Phylogenetic characterization of two novel <Emphasis Type="Italic">Kamalomyces</Emphasis> species in Tubeufiaceae (Tubeufiales)

Two novel species of bambusicolous fungi in the genus Kamalomyces, collected from northern Thailand, are described and illustrated herein. Kamalomyces bambusicola and K. thailandicus spp. nov. are typical of the genus Kamalomyces (Tubeufiaceae, Tubeufiales) and are morphologically distinct from known species with respect to their size of ascomata, asci and ascospores, ascospore septation and peridium structure, including the subiculum comprising hyphae on the host surface. Morphological examination reveals that the asexual morph of K. bambusicola is associated with its sexual morph in a subiculum forming dictyochlamydosporous conidia, which are similar to the asexual morph of Chlamydotubeufia. Phylogenetic analyses of combined LSU, ITS and TEF1-α sequence data also support these two species as distinct and confirm their phylogenetic affinities within the Tubeufiaceae. In particular, Kamalomyces shares a close phylogenetic relationship to Helicoma.     

Fungal cannons: explosive spore discharge in the Ascomycota

The ascomycetous fungi produce prodigious amounts of spores through both asexual and sexual reproduction. Their sexual spores (ascospores) develop within tubular sacs called asci that act as small water cannons and expel the spores into the air. Dispersal of spores by forcible discharge is important for dissemination of many fungal plant diseases and for the dispersal of many saprophytic fungi. The mechanism has long been thought to be driven by turgor pressure within the extending ascus; however, relatively little genetic and physiological work has been carried out on the mechanism. Recent studies have measured the pressures within the ascus and quantified the components of the ascus epiplasmic fluid that contribute to the osmotic potential. Few species have been examined in detail, but the results indicate diversity in ascus function that reflects ascus size, fruiting body type, and the niche of the particular species.     

Four new species and a new Chinese record of the nectrioid fungi

Four new species belonging to Bionectria, Calonectria, Haematonectria and Neonectria on plant substrates collected from nature reserves in southern and central China are described. Bionectria truncata has smooth perithecia of a flattened to shallow discoid apex, clavate asci with an apical ring, and ellipsoid, smooth to spinulose ascospores. Calonectria dicephalospora is characterized by pyriform perithecia with a warted surface, clavate asci with a simple apex and long, narrow stalk, and fusoid ascospores with a cap-like appendage at each end. Haematonectria lushanensis possesses warted perithecia which are laterally collapsing when dry, cylindrical asci with a simple apex, and ellipsoid, spinulose ascospores. Neonectria dinghushanica is distinguishable by subglobose perithecia with a warted surface, clavate asci, and striate ascospores. Morphological features of these new species are described comprehensively and compared with their related fungi. Neonectria castaneicola is recorded as new to China.     

Two new species of powdery mildew fungi from Japan

The following two new species of powdery mildew fungi were found onStephania cephalantha (Menispermaceae) andViburnum opulus var.calvescens (Caprifoliaceae) in Japan:

1. Erysiphe stephaniae onS. cephalantha has smaller cleistothecia and more asci present in the cleistothecium, which include remarkably larger ascospores, thanErysiphe pisi, which has been previously described on the same host plant from Taiwan.
2. Microsphaera shinanoensis onV. opulus var.calvescens is easily distinguishable fromMicrosphaera alni, hitherto known on this plant, and otherMicrosphaera fungi onViburnum plants by the shape of its appendages and the numbers of its asci and ascospores.

     

Genetic control of fungal differentiation: The three sporulation pathways of Neurospora crassa

Sporulation in the mold Neurospora crussa can proceed along three very different pathways, leading to the production of three types of spores. Two asexual sporulation pathways that lead to the formation of macroconidia and microconidia involve budding from hyphae by two different mechanisms. A much more complex sexual reproductive pathway involves the formation of a fruiting body called a perithecium, in which meiosis takes place and ascospores are formed in sac-like cells called asci. Numerous mutations exist that affect these developmental pathways, and genes have been isolated that are expressed preferentially during sporulation. The Neurospora sporulation pathways offer a simple system with which to study mechanisms and regulation of development that are usually obscured by complex cell-cell interactions involved in animal and plant development.     

