Gut-inhabiting yeasts from the gut of Australian passalid beetles

Passalidae (Polyphaga, Coleoptera) is a family of beetles with approximately 960 species that are distributed worldwide. Preliminary studies that characterized ascomycete and basidiomycete yeasts in the gut of these wood-eating beetles from the USA, Guatemala, and Thailand, demonstrated associations between certain yeast taxa and passalids. We extended the study to include yeasts and beetles from tropical forests near Cairns and Brisbane, Queensland, Australia. We isolated more than 1000 yeast strains from about 150 beetles belonging to 10 species. LSU and ITS rRNA markers were used to identify a subset of 250 yeast strains, which revealed that the gut of Australian passalids contained undescribed ascomycetes in the Debaryomyces, Dipodascus, Kazachstania, Ogataea, Scheffersomyces, Sugiyamaella, Spathaspora, Torulaspora, and Zygowilliopsis clades, as well as basidiomycetes in the genera Cryptococcus and Trichosporon. A close relative of Candida subhashii (Spathaspora clade) and the xylose-fermenting yeast Scheffersomyces stipitis were the most common species isolated in Queensland. These results agree with those of previous studies that showed a common association of xylose-fermenting yeasts in the gut of lignicolous insects. Species and higher taxa of yeasts, however, vary between Queensland passalids and those previously collected in distant regions.

Polypore assemblages in boreal old-growth forests, and associated Coleoptera

This thesis examines assemblages of wood-decaying fungi in Finnish old-growth forests, and patterns of species interactions between fruit bodies of wood-rotting Basidiomycetes and associated Coleoptera. The present work is a summary of four original publications and a manuscript, which are based on empirical observations and deal with the prevalence of polypores in old-growth forests, and fungicolous Coleoptera. The study area consists of eleven old-growth, mostly spruce- and pine-dominated, protected forests rich in dead wood in northern and southeastern Finland. Supplementary data on fungus beetle interactions were collected in southern Finland and the Åland Islands. 11251 observations of fruit bodies from 153 polypore species were made in 789 forest compartments. Almost a half of the polypore species demonstrated a distinct northern or southeastern trend of prevalence. Polypores with a northern prevalence profile were in extreme cases totally absent from the Southeast, although almost uniformly present in the North. These were Onnia leporina, Climacocystis borealis, Antrodiella pallasii, Skeletocutis chrysella, Oligoporus parvus, Skeletocutis lilacina, and Junghuhnia collabens. Species with higher prevalence in the southeastern sites were Bjerkandera adusta, Inonotus radiatus, Trichaptum pargamenum, Antrodia macra, and Phellinus punctatus. 198 (86%) species of Finnish polypores were examined for associated Coleoptera. Adult beetles were collected from polypore basidiocarps in the wild, while their larvae were reared to adulthood in the lab. Spatial and temporal parallels between the properties of polypore fruit body and the species composition of Coleoptera in fungus beetle interactions were discussed. New data on the biology of individual species of fungivorous Coleoptera were collected. 116 species (50% of Finnish polypore mycota) were found to host adults and/or larvae of 179 species from 20 Coleoptera families. Many new fungus beetle interactions were found among the 614 species pairs; these included 491 polypore fruit body adult Coleoptera species co-occurrences, and 122 fruit body larva interrelations. 82 (41%) polypore species were neither visited nor colonized by Coleoptera. The total number of polyporicolous beetles in Finland is expected to reach 300 species.

