Thomas J. Volk

Cytology of the life-cycle of Morchella

Various stages of the morel life-cycle are studied cytologically. Photomicrographic evidence demonstrates that the average number of nuclei per cellular compartment in vegetative hyphae of Morchella is 10–15 and that hyphal fusions are quite frequent. The resting structures, the sclerotia, are actually pseudosclerotia which form from the repeated branching and enlargement of terminal hyphae from either primary (homokaryotic) or secondary (heterokaryotic) hyphae. Photomicrographs also depict the development of fruiting body primordia. Photomicrographs of ascus development demonstrate autogamy rather than de novo heterokaryon formation by hyphal fusion in the subhymenial layer of the fruiting body. For the first time a comprehensive life-cycle diagram of the morel is introduced.

Ectomycorrhizal characterization of an American chestnut (Castanea dentata)-dominated community in Western Wisconsin

Circa 1900, a farmer from the eastern US planted 11 American chestnut (Castanea dentata) seeds on a newly established farm near West Salem in western Wisconsin. These trees were very successful, producing a large stand of over 6,000 trees. Since this area is well outside the natural range of chestnut, these trees remained free from chestnut blight until 1987. In the West Salem stand, chestnuts are the dominant species of a mixed forest community, reminiscent of the chestnut–oak ecosystems of pre-1900 Appalachia. To identify putative mycorrhizal associates of chestnut in this unique forest, our approach was twofold: (1) an extensive fruiting body survey was conducted for four seasons that yielded approximately 100 putative mycorrhizal species and (2) a belowground molecular approach was used to generate DNA sequences of the internal transcribed spacer region from ectomycorrhizae. Unexpectedly, chestnut did not appear to be the dominant underground ectomycorrhizal-forming plant species. This study highlights the need to identify the plant host species when conducting belowground molecular-based surveys and provides preliminary identification of ectomycorrhizal fungi associated with a disjunct stand of American chestnut.

Taxonomic revision of true morels (Morchella) in Canada and the United States

Recent molecular phylogenetic studies have revealed the existence of at least 50 species of Morchella worldwide and demonstrated a high degree of continental endemism within the genus. Here we describe 19 phylogenetic species of Morchella from North America, 14 of which are new (M. diminutiva, M. virginiana, M. esculentoides, M. prava, M. cryptica, M. frustrata, M. populiphila, M. sextelata, M. septimelata, M. capitata, M. importuna, M. snyderi, M. brunnea and M. septentrionalis). Existing species names (M. rufobrunnea, M. tomentosa, M. punctipes and M. angusticeps) are applied to four phylogenetic species, and formal description of one species (M. sp. “Mel-8”) is deferred pending study of additional material. Methods for assessing morphological features in Morchella are delineated, and a key to the known phylogenetic species of Morchella in North America is provided. Type studies of M. crassistipa, M. hotsonii, M. angusticeps and M. punctipes are provided. Morchella crassistipa is designated nomen dubium.

Experimental studies on the morel. I: Heterokaryon formation between monoascosporous strains of Morchella

Three lines of evidence are reported for heterokaryon formation in Morchella esculenta and related species. Cultural studies demonstrated a genetic basis for different types of interactions between mycelia from sister and non-sister spores. Cytological studies of the mycelial interaction between non-sister monoascosporous isolates demonstrated nuclear pairing in presumptive heterokaryons. Two different mutations isolated in this study were used to show genetic complementation in these heterokaryons.

Molecular phylogeny and morphology reveal three new species of Cantharellus within 20 m of one another in western Wisconsin, USA

Three new species, Cantharellus phasmatis, Cantharellus flavus and Cantharellus spectaculus, all previously considered Cantharellus cibarius, are described in this study. The circumscription of these three species from C. cibarius and other Cantharellus species is supported by morphological differences and nuclear DNA sequence data (nLSU, ITS, TEF1). All were found under Quercus spp. in a small plot in Hixon Forest Park in La Crosse, Wisconsin, emphasizing the need for further taxonomic study of even common and conspicuous genera in North America. In addition, a review of the current state of C. cibarius sensu lato systematics is presented, including a review of the recent elevation of C. cibarius var. roseocanus to the species rank. Taxonomic descriptions and photographs are provided for the newly described species.

Antibacterial Compounds from Mushrooms II: Lanostane Triterpenoids and an Ergostane Steroid with Activity Against Bacillus cereus Isolated from Fomitopsis pinicola

Anti- Bacillus cereus bioassay-guided fractionation of a crude extract of the American mushroom, Fomitopsis pinicola, was performed using thin-layer chromatography, Sephadex LH-20 column chromatography, and preparative-scale HPLC. Five lanostane triterpenoids (1-5) and one ergostane steroid (6) were isolated and identified. Compound 1 is a new lanostane triterpenoid, and its structure was determined using 1D and 2D NMR experiments, HR-MS, and physical data. Each of the purified compounds (1-6) was tested for antibacterial activity against B. cereus using standard MIC assays. Compounds 1-6 had MIC values of 32, 16, 32, 32, 128, and 64 microg/mL, respectively.

