复合感染建兰花叶病毒和齿兰环斑病毒的兰花超微结构观察及病原物快速鉴定

MOLECULAR IDENTIFICATION OF CYMBIDIUM MOSAIC POTEXVIRUS AND ODONTOGLOSSUM RINGSPOT TOBAMOVIRUS COMPLEX INFECTED PHALAENOPSIS AND ITS PATHOLOGICAL ULTRASTRUCTURAL ALTERATION

通过负染和超薄切片观察到蝴蝶兰(Phalaenopsis amabilis)病叶中线状和杆状病毒颗粒。组织切片观察,同时发现两种病毒粒子的典型聚集体:线状粒子的带状聚集体,粒子多层排列,层间呈一定角度或螺旋相叠;杆状粒子的平行、角层状或螺旋型排列聚集体。二种病毒聚集体均出现于薄壁细胞、细胞间隙和输导组织细胞中。感病细胞中叶绿体发育不全;线粒体增生、肿胀甚至空化;细胞核膨大、空化。进一步的多重RT-PCR与病毒核酸序列分析,同时扩增到建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)的外壳蛋白基因,与GenBank已知分离物同源性分别达到98%和99%-100%。从细胞和分子层面揭示了蝴蝶兰受CymMV和ORSV复合侵染并可能导致严重病症的事实,分析和明确了感病兰细胞超微结构的病变特征以及田间病症发生的细胞病理学依据。

Filamentous and rod-shaped virions,and aggregated crystals were observed in infected leaves of by negative staining and ultramicrotomy. Histologic study synchronously showed typical crystal forms of the two virions： the crystals from filamentous particles congregated in strips, arrayed in multilayer and piled in certain angles or helix between layers;while the crystals from rodshaped particles arrayed in parallel,angle-layer or helix. The two kinds of crystals both presented in parenchyma cells, plasmodesma, and vascular bundles; as an evidence that indicates the existence of short distance transport of viruses between ceils, plasmodesmata were produced through piercing the membrane around the reproducing viral crystals; the chloroplasts in the infected cells were hypoplastic, and the filamentous virion were observed within the chloroplasts; the mitochondrions were over-developed, swelled or even cavitated; the nucleus were also swelled and cavitated. Further multiplex RT-PCR and sequencing that the coat protein genes simultaneously expanded to Cymbidium mosaic potexvirus （CymMV） and Odontoglossum ringspot tobamovirus （ORSV） showed homology with available abstracts from GenBank, and the respective percentages of identity are 98% and 99%-100%. The instant and direct identification evidences of CymMV and ORSV complex infection on phalaenopsis are revealed at both cellular and molecular levels,and the character of its pathological uhrastructural alteration as the gist in cellular pathology for pathogenesis are also presented.