Response of fungal and actinobacterial communities to water-level drawdown in boreal peatland sites

"We used PCR-DGGE fingerprinting and direct sequencing to analyse the response of fungal and actinobacterial communities to changing hydrological conditions at 3 different sites in a boreal peatland complex in Finland. The experimental design involved a short-term (3 years; STD) and a long-term (43 years; LTD) water-level drawdown. Correspondence analyses of DGGE bands revealed differences in the communities between natural sites representing the nutrient-rich mesotrophic fen, the nutrient-poorer oligotrophic fen, and the nutrient-poor ombrotrophic bog. Still, most fungi and actinobacteria found in the pristine peatland seemed robust to the environmental variables. Both fungal and actinobacterial diversity was higher in the fens than in the bog. Fungal diversity increased significantly after STD whereas actinobacterial diversity did not respond to hydrology. Both fungal and actinobacterial communities became more similar between peatland types after LTD, which was not apparent after STD. Most sequences clustered equally between the two main fungal phyla Ascomycota and Basidiomycota. Sequencing revealed that basidiomycetes may respond more (either positively or negatively) to hydrological changes than ascomycetes. Overall, our results suggest that fungal responses to water-level drawdown depend on peatland type. Actinobacteria seem to be less sensitive to hydrological changes, although the response of some may similarly depend on peatland type. (C) 2009 Elsevier Ltd. All rights reserved."

Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.

华中地区多孔菌物种多样性和资源研究

多孔菌是指具有孔状子实层的木材腐朽菌，该类群隶属于担子菌门(Basidiomycota)，担子菌纲 (Basidiomycetes)，是一类数量较多、组成较为复杂的真菌。 多孔菌具有重要的生态功能和经济价值。该类群真菌能够降解木质素和纤维素，在森林生态系统中起着关键的降解还原作用。同时，多孔菌还是重要的生物资源。部分种类能够造成林木病害是林木病原菌；部分多孔菌具有药用、食用价值；有些种类的多孔菌还具有重要的工业应用价值。 华中地区是我国生物多样性最丰富的地区之一，该地区多孔菌资源丰富。然而华中地区的多孔菌研究非常薄弱，该地区的多孔菌资源的种类和分布仍不明确。本文对华中地区不同森林生态类型中的多孔菌进行了广泛调查和标本采集。通过形态学、分子生物学和遗传交配实验，对华中地区的多孔菌多样性与资源进行了系统的研究，并初步分析了华中地区多孔菌的区系特点。 通过本论文的研究，明确了华中地区多孔菌的种类与多孔菌资源的分布，丰富了我国木生真菌的种类，为控制林木病原菌、开发利用有益真菌资源提供了基础数据和科学依据。本论文的主要研究结论如下： 1.华中地区采集的近2000号标本，按照现代分类系统共鉴定出257种多孔 菌，包括5个新种：庄氏地花孔菌Albatrellus zhuangii Y.C. Dai & Juan Li，宽孢全缘孔菌Haploporus latisporus Juan Li & Y.C. Dai，河南纤孔菌Inonotus henanensis Juan Li & Y.C. Dai，覆瓦拟硬孔菌Rigidoporopsis tegularis Juan Li & Y.C. Dai，菌索干皮孔菌Skeletocutis fimbriata Juan Li & Y.C. Dai；4个中国新记录种：假斑点嗜蓝孢孔菌Fomitiporia pseudopunctata (A. David, Dequatre & Fiasson) Fiasson，仲氏针层孔菌Phellinus johnsonianus (Murrill) Ryvarden，合生坛担菌Sistotrema confluens Pers.，姬氏芮氏孔菌Wrightoporia gillesii A. David & Rajchenb.。 2.利用分子系统学方法，确定了两种通过形态学研究难鉴定的种类的分类学地位，并探讨了针层孔菌属和地花孔菌属的系统发育关系。 3.华中地区的异担子菌有岛生异担子菌和小孔异担子菌两种，不存在多年异担子菌这一严重的森林病原菌。该地区的岛生异担子菌为该复合种中的T生物种，为腐生菌，不对针叶树造成严重病害。 4.华中地区的257种多孔菌隶属于担子菌门、担子菌纲，共有8目、17科、75属。优势科依次为多孔菌科，锈革孔菌科和拟层孔菌科。这三科的种类构成了华中地区多孔菌区系的主体。针层孔菌属为华中地区多孔菌的优势属，其他依次为多孔菌属，波氏孔菌属，多年卧孔菌属，干皮孔菌属，栓孔菌属和薄孔菌属。 5.华中地区多孔菌的75个属共划分为6种地理成分：世界广布属，北温带分布属，热带–亚热带分布属，东亚–北美分布属，大洋洲–北美洲分布属和东亚分布属。除世界广布属外，北温带分布属和热带–亚热带分布属是华中地区多孔菌的主体。 6.华中地区的257种多孔菌划分为8种地理成分：世界广布成分，北温带分布成分，泛热带分布成分，亚–欧共有成分，东亚–北美共有成分，东亚–澳大利亚共有成分，东亚成分，中国特有种。除世界广布成分外，以北温带分布成分和泛热带成分为主。华中地区多孔菌区系基本上属温带性质，同时具有一定比例的泛热带成分，说明华中地区多孔菌区系具有明显的从温带到热带的过渡性质。 7.华中地区的多孔菌资源丰富，野生的食、药用多孔菌48种；工业用多孔菌有21种；林木病原菌有37种，其中新报道病原菌1种。 8.华中地区稀有多孔菌有22种，濒危多孔菌10种。

