Yield,size, and mushroom solids content of <Emphasis Type="Italic">Agaricus bisporus</Emphasis> produced on non-composted substrate and spent mushroom compost

Three crops of Agaricus bisporus were grown on non-composted substrate (NCS), spent mushroom compost (SMC), a 50/50 mixture of NSC/SMC, or pasteurized Phase II compost. NCS consisted of oak sawdust (28% oven dry wt), millet (29%), rye (8%), peat (8%), ground alfalfa (4%), ground soybean (4%), wheat bran (9%) and CaCO₃ (10%). Substrates were non-supplemented or supplemented with Target^® (a commercial delayed release nutrient for mushroom culture) or soybean meal at spawning or casing, or with Micromax^® (a mixture of nine micronutrients) at spawning. Mushroom yield (27.2 kg/m²) was greatest on a 50/50 mixture of NCS/SMC supplemented with 10% (dry wt) Target^® at casing. The same substrate supplemented with Target^® at spawning yielded 20.1 kg/m². By comparison, mushroom yield on Phase II compost supplemented at casing or at spawning with Target^® was 21.6 kg/m² and 20.6 kg/m², respectively. On NCS amended with 0.74% or 0.9% Micromax^® at spawning, yields increased by 51.8% (12.9 kg/m²) and 71.8% (14.6 kg/m²), respectively, over non-amended NCS (8.5 kg/m²). Conversely, mushroom yields were not affected when Micromax^® was added to a 50/50 mixture of NCS/SMC. Mushroom solids content was higher in mushrooms harvested from NCS amended with 0.74% Micromax^® (9.6%) compared to non-amended NCS (8.3%).     

Re-supplementing and re-casing mushroom (<Emphasis Type="Italic">Agaricus bisporus</Emphasis>) compost for a second crop

Experiments were performed to determine the effect of adding nutrient supplements to colonized mushroom compost (MC) for the production of a second crop of mushrooms. Mushrooms were harvested for 1, 2 or 3 flushes, the casing removed and the MC then was fragmented and re-supplemented with delayed release supplements treated or non-treated with fungicide (thiophanate-methyl; Topsin M 70WP) and re-cased. Overall double-crop yields were higher when MC was re-supplemented after 1st flush (1st flush MC) as compared to re-supplementation after the 2nd or 3rd flushes. Mean double-crop BEs were 128, 119 and 109% when 1st-, 2nd- and 3rd-flush MCs were used, respectively. Treatment of delayed release supplement with thiophanate-methyl fungicide did not affect mushroom yields. Soluble salts and potassium concentrations increased 350 and 900%, respectively, in the casing overlay through three flushes suggesting that removal of the casing would help to alleviate the build up of these potential growth-limiting materials. Re-supplementing and re-casing of MC represents a potential opportunity for growers to increase revenues and reduce costs associated with preparation and disposal of compost. The ability to double-crop mushroom compost would provide growers a chance to increase yields by 40% or more, depending on whether they re-supplement and re-case after 1st, 2nd or 3rd flush.     

Evaluation of the behavioural response of the flies Megaselia halterata and Lycoriella castanescens to different mushroom cultivation materials

A static‐air olfactometer was used to investigate the behavioural responses of adult female phorid [Megaselia halterata (Wood) (Diptera: Phoridae)] and sciarid [Lycoriella castanescens (Lengersdorf) (Diptera: Sciaridae)] flies to the commercial white mushroom, Agaricus bisporus (Lange) Imbach, grown on a standard pasteurised composted substrate. The attraction of the flies was measured in relation to four test materials: composted substrate spawned with A. bisporus mycelium for 4 days and 14 days, uncolonised composted substrate, and A. bisporus sporophores. The experiment was done according to a 4 × 4 × 4 Latin cube design, and the results were analysed using a generalised linear model. It was found that both the occasion on which a bioassay was run and the position of the olfactometer within a 4 × 4 array could affect the proportion of the fly population responding to a test material. Megalesia halterata preferred spawned compost to unspawned compost, and the level of response to compost spawned for 14 days was greater than to compost spawned for 4 days. In contrast, L. castanescens were attracted equally to all of the materials tested. Overall, L. castanescens showed a greater level of activity than M. halterata, and was more likely to enter the pitfall traps in the olfactometer. For both M. halterata and L. castanescens, the type of test material affected the numbers of adult flies of the F₁ generation that emerged from it following oviposition. The highest numbers of emerging M. halterata were obtained from a composted substrate spawned for 4 days, and none emerged from the unspawned compost. Emergence of L. castanescens was highest from the uncolonised composted substrate, and there was a negative relationship between emergence and the amount of mycelium in the composted substrate. The results are consistent with the use of volatiles in the detection of oviposition sites by both species; however, further studies of the materials will be necessary to determine precisely which oviposition cues the insects use.     

