Etiology And Biocontrol Screening Of Tobacco White Spot Disease

Phyllosticta nicotianae, also called tobacco fleck, is similar to the tobacco frogeye, tobacco Guignardia citricarpa and tobacco weather fleck, which makes it difficult to distinguish them and they often mixed easily. The pathogen of the Phyllosticta nicotianae is Phyllosticta nicotianae, which is considered as one of the genus of Phyllosticta, and Phyllosticta and Phoma both belong to the Sphaeropsidales, Coelomycetes, Deuteromycotina.Because of the morphological differences of the two genus are quite tiny and the definition standard of the classification is vague, which makes the classification more difficult. The basis of the traditional classification is mainly divided by the parasitic sites, namely, the parasitic sites that are on the leaves of plants are classified as Phyllosticta, that out of the leaves are classified as Phoma, however, in the practical identifications, the cases that the same genus of Phyllosticta or Phoma can parasitize on the leaves, stem, fruit and other parts of plants are often encountered, therefore, it is very difficult to identify the pathogen belongs to Phyllosticta or Phoma, what’s more, there are few research reports about this.In this study, the traditional identification of morphology and molecular biological identification were adopted, the samples that infected the pathogen were collected from 6 main tobacco production areas, the biological characteristics of the pathogen was studied and the antagonistic bacteria was also seclected. It is aimed to find out the taxonomic status of the pathogen of the Phyllosticta nicotianae, so the theoretical basis of prevention and cure of the Phyllosticta nicotianae can be provide. The research results are as follows:(1)The identification of the pathogen of the Phyllosticta nicotianae: the method of single spot separation of the infected leaves, testing the pathogenicity of the islotated strains, morphological identification and molecular biological identification were used in the research, the samples that infected the pathogen were collected from 6 main tobacco production areas, including Liaoning, Hei Longjiang, Guangxi, were isolated and identified. There were 31 suspected strains selected from more than 300 samples based on the morphological identification. The molecular biological identification indicated that: the differences of the ITS sequences of the strains were small and the similarities were high; the clustering analysis of the NCBI comparison showed that, 5strains among the 31 strains were Phoma.sp., 2 strains were Phomasis.sp., and 1 strain were Phyllosticta.sp. they were all indeterminate species, the result of sequencing the β-tubulin sequences suggested that 4 strains among the 31 strains were Phoma, the result of testing the pathogenicity of the islotated strains declared that they were all pathogenicity.(2)The research about the biological characteristics of the pathogen: the suitable medium for the Phyllosticta nicotianae was OA and PDA; the optimum pH was 5.0-7.0; the pathogen could grow at the temperature of 4-35℃, and at 25℃ the sporulation quantity and the germination rate of spores were maximum, when the temperature was over 40℃, there was no spores; the illumination had little influence on the diameter of the pathogen, but the alternation of light and darkness could promote the spore generation.(3)the selection of antagonistic bacteria and antibacterial effect: there were 20 strains of funus and 14 strains of bacteria separated and purified from tobacco rhizosphere soil, among them, 9 strains of fungus and 1 strains of bacteria had obvious effect of inhibition on the pathogen of Phyllosticta nicotianae. Plate confrontation was adopted to test the bacteriostatic activities of the 10 strains of fungus, 2 strains of bacteria and 5 kinds of pesticides, namely, Thiphanate methyl, Carbendazim, Duojunqing,Chlorothalonil and Hymexazol, the result manifested that: the fungus numbered asM7,M8,M9 and Duojunqing with the concentration of 25mg/μl had a better antibacterial effect, they could make the colonies of the pathogen not expend.