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.     

Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61–63 % and 51–55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59–60 % identity.Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.     

TRANSPIRATION FROM THE SPOROPHORE OF AGARICUS BISPORUS ‘WHITE’

The transpiration from normal, intact, growing sporophores of the cultivated mushroom, Agaricus bisporus cv ‘White’ was determined by a gravimetric method. A simple method was devised to estimate the surface area of a sporophore. Under different conditions of temperature and relative humidity, the quotient of transpiration/cm² sporophore surface area and evaporation/cm² free-water surface area did not significantly differ from 1. Transpiration from the underside of an open-veil mushroom was related to the planar area rather than to the total exposed gill area. Normally growing sporophores transpired up to 3 mg/cm²/hr. It was estimated that during development to the open-veil stage, a sporophore transpired a quantity of water equal to ca. one-half of its fresh weight. There was no evidence of factors other than environmental affecting the evaporation of water from the surface of the normally growing sporophore. Our data were not extensive enough, however, to provide evidence for or against Schütte's hypothesis that transpiration in a mature agaric fructification may be intimately linked with a physiological process.     

Salt tolerance of Agaricus bisporus in relation to water stress and toxicity of sodium ions

Using NaCl or polyethylene glycol (PEG) solutions to progressively decrease the external osmotic potential of the peat casing of the growing medium used to culture the mushroom Agaricus bisporus resulted in proportionately decreased yields of sporophores. Over the range of -0.07 to -0.37 MPa, the extent of decrease in yield was similar with both types of osmoticum. However, with further decrease in external osmotic potential (from -0.37 to -0.62 MPa) there was a further proportional decrease in sporophore yield with PEG but a complete suppression of sporophore production with NaCl. Treatments with both NaCl and PEG decreased the concentrations of P, Mg, K, Fe and Mn, but not N and Cu, in sporophore dry matter. Treatment with NaCl solutions increased the concentrations of Na and CI ions in sporophore dry matter and decreased the concentration of Ca; PEG solutions had no effect. Ion toxicity associated with excessive accumulation of Na and C1 ions, or ionic imbalance associated with the concomittant decrease in Ca ions appear to be additional factors to osmotic stress in decreasing yield of sporophores when the growing medium becomes highly saline. The critical concentration of NaCl which caused 10% reduction in sporophore yield was 28 mM; A. bisporus is, therefore, moderately salt-sensitive.     

Carbohydrate metabolism and respiration after harvest of the cultivated mushroom, Agaricus bisporus

The carbohydrate metabolism of the mushroom and the respiration of the sporophore after harvest at different stages of growth have been studied. Mannitol and trehalose were found to be the major soluble carbohydrates in the sporophore and mycelium respectively. Mannitol and trehalose levels fall during sporophore storage, and feeding experiments with¹⁴C-labelled sugars indicate that they are metabolised. The results were discussed in relation to the post-harvest development of the sporophore.     

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.     

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.     

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.     

The nature of the microbial stimulus affecting sporophore formation in Agaricus bisporus (Lange) Sing

An apparatus is described in which pure cultures of Agaricus bisporus were maintained on composted media in filtered atmospheres free from (a) noxious concentrations of carbon dioxide, and (b) contaminating microorganisms. When grown on compost alone, cultures of A. bisporus did not produce sporophores. Their formation was however stimulated by a covering layer of an unsterilized mixture of peat and chalk (=‘casing’ soil). Autoclaving or fumigating ‘casing’ with propylene oxide decreased populations of contaminating bacteria and prevented sporophore formation. Populations of micro-organisms isolated from unsterile ‘casing’ contained bacteria which when added to pure cultures of A. bisporus stimulated fruit-body formation. Numbers of these stimulators increased when cultured on a carbon-free liquid medium exposed to atmospheres with ethanol, ethyl acetate and acetone or containing the volatile metabolites of A. bisporus. The ability to utilize these volatile chemicals was exploited in a selective technique for isolating sporophore stimulators where aqueous suspensions of mixed bacterial populations were exposed to atmospheres of these materials for 5 days, before aliquots were added to agar media subsequently gelled. The stimulatory bacteria were identified as, or closely related to, Pseudomonas putida.     

Recycling spent larval food of Corcyra cephalonica Stainton for preparing spawn and sporophore of Pleurotus sajor-caju (Fr.) Singer

Large scale production of the rice moth, Corcyra cephalonica Stainton in pearl millet grain medium leads to a huge accumulation of spent larval medium in commercial insectaries. We attempted bioconversion of spent larval medium of C. cephalonica (CLM) for cultivation of the mushroom Pleurotus sajor-caju (Fr.) Singer, to increase the usage of these residues. Maximum efficiency limits of CLM for spawn run, sporophore cropping and as bed substrate were assessed with varying combinations of sorghum and rice straw. Sorghum grains and rice straw were the best substrates for spawn run and sporophore yield respectively. Having been crushed, macerated, heated and sterilized, CLM could also become a suitable substrate along with sorghum or rice straw. Sorghum and CLM at 16.7% + 83.3% and 33.3% + 66.7% combinations were very effective in supporting mycelial growth and quicker colonization of fungus, and mother spawn yield. The spawn that was obtained from these combinations yielded higher sporophore as well. The fungus did not rapidly colonize on other combinations (50% + 50%, 66.7% + 33.3% and 83.3% + 16.7%), and was completely unable to grow on CLM 100%. Combination of rice straw and CLM at 75% + 25% and 50% + 50% as bed substrate contributed higher sporophore yield. Analysis of the substrates indicated variation in their chemical and mineral composition, but they were good sources of N, P and Ca. The prospects of exploring CLM for the mushroom cultivation are discussed.     