中国产丝齿菌属Hyphodontia分类学研究

革菌是指平伏的具有光滑、齿状或瘤状子实层体表面的木材腐朽菌，该类群属于担子菌门（Basidiomycota），担子菌纲（Basidiomycetes），是一类数量较多、组成较为复杂的高等真菌。 革菌具有重要的生态学功能和经济价值。该类群真菌能够降解木质素和纤维素，在森林生态系统中起着关键的降解还原作用；同时，革菌还是重要的生物资源，部分种类能够造成林木病害是林木病原菌；部分革菌具有药用、食用价值；有些种类的革菌还具有重要的工业应用价值。 对采集自我国不同地区、不同森林生态类型中的，以及现保存于中国科学院沈阳应用生态研究所东北生物标本馆（IFP），中国科学院微生物研究所真菌标本馆（HMAS）等国内主要标本馆的非褶菌目木材腐朽菌 — 产丝齿菌属（Hyphodontia J. Erikss.）的真菌标本进行了全面系统的研究，其中自采标本约800余号，馆藏标本600余号。按照Donk，Eriksson和Parmasto提出的传统分类方法对该属的种类进行详细的描述和显微结构绘图，记载了每个种类的寄主、国内外分布及研究标本，并对每种与相似种的联系和区别进行了讨论。我国范围内共记录及描述产丝齿菌属（Hyphodontia）39种，其中共发现新种3个，分别是：头状囊产丝齿菌Hyphodontia capitatocystidiata H.X. Xiong, Y.C. Dai & Sheng H. Wu，异囊产丝齿菌H. heterocystidiata H.X. Xiong, Y.C. Dai & Sheng H. Wu 和 似土黄产丝齿菌H. subpallidula H.X. Xiong, Y.C. Dai & Sheng H. Wu；发现中国新记录种2个：冷杉产丝齿菌Hyphodontia abieticola (Bourdot & Galzin) J. Erikss.和弯孢产丝齿菌H. curvispora J. Erikss. & Hjortstam；大陆新记录种7个，分别是：台湾产丝齿菌Hyphodontia formosana Sheng H. Wu & Burds.，羊毛状产丝齿菌Hyphodontia lanata Burds. & Nakasone，膜质产丝齿菌Hyphodontia pelliculae (H. Furuk.) N. Maek.，无锁产丝齿菌Hyphodontia poroideoefibulata Sheng H. Wu，近球孢产丝齿菌Hyphodontia subglobasa Sheng H. Wu，热带产丝齿菌Hyphodontia tropica Sheng H. Wu和管形产丝齿菌Hyphodontia tubuliformis Sheng H. Wu。

Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi.

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15Delta strain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom.

