Exploration of bioactive metabolites from Streptomyces as natural insecticides against Helicoverpa armigera ABSTRACT Microorganisms produce a range of metabolites which plays key roles in agriculture including pest control properties. With this concept, 15 Streptomyces strains with insecticidal activity against the lepidopteran pests such as Helicoverpa armigera, Spodoptera litura, and Chilo partellus were identified. Based on the activity units, S. griseoplanus SAI-25 and Streptomyces sp., CAI-155 were further studied to identify the key insecticidal metabolites against H. armigera. An insecticidal compound called cyclo(Trp-Phe) belongs to the diketopiperazine class was purified from SAI-25 with antifeedant (70%), larvicidal (67%), and pupicidal (59%) activity. The LD50 and LD90 values were observed to be 619 and 2750 ppm, respectively. In the context of Streptomyces sp., CAI-155, a novel fatty acid amide derivative called N-(1-(2,2-dimethyl-5-undecyl-1,3-dioxolan-4-yl)-2-hydroxyethyl)stearamide with insecticidal activity of 70–78% was identified. The LD50 and LD90 values were found to be 627 and 2276 ppm respectively. Further characterization of these metabolites in the field conditions, will bring out the potentiality of microbial metabolites for the pod borer management. INTRODUCTION Legume pod borer, H. armigera, is one of the most important lepidopteran pests worldwide affecting more than 300 species of plants. Though there are many synthetic pesticides available as control measures, their indiscriminate use have developed insect resistance. Therefore, there is a need to do research on safer alternate products for pest management, where the microbial metabolites plays key role as they constitute an infinite pool for novel secondary metabolites. Hence, the present work was aimed to identify microbial metabolites with insecticidal properties from Streptomyces strains against H. armigera. RESULTS ESI-MS spectrum and structure of cyclo(Trp-Phe) EI-MS spectrum and structure of N-(1-(2,2-dimethyl-5-undecyl-1,3-dioxolan-4-yl)-2-hydroxyethyl)stearamide Larvicidal activity of extracellular extract Molecular formula: C20H20N3O2 Molecular mass: 333.3 Molecular formula: C36H71NO4 Molecular mass: 283.3 Each bar depicts the mean (n=3). Error bar indicates SE. Larvicidal activity of purified compounds Samples (ppm) Larvicidal activity (%) Cyclo(Trp-Phe) 250 23.3±2.7f 500 39.4±5.5def 1000 67.6±3.8bc N-(1-(2,2-dimethyl-5-undecyl-1,3-dioxolan-4-yl)-2-hydroxyethyl)stearamide 24.6±2.5ef 40.6±2.2de 78.3±1.2b Azadirachtin (Standard) 54.6±4.1cd 81.9±4.3ab 97.8±2.2a DISCUSSION MATERIALS AND METHODS LD50 and LD90 values of purified compounds Two microbial metabolites with insecticidal properties on H. armigera were identified. Cyclo(Trp-Phe) from , S. griseoplanus SAI-25 is the first report for its presence and biological activity from the genus Streptomyces. N-(1-(2,2-dimethyl-5-undecyl-1,3-dioxolan-4-yl)-2-hydroxyethyl)stearamide purified from Streptomyces sp., CAI-155 is a novel metabolite with insecticidal activity This study reveal the importance of microbial metabolites in the development of biopesticide formulation. Still, the study warrants further characterization in relation to its mode of action and efficacy trails under field conditions. Compounds LD50 ppm (95% LFL-UFL) LD90 ppm (95% LFL-UFL) χ2 (df) p value Cyclo(Trp-Phe) 619 (516-776) 2750 (1765-6431) 15.9(7) 0.026* N-(1-(2,2-dimethyl-5-undecyl-1,3-dioxolan-4-yl)-2-hydroxyethyl)stearamide 627 (535-760) 2276 (1589-4271) 15.7(7) 0.027* Azadirachtin 233 (164-286) 627 (511-895) 23.9(7) 0.001* S. griseoplanus SAI-25/Streptomyces sp. CAI-155 Diaion HP-20 FTIR, 1H and 13C NMR, HR-LCMS 1. Extraction Starch Casein Broth/8 Days/28 °C Extracellular extract 2. Purification C18 open column, HPLC Silica and C18 open column, TLC 3. Identification 1D and 2D NMR: 1H, 13C, COSY, NOESY, HMBC and HSQC; EI-MS SAI-25 CAI-155 Bioactivity guided fractionation * χ2 values are significant at p < 0.05; LFL – Lower Fiducial Limits; UFL – Upper Fiducial Limits; df – degree of freedom. Values are mean±SE (n=3). Values followed by different lower-case superscript letters are significantly different (p < 0.05) by Tukey test. REFERENCES/PUBLICATIONS Vijayabharathi R, RatnaKumari B, Sathya A, Srinivas V, Rathore A, Sharma HC,Gopalakrishnan S. Biological activity of entomopathogenic actinomycetes against lepidopteran insects (Noctuidae: Lepidoptera). Canadian Journal of Plant Science (2014) 94 (4): 759-769 Sathya A, Vijayabharathi R, Kumari BR, Srinivas V, Sharma HC, Sathyadevi P, Gopalakrishnan S. Assessment of a diketopiperazine, cyclo(Trp-Phe) from Streptomyces griseoplanus SAI-25 against cotton bollworm, Helicoverpa armigera. Applied Entomology and Zoology (2016) 51:11-20 Gopalakrishnan S, Vijayabharathi R, Sathya A, Sharma HC, Srinivas V, Bhimineni RK, Gonzalez SV, Melø TM, Simic N. Insecticidal activity of a novel fatty acid amide derivative from Streptomyces species against Helicoverpa armigera. Natural Products Research (2016) 8:1-10 Leveraging legumes to combat poverty, hunger, malnutrition and environmental degradation Funding from DBT, GOI for this study is greatly acknowledged. This work was undertaken as part of the CGIAR Research Program on Grain Legumes. ICRISAT is a member of CGIAR Consortium. Identification of metabolites through the support of NTNU, Norway is also greatly acknowledged. S. Gopalakrishnan1, Vijayabharathi R1, Sathya A1, Sharma HC1, Srinivas V1, Ratna Kumari B1, Gonzalez SV2, Melø TM3, Simic N2 1 ICRISAT, Patancheru, India; 2 Department of Chemistry, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; 3 Department of Biotechnology, NTNU, Trondheim, Norway.