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The enormous number of spores produced by fruiting bodies during cultivation of mushrooms can lead to allergic reactions of workers, reduction of commercial value, spread of mushroom disease, pollution of facilities, and depletion of genetic diversity in natural populations. A cultivar harboring a sporulation-deficient (sporeless) mutation would be very useful for preventing these problems, but sporeless commercial cultivars are very limited in usefulness because sporeless traits are often linked with traits that are unfavorable for commercial cultivation. Thus, identifying a causal gene of a sporeless phenotype not linked to the adverse traits in breeding and cultivation is crucial for the establishment of sporeless breeding using a strategy employing targeting induced local lesions in genomes (TILLING) in cultivated mushrooms. We used a Pleurotus pulmonarius (Fr.) Quél. sporeless strain to identify and characterize the single recessive gene controlling the mutation. The 3,853-bp stpp1 gene encodes a protein of 854 amino acids and belongs to the MutS homolog (MSH) family associated with mismatch repair in DNA synthesis or recombination in meiosis. Gene expression analysis of the fruiting body showed that this gene is strongly expressed in the gills. Phenotypic analysis of disruptants formed by gene targeting suggested a reproducible sporeless phenotype. Mutants deficient in a functional copy of this gene have no unfavorable traits for sporeless cultivar breeding, so this gene will be an extremely useful target for efficient and versatile sporeless breeding in P. pulmonarius and various other cultivated mushrooms.

The protective effect of autoclaved water extract from spent mushroom substrate (AWESMS) and autoclaved spent mushroom substrate (ASMS) of the edible mushrooms Lyophyllum decastes (hatakeshimeji) and Pleurotus eryngii (eringi) against fungal and bacterial diseases was investigated on cucumber plants. When the plants were treated with AWESMS by spraying the first true leaves and inoculated with the target pathogen 7 days later, AWESMS of hatakeshimeji significantly reduced powdery mildew by Podosphaera xanthii, angular leaf spot by Pseudomonas syringae pv. lachrymans, but not Corynespora leaf spot by Corynespora cassiicola and scab by Cladosporium cucumerinum. When the plants were grown in a mixture (1 : 2, v ⁄ v) of ASMS of hatakeshimeji and soil, a significant disease reduction was observed in powdery mildew, scab and angular leaf spot. The protective effect was also observed against anthracnose on plants treated with AWESMS or on plants grown in a mixture of ASMS of eringi (1 : 3, v ⁄ v). Our results indicated that AWESMS and ASMS, independently of the mushroom type, provide a protective effect against fungal and bacterial diseases. Therefore, SMS should be considered an easily available source of active compounds to protect plants from fungal and bacteria infections, helping alleviate the waste disposal problem in the mushroom industry and creating an environmentally friendly method to reduce plant pathogens.

In the cultivation of edible mushrooms, including Pleurotus pulmonarius (Fr.) Quel., the enormous number of spores produced by fruiting bodies can adversely affect mushroom growers’ health, mushroom cultivation facilities, and the genetic diversity of natural populations. In this study, we constructed a primary genetic linkage map and identified the locus associated with the sporulation-deficient (sporeless) mutation of P. pulmonarius using 150 progeny isolates derived from a cross between sporeless and wild-type isolates. Based on the segregation of 300 AFLP markers, two mating-type factors, and the sporeless trait, a linkage map was generated consisting of 12 linkage groups. The map covered a total genetic distance of 971 cM, with an average marker interval of 5.2 cM. The gene region responsible for the sporeless mutation was located in linkage group II including 40 AFLP markers and the A mating-type factor locus. Of these markers, the nearest marker to the sporeless locus was located 1.4 cM away. Construction of this P. pulmonarius genetic linkage map and identification of markers that are closely linked to the sporeless locus will facilitate marker-assisted selective breeding of a sporeless strain with economically important traits.

