Microbial diversity associated with <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> fruiting bodies

Endophytes play an important role in the growth and development of the host. However, the study of endophytes is mostly focused on plants and animals, and reports on microorganisms associated with fungus are relatively rare. We studied the microorganisms associated with Tricholoma matsutake fruiting bodies picked from three main T. matsutake-producing areas in Sichuan, China, by both culture-dependent and culture- independent methods. Altogether 13 fungus, 15 yeast and 14 bacterial strains were isolated from the T. matsutake fruiting bodies. The most abundant cultivable fungus, yeast and bacteria isolates were assigned as Fusarium solanis, Cryptococcus sp. and Pseudomonas sp., respectively. Terminal-restriction fragment length polymorphism analysis (T-RFLP) showed that the bacteria in T. matsutake were abundant and diverse. Betaand gamma-proteobacteria, Acidobacteria, Bacteroidetes and Sphingobacterium were found in samples from all collecting sites. Among these bacteria, we may find some strains that can promote the growth of T. matsutake.     

Cultivation and utility of <Emphasis Type="Italic">Piptoporus betulinus</Emphasis> fruiting bodies as a source of anticancer agents

Piptoporus betulinus is a wood-rotting basidiomycete used in medicine and biotechnology. However, to date, no indoor method for cultivation of this mushroom fruiting bodies has been developed. Here we present the first report of successful production of P. betulinus mature fruiting bodies in artificial conditions. Four P. betulinus strains were isolated from natural habitats and their mycelia were inoculated into birch sawdust substrate supplemented with organic additives. All the strains effectively colonized the medium but only one of them produced fruiting bodies. Moisture and organic supplementation of the substrate significantly determined the fruiting process. The biological efficiency of the P. betulinus PB01 strain cultivated on optimal substrate (moisture and organic substance content of 55 and 65 and 25 or 35 %, respectively) ranged from 12 to 16 %. The mature fruiting bodies reached weight in the range from 50 to 120 g. Anticancer properties of water and ethanol extracts isolated from both cultured and nature-derived fruiting bodies of P. betulinus were examined in human colon adenocarcinoma, human lung carcinoma and human breast cancer cell lines. The studies revealed antiproliferative and antimigrative properties of all the investigated extracts. Nevertheless the most pronounced effects demonstrated the ethanol extracts, obtained from fruiting bodies of cultured P. betulinus. Summarizing, our studies proved that P. betulinus can be induced to fruit in indoor artificial culture and the cultured fruiting bodies can be used as a source of potential anticancer agents. In this respect, they are at least as valuable as those sourced from nature.     

Dikaryotic fruiting body development in a single dikaryon of <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and the spectrum of monokaryotic fruiting types in its monokaryotic progeny

Using monokaryotic offspring from several dikaryotic parental strains, the phenomenon of monokaryotic fruiting has been previously analysed in the commercially cultivated high-quality edible mushroom Agrocybe aegerita, revealing a variety of monokaryotic fruiting types. Here, we report a single dikaryotic A. aegerita strain, A. aegerita AAE-3, and 40 monokaryons derived from it, which exhibit a wide spectrum of monokaryotic fruiting types, including a rare, previously unknown type. Advantageously, the selected parental strain A. aegerita AAE-3 completes its life cycle within three weeks by the formation of dikaryotic fruiting bodies of typical agaric morphology on malt extract agar plates. In order to morphologically compare normal dikaryotic fruiting to monokaryotic fruiting, histology was performed from all dikaryotic fruiting body development stages and all fruiting types of monokaryotic origin. No clamp connections or dikaryotic hyphae were observed within the plectenchyma of monokaryotic fruiting stages. Among the monokaryotic fruiting types of the A. aegerita AAE-3-derived monokaryons, we also characterised the rare ‘stipe type’ here described as ‘elongated initials type’ as no differentiation into a future cap and stipe was seen. The two mating-compatible monokaryotic strains representing the extremes of the fruiting type spectrum observed, A. aegerita AAE-3-13 (‘mycelium type’) and A. aegerita AAE-3-32 (‘abortive?+?true homokaryotic fruiting fruiter type, AHF?+?THF fruiter type’), were also found to readily produce oidia (arthrospores). In order to obtain a set of mating-compatible monokaryons covering the whole observed spectrum of monokaryotic fruiting, the two monokaryons A. aegerita AAE-3-40 (‘initials type’) and A. aegerita AAE-3-37 (‘elongated initials type’) have been selected for their mating compatibility with A. aegerita AAE-3-32 and A. aegerita AAE-3-13, respectively. Together with the parental dikaryotic strain A. aegerita AAE-3, this set of standard monokaryons could prove useful for studies exploring the factors regulating monokaryotic fruiting in comparison to dikaryotic mushroom formation.     

