Identification Of Sporulational Group And Analysis Of Genetic Polymorphism Of Botrytis Cinerea

Botrytis cinerea Pers.:Fr.(the anamorph of Botryotinia fuckeliana) is a broad host range necrotrophic phytopathogen that affects more than200kinds of plants. It not only affects plants in the field, but also causes serious loss of fruits and vegetables at the postharvest stage. Gray mold pathogens found in table grapes could make latent infection at low temperatures. Botrytis cinerea is a species complex of the genus Botrytis, with high rate of multiplication, genetic variabilityand fitness. At present, reliable uniform standards can not be available for the classification of the species of the genus Botrytis. As a species complex, Botrytis cinerea has been frequently repoted about the difference of biological characteristics, pathogenicity and resistant to fungicides between isolates. Hence, dividing the species complex into different groups and analysing the genetic polymorphism of different isolates of Botrytis cinerea will lay a solid foundation of classification of species and subspecies, and it will provide useful reference of making meatures towards the disease. In the present study, the gray mold pathogens isolated from diseased grape berries and other fruits were classified into two sporulational groups:Group A and Group B, based on the differences of the timing of conidiospores formation and the number of conidiospores produced. In addition, we analyzed other morphological characteristics (growth rate, conidiospores produced, sclerotia, pigment, conidiospore size,vegetative compatibility, spore ornamentation and so on) and pathogenicity among the isolates in these groups. We also used molecular methods (RAPD and ISSR) to analyse and compare the genetic polymorphism and evolutionary relationships between the two groups.Results obtained are as follows:1. Based on the timing of conidiospores formation and the number of conidiospores produced at25℃, under natural illumination on PDA medium, the gray mold pathogens isolated from table grapes and other fruits were classified into two groups (Group A and Group B), group A were strains which produced conidiospores quickly (4days after incubation) and numerously, group B contained isolates which could hardly produce conidiospores on PDA under natural illumination until14days. Group A produced visible layer of conidiospores on PDA medium at25℃after7days of growth and then developed a thick mycelial layer producing abundant conidiospores at14days after incubation in the absence of UV illumination. After30days of incubation, spores density reached105-106spores/cm2. However, it was quite difficult for Group B to produce conidiospores on PDA under natural illumination until7days. They needed continuous UV radiation for sporulation on PDA in3to4days. The density reached only102-103spores/cm2after30days of incubation and no visible layer of spores could be seen. Further more, we compared other biological characteristics of the two groups, and found that there were also difference between growth rate, sclerotia, pigment and so on.2. All of the test isolates of B.cinerea were capable of infecting grapes5days after inoculation. In Group A, Bot-0164was the most virulent isolate, with the lesion diameter of26.20±1.99mm(5days after inoculation), while Bot-0081was the most virulent isolate of Group B, with the lesion diameter of23.24±1.58mm(5days after inoculation). Bot-0023and Bot-0510made the highest Pericarp cracking percent. And all of the isolates of Group A could produce spores on grape, but Group B isolates did not have this ability.3. The host range test of B.cinerea found that all of the isolates could infect tomato and carrot, and only Bot-0025, Bot-0081and Bot-0167could not affect pepper. In addition, all of the isolates of Group A could produce spores on grape, but Group B isolates did not have this ability.4. Spore ornamentation of Group A and Group B were quite different from each other. There was longitudinal veins on spores of group A, instead, spore ornamentation of group B was smooth and light. As a consequence, the ornamentation of spore surface could be another classification standard for group A and group B.5. Using a primer set to amplify fragments specific to B. cinerea, the results showed that a single band of729bp was amplified in all the22strains. What's more, amplification of Bc-hch gene fragment (1171bp) in B. cinerea showed that both Group A and Group B contained this fragment. It means that both Group A and Group B are typical B.cinerea.6. Using RAPD-PCR method, we got a random primer E17, which could amplify a600-700bp band in all of the test isolates and this primer could be used for molecular marker designment of B.cinerea. ISSR cluster analysis showed genetic differences between strains from two groups. Genetic distance between Group Ⅰ and Group Ⅱ was0.62, Group Ⅰ include11isolates of Group A, and Group Ⅱ include all of the strains of Group B and5strains of Group A. We could draw a conclusion that there was no positive correlation between the biological characteristics and genetic variability. On the whole, the22isolates of B.cinerea could be divided into two groups, Group A produced conidiospores quickly and numerously, and it did not need UV to promote conidiospores formation. The pathogenicity of Group A was high, and it could easily produce spores on hosts and medium, because traditional method could not control the spread of this group, therefore, it is a new harmful group. Our result not only lay a foundation of classification of species and subspecies of B.cinerea, but also provide a useful reference of making new control methods.