耐苯并咪唑类杀菌剂木霉菌株的筛选及耐药机理研究
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摘要
本文进行了生防木霉菌株的筛选以及生防效果的测定，通过在含多菌灵平板培养基、紫外线照射和亚硝基胍对菌株进行诱导，筛选耐苯并咪唑类杀菌剂的木霉菌株，并对木霉菌耐病相关蛋白进行了分离纯化及理化特性测定。
自然界分离的木霉菌株，经离体筛选和温室活体筛选，共获得8株木霉生防菌株（Ty-10-2，LT-2，Tl-1-2，Tj-5-4，Ty-11-1，Tj-11-3，Ty-11-3，Tj-3-3）。这些菌株生长速度快、抑菌作用强，抑菌效果都在70%以上，活体筛选的防效达50%以上，能有效防治黄瓜灰霉病，且效果稳定。
将获得的8株木霉菌在含药平板培养基中培养、紫外线照射和用亚硝基胍诱导。在含药平板培养基中培养筛选得到Ty-11-3，Ty-10-2，Tj-5-4和LT-2 4株耐多菌灵木霉菌。用紫外线和亚硝基胍诱导没有筛选出耐药性菌株。供试菌株在含药平板培养基上培养24h，耐药菌株分生孢子萌发，不耐药菌株的分生孢子不萌发，且耐药菌株的菌丝前端畸形，较正常菌丝细短，生长紧凑。
可溶性蛋白和酯酶同工酶电泳分析表明，耐药菌株中可溶性蛋白种类及含量发生变化，细胞内可溶性蛋白总含量增加。究其原因可能诱导处理使原木霉菌株中一些基因发生突变，或者产生耐性基因，使耐药相关蛋白产物增多，同时一些基因转录活性降低或者不转录，导致相关蛋白减少。酯酶同工酶的测定结果显示，耐药菌株条带减少或者减弱，可能经过含药培养基的诱导处理，编码酯酶同工酶的基因发生突变，同时也有一些基因的转录活性减弱甚至消失。
The screening of biological isolates and the assay of biocontrol mechanism of Trichoderma were studied systematically in laboratory chamber and greenhouse in vivo. After the tolerant related proteins in Trichoderma strains purified, their physical and chemical characters were detected.
Eight biocontrol isolates were screened from isolates by comparing their biological activities in vitro and vivo in greenhouse, including Ty-10-2, LT-2, Tl-1-2, Tj-5-4, Ty-11-1, Tj-11-3, Ty-11-3, Tj-3-3, which had fast growth rates and higher inhibition to gray mould (Bortrytis cinerea), and had stable inhibition. The disease could be controlled by 70 percent and 50 percent in vivo.
Four isolates were screened from isolates by induced by the substrate with carbendazim, UV rays and nitrosoguanidine, including Tj-5-4, Tj-3-3, Ty-11-3, LT-2. None had screened from UV rays and nitrosoguanidine. After cultured for 24 hours, conidiospores of tolerant isolates were germinated but the ones of sensitive isolates didn't. Compared with the sensitive isolates, the hyphae front of the tolerant isolates is thinner and shorter.
Research of biocontrol mechanism showed the soluble proteins in tolerant isolates increased, some genes had mutated, transcripted in different levels and then translated into some tolerant related proteins, while some genes had opposite changes. On the whole the soluble proteins in the cells had increased. The esterase isoenzymes in the tolerant isolates decreased in different levels. After induced some genes had mutated and some genes transcription decreased and even diminished.
自然界分离的木霉菌株，经离体筛选和温室活体筛选，共获得8株木霉生防菌株（Ty-10-2，LT-2，Tl-1-2，Tj-5-4，Ty-11-1，Tj-11-3，Ty-11-3，Tj-3-3）。这些菌株生长速度快、抑菌作用强，抑菌效果都在70%以上，活体筛选的防效达50%以上，能有效防治黄瓜灰霉病，且效果稳定。
将获得的8株木霉菌在含药平板培养基中培养、紫外线照射和用亚硝基胍诱导。在含药平板培养基中培养筛选得到Ty-11-3，Ty-10-2，Tj-5-4和LT-2 4株耐多菌灵木霉菌。用紫外线和亚硝基胍诱导没有筛选出耐药性菌株。供试菌株在含药平板培养基上培养24h，耐药菌株分生孢子萌发，不耐药菌株的分生孢子不萌发，且耐药菌株的菌丝前端畸形，较正常菌丝细短，生长紧凑。
可溶性蛋白和酯酶同工酶电泳分析表明，耐药菌株中可溶性蛋白种类及含量发生变化，细胞内可溶性蛋白总含量增加。究其原因可能诱导处理使原木霉菌株中一些基因发生突变，或者产生耐性基因，使耐药相关蛋白产物增多，同时一些基因转录活性降低或者不转录，导致相关蛋白减少。酯酶同工酶的测定结果显示，耐药菌株条带减少或者减弱，可能经过含药培养基的诱导处理，编码酯酶同工酶的基因发生突变，同时也有一些基因的转录活性减弱甚至消失。
The screening of biological isolates and the assay of biocontrol mechanism of Trichoderma were studied systematically in laboratory chamber and greenhouse in vivo. After the tolerant related proteins in Trichoderma strains purified, their physical and chemical characters were detected.
Eight biocontrol isolates were screened from isolates by comparing their biological activities in vitro and vivo in greenhouse, including Ty-10-2, LT-2, Tl-1-2, Tj-5-4, Ty-11-1, Tj-11-3, Ty-11-3, Tj-3-3, which had fast growth rates and higher inhibition to gray mould (Bortrytis cinerea), and had stable inhibition. The disease could be controlled by 70 percent and 50 percent in vivo.
Four isolates were screened from isolates by induced by the substrate with carbendazim, UV rays and nitrosoguanidine, including Tj-5-4, Tj-3-3, Ty-11-3, LT-2. None had screened from UV rays and nitrosoguanidine. After cultured for 24 hours, conidiospores of tolerant isolates were germinated but the ones of sensitive isolates didn't. Compared with the sensitive isolates, the hyphae front of the tolerant isolates is thinner and shorter.
Research of biocontrol mechanism showed the soluble proteins in tolerant isolates increased, some genes had mutated, transcripted in different levels and then translated into some tolerant related proteins, while some genes had opposite changes. On the whole the soluble proteins in the cells had increased. The esterase isoenzymes in the tolerant isolates decreased in different levels. After induced some genes had mutated and some genes transcription decreased and even diminished.
引文
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