Identification And Molecular Biology Research Of Mulberry Wilt Pathogen

Mulberry is the only source of silkworm food.With the development of mulberry resources and its application in ecological management,mulberry industry has developed into an important part of modern sericulture industry system.Mulberry bacterial wilt is a disease that the whole plant death of mulberry caused by the infection of R.solanacearum or mulberry Enterobacter spp.In recent years,In Zhejiang and South China,about 10% mulberry garden area was changed to other economic crops due to the outbreak of mulberry bacterial wilt,which is a serious harm to the sericulture industry in China.The field symptoms of mulberry wilt caused by Enterobacter cloacae were similar and difficult to distinguish from those caused by R.solanacearum.Therefore,the study of the relationship between Enterobacter spp.and R.solanacearum provides ideas for the control of mulberry wilt.In this study,80 samples of mulberry wilt disease plants and rhizosphere soil were collected from Guangxi,Guangdong and Hainan.The samples were detected by PCR,and the pathogenic bacteria were isolated and identified.High-throughput sequencing technique was used to analyze the DNA of 8 samples from Liucheng County and Rongan County,and the whole genome of Enterobacter cloacae was sequenced.The results were as follows:(1)44 samples of DNA from Guangxi,24 samples from Guangdong Province,and12 samples from Hainan Province was performed by PCR of using self-designed primers5F/5R and Ralstonia specific primers AU749/AU750.44 samples of DNA in Guangxi,18 samples of DNA in Guangdong and 7 samples of DNA in Hainan were detected for R.solanacearum and E.cloacae.(2)The TTC culture medium was used to isolate and identify the pathogens detected by PCR.At the same time,R.solanacearum and E.cloacae were isolated;the rejuvenation experiment was carried out at 37° C.and 85% humidity,and the “wilting” symptoms ofmulberry trees caused by both R.solanacearum and E.cloacae were similar.Among them,E.cloacae can be used as the major pathogen of mulberry wilt disease;8 samples of total DNA of Fengshan Town,Liucheng County,and Rongan County Yikou Village were sequenced and analyzed using high-throughput sequencing technology.There is a content of the genus Ralstonia and Enterobacter.The above three results indicate that the “wilting”symptoms of mulberry may be related to the simultaneous mixing of E.cloacae and R.solanacearum.(3)8 samples of total DNA from Fengshan Town,,Liucheng County and Rongan County were analyzed by high-throughput sequencing data.The genome size of E.mori was4.73 M bp,GC ratio accounts for 56.05%,encoding 4600 genes.Based on the sequence of hsp 60,rpo B and gyr B gene,three strains of E.cloacae(LC01,XC01,KQ01)were constructed.The results showed that XC01,KQ01 and E.mori belong to the same branch and LC01 belong to the other branch.Based on the comparative analysis of the whole genome sequence of E.cloacae,the specific gene sequence of E.cloacae is2191639-2193771 bp.(4)Based on the specific gene sequence of E.cloacae is 2191639-2193771 bp.Lamp primer sets were designed and optimized,and LAMP method for detection of E.cloacae from mulberry was established.The results can be observed directly by naked eye and can be directly used for field detection.In this study,the high-throughput sequencing technique was used to analyze the species of mulberry wilt,and the whole genome was sequenced for the first time.The specific gene sequence was about 2100 bp.The LAMP detection technique for the detection of E.cloacae was established(Patent Application No.: 201711281274.2;Patent Application No.: 201810326627.4).