Fungi are essential components of soil microbial communities and have a crucial role in biogeochemical processes. Alpine regions are sensitive to climate change, and the importance of changes in fungal community composition along altitudinal gradients in alpine regions is hotly debated.We used 454 pyrosequencing approaches to investigate the fungal communities at 1600, 2300, 2800, 3000, and 3900 m above sea level along an altitudinal gradient on Mount Gongga.The results showed that Agaricomycetes, Sordariomycetes, and Tremellomycetes are the dominant classes at all sampling sites. Operational taxonomic unit richness decreased with increasing altitude, and the fungal communities were clustered into three groups that corresponded to altitudes of, i.e., 1600, 2300, and above 2800 m. The evenness of fungi was not significantly correlated with altitude, whereas beta diversities were significantly correlated with altitude. The distance-based redundancy analysis and Mantel test indicated that the composition of fungal assemblages was mostly driven by altitude and temperature.Our results indicated that ecological processes possibly related to altitude and temperature play an important role in structuring fungal biodiversity along the elevational gradient. Our results highlight that different microbes may respond differently to environmental gradients.

The alpine meadow has received mounting attention due to its degradation resulting from overgrazing on the Tibetan Plateau. However, belowground biotic characteristics under varied grazing stresses in this ecosystem are poorly understood.Here, the responses of soil protozoan abundance, community composition, microbial biomass, and enzyme activity to five grazing patterns including (1) artificial grassland without grazing (AG), (2) winter grazing (WG), (3) grazing for 7 months within a fence (GF), (4) continuous grazing for a whole year (CG), and (5) natural heavy grazing (HG) were investigated for two continuous years. Soil protozoan community composition was investigated using the most possible number (MPN) method, and soil microbial biomass and enzyme activity were analyzed using chloroform fumigation extraction and substrate utilization methods, respectively. Multivariate statistical analysis, the analysis of variance (ANOVA), multiple comparisons, and correlation analysis were together performed.The WG treatment had the highest abundance of total protozoa (2342–2524 cell g−1). Compared with AG treatment, HG treatment significantly reduced the abundance of soil total, flagellate and ciliate protozoa, and protease activities in 2012 and 2013. Significantly, lower soil microbial biomass nitrogen (MBN) was also observed in the HG (6.60 and 14.6 mg N kg−1) than those in other four treatments (22.3–82.9 mg N kg−1) both in 2012 and 2013, whereas significantly higher microbial biomass carbon (MBC) was observed in HG than that in AG treatment in 2012. Moreover, significantly positive correlations were detected between the abundance of soil protozoa and soil moisture, pH, organic C, total N, and MBN. Our results indicated that soil protozoa showed a negative response to increasing grazing intensities and therefore, suggesting that aboveground grazing practices also exerted strong impact on belowground protozoa, not only on soil microbial characteristics.Soil protozoan community composition was apparently different between the HG treatment and other four grazing patterns and was potentially impacted by altered soil properties and MBC and/or MBN. Our results suggested that moderate grazing may sustain better belowground biotic diversity and ecosystem functioning in this alpine meadow on the Tibetan Plateau.

The phylum Neocallimastigomycota contains eight genera (about 20 species) of strictly anaerobic fungi. The evolutionary relationships of these genera are uncertain due to insufficient sequence data to infer their phylogenies. Based on morphology and molecular phylogeny, thirteen isolates obtained from yak faeces and rumen digesta in China were assigned to Neocallimastix frontalis (nine isolates), Orpinomyces joyonii (two isolates) and Caecomyces sp. (two isolates), respectively. The phylogenetic relationships of the eight genera were evaluated using complete ITS and partial LSU sequences, compared to the ITS1 region which has been widely used in this phylum in the past. Five monophyletic lineages corresponding to six of the eight genera were statistically supported. Isolates of Caecomyces and Cyllamyces were present in a single lineage and could not be separated properly. Members of Neocallimastigomycota with uniflagellate zoospores represented by Piromyces were polyphyletic. The Piromyces-like genus Oontomyces was consistently closely related to the traditional Anaeromyces, and separated the latter genus into two clades. The phylogenetic position of the Piromyces-like genus Buwchfawromyces remained unresolved. Orpinomyces and Neocallimastix, sharing polyflagellate zoospores, were supported as sister genera in the LSU phylogeny. Apparently ITS, specifically ITS1 alone, is not a good marker to resolve the generic affinities of the studied fungi. The LSU sequences are easier to align and appear to work well to resolve generic relationships. This study provides a comparative phylogenetic revision of Neocallimastigomycota isolates known from culture and sequence data.

During a survey of fungicolous fungi, a novel taxon from the surface of stroma of an unidentified Xylaria species was collected. Phylogenetic analyses showed that this taxon clustered with Calcarisporium sp. and C. arbuscula isolates, but was resolved as a distinct species. A detailed morphological examination coupled with phylogenetic analysis indicated that the taxon represented a new species. Calcarisporium xylariicola sp. nov. is thus introduced. The new taxon is characterized by short conidiophores with swollen bases and less length/width ratio of conidia that distinguish it from other Calcarisporium species. Calcarisporium is presently placed in Hypocreales genera, incertae sedis genus. Species in the genus are largely fungicolous, or occasionally caulicolous or foliicolous, and have hyaline, erect, verticillate conidiophores and sympodial, polyblastic conidiation. A phylogenetic analysis of combined SSU, ITS, LSU, TEF and RPB2 sequence data from Calcarisporium species and other taxa in Hypocreales indicate that Calcarisporium is a distinct lineage from other families. Therefore, a new family, Calcarisporiaceae, in Hypocreales is introduced.

