甘蔗黑孢霉叶斑病病原菌分离鉴定及药剂筛选试验

Pathogen Isolation and Identification of Nigrospora Blight Disease on Sugarcane and Screening of Fungicides

【目的】明确引起甘蔗黑孢霉叶斑病的病原菌及其生物学特性,设计甘蔗黑孢霉叶斑病病原菌的检测引物,筛选能有效抑制病原菌Nigrospora sphaerica的杀菌剂,为甘蔗黑孢霉叶斑病的田间防治提供指导意见。【方法】对采自广东省广州市番禺区的发病黑皮甘蔗叶片采用组织分离法纯化培养,5 d后,光学显微镜下观察菌丝和孢子;对分离物进行致病性测定;基于ITS序列,采用MRGA7.0以最大似然法构建系统发育树并根据获得的ITS序列设计特异性检测引物Ng1F/Ng1R和Ng2F/Ng2R;采用菌丝生长速率法对7种不同杀菌剂对甘蔗黑孢霉叶斑病原菌N.sphaerica的室内抑菌效果进行筛选。【结果】通过病原菌形态和分子鉴定以及生物学特性研究,引起甘蔗黑孢霉叶斑病的病原菌为N.sphaerica,其最适培养条件为温度25℃和pH 7;Ng1F/Ng1R可用以甘蔗黑孢霉叶斑病原菌的检测;咪鲜胺药剂0.16 mg/L和施保功药剂2.4 mg/L对N.sphaerica的抑菌效果最显著,抑菌效果均可达到93%以上。【结论】引起甘蔗种传病害甘蔗黑孢霉叶斑病的病原菌为N.sphaerica,Ng1F/Ng1R可用于甘蔗黑孢霉叶斑病病原菌的检测,咪鲜胺和施保功药剂可作为甘蔗黑孢霉叶斑病的田间防治药剂或种苗脱毒处理药剂。

【Objective】The study was conducted to identify the pathogen causing Nigrospora blight on sugarcane,analyze the pathogen biological characteristics,design detection primers for the pathogen Nigrospora sphaerica,screen out fungicides which can effectively inhibit the pathogen N.sphaerica,and to provide guidance on field control of Nigrospora blight on sugarcane.【Method】The pathogen of Nigrospora blight on diseased sugarcane leaves collected from Panyu District,Guangzhou City,Guangdong Province was purified and cultured by tissue separation method.After 5 days of culture,the morphology of mycelium and spores was observed by using an optical microscope.The fungi were determined for pathogenicity,and the pathogenic strains were amplified and sequenced by PCR with the primers ITS1/ITS4;then,the software MRGA 7.0 was used to construct a phylogenetic tree based on ITS sequence with the maximum likelihood method and sequence-specific detection primers Ng1F/Ng1R and Ng2F/Ng2R were designed according to the obtained ITS sequence.The indoor control effects of 7 different fungicides on N.sphaerica were screened by the mycelium growth rate method.The antibacterial effect of N.sphaerica,which is the pathogen of Nigrospora leaf blight on sugarcane,was screened.【Result】Through the study on morphological and molecular identification as well as biological characteristics,it was determined that the pathogen causing Nigrospora blight on sugarcane was N.sphaerica,which had the fastest colony growth rate at the temperature of 25℃and pH of 7.The PCR method confirmed that the primers Ng1F/Ng1R could be used to detect the pathogen of sugarcane Nigrosporium blight.The results of the indoor antibacterial test showed that 0.16 mg/L of Prochloraz and 2.4 mg/L of Sporgon had the best antibacterial effect on N.sphaerica,and the antibacterial effect could reach over 93%.【Conclusion】The pathogen causing sugarcane seed-borne disease Nigrospora blight is N.sphaerica.Ng1F/Ng1R can be used to detect the pathogen of N.sphaerica.Prochloraz and Sporgon agents can be used as the optimal fungicides for N.sphaerica in the field or seedling detoxification treatment agent.