A Novel Gene,ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.Gibberella zeae (anamorph: Fusarium graminearum) causes Fusarium head blight in wheat, barley, and rice, as well as ear rot and stalk rot in maize (20, 23). The infected grains are frequently contaminated by mycotoxins, such as trichothecenes and zearalenone, which are harmful to humans and animals (6). The fungus overwinters in crop debris in the form of storage hyphae and develops ephemeral fruiting bodies (perithecia) at warmer temperatures. Ascospores formed within the perithecia are forcibly discharged into the air and are believed to serve as the primary inoculum of the disease (7, 27, 37, 39,–42). Therefore, sexual development and ascospore discharge are important factors in fungal survival and disease initiation.In fungi of the phylum Ascomycota, the sexual cycle is initiated when two genetically distinct nuclei combine to form a binucleate cell (31). As a homothallic fungus, G. zeae possesses the two mating type genes MAT1-1 and MAT1-2 in the haploid genome and therefore does not require a mating partner for sexual development (22, 46). Perithecium initials give rise to small, coiled initials that develop into perithecia filled with asci, tubular sacs of ascospores, which are the products of meiosis. Mature asci extend through the ostiole of perithecia and discharge their ascospores (40).Unique features of cell differentiation are involved in ascus and ascospore morphogenesis. Ascospore delimitation within the ascus and the development of a cell wall between the ascus and ascospore membranes are unique features of the process (31). Most studies of morphogenesis have described these changes in detail; however, much of these data have been limited to microscopic observations. Several genes involved in ascospore morphogenesis have been identified in Neurospora crassa (30), but the detailed mechanisms and genes involved in ascus and ascospore morphogenesis remain to be elucidated. The Round spore (R) mutant of N. crassa was shown to have round ascospores (24), and the gene responsible for this phenotype, rsp, was subsequently cloned (28). However, in G. zeae, no genes have been identified that are involved in ascus and ascospore morphogenesis.Although recent research has shed light on the physiological basis of ascospore discharge, the genetic basis remains largely unknown (38). The main force responsible for the observed shooting is turgor pressure within the extended asci. In G. zeae, a buildup of K⁺ and Cl⁻ ions drives the influx of water and causes turgor pressure that stretches the asci (41). Asci can accumulate polyols as well as ions. In a previous study, it was shown that the polyols are comprised mainly of mannitol and glucose; however, the concentration of these polyols is too low to make a significant contribution to turgor pressure (42). When the turgor pressure exceeds the threshold of the asci, apical pores rupture and ascospores are forcibly discharged (38). Trail et al. (41) estimated that the acceleration of ascospores in G. zeae is 8,500,000 m s⁻² using an iterative model to predict initial velocity. Recently, Yafetto et al. (44) used high-speed video photography to examine several large-spore fungi, including Ascobolus immerses, and to predict acceleration during dispersal. The asci of A. immerses are more than 12-fold larger in diameter than the asci of G. zeae (38). The size difference between these fungi greatly affects the behavior of their projectiles and results in an initial speed for G. zeae that is too great for application of the video photography method (for further discussion, see the supplemental material).To date, only one gene from G. zeae, the calcium ion channel gene cch1, has been shown to be involved in ascospore discharge (12). Deletion of this gene was shown to arrest ascospore discharge without affecting spore and ascus morphology. Since the genomic sequence of G. zeae is now available, the functional analysis of genes involved in sexual development has been accelerated. Random insertional mutagenesis is one strategy that has been used to identify novel genes associated with sexual development (13, 34). Previously, we produced a collection of more than 20,000 mutants from G. zeae by using the restriction enzyme-mediated integration (REMI) transformation procedure (13). In this study, the G. zeae mutant Z43R9901, which was isolated from a screening of REMI transformants, showed an unusual phenotype during sexual development. Further analysis demonstrated that the novel gene ROA is involved in ascospore morphogenesis and discharge in G. zeae. The results of this study increase our understanding of sexual development in the fungus.     

