Investigation into the production of bacteriostatic substances by fungi; preliminary examination of more of the larger Basidiomycetes and some of the larger Ascomycetes

This paper may be regarded as an addendum to previous work along the same lines, more particularly the work which dealt with some 700 of the larger Basidiomycetes. Both Ascomycetes and Basidiomycetes were tested for their antibiotic properties by means of sporophore extract. Of the ninety-nine fungi tested, twenty-eight showed a positive and seventy-one a negative reaction against the test bacteria, Bacterium coli and Staphylococcus aureus.     

<Emphasis Type="Italic">Monotropastrum humile</Emphasis> var. <Emphasis Type="Italic">humile</Emphasis> is associated with diverse ectomycorrhizal Russulaceae fungi in Japanese forests

Monotropastrum humile is nearly lacking in chlorophyll and obtains its nutrients, including carbon sources, from associated mycorrhizal fungi. We analyzed the mycorrhizal fungal affinity and species diversity of M. humile var. humile mycorrhizae to clarify how the plant population survives in Japanese forest ecosystems. We classified 78 samples of adult M. humile var. humile individuals from Hokkaido, Honshu, and Kyusyu Islands into 37 root mycorrhizal morphotypes. Of these, we identified 24 types as Russula or Lactarius fungal taxa in the Russulaceae, Basidiomycetes, but we could not identify the remaining 13 types as to their genus in the Basidiomycetes. The number of fungal species on M. humile var. humile was the highest in the plant subfamily. The diversity of fungal species revealed its increased trends in natural forests at the stand level, fagaceous vegetation, and cool-temperate climate. The most frequently observed fungus colonized mainly samples collected from sub-alpine forests; the second most frequently observed fungus colonized samples collected from sub-alpine to warm-temperate forests. These results suggest that Japanese M. humile populations are associated with specific but diverse fungi that are common ectomycorrhizal symbionts of various forest canopy trees, indicating a tripartite mycorrhizal relationship in the forest ecosystem.     

Antifungal cellobiose lipid secreted by the epiphytic yeast Pseudozyma graminicola

The yeast Pseudozyma graminicola isolated from plants inhibited growth of almost all ascomycetes and basidiomycetes tested (over 270 species of ca. 100 genera) including pathogenic species. This yeast secreted a fungicidal agent, which was identified as a glycolipid composed of cellobiose residue with two O-substituents (acetyl and 3-hydroxycaproic acid) and 2,15,16-trihydroxypalmitic acid. The release of ATP from the glycolipid-treated cells indicated that this glycolipid impaired the permeability of the cytoplasmic membrane. Basidiomycetes were more sensitive to the cellobiose lipid than ascomycetes.     

Epidemic Spread of the Rust Fungus Puccinia lagenophorae and its Impact on the Competitive Ability of Senecio vulgaris in Celeriac During Early Development

The 'system management approach' of biological weed control was applied in a small-scale field experiment with celeriac (root celery), intersown with an inbred line of the annual weed Senecio vulgaris L. The naturalized rust fungus Puccinia lagenophorae Cooke (Basidiomycetes: Uredinales), a common and widespread pathogen of S. vulgaris in Europe, was introduced into parts of the plots, and its impact on the competitive balance between the crop and weed in the presence and absence of an additional herbicide treatment was studied. Competition by S. vulgaris (at a realized density of only 50 plants/m2) during the first 10 weeks of growth was substantial, reducing the fresh weight of the celeriac bulbs by 28%. The epidemic spread of the rust fungus was relatively fast, and the time to infection was similar to that in full-area applications. Inoculation with the rust fungus strongly reduced crop losses due to competition with S. vulgaris . The fresh weight of the celeriac bulbs in plots with both S. vulgaris and the fungus was not statistically diVerent from the celeriac yield in plots without S. vulgaris . This eVect was mainly the result of the reduced biomass of S. vulgaris , and not reduced survival. Infected plants may, therefore, still contribute to soil cover and may help to suppress later germinating weed species. Older plant stages were found to be infected earlier than younger stages. No significant interactions were observed between the eVects of the fungal infection and a low-dose application of the herbicide chlorbromuron on weed performance. Basic studies necessary to develop the system management approach further are discussed.     

