Study On The Genetic Structure Of The Pathogenic Bacteria Of Guizhou Kiwifruit Canker

Kiwifruit is an important fruit.In Guizhou Province,a large number of kiwifruit are planted,which has become one of the most important fruit products in recent years.Kiwifruit canker is an important factor restricting the development of kiwifruit industry.It is a bacterial disease caused by Pseudomonas syringae pv.actinidiae(Psa).Because of its rapid transmission,great harm and difficult control,it has caused huge economic losses to the major kiwifruit planting areas in Guizhou Province.In order to understand the population genetic structure of Psas in Guizhou Province and find out the transmission and Epidemic Law of the disease,In this study,standard samples of kiwifruit canker were collected,and Psa was isolated,purified,identified and preserved.The biological type and pathogenicity of the preserved Psa were determined.At the same time,the Multiple Loci Variable Number of Tandem Repeats Analysis(MLVA)typing method was optimized by downloading the whole genome information of Psa and simulating PCR.The genetic structure of Psa population in Guizhou Province was analyzed by the optimized MLVA method,give the result as follows:1.More than 700 canker samples were collected in 31 counties(cities,districts),48towns(townships,sub-districts),and 74 orchards in 8 cities(prefectures)of Guizhou Province.The collected disease samples were isolated for pathogenic bacteria,identified using specific primers F1/R2 and 16 S sequencing methods and stored.A total of 377 Psa strains were collected,of which 2 were from Zhouzhi County,Shaanxi Province,and 375 were from Guizhou kiwifruit producing areas.2.The results showed that all the 377 strains of Psa were identified as Pseudomonas syringae pv.actinidiae biovar 3.The virulence of 377 strains of Psa was determined in vitro inoculation,the results showed that the average lesion length was 0.21-1.52 cm.The analysis of variance showed that the virulence was not related to geographical origin and host varieties.3.Using the simulated PCR method,34 pairs of primers which were reported to used for MLVA typing of genetic structure of Psa population and the whole genome information of 129 Psa strains downloaded from Gen Bank was analyzed and researched.22 pairs of primers were screened by MLVA data analysis.6 pairs of MLVA primer combinations were screened by Simpson index,and the number of multi locus genotypes and Simpson index were 52 and 0.9677978 respectively,which were the same as those of 22 primer combinations.The UPGMA clustering tree based on the MLVA data of the 6 primer pairs is almost the same as that of the MLVA data of the 22 primer pairs.The 6 pairs of primers can represent 22 pairs of primers and can be used to study the population genetic structure of Psa.4.The genetic structure of 377 strains of Psas was analyzed by using the combination of 6 pairs of MLVA primers.Combined with the MLVA data of 6 pairs of primers of 80 strains of Psas downloaded from Gen Bank,the results showed that the Psas in Guizhou Province could be divided into 9 groups,including group 4 and group 6,two groups with a large number,and 7 groups with a small number.Group 4 and group 6 were distributed in various regions of Guizhou Province,Among them,group 4 is the global epidemic group,which is also the first group introduced into Guizhou Province.5.The UPGMA cluster tree analysis showed that Psa had a long-distance transmission through pollen and seedlings.It is suggested that in the prevention and control of kiwifruit canker,not only orchard management should be standardized to prevent the short-distance spread of Psa among orchards,but also the Psa detection of pollen and seedlings in the market should be strengthened to prevent the long-distance spread of Psa.