Population Genetic Diversity Andmolecular Detection Of Phytophthora Nicotianae And Thielaviopsis Basicola In Henan Province

Tobacco Black Shank is one of the most devastating diseases for tobacco production,caused by Phytophthora parasitica var.nicotianae(Breda de Haan)TucKer.It is first reported in 1950,occurring in Huang-Huai region,but now it is universally distributed in the tobacco-planting areas of south,central,east and northwest China.It is also ubiquitous in south tobacco-growing areas,like Yunnan,Guizhou,Sichuan,Hunan,Guangdong,Guangxi,Fujian and so on.This disease is much more severe due to its co-occurrence with Tobacco bacterial wilt,especially in Anhui,Shandong,Henan.Black root rot is one of major tobacco root diseases,caused by Thielaviopsis basicola(Brek.et Br.)Ferr.,occurring in Yunnan,Guizhou,Hubei,etc.The incidence of this disease can research 30%in some serious conditions.It has become one of the main diseases affecting the tobacco production in Henan,Shandong,Anhui and some other provinces.It has cital significance to achieve the effective prevention and control of diseases,understanding the structure of population genetic diversity and the establishment of the molecular detection technology of the two main pathogens.In this paper,32 Phytophthora nicotianae strains from Henan tobacco-growing areas have been collected in 2011,and 160 corresponding DNA fragments have been cloned,and sequenced for 4 nuclear genes(including rDNA-ITS,β-tubulin,Ras and Gpi)and 1 mitochondrial gene(Cox1),Population evolution phylogenetic tree have been established based on these sequences.The results indicate that there is a high similarity between these 5 DNA sequences in the pathogen population and the reference DNA sequences,from 83%to 100%,average gene diversity index M=1.4227.The result indicates that 32 strains of Tobacco Black Shank can be divided into 6 groups by ITS sequence,but into 3 groups by β-tubulin,Ras,Gpi,Cox1.Population evolution phylogenetic tree have been established by using combined sequences of all 5 genes,revealing that 32 isolated strains from 14 countiescan be divided into 2 clear clusters,including 15 different combined genotypes with gene diversity index=2.35,reflecting the variation and genetic diversity in Phytophthoraparasitica var.nicotianae(Breda de Haan)TucKer.Among them,two most prevalent combined genotypes include 8 strains(c genotype)and 6 strains(e genotype),respectively.However,the total level of genetic differentiation between these genotypes is low,genetic distance between clusters is merely 0.0005,demonstrating that Henan strains may belong to same effective population.On the other hand,genetic differentiation(relationship)of these strains has no obvious correlation with geographic origin,indicating that there exists effective interflow between Henan Tobacco Black Shank population,and no clear subgroup differentiation has been formed.With the cost of next generation sequencing technique continues decreasing,the analysis of combined genotype in this article can be more widely applied to massive and dynamic detection of Tobacco Black Shank and the breeding of tobaccos with resistance to this fungus and rational arrangement of varieties.In this study,5Thielaviopsis basicola strains from Henan tobacco-growing areas have been collected in 2013,and purchased standard strains ATCC18723 from USA,and 18 corresponding DNA fragments have been cloned,and sequenced for 3 nuclear genes(including rDNA-ITS,β-tubulin,and TEF-1α),the results showed that there is a high similarity between these 3 DNA sequences in the pathogen population and the reference DNA sequences,from 99%to 100%.It indicates that the strains from Henan and ATCC18723 at a branch due to the phylogenetic tree established by these 3 genes sequences resbectly,the genetic distance less than 0.002 btween the strains from Henan and ATCC18723,for combined sequences of all 3 genes,show that genetic diversity level is low amang strains in Henan.The primers,specific for detection of Thielaviopsis basicola and Phytophthora nicotianae,were designed based on sequences sequenced in the experiment.The result indicates that the primers could only amplify the corresponding pathogen by Specific tests,and the corresponding strains(Fusarium oxysporum,Alternaria alternate,Botrytis sp.Onion,Nicotiana tabacum L,Pseudomonas syringae pv.angula(Frorme et al.)Holland,Cercospora nicotianae)is negotive.The results show that Phytophthora nicotianae primers YYI-F/R,YYH-F/R detection limit is 100 fg and 10 pg of total DNA,Thielaviopsis basicola primers TBITS 1419-F/R,TBβ 1419-F/R test a minimum of detection limit is 10 fg and 1 pg.The fluorescent quantitative PCR detection and double PCR detection is established,and can be applied to detect the field soil,and infected plants rapidly.It has great significance for early warning and study of infection rules as well as the effective control of the soil spread fungus diseases.