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PubMed2006

Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition.

Real-Time PCR has been applied to quantify extraradical soil mycelium of the edible ectomycorrhizal fungus Lactarius deliciosus in an interspecific competition experiment under greenhouse conditions. Couples of Pinus pinea seedlings inoculated with either L. deliciosus, Rhizopogon roseolus, or non-inoculated (control) were transplanted into pots filled with two types of soil in all the possible combinations. Total DNA was extracted from soil samples at 3 and 6 months after transplantation to perform real-time PCR analysis. DNA extractions from soil mixed with known amounts of mycelium of L. deliciosus were used as standards. Six months after transplantation, the percentage of mycorrhizas of L. deliciosus and seedling growth were significantly affected by the soil type. Extraradical soil mycelium of L. deliciosus was positively correlated with the final percentage of mycorrhizas and significantly affected by the sampling time and soil depth. The competition effect of R. roseolus was not significant for any of the measured parameters, probably due to the sharp decrease of the mycorrhizal colonization by this fungus. We conclude that real-time PCR is a powerful technique for extraradical mycelium quantification in studies aimed at evaluating the persistence of introduced strains of L. deliciosus in field plantations.

Agaricales↗
PubMed2005

Interaction of airway epithelial cells (A549) with spores and mycelium of Aspergillus fumigatus.

OBJECTIVE: To study the interaction of airway epithelial cell line A549 with fragments of mycelium, spores of Aspergillus fumigatus in vitro and to determine if toll-like receptors (TLRs) are involved in the process. METHODS: A549 cells were exposed to fragments of A. fumigatus mycelium, zymosan and inactivated A. fumigatus spores. Interleukin 6 (IL-6) and IL-8 released by A549 cells to the culture supernatant were measured by ELISA. Presence of TLR2 and TLR4 on A549 cells were studied by immuno-histochemistry. RESULTS: Mycelium fragments of A. fumigatus showed strong binding to epithelial cells but had limited effects on the release of IL-6 and IL-8 by A549 cells. Irradiated A. fumigatus spores were partly internalised by A549 cells and inhibited A549 cells to produce IL-6. TNF-alpha pre-incubated A549 cells produced increased IL-6 after exposure to zymosan and WIAF. Immuno-histochemistry showed a negative staining for TLR2 and TLR4. CONCLUSIONS: The low levels of cytokines produced by A549 cells after the firm binding of either mycelium or spores of A. fumigatus may lead to insufficient recruitment of inflammatory cells to the infected site, which may result in the escape of detection by the immune defence system. TLR2 and TLR4 are probably not or only in part involved in the above process, although very low expression cannot be excluded.

Aspergillosis↗
PubMed2005

Identification of genes with a biocontrol function in Trichoderma harzianum mycelium using the expressed sequence tag approach.

Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. But the integrated biocontrol mechanism of Trichoderma harzianum is not well explored at the genetic level. This study aimed to initiate the preliminary development of an expressed sequence tag (EST) database for T. harzianum and thereby gain potentially useful information on Trichoderma gene sequences in order to elucidate the integrated biocontrol mechanism. Partial sequencing of anonymous cDNA clones is a widely used technique for gene identification. A directional cDNA library has been constructed from mycelium of T. harzianum and 3298 clones have been randomly selected, subjected to single-pass sequencing from the 5' end of the vector, and identified by sequence similarity searches against gene sequences in international databases. Of the 3298 mycelium clones, 2174 exhibit similarity to known genes and 451 to known ESTs, while 673 represent novel gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes encoding proteins such as enzymes, structural proteins, and regulatory factors. A significant proportion of genes identified in the mycelium were involved in processes related to mycoparasitism and fungicidal metabolites, as would be expected in biocontrol fungus. These results present the successful application of EST analysis in T. harzianum and provide a preliminary indication of gene expression in mycelium.

Base Sequence↗
PubMed2002

Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii.

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-beta-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.

Ascomycota↗
PubMed2006

Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods.

BACKGROUND: Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods. RESULTS: The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray. CONCLUSION: The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants.

DNA, Fungal↗
PubMed1982

Antitumor effect of heat-killed Aspergillus fumigatus mycelium in a mouse model.

A suspension of heat-killed Aspergillus fumigatus mycelium inhibited the growth of a chemically-induced mouse bladder tumor (MBT). Tumor growth was inhibited when the mycelium was injected into mice in a mixture with the tumor cells, when injected into growing tumors, and when introduced IP at the time tumor cells were injected into the hind leg muscle. In the concentrations that affected tumor growth no toxicity of the fungus preparation was observed. The fungal suspension was more effective against MBT than a Corynebacterium parvum strain known to be a potent biologic response modifier. A significant increase in the number of mouse peritoneal exudate cells (PEC) was noted following inoculation with the mycelium. The induced PEC were cytotoxic to the tumor cells in vivo, suggesting that at least part of the tumor inhibition by the mycelium is host-mediated.

