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.     

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.     

Several pseudomonads,associated with the cultivated mushrooms Agaricus bisporus or Pleurotus sp., are hemolytic

Pseudomonas tolaasii, causing brown blotch disease on cultivated mushrooms, and yielding a white line precipitate towards P. “reactans”, has been shown to induce lysis of erythrocytes. Some Finnish strains isolated from diseased mushroom fruit bodies, although harboring the typical features of P. tolaasii, proved to be distinct, and have been allocated to a nov. sp. P. costantinii. We examined in these study whether all brown blotch causing agents were hemolytic. The induction of erythrocytes lysis seemed to be a rather common feature of mushroom associated-pseudomonads, especially for strains involved in the production of a white-line-in agar.     

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.     

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.     

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.     

Lecanicillium fungicola: causal agent of dry bubble disease in white‐button mushroom

Lecanicillium fungicola causes dry bubble disease in commercially cultivated mushroom. This review summarizes current knowledge on the biology of the pathogen and the interaction between the pathogen and its most important host, the white‐button mushroom, Agaricus bisporus. The ecology of the pathogen is discussed with emphasis on host range, dispersal and primary source of infection. In addition, current knowledge on mushroom defence mechanisms is reviewed. Taxonomy: Lecanicillium fungicola (Preuss) Zare and Gams: Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Sordariomycetes; Subclass Hypocreales; Order Hypocreomycetidae; Family Cordycipitaceae; genus Lecanicillium. Host range: Agaricus bisporus, Agaricus bitorquis and Pleurotus ostreatus. Although its pathogenicity for other species has not been established, it has been isolated from numerous other basidiomycetes. Disease symptoms: Disease symptoms vary from small necrotic lesions on the caps of the fruiting bodies to partially deformed fruiting bodies, called stipe blow‐out, or totally deformed and undifferentiated masses of mushroom tissue, called dry bubble. The disease symptoms and severity depend on the time point of infection. Small necrotic lesions result from late infections on the fruiting bodies, whereas stipe blow‐out and dry bubble are the result of interactions between the pathogen and the host in the casing layer. Economic importance: Lecanicillium fungicola is a devastating pathogen in the mushroom industry and causes significant losses in the commercial production of its main host, Agaricus bisporus. Annual costs for mushroom growers are estimated at 2–4% of total revenue. Reports on the disease originate mainly from North America and Europe. Although China is the main producer of white‐button mushrooms in the world, little is known in the international literature about the impact of dry bubble disease in this region. Control: The control of L. fungicola relies on strict hygiene and the use of fungicides. Few chemicals can be used for the control of dry bubble because the host is also sensitive to fungicides. Notably, the development of resistance of L. fungicola has been reported against the fungicides that are used to control dry bubble disease. In addition, some of these fungicides may be banned in the near future. Useful websites: http://www.mycobank.org ; http://www.isms.biz ; http://www.cbs.knaw.nl     

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.     

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.     

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.     

A note on ginger blotch, a new bacterial disease of the cultivated mushroom, Agaricus bisporus

Ginger blotch, a new bacterial disease of the cultivated mushroom, Agaricus bisporus , is described from farms in the UK. The symptoms are distinct from the classical blotch disease caused by Pseudomonas tolaasii. The causative organism has been isolated and identified as a new member of the Pseudomonas fluorescens complex which can be distinguished from Pseudomonas tolaasii by several simple tests.     

Advances in genetic analysis and biotechnology of the cultivated button mushroom, Agaricus bisporus

During the last decade several major breakthroughs have been achieved in mushroom biotechnology, which greatly enhanced classical mushroom breeding. DNA-based technologies such as restriction fragment length polymorphisms and randomly amplified polydisperse DNA sequences have allowed for a measure of genetic diversity, for the isolation of homokaryons, for the determination of inheritance of nuclear and mitochondrial markers, and for the production of a genetic linkage map. The recent availability of ready-to-use and affordable DNA technologies has resulted in a substantial increase in the number of Agaricus bisporus genes that have been identified and characterized. A major breakthrough was achieved in 1996 when the first successful and stable transformation system of A. bisporus was reported. Together, the availability of an increasing number of known genes and the possibility to produce transgenic mushrooms will result in a better understanding of the molecular, physiological and biochemical processes that are essential for mushroom production, shelf life and quality aspects such as flavor, texture and disease resistance. Some potential targets for strain improvement are discussed, such as the genes involved in brown discoloration, substrate utilization, carbon and nitrogen metabolism, and fruit body development. Received: 19 January 1999 / Received revision: 27 May 1999 / Accepted: 4 June 1999     

Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: numbers of the bacterium and symptom development on mushrooms grown in various environments after artificial inoculation

The recovery of Pseudomonas tolaasii applied to peat, limestone and mushroom caps, is very difficult, recovery rates being 0.2–16.0%. Without Agaricus bisporus mycelium, inoculated Ps.tolaasii disappears in the casing layer. As mushroom primordia grew in size on inoculated mushroom beds, the number of detectable cells of the pathogen increased. Symptoms of blotch disease became visible when 5.4 times 10⁶ cfu were detectable, when the mushroom primordia were 6 mm in diameter; 60% of mushrooms showed symptoms before they were 15 mm in diameter. Application of Ps.tolaasii cells as low as 20 cfu/cm² of bed gave epidemics of this severity. Neither size nor age of mushrooms affects their susceptibility. When Ps.tolaasii was placed directly onto caps, 6 times 10⁷ cfu were necessary to produce a blotch lesion (though only 3.5 times 10⁶ cfu could be recovered). Changes in r.h. and temperature did not affect the numbers of cells of Ps.tolaasii on inoculated caps; very frequent watering did so. Increased severity of the disease was seen only on over-watered mushrooms; this occurred by increase in the size of lesions seen at the primordium stage. The number of cells of Ps.tolaasii present on the early primordial stages of mushroom growth controls the extent of blotch disease seen at harvesting, whereas variations in r.h. or temperature during growing do not do so. An illustrated disease symptom measurement key (of general application for assessing severity of blotch disease) is included in the text.     

Classification of sequences expressed during the primordial and basidiome stages of the cultivated mushroom Agaricus bisporus

Two complementary DNA (cDNA) libraries were constructed from tissues isolated from primordia and basidiomes of Agaricus bisporus to characterize genes involved in mushroom development. Using single-pass sequencing of 869 cDNA clones, we found 477 expressed sequence tags (ESTs), including 466 not previously described in the databases for A. bisporus. A BLASTX search revealed that 374 ESTs had similarities with protein sequences available from databases; 193 of these ESTs were categorized according to their putative function. Most ESTs were assigned to one of four roles: metabolism (23%), cell structure (15%), cell growth and division (12%), and protein destination and storage (10%). The remaining ESTs with putative homologues were classified in 10 additional categories. Many ESTs could not be functionally assigned. Based on redundancy levels, at least 4 ESTs were preferentially expressed in each tissue type. Sequence analysis also suggested the presence of paralog tyrosinase genes in the A. bisporus genome.     

Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: effects of sodium hypochlorite on the bacterium and on blotch disease severity

Sodium hypochlorite killed Pseudomonas tolaasii in water in 30 s at pH 6.0 when 5 mg/1 free available chlorine (FAC) was used. On glass beads 62.5 mg/1 FAC was necessary to kill the pathogen in 30 s. Peat and limestone mixture ('casing') prevented some cells of the pathogen being killed by chlorine. Casing treated with 50 and 100 mg/1 FAC still contained some Ps. tolaasii cells which were later able to multiply. Although some viable cells of the pathogen survived the use of 150 mg/1 FAC these were apparently unable to multiply. Mushroom tissue is more 'disinfectant-wasting' than casing, the pathogen on it surviving 250 mg/1 FAC for 10 min. In controlled environmental experiments, use of 150 mg/1 FAC at mushroom 'pinning' (2.5 mm diameter primordia) gave as much control of blotch disease as was obtainable if chlorination began after casing. Delay in starting chlorination until the mushrooms were 10 to 15 mm in diameter resulted in blotch disease incidence and severity as severe as in unchlorinated controls. Disease incidence was not reduced when 50, 100 and 150 mg/1 FAC was used, but disease severity was significantly reduced when 150 mg/1 was used. Adjusting the pH of the water did not affect these results. On commercial farms, routine watering with 150 mg/1 FAC starting at pinning, checked frequently by the sodium arsenite titrimetric method, for 3 years, reduced the percentage of mushrooms discarded because of very severe Ps. tolaasii blotch from 5.2% to 0.6% on one farm and from 7.4% to 0.5% on another, but did not eliminate the disease completely.     

