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Establishment and validation of loop-mediated amplification for specific detection of tomato bacterial pathogen Clavibacter michiganensis subsp. Michiganensis

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Item Summary

Title:Establishment and validation of loop-mediated amplification for specific detection of tomato bacterial pathogen Clavibacter michiganensis subsp. Michiganensis
Authors:Yasuhara-Bell, Jarred Hideki
Keywords:Clavibacter michiganensis
Date Issued:Dec 2014
Publisher:[Honolulu] : [University of Hawaii at Manoa], [December 2014]
Abstract:Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial canker of tomato and pepper. The purpose of this study was to determine the potential role in disease outbreaks of seed-and plant-associated nonpathogenic Clavibacter strains, which gave false positives with currently available diagnostic tests. A loop-mediated amplification (LAMP) assay for specific detection of Cmm was designed to target the clvA gene, which is located in the clavicidin gene cluster that proved to be unique to and conserved in Cmm. PCR profiles of additional genes located on a pathogenicity island on the Cmm chromosome showed that a lack of some of these genes, but not all, resulted in a nonpathogenic phenotype. Strains with patterns 1-5 and 7 showed normal virulence, while strains with patterns 6 and 8 were non-pathogenic. LAMP detected all Cmm strains, even though some of the essential pathogenicity genes were missing. The clvA LAMP detected Cmm on tomato seed and infected tomato tissue, with and without an enrichment step. A collection of 348 Cmm strains, representing diversity in a worldwide population, was used to validate the assay. Included were two separate populations of seed-associated Clavibacter reclassified as two new Clavibacter subspecies, with proposed names of Clavibacter michiganensis subsp. chilensis subsp. nov. and Clavibactermichiganensis subsp. californiensis subsp. nov. These nonpathogenic strains were often associated with tomato tissue and seed, and cross-reacted with the standard Immunostrip® test, causing false positives. The LAMP assay discriminated them from Cmm, as well as other subspecies. In co-inoculation studies, these bacteria did not acquire virulence by gene exchange or synergistic complementation of their secreted enzyme repertoire. It is proposed that the LAMP assay be included in the standard seed testing regimen, with positives indicating contamination by true Cmm, and Immunostrip®-positive, LAMP-negative indicating potential presence of other C. michiganensis subspecies that should not pose a risk for international distribution of tomato seed. A positive LAMP result could be followed up by pathogenicity profiling using a panel of PCR tests described in this study. The LAMP assay is currently being used in greenhouse surveys for Cmm and may later be commercialized for tomato seed testing.
Description:Ph.D. University of Hawaii at Manoa 2014.
Includes bibliographical references.
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: Ph.D. - Molecular Biosciences and Bioengineering

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