Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus

Perinotto, Wendell Marcelo de Souza

Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus

Detalhes bibliográficos
Autor(a) principal:	Perinotto, Wendell Marcelo de Souza
Data de Publicação:	2013
Tipo de documento:	Tese
Idioma:	por
Título da fonte:	Biblioteca Digital de Teses e Dissertações da UFRRJ
Texto Completo:	https://tede.ufrrj.br/jspui/handle/jspui/3318
Resumo:	The fungus Metarhizium anisopliae sensu lato (s.l.) is one of the most promising micro-organisms to be used in the biological control of ticks. This fungus has been studied worldwide showing accurate acaricidal potential, especially in in vitro tests. However, a large variation on the virulence of different fungal isolates has been observed. From this perspective, the present study aimed to verify the pathogenic action of different fungal formulations (oil-based and aqueous) on ticks and also analyze the enzymatic action of M. anisopliae s.l. isolates from different Brazilian geographical regions in order to select the most virulent isolates for further use in programs of biological control of ticks. The presence of dsRNA viral particles (mycovirus) associated to the fungal enzymatic activities was also studied. Among the studied enzymes was the subtilisin Pr 1; whose enzymatic activity was associated with the percentage of tick control caused by the fungi aiming establish virulence markers specific for Rhipicephalus microplus ticks. Five M. anisopliae s.l. isolates from different Brazilian states were analyzed. First, a bioassay was performed on R. microplus engorged females to test the virulence of these fungal isolates. Concomitant assays were performed to quantify the enzymatic activity of lipases, proteases and chitinases secreted by the same M. anisopliae s.l. isolates. Fungi were grown in liquid medium containing ticks? cuticle and cholesteryl stearate; fluorescent and chromogenic substrates were used to quantify the enzymatic activity. Mycovirus survey was carried out following steps of total nucleic acid extraction before an endonuclease S1 and RNAse A digestion and consecutive visualization on agarose gel. Furthermore, a bioassay was conducted using different oil formulations (10 % vegetable oil and 10% mineral oil). In this experiment, R. microplus engorged females were immersed in the respective formulations for three minutes. The efficacy of the treatments was assessed through analysis in the females? reproductive parameters. The present study showed that there is variation in the virulence among M. anisopliae s.l. isolates against R. microplus, moreover, it was evident that the isolates that yielded the highest tick control percentages were also those which had the highest lipolytic, proteolytic and chitinolytic activities. Pr1 is known to be involved in the infection process of M. anisopliae s.l.; accordingly the present results demonstrate that this enzyme can be used as a molecular marker for R. microplus, since the isolates that showed higher Pr 1 activity were also the most virulent for this tick species. However, there was no evident analogy between the infection with dsRNA virulent particles and the enzymatic activity levels of the five isolates tested, considering that only one was not infected. Regarding the bioassay with oil-based formulations, it was clear that formulation containing mineral oil were more effective than those containing vegetable oil which provided low percentage of tick control. Furthermore, it can be concluded that the isolates GC 148 and GC 629 have a high potential to control R. microplus engorged females when formulated in mineral oil

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spelling	Bittencourt, V?nia Rita Elias Pinheiro505.198.676-53http://lattes.cnpq.br/3888832724995864Salles, Cristiane Martins Cardoso de035.399.287-90http://lattes.cnpq.br/3610279707231709Silva, Walter Orlando Beys dahttp://lattes.cnpq.br/1353100437268465Costa, Gisela Lara daReck J?nior, Jos?Famadas, K?tia MariaCoumendouros, Katherina306.937.188-46http://lattes.cnpq.br/1812730341180904Perinotto, Wendell Marcelo de Souza2020-02-17T12:59:37Z2013-03-05PERINOTTO, Wendell Marcelo de Souza. Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus. 2013. 