Commelinaceomyces, gen. nov., for four clavicipitaceous species misplaced in Ustilago that infect Commelinaceae.

A fungus causing false smut in the flowers of Murdannia keisak (Commelinaceae, Commelinales, Monocots) in Japan was morphologically identical to Ustilago aneilematis. The fungus infected ovaries of most flowers of host plants. Infected flowers were filled with yellow to orange thick-walled conidia that became olivaceous green at maturity. However, multilocus phylogenetic analysis of DNA sequences (18S, 28S, translation elongation factor 1α [TEF], the largest [RPB1] and the second largest [RPB2] subunit of RNA polymerase II) showed that the fungus belonged to the tribe Ustilaginoideae (Clavicipitaceae, Hypocreales, Ascomycota). Microscopic examination showed that the fungus developed conidia at the apex of conidiogenous cells, in contrast to other species in the Ustilaginoideae that develop conidia pleurogenously. A new genus, Commelinaceomyces, is formally proposed in the Ustilaginoideae to accommodate this fungus. Four species previously misplaced in Ustilago (Ustilaginales, Basidiomycota) are transferred to Commelinaceomyces, including the type of the genus, C. aneilematis, on Murdannia keisak. This is the first report of a clavicipitaceous species infecting host plants in the Commelinaceae.

Microbial Diversity in the Edible Gall on White Bamboo Formed by the Interaction between Ustilago esculenta and Zizania latifolia.

An edible gall is formed between the third and fourth nodes beneath the apical meristem near the base of Zizania latifolia shoots. This gall is harbored by and interacts with the smut fungus Ustilago esculenta. The gall is also a valuable vegetable called "white bamboo," jiaobai or gausun in China and makomotake in Japan. Five samples of the galls harvested at different stages of swelling were used to isolate microorganisms by culturing. Isolated fungal and bacterial colonies were identified by DNA sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, respectively. Several strains of U. esculenta as well as 6 other species of fungi and 10 species of bacteria were isolated. The microbiome was also evaluated by simple and outlined DNA profiling with automated rRNA intergenic spacer analysis (ARISA), and the amount of DNA of U. esculenta was determined by qPCR. At least 16 species of fungi and 40 species of bacteria were confirmed by ARISA of the overall sample. Interestingly, the greatest bacterial diversity, i.e., 18 species, was observed in the most mature sample, whereas the fungal diversity observed in this sample, i.e., 4 species, was rather poor. Based on qPCR, U. esculenta occurred in samples from all stages; however, the abundance of U. esculenta exhibited unique U-shaped relationships with growth. These results may explain why the interaction between U. esculenta and Z. latifolia also influences the unique microbial diversity observed throughout the growth stages of the swollen shoot, although the limited sample size does not allow conclusive findings.

Investigation on the differentiation of two Ustilago esculenta strains - implications of a relationship with the host phenotypes appearing in the fields.

BACKGROUND: Ustilago esculenta, a pathogenic basidiomycete fungus, infects Zizania latifolia to form edible galls named Jiaobai in China. The distinct growth conditions of U. esculenta induced Z. latifolia to form three different phenotypes, named male Jiaobai, grey Jiaobai and white Jiaobai. The aim of this study is to characterize the genetic and morphological differences that distinguish the two U. esculenta strains. RESULTS: In this study, sexually compatible haploid sporidia UeT14/UeT55 from grey Jiaobai (T strains) and UeMT10/UeMT46 from white Jiaobai (MT strains) were isolated. Meanwhile, we successfully established mating and inoculation assays. Great differences were observed between the T and MT strains. First, the MT strains had a defect in development, including lower teliospore formation frequency and germination rate, a slower growth rate and a lower growth mass. Second, they differed in the assimilation of nitrogen sources in that the T strains preferred urea and the MT strains preferred arginine. In addition, the MT strains were more sensitive to external signals, including pH and oxidative stress. Third, the MT strains showed an infection defect, resulting in an endophytic life in the host. This was in accordance with multiple mutated pathogenic genes discovered in the MT strains by the non-synonymous mutation analysis of the genome re-sequencing data between the MT and T strains (GenBank accession numbers of the genome re-sequencing data: JTLW00000000 for MT strains and SRR5889164 for T strains). CONCLUSION: The MT strains appeared to have defects in growth and infection and were more sensitive to external signals compared to the T strains. They displayed an absolutely stable endophytic life in the host without an infection cycle. Accordingly, they had multiple gene mutations occurring, especially in pathogenicity. In contrast, the T strains, as phytopathogens, had a complete survival life cycle, in which the formation of teliospores is important for adaption and infection, leading to the appearance of the grey phenotype. Further studies elucidating the molecular differences between the U. esculenta strains causing differential host phenotypes will help to improve the production and formation of edible white galls.

