Fungos endofíticos de soja: diversidade e prospecção enzimática
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações do UNIOESTE |
Texto Completo: | https://tede.unioeste.br/handle/tede/6430 |
Resumo: | Plants are inhabited by a microbiota composed of microorganisms in endophytic conditions and each endophyte-plant relation displays peculiar characteristics that vary according to environmental conditions. Studies related to the identification of endophytic populations associated with plant species allow using them as important tools for obtaining biotechnological products of agricultural and industrial interest. The objective of this study was to isolate and identify endophytic fungi from soybean plants, before and after fungicide application composed by Trifloxystrobin + Prothioconazole in the soybean crops, evaluate the sensitivity of the isolates in vitro against the same fungicide and also evaluate them regarding the biotechnological potential of enzyme production. The soybean plants used were cultivated at the Experimental Nucleus of Agricultural Engineering (NEEA) of the Western Paraná State University, in Cascavel, Paraná, Brazil. The leaves of soybean plants were collected at 48 days after sowing (prior to fungicide application) and at 62 days (14 days after application). The fungi were grouped and classified according to morphological and microscopic aspects, identified at the genera level and analyzed according to indexes such colonization frequency (FC%) and ecological diversity (Simpson (D e 1-D), Shannon-Wiener (H’) and Pielou (e). A total of 259 fungi (122 before the fungicide and 137 after the fungicide application) were obtained, which resulted in the identification of 11 genera, among which Diaporthe and Alternaria were the most abundant with FC of 38.6 and 25.5%, respectively. The frequency and diversity values showed that there was no significant difference between the two harvests carried out, revealing that the attainment of cultivable endophytic fungi from plants treated with chemical pesticides was not affected. The fungi were also tested in vitro for sensitivity to the fungicide at concentrations of 0.2, 2.0 and 3.8 mg L-1 . The results showed that all the fungi evaluated were able to grow in the presence of the fungicide in the solid culture medium, in all of the tested concentrations, with low sensitivity at the concentration of 0.2 mg L-1 . Only four fungi showed sensitivity at the concentration of 2.0 mg L-1 and, even at the highest concentration (3.8 mg L -1 ), the genera Cladosporium, Bipolaris, Cadophora, Rhizoctonia and Alternaria remained showing low sensitivity to the fungicide. The isolated fungi also underwent screening in order to detect the production of cellulase enzymes (CMCases), xylanases, proteases and amylases in solid media and quantify the xylanase activity in submerged cultivation, utilizing straw and sugarcane bagasse as substrate. Assays in solid media showed better results for xylanase with an enzyme index (IE) of 2.14 and for CMCase of 1.92. The submerged cultivation generated better results using 50% straw with 50% bagasse and was able to induce an activity of 383.63 U mL-1 of xylanase. Regarding the kinetic behavior of the crude xylanase produced by the fungus Colletrotrichum boninense, the optimal pH value found was 5.0 and the best temperature range was between 50 and 60 °C. At temperatures of 40 and 50 °C the enzyme preserved 87 and 76% of its activity when incubated for 30 min. The Km and VMax values found against xylan were 99.3 mg mL-1 and 3,333 µmol mL-1 min-1 , respectively. |
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Sene, LucianeSene, LucianeKadowaki, Marina KimikoGomes, Simone Damascenohttp://lattes.cnpq.br/3362104483832351 Coelho, Silvia Renata MachadoSilvia Renata Machado Coelho http://lattes.cnpq.br/3554106124561773 http://lattes.cnpq.br/8746354350047774Flores, Andressa Caroline2023-02-07T16:59:37Z2022-09-15Flores, Andressa Caroline. Fungos endofíticos de soja: diversidade e prospecção enzimática. 2022. 