Quantitative genetics to dissect the fungal-fungal interaction between Lecanicillium verticillium and the white button mushroom Agaricus bisporus

Lecanicillium fungicola (formerly Verticillium fungicola) is responsible for dry bubble disease in the white button mushroom Agaricus bisporus. Selection for resistance to this pathogen raises an important challenge for mushroom breeders. We have investigated the inheritance of resistance to dry bubble under artificial inoculation in three independent experiments, using a progeny of 89 hybrids derived from an intervarietal A. bisporus var. bisporus×A. bisporus var. burnettii cross. Overall, phenotypic correlations were highly significant between the different experiments. Principal component analysis, together with analysis of variance results stated that the disease reactions were accurately assessed using the percentage of bubbles (PB) and the percentage of spotty cap mushrooms (PS) separately rather than with the combination of both. An original contribution of this study lies in the effective use of area under the disease-progress curve (AUDPC) to describe the dry bubble resistance. The continuous phenotypic distribution observed for the resistance traits suggested that tolerance to dry bubble was under polygenic control. Heritability estimates for either PB or AUDPC were high (0.67-0.86) while it was inconsistent for PS (0.33-0.68) suggesting a strong impact of the environment on this latter trait. Earliness and latent period were found highly correlated with disease incidence. The earliest strains appeared to be the most resistant ones. These results contribute to disentangle the complex fungal-fungal A. bisporus / L. fungicola interaction and to provide genetic basis as a prerequisite for mushroom breeding program.     

Verticillium disease or "dry bubble" of cultivated mushrooms: the Agaricus bisporus lectin recognizes and binds the Verticillium fungicola cell wall glucogalactomannan

The step of recognition and (or) binding for the development of the disease of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola was studied by several approaches: agglutination of V. fungicola germinated spores by an A. bisporus extract from fruit body cell walls, immunofluorescence microscopy of A. bisporus hyphae from fruit bodies and vegetative mycelia pretreated with purified V. fungicola cell wall glucogalactomannan, and finally, by hemagglutination experiments carried out with an A. bisporus fruit body lectin in the presence and absence of the same glucogalactomannan. Hemagglutinating activity of the purified A. bisporus fruit body lectin was clearly inhibited by the V. fungicola glucogalactomannan, whereas in the A. bisporus vegetative mycelium such lectin was not encountered. All the results obtained make evident the recognition and binding of the A. bisporus fruit body lectin to the V. fungicola cell wall glucogalactomannan, clarifying why the mushrooms, but not the vegetative mycelium, become diseased.     

Comparison of partial sequence of the cap binding protein (eIF4E) isolated from Agaricus bisporus and its pathogen Verticillium fungicola

The 3' regions of the gene encoding the cap binding protein eIF4E were successfully isolated from Agaricus bisporus and Verticillium fungicola using a degenerate primer within the eIF4E gene and an anchored oligo d(T) primer. The deduced amino acid sequences contained 173 residues for A. bisporus and 171 residues V. fungicola. Analysis of these sequences shows that despite conserved regions of homology, centering around tryptophan residues, A. bisporus and V. fungicola are very diverse at the amino acid and DNA level. Percentage homology between the two fungi is low at the nucleotide, 35%, and amino acid level, 29%. The highest degree of similarity between the A. bisporus sequence and other published sequences is with the Homo sapiens eIF4E sequence (32%). V. fungicola exhibited highest homology with the eIF4E sequence from Caenorhabditis elegans (34%). Southern analysis of genomic DNA indicated a single copy of the gene within the A. bisporus genome.     

A polymerase chain reaction-based test for Verticillium fungicola causing dry bubble disease on the cultivated mushroom,Agaricus bisporus

A polymerase chain reaction (PCR)-based test is described for the specific detection of Verticillium fungicola var. aleophilum (Vfa), the fungal pathogen causing dry bubble disease on the cultivated button mushroom, Agaricus bisporus. PCR primers were tailored to target a 162-bp arbitrary sequence in the Vfa genome. In PCR amplifications using the primer pair, all of 20 isolates of Vfa that had been collected during a 29-year period at commercial mushroom operations located primarily in North America were found to generate the diagnostic 162-bp DNA product. Conversely, the primers failed to produce the specific amplicon with DNA from isolates representing 5 other species of Verticillium, the pathogenic subspecies V. fungicola var. fungicola from Europe, and 12 other fungal species commonly inhabiting mushroom compost. A protocol was designed enabling a confirmed diagnosis of dry bubble disease in less than 3 h. The PCR-based test should find application for the rapid diagnosis and detection of the fungal pathogen in disease management programs and, potentially, in screening for on-the-farm sources of infection.     

