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PubMed2001

Antifibrotic effect of extracellular biopolymer from submerged mycelial cultures of Cordyceps militaris on liver fibrosis induced by bile duct ligation and scission in rats.

The antifibrotic effects of hot water extract (WEC), intracellular biopolymer (IPC) and extracellular biopolymers (EPC) from mycelial liquid culture of Cordyceps militaris on liver fibrosis were studied. Liver fibrosis was induced by a bile duct ligation and scission (BDL/S) operation, duration of 4 weeks in rats. In BDL/S rats, the levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin in serum and hydroxyproline content in liver were dramatically increased. The WEC or IPC treatment (30 mg/kg/day for 4 weeks, p.o.) in BDL/S rats reduced the serum AST, ALT and ALP levels significantly (p<0.01). The EPC treatment (30 mg/kg/day for 4 weeks, p.o.) reduced the serum ALT, AST and ALP levels significantly (p<0.01). Malondialdehyde contents in liver treated with WEC, IPC or EPC were significantly reduced (p<0.05). But Liver hydroxyproline content was decreased only in EPC treated BDL/S rats to 55% that of BDL/S control rats (p<0.01). The morphological characteristics and expression of alpha smooth muscle like actin in fibrotic liver, which appeared in BDL/S control group were improved in EPC treated fibrotic liver. These results indicate that EPC (30 mg/kg/day for 4 weeks, p.o.) has an antifibrotic effect on fibrotic rats induced by BDL/S.

Animals↗
PubMed2002

Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences.

A persistent paradigm in insect pathology is one that relates the insect host to certain genetic groups of insect-pathogenic fungi. This paradigm assumes that the genotype of an insect-pathogenic fungus coevolves with a certain taxon of insect host that it infects. The insect-pathogenic fungus Beauveria bassiana shows a wide host range and is considered to be a facultative insect pathogen. In this study, a population genetics analysis of B. bassiana from forested and agricultural habitats as well as from the Canadian Arctic showed distinct genetic groups associated with the three different habitats. Within each group, recombining population structures and clonally reproducing lineages were observed. The B. bassiana isolates were also assessed for their abilities to grow at 8, 15, 25 and 37 degrees C and for their tolerances to UV exposure. The genetic groups from the Arctic and from the forested habitats grew at lower temperatures, while the genetic group from the agricultural habitat grew at 37 degrees C and was tolerant to UV exposure. There were no clear associations between the genetic group and the ability to infect coleopteran or lepidopteran insect larvae. There is increasing evidence that such studies represent a significant paradigm shift; habitat selection, not insect host selection, drives the population structure of deuteromycetous insect-pathogenic fungi. We suggest that adaptation to a certain habitat type is an important criterion for identifying insect-pathogenic fungal strains for use in insect biocontrol efforts.

Adaptation, Psychological↗
PubMed2005

Phylogenetic and exon-intron structure analysis of fungal subtilisins: support for a mixed model of intron evolution.

Phylogenetic and exon-intron structure analyses of intra- and interspecific fungal subtilisins in this study provided support for a mixed model of intron evolution: a synthetic theory of introns-early and introns-late speculations. Intraspecifically, there were three phase zero introns in Pr1A and its introns 1 and 2 located at the highly conserved positions were phylogentically congruent with coding region, which is in favor of the view of introns-early speculation, while intron 3 had two different sizes and was evolutionarily incongruent with coding region, the evidence for introns-late speculation. Noticeably, the subtilisin Pr1J gene from different strains of M. ansiopliae contained different number of introns, the strong evidence in support of introns-late theory. Interspecifically, phylogenetic analysis of 60 retrievable fungal subtilisins provided a clear relationship between amino acid sequence and gene exon-intron structure that the homogeneous sequences usually have a similar exon-infron structure. There were 10 intron positions inserted by highly biased phase zero introns across examined fungal subtilisin genes, half of these positions were highly conserved, while the others were species-specific, appearing to be of recent origins due to intron insertion, in favor of the introns-late theory. High conservations of positions 1 and 2 inserted by the high percentage of phase zero introns as well as the evidence of phylogenetic congruence between the evolutionary histories of intron sequences and coding region suggested that the introns at these two positions were primordial.

Amino Acid Sequence↗
PubMed2005

Microbial biomass, community structure and metal tolerance of a naturally Pb-enriched forest soil.