81 f. Tese( Doutorado em Engenharia Agrícola) - Universidade Estadual do Oeste do Paraná, Cascavel.https://tede.unioeste.br/handle/tede/6430Plants are inhabited by a microbiota composed of microorganisms in endophytic conditions and each endophyte-plant relation displays peculiar characteristics that vary according to environmental conditions. Studies related to the identification of endophytic populations associated with plant species allow using them as important tools for obtaining biotechnological products of agricultural and industrial interest. The objective of this study was to isolate and identify endophytic fungi from soybean plants, before and after fungicide application composed by Trifloxystrobin + Prothioconazole in the soybean crops, evaluate the sensitivity of the isolates in vitro against the same fungicide and also evaluate them regarding the biotechnological potential of enzyme production. The soybean plants used were cultivated at the Experimental Nucleus of Agricultural Engineering (NEEA) of the Western Paraná State University, in Cascavel, Paraná, Brazil. The leaves of soybean plants were collected at 48 days after sowing (prior to fungicide application) and at 62 days (14 days after application). The fungi were grouped and classified according to morphological and microscopic aspects, identified at the genera level and analyzed according to indexes such colonization frequency (FC%) and ecological diversity (Simpson (D e 1-D), Shannon-Wiener (H’) and Pielou (e). A total of 259 fungi (122 before the fungicide and 137 after the fungicide application) were obtained, which resulted in the identification of 11 genera, among which Diaporthe and Alternaria were the most abundant with FC of 38.6 and 25.5%, respectively. The frequency and diversity values showed that there was no significant difference between the two harvests carried out, revealing that the attainment of cultivable endophytic fungi from plants treated with chemical pesticides was not affected. The fungi were also tested in vitro for sensitivity to the fungicide at concentrations of 0.2, 2.0 and 3.8 mg L-1 . The results showed that all the fungi evaluated were able to grow in the presence of the fungicide in the solid culture medium, in all of the tested concentrations, with low sensitivity at the concentration of 0.2 mg L-1 . Only four fungi showed sensitivity at the concentration of 2.0 mg L-1 and, even at the highest concentration (3.8 mg L -1 ), the genera Cladosporium, Bipolaris, Cadophora, Rhizoctonia and Alternaria remained showing low sensitivity to the fungicide. The isolated fungi also underwent screening in order to detect the production of cellulase enzymes (CMCases), xylanases, proteases and amylases in solid media and quantify the xylanase activity in submerged cultivation, utilizing straw and sugarcane bagasse as substrate. Assays in solid media showed better results for xylanase with an enzyme index (IE) of 2.14 and for CMCase of 1.92. The submerged cultivation generated better results using 50% straw with 50% bagasse and was able to induce an activity of 383.63 U mL-1 of xylanase. Regarding the kinetic behavior of the crude xylanase produced by the fungus Colletrotrichum boninense, the optimal pH value found was 5.0 and the best temperature range was between 50 and 60 °C. At temperatures of 40 and 50 °C the enzyme preserved 87 and 76% of its activity when incubated for 30 min. The Km and VMax values found against xylan were 99.3 mg mL-1 and 3,333 µmol mL-1 min-1 , respectively.As plantas são habitadas por uma microbiota composta por microrganismos em condição endofítica e cada relação endófito-planta possui características peculiares e que variam conforme condições ambientais. Estudos relacionados à identificação das populações endofíticas associadas às espécies vegetais permitem utilizá-las como importantes ferramentas para obtenção de produtos biotecnológicos de interesse agrícola e industrial. O objetivo deste estudo foi o de isolar e identificar fungos endofíticos de plantas de soja, antes e após a aplicação do fungicida sistêmico composto por Trifloxistrobina + Protioconazol na lavoura de soja, avaliar a sensibilidade dos isolados in vitro frente ao mesmo fungicida e avaliá-los quanto ao potencial biotecnológico de produção de enzimas. As plantas de soja utilizadas foram cultivadas no Núcleo Experimental de Engenharia Agrícola (NEEA) da Universidade Estadual do Oeste do Paraná, região de Cascavel-PR. Foram coletadas folhas de plantas de soja, com 48 dias após semeadura (anterior à aplicação do fungicida) e com 62 dias (14 dias após a aplicação). Os fungos foram agrupados e classificados conforme aspectos morfológicos e microscópicos, identificados em nível de gênero e analisados de acordo com os índices de frequência de colonização (FC%) e de diversidade ecológica (Simpson (D e 1-D), Shannon-Wiener (H’) e Pielou (e). Um total de 259 fungos (122 antes do fungicida e 137 após aplicação do fungicida) foram obtidos que resultaram na identificação de 11 gêneros, dentre os quais Diaporthe e Alternaria foram os mais abundantes com FC de 38,6 e 25,5%, respectivamente. Os valores de frequência e diversidade demonstraram que não houve diferença significativa entre as duas coletas realizadas, revelando que a obtenção de fungos endofíticos cultiváveis a partir de plantas tratadas com defensivos químicos não foi afetada. Os fungos também foram testados in vitro quanto