67 f. Tese (Doutorado em Ci?ncias Veterin?rias) - Instituto de Veterin?ria, Universidade Federal Rural do Rio de Janeiro, Serop?dica, 2013.https://tede.ufrrj.br/jspui/handle/jspui/3318The fungus Metarhizium anisopliae sensu lato (s.l.) is one of the most promising micro-organisms to be used in the biological control of ticks. This fungus has been studied worldwide showing accurate acaricidal potential, especially in in vitro tests. However, a large variation on the virulence of different fungal isolates has been observed. From this perspective, the present study aimed to verify the pathogenic action of different fungal formulations (oil-based and aqueous) on ticks and also analyze the enzymatic action of M. anisopliae s.l. isolates from different Brazilian geographical regions in order to select the most virulent isolates for further use in programs of biological control of ticks. The presence of dsRNA viral particles (mycovirus) associated to the fungal enzymatic activities was also studied. Among the studied enzymes was the subtilisin Pr 1; whose enzymatic activity was associated with the percentage of tick control caused by the fungi aiming establish virulence markers specific for Rhipicephalus microplus ticks. Five M. anisopliae s.l. isolates from different Brazilian states were analyzed. First, a bioassay was performed on R. microplus engorged females to test the virulence of these fungal isolates. Concomitant assays were performed to quantify the enzymatic activity of lipases, proteases and chitinases secreted by the same M. anisopliae s.l. isolates. Fungi were grown in liquid medium containing ticks? cuticle and cholesteryl stearate; fluorescent and chromogenic substrates were used to quantify the enzymatic activity. Mycovirus survey was carried out following steps of total nucleic acid extraction before an endonuclease S1 and RNAse A digestion and consecutive visualization on agarose gel. Furthermore, a bioassay was conducted using different oil formulations (10 % vegetable oil and 10% mineral oil). In this experiment, R. microplus engorged females were immersed in the respective formulations for three minutes. The efficacy of the treatments was assessed through analysis in the females? reproductive parameters. The present study showed that there is variation in the virulence among M. anisopliae s.l. isolates against R. microplus, moreover, it was evident that the isolates that yielded the highest tick control percentages were also those which had the highest lipolytic, proteolytic and chitinolytic activities. Pr1 is known to be involved in the infection process of M. anisopliae s.l.; accordingly the present results demonstrate that this enzyme can be used as a molecular marker for R. microplus, since the isolates that showed higher Pr 1 activity were also the most virulent for this tick species. However, there was no evident analogy between the infection with dsRNA virulent particles and the enzymatic activity levels of the five isolates tested, considering that only one was not infected. Regarding the bioassay with oil-based formulations, it was clear that formulation containing mineral oil were more effective than those containing vegetable oil which provided low percentage of tick control. Furthermore, it can be concluded that the isolates GC 148 and GC 629 have a high potential to control R. microplus engorged females when formulated in mineral oilO fungo Metarhizium anisopliae sensu lato ? um dos micro-organismos mais promissores a ser utilizado no controle de carrapato, e por isso, vem sendo estudado em todo o mundo, demonstrando potencial acaricida, principalmente em testes in vitro. Entretanto, tem sido observada varia??o na virul?ncia entre diferentes isolados deste fungo. Nessa perspectiva, o presente estudo teve como objetivos verificar a a??o patog?nica em suspens?es aquosas e em diferentes formula??es oleosas e tamb?m analisar a a??o enzim?tica de isolados brasileiros de M. anisopliae s.l. com a finalidade de selecionar isolados mais virulentos para utiliza??o em futuros programas de biocontrole de carrapatos. Al?m disso, pesquisar a presen?a de part?culas virais de dsRNA e associar as atividades enzim?ticas, inclusive da subtilisina Pr 1 secretadas pelos isolados de M. anisopliae s.l. com os percentuais de controle, visando estabeler marcadores moleculares de virul?ncia espec?ficos para o carrapato R. microplus. Para tanto, cinco linhagens de M. anisopliae s.l. isolados de diferentes estados brasileiros foram analisadas. Primeiramente foi realizado um bioensaio para testar