一种葡萄贵腐灰葡萄孢的筛选鉴定

Isolation and identification of Botrytis cinerea for artificial noble rot grapes

为筛选可用于“人工贵腐葡萄酒”酿造的灰葡萄孢菌株,采用改良PDA培养基分离方法,从感染了灰霉病的果蔬组织中分离纯化出灰葡萄孢(Botrytis cinerea)疑似菌株,在乳酸石碳酸棉兰染色液染色下用显微镜对其进行形态学观察,并对其rDNA-ITS序列和RAPD片段进行扩增,对ITS序列进行测序,确定其灰葡萄孢的真菌分类地位。在不同pH值、不同侵染时间下,比较模拟葡萄汁中筛选菌株所产β-葡萄糖苷酶的活性,β-葡萄糖苷酶活性大小由模拟汁在400 nm处吸光值量化确定。研究发现,筛选的3株菌的β-葡萄糖苷酶活性均在pH值为6.0时最强,且 Botrytis-C菌株活性最高,Botrytis-T 所产β-葡萄糖苷酶的耐酸性较好;不同时间侵染的模拟汁中β-葡萄糖苷酶活性差异明显,Botrytis-C所产β-葡萄糖苷酶受侵染时间的影响最大,最不稳定。确定产孢能力强的两株灰葡萄孢适用于酿造“人工贵腐酒”的原料处理,菌株β-糖苷酶活性测试结果显示Botrytis-T用于葡萄“人工贵腐”的应用潜力更好。

To get appropriate Botrytis cinereas strain for artificial noble rot wine making,three suspected strains of Botrytis cinerea were isolated from fruit and vegetable samples inspected by gray mold. In this study,a classification of the genus was constructed based on morphological characters with modified PDA medium and microscopic observation,DNA sequence data of internal transcriped spacers (ITS) and electrophoretic bands of RAPD. In order to select the final strain adapted to artificial treating grapes,the β-glucosidase activity of strains at different levels of pH value and infection time in model grape juice were assayed. The three strains were identified as Botrytis cinerea strain. Three strains’β-glucosidase activity all showed highest level at pH 6.0,and Botrytis-C has the highest. Meanwhile, Botrytis-T behaved better at acid resistance. Clear differences existed in β-glucosidase activity of strains at different infection time,where Botrytis-C showed most unstable. The sporulation rate of Botrytis-T and Botrytis-C was higher. And the features of β-glucosidase activity indicated that Botrytis-T was the best to make artificial noble rot grapes.