Desenvolvimento de iniciadores para detecção e identificação de Diaporthe phaseolorum var. meridionalis em sementes de soja
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Data
2006-06-01
Autores
Vechiato, Marta Helena
Maringoni, Antonio Carlos [UNESP]
Martins, Elza Maria Frias
Título da Revista
ISSN da Revista
Título de Volume
Editor
Grupo Paulista de Fitopatologia
Resumo
Considerando a grande variabilidade apresentada pelo complexo Diaporthe/Phomopsis, os métodos rotineiros de sanidade de sementes, empregados na detecção do gênero Diaporthe, não são confiáveis visto que, para sua identificação são utilizadas as características morfológicas das colônias que se desenvolvem sobre as sementes. Diante disso, este trabalho teve como objetivo desenvolver iniciadores específicos, bem como um método confiável para detecção e identificação de Diaporthe phaseolorum var. meridionalis (Dphm), em sementes de soja. Amostra de sementes da cultivar IAS5, analisada previamente para sanidade, não portadoras de Diaporthe (SS) foram inoculadas com Dphm (SI), obtendo-se amostras com as seguintes proporções de SI:SS: 1:10, 1:50, 1:100, 1:200 e 1:400. Para a extração de DNA do micélio do patógeno das sementes foi necessário primeiro incubar as sementes por 7 dias, utilizando-se o método do papel de filtro modificado com 2,4 D e, em seguida submetê-las à lavagem em tampão de extração (1% de SDS, 10 mM de TRIS/HCL e 25 mM de EDTA) por 20 min, sob agitação a 100 rpm. em seguida, alíquotas de 350 miL do sobrenadante foram transferidas para novos microtubos contendo 350 miL de resina de sílica gel da Wizard/Promega permanecendo em contacto com a mesma por 1 minuto. Os DNAs extraídos foram utilizados como molde na reação de PCR com os iniciadores: DphLe (TCG GCC TTG GAA GTA GAA AG) e DphRi (ACT GAA TGC GTT GCG ATT CT) Utilizando-se estes iniciadores, foi possível detectar a presença de D. phaseolorum var meridionalis em sementes de soja, na proporção de uma semente infectada com o patógeno em amostras de 400 sementes (0,25% de incidência), utilizando-se o método do papel de filtro modificado com 2,4 D associado à técnica de PCR.
In spite of the great variability of the Diaporthe/Phomopsis complex, the routine seed health testing, used for detection of the Diaporthe, is not reliable, taking consideration into that for its identification morphological characteristics of the colonies are used. By considering, this work had as objective to develop an accurate, fast and reliable method for Diaporthe phaseolorum var. meridionalis (Dphm) detection and identification in soybean seeds. A sample of seeds from cultivar IAS5, analyzed previously for sanity, without Diaporthe (SS) was inoculated with Dphm (S.I.). Samples were collected with the following SI:SSrations: 1:10, 1:50, 1:100, 1:200 and 1:400. DNA mycelium extraction was necessary first to incubate, the seeds, during 3 to7 days, using blotter test modified with 2,4 D and washing them with buffer extraction (1% of SDS, 10 mM of TRIS/HCl and 25 mM of EDTA) for 20 min, on an orbital shaker (100 rpm). An aliquot of 350 muL of the supernatants was transferred to microcentrifuge tubes with 350 muL of silica resin gel of the Wizard kit (Promega) remaining in contact during one minute. DNA extracted was used as template in the reaction of PCR by using the following primers: DphLe (TCG GCC TTG GAA GTA GAA GAC) and DphRi (ACT GAA TGC GTT GCG ATT CT). Using these primers, it was possible to detect the presence of D.phaseolorum var meridionalis in soybean seeds, in the ratio of one infected seed with Dphm in samples of 400 seeds (0.25% of incidence), using blotter test modified with 2,4 D associated with the PCR technique.
In spite of the great variability of the Diaporthe/Phomopsis complex, the routine seed health testing, used for detection of the Diaporthe, is not reliable, taking consideration into that for its identification morphological characteristics of the colonies are used. By considering, this work had as objective to develop an accurate, fast and reliable method for Diaporthe phaseolorum var. meridionalis (Dphm) detection and identification in soybean seeds. A sample of seeds from cultivar IAS5, analyzed previously for sanity, without Diaporthe (SS) was inoculated with Dphm (S.I.). Samples were collected with the following SI:SSrations: 1:10, 1:50, 1:100, 1:200 and 1:400. DNA mycelium extraction was necessary first to incubate, the seeds, during 3 to7 days, using blotter test modified with 2,4 D and washing them with buffer extraction (1% of SDS, 10 mM of TRIS/HCl and 25 mM of EDTA) for 20 min, on an orbital shaker (100 rpm). An aliquot of 350 muL of the supernatants was transferred to microcentrifuge tubes with 350 muL of silica resin gel of the Wizard kit (Promega) remaining in contact during one minute. DNA extracted was used as template in the reaction of PCR by using the following primers: DphLe (TCG GCC TTG GAA GTA GAA GAC) and DphRi (ACT GAA TGC GTT GCG ATT CT). Using these primers, it was possible to detect the presence of D.phaseolorum var meridionalis in soybean seeds, in the ratio of one infected seed with Dphm in samples of 400 seeds (0.25% of incidence), using blotter test modified with 2,4 D associated with the PCR technique.
Descrição
Palavras-chave
Glycine max, cancro da haste e da vagem da soja, caracterização molecular, Glycine max, stem canker of soybean, molecular characterization
Como citar
Summa Phytopathologica. Grupo Paulista de Fitopatologia, v. 32, n. 2, p. 161-169, 2006.