RPF-I

PROFORMA FOR SUBMISSION OF RESEARCH PROJECTS

PART -I: GENERAL INFORMATION

200 Project code

2001 Institute Code No CP.3.1.2

2002 ICAR Code No:

201 Name of Institute and Division:

2011 Name & Address of Institute:

2012 Name of Division/ Section:

2013 Location of Project:

202 Project Title

Indian Grassland and Fodder Research

Institute (IGFRI), Jhansi 284 003

Division of Crop Production

CP Division, IGFRI, Jhansi

Development of Phosphate Solubilizing

Biofertilizers Suitable for Fodder Crops of Problem Soils

203 Priority Area Forage production in problematic soils

2031 Research Approach:

Applied res Basic Res Process/Techno logy Transfer ofDevelopment Technology

obi 02-../ 03 04204 Specific areas Development of Phosphate Solubilizing

Biofertilizers Suitable for Fodder Crops of acid and salt affected soils

2041 Previous projects in this specific area:NONE

205 Duration: Five years

2051 Date of Start July, 2013

2052 Likely date of completion June, 2018

206 Total cost of the project: Rs. 93.65 lakhs

2061 Foreign Exchange Component (if any): Nil

207 Project profile Summary:

Out of the 157 Mha of the cultivated area 49 mha is acidic, of which 26 mha is

having pH <5.6 and the rest vary from 5.6 to 6.5 and area under salt-affected

soils is 6.73 M ha (Sodic - 3.77 Mha & Saline - 2.96 Mha). All the acid and salt

affected soils are constrained by deficient supply of soluble P. The application of

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phosphatic fertilizers In acid soils form insoluble phosphate with AI3+, Fe3+,

while in calcareous soils, form insoluble calcium phosphate compounds.

Microbial solubilization of insoluble phosphates and RP is an environmentally

mild approach. The establishment and performance of PSMs is severely affected

by environmental factors especially under stressed conditions such as high salt,

pH and temperature prevalent in degraded ecosystems like alkaline soils.

Application of phosphate solubilizing microbial (PSM) biofertilizers prepared

out of strains isolated from normal soils do not perform well in problem soils due

to stress conditions. Very little information is available on the MPS

microorganisms of problem soils especially under acid and salt-affected soils.

Hence, it is highly necessary to study the MPS microorganisms of salt-affected

and acid soils to develop mineral phosphate solubilizing biofertilizers suitable

for fodder production in acid and salt affected soils.

208 Key words:

Acid soils, salt affected soils, forage crop, Phosphate solubilizers, biofertilizers,

Mineral phosphate solubilization.

Part -II: Investigator Profile

210 Principal Investigator

2101 Name : Dr Srinivasan R

: Senior Scientist

: Division of Crop Production

: IGFRI, Jhansi

: Indian Grassland and Fodder Research Institute, Jhansi

2102 Designation

2103 Division/ Section

2104 Location

2105 Institute Address

211 Co- Investigator

2111 Name

2112 Designation

2113 Division/Section

2114 Location

2115 Institute Address

212 Co-Investigator

2121 Name

: Mr. Manoj Chaudhary

: Scientist

: Division of Crop Production

: IGFRI, Jhansi

: Indian Grassland & Fodder Research Institute, Jhansi.

: Mr. Radhakrishna A

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Application of PSB had increased seed set (8.2 - 20.0%), seed filling efficiency

(1.3 - 1.9%), kernel yield (37.3 - 67.0%) and quality by reducing hull % (15.2 -

24.3) and by improving nutrient concentrations of sunflower seeds. Besides, PSB

application caused a substantial increase in all macro- and micro-nutrients in

seeds (Ekin, 2011). Inoculating thermo-tolerant PSMs into agricultural and

animal wastes represents a practical strategy for preparing multifunctional

Biofertilizer (Chang and Yang, 2009). Identification and phylogenetic analysis of

36 PSB isolates was done by 16S rDNA sequencing. four strains viz.,

Arthrobacter ureafaciens, Phyllobacterium myrsinacearum, Rhodococcus

erythropolis and Delftia sp. were reported for the first time as PSB (Chen et al.,

2006).