a virul?ncia destes isolados sobre f?meas alimentadas de R. microplus. Concomitantemente foram realizados ensaios enzim?ticos para quantificar as atividades de lipases, proteases e quitinases secretadas pelos diferentes isolados de M. anisopliae s.l. cultivados em meio l?quido contendo cut?cula de carrapato e estearato de colesteril, atrav?s da utiliza??o de substratos cromog?nicos e fluorescentes. Para a pesquisa de micov?rus, foi realizada a extra??o do ?cido nucl?ico total que foi submetido ? digest?o por uma endonuclease S1 e uma RNAse A e posteriormente visualizado em gel de agarose. Al?m desses ensaios, foi realizado um bioensaio utilizando diferentes formula??es oleosas, uma a base de ?leo vegetal e outra com ?leo mineral, ambas a 10%. Neste experimento, f?meas de R. microplus completamente alimentadas foram imersas por tr?s minutos nos respectivos tratamentos, a efic?cia dos tratamentos foi avaliada atrav?s de altera??es provocadas nos par?metros reprodutivos destas f?meas. O estudo demonstrou que h? varia??o na virul?ncia entre os isolados brasileiros de M. anisopliae s.l. para f?meas alimentadas de R. microplus, al?m disso, ficou evidenciado que os isolados que apresentaram os maiores percentuais de controle foram tamb?m os que tiveram as maiores atividade lipol?tica, proteol?tica e quitinol?tica. Pelo fato da enzima Pr 1 ser sabidamente envolvida no processo de infec??o de M. anisopliae s.l., os resultados aqui encontrados demonstraram a possibilidade de usar a atividade desta enzima como um potencial marcador molecular para R. microplus, visto que os isolados que apresentaram maior atividade de Pr 1 foram os mais virulentos para esta esp?cie de carrapato. Entretanto, n?o foi evidenciada nenhuma rela??o entre a virul?ncia com a infec??o por part?culas de dsRNA, sendo que dos cinco isolados testados, somente um n?o estava infectado. A partir do bioensaio utilizando formula??es oleosas, ficou evidenciada que as formula??es contendo ?leo mineral foram mais eficientes do que as que continham ?leo vegetal, proporciondo os melhores percentuais de controle. Al?m disso, pode-se concluir que os isolados CG 148 e CG 629 quando em formula??o de ?leo mineral apresentam alto potencial de controle para f?meas ingurgitadas de R. microplusSubmitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2020-02-17T12:59:37Z No. of bitstreams: 1 2013 - Wendell Marcelo de Souza Perinotto.pdf: 1424851 bytes, checksum: 6aab6cad0730f71ac3c9d5d95e788917 (MD5)Made available in DSpace on 2020-02-17T12:59:37Z (GMT). No. of bitstreams: 1 2013 - Wendell Marcelo de Souza Perinotto.pdf: 1424851 bytes, checksum: 6aab6cad0730f71ac3c9d5d95e788917 (MD5) Previous issue date: 2013-03-05Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPESapplication/pdfhttps://tede.ufrrj.br/retrieve/12844/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpghttps://tede.ufrrj.br/retrieve/17124/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpghttps://tede.ufrrj.br/retrieve/23584/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpghttps://tede.ufrrj.br/retrieve/29962/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpghttps://tede.ufrrj.br/retrieve/36336/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpghttps://tede.ufrrj.br/retrieve/42730/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpghttps://tede.ufrrj.br/retrieve/49110/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpghttps://tede.ufrrj.br/retrieve/55556/2013%20-%20Wendell%20Marcelo%20de%20Souza%20Perinotto.pdf.jpgporUniversidade Federal Rural do Rio de JaneiroPrograma de P?s-Gradua??o em Ci?ncias Veterin?riasUFRRJBrasilInstituto de Veterin?riaABDEL-SHAFY, S.; SOLIMAN, M.M.M. 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Screening of Beauveria bassiana (Deuteromycotina: Hyphomycetes) isolates against nymphs of Bemisia tabaci (Genn.) biotype B (Hemiptera: Aleyrodidae) with description of a new bioassay method. Neotropical Entomology. v. 30, p. 97?103, 2001. ZACHARUK, R.Y. Fine structure of the fungus Metarhizium anisopliae infecting three species of larval Elateridae (Coleoptera): II. Conidial germ tubes and appressoria. Journal of Invertebrate Pathology. v. 15, p. 81-91, 1970. ZHANG, D.; YANG, Y.; CASTLEBURY, L. A.; CERNIGLIA, C. E. A method for the large scale isolation of high transformation effiency fungal genomic DNA. FEMS Microbiology Letters. v. 145, p. 261-265, 1996. ZHANG, W.M.; LI, T.H.; CHEN, Y.Q.; QU, L.H. Cordyceps campsosterna, a new pathogen of Campsosternus auratus. Fungal Diversity. v. 17, p. 239-242, 2004. ZIMMERMANN, G. Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Science and Technology, v. 9, p. 553-596, 2007 a. ZIMMERMANN, G. Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Science and Technology. v. 17, p. 879-920, 2007 b.fungos artropodopatog?nicosvirul?nciacontrole biol?gicoand biological controlarthropod-pathogenic fungivirulenceMedicina Veterin?riaEstudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplusStudy of the enzymatic activity and pathogenic of Metarhizium anisopliae s.l. 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dc.title.por.fl_str_mv	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus
dc.title.alternative.eng.fl_str_mv	Study of the enzymatic activity and pathogenic of Metarhizium anisopliae s.l. Brazilian isolates targeting the biological control of Rhipicephalus (Boophilus) microplus
title	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus
spellingShingle	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus Perinotto, Wendell Marcelo de Souza fungos artropodopatog?nicos virul?ncia controle biol?gico and biological control arthropod-pathogenic fungi virulence Medicina Veterin?ria
title_short	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus
title_full	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus
title_fullStr	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus
title_full_unstemmed	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus
title_sort	Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus
author	Perinotto, Wendell Marcelo de Souza
author_facet	Perinotto, Wendell Marcelo de Souza
author_role	author
dc.contributor.advisor1.fl_str_mv	Bittencourt, V?nia Rita Elias Pinheiro
dc.contributor.advisor1ID.fl_str_mv	505.198.676-53
dc.contributor.advisor1Lattes.fl_str_mv	http://lattes.cnpq.br/3888832724995864
dc.contributor.advisor-co1.fl_str_mv	Salles, Cristiane Martins Cardoso de
dc.contributor.advisor-co1ID.fl_str_mv	035.399.287-90
dc.contributor.advisor-co1Lattes.fl_str_mv	http://lattes.cnpq.br/3610279707231709
dc.contributor.advisor-co2.fl_str_mv	Silva, Walter Orlando Beys da
dc.contributor.advisor-co2Lattes.fl_str_mv	http://lattes.cnpq.br/1353100437268465
dc.contributor.referee1.fl_str_mv	Costa, Gisela Lara da
dc.contributor.referee2.fl_str_mv	Reck J?nior, Jos?
dc.contributor.referee3.fl_str_mv	Famadas, K?tia Maria
dc.contributor.referee4.fl_str_mv	Coumendouros, Katherina
dc.contributor.authorID.fl_str_mv	306.937.188-46
dc.contributor.authorLattes.fl_str_mv	http://lattes.cnpq.br/1812730341180904
dc.contributor.author.fl_str_mv	Perinotto, Wendell Marcelo de Souza
contributor_str_mv	Bittencourt, V?nia Rita Elias Pinheiro Salles, Cristiane Martins Cardoso de Silva, Walter Orlando Beys da Costa, Gisela Lara da Reck J?nior, Jos? Famadas, K?tia Maria Coumendouros, Katherina
dc.subject.por.fl_str_mv	fungos artropodopatog?nicos virul?ncia controle biol?gico and biological control
topic	fungos artropodopatog?nicos virul?ncia controle biol?gico and biological control arthropod-pathogenic fungi virulence Medicina Veterin?ria
dc.subject.eng.fl_str_mv	arthropod-pathogenic fungi virulence
dc.subject.cnpq.fl_str_mv	Medicina Veterin?ria
description	The fungus Metarhizium anisopliae sensu lato (s.l.) is one of the most promising micro-organisms to be used in the biological control of ticks. This fungus has been studied worldwide showing accurate acaricidal potential, especially in in vitro tests. However, a large variation on the virulence of different fungal isolates has been observed. From this perspective, the present study aimed to verify the pathogenic action of different fungal formulations (oil-based and aqueous) on ticks and also analyze the enzymatic action of M. anisopliae s.l. isolates from different Brazilian geographical regions in order to select the most virulent isolates for further use in programs of biological control of ticks. The presence of dsRNA viral particles (mycovirus) associated to the fungal enzymatic activities was also studied. Among the studied enzymes was the subtilisin Pr 1; whose enzymatic activity was associated with the percentage of tick control caused by the fungi aiming establish virulence markers specific for Rhipicephalus microplus ticks. Five M. anisopliae s.l. isolates from different Brazilian states were analyzed. First, a