Changes in nitrogen resources with increases in temperature during production of mushroom compost

The rise in temperature is an important event during the composting of lignocellulosic materials and has to be controlled and regulated to improve the biodegradation. Phase I mushroom composts were incubated under environmentally controlled conditions. When the temperature was pre-set at 65°C and then later at around 80°C, the microbial respiration and the biodegradations were hardly affected. However residual activities due to thermoresistant bacteria were still measured after 68 h at a constant temperature of 80°C. Significant changes in nitrogen resources were observed: loss of nitrogen from microbial products, an increase in the proportion of nitrogen in non-hydrolysable and stable forms, and an increase in volatilisation of ammonia. These changes were mainly due to physico-chemical mechanisms associated with disturbances in the structure of the microbial community.The authors are with INRA, Unité de Recherches sur ies Champignons, BP 81, 33883 Villenave d'Ornon, France     

Stimulation of environmentally controlled mushroom composting by polysaccharidases

Polysaccharidases adsorbed on commercial amylodextrins were added to environmentally controlled composts of straw plus poultry manure. After 5 days of composting at 48°C, microbial enzyme activities and numbers of bacteria were higher in the treated compost than in the control. During the next phase at 80°C, between days 5 and 6, more C and N were solubilized in the treated compost. After introducing a microbial inoculum on day 6, and maintaining the substrate at 48°C, colonization by bacteria was faster in the treated compost and consequently, more fibre was degraded. Differences between composts in yields of Agaricus bisporus after 5 weeks of cropping were not significant (P=0.05).The authors are with INRA, Station de Recherches sur les Champignons, BP 81, 33883 Villenave d'Ornon, France     

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.     

Investigations into the taxonomy of the mushroom pathogen <Emphasis Type="Italic">Verticillium fungicola</Emphasis> and its relatives based on sequence analysis of nitrate reductase and ITS regions

The full sequence of the nitrate reductase gene was obtained from a type isolate of Verticillium fungicola var. fungicola and used for phylogenetic analysis against other ascomycete fungi. Sequencing obtained 2749 bp of coding region, 668 bp of 5′ flanking sequence and 731 bp of 3′ flanking sequence. In silico analysis indicated that the coding region contains a single intron and translates into an 893 amino acid protein, with BLAST analysis identifying five conserved nitrate reductase domains within the protein. The 5′ flanking sequence contains numerous conserved sites putatively involved in binding nitrogen regulatory proteins, indicating that the regulation of the gene is likely to be subject to the same regulation as that of model fungi such as Aspergillus nidulans. The central portion of this gene was amplified and sequenced from a number of V. fungicola isolates and related fungi and the resulting phylogenies compared to those obtained from analysis of the rDNA internal transcribed spacer regions for these fungi. Both nitrate reductase and ITS analyses provide additional evidence that reinforces previous findings that suggest the mushroom pathogenic Verticillium species are more related to other chitinolytic fungi such as the insect pathogens Verticillium lecanii and Beauveria bassiana than to the plant pathogenic Verticillia.     

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.     

