Pathogen Differentiation And Infection Dynamics Of The Pathogen Causing Grafted Chieh-qua Bacterial Wilt

In recent years,bacterial wilt has been reported on different Cucurbitaceae plants in succession,but the pathogenic characteristics and pathogenesis of the pathogen of disease are still unclear.Therefore,it is difficult to formulate effective control measures.In the previous work of this study,it was found that the bacterial wilt pathogen isolated from the grafted chieh-qua with pumpkin as rootstock exhibited strong pathogenicity to pumpkin and grafted chieh-qua with pumpkin as rootstock,and weak pathogenicity to chieh-qua seedling.The standard strain GMI1000 has no pathogenicity to Cucurbitaceae plants,although it belong to phylotype I and race 1 just like the strains from Cucurbitaceae bacterial wilt.In order to investigate the pathogenicity differentiation of different strains from grafted chieh-qua and their phytopathogenic characterizations with GMI1000 strain,the virulence of different strains from grafted chieh-qua to pumpkin and chieh-qua seedlings were determined,and the comparative genome analysis of grafted chieh-qua strain Cq01 were carried out.Meanwhile,the infection dynamics of Cq01 and GMI1000 strains on pumpkin were determinded.In addition,under the induction of secondary metabolites of pumpkin(As host signals),the differences of physiological and biochemical reactions related to pathogenicity between the two strains were compared.The main results were as follows:(1)Strains from grafted chieh-qua and nearby diseased crops(Balsam pear,pepper and peanut plants)were isolated from bacterial wilt samples in Wutang,Nanning,Guangxi.All strains were identified as belonging to phylotype I,namely Ralstonia pseudosolanacearum.Except for the peanut strain identified as sequevar 44,other strains belonged to sequevar 13.Cq01 and Cq03 strains from grafted chieh-qua had pathogenicity to 10 chieh-qua cultivars,but the virulence was weak.Ten strains from grafted chieh-qua had obvious virulence differentiation to pumpkin rootstock,with disease index of 7～94.These 10strains displayed low virulence toward chieh-qua seedling(Disease index<31),butthere were differences.(2)The genome sequences of Cq01 strain and other 9 strains(4 strains were pathogenic to Cucurbitaceae plants and 5 strains were not pathogenic to Cucurbitaceae plants)were determined by using high throughput sequencing technology.The genome of Cq01 strain was 5,882,659 bp in size,with a GC content of 67.72%,encoding a total of 5144 genes.The comparative genomic analysis revealed that the ratio of core genes to pan genes in pathogenic strains of Cucurbitaceae was close to that in non-pathogenic strains.However,the ratio of core genes to pan genes in all strains of two pathogenic types decreased by more than 10%compared with that of a single pathogenic type.Compared with non pathogenic strains,there were no genes shared or deleted only in all pathogenic strains.Phylogenetic analysis showed that the strains from Wutang town of Nanning city and pathogenic to pumkin had close genetic relationship,but these was no significant correlation between the genetic relationship of other pathogenic strains and the host or geographical origin.(3)Pumpkin roots were inoculated with gfp-labeled Cq01 and GMI1000strains.After 1 day,Cq01 invaded the root vascular bundle,and 2～3 days later,it entered the stem under the cotyledon,and the pumpkin began to show wilting symptoms.After 4 days of inoculation,Cq01 invaded the stem above cotyledon,and the disease reached peak.For GMI1000 strain,the invasion was only observed in the root hair,and the plants were healthy.The bacteria quantity in each part of plant was determined by using dilution-plate method.When the bacteria quantity in root reached 10~8CFU/g,the plant began to wilt;when the bacteria quantity in stem reached 10~8CFU/g,the plant was seriously affected.The highest infection bacteria quantity of GMI1000 in root was 10~6CFU/g.For the stem-injecting method,the population of Cq01 and GMI1000 strains multiplied extensively in the injection site,reaching the maximum value of 10~9CFU/g at 5 days after inoculating.Cq01 strain could expand from the injection site up and down,and the plant began to show symptoms after 3 days of inoculation,while the expansion of GMI1000 strain in the inoculated plant was limited and could not cause wilting symptoms.(4)Under the induction of pumpkin extract,the chemotactic response and extracellular polysaccharide production of Cq01 and GMI1000 strains were not affected.Although the motility of two strains was enhanced,the motility of Cq01 was stronger than that of GMI1000.The biofilm formation of Cq01 and GMI1000 strains increased significantly,and the low concentration of extracts could significantly promote the biofilm formation of GMI1000 strains.Low concentrations(12.5 and 6.25μg/m L)of pumpkin extract had no effect on the activities of cellulase and pectinase.When the concentration reached 25μg/m L,the pectinase activity of GMI1000 strain increased.In summary,the virulence of R.pseudosolanacearum strains from grafted chieh-qua on pumpkin rootstock were weak to chieh-qua seedlings,but strong to pumpkin.Different strains from grafted chieh-qua showed virulence differentiation.Compared with the genomes of non-pathogenic strains to Cucurbitaceae,specific genes were not found in the genomes of pathogenic strains.Cq01 strain from grafted chieh-qua,could infect pumpkin rapidly and propagate and expand in the plant.However,GMI1000 only invased into pumpkin tissue with a low amount and its expansion in the plant was limited.Under the induction of pumpkin extract,many pathogenic factors of Cq01 and GMI1000 strains were not inhibited.This study provided a certain reference for further study on geographical origin,host specificity and pathogenic mechanism of R.pseudosolanacearum strains from grafted chieh-qua,and laid a foundation for crop breeding and control of bacterial wilt.