A qPCR assay that specifically quantifies <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> biomass in natural soil

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, “matsutake” mushrooms along densely developed mycelia, called “shiro,” in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A₀ horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.     

Specialized pollination by fungus gnats in the introduced population of <Emphasis Type="Italic">Aspidistra elatior</Emphasis>

The monocot genus Aspidistra comprises rhizomatous perennials that are distributed in tropical to warm temperate regions of Asia. Little is known about the pollinators of almost all the species, probably due to the inconspicuous nature of Aspidistra flowers. Nevertheless, the unusual floral morphology suggests biotic pollination, since pollen grains are hidden under each flower’s stigma. Aspidistra elatior has been suspected to have a very peculiar pollination ecology. So far, pollination by mollusks, crustaceans, or collembolans has been suspected. However, a recent study showed that A. elatior is mainly pollinated by species of fungus gnats in Kuroshima Island, southern Japan, which is its natural habitat. Here, we investigated the pollination ecology of A. elatior in Shiga Prefecture, central Japan, which is the introduced population, to reveal whether fungus gnats are also the main pollinator in the introduced population. Our study confirmed fungus gnats pollination in the investigated pollination. Furthermore, the main pollinators (i.e., Cordyla sixi and Bradysia sp.) are the same in both Kuroshima and Shiga Prefecture. Therefore, A. elatior mainly depends on a narrow taxonomic group of fungus gnats for pollination. In contrast, we failed to document any terrestrial amphipods visiting the A. elatior flowers, in spite of a relatively high fruit set in natural conditions. This fact will refute the amphipod pollination hypothesis proposed by previous studies. We consider that A. elatior is pollinated by fungus gnats through fungal mimicry, due to its superficial similarity to mushroom fruiting bodies and strong, musky floral scent.     

Improved cordycepin production in a liquid surface culture of <Emphasis Type="Italic">Cordyceps militaris</Emphasis> isolated from wild strain

A Cordyceps militaris NBRC 10352-3 strain that was isolated from C. militaris NBRC10352 produced 68 mg of cordycepin from 100 mL of medium, which was the highest level of cordycepin among 60 isolates from three C. militaris (NBRC 9787, 100741 and 103752) strains. Interestingly, a liquid surface culture of C. militaris NBRC 103752-3 produced 2-fold cordycepin to that in a submerged culture. Cordycepin production was significantly affected by specific surface area (SSA) in the liquid surface culture, and 120.9 mg of cordycepin was produced on SSA of 1.57/cm (from 50 mL medium). The addition of glycine and adenine as an additive to its culture medium was optimized by an experimental design. When 6.75 g/L of adenine was added to the culture, 312.2 mg of cordycepin (apparent concentration: 6.2 g/L) was produced from 50 mL medium, improving the cordycepin production by 4.6-fold. In this study, the production and productivity of cordycepin were significantly improved in C. militaris wild type by a single cell colony isolation and additives without adopting any mutational technologies. This C. militaris NBRC 10352-3 strain can be used as a new cordycepin-hyperproducing one, instead of a cordycepin-hyperproducing mutant.     

Targeted Gene Deletion in <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Using the Split-Marker Approach

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.     

Why does the bioluminescent fungus <Emphasis Type="Italic">Armillaria mellea</Emphasis> have luminous mycelium but nonluminous fruiting body?

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin—the key component of fungal bioluminescent system—was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.     

Trypsin-Like Proteinases and Trypsin Inhibitors in Fruiting Bodies of Higher Fungi

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).     

The ectomycorrhizal status of a tropical black bolete, <Emphasis Type="Italic">Phlebopus portentosus</Emphasis>, assessed using mycorrhizal synthesis and isotopic analysis

Phlebopus portentosus is one of the most popular wild edible mushrooms in Thailand and can produce sporocarps in the culture without a host plant. However, it is still unclear whether Phlebopus portentosus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus. In this study, Phlebopus portentosus sporocarps were collected from northern Thailand and identified based on morphological and molecular characteristics. We combined mycorrhizal synthesis and stable isotopic analysis to investigate the trophic status of this fungus. In a greenhouse experiment, ECM-like structures were observed in Pinus kesiya at 1 year after inoculation with fungal mycelium, and the association of Phlebopus portentosus and other plant species showed superficial growth over the root surface. Fungus-colonized root tips were described morphologically and colonization confirmed by molecular methods. In stable isotope measurements, the δ¹³C and δ¹⁵N of natural samples of Phlebopus portentosus differed from saprotrophic fungi. Based on the isotopic patterns of Phlebopus portentosus and its ability to form ECM-like structures in greenhouse experiments, we conclude that Phlebopus portentosus could be an ECM fungus.     

