Lentinula edodes Genome Survey and Postharvest Transcriptome Analysis.

Lentinula edodes is a popular, cultivated edible and medicinal mushroom. Lentinula edodes is susceptible to postharvest problems, such as gill browning, fruiting body softening, and lentinan degradation. We constructed a de novo assembly draft genome sequence and performed gene prediction for Lentinula edodesDe novo assembly was carried out using short reads from paired-end and mate-paired libraries and by using long reads by PacBio, resulting in a contig number of 1,951 and an N50 of 1 Mb. Furthermore, we predicted genes by Augustus using transcriptome sequencing (RNA-seq) data from the whole life cycle of Lentinula edodes, resulting in 12,959 predicted genes. This analysis revealed that Lentinula edodes lacks lignin peroxidase. To reveal genes involved in the loss of quality of Lentinula edodes postharvest fruiting bodies, transcriptome analysis was carried out using serial analysis of gene expression (SuperSAGE). This analysis revealed that many cell wall-related enzymes are upregulated after harvest, such as ß-1,3-1,6-glucan-degrading enzymes in glycoside hydrolase (GH) families GH5, GH16, GH30, GH55, and GH128, and thaumatin-like proteins. In addition, we found that several chitin-related genes are upregulated, such as putative chitinases in GH family 18, exochitinases in GH20, and a putative chitosanase in GH family 75. The results suggest that cell wall-degrading enzymes synergistically cooperate for rapid fruiting body autolysis. Many putative transcription factor genes were upregulated postharvest, such as genes containing high-mobility-group (HMG) domains and zinc finger domains. Several cell death-related proteins were also upregulated postharvest.IMPORTANCE Our data collectively suggest that there is a rapid fruiting body autolysis system in Lentinula edodes The genes for the loss of postharvest quality newly found in this research will be targets for the future breeding of strains that keep fresh longer than present strains. De novoLentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding.

Mushroom Emergence Detected by Combining Spore Trapping with Molecular Techniques.

Obtaining reliable and representative mushroom production data requires time-consuming sampling schemes. In this paper, we assessed a simple methodology to detect mushroom emergence by trapping the fungal spores of the fruiting body community in plots where mushroom production was determined weekly. We compared the performance of filter paper traps with that of funnel traps and combined these spore trapping methods with species-specific quantitative real-time PCR and Illumina MiSeq to determine the spore abundance. Significantly more MiSeq proportional reads were generated for both ectomycorrhizal and saprotrophic fungal species using filter traps than were obtained using funnel traps. The spores of 37 fungal species that produced fruiting bodies in the study plots were identified. Spore community composition changed considerably over time due to the emergence of ephemeral fruiting bodies and rapid spore deposition (lasting from 1 to 2 weeks), which occurred in the absence of rainfall events. For many species, the emergence of epigeous fruiting bodies was followed by a peak in the relative abundance of their airborne spores. There were significant positive relationships between fruiting body yields and spore abundance in time for five of seven fungal species. There was no relationship between fruiting body yields and their spore abundance at plot level, indicating that some of the spores captured in each plot were arriving from the surrounding areas. Differences in fungal detection capacity by spore trapping may indicate different dispersal ability between fungal species. Further research can help to identify the spore rain patterns for most common fungal species.IMPORTANCE Mushroom monitoring represents a serious challenge in economic and logistical terms because sampling approaches demand extensive field work at both the spatial and temporal scales. In addition, the identification of fungal taxa depends on the expertise of experienced fungal taxonomists. Similarly, the study of fungal dispersal has been constrained by technological limitations, especially because the morphological identification of spores is a challenging and time-consuming task. Here, we demonstrate that spores from ectomycorrhizal and saprotrophic fungal species can be identified using simple spore traps together with either MiSeq fungus-specific amplicon sequencing or species-specific quantitative real-time PCR. In addition, the proposed methodology can be used to characterize the airborne fungal community and to detect mushroom emergence in forest ecosystems.

Early-diverging wood-decaying fungi detected using three complementary sampling methods.

