Simultaneous plasmid profiling of phytopathogenic <i>Rhizobium</i> species (former <i>Agrobacterium </i>species) using multiplex colony-direct PCR.

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  • SAWADA H.
    Genetic Resources Center, National Agriculture and Food Research Organization
  • SUZAKI K.
    Grape and Persimmon Research Division, Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization
  • KAWAGUCHI A.
    Department of Agriculture, Forestry and Fisheries, Okayama Prefectural Government Western Region Agricultural Research Center, National Agriculture and Food Research Organization

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Other Title
  • コロニーダイレクトのマルチプレックスPCRによる植物病原性<i>Rhizobium</i>属細菌（旧<i>Agrobacterium</i>属細菌）のプラスミド型別
  • コロニーダイレクトのマルチプレックスPCRによる植物病原性Rhizobium属細菌(旧Agrobacterium属細菌)のプラスミド型別
  • コロニーダイレクト ノ マルチプレックス PCR ニ ヨル ショクブツ ビョウゲンセイ Rhizobiumゾク サイキン(キュウ Agrobacteriumゾク サイキン)ノ プラスミド カタベツ

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Abstract

Among Rhizobium species, six species include plant pathogens as a member: R. radiobacter species complex (including R. nepotum and R. pusense), R. rhizogenes, R. vitis, R. rubi, R. larrymoorei, and R. skierniewicense. Based on their pathogenic states, the members belonging to the six species can be divided into three types, namely, crown gall bacteria carrying Ti plasmid, hairy root bacteria carrying Ri plasmid, and nonpathogenic bacteria carrying neither pathogenic plasmids. In this study, we developed a plasmid-profiling method using a multiplex colony-direct PCR to identify any pathogenic plasmid present in the respective strains and thus determine their pathogenic state. For that purpose, a universal primer set for both pathogenic plasmids and a specific primer set for the Ri plasmid were designed based on the sequences of virC1 and rolC, respectively. The universal primer set for 16S rRNA gene (16S rDNA), chosen as an internal control, was also added to the PCR mix to readily judge the success of the reaction and exclude false-negative reactions. Reliability of the method as a plasmid-profiling tool was then validated using the Rhizobium species containing plant pathogens and their relatives of 383 strains in total. As a result, two DNA fragments, one from virC1-specific amplification (278 bp) and the other (780–784 bp) from 16S rDNA as the internal control, were stably obtained from all crown gall bacteria used. For hairy root bacteria, three fragments derived from virC1 (278 bp), rolC (438 bp) and 16S rDNA were always observed. On the other hand, only a fragment derived from 16S rDNA was amplified from the other nonpathogenic bacteria. Therefore, this method is useful for rapid plasmid profiling of the Rhizobium species containing plant pathogens, which will improve the efficiency in areas such as surveillance and diagnosis of crown gall and hairy root diseases and epidemiological and ecological studies of the pathogens.

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