Comparative whole-genome analysis reveals artificial selection effects on Ustilago esculenta genome.

Ustilago esculenta, infects Zizania latifolia, and induced host stem swollen to be a popular vegetable called Jiaobai in China. It is the long-standing artificial selection that maximizes the occurrence of favourable Jiaobai, and thus maintaining the plant-fungi interaction and modulating the fungus evolving from plant pathogen to entophyte. In this study, whole genome of U. esculenta was sequenced and transcriptomes of the fungi and its host were analysed. The 20.2 Mb U. esculenta draft genome of 6,654 predicted genes including mating, primary metabolism, secreted proteins, shared a high similarity to related Smut fungi. But U. esculenta prefers RNA silencing not repeat-induced point in defence and has more introns per gene, indicating relatively slow evolution rate. The fungus also lacks some genes in amino acid biosynthesis pathway which were filled by up-regulated host genes and developed distinct amino acid response mechanism to balance the infection-resistance interaction. Besides, U. esculenta lost some surface sensors, important virulence factors and host range-related effectors to maintain the economic endophytic life. The elucidation of the U. esculenta genomic information as well as expression profiles can not only contribute to more comprehensive insights into the molecular mechanism underlying artificial selection but also into smut fungi-host interactions.

Genome-wide identification and analysis of the basic leucine zipper (bZIP) transcription factor gene family in Ustilaginoidea virens.

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the largest and most diverse TF families widely distributed across the eukaryotes. The bZIP TF family plays an important role in growth, development, and response to abiotic or biotic stresses, which have been well characterized in plants, but not in plant pathogenic fungi. In this study, we performed genome-wide and systematic bioinformatics analysis of bZIP genes in the fungus Ustilaginoidea virens, the causal agent of rice false smut disease. We identified 28 bZIP family members in the U. virens genome by searching for the bZIP domain in predicted genes. The gene structures, motifs, and phylogenetic relationships were analyzed for bZIP genes in U. virens (UvbZIP). Together with bZIP proteins from two other fungi, the bZIP genes can be divided into eight groups according to their phylogenetic relationships. Based on RNA-Seq data, the expression profiles of UvbZIP genes at different infection stages were evaluated. Results showed that 17 UvbZIP genes were up-regulated during the infection period. Furthermore, 11 infection-related UvbZIP genes were investigated under H2O2 stress and the expression level of eight genes were changed, which confirmed their role in stress tolerance and pathogenicity. In summary, our genome-wide systematic characterization and expression analysis of UvbZIP genes provided insight into the molecular function of these genes in U. virens and provides a reference for other pathogens.

Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production.

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis' itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5-fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.

Ustilago echinata: Infection in a Mixed Martial Artist Following an Open Fracture.

Ustilago, a common fungal parasite of grains, is infrequently isolated as a pathogen in humans. We describe a case of Ustilago echinata infection following an open distal tibia fracture, review the current literature of this genus as a cause of invasive fungal infection in humans, and discuss management issues.

Biodegradation of cefdinir by a novel yeast strain, Ustilago sp. SMN03 isolated from pharmaceutical wastewater.

