Comparison of fungal communities among ten macrophyte rhizospheres

The fungal community composition, size and several physico-chemical properties were individually investigated in ten macrophyte rhizospheric substrates using nested PCR-denaturing gradient gel electrophoresis and soil chemical methods. Results indicated that both Dothideomycetes and Sordariomycetes were dominant fungi in macrophyte rhizospheric substrates, and denitrifying fungi (Fusarium graminearum) was found in nine of ten macrophyte rhizospheres. Fungal Shannon-Wiener diversity index (H) and richness (S) in Thalia dealbata, Typha latifolia, Iris hexagona and Hemerocallis aurantiaca rhizospheres were higher than those in other six rhizospheres. Fungal number and biomass were 1.91 × 10³ CFUs g^?1 dw and 1.53 μg ergosterol g^?1 dw in Iris pseudacor rhizosphere, and were greater than in other nine rhizospheres. The correlation analysis showed that fungal number and biomass significantly and positively correlated to total soil phosphorus, while fungal H and S were significantly and negatively correlated to total organic carbon. The principal components analysis (PCA) showed that the fungal community significantly divided ten macrophyte rhizospheres into four groups, showing the significant difference of fungal communities among ten rhizospheric substrates. The current study revealed for the first time the importance of rhizospheric fungal community in distinguishing macrophyte rhizospheres, thus will undoubtedly widen our insight into fungal communities in aquatic rhizospheres.     

Cloning and overexpression of a maltase gene from Schizosaccharomyces pombe in Escherichia coli and characterization of the recombinant maltase

The Schizosaccharomyces pombe maltase structural gene (SPMAL1⁺) was amplified from genomic DNA of S. pombe by PCR. An open reading frame of 1740 bp, encoding a putative 579 amino-acid protein with a calculated molecular mass of 67.7 kDa was characterized in the genomic DNA insert of plasmid pQE30. The specific maltase activity in the induced transformants was 21 times higher than that in wild-type. However, the estimated molecular mass of the purified recombinant maltase was 44.3 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified recombinant maltase were 40 °C and 6, respectively. The recombinant maltase was weakly activated by Mg²⁺, Ca²⁺, Na⁺, and Ba²⁺, but was strongly inhibited by Hg²⁺, Ag⁺ and Cu²⁺, EDTA, and PMSF. The purified maltase could actively hydrolyse ρ-nitrophenyl glucoside (PNPG), maltose, dextrin, and soluble starch. The results demonstrate that maltase from S. pombe was different from that from other yeasts, and might be usefully exploited in the future by the biotechnology industry or lead to the development of new molecular genetic tools.     

Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects

A protocol for efficient extraction of fungal DNA from micromycetes colonising painted art objects was developed. Polymerase chain reaction (PCR) inhibitors were successfully removed by a combined application of a Chelex-100 adsorption resin and a Geneclean Kit for Ancient DNA. Universal fungal primers for PCR amplification of 28S rDNA (U1 and U2) were tested for their applicability in denaturing gradient gel electrophoresis (DGGE) analysis of fungal communities. Artificially produced mortar samples inoculated with fungal pure cultures isolated from mural paintings were used as model objects for DNA extractions and DGGE analysis. Good resolution in DGGE was achieved using 260-bp rDNA fragments amplified with U1/DGGE and U2 primers directly from model communities.     

Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation

The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. Moreover, in the last few years, B. cinerea has been adopted as an important model system in molecular phytopathology. In spite of these contributions, the molecular basis of the infection cycle remains unclear. Proteomic approaches have revealed significant information about the infective cycle of several pathogens, including B. cinerea. The main aim of this study is to make available a proteomic database containing a significant number of identified proteins from B. cinerea. In brief, three independent B. cinerea cultures supplemented with carboxymethylcellulose were used, and the extracted proteins were independently separated by 2‐D PAGE to obtain the proteome map from B. cinerea. Two hundred and sixty‐seven spots were selected for MALDI TOF/TOF MS analysis, resulting in 303 positive identifications, mostly representing unannotated proteins. Identified proteins were then classified into categories using the PANTHER classification system ( www.pantherdb.org ), showing the relevance of protein metabolism and modification process and oxidoreductase activity. Since cellulose is one of the major components of the plant cell wall, many of the identified proteins may have a crucial role in the pathogenicity process. In brief, this proteomic map of B. cinerea will be a useful basis for exploring the proteins involved in the infection cycle, which will in turn provide new targets for crop diagnosis and focused fungicide design.     

