A sensitive method for examining whole-cell biochemical composition in single cells of filamentous fungi using synchrotron FTIR spectromicroscopy

Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids at about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.     

A molecular phylogeny of thermophilic fungi

Sequences from 86 fungal genomes and from the two outgroup genomes Arabidopsis thaliana and Drosophila melanogaster were analyzed to construct a robust molecular phylogeny of thermophilic fungi, which are potentially rich sources of industrial enzymes. To provide experimental reference points, growth characteristics of 22 reported thermophilic or thermotolerant fungi, together with eight mesophilic species, were examined at four temperatures: 22 °C, 34 °C, 45 °C, and 55 °C. Based on the relative growth performances, species with a faster growth rate at 45 °C than at 34 °C were classified as thermophilic, and species with better or equally good growth at 34 °C compared to 45 °C as thermotolerant. We examined the phylogenetic relationships of a diverse range of fungi, including thermophilic and thermotolerant species, using concatenated amino acid sequences of marker genes mcm7, rpb1, and rpb2 obtained from genome sequencing projects. To further elucidate the phylogenetic relationships in the thermophile-rich orders Sordariales and Eurotiales, we used nucleotide sequences from the nuclear ribosomal small subunit (SSU), the 5.8S gene with internal transcribed spacers 1 and 2 (ITS 1 and 2), and the ribosomal large subunit (LSU) to include additional species for analysis. These phylogenetic analyses clarified the position of several thermophilic taxa. Thus, Myriococcum thermophilum and Scytalidium thermophilum fall into the Sordariales as members of the Chaetomiaceae, Thermomyces lanuginosus belongs to the Eurotiales, Malbranchea cinnamomea is a member of the Onygenales, and Calcarisporiella thermophila is assigned to the basal fungi close to the Mucorales. The mesophilic alkalophile Acremonium alcalophilum clusters with Verticillium albo-atrum and Verticillium dahliae, placing them in the recently established order Glomerellales. Taken together, these data indicate that the known thermophilic fungi are limited to the Sordariales, Eurotiales, and Onygenales in the Ascomycota and the Mucorales with possibly an additional order harbouring C. thermophila in the basal fungi. No supporting evidence was found for thermophilic species belonging to the Basidiomycota.     

Identification of fungi associated with rotylenchulus reniformis

The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.     

Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi

Termites, Coptotermes formosanus Shiraki, reared individually, were highly susceptible to entomopathogenic fungi, Paecilomyces fumosoroseus and Beauveria brongniartii and Metarhizium anisopliae, while termites reared in groups were highly resistant. Quantitative assays with an epifluoresent microscope revealed a significant difference in the number of conidia attachments among three entomopathogenic fungi. The conidia of B. brongniartii and P. fumosoroseus bound to termite cuticles more effectively than M. anisopliae conidia. Our results also suggested that self-grooming behavior is less effective, but mutual grooming is very effective in the removal of conidia from cuticles of their nestmates. Statistical analysis of removal rates indicated that conidia of P. fumosoroseus and B. brongniartii were removed more rapidly than M. anisopliae conidia from termite cuticles.     

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.     

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.     

Resistance of bioincised wood treated with wood preservatives to blue-stain and wood-decay fungi

Bioincising is a biotechnological process that aims at the improvement of wood preservative uptake in wood species with a low permeability, such as Norway spruce (Picea abies (L.) Karst). The process is based on a short-term pre-treatment with white-rot fungus Physisporinus vitreus. During incubation the membranes of bordered and half bordered pits are supposed to be degraded by fungal activity resulting in a better treatability of the wood structure for wood preservatives. In the present study, first of all the resistance of bioincised Norway spruce heartwood and untreated controls against blue-stain and wood-decay fungi (white- and brown-rot) was determined. Then, bioincised and untreated specimens were dipped or vacuum impregnated with six wood preservatives and substance uptake was assessed gravimetrically. Additionally, the penetration of 3-iodo-2-propynyl butylcarbamate (IPBC) into the wood was analyzed by high-pressure liquid chromatography (HPLC). Finally, wood resistance was assessed according to the European standards EN 152 and EN 113. Results showed no difference between bioincised wood without preservatives and the untreated wood against blue-stain discolouration. However, a significant (P < 0.05) increase in susceptibility against wood decay was recorded. In the bioincised wood samples a significantly higher uptake of all the different preservatives was determined and the HPLC-method revealed that IPBC penetrated deeper into bioincised wood than into control samples. The improved uptake of preservatives into bioincised wood resulted in a significantly higher resistance against white- and brown-rot fungi. However, only a slight protection against wood discolouration by blue-stain fungi was recorded. The results of this study show for the first time that the biotechnological process with P. vitreus can be used to improve wood durability by increasing the uptake and penetration of wood preservatives.     

