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.     

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit

BackgroundThe prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population.AimsTo evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method.MethodsBoth regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool.ResultsUsing ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions.ConclusionsIdentifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween® 80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.     

Mrakia arctica sp. nov., a new psychrophilic yeast isolated from an ice island in the Canadian High Arctic

Two strains of a psychrophilic basidiomycetous yeast species belonging to the genus Mrakia were isolated from a melt-pool mat community, on an ice island located in Disraeli Fjord, Ellesmere Island in the Canadian Arctic. Analysis of the large subunit rDNA D1/D2 domain and internal transcribed spacer region sequences indicated that these strains represent a novel species, and the name Mrakia arctica sp. nov. is proposed. This new species could grow at sub-zero temperatures and in vitamin-free media. Moreover, lipase and cellulase enzymes of M. arctica were strongly active even at ?3 °C. These results suggest an important role for M. arctica in the biogeochemical cycle of glacial ecosystems.     

Taxonomic and phenotypic characterization of yeasts isolated from worldwide cold rock-associated habitats

Yeast strains isolated from rock samples collected from worldwide cold regions were identified by sequence analysis of the D1/D2 domains of the 26S rDNA gene and the ITS region followed by molecular phylogeny. Over 77 % of yeasts isolates were Basidiomycota. Cryptococcus (orders Filobasidiales and Tremellales) and Rhodotorula (order Cystobasidiales) were the most frequent genera. About 40 % of yeast isolates belonged to undescribed species.     

26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法,为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性,为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株,从北京四家综合性医院收集临床酵母菌260余株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism,SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性,可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合,对260余株临床酵母菌进行了菌种鉴定,共鉴定有10个属20个种,优势属为念珠菌属(Candida),优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%),并发现过去从未或很少报道致病的酵母菌种,愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法;北京地区酵母菌临床分离株呈种群多样性分布,C. albicans虽然仍占优势,但其它念珠菌种的比例已达42%。     

Candida carvajalis sp. nov., an ascomycetous yeast species from the Ecuadorian Amazon jungle

In the course of a yeast biodiversity survey of different ecological habitats found in Ecuador, two yeast strains (CLQCA 20-011^T and CLQCA20-014) were isolated from samples of rotten wood and fallen leaf debris collected at separate sites in the central region of the Ecuadorian Amazonia. These strains were found to represent a novel yeast species based on the sequences of their D1/D2 domain of the large-subunit (LSU) rRNA gene and their physiological characteristics. Phylogenetic analysis based on LSU D1/D2 sequences revealed this novel species to be most closely related to Candida asparagi, Candida fructus, Candida musae and two as yet undescribed Candida species, with the six yeast taxa collectively forming a distinct species group within the Clavispora clade. The species name of Candida carvajalis sp. nov. is proposed to accommodate these strains, with CLQCA 20-011^T (NCYC 3509^T, CBS 11361^T) designated as the type strain.     

Species Richness of Yeast Communities in Floral Nectar of Southern Spanish Plants

Floral nectar of insect-pollinated plants often contains dense yeast populations, yet little quantitative information exists on patterns and magnitude of species richness of nectar-dwelling yeasts in natural plant communities. This study evaluates yeast species richness at both the plant community and plant species levels in a montane forest area in southern Spain, and also explores possible correlations between the incidence of different yeast species in nectar and their reported tolerance to high sugar concentrations, and between yeast diversity and pollinator composition. Yeast species occurring in a total of 128 field-collected nectar samples from 24 plant species were identified by sequencing the D1/D2 domain of the large subunit rDNA, and rarefaction-based analyses were used to estimate yeast species richness at the plant community and plant species levels, using nectar drops as elemental sampling units. Individual nectar samples were generally characterized by very low species richness (1.2 yeast species/sample, on average), with the ascomycetous Metschnikowia reukaufii and Metschnikowia gruessii accounting altogether for 84.7% of the 216 isolates identified. Other yeasts recorded included species in the genera Aureobasidium, Rhodotorula, Cryptococcus, Sporobolomyces, and Lecythophora. The shapes and slopes of observed richness accumulation curves were quite similar for the nectar drop and plant species approaches, but the two approaches yielded different expected richness estimates. Expected richness was higher for plant species-based than for nectar drop-based analyses, showing that the coverage of nectar yeast species occurring in the region would be improved by sampling additional host plant species. A significant correlation was found between incidence of yeast species in nectar and their reported ability to grow in a medium containing 50% glucose. Neither diversity nor incidence of yeasts was correlated with pollinator composition across plant species.     

