The Exploration Of The Biocontrol Mechanism Of Brevibacillus Laterosporus SN19-1 Against Rice Bacterial Leaf Blight

Rice bacterial leaf blight is a type of bacterial disease that causes serious harm to rice.Currently,the traditional prevention and control methods are the application of chemical pesticides and the selection of disease-resistant varieties.However,chemical pesticides will not only cause resistance to pathogens,but also damage the ecological environment,and it is easy to remain on rice,causing food safety issues.Rice disease resistant varieties are prone to losing their resistance with the variation of pathogenic bacteria,so it is imperative to research and develop safer,more environmentally friendly,and more efficient bio-control methods.We have isolated a strain of Brevibacillus laterosporus SN19-1 which has good antagonistic effects on various plant pathogenic bacteria and significant control effects on tomato bacterial wilt.We have completed the optimization of fermentation processes for biomass and spore rate and the establishment of microcapsule preparation processes.Whole-genome sequencing of this strain was also completed,which laid a solid foundation for its industrial application as a biocontrol agent.In order to further expand the biocontrol potential of B.laterosporus SN19-1,this study conducted a study on the control effect of B.laterosporus SN19-1 on rice bacterial leaf blight and explored its mechanism of inhibiting Xanthomonas oryzae pv.oryzae(Xoo).Firstly,it was found that B.laterosporus SN19-1 has excellent antagonistic effects against various pathogenic fungi and bacteria by plate antagonism method.The antibacterial spectrum of B.laterosporus SN19-1 was determined,and its inhibition rate against the target pathogen Xoo was as high as 75.56%.We have also found that B.laterosporus SN19-1 can produce various hydrolytic enzymes such as cellulase and protease,and has functions such as organic phosphorus hydrolysis,siderophore production,and IAA production,but it lacks the ability to hydrolyze inorganic phosphorus and produce ACC deaminase.The sterile supernatant of B.laterosporus SN19-1 after 48 hours of fermentation can effectively inhibit the growth of Xoo,and the semi inhibitory concentration has been determined.Then,we found that bacterial suspension containing B.laterosporus SN19-1 could promote the germination of rice seed,while the supernatant which B.laterosporus SN19-1 was removed by filtration had no effect on the germination of seeds.Interestingly,we found that the treatment of supernatant can increase the bud length and root number of seeds.Meanwhile,we found that B.laterosporus SN19-1 has no significant promoting effect on the growth of rice seedlings.Furthermore,we also found that the control effects of B.laterosporus SN19-1 on rice bacterial leaf blight were as high as 90.92%and 68.31%,respectively,through greenhouse pot experiments and leaf in vitro experiments.Finally,we applied the sterile supernatant of B.laterosporus SN19-1 to Xoo to study the inhibition mechanism of B.laterosporus SN19-1 on Xoo.According to a series of physiological changes produced by Xoo,we found that the metabolites of B.laterosporus SN19-1 can affect the normal physiological metabolism and pathogenicity of Xoo.In terms of pathogenic factors of Xoo,the metabolites of B.laterosporus SN19-1 have a significant impact on the hydrophobicity and biofilm formation ability of Xoo,reducing its hydrophobicity and inhibiting its biofilm formation ability.This can affect the colonization of Xoo on rice and invasion on vascular bundles of rice,thereby affecting its pathogenicity.As for physiological metabolism,scanning electron microscopy observation showed that after being treated with the sterile supernatant of B.laterosporus SN19-1,the bacterial body of Xoo showed wrinkling phenomenon.When the concentration of the supernatant increased,there were also"ribbons" of substances adhered between the bacterial bodies,which may be due to the stimulation of the production of poly-3-hydroxybutyrate(PHB)by the pathogenic bacteria after being treated with metabolites.By detecting the effects of metabolites on the respiration and energy metabolism of pathogenic bacteria,it was found that the metabolic activity,respiratory chain dehydrogenase activity,and succinate dehydrogenase(SDH)activity of Xoo were significantly inhibited,while the inhibitory effects on malate dehydrogenase(MDH)and ATPase activities were relatively weak.After being exposed to the supernatant,the level of reactive oxygen species(ROS)in Xoo also significantly increased.At the same time,the permeability of the cell membrane and the outer membrane significantly increased,and the fluidity of the cell membrane decreased,and the leakage of intracellular substances led to cell death.Under laser scanning confocal microscopy(LCSM),it can also be seen that the longer the supernatant acts on pathogenic bacteria,the higher the level of ROS and the permeability of the cell membrane.This study further confirms that B.laterosporus SN 19-1 has excellent biocontrol potential,which can produce various hydrolytic enzymes and has functions such as organic phosphorus hydrolysis,siderophore production and IAA production,and has significant control effects on rice bacterial leaf blight.It was clarified that the prevention and control of rice bacterial leaf blight by B.laterosporus SN19-1 mainly targets the pathogenic bacteria themselves.Metabolites of B.laterosporus SN19-1 can cause damage to the cell membrane and morphological changes of the pathogenic bacteria Xoo,resulting in the leakage of intracellular ions,imbalanced exchange of intracellular and extracellular substances,and the leakage of nutrients and some macromolecular substances.The normal function of proteins on the cell membrane is restricted,and it also affects TCA cycle and sugar metabolism,inhibits respiration and energy metabolism,and thus kills the pathogenic bacteria.At the same time,B.laterosporus SN19-1 also affects the pathogenic factors of pathogen Xoo,resulting in the decline of colonization ability of pathogenic bacteria when infecting rice,thus achieving efficient control of rice bacterial leaf blight.Overall,this study has laid the theoretical foundation for the development of B.laterosporus SN19-1 as a biocontrol agent against rice bacterial leaf blight.