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.

A New Screening Approach for Glycolipid-type Biosurfactant Producers Using MALDI-TOF/MS.

We applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) to screen for glycolipid-type biosurfactant (BS) producers. A crude extract of Pseudozyma antarctica, a well-known mannosylerythritol lipid (MEL) producer, was initially subjected to MALDI-TOF/MS. The spectrum of the extract showed the accumulation of diacylated MELs in culture. We then screened 80 environmental samples for BS-producing yeasts, and extracts from broth cultures of the selected five strains were examined using MALDI-TOF/MS. The results showed that all five strains produced MELs, whereas four strains also produced cellobiose lipids (CLs). By D1/D2 region sequence analysis, the MEL-producing strain was assigned to P. antarctica while the four MEL- and CL-producing strains were assigned to P. hubeiensis. These results demonstrate that MALDI-TOF/MS is a rapid and reliable tool to detect BS molecules in crude extracts of broth cultures to screen for glycolipid-type BS producers.

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.

The pathogenic mechanisms of Tilletia horrida as revealed by comparative and functional genomics.

Tilletia horrida is a soil-borne, mononucleate basidiomycete fungus with a biotrophic lifestyle that causes rice kernel smut, a disease that is distributed throughout hybrid rice growing areas worldwide. Here we report on the high-quality genome sequence of T. horrida; it is composed of 23.2 Mb that encode 7,729 predicted genes and 6,973 genes supported by RNA-seq. The genome contains few repetitive elements that account for 8.45% of the total. Evolutionarily, T. horrida lies close to the Ustilago fungi, suggesting grass species as potential hosts, but co-linearity was not observed between T. horrida and the barley smut Ustilago hordei. Genes and functions relevant to pathogenicity were presumed. T. horrida possesses a smaller set of carbohydrate-active enzymes and secondary metabolites, which probably reflect the specific characteristics of its infection and biotrophic lifestyle. Genes that encode secreted proteins and enzymes of secondary metabolism, and genes that are represented in the pathogen-host interaction gene database genes, are highly expressed during early infection; this is consistent with their potential roles in pathogenicity. Furthermore, among the 131 candidate pathogen effectors identified according to their expression patterns and functionality, we validated two that trigger leaf cell death in Nicotiana benthamiana. In summary, we have revealed new molecular mechanisms involved in the evolution, biotrophy, and pathogenesis of T. horrida.

Broad Genomic Sampling Reveals a Smut Pathogenic Ancestry of the Fungal Clade Ustilaginomycotina.

Ustilaginomycotina is home to a broad array of fungi including important plant pathogens collectively called smut fungi. Smuts are biotrophs that produce characteristic perennating propagules called teliospores, one of which, Ustilago maydis, is a model genetic organism. Broad exploration of smut biology has been hampered by limited phylogenetic resolution of Ustilaginiomycotina as well as an overall lack of genomic data for members of this subphylum. In this study, we sequenced eight Ustilaginomycotina genomes from previously unrepresented lineages, deciphered ordinal-level phylogenetic relationships for the subphylum, and performed comparative analyses. Unlike other Basidiomycota subphyla, all sampled Ustilaginomycotina genomes are relatively small and compact. Ancestral state reconstruction analyses indicate that teliospore formation was present at the origin of the subphylum. Divergence time estimation dates the divergence of most extant smut fungi after that of grasses (Poaceae). However, we found limited conservation of well-characterized genes related to smut pathogenesis from U. maydis, indicating dissimilar pathogenic mechanisms exist across other smut lineages. The genomes of Malasseziomycetes are highly diverged from the other sampled Ustilaginomycotina, likely due to their unique history as mammal-associated lipophilic yeasts. Despite extensive genomic data, the phylogenetic placement of this class remains ambiguous. Although the sampled Ustilaginomycotina members lack many core enzymes for plant cell wall decomposition and starch catabolism, we identified several novel carbohydrate active enzymes potentially related to pectin breakdown. Finally, ∼50% of Ustilaginomycotina species-specific genes are present in previously undersampled and rare lineages, highlighting the importance of exploring fungal diversity as a resource for novel gene discovery.

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.

Cryptic diversity in Tranzscheliella spp. (Ustilaginales) is driven by host switches.

Species of Tranzscheliella have been reported as pathogens of more than 30 genera of grasses (Poaceae). In this study, a combined morphological and molecular phylogenetic approach was used to examine 33 specimens provisionally identified as belonging to the T. hypodytes species complex. The phylogenetic analysis resolved several well-supported clades that corresponded to known and novel species of Tranzscheliella. Four new species are described and illustrated. In addition, a new combination in Tranzscheliella is proposed for Sorosporium reverdattoanum. Cophylogenetic analyses assessed by distance-based and event-cost based methods, indicated host switches are likely the prominent force driving speciation in Tranzscheliella.

