Akihiko Tsuneda

Phylogenetic diversity in shiitake inferred from nuclear ribosomal DNA sequences

Phylogenetic relationships of the edible shi? itake mushroom (Lentinula, Tricholomataceae) were studied using DNA sequences from the internal tran? scribed spacers (ITS) of nuclear ribosomal DNA. The ingroup consisted of seven isolates of L. edodes from Japan and Thailand, nine isolates of L. lateritia from Borneo, Papua New Guinea, and Tasmania, and five isolates of L. novaezelandieae from New Zealand. These species designations are based on morphological spe? cies concepts in Lentinula. However, because Lentinula isolates from throughout Asia-Australasia are mating compatible, some authors treat all of these as belong- ing to L. edodes. The outgroup included two isolates of L. boryana from Mexico. Parsimony, distance, and maximum likelihood analyses were performed, with various combinations of taxa, characters, and char? acter codings, and bootstrap and decay index mea- sures of robustness. Alternate topologies were evalu? ated in terms of tree lengths, maximum likelihood ratios, and Templetons nonparametric test of parsi? mony. Results suggest that there are four independent lineages of shiitake in Asia-Australasia, which provides partial support for the morphologically based species concepts. Lentinula novaezelandieae and L. edodes sensu

Comparative Morphology and Phylogenetic Placement of Two Microsclerotial Black Fungi from Sphagnum

Capnobotryella renispora and Scleroconidioma sphagnicola form black, irregularly shaped microsclerotia that are indistinguishable in gross morphology on leaves of Sphagnum fuscum. In culture, microsclerotia of these fungi were similar, in that mature component cells possessed thick, highly melanized cell walls, poorly defined organelles, large lipid bodies and simple septa. They were different in morphogenesis, in the way their component cells were organized and in disseminative propagules. Microsclerotia of S. sphagnicola formed phialidic conidiogenous cells on their surface, whereas in C. renispora, adjacent cells in mature microsclerotia often separated from each other by septum schizolysis and formed chlamydospores. The identification of C. renispora from Sphagnum is provisional despite a 100% ITS sequence match with data for a culture derived from the type strain. No holoblastic, reniform conidia typical of the species were formed in nature or in culture, and the SSU sequence for a separately preserved culture of the ex-type strain was markedly divergent. Parsimony analyses of nuclear ribosomal DNA sequences showed that these two fungi were related to separate orders of Dothideomycetes. Both SSU and ITS data supported a close relationship for S. sphagnicola to the Dothideales sensu stricto, while the closest ITS match was to Rhizosphaera spp. In the SSU analyses, C. renispora was nested within the Capnodiales.

Occurrence of Pseudomonas tolaasii on fruiting bodies of Lentinula edodes formed on Quercus logs

A bacterial disease occurred on fruiting bodies ofLentinula edodes that formed outdoors onQuercus bedlogs during winter. The pathogen was identified asPseudomonas tolaasii based on morphological and bacteriological characteristics. Symptoms exhibited by infected fruiting bodies ranged from mild browning to severe necrotic cavities that characteristically developed in the cap tissue along the periphery of the attachment area to the stalk. The mode of symptom development was greatly influenced by the internal tissue structure of fruiting bodies. Multiplication of bacterial cells within the fruiting bodies was strictly intercellular and thus differed from previously reported bacterial disease ofL. edodes incited by an unidentified rod-shaped bacterium. The present strain ofP. tolaasii was capable of attacking theL. edodes mycelium in the inner bark and outer sapwood regions and caused lysis of heavily infected hyphae.

Pathogenesis of bryophyte hosts by the ascomycete Atradidymella muscivora.

Atradidymella muscivora (Pleosporales) is a bryophyte pathogen that infects the mosses Aulacomnium palustre, Hylocomium splendens, and Polytrichum juniperinum. Light and scanning electron microscopy and extracellular enzyme production were used to characterize the interactions between this fungus and its native hosts and the model host Funaria hygrometrica. Penetration was direct via hyphae or appressoria, and hosts responded by forming layered, darkly pigmented deposits at penetration sites, similar to the papillae formed by vascular plants in response to fungal infection. Infected hosts gradually became chlorotic as hyphae grew intracellularly, presumably killing host cells. Pycnidia of the Phoma anamorph (P. muscivora) and uniloculate pseudothecia were initiated as tightly packed masses of stromatic dematiaceous hyphae within a single host cell. Mature pycnidia and pseudothecia were erumpent. A new microniche among bryophilous fungi is described, whereby A. muscivora supplants the gemmae of Aul. palustre and exploits the normal nutrient-flow of the moss gametophyte. Atradidymella muscivora produced both cellulases and soluble polyphenolic oxidases, allowing it to also function as a saprobe and degrade the cell walls of bryophytes. The saprophytic and pathogenic abilities of A. muscivora suggest it may play a role in nutrient cycling, population dynamics, and small-scale disturbances in boreal ecosystems.

Morphology and phylogenetic placement of Endoconidioma, a new endoconidial genus from trembling aspen.

