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林业科学 ›› 2018, Vol. 54 ›› Issue (9): 80-88.doi: 10.11707/j.1001-7488.20180910

• 论文与研究报告 • 上一篇    下一篇

石碌含笑枯梢病菌的鉴定及生物学特性

易润华, 陈玉娟, 韩静仪, 胡倩, 李慧琪, 吴海燕   

  1. 广东海洋大学农学院生物技术系 湛江 524088
  • 收稿日期:2017-02-14 修回日期:2018-02-16 出版日期:2018-09-25 发布日期:2018-09-10
  • 基金资助:
    广东省科技计划项目(20090215);广东省大学生创新创业训练计划项目(CXXL2015055)。

Identification and Biological Characteristics of Diaporthe ueckerae Causing Dieback Disease on Michelia shiluensis

Yi Runhua, Chen Yujuan, Han Jingyi, Hu Qian, Li Huiqi, Wu Haiyan   

  1. Department of Biotechnology, Agricultural College, Guangdong Ocean University Zhanjiang 524088
  • Received:2017-02-14 Revised:2018-02-16 Online:2018-09-25 Published:2018-09-10

摘要: [目的]对广东省徐闻县2~3年生石碌含笑嫁接苗枯梢病进行病原菌的分离鉴定,分析其致病性及生物学特性,为该病的诊断和防控提供理论基础。[方法]将病枝表皮削除后,取病组织接种到PDA培养基中,25~30℃黑暗培养后,得到纯培养物,将其接种到健康寄主,测定其致病性。将病原菌的ITS、组蛋白HIS、延长因子TEF和微管蛋白TUB的基因扩增测序后,在GenBank进行序列比对,采用邻接法(NJ)和贝叶斯法(BI)进行多基因位点系统发育分析,确定病原菌与近缘种的关系。根据系统发育分析以及形态学特征对病原菌进行种类鉴定。将菌饼接种到培养基中,测定不同培养基、温度、pH、碳氮源和光照处理对病原菌的菌丝生长和形成分生孢子座的影响。[结果]从病组织中分离到的菌株可引起健康植株发病,其形态学特性与间座壳菌一致。GenBank比对结果显示,病原菌ITS序列与间座壳菌几个种的序列同源性高于99%,而HIS、TEF和TUB基因序列与Diaporthe ueckerae的序列同源性为100%。系统发育分析结果显示石碌含笑病原菌与Diaporthe ueckerae以自展支持率99%和后验概率1.00聚集在同一分枝。病原菌在不同培养基、温度、碳氮源条件下,菌丝生长速度和产生分生孢子座数量差异显著,在pH 4-8时菌丝生长和产分生孢子座差异不明显,不同光处理对病原菌生长影响差异不显著,但对产生分生孢子座影响差异显著,12 h光照+12 h黑暗处理与12 h光照+12 h黑暗+10 min紫外线处理不利于形成分生孢子座。[结论]石碌含笑枯梢病的病原菌是D.ueckerae Udayanga et Castlebury 2015,对环境因子适应性广,仅在低于15℃和高于35℃时不产生分生孢子座。

关键词: 石碌含笑, 枯梢病, Diaporthe ueckerae, 生物学特性

Abstract: [Objective] In 2014, dieback disease was observed on the 2-3-year-old grafted twigs of Michelia shiluensis in Xuwen County, Guangdong Province. To understand the pathogen causing the dieback, the isolate was collected and identified, the pathogenicity was tested according to the Koch's postulates, and the biological characteristics were determined. This study aims to provide basic theoretical knowledge for diagnosis and control of this disease.[Method] To get the pathogen, the infested tissues were cut from symptomatic twigs of which the barks were removed, placed on potato dextrose agar (PDA), and incubated at 25-30℃ in dark. The isolates were inoculated on the healthy hosts to test the pathogenicity according to the Koch's postulates. The sequences of internal transcribed spacer (ITS) rDNA, translation elongation factor (TEF) gene, β-tubulin gene and histone (HIS) gene were amplified, sequenced and blasted in GenBank. Based on the multi-locus (ITS, HIS, TEF, TUB) phylogenetic analyses with Neighbor joining (NJ) and Bayesian inference (BI) method, the phylogenetic relationship of the pathogen with Diaporthe spp. was analyzed. The pathogen was identified according to the morphological and molecular characteristics. The effects of culture medium, temperature, pH, carbon and nitrogen source as well as light treatment on mycelial growth and conidiomata formation were determined.[Result] The isolate from infected tissues caused twigs dieback on healthy hosts, and the morphological characters was the same as Diaporthe sp. The Blast result showed that the ITS sequences of the pathogen were above 99% homology with several species of Diaporthe, and the sequences of HIS, TEF and TUB gene were 100% homology with D. ueckerae. The pathogen was clustered in the same branch with D. ueckerae in a well-supported clade with Bootstrap support values above 99% and Bayesian posterior probability values 1.00. Under the condition of culture medium, temperature, pH, carbon and nitrogen source and light treatment, the mycelial growth and conidiomata formation had significant difference. The pH values ranged from 4 to 8 had insignificant effects on mycelial growth and conidiomata formation. Different light treatments had insignificant effect on the mycelial growth, but significant effect on conidiomata formation, thus it was not conducive to form the conidiomata under two light treatments, i.e. alternation of 12 hillumination and 12 h darkness, and alternation of 12 hillumination, 12 h darkness and 10 minultraviolet radiation.[Conclusion] The pathogen causing dieback on M. shiluensis has been identified as D. ueckerae Udayanga et Castlebury 2015, with strong adaptation to various environmental factors. The fungus could not form conidiomata below 15℃ or above 35℃.

Key words: Michelia shiluensis, dieback, Diaporthe ueckerae, biological characteristics

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