Cefdinir, a semi-synthetic third generation cephalosporin antibiotic being considered as an emerging pollutant, demands removal from aquatic ecosystems. A yeast strain isolated from pharmaceutical wastewater which was identified as Ustilago sp. SMN03 by molecular techniques and was found to be capable of utilizing cefdinir as a sole carbon source. The isolate was found to degrade 81 % of cefdinir within 6 days under optimized conditions viz. pH 6.0, temperature 30 °C, a shaking speed of 120 rpm, an inoculum dosage of 4 % (w/v) and an initial cefdinir concentration of 200 mg L(-1). Kinetic studies revealed that cefdinir degradation followed the pseudo-first order model, a rate constant of 0.222 per day and a half-life period of 3.26 days. Using LC-MS analysis, six novel intermediates formed during the cefdinir degradation were identified and characterized. FT-IR analysis showed that the functional groups ranging from 1,766 to 1,519 cm(-1), characteristic for lactam ring were completely removed during the cefdinir degradation. The opening of the ß-lactam ring was one of the major steps in the cefdinir degradation process. Based on the results from the present study, a possible pathway of cefdinir degradation by Ustilago sp. SMN03 was proposed. To the best of our knowledge, this is the first report on microbial degradation of cefdinir by yeast.

Isolation and screening of glycolipid biosurfactant producers from sugarcane.

Forty-three fungal producers for glycolipid biosurfactants, mannosylerythritol lipids (MELs), were isolated from leaves and smuts of sugarcane plants. These isolates produced MELs with sugarcane juice as nutrient source. The strains were taxonomically categorized into the genera Pseudozyma and Ustilago on the basis of partial sequences of the ribosomal RNA gene.

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa.

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O-glycosidically linked to 3,15,16-trihydroxypalmitic acid. The sugar moiety is acylated with 2-hydroxy-octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl-transferase and is lacking a gene homologous to the α-hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl-transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl-transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.

Morphological and molecular differences in two strains of Ustilago esculenta.

Ustilago esculenta is a fungal endophyte of Zizania latifolia that plays an important agricultural role in this vegetable crop. The purpose of this study was to characterize sporidial (T) and mycelial (M-T) strains of U. esculenta isolated from sporulating and non-sporulating galls on plants growing in Zhejiang province, China. Morphological comparisons of the T strain and M-T strain were made by optical and scanning electron microscope observation. Genetic differences were examined by sequencing the ITS region of the fungus and examining differential protein expression by two-dimensional gel electrophoresis and MALDI-TOF-MS/MS. The sporidial (T) and mycelial (M-T) strains differed in morphological characteristics of their in vitro single colony formations and in cell shape. Alignment of ITS sequences of the T strain and M-T strain revealed a single mutation between the T strain and M-T strain, but the sequences were the same within strains. A total of 146 proteins were only expressed in the M-T strain, and 242 proteins were only expressed in the T strain isolated from infected plants. A total of 222 proteins were up-regulated or down-regulated in the T strain when compared with the M-T strain. Of these, 18 proteins were identified and eight were associated with processes involving energy metabolism and the cytoskeleton. Two morphology-related proteins, MAP kinase kinase and actin, were differentially expressed. The differences noted in the T strain and M-T strain may lead to a better understanding of the life cycle and morphogenesis in U. esculenta.

Flamingomyces and Parvulago, new genera of marine smut fungi (Ustilaginomycotina).

Teliospore walls, teliospore germinations, hyphal septations, cellular interactions, and nucleotide sequences from the D1/D2 region of the nuLSU rRNA gene of the marine smut fungi Melanotaenium ruppiae and Ustilago marina were examined and compared with findings in other Ustilaginomycotina. The data show that Melanotaenium ruppiae belongs to the Urocystaceae and Ustilago marina to the Ustilaginaceae. Within the Urocystaceae, Melanotaenium ruppiae is morphologically similar to Melanustilospora and Vankya. However, according to the molecular results Melanotaenium ruppiae can neither be ascribed to Melanustilospora nor to Vankya. Therefore, the new genus Flamingomyces is proposed for Melanotaenium ruppiae. Ustilago marina differs from the other Ustilaginaceae in the mode of sporulation, which exclusively occurs at the base of the host plant culms. Accordingly, the new genus Parvulago is proposed for Ustilago marina.

