MHRD-FAST-CoE on MICROBES TO FEED THE WORLD : Plant-Microbe interactions to boost Agricultural Production Our AIM • To identify and understand the significance of microbiome of whole plant by Omic approaches • To characterize functional capacity of microbiomes- proteins, small molecules, and volatile compounds involved in plant - microbe(s) interactions for plant health • To study these interactions on a molecular level and in real time and space, to determine the optimal community for plant productivity. • Field testing, evaluation of microbial inoculants, and outreach activities • Capacity building for faculty and research scholars on Proteomics, Quorum sensing, LC-MS, Pyrosequencing, Biosafety, etc Our TEAM Deliverable:
Progress made • Defense priming by • Protein and peptides-Flg22 • Translocator proteins-HpaG, HrpN • PGPR induced ISR activity • Metabolite induced plant nutrition by • ZSB for plant uptake of Zinc and fortification • Microbial gluconate based formulation for plant nutrition • Disease protection by • Fungal endophytes (Trichodermalongibrachiatum) • Bacillus amyloliquifaciensSR1 • Drought protection by • Bacillus megatheriumFD48
DEFENSE PRIMING Development of immunity and stress tolerance to plants Effector Proteins Plant Activating Proteins Identification & Development of protein product for sustained plant health Amplification of Gene(s) of interest SecretomeanalysisGrowthof microbes with & without inducer (M9, 0.5XM9, MME/XCM, XVM2, HIM) 1D/2D & MALDI analysis Xaflg22 (69bp) 150 Induced UI M 100 1 2 M Differential protein analysis MALDI-ToF 75 Expression of HpaG in Induced(I) cells. Uninduced (UI) as control. Colony PCR hpaG U1 50 37 I1 1 kb HpaG HR response npr1 transcript analysis Gene cloning & Protein Expression in E.coli I2 XahpaG Ra hrpZ (400bp) (230bp) flg22 hrpN (800bp) XaFlg22 2 XaFlg22 4MNA Homology modeling and interaction of fliC/flg22 with FLS2 1 Proteomics study of plant activating proteins Genome based method for obtaining microbial effector Bio-formulation &Testing on plants Sivakumar & team
DEFENSE PRIMING… PAPM-Plant Activating Proteins from Microbes Gram negative plant pathogens secrete proteins (Flagellin, hrp,etc) Growth response 1 2 Hypersensitive response Identify HR eliciting protein bacteria Nutrient uptake Photosynthesis Vigour • Improved quality • Increased yield • Enhanced shelf life (Flg22)Elicitor Plant reaction Identify HR inducing plant receptor Stress defense response FLS2 (Receptor) 24 h after infiltration 1-buffer and 2- protein Protein infiltration 1 Environmental-drought Physiological –ROS scavenging Pathogen –disease suppression 2
DEFENSE PRIMING… PGPR induced systemic resistance in rice • Xanthomonas oryzae pv oryzae inoculation triggered the SA-dependent genes, [OsPAL, OSNPR1] while the PGPR, Pseudomonas fluorescens (Pf1) altered JA-responsive genes such as OsLOX, OsAOS. • Both the strains induced OsPR1a, OsPR1b and OsPR10. • Azospirillum and PPFM have less influence on the expression of the defense genes and the rice plants inoculated with these strains were expressed at par with uninoculated control. Shobana et al. (2016) Arch. Microbiol.
METABOLITE INDUCED PLANT NUTRITION Microbial gluconate based formulation for mineral nutrition of crop plants HPLC spectrum of Crude broth • Zinc solubilization by the bacterial isolate • Media : Bunt and Rivera media • Zinc source: Zinc oxide, Zinc carbonate • And Zinc phosphate • The bacterial isolate capable of • synthesizing gluconic and acetic acid Gluconic acid Acetic acid GC-MS chromatograph indicating presence of gluconic acid in the form of gluconolactone Conversion of gluconic acid into metal gluconate by addition of potassium, sodium/ammonium carbonate HPLC chromatogram of potassium gluconate Gluconic acid M-1 Peak Potassium gluconate HPLC chromatogram of ammonium gluconate Gluconic acid HPLC chromatogram of sodium gluconate Ammonium gluconate Gluconic acid Sodium gluconate • To study the stability of metal gluconates converted from microbial derived gluconic acid and testing in crop trials Jayvel and Marimuthu , 2016
METABOLITE INDUCED PLANT NUTRITION… Zinc solubilizing bacteria,Enterobacter cloacae ZSB14 on expression of Zn transporters of rice Root ZIP transporters Shoot ZIP transporters • Fe sufficiency significantly reduced the root and shoot OsZIP1 expression, but not the OsZIP4 and OsZIP5 levels. • Zinc deficient conditions (no-Zn, zinc oxide alone controls) up-regulated all the assessed ZIP genes in root and shoot of rice seedlings. • When ZSB was inoculated along with insoluble zinc oxide in the growth medium, the expression of root and shoot OsZIP1, OsZIP4 and OsZIP5 was reduced. • In the absence of zinc oxide, ZSB inoculation controlled the OsZIP1 expression but not the OsZIP4 and OsZIP5. Leaf chlorosis Krithika & Balachandar (2016) Frontiers in Plant Sciences
DROUGHT PROTECTION Multifunctional Bacillus megatherium GA 2.38 µg/ml • Bacillus megatherium, a phyllosphere bacterium • Plant growth hormone IAA, GA & Cytokinin • Drought tolerance to plants-accD (Acc deaminase) • Antagonistic to fungal pathogens IAA Inverted plate accD Control Rhizoctonia solani Sharmila et al.,2016.Archives of Microbiol.
