IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

PrinciPal SPonSor: 

SPonSorS: 

Program Handbook 

HoSTED BY: 

SuPPorTED BY:

Welcome 

on behalf of the regional organising committee, i have great pleasure in welcoming you to the 17th 

international congress on nitrogen Fixation in Fremantle, Western australia. This congress, the first of 

which was held in 1974, provides the scientific community with the opportunity for a vital exchange of 

ideas on nitrogen fixation. let’s not forget that nitrogen fixation is as vital to our survival on the planet 

as photosynthesis! 

As always, the congress will feature 

high quality research across numerous 

disciplines and identify the emerging 

areas in the field. The strength of the 

international nitrogen fixation community 

lies in its people – our colleagues range 

from students and up-and-coming postdocs 

to the many experts in their field. If 

this is your first time attending this congress can I encourage you to 

take the opportunity to engage with as many colleagues as you can. 

Establishing research collaborations is what the meeting is all about. 

The 17th International Congress on Nitrogen Fixation also provides us 

with an opportunity to consider the questions– where are we now with 

nitrogen fixation and where are we going? On one hand we have the 

very exciting opportunities being provided by molecular technologies, 

with advances in these areas coming at an ever-increasing rate. 

On the other hand, many of the challenges confronting successful 

application of nitrogen fixation technology in agriculture remain 

the same e.g. developing new legumes and rhizobia for agriculture; 

achieving successful inoculation through improved technologies; and 

understanding the conundrums surrounding the life of rhizobia in soil 

and rhizosphere such as ineffective populations and competition. 

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Fremantle is the major port city of Western Australia, a state that 

is known for its magnificent coastline, incredible landscapes and 

unique wildlife. From tropical mangroves in the far north to the 

limestone cliffs pounded by the southern ocean, Western Australia 

offers a myriad of opportunities for travelers. Fremantle has been 

the gateway to Western Australia for well over 150 years and its 

multicultural history is reflected in its many cafes, restaurants, 

museums and other cultural sites. What better than a chance to browse 

the markets, or relax after the science in the cafes and restaurants? 

I sincerely hope that this will be a productive and memorable 

congress for all participants. 

associate Professor Graham o’Hara 

Director, Centre for Rhizobium Studies 

Murdoch University, Western Australia 

Congress Secretariat 

EEcW Pty ltd 

47 Hampden Road, Nedlands 

Phone: 08 9389 1488 

Fax: 08 9389 1499 

Emal: info@eecw.com.au

INVENTING THE FUTURE 

IN INOCULANTS 

Nodulaid N/T 

N/T stands for Nodulating Trigger 

Nodulaid N/T is unique. In addition to the nitrogen-fi xing rhizobia present in all 

inoculants, it also contains a Plant Growth Promoting Rhizobacteria (PGPR), Bacillus 

subtilis. This powerful active acts as a Nodulating Trigger. The combination of these 2 

ingredients adds an extra boost to the yield increases achieved by regular inoculants. 

Benefi ts of Inoculating with Nodulaid N/T 

The inclusion of the Nodulating Trigger results in more and healthier nitrogen-fi xing 

nodules per plant. Superior nodulation means a potential for increased nitrogen fi xation, 

producing greater, more vigorous plant root structure and resulting in greener, healthier 

early season plant foliage. This sets the crop up for a higher yield potential. 

Nodulaid N/T has been proven to increase crop performance in chickpeas, beans 

and lupins – even in paddocks that contain background or native rhizobia in the soil. 

Nodulaid N/T sets the crop up for optimal performance 

Nodulator Clay Granules 

Placing Nodulator Clay Granules into the soil adjacent to pulse seeds at sowing 

allows the roots to be inoculated as they emerge. Germinating seeds and developing 

root systems with easy access to higher levels of inoculant, result in better nodulation 

and better nitrogen fi xation. 

Nodulator Clay Granules are different, they provide: 

Dust and lump-free formulation 

Uniform size and smooth surface for superior fl ow 

High counts of live rhizobia 

Ideal for ‘dry seeding’ 

Toll Free: 1800 558 399 Email: info.au@beckerunderwood.com www.beckerunderwood.com.au

The International Congress on Nitrogen Fixation Series 

First 1974 Pullman, WA, USA (William E. Newton) 

Second 1976 Salamanca, Spain (Claudino Ridriguez-Barrueco) 

Third 1978 Madison, WI, USA (William H. Orme-Johnson) 

Fourth 1980 Canberra, Australia (Alan Gibson) 

Fifth 1983 Noordwijkerhout, The Netherlands (Cees Veeger) 

Sixth 1985 Corvallis, OR, USA (Harold J. Evans) 

Seventh 1988 Cologne, Germany (Hermann Bothe) 

Eighth 1990 Knoxville, TN, USA (Peter Gresshoff) 

Ninth 1992 Cancun, Mexico (Rafael Palacios) 

Tenth 1995 St. Petersburg, Russia (Igor Tikhonovich) 

Eleventh 1997 Paris, France (Claudine Elmerich) 

Twelfth 1999 Foz do Iguacu, Brazil (Fabio O. Pedrosa) 

Thirteenth 2001 Hamilton, ONT, Canada (Turlough M. Finan) 

Fourteenth 2004 Beijing, P.R. China (Yi Ping Wang) 

Fifteenth 2007 Cape Town, South Africa (Felix Dakora) 

Sixteenth 2009 Big Sky, MT, USA (John W. Peters) 

International Steering Committee 

(Provides continuity between congresses and decides among 

applicants for upcoming congresses) 

William Newton (USA) 

Felix Dakora (RSA) 

Ray Dixon (UK) 

Claudine Elmerich (France) 

Hauke Hennecke ( Switzerland) 

Fabio Pedrosa (Brazil) 

John Peters (USA) 

Igor Tikhonovich (Russia) 

Yi-Ping Wang (PRC) 

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17 ICNF Organising Committee 

Graham O’Hara, Chair, Murdoch University 

Lambert Brau, Murdoch University 

Michael Dilworth, Murdoch University 

John Howieson, Murdoch University 

Wayne Reeve, Murdoch University 

Elizabeth Watkin, Curtin University 

Ron Yates, Department of Agriculture and Food, Western Australia 

Lynne Greenaway, Congress Secretariat, EECW 

Renee Bennett, Congress Secretariat, EECW 

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17 ICNF Program Advisory Committee 

Ross Ballard (Australia) 

Ton Bisseling (The Netherlands) 

Robert Boddey (Brazil) 

Nantakorn Boonkerd (Thailand) 

Roz Deaker (Australia) 

Matthew Denton (Australia) 

Allan Downie (UK) 

Jean-Jacque Drevon (France) 

Hans-Martin Fischer (Switzerland) 

Ken Giller (The Netherlands) 

Rene Guerts (the Netherlands) 

David Herridge (Australia) 

Mariangela Hungria (Brazil) 

Michael Hynes (Canada) 

Didier Leseur (France/Thailand) 

Kristina Lindstrom (Finland) 

Kiwamu Minamisawa (Japan) 

Giles Oldroyd (UK) 

Mark Peoples (Australia) 

Xavier Perret (Switzerland) 

Phillip Poole (UK) 

Clive Ronson (New Zealand) 

Luis Rubio (Spain) 

Janet Sprent (UK) 

Jens Stougaard (Denmark) 

Michael Udvardi (USA) 

Murray Unkovich (Australia) 

Graham Walker (USA)

KeYNoTe SPeAKeRS 

Allan Downie 

After completing a PhD, post-doc and a fellowship in microbial bioenergetics, I moved from Canberra 

to the John Innes (Norwich) in 1981 and started working on Rhizobium-legume interactions. My work 

on identifying various nodulation genes included the characterisation of a secreted nodulation protein 

that forms cation-selective ion channels. This led me to analyse Ca++ changes in legume root hairs in 

response to Nod factors in WT and nodulation-defective mutants, leading to a proposed Nod-factor 

activated signalling pathway. More recently I have been trying to understand how Nod-factor-induced 

Ca++ signalling might be coupled to initiation of rhizobial infection of root hairs. 

Ken Giller 

Ken Giller is Professor Plant Production Systems at Wageningen University. He leads a group of scientists 

with profound experience in systems analysis and simulation modelling scenarios of change. Ken’s 

research has focused on nitrogen fixation in tropical legumes and he is author of the standard text 

“Nitrogen Fixation in Tropical Cropping Systems” published in second edition in 2001. He has 5 books, 

>200 papers in peer-reviewed, international journals (H factor=37) and >100 book chapters. He leads 

a US$19.2M project “N2Africa – Putting nitrogen fixation to work for smallholder farmers in Africa 

(www.N2Africa.org)” across 8 countries in Africa funded by the Bill & Melinda Gates Foundation. 

Giles Oldroyd 

Western agricultural systems are reliant on the application of inorganic nitrogen fertilisers that greatly 

enhance yield. However, production and application of nitrogen fertilisers account for a significant 

proportion of fossil fuel usage in food production and the major global source of nitrous oxide emissions, 

a very potent greenhouse gas. Prof Giles Oldroyd studies the mechanisms by which some species of 

plants are capable of forming beneficial interactions with nitrogen fixing bacteria, that provide a natural 

source of nitrogen for plant growth. A long term aim of this research is to reduce agricultural reliance on 

nitrogen fertilisers. Giles completed his PhD in 1998 at the University of California, Berkeley, studying 

plant/pathogen interactions and then moved to Stanford University to complete a postdoc on legume/ 

rhizobial interactions in the laboratory of Prof. Sharon Long. He has been an independent researcher 

at the John Innes Centre since 2002. From 2002 to 2007 he held a BBSRC David Philips Fellowship and 

since 2007 has been a tenured member of the JIC faculty. In 2008 he was made the head of the Institute 

Strategic Programme Grant “Plant sensing and responding to the Environment” and between 2010-2011 

he was the Deputy Director of the John Innes Centre. He has been recognised by a number of awards 

for his research: EMBO young investigator; European Research Council young investigator; Society of 

Experimental Biology Presidents medal; Royal Society Wolfson Research Merit award and the BBSRC 

David Phillips Fellowship. 

Phil Poole 

Phil Poole is originally from Perth but slipped up while on the typical Aussie European tour of Europe 

by staying to do post doctoral work in the biochemistry department at Oxford. From there he moved 

to Reading and then to the John Innes Centre in 2007. Phil works on the physiology and genetics of root 

nodule and rhizosphere/soil bacteria. He is particularly interested in: 

1) The regulation of nutrient exchange between Rhizobium and their host legumes 

2) Development and differentiation of the legume bacteroids 

3) The molecular basis of competitive success of bacteria in the plant rhizosphere 

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Cottesloe Beach © Tourism Western Australia 

Luis M. Rubio 

Luis M. Rubio is Assistant Professor at the Center for Plant Genomics and Biotechnology of the Technical 

University of Madrid (Spain). He obtained BS and PhD in Biology from the University of Seville. He 

worked as posdoctoral researcher at the University of Wisconsin-Madison and as Associate Scientist at 

the University of California-Berkeley. His main research interest is the biochemistry and biosynthesis of 

nitrogenase, with a focus on the study of iron-molybdenum cofactor biosynthesis. His ongoing research 

also includes improvement of biological hydrogen production by altered forms of nitrogenase. 

Janet Sprent 

Janet Sprent is Emeritus Professor of Plant Biology in the University of Dundee and Honorary Research 

Fellow at the James Hutton Institute, Dundee. She has spent decades working on nitrogen cycling and 

fixation, especially by legumes, working in all continents, including Antarctica. Current collaborations are in 

Europe, Africa, India and Australia and focus on nodulation processes in understudied legumes. Her talk will 

have a global coverage of the great variety of legumes, their nodule structure and nodulating bacteria and 

will speculate on how legumes may have evolved over time to fill particular geographic niches 

Jens Stougaard 

Jens Stougaard´s primary research activity is on genes regulating development of nitrogen fixing root 

nodules and mycorrhiza formation in legumes. Currently the mechanisms of Nod-factor perception, 

the function of receptors involved and the downstream signal transduction cascades are in focus. The 

plant model system used for this research is Lotus japonicus that is also used for investigating the long 

range signalling integrating root nodule development into the general developmental program of the 

plant. Genetics, genomics and biochemical methods are used to identify and characterise components of 

regulatory circuits. In order to improve the genetic analysis and to establish a system for reverse genetics, 

a large-scale insertion population based on the germ-line specific activity of the LORE1 retroelement is 

being established and made available to the community. 

Michael Udvardi 

Michael Udvardi: I earned my PhD at The Australian National University where I worked on the biochemistry 

of transporters of the symbiosome membrane that mediate traffic of nutrients between plant cells and 

rhizobia in nitrogen-fixing legume nodules. During my postdoc years, I used molecular biology approaches 

to isolate and characterize plant genes involved in nodule metabolism. Over the years, my research 

interests have broadened, although work on symbiotic nitrogen fixation remains a core activity of my 

lab. I am now a Professor at the Samuel Roberts Noble Foundation where we are developing and applying 

tools for functional genomics in Medicago truncatula. Using forward and reverse-genetic approaches, 

we are currently identifying new genes required for nodule development, differentiation, and symbiotic 

nitrogen fixation. 

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Amberley Estate © Tourism Western Australia

VeNUe 

The Esplanade Hotel Fremantle is privately owned. The Hotel offers 

a warm, friendly and professional service in the simple, elegant and 

relaxed surroundings. The uniqueness of the Heritage and Colonial 

architecture blends with the character of Fremantle’s historical Port 

and Fishing Boat Harbour. 

The Esplanade Hotel Fremantle is the first hotel in WA to be declared 

carbon neutral. 

The Esplanade Hotel Fremantle realises that climate change and 

greenhouse emissions present pressing global challenges that 

will only be successfully resolved if the majority of individuals 

and businesses get serious about reducing their damaging carbon 

footprint. The Hotel’s carbon-neutral status has been achieved by 

securing 9.6ha of rainforest in Ecuador through Rainforest Rescue 

to offset the Hotel’s remaining carbon emissions. 

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Esplanade Hotel Fremantle 

Corner Marine Tce & Essex St 

Fremantle, Western Australia 

Tel: +61 8 9432 4539 

Fax +61 8 9430 453 

www.esplanadehotelfremantle.com.au 

Orion 

Lift 

SouTHErn croSS Gala BallrooM 

Pleiades 

Atrium 

Garden 

Restaurant 

Prince Regent 

Main Foyer Entrance 

Sirius 

Southern Cross Lobby 

King Sound 

inDian ocEan SuiTE 

Admiralty Gulf

GeNeRAl INfoRmATIoN 

aTM’s 

Automatic Teller Machine is located next to Cafe Panache at the 

Esplanade hotel, alternatively various bank ATM’s are located on 

South Terrace (cappuccino strip) 

Banks 

Most major banks are located in the Fremantle City Centre. Please 

check with concierge staff for specific bank locations. 

car rental 

Budget Rent a Car: 13 27 27 

Hertz: 13 30 39 

Avis: 13 63 33 

child care 

Please note that no official arrangements have been made for child 

care during the conference. Please check with your hotel as they may 

be able to assist you further with babysitting services during your stay. 

congress Dinner 

Venue: The View Restaurant 

Time: 19:00 – 23:00 

Dress: Smart Casual 

Map of Fremantle 

A map of the City of Fremantle is included in your delegate bag. 

Meals 

All tea breaks will be served in the Southern Cross Foyer amongst the 

displays. Lunches each day will be served in the Atrium Restaurant. 

Special Dietary Requirements: Please see registration desk. The 

variety of selection on the Atrium buffet should be sufficient to 

cater for all dietary needs. 

Messages 

The Registration Desk will receive all messages which can be 

collected from the Registration Desk. 

Telephone: +61 (0) 439 912 333 

The Congress Secretariat will accept no responsibility for undelivered 

messages. 

name Badge 

It would be appreciated if delegates wear their name badge at all 

times as this identifies them as eligible for catering and entry to 

congress sessions. 

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Parking 

Collie street car park is a multi story, undercover facility located on 

Collie Street, behind the Esplanade Hotel. 

Valet parking for in-house guests at the Esplanade is provided at 

a $24.50 per day. For non hotel guests valet parking is available at 

$28 per day and must be pre-booked 48 hours in advance by calling 

9432 4815. 

Privacy Statement 

In registering for 17th International Nitrogen Fixation Congress 

relevant details may be incorporated into a delegate list for the 

benefit of all delegates, sponsors, exhibitors, EECW Pty Ltd and 

other parties directly related to the Congress. Should you wish for 

your details not to be included in the final list, please advise the 

registration desk. 

registration Desk 

The Registration Desk will be located in the Hotel foyer and will be 

open as follows: 

Sunday 27 November 14.00 – 17.00 

Monday 28 November 08.00 – 17.30 

Tuesday 29 November 08.00 – 17.30 

Wednesday 30 November 08.00 – 17.30 

Thursday 1 December 08.00 – 12.30 

Smoking Policy 

The Esplanade Hotel has a no smoking policy throughout the venue. 

Due to West Australian Government regulations this no smoking 

policy applies to most restaurants, bars and shopping centres 

throughout Western Australia. 

Shuttle Bus 

Shuttle Bus Services can be booked in advance for airport transfers 

to and from Fremantle. To receive this service, all pick-ups must be 

pre-booked at least 48 hours in advance, preferably between 8am – 

5pm Australian Western Standard Time Monday to Friday. To book 

contact Fremantle Shuttle Service: 

To Book: 

T: (08) 9457 7150 or 0437 197 240 (between 9am – 5.30pm AWST) 

Visitors to Fremantle 

Fremantle is one of the easiest cities in the world to explore by 

foot, bicycle or scooter. All leading attractions are located in close 

proximity and the ‘free’ Fremantle CAT bus completes regular 

circuits (every 15 minutes) around the City of Fremantle, stopping 

at popular places of interest and offering easy access to major 

attractions and venues. 

useful local Telephone numbers 

The Esplanade Hotel Fremantle: +61 8 9432 4000 

Quest Harbour Village Apartments: +61 8 9430 3888 

Rosie O’Grady’s Irish Pub: +61 8 9335 1645 

Swan Taxis: 13 13 30 

Bus & Rail Information: 13 62 13

PRoGRAm 

Sunday 27 November 2011 

14:00 - 17:00 Registration Desk Open 

17:30 - 19:30 Welcome Reception – Cicerello’s Fishing Boat Harbour Fremantle 

monday 28 November 2011 

08:00 - 17:00 Registration desk open 

09:00 - 10:30 coNfeReNce oPeNING ceRemoNY Chair: Graham O’Hara & Michael Dilworth, Room: Sirius 

10:30 - 11:00 Morning Tea 

convenor Welcome Graham o’Hara, Centre for Rhizobium Studies, Murdoch University, Australia 

Welcome to country Marie Taylor 

opening Address Peter roberts, Chair Western Panel, GRDC 

contemporary challenges for 

Symbiotic Nitrogen fixation 

John Howieson, Centre for Rhizobium Studies, Murdoch University, Australia 

11:00 - 12:30 PleNARY SeSSIoN Chair: Bill Newton, Room: Sirius 

11:00 - 11:30 allan Downie 

John Innes Centre, UK 

11:30 - 12:00 luis M. rubio 

Universidad Politecnica De Madrid, Spain 

12:00 - 12:30 Giles oldroyd 


12:30 - 13:30 Lunch – Atrium 

Characterisation of a legume pectate lyase required for initiation and growth 

of infection threads during nodule infection by rhizobia 

Changes in nif gene expression profiles during nitrogenase biogenesis 

Establishing beneficial interactions with the symbiosis signalling pathway 

13:30 - 15:00 PleNARY SeSSIoN Chair: Allan Downie, Room: Sirius 

13:30 - 14:00 Ken Giller 

Wageningen University, The Netherlands 

N2Africa – Putting Nitrogen Fixation to work for smallholder farmers in Africa 

14:00 - 14:30 Jens Stougaard 

Aarhus University, Denmark 

Nod factor perception, signal transduction and the role of LysM type receptors 

14:30 - 15:00 Kristina lindström 

University of Helsinki, Finland 

Rhizobium taxonomy and diversity – Is there anything more to learn? 

15:00 - 15:30 Afternoon Tea 

15:30 - 16:50 coNcURReNT SeSSIoN 1: 

coNcURReNT SeSSIoN 2: 


field Applications I 

function & control of Nitrogenase Taxonomy & evolution 

Chair David Herridge Michael Dilworth Kristina Lindström 

Room Sirius Pleiades Orion 

15:30 - 15:50 neil Ballard 

Bill newton 

nikolay Provorov 

Global Pasture Consultants, Australia 

Virginia Polytechnic Institute & 

All-Russia Research Institute For 

Application of Western Australian 

State University, USA 

Agricultural Microbiology, Russia 

legume and Rhizobium technologies Carbon monoxide adducts 

Mathematical simulation of 

in the developing world – 

of Azotobacter vinelandii 

evolutionary events in the 

a practitioner’s perspective 

Mo-nitrogenase 

N2-fixing plant-microbe symbioses 

15:50 - 16:10 raj Malik 

Stefan nordlund 

rene Guerts 

Department of Agriculture & Food 

Stockholm University, Sweden 


Western Australia, Australia 

Regulation of Nitrogenase activity in Parasponia to unravel genetic 

Impact of break crops on wheat Rhodospirillum rubrum – an interplay constraints underlying Rhizobium 

productivity in Western Australia of PII proteins, DRAT, DRAG and symbiosis 

cropping 

membrane sequestration 

16:10 - 16:30 Felipe Burgos 

Svetlana Yurgel 

Jelena Jalovaja 

Centre For Rhizobium Studies, 

Washington State University, USA 

Moscow Institute of Physics & 

Murdoch University, Australia 

GlnD and symbiosis: Control beyond 

Technology, Russia 

Use of remote sensing to assess the the nitrogen stress response? 

Comparative-genomic reconstruction 

symbiotic performance of Rhizobium 

of the NifA regulon evolution in 

leguminosarum var. vicieae strains 

and field pea 

alphaproteobacteria 

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16:30 - 16:50 Felix Dakora 

Tshwane University of Technology, 

South Africa 

Identifying cowpea genotypes that 

increase nitrogen contribution, 

household food security, and human 

nutrition/health in Africa 

Tuesday 29 November 2011 

Emilio Jiménez-Vicente 

Universidad Politécnica de Madrid, Spain 

The FdxN protein is required for the 

biosynthesis and activity of the NifDK 

and NifH nitrogenase components in 

Azotobacter vinelandii 

Tadashi Yokoyama 

Tokyo University of Agriculture and 

Technology, Japan 

Genetic diversity of native soybean 

and mungbean bradyrhizobia from 

different topographical regions 

along the southern slopes of the 

Himalayan Mountains in Nepal 


09:00 - 10:30 PleNARY SeSSIoN Chair: Clive Ronson, Room: Sirius 

09:00 - 09:30 Wayne reeve 

Centre for Rhizobium Studies, Murdoch 

University, Australia 

The GEBA - Root Nodule Bacteria Community Sequencing Project 

09:30 - 10:00 Michael Hynes 

Glycerol utilization by Rhizobium leguminosarum requires an ABC 

University of Calgary, Canada 

transporter and affects competition for nodulation 

10:00 - 10:30 Phil Poole 


Metabolic transitions of Rhizobium from rhizosphere to bacteroid 

10:30 - 11:00 Morning Tea 




Applications of New Technologies Bacteroids & Symbiosomes 

field Applications II 

Chair Ravi Tiwari Michael Hynes Felix Dakora 


11:00 - 11:20 Pascal Gamas 

Hauke Hennecke 

lori Phillips 

CNRS-INRA, France 

ETH, Institute of Microbiology, Swithzerland DPI-Vic, Australia 

Molecular dissection of the 

New facets of Bradyrhizobium 

Comparative diversity of 

rhizobium-legume interaction by japonicum carbon metabolism and rhizosphere and endophytic 

RNA seq and laser micro-dissection copper trafficking which support microbial communities in faba 

approaches 

bacteroid function 

cropping systems 

11:20 - 11:40 Xavier Perret 

David Day 

Kanjana chansa-ngavej 

University of Geneva, Switzerland 

Flinders University, Australia 

Chulalongkorn University, Thailand 

Profiling the symbiotic responses Identification of transport 

Selection and field trials of 

of Sinorhizobium fredii strain 

proteins on the symbiosome 

soybean rhizobium biofertilizers 

NGR234 with RNA-seq 

membrane in soybean 

with DNA fingerprints and can be 

kept at room temperature 

11:40 - 12:00 David chiasson 

Youguo li 

nicole Seymour 

The University of Adelaide, Australia 

Huazhong Agricultural University, China DEEDI, Australia 

The use of heterologous expression Identification of a novel symbiotic Nitrogen fertiliser reduces 

systems to characterise the function gene participating in synthesis of nodulation of mungbean but gives 

of the soybean transcription 

a bacteroid-specific electron carrier no yield advantage in central 

factor GmSAT1 

menaquinone 

Queensland trials 

12:00 - 12:20 Elena Dolgikh 

Vanessa Melino 

All-Russia Research Institute for 

Centre for Rhizobium Studies, 

Agricultural Microbiology (ARRIAM), Russia Murdoch University, Australia 

New application of N2-fixing organisms: Inside out: an in-depth characterisation 

heterologous expression of rhizobial of effective (Fix 

glycosylatransferases involved in 

chitin oligosaccharides synthesis 

+ ), sub-optimal 

(Fix partial ) and ineffective (Fix- ryan Farquharson 

CSIRO Land And Water, Australia 

Symbiotic performance of a 

herbicide tolerant Medicago littoralis 

) 

N2-fixation in Trifolium symbiosis 

12:20 - 13:30 Lunch – Atrium 

13:30 - 15:00 PleNARY SeSSIoN Chair: Craig Atkins, Room: Sirius 

13:30 - 14:00 Graham o’Hara 



Fifteen years of revolution in legume N-fixation in southern Australia 

14:00 - 14:30 Michael udvardi 

The Samuel Roberts Noble Foundation, USA 

Functional genomics of symbiotic nitrogen fixation in legumes 

14:30 - 15:00 clive ronson 

University of Otago, NZ 

The requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis 

15:00 - 16:00 PoSTeR SeSSIoN 1 (Even numbers) /Afternoon Tea 

11


field Applications III 


Plant Symbiotic Genes 


Native legume RNB 

Chair Ron Yates Michael Udvardi Elizabeth Watkin 


16:00 - 16:20 Elizabeth Drew 

Matthew Bellgard 

Hukam Singh Gelot 

SARDI, Australia 

CCG, Murdoch University, Australia 

Jai Narain Vyas University, India 

Impacts of pea genotype and soil Interpreting sub-optimal nitrogen Burkholderia phymatum isolated 

rhizobia on N2-fixation by field pea fixation in Lupinus angustifolius from root nodules of two species 

utilising the genome sequence of of Indigofera from alkaline soils of 

both symbiotic partners 

the Indian Thar desert 

16:20 - 16:40 Julie Grossman 

Brett Ferguson 

robert Walker 

North Carolina State University, USA 

University of Queensland, Australia 

Curtin University, Australia 

Effect of cover crop planting history Novel components involved in 

The phylogenetic diversity of 

on Rhizobium leguminosarum 

legume nodule development and Australian Burkholderia root 

ecology in organic farming systems autoregulation 

nodule bacteria 

16:40 - 17:00 amanda Bonython 

Michael Djordjevic 

Kerstin Huss-Danell 

SARDI, Australia 

Australian National University, Australia Swedish University of Agricultural Sciences, 

Improved nodulation of 

Nodule specific CLE peptides 

Sweden 

regenerating messina plants 

mediate crosstalk between local Nodulation and nitrogen fixation 

nodule development and systemic in native legumes in Scotland 

autoregulation of nodulation pathways and Sweden 

17:00 - 17:20 Keletso Mohale 

Senjuti Sinharoy 

Wen Feng chen 

Tshwane University of Technology 

Samuel Roberts Noble Foundation, USA China Agricultural University, China 

Symbiotic functioning and rhizobial Identification & functional 

Advances in rhizobial diversity 

biodiversity of Bambara groundnut characterization of Medicago truncatula and biogeography in China 

(Vigna subterranean L.Verdc) grown in transcription factor mutants with 

farmers’ field in South Africa 

impaired symbiotic nitrogen fixation 

Wednesday 30 November 2011 


09:00 - 10:30 PleNARY SeSSIoN Chair: Giles Oldroyd, Room: Sirius 

09:00 - 09:30 Janet Sprent 

Poles apart: Nodulation in native legumes from the Southern and 

University of Dundee, UK 

Northern hemispheres 

09:30 - 10:00 Erik limpens 


Formation of a symbiotic interface in rhizobial and mycorrhizal symbioses 

10:00 - 10:30 anton Hartman 

Molecular characterization of the active diazotrophic community in 

Helmholtz Zentrum Munich, Germany uninoculated and inoculated field-grown sugarcane (Saccharum sp.) 

10:30 - 11:00 Morning Tea 




ecology of RNB 

Nodule formation 

PGPR & Plant Production 

Chair Janet Sprent Jens Stougaard Anton Hartman 


11:00 - 11:20 Samson chimphango 

Shin okazaki 

Sharon Fox 

University of Cape Town, South Africa 

Nara Women’s University, Japan 

Centre for Rhizobium Studies, Murdoch 

Characterization of the cape floristic Hijacking the host nodulation 

University, Australia 

region rhizobia using 16S rRNA gene signaling by rhizobial type III 

Differential effects of Pseudomonas 

sequences and their distribution in secretion system 

fluorescens WSM3457 on enhanced 

different soil types 

Medicago nodulation 

11:20 - 11:40 Macarena Gerding 

christian Staehelin 

Khanok-on amprayn 

Universidad De Concepcion, Chile 

Sun Yat-Sen University, China 

University of Sydney Australia, Australia 

Mesorhizobium strains from the Characterization of type 3 effectors in Proteomics of rice-PGP 

South African herbaceous legume 

Lessertia spp. differ in their 

competitive ability in Australian soils 

the Rhizobium-legume symbiosis microorganisms’ interactions 

11:40 - 12:00 Julie ardley 

Kazuhiku Saeki 

Fernando Gonzales-andres 


Nara Women’s University, Japan 

University of Leon, Spain 


Mesorhizobium loti mutant lacking In vitro plant growth promotion 

The symbiosis between Listia spp. superoxide dismutase displays properties of endophitic bacteria 

and Methylobacterium and Microvirga Lotus-host accession-dependent isolated from banana (Musa sp.) 

rhizobia: specificity in epidermally 

infected legumes 

nitrogen fixation capacities 

roots in Dominican Republic 

12

Thursday 1 December 2011 


09:00 - 10:30 PleNARY SeSSIoN Chair: Ivan Kennedy, Room: Sirius 

09:00 - 09:30 robert Boddey 

Embrapa Agrobiologia, Brazil 

09:30 - 10:00 Perrin Beatty 

University of Alberta, Canada 

10:00 - 10:30 Jean-Jaques Drevon 

INRA, France 

10:30 - 11:00 Morning Tea 


Symbiotic Impacts & emissions 

Fremantle 

Legume N 2-fixation inputs preserve soil organic C stocks in no-till agriculture 

Environmental and economic impacts of biological N 2 fixing cereal crops 

Is phosphorus efficiency low for symbiotic nitrogen fixation? 


molecular characterization of 

N-fixing organisms 

The Vines Resort in the Swan Valley © Tourism Western Australia 

14 


P Nutrition & Symbioses 

Chair Robert Boddey Wayne Reeve Jean-Jaques Drevon 


11:00 - 11:20 Mark Peoples 

ana alexandre 

neera Garg 

CSIRO Plant Industry, Australia 

Universidade De Évora, Portugal 

Panjab University, India 

Effects of rhizobial strain and 

Major chaperone genes are highly Role of arbuscular mycorrhizal 

legume host on emissions of H2 induced in heat-tolerant rhizobia (AM) fungi and zinc in enhancing 

from nodules and the impact on 

nitrogen fixation in pigeon pea 

soil biology and plant growth 

under cadmium stress 

11:20 - 11:40 David Herridge 

nazalan najimudin 

andry andrianananjara 

University of New England, Australia 

Universiti Sains Malaysia, Malaysia 

LRI-SRA, Madagascar 

Nitrogen-fixing legumes in 

Characterization of nitrogen 

Genotypic variation for N2-fixation farming systems reduce greenhouse fixation genes from Paenibacillus in Voandzou (Vigna subterranea) 

gas emissions 

polymyxa ATCC 15970 

under P deficiency and P sufficiency 

11:40 - 12:00 Kiwamu Minamisawa 

Melissa corbett 

Sipho Maseko 

Tohoku University, Japan 

Curtin University, Australia 

Tshwane University of Technology, 

Mechanism of N2O emission from 

soybean nodule rhizosphere and its 

Identification and evolution of NIF 

genes in Leptospirillum species – 

South Africa 

Phosphorus nutrition in symbiotic 

mitigation 

an acidophilic chemolithoautotroph Cyclopia and Aspalathus species 

from the Cape Fynbos of South Africa 

12:00 - 12:20 Yen Thao Tran 

ni luh arpiwi 

Ghoulam cherki 

The Research Institute For Oil And Oil Plants, The University of Western Australia, 

Faculty of Sciences & Technique 

Vietnam 

Australia 

Gueliz-Marrakech, Morocco 

Change in farmer attitudes and Physiological and molecular 

Phosphorus deficiency affected 

practices in Vietnam in the use of 

inoculant compared with baseline 

characterization of the root nodule 

bacteria nodulating Millettia pinnata, 

conductance to O2 and ascorbate 

peroxydase gene expression in 

a biodiesel tree 

nodules of Phaseolus vulgarisrhizobia 

symbiosis 

12:20 - 12:40 coNfeReNce cloSING 

12:40 - 14:00 lUNch – ATRIUm 

*Committee reserves the right to alter the program as circumstances dictate

17 th International Congress on Nitrogen Fixation 


27 November – 1 December 2011 

Session Details: Monday 28 November 2011 

Plenary Session 1 

0900 - 1030 

Author: John G. Howieson 

Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology, Murdoch 

University, South Street, Murdoch 6150, Australia. 

Presentation Title: Contemporary Challenges for Symbiotic Nitrogen Fixation 

Presentation Time: 1000 - 1030 

The practical rhizobiologist now has a wide array of tools at his disposal with which to experiment. From whole genomes 

revealing multiple replicons in nodule bacteria that confer stress response, to horizontal gene transfer giving rise to new 

species of nodulating bacteria, to remote sensing from satellites that show differences in nitrogen fixation within a field, 

there are multiple opportunities for intervention. The challenge is to select the right tools with which to conduct research, and 

to seek the right questions to address. And moving outside the discipline, we must be mindful of how our work connects with 

global issues such as climate change and poverty. Life cycle analyses reveal that legume N fixation is better for the planet 

than the Haber-Bosch inspired burning of fossil fuels to create fertiliser – how do we make farmers, politicians and funders 

aware of this? The potential for domesticating new legumes that are better equipped to grow in a changing climate is high – 

but how do we educate those administrators responsible for dispensing funds for climate change adaptation of this 

potential? Along with these exigencies, we may also contemplate whether we should engineer nitrogenise to function in 

cereals, or endophytes to fix N with cereals. This presentation will explore these multiple considerations confronting today‟s 

rhizobiologists and legume scientists, to set the scene for an exciting week ahead. 

15 

2011






1100 - 1230 

Authors: Fang Xie, Giulia Morieri, Jeremy Murray, Anne L. Heckmann, Anne Edwards, Jiyoung Kim, 

Giles E. D. Oldroyd and J. Allan Downie 

John Innes Centre Norwich Research Park, Colney, Norwich, NR4 7UH, UK 

Presentation Title: Characterisation of a legume pectate lyase required for initiation and growth of infection 

threads during nodule infection by rhizobia 


It has long been recognised that nodule infection by rhizobia requires localised degradation of plant cell walls to 

enable infection threads to initiate and grow. In root hair infection foci, the cell wall is locally degraded enabling 

penetration by the bacteria, and this is coupled with the synthesis of a new infection thread wall, but relatively 

little is known about the proteins required for these processes. A microarray analysis identified Medicago 

truncatula genes more strongly induced by Nod factors from WT than a (nodL) mutant of Sinorhizobium meliloti, 

which is reduced for infection. Among the genes identified was a predicted pectate lyase. In parallel work we 

identified two allelic Lotus japonicus mutants, which are defective for infection thread growth and nodule 

infection. Fine mapping indicated that the mutation was located in a region of about 38 genes. Analysis of the 

equivalent region of M. truncatula showed that the pectate lyase gene identified from the transcriptomics was 

one of these 38 genes. Sequencing of the orthologous gene from the two L. japonicus mutants identified different 

mutations in each, and the cloned WT allele complemented the mutant phenotype. Therefore the mutations in 

this pectate lyase gene caused the infection defect. In vitro biochemical assays with the purified WT and mutant 

protein demonstrated that the gene encodes a plant pectinase and we conclude that this activity contributes to 

the cell-wall degradation that is required to initiate infection thread growth in root hairs. It also appears to be 

required for subsequent extension of the infection thread across cell-cell junctions. The pectate lyase gene is 

induced following Nod-factor activation of the common symbiosis pathway and requires the transcriptional 

regulators NIN and NSP1 for its normal induction. Chromosome immunoprecipitation and promoter binding 

experiments indicated that the pectate lyase gene is directly regulated by NIN. These results show that there is 

specific Nod-factor induction of genes that play a role in initiating root infection by rhizobia. 

16 

2011






1100 - 1230 

Authors: Luis M. Rubio, Emilio Jiménez-Vicente, César Poza-Carrión, Carlos Echavarri-Erasun, 

Alessandro Scandurra & Elena Moreno-Urbano 

Universidad Politécnica de Madrid. Centro de Biotecnología y Genómica de Plantas 

Pozuelo de Alarcón, 28223 Madrid (Spain). 

Presentation Title: Changes in nif gene expression profiles during nitrogenase biogenesis 

Presentation Time: 1130-1200 

A number of nitrogen fixation (nif) genes (10-20 depending on the organism) are involved in the assembly of a 

functional nitrogenase enzyme. Changes in the levels of nif gene products over time are required to obtain 

appropriate balances during nitrogenase biogenesis. In the model organism Azotobacter vinelandii nif gene 

expression exhibits exquisite regulation at least at the transcriptional and post-translational levels. 

We report here the changes over time of the cellular levels of nif mRNAs and Nif proteins with key functions in 

nitrogenase biosynthesis. Expression of nifH, nifD, nifK, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifA, nifB, FdxN, 

nifQ, and nafY genes was quantified by using real-time quantitative PCR. In addition, cellular accumulation of 

each one of the corresponding Nif proteins was determined by immunoblot analysis and quantified against 

standards obtained with purified protein preparations. The results will be analyzed in the context of observed in 

vivo nitrogenase activitiy development over time. The implications to constructing nitrogenase enzymes through 

synthetic biology techniques will be discussed. 

17 

2011






1100 - 1230 

Author: Giles Oldroyd 

John Innes Centre Norwich Research Park, Colney, Norwich, NR4 7UH, UK 

Presentation Title: Establishing beneficial interactions with the symbiosis signalling pathway 


The establishment of rhizobial and mycorrhizal symbioses requires the common symbiotic signalling pathway 

that utilises oscillations in calcium as a secondary messenger. This signalling pathway is differentially activated 

by rhizobial bacteria and mycorrhizal fungi and this is expressed as differential calcium oscillations. Specificity 

must be maintained in this common signalling pathway since alternate developmental processes need to be 

activated in order to accommodate rhizobial bacteria or mycorrhizal fungi. The calcium oscillations are decoded 

by a calcium and calmodulin dependent protein kinase (CCaMK). This protein is unique among calcium activated 

kinases in having the capability to respond independently to both free calcium and to calcium complexed with 

calmodulin. This dual modality of calcium activation appears to allow exquisite perception of calcium 

concentrations that is likely involved in the differential activation of CCaMK by the two symbionts. Lying 

downstream of CCaMK are a suite of transcription factors, with both rhizobial and mycorrhizal specific 

transcription factors. It appears that specificity in this pathway is encoded by the differential activation of these 

symbiosis specific transcription factors, that in turn coordinate the different processes necessary for these two 

symbioses. 

18 

2011






1330 - 1500 

Author: Ken Giller 

Plant Production Systems, Wageningen University, PO Box 430, 6700AT Wageningen, 

The Netherlands 

Presentation Title: N2Africa – Putting Nitrogen Fixation to work for smallholder farmers in Africa 


Maximal rates of N2-fixation recorded in the tropics reach an astonishing 5 kg N ha -1 day -1 . We have measured 

more than 250 kg N ha -1 of fixed N2 in soyabean in southern Africa with associated grain yields of more than 4 t 

ha -1 . But often less than 5 kg N ha -1 year -1 is fixed by legumes at farm scale in African smallholder systems. 

Increase of inputs from nitrogen fixation is required to achieve the increases in productivity required as part of the 

African green revolution that is gaining momentum. 

Successful N2-fixation by legumes in the field depends on the interaction: 

(GL � GR) � E � M 

that is (legume genotype � rhizobium genotype) � environment � management. Environment encompasses 

climate (temperature, rainfall, daylength etc) and soil stresses (acidity, aluminium toxicity, limiting nutrients etc). 

Management includes aspects of agronomic management (use of mineral fertilizers, sowing dates, plant density, 

weeding). Although much research is focused on identifying best combinations of GL and GR, the E and M factors 

often override the potential of the legume/rhizobium symbiosis for N2-fixation. Attention will be focused on 

identifying new socioecological niches for fitting grain, forage and tree legumes into existing farming systems, 

and the conditions necessary to achieve successful N2-fixation. 

The N2Africa project aims to increase inputs from N2-fixation on more than 225,000 smallholder farms across 

eight African countries within four years through: a) Increasing the area of land cropped with legumes; b) 

Increasing legume productivity through better agronomy and basal (P, K etc) fertilizer; c) Selecting and 

disseminating legume varieties with increased N2-fixation; d) Selecting better rhizobium strains and promoting 

high quality inoculants; e) Linking farmers to markets and creating new enterprises to increase demand for 

legumes. N2Africa has already reached more than 25,000 farmers and conducted more than 1,000 inoculation 

trials. The latest learnings will be discussed. 

19 

2011





Author: 


1330 - 1500 


Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, 

Aarhus University, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. 

Presentation Title: Nod factor perception, signal transduction and the role of LysM type receptors 


Formation of root nodules in legumes relies on a genetic program controlling and synchronising two processes 

running in parallel. Nodule primordia are formed from root cortical cells initiating cell divisions and simultaneously 

a bacterial infection process targets the primordia developing from the cell division foci. Plant receptors involved 

in perception of bacterial signal molecules are important components of these pathways and they are also 

involved in determining host specificity. The role of Lotus japonicus LysM type serine/threonine receptor kinases 

in perception of Nod-factor signals from bacterial microsymbionts during nodule initiation and nodule 

maintenance will be discussed. The extracellular domains of the trans-membrane kinases carry LysM domains 

suggesting that they are involved in perception of the rhizobial lipochitin-oligosaccharide signals and in 

deciphering the structure of lipochitin-oligosaccharides. Experiments and studies addressing these questions will 

be presented and the involvement of receptor kinases in the early physiological and cellular responses as well as 

later during nodule development will be illustrated. In addition to the NFR1 and NFR5 receptors, the Lotus 

japonicus genome encodes fifteen hitherto uncharacterised LysM type receptor kinases. The functional role of 

these receptors is currently investigated using a number or approaches including TILLING mutants and 

expression studies. 

20 

2011






1330 - 1500 

Author: Kristina Lindström 

University of Helsinki, Finland 

Presentation Title: Rhizobium taxonomy and diversity – Is there anything more to learn? 


The taxonomy and diversity of rhizobia have during the last few years deserved ample attention, and more new 

species than ever of legume-nodulating bacteria were described. 

With our collaborators we have recently studied collections of rhizobia from Galega orientalis and G. officinalis 

from the Caucasus, of bacteria from Chinese medicinal Glycyrrhiza species, and of bacteria isolated from 

Ethiopian Phaseolus beans. We also investigated diversity and evolutionary patterns in plant accessions of 

Caucasian Galega species and sequenced the genomes of two R. galegae strains. 

The phylogenetic tree of rhizobia and agrobacteria displays patterns of related species, “species compexes”. By 

an MLSA approach we try to sort out the “R. galegae species complex” in relation to genus Agrobacterium and 

other Rhizobium species. According to MLSA, the Ethiopian bean isolates mainly belong to the “R. 

leguminosarum species complex”, and represent R, leguminosarum, R. phaseoli and R. etli. In our study R. 

fabae and R. pisi seem to be one species, whereas current R. leguminosarum might be two. A sister clade to R. 

etli of Ethiopian strains might represent a new species, but do we want to be “splitters” or “lumpers”? 

Current criteria for the description of new species focus on doing a plethora of tests with little relevance to 

biology, evolution and symbiosis. Resources should be directed towards answering interesting questions in those 

fields instead. Our studies of the Glycyrrhiza isolates showed that the bacteria were taxonomically diverse but 

that symbiotically could be divided into true symbionts, sporadic symbionts and other endophytes. The true 

symbionts were Mesorhizobium species. 

Systematic sampling, biogeographical analyses, including aspects of symbiosis (host plants, effectiveness, Nod 

factors) in combination with molecular diversity and phylogenetic studies still hold promise and are likely to yield 

important information for biology and agronomy. 

21 

2011





Author: 

Concurrent Session 1 – Field Applications 1 

1530 - 1650 

Neil Ballard 

Global Pasture Consultants, Australia 

Presentation Title: Application of Western Australian legume and Rhizobium technologies in the developing 

world – a practitioner‟s perspective 

Presentation Time: 

1530 - 1550 

Western Australia has been at the leading edge of developing alternative pasture legumes and their rhizobial 

inoculants for infertile soils since the early 1990s. Using knowledge gained from applying these technologies to 

farming in WA on projects overseas has given me an understanding of the differences in agriculture in other 

countries, as well as the opportunities. It has also enabled me to help to show the farmers in those countries how 

the WA experience can help them to be more productive and more efficient. 

The widespread adoption of no till farming since the 1970‟s in WA is an object lesson for many other low rainfall 

countries to grow crops and pastures with minimum moisture loss. When this is coupled with the use of a wide 

range of pasture species and an understanding of the importance of developing hard seed banks, it opens up a 

completely new range of options for these farmers and communities, to produce the quantity and quality of 

forage they have never seen before. The Centre for Rhizobium Studies at Murdoch University at Murdoch 

University in Western Australia has been able to develop elite strains of Rhizobia to suit the range of forage 

species we have used in the overseas experience, and these have been critical to success. 

