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Development of a Specific Nested PCR Assay for the Detection of 16SrI Group Phytoplasmas Associated with Sisal Purple Leafroll Disease in Sisal Plants and Mealybugs

文献类型： 外文期刊

作者： Wang, Guihua 1 ; Wu, Weihuai 2 ; Tan, Shibei 2 ; Liang, Yanqiong 2 ; He, Chunping 2 ; Chen, Helong 2 ; Huang, Xing 2 ; Yi, Kexian 2 ;

作者机构： 1.Hainan Univ, Coll Ecol & Environm, Haikou 570228, Hainan, Peoples R China

2.Chinese Acad Trop Agr Sci, Environm & Plant Protect Inst, Haikou 571101, Hainan, Peoples R China

3.Hainan Univ, Coll Forestry, Haikou 570228, Hainan, Peoples R China

4.Minist Agr & Rural Affairs, Key Lab Integrated Pest Management Trop Crops, Haikou 571101, Hainan, Peoples R China

5.Chinese Acad Trop Agr Sci, Sanya Res Inst, Sanya 572025, Peoples R China

6.Hainan Key Lab Monitoring & Control Trop Agr Pest, Haikou 571101, Hainan, Peoples R China

关键词： sisal purple leafroll disease (SPLD); Dysmicoccus neobrevipes; phytoplasma; specific primers; 16Sr RNA gene

期刊名称：PLANTS-BASEL （ 影响因子：4.658； 五年影响因子：4.827 ）

ISSN：

年卷期： 2022 年 11 卷 21 期

页码：

收录情况： SCI

摘要： Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/mu L of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccus neobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays.

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