The Action Mechanism Of Bismerthiazol Against Rice Bacterial Bilght

Rice bacterial blight caused by Xanthomonas oryzae pv.oryzae(Xoo)is one of the most serious bacterial diseases of rice.Bismerthiazol is one of the commonly used bactericide to control this disease,but the difference between in vivo and in vitro efficacy of bismerthiazol and its mechanism against rice bacterial blight remain poorly understood.The objectives of this study were focused on 1)Photochemical degradation of bismerthiazol:structural characterization of the photoproducts and their inhibitory activities against Xoo;2)The mechanism of bismerthiazol and its homologue zinc thiazole on promoting rice defence against Xoo;3)The mechanism of bismerthiazol on reduction in virulence of Xoo;4)The study of genovariation of bismerthiazol-resistant Xoo 2-1-1.1.Photochemical degradation of bismerthiazol:structural characterization of the photoproducts and their inhibitory activities against XooAlthough chen et al.(1993)determined that bismerthiazol is susceptible to photolytic degradation in water and rice,the photodegradation pathway and degradation products have remained unknown.Determination of the inhibitory activities of its photoproducts is one unanswered question about the action mechaism of bismerthiazol against rice bacterial blight.In our study,the photodegradation of bismerthiazol was investigated with natural sunlight and lamplight.A yellow solid precipitate formed and was identified as elemental sulfur.Inhibition of Xoo was greater with a photolyzed solution than with a non-photolyzed solution of bismerthiazol whether the bismerthiazol was exposed to natural sunlight for 6 days or lamplight for 8 h.These results indicated that photodegradation increased the inhibitory activity of bismerthiazol against Xoo.We identified LC-MS chromatograms of a photolyzed bismerthiazol solution that had been irradiated in a solar simulator for 0,4 or 8 h,and three intermediate photoproducts and three end photoproducts of bismerthiazol were characterized by LC-MS.PP1 and PP6 were eluted at 2.23 min and 3.32 min respectively and identified as Tf-130 and ATDA.Because Tf-130 and ATDA were very toxic to animals and humans,these two compounds have been banned for many years.The present results indicate that bismerthiazol should not be exposed to light during storage and transportation.The results also suggest that,when used for BLB control,bismerthiazol should be applied to the seedling stage and the tillering stage so that Tf-130 and ATDA have sufficient time to degrade fully before harvest.PP3 was eluted at 1.81 min and not been studied so far.Li et al.(2015)found that 1,3,4-thiadiazole sulfone derivatives were more effective than bismerthiazol against Xoo and Xoc.We suspect that PP3,a 1,3,4-thiadiazole sulfone derivative,might also have high activity against these bacteria.Eluted at 5.95 min,PP5 was one intermediate photoproduct of bismerthiazol and identified as AMT.Based on these products,a photodegradation pathway was inferred.In addition,inhibition effects of bismerthiazol,PP2 and PP5 for Xoo and Xoc were determined on the basis of growth inhibition.Based on EC50 and MIC values,inhibition of Xoo and Xoc was significantly greater with bismerthiazol and AMT than with ATDA in vivo,suggesting that the sulfhydryl group was crucial for the inhibition of Xoo growth by bismerthiazol and its photoproducts.Xoo 2-1-1 was resistant to PP5 in vivo but sensitive in vitro.These results showed that bismerthiazol and AMT have a similar mode action against Xoo in vivo and in vitro,suggesting that these two thiadiazoles might have the same action mechaism against rice bacterial blight.2.The mechanism of bismerthiazol and its homologue zinc thiazole on promoting rice defence against XooThiazole,isothiazole,thiadiazole,and their derivatives are used to control various human,animal,and plant diseases.In addition to having direct anti-microbial properties,these compounds are thought to induce host defenses,but the mechanism of defense induction remains poorly understood.We assessed whether the expression of the five defence related genes(OsPR1a,OsPR1b,POX22.3,OsPAL,and LOX)and the generation of H2O2 in plants was affected by 200 mg L-1 bismerthiazol or zinc thiazole.The thiadiazoles of zinc thiazole and bismerthiazol induce H2O2 accumulation,up-regulation of defense-related genes,callose deposition,and hypersensitive response-like cell death in rice leaves infected with Xoo strain ZJ173 but not in non-infected leaves.These results show that bismerthiazol and zinc thiazole failed to induce defence responses in non-infected rice plants.These defense responses in Xoo-infected leaves were suppressed by exogenous application of catalase,which reduces H2O2 accumulation.The results indicate that H2O2 acts as a trigger and mediator of thiadiazole-induced defence responses.H2O2 production and inhibition were greater when zinc thiazole was applied 1 day after rather than 1 day before Xoo inoculation;moreover,when the concentration of zinc thiazole applied was increased from 200 to 300 mg L-1,H2O2 production decreased.These data suggest that thiadiazole-induced defence depends on the application time and concentration of thiadiazole.Streptomycin did not enhance the defence of Xoo-inoculated rice,indicating that this defence induction is specific for thiadiazole.Because it is needed for the production of bacterial biofilms,EPS is an important virulence factor,especially for vascular pathogens.Until now,no reports have evaluated the role of EPS in the susceptibility of rice to Xoo.we found that treatment of rice leaves with EPS extracted from Xoo inhibited the expression of defence genes in Xoo inoculated rice plants and enhanced the formation and expansion of BLB lesions.The EPS-deficient Xoo mutant ?gumH triggered a stronger defense than its parent,strain ZJ173.These results suggest that EPS produced by Xoo suppresses rice defence and enhances the susceptibility of rice to Xoo.Several recent studies have shown that some thiazole compounds have substantial anti-biofilm activity against Gram-positive and Gram-negative bacteria.Ma et al.