<Emphasis Type="Italic">Neurospora tetrasperma</Emphasis> crosses heterozygous for hybrid translocation strains produce rare eight-spored asci-bearing heterokaryotic ascospores

During ascogenesis in Neurospora, the ascospores are partitioned at the eight-nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previously created novel T ^Nt strains by introgressing four Neurospora crassa insertional translocations (EB4, IBj5, UK14-1, and B362i) into N. tetrasperma. We now show that crosses of all the T ^Nt strains with single-mating-type derivatives of the standard N. tetrasperma pseudohomothallic strain 85 (viz. T ^Nt a × 85A or T ^Nt A × 85a) can produce rare eight-spored asci that contain heterokaryotic ascospores, or ascospores with other unexpected genotypes. Our results suggest that these rare asci result from the interposition of additional mitoses between the post-meiotic mitosis and the partitioning of nuclei into ascospores, leading to the formation of supernumerary nuclei that then generate the heterokaryotic ascospores. The rare asci probably represent a background level of ascus dysgenesis wherein the partitioning of ascospores becomes uncoupled from the post-meiotic mitosis. Ordinarily, the severest effect of such dysgenesis, the production of mating-type heterokaryons, would be suppressed by the N. crassa tol (tolerant) gene, thus explaining why such dysgenesis remained undetected thus far.     

圆盘菌科无性型有性生殖能力评估

本文评估了分离自子囊孢子和分生孢子的圆盘菌科无性型有性生殖能力。结果表明,由子囊孢子和分生孢子萌发获得的菌株的有性生殖能力具有显著差异,这可能决定于不同的遗传特性。该试验支持了一种假说：仅能进行无性生殖的无性型菌株,很可能来源于逐渐丧失了有性生殖能力的全型菌株。     

New species and new Chinese records of Bionectriaceae (Hypocreales,Ascomycota)

Collections of bionectriaceous fungi from different areas of China were examined, in which 3 new species were encountered. Bionectria intermedia is characterized by smooth perithecia, 2-layered perithecial wall, cylindrical to clavate asci with an apical ring, ascospore striations composed of separate warts, and dimorphic conidiophores. Hydropisphaera yunnanensis has hairy ascomata which are cupulate when dry, clavate asci with a simple apex, and spinulose and very narrow ascospores fusiform and constricted at septum. Nectriopsis apiosporae possesses laterally pinched perithecia when dry, 1-layered perithecial wall, rough perithecial surface, clavate asci with a simple apex, spinulose ascospores with 3 septa, and on Apiospora sp. Four species, Bionectria epichloë, B. kowhaii, B. subquaternata and Hydropisphaera suffulta, are reported as new to China.     

Isolation ofGlomerella musae [teleomorph ofColletotrichum musae (Berk. & Curt.) Arx.] and segregation analysis of ascospore progeny

The sexual stage ofColletotrichum musae has been obtained under controlled laboratory conditions. Development of the sexual stage required light and suboptimal growth temperature. Meiosis was apparent as judged by the formation of perithecia typical of ascomycetes classified asGlomerella. Perithecia contained as many as 50–80 asci, each of which contained up to eight ascospores. The ascospores were larger than conidia and were easily dissected for segregation analysis of either random spores or tetrads. In order to determine if this meiotic system was homothallic or heterothallic, chlorate-resistant mutants were isolated and crossed with wild type. Chlorate resistance and susceptibility segregated in a 1:1 ratio, indicating that this system was heterothallic. Sexual recombination was observed by analyzing the segregation of several DNA markers identified by amplification of polymorphic fragments of genomic DNA using single oligonucleotide primers andTaq DNA polymerase. Segregation consistent with mendelian inheritance and sexual recombination of genetic markers indicated that it is feasible to construct a genetic map for this species. The complexity of sexual compatibility was analyzed by crossing several ascospore progeny which identified four distinct mating types.     

Two new lineages of aquatic ascomycetes: <Emphasis Type="Italic">Atractospora</Emphasis> gen. nov. and <Emphasis Type="Italic">Rubellisphaeria</Emphasis> gen. et sp. nov., and a sexual morph of <Emphasis Type="Italic">Myrmecridium montsegurinum</Emphasis> sp. nov.

