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.

Bioprospecting thermotolerant yeasts from distillery effluent and molasses for high-temperature ethanol production.

AIMS: Isolation, characterization and assessment of inhibitor tolerance of thermotolerant yeasts associated with distillery effluent and molasses, and their use in high-temperature ethanol production from alkali-treated rice straw. METHODS AND RESULTS: A total of 92 thermotolerant yeasts were isolated from seven different distillery effluent and molasses samples. Based on MSP-PCR, 34 yeasts were selected and identified by sequencing the D1/D2 domain of LSU rDNA. These yeasts belonged to eight genera and nine different species. We assessed the inhibitor tolerance of these 34 well-characterized yeasts against various pre-treatment-generated inhibitors (furfural, 5-hydroxymethyl furfural and acetic acid) and also evaluated their ethanol yields at 40, 45 and 50℃. Among selected strains, Pichia kudriavzevii DSA3.2 exhibited the highest ethanol production (24.5 g l-1 ) with an efficiency of 95.7% at 40℃ using 5% glucose. At 45℃, P. kudriavzevii DSA3.2 and Kluyveromyces marxianus MSS6.3 yielded maximum ethanol titres; 22.3 and 23 g l-1 with 87.4% and 90% efficiency, respectively. While using alkali-treated RS at 45℃, K. marxianus MSS6.3 produced 10.5 g l-1 of ethanol with 84.5% fermentation efficiency via separate hydrolysis and fermentation, and 10.9 g l-1 of ethanol with 85% efficiency via simultaneous saccharification and fermentation. Pichia kudriavzevii DSA3.2, DSA3.1 and K. marxianus MSS6.3 also exhibited significant tolerance against multiple inhibitors. CONCLUSIONS: Yeast isolates P. kudriavzevii DSA3.2 and K. marxianus MSS6.3 exhibited significant inhibitor tolerance and proved to be suitable for high-temperature ethanol fermentation. After additional optimization and scale-up experiments, these isolates can be exemplary candidates for industrial-scale ethanol production from lignocellulosic biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study recognizes distillery effluents and molasses as specialized niches for yeasts with a broad substrate range, capable of tolerating multiple inhibitors and yielding high levels of ethanol at elevated temperatures. These yeasts can further be exploited for bioethanol production through SSF/SHF at a larger scale.

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.

A Rare Stinkhorn Fungus Itajahya rosea Attract Drosophila by Producing Chemical Attractants.

Itajahya rosea was found growing in association with Leucaena leucocephala plants at Savitribai Phule Pune University campus in India. The species identity was confirmed by phylogenetic analysis based on ITS and LSU regions of rDNA, wherein, our fugus was placed along with I. rosea in phylogenetic tree. It represents first record of I. rosea from India. Frequent visitation by Drosophila species on I. rosea fruiting body particularly on gleba was observed. The Drosophila got attracted to the detached gleba under the laboratory conditions and even sometimes, they prefer to sit over the gleba as compare to their food banana. It suggested that I. rosea gleba or pseudostipe produces some compounds for attraction and feeding behavior of Drosophila species. Therefore, we characterized the volatile attractants produced by gleba and pseudostipe of I. rosea by GC-MS analysis. Nineteen compounds were identified from gleba while nine compounds were recovered from the pseudostipe. Out of them, blends of three abundant odor producing volatile compounds were reported namely, Hexadecane, Pentadecane and Nonadecane, which are responsible for attraction of Drosophila toward the gleba. Three fatty acids namely 9,12-octadecadienoic acid (Z,Z), hexadecanoic acid and benzoic acid ethyl ester produced are served as an appetitive signal through olfactory response of Drosophila, so the flies were feed on the gleba. Two pheromones' compounds, heneicosane and (+)-(5S,9S)-5,9-dimethylpentadecane, were also reported in pseudostipe and gleba, respectively, which play a role in Drosophila for breeding. Our study highlights an intriguing chemical ecology of fungus-Drosophila interaction.

Differential Physiological Prerequisites and Gene Expression Profiles of Conidial Anastomosis Tube and Germ Tube Formation in Colletotrichum gloeosporioides.

The conidia of a hemibiotrophic fungus, Colletotrichum gloeosporioides, can conventionally form a germ tube (GT) and develop into a fungal colony. Under certain conditions, they tend to get connected through a conidial anastomosis tube (CAT) to share the nutrients. CAT fusion is believed to be responsible for the generation of genetic variations in few asexual fungi, which appears problematic for effective fungal disease management. The physiological and molecular requirements underlying the GT formation versus CAT fusion remained underexplored. In the present study, we have deciphered the physiological prerequisites for GT formation versus CAT fusion in C. gloeosporioides. GT formation occurred at a high frequency in the presence of nutrients, while CAT fusion was found to be higher in the absence of nutrients. Younger conidia were found to form GT efficiently, while older conidia preferentially formed CAT. Whole transcriptome analysis of GT and CAT revealed highly differential gene expression profiles, wherein 11,050 and 9786 genes were differentially expressed during GT formation and CAT fusion, respectively. A total of 1567 effector candidates were identified; out of them, 102 and 100 were uniquely expressed during GT formation and CAT fusion, respectively. Genes coding for cell wall degrading enzymes, germination, hyphal growth, host-fungus interaction, and virulence were highly upregulated during GT formation. Meanwhile, genes involved in stress response, cell wall remodeling, membrane transport, cytoskeleton, cell cycle, and cell rescue were highly upregulated during CAT fusion. To conclude, the GT formation and CAT fusion were found to be mutually exclusive processes, requiring differential physiological conditions and sets of DEGs in C. gloeosporioides. This study will help in understanding the basic CAT biology in emerging fungal model species of the genus Colletotrichum.