Investigating the role of a Verticillium fungicola beta-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption

Studies on the mycopathogen Verticillium fungicola have shown the up-regulation of beta-1,6-glucanases when grown in the presence of host cell walls and host cell wall components including chitin. These cell-wall-degrading enzymes are hypothesized to contribute to the pathogenic ability of mycopathogens. A beta-1,6-glucanase gene, VfGlu1, showing high similarity to beta-1,6-glucanase genes from Hypocrea virens, Neotyphodium sp., and Trichoderma harzianum, was isolated using degenerate PCR from V. fungicola, a serious mycopathogen of the cultivated mushroom Agaricus bisporus. Agrobacterium-mediated transformation of V. fungicola using homologous DNA from VfGlu1 resulted in homologous integration at the VfGlu1 locus in 75% of transformants, generating mutants disrupted in the VfGlu1 gene. VfGlu1 mutants displayed reduced virulence and diminished ability to utilize chitin as a carbon source, implicating VfGlu1 in the disease process. Agrobacterium-mediated transformation affords an efficient technique for the disruption of genes associated with disease symptom development in the complex V. fungicola-A. bisporus interaction.     

Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures

Aims:  To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures.
Methods and Results:  Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus–Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus–Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms.
Conclusions:  The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma -mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes.
Significance and Impact of the Study:  Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures.     

Evidence for outcrossing via the Buller phenomenon in a substrate simultaneously inoculated with spores and mycelium of Agaricus bisporus

In Agaricus bisporus, traditional cultivars and most of the wild populations belong to A. bisporus var. bisporus, which has a predominantly pseudohomothallic life cycle in which most meiospores are heterokaryons (n + n). A lower proportion of homokaryotic (n) meiospores, which typify the heterothallic life cycle, also are produced. In wild populations, pseudohomothallism was thought previously to play a major role, but recent analyses have found that significant outcrossing also may occur. We inoculated a standard substrate for A. bisporus cultivation simultaneously with homokaryotic mycelium from one parent and spores from a second parent. Culture trays produced numerous sporocarps that could theoretically have resulted from five different reproductive modes (pseudohomothallism, selfing or outcrossing via heterothallism, and selfing or outcrossing via the Buller phenomenon [i.e., between a homokaryon and a heterokaryon]). Most or all of the sporocarps resulted from outcrossing between the inoculated homokaryon and the inoculated heterokaryotic spores (or mycelia that grew from them). These data broaden our understanding of population dynamics under field conditions and provide an outcrossing method that could be used in commercial breeding programs.     

Comparative effect of the fungicide Prochloraz-Mn on Agaricus bisporus vegetative-mycelium and fruit-body cell walls.

Fungicides to control mycopathogens of commercial Agaricus bisporus, a mushroom cultivated for human consumption, are a major field of study, since these chemicals are toxic to both the host and its fungal parasites. The fungicide Prochloraz-Mn, used at its LD50 for A. bisporus, partially inhibited protein biosynthesis in the vegetative mycelial cell walls of this mushroom and caused significant changes in cell-wall polysaccharide structure, as deduced by methylation analysis and gas liquid chromatography-mass spectrometry (GLC-MS). Furthermore, the aggregated mycelial walls showed distinct alterations in their overall chemical composition following the administration of Prochloraz-Mn at the LD50 and the LD50 x1000. As expected, GLC-MS studies indicated that the latter dose caused more appreciable differences in polysaccharide structure. The decrease in mushroom crop yields obtained from industrial cultures treated with Prochloraz-Mn to control V. fungicola infection depended on the dose of the fungicide employed, whereas fruit-body morphology was only slightly affected at the highest Prochloraz-Mn concentration used.     

Chitinase production during interaction of Trichoderma aggressivum and Agaricus bisporus

The competitor fungus Trichoderma aggressivum causes green mould disease, a potentially devastating problem of the commercial mushroom Agaricus bisporus. Due to the recent appearance of this problem, very little is known about the mechanisms by which T. aggressivum interacts with and inhibits A. bisporus. A mechanism generally used by Trichoderma species in the antagonism of other fungi is the secretion of cell wall degrading enzymes. In this study, we determined the activities of chitinases produced in dual cultures of these fungi over a 2 week period. Both intracellular and extracellular enzymes were studied. Agaricus bisporus produced N-acetylglucosaminidases with apparent molecular masses of 111, 105, and 96 kDa. Two resistant brown strains produced greater activities of the 96 kDa N-acetylglucosaminidase than susceptible off-white and white strains. This result suggested that this enzyme might have a role in the resistance of commercial brown strains to green mould disease. Trichoderma aggressivum produced three N-acetylglucosaminidases with apparent molecular masses of 131, 125, and 122 kDa, a 40 kDa chitobiosidase, and a 36 kDa endochitinase. The 122 kDa N-acetylglucosaminidase showed the greatest activity and may be an important predictor of antifungal activity.     

