Monitoring Of The Prevalence Of Seed-borne Acidovorax Citrulli In Commercial Watermelon And Other Cucurbitaceous Seed Lots In China And Pathogencity Functional Analysis Of T6SS In Erwinia Amylovora

Bacterial fruit blotch(BFB)is seed-borne and seed transmitted.To assess the prevalence of seed-borne Acidovorax citrulli in commercial watermelon and other cucurbitaceous seed lots in China,a 3-year survey was conducted between 2016 and 2018,a total of 1168 seed lots of watermelon and other cucurbitaceous species were collected from 16 provinces of China,and detected A.citrulli by PCR and Bio-PCR technique.The results showed that the bacterium was detected in 41.56%,17.43%and 31.43%of all commercial seed lots tested from the 2016,2017 and 2018 growing seasons,respectively.Among them,the prevalence of A.citru lli-infested seedlots was highest in Gansu province(36.17%)followed by Ningxia(33.71%)and Xinjiang(27.05%).The prevalence of A.citrulli in seedlots from the other 13 provinces combined was 22.13%.Previous work found that part of core genes of T6SS play a major role in pathogenicity-associated in Erwinia amylovora NCPPB1665.In this study,the rest of 20 core genes(tssA-1,tssA-2,tssA-3,tssE-1,tssE-2,tssF-1,tssF-2,tssJ-1,tssJ-2,tssL-1,tssL-2,tssL-3,tagH-1,tagH-2,vgrG-1,vgrG-2,vgrG-3,vgrG-4,clpV-1,clpV-2)within three T6SS clusters were studied by mutation.The results showed that all 20 mutants'antibacterial activities reduced against Escherichia coli as compared with the wild-type(WT)strain.And of the 20 mutants,10(?tssA-2,?tssA-3,?tssE-2,?tssF-2,?tssJ-2,?tssL-3,?tagH-1,?vgrG-1,?vgrG-2 and ?clpV-2)exhibited reduced virulence on immature pear fruit as compared with that of the WT strain.Nine mutants(?tssA-2,?tssA-3,?tssE-2,?tssF-2,?tssJ-2,?tssL-3,?vgrG-1,?vgrG-2 and ?clpV-2)produced less amylovoran and six(?tssA-1,?tssE-1,?tssF-1,?tssJ-2,?tagH-1 and ?tagH-2)produced less levan.These findings suggest that E.amylovora T6SS play a role in bacterial competition and virulence.By analyzing the transcriptome data,?tssG-1,one of the core genes of T6SS,was selected and verified.Fluorescence quantitative PCR showed that hrpN was up-regulated nearly 13 times.Western blot showed that the expression of HrpN was also increased.Management of fire blight is complicated because of the lack of effective pesticides.By applying plant resistance inducers,plants can be stimulated to produce "systemic acquired resistance"(SAR)so as to enhance their autoimmune ability to prevent and control fire blight.Four plant resistance inducers including potassium phosphites(PH),benzothiadiazole(BTH),Prohexadione-Ca and chitosan were studied in this experiment.The aim was to screen the suitable inducers for the manegement of fire blight.The results showed that all the four plant resistance inducers could induce the up-regulation of PR-1,PR-2 and PR-8 genes expression in plants at varying degrees.Prohexadione-Ca is the best effective one,which the gene expression of PR-1,PR-2 and PR-8 were about 5,7 and 3 fold higher,respectively.Subsequently,the effect of plant resistance inducer on fire blight in green house showed that the incidence of fire blight was reduced by about 34%compared with the control group after the treatment of Prohexadione-Ca,Benzothiadiazole,Chitosan and potassium phosphites can reduce 29%,20%and 12%,respectively.