Yeast identification: reassessment of assimilation tests as sole universal identifiers

Aims: To assess whether assimilation tests in isolation remain a valid method of identification of yeasts, when applied to a wide range of environmental and spoilage isolates. Methods and Results: Seventy‐one yeast strains were isolated from a soft drinks factory. These were identified using assimilation tests and by D1/D2 rDNA sequencing. When compared to sequencing, assimilation test identifications (MicroLog?) were 18·3% correct, a further 14·1% correct within the genus and 67·6% were incorrectly identified. The majority of the latter could be attributed to the rise in newly reported yeast species. Conclusions: Assimilation tests alone are unreliable as a universal means of yeast identification, because of numerous new species, variability of strains and increasing coincidence of assimilation profiles. Assimilation tests still have a useful role in the identification of common species, such as the majority of clinical isolates. Significance and Impact of the Study: It is probable, based on these results, that many yeast identifications reported in older literature are incorrect. This emphasizes the crucial need for accurate identification in present and future publications.     

青海东部土壤中酵母物种多样性研究

从青海的互助、民和、门源等10个州县收集土样分离得到98株酵母菌, 利用26S rDNA D1/D2区域序列分析并结合形态学和生理生化特性对这些菌株进行了分类学研究, 探讨了青海东部土壤中酵母的物种多样性及其分布。共鉴定出10属13种(其中有两个疑似新种), 其中 Galactomyces geotrichum和Rhodotorula mucilaginosa为该地的优势种。     

Isolation and identification of crude oil-degrading yeast strains from Khafji oil field,saud Arabia

Twenty five yeasts isolated were isolated from Khurais oil field in Saudi Arabia and assayed to evaluate their biodegradability. Only five isolates (namely, A1, A2, A3, A4 and A5) showed potential use of oil as sole carbon source. During incubation period, highest growth rate were recorded for A1, A2 and A3 isolates. Low growth distinguished A4 isolate; A5 isolate could not degrade oil.Spectrophotometrical analysis for four yeast isolates biodegradation activities indicated that, A1 isolate was superior for oil degradation (61%) comparing with A4 isolate which reflected lowest degradation % (33%). A2 and A3 isolates showed moderate biodegradation activity (56 and 51% respectively).D1/D2 domain of the 26S rRNA gene sequence was used as molecular marker to identify five yeast isolates. After comparing 26S rRNA gene sequences of five yeast isolates with highly similarity isolates, five yeast isolates (A1, A2, A3, A4 and A5)were submitted to database as Candida tropicalis (MW488263), Candida tropicalis (MW488264), Rhodotorula mucilaginosa (MW488265) and Rhodosporidium toruloides (MW488266) respectively. Using OXF1/ACR1 primer, specific lipase gene amplicon with 250 bp were detected with in all four yeast isolates.     

In Situ Analysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q²] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community.     

Comparison of redox and ligand binding behaviour of yeast and bovine cytochrome c oxidases using FTIR spectroscopy

Redox and CO photolysis FTIR spectra of yeast cytochrome c oxidase WT and mutants are compared to those from bovine and P. denitrificans CcOs in order to establish common functional features. All display changes that can be assigned to their E242 (bovine numbering) equivalent and to weakly H-bonded water molecules. The additional redox-sensitive band reported at 1736?cm^?1 in bovine CcO and previously assigned to D51 is absent from yeast CcO and couldn't be restored by introduction of a D residue at the equivalent position of the yeast protein. Redox spectra of yeast CcO also show much smaller changes in the amide I region, which may relate to structural differences in the region around D51 and the subunit I/II interface.     

Occurrence and Identification of Yeast Species in Fermented Liquid Feed for Piglets

The major objective of the present study was to investigate the occurrence and identity of yeast species in fermented liquid feed (FLF) used for feeding piglets. In total, 40 different Danish farms were included in the analysis. The preparation and composition of FLF was found to be very heterogeneous with high variations in both yeast counts and yeast species composition. The yeast population varied between 6.0?×?10³ and 4.2?×?10⁷?cfug^?1 with an average yeast count of 8.7?×?10⁶?±?1.1?×?10⁷?cfug^?1. A total of 766 yeasts were isolated and identified by conventional and/or molecular typing techniques. The predominant yeast species in the FLF samples were found to be Candida milleri (58.4%), Kazachstania exigua (17.5%), Candida pararugosa (6.40%) and Kazachstania bulderi (5.09%). No clear separation between isolates of C. milleri and Candida humilis could be obtained based on sequencing of the D1/D2 region of the 26S rRNA gene. The combined use of ITS-RFLP analysis and phenotypic criteria did meanwhile suggest a closer relationship with C. milleri than C. humilis.     

