The Pathogenicity Of Botrytis Cinerea Cutinase And The Gene Analysis Of Cutinase Mutants

Botrytis cinerea Pers. is an important plant pathogenic fungi, which could produce many kinds of degradative enzymes during the disease development. The pathogenic fungi could secrete cutinase to degrade the cutin of plant surface. Knowing the role of cutinase in the stage of the pathogen penetration, we could take more understanding about the pathogenic mechanisms of Botrytis cinerea.Two mutants H2-M1and H2-M2, with the cutinase activity enhancing were obtained using UV radiation from H2strain. The levels of cutinase activity of H2-M1and H2-M2were34.68U/mL and33.51U/mL, which were2.05and1.98times that the primary strain H2respectively. At one time the mutants H2-M3and H2-M4with activity declining of cutinase were obtained using the same method. The cutinase activity was3.80U/mL and3.98U/mL, which were22.5%and23.5%of the primary strain H2. The cutinase activity of the primary strain H2was16.92U/mL. Cutinase could be secreted by the germ-tube of H2, H2-M1, H2-M2, H2-M3and H2-M4during the conidia germination, but the activity was lower than that their mycelium. The cutinase activity by the germinating spores of H2-M1, H2-M2, H2-M3and H2-M4were0.20U/mL、0.18U/mL、0.01U/m and0.01U/mL respectively, while the enzyme activity of the primary strain H2was0.10U/mL. The pathogenic factors of these mutants were tested including pectinase, cellulose and toxin. The results exhibited that the activity of the pectinase, cellulose and toxin from these mutants was not remarkable different comparing with that of the primary strain.The pathogenicity of5mutants strains were tested by inoculating conidial suspension on the surface of tomato and strawberry leaves, and of mycelial cake on the fruits. The results showed that virulence to tomato leaf and fruit of the two mutant strains of H2-M1and H2-M2which had stronger ability of producing the enzyme was significantly higher than that of the primary strain. Meanwhile, it was observed that the markedly decrease in pathogenicity of the H2-M3and H2-M4with lower cutinase activity. The pathogenic variation of the mutants on the strawberries fruit and leaf was accordant with on the tomato fruit and leaf. The previous experiments exhibited that the cutinase of Botrytis cinerea played an important role in the pathogen penetrating plant and could increase the pathogen virulence to the host.Cutinase gene had been cloned from the mutants and primary strain. Although there were several different base in the cuinase gene of the mutants comparing with the primary strain by sequence analysis, those variance sites were not in the position of the enzyme activity center or in the combination position with cutin. Otherwise, some variation could not lead to the change of amino acid composing. According to Real-time qPCR, cutinase gene could be expressed in mutants, but there were some difference in different strains. The results that the expression quantity of H2-M1and H2-M2was more twice what the primary strain H2was. H2-M3and H2-M4cutinase gene expressed significantly declined, which the percentage was21%and19%of the primary strain. Basing on the above experiments we can concluded that the cutinase gene had no difference between the mutants and primary strain, and the variation of the cutinase activity could be caused by the change of transcription.