The effect of long-term elevated soil Pb levels on soil microbiota was studied at a forest site in Norway, where the soil has been severely contaminated with Pb since the last period of glaciation (several thousand years). Up to 10% Pb (total amount, w/w) has been found in the top layer. The microbial community was drastically affected, as judged from changes in the phospholipid fatty acid (PLFA) pattern. Specific PLFAs that were high in Pb-enriched soil were branched (especially br17:0 and br18:0), whereas PLFAs common in eukaryotic organisms such as fungi (18:2omega6,9 and 20:4) were low compared with levels at adjacent, uncontaminated sites. Congruent changes in the PLFA pattern were found upon analyzing the culturable part of the bacterial community. The high Pb concentrations in the soil resulted in increased tolerance to Pb of the bacterial community, measured using both thymidine incorporation and plate counts. Furthermore, changes in tolerance were correlated to changes in the community structure. The bacterial community of the most contaminated soils showed higher specific activity (thymidine and leucine incorporation rates) and higher culturability than that of control soils. Fungal colony forming units (CFUs) were 10 times lower in the most Pb-enriched soils, the species composition was widely different from that in control soils, and the isolated fungi had high Pb tolerance. The most commonly isolated fungus in Pb-enriched soils was Tolypocladium inflatum. Comparison of isolates from Pb-enriched soil and isolates from unpolluted soils showed that T. inflatum was intrinsically Pb-tolerant, and that the prolonged conditions with high Pb had not selected for any increased tolerance.

Bacteria↗
PubMed2006

Occurrence and genetic structure of the systemic grass endophyte Epichloë festucae in fine fescue populations.

Epichloë species are systemic fungal endophytes that usually specialize in a certain group of related grass species. We examined the infection frequency of Epichloë festucae in populations of two fine fescue species (Festuca rubra and F. ovina) in natural and seminatural habitats at 86 study sites (total=2514 plants) across Finland and northern Norway. Infection incidence varied significantly among grass species and populations. A substantial number of the F. rubra and F. ovina populations (53 out of 77 and 25 out of 30, respectively) were either endophyte-free or had very low (<20%) infection frequencies. The highest infection frequencies were found in subarctic areas. Moreover, infection incidence differed between habitats. In the area with the highest infection frequencies, we used microsatellite markers to study genetic diversity and the rates of gene flow of E. festucae among 12 F. rubra populations. Twenty out of the 25 fungal genotypes detected with four microsatellite markers were carrying multiple alleles in at least one locus, indicating multiple infections or vegetative hybridization of the fungus. One dominant genotype occurred in all 12 populations, representing 63.5% of all isolates. We found a moderate level of average genotypic variation and a low level of genetic differentiation (Fst=0.0814). There was no correlation between infection frequency and genotypic diversity. Although the existence of a dominant genotype and the detected linkage disequilibrium suggest that the fungus is mainly asexual and vertically transmitted, the multiallelic loci and variation of genetic diversity among populations indicate occasional contagious spread and sexual or parasexual recombination of the fungus in some populations. Furthermore, the genotypes carrying multiallelic loci suggest the possibility of multiple infections or hybridization of the endophyte.

DNA, Fungal↗
PubMed2006

Does an asexual endophyte symbiont alter life stage and long-term survival in a perennial host grass?

Asexual, seedborne endophytic fungi in perennial grasses are often viewed as strong mutualists because fitness of the symbiont and host grass are closely coupled. However, at least for some native grasses, the asexual endophyte, Neotyphodium, acts parasitically, yet remains at high frequencies in natural populations. Most previous studies of Neotyphodium effects on host survival have been short term relative to the long life span of the perennial grass host. We therefore tested the hypothesis that Neotyphodium alters the survival in various life stages and long-term survival of adult native Arizona fescue (Festuca arizonica). To test the former, we planted 40 infected (E+) and 40 uninfected (E-, endophyte removed) seeds from four different maternal plants in the field under ambient conditions. We followed survival of seeds, seedlings, and adult plants over a 5-year period. To test the latter, we determined the infection of 1633 adult plants and followed their survival over the next 5-7 years. E+ seeds did not differ from uninfected seeds in terms of overall survival from seed germination to seedling to adult. However, the shape of the survival curve differed, with E+ plants showing higher mortality in early life stages. E+ adult plants did not differ from E- plants in long-term survival. Survival was generally very high during the study, which included a severe and prolonged drought. Infection by asexual Neotyphodium does not increase survival in early life stages or that of adult plants. Because asexual, vertically transmitted symbionts are predicted by evolutionary theory to be strong mutualists, the persistence of high infection frequencies in natural populations without long-term benefits to the host remains enigmatic. One possible explanation is that the long life span of the perennial host and low seedling recruitment may obscure either the costs or benefits of endophyte infection.

Festuca↗
PubMed2006

Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana.

