2011<br />
08:00 - 12:40 Registration desk open<br />
09:00 - 10:30 PleNARY SeSSIoN Chair: Ivan Kennedy, Room: Sirius<br />
09:00 - 09:30 robert Boddey<br />
Embrapa Agrobiologia, Brazil<br />
09:30 - 10:00 Perrin Beatty<br />
University of Alberta, Canada<br />
10:00 - 10:30 Jean-Jaques Drevon<br />
<strong>IN</strong>RA, France<br />
10:30 - 11:00 Morning Tea<br />
11:00 - 12:20 coNcURReNT SeSSIoN 16:<br />
Symbiotic Impacts & emissions<br />
Fremantle<br />
Legume N 2-fixation inputs preserve soil organic C stocks in no-till agriculture<br />
Environmental and economic impacts of biological N 2 fixing cereal crops<br />
Is phosphorus efficiency low for symbiotic nitrogen fixation?<br />
coNcURReNT SeSSIoN 17:<br />
molecular characterization of<br />
N-fixing organisms<br />
The Vines Resort in the Swan Valley © Tourism Western Australia<br />
14<br />
coNcURReNT SeSSIoN 18:<br />
P Nutrition & Symbioses<br />
Chair Robert Boddey Wayne Reeve Jean-Jaques Drevon<br />
Room Sirius Pleiades Orion<br />
11:00 - 11:20 Mark Peoples<br />
ana alexandre<br />
neera Garg<br />
CSIRO Plant Industry, Australia<br />
Universidade De Évora, Portugal<br />
Panjab University, India<br />
Effects of rhizobial strain and<br />
Major chaperone genes are highly Role of arbuscular mycorrhizal<br />
legume host on emissions of H2 induced in heat-tolerant rhizobia (AM) fungi and zinc in enhancing<br />
from nodules and the impact on<br />
nitrogen fixation in pigeon pea<br />
soil biology and plant growth<br />
under cadmium stress<br />
11:20 - 11:40 David Herridge<br />
nazalan najimudin<br />
andry andrianananjara<br />
University of New England, Australia<br />
Universiti Sains Malaysia, Malaysia<br />
LRI-SRA, Madagascar<br />
<strong>Nitrogen</strong>-fixing legumes in<br />
Characterization of nitrogen<br />
Genotypic variation for N2-fixation farming systems reduce greenhouse fixation genes from Paenibacillus in Voandzou (Vigna subterranea)<br />
gas emissions<br />
polymyxa ATCC 15970<br />
under P deficiency and P sufficiency<br />
11:40 - 12:00 Kiwamu Minamisawa<br />
Melissa corbett<br />
Sipho Maseko<br />
Tohoku University, Japan<br />
Curtin University, Australia<br />
Tshwane University of Technology,<br />
Mechanism of N2O emission from<br />
soybean nodule rhizosphere and its<br />
Identification and evolution of NIF<br />
genes in Leptospirillum species –<br />
South Africa<br />
Phosphorus nutrition in symbiotic<br />
mitigation<br />
an acidophilic chemolithoautotroph Cyclopia and Aspalathus species<br />
from the Cape Fynbos of South Africa<br />
12:00 - 12:20 Yen Thao Tran<br />
ni luh arpiwi<br />
Ghoulam cherki<br />
The Research Institute For Oil And Oil Plants, The University of Western Australia,<br />
Faculty of Sciences & Technique<br />
Vietnam<br />
Australia<br />
Gueliz-Marrakech, Morocco<br />
Change in farmer attitudes and Physiological and molecular<br />
Phosphorus deficiency affected<br />
practices in Vietnam in the use of<br />
inoculant compared with baseline<br />
characterization of the root nodule<br />
bacteria nodulating Millettia pinnata,<br />
conductance to O2 and ascorbate<br />
peroxydase gene expression in<br />
a biodiesel tree<br />
nodules of Phaseolus vulgarisrhizobia<br />
symbiosis<br />
12:20 - 12:40 coNfeReNce cloS<strong>IN</strong>G<br />
12:40 - 14:00 lUNch – ATRIUm<br />
*Committee reserves the right to alter the program as circumstances dictate
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Plenary Session 1<br />
0900 - 1030<br />
Author: John G. Howieson<br />
Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology, Murdoch<br />
University, South Street, Murdoch 6150, Australia.<br />
Presentation Title: Contemporary Challenges for Symbiotic <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Presentation Time: 1000 - 1030<br />
The practical rhizobiologist now has a wide array of tools at his disposal with which to experiment. From whole genomes<br />
revealing multiple replicons in nodule bacteria that confer stress response, to horizontal gene transfer giving rise to new<br />
species of nodulating bacteria, to remote sensing from satellites that show differences in nitrogen fixation within a field,<br />
there are multiple opportunities for intervention. The challenge is to select the right tools with which to conduct research, and<br />
to seek the right questions to address. And moving outside the discipline, we must be mindful of how our work connects with<br />
global issues such as climate change and poverty. Life cycle analyses reveal that legume N fixation is better for the planet<br />
than the Haber-Bosch inspired burning of fossil fuels to create fertiliser – how do we make farmers, politicians and funders<br />
aware of this? The potential for domesticating new legumes that are better equipped to grow in a changing climate is high –<br />
but how do we educate those administrators responsible for dispensing funds for climate change adaptation of this<br />
potential? Along with these exigencies, we may also contemplate whether we should engineer nitrogenise to function in<br />
cereals, or endophytes to fix N with cereals. This presentation will explore these multiple considerations confronting today‟s<br />
rhizobiologists and legume scientists, to set the scene for an exciting week ahead.<br />
15<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Plenary Session 1<br />
1100 - 1230<br />
Authors: Fang Xie, Giulia Morieri, Jeremy Murray, Anne L. Heckmann, Anne Edwards, Jiyoung Kim,<br />
Giles E. D. Oldroyd and J. Allan Downie<br />
John Innes Centre Norwich Research Park, Colney, Norwich, NR4 7UH, UK<br />
Presentation Title: Characterisation of a legume pectate lyase required for initiation and growth of infection<br />
threads during nodule infection by rhizobia<br />
Presentation Time: 1100 - 1130<br />
It has long been recognised that nodule infection by rhizobia requires localised degradation of plant cell walls to<br />
enable infection threads to initiate and grow. In root hair infection foci, the cell wall is locally degraded enabling<br />
penetration by the bacteria, and this is coupled with the synthesis of a new infection thread wall, but relatively<br />
little is known about the proteins required for these processes. A microarray analysis identified Medicago<br />
truncatula genes more strongly induced by Nod factors from WT than a (nodL) mutant of Sinorhizobium meliloti,<br />
which is reduced for infection. Among the genes identified was a predicted pectate lyase. In parallel work we<br />
identified two allelic Lotus japonicus mutants, which are defective for infection thread growth and nodule<br />
infection. Fine mapping indicated that the mutation was located in a region of about 38 genes. Analysis of the<br />
equivalent region of M. truncatula showed that the pectate lyase gene identified from the transcriptomics was<br />
one of these 38 genes. Sequencing of the orthologous gene from the two L. japonicus mutants identified different<br />
mutations in each, and the cloned WT allele complemented the mutant phenotype. Therefore the mutations in<br />
this pectate lyase gene caused the infection defect. In vitro biochemical assays with the purified WT and mutant<br />
protein demonstrated that the gene encodes a plant pectinase and we conclude that this activity contributes to<br />
the cell-wall degradation that is required to initiate infection thread growth in root hairs. It also appears to be<br />
required for subsequent extension of the infection thread across cell-cell junctions. The pectate lyase gene is<br />
induced following Nod-factor activation of the common symbiosis pathway and requires the transcriptional<br />
regulators N<strong>IN</strong> and NSP1 for its normal induction. Chromosome immunoprecipitation and promoter binding<br />
experiments indicated that the pectate lyase gene is directly regulated by N<strong>IN</strong>. These results show that there is<br />
specific Nod-factor induction of genes that play a role in initiating root infection by rhizobia.<br />
16<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Plenary Session 1<br />
1100 - 1230<br />
Authors: Luis M. Rubio, Emilio Jiménez-Vicente, César Poza-Carrión, Carlos Echavarri-Erasun,<br />
Alessandro Scandurra & Elena Moreno-Urbano<br />
Universidad Politécnica de Madrid. Centro de Biotecnología y Genómica de Plantas<br />
Pozuelo de Alarcón, 28223 Madrid (Spain).<br />
Presentation Title: Changes in nif gene expression profiles during nitrogenase biogenesis<br />
Presentation Time: 1130-1200<br />
A number of nitrogen fixation (nif) genes (10-20 depending on the organism) are involved in the assembly of a<br />
functional nitrogenase enzyme. Changes in the levels of nif gene products over time are required to obtain<br />
appropriate balances during nitrogenase biogenesis. In the model organism Azotobacter vinelandii nif gene<br />
expression exhibits exquisite regulation at least at the transcriptional and post-translational levels.<br />
We report here the changes over time of the cellular levels of nif mRNAs and Nif proteins with key functions in<br />
nitrogenase biosynthesis. Expression of nifH, nifD, nifK, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifA, nifB, FdxN,<br />
nifQ, and nafY genes was quantified by using real-time quantitative PCR. In addition, cellular accumulation of<br />
each one of the corresponding Nif proteins was determined by immunoblot analysis and quantified against<br />
standards obtained with purified protein preparations. The results will be analyzed in the context of observed in<br />
vivo nitrogenase activitiy development over time. The implications to constructing nitrogenase enzymes through<br />
synthetic biology techniques will be discussed.<br />
17<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Plenary Session 1<br />
1100 - 1230<br />
Author: Giles Oldroyd<br />
John Innes Centre Norwich Research Park, Colney, Norwich, NR4 7UH, UK<br />
Presentation Title: Establishing beneficial interactions with the symbiosis signalling pathway<br />
Presentation Time: 1200 - 1230<br />
The establishment of rhizobial and mycorrhizal symbioses requires the common symbiotic signalling pathway<br />
that utilises oscillations in calcium as a secondary messenger. This signalling pathway is differentially activated<br />
by rhizobial bacteria and mycorrhizal fungi and this is expressed as differential calcium oscillations. Specificity<br />
must be maintained in this common signalling pathway since alternate developmental processes need to be<br />
activated in order to accommodate rhizobial bacteria or mycorrhizal fungi. The calcium oscillations are decoded<br />
by a calcium and calmodulin dependent protein kinase (CCaMK). This protein is unique among calcium activated<br />
kinases in having the capability to respond independently to both free calcium and to calcium complexed with<br />
calmodulin. This dual modality of calcium activation appears to allow exquisite perception of calcium<br />
concentrations that is likely involved in the differential activation of CCaMK by the two symbionts. Lying<br />
downstream of CCaMK are a suite of transcription factors, with both rhizobial and mycorrhizal specific<br />
transcription factors. It appears that specificity in this pathway is encoded by the differential activation of these<br />
symbiosis specific transcription factors, that in turn coordinate the different processes necessary for these two<br />
symbioses.<br />
18<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Plenary Session 2<br />
1330 - 1500<br />
Author: Ken Giller<br />
Plant Production Systems, Wageningen University, PO Box 430, 6700AT Wageningen,<br />
The Netherlands<br />
Presentation Title: N2Africa – Putting <strong>Nitrogen</strong> <strong>Fixation</strong> to work for smallholder farmers in Africa<br />
Presentation Time: 1330 - 1400<br />
Maximal rates of N2-fixation recorded in the tropics reach an astonishing 5 kg N ha -1 day -1 . We have measured<br />
more than 250 kg N ha -1 of fixed N2 in soyabean in southern Africa with associated grain yields of more than 4 t<br />
ha -1 . But often less than 5 kg N ha -1 year -1 is fixed by legumes at farm scale in African smallholder systems.<br />
Increase of inputs from nitrogen fixation is required to achieve the increases in productivity required as part of the<br />
African green revolution that is gaining momentum.<br />
Successful N2-fixation by legumes in the field depends on the interaction:<br />
(GL � GR) � E � M<br />
that is (legume genotype � rhizobium genotype) � environment � management. Environment encompasses<br />
climate (temperature, rainfall, daylength etc) and soil stresses (acidity, aluminium toxicity, limiting nutrients etc).<br />
Management includes aspects of agronomic management (use of mineral fertilizers, sowing dates, plant density,<br />
weeding). Although much research is focused on identifying best combinations of GL and GR, the E and M factors<br />
often override the potential of the legume/rhizobium symbiosis for N2-fixation. Attention will be focused on<br />
identifying new socioecological niches for fitting grain, forage and tree legumes into existing farming systems,<br />
and the conditions necessary to achieve successful N2-fixation.<br />
The N2Africa project aims to increase inputs from N2-fixation on more than 225,000 smallholder farms across<br />
eight African countries within four years through: a) Increasing the area of land cropped with legumes; b)<br />
Increasing legume productivity through better agronomy and basal (P, K etc) fertilizer; c) Selecting and<br />
disseminating legume varieties with increased N2-fixation; d) Selecting better rhizobium strains and promoting<br />
high quality inoculants; e) Linking farmers to markets and creating new enterprises to increase demand for<br />
legumes. N2Africa has already reached more than 25,000 farmers and conducted more than 1,000 inoculation<br />
trials. The latest learnings will be discussed.<br />
19<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Author:<br />
Plenary Session 2<br />
1330 - 1500<br />
Jens Stougaard<br />
Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology,<br />
Aarhus University, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark.<br />
Presentation Title: Nod factor perception, signal transduction and the role of LysM type receptors<br />
Presentation Time: 1400 - 1430<br />
Formation of root nodules in legumes relies on a genetic program controlling and synchronising two processes<br />
running in parallel. Nodule primordia are formed from root cortical cells initiating cell divisions and simultaneously<br />
a bacterial infection process targets the primordia developing from the cell division foci. Plant receptors involved<br />
in perception of bacterial signal molecules are important components of these pathways and they are also<br />
involved in determining host specificity. The role of Lotus japonicus LysM type serine/threonine receptor kinases<br />
in perception of Nod-factor signals from bacterial microsymbionts during nodule initiation and nodule<br />
maintenance will be discussed. The extracellular domains of the trans-membrane kinases carry LysM domains<br />
suggesting that they are involved in perception of the rhizobial lipochitin-oligosaccharide signals and in<br />
deciphering the structure of lipochitin-oligosaccharides. Experiments and studies addressing these questions will<br />
be presented and the involvement of receptor kinases in the early physiological and cellular responses as well as<br />
later during nodule development will be illustrated. In addition to the NFR1 and NFR5 receptors, the Lotus<br />
japonicus genome encodes fifteen hitherto uncharacterised LysM type receptor kinases. The functional role of<br />
these receptors is currently investigated using a number or approaches including TILL<strong>IN</strong>G mutants and<br />
expression studies.<br />
20<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Plenary Session 2<br />
1330 - 1500<br />
Author: Kristina Lindström<br />
University of Helsinki, Finland<br />
Presentation Title: Rhizobium taxonomy and diversity – Is there anything more to learn?<br />
Presentation Time: 1430 - 1500<br />
The taxonomy and diversity of rhizobia have during the last few years deserved ample attention, and more new<br />
species than ever of legume-nodulating bacteria were described.<br />
With our collaborators we have recently studied collections of rhizobia from Galega orientalis and G. officinalis<br />
from the Caucasus, of bacteria from Chinese medicinal Glycyrrhiza species, and of bacteria isolated from<br />
Ethiopian Phaseolus beans. We also investigated diversity and evolutionary patterns in plant accessions of<br />
Caucasian Galega species and sequenced the genomes of two R. galegae strains.<br />
The phylogenetic tree of rhizobia and agrobacteria displays patterns of related species, “species compexes”. By<br />
an MLSA approach we try to sort out the “R. galegae species complex” in relation to genus Agrobacterium and<br />
other Rhizobium species. According to MLSA, the Ethiopian bean isolates mainly belong to the “R.<br />
leguminosarum species complex”, and represent R, leguminosarum, R. phaseoli and R. etli. In our study R.<br />
fabae and R. pisi seem to be one species, whereas current R. leguminosarum might be two. A sister clade to R.<br />
etli of Ethiopian strains might represent a new species, but do we want to be “splitters” or “lumpers”?<br />
Current criteria for the description of new species focus on doing a plethora of tests with little relevance to<br />
biology, evolution and symbiosis. Resources should be directed towards answering interesting questions in those<br />
fields instead. Our studies of the Glycyrrhiza isolates showed that the bacteria were taxonomically diverse but<br />
that symbiotically could be divided into true symbionts, sporadic symbionts and other endophytes. The true<br />
symbionts were Mesorhizobium species.<br />
Systematic sampling, biogeographical analyses, including aspects of symbiosis (host plants, effectiveness, Nod<br />
factors) in combination with molecular diversity and phylogenetic studies still hold promise and are likely to yield<br />
important information for biology and agronomy.<br />
21<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Author:<br />
Concurrent Session 1 – Field Applications 1<br />
1530 - 1650<br />
Neil Ballard<br />
Global Pasture Consultants, Australia<br />
Presentation Title: Application of Western Australian legume and Rhizobium technologies in the developing<br />
world – a practitioner‟s perspective<br />
Presentation Time:<br />
1530 - 1550<br />
Western Australia has been at the leading edge of developing alternative pasture legumes and their rhizobial<br />
inoculants for infertile soils since the early 1990s. Using knowledge gained from applying these technologies to<br />
farming in WA on projects overseas has given me an understanding of the differences in agriculture in other<br />
countries, as well as the opportunities. It has also enabled me to help to show the farmers in those countries how<br />
the WA experience can help them to be more productive and more efficient.<br />
The widespread adoption of no till farming since the 1970‟s in WA is an object lesson for many other low rainfall<br />
countries to grow crops and pastures with minimum moisture loss. When this is coupled with the use of a wide<br />
range of pasture species and an understanding of the importance of developing hard seed banks, it opens up a<br />
completely new range of options for these farmers and communities, to produce the quantity and quality of<br />
forage they have never seen before. The Centre for Rhizobium Studies at Murdoch University at Murdoch<br />
University in Western Australia has been able to develop elite strains of Rhizobia to suit the range of forage<br />
species we have used in the overseas experience, and these have been critical to success.<br />
My presentation will cover application of these new technologies which have the potential to greatly increase<br />
productivity on marginal soils in developing countries. The role of sociology in implementing these technologies<br />
will be emphasised, beginning with the ACIAR funded project ECCAL in the eastern Cape of South Africa.<br />
22<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 1 – Field Applications 1<br />
1530 - 1650<br />
Authors: Malik R, Seymour M<br />
Presentation Title:<br />
Presentation Time: 1550 - 1610<br />
Department Of Agriculture & Food Western Australia,<br />
Impact of break crops on wheat productivity in Western<br />
Australian cropping system<br />
Cropping systems in Western Australia have become more intensive over the past 20 years – meaning there is<br />
more cereal after cereal, less lupin and pastures, but more canola. Each year farmers decide what crop to grow<br />
based on previous history and experience and market outlooks. So, what farmers often actually want to know is<br />
“what is the best crop to sow after the last one or two?” – I.e. what is the best crop sequence? Dynamic Crop<br />
Sequence trial at Katanning WA approaches this question, amongst others, in a different way. The trial is<br />
consisted of a range of cropping component options being grown in long plots in one direction in the first year<br />
(2008), and at right angles in the second year (2009) crossing all the first year plots. This allows the comparison<br />
of all possible two-year combinations (total 100) of those components. In the third year (2010) wheat was grown<br />
across the entire site. The results suggested that both 2008 and 2009 treatments had significant effects on third<br />
year wheat yield. The 2008 treatments leading to highest wheat yields were canola, fallow, field pea, lupin, and<br />
oaten hay; and the 2009 treatments leading to highest wheat yields were fallow, lupin and oaten hay. Not<br />
surprisingly, the second year effect of the 2008 treatments on 2010 wheat yield were smaller than the first year<br />
effects of the 2009 treatments. The only 2009 break crop that increased 2010 wheat yield significantly above<br />
wheat/wheat was lupin (wheat yield after lupin was 27% higher). This research can help identify crop sequence<br />
„synergisms‟ and „antagonisms‟, thereby providing the necessary foundation for developing strategies to<br />
sequence crops over a longer period of time as a long-term strategy of annual crop sequencing to attain<br />
production, economic, and resource conservation goals by using sound management practices.<br />
23<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 1 – Field Applications 1<br />
1530 - 1650<br />
Authors: Felipe A. Burgos 1 , Ron Yates 1,3 , Graham O‟Hara 1 , Halina Kobryn 2 & John Howieson 1<br />
1 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia.<br />
2 School of Environmental Science, Murdoch University, Murdoch, 6150, Western Australia.<br />
3 Department of Agriculture and Food of Western Australia<br />
Presentation Title: Use of remote sensing to assess the symbiotic performance of Rhizobium leguminosarum<br />
var. vicieae strains and field pea.<br />
Presentation Time: 1610 - 1630<br />
The symbiotic performance between legumes and rhizobia relies on the plant-bacteria genetic compatibility and<br />
on the symbiotic partner‟s capacity to overcome environmental stresses. Symbiosis contributes nitrogen to the<br />
plants, which, among other things, increases the number of chloroplasts, and the number and size of cells per<br />
leaf. Hyperspectral imagery can detect vegetation changes combining information stored in the image. The<br />
symbiotic performance is affected by some abiotic stress factors such as low clay content and low soil water<br />
holding capacity. These soil features can be estimated using ground penetrating radar (GPR), a geophysics<br />
instrument based on energy pulses interacting with soil layers. The aim of this work was to investigate whether<br />
integrated remote sensing techniques are able to estimate the interaction of field pea inoculated separately with<br />
five strains of Rhizobium leguminosarum bv. viceae with different nitrogen fixation effectiveness levels. The<br />
experiment was carried out firstly in a glasshouse to assess the pure symbiotic performance and then in an<br />
agricultural area to assess the interaction with abiotic factors. Hyperspectral images and GPR measurements<br />
were captured to cover the glasshouse and field site experiments. The plant sample analyses consisted of plant<br />
dry weight, nitrogen content and nodulation score.<br />
The plant samples showed significant differences in nitrogen levels, nodule score and dry weight across strains.<br />
The analyses of the spectral band combinations confirmed the presence of outstanding indices sensitive to the<br />
differential symbiotic performance and were correlated with plant analyses. The GPR data also revealed a mixed<br />
composition of soil properties associated with variable water availability that affected root and plant growth. It is<br />
concluded that remote sensing can be a valuable tool for estimating legume nitrogen fixation in fields, and GPR<br />
for estimating below ground properties that affect plant growth in field experiments.<br />
24<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 1 – Field Applications 1<br />
1530 - 1650<br />
Author: Felix Dakora<br />
Tshwane University of Technology, South Africa<br />
Presentation Title: Identifying cowpea genotypes that increase nitrogen contribution, household food security,<br />
and human nutrition/health in Africa<br />
Presentation Time: 1630 - 1650<br />
Cowpea is the most important food legume in Africa. In this study, over 30 cowpea genotypes have been<br />
evaluated for symbiotic N contribution, grain yield, mineral nutrient density and protein level in edible leaves and<br />
grain. The data revealed marked differences in both symbiotic contribution and nutritional attributes among the<br />
cowpea varieties. In all instances, plant growth (measured as biomass), grain yield and protein content, as well<br />
as trace element and macronutrient density were dictated by the efficacy of the species symbiosis. Thus, cowpea<br />
genotypes that were nodulated by high N2-fixing strains, fixed more N, grew better, and showed greater levels of<br />
dietary protein, macronutrients, and trace elements in leaves and grain than their counterparts nodulated by low<br />
N2-fixing bacterial strains. In cowpea, there is therefore potential to identify host/strain combinations that increase<br />
N contribution and grain yield in cropping systems, yet also accumulate high levels of protein and dietary<br />
nutrients for human nutrition and health. There is therefore a need to integrate these attributes into breeding<br />
programs in order to identify cowpea genotypes/varieties that improve N contribution and human nutrition/health.<br />
25<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 2 – Function & Control of <strong>Nitrogen</strong>ase<br />
1530 - 1650<br />
Authors: Lifen Yan 1 , Christie H. Dapper 2 , Simon J. George 1 , Hongxin Wang 1 , Devrani Mitra 1 ,<br />
Weibing Dong 1 , Stephen P. Cramer 1 & William E. Newton 2<br />
1 Department of Applied Science, University of California-Davis, Davis, CA 95616, USA.<br />
2 Department of Biochemistry, Virginia Polytechnic Institute & State University, Blacksburg,<br />
VA 24061, USA<br />
Presentation Title: Carbon monoxide adducts of Azotobacter vinelandii Mo-nitrogenase<br />
Presentation Time: 1530 - 1550<br />
Carbon monoxide (CO) is a non-competitive reversible inhibitor of all wild-type Mo-nitrogenase-catalyzed<br />
reactions except the reduction of protons to H2. An understanding of the CO interaction should give substantial<br />
insight into mechanism. Several CO-bound species are known; some reversibly interconvert. Which one is<br />
formed depends on the CO pressure (pCO) over Mo-nitrogenase during turnover. At pCO < 0.1 atm, “lo-CO” is<br />
formed, whereas under higher pCO, either “hi-CO” or “hi(5)-CO” or both are formed. Electron nuclear double<br />
resonance (ENDOR) spectroscopy suggests that: “lo-CO” has one bound CO bridging two Fe atoms; “hi-CO” has<br />
two CO ligands, each terminally bound to a different Fe atom; and “hi(5)-CO” has two bridging CO ligands. Both<br />
stopped-flow infrared and theoretical studies, however, appear inconsistent with these structures. To help<br />
resolve ambiguities, we investigated these species using photolysis under cryogenic conditions with Fourier<br />
transform-infrared (FT-IR) detection. Photolysis of “hi-CO” indicates loss of terminally bound (1973 cm -1 ) and<br />
bridging (1679 cm -1 ) CO molecules, with concomitant formation of a species with one bridging (1711 cm -1 ) CO<br />
molecule. These assignments were confirmed by using isotopically labeled CO. Our results are therefore only<br />
partly consistent with the ENDOR assignments. We extended these studies to two altered Mo-nitrogenases,<br />
both with a substitution at α-histidine-195 in the MoFe protein, namely α-H195Q and α-H195N. Surprisingly,<br />
although we expected a similar band at ca. 1973 cm -1 on photolysis of α-H195N, we saw a band at lower energy<br />
(1936 cm -1 ). More surprisingly, photolysis of α-H195Q showed both CO-related bands, at 1969 and 1932 cm -1 .<br />
Structures will be proposed for these species and the results will be discussed in terms of both CO inhibition and<br />
the catalytic mechanism.<br />
26<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 2 – Function & Control of <strong>Nitrogen</strong>ase<br />
1530 - 1650<br />
Authors: He Wang, Pedro Filipe Teixeira, Tiago T. Selão, Agneta Norén, Catrine L. Berthold, Martin<br />
Högbom, Stefan Nordlund<br />
Department of Biochemistry and Biophysics, Stockholm University, SE-10691, Sweden.<br />
Presentation Title: Regulation of <strong>Nitrogen</strong>ase activity in Rhodospirillum rubrum – an interplay of PII proteins,<br />
DRAT, DRAG and membrane sequestration<br />
Presentation Time: 1550 - 1610<br />
In the Rhodospirillum rubrum and some other phototrophic bacteria, nitrogenase activity is regulated at the<br />
metabolic level in response to changes in nitrogen availability or in energy supply, i.e. light/darkness. This<br />
regulation is also present in some species of Azospirillum, although in that case the energy supply is reflected as<br />
the concentration of oxygen. At the molecular level this regulation is due to reversible ADP-ribosylation of the Feprotein.<br />
DRAT catalyzes the addition of an ADP-ribose from NAD + and DRAG catalyzes the removal of ADPribose<br />
moiety, thereby restoring activity. One of the major questions has been the identity of the signal(s) in this<br />
system and some years ago we proposed that the association of DRAG to the chromatophore membrane plays a<br />
central role in the regulatory mechanism. Since then we and a number of other groups have provided evidence<br />
supporting this model and also demonstrated the involvement of AmtB1 and PII proteins in this regulation.<br />
There are three PII paralogs in R. rubrum, GlnB, GlnJ and GlnK. GlnB and GlnJ have both been shown to have<br />
specific functions in the regulation of nitrogen metabolism, whereas no specific function has yet been identifíed<br />
for GlnK.<br />
We have now furthered our studies on the role of the interaction of DRAG with a partner in the chromatophore<br />
membrane in the regulation of nitrogenase activity and the involvement of PII proteins in this interaction as well<br />
as the regulation of DRAT. We have also demonstrated the influence of different nitrogen sources during growth,<br />
N2 or glutamate, on the pathway leading to regulation of nitrogenase activity, both in ammonium and energy<br />
“switch-off”. Based on our studies we propose a model explaining the communication within the cell leading to a<br />
concerted regulation of nitrogen assimilation and nitrogen fixation.<br />
27<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 2 – Function & Control of <strong>Nitrogen</strong>ase<br />
1530 - 1650<br />
Authors: Svetlana N. Yurgel 1 , Jennifer Rice 1 , and Michael L. Kahn 1,2<br />
1 Institute of Biological Chemistry,Washington State University, Pullman, WA. 99164.<br />
2 School of Molecular Biosciences,Washington State University, Pullman, WA. 99164.<br />
Presentation Title: GlnD and symbiosis: Control beyond the nitrogen stress response?<br />
Presentation Time: 1610 - 1630<br />
To establish an effective symbiosis with alfalfa, S. meliloti Rm1021 needs normal operation of the GlnD protein, a<br />
bifunctional uridylyltransferase/uridylyl-cleavage enzyme that measures cellular nitrogen status and initiates a<br />
nitrogen stress response (NSR) (Yurgel & Kahn 2008). Both of the two known targets of GlnD modification, the<br />
PII proteins GlnB and GlnK, are not needed for effectiveness (Yurgel et al., 2010). However, the glnD-sm2<br />
mutation that leads to a Fix+Eff¯ phenotype is defective in uridylylation and cells are thought to only have<br />
unmodified PII proteins. To generate GlnB in the state expected in a glnD-sm2 mutant, we introduced a<br />
Tyr�Phe variant of GlnB that cannot be uridylylated into a glnBglnK background and found that this strain was<br />
Fix+Eff+. We also generated a glnBglnKglnD triple mutant and used this and other mutants to dissect the role of<br />
these proteins in regulating the free-living NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation<br />
was dominant to the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes. The data<br />
show that the GlnD protein has a role in free-living growth and in symbiotic nitrogen exchange that does not<br />
depend on the PII proteins, suggesting that S. meliloti GlnD can communicate information to the cell by alternate<br />
mechanisms.<br />
Yurgel SN & Kahn ML (2008). A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective<br />
Sinorhizobium meliloti symbiosis with alfalfa. Proc Natl Acad Sci U S A 105(48):18958-18963.<br />
Yurgel SN, Rice J, Mulder M & Kahn ML (2010). GlnB/GlnK PII proteins and regulation of the Sinorhizobium<br />
meliloti Rm1021 nitrogen stress response and symbiotic function. J Bacteriol 192:2473-2481.<br />
28<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 2 – Function & Control of <strong>Nitrogen</strong>ase<br />
1530 - 1650<br />
Authors: Emilio Jiménez-Vicente, César Poza-Carrión & Luis M Rubio<br />
Centre for Plant Biotechnology and Genomics U.P.M. – I.N.I.A. Parque Científico y<br />
Tecnológico de la U.P.M. Campus de Montegancedo 28223 Pozuelo de Alarcón<br />
(Madrid)<br />
Presentation Title: The FdxN protein is required for the biosynthesis and activity of the NifDK and NifH<br />
nitrogenase components in Azotobacter vinelandii<br />
Presentation Time: 1630 - 1650<br />
The molybdenum nitrogenase enzyme is composed of two [Fe-S] cluster containing proteins designated as<br />
dinitrogenase (NifDK) and dinitrogenase reductase (NifH). The biosynthesis of nitrogenase [Fe-S] clusters starts<br />
by the concerted activity of the NifS cysteine desulfurase and the NifU scaffolding protein. It is expected that the<br />
processes of [Fe4-S4] cluster formation on NifU and/or their transfer to apo-NifH and apo-NifDK require an<br />
electron donor. The FdxN ferredoxin is a likely candidate to perform such a role in A. vinelandii. This study shows<br />
the effects of deleting fdxN on NifH and NifDK.<br />
The fdxN mutant exhibited much lower diazotrophic growth rate and in vivo nitrogenase activity than the wild<br />
type. In vitro activ fdxN strain was defective in the activity of both component proteins,<br />
fdxN NifDK protein exhibited 20% acetylene reduction activity and had lower Fe<br />
content than wild- fdxN NifDK could not be reconstituted in vitro by FeMo-co suggesting<br />
that it is defective in [Fe4-S4] cluster incorporation or P-cluster synthesis.<br />
Time-course analysis of nifH, nifD and nifK fdxN strain<br />
had a profile of delayed nifHDK expression and lower nifD and nifK expression rates than wild type. Consistently,<br />
NifDK protein levels were lower in the strain. On the other hand, the strain accumulated higher levels<br />
of the FeMo-co biosynthetic proteins NifEN and NifB, a phenotype previously observed in mutants with defects in<br />
FeMo-co synthesis.<br />
In summary, fdxN deletion had a profound negative effect on NifDK expression, maturation and enzymatic<br />
activity, and a noticeable effect on cellular NifH activity level. The fact that fdxN deletion affects both NifH and<br />
NifDK suggests that FdxN could be involved in the synthesis or insertion of [Fe-S] clusters for both nitrogenase<br />
component proteins.<br />
29<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 3 – Taxonomy & Evolution<br />
1530 - 1650<br />
Authors: Nikolay Provorov & Nikolay Vorobyov<br />
All-Russia Research Institute for Agricultural Microbiology, Podbelsky Sh., 3, Pushkin,<br />
196608, St.-Petersburg, Russia<br />
Presentation Title: Mathematical simulation of evolutionary events in the N2-fixing plant-microbe symbioses<br />
Presentation Time: 1530 - 1550<br />
A series of mathematical models is constructed for simulating the evolution of N2-fixing nodular symbioses<br />
towards their improved ecological efficiency (Provorov & Vorobyov 2010a 2010b). By comparing the contrast<br />
forms of legume-rhizobia symbioses formed by plants from different phylogenetic groups of the Fabaceae we for<br />
the first time differentiate the modes and estimate the pressures of individual and group natural selection which<br />
supports the transitions from: (i) mixed to clonal infections of hosts by rhizobia; (ii) extra-cellular maintenance of<br />
rhizobia (inside infection/fixation threads) to their intra-cellular maintenance inside symbiosomes; (iii)<br />
unspecialized (multi-bacterial) symbiosomes to the specialized (mono-bacterial) symbiosomes in which the<br />
rhizobia are transformed into non-reproducible bacteroids. We also quantified the impacts of genotypic specificity<br />
of partners‟ beneficial interaction on the symbiosis evolution: the host-specific microsymbionts obtain a more<br />
pronounced selective support and remain stably mutualistic during a prolonged macroevolutionary process while<br />
the non-host-specific microsymbionts may be transformed readily into the hosts‟ antagonists. Therefore, nodular<br />
symbioses provide us the unique models to study the population-genetic background for progressive evolution<br />
which is poorly investigated in the free-living unitary organisms. The developed simulation techniques may be<br />
used for assessing the evolutionary processes in agriculturally important symbioses induced by: (i) introductions<br />
of cultivated legumes into the novel cropping areas; (ii) release of the genetically modified rhizobia strains into<br />
sustainable agricultural systems. Supported by RFBR grant 09-04-00907a.<br />
Provorov NA & Vorobyov NI (2010a). Evolutionary Genetics of Plant-Microbe Symbioses. Ed I Tikhonovich<br />
NOVA Sci Publ NY 290 p.<br />
Provorov NA & Vorobyov NI (2010b). Simulation of evolution implemented in the mutualistic symbioses towards<br />
enhancing their ecological efficiency, functional integrity and genotypic specificity. Theor Popul Biol 78: 259-269.<br />
30<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 3 – Taxonomy & Evolution<br />
1530 - 1650<br />
Authors: Rik op den Camp, Maryam Seifi Kalhor, Arend Streng, Qingqin Cao, Elena Federova, Elisa<br />
Polone, Ton Bisseling & René Geurts<br />
Wageningen University, The Netherlands<br />
Presentation Title: Parasponia to unravel genetic constraints underlying Rhizobium symbiosis<br />
Presentation Time: 1550 - 1610<br />
Ever since the discovery of Parasponia as the first, and till now only, non-legume species able to establish the<br />
nitrogen-fixing nodule symbiosis with rhizobial bacteria it has been clear that these „bridging‟ species will provide<br />
insight in the evolution of root nodule symbiosis. The Parasponia-rhizobium symbiosis evolved independently<br />
from legumes, but like in legumes, the interaction is set in motion by rhizobium lipo-chitooligosaccharides (LCOs)<br />
named Nod factors. We provide several lines of evidence that the Parasponia-rhizobium symbiosis has occurred<br />
rather recent. Subsequently, the symbiosis can be considered as primitive. Parasponia lacks mechanisms to<br />
select effective nitrogen fixing rhizobial symbionts. Also it is unable to repress nodulation when grown under<br />
relatively high external nitrogen concentrations. We used Parasponia to identify genetic constrains underlying<br />
evolution of the rhizobium symbiosis and provide evidence that it has recruited essential elements from the<br />
endomycorrhizal symbiosis.<br />
31<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 3 – Taxonomy & Evolution<br />
1530 - 1650<br />
Authors: Jelena Jalovaja 1 , Olga Tsoy 2,3 , Mikhail Gelfand 2,3<br />
1Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, 141700,<br />
Russia<br />
2 Institute for Information Transmission Problems RAS, Moscow, 127994,Russia<br />
3 Lomonosov Moscow State University, Department of Bioengineering and Bioinformatics,<br />
Moscow, 119992, Russia<br />
Presentation Title: Comparative-genomic reconstruction of the NifA regulon evolution in alphaproteobacteria<br />
Presentation Time: 1610 - 1630<br />
<strong>Nitrogen</strong> fixation is a complex redox process performed by a variety of bacteria. Since it requires a large amount<br />
of energy, and its main enzyme, nitrogenase, may be easily destroyed by oxygen, this process is tightly<br />
controlled at all levels, including the level of transcription. This study focuses on NifA, a major regulator of genes<br />
associated with nitrogen fixation. The NifA regulon has already been described for B.japonicum and R.etli [1, 2],<br />
but availability of complete genome sequences of other nitrogen fixing bacteria allows one to apply comparative<br />
genomics methods for accurate regulatory annotation. Analysis of 22 alpha-proteobacteria confirmed and further<br />
specified the NifA-binding motif TGT-N10-ACA. An evolutionary scenario of operon rearrangement and horizontal<br />
gene transfer was reconstructed. The conserved regulon core contains:<br />
� nifHDK, coding nitrogenase components,<br />
� iscNnifUS operon, coding nitrogenase FeS cluster assembly,<br />
� nifBfdxN coding Fe-Mo cofactor biosynthesis proteins,<br />
� fixABCX electron carrier<br />
Interestingly, NifA-RpoN sites were identified upstream of “broken” operon parts. The autoregulation of nifA in the<br />
Rhizobiales was confirmed, within operons fixABCX-nifA, fixR-nifA or ahpCD-nifA specific for individual taxa.<br />
New candidate regulon members are typically organism-specific and include molybdenum transporter genes,<br />
rpoN and nodulation genes.<br />
1. Salazar E, Díaz-Mejía JJ, Moreno-Hagelsieb G, et al. Characterization of the NifA-RpoN Regulon in<br />
Rhizobium etli in Free Life and in Symbiosis with Phaseolus vulgaris. Applied and environmental<br />
microbiology. 2010; 76(13):4510-4520.<br />
2. Hauser F, Pessi G, Friberg M, et al. Dissection of the Bradyrhizobium japonicum NifA+sigma54 regulon,<br />
and identification of a ferredoxin gene (fdxN) for symbiotic nitrogen fixation. Molecular genetics and<br />
genomics : MGG. 2007; 278(3):255-71.<br />
32<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 3 – Taxonomy & Evolution<br />
1530 - 1650<br />
Authors: Tadashi Yokoyama 1 , Chandra Prasad Risal 2 , Naoko Ohkama-Ohtsu 1 , Salem Djedidi 3 &<br />
Hitoshi Sekimoto 4<br />
1 Institute of Agriculture, Tokyo Univ. of Agri. and Tech., Saiwai-cho 3-5-8, Fuchu, Tokyo<br />
183-8509, Japan<br />
2 Soil Management Directorate, Dept. of Agriculture, Hariharbhawan 552-0314, Nepal<br />
3 Faculty of Agriculture, Tokyo Univ. of Agri. and Tech., Tokyo 183-8509, Japan<br />
4 Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan<br />
Presentation Title: Genetic diversity of native soybean and mungbean bradyrhizobia from different<br />
topographical regions along the southern slopes of the Himalayan Mountains in Nepal<br />
Presentation Time: 1630 - 1650<br />
Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study,<br />
the genetic diversity of native soybean and mungbean bradyrhizobia isolated from different topographical regions<br />
along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from<br />
three different topographical regions with contrasting climates. Local Glycine max cv. Cobb and Vigna radiata cv.<br />
Kalyan were used as trap plants to isolate bradyrhizobia. All the Nepalese soybean and mungbean isolates<br />
characterized in this study were slow growers and were assigned to the Bradyrhizobium genus based on their<br />
molecular characterization. We found that 46% of the isolates from soybean analyzed in the present study were<br />
phylogenetically related to B. elkanii, 21% were related to B. japonicum, and 8% were related to B.<br />
yuanmingense. Phylogenetic analyses also revealed three novel lineages comprising 25% of the analyzed<br />
population. Similarly, we found 50% of the isolates from the mungbean were phylogenetically related to B.<br />
yuanmingense, 13% were related to B. japonicum, 8% were related to B. elkanii, and 29% were found with novel<br />
phylogenetic origin. Furthermore, we found that most mungbean rhizobial genotypes were conserved across<br />
agro-ecological regions of Nepal. All the strains from tropical Terai region belonged to B. yuanmingense or a<br />
novel lineage of B. yuanmingense, and dominance of B. japonicum related strains observed in the Hill region.<br />
Our results indicate that there is higher genetic diversity of Bradyrhizobium strains in the temperate and subtropical<br />
region than in the tropical region. Though nifD genes are considered as high homology genes,<br />
considerable nifD gene sequence divergence of Nepalese strains with known reference strains were noticed.<br />
We, for the first time, analyzed nifD genes from B. yuanmingense in this study. We found nifD genes in B.<br />
yuanmingense isolated from soybean and mungbean are far different from other Bradyrhizobium species.<br />
33<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Plenary Session 3<br />
0900 - 1030<br />
Authors: Wayne G. Reeve 1 and Nikos Kyrpidis 2<br />
1 Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology,<br />
Murdoch University, South Street, Murdoch 6150, Australia.<br />
2 DOE Joint Genome Institute , Walnut Creek, CA, USA<br />
Presentation Title: The GEBA - Root Nodule Bacteria Community Sequencing Project<br />
Presentation Time: 0900 - 0930<br />
Genome sequencing has revolutionized understanding of microorganisms and the roles they play in important<br />
processes such as pathogenesis, energy production, bioremediation, global nutrient cycles, and the origins,<br />
evolution, and diversity of life. However, there are significant gaps in microbial genome sequence availability<br />
especially associated with environmental genomics. The currently available microbe genome sequences show a<br />
highly biased phylogenetic distribution compared to the extent of microbial diversity known today. This bias has<br />
resulted in a major gap in our knowledge of microbial genome complexity and our understanding of the evolution,<br />
physiology, and metabolic capacity of microbes. There is a strong need for a large-scale systematic effort to<br />
sequence genomes to fill in genomic gaps in the tree of life. To this end the Genomic Encyclopedia of Bacteria<br />
and Archaea – Root Nodule Bacteria (GEBA-RNB) initiative at the Joint Genome Institute aims to sequence 100<br />
RNB strains of commercial, genetic and ecological importance. This project will support the systematic<br />
sequence-based studies and understanding of the biogeographical effects on species evolution as well as the<br />
mechanisms of symbiotic nitrogen fixation by RNB. Shared genetic mechanisms between fungal and bacterial<br />
root endosymbioses exist and a detailed understanding of endosymbionts will be beneficial to drive bioenergy<br />
development from trees. The GEBA-RNB project is based on collaboration between JGI and an international<br />
consortium from 15 countries coordinated by the Centre for Rhizobium Studies at Murdoch University.<br />
