Isolation of 6-hydroxy-L-tryptophan from the fruiting body of Lyophyllum decastes for use as a tyrosinase inhibitor.

Tyrosinase is the key enzyme that controls melanin formation. We found that a hot water extract of the lyophilized fruiting body of the fungus Lyophyllum decastes inhibited tyrosinase from Agaricus bisporus. The extract was fractionated by ODS column chromatography, and an active compound was obtained by purification through successive preparative HPLC using an ODS and a HILIC column. Using spectroscopic data, the compound was identified to be an uncommon amino acid, 6-hydroxytryptophan. 6-Hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan were prepared through a Fenton reaction from L-tryptophan and D-tryptophan, respectively. The active compound was determined to be 6-hydroxy-L-tryptophan by comparison of their circular dichroism spectra and retention time on HPLC analysis of the Nα-(5-fluoro-2,4-dinitrophenyl)-L-leuciamide derivative with those of 6-hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan. A Lineweaver-Burk plot of the enzyme reaction in the presence of 6-hydroxy-L-tryptophan indicated that this compound was a competitive inhibitor. The IC50 values of 6-hydroxy-L-tryptophan was 0.23 mM.

An MSH4 homolog, stpp1, from Pleurotus pulmonarius is a "silver bullet" for resolving problems caused by spores in cultivated mushrooms.

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.

Construction of a genetic linkage map based on amplified fragment length polymorphism markers and development of sequence-tagged site markers for marker-assisted selection of the sporeless trait in the oyster mushroom (Pleurotus eryngii).

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.

Suppression of Alternaria brassicicola infection by volatile compounds from spent mushroom substrates.

Most commercially circulating mushrooms are produced via cultivation using artificially produced mushroom substrates. However, after mushroom harvesting, the disposal of spent mushroom substrates (SMSs) is a serious problem for the mushroom industry owing to the need for a disposal site and the cost involved. Thus, in view of the possibility of recycling SMSs as a soil modifier, we examined the effect of soil mixed with SMSs on the infection of Arabidopsis leaves by Alternaria brassicicola, the causal agent of cabbage leaf spot. The mixing of SMSs used for Hypsizygus marmoreus, Pholiota microspora, Lyophyllum decastes, and Auricularia polytricha into culture soil suppressed the lesion formation caused by A. brassicicola. The defense responses of Arabidopsis were not induced by the culturing of these seedlings in soils containing SMSs. Suppressed lesion formation was observed after the seedlings were treated with volatiles emitted from SMSs that were incubated with soil for 7 days and used for H. marmoreus, P. microspora, L. decastes, A. polytricha, Lentinula edodes, and Cyclocybe cylindracea. The volatiles from the SMSs reduced the elongation of A. brassicicola hyphae. GC-MS analyses of extracts from the SMS containing soils led to the detection of various volatile compounds; among these, skatole, 2,4-di-tert-butylphenol, γ-dodecalactone, butyric acid, guaiacol, 6-amyl-2-pyrone, and 1-octen-3-ol were examined for inhibitory activity on A. brassicicola and found to suppress hyphae elongation. These findings indicate that the antifungal volatile compounds emitted by the SMSs suppress A. brassicicola infection.

Morphological, cytological and genetic analyzes of the 'sango' mutant with the defects in basidiocarp development in edible mushroom Pleurotus pulmonarius.

A spontaneous, morphological variation 'sango' was observed in the progeny of a Pleurotus pulmonarius (Fr.) Quél. wild-type basidiocarp (also known as fruiting body) collected from the field. This variant developed wart- and coral-like structures instead of normal basidiocarps. Microscopic analysis showed that the sango phenotype had defects in the differentiation of the pileus and hymenium. Basidiocarp phenotypic data analysis in the progenies revealed that the sango trait is a heritable mutation character controlled by a single recessive gene. This mutation locus was mapped on linkage group III of a previously constructed genetic linkage map by amplified fragment length polymorphism (AFLP) technique in P. pulmonarius. Four AFLP markers identified by bulked segregant analysis showed linkage to the sango mutation locus, with the genetic distance ranging from 0 to 2.1 cM. Of these markers, one marker was co-segregated with the sango mutation locus. This knowledge will be a useful foundation for practical breeding as well as for elucidating molecular mechanisms in basidiocarp development of main edible mushrooms.

A genetic linkage map of Pleurotus pulmonarius based on AFLP markers, and localization of the gene region for the sporeless mutation.

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.