•Chitosan enhanced biocontrol efficacy of H. minnesotensis to parasitize SCNs.•Checking DNA amount of H. minnesotensis by real-time PCR.•Chitosan and H. minnesotensis changed the structure of rhizosphere microbial communities.•Chitosan and H. minnesotensis increased the relative abundance of suppressive bacteria and fungi.Chitosan, a deacetylated form of chitin, has been shown to enhance the biocontrol efficacy of nematophagous fungi to parasitize nematodes. However, the mechanism by which nematode endoparasitic fungi with chitosan suppresses disease caused by the soybean cyst nematode (SCN; Heterodera glycines) through the rhizosphere microbiota remains unclear. In this study, we performed a greenhouse experiment with soybean grown in five soil treatments. Strong synergetic suppression of the SCN egg density was observed using a combined treatment of H. minnesotensis and chitosan (SHg+Hm+Ch), followed by treatments with H. minnesotensis (SHg+Hm) or chitosan (SHg+Ch). The parasitism of SCN juveniles (J2) and amount of H. minnesotensis DNA were significantly increased by the presence of chitosan in soil. The bacterial and fungal communities of the soybean rhizosphere, profiled by the Illumina high-throughput pyrosequencing, showed that the rhizobacterial OTU richness was decreased by SHg (nematode infested), SHg+Hm, SHg+Ch, and SHg+Hm+Ch treatments, and fungal richness was increased by the same treatments except for SHg+Ch, compared to the mock-inoculated plants (S). Overall, the bacterial and fungal community structures were different among the different treatments, and soil containing chitosan was distinct from H. minnesotensis with and without chitosan as well as from nematode infested or uninfested soils. Except for the enhancement of H. minnesotensis, chitosan also increased the bacterial genus Streptomyces and fungal genera Purpureocillium and Pochonia, which have been documented to suppress SCN in the soybean rhizosphere. Combined application of H. minnesotensis and chitosan led to shifts in the abundance of the core rhizobacterial and rhizofungal taxa that respond to soil suppression of SCN. In this study, we provide evidence that chitosan shifts the abundance of resident and inoculated biocontrol agents in the rhizosphere to suppress pathogens.

Tristratiperidium microsporum gen. et sp. nov. (Xylariales) is introduced to accommodate a taxon isolated from dead leaves of Arundo plinii, collected in Italy. The sexual and asexual morphs are described and illustrated and compared with similar taxa. Phylogenetic analysis of combined Internal Transcribed Spacer (ITS) and Large Subunit (LSU) rRNA sequence data show the relationships of T. microsporum with other genera in the Xylariales. Tristratiperidium microsporum clusters with the hyphomycete Subramaniomyces fusisaprophyticus in the order Xylariales, in the clade comprising Apiosporaceae, Hyponectriaceae, Melogrammataceae, and Pseudomassariaceae. The phylogenetic relationships of T. microsporum and allied fungi are discussed and its placement in the Xylariales genera incertae sedis is suggested.

Repetitive DNA sequences make up a significant portion of all genomes and may occur in intergenic, regulatory, coding, or even intronic regions. Partial sequences of a serine protease gene csp1 was previously used as a population genetic marker of the Chinese caterpillar fungus Ophiocordyceps sinensis, but its first intron region was excluded due to ambiguous alignment. Here in this study, we report the presence of a minisatellite OsMin1 within this intron, where a 20(19)-bp repeat motif is duplicated two to six times in different isolates. Fourteen intron alleles and 13 OsMin1 alleles were identified among 125 O. sinensis samples distributed broadly on the Tibetan Plateau. Two OsMin1 alleles were prevalent, corresponding to either two or five repeats of the core sequence motif. OsMin1 appears to be a single locus marker in the O. sinensis genome, but its origin is undetermined. Abundant recombination signals were detected between upstream and downstream flanking regions of OsMin1, suggesting that OsMin1 mutate by unequal crossing over. Geographic distribution, fungal phylogeny, and host insect phylogeny all significantly affected intron distribution patterns but with the greatest influence noted for fungal genotypes and the least for geography. As far as we know, OsMin1 is the first minisatellite found in O. sinensis and the second found in fungal introns. OsMin1 may be useful in designing an efficient protocol to discriminate authentic O. sinensis from counterfeits.

The mutualism between fungus-growing animals and fungi is a classic example of a complex interspecies association. A handful of insects, notably the well-recognized fungus-farming ants, termites and beetles, have developed advanced agriculture, which includes seeding new gardens with crop propagules, improving growth conditions and protecting and harvesting the fungal crop. More examples, which could be called ‘proto-fungiculture’, involve fewer adaptations, as exemplified by marine snails that farm intertidal fungi on marsh grass. Recent work has indicated that the solitary leaf-rolling weevil Euops chinensis (family Attelabidae) has a protofarming symbiosis with the mycangial fungus Penicillium herquei (family Trichocomaceae). In this study, we investigated how the weevils create cradles (leaf-rolls) for their offspring and protect the fungal garden. We describe new specialized structures and behaviors that E. chinensis females use for leaf-rolling and fungus inoculation. The fungus P. herquei produces the antibiotic (+)-scleroderolide in laboratory culture and in leaf-rolls, which can serve to inhibit microbial ‘weeds’ and pests, thus protecting the fungal garden against potential infection. The fungiculture of E. chinensis differs from other advanced insect fungiculture systems because female weevils do not continuously tend the inoculated microbe and do not depend nutritionally on the fungus. The defensive role of the cultivated fungus makes the attelabid weevils exceptional in ‘proto-fungiculture’ animals.

Functional response is a key index in determining the population fluctuation in predation. However, the lack of operable research system limits the studies on functional response of fungal predators. Hirsutella rhossiliensis is a dominant parasite of the soybean cyst nematode, Heterodera glycines. In a soil microcosm bioassay, we determined fungal biomass at different days within 21 days after inoculation, and parasitism rate of H. glycines by the fungus was determined. The functional response of H. rhossiliensis to H. glycines was established and found to be Holling’s type III, which was influenced by mycelial densities. Meanwhile, we conducted anti-fungal analysis of metabolic fractions extracted from H. rhossiliensis to explain the potential mechanism of the intraspecific competition illustrated by functional response. The result of anti-fungal experiments indicated that the fungal predators had more complicated interaction at population level than expected, which might be regulated by self-inhibition metabolite(s). This study was the first functional response study of fungal predators in microcosm. With the increasing recognition of emerging fungal threats to animal, plant, and ecosystem health, the methodologies and hypotheses proposed in this study might inspire further research in fungal ecology.

