【作者】 吴小平；

【导师】 胡方平；

【作者基本信息】 福建农林大学 ， 植物病理学， 2008， 博士

【摘要】 对采自福建、浙江等省19个地方的95份木霉污染的食用菌菌筒和子实体样品进行分离,获得了49株木霉菌株,并对这49株木霉菌株进行了形态学、ITS/5.8S、RAPD鉴定分析以及生物学特性测定,调查了不同木霉侵染食用菌的能力以及食用菌菌株对木霉的抵抗力,探讨了枯草芽孢杆菌BS-2对木霉的抑制作用并对枯草芽孢杆菌产生的可用于防治食用菌真菌病害的活性物质进行了分析,同时了解了其活性物质的成分。具体结果如下:形态学方法以及ITS/5.8S测序结果表明,49株木霉菌株分别是哈茨木霉Trichoderma harzianum Rifai,长枝木霉T.longibrachiatum Rifai,深绿木霉T.atroviride Karsten和棘孢木霉T.asperellum Samuels 4种木霉菌。形态学与ITS鉴定结果一致;棘孢木霉首次在食用菌中发现,深绿木霉分离到少量菌株,哈茨木霉和长枝木霉出现频率最高,分别为59%和31%,似乎是食用菌栽培中优势污染种群;同时发现木霉病原菌的种类与采集地点、食用菌的种类有一定相关,如在浙江庆元香菇Lentinus edodes(Berk.)sing菌筒中分离的木霉主要是哈茨木霉,在广州刺芹侧耳(杏鲍菇)Pleurotus enyngii菌筒中分离的木霉主要是棘孢木霉,而从福建浦城香菇污染袋中分离的木霉主要是深绿木霉。哈茨木霉、长枝木霉、深绿木霉和棘孢木霉的ITS总长分别为:575bp-578bp、579bp-583bp、565bp-567bp、和560bp-561bp,GC含量分别为:55.6%-56.7%、57.2%-58.0%、55.6%-56.1%和55.8%-55.9%;所有木霉5.8S序列完全相同,总长为159bp,GC含量为46.5%。不同地理来源的49株木霉用5个RAPD引物扩增后分析结果表明,种间和种内具丰富的遗传多样性,菌株间差异与地理来源和寄主有一定相关。系统发育树反映的种间关系与形态分类结果基本一致,不同地理来源的相同种可以通过RAPD分析区分开来。木霉的生物学特性研究结果表明,木霉的最适温度、最适含水量和最适生长pH分别28℃、70%和5.7。木霉对35℃以上高温的适应性差异较大,木霉在高温干燥环境易产孢子,在潮湿环境孢子最易萌发,菌丝生长旺盛。在pH为11的培养基中不能生长。对不同木霉侵染食用菌的能力进行了研究,结果表明不同木霉对食用菌的侵染能力存在差异,侵染食用菌能力强的木霉对植物病原真菌的侵染能力并不一定强,这表现出木霉对真菌侵染的特异性。探讨了木霉侵染食用菌的机理,发现木霉可通过溶壁、缠绕等方式作用于食用菌菌丝细胞壁,也可通过产生挥发性物质、非挥发性物质抑制菌丝生长,但木霉产几丁质酶的能力以及木霉的生长速度与木霉的侵染能力没有直接相关。木霉经过食用菌菌丝诱导后,其发酵液对食用菌菌丝的抑制作用增强。通过PDA平板拮抗实验,评价了食用菌菌株的抗木霉能力,从408株不同的食用菌菌株中筛选到34株对木霉(63)有一定抵抗力的菌株,但经过菌袋复筛,发现经过初筛筛选出来的菌株对侵染力强的木霉(52)没有抵抗力。PDA平板筛选时还发现,检测的所有香菇Lentinus edodes、高温平菇Pleurotuscystidiosus、灰树花Grifola frondosa和杏鲍菇Pleurotus eryngi的菌株对木霉都没有抗性。通过对峙培养、发酵液的抑菌实验,发现枯草芽孢杆菌BS-2对脉孢菌Neurospora、木霉、疣孢霉Mycogone都有较强的抑制作用,而且BS-2还会产生挥发性物质抑制木霉孢子的萌发。通过对BS-2进行紫外线诱变,得到了一株对脉孢菌的抑菌活性增加215%的菌株(BS-2-E)。枯草芽孢杆菌是食用菌栽培中的污染菌,但可以利用枯草芽孢杆菌产生的活性物质来防治食用菌真菌病害。枯草芽孢杆菌产生的脂肽类抗生素有表面活性素(surfactin)、伊枯草菌素(iturin)和芬枯草菌素(fengycin),枯草芽孢杆菌BS-2以及BS-2的突变株BS-2-N和BS-2-E发酵液粗提样的LC-MS检测及比较分析,初步认为枯草芽孢杆菌BS-2的主要脂肽类抗生素是芬枯草菌素B和伊枯草菌素。更多还原

