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A culture-independent molecular phylogenetic analysis was carried out to study for the first time the diversity of bacterial ammonia monooxygenase subunit A (amoA) and nitrogenase reductase subunit H (nifH) genes from Urca inlet at Guanabara Bay in Rio de Janeiro, Brazil. Most bacterial amoA and nifH sequences exhibited identities of less than 95% to those in the GenBank database revealing that novel ammonia-oxidizing bacteria and nitrogen-fixing microorganisms may exist in this tropical marine environment. The observation of a large number of clones related to uncultured bacteria also indicates the necessity to describe these microorganisms and to develop new cultivation methodologies.

The effects of shrimp farm effluents on bacterial communities in mangroves have been infrequently reported. Classic and molecular biology methods were used to survey bacterial communities from four mangroves systems. Water temperature, salinity, pH, total heterotrophic bacteria and maximum probable numbers of Vibrio spp. were investigated. Genetic profiles of bacterial communities were also characterized by polymerase chain reaction (PCR) amplification of eubacterial and Vibrio 16S rDNA using denaturing gradient gel electrophoresis (DGGE). Highest heterotrophic counts were registered in the mangrove not directly polluted by shrimp farming. The Enterobacteriaceae and Chryseomonas luteola dominated the heterotrophic isolates. Vibrio spp. pathogenic to humans and shrimps were identified. Eubacterial genetic profiles suggest a shared community structure independent of mangrove system. Vibrio genetic profiles were mangrove specific. Neither microbial counts nor genetic profiling revealed a significant decrease in species richness associated with shrimp farm effluent. The complex nature of mangrove ecosystems and their microbial communities is discussed.

The survival and colonization patterns of Pseudomonas putida PRD16 and Enterobacter cowanii PRF116 in the rhizosphere of greenhouse-grown tomato plants and the effects of their inoculation on the indigenous bacterial community were followed by selective plating, molecular fingerprinting, and confocal laser scanning microscopy (CLSM) over 3 weeks. Both strains, which showed in vitro antagonistic activity against Ralstonia solanacearum, were previously tagged with gfp. Seed and root inoculation were compared. Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method. Analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and CLSM confirmed the decrease in the relative abundance of the inoculant strains. Pronounced differences in the Pseudomonas community patterns for plants inoculated with PRD16 compared to the control were detected 3 weeks after root inoculation, indicating a longer-lasting effect. Analysis by CLSM showed rather heterogeneous colonization patterns for both inoculant strains. In comparison with seed inoculation, root inoculation led to a much better colonization as evidenced by all three methods. The colonization patterns observed by CLSM provide important information on the sampling strategy required for monitoring inoculant strains in the rhizosphere.

Aims: Characterization of yeast populations and genetic polymorphism of Saccharomyces cerevisiae strains collected during the short fermentative cycles from the spontaneous fermentations during the artisanal cachaça production., Methods and Results: The prevalent S. cerevisiae strains were analysed by PFG and RAPD-PCR using primers EI1 and M13. The molecular analysis have showed a high degree of genetic polymorphism among the strains within a 24 h fermentative cycle., Conclusion: The genetic diversity observed in the S. cerevisiae strains may be occurring due to the existence of a large number of individual genotypes within the species. The unique characteristics of the cachaça fermentation process probably allows for a faster detection of molecular polymorphisms of yeast strains than other types of fermentations., Significance and Impact of the Study: Spontaneous fermentations to produce cachaça, due to their characteristics, are an excellent model for the study of molecular diversity of S. cerevisiae strains during the production of fermented beverages.

Yeast communities associated with sugarcane leaves, stems and rhizosphere during different phases of plant development were studied near Campos, in Rio de Janeiro, Brazil. Atmospheric temperature, soil granulometry and pH, and sugar cane juice degree Brix and pH were determined. Yeast communities associated with sugarcane were obtained after cellular extraction by shaking, blending and shaking plus sonication, and cultured on Yeast Nitrogen Base Agar plus glucose (0.5%) and Yeast Extract-Malt Extract Agar. No significant differences in yeast counts were found among the cellular extraction treatments and culture media. 230 yeast cultures were identified according to standard methods, and distinct yeast communities were found for each substrate studied. The prevalent species isolated from sugarcane were Cryptococcus laurentii, Cryptococcus albidus, Rhodotorula mucilaginosa and Debaryomyces hansenii.

