广东桉树黄化(丛枝)病植原体分子鉴定与检测

Molecular identification and detection of Eucalyptus yellowing and witches'-broom phytoplasma in Guangdong

从表现黄化(丛枝)症状的桉树上采集病叶,抽提主脉总 DNA,采用植原体通用引物与巢式引物进行 PCR 和巢式 PCR 扩增,对扩增产物进行克隆和序列测定,获得了植原体的近全长16S rRNA 基因及部分16～23S rRNA 基因间隔区序列。序列分析揭示,所获得的序列与已知植原体基因组相应区段的序列高度同源,与柳叶菜变叶植原体(epilobium phyllody)和白腊树丛枝植原体(ash witches'-broom)相应序列(GenBank 登录号:AY101386和 AY566302)同源率为99.9％,与白腊树黄化植原体(aster yellows BD2)相应序列和番茄巨芽植原体(tomato big bud)相应序列同源率分别为99.6％和99.3％。该序列构建的系统进化树表明,引起我国广州地区桉树黄化(丛枝)病的植原体属于16SrI 组(即翠菊黄化组),将其暂命名为桉树黄化(丛枝)植原体广东株系(Eucalyp-tus yellowing and witches'-broom phytoplasma strain Guangdong,EYWB-cd)。建立了桉树植原体巢式PCR 检测方法,对疑似病样及桉树组培苗进行了检测,多数疑似病样检测结果为阳性,供试的10株组培苗未发现阳性样品。

To identify and detect the phytoplasma infecting Eucalyptus spp. in South China, the diseased leaves of Eucalyptus sp. with symptoms of yellowing and witches＇-broom were collected from Guangdong Province and total DNA was extracted from their midrib. The universal and nested PCR primers were employed to amplify a DNA fragment of Eucalyptus phytoplasma, including the whole of 16S rDNA and apart of 16S -23S rDNA spacer. This nested PCR product was cloned and sequenced, the nucleotide sequence （ GenBank accession no. AY685054） was highly homologous with kwon phytoplasmas. The similarity was 99.9% with both epilobium phyllody （AY101386） and ash witches＇-broom （AY566302）, 96.6% and 99.3% with aster yellows BD2 （AY389822） and tomato big bud （ L33760）, respectively. Phylogenetic tree constructed by parsimony analysis of 16S rDNA sequences of phytoplasmas showed that the phytoplasma which caused eucalyptus yellowing and witches＇-broom （EYWB） disease in Guangdong area belongs to the group 16Sr Ⅰand was tentatively named as EYWB strain Guangdong （EYWB-Gd）. A rapid, sensitive method for EYWB detection has been established. Some samples with suspicious symptoms were detected and most of them showed positive results. No lets collected from a nursery in Guangzhou.