Anthostomella has long been regarded as a large, but polyphyletic genus in the family Xylariaceae, but species in this group generally lack phylogenetic data. In this study, 14 anthostomella-like taxa collected from Italy, were studied using both morphology and molecular data. Single ascospore isolates were obtained and the asexual morphs of five taxa established. The phylogenetic relationships of the xylariaceous taxa were inferred using combined ITS, RPB2, β tubulin and LSU gene regions. We introduce new sequence data for 24 with included the 14 new anthostomella-like taxa. The subfamilies Hypoxyloideae and Xylarioideae within Xylariaceae were recognized as the two major clades with high bootstrap support. Within the two clades 21 subclades were resolved and the anthostomella-like taxa clustered in five of these subclades indicating that the genus is polyphyletic. Anthostomella sensu stricto comprised A. forlicesenica, A. formosa, A. helicofissa, A. rubicola and A. obesa. The A. formosa and A. rubicola collections morphologically closely resemble the type specimens and therefore we designate reference specimens. Three new species Anthostomella helicofissa, A. forlicesenica and A. obesa are also introduced. Four distinct lineages of anthostomella-like taxa correspondent to four new genera, Anthocanalis, Brunneiperidium, Lunatiannulus and Pyriformiascoma, which are also introduced, while one clustered in Astrocystis and is introduced as a new species. Keys to the new anthostomella-like genera and species examined in this study are provided.

Species of Colletotrichum are associated with anthracnose of a wide range of host plants including cultivated and wild tropical fruits. The genetic and ecological diversity of species associated with wild fruits are poorly explored, as compared to those associated with pre and postharvest diseases of cultivated fruits. In the present study, isolates of Colletotrichum were obtained from commercially available cultivated fruits, wild fruits (from native trees in natural habitats) and a few herbaceous hosts collected in northern Thailand. These isolates were initially characterized based on analysis of complete sequences of nuclear ribosomal internal transcribed spacer (ITS), into the genetically defined species complexes of Colletotrichum gloeosporioides, C. acutatum, C. boninense and C. truncatum. The isolates were primarily identified in the C. gloeosporioides species complex, based on a strongly supported clade within the ITS gene tree and were further characterized using multi-gene phylogenetic analyses and morphology. Phylogenetic analyses of ITS, partial sequences of actin (ACT), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS) and β-tubulin (TUB2) genetic markers were performed individually and in combination. Colletotrichum gloeosporioides sensu stricto was identified from lime (Citrus aurantifolia) and rose apple (Syzygium samarangense). Colletotrichum fructicola was isolated from dragon fruit (Hylocerous undatus) and jujube (Ziziphus sp.). Colletotrichum endophytica was found only from an unknown wild fruit. We observed a considerable genetic and host diversity of species occurring on tropical fruits within the clade previously known as Colletotrichum siamense sensu lato. The clade consists of isolates identified as pre and postharvest pathogens on a wide range of fruits, including coffee (Coffea arabica), custard apple (Annona reticulata), Cerbera sp., figs (Ficus racemosa) mango (Mangifera indica), neem (Azadirachta indica) and papaya (Carica papaya) and was the dominant group of species among most wild fruits studied. With the exception of one isolate from banana, which grouped in the C. siamense clade, all the other isolates were identified as Colletotrichum musae. A new species, Colletotrichum syzygicola, associated with Syzygium samarangense in Thailand, is introduced with descriptions and illustrations. This study highlights the need to re-assess the evolutionary relationships of Colletotrichum species occurring on cultivated and wild fruits with emphasis on their ecology and cryptic diversification including sampling at regional and global scales.

Advances in next-generation sequencing technologies are providing longer nucleotide sequence reads that contain more information about phylogenetic relationships. We sought to use this information to understand the evolution and ecology of bacterioplankton at our long-term study site in the Western Sargasso Sea. A bioinformatics pipeline called PhyloAssigner was developed to align pyrosequencing reads to a reference multiple sequence alignment of 16S ribosomal RNA (rRNA) genes and assign them phylogenetic positions in a reference tree using a maximum likelihood algorithm. Here, we used this pipeline to investigate the ecologically important SAR11 clade of Alphaproteobacteria. A combined set of 2.7 million pyrosequencing reads from the 16S rRNA V1–V2 regions, representing 9 years at the Bermuda Atlantic Time-series Study (BATS) site, was quality checked and parsed into a comprehensive bacterial tree, yielding 929 036 Alphaproteobacteria reads. Phylogenetic structure within the SAR11 clade was linked to seasonally recurring spatiotemporal patterns. This analysis resolved four new SAR11 ecotypes in addition to five others that had been described previously at BATS. The data support a conclusion reached previously that the SAR11 clade diversified by subdivision of niche space in the ocean water column, but the new data reveal a more complex pattern in which deep branches of the clade diversified repeatedly across depth strata and seasonal regimes. The new data also revealed the presence of an unrecognized clade of Alphaproteobacteria, here named SMA-1 (Sargasso Mesopelagic Alphaproteobacteria, group 1), in the upper mesopelagic zone. The high-resolution phylogenetic analyses performed herein highlight significant, previously unknown, patterns of evolutionary diversification, within perhaps the most widely distributed heterotrophic marine bacterial clade, and strongly links to ecosystem regimes.

Whether fungal community structure depends more on historical factors or on contemporary factors is controversial. This study used culture-dependent and -independent (polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)) methods to assess the influence of historical and contemporary factors on the distributions of fungi in the wetland sediments at 10 locations along the Changjiang River and at 10 other locations in China. The culture-dependent approach detected greater species diversity (177 operational taxonomic units (OTUs)) than PCR-DGGE analysis (145 OTUs), and the species in the genera of Penicillium (relative frequency=16.8%), Fusarium (15.4%), Aspergillus (7.6%), Trichoderma (5.8%) and Talaromyces (4.2%) were dominant. On the basis of DGGE data, fungal diversity along the Changjiang River increased from upstream to downstream; altitude explained 44.8% of this variation in diversity. And based on the data from all 20 locations, the fungal communities were geographically clustered into three groups: Southern China, Northern China and the Qinghai-Tibetan Plateau. Multivariate regression tree analysis for data from the 20 locations indicated that the fungal community was influenced primarily by location (which explained 61.8% of the variation at a large scale), followed by total potassium (9.4%) and total nitrogen (3.5%) at a local scale. These results are consistent with the concept that geographic distance is the dominant factor driving variation in fungal diversity at a regional scale (1000–4000 km), whereas environmental factors (total potassium and total nitrogen) explain variation in fungal diversity at a local scale (<1000 km).

