Hanafi Marwa, Tahzima Rachid, Ben Kaab Sofiene, Tamisier Lucie, Roux Nicolas, Massart Sébastien
Integrated and Urban Plant Pathology Laboratory, Gembloux Agro-Bio Tech, University of Liège, 2, Passage des Déportés, 5030 Gembloux, Belgium.
Consultative Group on International Agricultural Research, 34090 Montpellier, France.
Pathogens. 2020 Dec 12;9(12):1045. doi: 10.3390/pathogens9121045.
(BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol.
香蕉温和花叶病毒（BanMMV）（线形病毒科，帚状病毒亚科，未分类种）是一种属于线形病毒科的丝状病毒。它感染多种植物，地理分布非常广泛。包括线形病毒科成员在内的植物病毒基因组变异性，使得通过特异性引物进行分子检测极具挑战性。在对植物种质资源进行常规检测时，发现一份无症状种质资源的电子显微镜检测结果与免疫捕获（IC）逆转录（RT）聚合酶链反应（PCR）检测结果存在差异。观察到丝状病毒粒子，但分子检测未能扩增出任何片段。对该种质资源进行了高通量测序，组装出了两个同一性为75.3%的香蕉温和花叶病毒完整基因组。基于这些序列以及GenBank中可获得的54个外壳蛋白序列，设计了一种与已用于诊断的寡聚dT逆转录引物Poty1兼容的新正向引物，命名为BanMMV CP9。对来自不同来源的110份不同种质资源进行了回顾性分析，比较了BanMMCP2和BanMMV CP9引物。在这110份种质资源中，16份用BanMMCP2和BanMMV CP9检测均为阳性，3份仅用BanMMCP2检测为阳性，2份仅用BanMMV CP9检测为阳性。否则，89份用这两种引物检测均为阴性且无弯曲病毒粒子。对纯化的PCR产物进行桑格测序，以确认5份结果不同的种质资源中香蕉温和花叶病毒序列的扩增情况。强烈建议依次使用这两种引物以提高检测方法的包容性。
