河北省大豆主要病原真菌鉴定及防治药剂筛选

Identification of Major Pathogenic Fungi of Soybean in Hebei Province and Screening of Control Fungicides

【目的】明确引起河北省大豆植株主要病害的病原真菌种类,筛选能有效抑制所鉴定病原菌的杀菌剂,为河北省大豆病害化学防治提供依据。【方法】对采自河北省的大豆主要病原真菌根据病症采用组织分离法纯化培养,初步利用超景深和正置光学显微镜分别进行菌落、菌丝和孢子形态学的观察鉴定,然后利用通用引物ITS1-ITS4对5种不同病症的致病菌株rDNA-ITS区进行PCR扩增、测序分析,采用MEGA 7.0中邻接法(neighbor-joining,NJ)构建系统发育树,分析病原菌种之间的亲缘关系,对具有特异性引物序列的病原菌进行分子鉴定,通过病害和病原菌宏观形态、显微特征与分子生物学技术相结合从而明确引起河北省大豆植株主要真菌病害的病原菌种类;采用菌丝生长速率法和不同种类杀菌剂在离体叶片或幼茎上对大豆主要病害的防治效果筛选防治药剂。【结果】通过病原菌的宏观形态、显微结构和分子序列综合分析,确定分离自河北省大豆主产区的病原菌分别为木贼镰孢(Fusarium equiseti)、大豆炭疽菌(Colletotrichum chlorophyti)、茎点霉菌(Phoma herbarum)、链格孢(Alternaria alternata)、嘴突凸脐蠕孢(Exserohilum rostratum),对应病害分别为大豆根腐病、炭疽病、茎点霉叶斑病、大豆黑斑病、凸脐蠕孢叶斑病。毒力测定结果显示,5种病原菌对三唑类杀菌剂苯醚甲环唑、氟菌唑、叶菌唑和甲氧基丙烯酸酯类杀菌剂吡唑醚菌酯均敏感,木贼镰孢对甲氧基丙烯酸酯类杀菌剂肟菌酯敏感,除木贼镰孢外的病原菌对酰胺类杀菌剂氟吡菌酰胺敏感,大豆炭疽菌和嘴突凸脐蠕孢对烷基多胺类杀菌剂辛菌胺敏感,大豆炭疽菌对有机硫类仿生杀菌剂乙蒜素敏感,EC50值均低于10μg·mL-1;杀菌剂对大豆主要病害的防治效果结果显示,苯醚甲环唑、氟菌唑、叶菌唑、吡唑醚菌酯和肟菌酯对上述大豆主要病害防治效果均显著,氟吡菌酰胺对除大豆根腐病外的病害防治效果显著,辛菌胺、乙蒜素对大豆炭疽病和凸脐蠕孢叶斑病防治效果显著,防治效果分别达到90%以上。【结论】共鉴定出河北省大豆病原真菌及其对应病症病害5种;推荐三唑类甾醇抑制剂苯醚甲环唑、氟菌唑、叶菌唑和甲氧基丙烯酸酯类呼吸抑制剂吡唑醚菌酯作为河北省大豆主要真菌病害的优选兼治药剂。

【Objective】The objective of this study is to identify the fungal pathogens causing the main diseases of soybean plants in Hebei Province,screen the fungicides which can effectively inhibit the identified pathogens,and to provide a basis for chemical control of soybean disease in Hebei Province.【Method】The main pathogens of soybean collected from Hebei Province were isolated and purified according to the disease symptom.The morphology of colony,mycelium and spore was observed and identified by using 3D ultra depth of field digital microscope and Olympus Microscope.The rDNA-ITS regions of five pathogenic strains were amplified by PCR and sequenced by universal primers ITS1-ITS4.Neighbor-joining method of software MEGA 7.0 was used to construct phylogenetic trees and analyze the relationship among different pathogens.The pathogens with specific primer sequences were identified and verified.The main pathogen kinds of soybean plants in Hebei Province were identified by the combination of macromorphology,microscopic characteristics and molecular biology techniques.The method of mycelium growth rate and the control effects of different fungicides on the major diseases in vitro leaves or young stems of soybean were used to screen the fungicides.【Result】Based on the analysis of macromorphology,microstructure and sequence,the pathogens isolated from the main soybean producing areas in Hebei Province were Fusarium equiseti,Colletotrichum chlorophyti,Phoma herbarum,Alternaria alternata and Exserohilum rostratum.The corresponding diseases were soybean root rot,anthracnose,leaf spot of P.herbarum,black spot and leaf spot of E.rostratum.The results of virulence test showed that the above pathogens were all sensitive to the triazole fungicides,such as difenoconazole,triflumizole,metconazole and Qoi fungicide pyraclostrobin.F.equiseti was sensitive to Qoi fungicide trifloxystrobin,the pathogens except F.equiseti were sensitive to amide succinate dehydrogenase inhibitors fluopyram,C.chlorophyti and E.rostratum were sensitive to alkyl polyamines fungicide xinjunan,C.chlorophyti was sensitive to organic sulfur bionic fungicide ethylicin,the EC50 values of the inhibitors were less than 10μg·mL-1.The control results of different fungicides on the major soybean diseases showed that the fungicides of difenoconazole,triflumizole,metconazole,pyraclostrobin,trifloxystrobin significantly inhibited above diseases,fluopyram significantly inhibited the diseases except soybean root rot,xinjunan and ethylicin significantly inhibited anthracnose and leaf spot of E.rostratum,the control effects were above 90%.【Conclusion】Five kinds of pathogen and disease of soybean in Hebei Province are identified.The triazole sterol biosynthesis inhibitors difenoconazole,triflumizole,metconazole and Qoi respiration inhibitor pyraclostrobin were recommended as the optimal fungicides for soybean fungal diseases in Hebei Province.