34<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Plenary Session 3<br />
0900 - 1030<br />
Authors: Hao Ding 1 , Cynthia Yip 1 , Barney A. Geddes 2 , Ivan J. Oresnik 2 , and Michael F. Hynes 1<br />
1 University of Calgary, Biological Sciences, Calgary AB, Canada T2N 1N4<br />
2 University of Manitoba, Microbiology, Winnipeg MB, Canada R3T 2N2<br />
Presentation Title: Glycerol utilization by Rhizobium leguminosarum requires an ABC transporter and affects<br />
competition for nodulation<br />
Presentation Time: 0930 - 1000<br />
Plasmid curing has shown that the ability to use glycerol as a carbon source is plasmid-encoded in Rhizobium<br />
leguminosarum (Baldani et al. 1992, Yost et al. 2006). We isolated the locus responsible for glycerol utilization<br />
from plasmid pRleVF39c in R. leguminosarum bv. viciae VF39. This region was analyzed by DNA sequencing<br />
and mutagenesis. The locus encompasses a gene encoding GlpR (a DeoR regulator), genes encoding an ABC<br />
transporter, and genes glpK and glpD, encoding a kinase and dehydrogenase. All the genes except the<br />
regulatory gene glpR were organized into a single operon, and were required for growth on glycerol. The glp<br />
operon was strongly induced by both glycerol and glycerol-3-phosphate, as well as by pea seed exudate. GlpR<br />
repressed the operon in the absence of inducer. Mutation of genes coding for the ABC transporter abolished all<br />
transport of glycerol in transport assays using radiolabelled glycerol. This confirms that, unlike in other<br />
organisms like Escherichia coli and Pseudomonas aeruginosa, which use facilitated diffusion, glycerol uptake<br />
occurs by an active process in R. leguminosarum. Since the glp locus is highly conserved in all sequenced R.<br />
leguminosarum and R. etli strains, as well as in Sinorhizobium spp. and Agrobacterium spp. and other<br />
alphaproteobacteria, this process for glycerol uptake is probably widespread. Mutants unable to use glycerol<br />
were deficient in competitiveness for nodulation of peas compared to wild-type, indicating that glycerol<br />
catabolism confers an advantage to the bacterium in the rhizosphere or in the infection thread.<br />
Baldani JI, Weaver R, Hynes MF, & Eardly B (1992) Utilization of carbon substrates, electrophoretic enzyme<br />
patterns, and symbiotic performance of clover rhizobia cured of plasmids. Appl Environ Microbiol 58:2308-2314<br />
Yost CK, Rath AM, Noel TC, & Hynes MF (2006) Characterization of genes involved in erythritol catabolism in<br />
Rhizobium leguminosarum bv. viciae. . Microbiology 152:2061-2074<br />
35<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Plenary Session 3<br />
0900 - 1030<br />
Authors: Philip Poole, Graham Hood, Alison East, Ramakrishnan Karunakaran, Vinoy<br />
Ramachandran, Jurgen Prell<br />
John Innes Centre, Norwich UK<br />
Presentation Title: Metabolic transitions of Rhizobium from rhizosphere to bacteroid<br />
Presentation Time: 1000 - 1030<br />
Comparative transcriptomics has been used to examine the changes when Rhizobium leguminosarum grows in<br />
the pea, alfalfa or sugar beet rhizospheres. This enabled the identification of genes that are expressed generally<br />
in the rhizosphere, as well as those that are legume (pea or alfalfa) or host (pea) specific. Carbon metabolism by<br />
bacteria in the rhizosphere all plants is dominated by organic acids with particularly strong induction of C1 and<br />
C2 metabolism. Gluconeogenesis is induced, indicating that while sugars are present, they are only minor<br />
carbon sources relative to the organic acids. Aromatic amino acids such as phenylalanine also appear to be<br />
particularly important. As expected nod genes were induced in the pea and alfalfa rhizosphere but not in the<br />
rhizosphere of sugar beet. Next we examined gene expression in early (7 d nodules) and mature bacteroids (15-<br />
28 d nodules). This enabled gene expression to be partitioned into developmental stages from the rhizosphere,<br />
probably infection thread/early development and mature bacteroids. Genes induced in infection threads and early<br />
development include; metal accumulation, oxidative stress and export systems for probable plant metabolites,<br />
GABA catabolism and a new terminal oxidase. To obtain a clear picture of genes induced in infection threads<br />
and early development it was crucial to subtract genes already induced in the rhizosphere. Bacteroid metabolism<br />
is characterized by dicarboxylate oxidation with very high expression of the decarboxylating arm of the TCAcycle.<br />
Amino acid synthesis is bacteroids is shut down with transcriptional as well as metabolite analysis showing<br />
almost all pools of amino acids have collapsed in bacteroids. This leads to amino acid auxotrophy for branched<br />
chain amino acids in pea and bean as demonstrated by a dependence on the Aap and Bra transporters.<br />
However, alfalfa bacteroids mutated in Aap and Bra still fix nitrogen so either they have an alternative transport<br />
system for branched chain amino acids or they retain synthesis of these amino acids.<br />
36<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Authors:<br />
Concurrent Session 4 – Applications of New Technologies<br />
1100 - 1230<br />
Brice Roux 1 , Erika Sallet 1 , Nathalie Rodde 1 , Laurent Sauviac 1 , Olivier Catrice 1 ,Ton<br />
Timmers 1 , Thomas Faraut 2 , Thomas Schiex 3 , Françoise Jardinaud 1 , Fernanda de<br />
Carvalho-Niebel 1 , Frédéric Debéllé 1 , Delphine Capela 1 , Claude Bruand 1 , Jérôme<br />
Gouzy 1 , Pascal Gamas 1<br />
1 . Laboratoire des Interactions Plantes Microorganismes (LIPM) CNRS-<strong>IN</strong>RA, Chemin<br />
de borde rouge BP 52627, 31326 Castanet-Tolosan cedex, France.<br />
2 . <strong>IN</strong>RA LGC Chemin de Borde Rouge BP52627, 31326 Castanet-Tolosan cedex,<br />
France.<br />
3 . <strong>IN</strong>RA UBIA Chemin de Borde Rouge BP52627, 31326 Castanet-Tolosan cedex,<br />
France.<br />
Presentation Title: Molecular dissection of the rhizobium-legume interaction by RNA seq and laser microdissection<br />
approaches<br />
Presentation Time: 1100 - 1120<br />
In order to have a comprehensive view of the genes involved in the symbiotic rhizobium-legume interaction, we<br />
set up integrated methods for simultaneous gene expression profiling of both partners, based upon RNA seq<br />
approaches. We analysed the plant and bacterial transcriptomes of whole and laser micro-dissected root nodules<br />
and compared them to transcriptomes from non inoculated roots or bacterial cultures, using Sinorhizobium<br />
meliloti 2011-Medicago truncatula as a model system. We thus combined genome-wide, quantitative and<br />
sensitive analyses of gene expression with technologies enabling tissue-level resolution. Importantly, we chose<br />
strand-specific RNA seq techniques that allowed us to identify, in addition to protein coding mRNAs, bacterial<br />
and plant short and long non-coding RNAs (ncRNAs), not previously predicted by automatic genome annotation.<br />
To provide a solid framework for quantitative transcriptome analyses, we sequenced and (re)annotated the<br />
genome of S. meliloti strain 2011. To do so, we developed bioinformatics tools for bacterial gene prediction that<br />
notably take advantage of RNA seq data. The results were validated by visual inspection and completed by<br />
manual annotation. We also completed the currently available M. truncatula genome sequence data by high<br />
depth Illumina sequencing.<br />
We thus found many bacterial ncRNAs previously described in other groups by different experimental<br />
approaches, which fully validated our approaches. We also discovered additional bacterial ncRNAs, as well as<br />
many plant ncRNAs (such as antisense RNAs), a number of which are differentially regulated when comparing<br />
roots and nodules. We are now using this extensive set of tools and data to (re)examine the sets of proteincoding<br />
and non coding genes involved in early and late stages of the rhizobium-legume interaction.<br />
37<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 4 – Applications of New Technologies<br />
1100 - 1230<br />
Authors: Antoine Huyghe, Nadia Bakkou, Maged Saad & Xavier Perret<br />
University of Geneva, Sciences III, Department of Botany and Plant Biology,<br />
Microbiology Unit, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.<br />
Presentation Title: Profiling the symbiotic responses of Sinorhizobium fredii strain NGR234 with RNA-seq<br />
Presentation Time: 1120 - 1140<br />
Sinorhizobium fredii strain NGR234 establishes proficient nitrogen-fixing symbioses with more than 150 plants<br />
that belong to distant genera and form nodules of either determinate (DN) or indeterminate (IDN) types (Pueppke<br />
and Broughton, 1999). Genome as well as functional analyses confirmed that genes of NGR234 required for<br />
nodule formation, infection of nodule cells and nitrogen (N2) fixation were not restricted to the 536 kb<br />
« symbiotic » plasmid pNGR234a as was previously thought. To identify within the 6.9 Mb genome of NGR234<br />
(Schmeisser et al. 2009) loci susceptible to contribute to broad host-range symbioses, we combined proteomic<br />
and transcriptomic datasets. In particular, RNA-seq was used to compare the transcriptomes of cells grown in<br />
free-living conditions (in the presence or absence of daidzein) or found inside nitrogen-fixing nodules of DN or<br />
IDN types. In parallel, an in vitro mutagenesis system designed to generate non-polar mutations as well as<br />
facilitate the functional analysis of NifA-regulated loci was successfully tested in NGR234 (Fumeaux et al. 2011).<br />
These combined approaches lead to the identification of new loci whose expression profiles were confirmed by<br />
qRT-PCR and were either modulated by flavonoids or bacteroid-specific. Amongst the novel flavonoid-inducible<br />
genes, the expression of a chromosomal locus was shown to be linked to a nod-box regulatory sequence but<br />
independent of the NodD1-TtsI-SyrM2-NodD2 regulatory cascade that controls nodulation and T3SS genes of<br />
pNGR234a (Kobayashi et al. 2004). The current status of the proteomic and transcriptomic approaches will be<br />
presented, with an emphasis on the distinct networks which control the flavonoid-dependent or bacteroid-specific<br />
expression of genes in the promiscous strain NGR234.<br />
38<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 4 – Applications of New Technologies<br />
1100 - 1230<br />
Authors: David Chiasson 1 , Patrick Loughlin 2 , Elena Fedorova 3 , Ton Bisseling 3 and Brent N.<br />
Kaiser 1<br />
1 School of Agriculture, Food, and Wine. The University of Adelaide, Urrbrae, SA, 5064,<br />
Australia.<br />
2 School of Biological Sciences, University of Sydney, Sydney New South Wales 2006,<br />
Australia.<br />
3 Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University,<br />
Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.<br />
Presentation Title: The use of heterologous expression systems to characterise the function of the soybean<br />
transcription factor GmSAT1<br />
Presentation Time: 1140 - 1200<br />
GmSAT1 (Glycine max Symbiotic Ammonium Transporter) was originally isolated in a screen for<br />
complementation of ammonium transport in the yeast mutant strain 26972c (Kaiser et al 1998). GmSAT1 has<br />
been localized to both the peribacteroid membrane (PBM) and the nucleus in soybean nodules by Western<br />
blotting and immunogold labeling. Recent work has revealed that GmSAT1 is critical for nodule organogenesis.<br />
Silencing GmSAT1 by RNA interference results in a reduced number of nodules, and a Fix- phenotype.<br />
Although the in planta function of GmSAT1 has not yet been elucidated, the use of heterologous systems has<br />
given us insight into a very dynamic protein. We have expressed GmSAT1 in yeast and onion epidermal peels.<br />
Expression in yeast has allowed us to confirm that GmSAT1 is indeed a transcription factor. GmSAT1 directly<br />
activates the transcription of a novel yeast ammonium channel (termed AMF) and binds the AMF promoter in<br />
vitro. Expression of GFP:GmSAT1 in yeast shows localization to punctate vesicles, the plasma membrane, and<br />
the nucleus. In soybean we have recently discovered new transcripts from the GmSAT1 locus. One transcript is<br />
an alternatively-spliced version of the original clone, and encodes for a nodule-enhanced N-terminal signal<br />
peptide. We have bombarded onion epidermal peels with various GFP-fusions of GmSAT1. These experiments<br />
have allowed us to determine the membrane-associated region of GmSAT1 and assign a localization function to<br />
the signal peptide. Lastly, we have made GFP fusions of both GmSAT1 orthologues from Medicago truncatula<br />
(MtSAT1 and MtSAT2). Confocal microscopy studies show that, similar to GmSAT1 in yeast and onions,<br />
MtSAT1 and MtSAT2 are localized to punctate vesicles and the nucleus. We conclude that GmSAT1 is a<br />
membrane-associated transcription factor.<br />
Kaiser, BN, Finnegan, PM, Tyerman, SD, Whitehead, LF, Bergersen, FJ, Day, DA, and Udvardi, MK (1998).<br />
Characterization of an Ammonium Transport Protein from the Peribacteroid Membrane of Soybean Nodules.<br />
Science 281:1202-1205.<br />
39<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Authors:<br />
Concurrent Session 4 – Applications of New Technologies<br />
1100 - 1230<br />
Tikhonovich I.A. 1,2 , Dolgikh E.A. 1 , Leppyanen I.V. 1 , Lopatin S.А. 3 , Varlamov V.P. 3<br />
1 All-Russia Research Institute for Agricultural Microbiology (ARRIAM), chausse<br />
Podbelskogo 3, Pushkin 8, Saint-Petersburg, 196608, Russia; 2 Saint Petersburg State<br />
University (SPbSU), Universitetskaya nab., 7-9, St. Petersburg, 199034, Russia;<br />
3 Center Bioengineering RAS, street of 60 th of October, 7/1, Moscow, 117312.<br />
Presentation Title: New application of N2-fixing organisms: heterologous expression of rhizobial<br />
glycosylatransferases involved in chitin oligosaccharides synthesis<br />
Presentation Time: 1200 - 1220<br />
Due to their unique properties like biocompatibility, low toxicity and simplicity of biodegradation, the chitin<br />
oligosaccharides (COs, chitin oligomers, chitosane oligomers and their derivatives) find a wide application. There<br />
is significant interest in application of COs in agriculture. Chitin oligosaccharides produced by pathogenic fungi,<br />
are effectively detected by many plant species as elicitors leading to defence reactions and inactivation or<br />
destruction of the potential pathogen. In plants COs activate a set of defence reactions such as activation of<br />
defence enzymes, production of active oxygen species (О2 - , Н2О2, OH - ), biosynthesis of phytoalexins and callose<br />
or lignin deposition. Therefore application of these compounds and their modified analogs for stimulation of plant<br />
resistance against pathogens looks like enough promising. The treatment of plants with COs is more preferential<br />
in comparison with chemical agents because they are not toxic and easily utilized. That is a reason why last<br />
years the interest in the synthesis of COs by enzymatic methods using either the biosynthetic<br />
glycosyltransferases or glycosidases is being increased. The possibility of enzymatic synthesis of chitin<br />
oligomeres by means of unique enzymes of soil bacteria rhizobia has been investigated in our experiments.<br />
Original approaches to analyse the structure of synthesized chitin oligosaccharides by methods of high<br />
performance liquid chromatography and mass-spectrometry have been developed. In our experiments the elicitor<br />
activity of synthesized chitin oligomers has been analysed using specific tests.<br />
This research was supported by the Ministry of Education and Science of the Russian Federation (state<br />
contracts ## 16.512.11.2183, 16.552.11.7047).<br />
40<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 5 – Bacteroids & Symbiosomes<br />
1100 - 1230<br />
Authors: Hauke Hennecke, Marion Koch, Fabio Serventi, Gabriella Pessi, Hans-Martin Fischer,<br />
Zeb Youard, Valérie Murset, Simona Huwiler, Doris Bühler, Olivier Braissant, Nathanaël<br />
Delmotte, Julia Vorholt, Francesco Danza & Barnali Padhi<br />
ETH, Institute of Microbiology, Department of Biology, CH-8093 Zurich, Switzerland<br />
Presentation Title: New facets of Bradyrhizobium japonicum carbon metabolism and copper trafficking<br />
which support bacteroid function<br />
Presentation Time: 1100 - 1120<br />
The use of transcriptomics and proteomics in culture-grown cells and root-nodule bacteroids of Bradyrhizobium<br />
japonicum has revealed new symbiotically important functions in metabolism which had not been addressed<br />
before. Two of them are reported here, one concerning carbon catabolism, the other related to copper<br />
acquisition. (i) B. japonicum was found to be an oxalotroph. Key genes for oxalate degradation were knocked<br />
out. The mutants were not only unable to use oxalate as the sole source of carbon but they were also partly<br />
defective in arabinose degradation. Several lines of evidence suggest that arabinose is degraded to pyruvate<br />
and glycolaldehyde, the latter being fully oxidized in a pathway via oxalate. The mutants also exhibited<br />
decreased nodule occupancy in competition with the wild type. Nodules of four B. japonicum host plants had<br />
higher oxalate content than root tissue. Oxalate degradation may thus be an advantageous trait in nodule<br />
colonization by B. japonicum. (ii) Copper starvation conditions led to the induction of an operon of five genes<br />
coding for putative or approved functions in copper acquisition and trafficking. Deletion of the operon resulted in<br />
strains with a pleiotropic phenotype, including defects in symbiotic nitrogen fixation, denitrification, and<br />
cytochrome oxidase biogenesis. The product of one gene (bll4880) was purified and shown to bind Cu(I) with<br />
high affinity and in a 1:1 stoichiometry. In vivo, it functions as a copper chaperone in the periplasm where it is<br />
involved in Cu delivery to the membrane-bound aa3-type cytochrome oxidase and perhaps to other targets. Why<br />
this protein and/or the other encoded proteins are symbiotically important is still enigmatic.<br />
41<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Authors:<br />
The symbiosome membrane (SM) is the interface between the plant and bacteria in the legume-rhizobium<br />
symbiosis. This plant-derived membrane separates the symbiotic form of the rhizobia, from the plant cytoplasm<br />
and controls both the type and amount of solutes exchanged between the two partners. In exchange for nitrogen<br />
fixed by the bacteroid, the plant provides reduced carbon, probably in the form of malate, to the bacteroids, as<br />
well as other solutes including iron, sulphate, zinc and possibly amino acids. We are investigating transport<br />
proteins on the SM that regulate this exchange in soybean nodules using a proteomic approach and nodule<br />
transcriptome data. Candidates that potentially encode malate, iron and amino acids are a major focus.<br />
Proteomic analysis of the SM involved trypsin digestion of total SM proteins, separation of peptide fragments by<br />
liquid chromotography (LC), and analysis with tandem mass spectrometry (MS/MS). Of the 71 proteins identified<br />
in all three biological replicates, 33 are predicted to be integral membrane proteins. A number of proteins known<br />
to be associated with the SM, such as nodulin 26 (an aquaporin), ATPase, symbiotic sulphur transporter (sst1),<br />
Rab7, nodulins 24 and 53, were identified. Many of the identified SM proteins are highly expressed in nodules<br />
compared to other tissues (Libault et al. 2010; Severin et al. 2010). We are currently assessing the importance of<br />
some integral membrane proteins of unknown function through an RNAi approach and confirming the location of<br />
proteins on the SM using reporter gene fusions. Candidate transporters confirmed to be localised to the SM will<br />
be functionally characterised in heterologous expression systems.<br />
We have also characterised a transporter, GmYSL1, with homology to Yellow Stripe Like (YSL) proteins that are<br />
implicated in iron transport in other plants. The gene complements growth of the yeast iron transport deficient<br />
mutant, fet3fet4, on media containing low concentrations of ferric citrate. Fusion of the coding region to GFP<br />
confirmed the localisation of this protein on the SM. Its expression in nodules is up-regulated in response to iron<br />
and this and its location suggest it may be involved in sequestration of iron in the symbiosome.<br />
Libault M Plant J. 2010 63:86<br />
Severin AJ BMC Plant Biol. 2010 10:160<br />
Concurrent Session 5 – Bacteroids & Symbiosomes<br />
1100 - 1230<br />
Patrick Loughlin 1 , Victoria Clarke 1 , Chi Chen 1 , Yihan Qu 1 , Joanne Castelli 3 , Martha<br />
Ludwig 3 , David Day 2 and Penelope Smith 1<br />
1 School of Biological Sciences, Macleay Bldg (A12), University of Sydney, Camperdown<br />
NSW, 2006. 2 School of Biological Sciences, Flinders University, SA, Australia. 3 School of<br />
Biomedical, Biomolecular and Chemical Sciences (M310), University of Western<br />
Australia, Crawley WA 6009.<br />
Presentation Title: Identification of transport proteins on the symbiosome membrane in soybean<br />
Presentation Time: 1120 - 1140<br />
42<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 5 – Bacteroids & Symbiosomes<br />
1100 - 1230<br />
Authors: Youguo Li, Fuli Xie, Guojun Cheng, Hui Xu, Zhi Wang & Lei Lei<br />
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,<br />
Wuhan, Hubei, 430070, P. R. China<br />
Presentation Title: Identification of a novel symbiotic gene participating in synthesis of a bacteroid-specific<br />
electron carrier menaquinone<br />
Presentation Time: 1140 - 1200<br />
Ubiquinone (UQ) has been considered an electron mediator in electron transfer that generates ATP in Rhizobium<br />
under both free-living and symbiosis conditions. When mutated, the dmtH gene has a symbiotic phenotype of<br />
forming ineffective nodules on Astragalus sinicus. The gene was isolated from a Mesorhizobium huakuii 7653R<br />
transposon-inserted mutant library. The DNA sequence and conserved protein domain analyses revealed that<br />
dmtH encodes demethylmenaquinone (DMK) methyltransferase, which catalyzes the terminal step of<br />
menaquinone (MK) biosynthesis. Comparative analysis indicated that dmtH homologs were present in only a few<br />
rhizobia. Real-time quantitative PCR showed dmtH is a bacteroid-specific gene. The highest expression was<br />
seen at 25 days after inoculation of strain 7653R. Gene disruption and complementation tests demonstrated that<br />
the dmtH gene was essential for bacteroid development and symbiotic nitrogen fixation ability. Menaquinone and<br />
ubiquinone were extracted from the wild type strain 7653R and mutant strain HK116. MK7 accumulated under<br />
microaerobic condition and UQ10 accumulated under aerobic condition in M. huakuii 7653R. The predicted<br />
function of DmtH protein was confirmed by the measurement of methyltransferase activity in vitro. These results<br />
revealed that MK7 was used as an electron carrier instead of UQ in M. huakuii 7653R bacteroids.<br />
43<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Authors:<br />
Concurrent Session 5 – Bacteroids & Symbiosomes<br />
1100 - 1230<br />
Vanessa J Melino 1 , Elizabeth A Drew 2 , Gordon Thomson 1 , Rosemary White 3 , Wayne G<br />
Reeve 1 , Ross A Ballard 2 , and Graham O‟Hara 1 .<br />
1 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia<br />
2 South Australian Research and Development Institute, Plant and Soil Health, Urrbrae,<br />
5064, South Australia<br />
3 Commonwealth Scientific and Industrial Research Organisation, Plant Industry,<br />
Canberra, 2601, ACT<br />
Presentation Title: Inside out: an in-depth characterisation of effective (Fix + ), sub-optimal (Fix partial ) and<br />
ineffective (Fix - ) N2-fixation in Trifolium symbiosis<br />
Presentation Time: 1200 - 1220<br />
A major challenge to the formation of a successful symbiotic partnership between Trifolium spp. and Rhizobium<br />
leguminosarum bv. trifolii (R.l.t) is the presence of naturalized soil bacteria which can cause the induction and<br />
infection of root nodules without the benefits of nitrogen fixation (ineffective) or which may alternatively result in<br />
poor nitrogen fixation (sub-optimal). Analysis of symbiotic performance and morphological examination of nodule<br />
and bacteroid development enabled the classification of each of the 16 symbiotic combinations into one of four<br />
groups: Effective ( Fix 90-100% ), Sub-optimal ( Fix 10-70% ), Ineffective-differentiated (Fix - ) and Ineffective-vegetative (Fix -<br />
). The effective combinations are each within the nodule function range of 90-100% containing infection threads,<br />
bacteroids in inter-zone II-III and III with 1-2 bacteroids per peribacteroid unit. Sub-optimal combinations result in<br />
a varied rate of fixation from 10 to 70% and their nodules contain an abundance of senescing bacteroids present<br />
either in an enlarged and developmentally early zone of senescence (IV) or where zonation is absent, they are<br />
found adjacent to bacteroid-dense plant cells. Although differentiated Y-shaped bacteroids of effective nodules<br />
were like-wise observed in sub-optimal nodules they were on average shorter in length. Ineffective-differentiated<br />
combinations produce nodules which are absent of zonation, void of acetylene reduction activity and their plant<br />
cells contain elongated but less swollen bacteroids alongside senescing bacteroids. Ineffective-vegetative<br />
combinations produce nodules which are absent of bacteroids despite the presence of rhizobia-filled infection<br />
threads. These findings demonstrate the diversity of symbiotic outcomes from combinations of symbionts within<br />
the same genus, and the range of failures that can occur during infection. Future functional genomic efforts will<br />
be directed towards studying genetic control of internalization of rhizobia by cortical cells (failed in the ineffectivevegetative<br />
nodules), abortion of bacteroid differentiation post-elongation (observed in the ineffectivedifferentiated<br />
nodules) and accelerated bacteroid senescence post-differentiation (observed in sub-optimal<br />
nodules).<br />
44<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 6 – Field Applications II<br />
1100 - 1230<br />
Authors: Lori Phillips 1 , Bernadette Carmody 2 , Sharon Fox 3 , Lambert Brau 3 , Graham O‟Hara,<br />
David Pearce 2<br />
Presentation Title:<br />
1 Department of Primary Industries Victoria, Bundoora Vic 3083;<br />
Lori.Phillips@dpi.vic.gov.au<br />
2 Department of Primary Industries Victoria, Rutherglen Vic 3685<br />
3 Murdoch University, School of Biological Sciences and Biotechnology, Murdoch,<br />
Western Australia 6150<br />
Presentation Time: 1100 – 1120<br />
Comparative diversity of rhizosphere and endophytic microbial communities in<br />
faba cropping systems<br />
Effective nodulation by rhizobia does not occur in an ecological void. Numerous and diverse microbial<br />
communities exist in the soil and develop in the rhizosphere and endosphere of the growing legume. These<br />
communities may influence effective nodulation, plant growth, and disease suppression, and represent a<br />
significant potential co-inoculant resource. The primary objective of this study was to examine the impacts of<br />
plant cultivar and inoculation treatment on the diversity of plant-associated bacteria. Faba bean cv. Farah and<br />
Nura were sown in a field trial in Rutherglen, Victoria. This site had not been cropped with legumes for over a<br />
decade. Treatments included non-inoculated controls, standard peat inoculation, peat inoculation at half the<br />
recommended rate, and co-inoculation with a Pseudomonas spp (potential plant growth promoting rhizobacteria).<br />
This trial is ongoing, and performance measurements to date include nodulation, root biomass, and shoot<br />
biomass. Plant cultivar and inoculation treatment effects on rhizosphere and endophytic microbial communities<br />
were evaluated using terminal restriction fragment length polymorphism (T-RFLP). Archaeal, bacterial, and<br />
fungal diversity were assessed. Inoculation type had no significant impact on either nodule number or plant<br />
biomass measures. Non-inoculated faba plants were well nodulated by resident rhizobial populations that<br />
persisted for more than a decade in the absence of a legume host. T-RFLP data is currently under analysis, and<br />
will further our understanding of the ecological parameters which help or hinder symbiotic success.<br />
45<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 6 – Field Applications II<br />
1100 - 1230<br />
Author: Kanjana Chansa-Ngavej<br />
Department of Microbiology, Faculty of Science, Chulalongkorn University, Thailand<br />
Presentation Title: Selection and Field Trials of Soybean Rhizobium Biofertilizers with DNA Fingerprints and<br />
can be Kept at Room Temperature<br />
Presentation Time: 1120 - 1140<br />
Soybean rhizobium biofertilizers are usually kept at low temperature or under refrigeration to prevent cell<br />
multiplication which leads to reduction in nodulation efficiency of soybean rhizobium strains. In 2009, the author<br />
applied for a Thai patent for the selection of soybean rhizobium strains with DNA fingerprints for the production of<br />
soybean rhizobium biofertilizers which can be kept at room temperature. Out of the 70 strains tested for growth<br />
at different temperatures in yeast extract mannitol (YM) broth at 200 rpm for 5 days, two strains, NA2 and NA82<br />
were selected. 16S rDNA sequences revealed they were Bradyrhizobium elkanii strains with different DNA<br />
fingerprints and bromthymol blue reactions. NA2 was found to secrete acidic products while NA82 was found to<br />
secrete alkali products when grown on YM agar with Bromthymol blue at 25 o C for 10 days. After incubating the<br />
biofertilizers produced by using each of the selected strains at 10 10 -10 12 CFU/gram biofertilizer at 37 o C and at 40<br />
o C for 4 weeks, the numbers of survival cells were 10 9 -10 10 CFU/ml and 10 4 -10 6 CFU/ml respectively. Field trials<br />
with soybean (Glycine max cv. Chiangmai 2) with each of the biofertilizers in 240 m 2 plots in three subdistricts in<br />
Phitsanulok province showed NA2 increased soybean yields by 18-42%. NA82 was found to increase soybean<br />
yield in Bung Kok subdistrict by 26%. Field trials with soybean (Glycine max cv. Chiangmai 60) with each of the<br />
biofertilizers in 240 m 2 plots in Sukhothai province showed the biofertilizers NA2 and NA82 could increase<br />
soybean yields 9-14%. PCR fingerprints of both NA2 and NA82 were detected in bacteria isolated from root<br />
nodules of soybeans in every experimental plot. DNA fingerprints of root nodule bacteria isolated from the control<br />
treatment subplots showed NA2 and NA82 were not indigenous rhizobia in the experimental plots.<br />
46<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 6 – Field Applications II<br />
1100 - 1230<br />
Author: Nicole Seymour 1 , Maurice Conway 2 , Andrew Erbacher 2 , Darren Aisthorpe 3 and Max<br />
Quinlivan 2<br />
1 Department of Employment, Economic Development and Innovation, Leslie Research<br />
Centre, Toowoomba, 4350, Queensland.<br />
2 Department of Employment, Economic Development and Innovation, Emerald, 4720,<br />
Queensland.<br />
3 Department of Employment, Economic Development and Innovation, Biloela, 4715,<br />
Queensland<br />
Presentation Title: <strong>Nitrogen</strong> fertiliser reduces nodulation of mungbean but gives no yield advantage in<br />
Central Queensland<br />
Presentation Time: 1140 - 1200<br />
Mungbean or green gram (Vigna radiata) is a short-seasoned tropical pulse crop used as a high protein human<br />
food. Australian production varies from 30000 to 60000 tonnes per year, 95% of which is exported. Mungbean<br />
production has increased in Central Queensland (CQ) due to high prices, improved yields and its valuable role in<br />
weed management.<br />
Low nodulation and N-fixation by commercial mungbean crops in the northern grain-growing region of Australia<br />
(Busby and Lawn 1992) suggested that inoculated mungbean was N-limited and current inoculation technology<br />
may be ineffectual (Herridge et al. 2005).<br />
Five field trials were conducted in 2010-11 across CQ to determine whether an economic response could be<br />
gained by applying fertiliser. Several N, P and Zn fertiliser rates were tested separately and in combination. All<br />
mungbean seed (cv. Crystal) was inoculated with the bradyrhizobial strain CB1015, as peat slurry except for one<br />
treatment (uninoculated with no fertiliser). At one site, on a self-mulching brown Vertosol at Wowan, Queensland,<br />
the impact of fertiliser on nodulation was assessed.<br />
In three of the five trials there was a significant yield response to inoculation but no consistent yield response to<br />
fertiliser treatments. At Wowan, nodulation scores indicated there was a suppressive effect of urea fertiliser at 20<br />
and 40 kg N/ha. There was no effect of any treatment on nodule dry weights. The results indicated that there<br />
was no economic advantage from added N fertiliser for inoculated mungbean cv. Crystal.<br />
Bushby HVA, Lawn RJ (1992) <strong>Nitrogen</strong> fixation in mungbeans – expectations and reality. In „Proceedings of the<br />
6 th Australian Society of Agronomy conference‟. Pp 161-164. (Australian Society of Agronomy: Armidale)<br />
Herridge DF, Robertson MJ, Cocks B, Peoples MB, Holland JF and Heuke L (2005). Low nodulation and nitrogen<br />
fixation of mungbean reduce biomass and grain yields. Aust J Exp Agric. 45:269-277<br />
47<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 6 – Field Applications II<br />
1100 - 1230<br />
Authors: Ryan Farquharson 1 , Ross Ballard 2 , Nigel Charman 2 , Jeff Baldock 1<br />
1 CSIRO Land and Water. PMB 2 Glen Osmond, South Australia 5064.<br />
2 South Australian Research and Development Institute. GPO Box 397, Adelaide, South<br />
Australia 5001.<br />
Presentation Title: Symbiotic performance of a herbicide tolerant Medicago littoralis<br />
Presentation Time: 1200 – 1220<br />
Group B herbicides inhibit the acetohydroxyacid synthase (AHAS – also known as acetolactate synthase)<br />
enzyme in the pathway of branched chain amino acid synthesis. These herbicides have gained widespread use<br />
worldwide; however the potential impacts on nitrogen fixation are underappreciated. A new strand medic<br />
(Medicago littoralis) cultivar (FEH-1) was developed by chemical mutagenesis of seed of the Herald variety<br />
(Heap, 2000). FEH-1 displayed tolerance to various group B herbicides including the sulfonylurea class,<br />
however this work had only analysed biomass production and yield with no consideration of nitrogen fixation<br />
(Heap, 2000; Howie and Bell, 2005). Through a targeted approach, a single mutation in the AHAS gene was<br />
identified as the mechanism of herbicide tolerance (Oldach et al., 2008). An experiment comparing the symbiotic<br />
performance of FEH-1 and Herald showed that FEH-1 had useful tolerance to chlorsulfuron, with higher biomass<br />
production, nodulation and nitrogen fixation than Herald after application of that herbicide. However it was noted<br />
that in the absence of herbicide, Herald had greater biomass production, nodulation and nitrogen fixation than<br />
FEH-1. Differences in seed mass and nitrogen could not explain the disparity and a „tolerance penalty‟ was<br />
proposed. A subsequent experiment measuring the growth of Herald and FEH-1 with different strains of rhizobia<br />
showed symbiotic compatibility had changed in FEH-1. This may be contributing to the tolerance penalty<br />
observed when FEH-1 is grown in the absence of herbicide. Although there is scope to better understand the<br />
complexities surrounding group B herbicides and nitrogen fixation, it is clear that where in-crop or residual<br />
herbicides are of concern, herbicide tolerant varieties will provide a useful option to maintain inputs of biologically<br />
fixed nitrogen.<br />
Heap J (2000). Increasing Medicago resistance to soil residues of ALS-inhibiting herbicides. Thesis, University of<br />
Adelaide.<br />
Howie J & Bell CA (2005) Field performance of an annual medic tolerant of sulfonylurea herbicide residues.<br />
Proceedings of the XX <strong>International</strong> Grassland Congress, Dublin, Ireland.<br />
Oldach KH, Peck DM, Cheong J, Williams KJ, Nair RM (2008) Identification of a chemically induced point<br />
mutation mediating herbicide tolerance in annual medics (Medicago spp.)<br />
48<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Plenary Session 4<br />
1330 - 1500<br />
Authors Graham W. O‟Hara 1 Ron J. Yates 1, 2 & John G. Howieson 1<br />
1 Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology,<br />
Murdoch University, South Street, Murdoch 6150, Australia.<br />
2 Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South<br />
Perth, Western Australia 6151<br />
Presentation Title: Fifteen years of revolution in legume N-fixation in southern Australia<br />
Presentation Time: 1330 – 1400<br />
Legumes in southern Australian agriculture have been, and continue to be, introduced from exotic centres of<br />
origin. Inoculation is essential because Australian soils do not naturally contain rhizobia that form effective<br />
nitrogen-fixing symbioses with these legumes. During the past 15 years the Centre for Rhizobium Studies & the<br />
National Rhizobium Program have developed 15 strains of rhizobia for commercial production, and contributed to<br />
development of new legumes which, together with their rhizobia, have been sown over 5 million ha. in southern<br />
Australia. In addition, new insights have been provided into phenomena affecting symbiotic performance e.g. the<br />
role of horizontal gene transfer in development of poorly effective soil populations of rhizobia, and selective<br />
nodulation.<br />
Selective nodulation describes an active phenomenon that results in the establishment of a symbiosis between a<br />
legume and an effective strain of rhizobia in the presence of ineffective strains. Selective nodulation can<br />
overcome a numerical disadvantage in the effective microsymbiontt when there is a population size window of<br />
between 1,000 and 100,000 cells.<br />
Alternative species of herbaceous perennial legumes (e.g. Lebeckia spp., Rynchosia spp.) originating in<br />
southern Africa and nodulated by novel Burkholderia spp. are being evaluated for their potential role in providing<br />
green feed in autumn and late spring. Target soils do not sustain traditional legumes (e.g. lucerne, white clover,<br />
lotus) because of abiotic stresses such as low pH, low clay content, low nutrient status and low rainfall.<br />
Overcoming the challenges of establishing symbioses with these new legumes will be discussed.<br />
The Grain Research and Development Corporation (GRDC) of Australia support the National Rhizobium<br />
Program.<br />
49<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Plenary Session 4<br />
1330 - 1500<br />
Authors: Michael Udvardi 1 , Catalina Pislariu 1 , Igor Kryvoruchko 1 , Senjuti Sinharoy 1 , Mingyi<br />
Wang 1 , Rajasekhara R. Duvvuru Muni 1 , Ivone Torres-Jerez 1 , Mark Taylor 1 , Shulan<br />
Zhang 1 , Xiaofei Cheng 1 , Jiangqi Wen 1 , Ji He 1 , Xinbin Dai 1 , Patrick X Zhao 1 , Yuhong<br />
Tang 1 , Rujin Chen 1 , Kirankumar Mysore 1 , Pascal Ratet 2 , Vagner A Benedito 3 , Giles<br />
Oldroyd 4 , and Jeremy D Murray 4<br />
1 Samuel Roberts Noble Foundation, Ardmore, OK, USA; 2 ISV-CNRS, Gif sur Yvette,<br />
France; 3 West Virginia University, USA; 4 John Innes Centre, Norwich, UK;<br />
Presentation Title: Functional genomics of symbiotic nitrogen fixation in legumes<br />
Presentation Time: 1400 – 1430<br />
The past few years have seen tremendous development of tools and resources for functional genomics in<br />
legumes, including completion or near-completion of several legume genomes, comprehensive gene expression<br />
databases for model and crop species, and production of near-saturating mutant populations for efficient forward<br />
and reverse-genetic studies of gene function. This presentation will highlight some of these advances in various<br />
legumes species before focusing on resource development in the model species, Medicago truncatula and how<br />
these resources are accelerating research on symbiotic nitrogen fixation.<br />
Phenotypic screening of several mutant populations of Medicago followed by map-based and other cloning<br />
approaches have identified several genes that are required for nodule development, differentiation, and/or<br />
symbiotic nitrogen fixation (SNF). However, it is clear from transcriptomic studies that hundreds, if not thousands<br />
of plant genes are involved in SNF. Anticipating the completion of the Medicago genome sequence, we invested<br />
heavily in the development of tools and resources that will enable us and others to decipher the functions of<br />
many Medicago genes in the coming years. These resources include a gene expression atlas/database that can<br />
be used to obtain developmental and other types of expression data for most Medicago genes, and two large<br />
mutant populations, a fast-neutron-bombardment-deletion and a tobacco retro-transposonTnt1-insertion<br />
population, which can be used for both forward and high-throughput reverse genetics. About 20,000 Tnt1<br />
insertion lines have been generated in the R108 genotype and phenotypic screens of about half of the population<br />
revealed 172 lines defective in SNF. These were sorted into six distinct phenotypic categories. Thermal<br />
Asymmetric InterLaced PCR (TAIL-PCR) was used to generate 2,422 flanking sequence tags (FSTs) from these<br />
SNF mutant lines. FST analysis identified 33 insertion alleles of the following essential symbiotic genes:<br />
DMI1,2,3; NSP1,2; ERN1; N<strong>IN</strong>; LYK3; FLOT1,2; ENOD40; SYMREM1; and SUNN. High-throughput 454<br />
sequencing using a 2D-pooling strategy is being used to accelerate our current FST sequencing efforts. This will<br />
lead to a comprehensive FST database for all Tnt1 insertion lines. In parallel, a PCR-based reverse-screening<br />
strategy was established to identify Tnt1 insertions in specific genes of interest. As a result, we have identified<br />
additional insertion alleles of known, essential symbiosis genes. The mutant resources and gene expression<br />
atlas have also been used to identify novel symbiotic genes, including VAPYR<strong>IN</strong>, which is required for<br />
intracellular accommodation of rhizobia in nodules and of arbuscular mycorrhizal fungi in root cells. Reversegenetics,<br />
using the Tnt1-insertion population is currently being used to identify and characterize transcription<br />
factors and transporters with essential roles in SNF.<br />
This work was supported by the National Science Foundation, the USDA CSREES-NRI, and the Samuel Roberts<br />
Noble Foundation.<br />
50<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Plenary Session 4<br />
1330 - 1500<br />
Authors: Simon Kelly 1,2 , Yasuyuki Kawaharada 2,3 , Artur Muszynski4, Andree Hubber 1,2 , John<br />
Sullivan 1,2 , Niels Sandal 2,3 , Russell Carlson 4 , Jens Stougaard 2,3, and Clive Ronson 1,2.<br />
1 Department of Microbiology and Immunology, University of Otago, Dunedin, New<br />
Zealand<br />
2 Centre for Carbohydrate Recognition and Signalling, University of Aarhus, Aarhus,<br />
Denmark<br />
3 Department of Molecular Biology, University of Aarhus, Aarhus, Denmark<br />
4 Complex Carbohydrate Research Center, University of Georgia, Athens, USA<br />
Presentation Title: The requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis<br />
Presentation Time: 1430 – 1500<br />
To investigate the requirement for exopolysaccharide (EPS) in the formation of determinate nodules on Lotus<br />
species, Mesorhizobium loti strain R7A mutants affected in early (exoA, exoB) or mid/late-stages (exoO, exoU,<br />
exoK, mlr5265 and mlr5266) of the EPS biosynthetic pathway were isolated. Elution profiles of EPS extracts<br />
showed that R7A produced both HMW and LMW fractions of EPS whilst mutant strains produced only a LMW<br />
fraction. Early-stage affected mutants formed nitrogen-fixing nodules on L. japonicus cv. Gifu at rates<br />
comparable to wild-type R7A, whereas the mid/late-stage mutants formed uninfected nodule primordia, a few of<br />
which developed into nodules following a lengthy delay. Examination of the symbiotic proficiency of the exoU<br />
mutant on various L. japonicus ecotypes and under differing environmental conditions revealed host and<br />
environment interactions in the requirement for EPS. Analysis of fluorescent protein-tagged strains revealed that<br />
the ineffective mutants were disrupted at the stage of infection thread (IT) development. Symbiotically-defective<br />
EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority<br />
of full-length ITs observed harboured only the EPS mutant strain and did not show bacterial release, whereas all<br />
nitrogen-fixing nodules contained both mutants. These results suggest that EPS is required at both the stages of<br />
IT initiation and bacterial release and reveal a complex role and requirement for M. loti EPS in determinate<br />
nodule formation. These and other results also suggest that EPS plays a signalling rather than structural role in<br />
the symbiosis. To identify the putative EPS receptor and the downstream signalling pathway, we have carried out<br />
a screen for plant suppressor mutants that form nodules with the exoU mutant. Progress towards the<br />
characterisation of these mutants will be presented.<br />
51<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 7 – Field Applications III<br />
1600 - 1720<br />
Authors: Elizabeth Drew 1 , Victor Sadras 1,2 , Lachlan Lake 1 , Matthew Denton 2 & Ross Ballard 1<br />
1 Plant and Soil Health, South Australian Research and Development Institute, GPO<br />
Box 397, Adelaide, SA, 5001, Australia.<br />
2. School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA,<br />
5064, Australia<br />
Presentation Title: Impacts of pea genotype and soil rhizobia on N2-fixation by field pea<br />
Presentation Time: 1600 – 1620<br />
Enhancing the fixed N contribution from legumes into agricultural systems will become increasingly important as<br />
higher N fertiliser costs erode farm profitability. This paper examines factors affecting the N2-fixation potential of<br />
field pea (Pisum sativum).<br />
Having previously shown that about 70% of southern Australian cropping systems with a history of pea<br />
cultivation contain adequate numbers of rhizobia for prompt nodulation (>100 rhizobia per g soil), recent efforts<br />
have been directed at understanding the intrinsic compatibility (N2-fixation capacity) of pea genotypes with soil<br />
rhizobia. Contributions of fixed N by different pea genotypes have also been quantified in the field.<br />
The greenhouse assessment of commercial pea cultivars and advanced breeding lines with soil rhzobia included<br />
material with diverse maturity times and growth habits. Effective symbioses formed with most communities of soil<br />
rhizobia, but some variation occurred between pea genotypes. For example, the symbiotic performance of cv.<br />
Kaspa was on average 10% lower than that of cv. Parafield (85%) when nitrogen fixation resulting from 82 soil<br />
inoculants was compared to the commercial inoculant strain (SU303).<br />
When five pea genotypes were grown in the field, differences were measured in nodulation pattern and<br />
partitioning of dry matter, which contributed to large differences in N benefit. Pea genotypes were sown with and<br />
without N fertiliser at Roseworthy SA and were reliant on soil rhizobia for nodulation. Nodule number and<br />
distribution on the roots eight weeks after sowing varied significantly with pea genotype and nitrogen treatment.<br />
