A polyphasic approach to study the diversity of nonfluorescent pseudomonads causing pith necrosis of tomato in Japan

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  • SAWADA H.
    Genetic Resources Center, National Agriculture and Food Research Organization
  • TAKIKAWA Y.
    Graduate School of Science and Technology, Shizuoka University

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  • トマト茎えそ細菌病を引き起こす蛍光色素非産生の日本産<i>Pseudomonas</i>属細菌に関する多相分類学的解析
  • トマト茎えそ細菌病を引き起こす蛍光色素非産生の日本産Pseudomonas属細菌に関する多相分類学的解析
  • トマト ケイエソサイキンビョウ オ ヒキオコス ケイコウ シキソ ヒサンセイ ノ ニホンサン Pseudomonasゾク サイキン ニ カンスル タソウ ブンルイガクテキ カイセキ

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Abstract

<p>To understand the diversity of nonfluorescent isolates of the tomato pith necrosis pathogens distributed in Japan, we used a polyphasic approach to analyze 21 isolates, obtained from eight prefectures (Iwate, Miyagi, Tochigi, Saitama, Chiba, Shizuoka, Ishikawa, and Oita) between 1980 and 2002. All isolates were demonstrated to reproduce the typical pith necrosis symptoms on tomato after inoculation of stems, and no significant differences in symptoms and virulence were found among the isolates. They were gram-negative, aerobic rods with one to three polar flagella, and did not produce fluorescent pigments on King’s B medium. On the other hand, in the phylogenetic tree based on the concatenated sequences of the 16S rRNA gene and rpoD and in the dendrogram based on a MALDI-TOF MS analysis using the Bruker MALDI Biotyper system, these isolates and reference strains were classified into the following three groups: Group 1 (six isolates and the type strain of Pseudomonas corrugata [Pc]), Group 2 (seven isolates and the type strain of P. mediterranea [Pm]), and Group 3 (eight isolates). Group 1 and Group 2 were identified as Pc and Pm, respectively, based on biochemical and physiological characterizations and PCR assays using species-specific primer sets designed in this study. To our knowledge, this report is the first in Japan of domestic isolates identified as Pm. On the other hand, Group 3 formed a monophyletic clade separated from Pc and Pm in the phylogenetic analysis, utilized 2-ketogluconate and d-aspartic acid, did not utilize meso-tartrate and histamine, and gave different PCR results from Pc and Pm, suggesting that it might represent a novel taxon (new species, or new subspecies of Pc or Pm). Therefore, we propose the tentative treatment of Group 3 as “Pseudomonas sp.” and to add Pm and Pseudomonas sp. as new causal agents of tomato pith necrosis disease in Japan. Pm and Pseudomonas sp. (Group 3), together with Pc, may have been involved in the outbreak of this disease in Japan from the outset, but there is no information on their origins or the history of their invasion in Japan. Thus, we need to establish reliable detection methods based on the results obtained in this study to clarify the origins, ecology and distribution of these three pathogens responsible for this disease.</p>

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