BFNet - ISCB

Indo-Swiss Collaboration in Biotechnology
Biofertilizer Network Project
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DEPARTMENT OF BIOTECHNOLOGY, DBT
MINISTRY OF SCIENCE AND TECHNOLOGY
GOVERNMENT OF INDIA
Organism
From Field field to lab Lab and back to field
Field
Industry
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Extraradical mycelium and spores of AMF
Black gram, wheat and rice seeds coated with
AMF & PGPR formulations
Field trial
ISCB Biofertilizer Network Project
Agricultural production worldwide is faced with severe problems
caused by climate change. Modern bio-technologies such as
biofertilizers consisting of arbuscular mycorrhizal fungi (AMF) and
plant growth-promoting rhizobacteria (PGPR) are a powerful tool
to improve soil fertility and sustainable production, rendering agrisystems
more stress-tolerant. Moreover, they help to reduce the
amount of energy demanding inputs, such as chemical fertilizers.
Biofertilizers contribute to mitigation of and adaptation to
climate change.
This project is part of an Indo-Swiss Collaboration in Biotechnology
(ISCB), dedicated to biofertilizers. It is referred to as ISCB Biofertilizer
project phase III, as it is a continuation of two previous phases within
this programme. It essentially aims to deliver a tested and validated
combined bio-inoculant product – based on AMF and PGPR - for
application in sustainable and low-input agriculture especially for
wheat-based cropping systems in India.
The project involves the core competencies of multi-institutions
of India (TERI, GBPUAT, BU, and IIT-D) and Switzerland (UB, UN
and FiBL) to develop, test, and monitor the combined bio-inoculant
product in various multi-location fields, validation and demonstration
trials– in order to increase their practical use in tune with existing
agricultural practices as well as some fundamental research. It also
includes the unique capability for identification and tracking of the
bio-inoculant’s components after application in the field developed
during phase II of ISCB activity. The following institutes are/were
involved:
• The Energy and Resources Institute (TERI), New Delhi, India
• GB Pant University for Agriculture and Technology (GBPUAT),
Pantnagar, India
• Barkatullah University (BU), Bhopal, India
• Indian Institute of Technology, Delhi (IIT-D), India
• University of Basel (UB), Switzerland
• University of Neuchâtel (UN), Switzerland
• Research Institute of Organic Agriculture (FiBL), Switzerland

University of Basel
Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria
(PGPR) are in the focus of ISCB as potential “biofertilizers” in sustainable agriculture.
In order to make use of such biofertilizers in a reliable fashion, it is necessary to have
molecular diagnostic tools for their identification and quantification. In this context,
the team of Andres Wiemken of the Botanical Institute of the University of Basel has
worked for many years to develop and apply microsatellite markers for AMF strains,
and the team of Thomas Boller of the same institute has joined in more recently to
produce similar markers for PGPR, based on preceding work by the group of Michel
Aragno of the University of Neuchâtel.
The Energy and Resources Institute
This project utilizes TERI’s core competency for mass production of AMF using
Root Organ Culture. This mass production system uses root-inducing transfer-DNAtransformed
roots of a host plant to develop the symbiosis on a specific medium
in vitro, which provides pure, viable, contamination-free inoculum using least space.
TERI team led by Alok Adholeya has collected (139 isolates), maintained, and selected
functionally efficient AMF for wheat cropping system (LL2 consortia, TERI commercial)
in the first phase. TERI team then tested the selected AMF along with selected PGPR
from GBPUAT in multilocation field research trials (of wheat-legume cropping system)
in the second phase and has developed the formulation for AMF for field application.
In the third phase, TERI is continuing testing AMF & PGPR in research trials, validation
trials, and farmers’ field demonstration trials. TERI is also involved in developing joint
formulation of AMF & PGPR using its well known expertise in lyophilization.
Research Institute of Organic Agriculture
The Soil Sciences Division at the Research Institute of Organic Agriculture (FiBL)
developed its agronomic and conceptual expertise over several decades of applied
research. Since the beginning of ISCB Phase II, it acts as a project partner for
important technical and scientific questions, including regular field visits in India,
within the Biofertilizer network. FiBL develops the concepts in collaboration with the
project partners, prepares the planning documents for the field trials and organises
the compilation, validation, statistical analysis and evaluation of the pooled data
obtained from the field trials by the Indian partners.
Indian Institute of Technology, Delhi
The IIT-D group joined the Biofertilizer project in Phase II. It was jointly led by
Prof. Vikram Sahai (PI till September 2010 and Project Consultant thereafter)
and Prof Virendra S Bisaria (PI since October 2010). The group developed mass
Prof. Andres Wiemken, BIB (PI)
“The current high-input agriculture
is not sustainable worldwide. Now,
India has the chance to introduce
environmentally friendly agriculture
based on biofertilizers and mixed
cropping fostering agrobiodiversity.”
Prof. Thomas Boller, BIB (PI)
“Basic research is urgently needed to
establish reliable molecular markers for
individual biofertilizer strains in order to
apply them in sustainable agriculture.”
Dr Alok Adholeya, TERI
(PI and Indian Coordinator)
“Comparatively low yielding cropping
systems and deteriorating soil fertility
needed a biological solution, which is
now developed and amply evaluated.
A new era of biological agents is on the
offering.”
Dr Paul Mäder, FiBL
(PI and Swiss Coordinator)
“Biofertilizers…a good option for
sustainable agriculture less dependent
on limited phosphorus stocks.”
Prof. V S Bisaria,IIT-D (PI)
“Bioprocessing – a sustainable
way of life.”