中国菌物已知种数

据作者对1978年以来国内外主要菌物学期刊和专著进行的系统搜集,中国大陆发表的菌物累计有2,849新种,129新变种,5,260新记录种。若加上戴芳澜编著的《中国真菌总汇》所记载的6,737种和168变种,中国大陆已知菌物计14,846种297变种。据不完全统计,香港和台湾报道的菌物种类中分别约有800种和400种在中国大陆未曾记载,因此全国总计已知菌物应为16,046种297变种。假设其中有10%为同物异名,则目前我国菌物已知种数约为14,700种,其中管毛生物界(主要是卵菌)约300种,原生动物界(主要是黏菌)约有340种,真菌约14,060种。     

Endophyte research: going beyond isolation and metabolite documentation

Many fungi belonging to mostly Ascomycota inhabit living tissues of plants of all major lineages without causing any visible symptoms. Termed horizontally transmitted endophytes, they have been investigated mostly for their capacity to produce bioactive secondary metabolites. However, many questions regarding the interactions between endophytes and their plant hosts, phytophagous insects and other fungi remain unanswered. This review highlights some of these areas of endophyte biology about which very little or no knowledge exists. Information garnered' using modern methodologies' on these grey areas of ‘endophytism’ (endophytic mode of lifestyle) would help immensely in understanding the evolution of endophytes of aerial plant tissues and in exploiting endophytes in various fields of biotechnology.     

Orchestration of morphogenesis in filamentous fungi: conserved roles for Ras signaling networks

Filamentous fungi undergo complex developmental programs including conidial germination, polarized morphogenesis, and differentiation of sexual and asexual structures. For many fungi, the coordinated completion of development is required for pathogenicity, as specialized morphological structures must be produced by the invading fungus. Ras proteins are highly conserved GTPase signal transducers and function as major regulators of growth and development in eukaryotes. Filamentous fungi typically express two Ras homologs, comprising distinct groups of Ras1-like and Ras2-like proteins based on sequence homology. Recent evidence suggests shared roles for both Ras1 and Ras2 homologs, but also supports the existence of unique functions in the areas of stress response and virulence. This review focuses on the roles played by both Ras protein groups during growth, development, and pathogenicity of a diverse array of filamentous fungi.     

Fungal pathogenesis: Past,present and future

Over the last 20 years a record number of fungal and fungal-like diseases have jeopardized wild species the world over, causing several of the most severe population declines and extinctions ever witnessed (Fisher et al. 2012). Such events include the devastating impact of Batrachochytrium dendrobatidis on amphibian populations and the extinction of bat populations as a result of Geomyces destructans infection. This commentary focusses on two human-infecting fungal pathogens causing much scientific interest, that is, Cryptococcus gattii and Trichophyton rubrum. It summarises recent research findings into their pathogenic evolution and adaptive strategies and highlights key gaps in our knowledge. Finally, the prose attempts to fuse such data with the work of Casadevall, exploiting his theories to predict the future of fungal pathogenesis, that is, where pathogenesis refers to the mechanism that results in disease (Casadevall 2012).     

Diversity of rhizosphere associated entomopathogenic fungi of perennial herbs, shrubs and coniferous trees

Understanding habitat selection of fungal entomopathogens is critical to improve the efficacy, persistence and cost of these fungi as microbial insecticides. This study sought to determine the prevalence of Metarhizium and Beauveria spp. isolated from the rhizosphere of strawberry, blueberry, grape and Christmas tree crops in the Willamette Valley of Oregon. Entomopathogenic fungi were assigned to thirteen species based on molecular phylogenetic criteria. Four species of Metarhizium were isolated including Metarhizium brunneum, Metarhizium guizhouense, Metarhizium robertsii, and Metarhizium flavoviride var. pemphigi. Nine Beauveria species were isolated including, Beauveria brongniartii, an undescribed species referred to as Clade C and seven phylogenetic species of Beauveria bassiana. Strawberries and blueberries were significantly associated with M. brunneum and Christmas trees with M. guizhouense and M. robertsii. Grapes were significantly associated with B. bassiana phylogenetic species Bbas-16. All of the Metarhizium isolates screened were pathogenic to Otiorhynchus sulcatus larvae in laboratory bioassays but only M. brunneum and M. robertsii caused significant levels of infection. The study results suggest that certain species of Metarhizium and Beauveria are significantly associated with the strawberry, blueberry and Christmas tree rhizosphere and could potentially provide better control of O. sulcatus.     

