Endophytic yeast diversity in leaf tissue of rice,corn and sugarcane cultivated in Thailand assessed by a culture-dependent approach

Endophytic yeasts are yeast that can colonize healthy plant tissues without causing any damage to the host plant. This work aimed to explore the diversity of endophytic yeasts in leaf tissue of main agricultural crops (rice, corn and sugarcane) in Thailand, by a culture-dependent approach. A total of 311 leaf samples, consisting of rice (n = 100), corn (n = 109) and sugarcane (n = 102). From the tissue of rice (n = 92), corn (n = 76) and sugarcane (n = 78) leaf samples, 117, 118 and 123 yeast strains were respectively isolated and identified based on the D1/D2 region of the large subunit (LSU) rRNA gene sequence analysis to be yeast species in both the phyla Basidiomycota and Ascomycota. Higher numbers of basidiomycetous yeast than ascomycetous yeast were detected in the leaf tissue of the three crops. Pseudozyma (Dirkmeia) churashimaensis (Ustilaginales) was the most prevalent yeast species in the rice and corn leaves with relative frequencies (RF) of 35.9 % and 17.8 %, respectively. Whereas the predominant species in the sugarcane leaves was Meyerozyma caribbica (Saccharomycetales) with an RF of 14.6 %. In addition, six new yeast species and one new yeast genus were proposed. Our findings suggest that these plant species are good sources from which new yeast species may be isolated.     

Candida siamensis sp. nov., an anamorphic yeast species in the Saturnispora clade isolated in Thailand

Three strains of a novel anamorphic yeast species were isolated from natural samples collected in Thailand, including strain EF10 isolated from sediment in a mangrove forest, strain ST-473 from rotten wood, and strain ST-479 from a wild mushroom ( Hygrophorus sp.). Analysis of the D1/D2 domains of the large-subunit rRNA gene sequence revealed that the sequence of strain EF10 differed from the other two strains (ST-473 and ST-479), which had identical sequences, by only one nucleotide (nt) substitution in 535 nt. The closest species in terms of pairwise sequence similarity was Saturnispora mendoncae , with 32 nt substitutions and seven gaps in 535 nt. Phylogenetic analysis demonstrated that the three strains form a cluster with S. mendoncae and the other eight species in the Saturnispora clade. Phenotypic characteristics of the three strains were the same including proliferation by multilateral budding, absence of ascospores and negative diazonium blue B and urease reactions. The major ubiquinone was Q-7. On this basis, the three strains were assigned to a single novel species of the genus Candida , for which the name Candida siamensis sp. nov. is proposed. The type strain is EF10^T (=BCC 29901^T=NBRC 104878^T=CBS 11022^T).     

Pseudozyma vetiver sp. nov., a novel anamorphic ustilaginomycetous yeast species isolated from the phylloplane in Thailand

Three strains representing one novel yeast species were isolated from the phylloplanes of the vetiver grasses (DMKU-LV90 and DMKU-LV99^T) and sugarcane (DMKU-SP260) collected in Thailand by leaf washing followed by a plating technique. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer region (ITS), the three strains were found to represent a single novel anamorphic ustilaginomycetous yeast species in the genus Pseudozyma. The name Pseudozyma vetiver sp. nov. is proposed for this novel species. The type strain is DMKU-LV99^T (BCC 61021 = CBS 12824). The novel species showed phylogenetic relationships to the other members of the genus Pseudozyma and to teleomorphic fungal genera, namely Ustilago, Sporisorium and Anomalomyces in Ustilaginaceae, Ustilaginales. The three strains showed identical sequences both in the D1/D2 and ITS regions. The Pseudozyma species closest to the novel species in terms of pairwise sequence similarity in the D1/D2 region was Pseudozyma pruni but with 2.3 % nucleotide substitutions (14 nucleotide substitutions and no gaps out of 606 nt). The novel species and P. pruni differed by 10.9 % nucleotide substitutions (75 nucleotide substitutions and 31 gaps out of 691 nt) in the ITS region. The phylogenetic analysis based on the combined sequences of the ITS region and the D1/D2 region of the LSU rRNA gene showed that the novel species was found to be most closely related to Pseudozyma fusiformata but with 2.9 % nucleotide substitutions in the D1/D2 region and 7.4 % nucleotide substitutions in the ITS region.     