My presentation will cover application of these new technologies which have the potential to greatly increase 

productivity on marginal soils in developing countries. The role of sociology in implementing these technologies 

will be emphasised, beginning with the ACIAR funded project ECCAL in the eastern Cape of South Africa. 

22 

2011






1530 - 1650 

Authors: Malik R, Seymour M 

Presentation Title: 


Department Of Agriculture & Food Western Australia, 

Impact of break crops on wheat productivity in Western 

Australian cropping system 

Cropping systems in Western Australia have become more intensive over the past 20 years – meaning there is 

more cereal after cereal, less lupin and pastures, but more canola. Each year farmers decide what crop to grow 

based on previous history and experience and market outlooks. So, what farmers often actually want to know is 

“what is the best crop to sow after the last one or two?” – I.e. what is the best crop sequence? Dynamic Crop 

Sequence trial at Katanning WA approaches this question, amongst others, in a different way. The trial is 

consisted of a range of cropping component options being grown in long plots in one direction in the first year 

(2008), and at right angles in the second year (2009) crossing all the first year plots. This allows the comparison 

of all possible two-year combinations (total 100) of those components. In the third year (2010) wheat was grown 

across the entire site. The results suggested that both 2008 and 2009 treatments had significant effects on third 

year wheat yield. The 2008 treatments leading to highest wheat yields were canola, fallow, field pea, lupin, and 

oaten hay; and the 2009 treatments leading to highest wheat yields were fallow, lupin and oaten hay. Not 

surprisingly, the second year effect of the 2008 treatments on 2010 wheat yield were smaller than the first year 

effects of the 2009 treatments. The only 2009 break crop that increased 2010 wheat yield significantly above 

wheat/wheat was lupin (wheat yield after lupin was 27% higher). This research can help identify crop sequence 

„synergisms‟ and „antagonisms‟, thereby providing the necessary foundation for developing strategies to 

sequence crops over a longer period of time as a long-term strategy of annual crop sequencing to attain 

production, economic, and resource conservation goals by using sound management practices. 

23 

2011






1530 - 1650 

Authors: Felipe A. Burgos 1 , Ron Yates 1,3 , Graham O‟Hara 1 , Halina Kobryn 2 & John Howieson 1 

1 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia. 

2 School of Environmental Science, Murdoch University, Murdoch, 6150, Western Australia. 

3 Department of Agriculture and Food of Western Australia 

Presentation Title: Use of remote sensing to assess the symbiotic performance of Rhizobium leguminosarum 

var. vicieae strains and field pea. 


The symbiotic performance between legumes and rhizobia relies on the plant-bacteria genetic compatibility and 

on the symbiotic partner‟s capacity to overcome environmental stresses. Symbiosis contributes nitrogen to the 

plants, which, among other things, increases the number of chloroplasts, and the number and size of cells per 

leaf. Hyperspectral imagery can detect vegetation changes combining information stored in the image. The 

symbiotic performance is affected by some abiotic stress factors such as low clay content and low soil water 

holding capacity. These soil features can be estimated using ground penetrating radar (GPR), a geophysics 

instrument based on energy pulses interacting with soil layers. The aim of this work was to investigate whether 

integrated remote sensing techniques are able to estimate the interaction of field pea inoculated separately with 

five strains of Rhizobium leguminosarum bv. viceae with different nitrogen fixation effectiveness levels. The 

experiment was carried out firstly in a glasshouse to assess the pure symbiotic performance and then in an 

agricultural area to assess the interaction with abiotic factors. Hyperspectral images and GPR measurements 

were captured to cover the glasshouse and field site experiments. The plant sample analyses consisted of plant 

dry weight, nitrogen content and nodulation score. 

The plant samples showed significant differences in nitrogen levels, nodule score and dry weight across strains. 

The analyses of the spectral band combinations confirmed the presence of outstanding indices sensitive to the 

differential symbiotic performance and were correlated with plant analyses. The GPR data also revealed a mixed 

composition of soil properties associated with variable water availability that affected root and plant growth. It is 

concluded that remote sensing can be a valuable tool for estimating legume nitrogen fixation in fields, and GPR 

for estimating below ground properties that affect plant growth in field experiments. 

24 

2011






1530 - 1650 

Author: Felix Dakora 

Tshwane University of Technology, South Africa 

Presentation Title: Identifying cowpea genotypes that increase nitrogen contribution, household food security, 

and human nutrition/health in Africa 


Cowpea is the most important food legume in Africa. In this study, over 30 cowpea genotypes have been 

evaluated for symbiotic N contribution, grain yield, mineral nutrient density and protein level in edible leaves and 

grain. The data revealed marked differences in both symbiotic contribution and nutritional attributes among the 

cowpea varieties. In all instances, plant growth (measured as biomass), grain yield and protein content, as well 

as trace element and macronutrient density were dictated by the efficacy of the species symbiosis. Thus, cowpea 

genotypes that were nodulated by high N2-fixing strains, fixed more N, grew better, and showed greater levels of 

dietary protein, macronutrients, and trace elements in leaves and grain than their counterparts nodulated by low 

N2-fixing bacterial strains. In cowpea, there is therefore potential to identify host/strain combinations that increase 

N contribution and grain yield in cropping systems, yet also accumulate high levels of protein and dietary 

nutrients for human nutrition and health. There is therefore a need to integrate these attributes into breeding 

programs in order to identify cowpea genotypes/varieties that improve N contribution and human nutrition/health. 

25 

2011





Concurrent Session 2 – Function & Control of Nitrogenase 

1530 - 1650 

Authors: Lifen Yan 1 , Christie H. Dapper 2 , Simon J. George 1 , Hongxin Wang 1 , Devrani Mitra 1 , 

Weibing Dong 1 , Stephen P. Cramer 1 & William E. Newton 2 

1 Department of Applied Science, University of California-Davis, Davis, CA 95616, USA. 

2 Department of Biochemistry, Virginia Polytechnic Institute & State University, Blacksburg, 

VA 24061, USA 

Presentation Title: Carbon monoxide adducts of Azotobacter vinelandii Mo-nitrogenase 


Carbon monoxide (CO) is a non-competitive reversible inhibitor of all wild-type Mo-nitrogenase-catalyzed 

reactions except the reduction of protons to H2. An understanding of the CO interaction should give substantial 

insight into mechanism. Several CO-bound species are known; some reversibly interconvert. Which one is 

formed depends on the CO pressure (pCO) over Mo-nitrogenase during turnover. At pCO < 0.1 atm, “lo-CO” is 

formed, whereas under higher pCO, either “hi-CO” or “hi(5)-CO” or both are formed. Electron nuclear double 

resonance (ENDOR) spectroscopy suggests that: “lo-CO” has one bound CO bridging two Fe atoms; “hi-CO” has 

two CO ligands, each terminally bound to a different Fe atom; and “hi(5)-CO” has two bridging CO ligands. Both 

stopped-flow infrared and theoretical studies, however, appear inconsistent with these structures. To help 

resolve ambiguities, we investigated these species using photolysis under cryogenic conditions with Fourier 

transform-infrared (FT-IR) detection. Photolysis of “hi-CO” indicates loss of terminally bound (1973 cm -1 ) and 

bridging (1679 cm -1 ) CO molecules, with concomitant formation of a species with one bridging (1711 cm -1 ) CO 

molecule. These assignments were confirmed by using isotopically labeled CO. Our results are therefore only 

partly consistent with the ENDOR assignments. We extended these studies to two altered Mo-nitrogenases, 

both with a substitution at α-histidine-195 in the MoFe protein, namely α-H195Q and α-H195N. Surprisingly, 

although we expected a similar band at ca. 1973 cm -1 on photolysis of α-H195N, we saw a band at lower energy 

(1936 cm -1 ). More surprisingly, photolysis of α-H195Q showed both CO-related bands, at 1969 and 1932 cm -1 . 

Structures will be proposed for these species and the results will be discussed in terms of both CO inhibition and 

the catalytic mechanism. 

26 

2011






1530 - 1650 

Authors: He Wang, Pedro Filipe Teixeira, Tiago T. Selão, Agneta Norén, Catrine L. Berthold, Martin 

Högbom, Stefan Nordlund 

Department of Biochemistry and Biophysics, Stockholm University, SE-10691, Sweden. 

Presentation Title: Regulation of Nitrogenase activity in Rhodospirillum rubrum – an interplay of PII proteins, 

DRAT, DRAG and membrane sequestration 


In the Rhodospirillum rubrum and some other phototrophic bacteria, nitrogenase activity is regulated at the 

metabolic level in response to changes in nitrogen availability or in energy supply, i.e. light/darkness. This 

regulation is also present in some species of Azospirillum, although in that case the energy supply is reflected as 

the concentration of oxygen. At the molecular level this regulation is due to reversible ADP-ribosylation of the Feprotein. 

DRAT catalyzes the addition of an ADP-ribose from NAD + and DRAG catalyzes the removal of ADPribose 

moiety, thereby restoring activity. One of the major questions has been the identity of the signal(s) in this 

system and some years ago we proposed that the association of DRAG to the chromatophore membrane plays a 

central role in the regulatory mechanism. Since then we and a number of other groups have provided evidence 

supporting this model and also demonstrated the involvement of AmtB1 and PII proteins in this regulation. 

There are three PII paralogs in R. rubrum, GlnB, GlnJ and GlnK. GlnB and GlnJ have both been shown to have 

specific functions in the regulation of nitrogen metabolism, whereas no specific function has yet been identifíed 

for GlnK. 

We have now furthered our studies on the role of the interaction of DRAG with a partner in the chromatophore 

membrane in the regulation of nitrogenase activity and the involvement of PII proteins in this interaction as well 

as the regulation of DRAT. We have also demonstrated the influence of different nitrogen sources during growth, 

N2 or glutamate, on the pathway leading to regulation of nitrogenase activity, both in ammonium and energy 

“switch-off”. Based on our studies we propose a model explaining the communication within the cell leading to a 

concerted regulation of nitrogen assimilation and nitrogen fixation. 

27 

2011






1530 - 1650 

Authors: Svetlana N. Yurgel 1 , Jennifer Rice 1 , and Michael L. Kahn 1,2 

1 Institute of Biological Chemistry,Washington State University, Pullman, WA. 99164. 

2 School of Molecular Biosciences,Washington State University, Pullman, WA. 99164. 

Presentation Title: GlnD and symbiosis: Control beyond the nitrogen stress response? 


To establish an effective symbiosis with alfalfa, S. meliloti Rm1021 needs normal operation of the GlnD protein, a 

bifunctional uridylyltransferase/uridylyl-cleavage enzyme that measures cellular nitrogen status and initiates a 

nitrogen stress response (NSR) (Yurgel & Kahn 2008). Both of the two known targets of GlnD modification, the 

PII proteins GlnB and GlnK, are not needed for effectiveness (Yurgel et al., 2010). However, the glnD-sm2 

mutation that leads to a Fix+Eff¯ phenotype is defective in uridylylation and cells are thought to only have 

unmodified PII proteins. To generate GlnB in the state expected in a glnD-sm2 mutant, we introduced a 

Tyr�Phe variant of GlnB that cannot be uridylylated into a glnBglnK background and found that this strain was 

Fix+Eff+. We also generated a glnBglnKglnD triple mutant and used this and other mutants to dissect the role of 

these proteins in regulating the free-living NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation 

was dominant to the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes. The data 

show that the GlnD protein has a role in free-living growth and in symbiotic nitrogen exchange that does not 

depend on the PII proteins, suggesting that S. meliloti GlnD can communicate information to the cell by alternate 

mechanisms. 

Yurgel SN & Kahn ML (2008). A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective 

Sinorhizobium meliloti symbiosis with alfalfa. Proc Natl Acad Sci U S A 105(48):18958-18963. 

Yurgel SN, Rice J, Mulder M & Kahn ML (2010). GlnB/GlnK PII proteins and regulation of the Sinorhizobium 

meliloti Rm1021 nitrogen stress response and symbiotic function. J Bacteriol 192:2473-2481. 

28 

2011






1530 - 1650 

Authors: Emilio Jiménez-Vicente, César Poza-Carrión & Luis M Rubio 

Centre for Plant Biotechnology and Genomics U.P.M. – I.N.I.A. Parque Científico y 

Tecnológico de la U.P.M. Campus de Montegancedo 28223 Pozuelo de Alarcón 

(Madrid) 

Presentation Title: The FdxN protein is required for the biosynthesis and activity of the NifDK and NifH 

nitrogenase components in Azotobacter vinelandii 


The molybdenum nitrogenase enzyme is composed of two [Fe-S] cluster containing proteins designated as 

dinitrogenase (NifDK) and dinitrogenase reductase (NifH). The biosynthesis of nitrogenase [Fe-S] clusters starts 

by the concerted activity of the NifS cysteine desulfurase and the NifU scaffolding protein. It is expected that the 

processes of [Fe4-S4] cluster formation on NifU and/or their transfer to apo-NifH and apo-NifDK require an 

electron donor. The FdxN ferredoxin is a likely candidate to perform such a role in A. vinelandii. This study shows 

the effects of deleting fdxN on NifH and NifDK. 

The fdxN mutant exhibited much lower diazotrophic growth rate and in vivo nitrogenase activity than the wild 

type. In vitro activ fdxN strain was defective in the activity of both component proteins, 

fdxN NifDK protein exhibited 20% acetylene reduction activity and had lower Fe 

content than wild- fdxN NifDK could not be reconstituted in vitro by FeMo-co suggesting 

that it is defective in [Fe4-S4] cluster incorporation or P-cluster synthesis. 

Time-course analysis of nifH, nifD and nifK fdxN strain 

had a profile of delayed nifHDK expression and lower nifD and nifK expression rates than wild type. Consistently, 

NifDK protein levels were lower in the strain. On the other hand, the strain accumulated higher levels 

of the FeMo-co biosynthetic proteins NifEN and NifB, a phenotype previously observed in mutants with defects in 

FeMo-co synthesis. 

In summary, fdxN deletion had a profound negative effect on NifDK expression, maturation and enzymatic 

activity, and a noticeable effect on cellular NifH activity level. The fact that fdxN deletion affects both NifH and 

NifDK suggests that FdxN could be involved in the synthesis or insertion of [Fe-S] clusters for both nitrogenase 

component proteins. 

29 

2011





Concurrent Session 3 – Taxonomy & Evolution 

1530 - 1650 

Authors: Nikolay Provorov & Nikolay Vorobyov 

All-Russia Research Institute for Agricultural Microbiology, Podbelsky Sh., 3, Pushkin, 

196608, St.-Petersburg, Russia 

Presentation Title: Mathematical simulation of evolutionary events in the N2-fixing plant-microbe symbioses 


A series of mathematical models is constructed for simulating the evolution of N2-fixing nodular symbioses 

towards their improved ecological efficiency (Provorov & Vorobyov 2010a 2010b). By comparing the contrast 

forms of legume-rhizobia symbioses formed by plants from different phylogenetic groups of the Fabaceae we for 

the first time differentiate the modes and estimate the pressures of individual and group natural selection which 

supports the transitions from: (i) mixed to clonal infections of hosts by rhizobia; (ii) extra-cellular maintenance of 

rhizobia (inside infection/fixation threads) to their intra-cellular maintenance inside symbiosomes; (iii) 

unspecialized (multi-bacterial) symbiosomes to the specialized (mono-bacterial) symbiosomes in which the 

rhizobia are transformed into non-reproducible bacteroids. We also quantified the impacts of genotypic specificity 

of partners‟ beneficial interaction on the symbiosis evolution: the host-specific microsymbionts obtain a more 

pronounced selective support and remain stably mutualistic during a prolonged macroevolutionary process while 

the non-host-specific microsymbionts may be transformed readily into the hosts‟ antagonists. Therefore, nodular 

symbioses provide us the unique models to study the population-genetic background for progressive evolution 

which is poorly investigated in the free-living unitary organisms. The developed simulation techniques may be 

used for assessing the evolutionary processes in agriculturally important symbioses induced by: (i) introductions 

of cultivated legumes into the novel cropping areas; (ii) release of the genetically modified rhizobia strains into 

sustainable agricultural systems. Supported by RFBR grant 09-04-00907a. 

Provorov NA & Vorobyov NI (2010a). Evolutionary Genetics of Plant-Microbe Symbioses. Ed I Tikhonovich 

NOVA Sci Publ NY 290 p. 

Provorov NA & Vorobyov NI (2010b). Simulation of evolution implemented in the mutualistic symbioses towards 

enhancing their ecological efficiency, functional integrity and genotypic specificity. Theor Popul Biol 78: 259-269. 

30 

2011






1530 - 1650 

Authors: Rik op den Camp, Maryam Seifi Kalhor, Arend Streng, Qingqin Cao, Elena Federova, Elisa 

Polone, Ton Bisseling & René Geurts 


Presentation Title: Parasponia to unravel genetic constraints underlying Rhizobium symbiosis 


Ever since the discovery of Parasponia as the first, and till now only, non-legume species able to establish the 

nitrogen-fixing nodule symbiosis with rhizobial bacteria it has been clear that these „bridging‟ species will provide 

insight in the evolution of root nodule symbiosis. The Parasponia-rhizobium symbiosis evolved independently 

from legumes, but like in legumes, the interaction is set in motion by rhizobium lipo-chitooligosaccharides (LCOs) 

named Nod factors. We provide several lines of evidence that the Parasponia-rhizobium symbiosis has occurred 

rather recent. Subsequently, the symbiosis can be considered as primitive. Parasponia lacks mechanisms to 

select effective nitrogen fixing rhizobial symbionts. Also it is unable to repress nodulation when grown under 

relatively high external nitrogen concentrations. We used Parasponia to identify genetic constrains underlying 

evolution of the rhizobium symbiosis and provide evidence that it has recruited essential elements from the 

endomycorrhizal symbiosis. 

31 

2011






1530 - 1650 

Authors: Jelena Jalovaja 1 , Olga Tsoy 2,3 , Mikhail Gelfand 2,3 

1Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, 141700, 

Russia 

2 Institute for Information Transmission Problems RAS, Moscow, 127994,Russia 

3 Lomonosov Moscow State University, Department of Bioengineering and Bioinformatics, 

Moscow, 119992, Russia 

Presentation Title: Comparative-genomic reconstruction of the NifA regulon evolution in alphaproteobacteria 


Nitrogen fixation is a complex redox process performed by a variety of bacteria. Since it requires a large amount 

of energy, and its main enzyme, nitrogenase, may be easily destroyed by oxygen, this process is tightly 

controlled at all levels, including the level of transcription. This study focuses on NifA, a major regulator of genes 

associated with nitrogen fixation. The NifA regulon has already been described for B.japonicum and R.etli [1, 2], 

but availability of complete genome sequences of other nitrogen fixing bacteria allows one to apply comparative 

genomics methods for accurate regulatory annotation. Analysis of 22 alpha-proteobacteria confirmed and further 

specified the NifA-binding motif TGT-N10-ACA. An evolutionary scenario of operon rearrangement and horizontal 

gene transfer was reconstructed. The conserved regulon core contains: 

� nifHDK, coding nitrogenase components, 

� iscNnifUS operon, coding nitrogenase FeS cluster assembly, 

� nifBfdxN coding Fe-Mo cofactor biosynthesis proteins, 

� fixABCX electron carrier 

Interestingly, NifA-RpoN sites were identified upstream of “broken” operon parts. The autoregulation of nifA in the 

Rhizobiales was confirmed, within operons fixABCX-nifA, fixR-nifA or ahpCD-nifA specific for individual taxa. 

New candidate regulon members are typically organism-specific and include molybdenum transporter genes, 

rpoN and nodulation genes. 

1. Salazar E, Díaz-Mejía JJ, Moreno-Hagelsieb G, et al. Characterization of the NifA-RpoN Regulon in 

Rhizobium etli in Free Life and in Symbiosis with Phaseolus vulgaris. Applied and environmental 

microbiology. 2010; 76(13):4510-4520. 

2. Hauser F, Pessi G, Friberg M, et al. Dissection of the Bradyrhizobium japonicum NifA+sigma54 regulon, 

and identification of a ferredoxin gene (fdxN) for symbiotic nitrogen fixation. Molecular genetics and 

genomics : MGG. 2007; 278(3):255-71. 

32 

2011






1530 - 1650 

Authors: Tadashi Yokoyama 1 , Chandra Prasad Risal 2 , Naoko Ohkama-Ohtsu 1 , Salem Djedidi 3 & 

Hitoshi Sekimoto 4 

1 Institute of Agriculture, Tokyo Univ. of Agri. and Tech., Saiwai-cho 3-5-8, Fuchu, Tokyo 

183-8509, Japan 

2 Soil Management Directorate, Dept. of Agriculture, Hariharbhawan 552-0314, Nepal 

3 Faculty of Agriculture, Tokyo Univ. of Agri. and Tech., Tokyo 183-8509, Japan 

4 Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan 

Presentation Title: Genetic diversity of native soybean and mungbean bradyrhizobia from different 

topographical regions along the southern slopes of the Himalayan Mountains in Nepal 


Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, 

the genetic diversity of native soybean and mungbean bradyrhizobia isolated from different topographical regions 

along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from 

three different topographical regions with contrasting climates. Local Glycine max cv. Cobb and Vigna radiata cv. 

Kalyan were used as trap plants to isolate bradyrhizobia. All the Nepalese soybean and mungbean isolates 

characterized in this study were slow growers and were assigned to the Bradyrhizobium genus based on their 

molecular characterization. We found that 46% of the isolates from soybean analyzed in the present study were 

phylogenetically related to B. elkanii, 21% were related to B. japonicum, and 8% were related to B. 

yuanmingense. Phylogenetic analyses also revealed three novel lineages comprising 25% of the analyzed 

population. Similarly, we found 50% of the isolates from the mungbean were phylogenetically related to B. 

yuanmingense, 13% were related to B. japonicum, 8% were related to B. elkanii, and 29% were found with novel 

phylogenetic origin. Furthermore, we found that most mungbean rhizobial genotypes were conserved across 

agro-ecological regions of Nepal. All the strains from tropical Terai region belonged to B. yuanmingense or a 

novel lineage of B. yuanmingense, and dominance of B. japonicum related strains observed in the Hill region. 

Our results indicate that there is higher genetic diversity of Bradyrhizobium strains in the temperate and subtropical 

region than in the tropical region. Though nifD genes are considered as high homology genes, 

considerable nifD gene sequence divergence of Nepalese strains with known reference strains were noticed. 

We, for the first time, analyzed nifD genes from B. yuanmingense in this study. We found nifD genes in B. 

yuanmingense isolated from soybean and mungbean are far different from other Bradyrhizobium species. 

33 

2011




Session Details: Tuesday 29 November 2011 


0900 - 1030 

Authors: Wayne G. Reeve 1 and Nikos Kyrpidis 2 

1 Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology, 

Murdoch University, South Street, Murdoch 6150, Australia. 

2 DOE Joint Genome Institute , Walnut Creek, CA, USA 

Presentation Title: The GEBA - Root Nodule Bacteria Community Sequencing Project 


Genome sequencing has revolutionized understanding of microorganisms and the roles they play in important 

processes such as pathogenesis, energy production, bioremediation, global nutrient cycles, and the origins, 

evolution, and diversity of life. However, there are significant gaps in microbial genome sequence availability 

especially associated with environmental genomics. The currently available microbe genome sequences show a 

highly biased phylogenetic distribution compared to the extent of microbial diversity known today. This bias has 

resulted in a major gap in our knowledge of microbial genome complexity and our understanding of the evolution, 

physiology, and metabolic capacity of microbes. There is a strong need for a large-scale systematic effort to 

sequence genomes to fill in genomic gaps in the tree of life. To this end the Genomic Encyclopedia of Bacteria 

and Archaea – Root Nodule Bacteria (GEBA-RNB) initiative at the Joint Genome Institute aims to sequence 100 

RNB strains of commercial, genetic and ecological importance. This project will support the systematic 

sequence-based studies and understanding of the biogeographical effects on species evolution as well as the 

mechanisms of symbiotic nitrogen fixation by RNB. Shared genetic mechanisms between fungal and bacterial 

root endosymbioses exist and a detailed understanding of endosymbionts will be beneficial to drive bioenergy 

development from trees. The GEBA-RNB project is based on collaboration between JGI and an international 

consortium from 15 countries coordinated by the Centre for Rhizobium Studies at Murdoch University. 

34 

2011






0900 - 1030 

Authors: Hao Ding 1 , Cynthia Yip 1 , Barney A. Geddes 2 , Ivan J. Oresnik 2 , and Michael F. Hynes 1 

1 University of Calgary, Biological Sciences, Calgary AB, Canada T2N 1N4 

2 University of Manitoba, Microbiology, Winnipeg MB, Canada R3T 2N2 

Presentation Title: Glycerol utilization by Rhizobium leguminosarum requires an ABC transporter and affects 

competition for nodulation 


Plasmid curing has shown that the ability to use glycerol as a carbon source is plasmid-encoded in Rhizobium 

leguminosarum (Baldani et al. 1992, Yost et al. 2006). We isolated the locus responsible for glycerol utilization 

from plasmid pRleVF39c in R. leguminosarum bv. viciae VF39. This region was analyzed by DNA sequencing 

and mutagenesis. The locus encompasses a gene encoding GlpR (a DeoR regulator), genes encoding an ABC 

transporter, and genes glpK and glpD, encoding a kinase and dehydrogenase. All the genes except the 

regulatory gene glpR were organized into a single operon, and were required for growth on glycerol. The glp 

operon was strongly induced by both glycerol and glycerol-3-phosphate, as well as by pea seed exudate. GlpR 

repressed the operon in the absence of inducer. Mutation of genes coding for the ABC transporter abolished all 

transport of glycerol in transport assays using radiolabelled glycerol. This confirms that, unlike in other 

organisms like Escherichia coli and Pseudomonas aeruginosa, which use facilitated diffusion, glycerol uptake 

occurs by an active process in R. leguminosarum. Since the glp locus is highly conserved in all sequenced R. 

leguminosarum and R. etli strains, as well as in Sinorhizobium spp. and Agrobacterium spp. and other 

alphaproteobacteria, this process for glycerol uptake is probably widespread. Mutants unable to use glycerol 

were deficient in competitiveness for nodulation of peas compared to wild-type, indicating that glycerol 

catabolism confers an advantage to the bacterium in the rhizosphere or in the infection thread. 

Baldani JI, Weaver R, Hynes MF, & Eardly B (1992) Utilization of carbon substrates, electrophoretic enzyme 

patterns, and symbiotic performance of clover rhizobia cured of plasmids. Appl Environ Microbiol 58:2308-2314 

Yost CK, Rath AM, Noel TC, & Hynes MF (2006) Characterization of genes involved in erythritol catabolism in 

Rhizobium leguminosarum bv. viciae. . Microbiology 152:2061-2074 

35 

2011






0900 - 1030 

Authors: Philip Poole, Graham Hood, Alison East, Ramakrishnan Karunakaran, Vinoy 

Ramachandran, Jurgen Prell 

John Innes Centre, Norwich UK 

Presentation Title: Metabolic transitions of Rhizobium from rhizosphere to bacteroid 


Comparative transcriptomics has been used to examine the changes when Rhizobium leguminosarum grows in 

the pea, alfalfa or sugar beet rhizospheres. This enabled the identification of genes that are expressed generally 

in the rhizosphere, as well as those that are legume (pea or alfalfa) or host (pea) specific. Carbon metabolism by 

bacteria in the rhizosphere all plants is dominated by organic acids with particularly strong induction of C1 and 

C2 metabolism. Gluconeogenesis is induced, indicating that while sugars are present, they are only minor 

carbon sources relative to the organic acids. Aromatic amino acids such as phenylalanine also appear to be 

particularly important. As expected nod genes were induced in the pea and alfalfa rhizosphere but not in the 

rhizosphere of sugar beet. Next we examined gene expression in early (7 d nodules) and mature bacteroids (15- 

28 d nodules). This enabled gene expression to be partitioned into developmental stages from the rhizosphere, 

probably infection thread/early development and mature bacteroids. Genes induced in infection threads and early 

development include; metal accumulation, oxidative stress and export systems for probable plant metabolites, 

GABA catabolism and a new terminal oxidase. To obtain a clear picture of genes induced in infection threads 

and early development it was crucial to subtract genes already induced in the rhizosphere. Bacteroid metabolism 

is characterized by dicarboxylate oxidation with very high expression of the decarboxylating arm of the TCAcycle. 

Amino acid synthesis is bacteroids is shut down with transcriptional as well as metabolite analysis showing 

almost all pools of amino acids have collapsed in bacteroids. This leads to amino acid auxotrophy for branched 

chain amino acids in pea and bean as demonstrated by a dependence on the Aap and Bra transporters. 

However, alfalfa bacteroids mutated in Aap and Bra still fix nitrogen so either they have an alternative transport 

system for branched chain amino acids or they retain synthesis of these amino acids. 

36 

2011





Authors: 

Concurrent Session 4 – Applications of New Technologies 

1100 - 1230 

Brice Roux 1 , Erika Sallet 1 , Nathalie Rodde 1 , Laurent Sauviac 1 , Olivier Catrice 1 ,Ton 

Timmers 1 , Thomas Faraut 2 , Thomas Schiex 3 , Françoise Jardinaud 1 , Fernanda de 

Carvalho-Niebel 1 , Frédéric Debéllé 1 , Delphine Capela 1 , Claude Bruand 1 , Jérôme 

Gouzy 1 , Pascal Gamas 1 

1 . Laboratoire des Interactions Plantes Microorganismes (LIPM) CNRS-INRA, Chemin 

de borde rouge BP 52627, 31326 Castanet-Tolosan cedex, France. 

2 . INRA LGC Chemin de Borde Rouge BP52627, 31326 Castanet-Tolosan cedex, 

France. 

3 . INRA UBIA Chemin de Borde Rouge BP52627, 31326 Castanet-Tolosan cedex, 

France. 

Presentation Title: Molecular dissection of the rhizobium-legume interaction by RNA seq and laser microdissection 

approaches 


In order to have a comprehensive view of the genes involved in the symbiotic rhizobium-legume interaction, we 

set up integrated methods for simultaneous gene expression profiling of both partners, based upon RNA seq 

approaches. We analysed the plant and bacterial transcriptomes of whole and laser micro-dissected root nodules 

and compared them to transcriptomes from non inoculated roots or bacterial cultures, using Sinorhizobium 

meliloti 2011-Medicago truncatula as a model system. We thus combined genome-wide, quantitative and 

sensitive analyses of gene expression with technologies enabling tissue-level resolution. Importantly, we chose 

strand-specific RNA seq techniques that allowed us to identify, in addition to protein coding mRNAs, bacterial 

and plant short and long non-coding RNAs (ncRNAs), not previously predicted by automatic genome annotation. 

To provide a solid framework for quantitative transcriptome analyses, we sequenced and (re)annotated the 

genome of S. meliloti strain 2011. To do so, we developed bioinformatics tools for bacterial gene prediction that 

notably take advantage of RNA seq data. The results were validated by visual inspection and completed by 

manual annotation. We also completed the currently available M. truncatula genome sequence data by high 

depth Illumina sequencing. 

We thus found many bacterial ncRNAs previously described in other groups by different experimental 

approaches, which fully validated our approaches. We also discovered additional bacterial ncRNAs, as well as 

many plant ncRNAs (such as antisense RNAs), a number of which are differentially regulated when comparing 

roots and nodules. We are now using this extensive set of tools and data to (re)examine the sets of proteincoding 

and non coding genes involved in early and late stages of the rhizobium-legume interaction. 

37 

2011






1100 - 1230 

Authors: Antoine Huyghe, Nadia Bakkou, Maged Saad & Xavier Perret 

University of Geneva, Sciences III, Department of Botany and Plant Biology, 

Microbiology Unit, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland. 

Presentation Title: Profiling the symbiotic responses of Sinorhizobium fredii strain NGR234 with RNA-seq 


Sinorhizobium fredii strain NGR234 establishes proficient nitrogen-fixing symbioses with more than 150 plants 

that belong to distant genera and form nodules of either determinate (DN) or indeterminate (IDN) types (Pueppke 

and Broughton, 1999). Genome as well as functional analyses confirmed that genes of NGR234 required for 

nodule formation, infection of nodule cells and nitrogen (N2) fixation were not restricted to the 536 kb 

« symbiotic » plasmid pNGR234a as was previously thought. To identify within the 6.9 Mb genome of NGR234 

(Schmeisser et al. 2009) loci susceptible to contribute to broad host-range symbioses, we combined proteomic 

and transcriptomic datasets. In particular, RNA-seq was used to compare the transcriptomes of cells grown in 

free-living conditions (in the presence or absence of daidzein) or found inside nitrogen-fixing nodules of DN or 

IDN types. In parallel, an in vitro mutagenesis system designed to generate non-polar mutations as well as 

facilitate the functional analysis of NifA-regulated loci was successfully tested in NGR234 (Fumeaux et al. 2011). 

These combined approaches lead to the identification of new loci whose expression profiles were confirmed by 

qRT-PCR and were either modulated by flavonoids or bacteroid-specific. Amongst the novel flavonoid-inducible 

genes, the expression of a chromosomal locus was shown to be linked to a nod-box regulatory sequence but 

independent of the NodD1-TtsI-SyrM2-NodD2 regulatory cascade that controls nodulation and T3SS genes of 

pNGR234a (Kobayashi et al. 2004). The current status of the proteomic and transcriptomic approaches will be 

presented, with an emphasis on the distinct networks which control the flavonoid-dependent or bacteroid-specific 

expression of genes in the promiscous strain NGR234. 

38 

2011






1100 - 1230 

Authors: David Chiasson 1 , Patrick Loughlin 2 , Elena Fedorova 3 , Ton Bisseling 3 and Brent N. 

Kaiser 1 

1 School of Agriculture, Food, and Wine. The University of Adelaide, Urrbrae, SA, 5064, 

Australia. 

2 School of Biological Sciences, University of Sydney, Sydney New South Wales 2006, 

Australia. 

3 Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, 

Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands. 

Presentation Title: The use of heterologous expression systems to characterise the function of the soybean 

transcription factor GmSAT1 


GmSAT1 (Glycine max Symbiotic Ammonium Transporter) was originally isolated in a screen for 

complementation of ammonium transport in the yeast mutant strain 26972c (Kaiser et al 1998). GmSAT1 has 

been localized to both the peribacteroid membrane (PBM) and the nucleus in soybean nodules by Western 

blotting and immunogold labeling. Recent work has revealed that GmSAT1 is critical for nodule organogenesis. 

Silencing GmSAT1 by RNA interference results in a reduced number of nodules, and a Fix- phenotype. 

Although the in planta function of GmSAT1 has not yet been elucidated, the use of heterologous systems has 

given us insight into a very dynamic protein. We have expressed GmSAT1 in yeast and onion epidermal peels. 

Expression in yeast has allowed us to confirm that GmSAT1 is indeed a transcription factor. GmSAT1 directly 

activates the transcription of a novel yeast ammonium channel (termed AMF) and binds the AMF promoter in 

vitro. Expression of GFP:GmSAT1 in yeast shows localization to punctate vesicles, the plasma membrane, and 

the nucleus. In soybean we have recently discovered new transcripts from the GmSAT1 locus. One transcript is 

an alternatively-spliced version of the original clone, and encodes for a nodule-enhanced N-terminal signal 

peptide. We have bombarded onion epidermal peels with various GFP-fusions of GmSAT1. These experiments 

have allowed us to determine the membrane-associated region of GmSAT1 and assign a localization function to 

the signal peptide. Lastly, we have made GFP fusions of both GmSAT1 orthologues from Medicago truncatula 

(MtSAT1 and MtSAT2). Confocal microscopy studies show that, similar to GmSAT1 in yeast and onions, 

MtSAT1 and MtSAT2 are localized to punctate vesicles and the nucleus. We conclude that GmSAT1 is a 

membrane-associated transcription factor. 

Kaiser, BN, Finnegan, PM, Tyerman, SD, Whitehead, LF, Bergersen, FJ, Day, DA, and Udvardi, MK (1998). 

Characterization of an Ammonium Transport Protein from the Peribacteroid Membrane of Soybean Nodules. 

Science 281:1202-1205. 

39 

2011





Authors: 


1100 - 1230 

Tikhonovich I.A. 1,2 , Dolgikh E.A. 1 , Leppyanen I.V. 1 , Lopatin S.А. 3 , Varlamov V.P. 3 

1 All-Russia Research Institute for Agricultural Microbiology (ARRIAM), chausse 

Podbelskogo 3, Pushkin 8, Saint-Petersburg, 196608, Russia; 2 Saint Petersburg State 

University (SPbSU), Universitetskaya nab., 7-9, St. Petersburg, 199034, Russia; 

3 Center Bioengineering RAS, street of 60 th of October, 7/1, Moscow, 117312. 

Presentation Title: New application of N2-fixing organisms: heterologous expression of rhizobial 

glycosylatransferases involved in chitin oligosaccharides synthesis 


Due to their unique properties like biocompatibility, low toxicity and simplicity of biodegradation, the chitin 

oligosaccharides (COs, chitin oligomers, chitosane oligomers and their derivatives) find a wide application. There 

is significant interest in application of COs in agriculture. Chitin oligosaccharides produced by pathogenic fungi, 

are effectively detected by many plant species as elicitors leading to defence reactions and inactivation or 

destruction of the potential pathogen. In plants COs activate a set of defence reactions such as activation of 

defence enzymes, production of active oxygen species (О2 - , Н2О2, OH - ), biosynthesis of phytoalexins and callose 

or lignin deposition. Therefore application of these compounds and their modified analogs for stimulation of plant 

resistance against pathogens looks like enough promising. The treatment of plants with COs is more preferential 

in comparison with chemical agents because they are not toxic and easily utilized. That is a reason why last 

years the interest in the synthesis of COs by enzymatic methods using either the biosynthetic 

glycosyltransferases or glycosidases is being increased. The possibility of enzymatic synthesis of chitin 

oligomeres by means of unique enzymes of soil bacteria rhizobia has been investigated in our experiments. 

Original approaches to analyse the structure of synthesized chitin oligosaccharides by methods of high 

performance liquid chromatography and mass-spectrometry have been developed. In our experiments the elicitor 

activity of synthesized chitin oligomers has been analysed using specific tests. 

This research was supported by the Ministry of Education and Science of the Russian Federation (state 

contracts ## 16.512.11.2183, 16.552.11.7047). 

40 

2011





Concurrent Session 5 – Bacteroids & Symbiosomes 

1100 - 1230 

Authors: Hauke Hennecke, Marion Koch, Fabio Serventi, Gabriella Pessi, Hans-Martin Fischer, 

Zeb Youard, Valérie Murset, Simona Huwiler, Doris Bühler, Olivier Braissant, Nathanaël 

Delmotte, Julia Vorholt, Francesco Danza & Barnali Padhi 

ETH, Institute of Microbiology, Department of Biology, CH-8093 Zurich, Switzerland 

Presentation Title: New facets of Bradyrhizobium japonicum carbon metabolism and copper trafficking 

which support bacteroid function 


The use of transcriptomics and proteomics in culture-grown cells and root-nodule bacteroids of Bradyrhizobium 

japonicum has revealed new symbiotically important functions in metabolism which had not been addressed 

before. Two of them are reported here, one concerning carbon catabolism, the other related to copper 

acquisition. (i) B. japonicum was found to be an oxalotroph. Key genes for oxalate degradation were knocked 

out. The mutants were not only unable to use oxalate as the sole source of carbon but they were also partly 

defective in arabinose degradation. Several lines of evidence suggest that arabinose is degraded to pyruvate 

and glycolaldehyde, the latter being fully oxidized in a pathway via oxalate. The mutants also exhibited 

decreased nodule occupancy in competition with the wild type. Nodules of four B. japonicum host plants had 

higher oxalate content than root tissue. Oxalate degradation may thus be an advantageous trait in nodule 

colonization by B. japonicum. (ii) Copper starvation conditions led to the induction of an operon of five genes 

coding for putative or approved functions in copper acquisition and trafficking. Deletion of the operon resulted in 

strains with a pleiotropic phenotype, including defects in symbiotic nitrogen fixation, denitrification, and 

cytochrome oxidase biogenesis. The product of one gene (bll4880) was purified and shown to bind Cu(I) with 

high affinity and in a 1:1 stoichiometry. In vivo, it functions as a copper chaperone in the periplasm where it is 

involved in Cu delivery to the membrane-bound aa3-type cytochrome oxidase and perhaps to other targets. Why 

this protein and/or the other encoded proteins are symbiotically important is still enigmatic. 

41 

2011





Authors: 

The symbiosome membrane (SM) is the interface between the plant and bacteria in the legume-rhizobium 

symbiosis. This plant-derived membrane separates the symbiotic form of the rhizobia, from the plant cytoplasm 

and controls both the type and amount of solutes exchanged between the two partners. In exchange for nitrogen 

fixed by the bacteroid, the plant provides reduced carbon, probably in the form of malate, to the bacteroids, as 

well as other solutes including iron, sulphate, zinc and possibly amino acids. We are investigating transport 

proteins on the SM that regulate this exchange in soybean nodules using a proteomic approach and nodule 

transcriptome data. Candidates that potentially encode malate, iron and amino acids are a major focus. 

Proteomic analysis of the SM involved trypsin digestion of total SM proteins, separation of peptide fragments by 

liquid chromotography (LC), and analysis with tandem mass spectrometry (MS/MS). Of the 71 proteins identified 

in all three biological replicates, 33 are predicted to be integral membrane proteins. A number of proteins known 

to be associated with the SM, such as nodulin 26 (an aquaporin), ATPase, symbiotic sulphur transporter (sst1), 

Rab7, nodulins 24 and 53, were identified. Many of the identified SM proteins are highly expressed in nodules 

compared to other tissues (Libault et al. 2010; Severin et al. 2010). We are currently assessing the importance of 

some integral membrane proteins of unknown function through an RNAi approach and confirming the location of 

proteins on the SM using reporter gene fusions. Candidate transporters confirmed to be localised to the SM will 

be functionally characterised in heterologous expression systems. 

We have also characterised a transporter, GmYSL1, with homology to Yellow Stripe Like (YSL) proteins that are 

implicated in iron transport in other plants. The gene complements growth of the yeast iron transport deficient 

mutant, fet3fet4, on media containing low concentrations of ferric citrate. Fusion of the coding region to GFP 

confirmed the localisation of this protein on the SM. Its expression in nodules is up-regulated in response to iron 

and this and its location suggest it may be involved in sequestration of iron in the symbiosome. 

Libault M Plant J. 2010 63:86 

Severin AJ BMC Plant Biol. 2010 10:160 


1100 - 1230 

Patrick Loughlin 1 , Victoria Clarke 1 , Chi Chen 1 , Yihan Qu 1 , Joanne Castelli 3 , Martha 

Ludwig 3 , David Day 2 and Penelope Smith 1 

1 School of Biological Sciences, Macleay Bldg (A12), University of Sydney, Camperdown 

NSW, 2006. 2 School of Biological Sciences, Flinders University, SA, Australia. 3 School of 

Biomedical, Biomolecular and Chemical Sciences (M310), University of Western 

Australia, Crawley WA 6009. 

Presentation Title: Identification of transport proteins on the symbiosome membrane in soybean 


42 

2011






1100 - 1230 

Authors: Youguo Li, Fuli Xie, Guojun Cheng, Hui Xu, Zhi Wang & Lei Lei 

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 

Wuhan, Hubei, 430070, P. R. China 

Presentation Title: Identification of a novel symbiotic gene participating in synthesis of a bacteroid-specific 

electron carrier menaquinone 


Ubiquinone (UQ) has been considered an electron mediator in electron transfer that generates ATP in Rhizobium 

under both free-living and symbiosis conditions. When mutated, the dmtH gene has a symbiotic phenotype of 

forming ineffective nodules on Astragalus sinicus. The gene was isolated from a Mesorhizobium huakuii 7653R 

transposon-inserted mutant library. The DNA sequence and conserved protein domain analyses revealed that 

dmtH encodes demethylmenaquinone (DMK) methyltransferase, which catalyzes the terminal step of 

menaquinone (MK) biosynthesis. Comparative analysis indicated that dmtH homologs were present in only a few 

rhizobia. Real-time quantitative PCR showed dmtH is a bacteroid-specific gene. The highest expression was 

seen at 25 days after inoculation of strain 7653R. Gene disruption and complementation tests demonstrated that 

the dmtH gene was essential for bacteroid development and symbiotic nitrogen fixation ability. Menaquinone and 

ubiquinone were extracted from the wild type strain 7653R and mutant strain HK116. MK7 accumulated under 

microaerobic condition and UQ10 accumulated under aerobic condition in M. huakuii 7653R. The predicted 

function of DmtH protein was confirmed by the measurement of methyltransferase activity in vitro. These results 

revealed that MK7 was used as an electron carrier instead of UQ in M. huakuii 7653R bacteroids. 

43 

2011





Authors: 


1100 - 1230 

Vanessa J Melino 1 , Elizabeth A Drew 2 , Gordon Thomson 1 , Rosemary White 3 , Wayne G 

Reeve 1 , Ross A Ballard 2 , and Graham O‟Hara 1 . 

1 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, Western Australia 

2 South Australian Research and Development Institute, Plant and Soil Health, Urrbrae, 

5064, South Australia 

3 Commonwealth Scientific and Industrial Research Organisation, Plant Industry, 

Canberra, 2601, ACT 

Presentation Title: Inside out: an in-depth characterisation of effective (Fix + ), sub-optimal (Fix partial ) and 

ineffective (Fix - ) N2-fixation in Trifolium symbiosis 


A major challenge to the formation of a successful symbiotic partnership between Trifolium spp. and Rhizobium 

leguminosarum bv. trifolii (R.l.t) is the presence of naturalized soil bacteria which can cause the induction and 

infection of root nodules without the benefits of nitrogen fixation (ineffective) or which may alternatively result in 

poor nitrogen fixation (sub-optimal). Analysis of symbiotic performance and morphological examination of nodule 

and bacteroid development enabled the classification of each of the 16 symbiotic combinations into one of four 

groups: Effective ( Fix 90-100% ), Sub-optimal ( Fix 10-70% ), Ineffective-differentiated (Fix - ) and Ineffective-vegetative (Fix - 

). The effective combinations are each within the nodule function range of 90-100% containing infection threads, 

bacteroids in inter-zone II-III and III with 1-2 bacteroids per peribacteroid unit. Sub-optimal combinations result in 

a varied rate of fixation from 10 to 70% and their nodules contain an abundance of senescing bacteroids present 

either in an enlarged and developmentally early zone of senescence (IV) or where zonation is absent, they are 

found adjacent to bacteroid-dense plant cells. Although differentiated Y-shaped bacteroids of effective nodules 

were like-wise observed in sub-optimal nodules they were on average shorter in length. Ineffective-differentiated 

combinations produce nodules which are absent of zonation, void of acetylene reduction activity and their plant 

cells contain elongated but less swollen bacteroids alongside senescing bacteroids. Ineffective-vegetative 

combinations produce nodules which are absent of bacteroids despite the presence of rhizobia-filled infection 

threads. These findings demonstrate the diversity of symbiotic outcomes from combinations of symbionts within 

the same genus, and the range of failures that can occur during infection. Future functional genomic efforts will 

be directed towards studying genetic control of internalization of rhizobia by cortical cells (failed in the ineffectivevegetative 

nodules), abortion of bacteroid differentiation post-elongation (observed in the ineffectivedifferentiated 

nodules) and accelerated bacteroid senescence post-differentiation (observed in sub-optimal 

nodules). 

