Communities of culturable yeasts and yeast-like fungi in oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar.

This report is the first investigation of yeast biodiversity from the oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar. Yeasts and yeast-like fungi, were cultured from seawater sampled at 13 coastal areas surrounding Qatar over a period of 2 years (December 2013-September 2015). Eight hundred and forty-two isolates belonging to 82 species representing two phyla viz., Ascomycota (23 genera) and Basidiomycota (16 genera) were identified by molecular sequencing. The results indicated that the coastal waters of the Qatari oligotrophic marine environment harbor a diverse pool of yeast species, most of which have been reported from terrestrial, clinical and aquatic sources in various parts of the world. Five species, i.e., Candida albicans, C. parapsilosis, C. tropicalis, Pichia kudriavzevii and Meyerozyma guilliermondii (n = 252/842; 30% isolates) are known as major opportunistic human pathogens. Fifteen species belonging to nine genera (n = 498/842; 59%) and 12 species belonging to seven genera (n = 459/842; 55%) are hydrocarbon degrading yeast and pollution indicator yeast species, respectively. Ascomycetous yeasts were predominant (66.38%; 559/842) as compared to their basidiomycetous counterparts (33.6%; 283/842). The most isolated yeast genera were Candida (28%; 236/842) (e.g., C. aaseri, C. boidinii, C. glabrata, C. intermedia, C. oleophila, C. orthopsilosis, C. palmioleophila, C. parapsilosis, C. pseudointermedia, C. rugopelliculosa, C. sake, C. tropicalis and C. zeylanoides), Rhodotorula (12.7%; 107/842), Naganishia (8.4%; 71/842), Aureobasidium (7.4%; 62/842), Pichia (7.3%; 62/842), and Debaryomyces (6.4%; 54/842). A total of eleven yeast species ( n = 38) isolated in this study are reported for the first time from the marine environment. Chemical testing demonstrated that seven out of the 13 sites had levels of total petroleum hydrocarbons (TPH) ranging from 200 to 900 µg/L, whereas 6 sites showed higher TPH levels (> 1000-21000 µg/L). The results suggest that the yeast community structure and density are impacted by various physico-chemical factors, namely total organic carbon, dissolved organic carbon and sulphur.

Vishniacozyma alagoana sp. nov. a tremellomycetes yeast associated with plants from dry and rainfall tropical forests.

Plants are important reservoirs of described and undescribed species of yeast. During a study of yeasts associated with bromeliads from the Northeast region of Brazil (collected in 2013-2017), analysis of the D1/D2 domain of the LSU rRNA and internal transcribed spacer (ITS) region identified eleven strains of yeasts as representing an unknown species of the genus Vishniacozyma. The species may have a diverse habitat in Brazil as a strain was collected from a flowering plant (Acanthaceae) in 1994. As a consequence, we propose Vishniacozyma alagoana sp. nov. as a member of the tremellomycetes yeasts (Agaricomycotina, Basidiomycota). Vishniacozyma alagoana sp. nov. was found in Atlantic Forest (a tropical rainforest) and the Caatinga (a seasonally dry tropical forest) associated with bromeliads in northeast and southeastern Brazil. The proposed novel species is related to Vishniacozyma taibaiensis and distinguished by eight nucleotide substitutions in the D1/D2 domain and seventeen in the ITS region. In addition, Vishniacozyma alagoana sp. nov. differs from V. taibaiensis by the ability to assimilate ribitol. The holotype is CBS 15966T.

Cystobasidium halotolerans sp. nov., a novel basidiomycetous yeast species isolated from the Arabian Gulf.