Some DNA sequences in the International Nucleotide Sequence Databases (INSD) are erroneously annotated, which has lead to misleading conclusions in publications. Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a fungus endemic to the Tibetan Plateau, and more than 100 populations covering almost its distribution area have been examined by us over recent years. In this study, using the data from authentic materials, we have evaluated the reliability of nucleotide sequences annotated as O. sinensis in the INSD. As of October 15, 2012, the INSD contained 874 records annotated as O. sinensis, including 555 records representing nuclear ribosomal DNA (63.5 %), 197 representing protein-coding genes (22.5 %), 92 representing random markers with unknown functions (10.5 %), and 30 representing microsatellite loci (3.5 %). Our analysis indicated that 39 of the 397 internal transcribed spacer entries, 27 of the 105 small subunit entries, and five of the 53 large subunit entries were incorrectly annotated as belonging to O. sinensis. For protein-coding sequences, all records of serine protease genes, the mating-type gene MAT1-2-1, the DNA lyase gene, the two largest subunits of RNA polymerase II, and elongation factor-1α gene were correct, while 14 of the 73 β-tubulin entries were indeterminate. Genetic diversity analyses using those sequences correctly identified as O. sinensis revealed significant genetic differentiation in the fungus although the extent of genetic differentiation varied with the gene. The relationship between O. sinensis and some other related fungal taxa is also discussed.

The new naphthalenone derivatives perenniporides A–D (1–4) were isolated from solid cultures of a fungus Perenniporia sp. inhabiting the larva of Euops chinesis, a phytophagous weevil with high host specificity to the medicinal plant Fallopia japonica. The structures of 1–4 were elucidated primarily by NMR experiments, and 1 was confirmed by X-ray crystallography. The absolute configuration of 1 and 2 was assigned by electronic circular dichroism (ECD) calculations, whereas that of the C-10 tertiary alcohol in 3 was deduced via the CD data of the in situ formed [Rh2(OCOCF3)4] complex and supported by the ECD data. Compound 1 showed antifungal activity against five plant pathogens.

Carnivorism is one of the basic life strategies of fungi. Carnivorous fungi possess the ability to trap and digest their preys by sophisticated trapping devices. However, the origin and development of fungal carnivorism remains a gap in evolution biology. In this study, five protein-encoding genes were used to construct the phylogeny of the carnivorous fungi in the phylum Ascomycota; these fungi prey on nematodes by means of specialized trapping structures such as constricting rings and adhesive traps. Our analysis revealed a definitive pattern of evolutionary development for these trapping structures. Molecular clock calibration based on two fossil records revealed that fungal carnivorism diverged from saprophytism about 419 Mya, which was after the origin of nematodes about 550–600 Mya. Active carnivorism (fungi with constricting rings) and passive carnivorism (fungi with adhesive traps) diverged from each other around 246 Mya, shortly after the occurrence of the Permian–Triassic extinction event about 251.4 Mya. The major adhesive traps evolved around 198–208 Mya, which was within the time frame of the Triassic–Jurassic extinction event about 201.4 Mya. However, no major carnivorous ascomycetes divergence was correlated to the Cretaceous–Tertiary extinction event, which occurred more recently (about 65.5 Mya). Therefore, a causal relationship between mass extinction events and fungal carnivorism evolution is not validated in this study. More evidence including additional fossil records is needed to establish if fungal carnivorism evolution was a response to mass extinction events.

Predacious fungi form specialized hyphae structures to trap nematodes and other microscopic animals. Among the six kinds of trapping devices, the constricting ring is the only one that actively captures nematodes. When a nematode enters the aperture of the ring, which is formed by three cells, the cells rapidly triple their volume, close the aperture and hold the nematode in place. Hyphae then penetrate and consume the nematode. This paper reviews the data and hypotheses on conserving the evolution of constricting rings and their cytological and molecular mechanisms.

The Chinese caterpillar fungus Ophiocordyceps sinensis, endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. Genetic differentiation within this fungus was observed; however, due to lack of highly efficient molecular markers, the overall genetic structure of this fungus has not been clarified. In this study, a shotgun genomic library of O. sinensis was constructed, and >181,848 nt were analyzed from >250 random clones. Primers from 33 sequenced fragments were then designed to amplify O. sinensis samples collected from widely separated regions on the Tibetan Plateau. Ten of the 33 fragments had no amplification or poor sequencing quality from all or certain samples. Sequence variations of the remaining 23 fragments among different samples were investigated in detail. Three fragments (OSRC14, OSRC19, and OSRC32) were the most variable with 7–43 single-nucleotide polymorphism (SNP) sites, representing the SNP frequency of 1.2–6.7 % per nucleotide site. These three fragments have the potential to be useful molecular markers for studying the population genetics of O. sinensis. These results also showed that constructing and screening a shotgun genomic library was an efficient approach to identify novel molecular markers from non-model organisms.

The rice blast fungus Magnaporthe oryzae forms a specialized infection structure called an appressorium to breach the host-plant epidermis for successful infection. In this study, a mutant defective in appressorial penetration was isolated by a mutagenesis approach, in which an exogenous DNA fragment was found to be inserted into the first exon of MoCRC1. This gene encodes a putative carnitine–acylcarnitine carrier protein that is widely conserved among eukaryotic organisms. Deletion of MoCRC1 severely reduces appressorium turgor generation, appressorial penetration, and development of infection hyphae. The null mutant of MoCRC1 lost pathogenicity on intact and abraded host leaves. MoCRC1 was also found to be required for growth on minimal medium containing sodium acetate or olive oil. Moreover, the transformed MoCrc1–eGFP fusion protein was expressed throughout the infection process. Our results suggest that the carnitine–acylcarnitine carrier protein plays vital roles in appressorium-mediated infection and is essential for pathogenesis of M. oryzae and perhaps other phytopathogenic fungi.