A novel system was developed for efficient transformation of the fungal biocontrol agent Beauveria bassiana. Competent blastospores were prepared and stored in LiAc- and glycerol-inclusive suspension at -76 degrees C for sequential use in transformation. The system was successfully applied to integrating phosphinothricin resistance gene bar and enhanced green fluorescence protein gene egfp into B. bassiana via blastospore absorption of a plasmid vectoring bar and egfp. A frequency of 24 transformants per microgram of DNA was achieved. The blastospore-based transformation system has proven to be very convenient and would be highly potential for use in genetic manipulation of B. bassiana and other filamentous species.

Aminobutyrates↗
PubMed2003

Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae.

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS-PAGE.

Animals↗
PubMed2004

Distribution of chitinases in the entomopathogen Metarhizium anisopliae and effect of N-acetylglucosamine in protein secretion.

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.

Acetylglucosamine↗
PubMed2004

Nucleotide sequence variation does not relate to differences in kinetic properties of neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

Genetic variability in a putative virulence factor, the neutral trehalase ( Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., Km, Vmax, and Kcat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower Kcat/Km. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.

Amino Acid Substitution↗
PubMed2004

The Metarhizium anisopliae trp1 gene: cloning and regulatory analysis.

The trp1 gene from the entomopathogenic fungus Metarhizium anisopliae, cloned by heterologous hybridization with the plasmid carrying the trpC gene from Aspergillus nidulans, was sequence characterized. The predicted translation product has the conserved catalytic domains of glutamine amidotransferase (G domain), indoleglycerolphosphate synthase (C domain), and phosphoribosyl anthranilate isomerase (F domain) organized as NH2-G-C-F-COOH. The ORF is interrupted by a single intron of 60 nt that is position conserved in relation to trp genes from Ascomycetes and length conserved in relation to Basidiomycetes species. RT-PCR analysis suggests constitutive expression of trp1 gene in M. anisopliae.

Amino Acid Sequence↗
PubMed2006

Biotransformation of 1-naphthol by a strictly aquatic fungus.

The aquatic hyphomycete Heliscus lugdunensis belongs to a group of exclusively aquatic mitosporic fungi with an only scarcely explored potential to oxidatively attack xenobiotic compounds, and was used to study the biotransformation of the environmental pollutant metabolite 1-naphthol. H. lugdunensis metabolized approximately 74% of 1-naphthol within 5 days. The identification and quantification of degradation products using gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and high performance liquid chromatography revealed that approximately 12% of the parent compound was converted into 1-naphthylsulfate, 3% was transformed into 1-methoxy-naphthalene, and less than 1% was converted into 1,4-naphthoquinone. A further metabolite, most likely 4-hydroxy-1-naphthylsulfate, was also detected. In contrast to sulfate conjugate metabolites, no glucuronide and glucoside conjugates of 1-naphthol were found, and neither UDP-glucuronyltransferase nor UDP-glucosyltransferase present in H. lugdunensis showed activity towards 1-naphthol. These results support a role of fungi adapted to aquatic environments in affecting the environmental fate of pollutants in aquatic ecosystems.

Biotransformation↗
PubMed2006

Cloning a cuticle-degrading serine protease gene with biologic control function from Beauveria brongniartii and its expression in Escherichia coli.

Beauveria brongniartii extracellular subtilisin-like serine protease (Pr1) is one of the most virulent factors by virtue of its activity against insect cuticles. The Pr1 cDNA was cloned using the switching mechanism at the 5' end of the RNA transcript and rapid amplification of cDNA ends. The 1732-bp fragment of genomic DNA containing the predicted open-reading frame of the Pr1 gene was cloned by polymerase chain reaction and sequenced. The Pr1 cDNA is 1550 bp and contains an 1140-bp ORF. The deduced amino-acid sequence of the protein shows identity to that of proteinase K from Tritirachium album (62%), Pr1 from Metarhizium nisopliae (67%), and Pr1 from B. bassiana (76%). The Pr1 protein with an N-terminal fusion to the six-histidine tag was expressed in Escherichia coli as inclusion bodies with the expression vector pBV220. Sodium dodecylsulsulfate-polyacrylamide gel electrophoresis clearly revealed expressed product. The Pr1 protein was purified and refolded and had proteolytic activity of 0.288 U mg(-1).

Amino Acid Sequence↗
PubMed2006

Isolation, characterization, and transcriptional analysis of the chitinase chi2 Gene (DQ011663) from the biocontrol fungus Metarhizium anisopliae var. anisopliae.