Prof. Vikram Sahai
(Consultant & former PI)
“Well researched biofertilizers have
paved the way for safe and sustainable
agriculture”
Prof. Michel Aragno
(Consultant and former PI)
“The main issue of this project is not
just to have developed a product, but
to have shown the way to scientists and
agronomists in the future of biofertilizer
research. As Louis Pasteur said, ‘there
are no applied sciences, but there are
applications of science.’”
Dr Padruot Fried
(Advisor, BF Net & Member
Steering Committee, ISCB)
“Positive plant-microbe interactions will
also be found in other crops, increasing
their nutritional value and fostering
environmentally friendly production.”
Prof. B N Johri
(Consultant and former PI)
“Selection of rhizobacteria R62 and
R81 has been the hallmark of this
collaboration. What team work can do, is
the end result in the form of a product !”
Dr A K Sharma, GBPUAT (PI)
"Research on biofertilizer should
be strengthened with strong basic
understanding of functionality
of organisms. Second generation
biofertilizers having robust nature to
sustain even under stressed environments
need to be searched"
multiplication technology of PGPR strains at bench and pilot scale through submerged
cultivation in bioreactors, and also developed appropriate bio-inoculant’s formulations.
The group has modified and optimized Schlegel’s synthetic medium for cultivation of
fluorescent pseudomonads R62 and R81 to produce higher biomass and other desired
metabolites, such as siderophore and 2,4- diacetylphloroglucinol (DAPG) in shake flask
as well as in bioreactors. The group has developed a fed-batch process strategy and
process control software for enhanced production of cell mass and DAPG for the
PGPR strains. The output of one fed - batch run produces approximately twenty times
more carrier based bio-inoculant’s formulations compared to that of a conventional
batch run.
University of Neuchâtel
Phase 1: lsolation, selection, and identification of promising PGPR strains (in
collaboration with GBPUAT), based on plant-growth promoting activities; responses
of bacterial communities to the presence of AMF in the mycorhizosphere and
hyphosphere; in vitro study of interactions between the selected strains and AMF (in
collaboration with TERI); study of AMF-spores associated bacteria (in collaboration
with BIB)
Phase 2: Molecular characterization, at strain level, of the selected R62 and R81
strains (Pseudomonas jessenii, resp. P. synxantha); definition of strain-specific primers
(R62) allowing field monitoring; establishment of strain-specific PFGE (Pulsed-Field
Gel Electrophoresis); batch cultivation of both strains in synthetic media, and test of
potential C-sources (in collaboration with IIT)
Phase 3: Preparation of phase III application, taken up by UB after M Aragno’s
retirement; M Aragno continued to be associated with the programme as ISCB
consultant.
Govind Ballabh Pant University of
Agriculture and Technology
Phase I: The GBPUAT group led by Prof. B N Johri has played a key role in the
selection of PGPR strains along with UN group. The strains were identified and
characterized for their functionality and tested at a very small scale for the plant
growth promotion.
Phase II: The group led by Dr A K Sharma developed the inoculums of PGPRs and set
up the field trials at different level at farmers’ fields under rice – wheat and wheat –
pulse system both in plains and hills where the conditions are rainfed especially for
wheat – pulse system.
Phase III: AMF and PGPRs inoculum developed by TERI and IIT was further tested
in validation and demonstration trials. Genetic stability and marker development
was carried out in collaboration with UN and continued with UB. GBPUAT has also
developed formulation of PGPRs.