Nodule mass/g root was weakly (R 2 =0.17) but significantly (P
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 7 – Field Applications III<br />
1600 - 1720<br />
Authors: Julie Grossman 1 , Nape Mothapo 1 , Mary Parr 1 , Malik Oliver 1<br />
1 North Carolina State University, Department of Soil Science, Raleigh, North Carolina,<br />
27695.<br />
Presentation Title: Effect of cover crop planting history on Rhizobium leguminosarum ecology in organic<br />
farming systems<br />
Presentation Time: 1620 – 1640<br />
Biological nitrogen fixation (BNF) is a major contributor of nitrogen to certified organic farming systems in the<br />
United States (U.S.), with presence of effective rhizobia strains being essential for optimal nodulation of legume<br />
cover crops and green manures. Organic farmers have particular interest in the winter annual cover crop species<br />
hairy vetch (HV; Vicia villosa Roth). Our recent work in the Southeastern U.S. shows V. villosa to have high<br />
biomass production and BNF capacity, with many varieties producing over 200 kg N ha -1 , and more than 170 kg<br />
N ha -1 derived from BNF. In this study we determined the impact of past hairy vetch cultivation on<br />
resident Rhizobium leguminosarum biovar viciae (Rlv) populations. Organic farm soils with and without history of<br />
HV cultivation were used to assess effect of planting history on nodulation of ten HV trap-host genotypes and<br />
diversity of Rlv. Paired soils from each field type were collected from three organic farms across North Carolina.<br />
Plants were inoculated with soil dilutions from the six fields and used to trap Rlv in a growth chamber. V. villosa<br />
inoculated with HV+ soil dilutions had 60% more nodules with 70% greater total mass than HV- treatments. Two<br />
of three HV+ soils produced greater plant biomass and plant tissue N than those inoculated with soil dilutions<br />
from HV- fields, suggesting improved BNF. Molecular analysis of the 473 Rlv isolates using BOX-PCR produced<br />
more than 35 genetic groupings across the three sites, and indicated that rhizobia diversity was most impacted<br />
by site, followed by hairy vetch field history. Results suggest that hairy vetch cultivation appears to increase<br />
population size of resident Rlv capable of nodulation and BNF with hairy vetch cover crops. Current work is<br />
underway to quantify Rlv populations in each field type using qPCR and Most Probable Number methodologies.<br />
53<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 7 – Field Applications III<br />
1600 - 1720<br />
Authors: Amanda Bonython 1,2 , Ross Ballard 1,2 , Nigel Charman 1,2 & Andrew Craig 1,2<br />
1 South Australian Research and Development Institute, GPO Box 397, Adelaide, SA,<br />
5001, Australia.<br />
2 Cooperative Research Centre for Future Farm Industries. Crawley, WA, 6009.<br />
Presentation Title: Improved nodulation of regenerating messina plants<br />
Presentation Time: 1640 – 1700<br />
Messina (Melilotus siculus syn. M. messanensis) is a promising annual pasture legume for areas affected by<br />
dryland salinity. Sinorhizobium medicae strain, WSM1115, was used during initial species evaluation. Prompt<br />
nodulation occurred in year of sowing but nodulation failure was commonplace in regenerating pastures. Strain<br />
WSM1115 failed to persist in the saline soils over summer. Alternative strains of rhizobia and agronomic<br />
practices were evaluated to improve nodulation.<br />
Six agronomic treatments and two inoculation treatments were applied in a factorial design. Messina seed was<br />
inoculated with either a mixture of experimental strains (SRDI554, WSM4118 and WSM4194) or WSM1115.<br />
Agronomic treatments were applied to modify the soil micro-environment and improve rhizobial persistence.<br />
They comprised sowing a perennial companion species, either Cichorium intybus or Puccinellia ciliata;<br />
incorporating soil additives at sowing, gypsum (2.5 t/ha) or clay (75t/ha); applying an organic mulch over the<br />
summer months, or applying a soil containing medic rhizobia prior to regeneration. Plots were sown in 2008 at<br />
two trial sites near Keith, South Australia. The soils were alkaline sands with summer soil surface salinity levels<br />
of 14.6 ECe dS/m (Site 1) and 5.3 ECe dS/m (Site 2). The sites were free of medic rhizobia prior to sowing.<br />
In 2008, all treatments had >97% plants nodulated. In 2009, ten regenerating plants from each plot were<br />
sampled and percentage nodulation and nodule score per plant determined (Score 0=0 nodules; 1=1-4 nodules;<br />
2=5-9 nodules; 3=10-14 nodules, 4=15-19 nodules, 5=20-29 nodules, 6=30+ nodules). At Site 1 (more saline)<br />
plant nodulation and nodule score increased from 35% and 0.6 (WSM1115 treatment) to 95% and 3.0 (strain<br />
mixture treatment). At Site 2, most regenerating plants in the WSM1115 treatment were nodulated (80%), but<br />
had few nodules (score 1.6). Nodule score increased to 3.9 in the strain mixture treatment. At both sites, the<br />
application of soil containing medic rhizobia resulted in levels of nodulation approaching the strain mixture<br />
treatment. Other agronomic treatments were less effective.<br />
The mixture of rhizobia strains improved the nodulation of regenerating messina plants compared to WSM1115.<br />
Strain of rhizobia influenced messina nodulation more than the agronomic treatments.<br />
54<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 7 – Field Applications III<br />
1600 – 1720<br />
Authors: Keletso Mohale 1 , Alphonsus Belane 1 , Flora Pule-Meulenburg 2 and Felix D Dakora 3<br />
1 Department of Crop Sciences, Tshwane University of Technology, 175 Nelson<br />
Mandela Drive, Private Bag X680, Pretoria 0001, South Africa<br />
2 Department of Biotechnology and Food Technology, Tshwane University of<br />
Technology, 175 Nelson Mandela Drive, Private Bag X680, Pretoria 0001, South Africa<br />
3 Department of Chemistry, Tshwane University of Technology, 175 Nelson Mandela<br />
Drive, Private Bag X680, Pretoria 0001, South Africa<br />
Presentation Title: Symbiotic functioning and rhizobial biodiversity of Bambara groundnut (Vigna<br />
subterranean L.Verdc) grown in farmers‟ field in South Africa<br />
Presentation Time: 1700 – 1720<br />
Although Bambara groundnut (Vigna subterranea L.Verdc) is an important food legume in Africa, little is known<br />
about its symbiotic N nutrition. This study assessed N2 fixation in Bambara groundnut plants grown in 26 farmers‟<br />
fields, and characterized their bacterial symbionts isolated from root nodules. Isotopic analysis showed that N<br />
derived from fixation in Bambara groundnut varied from 55% to 97%. Actual amounts of N-fixed also varied for<br />
different farms and villages, and ranged from 3.7 to 212.7 kg N.ha -1 . Bacterial isolates from root nodules showed<br />
phenotypic differences in growth rate, colony appearance, shape, and texture. Following Koch‟s postulate, 19<br />
slow-growers and 31 fast-growers were tested for their nodule-forming ability on Bambara groundnut. The<br />
bacterial isolates showed marked differences in their ability to elicit nodule formation and promote plant growth in<br />
Bambara groundnut. However four slow-growers and five fast-growers formed ineffective nodules on Bambara<br />
groundnut. Analysis of data from 16S rDNA sequencing showed that Bambara groundnut isolates formed three<br />
distinct clades, one with Bradyrhizobium liaoningense, another with Burkholderia tuberum CIP 108238, while the<br />
third clade clustered with Rhizobium sp. CCNNYC119 (in a sub-clade) and Mesorhizobium sp. W39 (in another<br />
sub-clade). Taken together, the data revealed that Bambara groundnut depends on N2 fixation for its N nutrition<br />
and is nodulated by diverse microsymbionts that belong to both alpha and beta-proteobacteria.<br />
55<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 8 – Plant Symbiotic Genes<br />
1600 – 1720<br />
Authors: Matthew Bellgard1 , Wayne Reeve2 , Huaan Yang3 , Jingjuan Zhang4 , Roberto Barrero1 and John Howieson2, 4<br />
1 Centre for Comparative Genomics, Murdoch University, South Street, Murdoch 6150,<br />
Australia.<br />
2 Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology,<br />
Murdoch University, South Street, Murdoch 6150, Australia.<br />
3 Department of Agriculture and Food, Western Australia (DAFWA), Baron Hay Court,<br />
South Perth, 6151, Western Australia.<br />
4 Crop and Plant Research Institute, Murdoch University<br />
Presentation Title: Interpreting sub-optimal nitrogen fixation in Lupinus angustifolius utilising the<br />
genome sequence of both symbiotic partners.<br />
Presentation Time: 1600 – 1620<br />
WSM1417, WSM1253 and WSM471 are nodule bacteria that infect Lupinus angustifolius (NLL narrow-leaf lupin)<br />
and which have different nodulation and nitrogen fixation profiles. These three strains were sequenced within the<br />
JGI GEBA project through the US Department of Energy. To understand the genetic control of the relationship<br />
between these three micro-symbionts and their legume host we required both the lupin genome sequence, and<br />
transcriptome information. We thus sequenced NLL cv Tanjil using a WGS approach and achieved a 30 fold<br />
coverage of the whole genome. We obtained 31 billion base pair high quality sequencing data and are<br />
undertaking the lupin genome assembly and annotation with a view to identify candidate genes in nodule<br />
formation and function. The transcriptome data was of nodules of three NLL plants inoculated with the three<br />
sequenced strains. From the NLL genome sequence we obtained 23,000 contigs, which were assembled into<br />
12,500 scaffolds. The largest contig we have is 149Mb, the N50 size of 13.6 Kb and there is ~80Mb of repeat<br />
sequences. Further analysis of the genome sequence data will be provided.<br />
56<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 8 – Plant Symbiotic Genes<br />
1600 – 1720<br />
Authors: Brett Ferguson 1 , Dongxue Li 1 , Meng-Han Lin 1 , Satomi Hayashi 1 , Yu-Hsiang Lin 1 , Dugald<br />
Reid 1 , Saeid Mirzaei 1 , Alina Tollenaere 1 , Peter Gresshoff 1<br />
1 Australian Research Council Center of Excellence for Integrative Legume Research,<br />
School of Agriculture and Food Sciences, University of Queensland, St. Lucia, Brisbane,<br />
QLD, 4072, Australia<br />
Presentation Title: Novel components involved in legume nodule development and autoregulation<br />
Presentation Time: 1620 – 1640<br />
To discover novel legume components involved in nodule formation (Ferguson et al. 2010), we used nextgeneration<br />
sequencing (Illumina) and the available soybean genome (www.phytozome.net/soybean) to identify<br />
genes that are differentially expressed in the zone of root hair emergence following rhizobia-inoculation.<br />
Legumes regulate the number of nodules they form via the Autoregulation Of Nodulation (AON). Following<br />
rhizobia-inoculation, AON commences with the production of a root-derived signal called Q (Reid et al. 2011a).<br />
We have identified soybean Q candidate genes encoding CLAVATA3/ESR related (CLE) peptides that exhibit<br />
increased expression following rhizobia inoculation or inhibitory nitrate treatment (Reid et al. 2011b). Overexpression<br />
of these genes significantly reduces soybean nodule numbers. The nitrate-induced Q peptide<br />
appears to act locally, whereas the rhizobia-induced Q peptide is transported to the shoot where it, or a product<br />
of its action, is perceived by a LRR receptor kinase called NARK. We used next-generation sequencing to<br />
identify soybean components acting downstream of NARK in the leaf. Candidate genes identified can now be<br />
used in our recently established bioassay to confirm the presence of Q in various samples and biological extracts<br />
(e.g., xylem sap). We also isolated and characterized the phenotype of a soybean line mutated in the<br />
homeologous gene of GmNARK, GmCLAVATA1A. Following the perception of Q by NARK in the leaf, a number<br />
of factors are produced including a novel signaling compound called the Shoot-Derived Inhibitor (SDI). SDI is<br />
predicted to be transported from the shoot to the root where it inhibits continued nodule development. We found<br />
SDI to be NARK- and Nod factor-dependent, heat stable, small, and likely not a peptide or RNA molecule (Lin et<br />
al. 2010, 2011). Findings regarding our progress in identifying and characterising the abovementioned<br />
nodulation factors will be presented.<br />
Ferguson BJ, Indrasumunar A, Hayashi S, Lin Y-H, Lin M-H, Reid D, Gresshoff PM (2010) Molecular analysis of legume nodule<br />
development and autoregulation. Journal of Integrative Plant Biology 52: 61-76<br />
Lin Y-H, Ferguson BJ, Kereszt A, Gresshoff PM (2010) Suppression of hypernodulation in soybean by a leaf-extracted, NARK- and Nod<br />
factor-dependent, low molecular mass fraction. New Phytologist 185: 1074-1086<br />
Lin Y-H, Lin M-H, Gresshoff PM, Ferguson BJ (2011) An efficient petiole-feeding bioassay for introducing aqueous solutions into<br />
dicotyledonous plants. Nature Protocols 6: 36-45<br />
Reid DE, Ferguson BJ, Hayashi S, Lin Y-H, Gresshoff PM (2011a) Molecular mechanisms controlling legume autoregulation of nodulation.<br />
Annals of Botany 108: 789-795<br />
Reid DE, Ferguson BJ, Gresshoff PM (2011b) Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule<br />
formation. Molecular Plant-Microbe Interactions 24: 606-618<br />
57<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 8 – Plant Symbiotic Genes<br />
1600 – 1720<br />
Authors: Nijat Imin 1 , Isabel Saur 1 , Nadiatul Radzman 1 , Marie Oakes 1 , Michael A Djordjevic 1<br />
1 Plant Science Division and A. R. C. Centre of Excellence for Integrative Legume<br />
Research , Research School of Biology, Canberra, 0200, Australia.<br />
Presentation Title: Nodule specific CLE peptides mediate crosstalk between local nodule development and<br />
systemic autoregulation of nodulation pathways<br />
Presentation Time: 1640 – 1700<br />
Root nodulation is controlled by the local Nod Factor and cytokinin-dependent pathways and systemically by the<br />
autoregulation of nodulation (AON) pathway (1). The AON pathway controls nodule number by suppressing nodulation<br />
competency in yet-to-be-infected roots via the shoot-expressing SUNN receptor kinase (1-3).<br />
Recently, MtCLE12 and MtCLE13, which show nodule-specific expression and encode CLE (CLAVATA3/ESR) peptide<br />
ligands, have been shown to regulate AON (3). The ectopic expression of MtCLE12 or MtCLE13 (but not MtCLE26 which<br />
does not have nodule specific expression) abolishes nodulation in wild type roots but not in the super nodulating null mutant<br />
sunn-4. This suggests that root MtCLE12 or 13 over-expression triggers constitutive AON which suppresses nodulation<br />
competency in a SUNN-dependent manner. Using qRT-PCR, we show that three local nodulation pathway genes: NODULE<br />
<strong>IN</strong>CEPTION (N<strong>IN</strong>, a common regulator of the Nod Factor and cytokinin pathways), MtEFD (ethylene response factor and<br />
negative regulator of nodule differentiation) and MtRR8 (a type-A response regulator involved in negatively regulating<br />
cytokinin signaling) are each regulated specifically by MtCLE12 in a SUNN-dependent manner. This suggests that MtCLE12<br />
expression in the first formed nodules influences shoot SUNN to produce the AON signal that travels to the yet-to-be<br />
infected roots to up regulate MtEFD and MtRR8 to reduce nodulation competency by down regulating N<strong>IN</strong>. This would<br />
suppress local Nod Factor and cytokinin signaling at a common point: N<strong>IN</strong>. This proposed model links for the first time the<br />
pathways for Nod factor signaling, cytokinin perception and AON and provides a means for the plant to exert a mechanism<br />
to regulate nodule numbers in tune with its physiological status.<br />
58<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 8 – Plant Symbiotic Genes<br />
1600 – 1720<br />
Authors: Senjuti Sinharoy 1 , Ivone Torres-Jerez 1 , Catalina Pislariu 1 , Mingyi Wang 1 , Vagner<br />
Benedito 2 and Michael Udvardi 1<br />
1. Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, Oklahoma 73401<br />
2. West Virginia University, Division of Plant & Soil Sciences, Morgantown, West Virginia<br />
26506<br />
Presentation Title: Identification & functional characterization of Medicago truncatula transcription factor<br />
Presentation Time: 1700 – 1720<br />
mutants with impaired symbiotic nitrogen fixation<br />
Transcriptomic studies have revealed that thousands of plant genes are involved in symbiotic nitrogen fixation<br />
(SNF). Approximately two hundred transcription factor (TF) genes are under spatial control during nodule<br />
development (Pislariu et al.,unpublished data), indicating that they may have an important roles in establishing<br />
successful symbiosis. Very few TFs have been described that are required for SNF. To understand nodule<br />
organogenesis more deeply we need to characterize many more nodule TFs and the gene networks that they<br />
control. We constructed hypothetical gene regulatory networks consisting of TFs and their putative targets,<br />
based on correlation analysis of spatio-temporal gene expression data. Subsets of nodule-expressed TFs were<br />
chosen for further characterization. Medicago mutants with Tnt1-insertions in the chosen TFs were identified by a<br />
PCR-based screen of DNA pooled from the mutant population. Thirty one mutant lines with insertions in thirteen<br />
different TF genes were identified and phenotyped. Several mutants were found to have altered SNF.<br />
Mutants affected in a C2H2-TF gene were characterized in detail. Microscopical analysis of mutant nodules 12<br />
days post inoculation (DPI) indicated that bacteria were released from infection threads and surrounded by the<br />
symbiosome membrane, but remained small and did not elongate like bacteroids in wild type nodules. At 15 DPI,<br />
mutant nodules were dark/black in color, possibly the result of defense responses triggered after bacterial<br />
endocytosis, based on microscopical observations. Expression of the C2H2-TF gene in wild-type nodules was<br />
detected first at 4-DPI and increased around 1600 fold by 8-DPI, with highest expression in nodule zone II<br />
(invasion-zone) followed by zone III (inter-zone), consistent with the phenotype of the mutant. To identify<br />
possible target genes of the C2H2 TF, transcriptome analysis of mutant and wild-type nodules and of<br />
p35S::C2H2-TF and p35S::GFP-transformed roots was carried out, using the Affymetrix Medicago GeneChip.<br />
Detailed results and analysis of this work will be presented.<br />
59<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 9 – Native Legume RNB<br />
1600 - 1720<br />
Authors: Rashmita Parihar 1 , Neetu Poonar 1 , Nisha Tak 1 , Alkesh Tak 1 , Indu Sankhla 1 , Anil<br />
Tripathi 2 , Ravi Tiwari 3 , Euan K. James 4 , Janet I. Sprent 5 and H.S.Gehlot 1<br />
1 Department of Botany, Jai Narain Vyas University, Jodhpur, 342001, India<br />
2 School of Biotechnolgy, Banaras Hindu University, Varanasi, 221005, India<br />
3 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, W Australia<br />
4 James Hutton Institute, EPI Division, Invergowrie, Dundee DD2 5DA, UK<br />
5 Division of Plant Sciences, University of Dundee at JHI, Invergowrie, Dundee DD2<br />
5DA, UK<br />
Presentation Title: Burkholderia phymatum isolated from root nodules of two species of Indigofera from<br />
alkaline soils of the Indian Thar desert<br />
Presentation Time: 1600 – 1620<br />
It is long established that many species in the large Mimosoid genus Mimosa are nodulated by Beta-rhizobia<br />
(which so far include strains of Cupriavidus and Burkholderia), and that in its major centre of diversification in<br />
central Brazil there has probably been co-evolution between Mimosa and its Burkholderia symbionts (Bontemps<br />
et al. 2010; dos Reis Junior et al. 2010). Recently there has also been increasing evidence of nodulation and<br />
nitrogen fixation by Burkholderia in symbiosis with legumes outside the genus Mimosa. These include species of<br />
Cyclopia (Podalyrieae) and Rhynchosia (Phaseoleae) native to South Africa, and the agriculturally important<br />
legume Phaseolus vulgaris has also been shown to harbor B. phymatum as a symbiont in Morocco. In the<br />
present study we have isolated four strains (IL24, IL26, IC12 and IC14) of Burkholderia from root nodules of two<br />
Indian endemic species from the Papilionoid genus Indigofera (I. cordifolia and I. linnaei) growing in alkaline soils<br />
in the semi-arid Thar Desert in Western Rajasthan. Based on 16S rRNA gene sequences the strains are very<br />
close to those isolated from Mimosa spp., P. vulgaris and Indigofera suffruticosa. An ARDRA pattern obtained<br />
using AluI and Sau3AI, as well as an RPO1-based RAPD profile, showed that isolates IL24, IL26, IC12 and IC14<br />
from Indigofera were genetically identical to five isolates of Burkholderia from M. pudica (MP17, MP20, MP21,<br />
MP22 and MP23). Moreover, phenotypic characteristics (eg. colony morphology, pH range, NaCl tolerance,<br />
antibiotic sensitivity and C-utilization patterns) were also similar. These data are discussed in terms of the<br />
legume hosts and their various ecological niches within India, including such characteristics as climate and soils.<br />
Bontemps et al. 2010. Burkholderia species are ancient symbionts of legumes. Mol. Ecol. 19:44-52.<br />
dos Reis Junior et al. 2010. Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga<br />
biomes of Brazil. New Phytol 186:934-946.<br />
Acknowledgement: Authors acknowledge the financial support from ATSE Crawford Training Fund; Centre for<br />
Rhizobium Studies, Murdoch University and DBT, Govt. of India (BT/PR11461/AGR/21/270/2008).<br />
60<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 9 – Native Legume RNB<br />
1600 - 1720<br />
Authors: Robert Walker 1,2 , Graham O‟Hara 2 , Lesley Mutch 1 , Lambert Bräu 2 & Elizabeth Watkin 1<br />
1 School of Biomedical Sciences, Curtin University, Bentley, 6845, Western Australia.<br />
2 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia.<br />
Presentation Title: The phylogenetic diversity of australian Burkholderia root nodule bacteria<br />
Presentation Time: 1620 – 1640<br />
Recently, members of the beta-proteobacteria were described as novel root nodule bacteria (coined betarhizobia)<br />
with many species represented by the genus Burkholderia and some Cupriavidus. Endosymbionts<br />
isolated from nodules of Mimosa spp. are predominantly Burkholderia spp. and this appears to be a preferential<br />
association however, Burkholderia spp. have also been isolated from Papillionoideae and other members of the<br />
Mimosoideae subfamily of legumes. Beta-rhizobia from Australia have been isolated from Acacia spp. and from<br />
members of the tribes Kennediinae and the endemic Mirbeliaea, however in glasshouse trials these isolates form<br />
ineffective nodules on many other Australian legume species. This study examined twelve authenticated<br />
Burkholderia sp. isolated from both native and invasive legumes from New South Wales, Western Australia, the<br />
Northern Territory, and two South African isolates from Rhynchosia ferulifolia. The type strains Burkholderia<br />
phymatum STM815 and Cupriavidus taiwanensis LMG19424 were also included. The phylogenetic diversity of<br />
these isolates was explored by examining the partial sequences from coding regions of the 16S rRNA, recA,<br />
dnaK and atpD genes. To determine the phylogenetic distribution within Australian isolates only, a concatenated<br />
neighbour-joining tree was constructed and most isolates fell into geographical clades. When including non-<br />
Australian isolates, Australian Burkholderia root nodule bacteria appear to form a distinct monophyletic group,<br />
separate to the Burkholderia cepacia complex (BCC) and from non-Australian isolates. Nodulation and nitrogen<br />
fixation genes were also partially sequenced and strongly align to nod and nif genes from Australian<br />
Bradyrhizobium spp. however, very few sequence data exist for the nodulation and nitrogen fixation genes of<br />
Australian Burkholderia root nodule bacteria. Australian root nodule Burkholderia may have evolved from a<br />
distinct monophyletic lineage from other beta-rhizobia allowing them to nodulate Australian legumes with varying<br />
effectiveness providing a competitive advantage over other soil saprophytes however, Australian beta-rhizobia<br />
may not be useful as effective nitrogen fixing endosymbionts.<br />
61<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Authors<br />
Concurrent Session 9 – Native Legume RNB<br />
1600 - 1720<br />
Euan K. James 1 , Osei Y. Ampomah 2 , Pietro P.M. Iannetta 1 , Gregory Kenicer 3 , Geoff<br />
Squire 1 , Janet I. Sprent 4 & Kerstin Huss-Danell 2<br />
1 James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK<br />
2 Department of Agricultural Research for Northern Sweden, Swedish University of<br />
Agricultural Sciences (SLU), SE-90183 Umeả, Sweden<br />
3 Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK<br />
4 Division of Plant Sciences, College of Life Sciences, University of Dundee at James<br />
Hutton Institute, Dundee DD2 5DA, UK<br />
Presentation Title: Nodulation and nitrogen fixation in native legumes in Scotland and Sweden<br />
Presentation Time: 1640 – 1700<br />
Native legumes in temperate and northern areas have received surprisingly little research attention. Our aim was<br />
to get an overview of the nodulation of native legumes in Scotland and Sweden. We collected 24 out of the 47<br />
native legume species in Scotland and 30 out of the 90 in Sweden; 17 species were from both countries. A wide<br />
range of habitats were visited. A majority of our collected legumes, e.g. Lathyrus pratensis, Lotus corniculatus,<br />
Medicago lupulina, Trifolium spp. and Vicia spp. occurred in many habitats such as meadows, edges of<br />
cultivated land, waste ground and road sides. In contrast, other species grew in more specific habitats:<br />
Astragalus danicus, Lathyrus japonicus, L. palustris, Tetragonolobus maritimus and Vicia lutea on sea shores<br />
and A. alpinus, A. frigidus, Oxytropis campestris and O. lapponica in northern and mountain areas. All collected<br />
species were nodulated. Indeterminate (Astragalus, Cytisus, Lathyrus, Medicago, Melilotus, Ononis, Oxytropis,<br />
Trifolium, Ulex and Vicia, spp.) and determinate (Anthyllis, Lotus and Tetragonolobus spp.) nodule types were<br />
represented. <strong>Nitrogen</strong> fixation was deduced from pink nodule interior. Nodule anatomy, immunolocalization of<br />
nitrogenase and foliar 15 N analysis gave further support for active nitrogen fixation at time of collection. Some<br />
native species are closely related to cultivated species of Lotus, Medicago, Trifolium and Vicia. Root nodule<br />
bacteria from cultivated and native plants of Lotus corniculatus in Sweden mainly belonged to Mesorhizobium loti<br />
even though other Mesorhizobium spp. were associated with some of the naturally growing L. corniculatus plants<br />
(Ampomah & Huss-Danell 2011). It seems that native legumes are important to the N cycle at their sites and, in<br />
addition, some of their root nodule bacteria may be of interest as inoculants in agriculture.<br />
Ampomah OY& Huss-Danell K (2011). Genetic diversity of root nodule bacteria nodulating Lotus corniculatus<br />
and Anthyllis vulneraria in Sweden. Syst Appl Microbiol 34:267-275.<br />
62<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Tuesday 29 November 2011<br />
Concurrent Session 9 – Native Legume RNB<br />
1600 - 1720<br />
Authors: Wen Feng Chen 1 , Chang Fu Tian 1 , Yan Ming Zhang 1 , Qin Qin Li 1 , Jun Jie Zhang 1 , Mao<br />
Li 1 , Tian Yan Liu 1 , En Tao Wang 1,2 , Xing Hua Sui 1 , & Wen Xin Chen 1*<br />
1 State Key Laboratory for Agrobiotechnology/College of Biological Sciences, China<br />
Agricultural University, Beijing, 100193, China.<br />
2 Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto<br />
Politécnico Nacional, 11340 México D. F., México<br />
Presentation Title: Advances in rhizobial diversity and biogeography in China<br />
Presentation Time: 1700 – 1720<br />
Besides the common genera, including the Rhizobium, Bradyrhizobium, Ensifer (former Sinorhizobium),<br />
Mesorhizobium which are widespread in China, nodule bacteria associated with leguminous plants are also<br />
found in the genera of Burkholderia, Cupriavidus, Shinella and Iniquilinus. By using the polyphasic taxonomy and<br />
multilocus sequence analysis (MLSA), nearly 30 novel species were described and published in China since<br />
2006, accounting for more than half of the total novel species published around the world during the same<br />
period. Some rhizobial species were only found and reported until now in China though their symbiotic genes<br />
were found worldwide. The rhizobial chromosomal core genes and the accessorial symbiotic genes may evolve<br />
in different ways to ensure the rhizobia to survive in the environments and to invade the host plant respectively.<br />
The increased reports on the non-symbiotic rhizobia isolated from root nodules will be the hot spots for the study<br />
of rhizobial diversity in China. The origin of the symbiotic genes and the transferring of them from symbiotic<br />
rhizobia to non-symbiotic ones may depend on the mechanism of “loss and gain”. To study the soil factors<br />
affecting the diversity and distribution of rhizobia in different regions, biogeography of the rhizobia associated<br />
with soybean (Glycine max), as example, were further extensively investigated. We will discuss the crucial<br />
factors that determine the distribution of genera of Bradyrhizobium, Sinorhizobium and Mesorhizobium<br />
associated with the soybean and the relationship between rhizobia and host plants.<br />
63<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Plenary Session 5<br />
0900 - 1030<br />
Author: Janet Sprent<br />
University of Dundee, UK<br />
Presentation Title: Poles apart: Nodulation in native legumes from the Southern and Northern hemispheres<br />
Presentation Time: 0900 – 0930<br />
Publications on legume biogeography generally ignore nodulation parameters and are usually discussed from a<br />
longitudinal perspective, with a bias towards the Old and New worlds (1). However, it has long been known that<br />
nodulation parameters can be useful taxonomic ones (2). Recent evidence on the evolution of nodulation (3), on<br />
the functions of different cells inside nodules (4) and on both the nature of nodulating bacteria (5) and whether or<br />
not they become terminally differentiated (6) suggests that there may be a latitudinal dimension to nodule<br />
evolution. This paper discusses nodulated legumes north and south of the equator pointing out both differences<br />
and similarities. Some of the former may be related to soil nutrient levels, which in much of the Northern<br />
hemisphere are relatively rich and much of the Southern hemisphere relatively poor, leading in the latter to many<br />
legumes evolving a broader suite of nutrient uptake systems (dual mycorrhizas, cluster roots) than those in the<br />
North (7). Further, nodulated caesalpinioid legumes and most of the nodulated mimosoids are usually in low<br />
latitudes on either side of the equator. On the other hand, certain groups of legumes in the North have extended<br />
into the Arctic Circle and these too may have particular characters. Bridging the N/S divide are many legume<br />
genera, for example Indigofera, which are adapted to dry environments (8). Major questions remain to be<br />
answered, including the reasons why nodulation evolved on several different occasions, why some legumes that<br />
lack uninfected cells in the infected region appear to function well, why (in the Southern hemisphere) legumes<br />
growing side-by-side have a preference for either alpha or beta proteobacteria and cases where current<br />
taxonomy does not align with nodule characters.<br />
References numbered (1) to (8) will be given in full in the talk and made available to anyone interested.<br />
64<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Plenary Session 5<br />
0900 - 1030<br />
Authors: Erik Limpens 1 , Sergey Ivanov 1 , Evgenia Ovchinnikova 1,2 , Alexey Borisov 2 , Rik op den<br />
Camp 1 , Stephane de Mita 3 , René Geurts 1 , Elena Fedorova 1,4 , Ton Bisseling 1<br />
1 Laboratory of Molecular Biology, Wageningen University, The Netherlands.<br />
2 All-Russia Research Institute for Agricultural Microbiology, Laboratory of Genetics of<br />
Plant-Microbe Interactions, St. Petersburg, Russia.<br />
3 Plant Diversity and Adaptation, Institute for Research and Development, Montpellier,<br />
France.<br />
4 Timiryazev Institute of Plant Physiology RAS, Moscow, Russia.<br />
Presentation Title: Formation of a symbiotic interface in rhizobial and mycorrhizal symbioses<br />
Presentation Time: 0930 – 1000<br />
In both the rhizobium-legume symbiosis and the arbuscular mycorrhizal symbiosis the microbes are hosted<br />
intracellularly in a novel, specialized membrane compartment that forms a symbiotic interface. The formation of<br />
this interface is at the heart of symbiosis. In the case of rhizobia they are accommodated as novel nitrogen-fixing<br />
organelles, called symbiosomes, inside infected cells of the root nodule. Arbuscular mycorrhizal (AM) fungi form<br />
highly branched hyphal structures, called arbuscules, in infected root cortical cells. In both cases the microbial<br />
symbiont is surrounded by a plant derived membrane that has a special plasma membrane identity and where<br />
generally a structured plant cell wall is lacking. It has recently become clear that the signaling molecules and<br />
signaling pathway that trigger these different symbioses are strikingly similar. We show that this signaling<br />
pathway also controls the formation of the symbiotic interfaces in both symbioses. The observed plasma<br />
membrane identity of both interface membranes indicates a major role for exocytotic vesicle traffic. By studying<br />
plasma membrane (PM) SNARE proteins we show that knock-down of specific PM SNARE members blocks the<br />
release of the bacteria from cell wall bound infection threads and/or subsequent symbiosome development.<br />
Furthermore, two specific SNARE proteins required for symbiosome formation are also essential for arbuscule<br />
formation by AM fungi. These results suggest that in evolution the ability to produce Nod factors gave rhizobia<br />
the ability to use the ancient AM machinery to recruit a specific exocytotic pathway for the establishment of a<br />
symbiotic interface.<br />
65<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Plenary Session 5<br />
0900 - 1030<br />
Authors: Doreen Fischer 1 , Barbara Pfitzner 1 , Veronica M. Reis 2 , Jean L. Simoes-Araújo 2 , Michael<br />
Schmid 1 , Esperanza Martinez-Romero 3 , Jose I. Baldani 2 , Anton Hartmann 1<br />
1 Helmholtz Zentrum Munich, German Research Center for Environmental Health,<br />
Research Unit Microbe-Plant Interactions, Neuherberg, Germany<br />
2 EMBRAPA-CNPAB Seropedica, Seropédica, RJ, Brazil<br />
3 Centro de Ciencias Genomicas, Universidad Nacional Autonoma de Mexico, UNAM,<br />
Cuernavaca, Morelos, Mexico<br />
Presentation Title: Molecular characterization of the active diazotrophic community in uninoculated and<br />
inoculated field-grown sugarcane (Saccharum sp.)<br />
Presentation Time: 1000 – 1030<br />
Certain cultivars of sugar cane are known for their high potential of supporting biological nitrogen fixation.<br />
Although many different diazotrophic bacteria have been isolated from sugar cane and demonstrated to colonize<br />
roots, stems and leaves of these plants in considerable number, no clear evidence exists about the type of<br />
diazotrophic bacteria which fixes nitrogen in planta and about the locations of nitrogen fixation activity. To identify<br />
active diazotrophs in field grown sugarcane, 16S rRNA and nifH transcript analyses were applied. This should<br />
help to better understand the basis of the biological nitrogen fixation (BNF) activity in a high nitrogen fixing<br />
sugarcane variety. A field experiment using the sugarcane variety RB 867515 was conducted in Seropédica, RJ,<br />
Brazil, receiving the following treatments: unfertilised and fertilised controls without inoculation, unfertilised with<br />
inoculation. The five-strain mixture of diazotrophic bacteria containing Gluconacetobacter diazotrophicus,<br />
Burkholderia tropica, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans and Azospirillum amazonense,<br />
developed by EMBRAPA-CNPAB, was used as inoculum. Root and leaf sheath samples were harvested in the<br />
third year of cultivation (after receiving an inoculum at the beginning of the season) to analyse the 16S rRNA and<br />
nifH transcript diversity. In addition to nifH expression from Gluconacetobacter spp. and Burkholderia spp., a<br />
wide diversity of nifH transcripts from previously uncharacterised Ideonella / Herbaspirillum related phylotypes in<br />
sugarcane shoots as well as Bradyrhizobium sp. and Rhizobium sp. in roots were found. These results were<br />
confirmed using 16S cDNA analysis. From the inoculated bacteria, only nifH transcripts from G. diazotrophicus<br />
and B. tropica were detected in leaf sheaths and roots at the time of harvest in late summer, respectively. Thus,<br />
well known as well as yet uncultivated diazotrophs were found active in sugarcane roots and stems using<br />
molecular analyses. Two strains of the inoculum mix were found active at the late summer harvest.<br />
66<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
The Cape Floristic Region (CFR) has a mosaic of soil types derived from parent materials including sandstone,<br />
granite, limestone and shale. It has high species diversity, varying among soil types and environmental<br />
gradients. Among legumes, particular lineages appear to be restricted to one or few substrates, raising questions<br />
on whether their species distribution is predetermined by symbiotic bacteria. We hypothesized that rhizobial<br />
isolates from indigenous legumes would cluster phylogenetically according to soil types and/or host legume.<br />
Rhizobia were isolated from nodules collected from 87 species in over 12 genera belonging to tribes<br />
Crotalarieae, Indigofereae, Phaseoleae, Podalyrieae and Psoraleae. Sequenced data using the 16S rRNA gene<br />
revealed the presence of both α- (Bradyrhizobium, Mesorhizobium, Rhizobium, Synorhizobium) and β-rhizobia<br />
(Burkholderia), the latter nearly exclusive to the tribe Podalyrieae. Mesorhizobium was isolated from Aspalathus,<br />
Otholobium, Psoralea, Indigofera and Argyrolobium species collected from all substrates, while Bradyrhizobium<br />
species were isolated from Indigofera and Tephrosia species collected from granite and sandstone-derived soils.<br />
Burkholderia species were isolated from Podalyria, Amphithalia and Rafnia species collected from granite,<br />
coastal sand and limestone derived soils. These data were confirmed by immunogold labeling of the bacteria in<br />
nodule sections with an antibody specific to Burkholderia (Elliott et al. 2007a). Furthermore, a GFP-tagged strain<br />
of B. tuberum STM678, which has previously been shown to nodulate species of Cyclopia (tribe Podalyrieae<br />
native to the CFR; Elliott et al. 2007b), could effectively nodulate species of Podalyria and Virgilia. The<br />
phylogenies of the rhizobia isolates from the 16S rRNA gene sequence and of the host legumes from literature<br />
are discussed in relation to the evolutionary relationships of the symbiotic partners. The available data suggest<br />
that the soil types do not influence the distribution of rhizobia in the CFR.<br />
Elliott, G. N. et al. 2007a. New Phytol. 173:168-180.<br />
Elliott, G. N. et al. 2007b Ann. Bot. 100:1403-1411.<br />
Concurrent Session 10 – Ecology of RNB<br />
1100 – 1230<br />
Authors: Oscar Dlodlo 1 , A. Muthama Muasya 1 , Janet I. Sprent 2 , Euan K. James 3 , Wen-Ming<br />
Chen 4 , Samson B.M. Chimphango 1<br />
1 University of Cape Town, Botany Department, Private Bag X3 Rondebosch, South<br />
Africa.<br />
2 Division of Plant Sciences, University of Dundee at JHI, Invergowrie, Dundee DD2<br />
5DA, UK<br />
3 James Hutton Institute, EPI Division, Invergowrie, Dundee DD2 5DA, UK<br />
4 Dept. of Seafood Science, National Kaohsiung Marine University, Kaohsiung City 811,<br />
Taiwan<br />
Presentation Title: Characterization of the cape floristic region rhizobia using 16S rRNA gene sequences<br />
and their distribution in different soil types<br />
Presentation Time: 1100 – 1120<br />
67<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 10 – Ecology of RNB<br />
1100 – 1230<br />
Authors: Macarena Gerding 1,2 , Graham O‟Hara 1 , Lambert Bräu 1 & John Howieson 1<br />
1 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia.<br />
2 Facultad de Agronomía, Universidad de Concepción, Casilla 537, Chillán, Chile.<br />
Presentation Title: Mesorhizobium strains from the South African herbaceous legume Lessertia spp.<br />
Presentation Time: 1120 – 1140<br />
differ in their competitive ability in Australian soils<br />
Legumes of the South African genus Lessertia, along with their microsymbionts, were introduced into different<br />
sites of the Western Australia wheatbelt during 2007-2009. Four Lessertia spp. achieved only poor establishment<br />
followed by weak summer survival. This was caused in part by low levels of nodulation with the inoculant strains,<br />
and by competitive ineffective nodulation from naturalized strains of Rhizobium leguminosarum bv. trifolii. To<br />
improve the adaptation of the legume and to assess the competitive ability of inoculant strains, two field<br />
experiments were set up at Karridale, Western Australia. The aims were to assess the effect of increased doses<br />
of an effective inoculant strain (WSM3565) with L. herbacea, and to study the competitive ability and symbiotic<br />
performance of different Mesorhizobium strains nodulating L. diffusa.<br />
Increasing the inoculation dose of L. herbacea with WSM3565 did not improve establishment and survival of the<br />
legume. Although WSM3565 nodule occupancy improved from 28 to 54% with higher doses of inoculation, none<br />
of the treatments increased L. herbacea yield over the inoculated control.<br />
The inoculation of L. diffusa with the strains WSM3598, 3636, 3626 and 3565 resulted in greater biomass<br />
production than the uninoculated control. These strains were able to outcompete resident rhizobia and to occupy<br />
a high (>60%) proportion of lateral root nodules. Low rainfall in 2009 caused an early senescence of tap root<br />
nodules and plants had to rely on nitrogen fixation from nodules formed later in the season (i.e. lateral root<br />
nodules). This possibly explained the increased dry weight achieved by these strains.<br />
The high numbers of resident rhizobia in Western Australian soils, and their ability to rapidly nodulate Lessertia<br />
spp. are likely to be the main reasons for the low nodule occupancy achieved by some effective inoculant strains<br />
with L. diffusa and L. herbacea. The naturalised strains that achieved nodulation were identified as R.<br />
leguminosarum bv. trifolii, whereas in the South African soils Lessertia is nodulated by mesorhizobia. This<br />
phenomenon represents a barrier to the successful introduction of the exotic legume genus Lessertia to WA<br />
soils.<br />
68<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 10 – Ecology of RNB<br />
1100 – 1230<br />
Authors: Julie K. Ardley 1 , Graham O‟Hara 1 , Wayne Reeve 1 , Ron Yates 1,2 , Michael Dilworth 1 , and<br />
John Howieson 1<br />
1 Centre for Rhizobium Studies, Murdoch University, Murdoch W. A. 6150, Australia<br />
2 Department of Agriculture Western Australia, Baron Hay Court, South Perth, 6151,<br />
Western Australia<br />
Presentation Title: The symbiosis between Listia spp. and Methylobacterium and Microvirga rhizobia:<br />
specificity in epidermally infected legumes<br />
Presentation Time: 1140 – 1200<br />
Lotononis is a genus in the Crotalarieae tribe with a centre of origin in South Africa. The taxonomy has recently<br />
been revised and the three distinct clades of this genus are now recognised at the generic level as Listia,<br />
Leobordea and Lotononis sensu stricto (Boatwright et al., 2011). Different symbiotic specificity groups exist<br />
within Lotononis sensu lato, which is nodulated by a remarkable diversity of rhizobia. Leobordea and Lotononis<br />
s.str. species form indeterminate nodules and are more or less promiscuous. Rhizobia associated with these<br />
legumes are related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and<br />
Methylobacterium nodulans, based on sequence analysis of the 16S rRNA gene. Listia species are waterlogging<br />
tolerant, have adventitious roots and form lupinoid nodules. They are specifically nodulated by pink-pigmented<br />
methylobacteria that are effective nitrogen fixers on all studied species, with the exception of Listia angolensis,<br />
which forms ineffective nodules with these rhizobia (Yates et al., 2007). L. angolensis forms effective nodules<br />
only with novel rhizobial species of the Alphaproteobacterial genus Microvirga (Ardley et al, submitted paper).<br />
The nodA sequences of the Lotononis s. l. rhizobia are polyphyletic and group according to microbial<br />
chromosomal background rather than host plant taxonomy. L. angolensis and Listia bainesii appear to be<br />
infected via epidermal entry, with no formation of infection threads, and are examples of symbiotic specificity in a<br />
non root-hair-mediated infection process. The genomes of Methylobacterium sp. 4-46 and Microvirga strain<br />
WSM3557 have been sequenced and will greatly aid an understanding of the genetic underpinnings of these<br />
symbioses. A preliminary analysis of the WSM3557 genome compared with that of M. sp. 4-46 will also be<br />
presented.<br />
Boatwright, J. S., Wink, M. & van Wyk, B.-E. (2011). The generic concept of Lotononis (Crotalarieae,<br />
Fabaceae): Reinstatement of the genera Euchlora, Leobordea and Listia and the new genus Ezoloba. Taxon 60,<br />
161-177.<br />
Yates, R. J., Howieson, J. G., Reeve, W. G., Nandasena, K. G., Law, I. J., Bräu, L., Ardley, J. K.,<br />
Nistelberger, H. M., Real, D. & O'Hara, G. W. (2007). Lotononis angolensis forms nitrogen fixing, lupinoid<br />
nodules with phylogenetically unique, fast-growing, pink-pigmented bacteria, which do not nodulate L. bainesii or<br />
L. listii. Soil Biology & Biochemistry 39, 1680-1688.<br />
69<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 10 – Ecology of RNB<br />
1100 – 1230<br />
Authors: Tomasz Stępkowski 1 , Alison McInnes 2 , Elizabeth Watkin 3 , Dorota Narożna 4 , Magdalena<br />
Mantaj 1 , Justyna Rudnicka 1 , Graham O‟Hara 5 , Emma T. Steenkamp 6<br />
1 Institute of Bioorganic Chemistry Polish Academy of Sciences, Poznań , Poland;<br />
2 Centre for Plant and Food Science, University of Western Sydney, Penrith South DC,<br />
Australia;<br />
3 School of Biomedical Sciences, Curtin University, Perth, Australia<br />
; 4 Department of Biochemistry and Biotechnology, Poznan University of Life Sciences,<br />
Poznan , Poland;<br />
5 Centre for Rhizobium Studies, Murdoch University, Murdoch, Australia<br />
; 6 Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria,<br />
South Africa<br />
Presentation Title: Diverse origin of Bradyrhizobium strains nodulating legumes native to the tropicalmonsoon<br />
part of Australia<br />
Presentation Time: 1200 – 1220<br />
The geographical isolation of Australia, together with the formation of an arid climate in its central regions and a<br />
tropical-monsoon climate in its northern part impacted significantly on the evolution of legumes native to the<br />
continent. Additionally, the northward drift of the Australian plate and its collision with the Euroasian plate<br />
facilitated the colonization of Australia by many alien legume taxa during the last ten million years. Conceivably,<br />
