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Spent mushroom compost (SMC), a byproduct of commercial mushroom cultivation, poses serious environmental problems that have hampered the growth of this important agro-industry. In an effort to develop new applications for SMC, we explored its use as a feedstock for bioethanol production. SMC constitutes approximately 30%w/w polysaccharides, 66% of which is glucan. Following dilute-acid pretreatment and enzymatic hydrolysis, both in the presence of PEG 6000, 97% of glucan and 44% of xylan in SMC were converted into the corresponding monosaccharides. Incorporation of PEG 6000 reduced the cellulase requirement by 77%. Zwittergent 3-12 and 3-14 also significantly increased the efficacy of acid pretreatment and enzymatic hydrolysis. The use of SMC in bioethanol production represents a potential mitigation solution for the critical environmental issues associated with the stockpiling of the major byproduct of the mushroom industry., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement.

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 degrees 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.

We have devised an easy and effective genetic transformation method for the preeminent edible mushroom, Agaricus bisporus. Our method exploits the T-DNA transfer mechanism in Agrobacterium tumefaciens and relies on the reproductive fruiting body as the recipient tissue. The use of fruiting body explants, particularly the gill, provided high-frequency transformation, overcoming the inefficacy of Agrobacterium-based methods targeting fungal spores or vegetative mycelium. The protocol entails incubation of A. tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin. Between 7 and 28 d on the selection medium, upwards of 95% of the gill explants generate hygromycin-resistant colonies. About 75% of the mushroom transformants show a single-copy of the hygromycin-resistant gene integrated at random sites in the genome.

Since the early 1990s, the epidemic of green mold on the cultivated mushroom Agaricus bisporus in North America has been caused by Trichoderma aggressivum f. aggressivum. The findings of earlier research suggested that the microevolutionary emergence of T. aggressivum f. aggressivum coincided with the onset of the epidemic. This hypothesis was tested further by determining the disease susceptibility of mushroom strains grown widely before the epidemic manifested. The results of complementary methods of analysis, which entailed a grain protection assay and cropping trials, established that two pre-epidemic strains were more susceptible to green mold than three post-epidemic strains being cultivated at the time of the epidemic. Thus, if T. aggressivum f. aggressivum had been present within cultivated mushrooms prior to the epidemic, it should have been detected. It still appears to be true that T. aggressivum f. aggressivum emerged during the 1990s in a manner that remains unclear.

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.

ABSTRACT Molecular phylogenetic analyses were performed on 40 isolates of Verticillium fungicola collected from various Pennsylvania mushroom farms in 1999 and 28 isolates of Verticillium spp. collected during the last 50 years from various geographic locations. Sequence analysis of internal transcribed spacers 1 and 2 (ITS1 and ITS2) and 5.8S regions of the nuclear ribosomal DNA (rDNA) transcriptional unit and analysis of random amplified polymorphic DNA (RAPD) data were performed for the 68 isolates of Verticillium spp. Identical rDNA sequences were obtained for all 40 Pennsylvania isolates collected during 1999, 13 North American isolates collected during the last 50 years, and the ex-type strain of V. fungicola var. aleophilum. Sequence analysis of European isolates revealed a close relationship to the ex-type strain V. fungicola var. fungicola. No European-like isolates of V. fungicola var. fungicola were detected in the collection of North American isolates examined. Results from six decamer RAPD primers strongly indicate the presence of a clonal population of V. fungicola among Pennsylvania isolates. In addition, RAPD data delineated a Korean isolate (DC130) and ex-type strain V. fungicola var. aleophilum from the North American group. Virulence assays, based on spore inoculation of mushroom pilei, revealed variation corresponding to each neighbor-joining and RAPD grouping. All isolates with rDNA sequence and RAPD grouping similarity to ex-type strains V. fungicola var. aleophilum and V. fungicola var. fungicola displayed the highest level of virulence. Based on rDNA sequence and RAPD analyses, isolates displaying reduced or no virulence were distantly related to these two varieties. All results obtained for the analyses of ex-type strain V. fungicola var. flavidum suggested that this fungal isolate should not be considered a variety of V. fungicola, but rather a distinct species.

We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species.

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.

We used randomly amplified polymorphic DNA (RAPD)-PCR to estimate genetic variation among isolates of Trichoderma associated with green mold on the cultivated mushroom Agaricus bisporus. Of 83 isolates examined, 66 were sampled during the recent green mold epidemic, while the remaining 17 isolates were collected just prior to the epidemic and date back to the 1950s. Trichoderma harzianum biotype 4 was identified by RAPD analysis as the cause of almost 90% of the epidemic-related episodes of green mold occurring in the major commercial mushroom-growing region in North America. Biotype 4 was more closely allied to T. harzianum biotype 2, the predominant pathogenic genotype in Europe, than to the less pathogenic biotype 1 and Trichoderma atroviride (formerly T. harzianum biotype 3). No variation in the RAPD patterns was observed among the isolates within biotype 2 or 4, suggesting that the two pathogenic biotypes were populations containing single clones. Considerable genetic variation, however, was noted among isolates of biotype 1 and T. atroviride from Europe. Biotype 4 was not represented by the preepidemic isolates of Trichoderma as determined by RAPD markers and PCR amplification of an arbitrary DNA sequence unique to the genomes of biotypes 2 and 4. Our findings suggest that the onset of the green mold epidemic in North America resulted from the recent introduction of a highly virulent genotype of the pathogen into cultivated mushrooms.