Beetles (Coleoptera) associated with the birch fungus Piptoporus betulinus (Bull.: Fr.) P. Karst. (Basidiomycetes, Aphyllophorales) in forests of the Urals and Transurals

Detailed characteristics of the fauna of beetles associated with the birch fungus Piptoporus betulinus (Bull.: Fr.) (Basidiomycetes, Aphyllophorales) in the Urals and Transurals are given. A total of 37 species of 17 families were found, with dominating species including Diaperis boleti (Linnaeus, 1758) (Tenebrionidae), Thymalus oblongus Reitter, 1889 (Trogossitidae), and Dacne bipustulata Thunberg, 1781 (Erotylidae). The main trends in the ecological and trophic specialization of mycetophagous beetles are analyzed. Some characteristics concerning the formation of beetle assemblages at different stages of the fruit body development are revealed.     

The interaction among evolutionary forces in the pathogenic fungus Mycosphaerella graminicola

The population genetic dynamic of a species is driven by interactions among mutation, migration, drift, mating system, and selection, but it is rare to have sufficient empirical data to estimate values for all of these forces and to allow comparison of the relative magnitudes of these evolutionary forces. We combined data from a mark-release-recapture experiment, extensive population surveys, and computer simulations to evaluate interactions among these evolutionary forces in the pathogenic fungus Mycosphaerella graminicola. The results from these studies showed that, on average, the immigration rate was 0.027, the fraction of outcrossing individuals was 0.035, and the selection coefficient associated with immigrants was 0.106 each generation. We also estimated that effective population sizes for this fungus were larger than 24,000 and the mutation rate for the RFLP markers used in surveys and field experiments was approximately 4 x 10(-5). Computer simulations based on these estimates indicate that, on average, the global population of M. graminicola has reached equilibrium. Population genetic parameters including number of alleles, gene diversity, and population subdivision estimated from the computer simulations were surprisingly close to empirical estimates. Simulations also revealed that random drift is the major evolutionary force decreasing genetic variation in this fungus, followed by natural selection. The major force adding to genetic variation was mutation, followed by gene flow and sexual recombination. Gene flow played the leading role in decreasing population subdivision while natural selection was the major factor increasing population subdivision.     

Chemical constituents of Stereum subtomentosum and two other birch-associated basidiomycetes: an interspecies comparative study

Metabolites of the wood-rotting fungus Stereum subtomentosum Pouzar (Basidiomycetes, order Russulales, family Stereaceae) occurring on birch (Betula pendula Roth) trees were phytochemically investigated for the first time. Three main metabolite chemotypes present in MeOH extracts of the fruit bodies, viz. steroids, fatty acids, and water-soluble sugars, were fractionated, isolated, and identified by 1D/2D NMR-spectroscopic analyses, NMR data comparisons, and chemical correlations combined with GC/MS experiments. Thirteen compounds including two 5 alpha,8 alpha-epidioxy steroids, alpha,alpha'-trehalose, D-arabinitol, D-mannitol, and saturated/unsaturated fatty acids, were identified. Differences among S. subtomentosum and two other birch-associated fungal species, Trametes versicolor (L.: Fr.) Pilát, and Piptoporus betulinus (Bull.: Fr.) P. Karst (Basidiomycetes, order Polyporales, family Polyporaceae) were evaluated as regards the richness and abundance relationships in metabolite profiling.     

Host Range of Mycoleptodiscus terrestris, a Microbial Herbicide Candidate for Eurasian Watermilfoil, Myriophyllum spicatum

A native strain of the fungal plant pathogen Mycoleptodiscus terrestris is capable of causing under experimental conditions necrotic shoot lesions and a generalized decline and disintegration of Eurasian watermilfoil (Myriophyllum spicatum), an exotic submerged aquatic weed in North America. The potential of this fungus to cause disease on nontarget plants was evaluated on 33 species and cultivars in 11 families. The nontarget selection, intended as a Tier I group, was biased toward probable suscepts consisting of submerged, floating, and emergent aquatic species and terrestrial crop plants. The plants were exposed to the fungal mycelium formulated in alginate beads. Pathogenicity rather than phytotoxicity was the primary mode of attack by the fungus. Of the 16 nontarget aquatic species tested, the fungus was pathogenic to Hydrilla verticillata (hydrilla), Myriophyllum aquaticum (parrotfeather), and Ceratophyllum demersum (coontail), but only in hydrilla did it cause plant mortality comparable to that in Eurasian watermilfoil. The remaining 13 submerged, floating, and emergent aquatic species were unaffected. Of the 17 terrestrial species screened, none was significantly affected by the fungus in germination studies, but postemergent disease symptoms developed on seedlings of 10 species. Four of the 10, Medicago sativa (alfalfa), Lotus corniculatus (birdsfoot trefoil), Trifolium hybridum (alyce clover), and T. repens (white clover), developed disease affecting 26-50% of their tissues. Thus, this host range testing scheme, based on the concept of screening plants under the maximum hazard potential, helped to identify susceptible species. Nonetheless, it exaggerated the nontarget risk due to the small number of host species tested. Screening a taxonomically diverse and larger selection of plants as well as testing under conditions of less severe exposure to the fungal inoculum are necessary to obtain a more realistic view of the host range than presently indicated.     