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus

Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 degrees C, produced high concentrations of ethanol at both 40 and 45 degrees C. Ethanol production by this strain in shaking flask cultivation in sugar cane juice media at 37 degrees C was highest in a medium containing 22% total sugars, 0.05% (NH(4))(2)SO(4), 0.05% KH(2)PO(4), and 0.15% MgSO(4).7H(2)O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 degrees C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a final ethanol concentration of 6.43% (w/v), a productivity of 1.3g/l/h and a yield of 57.1% of theoretical yield.     

Candida ratchasimensis sp. nov. and Candida khaoyaiensis sp. nov., two anamorphic yeast species isolated from flowers in Thailand

Two yeast strains of the genus Candida were isolated from wild flowers collected in Khao Yai National Park, Nakhonratchasima Province, Thailand. Based on morphological, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 domain of the 26S rRNA gene, strains BCC 7722^T (=NBRC 102563^T=CBS 10611^T) and BCC 7729^T (=NBRC 102565^T=CBS 10839^T) were found to represent two distinct novel Candida species, for which the names Candida ratchasimensis sp. nov. and Candida khaoyaiensis sp. nov. are proposed, respectively. In the phylogenetic tree constructed according to the neighbour-joining method based on sequences of the D1/D2 domain of the 26S rRNA gene, strains BCC 7722^T (GenBank accession no. AY228492 ) and BCC 7729^T (accession no. DQ400367 ) constituted a cluster with Candida cellae that was connected with a clade with Starmerella meliponinorum and Candida etschellsii . Within the D1/D2 domain, C. ratchasimensis and C. khaoyaiensis differ from C. cellae in 25 nucleotide substitutions with five gaps and 29 nucleotide substitutions with one gap, respectively.     

Yamadazyma ubonensis f.a., sp. nov., a novel xylitol-producing yeast species isolated in Thailand

Three hundred and thirty-seven xylose-utilizing yeast strains were isolated from various natural samples. Among these, 68 strains produced xylitol in the range of 0.1–0.69 g xylitol/g xylose. Thirty-nine xylitol-producing strains were identified to be Candida tropicalis. Ten strains were found belonging to 14 known species in the genus Candida, Cyberlindnera, Meyerozyma, Pichia, Wickerhamomyces, Yamadazyma and Cryptococcus. Two strains were identified to be two Candida species and two strains (DMKU-XE142^T and DMKU-XE332) were found to be a novel species. Strain DMKU-XE142^T was isolated from tree bark and DMKU-XE332 was obtained from decaying plant leaf collected in Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 region of the large subunit rRNA gene (LSU) and the internal transcribed spacer (ITS) region, the two strains were determined to represent a novel Yamadazyma species although formation of ascospores was not observed. The sequences of the D1/D2 region of the LSU rRNA gene and the ITS region of the two strains were identical but differed from Yamadazyma phyllophila, the closest species in terms of pairwise sequence similarity of the D1/D2 region, by 1.7 % nucleotide substitutions and 3.5 % nucleotide substitutions in the ITS region. The name Yamadazyma ubonensis f.a., sp. nov. is proposed (type strain is DMKU-XE142^T = BCC 61020^T = CBS 12859^T).     

Comparison of the gene expression patterns of alcohol dehydrogenase isozymes in the thermotolerant yeast Kluyveromyces marxianus and their physiological functions

Four genes encoding alcohol dehydrogenase (Adh) isozymes in the thermotolerant yeast Kluyveromyces marxianus, a potent candidate for ethanol production at high temperatures, were investigated. Of these, KmADH3 and KmADH4 were cloned and sequenced, and their deduced amino acid sequences were compared with those of KmAdh1 and KmAdh2 and other Adhs of Kluyveromyces lactis and Saccharomyces cerevisiae. The four KmAdhs had high sequence similarity, though KmAdh3 and KmAdh4 possessed an amino-terminal extension as a mitochondrial targeting sequence, and appear to belong to the zinc-containing Adh family. These results and the results of Southern blot experiments suggest that there are at least four Adh isozymes in K. marxianus, two cytoplasmic enzymes and two mitochondrial enzymes. The expression profile revealed that KmADH genes are differently expressed depending on growth phase and carbon source, suggesting that these highly homologous Adhs play distinctive roles in cells.