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Scientia Silvae Sinicae ›› 2006, Vol. 42 ›› Issue (2): 63-72.doi: 10.11707/j.1001-7488.20060211

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

Tian Guozhong1,Li Yong1,Zhu Shuifang2,Jia Ruixiang3,Wang Huimin3   

  1. 1. Institute of Forest Ecology, Environment and Protection, CAF Beijing 100091; 2.Institute of Animal and Plant Quarantine, CAIQ Beijing 100029;3.Department of Plant Pathology, China Agricultural University Beijing 100094
  • Received:2003-12-31 Revised:1900-01-01 Online:2006-02-25 Published:2006-02-25

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Abstract:

Based on the conserve sequences of Ti plasmid of Agrobacterium tumefaciens, two pairs of primers, CYTCYT' and VirD2A/VirD2E 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.

Key words: Agrobacterium spp., polymerase chain reaction(PCR), ipt and VirD2 gene, nucleic acid hybridization, paulownia witches' broom phytoplasma