these factors also shaped the evolution of root-nodule bacteria associated with these legumes, and contributed<br />
to the high diversity of indigenous rhizobium communities and the presence of many groups unique to Australia.<br />
By making use of multilocus sequence analysis we investigated 132 strains of Bradyrhizobium, which originated<br />
from the tropical-monsoon part of the Northern Territory. All strains were obtained from root nodules of legumes<br />
belonging to endemic or native Australian tribes, genera or species. Phylogenetic analyses based on six<br />
housekeeping genes (atpD, dnaK, glnII, gyrB, recA and rpoB) separated the strains into 16 lineages, of which<br />
most were different from the known species of Bradyrhizobium. Phylogenies based on the symbiotic nodA and<br />
nifD genes separated the strains into six clades (i.e., Clade I (subgroup I.2), III (subgroup III.3), IV, V, VII and<br />
VIII), out of the eight clades that have been described thus far for Bradyrhizobium. Legumes with centers of<br />
diversification located outside Australia were nodulated predominantly by strains belonging to Clade III, which is<br />
regarded as a cosmopolitan, pantropical group. Surprisingly, this was also true for all strains from Clade IV,<br />
which suggests that this apparently Australian clade may have originally diversified outside this continent.<br />
Conversely, the legumes with Australian centers of diversity (e.g. phylodinous Acacia spp.) were nodulated by<br />
strains representing the Australian Clade I and Clade VIII, but also by Clade V, which also occurs in South<br />
America.<br />
70<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 11 – Nodule Formation<br />
1100 – 1230<br />
Authors: Shin Okazaki and Kazuhiko Saeki<br />
Department of Biological Sciences, Faculty of Science, Nara Women’s University, Nara<br />
630-8506, Japan<br />
Presentation Title: Hijacking the host nodulation signaling by rhizobial type III secretion system<br />
Presentation Time: 1100 - 1120<br />
Root nodule symbiosis between leguminous plants and nitrogen-fixing bacteria (rhizobia) requires molecular<br />
communication between both partners. Key components for the establishment of symbiosis are host plantderived<br />
flavonoids that induce the transcription of rhizobial nodulation (nod) genes and rhizobium-produced<br />
lipochitooligo-saccharides (Nod-factors) that initiate nodule development and bacterial entry. Besides the Nodfactors<br />
there are other determinants that influence the extent of the symbiosis. Among them, we have focused on<br />
a rhizobial protein secretion system, called type III secretion system (T3SS).<br />
T3SSs play an essential role in the pathogenicity of many bacteria infecting humans, animals and plants.<br />
Pathogenic bacteria use the T3SS to deliver effector proteins directly into eukaryotic cells or the external<br />
environment, where they manipulate host cellular processes to promote pathogenicity. The T3SSs have been<br />
identified in rhizobia and were shown to affect symbiosis with leguminous hosts. In this study, we analyzed the<br />
role of T3SS in the interaction between Bradyrhizobium elkanii and soybean (Glycine max (L.) Merr.).<br />
Mutational analysis and inoculation tests of B. elkanii USDA61 revealed that the presence of T3SS affected<br />
symbiotic capacity either positively or negatively depending on host genotype. On G. max cv. Enrei, wild-type<br />
USDA61 induced more nodules than T3SS mutant. On the other hands, cultivar Hill interdicted nodulation by the<br />
wild type but was nodulated by the T3SS mutant. Intriguingly, when infected to the soybean mutant En1282 that<br />
has defective Nod factor receptor 1 (NFR1) and show non-nodulating phenotype with B. japonicum and other<br />
rhizobial strains, USDA61 but not its T3SS mutant induced effective nodules. Transcriptional analysis revealed<br />
that the expression of early nodulation gene ENOD40 and N<strong>IN</strong> was increased in the root of En1282 inoculated<br />
with wild type but not with T3SS mutant. These results suggest that T3SS of USDA61 has functions to enforce<br />
legume host to initiate symbiotic programs by bypassing Nod-factor recognition.<br />
71<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 11 – Nodule Formation<br />
1100 – 1230<br />
Authors: Christian Staehelin, Ling Zhang, Ying-Ying Ge, Da-Wei Xin, Sha Liao and Zhi-Ping Xie<br />
State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-Sen (Zhongshan)<br />
University, East Campus, Guangzhou 510006, China<br />
Presentation Title: Characterization of type 3 effectors in the Rhizobium-legume symbiosis<br />
Presentation Time: 1120 - 1140<br />
Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) into<br />
eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation<br />
in the interaction with various host legumes. Here, we characterized NopL and NopM, two type 3 effectors of<br />
Rhizobium sp. strain NGR234. Nodulation tests and microscopic analysis showed that distinct necrotic areas<br />
were rapidly formed in ineffective nodules of Phaseolus vulgaris (cv. Tendergreen) induced by a nopL mutant,<br />
indicating that NopL antagonized nodule senescence. Further experiments revealed that NopL interfered with<br />
mitogen-activated protein kinase (MAPK) signaling in yeast and plant cells (Nicotiana tabacum). Expression of<br />
nopL in yeast disrupted the mating pheromone (α-factor) response pathway, whereas nopL expression in N.<br />
tabacum suppressed cell death induced by over-expression of the MAPK gene SIPK (salicylic acid-induced<br />
protein kinase). NopL was multiply phosphorylated either in yeast or N. tabacum cells that expressed nopL. Four<br />
phosphorylated serines were confirmed by mass spectrometry. All four phosphorylation sites exhibit a Ser-Pro<br />
pattern, a typical motif in MAPK substrates. These data suggest that NopL is a suppressor of MAPK signaling.<br />
NopM, another type 3 effector of Rhizobium sp. NGR234 displayed E3 ubiquitin ligase activity in vitro, whereas<br />
the mutant protein NopM-C338A was inactive. Corresponding mutant analysis indicated that NopM also acts as<br />
an E3 ubiquitin ligase during symbiosis with Lablab purpureus, providing evidence that NopM is delivered into<br />
legume host cells and targets host proteins. Finally, effects of NopM on eukaryotic cells will be reported.<br />
Zhang L, Chen X-J, Lu H-B, Xie Z-P, & Staehelin C (2011). Functional analysis of the type 3 effector NopL from<br />
Rhizobium sp. NGR234: symbiotic effects, phosphorylation and interference with MAPK signaling. J. Biol. Chem.<br />
in press<br />
72<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 11 – Nodule Formation<br />
1100 – 1230<br />
Authors: Masaki Hanyu 1,2 , Shin Okazaki 1 , Rie Shirai 1 and Kazuhiko Saeki 1<br />
1 Department of Biological Sciences, Faculty of Science, Nara Women’s University,<br />
Nara630-8506<br />
2 Department of Biological Sciences, Graduate School of Science, Osaka University,<br />
Toyonaka, Osaka 560-0043, Japan<br />
Presentation Title: Mesorhizobium loti mutant lacking superoxide dismutase displays Lotus-host accessiondependent<br />
nitrogen fixation capacities<br />
Presentation Time: 1140 - 1200<br />
Protection against reactive oxygen species (ROS) is important for legume-nodulating rhizobia during the<br />
establishment and maintenance of symbiosis as well as under free-living conditions because legume hosts might<br />
assail incoming microbes with ROS and because nitrogenase is extremely sensitive to ROS. We generated a<br />
deletion mutant of the putative superoxide dismutase (SOD) gene, with locus tag mlr7636, in Mesorhizobium loti<br />
MAFF303099 to investigate physiological significance. Under free-living conditions, the mutant displayed a<br />
number of properties typical of SOD-lacking strains. Its cell extract showed no general SOD activity under the<br />
assay conditions that enabled to detect the activity of the wild-type. It proliferated very slowly and showed<br />
elevated sensitivity to exogenous superoxide generators. Its spontaneous mutation rate was approximately 20times<br />
of that of wild-type. These defects were complemented by re-introduction of the wild-type gene. Based on<br />
the result and sequence similarity with Escherichia coli enzyme, we named the gene sodA. With regard to<br />
symbiotic capacities with Lotus japonicus, the sodA mutant showed quasi-wild-type nodulation and nitrogen<br />
fixation efficiencies with the host accession Gifu B-129, whereas it showed low nodulation efficiency and poor<br />
support of plant growth with the host accession Miyakojima MG-20.<br />
73<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Rhizobium-legume symbiosis is an ecologically friendly source of nitrogen to improve soil fertility. <strong>Nitrogen</strong>-fixing<br />
Rhizobia are situated in root nodule in a specialized endosomes called symbiosomes (SBs). Host plant derived<br />
SB membrane (SM) is the interface providing the metabolic exchange between two partners. During the<br />
development SBs are multiplying and growing in volume more than 40 times. We have performed the<br />
quantitative estimation of nodule infected cell structures in the course of symbiosis using 3D reconstruction of<br />
confocal images of Medicago truncatula. We have specified the particular cell layer with the highest level of<br />
symbiosomes growth. Surprisingly in this cell layer host cell vacuole volume was drastically diminished. We<br />
hypothesized that transport of water is redirected in this cell layer from vacuole to bacteroids and potential<br />
symbiosome membrane aquaporins should be involved. Q-PCR analysis of 6 tonoplast aquaporins expression<br />
level has revealed that MtTIP1g is significantly upregulated. The localization of GFP-tagged MtTIP1g has shown<br />
host tonoplast labeling in all nodule cells, but SMs acquired GFP only in the cell layer where most rapid growth of<br />
SBs happens. Functional analysis of MtTIP1g in root nodules using an RNAi method showed that partial<br />
silencing of MtTIP1g is causing the arrest of SBs development and preventing the formation of nitrogen-fixing<br />
SBs.<br />
According to our previous data symbiosome membrane has mixed identity and coopting the molecular markers<br />
of exo- and endocytotic pathway (Limpens et al., 2009). It is established that SBs are accepting plasma<br />
membrane identity immediately after being released to host cell, but the identity of young vacuole it obtains much<br />
later. The functional significance of this identity change was not known. Our data are providing the clue to<br />
functionality of SM identity. The acceptance of vacuole identity is crucial as it permits to recruit vacuolar proteins<br />
like MtTIP1g and promote an extremely rapid and robust SBs growth.<br />
Limpens et al., Plant Cell 21: 2811–2828 (2009).<br />
Concurrent Session 11 – Nodule Formation<br />
1100 – 1230<br />
Authors: Aleksandr Gavrin 1 , Sergey Ivanov 1 , Ton Bisseling 1 , Elena Fedorova 1<br />
1 Laboratory of Molecular Biology, Wageningen University, Wageningen, 6708PB, The<br />
Netherlands<br />
Presentation Title: The role of symbiosome membrane aquaporin MtTIP1g in bacteroids growth and<br />
maturation<br />
Presentation Time: 1200 - 1220<br />
74<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 12 – PGPR & Plant Production<br />
1100 – 1230<br />
Authors: Sharon Fox 1 , Graham O‟Hara 1 and Lambert Bräu 1<br />
1 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia.<br />
Presentation Title: Differential effects of Pseudomonas fluorescens WSM3457 on enhanced<br />
Medicago nodulation<br />
Presentation Time: 1100 - 1120<br />
Pseudomonas fluorescens WSM3457 has been studied as a nodulation increasing bacteria (NIB) since 2006<br />
due to increases in the nodulation of subterranean clover reported in glasshouse and field trials. We have<br />
demonstrated a similar phenomenon on the Medicago truncatula / Sinorhizobium medicae WSM419 symbiosis.<br />
During glasshouse trials it was observed that co-inoculation results in larger, pinker nodules, located higher on<br />
the root system, compared to M. truncatula inoculated with only S. medicae WSM419.<br />
Nodule location suggested that nodules were elicited earlier on co-inoculated plants and this was confirmed<br />
during studies comparing the rate of nodule initiation on M. truncatula inoculated with either S. medicae WSM419<br />
or both WSM419 and P. fluorescens WSM3457. The results also demonstrated that there was a significant<br />
increase in symbiotic effectiveness, both in terms of plant mass and total fixed N.<br />
Following those results it was investigated whether co-inoculation increased the effectiveness of a partially<br />
effective symbioisis, namely that of the symbiosis between S. meliloti 1021 with M. truncatula. The results show<br />
that there was no significant increase in shoot mass with co-inoculation with P. fluorescens WSM3457. However,<br />
the nodulation data tells a conflicting story, with enhanced nodulation scores on M. truncatula when P.<br />
fluorescens WSM3457 was co-inoculated with S. meliloti 1021.<br />
75<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 12 – PGPR & Plant Production<br />
1100 – 1230<br />
Authors: Khanok-on Amprayn and Ivan Kennedy<br />
SUNFix Centre for <strong>Nitrogen</strong> <strong>Fixation</strong><br />
University of Sydney, NSW 2006, Australia<br />
Presentation Title: Proteomics of rice-PGP microorganisms' interactions<br />
Presentation Time: 1120 - 1140<br />
A plant growth-promoting (PGP) bacterium Pseudomonas fluorescens 1N (Pf1N) and a soil yeast Candida<br />
tropicalis HY (CtHY) have been used as two of the components of a commercial biofertiliser named BioGro to<br />
enhance growth and yield of rice allowing reduce chemical applications in Vietnam for many years. The present<br />
study verified the colonisation on rice roots and the effects of the strains on rice root mechanisms as observable<br />
morphological changes were correlated with results by proteomics. In addition, bacterial proteins mediating the<br />
interaction between rice root and Pf1N were investigated.<br />
To explain the strains influence on rice root growth, gel-based comparative proteomics and mass spectrometry<br />
were employed. A comparison of 2-DE protein profiles between untreated control and PGP microbes treated<br />
roots revealed that the inoculation of the Pf1N contributed 11 differentially expressed proteins in rice roots, while<br />
20 and 18 root proteins were differentially expressed in CtHY and co-inoculation of Pf1N and CtHY treatment. Of<br />
these changed proteins, the identity of 20 proteins was determined by MALDI TOF MS. The protein identification<br />
revealed that responsive plant proteins belonged to eight major metabolic processes including catabolism and<br />
respiration (energy production), transcription, protein synthesis, protein destination and storage, secondary<br />
metabolism, membrane transporters, disease-defense, and unknown.<br />
Conversely, the bacterial proteome alteration in presence of root exudates was also profiled using 2-DE and MS<br />
to examine of bacterial adaptation in association with rice. The bacterial protein expressions revealed several<br />
mechanisms involved in protein transcription and synthesis, primary metabolism, cell envelope development,<br />
siderophore production, nutrient transportation, general stress response, and unknown. The patterns of protein<br />
expression of Pf1N such as elevated iron uptake mechanisms, repressed essential growth-related proteins and<br />
ABC transporter suggested that the bacterium was adapted to form biofilm.<br />
76<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 12 – PGPR & Plant Production<br />
1100 – 1230<br />
Authors: Iris Marcano 1 , César A. Díaz-Alcántara 1 , Beatriz Urbano 2 , Encarna Velázquez 3 , Fernando<br />
González-Andrés 4 .<br />
1 Facultad de CC. Agronómicas y Veterinarias. Univ. Autónoma de Santo Domingo.<br />
Dominican Republic.<br />
2 Departamento de Ingeniería Agrícola y Forestal. Universidad de Valladolid. Spain.<br />
3 Departamento de Microbiología y Genética. Universidad de Salamanca. Spain.<br />
4 Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad. Universidad de León.<br />
Spain.<br />
Presentation Title: In vitro plant growth promotion properties of endophitic bacteria isolated from banana<br />
(Musa sp.) roots in Dominican Republic<br />
Presentation Time: 1140 - 1200<br />
Organic agriculture, using rizospheric or endophytic bacteria which promote plants growth (PGPR) is one of the<br />
chief strategies to reduce the use of Chemicals in agriculture. The present study had the objective of evaluates<br />
endophytic rhizobacteria isolated from banana roots (Musa sp) and other crops, from four provinces in<br />
Dominican Republic (Mao, Azua, Montecristi and Dajabón), in order to make a preselection of bacterial strains to<br />
elaborate biofertilisers for banano organic crops in Dominican Republic. The total number of isolates was 160.<br />
The RAPD pattern obtained after amplification of the DNA with the primer M13 was used to identify possible<br />
clones, and the collection was reduced to 144 different strains. The 144 strains were evaluated in vitro by their<br />
efficiency producing the hormone IAA, the evidence of siderophores production and the ability of solubilise<br />
insoluble mineral phosphate. Sixteen per cent of the 144 strains produced significant rates of IAA, 22% produced<br />
siderophores and 11% solubilized P. Only 14 strains solubilised inorganic P and produced siderophores at the<br />
same time, and none of them were among the best producers of IAA, indicating that it is not common to find<br />
strains with several in vitro plant growth promotion properties simultaneously. The strains with highest plant<br />
growth promotion properties in vitro have been identified by 16S rRNA sequencing and comparison with<br />
international databases, to ensure that they belong to non-pathogenic taxa, following the phase of in vivo<br />
selection. By the moment, 6 of the 14 strains that solubilize inorganic P and produce siderophores, were<br />
identified, and 4 belonged to the species Pseudomonas taiwanensis, one to P. mosselii and one to Enterobacter<br />
absuriae, all of them common endophytic bacterial taxa (Dobbelaere et al., 2003).<br />
Dobbelaere, S. Vanderleyden, J., Okon, Y. 2003. Plant Growth-Promotign Effects of Diazotrophs in the<br />
Rhizosphere. Critical Reveiws in Plant Sciences, 22 (2): 107-149.<br />
77<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 12 – PGPR & Plant Production<br />
1100 – 1230<br />
Author: Ivan Kennedy<br />
SUNFix Centre for <strong>Nitrogen</strong> <strong>Fixation</strong>, University of Sydney, Australia<br />
Presentation Title: Beneficial biofilms on plant root surfaces – achieving more sustainable crop production<br />
Presentation Time: 1200 - 1220<br />
Plant growth promotion (PGP) for crops by microorganisms have been recognised for some time in biological<br />
nitrogen fixation (BNF) in legumes, shown in Germany in the late 19 th century in the face of opposition by Justus<br />
Von Liebig. More subtle are the beneficial effects of the PGP microbes in cereals and other crops. These<br />
involve a complex of phytohormonal effects, nutrient mobilisation, BNF and biocontrol that benefit plant growth<br />
and overall yield (Kennedy et al. 2008). Essentially, it is possible to grow crops such as rice inoculated with<br />
microbes forming biofilms on the surfaces of their roots while that allow significantly reduced inputs of chemical<br />
fertilisers, seed, pesticides and water, as well as reduced harvest losses.<br />
This presentation will focus on properties of a successful biofertiliser, BioGro ─ a commercial product invented in<br />
Vietnam consisting of four strains (Pseudomonas, Bacillus and a yeast) inoculated to the rhizosphere of rice<br />
seedlings. It is remarkable that BioGro contains strains selected empirically on the basis of their effectivness for<br />
rice farmers later identified in Australia as including species recognised around the world in research laboratories<br />
as having a strong basis for beneficial effects as biofertilisers.<br />
As was the case about a century ago with legumes and the rhizobia that form N2-fixing nodules on their roots,<br />
obtaining an effective outcome from applying these biofilm-forming organisms to crops in the field is challenging.<br />
It may be important to match microbial strains with plant species but it is certainly important that inoculants of<br />
high quality in terms of particular microbial strains in sufficient numbers (ca. 10 8 cells per g of inert carrier).<br />
Furthermore, convincing farmers of the beneficial effects and the scale of economic benefits possible is also<br />
important. The design of an efficient supply chain that includes quality control and delivery of economic and<br />
environmental benefits from biofertilisers to farmers is equally challenging.<br />
In this presentation, a strategy for simultaneously achieving environment-friendly and economic benefits being<br />
developed in a World Bank Development Marketplace project Sustaining nitrogen-efficient rice production, will be<br />
described. Lessons learnt from this project found to be important for implementation of new technology based<br />
on these microbial biofilms in both developing and developed countries will be discussed.<br />
Efficient nutrient use in rice production in Vietnam achieved using inoculant biofertilisers, I.R. Kennedy et al.,<br />
eds. ACIAR Proceedings 130, 2008, book downloadable from www.aciar.gov.au.<br />
78<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Plenary Session 6<br />
1330 - 1500<br />
Authors: Ross Ballard 1,5 , Nigel Charman 1,5 , Alan Humphries 1,5 , Matthew Denton 2,3,5 , Lori<br />
Phillips 2,5 , David Pearce 2 , Shoba Venkatanagappa 4,5 & Tim O‟Brien 4<br />
1 South Australian Research and Development Institute, GPO Box 397, Adelaide, South<br />
Australia, 5001.<br />
2 Department of Primary Industries Victoria, Rutherglen, Vic 3685.<br />
3 The University of Adelaide, Waite Campus, Glen Osmond SA 5064<br />
4 NSW Department of Primary Industries. Tamworth Agricultural Institute, Tamworth<br />
NSW 2340.<br />
5 Cooperative Research Centre for Future Farm Industries. Crawley, WA, 6009.<br />
Presentation Title: Limits to lucerne nodulation in acidic field soils<br />
Presentation Time: 1330 – 1400<br />
Lucerne is valued for its supply of high quality feed throughout the year, N2-fixation and environmental benefits. A<br />
constraint to the wider use of lucerne is the sensitivity of the symbiosis and in particular the rhizobia to low pH.<br />
Hence, lucerne is not recommended for soils below pH 5 (CaCl2). Ten strains of rhizobia that resulted in large<br />
improvements in lucerne nodulation in solution culture at pH 4.8 are being evaluated at four field sites ranging in<br />
pH (CaCl2) from 4.1 to 4.7. Lucerne nodulation, and plant survival and production were measured in the two<br />
years after establishment. Saprophytic competence of the strains has also been determined.<br />
Large differences in plant nodulation 10 months after sowing occurred at the different sites. At the site with soil<br />
pH 4.7, 64% of plants were nodulated compared to 8% at the site with soil pH 4.1. Differences in nodulation by<br />
the strains of rhizobia were smaller. Strains SRDI684, SRDI672 and SRDI736 ranked highest overall. Strain<br />
SRDI736 was also shown to have greater saprophytic competence at pH 4.7 than RRI128 which is used in<br />
commercial inoculants.<br />
Density of inoculated plants declined from 127 to 76 plants/m 2 (pH 4.7 site) and from 103 to 21 plants/m 2 (pH 4.1<br />
site). To date strain of rhizobia has not significantly (P
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Plenary Session 6<br />
1330 - 1500<br />
Author: Murray Unkovich<br />
Agriculture, Food and Wine, University of Adelaide, PMB 1, Glen Osmond, SA 5064<br />
Presentation Title: <strong>Nitrogen</strong> fixation and nitrogen isotope fractionation in legumes<br />
Presentation Time: 1400 - 1430<br />
Isotope fractionation occurs when there are changes in the partitioning of isotopes between a source and a<br />
product. For biological N2 fixation it equates to differences in the relative abundance of N isotopes between<br />
atmospheric N2 and fixed NH4 + in bacteria. The literature is somewhat confusing on this topic and a range of<br />
fractionation factors has been proposed. For N2 fixing legume symbioses, this fractionation becomes important<br />
when using natural variations in 15 N abundance to quantify N2 fixation where there is a need to be clear about the<br />
extent and source of fractionation. On a whole plant basis there is very little evidence for any isotope<br />
fractionation, and indeed if one considers the biochemistry and kinetics of the N2 fixation process one would not<br />
expect any fractionation. Evidence is presented that sources of variation in apparent isotope fractionation may<br />
have arisen from a number of factors including errors in measurement and processes other than N2 fixation<br />
contributing to apparent fractionation. For legumes, differences in the efficiency of symbiont bacteria and<br />
differences in plant age appear to influence shoot 15 N. It is important that these factors are teased out so that<br />
isotope fractionation can be reliably measured and better exploited in N cycle studies at plant and ecosystem<br />
scales. Careful attention needs to be paid to standardisation of mass spectrometric analysis, particularly since<br />
the adoption of continuous flow machines which have tended to divert focus to relative and precise rather than<br />
accurate measurement, such that measurement aganst atmospheric N2, the primary standard is no longer<br />
undertaken, even though it is readily available. More accurate rather than more precise measurement of 15 N will<br />
permit a better understanding of the N exchanges which occur between between legumes and their<br />
microsymbionts, which are still only partly understood, and also between ecosystem components.<br />
80<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Plenary Session 6<br />
1330 - 1500<br />
Authors: Xi Chen, Theja Shidore, Theresa Dinse, Sabrina Gemmer, Thomas Hurek and Barbara<br />
Reinhold-Hurek<br />
Department of Microbe-Plant Interactions, University of Bremen, Postfach 33 04 40,<br />
28334 Bremen.<br />
Presentation Title: Functional genomic analyses of endophyte – rice Interactions<br />
Presentation Time: 1430 -1500<br />
Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agro-biotechnological interest<br />
because it supplies fixed nitrogen to its host and colonises plants in remarkably high numbers without eliciting<br />
disease symptoms. This raises the question of mechanisms of compatible interactions between host and<br />
bacterium. The complete genome of strain BH72 was sequenced (1), and the rice genome is also available. This<br />
allows application of functional genomic analyses of both partners during interaction. For symbiotic interactions<br />
such with rhizobia and arbuscular mykorrhiza (AM), common plant signalling cascades are now well<br />
characterized. The results of our analysis of differential gene expression in Azoarcus-infected rice roots and<br />
mutational analyses suggest that the cascades are not operating during endophytic interactions. Nevertheless,<br />
transcriptomic analysis demonstrated that both partners show extensive metabolic adaptations during endophytic<br />
interaction. Transcriptomic analyses were carried out for both partners, rice as well as exudates-exposed<br />
Azoarcus sp. They allowed identification of candidate genes which were shown by mutational analysis to be<br />
required for optimal rhizosphere fitness in Azoarcus sp.<br />
(1) Krause et al. 2006. Genomic insights into the lifestyle of the mutualistic, N2-fixing grass endophyte Azoarcus<br />
sp. strain BH72. Nature Biotechnol. 24: 1385-1391.<br />
81<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 13 - Inoculant Quality & Application<br />
1600 - 1740<br />
Authors: Rosalind Deaker, Andrea Casteriano, Elizabeth Hartley and Greg Gemell<br />
University of Sydney, Australia<br />
Presentation Title: Making the most of high quality inoculants: survival of rhizobia during application to<br />
legume crops<br />
Presentation Time: 1600 – 1620<br />
Despite developments in inoculant technology and a high level of compliance of commercial legume inoculants<br />
to quality standards, poor survival of rhizobia during delivery to legume crops and pastures is still a major issue.<br />
Loss of viability of inoculant microorganisms limits the potential for maximum nodulation by elite strains and thus<br />
nitrogen fixation and yield. Legume inoculants are most commonly applied to seed prior to sowing; however, the<br />
seed surface presents a hostile environment for rhizobia exposing cells to desiccation stress and potentially toxic<br />
chemicals. Inoculant technology balances an incomplete scientific understanding of growth and survival with<br />
production and market demands. As a result, the potential for rhizobia to survive seed coating operations both<br />
commercially and on farm is not maximised.<br />
Polymer adhesives are typically used to fasten inoculants, growth enhancing and pelleting materials to seed.<br />
However, polymer properties that enhance survival of inoculant microorganisms are poorly defined. Preference<br />
for polymer latexes in the pre-inoculated seed industry is largely based on pellet quality and ease of handling but<br />
little attention has been given to compatibility and protection of rhizobia. Polymer latexes are not only likely to<br />
contain incompatible chemical constituents such as excess polymerisation initiator, surfactants and biocides but<br />
are also faster drying than solution polymers. Rhizobial survival is substantially improved when the rate of<br />
dehydration is reduced.<br />
In addition to external protectants, intracellular physiological changes after growth of rhizobia in different media<br />
prior to drying significantly affect survival and may be responsible for improved desiccation tolerance. A better<br />
understanding of how to induce physiological mechanisms involved in desiccation tolerance will provide new<br />
direction for inoculant technology development. It is expected that combining targeted physiological conditioning<br />
during production of inoculants with optimised external protection during application will maximise the potential of<br />
microbial inoculants to enhance agricultural production systems.<br />
82<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 13 - Inoculant Quality & Application<br />
1600 - 1740<br />
Authors: Ron Yates 1,2 , Angelo Loi 1 , Brad Nutt 1 & John Howieson 2<br />
1 Department of Agriculture and Food, Western Australia (DAFWA), Baron Hay Court,<br />
South Perth, 6151, Western Australia.<br />
2 Centre for Rhizobium Studies (CRS), Murdoch University, Murdoch, 6150, Western<br />
Australia.<br />
Presentation Title: How do we ensure nodulation of annual pasture legumes sown out of season into hot,<br />
dry soil?<br />
Presentation Time: 1620 – 1640<br />
Biological nitrogen (N) fixation from effectively nodulated legumes are an integral component of sustainable<br />
agriculture. Agricultural legumes not only provide N for subsequent crops and high value feed for animals, they<br />
also assist in spreading the risk in the production system through the management of weeds, pests and<br />
diseases. In Australia, it is essential legumes are inoculated with the correct and current commercial rhizobia<br />
strain (or Group) for maximum N fixation. Commercial inoculant strains go through an extensive selection<br />
process, in which the strains must possess the ability to maintain high N fixation over a broad host range and<br />
adapt to the anticipated soil niche of the host legume. However, an ongoing challenge is to successfully deliver<br />
high numbers of the commercial inoculant. If this is not achieved it leads to failed nodulation, or nodulation of the<br />
legume with resident soil-borne strains that are usually sub-optimal in N fixation.<br />
Inoculants in Australia are available in four different carriers: (i) peat; (ii) freeze dried powders; (iii) granular; and<br />
(iv) a pre-coated seed form, with inoculum as part of the pellet. The efficiencies of these products are continually<br />
being evaluated for use in Western Australian agriculture. However, current research is largely focused on<br />
providing best practices for inoculating pasture legumes when they are introduced by “Summer sowing” in the<br />
Mediterranean climates of southern Australia (i.e. when the soil is hot and dry). This technique has been<br />
designed to cheaply introduce legume species into paddocks at a time when farm labour is not limiting, assisted<br />
by strategically harnessing inherent hard-seed breakdown in the legume cultivars. However this presents a<br />
challenge in terms of keeping the inoculant rhizobia alive until a rainfall event that induces germination, and<br />
identifying which carrier is the most efficient at doing so. Summer sowing requires the bacteria to survive through<br />
dry, hot, soil conditions in sufficient number to out-compete resident strains for nodulation when the seeds<br />
germinate, which may be up to four months after sowing. Recent findings will be presented that suggest<br />
surprising outcomes in rhizobial survival.<br />
83<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Authors:<br />
Concurrent Session 13 - Inoculant Quality & Application<br />
1600 - 1740<br />
Didier Lesueur 1,2 , Laetitia Herrmann 2 , Moses Thuita 2, 3 , Mary Atieno 2, 3 , Edwin Mutegi 2, ,<br />
Keziah Ndung‟u 2, 3 4 , Aliou Faye 2, 5 , Mary Kamaa 2 , Pieter Pypers 2 & Robert Okalebo 3<br />
1 CIRAD, UMR Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols &<br />
Agroécosystèmes (SupAgro-CIRAD-<strong>IN</strong>RA-IRD), 2 Place Viala, F34060 Montpellier,<br />
France. <br />
2 Tropical Soil Biology and Fertility Institute (TSBF) / CIAT - World Agroforestry Centre<br />
(ICRAF), P.O. Box 30677, Nairobi, Kenya.<br />
3 Moi University, Department of Soil Science, PO Box 1125, Eldoret, Kenya.<br />
4 Kenya Agricultural Research Institute (KARI – Kitale), P.O. Box 450 - 30200, Kitale,<br />
Kenya.<br />
5 Institut Sénégalais de Recherche Agricole, Route des hydrocarbures, Dakar, Sénégal<br />
Presentation Title: How commercial rhizobial inoculants can contribute to improved livelihoods of resource<br />
poor African farmers.<br />
Presentation Time: 1640 – 1700<br />
There is a proliferation of commercial bioinoculant products appearing on the market that claim major impact in<br />
increasing crop productivity without any scientific assessments about their effectiveness in the field. TSBF-CIAT<br />
has been mandated by Bill and Melinda Gate Foundation to scientifically evaluate and select effective<br />
commercial products for improving and sustaining crop yields in selected agro ecological zones in 3 African<br />
countries.<br />
Our results on about 120 products showed that many private companies commercialize a mix of non-defined<br />
microorganisms and thus do not ensure the quality of their inoculants. This may explain their lack of impact on<br />
the plant growth even under controlled greenhouse conditions. Generally, high level of contamination was<br />
observed as pure products represented only 33% of the products, 41% of the products contained all the<br />
expected strains and 52% of the products contain none or part of the expected contaminants. We assessed<br />
under greenhouse conditions the possible effect of the formulation of effective rhizobial inoculants on both<br />
soybean growth and nodulation. Our results showed that there was no effect and the association of the rhizobia<br />
with a Bacillus strain didn‟t induce any significant stimulation of soybean growth. However, through field<br />
demonstration trials combining rhizobia with mycorrhiza and P solubilizing bacteria, our results suggested some<br />
relevant positive interactions on soybean yield. In Kenya, we tested one rhizobial inoculant in 3 mandates areas<br />
(about 50 farms in each) and our results demonstrated a significant effect of the inoculation on soybean yield.<br />
The next step will consist of making such effective inoculants cheap and available on the local markets.<br />
Administrative issues shall be sorted out for each country as the national regulations are not similar and of<br />
course the quality control issue shall be taken into account to ensure the viability of the inoculants to farmers who<br />
purchase them.<br />
84<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 13 - Inoculant Quality & Application<br />
1600 - 1740<br />
Authors: Matthew D Denton 1 David J Pearce 2 and Mark B Peoples 3<br />
1 The University of Adelaide, School of Agriculture, Food and Wine, Adelaide SA 5005<br />
Australlia<br />
2 Department of Primary Industries, RMB 1145 Chiltern Valley Road, Rutherglen, VIC<br />
3685, Australia<br />
3 CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601 Australia<br />
Presentation Title: The influence of rhizobial inoculant application rate on nitrogen fixation in faba bean<br />
(Vicia faba l.) In south eastern Australia<br />
Presentation Time: 1700 – 1720<br />
Nodulation of legumes is essential to create effective nitrogen fixing symbioses, yet the need to inoculate pulses<br />
with large numbers of effective rhizobia is often not appreciated. In this study, we tested the influence of seven<br />
rates of rhizobia inoculation, from no inoculant to 100 times the normal rate of inoculation, on the responses of<br />
faba bean in two fields in south-eastern Australia: one site with no detectable rhizobia and one with 2900 rhizobia<br />
g -1 soil. Rhizobia inoculation increased nodule numbers from 0 to 55 nodules per plant and nodule dry matter<br />
(DM) from 0 to 330 mg. The increase in nodule numbers directly impacted on fixed N, which increased from 20 to<br />
218 kg shoot N ha -1 , and on grain N concentrations, which increased from 3.2 to 4.5%. An increase in N2 fixation<br />
was even observed in the presence of a soil population when a high rate of inoculation was provided. Increases<br />
in nodulation improved grain yields by around 1.0 t ha -1 in well-nodulated treatments (2.6 t ha -1 ) compared with<br />
those of un-inoculated faba bean (1.6 t ha -1 ) at the site without rhizobia present in soil. Differences in nodulation<br />
also influenced the contributions of legume N to the system, which varied from the removal of 20 kg N ha -1 from<br />
the system to an input of 199 kg N ha -1 . This study illustrates the importance of inoculation to N2 fixation, grain<br />
yield, grain N concentration and the potential contributions of legume cropping to soil N fertility. The study also<br />
identifies the need for technologies to increase inoculant numbers supplied to legumes as a pathway to improve<br />
N2 fixation by legumes that are sown into fields with existing rhizobial populations.<br />
85<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 13 - Inoculant Quality & Application<br />
1600 - 1740<br />
Authors: Laetitia Herrmann 1 , Collins Majengo 2 , Edwin Mutegi 1 , Martin Kimanthi 1 , Paul Onyango 1 ,<br />
Robert Okalebo 2 & Didier Lesueur 1,3<br />
1 Tropical Soil Biology and Fertility institute of CIAT, World Agroforestry Center, PO Box<br />
30677 Nairobi, Kenya.<br />
2 Moi University, School of Agriculture and Biotechnology, PO Box 1125 Eldoret, Kenya.<br />
3 CIRAD, UMR Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols &<br />
Agroécosystèmes (SupAgro-CIRAD-<strong>IN</strong>RA-IRD), 2 Place Viala, F34060 Montpellier,<br />
France.<br />
Presentation Title: Field evaluation of rhizobial inoculant: high nodule occupancy by the strain doesn‟t lead<br />
to improved soybean yields in Kenya<br />
Presentation Time: 1720 – 1740<br />
A range of soybean commercial inoculants were tested under greenhouse and field conditions in different<br />
locations in Kenya. One rhizobial (Legumefix) and one mycorrhizal (Rhizatech) inoculants were selected<br />
according to the promising results obtained. The efficiency of these 2 products (single or combined inoculation)<br />
was assessed in 150 farms in 3 mandate areas in Kenya presenting different soil characteristics and<br />
environmental conditions. Both biomass and grain yields were measured and nodules occupancy of the rhizobial<br />
strain was assessed by ELISA using specific monoclonal antibodies. Application of the rhizobial inoculant, alone<br />
or in combination with the mycorrhizal product significantly increased the yields in all mandate areas (about 75%<br />
of the farms). However, the co-inoculation didn‟t perform significantly better than the rhizobial product alone.<br />
Controls plots gave poor results, as well as the mycorrhizal product, though that was not expected given the<br />
good performances observed under greenhouse conditions. Nodule occupancy analysis showed that a high<br />
number of nodules occupied by the inoculated strain did not obviously lead to an increase of soybean production.<br />
Soil factors (pH, P, C, N…) seemed to affect the inoculant efficiency whether the strain is occupying the nodules<br />
or not. Our statistic analysis showed that soil pH significantly affected nodulation and yield, though the effect was<br />
variable depending on the region. P content also positively correlated with nodulation and yields. This study<br />
provided relevant information on field efficiency of rhizobial inoculants on soybean yield in Kenya and showed<br />
that the competitiveness of rhizobial strains might not be the main factor explaining the effect (or lack of) of<br />
legumes inoculation in the field.<br />
86<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 14 – Cyanobacteria & other organisms<br />
1600 - 1740<br />
Authors: Faten Mohamed, Soha Mostafa, & Nabil Omar<br />
Soils, Water and Environ. Res. Inst., Agric. Microbiol. Res. Dept., ARC, Giza,<br />
Egypt.<br />
Presentation Title: Influence of beneficial microbes and proline treatments on sugarbeet under nitrogen<br />
limitation in saline-sodic soil<br />
Presentation Time: 1600 – 1620<br />
A field experiment on sugar beet (Beta vulgaris L.) yield and yield quality under saline-sodic soil conditions<br />
was conducted at Sahl El-Hussinia Res. station, El-Sharkia Governorate, Egypt, during the winter season of<br />
2008/2009. The influence of cyanobacteria and N2-fixing bacteria were compared either individually or in<br />
combination to supplementation with proline amino acid under two levels of mineral nitrogen fertilization (50<br />
and 75% of nitrogen recommended dose). Soil enzymatic activities (dehydrogenase and nitrogenase), total<br />
bacterial counts, total cyanobacteria counts and total nitrogen fixing bacteria counts were enhanced by the<br />
biofertilizers compared to proline treatment and control, particularly when the combined inoculum of<br />
cyanobacteria and N2-fixing bacteria was applied in the presence of 75% N. In addition, inoculation with<br />
cyanobacteria and N2-fixing bacteria, either individually or in combination with 75%N, led to a slight decrease<br />
of pH and EC values of saline soil, while there was an increase in the availability of NPK as compared with<br />
control plots.Proline and biofertilizers showed a significant positive impact on some physiological properties of<br />
plants drown at 75% nitrogen level, such as chlorophyll in leaves, proline and phenolic compounds in roots.<br />
The highest responses for these traits were in proline-treated plots followed by the combined inoculation of<br />
cyanobacteria and N2-fixing bacteria. While, there was no significant difference in root yield productivity<br />
between proline treatment and the combined inoculum of cyanobacteria and N2-fixing bacteria with 75%<br />
nitrogen fertilizer. The combined inoculation positively increased N, P and K uptake and decreased the uptake<br />
of Na in roots. The combined inoculum of cyanobacteria and N2-fixing bacteria with 75% nitrogen led to a<br />
significant increase in shoot and root dry weight as well as root yield quality (sucrose and purity). Results<br />
suggest that the beneficial effect of the cyanobacteria and N2-fixing bacteria on sugar beet growth, yield and<br />
yield quality was attributed to the biologically active substances produced by these microbial strains besides<br />
the nitrogen fixation of the diazotrophs which compensate the reduction of the costly and the environmentally<br />
polluted mineral nitrogen fertilizers in the new reclaimed saline-sodic soil.<br />
87<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 14 – Cyanobacteria & other organisms<br />
1600 - 1740<br />
Authors: Radziah Othman 13 , Umme Aminun Naher 1 , Zulkifli Hj. Shamsuddin 1 , Halimi Mohd Saud 2<br />
, Mohd Razi Ismail 3 and Khairudin Abdul Rahim 4<br />
1 Department of Land Management, 2 Department of Agriculture Technology, Faculty of<br />
Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia<br />
3 Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang,<br />
Selangor, Malaysia<br />
4 Agrotechnology and Biosciences Division, Nuclear Agency Malaysia, Ministry of<br />
Science Technology and Innovation (MOSTI), Bangi, 43000 Kajang, Malaysia<br />
Presentation Title: Effect of root exuded sugars on biological nitrogen fixation and growth of rice (Oryza<br />
sativa)<br />
Presentation Time: 1620 – 1640<br />
Biological <strong>Nitrogen</strong> <strong>Fixation</strong> (BNF) is an important process in wetland rice ecosystem requiring high energy input.<br />
A 15 N tracer study was conducted under glasshouse condition to determine the effect of rice exuded sugars<br />
(glucose, galactose and arabinose) on BNF of two locally isolated diazotrophs, Rhizobium sp. Sb16 and<br />
Corynebacterium sp. Sb26 in association with two rice genotypes (traditional variety Mayang Segumpal and<br />
cultivated variety MR219). Both diazotrophs showed preferences for specific sugars and genotype association.<br />
Isolate Sb16 showed high preference for galactose and isolate Sb26 for arabinose. Application of 10 mM sugars<br />
either galactose or arabinose in pot experiment (5 kg soil) to the respective rice genotypes enhanced the<br />
diazotroph populations, N2 fixation activity and subsequently plant growth. Mayang Segumpal genotype<br />
inoculated with Rhizobium Sb16 and applied with galactose increased plant N concentration with 42 ± 1.06 % of<br />
the N derived from the atmosphere. MR219 genotype inoculated with Corynebacterium Sb26 and applied with<br />
arabinose obtained 40 ± 1.29 % of the N concentration from BNF. Comparing with the uninoculated control, the<br />
association between Mayang Segumpal with Sb16 increased plant biomass by 195 ± 40 % and the association<br />
between MR219 with Sb26 resulted in biomass increase by 108 ± 37.07 %. In plants fertilized with 60 kg ha -1 of<br />
N, inoculated MR219 genotype showed higher biomass increment compared to that of Mayang Segumpal. The<br />
association between the plant and diazotrophs with the respective sugars significantly increased plant<br />
photosynthetic activity. The study indicated that N2 fixation activity and growth of different rice genotypes can be<br />
increased by increasing the availability of specific sugars in the rhizosphere.<br />
88<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 14 – Cyanobacteria & other organisms<br />
1600 - 1740<br />
Authors: Cuddy, W. S. 1 , Forster, B. 2 , Deaker, R. 3 , Summerell, B. A. 4 , Neilan, B. A. 1 ,Gehringer, M.<br />
M. 1<br />
1 . School of Biotechnology and Biomolecular Sciences, University of New South Wales,<br />
Sydney, 2052, New South Wales.<br />
2. Research School of Biology, Plant Sciences Division, Australian National University,<br />
Canberra, 0200, Australian Capital Territory.<br />
3 . Faculty of Agriculture, Food and Natural Resources, University of Sydney, Sydney,<br />
2006, New South Wales.<br />
4. Science and Public Programs, Botanic Gardens Trust, Sydney, 2000, New South<br />
Wales.<br />
