Mycosarcoma aegyptiacum sp. nov., an antagonistic polymorphic basidiomycetous yeast related to smut fungi.

Six polymorphic yeast strains with strong antifungal activities isolated from dicot plants in an alkaline-lake desert region were subjected to taxonomic examination. The phylogenetic trees reconstructed by using neighbour-joining, maximum-likelihood and Bayesian methods from concatenated D1/D2 and ITS-5.8S-ITS2 sequences revealed phylogenetic affinity to Ustilaginaceae, but the large phylogenetic distance separating the isolates from the most closely related groups of species indicates that they represent a separate species. The sequences of the genes coding for the LSU rDNA, act1, rpb2 and a protein of unknown function corroborate this position. The isolates can easily be distinguished from their closest relatives by physiological tests (utilisation of carbon and nitrogen sources). Based on these results, a new species, Mycosarcoma aegyptiacum sp. nov., is proposed to accommodate the isolates. All isolates are polymorphic. Transitions between budding-yeast and pseudohyphal morphologies which take place during colony formation result in morphologically different colony sectors and invasive growth into the medium. Neither sexual mating nor sporulation was observed in cultures growing on laboratory media.

Antagonistic yeasts from a salt-lake region in Egypt: identification of a taxonomically distinct group of phylloplane strains related to Sporisorium.

Non-pathogenic yeasts antagonising microorganisms that cause pre- and postharvest diseases of plants have been found in diverse habitats. Their practical applicability as biocontrol agents (BCAs) depends on the strength of their antagonistic activity and/or spectrum of sensitive target microorganisms. In this study, yeasts were isolated from the phylloplane and fruits of plants growing in the alkaline water lake region Wadi El-Natrun, Egypt, and tested for antifungal and antibacterial activity. All phylloplane yeast isolates belonged to the Basidiomycota and most of them could antagonise at least certain test organisms. One group of isolates showing strong antagonism against almost all fungi and yeasts appears to represent a hitherto undescribed species distantly related to the smut genus Sporisorium. This is the first report of antagonistic activity in Sporisorium. The isolates assigned to Naganishia and Papiliotrema were more effective against bacteria. The broadest range and intensity of antagonism was observed in the fruit-associated strains belonging to the ascomycetous species Wickerhamomyces subpelliculosus. The Wickerhamomyces strains are good broad-spectrum BCA candidates, the Sporisorium strains could be used as efficient antifungal BCAs, whereas the Papiliotrema isolate can be exploited as an antibacterial biocontrol agent.

A teleomorph of the ustilaginalean yeast Moesziomyces antarcticus on barnyardgrass in Japan provides bioresources that degrade biodegradable plastics.

The basidiomycetous yeast Moesziomyces antarcticus (often cited as Pseudozyma antarctica), originally isolated from a sediment sample obtained from Lake Vanda in Antarctica, was asexually typified but closely related to the smut fungus Moesziomyces bullatus (Ustilaginales). We found a smut fungus on an ovary of barnyardgrass (Echinochloa crus-galli) in Japan, which had been identified as M. bullatus. The teliospores germinated and formed yeast-like colonies. Physiological and phylogenetic studies revealed that the characteristics of the yeast-like isolates coincided with those of "P. antarctica." We thus recognised the smut fungus as the teleomorph of M. antarcticus, and then emended the description of M. antarcticus based on the holomorph. The identified fungus could degrade certain biodegradable plastics and produce mannosylerythritol lipids (MELs) in similar qualities as the "P. antarctica" type strain. This discovery provides a significant bioresource, as genetically diverse M. antarcticus isolates could be obtained from the smut fungus.

Morphological differences between aerial and submerged sporidia of bio-fongicide Pseudozyma flocculosa CBS 16788.

