Desenvolvimento de marcadores scar para identificação de espécies de Meloidogyne do cafeeiro e variabilidade genética e agressividade de populações de M. paranaensis a genótipos de Coffea spp.
Data
2016-06-08
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Editor
Universidade de Brasília
Resumo
Os nematoides das galhas (NGs), Meloidogyne spp., estão entre os patógenos mais importantes economicamente por infectarem raízes de cafeeiro em vários países das Américas. Os objetivos do estudo foram desenvolver marcadores específicos do tipo SCAR para a detecção de duas espécies importantes do cafeeiro na América Central: Meloidogyne arabicida e M. izalcoensis (Tylenchida: Meloidogynidae), e avaliar a variabilidade genética e a agressividade de sete populações de M. paranaensis, uma das espécies de NGs mais destrutivas para o cafeeiro, em Coffea spp., com genes de resistência a Meloidogyne spp. No primeiro estudo, fragmentos polimórficos amplificados através de reações de PCR-RAPD foram selecionados e transformados em marcadores específicos do tipo SCAR. A amplificação dos primers desenvolvidos produziram fragmentos específicos de 300 pb e 670 pb para as espécies M. arabicida e M. izalcoensis, respectivamente. A especificidade dos primers também foi validada para um único indivíduo J2, macho ou fêmea, além de populações de campo, contendo mistura de espécies. Portanto, estes resultados demonstraram a eficácia destes marcadores SCAR para a identificação das espécies M. arabicida e M. izalcoensis, contribuindo para o diagnóstico específico dos NGs nas Américas. No segundo estudo, sete populações de M. paranaensis foram identificadas pela caracterização bioquímica e molecular. Os estudos filogenéticos mostraram que apesar da existência de três fenótipos de esterase (Est P1, P2 e P2a), uma baixa variabilidade genética foi observada, até mesmo em regiões distintas do DNA. A análise molecular mostrou que existe divergência genética na população de perfil P2a da Guatemala, em relação às populações do Brasil (Est P1 e P2). Essa população nos estudos de patogenicidade às cultivares suscetíveis de C. arabica mostrou-se uma das mais agressivas. Nos estudos de agressividade/virulência de M. paranaensis em Coffea spp. foram realizados dois ensaios. No primeiro, foram usadas duas cultivares, C. arabica (cv. Catuaí IAC 81) e C. canephora (cv. Clone 14), e foi confirmada a alta suscetibilidade da cv. Catuaí IAC 81 v com FR > 30 e alta resistência da variedade clonal “Clone 14” com FR < 0,2, em relação a diferentes populações de M. paranaensis. Ou seja, nenhuma população foi virulenta ao ‘Clone 14’ e as populações Par 3 da Guatemala, Par 5 Piumbí-MG e Par 4 Rolândia- PR foram as mais agressivas ao ‘Catuaí IAC 81’. No segundo ensaio foram testados quatro padrões de resistência em relação a sete populações de M. paranaensis: Catuaí Vermelho x Amphillo MR2161 (C. arabica), porta enxerto Apoatã IAC 2258 (C. canephora), Híbrido do Timor UFV 408-01(E1 6-6 II) e cv. IPR 100 (C. arabica), e o controle suscetível cv. ‘Mundo Novo 379-19’. Quanto à agressividade na cultivar Mundo Novo, as populações Par 2 Herculândia-SP, Par 3 da Guatemala e Par 5 PiumbíMG foram as mais agressivas. Nenhuma população de M. paranaensis foi virulenta às quatro cultivares resistentes: Apoatã IAC 2258, Catuaí Vermelho x Amphillo MR2161(E1 16-5 III), IPR 100 e Híbrido do Timor UFV 408-01 (E1 6-6 II), apresentando uma segregação de 15%, 0%, 26% e 31%, respectivamente. Esses resultados são promissores, pois validam a resistência de diferentes fontes genéticas de Coffea spp., para a espécie M. paranaensis.
Root-knot nematodes (RKN), Meloidogyne spp., are amongst the most economically important plant parasitic nematodes infecting coffee (Coffea spp.) in several countries in the Americas. The objectives of this study were to developed a polymerase chain reaction (PCR)-based assay for specific detection of two RKN species, Meloidogyne arabicida and M. izalcoensis (Tylenchida: Meloidogynidae), major pathogens of coffee crops in Central America and to assess the genetic variability and aggressiveness of seven populations of M. parananesis, one of the most destructive RKN species in coffea, Coffea spp., which harbors resistance to Meloidogyne spp. In the first study Random Amplified Polymorphic DNA (RAPD) fragments specific for these two species were converted into sequence characterized amplified region (SCAR marker). PCR amplification using SCAR primers produced a specific fragment of expected size (e.g., 300 base pairs and 670 bp) in M. arabicida and M. izalcoensis, respectively. SCAR primers also allowed successful amplification of DNA from single infective juveniles, males, females and on field-isolated populations, containing mixtures of species. Therefore, these results demonstrate the effectiveness of these SCAR markers developed for identification M. arabicida and M. izalcoensis species and may contribute to specific diagnosis of these RKN in the Americas. In the second study, seven populations of M. paranaensis were identified by biochemical and molecular characterization. Phylogenetic studies have shown that despite the existence of three esterase phenotypes (Est P1, P2, P2a), a low genetic variability was observed, even in different DNA regions. Molecular analysis showed that there is a genetic difference between population P2a from Guatemala compared to other M. paranaensis populations from Brazil containing Est P1 and P2 profiles. In addition, population P2a was the most aggressive in pathogenicity assays with susceptible C. arabica cultivars. Two assays were carried out to study the aggressiveness/virulence among M. paranaensis populations against Coffea genotypes. In the first assay using different M. paranaensis vii populations against C. arabica (cv. Catuaí IAC 81) and C. canephora (cv. Clone 14), a high susceptibility pattern of cv. Catuaí IAC 81 was confirmed with RF> 30, as well as a high resistance phenotype for the clonal variety "Clone 14" with RF <0.2 was observed. None of the populations was virulent to the resistant Coffea genotype 'Clone 14' and populations Par 3 Guatemala, Par 5 Piumbí-MG and Par 4 Rolândia- PR were the most aggressive against the susceptible control ‘Catuaí IAC 81’. In the second assay, four resistant genotypes were tested against seven M. paranaensis populations: Catuaí Vermelho x Amphillo MR2161 (E1 16-5 III), C. canephora rootstock Apoatã IAC 2258, Timor Hybrid UFV 408-01 (E1 6-6 II) and IPR 100 (C. arabica), plus the susceptible control C. arabica cv. Mundo Novo 379-19. Populations Par 2 HerculândiaSP, Par 3 Guatemala and Par 5 Piumbí-MG were the most aggressive against the susceptible control cv. Mundo Novo 379-19. Not a single M. paranaensis population was virulent to all four resistant cultivars: Apoatã IAC 2258, Catuaí Vermelho x Amphillo MR2161 (E1 16-5 III), IPR 100 and Timor Hybrid UFV 408-01 (E1 6-6 II), exhibiting segregation rates of 15%, 0%, 26% and 31%, respectively. These are promising results due to the validation of resistance from different genetic sources in Coffea spp. against M. paranaensis.
