Diversidade de bactérias endofíticas em frutos de café
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2008
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Universidade Federal de Viçosa
Resumo
O objetivo do presente trabalho foi estudar a diversidade de bactérias endofíticas associadas aos frutos em quatro cultivares de Coffea arabica L., em diferentes altitudes na Zona da Mata Norte, Minas Gerais, Brasil. As amostras de frutos sadios, no estádio cereja, foram coletadas em lavouras de Catuaí Amarelo, Catuaí Vermelho, Bourbon Amarelo e Bourbon Vermelho. O isolamento e a quantificação de bactérias dos frutos foram realizados em meio de cultura R2A, estabelecendo-se uma coleção de culturas de bactérias endofíticas e epifíticas de frutos de café no Laboratório de Ecologia Microbiana (LEM) do Departamento de Microbiologia com 381 isolados, dos quais 134 de endofíticas. Entre essas foram identificados e descritos 48 morfotipos, que constituíram o universo-base para o presente estudo. A identificação fenotípica foi por características culturais e análises de ésteres metílicos de ácidos graxos (FAME) pelo sistema de identificação Sherlock®(MIDI). O estudo das endofíticas cultiváveis foi realizado com dados do seqüenciamento direto de amplicons obtidos por PCR, enquanto que para o das não-cultiváveis foram utilizados primer específicos para grupos de bactérias de três filos, Proteobacteria classes, [alfa], [Beta] e y, Firmicutes e Actinobacteria, sendo os produtos do Nested-PCR analisados por eletroforese em gel de gradiente desnaturante (DGGE). Nas amostras de café cereja a altitudes entre 676 até 1.187 m, as densidades de endofíticas variaram, em UFC.fruto-1, de 2,93 x 104 a 6,2 x 106 em Catuaí Amarelo; 7,6 x 104 a 6,0 x 106 em Catuaí Vermelho; 1,24 x 104 a 1,35 x 106 em Bourbon Amarelo e 4,6 x 104 a 2,69 x 106 em Bourbon Vermelho. As médias de densidades populacionais de bactérias endofíticas, em relação a cultivares e à altitude, diferiram (p<0,05) entre si. Os maiores valores das médias a altitudes superiores a 1.000 m, em Catuaí Vermelho e Bourbon Vermelho, diferiram (p<0,05) das de Catuaí, Amarelo e Vermelho à 676 m . A correlação simples por Pearson mostrou relação positiva (p<0,05) entre as contagens e altitude. As endofíticas cultiváveis foram identificadas com base no perfil FAME/MIDI como sendo de isolados de Curtubacterium flaccumfaciens betae/oortii (LEM CA16, LEM CV20, LEM CA25 e LEM CA28), Brevibacterium epidermis/oidium (LEM BV41), Microbacterium barkeri (LEM CA26 e LEM BV37), Microbacterium luteolum (LEM CA01), Pectinobacterium carotovorum/carotovorum (LEM CA30, LEM CV35, LEM BV38, LEM BA43 e LEM BA45), Pseudomonas putida biótipo A (LEM BA47), Salmonella typhymurium GC grupo B (LEM CV19), Staphylococcus simulans (LEM CV23) e Bacillus sp. (LEM CV24). A análise filogenética com uso de seqüências de rDNA 16S mostrou, para o isolado LEM CV24 uma maior identidade com Bacillus niacini AJ21160.1, enquanto o LEM BV39 correspondeu a Enterobacter amnigenus EU340927.1, e os isolados LEM CA01 e BV37 a Microbacterium sp. Das bactérias endofíticas associadas a cultivares de café, P. carotovorum/carotovorum, S. typhymurium, B. epidermis/oidium, S. simulans, B. niacini e E. amnigenus, são relatadas pela primeira vez como endofíticas em frutos de cafeeiros arábica. As análises de diversidade de bactérias totais, endofíticas e epifíticas, por PCR-DGGE utilizando o primer universal (F948GC/R1378), revelaram a presença de 80 unidades taxonômicas operacionais (UTOs), enquanto a riqueza de endofíticas correspondeu a 64 UTOs. Entretanto, com a utilização de grupos de primer específicos, Nested-PCR, a diversidade de bactérias endofíticas correspondeu a uma riqueza de 110 UTOS para o filo Firmicutes, 71 para y-Proteobacteria e 50 UTOs para Actinobacteria. A constatação da existência de diversidade de endofíticas em C. arábica evidencia a urgência de estudos funcionais desta microbiota, especialmente com relação a compostos precursores de aroma, sabor e acidez, de interesse para produção comercial de cafés de qualidade superior.
