Objectives
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Workplan
  • Objective 1. Diversity analysis and identification of Bacillus and other predominant genera from extreme conditions of salinity, drought, acidity and mangrove.
Time schedule of activities and activity milestones
S no. Objective/
activity		 Activity milestones Milestone and when to be attained Expected output Responsibility
1(2009-10) 2(2010-11) 3(2011-12) 4
1 Selection of different extreme environment for survey and collection of soil, water, sediment and plant samples. The sites proposed to be surveyed arewith regards to extreme conditions of salinity, drought, acidity and mangrove. Survey and collection of soil samples from extreme environments.       Samples from different sites will be available for isolation of Bacillus; Bacillus derived genera and other predominant genera. NBAIM, Mau Nath Bhanjan, IARI, New Delhi, NRC Groundnut, Junagarh
2 Analysis of diversity Bacillus and other predominant genera from various extreme environments Isolation of Bacillus and other predominant genera from samples collected from extreme environments of the country ?     Development of database of Bacillus and other predominant genera isolated from extreme environments. Generation of baseline information in the country. NBAIM, Mau Nath Bhanjan, IARI, New Delhi, NRC Groundnut, Junagarh
3 Morphological and cultural characterization of Bacillus and other predominat genera isolates. Isolates will be characterized morphologically and culturally.     Tentaive identification of the isolates obtained from soil samples based on enrichment techniques. NBAIM, Mau Nath Bhanjan,IARI, New Delhi
4 Isolation of genomic DNA from isolates and PCR amplification of 16S and 16-23S rDNA using universal primers. Pure genomic DNA isolation and amplified product of 16S rDNA and 16-23S rDNA sequences development. PCR amplification.
       The 16S and 16-23 S rDNA amplified products will be used to develop molecular fingerprints and for identification of isolates up to species level.
 NBAIM, Mau Nath Bhanjan, IARI, New Delhi
5 RFLP analysis of PCR amplified 16S and 16-23S rDNA gene sequences followed by phylogenetic analysis. RFLP profiles of PCR amplified 16S and 16-23S rDNA gene sequences will be generated. Development of phylogenetic trees.       Molecular fingerprints of Bacillus species, Bacillus derived genera and other predominant genera will be developed. NBAIM, Mau Nath Bhanjan, IARI, New Delhi
6 PCR -RFLP analysis of house keeping genes like rpo1 and gyrB
 Phylogenetic relationship and identification of isolates based on house keeping genes like rpo1 and gyr.B       Development of phylogenetic relationship and differentiation of closely related species other wise not distinguished through rDNA analysis. NBAIM, Mau Nath Bhanjan, IARI, New Delhi
7 Sequencing of 16S rDNA genes for identification of isolates/strains. Generation of sequence database of native isolates and identification of isolates. Submission of DNA sequences in GenBank of native isolates.Identification and distribution of species will be deciphered IARI, New Delhi,
NBAIM, Mau
AKS: A K Saxena, DOM, IARI, New Delhi; KKP: K.K. Pal, NRC Groundnut, Junagarh; RK: Rajeev Kaushik, NBAIM, Mau

