白三叶草一种叶斑病病原菌的分离鉴定

doi:10.11924/j.issn.1000-6850.casb2021-0031

中国农学通报 ›› 2022, Vol. 38 ›› Issue (4): 81-85.doi: 10.11924/j.issn.1000-6850.casb2021-0031

所属专题：生物技术；植物保护

白三叶草一种叶斑病病原菌的分离鉴定

商娜¹^,²(), 任爱芝¹, 刘冰¹, 赵培宝¹()

¹聊城大学农学院,山东聊城 252000
²聊城市农业科学院,山东聊城 252000

收稿日期:2021-01-13 修回日期:2021-11-26 出版日期:2022-02-05 发布日期:2022-03-16
通讯作者: 赵培宝
作者简介:商娜,女,1989年出生,山东济宁人,农艺师,硕士研究生,主要从事植物病理学研究。通信地址：252000 山东省聊城市东昌府区聊大西路11号,E-mail： shangna126@126.com。
基金资助:
山东省自然科学基金委“长枝木霉菌与植物互作效应子(effector)的分离鉴定”(ZR2020MC125);山东省科技厅“聊红椿果色遗传机制、组培快繁及高效栽培技术集成”(2019GNC106018);山东省科技发展计划“拟康氏木霉及其产生的多肽抗菌素peptaibols在植物病害防治上的应用研究”(2014GNC110020)

Isolation and Identification of the Pathogen Causing Leaf Spot on Trifolium repens

SHANG Na¹^,²(), REN Aizhi¹, LIU Bing¹, ZHAO Peibao¹()

¹College of Agronomy, Liaocheng University, Liaocheng, Shandong 252000
²Liaocheng Academy of Agricultural Sciences, Liaocheng, Shandong 252000

Received:2021-01-13 Revised:2021-11-26 Online:2022-02-05 Published:2022-03-16
Contact: ZHAO Peibao

摘要/Abstract

摘要：

明确在山东省聊城市白三叶草上发现的一种叶斑病的病原菌,以期为该病害的防控提供参考。2019年采集山东省聊城市东昌府区具有叶斑病的白三叶叶片,利用组织分离法分离病原菌,并依据柯赫式法则验证致病性。在形态学观察的基础上,对病原菌进行分子生物学鉴定,构建系统发育树,分析其遗传多样性。从发病叶片中分离得到病原菌（标记为SY1）,具有致病性,且与田间发病症状一致。利用真菌通用引物ITS4/ITS5进行PCR扩增和测序,序列分析表明SY1菌株与乔木链格孢(Alternaria arborescens)（序列号KC464334.1）的相似性为100%,且在系统发育树上聚在一起。根据形态特征和ITS基因序列鉴定结果,将白三叶草叶斑病病原菌鉴定为乔木链格孢,为国内首次报道乔木链格孢引起白三叶草叶斑病。

关键词: 白三叶草, 乔木链格孢, 分子生物学鉴定, 形态学鉴定, 叶斑病, 病原菌

Abstract:

An investigation in Liaocheng of Shandong Province shows that a pathogen causing leaf spot was found on Trifolium repens. Therefore, it is of great significance to find out the pathogen species and provide reference for the prevention and control of the disease. The leaves of T. repens with spot disease collected from Liaocheng were used as materials. The pathogenic bacteria were separated by tissue separation method, and the pathogenicity was verified by Koch's rule. Based on morphological observation, molecular biological method was used to identify pathogenic bacteria. The phylogenetic tree was constructed to analyze the fungus genetic diversity. The pathogenic bacteria (labelled SY1) isolated from diseased leaves were pathogenic and consistent with the field symptoms. To further classify the taxonomic status of the pathogen, the generic fungal primers ITS4/ITS5 were used for PCR amplification and sequencing. Sequence analysis showed that the SY1 was 100% similar to Alternaria arborescens (serial number: KC464334.1), and gathered together on the phylogenetic tree. According to the morphological characteristics and gene sequence identification results, the pathogen of T. repens leaf spot was identified as A. arborescens, which was the first report of A. arborescens causing T. repens leaf spot in China.

Key words: Trifolium repens, Alternaria arborescens, molecular biological identification, morphological identification, leaf spot, pathogen

中图分类号:

S436.8

商娜, 任爱芝, 刘冰, 赵培宝. 白三叶草一种叶斑病病原菌的分离鉴定[J]. 中国农学通报, 2022, 38(4): 81-85.

SHANG Na, REN Aizhi, LIU Bing, ZHAO Peibao. Isolation and Identification of the Pathogen Causing Leaf Spot on Trifolium repens[J]. Chinese Agricultural Science Bulletin, 2022, 38(4): 81-85.

图/表 4

图1. 山东省聊城市白三叶草叶斑病症状

图2. 病原菌菌落、菌丝和分生孢子形态特征 A.菌落,B.菌丝,C.分生孢子

图3. 病原菌rDNA-ITS扩增片段电泳 M为DL2000 plus,SY1为rDNA-ITS的PCR产物

图4. 基于rDNA-ITS序列采用邻接法构建病原菌与其相关菌株的系统发育树括号中的字母和数字为该菌株在GenBank中的登录号

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白三叶草一种叶斑病病原菌的分离鉴定

Isolation and Identification of the Pathogen Causing Leaf Spot on Trifolium repens

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