The sister-group relationships of the largest family of lichenized fungi,Parmeliaceae (Lecanorales, Ascomycota)

Parmeliaceae is the largest family of lichen-forming fungi. In spite of its importance for fungal diversity, its relationships with other families in Lecanorales remain poorly known. To better understand the evolutionary history of the diversification of lineages and species richness in Parmeliaceae it is important to know the phylogenetic relationships of the closest relatives of the family. A recent study based on two molecular loci suggested that either Protoparmelia s. str. or a group consisting of Gypsoplaca and Protoparmelia s. str. were the possible sister-group candidates of Parmeliaceae, but that study could not distinguish between these two alternatives. Here, we used a four-locus phylogeny (nuLSU, ITS, RPB1, MCM7) to reveal relationships of Parmeliaceae with other potential relatives in Lecanorales. Maximum likelihood and Bayesian analyses showed that Protoparmelia is polyphyletic, with Protoparmelia s. str. (including Protoparmelia badia and Protoparmelia picea) being most closely related to Parmeliaceae s. str., while the Protoparmelia atriseda-group formed the sister-group to Miriquidica. Gypsoplaca formed the sister-group to the Parmeliaceae s. str. + Protoparmelia s. str. clade. Monophyly of Protoparmelia as currently circumscribed, and Gypsoplaca as sister-group to Parmeliaceae s. str. were both significantly rejected by alternative hypothesis testing.     

Biotransformations of ent-18-acetoxy-6-ketomanoyl oxides epimers at C-13 with filamentous fungi

Two ent-18-acetoxy-6-oxomanoyl oxides, epimers at C-13, have been prepared from ent-6alpha,8alpha,18-trihydroxylabda-13(16),14-diene (andalusol), isolated from Sideritis foetens, by means of several chemical pathways and a regioselective acylation with Candida cylindracea lipase (CCL). Biotransformation of these 13-epimeric ent-manoyl oxides by Fusarium moniliforme and Neurospora crassa produced mainly ent-1beta- or ent-11alpha-hydroxylations, as well as their deacetylated derivatives, in both epimers. In addition, with the 13-epi substrate N. crassa originated other minor hydroxylations by the ent-alpha face at C-1 or at C-12, whereas an ent-11beta-hydroxyl group, probably originated by reduction of an 11-oxo derivative also isolated, was achieved with the 13-normal substrate.     

The genetics and molecular biology of marine fungi

Interest in the genetics of marine fungi has focused upon the basis of stress adaptation and the control of the production of secondary metabolites and enzymes. Analysis by molecular genetics has been applied to marine fungal taxonomy, phylogeny and species identification. The advent of the Debaryomyces hansenii genome project and the influence of climate change on this research are discussed.     

3-hydroxypropionic acid as a nematicidal principle in endophytic fungi

3- Hydroxypropionic acid was isolated by bioactivity-guided fractionation of extracts obtained from submerged cultures of several endophytic fungi isolated from above-ground plant organs. This compound showed selective nematicidal activity against the plant-parasitic nematode Meloidogyne incognita with LD50 values of 12.5-15 microg/ml. Activity against the saprophytic Caenorhabditis elegans was fivefold lower. No antimicrobial, cytotoxic or phytotoxic effects were observed. Propionic acid and D- and L-lactic acids were not active against either nematode species. Based on morphological features and ITS, 18S and 28S rDNA analyses, the producing strains were identified as Phomopsis phaseoli isolated from the leaf of a tropical tree, and four strains of Melanconium betulinum isolated from twigs of Betula pendula and B. pubescens in Germany. This is the first report of 3-hydroxypropionic acid in fungi, and of the nematicidal activity of this metabolite.     

