我国木本植物致病性土壤杆菌的分子检测和比较鉴定

根据根癌土壤杆菌Ti质粒的保守序列设计了2对引物,对我国不同木本植物上分离鉴定的10株致病性根癌土壤杆菌进行PCR,致瘤性根癌土壤杆菌(Agrobacterium tumefaciens)扩增出427 bp和338 bp的特异性DNA片段,而发根土壤杆菌(A.rhizogenes)仅扩增出338 bp片段,放线土壤杆菌(A.radiobacter)、丁香假单胞菌(Pseudomonas syringae)和泡桐丛枝病植原体(phytoplasma)皆未有特异扩增带.用CYT/CYT'引物对也可鉴定T-DNA遗传转化瘤组织,而VirD2A/VirD2E可用于工程土壤杆菌株侵染能力鉴定.从北京通州杨树苗圃和河北廊坊桃园的病树冠瘿组织和土壤中经选择培养基分离菌株,然后PCR筛选出6个致病性根癌土壤杆菌菌株,而未能从施用过抗根癌生防制剂的北京玉渊潭公园樱花根癌病园中分离和鉴定出典型的致病根癌土壤杆菌株.将杨树根癌土壤杆菌菌株(CFCC1001)异戊烯基转移酶基因(ipt)片段克隆和序列测定结果显示与A.tumefaciens Ti15955菌株的ipt基因序列同源性为83.64%.用此片段制备的ipt cRNA基因探针进行斑点杂交和Northern blot,结果显示此探针与杨树根癌土壤杆菌以及玫瑰、樱花、海棠、桃树等木本植物上分离鉴定的致病土壤杆菌菌株所扩增出的427 bp片段都有明显杂交信号,但与引起泡桐丛枝病植原体染色质和染色质外DNA均未有明显杂交信号.

Molecular Detection and Identification of Pathogenic Agrobacterium spp. Isolates Associated with Woody Plant Crown Gall Diseases in China

Based on the conserve sequences of Ti plasmid of Agrobacterium tumefaciens, two pairs of primers, CYTCYT' and VirD2AVirD2E were designed and synthesized. PCR was used for the identification and differentiation of several isolates of Agrobacterium spp. and other bacteria. Both 427 bp and 338 bp DNA products were amplified from Agrobacterium tumefaciens isolates causing crown gall of woody plants. Only 338 bp fragment was amplified from A. rhizogenes, while no specific DNA band was amplified from A. radiobacter, Pseudomonas syringae and Paulownia witches' broom phytoplasma. The PCR using CYTCYT' primer pairs could be used for determining the T-DNA transformed plant gall tissues, while VirD2A VirD2E could be also used for the detection of the infection capacity of engineering A. tumefaciens strains. Six A. tumefaciens strains were isolated and identified from the poplar nursery in Tongzhou, Beijing and peach orchard in Langfang, Hebei Province. However, no typical pathogenic Agrobacterium strain was identified from the Yoshino cherry crown gall tissues and infested soil of Yuyuantan Park, Beijing, where the anti-crown gall disease agents were applied to the disease control for several years. The 427 bp isopentenyl adenosine transferase gene(ipt) fragment from poplar crown gall A. tumefaciens strains CFCC 1001 was cloned and sequenced, finding that CFCC1001 shared 83.64% nucleotide homology with A. tumefaciens Ti15955. Dot blot and northern blot analysis using the cRNA probe prepared by 427 bp DNA labelled with digoxigenin demonstrated that this probe had strong hybridization with the A. tumefacines strains from poplar crown gall disease as well as ones from rose, Yoshino cherry, peach, grapevine etc. Northern blot analysis showed that the ipt gene probe had no distinct hybridization signal with the chromosomal and extrochromosomal DNA of paulownia witches' broom phytoplasma.