In the cultivation of edible mushrooms including Pleurotus pulmonarius, the enormous number of spores produced by fruiting bodies adversely affects the health of mushroom farmers, mushroom cultivation facilities and the genetic diversity of natural populations. A sporeless mutant of P. pulmonarius (TMIC-30058) has been introduced with a high potential for breeding sporeless cultivars to reduce these adverse effects. We report here the conversion of the amplified fragment length polymorphism (AFLP) marker, closely linked to a sporeless locus, to a sequence tagged site (STS) marker for use in marker-assisted selection (MAS). By mainly bulked segregant analysis-based AFLP (BSA-AFLP), a total of 18 markers were identified the linkage to the sporeless locus with genetic distances ranging from 0 (co-segregation) from 3.4 cM. Out of them, 2 AFLP markers could be converted into STS markers, SD192 and SD296, and their implementations in various genetic backgrounds were validated by the amplification test against 15 wild type isolates from different geographical origin. Furthermore, we could amplify these 2 STS markers’ fragments by using multiplex PCR reproducibly. These results indicated that 2 STS markers can be used in MAS targeted at the sporeless trait in P. pulmonarius breeding.

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny.

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.

B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes.

The Hyphozyma synanamorph of Eleuther- omyces subulatus was found to be the cause of black spot disease of Lentinula edodes fruiting bodies. Mul- tiplication of yeastlike cells and hyphae of Hyphozyma resulted in blackening and mild lysis of the infected host cap tissues where partially degraded skeletal mi- crofibrils of host cell walls were abundant. In general, host tissue lysis remained superficial; however in some cases, Hyphozyma caused severe lysis and formed deep cavities containing remnants of highly dissolved host cell walls and numerous yeastlike cells of the pathogen. Enzyme assays demonstrated the ability of Hyphozyma to produce both chitinase and (3-(1,3) glucanase enzymes, requirements for hyphal wall degradation. On agar, conidiogenesis is enter- oblastic in the Hyphozyma morph, whereas it is ap- parently holoblastic in the Eleutheromyces morph. No evidence of phialidic conidiogenesis was obtained.

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.

A large number of spores from fruiting bodies can lead to allergic reactions and other problems during the cultivation of edible mushrooms, including Pleurotus eryngii (DC.) Quél. A cultivar harboring a sporulation-deficient (sporeless) mutation would be useful for preventing these problems, but traditional breeding requires extensive time and labor. In this study, using a sporeless P. eryngii strain, we constructed a genetic linkage map to introduce a molecular breeding program like marker-assisted selection. Based on the segregation of 294 amplified fragment length polymorphism markers, two mating type factors, and the sporeless trait, the linkage map consisted of 11 linkage groups with a total length of 837.2 centimorgans (cM). The gene region responsible for the sporeless trait was located in linkage group IX with 32 amplified fragment length polymorphism markers and the B mating type factor. We also identified eight markers closely linked (within 1.2 cM) to the sporeless locus using bulked-segregant analysis-based amplified fragment length polymorphism. One such amplified fragment length polymorphism marker was converted into two sequence-tagged site markers, SD488-I and SD488-II. Using 14 wild isolates, sequence-tagged site analysis indicated the potential usefulness of the combination of two sequence-tagged site markers in cross-breeding of the sporeless strain. It also suggested that a map constructed for P. eryngii has adequate accuracy for marker-assisted selection.

We investigated nuclear behavior during basidiosporogenesis in Lentinw, Panus, and Neolenti- nus in an attempt to discover characters for taxonomic segregation of lentinoid-pleurotoid fungi. In Lentinus tigrinus and Panus lecomtei somatic hyphae are binu- cleate. Karyogamy and meiosis occur in the basidia, meiotic products migrate into spores and postmeiotic mitosis occurs; this is followed by back-migration of one nucleus from each spore into the basidium. Ma- ture spores are consequently uninucleate and dis- charged basidia are quadrinucleate. In Neolentinus le- pideus many somatic cells are multinucleate. Karyogamy, meiosis, and postmeiotic mitosis occur as in L. tigrinus and P. lecomtei, but there is no back- migration, mature spores are binucleate, and dis- charged basidia are anucleate. The patterns of nuclear behavior in L. tigrinus and P. lecomtei correspond to type C nuclear behavior as defined by Duncan and Galbraith, whereas that of Neolentinus corresponds to type D nuclear behavior. Lentinus and Panus cannot be distinguished on the basis of postmeiotic nuclear behavior, but both can be distinguished from Pleurotus

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...

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...

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.