Biotransformation of betulinic and betulonic acids by fungi

Betulinic acid (1), a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties, such as anti-cancer and anti-HIV activities. The closely related, betulonic acid (2) also has similar properties. In order to obtain derivatives potentially useful for detailed pharmacological studies, both compounds were submitted to incubations with selected microorganisms. In this work, both were individually metabolized by the fungi Arthrobotrys, Chaetophoma and Dematium, isolated from the bark of Platanus orientalis as well as with Colletotrichum, obtained from corn leaves; such fungal transformations are quite rare in the scientific literature. Biotransformations with Arthrobotrys converted betulonic acid (2) into 3-oxo-7beta-hydroxylup-20(29)-en-28-oic acid (3), 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4) and 3-oxo-7beta,30-dihydroxylup-20(29)-en-28-oic acid (5); Colletotrichum converted betulinic acid (1) into 3-oxo-15alpha-hydroxylup-20(29)-en-28-oic (6) acid whereas betulonic acid (2) was converted into the same product and 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4); Chaetophoma converted betulonic acid (2) into 3-oxo-25-hydroxylup-20(29)-en-28-oic acid (7) and both Chaetophoma and Dematium converted betulinic acid (1) into betulonic acid (2). Those fungi, therefore, are useful for mild, selective oxidations of lupane substrates at positions C-3, C-7, C-15, C-25 and C-30.     

Thermogravimetric kinetics of corn stalk pretreated by oleaginous fungi Cunninghamella echinulata

The thermogravimetric and composition of corn stalk pretreated by oleaginous fungi Cunninghamella echinulata had been studied in this paper. Results indicated that pretreatment by oleaginous fungi C. echinulata could decrease the activation energy and make the pyrolysis more efficient and energy-saving. By bio-pretreatment, the contents of elements agreed with the weight loss, sugar content, and oil contents, especially the sulfur content was greatly decreased, greatly eliminating the inventory of gas contamination such as the emission of SOx and making the pyrolysis more environmentally friendly. Therefor, corn stalk with sugar pretreated by oleaginous fungi C. echinulata should be a good pyrolysis material to obtain high quality bio-oil.     

云南会泽铅锌矿废弃矿渣堆常见植物内生真菌多样性

从云南会泽铅锌矿废弃矿渣堆上的常见植物硬毛南芥(Arabis hirsuta)、毛萼香茶菜(Rabbosia eriocalyx)和倒挂刺(Rosa longicuspis)等6种植物的690个组织块中共分离得到内生真菌495株,内生真菌的分离频率在0.42—0.93之间,平均为0.72,所有植物茎内生真菌的分离频率都明显高于叶(P<0.05)。经形态学鉴定,内生真菌分属于茎点霉属(Phoma)、交链孢属(Alternaria)和派伦霉属(Peyronellaea)等20个分类单元,其中茎点霉属和派伦霉属为该废弃矿渣堆上常见植物的优势内生真菌属。6种植物内生真菌的多样性指数在1.05—2.29之间,与其它非重金属污染环境植物内生真菌的多样性指数相似,说明在重金属污染地区仍然存在多种重金属耐受的内生真菌种类。6种植物内生真菌的相似性系数(0.455—0.833)表明,会泽铅锌矿区植物内生真菌的宿主专一性较小。     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Taxonomical re-evaluation of Phoma-like soybean pathogenic fungi

Coelomycetous fungi classified in Ascochyta, Phoma, and Phyllosticta have been recorded from spots on leaves and pods of soybeans. Based on the Genealogical Concordance Phylogenetic Species Concept, the authors suggest the re-evaluation of the taxonomic status of Phoma sojicola (syn. = Ascochyta sojicola) and Phyllosticta sojicola. In spite of the former delimitation of Phoma sojicola based on small differences in morphological features, it has proved to be identical to Phoma pinodella. Similarly, it was also confirmed that Phyllosticta sojicola was identical to Phoma exigua var. exigua. The authors supply tools for identification of Phoma-like fungi by combined conventional and molecular methods. Protein-encoding genes (tef1 and β-tubulin) were successfully applied within the Phoma genus to infer phylogenetic relationships.     

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.     

Laboratory bioassays of entomopathogenic fungi for control of Delia radicum (L.) larvae

Laboratory soil bioassays were performed at economic field rates for in-furrow (3.85 x 10(6)spores/g dry soil) and broadcast (3.85 x 10(5)spores/g dry soil) applications with three isolates of Metarhizium anisopliae (F52, ATCC62176, and ARSEF5520) and one isolate of Beauveria bassiana (GHA). All isolates tested were infective to second instar Delia radicum (L.). The conditionally registered M. anisopliae isolate (F52) performed best killing an average of 85 and 72% of D. radicum larvae at the high and low concentration, respectively. The mean LC50 and LC95 of F52 against second instar D. radicum was 2.7 x 10(6) and 1.8 x 10(8)spores/g dry soil, respectively. The use of F52 in an integrated management program is discussed.