Against the naming of fungi

The use of molecular bar-coding and consensus on nomenclatural practices has encouraged optimism about the future of fungal taxonomy and systematics. There are, however, profound deficiencies in our understanding of fungal diversity and broader problems with the taxonomic enterprise that deserve greater attention. For 250 years mycologists have tried to reconcile fungal diversity with the Linnean fantasy of a divine order throughout nature that included unambiguous species. This effort has failed and today's taxonomy rests on an unstable philosophical foundation. Rather than persisting with the present endeavour, it may be more fruitful to abandon the notion of fungal species pending further basic research. In the meantime, mycologists should consider tagging collections with digital codes and assigning these operational taxonomic units to higher taxonomic ranks whose objective reality is corroborated by strong phylogenetic evidence.     

DNA sequence incongruence and inconsistent morphology obscure species boundaries in the Teratosphaeria suttonii species complex

Teratosphaeria suttonii (=Kirramyces epicoccoides) is a leaf pathogen that can cause premature defoliation, reduced growth and vigor, and subsequent tree death of many Eucalyptus species. Although the fungus primarily infects mature leaves in the lower canopy, infections can spread to younger leaves during continued epidemics or when trees are stressed. Teratosphaeria suttonii has a wide distribution in Australia and has been introduced to many other parts of the world, most probably with germplasm used to establish plantations. The aim of this study was to establish the phylogenetic relationships between T. suttonii isolates from different countries and to consider whether cryptic species exist in a species complex. DNA from parts of the nuclear ribosomal internal transcribed spacer, β-tubulin, and elongation factor-1α genes was sequenced and analyzed for isolates from throughout the range of T. suttonii in Australia, and from six countries (China, Indonesia, South Africa, Uruguay, United States, and Vietnam) where the pathogen is introduced. Morphometrics of conidia produced both in vivo and in vitro were also considered. Analysis of the sequence data resulted in incongruent genealogies. Furthermore, groups of isolates in the genealogies could not be linked to area of origin. Similarly, differences in conidial morphology could not be linked to any of the phylogenetic groups. There was no evidence of distinct species boundaries, and isolates from Australia were closely related to those from other parts of the world. The results of this study support the treatment of T. suttonii as a morphologically and genetically diverse species in its natural range in Australia. The diversity is reflected in introduced populations.     

肝硬化患者无菌腹水中真菌DNA的检测及易位研究

目的检测和分析肝硬化患者无菌腹水中真菌DNA存在状况。方法选取118例无菌腹水的肝硬化患者为研究对象,分别采集患者刚入院时的腹水、血液和粪便标本。应用PCR技术直接检测标本的真菌DNA并克隆测序。并收集患者临床资料,对患者临床症状及检验结果进行分析比较。结果1名患者的腹水、血液、粪便中同时检测到真菌DNA,14名患者的腹水和粪便中同时检测到真菌DNA,3名患者的血液和粪便中同时检测到真菌DNA,大部分序列间的相似性高于99％。测序检测到最多的菌种为Candidaalbicans。结论肝硬化患者无菌腹水中能够检测到真菌DNA,并且提示在肝硬化患者腹水和血清中检测到的真菌DNA易位于肠道。     

Delving into the dark ecology: A continent-wide assessment of patterns of composition in soil fungal communities from Australian tussock grasslands

Soil fungi play essential roles in many terrestrial processes, but our knowledge of the forces governing fungal distribution and community composition along broad-scale environmental gradients is still limited. In this study, we explored biogeographic distribution and composition of soil fungal communities associated with 62 tussock grasslands across different regions of Australia. Climatic parameters had only a limited correlation with fungal community structure, while edaphic variables and spatial distance were significantly associated with changes in fungal community composition. We also observed high variations in composition among fungal assemblages from different ecological regions, suggesting some regional endemism in these communities. The discrete distribution of fungi in soil was further confirmed by indicator analysis, which identified distinct indicator operational taxonomic units associated with grasslands from different climatic regions. Finally, fungi with flexible trophic interactions had a central role in the network architecture of both arid and temperate communities. Taken together, the results from our study confirm the prominent role of soil physico-chemical status and geographic location in determining fungal biogeographic patterns over large scales in Australia.     

