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林业科学 ›› 2021, Vol. 57 ›› Issue (5): 195-201.doi: 10.11707/j.1001-7488.20210519

• 研究简报 • 上一篇    下一篇

羽脉山黄麻丛枝植原体的分子鉴定及病害调查

万琼莲1,2,王连春1,*,王泉1,许杏萍2,苏帆2,赵静1,蔡红2   

  1. 1. 玉溪师范学院化学生物与环境学院 玉溪 653100
    2. 云南农业大学农业生物多样性与病虫害控制教育部重点实验室 昆明 650201
  • 收稿日期:2019-12-25 出版日期:2021-05-25 发布日期:2021-07-09
  • 通讯作者: 王连春
  • 基金资助:
    云南省教育厅科学研究基金项目(2017ZZX176);云南省教育厅科学研究基金项目(2017ZDX136)

Molecular Identification and Disease Investigation of Trema levigata Witches'-Broom Phytoplasma

Qionglian Wan1,2,Lianchun Wang1,*,Quan Wang1,Xingping Xu2,Fan Su2,Jing Zhao1,Hong Cai2   

  1. 1. School of Chemistry, Biology & Environment, Yuxi Normal University Yuxi 653100
    2. Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University Kunming 650201
  • Received:2019-12-25 Online:2021-05-25 Published:2021-07-09
  • Contact: Lianchun Wang

摘要:

目的: 了解羽脉山黄麻丛枝病在云南省玉溪市新平县的发生情况和危害程度,通过分子生物学方法确定病原物及其分类地位,为本地区植原体防控提供参考。方法: 利用普查法获得病害发病率,评估病害危害程度。利用植原体通用引物P1/P7及R16F2n/R16R2对感病植株总DNA进行巢式PCR扩增、克隆和测序,得到16S rDNA序列。再用植原体在线分类鉴定工具iPhyClassifier以及MegaX软件分别进行序列分析和构建基于16S rDNA序列的系统进化树,确定病害病原物及其分类地位,通过在线分析软件MUSCLE比较相关序列的同源性。结果: 根据调查发现羽脉山黄麻丛枝病发生于大约101°35'-101°53'E和23°56'-24°20'N之间,主要是在低海拔400~600 m温度相对较高的干热河谷地区,说明高温有利于植原体病害的发生。该处连续3年发生此病害,2018年7月调查得知其平均发病率为38.9%,属中度危害。PCR扩增获得约1 246 bp的羽脉山黄麻丛枝病植原体16S rDNA序列(株系:TLWBYN01,登录号:MN513329)。分析表明TLWBYN01与翠菊黄化植原体(Candidatus Phytoplasma asteris)的参考菌株(M30790)相似度为99.8%,因此该植原体为‘Candidatus Phytoplasma asteris’相关菌株,属于16S rI组成员。TLWBYN01的F2nR2片段虚拟RFLP模型与16S rI-X亚组的参考模型番木瓜束顶植原体(登录号:JF781308)最为相似,相似系数为0.98,17种限制性内切酶的酶切图谱显示与16S rI-X参考株只有MseI不同,因此该植原体属于16S rI-X亚组的变种。比较得出TLWBYN01与16S rI-X亚组成员番木瓜束顶植原体(JF781308)和印度葫芦植原体(LT594117、LT594118)同源性分别为99.2%和99.6%。结论: 玉溪市新平县发现的羽脉山黄麻丛枝病属局部常发病害,中度危害,但一旦感染,就会严重影响植物生长。羽脉山黄麻丛枝植原体属16 SrI组成员,也是报道较少的植原体16S rI-X亚组的一个变种,且羽脉山黄麻为新发现的植原体新寄主。

关键词: 植原体, 羽脉山黄麻, 病害调查, 16S rDNA, 分子鉴定

Abstract:

Objective: Based on the investigation of Trema levigata broom in Xinping county, Yuxi city, Yunnan province, this study aimed to understand the occurrence and damage degree of the disease, and further determine the pathogens and their classification status by molecular biological method. Method: The incidence of the disease was obtained by general survey method, and its damage degree was evaluated. The total DNA of the affected plants was extracted, and the 16S rDNA sequences were amplified by using nested-PCR from the total DNA with phytoplasma universal primer pair P1/P7 and R16F2n/R16R2, then the amplified fragments were cloned and sequenced. A sequence analysis was performed with iPhyClassifier, an online classification and identification tool of phytoplasma, and a phylogenetic tree based on the 16S rDNA sequence was constructed by using Megax software to determine the pathogens and their classification status. The homology of related sequences were compared by online analysis software MUSCLE. Result: The survey result indicated that T. levigata witches' broom approximately occurred in east longitude 101°35'-101°53' and north latitude 23°56'-24°20'. It was mainly in the dry-hot valley region at the low altitudes from 600 to 400 m with relatively high temperature, which also showed that high temperature was conducive to the occurrence of phytoplasma disease. In this region, the disease occurred for three consecutive years. The average incidence rate was 38.9% with moderate damage in July 2018. The 1 246 bp 16S rDNA sequence of T. levigata witches' broom phytoplasma was amplified by PCR amplification (strain: TLWBYN0, GenBank accession: MN513329). Analysis showed that TLWBYN01 shared 99.8% similarity with the reference strain of Candidatus Phytoplasma asteris (GenBank accession: M30790), thus the phytoplasma under study was a 'Candidatus Phytoplasma asteris' related strain and belonged to a member of 16S rI group. The virtual RFLP pattern derived from the F2nR2 fragment of TLWBYN01 was most similar to the reference pattern of the 16SrⅠ, subgroup X (GenBank accession: JF781308), with a pattern similarity coefficient of 0.98. The enzymatic profiling of 17 restriction enzymes showed that only MseI was different from the 16S rI-X reference strain. Thus, the phytoplasma was a variant of 16S rI-X. TLWBYN01 had 99.2% and 99.6% homology respectively with phytoplasma of papaya bunchy top (JF781308) and lagenaria siceraria yellow little leaf (LT594117、LT594118), two members of 16S rI-X subgroup. Conclusion: Trema levigata witches' broom from Xinping county of Yuxi city is a local common disease with moderate damage. But once being infected, the plant growth could be seriously affected. T. levigata witches' broom phytoplasma belongs to the member of 16S rI group and is a variant of 16S rI-X subgroup with being less reported so far, and T. levigata is a new host of phytoplasma. These findings are of great significance for further study of phytoplasma. At the same time the plant is the pioneer tree species for greening and widely distributed, thus the investigation and study on the disease could provide a basis for prevention and control of forest diseases in this region.

Key words: phytoplasma, Trema levigata, witches'-broom, 16S rDNA, molecular identification

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