Molecular Epidemiology of Ralstonia pseudosolanacearum Phylotype I Strains in the Southwest Indian Ocean Region and Their Relatedness to African Strains.

The increasing requirement for developing tools enabling fine strain traceability responsible for epidemics is tightly linked with the need to understand factors shaping pathogen populations and their environmental interactions. Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is one of the most important plant diseases in tropical and subtropical regions. Sadly, little, outdated, or no information on its epidemiology is reported in the literature, although alarming outbreaks are regularly reported as disasters. A large set of phylotype I isolates (n = 2,608) was retrieved from diseased plants in fields across the Southwest Indian Ocean (SWIO) and Africa. This collection enabled further assessment of the epidemiological discriminating power of the previously published RS1-MLVA14 scheme. Thirteen markers were validated and characterized as not equally informative. Most had little infra-sequevar polymorphism, and their performance depended on the sequevar. Strong correlation was found with a previous multilocus sequence typing scheme. However, 2 to 3% of sequevars were not correctly assigned through endoglucanase gene sequence. Discriminant analysis of principal components (DAPC) revealed four groups with strong phylogenetic relatedness to sequevars 31, 33, and 18. Phylotype I-31 isolates were highly prevalent in the SWIO and Africa, but their dissemination pathways remain unclear. Tanzania and Mauritius showed the greatest diversity of RSSC strains, as the four DAPC groups were retrieved. Mauritius was the sole territory harboring a vast phylogenetic diversity and all DAPC groups. More research is still needed to understand the high prevalence of phylotype I-31 at such a large geographic scale.

Sequevar Diversity and Virulence of Ralstonia solanacearum Phylotype I on Mayotte Island (Indian Ocean).

The genetic and phenotypic diversity of the Ralstonia solanacearum species complex, which causes bacterial wilt to Solanacae, was assessed in 140 strains sampled from the main vegetable production areas of the Mayotte island. Only phylotype I strains were identified in the five surveyed areas. The strains were distributed into the following 4 sequevars: I-31 (85.7%), I-18 (5.0%), I-15 (5.7%), and I-46 (3.6%). The central area of Mayotte was the most diverse region, harboring 4 sequevars representing 47.1% of the collected strains. Virulence tests were performed under field and controlled conditions on a set of 10 tomato breeding line accessions and two commercial hybrid tomato cultivars. The strains belonging to sequevar I-31 showed the highest virulence on the tomatoes (pathotypes T-2 and T-3), whereas sequevars I-18, I-15, and I-46 were grouped into the weakly T-1 pathotype. When the tomato accessions were challenged in the field and growth chambers, the highest level of resistance were observed from the genetically related accessions Hawaii 7996, R3034, TML46, and CLN1463. These accessions were considered moderately to highly resistant to representative strains of the most virulent and prevalent sequevar (I-31). Interestingly, the Platinum F1 cultivar, which was recently commercialized in Mayotte for bacterial wilt resistance, was highly or moderately resistant to all strains. This study represents the first step in the rationalization of resistance deployment strategies against bacterial wilt-causing strains in Mayotte.

Genetic Diversity of the Ralstonia solanacearum Species Complex in the Southwest Indian Ocean Islands.

Epidemiological surveillance of plant pathogens based on genotyping methods is mandatory to improve disease management strategies. In the Southwest Indian Ocean (SWIO) islands, bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is hampering the production of many sustainable and cash crops. To thoroughly analyze the genetic diversity of the RSSC in the SWIO, we performed a wide sampling survey (in Comoros, Mauritius, Reunion, Rodrigues, and Seychelles) that yielded 1,704 isolates from 129 plots, mainly from solanaceous crops. Classification of the isolates to the four major RSSC phylogenetic groups, named phylotypes, showed that 87% were phylotype I, representing the most prevalent strain in each of the SWIO islands. Additionally, 9.7% were phylotype II, and 3.3% were phylotype III; however, these isolates were found only in Reunion. Phylotype IV (2 isolates), known to be restricted to Indonesia-Australia-Japan, was reported in Mauritius, representing the first report of this group in the SWIO. Partial endoglucanase (egl) sequencing, based on the selection of 145 isolates covering the geographic and host diversity in the SWIO (also including strains from Mayotte and Madagascar), revealed 14 sequevars with Reunion and Mauritius displaying the highest sequevar diversity. Through a multilocus sequence analysis (MLSA) scheme based on the partial sequencing of 6 housekeeping genes (gdhA, gyrB, rplB, leuS, adk, and mutS) and 1 virulence-associated gene (egl), we inferred the phylogenetic relationships between these 145 SWIO isolates and 90 worldwide RSSC reference strains. Phylotype I was the most recombinogenic, although recombination events were detected among all phylotypes. A multilocus sequence typing (MLST) scheme identified 29 sequence types (STs) with variable geographic distributions in the SWIO. The outstanding epidemiologic feature was STI-13 (sequevar I-31), which was overrepresented in the SWIO and obviously reflected a lineage strongly adapted to the SWIO environment. A goeBURST analysis identified eight clonal complexes (CCs) including SWIO isolates, four CCs being geographically restricted to the SWIO, and four CCs being widespread beyond the SWIO. This work, which highlights notable genetic links between African and SWIO strains, provides a basis for the epidemiological surveillance of RSSC and will contribute to BW management in the SWIO.

Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates.

Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats.