【作者】 刘群；

【导师】 李向东；

【作者基本信息】 山东农业大学 ， 植物保护（专业学位）， 2015， 硕士

【摘要】 玉米是我国主要的粮食作物之一,又是重要的饲料作物和工业原料。目前其播种面积和总产量已超过水稻和小麦。然而病害一直是影响玉米生产的重要因素,其中小斑病(Bipolaris maydis Nisik&Miyake)和弯孢霉叶斑病(Curvularia lunata Boedijn)是玉米重要的叶部病害,危害逐年加重。特别是玉米弯孢霉叶斑病在我国玉米主产区发生面积逐年扩大,病情逐年加重,已由过去的次要病害逐渐发展成为主要病害。本研究分离得到山东玉米小斑病和弯孢霉叶斑病病菌,研究了其生物学特性,筛选到有效的化学药剂和生防菌株,为玉米小斑病和弯孢霉叶斑病的防治提供理论依据。本论文的主要研究结果如下:从枣庄、泰安、临沂、聊城等地采集病叶,分离得到病原真菌,根据分生孢子形态学特征和ITS聚类分析结果发现,引起山东玉米小斑病和弯孢霉叶斑病的病原分别是Bipolaris maydis Nisik&Miyake和Curvularia lunata Boedijn。玉米小斑病菌分生孢子萌发对温度的要求不严格,在5~35℃之间均可以萌发,其中25℃最适宜萌发,萌发率达到100%。RH=100且保湿时间2h以上,玉米小斑病菌分生孢子即可萌发,保湿8h时,孢子萌发率达到100%。光照对玉米小斑病菌分生孢子萌发的影响差异不大。温度在15~35℃之间玉米小斑病菌菌丝均可生长,其中25~30℃最适合生长。玉米弯孢霉叶斑病菌分生孢子在10~35℃之间均可以萌发,其中25~30℃萌发率均达到100%。RH=100且保湿时间2h以上,玉米弯孢霉叶斑病菌分生孢子即可萌发,保湿10h时,孢子萌发率达到100%。光照对玉米弯孢霉叶斑病菌分生孢子萌发的影响差异不大。温度在15~35℃之间菌丝均可生长,其中30℃最适合菌丝生长。分别测定了14种和16种药剂对玉米小斑和弯孢霉叶斑病菌的抑制效果,发现30%氟菌唑可湿性粉剂、30%苯甲醚菌酯悬浮剂、22.5%啶氧菌酯悬浮剂、42.8%氟菌肟菌酯悬浮剂、75%肟菌戊唑醇水分散粒剂、500g/L异菌脲悬浮剂、250g/L吡唑醚菌酯乳油、500g/L氟啶胺悬浮剂能有效抑制两种病菌菌丝的生长,其中30%氟菌唑可湿性粉剂、30%苯甲醚菌酯悬浮剂抑菌效果最好,对玉米小斑病菌和弯孢霉叶斑病菌的EC50分别为0.0277mg/L和0.058mg/L、0.1987mg/L和0.1279mg/L。初步筛选到32株生防菌,通过生防菌的复筛,共筛选到5株对玉米小斑病菌和6株对玉米弯孢霉叶斑病抑制作用较好的生防菌,其中CLS4、CLS11、CLS20、CLS23四株生防菌对玉米小斑病和弯孢霉叶斑病的的抑菌效果最好,通过16s rDNA序列测定鉴定,CLS23为解淀粉类芽孢杆菌(Bacillus amyloliquefaciens),CLS11为洋葱伯克霍尔德氏菌(Burkholderia cepacia),CLS20为新洋葱伯克霍尔德氏菌(Burkholderia cenocepacia),CLS4为类芽孢杆菌(Paenibacillus jamilae)。基于玉米小斑病菌5.8SrDNA-ITS序列和玉米弯孢霉叶斑病菌Clg2p基因序列,设计了玉米小斑病菌和弯孢霉叶斑病菌的特异性引物对BMF-3、BMR-3和CLF-4、CLR-4,利用CTAB法提取玉米小斑病菌和弯孢霉叶斑病菌的DNA,以玉米弯孢霉叶斑病菌、玉米小斑病菌和玉米大斑病菌的DNA为模板对所涉及的引物进行了特异性检测,电泳结果显示以引物对BMF-3、BMR-3为引物时,只有以玉米小斑病菌的DNA为模板时可扩增产生目的片段。以引物对CLF-4、CLR-4为引物时,只有以玉米弯孢霉叶斑病菌DNA为模板时可扩增产生目的片段。更多还原