Pseudozyma flocculosa is a fungus very useful and highly efficient as a biocontrol agent against powdery mildew. The reproduction of this fungus occurs exclusively by asexual production of conidia or sporidia that are the most suitable form for agricultural use and seems to be the most resistant to storage conditions. Despite the advantages offered by P. flocculosa in biological control, the use of this fungus use remains largely limited compared to that of chemical fungicides, at least partly due to the difficulty to obtain sporidia resistant to adverse environmental stresses in submerged culture conditions. Under solid-state and submerged-state cultivation, P. flocculosa strain CBS 16788 produced different types of sporidia. The submerged sporidia (SS) appeared relatively uniform in size, which was 15,4 ± 1,6 µm µm long, and 2,8 ± 0.8 µm wide. The aerial sporidia (AS) varied in shape and size, with a mean length of 8,2 ± 3 µm and width of 2,3 ± 0.6 µm. Under scanning and transmission electron microscopy, the cell wall of submerged sporidia was thinner than that of aerial spores, and the surface was smooth in contrast to the aerial sporidia that had a tendency to have verrucous, brittle surface characteristics. The thickness of the aerial sporidia wall is due to the presence of an outer layer rich in melanin. The sporidia germination was compared on YMPD (yeast extract, malt extract, soy peptone, dextrose and agar) coated coverslips. The aerial sporidia did not show germ tubes until 5 h of incubation, while the submerged sporidia showed many germ tubes after the same time. The resistance against the adverse environmental conditions in relation to the type of sporidia of P. flocculosa is discussed.

Metabolism-dependent bioaccumulation of uranium by Rhodosporidium toruloides isolated from the flooding water of a former uranium mine.

Remediation of former uranium mining sites represents one of the biggest challenges worldwide that have to be solved in this century. During the last years, the search of alternative strategies involving environmentally sustainable treatments has started. Bioremediation, the use of microorganisms to clean up polluted sites in the environment, is considered one the best alternative. By means of culture-dependent methods, we isolated an indigenous yeast strain, KS5 (Rhodosporidium toruloides), directly from the flooding water of a former uranium mining site and investigated its interactions with uranium. Our results highlight distinct adaptive mechanisms towards high uranium concentrations on the one hand, and complex interaction mechanisms on the other. The cells of the strain KS5 exhibit high a uranium tolerance, being able to grow at 6 mM, and also a high ability to accumulate this radionuclide (350 mg uranium/g dry biomass, 48 h). The removal of uranium by KS5 displays a temperature- and cell viability-dependent process, indicating that metabolic activity could be involved. By STEM (scanning transmission electron microscopy) investigations, we observed that uranium was removed by two mechanisms, active bioaccumulation and inactive biosorption. This study highlights the potential of KS5 as a representative of indigenous species within the flooding water of a former uranium mine, which may play a key role in bioremediation of uranium contaminated sites.

Efficient production of tri-acetylated mono-acylated mannosylerythritol lipids by Sporisorium sp. aff. sorghi SAM20.

AIMS: The aim of this study was to isolate a novel yeast strain, evaluate biosurfactant production by the strain and characterize the major product. METHODS AND RESULTS: The strain SAM20, isolated from grass, identified as Sporisorium sp. aff. sorghi based on phylogenetic analyses. The strain produced approximately 32 g l-1 glycolipid biosurfactants from 40 g l-1 soybean oil after 7 days at 28°C. The glycolipids showed a unique pattern of mannosylerythritol lipids (MELs) on thin layer chromatography plate compared to those hitherto reported. Structural characterization of the major product, called GL-A, revealed that it was mainly tri-acetylated mono-acylated MELs (MEL-A2) with C14:0, C16:0, C12:0 or C14:1 as the hydrophobic chain. The critical micelle concentration (CMC), the surface tension at CMC and hydrophilic-lipophilic balance value for GL-A were estimated to be 20 mg l-1 , 30·0 mN m-1 and 8·7, respectively. CONCLUSIONS: A MEL-A2 with novel composition and surface activities was efficiently produced from a novel MEL producer. This is the first report on production of MEL-A2 as the major product and from soybean oil. The biosurfactant has potential application as a wetting agent and oil-in-water emulsifier. SIGNIFICANCE AND IMPACT OF THE STUDY: Discovery of novel structures and novel strains is valuable for further commercial development and application of MELs. Sporisorium sp. aff. sorghi SAM20 can be considered as a potential candidate for commercial production of biosurfactants.

Ten new species of Macalpinomyces on Eriachne in northern Australia.

Macalpinomyces was established in 1977, with the type species M. eriachnes described from a specimen collected in northern Australia on the grass Eriachne sp. in 1855. Subsequently, M. eriachnes has been reported on more than 21 species of Eriachne in northern Australia. In this study, a polyphasic approach was employed to determine whether M. eriachnes masked cryptic diversity. On the basis of morphology, multilocus phylogeny, and coalescent methods of generalized mixed Yule-coalescent (GMYC) and Poisson tree processes (PTP) models, 26 specimens of Macalpinomyces on 13 species of Eriachne held in Australian herbaria were studied. Consequently, 10 new species of Macalpinomyces that satisfied the phylogenetic species recognition criteria are described.