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.

Quorum sensing-mediated inter-specific conidial anastomosis tube fusion between Colletotrichum gloeosporioides and C. siamense.

Many plant pathogenic filamentous fungi undergo fusion of conidia through conidial anastomosis tubes (CATs), which is believed to facilitate horizontal gene transfer between species. We discovered a remarkable inter-specific CAT fusion between two important plant fungal pathogens Colletotrichum gloeosporioides and C. siamense. In an invitro assay, under no selection pressure, the inter-specific CAT fusion was preferred with higher frequency (25% ± 5%) than intra-specific CAT fusion (11% ± 3.6%). Different stages of CAT fusion viz. CAT induction, homing, and fusion were observed during this inter-specific CAT fusion. The CAT fusion was found to be higher in absence of nutrients and under physiological stresses. This CAT fusion involved a quorum sensing phenomenon, wherein the CAT induction was dependent on conidial density and the putative quorum sensing molecule was extractable in chloroform. Movement of nuclei, mitochondria, and lipid droplets were observed during the CAT fusion. Post CAT fusion, the resulting conidia gave rise to putative heterokaryotic progenies with variable colony characteristics as compared to their parental strains. Few heterokaryons showed variable AFLP banding pattern compared to their parental strains, thereby suggesting a possible genetic exchange between the two species through CAT fusion. The heterokaryotic progenies exhibited varied fitness under different stress conditions. Our study illustrated a possible role of inter-specific CAT fusion in generation of genetic and phenotypic diversity in these fungal pathogens.

In Vitro Assessment of Probiotic Potential of Saccharomyces Cerevisiae DABRP5 Isolated from Bollo Batter, a Traditional Goan Fermented Food.

Bollo is a traditional Goan fermented food in which coarse wheat/wheat and finger millet is leavened with toddy. We here isolated 42 yeast strains from Bollo batter. Initial screening of the isolates with probiotic properties yielded four yeast isolates (DABRP1, DABRP2, DABRP5 and DABRP12). These isolates exhibited tolerance to high bile salt concentration and acidic pH and resistance to various antibiotics, which indicated their probiotic nature. All these yeast isolates were identified as Saccharomyces cerevisiae through D1D2-LSU-rDNA sequencing. These yeast isolates also showed higher percent hydrophobicity towards chloroform followed by n-hexadecane and o-xylene indicating their mucosal surface-adhesive property. To evaluate the safety of the isolates for them to be called as generally recognized as safe, the pathogenic behavior of the isolates determined through the in vitro hemolysis assay and evaluation of DNase and gelatinase activities. None of the isolates exhibited hemolysis or produced DNase or gelatinase and thus were considered potentially safe. In terms of beneficial effects, the most potent isolate S. cerevisiae DABRP5 showed antibacterial activity against the test pathogens. It also showed excellent antioxidant activity with DPPH free radical scavenging activity of 68.85 ± 0.69%, anti-inflammatory activity with 60.39 ± 0.34% inhibition of protein denaturation, and antidiabetic activity with 71.75 ± 0.45% inhibition of α-amylase activity. The isolate produced α-amylase, lipase, and ß-galactosidase. The probiotic potential of the isolate S. cerevisiae DABRP5 was similar to that of the reference strain (Saccharomyces boulardii CNCM I-745) used in this study. The results thus indicate that yeast isolates from Bollo batter have probiotic potential.

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.

Wickerhamiella shivajii sp. nov., a thermotolerant yeast isolated from distillery effluent.

Ethanol production at high temperatures has garnered much interest in recent years and a key factor is the availability of thermotolerant yeasts. During an investigation on the diversity of thermotolerant yeasts from different habitats, a novel yeast species from the spent wash of a distillery unit associated with a sugar factory was isolated. Phylogenetic analysis of D1/D2 large subunit and ITS rRNA genes placed this species in the ascomycetous genus Wickerhamiella. The novel species can be distinguished from the closely related species Wickerhamiella pararugosa using these rRNA gene regions. The cells of the new species are ovoid to ellipsoid with a diameter of 3.5-6.0×2.4-3.10 µm, while W. pararugosa cells are cylindrical with a cell diameter of 1.5-3.0×6-23 µm. This novel species represents, together with Wickerhamiella cacticola, one of the two most thermotolerant yeast species in the genus Wickerhamiella, able to grow at 42 °C. Wickerhamiella shivajii sp. nov. is proposed during this study.

