GENETIC VARIABILITY OF Metarhizium anisopliae var. ANISOPLIAE ISOLATED FROM Dendroctonus mexicanus sp. AT FOUR SITES IN DURANGO, MEXICO WITH ISSR MARKERS
JESÚS GARCÍA-PEREYRA *
Tecnológico Nacional de México, Campus Valle del Guadiana Km 22.5 Car. Durango-México. C.P. 34371, Durango, Dgo, México.
GABRIEL NICOLÁS AVIÑA-MARTÍNEZ
Tecnológico Nacional de México, Campus Valle del Guadiana Km 22.5 Car. Durango-México. C.P. 34371, Durango, Dgo, México.
SERGIO DE LOS SANTOS -VILLALOBOS
Instituto Tecnológico de Sonora, 5 de Febrero 818 Sur, 85000, Cd. Obregón, Sonora, México.
ROSA BERTHA RUBIO-GRACIANO
Tecnológico Nacional de México, Campus Valle del Guadiana Km 22.5 Car. Durango-México. C.P. 34371, Durango, Dgo, México.
MÓNICA GARCÍA-MONTELONGO
Centro de Investigación y de Estudios Avanzados del IPN (Cinvestav) Unidad Irapuato, Libramiento Norte Carretera Irapuato León Kilómetro 9.6, Car. Panamericana Irapuato León, 36821 Irapuato, Gto, México.
GABRIEL ALEJANDRE-ITURBIDE
CIIDIR Durango IPN, Mexico.
HIRAM MEDRANO-ROLDAN
Tecnologico Nacional de México, Campus Tecnológico de Durango, México.
*Author to whom correspondence should be addressed.
Abstract
The intraspecific genetic variability among entomopathogenic fungi Metarhizium anisopliae var. anisopliae isolated from the Mexican pine beetle Dendroctonus mexicanus sp. was analyzed. Specimens were collected from four regions of Otinapa Durango, Mexico (geographic coordinates: -105.008333 longitude, 24.053889 latitude). Strains collected from the specimens were compared to commercial Metarhizium anisopliae (Metatron®) using inter simple sequence repeat (ISSR) polymerase chain reaction (PCR). M. anisopliae DNA was extracted employing two protocols: plant DNAzol and SDS (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Extracted DNA was further analyzed and used for PCR amplification employing commercial ISSR primers: ISSR1, ISSR4 and ISSR7. Statistical analysis to evaluate strain variability was conducted using Shannon´s diversity index (CI=0.95) while efficiency of DNA extraction methods was measured using variance analysis (p<0.05) and Tukey´s range test. Statistically significant differences were found between the two DNA extraction methods employed. Genetic variability among M. anisopliae strains was observed, high levels of homozygosis were identified between M. anisopliae strains collected from collection sites 3 and 1, while strains collected from collection site 2 exhibited similar behavior with the commercial Metatron® strain.
Keywords: Metarhizium anisopliae, Dendroctonus mexicanus sp, ISSR molecular markers