【Abstract】 Maize is important for crop production, industrial raw materials and animal feeds in China. The maize’s growing area and yield is the first one followed by rice and wheat. Maize disease especially the Bipolaris maydis Nisik & Miy and Curvularia lunata Boedijn are the important factors affecting maize quality and production. The problem became worse because of knowing little about the pathogen and lacking effective control methods.In this study, Pathogen Bipolaris maydis and Curvularia lunata were isolated and characterized. Effective agrochemicals and bio-control bacterial strains were screened. The main results obtained in this study are listed as follows:Diseased leaf and rhizosphere samples were collected from Zaozhuang, Taian, Linyi, Liaocheng. According to the morphological and ITS cluster analysis, Pathogen were Bipolaris maydis Nisik & Miyake and Curvularia lunata Boedijn.Pathogen Bipolaris maydis and Curvularia lunata were isolated and their biological properties were characterized. Spores of the Bipolaris maydis could germinate between 5-35°C, in which the germination rate could reach 100% at 25°C. With 100% relative humidity(RH), spores started to germinate after 2 hours and germination rate reached 100% after 8 hours. No significant difference was found with different light treatment on the spores’ germination. Hyphal could grow between 15-35°C, moreover, 25-30°C was the most suitable temperature for hyphal growth.Curvularia lunata could germinate between 10-35°C; the germination rate could reach 100% at temperature between 25-30°C. Similar to Corn southern leaf blight, spores were starting germination at RH 100% for 2 hours. The germination rate reaches 100% after incubating 10 hours at RH 100%. No obvious difference was found with different light treatment on the spores’ germination. Hyphal could grow between 15-35°C, in which 30°C was the most suitable temperature for hyphal growth.Nine agrochemicals were found which can suppress the hyphal growth of 35°C, in which 30°. In which 30% Triflumizole wettable powders aqueous solutions showed the best suppression effect with EC50 value of 0.0277mg/L. The second one was 42.4% Imidazole fluorine amide suspending agent with EC50 value of 0.1987mg/L.Eight agrochemicals showed suppression ability on hyphal growth of Curvularia lunata. The 30% Triflumizole wettable powders aqueous solutions Curvularia lunata. The 30% Triflumizole wettable powders aqueous solutions has the best performance with EC50 value of 0.058mg/L, followed by 30% benzyl-kresoxim-methyl suspending agent with EC50 value of 0.1279mg/L.Five and six bacterial species have shown ability to inhibit the hyphal growth of Corn southern leaf blight and Curvularia lunata, respectively. Four(designated as CLS23, CLS11, CLS20 and CLS4) which have the best performance for both pathogen were Bacillus amyloliquefaciens, Burkholderia cepacia, Burkholderia cenocepacia and Paenibacillus jamilae.Based on the sequence of 5.8S and ITS of Bipolaris maydis Nisik & Miy and Clg2 p of Curvularia lunata Boedijn,we designed specific primer pairs BMF-3,BMR-3 and CLF-4,CLR-4. Bipolaris maydis Nisik & Miy,Clg2 p of Curvularia lunata Boedijn and Exserohilum turcicum(Pass.)Leonard et Suggs were used as templates to detect the specific of the primer pairs,conventional PCR test results showed that the specific primers designed could effectively amplify the DNA of Bipolaris maydis Nisik & Miy and Curvularia lunata Boedijn and gained about 200 bp fragments.更多还原