Molecular weights of the ribosomal ribonucleic acid of fungi

Summary The molecular weights of the 18s and 25s ribosomal RNA components of fungi from all major classes were determined by electrophoresis in polyacrylamide gels. The molecular weight of the 18s RNA was found to be very similar for all fungi (range 0.71–0.75 million) and about 4–5% larger than the 18s RNA of HeLa cells and soybean. The molecular weight of the 25s RNA ranged between 1.45 million in the Myxomycetes and 1.30–1.31 million in the Ascomycetes and Basidiomycetes. The differences in the 25s RNA molecular weights between various classes of fungi were interpreted as being in agreement with a monophyletic origin of the Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomycetes, and independent origins for the Myxomycetes and the Oomycetes. The Hyphochytridiomycete examined could not be placed unequivocally in any group on the basis of its 25s RNA. Fungal RNA extracted with a p-aminosalicylate-triisopropylnaphthalene sulfonate-phenol mixture at 40–60°C contained a high molecular weight aggregate of the 18s and 25s ribosomal RNA; this suggested significant base sequence homology between the two ribosomal RNA species in fungi.     

An exceptional family: Ophiocordyceps‐allied fungus dominates the microbiome of soft scale insects (Hemiptera: Sternorrhyncha: Coccidae)

Hemipteran insects of the suborder Sternorrhyncha are plant sap feeders, where each family is obligately associated with a specific bacterial endosymbiont that produces essential nutrients lacking in the sap. Coccidae (soft scale insects) is the only major sternorrhynchan family in which obligate symbiont(s) have not been identified. We studied the microbiota in seven species from this family from Israel, Spain and Cyprus, by high‐throughput sequencing of ribosomal genes, and found that no specific bacterium was prevalent and abundant in all the tested species. In contrast, an Ophiocordyceps‐allied fungus sp.—a lineage widely known as entomopathogenic—was highly prevalent. All individuals of all the tested species carried this fungus. Phylogenetic analyses showed that the Ophiocordyceps‐allied fungus from the coccids is closely related to fungi described from other hemipterans, and they appear to be monophyletic, although the phylogenies of the Ophiocordyceps‐allied fungi and their hosts do not appear to be congruent. Microscopic observations show that the fungal cells are lemon‐shaped, are distributed throughout the host's body and are present in the eggs, suggesting vertical transmission. Taken together, the results suggest that the Ophiocordyceps‐allied fungus may be a primary symbiont of Coccidae—a major evolutionary shift from bacteria to fungi in the Sternorrhyncha, and an important example of fungal evolutionary lifestyle switch.     

Phylogenetic relationships among strains of the entomopathogenic fungus, Nomuraea rileyi, as revealed by partial beta-tubulin sequences and inter-simple sequence repeat (ISSR) analysis