Aspects of the pathology and etiology of 'drippy gill' disease of the cultivated mushroom Agaricus bisporus

Agaricus bisporus sporocarps exhibiting characteristic 'drippy gill' symptoms from a natural outbreak were examined. Discrete bacterial droplets on the hymenial lamellae often coalesced to form ribbons of bacterial ooze. Longitudinal splits on the stipe were lined with a similar bacterial ooze. Bacteria isolated from both the hymenium and stipe were identified as Pseudomonas agarici, and were confirmed to be the causal organism by satisfying Koch's postulates. By light and transmission electron microscopy, the causal bacteria were found to colonize the extrahyphal spaces and degrade the extracellular matrix within affected sporocarps. Degradation of the extracellular matrix was shown to reduce the integrity of the sporocarp, and result in stipe splitting and hymenium disruption. In artificial inoculations of the pileus, bacteria were shown to exist predominantly in sporocarp tissue below the point of inoculation and above affected areas of the hymenium, indicating an approximately vertical passage through the sporocarp via the extracellular spaces. The dissolution of the extracellular matrix, and the observed failure of the bacterium to produce a toxin active against A. bisporus, allow the bacteria to pass through protective membranes unnoticed, and infect the stipe and hymenium prior to veil break. These observations dispel previous assumptions of intrahyphal existence and transmission. In the few instances in which the bacteria were observed to be intrahyphal, the host fungal cell wall was often broken, suggesting intrahyphal existence was opportunistic rather than obligatory. The taxonomic position of a bacterium isolated previously from sporocarps exhibiting symptoms similar to those of drippy gill was determined by examining the biochemical and nutritional profiles of the bacterium, and comparing them with other Pseudomonas agarici isolates.     

Chemical components and their locations in the Verticillium fungicola cell wall

The chemical structure of cell walls and fractions of Verticillium fungicola, a pathogen of Agaricus bisporus, as well as their corresponding ultrastructures were studied. There are at least three chemically distinct types of carbohydrate polymers: one yielding mannose with lower amounts of galactose and glucose (glucogalactomannan), another one composed mainly of glucose (glucan), and a third one containing only N-acetylglucosamine (chitin). Attempts were made to locate these materials in situ by comparing electron micrographs of shadowed and sectioned cell walls, and also by indirect immunofluorescence. It was shown that none of these polymers constituted a completely physically distinct layer, but there seem to be different solubility properties in the outer, inner, and intermediate layers. It was also shown that fibrillar material (chitin) embedded in cementing glucan constituted the residual inner fraction of the original wall material. Indirect immunofluorescence showed the location of a significant amount of glucogalactomannan on the surface of the walls in which rodlet structures were visualized by electron microscopy.     

Properties of a hydrophobin isolated from the mycoparasitic fungus Verticillium fungicola

Verticillium fungicola, isolated from Agaricus bisporus (commercial mushroom), produced significant extracellular hydrophobin when grown for 7 days in a static liquid culture of synthetic minimal medium. The hydrophobin was purified by precipitation with ammonium sulphate (80% saturation), Sephadex G-100 gel filtration, and hydroxyapatite column chromatography. The purified protein yielded a single band in polyacrylamide gel electrophoresis under native conditions, with an apparent molecular mass of 70 +/- 4 kDa, and also another single band in SDS-PAGE, with a molecular mass of 7 +/- 3 kDa. Molecular mass determined with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) resulted in 7563.9 m/z. The same protein was extracted from the V. fungicola mycelium. Analysis of the amino acid composition revealed the presence of about 50% hydrophobic residues, detecting at least six cysteines, evaluated as cystines, and no free sulfhydryl groups. The protein did not show any glycosylation. On the basis of similarities in hydropathy patterns and solubility characteristics, V. fungicola hydrophobin can be included as a new member of Class II hydrophobins.     

