Evolutionary relationships and molecular diagnostics of Ralstonia solanacearum species complex associated with declining ironwood trees in Guam

Date
2020
Authors
Paudel , Sujan
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Arif, Mohammad
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Tropical Plant Pathology
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Abstract
Ironwood (Casuarina equisetifolia) is an important component of the island of Guam’s culture and ecology. Different biotic and abiotic factors have been associated with the declining ironwood trees on Guam. The bacterial wilt disease caused by Ralstonia solanacearum species complex (RSSC) was considered an associated factor after the positive test results of RSSC specific immunostrips with ooze from declining trees. The long-range spread, broad host range along with emerging new hosts of the bacterium necessitated the characterization of RSSC ironwood decline strains to understand the diversity, evolutionary relationships among the strains and predict the future movement pattern. In this study, we developed an efficient protocol for the isolation and characterization of RSSC strains from the declining trees. We also used the MLST approach to study the diversity, evolutionary relationships and predict the genealogies of the isolated strains. The presence of fast-growing saprophytes in the declining trees necessitated the development of field deployable and rapid Exo-RPA assay in our study. Furthermore, we also developed a five-plex multiplex PCR assay to detect and differentiate the genospecies of RSSC strains associated with ironwood and all other hosts including the Select Agent (SA) group. The isolation of RSSC from the declining ironwood tree was found to be highly effective with the use of modified SMSA media from the root slices. Both R. pseudosolanacearum (Rps) and R. solanacearum (Rs) were found to be associated with decline although the later species was found to be much lesser in number (3) compared to the former species (35). The phenotypic characterization assays showed the similar utilization pattern for the Rps strains and for the Rs strains. The ironwood Rps population in Guam was found to be highly clonal with least nucleotide diversity and contracting population structure. Our analysis showed North and Eastern Asia and Indonesia and Northern Australia region as the potential origin of ironwood Phylotype I strains whereas Central America and South-Eastern USA and Northern Latin America and Caribbean clade may be the potential origin of Phylotype II ironwood strains. The unique target genomic region for the molecular diagnostics of RSSC ironwood strains was determined using whole genome based comparative genomics approach and ORTHOMCL. The developed RPA assay was found to be highly specific, rapid, field- deployable, and efficient in detection of target directly from the infected plant material with least or no effect of plant inhibitors. When tested with a total of 110 RSSC strains from all the genospecies and SA group, the developed multiplex assay gave accurate results with no cross-amplification. The multiplex PCR assay also successfully differentiated the genospecies from the artificially inoculated plant DNA proving its high usefulness in culture characterization, routine diagnostics, and surveys. Microscopy, labelling studies and whole-genome based studies will provide better understanding about the role of secondary saprophytes in ironwood decline and unlock the questions about role of the bacterium as a primary or an opportunistic pathogen.
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Plant pathology, Epidemiology, characterization, diversity, genealogies, Ironwood decline, molecular diagnostics
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173 pages
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