The genus Diaporthe (Phomopsis) includes important plant pathogenic fungi with wide host ranges and geographic distributions. In the present study, phylogenetic species recognition in Diaporthe is re-evaluated using a multi-locus phylogeny based on a combined data matrix of rDNA ITS, and partial sequences from the translation elongation factor 1-α (EF 1-α), β tubulin (TUB) and calmodulin (CAL) molecular markers. DNA sequences of available ex-type cultures have been included, providing a multi-locus backbone tree for future studies on Diaporthe. Four utilizable loci were analyzed individually and in combination, and ITS, EF 1-α and multi-locus phylogenetic trees are presented. The phylogenetic tree inferred by combined analysis of four loci provided the best resolution for species as compared to single gene analysis. Notes are provided for nine species previously known in Phomopsis that are transferred to Diaporthe in the present study. The unraveling of cryptic species complexes of Diaporthe based on Genealogical Concordance Phylogenetic Species Recognition (GCPSR) is emphasized.

Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a highly valued medicinal fungus. This entomopathogen has a limited distribution, has been overharvested in the wild, and its stromata have not been artificially cultivated. Another entomopathogenic fungus, Cordyceps militaris (commonly known as orange caterpillar fungus), has chemical capacities similar to those of O. sinensis, but unlike O. sinensis, its stromata can be easily cultivated. Consequently, C. militaris is being studied as an alternative to O. sinensis, and the large-scale production of stromata is receiving substantial attention. Significant research has been conducted on the genetic resources, nutritional and environmental requirements, mating behavior, and biochemical and pharmacological properties of C. militaris. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biology. The current paper reviews the biological aspects of the fungus including host range, mating system, cytology and genetics, insect- and non-insect nutritional requirements, environmental influence on stroma development, and commercial development.

During a recent survey of fungi colonizing juveniles of the soybean cyst nematode, Heterodera glycines, a fungus producing spiny chlamydospores and monosporous sporangia was isolated. Based on morphology and sequences of the rDNA ITS region and 18S and 28S genes, the fungus greatly resembles some species of Mortierella. Although the new isolate’s spiny chlamydospores and delicate arachnoid-like mycelium are very similar to those of Mortierella, it differs significantly from Mortierella in its overall colony appearance and odour, unispored sporangium with a small columella, chlamydospores born laterally on short hyphae, and especially in its phylogenetic position. A new genus and new species, Echinochlamydosporium variabile, in Mortierellaceae (Mortierellales, Zygomycota) are proposed. A key to the genera of Mortierellaceae is provided.

Bioactive proteins represent an important group of functional agents in medicinal mushrooms. Trametes versicolor (L.) Lloyd is a mushroom frequently used in traditional Chinese medicine for its anti-tumor and immunomodulatory activities. A new immunomodulatory protein from T. versicolor, named TVC, was purified by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed a single band with a molecular weight of 15.0 kD. Native polyacrylamide gel analysis revealed a band at 30 kD, indicating that TVC exists in solution as a homodimer. Isoelectric focusing showed that TVC was an acidic protein with an isoelectric point of 4.0. TVC was found to lack carbohydrate modifications (based on periodic acid/Schiff staining) and it does not agglutinate mouse red blood cells, suggesting that TVC is not a lectin-like protein. Biological activity assays demonstrated that TVC can enhance the proliferation of splenocytes, while it has no stimulatory effects on CD4+ and CD8+ T cells. TVC markedly increases the proliferation of human peripheral blood lymphocytes in a dose-dependent manner and enhances the production of both nitric oxide and tumor necrosis factor-alpha by lipopolysaccharide-induced murine macrophages. The results indicate that TVC is an immunostimulant that can boost immune response. Comparison of the N-terminal amino acid residues and mass spectrometry results with the protein database revealed no homologous proteins.

The development of fungal biopesticides requires the efficient production of large numbers spores or other propagules. The current study used published information concerning carbon concentrations and C:N ratios to evaluate the effects of carbon and nitrogen sources on sporulation of Paecilomyces lilacinus (IPC-P and M-14) and Metarhizium anisopliae (SQZ-1-21 and RS-4-1) in a two-stage cultivation system. For P. lilacinus IPCP, the optimal sporulation medium contained urea as the nitrogen source, dextrin as the carbon source at 1 g/L, a C:N ratio of 5:1, with ZnSO4·7H2O at 10 mg/L and CaCl2 at 3 g/L. The optimal sporulation medium for P. lilacinus M-14 contained soy peptone as the nitrogen source and maltose as the carbon source at 2 g/L, a C:N ratio of 10:1, with ZnSO4·7H2O at 250 mg/L, CuSO4·5H2O at 10 mg/L, H3BO4 at 5 mg/L, and Na2MoO4·2H2O at 5 mg/L. The optimum sporulation medium for M. anisopliae SQZ-1-21 contained urea as the nitrogen source, sucrose as the carbon source at 16 g/ L, a C:N ratio of 80:1, with ZnSO4·7H2O at 50 mg/L, CuSO4·5H2O at 50 mg/L, H3BO4 at 5 mg/L, and MnSO4·H2O at 10 mg/L. The optimum sporulation medium for M. anisopliae RS-4-1 contained soy peptone as the nitrogen source, sucrose as the carbon source at 4 g/L, a C:N ratio of 5:1, with ZnSO4·7H2O at 50 mg/L and H3BO4 at 50 mg/L. All sporulation media contained 17 g/L agar. While these results were empirically derived, they provide a first step toward low-cost mass production of these biocontrol agents.

