Inhibitory Mechanism Of Bacillus Tequilensis GYLH-1 On Magnaporthe Oryzae And Isolation Of Secondary Metabolites

Rice blast is one of the three major diseases of rice,which seriously affects the sustainable development of rice industry and poses a major threat to global grain yield and safety.At present,cultivation of resistant varieties and application of chemical pesticides are the main methods to control rice blast.Because Magnaporthe oryzae is easy to mutate,the cultivation period of resistant rice is too long,and chemical pesticides are seriously harmful to human health and the environment,it is very important to find effective measures to prevent and control rice blast for food security.Numerous studies have shown that Bacillus has good bacteriostasis and the ability to promote plant growth,and has been widely used in the field of biological control.In this study,Bacillus tequilensis GYLH-1 was used as the research material and rice blast was used as the target disease to study the inhibition mechanism of B.tequilensis GYLH-1 on M.oryzae Guy11,including the inhibition of hypha,conidia,germination of germ tube and formation of attachment spore.The control effect of GYLH-1 strain on rice blast was evaluated through in vitro leaf experiment and field experiment.The bacteriostatic substances in the fermentation broth of strain GYLH-1 were preliminarily investigated,and it was expected that new substances with anti-M.oryzae activity would be isolated from GYLH-1 strain.The inhibitory effect of fermentation broth of strain GYLH-1 on M.oryzae Guy11 was investigated.Through scanning electron microscope,it was found that GYLH-1 fermentation broth could affect the morphology of Guy11 hypha,causing wrinkles,deformities,swelling and shrinkage.When the fermentation broth was added to the culture broth of M.oryzae Guy11,the macromolecular contents of DNA and RNA in the culture broth of M.oryzae increased and the content of soluble sugar increased,which indicated thatcertain antibacterial substances produced by Strain GYLH-1 damaged the cell membrane of M.oryzae Guy11,causing the leakage of substances in the bacteria,thus causing the death of the bacteria.FDA-PI staining confirmed that GYLH-1 fermentation broth can destroy hypha cell membrane and cause cell membrane damage.By observing the morphology of conidia of M.oryzae Guy11-GFP,it was found that strain GYLH-1 had significant inhibitory effect on conidia germination and attachment sporulation,and changed the growth state of conidia.In addition,GYLH-1 fermentation broth contains some bacteriostatic substance which has teratogenic effect on conidial bud tube,and the bud tube malformation cannot normally form attachment spore.In vitro leaf experiment proved that the conidia infectivity treated with GYLH-1fermentation broth was significantly lower than that of the control group,thus achieving the purpose of controlling M.oryzae.In vitro leaf experiment shows that strain GYLH-1 has strong control effect on rice blast,and the control effect of bacterial liquid is better than that of fermentation liquid,and the prevention effect is better than the treatment effect.On the premise that the fermentation liquid and bacterial liquid of Strain GYLH-1 have stable and high activity,spraying the fermentation liquid and bacterial liquid to treat rice leaves shows better control effect on leaf blast.Spraying 1X fermentation liquid and bacterial liquid has the best effect,effectively reducing the incidence of rice blast,and is similar to the control effect of 0.1mg/mL carbendazim.In addition,research also proves that the prevention effect of strain GYLH-1 is better than the treatment effect.It may be due to preferential spraying of strain GYLH-1,which colonizes a large number of rice leaves,occupies favorable ecological sites,forms a competitive effect with M.oryzae,and further prevents the infection of M.oryzae on leaves.The active substance was separated and purified by polarity detection,column chromatography and high performance liquid chromatography.Experiments show that the active part is a strong polar substance,which is easily soluble in polar solutions such as methanol and water.The active ingredient is further separated from magazines by column chromatography.Finally,the collected active part is separated and purified again by high performance liquid chromatography.Finally,we obtained a pure substance with strong activity,and its molecular weight was about 475.32 through mass spectrometry analysis.