Absence of induced resistance in Agaricus bisporus against Lecanicillium fungicola

Lecanicillium fungicola causes dry bubble disease and is an important problem in the cultivation of Agaricus bisporus. Little is known about the defense of mushrooms against pathogens in general and L. fungicola in particular. In plants and animals, a first attack by a pathogen often induces a systemic response that results in an acquired resistance to subsequent attacks by the same pathogen. The development of functionally similar responses in these two eukaryotic kingdoms indicates that they are important to all multi-cellular organisms. We investigated if such responses also occur in the interaction between the white button mushroom and L. fungicola. A first infection of mushrooms of the commercial A. bisporus strain Sylvan A15 by L. fungicola did not induce systemic resistance against a subsequent infection. Similar results were obtained with the A. bisporus strain MES01497, which was demonstrated to be more resistant to dry bubble disease. Apparently, fruiting bodies of A. bisporus do not express induced resistance against L. fungicola.     

Effect of the fungal pathogen Verticillium fungicola on fruiting initiation of its host, Agaricus bisporus

Dry bubble disease caused by the fungal pathogen Verticillium fungicola¹ is responsible for large losses to the mushroom (Agaricus bisporus) industry. The pathogen induces various symptoms on the host, bubbles (undifferentiated spherical masses), bent and/or split stipes (blowout) and spotty caps. Inoculation of A. bisporus crops with isolates of V. fungicola var. fungicola of various degrees of aggressiveness showed that the more aggressive isolates induced higher numbers of bubbles. The production of other symptoms did not vary with the isolate of pathogen. The total weight of the crop (healthy and diseased mushrooms) was not significantly affected by the disease, but inoculation with highly aggressive isolates resulted in a significant increase in the total numbers of mushrooms. Two hypotheses are proposed to explain the effect of the pathogen on fruiting initiation in relation to aggressiveness.     

Effects of the mushroom-volatile 1-octen-3-ol on dry bubble disease

Dry bubble disease caused by Lecanicillium fungicola is a persistent problem in the cultivation of the white button mushroom (Agaricus bisporus). Because control is hampered by chemicals becoming less effective, new ways to control dry bubble disease are urgently required. 1-Octen-3-ol is a volatile that is produced by A. bisporus and many other fungi. In A. bisporus, it has been implicated in self-inhibition of fruiting body formation while it was shown to inhibit spore germination in ascomycetes. Here, we show that 1-octen-3-ol inhibits germination of L. fungicola and that enhanced levels of 1-octen-3-ol can effectively control the malady. In addition, application of 1-octen-3-ol stimulates growth of bacterial populations in the casing and of Pseudomonas spp. specifically. Pseudomonas spp. and other bacteria have been demonstrated to play part in both the onset of mushroom formation in A. bisporus, as well as the inhibition of L. fungicola spore germination. A potential role of 1-octen-3-ol in the ecology of L. fungicola is discussed.     

Tolerance to dry bubble disease (Lecanicillium fungicola) in Iranian wild germplasm of button mushroom (Agaricus bisporus)

Agaricus bisporus is the most widely cultivated mushroom. The mushroom crop is subjected to several fungal diseases. Dry bubble disease caused by Lecanicillium fungicola is among notorious diseases of A. bisporus. This study aimed to assess phenotypic resistance to dry bubble disease among A. bisporus wild strains, collected from Iran regions. The reliability of resistance evaluations regarding disease incidence and intensity was well documented. The extraordinary tolerance of some wild strains to even high degrees of inoculum concentrations (10⁷ and 10⁸ spore/m² mushroom growth bed) of the pathogen in compare to commercial cultivars approved potentials of the wild germplasm in breeding programs for resistance. Also, the potential of some Microsatellite loci for the molecular-based rapid screening of tolerance was established by attributing SSR loci of phenotypically tolerant strains to QTLs for dry-bubble resistance-related traits.     

Some Properties of an Extracellular Proteolytic Enzyme of Verticillium fungicola, a Pathogen of the Cultivated Mushroom Agaricus bisporus

In a casein nutrient solution, Verticillium fungiocla, the causal agent of the dry bubble disease of the cultivated mushroom Agaricus bisporus, produces a proteolytic enzyme. The effects of the pH and of inhibitors on the protease activity and the heat stability of the enzyme are described. The protease is of interest in connection with the attacking mechanism of Verticillium fungicola.     

Microbially induced diseases of Agaricus bisporus: biochemical mechanisms and impact on commercial mushroom production

The button mushroom, Agaricus bisporus (Lange) Imbach, the most common cultivated mushroom, is susceptible to a wide range of virus, bacterial, and fungal diseases. However, only some diseases were studied for the mechanisms involved in the host–microorganism interaction. This review deals with biochemical mechanisms related to cavity disease (Burkholderia gladioli) and to the interaction between A. bisporus and the causal agents responsible for the most severe diseases, namely the bacteria Pseudomonas tolaasii and Pseudomonas reactans and the fungi Trichoderma aggressivum and Lecanicillium fungicola.     