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.

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.

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.

A novel calb-type lipase discovered by fungal genomes mining.

The fungus Pseudozyma antarctica produces a lipase (CalB) with broad substrate specificity, stability, high regio- and enantio-selectivity. It is active in non-aqueous organic solvents and at elevated temperatures. Hence, CalB is a robust biocatalyst for chemical conversions on an industrial scale. Here we report the in silico mining of public metagenomes and fungal genomes to discover novel lipases with high homology to CalB. The candidates were selected taking into account homology and conserved motifs criteria, as well as, phylogeny and 3D model analyses. The most promising candidate (PlicB) presented interesting structural properties. PlicB was expressed in a heterologous host, purified and partially characterized. Further experiments will allow finding novel catalytic properties with biotechnological interest.

Mannosylerythritol lipids: production and applications.

Mannosylerythritol lipids (MELs) are a glycolipid class of biosurfactants produced by a variety yeast and fungal strains that exhibit excellent interfacial and biochemical properties. MEL-producing fungi were identified using an efficient screening method for the glycolipid production and taxonomical classification on the basis of ribosomal RNA sequences. MEL production is limited primarily to the genus Pseudozyma, with significant variability among the MEL structures produced by each species. Outside of Pseudozyma, one recently isolated strain, Ustilago scitaminea, has been shown to exhibit abundant MEL-B production from sugarcane juice. Structural analyses of these compounds suggest a role for MELs in numerous cosmetic applications. MELs act as effective topical moisturizers and can repair damaged hair. Furthermore, these compounds have been shown to exhibit both protective and healing activities, to activate fibroblasts and papilla cells, and to act as natural antioxidants. In this review, we provide a brief summary of MEL research over the past few decades, focusing on the identification of MEL-producing fungi, the structural characterization of MELs, the use of alternative compounds as a primary carbon source, and the use of these compounds in cosmetic applications.

Characteristics of mannosylerythritol lipids and their environmental potential.

Mannosylerythritol lipids (MELs) are promising biosurfactants containing two glycosyl derivatives and various fatty acids, which are mainly secreted by Pseudozyma as well as Ustilago. In this review, the latest research is demonstrated on production conditions, structural diversity, self-assembling properties and versatile biochemical functions of MELs. The genetic study and synthetic pathways, which mainly influence the type and yield of MELs production. Due to the excellent surface activity, biocompatibility and restorative function, MELs can be used in enviornmental industry, which has not been widely noted. In this paper, the current status of research on enviornmental potential of MELs has been discussed including petroleum degradation, bioconversion of chemical wastes and enhanced bioremediation of amphiphilic wastes.

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.

Selective production of two diastereomers of disaccharide sugar alcohol, mannosylerythritol by Pseudozyma yeasts.

Mannosylerythritol (ME) is the hydrophilic backbone of mannosylerythritol lipids as the most promising biosurfactants produced by different Pseudozyma yeasts, and has been receiving attention as a new sugar alcohol. Different Pseudozyma yeasts were examined for the sugar alcohol production using glucose as the sole carbon source. P. hubeiensis KM-59 highly produced a conventional type of ME, i.e., 4-O-ß-D-mannopyranosyl-D-erythritol (4-ME). Interestingly, P. tsukubaensis KM-160 produced a diastereomer of 4-ME, i.e., 1-O-ß-D-mannopyranosyl-D-erythritol (1-ME). In shake flask culture with 200 g/l of glucose, strain KM-59 produced 4-ME at a yield of 33.2 g/l (2.2 g/l/day of the productivity), while strain KM-160 produced 1-ME at 30.0 g/l (2.0 g/l/day). Moreover, the two strains were found to produce ME from glycerol; the maximum yields of 4-ME and 1-ME from 200 g/l of glycerol were 16.1 g/l (1.1 g/l/day) and 15.8 g/l (1.1 g/l/day), respectively. The production of 1-ME as the new diastereomer was further investigated in fed batch culture using a 5-l jar-fermenter. Compared to the flask culture, strain KM-160 gave three times higher productivity of 1-ME at 38.0 g/l (6.3 g/l/day) from glucose and at 31.1 g/l (3.5 g/l/day) from glycerol, respectively. This is the first report on the selective production of two diastereomers of ME, and should thus facilitate the functional development and application of the disaccharide sugar alcohol in the food and relative industries.

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.

Pulmonary infection due to Pseudozyma aphidis in a patient with Burkitt lymphoma: first case report.

Fungal infections are being increasingly reported in patients with malignancies. Pseudozyma aphidis is an opportunistic yeast usually isolated from plants and rarely from human samples. In this study, we report the first case of pulmonary infection due to P. aphidis in a Burkitt lymphoma patient.