双孢蘑菇酪氨酸酶基因在酿酒酵母中的异源表达及其酶学特性

【目的】在酿酒酵母中异源表达双孢蘑菇来源的酪氨酸酶基因PPO2,并研究酪氨酸酶在酿酒酵母胞内及胞外的酶学特性。【方法】提取双孢蘑菇总RNA,通过RT-PCR克隆酪氨酸酶基因PPO2,构建表达载体pSP-G1-PPO2,并转化至酿酒酵母进行表达,采用镍亲和层析纯化蛋白并研究其酶学性质。【结果】在酿酒酵母中正确表达了大小为65 kDa的酪氨酸酶蛋白。重组酶能催化底物酪氨酸产生黑色素。体外活性测定表明,酪氨酸酶催化最适温度为45°C,以酪氨酸和多巴为底物时最适pH分别为7.0和8.0。在酿酒酵母中测得底物酪氨酸浓度低于2.5 mg/mL时,黑色素的产量与底物浓度呈现正相关性。【结论】来源于双孢蘑菇的酪氨酸酶基因PPO2在酿酒酵母中成功表达,重组酶具有良好的酶学特性。利用酪氨酸酶产物黑色素的产量与底物浓度呈现正相关性这一特性,可将其作为细胞酪氨酸产量的传感器,为高通量筛选酪氨酸高产菌株提供了思路。     

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65°C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a K _m for p-nitrophenylphosphate and fructose-6-phosphate of 370 M and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and -glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.     

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.     

Properties of peat-based casing soils and their influence on the water relations and growth of the mushroom (Agaricus bisporus)

Different combinations of peat and chalk or lime sources with differing moisture contents were used to determine how specific physical and chemical properties of the casing soil relate to the growth and water relations of the mushroom. The peat types varied in terms of decomposition and extraction method; the lime addition varied in terms of rate and type (chalk or sugar beet lime). During the colonisation of the casing soil before fruiting, the extension growth rate of mushroom mycelium was most closely correlated (negatively) with the volumetric moisture content of the casing soil. Scanning electron microscopy showed that mycelium growing at a lower casing soil matric potential (Ψ_m) had a much finer and branched structure than mycelium growing at a higher Ψ_m. Across all the peat and lime source treatments, a relationship was found between the mean Ψ_m of the casing soil and mushroom yield, with an optimum Ψ_m of -7.9 to -9.4 kPa. Mushrooms are produced in ‘flushes’ at about 8-day intervals and during the development of each flush of mushrooms, there was a significant decrease in casing soil Ψ_m . This decrease (to below -40 kPa) was greatest in the second flush, which was the highest yielding. There were no relationships between mushroom yield and casing soil osmotic potential Ψ_π within the range -93 to -154 kPa or any of the other chemical properties and water and air holding characteristics of the casing soils which were determined. Across different casing soil treatments, mushroom dry matter content was negatively correlated with mushroom yield and positively correlated with mushroom tissue osmotic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Supplementation of first break mushroom compost with hydrolyzed protein, commercial supplements and crystalline amino acids

Three mushroom (Agaricus bisporus) crops (Crops 1, 2, 3) were grown to evaluate the effects of re-supplementing “spent” first break compost [mushroom compost (MC)] on mushroom yield. Mushrooms were produced for one break at the Mushroom Test Demonstration Facility, the casing layer was removed and the MC was re-supplemented with hydrolyzed protein, commercial supplements and crystalline amino acids and then re-cased at the Mushroom Research Center. Sixteen supplements, including five crystalline amino acids, one amino acid blend, one egg white and four hydrolyzed proteins, Micromax^® (a micronutrient containing nine minerals) and four commercial supplements were evaluated for their effect on mushroom yield and biological efficiency. In Crop 1, mushroom yields were stimulated (49–61%) when MC was re-supplemented with 3.6% (dry wt) Pro-Fam^® H200 FG hydrolyzed soy protein, Remo’s commercial supplement, l-isoleucine (ile), egg white protein, amino blend HLA-198 and hydrolyzed whey. Significant yield reductions were observed for MC re-supplemented with 3.6% l-tyrosine, dl-methionine or l-arginine compared to the non-supplemented control. In Crop 2, mushroom yield ranged from a high of 31.3 kg/m² on MC supplemented with 3.3% Remo’s + 0.3% ile (oven dry MC) to a low of 22.6 kg/m² on non-supplemented (control) MC (38.5% difference). In Crop 3, a response surface model was used in an attempt to optimize combinations of Remo’s commercial supplement, ile and Micromax. The response surface solution for optimal yield was 2.9% Remo’s, 0.16% ile and 0.4% Micromax. Because many of the products tested performed equally well but varied substantially in their amino acid profiles, A. bisporus appears adaptable to different supplements containing both balanced and unbalanced amino acid contents, especially those rich in the branched chain amino acids. Development and improvement of supplements designed specifically for MC may allow further increases in productivity. Double cropping would ultimately lower the cost of mushroom production by reducing labor, raw materials and time required to prepare fresh Phase II compost.     

