Diversidade genética de isolados de Ralstonia solanacearum e caracterização molecular quanto a filotipos e sequevares

Abstract

Resumo -O objetivo deste trabalho foi identificar isolados brasileiros de Ralstonia solanacearum quanto a filotipos e sequevares, determinar sua diversidade genética, realizar a associação da estrutura genética do patógeno com sua classificação e origem geográfica e identificar um marcador molecular para a diagnose do moko-da-bananeira. Um grupo de 33 isolados de R. solanacearum, da coleção da Embrapa Tabuleiros Costeiros, coletado de diversos hospedeiros, foi caracterizado por meio de PCR em sequência palindrômica extragênica repetitiva (rep-PCR) e RAPD. Deste grupo, 19 perteciam à raça 2 do patógeno e 14 à raça 1, e 15 isolados eram associados à cultura da bananeira. Os filotipos e sequevares foram caracterizados e determinados por PCR Multiplex. Verificou-se que 82% dos isolados pertencem ao filotipo II, e 12% ao filotipo III. Todos os isolados da bananeira pertencem ao filotipo II. A técnica de RAPD foi eficiente em agrupar os isolados de acordo com sua origem geográfica; entretanto, ela requer um número elevado de marcas moleculares. Foi possível relacionar os isolados pela análise rep-PCR. O iniciador com sequências repetitivas enterobacterianas intergênicas de consenso (ERIC) possibilitou a separação dos isolados de acordo com a raça, e o iniciador REP permitiu a discriminação entre os filotipos. Estas duas análises foram as mais informativas.Termos para indexação: Musa, marcador molecular, moko-da-bananeira, murcha bacteriana, rep-PCR, variabilidade genética. Genetic diversity of Ralstonia solanacearum isolates and molecular characterization as to phylotypes and sequevarsAbstract -The objective of this work was to identify Brazilian isolates of Ralstonia solanacearum according to phylotypes and sequevars, to determine their genetic diversity, to associate the pathogen genetic structure with its taxonomy and geographical origin, and to identify a specific molecular marker to diagnose banana moko disease. A group of 33 isolates of R. solanacearum, from the collection of Embrapa Tabuleiros Costeiros, collected from different plant hosts, was characterized using the repetitive extragenic palindromic sequence-based PCR (rep-PCR) and RAPD. From this group, 19 belonged to the pathogen race 2 and 14 to the race 1, and 15 isolates were associated with banana crop. Phylotypes and sequevars were characterized and determined by Multiplex PCR. It was verified that the isolates belonged to phylotypes II (82%) and III (12%). All isolates from banana plants belonged to phylotype II. The RAPD technique was efficient in grouping these isolates according to their geographical origin; however, it requires a large number of molecular markers. It was possible to establish the relationships among the isolates by rep-PCR. The enterobacterial repetitive intergenic consensus primer (ERIC) made it possible to separate the isolates according to the race, and the REP primer allowed for the discrimination among phylotypes. These were the two most informative analyses.

Phylotype and sequevar variability of Ralstonia solanacearum in Brazil, an ancient centre of diversity of the pathogen

“…This study also rectified some incorrect isolate assignments. A previous study characterized six Brazilian isolates of R. solanacearum (two from tomato isolated in Guaraí‐TO, two from Nova Friburgo‐RJ, an isolate of aubergine from Gurupi‐TO, and one from bell pepper from Camocin de São Felix‐PE) as belonging to phylotype III (Pinheiro et al ., ). However, an amplicon size analysis conducted in the present study indicated that all of these isolates belonged to phylotype I.…”

Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II

“…Unfortunately, the strains that are assigned to the obsolete race 1 classification are distributed across all four of the phylotypes. To our knowledge and based on the important role of the banana industry in Brazil, it is surprising that only one study has phylogenetically typed 19 Moko diseasecausing strains by Mmx-PCR (20). Typical Moko disease symptoms in the wild include yellowing and wilting of the inner leaves caused by the infection, which initiates in the rhizomes and moves toward the pseudostem; fruit are then deformed, turn black, and shrivel up.…”

“…Determining the genetic and phenotypic variability of the Moko disease-causing strains is of the upmost importance; however, this variability remains to be described in South America (4), especially in Brazil (20). The aim of this study is to explore the pathogenic and phylogenetic diversity of R. solanacearum strains that originates in Brazil where recent wilting has appeared on banana (Musa sp.)…”

Occurrence and diversity of Ralstonia solanacearum populations in Brazil

“…For instance, from 33 R. solanacearum strains collected from several hosts (19 strains from race 2, 14 from race 1, and 15 strains associated to banana plants), 82% were classified as phylotype II (including all strains from banana). However, it was not possible to characterize most strains into sequevars, and, possibly, the banana strains belong to a not yet determined sequevar (PINHEIRO et al, 2011). This observation was also reported by Albuquerque et al (2014) that described a new sequevar associated to Moko, named IIA-53.…”

“…Mistakenly, Pinheiro et al (2011) published the characterization of four R. solanacearum strains (two from tomato plants from Guaraí-TO and Nova Friburgo-RJ, one from eggplants from Gurupi-TO, and one from bell pepper from Camocin S. Felix-PE) as positive for phylotype III. However, the analysis of the amplicon size reveals correspondence to phylotype I, of 144 bp (and not 91 bp as mentioned by the authors).…”