Occurrence of Verticillium fungicola var. fungicola on Agaricus bitorquis Mushroom Crops in Spain

Diseased fruit bodies of Agaricus bitorquis, with similar symptoms to those caused by dry bubble on Agaricus bisporus, were observed in some Spanish crops during summer 1999. Isolates of Verticillium fungicola from A. bitorquis and A. bisporus were submitted to different temperatures and to prochloraz–Mn sensitivity tests. All the isolates collected from A. bitorquis and A. bisporus were identified as V. fungicola var. fungicola. Artificial infections of A. bisporus and A. bitorquis with V. fungicola var. fungicola are also described in the present study. The appearance of natural infections of V. fungicola var. fungicola in A. bitorquis crops could well be due to the growing temperatures used in Spain, which are considerably below those used in other countries.     

Molecular and physiological diversity among Verticillium fungicola var. fungicola

The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible for Agaricus bisporus dry bubble disease in North America is well documented but little is known about the var. fungicola affecting European crops. Variability was assessed within this variety and compared with that reported for the var. aleophilum. Eighteen isolates of V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymorphism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homogeneous group containing the other var. fungicola isolates, but no correlation could be drawn between DNA polymorphism and the various traits studied. The var. fungicola isolates were more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only mycelial growth rate at 23 °C could explain the variability in aggressiveness among the European isolates. The putative effect of the post-incubation temperature on contamination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices.     

The effect of tibia morphology on vector competency of mushroom sciarid flies

Mushroom sciarid flies Lycoriella ingenua (Dufour) and Bradysia ocellaris (Comstock) are major pests of cultivated mushrooms, Agaricus bisporus (Lange) Imbach. The economic threshold of these pests is very low because they vector pathogens across mushroom beds, e.g. Verticillium fungicola which causes ‘dry bubble’ disease. Under controlled conditions, B. ocellaris transported more V. fungicola spores than L. ingenua from infected to sterile culture plates. Similar results were obtained when L. ingenua and B. ocellaris were collected from a growing room infected with V. fungicola then introduced onto sterile culture plates for 90 min. The external morphology of B. ocellaris and L. ingenua was examined using scanning electron microscopy. The micrographs showed clusters of V. fungicola spores attached to the inner side of a comb‐like row of bristles on the fore tibia of B. ocellaris whereas L. ingenua does not possess an equivalent structure on the fore tibia. These morphological differences are the most probable explanation for the greater competence of B. ocellaris as a vector of V. fungicola compared with L. ingenua.     

The effect of spent mushroom compost on Lecanicillium fungicola in vivo and in vitro

Leached spent mushroom compost (SMC) and its extract were tested to suppress Lecanicillium fungicola in white button mushroom. Sterile and non-sterile mixture of SMC and peat were used to assess suppressiveness against L. fungicola in greenhouse experiments. The extract of SMC was prepared with sterile, non-sterile, filtered, supplied with nystatin, streptomycin and penicillin antibiotics to evaluate their effect in suppression of pathogen in vitro. Isolated bacteria from SMC extract were tested for antagonism rate against Lecanicillium fungicola. The results of the experiments showed that all applications rate of none-sterile SMC were effective in control of pathogen. However, the sterile SMC amendments did not have a positive effect on the pathogen suppression in vitro or in vivo, as was expected. The treatments amended with SMC 100% and 60% showed the most suppressive effect in the control of pathogen. Using of non-sterile SMC 20%, 40%, 60% and peat soil were most effective in mushroom yield. The extract of leached SMC showed inhibition of L. fungicola in petri dishes. Three bacteria isolated from extract, Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefacien identified using 16s rRNA, showed an antagonistic effect with the fungal growth.     

Biochemical Induction of Fruiting in Schizophyllum: Isolation and Preliminary Purification of an Inducing Substance from Agaricus bisporus Mushrooms

A diffusible substance was isolated from Agaricus bisporus mushrooms that induced haploid fruiting body formation in Schizophyllum commune. An efficient isolation procedure was developed. The fruiting-inducing substance was shown to be present in all stages of Agaricus mushroom development and there was no preferential accumulation of the activity in gill, cap, or stipe tissues. Some chemical and physical properties of the inducing factor are described.     

Attraction of female fungus gnats,Lycoriella ingenua,to mushroom‐growing substrates and the green mold Trichoderma aggressivum

To evaluate the attractiveness of several mushroom‐growing substrates to the female mushroom fly Lycoriella ingenua (Dufour) (Diptera: Sciaridae), a pest of the cultivated white button mushroom, Agaricus bisporus (JE Lange) Emil J Imbach (Agaricales), we developed a two‐choice, static‐flow olfactometer. Behavioral assays using this olfactometer indicated that mushroom compost with A. bisporus mycelia growing in it was not more attractive than compost lacking growing mycelia. We also found that female flies were more attracted to compost lacking A. bisporus mycelia than to the actual commodity, the white button mushroom fruiting bodies. Flies were not, however, attracted to sterilized compost, suggesting the attraction is due to volatiles produced by microbial metabolism in the compost. We also found that female L. ingenua flies were attracted to the mycoparasitic green mold Trichoderma aggressivum Samuels & W Gams (Hypocreales). Flies preferred mushroom compost that had T. aggressivum growing in it over compost lacking T. aggressivum, providing an experimental outcome consistent with the anecdotal belief that L. ingenua flies are vectors of T. aggressivum spores that can infest mushroom‐growing houses.     