This study measured trap induction and trapping on agar disks as affected by juvenile stages (J1, J2, J3, and J4) of the nematode Caenorhabditis elegans and by species of nematode-trapping fungi. Eight species of nematode-trapping fungi belonging to the family Orbiliaceae and producing four kinds of traps were studied: adhesive network-forming Arthrobotrys oligospora, A. vermicola, and A. eudermata, constricting ring-forming Drechslerella brochopaga, and Dr. stenobrocha, adhesive column-forming Dactylellina cionopaga, and adhesive knob-forming Da. ellipsospora, and Da. drechsleri. The number of traps induced generally increased with increasing juvenile stages of C. elegans. The ability to capture the juveniles tended to be similar among isolates that produced the same kind of trap but differed among species that produced different kinds of traps. Trapping by Dr. stenobrocha and Da. cionopaga was correlated with trap number and with juvenile stage. A. oligospora and A. vermicola respectively captured more than 92 and 88% of the J1, J3, and J4 but captured a lower percentage of J2. The knob-producing isolates captured more younger than elder juveniles. Partial correlation analyses demonstrated that the trap induction of the most fungal species positively correlated with the juvenile size and motility, which was juvenile stage dependent. Overall, trap induction and trapping correlated with C. elegans juvenile stage (size and motility) in six species of trapping fungi.

The infection structures, trophism, and ecological character of nematophagous fungi are reviewed in this article on the basis of data extracted from the literature and the most recent experiments conducted in this area. Traditionally, nematophagous fungi are classified into four groups according to their modes of attacking nematodes: nematode-trapping fungi using adhesive or mechanical hyphal traps, endoparasitic fungi using their spores, eggparasitic fungi invading nematode eggs or females with their hyphal tips, and toxin-producing fungi immobilizing nematodes before invasion. In the present review, we focus on the first two groups. The living strategies of these nematophagous fungi depend on the diversity of their infection structures, such as different traps and spore types, which determine the modes of infecting nematodes. The diversity of trophic modes of nematophagous fungi is an important prerequisite for fungal survival and activity in soil. The abundance and activity of Hirsutella rhossiliensis and H. minnesotensis, representatives of endoparasites and potential biocontrol agents against nematodes, are highly dependent on environmental factors. Comprehensive understanding of the survival and activity of nematophagous fungi in soil is fundamental for the exploitation of these fungi as successful biocontrol agents.

To supply essential information for improving mass production and biocontrol efficacy, two-stage cultivation on agar plates was used to evaluate the environmental conditions affecting mycelial growth and sporulation of seven biocontrol fungi. Maximum growth and sporulation occurred on acid media for Paecilomyces (Pa.) lilacinus IPC-P, Pochonia (Po.) chlamydosporia HSY-12-14, and Lecanicillium lecanii CA-1-G, and on alkaline media for Metarhizium anisopliae isolates. All fungi preferred a certain water potential and temperature for sporulation. Light greatly inhibited the growth of P. lilacinus IPC-P, M. anisopliae SQZ-1-21, and L. lecanii CA-1-G but enhanced the sporulation of P. lilacinus M-14, P. chlamydosporia HSY-12-14, and L. lecanii CA-1-G.

Ophiocordyceps sinensis (syn. Cordyceps sinensis), endemic to alpine regions on the Tibetan plateau, is one of the most valuable medicinal fungi in the world. Huge commercial demand has led to excessive harvest and a dramatic decline in its numbers. The diversity of terrains and climates on the Tibetan Plateau and the broad insect host range (more than 50 species in the family Hepialidae) may have resulted in substantial intraspecific genetic diversity for this fungus. The objective of this study was to evaluate the population distribution of O. sinensis from geographically diverse regions of the Tibetan Plateau based on nrDNA ITS and MAT1-2-1 gene sequences. Understanding of the genetic diversity and genesis of O. sinensis will provide important information for the evolution and conservation of this fungus.Significant sequence variations in the ITS and MAT1-2-1 genes (27 and 23 informative sites, eight and seven haplotypes, respectively) were observed. Phylogenetic analysis based on ITS sequences, MAT1-2-1 sequences, or their combined data set, clustered isolates from northern regions in one clade (clade I), whereas isolates from southern regions were dispersed in all four clades (clade I-IV). Single-strand conformation polymorphism (SSCP) analyses of 2639 ITS clones from seven samples revealed 91 different SSCP patterns that were subsequently sequenced. ITS heterogeneity was found in XZ-LZ07-H1 (Nyingchi population), and 17 informative sites and five haplotypes were detected from 15 clones. The five haplotypes clustered into three clades (clade I, II, and IV).Significant genetic divergence in O. sinensis was observed and the genetic diversification was greater among southern isolates than that among northern isolates. The polymorphism of nrDNA ITS sequences suggested that O. sinensis spread from a center of origin (the Nyingchi District) to southern regions and subsequently to northern areas. These results suggest that southern populations are important reservoirs of genetic diversity and should be taken into account in conservation programs.

A two-stage solid cultivation method was used to determine the precise requirements of carbon concentration (1–16 g/L) and C: N ratio (0.625: 1 to 80: 1) for the sporulation of six biocontrol fungi. The C concentration and C: N ratio producing the highest conidia yield were 1 g/L and 5: 1 for Paecilomyces lilacinus IPC-P; 2 g/L and 10: 1 for P. lilacinus M-14; 16 g/L and 80: 1 for Metarhizium anisopliae SQZ-1-21; 4 g/L and 5: 1 for M. anisopliae RS-4-1 and Lecanicillium lecanii CA-1-G; and 2 g/L and 10: 1 for Trichoderma viride TV-1. Sporulation was more affected by C: N ratio than by C and N concentration per se. More spores per colony were produced by the two-stage method than by a conventional, single-stage cultivation method. These results should be useful for improving the mass production of these biocontrol agents.

An aerobic bacterial strain J5 capable of effectively degrading nicotine was isolated from the rhizosphere of tobacco in Yunnan, China. This strain was identified asPseudomonas putida based on morphology, physiological characteristics, and 16S rDNA sequence analysis. The optimum nicotine concentration for the growth of strain J5 was 2.0 g/l. There was no more nicotine detected in the medium containing 3.0 g of nicotine/l after J5 growth for 24 h and less than 0.2% of the nicotine in a medium containing 4.0 g of nicotine/l after J5 growth for 48 h. There was a statistically significant linear relationship between nicotine degradation and biomass of strain J5. When a J5 cell suspension (108 CFU/ml) was applied to tobacco leaves, the nicotine concentration was decreased by 11.7%. These data suggest that the novel strain J5 ofP. putida may be useful for nicotine biodegradation.