bioassay was performed on R. microplus engorged females to test the virulence of these fungal isolates. Concomitant assays were performed to quantify the enzymatic activity of lipases, proteases and chitinases secreted by the same M. anisopliae s.l. isolates. Fungi were grown in liquid medium containing ticks? cuticle and cholesteryl stearate; fluorescent and chromogenic substrates were used to quantify the enzymatic activity. Mycovirus survey was carried out following steps of total nucleic acid extraction before an endonuclease S1 and RNAse A digestion and consecutive visualization on agarose gel. Furthermore, a bioassay was conducted using different oil formulations (10 % vegetable oil and 10% mineral oil). In this experiment, R. microplus engorged females were immersed in the respective formulations for three minutes. The efficacy of the treatments was assessed through analysis in the females? reproductive parameters. The present study showed that there is variation in the virulence among M. anisopliae s.l. isolates against R. microplus, moreover, it was evident that the isolates that yielded the highest tick control percentages were also those which had the highest lipolytic, proteolytic and chitinolytic activities. Pr1 is known to be involved in the infection process of M. anisopliae s.l.; accordingly the present results demonstrate that this enzyme can be used as a molecular marker for R. microplus, since the isolates that showed higher Pr 1 activity were also the most virulent for this tick species. However, there was no evident analogy between the infection with dsRNA virulent particles and the enzymatic activity levels of the five isolates tested, considering that only one was not infected. Regarding the bioassay with oil-based formulations, it was clear that formulation containing mineral oil were more effective than those containing vegetable oil which provided low percentage of tick control. Furthermore, it can be concluded that the isolates GC 148 and GC 629 have a high potential to control R. microplus engorged females when formulated in mineral oil
publishDate	2013
dc.date.issued.fl_str_mv	2013-03-05
dc.date.accessioned.fl_str_mv	2020-02-17T12:59:37Z
dc.type.status.fl_str_mv	info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv	info:eu-repo/semantics/doctoralThesis
format	doctoralThesis
status_str	publishedVersion
dc.identifier.citation.fl_str_mv	PERINOTTO, Wendell Marcelo de Souza. Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus. 2013. 67 f. Tese (Doutorado em Ci?ncias Veterin?rias) - Instituto de Veterin?ria, Universidade Federal Rural do Rio de Janeiro, Serop?dica, 2013.
dc.identifier.uri.fl_str_mv	https://tede.ufrrj.br/jspui/handle/jspui/3318
identifier_str_mv	PERINOTTO, Wendell Marcelo de Souza. Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus. 2013. 67 f. Tese (Doutorado em Ci?ncias Veterin?rias) - Instituto de Veterin?ria, Universidade Federal Rural do Rio de Janeiro, Serop?dica, 2013.
url	https://tede.ufrrj.br/jspui/handle/jspui/3318
dc.language.iso.fl_str_mv	por
language	por
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Two new Beauveria spp. from South America. Journal of Invertebrate Pathology. v. 39, p. 93-97, 1982. 64 SANGORR?N, M.P.; FOLCO, E.J.; MARTONE, C.M.; S?NCHEZ, J.J. Purification and characterization of a protease inhibitor from white croaker skeletal muscle (Micropogon opercularis). International Journal of Biochemistry & Cell Biology. v. 33, p. 691-699, 2001. SANTI, L.; BEYS DA SILVA, W.O.; BERGER, M.; GUIMAR?ES, J.A.; SCHRANK, A.; VAINSTEIN, M.H. Conidial surface proteins of Metarhizium anisopliae s.l.: Source of activities related with toxic effects, host penetration and pathogenesis. Toxicon. v. 55, p. 874-880, 2010 a. SANTI, L.; BEYS-DA-SILVA, W.O.; PINTO, A.F.M.; SCHRANK, A.; VAINSTEIN, M.H. Metarhizium anisopliae host-pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthorpods. Fungal Biology. v. 114, p. 312-319, 2010 b. SANTI, L.; SILVA, L.A.D.; SILVA, W.O.B.; CORR?A, A.P.F.; RANGEL, D.E.N.; CARLINI, C.R.; SCHRANK, A. Virulence of the entomopathogenic fungus Metarhizium anisopliae using soyb
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Estudo da atividade enzim?tica e patog?nica de isolados brasileiros de Metarhizium anisopliae s.l. visando o uso no controle biol?gico de Rhipicephalus (Boophilus) microplus

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