The highest records of all tested parameters were recorded with rock phosphate

inoculated with Paenibacillus polymyxa plus Bacillus megaterium treatment (El-

Yazeid and Abou Aly, 2011). Seed inoculation by PSMs and different levels of

phosphorus chemical fertilizer on forage and grain quality of barley had

decreased neutral detergent fibers (NDF) (57.4%) in barley forage (Mehrvarz

and Chaichi, 2008).

Biofertilizer named BioPower has exerted a positive influence by increasing the

accumulation of K+and Ca2+ ions for minimizing the deteriorating effect ofNaCl

salinity (Chugtai et al., 2003). Another Biofertilizer, Biogene, mixed to the soil,

improved the growth, performance and chemical constituents of both salt-

stressed and unstressed soyabean plants (Moftah, 2000).

221 Project Technical profile:

2211 Organization of work elements (for objective and participating investigator

giving man-months involved):

S. Name of the % time Work to be done

No. Scientist (man

months)

8

Dr. Srinivasan R 30 (4) Overall responsibility of project planning,

implementation of technical programme and

reporting. Microbiological aspects of

complete programme.

2. Mr. Manoj 20 (2.5)

Chaudhary

Soil analytical aspects of soil samples, Pot

and field experiment and analysis of soil and

plants sample for the mentioned

observations.

Molecular characterization for identification3. Mr. 20 (2.5)

Radhakrishna A of PSB and PSF; and Elucidation of possible

mechanism of MPS activity and stress

tolerance of the selected isolate

2212 Methodology:

Different salt affected and acid soils will be collected from various parts of India and

isolation of phosphate solubilizing microorganisms will be isolated. The isolates will be

screened for their phosphate solubilizing ability by using various P sources. About 20

PSB and PSF isolates will be selected for screening for various stress tolerance abilities

like acid tolerance, salt tolerance, drought tolerance, temperature stress tolerance. The

selected isolates will be also screened for any additional plant growth promoting.

substances production potential. Selected isolates will be characterized and identified by

morphological, biochemical and molecular based methods. Attempt will also be made to

elucidate possible mechanism of MPS activity and stress tolerance of the selected

isolate. 5 efficient salt and acid tolerant PSB and PSF isolates will be selected and tested

for its effect on growth of Fodder crop in salt affected soil (Barley) and in acid soil

(Oats) under Pot culture condition.

2213 Plan of action

2214 Time schedule of Activities (Milestones):

9

SI. Activities Responsible Years*Milestones

No organization 1st 2nd 3rd 4th 5th

1 Isolation of MPS micro Literature search and project out line Crop ~

organisms from the acid ProductionCollection of soil samples and soil ~ ~

and salt affected .soils Division 0

characterization and isolation ofPSMsIGFRIfrom different regions

~ Screening the efficiency Screening for mineral phosphate ~ ~

of the isolated strains for solubilizing activity

MPS activity, stress Screening of MPS isolates for various stress ~ ~ ~

olerance and other olerancebeneficial traits Screening selected isolates for possession of ~ ~

additional PGPR traits

~ Characterization and Characterization and identification of ~ ~ ~

identification of selected selected isolates

isolates by Elucidation of possible mechanism of MPS ~ ~ ~ ~.morphological, activity and stress tolerance of the selectedbiochemical and isolatemolecular methods

4 Testing the performance Effect on plant growth ~ ~

of selected efficient

strains for productivity of iAnalysis of soil and plant samples ~ ~

forage crops In in-vitro Field experiment ~ ~

conditionAnalysis of soil and plant samples ~ ~

Reporting the results ~

* In case of the unforeseen reasons if some of the activities are left duration may be

extended to the 6th year.

2215 Annual target of each activity:

Year wise plan of activity is given below and as mentioned above

10

First year:

Isolation of MPS microorganisms

Collection of soil samples for isolation of PSM

• Salt affected soils from Haryana (Kamal), Rajasthan / M. P. (Indore), T.

N. (Coastal areas, Chennai) - about 10 samples from each location

Acid soils from Kerala / Karnataka (Western Ghats), Himachal/Greater

Himalayas, West Bengal (Sundarbans) / NEHR.