à sensibilidade ao fungicida nas concentrações de 0,2, 2,0 e 3,8 mg L-1 . Os resultados demonstraram que todos os fungos avaliados foram capazes de crescer na presença do fungicida no meio de cultivo sólido, em todas as concentrações testadas, com baixa sensibilidade na concentração de 0,2 mg L-1 . Somente quatro fungos apresentaram sensibilidade na concentração de 2,0 mg L-1 e, mesmo na maior concentração (3,8 mg L-1 ), os gêneros Cladosporium, Bipolaris, Cadophora, Rhizoctonia e Alternaria permaneceram demonstrando baixa sensibilidade ao fungicida. Os fungos isolados também passaram por triagem para detecção de produção das enzimas celulases (CMCases), xilanases, proteases e amilases em meio sólido e quantificação da atividade de xilanase em cultivo submerso, utilizando palha e bagaço de cana-de-açúcar como substrato. Os ensaios em meio sólido demonstraram melhores resultados para xilanase com índice enzimático (IE) de 2,14 e para CMCase de 1,92. O cultivo submerso gerou melhores resultados utilizando 50% de palha com 50% de bagaço e foi capaz de induzir uma atividade de 383,63 U mL-1 de xilanase. Com relação ao comportamento cinético da xilanase bruta produzida pelo fungo Colletrotrichum boninense, o valor de pH ótimo encontrado foi de 5,0 e melhor faixa de temperatura entre 50 e 60 °C. Nas temperaturas de 40 e 50 °C a enzima preservou 87 e 76% de sua atividade quando incubada por 30 min. Os valores de Km e de VMáx encontrados frente ao xilano foram de 99,3 mg mL-1 e 3.333 µmol mL-1 min-1 , respectivamente.Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2023-02-07T16:59:36Z No. of bitstreams: 2 Andressa_Flores.2022.pdf: 2369829 bytes, checksum: bcc3f9a3e4822488933024a367def271 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2023-02-07T16:59:37Z (GMT). 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dc.title.por.fl_str_mv |
Fungos endofíticos de soja: diversidade e prospecção enzimática |
dc.title.alternative.eng.fl_str_mv |
Endophytic fungi from soybean: diversity and enzymatic prospection |
title |
Fungos endofíticos de soja: diversidade e prospecção enzimática |
spellingShingle |
Fungos endofíticos de soja: diversidade e prospecção enzimática Flores, Andressa Caroline Endófito Enzimas Celulase Xilanase Fungicida Potencial biotecnológico Endophyte Enzymes Cellulase Xylanase Fungicide Biotechnological potential RECURSOS HÍDRICOS E SANAEAMENTO AMBIENTAL |
title_short |
Fungos endofíticos de soja: diversidade e prospecção enzimática |
title_full |
Fungos endofíticos de soja: diversidade e prospecção enzimática |
title_fullStr |
Fungos endofíticos de soja: diversidade e prospecção enzimática |
title_full_unstemmed |
Fungos endofíticos de soja: diversidade e prospecção enzimática |
title_sort |
Fungos endofíticos de soja: diversidade e prospecção enzimática |
author |
Flores, Andressa Caroline |
author_facet |
Flores, Andressa Caroline |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Sene, Luciane |
dc.contributor.referee1.fl_str_mv |
Sene, Luciane |
dc.contributor.referee2.fl_str_mv |
Kadowaki, Marina Kimiko |
dc.contributor.referee4.fl_str_mv |
Gomes, Simone Damasceno |
dc.contributor.referee4Lattes.fl_str_mv |
http://lattes.cnpq.br/3362104483832351 |
dc.contributor.referee5.fl_str_mv |
Coelho, Silvia Renata Machado |
dc.contributor.referee5Lattes.fl_str_mv |
Silvia Renata Machado Coelho http://lattes.cnpq.br/3554106124561773 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/8746354350047774 |
dc.contributor.author.fl_str_mv |
Flores, Andressa Caroline |
contributor_str_mv |
Sene, Luciane Sene, Luciane Kadowaki, Marina Kimiko Gomes, Simone Damasceno Coelho, Silvia Renata Machado |
dc.subject.por.fl_str_mv |
Endófito Enzimas Celulase Xilanase Fungicida Potencial biotecnológico |
topic |
Endófito Enzimas Celulase Xilanase Fungicida Potencial biotecnológico Endophyte Enzymes Cellulase Xylanase Fungicide Biotechnological potential RECURSOS HÍDRICOS E SANAEAMENTO AMBIENTAL |
dc.subject.eng.fl_str_mv |
Endophyte Enzymes Cellulase Xylanase Fungicide Biotechnological potential |
dc.subject.cnpq.fl_str_mv |
RECURSOS HÍDRICOS E SANAEAMENTO AMBIENTAL |
description |