Wood-decaying fungi are essential components of degradation systems in forest ecosystems. However, their species diversity and ecological features are largely unknown. Three methods are commonly used to investigate fungal diversity: fruiting body collection, culturing, and environmental DNA analysis. Because no single method fully characterises fungal diversity, complementary approaches using two or more methods are required. However, few studies have compared the different methods and determined the best way to characterise fungal diversity. To this end, we investigated wood-decomposing Dacrymycetes (Agaricomycotina, Basidiomycota) using a complementary approach combining fruiting body collection, culturing, and environmental DNA analysis, thereby offering an effective approach for investigating the diversity of saprotrophic mushrooms. Fruiting body collection, culturing, and environmental DNA analysis detected 11, 10, and 16 operational taxonomic units (OTUs; 25 OTUs in total) and identified three, seven, and seven novel lineages, respectively. The three methods were complementary to each other to detect greater Dacrymycetes diversity. The culturing and environmental DNA analysis identified three early-diverging lineages that were not identified in the fruiting body collection suggesting that diverse lineages lacking observable fruiting bodies remain undiscovered. Such lineages may be important to understand Dacrymycetes evolution. To detect early branches of Dacrymycetes more efficiently, we recommend a combined approach consisting of a primary environmental DNA survey to detect novel lineages and a secondary culture survey to isolate their living mycelia. This approach would be helpful for identifying otherwise-undetectable lineages, and could thus uncover missing links that are important for understanding the evolution of mushroom-forming fungi.

Multiplex real-time PCR assay for simultaneous detection of Omphalotus guepiniformis and Lentinula edodes.

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.

A new Cortinarius of section Calochroi (Basidiomycota, Agaricomycetes) from Mediterranean Quercus woodlands (Italy).

A new species of Cortinarius, C. flavoaurantians sp. nov., is described from Italian Quercus woods based on both morphological and ITS rDNA data. This taxon is characterized by a yellowish pileus and cortina, a white universal veil and a pileipellis that reacts yellow-orange with KOH. Illustrations of the key micromorphological features and fresh basidiomata in situ are provided. Closely related species are discussed.

Two new species of Hydnum with ovoid basidiospores: H. ovoideisporum and H. vesterholtii.

Two new species of Hydnum, characterized by slender Hydnum rufescens-like basidiomes and ovoid to broadly ellipsoid basidiospores, are described from the Iberian Peninsula based on morphological and ITS molecular data. Hydnum ovoideisporum is distinguished by pilei with deep orange tones and strong preference for calcareous soil. It is widespread in the Iberian-Mediterranean area. Hydnum vesterholtii is characterized by its ocher to light ocher pileus, and nearly all the collections were made in the Pyrenees. Both ovoid-spored species are monophyletic well supported groups in the maximum parsimony and Bayesian ITS phylogenies, while the remainder of the samples assigned to H. rufescens s.l. and having globose basidiospores split into six well supported clades. The need to typify the name Hydnum rufescens is discussed, and a provisional key is given for the European taxa of Hydnum.

Lactarius subg. Plinthogalus: the European taxa and American varieties of L. lignyotus re-evaluated.

The European species Lactarius subg. Plinthogalus were subjected to a molecular phylogenetic analysis based on ITS, LSU and rpb2 sequences. Morphological characters of the species are discussed in the light of the phylogenetic results. In addition to a broad sampling within Europe, some Asian and North American taxa also were included in the analysis. Eight European species are confirmed molecularly: L. lignyotus, L. acris, L. azonites, L. pterosporus, L. ruginosus, L. romagnesii, L. fuliginosus and L. picinus. Except the sibling species L. fuliginosus and L. picinus, all are morphologically distinct. Our results suggest that L. fuliginosus is associated exclusively with broadleaf trees and L. picinus with conifers, but this putative difference in host specificity needs to be investigated further. Lactarius subruginosus turns out to be a synonym of either L. pterosporus or L. ruginosus. The position of Lactarius terenopus remains to be clarified. The North American taxa that are closely related to the European L. lignyotus (L. fallax, L. lignyotus var. canadensis, var. nigroviolascens, var. marginatus) are not resolved. Intercontinental conspecificity was demonstrated between Europe and northern Asia but was not found between Europe and southern Asia or between Europe and North America. A taxonomic subdivision of L. subg. Plinthogalus based on the height of the spore ornamentation should be rejected.

Cantharellaceae of Guyana I: new species, combinations and distribution records of Craterellus and a synopsis of known taxa.