  • Objective 2. To understand the mechanisms of adaptation in Bacillus and mining of relevant genes.
Time schedule of activities and activity milestones
S no. Objective/
activity		 Activity milestones Milestone and when to be attained Expected output Responsibility
1(2009-10) 2(2010-11) 3(2011-12)
1 Determination and identification of organic osmolytes sythesized de novo or accumulated by the extreme halophilic Bacillus species at different level of osmolarity Detection, quantification and identification of organic osmolytes.     Organic osmolytes mechanism of osmotolerance of extreme halophilic Bacillus species will be idetified. NRC Groundnit, Junagarh
2 Analysis of diversity Bacillus and other predominant genera from various extreme environments. Isolation of Bacillus and other predominant genera from samples collected from extreme environments of the country.     Development of database of Bacillus and other predominant genera isolated from extreme environments. Generation of baseline information in the country. NBAIM, Mau Nath Bhanjan, IARI, New Delhi, NRC Groundnut, Junagarh
3 Studying the interrelationship between organic osmolytes and inorganic ions sythesized or accumulated at different osmolarity. Studying the inter-relationship between organic compatible solutes and inorganic ions.     Inter-relationship between organic compatible solutes and inorganic ions will be established NRC Groundnit, Junagarh
4 To study whether de novo biosynthesis or accumulation of organic osmolyte is a chloride dependent process
 Studying the role of chloride on the synthesis/
accumulation of
organic compatible solutes
     Role of chloride on the synthesis/ accumulation of organic compatible solutes will be established
 NRC Groundnit, Junagarh
5 Studying the bioenergetics of the adapted strategies of osmoadaptation. Bioenergetics of inorganic ions vs organic solutes in osmotolerance.     Bioenergetically efficient strategy of osmoadaptation will be elucidated. NRC Groundnit, Junagarh
6 Studying the role of osmoprotectants in providing cross protection to high temperature tolerance in extreme halophilic Bacillus species, if any.
 Determination of the role of osmolytes in thermo-tolerance.     The role of osmolytes in providing thermotolerance, if any, will be identified. NRC Groundnit, Junagarh
7 Determination of the presence of known genes, if any, involved in biosynthesis, and accumulation of compatible solutes and inorganic ions in selected extreme halophilic Bacillus species. Detection of the presence of genes involved in biosynthesis, accumulation and transportation of osmolytes and inorganic ions.   The possible gene(s) involved in biosynthesis and accumulation of compatible solutes and inorganic ions in extreme halophilic Bacillus species will be detected. NRC Groundnit, Junagarh
8 Identification of probable genes involved in osmoregulation and transport of osmolytes in extreme halophilic Bacillus species Detection and identification of genes involved in osmoregulation and transportaion of osmolytes     Identification of genes involved in osmoregulation and transportation of osmolytes in extreme halophilic Bacillus species will be made NRC Groundnit, Junagarh
9 Diversity of the osmoregulatory and transporter genes in extreme haolphilic Bacillus species Studying diversity of osmoregulatory and transporter genes across different species of extreme halophilic Bacillus species     Diversity of the osmoregulator and transporter genes will be illustrated NRC Groundnit, Junagarh
10 Developemnt of possible knock down mutants of important osmotransporters and regulators to decipher the functionality in selected Bacillus species. Development of knock down mutants to decipher the functionality     Deciphering the functionality of the possible osmoregulator and transporter genes NRC Groundnit, Junagarh
KKP: K.K. Pal, NRC Groundnit, Junagarh

  • Objective 3. Study of the diversity of Bacillus and other predominant genera associated with plant species under extreme environments and evaluating their role as ameliorating agents for crops grown in deteriorated environments.
Time schedule of activities and activity milestones
S no. Objective/
activity		 Activity milestones Milestone and when to be attained Expected output Responsibility
1(2009-10) 2(2010-11) 3(2011-12) 4
1 Collection of rhizosphere soil from chickpea rhizosphere grown is saline-sodic soils of Eastern U.P. and isolation of Bacillus and other predominant genera. Isolation of Bacillus and other predominant genera isolates specifically from the rhizosphere of chickpea..       Development of inventory of Bacillus and other predominant genera isolates proliferating in chickpea rhizosphere under saline conditions.. NBAIM, Mau Nath Bhanjan.
2 Collection of rhizosphere soil from coconut, arecanut, cocoa and vanilla from Kerala, Dakshin Karnataka and      Maharashtra and analyzing the Bacillus spp. And other predominat genera. Isolation of Bacillus and other predominat genera isolates specifically from the rhizosphere of coconut, arecanut, cocoa and vanilla.       Development of inventory of Bacillus and other predominat genera isolates proliferating in rhizosphere of coconut, arecanut, cocoa and vanilla under acidic soil conditions. CPCRI, Kasargod.
3 Characterizing and identifying the isolates for plant growth promoting attributes like production of  IAA, GA, cytokinin, ACC deaminase, siderophore and mineral phosphate solubilization. Identification of plant growth promoting species.     A collection of crop specific specieswith potential for plant growth promotion will be available after these studies are completed.  Scientific information on the population ratio of plant growth promoting Bacillus spp possessing different mechanisms to non plant growth promoting Bacillus spp residing in the rhizosphere of coconut, arecanut, cocoa and vanilla will also be known. NBAIM, Mau Nath Bhanjan. CPCRI, Kasargod.
4 Conducting seedlings-acid soil microcosm bioassay in poly-bags and identifying the putative isolates for plant growth promotion potentials for horticultural crops.
 Selection of PGPR isolates for horticultural crops.
     Development of Microbial inoculant for horticultural crops.
 CPCRI, Kasargod
5 Analyzing the effect of rhizospheric inoculation on endophytic Bacillus for plant growth promotion activity (Horticultural crops) in acidic soils. Selection of PGPR isolates for horticultural crops.     The scientific data on the correlation between rhzospheric and endophytic Bacillus spp in promoting the growth of seedlings will also be known. CPCRI, Kasargod
6 To evaluate the influence of selected salt tolerant PGPR along with Rhizobium specific to chickpea on growth and nodulation of chickpea under green house conditions.
 Selection of combination of PGPR isolates and Rhizobium for chickpea inoculation and alleviation of salt stress.       PGPR and Rhizobium combinations will be developed. NBAIM, Mau Nath Bhanjan
7 Field evaluation of combinations of PGPR and Rhizobium to alleviate the effect of salt stress on growth and yield of chickpea. Combinations of PGPR and Rhizobium for alleviating salt stress and enhanced growth and yield.   Microbe based technology for alleviation of salt stress for growth and yield of chickpea under saline conditions. NBAIM, Mau Nath Bhanjan
KKP: K.K. Pal, NRC Groundnit, Junagarh