The presence of killer and proteolytic yeasts was studied among 944 isolates representing 105 species from tropical yeast communities. We found 13 killer toxin producing species, with Pichia kluyveri being the most frequent. Other killer yeast isolates were Candida apis, Candida bombicola, Candida fructus, Candida krusei, Candida sorbosa, Hanseniaspora uvarum, Issatchenkia occidentalis, Kloeckera apis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia ohmeri-like, and Sporobolomyces roseus. The communities from which killer yeasts were isolated had strains sensitive to them, and there were interspecific and intraspecific differences in the spectra of their killer activities. Pichia kluyveri had the broadest spectra of activity against sensitive isolates, and it apparently produced different toxins. The coexistence of sensitive and killer yeasts using the same substrate suggests that there is spatial separation in microhabitats or temporal separation in different stages of successions. Basidiomycetous yeasts were more frequently proteolytic than ascomycetous yeasts. Extracellular proteases could be important for the yeasts to have access to more nitrogen nutrients and obtain a better balance with available carbon sources.

The size of the internal transcribed spacer (ITS) region as measured by gel electrophoresis of PCR products, amplified by primers ITS1 and ITS4, was over 800 bp for all Saccharomyces sensu stricto species, but yeasts belonging to other Saccharomyces species had a shorter ITS region, making this characteristic potentially useful in the identification of Saccharomyces isolates. The ITS product length was homogeneous within the species Saccharomyces cerevisiae.

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.

Genetic and karyotypic studies of some Saccharomyces sensu stricto yeasts from Brazil revealed a genetically isolated population which apparently represents a new sibling species of S. cerevisiae.

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the moth Sigelgaita sp. in the cactus Pilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactus Pilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent species Pichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp., Myxozyma mucilagina and Sporopachydermia sp. A, representing about 80% of the total isolates. Pichia sp. A and a Candida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genus Kloeckera, basidiomicetous anamorphs of the genus Cryptococcus, the black yeast Aureobasidium pullulans, Pichia sp. A, a Candida domercqii-like species, and some cactophilic yeasts, especially Clavispora opuntiae. The feeding site of Sigelgaita sp. larvae had Clavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.

Yeasts and coliform bacteria were isolated from water that accumulated in the central cups and adjacent leaf axilae of two bromeliads, Neoregelia cruenta of a coastal sand dune and Quesnelia quesneliana of a mangrove ecosystem near the city of Rio de Janeiro, Brazil. The mean total coliform counts were above 10,000 per 100 mL for waters of both plants, but the mean fecal coliform counts were only 74 per 100 mL for Q. quesneliana and mostly undetected in water from N. cruenta. Of 90 fecal coliform isolates, 51 were typical of Escherichia coli in colony morphology and indol, methyl red, Volges-Proskauer, and citrate (IMViC) tests. Seven representatives of the typical E. coli cultures were identified as this species, but the identifications of nine other coliform bacteria were mostly dubious. The yeast community of N. cruenta was typical of plant surfaces with basidiomycetous yeasts anamorphs, and the black yeast Aureobasidium pullulans was prevalent. Quesnelia quesneliana had a substantial proportion of ascomycetous yeasts and their anamorphs, including a probable new biotype of Saccharomyces unisporus. Our results suggested that the microbial communities in bromeliad waters are typically autochtonous and not contaminants.