Characterization of soil samples by standard analytical methods

pH,EC,ESP

• Isolation ofPSM using Pikovskaya's (1948) medium (PSB and PSF)

Second-third year:

Continuation of first year activity

Screening for MPS activity of isolates

• In Pikovskaya's broth medium modified with different P sources following

standard method (Jackson, 1973)

• TCP

• Al P04

FeP04

• Rock Phosphate (URP)

Select a total of about 20 PSB and 20 PSF having (High, Medium and low activities)

Screening for various stress tolerance of selected PSM isolates by testing the growth in

broth medium amended with salt or acid or PEG6000

• Salt tolerance range (Low, Medium, High)

• Acid tolerance range (Low, Medium, High)

• Drought tolerance (Low, Medium, High)

• Temperature tolerance (Low, Medium, High)

Characterization and identification of selected isolates

• Morphological (colony morphology, cell morphology (Gram staining and otherstaining methods)

• Biochemical (Different sugar utilization, and standard biochemical methods asper Bergey's manual and/or FAME analysis for PSB)

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• Molecular methods (16S rDNA gene sequencing/ ARDRA for PSB and 18SrDNA gene sequencing! ARDRA / ITS gene based identification for PSF)

Screening of selected isolates for possession of additional PGPR traits if any,

Production of plant growth promoting substances like IAA, GA production using

standard methods

Elucidation of possible mechanism of MPS activity and stress tolerance of the selected

isolate

Biochemical (Chromatography / protein profiling) and/or

Molecular methods (based on identity of isolate, and its reported possible

expressed gene sequences, by PCR based method)

Fourth/fifth Year

••

Effect on plant growth

• Selection of 5 efficient salt tolerant PSB and PSF isolates for Pot cultureexperiment to test for effect on growth of Fodder crop in salt affected soil(Sorghum/Barley)

• Selection of 5 efficient acid tolerant PSB and PSF isolates for Pot cultureexperiment to test for effect on growth of Fodder crop in acid soil(Sorghum/Oats)

Treatments

Testing the performance of selected efficient strain(s) for P uptake and growth of

selected fodder crop under in vitro/in vivo condition.

In Salt affected soil - fodder crop (SorghumlBarley)

PSB-5

PSF -5

Reference culture - 2

VIC Control- 3 (RP, SSP, Absolute control)

In Acid soil - fodder crop (Sorghum/Oats)

PSB ., 5

PSF-5

Reference culture - 2

VIC Control- 3 (RP, SSP, Absolute control)

Observations

12

J

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• Soil parameters to be studied - pH, EC, CEC, ESP, Ca, Mg, Na, K, organic C,moisture content, bulk density, available and total P

• Microbial population dynamics of soil

• Plant parameters - plant height, weight, shoot length, root length, girth, drymatter, plant P

Reporting the results

2216 Estimated Man months

man months

a. Scientific 9

b. Technical 4

c. Supporting 4

Proposed Research Details

Importance of the proposed project (Gaps in Knowledge / products/ process

technology) to the Institute mandate:

Proposed project will address the issues related to performance of PSM

biofertilizers in forage production under the problem soils, an important component of

the Institute mandate. Forage production under acid and salt affected soil. conditions are

constrained by low P availability, due to high P fixation. Such factors led to high

application of costly fertilizers to get desired level of production. In the proposed project

man months

man months

attempt will be made to develop the PSM biofertilizer to increase the bioavailability of

the native soil P reserve and to exploit the use of rock phosphate as P fertilizer along

with PSM biofertilizer.

Questions Attempted to be answered:

• How to increase the bioavailability of the native soil Preserve?

• How the efficiency of P sources can be increased in forage based cropping

system?

• How to address the problem of less/non performance of Biofertilizer of normal

soil origin in problem soil?

• What is the possible mechanism of P solubilization and stress tolerance in such

organisms?

• What are the possibilities of utilizing indigenous P sources or rock phosphate to

maintain desirable P balance in soil?

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PART - V: DECLARATION

This is to certify that

the research work proposed in the scheme / project does not in any way duplicate

the work already done or being carried out in the Institute on the subject.

the same project has been / has not been submitted to any other agency (ies) for

financial support.

the investigator/ Co investigators have been fully consulted in the development

of project and have fully undertaken the responsibility to carry out the

programme as per the technical programme.

~~'~ l' \~. . \~Srinivasan R)

Principal Investigato

( adhakrishna A)Co - Investigators

~~'1\\~(Manoj Choudhary) .

Co - Investigators

Signature & Comments of theJoint Director (Research)

~~~c:\,~~\~ t

Signature & Comments of theDirector