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Doctoral Thesis
DOI
10.11606/T.11.2003.tde-15072003-141752
Document
Author
Full name
Ricardo Henri Rodrigues Destefano
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2003
Supervisor
Committee
Messias, Claudio Luiz (President)
Arrigoni, Enrico de Beni
Azevedo, Joao Lucio de
Duarte, Marta Cristina Teixeira
Kleiner, Aline Aparecida Pizzirani
Title in Portuguese
Detecção e identificação de Metarhizium anisopliae em larvas de Diatraea saccharalis por Primers específicos.
Keywords in Portuguese
broca (inseto nocivo)
controle biológico
fungo entomopatogênico.
Abstract in Portuguese
A região espaçadora ITS rDNA tem sido utilizada como uma importante ferramenta molecular para a identificação de fungos. Neste estudo a região ITS1 - 5.8S - ITS2 foi analisada em diferentes espécies do fungo entomopatogênico Metarhizium incluindo M. anisopliae, M. album e M. flavoviride com o objetivo de se construir primers específicos para a detecção e identificação do fungo no interior de larvas infectadas de Diatraea saccharalis. A amplificação desta região produziu um fragmento único de aproximadamente 540 pb para as linhagens E9, B/Vi e C, e de 600 pb para a linhagem 14 de M. anisopliae var. anisopliae; de 650 bp para M. album e 600 pb para M. flavoviride. Os produtos de PCR obtidos foram digeridos com diferentes enzimas de restrição Afa I, Alu I, Dde I, Hae III, Hpa II e Sau 3A I; e os perfis de PCR-RFLP mostraram nítidas diferenças entre as espécies analisadas. O sequenciamento da região ITS1 - 5.8S - ITS2 permitiu o desenho de primers específicos para as linhagens de M.a. var. anisopliae do Brasil e da Austrália. A amplificação não ocorreu em linhagens de M. álbum, M. flavoviride e Beauveria bassiana. Os DNAs extraídos de larvas infectadas pelas linhagens E9, B/Vi e C do Brasil e linhagem 14, da Austrália, foram testados utilizando-se os primers desenvolvidos. Em todos os experimentos o fungo M.a. var. anisopliae foi detectado 48 horas após a inoculação. A técnica molecular empregada neste estudo permite a rápida e segura detecção e identificação de M.a. var. anisopliae em bioensaios de laboratório e de campo, programas de manejo de pragas e estudos de epizootiologia.
Title in English
Detection and identification of metarhizium anisopliae within infected sugarcane borer diatraea saccharalis larvae using specific primers.
Keywords in English
biological control
entomopathogenic fungus.
sugarcane borer
Abstract in English
The ITS rDNA have been used as an important molecular tool for fungi identification. In this study the ITS1 - 5.8S - ITS2 rDNA regions were analyzed in different species of the entomopathogenic fungus Metarhizium including M. anisopliae, M. album and M. flavoviride, in order to construct specific primers for their detection and identification within infected Diatraea saccharalis larvae. The amplification of this region yielded a unique fragment of approximately 540 bp for E9, B/Vi and C, and 600 bp for strain 14 of M. anisopliae var. anisopliae; of 650 bp for M. album and 600 bp for M. flavoviride. The PCR products were digested with the different restriction endonucleases Afa I, Alu I, Dde I, Hae III, Hpa II and Sau 3A I; and the PCR-RFLP profiles showed clear differences amongst the species. The sequencing of the ITS-5.8S rDNA regions allowed for the design of specific primers for M. anisopliae var. anisopliae. The amplification was not observed with M. album, M flavoviride and Beauveria bassiana strains. DNAs extracted from infected larvae by E9 and C strains from Brazil and the strain 14 from Australia in individual bioassays were tested using previously designed specific primers. In all experiments, the fungus was detected after 48 hours of post-inoculation. This molecular technique allows a fast and secure detection and identification of the entomopathogen in bioassays, in pest management programs and epizootiology.
 
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Publishing Date
2003-07-17
 
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