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.     

Effect of 1-aminocyclopropane-1-carboxylic acid deaminase producing bacteria on the hyphal growth and primordium initiation of Agaricus bisporus

The mechanism of casing soil stimulating the primordium formation of Agaricus bisporus is not well understood so far. Our results showed that 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (AcdS)-producing bacteria were abundant in the casing soil of A. bisporus and accounted for up to 20 % of total culturable bacteria. A. bisporus produced ACC and ethylene. The supplement of methionine increased the ACC concentrations within the hyphae, and aminooxyacetic acid displayed an opposite effect. Methionine and ACC promoted the ethylene production while CoCl₂ suppressed the production. The AcdS-producing bacterial strain Pseudomonas putida UW4 co-cultured with A. bisporus could attach to hyphae, stimulate the hyphal growth, and reduce the ethylene production of A. bisporus. Added in sterilized casing soil, it induced the primordium formation of A. bisporus. In comparison, its AcdS-deficient mutant UW4-AcdS^? displayed the opposite effects. These results indicated that the inhibitor to the primordium formation of A. bisporus was ethylene; the AcdS-producing bacteria within the casing layer cleaved ACC, lowered the ethylene level in mushroom hyphae, and relieved the inhibition of ethylene. This is a new model of the synergism between bacteria and fungi.     

Physiologic response of Agaricus subrufescens using different casing materials and practices applied in the cultivation of Agaricus bisporus

Casing materials and practices used in the cultivation of Agaricus bisporus were evaluated in the cultivation of Agaricus subrufescens, using the best techniques for optimization of production, including the possibility of re-casing of the compost for the production of a second crop of mushroom. Casing based on peat moss, loam soil or coir was compared to casing material mixed with or without spawn-run compost. Based on the results, we conclude that the casing layer used in the cultivation of A. subrufescens should not necessarily be the same as that used in the cultivation of A. bisporus. For the tested strain cultivated with loam soil as casing layer, the ruffling technique is highly superior to CACing and should be pursued in further research. The re-casing of compost in new cycles showed good results suggesting that the currently used compost could be improved.     

Incidence,identification and pathogenicity of Cladobotryum mycophilum,causal agent of cobweb disease on Agaricus bisporus mushroom crops in Spain

Between 2008 and 2011, outbreaks of cobweb were observed in commercial white button mushroom (Agaricus bisporus) crops in Castilla‐La Mancha (Spain). In the last 3 years, the presence of the disease has notably increased resulting in serious economic losses. Based on morphological and genetic analyses, the casual agent of cobweb was identified as Cladobotryum mycophilum. A. bisporus mushroom crops were surveyed over a 2‐year period to estimate the incidence of cobweb. The presence of the disease was detected in 32% of the mushroom crops observed, being of particular concern in autumn (44% of crops infected) and winter (37%). As regards the casing material, the percentage of crops affected by cobweb was 34% in crops using mineral casing and 29% in those cased with a peat‐based casing, with no statistical relationship between the casing and the presence of cobweb. Two cropping trials inoculated with C. mycophilum were set up to evaluate the pathogenicity of the causal agent of cobweb in three peat‐based casings (C1, C2 and C3). The effect of cobweb on mushroom productivity was evaluated by comparing mushroom production and the cobweb patches detected in the casing soil. The decrease in total yield of mushrooms attributed to cobweb reached 12.9% with C2, and the crop area colonised by cobweb reached a final percentage of 36% with C3.     