Temperature and water potential relations of tropical Trametes and other wood-decay fungi from the indigenous forests of Zimbabwe

Three isolates each, of nine different Trametes and five other wood inhabiting basidiomycetes, were collected from the indigenous forests of Zimbabwe, and the impact of temperature (20-60 degrees C), osmotic and matric potential (-0.5 to - 8.0 MPa), and their interactions on in vitro growth compared. Generally, there was no significant difference between growth of isolates of the same species in relation to temperature. Temperature relationships of the species studied correlated well with their geographic distributions. Species occurring in hot, dry regions tolerated a wide temperature range, with some showing unusually high thermotolerance (55 degrees, T. socotrana, T. cingulata and T. cervina). There were significant intra-strain differences for individual species in relation to solute potential on glycerol-modified media. Generally, growth of ail species was better on glycerol- and KCl-modified osmotic media than on a metrically-modified medium (PEG 8000) at 25, 30 and 37 degrees. The limits for growth on the osmotic media were significantly wider than matric medium, being - 4.5 to - 5.0 and - 2.5 to - 4.5 MPa, respectively. An Irpex sp. grew at lower water potentials than all other species, with good growth at - 7.0 MPa. This study suggests that the capacity of these fungi for effective growth over a range of temperatures, osmotic and matric potentials contributes to their rapid wood decay capacities in tropical climates.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

The continually growing worldwide hazardous waste problem is receiving much attention lately. The development of cost effective, yet efficient methods of decontamination are vital to our success in solving this problem.Bioremediation using white rot fungi, a group of basidiomycetes characterized by their ability to degrade lignin by producing extracellular LiP, MnP and laccase have come to be recognized globally which is described in detail in Chapter 1.These features provide them with tremendous advantages over other micro-organisms.Chapter 2 deals with the isolation and screening of lignin degrading enzyme producing micoro-organisms from mangrove area. Marine microbes of mangrove area has great capacity to tolerate wide fluctuations of salinitie.Primary and secondary screening for lignin degrading enzyme producing halophilic microbes from mangrove area resulted in the selection of two fungal strains from among 75 bacteria and 26 fungi. The two fungi, SIP 10 and SIP ll, were identified as penicillium sp and Aspergillus sp respectively belonging to the class Ascomycetes .Specific activity of the purified LiP was 7923 U/mg protein. The purification fold was 24.07 while the yield was 18.7%. SDS PAGE of LiP showed that it was a low molecular weight protein of 29 kDa.Zymogram analysis using crystal violet dye as substrate confirmed the peroxidase nature of the purified LiP.The studies on the ability of purified LiP to decolorize different synthetic dyes was done. Among the dyes studied, crystal violet, a triphenyl methane dye was decolorized to the greatest extent.

Phosphate supply and arsenate toxicity in ectomycorrhizal fungi

Three species of ectomycorrhizal fungi (Hebeloma crustuliniforme, Suillus variegatus and Cenococcum geophilum) were grown in axenic culture amended with range of AsO43– concentration under three different PO43– regimes. The fungi exhibited different growth responses to AsO43– that varied with PO43– concentration. Suillus variegatus showed the greatest sensitivity to AsO43–, with growth almost completely inhibited in the presence of AsO43– under the lower two PO43– treatments. Under the highest PO43– treatment however, growth was enhanced and S. variegatus was able to persist at AsO43– concentrations of up to 4 mM. Hebeloma crustuliniforme also showed high sensitivity to AsO43– especially at low PO43– concentration. The two higher PO43– treatments had an ameliorating effect on AsO43– toxicity in H. crustuliniforme. This demonstrates the ability of PO43– to alleviate AsO43– toxicity. The response from S. variegatus and H. crustuliniforme, both basidiomycetes, was in contrast to the ascomycete C. geophilum. This fungus demonstrated tolerance to AsO43– when grown in culture solution and PO43– did not have an ameliorating effect on AsO43– toxicity in C. geophilum.