Phylogenetic relationships of species assigned to the genus Metschnikowia were estimated from the extents of divergence among partial sequences of rRNA. The data suggest that the aquatic species (Metschnikowia australis, Metschnikowia bicuspidata, Metschnikowia krissii, and Metschnikowia zobellii) and the terrestrial species (Metschnikowia hawaiiensis, Metschnikowia lunata, Metschnikowia pulcherrima, and Metschnikowia reukaufii) form two groups within the genus. M. lunata and M. hawaiiensis are well separated from other members of the genus, and M. hawaiiensis may be sufficiently divergent that it could be placed in a new genus. Species of the genus Metschnikowia are unique compared with other ascomycetous yeasts because they have a deletion in the large-subunit rRNA sequence that includes nucleotides 434 to 483.

Clavispora opuntiae was the prevalent yeast associated with the feeding sites of Sigelgaita sp. larvae in the cactus Pilosocereus arrabidae. Also associated with this habitat were Candida sonorensis, Pichia cactophila, Pichia barkeri, Candida sp. A, Geotrichum sp., Geotrichum sericeum and the yeast like organisms Prototheca zopfii and Acremonium sp. Atypical yeast biotypes that may represent new species of Pichia, Sporopachydermia and Candida were isolated. Mating types of Clavispora opuntiae were at a ratio 70 h- to 3 h- and reduced levels of sporulation suggested low pressure for sexual reproduction in this habitat. Sigelgaita sp. probably was not an important vector for Clavispora opuntiae because it was not isolated from an adult or eggs of this moth.

Sixty two strains of Rhodotorula rubra which were all isolated from marine and estuarine waters of Rio de Janeiro were found variable for 19 to 32 carbon assimilation tests used in yeast taxonomy. Two R. rubra strains which had latent assimilation of maltose and melizitose appeared to be intermediate with R. pilimanae. Strains of R. glutinis var dairenensis which differed from R. rubra only by weak assimilation of nitrate appeared to be intermediated between these two species. Our physiological tests suggested that R. rubra, R. pilimanae, and part of R. glutinis should be combined and this was supported by DNA base composition and coezyme Q data existing in the literature.

Yeast counts were made at 24 marine and estuarine sites in the vicinity of Rio de Janeiro, Brazil. Mean salinities of estuarine sites ranged from 14.2 to 27.4 per thousand, and mean temperatures ranged from 25 to 28 degrees C. Total coliform counts varied from 80% above 100,000 colony-forming units (CFU)/100 ml at heavily polluted sites to 100% below 100 CFU/100 ml at unpolluted sites. Total yeast counts above 100 CFU/100 ml were typical of heavily and moderately polluted water but atypical of lightly polluted and unpolluted water. Mean total yeast counts were 2,880 CFU/100 ml for heavily polluted sites, 202 CFU/100 ml for moderately polluted sites, and 3 CFU/100 ml for lightly polluted and unpolluted sites. Total yeast counts had a positive response to increased pollution levels, and Candida krusei and phenotypically similar yeasts as a group were prevalent in polluted estuarine water but rare in unpolluted seawater. The 549 strains of yeasts and yeast-like organisms isolated were grouped into 67 species, of which the 21 most prevalent made up 86% of the total yeast population. The prevalent genera in the polluted estuary were Candida, Rhodotorula, Torulopsis, Hanseniaspora, Debaryomyces, and Trichosporon.

Yeasts were found concentrated at the sediment surface and with the highest counts at the most polluted site. Candida krusei, Pichia membranaefaciens and similar species typically forming rugose colonies with radiating ridges were the prevalent yeasts in these sediments, and species like Rhodotorula rubra related to basidiomycetous fungi were found in relatively low numbers.

The specificity of indicators that depend on elevated incubation temperature as a selective factor for their enumeration was questioned because of the possibility of interference from autochthonous microorganisms adapted to high ambient temperatures. Lactose-fermenting cultures isolated from fecal coliform tests of tropical marine surface waters were identified as consisting of about 70% Escherichia coli, and most of the remaining cultures being Klebsiella, Enterobacter or Citrobacter species. This confirmed the taxonomic specificity of fecal coliform tests for these waters. Fecal and total coliforms, fecal streptococcus, heterotrophic bacteria and yeast counts had correlations of above 99% confidence levels with most other microbial and chemical parameters studied. Waters with fecal coliform counts above 1000 per 100 ml had increased incidence of presumptive pathogenic yeasts, Pseudomonas aeruginosa and Salmonella. Our data support the use of coliforms or fecal streptococci as indicators of recent fecal pollution in tropical marine waters and yeast or heterotrophic bacteria counts as complementary indicator methods for these waters.