Effect of medium composition on 1-octen-3-ol formation in submerged cultures of Pleurotus pulmonarius

The effect of nitrogen and fatty-acid-rich substrates on the production of 1-octen-3-ol by the edible fungus Pleurotus pulmonarius, during growth in both shaken flask and fermentor cultures, and in-vitro, in post-harvested mycelium, was studied. Addition of soybean flour and soybean oil to the growth medium enhanced 1-octen-3-ol production about sevenfold and doubled the fungal biomass, as compared to that obtained from P. pulmonarius cultured on a defined synthetic medium. A clear relationship between the production of 1-octen-3-ol and lipoxygenase activity was found during the growth of mushroom pellets. The highest in-vitro generation of 1-octen-3-ol was obtained upon addition of exogenous linoleic acid and pure O₂ to pellets grown with soybean fluor and soybean oil. This generation was even higher than that of fruiting bodies exposed to the same conditions. These results suggest that lipoxygenase activity and, subsequently, 1-octen-3-ol biosynthesis in P. pulmonarius are enhanced by the presence of substrates containing fatty acids in the growth medium. Correspondence to: D. Levanon     

Scanning Electron Microscope Studies of Interactions between Agaricus bisporus (Lang) Sing Hyphae and Bacteria in Casing Soil

Relationships between the hyphae of Agaricus bisporus (Lang) Sing and bacteria from the mushroom bed casing layer were examined with a scanning electron microscope. Hyphae growing in the casing layer differed morphologically from compost-grown hyphae. Whereas the compost contained thin single hyphae surrounded by calcium oxalate crystals, the casing layer contained mainly wide hyphae or mycelial strands without crystals. The bacterial population in the hyphal environment consisted of several types, some attached to the hyphae with filamentlike structures. This attachment may be important in stimulation of pinhead initiation.     

Bioassay for evaluating attraction of the phorid fly,Megaselia halterata to compost colonized by the commercial mushroom,Agaricus bisporus and to 1-octen-3-ol and 3-octanone

A bioassay chamber was developed to evaluate the attraction of the phorid fly pest,Megaselia halterata (Wood) (Diptera, Phoridae), to volatiles from mushroom compost colonized by the commercial mushroom,Agaricus bisporus (Fungi, Agaricaceae) and to its volatile constituents, 1-octen-3-ol and 3-octanone. The bioassay chamber consisted of a large round container which was attached, via glass tubes, to three removable sample cups situated below the container. Once a fly had entered a sample cup, the flatly cut glass tubes prevented the flies from readily returning to the release chamber and, in effect, was a pit fall trap which kept the flies in the cups until the flies were counted. Holes in the bottom of the cups and removal of 20 ml of air from the chamber to fill the entrance tubes with volatiles from the cups were necessary for the success of the bioassay. Mated and unmated femaleM. halterata and, to a lesser extent, mated males preferred spawned compost (compost well colonized by fungal mycelia) relative to unspawned compost or blank cups. Unmated males were not attracted to spawned compost. Males and females were not attracted to unspawned compost. No attraction of male or femaleM. halterata to the spawned compost volatiles, 1-octen-3-ol and 3-octanone, could be demonstrated under various experimental conditions. These two compounds seemed to deter females at high concentrations.     

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.     

Biological control of mushroom brown blotch disease using antagonistic bacteria

Button mushroom brown blotch disease is one of the most important and devastating diseases in Iran which is caused by Pseudomonas tolaasii. To screen antagonistic bacteria against this pathogen, major mushroom cultivation centres in Iran were surveyed and samples were taken from compost, soil cover and button caps with or without visible symptoms. In total, 120 bacteria were isolated on the basis of their morphology and pathology on excised tissue blocks of the fresh Agaricus bisporus. Among all tested bacteria, thirty-six isolates produced variable inhibition zones and reduced the symptom incidence on tissue blocks of A. bisporus. The most effective antagonistic bacteria caused almost 100% inhibition of pathogenic bacterium. To confirm the identification of antagonistic bacteria, RNA polymerase beta-subunit gene (rpoB) of five antagonistic strains (A1, A2, A3, A4 and A6) were amplified using primer sets of long amplicon primers (LAPS) and LAP27. The polymerase chain reaction products of the strains A1, A3 and A6 were sequenced. Based on phenotypic, biochemical and molecular characteristics, the bacterial antagonists were identified as P. putida (A1), P. reactants (A2 and A6), P. fluorescens (A3 and A4) and Bacillus subtilis (A5), respectively. In all four criteria including weight of the diseased and healthy caps, per cent disease severity and per cent disease incidence, the treatment 2 (P. fluorescens A4) was the most effective from among T1, T3, T4 and T5 treatments. Overall results of this study suggest that bacterial antagonists may be potential biocontrol agents for biological promotion of the health and growth of button mushroom.     