Distribution and ecological traits of a rare and threatened fungus Hericium flagellum in Poland with the prediction of its potential occurrence in Europe

Hericium flagellum is a highly host-dependent wood-inhabiting fungus in Europe. Its occurrence is strongly connected to the distribution of silver fir (Abies alba). We analysed available data describing ecological factors, especially habitat, substrate preferences and phenology, which are regarded as drivers of H. flagellum occurrence. We also implemented ecological niche modelling to determine the potential range of the fungus. More than half of H. flagellum records (57%) were found in high conservation value areas, on fallen trunks of silver fir trees. The basidiomata were predominantly recorded between August and late November. Distribution of the tree host, precipitation in the driest month, isothermality and annual mean temperature were the most decisive factors influencing H. flagellum occurrence. We conclude that the disjunctive range at present, the risk of tree host extinction linked to habitat loss, and the limited dispersal of H. flagellum propagules are the main threats to this species.     

Fungal mycelium--the source of chitosan for chromatography

Mycelium of the mold Aspergillus niger was used as a raw material for the preparation of microbial chitosan. Aspergillus niger, the mold used for the production of citric acid, contains approx. 15% of chitin, which can be separated, transformed into chitosan, and used as a sorbent for chromatography. The main advantage of this material in comparison with krill chitosan is the uniformity of particle size leading to the low back-pressure in the column. The other advantage is the fact, that original fibrous structure of mycelial pellets could be stabilized before chitosan preparation by cross-linking with glutaraldehyde. The product prepared by this way -- crosslinked chitosan of uniform particle size, is highly porous, with high water regain and, as a result, low sedimentation velocity. Low sedimentation velocity is not disadvantage in chromatographic application, but may form some problems in batchwise operation. Chitosan as a polymer of glucosamine is anion exchanger in nature and the chromatographic properties of this anion exchanger was demonstrated by the chromatography of bovine blood plasma, glucose oxidase, and chicken pepsinogen. In all cases, the course of chromatography on crosslinked chitosan was compared with the chromatography on MONO Q (bovine blood plasma) or DEAE-cellulose (glucose oxidase, chicken pepsinogen) under the same protocol.     

Gene expression varies within and between enzootic and epizootic lineages of Batrachochytrium dendrobatidis (Bd) in the Americas

While much research focus is paid to hypervirulent fungal lineages during emerging infectious disease outbreaks, examining enzootic pathogen isolates can be equally fruitful in delineating infection dynamics and determining pathogenesis. The fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), exhibits markedly different patterns of disease in natural populations, where it has caused massive amphibian declines in some regions, yet persists enzootically in others. Here we compare in vitro gene expression profiles of a panel of Bd isolates representing both the enzootic Bd-Brazil lineage, and the more recently diverged, panzootic lineage, Bd-GPL. We document significantly different lineage-specific and intralineage gene expression patterns, with Bd-Brazil upregulating genes with aspartic-type peptidase activity, and Bd-GPL upregulating CBM18 chitin-binding genes, among others. We also find pronounced intralineage variation in membrane integrity and transmembrane transport ability within our Bd-GPL isolates. Finally, we highlight unexpectedly divergent expression profiles in sympatric panzootic isolates, underscoring microgeographic functional variation in a largely clonal lineage. This variation in gene expression likely plays an important role in the relative pathogenesis and host range of Bd-Brazil and Bd-GPL isolates. Together, our results demonstrate that functional genomics approaches can provide information relevant to studies of virulence evolution within the Bd clade.     