【Abstract】 Forty-nine strains of Trichoderma spp.were isolated from 95 samples of spawn and fruiting bodies of edible fungi collected from nineteen regions in Fujian and Zhejiang provinces.The taxonomy,biological characteristics and biocontrol of the strains were investigated,The results were as followes.The morphological characteristics showed that the strains could be divited into four groups,belonging to T.harzianum,T.longibrachiatum,T.atroviride and T. asperellum respectively.The result of ITS analysis was coincident with the morphological classification.The species T.asperellum was first found to contaminate edible fungi,The species,T.atroviride was rarely present,and the frequencies of appearance of species,T.harzianum and T.longibrachiatum were 59%and 31%respectively,seeming to be the dominant contaminative fungi in mushroom cultivation.The species of Trichoderma varied with collective location and mushroom species.The strains isolated from spawn of Lentinula edodes in Qingyuan country of Zhejiang province,were most of the species,T.harzianum.The strains isolated from spawn of Pleurotus eryngii in Guangzhou,were most of the species,T.asperellum,but that isolated from lentinula edodes in Pucheng of Fujian province,were most of the species,T.atroviride.The lengths of the ITS of the tested strains of T.harzianum,T.longibrachiatum, T.atroviride and T.asperellum were 575bp-578bp,579bp-583bp,565bp-567bp and 560bp-561bp,respectively.Their G+C mol%were 55.6%-56.7%,57.2%-58.0%, 55.6%-56.1%and 55.8%-55.9%.The length of 5.8 S of T.spp.was159 bp,and G+C mol%was 46.5%.The forty-nine strains were PCR using five RAPD primers.The results showed that among the strains there was genetic diversity.In the constructed phylogenetic tree,these strains were not clustered according to their geographical origins or hosts. The relationships of interspecies showed in phylogenetic tree were basically consistent with the results of morphological study.The strains of the same species isolated from diferent geographic origins could be clearly distinguished in the tree.Biological characteristics study of Trichoderma spp.showed their growth optimum temperature,water content and pH was 28℃,70%and5.7,respectively. Some of them could grow from 35℃to 40℃,and the lethal pH for mycelia was 11. They could produce conidia easily at high temperatures and under dry conditions, while the mycelia grow well in the wet environments.The infective ability of Trichoderma hyphae to the edible fungi was investigated, the results indicated that different Trichoderma strains displayed different infection ability.It was also found that the fungi Trichoderma had strong infective ability to mushroom but not to phytopathogenic fungi,which also showed that there was infective specificity to Trichoderma towards different fungi.The infective mechanisms of the fungi to mushroom were,that they could infect edible mushroom by lysing or winding the cell walls of mushroom mycelia,or inhibit hyphal growth based on the volatile substances or non-volatile substances generated.However,no direct relationships were proved between the chitinaseoproducing capacity or growth velocity and the invasiveness of the fungi.Inhibitory effects of the fungi Trichoderma could be induced by mushroom mycelia.408 strains of edible fungi were evaluated for the resistance to the fungi Trichoderma.The results indicated 34 strains of Pleurotus ostreatus were resistance to Trichoderma sp No63 by the screening with PDA Plate,but had no resistance to Trichoderma sp No52 with high infective ability by the cultivation re-screening.The results of the screening with PDA Plate also showed that Lentinus edodes,Pleurotus cystidiosus,Grifola frondosa and Pleurotus eryngi were no resistance to the fungi Trichoderma.The results of confrontation culture and anti-bacterial experiment indicated that Bacillus subtilis BS-2 had strong inhibition to the fungi Neurospora,Trichoderma and Mycogone,and could produce the volatile substance to inhibit the germination of the spores of the fungi Trichoderma.By the UV mutagenesis for BS-2,the anti-bacterial activity of the mutant,BS-2-E,increased up to 215%.The substance produced by B. subtilis could be used to control fungal diseases of edible fungi although B.subtilis might cause the pollution of edible fungi cultivation.The lipopeptides produced by B.subtilis included surfactin,iturin and fengycin. Fengycin B and iturin was the main based on the analysis of LC-MS detection of the crude extracts from the fermentation liquids of B.subtilis BS-2 and its two mutants-BS-2-N and BS-2-E.更多还原