Achievements from Phase I (2000–2004)
Wheat field
• Collection of 3000 PGPR and 139
AMF isolates from wheat fields of
six different sites of India
• Maintenance of collected PGPR and
AMF germplasm
• Testing of AMF and PGPR for their
functional and synergistic properties
• Screening of AMF and PGPR in
greenhouse for the plant growth
promotion
• Selection of AMF (natural
consortium consisting of 4 or more
AMF species) and PGPR strains
(R62-Pseudomonas jessenii & R81-
Pseudomonas synxantha)
• Development of Root Organ
Culture and in vitro mass production
technology for AMF single strains
and consortia.
Trap culture of AMF
Root Organ Culture of AMF
single strain
In vitro mass production
technology for AMF
Testing of PGPR functions Pot trials AMF and PGPR synergism

Achievements from Phase II (2005–2008)
Multi-location and multi-year field testing
Wheat field (March 2007)
Mass production of PGPR
Black Gram field (September 2008) Rice field (October 2007)
Treatments in 4 replicates
T1 No inoculation of AMF and PGPR
T2 Natural AMF consortium produced via trap
cultures and containing at least 4 Glomus species
T3 Single Glomus species isolate obtained from
natural consortium and successfully produced invitro
T4 Pf = fluorescent Pseudomonas (R62 + R81)
T5 Natural consortium of AMF (T2) and fluorescent
Pseudomonas (T4) together
T6 Single Glomus species isolate (T3) and
fluorescent Pseudomonas (T4) together
T7 Pre-commercialized AMF by TERI produced in
vitro as reference product
Crops
Wheat Triticum aestivum (L.) variety UP2338
Rice Oryza sativa (L.) variety Pant Dhan 4
Black gram Vigna mungo (L.) Hepper,
variety Pant Urd 35
Fertilization
Zero and farmers practice
Field locations in India:
1. Ujhani, Budaun (Uttar
Pradesh)
2. Bhavanipur, Budaun (Uttar
Pradesh)
3. Salari I, Nainital
(Uttarakhand)
4. Salari II, Nainital
(Uttarakhand)
5. Shyampur, Haridwar,
(Uttarakhand)
6. Mandori, Palwal (Haryana)
• Biofertilizer growth response of crops was highest at marginal site and biofertilizers were also effective at
farmers practice fertilization.
• Yield increase of wheat after dual inoculation of AMF (Natural AMF consortium produced via trap cultures and
containing at least 4 Glomus species) and PGPR (R61+R81) was 41% (n=11).
• Biofertilizers in rice and black gram were only effective in the second year of application.
• Sesbania as a leguminous green manure plant substantially increased yields of rice and wheat (25%–27%).
• P, K, Zn, Cu, and Mn of wheat and rice and crude protein content in wheat were significantly higher after
inoculation of AMF and PGPR. This is most relevant with respect to the diet of the Indian population, since
these plant species are staple food crops.
• Fermenter based technology for mass cultivation of PGPR strains was developed.

