Nitaro Maekawa

Direct ethanol production from starch, wheat bran and rice straw by the white rot fungus Trametes hirsuta

The white rot fungus Trametes hirsuta produced ethanol from a variety of hexoses: glucose, mannose, cellobiose and maltose, with yields of 0.49, 0.48, 0.47 and 0.47 g/g of ethanol per sugar utilized, respectively. In addition, this fungus showed relatively favorable xylose consumption and ethanol production with a yield of 0.44 g/g. T. hirsuta was capable of directly fermenting starch, wheat bran and rice straw to ethanol without acid or enzymatic hydrolysis. Maximum ethanol concentrations of 9.1, 4.3 and 3.0 g/l, corresponding to 89.2%, 78.8% and 57.4% of the theoretical yield, were obtained when the fungus was grown in a medium containing 20 g/l starch, wheat bran or rice straw, respectively. The fermentation of rice straw pretreated with ball milling led to a small improvement in the ethanol yield: 3.4 g ethanol/20 g ball-milled rice straw. As T. hirsuta is an efficient microorganism capable of hydrolyzing biomass to fermentable sugars and directly converting them to ethanol, it may represent a suitable microorganism in consolidated bioprocessing applications.

Phylogeography of Hyphoderma setigerum (Basidiomycota) in the Northern Hemisphere

Previous studies of morphological variation in the homobasidiomycete Hyphoderma setigerum have lead to suspicions of a species complex. This study explores variation in DNA sequences from the nuclear ribosomal ITS region of 45 specimens from America, Asia, and Europe in a phylogeographic context. Based on molecular analysis, morphological studies, and crossing tests, nine preliminary taxa are shown to exist inside the species complex, and the two previously described segregate species H. subsetigerum and H. nudicephalum are confirmed. The molecular analysis shows evidence of allopatric differentiation over intercontinental distances. Only one of the nine well-supported clades has a geographic distribution spanning more than one continent, probably indicating the importance of vicariance in the evolution of this species complex. The basionym of H. setigerum, Thelephora setigera, is neotypified to fix the application of that name.

Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR

ABSTRACT Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

The sequestrate genus Rosbeeva T.Lebel & Orihara gen. nov. (Boletaceae) from Australasia and Japan: new species and new combinations

The sequestrate genus Chamonixia has been shown to have affinities to the Boletales, in particular the genus Leccinum. Australasian and Japanese species of Chamonixia were examined using morphological and molecular (ITS and nLSU rDNA) data and found to also have affinities with Leccinum and Leccinellum, however they form a distinct clade separate from the European type species C. caespitosa Rolland and North American species. A new genus, Rosbeeva T.Lebel & Orihara gen. nov., is proposed for the Australasian, Japanese and Chinese taxa. The species R. mucosa (Petri) T.Lebel comb. nov. is restricted in distribution to Singapore and Borneo, and R. pachyderma (Zeller & C.W. Dodge) T.Lebel comb. nov. to New Zealand, with Australian collections considered to belong to a revised R. vittatispora (G.W.Beaton, Pegler & T.W.K.Young) T.Lebel comb. nov. or a new species R. westraliensis T. Lebel sp. nov. The Chinese species R. bispora (B.C.Zhang & Y.N.Yu) T.Lebel & Orihara comb. nov is transferred to the new genus based upon morphological data. Two new species from Japan, Rosbeeva eucyanea Orihara and R. griseovelutina Orihara, are also described and illustrated. A key to all species of Rosbeeva is provided. Due to the highly modified gastroid sporocarp forms of both Chamonixia and Rosbeeva, many macroscopic characters of use in agaricoid taxonomy are difficult to interpret. However, color change and texture of sporocarps are of some use to distinguish genera and species. Microscopic characters such as spore shape, dimensions, and ornamentation, and pileipellis and hymenophoral trama structure, are essential for determining genera and species.