Presentation Title: Cyanobacterial salt physiology and their impact on wheat seedling salt tolerance<br />
Presentation Time: 1640 – 1700<br />
<strong>Nitrogen</strong>-fixing cyanobacteria have been identified in soils and biological soil crusts worldwide. Understanding<br />
their potential to support cereal growth, particularly under adverse conditions, is of great interest to crop<br />
management and breeding. We assessed the potential benefits of cyanobacteria on growth of wheat seedlings<br />
under salt stress. First, three cyanobacterial isolates from wheat fields: a heterocyst-producing Nostoc sp., a<br />
Microcoleus vaginatus and a Leptolyngbya sp. were cultured in salinised medium at 5 and 15 dS m -1 and<br />
sampled at mid-exponential and stationary growth phases to determine photosynthetic and nitrogen fixation rates<br />
using Pulse Amplitude Modulation (PAM) fluorometry and Acetylene Reduction Assays (ARA), respectively.<br />
These isolates were then established as soil crusts on sterilized sand into which germinated wheat seeds were<br />
planted 1 cm below the surface. Sodium chloride was applied to generate salt stress with electrical conductivities<br />
(ECe) of 6 and 13 dS m -1 . Pots were watered to field capacity daily, and the electrical conductivities monitored<br />
during seedling development for either 14 days at 6 dS m -1 or 21 days at 13 dS m -1 . Quenching analysis using<br />
PAM fluorometry was done on wheat leaves before sampling. Inductively Coupled Plasma-Optical Emission<br />
Spectroscopy (ICP-OES) was conducted on separated root and shoot material. Both the Nostoc and the nonheterocystous<br />
cyanobacteria Microcoleus vaginatus isolates fixed nitrogen at 8 and 6 n moles C2H4 μg Chl a -1 h -1<br />
respectively, without affecting photosynthetic efficiency. The cyanobacterial soil crusts induced significant effects<br />
on root and shoot nutrient concentrations, impairing plant salt tolerance. This study provides new evidence for<br />
the ability of Microcoleus vaginatus to fix nitrogen, which suggests its contribution to soil ecology may currently<br />
be undervalued. We were able to demonstrate that the cyanobacterial isolates used in this study in fact<br />
enhanced the salt stress of wheat seedlings, thereby decreasing plant growth.<br />
89<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 14 – Cyanobacteria & other organisms<br />
1600 - 1740<br />
Authors: Zulkifli Hj. Shamsuddin, Tan Kee Zuan and Puteri Aminatulhawa Megat Amaddin<br />
Department of Land Management, Faculty of Agriculture, Universiti Putra<br />
Malaysia, 43400 UPM Serdang, Selangor, Malaysia<br />
Presentation Title: Development and application of liquid biofertilizer inoculum for non-legume and<br />
vegetable soybean intercrop<br />
Presentation Time: 1700 – 1720<br />
Solid substrate inoculant is more widely used than liquid inoculant in legume and non-legume cultivation. It is<br />
more pronounced in developing countries. In Malaysia, the application of liquid inoculant with a consequential<br />
reduction in chemical fertilizer consumption could be more beneficial due to the hilly terrain, reduction in<br />
transport and fertilizer cost, and minimal hazardous effect on the environment. Locally used liquid inoculant<br />
technology is now emerging using various organic sources and stimulant in the culture medium to prolong shelflife<br />
and effectiveness of the bacterial inoculum. An organic fertilizer-based amendment has been used to sustain<br />
the shelf-life of a Plant Growth-Promoting Rhizobacteria ( PGPR ) for more than nine months. This locally<br />
isolated non-pathogenic PGPR from the roots of oil palm has N2 fixing and PO4 -- solubilizing properties besides<br />
producing phytohormones which enhance root development and increase water and nutrient uptake (<br />
Shamsuddin et al., 2009 ). The PGPR is protected from any adverse soil environmental conditions due to its<br />
endophytic habitat in the plant roots. Inoculated plants showed increased shoot and root growth ( rice, oil palm,<br />
banana )( Kok-Ang et al., 2010 ), are drought and disease tolerant ( Fusarium oxysporum cubense on banana )<br />
and high yielding but with 65% less fertilizer-N input ( banana, sweet potato )( Mia et al., 2010 ). Inoculated<br />
herbal plant, Safed Musli ( Chlorophytum borivilianum) have also shown increased number of tuber and content<br />
of its bioactive compound, saponin. This liquid inoculant will be commercially prepared in concentrated form to<br />
reduce bulk and storage space relative to solid inoculum and used in the field at 100 fold dilution while reducing<br />
transport and labor cost in the field application. The inoculum can be used by mixing it with the seeds prior to<br />
planting and sprayed around the base of non- leguminous plant or in the interrow of the young rice seedling and<br />
vegetable soybean crop after four weeks of growth. These beneficial effects of the environmental friendly liquid<br />
biofertilizer can minimize the use of inorganic fertilizer in sustainable crop production system.<br />
90<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details:<br />
Wednesday 30 November 2011<br />
Concurrent Session 14 – Cyanobacteria & other organisms<br />
1600 - 1740<br />
Authors: Junko Tazawa 1 , Yoshinari Ohwaki 1 , Tadashi Yokoyama 2 , Yoshiaki Shizukawa 3 , Ui<br />
Ono 3 & Masami Yoshikawa 3<br />
1 NARO Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan<br />
2 Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan<br />
3 Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and<br />
Fisheries Technology Center, Seika, Kyoto 619-0244, Japan<br />
Presentation Title: Endophytic occupation of soybean nodules by Rhizobium and other bacterial species<br />
under continuous cultivating conditions<br />
Presentation Time: 1720 – 1740<br />
Rhizobial bacteria can form nodules and establish symbiosis on the roots of their leguminous host plants.<br />
Recently, several reports have stated that some non-symbiotic bacteria occupy legume nodules endophyticly,<br />
although the function of these bacteria in the nodules has not been understood. We have isolated non-symbiotic<br />
bacteria from root nodules of soybean (cv: Hatayutaka) grown in a continuously cultivated field. Fast-growing<br />
bacterial strains were isolated from surface-sterilized nodules on YMA medium as non-symbiotic endophytic<br />
bacteria. Phylogenetic analysis based on the partial sequence of 16S rDNA gene demonstrated that most of<br />
these isolates were close to the genera Rhizobium/Agrobacterium, Bacillus, Paenibacillus, Enterobacter, and<br />
Pseudomonas. The percentage of nodules that coexisted with symbiotic Bradyrhizobium strains and nonsymbiotic<br />
bacteria was very low (
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 15 – Quantification of N-<strong>Fixation</strong><br />
1600 – 1740<br />
Authors: Georg Carlsson 1 & Kerstin Huss-Danell 2<br />
1 Department of Agrosystems, Swedish University of Agricultural Sciences, SE-23053<br />
Alnarp, Sweden.<br />
2 Department of Agricultural Research for Northern Sweden, Swedish University of<br />
Agricultural Sciences, SE-90183 Umeå, Sweden.<br />
Presentation Title: Does nitrogen transfer confound 15 N-based quantifications of N2 fixation?<br />
Presentation Time: 1600 – 1620<br />
<strong>Nitrogen</strong> transfer between neighbouring plants is a well-known phenomenon, mediated via e.g. rhizodeposition<br />
and mycorrhizae. Transfer of fixed N from legumes to non-legume reference plants has been suggested to<br />
confound N2 fixation estimates obtained by the 15N natural abundance and isotope dilution methods, because a<br />
reference plant taking up fixed N will supposedly not correctly reflect the 15 N signature of plant-available soil N.<br />
On the other hand, experiments using direct leaf-feeding of 15 N, which allows for direct detection of N transfer<br />
with high precision, have shown that N transfer also occurs in the direction from non-legume to legume. In order<br />
to establish whether N transfer causes problems in N2 fixation measurements with 15 N-based methods when<br />
using reference plants grown together with the legume, we labelled both legumes and non-legumes with 15 N and<br />
performed detailed measurements of N transfer in mixed plant communities in the field, including measurements<br />
of potential N transfer from legume to legume. N transfer occurred in all directions: from legume to non-legume,<br />
from non-legume to legume and from legume to legume. These results, together with analyses of previously<br />
published data on N transfer, were used to calculate nitrogen fixation with different N transfer scenarios, showing<br />
the effects of using reference plants in mixture with the legume versus in pure stand. We conclude that the most<br />
important mechanism of N transfer is likely to be indirect, via rhizodeposition and litter degradation, and that fixed<br />
N transferred to neighbouring reference plants also modifies the 15 N signature of the soil N available to the N2fixing<br />
legume. This provides strong support for using reference plants growing as close as possible to the N2fixing<br />
legume for reliable N2 fixation quantifications.<br />
92<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 15 – Quantification of N-<strong>Fixation</strong><br />
1600 – 1740<br />
Authors: Xinhua He 1, 2, 3, 4 , Christa Critchley 4 , Caroline Bledsoe 5<br />
1, 2 State Centre of Excellence for Ecohydrology, Centre for Ecosystem Management,<br />
1 School of Natural Resources, Edith Cowan University, Joondalup, WA 6207, Australia<br />
2 School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia<br />
3 Key Laboratory of Crop Nutrition and Fertilization, Institute of Agricultural Resources<br />
and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081,<br />
China<br />
4 School of Agriculture & Food Sciences, University of Queensland, Brisbane, QLD 4072<br />
Australia<br />
5 Department of Land, Air and Water Resources, University of California, Davis, CA<br />
95616 USA<br />
Presentation Title: Reciprocal two-way n ( 15 NH4 or 15 NO3) transfer between non-N2-fixer Eucalyptus<br />
maculata and Frankia N2-fixer Casuarina cunninghamiana<br />
Presentation Time: 1620 – 1640<br />
<strong>Nitrogen</strong> (N) movement or transfer from one plant to another could have important implications for nutrient<br />
cycling and eco-physiological significance in terrestrial and agricultural ecosystems. In controlled pots with or<br />
without Frankia inoculation to Casuarina, two-way N transfers through an ectomycorrhizal fungus Pisolithus sp.<br />
were examined by excluding root contact and supplying 15 NH4 + or 15 NO3 � to 5 or 11-month-old Eucalyptus<br />
maculata or Casuarina cunninghamiana pairs grown in 2-chambered-pots separated by 37 µm nylon screens.<br />
With either Eucalyptus or Casuarina as the N-donor, the three pairs were non-nodulated nonmycorrhizal/nonmycorrhizal,<br />
non-nodulated mycorrhizas/mycorrhizal and nodulated mycorrhizal/mycorrhizal.<br />
Using an environmental scanning electron microscope, living mycorrhizal hyphae were observed to interconnect<br />
Eucalyptus and Casuarina. Results showed that N2-fixation supplied ~35% of the N in Casuarina and was<br />
enhanced by mycorrhization, which was 67% in Eucalyptus and 36% in Casuarina. Biomass, N and 15 N contents<br />
were lowest in the non-mycorrhizal and greatest in the nodulated/mycorrhizal plants. <strong>Nitrogen</strong> transfer was<br />
enhanced by nodulation and/or mycorrhization, and greater when N was supplied as 15 NH4 + than 15 NO3 � .<br />
<strong>Nitrogen</strong> transfer from either 15 N-source was lowest in the non-mycorrhizal treatment and greatest in the<br />
nodulated mycorrhizal treatment, and also was greater to Casuarina than to Eucalyptus and where NH4 + than<br />
NO3 � supplied. Irrespective of the 15 N-source and of whether Casuarina or Eucalyptus was the N sink, net N<br />
transfer was low and similar in non-nodulated treatments, but was much greater when Casuarina was the N sink<br />
in the nodulated mycorrhizal treatment. High-N-demand by Casuarina resulted in greater net N transfer from the<br />
less N-demand Eucalyptus. Net N transfer from a non-N2-fixing to an N2-fixing plant may reflect the very high N<br />
demand of N2-fixing species. Our research may provide insights into our understanding of complex interactions<br />
among plants, Frankia, mycorrhizal fungi, and soil resources in both terrestrial and agricultural ecosystems.<br />
93<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 15 – Quantification of N-<strong>Fixation</strong><br />
1600 – 1740<br />
Authors: Alejandro del Pozo 1 , Carlos Ovalle 2 & Soledad Espinoza 2<br />
1 Facultad de Ciencias Agrarias, Universidad de Talca, Talca, Chile.<br />
2 CRI-Quilamapu, <strong>IN</strong>IA, Chillán, Chile.<br />
Presentation Title: <strong>Nitrogen</strong> fixation of grain legumes in two contrasting mediterranean environments of<br />
Chile<br />
Presentation Time: 1640 – 1700<br />
Grain legumes are important components of Mediterranean farming systems due to their capacity to fix nitrogen<br />
and to improve soil fertility, as well as to reduce weeds in legume-cereal rotations. In this work we evaluate the<br />
nitrogen fixation capacity of four grain legumes, Pisum sativum, Lupinus angustifolius, L. luteus and L. albus, in<br />
rotation with wheat, by using the 15 N natural abundance technique. Field experiments were conducted in two<br />
contrasting Mediterranean environments and in two years (2008 and 2009). In the interior dryland (granitic<br />
Alfisol; 650 mm of annual rainfall), total dry matter production was higher in P. sativum (average 10.5 ton ha -1 )<br />
than in L. angustifolius and L. luteus (average 5.5 ton ha -1 ). The N derived from the atmosphere (%Ndfa) was<br />
significant (p
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 15 – Quantification of N-<strong>Fixation</strong><br />
1600 – 1740<br />
Authors: Salmina N. Mokgehle 1 , Cherian Mathews 2 and Felix D. Dakora 3<br />
1 Department of Crop Sciences, Tshwane University of Technology, Private Bag X680,<br />
Pretoria 0001, South Africa.<br />
2 Department of Agriculture, Rural Development and Land Administration, Private Bag<br />
X11318, Nelspruit, 1200<br />
3 Department of Chemistry, Tshwane University of Technology, Private Bag X680,<br />
Pretoria 0001, South Africa.<br />
Presentation Title: Selecting field plants for drought tolerance and N2 fixation in 25 groundnut (Arachis<br />
hypogaea<br />
13C and 15N natural abundance<br />
Presentation Time: 1700 – 1720<br />
Groundnut is a major commercial grain legume in Africa, often cultivated for its seed oil and high protein content.<br />
Although ICRISAT has historically selected groundnut genotypes for grain yield and disease resistance in Africa,<br />
little is known about their N contribution and drought tolerance under local conditions. In this study, field trials<br />
were conducted at three sites (namely, Nelspruit, Mzinti and Kliplaatdrift) within Mpumalanga Province of South<br />
Africa in 2009. The data revealed significant differences in plant biomass, N concentration, and N content at all<br />
three sites. The δ 15 N values of groundnut genotypes ranged from -0.08‰ to +1.06‰ at Nelspruit, +0.41‰ to<br />
+0.95‰ at Mzinti, and +0.73‰ to +1.96‰ at Kliplaatdrift. About 24 out of the 25 groundnut genotypes obtained<br />
over 50% of their N nutrition from symbiotic fixation at Mzinti, while at Kliplaatdrift only five genotypes derived<br />
50% or more of their N nutrition from symbiotic fixation. The amounts of N-fixed at Nelspruit ranged from 76<br />
kg/ha for ICGV99033 and PC327K31 to 188 kg/ha for ICGV00362. At Mzinti, 10 genotypes contributed over 100<br />
kg N /ha, while at Kliplaatdrift only 5 genotypes fixed more than 100 kg/ha. Data on ∂ 13 C values also revealed<br />
marked difference in drought tolerance. Genotypes ICGV03157 and ICGV00369, for example, showed much<br />
lower 13 C discrimination at all three sites, indicating greater water-use efficiency compared to the remaining<br />
genotypes.<br />
95<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Wednesday 30 November 2011<br />
Concurrent Session 15 – Quantification of N-<strong>Fixation</strong><br />
1600 – 1740<br />
Authors: Rachel Pipai 1,2 Murray Unkovich 1 Ann McNeill 1 Murom Banabas 2 Mike Webb 3 David<br />
Herridge 4 Paul Nelson 5<br />
1 University of Adelaide Waite Campus Glen Osmond SA5064<br />
2 PNG Oil Palm Research Association Inc DAMI Research Station PO Box 97 Kimbe<br />
West New Britain PNG<br />
3 CSIRO Land & Water Davies Laboratory, Discovery Drive, Douglas, Queensland<br />
4814, Australia.CSIRO Townsville<br />
4 UNE–NSW DPI Primary Industries Innovation Centre University of New England<br />
Armidale NSW 2351<br />
5 James Cook University PO Box 6811 Cairns, Qld 4870<br />
Presentation Title: <strong>Nitrogen</strong> fixation by legumes under oil palm plantations in Papua New Guinea<br />
Presentation Time: 1720 – 1740<br />
There are currently no published estimates for the amount of nitrogen that is fixed by tropical legume cover<br />
plants in oil palm plantations on the volcanic ash soils of PNG. Such estimates require knowledge of the<br />
proportional dependence of plants on N2 fixation (%Ndfa), as well as data for dry matter production. A field study<br />
is planned to measure these variables for legume cover plants (Pueraria phaseoloides, Calopogonium caerulum,<br />
and Mucuna bracteata) in plantations of different ages, since shading increases as the palms grow and legume<br />
cover changes in composition and growth rate. Two methods will be used for assessing %Ndfa, the 15 N natural<br />
abundance and the xylem sap techniques. Initially a glasshouse experiment using 15 N-labelled nutrient solutions<br />
will be undertaken to calibrate the xylem sap technique for estimating %Ndfa for each of the legume cover crops.<br />
Results from the glasshouse study and a preliminary field survey will be presented at the conference.<br />
96<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Plenary Session<br />
0900 – 1030<br />
Authors: Robert M. Boddey 1 , Lincoln Zotarelli 2 , Natalia P. Zatorre 3 , Segundo Urquiaga 1 , Claudia<br />
P. Jantalia 1 , Julio C. Franchini 4 , Bruno J. R. Alves 1<br />
1 Embrapa Agrobiologia, Seropédica, 23890-000, Rio de Janeiro, Brazil.<br />
2 Horticultural Sciences Dept., University of Florida, Gainesville, FL 32611-0690, USA.<br />
3 Departamento de Solos, Universidade Federal Rural do Rio de Janeiro, Seropédica,<br />
23890-000, RJ, Brazil.<br />
4 Embrapa Soja, Rod. Carlos João Strass , Warta, 86001-970, Londrina, PR, Brazil.<br />
Presentation Title: Legume N2-fixation inputs preserve soil organic C stocks in no-till agriculture<br />
Presentation Time: 0900 - 0930<br />
In Brazil, no-tillage is widely adopted for soybean-based cropping systems. In the Southern region, soybean<br />
alternates with maize in the summer, and oats or green-manure legumes in the winter to break the continuous<br />
use of wheat in this season. Results from long-term experiments comparing conventional plough tillage (CT) and<br />
no-till (NT) in this region have shown that if the only legume in the rotation is soybean there is very little increase<br />
in soil C stocks, which has been attributed to lack of significant residual N in the system to build soil organic<br />
matter owing to the large export of N in soybean grain. Here we present the results of a study with different crop<br />
rotations of soybean, maize, wheat, oats and lupins managed under NT or CT which differed from each other in<br />
the frequency of soybean and lupins in the crop sequences. Soil C and N stocks to a depth of 80 cm were<br />
evaluated and the contributions of N2 fixation (BNF) to all the legumes in the systems were quantified using the<br />
15 N natural abundance technique. Reliance on BNF of both soybean and lupins was higher under NT than under<br />
CT. The use of lupins as a green manure represented an extra contribution to soil N of up to 300 kg N ha -1 , which<br />
allowed high maize yields, comparable to N-fertilized maize following oats, and was essential to maintain a<br />
positive cropping system N balance. Comparison of soil C stocks between 1997 and 2009 revealed almost no<br />
gain in soil C under no-tillage, but a C loss of 19 Mg C ha -1 after 12 years of conventional till. The results highlight<br />
the value of green manure legumes as a significant source of N for cropping systems which are a key aid to<br />
preserving or increasing soil C stocks under NT.<br />
97<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Plenary Session<br />
0900 – 1030<br />
Authors: Perrin H Beatty 1 , and Allen G Good 1<br />
1 Department of Biological Sciences, Biological Sciences Building, Edmonton, Alberta,<br />
Canada, T6G 2E9<br />
Presentation Title: Environmental and economic impacts of biological N2 fixing cereal crops<br />
Presentation Time: 0930 - 1000<br />
We will need to feed a large future global population using less arable land while coping with rising fuel prices<br />
and environmental pollution inherent to the increase in N fertilizer use in intensive farming practices. Cereal<br />
crops tend to have low nitrogen use efficiency and require N fertilizer application (synthetic or organic) for high<br />
yields. A portion of the applied N fertilizer will be lost to the plant by either uptake competition from soil microbes<br />
resulting in nitrification/denitrification and eventually loss back to the atmosphere, leaching into waterways and<br />
ammonia volatization. However, cereal crops that can fix their own nitrogen would bypass the N losses to the<br />
environment while still providing N to the plants.<br />
The engineering of N2 fixing cereal crops, by introducing either diazotrophic bacterial nodulation within cereal<br />
crop roots or nitrogenase genes (nif genes) directly into plant chloroplasts or mitochondria, is an attainable future<br />
goal (Beatty and Good, 2011). Both approaches are expected to take multiple years to completion, although<br />
step-wise advances in the ability of cereal crops to fix N2 are expected along the way. The rate of N2 fixation<br />
using either approach is predicted to be similar to the rate measured in rhizobial-legume symbiosis, or higher.<br />
This suggests that, if we can successfully engineer BNF cereal crops, the requirement for cereal crop N fertilizer<br />
application will be reduced, or potentially, even eliminated.<br />
The environmental and economic impacts of N2 fixing cereal crops are assessed in this paper, such as the<br />
environmental effects of using less N fertilizer on the atmosphere and aquatic ecosphere, the economic<br />
implications for farmers, as well as the potential implications to the plants‟ N economy.<br />
Beatty PH & Good AG (2011). Cereal crops and biological nitrogen fixation; future prospects. Science, July 24.<br />
98<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Plenary Session<br />
0900 – 1030<br />
Authors: Jean-Jacques Drevon 1 , Hesham Attar 2 , Adnane Bargaz 3 , Mustapha Faghire 3 , Cherki<br />
Ghoulam 3 , Mohamed Lazali 4 , Mohamed Ounan 1 , Paula Rodino 5<br />
1 <strong>IN</strong>RA, Eco&Sols, 2 Place Viala, 34000 Montpellier, France<br />
2 NRC Cairo, Egypt<br />
3 Faculté des Sciences et Techniques, Gueliz, Marrakech, Maroc<br />
4 Ecole Nationale Supérieure Agronomique, El Harrach, Alger, Algérie.<br />
5 Plant Genetics and Breeding, CSIC, 36080-Pontevedra, Spain.<br />
Presentation Title: Is phosphorus efficiency low for symbiotic nitrogen fixation?<br />
Presentation Time: 1000 - 1030<br />
Phosphorus is the limiting nutrient for legume symbiotic nitrogen fixation in most soils of the planet. Thus the<br />
yield of legumes generally responds to phosphorus fertilization. The P requirement of legumes is generally<br />
higher when their growth depends upon atmospheric nitrogen than upon ammonium or nitrate. This<br />
communication will revise the metabolic and structural utilizations of P that may limit the P efficiency for N2<br />
reduction in legume nodules and their genetic variation among legume species. The interaction with P<br />
metabolism will be illustrated with our results on nodule phytase activity (Araujo et al. 2008), and the tissue<br />
expression of the corresponding gene by in situ RT-PCR on nodule sections. The later will be compared with that<br />
of phosphoenol pyruvate phosphatase, and other candidate genes like trehalose 6-P phosphatase, that have<br />
been identified by deep super SAGE on nodule mRNA populations (Molina et al. 2011). The selection procedure<br />
of contrasting genotypes in P utilization for N2 fixation will be proposed to assess the functions involved in the<br />
adaptation of N2-fixing legumes in agro-ecosystems with low P soils, and to improve the yield of legumes and<br />
the sustainability of agricultural systemsdepending upon N2-fixation.<br />
Araujo AP, Plassard C & Drevon JJ (2008) Phosphatase and phytase activities in nodules of common bean<br />
genotypes at different levels of phosphorus supply. Plant Soil 312, 129-138.<br />
Molina C, Zaman-Allah M, Faheema K, Fatnassi N, Horres R, Rotter B, Steinhauer D, Drevon JJ, Winter P, Kahl<br />
G (2011) The salt-responsive transcriptome of chickpea root and nodules via deep super SAGE. BMC Plant<br />
Biology 11, 11-31.<br />
99<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 16 – Symbiotic Impacts & Emissions<br />
1100 - 1230<br />
Authors: Mark B Peoples 1 , John Brockwell 1 , John F Angus 1 , B Smith 1 , A Swan 1 and Catherine A<br />
Osborne 2<br />
1 CSIRO Sustainable Agriculture Flagship, CSIRO Plant Industry, GPO Box 1600,<br />
Canberra, ACT 2601.<br />
2 Department of Civil Engineering, Monash University, Clayton, Victoria 3800 (formerly<br />
Microbiology Department, University of Melbourne)<br />
Presentation Title: Effects of rhizobial strain and legume host on emissions of H2 from nodules and the<br />
impact on soil biology and plant growth<br />
Presentation Time: 1100 – 1120<br />
Hydrogen (H2) is produced as an obligate by-product of nitrogenase activity in legume nodules. Some<br />
symbioses possess a hydrogenase-uptake system (designated Hup + ) that is able to recycle almost all of the H2<br />
evolved. However, many strains of root-nodule bacteria (rhizobia) lack the hydrogenase enzyme (Hup ─ ), or have<br />
low Hup activity. In both these latter cases, the H2 diffuses out of the nodules into the soil. Within a very short<br />
period, this H2 is consumed by microorganisms residing close to the nodule ─ and none of it escapes into the<br />
atmosphere.<br />
Experimentation undertaken to assess the Hup status of various nodulated legumes indicated that, of all the<br />
legume x rhizobial strain combinations examined, only (i) soybean x CB1809 (Australian inoculant strain) and (ii)<br />
soybean x USDA110 (former US inoculant strain) were Hup + . All other associations emitted H2 from their<br />
nodules to a greater or lesser extent. Interestingly, the rates of H2 evolution from the nodules of faba bean, lupin<br />
and subterranean clover (mean: >450 μmol H2 evolution per gram nodule dry weight per hour) were consistently<br />
two- to five-fold greater than those observed for other legume x rhizobia combinations.<br />
Measurements of H2 evolution from Hup ─ nodules in the field indicate that >200,000 litres per hectare of H2 gas<br />
may be released into the soil during the life of a legume crop fixing around 200 kg nitrogen per hectare (Peoples<br />
et al. 2008). In addition, we observed increased populations of certain species of actinomycetes in the<br />
immediate vicinity of field-grown Hup ─ soybean nodules (Osborne et al. 2010). Some of these organisms are<br />
known to have growth-promoting characteristics.<br />
A number of studies in controlled-growth facilities and in the field in Australia and Canada have indicated<br />
improvements in plant growth and yield of 10-33% when plants are grown in soil exposed to H2. These findings<br />
need to be confirmed in other environments. If confirmation is obtained, there would be strong reason to<br />
recommend that future decisions on the choice of rhizobial strains for inoculants should involve ensuring that the<br />
resultant symbioses are Hup ─ in order to confer yield advantages on subsequent crops.<br />
Osborne CA, Peoples MB, Janssen PH (2010). Detection of a reproducible, single-member<br />
shift in soil bacterial communities exposed to low levels of hydrogen. Applied and<br />
Environmental Microbiology 76, 1471-1479.<br />
Peoples MB, McLennan PD, Brockwell J (2008). Hydrogen emission from nodulated<br />
soybeans [Glycine max (L.) Merr.] and consequences for the productivity of a subsequent<br />
maize (Zea mays L.) crop. Plant and Soil 307, 67-82.<br />
100<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 16 – Symbiotic Impacts & Emissions<br />
1100 - 1230<br />
Authors: Graeme Schwenke 1 , Pip Brock 2 & David Herridge 3<br />
1 NSW Department of Primary Industries, Marsden Park Road, Calala, 2340, New South<br />
Wales.<br />
2 NSW Department of Primary Industries, Port Stephens Fisheries Centre, Locked Bag<br />
1, Nelson Bay, 2315, New South Wales.<br />
3 University of New England, Primary Industries Innovation Centre, Armidale, 2351, New<br />
South Wales.<br />
Presentation Title: <strong>Nitrogen</strong>-fixing legumes in farming systems reduce greenhouse gas emissions<br />
Presentation Time: 1120 – 1140<br />
Nitrous oxide (N2O) is a greenhouse gas with close to 300 times the global warming potential of carbon dioxide<br />
(CO2). A major source of N2O emissions (50–60%) is associated with the application of nitrogenous fertilisers to<br />
agricultural soils. However, the direct emissions of N2O from soil, as a by-product of nitrification and an endproduct<br />
of denitrification, represent only a fraction of total greenhouse gas emissions related to N fertiliser use.<br />
Greenhouse gases (principally CO2) are also emitted in the production, transport and application of nitrogenous<br />
fertilisers and can be quantified using Life Cycle Assessment (LCA). Emissions of greenhouse gases from<br />
agricultural systems may be mitigated through partial substitution of fertiliser nitrogen (N) inputs with biologicallyfixed<br />
legume N because the latter is produced in the soil in situ and is of a dispersed, i.e. non point-source, and<br />
slow-release nature. To compare total greenhouse gas emissions from crop sequences with contrasting inputs of<br />
fertiliser- and legume-N in Australia‟s northern grains region, we monitored N2O emissions from the sequences<br />
for two years and conducted cradle-to-gate, i.e. pre-farm plus on-farm but not post-farm, LCA. We used<br />
automated greenhouse gas (N2O, CO2, CH4) measuring chambers to monitor soil emissions 7-8 times per day.<br />
During two years of measurement, cumulative soil N2O emissions differed fourfold between the rotation<br />
treatments, with canola (+N)–wheat (+N) emitting 1.32 kg N2O-N/ha, compared to 0.71 kg N2O-N/ha from the<br />
chickpea–wheat (+N) treatment and only 0.34 kg N2O-N/ha from the chickpea–wheat (no N) treatment. These<br />
treatment differences were increased further in the LCAs when all emissions associated with fertiliser N were<br />
included. We conclude that substitution of fertiliser N inputs with biologically-fixed legume N can substantially<br />
reduce greenhouse emissions from grain production systems.<br />
101<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Authors:<br />
Concurrent Session 16 – Symbiotic Impacts & Emissions<br />
1100 - 1230<br />
Kiwamu Minamisawa, Manabu Itakura, Shoko Inaba, and Yoko Shiina.<br />
Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aobaku, Sendai,<br />
980-8577, Japan<br />
Presentation Title: Mechanism of N2O emission from soybean nodule rhizosphere and its mitigation<br />
Presentation Time: 1140 – 1200<br />
Nitrous oxide (N2O) is a greenhouse gas that also degrades stratosphere ozone. Agricultural land is a major<br />
source of N2O through microbial transformation of nitrogen in plant-soil ecosystems. In particular, more N2O is<br />
emitted from legumes than that with non-legumes. Marked N2O emission were detected from root systems with<br />
degraded nodules during late growth stage in field-grown soybeans. To clarify the mechanism of N2O emission, a<br />
model system was developed in laboratory. Thirty-days after soybean plants inoculated with B. japonicum were<br />
cultivated in Leonard‟s jars, treatments of shoot decapitation (D) and/or soil addition (S) were conducted to<br />
simulate the nodule degradation and subsequent N2O emission. Double treatment (DS) resulted in the<br />
degradation and N2O emission from the nodules formed with B. japonicum lacking nosZ. These results<br />
suggested that soil microbes are required for the N2O emission from degraded soybean nodules. To evaluate<br />
bradyrhizobial contribution, N2O emission was compared between nirK mutant (∆nirK) and wild-type USDA110<br />
under identical nosZ genetic backgrounds. N2O emission from the nodules formed with ∆nirK∆nosZ mutant was<br />
significantly lower than that from ∆nosZ mutant under DS treatment, but retained approximately a half amount of<br />
N2O emission in ∆nosZ mutant (30-60%), suggesting that nitrate reduction to N2O is due to both B. japonicum<br />
and other soil microorganisms. On the other hand, it is likely N2O reduction to N2 was mainly mediated by B.<br />
japonicum cells carrying nosZ, which was consistently supported by the comparisons between wild-type<br />
USDA110 and nosZ mutants. Thus, B. japonicum plays an important role in determining N2O flux from soybean<br />
rhizosphere as well as unknown soil microorganisms. To mitigate N2O emission from soybean rhizosphere, we<br />
developed B. japonicum mutants with higher N2OR activity, and inoculated them to field soils where nosZ minus<br />
strains of B. japonicum are dominant. As a result, N2O emission from soybean rhizosphere significantly reduced<br />
in Leonard‟s jar system.<br />
102<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 16 – Symbiotic Impacts & Emissions<br />
1100 - 1230<br />
Authors: Tran Yen Thao 1 , David Herridge 2 , Rosalind Deaker 3 , Le Nhu Kieu 4 , Phan Thi Cong 5<br />
1 Research Institute for Oil and Oil Plants, 171-175 Ham Nghi Street, District 1, Ho Chi<br />
Minh City, Vietnam<br />
2 University of New England, Primary Industries Innovation Centre, Tamworth<br />
Agricultural Institute, 4 Marsden Park Road, Calala, New South Wales 2340<br />
3 Faculty of Agriculture, Food and Natural Resources, Ross St. Building A03, University<br />
of Sydney, NSW 2006<br />
4 National Soils and Fertilizers Institute, Dong Ngac Village, Tu Liem, Hanoi, Vietnam<br />
5 Institute of Agricultural Science, 121 Nguyen Binh Khiem Street, District 1, Ho Chi Minh<br />
City, Vietnam<br />
Presentation Title: Change in farmer attitudes and practices in Vietnam in the use of inoculant compared<br />
with baseline<br />
Presentation Time: 1200 – 1220<br />
The survey was constructed to provide the critical information that could be evaluated against baseline attitudes<br />
established from a similar survey carried out at the beginning of the project. It targeted farmers, extension<br />
workers as well as local agricultural technicians who are responsible for extending technological advances and<br />
innovations at agricultural localities. We conclude from this survey that there has been an increase in farmer<br />
awareness of inoculants and their role in legume growth promotion through biological N fixation. Almost all<br />
farmers in the final survey knew about inoculants and understood what they do. This was a result of the training<br />
and extension programe. Their knowledge mainly came from workshops and demonstrations. This survey also<br />
indicated a great interest by farmers and extension officers in future use of legume inoculants for soybean and<br />
groundnut in the target areas in Vietnam mostly because of economic reasons and because of their interest and<br />
desire to utilise new and novel technologies. The lack of use at the time of the second survey largely reflects<br />
lack of availability in the market place. The survey indicated that legume inoculants would be adopted readily in<br />
Vietnam provided they were accessible and easy to apply. Increasing production and supply of high quality<br />
legume inoculants in Vietnam, coupled with an effective extension program, should result in high adoption of<br />
inoculants. The further extension program would still need to emphasise the replacement of fertiliser N inputs,<br />
which represent a substantial part of the cost of growing these crops. The whole package should lead to<br />
increased farmer incomes and the relieving of poverty in many agricultural areas.<br />
103<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 17 – Molecular Characterization of N-fixing organisms<br />
1100 - 1230<br />
Authors: Ana Alexandre 1,2 Solange Oliveira 1<br />
1 ICAAM - Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de<br />
Évora, 7002-554 Évora Portugal.<br />
2 IIFA - Instituto de Investigação e Formação Avançada, Universidade de Évora, 7002-<br />
554 Évora, Portugal.<br />
Presentation Title: Major chaperone genes are highly induced in heat-tolerant rhizobia<br />
Presentation Time: 1100 – 1120<br />
The demand for more effective utilization of biologically-fixed N in agricultural systems has prompted studies on<br />
rhizobia tolerance to abiotic factors. Rhizobia ability to endure environmental stresses, such as soil pH, salinity<br />
and temperature is particularly important in legume-rhizobia symbioses under suboptimal conditions. The present<br />
work aimed at evaluating the temperature stress tolerance of native chickpea rhizobia and investigating if<br />
tolerance is related to isolates„ species or origin site. Another aim was to investigate the molecular bases of<br />
temperature stress tolerance, by comparing the expression levels of major chaperone genes, in thermotolerant<br />
and thermosensitive isolates.<br />
A set of 53 chickpea mesorhizobia, previously isolated from several provinces of Portugal and characterized in<br />
terms of symbiotic effectiveness, plasmid profiles and species affiliation, was used (Alexandre et al 2009).<br />
Temperature stress tolerance was evaluated under cold, heat and heat shock conditions. Mesorhizobia showed<br />
high diversity in their ability to grow under temperature stress; nevertheless most isolates tolerate heat shock or<br />
cold stress better than continuous heat. Distinct species groups were found to differ significantly in their ability to<br />
tolerate temperature stress. An association was found between some provinces of origin and stress tolerance of<br />
the isolates.<br />
In order to study the molecular bases of temperature stress tolerance, the mRNA levels of chaperone genes<br />
were analysed upon stress, using tolerant and sensitive chickpea rhizobia. Analysis of dnaK and groESL genes<br />
expression by northern hybridisation, using isolates from several species groups, showed an increase in the<br />
transcripts levels with heat but not with cold stress. Interestingly, following temperature upshifts, the induction of<br />
chaperone genes was higher in tolerant than in sensitive isolates from the same species (Alexandre & Oliveira,<br />
2011). Overall, these results suggest that higher transcriptional induction of the major chaperone genes could be<br />
related with a higher tolerance to heat in rhizobia.<br />
Alexandre A, Brígido C, Laranjo M, Rodrigues S & Oliveira S (2009). Survey of chickpea rhizobia diversity in Portugal reveals the<br />
predominance of species distinct from Mesorhizobium ciceri and Mesorhizobium mediterraneum. Microb Ecol 58: 930-841.<br />
Alexandre A & Oliveira S (2011). Most heat-tolerant rhizobia show high induction of major chaperone genes upon stress. FEMS Microbiol<br />
Ecol 75: 28-36.<br />
This work was supported by Fundação para a Ciência e Tecnologia: project FCOMP-01-0124-FEDER-007091 and fellowship to A. A.<br />
(SFRH/BPD/73243/2010).<br />
104<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 17 – Molecular Characterization of N-fixing organisms<br />
1100 - 1230<br />
Authors: Nazalan Najimudin 1 , Hok-Chai Yam 1 , Mohd-Razip Samian 1 and Suriani Mohamad 1<br />
1 School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Penang,<br />
Malaysia.<br />
Presentation Title: Characterization of nitrogen fixation genes from Paenibacillus polymyxa ATCC 15970<br />
Presentation Time: 1120 – 1140<br />
Paenibacilus polymyxa ATCC 15970 is a Gram positive bacterium capable of converting dinitrogen (N2) to<br />
ammonia. The nitrogen fixation operon of this strain was determined to have the gene arrangement of nifBHDK.<br />
The deduced NifBHDK amino acid sequences were 499 aa, 255 aa, 482 aa and 509 aa, respectively. The<br />
putative nifD and nifK ORFs were overlapping by 4 nucleotides. The transcriptional start sites of nifB and nifH<br />
were determined by cRACE and cmRACE methods. Two different transcriptional start sites were obtained -<br />
upstream of nifB and another upstream of nifH. Therefore, the nifH gene could possibly be expressed from two<br />
different mRNA molecules transcribed from two different promoters located upstream of nifB and nifH,<br />
respectively. Based on their transcriptional start sites, the promoter regions of nifB and nifH revealed consensus<br />
sequences that were distinguishable from the traditional nif promoter. This suggested that the expression of nif<br />
genes in P. polymyxa was possibly controlled by a unique regulation system. The nifB gene is possibly coexpressed<br />
with the structural genes nifHDK. The promoter sequences of nifB and nifH were different from the<br />
typical nif promoter motif. Two highly consensus sequences were found at the presumptive promoter regions of<br />
nifB and nifH located roughly at positions -8 (GTAAGGG) and -28 (AGACAAA) with respect to their<br />
transcriptional start sites. This novel promoter motif merits future investigation.<br />
105<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 17 – Molecular Characterization of N-fixing organisms<br />
1100 - 1230<br />
Authors: Melissa K. Corbett, Lesley A. Mutch and Elizabeth L.J. Watkin<br />
School of Biomedical Sciences, Curtin University, Kent Street, 6845, Western Australia<br />
Presentation Title: Identification and evolution of NIF genes in Leptospirillum species – an acidophilic<br />
chemolithoautotroph<br />
Presentation Time: 1140 – 1200<br />
Leptospirillum are gram negative, chemolithoautotrophic extremophiles found in environments where sulfide<br />
and iron bearing minerals are exposed to the air. Their natural ability to oxidize ferrous iron for energy, leaving<br />
behind other liberated minerals has been industrialized in the form of biomining. Biomining operations rely on<br />
this microbial aided extraction of metals from solid minerals, but mineral recovery rates can be affected if<br />
nutrient levels fluctuate and gradients form. The addition of ammonium based chemicals to meet<br />
Leptospirillum growth needs can be expensive, and equal distribution is not guaranteed. Genomic research<br />
has uncovered that two of the three documented species, L. ferrooxidans and L. ferrodiazotrophum encode<br />
genes for nitrogen fixation. To ascertain whether the third species, L. ferriphilum, also has the potential for<br />
diazotrophy, degenerate nifH primers were tested. A single PCR product of approximately ~900bp was<br />
amplified and subsequent sequence analysis revealed significant identity (84%) to the nifH genes in the other<br />
Leptospirillum species. Phylogenetic analysis of the single nifH genes placed all Leptospirillum species in the<br />
conventional Mo-containing nifH cluster I, closely related to the nifH genes of �-proteobacterium,<br />
Acidithiobacillus, a known nitrogen fixing biomining microbe. Concatenated super-gene alignment of the nifH<br />
gene with 16SrRNA, gyr B, nifD and nifK provided a greater resolution of evolution amongst the closely<br />
related strains. The use of maximum likelihood and maximum parsimony phylogenetic trees further elucidated<br />
the evolutionary relationships between the three Leptospirillum species. Discovery of these genes in all<br />
identified Leptospirillum species aids in cementing the status of the Leptospirillum genus as free living<br />
diazotrophs whose presence in biomining operations can help sustain mineral leaching rates when soluble<br />
nitrogen levels are low, without the incursion of additional operational costs.<br />
106<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 17 – Molecular Characterization of N-fixing organisms<br />
1100 - 1230<br />
Authors: Ni Luh Arpiwi1 , Elizabeth LJ Watkin2 , Guijun Yan1 , Elizabeth L Barbour, 1, 3<br />
and Julie A Plummer 1<br />
1 School of Plant Biology, University of Western Australia, 35 Stirling Hwy Crawley WA<br />
6009<br />
2 School of Biomedical Science, Curtin University, Bentley WA 6102<br />
3 Forest Products Commission of Western Australia, 117 Great Eastern Highway<br />
Rivervale, WA 6103, Australia.<br />
Presentation Title: Physiological and molecular characterization of the root nodule bacteria nodulating<br />
Millettia pinnata, a biodiesel tree<br />
Presentation Time: 1200 – 1220<br />
Milletia pinnata is a leguminous tropical tree that produces seed oil suitable for biodiesel. By optimizing the<br />
symbiotic relationship with nitrogen fixing bacteria reduced fertilizer inputs would be required, which would be<br />
advantageous for this second generation biofuel crop, especially for production on marginal land. This study<br />
aimed to isolate root nodule bacteria from soil around Millettia trees, confirm their symbiotic potential, examine<br />
their physiological characteristics and to identify elite strains.<br />
Soil samples were collected from the base of Millettia pinnata grown in plantations in northern, tropical<br />
Western Australia. Seeds were planted in pots layered with 3 cm of sterile coarse sand then 3 cm soil, 2 cm<br />
sterile coarse sand and 1 cm sterile plastic beads on the top. Seeds were placed in the upper coarse sand<br />
layer and grown for 18 – 20 weeks. Root nodules were harvested and crushed, and the contents streaked<br />
onto a half-lupin agar plates and incubated at 29 o C. Forty pure isolates were obtained which produced single<br />
colonies in 5-9 days. Optimum growth conditions for these isolates were pH 7 – 9 and 29 – 37 o C. Growth of<br />
all isolates was reduced at 1 – 2% NaCl and no isolates grew at 3% NaCl. Most isolates had optimal growth<br />
on mannitol, arabinose or glutamate as a single carbon source, only a few grew on sucrose and none grew on<br />
lactose. Cluster analysis of isolates based on physiological characteristics indicated phenotypic diversity.<br />
Isolates were re-authenticated and the effectiveness of the symbiotic association assessed based on shoot<br />
dry weight compared to that of the nitrogen-fed control plants. Genetic diversity of isolates was analyzed and<br />
confirmed using the sequence of the 16S RNA gene. The outcomes of this research will promote the growth<br />
of Millettia plantations in nutrient poor soils which alleviates competition on arable lands for food production.<br />
107<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 18 – P Nutrition & Symbioses<br />
1100 - 1230<br />
Authors: Neera Garg 1 and Harmanjit Kaur 2<br />
Department of Botany, Panjab University, Chandigarh 160014, India<br />
Presentation Title: Role of arbuscular mycorrhizal (am) fungi and zinc in enhancing nitrogen fixation in<br />
pigeon pea under cadmium stress<br />
Presentation Time: 1100 – 1120<br />
AM fungi are known to alleviate heavy metal stress in plants (Hildebrandt et al 2007). Cadmium (Cd) is a toxic<br />
non essential element, whereas Zinc (Zn) is an essential element but regarded toxic at high concentrations.<br />
Cd and Zn compete for transport via common carrier at root plasma membrane and Zn interferes with Cd<br />
uptake in plants (Hassan et al 2005). The intent of present work was to analyze accumulation of heavy metals<br />
