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Botrytis cinerea causes gray mold diseases in a broad range of plant species, in addition to its cold tolerance and fungicide resistance, Botrytis cinerea is one of the most necrotrophic pathogens. Botrytis cinerea 0023 is a strain isolated from grapes, typical for its mass sporulation. Our previous work showed that there is a certain relationship between ethylene and pathogenesis of strain 0023, and the strain can produce ethylene itself both in vivo and in vitro. In order to find the role of ethylene in the grey mould development, relative mutants experiment is necessary, in which the appropriate transformation method is the key technology. Taking 0023 as the material, established optimal condition of polyethylene glycol (PEG)-mediated protoplast transformation method, the transformation progress has been described in detail. In this experiment, we have screened nine transformants with fluorescence labeled, to bring them to infect grape and Physcomitrella patens, three of them appeared good disease-causing capability. On the other hand, Physcomitrella patens has many advantages for interaction reaction study, such as easy to culture and plenty genetic information. In this work, we have established a Botrytis cinerea-Physcomitrella patens disease reaction system. The results obtained were summarized as follows:1. PEG-mediated protoplast transformation method is suit for the Botrytis cinerea 0023 transformation. Concentration of 15mg/mL Lysing enzyme (Sigma L1412-25G) enzyme solution,25℃,100rpm,2h is the optimal condition.2.40% PEG 3350,0.6M KCl and 20% sucrose solution were used in transformation. SHA medium used in protoplast recovery, recovery time is 2 weeks, the recovery rate is 30%.3. Promoter Olic and terminator Trpc assist interested gene expression in strain 0023, each 1μg gene for 107/mL protoplast. Transformants obtained as the ratio of 1/106. 4. Transformant Gl showed green fluorescence, covered with petri dish in 5d, sporulation was 4.25104 spores/cm 2, the quantity of ethylene is 1.19μL/h, good cold tolerance, can infect grapes and Physcomitrella patens.5. Physcomitrella patens cultured in BCDATG medium, dropping 10μL spore suspension, the concentration of 107 spores/mL, result in the host disease development rapidly.6. Mucor racemosus cause moss wilting, which is the first report of Mucor racemosus as a moss pathogen.7. Established a Botrytis cinerea-Physcomitrella patens disease interaction system.This study established a reliable transformation method for strain 0023, and found the Botrytis cinerea-Physcomitrella patens interaction system is very useful in the pathogen reaction study.
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