Two strains, 1Y129 and 3Y383, which represent a single novel anamorphic yeast species, were isolated from the Arabian Gulf surrounding Qatar. The two strains have identical sequences in the D1/D2 domains of the large subunit (LSU) and the internal transcribed spacer (ITS) regions of the rRNA gene. On the basis of this sequence analysis the two strains were found to be closely related to Cystobasidium slooffiae CBS5706T with 97. 3 % nucleotide substitutions (12 nt; four gaps) in D1/D2 regions of the LSU rRNA and 97.0 % nucleotide substitutions (14 nt; two gaps) in ITS regions. In contrast to Cystobasidium halotolerans sp. nov., the related species Cystobasidium slooffiae, Cystobasidium minutum and Cystobasidium fimetarium were unable to assimilate d-galactose, soluble starch, galactitol, methanol or nitrate, and grew at 37 °C. The name proposed for this undescribed species is Cystobasidium halotolerans sp. nov. The ex-type strain is QCC/Y31/17 (=MUCL057192=CGMCC2.5570).

Kondoa qatarensis f.a., sp. nov., a novel yeast species isolated from marine water in Qatar.

During a study of yeast diversity in marine waters of the Arabian Gulf surrounding Qatar, 30 strains were isolated that represent a novel species of Kondoa. The isolates were obtained from five locations along the Qatari coast. Species identifications were based on sequence analysis of the internal transcribed spacer (ITS) regions and D1/D2 domains of the large subunit (LSU) rRNA gene. Five strains were selected for further study. The novel species was closely related to Kondoa aeria (CBS 8352T) and Kondoa malvinella (CBS 6082T) but differed from the two species by 5.6 % sequence divergence (16 substitutions and 14 gaps) in the ITS region. On the basis of D1/D2 domains of the LSU rRNA gene, the novel species differed from K. aeria by only >0.35 % sequence divergence (2 substitutions and 0 gaps) and 1.5 % sequence divergence (9 substitutions and 1 gap) to that of K. malvinella. Although the novel species showed a close similarity in the D1/D2 domains to K. aeria (CBS 8352T), based on the significant differences (16 substitutions) in ITS regions and on the basis of physiological and biochemical tests, viz ability to grow at 35 °C, and inability to assimilate, sucrose, raffinose, d,l-lactate, succinate, citrate and nitrite, these strains are considered as novel species of Kondoa. The species name of Kondoa qatarensis f.a., sp. nov. is proposed with specimen 2Y109 as the holotype.

Naganishia qatarensis sp. nov., a novel basidiomycetous yeast species from a hypersaline marine environment in Qatar.

Two yeast strains (INY29 and INY13) representing a novel yeast species were isolated from the hypersaline marine environment of the Inland Sea, Qatar. Phylogenetic analysis based on the D1/D2 domains of the large subunit (LSU) regions and internal transcribed spacer (ITS1 and ITS2) regions showed that the two strains represent a single species in the genus Naganishia that is distinct from other species. These two strains were classified as members of the genus Naganishia and clustered in a strongly supported clade represented by Naganishia albidus in the Filobasidiales order in the phylogenetic tree drawn from ITS and D1/D2 sequences. The novel species was most closely related to the type strain of Naganishia cerealis but the two species differed by 1 % sequence divergence (four substitutions and one gap) in the D1/D2 domains and (five substitutions and one gap) in the ITS regions. In contrast to the closest relative, N. cerealis, the novel yeast species assimilated melibiose, glycerol, meso-erythritol, dl-lactate, methanol, propane 1-2-diol, butane 2-3-diol, and grew at 35 °C. The name Naganishia qatarensis sp. nov. is proposed to accommodate these strains, with INY29 as the holotype.

Papiliotrema leoncinii sp. nov. and Papiliotrema miconiae sp. nov., two tremellaceous yeast species from Brazil.

