| 金叶女贞棒孢叶斑病菌的生物学特性、致病性及系统发育 |
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| 引用本文： | 王树和,周彩勤,张奎望,刘慧迪.金叶女贞棒孢叶斑病菌的生物学特性、致病性及系统发育[J].浙江农林大学学报,2019,36(6):1174-1181. |
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| 作者姓名： | 王树和 周彩勤 张奎望 刘慧迪 |
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| 作者单位： | 河南科技大学 林学院, 河南 洛阳 471023 |
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| 基金项目： | 河南科技大学博士启动基金13480059 |
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| 摘 要： | 对新见的金叶女贞Ligustrum×vicaryi棒孢叶斑病病原多主棒孢Corynespora cassiicola进行形态学、生物学特性、致病性及分子系统发育研究，为该病的诊断和防控提供理论基础。对病叶进行保湿培养，观察病原菌孢子形态和大小；采用菌丝生长速率法测定不同温度、pH值、光照及碳氮源对病原菌菌丝生长的影响；采用离体叶片接种方法测定其对不同寄主致病性；将病原菌的rDNA-ITS，β-tubulin基因和ef-1α基因扩增测序后，在GenBank进行序列比对，用MEGA软件进行系统发育进化分析，采用邻接法构建系统发育进化树。结果表明:多主棒孢在自然发病叶片上形成的分生孢子比马铃薯葡萄糖琼脂培养基上形成的分生孢子具有更多的隔膜且更长。多主棒孢生长的最适温度为25℃，5℃不能生长；多主棒孢在pH值为4~11时都能生长，适宜生长的pH值为6~8；光暗交替（12 h光照/12 h黑暗）有利于菌丝生长。多主棒孢能够利用多种碳氮源，对麦芽糖利用率最高，蛋白胨和酵母膏是最适合的氮源。致病性测定表明:金叶女贞上分离C.cassiicola除了侵染金叶女贞外，还可侵染黄瓜Cucumis sativus，番茄Solanum lycopersicum，辣椒Capsicum annuum和茄S.melongena，不同植物叶片上的显症时间及病斑大小存在差异，金叶女贞表现最感病。基于rDNA-ITS序列、β-tubulin基因序列和ef-1α基因序列构建的系统发育树显示金叶女贞上分离菌株与黄瓜上分离的菌株归在一个分支，自展支持率为100%。
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| 关 键 词： | 微生物学 金叶女贞 叶斑病 多主棒孢 生物学特性 系统发育分析 |
| 收稿时间： | 2018-12-06 |
Characterization of Corynespora cassiicola from golden vicary privet based on phylogeny,biology, and pathogenicity |
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| WANG Shuhe,ZHOU Caiqin,ZHANG Kuiwang,LIU Huidi.Characterization of Corynespora cassiicola from golden vicary privet based on phylogeny,biology, and pathogenicity[J].Journal of Zhejiang A&F University,2019,36(6):1174-1181. |
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| Authors: | WANG Shuhe ZHOU Caiqin ZHANG Kuiwang LIU Huidi |
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| Institution: | College of Forestry, Henan University of Science and Technology, Luoyang 471023, Henan, China |
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| Abstract: | A new golden vicary privet (Ligustrum×vicaryi) leaf spot disease caused by Corynespora cassiicola was found in Luoyang, Henan Province. To provide basic theoretical knowledge for diagnosis and control of this disease, Corynespora cassiicola isolates collected from L.×vicaryi were investigated by morphology, biological characteristics, pathogenicity, and molecular phylogenetics. Morphological examinations were made from cultures sporulating on potato dextrose agar (PDA), as well as on host material. Diseased leaves were incubated in moist chambers to enhance sporulation for morphological observations. The effects of temperature, pH, and light treatment, as well as carbon and nitrogen sources on mycelial growth were determined. Pathogenicity tests were performed by inoculating detached leaves from L.×vicaryi, Cucumis sativus, Solanum lycopersicum, Capsicum annuum, and Solanum melongena using one representative isolate. Sequences of internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA), the β-tubulin (tub 2) gene, and the translation elongation factor 1α (ef-1α) gene were amplified, sequenced, and blasted in GenBank. Phylogenetic analysis was performed with the concatenated sequence alignment of three genes, (ITS, tub 2, and ef-1α) using MEGA 6.0 software. A multilocus phylogenetic tree was obtained using neighbor-joining analysis. Results showed variations in morphology among origin of the conidia of C. cassiicola. Compared to those on PDA, conidia obtained from infected leaves had higher numbers of pseudosepta, and were longer. Biological characteristics analysis showed that the fungus grew at temperatures ranging from 10℃ to 40℃ with optimum growth at 25℃ (P < 0.05) and no growth being observed at 5℃. The fungus grew at pH 4-11 and optimum growth was observed at pH 6-8. The light period of 12 h (light)/12 h (dark) promoted mycelial growth of C. cassiicola compared with dark treatment of all 24 h (P < 0.05). Maltose supplemented media showed maximum growth among the tested carbon sources (P < 0.05). Among several nitrogen sources tested, peptone and yeast extract paste were found superior for growth (P < 0.05). Pathogenicity tests revealed that the isolates from L.×vicaryi could infect L.×vicaryi, C. sativus, S. lycopersicum, C. annuum, and S. melongena, and that there were differences in the time of appearance and size of lesions according to the hosts. Ligustrum×vicaryi was most susceptible to the isolates that originated from L.×vicaryi. The phylogenetic tree showed that the three isolates of C. cassiicola from L.×vicaryi and all the isolates from C. sativus could be put into one clade (bootstrap value of 100%). These analyses showed that the phylogenetic lineages among the tested isolates correlated with host of origin and pathogenicity. |
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