Bacillus megatherium;growth, improved seed germination, phytohormone regulation under drought stressed condition Cytokinin bioassay with cucumber cotyledon Drought tolerance by isolates (OD at 660 nm) Quantitative estimation of IAA,Gibberellin and Cytokinin Effect of PEG induced drought stress on germination of rice seeds after 72 hrs of germination Cytokinin FD48 showed 90% germination under 20% of PEG 1.03 µg/ml 1 2 3 4 1. Control 2.FD48 3.FS20 4.MD02 FD48 treated seedlings showed more tolerance to drought when compared to control and other treatments. Ashwathy and Srithar, 2017.
DISEASE PROTECTION Fungal Endophytes from rice as Phytoprotectant • EF 1 - Paecilomycestenuis • EF 2 - Talaromyces sp. • EF 3 - Nigrosporaoryzae • EF 4 - Nigrosporasphaerica • EF 5 - Trichodermalongibrachiatum • EF 6 and EF 7 3.0 1.5 1000 500 EF1 EF2 EF3 EF4 EF5 EF6 EF7 Seven fungal endophytes were isolated from rice leaves Growth curve of fungal endophytes Antagonistic activity – Dual plate screening T. longibrachiatum Antagonistic activity R. solaniPythium sp. Xanthomonas sp. Efiiciency of T. longibrachiatum FTIR profile of T. longibrachiatumagainst fungal pathogens Inverted plate assay for volatiles - T. longibrachiatum Inverted plate T. longibrachiatum R. solani M. phaseolina T. longibrachiatum + R. solani T. longibrachiatum + M. phaseolina Growth in inverted plate Inverted plate control Growth in control plate M. phaseolinaR. solaniPythiumsp. T.longibrachiatum Archana and Sivakumar (2016)
DISEASE PROTECTION… Inhibition of sclerotium formation in Sclerotiumrolfsii • Formulation of elite Bacillus isolate exhibiting antifungal activity F. oxysporum M. phaseolina R. solani C. falcatum 1 2 3 4 Inhibition of resting spore formation in Rhizoctonia solani • Characterization of surfactant property Haemolytic activity Antimicrobial activity Oil spreading activity 1 2 3 4 • 1- Bacillus isolate showing inhibition of both growth and sclerotioum 2-4 – Other isolates showing growth inhibition alone FTIR profile of Bacillus indicating presence of surfactin HPLC profile of Bacillus amyloliquifaciens indicating presence of surfactin C15- Surfactin C14- Surfactin C13- Surfactin C=O CH3, alkyl chains Peptide group Jayvel and Marimuthu , 2016
Salient findings • BacillusamyloliquifaciesnSR1 . possessing antimicrobial activity against fungal plant pathogens viz., Fusariumoxysporum, Rhizoctoniasolani, Macrophominaphaseolina, Colletotrichumfalcatum was isolated- Lipopeptides with antimicrobial action against plant pathogens are being identified • MultifinctionalBacillus megatheriumFD48 isreported to give drought protection and disease protection to the crop plants. • Mechanism of Zinc nutrition (by Enterobactorsp., Bacillus sp.) in rice for enhanced plant growth and nutrient solubilization by gluconate synthesis by microbes. • Proteome of bacterial secretome yielded a Plant Activating Proteins for defence priming identified (flg22), HpaG and interaction studies with plant receptor FLS2 is under progress • Fungal endophytes (Trichodermalongibrachiatum) is effective against (M. phaseolina, R. Solani, F. oxysporum, C. falcatumwith highest growth inhibition of 82.35, 64.71 and 74.94, 71.40 per cent, respectively.) by exudate secretion and volatiles as well.
Future programme of work • Plant Activating Proteins from Microbes (flg22)- Secretome & Metaproteome: Proteins on Plant signaling and multi-functions • hrp based protein and plant hormone synthesis by PGPM and their role on plant health • Metabolite profiling of drought tolerant crops as influenced by microbes (Bacillus megatherium, Methylobacterium) • Plant defense by ISR and hormonal regulation (SA, JA, Ub)- PGPR induced systemic resistance in rice - PGPM-Phytopathogen-Plant interactions • Rhizobia-nonrhizobial endophytes interactions for symbiotic efficiency and crop productivity (Blackgram) PGPR-and metabolite induced rice plant with consonant levels of immunity against pathogens, tolerance to drought and nutrient acquisition