Although the petroleum degrading ability of Candida lipolytica is well known, its ability to grow in seawater is questionable. Sixteen strains of C. lipolytica were isolated from marine sites in Rio de Janeiro. Some variations from the standard description were noted including strains intermediate between the varieties lipolytica and deformans. A representative strain which grew well on petroleum was found to grow well in marine and estuary conditions which reinforces its potential as a seed organism for marine oil spills.

Deoxyribonucleic acid (DNA) base composition, as measured by guanine plus cytosine (G + C) content, was determined in 17 strains of Sporothrix schenckii and Ceratocystis species. The average G + C content for S. schenckii was 54.7 +/- 0.2 mol%. No strain of Ceratocystis, with the exception of C. minor, gave values for G + C content similar to those obtained for S. schenckii. All DNA samples from strains of S. schenckii cross-hybridized with high percentages of relative binding. DNA samples from C. stenoceras and C. pilifera showed low degrees of homology with S. schenckii DNA. The DNA from C. minor hybridized with 75% binding with that from a strain of S. schenckii, as compared with the homologous reaction.

DNA hybridization studies were conducted to determine the taxonomic status of the aquatic group of Metschnikowia species and their varieties. Among the DNAs of the four varieties of Metschnikowia bicuspidata, that of Metschnikowia bicuspidata var. australis showed 37 to 51% relative binding with the DNAs of Metschnikowia bicuspidata var. bicuspidata and of the varieties chathamia and californica. On this basis, low intervarietal fertility, and unique habitat in antarctic seawater, we have proposed to raise M. bicuspidata var. australis to the rank of species, Metschnikowia australis, comb. nov. DNA complementarity of Metschnikowia bicuspidata var. californica and Metschnikowia bicuspidata var. chathamia DNA was greater than 80% with that of M. bicuspidata var. bicuspidata. Metschnikowia australis can be differentiated from other Metschnikowia species and varieties by its inability to form chlamydospores, the formation of two needle-shaped ascospores per ascus, lack of glucose fermentation, and lack of assimilation of both methyl-alpha-D-glucoside and glucono-delta-lactone. Other DNA-DNA reassociation experiments showed that Metschnikowia zobellii is a distinct species when compared with both aquatic and terrestrial species of the genus.

Samples were collected in a polluted site with mean fecal coliform counts of the water being 9.0 x 10(2)/100 ml and less polluted areas including commercial fishing areas with fecal coliforms typically below 2.0 x 10(1)/100 ml. Yeast populations from the polluted site were 4.6 x 10(2) to 9.2 x 10(3)/g of shrimp intestine, 1.0 x 10(1) to 2.4 x 10(2)/g of sediment and 4.0 x 10(-1) to 4.6 x 10(1)/ml of water, and in the less polluted site 3.0 x 10(1) to 2.3 x 10(2)/g of intestines and 3.0 x 10(-1) to 2.4 x 10(1)/ml of water. About half of these yeast populations were fermentative. Prevalent yeasts isolated from shrimp were Candida famata, Candida intermedia, Candida tropicalis, Candida krusei, Geotrichum sp., Trichosporon sp., Debaryomyces hansenii, Rhodotorula rubra, Kloeckera apiculata, Cryptococcus sp., Hansenula anomala, and Candida spp. The species found in water and sediment were similar to those of shrimp except that C. tropicalis was more prevalent in sediment with 25% of the population and D. hansenii in water with 12%. Yeasts normally associated with warm blooded animals and sewage were predominant in the shrimp from the polluted site, but yeasts from the less polluted site were more typical of clean water. Yeasts were concentrated in the shrimp intestine suggesting that this is a favorable environment for them but no species were found to be selectively associated with this niche.