Two new genera, Atractospora and Rubellisphaeria, are described for perithecial ascomycetes occurring on decaying wood submerged in freshwater habitats. Their relationships with other morphologically similar fungi were investigated utilising four nuclear ribosomal and protein-coding loci. They are nested together with members of five orders and families and numerous genera incertae sedis in the Sordariomycetidae in a clade supported by Maximum Likelihood and Bayesian inference methods. Their closest relative is Lentomitella. Atractospora includes species characterised by dark, immersed to semi-immersed beaked ascomata lying horizontally to the host, stipitate asci with a pronounced non-amyloid apical annulus and hyaline, fusiform, septate, thick-walled ascospores with smooth or ornamented wall. Three novel species are introduced (A. decumbens, A. reticulata and A. verruculosa). A new combination for Aquaticola ellipsoidea is proposed in Atractospora based on molecular data and morphological characters. Rubellisphaeria, with a new species R. abscondita, is distinct by subhyaline to reddish brown, immersed ascomata with a lateral neck, short-stipitate asci and ellipsoidal smooth ascospores with a delayed formation of a middle septum. The sexual morph of the dematiaceous hyphomycete genus Myrmecridium was encountered for the first time on decaying wood submerged in a stream. It is nested in a strongly supported monophyletic clade of the Myrmecridiales. The novel species M. montsegurinum is described; it possesses papillate ascomata immersed beneath a clypeus with subhyaline to weakly pigmented ascomatal wall, long-stipitate asci with a non-amyloid apical annulus and three-septate, hyaline, ellipsoidal, delicately verruculose ascospores. A morphology-based key is provided to facilitate identification of species of Atractospora.     

A new species of <Emphasis Type="Italic">Cosmospora</Emphasis> and the first record of sexual state of <Emphasis Type="Italic">C. lavitskiae</Emphasis>

A new fungicolous species, Cosmospora inonoticola, is described and illustrated based on a specimen collected from northeast China. It is characterized by the occurrence of fruitbodies of Inonotus sp.; perithecia nonstromatic, subglobose to pyriform, not collapsing when dry; clavate asci containing (3–)6(?8) spores; ascospores ellipsoidal to broadly ellipsoidal, pale brown, finely warted, bicellular, conspicuously constricted at septum; and producing a verticillium-like asexual state. A multi-locus analysis of the combined ITS, rpb1 and tub sequences confirmed its taxonomic position, and revealed the distinction from its morphologically similar species including the type species, C. coccinea, which occurs also on Inonotus. A specimen with sexual state of C. lavitskiae is reported for the first time, and a reference specimen is indicated.     

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora Crassa

In previous work, the asd-1 (ascus development) gene of the filamentous fungus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-1 gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-1 protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-1 crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-1 asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-1 protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

New species of <Emphasis Type="Italic">Clohiesia</Emphasis> and <Emphasis Type="Italic">Paraniesslia</Emphasis> collected from freshwater habitats in China

Two fungi collected from submerged woody debris were found to represent hitherto undescribed species of the ascomycete genera Clohiesia and Paraniesslia. They are described as Clohiesia curvispora sp. nov. and Paraniesslia aquatica sp. nov. based on morphological characters. Clohiesia curvispora is characterized by immersed ascomata under a clypeus, and unitunicate, cylindrical asci containing one-celled, curved, elongate-fusiform ascospores. Paraniesslia aquatica is characterized by small, superficial, setose ascomata, and unitunicate, clavate asci containing verrucose, brown ascospores. Each species is illustrated with light micrographs and compared with similar taxa in this article.     

Four new species of the genus Hymenoscyphus (fungi) based on morphology and molecular data

Four new species of Hymenoscyphus (H. brevicellulus, H. hyaloexcipulus, H. microcaudatus, and H. subsymmetricus) and a new Chinese record (H. subpallescens) are described. These five species share common characteristics: small apothecia (<3mm in diameter); hymenium whitish, pale yellow, to yellow in color; ectal excipulum of textura prismatica; asci arising from simple septa; ascospores scutuloid and guttulate; saprophytic nutrition; and leaf habitats, except for H. subsymmetricus, which grows on herbaceous stems. Phylogenetic analyses of internal transcribed spacer nuclear ribosomal DNA sequences, the universal DNA barcode for fungi, for 16 species in the genus indicated that these taxa were closely related to H. microserotinus, in accordance with their morphological features, but represented independent species. The distinguishing features of each new species from its relatives are discussed, and their phylogenetic relationships explored.