Diversity and antifungal activity of endophytic bacteria associated with <Emphasis Type="Italic">Panax ginseng</Emphasis> seedlings

Ginseng (Panax ginseng C.A. Meyer) is a medicinal crop that requires a long culture time before it is ready to harvest, thus generating high economic and environmental costs. Symbiotic bacteria that live within the plant provide the host plant with many advantages in terms of metabolism and disease resistance. Here, we isolated endophytic bacteria from various tissues of P. ginseng seedlings using a culture-dependent method and we compared their tissue distribution. In addition, their antimicrobial activity against two fungal pathogens was investigated. Based on 16S rRNA sequencing, we identified 21 bacterial strains from ginseng seedlings. Leaves and rhizomes showed higher bacterial species diversity than root bodies and tails. While Bacillus strains were detected in all tissues, Xanthomonas and Micrococcaceae strains were specifically isolated from rhizome and leaf tissues, respectively. Fourteen bacterial strains showed antimicrobial activities against Cylindrocarpon destructans and/or Botrytis cinerea, with different activities. Among them, two strains (PgKB29 and PgKB35) showed strong antimicrobial activities against both fungi. Taken together, these results provide a better understanding of endophytic bacteria in P. ginseng seedlings and suggest the possibility of biological control of fungal pathogens using endophytic bacteria.     

Fungal load in <Emphasis Type="Italic">Bradysia agrestis</Emphasis>, a phytopathogen-transmitting insect vector

Larvae of Bradysia agrestis, a phytopathogen-transmitting insect vector in East Asia, were sampled from geographically (ecologically) segregated regions to identify their intestinal fungal flora. A total of 24 fungal strains were isolated from the insect vectors and selected based on morphological differences. In addition, 38 fungal strains were isolated from the ulcerated parts of invaded host plants by the same method, revealing the impact of vector fungal flora on their host plants. For molecular identification of the fungi, internal transcribed spacer (ITS) regions were amplified and sequenced. Their sequences were compared with sequences of other fungal strains obtained from NCBI GenBank, and their phylogeny was determined. The dominant fungal genera in the insect vector were Penicillium (25%), Aspergillus (21%), and Cladosporium (13%). In plant scar lesions, most fungal isolates belonged to the genera Fusarium (31.6%), Phoma (7.8%), Didymella (7.8%), and Epicoccum (7.8%). Fungal genera in vectors or host plant lesions differed by study site. Furthermore, diversity indices by study site showed clear differences based on Margalef’s richness (2.06, 2.40, 3.04), and Menhinick’s (1.89, 2.12, 2.53), and Simpson’s indices (0.14, 0.07, 0.07). In addition, common fungal strains in insect vectors were found to be closely related to members of the genera Cladosporium, Penicillium, or Aspergillus. Among these strains, those showing the highest homology with Aspergillus terreus, which regarded as beneficial fungal genera could be considered ideal paratransgenesis candidates. Some other fungal strains from vectors or ulcerated plant parts from each study site after B. agrestis invasion may be harmful in terms of plant disease or agrifood safety. This study provides information on the fungal microbiota of B. agrestis, an emerging problem in East Asia, and proposes paratransgenesis candidates to control this insect vector. Furthermore, potential transferable pathogens or commensal fungi were revealed by comparing the fungal biota between the insect gut and the ulcerated parts of the invaded host plants.     

Antibiotic activity of bacterial endobionts of basidiomycete fruit bodies

Bacterial strains (93 isolates) capable of growth on full-strength nutrient media were isolated from 86 fungal fruit bodies collected in the Moscow region. Antimicrobial activity of the endobiont isolates against 12 bacterial and fungal test strains (including drug-resistant ones) was studied in submerged cultures. Most of the strains (84.9%) were found to produce antibiotic compounds with different antimicrobial properties, including antifungal activity in 18.3% of the strains. Morphological characteristics and analysis of the 16S rRNA gene sequences were used to determine the taxonomic position of 16 bacterial strains of the following 10 species: Bacillus subtilis, Ewingella americana, Pseudomonas sp., Stenotrophomonas maltophilia, as well as Achromobacter spanius, B. licheniformis, Hafnia paralvei, Micrococcus terreus, Nocardia coeliaca, and St. rhizophila, which have not been previously known to be endobionts of basidiomycete fruit bodies. Antimicrobial activity of A. spanius, E. americana, H. paralvei, M. terreus, N. coeliaca, and St. rhizophila has not been reported previously. Complex mechanisms of symbiotic relations between fungi and bacteria, including those associated with antibiotic formation, probably developed in the course of co-evolution.