Relative contribution of nematodes (Caenorhabditis elegans) and bacteria towards the disruption of flushing patterns and losses in yield and quality of mushrooms (Agaricus bisporus)

Laboratory tests of bacteria isolated from the body surface, or from the gut, of a saprophagous rhabditid nematode Caenorhabditis elegans infesting mushrooms (Agaricus bisporus) showed that some bacteria enhanced nematode reproduction and that others inhibited it. As some bacteria were shown to inhibit mycelial growth of the mushroom, the effects of Acinetobacter calcoaceticus var. anitratus, Enterobacter cloacae and Serratia liquefaciens, either alone or in combination with C. elegans, on the flushing patterns, quality and yield of A. bisporus (strain Horst U3) were studied. Bacteria alone had little effect on flushing patterns whereas C. elegans delayed the onset of mushroom production and significantly disrupted the growth pattern of crops, with mushrooms appearing more regularly and not within obvious flushes. Inoculation with bacteria resulted in ‘browning’ of mushrooms that was even more pronounced in C. elegans treatments. Characteristic distortion of sporophores was observed only in the presence of C. elegans. Nematodes commonly colonised sporophores. Bacteria affected the size of nematode populations both on the sporophores and in the casing. Significant yield loss occurred; up to 10% when bacteria were inoculated, up to 27.8% when C. elegans was inoculated, and up to 35% with both bacteria and nematodes. Synergism between C. elegans and A. calcoaceticus var. anitratus was observed; the combination resulted in significantly greater reduction in mushroom yield than any other treatment. It is concluded that bacteria contribute to yield loss and quality deterioration in A. bisporus but that the effects are far greater in the presence of C. elegans.     

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.     

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     

Comparative effects of Steinernema feltiae (Nematoda: Steinernematidae) and insecticides on yield and cropping of the mushroom Agaricus bisporus

In a mushroom crop (Agaricus bisporus) affected by a very low level of sciarid fly (Lycoriella auripila) infestation, the effects of an indigenous isolate of insect-parasitic nematode (Steinernema feltiae) and of two commonly used insecticides (diazinon and diflubenzuron) were studied. When compared with untreated plots, nematodes applied to the casing had no adverse effects on mushroom yields whereas insecticides decreased yields. At a rate of 3 × 10⁶ infective juveniles per tray (surface area = 0.56 m²), S. feltiae elicited increases of 28.5% and 19% in the mean total numbers and weights of mushrooms respectively. Treatment only with diflubenzuron resulted in 14.6% and 6% reductions in mean total numbers and weights of mushrooms, respectively; treatment with both diazinon and diflubenzuron caused 18.5% and 9.4% losses. Application of nematodes generally reduced the mean weight per mushroom whereas insecticides increased it; nematodes delayed the onset of mushroom production (first flush) whereas diflubenzuron delayed the third and fourth flushes. Nematode contamination of sporophores was minimal when S. feltiae was applied at casing. Although their numbers declined with time, the nematodes persisted, in the casing layer, throughout the cropping period of seven weeks. It is concluded that yield benefits associated with nematode application can result mainly from nematode effects on A. bisporus and not solely from suppression of a damaging pest population.     

