摘 要 ：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16Sr I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(Eucalyptus yellowing and witches-broom phytoplasma strain Guangdong, EYWB-Gd)。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。
Abstract: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 16SrⅠand 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 positive sample was found in tissue-culture plant- lets collected from a nursery in Guangzhou.