Foliar application of the leaf-colonizing yeast Pseudozyma churashimaensis elicits systemic defense of pepper against bacterial and viral pathogens.

Yeast associates with many plant parts including the phyllosphere, where it is subject to harsh environmental conditions. Few studies have reported on biological control of foliar pathogens by yeast. Here, we newly isolated leaf-colonizing yeasts from leaves of field-grown pepper plants in a major pepper production area of South Korea. The yeast was isolated using semi-selective medium supplemented with rifampicin to inhibit bacterial growth and its disease control capacity against Xanthomonas axonopodis infection of pepper plants in the greenhouse was evaluated. Of 838 isolated yeasts, foliar spray of Pseudozyma churashimaensis strain RGJ1 at 108 cfu/mL conferred significant protection against X. axonopodis and unexpectedly against Cucumber mosaic virus, Pepper mottle virus, Pepper mild mottle virus, and Broad bean wilt virus under field conditions. Direct antagonism between strain RGJ1 and X. axonopodis was not detected from co-culture assays, suggesting that disease is suppressed via induced resistance. Additional molecular analysis of the induced resistance marker genes Capsicum annuum Pathogenesis-Related (CaPR) 4 and CaPR5 indicated that strain RGJ1 elicited plant defense priming. To our knowledge, this study is the first report of plant protection against bacterial and viral pathogens mediated by a leaf-colonizing yeast and has potential for effective disease management in the field.

Pseudozyma and other non-Candida opportunistic yeast bloodstream infections in a large stem cell transplant center.

Non-Candida opportunistic yeasts are emerging causes of bloodstream infection (BSI) in immunocompromised hosts. However, their clinical presentation, management, and outcomes in stem cell transplant (SCT) recipients are not well described. We report the first case to our knowledge of Pseudozyma BSI in a SCT recipient. He had evidence of cutaneous involvement, which has not been previously described in the literature. He became infected while neutropenic and receiving empiric micafungin, which is notable because Pseudozyma is reported to be resistant to echinocandins. He was successfully treated with the sequential use of liposomal amphotericin B and voriconazole. A review of the literature revealed nine reported instances of Pseudozyma fungemia. We performed a retrospective review of 3557 SCT recipients at our institution from January 2000 to June 2015 and identified four additional cases of non-Candida yeast BSIs. These include two with Cryptococcus, one with Trichosporon, and one with Saccharomyces. Pseudozyma and other non-Candida yeasts are emerging pathogens that can cause severe and disseminated infections in SCT recipients and other immunocompromised hosts. Clinicians should have a high degree of suspicion for echinocandin-resistant yeasts, if patients develop breakthrough yeast BSIs while receiving echinocandin therapy.

Molecular variability and genetic relationship among Brazilian strains of the sugarcane smut fungus.

Sporisorium scitamineum is the fungus that causes sugarcane smut disease. Despite of the importance of sugarcane for Brazilian agribusiness and the persistence of the pathogen in most cropping areas, genetic variation studies are still missing for Brazilian isolates. In this study, sets of isolates were analyzed using two molecular markers (AFLP and telRFLP) and ITS sequencing. Twenty-two whips were collected from symptomatic plants in cultivated sugarcane fields of Brazil. A total of 41 haploid strains of compatible mating types were selected from individual teliospores and used for molecular genetic analyses. telRFLP and ITS analyses were expanded to six Argentine isolates, where the sugarcane smut was first recorded in America. Genetic relationship among strains suggests the human-mediated dispersal of S. scitamineum within the Brazilian territory and between the two neighboring countries. Two genetically distinct groups were defined by the combined analysis of AFLP and telRFLP. The opposite mating-type strains derived from single teliospores were clustered together into these main groups, but had not always identical haplotypes. telRFLP markers analyzed over two generations of selfing and controlled outcrossing confirmed the potential for emergence of new variants and occurrence of recombination, which are relevant events for evolution of virulence and environmental adaptation.

Yeast diversity and novel yeast D1/D2 sequences from corn phylloplane obtained by a culture-independent approach.