(1997)has indicated that bismerthiazol significantly decreases EPS production.The current study confirmed these results in that bismerthiazol and zinc thiazole suppressed EPS synthesis genes and inhibited EPS production by Xoo ZJ173.Exogenous EPS did not affect the inhibitory effects of either compound in vitro,whereas EPS addition to the plants strongly decreased the induction of defence responses.In addition,the thiadiazole treatments did not reduced EPS production by the thiadiazole-resistant strain 2-1-1,which is thiadiazole-resistant in vivo but not in vitro;moreover,enhanced defense was not detected in thiadiazole-treated rice inoculated with 2-1-1.Based on these data,we infer that bismerthiazol and its homologue zinc thiazole promote rice defense against Xoo by inhibiting the production of bacterial EPS.3.The mechanism of bismerthiazol on reduction in virulence of XooBismerthiazol provides excellent control of bacterial rice leaf blight(BLB)caused by Xoo but does not greatly inhibit Xoo growth in vitro.Genome-wide mRNAs in Xoo were sequenced to monitor the global changes in gene expression of Xoo exposed to 30 mg L-1 bismerthiazol for 4.5 h and 9 h.The transcriptome profiles of Xoo were analyzed using heat maps that included genes with a two-fold change with respect to the control,i.e.,Xoo without bismerthiazol treatment.The heat maps revealed clear differences in the expression pattern between bismerthiazol-treated and control samples at 4.5 h;differences between the profiles of bismerthiazol-treated and control samples were less at 9 h than at 4.5 h.After 4.5 h of bismerthiazol treatment,217 genes were significantly up-regulated and 192 genes were significantly down-regulated;after 9 h of treatment,31 genes were significantly up-regulated and 19 genes were significantly down-regulated.All genes showed the same expression patterns in the qRT-PCR assays as in the RNA-Seq data,suggesting that the RNA-Seq data were highly reliable.Among the DEGs in the bismerthiazol-treated samples,32 were the same in the 4.5-h and the 9-h samples,indicating that the expression of these genes was continuously affected by bismerthiazol.Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed to determine the biological functions of the 32 DEGs.6 DEGs(hutC,hutG,hutH,hutI,hutU,and sdeB)were mapped to the Hut pathway and was repressed by bismerthiazol.Following bismerthiazol treatment,the expression of all of these genes was significantly down-regulated in both NB medium and plant cell-mimicking XOM3 medium,indicating that bismerthiazol might inhibits the Hut pathway of Xoo in vitro and in vivo.AMT but not ATDA or BTH also repressed the Hut pathway of Xoo,indicating that inhibition by bismerthiazol depends on its sulfhydryl group,which BTH and ATDA lack.To study how inhibition of the Hut pathway by bismerthiazol affects Xoo,we compared hutG and hutU deletion mutants with the wild-type strain.The Hut pathway is not involved in the inhibition of Xoo in vitro growth by bismerthiazol.The ?hutG and ?hutU mutants formed readily observable aggregates or clumps at the late growth stage while the wild type ZJ173 and the complemented strains did not form aggregates but grew in a dispersed fashion.Deletion mutants of hutG or hutU were more motile,produced less biofilm,and were less virulent than the wild type,indicating that the Hut pathway is involved in quorum sensing(QS)and contributes to virulence.Some 1,3,4-thiadiazole derivatives demonstrate good anti-QS activity in Gram-negative bacteria.As a 1,3,4-thiadiazole molecule,bismerthiazol inhibits the Hut pathway in Xoo and thereby interferes with the bacterium's QS.At a dosage that resulted in the same degree of growth inhibition as that caused by streptomycin and shenqinmycin,bismerthiazol but not streptomycin or shenqinmycin increased the motility and reduced the biofilm production of ZJ173.Moreover,we found 13 other QS-related DEGs in the 4.5-h bismerthiazol-treated sample.These QS-related genes regulate DSF synthesis,EPS synthesis,chemotaxis,and stress response in Xoo.Bismerthiazol did not inhibit transcription of Hut pathway genes,QS,or virulence of the bismerthiazol-resistant strain 2-1-1.The results indicate that bismerthiazol reduces Xoo virulence by inhibiting the Hut pathway and QS.4.The study of genovariation of bismerthiazol-resistant Xoo 2-1-1The resistance mechanism of pathogen is closely related with the action mechanism of bactericide agasint pathogen.So we might find out the specific target of bactericide by studying the resistance mechanism of pathogen.The mutant Xoo 2-1-1,which was selected on bismerthiazol-treated rice inoculated with wild-type Xoo ZJ173,shows cross-resistance to zinc thiazole in vivo,but not in vitro.We preliminarily studied the genovariation of bismerthiazol-resistant Xoo 2-1-1 using whole-genome sequencing.A 350-bp paired-end library was constructed andsequencing was performed on the Illumina Hiseq 2000 aftercluster generation.The sequencing depth was more than 100×.The sequencing results showed that bismerthiazol-resistant Xoo 2-1-1 and wild type ZJ173 have 99.37%homology in its nucleotide sequence with MAFF 311018.Several point mutations were found in the genomes between ZJ173 and 2-1-1 using the analysis of single nucleotide polymorphism(SNP).Then we used PCR to confirm these point mutations and chose Xoo 3738 gene containg missense mutationto the next study.