Characterization of Streptomyces sp Characterization of Streptomyces sp. and its antagonistic activity against Pyricularia oryzae causing rice blast Hayman kaka khan Awla1, Jugah Kadir1, Radziah Othman2,3 and , Wong Mui Yun1,2*     1Faculty of Agriculture, Department of Plant Protection, 2Institute of Tropical Agriculture Department of Plant Protection, 3 Faculty of Agriculture, Department of land management, Universiti Putra Malaysia, 43400 (UPM) Serdang, Selangor, Malaysia.   *Corresponding author email: muiyun@upm.edu.my Blast disease caused by Pyricularia oryzae is a major fungal disease of rice. To reduce the usage of chemicals, an alternative sustainable control method is necessary. Actinomycetes have been known to produce antimicrobial compounds. The aim of this study was to screen for bioactive compounds from an actinomycete isolated from healthy paddy rhizosphere taken from Tanjung Karang Selangor in 2014. Morphological characterization, 16S rDNA gene sequencing and phylogenetic analysis suggested that the isolate, UPM-RS4, belongs to Streptomyces sp. Preliminary screening using dual culture assay revealed that the isolate highly inhibited the mycelial growth of P. oryzae in vitro with 98.32% PIRG. Ethyl acetate (EtOAc) was the best extraction solvent for the isolation of the antimicrobial compounds and 100 µg/ml of crude extract was found to inhibit 99% of the mycelial growth of P. oryzae using well diffusion method. Biochemical characterization indicated that isolate UPM-RS4 was positive for ammonia, urea and sidrophore production, and cellullase, amylase, protease and lipase lytic enzymes activities test which are involved in plant growth promotion. GC-MS chromatogram analysis showed that isolate UPM-RS4 produced 22 compounds with five high peaks identified as glycyl-L-proline, N-Valery l,1,4-dione, ergotaman-3,6,18-trion, benzyl-hexahdropyrrolo which were reported to be antimicrobial compounds. These results suggest that Streptomyces sp. UPM-RS4 could be a potential bio-control agent to be used for the development of a bio-fungicide against rice blast disease. ABSTRACT RESULTS INTRODUCTION Rice blast caused by Pyricularia oryzae Cav. (Kato, 2001) is one of the most important fungal diseases on rice that may result in apparent yield loss. The yield losses resulted from the outbreak of blast disease varied according to the geographical condition. The disease may causes the yield loss up to 100% (Kato, 2001) in Japan, up to 70% in China, and varied from 21% to 37% in Bali and Indonesia (Suprapta, 2012). Biological control of fungal diseases by an applied bacterial inoculum is now seen as a viable method of controlling some diseases and several commercial biological control agents are available. There are several mechanism by which biological control is thought to work including the production of antifungal antibiotics, competition for nutrients and rhizosphere colonization (Saikia,2008). Streptomycetes are Gram-positive aerobic members of the order Actinomycetales within the classis Actinobacteria (Stackebrandt et al., 1997). Streptomycetes are considered as one of the important bacteria due to their ability to produce several substances as secondary metabolites, found to be effective in the control of some plant pathogens (Mohamed Sonya, et al., 2012). Figure 1: Blast Symptoms Figure 2: P. oryzae culture on PDA Figure 3: UPMRS4 strain under SEM Figure 4: Inhibition growth by ethyl acetate crude extract using well diffusion method Figure 5: phylogenetic tree of Streptomyces sp. using MEGA6. Characterizes UPMRS4 Chitinase + Glucanase Cellulose Protase Phosphate solabization -- Sidrophor Ammonia production IAA Urea ++ Amylase HCN Nitrogen production METHODOLOGY Isolated from healthy paddy rhizosphere 16s rDNA Identification Biochemical characterizations (Remya&Viiayakuma,2008) Antagonistic essay in vitro GC-MS Analysis Ara(2012) Rotary Evaporation Crude extraction Streptomyces sp. Table 1: biochemical characteristics of Streptomyces sp. (+ positive result, - negative result) reaction. Figure 6: Mycelium Growth Inhibition by UPMRS4 strain using dual culture method Benzylhexahdropyrrolo Ergotaman-3,6,18-trion Glycyl-L- proline 1,4-dione N- Valeryl Mohamed Sonya, H., Omran, W., Shimaa, M. A.-S., Al-Shehri, A., & Sadik, A. (2012). Isolation and identification of some halotolerant actinomycetes having antagonistic activities against some plant pathogens (ie, Tobacco mosaic virus, Aspergillus sp., Fusarium sp.) from soil of Taif Governorate, KSA. Pakistan Journal of Biotechnology, 9(1), 1-12. Ara, I., Bukhari, N., Perveen, K., & Bakir, M. (2012). Antifungal activity of some actinomycetes isolated from Riyadh soil, Saudi Arabia: An evaluation for their ability to control Alternaria caused tomato blight in green house pot trial. African Journal of Agricultural Research, 7(13), 2042-2050. Saikia, R.(2008). Microbial Biotechnology (p.422) New India publishing Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491. REFERENCES Figure 7: GC-MS chromatogram of the ethyl acetate crude extract of strain UPMRS4. Showed that 22 compounds available with 5 high peaks active compounds probably are responsible of inhibition of the growth of P. oryzae . In the present study, it was demonstrated that paddy rhizosphere is a rich source of Streptomycetes. Streptomyces sp. had the ability to exhibit antifungal activity in vitro against hyphal growth of P. oryzae. Streptomyces sp. was found to produce ammonia, cellulose, amylase, urea, lipase, and siderophore. However, According to GCMS active strain (UPMRS4) can produce many compounds which are antifungal compounds. further studies are necessary to purify and characterize the bioactive compounds produced by this strain for the biocontrol of pathogenic fungi which then might be useful in agricultural practice. CONCLUSION Acknowledgements : This study was partially supported by Fundamental Research Grant Scheme (FRGS) provided by the Ministry of Education, Malaysia. We thank the technical staff of the Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia for their excellent technical and sampling assistance.