44 

2011





Concurrent Session 6 – Field Applications II 

1100 - 1230 

Authors: Lori Phillips 1 , Bernadette Carmody 2 , Sharon Fox 3 , Lambert Brau 3 , Graham O‟Hara, 

David Pearce 2 

Presentation Title: 

1 Department of Primary Industries Victoria, Bundoora Vic 3083; 

Lori.Phillips@dpi.vic.gov.au 

2 Department of Primary Industries Victoria, Rutherglen Vic 3685 

3 Murdoch University, School of Biological Sciences and Biotechnology, Murdoch, 

Western Australia 6150 

Presentation Time: 1100 – 1120 

Comparative diversity of rhizosphere and endophytic microbial communities in 

faba cropping systems 

Effective nodulation by rhizobia does not occur in an ecological void. Numerous and diverse microbial 

communities exist in the soil and develop in the rhizosphere and endosphere of the growing legume. These 

communities may influence effective nodulation, plant growth, and disease suppression, and represent a 

significant potential co-inoculant resource. The primary objective of this study was to examine the impacts of 

plant cultivar and inoculation treatment on the diversity of plant-associated bacteria. Faba bean cv. Farah and 

Nura were sown in a field trial in Rutherglen, Victoria. This site had not been cropped with legumes for over a 

decade. Treatments included non-inoculated controls, standard peat inoculation, peat inoculation at half the 

recommended rate, and co-inoculation with a Pseudomonas spp (potential plant growth promoting rhizobacteria). 

This trial is ongoing, and performance measurements to date include nodulation, root biomass, and shoot 

biomass. Plant cultivar and inoculation treatment effects on rhizosphere and endophytic microbial communities 

were evaluated using terminal restriction fragment length polymorphism (T-RFLP). Archaeal, bacterial, and 

fungal diversity were assessed. Inoculation type had no significant impact on either nodule number or plant 

biomass measures. Non-inoculated faba plants were well nodulated by resident rhizobial populations that 

persisted for more than a decade in the absence of a legume host. T-RFLP data is currently under analysis, and 

will further our understanding of the ecological parameters which help or hinder symbiotic success. 

45 

2011






1100 - 1230 

Author: Kanjana Chansa-Ngavej 

Department of Microbiology, Faculty of Science, Chulalongkorn University, Thailand 

Presentation Title: Selection and Field Trials of Soybean Rhizobium Biofertilizers with DNA Fingerprints and 

can be Kept at Room Temperature 


Soybean rhizobium biofertilizers are usually kept at low temperature or under refrigeration to prevent cell 

multiplication which leads to reduction in nodulation efficiency of soybean rhizobium strains. In 2009, the author 

applied for a Thai patent for the selection of soybean rhizobium strains with DNA fingerprints for the production of 

soybean rhizobium biofertilizers which can be kept at room temperature. Out of the 70 strains tested for growth 

at different temperatures in yeast extract mannitol (YM) broth at 200 rpm for 5 days, two strains, NA2 and NA82 

were selected. 16S rDNA sequences revealed they were Bradyrhizobium elkanii strains with different DNA 

fingerprints and bromthymol blue reactions. NA2 was found to secrete acidic products while NA82 was found to 

secrete alkali products when grown on YM agar with Bromthymol blue at 25 o C for 10 days. After incubating the 

biofertilizers produced by using each of the selected strains at 10 10 -10 12 CFU/gram biofertilizer at 37 o C and at 40 

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 

with soybean (Glycine max cv. Chiangmai 2) with each of the biofertilizers in 240 m 2 plots in three subdistricts in 

Phitsanulok province showed NA2 increased soybean yields by 18-42%. NA82 was found to increase soybean 

yield in Bung Kok subdistrict by 26%. Field trials with soybean (Glycine max cv. Chiangmai 60) with each of the 

biofertilizers in 240 m 2 plots in Sukhothai province showed the biofertilizers NA2 and NA82 could increase 

soybean yields 9-14%. PCR fingerprints of both NA2 and NA82 were detected in bacteria isolated from root 

nodules of soybeans in every experimental plot. DNA fingerprints of root nodule bacteria isolated from the control 

treatment subplots showed NA2 and NA82 were not indigenous rhizobia in the experimental plots. 

46 

2011






1100 - 1230 

Author: Nicole Seymour 1 , Maurice Conway 2 , Andrew Erbacher 2 , Darren Aisthorpe 3 and Max 

Quinlivan 2 

1 Department of Employment, Economic Development and Innovation, Leslie Research 

Centre, Toowoomba, 4350, Queensland. 

2 Department of Employment, Economic Development and Innovation, Emerald, 4720, 

Queensland. 

3 Department of Employment, Economic Development and Innovation, Biloela, 4715, 

Queensland 

Presentation Title: Nitrogen fertiliser reduces nodulation of mungbean but gives no yield advantage in 

Central Queensland 


Mungbean or green gram (Vigna radiata) is a short-seasoned tropical pulse crop used as a high protein human 

food. Australian production varies from 30000 to 60000 tonnes per year, 95% of which is exported. Mungbean 

production has increased in Central Queensland (CQ) due to high prices, improved yields and its valuable role in 

weed management. 

Low nodulation and N-fixation by commercial mungbean crops in the northern grain-growing region of Australia 

(Busby and Lawn 1992) suggested that inoculated mungbean was N-limited and current inoculation technology 

may be ineffectual (Herridge et al. 2005). 

Five field trials were conducted in 2010-11 across CQ to determine whether an economic response could be 

gained by applying fertiliser. Several N, P and Zn fertiliser rates were tested separately and in combination. All 

mungbean seed (cv. Crystal) was inoculated with the bradyrhizobial strain CB1015, as peat slurry except for one 

treatment (uninoculated with no fertiliser). At one site, on a self-mulching brown Vertosol at Wowan, Queensland, 

the impact of fertiliser on nodulation was assessed. 

In three of the five trials there was a significant yield response to inoculation but no consistent yield response to 

fertiliser treatments. At Wowan, nodulation scores indicated there was a suppressive effect of urea fertiliser at 20 

and 40 kg N/ha. There was no effect of any treatment on nodule dry weights. The results indicated that there 

was no economic advantage from added N fertiliser for inoculated mungbean cv. Crystal. 

Bushby HVA, Lawn RJ (1992) Nitrogen fixation in mungbeans – expectations and reality. In „Proceedings of the 

6 th Australian Society of Agronomy conference‟. Pp 161-164. (Australian Society of Agronomy: Armidale) 

Herridge DF, Robertson MJ, Cocks B, Peoples MB, Holland JF and Heuke L (2005). Low nodulation and nitrogen 

fixation of mungbean reduce biomass and grain yields. Aust J Exp Agric. 45:269-277 

47 

2011






1100 - 1230 

Authors: Ryan Farquharson 1 , Ross Ballard 2 , Nigel Charman 2 , Jeff Baldock 1 

1 CSIRO Land and Water. PMB 2 Glen Osmond, South Australia 5064. 

2 South Australian Research and Development Institute. GPO Box 397, Adelaide, South 

Australia 5001. 

Presentation Title: Symbiotic performance of a herbicide tolerant Medicago littoralis 


Group B herbicides inhibit the acetohydroxyacid synthase (AHAS – also known as acetolactate synthase) 

enzyme in the pathway of branched chain amino acid synthesis. These herbicides have gained widespread use 

worldwide; however the potential impacts on nitrogen fixation are underappreciated. A new strand medic 

(Medicago littoralis) cultivar (FEH-1) was developed by chemical mutagenesis of seed of the Herald variety 

(Heap, 2000). FEH-1 displayed tolerance to various group B herbicides including the sulfonylurea class, 

however this work had only analysed biomass production and yield with no consideration of nitrogen fixation 

(Heap, 2000; Howie and Bell, 2005). Through a targeted approach, a single mutation in the AHAS gene was 

identified as the mechanism of herbicide tolerance (Oldach et al., 2008). An experiment comparing the symbiotic 

performance of FEH-1 and Herald showed that FEH-1 had useful tolerance to chlorsulfuron, with higher biomass 

production, nodulation and nitrogen fixation than Herald after application of that herbicide. However it was noted 

that in the absence of herbicide, Herald had greater biomass production, nodulation and nitrogen fixation than 

FEH-1. Differences in seed mass and nitrogen could not explain the disparity and a „tolerance penalty‟ was 

proposed. A subsequent experiment measuring the growth of Herald and FEH-1 with different strains of rhizobia 

showed symbiotic compatibility had changed in FEH-1. This may be contributing to the tolerance penalty 

observed when FEH-1 is grown in the absence of herbicide. Although there is scope to better understand the 

complexities surrounding group B herbicides and nitrogen fixation, it is clear that where in-crop or residual 

herbicides are of concern, herbicide tolerant varieties will provide a useful option to maintain inputs of biologically 

fixed nitrogen. 

Heap J (2000). Increasing Medicago resistance to soil residues of ALS-inhibiting herbicides. Thesis, University of 

Adelaide. 

Howie J & Bell CA (2005) Field performance of an annual medic tolerant of sulfonylurea herbicide residues. 

Proceedings of the XX International Grassland Congress, Dublin, Ireland. 

Oldach KH, Peck DM, Cheong J, Williams KJ, Nair RM (2008) Identification of a chemically induced point 

mutation mediating herbicide tolerance in annual medics (Medicago spp.) 

48 

2011






1330 - 1500 

Authors Graham W. O‟Hara 1 Ron J. Yates 1, 2 & John G. Howieson 1 



2 Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South 

Perth, Western Australia 6151 

Presentation Title: Fifteen years of revolution in legume N-fixation in southern Australia 


Legumes in southern Australian agriculture have been, and continue to be, introduced from exotic centres of 

origin. Inoculation is essential because Australian soils do not naturally contain rhizobia that form effective 

nitrogen-fixing symbioses with these legumes. During the past 15 years the Centre for Rhizobium Studies & the 

National Rhizobium Program have developed 15 strains of rhizobia for commercial production, and contributed to 

development of new legumes which, together with their rhizobia, have been sown over 5 million ha. in southern 

Australia. In addition, new insights have been provided into phenomena affecting symbiotic performance e.g. the 

role of horizontal gene transfer in development of poorly effective soil populations of rhizobia, and selective 

nodulation. 

Selective nodulation describes an active phenomenon that results in the establishment of a symbiosis between a 

legume and an effective strain of rhizobia in the presence of ineffective strains. Selective nodulation can 

overcome a numerical disadvantage in the effective microsymbiontt when there is a population size window of 

between 1,000 and 100,000 cells. 

Alternative species of herbaceous perennial legumes (e.g. Lebeckia spp., Rynchosia spp.) originating in 

southern Africa and nodulated by novel Burkholderia spp. are being evaluated for their potential role in providing 

green feed in autumn and late spring. Target soils do not sustain traditional legumes (e.g. lucerne, white clover, 

lotus) because of abiotic stresses such as low pH, low clay content, low nutrient status and low rainfall. 

Overcoming the challenges of establishing symbioses with these new legumes will be discussed. 

The Grain Research and Development Corporation (GRDC) of Australia support the National Rhizobium 

Program. 

49 

2011






1330 - 1500 

Authors: Michael Udvardi 1 , Catalina Pislariu 1 , Igor Kryvoruchko 1 , Senjuti Sinharoy 1 , Mingyi 

Wang 1 , Rajasekhara R. Duvvuru Muni 1 , Ivone Torres-Jerez 1 , Mark Taylor 1 , Shulan 

Zhang 1 , Xiaofei Cheng 1 , Jiangqi Wen 1 , Ji He 1 , Xinbin Dai 1 , Patrick X Zhao 1 , Yuhong 

Tang 1 , Rujin Chen 1 , Kirankumar Mysore 1 , Pascal Ratet 2 , Vagner A Benedito 3 , Giles 

Oldroyd 4 , and Jeremy D Murray 4 

1 Samuel Roberts Noble Foundation, Ardmore, OK, USA; 2 ISV-CNRS, Gif sur Yvette, 

France; 3 West Virginia University, USA; 4 John Innes Centre, Norwich, UK; 

Presentation Title: Functional genomics of symbiotic nitrogen fixation in legumes 


The past few years have seen tremendous development of tools and resources for functional genomics in 

legumes, including completion or near-completion of several legume genomes, comprehensive gene expression 

databases for model and crop species, and production of near-saturating mutant populations for efficient forward 

and reverse-genetic studies of gene function. This presentation will highlight some of these advances in various 

legumes species before focusing on resource development in the model species, Medicago truncatula and how 

these resources are accelerating research on symbiotic nitrogen fixation. 

Phenotypic screening of several mutant populations of Medicago followed by map-based and other cloning 

approaches have identified several genes that are required for nodule development, differentiation, and/or 

symbiotic nitrogen fixation (SNF). However, it is clear from transcriptomic studies that hundreds, if not thousands 

of plant genes are involved in SNF. Anticipating the completion of the Medicago genome sequence, we invested 

heavily in the development of tools and resources that will enable us and others to decipher the functions of 

many Medicago genes in the coming years. These resources include a gene expression atlas/database that can 

be used to obtain developmental and other types of expression data for most Medicago genes, and two large 

mutant populations, a fast-neutron-bombardment-deletion and a tobacco retro-transposonTnt1-insertion 

population, which can be used for both forward and high-throughput reverse genetics. About 20,000 Tnt1 

insertion lines have been generated in the R108 genotype and phenotypic screens of about half of the population 

revealed 172 lines defective in SNF. These were sorted into six distinct phenotypic categories. Thermal 

Asymmetric InterLaced PCR (TAIL-PCR) was used to generate 2,422 flanking sequence tags (FSTs) from these 

SNF mutant lines. FST analysis identified 33 insertion alleles of the following essential symbiotic genes: 

DMI1,2,3; NSP1,2; ERN1; NIN; LYK3; FLOT1,2; ENOD40; SYMREM1; and SUNN. High-throughput 454 

sequencing using a 2D-pooling strategy is being used to accelerate our current FST sequencing efforts. This will 

lead to a comprehensive FST database for all Tnt1 insertion lines. In parallel, a PCR-based reverse-screening 

strategy was established to identify Tnt1 insertions in specific genes of interest. As a result, we have identified 

additional insertion alleles of known, essential symbiosis genes. The mutant resources and gene expression 

atlas have also been used to identify novel symbiotic genes, including VAPYRIN, which is required for 

intracellular accommodation of rhizobia in nodules and of arbuscular mycorrhizal fungi in root cells. Reversegenetics, 

using the Tnt1-insertion population is currently being used to identify and characterize transcription 

factors and transporters with essential roles in SNF. 

This work was supported by the National Science Foundation, the USDA CSREES-NRI, and the Samuel Roberts 

Noble Foundation. 

50 

2011






1330 - 1500 

Authors: Simon Kelly 1,2 , Yasuyuki Kawaharada 2,3 , Artur Muszynski4, Andree Hubber 1,2 , John 

Sullivan 1,2 , Niels Sandal 2,3 , Russell Carlson 4 , Jens Stougaard 2,3, and Clive Ronson 1,2. 

1 Department of Microbiology and Immunology, University of Otago, Dunedin, New 

Zealand 

2 Centre for Carbohydrate Recognition and Signalling, University of Aarhus, Aarhus, 

Denmark 

3 Department of Molecular Biology, University of Aarhus, Aarhus, Denmark 

4 Complex Carbohydrate Research Center, University of Georgia, Athens, USA 

Presentation Title: The requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis 


To investigate the requirement for exopolysaccharide (EPS) in the formation of determinate nodules on Lotus 

species, Mesorhizobium loti strain R7A mutants affected in early (exoA, exoB) or mid/late-stages (exoO, exoU, 

exoK, mlr5265 and mlr5266) of the EPS biosynthetic pathway were isolated. Elution profiles of EPS extracts 

showed that R7A produced both HMW and LMW fractions of EPS whilst mutant strains produced only a LMW 

fraction. Early-stage affected mutants formed nitrogen-fixing nodules on L. japonicus cv. Gifu at rates 

comparable to wild-type R7A, whereas the mid/late-stage mutants formed uninfected nodule primordia, a few of 

which developed into nodules following a lengthy delay. Examination of the symbiotic proficiency of the exoU 

mutant on various L. japonicus ecotypes and under differing environmental conditions revealed host and 

environment interactions in the requirement for EPS. Analysis of fluorescent protein-tagged strains revealed that 

the ineffective mutants were disrupted at the stage of infection thread (IT) development. Symbiotically-defective 

EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority 

of full-length ITs observed harboured only the EPS mutant strain and did not show bacterial release, whereas all 

nitrogen-fixing nodules contained both mutants. These results suggest that EPS is required at both the stages of 

IT initiation and bacterial release and reveal a complex role and requirement for M. loti EPS in determinate 

nodule formation. These and other results also suggest that EPS plays a signalling rather than structural role in 

the symbiosis. To identify the putative EPS receptor and the downstream signalling pathway, we have carried out 

a screen for plant suppressor mutants that form nodules with the exoU mutant. Progress towards the 

characterisation of these mutants will be presented. 

51 

2011





Concurrent Session 7 – Field Applications III 

1600 - 1720 

Authors: Elizabeth Drew 1 , Victor Sadras 1,2 , Lachlan Lake 1 , Matthew Denton 2 & Ross Ballard 1 

1 Plant and Soil Health, South Australian Research and Development Institute, GPO 

Box 397, Adelaide, SA, 5001, Australia. 

2. School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA, 

5064, Australia 

Presentation Title: Impacts of pea genotype and soil rhizobia on N2-fixation by field pea 


Enhancing the fixed N contribution from legumes into agricultural systems will become increasingly important as 

higher N fertiliser costs erode farm profitability. This paper examines factors affecting the N2-fixation potential of 

field pea (Pisum sativum). 

Having previously shown that about 70% of southern Australian cropping systems with a history of pea 

cultivation contain adequate numbers of rhizobia for prompt nodulation (>100 rhizobia per g soil), recent efforts 

have been directed at understanding the intrinsic compatibility (N2-fixation capacity) of pea genotypes with soil 

rhizobia. Contributions of fixed N by different pea genotypes have also been quantified in the field. 

The greenhouse assessment of commercial pea cultivars and advanced breeding lines with soil rhzobia included 

material with diverse maturity times and growth habits. Effective symbioses formed with most communities of soil 

rhizobia, but some variation occurred between pea genotypes. For example, the symbiotic performance of cv. 

Kaspa was on average 10% lower than that of cv. Parafield (85%) when nitrogen fixation resulting from 82 soil 

inoculants was compared to the commercial inoculant strain (SU303). 

When five pea genotypes were grown in the field, differences were measured in nodulation pattern and 

partitioning of dry matter, which contributed to large differences in N benefit. Pea genotypes were sown with and 

without N fertiliser at Roseworthy SA and were reliant on soil rhizobia for nodulation. Nodule number and 

distribution on the roots eight weeks after sowing varied significantly with pea genotype and nitrogen treatment. 

Nodule mass/g root was weakly (R 2 =0.17) but significantly (P






1600 - 1720 

Authors: Julie Grossman 1 , Nape Mothapo 1 , Mary Parr 1 , Malik Oliver 1 

1 North Carolina State University, Department of Soil Science, Raleigh, North Carolina, 

27695. 

Presentation Title: Effect of cover crop planting history on Rhizobium leguminosarum ecology in organic 

farming systems 


Biological nitrogen fixation (BNF) is a major contributor of nitrogen to certified organic farming systems in the 

United States (U.S.), with presence of effective rhizobia strains being essential for optimal nodulation of legume 

cover crops and green manures. Organic farmers have particular interest in the winter annual cover crop species 

hairy vetch (HV; Vicia villosa Roth). Our recent work in the Southeastern U.S. shows V. villosa to have high 

biomass production and BNF capacity, with many varieties producing over 200 kg N ha -1 , and more than 170 kg 

N ha -1 derived from BNF. In this study we determined the impact of past hairy vetch cultivation on 

resident Rhizobium leguminosarum biovar viciae (Rlv) populations. Organic farm soils with and without history of 

HV cultivation were used to assess effect of planting history on nodulation of ten HV trap-host genotypes and 

diversity of Rlv. Paired soils from each field type were collected from three organic farms across North Carolina. 

Plants were inoculated with soil dilutions from the six fields and used to trap Rlv in a growth chamber. V. villosa 

inoculated with HV+ soil dilutions had 60% more nodules with 70% greater total mass than HV- treatments. Two 

of three HV+ soils produced greater plant biomass and plant tissue N than those inoculated with soil dilutions 

from HV- fields, suggesting improved BNF. Molecular analysis of the 473 Rlv isolates using BOX-PCR produced 

more than 35 genetic groupings across the three sites, and indicated that rhizobia diversity was most impacted 

by site, followed by hairy vetch field history. Results suggest that hairy vetch cultivation appears to increase 

population size of resident Rlv capable of nodulation and BNF with hairy vetch cover crops. Current work is 

underway to quantify Rlv populations in each field type using qPCR and Most Probable Number methodologies. 

53 

2011






1600 - 1720 

Authors: Amanda Bonython 1,2 , Ross Ballard 1,2 , Nigel Charman 1,2 & Andrew Craig 1,2 

1 South Australian Research and Development Institute, GPO Box 397, Adelaide, SA, 

5001, Australia. 

2 Cooperative Research Centre for Future Farm Industries. Crawley, WA, 6009. 

Presentation Title: Improved nodulation of regenerating messina plants 


Messina (Melilotus siculus syn. M. messanensis) is a promising annual pasture legume for areas affected by 

dryland salinity. Sinorhizobium medicae strain, WSM1115, was used during initial species evaluation. Prompt 

nodulation occurred in year of sowing but nodulation failure was commonplace in regenerating pastures. Strain 

WSM1115 failed to persist in the saline soils over summer. Alternative strains of rhizobia and agronomic 

practices were evaluated to improve nodulation. 

Six agronomic treatments and two inoculation treatments were applied in a factorial design. Messina seed was 

inoculated with either a mixture of experimental strains (SRDI554, WSM4118 and WSM4194) or WSM1115. 

Agronomic treatments were applied to modify the soil micro-environment and improve rhizobial persistence. 

They comprised sowing a perennial companion species, either Cichorium intybus or Puccinellia ciliata; 

incorporating soil additives at sowing, gypsum (2.5 t/ha) or clay (75t/ha); applying an organic mulch over the 

summer months, or applying a soil containing medic rhizobia prior to regeneration. Plots were sown in 2008 at 

two trial sites near Keith, South Australia. The soils were alkaline sands with summer soil surface salinity levels 

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. 

In 2008, all treatments had >97% plants nodulated. In 2009, ten regenerating plants from each plot were 

sampled and percentage nodulation and nodule score per plant determined (Score 0=0 nodules; 1=1-4 nodules; 

2=5-9 nodules; 3=10-14 nodules, 4=15-19 nodules, 5=20-29 nodules, 6=30+ nodules). At Site 1 (more saline) 

plant nodulation and nodule score increased from 35% and 0.6 (WSM1115 treatment) to 95% and 3.0 (strain 

mixture treatment). At Site 2, most regenerating plants in the WSM1115 treatment were nodulated (80%), but 

had few nodules (score 1.6). Nodule score increased to 3.9 in the strain mixture treatment. At both sites, the 

application of soil containing medic rhizobia resulted in levels of nodulation approaching the strain mixture 

treatment. Other agronomic treatments were less effective. 

The mixture of rhizobia strains improved the nodulation of regenerating messina plants compared to WSM1115. 

Strain of rhizobia influenced messina nodulation more than the agronomic treatments. 

54 

2011






1600 – 1720 

Authors: Keletso Mohale 1 , Alphonsus Belane 1 , Flora Pule-Meulenburg 2 and Felix D Dakora 3 

1 Department of Crop Sciences, Tshwane University of Technology, 175 Nelson 

Mandela Drive, Private Bag X680, Pretoria 0001, South Africa 

2 Department of Biotechnology and Food Technology, Tshwane University of 

Technology, 175 Nelson Mandela Drive, Private Bag X680, Pretoria 0001, South Africa 

3 Department of Chemistry, Tshwane University of Technology, 175 Nelson Mandela 

Drive, Private Bag X680, Pretoria 0001, South Africa 

Presentation Title: Symbiotic functioning and rhizobial biodiversity of Bambara groundnut (Vigna 

subterranean L.Verdc) grown in farmers‟ field in South Africa 


Although Bambara groundnut (Vigna subterranea L.Verdc) is an important food legume in Africa, little is known 

about its symbiotic N nutrition. This study assessed N2 fixation in Bambara groundnut plants grown in 26 farmers‟ 

fields, and characterized their bacterial symbionts isolated from root nodules. Isotopic analysis showed that N 

derived from fixation in Bambara groundnut varied from 55% to 97%. Actual amounts of N-fixed also varied for 

different farms and villages, and ranged from 3.7 to 212.7 kg N.ha -1 . Bacterial isolates from root nodules showed 

phenotypic differences in growth rate, colony appearance, shape, and texture. Following Koch‟s postulate, 19 

slow-growers and 31 fast-growers were tested for their nodule-forming ability on Bambara groundnut. The 

bacterial isolates showed marked differences in their ability to elicit nodule formation and promote plant growth in 

Bambara groundnut. However four slow-growers and five fast-growers formed ineffective nodules on Bambara 

groundnut. Analysis of data from 16S rDNA sequencing showed that Bambara groundnut isolates formed three 

distinct clades, one with Bradyrhizobium liaoningense, another with Burkholderia tuberum CIP 108238, while the 

third clade clustered with Rhizobium sp. CCNNYC119 (in a sub-clade) and Mesorhizobium sp. W39 (in another 

sub-clade). Taken together, the data revealed that Bambara groundnut depends on N2 fixation for its N nutrition 

and is nodulated by diverse microsymbionts that belong to both alpha and beta-proteobacteria. 

55 

2011





Concurrent Session 8 – Plant Symbiotic Genes 

1600 – 1720 

Authors: Matthew Bellgard1 , Wayne Reeve2 , Huaan Yang3 , Jingjuan Zhang4 , Roberto Barrero1 and John Howieson2, 4 

1 Centre for Comparative Genomics, Murdoch University, South Street, Murdoch 6150, 

Australia. 



3 Department of Agriculture and Food, Western Australia (DAFWA), Baron Hay Court, 

South Perth, 6151, Western Australia. 

4 Crop and Plant Research Institute, Murdoch University 

Presentation Title: Interpreting sub-optimal nitrogen fixation in Lupinus angustifolius utilising the 

genome sequence of both symbiotic partners. 


WSM1417, WSM1253 and WSM471 are nodule bacteria that infect Lupinus angustifolius (NLL narrow-leaf lupin) 

and which have different nodulation and nitrogen fixation profiles. These three strains were sequenced within the 

JGI GEBA project through the US Department of Energy. To understand the genetic control of the relationship 

between these three micro-symbionts and their legume host we required both the lupin genome sequence, and 

transcriptome information. We thus sequenced NLL cv Tanjil using a WGS approach and achieved a 30 fold 

coverage of the whole genome. We obtained 31 billion base pair high quality sequencing data and are 

undertaking the lupin genome assembly and annotation with a view to identify candidate genes in nodule 

formation and function. The transcriptome data was of nodules of three NLL plants inoculated with the three 

sequenced strains. From the NLL genome sequence we obtained 23,000 contigs, which were assembled into 

12,500 scaffolds. The largest contig we have is 149Mb, the N50 size of 13.6 Kb and there is ~80Mb of repeat 

sequences. Further analysis of the genome sequence data will be provided. 

56 

2011






1600 – 1720 

Authors: Brett Ferguson 1 , Dongxue Li 1 , Meng-Han Lin 1 , Satomi Hayashi 1 , Yu-Hsiang Lin 1 , Dugald 

Reid 1 , Saeid Mirzaei 1 , Alina Tollenaere 1 , Peter Gresshoff 1 

1 Australian Research Council Center of Excellence for Integrative Legume Research, 

School of Agriculture and Food Sciences, University of Queensland, St. Lucia, Brisbane, 

QLD, 4072, Australia 

Presentation Title: Novel components involved in legume nodule development and autoregulation 


To discover novel legume components involved in nodule formation (Ferguson et al. 2010), we used nextgeneration 

sequencing (Illumina) and the available soybean genome (www.phytozome.net/soybean) to identify 

genes that are differentially expressed in the zone of root hair emergence following rhizobia-inoculation. 

Legumes regulate the number of nodules they form via the Autoregulation Of Nodulation (AON). Following 

rhizobia-inoculation, AON commences with the production of a root-derived signal called Q (Reid et al. 2011a). 

We have identified soybean Q candidate genes encoding CLAVATA3/ESR related (CLE) peptides that exhibit 

increased expression following rhizobia inoculation or inhibitory nitrate treatment (Reid et al. 2011b). Overexpression 

of these genes significantly reduces soybean nodule numbers. The nitrate-induced Q peptide 

appears to act locally, whereas the rhizobia-induced Q peptide is transported to the shoot where it, or a product 

of its action, is perceived by a LRR receptor kinase called NARK. We used next-generation sequencing to 

identify soybean components acting downstream of NARK in the leaf. Candidate genes identified can now be 

used in our recently established bioassay to confirm the presence of Q in various samples and biological extracts 

(e.g., xylem sap). We also isolated and characterized the phenotype of a soybean line mutated in the 

homeologous gene of GmNARK, GmCLAVATA1A. Following the perception of Q by NARK in the leaf, a number 

of factors are produced including a novel signaling compound called the Shoot-Derived Inhibitor (SDI). SDI is 

predicted to be transported from the shoot to the root where it inhibits continued nodule development. We found 

SDI to be NARK- and Nod factor-dependent, heat stable, small, and likely not a peptide or RNA molecule (Lin et 

al. 2010, 2011). Findings regarding our progress in identifying and characterising the abovementioned 

nodulation factors will be presented. 

Ferguson BJ, Indrasumunar A, Hayashi S, Lin Y-H, Lin M-H, Reid D, Gresshoff PM (2010) Molecular analysis of legume nodule 

development and autoregulation. Journal of Integrative Plant Biology 52: 61-76 

Lin Y-H, Ferguson BJ, Kereszt A, Gresshoff PM (2010) Suppression of hypernodulation in soybean by a leaf-extracted, NARK- and Nod 

factor-dependent, low molecular mass fraction. New Phytologist 185: 1074-1086 

Lin Y-H, Lin M-H, Gresshoff PM, Ferguson BJ (2011) An efficient petiole-feeding bioassay for introducing aqueous solutions into 

dicotyledonous plants. Nature Protocols 6: 36-45 

Reid DE, Ferguson BJ, Hayashi S, Lin Y-H, Gresshoff PM (2011a) Molecular mechanisms controlling legume autoregulation of nodulation. 

Annals of Botany 108: 789-795 

Reid DE, Ferguson BJ, Gresshoff PM (2011b) Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule 

formation. Molecular Plant-Microbe Interactions 24: 606-618 

57 

2011






1600 – 1720 

Authors: Nijat Imin 1 , Isabel Saur 1 , Nadiatul Radzman 1 , Marie Oakes 1 , Michael A Djordjevic 1 

1 Plant Science Division and A. R. C. Centre of Excellence for Integrative Legume 

Research , Research School of Biology, Canberra, 0200, Australia. 

Presentation Title: Nodule specific CLE peptides mediate crosstalk between local nodule development and 

systemic autoregulation of nodulation pathways 


Root nodulation is controlled by the local Nod Factor and cytokinin-dependent pathways and systemically by the 

autoregulation of nodulation (AON) pathway (1). The AON pathway controls nodule number by suppressing nodulation 

competency in yet-to-be-infected roots via the shoot-expressing SUNN receptor kinase (1-3). 

Recently, MtCLE12 and MtCLE13, which show nodule-specific expression and encode CLE (CLAVATA3/ESR) peptide 

ligands, have been shown to regulate AON (3). The ectopic expression of MtCLE12 or MtCLE13 (but not MtCLE26 which 

does not have nodule specific expression) abolishes nodulation in wild type roots but not in the super nodulating null mutant 

sunn-4. This suggests that root MtCLE12 or 13 over-expression triggers constitutive AON which suppresses nodulation 

competency in a SUNN-dependent manner. Using qRT-PCR, we show that three local nodulation pathway genes: NODULE 

INCEPTION (NIN, a common regulator of the Nod Factor and cytokinin pathways), MtEFD (ethylene response factor and 

negative regulator of nodule differentiation) and MtRR8 (a type-A response regulator involved in negatively regulating 

cytokinin signaling) are each regulated specifically by MtCLE12 in a SUNN-dependent manner. This suggests that MtCLE12 

expression in the first formed nodules influences shoot SUNN to produce the AON signal that travels to the yet-to-be 

infected roots to up regulate MtEFD and MtRR8 to reduce nodulation competency by down regulating NIN. This would 

suppress local Nod Factor and cytokinin signaling at a common point: NIN. This proposed model links for the first time the 

pathways for Nod factor signaling, cytokinin perception and AON and provides a means for the plant to exert a mechanism 

to regulate nodule numbers in tune with its physiological status. 

58 

2011






1600 – 1720 

Authors: Senjuti Sinharoy 1 , Ivone Torres-Jerez 1 , Catalina Pislariu 1 , Mingyi Wang 1 , Vagner 

Benedito 2 and Michael Udvardi 1 

1. Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, Oklahoma 73401 

2. West Virginia University, Division of Plant & Soil Sciences, Morgantown, West Virginia 

26506 

Presentation Title: Identification & functional characterization of Medicago truncatula transcription factor 


mutants with impaired symbiotic nitrogen fixation 

Transcriptomic studies have revealed that thousands of plant genes are involved in symbiotic nitrogen fixation 

(SNF). Approximately two hundred transcription factor (TF) genes are under spatial control during nodule 

development (Pislariu et al.,unpublished data), indicating that they may have an important roles in establishing 

successful symbiosis. Very few TFs have been described that are required for SNF. To understand nodule 

organogenesis more deeply we need to characterize many more nodule TFs and the gene networks that they 

control. We constructed hypothetical gene regulatory networks consisting of TFs and their putative targets, 

based on correlation analysis of spatio-temporal gene expression data. Subsets of nodule-expressed TFs were 

chosen for further characterization. Medicago mutants with Tnt1-insertions in the chosen TFs were identified by a 

PCR-based screen of DNA pooled from the mutant population. Thirty one mutant lines with insertions in thirteen 

different TF genes were identified and phenotyped. Several mutants were found to have altered SNF. 

Mutants affected in a C2H2-TF gene were characterized in detail. Microscopical analysis of mutant nodules 12 

days post inoculation (DPI) indicated that bacteria were released from infection threads and surrounded by the 

symbiosome membrane, but remained small and did not elongate like bacteroids in wild type nodules. At 15 DPI, 

mutant nodules were dark/black in color, possibly the result of defense responses triggered after bacterial 

endocytosis, based on microscopical observations. Expression of the C2H2-TF gene in wild-type nodules was 

detected first at 4-DPI and increased around 1600 fold by 8-DPI, with highest expression in nodule zone II 

(invasion-zone) followed by zone III (inter-zone), consistent with the phenotype of the mutant. To identify 

possible target genes of the C2H2 TF, transcriptome analysis of mutant and wild-type nodules and of 

p35S::C2H2-TF and p35S::GFP-transformed roots was carried out, using the Affymetrix Medicago GeneChip. 

Detailed results and analysis of this work will be presented. 

59 

2011





Concurrent Session 9 – Native Legume RNB 

1600 - 1720 

Authors: Rashmita Parihar 1 , Neetu Poonar 1 , Nisha Tak 1 , Alkesh Tak 1 , Indu Sankhla 1 , Anil 

Tripathi 2 , Ravi Tiwari 3 , Euan K. James 4 , Janet I. Sprent 5 and H.S.Gehlot 1 

1 Department of Botany, Jai Narain Vyas University, Jodhpur, 342001, India 

2 School of Biotechnolgy, Banaras Hindu University, Varanasi, 221005, India 

3 Centre for Rhizobium Studies, Murdoch University, Murdoch, 6150, W Australia 

4 James Hutton Institute, EPI Division, Invergowrie, Dundee DD2 5DA, UK 

5 Division of Plant Sciences, University of Dundee at JHI, Invergowrie, Dundee DD2 

5DA, UK 

Presentation Title: Burkholderia phymatum isolated from root nodules of two species of Indigofera from 

alkaline soils of the Indian Thar desert 


It is long established that many species in the large Mimosoid genus Mimosa are nodulated by Beta-rhizobia 

(which so far include strains of Cupriavidus and Burkholderia), and that in its major centre of diversification in 

central Brazil there has probably been co-evolution between Mimosa and its Burkholderia symbionts (Bontemps 

et al. 2010; dos Reis Junior et al. 2010). Recently there has also been increasing evidence of nodulation and 

nitrogen fixation by Burkholderia in symbiosis with legumes outside the genus Mimosa. These include species of 

Cyclopia (Podalyrieae) and Rhynchosia (Phaseoleae) native to South Africa, and the agriculturally important 

legume Phaseolus vulgaris has also been shown to harbor B. phymatum as a symbiont in Morocco. In the 

present study we have isolated four strains (IL24, IL26, IC12 and IC14) of Burkholderia from root nodules of two 

Indian endemic species from the Papilionoid genus Indigofera (I. cordifolia and I. linnaei) growing in alkaline soils 

in the semi-arid Thar Desert in Western Rajasthan. Based on 16S rRNA gene sequences the strains are very 

close to those isolated from Mimosa spp., P. vulgaris and Indigofera suffruticosa. An ARDRA pattern obtained 

using AluI and Sau3AI, as well as an RPO1-based RAPD profile, showed that isolates IL24, IL26, IC12 and IC14 

from Indigofera were genetically identical to five isolates of Burkholderia from M. pudica (MP17, MP20, MP21, 

MP22 and MP23). Moreover, phenotypic characteristics (eg. colony morphology, pH range, NaCl tolerance, 

antibiotic sensitivity and C-utilization patterns) were also similar. These data are discussed in terms of the 

legume hosts and their various ecological niches within India, including such characteristics as climate and soils. 

Bontemps et al. 2010. Burkholderia species are ancient symbionts of legumes. Mol. Ecol. 19:44-52. 

dos Reis Junior et al. 2010. Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga 

biomes of Brazil. New Phytol 186:934-946. 

Acknowledgement: Authors acknowledge the financial support from ATSE Crawford Training Fund; Centre for 

Rhizobium Studies, Murdoch University and DBT, Govt. of India (BT/PR11461/AGR/21/270/2008). 

60 

2011






1600 - 1720 

Authors: Robert Walker 1,2 , Graham O‟Hara 2 , Lesley Mutch 1 , Lambert Bräu 2 & Elizabeth Watkin 1 

1 School of Biomedical Sciences, Curtin University, Bentley, 6845, Western Australia. 


Presentation Title: The phylogenetic diversity of australian Burkholderia root nodule bacteria 


Recently, members of the beta-proteobacteria were described as novel root nodule bacteria (coined betarhizobia) 

with many species represented by the genus Burkholderia and some Cupriavidus. Endosymbionts 

isolated from nodules of Mimosa spp. are predominantly Burkholderia spp. and this appears to be a preferential 

association however, Burkholderia spp. have also been isolated from Papillionoideae and other members of the 

Mimosoideae subfamily of legumes. Beta-rhizobia from Australia have been isolated from Acacia spp. and from 

members of the tribes Kennediinae and the endemic Mirbeliaea, however in glasshouse trials these isolates form 

ineffective nodules on many other Australian legume species. This study examined twelve authenticated 

Burkholderia sp. isolated from both native and invasive legumes from New South Wales, Western Australia, the 

Northern Territory, and two South African isolates from Rhynchosia ferulifolia. The type strains Burkholderia 

phymatum STM815 and Cupriavidus taiwanensis LMG19424 were also included. The phylogenetic diversity of 

these isolates was explored by examining the partial sequences from coding regions of the 16S rRNA, recA, 

dnaK and atpD genes. To determine the phylogenetic distribution within Australian isolates only, a concatenated 

neighbour-joining tree was constructed and most isolates fell into geographical clades. When including non- 

Australian isolates, Australian Burkholderia root nodule bacteria appear to form a distinct monophyletic group, 

separate to the Burkholderia cepacia complex (BCC) and from non-Australian isolates. Nodulation and nitrogen 

fixation genes were also partially sequenced and strongly align to nod and nif genes from Australian 

Bradyrhizobium spp. however, very few sequence data exist for the nodulation and nitrogen fixation genes of 

Australian Burkholderia root nodule bacteria. Australian root nodule Burkholderia may have evolved from a 

distinct monophyletic lineage from other beta-rhizobia allowing them to nodulate Australian legumes with varying 

effectiveness providing a competitive advantage over other soil saprophytes however, Australian beta-rhizobia 

may not be useful as effective nitrogen fixing endosymbionts. 

61 

2011





Authors 


1600 - 1720 

Euan K. James 1 , Osei Y. Ampomah 2 , Pietro P.M. Iannetta 1 , Gregory Kenicer 3 , Geoff 

Squire 1 , Janet I. Sprent 4 & Kerstin Huss-Danell 2 

1 James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK 

2 Department of Agricultural Research for Northern Sweden, Swedish University of 

Agricultural Sciences (SLU), SE-90183 Umeả, Sweden 

3 Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK 

4 Division of Plant Sciences, College of Life Sciences, University of Dundee at James 

Hutton Institute, Dundee DD2 5DA, UK 

Presentation Title: Nodulation and nitrogen fixation in native legumes in Scotland and Sweden 


Native legumes in temperate and northern areas have received surprisingly little research attention. Our aim was 

to get an overview of the nodulation of native legumes in Scotland and Sweden. We collected 24 out of the 47 

native legume species in Scotland and 30 out of the 90 in Sweden; 17 species were from both countries. A wide 

range of habitats were visited. A majority of our collected legumes, e.g. Lathyrus pratensis, Lotus corniculatus, 

Medicago lupulina, Trifolium spp. and Vicia spp. occurred in many habitats such as meadows, edges of 

cultivated land, waste ground and road sides. In contrast, other species grew in more specific habitats: 

Astragalus danicus, Lathyrus japonicus, L. palustris, Tetragonolobus maritimus and Vicia lutea on sea shores 

and A. alpinus, A. frigidus, Oxytropis campestris and O. lapponica in northern and mountain areas. All collected 

species were nodulated. Indeterminate (Astragalus, Cytisus, Lathyrus, Medicago, Melilotus, Ononis, Oxytropis, 

Trifolium, Ulex and Vicia, spp.) and determinate (Anthyllis, Lotus and Tetragonolobus spp.) nodule types were 

represented. Nitrogen fixation was deduced from pink nodule interior. Nodule anatomy, immunolocalization of 

nitrogenase and foliar 15 N analysis gave further support for active nitrogen fixation at time of collection. Some 

native species are closely related to cultivated species of Lotus, Medicago, Trifolium and Vicia. Root nodule 

bacteria from cultivated and native plants of Lotus corniculatus in Sweden mainly belonged to Mesorhizobium loti 

even though other Mesorhizobium spp. were associated with some of the naturally growing L. corniculatus plants 

(Ampomah & Huss-Danell 2011). It seems that native legumes are important to the N cycle at their sites and, in 

addition, some of their root nodule bacteria may be of interest as inoculants in agriculture. 

Ampomah OY& Huss-Danell K (2011). Genetic diversity of root nodule bacteria nodulating Lotus corniculatus 

and Anthyllis vulneraria in Sweden. Syst Appl Microbiol 34:267-275. 

62 

2011






1600 - 1720 

Authors: Wen Feng Chen 1 , Chang Fu Tian 1 , Yan Ming Zhang 1 , Qin Qin Li 1 , Jun Jie Zhang 1 , Mao 

Li 1 , Tian Yan Liu 1 , En Tao Wang 1,2 , Xing Hua Sui 1 , & Wen Xin Chen 1* 

1 State Key Laboratory for Agrobiotechnology/College of Biological Sciences, China 

Agricultural University, Beijing, 100193, China. 

2 Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto 

Politécnico Nacional, 11340 México D. F., México 

Presentation Title: Advances in rhizobial diversity and biogeography in China 


Besides the common genera, including the Rhizobium, Bradyrhizobium, Ensifer (former Sinorhizobium), 

Mesorhizobium which are widespread in China, nodule bacteria associated with leguminous plants are also 

found in the genera of Burkholderia, Cupriavidus, Shinella and Iniquilinus. By using the polyphasic taxonomy and 

multilocus sequence analysis (MLSA), nearly 30 novel species were described and published in China since 

2006, accounting for more than half of the total novel species published around the world during the same 

period. Some rhizobial species were only found and reported until now in China though their symbiotic genes 

were found worldwide. The rhizobial chromosomal core genes and the accessorial symbiotic genes may evolve 

in different ways to ensure the rhizobia to survive in the environments and to invade the host plant respectively. 

The increased reports on the non-symbiotic rhizobia isolated from root nodules will be the hot spots for the study 

of rhizobial diversity in China. The origin of the symbiotic genes and the transferring of them from symbiotic 

rhizobia to non-symbiotic ones may depend on the mechanism of “loss and gain”. To study the soil factors 

affecting the diversity and distribution of rhizobia in different regions, biogeography of the rhizobia associated 

with soybean (Glycine max), as example, were further extensively investigated. We will discuss the crucial 

factors that determine the distribution of genera of Bradyrhizobium, Sinorhizobium and Mesorhizobium 

associated with the soybean and the relationship between rhizobia and host plants. 

