The causal agents of witches’ broom of bamboo plants in East Asia, Epichloë bambusae and E. sasae, were morphologically and phylogenetically examined. The phylogenetic studies were conducted using ITS 1, 2, and 5.8 S rDNA regions. Both Epichloë species produce Ephelis-type conidia in artificial medium and are phylogenetically situated in different clades from Epichloë and Parepichloë. Here, we propose a new genus Heteroepichloë for these two bambsicolous Epichloë species.

In the fourth report of the present series, five species of the genus Cercospora and allied genera were added to the Japanese fungus flora: Cercospora brunkii Ellis et Galloway, C. richardiaecola Atkinson, Pseudocercospora annonicola Hsieh et Goh, P. xenosyzygiicola Crous, and P. celosiarum (Kar et Mandal) Deighton. Three species names recorded in the early report were revised based on their nomenclature.

Exposure of Neurospora crassa cells to heat shock and oxidative stress results in the synthesis of several stress-related proteins, including a peroxidase. Northern blot analysis of total RNA revealed a heat-inducible (HI)-peroxidase transcript of ～10 kb, induced in response to heat shock and oxidative stress. The HI peroxidase was isolated from heat-shocked mycelium and purified to near homogeneity, and its properties were examined. Chromatography in size-exclusion matrices yielded an apparent molecular mass of ～116 kDa for the native enzyme, whereas the estimate obtained by SDS-PAGE was 90–95 kDa. Studies of substrate saturation kinetics were conducted using the purified enzyme with ABTS [2,2'-azino-bis (3- ethylbenzthiazole-6-sulfonic acid)] and H₂O₂ as substrates. The experimentally estimated K_m, V_max, and K_cat values for ABTS were ～36 μM, 5200 nmolmg^-1, and 8 s^-1, respectively, and those for H₂O₂ were 44 μM, 6640 nmolmg^-1, and 10 s^-1. O-dianisidine was a substrate for this enzyme, but guaiacol was not. HI peroxidase was found to be a glycoprotein, stable at temperatures up to 60°C.

Pholiota nameko is a wood-rotting edible mushroom that carries a bipolar A incompatibility factor gene. The linkage analysis of the multiple allelomorphic A factor gene demonstrated that sexual reproduction produced a monospore isolate carrying a new A factor gene in addition to two parental mating types of isolates. However, 10%-30% of the modified monospore isolates could not produce a dikaryon with both of the parental monokaryons by crossing. It is concluded that the bipolar A incompatibility factor gene of P. nameko is constituted of two functional subunits, Aα and Aβ, which might be successively located beside each other with an apparent genetic distance of 0.3 centi-Morgan between them on the same chromosome. Further, some monospore isolates that did not conjugate with both parental monokaryons could produce dikaryons with different monokaryotic stocks with either one of the parental mating types. This result suggests that the crossing capability of these isolates were essentially those for one of the mating types of the parental monokaryons, but that their function for mating activity was made partially by unequal crossing-over in the process of sexual recombination.

Haptoglossa spp. (Lagenidiales, Oomycetes) have been known to parasitize microscopic animals by means of a “gun” cell that shoots an infection cell, named the sporidium, into the body of the animal. A thallus grown from the sporidium changes into a zoosporangium at maturation to produce a number of zoospores that encyst after a swarming period, and the resulting cysts germinate to produce gun cells. In Haptoglossa zoospora, endoparasitic in nematodes, the cysts of primary zoospores that swam for about 5 min did not develop gun cells but produced secondary zoospores that swam for about 3 h. After encystment of the secondary zoospores, each secondary cyst germinated to produce a gun cell. In the present study, the secondary zoospores of the genus Haptoglossa could be recorded with a videocassette recorder for the first time. The videocassette recording also revealed the infection of a nematodes by H. zoospora and H. heterospora to be composed of two steps of injection of a sporidium by the gun cell, in which the gun cell came in contact with the cuticle of a nematode and produced a spherical adhesorium on the tip of the cell in 0.07–0.1 s in both species. The adhesorium was ～2 μm in H. zoospora and ～4 μm in H. heterospora. When the adhesorium inflated to full size, it shot the sporidium into the nematode’s body in 0.5-0.65 s and in 0.2-0.5 (or rarely 1.0) s in H. zoospora and H. heterospora, respectively. After shooting, the empty gun cell with an empty cyst case was separated from the cuticle immediately in both species.

A fungal strain, FOM-8108, that was isolated from sea sand was found to produce gentisylquinone and chlorogentisylquinone, inhibitors of neutral sphingomyelinase. The fungus grew well under normal conditions, in the darkness, or on various agar media, but typical morphological characteristics were not observed to determine its taxonomy. Therefore, culture conditions were studied extensively, resulting in formation of a number of pycnidia by the fungus on the media containing seawater or on natural substrates such as hydrangea leaves, gardenia leaves, and rice straw under natural light or near-ultraviolet radiation exposure. Eventually, from the morphological characteristics of pycnidia, conidiogenous cells, and conidia, strain FOM-8108 was considered to belong to the genus Phoma. Culture studies showed seawater in the fermentation medium was necessary for the strain to produce gentisylquinones. Particularly, a full production of chlorogentisylquinone, which has a chloride ion in the structure, was performed in the medium with higher concentration (75%–100%) of seawater.