•Rhizosphere bacterial communities of healthy and SCN-infected soybean plants were different.•Occurrence of SCN disease was related to soybean rhizosphere bacterial community structure.•Rhizosphere bacterial community structure of SCN-infected plants was affected by the biocontrol agent Purpureocillium lilacinus.Bacterial communities in rhizosphere soil of soybean plants that were healthy, infected with soybean cyst nematode (SCN, Heterodera glycines), and infected with SCN but treated with Purpureocillium lilacinus YES-2 were investigated with community-level physiological profile (Biolog) and 16S rDNA clone library analyses. Biolog data indicated significant differences in substrate utilization patterns of the rhizosphere bacterial communities associated with healthy, SCN-infected, and SCN-infected plus P. lilacinus-treated plants; among the three treatments, substrate richness and catabolic diversity were lowest in the rhizosphere of healthy soybeans. Analysis of 16S rDNA profiles placed the soybean rhizosphere bacteria into seven groups: Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and an unclassified bacterial clade. The percentages of Rhizobiales and Actinobacteria clones were greater in the rhizosphere of healthy plants than SCN-infected plants, while the opposite was true for the proportions of Bacteroidetes and Firmicutes clones. Addition of P. lilacinus did significantly affect the rhizosphere bacterial community of SCN-infected plants. These results suggest that rhizosphere bacterial community may play an important role in the changes of soybean rhizosphere biological conditions during the infection process. Further studies will identify more specific changes in the rhizosphere bacterial community during the establishment and progression of SCN disease, and relate these changes to potential effects on disease management, soybean health, and soybean productivity.

To supply essential information for improving mass production and biocontrol efficacy, two-stage cultivation on agar plates was used to evaluate the environmental conditions affecting mycelial growth and sporulation of seven biocontrol fungi. Maximum growth and sporulation occurred on acid media for Paecilomyces (Pa.) lilacinus IPC-P, Pochonia (Po.) chlamydosporia HSY-12-14, and Lecanicillium lecanii CA-1-G, and on alkaline media for Metarhizium anisopliae isolates. All fungi preferred a certain water potential and temperature for sporulation. Light greatly inhibited the growth of P. lilacinus IPC-P, M. anisopliae SQZ-1-21, and L. lecanii CA-1-G but enhanced the sporulation of P. lilacinus M-14, P. chlamydosporia HSY-12-14, and L. lecanii CA-1-G.

The infection structures, trophism, and ecological character of nematophagous fungi are reviewed in this article on the basis of data extracted from the literature and the most recent experiments conducted in this area. Traditionally, nematophagous fungi are classified into four groups according to their modes of attacking nematodes: nematode-trapping fungi using adhesive or mechanical hyphal traps, endoparasitic fungi using their spores, eggparasitic fungi invading nematode eggs or females with their hyphal tips, and toxin-producing fungi immobilizing nematodes before invasion. In the present review, we focus on the first two groups. The living strategies of these nematophagous fungi depend on the diversity of their infection structures, such as different traps and spore types, which determine the modes of infecting nematodes. The diversity of trophic modes of nematophagous fungi is an important prerequisite for fungal survival and activity in soil. The abundance and activity of Hirsutella rhossiliensis and H. minnesotensis, representatives of endoparasites and potential biocontrol agents against nematodes, are highly dependent on environmental factors. Comprehensive understanding of the survival and activity of nematophagous fungi in soil is fundamental for the exploitation of these fungi as successful biocontrol agents.

The in vitro effects of abscisic acid (ABA) and nitric oxide (NO) on the nematode-trapping fungus Drechslerella stenobrocha AS6.1 were examined. The average number of traps (constricting rings) per colony and the percentage of nematodes (Caenorhabditis elegans) trapped were greatly increased by addition of ABA but greatly suppressed by addition of sodium nitroprusside (SNP, an NO donor) to corn meal agar. The suppressive effect of SNP was not negated by addition of an NO synthase competitive inhibitor (l-naphthylacetic acid, L-NNA) or an NO-specific scavenger [2-(4-carboxyphenyl)-4,4, 5,5-tetramethylimidazoline-1-oxyl-3-oxide, cPTIO]. When added without SNP, however, L-NNA and cPTIO caused moderate increases in trap number and trapping. The results indicate that the trap formation and nematode-trapping ability of D. stenobrocha were enhanced by ABA but decreased by exogenous NO.

A real-time PCR assay was developed to quantify in soil the fungus Hirsutella minnesotensis, an important parasite of secondary-stage juvenile (J2) of the soybean cyst nematode. A primer pair 5′-GGGAGGCCCGGTGGA-3′ and 5′-TGATCCGAGGTCAACTTCTGAA-3′ and a TaqMan probe 5′-CGTCCGCCGTAAAACGCCCAAC-3′ were designed based on the sequence of the ITS region of the rRNA gene. The primers were highly species-specific. The PCR reaction system was very sensitive and able to detect as few as 4 conidia g−1 soil. Regression analysis showed similar slopes and efficiency on DNA from pure culture (y = −3.587x + 41.017, R2 = 0.9971, E = 0.9055) and from Log conidia g−1 soil (y = −3.855x + 37.669, R2 = 0.9139, E = 0.8172), indicating that the real-time PCR protocol can reliably quantify H. minnesotensis in the soil. The real-time PCR assay was applied to 20 soil samples from soybean fields, and compared with a parasitism assay. The real-time PCR assay detected H. minnesotensis in six of the soils, whereas the parasitism assay detected H. minnesotensis in the same six soils and three additional soils. The real-time PCR assay was weakly correlated (R2 = 0.49) with the percentage of parasitized J2 in the six soils, indicating that different types of soil may interfere the efficiency of the real-time PCR assay, possibly due to the effect of soil types on efficacy of DNA extraction. The parasitism assay appeared to be more sensitive than real-time PCR in detecting presence of H. minnesotensis, but real-time PCR was much faster and less costly and provided a direct assessment of fungal biomass. Using the two assays in combination can obtain more complete information about the fungus in soil than either assay alone. Hirsutella parasitism was widespread and detected in 13 of the 20 field soils, indicating that these fungi may contribute to suppressiveness of soybean cyst nematode in nature and likely have high biological control potential for the nematode.