Colonization of hydrophilic contact lenses by yeast

The growth of six strains of yeast was analyzed in vitro in order to assess their capacity for colonizing (adhesion and invasion) hydrophilic contact lenses. Lenses with different water content were cultured in two culture media for 3, 7, 14, and 21 days. Only strain 93150 of Candida albicans could adhere to and invade the polymers. Specifically, fungal growth was observed in cultures with Sabourauds broth. The degree of yeast colonization of contact lenses was significantly related to the species, the strain, and the culture medium in which the yeast and lenses were cultured. The results here obtained were compared with those reported for the filamentous fungus Aspergillus niger 2700. For both microorganisms, the strain and the medium in which the lenses and microorganism were cultured influenced the colonization, but the percentage of colonized lenses, the degree of colonization, and the density and size of the internalized colonies were always noticeably lower for C. albicans 93150. Colonization by A. niger 2700 was also related to the type of material of the lenses and the incubation period. For both microorganisms, when the strain is right and the growth and development are correct, colonization of hydrophilic contact lenses occurs.     

G试验联合真菌培养在假丝酵母菌血流感染诊治中的价值

目的了解假丝酵母菌血流感染患者常见病原菌分布及药物敏感性情况,评估(1,3)-β-D-葡聚糖检测(G试验)联合真菌培养在假丝酵母菌血流感染诊治中的应用价值。方法回顾性分析浙江省人民医院2014年1月至2018年12月假丝酵母菌血流感染患者的病原菌分布及药物敏感状况,同步分析G试验检测结果。结果假丝酵母菌血流感染病原菌以白假丝酵母菌为主。所检出的90株假丝酵母菌对两性霉素B最为敏感,敏感率为100.00%;对5-氟胞嘧啶、伏立康唑、氟康唑和伊曲康唑的敏感率分别为98.89%、90.00%、87.78%和77.78%。热带假丝酵母菌对伏立康唑、氟康唑和伊曲康唑的敏感率分别为56.25%、50.00%和43.75%,光滑假丝酵母菌对伊曲康唑敏感率仅为58.33%。假丝酵母菌血流感染患者、局部假丝酵母菌感染患者血清(1,3)-β-D-葡聚糖水平高于对照组,差异有统计学意义(t=6.576、P0.001;t=1.988、P=0.049)。假丝酵母菌血流感染患者血清(1,3)-β-D-葡聚糖水平高于局部假丝酵母菌感染患者,差异有统计学意义(t=4.738,P0.001)。90例患者中有54例进行了G试验,其中13例患者G试验先于血培养出现阳性结果。G试验阴性患者(54例中有17例G试验为阴性)中有8例第1次送检G试验晚于血培养报阳时间。结论 G试验可作为假丝酵母菌血流感染的诊断参考依据,血清(1,3)-β-D-葡聚糖水平与假丝酵母菌感染的严重程度有关。G试验联合真菌培养在假丝酵母菌血流感染诊治中具有重要作用,有助于临床早期诊断和监测治疗。     

Schistosomicidal activity of kaurane,labdane and clerodane-type diterpenes obtained by fungal transformation

Schistosomiasis continues to be a huge challenge for researchers, pharmaceutical companies, and governments in developing countries. Diterpene compounds are good source for the development of novel potential leading compounds to treat schistosomiasis. We are reporting herein the schistosomicidal activity of ent-kaurenoic acid, ent-copalic acid, ent-hardwickiic acid, isolated from oleoresins of Copaifera spp, and of their derivatives obtained by fungal transformation with strains of Aspergillus fumigatus, A. terreus, A. phoenicis and A. ochraceus, and of Cunninghamella echinulata e C. elegans. The in vitro antiparasitical assays were performed using adult worm pairs of Schistosoma mansoni for the evaluation of the worm pairing, egg production, and eggs development. Ten kaurane, labdane and clerodane-type diterpenes were obtained by fungal transformation and 7α-acetoxy-ent-kaur-16-en-19-oic acid and ent-hardwickiic acid were the most active ones by causing mortality of 100 % of the parasites within 24 h (concentrations of 100.0 and 200.0 μM) and displaying respective IC₅₀ values for 24, 48 and 72 h of 56.7, 37.6 and 29.2 μM and 29.6, 30.8 and 25.7 μM. Additionally, 7α-acetoxy-ent-kaur-16-en-19-oic acid and ent-hardwickiic acid highly reduced the number of laid eggs at 6.25 and 12.5 μM, respectively. These diterpenes should be further investigated as potential candidates for antiparasitic drug discovery.