Cordyceps owariensis f. viridescens forma nov. (Nom. Jap., Amami-yarinohosemitake) is described from an underground nymph of Platypleura kuroiwae collected in Amami-oshima Island, southwestern Japan. Cultural isolations were made from ascospores of the fresh material of this fungus, and consequently a new hyphomycete was developed as its anamorph. Nomuraea owariensis is described to accommodate this anamorphic state. This is the first report on the association of a Nomuraea anamorph with cicadicolous Cordyceps species.

Recovery of 66 fungus stock cultures including Oomycota, Zygomycota, Ascomycota, Basidiomycota, and mitosporic mycetes were examined after cryopreservation. Almost all the stock cultures remained viable when the mycelia that had grown over the sawdust medium containing 10% glycerol as the cryoprotectant (65% moisture content, W/W) were frozen rapidly at -85°C and then allow to thaw naturally at room temperature. Test stock cultures were preserved for more than 10 years by this preservation method without any programmed precooling and rapid thawing for their cryopreservation. Most of the test fungi could survive for 5 years in medium containing 10% glycerol even after alternate freezing and thawing at intervals of 6 months. When a strain of Flammulina velutipes was tested for mycelial growth rate and productivity of fruit-bodies after cryopreservation for 3 years, the fungus reproduced with its initial capability. These results demonstrate that the sawdust-freezing method using a cryoprotectant is expected to be a reliable and easy preservation method for fungus stock cultures.

Two species of Ophiostoma were isolated from four bark beetles (i.e., Cryphalus montatus, C. piceae, Dryocoetes hectographus, and Polygraphus proximus) infesting Abies veitchii and from their galleries in the wave regenerated forests in the central part of the main island (Honshu) of Japan. One of them is described here as a new species, Ophiostoma subalpinum, and the other is identified as O. davidsonii, newly reported in Japan. Ophiostoma subalpinum is characterized by short ostiolar hyphae, oblong or allantoid ascospores enclosed in a thin, hyaline sheath, and a Pesotum anamorph.

Aeciospore-surface structures of Phragmidium species parasitic on roses were investigated by scanning electron microscopy. Seven kinds of structures were distinguished according to gross shape of ornamentations and their distribution on the aeciospore wall. The seven different structures were categorized into three major types: echinulate, annulate, and verrucose. The echinulate type was further classified into five types and designated in a numerical series, echinulate types 1–5. Application of the aeciospore-surface structures in the classification of Phragmidium species is discussed.

To estimate the phylogenetic position of the strictly anamorphic genus Thysanophora among the class Ascomycetes sensu Kirk et al. and to examine the phylogenetic relationships among T. penicillioides and other Thysanophora species, 18S and 28S rDNA (D1 and D2 regions) sequences of 22 strains of four known and two unidentified Thysanophora species were determined and phylogenetically analyzed. The 18S rDNA analysis suggested that all Thysanophora species examined were members of Eurotiomycetidae, Eurotiales, Trichocomaceae. The 28S rDNA analysis indicated that these species were clustered together with Chromocleista, Eupenicillium, Geosmithia, and Penicillium assignable to three subgenera – Aspergilloides, Furcatum, and Penicillium. In the Eupenicillium lineage, a monophyly of T. penicillioides, T. longispora, T. taxi, T. canadensis, and T. cf. canadensis was supported by comparatively high bootstrap values. However, the ex-type strain and two strains of T. longispora isolated in Japan were of different phylogenetic positions. Thysanophora sp. was positioned at the base of the Thysanophora clade, although it was not supported by significant bootstrap values. From the results of this study, we consider that two anamorphic genera, Penicillium and Thysanophora, are clearly distinct in morphology but that they are not phylogenetically separable.

Three Cylindrosporella species on the leaves of betulaceous trees – C. carpini, C. coryli, and C. microsperma – were first reported from Japan. The genus Cylindrosporella is sometimes treated as congeneric with Asteroma; however, we considered these to differ based on conidial morphology following the concept of Arx. The genus Cylindrosporella is characterized by one-celled, filiform or fusiform, small conidia that are often curved, have hyaline, thin walls, and are produced from phialidic conidiogenous cells in subcuticular, flat acervuli. The three species are distinguished from each other on the basis of conidium size.

Specifically primed polymerase chain reaction (PCR) analysis was used for direct detection and identification of Rhizoctonia solani isolates belonging to AG 1 subgroups (IA, IB, and IC) and AG 2 subgroups (2-1 and 2-2). A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract PCR templates. PCR specific primer sets for each group were designed from sequences in the regions of the 28S ribosomal DNA of this fungus. The results of specifically primed PCR analysis showed that AG 1-IA, AG 1-IB, AG 1-IC, AG 2-1, and AG 2-2 primers sets contributed detection from the same AG isolates and could escape detection from different AG isolates at a high level of frequency. In this experiment, we suggested that our synthesized primer sets might provide a method for the direct detection and identification of AGs of R. solani.

Dark septate endophytic fungi (DSE) may have an important functional relationship with host plants, but these functions and the colonization process remain unknown. We made microscopic observations of the growth of an endophytic hyphomycete in Chinese cabbage roots to understand its colonization process. This hyphomycete was Heteroconium chaetospira, a suspected DSE. Three weeks post inoculation, some hyphae became irregularly lobed and formed microsclerotia within host epidermal cells of healthy plants. In stunted plants, hyphae formed closely packed masses of fungal cells within host epidermal cells, but conidiophores rarely broke through the cell walls to produce conidia.