Bacterial blotch disease of the cultivated mushroom Agaricus bisporus: screening, isolation and characterization of bacteria antagonistic to the pathogen (Pseudomonas tolaasii)

Bacteria, mainly pseudomonads, were isolated from mushroom farms and from soil and plant materials. They were screened for antagonism to Pseudomonas tolaasii , the cause of bacterial blotch of mushroom, using an exclusion zone assay against a bacterial lawn of the pathogen. Selected potential antagonists were identified by the API system and whole cell fatty acid profiles. These strains were tested further in the white line test and host pathogenicity test with mushroom caps. Some of the antagonists have been stable in their aggressiveness over 1 year and several transfers during storage on nutrient agar.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on yield and quality of the cultivated mushroom Agaricus bisporus

Thirteen species of saprobic rhabditid nematodes (11 genera) were identified from samples of compost and casing material collected from mushroom farms in the British Isles. Caenorhabditis elegans, the most frequently found saprobe, was mass-produced monoxenically and its effects on the cultivated mushroom, Agaricus bisporus (strain U3) were studied. C. elegans did not multiply in well-prepared, pasteurised, spawned compost, whereas casing material proved to be a highly suitable environment for its reproduction. An initial casing inoculum of 10⁶ nematodes/crate of compost (7.5 kg), caused a significant reduction in mushroom yield. Losses in total mushroom yields of 11%, 20% and 26% were caused by initial inoculum rates of 10⁶, 10⁷and 2 × 10⁷ nematodes/crate, respectively. Yields were negatively correlated with the initial nematode inoculation level and regression equations were derived. The nematode treatments caused fewer mushrooms to be produced and an absence of the usual distinctive flushing patterns. C. elegans caused considerable deterioration in mushroom quality and characteristic distortion of mushrooms. Individual sporophores were mis-shapen, notched and had brown or violet coloured grills. Up to 3.8%, 6.7% and 10.8% of total weight and 3.5%, 5.4% and 8% of total numbers of mushrooms were distorted at the three highest nematode inoculum rates tested. Weights and numbers of distorted mushrooms were positively correlated with the initial nematode population. C. elegans commonly colonised sporophores.     

Effects of cultural conditions on the mycelial growth of healthy and virus-infected cultivated mushroom, Agaricus bisporus

There was a significant inverse correlation (P= 0=001) between concentrations of mushroom viruses 1 and 2 in sporophores and amounts of mycelial growth on malt agar of isolates taken from them. Increasing virus concentrations decreased linear growth of one mushroom strain from 76 mm (healthy) to 35 mm (mildly infected) and 7 mm (severely infected) when incubated at 25°C for 3 wk. Mycelial growth rates of isolates from healthy and from virus-infected mushrooms were compared on eleven agar media. All media clearly differentiated between healthy and severely infected isolates, but fewer separated healthy from mildly infected isolates. Those that did contained maltose, sucrose or starch as carbon source. Media containing peptone usually gave better differentiation than those with other sources of nitrogen, but the best differentiation was obtained with malt agar. Growing healthy and infected isolates on a range of media affected their subsequent growth on malt agar, the growth of some isolates apparently being changed permanently after 2 months on some of the different media. Whereas none of the infected isolates grew less rapidly after this treatment, the growth of some of the mildly infected isolates improved to such an extent that they could no longer be distinguished from healthy isolates. After heat-treatment (1–6 wk at 33°C), mycelial growth rates of infected isolates were increased, but viruses 1 and 2 were not always eliminated unless the heat-treatment was begun immediately after subculture. Mycelial growth rate and colony characters are not infallible criteria of the presence or absence of virus, a feature of particular significance when checking the health of mushroom spawn.