Members of the Cantharellaceae (Cantharellales, Basidiomycota) are common ectomycorrhizal associates of the leguminous genus Dicymbe in the Pakaraima Mountains of Guyana. Eight distinct species or morphospecies currently are recognized in Craterellus Pers. or Cantharellus Adans. ex Fr. from Guyanese Dicymbe-dominated forests. We evaluated the systematics of these taxa with phylogenetic analyses of DNA sequence data from the nuclear ribosomal regions of the internal transcribed spacer (ITS) and 28S large subunit (LSU). The results of these analyses along with careful assessment of morphology let us described two new species, Craterellus atratoides sp. nov. and Craterellus strigosus sp. nov., redescribe Craterellus atratus (Corner) Yomyart et al. based on new material from Guyana, and propose a new combination in Craterellus for Cantharellus pleurotoides T.W. Henkel, Aime & S.L. Mill. Macroscopic illustrations are provided for two additional cantharelloid morphospecies confirmed in Craterellus, as well as the regionally endemic Cantharellus guyanensis Mont. Macromorphological, micromorphological and habitat data are provided for C. atratoides, C. strigosus and C. atratus, and ITS and LSU sequence data are provided for each of the eight known Guyanese taxa.

Three new ascomycetes from freshwater in China.

Three new freshwater ascomycetes, Diaporthe aquatica sp. nov. (Diaporthaceae), Ophioceras aquaticus sp. nov. (Magnaporthaceae) and Togninia aquatica sp. nov. (Togniniaceae), are described and illustrated based on morphological and molecular data (ITS, 18S, 28S rDNA sequences). Diaporthe aquatica is characterized by globose to subglobose, black ascomata with long necks, broadly cylindrical to obclavate asci, and small, ellipsoidal to fusiform, one-septate, hyaline ascospores; it is unusual among Diaporthe species in the fact that it lacks a stroma and has freshwater habitat. Ophioceras aquaticus is characterized by globose ascomata with a long beak, cylindrical, eight-spored asci with J- subapical rings and 3-5-septate filiform ascospores with slightly acute ends. Togninia aquatica is characterized by globose ascomata with long necks, clavate and truncate asci clustered on distinct ascogenous hyphae, and small, reniform, hyaline ascospores. Differences among the new taxa and similar species are discussed.

The ectomycorrhizal fungus Tricholoma matsutake is a facultative saprotroph in vitro.

Tricholoma matsutake is an economically important ectomycorrhizal fungus of coniferous woodlands. Mycologists suspect that this fungus is also capable of saprotrophic feeding. In order to evaluate this hypothesis, enzyme and chemical assays were performed in the field and laboratory. From a natural population of T. matsutake in southern Finland, samples of soil-mycelium aggregate (shiro) were taken from sites of sporocarp formation and nearby control (PCR-negative) spots. Soil organic carbon and activity rates of hemicellulolytic enzymes were measured. The productivity of T. matsutake was related to the amount of utilizable organic carbon in the shiro, where the activity of xylosidase was significantly higher than in the control sample. In the laboratory, sterile pieces of bark from the roots of Scots pine were inoculated with T. matsutake and the activity rates of two hemicellulolytic enzymes (xylosidase and glucuronidase) were assayed. Furthermore, a liquid culture system showed how T. matsutake can utilize hemicellulose as its sole carbon source. Results linked and quantified the general relationship between enzymes secreted by T. matsutake and the degradation of hemicellulose. Our findings suggest that T. matsutake lives mainly as an ectomycorrhizal symbiont but can also feed as a saprotroph. A flexible trophic ecology confers T. matsutake with a clear advantage in a heterogeneous environment and during sporocarp formation.

First report of the ectomycorrhizal status of boletes on the Northern Yucatan Peninsula, Mexico determined using isotopic methods.

Despite their prominent role for tree growth, few studies have examined the occurrence of ectomycorrhizal fungi in lowland, seasonally dry tropical forests (SDTF). Although fruiting bodies of boletes have been observed in a dry tropical forest on the Northern Yucatan Peninsula, Mexico, their occurrence is rare and their mycorrhizal status is uncertain. To determine the trophic status (mycorrhizal vs. saprotrophic) of these boletes, fruiting bodies were collected and isotopically compared to known saprotrophic fungi, foliage, and soil from the same site. Mean δ(15)N and δ(13)C values differed significantly between boletes and saprotrophic fungi, with boletes 8.0‰ enriched and 2.5‰ depleted in (15)N and (13)C, respectively relative to saprotrophic fungi. Foliage was depleted in (13)C relative to both boletes and saprotrophic fungi. Foliar δ(15)N values, on the other hand, were similar to saprotrophic fungi, yet were considerably lower relative to bolete fruiting bodies. Results from this study provide the first isotopic evidence of ectomycorrhizal fungi in lowland SDTF and emphasize the need for further research to better understand the diversity and ecological importance of ectomycorrhizal fungi in these forested ecosystems.