Trichoderma mienum sp. nov., isolated from mushroom farms in Japan

During an investigation of Hypocrea/Trichoderma species inhabiting mushroom bedlogs, we found five strains of an undescribed species from a culture collection. These were analyzed using a combined approach, including morphology of holomorph, cultural studies, and phylogenetic analyses of the rRNA gene cluster of the internal transcribed spacer region, translation elongation factor 1-α, and RNA polymerase subunit II gene sequences. Distinctive morphological characters include stromata with green ascospores produced on potato dextrose agar medium, and Gliocladium-like to irregularly Verticillium-like conidiophores. In phylogenetic analyses, this species belongs to the Semiorbis clade, but its morphological characteristics do not match the other members of this clade. Based on morphological observations and phylogenetic analyses, we describe this as a new species, Trichoderma mienum, representing its Hypocrea teleomorph and Trichoderma anamorph.

Use of ionic liquid in fungal taxonomic study of ultrastructure of basidiospore ornamentation

An ionic liquid (IL) is a kind of salt that stays in a molten state even at room temperature. Since ILs do not vaporize even under vacuum conditions and show high ionic conductivity, they can be used in scanning electron microscopy (SEM) studies. The ultrastructural features of basidiospore ornamentation are considered to be important in the delimitation of taxa for fungi. In the present study, we carried out SEM observations on basidiospores that were subjected to an IL treatment, and evaluated the usefulness of this method in comparison with a conventional preparation method in which dehydrating, drying and platinum (Pt) coating were used. Using the conventional method, a considerable number of basidiospores was lost from the gill tissues; however, using the IL method, the decrease in basidiospores was extremely small. No significant differences in ultrastructural morphology or basidiospore size were found between Pt-coated basidiospores and IL-treated ones. SEM images of Pt-coated basidiospores tended to have higher contrast than those of IL-treated ones. Charging effects were observed with Pt-coated basidiospores, especially at the tips of the ornaments, whereas no such effects occurred for the IL-treated ones. In addition, small crinkles were observed in the Pt-coated basidiospores, but not in the IL-treated ones. These results suggest that the IL method is useful for fungal taxonomic studies.

Specific detection of a basidiomycete, Phlebia brevispora associated with butt rot of Chamaecyparis obtusa, by PCR-based analysis

In order to monitor the basidiomycetous fungus Phlebia brevispora isolated from butt rot of Chamaecyparis obtusa (Japanese cypress) in 1997 in Nagasaki Prefecture, a sensitive polymerase chain reaction (PCR)-based assay was developed to specifically detect the fungus on-site. A species-specific primer for P. brevispora was derived from the internal transcribed spacer (ITS) region (containing 5.8S ribosomal DNA, ITS1 and ITS2) sequences of the fungus. The PCR assay was able to detect down to 1 fg DNA (per 1 µl PCR reaction mixture) and down to 0.2 mg mycelium of P. brevispora (per 1 g of decayed wood). The samples for on-site monitoring were collected in 2002 from the decayed tree stump in which P. brevispora had first been isolated. From the decayed tree tissue, P. brevispora could be detected by PCR assay even when its mycelium could not isolated from the tree tissue by culturing. This indicates that the PCR amplification using the specific primer developed here is a useful method for monitoring P. brevispora on-site.

Identification of Trichoderma, a Competitor of Shiitake Mushroom (Lentinula edodes), and Competition between Lentinula edodes and Trichoderma species in Korea

