Identification Of Pathogen And The Association Of Mmicrobial Community In Root And Rhizosphere Soil With Mulberry Bacterial Wilt In Hainan

Mulberry bacterial wilt is a soil borne disease and usually caused by Ralstonia solanacearum and other bacteria,which was mainly occurred in tropical and subtropical areas with high temperature and humidity.The vascular bundles are blocked by the proliferation of bacteria and then inhibit the transportation of water and nutrients in plants.The leaves lose water and wilt but always keep green,and the plant is eventually to death due to lack of water and nutrition.Further more,the bacterial wilt could spread rapidly and there is no effective control agents.Physical isolation or plant digging are used as the main prevention and control methods.The bacterial wilt could result in a large reduction of mulberry production and seriously restrict the development of sericulture industry in tropical and subtropical areas.The microbial community structure plays an important role in the occurrence of soil borne diseases.In this study,the occurrence regularity of mulberry bacterial wilt,and the isolation and identification of pathogenic bacteria in Hainan were completed;the difference in rhizosphere microbial community and endophytic microbial community in different infection stages of mulberry bacterial wilt were analyzed by high-throughput microbial sequencing method;meanwhile the changes in rhizosphere microbial community in resistant and susceptible mulberry varieties were also analyzed;further more,the soil physical and chemical properties of resistant and susceptible mulberry seedlings in different infection stages were detected,and the correlation with rhizosphere microbial community was analyzed.The main results are shown as follows:(1)Epidemiology of mulberry bacterial wilt and identification of pathogenic bacteria in HainanThe survey found that the morbidity of bacterial wilt in 3 years mulberry fields was over 80%,and it occurred mostly in the rainy season in 6-10 month.Three high pathogenicity strains were isolated from roots and soil of naturally occurring plants.The physiology and biochemistry analysis showed that the three strains were belonged to race 5 and biovar Ⅴ of R.solanacearum;intraspecific genetic evolution analysis showed that they belonged to evolution type I(Asian type);EGL gene cluster analysis showed that they belonged to sequevar 12.(2)Microbial diversity assay of rhizosphere soil at different infection stagesThere was a significant difference in microbial community structure in rhizosphere soil of different infection stages of mulberry bacterial wilt.The bacterial abundance in rhizosphere soil increased significantly at the earlier infection stage and reached saturation at the middle stage,while the bacterial diversity increased significantly at the last infection stage.The abundance of Ralstonia and other genus of Proteobacteria were significantly increased during the infection process,while Sphingomonas and other probiotics decreased gradually.The abundance of fungi were increased gradually during the infection process,but the diversity was not significantly increased.The abundance of pathogenic fungi,such as Pyrenochaeta,was increased significantly and reached the peak at the earlier infection stage,while Fusarium at the middle infection stage;the abundance of beneficial fungi,such as Cladosporium and Trichoderma,were increased significantly at the last infection stage.(3)Microbial diversity assay of roots at different infection stagesThe ACE and Shannon index of bacterial diversity in root were firstly decreased and then increased during the infection process;the Shannon index at the last infection stage was significantly higher than the early stage.The ACE index of fungal diversity was similar to the bacteria,while the Shannon index was not significantly changed.There were significant differences in microbial abundance in roots at different stages.The relative abundance of Ralstonia was increased from 18.90% to 71.11% during the infection process;interestingly,the relative abundance of beneficial bacteria,such as Sphingomonas and bradyrizbium,were also significantly increased at the last infection stage.In addition,the relative abundance of pathogenic fungi,such as Fusarium,Lasiodiplodia and Gibellulopsis,were increased significantly at the last infection stage.(4)Microbial diversity assay and physicochemical properties assay of rhizosphere soil of resistant and susceptible mulberry varietiesThe diversity of bacteria in rhizosphere soil of resistant varieties was significantly higher than that of susceptible varieties,but the abundance was lower.Ralstonia of the susceptible varieties was increased by 2.31 times than resistant varieties.The change of the diversity of fungi in rhizosphere soil of resistant and susceptible varieties was not significant,but the abundance of fungi in resistant varieties was significantly higher than susceptible varieties.Moreover,the p H,total potassium and exchangeable ions of resistant mulberry varieties were significantly higher than susceptible varieties,while the contents of total nitrogen and phosphorus were significantly lower than susceptible varieties.There was a significant correlation between rhizosphere microbial diversity and mulberry bacterial wilt.We speculate that the resistant mulberry varieties may change the structure of soil rhizosphere microbial community directly or indirectly by changing soil physical and chemical properties to resist bacterial wilt.The results above have a scientific significance for early diagnosis of bacterial wilt using microbial diversity assay,and yield insights to the prevention and control methods of bacterial wilt by adjusting microbial community structure in soil.