Yeast Diversity Associated with Invasive Dendroctonus valens Killing Pinus tabuliformis in China Using Culturing and Molecular Methods

Bark beetle-associated yeasts are much less studied than filamentous fungi, yet they are also considered to play important roles in beetle nutrition, detoxification, and chemical communication. The red turpentine beetle, Dendroctonus valens, an invasive bark beetle introduced from North America, became one of the most destructive pests in China, having killed more than 10 million Pinus tabuliformis as well as other pine species. No investigation of yeasts associated with this bark beetle in its invaded ranges has been conducted so far. The aim of this study was to assess the diversity of yeast communities in different microhabitats and during different developmental stages of Den. valens in China using culturing and denaturing gradient gel electrophoresis (DGGE) approaches and to compare the yeast flora between China and the USA. The yeast identity was confirmed by sequencing the D1/D2 domain of LSU ribosomal DNA (rDNA). In total, 21 species (13 ascomycetes and eight basidiomycetes) were detected by culturing method, and 12 species (11 ascomycetes and one basidiomycetes) were detected by molecular methods from China. The most frequent five species in China were Candida piceae (Ogataea clade), Cyberlindnera americana, Candida oregonensis (Metschnikowia clade), Candida nitratophila (Ogataea clade) and an undescribed Saccharomycopsis sp., detected by both methods. Seven species were exclusively detected by DGGE. Ca. oregonensis (Metschnikowia clade) was the most frequently detected species by DGGE method. Eight species (all were ascomycetes) from the USA were isolated; seven of those were also found in China. We found significant differences in yeast total abundance as well as community composition between different developmental stages and significant differences between the surface and the gut. The frass yeast community was more similar to that of Den. valens surface or larvae than to the community of the gut or adults. Possible functions of the yeast associates are discussed.     

Cryptococcus nanyangensis sp. nov., A New Basidiomycetous Yeast Isolated from the Gut of Wood-Boring Larvae

Two strains of a novel basidiomycetous yeast species were isolated from the gut of wood-boring larvae collected in the Baotianman Nature Reserve, the central China. Sequence analysis of the D1/D2 domains of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) regions showed that these yeasts belong to the Bulleromyces clade and formed a cluster together with eleven undescribed Cryptococcus species. The novel species differed from its closest known species, Cryptococcus rajasthanensis, by 3.3 % divergence (15 substitutions and 6 gaps over 630 bases) in the D1/D2 domains, and by 13.4 % divergence (41 substitutions and 27 gaps over 508 bases) in the ITS regions. Physiologically, the fermentation of glucose, galactose, sucrose, trehalose, and raffinose in Durham tubes was observed for the strains of this new yeast. Based on the phenotypical and molecular characteristics presented, the two strains are proposed as a new species, Cryptococcus nanyangensis sp. nov., with the type strain KCY-1^T (=CICC 1976^T = CBS 12474^T).     

葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株,利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究,探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布.共鉴定出13属26种,其中优势属为汉逊酵母属Hanseniaspora(5种),假丝酵母属Candida(4种),毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种).对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较,以探讨不同地理起源地酵母种内序列稳定性及其变异.     

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.     

葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株, 利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究, 探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布。共鉴定出13属26种, 其中优势属为汉逊酵母属Hanseniaspora(5种), 假丝酵母属Candida(4种), 毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种)。对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较, 以探讨不同地理起源地酵母种内序列稳定性及其变异。     

Yeasts from phylloplane and their capability to produce indole-3-acetic acid

Yeasts were isolated from the phylloplane of various plant species collected from seven provinces in Thailand. A total of 114 yeast strains and 10 strains of a yeast-like fungus were obtained by enrichment isolation from 91 out of 97 leaf samples (93.8?%). On the basis of the D1/D2 domain of the large subunit rRNA gene sequence similarity, 98 strains were identified to be of 36 yeast species in 18 genera belonging to Ascomycota viz. Candida, Clavispora, Cyberlindnera, Debaryomyces, Hanseniaspora, Hyphopichia, Kazachstania, Kluyveromyces, Kodamaea, Lachancea, Metschnikowia, Meyrozyma, Pichia, Starmerella, Torulaspora and Wickerhamomyces, and to Basidiomycota viz. Sporidiobolus and Trichosporon. Three strains were found to represent two novels Candida species which were previously described as C. sirachaensis and C. sakaeoensis. Ten strains of yeast-like fungus were identified as Aureobasidium pullulans of the phylum Ascomycota. Ascomycetous yeast species accounted altogether for 98.0?% of the 98 strains. The prevalent species was Candida tropicalis with a low frequency of isolation (14.3?%). Diversity of yeasts other than ballistoconidium-forming yeast in phylloplane in a tropical country in Asia has been reported for the first time. All strains obtained were accessed for the capability to produce IAA and result revealed that 39 strains in 20 species, one strain each of an undescribed and a novel species, and two unidentified strains showed the capability of producing IAA when cultivated in yeast extract peptone dextrose broth supplemented with 0.1?% l-tryptophan. All five strains of Candida maltosa produced relatively high concentrations of IAA.