An axenic culture system for fruiting body formation by an edible bolete phylogenetically related to culinary-medicinal penny bun mushroom, Boletus edulis Bull.:Fr. strains from China.

The ability of two freshly isolated Boletus stains to fruit under axenic conditions was tested using different solid and liquid nutrient media. One strain (YNCX04) produced numerous primordia from which fruiting bodies, 12 mm and 10 mm in length, with grey, convex pilei, and yellow-white, clavate stipes developed between 15 and 30 d after inoculation of fungal mycelium onto a solid medium consisting of mineral salts, thiamine, glucose, potato, an extract of Cunninghamia lanceolata root, and agar. The other strain (YNB200) produced numerous primordia but no sporophores. Strain YNCX04 lost the ability to form fruiting bodies in axenic culture 6 mo after initial isolation but retained the ability to form primordia for up to 18 mo. Based on internal transcribed spacer sequencing data, strains YNB200 and YNCX04 formed a sub-cluster together with four previously designated Boletus edulis strains from China. Phylogenetic analysis placed the Chinese strains closer to B. aestivalis than to European and North American strains of B. edulis, although a 29-bp fragment specific to all the B. aestivalis strains was absent from all the Chinese strains. Furthermore, partial 18S rDNA sequences from strains YNB200 and YNCX04 exhibited 98% similarity with an 18S rDNA sequence from B. edulis strain Be3. Further molecular studies are indicated to more accurately establish the taxonomic positions ofF3 and F4-3, as well as the Chinese strains designated as B. edulis.

Purification, characterization and anti-atherosclerotic effects of the polysaccharides from the fruiting body of Cordyceps militaris.

Hyperlipidemia is one major cause of atherosclerosis, which is a basic pathological change of cardiovascular diseases. Polysaccharide is a water-soluble component with lipid-lowering effects. In this study, alkaline-extracted polysaccharides were obtained from the fruiting body of C. militaris. Polysaccharides were purified via anion exchange and size exclusion chromatography. Their structural characteristics were investigated via chemical and spectroscopic methods. CM3I was mainly composed of →4)α-D-Glcp(1 → glycosyls and differed from starch due to the presence of →4,6)ß-D-Glcp(1 → glycosyls. CM3II was characterized by its backbone, which was composed of →4)-ß-D-Manp(1 → 6)-α-D-Manp(1 → 6)-ß-D-Manp(1 → linked glycosyls, and especially the presence of O-methyl. Moreover, CM3II exhibited powerful anti-atherosclerotic effects via lowering plasma lipid levels in apolipoprotein E-deficient mice. The underlying mechanisms were attributed to its promoting effect on LXRα and inhibitory effect on SREBP-2. Collectively, CM3I and CM3II are different from the previously reported polysaccharides from C. militaris, and CM3II has a potential application in hypolipidemia and anti-atherosclerosis.

Neoaleurodiscus fujii, a new genus and new species found at the timberline in Japan.

A new genus, Neoaleurodiscus, is proposed as a segregate from Aleurodiscus (Basidiomycota), and the new species, Neoaleurodiscus fujii, is described. It was collected at the timberline of Mount Fuji, Japan. Specimens were found on trunks of Rhododendron sp. Morphological study and phylogenetic analysis based on sequence data derived from LSU nrDNA indicated that Neoaleurodiscus is separate from other segregate genera from Aleurodiscus s.l. Neoaleurodiscus is characterized by having a disciform basidiocarp; microscopically it has moniliform gloeocystidia, absence of acanthophyses, nodose-septate hyphae, hyphidia, and basidiospores, which are amyloid, smooth and +/- thick-walled. Among genera studied Acanthobasidium and Aleurodiscus (s.s.) are most closely related to Neoaleurodiscus, but these genera have warted or spiny basidiospores. Two new combinations, Acanthobasidium penicillatum and Neoaleurodiscus monilifer, are proposed.

Characterization of α-glucosidase inhibitory constituents of the fruiting body of lion's mane mushroom (Hericium erinaceus).

