Identification And Analysis Of Active Components And Related Gene Clusters Of Pseudomonas Mosselii BS011 Against Bacterial Leaf Blight In Rice

The rice bacterial leaf blight caused by Xanthomonas oryzae pv.Oryzae,is an important bacterial disease,which results in serious losses of crop yield.Currently crop breeding and chemical pesiticide were used for controlling rice bacterial leaf blight.However,the resistant varieties lose resistance easily with evolution of physiological races of rice bacterial leaf blight.Chemical pesiticide caused serious environmental pollutions which were seriously threatened to human health.Thus,a sustainable control measures is necessary for controlling rice diseases.Microorganism metabolite,has attracted wide attention for controlling rice disease in recent years,and they have high control efficiency,biodegradable and environment friendly.In this study,the rhizosphere microorganisms of rice which inhibit bacterial blight were isolated and screened,and study on the mechanisms of resistance to bacterial leaf blight.The resluts are summarized as follows:1.1052 strains of rice rhizosphere microorganisms were isolated from soil and rice roots in rice field.Four strains showed strong inhibitory activity against important rice diseases by plate inhibition experiments.16 S rRNA sequencing and phylogenetic analysis demonstrate that JP2-270 is Burkholderia sp.JP2-207 and BS011 are Pseudomonas mosselii,and JP2-74 is Chromobacterium rhizoryzae,BS011 can inhibit the growth of Xanthomonas oryzae pv.2.Crude extract was separated from BS011 fermentation broth by acid precipitation.The crude extract was able to inhibit bacterial leaf blight(the average antibacterial diameter is 1.6 cm)at 20 ?g/ml;the pure active compound 4-H was isolated by preparative liquid chromatography and high performance liquid chromatography.The petri dish assay and pot experiment to inhibit bacterial leaf blight(the average antibacterial diameter is 1.7 cm)showed that the MIC of compound 4-H was 10 ?g/ml;HRMS and NMR analysis showed that compound 4-H was xantholysinA.3.Whole genome sequencing analysis showed that the BS011 genome consists of a circular chromosome with a size of 5.75 Mb,encoding 5170 genes,containing 23 rRNA and 78 tRNA operons.Bioinformatics analysis revealed that seven gene clusters may be involved in the biosynthesis of metabolites.c-xtl,c-pms,c-pvd1,and c-pvd2 belong to the NRPS type gene cluster,c-ppyS and c-mbo belong to the Bacteriocin gene cluster,and c-hcn is other types of gene clusters.The c-xtl shows high homology to the xantholysin biosynthetic gene cluster.We inferred that the c-xtl gene cluster synthesizes xantholysinA.4.The mutant strain BS011?c-xtl(gene cluster c-xt1,deletion fragment 430746bp-476301bp)was constructed by gene knockout experiment,BS011?c-xtl lost the ability to inhibit rice bacterial blight,the resut demonstrate that c-xt1 is necessary to inhibit rice bacterial blight.Further knock-out experiments showed that the xtlA gene in the gene cluster c-xt1 inhibited bacterial blight,and the complement experiment confirmed that BS011?xtlA was recovered the antagonsitic activity of bacterial blight by introducing gene cluster xt1 A into BS011?xtlA.HPLC analysis confirmed that BS011?xtlA,mutant of the deletion of gene xtlA,could not produce xantholysinA,demonstrating that the gene xtlA in P.mosselii BS011 is involved in the biosynthesis of xantholysinA.