Antibacterial Compounds from Mushrooms I: A Lanostane-Type Triterpene and Prenylphenol Derivatives from Jahnoporus hirtus and Albatrellus flettii and Their Activities Against Bacillus cereus and Enterococcus faecalis

Antibacterial bioassay-guided fractionation of two American mushroom species, Jahnoporus hirtus and Albatrellus flettii, led to the isolation and identification of their major antibacterial constituents: 3,11-dioxolanosta-8,24( Z)-diene-26-oic acid (1) from J. hirtus and confluentin (2), grifolin (3), and neogrifolin (4) from A. flettii. Compound 1 is a new lanostane-type triterpene. All purified compounds were evaluated for their ability to inhibit the growth of Bacillus cereus and Enterococcus faecalis using standard MIC assays. Compounds 1- 4 demonstrated MIC values of 40, 20, 10, and 20 microg/mL, respectively, against B. cereus and MIC values of 32, 1.0, 0.5, and 0.5 microg/mL, respectively, against E. faecalis. Thus, one novel compound and three others were shown to possess antimicrobial activities against these gram-positive bacteria employed as surrogates for more virulent and dangerous pathogens.

Screening a mushroom extract library for activity against Acinetobacter baumannii and Burkholderia cepacia and the identification of a compound with anti-Burkholderia activity

BackgroundAcinetobacter baumannii and species within the Burkholderia cepacia complex (BCC) are significant opportunistic bacterial pathogens of humans. These species exhibit a high degree of antibiotic resistance, and some clinical isolates are resistant to all currently available antimicrobial drugs used for treatment. Thus, new drugs are needed to treat infections by these species. Mushrooms could be a potential source for new drugs to treat A. baumannii and BCC infections.MethodsThe aim of this study was to screen a library of crude extracts from 330 wild mushrooms by disk diffusion assays for antibacterial activity against A. baumannii and Burkholderia cepacia in the hope of identifying a novel natural drug that could be used to treat infections caused by these species. Once positive hits were identified, the extracts were subjected to bioassay-guided separations to isolate and identify the active drug molecules. MICs were performed to gauge the in vitro activity of the purified compounds.ResultsOnly three crude extracts (0.9%) had activity against A. baumannii and B. cepacia. Compounds from two of these extracts had MICs greater than 128 μg/ml, and further analyses were not performed. From the third extract, prepared from Leucopaxillus albissimus, 2-aminoquinoline (2-AQ) was isolated. This compound exhibited a modest MIC in vitro against strains from nine different BCC species, including multi-drug resistant clinical isolates (MIC = 8-64 μg/ml), and a weak MIC (128 μg/ml) against A baumannii. The IC50 against a murine monocyte line was 1.5 mg/ml.ConclusionThe small number of positive hits in this study suggests that finding a new drug from mushrooms to treat Gram-negative bacterial infections may be difficult. Although 2-AQ was identified in one mushroom, and it was shown to inhibit the growth of multi-drug resistant BCC isolates, the relatively high MICs (8-128 μg/ml) for both A. baumannii and BCC strains suggests that 2-AQ is not suitable for further drug development in its current form.

Field Observations and Inoculation Experiments to Determine the Nature of the Carpophoroids Associated with Entoloma abortivum and Armillaria

Carpophoroids traditionally attributed to Entoloma abortivum (“Aborted Entolomas”) represent malformed Armillaria fruiting bodies permeated by E. abortivum hyphae, as shown by our field observations and preliminary laboratory work. This contradicts the generally accepted hypothesis that carpophoroids are E. abortivum fruiting bodies colonized by Armillaria. Carpophoroids possess many of the structural characteristics of Armillaria fruiting bodies, including growth and development from rhizomorphs and the production of Armillaria-like spores on basidia. Our inoculation experiments demonstrate the ability of E. abortivum to abort the development of A. tabescens fruiting structures in vitro. In rare instances the introduction of E. abortivum disrupts A. tabescens fruiting structures to the point where they macroscopically resemble immature carpophoroids as observed in nature. If E. abortivum is a parasite of Armillaria species under natural conditions, E. abortivum may contribute to the regulation of Armillaria populations and could be investigated as a candidate for the biological control of destructive Armillaria species. We recommend “Abortive Entoloma” be used as the common name for E. abortivum.

Production of Specialty Mushrooms in North America: Shiitake and Morels

There is increasing interest in the American marketplace for mushrooms other than the common white button mushroom. The trend is toward species with more flavor. Among the new mushrooms making common appearances are the oyster mushroom and shiitake, more formally known as Pleurotus spp. and Lentinula (=Lentinus) edodes, respectively. A third type of mushroom, although less common, is the morel, Morchella spp, which is just beginning to be developed commercially. Since morels and shiitake are the more flavorful of the three mushrooms and are more difficult to produce, we focus our discussion on commercial cultivation practices for these two mushrooms and the challenges ahead for making them more readily available.