In vitro sensitivity of Verticillium fungicola to selected fungicides

Twenty isolates of Verticillium fungicola var. fungicola collected from diseased fruit-bodies of Agaricus bisporus from prochloraz-treated crops, were exposed to a range of concentrations of six chemicals (benomyl, chlorothalonil, formaldehyde, iprodione, prochloraz-Mn-complex and prochloraz + carbendazim) in vitro. EC₅₀ values were determined for each fungus-fungicide combination. All isolates were more sensitive to prochloraz-Mn-complex (EC₅₀ values less than 5 mg 1^–1) than to the remainder fungicides, and only seven isolates were moderately sensitive (EC₅₀ values between 5 and 50 mg 1^–1) to prochloraz + carbendazim. All isolates were moderately sensitive to formaldehyde, whereas the majority of isolates were very resistant to the other three fungicides (benomyl, chlorothalonil and iprodione).     

Transformation of the cultivated mushroom,Agaricus bisporus, to hygromycin B resistance

Application of biotechnology to the cultivated mushroom,Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain ofA. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into theA. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomyceteAspergillus nidulans and fromA. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from theA. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.     

Effect of five fungicides with different modes of action on cobweb disease (Cladobotryum mycophilum) and mushroom yield

The fungicides chlorothalonil, metrafenone, prochloraz‐Mn, thiabendazole and thiophanate‐methyl were tested in vitro and in vivo for their effect on Cladobotryum mycophilum, the mycoparasite that causes cobweb disease in white button mushroom. In vitro experiments showed that metrafenone (EC₅₀= 0.025 mg L^?1) and prochloraz‐Mn (EC₅₀= 0.045 mg L^?1) were the most effective fungicides for inhibiting the mycelial growth of C. mycophilum. Selectivity indexes of the tested fungicides on both C. mycophilum and Agaricus bisporus indicated that metrafenone was also the most selective fungicide, while chlorothalonil was the most toxic fungicide against A. bisporus mycelium. The in vivo efficacy of fungicides for controlling cobweb was evaluated in three mushroom cropping trials, which were artificially inoculated with C. mycophilum (10⁶ conidia m^?2). Prochloraz‐Mn provided good control, although the surface colonised by cobweb reached 12% by the end of the crop cycles. None of the inoculated cropping trials treated with metrafenone showed any cobweb disease symptoms, and neither were any significant phytotoxic effects on mushroom yield recorded. These results indicated that metrafenone can be used as an alternative to prochloraz‐Mn in the control of cobweb disease.     

Expression of the urease gene of Agaricus bisporus: a tool for studying fruit body formation and post-harvest development

Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality of the mushroom, i.e. by the formation of ammonia from urea through the action of urease (EC 3.5.1.5). Despite the abundance of urea in the edible mushroom A. bisporus, little is known about its physiological role. The urease gene of A. bisporus and its promoter region were identified and cloned. The coding part of the genomic DNA was interrupted by nine introns as confirmed by cDNA analysis. The first full homobasidiomycete urease protein sequence obtained comprised 838 amino acids (molecular mass 90,694 Da, pI 5.8). An alignment with fungal, plant and bacterial ureases revealed a high conservation. The expression of the urease gene, measured by Northern analyses, was studied both during normal development of fruit bodies and during post-harvest senescence. Expression in normal development was significantly up-regulated in developmental stages 5 and 6. During post-harvest senescence, the expression of urease was mainly observed in the stipe tissue; expression decreased on the first day and remained at a basal level through the remaining sampling period.     

Chemical Analysis of the Lamella Walls of Agaricus bisporus Fruit Bodies

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus. Received: 30 November 1998 / Accepted: 29 January 1999     

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.This revised version was published online in October 2005 with corrections to the Cover Date.     

Stimulation of yield in the cultivated mushroom by vegetable oils

Supplementation of mushroom compost at spawning and at casing with various refined and crude seed oils resulted in 1 to 1.5 lb/ft² increases in mushroom yield. Supplementation at casing with ground seeds or protein-oil combinations caused 2 to 2.5 lb/ft² increases in mushroom yield. Further evidence is presented for a relationship between lipid metabolism and the initiation of fruiting in the cultivated mushroom, Agaricus bisporus (Lange) Sing. Preliminary results suggest the possible involvement of sterols in the fruiting stimulation.