Comparative growth of ectomycorrhizal fungi (Hebeloma spp.) on organic and inorganic nitrogen

In the largely organic soils in which ectomycorrhizas are commonly found, a preference for absorbing organic nitrogen over mineral forms is likely to be an advantage, especially where mineralisation rates are low. To determine rates of both independent and preferential growth of ectomycorrhizal basidiomycetes on organic and inorganic nitrogen, strains of Hebeloma were grown on nutrient agar media containing either NH4+ or glutamic acid as the sole source of nitrogen, on both single medium and split plate Petri dishes. Growth rates on the split plate Petri dishes, where the fungi had access to both nitrogen sources, were generally greater than on the single medium dishes. Growth on glutamic acid was at least equal to, and usually greater than, that on NH4+. In some cases growth on NH4+ alone appeared severely inhibited, a condition that was partially alleviated by access to glutamic acid on the split plates Petri dishes. This highlights a potential pitfall of single nitrogen source growth studies. The greater growth of most strains on glutamic acid suggests an adaptation to organic nitrogen utilisation in these strains. If this is so in soils with low mineralisation rates, direct uptake of amino acids by ectomycorrhizal plants could by-pass the bottle neck that requires mineral nitrogen to be made available for plant uptake.

A new topology of ACBP from Moniliophthora perniciosa

Acyl-CoA binding protein (ACBP) is a housekeeping protein and is an essential protein in human cell lines and in Trypanosoma brucei. The ACBP of Moniliophthora perniciosa is composed of 104 amino acids and is possibly a non-classic isoform exclusively from Basidiomycetes. The M. perniciosa acbp gene was cloned, and the protein was expressed and purified. Acyl-CoA ester binding was analyzed by isoelectric focusing, native gel electrophoresis and isothermal titration calorimetry. Our results suggest an increasing affinity of ACBP for longer acyl-CoA esters, such as myristoyl-CoA to arachidoyl-CoA, and best fit modeling indicates two binding sites. ACBP undergoes a shift from a monomeric to a dimeric state, as shown by dynamic light scattering, fluorescence anisotropy and native gel electrophoresis in the absence and presence of the ligand. The protein`s structure was determined at 1.6 angstrom resolution and revealed a new topology for ACBP, containing five a-helices instead of four. alpha-helices 1, 2, 3 and 4 adopted a bundled arrangement that is unique from the previously determined four-helix folds of ACBP, while alpha-helices 1, 2, 4 and 5 formed a classical four-helix bundle. A MES molecule was found in the CoA binding site, suggesting that the CoA site could be a target for small compound screening. (C) 2009 Elsevier B.V. All rights reserved.

EVALUATION OF METAL TOXICITY BY A MODIFIED METHOD BASED ON THE FUNGUS GERRONEMA VIRIDILUCENS BIOLUMINESCENCE IN AGAR MEDIUM

Metal cation toxicity to basidiomycete fungi is poorly understood, despite its well-known importance in terrestrial ecosystems. Moreover, there is no reported methodology for the routine evaluation of metal toxicity to basidiomycetes. In the present study, we describe the development of a procedure to assess the acute toxicity of metal cations (Na(+), K(+), Li(+), Ca(2+), Mg(2+), Co(2+), Zn(2+), Ni(2+), Mn(2+), Cd(2+), and Cu(2+)) to the bioluminescent basidiomycete fungus Gerronema viridilucens. The method is based on the decrease in the intensity of bioluminescence resulting from injuries sustained by the fungus mycelium exposed to either essential or nonessential metal toxicants. The assay described herein enables LIS to propose a metal toxicity series to Gerronenia viridilucens based on data obtained from the bioluminescence intensity (median effective concentration [EC50] values) versus metal concentration: Cd(2+) > Cu(2+) > Mn(2+) approximate to Ni(2+) approximate to Co(2+) > Zn(2+) > Mg(2+) > Li(+) > K(+) approximate to Na(+) > Ca(2+), and to shed some li-ht on the mechanism of toxic action of metal cations to basidiomycete fungi. Environ. Toxicol. Chem. 2010;29:320-326. (C) 2009 SETAC