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【目的】检测湖南地区柑橘所携带的柑橘类病毒类型,并鉴定柑橘类病毒的亚型。【方法】以田间柑橘枝条韧皮部为试材,提取总RNA,反转录成cDNA,再用不同类型类病毒的特异引物进行PCR扩增,鉴定出各个样品所携带的柑橘类病毒类型;通过测序分析各类型的序列,并与国内外报道的序列进行同源性分析,确定所测样品的亚型。【结果】建立了CEVd、CVdII、CVdⅢ等柑橘类病毒的RT-PCR检测方法。在5个供试样品中检测出3种类型的柑橘类病毒,即柑橘裂皮病类病毒(citrus exocortis viroid,CEVd)、柑橘类病毒Ⅱ型(citrus viriodⅡ,CVdⅡ,又名hop stunt viroid,HSVd)和柑橘类病毒Ⅲ型(citrus viriodⅢ,CVdⅢ),其中CVdⅡ和CVdⅢ为国内首次发现。亚型分析表明CVdⅡ可能为新的亚型;CVdⅢ为b亚型,可能具有矮化功能。【结论】在中国鉴定出3种类型的柑橘类病毒,CEVd与国内已报道的序列完全一致,其中CVdⅡ和CVdⅢ为首次在中国发现;检测到的CVdⅡ可能为新的亚型。
【Objective】 To detect the type of citrus virus carried by citrus in Hunan and to identify the subtype of citrus virus. 【Method】 The total RNA was extracted from the phloem of citrus branches in the field and reverse transcribed into cDNA. The specific primers of different types of viruses were used for PCR amplification to identify the types of citrus viruses carried by each sample. Analysis of various types of sequences, and homologous analysis of the sequence reported at home and abroad to determine the subtype of the measured samples. 【Result】 The RT-PCR method for detecting citrus viruses such as CEVd, CVdII and CVdⅢ was established. Three types of citrus viruses, citrus exocortis viroid (CEVd), citrus viriod II (CVDII), also known as hop stunt viroid, HSVd ) And citrus viriodⅢ (CVdⅢ), of which CVdⅡand CVdⅢwere first found in China. Subtype analysis showed that CVdII may be a new subtype; CVdIII is subtype b, which may have dwarfing function. 【Conclusion】 Three types of citrus viruses were identified in China. CEVd was completely identical with the reported sequences in China. Among them, CVdⅡ and CVdⅢ were found in China for the first time. The detected CVdⅡ may be a new subtype.