63 

2011




Session Details: Wednesday 30 November 2011 


0900 - 1030 

Author: Janet Sprent 

University of Dundee, UK 

Presentation Title: Poles apart: Nodulation in native legumes from the Southern and Northern hemispheres 


Publications on legume biogeography generally ignore nodulation parameters and are usually discussed from a 

longitudinal perspective, with a bias towards the Old and New worlds (1). However, it has long been known that 

nodulation parameters can be useful taxonomic ones (2). Recent evidence on the evolution of nodulation (3), on 

the functions of different cells inside nodules (4) and on both the nature of nodulating bacteria (5) and whether or 

not they become terminally differentiated (6) suggests that there may be a latitudinal dimension to nodule 

evolution. This paper discusses nodulated legumes north and south of the equator pointing out both differences 

and similarities. Some of the former may be related to soil nutrient levels, which in much of the Northern 

hemisphere are relatively rich and much of the Southern hemisphere relatively poor, leading in the latter to many 

legumes evolving a broader suite of nutrient uptake systems (dual mycorrhizas, cluster roots) than those in the 

North (7). Further, nodulated caesalpinioid legumes and most of the nodulated mimosoids are usually in low 

latitudes on either side of the equator. On the other hand, certain groups of legumes in the North have extended 

into the Arctic Circle and these too may have particular characters. Bridging the N/S divide are many legume 

genera, for example Indigofera, which are adapted to dry environments (8). Major questions remain to be 

answered, including the reasons why nodulation evolved on several different occasions, why some legumes that 

lack uninfected cells in the infected region appear to function well, why (in the Southern hemisphere) legumes 

growing side-by-side have a preference for either alpha or beta proteobacteria and cases where current 

taxonomy does not align with nodule characters. 

References numbered (1) to (8) will be given in full in the talk and made available to anyone interested. 

64 

2011






0900 - 1030 

Authors: Erik Limpens 1 , Sergey Ivanov 1 , Evgenia Ovchinnikova 1,2 , Alexey Borisov 2 , Rik op den 

Camp 1 , Stephane de Mita 3 , René Geurts 1 , Elena Fedorova 1,4 , Ton Bisseling 1 

1 Laboratory of Molecular Biology, Wageningen University, The Netherlands. 

2 All-Russia Research Institute for Agricultural Microbiology, Laboratory of Genetics of 

Plant-Microbe Interactions, St. Petersburg, Russia. 

3 Plant Diversity and Adaptation, Institute for Research and Development, Montpellier, 

France. 

4 Timiryazev Institute of Plant Physiology RAS, Moscow, Russia. 

Presentation Title: Formation of a symbiotic interface in rhizobial and mycorrhizal symbioses 


In both the rhizobium-legume symbiosis and the arbuscular mycorrhizal symbiosis the microbes are hosted 

intracellularly in a novel, specialized membrane compartment that forms a symbiotic interface. The formation of 

this interface is at the heart of symbiosis. In the case of rhizobia they are accommodated as novel nitrogen-fixing 

organelles, called symbiosomes, inside infected cells of the root nodule. Arbuscular mycorrhizal (AM) fungi form 

highly branched hyphal structures, called arbuscules, in infected root cortical cells. In both cases the microbial 

symbiont is surrounded by a plant derived membrane that has a special plasma membrane identity and where 

generally a structured plant cell wall is lacking. It has recently become clear that the signaling molecules and 

signaling pathway that trigger these different symbioses are strikingly similar. We show that this signaling 

pathway also controls the formation of the symbiotic interfaces in both symbioses. The observed plasma 

membrane identity of both interface membranes indicates a major role for exocytotic vesicle traffic. By studying 

plasma membrane (PM) SNARE proteins we show that knock-down of specific PM SNARE members blocks the 

release of the bacteria from cell wall bound infection threads and/or subsequent symbiosome development. 

Furthermore, two specific SNARE proteins required for symbiosome formation are also essential for arbuscule 

formation by AM fungi. These results suggest that in evolution the ability to produce Nod factors gave rhizobia 

the ability to use the ancient AM machinery to recruit a specific exocytotic pathway for the establishment of a 

symbiotic interface. 

65 

2011






0900 - 1030 

Authors: Doreen Fischer 1 , Barbara Pfitzner 1 , Veronica M. Reis 2 , Jean L. Simoes-Araújo 2 , Michael 

Schmid 1 , Esperanza Martinez-Romero 3 , Jose I. Baldani 2 , Anton Hartmann 1 

1 Helmholtz Zentrum Munich, German Research Center for Environmental Health, 

Research Unit Microbe-Plant Interactions, Neuherberg, Germany 

2 EMBRAPA-CNPAB Seropedica, Seropédica, RJ, Brazil 

3 Centro de Ciencias Genomicas, Universidad Nacional Autonoma de Mexico, UNAM, 

Cuernavaca, Morelos, Mexico 

Presentation Title: Molecular characterization of the active diazotrophic community in uninoculated and 

inoculated field-grown sugarcane (Saccharum sp.) 


Certain cultivars of sugar cane are known for their high potential of supporting biological nitrogen fixation. 

Although many different diazotrophic bacteria have been isolated from sugar cane and demonstrated to colonize 

roots, stems and leaves of these plants in considerable number, no clear evidence exists about the type of 

diazotrophic bacteria which fixes nitrogen in planta and about the locations of nitrogen fixation activity. To identify 

active diazotrophs in field grown sugarcane, 16S rRNA and nifH transcript analyses were applied. This should 

help to better understand the basis of the biological nitrogen fixation (BNF) activity in a high nitrogen fixing 

sugarcane variety. A field experiment using the sugarcane variety RB 867515 was conducted in Seropédica, RJ, 

Brazil, receiving the following treatments: unfertilised and fertilised controls without inoculation, unfertilised with 

inoculation. The five-strain mixture of diazotrophic bacteria containing Gluconacetobacter diazotrophicus, 

Burkholderia tropica, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans and Azospirillum amazonense, 

developed by EMBRAPA-CNPAB, was used as inoculum. Root and leaf sheath samples were harvested in the 

third year of cultivation (after receiving an inoculum at the beginning of the season) to analyse the 16S rRNA and 

nifH transcript diversity. In addition to nifH expression from Gluconacetobacter spp. and Burkholderia spp., a 

wide diversity of nifH transcripts from previously uncharacterised Ideonella / Herbaspirillum related phylotypes in 

sugarcane shoots as well as Bradyrhizobium sp. and Rhizobium sp. in roots were found. These results were 

confirmed using 16S cDNA analysis. From the inoculated bacteria, only nifH transcripts from G. diazotrophicus 

and B. tropica were detected in leaf sheaths and roots at the time of harvest in late summer, respectively. Thus, 

well known as well as yet uncultivated diazotrophs were found active in sugarcane roots and stems using 

molecular analyses. Two strains of the inoculum mix were found active at the late summer harvest. 

66 

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The Cape Floristic Region (CFR) has a mosaic of soil types derived from parent materials including sandstone, 

granite, limestone and shale. It has high species diversity, varying among soil types and environmental 

gradients. Among legumes, particular lineages appear to be restricted to one or few substrates, raising questions 

on whether their species distribution is predetermined by symbiotic bacteria. We hypothesized that rhizobial 

isolates from indigenous legumes would cluster phylogenetically according to soil types and/or host legume. 

Rhizobia were isolated from nodules collected from 87 species in over 12 genera belonging to tribes 

Crotalarieae, Indigofereae, Phaseoleae, Podalyrieae and Psoraleae. Sequenced data using the 16S rRNA gene 

revealed the presence of both α- (Bradyrhizobium, Mesorhizobium, Rhizobium, Synorhizobium) and β-rhizobia 

(Burkholderia), the latter nearly exclusive to the tribe Podalyrieae. Mesorhizobium was isolated from Aspalathus, 

Otholobium, Psoralea, Indigofera and Argyrolobium species collected from all substrates, while Bradyrhizobium 

species were isolated from Indigofera and Tephrosia species collected from granite and sandstone-derived soils. 

Burkholderia species were isolated from Podalyria, Amphithalia and Rafnia species collected from granite, 

coastal sand and limestone derived soils. These data were confirmed by immunogold labeling of the bacteria in 

nodule sections with an antibody specific to Burkholderia (Elliott et al. 2007a). Furthermore, a GFP-tagged strain 

of B. tuberum STM678, which has previously been shown to nodulate species of Cyclopia (tribe Podalyrieae 

native to the CFR; Elliott et al. 2007b), could effectively nodulate species of Podalyria and Virgilia. The 

phylogenies of the rhizobia isolates from the 16S rRNA gene sequence and of the host legumes from literature 

are discussed in relation to the evolutionary relationships of the symbiotic partners. The available data suggest 

that the soil types do not influence the distribution of rhizobia in the CFR. 

Elliott, G. N. et al. 2007a. New Phytol. 173:168-180. 

Elliott, G. N. et al. 2007b Ann. Bot. 100:1403-1411. 

Concurrent Session 10 – Ecology of RNB 

1100 – 1230 

Authors: Oscar Dlodlo 1 , A. Muthama Muasya 1 , Janet I. Sprent 2 , Euan K. James 3 , Wen-Ming 

Chen 4 , Samson B.M. Chimphango 1 

1 University of Cape Town, Botany Department, Private Bag X3 Rondebosch, South 

Africa. 

2 Division of Plant Sciences, University of Dundee at JHI, Invergowrie, Dundee DD2 

5DA, UK 

3 James Hutton Institute, EPI Division, Invergowrie, Dundee DD2 5DA, UK 

4 Dept. of Seafood Science, National Kaohsiung Marine University, Kaohsiung City 811, 

Taiwan 

Presentation Title: Characterization of the cape floristic region rhizobia using 16S rRNA gene sequences 

and their distribution in different soil types 


67 

2011






1100 – 1230 

Authors: Macarena Gerding 1,2 , Graham O‟Hara 1 , Lambert Bräu 1 & John Howieson 1 


2 Facultad de Agronomía, Universidad de Concepción, Casilla 537, Chillán, Chile. 

Presentation Title: Mesorhizobium strains from the South African herbaceous legume Lessertia spp. 


differ in their competitive ability in Australian soils 

Legumes of the South African genus Lessertia, along with their microsymbionts, were introduced into different 

sites of the Western Australia wheatbelt during 2007-2009. Four Lessertia spp. achieved only poor establishment 

followed by weak summer survival. This was caused in part by low levels of nodulation with the inoculant strains, 

and by competitive ineffective nodulation from naturalized strains of Rhizobium leguminosarum bv. trifolii. To 

improve the adaptation of the legume and to assess the competitive ability of inoculant strains, two field 

experiments were set up at Karridale, Western Australia. The aims were to assess the effect of increased doses 

of an effective inoculant strain (WSM3565) with L. herbacea, and to study the competitive ability and symbiotic 

performance of different Mesorhizobium strains nodulating L. diffusa. 

Increasing the inoculation dose of L. herbacea with WSM3565 did not improve establishment and survival of the 

legume. Although WSM3565 nodule occupancy improved from 28 to 54% with higher doses of inoculation, none 

of the treatments increased L. herbacea yield over the inoculated control. 

The inoculation of L. diffusa with the strains WSM3598, 3636, 3626 and 3565 resulted in greater biomass 

production than the uninoculated control. These strains were able to outcompete resident rhizobia and to occupy 

a high (>60%) proportion of lateral root nodules. Low rainfall in 2009 caused an early senescence of tap root 

nodules and plants had to rely on nitrogen fixation from nodules formed later in the season (i.e. lateral root 

nodules). This possibly explained the increased dry weight achieved by these strains. 

The high numbers of resident rhizobia in Western Australian soils, and their ability to rapidly nodulate Lessertia 

spp. are likely to be the main reasons for the low nodule occupancy achieved by some effective inoculant strains 

with L. diffusa and L. herbacea. The naturalised strains that achieved nodulation were identified as R. 

leguminosarum bv. trifolii, whereas in the South African soils Lessertia is nodulated by mesorhizobia. This 

phenomenon represents a barrier to the successful introduction of the exotic legume genus Lessertia to WA 

soils. 

68 

2011






1100 – 1230 

Authors: Julie K. Ardley 1 , Graham O‟Hara 1 , Wayne Reeve 1 , Ron Yates 1,2 , Michael Dilworth 1 , and 

John Howieson 1 

1 Centre for Rhizobium Studies, Murdoch University, Murdoch W. A. 6150, Australia 

2 Department of Agriculture Western Australia, Baron Hay Court, South Perth, 6151, 

Western Australia 

Presentation Title: The symbiosis between Listia spp. and Methylobacterium and Microvirga rhizobia: 

specificity in epidermally infected legumes 


Lotononis is a genus in the Crotalarieae tribe with a centre of origin in South Africa. The taxonomy has recently 

been revised and the three distinct clades of this genus are now recognised at the generic level as Listia, 

Leobordea and Lotononis sensu stricto (Boatwright et al., 2011). Different symbiotic specificity groups exist 

within Lotononis sensu lato, which is nodulated by a remarkable diversity of rhizobia. Leobordea and Lotononis 

s.str. species form indeterminate nodules and are more or less promiscuous. Rhizobia associated with these 

legumes are related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and 

Methylobacterium nodulans, based on sequence analysis of the 16S rRNA gene. Listia species are waterlogging 

tolerant, have adventitious roots and form lupinoid nodules. They are specifically nodulated by pink-pigmented 

methylobacteria that are effective nitrogen fixers on all studied species, with the exception of Listia angolensis, 

which forms ineffective nodules with these rhizobia (Yates et al., 2007). L. angolensis forms effective nodules 

only with novel rhizobial species of the Alphaproteobacterial genus Microvirga (Ardley et al, submitted paper). 

The nodA sequences of the Lotononis s. l. rhizobia are polyphyletic and group according to microbial 

chromosomal background rather than host plant taxonomy. L. angolensis and Listia bainesii appear to be 

infected via epidermal entry, with no formation of infection threads, and are examples of symbiotic specificity in a 

non root-hair-mediated infection process. The genomes of Methylobacterium sp. 4-46 and Microvirga strain 

WSM3557 have been sequenced and will greatly aid an understanding of the genetic underpinnings of these 

symbioses. A preliminary analysis of the WSM3557 genome compared with that of M. sp. 4-46 will also be 

presented. 

Boatwright, J. S., Wink, M. & van Wyk, B.-E. (2011). The generic concept of Lotononis (Crotalarieae, 

Fabaceae): Reinstatement of the genera Euchlora, Leobordea and Listia and the new genus Ezoloba. Taxon 60, 

161-177. 

Yates, R. J., Howieson, J. G., Reeve, W. G., Nandasena, K. G., Law, I. J., Bräu, L., Ardley, J. K., 

Nistelberger, H. M., Real, D. & O'Hara, G. W. (2007). Lotononis angolensis forms nitrogen fixing, lupinoid 

nodules with phylogenetically unique, fast-growing, pink-pigmented bacteria, which do not nodulate L. bainesii or 

L. listii. Soil Biology & Biochemistry 39, 1680-1688. 

69 

2011






1100 – 1230 

Authors: Tomasz Stępkowski 1 , Alison McInnes 2 , Elizabeth Watkin 3 , Dorota Narożna 4 , Magdalena 

Mantaj 1 , Justyna Rudnicka 1 , Graham O‟Hara 5 , Emma T. Steenkamp 6 

1 Institute of Bioorganic Chemistry Polish Academy of Sciences, Poznań , Poland; 

2 Centre for Plant and Food Science, University of Western Sydney, Penrith South DC, 

Australia; 

3 School of Biomedical Sciences, Curtin University, Perth, Australia 

; 4 Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, 

Poznan , Poland; 

5 Centre for Rhizobium Studies, Murdoch University, Murdoch, Australia 

; 6 Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 

South Africa 

Presentation Title: Diverse origin of Bradyrhizobium strains nodulating legumes native to the tropicalmonsoon 

part of Australia 


The geographical isolation of Australia, together with the formation of an arid climate in its central regions and a 

tropical-monsoon climate in its northern part impacted significantly on the evolution of legumes native to the 

continent. Additionally, the northward drift of the Australian plate and its collision with the Euroasian plate 

facilitated the colonization of Australia by many alien legume taxa during the last ten million years. Conceivably, 

these factors also shaped the evolution of root-nodule bacteria associated with these legumes, and contributed 

to the high diversity of indigenous rhizobium communities and the presence of many groups unique to Australia. 

By making use of multilocus sequence analysis we investigated 132 strains of Bradyrhizobium, which originated 

from the tropical-monsoon part of the Northern Territory. All strains were obtained from root nodules of legumes 

belonging to endemic or native Australian tribes, genera or species. Phylogenetic analyses based on six 

housekeeping genes (atpD, dnaK, glnII, gyrB, recA and rpoB) separated the strains into 16 lineages, of which 

most were different from the known species of Bradyrhizobium. Phylogenies based on the symbiotic nodA and 

nifD genes separated the strains into six clades (i.e., Clade I (subgroup I.2), III (subgroup III.3), IV, V, VII and 

VIII), out of the eight clades that have been described thus far for Bradyrhizobium. Legumes with centers of 

diversification located outside Australia were nodulated predominantly by strains belonging to Clade III, which is 

regarded as a cosmopolitan, pantropical group. Surprisingly, this was also true for all strains from Clade IV, 

which suggests that this apparently Australian clade may have originally diversified outside this continent. 

Conversely, the legumes with Australian centers of diversity (e.g. phylodinous Acacia spp.) were nodulated by 

strains representing the Australian Clade I and Clade VIII, but also by Clade V, which also occurs in South 

America. 

70 

2011





Concurrent Session 11 – Nodule Formation 

1100 – 1230 

Authors: Shin Okazaki and Kazuhiko Saeki 

Department of Biological Sciences, Faculty of Science, Nara Women’s University, Nara 

630-8506, Japan 

Presentation Title: Hijacking the host nodulation signaling by rhizobial type III secretion system 


Root nodule symbiosis between leguminous plants and nitrogen-fixing bacteria (rhizobia) requires molecular 

communication between both partners. Key components for the establishment of symbiosis are host plantderived 

flavonoids that induce the transcription of rhizobial nodulation (nod) genes and rhizobium-produced 

lipochitooligo-saccharides (Nod-factors) that initiate nodule development and bacterial entry. Besides the Nodfactors 

there are other determinants that influence the extent of the symbiosis. Among them, we have focused on 

a rhizobial protein secretion system, called type III secretion system (T3SS). 

T3SSs play an essential role in the pathogenicity of many bacteria infecting humans, animals and plants. 

Pathogenic bacteria use the T3SS to deliver effector proteins directly into eukaryotic cells or the external 

environment, where they manipulate host cellular processes to promote pathogenicity. The T3SSs have been 

identified in rhizobia and were shown to affect symbiosis with leguminous hosts. In this study, we analyzed the 

role of T3SS in the interaction between Bradyrhizobium elkanii and soybean (Glycine max (L.) Merr.). 

Mutational analysis and inoculation tests of B. elkanii USDA61 revealed that the presence of T3SS affected 

symbiotic capacity either positively or negatively depending on host genotype. On G. max cv. Enrei, wild-type 

USDA61 induced more nodules than T3SS mutant. On the other hands, cultivar Hill interdicted nodulation by the 

wild type but was nodulated by the T3SS mutant. Intriguingly, when infected to the soybean mutant En1282 that 

has defective Nod factor receptor 1 (NFR1) and show non-nodulating phenotype with B. japonicum and other 

rhizobial strains, USDA61 but not its T3SS mutant induced effective nodules. Transcriptional analysis revealed 

that the expression of early nodulation gene ENOD40 and NIN was increased in the root of En1282 inoculated 

with wild type but not with T3SS mutant. These results suggest that T3SS of USDA61 has functions to enforce 

legume host to initiate symbiotic programs by bypassing Nod-factor recognition. 

71 

2011






1100 – 1230 

Authors: Christian Staehelin, Ling Zhang, Ying-Ying Ge, Da-Wei Xin, Sha Liao and Zhi-Ping Xie 

State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-Sen (Zhongshan) 

University, East Campus, Guangzhou 510006, China 

Presentation Title: Characterization of type 3 effectors in the Rhizobium-legume symbiosis 


Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) into 

eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation 

in the interaction with various host legumes. Here, we characterized NopL and NopM, two type 3 effectors of 

Rhizobium sp. strain NGR234. Nodulation tests and microscopic analysis showed that distinct necrotic areas 

were rapidly formed in ineffective nodules of Phaseolus vulgaris (cv. Tendergreen) induced by a nopL mutant, 

indicating that NopL antagonized nodule senescence. Further experiments revealed that NopL interfered with 

mitogen-activated protein kinase (MAPK) signaling in yeast and plant cells (Nicotiana tabacum). Expression of 

nopL in yeast disrupted the mating pheromone (α-factor) response pathway, whereas nopL expression in N. 

tabacum suppressed cell death induced by over-expression of the MAPK gene SIPK (salicylic acid-induced 

protein kinase). NopL was multiply phosphorylated either in yeast or N. tabacum cells that expressed nopL. Four 

phosphorylated serines were confirmed by mass spectrometry. All four phosphorylation sites exhibit a Ser-Pro 

pattern, a typical motif in MAPK substrates. These data suggest that NopL is a suppressor of MAPK signaling. 

NopM, another type 3 effector of Rhizobium sp. NGR234 displayed E3 ubiquitin ligase activity in vitro, whereas 

the mutant protein NopM-C338A was inactive. Corresponding mutant analysis indicated that NopM also acts as 

an E3 ubiquitin ligase during symbiosis with Lablab purpureus, providing evidence that NopM is delivered into 

legume host cells and targets host proteins. Finally, effects of NopM on eukaryotic cells will be reported. 

Zhang L, Chen X-J, Lu H-B, Xie Z-P, & Staehelin C (2011). Functional analysis of the type 3 effector NopL from 

Rhizobium sp. NGR234: symbiotic effects, phosphorylation and interference with MAPK signaling. J. Biol. Chem. 

in press 

72 

2011






1100 – 1230 

Authors: Masaki Hanyu 1,2 , Shin Okazaki 1 , Rie Shirai 1 and Kazuhiko Saeki 1 

1 Department of Biological Sciences, Faculty of Science, Nara Women’s University, 

Nara630-8506 

2 Department of Biological Sciences, Graduate School of Science, Osaka University, 

Toyonaka, Osaka 560-0043, Japan 

Presentation Title: Mesorhizobium loti mutant lacking superoxide dismutase displays Lotus-host accessiondependent 

nitrogen fixation capacities 


Protection against reactive oxygen species (ROS) is important for legume-nodulating rhizobia during the 

establishment and maintenance of symbiosis as well as under free-living conditions because legume hosts might 

assail incoming microbes with ROS and because nitrogenase is extremely sensitive to ROS. We generated a 

deletion mutant of the putative superoxide dismutase (SOD) gene, with locus tag mlr7636, in Mesorhizobium loti 

MAFF303099 to investigate physiological significance. Under free-living conditions, the mutant displayed a 

number of properties typical of SOD-lacking strains. Its cell extract showed no general SOD activity under the 

assay conditions that enabled to detect the activity of the wild-type. It proliferated very slowly and showed 

elevated sensitivity to exogenous superoxide generators. Its spontaneous mutation rate was approximately 20times 

of that of wild-type. These defects were complemented by re-introduction of the wild-type gene. Based on 

the result and sequence similarity with Escherichia coli enzyme, we named the gene sodA. With regard to 

symbiotic capacities with Lotus japonicus, the sodA mutant showed quasi-wild-type nodulation and nitrogen 

fixation efficiencies with the host accession Gifu B-129, whereas it showed low nodulation efficiency and poor 

support of plant growth with the host accession Miyakojima MG-20. 

73 

2011





Rhizobium-legume symbiosis is an ecologically friendly source of nitrogen to improve soil fertility. Nitrogen-fixing 

Rhizobia are situated in root nodule in a specialized endosomes called symbiosomes (SBs). Host plant derived 

SB membrane (SM) is the interface providing the metabolic exchange between two partners. During the 

development SBs are multiplying and growing in volume more than 40 times. We have performed the 

quantitative estimation of nodule infected cell structures in the course of symbiosis using 3D reconstruction of 

confocal images of Medicago truncatula. We have specified the particular cell layer with the highest level of 

symbiosomes growth. Surprisingly in this cell layer host cell vacuole volume was drastically diminished. We 

hypothesized that transport of water is redirected in this cell layer from vacuole to bacteroids and potential 

symbiosome membrane aquaporins should be involved. Q-PCR analysis of 6 tonoplast aquaporins expression 

level has revealed that MtTIP1g is significantly upregulated. The localization of GFP-tagged MtTIP1g has shown 

host tonoplast labeling in all nodule cells, but SMs acquired GFP only in the cell layer where most rapid growth of 

SBs happens. Functional analysis of MtTIP1g in root nodules using an RNAi method showed that partial 

silencing of MtTIP1g is causing the arrest of SBs development and preventing the formation of nitrogen-fixing 

SBs. 

According to our previous data symbiosome membrane has mixed identity and coopting the molecular markers 

of exo- and endocytotic pathway (Limpens et al., 2009). It is established that SBs are accepting plasma 

membrane identity immediately after being released to host cell, but the identity of young vacuole it obtains much 

later. The functional significance of this identity change was not known. Our data are providing the clue to 

functionality of SM identity. The acceptance of vacuole identity is crucial as it permits to recruit vacuolar proteins 

like MtTIP1g and promote an extremely rapid and robust SBs growth. 

Limpens et al., Plant Cell 21: 2811–2828 (2009). 


1100 – 1230 

Authors: Aleksandr Gavrin 1 , Sergey Ivanov 1 , Ton Bisseling 1 , Elena Fedorova 1 

1 Laboratory of Molecular Biology, Wageningen University, Wageningen, 6708PB, The 

Netherlands 

Presentation Title: The role of symbiosome membrane aquaporin MtTIP1g in bacteroids growth and 

maturation 


74 

2011





Concurrent Session 12 – PGPR & Plant Production 

1100 – 1230 

Authors: Sharon Fox 1 , Graham O‟Hara 1 and Lambert Bräu 1 


Presentation Title: Differential effects of Pseudomonas fluorescens WSM3457 on enhanced 

Medicago nodulation 


Pseudomonas fluorescens WSM3457 has been studied as a nodulation increasing bacteria (NIB) since 2006 

due to increases in the nodulation of subterranean clover reported in glasshouse and field trials. We have 

demonstrated a similar phenomenon on the Medicago truncatula / Sinorhizobium medicae WSM419 symbiosis. 

During glasshouse trials it was observed that co-inoculation results in larger, pinker nodules, located higher on 

the root system, compared to M. truncatula inoculated with only S. medicae WSM419. 

Nodule location suggested that nodules were elicited earlier on co-inoculated plants and this was confirmed 

during studies comparing the rate of nodule initiation on M. truncatula inoculated with either S. medicae WSM419 

or both WSM419 and P. fluorescens WSM3457. The results also demonstrated that there was a significant 

increase in symbiotic effectiveness, both in terms of plant mass and total fixed N. 

Following those results it was investigated whether co-inoculation increased the effectiveness of a partially 

effective symbioisis, namely that of the symbiosis between S. meliloti 1021 with M. truncatula. The results show 

that there was no significant increase in shoot mass with co-inoculation with P. fluorescens WSM3457. However, 

the nodulation data tells a conflicting story, with enhanced nodulation scores on M. truncatula when P. 

fluorescens WSM3457 was co-inoculated with S. meliloti 1021. 

75 

2011






1100 – 1230 

Authors: Khanok-on Amprayn and Ivan Kennedy 

SUNFix Centre for Nitrogen Fixation 

University of Sydney, NSW 2006, Australia 

Presentation Title: Proteomics of rice-PGP microorganisms' interactions 


A plant growth-promoting (PGP) bacterium Pseudomonas fluorescens 1N (Pf1N) and a soil yeast Candida 

tropicalis HY (CtHY) have been used as two of the components of a commercial biofertiliser named BioGro to 

enhance growth and yield of rice allowing reduce chemical applications in Vietnam for many years. The present 

study verified the colonisation on rice roots and the effects of the strains on rice root mechanisms as observable 

morphological changes were correlated with results by proteomics. In addition, bacterial proteins mediating the 

interaction between rice root and Pf1N were investigated. 

To explain the strains influence on rice root growth, gel-based comparative proteomics and mass spectrometry 

were employed. A comparison of 2-DE protein profiles between untreated control and PGP microbes treated 

roots revealed that the inoculation of the Pf1N contributed 11 differentially expressed proteins in rice roots, while 

20 and 18 root proteins were differentially expressed in CtHY and co-inoculation of Pf1N and CtHY treatment. Of 

these changed proteins, the identity of 20 proteins was determined by MALDI TOF MS. The protein identification 

revealed that responsive plant proteins belonged to eight major metabolic processes including catabolism and 

respiration (energy production), transcription, protein synthesis, protein destination and storage, secondary 

metabolism, membrane transporters, disease-defense, and unknown. 

Conversely, the bacterial proteome alteration in presence of root exudates was also profiled using 2-DE and MS 

to examine of bacterial adaptation in association with rice. The bacterial protein expressions revealed several 

mechanisms involved in protein transcription and synthesis, primary metabolism, cell envelope development, 

siderophore production, nutrient transportation, general stress response, and unknown. The patterns of protein 

expression of Pf1N such as elevated iron uptake mechanisms, repressed essential growth-related proteins and 

ABC transporter suggested that the bacterium was adapted to form biofilm. 

76 

2011






1100 – 1230 

Authors: Iris Marcano 1 , César A. Díaz-Alcántara 1 , Beatriz Urbano 2 , Encarna Velázquez 3 , Fernando 

González-Andrés 4 . 

1 Facultad de CC. Agronómicas y Veterinarias. Univ. Autónoma de Santo Domingo. 

Dominican Republic. 

2 Departamento de Ingeniería Agrícola y Forestal. Universidad de Valladolid. Spain. 

3 Departamento de Microbiología y Genética. Universidad de Salamanca. Spain. 

4 Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad. Universidad de León. 

Spain. 

Presentation Title: In vitro plant growth promotion properties of endophitic bacteria isolated from banana 

(Musa sp.) roots in Dominican Republic 


Organic agriculture, using rizospheric or endophytic bacteria which promote plants growth (PGPR) is one of the 

chief strategies to reduce the use of Chemicals in agriculture. The present study had the objective of evaluates 

endophytic rhizobacteria isolated from banana roots (Musa sp) and other crops, from four provinces in 

Dominican Republic (Mao, Azua, Montecristi and Dajabón), in order to make a preselection of bacterial strains to 

elaborate biofertilisers for banano organic crops in Dominican Republic. The total number of isolates was 160. 

The RAPD pattern obtained after amplification of the DNA with the primer M13 was used to identify possible 

clones, and the collection was reduced to 144 different strains. The 144 strains were evaluated in vitro by their 

efficiency producing the hormone IAA, the evidence of siderophores production and the ability of solubilise 

insoluble mineral phosphate. Sixteen per cent of the 144 strains produced significant rates of IAA, 22% produced 

siderophores and 11% solubilized P. Only 14 strains solubilised inorganic P and produced siderophores at the 

same time, and none of them were among the best producers of IAA, indicating that it is not common to find 

strains with several in vitro plant growth promotion properties simultaneously. The strains with highest plant 

growth promotion properties in vitro have been identified by 16S rRNA sequencing and comparison with 

international databases, to ensure that they belong to non-pathogenic taxa, following the phase of in vivo 

selection. By the moment, 6 of the 14 strains that solubilize inorganic P and produce siderophores, were 

identified, and 4 belonged to the species Pseudomonas taiwanensis, one to P. mosselii and one to Enterobacter 

absuriae, all of them common endophytic bacterial taxa (Dobbelaere et al., 2003). 

Dobbelaere, S. Vanderleyden, J., Okon, Y. 2003. Plant Growth-Promotign Effects of Diazotrophs in the 

Rhizosphere. Critical Reveiws in Plant Sciences, 22 (2): 107-149. 

77 

2011






1100 – 1230 

Author: Ivan Kennedy 

SUNFix Centre for Nitrogen Fixation, University of Sydney, Australia 

Presentation Title: Beneficial biofilms on plant root surfaces – achieving more sustainable crop production 


Plant growth promotion (PGP) for crops by microorganisms have been recognised for some time in biological 

nitrogen fixation (BNF) in legumes, shown in Germany in the late 19 th century in the face of opposition by Justus 

Von Liebig. More subtle are the beneficial effects of the PGP microbes in cereals and other crops. These 

involve a complex of phytohormonal effects, nutrient mobilisation, BNF and biocontrol that benefit plant growth 

and overall yield (Kennedy et al. 2008). Essentially, it is possible to grow crops such as rice inoculated with 

microbes forming biofilms on the surfaces of their roots while that allow significantly reduced inputs of chemical 

fertilisers, seed, pesticides and water, as well as reduced harvest losses. 

This presentation will focus on properties of a successful biofertiliser, BioGro ─ a commercial product invented in 

Vietnam consisting of four strains (Pseudomonas, Bacillus and a yeast) inoculated to the rhizosphere of rice 

seedlings. It is remarkable that BioGro contains strains selected empirically on the basis of their effectivness for 

rice farmers later identified in Australia as including species recognised around the world in research laboratories 

as having a strong basis for beneficial effects as biofertilisers. 

As was the case about a century ago with legumes and the rhizobia that form N2-fixing nodules on their roots, 

obtaining an effective outcome from applying these biofilm-forming organisms to crops in the field is challenging. 

It may be important to match microbial strains with plant species but it is certainly important that inoculants of 

high quality in terms of particular microbial strains in sufficient numbers (ca. 10 8 cells per g of inert carrier). 

Furthermore, convincing farmers of the beneficial effects and the scale of economic benefits possible is also 

important. The design of an efficient supply chain that includes quality control and delivery of economic and 

environmental benefits from biofertilisers to farmers is equally challenging. 

In this presentation, a strategy for simultaneously achieving environment-friendly and economic benefits being 

developed in a World Bank Development Marketplace project Sustaining nitrogen-efficient rice production, will be 

described. Lessons learnt from this project found to be important for implementation of new technology based 

on these microbial biofilms in both developing and developed countries will be discussed. 

Efficient nutrient use in rice production in Vietnam achieved using inoculant biofertilisers, I.R. Kennedy et al., 

eds. ACIAR Proceedings 130, 2008, book downloadable from www.aciar.gov.au. 

78 

2011






1330 - 1500 

Authors: Ross Ballard 1,5 , Nigel Charman 1,5 , Alan Humphries 1,5 , Matthew Denton 2,3,5 , Lori 

Phillips 2,5 , David Pearce 2 , Shoba Venkatanagappa 4,5 & Tim O‟Brien 4 

1 South Australian Research and Development Institute, GPO Box 397, Adelaide, South 

Australia, 5001. 

2 Department of Primary Industries Victoria, Rutherglen, Vic 3685. 

3 The University of Adelaide, Waite Campus, Glen Osmond SA 5064 

4 NSW Department of Primary Industries. Tamworth Agricultural Institute, Tamworth 

NSW 2340. 

5 Cooperative Research Centre for Future Farm Industries. Crawley, WA, 6009. 

Presentation Title: Limits to lucerne nodulation in acidic field soils 


Lucerne is valued for its supply of high quality feed throughout the year, N2-fixation and environmental benefits. A 

constraint to the wider use of lucerne is the sensitivity of the symbiosis and in particular the rhizobia to low pH. 

Hence, lucerne is not recommended for soils below pH 5 (CaCl2). Ten strains of rhizobia that resulted in large 

improvements in lucerne nodulation in solution culture at pH 4.8 are being evaluated at four field sites ranging in 

pH (CaCl2) from 4.1 to 4.7. Lucerne nodulation, and plant survival and production were measured in the two 

years after establishment. Saprophytic competence of the strains has also been determined. 

Large differences in plant nodulation 10 months after sowing occurred at the different sites. At the site with soil 

pH 4.7, 64% of plants were nodulated compared to 8% at the site with soil pH 4.1. Differences in nodulation by 

the strains of rhizobia were smaller. Strains SRDI684, SRDI672 and SRDI736 ranked highest overall. Strain 

SRDI736 was also shown to have greater saprophytic competence at pH 4.7 than RRI128 which is used in 

commercial inoculants. 

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 

site). To date strain of rhizobia has not significantly (P






1330 - 1500 

Author: Murray Unkovich 

Agriculture, Food and Wine, University of Adelaide, PMB 1, Glen Osmond, SA 5064 

Presentation Title: Nitrogen fixation and nitrogen isotope fractionation in legumes 


Isotope fractionation occurs when there are changes in the partitioning of isotopes between a source and a 

product. For biological N2 fixation it equates to differences in the relative abundance of N isotopes between 

atmospheric N2 and fixed NH4 + in bacteria. The literature is somewhat confusing on this topic and a range of 

fractionation factors has been proposed. For N2 fixing legume symbioses, this fractionation becomes important 

when using natural variations in 15 N abundance to quantify N2 fixation where there is a need to be clear about the 

extent and source of fractionation. On a whole plant basis there is very little evidence for any isotope 

fractionation, and indeed if one considers the biochemistry and kinetics of the N2 fixation process one would not 

expect any fractionation. Evidence is presented that sources of variation in apparent isotope fractionation may 

have arisen from a number of factors including errors in measurement and processes other than N2 fixation 

contributing to apparent fractionation. For legumes, differences in the efficiency of symbiont bacteria and 

differences in plant age appear to influence shoot 15 N. It is important that these factors are teased out so that 

isotope fractionation can be reliably measured and better exploited in N cycle studies at plant and ecosystem 

scales. Careful attention needs to be paid to standardisation of mass spectrometric analysis, particularly since 

the adoption of continuous flow machines which have tended to divert focus to relative and precise rather than 

accurate measurement, such that measurement aganst atmospheric N2, the primary standard is no longer 

undertaken, even though it is readily available. More accurate rather than more precise measurement of 15 N will 

permit a better understanding of the N exchanges which occur between between legumes and their 

microsymbionts, which are still only partly understood, and also between ecosystem components. 

80 

2011






1330 - 1500 

Authors: Xi Chen, Theja Shidore, Theresa Dinse, Sabrina Gemmer, Thomas Hurek and Barbara 

Reinhold-Hurek 

Department of Microbe-Plant Interactions, University of Bremen, Postfach 33 04 40, 

28334 Bremen. 

Presentation Title: Functional genomic analyses of endophyte – rice Interactions 

Presentation Time: 1430 -1500 

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agro-biotechnological interest 

because it supplies fixed nitrogen to its host and colonises plants in remarkably high numbers without eliciting 

disease symptoms. This raises the question of mechanisms of compatible interactions between host and 

bacterium. The complete genome of strain BH72 was sequenced (1), and the rice genome is also available. This 

allows application of functional genomic analyses of both partners during interaction. For symbiotic interactions 

such with rhizobia and arbuscular mykorrhiza (AM), common plant signalling cascades are now well 

characterized. The results of our analysis of differential gene expression in Azoarcus-infected rice roots and 

mutational analyses suggest that the cascades are not operating during endophytic interactions. Nevertheless, 

transcriptomic analysis demonstrated that both partners show extensive metabolic adaptations during endophytic 

interaction. Transcriptomic analyses were carried out for both partners, rice as well as exudates-exposed 

Azoarcus sp. They allowed identification of candidate genes which were shown by mutational analysis to be 

required for optimal rhizosphere fitness in Azoarcus sp. 

(1) Krause et al. 2006. Genomic insights into the lifestyle of the mutualistic, N2-fixing grass endophyte Azoarcus 

sp. strain BH72. Nature Biotechnol. 24: 1385-1391. 

81 

2011





Concurrent Session 13 - Inoculant Quality & Application 

1600 - 1740 

Authors: Rosalind Deaker, Andrea Casteriano, Elizabeth Hartley and Greg Gemell 

University of Sydney, Australia 

Presentation Title: Making the most of high quality inoculants: survival of rhizobia during application to 

legume crops 


Despite developments in inoculant technology and a high level of compliance of commercial legume inoculants 

to quality standards, poor survival of rhizobia during delivery to legume crops and pastures is still a major issue. 

Loss of viability of inoculant microorganisms limits the potential for maximum nodulation by elite strains and thus 

nitrogen fixation and yield. Legume inoculants are most commonly applied to seed prior to sowing; however, the 

seed surface presents a hostile environment for rhizobia exposing cells to desiccation stress and potentially toxic 

chemicals. Inoculant technology balances an incomplete scientific understanding of growth and survival with 

production and market demands. As a result, the potential for rhizobia to survive seed coating operations both 

commercially and on farm is not maximised. 

Polymer adhesives are typically used to fasten inoculants, growth enhancing and pelleting materials to seed. 

However, polymer properties that enhance survival of inoculant microorganisms are poorly defined. Preference 

for polymer latexes in the pre-inoculated seed industry is largely based on pellet quality and ease of handling but 

little attention has been given to compatibility and protection of rhizobia. Polymer latexes are not only likely to 

contain incompatible chemical constituents such as excess polymerisation initiator, surfactants and biocides but 

are also faster drying than solution polymers. Rhizobial survival is substantially improved when the rate of 

dehydration is reduced. 

In addition to external protectants, intracellular physiological changes after growth of rhizobia in different media 

prior to drying significantly affect survival and may be responsible for improved desiccation tolerance. A better 

understanding of how to induce physiological mechanisms involved in desiccation tolerance will provide new 

direction for inoculant technology development. It is expected that combining targeted physiological conditioning 

during production of inoculants with optimised external protection during application will maximise the potential of 

microbial inoculants to enhance agricultural production systems. 

82 

2011






1600 - 1740 

Authors: Ron Yates 1,2 , Angelo Loi 1 , Brad Nutt 1 & John Howieson 2 

1 Department of Agriculture and Food, Western Australia (DAFWA), Baron Hay Court, 

South Perth, 6151, Western Australia. 

2 Centre for Rhizobium Studies (CRS), Murdoch University, Murdoch, 6150, Western 

Australia. 

Presentation Title: How do we ensure nodulation of annual pasture legumes sown out of season into hot, 

dry soil? 


Biological nitrogen (N) fixation from effectively nodulated legumes are an integral component of sustainable 

agriculture. Agricultural legumes not only provide N for subsequent crops and high value feed for animals, they 

also assist in spreading the risk in the production system through the management of weeds, pests and 

diseases. In Australia, it is essential legumes are inoculated with the correct and current commercial rhizobia 

strain (or Group) for maximum N fixation. Commercial inoculant strains go through an extensive selection 

process, in which the strains must possess the ability to maintain high N fixation over a broad host range and 

adapt to the anticipated soil niche of the host legume. However, an ongoing challenge is to successfully deliver 

high numbers of the commercial inoculant. If this is not achieved it leads to failed nodulation, or nodulation of the 

legume with resident soil-borne strains that are usually sub-optimal in N fixation. 

Inoculants in Australia are available in four different carriers: (i) peat; (ii) freeze dried powders; (iii) granular; and 

(iv) a pre-coated seed form, with inoculum as part of the pellet. The efficiencies of these products are continually 

being evaluated for use in Western Australian agriculture. However, current research is largely focused on 

providing best practices for inoculating pasture legumes when they are introduced by “Summer sowing” in the 

Mediterranean climates of southern Australia (i.e. when the soil is hot and dry). This technique has been 

designed to cheaply introduce legume species into paddocks at a time when farm labour is not limiting, assisted 

by strategically harnessing inherent hard-seed breakdown in the legume cultivars. However this presents a 

challenge in terms of keeping the inoculant rhizobia alive until a rainfall event that induces germination, and 

identifying which carrier is the most efficient at doing so. Summer sowing requires the bacteria to survive through 

dry, hot, soil conditions in sufficient number to out-compete resident strains for nodulation when the seeds 

germinate, which may be up to four months after sowing. Recent findings will be presented that suggest 

surprising outcomes in rhizobial survival. 

83 

2011





Authors: 


1600 - 1740 

Didier Lesueur 1,2 , Laetitia Herrmann 2 , Moses Thuita 2, 3 , Mary Atieno 2, 3 , Edwin Mutegi 2, , 

Keziah Ndung‟u 2, 3 4 , Aliou Faye 2, 5 , Mary Kamaa 2 , Pieter Pypers 2 & Robert Okalebo 3 

1 CIRAD, UMR Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols & 

Agroécosystèmes (SupAgro-CIRAD-INRA-IRD), 2 Place Viala, F34060 Montpellier, 

France.  

2 Tropical Soil Biology and Fertility Institute (TSBF) / CIAT - World Agroforestry Centre 

(ICRAF), P.O. Box 30677, Nairobi, Kenya. 

3 Moi University, Department of Soil Science, PO Box 1125, Eldoret, Kenya. 

4 Kenya Agricultural Research Institute (KARI – Kitale), P.O. Box 450 - 30200, Kitale, 

Kenya. 

5 Institut Sénégalais de Recherche Agricole, Route des hydrocarbures, Dakar, Sénégal 

Presentation Title: How commercial rhizobial inoculants can contribute to improved livelihoods of resource 

poor African farmers. 


There is a proliferation of commercial bioinoculant products appearing on the market that claim major impact in 

increasing crop productivity without any scientific assessments about their effectiveness in the field. TSBF-CIAT 

has been mandated by Bill and Melinda Gate Foundation to scientifically evaluate and select effective 

commercial products for improving and sustaining crop yields in selected agro ecological zones in 3 African 

countries. 

Our results on about 120 products showed that many private companies commercialize a mix of non-defined 

microorganisms and thus do not ensure the quality of their inoculants. This may explain their lack of impact on 

the plant growth even under controlled greenhouse conditions. Generally, high level of contamination was 

observed as pure products represented only 33% of the products, 41% of the products contained all the 

expected strains and 52% of the products contain none or part of the expected contaminants. We assessed 

under greenhouse conditions the possible effect of the formulation of effective rhizobial inoculants on both 

soybean growth and nodulation. Our results showed that there was no effect and the association of the rhizobia 

with a Bacillus strain didn‟t induce any significant stimulation of soybean growth. However, through field 

demonstration trials combining rhizobia with mycorrhiza and P solubilizing bacteria, our results suggested some 

relevant positive interactions on soybean yield. In Kenya, we tested one rhizobial inoculant in 3 mandates areas 

(about 50 farms in each) and our results demonstrated a significant effect of the inoculation on soybean yield. 

The next step will consist of making such effective inoculants cheap and available on the local markets. 

Administrative issues shall be sorted out for each country as the national regulations are not similar and of 

course the quality control issue shall be taken into account to ensure the viability of the inoculants to farmers who 

purchase them. 