ETHNOPHARMACOLOGICAL RELEVANCE: Hericium erinaceus, commonly called lion's mane mushroom, is an edible and medicinal mushroom that has been traditionally used for the treatment of metabolic disorders, gastrointestinal diseases and memory impairment. In this study, potential anti-hyperglycemic constituents were identified to support the traditional usage of H. erinaceus. MATERIALS AND METHODS: The components of H. erinaceus were purified using various column chromatography techniques. The structure of the separated compounds was determined based on spectroscopic data analysis, i.e., 1D and 2D NMR analysis. The anti-hyperglycemic activity of the isolated compounds was evaluated by measuring the inhibitory effects on α-glucosidase activity. Molecular docking analysis was also conducted for elucidation of α-glucosidase inhibitory activity of isolated compounds. RESULTS: Ten compounds including four new compounds, erinacenols A-D (1-4), were isolated from the fruiting bodies of H. erinaceus. Investigation of the anti-hyperglycemic effect of isolated compounds demonstrated that erinacenol D (4), 4-[3',7'-dimethyl-2',6'-octadienyl]-2-formyl-3-hydroxy-5-methyoxybenzylalcohol (6), hericene A (7), hericene D (8) and hericenone D (9) strongly inhibited α-glucosidase activity with IC50 values of <20 µM. The structure activity relationship suggested the importance of long side chain for α-glucosidase inhibitory activity. Further analysis by molecular docking demonstrated the interaction of α-glucosidase and isolated compounds, which supported the inhibitory activity of α-glucosidase. CONCLUSION: Our present study demonstrated the beneficial effect of H. erinaceus by characterization of α-glucosidase inhibitory compounds, including four new compounds. This approach can be valuable support for the traditional use of H. erinaceus for the treatment of diabetes and metabolic diseases, which needs to be clarified by further in-vivo study.

Morphological and molecular identification of three new species of Tomentella from Finland.

Three new species of Tomentella (Thelephorales) from Finland, T. globosa, T. lammiensis, and T. longisterigmata, are described and illustrated with morphological characteristics and nuc rDNA ITS1-5.8S-ITS2 sequences. T. globosa is characterized by mucedinoid, pale to dark brown basidiocarps adherent to the substrate, generative hyphae with clamps and rarely with simple septa, and echinulate, globose basidiospores (echinuli up to 1.5 µm long). T. lammiensis is characterized by mucedinoid, oxide yellow to golden brown basidiocarps adherent to the substrate, generative hyphae with clamps and rarely with simple septa, and echinulate, ellipsoid, triangular, or lobbed basidiospores (echinuli up to 2 µm long). T. longisterigmata is characterized by mucedinoid, dark brown to chestnut basidiocarps separable from the substrate, generative hyphae clamped and rarely with simple septa, the long basidial sterigmata (7-11 µm long), and echinulate, globose basidiospores (echinuli up to 2 µm long). An absence of rhizomorphs and cystidia is their common morphological feature. Molecular analyses by maximum likelihood, maximum parsimony, and Bayesian analyses confirm the phylogenetic position of these three new species. The discriminating characters of these new species and their closely related species are discussed in this study, and a key to the species from Finland is provided.

The genome and microbiome of a dikaryotic fungus (Inocybe terrigena, Inocybaceae) revealed by metagenomics.

Recent advances in molecular methods have increased our understanding of various fungal symbioses. However, little is known about genomic and microbiome features of most uncultured symbiotic fungal clades. Here, we analysed the genome and microbiome of Inocybaceae (Agaricales, Basidiomycota), a largely uncultured ectomycorrhizal clade known to form symbiotic associations with a wide variety of plant species. We used metagenomic sequencing and assembly of dikaryotic fruiting-body tissues from Inocybe terrigena (Fr.) Kuyper, to classify fungal and bacterial genomic sequences, and obtained a nearly complete fungal genome containing 93% of core eukaryotic genes. Comparative genomics reveals that I. terrigena is more similar to ectomycorrhizal and brown rot fungi than to white rot fungi. The reduction in lignin degradation capacity has been independent from and significantly faster than in closely related ectomycorrhizal clades supporting that ectomycorrhizal symbiosis evolved independently in Inocybe. The microbiome of I. terrigena fruiting-bodies includes bacteria with known symbiotic functions in other fungal and non-fungal host environments, suggesting potential symbiotic functions of these bacteria in fungal tissues regardless of habitat conditions. Our study demonstrates the usefulness of direct metagenomics analysis of fruiting-body tissues for characterizing fungal genomes and microbiome.

Antiproliferative Activity and Cytotoxicity of Some Medicinal Wood-Destroying Mushrooms from Russia.