Endoconidioma populi gen. et sp. nov. is described from black subicula on twigs of trembling aspen, Populus tremuloides, in Alberta, Canada. Pycnidium-like conidiomata are produced on twigs and in culture, but, unlike pycnidia, conidiomata of E. populi have a closed peridium and a locule filled with conidiogenous cells that form conidia endogenously. These endoconidia are hyaline, unicellular and released by the dissolution of the peridial cell wall. In addition to endoconidia, mostly two-celled conidia that form blastically from undifferentiated hyphae occur often in culture but are observed only occasionally on Populus twigs. No coelomycetous taxa have been reported to produce endoconidia, and both the morphological features and DNA sequence data demonstrate that Endoconidioma is distinct from the previously established endoconidial genera. Parsimony analyses of portions of the nuclear ribosomal RNA gene (SSU and ITS) suggest that Endoconidioma is closely related phylogenetically to members of the Dothideales and allied anamorphs in Hormonema and Kabatina.

Cytology and genetics of a sporeless mutant of Lentinus edodes

A sporulation-deficient (sporeless) mutant in Lentinus edodes gave rise to basidiocarps visibly indistinguishable from those of wild-type dikaryons. In the basidia, karyogamy and meiosis occurred n...

Molecular genetic characterization of bacterial isolates causing brown blotch on cultivated mushrooms in Japan

The polymerase chain-reaction (PCR) was used to amplify 16S ribosomal DNA (16S rDNA) from bacteria, identified asPseudomonas tolaasii orP. fluorescens, causing brown blotch on cultivated mushrooms in Japan. PCR-amplified 16S rDNA was analyzed on the basis of nucleotide sequence and restriction fragment length polymorphisms (RFLP) to determine the specific identity of isolates. Banding patterns obtained through PCR using primers corresponding to repetitive extragenic palindromic sequences of enteric bacteria (REP-PCR) were used to determine the relatedness of conspecific isolates. AllP. tolaasii isolates and a mushroom pathogen identified asP. fluorescens had identical RFLP patterns and partial 16S sequences, and are considered conspecific. An isolate ofP. fluorescens from creamery wastes (IFO 3507) differed slightly from isolates ofP. tolaasii in both 16S sequence (0.8%) and RFLP patterns (d=0.08), and had almost entirely different REP-PCR bands (d=0.88–1.0). Phylogenetic analyses based on 16S sequences indicated thatP. tolaasii andP. fluorescens are close members ofPseudomonas sensu stricto. REP-PCR shows promise in characterizing isolates pathogenic on different mushroom crops. Two isolates ofP. tolaasii pathogenic onPleurotus ostreatus had identical banding patterns, but three isolates fromLentinula edodes showed the greatest diversity.

Endoconidium development and release in the hyphomycete Phaeotheca fissurella

On agar culture media, conidia of Phaeotheca fissurella did not germinate but enlarged to form small mother cells within four da. Division of the small mother cells occurred by development of multilayered, simple septa to form two to several daughter cells. Schizolysis ofthe cross walls resulted in separation ofthe daughter cells to form endoconidia which were subsequently released by rupture ofthe mother cell wall. After a 2-3-week incubation, colonies were elevated and consisted of numerous sclerotic cells. Active endosporulation was restricted to one to several cell layers from the exposed surface of these colonies where large mother cells containing many endoconidia were abundant. Release ofthe endoconidia from the larger mother cells often was by dissolution ofthe mother cell wall. The liberated endoconidia were uninucleate and contained well-defined mitochondria, ER, ribosomes, lipid bodies and vac? uoles.

Hymenophore development and evolution in Lentinus

Morphological evolution of the Lentinus hymenophore was investigated through scanning electron microscopic observations of development in cultured sporocarps of Lentinus tigrinus, L. crinitus, L. s...

The Peridial Development and Dehiscence Mechanism of Cryptendoxyla hypophloia, a Cleistothecial Ascomycete Isolated from the Bodies of Arthropods

Cryptendoxyla hypophloia is a rarely reported cleistothecial ascomycete that has a cephalothecoid peridium comprising six to eight plates that split apart at maturity to expose the ascospores. Three new isolates from the bodies of live‐trapped insects represent circumstantial evidence supporting an older hypothesis that this fungus is dispersed by arthropods and provided fresh material in which to examine the development of the ascocarp and its unusual active dehiscence mechanism. Vegetative hyphae envelop coiled gametangia to form the earliest stages of the development of the peridium and then accommodate the increasing volume of the expanding centrum tissues by branching and tip growth. Peridial plates are defined early in ascocarp ontogeny and at maturity consist of thick‐walled, radially arranged hyphae that have transverse wall thickenings. Cells between plates and underlying dehiscence lines are thin walled. On drying, the radially arranged hyphae of the outer peridial layer contract, rupturing the thin‐walled cells around the periphery of each plate along previously formed dehiscence lines. As plates flatten and evert, the ascocarp opens to expose the ascospores, ostensibly for pickup by arthropod carriers. Meristematic growth could not be confirmed in the developing ascocarps of C. hypophloia even though it has been implicated in the growth of the peridium in other cephalothecoid taxa.