84 

2011






1600 - 1740 

Authors: Matthew D Denton 1 David J Pearce 2 and Mark B Peoples 3 

1 The University of Adelaide, School of Agriculture, Food and Wine, Adelaide SA 5005 

Australlia 

2 Department of Primary Industries, RMB 1145 Chiltern Valley Road, Rutherglen, VIC 

3685, Australia 

3 CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601 Australia 

Presentation Title: The influence of rhizobial inoculant application rate on nitrogen fixation in faba bean 

(Vicia faba l.) In south eastern Australia 


Nodulation of legumes is essential to create effective nitrogen fixing symbioses, yet the need to inoculate pulses 

with large numbers of effective rhizobia is often not appreciated. In this study, we tested the influence of seven 

rates of rhizobia inoculation, from no inoculant to 100 times the normal rate of inoculation, on the responses of 

faba bean in two fields in south-eastern Australia: one site with no detectable rhizobia and one with 2900 rhizobia 

g -1 soil. Rhizobia inoculation increased nodule numbers from 0 to 55 nodules per plant and nodule dry matter 

(DM) from 0 to 330 mg. The increase in nodule numbers directly impacted on fixed N, which increased from 20 to 

218 kg shoot N ha -1 , and on grain N concentrations, which increased from 3.2 to 4.5%. An increase in N2 fixation 

was even observed in the presence of a soil population when a high rate of inoculation was provided. Increases 

in nodulation improved grain yields by around 1.0 t ha -1 in well-nodulated treatments (2.6 t ha -1 ) compared with 

those of un-inoculated faba bean (1.6 t ha -1 ) at the site without rhizobia present in soil. Differences in nodulation 

also influenced the contributions of legume N to the system, which varied from the removal of 20 kg N ha -1 from 

the system to an input of 199 kg N ha -1 . This study illustrates the importance of inoculation to N2 fixation, grain 

yield, grain N concentration and the potential contributions of legume cropping to soil N fertility. The study also 

identifies the need for technologies to increase inoculant numbers supplied to legumes as a pathway to improve 

N2 fixation by legumes that are sown into fields with existing rhizobial populations. 

85 

2011






1600 - 1740 

Authors: Laetitia Herrmann 1 , Collins Majengo 2 , Edwin Mutegi 1 , Martin Kimanthi 1 , Paul Onyango 1 , 

Robert Okalebo 2 & Didier Lesueur 1,3 

1 Tropical Soil Biology and Fertility institute of CIAT, World Agroforestry Center, PO Box 

30677 Nairobi, Kenya. 

2 Moi University, School of Agriculture and Biotechnology, PO Box 1125 Eldoret, Kenya. 

3 CIRAD, UMR Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols & 

Agroécosystèmes (SupAgro-CIRAD-INRA-IRD), 2 Place Viala, F34060 Montpellier, 

France. 

Presentation Title: Field evaluation of rhizobial inoculant: high nodule occupancy by the strain doesn‟t lead 

to improved soybean yields in Kenya 


A range of soybean commercial inoculants were tested under greenhouse and field conditions in different 

locations in Kenya. One rhizobial (Legumefix) and one mycorrhizal (Rhizatech) inoculants were selected 

according to the promising results obtained. The efficiency of these 2 products (single or combined inoculation) 

was assessed in 150 farms in 3 mandate areas in Kenya presenting different soil characteristics and 

environmental conditions. Both biomass and grain yields were measured and nodules occupancy of the rhizobial 

strain was assessed by ELISA using specific monoclonal antibodies. Application of the rhizobial inoculant, alone 

or in combination with the mycorrhizal product significantly increased the yields in all mandate areas (about 75% 

of the farms). However, the co-inoculation didn‟t perform significantly better than the rhizobial product alone. 

Controls plots gave poor results, as well as the mycorrhizal product, though that was not expected given the 

good performances observed under greenhouse conditions. Nodule occupancy analysis showed that a high 

number of nodules occupied by the inoculated strain did not obviously lead to an increase of soybean production. 

Soil factors (pH, P, C, N…) seemed to affect the inoculant efficiency whether the strain is occupying the nodules 

or not. Our statistic analysis showed that soil pH significantly affected nodulation and yield, though the effect was 

variable depending on the region. P content also positively correlated with nodulation and yields. This study 

provided relevant information on field efficiency of rhizobial inoculants on soybean yield in Kenya and showed 

that the competitiveness of rhizobial strains might not be the main factor explaining the effect (or lack of) of 

legumes inoculation in the field. 

86 

2011





Concurrent Session 14 – Cyanobacteria & other organisms 

1600 - 1740 

Authors: Faten Mohamed, Soha Mostafa, & Nabil Omar 

Soils, Water and Environ. Res. Inst., Agric. Microbiol. Res. Dept., ARC, Giza, 

Egypt. 

Presentation Title: Influence of beneficial microbes and proline treatments on sugarbeet under nitrogen 

limitation in saline-sodic soil 


A field experiment on sugar beet (Beta vulgaris L.) yield and yield quality under saline-sodic soil conditions 

was conducted at Sahl El-Hussinia Res. station, El-Sharkia Governorate, Egypt, during the winter season of 

2008/2009. The influence of cyanobacteria and N2-fixing bacteria were compared either individually or in 

combination to supplementation with proline amino acid under two levels of mineral nitrogen fertilization (50 

and 75% of nitrogen recommended dose). Soil enzymatic activities (dehydrogenase and nitrogenase), total 

bacterial counts, total cyanobacteria counts and total nitrogen fixing bacteria counts were enhanced by the 

biofertilizers compared to proline treatment and control, particularly when the combined inoculum of 

cyanobacteria and N2-fixing bacteria was applied in the presence of 75% N. In addition, inoculation with 

cyanobacteria and N2-fixing bacteria, either individually or in combination with 75%N, led to a slight decrease 

of pH and EC values of saline soil, while there was an increase in the availability of NPK as compared with 

control plots.Proline and biofertilizers showed a significant positive impact on some physiological properties of 

plants drown at 75% nitrogen level, such as chlorophyll in leaves, proline and phenolic compounds in roots. 

The highest responses for these traits were in proline-treated plots followed by the combined inoculation of 

cyanobacteria and N2-fixing bacteria. While, there was no significant difference in root yield productivity 

between proline treatment and the combined inoculum of cyanobacteria and N2-fixing bacteria with 75% 

nitrogen fertilizer. The combined inoculation positively increased N, P and K uptake and decreased the uptake 

of Na in roots. The combined inoculum of cyanobacteria and N2-fixing bacteria with 75% nitrogen led to a 

significant increase in shoot and root dry weight as well as root yield quality (sucrose and purity). Results 

suggest that the beneficial effect of the cyanobacteria and N2-fixing bacteria on sugar beet growth, yield and 

yield quality was attributed to the biologically active substances produced by these microbial strains besides 

the nitrogen fixation of the diazotrophs which compensate the reduction of the costly and the environmentally 

polluted mineral nitrogen fertilizers in the new reclaimed saline-sodic soil. 

87 

2011






1600 - 1740 

Authors: Radziah Othman 13 , Umme Aminun Naher 1 , Zulkifli Hj. Shamsuddin 1 , Halimi Mohd Saud 2 

, Mohd Razi Ismail 3 and Khairudin Abdul Rahim 4 

1 Department of Land Management, 2 Department of Agriculture Technology, Faculty of 

Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 

3 Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, 

Selangor, Malaysia 

4 Agrotechnology and Biosciences Division, Nuclear Agency Malaysia, Ministry of 

Science Technology and Innovation (MOSTI), Bangi, 43000 Kajang, Malaysia 

Presentation Title: Effect of root exuded sugars on biological nitrogen fixation and growth of rice (Oryza 

sativa) 


Biological Nitrogen Fixation (BNF) is an important process in wetland rice ecosystem requiring high energy input. 

A 15 N tracer study was conducted under glasshouse condition to determine the effect of rice exuded sugars 

(glucose, galactose and arabinose) on BNF of two locally isolated diazotrophs, Rhizobium sp. Sb16 and 

Corynebacterium sp. Sb26 in association with two rice genotypes (traditional variety Mayang Segumpal and 

cultivated variety MR219). Both diazotrophs showed preferences for specific sugars and genotype association. 

Isolate Sb16 showed high preference for galactose and isolate Sb26 for arabinose. Application of 10 mM sugars 

either galactose or arabinose in pot experiment (5 kg soil) to the respective rice genotypes enhanced the 

diazotroph populations, N2 fixation activity and subsequently plant growth. Mayang Segumpal genotype 

inoculated with Rhizobium Sb16 and applied with galactose increased plant N concentration with 42 ± 1.06 % of 

the N derived from the atmosphere. MR219 genotype inoculated with Corynebacterium Sb26 and applied with 

arabinose obtained 40 ± 1.29 % of the N concentration from BNF. Comparing with the uninoculated control, the 

association between Mayang Segumpal with Sb16 increased plant biomass by 195 ± 40 % and the association 

between MR219 with Sb26 resulted in biomass increase by 108 ± 37.07 %. In plants fertilized with 60 kg ha -1 of 

N, inoculated MR219 genotype showed higher biomass increment compared to that of Mayang Segumpal. The 

association between the plant and diazotrophs with the respective sugars significantly increased plant 

photosynthetic activity. The study indicated that N2 fixation activity and growth of different rice genotypes can be 

increased by increasing the availability of specific sugars in the rhizosphere. 

88 

2011






1600 - 1740 

Authors: Cuddy, W. S. 1 , Forster, B. 2 , Deaker, R. 3 , Summerell, B. A. 4 , Neilan, B. A. 1 ,Gehringer, M. 

M. 1 

1 . School of Biotechnology and Biomolecular Sciences, University of New South Wales, 

Sydney, 2052, New South Wales. 

2. Research School of Biology, Plant Sciences Division, Australian National University, 

Canberra, 0200, Australian Capital Territory. 

3 . Faculty of Agriculture, Food and Natural Resources, University of Sydney, Sydney, 

2006, New South Wales. 

4. Science and Public Programs, Botanic Gardens Trust, Sydney, 2000, New South 

Wales. 

Presentation Title: Cyanobacterial salt physiology and their impact on wheat seedling salt tolerance 


Nitrogen-fixing cyanobacteria have been identified in soils and biological soil crusts worldwide. Understanding 

their potential to support cereal growth, particularly under adverse conditions, is of great interest to crop 

management and breeding. We assessed the potential benefits of cyanobacteria on growth of wheat seedlings 

under salt stress. First, three cyanobacterial isolates from wheat fields: a heterocyst-producing Nostoc sp., a 

Microcoleus vaginatus and a Leptolyngbya sp. were cultured in salinised medium at 5 and 15 dS m -1 and 

sampled at mid-exponential and stationary growth phases to determine photosynthetic and nitrogen fixation rates 

using Pulse Amplitude Modulation (PAM) fluorometry and Acetylene Reduction Assays (ARA), respectively. 

These isolates were then established as soil crusts on sterilized sand into which germinated wheat seeds were 

planted 1 cm below the surface. Sodium chloride was applied to generate salt stress with electrical conductivities 

(ECe) of 6 and 13 dS m -1 . Pots were watered to field capacity daily, and the electrical conductivities monitored 

during seedling development for either 14 days at 6 dS m -1 or 21 days at 13 dS m -1 . Quenching analysis using 

PAM fluorometry was done on wheat leaves before sampling. Inductively Coupled Plasma-Optical Emission 

Spectroscopy (ICP-OES) was conducted on separated root and shoot material. Both the Nostoc and the nonheterocystous 

cyanobacteria Microcoleus vaginatus isolates fixed nitrogen at 8 and 6 n moles C2H4 μg Chl a -1 h -1 

respectively, without affecting photosynthetic efficiency. The cyanobacterial soil crusts induced significant effects 

on root and shoot nutrient concentrations, impairing plant salt tolerance. This study provides new evidence for 

the ability of Microcoleus vaginatus to fix nitrogen, which suggests its contribution to soil ecology may currently 

be undervalued. We were able to demonstrate that the cyanobacterial isolates used in this study in fact 

enhanced the salt stress of wheat seedlings, thereby decreasing plant growth. 

89 

2011






1600 - 1740 

Authors: Zulkifli Hj. Shamsuddin, Tan Kee Zuan and Puteri Aminatulhawa Megat Amaddin 

Department of Land Management, Faculty of Agriculture, Universiti Putra 

Malaysia, 43400 UPM Serdang, Selangor, Malaysia 

Presentation Title: Development and application of liquid biofertilizer inoculum for non-legume and 

vegetable soybean intercrop 


Solid substrate inoculant is more widely used than liquid inoculant in legume and non-legume cultivation. It is 

more pronounced in developing countries. In Malaysia, the application of liquid inoculant with a consequential 

reduction in chemical fertilizer consumption could be more beneficial due to the hilly terrain, reduction in 

transport and fertilizer cost, and minimal hazardous effect on the environment. Locally used liquid inoculant 

technology is now emerging using various organic sources and stimulant in the culture medium to prolong shelflife 

and effectiveness of the bacterial inoculum. An organic fertilizer-based amendment has been used to sustain 

the shelf-life of a Plant Growth-Promoting Rhizobacteria ( PGPR ) for more than nine months. This locally 

isolated non-pathogenic PGPR from the roots of oil palm has N2 fixing and PO4 -- solubilizing properties besides 

producing phytohormones which enhance root development and increase water and nutrient uptake ( 

Shamsuddin et al., 2009 ). The PGPR is protected from any adverse soil environmental conditions due to its 

endophytic habitat in the plant roots. Inoculated plants showed increased shoot and root growth ( rice, oil palm, 

banana )( Kok-Ang et al., 2010 ), are drought and disease tolerant ( Fusarium oxysporum cubense on banana ) 

and high yielding but with 65% less fertilizer-N input ( banana, sweet potato )( Mia et al., 2010 ). Inoculated 

herbal plant, Safed Musli ( Chlorophytum borivilianum) have also shown increased number of tuber and content 

of its bioactive compound, saponin. This liquid inoculant will be commercially prepared in concentrated form to 

reduce bulk and storage space relative to solid inoculum and used in the field at 100 fold dilution while reducing 

transport and labor cost in the field application. The inoculum can be used by mixing it with the seeds prior to 

planting and sprayed around the base of non- leguminous plant or in the interrow of the young rice seedling and 

vegetable soybean crop after four weeks of growth. These beneficial effects of the environmental friendly liquid 

biofertilizer can minimize the use of inorganic fertilizer in sustainable crop production system. 

90 

2011




Session Details: 

Wednesday 30 November 2011 


1600 - 1740 

Authors: Junko Tazawa 1 , Yoshinari Ohwaki 1 , Tadashi Yokoyama 2 , Yoshiaki Shizukawa 3 , Ui 

Ono 3 & Masami Yoshikawa 3 

1 NARO Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan 

2 Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan 

3 Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and 

Fisheries Technology Center, Seika, Kyoto 619-0244, Japan 

Presentation Title: Endophytic occupation of soybean nodules by Rhizobium and other bacterial species 

under continuous cultivating conditions 


Rhizobial bacteria can form nodules and establish symbiosis on the roots of their leguminous host plants. 

Recently, several reports have stated that some non-symbiotic bacteria occupy legume nodules endophyticly, 

although the function of these bacteria in the nodules has not been understood. We have isolated non-symbiotic 

bacteria from root nodules of soybean (cv: Hatayutaka) grown in a continuously cultivated field. Fast-growing 

bacterial strains were isolated from surface-sterilized nodules on YMA medium as non-symbiotic endophytic 

bacteria. Phylogenetic analysis based on the partial sequence of 16S rDNA gene demonstrated that most of 

these isolates were close to the genera Rhizobium/Agrobacterium, Bacillus, Paenibacillus, Enterobacter, and 

Pseudomonas. The percentage of nodules that coexisted with symbiotic Bradyrhizobium strains and nonsymbiotic 

bacteria was very low (





Concurrent Session 15 – Quantification of N-Fixation 

1600 – 1740 

Authors: Georg Carlsson 1 & Kerstin Huss-Danell 2 

1 Department of Agrosystems, Swedish University of Agricultural Sciences, SE-23053 

Alnarp, Sweden. 

2 Department of Agricultural Research for Northern Sweden, Swedish University of 

Agricultural Sciences, SE-90183 Umeå, Sweden. 

Presentation Title: Does nitrogen transfer confound 15 N-based quantifications of N2 fixation? 


Nitrogen transfer between neighbouring plants is a well-known phenomenon, mediated via e.g. rhizodeposition 

and mycorrhizae. Transfer of fixed N from legumes to non-legume reference plants has been suggested to 

confound N2 fixation estimates obtained by the 15N natural abundance and isotope dilution methods, because a 

reference plant taking up fixed N will supposedly not correctly reflect the 15 N signature of plant-available soil N. 

On the other hand, experiments using direct leaf-feeding of 15 N, which allows for direct detection of N transfer 

with high precision, have shown that N transfer also occurs in the direction from non-legume to legume. In order 

to establish whether N transfer causes problems in N2 fixation measurements with 15 N-based methods when 

using reference plants grown together with the legume, we labelled both legumes and non-legumes with 15 N and 

performed detailed measurements of N transfer in mixed plant communities in the field, including measurements 

of potential N transfer from legume to legume. N transfer occurred in all directions: from legume to non-legume, 

from non-legume to legume and from legume to legume. These results, together with analyses of previously 

published data on N transfer, were used to calculate nitrogen fixation with different N transfer scenarios, showing 

the effects of using reference plants in mixture with the legume versus in pure stand. We conclude that the most 

important mechanism of N transfer is likely to be indirect, via rhizodeposition and litter degradation, and that fixed 

N transferred to neighbouring reference plants also modifies the 15 N signature of the soil N available to the N2fixing 

legume. This provides strong support for using reference plants growing as close as possible to the N2fixing 

legume for reliable N2 fixation quantifications. 

92 

2011






1600 – 1740 

Authors: Xinhua He 1, 2, 3, 4 , Christa Critchley 4 , Caroline Bledsoe 5 

1, 2 State Centre of Excellence for Ecohydrology, Centre for Ecosystem Management, 

1 School of Natural Resources, Edith Cowan University, Joondalup, WA 6207, Australia 

2 School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia 

3 Key Laboratory of Crop Nutrition and Fertilization, Institute of Agricultural Resources 

and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, 

China 

4 School of Agriculture & Food Sciences, University of Queensland, Brisbane, QLD 4072 

Australia 

5 Department of Land, Air and Water Resources, University of California, Davis, CA 

95616 USA 

Presentation Title: Reciprocal two-way n ( 15 NH4 or 15 NO3) transfer between non-N2-fixer Eucalyptus 

maculata and Frankia N2-fixer Casuarina cunninghamiana 


Nitrogen (N) movement or transfer from one plant to another could have important implications for nutrient 

cycling and eco-physiological significance in terrestrial and agricultural ecosystems. In controlled pots with or 

without Frankia inoculation to Casuarina, two-way N transfers through an ectomycorrhizal fungus Pisolithus sp. 

were examined by excluding root contact and supplying 15 NH4 + or 15 NO3 � to 5 or 11-month-old Eucalyptus 

maculata or Casuarina cunninghamiana pairs grown in 2-chambered-pots separated by 37 µm nylon screens. 

With either Eucalyptus or Casuarina as the N-donor, the three pairs were non-nodulated nonmycorrhizal/nonmycorrhizal, 

non-nodulated mycorrhizas/mycorrhizal and nodulated mycorrhizal/mycorrhizal. 

Using an environmental scanning electron microscope, living mycorrhizal hyphae were observed to interconnect 

Eucalyptus and Casuarina. Results showed that N2-fixation supplied ~35% of the N in Casuarina and was 

enhanced by mycorrhization, which was 67% in Eucalyptus and 36% in Casuarina. Biomass, N and 15 N contents 

were lowest in the non-mycorrhizal and greatest in the nodulated/mycorrhizal plants. Nitrogen transfer was 

enhanced by nodulation and/or mycorrhization, and greater when N was supplied as 15 NH4 + than 15 NO3 � . 

Nitrogen transfer from either 15 N-source was lowest in the non-mycorrhizal treatment and greatest in the 

nodulated mycorrhizal treatment, and also was greater to Casuarina than to Eucalyptus and where NH4 + than 

NO3 � supplied. Irrespective of the 15 N-source and of whether Casuarina or Eucalyptus was the N sink, net N 

transfer was low and similar in non-nodulated treatments, but was much greater when Casuarina was the N sink 

in the nodulated mycorrhizal treatment. High-N-demand by Casuarina resulted in greater net N transfer from the 

less N-demand Eucalyptus. Net N transfer from a non-N2-fixing to an N2-fixing plant may reflect the very high N 

demand of N2-fixing species. Our research may provide insights into our understanding of complex interactions 

among plants, Frankia, mycorrhizal fungi, and soil resources in both terrestrial and agricultural ecosystems. 

93 

2011






1600 – 1740 

Authors: Alejandro del Pozo 1 , Carlos Ovalle 2 & Soledad Espinoza 2 

1 Facultad de Ciencias Agrarias, Universidad de Talca, Talca, Chile. 

2 CRI-Quilamapu, INIA, Chillán, Chile. 

Presentation Title: Nitrogen fixation of grain legumes in two contrasting mediterranean environments of 

Chile 


Grain legumes are important components of Mediterranean farming systems due to their capacity to fix nitrogen 

and to improve soil fertility, as well as to reduce weeds in legume-cereal rotations. In this work we evaluate the 

nitrogen fixation capacity of four grain legumes, Pisum sativum, Lupinus angustifolius, L. luteus and L. albus, in 

rotation with wheat, by using the 15 N natural abundance technique. Field experiments were conducted in two 

contrasting Mediterranean environments and in two years (2008 and 2009). In the interior dryland (granitic 

Alfisol; 650 mm of annual rainfall), total dry matter production was higher in P. sativum (average 10.5 ton ha -1 ) 

than in L. angustifolius and L. luteus (average 5.5 ton ha -1 ). The N derived from the atmosphere (%Ndfa) was 

significant (p






1600 – 1740 

Authors: Salmina N. Mokgehle 1 , Cherian Mathews 2 and Felix D. Dakora 3 

1 Department of Crop Sciences, Tshwane University of Technology, Private Bag X680, 

Pretoria 0001, South Africa. 

2 Department of Agriculture, Rural Development and Land Administration, Private Bag 

X11318, Nelspruit, 1200 

3 Department of Chemistry, Tshwane University of Technology, Private Bag X680, 

Pretoria 0001, South Africa. 

Presentation Title: Selecting field plants for drought tolerance and N2 fixation in 25 groundnut (Arachis 

hypogaea 

13C and 15N natural abundance 


Groundnut is a major commercial grain legume in Africa, often cultivated for its seed oil and high protein content. 

Although ICRISAT has historically selected groundnut genotypes for grain yield and disease resistance in Africa, 

little is known about their N contribution and drought tolerance under local conditions. In this study, field trials 

were conducted at three sites (namely, Nelspruit, Mzinti and Kliplaatdrift) within Mpumalanga Province of South 

Africa in 2009. The data revealed significant differences in plant biomass, N concentration, and N content at all 

three sites. The δ 15 N values of groundnut genotypes ranged from -0.08‰ to +1.06‰ at Nelspruit, +0.41‰ to 

+0.95‰ at Mzinti, and +0.73‰ to +1.96‰ at Kliplaatdrift. About 24 out of the 25 groundnut genotypes obtained 

over 50% of their N nutrition from symbiotic fixation at Mzinti, while at Kliplaatdrift only five genotypes derived 

50% or more of their N nutrition from symbiotic fixation. The amounts of N-fixed at Nelspruit ranged from 76 

kg/ha for ICGV99033 and PC327K31 to 188 kg/ha for ICGV00362. At Mzinti, 10 genotypes contributed over 100 

kg N /ha, while at Kliplaatdrift only 5 genotypes fixed more than 100 kg/ha. Data on ∂ 13 C values also revealed 

marked difference in drought tolerance. Genotypes ICGV03157 and ICGV00369, for example, showed much 

lower 13 C discrimination at all three sites, indicating greater water-use efficiency compared to the remaining 

genotypes. 

95 

2011






1600 – 1740 

Authors: Rachel Pipai 1,2 Murray Unkovich 1 Ann McNeill 1 Murom Banabas 2 Mike Webb 3 David 

Herridge 4 Paul Nelson 5 

1 University of Adelaide Waite Campus Glen Osmond SA5064 

2 PNG Oil Palm Research Association Inc DAMI Research Station PO Box 97 Kimbe 

West New Britain PNG 

3 CSIRO Land & Water Davies Laboratory, Discovery Drive, Douglas, Queensland 

4814, Australia.CSIRO Townsville 

4 UNE–NSW DPI Primary Industries Innovation Centre University of New England 

Armidale NSW 2351 

5 James Cook University PO Box 6811 Cairns, Qld 4870 

Presentation Title: Nitrogen fixation by legumes under oil palm plantations in Papua New Guinea 


There are currently no published estimates for the amount of nitrogen that is fixed by tropical legume cover 

plants in oil palm plantations on the volcanic ash soils of PNG. Such estimates require knowledge of the 

proportional dependence of plants on N2 fixation (%Ndfa), as well as data for dry matter production. A field study 

is planned to measure these variables for legume cover plants (Pueraria phaseoloides, Calopogonium caerulum, 

and Mucuna bracteata) in plantations of different ages, since shading increases as the palms grow and legume 

cover changes in composition and growth rate. Two methods will be used for assessing %Ndfa, the 15 N natural 

abundance and the xylem sap techniques. Initially a glasshouse experiment using 15 N-labelled nutrient solutions 

will be undertaken to calibrate the xylem sap technique for estimating %Ndfa for each of the legume cover crops. 

Results from the glasshouse study and a preliminary field survey will be presented at the conference. 

96 

2011




Session Details: Thursday 1 December 2011 

Plenary Session 

0900 – 1030 

Authors: Robert M. Boddey 1 , Lincoln Zotarelli 2 , Natalia P. Zatorre 3 , Segundo Urquiaga 1 , Claudia 

P. Jantalia 1 , Julio C. Franchini 4 , Bruno J. R. Alves 1 

1 Embrapa Agrobiologia, Seropédica, 23890-000, Rio de Janeiro, Brazil. 

2 Horticultural Sciences Dept., University of Florida, Gainesville, FL 32611-0690, USA. 

3 Departamento de Solos, Universidade Federal Rural do Rio de Janeiro, Seropédica, 

23890-000, RJ, Brazil. 

4 Embrapa Soja, Rod. Carlos João Strass , Warta, 86001-970, Londrina, PR, Brazil. 

Presentation Title: Legume N2-fixation inputs preserve soil organic C stocks in no-till agriculture 


In Brazil, no-tillage is widely adopted for soybean-based cropping systems. In the Southern region, soybean 

alternates with maize in the summer, and oats or green-manure legumes in the winter to break the continuous 

use of wheat in this season. Results from long-term experiments comparing conventional plough tillage (CT) and 

no-till (NT) in this region have shown that if the only legume in the rotation is soybean there is very little increase 

in soil C stocks, which has been attributed to lack of significant residual N in the system to build soil organic 

matter owing to the large export of N in soybean grain. Here we present the results of a study with different crop 

rotations of soybean, maize, wheat, oats and lupins managed under NT or CT which differed from each other in 

the frequency of soybean and lupins in the crop sequences. Soil C and N stocks to a depth of 80 cm were 

evaluated and the contributions of N2 fixation (BNF) to all the legumes in the systems were quantified using the 

15 N natural abundance technique. Reliance on BNF of both soybean and lupins was higher under NT than under 

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 

allowed high maize yields, comparable to N-fertilized maize following oats, and was essential to maintain a 

positive cropping system N balance. Comparison of soil C stocks between 1997 and 2009 revealed almost no 

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 

the value of green manure legumes as a significant source of N for cropping systems which are a key aid to 

preserving or increasing soil C stocks under NT. 

97 

2011






0900 – 1030 

Authors: Perrin H Beatty 1 , and Allen G Good 1 

1 Department of Biological Sciences, Biological Sciences Building, Edmonton, Alberta, 

Canada, T6G 2E9 

Presentation Title: Environmental and economic impacts of biological N2 fixing cereal crops 


We will need to feed a large future global population using less arable land while coping with rising fuel prices 

and environmental pollution inherent to the increase in N fertilizer use in intensive farming practices. Cereal 

crops tend to have low nitrogen use efficiency and require N fertilizer application (synthetic or organic) for high 

yields. A portion of the applied N fertilizer will be lost to the plant by either uptake competition from soil microbes 

resulting in nitrification/denitrification and eventually loss back to the atmosphere, leaching into waterways and 

ammonia volatization. However, cereal crops that can fix their own nitrogen would bypass the N losses to the 

environment while still providing N to the plants. 

The engineering of N2 fixing cereal crops, by introducing either diazotrophic bacterial nodulation within cereal 

crop roots or nitrogenase genes (nif genes) directly into plant chloroplasts or mitochondria, is an attainable future 

goal (Beatty and Good, 2011). Both approaches are expected to take multiple years to completion, although 

step-wise advances in the ability of cereal crops to fix N2 are expected along the way. The rate of N2 fixation 

using either approach is predicted to be similar to the rate measured in rhizobial-legume symbiosis, or higher. 

This suggests that, if we can successfully engineer BNF cereal crops, the requirement for cereal crop N fertilizer 

application will be reduced, or potentially, even eliminated. 

The environmental and economic impacts of N2 fixing cereal crops are assessed in this paper, such as the 

environmental effects of using less N fertilizer on the atmosphere and aquatic ecosphere, the economic 

implications for farmers, as well as the potential implications to the plants‟ N economy. 

Beatty PH & Good AG (2011). Cereal crops and biological nitrogen fixation; future prospects. Science, July 24. 

98 

2011






0900 – 1030 

Authors: Jean-Jacques Drevon 1 , Hesham Attar 2 , Adnane Bargaz 3 , Mustapha Faghire 3 , Cherki 

Ghoulam 3 , Mohamed Lazali 4 , Mohamed Ounan 1 , Paula Rodino 5 

1 INRA, Eco&Sols, 2 Place Viala, 34000 Montpellier, France 

2 NRC Cairo, Egypt 

3 Faculté des Sciences et Techniques, Gueliz, Marrakech, Maroc 

4 Ecole Nationale Supérieure Agronomique, El Harrach, Alger, Algérie. 

5 Plant Genetics and Breeding, CSIC, 36080-Pontevedra, Spain. 

Presentation Title: Is phosphorus efficiency low for symbiotic nitrogen fixation? 


Phosphorus is the limiting nutrient for legume symbiotic nitrogen fixation in most soils of the planet. Thus the 

yield of legumes generally responds to phosphorus fertilization. The P requirement of legumes is generally 

higher when their growth depends upon atmospheric nitrogen than upon ammonium or nitrate. This 

communication will revise the metabolic and structural utilizations of P that may limit the P efficiency for N2 

reduction in legume nodules and their genetic variation among legume species. The interaction with P 

metabolism will be illustrated with our results on nodule phytase activity (Araujo et al. 2008), and the tissue 

expression of the corresponding gene by in situ RT-PCR on nodule sections. The later will be compared with that 

of phosphoenol pyruvate phosphatase, and other candidate genes like trehalose 6-P phosphatase, that have 

been identified by deep super SAGE on nodule mRNA populations (Molina et al. 2011). The selection procedure 

of contrasting genotypes in P utilization for N2 fixation will be proposed to assess the functions involved in the 

adaptation of N2-fixing legumes in agro-ecosystems with low P soils, and to improve the yield of legumes and 

the sustainability of agricultural systemsdepending upon N2-fixation. 

Araujo AP, Plassard C & Drevon JJ (2008) Phosphatase and phytase activities in nodules of common bean 

genotypes at different levels of phosphorus supply. Plant Soil 312, 129-138. 

Molina C, Zaman-Allah M, Faheema K, Fatnassi N, Horres R, Rotter B, Steinhauer D, Drevon JJ, Winter P, Kahl 

G (2011) The salt-responsive transcriptome of chickpea root and nodules via deep super SAGE. BMC Plant 

Biology 11, 11-31. 

99 

2011





Concurrent Session 16 – Symbiotic Impacts & Emissions 

1100 - 1230 

Authors: Mark B Peoples 1 , John Brockwell 1 , John F Angus 1 , B Smith 1 , A Swan 1 and Catherine A 

Osborne 2 

1 CSIRO Sustainable Agriculture Flagship, CSIRO Plant Industry, GPO Box 1600, 

Canberra, ACT 2601. 

2 Department of Civil Engineering, Monash University, Clayton, Victoria 3800 (formerly 

Microbiology Department, University of Melbourne) 

Presentation Title: Effects of rhizobial strain and legume host on emissions of H2 from nodules and the 

impact on soil biology and plant growth 


Hydrogen (H2) is produced as an obligate by-product of nitrogenase activity in legume nodules. Some 

symbioses possess a hydrogenase-uptake system (designated Hup + ) that is able to recycle almost all of the H2 

evolved. However, many strains of root-nodule bacteria (rhizobia) lack the hydrogenase enzyme (Hup ─ ), or have 

low Hup activity. In both these latter cases, the H2 diffuses out of the nodules into the soil. Within a very short 

period, this H2 is consumed by microorganisms residing close to the nodule ─ and none of it escapes into the 

atmosphere. 

Experimentation undertaken to assess the Hup status of various nodulated legumes indicated that, of all the 

legume x rhizobial strain combinations examined, only (i) soybean x CB1809 (Australian inoculant strain) and (ii) 

soybean x USDA110 (former US inoculant strain) were Hup + . All other associations emitted H2 from their 

nodules to a greater or lesser extent. Interestingly, the rates of H2 evolution from the nodules of faba bean, lupin 

and subterranean clover (mean: >450 μmol H2 evolution per gram nodule dry weight per hour) were consistently 

two- to five-fold greater than those observed for other legume x rhizobia combinations. 

Measurements of H2 evolution from Hup ─ nodules in the field indicate that >200,000 litres per hectare of H2 gas 

may be released into the soil during the life of a legume crop fixing around 200 kg nitrogen per hectare (Peoples 

et al. 2008). In addition, we observed increased populations of certain species of actinomycetes in the 

immediate vicinity of field-grown Hup ─ soybean nodules (Osborne et al. 2010). Some of these organisms are 

known to have growth-promoting characteristics. 

A number of studies in controlled-growth facilities and in the field in Australia and Canada have indicated 

improvements in plant growth and yield of 10-33% when plants are grown in soil exposed to H2. These findings 

need to be confirmed in other environments. If confirmation is obtained, there would be strong reason to 

recommend that future decisions on the choice of rhizobial strains for inoculants should involve ensuring that the 

resultant symbioses are Hup ─ in order to confer yield advantages on subsequent crops. 

Osborne CA, Peoples MB, Janssen PH (2010). Detection of a reproducible, single-member 

shift in soil bacterial communities exposed to low levels of hydrogen. Applied and 

Environmental Microbiology 76, 1471-1479. 

Peoples MB, McLennan PD, Brockwell J (2008). Hydrogen emission from nodulated 

soybeans [Glycine max (L.) Merr.] and consequences for the productivity of a subsequent 

maize (Zea mays L.) crop. Plant and Soil 307, 67-82. 

100 

2011






1100 - 1230 

Authors: Graeme Schwenke 1 , Pip Brock 2 & David Herridge 3 

1 NSW Department of Primary Industries, Marsden Park Road, Calala, 2340, New South 

Wales. 

2 NSW Department of Primary Industries, Port Stephens Fisheries Centre, Locked Bag 

1, Nelson Bay, 2315, New South Wales. 

3 University of New England, Primary Industries Innovation Centre, Armidale, 2351, New 

South Wales. 

Presentation Title: Nitrogen-fixing legumes in farming systems reduce greenhouse gas emissions 


Nitrous oxide (N2O) is a greenhouse gas with close to 300 times the global warming potential of carbon dioxide 

(CO2). A major source of N2O emissions (50–60%) is associated with the application of nitrogenous fertilisers to 

agricultural soils. However, the direct emissions of N2O from soil, as a by-product of nitrification and an endproduct 

of denitrification, represent only a fraction of total greenhouse gas emissions related to N fertiliser use. 

Greenhouse gases (principally CO2) are also emitted in the production, transport and application of nitrogenous 

fertilisers and can be quantified using Life Cycle Assessment (LCA). Emissions of greenhouse gases from 

agricultural systems may be mitigated through partial substitution of fertiliser nitrogen (N) inputs with biologicallyfixed 

legume N because the latter is produced in the soil in situ and is of a dispersed, i.e. non point-source, and 

slow-release nature. To compare total greenhouse gas emissions from crop sequences with contrasting inputs of 

fertiliser- and legume-N in Australia‟s northern grains region, we monitored N2O emissions from the sequences 

for two years and conducted cradle-to-gate, i.e. pre-farm plus on-farm but not post-farm, LCA. We used 

automated greenhouse gas (N2O, CO2, CH4) measuring chambers to monitor soil emissions 7-8 times per day. 

During two years of measurement, cumulative soil N2O emissions differed fourfold between the rotation 

treatments, with canola (+N)–wheat (+N) emitting 1.32 kg N2O-N/ha, compared to 0.71 kg N2O-N/ha from the 

chickpea–wheat (+N) treatment and only 0.34 kg N2O-N/ha from the chickpea–wheat (no N) treatment. These 

treatment differences were increased further in the LCAs when all emissions associated with fertiliser N were 

included. We conclude that substitution of fertiliser N inputs with biologically-fixed legume N can substantially 

reduce greenhouse emissions from grain production systems. 

101 

2011





Authors: 


1100 - 1230 

Kiwamu Minamisawa, Manabu Itakura, Shoko Inaba, and Yoko Shiina. 

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aobaku, Sendai, 

980-8577, Japan 

Presentation Title: Mechanism of N2O emission from soybean nodule rhizosphere and its mitigation 


Nitrous oxide (N2O) is a greenhouse gas that also degrades stratosphere ozone. Agricultural land is a major 

source of N2O through microbial transformation of nitrogen in plant-soil ecosystems. In particular, more N2O is 

emitted from legumes than that with non-legumes. Marked N2O emission were detected from root systems with 

degraded nodules during late growth stage in field-grown soybeans. To clarify the mechanism of N2O emission, a 

model system was developed in laboratory. Thirty-days after soybean plants inoculated with B. japonicum were 

cultivated in Leonard‟s jars, treatments of shoot decapitation (D) and/or soil addition (S) were conducted to 

simulate the nodule degradation and subsequent N2O emission. Double treatment (DS) resulted in the 

degradation and N2O emission from the nodules formed with B. japonicum lacking nosZ. These results 

suggested that soil microbes are required for the N2O emission from degraded soybean nodules. To evaluate 

bradyrhizobial contribution, N2O emission was compared between nirK mutant (∆nirK) and wild-type USDA110 

under identical nosZ genetic backgrounds. N2O emission from the nodules formed with ∆nirK∆nosZ mutant was 

significantly lower than that from ∆nosZ mutant under DS treatment, but retained approximately a half amount of 

N2O emission in ∆nosZ mutant (30-60%), suggesting that nitrate reduction to N2O is due to both B. japonicum 

and other soil microorganisms. On the other hand, it is likely N2O reduction to N2 was mainly mediated by B. 

japonicum cells carrying nosZ, which was consistently supported by the comparisons between wild-type 

USDA110 and nosZ mutants. Thus, B. japonicum plays an important role in determining N2O flux from soybean 

rhizosphere as well as unknown soil microorganisms. To mitigate N2O emission from soybean rhizosphere, we 

developed B. japonicum mutants with higher N2OR activity, and inoculated them to field soils where nosZ minus 

strains of B. japonicum are dominant. As a result, N2O emission from soybean rhizosphere significantly reduced 

in Leonard‟s jar system. 

102 

2011






1100 - 1230 

Authors: Tran Yen Thao 1 , David Herridge 2 , Rosalind Deaker 3 , Le Nhu Kieu 4 , Phan Thi Cong 5 

1 Research Institute for Oil and Oil Plants, 171-175 Ham Nghi Street, District 1, Ho Chi 

Minh City, Vietnam 

2 University of New England, Primary Industries Innovation Centre, Tamworth 

Agricultural Institute, 4 Marsden Park Road, Calala, New South Wales 2340 

3 Faculty of Agriculture, Food and Natural Resources, Ross St. Building A03, University 

of Sydney, NSW 2006 

4 National Soils and Fertilizers Institute, Dong Ngac Village, Tu Liem, Hanoi, Vietnam 

5 Institute of Agricultural Science, 121 Nguyen Binh Khiem Street, District 1, Ho Chi Minh 

City, Vietnam 

Presentation Title: Change in farmer attitudes and practices in Vietnam in the use of inoculant compared 

with baseline 


The survey was constructed to provide the critical information that could be evaluated against baseline attitudes 

established from a similar survey carried out at the beginning of the project. It targeted farmers, extension 

workers as well as local agricultural technicians who are responsible for extending technological advances and 

innovations at agricultural localities. We conclude from this survey that there has been an increase in farmer 

awareness of inoculants and their role in legume growth promotion through biological N fixation. Almost all 

farmers in the final survey knew about inoculants and understood what they do. This was a result of the training 

and extension programe. Their knowledge mainly came from workshops and demonstrations. This survey also 

indicated a great interest by farmers and extension officers in future use of legume inoculants for soybean and 

groundnut in the target areas in Vietnam mostly because of economic reasons and because of their interest and 

desire to utilise new and novel technologies. The lack of use at the time of the second survey largely reflects 

lack of availability in the market place. The survey indicated that legume inoculants would be adopted readily in 

Vietnam provided they were accessible and easy to apply. Increasing production and supply of high quality 

legume inoculants in Vietnam, coupled with an effective extension program, should result in high adoption of 

inoculants. The further extension program would still need to emphasise the replacement of fertiliser N inputs, 

which represent a substantial part of the cost of growing these crops. The whole package should lead to 

increased farmer incomes and the relieving of poverty in many agricultural areas. 

103 

2011





Concurrent Session 17 – Molecular Characterization of N-fixing organisms 

1100 - 1230 

Authors: Ana Alexandre 1,2 Solange Oliveira 1 

1 ICAAM - Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de 

Évora, 7002-554 Évora Portugal. 

2 IIFA - Instituto de Investigação e Formação Avançada, Universidade de Évora, 7002- 

554 Évora, Portugal. 

Presentation Title: Major chaperone genes are highly induced in heat-tolerant rhizobia 


The demand for more effective utilization of biologically-fixed N in agricultural systems has prompted studies on 

rhizobia tolerance to abiotic factors. Rhizobia ability to endure environmental stresses, such as soil pH, salinity 

and temperature is particularly important in legume-rhizobia symbioses under suboptimal conditions. The present 

work aimed at evaluating the temperature stress tolerance of native chickpea rhizobia and investigating if 

tolerance is related to isolates„ species or origin site. Another aim was to investigate the molecular bases of 

temperature stress tolerance, by comparing the expression levels of major chaperone genes, in thermotolerant 

and thermosensitive isolates. 

A set of 53 chickpea mesorhizobia, previously isolated from several provinces of Portugal and characterized in 

terms of symbiotic effectiveness, plasmid profiles and species affiliation, was used (Alexandre et al 2009). 

Temperature stress tolerance was evaluated under cold, heat and heat shock conditions. Mesorhizobia showed 

high diversity in their ability to grow under temperature stress; nevertheless most isolates tolerate heat shock or 

cold stress better than continuous heat. Distinct species groups were found to differ significantly in their ability to 

tolerate temperature stress. An association was found between some provinces of origin and stress tolerance of 

the isolates. 

In order to study the molecular bases of temperature stress tolerance, the mRNA levels of chaperone genes 

were analysed upon stress, using tolerant and sensitive chickpea rhizobia. Analysis of dnaK and groESL genes 

expression by northern hybridisation, using isolates from several species groups, showed an increase in the 

transcripts levels with heat but not with cold stress. Interestingly, following temperature upshifts, the induction of 

chaperone genes was higher in tolerant than in sensitive isolates from the same species (Alexandre & Oliveira, 

2011). Overall, these results suggest that higher transcriptional induction of the major chaperone genes could be 

related with a higher tolerance to heat in rhizobia. 

Alexandre A, Brígido C, Laranjo M, Rodrigues S & Oliveira S (2009). Survey of chickpea rhizobia diversity in Portugal reveals the 

predominance of species distinct from Mesorhizobium ciceri and Mesorhizobium mediterraneum. Microb Ecol 58: 930-841. 

Alexandre A & Oliveira S (2011). Most heat-tolerant rhizobia show high induction of major chaperone genes upon stress. FEMS Microbiol 

Ecol 75: 28-36. 

This work was supported by Fundação para a Ciência e Tecnologia: project FCOMP-01-0124-FEDER-007091 and fellowship to A. A. 

(SFRH/BPD/73243/2010). 

104 

2011






1100 - 1230 

Authors: Nazalan Najimudin 1 , Hok-Chai Yam 1 , Mohd-Razip Samian 1 and Suriani Mohamad 1 

1 School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Penang, 

Malaysia. 

Presentation Title: Characterization of nitrogen fixation genes from Paenibacillus polymyxa ATCC 15970 


Paenibacilus polymyxa ATCC 15970 is a Gram positive bacterium capable of converting dinitrogen (N2) to 

ammonia. The nitrogen fixation operon of this strain was determined to have the gene arrangement of nifBHDK. 

The deduced NifBHDK amino acid sequences were 499 aa, 255 aa, 482 aa and 509 aa, respectively. The 

putative nifD and nifK ORFs were overlapping by 4 nucleotides. The transcriptional start sites of nifB and nifH 

were determined by cRACE and cmRACE methods. Two different transcriptional start sites were obtained - 

upstream of nifB and another upstream of nifH. Therefore, the nifH gene could possibly be expressed from two 

different mRNA molecules transcribed from two different promoters located upstream of nifB and nifH, 

respectively. Based on their transcriptional start sites, the promoter regions of nifB and nifH revealed consensus 

sequences that were distinguishable from the traditional nif promoter. This suggested that the expression of nif 

genes in P. polymyxa was possibly controlled by a unique regulation system. The nifB gene is possibly coexpressed 

with the structural genes nifHDK. The promoter sequences of nifB and nifH were different from the 

typical nif promoter motif. Two highly consensus sequences were found at the presumptive promoter regions of 

nifB and nifH located roughly at positions -8 (GTAAGGG) and -28 (AGACAAA) with respect to their 

transcriptional start sites. This novel promoter motif merits future investigation. 