Root-knot nematodes (RKN), Meloidogyne spp., are amongst the most economically important plant parasitic nematodes infecting coffee (Coffea spp.) in several countries in the Americas. The objectives of this study were to developed a polymerase chain reaction (PCR)-based assay for specific detection of two RKN species, Meloidogyne arabicida and M. izalcoensis (Tylenchida: Meloidogynidae), major pathogens of coffee crops in Central America and to assess the genetic variability and aggressiveness of seven populations of M. parananesis, one of the most destructive RKN species in coffea, Coffea spp., which harbors resistance to Meloidogyne spp. In the first study Random Amplified Polymorphic DNA (RAPD) fragments specific for these two species were converted into sequence characterized amplified region (SCAR marker). PCR amplification using SCAR primers produced a specific fragment of expected size (e.g., 300 base pairs and 670 bp) in M. arabicida and M. izalcoensis, respectively. SCAR primers also allowed successful amplification of DNA from single infective juveniles, males, females and on field-isolated populations, containing mixtures of species. Therefore, these results demonstrate the effectiveness of these SCAR markers developed for identification M. arabicida and M. izalcoensis species and may contribute to specific diagnosis of these RKN in the Americas. In the second study, seven populations of M. paranaensis were identified by biochemical and molecular characterization. Phylogenetic studies have shown that despite the existence of three esterase phenotypes (Est P1, P2, P2a), a low genetic variability was observed, even in different DNA regions. Molecular analysis showed that there is a genetic difference between population P2a from Guatemala compared to other M. paranaensis populations from Brazil containing Est P1 and P2 profiles. In addition, population P2a was the most aggressive in pathogenicity assays with susceptible C. arabica cultivars. Two assays were carried out to study the aggressiveness/virulence among M. paranaensis populations against Coffea genotypes. In the first assay using different M. paranaensis vii populations against C. arabica (cv. Catuaí IAC 81) and C. canephora (cv. Clone 14), a high susceptibility pattern of cv. Catuaí IAC 81 was confirmed with RF> 30, as well as a high resistance phenotype for the clonal variety "Clone 14" with RF <0.2 was observed. None of the populations was virulent to the resistant Coffea genotype 'Clone 14' and populations Par 3 Guatemala, Par 5 Piumbí-MG and Par 4 Rolândia- PR were the most aggressive against the susceptible control ‘Catuaí IAC 81’. In the second assay, four resistant genotypes were tested against seven M. paranaensis populations: Catuaí Vermelho x Amphillo MR2161 (E1 16-5 III), C. canephora rootstock Apoatã IAC 2258, Timor Hybrid UFV 408-01 (E1 6-6 II) and IPR 100 (C. arabica), plus the susceptible control C. arabica cv. Mundo Novo 379-19. Populations Par 2 HerculândiaSP, Par 3 Guatemala and Par 5 Piumbí-MG were the most aggressive against the susceptible control cv. Mundo Novo 379-19. Not a single M. paranaensis population was virulent to all four resistant cultivars: Apoatã IAC 2258, Catuaí Vermelho x Amphillo MR2161 (E1 16-5 III), IPR 100 and Timor Hybrid UFV 408-01 (E1 6-6 II), exhibiting segregation rates of 15%, 0%, 26% and 31%, respectively. These are promising results due to the validation of resistance from different genetic sources in Coffea spp. against M. paranaensis.
Descrição
Tese de Doutorado defendida na Universidade de Brasília.
Palavras-chave
marcadores moleculares, Meloidogyne spp, nematoides das galhas, resistência, molecular markers, resistance, root-knot nematodes
Citação
SANTOS, Marcilene Fernandes Almeida dos. Desenvolvimento de marcadores scar para identificação de espécies de Meloidogyne do cafeeiro e variabilidade genética e agressividade de populações de M. paranaensis a genótipos de Coffea spp. 2016. vii, 124 f., il. Tese (Doutorado em Agronomia)-Universidade de Brasília, Brasília, 2016.