Abstract
The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.
Highlights
Ralstonia solanacearum is a Gram-negative soil-borne betaproteobacterium that is the causal agent of bacterial wilt disease, or potato brown-rot disease [1,2]
Based on phylogenetic grouping and deeply separated evolutionary lineages, R. solanacearum is divided into four phylotypes that reflect its geographic isolation and spatial distances: phylotypes I and II are composed of Asian and American strains, respectively, phylotype III members are of African origin, and phylotype IV isolates originate from Indonesia, Japan, and Australia, and include R. syzigii and blood disease bacteria (BDB)
The optimized fliC Loop-mediated isothermal amplification (LAMP) assay showed a better performance than the 16S rRNA LAMP assay in terms of specificity, sensitivity, and speed, but it did not detect several economically important R. solanacearum strains, and this was not selected for on-site application
Summary
Ralstonia solanacearum is a Gram-negative soil-borne betaproteobacterium that is the causal agent of bacterial wilt disease, or potato brown-rot disease [1,2]. Based on phylogenetic grouping and deeply separated evolutionary lineages, R. solanacearum is divided into four phylotypes that reflect its geographic isolation and spatial distances: phylotypes I and II are composed of Asian and American strains, respectively, phylotype III members are of African origin, and phylotype IV isolates originate from Indonesia, Japan, and Australia, and include R. syzigii and blood disease bacteria (BDB). These phylotypes are further subdivided in sequevars that are based on differences in the sequences of a portion of the endoglucanase (egl) gene [9,10].
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