  • Objective 4. Selection of novel strains of Bacillus thuriengiensis and other Bacillus species with insecticidal property and isolation of cry and other new insecticidal genes.

Time schedule of activities and activity milestones
S no. Objective/
activity		 Activity milestones Milestone and when to be attained Expected
output		 Responsibility
1(2009-10) 2(2010-11) 3(2011-12) 4(2012)
1 Collection of soil samples and isolation of native Bt isolates. Isolation of native strains of Bt.       Database of native Bt isolates. NRCPB, New Delhi
2 Molecular characterization of native Bt isolates and PCR screening for cry genes using specific primers PCR amplification of cry genes using specific primers       Database of Bt strains with cry genes. NRCPB, New Delhi
3 Bioassays against important pests. Identification of potent Bt against insect pests     Potent Bt isolates toxic to insect pests. Division of Entomology, IARI, New Delhi
4 Development of blends/
formulations.		 Development of blends/
formulations.
     The 16S and
16-23 S rDNA amplified products
will be used to develop molecular fingerprints and for identification of isolates up to
species level.
 NBAIM, Mau Nath Bhanjan, IARI, New Delhi
5 Development of fermentation technology. Fermentation technology standardized.     Standardized protocols for fermentation. Division of Entomology, IARI, New Delhi
6 Development of formulation technology
 Formulations developed       Novel effective formulations. NBAIM, Mau Nath Bhanjan, IARI, New Delhi
7 Isolation and cloning of cry genes.

1. Purification of PCR amplified cry genes.

2. Cloning of genes in suitable vector for sequencing identification of isolates.		   Cloned cry genes from native isolates. NRCPB, New Delhi
8 Sequence analysis of these genes 1. DNA sequencing of cloned genes.       Database of sequences of cry genes. NRCPB, New Delhi
9 Cloning of variant genes into expression vector.

1. Alignmnet of sequences.
2. BLAST search to look for homologies in the NCBI database.

3. Selection of variants.		       Cloned cry genes variants in expression vector NRCPB, New Delhi
10 Expression of cry proteins in E. coli and SDS-PAGE analysis of the expressed proteins. SDS-PAGE profiles with expressed proteins       Identification of new cry genes that can be used for developing transgenics. NRCPB, New Delhi
11 Evaluation of toxicity of proteins towards H. armigera Insect assay to look for toxicity of crystal protein       Confirmation of virulence of cloned genes. NRCPB, New Delhi

SK: Sarvjeet Kaur, NRCPB, New Delhi
GTG: G. T. Gujar, Division of Entomology, IARI, New Delhi –110012.