The Study Of Physicochemical Properties Of Toxin And Comparative Genomic Analysis Of Pathogen Of Tobacco Brown Spot

The pathogen of tobacco brown spot disease in Hunan Province were isolated and identified by morphological and molecular biological methods.On this basis,we did pilot study on the the biological characteristics,pathogenicity difference,toxin and comparative genomic analysis of DZ12 and Y1.The main results are as follows:1.Ten isolates,including N1,N2,N3,Y1,Y2,Y3,Y4,DB1,DB2 and DB3,were isolated from tobacco brown spot samples from three tobacco-producing areas in Changsha,Ningxiang and Yongzhou of Hunan Province.All of the isolates could be inoculated on the tobacco leaves and could develop into typical symptom of tobacco brown spot.We identified the 10 isolates from Hunan Province as Alternaria alternata by morphology and molecular evidence based on r DNA-ITS sequences.2.Alternaria altternata could grow at 15～37°C,and the most suitable temperature was 25～30°C.The sporulation of Alternaria alternata has a positive correlation with temperature,the growth was extremely suppressed and did not produce spores at 37°C.Alternaria anternata can utilize multiple carbon sources,such as glucose,lactose,maltose,sucrose and soluble starch.Soluble starch was selected as suitable carbon source for mycelial growth and sporulation of Alternaria alternata.The pathogenicity varied in different strains with different inoculation methods.We compared three inoculation methods,including mycelial pellets inoculation,spore suspension injection and broth immersion,and the results showed that spore suspension injection was the best inoculation method for tobacco brown spot disease.3.Pilot study on Alternaria alternata toxin showed that DZ12 had strong pathogenicity.In order to determine the pigment concentration of fermentation broth,the OD of fermentation broth under different wavelengths was measured,and the maximμm absorbance wavelength was set as 520 nm.The study showed that the pigment produced by DZ12 and the virulence was positively correlated with its OD.The broth have virulence when the OD₅₂₀was above 0.0772.The toxin was high stability and it still had obvious virulence after UV irradiation for 2 h,90°C water bath heating for 1 h and ultrasonic treatment for 1 h.4.The complete genome sequences of two tobacco brown spot pathogen strains DZ12 and Y1 were determined.A total of 10 chromosomes for each strain were assembled and they were highly collinear with other Alternaria alternata strains from Citrus,pear and strawberry.No conditionally dispensable chromosome（CDC）in the genome of pathogen of tobacco brown spot could be identified by comparing with the genome of other strains.Pathogens of Alternaria alternata on other plants usually contain CDC,and their host-specific toxin synthesis gene clusters are also generally located on CDC.However,no CDC was found in the genome of Alternaria alternata on tobacco,and no secondary metabolites synthesis gene clusters specific to DZ12and Y1.