The vtc4 gene influences polyphosphate storage, morphogenesis, and virulence in the maize pathogen Ustilago maydis.

The maize pathogen Ustilago maydis switches from budding to filamentous, dikaryotic growth in response to environmental signals including nutrient status, growth in the host, and the presence of mating pheromones. The filamentous dikaryon is capable of proliferating within host tissue to cause disease symptoms including tumors. The transition from yeast cells to hyphal filaments is regulated by a mitogen-activated protein kinase cascade and a cyclic-AMP-protein kinase A (PKA) pathway. Serial analysis of gene expression with PKA mutants identified orthologs of components of the PHO phosphate acquisition pathway as transcriptional targets of the PKA pathway, and these included genes for Pho84, an acid phosphatase, and the vacuolar transport chaperones Vtc1 and Vtc4. In Saccharomyces cerevisiae, Vtc4p is required during the fusion of inorganic-phosphate-containing vesicles to the vacuolar membrane and the consequent accumulation of phosphate stored as polyphosphate (polyP) in the vacuole. We found that deletion of vtc4 in U. maydis also reduced polyP stored in vacuoles. Intriguingly, Deltavtc4 mutants possessed a filamentous cellular morphology, in contrast to the budding, yeast-like growth of the wild-type parent. The Deltavtc4 mutants also displayed decreased symptom development and reduced proliferation in planta. The interaction with PKA signaling was further investigated by the generation of Deltavtc4 ubc1 double mutants. Deletion of vtc4 completely suppressed the multiple-budded phenotype of a Deltaubc1 mutant, indicating that polyP stores are essential for this PKA-induced trait. Overall, this study reveals a novel role for PKA-regulated polyP accumulation in the control of fungal morphogenesis and virulence.

Molecular phylogeny of Ustilago, Sporisorium, and related taxa based on combined analyses of rDNA sequences.

Combined analyses of ITS and LSU rDNA sequences were utilized to resolve the phylogenetic relationships of 98 members of the smut genera Lundquistia, Melanopsichium, Moesziomyces, Macalpinomyces, Sporisorium, and Ustilago (Basidiomycota: Ustilaginales). Minimum Evolution and Bayesian inference of phylogeny resolve three major groups of almost identical composition: Sporisorium, Ustilago, and a basal assemblage of both Ustilago and Sporisorium species. Macalpinomyces deserves generic rank regarding its type species M. eriachnes; all other Macalpinomyces species of our study clearly turn out to be part of Ustilago or Sporisorium. Lundquistia evidently belongs to Sporisorium. Moesziomyces, probably paraphyletic, stands basal to all other genera. Interestingly, Melanopsichium belongs to the Ustilago clade, being the only member of the ingroup not parasitizing on Poaceae. The patchy distribution of commonly used morphological characters along our phylograms points to their variability and dependence on the host's morphological traits instead of being valuable for resolving parasite phylogeny. The new combination: Sporisorium fascicularis comb. nov. (syn. Lundquistia.fascicularis) is made.

The genus Anthracoidea (Basidiomycota, Ustilaginales): a molecular phylogenetic approach using LSU rDNA sequences.

The phylogenetic relationship of 52 specimens representing 30 species of Anthracoidea (Ustilaginales) was investigated by molecular analyses using sequence data from the large subunit (LSU) of nuclear rDNA. Phylogenetic trees were inferred with neighbour-joining (NJ), maximum parsimony (MP), and Bayesian Markov chain Monte Carlo (MCMC) methods. The results are discussed with respect to the species concept and the subdivision of the genus into subgenera and sections. Collections from different hosts and localities were compared. Our analyses can neither support nor significantly reject the hypothesis of the bipartition of the genus Anthracoidea. Thus, the representatives of the subgenus Proceres appeared in the NJ analysis as a moderately supported monophylum, whereas MCMC analysis revealed a polyphyletic topology for this group. Paraphyly of the subgenus Anthracoidea was supported by all methods used. Sections Echinosporae and Leiosporae were each represented by two species in our analyses which grouped together with high support. Section Anthracoidea should be restricted to a highly supported group with extremely irregular to angular teliospore shape. However, these three sections do not cover the whole diversity of the subgenus Anthracoidea. Molecular data largely supported the traditional circumscription of species, and species delimitations are discussed.

