【作者】 张爱萍；

【导师】 于金凤；

【作者基本信息】 山东农业大学 ， 植物病理学， 2010， 硕士

【摘要】 随着城市化进程的不断加快,草坪在城市绿化中的作用也越来越明显。然而由于不当的水肥管理和修剪,草坪丝核菌病害的发生也越来越严重。目前,在对病害的防治中,提倡综合防治,而生物防治,尤其是拮抗木霉在土传病害防治中的作用尤为显著。本研究对山东省青岛、东营、泰安、上海市、广东省广州等地的草坪丝核菌进行了系统研究并筛选出了对优势融合群具有明显拮抗作用的生防木霉菌株。具体结果有如下几个方面:1.从山东省青岛、东营、泰安、上海市、广东省广州等地采集草坪丝核菌病害标本,采用常规组织分离法分离菌株获得48个丝核菌菌株。融合群测定结果表明,这些菌株分别属于多核丝核菌的WAG-Z、AG1-IA、AG4-HG-I融合群及双核丝核菌的AG-A、AG-P、AG-L融合群构成,其中WAG-Z群出现频率最高,占全部菌株的70.83%,为优势融合群;其次是AG-A群,占14.59%;再依次为AG1-IA群,占分离菌株的8.34%;AG4-HG-I、AG-P和AG-L群均有1个菌株,均占分离菌株的2.08%。另外AG-P群、AG-L群和AG-A群是国内外首次从草坪上分离得到的三个融合群。2.分离获得的草坪丝核菌,其菌丝经DAPI染色后制成玻片,置于荧光显微镜下观察,发现草坪丝核菌的细胞核相大致分两种:多核和双核,其中AG1-IA融合群的菌株细胞核数目多为5-11个;AG4-HG-I融合群的菌株细胞核数目为3-10个;WAG-Z融合群的菌株核相较为复杂,有单核细胞、双核细胞及多核细胞,其中以多核为主;AG-P、AG-L和AG-A融合群的菌株均为双核。3.利用5.8SrDNA-ITS区序列分析法,对草坪丝核菌不同融合群菌株进行了分子鉴定。结果表明,所有测试菌株其5.8S rDNA序列高度保守,ITS1、ITS2区存在不同程度的差异,隶属不同融合群或不同亚群的菌株其5.8S rDNA-ITS区序列存在较大的差异,而相同融合群或亚群菌株的一致性较高,利用此基因序列对丝核菌不同融合群或亚群的菌株可以进行有效区分和鉴定。4.运用灰色关联度分析法,对10个初筛木霉菌株的生长速度、耐性、产孢量、抗生作用等8个主要性状指标进行综合评价和分析。结果表明,木霉菌株T05-037的灰色关联度ri=0.9064最大,其次是T05-066和T05-048菌株,其灰色关联度分别为0.8714和0.8712。表明T05-037、T05-066、T05-048这三个木霉菌株的综合性状与构建的理想的参考菌株接近程度大,是比较理想的拮抗丝核菌的木霉生防菌株。其中,T05-037菌株的综合评价结果最好。5.本试验筛选获得的优良木霉菌株T05– 037,利用形态分类学的方法初步将其鉴定为绿色木霉(Trichoderma viride)。利用室内平板对峙方法评价木霉菌株T05-037对隶属不同融合群丝核菌的抑制作用。结果表明,T05-037木霉菌对供试的分属4个不同融合群的丝核菌菌株都有较强的抑制作用(抑制率为55.52%-64.49%),其中T05-037菌株对属于WAG-Z融合群的丝核菌菌株抑制率最高,说明木霉菌株T05–037对草坪丝核菌病害具有良好的生防潜力。更多还原

【Abstract】 Turfgrasses are more and more impotant in the urban afforestation, and now the disease of turfgrasses caused by Rhizoctonia spp is a common and important disease. In this study, we indentified the anastomosis group obtained from turfgrass in Qingdao, Taian, Dongying of Shandong Province, Shanghai and Guangzhou of Guangdong Province. Also we selected a high efficient antagonistic Trichoderma isolate to the the major anastomosis group of turfgrass Rhizoctonia. The results gained are summarized as following :1.Fourty-eight isolates were obtained from turfgrasses in several areas of China (including Qingdao, Taian, Dongying, Shanghai and Guangzhou). Anastomosis group indentification showed that the isolates belonged to multinucleat Rhizoctonia WAG-Z、AG1-IA、AG4-HG-I and binucleate Rhizoctonia AG-A、AG-P、AG-L.Of these, WAG-Z was the major anastomosis group(70.83% of total isolates),AG-A was the second anastomosis group ( 14.59% of total isolates) , AG1-IA was the third(8.34% of total isolates), there were all one strain of AG4-HG-I、AG-P and AG-L(2.08% of total isolates). AG-P、AG-L and AG-A were isolated for the first time from turfgrass in China.2.Putting the mycelium on the slide after DAPI, obsorbed under the microscope. The results showed that there were multinucleate and binucleate of the nucleus of the mycelium cell of Rhizoctonia from turfgrass. There were 5-11 nucleus of AG1-IA and 3-10 nucleus of AG4-HG-I. The nucleus of WAG-Z were complex, and the main was multinucleat Rhizoctonia. AG-P, AG-L and AG-A were binucleate Rhizoctonia.3.Phylogenetic analysis among isolates belonging to different AGs was studied based upon 5.8S rDNA-ITS sequences. The results obtained from nuclear and mitochondrial sequence analyses were similar. The sequence alignment of 5.8S rDNA-ITS revealed that the isolates of the same AGs had a high sequence similarity.The isolates of the different AGs had a low sequence similarity. So using 5.8sDNA-ITS sequence analysis was a effective method of the AGs identification.4.There were 8 biocontrol characters of 10 Trichoderma isolates (including growth, inhibitionrate, amount of sporulation, tolerate to low temperature, tolerate to high temperature, tolerate to acid, tolerate to alkaline and Tolerate to Carbendazim).They were evaluated and analyzed by Grey related degree.The results showed that the biggest Grey related degree was T05-037, the second one was T05-066 and the third was T05-048. The biocontrol characters of these three isolates were approximative to the reference. T05-037 was the best among these isolates.5.Using morphology analysis, we identified that isolate T05-037 that we selected in this study was Trichoderma viride. We studied the inhibition percentage of T05-037 to different anastomosis group by cultivated on the plate. The results showed that the isolate T05-037 is effectivity to the 4 isolates that belonged to different anastomosis group.The inhibitionrate to WAG-Z is the biggest. So we can conclude that T05-037 is potential to prevente the disease of turfgrass caused by Rhizoctonia spp.更多还原