The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"

A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans". This interaction has been considered as highly specific. However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T. Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P. tolaasii(T), produced a typical white precipitate when streaked towards P. "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P. tolaasii. We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P. tolaasii.     

Evaluating the addition of activated carbon to heat-treated mushroom casing for grain-based and compost-based substrates

Two substrates, a non-composted grain spawn substrate and a traditional composted substrate, each covered with peat-based casing that contained varying amounts of activated carbon (AC) and each receiving different heat-treatment durations, were tested for Agaricus bisporus mushroom production. The amounts of AC were 0, 5, 10, 15, and 20% v/v, and the heat treatments were 0, 60, and 180 min at 121 °C and 103.4 kPa. Overall, the addition of AC up to 10–15% of casing for a grain spawn substrate increased mushroom yield. However, the addition of AC to the casing for compost substrates had no significant effect on yield, whereas heat-treating the casing increased yield. The onset of fruiting was retarded in grain spawn treatments not receiving AC with heat-treatment durations of 60 and 180 min, whereas this effect was not as apparent for the compost substrates. On average, mushroom yield was greater for the grain spawn substrate (366 g) than for compost substrate (287 g). For grain spawn substrate, the results show that the addition of AC ranging from 5% to 10% was adequate for maximum mushroom production.     

Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii,cause of brown blotch disease of the cultivated mushroom Agaricus bisporus

Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized. Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329. These 16 isolates of P. tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates. Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping. Ribotyping differentiated P. tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes. A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P. tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2). Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T. Sequence determination and analysis of the internally transcribed spacer region ITSI for P. tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars. It is concluded that considerable genotypic differences exist among Finnish isolates of P. tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies.     

蘑菇渣堆肥对华北落叶松移植容器苗生长和营养积累的影响

为缓解草炭大量使用带来的环境问题,在华北落叶松容器育苗过程中添加蘑菇渣堆肥以替代草炭,添加蘑菇渣堆肥体积比例分别为0%(T₀)、15%(T₁)、18.75%(T₂)、25%(T₃)、37.50%(T₄)、50%(T₅)、56.25%(T₆)、60%(T₇),研究不同配比下基质的理化性质以及对华北落叶松1年生移植容器苗生长和营养积累的影响.结果表明: 当蘑菇渣堆肥替代草炭的比例≤50%时,苗木苗高、地径和生物量与常用草炭处理无显著差异,且处理T₂、T₄、T₅苗木体内养分浓度显著高于对照,基质pH值处于微酸性或中性范围,适宜苗木生长;当蘑菇渣添加比例＞50%时,基质pH值偏碱性,苗木生长受到不利影响,苗木质量下降.当蘑菇渣添加比例为15%时,苗木质量最好,苗高、地径和单株生物量达到最大.因此,蘑菇渣替代草炭培育华北落叶松移植容器苗是完全可行的,替代比例最高可达到50%,当添加比例为15%时,可培育出高质量的苗木.     

二氧化氯浸泡对双孢蘑菇褐变的抑制效应及其机理分析

以‘W2000’双孢蘑菇为试验材料,设清水(CK)和ClO2浸泡(120mg·L-1)2个处理,研究ClO2浸泡处理在低温贮藏条件下对双孢蘑菇采后品质、生理及相关酶活性的影响。结果显示:ClO2浸泡处理配合0℃低温贮藏可显著降低双孢蘑菇的呼吸强度,推迟呼吸高峰的出现时间,呼吸强度较CK降低29.2%,并使呼吸高峰推迟5d出现;同时能够有效抑制子实体多酚氧化酶(PPO)和过氧化物酶(POD)的活性,控制酚类氧化产物的积累,减缓子实体褐化进程,贮藏20d时,处理的总酚含量仅为0.81μmol/mg。另外,ClO2处理还可延缓子实体硬度的下降,保持其可溶性固形物的含量,并抑制开伞,有效延长双孢蘑菇的贮藏期。研究表明,ClO2处理+低温贮藏对双孢蘑菇具有较理想的保鲜效应,可有效保持双孢蘑菇的外观和营养品质,提高商品价值,具有一定的实际应用价值。