Effect of Metabolites Produced by Trichoderma harzianum Biotypes and Agaricus bisporus on Their Respective Growth Radii in Culture

Trichoderma harzianum biotypes Th1, Th2, and Th3 produced volatile metabolites in vitro which had similar fungistatic effects on the growth of Agaricus bisporus. Metabolites present in agar colonized by these strains also inhibited mycelial growth of A. bisporus, although the reduction in growth was less in the presence of metabolites produced by biotype Th2 than that in the presence of metabolites produced by Th1 or Th3. A. bisporus produced metabolites in liquid culture that inhibited the growth of Th1 and Th3 but stimulated the growth of Th2. A compound(s) responsible for the inhibition and stimulation was extracted from A. bisporus culture filtrate and from compost-grown fruit bodies with n-butanol, but the identity of the compound(s) was not determined. We suggest that the stimulation of Th2 by metabolites produced by A. bisporus and the relatively low level of inhibition of A. bisporus by Th2 facilitate colonization of compost by both fungi. However, as compost colonization reaches a maximum, a change in the competitive balance in favor of Th2 results in the inhibition of fruit body production by A. bisporus and the devastating green mold epidemics affecting mushroom production.     

The medicinal <Emphasis Type="Italic">Agaricus</Emphasis> mushroom cultivated in Brazil: biology,cultivation and non-medicinal valorisation

Sun mushroom is a cultivated mushroom extensively studied for its medicinal properties for several years and literature abounds on the topic. Besides, agronomical aspects were investigated in Brazil, the country the mushroom comes from, and some studies focus on the biology of the fungus. This review aimed to present an overview of the non-medicinal knowledge on the mushroom. Areas of commercial production and marketing trends are presented. Its specific fragrance, taste, nutritional value and potential use of extracts as food additives are compared to those of the most cultivated fungi and laboratory models. The interest of the mushroom for lignocellulosic enzyme production and source of biomolecules for the control of plant pathogens are shown. Investigation of genetic variability among cultivars is reported. Growing and storage of mycelium, as well as cultivation conditions (substrate and casing generally based on local products; indoor and outdoor cultivation; diseases and disorders) are described and compared to knowledge on Agaricus bisporus.     

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.     

Differentiation between Commercial Strains of Oyster and Button Mushrooms Using Molecular Markers

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. bisporusshowed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.     

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.     

Plant growth-promoting rhizobacteria modulate root-system architecture in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> through volatile organic compound emission

Extensive communication occurs between plants and microorganisms during different stages of plant development in which signaling molecules from the two partners play an important role. Volatile organic compounds (VOCs) emission by certain plant-growth promoting rhizobacteria (PGPR) has been found to be involved in plant growth. However, little is known about the role of bacterial VOCs in plant developmental processes. In this work, we investigated the effects of inoculation with twelve bacterial strains isolated from the rhizosphere of lemon plants (Citrus aurantifolia) on growth and development of Arabidopsis thaliana seedlings. Several bacterial strains showed a plant growth promoting effect stimulating biomass production, which was related to differential modulation of root-system architecture. The isolates L263, L266, and L272a stimulated primary root growth and lateral root development, while L254, L265a and L265b did not significantly alter primary root growth but strongly promoted lateral root formation. VOC emission analysis by SPME-GC-MS identified aldehydes, ketones and alcohols as the most abundant compounds common to most rhizobacteria. Other VOCs, including 1-octen-3-ol and butyrolactone were strain specific. Characterization of L254, L266 and L272a bacterial isolates by 16S rDNA analysis revealed the identity of these strains as Bacillus cereus, Bacillus simplex and Bacillus sp, respectively. Taken together, our data suggest that rhizospheric bacterial strains can modulate both plant growth promotion and root-system architecture by differential VOC emission.     

DNA polymorphisms in commercial and wild strains of the cultivated mushroom,Agaricus bisporus

Summary DNA from the cultivated mushroom, Agaricus bisporus, was cloned into the bacteriophage lambda vector EMBL3 creating a partial genomic library. Ten random clones from the library were used to probe for restriction fragment length polymorphisms (RFLPs). Six of the ten probes detected polymorphisms and were used to demonstrate variation in wild and cultivated strains of the mushroom. These results suggest that RFLPs could form a basis for genetic finger-printing and subsequent strain protection in A. bisporus. In single spore progeny, RFLPs were used to demonstrate normal meiotic segregation and to differentiate between homokaryons and heterokaryons. RFLPs therefore have great potential in the development of the genetics and breeding of this commercially important species.