Metschnikowia ahupensis f.a., sp. nov., a new yeast species isolated from the gut of the wood-feeding termite Nasutitermes sp.

The gut of xylophagous insects such as termites harbours various symbiotic micro-organisms, including many yeast species. In a taxonomic study of gut-associated yeasts, two strains (ATS2.16 and ATS2.18) were isolated from the gut of the wood-feeding termite Nasutitermes sp. in Maharashtra, India. Morphological and physiological characteristics and sequence analyses of the ITS and D1/D2 region of the large subunit rRNA gene revealed that these two strains represent a novel asexual ascomycetous yeast species in the genus Metschnikowia. The species differs from some of its close affiliates in the genus in its inability to utilize ethanol and succinate as the sole carbon source and growth in high sugar concentrations (up to 50 % glucose). In contrast to most members of Metschnikowia, the formation of ascospores was not observed on various sporulation media. Moreover, whole-genome sequencing was used to further confirm the novelty of this species. When compared with other large-spored Metschnikowia species, average nucleotide identity values of 79-80 % and digital DNA-DNA hybridization values of 16-17 % were obtained. The name Metschnikowia ahupensis f.a., sp. nov. is proposed to accommodate this novel yeast species, with ATS2.16 as the holotype and strains NFCCI 4949, MTCC 13085 and PYCC 9152 as isotypes. The MycoBank number is MB 844210.

Nakazawaea odontotermitis f.a., sp. nov., a novel yeast isolated from the gut of Odontotermes horni in India.

Three strains, SMT1.3, SMT1.10, and SMT2.2, representing a novel asexual ascomycetous yeast species, were isolated from the gut of a termite Odontotermes horni in Maharashtra, India. Phylogenetic analyses of the LSU, ITS, and SSU sequences revealed that they belonged to the genus Nakazawaea, with N. siamensis as the closest relative. The new species differed from the type strain of N. siamensis (DMKU-RK467T) by 11 substitutions in the D1/D2 region of the large subunit (LSU) rRNA gene and by 8 substitutions and one gap in the small subunit (SSU) rRNA gene. Notable biochemical and physiological differences were also observed between N. siamensis and the new species. Hence, the species Nakazawaea odontotermitis f.a., sp. nov. is proposed. The type strain is SMT1.3 T (MTCC 13,105 = NFCCI 5011 = PYCC 9153). GenBank accession numbers of the LSU, ITS and SSU sequences of Nakazawaea odontotermitis f.a., sp. nov. are MZ234240, MZ234239, and OK384663. The MycoBank number is MB 841926.

Nectar Yeast Community of Tropical Flowering Plants and Assessment of Their Osmotolerance and Xylitol-Producing Potential.

Floral nectar is colonised by microbes, especially yeasts which alter the scent, temperature, and chemical composition of nectar, thereby playing an essential role in pollination. The yeast communities inhabiting the nectar of tropical flowers of India are not well explored. We isolated 48 yeast strains from seven different tropical flowering plants. Post MSP-PCR-based screening, 23 yeast isolates and two yeast-like fungi were identified, which belonged to 16 species of 12 genera viz. Candida (2 species), Aureobasidium (2 species), Metschnikowia (2 species), Meyerozyma (1 species), Saitozyma (1 species), Wickerhamomyces (1 species), Kodamaea (2 species), Pseudozyma (1 species), Starmerella (1 species), Hanseniaspora (1 species), Rhodosporidiobolus (1 species), Moesziomyces (1 species), and two putative novel species. All yeast strains were assessed for their osmotolerance abilities in high salt and sugar concentration. Among all the isolates, C. nivariensis (SRA2.2, SRA1.1 and SRA2.1), M. caribbica (SRA4.8 and SRA4.6), S. flava SRA4.2, and M. reukaufii SRA3.2 showed significant growth in high concentrations of sugar (40-50% glucose), as well as salt (12-15% NaCl). All 25 strains were also screened for their ability to utilise xylose to produce xylitol. Meyerozyma caribbica was the most efficient xylitol producer, wherein three strains of this species (SRA4.6, SRA4.1, and SRA4.8) generated 18.61 to 21.56 g l-1 of xylitol, with 0.465-0.539 g g-1 yields. Through this study, we draw attention towards the tropical floral nectar as a potential niche for the isolation of diverse, osmotolerant, and xylitol-producing yeasts. Such osmotolerant yeasts have potential applications in food industries and biofuel production.

Rhodotorula sampaioana f.a., sp. nov., a novel red yeast of the order Sporidiobolales isolated from Argentina and India.

A set of four strains representing a novel basidiomycetous yeast species Rhodotorula sampaioana f. a., sp. nov. were isolated from two different habitats, subsurface waters of Lake Negra in Argentina, and the gut of a xylophagous termite in India. Phylogenetic analyses of LSU and ITS sequences showed that they belonged to the genus Rhodotorula of the order Sporidiobolales (subphylum Pucciniomycotina) and the closest known relative being R. kratochvilovae. The new species differed from R. kratochvilovae CBS 7436 (AF071436, AF444520) by nine nucleotide substitutions and one deletion (1.7 % sequence variation) in a 593 bp D1/D2 region, and by five nucleotide substitutions and three deletions (1.3 %) in a 592 bp ITS region, respectively. Several morphological and physiological differences were also observed between R. kratochvilovae and the strains obtained during this study. These data support the proposal of Rhodotorula sampaioana as a novel species, with CRUB 1124 as the holotype, CBS 10798 as ex-type, and NFCCI 4872 as an additional strain. The GenBank accession numbers of the LSU and ITS sequences of Rhodotorula sampaioana f. a., sp. nov. are EF595748 and MW879331. The MycoBank number is MB 838533.