Mean grain yield of wheat, rice, and black gram from two cropping seasons
Inoculation Wheat Rice Black Gram
LS Mean
(t/ha*)
% LS Mean
(t/ha*)
% LS Mean
(t/ha*)
T1 No inoculation of AMF and PGPR 2.36 c 100 2.90 a 100 0.307 b 100
T2 Natural AMF consortium produced via trap cultures
and containing at least 4 Glomus species
3.05 b 129 3.05 a 105 0.382 a 124
T4 Pf = fluorescent Pseudomonas (R62 + R81) 3.09 ab 131 3.15 a 109 0.357 ab 116
T5 Natural consortium of AMF (T2) and fluorescent
Pseudomonas (T4) together
T7 Pre-commercialized AMF by TERI produced in vitro
as reference product
3.35 a 141 3.14 a 108 0.345 ab 112
3.03 b 128 2.99 a 103 0.372 a 121
*Mean grain yield of wheat, rice, and black gram from two cropping seasons. Grain yield of wheat grown in 2006 and 2007
and of rice and black gram grown in 2005 and 2006 after four inoculation treatments at two fertilization levels at three to seven
locations. Calculated means of 14 harvests of wheat and six harvests each of rice and black gram.
Food quality of wheat, rice, and black gram from two cropping seasons
a) Wheat b) Rice c) Black Gram
Crude protein***
150
Crude protein*
150
Crude protein
150
Ash*** 125 P*** Ash*** 125 P*** Ash 125
P***
100 100 100
75 75 75
Mn** 50 K*** Mn** 50 K** Mn
50
K***
Zn*** Cu*** Zn** Cu* Zn
Cu
Fe** Fe Fe
Food quality as indicated by crude protein, macro- and microelement concentration of wheat, rice, and black gram
grains. a) Wheat grown in 2006 and 2007, b) rice and c) black gram grown in 2005 and 2006 after four inoculation
treatments across two fertilizer levels. Calculated means in percent (control = 100%) of four harvests of wheat and
two harvests of rice and black gram at two locations.
Reference
Paul Mäder, Franziska Kaiser, Alok Adholeya, Reena Singh, Harminder S. Uppal, Anil K. Sharma, Rashmi Srivastava, Vikram
Sahai, Michel Aragno, Andres Wiemken, Bhavdish N. Johri, Padruot M. Fried (2011), Soil Biology & Biochemistry 43, 609-619
Research trials were initiated during Phase II and terminated (after five cropping
seasons) in Phase III
Wheat yield increase by around 40% was confirmed.
Although AMF and PGPR were selected for wheat, yields was also found to be increased in rice and black gram.
Crop quality and soil quality was found to be improved with the co-inoculation of AMF & PGPR.
%
T1
T2
T4
T5
T7

Achievements from Phase III (2008–2012)
Crop rotation
Wheat – Rice, Wheat – Cotton,
Wheat – Pulse – Rice
Varieties
PBW-343, PBW-373, PBW-550, DBW-17,
HD-2258, HD-2851, HD-2894, JW-273 and
WH-711
Inputs
100% RDF*, 75% RDF*, 50% RDF* treatments
with Bioinoculants (AMF + PGPR) in
comparison with Control Plots of 100% RDF* (*
State Agricultural University’s Recommended
Dose of Chemical Fertilizer)
Findings
• Co-inoculation of AMF and PGPR at
100% RDF improved wheat grain yield
up to 12%.
• Inoculation was found to be effective in all
the tested agro-climatic regions and wheat
based crop rotations.
• No yield decline at 75% RDF (& at 50% in
some fields) was observed when
co- inoculation was applied to wheat,
compare to 100% RDF without inoculation.
Formulation
AMF has long shelf life (up to 5 years) at low
moisture (5%) at ambient temperatures
(upto 45 °C)
Since PGPR has shorter shelf life being
non-sporulating isolates especially under hot
climate which very much prevails under Indian
conditions, two techniques (flocculation and
lyophilization) have been attempted which
could sustain the shelf life of PGPRs (R62 and
R81) until 6 months without significant loss in
the cell count. The further refinement of these
leads is necessary to develop finished product
packaged appropriately for prolong shelf
life and effective delivery on seeds.
For joint formulation, two approaches were
tested extensively
• Lyophilization
• Flocculation
For further information, contact
Dr Alok Adholeya, Director
Biotechnology and Management of Bioresources Division
TERI, Darbari Seth Block, Lodhi Road
New Delhi 110 003, INDIA
Tel. +91 11 24682100 or +91 11 41504900
E-mail aloka@teri.res.in • Web www.teriin.org
Field Trial Locations in Phase III
Rajasthan
Punjab
Haryana
Madhya Pradesh
Uttar Pradesh
Bihar
Middle Gangetic Plains Region: UP, Bihar
Upper Gangetic Plains Region: UP
Trans-Gangetic Plains Region: Punjab, Haryana and Rajasthan
Central Plateau and Hills Region: MP, Rajasthan
Dr Paul Mäder, Head of Soil Sciences Division
Research Institute of Organic Agriculture (FiBL)
Ackerstrasse 21
5070 Frick, Switzerland
Tel. +41 62 865 72 32
E-mail paul.maeder@fibl.org
Web www.fibl.org