Animals↗
PubMed1975

Production of aflatoxin and its partition between the medium and the mycelium of Aspergillus parasiticus during incubation under various conditions.

Spores of an aflatoxigenic strain of Aspergillus parasiticus were inoculated into a glucose-salts medium which was incubated with and without shaking at 28 degrees C for 15 days. Without shaking, maximal production of total aflatoxin and aflatoxins B1, G1, and G2 occurred at 5 days, whereas the maximal amount of B2 appeared after 7 days. Initially approximately 5% of the total toxins appeared in the mycelium but this increased to more than 60% after 5 days. Shaking of cultures during incubation served to reduce production of total aflatoxin and of each of the individual toxins. The maximal amount of total aflatoxin and of toxins B1 and G1 appeared in shaken cultures after 5 days, whereas 8 and 11 days were needed to obtain maximal amounts of B2 and G2, respectively. The mycelium of shaken cultures initially retained approximately 50% of the total aflatoxin and this increased to about 80% as the incubation progressed. Very little aflatoxin was synthesized at 35 and 45 degrees C and production of total aflatoxin and of each individual toxin was less at 15 degrees C than at 25 or 28 degrees C. When the medium contained 0.5 to 50% glucose, maximal amounts of total aflatoxin and of aflatoxins B1, G1 and G2 appeared in the presence of 30% glucose; only 20% glucose was needed to obtain the greatest amount of B2. The mycelium retained approximately 50% of total aflatoxin when the medium contained 5 to 20%. Neither aflatoxin G1 nor G2 were detected when the medium contained 0.05% ammonium sulfate and only B1, B2, and G1 appeared in the medium with 0.1% of the salt. Maximal production of each individual aflatoxin and of total aflatoxin occured with 1% of ammonium sulfate in the medium. The proportion of total aflatoxin retained by the mycelium decreased from 83 to 37% as the amount of ammonium sulfate in the medium was increased from 0,05 to 10%.

Aflatoxins↗
PubMed1975

Aflatoxin can be degraded by the mycelium of aspergillus parasiticus.

Aflatoxins B1, B2, G1, and G2 were degraded by the 8- and 16-day old but not by the 4-day old mycelium of a toxigenic strain of Aspergillus parasiticus. The 16-day old mycelium degraded the toxin more rapidly than did the 8-day old mycelium. Degradation of toxin by the mycelium was similar at pH 2.5 and 6.0.

Aflatoxins↗
PubMed2000

Fungal spore germination into yeast or mycelium: possible implications of dimorphism in evolution and human pathogenesis.

The ability of dimorphism in fungi is conventionally regarded as a reversible change between the two vegetative forms, yeast and mycelium, in response to environmental change. A zygomycetous isolate, Benjaminiella poitrasii, exhibited yeast-mycelium transition in response to the change in temperature (37-28 degrees C) and decrease in glucose concentration. For the first time the presence of dimorphic response during asexual and sexual spore germination is reported under the dimorphism-triggering conditions in B. poitrasii. The zygospores germinated into budding yeast when subjected to yeast-form supporting conditions. The mycelium-form favoring conditions gave rise to true mycelium. Similarly, the asexual spores displayed a dimorphic response during germination. Our observations suggest that dimorphism is an intrinsic ability present in the vegetative, asexual, and sexual forms of the fungus. As dimorphic fungi are intermediate to the unicellular yeast and the filamentous forms, understanding of the dimorphic character could be useful to trace the evolutionary relationships among taxonomically different fungi. Moreover, the implications of spore germination during the onset of pathogenesis and in drug development for human health care are discussed.

Biological Evolution↗
PubMed1996

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2).

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network.

4-Butyrolactone↗
PubMed1977

[Biodegradation of the mycelium of Pyrenochaeta lycopersici Schneider et Gerlach in the soil. Ultrastructural study].

Under natural conditions, Pyrenochaeta lycopersici forms a sterile mycelium. Some strains form chlamydospores, while others form micro-sclerotia. The fungus survives by hyphae cells which are present in soil either free-living or momentarily protected by the tissue of the infected root. In a model experiment, the behaviour of the free mycelium was tested in a soil which had been supplied with asparagine and manure, respectively. A known quantity of living mycelium was added to the prepared soils. Samples were taken after 1, 3, 6, and 10 months. They were examined on loss of weight of the mycelium, inoculum potential, and ultra-structure of the hyphae before and during degradation by transmission electron microscopy.

Biodegradation, Environmental↗
PubMed2003

Cloning of the conserved regulatory operon by its aerial mycelium-inducing activity in an amfR mutant of Streptomyces griseus.

