Etiology And Genetic Diversity Analysis Of Pathogen Causing Tobacco Brown Spot Disease In Hubei Province

Tobacco is an important economic crop in Hubei Province and Tobacco cultivation is one of the important ways for tobacco farmers to get rid of poverty and become rich.In recent years,tobacco brown spot disease in Hubei Province has been observed severely,which has reduced the quality and yield,affected the development of the tobacco planting industry.In this study,the pathogens of tobacco brown spot disease in Hubei Province were identified by morphological and molecular methods.The biological characteristics and genetic diversity of pathogenic fungi were studied.The main results are shown as follows :Eighty-two strains of Alternaria spp.were isolated from four tobacco producing areas in Enshi,Shiyan,Xiangyang and Yichang of Hubei Province.Morphological identification and molecular assisted identification were performed by rDNA-ITS,Calmodulin and Plasma membrane ATPase gene sequences.The results showed that the pathogens causing tobacco brown spot disease in Hubei Province are A.tenuissima,A.alternata,A.longipes and A.yaliinficiens.Sporangial chains of A.longipes and A.tenuissima were linear and had few branche.However,most of the spores often extend to form a 40~70um false pupa,while the middle of the mature conidiophores of A.tenuissima had a deepened diaphragm and obvious shrinks.Both the A.alternata and A.yaliinficiens had a large number of branches.The sporangial chains of A.alternata were mostly short-branched with 3 to 12 spores in the main chain and 1 to 5 spores in the branch and the spores were most elliptic,oval and retrograded.A.yaliinficiens had 8 to 18 spores in the main chain,and 1 to 4 spores in the branch.The main chain spores were significantly more than the branched spores,and the spores were mostly oval or elliptical shape.Morphological identification results showed that the ITS and Calmodulin gene sequence were not able to distinguish the A.alternata,A.tenuissima,A.longipes and A.yaliinficiens effectively.The ATPase gene could be used to identify Alternaria spp.,and the identification results were consistent with the morphological identification results.The distribution of the four species in Hubei is not the same,and A.longipes was the dominant species,with rate of 67% of the total isolates.Biological characteristics of representative strains of four Alternaria spp.Were studied.The results showed that the four species of Alternaria spp.strains can grow at a temperature of 5~30? and a pH of 4~10.The most suitable growth temperature was 25~30?.In addition to A.longipes,other species of Alternaria spp.were the suitable for the growth of pH6~8.All four species can use multiple carbon sources and nitrogen sources,but different species of Alternaria spp.had different optional carbon and nitrogen sources.Among them,When mannitol and galactose were used as carbon sources,A.yaliinficiens grew fastest,and five carbon sources,such as lactose and glucose were most suitable for the growth of A.alternata,The most suitable carbon source for A.longipes is sucrose and grew slowly with all carbon sources.The effect of different nitrogen sources on the growth showed that the A.longipes grew fastest when ammonium chloride was used as nitrogen source,and the other three species grew fastest when peptone was used as nitrogen source.Virulence of four species was tested,and the results showed that the virulence of four species of Alternaria spp.were different.Among them,A.tenuissima A.alternata and A.longipes had strong virulence strains,while the strains of A.yaliinficiens showed weak virulence.Different temperatures and wetness duration affected virulence of A.longipes.The optimal temperature for disease is 25~30?,and the temperature is not suitable when lower than 15? or higher than 35?,does not develop at temperatures below 15? or above 35?.Wetness duration was positively correlated with disease severity.ISSR analysis technique was used to study the genetic diversity of A.longipes.The results showed that the Nei's diversity index was between 0.1118-0.2061,and the Shnanon's information index was between 0.1972~0.3177.The Nei's diversity index and Shnanon's information index of the Shiyan strains were the highest among the four geographical populations,and the Nei's diversity index and Shnanon's information index of the Yichang strain were relatively low compared to other geographical populations.The results showed there was abundant genetic diversity among the species.From the results of population clustering,Shiyan and Xiangyang,Enshi and Yichang are closely related to each other.The gene flow(Nm)of 4 different geographic populations was 3.2740,indicating that there were gene flow among populations of different geographical origins of A.longipes in Hubei Province.The UPGAM cluster analysis of 34 strains can divide the tested strains into 4 groups,of which group 2 is the dominant group including 31 strains.Cluster analysis showed that there was no significant correlation between the genetic polymorphisms and their geographical origin.