PGPR in Groundnut: Opportunities and Challenges

1. PGPR in Groundnut: Opportunities and Challenges Hari K. Sudini1, C.L.L. Gowda1, and M.S.Reddy2 International Crops Research Institute for the Semi Arid Tropics, India Department of Entomology and Plant Pathology, Auburn University, USA

2. Top five producers of Groundnut and their important constraints A. flavus infection and subsequent aflatoxin contamination is a worldwide qualitative issue

3. PGPR Growth Promotion Mechanisms in Groundnut Direct Indirect Production of plant growth regulators (Kishore et al., 2005, Dey et al. 2004) Prevent the deleterious effect of phytopathogens (Anjaiah et al. 2006, Abd-Allah and El Didamony 2007) Nitrogen fixation (NPR, Kloepper et al., 1991) ISR (Priming) Positive-Maria Laura Tonelli et al. 2011 Negative-Zhang et al. 2001 Uptake of minerals (Increase in available P, Dey et al 2004) Root colonization (Kishore et al. 2005)

4. Groundnut PGPR Research @ ICRISAT • Biocontrol-PGPR • Pre-harvest aflatoxin contamination in Groundnut • fluorescent Pseudomonads and Bacillus strains (Anjaiah et al., 2006) • Late leaf spot management • Chitinolytic bacteria (Kishore et al., 2005) • Chlorothalonil-tolerant endophytic isolates of Pseudomonas aeruginosa (Kishore et al., 2005) • Collar rot disease management • Pseudomonas aeruginosa GSE 18 (Kishore et al., 2005)

5. Opportunities/Thrust areas for Groundnut PGPR Research Microbial Ecology-PGPR Metagenomics-PGPR Climate Change-PGPR • Low-input agricultural production systems • Intensive agricultural production systems • BNF • Interaction of Rhizobial and PGPR strains

6. MicrobialEcology-PGPR Cultivation practices Soil type PGPR communities Plant community structure

7. Methods to assess PGPR community structure and diversity Low-resolution techniques Intermediate-resolution High-resolution • Bacterial counts/plating • methods • Microbial biomass • measurements • Community-level • physiological profiling • (CLPP) • Phospholipid fatty acid • analysis (PLFA) • Fatty acid methyl ester • analysis (FAME) • DGGE/TGGE • SSCP • T-RFLP • RISA/ARISA • ARDRA & T-ARDRA • Cloning & Sequencing • Metagenomics • Taxonomic microarrays These techniques have their own limitations but the information obtained from complex environmental samples is more important.

8. Microbial Ecology in Groundnut Ecosystems with reference to PGPR • Soil genomic DNA from rhizosphere samples • PCR amplification with universal bacterial specific primers (ITSF & ITSR eub) • PCR amplicons were run on the NEN Global Edition Li-Cor 4300 DNA Sequencer • Results • Similarities up to (90%) between profiles of same cropping sequence • Time of sampling has more profound effect on bacterial community groupings • Similarities and dissimilarities are attributed to crop diversity and time of sampling Phytopathology. 2011 Jul;101(7):819-27. Exploring soil bacterial communities in different peanut-cropping sequences using multiple molecular approaches. Sudini H, Liles MR, Arias CR, Bowen KL, Huettel RN.

9. Microbial Ecology in Groundnut Ecosystems with reference to PGPR • PCR-Cloning and Sequencing • 16S rRNA library construction • PCR amplification of genomic DNA • Universal bacterial primers (27F and 1492R) • PCR product purification by Centrifugation (Wizard SV Gel and PCR Clean-Up System) • Ligated into a TOPO-TA pCR2.1 vector • Transformation into E. coli and were grown on LB agar plates containing 25 microgram per ml kanamycin

10. Microbial Ecology in Groundnut Ecosystems with reference to PGPR • 16S rRNA gene Sequencing • 288 different rRNA genes from each soil sample were sequenced using Rolling Circle Amplification of plasmid DNA and unidirectional sequencing with a vector primer (Symbio Corp., CA) • Divisional level allocation was annotated based upon BLASTn analysis with the nr/nt GenBank database

11. PGPR Ecology Divisional level affiliation of bacterial groups in Groundnut ecosystems (Sudini et al.,2011)

12. PGPR Ecology Bacterial divisions in peanut rotation sequences (Sudini et al., 2011)

13. PGPR Ecology Correlations between bacterial divisions (Sudini et al., 2011)

14. Metagenomics-PGPR • Advances in • Environmental DNA extraction protocols • Metagenomic library construction • High-throughput sequencing methods • Possible contributions to PGPR • The discovery of novel PGPR genes and gene products • The characterization of not-yet cultivable PGPRs Susannah Green Tringe and Edward M. Rubin, 2005

15. Expected benefits with reference to Groundnut rhizosphere PGPR Identify biologically different PGPR communities associated with Groundnut ecosystems Insights into “inaccessible” PGPR Factors responsible for disease suppressive soils

16. Climate Change-PGPR • Increased burden of arid and semi-arid land areas • Elevated temperature, CO2, Erratic weather, floods, droughts • PGPR could be a viable option • Concept proved in model plant Arabidopsis (Cohen et al., 2008) • Induction of plant tolerance to semi-arid environments • Stress relievers-PGPR

17. PGPR modify plant hormonal contents in order to increase drought tolerance R. Aroca and J.M. Ruiz-Lozano, 2009

18. Interaction of Rhizobial and PGPR strains • Important area of research • Co-inoculation (PGPR strains + Rhizobia) • Nodulation promoting rhizobacteria (NPR, Kloepper et al., 1991) • Growth enhancement (Madhaiyan et al 2006, Vikram et al 2007) • Exact mechanisms still need to be explored

19. Challenges for Groundnut PGPR Research • Contradictory reports on Induced Systemic Resistance • Interaction between associative PGPR and Groundnut unstable • Reproducibility obstacles between in vitro and field studies • Influence of environmental factors

20. Way Forward/Future Research • Better understanding of Plant-PGPR interactions • Insight into PGPR mode of actions and adaptability to extreme environmental conditions • Ways and means for better utilization of PGPR in the farmers fields • PGPR & Omics approaches • Translational Research on PGPR: From “Omics to the Field”

21. Thank you