(Cd and Zn) in nodules of two Cajanus cajan (L.) Millsp. (pigeonpea) genotypes and their subsequent impact<br />
on nitrogen fixation, oxidative stress and non protein thiols (glutathione and phytochelatins) with and without<br />
AM fungus. Two levels of Cd (25 and 50 mg kg -1 dry soil) and two levels of Zn (500 and 1000 mg kg -1 dry soil)<br />
were applied singly and in combinations to 15 day old seedlings. Accumulation of Cd and Zn resulted in sharp<br />
reduction in nodule number (NN), nodule dry mass (NDM) as well as nitrogen fixation {leghemoglobin (LHb)<br />
and nitrogenase (N2ase)} of pigeonpea plants, although Cd had more pronounced effects than Zn. Cd induced<br />
ROS generation, lipid peroxidation and altered non protein thiols were largely reversed by Zn<br />
supplementation, suggesting antagonistic behaviour of Zn. Remarkable genotypic variation was found, with<br />
much more severe response of both the metals in P792 than Sel 85N. Glomus mosseae along with Zn further<br />
attenuated the phytotoxic effects of metals in nodules by significantly decreasing metal uptake and oxidative<br />
stress ultimately leading to better nitrogen-fixation in pigeonpea.<br />
Hassan MJ, Zhang G, Wu F, Wei K & Chen Z (2005). Zinc alleviates growth inhibition and oxidative stress<br />
caused by cadmium in rice J Plant Nutr Soil Sci 168: 255-261.<br />
Hildebrandt U, Regvar M & Bothe H (2007). Arbuscular Mycorrhiza and Heavy Metal Tolerance.<br />
Phytochemistry 68: 139-146.<br />
108<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 18 – P Nutrition & Symbioses<br />
1100 - 1230<br />
Authors: Andry Andriamananjara 1,2,* , Mahamadou Malam Abdou 3 , Lilia Rabeharisoa 1 , Laurie<br />
Amenc 5 , Hélène Vailhe 5 , Dominique Masse 4 , Jean-Jacques Drevon 5<br />
1 LRI-SRA, Laboratoire des Radio-isotopes, Université d'Antananarivo, Route<br />
d'Andraisoro, BP 3383, 101 Antananarivo, Madagascar ;<br />
2 Ecole Supérieure des Sciences Agronomiques d’Antananarivo, Madagascar.<br />
3 Laboratoire Banques de gènes CERRA / KOLLO, Institut National de Recherche<br />
Agronomique du Niger (<strong>IN</strong>RAN), BP 429 Niamey, NIGER.<br />
4 Institut de Recherche pour le Développement, UMR Eco&Sols, Montpellier- France .<br />
5 Institut National de la Recherche Agronomique, UMR Eco&Sols, Montpellier-France.<br />
Presentation Title: Genotypic variation for N2-fixation in Voandzou (Vigna subterranea) under P deficiency<br />
and P sufficiency<br />
Presentation Time: 1120 – 1140<br />
Genetic variation associated with N2 fixation exists in numerous legume species (Graham, 2004). High<br />
symbiotic N2 fixation under P deficiency is related closely to nodulation which was used in legume selection<br />
for N2 fixation (Herridge and Rose, 2000). Until now, study of genetic potential of neglected crops like Vigna<br />
subterranea (bambara groundnut or voandzou) is often limited while its agronomic properties is interesting for<br />
the farmers of Africa. In order to assess the genotypic variation of voandzou for tolerance to phosphorus<br />
deficiency, a physiological approach of cultivar selection was performed with 54 cultivars from Madagascar,<br />
Niger and Mali in hydroponic culture under P deficiency and P sufficiency and inoculated with the reference<br />
strain of Bradyrhizobium sp. Vigna CB756. The results of nodulation and plant biomass, which are closely<br />
related, showed a large dispersion between cultivars (0.05-0.43 g nodule dry weight per plant and 0.50-5.51 g<br />
shoot dry weight per plant). The cultivars which presented the maximum growth during the experiment<br />
presented a high efficiency in use of the rhizobial symbiosis calculated as the slope of plant biomass<br />
regression as a function of nodulation. A large increase in nodulated-root O2 consumption under P deficiency<br />
was observed for the two most tolerant cultivars. The microscopic analysis with in situ RT-PCR of the nodule<br />
sections showed an increase of a phytase gene expression with tolerance of cultivars to P deficiency. These<br />
results highlight the genotypic variability among voandzou cultivars for the phosphorus use efficiency for N2<br />
fixation as a mechanism of adaptation to phosphorus deficiency.<br />
Herridge D, Rose I, 2000. Breeding for enhanced nitrogen fixation in crop legumes. Field Crops Research 65.<br />
229-248.<br />
Graham PH., Hungria M, Tlusty B., 2004. Breeding for better nitrogen fixation in grain legumes: Where do the<br />
rhizobia fit in? On line Crop Management.<br />
109<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 18 – P Nutrition & Symbioses<br />
1100 - 1230<br />
Authors: Sipho Maseko 1 and Felix D. Dakora 2<br />
1 Department of Crop Science, and 2 Department of Chemistry, Tshwane University of<br />
Technology, 175 Nelson Mandela Drive, Private Bag X680, Pretoria 0001, South Africa<br />
Presentation Title: Phosphorus nutrition in symbiotic Cyclopia and Aspalathus species from the Cape<br />
fynbos of South Africa<br />
Presentation Time: 1140 – 1200<br />
<strong>Nitrogen</strong> and phosphorus are the most limiting nutrients affecting plant growth in the Cape fynbos. In this study,<br />
acid and alkaline phosphatase (APase) activity of rhizosphere soils from Cyclopia genistoides, C. subternata,<br />
Aspalathus caledonensis and A. aspalathoides were assayed as indicators of P nutrition in these legumes. The<br />
results showed a significantly (p≤ 0.05) higher APase activity in rhizosphere soils of the four legumes and the<br />
non-legumes Leucadendron strictum, Eleiga thyrsoidea and Mimetes cucullatus relative to bulk soil. Furthermore,<br />
rhizosphere P and shoot P concentrations closely mirrored the phosphatase-enzyme activity in the rhizosphere<br />
of each species, suggesting that rhizosphere enzyme activity can serve as a good indicator of P nutrition in<br />
Cyclopia and Aspalathus species in the Cape fynbos. The relationship between APase activity in organs and P<br />
concentration of those organs in three Cyclopia and two Aspalathus species was also assessed at the same<br />
sites in the fynbos. At all three sites, APase activity was greater in organs of the legumes compared to the nonlegume<br />
Leucadendron strictum. At Koksrivier, leaf APase activity was highest in Cyclopia genistoides, followed<br />
by Aspalathus caledonensis, and least in A. aspalathoides. At Kleinberg and Kanetberg, Cyclopia subternata and<br />
C. longifolia also showed highest APase activity in leaves, followed by stems, and lowest in roots. The P<br />
concentration of each organ closely mirrored its APase activity, and APase activity was positively correlated with<br />
P concentration in leaves, stems and roots of C. genistoides, C. subternata, and C. longifolia. This indicates that<br />
in the Cape fynbos, APase activity can be used as a good indicator of P nutrition in Cyclopia and to, some<br />
extent, Aspalathus species.<br />
110<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Thursday 1 December 2011<br />
Concurrent Session 18 – P Nutrition & Symbioses<br />
1100 – 1230<br />
Authors: Bargaz Adnane 1,2 , Faghire Mustapha 1 , Mandri Btissam 1 , Farissi Mohamed 1 , Jean<br />
Jacques Drevon 2 , and Ghoulam Cherki 1<br />
1 Equipe de Biotechnologie Végétale et Agrophysiologie des Symbioses, Faculté des<br />
Sciences et Techniques Guéliz-Marrakech, BP 549, 40000, Marrakech, Morocco.<br />
2 <strong>IN</strong>RA-IRD-SupAgro, UMR Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols<br />
& Agroécosystèmes 2 Place Viala, F34060, Montpellier, France.<br />
Presentation Title: Phosphorus deficiency affected conductance to O2 and ascorbate peroxydase gene<br />
expression in nodules of Phaseolus vulgaris-rhizobia symbiosis<br />
Presentation Time: 1200 – 1220<br />
Although recent studies have addressed the effects of phosphorus (P) deficiency on nodule respiration and<br />
oxidative stress, little attention has been given to elucidate the relationships between nodule permeability, nodule<br />
P status, and antioxidative responses. To study these traits, two recombinant inbred lines, namely RILs L115 and<br />
L147 were inoculated with Rhizobium tropici CIAT899, and grown in hydroaeroponic culture under P-sufficiency<br />
(250) versus P-deficiency (75 µmol P plant -1 week -1 ) conditions. At the flowering stage, plants and nodules<br />
biomasses, P contents and localization of nodule ascorbate peroxidase (APX) transcripts were determined after<br />
measuring O2 uptake by nodulated roots and nodule conductance to O2 diffusion (gn). The results showed that Pdeficiency<br />
significantly decreased plant growth and nodulation, with differences between bean genotypes. Pdeficiency<br />
also induced a decrease in nodule P content (31%) in the sensitive L147 and tolerant line L115, a 42<br />
and 27% reduction in shoots of sensitive and tolerant lines, respectively. Under P-deficiency, gn increased more<br />
for the sensitive (39%) than for the tolerant lines (27%). This increase was linked to a rise both in the P levels in<br />
nodules and shoots, as well as in the efficiency of symbiotic nitrogen fixation as determined by nodule-dependent<br />
biomass production for the sensitive lines. Furthermore, positive correlations were found between O2<br />
permeability, gn and P content both in nodules and shoots (r 2 = 0.94** and r 2 = 0.96**). Additionally, P-deficiency<br />
increased electrolyte leakage, the oxidative stress and the transcripts for ascorbate peroxidase gene in nodules.<br />
The later was more abundant in the inner cortex and the infected cells close to inner cortex. We conclude that gn<br />
increased with increases in both nodule P content and antioxidative responses.<br />
111<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Poster Board<br />
Poster Title Presenting<br />
Number<br />
Author<br />
1 Symbiotic nitrogen contribution by a long-duration pigeonpea<br />
genotype intercropped with maize in South Africa<br />
Cherian Mathews<br />
2 Growth inhibition of Sinorhizobium medicae in the presence of<br />
amino acids and the absence of supplemented calcium is<br />
alleviated by a mutation in the low pH inducible gene lpiA.<br />
3 Rhizobia nodulating Vicia faba in Tunisia constitute a new branch<br />
inside the genus Rhizobium<br />
4 Characterization of two splice variants of SIP1 and their functions<br />
during nodule development in Lotus japonicus<br />
5 Characterization of rhizobia isolates using molecular methods<br />
6 The contribution of N-fixing legumes to the productivity of wheat in<br />
mediterranean agriculture systems of Chile.<br />
7 Crosstalk between GmSAT1 and yeast unveils a novel family of<br />
eukaryotic ammonium transport proteins<br />
8 Co-inoculation of rhizobia with highly induced ACC deaminase<br />
bacteria could alleviate the stress of legumes growing under stress<br />
conditions<br />
9 Rice growth promotion after inoculation of Sinorhizobium melilotii<br />
1021 and its mechanism<br />
10 Differential activation of CCaMK between root nodule and<br />
arbuscular mycorrhizal symbioses<br />
11 Determination of optimal phosphorus concentrations which<br />
enhance the establishment of both mycorrhizal and rhizobial<br />
symbiosis<br />
12 Comparative analysis of genome sequences of Rhizobium<br />
galegae HAMBI 540 and HAMBI 1141<br />
13 Denitrification and nitrous oxide emissions by soybean<br />
bradyrhizobia<br />
14 Proteomic profile of the soybean symbiosome membrane<br />
15 Nodulation studies of cowpea and its response to phosphorus<br />
application in Botswana under glasshouse conditions<br />
112<br />
Wan Adnawani<br />
Meor Osman<br />
Ridha Mhamdi<br />
Hui Zhu<br />
Bakhtiyor Umarov<br />
Carlos Ovalle<br />
Danielle<br />
Mazurkiewicz<br />
Panlada Tittabutr<br />
Yuxiang Jing<br />
Yoshikazu<br />
Shimoda<br />
Manijeh<br />
Mohammadi-<br />
Dehcheshmeh<br />
Janina Osterman<br />
2011<br />
Presenting Day<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
Robert M. Boddey 30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
Victoria Clarke 29 th November<br />
Tuesday<br />
Flora Pule-<br />
Meulenberg<br />
16 Lotus japonicus nodulates when it sees red Akihiro Suzuki<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Poster Board<br />
Number<br />
Poster Title<br />
17 Role of the Cpx two component regulatory system and a MFS<br />
transporter in the symbiosis between Sinorhizobium meliloti and<br />
leguminous plants<br />
18 Compatibility with rhizobia of polymer adhesives and colourants<br />
used in preinoculated legume pasture seed<br />
19 Efficiency of Bradyrhizobium japonicum in different formulations<br />
and when co-inoculated with bacillus subtilis on soybean in a<br />
Kenyan soil<br />
20 Potential of indigenous bradyrhizobia versus commercial<br />
inoculants to improve cowpea and green gram yields in Kenya<br />
21 Isolation and characterization of diazotrophic microsymbionts<br />
from root nodules of Mucuna bracteata<br />
22 Lipopolysaccharide binding protein, a new requirement for<br />
legume-rhizobium symbiosis<br />
23 The role of 1-aminocyclopropane-1-carboxylate (ACC)<br />
deaminase enzyme on leguminous nodule senescence<br />
24 Gamma irradiation and autoclave sterilization of peat and<br />
compost as the carrier for rhizobial inoculant production<br />
25 Effect of nitrogen, phosphorus, magnesium and calcium nutrition<br />
on plant growth and levels of macronutrients in the honeybush<br />
tea plant, Cyclopia longifolia (Vogel l.)<br />
26 Root-nodule bacteria isolated from cowpea and bambara<br />
groundnut enhance mineral nutrition in their homologous hosts<br />
27 Evaluation of elite commercial soybean varieties for N2 fixation<br />
and grain yield in South Africa<br />
28 Ecophysiological studies and biodiversity of root-nodule bacteria<br />
nodulating Psoralea species in the cape fynbos of South Africa<br />
29 <strong>Nitrogen</strong> fixation promotes accumuation of dietarily-important<br />
mineral nutrients in edible leaves of cowpea (Vigna unguiculata l.<br />
Walp.)<br />
30 Diversity and phylogeny of Bradyrhizobium spp. Isolated from<br />
shrub and tree legumes in Ethiopia<br />
113<br />
Presenting Author<br />
2011<br />
Presenting Day<br />
Mário Santos 30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
Elizabeth Hartley<br />
Mary Atieno<br />
Samuel Mathu<br />
Amir Ghazali<br />
Toshiki Uchiumi<br />
Neung Teaumroong<br />
Nantakorn Boonkerd<br />
Buhlebelive<br />
Mndzebele<br />
Thabo Makhubedu<br />
Nyamande Mapope<br />
Sheku Kanu<br />
Alphonsus Belane<br />
Aregu Amsalu<br />
Aserse<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Poster Board<br />
Number<br />
Poster Title<br />
31 Phylogenetic position of Rhizobium galegae<br />
32 Study on soybean nodule symbiosome membrane localised<br />
transporters<br />
33 Development of an inoculant for Acacia acuminata to improve<br />
growth of Santalum spicatum<br />
34 Endophytic colonization of sweet potato by a diazotrophic<br />
bradyrhizobia<br />
35 Functional Analysis of STM Mutants Concerning Amino Acid<br />
Metabolism of Mesorhizobium loti<br />
36 Establishment of retrotransposon-mutagenized population of the<br />
model legume Lotus japonicus and high throughput, deep<br />
sequencing-based insertion site identification.<br />
37 Rhizobium delivery systems for grain legumes in southern<br />
Australia<br />
38 BNF efficiency of rhizobia isolates from Phaseolus vulgaris in<br />
subsistence agrosystems in Dominican Republic.<br />
39 Protection of plant health by Micromonospora isolated from<br />
alfalfa root nodules.<br />
40 Identification by MALDI-TOF MS spectrometry of rhizobia<br />
isolated from nodules of different legumes<br />
41 Expression of an abscisic acid-responsive ƒÀ-1,3-glucanase<br />
gene in autoregulation of nodulation.<br />
42 Conservation of legume-rhizobium symbiosis genes in nonlegume.<br />
43 Analysis of nif gene derepression in Azotobacter vinelandii by<br />
quantitative real-time PCR<br />
44 Nodule-specific cystein-rich peptides found in actinorhizal plant<br />
Datisca glomerata<br />
45 N2O emission from degraded soybean nodules by denitrification<br />
of Bradyrhizobim japonicum and other soil microorganisms<br />
114<br />
Author<br />
Seyed Abdollah<br />
Mousavi<br />
2011<br />
Presenting Day<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
Chi Chen 30 th November<br />
Wednesday<br />
Elizabeth Watkin<br />
Yoshinari Ohwaki<br />
Shigeyuki Tajima<br />
Jens Stougaard<br />
David Pearce<br />
Beatriz Urbano<br />
Pilar Martínez-<br />
Hidalgo<br />
Eustoquio Martinez-<br />
Molina<br />
Ken-Ichi Osuki<br />
Keisuke Yokota<br />
Cesar Poza Carrion<br />
Irina Demina<br />
Fumio Ikenishi<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Poster Board<br />
Number<br />
Poster Title<br />
46 Functional characterization and interactions of the azorhizobial<br />
parA and parB proteins<br />
47 Trehalose accumulation in osmotically challenged rhizobia and<br />
its effect on desiccation tolerance.<br />
48 A MYB coiled-coil type transcription factor interacts with NSP2<br />
and is essential for nodulation in Lotus japonicus<br />
49 Genome sequence of Mesorhizobium huakuii 7653r which<br />
establishs a highly specific symbiosis with Astragalus sinicus in<br />
China<br />
50 Identification of the regulatory genes required for the acid<br />
activation of the low pH inducible gene lpiA in Sinorhizobium<br />
medicae<br />
51 Selection of plant growth promoting rhizobacteria (PGPR) to<br />
enhance nodulation of grain legumes by rhizobia<br />
115<br />
Chi-Te Liu<br />
Author<br />
Andrea Casteriano<br />
Zhongming Zhang<br />
Youguo Li<br />
Tian Rui<br />
2011<br />
Presenting Day<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
30 th November<br />
Wednesday<br />
15:00 – 16:00<br />
29 th November<br />
Tuesday<br />
15:00 – 16:00<br />
Liza Parkinson 30 th November<br />
Wednesday<br />
15:00 – 16:00
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Symbiotic nitrogen contribution by a long-duration pigeonpea genotype intercropped with<br />
maize in South Africa<br />
Authors<br />
Poster Board Number 1<br />
1 Cherian Mathews, 2 Wim van Averbeke and 2 Felix Dakora<br />
1 Department of Agriculture, P/Bag X11318, Nelspruit-1200<br />
2 Tshwane University of Technology P/Bag X680, Pretoria-0001<br />
As a crop, pigeonpea has a huge market value in South Africa due to the growing Asian population. Large<br />
quantities of pigeonpea grown is currently imported to meet local demand. This indicates the need to evaluate<br />
pigeonpea genotypes for growth, grain yield and symbiotic performance in order to identify genotypes for use in<br />
local cropping systems.<br />
Field experiments were conducted at Nelspruit during 2007, 2008 and 2009 cropping seasons to assess N2<br />
fixation and yield potential of pigeonpea (Cajanus cajan L. Millspaugh) in intercropping systems used by<br />
resource-poor farmers in Africa. Three intercropping systems (within-rows, alternate rows, and paired rows) and<br />
their sole cropped pigeonpea (cv. ICEAP00040) and maize (cv. ZM521) were used in a randomized complete<br />
block design with five replications. Destructive sampling was done at four stages of legumes (i.e. maize<br />
tasseling, maize harvest, pigeonpea flowering, and pigeonpea harvest) for dry matter yield and measurement of<br />
N2 fixation using 15 N natural abundance. The results of two cropping seasons (2007-08 and 2008-09) are<br />
presented here. Dry matter yield was significantly (p=0.01) lower in intercropped pigeonpea relative to<br />
monoculture, but was positively correlated with N content, δ 15 N and amount of N-fixed. The N content per plant<br />
was highest in monocultured pigeonpea as a result of its larger dry matter yield. In both seasons, the δ 15 N values<br />
of intercropped pigeonpea were lower than monoculture, and were lowest in plants on same-row intercropping<br />
and/or alternate row intercroping.<br />
As a result, %Ndfa values were significantly greater under these cropping systems. The average amount of Nfixed<br />
was up to 173 kg ha -1 in monoculture and 153 kg ha -1 in intercrop, levels which peaked at pigeonpea<br />
flowering in both seasons. The results show that pigeonpea can contribute substantial amounts of symbiotic N<br />
and thus enhance soil productivity under traditional cropping systems.<br />
116<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Growth inhibition of Sinorhizobium medicae in the presence of amino acids and the absence<br />
of supplemented calcium is alleviated by a mutation in the low pH inducible gene lpiA.<br />
Author Wan Adnawani Meor Osman 1,2 , Ravi Tiwari 1 , Lambert Brau 1 , Vanessa Melino 1 & Wayne<br />
Reeve 1 .<br />
Poster Board Number 2<br />
1 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia.<br />
Australia<br />
2 Department of Biological Sciences, Faculty of Sciences and Technology, University<br />
Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia.<br />
Sinorhizobium medicae WR101 was identified as a mTn5-GNm induced mutant of WSM419 that expressed acid<br />
inducible GUS activity. Characterisation of the mutation revealed that the promoterless gusA gene in mTn5-GNm<br />
was fused to the low pH inducible gene lpiA. The LpiA protein contains a COG0392 signature (indicative of an<br />
integral membrane protein) domain revealing that this protein is most likely a transmembrane protein. In addition,<br />
LpiA also contains three other domains of unknown function including DUF470, DUF471 and DUF472 which<br />
share 83%, 62% and 58% similarity to proteins found in animal pathogens, plant pathogens and other symbionts,<br />
respectively (Reeve et al., 2006). An in depth characterisation of the phenotype of the mutant cells revealed that<br />
the lpiA mutation disrupted the acid-tolerance response (ATR) associated with S. medicae WSM419. In addition<br />
to this, we have now found that the lpiA mutation enables the mutant to grow on amino acid rich media devoid of<br />
calcium. In contrast, the wild-type strain WSM419 cannot grow in this condition and requires calcium to<br />
overcome the growth inhibition found with amino acid containing rich media. Complementation analysis has<br />
revealed that mutant growth can be severely inhibited on media containing amino acids in the absence of<br />
sufficient calcium. These observations have demonstrated that the absence of LpiA is essential for growth on<br />
calcium deficient media containing amino acids. Potential reasons for the growth inhibition of S. medicae by<br />
amino acids in the absence of calcium will be presented.<br />
117<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Rhizobia nodulating Vicia faba in Tunisia constitute a new branch inside the genus<br />
Rhizobium<br />
Authors Sabrine Saïdi, Sabrine Chaïbi & Ridha Mhamdi<br />
Poster Board Number 3<br />
Centre of Biotechnology of Borj-Cedria, Laboratory of Legumes, Hammam-lif, Tunisia.<br />
Faba bean is a major leguminous crop cultivated in many countries around the world. The nodulating rhizobia<br />
were usually associated to Rhizobium leguminosarum bv. viciae. However, some recent studies revealed that<br />
this crop is also nodulated by R. pisi and R. fabae. Nevertheless, R. pisi was described on the basis of a unique<br />
isolate with an ambiguous origin. The phylogenetic analysis of the 16S rDNA and housekeeping genes showed<br />
that these novel described species are closely related and would be synonymous. The aim of this work is to<br />
study the genetic diversity and symbiotic effectiveness of rhizobia nodulating faba bean in Tunisia. Rhizobia<br />
nodulating Vicia faba in various Tunisian soils from different geographical regions were isolated using standard<br />
procedures. More than 50% of the nodule isolates were endophytes that failed to re-nodulate their original host.<br />
The nodulating isolates were assigned all to Rhizobium leguminosarum on the basis of PCR-RFLP analysis of<br />
16S rDNA. Ten representative isolates were selected based on their REP-PCR and PCR-RFLP of nodC and<br />
nifDK patterns. Their taxonomic position was further assessed by sequence analysis of 16S rDNA, recA, atpD,<br />
glnII, nodA, nodC and nifH genes. The results indicated that all the isolates formed a new distinct branch inside<br />
the genus Rhizobium and would probably constitute a new species. Nodulation and effectiveness tests on V.<br />
faba vars. equina and minor and Lens culinaris showed different host-specific behaviors with variable<br />
efficiencies.<br />
118<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Hui Zhu, Chao Wang, Liping Jin, Tao Chen, Longxiang Wang, Zhongming Zhang*<br />
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University<br />
Authors Characterization of two splice variants of SIP1 and their functions during nodule<br />
development in Lotus japonicas<br />
Poster Board Number 4<br />
Lotus japonicus SymRK and N<strong>IN</strong> genes are both required for the successful establishment of root nodule<br />
symbiosis. Previously, we discovered that ARID family transcription factor SIP1 interacted with SymRK and<br />
specifically bound to the AT-rich motif of N<strong>IN</strong> promoter (Zhu et al., 2008). However, the biological function of<br />
SIP1 wasn‟t fully understood. Here, an isoform of SIP1 (called SIP1S in this work) was isolated, referred to as<br />
SIP1L, which is produced by alternative splicing. SIP1L contains an ARID domain and an Hsp20 domain, while<br />
SIP1S lacks the complete Hsp20 domain with 17 amino acids deletion. SIP1L presents a more abundant<br />
expression than SIP1S and can‟t interact with SymRK in yeast, suggesting some characterizations of the two<br />
splice variants are altered. However, SIP1L and SIP1S colocalize in nuclei in onion epidermal cells and form<br />
homo- and heterocomplexes with itself and with each other, respectively, which is verified via yeast-two hybrid, in<br />
vitro pull-down and BiFC assay in Nicotiana benthamiana. Overexpression of the less abundant SIP1S leads to<br />
the increasing of the nodule number and RNA interference of SIP1 gene results in the significant impairment of<br />
nodulation and the interrupted nodule formation during nodule primordial stage. Spatial expression by<br />
SIP1p::GUS shows the centralized expression in the vascular tissue of developing nodule primordia. We propose<br />
that SIP1L and SIP1S may form polymers and be involved in nodule development process. On the other hand,<br />
we surprisedly find that suppressed SIP1 expression affects arbuscule formation, implicating the possible<br />
function of SIP1 gene in arbuscular mycorrhizal symbiosis.<br />
Zhu, H, Chen, T, Zhu, M, Fang, Q, Kang, H, Hong, Z, and Zhang, Z (2008). A novel ARID DNA-binding protein<br />
interacts with SymRK and is expressed during early nodule development in Lotus japonicus. Plant Physiol<br />
148:337-347.<br />
119<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Characterization of Rhizobia isolates using molecular methods<br />
Authors Bakhtiyor Umarov 1,2 , Zair Shakirov 1 , Khojiakbar Yadgarov 2 , Zufarjon Khojiev 2<br />
Poster Board Number 5<br />
1 Institute of Microbiology, Uzbekistan,<br />
2 Institute of Genetics and Experimental Biology of Plants, Uzbekistan<br />
The rhizobia/legume root nodule symbiosis is the result of a complex interaction between the host plant and the<br />
bacteria. Within the rhizosphere, bacteria have access to carbon and nitrogen sources originating from the plant.<br />
In response, rhizobia themselves secrete molecules (Nod factors) that signal to the plant the presence of suitable<br />
symbiotic partners. The rhizobia are then able to enter the root in the region of emerging root hairs. Finally, the<br />
bacteria are wrapped by a plant-derived membrane and enter the cytosol of plant cells. (DW Ehrhardt, et al 1992)<br />
. The bacteria are now referred to as bacteroids and are able to fix molecular nitrogen, which is released into the<br />
plant cells. We are isolate new effective strains from nodule plants Onobrycis tr., grow in the Arid zones Central<br />
Asia. For the characterize and identify rhizobial strains and to study the diversity of indigenous Rhizobium strains<br />
we are used the PCR. PCR can be performed rapidly with strains, species or genus specific primers that<br />
generate fingerprint characteristics of each strain. DNA primers corresponding to repetitive sequences that<br />
present in multiple copies of the genomes of most Gram-negative and Gram-positive bacteria can be used to<br />
fingerprint the genomes of rhizobial strains. Three families of repetitive sequences have been identified, including<br />
(REP), (ERIC) and BOX element. The REP-PCR genomic fingerprinting protocols have been successfully used<br />
in a wide variety of Eubacteria for typing strains and studying their diversity. We have used this highly<br />
discriminative and reproducible technique to study the genetic diversity of rhizobial strains.<br />
DW Ehrhardt, EM Atkinson and Long, SR. 1992: Depolarization of alfalfa root hair membrane potential by<br />
Rhizobium meliloti Nod factors Science. Vol. 256 no. 5059 pp. 998-1000 ,DOI: 10.1126/science.10744524<br />
120<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Titles The contribution of N-fixing legumes to the productivity of wheat in Mediterranean agriculture<br />
systems of Chile.<br />
Authors Carlos Ovalle 1 , Alejandro del Pozo 2 & Soledad Espinoza 1<br />
Poster Board Number 6<br />
1 <strong>IN</strong>IA-Quilamapu, Chillán, Chile.<br />
2 Facultad de Ciencias Agrarias, Universidad de Talca, Talca, Chile.<br />
In highly intensified cereal farming systems in Chile fertilization is approximately 40% of the production costs,<br />
and N represents over 60% of this cost. The aim of this work was to evaluate the recovery of N by wheat grown<br />
after Lupinus angustifolius, L. luteus, Pisum sativum or two mixtures of annual legumes. As a control we used a<br />
oat-wheat rotation without N fertilization. The study was conducted in two contrasting Mediterranean<br />
environments: interior dryland ( granitic Alfisol, pH 6.2, organic matter (OM) 1.5%; 650 mm of annual rainfall)<br />
and Andes foothill (volcanic Andisol, pH 5.5, OM 13%; 1200 mm of annual rainfall), during 2008 and 2009. The<br />
determination of the recovery of N in the cereal was performed by isotope dilution method with 15 N; after sowing<br />
the wheat crop. A microplot of 1 m 2 was enriched with the equivalent of 40 kg N ha -1 of (NH4)2SO4 10% enriched<br />
15 N atom excess. In the interior dryland, the yield of wheat ranged 2.4-2.6 ton ha -1 after grain legumes, and 2.3-<br />
2.4 ton ha -1 after annual legume mixtures. The recovery of N by wheat from the N fixed by grain legumes ranged<br />
2.5- 2.9 kg N ha -1 (representing 6- 7.5% of the total N extracted by the wheat) and 2.7 - 2.8 kg N ha -1 (7.5% of<br />
total N) in the case of annual legumes . In the Andes foothill, wheat yield ranged 5.5-6.4 ton ha -1 after grain<br />
legumes and 6.9-7.4 ton ha -1 after annual legume mixtures.The recovery of N was 1.4 kg N ha -1 from P. sativum<br />
and between 3.4 to 4.3 kg N ha -1 in the rotation with annual legumes. With the other grain legumes the N<br />
recovery by wheat was not detected.<br />
121<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Crosstalk between GmSAT1 and yeast unveils a novel family of eukaryotic ammonium<br />
transport proteins<br />
Authors Danielle Mazurkiewicz 1 , Patrick Loughlin 2 , David Chiasson 1 , Mamoru Okamoto 3 , David A.<br />
Day 4 , Stephen D. Tyerman 1 & Brent N. Kaiser 1<br />
Poster Board Number 7<br />
1 School of Agriculture, Food and Wine. The University of Adelaide,<br />
2 School of Biological Sciences. The University of Sydney,<br />
3 Australian Centre For Plant Functional Genomics. The University of Adelaide,<br />
4 Flinders University<br />
GmSAT1 is a soybean basic helix-loop-helix (bHLH) transcription factor localised to the peribacteroid membrane<br />
of nitrogen fixing nodules 1 . Overexpression of GmSAT1 restores the growth of a yeast ammonium (NH4 + )<br />
transport mutant 26972c on low (1 mM) NH4 + concentrations (Kaiser et al., 1998). GmSAT1 overexpression in<br />
yeast also increases the uptake of methylammonium (MA), a toxic NH4 + analogue. The objective of this study<br />
was to identify and characterise the NH4 + /MA transporter(s) regulated by GmSAT1 in yeast. We have evaluated<br />
the transcriptional activity of GmSAT1 in 26972c cells grown for 12 hours in media containing 1mM NH4 + , using<br />
microarray (Affymetrix Yeast Genome 2.0 Array) and quantitative RT-PCR. We have characterised one of the<br />
genes, ScAMF1, found to be upregulated by GmSAT1. ScAMF1 (Saccharomyces cerevisiae Ammonium<br />
Facilitator 1), previously uncharacterised, shows sequence similarity to predicted drug:H + antiporters of the DHA2<br />
family of major facilitator proteins. Overexpression of ScAMF1 in 26972c and a second NH4 + transport mutant<br />
yeast strain, 31019b, enhanced 14 C-MA uptake and established a related toxicity phenotype when grown at<br />
elevated MA (0.1M) concentrations. The loss of ScAMF1 activity in 26972c:Δamf1 and 31019b:Δamf1 mutants<br />
eliminated the ability of GmSAT1 to accumulate 14 C-MA and instate the sensitivity phenotype. The MA<br />
associated phenotypes were also lost when the bHLH region of GmSAT1 was mutated. Mutagenesis of the<br />
bHLH region of GmSAT1 was shown to abolish the expression of ScAMF1. Stopped-flow spectroscopy<br />
demonstrated the ability of ScAMF1 to transport NH4 + and MA into yeast spheroplasts. Parallel experiments with<br />
a soybean homolog (GmAMF1) also showed enhanced accumulation of 14 C-MA and transport of NH4 + and MA<br />
into spheroplasts. The functional similarity of ScAMF1 homologues identified in Arabidopsis and Medicago<br />
truncatula are currently being investigated.<br />
Kaiser, B.N., Finnegan, P.M., Tyerman, S.D., Whitehead, L.F., Bergersen, F.J., Day, D.A. and Udvardi, M.K.<br />
1998. Characterisation of an ammonium transport protein from the peribacteroid membrane of soybean nodules.<br />
Science 281: 1202-6.<br />
122<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Titles Co-inoculation of rhizobia with highly induced acc deaminase bacteria could alleviate the<br />
stress of legumes growing under stress conditions<br />
Authors Panlada Tittabutr, Pongdaj Piromyou, Nantakorn Boonkerd & Neung Teaumroong<br />
Poster Board Number 8<br />
School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000,<br />
Thailand<br />
Since it was previously found that silver thiosulfate (STS) could lower the level of ethylene production in<br />
mungbean and resulted in increasing plant growth under both normal and various stress conditions. It is<br />
interesting to investigate whether highly 1-amino-cyclopropane-1-carboxylate (ACC) deaminase activity<br />
producing bacteria promote the growth of mungbean under various stress conditions. ACC deaminase<br />
producing bacteria were isolated from mungbean root growing in the field, and three isolates ACC1, ACC2, and<br />
ACC3, which are highly homology to Enterobacter sp. ZJUPD4, Enterobacter sp. M.D.E.NA4-3, and<br />
Chryseobacterium sp. KR200, respectively were obtained with high ACC deaminase activity. The experiments<br />
of co-inoculation with mungbean bradyrhizobia PRC008 were tested under each condition of high temperature-,<br />
water-, salt-stress, and under normal condition. The results showed that ethylene was highly produced by plant<br />
growing under all stress conditions. The amount of ethylene production in plant directly influenced mungbean<br />
growth and nodulation efficiency under stress conditions. However, co-inoculation of PRC008 with high ACC<br />
deaminase activity producing bacteria showed the better plant growth and nodulation than those inoculated with<br />
PRC008 alone. Co-inoculation with ACC3 obviously alleviated the stress of legumes growing under water- and<br />
high temperature-stress conditions, which coincided with high inducton of ACC deaminase activity in the cell<br />
cultured under stress conditions even ACC3 produced lower ACC deaminase activity when compared with<br />
ACC1 and ACC2 under normal condition. These results revealed the role and relationship of ethylene and the<br />
ACC deaminase producing bacteria on the growth of leguminous plant under stress conditions. This<br />
information will be useful for further development of high efficient bacterial inoculant using in agriculture under<br />
global warming situation.<br />
123<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Rice growth promotion after inoculation of Sinorhizobium melilotii 1021 and its mechanism<br />
Authors Mingfeng Yang, Wenpeng Zhang, Shihua Shen and Yuxiang Jing<br />
Poster Board Number 9<br />
Key Laboratory of Plant Resource Research and Development, the Chinese Academy of<br />
Sciences, Beijing 100093, China.<br />
The studies have shown that the endophytic association of rhizobia with non-legumes, such as rice, wheat,<br />
maize, rape, lettuce, etc is of benefit to their growth with increased grains or/and biomass. Our previous study<br />
indicated that the bacteria of Sinorhizobium melilotii 1021, etc which were inoculated to rice seedlings not only<br />
entered into root interior through lateral root emergence and root hairs, but also ascended into stem base, leave<br />
shealth and leaves where they developed high population. Finally, they made rice increase its biomass and<br />
grains (Chi F. et al, 2005). Recently, we found the key point time that the rice seedlings inoculated with rhizobia<br />
grew more significantly than the controls based on rice morphology was at 5 days postinoculation(dpi). The<br />
observation under laser confocal microscope pointed out that the gfp-tagged rhizobia moved so fast that they<br />
arrived in rice leaves at the 5-8 dpi, and located in the intercellular space, and some of them even got into<br />
diachyma cells after isolation of leaf cell protoplasm. Microarray-gene chip and quantitative real-time PCR<br />
determined the gene expression of rice seedlings, showing that the related genes of DNA replication, cell division<br />
and cell wall formation, etc were up-reguated at 2 dpi, then some of photosynthetic genes, carbohydrate<br />
anabolism genes, defense and resistant genes were also up-regulated at 5, 8 dpi. These results indicate the<br />
molecular basis and the mechanism that the rhizobia could promote rice growth. The following step in future is to<br />
look for the reasons of molecular interaction between rhizobia and rice plants through metabolomics.<br />
Feng Chi, Shi-Hua Shen, Hai-Ping Cheng, Yu-Xiang Jing,* Youssef G. Yanni, and Frank B. Dazzo. Ascending<br />
Migration of Endophytic Rhizobia, from Roots to Leaves, inside Rice Plants and Assessment of Benefits to Rice<br />
Growth Physiology. AEM, 2005, 71: 7271–7278.<br />
124<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Titles Differential activation of CCAMK between root nodule and arbuscular mycorrhizal symbioses<br />
Authors Yoshikazu Shimoda 1 , Lu Han 1 , Makoto Hayashi 1 , Haruko Imaizumi-Anraku 1<br />
Poster Board Number 10<br />
1 National Institute of Agrobiological Sciences, Division of Plant Sciences, Tsukuba, Ibaraki<br />
305-8602, Japan<br />
Leguminous plants can form mutually beneficial endosymbioses with rhizobial bacteria and arbuscular<br />
mycorrhizal (AM) fungi. Recent genetic studies in model legumes have revealed that the two symbiotic systems<br />
share a common signaling pathway (CSP), which is essential for the induction of cytosolic Ca 2+ spiking in<br />
response to infection signal molecules derived from rhizobia or AM fungi. Among the components of CSP,<br />
Ca 2+ /calmodulin(CaM)-dependent protein kinase (CCaMK) is downstream of Ca 2+ spiking and thought to be an<br />
ideal decoder for microsymbionts-induced Ca 2+ signals, because of its domain structure including CaM binding<br />
domain and EF hand motifs. Recent studies have identified crucial roles of CCaMK for bacterial/fungal<br />
infections and nodule organogenesis. However, it remains unknown how CCaMK is activated differentially in<br />
response to Ca 2+ signals induced by rhizobia or AM fungi.<br />
In order to elucidate the difference of CCaMK activation mechanisms during RN and AM symbioses, we carried<br />
out a detailed complementation analysis of ccamk mutant of Lotus japonicus with various kinds of mutated<br />
CCaMKs. We also analyzed epistatic relationships among CCaMK functional domains by combining the<br />
mutations of each domain to clarify the involvement of the domains in Ca 2+ -dependent activation of CCaMK. In<br />
this presentation, we propose an activation mechanism of CCaMK in which RN and AM symbioses are<br />
distinguished by differential regulation of CCaMK by Ca 2+ signals.<br />
125<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Determination of optimal phosphorus concentrations which enhance the establishment of<br />
both mycorrhizal and rhizobial symbiosis<br />
Authors Manijeh Mohammadi-Dehcheshmeh, Sally E Smith, Steve D. Tyerman<br />
Poster Board Number 11<br />
and Brent N Kaiser<br />
School of Agriculture, Food and Wine, Waite Campus, University of Adelaide<br />
Legume growth and yield productivity is influenced through symbiotic interactions with both rhizobial bacteria and<br />
mycorrhizal fungi. Symbiosis with Rhizobium sp. enables access to atmospherically reduced N2, through<br />
nitrogen fixation, while the mycorrhizal symbiosis strongly enhances mineral uptake especially phosphorus (P)<br />
and zinc(1). P is an essential nutrient required for plant growth. The availability of P in the soil and internally<br />
within the plant has been shown to have a contrasting influence on the impact of either the rhizobial or<br />
mycorrhizal symbiosis (2-5). At high levels of available plant P, symbiotic nitrogen fixation is enhanced while high<br />
concentrations of P can hinder mycorrhizal colonization. The impact of P on either nodulation or mycorrhizal<br />
colonization has been studied widely, however little information is available on how P affectsthe interactions<br />
between the two symbiosis. In this study, we examined the effects of different concentrations of soil P on both<br />
the rhizobial and mycorrhizal symbiosis in soybean. At high P, mycorrhizal colonization (percent root length)<br />
decreased while nodule development increased. At low P the opposite response occurred. We are currently<br />
evaluating the combined symbiotic response to P in order to identify P concentration ranges that satisfy the<br />
needs of both symbiosis. This work will be discussed in the context of maximizing P efficiency under symbiotic<br />
conditions and the competing symbiotic relationships regulating P availability.<br />
1. S. E. Smith, F. A. Smith, Annual Review of Plant Biology 62, 227 (2011).<br />
2. D. W. Israel, Plant Physiology 84, 835 (1987).<br />
3. M. Chaudhary et al., Acta Physiologiae Plantarum 30, 537 (2008).<br />
4. J. A. Menge, D. Steirle, D. J. Bagyaraj, E. L. V. Johnson, R. T. Leonard, New Phytologist 80, 575<br />
(1978).<br />
5. P. G. Braunberger, M. H. Miller, R. L. Peterson, New Phytologist 119, 107 (1991).<br />
126<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Comparative analysis of genome sequences of Rhizobium galegae HAMBI 540 and<br />
HAMBI 1141<br />
Authors Janina Österman 1,3 , Lars Paulin 2 , Edward Alatalo 2 , J. Peter W. Young 3 , Kristina<br />
Lindström 1<br />
Poster Board Number 12<br />
1 Department of Food and Environmental Sciences and<br />
2 Institute of Biotechnology, University of Helsinki<br />
3 Department of Biology, University of York<br />
Rhizobium galegae (Lindström 1989) is taxonomically distinct and known for its strict host specificity both<br />
regarding nodulation and nitrogen fixation. Our aim is to gain insight into molecular mechanisms behind the<br />
specificity of nitrogen fixation. As a first step towards this goal we have performed whole genome sequencing of<br />
R. galegae symbiovar orientalis strain HAMBI 540 (effective on Galega orientalis) and R. galegae symbiovar<br />
officinalis strain HAMBI 1141 (effective on G. officinalis). The draft assembly indicates that both genomes are<br />
about 6.4 Mb in size. Preliminary comparison of the genomes has been performed, with a more specific<br />
comparison of the symbiotic gene regions of the two strains. Although the two genomes are of approximately the<br />
same size, there are important genetic differences between the strains. The most obvious difference is that the<br />
HAMBI 1141 genome harbours three replicons, while the corresponding number in HAMBI 540 is two.<br />
Furthermore, HAMBI 1141 harbours conjugal transfer systems not present in HAMBI 540. The genes present in<br />
the symbiotic regions are the same except for one gene, rpoN, the function of which will be further studied.<br />
Annotation of the symbiotic region also led us to the identification of a candidate gene for acetylation of the<br />
penultimate N-acetylglucosamine in the R. galegae Nod factor. Differences detected in the comparative analyses<br />
will be discussed.<br />
127<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Denitrification and nitrous oxide emissions by soybean bradyrhizobia<br />
Authors Elisamara C. do Nascimento 1 , Jean L.S. de Araújo 2 , Claudia P. Jantalia 2 , Segundo<br />
Urquiaga 2 , Robert M. Boddey 2 , Bruno J. R. Alves 2<br />
Poster Board Number 13<br />
1 Departamento de Fitotecnia, Universidade Federal Rural do Rio de Janeiro, Seropédica,<br />
23890-000, RJ, Brazil.<br />
2 Embrapa Agrobiologia, Seropédica, 23890-000, Rio de Janeiro, Brazil.<br />
Soil denitrifiers include several Bradyrhizobium spp. strains, but just some of them are genetically capable of<br />
using all of the N oxides from nitrate to N2O. Soybean is planted on more than 22 million ha in Brazil, and<br />
commercial inoculants for this crop are manufactured from four selected strains of B. japonicum and B. elkanii.<br />
There are still doubts concerning the impact of nodulated legumes on emissions of N2O, a powerful greenhouse<br />
gas. Firstly the ability of 12 strains of Bradyrhizobium japonicum and B. elkanii from Brazil and USA to produce<br />
N2O from nitrate in pure culture of was investigated. Secondly a study was made to quantify the N2O emissions<br />
from soybean grown in soil never planted to soybean and inoculated with the four strains used in Brazilian<br />
commercial inoculants. Subsequently nodules from these plants were transferred to a sealed vials containing 2<br />
ml of a 3 ppm solution of N-nitrate and incubated under N2 or Ar plus 1.2 ppm of N2O. The assays with plant<br />
nodules were performed to evaluate the denitrifying ability of different N oxides by the strains. A DGGE analysis<br />
was performed using specific primers for each enzyme involved in the denitrification process. Only B. japonicum<br />
strains grown in pure culture showed the ability to reduce NO3 - to N2O. In the pot experiment, plants inoculated<br />
with B. japonicum presented the highest N2O emissions, whilst soybean nodulated with B. elkanii presented low<br />
emissions, similar to those non-inoculated plants. The production of N2O from nodules of plants inoculated with<br />
B. elkanii suggests soil rhizobia also occupied the nodules. However, nodules from plants inoculated with B.<br />
japonicum strain BR86 (SEMIA 5079) showed the ability to convert all N2O into N2, which was not observed for<br />
any other treatment.<br />
128<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Proteomic profile of the soybean symbiosome membrane<br />
Authors Clarke, V.C. 1 , Loughlin, P.C. 1 , Taylor, N.L. 2 , Millar, A.H. 2 , Day, D.A. 3 and Smith, P.M.C. 1<br />
Poster Board Number 14<br />
1 School of Biological Sciences, The University of Sydney, NSW, Australia; 2 ARC Center of<br />
Excellence in Plant Energy Biology, The University of Western Australia, WA, Australia;<br />
3 Flinders University, SA, Australia.<br />
Symbiotic nitrogen fixation in legumes is characterised by the formation of a novel root organ called the nodule,<br />
within which unique organelle-like structures termed symbiosomes develop. Free living rhizobia are engulfed<br />
within plant cells and are surrounded by a membrane of plant origin termed the symbiosome membrane (SM). It<br />
