title: Specific detection of Pectobacterium carotovorum by loop-mediated isothermal amplification.
abstract: Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays.
authors.affiliation: Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawai'i at Mānoa, 3190 Maile Way, St. John Room 315, Honolulu, HI, 96822, USA.
authors.forename: Jarred
authors.initials: J
authors.lastname: Yasuhara-Bell
authors.affiliation: Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawai'i at Mānoa, 3190 Maile Way, St. John Room 315, Honolulu, HI, 96822, USA.
authors.forename: Glorimar
authors.initials: G
authors.lastname: Marrero
authors.affiliation: Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawai'i at Mānoa, 3190 Maile Way, St. John Room 315, Honolulu, HI, 96822, USA.
authors.forename: Asoka
authors.initials: A
authors.lastname: De Silva
authors.affiliation: Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawai'i at Mānoa, 3190 Maile Way, St. John Room 315, Honolulu, HI, 96822, USA.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Alvarez
mesh_terms: D017931:DNA Primers / Q000378:metabolism; D009391:Nephelometry and Turbidimetry; D016973:Pectobacterium carotovorum / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011198:Solanum tuberosum / Q000382:microbiology
chemical_list: D017931:DNA Primers
keywords: Pectobacterium carotovorum; blackleg; detection; loop-mediated isothermal amplification; potato; soft rot
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