A nicotine-sensitive mutant was generated from the nicotine-degrading bacterium, Pseudomonas putida strain J5, by mini-Tn5 transposon mutagenesis. This mutant was unable to grow with nicotine as the sole carbon source but could grow with glucose. Sequence analysis showed that the Tn5 transposon inserted at the site of the ketopantoate hydroxymethyltransferase gene (panB), which had 54% identity to PanB in Escherichia coli K-12. In-frame deletion of the panB gene abolished the nicotine-degrading ability of strain J5, while complementation with panB from P. putida J5 and E. coli K-12 restored the degrading activity of the mutant to the wild-type level. These results suggest that ketopantoate hydroxymethyltransferase is a crucial enzyme in nicotine metabolism in P. putida J5.

•Hm is an invasive species in China.•Hm has undergone a founding event during the invasion.•Phenotypic divergence occurred with a simple genetic background in Chinese Hm.•Invasive fungi can develop phenotypic diversity even with simple genetic background.The fungal parasitoid, Hirsutella minnesotensis, is a dominant parasitoid of the soybean cyst nematode, which is a destruction pest of soybean crops. We investigated population structure and parasitism pattern in samples of H. minnesotensis in China to reveal the spreading pattern of this fungal species and the underlying mechanism generating the parasitization-related ability variability in Chinese population. In cross-inoculation experiments using different combinations of H. minnesotensis and soybean cyst nematode samples from China, most H. minnesotensis isolates fitted the criterion for “local versus foreign” parasitism profile, exhibiting local adaptation pattern to the SCN host. However, the genetic analysis of the single nucleotide polymorphisms with clone-corrected samples based on ten DNA fragments in 56 isolates of H. minnesotensis from China revealed that the Chinese H. minnesotensis population was a clonal lineage that underwent a founder event. The results demonstrated that the Chinese H. minnesotensis population had generated parasitization-related ability diversity after a founder event through individual variation or phenotypic plasticity other than local adaptation. The rapid divergence of parasitization-related abilities with simple genetic structure in Chinese H. minnesotensis population indicates a fundamental potential for the establishment of invasive fungal species, which is a prerequisite for biological control agents.

The entomopathogenic fungus Ophiocordyceps sinensis has been important in traditional Chinese medicine but has yet to be commercially cultivated. One bottleneck is the very low frequency of stromata formation from artificially infected moth larvae. The mating system of fungi is the determining factor for sexual reproduction, but mating-type genes of O. sinensis have not been previously investigated. In this study, the putative mating-type gene MAT1-2-1 within the MAT1-2 idiomorph was amplified by polymerase chain reaction (PCR) and was determined to consist of 859 nucleotides that encode 249 amino acids; genes within the MAT1-1 idiomorph, however, were not determined. The MAT1-2-1 gene contained the conserved high-mobility group (HMG) box, and MAT1-2-1 flanking sequences were subsequently obtained. Although no putative open reading frames of the MAT1-1 idiomorph were detected within the ca. 8-kb flanking sequences of MAT1-2-1, a putative DNA lyase gene (which is present next to both idiomorphs in some heterothallic fungi) was found ca. 3.0 kb downstream of MAT1-2-1. The intervening distance between MAT1-2-1 and the DNA lyase gene in O. sinensis is larger than that in Cordyceps militaris and Cordyceps takaomontana. In addition, O. sinensis showed low sequence similarities with C. militaris and C. takaomontana in both MAT1-2-1 and the DNA lyase gene. In the phylogenetic tree, different MAT1-2-1 haplotypes of O. sinensis clustered together with high bootstrap support. As a single-copy gene, MAT1-2-1 was detected in all examined O. sinensis isolates including tissue cultures and single-ascospore cultures. This report describes, for the first time, a mating-type gene of O. sinensis.Highlights► The MAT1-2-1 gene within the MAT1-2 idiomorph was amplified for O. sinensis. ► A putative DNA lyase gene was detected in flanking sequences of MAT1-2-1. ► O. sinensis showed low sequence similarities with C. militaris and C. takaomontana. ► As a single-copy gene, MAT1-2-1 existed in all tested O. sinensis isolates. ► This report describes, for the first time, a mating-type gene of O. sinensis.

•C. elegans cell-based MTS assay facilitates high-throughput cytotoxicity screening.•There is positive correlation between cell viability and nematode viability.•A two-step method based on cell assay coupled with larval assay was established.•The method can be used for high-throughput screening of potential nematicides.Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides’ test, high correlations were obtained between cell viability and larval viability and “r” values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r = 0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r = 0.48) and pine wood nematode Bursaphelenchus xylophilus (r = 0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8–500 μg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.Download full-size image

Homology-based cloning was used to obtain a new gene (named FIP-tvc) from the genomic DNA of the Chinese traditional medicinal mushroom Trametes versicolor. FIP-tvc is a member of the fungal immunomodulatory protein family, is composed of 336 bp encoding 111 amino acids, and is highly homologous with other fungal immunomodulatory proteins. In addition, an expression system for FIP-tvc was constructed. The results indicated that recombinant protein FIP-tvc could be expressed in Escherichia coli and that about 20% of the expressed protein was in soluble form. Recombinant FIP-tvc was capable of agglutinating mouse and rat red blood cells. Furthermore, recombinant protein FIP-tvc could selectively enhance the expression of interleukin (IL)-1α, IL-2, IL-5, IL-6, tumor necrosis factor-alpha (TNF-α), and lympotoxin (LT) in mouse spleen cells.Highlights► A new gene FIP-tvc was cloned from Trametes versicolor. ► An expression system for FIP-tvc was constructed in Escherichia coli. ► FIP-tvc expressed by E. coli shows good immunomodulating activities.