is this membrane that regulates the movement of solutes from plant to bacteroid (the symbiotic form of the<br />
rhizobium) and vice versa. The SM is a unique structure containing an array of plant-derived proteins through<br />
which the plant can regulate the symbiosis. Previous attempts to characterise the protein complement of this<br />
membrane have been hindered by its hydrophobic nature and the absence of a complete genome for reference.<br />
In this study, SM was isolated from mature nitrogen-fixing soybean root nodules and analysed using shotgun<br />
proteomic techniques. The recent release of the complete soybean genome has allowed for the identification of<br />
peptide sequences. Our proteomic analysis of soybean SM has identified eighty putative SM-localised proteins,<br />
including ten previously localised to the SM such as nodulin-26, an aquaporin. We have focused on two classes<br />
of proteins identified in the proteomics for further studies: amino acid transporters and ABC family transporters.<br />
Gene expression of these candidates has been confirmed by qRT-PCR and is specific to nodule tissue.<br />
Expression profiles across nodule development have also been completed with all candidates showing<br />
increasing gene expression levels correlated with the onset of nitrogen fixation. This suggests these genes may<br />
have a role in maintaining the mature symbiosis, consistent with what would be expected of a SM transporter.<br />
amiRNA silencing vectors have been constructed to reduce expression of candidates in nodules. Further<br />
localisation and functional studies are planned for these candidates with the aim of characterising novel<br />
transporters on the soybean SM.<br />
129<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Titles Nodulation studies of cowpea and its response to phosphorus application in<br />
Botswana under glasshouse conditions<br />
Authors Kenneth K. Keakile and Tebogo Balone, Flora Pule-Meulenberg<br />
Poster Board Number 15<br />
Department of Crop Science and Production, Botswana College of Agriculture, Private Bag<br />
0027, Gaborone, Botswana<br />
Cowpea (Vigna unguiculata L. Walp) is an indigenous leguminous plant grown mostly in tropical Africa as a food<br />
crop. Its fresh young leaves and fresh pods are consumed as vegetables by many Africans whereas the seed is<br />
used in many food preparations. It is known to form symbiotic partnerships with some members of Rhizobiales as<br />
well as some β-proteobacteria ( “rhizobia”), where the legume provides the microsymbiont with carbon for its<br />
energy needs and in turn receives fixed-N for its nutrition from the rhizobia. In Botswana, nodulation status of<br />
many economically important legumes such as cowpea is not known. This study assessed if soils of Sebele<br />
harboured bacteria that nodulates cowpea. The study also assessed the response of cowpea to phosphorus<br />
application under glasshouse conditions. Two cowpea genotypes, Black eye and Tswana, were planted in a<br />
completely randomised design using pots in a glasshouse at the Botswana College of Agriculture in Sebele. In<br />
the first experiment which tested for nodulation, all plants received a basal application of P equivalent to 130 kg<br />
P/ha. In the second experiment, treatments consisted of four levels of P equivalent to 0, 65, 130 and 195 kg P/ha<br />
using single superphosphate. Both genotypes formed effective nodules when grown in the soil from Sebele.<br />
Black eye produced significantly more nodules and higher nodule dry matter compared to Tswana, which was<br />
reflected in significantly higher shoot N concentration in this genotype. The highest P fertilisation resulted in<br />
significantly higher nodule number, shoot and root dry matter as well as shoot N. It was also observed that Black<br />
eye plants that received 195 kg P/ha flowered significantly earlier than the rest. In conclusion, the Sebele soil<br />
harboured rhizobia that nodulated cowpea genotypes Black eye and Tswana. Further studies will include<br />
evaluating a wider selection of legumes including potential forage plants grown on different soil types. Levels of<br />
N-fixed will be measured, root-nodule bacteria isolated and cross-infectivity studies conducted.<br />
130<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Lotus japonicus nodulates when it sees red.<br />
Authors Akihiro Suzuki 1 , Lalith Suriyagoda 1 , Tamaki Shigeyama 1 , Akiyoshi Tominaga 1 , Masayo<br />
Sasaki 1 , Yoshimi Hiratsuka 1 , Aya Yoshinaga 1 , Susumu Arima 1 , Sakae Agarie 1 , Tatsuya<br />
Sakai 2 , Sayaka Inada 2 , Yusuke Jikumaru 2 , Yuji Kamiya 2 , Toshiki Uchiumi 3 , Mikiko Abe 3 ,<br />
Masatsugu Hashiguchi 4 , Ryo Akashi 4 , Shusei Sato 5 , Takakazu Kaneko 5 , Satoshi Tabata 5 &<br />
Ann M. Hirsch 6<br />
Poster Board Number 16<br />
1 Faculty of Agriculture, Saga University, Honjyo-machi, Saga, Saga 840-8502, Japan.<br />
2 RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan.<br />
3 Faculty of Science, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan.<br />
4 Frontier Science Research Center, University of Miyazaki, Miyazaki, Miyazaki 889-2192,<br />
Japan.<br />
5 Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan.<br />
6 Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute,<br />
University of California-Los Angeles, California, USA.<br />
Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes<br />
and rhizobia. In this study, we show that Phytochrome B (PHYB) is part of the monitoring system to detect<br />
suboptimal light conditions, which normally suppress Lotus japonicus nodule development in response to<br />
inoculation of Mesorhizobium loti. We found that the number of nodules produced by phyB mutants of L.<br />
japonicus is significantly reduced compared to that of wild-type MG20. To explore another cause other than<br />
photoassimilates, the possibility of local control was investigated by grafting experiments. The results showed<br />
that shoot genotype, and not the root genotype is responsible for root nodule formation. To explore causes by<br />
systemic regulation, we moved wild-type MG20 plants from white light to conditions differing in the ratios of low<br />
or high red (R)/far-red (FR) light. In low R/FR light, the number of root nodules on MG20 plants dramatically<br />
decreased compared to plants grown in high R/FR even though photoassimilate content was higher for plants<br />
grown under low R/FR. We found that the expression of jasmonic acid (JA)-responsive genes was decreased in<br />
both low R/FR light-grown MG20 and in white light-grown phyB mutant plants. Indeed Endogenous concentration<br />
of JA-Ile was decreased in phyB mutant. Moreover, both infection thread formation and root nodule formation<br />
were positively influenced by JA treatment of wild-type plants grown in low R/FR light and of white light-grown<br />
phyB mutants. These results indicate that root nodule formation is photomorphogenetically controlled by sensing<br />
the R/FR ratio through JA signaling.<br />
131<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Role of the Cpx two component regulatory system and a MFS transporter in the symbiosis<br />
between Sinorhizobium meliloti and leguminous plants<br />
Authors Mário R. Santos, Andreia T. Marques, Leonilde M. Moreira<br />
Poster Board Number 17<br />
IBB-Institute for Biotechnology and Bioengineering, Centro de Engenharia Biológica e<br />
Química, Instituto Superior Técnico<br />
Sinorhizobium meliloti establishes a symbiotic nitrogen fixation relationship with Medicago sativa in a process<br />
involving signalling between rhizobia and the legume plant. There are several bacterial molecular determinants<br />
of symbiosis and we recently added protein TolC to those determinants [1]. A S. meliloti tolC mutant was strongly<br />
affected in the resistance to antimicrobial agents, presented higher susceptibility to osmotic/oxidative stresses<br />
and also had affected secretion of Ca 2+ -binding proteins, of exopolysaccharides and Nod-factors. Furthermore,<br />
S. meliloti tolC mutant induced a reduced number of nodules, which were unable to fix nitrogen in plant roots.<br />
The comparison of the transcriptome of the tolC mutant to that of the wild-type strain identified 1809 genes<br />
differently expressed, including those encoding proteins involved in central metabolism, protection against<br />
stresses and transport. From these genes, the ones with the highest expression in the tolC mutant included the<br />
homologues of the sensor histidine kinase CpxA, the response regulator CpxR and the MFS (Major Facilitor<br />
Superfamily) transporter encoded by genes SMc03167 and SMc03168 [2]. To determine whether these genes<br />
are involved in the symbiotic process, insertion mutants of cpx homologues and deletion mutants of the MFS<br />
genes were constructed. Phenotypic tests performed with cpx insertion mutants showed minor differences in<br />
motility and in symbiosis when compared to the wild-type strain. Deletion of MFS encoding genes had an effect<br />
in stress resistance, pH tolerance, and a slight effect in the number of nodules. Further studies are being<br />
conducted to determine other functions for these gene products in S. meliloti biology.<br />
132<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Compatibility with rhizobia of polymer adhesives and colourants used in preinoculated<br />
legume pasture seed<br />
Authors Elizabeth Hartley, Greg Gemell & Jade Hartley<br />
Poster Board Number 18<br />
Australian Inoculants Research Group, NSW Department of Primary Industries, Ourimbah<br />
Campus, University of Newcastle<br />
In recent years there has been an increase in commercially-produced preinoculated pasture legume seed.<br />
Nowadays, preinoculation is the incorporation of ingredients beneficial to seedlings, into a sticker solution<br />
containing rhizobia. Seed is coated then dried. Preinoculated seed is often stored for weeks or months before<br />
use. Data show that survival of rhizobia on preinoculated seed varies and is generally poor after a few weeks.<br />
Sowing preinoculated seed with nil or very few rhizobia may contribute to inadequate nodulation and possible<br />
crop failure costing the farmer in lost production.<br />
Many physical and chemical factors affect the ability of rhizobia to survive in high numbers when inoculated onto<br />
seed.<br />
This presentation focuses on ingredients such as polymers, stickers, dyes, pigments and insecticides and their<br />
compatibility with rhizobial strains when used in the commercial seed coating process.<br />
Chemical and agricultural companies who manufacture and supply such materials contributed 60 ingredients for<br />
assessment.<br />
To determine the compatibility with rhizobia, and their suitability as an ingredient in the seed coating process,<br />
each was applied at recommended rates to rhizobial broth cultures of 3 commercial strains, TA1 (white clover),<br />
WSM1325 (subterranean clover) and CB1809 (soybean). Viable rhizobia were counted after 0h, 3h, 24h and 72h<br />
incubation. 29 ingredients that were compatible with rhizobia were screened to determine their effect on survival<br />
of rhizobia when incorporated into peat slurries used to inoculate polyethylene beads as seed substitutes. Viable<br />
rhizobia on beads were counted 1h, 24h and 48h after coating. Results varied. The affect of each of the<br />
ingredients to rhizobial survival depended upon the strain, and the method of exposure of the cells to the<br />
ingredients. After 24h, 6 ingredient treatments supported better survival of rhizobia on beads than the control<br />
treatment. However, after 48h, only one ingredient promoted better survival than the control.<br />
133<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Efficiency of Bradyrhizobium japonicum in different formulations and when co-inoculated with<br />
bacillus subtilis on soybean in a Kenyan soil<br />
Authors Mary Atieno 1, 2 , Laetitia Herrmann 2 , Robert Okalebo1 , Didier Lesueur 2, 3<br />
Poster Board Number 19<br />
1 Moi University, Department of Soil Science, PO Box 1125-30100, Eldoret, Kenya<br />
2 Tropical Soil Biology and Fertility Institute of the <strong>International</strong> Centre for Tropical Agriculture<br />
(TSBF-CIAT), United Nations Avenue, PO Box 30677-00100, Nairobi, Kenya<br />
3 CIRAD, UMR Eco&Sols (CIRAD-IRD-<strong>IN</strong>RA-SupAgro), Land Development Department,<br />
2003/61 Paholyothin Road, Lardyao Chatuchak, Bangkok 10900 Thailand<br />
The major setback in successfully obtaining an effective inoculant is overcoming difficulties in formulating a<br />
viable and user-friendly final product as the live nature of the active ingredient underscores the importance of<br />
formulation in maintaining the microbial cells in a competent state. Co-cultures of rhizobia and PGPR (Plant<br />
Growth Promoting Rhizobacteria) are also known to influence the efficacy of the symbiotic bacteria on plant<br />
biological nitrogen fixation. A greenhouse experiment was set to assess the formulation effect of one strain i.e.<br />
Bradyrhizobium japonicum, 532c (granules, liquid and broth) and to determine the efficiency of co-inoculation of<br />
Bacillus subtilis with two strains of Bradyrhizobium japonicum (532c and RCR 3407). The objectives were<br />
evaluated on 2 soybean (Glycine max L.) varieties: Nyala, a non-promiscuous variety and TGx1740-2F, a<br />
promiscuous test variety. A non sterile soil from Central Kenya (Chuka) classified as a Nitisol was used. Nodule<br />
occupancy was determined by PCR-RFLP. Most of the inoculants showed increased nodulation and biomass<br />
yields as compared to the un-inoculated controls with a higher response seen in the promiscuous TGx1740-2F<br />
variety as compared to the non-promiscuous variety. The liquid and granule-based inoculants had higher<br />
biomass yields suggesting an impact of formulation on the effectiveness of the inoculants. The co-inoculants also<br />
gave higher yields but showed no significant differences to the rhizobial inoculants alone. Nodule occupancy was<br />
100 % for all the rhizobial inoculants as well as the co-inoculants emphasizing the infectivity and high<br />
competitiveness of 532c and RCR 3407 strains even in the presence of indigenous strains (80-113 cell/g of soil).<br />
These inoculants, though not initially made for SSA countries, showed promising increased yields in a Kenyan<br />
soil containing significant populations of native rhizobia nodulating soybean, signifying a possibility of their<br />
adoption in increasing soil fertility and crop yields in the poor SSA soils.<br />
134<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Potential of indigenous Bradyrhizobia versus commercial inoculants to improve cowpea and<br />
green gram yields in Kenya<br />
Author Samuel Mathu 1, 2 , Pieter Pypers 1 , Laetitia Herrmann 1 , Viviene Matiru 2 Romano Mwirichia 2<br />
and Didier Lesueur 1,3<br />
Poster Board Number 20<br />
1. Tropical Soil Biology and Fertility Institute of CIAT, World Agroforestry Center,<br />
P.O. Box 30677-00100 Nairobi – Kenya.<br />
2. Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200<br />
Nairobi- Kenya.<br />
3. CIRAD, UMR Eco&Sols Land Development Department, 2003/61 Paholyothin<br />
Road, Lardyao Chatuchak, Bangkok 10900 Thailand.<br />
Limited information is available on reduced cowpea and green gram yields in Kenya. Declining soil fertility or<br />
presence of ineffective indigenous rhizobia? In regards to this, soils were collected from Western province<br />
(Bondo, Bungoma) to Eastern (Isiolo), Central (Meru) and at the Coast (Kilifi) to trap indigenous rhizobia<br />
nodulating both legumes under greenhouse condition. Highest nodule fresh weights of 4.63 and 3.32 g plant -1 for<br />
cowpea and green gram were observed in soil from Isiolo site A and Kilifi site A respectively suggesting<br />
significant populations of indigenous strains in such soils. Lowest nodule fresh weights of 2.17 and 0.72 g plant -1<br />
were observed in soil from Bungoma site B for cowpea and green gram respectively. Genetic diversity of<br />
indigenous strains nodulating both legumes was assayed using PCR-RFLP of the 16S-23S rDNA IGS and 19<br />
IGS groups were identified with I and II predominating for both legumes. A second greenhouse experiment was<br />
set up to evaluate if commercial inoculants significantly improve cowpea and green gram yields in soils with<br />
significant populations of native rhizobia. Rhizobial inoculation did not significantly (p
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Isolation and characterization of diazotrophic microsymbionts from root nodules of mucuna<br />
bracteata<br />
Authors Salwani Shaffie ,Amir Ghazali, and Nazalan Najimudin<br />
Poster Board Number 21<br />
School of Biological Sciences, Universiti Sains Malaysia<br />
An increasing number of bacteria that can nodulate and fix N2 in legumes which do not belong to the genus of<br />
Rhizobium or Bradyrhizobium was identified. This study was conducted to determine indigenous microsymbiont<br />
strains which could further promote symbiotic N2-fixation activities for Mucuna bracteata (an important<br />
leguminous cover crop) and to verify the identity of the isolates based on nitrogenase enzyme assay, nifH gene<br />
isolation and partial 16S rDNA sequence analysis. Our findings indicated that the isolated microsymbionts could<br />
nodulate and promote N2-fixation activity in M. bracteata. The isolates also contributed to enhanced plant growth<br />
in terms of leaf protein and chlorophyll content and higher plants and nodule biomass. Additionally, the nifH gene<br />
fragments were successfully amplified for all of the isolates. The 16S rDNA sequencing results suggested that<br />
bacteria which were able to form N2-fixing symbioses with root known as rhizobia is not only from the α-class of<br />
proteobacteria but also from β-class of proteobacteria (Burkholderia sp. and Achromobacter sp., 99-100% of<br />
similarity) and γ-class of proteobacteria (Stenotrophomonas sp., 99% of similarity). The findings indicate the<br />
diversity of potentially-beneficial diazotrophic microsymbionts active in this emerging legume species.<br />
136<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Lipopolysaccharide binding protein, a new requirement for legume-rhizobium symbiosis<br />
Author Ei-ichi Murakami 1 , Hitomi Takayama 1 , Ken-ichi Osuki 1 , Maki Nagata 1 , Yoshina Hori 2 ,<br />
Mayumi Shigeoka 2 , Mana Sahara 2 , Yoshikazu Shimoda 3 Sayaka Muto 4 , Yukio Nagano 4 ,<br />
Ken-ichi Kucho 1 , Mikiko Abe 1 , Shusei Sato 5 , Shiro Higashi 2 , Toshiki Uchiumi 1<br />
Poster Board Number 22<br />
1 Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan.<br />
2 Faculty of Science, Kagoshima University, Kagoshima, Japan.<br />
3 National Institute of Agrobiological Sciences, Ibaraki, Japan.<br />
4 The United Graduate School of Agricultural Sciences, Kagoshima University, Kaogoshima,<br />
Japan.<br />
5 Kazusa DNA Research Institute, Chiba, Japan<br />
Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. LPS induces the<br />
plant resistance in pathogenic combination and is also involved in the establishment of symbiosis between<br />
legumes and rhizobia. Thus the elucidation of LPS recognition system is essential for understanding the plantbacteria<br />
interaction. In mammals, LPS binding protein (LBP) has already been identified and signal transduction<br />
system via Toll-like receptor TLR4 is well investigated. However, LPS recognition system is still unknown in<br />
plants. Referring LBP of mammals, four LBP-candidates, LjLBP1, LjLBP2, LjLBP3 and LjLBP4, were identified<br />
on the genome of Lotus japonicus. N-terminal barrel of LjLBP1, LjLBP2 and LjLBP3 bound with LPS of<br />
Escherichia coli. The expression levels of LjLBP1 and LjLBP2 were low and stable, whereas LjLBP3/4 was<br />
inducible in responding to rhizobial inoculation. In the nodules on the hairy roots of LjLBP1-RNAi, nodule cells<br />
were broken and no symbiosome was observed. The shape of bacteroids of LjLBP2-RNAi was abnormal.<br />
Symbiosomes of LjLBP3/4-RNAi were larger than those of the control. These results suggest that LjLBPs are<br />
required for the symbiosis between L. japonicus and M. loti.<br />
137<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title The role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme on leguminous<br />
nodule senescence<br />
Author Sudarat Sripakdi 1 , Panlada Tittabutr 1 , Nantakorn Boonkerd 1 & Neung Teaumroong 1<br />
Poster Board Number 23<br />
1 School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima<br />
30000, Thailand.<br />
The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of<br />
Ensifer sp. (Sinorhizobium sp.) strain BL3 on leguminous nodule senescence. The acdRS genes encoding ACC<br />
deaminase were cloned from BL3 into multiple copy plasmids (pRK404A), and transferred to wild type. The BL3<br />
transconjugant containing of multiple copy number of acdRS (BL3 + ) greatly enhanced ACC deaminase activity<br />
compared to BL3 (1.634 and 0.447 μmol of �-ketobutyrate mg -1 protein h -1 , respectively). ACC deaminase<br />
activity of BL3 was also higher than mungbean nodulating strain as Bradyrhizobium sp. PRC008 (0.126 μmol of<br />
�-ketobutyrate mg -1 protein h -1 ). Moreover, BL3 + retarded nodule senescence (60.25%) greater than BL3<br />
(39.75%) after 5 weeks planting. In the meantime, the kanamycin resistance gene (Km r ) were inserted into the<br />
acdRS fragment and transferred into BL3 in order to construct BL3 mutant, which was interrupted at acdR and<br />
acdS (BL3 - ). All strains were inoculated to the host plant, Vigna radiata cultivar SUT1. Inoculation of BL3, and<br />
BL3 + were not significantly effected on plant growth and root nodule number whilst BL3 - showed significantly<br />
lower than both strains. Whereas flowering period on BL3 + was longer than wild type and mutant strains.<br />
138<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Gamma irradiation and autoclave sterilization of peat and compost as the carrier for rhizobial<br />
inoculant production<br />
Author Panlada Tittabutr 1 , Kamonluck Teamthisong 2 , Neung Teaumroong 1 &<br />
Poster Board Number 24<br />
Nantakorn Boonkerd 1<br />
1 School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000,<br />
Thailand<br />
2 The Center for Scientific and Technological Equipment, Suranaree University of<br />
Technology, Nakhon Ratchasima 30000, Thailand<br />
Although several alternative carriers have been investigated and successfully used instead of peat, the main<br />
problem found in rhizobial inoculant production is contamination by other bacteria or molds that cause reduction<br />
of rhizobial cell number. This study aimed to elucidates the efficient sterilization process of two carriers, peat<br />
and compost before using as rhizobial inoculant. Peat and compost could be efficiently sterilized by irradiation.<br />
The carrier with 10% moisture content could be sterilized by irradiation at 10 kGy, while carrier with 30%<br />
moisture content must be sterilized by irradiation at 25 kGy. Penetration of irradiation through polypropylene (PP)<br />
bag tend to be better than polyethylene (PE) bag, since lower dose of irradiation was needed for carrier<br />
sterilization. However, PE bag appear to be more durable than PP bag after gamma irradiation at high doses.<br />
Nevertheless, contaminants could be detected in irradiated carrier after storage at room temperature for two<br />
months. Thus, autoclaving with tyndallization approach was applied in order to minimize the contaminant<br />
microorganisms in the carrier. Carriers with 10% moisture were autoclaved two times in a row at 121ºC for 60<br />
min, with the waiting period during each time after autoclaving for 18 h could eliminate the fungal contaminants,<br />
while bacterial contaminants still remained about 10 2 cfu/g carrier. The number of Bradyrhizobium sp. PRC008<br />
was in the range of 10 8 -10 9 cfu/g in both irradiated and autoclaved peat after 6 months storage. However, the<br />
numbers of bradyrhizobial cell were reduced in compost sterilized by both methods after one month storage.<br />
These results indicated that carrier material had an important influence on inoculant quality, while sterilization<br />
processes using gamma irradiation and autoclaving with tyndallization approach could be used for efficient<br />
rhizobial inoculant production with peat based carrier.<br />
139<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Effect of nitrogen, phosphorus, magnesium and calcium nutrition on plant growth and levels<br />
of macronutrients in the honeybush tea plant, Cyclopia longifolia (Vogel l.)<br />
Authors Buhlebelive Mndzebele 1 and FD Dakora 2<br />
Poster Board Number 25<br />
1 Department of Crop Sciences, Tshwane University of Technology,<br />
2 Department of Chemistry, Faculty of Science, Tshwane University of Technology<br />
Cyclopia longifolia (Leguminosae) is endemic to the fynbos of the Cape Floristic Region of South Africa.<br />
Together with eight other Cyclopia species, C. longifolia is used for making Honeybush tea, a herbal beverage<br />
that is contributing to the South African economy. However, the effect of nutrient supply on plant growth, and the<br />
concentration, and amounts of major minerals are not well understood in this legume. Because the shoots are<br />
harvested annually as tea, establishing nutrient requirements for this species is necessary to minimize nutrient<br />
mining. The aim of this study was to assess plant growth, as well as the concentration and amounts of major<br />
elements in C. longifolia supplied with N, Mg, Ca and P as ammonium nitrate, magnesium chloride hexahydrate<br />
and calcium chloride dehydrate dipotassium and hypophosphate at four levels (i.e. 0, 5, 25 and 50 mM). About<br />
100 mL of each nutrient concentration was applied per plant at three months interval for 9 months. Plants were<br />
harvested at 300 days after treatment in the field trial. Shoots were oven-dried (60 º C), weighed, and ground to<br />
fine powder (0.85 mm) for tissue analysis using inductively coupled plasma-mass spectrometry. The data<br />
showed that supplying of N, Mg, Ca and P to C. longifolia increased biomass and the concentration, and<br />
amounts of major nutrients in plant tissue. The provision of N to C. longifolia induced uptake and accumulation of<br />
N, P, Mg, Ca and Na in the field. Phosphorus supply also led to higher tissue concentrations of N, P, Ca, Mg and<br />
Na just as the addition of Mg increased the concentration of Mg, P, Ca and Na in C. longifolia. Applying Ca to C.<br />
longifolia plants also caused the accumulation of Ca, P, Mg and Na in shoots, but decreased N concentration<br />
and content in field experiments. These data suggest that a moderate supply of N, P, Mg and Ca to C. longifolia<br />
can stimulate plant growth and accumulation, and thus increase tea yields in farmers‟ fields.<br />
140<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Root-nodule bacteria isolated from cowpea and bambara groundnut enhance mineral<br />
nutrition in their homologous hosts<br />
Author Irvin Makhubedu 1 , Keletso Mohale 1 , Flora Pule-Meulenberg 2 and Felix Dakora* 3<br />
Poster Board Number 26<br />
1 Department of Crop Science, Tshwane University of Technology<br />
2 Department of Biotechnology, Tshwane University of Technology,<br />
3 Department of Chemistry, Tshwane University of Technology<br />
Little is currently known about the role of symbiotic root-nodule bacteria in accumulating mineral nutrients in<br />
legumes. This study screened cowpea and Bambara groundnut bacterial isolates for their ability to induce<br />
nutrient uptake under sterile conditions in Leonard jars. Seedlings of cowpea (V. unguiculata L. Walp. cv.<br />
TVu11424), and Bambara groundnut (Vigna subterranea L. Verdc.) were raised in sterile Leonard jars containing<br />
¼ strength Hoagland nutrient solution, and inoculated (in four replicates) with broth cultures of their respective<br />
bacterial microsymbionts. NO3-fed plants (0.5 mM) were included as control. All plants were harvested at 30<br />
days after planting, separated into shoots and roots, and oven-dried (60 o C). The shoots were weighed, ground<br />
into fine powder (0.85 mm), and measured for mineral nutrients using inductively coupled plasma massspectrometry.<br />
The cowpea data revealed marked differences in bacterial strain effect on shoot and whole-plant<br />
biomass. All nodulated cowpea plants showed much higher concentrations of trace elements and macronutrients<br />
in shoots compared to 0.5 mM NO3-fed plants. Where strains TUT13d1vu and TUT26a1vu nodulated cowpea,<br />
they consistently showed increased concentrations of P, K, Ca, Mg, S, Fe, Cu, Zn, Mn and B in shoots when<br />
compared to other strains. However, strains TUT53b2vu and TUT33b4vu (which were the highest fixers)<br />
accumulated much greater amounts of P, K, Ca, Mg, S, Fe, Cu, Zn, Mn and B in cowpea shoots than any of the<br />
other strains. Re-testing strains TUT13d1vu, TUT26a1vu, TUT53b2vu and TUT33b4vu and 12 other N2-fixing<br />
cowpea isolates again revealed similar results for strains TUT26a1vu and TUT13d1vu. Strain TUT53b2vu and<br />
TUT33b4vu together with three other strains (which were the highest fixers) accumulated greater amounts of the<br />
major and minor elements in shoots of cowpea. Nodulating Bambara groundnut with its bacterial isolates also<br />
revealed marked strain differences in inducing mineral accumulation in shoots.<br />
141<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Evaluation of elite commercial soybean varieties for N2 fixation and grain yield in South<br />
Africa<br />
Author Nyamande Mapope 1 and Felix D Dakora 2<br />
Poster Board Number 27<br />
1 Department of Crop Science, Tshwane University of Technology<br />
2 Department Chemistry, Faculty of Science, Tshwane University of Technology<br />
South Africa is currently a net importer of soybean which is much needed for poultry production and the livestock<br />
industry. There is therefore an urgency to increase local production of soybean through breeding of improved<br />
varieties, their evaluation for adaptability, and selection for high grain yield and symbiotic performance. This<br />
study assessed plant growth, N2 fixation, and grain yield in thirteen commercially-bred, elite soybean genotypes<br />
under field conditions in the three soybean-producing provinces of South Africa. The varieties used originated<br />
from Pioneer, Pannar and Linkseed in South Africa. The experiments were laid out in a randomized block design<br />
with four replications. At pod filling (R6) stage, 10 soybean plants were randomly sampled from each plot, and<br />
oven-dried (60oC) separately, weighed and ground into fine powder (0.85mm) for 15N isotope analysis. The data<br />
obtained revealed marked differences in dry matter yield, N concentration, N content, δ15N, %Ndfa and actual<br />
amounts of N fixed among the thirteen soybean genotypes at each of the three sites. Dry matter yield ranged<br />
from 26 g.plant-1 for PAN1454 to 50 g.plant-1 for PAN737 at Balfour in Mpumalanga, and 33 to 73 g.plant-1 at<br />
Herman in the North West Province. The N concentrations of shoots also differed between and among<br />
genotypes at each site, and ranged from 2.2 % for 95Y20 to 3.5 % for PAN1666 at Balfour in Mpumalanga. The<br />
δ15N values ranged from -0.73 ‰ for PAN1454 to 2.33 ‰ for PAN1666 at Balfour in Mpumalanga. Cross site<br />
analysis also revealed significant interaction between site and varieties. PAN1666 showed the lowest %Ndfa<br />
(64%) and 95Y40 the highest %Ndfa (85%) across all sites. The actual amounts of N-fixed ranged from 114<br />
kg.ha-1 for 95Y20 to 215 kg.ha-1 for PAN1666 across the sites. Soil N uptake was generally low across the<br />
sites, indicating greater dependence of these elite varieties on N2 fixed for their N nutrition. PAN1666 for<br />
accumulated greater dry matter and amount of N-fixed across all sites and was therefore the best variety for use<br />
by farmers.<br />
142<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Ecophysiological studies and biodiversity of root-nodule bacteria nodulating Psoralea<br />
species in the cape fynbos of South Africa<br />
Authors Sheku A. Kanu 1 & Felix D. Dakora 2<br />
Poster Board Number 28<br />
1 Crop Science and 2 Chemistry Department, Tshwane University of Technology<br />
The tribe Psoraleae has 51 species, which are endemic to the Cape fynbos. However, little is known about the N<br />
contribution and microsymbionts associated with the nodulation of members of the tribe Psoraleae. In this study<br />
eight Psoralea species (namely, P. pinnata, P. repens, P. aphylla, P. asarina, P. monophylla, P. aculeata, P.<br />
restioides and P. laxa) growing in the fynbos were assessed for their symbiotic N nutrition and biodiversity of<br />
bacteria nodulating this tribe. Isotopic analysis of young branches with leaves showed that Psoralea species<br />
were highly dependent on N2 fixation for their N nutrition. The � 15 N and %Ndfa values of plant parts differed<br />
between species, and across the study sites. For example, at Betty‟s Bay, four Psoralea species (namely P.<br />
aculeata, P. aphylla, P. laxa and P. pinnata) showed strong differences in the � 15 N values of young branches<br />
with P. pinnata exhibiting the lowest stem � 15 N value of -2.00 ‰ and the highest %Ndfa value of 84%.<br />
Excavation of plants showed that all eight Psoralea species had round determinate root nodules. Surface<br />
sterilization, followed by streaking of nodule homogenates onto yeast-mannitol agar medium revealed bacterial<br />
isolates that differed in their growth rate, colony appearance, shape and texture. When 72 single-colony isolates<br />
from eight members of the tribe Psoraleae were tested for their ability nodulate P. pinnata, only 5 isolates<br />
(TUT54pp, TUT55pp, TUT57pp, TUT58pp and TUT60pp) effectively nodulated their original host together with 3<br />
other isolates from P. asarina (TUT71pas), P. restioides (TUT23prt) and P. aculeata (TUT17pac). Although 7<br />
isolates formed root nodules on siratro, only 5 (TUT1pm, TUT23prt, TUT32pap, TUT43pap and TUT45pap) were<br />
effective. The results from 16S rDNA sequencing revealed considerable microsymbiont diversity associated with<br />
the tribe Psoraleae with strains belonging to both α and β-proteobacteria. Taken together, these results suggest<br />
that Psoralea species have the potential to contribute to the N economy of the nutrient-poor soils of the Cape<br />
fynbos when associated with both .α and β-proteobacteria.<br />
143<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title <strong>Nitrogen</strong> fixation promotes accumulation of dietarily-important mineral nutrients in edible<br />
leaves of cowpea (Vigna unguiculata l. Walp.)<br />
Authors Alphonsus K. Belane 1 and Felix D. Dakora 2*<br />
Poster Board Number 29<br />
1 Department of Crop Science, and 2 Department of Chemistry, Tshwane University of<br />
Technology<br />
Cowpea leaves are a major source of dietary mineral nutrients for many households in rural Africa. Little is<br />
however known of the relationship between cowpea symbiosis and mineral accumulation in its edible leaves.<br />
This study evaluated 30 field-grown cowpea genotypes at Wa and Manga in Ghana, and Taung in South Africa,<br />
for N2 fixation and mineral density using the 15 N natural abundance technique and inductively coupled plasma<br />
mass spectrometry respectively. The mean levels of leaf minerals presented here are for the highest and lowest<br />
N2-fixing cowpea material taken from a study of 30 genotypes. The results showed that, at Wa in Ghana, IT90K-<br />
76, Bensogla and Glenda produced the largest biomass per hectare, fixed the highest amount of symbiotic N,<br />
and showed much higher concentrations and amounts of trace elements and macronutrients in edible cowpea<br />
leaves. In contrast, genotype ITH98-46, which recorded the least biomass, fixed the lowest amount of symbiotic<br />
N, and exhibited the lowest concentrations and amounts of mineral nutrients in its leaves. The data for Manga in<br />
Ghana and Taung in South Africa showed a similar pattern; the highest N2-fixing genotypes indicated better plant<br />
growth and greater mineral accumulation compared to their counterparts with little biomass and low N2 fixation.<br />
This observation was confirmed by correlation analysis which revealed a significantly positive relationship<br />
between mineral nutrients and symbiotic parameters such as δ 15 N, %Ndfa and amount of N-fixed. Thus, in<br />
addition to N contribution in cropping systems, the cowpea symbiosis also enhances mineral density in edible<br />
leaves for improved human nutrition and health.<br />
144<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Diversity and phylogeny of Bradyrhizobium spp. Isolated from shrub and tree legumes in<br />
Ethiopia<br />
Authors Aregu Amsalu Aserse 1,2 , Leena.A.Räsänen 1 ,Fassil Assefa 2 , Asfaw Hailemariam 3 and<br />
Kristina Lindström 1<br />
Poster Board Number 30<br />
1 University of Helsinki, Department of Food and Environmental Sciences,<br />
2 Addis Ababa University, Cellular, Microbial and Molecular Biology Program Unit<br />
3 National Soil Testing Centre,<br />
In Eastern Africa including Ethiopia, crop production has been affected by recurrent drought, deforestation and<br />
losses of soil fertility. Soil fertility is the major constraint and nitrogen is considered as one of the most limiting<br />
nutrients in the region. <strong>Nitrogen</strong>-fixing shrubs and tree legumes, such as Crotalaria, Erythrina and Indigofera<br />
species have been used in agroforestry systems (home-gardens, fallows and land reclamation) to improve the<br />
availability of nitrogen in the soil. Soybean is an exotic crop legume in Ethiopia and its symbionts capable of<br />
forming nitrogen-fixing nodules do not exist in the soil. Inoculation of soybean has not always been successful.<br />
Therefore, effective rhizobial strains that are adapted to their local environments are required. Interestingly,<br />
indigenous rhizobial strains, which nodulate leguminous shrubs and trees have been found to infect soybean as<br />
well. However, knowledge on the symbionts of different leguminous plants in Ethiopia is scarce. Therefore, the<br />
aim of this study was to explore the diversity and phylogeny of indigenous Bradyrhizobium strains isolated from<br />
Crotalaria incana, Erythrina brucei, Indigofera arrecta and Gycine max. Fifty-five strains were isolated from<br />
nodules collected at 29 sites located mainly in southwestern Ethiopia. The diversity of the bacterial collection was<br />
studied using amplified fragment length polymorphism (AFLP) fingerprinting. The strains were preliminary<br />
identified by comparing the partial sequences of the 16S rRNA gene against a nucleotide database. For further<br />
phylogenetic studies, the housekeeping genes recA, rpoB and glnII were sequenced. Phylogenetic trees of these<br />
genes as well as phylogenies of the symbiotic genes nodA and nifH will be presented together with strains<br />
symbiotic capacity and plant growth promoting activities.<br />
145<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Phylogenetic position of Rhizobium galegae<br />
Authors Seyed Abdollah Mousavi 1 , Janina Österman 1 , Lars Paulin 2 & Kristina Lindström 1<br />
Poster Board Number 31<br />
1 Department of Food and Environmental Sciences<br />
2 Institute of Biotechnology<br />
Rhizobia are defined as bacteria that can enter nitrogen-fixing symbioses by inducing nodules in legumes<br />
(Lindström & Mousavi 2010), whilst agrobacteria induce tumours in plants. Rhizobium galegae is fixing nitrogen<br />
in Galega plants, and is one of the species of the family Rhizobiaceae. The phylogenetic placement of R.<br />
galegae is not stable in phylogenetic trees of rhizobia and it does not fit with most other rhizobia in some gene<br />
trees. R. galegae was e.g. placed in the Rhizobium rhizogenes - Rhizobium cluster in dnaK nucleotide sequence<br />
trees; whereas it was placed with Agrobacterium species in the 16S rRNA tree (Eradly at al 2005). Construction<br />
ofa concatenated tree of eight housekeeping genes, recA, dnaK, thrC, gltA, rpoB, atpD, pnp and gyrB, for twenty<br />
whole-genome sequenced rhizobia and agrobacteria showed that R. galegae could be placed in a cluster with A.<br />
vitis and A. tumefaciens, whereas Rhizobium rhizogenes K84 (formerly A. radiobacter K84) was situated in the<br />
Rhizbium clade, wihch included R. etli and R. leguminosarum. To get a robust estimate of the phylogenetic<br />
placement of R. galegae, four house-keeping genes (recA, thrC, atpD and rpoB), and glnA and glnII genes of<br />
forty-four strains of R. galegae and more than forty strains of related rhizobia and agrobacteria were sequenced.<br />
Results of Multilocus Sequence Analysis of rhizobia and agrobacteria will be presented.<br />
Eardly BD, Nour SM, van Berkum P & Selander RK (2005) Rhizobial 16S rRNA and dnaK genes: Mosaicism and<br />
the Uncertain Phylogenetic Placement of Rhizobium galegae, Appl Environ Microbiol 71: 1328-1335.<br />
Lindström K & Mousavi SA (2010) Rhizobium and Other N-fixing Symbioses. In: Encyclopedia of Life Sciences<br />
(ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0021157.<br />
146<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Study on soybean nodule symbiosome membrane localised transporters<br />
Authors Chen C., Loughlin P., Day D., Smith P.<br />
Poster Board Number 32<br />
In nodules, the symbiosome membrane (SM) shares properties of both the plasma membrane and vacuolar<br />
membrane, however the unique nature of the SM suggests specific mechanism(s) for targeting proteins is<br />
essential. Recent work has indentified the first 24 amino acids from the N-terminus of a Medicago symbiosome<br />
protein as an address label (signal peptide) for its targeting (Hohjnec et al., 2009), however this signal peptide is<br />
not present on many known SM proteins. We are focusing on clarifying the targeting mechanism of soybean<br />
nodule SM-localised GmZIP1 (zinc transporter) and GmYSL1 (iron transporter). In silico analysis predicts the first<br />
28 amino acids of GmZIP1 may be a signal peptide. Full length GmZIP1 with GFP fused internally, immediately<br />
after the last predicted transmembrane domain is targeted to the SM. Deletion of the predicted signal peptide<br />
appears to disrupt the expression and/or localization of the fusion protein. GmYSL1 is not predicted to have an<br />
N-terminal signal peptide and N-terminal fusion of GFP does not disrupt its SM localisation. We are currently<br />
examining the localisation of a C-terminally truncated GmYSL1 construct to determine whether this is important<br />
for SM targeting. Furthermore we have characterised 4 novel soybean promoters that can drive high and specific<br />
expression in soybean nodule infected cells. Role of other soybean ZIP homologues are being characterised<br />
through expression, function and localisation studies to elucidate the zinc transporter family.<br />
147<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Development of an inoculant for Acacia acuminata to improve growth of Santalum spicatum<br />
Authors Elizabeth Watkin 1 , Len Norris 2, 3 , Liz Barbour 2,4<br />
Poster Board Number 33<br />
1 School of Biomedical Sciences, Curtin University<br />
2 Forest Products Commission<br />
3 Department of Agriculture & Food WA<br />
4 Faculty of Natural and Agricultural Sciences, The University of Western Australia<br />
Acacia acuminata is the most widely used host species within commercial plantations of the hemi-parasitic<br />
species Santalum spicatum (West Australian sandalwood). Some 10 thousand hectares of WA sandalwood<br />
plantations have been established in the 400 to 600 mm rainfall zone of the wheat-belt to supplement future<br />
shortfalls in West Australian sandalwood supply resulting from native harvest restrictions and increase in world<br />
demand. Host selection trials in WA demonstrated that leguminous N-fixing species are superior compared to<br />
non N-fixing species, with A. acuminata being a superior choice across a range of environments. Improving Nfixation<br />
of A. acuminata was identified as a potential factor to increase sandalwood productivity.<br />
This study aimed to develop a high-performing N-fixing inoculant for application to A. acuminata seedlings in<br />
nurseries or directly onto seeds. Seventeen root nodule bacteria isolates previously identified as being effective<br />
on A. acuminata were used to inoculate five different A. acuminata provenances to test their effectiveness in a<br />
containerised nursery environment. Isolates were ranked on their ability to increase seedling growth over an uninoculated<br />
control. Based on the results of this trial, five inoculum treatments were prepared with combinations of<br />
the most effective isolates. All five treatments produced significantly larger seedlings than the control. Higher<br />
total nodule mass had a stronger relationship with seedling growth, than nodule size alone. All isolates were<br />
demonstrated to be Bradyrhizobium spp. based on 16 S rRNA gene sequencing.<br />
These isolates are a valuable resource to further investigate the impact of efficient N-fixation in A. acuminata on<br />
the vigour and heartwood production in WA sandalwood. Greater N-fixation efficiency through a seed or nurseryapplied<br />
inoculant may enable the host to survive greater parasitic loading. Thus superior inoculant treatment<br />
could improve the supply of nutrients for increased sandalwood growth or reduce host mortality within a<br />
plantation and subsequent sandalwood death.<br />
148<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Endophytic colonization of sweet potato by a diazotrophic bradyrhizobia<br />
Authors Yoshinari Ohwaki 1 and Junko Terakado-Tonooka 1,2<br />
Poster Board Number 34<br />
1 NARO Agricultural Research Centre, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan<br />
2 JSPS Research Fellow, 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan<br />
Biological nitrogen fixation has been suggested as a potential source of nitrogen to sweet potato. In the present<br />
study, endophytic diazotrophs were isolated from surface-sterilized stems and tubers of field-grown sweet potato<br />
and characterized. 16S rRNA gene sequence analysis revealed that the isolates belong to the genera of<br />
Bradyrhizobium, Pseudomonas and Paenibacillus. <strong>Nitrogen</strong>-fixing capacity of the isolates was confirmed by<br />
sequence analysis of the PCR-amplified fragment of nifH gene and acetylene reduction activity in the semi-solid<br />
Rennie medium. The phylogenetic tree based on nifH gene sequences was consistent with 16s rRNA gene<br />
phylogeny, and all the isolates exhibited nitrogenase activity. Re-colonization ability of Bradyrhizobium as<br />
endophyte was assessed in the laboratory conditions. Further, the influence of nitrogen on the endophytic<br />
colonization of the isolate was examined. Internal population of the Bradyrhizobium in surface-sterilized leaves,<br />
stems, petioles and roots of sweet potato was in the range of 10 3 to 10 5 CFU per g fresh weight by 24 days after<br />
inoculation to the roots. In contrast, none of the uninoculated control plants showed any infection of the bacteria.<br />
The amount of nitrogen applied to the media had only a small effect on the internal population of the endophytes<br />
in sweet potato. Colonization in the stems after inoculation of the Bradyrhizobium to the roots as assessed by<br />
PCR-amplification of the nifH gene fragments was also observed in sunflower, rice, soybean and maize.<br />
149<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Functional Analysis of STM Mutants Concerning Amino Acid Metabolism of Mesorhizobium<br />
loti<br />
Authors Shigeyuki Tajima 1 , Mika Nomura 1 , Nanthipak Thapanapongworakul 2 , Ayao Enoki 1 and<br />
Hiroyuki Matsuura 1<br />
Poster Board Number 35<br />
1 Dept of Applied Life Sciences, Kagawa Universit<br />
2 Dept of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University<br />