Two yeast species, Papiliotrema leoncinii sp. nov. and Papiliotrema miconiae sp. nov., in the family Rhynchogastremataceae of the Tremellales are proposed. The two species are related to six species of the genus Papiliotrema: Papiliotrema aureus, P. flavescens, P. terrestris, P. baii, P. ruineniae and P. wisconsinensis. The novel species are proposed on the basis of the sequence-based phylogenetic species concept with analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region. A total of 16 strains of Papiliotrema leoncinii sp. nov. were obtained from freshwater and bromeliad leaves collected in Brazil. Papiliotrema leoncinii sp. nov. differs by 11, 12, 16, 14, 11 and 13 substitutions in the D1/D2 domain from the related species P. aureus, P. flavescens, P. terrestris, P. baii, P. ruineniae and P. wisconsinensis, respectively. Differences of 11 substitutions and 21 or more substitutions in ITS regions were found when the sequences of Papiliotrema leoncinii sp. nov. were compared with P. wisconsinensis and its closest relatives. The type strain of Papiliotrema leoncinii sp. nov. is UFMG-CM-Y374T (=CBS 13918T). Papiliotrema miconiae sp. nov. is represented by two strains isolated from a flower of Miconia sp. and a water sample in Brazil. Papiliotrema miconiae sp. nov. differs from the related species P. aureus and P. ruineniae by eight substitutions, from P. flavescens and P. terrestris by 11 substitutions, from P. baii by 10 substitutions and from P. wisconsinensis by 6 substitutions in the D1/D2 domain, and by 7 substitutions from P. wisconsinensis and more than 19 substitutions in the ITS region from its closest relatives. The type strain of Papiliotrema miconiae sp. nov. is CBS 8358T (ML 3666T=DBVPG-4492T). The MycoBank numbers for Papiliotrema leoncinii sp. nov. and Papiliotrema miconiae sp. nov. are MB 813594 and MB 814882, respectively.

Description of the Halophile Euplotes qatarensis nov. spec. (Ciliophora, Spirotrichea, Euplotida) Isolated from the Hypersaline Khor Al-Adaid Lagoon in Qatar.

The morphology, ontogenesis, and phylogenetic relationships of a halophile euplotid ciliates, Euplotes qatarensis nov. spec., isolated from the Khor Al-Adaid Lagoon in Qatar were investigated based on live observation as well as protargol- and silver nitrate-impregnated methods. The new species is characterised by a combination of features: the halophile habitat, a cell size of 50-65 × 33-40 µm, seven dorsal ridges, 10 commonly sized frontoventral cirri, two widely spaced marginal cirri, 10 dorsolateral kineties, and a double silverline pattern. The morphogenesis is similar to that of its congeners: (i) the oral primordium develops hypoapokinetally and the parental oral apparatus is retained; (ii) the frontoventral-transverse field of five streaks gives rise to the frontal, ventral, and transverse cirri, but not to the cirri I/1 and the marginal cirri; (iii) the dorsal somatic ciliature develops by intrakinetal proliferation of basal bodies in two anlagen per kinety that are just anterior and posterior to the future division furrow; (iv) the caudal cirri are formed by the two rightmost dorsolateral kineties. The SSU rDNA sequence of E. qatarensis branches with full support in the Euplotopsis elegans-Euplotes nobilii-Euplotopsis raikovi clade. The closest related publicly available SSU rDNA sequence is the one of E. nobilii, with which E. qatarensis has 93.4% sequence similarity. Euplotes parawoodruffi Song & Bradbury, 1997 is transferred to the genus Euplotoides based on the absence of frontoventral cirrus VI/3.

Hannaella pagnoccae sp. nov., a tremellaceous yeast species isolated from plants and soil.

Several independent surveys of yeasts associated with different plant materials and soil led to the proposal of a novel yeast species belonging to the Tremellales clade (Agaricomycotina, Basidiomycota). Analysis of the sequences of the D1/D2 domains and internal transcribed spacer region of the large subunit of the rRNA gene suggested affinity to a phylogenetic lineage that includes Hannaella coprosmaensis, Hannaella oryzae and Hannaella sinensis. Thirty-two isolates were obtained from different sources, including bromeliads, nectar of Heliconia psittacorum (Heliconiaceae), flowers of Pimenta dioica (Myrtaceae), roots and leaves of sugar cane (Saccharum spp.) in Brazil, leaves of Cratoxylum maingayi, Arundinaria pusilla and Vitis vinifera in Thailand, soil samples in Taiwan, and prairie soil in the USA. Sequence analysis of the D1/D2 domains of the large subunit of the rRNA gene showed that the novel species differs from Hannaella coprosmaensis and Hannaella oryzae by 36 and 46 nt substitutions, respectively. A novel species is suggested to accommodate these isolates, for which the name Hannaella pagnoccae sp. nov. is proposed. The type strain is BI118(T) ( = CBS 11142(T) = ATCC MYA-4530(T)).