We report cloning and characterization of a 2.8 kb DNA fragment that suppressed the aerial mycelium-deficient phenotype of an amfR mutant of Streptomyces griseus when it was introduced on a high-copy-number plasmid. Nucleotide sequencing revealed that the cloned DNA fragment contained a part of a regulatory operon homologous to one of the conserved operons identified in the genome of Streptomyces coelicolor A3(2). The operon appeared to consist of 5 CDSs (rarA-E; restoration of aerial mycelium formation in an amfR mutant): rarA encoded a membrane protein with weak similarity to the histidine kinase of the two-component regulatory system; rarB and rarC products did not show marked similarity to other proteins with known function; rarD encoded a G-protein carrying two GTP-binding consensus sequences conserved in the eukaryotic Ras-like proteins; rarE product showed end-to-end homology to cytochrome P450. The 2.8 kb fragment contained a 5'-end incomplete rarA and complete rarB-D in the downstream from the promoter region of mel operon of the vector plasmid. Subcloning showed that the region containing rarA only is sufficient for the aerial mycelium-inducing activity. The truncation of rarA at its 5' terminus was essential for the restoration activity, which implied that the mutated rarA product causes unusual signaling that directs the onset of morphogenesis without amfR function. Inactivation of both rarA in Streptomyces griseus and cvnD9, a rarD ortholog in S. coelicolor resulted in precocious and glucose-resistant formation of aerial mycelium and secondary metabolites, which suggested that the operon negatively regulates the onset of differentiation. S1 nuclease protection analysis showed that the transcriptional activity of the promoter preceding rarA is developmentally regulated in an amfR- and glucose-dependent manner.

Amino Acid Sequence↗
PubMed1983

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber.

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.

Candida albicans↗
PubMed1991

Phenotypic variation of Pseudomonas putida and P. tolaasii affects attachment to Agaricus bisporus mycelium.

The effect of phenotypic variation on attachment of Pseudomonas tolaasii and P. putida to Agaricus bisporus mycelium was investigated. Quantitative studies demonstrated the ability of each isolate to attach rapidly and firmly to A. bisporus mycelium and significant differences in attachment of wild-type and phenotypic variant strains were observed. This was most pronounced in P. tolaasii, where the percentage attachment of the wild-type form was always greater than that of the phenotypic variant. The medium upon which the bacteria were cultured, prior to conducting an attachment assay, had a significant effect on their ability to attach. Attachment of the wild-type form of P. putida was enhanced when the assay was performed in the presence of CaCl2, suggesting the involvement of electrostatic forces. No correlation was observed between bacterial hydrophobicity and ability to attach to A. bisporus mycelium. Scanning electron microscopy confirmed the results obtained from the quantitative studies and provided further evidence for marked differences in the ability of the pseudomonads to attach to mycelium. Fibrillar structures and amorphous material were frequently associated with attached cells and appeared to anchor bacteria to each other and to the hyphal surface. A time-course study of attachment using transmission electron microscopy revealed the presence of uneven fibrillar material on the surface of cells. This material stained positive for polysaccharide and may be involved in ensuring rapid, firm attachment of the cells.

Agaricus↗
PubMed1999

Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid pIJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody.

4-Butyrolactone↗
PubMed1995

Double-stranded RNA mycoviruses in mycelium of Pleurotus ostreatus.

Mycelium of Pleurotus ostreatus var. florida with a decreased growth rate contained seven double-stranded RNA segments and isometrical virus particles with diameters of 24 and 30 nm. Mycelium with a normal growth rate lacked dsRNA. Protoclones from virus-containing mycelium contained one to seven of these dsRNA segments in varying concentrations. The exact correlation between slow growth and the presence of dsRNA molecules could not be established. Infection of virus-free protoplasts with PEG-precipitated virus particles resulted in mycelium that stably maintained the 2.4 kbp dsRNA.

Electrophoresis, Agar Gel↗
PubMed1999

Effect of tyrosinase inhibitors on Tuber borchii mycelium growth in vitro.

This paper reports the effect of the tyrosinase (monophenol o-diphenol:oxygen oxidoreductase; EC 1.14.18.1) inhibitors diethyldithiocarbamate (DETC), L-tropolone, kojic acid, phenylthiourea (PTU) and L-mimosine on the in vitro growth of Tuber borchii (a white truffle) mycelium. A significant inhibitory effect on mycelium growth was observed for DETC, PTU and L-tropolone (0% growth compared to control at 100 microg ml(-1) DETC, PTU and L-tropolone and at 10 microg ml(-1) DETC and L-tropolone). As a comparison the action of the same inhibitors was also tested on the growth and pigmentation of the mould Cladosporium sphaerospermum. In the presence of CuSO(4) 10(-6) M T. borchii mycelium acquired pigmentation (as rounded aggregates compared to control revealed by SEM microscopy). Tyrosinase activity in the extract from T. borchii mycelium (18-day culture) was detected spectrophotometrically.

Ascomycota↗