AIMS: To elucidate the phyletic relationships among three members of the entomogenous fungal genus, Nomuraea, with an emphasis on N. rileyi. METHODS AND RESULTS: Relationships were evaluated by analysis of the beta-tubulin gene and of inter-simple sequence repeats (ISSR). The amplification product of the partial beta-tubulin gene was larger for N. atypicola than for N. rileyi, and sequencing of this gene fragment confirmed that N. atypicola possesses approximately 25 more nucleotides than N. rileyi and N. anemonoides. Based on neighbor joining and bootstrap analysis of the partial beta-tubulin gene, N. atypicola failed to form a monophyletic grouping with the other two species of Nomuraea. In contrast, the single isolate of N. anemonoides clustered with the N. rileyi isolates, and both taxa grouped with Epichloe typhina (Hypocreales: Clavicipitaceae). Results from this study suggested that N. rileyi and N. anemonoides are closely related to the Clavicipitaceae. In contrast, evidence indicated that N. atypicola is not closely related to this family, and that this taxon is not a Nomuraea. Based on the 83 polymorphic loci of ISSR, it was observed that isolates of N. rileyi from diverse geographical origins were distinctly different from both N. atypicola and N. anemonoides. Considerable heterogeneity was observed in the 18 isolates of N. rileyi tested, and several clusters contained isolates from disparate geographical locations and hosts. However, three isolates from the Philippines (three host species) and three strains isolated from velvetbean caterpillar (Anticarsia gemmatalis) larvae in South America did cluster together. Two other strains from Brazil (isolated from Spodoptera spp.) were distinct from the velvetbean caterpillar isolates from South America. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-tubulin gene was generally too conserved to resolve intraspecies variability. However, ISSR did identify polymorphisms among the isolates of N. rileyi tested. The results of this study indicate that ISSR may be used as robust molecular markers for studying the population genetics of this entomopathogenic fungus.     

Stipitate stereoid basidiocarps have evolved multiple times

Stipitate stereoid fungi are Basidiomycetes with a stipe, a spathulate-to funnel-shaped pileus, a smooth hymenophore, and hyaline, smooth spores. Representatives of the genera Cotylidia, Cymatoderma, Muscinupta, Podoscypha and Stereopsis were subjected to molecular phylogenetic analyses based on nuclear ribosomal large subunit, 5.8S and ITS sequences. For four of the genera the type species was included in analyses. Stereopsis radicans, the type species of Stereopsis, forms a lineage with the corticioid species Clavulicium globosum but could not be placed in any of the presently accepted orders within Agaricomycotina. Stereopsis vitellina falls within the Atheliales, making it the first pileus- and stipe-forming fungus recovered in this order. Cotylidia and Muscinupta again are shown to be members of the Hymenochaetales, whereas Cymatoderma and Podoscypha belong in the Polyporales. Cymatoderma is polyphyletic and Cymatoderma sensu stricto is separated from other stipitate stereoid fungi in the Polyporales, whereas the remaining Cymatoderma species are nested within a well supported clade holding all Podoscypha species but also Abortiporus biennis.     

Necrotrophic mycoparasitism of Botrytis cinerea by cellulolytic and ligninocellulolytic Basidiomycetes

Twenty-six isolates representing 17 species of aphyllophoraceous, wood-decaying Basidiomycetes and five species of agaricoid, turf-borne, thatch-decaying Basidiomycetes were screened for their abilities to degrade cellulose, lignin, and melanin by using colorimetric degradation assays on agar media. Selected ligninocellulolytic Basidiomycetes capable of degrading melanin were screened for antagonism of Botrytis cinerea Per.:Fr. The greatest inhibition of Botrytis colony and hyphal growth in vitro was observed in confrontations with Irpex lacteus (Fr.) Fr., Trametes versicolor (L.:Fr.) Pilat, and Chondrostereum purpureum (Pers.:Fr.) Pouzar. Hyphal interference and necrotrophic mycoparasitism by these ligninocellulolytic Basidiomycetes were recognized microscopically as coagulation and degeneration of Botrytis cytoplasm and as coiling and invasion of hyphae, conidiophores, and conidia, respectively. Sclerotia of B. cinerea were killed and parasitized in agar media, straw mulch, or moist sand infested separately with these three mycoparasites.     

Molecular classification and phylogenetic relationships of selected edible Basidiomycetes species