Five new species of yeasts from fresh water and marine habitats in the Florida Everglades.

Yeast populations in the Shark River Slough of the Florida Everglades, USA, were examined during a 3-year period (2002-2005) at six locations ranging from fresh water marshes to marine mangroves. Seventy-four described species (33 ascomycetes and 41 basidiomycetes) and an approximately equal number of undescribed species were isolated during the course of the investigation. Serious human pathogens, such as Candida tropicalis, were not observed, which indicates that their presence in coastal waters is due to sources of pollution. Some of the observed species were widespread throughout the fresh water and marine habitats, whereas others appeared to be habitat restricted. Species occurrence ranged from prevalent to rare. Five representative unknown species were selected for formal description. The five species comprise two ascomycetes: Candida sharkiensis sp. nov. (CBS 11368(T)) and Candida rhizophoriensis sp. nov. (CBS 11402(T)) (Saccharomycetales, Metschnikowiaceae), and three basidiomycetes: Rhodotorula cladiensis sp. nov. (CBS 10878(T)) in the Sakaguchia clade (Cystobasidiomycetes), Rhodotorula evergladiensis sp. nov. (CBS 10880(T)) in the Rhodosporidium toruloides clade (Microbotryomycetes, Sporidiobolales) and Cryptococcus mangaliensis sp. nov. (CBS 10870(T)) in the Bulleromyces clade (Agaricomycotina, Tremellales).

Nomenclatural issues concerning cultured yeasts and other fungi: why it is important to avoid unneeded name changes.

The unambiguous application of fungal names is important to communicate scientific findings. Names are critical for (clinical) diagnostics, legal compliance, and regulatory controls, such as biosafety, food security, quarantine regulations, and industrial applications. Consequently, the stability of the taxonomic system and the traceability of nomenclatural changes is crucial for a broad range of users and taxonomists. The unambiguous application of names is assured by the preservation of nomenclatural history and the physical organisms representing a name. Fungi are extremely diverse in terms of ecology, lifestyle, and methods of study. Predominantly unicellular fungi known as yeasts are usually investigated as living cultures. Methods to characterize yeasts include physiological (growth) tests and experiments to induce a sexual morph; both methods require viable cultures. Thus, the preservation and availability of viable reference cultures are important, and cultures representing reference material are cited in species descriptions. Historical surveys revealed drawbacks and inconsistencies between past practices and modern requirements as stated in the International Code of Nomenclature for Algae, Fungi, and Plants (ICNafp). Improper typification of yeasts is a common problem, resulting in a large number invalid yeast species names. With this opinion letter, we address the problem that culturable microorganisms, notably some fungi and algae, require specific provisions under the ICNafp. We use yeasts as a prominent example of fungi known from cultures. But viable type material is important not only for yeasts, but also for other cultivable Fungi that are characterized by particular morphological structures (a specific type of spores), growth properties, and secondary metabolites. We summarize potential proposals which, in our opinion, will improve the stability of fungal names, in particular by protecting those names for which the reference material can be traced back to the original isolate.

Cryptococcus randhawai sp. nov., a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of Ficus religiosa (peepal tree) from New Delhi, India.

A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37degrees C), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, L: -rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate L: -sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).

Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology.

Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

Electrochemical detection of harmful algae and other microbial contaminants in coastal waters using hand-held biosensors.