105 

2011






1100 - 1230 

Authors: Melissa K. Corbett, Lesley A. Mutch and Elizabeth L.J. Watkin 

School of Biomedical Sciences, Curtin University, Kent Street, 6845, Western Australia 

Presentation Title: Identification and evolution of NIF genes in Leptospirillum species – an acidophilic 

chemolithoautotroph 


Leptospirillum are gram negative, chemolithoautotrophic extremophiles found in environments where sulfide 

and iron bearing minerals are exposed to the air. Their natural ability to oxidize ferrous iron for energy, leaving 

behind other liberated minerals has been industrialized in the form of biomining. Biomining operations rely on 

this microbial aided extraction of metals from solid minerals, but mineral recovery rates can be affected if 

nutrient levels fluctuate and gradients form. The addition of ammonium based chemicals to meet 

Leptospirillum growth needs can be expensive, and equal distribution is not guaranteed. Genomic research 

has uncovered that two of the three documented species, L. ferrooxidans and L. ferrodiazotrophum encode 

genes for nitrogen fixation. To ascertain whether the third species, L. ferriphilum, also has the potential for 

diazotrophy, degenerate nifH primers were tested. A single PCR product of approximately ~900bp was 

amplified and subsequent sequence analysis revealed significant identity (84%) to the nifH genes in the other 

Leptospirillum species. Phylogenetic analysis of the single nifH genes placed all Leptospirillum species in the 

conventional Mo-containing nifH cluster I, closely related to the nifH genes of �-proteobacterium, 

Acidithiobacillus, a known nitrogen fixing biomining microbe. Concatenated super-gene alignment of the nifH 

gene with 16SrRNA, gyr B, nifD and nifK provided a greater resolution of evolution amongst the closely 

related strains. The use of maximum likelihood and maximum parsimony phylogenetic trees further elucidated 

the evolutionary relationships between the three Leptospirillum species. Discovery of these genes in all 

identified Leptospirillum species aids in cementing the status of the Leptospirillum genus as free living 

diazotrophs whose presence in biomining operations can help sustain mineral leaching rates when soluble 

nitrogen levels are low, without the incursion of additional operational costs. 

106 

2011






1100 - 1230 

Authors: Ni Luh Arpiwi1 , Elizabeth LJ Watkin2 , Guijun Yan1 , Elizabeth L Barbour, 1, 3 

and Julie A Plummer 1 

1 School of Plant Biology, University of Western Australia, 35 Stirling Hwy Crawley WA 

6009 

2 School of Biomedical Science, Curtin University, Bentley WA 6102 

3 Forest Products Commission of Western Australia, 117 Great Eastern Highway 

Rivervale, WA 6103, Australia. 

Presentation Title: Physiological and molecular characterization of the root nodule bacteria nodulating 

Millettia pinnata, a biodiesel tree 


Milletia pinnata is a leguminous tropical tree that produces seed oil suitable for biodiesel. By optimizing the 

symbiotic relationship with nitrogen fixing bacteria reduced fertilizer inputs would be required, which would be 

advantageous for this second generation biofuel crop, especially for production on marginal land. This study 

aimed to isolate root nodule bacteria from soil around Millettia trees, confirm their symbiotic potential, examine 

their physiological characteristics and to identify elite strains. 

Soil samples were collected from the base of Millettia pinnata grown in plantations in northern, tropical 

Western Australia. Seeds were planted in pots layered with 3 cm of sterile coarse sand then 3 cm soil, 2 cm 

sterile coarse sand and 1 cm sterile plastic beads on the top. Seeds were placed in the upper coarse sand 

layer and grown for 18 – 20 weeks. Root nodules were harvested and crushed, and the contents streaked 

onto a half-lupin agar plates and incubated at 29 o C. Forty pure isolates were obtained which produced single 

colonies in 5-9 days. Optimum growth conditions for these isolates were pH 7 – 9 and 29 – 37 o C. Growth of 

all isolates was reduced at 1 – 2% NaCl and no isolates grew at 3% NaCl. Most isolates had optimal growth 

on mannitol, arabinose or glutamate as a single carbon source, only a few grew on sucrose and none grew on 

lactose. Cluster analysis of isolates based on physiological characteristics indicated phenotypic diversity. 

Isolates were re-authenticated and the effectiveness of the symbiotic association assessed based on shoot 

dry weight compared to that of the nitrogen-fed control plants. Genetic diversity of isolates was analyzed and 

confirmed using the sequence of the 16S RNA gene. The outcomes of this research will promote the growth 

of Millettia plantations in nutrient poor soils which alleviates competition on arable lands for food production. 

107 

2011





Concurrent Session 18 – P Nutrition & Symbioses 

1100 - 1230 

Authors: Neera Garg 1 and Harmanjit Kaur 2 

Department of Botany, Panjab University, Chandigarh 160014, India 

Presentation Title: Role of arbuscular mycorrhizal (am) fungi and zinc in enhancing nitrogen fixation in 

pigeon pea under cadmium stress 


AM fungi are known to alleviate heavy metal stress in plants (Hildebrandt et al 2007). Cadmium (Cd) is a toxic 

non essential element, whereas Zinc (Zn) is an essential element but regarded toxic at high concentrations. 

Cd and Zn compete for transport via common carrier at root plasma membrane and Zn interferes with Cd 

uptake in plants (Hassan et al 2005). The intent of present work was to analyze accumulation of heavy metals 

(Cd and Zn) in nodules of two Cajanus cajan (L.) Millsp. (pigeonpea) genotypes and their subsequent impact 

on nitrogen fixation, oxidative stress and non protein thiols (glutathione and phytochelatins) with and without 

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) 

were applied singly and in combinations to 15 day old seedlings. Accumulation of Cd and Zn resulted in sharp 

reduction in nodule number (NN), nodule dry mass (NDM) as well as nitrogen fixation {leghemoglobin (LHb) 

and nitrogenase (N2ase)} of pigeonpea plants, although Cd had more pronounced effects than Zn. Cd induced 

ROS generation, lipid peroxidation and altered non protein thiols were largely reversed by Zn 

supplementation, suggesting antagonistic behaviour of Zn. Remarkable genotypic variation was found, with 

much more severe response of both the metals in P792 than Sel 85N. Glomus mosseae along with Zn further 

attenuated the phytotoxic effects of metals in nodules by significantly decreasing metal uptake and oxidative 

stress ultimately leading to better nitrogen-fixation in pigeonpea. 

Hassan MJ, Zhang G, Wu F, Wei K & Chen Z (2005). Zinc alleviates growth inhibition and oxidative stress 

caused by cadmium in rice J Plant Nutr Soil Sci 168: 255-261. 

Hildebrandt U, Regvar M & Bothe H (2007). Arbuscular Mycorrhiza and Heavy Metal Tolerance. 

Phytochemistry 68: 139-146. 

108 

2011






1100 - 1230 

Authors: Andry Andriamananjara 1,2,* , Mahamadou Malam Abdou 3 , Lilia Rabeharisoa 1 , Laurie 

Amenc 5 , Hélène Vailhe 5 , Dominique Masse 4 , Jean-Jacques Drevon 5 

1 LRI-SRA, Laboratoire des Radio-isotopes, Université d'Antananarivo, Route 

d'Andraisoro, BP 3383, 101 Antananarivo, Madagascar ; 

2 Ecole Supérieure des Sciences Agronomiques d’Antananarivo, Madagascar. 

3 Laboratoire Banques de gènes CERRA / KOLLO, Institut National de Recherche 

Agronomique du Niger (INRAN), BP 429 Niamey, NIGER. 

4 Institut de Recherche pour le Développement, UMR Eco&Sols, Montpellier- France . 

5 Institut National de la Recherche Agronomique, UMR Eco&Sols, Montpellier-France. 

Presentation Title: Genotypic variation for N2-fixation in Voandzou (Vigna subterranea) under P deficiency 

and P sufficiency 


Genetic variation associated with N2 fixation exists in numerous legume species (Graham, 2004). High 

symbiotic N2 fixation under P deficiency is related closely to nodulation which was used in legume selection 

for N2 fixation (Herridge and Rose, 2000). Until now, study of genetic potential of neglected crops like Vigna 

subterranea (bambara groundnut or voandzou) is often limited while its agronomic properties is interesting for 

the farmers of Africa. In order to assess the genotypic variation of voandzou for tolerance to phosphorus 

deficiency, a physiological approach of cultivar selection was performed with 54 cultivars from Madagascar, 

Niger and Mali in hydroponic culture under P deficiency and P sufficiency and inoculated with the reference 

strain of Bradyrhizobium sp. Vigna CB756. The results of nodulation and plant biomass, which are closely 

related, showed a large dispersion between cultivars (0.05-0.43 g nodule dry weight per plant and 0.50-5.51 g 

shoot dry weight per plant). The cultivars which presented the maximum growth during the experiment 

presented a high efficiency in use of the rhizobial symbiosis calculated as the slope of plant biomass 

regression as a function of nodulation. A large increase in nodulated-root O2 consumption under P deficiency 

was observed for the two most tolerant cultivars. The microscopic analysis with in situ RT-PCR of the nodule 

sections showed an increase of a phytase gene expression with tolerance of cultivars to P deficiency. These 

results highlight the genotypic variability among voandzou cultivars for the phosphorus use efficiency for N2 

fixation as a mechanism of adaptation to phosphorus deficiency. 

Herridge D, Rose I, 2000. Breeding for enhanced nitrogen fixation in crop legumes. Field Crops Research 65. 

229-248. 

Graham PH., Hungria M, Tlusty B., 2004. Breeding for better nitrogen fixation in grain legumes: Where do the 

rhizobia fit in? On line Crop Management. 

109 

2011






1100 - 1230 

Authors: Sipho Maseko 1 and Felix D. Dakora 2 

1 Department of Crop Science, and 2 Department of Chemistry, Tshwane University of 

Technology, 175 Nelson Mandela Drive, Private Bag X680, Pretoria 0001, South Africa 

Presentation Title: Phosphorus nutrition in symbiotic Cyclopia and Aspalathus species from the Cape 

fynbos of South Africa 


Nitrogen and phosphorus are the most limiting nutrients affecting plant growth in the Cape fynbos. In this study, 

acid and alkaline phosphatase (APase) activity of rhizosphere soils from Cyclopia genistoides, C. subternata, 

Aspalathus caledonensis and A. aspalathoides were assayed as indicators of P nutrition in these legumes. The 

results showed a significantly (p≤ 0.05) higher APase activity in rhizosphere soils of the four legumes and the 

non-legumes Leucadendron strictum, Eleiga thyrsoidea and Mimetes cucullatus relative to bulk soil. Furthermore, 

rhizosphere P and shoot P concentrations closely mirrored the phosphatase-enzyme activity in the rhizosphere 

of each species, suggesting that rhizosphere enzyme activity can serve as a good indicator of P nutrition in 

Cyclopia and Aspalathus species in the Cape fynbos. The relationship between APase activity in organs and P 

concentration of those organs in three Cyclopia and two Aspalathus species was also assessed at the same 

sites in the fynbos. At all three sites, APase activity was greater in organs of the legumes compared to the nonlegume 

Leucadendron strictum. At Koksrivier, leaf APase activity was highest in Cyclopia genistoides, followed 

by Aspalathus caledonensis, and least in A. aspalathoides. At Kleinberg and Kanetberg, Cyclopia subternata and 

C. longifolia also showed highest APase activity in leaves, followed by stems, and lowest in roots. The P 

concentration of each organ closely mirrored its APase activity, and APase activity was positively correlated with 

P concentration in leaves, stems and roots of C. genistoides, C. subternata, and C. longifolia. This indicates that 

in the Cape fynbos, APase activity can be used as a good indicator of P nutrition in Cyclopia and to, some 

extent, Aspalathus species. 

110 

2011






1100 – 1230 

Authors: Bargaz Adnane 1,2 , Faghire Mustapha 1 , Mandri Btissam 1 , Farissi Mohamed 1 , Jean 

Jacques Drevon 2 , and Ghoulam Cherki 1 

1 Equipe de Biotechnologie Végétale et Agrophysiologie des Symbioses, Faculté des 

Sciences et Techniques Guéliz-Marrakech, BP 549, 40000, Marrakech, Morocco. 

2 INRA-IRD-SupAgro, UMR Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols 

& Agroécosystèmes 2 Place Viala, F34060, Montpellier, France. 

Presentation Title: Phosphorus deficiency affected conductance to O2 and ascorbate peroxydase gene 

expression in nodules of Phaseolus vulgaris-rhizobia symbiosis 


Although recent studies have addressed the effects of phosphorus (P) deficiency on nodule respiration and 

oxidative stress, little attention has been given to elucidate the relationships between nodule permeability, nodule 

P status, and antioxidative responses. To study these traits, two recombinant inbred lines, namely RILs L115 and 

L147 were inoculated with Rhizobium tropici CIAT899, and grown in hydroaeroponic culture under P-sufficiency 

(250) versus P-deficiency (75 µmol P plant -1 week -1 ) conditions. At the flowering stage, plants and nodules 

biomasses, P contents and localization of nodule ascorbate peroxidase (APX) transcripts were determined after 

measuring O2 uptake by nodulated roots and nodule conductance to O2 diffusion (gn). The results showed that Pdeficiency 

significantly decreased plant growth and nodulation, with differences between bean genotypes. Pdeficiency 

also induced a decrease in nodule P content (31%) in the sensitive L147 and tolerant line L115, a 42 

and 27% reduction in shoots of sensitive and tolerant lines, respectively. Under P-deficiency, gn increased more 

for the sensitive (39%) than for the tolerant lines (27%). This increase was linked to a rise both in the P levels in 

nodules and shoots, as well as in the efficiency of symbiotic nitrogen fixation as determined by nodule-dependent 

biomass production for the sensitive lines. Furthermore, positive correlations were found between O2 

permeability, gn and P content both in nodules and shoots (r 2 = 0.94** and r 2 = 0.96**). Additionally, P-deficiency 

increased electrolyte leakage, the oxidative stress and the transcripts for ascorbate peroxidase gene in nodules. 

The later was more abundant in the inner cortex and the infected cells close to inner cortex. We conclude that gn 

increased with increases in both nodule P content and antioxidative responses. 

111 

2011




Poster Board 

Poster Title Presenting 

Number 

Author 

1 Symbiotic nitrogen contribution by a long-duration pigeonpea 

genotype intercropped with maize in South Africa 

Cherian Mathews 

2 Growth inhibition of Sinorhizobium medicae in the presence of 

amino acids and the absence of supplemented calcium is 

alleviated by a mutation in the low pH inducible gene lpiA. 

3 Rhizobia nodulating Vicia faba in Tunisia constitute a new branch 

inside the genus Rhizobium 

4 Characterization of two splice variants of SIP1 and their functions 

during nodule development in Lotus japonicus 

5 Characterization of rhizobia isolates using molecular methods 

6 The contribution of N-fixing legumes to the productivity of wheat in 

mediterranean agriculture systems of Chile. 

7 Crosstalk between GmSAT1 and yeast unveils a novel family of 

eukaryotic ammonium transport proteins 

8 Co-inoculation of rhizobia with highly induced ACC deaminase 

bacteria could alleviate the stress of legumes growing under stress 

conditions 

9 Rice growth promotion after inoculation of Sinorhizobium melilotii 

1021 and its mechanism 

10 Differential activation of CCaMK between root nodule and 

arbuscular mycorrhizal symbioses 

11 Determination of optimal phosphorus concentrations which 

enhance the establishment of both mycorrhizal and rhizobial 

symbiosis 

12 Comparative analysis of genome sequences of Rhizobium 

galegae HAMBI 540 and HAMBI 1141 

13 Denitrification and nitrous oxide emissions by soybean 

bradyrhizobia 

14 Proteomic profile of the soybean symbiosome membrane 

15 Nodulation studies of cowpea and its response to phosphorus 

application in Botswana under glasshouse conditions 

112 

Wan Adnawani 

Meor Osman 

Ridha Mhamdi 

Hui Zhu 

Bakhtiyor Umarov 

Carlos Ovalle 

Danielle 

Mazurkiewicz 

Panlada Tittabutr 

Yuxiang Jing 

Yoshikazu 

Shimoda 

Manijeh 

Mohammadi- 

Dehcheshmeh 

Janina Osterman 

2011 

Presenting Day 

30 th November 

Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 

Robert M. Boddey 30 th November 

Wednesday 

15:00 – 16:00 

Victoria Clarke 29 th November 

Tuesday 

Flora Pule- 

Meulenberg 

16 Lotus japonicus nodulates when it sees red Akihiro Suzuki 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00




Poster Board 

Number 

Poster Title 

17 Role of the Cpx two component regulatory system and a MFS 

transporter in the symbiosis between Sinorhizobium meliloti and 

leguminous plants 

18 Compatibility with rhizobia of polymer adhesives and colourants 

used in preinoculated legume pasture seed 

19 Efficiency of Bradyrhizobium japonicum in different formulations 

and when co-inoculated with bacillus subtilis on soybean in a 

Kenyan soil 

20 Potential of indigenous bradyrhizobia versus commercial 

inoculants to improve cowpea and green gram yields in Kenya 

21 Isolation and characterization of diazotrophic microsymbionts 

from root nodules of Mucuna bracteata 

22 Lipopolysaccharide binding protein, a new requirement for 

legume-rhizobium symbiosis 

23 The role of 1-aminocyclopropane-1-carboxylate (ACC) 

deaminase enzyme on leguminous nodule senescence 

24 Gamma irradiation and autoclave sterilization of peat and 

compost as the carrier for rhizobial inoculant production 

25 Effect of nitrogen, phosphorus, magnesium and calcium nutrition 

on plant growth and levels of macronutrients in the honeybush 

tea plant, Cyclopia longifolia (Vogel l.) 

26 Root-nodule bacteria isolated from cowpea and bambara 

groundnut enhance mineral nutrition in their homologous hosts 

27 Evaluation of elite commercial soybean varieties for N2 fixation 

and grain yield in South Africa 

28 Ecophysiological studies and biodiversity of root-nodule bacteria 

nodulating Psoralea species in the cape fynbos of South Africa 

29 Nitrogen fixation promotes accumuation of dietarily-important 

mineral nutrients in edible leaves of cowpea (Vigna unguiculata l. 

Walp.) 

30 Diversity and phylogeny of Bradyrhizobium spp. Isolated from 

shrub and tree legumes in Ethiopia 

113 

Presenting Author 

2011 


Mário Santos 30 th November 

Wednesday 

15:00 – 16:00 

Elizabeth Hartley 

Mary Atieno 

Samuel Mathu 

Amir Ghazali 

Toshiki Uchiumi 

Neung Teaumroong 

Nantakorn Boonkerd 

Buhlebelive 

Mndzebele 

Thabo Makhubedu 

Nyamande Mapope 

Sheku Kanu 

Alphonsus Belane 

Aregu Amsalu 

Aserse 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00




Poster Board 

Number 

Poster Title 

31 Phylogenetic position of Rhizobium galegae 

32 Study on soybean nodule symbiosome membrane localised 

transporters 

33 Development of an inoculant for Acacia acuminata to improve 

growth of Santalum spicatum 

34 Endophytic colonization of sweet potato by a diazotrophic 

bradyrhizobia 

35 Functional Analysis of STM Mutants Concerning Amino Acid 

Metabolism of Mesorhizobium loti 

36 Establishment of retrotransposon-mutagenized population of the 

model legume Lotus japonicus and high throughput, deep 

sequencing-based insertion site identification. 

37 Rhizobium delivery systems for grain legumes in southern 

Australia 

38 BNF efficiency of rhizobia isolates from Phaseolus vulgaris in 

subsistence agrosystems in Dominican Republic. 

39 Protection of plant health by Micromonospora isolated from 

alfalfa root nodules. 

40 Identification by MALDI-TOF MS spectrometry of rhizobia 

isolated from nodules of different legumes 

41 Expression of an abscisic acid-responsive ƒÀ-1,3-glucanase 

gene in autoregulation of nodulation. 

42 Conservation of legume-rhizobium symbiosis genes in nonlegume. 

43 Analysis of nif gene derepression in Azotobacter vinelandii by 

quantitative real-time PCR 

44 Nodule-specific cystein-rich peptides found in actinorhizal plant 

Datisca glomerata 

45 N2O emission from degraded soybean nodules by denitrification 

of Bradyrhizobim japonicum and other soil microorganisms 

114 

Author 

Seyed Abdollah 

Mousavi 

2011 



Wednesday 

15:00 – 16:00 

Chi Chen 30 th November 

Wednesday 

Elizabeth Watkin 

Yoshinari Ohwaki 

Shigeyuki Tajima 


David Pearce 

Beatriz Urbano 

Pilar Martínez- 

Hidalgo 

Eustoquio Martinez- 

Molina 

Ken-Ichi Osuki 

Keisuke Yokota 

Cesar Poza Carrion 

Irina Demina 

Fumio Ikenishi 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00




Poster Board 

Number 

Poster Title 

46 Functional characterization and interactions of the azorhizobial 

parA and parB proteins 

47 Trehalose accumulation in osmotically challenged rhizobia and 

its effect on desiccation tolerance. 

48 A MYB coiled-coil type transcription factor interacts with NSP2 

and is essential for nodulation in Lotus japonicus 

49 Genome sequence of Mesorhizobium huakuii 7653r which 

establishs a highly specific symbiosis with Astragalus sinicus in 

China 

50 Identification of the regulatory genes required for the acid 

activation of the low pH inducible gene lpiA in Sinorhizobium 

medicae 

51 Selection of plant growth promoting rhizobacteria (PGPR) to 

enhance nodulation of grain legumes by rhizobia 

115 

Chi-Te Liu 

Author 

Andrea Casteriano 

Zhongming Zhang 

Youguo Li 

Tian Rui 

2011 



Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 


Wednesday 

15:00 – 16:00 


Tuesday 

15:00 – 16:00 

Liza Parkinson 30 th November 

Wednesday 

15:00 – 16:00




Title Symbiotic nitrogen contribution by a long-duration pigeonpea genotype intercropped with 

maize in South Africa 

Authors 

Poster Board Number 1 

1 Cherian Mathews, 2 Wim van Averbeke and 2 Felix Dakora 

1 Department of Agriculture, P/Bag X11318, Nelspruit-1200 

2 Tshwane University of Technology P/Bag X680, Pretoria-0001 

As a crop, pigeonpea has a huge market value in South Africa due to the growing Asian population. Large 

quantities of pigeonpea grown is currently imported to meet local demand. This indicates the need to evaluate 

pigeonpea genotypes for growth, grain yield and symbiotic performance in order to identify genotypes for use in 

local cropping systems. 

Field experiments were conducted at Nelspruit during 2007, 2008 and 2009 cropping seasons to assess N2 

fixation and yield potential of pigeonpea (Cajanus cajan L. Millspaugh) in intercropping systems used by 

resource-poor farmers in Africa. Three intercropping systems (within-rows, alternate rows, and paired rows) and 

their sole cropped pigeonpea (cv. ICEAP00040) and maize (cv. ZM521) were used in a randomized complete 

block design with five replications. Destructive sampling was done at four stages of legumes (i.e. maize 

tasseling, maize harvest, pigeonpea flowering, and pigeonpea harvest) for dry matter yield and measurement of 

N2 fixation using 15 N natural abundance. The results of two cropping seasons (2007-08 and 2008-09) are 

presented here. Dry matter yield was significantly (p=0.01) lower in intercropped pigeonpea relative to 

monoculture, but was positively correlated with N content, δ 15 N and amount of N-fixed. The N content per plant 

was highest in monocultured pigeonpea as a result of its larger dry matter yield. In both seasons, the δ 15 N values 

of intercropped pigeonpea were lower than monoculture, and were lowest in plants on same-row intercropping 

and/or alternate row intercroping. 

As a result, %Ndfa values were significantly greater under these cropping systems. The average amount of Nfixed 

was up to 173 kg ha -1 in monoculture and 153 kg ha -1 in intercrop, levels which peaked at pigeonpea 

flowering in both seasons. The results show that pigeonpea can contribute substantial amounts of symbiotic N 

and thus enhance soil productivity under traditional cropping systems. 

116 

2011




Title Growth inhibition of Sinorhizobium medicae in the presence of amino acids and the absence 

of supplemented calcium is alleviated by a mutation in the low pH inducible gene lpiA. 

Author Wan Adnawani Meor Osman 1,2 , Ravi Tiwari 1 , Lambert Brau 1 , Vanessa Melino 1 & Wayne 

Reeve 1 . 



Australia 

2 Department of Biological Sciences, Faculty of Sciences and Technology, University 

Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. 

Sinorhizobium medicae WR101 was identified as a mTn5-GNm induced mutant of WSM419 that expressed acid 

inducible GUS activity. Characterisation of the mutation revealed that the promoterless gusA gene in mTn5-GNm 

was fused to the low pH inducible gene lpiA. The LpiA protein contains a COG0392 signature (indicative of an 

integral membrane protein) domain revealing that this protein is most likely a transmembrane protein. In addition, 

LpiA also contains three other domains of unknown function including DUF470, DUF471 and DUF472 which 

share 83%, 62% and 58% similarity to proteins found in animal pathogens, plant pathogens and other symbionts, 

respectively (Reeve et al., 2006). An in depth characterisation of the phenotype of the mutant cells revealed that 

the lpiA mutation disrupted the acid-tolerance response (ATR) associated with S. medicae WSM419. In addition 

to this, we have now found that the lpiA mutation enables the mutant to grow on amino acid rich media devoid of 

calcium. In contrast, the wild-type strain WSM419 cannot grow in this condition and requires calcium to 

overcome the growth inhibition found with amino acid containing rich media. Complementation analysis has 

revealed that mutant growth can be severely inhibited on media containing amino acids in the absence of 

sufficient calcium. These observations have demonstrated that the absence of LpiA is essential for growth on 

calcium deficient media containing amino acids. Potential reasons for the growth inhibition of S. medicae by 

amino acids in the absence of calcium will be presented. 

117 

2011




Title Rhizobia nodulating Vicia faba in Tunisia constitute a new branch inside the genus 

Rhizobium 

Authors Sabrine Saïdi, Sabrine Chaïbi & Ridha Mhamdi 


Centre of Biotechnology of Borj-Cedria, Laboratory of Legumes, Hammam-lif, Tunisia. 

Faba bean is a major leguminous crop cultivated in many countries around the world. The nodulating rhizobia 

were usually associated to Rhizobium leguminosarum bv. viciae. However, some recent studies revealed that 

this crop is also nodulated by R. pisi and R. fabae. Nevertheless, R. pisi was described on the basis of a unique 

isolate with an ambiguous origin. The phylogenetic analysis of the 16S rDNA and housekeeping genes showed 

that these novel described species are closely related and would be synonymous. The aim of this work is to 

study the genetic diversity and symbiotic effectiveness of rhizobia nodulating faba bean in Tunisia. Rhizobia 

nodulating Vicia faba in various Tunisian soils from different geographical regions were isolated using standard 

procedures. More than 50% of the nodule isolates were endophytes that failed to re-nodulate their original host. 

The nodulating isolates were assigned all to Rhizobium leguminosarum on the basis of PCR-RFLP analysis of 

16S rDNA. Ten representative isolates were selected based on their REP-PCR and PCR-RFLP of nodC and 

nifDK patterns. Their taxonomic position was further assessed by sequence analysis of 16S rDNA, recA, atpD, 

glnII, nodA, nodC and nifH genes. The results indicated that all the isolates formed a new distinct branch inside 

the genus Rhizobium and would probably constitute a new species. Nodulation and effectiveness tests on V. 

faba vars. equina and minor and Lens culinaris showed different host-specific behaviors with variable 

efficiencies. 

118 

2011




Title Hui Zhu, Chao Wang, Liping Jin, Tao Chen, Longxiang Wang, Zhongming Zhang* 

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University 

Authors Characterization of two splice variants of SIP1 and their functions during nodule 

development in Lotus japonicas 


Lotus japonicus SymRK and NIN genes are both required for the successful establishment of root nodule 

symbiosis. Previously, we discovered that ARID family transcription factor SIP1 interacted with SymRK and 

specifically bound to the AT-rich motif of NIN promoter (Zhu et al., 2008). However, the biological function of 

SIP1 wasn‟t fully understood. Here, an isoform of SIP1 (called SIP1S in this work) was isolated, referred to as 

SIP1L, which is produced by alternative splicing. SIP1L contains an ARID domain and an Hsp20 domain, while 

SIP1S lacks the complete Hsp20 domain with 17 amino acids deletion. SIP1L presents a more abundant 

expression than SIP1S and can‟t interact with SymRK in yeast, suggesting some characterizations of the two 

splice variants are altered. However, SIP1L and SIP1S colocalize in nuclei in onion epidermal cells and form 

homo- and heterocomplexes with itself and with each other, respectively, which is verified via yeast-two hybrid, in 

vitro pull-down and BiFC assay in Nicotiana benthamiana. Overexpression of the less abundant SIP1S leads to 

the increasing of the nodule number and RNA interference of SIP1 gene results in the significant impairment of 

nodulation and the interrupted nodule formation during nodule primordial stage. Spatial expression by 

SIP1p::GUS shows the centralized expression in the vascular tissue of developing nodule primordia. We propose 

that SIP1L and SIP1S may form polymers and be involved in nodule development process. On the other hand, 

we surprisedly find that suppressed SIP1 expression affects arbuscule formation, implicating the possible 

function of SIP1 gene in arbuscular mycorrhizal symbiosis. 

Zhu, H, Chen, T, Zhu, M, Fang, Q, Kang, H, Hong, Z, and Zhang, Z (2008). A novel ARID DNA-binding protein 

interacts with SymRK and is expressed during early nodule development in Lotus japonicus. Plant Physiol 

148:337-347. 

119 

2011




Title Characterization of Rhizobia isolates using molecular methods 

Authors Bakhtiyor Umarov 1,2 , Zair Shakirov 1 , Khojiakbar Yadgarov 2 , Zufarjon Khojiev 2 


1 Institute of Microbiology, Uzbekistan, 

2 Institute of Genetics and Experimental Biology of Plants, Uzbekistan 

The rhizobia/legume root nodule symbiosis is the result of a complex interaction between the host plant and the 

bacteria. Within the rhizosphere, bacteria have access to carbon and nitrogen sources originating from the plant. 

In response, rhizobia themselves secrete molecules (Nod factors) that signal to the plant the presence of suitable 

symbiotic partners. The rhizobia are then able to enter the root in the region of emerging root hairs. Finally, the 

bacteria are wrapped by a plant-derived membrane and enter the cytosol of plant cells. (DW Ehrhardt, et al 1992) 

. The bacteria are now referred to as bacteroids and are able to fix molecular nitrogen, which is released into the 

plant cells. We are isolate new effective strains from nodule plants Onobrycis tr., grow in the Arid zones Central 

Asia. For the characterize and identify rhizobial strains and to study the diversity of indigenous Rhizobium strains 

we are used the PCR. PCR can be performed rapidly with strains, species or genus specific primers that 

generate fingerprint characteristics of each strain. DNA primers corresponding to repetitive sequences that 

present in multiple copies of the genomes of most Gram-negative and Gram-positive bacteria can be used to 

fingerprint the genomes of rhizobial strains. Three families of repetitive sequences have been identified, including 

(REP), (ERIC) and BOX element. The REP-PCR genomic fingerprinting protocols have been successfully used 

in a wide variety of Eubacteria for typing strains and studying their diversity. We have used this highly 

discriminative and reproducible technique to study the genetic diversity of rhizobial strains. 

DW Ehrhardt, EM Atkinson and Long, SR. 1992: Depolarization of alfalfa root hair membrane potential by 

Rhizobium meliloti Nod factors Science. Vol. 256 no. 5059 pp. 998-1000 ,DOI: 10.1126/science.10744524 

120 

2011




Titles The contribution of N-fixing legumes to the productivity of wheat in Mediterranean agriculture 

systems of Chile. 

Authors Carlos Ovalle 1 , Alejandro del Pozo 2 & Soledad Espinoza 1 


1 INIA-Quilamapu, Chillán, Chile. 

2 Facultad de Ciencias Agrarias, Universidad de Talca, Talca, Chile. 

In highly intensified cereal farming systems in Chile fertilization is approximately 40% of the production costs, 

and N represents over 60% of this cost. The aim of this work was to evaluate the recovery of N by wheat grown 

after Lupinus angustifolius, L. luteus, Pisum sativum or two mixtures of annual legumes. As a control we used a 

oat-wheat rotation without N fertilization. The study was conducted in two contrasting Mediterranean 

environments: interior dryland ( granitic Alfisol, pH 6.2, organic matter (OM) 1.5%; 650 mm of annual rainfall) 

and Andes foothill (volcanic Andisol, pH 5.5, OM 13%; 1200 mm of annual rainfall), during 2008 and 2009. The 

determination of the recovery of N in the cereal was performed by isotope dilution method with 15 N; after sowing 

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 

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- 

2.4 ton ha -1 after annual legume mixtures. The recovery of N by wheat from the N fixed by grain legumes ranged 

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 

total N) in the case of annual legumes . In the Andes foothill, wheat yield ranged 5.5-6.4 ton ha -1 after grain 

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 

and between 3.4 to 4.3 kg N ha -1 in the rotation with annual legumes. With the other grain legumes the N 

recovery by wheat was not detected. 

121 

2011




Title Crosstalk between GmSAT1 and yeast unveils a novel family of eukaryotic ammonium 

transport proteins 

Authors Danielle Mazurkiewicz 1 , Patrick Loughlin 2 , David Chiasson 1 , Mamoru Okamoto 3 , David A. 

Day 4 , Stephen D. Tyerman 1 & Brent N. Kaiser 1 


1 School of Agriculture, Food and Wine. The University of Adelaide, 

2 School of Biological Sciences. The University of Sydney, 

3 Australian Centre For Plant Functional Genomics. The University of Adelaide, 

4 Flinders University 

GmSAT1 is a soybean basic helix-loop-helix (bHLH) transcription factor localised to the peribacteroid membrane 

of nitrogen fixing nodules 1 . Overexpression of GmSAT1 restores the growth of a yeast ammonium (NH4 + ) 

transport mutant 26972c on low (1 mM) NH4 + concentrations (Kaiser et al., 1998). GmSAT1 overexpression in 

yeast also increases the uptake of methylammonium (MA), a toxic NH4 + analogue. The objective of this study 

was to identify and characterise the NH4 + /MA transporter(s) regulated by GmSAT1 in yeast. We have evaluated 

the transcriptional activity of GmSAT1 in 26972c cells grown for 12 hours in media containing 1mM NH4 + , using 

microarray (Affymetrix Yeast Genome 2.0 Array) and quantitative RT-PCR. We have characterised one of the 

genes, ScAMF1, found to be upregulated by GmSAT1. ScAMF1 (Saccharomyces cerevisiae Ammonium 

Facilitator 1), previously uncharacterised, shows sequence similarity to predicted drug:H + antiporters of the DHA2 

family of major facilitator proteins. Overexpression of ScAMF1 in 26972c and a second NH4 + transport mutant 

yeast strain, 31019b, enhanced 14 C-MA uptake and established a related toxicity phenotype when grown at 

elevated MA (0.1M) concentrations. The loss of ScAMF1 activity in 26972c:Δamf1 and 31019b:Δamf1 mutants 

eliminated the ability of GmSAT1 to accumulate 14 C-MA and instate the sensitivity phenotype. The MA 

associated phenotypes were also lost when the bHLH region of GmSAT1 was mutated. Mutagenesis of the 

bHLH region of GmSAT1 was shown to abolish the expression of ScAMF1. Stopped-flow spectroscopy 

demonstrated the ability of ScAMF1 to transport NH4 + and MA into yeast spheroplasts. Parallel experiments with 

a soybean homolog (GmAMF1) also showed enhanced accumulation of 14 C-MA and transport of NH4 + and MA 

into spheroplasts. The functional similarity of ScAMF1 homologues identified in Arabidopsis and Medicago 

truncatula are currently being investigated. 

Kaiser, B.N., Finnegan, P.M., Tyerman, S.D., Whitehead, L.F., Bergersen, F.J., Day, D.A. and Udvardi, M.K. 

1998. Characterisation of an ammonium transport protein from the peribacteroid membrane of soybean nodules. 

Science 281: 1202-6. 

122 

2011




Titles Co-inoculation of rhizobia with highly induced acc deaminase bacteria could alleviate the 

stress of legumes growing under stress conditions 

Authors Panlada Tittabutr, Pongdaj Piromyou, Nantakorn Boonkerd & Neung Teaumroong 


School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, 

Thailand 

Since it was previously found that silver thiosulfate (STS) could lower the level of ethylene production in 

mungbean and resulted in increasing plant growth under both normal and various stress conditions. It is 

interesting to investigate whether highly 1-amino-cyclopropane-1-carboxylate (ACC) deaminase activity 

producing bacteria promote the growth of mungbean under various stress conditions. ACC deaminase 

producing bacteria were isolated from mungbean root growing in the field, and three isolates ACC1, ACC2, and 

ACC3, which are highly homology to Enterobacter sp. ZJUPD4, Enterobacter sp. M.D.E.NA4-3, and 

Chryseobacterium sp. KR200, respectively were obtained with high ACC deaminase activity. The experiments 

of co-inoculation with mungbean bradyrhizobia PRC008 were tested under each condition of high temperature-, 

water-, salt-stress, and under normal condition. The results showed that ethylene was highly produced by plant 

growing under all stress conditions. The amount of ethylene production in plant directly influenced mungbean 

growth and nodulation efficiency under stress conditions. However, co-inoculation of PRC008 with high ACC 

deaminase activity producing bacteria showed the better plant growth and nodulation than those inoculated with 

PRC008 alone. Co-inoculation with ACC3 obviously alleviated the stress of legumes growing under water- and 

high temperature-stress conditions, which coincided with high inducton of ACC deaminase activity in the cell 

cultured under stress conditions even ACC3 produced lower ACC deaminase activity when compared with 

ACC1 and ACC2 under normal condition. These results revealed the role and relationship of ethylene and the 

ACC deaminase producing bacteria on the growth of leguminous plant under stress conditions. This 

information will be useful for further development of high efficient bacterial inoculant using in agriculture under 

global warming situation. 

123 

2011




Title Rice growth promotion after inoculation of Sinorhizobium melilotii 1021 and its mechanism 

Authors Mingfeng Yang, Wenpeng Zhang, Shihua Shen and Yuxiang Jing 


Key Laboratory of Plant Resource Research and Development, the Chinese Academy of 

Sciences, Beijing 100093, China. 

The studies have shown that the endophytic association of rhizobia with non-legumes, such as rice, wheat, 

maize, rape, lettuce, etc is of benefit to their growth with increased grains or/and biomass. Our previous study 

indicated that the bacteria of Sinorhizobium melilotii 1021, etc which were inoculated to rice seedlings not only 

entered into root interior through lateral root emergence and root hairs, but also ascended into stem base, leave 

shealth and leaves where they developed high population. Finally, they made rice increase its biomass and 

grains (Chi F. et al, 2005). Recently, we found the key point time that the rice seedlings inoculated with rhizobia 

grew more significantly than the controls based on rice morphology was at 5 days postinoculation(dpi). The 

observation under laser confocal microscope pointed out that the gfp-tagged rhizobia moved so fast that they 

arrived in rice leaves at the 5-8 dpi, and located in the intercellular space, and some of them even got into 

diachyma cells after isolation of leaf cell protoplasm. Microarray-gene chip and quantitative real-time PCR 

determined the gene expression of rice seedlings, showing that the related genes of DNA replication, cell division 

and cell wall formation, etc were up-reguated at 2 dpi, then some of photosynthetic genes, carbohydrate 

anabolism genes, defense and resistant genes were also up-regulated at 5, 8 dpi. These results indicate the 

molecular basis and the mechanism that the rhizobia could promote rice growth. The following step in future is to 

look for the reasons of molecular interaction between rhizobia and rice plants through metabolomics. 

Feng Chi, Shi-Hua Shen, Hai-Ping Cheng, Yu-Xiang Jing,* Youssef G. Yanni, and Frank B. Dazzo. Ascending 

Migration of Endophytic Rhizobia, from Roots to Leaves, inside Rice Plants and Assessment of Benefits to Rice 

Growth Physiology. AEM, 2005, 71: 7271–7278. 

124 

2011




Titles Differential activation of CCAMK between root nodule and arbuscular mycorrhizal symbioses 

Authors Yoshikazu Shimoda 1 , Lu Han 1 , Makoto Hayashi 1 , Haruko Imaizumi-Anraku 1 


1 National Institute of Agrobiological Sciences, Division of Plant Sciences, Tsukuba, Ibaraki 

305-8602, Japan 

Leguminous plants can form mutually beneficial endosymbioses with rhizobial bacteria and arbuscular 

mycorrhizal (AM) fungi. Recent genetic studies in model legumes have revealed that the two symbiotic systems 

share a common signaling pathway (CSP), which is essential for the induction of cytosolic Ca 2+ spiking in 

response to infection signal molecules derived from rhizobia or AM fungi. Among the components of CSP, 

Ca 2+ /calmodulin(CaM)-dependent protein kinase (CCaMK) is downstream of Ca 2+ spiking and thought to be an 

ideal decoder for microsymbionts-induced Ca 2+ signals, because of its domain structure including CaM binding 

domain and EF hand motifs. Recent studies have identified crucial roles of CCaMK for bacterial/fungal 

infections and nodule organogenesis. However, it remains unknown how CCaMK is activated differentially in 

response to Ca 2+ signals induced by rhizobia or AM fungi. 

In order to elucidate the difference of CCaMK activation mechanisms during RN and AM symbioses, we carried 

out a detailed complementation analysis of ccamk mutant of Lotus japonicus with various kinds of mutated 

CCaMKs. We also analyzed epistatic relationships among CCaMK functional domains by combining the 

mutations of each domain to clarify the involvement of the domains in Ca 2+ -dependent activation of CCaMK. In 

this presentation, we propose an activation mechanism of CCaMK in which RN and AM symbioses are 

distinguished by differential regulation of CCaMK by Ca 2+ signals. 

125 

2011




Title Determination of optimal phosphorus concentrations which enhance the establishment of 

both mycorrhizal and rhizobial symbiosis 

Authors Manijeh Mohammadi-Dehcheshmeh, Sally E Smith, Steve D. Tyerman 


and Brent N Kaiser 

School of Agriculture, Food and Wine, Waite Campus, University of Adelaide 

Legume growth and yield productivity is influenced through symbiotic interactions with both rhizobial bacteria and 

mycorrhizal fungi. Symbiosis with Rhizobium sp. enables access to atmospherically reduced N2, through 

nitrogen fixation, while the mycorrhizal symbiosis strongly enhances mineral uptake especially phosphorus (P) 

and zinc(1). P is an essential nutrient required for plant growth. The availability of P in the soil and internally 

within the plant has been shown to have a contrasting influence on the impact of either the rhizobial or 

mycorrhizal symbiosis (2-5). At high levels of available plant P, symbiotic nitrogen fixation is enhanced while high 

concentrations of P can hinder mycorrhizal colonization. The impact of P on either nodulation or mycorrhizal 

colonization has been studied widely, however little information is available on how P affectsthe interactions 

between the two symbiosis. In this study, we examined the effects of different concentrations of soil P on both 

the rhizobial and mycorrhizal symbiosis in soybean. At high P, mycorrhizal colonization (percent root length) 

decreased while nodule development increased. At low P the opposite response occurred. We are currently 

evaluating the combined symbiotic response to P in order to identify P concentration ranges that satisfy the 

needs of both symbiosis. This work will be discussed in the context of maximizing P efficiency under symbiotic 

conditions and the competing symbiotic relationships regulating P availability. 

1. S. E. Smith, F. A. Smith, Annual Review of Plant Biology 62, 227 (2011). 

2. D. W. Israel, Plant Physiology 84, 835 (1987). 

3. M. Chaudhary et al., Acta Physiologiae Plantarum 30, 537 (2008). 

4. J. A. Menge, D. Steirle, D. J. Bagyaraj, E. L. V. Johnson, R. T. Leonard, New Phytologist 80, 575 

(1978). 

5. P. G. Braunberger, M. H. Miller, R. L. Peterson, New Phytologist 119, 107 (1991). 

126 

2011




Title Comparative analysis of genome sequences of Rhizobium galegae HAMBI 540 and 

HAMBI 1141 

Authors Janina Österman 1,3 , Lars Paulin 2 , Edward Alatalo 2 , J. Peter W. Young 3 , Kristina 

Lindström 1 


1 Department of Food and Environmental Sciences and 

2 Institute of Biotechnology, University of Helsinki 

3 Department of Biology, University of York 

Rhizobium galegae (Lindström 1989) is taxonomically distinct and known for its strict host specificity both 

regarding nodulation and nitrogen fixation. Our aim is to gain insight into molecular mechanisms behind the 

specificity of nitrogen fixation. As a first step towards this goal we have performed whole genome sequencing of 

R. galegae symbiovar orientalis strain HAMBI 540 (effective on Galega orientalis) and R. galegae symbiovar 

officinalis strain HAMBI 1141 (effective on G. officinalis). The draft assembly indicates that both genomes are 

about 6.4 Mb in size. Preliminary comparison of the genomes has been performed, with a more specific 

comparison of the symbiotic gene regions of the two strains. Although the two genomes are of approximately the 

same size, there are important genetic differences between the strains. The most obvious difference is that the 

HAMBI 1141 genome harbours three replicons, while the corresponding number in HAMBI 540 is two. 

Furthermore, HAMBI 1141 harbours conjugal transfer systems not present in HAMBI 540. The genes present in 

the symbiotic regions are the same except for one gene, rpoN, the function of which will be further studied. 

Annotation of the symbiotic region also led us to the identification of a candidate gene for acetylation of the 

penultimate N-acetylglucosamine in the R. galegae Nod factor. Differences detected in the comparative analyses 

will be discussed. 

127 

2011




Title Denitrification and nitrous oxide emissions by soybean bradyrhizobia 

Authors Elisamara C. do Nascimento 1 , Jean L.S. de Araújo 2 , Claudia P. Jantalia 2 , Segundo 

Urquiaga 2 , Robert M. Boddey 2 , Bruno J. R. Alves 2 


1 Departamento de Fitotecnia, Universidade Federal Rural do Rio de Janeiro, Seropédica, 

23890-000, RJ, Brazil. 

2 Embrapa Agrobiologia, Seropédica, 23890-000, Rio de Janeiro, Brazil. 

Soil denitrifiers include several Bradyrhizobium spp. strains, but just some of them are genetically capable of 

using all of the N oxides from nitrate to N2O. Soybean is planted on more than 22 million ha in Brazil, and 

commercial inoculants for this crop are manufactured from four selected strains of B. japonicum and B. elkanii. 