We analyzed the antiproliferative activity of 6 medicinal wood-destroying mushrooms (Fomes fomentarius, Fomitopsis pinicola, Trametes versicolor, Trichaptum biforme, Inonotus obliquus, and Coniophora puteana) that are common in deciduous and mixed coniferous forests in Central Russia. Morphological identification of strains collected from the wild was confirmed based on ribosomal DNA internal transcribed spacer phylogenetic analysis. We observed cytotoxic and cell growth-inhibitory effects of hot water extracts from mycelial biomass of 5 species-T. versicolor, C. puteana, F. fomentarius, F. pinicola, and I. obliquus-on leukemia cell lines (Jukart, K562, and THP-1); the effective extract concentrations were mostly less than 50 µg · mL-1. However, we observed no antiproliferative activity of dry biomass from methanol-chloroform (1:1) extracts of C. puteana and F. fomentarius. A chemosensitivity assay showed that the most effective polypore mushroom extract was the methanol extract of T. versicolor (strain It-1), which inhibited the growth of 6 various solid tumors (A-549 and SWi573 [lung], HBL-100 and T-47D [breast], HeLa [cervix], and WiDr [colon]) at concentrations below 45 µg · mL-1, with a concentration as low as 0.7-3.6 µg · mL-1 causing 50% reduction in the proliferation of cancer cells in lung and cervix tumors. Methanol extracts of F. pinicola and I. obliquus were less effective, with proliferation-inhibiting capacities at concentrations below 70 and 200 µg · mL-1, respectively. Thus, T. versicolor is a prospective candidate in the search for and production of new antiproliferative chemical compounds.

Selection of a set of specific primers for the identification of Tuber rufum: a truffle species with high genetic variability.

Tuber rufum is a truffle widely distributed throughout Europe, which forms mycorrhizal associations with numerous species of broadleaf and coniferous trees. The possibility of T. rufum contamination in commercial truffle-infected plants makes its detection important. To facilitate the identification of T. rufum from mycorrhiza and fruitbodies, species-specific primers were designed and tested. To overcome the high intraspecific genetic variability within the internal transcribed spacer (ITS) regions of T. rufum, as demonstrated by phylogenetic analysis, two forward primers, Ru1f and Ru2f, located on the ITS1 region were designed to be used in concert with the reverse primer ITS4. Only T. rufum was amplified with this primer combination, while DNA of Tuber magnatum, Tuber brumale, Tuber maculatum, Tuber borchii, Tuber excavatum and Tuber melanosporum was not. These primers give a specific amplicon ranging between 566 and 572 bp and are able to discriminate between T. rufum, T. borchii and T. magnatum in multiplex PCR. In addition, T. rufum-specific amplicons were obtained from both spore suspensions and mycorrhiza by direct PCR. Tuber rufum mycorrhiza obtained in the greenhouse using mycelial inoculation techniques had morphological features similar to those of other species of Tuber, stressing the importance of molecular tools for their identification.

A method to construct cDNA library of the entomopathogenic fungus, Metarhizium anisopliae, in the hemolymph of the infected locust.

A method was developed to construct cDNA library of pathogenic fungus in the blood of the infected insect for cloning the fungal genes expressed in the host. This method is designed to take advantage of the obvious difference between the cell structures and components of the pathogen cells and that of the host cells. The host blood cells only have cell membrane, which can be disrupted by using SDS/proteinase K (PK). The fungal cells grown in the animal blood have cell wall, which can protect the fungal cell from the disruption of SDS/proteinase K (PK). By this method, the blood cells were disrupted by SDS/proteinase K (PK) and then the released animal RNA and DNA were digested completely with RNase and DNase. Therefore, the fungi grown in the blood were harvested without any contamination of host RNA and DNA. The pure fungi harvested from the infected blood can be used for mRNA extraction and cDNA library construction. The purity of the fungal mRNA was confirmed by PCR and RT-PCR with specific primer pairs for the host and specific primer pairs for the fungus, respectively, and the clones of cDNA library constructed by using the fungal mRNA was also analyzed. The results showed that there was no detectable contaminated insect DNA or RNA existing in the fungal mRNA. Randomly selected cDNA clones from cDNA library were sequenced and analyzed against GenBank using Blastx; no selected sequences had significant similarity with insects' genes in comparison with the data of GenBank. The results further confirmed that the method to purify the pathogenic fungus from the host animal is reliable and the mRNA extracted from the fungus is eligible for cDNA library construction, and other molecular analysis including RT-PCR. This method may be applied to other pathogenic fungi and their host animals.