Analysis of Cytology and Expression of Resistance Genes in Maize Infected with Sporisorium reilianum.

Head smut, caused by the fungus Sporisorium reilianum, is a devastating global disease of maize (Zea mays). In the present study, maize seedlings were artificially inoculated with compatible mating-type strains of S. reilianum by needle inoculation of mesocotyls (NIM) or by soaking inoculation of radicles (SIR). After NIM or SIR, Huangzao4 mesocotyls exhibited severe damage with brownish discoloration and necrosis, whereas Mo17 mesocotyls exhibited few lesions. Fluorescence and electron microscopy showed that S. reilianum infected maize within 0.5 day after SIR and mainly colonized the phloem. With longer incubation, the density of S. reilianum hyphae increased in the vascular bundles, concentrated mainly in the phloem. In Mo17, infected cells exhibited apoptosis-like features, and hyphae became sequestered within dead cells. In contrast, in Huangzao4, pathogen invasion resulted in autophagy that failed to prevent hyphal spreading. The growth of S. reilianum hyphae diminished at 6 days after inoculation when expression of the R genes ZmWAK and ZmNL peaked. Thus, 6 days after SIR inoculation might be an important time for inhibiting the progress of S. reilianum infection in maize. The results of this study will provide a basis for further analysis of the mechanisms of maize resistance to S. reilianum.

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.

Biotrophic interaction of Sporisorium scitamineum on a new host--Saccharum spontaneum.

Sporisorium scitamineum is a biotrophic smut fungus harbored inside the smut gall on the top internodal region of Saccharum spontaneum, a wild relative of sugarcane (Saccharum officinarum). The interactions of spined conidia of S. scitamineum with S. spontaneum were examined during the different stages of plant growth starting from the bud stage to the decaying stage. The spores in the soil from the polyetic inocula grew into confined epidermal cells of the buds and finally sporulated in the topmost internodal region. Hyphae invasion of the plant tissues were restricted to the point of infection. Culms of infected plants in late October sporulated, notably; hyphal sporulation produced shorter hyphal stolons. Remarkably, the nodal regions of infected plants had no spores and fragmented hyphae. On the basis of microscopic analyses, hyphae and spores were absent in all internodes above the ground till the topmost smut gall region. This result indicated that, S. scitamineum undergoes tissue-confined invasion of S. spontaneum. By associating culture medium method with polymerase chain reaction (PCR) on plant portions void of smut gall, S. scitamineum was not detected, indicating that colonization was not systemic. It was observed that the biotrophic interaction resulted in structural reorganization in the restricted region of infection forming erect cylindrical structure, in which the fungus was sandwiched between the central stalk and sheath, and possibly played a key role in preventing inflorescence. Comparatively, a significant difference in the rate of teliospores germination between reference Ustilago esculenta (26.6%, P<0.05) and S. scitamineum (62.9%, P<0.05) at 20° C was observed. This study also provides insights on the effect of different temperature regimes on the germination of S. scitamineum teliospores in vitro.

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.

Mannosylerythritol lipids secreted by phyllosphere yeast Pseudozyma antarctica is associated with its filamentous growth and propagation on plant surfaces.

The biological function of mannosylerythritol lipids (MELs) towards their producer, Pseudozyma antarctica, on plant surfaces was investigated. MEL-producing wild-type strain and its MEL production-defective mutant strain (ΔPaEMT1) were compared in terms of their phenotypic traits on the surface of plastic plates, onion peels, and fresh leaves of rice and wheat. While wild-type cells adhering on plastic surfaces and onion peels changed morphologically from single cells to elongated ones for a short period of about 4 h and 1 day, respectively, ΔPaEMT1 cells did not. Microscopic observation of both strains grown on plant leaf surfaces verified that the wild type colonized a significantly bigger area than that of ΔPaEMT1. However, when MELs were exogenously added to the mutant cells on plant surfaces, their colonized area became enlarged. High-performance liquid chromatography analysis revealed a secretion of higher amount of MELs in the cell suspension incubated with wheat leaf cuttings compared to that in the suspension without cuttings. Transcriptional analysis by real-time reverse transcriptase PCR verified that the expression of erythritol/mannose transferase gene and MELs transporter gene of P. antarctica increased in the cells inoculated onto wheat leaves at 4, 6, and 8 days of incubation, indicating a potential of P. antarctica to produce MELs on the leaves. These findings demonstrate that MELs produced by P. antarctica on plant surfaces could be expected to play a significant role in fungal morphological development and propagation on plant surfaces.

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica.

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast.