Plants are inhabited by a microbiota composed of microorganisms in endophytic conditions and each endophyte-plant relation displays peculiar characteristics that vary according to environmental conditions. Studies related to the identification of endophytic populations associated with plant species allow using them as important tools for obtaining biotechnological products of agricultural and industrial interest. The objective of this study was to isolate and identify endophytic fungi from soybean plants, before and after fungicide application composed by Trifloxystrobin + Prothioconazole in the soybean crops, evaluate the sensitivity of the isolates in vitro against the same fungicide and also evaluate them regarding the biotechnological potential of enzyme production. The soybean plants used were cultivated at the Experimental Nucleus of Agricultural Engineering (NEEA) of the Western Paraná State University, in Cascavel, Paraná, Brazil. The leaves of soybean plants were collected at 48 days after sowing (prior to fungicide application) and at 62 days (14 days after application). The fungi were grouped and classified according to morphological and microscopic aspects, identified at the genera level and analyzed according to indexes such colonization frequency (FC%) and ecological diversity (Simpson (D e 1-D), Shannon-Wiener (H’) and Pielou (e). A total of 259 fungi (122 before the fungicide and 137 after the fungicide application) were obtained, which resulted in the identification of 11 genera, among which Diaporthe and Alternaria were the most abundant with FC of 38.6 and 25.5%, respectively. The frequency and diversity values showed that there was no significant difference between the two harvests carried out, revealing that the attainment of cultivable endophytic fungi from plants treated with chemical pesticides was not affected. The fungi were also tested in vitro for sensitivity to the fungicide at concentrations of 0.2, 2.0 and 3.8 mg L-1 . The results showed that all the fungi evaluated were able to grow in the presence of the fungicide in the solid culture medium, in all of the tested concentrations, with low sensitivity at the concentration of 0.2 mg L-1 . Only four fungi showed sensitivity at the concentration of 2.0 mg L-1 and, even at the highest concentration (3.8 mg L -1 ), the genera Cladosporium, Bipolaris, Cadophora, Rhizoctonia and Alternaria remained showing low sensitivity to the fungicide. The isolated fungi also underwent screening in order to detect the production of cellulase enzymes (CMCases), xylanases, proteases and amylases in solid media and quantify the xylanase activity in submerged cultivation, utilizing straw and sugarcane bagasse as substrate. Assays in solid media showed better results for xylanase with an enzyme index (IE) of 2.14 and for CMCase of 1.92. The submerged cultivation generated better results using 50% straw with 50% bagasse and was able to induce an activity of 383.63 U mL-1 of xylanase. Regarding the kinetic behavior of the crude xylanase produced by the fungus Colletrotrichum boninense, the optimal pH value found was 5.0 and the best temperature range was between 50 and 60 °C. At temperatures of 40 and 50 °C the enzyme preserved 87 and 76% of its activity when incubated for 30 min. The Km and VMax values found against xylan were 99.3 mg mL-1 and 3,333 µmol mL-1 min-1 , respectively. |
publishDate |
2022 |
dc.date.issued.fl_str_mv |
2022-09-15 |
dc.date.accessioned.fl_str_mv |
2023-02-07T16:59:37Z |
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 |
Flores, Andressa Caroline. Fungos endofíticos de soja: diversidade e prospecção enzimática. 2022. 81 f. Tese( Doutorado em Engenharia Agrícola) - Universidade Estadual do Oeste do Paraná, Cascavel. |
dc.identifier.uri.fl_str_mv |
https://tede.unioeste.br/handle/tede/6430 |
identifier_str_mv |
Flores, Andressa Caroline. Fungos endofíticos de soja: diversidade e prospecção enzimática. 2022. 81 f. Tese( Doutorado em Engenharia Agrícola) - Universidade Estadual do Oeste do Paraná, Cascavel. |
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https://tede.unioeste.br/handle/tede/6430 |
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por |
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por |
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-5347692450416052129 |
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600 600 600 |
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2214374442868382015 |
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http://creativecommons.org/licenses/by/4.0/ |
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openAccess |
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application/pdf |
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Universidade Estadual do Oeste do Paraná Cascavel |
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Programa de Pós-Graduação em Engenharia Agrícola |
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UNIOESTE |
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Brasil |
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Centro de Ciências Exatas e Tecnológicas |
publisher.none.fl_str_mv |
Universidade Estadual do Oeste do Paraná Cascavel |
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UNIOESTE |
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