Blastobotrys bombycis sp. nov., a d-xylose-fermenting yeast isolated from the gut of the silkworm larva Bombyx mori.

The gut of insects harbors a yeast community that is still poorly understood. Here, a novel species of the ascomycetous genus Blastobotrys is proposed based on a yeast strain isolated from the larval gut of the silkworm Bombyx mori (Order Lepidoptera). The novel species is closely related to Blastobotrys aristata and Blastobotrys elegans on the basis of the results of molecular phylogenetic analyses. A preliminary screening revealed that it produces 1.5 g l-1 ethanol by fermenting 5 % d-xylose. The novel species, that represents the first report, to our knowledge, of yeast isolation from silkworms, is described as Blastobotrys bombycis sp. nov. (type strain RAAB001T=CBS 15274T=PYCC 8105T=MCC 1427T; MycoBank accession number MB 825095).

Development of a High-Resolution Multi-Locus Microsatellite Typing Method for Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is an economically important fungal pathogen causing substantial yield losses indifferent host plants. To understand the genetic diversity and molecular epidemiology of this fungus, we have developed a novel, high-resolution multi-locus microsatellite typing (MLMT) method. Bioinformatic analysis of C. gloeosporioides unannotated genome sequence yielded eight potential microsatellite loci, of which five, CG1 (GT)n, CG2 (GT1)n, CG3 (TC)n, CG4 (CT)n, and CG5 (CT1)n were selected for further study based on their universal amplification potential, reproducibility, and repeat number polymorphism. The selected microsatellites were used to analyze 31 strains of C. gloeosporioides isolated from 20 different host plants from India. All microsatellite loci were found to be polymorphic, and the approximate fragment sizes of microsatellite loci CG1, CG2, CG3, CG4, and CG5 were in ranges of 213-241, 197-227, 231-265, 209-275, and 132-188, respectively. Among the 31 isolates, 55 different genotypes were identified. The Simpson's index of diversity (D) values for the individual locus ranged from 0.79 to 0.92, with the D value of all combined five microsatellite loci being 0.99. Microsatellite data analysis revealed that isolates from Ocimum sanctum, Capsicum annuum (chili pepper), and Mangifera indica (mango) formed distinct clusters, therefore exhibited some level of correlation between certain genotypes and host. The developed MLMT method would be a powerful tool for studying the genetic diversity and any possible genotype-host correlation in C. gloeosporioides.

New species of xerocomoid boletes (Boletaceae) from Himalayan India based on morphological and molecular evidence.

Xerocomus doodhcha and Hortiboletus indorubellus (Boletaceae) from broadleaf montane forest in Sikkim, India, are proposed as new. They are described in detail with supporting morphological illustrations and compared with related taxa using molecular phylogenetic analysis of ITS and 28S rDNA sequences. Xerocomus doodhcha is characterized by a pale brown pileus, basidiospores with a finely bacillate surface under SEM, and phylogenetic proximity to the type species of Xerocomus, X. subtomentosus Hortiboletus indorubellus is characterized by a dark brown to reddish brown pileus, context that turns brownish to brownish orange on bruising, and phylogenetic proximity to Hortiboletus rubellus.

Multilocus microsatellite typing for Rhizopus oryzae.

Rhizopus oryzae is the most frequent causative agent of zygomycosis. Although zygomycosis causes considerable morbidity and mortality in immunocompromised patients, the epidemiology of the disease is not well studied and no standard molecular typing method has been described for any of the causative agents. Here we describe a multilocus microsatellite typing (MLMT) method for R. oryzae. R. oryzae genome sequences were downloaded from the Fungal Genome Initiative database (Broad Institute). The intergenic regions and ORFs of approximately 5.7 Mb were screened for repeat regions with the help of the online repeat search tool Repeat Masker. Of the 30 microsatellite loci identified, 3 microsatellites [RO3, (CCT)(n); RO4, (TA)(n); and RO8, (GAA)(GGA)(n)] were selected after validation of the ability to amplify them and their size variation in 8 randomly selected clinical isolates of R. oryzae. Nucleotide sequence analysis of these loci demonstrated polymorphism in the microsatellite repeat number. The capabilities of these microsatellite loci were assessed for strain differentiation on 30 clinical isolates, based on fragment size determination in an automated capillary electrophoresis using fluorescent labelled primers. These three polymorphic microsatellite loci were found to have good discriminatory power (D) (RO3, D=0.846; RO4, D=0.747; RO8, D=0.742; with a combined D=0.986) and stability for seven subcultures. It was also confirmed that the MLMT method may be applied to both R. oryzae and Rhizopus delemar (a proposed new species), although MLMT analysis could not differentiate them into two clusters. The MLMT system, described here for what is believed to be the first time for a zygomycotic fungus, holds promise as a powerful tool for the strain typing of R. oryzae.

Cavitary pulmonary zygomycosis caused by Rhizopus homothallicus.

We report the first two proven cases of cavitary pulmonary zygomycosis caused by Rhizopus homothallicus. The diagnosis in each case was based on histology, culture of the causal agent, and the nucleotide sequence of the D1/D2 region of the 28S ribosomal DNA.