Fungus/Mushroom Resource and Research Center, Tottori University, Tottori 680-8553, Japan(Received on February 7, 2011; Revised on February 1, 2012; Accepted on February 15, 2012)During investigating of shiitake mushroom competitors,289 isolates of Trichoderma spp. were collected fromshiitake mushroom farms in different districts and theForest Mushroom Research Center of Korea, amongwhich 29 representative strains were selected. Based onthe DNA sequences of the rpb2 and tef1 genes and theITS rDNA, and their morphological characteristics,they were identified as T. atroviride, T. citrinoviride, T.harzianum, T. longibrachiatum, and two undescribedspecies, Trichoderma spp. 1 and 2, which are consideredto be the candidate of new species. Competition testsbetween Lentinula edodes (Sanjo302) and the Tricho-derma species indicated that the six species of Tricho-derma were significantly different from each other interms of their ability to invade the mycelial blocks ofshiitake. In both of dual cultures on potato dextroseagar and sawdust media, Trichoderma spp. 1 and 2strongly invaded the mycelial blocks of shiitake. Ourresults suggest that the two Trichoderma species maycause potentially serious economic losses in shiitakecultivation of Korea.Keywords : competition test, ITS, phylogenetic analysis,rpb2, tef1Shiitake mushrooms (Lentinula edodes) are widely culti-vated as a food source in East Asia and are dried andexported to many countries because of its special flavor andaroma (Chen, 2005; Luo, 2004). Recent research has indi-cated that the shiitake mushroom also has useful clinicaleffects, including an immunostimulant (Yamamoto et al.,1997). In Korea, the consumption of shiitake mushrooms isincreasing annually since 1999, and there are now about 20cultivars of shiitake, which were promoted and disseminat-ed by the Forest Mushroom Research Center.The genus Trichoderma is one of the most importantpathogens in the cultivation of the shiitake mushroom, andoften causes severe damage during its production (Miyazakiet al., 2009). Trichoderma species mainly attack the myceliaof L. edodes in bed logs and sawdust cultures. The identi-fication of Trichoderma at the species level has proveddifficult because of their interspecific morphological simi-larities (Chaverri and Samuels, 2003). It led to the establi-shment of “aggregate” species concept by Rifai (1969) thatall Trichoderma species could be distinguished to nineaggregates. Later, Bissett (1984, 1991a, b, c) established anew system of Trichoderma classification based on thebranching pattern of conidiophores and the characteristicsof phialides and conidia. After the introduction of mole-cular methods in Trichoderma taxonomy, the species con-cept of Trichoderma has changed dramatically (Chaverriand Samuels, 2003; Jaklitsch, 2009). In addition, the phylo-genetic data helped to establish the relationship betweenanamorph Trichoderma and their related teleomorph Hypocrea(Chaverri and Samuels, 2003; Samuels et al., 1998, 2002).In recent years, Park et al. (2005, 2006) identified sevendistinct species of Trichoderma from that Trichodermaisolates from green mold of oyster mushroom were identi-fied as seven distinct species (T. pleuroticola, T. pleurotum,T. atroviride, T. citrinoviride, T. harzianum, T. longibrachi-atum and T. virens). However, little is known about thespecies of Trichoderma associated with the green moldobserved on the shiitake mushrooms in Korea, and onlyfive species of Trichoderma (T. citrinoviride, T. harzianum,T. polysporum, T. longibrachiatum and T. viride) were

Functional analysis of the melanin biosynthesis genes ALM1 and BRM2-1 in the tomato pathotype of Alternaria alternata

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.

Degradation of chlorinated pesticide DDT by litter-decomposing basidiomycetes

One hundred and two basidiomycete strains (93 species in 41 genera) that prefer a soil environment were examined for screening of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) biodegradation. Three strains within two litter-decomposing genera, Agrocybe and Marasmiellus, were selected for their DDT biotransformation capacity. Eight metabolites; 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), two monohydroxy-DDTs, monohydroxy-DDD, 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol, putative 2,2-bis(4-chlorophenyl)ethanol and two unidentified compounds were detected from the culture with Marasmiellus sp. TUFC10101. A P450 inhibitor, 1-ABT, inhibited the formation of monohydroxy-DDTs and monohydroxy-DDD from DDT and DDD, respectively. These results indicated that oxidative pathway which was catalyzed by P450 monooxygenase exist beside reductive dechlorination of DDT. Monohydroxylation of the aromatic rings of DDT (and DDD) by fungal P450 is reported here for the first time.