Solopathogenic strain formation strongly differs among Ustilaginaceae species.

The pathogenicity of smut fungi is initiated by the fusion of two compatible saprotrophic yeasts that give rise to the formation of dikaryotic pathogenic hyphae. It has been described in the literature that complementation assays of auxotrophic yeasts of Ustilago maydis have allowed the isolation of diploid strains that are solopathogenic, i.e. pathogenic in the absence of mating. The occurrence of such strains from germinating teliospores was not investigated. We evaluated the ability of teliospores to generate solopathogenic strains in three species of smut fungi: Sporisorium reilianum f.sp. zeae, U. maydis and Moesziomyces penicillariae. Using an approach based on the stability of pseudohyphae of solopathogenic strains, we isolated the strain SRZS1 from teliospores of S. reilianum. Microscopic observations and analyses of mating-type alleles showed that SRZS1 is monokaryotic and diploid. Inoculation tests on maize plantlets indicated that SRZS1 is infectious. The same protocol was applied to polyteliosporal isolates from M. penicillariae, U. maydis and S. reilianum of diverse geographic origin. Surprisingly, all strains from teliospores of M. penicillariae were solopathogenic, whereas only few solopathogenic strains were obtained from the other two species. The possible incidence of solopathogenic strain production in the biology of these species is discussed.

Recombinant protein secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a novel signal peptide.

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.

Anther smut fungi on monocots.

Teliospores, hyphal septa, cellular interactions, and nucleotide sequences from the ITS and LSU region of the rRNA gene of specimens of Ustilago vaillantii s. lat. on Muscari and Scilla species were examined and compared with findings in other Ustilaginomycotina. The data show that U. vaillantii s. lat. specimens belong to the Urocystales and represent the sister group of the Urocystaceae, standing well apart from Vankya heufleri and V. ornithogali. Within the Urocystales, U. vaillantii s. lat. is unique in sporulating in the anthers of the host plants. Accordingly, the new genus Antherospora is proposed for the anther smuts on Hyacinthaceae. In addition, our data show that there is a stringent phylogenetic correlation between the specimens of Antherospora and their respective hosts. Thus, the specimens on Scilla spp. as well as those on Muscari spp. form highly supported monophyla. Furthermore, on Scilla a phylogenetic dichotomy exists between the specimens infecting Scilla bifolia and those infecting S. vindobonensis, with the specimens of the two host species showing a difference of 17bp in the ITS nucleotide sequences. Therefore, A. vindobonensis is described as a new species, and A. scillae and A. vaillantii are proposed as new combinations. Consequently, because of their sporulation in anthers and their parasitism on species of other genera of the Hyacinthaceae, Ustilago albucae, U. peglerae, U. tourneuxii, and U. urgineae are also ascribed to Antherospora as new combinations. Descriptions are given for all species.

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa.

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.

Efficient production of mannosylerythritol lipids with high hydrophilicity by Pseudozyma hubeiensis KM-59.

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known because of their multifunctionality and biocompatibility. A previously isolated yeast strain, Pseudozyma sp. KM-59, mainly produced a hydrophilic MEL, namely MEL-C (4-O-[4'-O-acetyl-2',3'-di-O-alka(e)noyl-beta-D: -mannopyranosyl]-D: -erythritol). In this study, we taxonomically characterize the strain in detail and investigate the culture conditions. The genetic, morphological, and physiological characteristics of the strain coincided well with those of Pseudozyma hubeiensis. On batch culture for 4 days under optimal conditions, the yield of all MELs was 21.8 g/l; MEL-C comprised approximately 65% of the all MELs. Consequently, on fed-batch culture for 16 days, the yield reached 76.3 g/l; the volumetric productivity was approximately 4.8 g l(-1) day(-1). We further examined the surface-active and self-assembling properties of the hydrophilic MELs produced by the yeast strain. They showed higher emulsifying activities against soybean oil and a mixture of hydrocarbons (2-methylnaphtarene and hexadecane, 1:1) than the synthetic surfactants tested. On water penetration scans, they efficiently formed lyotropic liquid crystalline phases such as myelines and lamella (L alpha) in a broad range of their concentrations, indicating higher hydrophilicity than conventional MELs. More interestingly, there was little difference in the liquid crystal formation between the crude product and purified MEL-C. The present glycolipids with high hydrophilicity are thus very likely to have practical potential without further purification and to expand the application of MELs especially their use in washing detergents and oil-in-water-type emulsifiers.

Pseudozyma jejuensis sp. nov., a novel cutinolytic ustilaginomycetous yeast species that is able to degrade plastic waste.

