基于ITS和β-tubulin基因分析的居间疫霉菌系统发育

doi:10.11707/j.1001-7488.20180308

摘要/Abstract

摘要： [目的]对我国亚热带部分森林中的疫霉菌及其所致病害进行了系统调查和分析，研究疫霉菌的种类和遗传多样性，探讨疫霉菌的系统发育关系，为林木疫病的防治提供理论依据。[方法]利用健康叶片诱捕林间溪流里的疫霉菌，对有症状的叶片组织进行分离纯化，显微镜下根据其菌丝分枝和结构特征，初步判定为疫霉菌。对菌株的ITS和β-tubulin基因进行PCR扩增和测序，将序列拼接后，用MAFFT 7.0、PAUP 4.0 beta10、MrBayes 3.2.6及PhyML 3.0等软件进行基因序列系统发育分析。[结果]分离鉴定得到46株中国新记录种，通过进一步培养和显微镜下观察，其形态特征与居间疫霉菌Phytophthora intercalaris相吻合。拼接之后得到完整的ITS序列为847~849 bp，与参考菌株（KT163268）序列一致性为99.29%~99.53%；β-tubulin序列均为882 bp，与参考菌株（KT163336）序列一致性为99.43%~99.66%。系统发育分析结果显示供试菌株与居间疫霉菌以100%的支持率聚为一支。本研究不仅增加了居间疫霉菌的菌株数量，也扩大了该菌的分布范围，同时也增加了中国疫霉菌的种类。[结论]居间疫霉菌种内不同地理来源的菌株之间具有较高的序列一致性，但是也存在一些碱基的差异，从而形成不同的基因型。

关键词: 林木疫霉菌, 居间疫霉菌, ITS, β-tubulin, 系统发育

Abstract: [Objective] A survey of diseases caused by the genus of Phytophthora were carried out in the subtropical forests of China. This study aimed at exploring the genetic diversity of Phytophthora and providing a theory basis for controlling the disease caused by Phytophthora.[Method] The healthy leaves were used to bait Phytophthora isolates from stream water.The symptomatic leaf tissues were isolated and Phytophthora was preliminarily determined based on its hyphal branching structure under a microscope,then ITS and β-tubulin gene of strains were amplified with PCR and sequenced.The spliced sequence was used for phylogenetic analysis with MAFFT 7,PAUP 4 beta10,MrBayes 3.2.6 and PhyML 3 software.[Result]Forty-six isolates of Phytophthora were obtained, and their morphological characteristics were consistent with P. intercalaris, a new record species in China. All isolates produced ITS amplicons of 847 to 849 bp in size, which differed from each other in one to three positions. These sequences had identity from 99.29% to 99.53% with the reference sequences of P. intercalaris type culture (KT163268). All the β-tubulin amplicons were 882 bp, which shared 99.43% to 99.66% identity with reference sequences of P. intercalaris (KT163336). Phylogenetic analysis showed that the tested strains were clustered with P. intercalaris with a high bootstrap support value (100%). This study not only increased the number of P. intercalaris isolates, but also expanded the distribution range of P. intercalaris, and enriched the number of Phytophthora species in China as well.[Conclusion]The result of gene sequence analysis showed that there was high sequence consistency among isolates of P. intercalaris from different regions, but there were variations in the base site, which formed different gene types. The present study provides a theoretical basis for the further development of the epidemiology and control strategy for the tree blight diseases.

Key words: Forest Phytophthora, Phytophthora intercalaris, ITS, β-tubulin, phylogenetic analysis

中图分类号:

S718.81

李雯雯, 赵文霞, 林若竹, 姚艳霞, 李娟, 淮稳霞. 基于ITS和β-tubulin基因分析的居间疫霉菌系统发育[J]. 林业科学, 2018, 54(3): 73-82.

Li Wenwen, Zhao Wenxia, Lin Ruozhu, Yao Yanxia, Li Juan, Huai Wenxia. Phylogenetic Relationships of Phytophthora intercalaris Based on ITS and β-tubulin Gene Sequence[J]. Scientia Silvae Sinicae, 2018, 54(3): 73-82.

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