Xylanolytic and Ethanologenic Potential of Gut Associated Yeasts from Different Species of Termites from India.

Xylophagous termites are capable of degrading lignocellulose by symbiotic gut microorganisms along with the host's indigenous enzymes. Therefore, the termite gut might be a potential niche to obtain natural yeasts with celluloytic, xylanolytic and ethanologenic traits required for bioethanol production from lignocellulosic biomass. In this study, we cultured 79 yeasts from three different termites viz. Coptotermes heimi, Odontotermes javanicus and Odontotermes obesus. After suitable screening methods, we identified 53 yeasts, which belonged to 10 genera and 16 different species of both ascomycetous and basidiomycetous yeasts. Most yeasts in the present study represent their first-ever isolation from the termite gut. Representative strains of identified yeasts were evaluated for their cellulolytic, xylanolytic, and ethanologenic abilities. None of the isolates showed cellulase activity; 22 showed xylanolytic activity, while six produced substantial quantities of ethanol. Among xylanolytic cultures, Pseudozyma hubeiensis STAG 1.7 and Hannaella pagnoccae STAG 1.14 produced 1.31 and 1.17 IU of xylanase. Among ethanologenic yeasts, the strains belonging to genera Candida and Kodamaea produced high amount of ethanol. Overall, highest ethanol level of 4.42 g/L was produced by Candida tropicalis TS32 using 1% glucose, which increased up to 22.92 g/L at 35 °C, pH 4.5 with 5% glucose. Fermentation of rice straw hydrolysate gave 8.95 g/l of ethanol with a yield of 0.42 g/g using the strain TS32. Our study highlights the gut of wood-feeding termites as a potential source of diverse yeasts that would be useful in the production of xylanase and bioethanol.

Suhomyces drosophilae sp. nov., isolated from Drosophila flies feeding on a stinkhorn mushroom.

The majority of Suhomyces species have been isolated from fungus-feeding insects and particularly from the gut of beetles. In the present study, seven yeast strains were isolated from the gut of Drosophila species feeding on gleba, the spore-bearing inner mass, of a stinkhorn mushroom belonging to the family Phallaceae. Based on phenotypic, biochemical characterization and sequence analysis of the D1/D2 region of the large subunit rRNA gene and the internal transcribed spacer (ITS) region, two of these yeast strains, DGY3 and DGY4, represented a novel species of the genus Suhomyces. The novel species is closely related to an undescribed species of Candida ST-370 (DQ404513) and with Suhomyces canberraensis, wherein, the novel species differs from S. canberraensis by 40 nucleotide substitutions and three gaps (7.7 % sequence variation) in the D1/D2 region and 50 nucleotide substitutions and seven gaps (13.7 % sequence variation) in the ITS region. Several morphological and physiological differences were also observed between S. canberraensis and the strains obtained during this study. These data support the proposal of Suhomyces drosophilae as a novel species, with DGY3T as the holotype and CBS 16329T and MCC 1871T as ex-type strains.

Isaria tenuipes Peck, an entomopathogenic fungus from Darjeeling Himalaya: Evaluation of in-vitro antiproliferative and antioxidant potential of its mycelium extract.

BACKGROUND: Isaria tenuipes is one of the potent species in the members of the genus Isaria, which is well reported to possess multiple bioactive substances of therapeutic importance. Therefore, an in vitro experimental study was carried to evaluate the bioactivities of the crude methanolic extract from the mycelium of this fungus. METHODS: The fungus was authenticated through morphological characters and the species discrepancy was resolved using the nuclear rDNA ITS sequence. The methanolic extract was fingerprinted by FTIR. The antioxidant components in terms of total phenols and flavonoids were determined as gallic acid and quercetin equivalents respectively. Antioxidant activities of the methanolic extract was assessed using 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2/-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) radical cation (ABTS0+), Fe2+chelating activity, and hydroxyl radical scavenging assays. Cytotoxicity of the extract was determined by [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (MTT) assay on three cancer cell lines: HeLa, HepG2, and PC3. Apoptosis was further studied by propidium iodide (PI) and Annexin-V/PI staining flow cytometric analysis. Anti-proliferation capacity was studied by colony-forming assay. RESULTS: In the present study total phenol content of the dried methanol extract was 148.09 ± 3.51µg gallic acid equivalent/mg and flavonoid was 9.02±0.95 µg quercetin/mg. The antioxidant activities of methanol-water extract (8:2 v/v) from cultured mycelia of I. tenuipes investigated and evaluated with 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay revealed IC50 value of 5.04mg/ml with an inhibition rate of 74.77% at 10mg/ml and with an iron-chelating assay the chelating ability was recorded to be 86.76% where the IC50 value was 4.43 mg/ml. In comparison among the antioxidant assays, 2,2/-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) radical cation (ABTS0+) and hydroxyl assay exhibited radical scavenging rate of 44.42% and 49.82% respectively at a concentration of 10 mg/ml. The IC50 value of the extract in MTT assay was 43.45µg/ml with HeLa cells, 119.33µg/ml with PC3 cells, and 125.55µg/ml with HepG2 cells. CONCLUSION: In this study, it can be concluded that the crude methanolic extract exhibited potent antioxidant and antiproliferative activities suggesting natural antioxidative and antiproliferative agents.