An ustilaginomycetous anamorphic yeast, isolated from orange leaves on Jeju island in South Korea, represents a novel Pseudozyma species according to morphologic and physiologic findings and molecular taxonomic analysis using the D1/D2 domains of the large subunit (26S) rRNA gene and the internally transcribed spacer (ITS) 1+2 regions. The name Pseudozyma jejuensis sp. nov. is proposed for this novel species, with OL71(T) (=KCTC 17482(T)=CBS 10454(T)) as type strain. In the present study, we have also demonstrated that Pseudozyma jejuensis OL71 is capable of producing cutinase and degrading polycaprolactone. These results suggest that Pseudozyma jejuensis or its cutinase may be useful for the biological degradation of plastic waste.

Molecular signaling in pathogenicity and host recognition in smut fungi taking Karnal bunt as a model system.

Karnal bunt of wheat, incited by a phytopathogen Tilletia indica (Syn. Neovossia indica) is a floret infecting disease. In the floral tissues fungus proliferates and produces massive amount of black spores. In smut fungi, belonging to order Ustilaginales, communication between cells is necessary to regulate growth, differentiation and monokaryotic to dikaryotic transition during pathogenic and sexual development. Neighbouring cells are able to communicate with each other by direct cell to cell contact through plasma membrane bound signaling molecules or through formation of gap junctions and alternatively through secretion of chemical signals if cells are some distance away. Current research efforts toward understanding of pathogenic and sexual development in phytopathogenic fungi, offer a number of opportunities. These include the analysis of molecular signal(s) for direct contribution of sexual interactions to ability of smut and bunt pathogens to cause disease. These efforts will provide not only to explore the mechanisms of pathogenesis, but also to enhance knowledge of basic cellular biology of an economically important group of fungi.

Significance of ribosomal ribonucleic acid synthesis for control of the G1 period in the cell cycle of the heterobasidiomycetous yeast Rhodosporidium toruloides.

A cell cycle mutant strain which is defective in the G1 period, B2-39, was selected from 1,200 temperature-sensitive mutants of the heterobasidiomycetous yeast Rhodosporidium toruloides M-1057. In the mutant cells, ribosomal ribonucleic acid synthesis was initially inhibited upon temperature shift-up from a permissive (25 degrees C) to a restrictive (36 degrees C) temperature. Moreover, the mutant was found to be temperature sensitive in deoxyribonucleic acid-dependent ribonucleic acid polymerase I activity in vitro. In a revertant-mutant strain, B2-39-R-2, both ribosomal ribonucleic acid synthesis in vivo and enzyme activity in vitro were simultaneously recovered. These results indicate that the mutant has a temperature-sensitive, deoxyribonucleic acid-dependent ribonucleic acid polymerase I and suggest that ribosomal ribonucleic acid synthesis acts as one of the control factors for initiation of both deoxyribonucleic acid synthesis and bud emergence.

Morphogenic effect of 2-deoxy-D-arabino-hexose on Rhodosporidium toruloides.

The presence of 2-deoxy-D-arabino-hexose in the growth medium caused marked morphological changes in the cells of Rhodosporidium toruloides. The originally elongated ellipsoidal cells grew spherically in the presence of the deoxy-sugar, displayed differences in cell division and separation, and were larger than the control cells. After exhaustion of glucose from the medium the cells died, although no lysis was observed. The morphological changes were accompanied by significant alterations in the carbohydrate composition of the cell wall. The wall of R. toruloides grown in the presence of the deoxy-sugar contains higher proportions of chitin and glucan, while the relative contents of mannose and galactose polymers decreased drastically in comparison to normal cells.

Morphological change in the early stages of the mating process of Rhodosporidium toruloides.

The events which occur in the early stages of the mating process of the yeast Rhodosporidium toruloides between strains M-919 (mating type A) and M-1057 (mating type a) were investigated. In preliminary experiments we determined the frequency of mating by two newly designed methods: the liquid culture method and the membrane-filter microculture method. The mating frequencies of strains M-919 and M-1057 were 89% in the liquid culture method and 62% in the membrane-filter microculture method. The early stages in the mating process included the following events: (i) M-919 cells produce constitutively the extracellular inducing substance (A factor), (ii) M-1057 cells receive A factor, and in response to it they form mating tubes and secrete another inducing substance (a factor), (iii) M-919 cells receive a factor, and in response to it they form mating tubes, (iv) mating tubes elongate to the cells or the tubes of mating partner, (v) tips of the growing tubes recognize the opposite mating type cells or their tubes, followed by cell-to-cell fusion.