广东桉树黄化(丛枝)病植原体分子鉴定与检测

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-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.

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

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 em- ployed 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 phytoplas- ma which caused eucalyptus yellowing and witches'-broom (EYWB) disease in Guangdong area belongs to the group 16Sr I and was tentatively named as EYWB strain Guangdong (EYWB-Gd).A rapid,sensi- tive 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.