Production and purification of a chitinase from Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus

The chitinolytic properties of Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus, are reported. E. americana was shown to produce chitinolytic activity in the absence of chitin and in the presence of glucose and N-acetylglucosamine, indicating constitutive synthesis by these strains. A single 33-kDa protein with chitinolytic activity was purified to homogeneity from culture filtrates, by hydrophobic interaction chromatography using a phenyl-group substituted matrix. This enzyme, by virtue of differential activity against chromogenic chitooligosaccharides and against dye-labelled soluble carboxymethylated chitin (CM-chitin-RBV), was demonstrated to be an endochitinase. Our data suggest this 33-kDa chitinase appeared to be the only chitinolytic enzyme produced by E. americana, strains of which do not grow using chitin as a carbon source. The significance of these findings in the context of mushroom disease is discussed.     

Diversity in the ability of Agaricus bisporus wild isolates to fruit at high temperature (25°C)

The button mushroom Agaricus bisporus commercially cultivated requires 16-19 °C during the fruiting period. Wild strains are also present in natural habitat, and in light of their wide range of geographic distribution reported, from boreal region to tropical region, questions on the development adaptation to temperature arose. Isolates from various geographic areas were screened for their ability to fruit at higher temperature (FHT ability) than commercial cultivars. The FHT trait discriminated at the varietal rank. Agaricus bisporus var. eurotetrasporus was unable to develop any sporophores whilst A. bisporus var. burnettii adapted perfectly to 25 °C for fruiting, suggesting that the FHT ability is a fixed trait in these varieties. In contrast, FHT ability of A. bisporus var. bisporus appeared variable and correlated neither with climate/microclimate nor with habitat. However, FHT ability taken as a whole appeared higher in North American populations than in European ones. Some A. bisporus var. bisporus isolates revealed a good potential for cultivation at 25 °C.     

Effects of bacteria isolated from a saprophagous rhabditid nematode Caenorhabditis elegans on the mycelial growth of Agaricus bisporus

P. S. GREWAL AND P. HAND. 1992. The effects of 10 species of bacteria isolated from a saprophagous rhabditid nematode Caenorhabditis elegans on mycelial growth of the cultivated mushroom Agaricus bisporus were studied in agar cultures. Bacterial species showed differential effects on the mycelial growth of A. bisporus and the effects also depended upon the mushroom strain (C43, C54 and U3). Bacillus cereus, Bacillus sp. and Enterobacter amnigenus caused significant inhibition in mycelial growth of all three strains of A. bisporus. Pseudomonas aeruginosa, Ps. fluorescens biovar reactans and Ps. maltophilia resulted in a significant increase in mycelial growth of C54 strain. Enterobacter cloacae caused a mean inhibition of about 83% in the linear mycelial extension of the most commonly cultivated mushroom strain U3. Bacillus cereus, Ent. amnigenus and Ent. cloacae produced volatile inhibitory substance(s). This is believed to be the first report about the inhibitory effects of specific bacteria isolated from a saprophagous nematode on the mycelial growth of A. bisporus.     

Screening of basidiomycete fungi for the quinone-dependent sugar C-2/C-3 oxidoreductase, pyranose dehydrogenase, and properties of the enzyme from Macrolepiota rhacodes

Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.     

利用培养特征和生理特性鉴别担子菌菌种的研究

担子菌菌种多是菌丝型的培养物,在长期保藏中如发生错误也较难辨别。本文报道了以培养特征和生理特性作为保藏的担子菌菌种的特征集要,并根据集要制定出鉴别他们的检索表。通过对48属、71种、142株菌种的实验结果,利用培养特征和生理特性鉴别保藏的担子菌菌种是可行的。     

Use of molecular markers for differentiation of cultivated strains of oyster and button mushrooms

Analysis of commercial strains of two edible mushrooms, Pleurotus ostreatus and Agaricus bisporus, using PCR and isozyme electrophoresis techniques allowed us to differentiate groups of genetically similar and distant strains. Among the commercial strains of P. ostreatus, the level of genetic variation was higher suggesting a broader genetic basis employed in breeding of this mushroom. The cultivars and hybrids of, A. bisporus, showed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.     

双孢蘑菇耐热相关基因的表达载体构建及转化研究

构建了双孢蘑菇Agaricus bisporus耐热相关基因028-1全长cDNA序列的双元表达载体,通过农杆菌介导转化双孢蘑菇非耐热菌株8213,经潮霉素抗性筛选和PCR鉴定,获得了一批双孢蘑菇转基因菌株。对10株转基因菌株进行了不同温度下的草管走菌试验,结果显示大部分转基因菌株的耐热性能有较为明显的提高。