Standard methods to identify microbial contaminants in the environment are slow, laborious, and can require specialized expertise. This study investigated electrochemical detection of microbial contaminants using commercially available, hand-held instruments. Electrochemical assays were developed for a red tide dinoflagellate (Karenia brevis), fecal-indicating bacteria (Enterococcus spp.), markers indicative of human sources of fecal pollution (human cluster Bacteroides and the esp gene of Enterococcus faecium), bacterial pathogens (Escherichia coli 0157:H7, Salmonella spp., Campylobacter jejuni, Staphylococcus aureus), and a viral pathogen (adenovirus). For K. brevis, two assay formats (Rapid PCR-Detect and Hybrid PCR-Detect) were tested and both provided detection limits of 10 genome equivalents for DNA isolated from K. brevis culture and amplified by PCR. Sensitivity with coastal water samples was sufficient to detect K. brevis that was "present" (

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand.

Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex 100 system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli and Shigella spp., Bacteroides distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.

Microcoding and flow cytometry as a high-throughput fungal identification system for Malassezia species.

Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in humans and domestic animals. With the recent recognition of new members of the genus, new questions are emerging with regard to the pathogenesis and epidemiology of the new species. As new species are recognized, a precise and comprehensive identification system is needed. Herein is described a bead suspension culture-based array that combines the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of the Luminex analyser. The developed 16-plex array consisted of species- and group-specific capture probes that acted as 'microcodes' for species identification. The probes, which were designed from sequence analysis in the D1/D2 region of rRNA and internal transcribed spacer (ITS) regions, were covalently bound to unique sets of fluorescent beads. Upon hybridization, the biotinylated amplicon was detected by the addition of a fluorochrome coupled to a reporter molecule. The hybridized beads were subsequently analysed by flow cytometric techniques. The developed array, which allowed the detection of species in a multiplex and high-throughput format, was accurate and fast, since it allowed precise identification of species and required less than 1 h following PCR amplification. The described protocol, which can integrate uniplex or multiplex PCR reactions, permitted the simultaneous detection of target sequences in a single reaction, and allowed single mismatch discrimination between probe and non-target sequences. The assay has the capability to be expanded to include other medically important pathogenic species in a single or multiplex array format.

An electrochemical RNA hybridization assay for detection of the fecal indicator bacterium Escherichia coli.

Monitoring waters for indicator bacteria is required to protect the public from exposure to fecal pollution. Our proof-of-concept study describes a method for detecting fecal coliforms. The coliform Escherichia coli was used as a model fecal indicator. DNA probe-coated magnetic beads in combination with the electrochemical monitoring of the oxidation state of guanine nucleotides should allow for direct detection of bacterial RNA. To demonstrate this concept, we used voltammetry in connection with pencil electrodes to detect isolated E. coli 16S rRNA. Using this approach, 10(7) cells of E. coli were detected in a quantitative, reproducible fashion in 4h. Detection was achieved without a nucleic acid amplification step. The specificity of the assay for coliforms was demonstrated by testing against a panel of bacterial RNA. We also show that E. coli RNA can be detected directly from cell extracts. The method could be used for on-site detection and shows promise for adaptation into automated biosensors for water-quality monitoring.

Rapid identification of adult and naupliar stages of copepods using DNA hybridization methodology.

Larval stages of common marine invertebrates and their ecological roles within their respective communities are frequently ignored because they are hard to identify. Morphological characters are often insufficient to differentiate between genera, much less species. To overcome the obstacles associated with species identification of copepod larvae, we developed a microtiter plate-based hybridization assay. Species-specific probes based on rDNA sequences were bound to microplates and used to capture target DNA. A novel method of linking the probes to the plate with poly-T tail ensured the probes were positioned above the plate surface and available for hybridization; this significantly increased the sensitivity of the assay. Target DNA extracted from individual copepods was amplified with biotin-labeled primers. The labeled target DNA bound to the probe specific for that species and produced a colorimetric change in the assay. The assay can be rapidly performed on freshly caught or ethanol preserved samples and the results visually interpreted.