Diversidade de bactérias endofíticas em frutos de café

• Autor(a) principal: Cordero, Alexander Francisco Perez
• Data de Publicação: 2008
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1528
• topic: Café; Bactérias cultiváveis; Bactérias não-cultiváveis; Microbiologia; Microrganismos endofíticos; Coffee; Culturable bacteria; Non-culturable bacteria; Microbiology; Endophytic microrganisms; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA

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, &#945;, &#946;, and &#947; 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 &#947;-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.