4 th Asian PGPR Conference, May 3-6, 2015; Hanoi, Vietnam

Transcription

1 Streptomyces flocculus (RP1A-12): A Potential Actinomycete against Groundnut Stem Rot Disease (Sclerotium rolfsii) Simi Jacob and H. Sudini Groundnut Pathology, Research Program-Grain Legumes, ICRISAT, India 4 th Asian PGPR Conference, May 3-6, 2015; Hanoi, Vietnam

2 Groundnut Important food legume in >100 countries 96% of its area in Asia & Africa Soilborne diseases as major bottlenecks Stem rot, Collar rot, Sclerotinia blight, & Bacterial wilt

3 Why difficult to manage: Key issues: Suppressive & Conducive soils Population dynamics of pathogens Impact of climate change

4 STEM ROT/White mold/southern blight Sclerotium rolfsii 25% annual yield loss. Sometimes reaches 80-90%

5 Rationale: Crop Rotation & Host range Resistance in cultivars Resource poor farmers Hypotheses: Actinomycetes as viable alternatives to fungicides Specific metabolite producers are effective on long term basis

6 Immediate Goals Potential actinomycete against S. rolfsii Establish its efficacy in GH & Field Identification of specific secondary metabolite Long term goal Metabolite based actinomycete formulation X stem rot

7 METHODOLOGY ACTINOMYCETES: rhizosphere ANTIFUNGAL ASSAYS: Organism based Dual culture assay (108 isolates) Metabolite based Culture filtrate (10 isolates ; 12.5%, 15% and 50%) Crude extract (5 isolates ; 1%) BIOCHEMICAL ASSAYS (5 isolates) IAA, Siderophore, lipase, protease, HCN, chitinase β-1,3 glucanase & phosphate solubilization PATHOGENICITY FACTOR: (RP1A-12) Reduction in Oxalic acid production of S. rolfsii Only crude extracts used (0.5% and 1%)

8 METHODOLOGY GH & FIELD EVALUATION (RP1A-12) Talc formulation of whole 10 8 CFU ml -1 CE mixed in water (1:100)

9 METHODOLOGY MOLECULAR CHARACTERIZATION (RP1A-12) 16S rdna gene sequencing BLAST analysis and phylogenetic tree construction SECONDARY METABOLITE PURIFICATION Silica gel flash column chromatography Bioassay of fractions HPLC on the fractions

10 RESULTS Dual culture assays: Ten isolates effective RP1A-10 RP1A-12 RP1A-15 RCE-10 RP9A-8 CONTROL

11 Culture Filtrate Assays Actinomycetes % Inhibition of S rolfsii & Culture filtrate concentration (12.5%) (25%) (50%) RCE RCE RCE RP1A RP1A RP1A RP9A RP9A RP9A RP9A LSD (5%)

12 Crude extract assay

13 IAA production IAA (µg/ml) RCE-10 RP1A-10 RP9A-8 RP1A-12 RP1A-15 Streptomyces isolates

14 BIOCHEMICAL STUDIES STRAINS* +ve for production RCE-10 Lipase, HCN RP1A-12 Lipase, HCN, siderophore RP1A-10 Lipase, HCN, siderophore RP1A-15 Lipase, HCN RP9A-8 Lipase * ve for protease, chitinase, β1-3, glucanase, phosphate solubilization

15 Oxalic acid

16 Greenhouse Studies Treatments Disease Severity Yield (g/pot) Shoot length (cm) Root length (cm) Vigor index (ST ) T f (ST) C f SA T f SA C f (ST+SA) T f (ST+SA) C f ml/l Inoculated control Trichoderma viride Uninoculate Control LSD (5%) ST- Seed treatments; SA- Soil application T f Talc formulations; C f Crude formulations

17 A B C D ST (RP1A-12); ST (crude extract) Inoculated Healthy control control

18 Field studies Treatment Disease severity Yield (kg/ha) % Mortality ST T f ST C f SA T f SA C f ST+SA T f ST+SA C f ml/l Inoculated control Trichoderma viride Untreated control LSD (P 0.05) ST- Seed treatments; SA- Soil application; T f Talc formulations; C f Crude formulations RP1A-12 strain

19 Molecular characterization RP1A-12 (Streptomyces flocculus)

20 METABOLITE PURIFICATION: Bioassay of fractions of RP1A-12 on S. rolfsii PURE FRACTIONS

21 METABOLITE PURIFICATION: HPLC chromatogram of Fraction I with Retention time min

22 METABOLITE PURIFICATION: HPLC chromatogram of Fraction II with Retention time min

23 METABOLITE PURIFICATION: HPLC chromatogram of Fraction IIIwith Retention time min and 20.01min

24 CONCLUSIONS Crude Extract of RP1A-12 is antagonistic to S. rolfsii RP1A-12 effective in GH & Field as whole organism Crude extracts alone of RP1A-12 also effective Streptomyces flocculus (RP1A-12) has potential to reduce Stem Rot In process: Elucidation of antifungal compound

25 Way Forward Characterization of antibiotic producing genes of RP1A-12 Metabolite based formulation of RP1A-12 Acknowledgements Thank you! BIRAC, Govt. of India for providing financial support under Biotechnology Industry Partnership Program (BIPP) Sri Biotech Laboratories, Hyderabad, India