Contribuição ao controle biológico de Rhynchophorus palmarum - atividade de vôo, eficiência de autoinoculação e caracterização molecular de isolados de Beauveria bassiana

• Autor(a) principal: Mendonça, Marcelo da Costa
• Data de Publicação: 2007
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da Universidade Federal de Alagoas (UFAL)
• Texto Completo: http://www.repositorio.ufal.br/handle/riufal/2405
• topic: Broca-do-olho-do-coqueiro; Cocos nucifera; Fungos entomopatogênico; Palm weevil; Entomopathogenic fungi; CNPQ::CIENCIAS EXATAS E DA TERRA::QUIMICA

The coleoptera Rhynchophorus palmarum (L.), vulgarly known as palm weevil is considered on of mainly pest of several palm trees of economic importance. In coconut tree their larvae destroy the plant culms and their adults act like a vector of nematode Bursaphelenchus cocophilus that cause the red ring disease. With the objective of subside the practices of biological control of R. palmarum already utilized it was carried out tests aiming to determine the flight activity of the insect in coconut fields; to validate the technique of autoinoculation of adults of R. palmarum using traps like vegetal traps bait inoculated with the Beauveria bassiana enthomopatogenic fungi; and to characterize genetically isolates of the fungi by molecular makers of RAPD, microsatellites and ITS-DNAr. To determine the flight activity of the insect it was installed, around the commercial field, traps of capture/monitoring for population of R. palmarum adult’s insect, containing attractive feed (culms of sugar cane) added with pheromone of aggregation. For the autoinoculation tests it was used the randomized statistical design, in a factorial scheme 3x4 (3 spores concentration of the fungi and 4 periods of autoinoculation), using four replications constituted by 5 pairs of males and females. The insects were separated, cleaned with distilled water and transfer to buckets with sugar cane culms inoculated with B. bassiana on concentrations 107, 108 and 109 spores/mL of suspension), keeping in contact with the inoculums for 45min.; 90min.; 3h00 and 6h00. After this period of exposition the insects were individualized being the mortality/infection evaluated for 20 days. For genetic characterization it was used 11 isolates obtained from monospores cultures that posterior were inoculated in a liquid medium at 25ºC±1ºC under constant shaking in dark conditions. After seven days the mycelium were filtered, lyophilized and after DNA extracted in SDS buffer at 10%. It was tested 20 decamer primers of arbitrary sequence of IDT Company. The amplifications were carried out in termocycler (Biometra/Unisciense) programmed for initial temperature of 94°C for 5min. and 45 cycles of: 94°C for 1min., 36°C for 2min. and 72°C for 1min.. For microsatellites analyses it was used four pairs of primers. The amplification reactions were carried out using a volume of 25µL. The amplifications consisted by one initial cycle of 94ºC and 30 cycles of 94ºC for 1min.; 50ºC for 1min. and 72ºC for 2min., and a end cycle of 72ºC for 7min.. The amplified fragments were separated by electrophoresis in agarose gel using TBE buffer, and the gel stained with ethidium bromete (0.5µg/mL) after visualized under UV light. The insect initiates flight activity at 5h00 am through 6h00 pm o’clock occurring the population maximum at 9h00 am to11h00 am. It was verified the mortality increase of adults of R. palmarum in accord to fungi concentration and period of exposition of the inoculums. The higest index of mortality (53%) occurred nine days after the autoinoculation by B. bassiana. The RAPD analysis generated 138 polymorphic and 14 monomorphic bands being the patterns used to calculate the genetic similarity that varies of 2% to 77%. The 57.99 (Betume2-SE) and 53.96 (Mojú-PA) isolates presented high similarity (77%), followed by 64.99 (Egypt) and 53.96 (Mojú-PA), with similarity of 73% by RAPD test. For microssatellite the BC.05 and 32.91 isolates presented genetic similarity of 82%, followed by 64.99 and 57.99 with genetic similarity of 80%. It was not observed amplifications variations using ITS regions for B. bassiana isolates.