There are still doubts concerning the impact of nodulated legumes on emissions of N2O, a powerful greenhouse 

gas. Firstly the ability of 12 strains of Bradyrhizobium japonicum and B. elkanii from Brazil and USA to produce 

N2O from nitrate in pure culture of was investigated. Secondly a study was made to quantify the N2O emissions 

from soybean grown in soil never planted to soybean and inoculated with the four strains used in Brazilian 

commercial inoculants. Subsequently nodules from these plants were transferred to a sealed vials containing 2 

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 

nodules were performed to evaluate the denitrifying ability of different N oxides by the strains. A DGGE analysis 

was performed using specific primers for each enzyme involved in the denitrification process. Only B. japonicum 

strains grown in pure culture showed the ability to reduce NO3 - to N2O. In the pot experiment, plants inoculated 

with B. japonicum presented the highest N2O emissions, whilst soybean nodulated with B. elkanii presented low 

emissions, similar to those non-inoculated plants. The production of N2O from nodules of plants inoculated with 

B. elkanii suggests soil rhizobia also occupied the nodules. However, nodules from plants inoculated with B. 

japonicum strain BR86 (SEMIA 5079) showed the ability to convert all N2O into N2, which was not observed for 

any other treatment. 

128 

2011




Title Proteomic profile of the soybean symbiosome membrane 

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 


1 School of Biological Sciences, The University of Sydney, NSW, Australia; 2 ARC Center of 

Excellence in Plant Energy Biology, The University of Western Australia, WA, Australia; 

3 Flinders University, SA, Australia. 

Symbiotic nitrogen fixation in legumes is characterised by the formation of a novel root organ called the nodule, 

within which unique organelle-like structures termed symbiosomes develop. Free living rhizobia are engulfed 

within plant cells and are surrounded by a membrane of plant origin termed the symbiosome membrane (SM). It 

is this membrane that regulates the movement of solutes from plant to bacteroid (the symbiotic form of the 

rhizobium) and vice versa. The SM is a unique structure containing an array of plant-derived proteins through 

which the plant can regulate the symbiosis. Previous attempts to characterise the protein complement of this 

membrane have been hindered by its hydrophobic nature and the absence of a complete genome for reference. 

In this study, SM was isolated from mature nitrogen-fixing soybean root nodules and analysed using shotgun 

proteomic techniques. The recent release of the complete soybean genome has allowed for the identification of 

peptide sequences. Our proteomic analysis of soybean SM has identified eighty putative SM-localised proteins, 

including ten previously localised to the SM such as nodulin-26, an aquaporin. We have focused on two classes 

of proteins identified in the proteomics for further studies: amino acid transporters and ABC family transporters. 

Gene expression of these candidates has been confirmed by qRT-PCR and is specific to nodule tissue. 

Expression profiles across nodule development have also been completed with all candidates showing 

increasing gene expression levels correlated with the onset of nitrogen fixation. This suggests these genes may 

have a role in maintaining the mature symbiosis, consistent with what would be expected of a SM transporter. 

amiRNA silencing vectors have been constructed to reduce expression of candidates in nodules. Further 

localisation and functional studies are planned for these candidates with the aim of characterising novel 

transporters on the soybean SM. 

129 

2011




Titles Nodulation studies of cowpea and its response to phosphorus application in 

Botswana under glasshouse conditions 

Authors Kenneth K. Keakile and Tebogo Balone, Flora Pule-Meulenberg 


Department of Crop Science and Production, Botswana College of Agriculture, Private Bag 

0027, Gaborone, Botswana 

Cowpea (Vigna unguiculata L. Walp) is an indigenous leguminous plant grown mostly in tropical Africa as a food 

crop. Its fresh young leaves and fresh pods are consumed as vegetables by many Africans whereas the seed is 

used in many food preparations. It is known to form symbiotic partnerships with some members of Rhizobiales as 

well as some β-proteobacteria ( “rhizobia”), where the legume provides the microsymbiont with carbon for its 

energy needs and in turn receives fixed-N for its nutrition from the rhizobia. In Botswana, nodulation status of 

many economically important legumes such as cowpea is not known. This study assessed if soils of Sebele 

harboured bacteria that nodulates cowpea. The study also assessed the response of cowpea to phosphorus 

application under glasshouse conditions. Two cowpea genotypes, Black eye and Tswana, were planted in a 

completely randomised design using pots in a glasshouse at the Botswana College of Agriculture in Sebele. In 

the first experiment which tested for nodulation, all plants received a basal application of P equivalent to 130 kg 

P/ha. In the second experiment, treatments consisted of four levels of P equivalent to 0, 65, 130 and 195 kg P/ha 

using single superphosphate. Both genotypes formed effective nodules when grown in the soil from Sebele. 

Black eye produced significantly more nodules and higher nodule dry matter compared to Tswana, which was 

reflected in significantly higher shoot N concentration in this genotype. The highest P fertilisation resulted in 

significantly higher nodule number, shoot and root dry matter as well as shoot N. It was also observed that Black 

eye plants that received 195 kg P/ha flowered significantly earlier than the rest. In conclusion, the Sebele soil 

harboured rhizobia that nodulated cowpea genotypes Black eye and Tswana. Further studies will include 

evaluating a wider selection of legumes including potential forage plants grown on different soil types. Levels of 

N-fixed will be measured, root-nodule bacteria isolated and cross-infectivity studies conducted. 

130 

2011




Title Lotus japonicus nodulates when it sees red. 

Authors Akihiro Suzuki 1 , Lalith Suriyagoda 1 , Tamaki Shigeyama 1 , Akiyoshi Tominaga 1 , Masayo 

Sasaki 1 , Yoshimi Hiratsuka 1 , Aya Yoshinaga 1 , Susumu Arima 1 , Sakae Agarie 1 , Tatsuya 

Sakai 2 , Sayaka Inada 2 , Yusuke Jikumaru 2 , Yuji Kamiya 2 , Toshiki Uchiumi 3 , Mikiko Abe 3 , 

Masatsugu Hashiguchi 4 , Ryo Akashi 4 , Shusei Sato 5 , Takakazu Kaneko 5 , Satoshi Tabata 5 & 

Ann M. Hirsch 6 


1 Faculty of Agriculture, Saga University, Honjyo-machi, Saga, Saga 840-8502, Japan. 

2 RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan. 

3 Faculty of Science, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan. 

4 Frontier Science Research Center, University of Miyazaki, Miyazaki, Miyazaki 889-2192, 

Japan. 

5 Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan. 

6 Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, 

University of California-Los Angeles, California, USA. 

Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes 

and rhizobia. In this study, we show that Phytochrome B (PHYB) is part of the monitoring system to detect 

suboptimal light conditions, which normally suppress Lotus japonicus nodule development in response to 

inoculation of Mesorhizobium loti. We found that the number of nodules produced by phyB mutants of L. 

japonicus is significantly reduced compared to that of wild-type MG20. To explore another cause other than 

photoassimilates, the possibility of local control was investigated by grafting experiments. The results showed 

that shoot genotype, and not the root genotype is responsible for root nodule formation. To explore causes by 

systemic regulation, we moved wild-type MG20 plants from white light to conditions differing in the ratios of low 

or high red (R)/far-red (FR) light. In low R/FR light, the number of root nodules on MG20 plants dramatically 

decreased compared to plants grown in high R/FR even though photoassimilate content was higher for plants 

grown under low R/FR. We found that the expression of jasmonic acid (JA)-responsive genes was decreased in 

both low R/FR light-grown MG20 and in white light-grown phyB mutant plants. Indeed Endogenous concentration 

of JA-Ile was decreased in phyB mutant. Moreover, both infection thread formation and root nodule formation 

were positively influenced by JA treatment of wild-type plants grown in low R/FR light and of white light-grown 

phyB mutants. These results indicate that root nodule formation is photomorphogenetically controlled by sensing 

the R/FR ratio through JA signaling. 

131 

2011




Title Role of the Cpx two component regulatory system and a MFS transporter in the symbiosis 

between Sinorhizobium meliloti and leguminous plants 

Authors Mário R. Santos, Andreia T. Marques, Leonilde M. Moreira 


IBB-Institute for Biotechnology and Bioengineering, Centro de Engenharia Biológica e 

Química, Instituto Superior Técnico 

Sinorhizobium meliloti establishes a symbiotic nitrogen fixation relationship with Medicago sativa in a process 

involving signalling between rhizobia and the legume plant. There are several bacterial molecular determinants 

of symbiosis and we recently added protein TolC to those determinants [1]. A S. meliloti tolC mutant was strongly 

affected in the resistance to antimicrobial agents, presented higher susceptibility to osmotic/oxidative stresses 

and also had affected secretion of Ca 2+ -binding proteins, of exopolysaccharides and Nod-factors. Furthermore, 

S. meliloti tolC mutant induced a reduced number of nodules, which were unable to fix nitrogen in plant roots. 

The comparison of the transcriptome of the tolC mutant to that of the wild-type strain identified 1809 genes 

differently expressed, including those encoding proteins involved in central metabolism, protection against 

stresses and transport. From these genes, the ones with the highest expression in the tolC mutant included the 

homologues of the sensor histidine kinase CpxA, the response regulator CpxR and the MFS (Major Facilitor 

Superfamily) transporter encoded by genes SMc03167 and SMc03168 [2]. To determine whether these genes 

are involved in the symbiotic process, insertion mutants of cpx homologues and deletion mutants of the MFS 

genes were constructed. Phenotypic tests performed with cpx insertion mutants showed minor differences in 

motility and in symbiosis when compared to the wild-type strain. Deletion of MFS encoding genes had an effect 

in stress resistance, pH tolerance, and a slight effect in the number of nodules. Further studies are being 

conducted to determine other functions for these gene products in S. meliloti biology. 

132 

2011




Title Compatibility with rhizobia of polymer adhesives and colourants used in preinoculated 

legume pasture seed 

Authors Elizabeth Hartley, Greg Gemell & Jade Hartley 


Australian Inoculants Research Group, NSW Department of Primary Industries, Ourimbah 

Campus, University of Newcastle 

In recent years there has been an increase in commercially-produced preinoculated pasture legume seed. 

Nowadays, preinoculation is the incorporation of ingredients beneficial to seedlings, into a sticker solution 

containing rhizobia. Seed is coated then dried. Preinoculated seed is often stored for weeks or months before 

use. Data show that survival of rhizobia on preinoculated seed varies and is generally poor after a few weeks. 

Sowing preinoculated seed with nil or very few rhizobia may contribute to inadequate nodulation and possible 

crop failure costing the farmer in lost production. 

Many physical and chemical factors affect the ability of rhizobia to survive in high numbers when inoculated onto 

seed. 

This presentation focuses on ingredients such as polymers, stickers, dyes, pigments and insecticides and their 

compatibility with rhizobial strains when used in the commercial seed coating process. 

Chemical and agricultural companies who manufacture and supply such materials contributed 60 ingredients for 

assessment. 

To determine the compatibility with rhizobia, and their suitability as an ingredient in the seed coating process, 

each was applied at recommended rates to rhizobial broth cultures of 3 commercial strains, TA1 (white clover), 

WSM1325 (subterranean clover) and CB1809 (soybean). Viable rhizobia were counted after 0h, 3h, 24h and 72h 

incubation. 29 ingredients that were compatible with rhizobia were screened to determine their effect on survival 

of rhizobia when incorporated into peat slurries used to inoculate polyethylene beads as seed substitutes. Viable 

rhizobia on beads were counted 1h, 24h and 48h after coating. Results varied. The affect of each of the 

ingredients to rhizobial survival depended upon the strain, and the method of exposure of the cells to the 

ingredients. After 24h, 6 ingredient treatments supported better survival of rhizobia on beads than the control 

treatment. However, after 48h, only one ingredient promoted better survival than the control. 

133 

2011




Title Efficiency of Bradyrhizobium japonicum in different formulations and when co-inoculated with 

bacillus subtilis on soybean in a Kenyan soil 

Authors Mary Atieno 1, 2 , Laetitia Herrmann 2 , Robert Okalebo1 , Didier Lesueur 2, 3 


1 Moi University, Department of Soil Science, PO Box 1125-30100, Eldoret, Kenya 

2 Tropical Soil Biology and Fertility Institute of the International Centre for Tropical Agriculture 

(TSBF-CIAT), United Nations Avenue, PO Box 30677-00100, Nairobi, Kenya 

3 CIRAD, UMR Eco&Sols (CIRAD-IRD-INRA-SupAgro), Land Development Department, 

2003/61 Paholyothin Road, Lardyao Chatuchak, Bangkok 10900 Thailand 

The major setback in successfully obtaining an effective inoculant is overcoming difficulties in formulating a 

viable and user-friendly final product as the live nature of the active ingredient underscores the importance of 

formulation in maintaining the microbial cells in a competent state. Co-cultures of rhizobia and PGPR (Plant 

Growth Promoting Rhizobacteria) are also known to influence the efficacy of the symbiotic bacteria on plant 

biological nitrogen fixation. A greenhouse experiment was set to assess the formulation effect of one strain i.e. 

Bradyrhizobium japonicum, 532c (granules, liquid and broth) and to determine the efficiency of co-inoculation of 

Bacillus subtilis with two strains of Bradyrhizobium japonicum (532c and RCR 3407). The objectives were 

evaluated on 2 soybean (Glycine max L.) varieties: Nyala, a non-promiscuous variety and TGx1740-2F, a 

promiscuous test variety. A non sterile soil from Central Kenya (Chuka) classified as a Nitisol was used. Nodule 

occupancy was determined by PCR-RFLP. Most of the inoculants showed increased nodulation and biomass 

yields as compared to the un-inoculated controls with a higher response seen in the promiscuous TGx1740-2F 

variety as compared to the non-promiscuous variety. The liquid and granule-based inoculants had higher 

biomass yields suggesting an impact of formulation on the effectiveness of the inoculants. The co-inoculants also 

gave higher yields but showed no significant differences to the rhizobial inoculants alone. Nodule occupancy was 

100 % for all the rhizobial inoculants as well as the co-inoculants emphasizing the infectivity and high 

competitiveness of 532c and RCR 3407 strains even in the presence of indigenous strains (80-113 cell/g of soil). 

These inoculants, though not initially made for SSA countries, showed promising increased yields in a Kenyan 

soil containing significant populations of native rhizobia nodulating soybean, signifying a possibility of their 

adoption in increasing soil fertility and crop yields in the poor SSA soils. 

134 

2011




Title Potential of indigenous Bradyrhizobia versus commercial inoculants to improve cowpea and 

green gram yields in Kenya 

Author Samuel Mathu 1, 2 , Pieter Pypers 1 , Laetitia Herrmann 1 , Viviene Matiru 2 Romano Mwirichia 2 

and Didier Lesueur 1,3 


1. Tropical Soil Biology and Fertility Institute of CIAT, World Agroforestry Center, 

P.O. Box 30677-00100 Nairobi – Kenya. 

2. Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200 

Nairobi- Kenya. 

3. CIRAD, UMR Eco&Sols Land Development Department, 2003/61 Paholyothin 

Road, Lardyao Chatuchak, Bangkok 10900 Thailand. 

Limited information is available on reduced cowpea and green gram yields in Kenya. Declining soil fertility or 

presence of ineffective indigenous rhizobia? In regards to this, soils were collected from Western province 

(Bondo, Bungoma) to Eastern (Isiolo), Central (Meru) and at the Coast (Kilifi) to trap indigenous rhizobia 

nodulating both legumes under greenhouse condition. Highest nodule fresh weights of 4.63 and 3.32 g plant -1 for 

cowpea and green gram were observed in soil from Isiolo site A and Kilifi site A respectively suggesting 

significant populations of indigenous strains in such soils. Lowest nodule fresh weights of 2.17 and 0.72 g plant -1 

were observed in soil from Bungoma site B for cowpea and green gram respectively. Genetic diversity of 

indigenous strains nodulating both legumes was assayed using PCR-RFLP of the 16S-23S rDNA IGS and 19 

IGS groups were identified with I and II predominating for both legumes. A second greenhouse experiment was 

set up to evaluate if commercial inoculants significantly improve cowpea and green gram yields in soils with 

significant populations of native rhizobia. Rhizobial inoculation did not significantly (p




Title Isolation and characterization of diazotrophic microsymbionts from root nodules of mucuna 

bracteata 

Authors Salwani Shaffie ,Amir Ghazali, and Nazalan Najimudin 


School of Biological Sciences, Universiti Sains Malaysia 

An increasing number of bacteria that can nodulate and fix N2 in legumes which do not belong to the genus of 

Rhizobium or Bradyrhizobium was identified. This study was conducted to determine indigenous microsymbiont 

strains which could further promote symbiotic N2-fixation activities for Mucuna bracteata (an important 

leguminous cover crop) and to verify the identity of the isolates based on nitrogenase enzyme assay, nifH gene 

isolation and partial 16S rDNA sequence analysis. Our findings indicated that the isolated microsymbionts could 

nodulate and promote N2-fixation activity in M. bracteata. The isolates also contributed to enhanced plant growth 

in terms of leaf protein and chlorophyll content and higher plants and nodule biomass. Additionally, the nifH gene 

fragments were successfully amplified for all of the isolates. The 16S rDNA sequencing results suggested that 

bacteria which were able to form N2-fixing symbioses with root known as rhizobia is not only from the α-class of 

proteobacteria but also from β-class of proteobacteria (Burkholderia sp. and Achromobacter sp., 99-100% of 

similarity) and γ-class of proteobacteria (Stenotrophomonas sp., 99% of similarity). The findings indicate the 

diversity of potentially-beneficial diazotrophic microsymbionts active in this emerging legume species. 

136 

2011




Title Lipopolysaccharide binding protein, a new requirement for legume-rhizobium symbiosis 

Author Ei-ichi Murakami 1 , Hitomi Takayama 1 , Ken-ichi Osuki 1 , Maki Nagata 1 , Yoshina Hori 2 , 

Mayumi Shigeoka 2 , Mana Sahara 2 , Yoshikazu Shimoda 3 Sayaka Muto 4 , Yukio Nagano 4 , 

Ken-ichi Kucho 1 , Mikiko Abe 1 , Shusei Sato 5 , Shiro Higashi 2 , Toshiki Uchiumi 1 


1 Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan. 

2 Faculty of Science, Kagoshima University, Kagoshima, Japan. 

3 National Institute of Agrobiological Sciences, Ibaraki, Japan. 

4 The United Graduate School of Agricultural Sciences, Kagoshima University, Kaogoshima, 

Japan. 

5 Kazusa DNA Research Institute, Chiba, Japan 

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. LPS induces the 

plant resistance in pathogenic combination and is also involved in the establishment of symbiosis between 

legumes and rhizobia. Thus the elucidation of LPS recognition system is essential for understanding the plantbacteria 

interaction. In mammals, LPS binding protein (LBP) has already been identified and signal transduction 

system via Toll-like receptor TLR4 is well investigated. However, LPS recognition system is still unknown in 

plants. Referring LBP of mammals, four LBP-candidates, LjLBP1, LjLBP2, LjLBP3 and LjLBP4, were identified 

on the genome of Lotus japonicus. N-terminal barrel of LjLBP1, LjLBP2 and LjLBP3 bound with LPS of 

Escherichia coli. The expression levels of LjLBP1 and LjLBP2 were low and stable, whereas LjLBP3/4 was 

inducible in responding to rhizobial inoculation. In the nodules on the hairy roots of LjLBP1-RNAi, nodule cells 

were broken and no symbiosome was observed. The shape of bacteroids of LjLBP2-RNAi was abnormal. 

Symbiosomes of LjLBP3/4-RNAi were larger than those of the control. These results suggest that LjLBPs are 

required for the symbiosis between L. japonicus and M. loti. 

137 

2011




Title The role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme on leguminous 

nodule senescence 

Author Sudarat Sripakdi 1 , Panlada Tittabutr 1 , Nantakorn Boonkerd 1 & Neung Teaumroong 1 


1 School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 

30000, Thailand. 

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of 

Ensifer sp. (Sinorhizobium sp.) strain BL3 on leguminous nodule senescence. The acdRS genes encoding ACC 

deaminase were cloned from BL3 into multiple copy plasmids (pRK404A), and transferred to wild type. The BL3 

transconjugant containing of multiple copy number of acdRS (BL3 + ) greatly enhanced ACC deaminase activity 

compared to BL3 (1.634 and 0.447 μmol of �-ketobutyrate mg -1 protein h -1 , respectively). ACC deaminase 

activity of BL3 was also higher than mungbean nodulating strain as Bradyrhizobium sp. PRC008 (0.126 μmol of 

�-ketobutyrate mg -1 protein h -1 ). Moreover, BL3 + retarded nodule senescence (60.25%) greater than BL3 

(39.75%) after 5 weeks planting. In the meantime, the kanamycin resistance gene (Km r ) were inserted into the 

acdRS fragment and transferred into BL3 in order to construct BL3 mutant, which was interrupted at acdR and 

acdS (BL3 - ). All strains were inoculated to the host plant, Vigna radiata cultivar SUT1. Inoculation of BL3, and 

BL3 + were not significantly effected on plant growth and root nodule number whilst BL3 - showed significantly 

lower than both strains. Whereas flowering period on BL3 + was longer than wild type and mutant strains. 

138 

2011




Title Gamma irradiation and autoclave sterilization of peat and compost as the carrier for rhizobial 

inoculant production 

Author Panlada Tittabutr 1 , Kamonluck Teamthisong 2 , Neung Teaumroong 1 & 


Nantakorn Boonkerd 1 

1 School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, 

Thailand 

2 The Center for Scientific and Technological Equipment, Suranaree University of 

Technology, Nakhon Ratchasima 30000, Thailand 

Although several alternative carriers have been investigated and successfully used instead of peat, the main 

problem found in rhizobial inoculant production is contamination by other bacteria or molds that cause reduction 

of rhizobial cell number. This study aimed to elucidates the efficient sterilization process of two carriers, peat 

and compost before using as rhizobial inoculant. Peat and compost could be efficiently sterilized by irradiation. 

The carrier with 10% moisture content could be sterilized by irradiation at 10 kGy, while carrier with 30% 

moisture content must be sterilized by irradiation at 25 kGy. Penetration of irradiation through polypropylene (PP) 

bag tend to be better than polyethylene (PE) bag, since lower dose of irradiation was needed for carrier 

sterilization. However, PE bag appear to be more durable than PP bag after gamma irradiation at high doses. 

Nevertheless, contaminants could be detected in irradiated carrier after storage at room temperature for two 

months. Thus, autoclaving with tyndallization approach was applied in order to minimize the contaminant 

microorganisms in the carrier. Carriers with 10% moisture were autoclaved two times in a row at 121ºC for 60 

min, with the waiting period during each time after autoclaving for 18 h could eliminate the fungal contaminants, 

while bacterial contaminants still remained about 10 2 cfu/g carrier. The number of Bradyrhizobium sp. PRC008 

was in the range of 10 8 -10 9 cfu/g in both irradiated and autoclaved peat after 6 months storage. However, the 

numbers of bradyrhizobial cell were reduced in compost sterilized by both methods after one month storage. 

These results indicated that carrier material had an important influence on inoculant quality, while sterilization 

processes using gamma irradiation and autoclaving with tyndallization approach could be used for efficient 

rhizobial inoculant production with peat based carrier. 

139 

2011




Title Effect of nitrogen, phosphorus, magnesium and calcium nutrition on plant growth and levels 

of macronutrients in the honeybush tea plant, Cyclopia longifolia (Vogel l.) 

Authors Buhlebelive Mndzebele 1 and FD Dakora 2 


1 Department of Crop Sciences, Tshwane University of Technology, 

2 Department of Chemistry, Faculty of Science, Tshwane University of Technology 

Cyclopia longifolia (Leguminosae) is endemic to the fynbos of the Cape Floristic Region of South Africa. 

Together with eight other Cyclopia species, C. longifolia is used for making Honeybush tea, a herbal beverage 

that is contributing to the South African economy. However, the effect of nutrient supply on plant growth, and the 

concentration, and amounts of major minerals are not well understood in this legume. Because the shoots are 

harvested annually as tea, establishing nutrient requirements for this species is necessary to minimize nutrient 

mining. The aim of this study was to assess plant growth, as well as the concentration and amounts of major 

elements in C. longifolia supplied with N, Mg, Ca and P as ammonium nitrate, magnesium chloride hexahydrate 

and calcium chloride dehydrate dipotassium and hypophosphate at four levels (i.e. 0, 5, 25 and 50 mM). About 

100 mL of each nutrient concentration was applied per plant at three months interval for 9 months. Plants were 

harvested at 300 days after treatment in the field trial. Shoots were oven-dried (60 º C), weighed, and ground to 

fine powder (0.85 mm) for tissue analysis using inductively coupled plasma-mass spectrometry. The data 

showed that supplying of N, Mg, Ca and P to C. longifolia increased biomass and the concentration, and 

amounts of major nutrients in plant tissue. The provision of N to C. longifolia induced uptake and accumulation of 

N, P, Mg, Ca and Na in the field. Phosphorus supply also led to higher tissue concentrations of N, P, Ca, Mg and 

Na just as the addition of Mg increased the concentration of Mg, P, Ca and Na in C. longifolia. Applying Ca to C. 

longifolia plants also caused the accumulation of Ca, P, Mg and Na in shoots, but decreased N concentration 

and content in field experiments. These data suggest that a moderate supply of N, P, Mg and Ca to C. longifolia 

can stimulate plant growth and accumulation, and thus increase tea yields in farmers‟ fields. 

140 

2011




Title Root-nodule bacteria isolated from cowpea and bambara groundnut enhance mineral 

nutrition in their homologous hosts 

Author Irvin Makhubedu 1 , Keletso Mohale 1 , Flora Pule-Meulenberg 2 and Felix Dakora* 3 


1 Department of Crop Science, Tshwane University of Technology 

2 Department of Biotechnology, Tshwane University of Technology, 

3 Department of Chemistry, Tshwane University of Technology 

Little is currently known about the role of symbiotic root-nodule bacteria in accumulating mineral nutrients in 

legumes. This study screened cowpea and Bambara groundnut bacterial isolates for their ability to induce 

nutrient uptake under sterile conditions in Leonard jars. Seedlings of cowpea (V. unguiculata L. Walp. cv. 

TVu11424), and Bambara groundnut (Vigna subterranea L. Verdc.) were raised in sterile Leonard jars containing 

¼ strength Hoagland nutrient solution, and inoculated (in four replicates) with broth cultures of their respective 

bacterial microsymbionts. NO3-fed plants (0.5 mM) were included as control. All plants were harvested at 30 

days after planting, separated into shoots and roots, and oven-dried (60 o C). The shoots were weighed, ground 

into fine powder (0.85 mm), and measured for mineral nutrients using inductively coupled plasma massspectrometry. 

The cowpea data revealed marked differences in bacterial strain effect on shoot and whole-plant 

biomass. All nodulated cowpea plants showed much higher concentrations of trace elements and macronutrients 

in shoots compared to 0.5 mM NO3-fed plants. Where strains TUT13d1vu and TUT26a1vu nodulated cowpea, 

they consistently showed increased concentrations of P, K, Ca, Mg, S, Fe, Cu, Zn, Mn and B in shoots when 

compared to other strains. However, strains TUT53b2vu and TUT33b4vu (which were the highest fixers) 

accumulated much greater amounts of P, K, Ca, Mg, S, Fe, Cu, Zn, Mn and B in cowpea shoots than any of the 

other strains. Re-testing strains TUT13d1vu, TUT26a1vu, TUT53b2vu and TUT33b4vu and 12 other N2-fixing 

cowpea isolates again revealed similar results for strains TUT26a1vu and TUT13d1vu. Strain TUT53b2vu and 

TUT33b4vu together with three other strains (which were the highest fixers) accumulated greater amounts of the 

major and minor elements in shoots of cowpea. Nodulating Bambara groundnut with its bacterial isolates also 

revealed marked strain differences in inducing mineral accumulation in shoots. 

141 

2011




Title Evaluation of elite commercial soybean varieties for N2 fixation and grain yield in South 

Africa 

Author Nyamande Mapope 1 and Felix D Dakora 2 


1 Department of Crop Science, Tshwane University of Technology 

2 Department Chemistry, Faculty of Science, Tshwane University of Technology 

South Africa is currently a net importer of soybean which is much needed for poultry production and the livestock 

industry. There is therefore an urgency to increase local production of soybean through breeding of improved 

varieties, their evaluation for adaptability, and selection for high grain yield and symbiotic performance. This 

study assessed plant growth, N2 fixation, and grain yield in thirteen commercially-bred, elite soybean genotypes 

under field conditions in the three soybean-producing provinces of South Africa. The varieties used originated 

from Pioneer, Pannar and Linkseed in South Africa. The experiments were laid out in a randomized block design 

with four replications. At pod filling (R6) stage, 10 soybean plants were randomly sampled from each plot, and 

oven-dried (60oC) separately, weighed and ground into fine powder (0.85mm) for 15N isotope analysis. The data 

obtained revealed marked differences in dry matter yield, N concentration, N content, δ15N, %Ndfa and actual 

amounts of N fixed among the thirteen soybean genotypes at each of the three sites. Dry matter yield ranged 

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 

Herman in the North West Province. The N concentrations of shoots also differed between and among 

genotypes at each site, and ranged from 2.2 % for 95Y20 to 3.5 % for PAN1666 at Balfour in Mpumalanga. The 

δ15N values ranged from -0.73 ‰ for PAN1454 to 2.33 ‰ for PAN1666 at Balfour in Mpumalanga. Cross site 

analysis also revealed significant interaction between site and varieties. PAN1666 showed the lowest %Ndfa 

(64%) and 95Y40 the highest %Ndfa (85%) across all sites. The actual amounts of N-fixed ranged from 114 

kg.ha-1 for 95Y20 to 215 kg.ha-1 for PAN1666 across the sites. Soil N uptake was generally low across the 

sites, indicating greater dependence of these elite varieties on N2 fixed for their N nutrition. PAN1666 for 

accumulated greater dry matter and amount of N-fixed across all sites and was therefore the best variety for use 

by farmers. 

142 

2011




Title Ecophysiological studies and biodiversity of root-nodule bacteria nodulating Psoralea 

species in the cape fynbos of South Africa 

Authors Sheku A. Kanu 1 & Felix D. Dakora 2 


1 Crop Science and 2 Chemistry Department, Tshwane University of Technology 

The tribe Psoraleae has 51 species, which are endemic to the Cape fynbos. However, little is known about the N 

contribution and microsymbionts associated with the nodulation of members of the tribe Psoraleae. In this study 

eight Psoralea species (namely, P. pinnata, P. repens, P. aphylla, P. asarina, P. monophylla, P. aculeata, P. 

restioides and P. laxa) growing in the fynbos were assessed for their symbiotic N nutrition and biodiversity of 

bacteria nodulating this tribe. Isotopic analysis of young branches with leaves showed that Psoralea species 

were highly dependent on N2 fixation for their N nutrition. The � 15 N and %Ndfa values of plant parts differed 

between species, and across the study sites. For example, at Betty‟s Bay, four Psoralea species (namely P. 

aculeata, P. aphylla, P. laxa and P. pinnata) showed strong differences in the � 15 N values of young branches 

with P. pinnata exhibiting the lowest stem � 15 N value of -2.00 ‰ and the highest %Ndfa value of 84%. 

Excavation of plants showed that all eight Psoralea species had round determinate root nodules. Surface 

sterilization, followed by streaking of nodule homogenates onto yeast-mannitol agar medium revealed bacterial 

isolates that differed in their growth rate, colony appearance, shape and texture. When 72 single-colony isolates 

from eight members of the tribe Psoraleae were tested for their ability nodulate P. pinnata, only 5 isolates 

(TUT54pp, TUT55pp, TUT57pp, TUT58pp and TUT60pp) effectively nodulated their original host together with 3 

other isolates from P. asarina (TUT71pas), P. restioides (TUT23prt) and P. aculeata (TUT17pac). Although 7 

isolates formed root nodules on siratro, only 5 (TUT1pm, TUT23prt, TUT32pap, TUT43pap and TUT45pap) were 

effective. The results from 16S rDNA sequencing revealed considerable microsymbiont diversity associated with 

the tribe Psoraleae with strains belonging to both α and β-proteobacteria. Taken together, these results suggest 

that Psoralea species have the potential to contribute to the N economy of the nutrient-poor soils of the Cape 

fynbos when associated with both .α and β-proteobacteria. 

143 

2011




Title Nitrogen fixation promotes accumulation of dietarily-important mineral nutrients in edible 

leaves of cowpea (Vigna unguiculata l. Walp.) 

Authors Alphonsus K. Belane 1 and Felix D. Dakora 2* 


1 Department of Crop Science, and 2 Department of Chemistry, Tshwane University of 

Technology 

Cowpea leaves are a major source of dietary mineral nutrients for many households in rural Africa. Little is 

however known of the relationship between cowpea symbiosis and mineral accumulation in its edible leaves. 

This study evaluated 30 field-grown cowpea genotypes at Wa and Manga in Ghana, and Taung in South Africa, 

for N2 fixation and mineral density using the 15 N natural abundance technique and inductively coupled plasma 

mass spectrometry respectively. The mean levels of leaf minerals presented here are for the highest and lowest 

N2-fixing cowpea material taken from a study of 30 genotypes. The results showed that, at Wa in Ghana, IT90K- 

76, Bensogla and Glenda produced the largest biomass per hectare, fixed the highest amount of symbiotic N, 

and showed much higher concentrations and amounts of trace elements and macronutrients in edible cowpea 

leaves. In contrast, genotype ITH98-46, which recorded the least biomass, fixed the lowest amount of symbiotic 

N, and exhibited the lowest concentrations and amounts of mineral nutrients in its leaves. The data for Manga in 

Ghana and Taung in South Africa showed a similar pattern; the highest N2-fixing genotypes indicated better plant 

growth and greater mineral accumulation compared to their counterparts with little biomass and low N2 fixation. 

This observation was confirmed by correlation analysis which revealed a significantly positive relationship 

between mineral nutrients and symbiotic parameters such as δ 15 N, %Ndfa and amount of N-fixed. Thus, in 

addition to N contribution in cropping systems, the cowpea symbiosis also enhances mineral density in edible 

leaves for improved human nutrition and health. 

144 

2011




Title Diversity and phylogeny of Bradyrhizobium spp. Isolated from shrub and tree legumes in 

Ethiopia 

Authors Aregu Amsalu Aserse 1,2 , Leena.A.Räsänen 1 ,Fassil Assefa 2 , Asfaw Hailemariam 3 and 

Kristina Lindström 1 


1 University of Helsinki, Department of Food and Environmental Sciences, 

2 Addis Ababa University, Cellular, Microbial and Molecular Biology Program Unit 

3 National Soil Testing Centre, 

In Eastern Africa including Ethiopia, crop production has been affected by recurrent drought, deforestation and 

losses of soil fertility. Soil fertility is the major constraint and nitrogen is considered as one of the most limiting 

nutrients in the region. Nitrogen-fixing shrubs and tree legumes, such as Crotalaria, Erythrina and Indigofera 

species have been used in agroforestry systems (home-gardens, fallows and land reclamation) to improve the 

availability of nitrogen in the soil. Soybean is an exotic crop legume in Ethiopia and its symbionts capable of 

forming nitrogen-fixing nodules do not exist in the soil. Inoculation of soybean has not always been successful. 

Therefore, effective rhizobial strains that are adapted to their local environments are required. Interestingly, 

indigenous rhizobial strains, which nodulate leguminous shrubs and trees have been found to infect soybean as 

well. However, knowledge on the symbionts of different leguminous plants in Ethiopia is scarce. Therefore, the 

aim of this study was to explore the diversity and phylogeny of indigenous Bradyrhizobium strains isolated from 

Crotalaria incana, Erythrina brucei, Indigofera arrecta and Gycine max. Fifty-five strains were isolated from 

nodules collected at 29 sites located mainly in southwestern Ethiopia. The diversity of the bacterial collection was 

studied using amplified fragment length polymorphism (AFLP) fingerprinting. The strains were preliminary 

identified by comparing the partial sequences of the 16S rRNA gene against a nucleotide database. For further 

phylogenetic studies, the housekeeping genes recA, rpoB and glnII were sequenced. Phylogenetic trees of these 

genes as well as phylogenies of the symbiotic genes nodA and nifH will be presented together with strains 

symbiotic capacity and plant growth promoting activities. 

145 

2011




Title Phylogenetic position of Rhizobium galegae 

Authors Seyed Abdollah Mousavi 1 , Janina Österman 1 , Lars Paulin 2 & Kristina Lindström 1 


1 Department of Food and Environmental Sciences 

2 Institute of Biotechnology 

Rhizobia are defined as bacteria that can enter nitrogen-fixing symbioses by inducing nodules in legumes 

(Lindström & Mousavi 2010), whilst agrobacteria induce tumours in plants. Rhizobium galegae is fixing nitrogen 

in Galega plants, and is one of the species of the family Rhizobiaceae. The phylogenetic placement of R. 

galegae is not stable in phylogenetic trees of rhizobia and it does not fit with most other rhizobia in some gene 

trees. R. galegae was e.g. placed in the Rhizobium rhizogenes - Rhizobium cluster in dnaK nucleotide sequence 

trees; whereas it was placed with Agrobacterium species in the 16S rRNA tree (Eradly at al 2005). Construction 

ofa concatenated tree of eight housekeeping genes, recA, dnaK, thrC, gltA, rpoB, atpD, pnp and gyrB, for twenty 

whole-genome sequenced rhizobia and agrobacteria showed that R. galegae could be placed in a cluster with A. 

vitis and A. tumefaciens, whereas Rhizobium rhizogenes K84 (formerly A. radiobacter K84) was situated in the 

Rhizbium clade, wihch included R. etli and R. leguminosarum. To get a robust estimate of the phylogenetic 

placement of R. galegae, four house-keeping genes (recA, thrC, atpD and rpoB), and glnA and glnII genes of 

forty-four strains of R. galegae and more than forty strains of related rhizobia and agrobacteria were sequenced. 

Results of Multilocus Sequence Analysis of rhizobia and agrobacteria will be presented. 

Eardly BD, Nour SM, van Berkum P & Selander RK (2005) Rhizobial 16S rRNA and dnaK genes: Mosaicism and 

the Uncertain Phylogenetic Placement of Rhizobium galegae, Appl Environ Microbiol 71: 1328-1335. 

Lindström K & Mousavi SA (2010) Rhizobium and Other N-fixing Symbioses. In: Encyclopedia of Life Sciences 

(ELS). John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0021157. 

146 

2011




Title Study on soybean nodule symbiosome membrane localised transporters 

Authors Chen C., Loughlin P., Day D., Smith P. 


In nodules, the symbiosome membrane (SM) shares properties of both the plasma membrane and vacuolar 

membrane, however the unique nature of the SM suggests specific mechanism(s) for targeting proteins is 

essential. Recent work has indentified the first 24 amino acids from the N-terminus of a Medicago symbiosome 

protein as an address label (signal peptide) for its targeting (Hohjnec et al., 2009), however this signal peptide is 

not present on many known SM proteins. We are focusing on clarifying the targeting mechanism of soybean 

nodule SM-localised GmZIP1 (zinc transporter) and GmYSL1 (iron transporter). In silico analysis predicts the first 

28 amino acids of GmZIP1 may be a signal peptide. Full length GmZIP1 with GFP fused internally, immediately 

after the last predicted transmembrane domain is targeted to the SM. Deletion of the predicted signal peptide 

appears to disrupt the expression and/or localization of the fusion protein. GmYSL1 is not predicted to have an 

N-terminal signal peptide and N-terminal fusion of GFP does not disrupt its SM localisation. We are currently 

examining the localisation of a C-terminally truncated GmYSL1 construct to determine whether this is important 

for SM targeting. Furthermore we have characterised 4 novel soybean promoters that can drive high and specific 

expression in soybean nodule infected cells. Role of other soybean ZIP homologues are being characterised 

through expression, function and localisation studies to elucidate the zinc transporter family. 

147 

2011




Title Development of an inoculant for Acacia acuminata to improve growth of Santalum spicatum 

Authors Elizabeth Watkin 1 , Len Norris 2, 3 , Liz Barbour 2,4 


1 School of Biomedical Sciences, Curtin University 

2 Forest Products Commission 

3 Department of Agriculture & Food WA 

4 Faculty of Natural and Agricultural Sciences, The University of Western Australia 

Acacia acuminata is the most widely used host species within commercial plantations of the hemi-parasitic 

species Santalum spicatum (West Australian sandalwood). Some 10 thousand hectares of WA sandalwood 

plantations have been established in the 400 to 600 mm rainfall zone of the wheat-belt to supplement future 

shortfalls in West Australian sandalwood supply resulting from native harvest restrictions and increase in world 

demand. Host selection trials in WA demonstrated that leguminous N-fixing species are superior compared to 

non N-fixing species, with A. acuminata being a superior choice across a range of environments. Improving Nfixation 

of A. acuminata was identified as a potential factor to increase sandalwood productivity. 

This study aimed to develop a high-performing N-fixing inoculant for application to A. acuminata seedlings in 

nurseries or directly onto seeds. Seventeen root nodule bacteria isolates previously identified as being effective 

on A. acuminata were used to inoculate five different A. acuminata provenances to test their effectiveness in a 

containerised nursery environment. Isolates were ranked on their ability to increase seedling growth over an uninoculated 

control. Based on the results of this trial, five inoculum treatments were prepared with combinations of 

the most effective isolates. All five treatments produced significantly larger seedlings than the control. Higher 

total nodule mass had a stronger relationship with seedling growth, than nodule size alone. All isolates were 

demonstrated to be Bradyrhizobium spp. based on 16 S rRNA gene sequencing. 

These isolates are a valuable resource to further investigate the impact of efficient N-fixation in A. acuminata on 

the vigour and heartwood production in WA sandalwood. Greater N-fixation efficiency through a seed or nurseryapplied 

inoculant may enable the host to survive greater parasitic loading. Thus superior inoculant treatment 

could improve the supply of nutrients for increased sandalwood growth or reduce host mortality within a 

plantation and subsequent sandalwood death. 

148 

2011




Title Endophytic colonization of sweet potato by a diazotrophic bradyrhizobia 

Authors Yoshinari Ohwaki 1 and Junko Terakado-Tonooka 1,2 


1 NARO Agricultural Research Centre, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan 

2 JSPS Research Fellow, 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan 

Biological nitrogen fixation has been suggested as a potential source of nitrogen to sweet potato. In the present 

study, endophytic diazotrophs were isolated from surface-sterilized stems and tubers of field-grown sweet potato 

and characterized. 16S rRNA gene sequence analysis revealed that the isolates belong to the genera of 

Bradyrhizobium, Pseudomonas and Paenibacillus. Nitrogen-fixing capacity of the isolates was confirmed by 

sequence analysis of the PCR-amplified fragment of nifH gene and acetylene reduction activity in the semi-solid 

Rennie medium. The phylogenetic tree based on nifH gene sequences was consistent with 16s rRNA gene 

phylogeny, and all the isolates exhibited nitrogenase activity. Re-colonization ability of Bradyrhizobium as 

endophyte was assessed in the laboratory conditions. Further, the influence of nitrogen on the endophytic 

colonization of the isolate was examined. Internal population of the Bradyrhizobium in surface-sterilized leaves, 

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 

inoculation to the roots. In contrast, none of the uninoculated control plants showed any infection of the bacteria. 

The amount of nitrogen applied to the media had only a small effect on the internal population of the endophytes 

in sweet potato. Colonization in the stems after inoculation of the Bradyrhizobium to the roots as assessed by 

PCR-amplification of the nifH gene fragments was also observed in sunflower, rice, soybean and maize. 

149 

2011




Title Functional Analysis of STM Mutants Concerning Amino Acid Metabolism of Mesorhizobium 

loti 

Authors Shigeyuki Tajima 1 , Mika Nomura 1 , Nanthipak Thapanapongworakul 2 , Ayao Enoki 1 and 

Hiroyuki Matsuura 1 


1 Dept of Applied Life Sciences, Kagawa Universit 

2 Dept of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University 

The soil bacterium Mesorhizobium loti is able to induce the formation of nitrogen-fixing nodules on the root of a 

determinate-type legume plant, Lotus japonicus. The research on various metabolites during symbiosis has been 

elucidated that several bacteroid metabolic pathways are essential for maintaining efficient symbiotic interaction 

and for enhancing the in vivo nitrogenase activity of the nodules. 

To elucidate the molecular mechanism of such metabolic bacteroid differentiation in determinate nodules, protein 

profiles and functions of Mesorhizobium loti for Lotus japonicus were compared between cultured bacteria and 

nodule bacteroids. 

The transposon insertion mutant strains of M. loti were generated using the signature-tagged mutagenesis (STM) 

technique. To determine the functions of the up-regulated proteins in the bacteroids, 130 STM mutants were 

inoculated with Lotus plants. 

We focused on the four STM mutants (STM5, 30, 42, 130) which were related to the amino acid metabolism. M. 

loti mutant (STM5) that was inserted a transposon in the PHGDH gene, mll3875, showed an absolute 

dependence on serine or glycine in the minimal medium for the growth. When L. japonicus plant was infected 

with STM5, the root formed nodules with comparable number with that of wild type M. loti. However, the nodules 

showed very low acetylene reduction activity and significant starch granule accumulation was observed in the 

uninfected cells. In addition, STM42 that was inserted in the amino acid transporter showed the 70 % acetylene 

reduction activity of wild type nodule. This amino acid transporter showed high homology to aapJ in R. 

leguminosarum. However, STM42 bacteroid showed lower concentration in some amino acids, especially Ala, 

Val, Leu, Lys, Arg, and Orn, but the concentrations of Glu and Asp did not change. This data suggested that this 

amino acid transporter was different from aap/bra amino acid transporter in R. leguminosarum. Other amino acid 

synthetic proteins, STM30 and STM130, were also discussed. 

150 

2011




Title Establishment of retrotransposon-mutagenized population of the model legume Lotus 

japonicus and high throughput, deep sequencing-based insertion site identification. 