Random amplified polymorphic DNA analysis of Ustilago violacea.

Isolates of the two mating type strains of the basidiomycete phytopathogen Ustilago violacea (Pers.) Roussel [a.k.a Microbotryum violaceum (Pers.:Pers.) Deml and Oberw] are restricted in their host range to one or a few species of Caryophyllaceae (Pinks). Molecular genetics maps in this species are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross and more recently through electrophoretic karyotypes and chromosomal polymorphism using CHEF gel analysis. However, currently, polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains of almost any organism, which is useful in genetic mapping. The objective of this project was to use PCR technology, 40 Operon 10-mer primers, and 5 simple sequence repeat (SSR) primers, designed on microsatellite sequences to determine their efficiency in detecting intraspecific differences between the genomic DNA of the two mating type strains of U. violacea (a1 and a2). Polymorphism in the banding patterns of the DNA samples was detected after PCR and electrophoresis in 1.4% agarose gels. This polymorphic intraspecific variation will be utilized to detect cryptic and trans-active transposable elements in U. violacea.

Huitlacoche (Ustilago maydis) as a food source--biology, composition, and production.

Huitlacoche is the ethnic name applied to the young fruiting bodies (galls) of the fungus Ustilago maydis, which causes common smut of maize (Zea mays L). Biologists and agronomists have historically used U. maydis as a model to study a wide array of genetic, physiological, ecological, and phytopathological phenomena. In Mexico and other Latin American countries, huitlacoche has been used traditionally as human food, being highly regarded as an interesting dish or condiment. The food potential of huitlacoche is described here in terms of its chemical composition, which includes carbohydrates, proteins, fats, vitamins, and minerals. In addition, essential amino acids (especially lysine) and fatty acids (linoleate) are present in huitlacoche in considerable levels, adding to its nutritional attributes. The feasibility of growing U. maydis in submerged agitated culture has yielded a variety of fermentation products, including essential amino acids, proteins, vitamins, and flavorings, among others. Recent interest in developing huitlacoche as a cash crop has come from increasing acceptance by the North American public, who prize it as a new delicacy. However, research efforts are still needed to determine the biological factors involved in the establishment of U. maydis as a pathogen on the maize plant. This review deals with the role of huitlacoche as a food source, implicating the biological components that will determine the development of technologies for large scale production.

Gunacin, a new quinone antibiotic from Ustilago sp.

In a screening program for antibiotics which were antagonized by cysteine, a strain, which was characterized as Ustilago sp., was found to produce a new quinone antibiotic, gunacin. The molecular weight M+ = 348.084 determined by mass spectroscopy, corresponds to a molecular formula of C17H16O8. Further spectroscopic data prove that gunacin is a new antibiotic. The antibiotic possesses a good inhibitory effect against mycoplasmas and Gram-positive bacteria including multi-resistant strains. It also possesses a weak activity against Gram-negative bacteria with the exception of Proteus vulgaris, which is more strongly inhibited. The main activity against fungi is found against Trichophyton mentagrophytes. Gunacin shows an inhibition of the DNA synthesis in vivo, is antagonized by mercapto compounds and possesses an acute toxicity of LD50 = 16 mg/kg i.p. and LD50 = 12 mg/kg i.v. in mice. Against HeLa-cell the antibiotic shows an ED50 = 12.11 microgram/ml. Thirty five microgram/ml of gunacin induces 1,063 interferon units.