The objective of this work was to study the diversity of endophytic bacteria in Coffea arabica L. cherries collected at different altitudes in the Zona da Mata Norte, Minas Gerais State, Brazil. Samples of healthy ripe fruits were collected in areas planted with Catuaí Amarelo, Catuaí Vermelho, Bourbon Amarelo, and Bourbon Vermelho. The isolation and quantification of bacteria were done on R2A medium, with the establishment of a culture collection of epiphytic and endophytic bacteria containing 381 isolates. Among the total of isolates obtained, 134 were endophytic. Forty-eight distinct morphotypes of endophytic bacteria were identified, described, and further characterized in this study. The phenotypic identification was done by analyzing cultural traits and the fatty acid methyl ester profiles (FAME) by the Sherlock® (MIDI) identification system. The study of the culturable endophytic bacteria was done based on the direct sequencing of PCR amplicons, while non-culturable bacteria were analyzed using specific primers for the bacterial groups corresponding to Proteobacteria, [alpha], [Beta], and y classes, Firmicutes, and Actinobacteria. Nested-PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE). In the coffee cherry samples from altitudes ranging from 676 to 1,187 m, the density of endophytic bacteria varied, in CFU fruit-1, from 2.93 x 104 to 6.2 x 106 for Catuaí Amarelo; from 7.6 x 104 to 6.0 x 108 for Catuaí Vermelho; from 1.24 x 104 to 1.35 x 106 for Bourbon Amarelo; and from 4.6 x 104 to 2.69 x 106 for Bourbon Vermelho. Population density means of endophytic bacteria for the coffee cultivars and sampling altitudes were statistically different (p < 0.05). The largest values obtained for altitudes higher than 1,000 m, for Catuaí Vermelho and Bourbon Vermelho, were statistically distinct (p < 0.05) from those for Catuaí Vermelho and Catuaí Amarelo at 676 m. Pearson`s correlation showed a positive relation (p<0.05) between bacterial counts and altitude. The culturable endophytic bacteria were identified based on the FAME/MIDI profile and corresponded to Curtubacterium flaccumfaciens betae/oortii (LEM CA16, LEM CV20, LEM CA25, and LEM CA28), Brevibacterium epidermis/oidium (LEM BV41), Microbacterium barkeri (LEM CA26 and LEM BV37), Microbacterium luteolum (LEMCA01), Pectinobacterium carotovorum/ carotovorum (LEM CA30, LEM CV35, LEM BV38, LEM BA43, and LEM BA45 ), Pseudomonas putida biotype A (LEM BA47), Salmonella typhymurium GC group B (LEM CV19), Staphylococcus simulans (LEM CV23), and Bacillus sp. (LEM CV24). Phylogenetic analyses using 16S rDNA sequences showed a higher identity to Bacillus niacin AJ21160.1 for isolate LEM CV24, while LEM BV 39 corresponded to Enterobacter amnigenus EU340927.1. The isolates LEM CA01 and BV37 corresponded to Microbacterium sp. Among the endophytic bacteria associated to coffee fruits, P. carotovorum/carotovorum, S. typhymurium, B. epidermis/oidium, S. simulans, B. niacini, and E. amnigenus, are reported for the first time as endophytic in C. arabica. The diversity analyses of total bacteria (endophytic + epiphytic) by PCR-DGGE using universal primers (F948GC/R1378), revealed 80 operational taxonomic units (OTU) in the coffee cherries, while species richness of endophytic bacteria corresponded to 64 OTUs. The use of specific primers for the diversity of endophytic bacteria during Nested-PCR revealed a richness of 110 OTUs for Firmicutes, 71 OTUs for γ-Proteobacteria, and 50 OTUs for Actinobacteria. The existence of such diversity of endophytic bacteria in C. arabica evidences the urgency for further functional studies on the microbiota of coffee plants, and, especially, on the precursor compounds that may be determinant to flavor and acidity, two traits of great interest for the production of high quality coffee.