The soil bacterium Mesorhizobium loti is able to induce the formation of nitrogen-fixing nodules on the root of a<br />
determinate-type legume plant, Lotus japonicus. The research on various metabolites during symbiosis has been<br />
elucidated that several bacteroid metabolic pathways are essential for maintaining efficient symbiotic interaction<br />
and for enhancing the in vivo nitrogenase activity of the nodules.<br />
To elucidate the molecular mechanism of such metabolic bacteroid differentiation in determinate nodules, protein<br />
profiles and functions of Mesorhizobium loti for Lotus japonicus were compared between cultured bacteria and<br />
nodule bacteroids.<br />
The transposon insertion mutant strains of M. loti were generated using the signature-tagged mutagenesis (STM)<br />
technique. To determine the functions of the up-regulated proteins in the bacteroids, 130 STM mutants were<br />
inoculated with Lotus plants.<br />
We focused on the four STM mutants (STM5, 30, 42, 130) which were related to the amino acid metabolism. M.<br />
loti mutant (STM5) that was inserted a transposon in the PHGDH gene, mll3875, showed an absolute<br />
dependence on serine or glycine in the minimal medium for the growth. When L. japonicus plant was infected<br />
with STM5, the root formed nodules with comparable number with that of wild type M. loti. However, the nodules<br />
showed very low acetylene reduction activity and significant starch granule accumulation was observed in the<br />
uninfected cells. In addition, STM42 that was inserted in the amino acid transporter showed the 70 % acetylene<br />
reduction activity of wild type nodule. This amino acid transporter showed high homology to aapJ in R.<br />
leguminosarum. However, STM42 bacteroid showed lower concentration in some amino acids, especially Ala,<br />
Val, Leu, Lys, Arg, and Orn, but the concentrations of Glu and Asp did not change. This data suggested that this<br />
amino acid transporter was different from aap/bra amino acid transporter in R. leguminosarum. Other amino acid<br />
synthetic proteins, STM30 and STM130, were also discussed.<br />
150<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Establishment of retrotransposon-mutagenized population of the model legume Lotus<br />
japonicus and high throughput, deep sequencing-based insertion site identification.<br />
Authors Dorian Fabian Urbański, Anna Małolepszy, Stig Uggerhøj Andersen, Jens Stougaard<br />
Poster Board Number 36<br />
Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology,<br />
Aarhus University, 8000 Aarhus C, Denmark<br />
Targeted gene knockout by homologues recombination is not yet a standard technique for reverse genetics in<br />
plants. As an alternative we have taken advantage of inducible de-repression of Lotus retrotransposon 1<br />
(LORE1), a member of small family belonging to the Ty3-Gypsy class of LTR retro-elements. LORE1 has a<br />
unique feature of being transcriptionally active in the male gametophyte 4, 5 , producing a small number of new<br />
insertions at random sites only during transition to a new generation. We are currently using a founder line of<br />
Lotus japonicus, with active LORE1 to generate a large, mutagenized population. For robust identification of<br />
novel insertion sites we are using a 2D pooling strategy and taking advantage of the known LORE1 LTR<br />
sequence to simultaneously amplify LORE1 flanking sequence tags (FSTs) in dozens of pooled plants. Illumina<br />
deep sequencing technology, an extremely high degree of multiplexing and custom-designed bioinformatics‟<br />
pipeline led us to establishing high throughput detection method for robust identification of those insertions.<br />
Study on a test set of 3744 plants, enabled identification of app. 8000 novel insertions with an average of 2,4 per<br />
plant. Novel LORE1 copies do not show tendency to cluster and are evenly distributed along Lotus<br />
chromosomes. Moreover, genes were preferentially targeted and insertions in exons were 5.7 times more<br />
frequent than in intergenic regions (P
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Rhizobium delivery systems for grain legumes in southern Australia<br />
Author David Pearce 1 , Bernadette Carmody 1 , Matthew Denton 2 , Mark Peoples 3 , Lori Phillips 4<br />
Poster Board Number 37<br />
1 Department of Primary Industries<br />
2 University of Adelaide<br />
3 CSIRO Plant Industry, Canberra<br />
4 Department of Primary Industries<br />
Grain legumes are an integral component in Australian cropping systems. Due to low soil Rhizobium populations,<br />
inoculation is often required to provide sufficient numbers of suitable rhizobia on or near the germinating seed.<br />
Traditionally, the main inoculation delivery system has been peat slurry application, where the seed is coated<br />
with the appropriate rhizobia just prior to sowing. Although very effective, this method requires strict adherence to<br />
application recommendations and may be too troublesome for growers during sowing. New inoculation delivery<br />
technologies are much easier to use and have the potential to increase inoculation flexibility at sowing. However,<br />
there is still a need for field evaluation to ensure that optimal nodulation, crop performance, and nitrogen (N)<br />
fixation occurs. In this study, we evaluated the performance of different delivery systems in Southern New South<br />
Wales. Faba bean cv. Farah and Lupin cv. Jindalee were sown using three delivery systems (peat slurry on<br />
seed, peat granules, peat slurry injection) and non-inoculated controls. Performance was measured with respect<br />
to nodulation, herbage biomass, grain yield, N content, and total N fixed. Inoculation with any method was<br />
significantly better than no inoculation. The peat slurry on seed method also generally outperformed the other<br />
methods. For example, Faba inoculated with this method yielded 3.69 t/ha of grain (4.37% N) and 11.61 t/ha of<br />
herbage (2.79% N). In comparison, Faba inoculated using peat slurry injection yielded 2.70 t/ha of grain (4.03%<br />
N) and 8.40 t/ha of herbage (2.48% N). Greater crop biomass with higher N content resulted in increased N fixed<br />
within the cropping system as a whole. Peat-seed inoculation systems fixed 316 kgN/ha, while peat-injection<br />
systems fixed 188 kgN/ha. Our results highlight not only the value of inoculation at sowing, but also the<br />
importance of choosing the appropriate inoculation delivery system for local conditions.<br />
152<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title BNF efficiency of rhizobia isolates from Phaseolus vulgaris in subsistence agrosystems in<br />
Dominican Republic.<br />
Authors César A. Díaz-Alcántara 1 , Beatriz Urbano 2 , Daniel Mulas 3 , Encarna Velázquez 4 , Fernando<br />
González-Andrés 3 .<br />
Poster Board Number 38<br />
1 Facultad de CC. Agronómicas y Veterinarias. Univ. Autónoma de Santo Domingo.<br />
Dominican Republic.<br />
2 Departamento de Ingeniería Agrícola y Forestal. Universidad de Valladolid. Spain.<br />
3 Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad. Universidad de León.<br />
Spain.<br />
4 Departamento de Microbiología y Genética. Universidad de Salamanca. Spain.<br />
Biofertilisation of common bean (Phaseolus vulgaris L.) using selected native rhizobial strains can improve the crop yield in<br />
subsistence agrosystems of Dominican Republic. With this objective 5 rhizobial strains were preselected in the basis of N<br />
fixation efficiency in axenic conditions, from an initial collection consisting of 23 strains isolated from root nodules of<br />
common bean (Phaseolus vulgaris L.) growing in subsitence agrosystems in 3 provinces of Dominican Republic (Elías Piña,<br />
San José de Ocoa and La Vega). On the basis of the 16S rRNA and housekeeping genes (recA and atpD) sequence<br />
analysis, the 5 strains belonged to the Rhizobium genus, although the identity with the previously described species is too<br />
low to precisely identify them, so they could be new species, which needs further research (Díaz-Alcántara et al., 2008). In<br />
order to assess the potential benefit of the inoculation with the preselected strains for the farmers, a microcosm experiment<br />
was carried out in 3 different soils form Elías Piña, San José de Ocoa and La Vega respectively, with the purpose of<br />
compare the effect of inoculation vs. the conventional N fertilisation. In spite of the limiting conditions of microcosm for<br />
nodulation and BNF because of the high temperatures inside the pots, 2 out of the 5 strains produced significantly higher<br />
aerial biomass than the non fertilised control. The interaction between soil and strain was not significant for p
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Protection of plant health by Micromonospora isolated from alfalfa root nodules.<br />
Authors Pilar Martínez-Hidalgo 1,3 , Pablo García-Benavides 2 ,José Maria Díaz-<br />
Mínguez 1 , Ernesto Pérez Benito 1 , Martha E. Trujillo 1, and Eustoquio<br />
Martínez-Molina 1 .<br />
Poster Board Number 39<br />
1 Departamento de Microbiología y Genética, Universidad de Salamanca<br />
2 Departmento de Construcción y Agronomía, Universidad de Salamanca<br />
3 GIR "Interacciones mutualistas planta microorganismo", Unidad<br />
Asociada al IRNASA. CSIC,<br />
The studies of new interactions that allow us to design efficient biofertilizers, to reduce the use of pesticides and<br />
chemical fertilizers are very important nowadays. The endophytic actinobacteria have a very high<br />
biotechnological potential in agriculture and environment. The activity of endophytic actinobacteria can affect (A)<br />
the development of plants in a direct way (B) their nutrition, making it easier to get essential elements like<br />
nitrogen, iron or phosphore (C) the health of the plants by means of direct mechanisms, like antibiotic or<br />
siderophore synthesis or inducing plant defence responses and (D) improving adverse environmental factors<br />
tolerance.<br />
In this work we study the PGPR effect of actinobacteria from Micromonospora genus that were isolated from<br />
alfalfa nodules. Strains that promote plant growth, improve nutrient uptake and nodulation were selected. The<br />
selected strains were used, in this work, for studies focused on the protection of plants from pathogens.<br />
Protection of plant health by means of direct mechanisms,<br />
In vitro studies of growth inhibition of plant pathogens: The following plant pathogens were used for this study:<br />
Fusarium circinatum, Sclerotinia sclerotionum, Botrytis cinerea, Rhizoctonia solani, Clavivacter miichiganensis,<br />
Erwinia amylovora, Pectobacterium chrisanthemi, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv.<br />
syringae, Ralstonia solanacearum, Xanthomonas vesicatoria<br />
Micromonospora strains were tested for siderophore production on CAS medium.<br />
Protection of plant health by induction of plant defence responses: These studies have been performed on tomato<br />
plants using two Micromonospora strains (ALFb5 and ALFpr18c) and two plant pathogens: Botrytis cinerea and<br />
Fusarium oxysporum<br />
The results obtained point out that the selected strains of Micromonospora may be used to protect plants from<br />
pathogens. (i) Some of the tested strains inhibit fungal growth in vitro. (ii) No effect was detected against the<br />
tested bacteria. (iii) Many of the strains tested produce siderophores. (iv) Micromonospora induces plant defence<br />
response.<br />
154<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Titles Identification by MALDI-TOF MS spectrometry of rhizobia isolated from nodules of different<br />
legumes<br />
Authors Pilar Martínez-Hidalgo 1 , Laura Ferreira 2 , Martha-Helena Ramírez-Bahena 3 , Lina P. Rivera,<br />
Fernando Sánchez-Juanes 2 , Paula García-Fraile 1 , Raúl Rivas 1 , Pedro F. Mateos 1 , José<br />
Manuel González-Buitrago 2,4 , Alvaro Peix 3 , Eustoquio Martínez-Molina 1 , , Encarna Velázquez 1<br />
Poster Board Number 40<br />
1 Departamento de Microbiología y Genética, Universidad de Salamanca<br />
2 Unidad de Investigación. Hospital Universitario de Salamanca<br />
3 IRNASA-CSIC.<br />
4 Departamento de Bioquímica y Biología Molecular. Universidad de Salamanca<br />
The identification of members from family Rhizobiaceae currently arranged into the genera Rhizobium, Ensifer<br />
and Shinella, is not possible on the basis of physiological or biochemical traits and should be based on<br />
sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of<br />
bacteria isolated from legume nodules. MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time-of-<br />
Flight Mass Spectrometry) is a reliable method for bacterial identification, but it was necessary to extend the<br />
database Biotyper 2.0 to include the species from family Rhizobiaceae (Ferreira et al., 2011). After database<br />
extension, in this work, several strains isolated from nodules of alfalfa, clover and common beans isolated in<br />
Spain were subjected to MALDI TOF MS analysis. The results obtained showed a correct identification for most<br />
of the strains analysed. Some strains were not identified as any of species from the extended database<br />
indicating they can belong to undescribed species. Therefore MALDI-TOF MS is an excellent tool for<br />
identification of fast growing rhizobia and detection of new ones applicable to large populations of isolates in<br />
ecological and taxonomic studies.<br />
Ferreira L, Sánchez-Juanes F, García-Fraile P, Rivas R, Mateos PF, Martínez-Molina E, González-Buitrago JM &<br />
Velázquez E (2011) Maldi-tof mass spectrometry is a fast and reliable platform for identification and ecological<br />
studies of species from family Rhizobiaceae. PLoS One. 6: e20223.<br />
155<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Expression of an abscisic acid-responsive ƒÀ-1,3-glucanase gene in autoregulation of<br />
nodulation.<br />
Author Keni-chi Osuki 1 , Akihiro Suzuki 2 , Hisatoshi Hara 1 , Akihito Takahara 1 , Masato Araragi 1 ,Tadao<br />
Asami 3 , Keni-chi Kucho 1 , Shiro Higashi 1 , Mikiko Abe 1 and Toshiki Uchiumi 1<br />
Poster Board Number 41<br />
1 Graduate School of Science and Engineering, Kagoshima University,<br />
2 Department of Environmental Science, Saga University<br />
3 Graduate School of Agriculture and Life Science, The University of Tokyo<br />
Host legumes control nodule number by autoregulation of nodulation (AUT), in which the presence of root<br />
nodules inhibits further root nodule formation. AUT will consist of at least two long-distance signals, i. e. a rootderived<br />
infection signal and a shoot-derived signal that inhibits nodulation. In Lotus japonicus, HAR1 mediates<br />
AUT and, LjCLE-RS1 and LjCLE-RS2 have been identified as strong candidates as the infection signal. We<br />
employed split-root system of L. japonicus to investigate plant molecules involved in inhibition of nodule<br />
formation in AUT. Abscisic acid (ABA) and ABA responsible β-1,3-glucanase gene (designated as LjGlu1) were<br />
focused in this study.<br />
Plants were set up for split-root system. One side of root systems was inoculated with Mesorhizobium loti<br />
MAFF303099. At 1, 2, 3, 5, 7, 9 and 14 days after inoculation, expression of LjGlu1 in leaves, and inoculated-<br />
and uninoculated roots, were analyzed by quantitative RT-PCR. In the leaves, expression of LjGlu1 increased at<br />
the 7 days after inoculation. In the inoculated- and uninoculated roots, expression of LjGlu1 increased at the 5, 7<br />
and 9 day after inoculation. AUT response of L.japonicus is detectable 3 days after the first inoculation and that<br />
full induction of the response occurs after 5 days (Suzuki et al 2008).These results suggest that LjGlu1 increased<br />
in the root, when the full induction of the AUT response occurs.<br />
Hairy roots transformed with LjCLE-RS1 driven by the Cauliflower mosaic virus 35S promoter were prepared. In<br />
the plant that has both the transgenic and untransgenic roots, LjGlu1 was induced in not only the transgenic<br />
roots but also untransgenic roots. These findings indicate that constitutive expression of LjCLE-RS1 also induced<br />
expression of LjGlu1 systemically. These results suggest that LjGlu1 is functional involved in inhibition of nodule<br />
formation in AUT.<br />
Akihiro Suzuki , Hisatoshi Hara ,Tomoyo Kinoue ,Mikiko Abe , Toshiki Uchiumi , Ken-ichi Kucho ,Shiro Higashi<br />
,Ann M. Hirsch ,Susumu Arima (2008).Split-root study of autoregulation of nodulation in the model legume Lotus<br />
japonicus. J Plant Res 121:245–249<br />
156<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Conservation of legume-rhizobium symbiosis genes in non-legume.<br />
Author Keisuke Yokota, Takashi Soyano, Hiroshi Kouchi, Makoto Hayashi<br />
Poster Board Number 42<br />
National Institute of Agrobiological Sciences<br />
In recent years, a number of legume genes involved in the root nodule symbiosis has been identified in the<br />
model legumes, such as Lotus japonicus and Medicago truncatula. Among them, the distinct set of genes has<br />
been characterized as common sym, because they are also essential for another mutual interaction, the<br />
arbuscular mycorrhiza symbiosis. The non-legume homologs of common sym genes, such as in rice, have been<br />
well documented to be not only essential for the arbuscular mycorrhiza symbiosis in non-legume mycorrhizal<br />
plants but also functional in both the root nodule symbiosis and the arbuscular mycorrhiza symbiosis in legumes.<br />
In contrast, it has not been investigated in detail whether the root nodule symbiosis-specific genes, which are not<br />
essential for the arbuscular mycorrhiza symbiosis, are functionally conserved in non-legumes.<br />
In this presentation, we will report that the functional conservation analyses of the root nodule symbiosis-specific<br />
gene homologs from rice in L. japonicus.<br />
References<br />
Yokota K, Hayashi M. Function and evolution of nodulation genes in legumes. (2011) Cell Mol Life Sci.<br />
68(8):1341-1351.<br />
Yokota K, Soyano T, Kouchi H, Hayashi M. Function of GRAS proteins in root nodule symbiosis is retained in<br />
homologs of a non-legume, rice. (2010) Plant Cell Physiol. 51(9):1436-1442.<br />
157<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Analysis of nif gene derepression in Azotobacter vinelandii by quantitative real-time PCR<br />
Author César Poza-Carrión, Emilio Jiménez-Vicente, & Luis M. Rubio<br />
Poster Board Number 43<br />
Universidad Politécnica de Madrid. Centro de Biotecnología y Genómica de Plantas Pozuelo<br />
de Alarcón, 28223 Madrid (Spain). cesar.poza@upm.es<br />
Azotobacter vinelandii serves as a model to study the biochemistry and regulation of nitrogenase. In Azotobacter<br />
vinelandii, the nitrogen fixation (nif) genes are clustered in two chromosomal regions designated as the major<br />
and the minor nif clusters. Apart from the nitrogenase structural genes (nifHDK), a number of nif gene products<br />
are required for the assembly of active nitrogenase component proteins. Not all gene products act at the same<br />
time during nitrogenase biogenesis; therefore their expression levels and profile over time must be strickly<br />
coordinated.<br />
In this work, we study the time-dependent expression of fifteen nif genes involved in biosynthesis and regulation<br />
of nitrogenase (nifH, nifD, nifK, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifA, nifB, FdxN, nifQ, and nafY) under<br />
nitrogenase derepressing conditions by using real-time quantitative PCR. Nif gene expresion is observed as fast<br />
as 10 minutes after ammonium removal from the medium. Expression of genes whose products are involved in<br />
early steps of iron-molybdenum cofactor (FeMo-co) biosynthesis, such as nifB, reached maximum levels before<br />
nitrogenase structural genes. In general, it was observed that mRNA levels decreased rapidly as soon as<br />
nitrogenase activity appeared in the cultures, indicating tight feedback regulation of nif gene expression by<br />
nitrogenase activity.<br />
Applying synthetic biology to generate artificial nitrogenase systems is a promosing avenue to transfer nitrogen<br />
fixation capability to organims of interest. However, to build a synthetic nitrogenase, researchers must first<br />
understand the appropriate balance of gene expression in quantitative and temporal terms. Results from this<br />
work provide an important step towards elucidating these parameters in the model nitrogen-fixing organism<br />
Azotobacter vinelandii.<br />
158<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Nodule-specific cystein-rich peptides found in actinorhizal plant Datisca glomerata<br />
Author Irina V Demina, Tomas Persson, Marian Plaszczyca, and Katharina Pawlowski<br />
Poster Board Number 44<br />
Stockholm University, Department of Botany<br />
The actinorhizal plant Datisca glomerata enters a symbiosis with nitrogen-fixing actinomycetes of the genus<br />
Frankia. The bacteria are hosted in root nodules. In the course of root and nodule transcriptome studies of D.<br />
glomerata, full-size cDNA sequences of two genes encoding small nodule-specific cysteine-rich (NCR) peptides<br />
were obtained. One of the cDNAs, DgDEF1, shows amino acid homology with defensins; the other cDNA<br />
encodes a protein for which no homolog has been found in the databases. The predicted sizes of the mature<br />
peptides are rather large for defensins, 86 and 94 aa, respectively. This finding is the first report on NCR<br />
peptides in actinorhizal plants.<br />
Previously NCR peptides have been characterized in legumes, where they were demonstrated to govern the<br />
terminal differentiation of the rhizobial microsymbionts in symbiosis (Van de Velde et al. 2010) and to show<br />
antimicrobial activity toward a diverse group of bacteria (WO2010/146067). Hence, based on the homology<br />
between actinorhizal and legume symbioses, it seems likely that actinorhizal plants control the differentiation of<br />
their bacterial endosymbionts in a similar manner. The fact that DgDEF1 shows high homology with defensins of<br />
class A3, which reduce hyphal elongation while increasing hyphal branching in fungi, suggests that this peptide<br />
is responsible for the induction of hyphal branching in symbiotic Frankia in nodule cells.<br />
To determine in situ localization of the D. glomerata NCR peptides in nodules, Agrobacterium rhizogenes<br />
transformation was used. In transgenic nodules transcriptional fusions of the promoters of DgDEF1 and DgCRP1<br />
with the GUS coding region were expressed. The expression patterns of the transgenes will be discussed in<br />
comparison to the expression patterns of NCR peptide genes in legumes.<br />
Van de Velde et al. (2010). Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327:<br />
1122-1126<br />
159<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title N2O emission from degraded soybean nodules by denitrification of Bradyrhizobim japonicum<br />
and other soil microorganisms<br />
Authors Fumio Ikenishi, Shoko Inaba, Manabu Itakura, Shima Eda, Hisayuki Mitsui, and Kiwamu<br />
Minamisawa<br />
Poster Board Number 45<br />
Graduate School of Life Sciences, Tohoku University<br />
To clarify mechanism of N2O emission from degraded soybean nodules (Inaba et al. 2009), a model system was<br />
developed to reproduce N2O emission from degrading soybean nodules in laboratory. Thirty-days after soybean<br />
plants inoculated with Bradyrhizobium japonicum were cultivated in Leonard‟s jars, treatments of shoot<br />
decapitation (D) and/or soil addition (S) were conducted to simulate the nodule degradation and subsequent N2O<br />
emission. Double treatment (DS) resulted in the degradation and N2O emission from the nodules formed with B.<br />
japonicum lacking nosZ. These results suggested that soil microbes are required for the N2O emission from<br />
degraded soybean nodules. To evaluate bradyrhizobial contribution, N2O emission was compared between nirK<br />
mutant (∆nirK) and wild-type B. japonicum USDA110 under identical nosZ genetic backgrounds. N2O emission<br />
from the nodules formed with ∆nirK∆nosZ mutant was significantly lower than that from ∆nosZ mutant under DS<br />
treatment, but retained approximately a half amount of N2O emission in ∆nosZ mutant (30-60%), suggesting that<br />
nitrate reduction to N2O is due to both B. japonicum and other soil microorganisms. On the other hand, it is likely<br />
N2O reduction to N2 was mainly mediated by B. japonicum cells carrying nosZ, which was consistently supported<br />
by the comparisons between wild-type USDA110 and nosZ mutants. Thus, B. japonicum plays an important role<br />
in determining N2O flux from soybean rhizosphere as well as unknown soil microorganisms. It has been reported<br />
that fungi emit N2O in various fields. Thus, we isolated fungi spores from the model system, and evaluated their<br />
N2O production. As a result, many of the isolates produced N2O from nitrite in culture.<br />
Inaba S, Tanabe K, Eda S. Ikeda S, Higashitani A, Mitsui H & Minamisawa K (2009) Nitrous oxide emissions and<br />
microbial community in the rhizosphere of nodulated soybeans during the late growth period. Microbes Environ.<br />
24: 64-67.<br />
160<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Functional characterization and interactions of the azorhizobial parA and parB proteins<br />
Author Min-Hua Peng 1 , Yu-Sheng Wang 1 , Ming-Yen Tsai 2 , Hisao-Lin Chien 2 , Kung-Ta Lee 1 ,<br />
Poster Board Number 46<br />
Chi-Te Liu 2,3, *<br />
1 Department of Biochemical Science and Technology, National Taiwan University, Taipei<br />
106, Taiwan<br />
2 Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan<br />
3 Agricultural Biotechnology Research Centre, Academia Sinica, Taipei, 115, Taiwan<br />
Bacteroid development consists of several stages, and a number of bacterial genes that are involved in this<br />
process have been identified. However, the genes and factors that are engaged in the initiation of bacteroid<br />
formation, and those that regulate it, are incompletely identified. Our experimental evidence has demonstrated<br />
that the chromosome-partitioning gene (parA) gene of Azorhizobium caulinodans ORS571 not only plays crucial<br />
roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in<br />
Sesbania rostrata stem nodules. In this study, we will clarify the functions of the recombinant ParA and ParB<br />
proteins of A. caulinodans, and the interactions between the par system and bacteroid differentiation related<br />
genes. We noticed that the ATPase activity of ParA can be enhanced in the presence of ParB protein. In<br />
addition, ParB bound to a centromere-like parS DNA fragment, and was stimulated by the increasing amounts of<br />
ParA protein. Interactions between ParA and ParB were confirmed by in vitro immunoprecipitation assay. Using<br />
gel mobility shift assay, we found that ParB bound to the bacteroid formation associative gene (bacA) and the<br />
promoter region of par operon. This binding seems to be regulated by mole ratio of ParB/ ParA . We proposed a<br />
mechanism for regulation of bacteroid differentiation of A. caulinodans. Interactions between azorhizobial ParA<br />
and ParB proteins may function as a checkpoint, which couples the chromosome partitioning to the onset of<br />
bacteroid formation in symbiosis.<br />
Liu, C.-T., Lee, K.-B., Wang, Y.-S., Peng, M.-H., Lee, K.-T., Suzuki, S., Suzuki, T. & Oyaizu, H. (2011).<br />
Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during<br />
Sesbania rostrata stem nodule development. Appl Environ Microbiol 77, 4371-4382.<br />
161<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Trehalose accumulation in osmotically challenged rhizobia and its effect on desiccation<br />
tolerance.<br />
Authors Andrea Casteriano and Rosalind Deaker<br />
Poster Board Number 47<br />
University of Sydney - Faculty of Agriculture, Food and Natural Resources<br />
Pre-inoculated seeds are a convenient and much sought after legume inoculant product. However, rhizobia is<br />
known to survive poorly on seed mainly due to desiccation stress encountered during the seed coating process.<br />
Rhizobia is also known to possess inherent desiccation tolerance mechanisms including the production of<br />
compatible solutes such as trehalose through the de novo synthesis.<br />
Batch cultures of Bradyrhizobium japonicum (CB1809) and Rhizobium leguminosarum bv. trifolii (TA1) were<br />
grown in a defined medium with altered osmotic pressure through the addition of solutes. Increasing the osmotic<br />
pressure (1atm to 2.5atm) of the medium resulted in cells with a higher amount of intracellular trehalose. Vacuum<br />
drying those cells in the absence of an external protectant showed an improvement in % survival for CB1809<br />
immediately after drying and during storage at RH ≤ 9%. However the accumulation of intracellular trehalose did<br />
not result in improved desiccation tolerance for TA1.<br />
Cells of CB1809 extracted from peat and dried under vacuum in the absence of an external protectant show a<br />
much greater rate of survival not only immediately after drying but also during prolonged storage. Also, cells of<br />
TA1 and CB1809 grown in a peat extract have shown a much greater rate of survival after vacuum drying than<br />
those cells grown in a liquid medium.<br />
In conclusion, osmotically challenging CB1809 and TA1 during growth appears to increase their ability to<br />
accumulate intracellular trehalose. However, trehalose accumulation only improved desiccation tolerance of<br />
CB1809 and not TA1. Cells were better able to tolerate desiccation after growth in peat and peat extract<br />
indicating that not only conditions of nutrient and oxygen limitation play a role but also soluble constituents of<br />
peat.<br />
162<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title A MYB coiled-coil type transcription factor interacts with NSP2 and is essential for nodulation<br />
in Lotus japonicus<br />
Author Zhongming Zhang, Heng Kang, Xiaojie Chu, Chao Wang, Hui Zhu, Songli Yuan, Dunqiang<br />
Yu, Zhenzhen Yang<br />
Poster Board Number 48<br />
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University<br />
Transcription factor complex formation is a central step in regulating gene expression. In Medicago truncatula,<br />
two GRAS family transcription factors, Nodulation Signaling Pathway 1 (NSP1) and NSP2 form a DNA-binding<br />
complex to induce specific changes of gene expression, which is essential for the root nodule symbiosis (Hirsch<br />
et al., 2009). Using yeast two-hybrid (Y2H) screening, we identified a novel MYB coiled-coil transcription factor,<br />
referred as IPN2, which interacts with NSP2 in Lotus japonicus. This interaction was confirmed by protein pulldown<br />
assay and BiFC assay in Nicotiana benthamiana. Moreover, IPN2 and NSP2 were co-localized in nuclei of<br />
L. japonicus hairy roots. The Y2H assay showed that the GRAS domain of NSP2 was required for interacting<br />
with IPN2, while the coiled-coil domain of IPN2 was necessary and sufficient for interacting with NSP2. IPN2<br />
showed strong transcriptional activation activity in yeast cells, and it bound to the N<strong>IN</strong> promoter in vivo and in<br />
vitro. IPN2 was widely expressed in various organs, and showed phloem-specific expression within the<br />
vasculature of transgenic hairy roots. Overexpression of IPN2 promoted nodulation, while RNA interference<br />
(RNAi) knockdown of IPN2 expression led to decreased nodule formation in L. japonicus. Take together, our<br />
results strongly suggest that IPN2 plays an essential role in nodulation.<br />
Hirsch, S, Kim, J, Munoz, A, Heckmann, AB, Downie, JA, and Oldroyd, GE (2009). GRAS proteins form a DNA<br />
binding complex to induce gene expression during nodulation signaling in Medicago truncatula. Plant Cell<br />
21:545-557.<br />
163<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Genome sequence of Mesorhizobium huakuii 7653r which establishes a highly specific<br />
symbiosis with Astragalus sinicus in China<br />
Author Youguo Li, Shanming Wang, Jieli Peng, Baohai Hao & Liu Liu<br />
Poster Board Number 49<br />
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan,<br />
Hubei, 430070, P. R. China<br />
M. huakuii 7653R, isolated from a rice-growing field in southern China, can establish a highly specific and<br />
effective symbiosis with A. sinicus via forming indeterminate-type nitrogen-fixing nodules. To date, there is very<br />
limited information on the mechanism of its bacteroid development, nodulation specificity and molecular<br />
interactions with Chinese milk vetch. In the present work, the complete genome sequence of M. huakuii 7653R<br />
has been established and the genome structure and phylogeneticas signment of the organism was analysed.<br />
For de novo sequencing of the M. huakuii 7653R genome, a combined strategy comprising Solexa-sequencing<br />
on the Illumina GAIIx platform and PCR-based amplicon sequencing for gap closure was applied. The finished<br />
genome consists of three replicons and comprises 6,952,365 bases. The genome sequence of M. huakuii 7653R<br />
totals 6.95 Mbp with a GC content of 62.9%. It contains 6,792 predicted open reading frames (ORFs), located on<br />
a chromosome and two plasmids. The 6.2 Mbp circular chromosome encodes 6,150 ORFs with a mean GC<br />
content of 63.1%. The 320,051-bp pSym pMh7653Rb comprises 282 ORFs with a mean GC content of 62.6%.<br />
The plasmid pMh7653Ra (170,165 bp) has a relatively lower GC content (59.5%).<br />
Comparative analysis of the assembled genome sequence of M. huakuii 7653R revealed high homology and<br />
extensive synteny with M. loti MAFF303099, and partly to other rhizobial genomes, in particular to Sinorhizobium<br />
meliloti 1021 and to Rhizobium sp. NGR234.<br />
164<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Identification of the regulatory genes required for the acid activation of the low pH inducible<br />
gene lpiA in Sinorhizobium medicae<br />
Author Rui Tian 1 , Ravi Tiwari 1 , Lambert Bräu 1 , John Howieson 1 , Graham O‟Hara 1 & Wayne Reeve 1 .<br />
Poster Board Number 50<br />
1 Center for Rhizobium studies, Murdoch University, South Street, Murdoch, Western<br />
Australia, 6150.<br />
The acid tolerance response of Sinorhizobium medicae (S. medicae) WSM419 enables cell adaptation to lethal<br />
acid after cell exposure to mild acidity. The expression of lpiA (low pH induced gene A) gene is critical for this<br />
response and is acid-activated at least 20-fold in mild acidic conditions. Inactivation of an upstream gene fsrR<br />
(fused sensor regulator) reduces lpiA expression to just 3-fold of its maximum level. This observation suggested<br />
that other regulatory proteins are involved in complete activation of the lpiA gene in acidic conditions.<br />
Inactivation of putative regulators of lpiA located upstream (tcrA and tcsA) or downstream (acvB) genes and the<br />
sigma-54 factor encoded by rpoN was achieved by single crossover mutation in WSM419. Expression studies<br />
(GUS-linked assays and qRT-PCR) revealed that the two component system TcsA/TcrA and a fused sensor<br />
regulator FsrR (encoded by a gene directly upstream from lpiA) are controlling expression of lpiA and<br />
demonstrated that RpoN is essential for acid activation. A putative RpoN enhancer binding protein (Smed_5956)<br />
was located upstream of tcsA. RACE analysis located the transcriptional start site 14 bp downstream of a<br />
classical -24 and -12 RpoN binding motif upstream of the lpiA start codon demonstrating that lpiA and acvB are<br />
co-transcribed as an operon. The expression of acvB was determined by qRT-PCR and was found to be induced<br />
18-fold by acid which is consistent with the finding that both lpiA and acvB share the same transcription start site.<br />
In contrast, fsrR, tcrA, tcsA, and rpoN were constitutively expressed with respect to pH.<br />
While mutations in tcsA, tcrA, fsrR and acvB affect acid induction of lpiA, these genes are not essential for stress<br />
tolerance or symbiotic nitrogen fixation. In contrast, rpoN is essential for symbiotic nitrogen fixation with<br />
Medicago. This study has therefore identified for the first time an alternative sigma factor which is essential for<br />
pH response as well as symbiosis in S. medicae.<br />
165<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title<br />
Selection of plant growth promoting rhizobacteria (PGPR) to enhance nodulation of<br />
grain legumes by rhizobia<br />
Author Liza Parkinson 1 , Graham O‟Hara 1 , Ron Yates 1, 2 , Paul Harvey 3 and Lambert Brau 1<br />
Poster Board Number 51<br />
1 Murdoch University, Centre for Rhizobium Studies, Murdoch, Western Australia, 6150<br />
2 Department of Agriculture and Food Western Australia, South Perth, Western Australia,<br />
6151<br />
3 CSIRO, Urrbrae, South Australia, 5064<br />
Plant growth-promoting rhizobacteria (PGPRs) are soil bacteria associated with plant roots that have been<br />
shown to stimulate plant growth and crop yield. The mechanisms by which PGPRs increase plant growth include<br />
the production of plant hormones, suppression of disease–causing microbes as well as improved plant nutrient<br />
availability and assimilation. It has also been shown that the formation of nodules on legumes induced by<br />
rhizobia can be enhanced by co-inoculation with PGPRs.<br />
To examine the benefits of co-inoculating rhizobia with PGPRs, six newly identified PGPRs will be screened in<br />
glasshouse trials on Lupins (Lupinus angustifolius), Peas (Pisum sativum), Lentils (Lens culinaris), Beans<br />
(Phaseolus vulgaris), Chickpeas (Cicer arietinum) and Soybeans (Glycine max) in addition to the current<br />
commercial rhizobia. PGPRs used in this study include five Pseudomonas species, which have previously been<br />
shown to enhance nodulation in legumes and one gram-positive strain isolated from a Lupinus nodule. To<br />
evaluate the efficacy of the PGPRs on enhancing the legume-rhizobia symbiosis, the following will be measured:<br />
nodule numbers, nodule biomass, shoot and root biomass and shoot nitrogen content. In addition, nodule<br />
occupancy will be analyzed using PCR fingerprinting techniques to establish whether PGPRs are able to colocalize<br />
with rhizobia in the nodules.<br />
Promising isolates from these trails will be selected to elucidate the mechanism by which they enhance<br />
nodulation and/or nitrogen fixation. This will include analysis of expression of putative PGP genes, targeted<br />
gene knock-outs, and population genetic studies on the microbial community to determine the impact of<br />
inoculants on rhizobium populations in the rhizosphere.<br />
166<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Abdou, MM 109 Cao, Q 31 do Nascimento, EC 128 Hartley, E 82, 133 Keakile, KK 130<br />
Abe, M 131, 137,156 Capela, D 37 Dolgikh, EA, 40 Hartley, J 133 Kelly, S 51<br />
Adnane, B 111 Carlson, R 51 Dong, W 26 Hartmann, A 66 Kenicer, G 62<br />
Agarie, S 131 Carlsson , G 101 Downie JA 16 Harvey, P 166 Kennedy, I 76, 78<br />
Aisthorpe, D 47 Carmody, B 45, 152 Drevon, JJ 99, 109, 111 Hashiguchi, M 131 Khojiev, Z 120<br />
Akashi, R 131 Castelli, J 42 Drew, EA 44, 52 Hayashi, M 125, 158 Kieu, LN 103<br />
Alatalo, E 127 Casteriano, A 81, 162 East, A 36 Hayashi, S 57 Kim, J 16<br />
Alexandre, A 104 Catrice, O 37 Echavarri-Erasun, C 17 He, J 50 Kimanthi, M 86<br />
Alves, BJR 97, 128 Chaïbi, S 118 Eda, S 160 He, X 93 Kobryn, H 24<br />
Amaddin, PAM 90 Chansa-Ngavej, K 46 Edwards, A 16 Heckmann, AL 16 Koch, M 41<br />
Amenc, L 109 Charman, N 48, 54, 79 Enoki, A 150 Hennecke, H 41 Kouchi, H 157<br />
Ampomah, OY 62 Chen W-M 67 Erbacher, A 47 Herridge, D 96, 101, 103 Kryvoruchko, I 50<br />
Amprayn, K 76 Chen, C 42, 147 Espinoza, S 94, 121 Herrmann, L 84, 86, 134, Kucho, K 137, 156<br />
Andersen, SU 151 Chen, R 50 Faghire, M 99 Higashi, S 137,156 Kyrpidis, N 34<br />
Andrianananjara, A 109 Chen, T 119 Faraut, T 37 Hiratsuka, Y 131 Lake, L 52<br />
Angus, JF 100 Chen, WF 63 Farquharson, R 48 Hirsch, AM 131 Lazali, M 99<br />
Araragi, M 156 Chen, WX 63 Faye, A 84 Högbom, M 27 Lee, K-T 161<br />
Ardley, JK 69 Chen, X 81 Federova, E 31, 39, 65, 74 Hood, G 36 Lei, L 43<br />
Arima, S 131 Cheng, G 43 Ferguson, B 57 Hori, Y 137 Leppyanen, IV, 40<br />
Arpiwi, NL 107 Cheng, Z 50 Ferreira, L 155 Howieson, JG 15, 24, 49, Lesueur, D 84, 86, 134, 135<br />
167<br />
135<br />
56, 68, 69, 83, 165<br />
Asami, T 156 Chiasson, D 39, 122 Fischer, D 66 Hubber, A 51 Li, D 57<br />
Aserse, AA 145 Chien, H-L 161 Fischer, H-M, 41 Humphries, A 79 Li, M 63<br />
Assefa, F 145 Chimphango, SBM 67 Forster, B 89 Hurek, T 81 Li, QQ 63<br />
Atieno, M 84, 134 Chu, X 163 Fox, S 45, 75 Huss-Danell, K 62, 92 Li, Y 43, 164<br />
Attar, H 99 Clarke, VC 42, 129 Franchini, JC 97 Huwiler, S 41 Liao, S 72<br />
Bakkou, N 38 Cong, PT 103 Gamas, P 37 Huyghe, A 38 Limpens, E 65<br />
Baldani, JI 66 Conway, M 47 García-Benavides, P 154 Hynes, MF 35 Lin, M-H 57<br />
Baldock, J 48 Corbett, MK 106 García-Fraile, P 155 Iannetta, PPM 62 Lin, Y-H 57<br />
Ballard, N 22 Craig, A 54 Garg, N 108 Ikenishi, F 160 Lindström, K,21, 127, 145, 146<br />
Ballard, RA 44, 48, 52, 54, 79 Cramer, SP 26 Gavrin, A 74 Imaizumi-Anraku, H 125 Liu, C-T 161<br />
Balone, T 130 Critchley, C 93 Ge, Y-Y 72 Imin, N 58 Liu, L 164<br />
Banabas, M 96 Cuddy, W 89 Geddes, BA 35 Inaba, S 102, 160 Liu, TY 63<br />
Barbour, EL 107, 148 Dai, X 50 Gehlot, HS 60 Inada, S 131 Loi, A 83<br />
Bargaz, A 99 Dakora, FD 25, 55, 95, 110, Gehringer, MM 89 Ismail, MR 88 Lopatin, SA 40<br />
116, 140, 141, 142, 143, 144<br />
Barrero, R 56 Danza, F 41 Gelfand, M 32 Itakura, M 102, 160 Loughlin, P 39, 42, 122, 129,<br />
147<br />
Beatty, P 98 Dapper, CH 26 Gemell, G 82, 133 Ivanov, S 65, 74 Ludwig, M 42<br />
Belane, A 55, 144 Day, DA 42, 122, 129, 167 Gemmer, S 81 Jalovaja, J 32 Majengo, C 86<br />
Bellgard, M 56 de Araújo, JLS 128 George, SJ 26 James, EK 60, 62, 67 Makhubedu, I 141<br />
Benedito, VA 50, 59 De Carvalho-Niebel, F 37 Gerding, M 68 Jantalia, CP 97, 128 Malik, R 23<br />
Benito, EP 154 de Mita, S 65 Ghazali,, A 136 Jardinaud, F 37 Małolepszy, A 151<br />
Berthold, CL 27 Deaker, R 82, 89, 103, 162 Ghoulam, C 99, 111 Jikumaru, Y 131 Mantaj, M 70<br />
Bisseling, T 31, 39, 65, 74 Debéllé, F 37 Giller, K,19 Jiménez-Vicente, E 17, 29, Mapope, N 142<br />
Bledsoe, C 93 Del Pozo, A 94, 121 Gonzales-Andres, F 77, 153 Jin, L 119 Marcano, I 77<br />
Boddey, RM 97, 128 Delmotte, N 41 González-Buitrago, JM 155 Jing, Y 124 Marques, AT 132<br />
Bonython, A 54 Demina, IV 159 Good, AO 98 Kahn, ML 28 Martínez-Hidalgo, P 154,<br />
155<br />
Boonkerd, N 123, 138, 140 Denton, MD 52, 79, 85, 152 Gouzy, J 37 Kaiser, BN 39, 122, 126 Martínez-Molina, E 154,<br />
155<br />
Borisov, A 65 Díaz-Alcántara, CA 77, 153 Gresshoff, P 57 Kalhor, MS 31 Martinez-Romero, E 66<br />
Braissant, O 41 Díaz-Mínguez, JM 154 Grossman, J 53 Kamaa, M 84 Maseko, S 110<br />
Brau, L 45, 61, 68, 75, 117, Dilworth, M 69 Guerts, R 31, 65 Kamiya, Y 131 Masse, D 109<br />
165, 166<br />
Brock, P 101 Ding, H 35 Hailemariam, A 145 Kaneko, T 131 Mateos, PF 155<br />
Brockwell, J 100 Dinse, T 81 Han, L 125 Kang, H 163 Mathews, C 95, 116<br />
Bruand, C 37 Djedidi, S 33 Hanyu, M 73 Kanu, SA 143 Mathu, S 135<br />
Btissam, M 111 Djordjevic, MA 58 Hao, B 164 Karunakaran, R 36 Matiru, V 135<br />
Bühler, D 41 Dlodlo, O 67 Hara, H 156 Kaur, H 106 Matsuura, H 150<br />
Burgos, FA 24 Kawaharada, Y 51<br />
158<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Mazurkiewicz, D 122 Ohwaki, Y 91, 149 Räsänen, LA 145 Soyano, T 158 van Averbeke, W 116<br />
McInnes, A 70 Okalebo, R 84, 86, 134 Ratet, P 50 Sprent, JI 60,62,64,67 Varlamov, VP 40<br />
McNeill, A 96 Okamoto, M 122 Reeve, WG 34, 44, 56, 69,<br />
117, 166<br />
Squire, G 62 Velázquez, E 77, 153, 155<br />
Melino, V 44, 117 Okazaki, S 71, 73 Reid, D 57 Sripakdi, S 138 Venkatanagappa, S 79<br />
Mhamdi, R 118 Oldroyd, GED 16, 18, 50 Reinhold-Hurek, B 81 Staehelin, C 72 Vorholt, J 41<br />
Millar, AH 129 Oliveira, S 104 Reis, VM 66 Steenkamp, ET 70 Vorobyov, N 30<br />
Minamisawa, K 102, 160 Oliver, M 53 Rice, J 28 Stepkowski, T 70 Walker, R 61<br />
Mirzaei, S 57 Omar, N 87 Rodde, N 37 Stougaard, J 20, 51, 151 Wang Y-S 161<br />
Mitra, D 26 Onyango, P 86 Rodino, N 99<br />
Streng, A 31 Wang, C 119, 163<br />
Ronson, C 51<br />
Mitsui, H 160 op den Camp, R 31, 65 Roux, B 37 Sui, XH 63 Wang, ET 63<br />
Mndzebele, B 140 Oresnik, IJ 35 Rubio, LM 17, 29, 158 Sullivan, J 51 Wang, He 27<br />
Mohale, K 55, 141 Osbourne, CA 100 Rudnicka, J 70 Summerell, BA 89 Wang, Ho 26<br />
Mohamad, S 105 Osman WAM, 117 Saad, M 38 Suriyagoda, L 131 Wang, L 119<br />
Mohamed, Far. 111 Österman, J 127, 146 Sadras, V 52 Suzuki, A 131, 156 Wang, M 50, 59<br />
Mohamed, Fat. 87 Osuki, K 137, 156 Saeki, K 71, 73 Swan, A 100 Wang, S 164<br />
Mohammadi-Dehcheshmeh, M<br />
126<br />
Othman, R 88 Sahara, M 137 Tabata, S 131 Wang, Z 43<br />
Mokgehle, S 95 Ounan, M 99 Saïdi, S 118 Tajima, S 150 Watkin, E 61, 70, 106, 107,<br />
148<br />
Moreira, LM 132 Ovalle, C 94, 121 Sakai, T 131 Tak, N 60 Webb, M 96<br />
Moreno-Urbano, E 17 Ovchinnikova, E 65 Sallet, E 37 Takahara, A 156 Wen, J 50<br />
Morieri, G 16 Padhi, B 41 Samian, M-R 105 Takayama, H 137 White, R 44<br />
Mostafa, S 87 Parihar, R 60 Sánchez-Juanes, F 155 Tang, Y 50 Xie, Fu. 43<br />
Mothapo, N 53 Parkinson, L 166 Sandal, N 51 Taylor, M 50 Xie, Fa. 16<br />
Mousavi, SA 146 Parr, M 53 Sankhla, I 60 Taylor, NL 129 Xie, Z-P 72<br />
Muasya, AM 67 Paulin, L 127, 146 Santos, MR 132 Tazawa, J 91 Xin, D-W 72<br />
Mulas, D 153 Pawlowski, K 159 Sasaki, M 131 Teamthisong, K 139 Xu, H 43<br />
Muni, RRD 50 Pearce, DJ 45, 79, 85, 152 Sato, S 131, 137 Teaumroong, N 123, 138, 139 Yadgarov, K 120<br />
Murakami, E 137 Peix, A 155 Saud, HM 88 Teixeira, PF 27 Yam, H-C 105<br />
Murray, J 16, 50 Peng J 164 Saur, I 58 Terakado-Tonooka, J 149 Yan, G 107<br />
Murset, V 41 Peng, M-H 161 Sauviac, l 37 Thapanapongworaku, N 150 Yan, L 26<br />
Mustapha, F 111 Peoples, MB 85, 100, 152 Scandurra, A 17 Thomson, G 44 Yang, H 56<br />
Muszynski, A 51 Perret, X 38 Schiex, T 37 Thuita, M 84 Yang, M 124<br />
Mutch, L 61, 106 Persson, T 159 Schmid, M 66 Tian, CF 63 Yang, Z 163<br />
Mutegi, E 84, 86 Pfitzner, B 66 Schwenke, G 101 Tian, R, 165 Yates, RJ 24, 49, 69, 83, 166<br />
Muto, S 137 Phillips, L 45, 79, 152 Sekimoto, H 33 Tikhonovich, IA, 40 Yip, C 35<br />
Mwirichia, R 135 Pipai, R 96 Selão, TT 27 Timmers, T 37 Yokota, K 157<br />
Mysore, K 50 Piromyou, P 123 Serventi, G 41 Tittabutr, P 123, 138, 139 Yokoyama, T 33, 91<br />
Nagano, Y 137 Pislariu, C 50, 59 Seymour, M 23 Tiwari, RT 60, 117, 165 Yoshikawa, M 91<br />
Nagata, M 137 Plaszczyca,, M 159 Seymour, N 47 Tollenaere, A 57 Yoshinaga, A 131<br />
Naher, UA 88 Plummer, JA 107 Shaffie, S 136 Tominaga, A 131 Youard, Z 41<br />
Najimudin, N 105, 136 Polone, E 31 Shakirov, Z 120 Torres-Jerez, I 50, 59 Young, PW 127<br />
Narozna, D 70 Poole, P 36 Shamsuddin, ZH 88, 90 Tran, YT 103 Yu, D 163<br />
Ndung‟u, K 84 Poonar, N 60 Shen, S 124 Tripathi, A 60 Yuan, S 163<br />
Neilan, BA 89 Poza-Carrión, C 17, 29, 158 Shidore, T 81 Trujillo, ME 154 Yurgel, S 28<br />
Nelson, P 96 Prell, J 36 Shigeoka, M 137 Tsai, M-Y 161 Zatorre, NP 97<br />
Newton, W 26 Provorov, N 30 Shigeyama, T 131 Tsoy, O 32 Zhang, J 56<br />
Nomura, M 150 Pule-Meulenburg, F 55, 130, 141 Shimoda, Y 125, 137 Tyerman, SD 122, 126 Zhang, JJ 63<br />
Nordlund, S 27 Pypers, P 84, 135 Shina, Y 102 Uchiumi, T 132, 138, 157 Zhang, L 72<br />
Norén, A 27 Qu, Y 42 Shirai, R 73 Udvardi, M 50, 59 Zhang, S 50<br />
Norris, L 148 Quinlivan, M 47 Shizukawa, Y 91 Umarov, B 120 Zhang, W 124<br />
Nutt, B 83 Rabeharisoa, L 109 Simoes-Araújo, JL 66 Unkovich, M 80, 96 Zhang, YM 63<br />
O‟Brien, T 79 Radzman, N 58 Sinharoy, S 50, 59 Uno, U 91 Zhang, Z 119, 163<br />
O‟Hara, GW 24, 44, 45, 49, 61,<br />
68, 69, 70, 75, 165, 166<br />
Rahim, KA 88 Smith, B 100 Urbano, B 77, 153 Zhao, PX 50<br />
Oakes, M 58 Ramachandran, V 36 Smith, PMC 42, 129, 147 Urbański, DF 151 Zhu, H 119, 163<br />
Ohkama-Ohtsu, N 33 Ramírez-Bahena, M-H 155 Smith, SE 126 Urquiaga, S 97, 128 Zotarelli, L 97<br />
Vailhe, H 109 Zuan, TK 90<br />
168<br />
2011
17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
169<br />
2011