Evalution Of Endophytic Streptomyce OsiSh-2 From Rice With Potential Application For Biocontrol Of Rice Blast Disease

Rice blast, which is caused by Magnaporthe grisea, is a widespread and highly destructive disease. It affect rice production and food security seriously. At present, the most generally used methods to control rice blast are chemical fungicides and resistant breeding. However, perpetual use of these chemical agents has caused several problems such as polluting the environment and doing harm to human health. Resistant breeding may causing emergence of new physiological races of fungal strains which can rapidly overcome plant resistance. In recent years, a growing number of scientists have increased interest in searching for microbial resources to control plant disease since they are generally recognized to be more environmentally friendly, having low toxicity and lack of pathogen resistance. As a significantly important part of microbial ecosystem in plants, studies have shown that endophytes colonized interior of plants could compete with pathogens for living spaces and nutrients. In addition, the antibiotics and other bioactive substances produced by endophytes could kill the pathogens directly. Lastly, endophytes can through promote growing ability of the host plant to improved the plant disease resistance ability. In previous studies, our lab has isolated an endophytic actinomyces OsiSh-2 which has antagonistic effect on the rice blast fungus from rice, the aim of this study is to investigate the antaganistic ability of OsiSh-2 against Magnaporthe grisea, researching the bioactive substances, thus providing theoretical basis for the development and practical application of biopesticides.1 Basic training experiments showed that strain OsiSh-2 grew on 10 different agar medium, showing morphological characteristics of Streptomyces. The substrate mycelium was brown, pale yellow or yellow and the aerial mycelium was white, grey or black. Except ISP5, OsiSh-2 could produce abundant spores on the other agar medium. No soluble pigment was observed on any of the media. Its aerial hyphae was differentiated into spiral spore chains. It could use D-glucose, L-arabinose, L-rhamnose and D-fructose as sole carbon source. Catalase were produced, starch and casein were hydrolysed, melanin and H2S were not produced. The 16S rRNA sequence alignment results indicated that strain OsiSh-2 showed 100% homology similarity with Streptomyces endus. Based on its morphological、cultural、physiological and genetic characteristics, we identified OsiSh-2 as a strain of Streptomyces endus.2 The antagonistic activity of OsiSh-2 was evaluated against different physiological races of Magnaporthe grisea (Guy 11,RB3, SO7,62) by using dual plate assay method. Results showed that OsiSh-2 had strong antaganism to four target races and the best antaganism effect is against RB3 withthe maximum inhibitory rate 75.3%. Futher study showed that OsiSh-2 do not produce volatile antaganism substances, its cell-free fermentation also significantly inhibited fungal mycelia growth. SEM observations of Magnaporthe grisea hyphal structure show that compared with the control, Magnaporthe grisea exposed to OsiSh-2 showed the cellular changes in hyphal morphology including hyphal swelling and cytoplasm aggregation. These results demonstrated that certain components secreted by OsiSh-2 showed antifungal activity against Magnaporthe grisea. Field experiment results showed that when rice leaves were treated with 107CFU/mL of OsiSh-2 spore solution prior to rice blast infection, the disease severity reduced by 48.19%. Furthermore, the shoot length treated with OsiSh-2 was significantly 11.5cm higher than the untreated rice shoot length. Indicate that a certain concentration of OsiSh-2 spore solution sprayed on rice leave can control the rice blast.3 This paper have researched the bioactive substances produced by OsiSh-2. Qualitative testing found that OsiSh-2 can produce ACC deaminase, plant hormone IAA and siderophore that are related to promote plant growth and produce protease, cellulase, gelatinase and peroxidase that are related to antagonistic fungi. Methanol extract of OsiSh-2 strains, methanol and ethyl acetate extract of the PDA solid culture medium both have antagonistic effect on Magnaporthe grisea, the antagonistic effect of methanol extract is better than ethyl acetate. Methanol extract of PDA was separated into four spots by using thin-layer chromatography (TLC) techniques and only the second spot have antagonistic effects. Wavelength scanning found the second spot have maximum absorption at 200-300nm. We determined OsiSh-2 may have produced the polyether compounds. PCR test result showed OsiSh-2 genome containing nitrogen-related genes NifU, nonribosomal peptide synthesis genes NRPS and polyketide synthesis genes PKS. This indicated that OsiSh-2 may through comprehensive mechanisms such as producing various antifungal enzymes and metabolites to improve the disease resistance of rice.