The objective of this work was to study the diversity of endophytic bacteria in Coffea arabica L. cherries collected at different altitudes in the Zona da Mata Norte, Minas Gerais State, Brazil. Samples of healthy ripe fruits were collected in areas planted with Catuaí Amarelo, Catuaí Vermelho, Bourbon Amarelo, and Bourbon Vermelho. The isolation and quantification of bacteria were done on R2A medium, with the establishment of a culture collection of epiphytic and endophytic bacteria containing 381 isolates. Among the total of isolates obtained, 134 were endophytic. Forty-eight distinct morphotypes of endophytic bacteria were identified, described, and further characterized in this study. The phenotypic identification was done by analyzing cultural traits and the fatty acid methyl ester profiles (FAME) by the Sherlock® (MIDI) identification system. The study of the culturable endophytic bacteria was done based on the direct sequencing of PCR amplicons, while non-culturable bacteria were analyzed using specific primers for the bacterial groups corresponding to Proteobacteria, [alpha], [Beta], and y classes, Firmicutes, and Actinobacteria. Nested-PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE). In the coffee cherry samples from altitudes ranging from 676 to 1,187 m, the density of endophytic bacteria varied, in CFU fruit-1, from 2.93 x 104 to 6.2 x 106 for Catuaí Amarelo; from 7.6 x 104 to 6.0 x 108 for Catuaí Vermelho; from 1.24 x 104 to 1.35 x 106 for Bourbon Amarelo; and from 4.6 x 104 to 2.69 x 106 for Bourbon Vermelho. Population density means of endophytic bacteria for the coffee cultivars and sampling altitudes were statistically different (p < 0.05). The largest values obtained for altitudes higher than 1,000 m, for Catuaí Vermelho and Bourbon Vermelho, were statistically distinct (p < 0.05) from those for Catuaí Vermelho and Catuaí Amarelo at 676 m. Pearson`s correlation showed a positive relation (p<0.05) between bacterial counts and altitude. The culturable endophytic bacteria were identified based on the FAME/MIDI profile and corresponded to Curtubacterium flaccumfaciens betae/oortii (LEM CA16, LEM CV20, LEM CA25, and LEM CA28), Brevibacterium epidermis/oidium (LEM BV41), Microbacterium barkeri (LEM CA26 and LEM BV37), Microbacterium luteolum (LEMCA01), Pectinobacterium carotovorum/ carotovorum (LEM CA30, LEM CV35, LEM BV38, LEM BA43, and LEM BA45 ), Pseudomonas putida biotype A (LEM BA47), Salmonella typhymurium GC group B (LEM CV19), Staphylococcus simulans (LEM CV23), and Bacillus sp. (LEM CV24). Phylogenetic analyses using 16S rDNA sequences showed a higher identity to Bacillus niacin AJ21160.1 for isolate LEM CV24, while LEM BV 39 corresponded to Enterobacter amnigenus EU340927.1. The isolates LEM CA01 and BV37 corresponded to Microbacterium sp. Among the endophytic bacteria associated to coffee fruits, P. carotovorum/carotovorum, S. typhymurium, B. epidermis/oidium, S. simulans, B. niacini, and E. amnigenus, are reported for the first time as endophytic in C. arabica. The diversity analyses of total bacteria (endophytic + epiphytic) by PCR-DGGE using universal primers (F948GC/R1378), revealed 80 operational taxonomic units (OTU) in the coffee cherries, while species richness of endophytic bacteria corresponded to 64 OTUs. The use of specific primers for the diversity of endophytic bacteria during Nested-PCR revealed a richness of 110 OTUs for Firmicutes, 71 OTUs for γ-Proteobacteria, and 50 OTUs for Actinobacteria. The existence of such diversity of endophytic bacteria in C. arabica evidences the urgency for further functional studies on the microbiota of coffee plants, and, especially, on the precursor compounds that may be determinant to flavor and acidity, two traits of great interest for the production of high quality coffee.
Descrição
Tese de Doutorado defendida na Universidade Federal de Viçosa
Palavras-chave
Café Bactérias cultiváveis Bactérias não-cultiváveis Microbiologia Microrganismos endofíticos, Coffee Culturable bacteria Non culturable bacteria Microbiology Endophytic microrganisms
Citação
Perez Cordero, Alexander Francisco. Diversidade de bactérias endofíticas em frutos de café. Viçosa : UFV, 2007. 78 f. : il. (Tese - Doutorado em Microbiologia Agrícola). Orientador: Arnaldo Chaer Borges. T 577.852012 P438d 2008