Culture-independent techniques have recently been used for evaluation of microbial diversity in the environment since it addresses the problem of unculturable microorganisms. In this study, the diversity of epiphytic yeasts from corn (Zea mays Linn.) phylloplanes in Thailand was investigated using this technique and sequence-based analysis of the D1/D2 domains of the large subunit ribosomal DNA sequences. Thirty-seven samples of corn leaf were collected randomly from 10 provinces. The DNA was extracted from leaf washing samples and the D1/D2 domains were amplified. The PCR products were cloned and then screened by colony PCR. A total of 1049 clones were obtained from 37 clone libraries. From this total, 329 clones (213 sequences) were closely related to yeast strains in the GenBank database, and they were clustered into 77 operational taxonomic units (OTUs) with a similarity threshold of 99 %. The majority of sequences (98.5 %) were classified into the phylum Basidiomycota. Sixteen known yeast species were identified. Interestingly, more than 65 % of the D1/D2 sequences obtained by this technique were suggested to be sequences from new yeast taxa. The predominant yeast sequences detected belonged to the order Ustilaginales with relative frequency of 68.0 %. The most common known yeast species detected on the leaf samples were Pseudozyma hubeiensis pro tem. and Moesziomyces antarcticus with frequency of occurrence of 24.3 and 21.6 %, respectively.

Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane.

Smut is a fungal disease with widespread prevalence in sugarcane planting areas. Early detection and proper identification of Sporisorium scitamineum are essential in smut management practices. In the present study, four specific primers targeting the core effector Pep1 gene of S. scitamineum were designed. Optimal concentrations of Mg(2+), primer and Bst DNA polymerase, the three important components of the loop-mediated isothermal amplification (LAMP) reaction system, were screened using a single factor experiment method and the L16(4(5)) orthogonal experimental design. Hence, a LAMP system suitable for detection of S. scitamineum was established. High specificity of the LAMP method was confirmed by the assay of S. scitamineum, Fusarium moniliforme, Pestalotia ginkgo, Helminthospcrium sacchari, Fusarium oxysporum and endophytes of Yacheng05-179 and ROC22. The sensitivity of the LAMP method was equal to that of the conventional PCR targeting Pep1 gene and was 100 times higher than that of the conventional PCR assay targeting bE gene in S. scitamineum. The results suggest that this novel LAMP system has strong specificity and high sensitivity. This method not only provides technological support for the epidemic monitoring of sugarcane smut, but also provides a good case for development of similar detection technology for other plant pathogens.

Pseudozyma aphidis fungaemia with invasive fungal pneumonia in a patient with acute myeloid leukaemia: case report and literature review.

Pseudozyma species rarely cause invasive diseases in humans, which are usually isolated from plants. There have been anecdotal reports regarding Pseudozyma species infections in patients with underlying diseases or in neonates. However, clinical data and the pathogenicity in humans are still insufficient. We experienced a case of Pseudozyma aphidis fungaemia with invasive fungal pneumonia that developed during reinduction chemotherapy in a 51-year-old male with acute myeloid leukaemia (AML). P. aphidis was suspected based on the morphology of the yeast isolated from the blood and was confirmed via rDNA gene sequencing analysis. The patient successfully underwent stem cell transplantation with continuing antifungal treatment and finally completely recovered from both the AML and infectious complications. Here, we report a case of P. aphidis infection that developed during neutropenia in an AML patient and review the global literature.

High Production of Squalene Using a Newly Isolated Yeast-like Strain Pseudozyma sp. SD301.

A yeast-like fungus, termed strain SD301, with the ability to produce a high concentration of squalene, was isolated from Shuidong Bay, China. The nucleotide sequence analysis of the internal transcribed spacer (ITS) region of SD301 indicated the strain belonged to Pseudozyma species. The highest biomass and squalene production of SD301 were obtained when glucose and yeast extracts were used as the carbon and nitrogen sources, respectively, with a C/N ratio of 3. The optimal pH and temperature were 6 and 25 °C, with 15 g L(-1) of supplemented sea salt. The maximum squalene productivity reached 0.039 g L(-1) h(-1) in batch fermentation, while the maximum squalene yield of 2.445 g L(-1) was obtained in fed-batch fermentation. According to our knowledge, this is the highest squalene yield produced thus far using fermentation technology, and the newly isolated strain Pseudozyma sp. SD301 is a promising candidate for commercial squalene production.

First Case of Fungemia Due to Pseudozyma aphidis in a Pediatric Patient with Osteosarcoma in Latin America.

We report the first case of blood infection due to Pseudozyma aphidis in Latin America. We contribute evidence showing this organism to be a potential human pathogen, and we provide new data about its identification, drug susceptibility, and treatment outcome.

Detection of Tilletia controversa using immunofluorescent monoclonal antibodies.

