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Previous issue date: 2012-03-30 / FAPERJ / Aiming to decrease the chemicals acaricide use and their damages, new alternatives for ticks control has been studied. Metarhizium anisopliae s.l. is one of the most studied fungi in agricultural pest management programs, since it has great acaricide potential. Therefore, this study aimed to characterize molecular and morphologically, as well as evaluate the virulent potential of 30 M. anisopliae s.l. isolates from different geographical regions, hosts or substrates allowing the selection of virulent isolates in order to be further investigated for field programs of microbial control of pests. Initially, the analyses of morphological characterizations of the isolates were made to confirm their identification. Each isolate had its conidial potential production evaluated. The colonies studied showed morphological characteristics consistent with those described in the literature. The colonies diameter varied between 29.66 mm and 51.33 mm among isolates. There was both length and width variation in the conidia and phialides in the same isolate, as well as the presence of grouped and solitary phialides. The conidial production potential was variable among isolates, but both conidial size and colonies diameter did not influence the conidial production; isolates with low conidial production showed similar colony size in comparison to isolates with high potential. In a second stage of the study, the virulence of these isolates was evaluated to Rhipicephalus microplus larvae treated with one of the four different conidial concentrations (105, 106, 107 or 108 conidia.mL-1). The lethal action of Brazilian M. anisopliae s.l isolates to R. microplus larvae were confirmaded with high mortality among the isolates, which in general was proportional to the conidia concentration of the treatments. Most isolates killed larvae population with 107 conidia.mL-1 concentration, however the most virulent isolates presented lethal concentration of 106 conidia.mL-1 with main percentages of mortality nearly 100% at day 20 after treatment. In addition, the genetic variability of these isolates was performed to evaluate their relationship with other species of Metarhizium sp. through RFLP-PCR analysis and ITS1-5.8S-ITS2 rDNA sequencing. No specificity pattern was observed when isolates from the same region, host or substrate were grouped. Low genetic variability was observed among isolates, which were basically grouped into two groups. The CG 344 isolate was shown to be genetically distant from the remaining Brazilian isolates studied, but according to the ?GenBank? sequences comparison, it was related to the Metarhizium genus. It is suggested that this variation occured owing the lack of procedures that could generated morphological and molecular changes, which probably contribute to this low genetic variability. The present study allowed the detection of M. anisopliae s.l. isolates with highly virulence to R. microplus larvae, that may be considered potential biocontrol agents for this tick species, emphasizing the importance of molecular tools for identification and characterization of fungal isolates, ensuring the product quality, their success implement and the environmental track of the fungi at field biological control programs. / Na tentativa de diminuir a utiliza??o de produtos qu?micos e os danos por eles causados, novas alternativas para o controle de carrapatos vem sendo estudadas. O fungo Metarhizium anisopliae ? um dos mais estudados em programas agropecu?rios de manejo de pragas, pois apresenta grande potencial acaricida. Baseado nisso, o presente estudo objetivou a caracteriza??o morfol?gica, molecular e a avalia??o da virul?ncia de 30 isolados brasileiros de M. anisopliae s.l. provenientes de diferentes regi?es geogr?ficas, hospedeiros ou substratos, com a finalidade de selecionar isolados mais virulentos para utiliza??o em futuros programas de biocontrole de carrapatos. Inicialmente os isolados foram caracterizados morfologicamente para confirma??o de sua identifica??o, tamb?m sendo avaliado o potencial de produ??o de con?dios de cada isolado. As col?nias estudadas apresentaram caracter?sticas morfol?gicas compat?veis com as descritas na literatura. O tamanho das col?nias variou entre 29,66 mm e 51,33 mm de di?metro. Houve varia??o no comprimento e na largura de con?dios e fi?lides num mesmo isolado, assim como a presen?a de fi?lides agrupadas e solit?rias. O potencial de produ??o de con?dios foi vari?vel entre os isolados, por?m tanto o tamanho dos con?dios quanto o di?metro das col?nias n?o influenciaram a produ??o de con?dios. Numa segunda etapa do estudo, foi avaliada a virul?ncia destes isolados sobre larvas de Rhipicephalus microplus tratadas com uma das quatro diferentes concentra??es de con?dios (105, 106, 107 ou 108 con?dios/mL). Foi confirmada a a??o letal dos isolados brasileiros de M. anisopliae s.l. sobre larvas de R. microplus, geralmente ocorrendo de forma diretamente proporcional a concentra??o conidial dos tratamentos. A maioria dos isolados ocasionou a morte de metade da popula??o de larvas com a concentra??o de 107 con?dios/mL; os isolados mais virulentos apresentaram esta concentra??o letal com 106 con?dios/mL, com percentuais m?dios de mortalidade de larvas pr?ximos de 100% ao 20? dia ap?s tratamento. Al?m disso, buscou-se avaliar a variabilidade gen?tica destes isolados e sua rela??o com outras esp?cies do g?nero Metarhizium atrav?s da an?lise de RFLP-PCR e do sequenciamento da regi?o ITS1-5.8S-ITS2 do rDNA. N?o foi observado um padr?o de especificidade para o agrupamento entre isolados oriundos de mesma regi?o, hospedeiro ou substrato. Foi observada variabilidade gen?tica entre os isolados que basicamente se agruparam em dois grupos. O isolado CG 344 mostrou-se geneticamente distante de todos os outros, mas de acordo com a compara??o com sequ?ncias obtidas do ?GenBank? mostrou-se relacionado ao g?nero Metarhizium. Esta varia??o pode ser devido ao fato deste isolado ter sido poupado de processos que gerassem altera??es morfol?gicas e moleculares, o que possivelmente contribuiu para a pequena variabilidade gen?tica obsevada. O presente estudo possibilitou a detec??o de isolados brasileiros de M. anisopliae s.l. com elevada virul?ncia para larvas de R. microplus, podendo ser considerados potenciais agentes no biocontrole desta esp?cie de carrapato, ressaltando a import?ncia da utiliza??o de ferramentas moleculares para identifica??o e caracteriza??o destes isolados, contribuindo para a qualidade do produto, o sucesso de sua aplica??o e o monitoramento de um isolado introduzido no ambiente com finalidade de controle biol?gico.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/1559 |
| Date | 30 March 2012 |
| Creators | BEZERRA, Simone Quinelato |
| Contributors | Bittencourt, V?nia Rita Elias Pinheiro, Moraes, Aurea Maria Lage de, Zahner, Viviane, Bittencourt, V?nia Rita Elias Pinheiro, Borba, C?ntia de Moraes, Fernades, ?verton Kort Kamp, Costa, Gisela Lara da, Prata, Maria Cristina de Azevedo |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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