Morphological identification of edible mushrooms can sometimes prove troublesome, because phenotypic variation in fungi can be affected by substrate and environmental factors. One of the most important problems for mushroom breeders is the lack of a systematic consensus tool to distinguish different species, which are sometimes morphologically identical. Basidiomycetes as one of the largest groups of edible mushrooms have become more important in recent times for their medicinal and nutritional properties. Partial rDNA sequences, including the Internal Transcribed Spacer I-5.8SrDNA-Internal Transcribed Spacer II, were used in this study for molecular identification and assessment of phylogenetic relationships between selected edible species of the Basidiomycetes. Phylogenetic trees showed five distinct clades; each clade belonging to a separate family group. The first clade included all the species belonging to the Pleurotaceae (Pleurotus spp.) family; similarly, the second, third, fourth, and fifth clades consist of species from the Agaricaceae (Agaricus sp.), Lyophllaceae (Hypsigygus sp.), Marasmiaceae (Lentinula edodes sp.) and Physalacriaceae (Flammulina velutipes sp.) families, respectively. Moreover, different species of each family were clearly placed in a distinct sub-cluster and a total of 13 species were taken for analysis. Species differentiation was re-confirmed by AMOVA analysis (among the populations: 99.67%; within: 0.33%), nucleotide divergence, haplotyping and P value. Polymorphism occurred throughout the ITS regions due to insertion-deletion and point mutations, and can be clearly differentiated within the families as well as genera. Moreover, this study proves that the sequence of the ITS region is a superior molecular DNA barcode for taxonomic identification of Basidiomycetes.     

Genus-specific host range of Phomopsis amaranthicola (Sphaeropsidales), a bioherbicide agent for Amaranthus spp.

Phomopsis amaranthicola, a newly described species discovered in Florida, is under development as a biological control agent for weeds belonging to the genus Amaranthus. Host-range testing was performed on the fungus, using the centrifugal phylogenetic scheme, with Amaranthus hybridus as the focal plant. Forty-five accessions of 21 species in the genus Amaranthus were tested for susceptibility to infection by this fungus. Fifty-six plant species outside the genus Amaranthus were also tested. The latter included crops in which the fungus might be used as a bioherbicide, members of genera that are closely related to Amaranthus, and selections of species that have been reported as hosts of other species of the genera Phomopsis or Diaporthe. Phomopsis amaranthicola did not infect any of the plants from outside the genus Amaranthus but was highly pathogenic to several species in the genus Amaranthus. All Amaranthus species tested were infected to varying degrees by P. amaranthicola; the resulting mortality levels were also variable. Thus, P. amaranthicola appears to be host-specific to the members of the genus Amaranthus. The generic level of specificity of this pathogen renders it not only a safe bioherbicide agent but also one that could be used against several weedy Amaranthus species.     

Large-scale occurrence patterns of red-listed lichens and fungi on old oaks are influenced both by current and historical habitat density

Current occurrence patterns of species associated with ancient trees may reflect higher historical habitat densities, because the dynamics of the habitat and the colonisation-extinction processes for many inhabiting species are expected to be slow. We tested this hypothesis in southeast Sweden by analysing species occurrence per parish for twelve redlisted lichen species and nine redlisted fungus species in relation with current density of big oaks, the density of oaks in the 1830s and connectivity with parishes with the species present. For most species, the occurrence was positively related with current density of habitat (for 18 species out of 21) and parish area (for 16 species). Historical habitat density was positively related with occurrence for 11 species, while connectivity with current occurrences in the surroundings was positive for the occurrence of 12 species and negative for the occurrence of two. For lichen species the connectivity measure that best explained the variation was at a larger spatial scale as compared to fungus species. Even if the density of old oaks remains in the future, inhabiting species will most likely decline because their distribution patterns are not in equilibrium with the current habitat density. Therefore, to allow long-term persistence of inhabiting species the number of old oaks should be increased. Areas where such an increase is most urgent could be identified based on species occurrence data and current habitat density, but because species data will always be incomplete data on the historical habitat distribution is valuable.     

Chitin and chitosan from Basidiomycetes

Chitinous material was isolated from the mycelium of seven species of Basidiomycetes to evaluate the possibility of using fungal biomass as a source of chitin and chitosan. Such material was characterised for its purity, degree of acetylation and crystallinity. Chitin yields ranged between 8.5 and 19.6% dry weight and the chitosan yield was approximately 1%. The characteristics of the fungal chitins were similar to those of commercial chitin. Chitosans, with a low degree of acetylation, comparable with that of commercial chitosan, were obtained by the chemical deacetylation of fungal chitins.     

Studies on the Oxidation of Steroids by Fungi

As a result of seeking for suitable microorganisms which catalize the hydroxylation of steroids at C-11 position, Corticium sasakii, the fungus of sheath spot disease of rice, was found among the culture specimens of 28 genera of Basidiomycetes. This organism converted Reich-stein’s compound S to hydrocortisone, epi-hydrocortisone and 17α, 19-dihydroxydesoxycorticosterone.