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.     

Biology, life table and host specificity of the mushroom pest, Brennandania lambi (Acari: Pygmephoroidea)

Biology and life table parameters of Brennandania lambi (Krczal) were studied at different temperatures while feeding on white mushroom (Agaricus bisporus) mycelium cultured on mushroom compost. The duration of egg and larva development, preoviposition and oviposition period, female longevity, and the time to 50% mortality declined as temperature increased from 16 to 28°C. The threshold temperature of development (female) was 9°C and the thermal constant for completion of development (female) was 195 day-degrees. At 16, 20, 24 and 28°C, the total fecundity (eggs/female) was 71, 67, 66 and 57, respectively and the daily fecundity rate (eggs/female/day) was 5.6, 8.7, 8.7 and 9.1, respectively. The sex ratio (female/male) ranged from 1.9 to 2.1 at 16–28°C. At 16, 20, 24 and 28°C, the intrinsic rate of natural increase (r _m) was 0.11, 0.18, 0.22 and 0.27, respectively, and the population doubling time was 6.1, 3.9, 3.2 and 2.5 days, respectively. All life stages of the mite died when exposed to 35°C constant temperature for 24h, or to 32°C constant temperature for 12 days or to 31–35°C (average 32.9°C) ambient temperature for 4 days. Brennandania lambi completed development only when fed on Ag. bisporus mycelium growing on mushroom compost. It could not survive on mushroom mycelia of Auricularia auricula, Au. polytricha, Ganoderma lucidum, Hericium erinaceus, Lentinus edodes, Pleurotus ostreatus, P. sajor-caju and Tremella fuciformis.     

Protease activity inAgaricus bisporus during periodic fruiting (flushing) and sporophore development

Protease activity from sporophores and mycelium of the mushroomAgaricus bisporus was assayed during periodic cropping (flushing) and from sporophores during maturation. When the sporophores were harvested at the same developmental stages (pins or buttons) during cropping, proteolytic activity of the sporophores was found to oscillate with the same periodicity as the flushing cycle. For pin mushrooms (an early stage of development), peaks of activity occurred during the interflush periods, whereas for button mushrooms (a later stage of development) peak proteolytic activity coincided with the periods of maximum production. The proteolytic activity in the mycelium remained low and varied little with time. Of the tissues within the sporophore, gill tissue had a higher activity than the stipe or pileus. The changes in activity during sporophore development or maturation depended on the period in the flushing cycle when the sporophore was initiated. The results are discussed in relation to the possible role and regulation of flush co-ordinated proteases.     

The Production of Marker-Free Genetically Engineered Broccoli with Sense and Antisense ACC synthase 1 and ACC oxidases 1 and 2 to Extend Shelf-Life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri T_L-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T₀ broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T₀ lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 °C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T₀ plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 °C by reducing post-harvest ethylene production.     

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.     

Isolation and characterization of membranes from the cultivated mushroom

The membranes of sporophore cap tissue from the cultivated mushroom, Agaricus bisporus (Lange) Sing., were isolated using discontinuous sucrose gradient ultracentrifugation of a tissue homogenate. A membrane-rich fraction was concentrated at the 1.16/1.18 g/cc interface and a mitochondria-rich fraction at the 1.18/1.20 g/cc interface. The membrane fraction was judged to be greater than 90% membrane vesicles by electron microscopy. The protein to lipid ratio of the membrane fraction was 1.1; the molar ratio of sterol to phospholipid was 0.77. The specific radioactivity of a Mg-activated ATPase was 2.5 times greater in the membrane fraction than in the homogenate. No 5′-nucleotidase or Na-K-Mg-activated ATPase activity was observed.     

Novel microsatellite markers suitable for genetic studies in the white button mushroom <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Co-dominant microsatellite molecular markers were obtained from the Agaricus bisporus cultivated mushroom. Their potential for both the molecular characterisation of commercial strains and the monitoring of the intraspecific genetic variation was demonstrated. The analysis of 673 unique sequences issued from public database and 59 from an enriched A. bisporus genomic library resulted in the development of a total of 33 single sequence repeat or microsatellite (SSR) markers. Their usefulness for genetic analysis was assessed on 28 strains, which include six cultivars representative of traditional lineage, two hybrids and 20 strains originating from wild populations. A. bisporus SSR markers displayed each from two to ten alleles, with an average of 5.6 alleles per locus. The observed heterozygosity ranged from 0 to 0.88. Cluster analysis resulting from SSR fingerprintings was in agreement with published A. bisporus population structure. A combination of only three selected SSR markers was sufficient to discriminate unambiguously 27 out of 28 distinct genotypes. However, the two genetically related hybrids were not distinguishable. Multiplexing was tested, and up to seven loci could be genotyped simultaneously. We are therefore reporting the first development in A. bisporus of a set of microsatellite markers powerful and suitable for genetic analysis.     

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.