Metarhizium anisopliae infects arthropods via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the host penetration step and include chitinases. The characterization of the chi2 chitinase gene from M. anisopliae var. anisopliae is reported. The chi2 gene is interrupted by two short introns and is 1,542-bp long, coding a predicted protein of 419 amino acids with a stretch of 19 amino acid residues displaying characteristics of signal peptide. The predicted chitinase molecular mass is 44 kDa with a mature protein of 42 kDa and a theoretical pI of 4.8. The comparison of the CHI2 predicted protein to fungal orthologues revealed similarity to the glycohydrolase family 18 and a phylogenetic analysis was conducted. The chi2 gene is up-regulated by chitin as a carbon source and in conditions of fungus autolysis, and is down-regulated by glucose. This regulation is consistent with the presence of putative CreA/Crel/Crr1 carbon catabolic repressor binding domains on the regulatory sequence.

Base Sequence↗
PubMed2002

Nuclear small subunit rRNA group I intron variation among Beauveria spp provide tools for strain identification and evidence of horizontal transfer.

An optional group I intron was characterized at a single insertion point in nuclear small subunit rRNA (nuSSU rRNA) genes of the imperfect entomopathogenic fungi, Beauveria bassiana and B. brongniartii. Insertion points were conserved among nuSSU rRNA genes from 35 Beauveria isolates. PCR-RFLP and DNA sequencing identified 12 group I intron variants and were applied to the identification of strains isolated from insect hosts. Alignment of 383-404-nt subgroup IB3 group I introns indicated that four insertion/deletion (indel) mutations were the main basis of fragment length variation. Phylogeny reconstruction using parsimony and neighbor-joining methods suggested six lineages may be present among nuSSU rRNA group I intron sequences from Beauveria and related ascomycete fungi. Terminal node placement of Beauveria introns conflicted with previously published phylogenies constructed from gene sequences, suggesting horizontal transfer of group I introns. PCR-RFLP among introns provided a means for the differentiation of Beauveria isolates.

Base Sequence↗
PubMed2003

Expression profiles of pea pathogenicity ( PEP) genes in vivo and in vitro, characterization of the flanking regions of the PEP cluster and evidence that the PEP cluster region resulted from horizontal gene transfer in the fungal pathogen Nectria haematococca.

A cluster of pathogenicity genes ( PEP1, PEP2, PDA1, PEP5), termed the pea pathogenicity ( PEP) cluster and located on a 1.6-Mb conditionally dispensable (CD) chromosome, was identified in the fungal pathogen Nectria haematococca. Studies determined that the expression of PDA1 is induced in both infected pea tissues and in vitro by the phytoalexin pisatin. The present study reports the use of real-time quantitative RT-PCR to monitor the expression of each PEP gene and PDA1. In mycelia actively growing in culture, the mRNA levels of PEP1, PEP5 and PDA1 were very low and the PEP2 transcript was undetectable. In planta, PDA1 and PEP2 were strongly induced, while PEP1 and PEP5 were moderately induced. Starvation slightly enhanced the expression of PEP1, PDA1 and PEP5, while the expression of PEP2 remained undetectable. Exposure to pisatin in culture stimulated the expression of PDA1 and each PEP gene to a similar level as occurred in planta. In addition, all four pathogenicity genes displayed similar temporal patterns of expression in planta and in vitro, consistent with a coordinated regulation of these genes by pisatin during pea pathogenesis. In the flanking regions of the PEP cluster, six open reading frames (ORFs) were identified and all were expressed during infection of pea. Comparison of the codon preferences of these ORFs and seven additional genes from CD chromosomes with the codon preferences of 21 genes from other chromosomes revealed there is a codon bias that correlates with the source of the genes. This difference in codon bias is consistent with the hypothesis that genes on the CD chromosome have a different origin from genes of normal chromosomes, suggesting that horizontal gene transfer may have played a role in the evolution of pathogenesis in N. haematococca.

Actins↗
PubMed2003

Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana.

Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome. The transformation protocol was optimized to generate a library of insertion mutants of Beauveria. Transformation frequencies around 10(-4) and suppression of background growth were achieved. Over 90% of the AIM mutants investigated contained single-copy T-DNA integrations at different chromosomal locations. Integrated T-DNAs were re-isolated from ten transformants by a marker rescue approach. When the sequences flanking these T-DNAs were compared with the corresponding locations of the wild-type genome, truncations of T-DNA borders were found to be common, while none of the sites of integration had suffered deletion or rearrangement. Thus, AIM can be considered a promising tool for insertional mutagenesis studies of entomopathogenic filamentous fungi.

Agrobacterium tumefaciens↗