Authors Dorian Fabian Urbański, Anna Małolepszy, Stig Uggerhøj Andersen, Jens Stougaard 


Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, 

Aarhus University, 8000 Aarhus C, Denmark 

Targeted gene knockout by homologues recombination is not yet a standard technique for reverse genetics in 

plants. As an alternative we have taken advantage of inducible de-repression of Lotus retrotransposon 1 

(LORE1), a member of small family belonging to the Ty3-Gypsy class of LTR retro-elements. LORE1 has a 

unique feature of being transcriptionally active in the male gametophyte 4, 5 , producing a small number of new 

insertions at random sites only during transition to a new generation. We are currently using a founder line of 

Lotus japonicus, with active LORE1 to generate a large, mutagenized population. For robust identification of 

novel insertion sites we are using a 2D pooling strategy and taking advantage of the known LORE1 LTR 

sequence to simultaneously amplify LORE1 flanking sequence tags (FSTs) in dozens of pooled plants. Illumina 

deep sequencing technology, an extremely high degree of multiplexing and custom-designed bioinformatics‟ 

pipeline led us to establishing high throughput detection method for robust identification of those insertions. 

Study on a test set of 3744 plants, enabled identification of app. 8000 novel insertions with an average of 2,4 per 

plant. Novel LORE1 copies do not show tendency to cluster and are evenly distributed along Lotus 

chromosomes. Moreover, genes were preferentially targeted and insertions in exons were 5.7 times more 

frequent than in intergenic regions (P




Title Rhizobium delivery systems for grain legumes in southern Australia 

Author David Pearce 1 , Bernadette Carmody 1 , Matthew Denton 2 , Mark Peoples 3 , Lori Phillips 4 


1 Department of Primary Industries 

2 University of Adelaide 

3 CSIRO Plant Industry, Canberra 

4 Department of Primary Industries 

Grain legumes are an integral component in Australian cropping systems. Due to low soil Rhizobium populations, 

inoculation is often required to provide sufficient numbers of suitable rhizobia on or near the germinating seed. 

Traditionally, the main inoculation delivery system has been peat slurry application, where the seed is coated 

with the appropriate rhizobia just prior to sowing. Although very effective, this method requires strict adherence to 

application recommendations and may be too troublesome for growers during sowing. New inoculation delivery 

technologies are much easier to use and have the potential to increase inoculation flexibility at sowing. However, 

there is still a need for field evaluation to ensure that optimal nodulation, crop performance, and nitrogen (N) 

fixation occurs. In this study, we evaluated the performance of different delivery systems in Southern New South 

Wales. Faba bean cv. Farah and Lupin cv. Jindalee were sown using three delivery systems (peat slurry on 

seed, peat granules, peat slurry injection) and non-inoculated controls. Performance was measured with respect 

to nodulation, herbage biomass, grain yield, N content, and total N fixed. Inoculation with any method was 

significantly better than no inoculation. The peat slurry on seed method also generally outperformed the other 

methods. For example, Faba inoculated with this method yielded 3.69 t/ha of grain (4.37% N) and 11.61 t/ha of 

herbage (2.79% N). In comparison, Faba inoculated using peat slurry injection yielded 2.70 t/ha of grain (4.03% 

N) and 8.40 t/ha of herbage (2.48% N). Greater crop biomass with higher N content resulted in increased N fixed 

within the cropping system as a whole. Peat-seed inoculation systems fixed 316 kgN/ha, while peat-injection 

systems fixed 188 kgN/ha. Our results highlight not only the value of inoculation at sowing, but also the 

importance of choosing the appropriate inoculation delivery system for local conditions. 

152 

2011




Title BNF efficiency of rhizobia isolates from Phaseolus vulgaris in subsistence agrosystems in 


Authors César A. Díaz-Alcántara 1 , Beatriz Urbano 2 , Daniel Mulas 3 , Encarna Velázquez 4 , Fernando 

González-Andrés 3 . 


1 Facultad de CC. Agronómicas y Veterinarias. Univ. Autónoma de Santo Domingo. 


2 Departamento de Ingeniería Agrícola y Forestal. Universidad de Valladolid. Spain. 

3 Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad. Universidad de León. 

Spain. 

4 Departamento de Microbiología y Genética. Universidad de Salamanca. Spain. 

Biofertilisation of common bean (Phaseolus vulgaris L.) using selected native rhizobial strains can improve the crop yield in 

subsistence agrosystems of Dominican Republic. With this objective 5 rhizobial strains were preselected in the basis of N 

fixation efficiency in axenic conditions, from an initial collection consisting of 23 strains isolated from root nodules of 

common bean (Phaseolus vulgaris L.) growing in subsitence agrosystems in 3 provinces of Dominican Republic (Elías Piña, 

San José de Ocoa and La Vega). On the basis of the 16S rRNA and housekeeping genes (recA and atpD) sequence 

analysis, the 5 strains belonged to the Rhizobium genus, although the identity with the previously described species is too 

low to precisely identify them, so they could be new species, which needs further research (Díaz-Alcántara et al., 2008). In 

order to assess the potential benefit of the inoculation with the preselected strains for the farmers, a microcosm experiment 

was carried out in 3 different soils form Elías Piña, San José de Ocoa and La Vega respectively, with the purpose of 

compare the effect of inoculation vs. the conventional N fertilisation. In spite of the limiting conditions of microcosm for 

nodulation and BNF because of the high temperatures inside the pots, 2 out of the 5 strains produced significantly higher 

aerial biomass than the non fertilised control. The interaction between soil and strain was not significant for p




Title Protection of plant health by Micromonospora isolated from alfalfa root nodules. 

Authors Pilar Martínez-Hidalgo 1,3 , Pablo García-Benavides 2 ,José Maria Díaz- 

Mínguez 1 , Ernesto Pérez Benito 1 , Martha E. Trujillo 1, and Eustoquio 

Martínez-Molina 1 . 


1 Departamento de Microbiología y Genética, Universidad de Salamanca 

2 Departmento de Construcción y Agronomía, Universidad de Salamanca 

3 GIR "Interacciones mutualistas planta microorganismo", Unidad 

Asociada al IRNASA. CSIC, 

The studies of new interactions that allow us to design efficient biofertilizers, to reduce the use of pesticides and 

chemical fertilizers are very important nowadays. The endophytic actinobacteria have a very high 

biotechnological potential in agriculture and environment. The activity of endophytic actinobacteria can affect (A) 

the development of plants in a direct way (B) their nutrition, making it easier to get essential elements like 

nitrogen, iron or phosphore (C) the health of the plants by means of direct mechanisms, like antibiotic or 

siderophore synthesis or inducing plant defence responses and (D) improving adverse environmental factors 

tolerance. 

In this work we study the PGPR effect of actinobacteria from Micromonospora genus that were isolated from 

alfalfa nodules. Strains that promote plant growth, improve nutrient uptake and nodulation were selected. The 

selected strains were used, in this work, for studies focused on the protection of plants from pathogens. 

Protection of plant health by means of direct mechanisms, 

In vitro studies of growth inhibition of plant pathogens: The following plant pathogens were used for this study: 

Fusarium circinatum, Sclerotinia sclerotionum, Botrytis cinerea, Rhizoctonia solani, Clavivacter miichiganensis, 

Erwinia amylovora, Pectobacterium chrisanthemi, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv. 

syringae, Ralstonia solanacearum, Xanthomonas vesicatoria 

Micromonospora strains were tested for siderophore production on CAS medium. 

Protection of plant health by induction of plant defence responses: These studies have been performed on tomato 

plants using two Micromonospora strains (ALFb5 and ALFpr18c) and two plant pathogens: Botrytis cinerea and 

Fusarium oxysporum 

The results obtained point out that the selected strains of Micromonospora may be used to protect plants from 

pathogens. (i) Some of the tested strains inhibit fungal growth in vitro. (ii) No effect was detected against the 

tested bacteria. (iii) Many of the strains tested produce siderophores. (iv) Micromonospora induces plant defence 

response. 

154 

2011




Titles Identification by MALDI-TOF MS spectrometry of rhizobia isolated from nodules of different 

legumes 

Authors Pilar Martínez-Hidalgo 1 , Laura Ferreira 2 , Martha-Helena Ramírez-Bahena 3 , Lina P. Rivera, 

Fernando Sánchez-Juanes 2 , Paula García-Fraile 1 , Raúl Rivas 1 , Pedro F. Mateos 1 , José 

Manuel González-Buitrago 2,4 , Alvaro Peix 3 , Eustoquio Martínez-Molina 1 , , Encarna Velázquez 1 


1 Departamento de Microbiología y Genética, Universidad de Salamanca 

2 Unidad de Investigación. Hospital Universitario de Salamanca 

3 IRNASA-CSIC. 

4 Departamento de Bioquímica y Biología Molecular. Universidad de Salamanca 

The identification of members from family Rhizobiaceae currently arranged into the genera Rhizobium, Ensifer 

and Shinella, is not possible on the basis of physiological or biochemical traits and should be based on 

sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of 

bacteria isolated from legume nodules. MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time-of- 

Flight Mass Spectrometry) is a reliable method for bacterial identification, but it was necessary to extend the 

database Biotyper 2.0 to include the species from family Rhizobiaceae (Ferreira et al., 2011). After database 

extension, in this work, several strains isolated from nodules of alfalfa, clover and common beans isolated in 

Spain were subjected to MALDI TOF MS analysis. The results obtained showed a correct identification for most 

of the strains analysed. Some strains were not identified as any of species from the extended database 

indicating they can belong to undescribed species. Therefore MALDI-TOF MS is an excellent tool for 

identification of fast growing rhizobia and detection of new ones applicable to large populations of isolates in 

ecological and taxonomic studies. 

Ferreira L, Sánchez-Juanes F, García-Fraile P, Rivas R, Mateos PF, Martínez-Molina E, González-Buitrago JM & 

Velázquez E (2011) Maldi-tof mass spectrometry is a fast and reliable platform for identification and ecological 

studies of species from family Rhizobiaceae. PLoS One. 6: e20223. 

155 

2011




Title Expression of an abscisic acid-responsive ƒÀ-1,3-glucanase gene in autoregulation of 

nodulation. 

Author Keni-chi Osuki 1 , Akihiro Suzuki 2 , Hisatoshi Hara 1 , Akihito Takahara 1 , Masato Araragi 1 ,Tadao 

Asami 3 , Keni-chi Kucho 1 , Shiro Higashi 1 , Mikiko Abe 1 and Toshiki Uchiumi 1 


1 Graduate School of Science and Engineering, Kagoshima University, 

2 Department of Environmental Science, Saga University 

3 Graduate School of Agriculture and Life Science, The University of Tokyo 

Host legumes control nodule number by autoregulation of nodulation (AUT), in which the presence of root 

nodules inhibits further root nodule formation. AUT will consist of at least two long-distance signals, i. e. a rootderived 

infection signal and a shoot-derived signal that inhibits nodulation. In Lotus japonicus, HAR1 mediates 

AUT and, LjCLE-RS1 and LjCLE-RS2 have been identified as strong candidates as the infection signal. We 

employed split-root system of L. japonicus to investigate plant molecules involved in inhibition of nodule 

formation in AUT. Abscisic acid (ABA) and ABA responsible β-1,3-glucanase gene (designated as LjGlu1) were 

focused in this study. 

Plants were set up for split-root system. One side of root systems was inoculated with Mesorhizobium loti 

MAFF303099. At 1, 2, 3, 5, 7, 9 and 14 days after inoculation, expression of LjGlu1 in leaves, and inoculated- 

and uninoculated roots, were analyzed by quantitative RT-PCR. In the leaves, expression of LjGlu1 increased at 

the 7 days after inoculation. In the inoculated- and uninoculated roots, expression of LjGlu1 increased at the 5, 7 

and 9 day after inoculation. AUT response of L.japonicus is detectable 3 days after the first inoculation and that 

full induction of the response occurs after 5 days (Suzuki et al 2008).These results suggest that LjGlu1 increased 

in the root, when the full induction of the AUT response occurs. 

Hairy roots transformed with LjCLE-RS1 driven by the Cauliflower mosaic virus 35S promoter were prepared. In 

the plant that has both the transgenic and untransgenic roots, LjGlu1 was induced in not only the transgenic 

roots but also untransgenic roots. These findings indicate that constitutive expression of LjCLE-RS1 also induced 

expression of LjGlu1 systemically. These results suggest that LjGlu1 is functional involved in inhibition of nodule 

formation in AUT. 

Akihiro Suzuki , Hisatoshi Hara ,Tomoyo Kinoue ,Mikiko Abe , Toshiki Uchiumi , Ken-ichi Kucho ,Shiro Higashi 

,Ann M. Hirsch ,Susumu Arima (2008).Split-root study of autoregulation of nodulation in the model legume Lotus 

japonicus. J Plant Res 121:245–249 

156 

2011




Title Conservation of legume-rhizobium symbiosis genes in non-legume. 

Author Keisuke Yokota, Takashi Soyano, Hiroshi Kouchi, Makoto Hayashi 


National Institute of Agrobiological Sciences 

In recent years, a number of legume genes involved in the root nodule symbiosis has been identified in the 

model legumes, such as Lotus japonicus and Medicago truncatula. Among them, the distinct set of genes has 

been characterized as common sym, because they are also essential for another mutual interaction, the 

arbuscular mycorrhiza symbiosis. The non-legume homologs of common sym genes, such as in rice, have been 

well documented to be not only essential for the arbuscular mycorrhiza symbiosis in non-legume mycorrhizal 

plants but also functional in both the root nodule symbiosis and the arbuscular mycorrhiza symbiosis in legumes. 

In contrast, it has not been investigated in detail whether the root nodule symbiosis-specific genes, which are not 

essential for the arbuscular mycorrhiza symbiosis, are functionally conserved in non-legumes. 

In this presentation, we will report that the functional conservation analyses of the root nodule symbiosis-specific 

gene homologs from rice in L. japonicus. 

References 

Yokota K, Hayashi M. Function and evolution of nodulation genes in legumes. (2011) Cell Mol Life Sci. 

68(8):1341-1351. 

Yokota K, Soyano T, Kouchi H, Hayashi M. Function of GRAS proteins in root nodule symbiosis is retained in 

homologs of a non-legume, rice. (2010) Plant Cell Physiol. 51(9):1436-1442. 

157 

2011




Title Analysis of nif gene derepression in Azotobacter vinelandii by quantitative real-time PCR 

Author César Poza-Carrión, Emilio Jiménez-Vicente, & Luis M. Rubio 


Universidad Politécnica de Madrid. Centro de Biotecnología y Genómica de Plantas Pozuelo 

de Alarcón, 28223 Madrid (Spain). cesar.poza@upm.es 

Azotobacter vinelandii serves as a model to study the biochemistry and regulation of nitrogenase. In Azotobacter 

vinelandii, the nitrogen fixation (nif) genes are clustered in two chromosomal regions designated as the major 

and the minor nif clusters. Apart from the nitrogenase structural genes (nifHDK), a number of nif gene products 

are required for the assembly of active nitrogenase component proteins. Not all gene products act at the same 

time during nitrogenase biogenesis; therefore their expression levels and profile over time must be strickly 

coordinated. 

In this work, we study the time-dependent expression of fifteen nif genes involved in biosynthesis and regulation 

of nitrogenase (nifH, nifD, nifK, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifA, nifB, FdxN, nifQ, and nafY) under 

nitrogenase derepressing conditions by using real-time quantitative PCR. Nif gene expresion is observed as fast 

as 10 minutes after ammonium removal from the medium. Expression of genes whose products are involved in 

early steps of iron-molybdenum cofactor (FeMo-co) biosynthesis, such as nifB, reached maximum levels before 

nitrogenase structural genes. In general, it was observed that mRNA levels decreased rapidly as soon as 

nitrogenase activity appeared in the cultures, indicating tight feedback regulation of nif gene expression by 

nitrogenase activity. 

Applying synthetic biology to generate artificial nitrogenase systems is a promosing avenue to transfer nitrogen 

fixation capability to organims of interest. However, to build a synthetic nitrogenase, researchers must first 

understand the appropriate balance of gene expression in quantitative and temporal terms. Results from this 

work provide an important step towards elucidating these parameters in the model nitrogen-fixing organism 

Azotobacter vinelandii. 

158 

2011




Title Nodule-specific cystein-rich peptides found in actinorhizal plant Datisca glomerata 

Author Irina V Demina, Tomas Persson, Marian Plaszczyca, and Katharina Pawlowski 


Stockholm University, Department of Botany 

The actinorhizal plant Datisca glomerata enters a symbiosis with nitrogen-fixing actinomycetes of the genus 

Frankia. The bacteria are hosted in root nodules. In the course of root and nodule transcriptome studies of D. 

glomerata, full-size cDNA sequences of two genes encoding small nodule-specific cysteine-rich (NCR) peptides 

were obtained. One of the cDNAs, DgDEF1, shows amino acid homology with defensins; the other cDNA 

encodes a protein for which no homolog has been found in the databases. The predicted sizes of the mature 

peptides are rather large for defensins, 86 and 94 aa, respectively. This finding is the first report on NCR 

peptides in actinorhizal plants. 

Previously NCR peptides have been characterized in legumes, where they were demonstrated to govern the 

terminal differentiation of the rhizobial microsymbionts in symbiosis (Van de Velde et al. 2010) and to show 

antimicrobial activity toward a diverse group of bacteria (WO2010/146067). Hence, based on the homology 

between actinorhizal and legume symbioses, it seems likely that actinorhizal plants control the differentiation of 

their bacterial endosymbionts in a similar manner. The fact that DgDEF1 shows high homology with defensins of 

class A3, which reduce hyphal elongation while increasing hyphal branching in fungi, suggests that this peptide 

is responsible for the induction of hyphal branching in symbiotic Frankia in nodule cells. 

To determine in situ localization of the D. glomerata NCR peptides in nodules, Agrobacterium rhizogenes 

transformation was used. In transgenic nodules transcriptional fusions of the promoters of DgDEF1 and DgCRP1 

with the GUS coding region were expressed. The expression patterns of the transgenes will be discussed in 

comparison to the expression patterns of NCR peptide genes in legumes. 

Van de Velde et al. (2010). Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327: 

1122-1126 

159 

2011




Title N2O emission from degraded soybean nodules by denitrification of Bradyrhizobim japonicum 

and other soil microorganisms 

Authors Fumio Ikenishi, Shoko Inaba, Manabu Itakura, Shima Eda, Hisayuki Mitsui, and Kiwamu 

Minamisawa 


Graduate School of Life Sciences, Tohoku University 

To clarify mechanism of N2O emission from degraded soybean nodules (Inaba et al. 2009), a model system was 

developed to reproduce N2O emission from degrading soybean nodules in laboratory. Thirty-days after soybean 

plants inoculated with Bradyrhizobium japonicum were cultivated in Leonard‟s jars, treatments of shoot 

decapitation (D) and/or soil addition (S) were conducted to simulate the nodule degradation and subsequent N2O 

emission. Double treatment (DS) resulted in the degradation and N2O emission from the nodules formed with B. 

japonicum lacking nosZ. These results suggested that soil microbes are required for the N2O emission from 

degraded soybean nodules. To evaluate bradyrhizobial contribution, N2O emission was compared between nirK 

mutant (∆nirK) and wild-type B. japonicum USDA110 under identical nosZ genetic backgrounds. N2O emission 

from the nodules formed with ∆nirK∆nosZ mutant was significantly lower than that from ∆nosZ mutant under DS 

treatment, but retained approximately a half amount of N2O emission in ∆nosZ mutant (30-60%), suggesting that 

nitrate reduction to N2O is due to both B. japonicum and other soil microorganisms. On the other hand, it is likely 

N2O reduction to N2 was mainly mediated by B. japonicum cells carrying nosZ, which was consistently supported 

by the comparisons between wild-type USDA110 and nosZ mutants. Thus, B. japonicum plays an important role 

in determining N2O flux from soybean rhizosphere as well as unknown soil microorganisms. It has been reported 

that fungi emit N2O in various fields. Thus, we isolated fungi spores from the model system, and evaluated their 

N2O production. As a result, many of the isolates produced N2O from nitrite in culture. 

Inaba S, Tanabe K, Eda S. Ikeda S, Higashitani A, Mitsui H & Minamisawa K (2009) Nitrous oxide emissions and 

microbial community in the rhizosphere of nodulated soybeans during the late growth period. Microbes Environ. 

24: 64-67. 

160 

2011




Title Functional characterization and interactions of the azorhizobial parA and parB proteins 

Author Min-Hua Peng 1 , Yu-Sheng Wang 1 , Ming-Yen Tsai 2 , Hisao-Lin Chien 2 , Kung-Ta Lee 1 , 


Chi-Te Liu 2,3, * 

1 Department of Biochemical Science and Technology, National Taiwan University, Taipei 

106, Taiwan 

2 Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan 

3 Agricultural Biotechnology Research Centre, Academia Sinica, Taipei, 115, Taiwan 

Bacteroid development consists of several stages, and a number of bacterial genes that are involved in this 

process have been identified. However, the genes and factors that are engaged in the initiation of bacteroid 

formation, and those that regulate it, are incompletely identified. Our experimental evidence has demonstrated 

that the chromosome-partitioning gene (parA) gene of Azorhizobium caulinodans ORS571 not only plays crucial 

roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in 

Sesbania rostrata stem nodules. In this study, we will clarify the functions of the recombinant ParA and ParB 

proteins of A. caulinodans, and the interactions between the par system and bacteroid differentiation related 

genes. We noticed that the ATPase activity of ParA can be enhanced in the presence of ParB protein. In 

addition, ParB bound to a centromere-like parS DNA fragment, and was stimulated by the increasing amounts of 

ParA protein. Interactions between ParA and ParB were confirmed by in vitro immunoprecipitation assay. Using 

gel mobility shift assay, we found that ParB bound to the bacteroid formation associative gene (bacA) and the 

promoter region of par operon. This binding seems to be regulated by mole ratio of ParB/ ParA . We proposed a 

mechanism for regulation of bacteroid differentiation of A. caulinodans. Interactions between azorhizobial ParA 

and ParB proteins may function as a checkpoint, which couples the chromosome partitioning to the onset of 

bacteroid formation in symbiosis. 

Liu, C.-T., Lee, K.-B., Wang, Y.-S., Peng, M.-H., Lee, K.-T., Suzuki, S., Suzuki, T. & Oyaizu, H. (2011). 

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during 

Sesbania rostrata stem nodule development. Appl Environ Microbiol 77, 4371-4382. 

161 

2011




Title Trehalose accumulation in osmotically challenged rhizobia and its effect on desiccation 

tolerance. 

Authors Andrea Casteriano and Rosalind Deaker 


University of Sydney - Faculty of Agriculture, Food and Natural Resources 

Pre-inoculated seeds are a convenient and much sought after legume inoculant product. However, rhizobia is 

known to survive poorly on seed mainly due to desiccation stress encountered during the seed coating process. 

Rhizobia is also known to possess inherent desiccation tolerance mechanisms including the production of 

compatible solutes such as trehalose through the de novo synthesis. 

Batch cultures of Bradyrhizobium japonicum (CB1809) and Rhizobium leguminosarum bv. trifolii (TA1) were 

grown in a defined medium with altered osmotic pressure through the addition of solutes. Increasing the osmotic 

pressure (1atm to 2.5atm) of the medium resulted in cells with a higher amount of intracellular trehalose. Vacuum 

drying those cells in the absence of an external protectant showed an improvement in % survival for CB1809 

immediately after drying and during storage at RH ≤ 9%. However the accumulation of intracellular trehalose did 

not result in improved desiccation tolerance for TA1. 

Cells of CB1809 extracted from peat and dried under vacuum in the absence of an external protectant show a 

much greater rate of survival not only immediately after drying but also during prolonged storage. Also, cells of 

TA1 and CB1809 grown in a peat extract have shown a much greater rate of survival after vacuum drying than 

those cells grown in a liquid medium. 

In conclusion, osmotically challenging CB1809 and TA1 during growth appears to increase their ability to 

accumulate intracellular trehalose. However, trehalose accumulation only improved desiccation tolerance of 

CB1809 and not TA1. Cells were better able to tolerate desiccation after growth in peat and peat extract 

indicating that not only conditions of nutrient and oxygen limitation play a role but also soluble constituents of 

peat. 

162 

2011




Title A MYB coiled-coil type transcription factor interacts with NSP2 and is essential for nodulation 

in Lotus japonicus 

Author Zhongming Zhang, Heng Kang, Xiaojie Chu, Chao Wang, Hui Zhu, Songli Yuan, Dunqiang 

Yu, Zhenzhen Yang 


State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University 

Transcription factor complex formation is a central step in regulating gene expression. In Medicago truncatula, 

two GRAS family transcription factors, Nodulation Signaling Pathway 1 (NSP1) and NSP2 form a DNA-binding 

complex to induce specific changes of gene expression, which is essential for the root nodule symbiosis (Hirsch 

et al., 2009). Using yeast two-hybrid (Y2H) screening, we identified a novel MYB coiled-coil transcription factor, 

referred as IPN2, which interacts with NSP2 in Lotus japonicus. This interaction was confirmed by protein pulldown 

assay and BiFC assay in Nicotiana benthamiana. Moreover, IPN2 and NSP2 were co-localized in nuclei of 

L. japonicus hairy roots. The Y2H assay showed that the GRAS domain of NSP2 was required for interacting 

with IPN2, while the coiled-coil domain of IPN2 was necessary and sufficient for interacting with NSP2. IPN2 

showed strong transcriptional activation activity in yeast cells, and it bound to the NIN promoter in vivo and in 

vitro. IPN2 was widely expressed in various organs, and showed phloem-specific expression within the 

vasculature of transgenic hairy roots. Overexpression of IPN2 promoted nodulation, while RNA interference 

(RNAi) knockdown of IPN2 expression led to decreased nodule formation in L. japonicus. Take together, our 

results strongly suggest that IPN2 plays an essential role in nodulation. 

Hirsch, S, Kim, J, Munoz, A, Heckmann, AB, Downie, JA, and Oldroyd, GE (2009). GRAS proteins form a DNA 

binding complex to induce gene expression during nodulation signaling in Medicago truncatula. Plant Cell 

21:545-557. 

163 

2011




Title Genome sequence of Mesorhizobium huakuii 7653r which establishes a highly specific 

symbiosis with Astragalus sinicus in China 

Author Youguo Li, Shanming Wang, Jieli Peng, Baohai Hao & Liu Liu 


State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 

Hubei, 430070, P. R. China 

M. huakuii 7653R, isolated from a rice-growing field in southern China, can establish a highly specific and 

effective symbiosis with A. sinicus via forming indeterminate-type nitrogen-fixing nodules. To date, there is very 

limited information on the mechanism of its bacteroid development, nodulation specificity and molecular 

interactions with Chinese milk vetch. In the present work, the complete genome sequence of M. huakuii 7653R 

has been established and the genome structure and phylogeneticas signment of the organism was analysed. 

For de novo sequencing of the M. huakuii 7653R genome, a combined strategy comprising Solexa-sequencing 

on the Illumina GAIIx platform and PCR-based amplicon sequencing for gap closure was applied. The finished 

genome consists of three replicons and comprises 6,952,365 bases. The genome sequence of M. huakuii 7653R 

totals 6.95 Mbp with a GC content of 62.9%. It contains 6,792 predicted open reading frames (ORFs), located on 

a chromosome and two plasmids. The 6.2 Mbp circular chromosome encodes 6,150 ORFs with a mean GC 

content of 63.1%. The 320,051-bp pSym pMh7653Rb comprises 282 ORFs with a mean GC content of 62.6%. 

The plasmid pMh7653Ra (170,165 bp) has a relatively lower GC content (59.5%). 

Comparative analysis of the assembled genome sequence of M. huakuii 7653R revealed high homology and 

extensive synteny with M. loti MAFF303099, and partly to other rhizobial genomes, in particular to Sinorhizobium 

meliloti 1021 and to Rhizobium sp. NGR234. 

164 

2011




Title Identification of the regulatory genes required for the acid activation of the low pH inducible 

gene lpiA in Sinorhizobium medicae 

Author Rui Tian 1 , Ravi Tiwari 1 , Lambert Bräu 1 , John Howieson 1 , Graham O‟Hara 1 & Wayne Reeve 1 . 


1 Center for Rhizobium studies, Murdoch University, South Street, Murdoch, Western 

Australia, 6150. 

The acid tolerance response of Sinorhizobium medicae (S. medicae) WSM419 enables cell adaptation to lethal 

acid after cell exposure to mild acidity. The expression of lpiA (low pH induced gene A) gene is critical for this 

response and is acid-activated at least 20-fold in mild acidic conditions. Inactivation of an upstream gene fsrR 

(fused sensor regulator) reduces lpiA expression to just 3-fold of its maximum level. This observation suggested 

that other regulatory proteins are involved in complete activation of the lpiA gene in acidic conditions. 

Inactivation of putative regulators of lpiA located upstream (tcrA and tcsA) or downstream (acvB) genes and the 

sigma-54 factor encoded by rpoN was achieved by single crossover mutation in WSM419. Expression studies 

(GUS-linked assays and qRT-PCR) revealed that the two component system TcsA/TcrA and a fused sensor 

regulator FsrR (encoded by a gene directly upstream from lpiA) are controlling expression of lpiA and 

demonstrated that RpoN is essential for acid activation. A putative RpoN enhancer binding protein (Smed_5956) 

was located upstream of tcsA. RACE analysis located the transcriptional start site 14 bp downstream of a 

classical -24 and -12 RpoN binding motif upstream of the lpiA start codon demonstrating that lpiA and acvB are 

co-transcribed as an operon. The expression of acvB was determined by qRT-PCR and was found to be induced 

18-fold by acid which is consistent with the finding that both lpiA and acvB share the same transcription start site. 

In contrast, fsrR, tcrA, tcsA, and rpoN were constitutively expressed with respect to pH. 

While mutations in tcsA, tcrA, fsrR and acvB affect acid induction of lpiA, these genes are not essential for stress 

tolerance or symbiotic nitrogen fixation. In contrast, rpoN is essential for symbiotic nitrogen fixation with 

Medicago. This study has therefore identified for the first time an alternative sigma factor which is essential for 

pH response as well as symbiosis in S. medicae. 

165 

2011




Title 

Selection of plant growth promoting rhizobacteria (PGPR) to enhance nodulation of 

grain legumes by rhizobia 

Author Liza Parkinson 1 , Graham O‟Hara 1 , Ron Yates 1, 2 , Paul Harvey 3 and Lambert Brau 1 


1 Murdoch University, Centre for Rhizobium Studies, Murdoch, Western Australia, 6150 

2 Department of Agriculture and Food Western Australia, South Perth, Western Australia, 

6151 

3 CSIRO, Urrbrae, South Australia, 5064 

Plant growth-promoting rhizobacteria (PGPRs) are soil bacteria associated with plant roots that have been 

shown to stimulate plant growth and crop yield. The mechanisms by which PGPRs increase plant growth include 

the production of plant hormones, suppression of disease–causing microbes as well as improved plant nutrient 

availability and assimilation. It has also been shown that the formation of nodules on legumes induced by 

rhizobia can be enhanced by co-inoculation with PGPRs. 

To examine the benefits of co-inoculating rhizobia with PGPRs, six newly identified PGPRs will be screened in 

glasshouse trials on Lupins (Lupinus angustifolius), Peas (Pisum sativum), Lentils (Lens culinaris), Beans 

(Phaseolus vulgaris), Chickpeas (Cicer arietinum) and Soybeans (Glycine max) in addition to the current 

commercial rhizobia. PGPRs used in this study include five Pseudomonas species, which have previously been 

shown to enhance nodulation in legumes and one gram-positive strain isolated from a Lupinus nodule. To 

evaluate the efficacy of the PGPRs on enhancing the legume-rhizobia symbiosis, the following will be measured: 

nodule numbers, nodule biomass, shoot and root biomass and shoot nitrogen content. In addition, nodule 

occupancy will be analyzed using PCR fingerprinting techniques to establish whether PGPRs are able to colocalize 

with rhizobia in the nodules. 

Promising isolates from these trails will be selected to elucidate the mechanism by which they enhance 

nodulation and/or nitrogen fixation. This will include analysis of expression of putative PGP genes, targeted 

gene knock-outs, and population genetic studies on the microbial community to determine the impact of 

inoculants on rhizobium populations in the rhizosphere. 

166 

2011




Abdou, MM 109 Cao, Q 31 do Nascimento, EC 128 Hartley, E 82, 133 Keakile, KK 130 

Abe, M 131, 137,156 Capela, D 37 Dolgikh, EA, 40 Hartley, J 133 Kelly, S 51 

Adnane, B 111 Carlson, R 51 Dong, W 26 Hartmann, A 66 Kenicer, G 62 

Agarie, S 131 Carlsson , G 101 Downie JA 16 Harvey, P 166 Kennedy, I 76, 78 

Aisthorpe, D 47 Carmody, B 45, 152 Drevon, JJ 99, 109, 111 Hashiguchi, M 131 Khojiev, Z 120 

Akashi, R 131 Castelli, J 42 Drew, EA 44, 52 Hayashi, M 125, 158 Kieu, LN 103 

Alatalo, E 127 Casteriano, A 81, 162 East, A 36 Hayashi, S 57 Kim, J 16 

Alexandre, A 104 Catrice, O 37 Echavarri-Erasun, C 17 He, J 50 Kimanthi, M 86 

Alves, BJR 97, 128 Chaïbi, S 118 Eda, S 160 He, X 93 Kobryn, H 24 

Amaddin, PAM 90 Chansa-Ngavej, K 46 Edwards, A 16 Heckmann, AL 16 Koch, M 41 

Amenc, L 109 Charman, N 48, 54, 79 Enoki, A 150 Hennecke, H 41 Kouchi, H 157 

Ampomah, OY 62 Chen W-M 67 Erbacher, A 47 Herridge, D 96, 101, 103 Kryvoruchko, I 50 

Amprayn, K 76 Chen, C 42, 147 Espinoza, S 94, 121 Herrmann, L 84, 86, 134, Kucho, K 137, 156 

Andersen, SU 151 Chen, R 50 Faghire, M 99 Higashi, S 137,156 Kyrpidis, N 34 

Andrianananjara, A 109 Chen, T 119 Faraut, T 37 Hiratsuka, Y 131 Lake, L 52 

Angus, JF 100 Chen, WF 63 Farquharson, R 48 Hirsch, AM 131 Lazali, M 99 

Araragi, M 156 Chen, WX 63 Faye, A 84 Högbom, M 27 Lee, K-T 161 

Ardley, JK 69 Chen, X 81 Federova, E 31, 39, 65, 74 Hood, G 36 Lei, L 43 

Arima, S 131 Cheng, G 43 Ferguson, B 57 Hori, Y 137 Leppyanen, IV, 40 

Arpiwi, NL 107 Cheng, Z 50 Ferreira, L 155 Howieson, JG 15, 24, 49, Lesueur, D 84, 86, 134, 135 

167 

135 

56, 68, 69, 83, 165 

Asami, T 156 Chiasson, D 39, 122 Fischer, D 66 Hubber, A 51 Li, D 57 

Aserse, AA 145 Chien, H-L 161 Fischer, H-M, 41 Humphries, A 79 Li, M 63 

Assefa, F 145 Chimphango, SBM 67 Forster, B 89 Hurek, T 81 Li, QQ 63 

Atieno, M 84, 134 Chu, X 163 Fox, S 45, 75 Huss-Danell, K 62, 92 Li, Y 43, 164 

Attar, H 99 Clarke, VC 42, 129 Franchini, JC 97 Huwiler, S 41 Liao, S 72 

Bakkou, N 38 Cong, PT 103 Gamas, P 37 Huyghe, A 38 Limpens, E 65 

Baldani, JI 66 Conway, M 47 García-Benavides, P 154 Hynes, MF 35 Lin, M-H 57 

Baldock, J 48 Corbett, MK 106 García-Fraile, P 155 Iannetta, PPM 62 Lin, Y-H 57 

Ballard, N 22 Craig, A 54 Garg, N 108 Ikenishi, F 160 Lindström, K,21, 127, 145, 146 

Ballard, RA 44, 48, 52, 54, 79 Cramer, SP 26 Gavrin, A 74 Imaizumi-Anraku, H 125 Liu, C-T 161 

Balone, T 130 Critchley, C 93 Ge, Y-Y 72 Imin, N 58 Liu, L 164 

Banabas, M 96 Cuddy, W 89 Geddes, BA 35 Inaba, S 102, 160 Liu, TY 63 

Barbour, EL 107, 148 Dai, X 50 Gehlot, HS 60 Inada, S 131 Loi, A 83 

Bargaz, A 99 Dakora, FD 25, 55, 95, 110, Gehringer, MM 89 Ismail, MR 88 Lopatin, SA 40 

116, 140, 141, 142, 143, 144 

Barrero, R 56 Danza, F 41 Gelfand, M 32 Itakura, M 102, 160 Loughlin, P 39, 42, 122, 129, 

147 

Beatty, P 98 Dapper, CH 26 Gemell, G 82, 133 Ivanov, S 65, 74 Ludwig, M 42 

Belane, A 55, 144 Day, DA 42, 122, 129, 167 Gemmer, S 81 Jalovaja, J 32 Majengo, C 86 

Bellgard, M 56 de Araújo, JLS 128 George, SJ 26 James, EK 60, 62, 67 Makhubedu, I 141 

Benedito, VA 50, 59 De Carvalho-Niebel, F 37 Gerding, M 68 Jantalia, CP 97, 128 Malik, R 23 

Benito, EP 154 de Mita, S 65 Ghazali,, A 136 Jardinaud, F 37 Małolepszy, A 151 

Berthold, CL 27 Deaker, R 82, 89, 103, 162 Ghoulam, C 99, 111 Jikumaru, Y 131 Mantaj, M 70 

Bisseling, T 31, 39, 65, 74 Debéllé, F 37 Giller, K,19 Jiménez-Vicente, E 17, 29, Mapope, N 142 

Bledsoe, C 93 Del Pozo, A 94, 121 Gonzales-Andres, F 77, 153 Jin, L 119 Marcano, I 77 

Boddey, RM 97, 128 Delmotte, N 41 González-Buitrago, JM 155 Jing, Y 124 Marques, AT 132 

Bonython, A 54 Demina, IV 159 Good, AO 98 Kahn, ML 28 Martínez-Hidalgo, P 154, 

155 

Boonkerd, N 123, 138, 140 Denton, MD 52, 79, 85, 152 Gouzy, J 37 Kaiser, BN 39, 122, 126 Martínez-Molina, E 154, 

155 

Borisov, A 65 Díaz-Alcántara, CA 77, 153 Gresshoff, P 57 Kalhor, MS 31 Martinez-Romero, E 66 

Braissant, O 41 Díaz-Mínguez, JM 154 Grossman, J 53 Kamaa, M 84 Maseko, S 110 

Brau, L 45, 61, 68, 75, 117, Dilworth, M 69 Guerts, R 31, 65 Kamiya, Y 131 Masse, D 109 

165, 166 

Brock, P 101 Ding, H 35 Hailemariam, A 145 Kaneko, T 131 Mateos, PF 155 

Brockwell, J 100 Dinse, T 81 Han, L 125 Kang, H 163 Mathews, C 95, 116 

Bruand, C 37 Djedidi, S 33 Hanyu, M 73 Kanu, SA 143 Mathu, S 135 

Btissam, M 111 Djordjevic, MA 58 Hao, B 164 Karunakaran, R 36 Matiru, V 135 

Bühler, D 41 Dlodlo, O 67 Hara, H 156 Kaur, H 106 Matsuura, H 150 

Burgos, FA 24 Kawaharada, Y 51 

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Mazurkiewicz, D 122 Ohwaki, Y 91, 149 Räsänen, LA 145 Soyano, T 158 van Averbeke, W 116 

McInnes, A 70 Okalebo, R 84, 86, 134 Ratet, P 50 Sprent, JI 60,62,64,67 Varlamov, VP 40 

McNeill, A 96 Okamoto, M 122 Reeve, WG 34, 44, 56, 69, 

117, 166 

Squire, G 62 Velázquez, E 77, 153, 155 

Melino, V 44, 117 Okazaki, S 71, 73 Reid, D 57 Sripakdi, S 138 Venkatanagappa, S 79 

Mhamdi, R 118 Oldroyd, GED 16, 18, 50 Reinhold-Hurek, B 81 Staehelin, C 72 Vorholt, J 41 

Millar, AH 129 Oliveira, S 104 Reis, VM 66 Steenkamp, ET 70 Vorobyov, N 30 

Minamisawa, K 102, 160 Oliver, M 53 Rice, J 28 Stepkowski, T 70 Walker, R 61 

Mirzaei, S 57 Omar, N 87 Rodde, N 37 Stougaard, J 20, 51, 151 Wang Y-S 161 

Mitra, D 26 Onyango, P 86 Rodino, N 99 

Streng, A 31 Wang, C 119, 163 

Ronson, C 51 

Mitsui, H 160 op den Camp, R 31, 65 Roux, B 37 Sui, XH 63 Wang, ET 63 

Mndzebele, B 140 Oresnik, IJ 35 Rubio, LM 17, 29, 158 Sullivan, J 51 Wang, He 27 

Mohale, K 55, 141 Osbourne, CA 100 Rudnicka, J 70 Summerell, BA 89 Wang, Ho 26 

Mohamad, S 105 Osman WAM, 117 Saad, M 38 Suriyagoda, L 131 Wang, L 119 

Mohamed, Far. 111 Österman, J 127, 146 Sadras, V 52 Suzuki, A 131, 156 Wang, M 50, 59 

Mohamed, Fat. 87 Osuki, K 137, 156 Saeki, K 71, 73 Swan, A 100 Wang, S 164 

Mohammadi-Dehcheshmeh, M 

126 

Othman, R 88 Sahara, M 137 Tabata, S 131 Wang, Z 43 

Mokgehle, S 95 Ounan, M 99 Saïdi, S 118 Tajima, S 150 Watkin, E 61, 70, 106, 107, 

148 

Moreira, LM 132 Ovalle, C 94, 121 Sakai, T 131 Tak, N 60 Webb, M 96 

Moreno-Urbano, E 17 Ovchinnikova, E 65 Sallet, E 37 Takahara, A 156 Wen, J 50 

Morieri, G 16 Padhi, B 41 Samian, M-R 105 Takayama, H 137 White, R 44 

Mostafa, S 87 Parihar, R 60 Sánchez-Juanes, F 155 Tang, Y 50 Xie, Fu. 43 

Mothapo, N 53 Parkinson, L 166 Sandal, N 51 Taylor, M 50 Xie, Fa. 16 

Mousavi, SA 146 Parr, M 53 Sankhla, I 60 Taylor, NL 129 Xie, Z-P 72 

Muasya, AM 67 Paulin, L 127, 146 Santos, MR 132 Tazawa, J 91 Xin, D-W 72 

Mulas, D 153 Pawlowski, K 159 Sasaki, M 131 Teamthisong, K 139 Xu, H 43 

Muni, RRD 50 Pearce, DJ 45, 79, 85, 152 Sato, S 131, 137 Teaumroong, N 123, 138, 139 Yadgarov, K 120 

Murakami, E 137 Peix, A 155 Saud, HM 88 Teixeira, PF 27 Yam, H-C 105 

Murray, J 16, 50 Peng J 164 Saur, I 58 Terakado-Tonooka, J 149 Yan, G 107 

Murset, V 41 Peng, M-H 161 Sauviac, l 37 Thapanapongworaku, N 150 Yan, L 26 

Mustapha, F 111 Peoples, MB 85, 100, 152 Scandurra, A 17 Thomson, G 44 Yang, H 56 

Muszynski, A 51 Perret, X 38 Schiex, T 37 Thuita, M 84 Yang, M 124 

Mutch, L 61, 106 Persson, T 159 Schmid, M 66 Tian, CF 63 Yang, Z 163 

Mutegi, E 84, 86 Pfitzner, B 66 Schwenke, G 101 Tian, R, 165 Yates, RJ 24, 49, 69, 83, 166 

Muto, S 137 Phillips, L 45, 79, 152 Sekimoto, H 33 Tikhonovich, IA, 40 Yip, C 35 

Mwirichia, R 135 Pipai, R 96 Selão, TT 27 Timmers, T 37 Yokota, K 157 

Mysore, K 50 Piromyou, P 123 Serventi, G 41 Tittabutr, P 123, 138, 139 Yokoyama, T 33, 91 

Nagano, Y 137 Pislariu, C 50, 59 Seymour, M 23 Tiwari, RT 60, 117, 165 Yoshikawa, M 91 

Nagata, M 137 Plaszczyca,, M 159 Seymour, N 47 Tollenaere, A 57 Yoshinaga, A 131 

Naher, UA 88 Plummer, JA 107 Shaffie, S 136 Tominaga, A 131 Youard, Z 41 

Najimudin, N 105, 136 Polone, E 31 Shakirov, Z 120 Torres-Jerez, I 50, 59 Young, PW 127 

Narozna, D 70 Poole, P 36 Shamsuddin, ZH 88, 90 Tran, YT 103 Yu, D 163 

Ndung‟u, K 84 Poonar, N 60 Shen, S 124 Tripathi, A 60 Yuan, S 163 

Neilan, BA 89 Poza-Carrión, C 17, 29, 158 Shidore, T 81 Trujillo, ME 154 Yurgel, S 28 

Nelson, P 96 Prell, J 36 Shigeoka, M 137 Tsai, M-Y 161 Zatorre, NP 97 

Newton, W 26 Provorov, N 30 Shigeyama, T 131 Tsoy, O 32 Zhang, J 56 

Nomura, M 150 Pule-Meulenburg, F 55, 130, 141 Shimoda, Y 125, 137 Tyerman, SD 122, 126 Zhang, JJ 63 

Nordlund, S 27 Pypers, P 84, 135 Shina, Y 102 Uchiumi, T 132, 138, 157 Zhang, L 72 

Norén, A 27 Qu, Y 42 Shirai, R 73 Udvardi, M 50, 59 Zhang, S 50 

Norris, L 148 Quinlivan, M 47 Shizukawa, Y 91 Umarov, B 120 Zhang, W 124 

Nutt, B 83 Rabeharisoa, L 109 Simoes-Araújo, JL 66 Unkovich, M 80, 96 Zhang, YM 63 

O‟Brien, T 79 Radzman, N 58 Sinharoy, S 50, 59 Uno, U 91 Zhang, Z 119, 163 

O‟Hara, GW 24, 44, 45, 49, 61, 

68, 69, 70, 75, 165, 166 

Rahim, KA 88 Smith, B 100 Urbano, B 77, 153 Zhao, PX 50 

Oakes, M 58 Ramachandran, V 36 Smith, PMC 42, 129, 147 Urbański, DF 151 Zhu, H 119, 163 

Ohkama-Ohtsu, N 33 Ramírez-Bahena, M-H 155 Smith, SE 126 Urquiaga, S 97, 128 Zotarelli, L 97 

Vailhe, H 109 Zuan, TK 90 

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IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

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