AIMS: Tilletia controversa is an internationally quarantined pathogenic fungus that causes dwarf bunt of wheat and is similar to Tilletia caries in both teliospore morphology and genetic structure. This study developed a rapid and sensitive immunofluorescence method for differentiating the teliospores of T. controversa from T. caries. METHODS AND RESULTS: The method utilizes monoclonal antibody D-1 against teliospores of T. controversa as well as a PE-Cy3-conjugated goat anti-mouse antibody (overlapping light excitation of 495 and 555 nm). The orange cycle fluorescent signal was stronger against T. controversa teliospores in the outer spore wall and net ridge, whereas only the green signal was observed for the protoplasm of T. caries teliospores. The detection limit of this method was 2.0 µg ml(-1) of the D-1 monoclonal antibody. CONCLUSION: This study describes the production and diagnostic application of a novel mouse monoclonal antibody specific to T. controversa teliospores. SIGNIFICANCE AND IMPACT OF THE STUDY: This method could be used for the on-site identification of T. controversa teliospores in the near future and will help in selecting fungicides to control dwarf bunt of wheat as further technical developments are achieved.

Isolation of oleaginous yeast (Rhodosporidium toruloides) mutants tolerant of sugarcane bagasse hydrolysate.

Rhodosporidium toruloides is a lipid-producing yeast, the growth of which is severely suppressed when hydrolysates of lignocellulosic biomass are used as carbon source. This is probably due to the toxic substances, such as organic acids, furans, and phenolic compounds produced during the preparation of the hydrolysates. In order to solve this problem, R. toruloides cultures were subjected to atmospheric room-temperature plasma mutagenesis, resulting in the isolation of mutants showing tolerance to sugarcane bagasse hydrolysate (SBH). Three mutant strains, M11, M13, and M18, were found to grow with producing lipids with SBH as carbon source. M11 in particular appeared to accumulate higher levels (up to 60% of dry cell weight) of intracellular lipids. Further, all three mutant strains showed tolerance of vanillin, furfural, and acetic acid, with different spectra, suggesting that different genetic determinants are involved in SBH tolerance.

Pseudozyma brasiliensis sp. nov., a xylanolytic, ustilaginomycetous yeast species isolated from an insect pest of sugarcane roots.

A novel ustilaginomycetous yeast isolated from the intestinal tract of an insect pest of sugarcane roots in Ribeirão Preto, São Paulo State, Brazil, represents a novel species of the genus Pseudozyma based on molecular analyses of the D1/D2 rDNA large subunit and the internal transcribed spacer (ITS1+ITS2) regions. The name Pseudozyma brasiliensis sp. nov. is proposed for this species, with GHG001(T) ( = CBS 13268(T) = UFMG-CM-Y307(T)) as the type strain. P. brasiliensis sp. nov. is a sister species of Pseudozyma vetiver, originally isolated from leaves of vetiver grass and sugarcane in Thailand. P. brasiliensis sp. nov. is able to grow well with xylan as the sole carbon source and produces high levels of an endo-1,4-xylanase that has a higher specific activity in comparison with other eukaryotic xylanases. This enzyme has a variety of industrial applications, indicating the great biotechnological potential of P. brasiliensis.

First neonatal case of fungaemia due to Pseudozyma aphidis and a global literature review.

The Ustilaginomycetous basidiomycete yeast, Pseudozyma aphidis has recently been implicated in potentially fatal disorders ranging from subcutaneous mycoses to disseminated infections. Till date a solitary case of P. aphidis fungaemia in a paediatric patient has been reported. We present a case of fungaemia due to P. aphidis in a rhesus factor-isoimmunised, low-birth-weight neonate. The isolate was identified by sequencing the D1/D2 domain of the LSU region. Antifungal susceptibility of the isolate revealed susceptibility to amphotericin B, voriconazole, itraconazole, isavuconazole and posaconazole. It had high minimum inhibitory concentrations of fluconazole and was resistant to flucytosine and echinocandins. Consequently, the patient was successfully treated with intravenous amphotericin B. Although the source of infection could not be traced, as the neonate developed fungaemia on the first day of life, it could possibly be from the maternal urogenital tract or intrahospital transmission. A review of previously published cases revealed that risk factors for invasive Pseudozyma spp. infections were similar to those previously reported for non-albicans Candida spp. Pseudozyma species are underreported due to the difficulty of identifying this rare yeast pathogen by commercial identification systems. Considering that Pseudozyma spp. cause invasive fungal infections globally and are resistant to flucytosine, fluconazole and echinocandins, this pathogen assumes a greater clinical significance.

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.