ABSTRACT A polymerase chain reaction-amplified DNA containing the internal transcribed spacer (ITS)-1, 5.8S, and ITS-2 regions of the nuclear ribosomal DNA transcriptional unit was sequenced for 81 isolates of Trichoderma spp. associated with mushroom culture or used for biological control of plant pathogens. Phylogenetic analyses revealed that the biocontrol isolates were more closely related to an isolate of T. harzianum biotype 1 (Th1) than to the aggressive biotypes 2 and 4. Th1 has been isolated from mushroom compost but is not the cause of widespread green mold epidemics that have occurred during the last 12 years in Europe and North America. Three isolates of T. harzianum obtained from shiitake (Lentinula edodes; Shi1B and S3-96) and maitake (Grifola frondosa; Mai1) substrates were placed within the biocontrol group. We also found evidence suggesting that some isolates of T. harzianum originally identified as Th4 from Pennsylvania are more closely related to Th2 from Europe. Finally, considering the wide range in sequence distribution of our samples, we propose that the consensus sequence found in this investigation be used as the reference sequence for further studies involving the identification and taxonomy of T. harzianum.

Statistical analyses were performed on spatial distributions of mushroom green mold foci caused by Trichoderma spp. in 30 standard Pennsylvania doubles (743 m 2 production surface) selected at random from over 900 total crops mapped. Mapped production houses were divided into four tiers of six beds each with 16 sections per bed (total = 384 sections per double). Each section contained approximately 2 m 2 . Green mold foci were mapped according to presence or absence in each section as they became visible during the course of the mushroom production. There was a trend toward higher disease incidence at the ends of the doubles, although this was not consistent from level to level. Spatial analysis revealed that green mold foci were more likely to occur in neighboring sections along the beds rather than above, below, or across from each other. Cultural practices that were associated with movement along the beds, i.e., nutrient supplementation, spawning, bed tamping, surface covering, etc., were considered the most likely factors influencing the incidence of green mold in spawned compost. Airborne contamination was considered a less likely source of inocula contributing to epidemic development. Sanitation practices that reduce spore loads along the beds are expected to provide the greatest degree of green mold control.

Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or beta-mercaptoethanol), and Mg2+ and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, alpha-amanitin, and rifampin). The radiolabeled enzyme reaction products were predominantly (95%) single-stranded RNA (ssRNA) as determined by cellulose column chromatography and ionic-strength-dependent sensitivity to hydrolysis by RNase A. Three major size classes of ssRNA transcripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electrophoresis, although the transcripts hybridized to all nine of the virion-associated dsRNAs. The RNA products synthesized in vitro appeared to be of a single polarity, as they hybridized to an ssDNA corresponding to one strand of a genomic dsRNA and not to the complementary strand. Similarly, reverse transcription-PCR with total cellular ssRNA as a template and strand-specific primers targeting a genomic dsRNA during synthesis of cDNA suggested that only the coding strand was transcribed in vivo. Our data indicate that the RDRP activity associated with virions of LIV is probably a transcriptase engaged in the synthesis of ssRNA transcripts corresponding to each of the virion-associated dsRNAs.

Reverse transcription PCR analysis was used to investigate the involvement of two RNA-genome viruses, La France isometric virus (LIV) and mushroom bacilliform virus (MBV), in the etiology of La France disease of the cultivated mushroom Agaricus bisporus. Reverse transcription PCR amplification of sequences targeted to the genomes of LIV and MBV, with a sensitivity of detection of < 10 fg of viral RNA, showed diseased mushrooms to be either singly infected by LIV or doubly infected by LIV and MBV. Of 70 geographically diverse diseased mushroom isolates, 100% were infected by LIV, whereas almost 60% of these isolates were coinfected by MBV. Of 58 mushroom isolates determined to be free of infection by LIV, 3 were found to be infected by MBV. This represents the first documented report of the independent replication of these two viruses. Our data support the hypothesis that La France disease is associated with infection by two autonomously replicating viruses in which LIV is the primary causal agent and MBV, although possibly pathogenic and capable of modulating symptoms, is not required for pathogenesis.

Double-stranded ribonucleic acids (dsRNAs) were isolated from fruit bodies of commercial strains of the cultivated mushroom (Agaricus bisporus) by polyethylene glycol-NaCl precipitation, differential centrifugation, rate-zonal centrifugation in sucrose, and equilibrium centrifugation in cesium sulphate. In all seven of the mushroom isolates examined, three dsRNAs were identified: two major dsRNA segments of > 13.1-kb (L-RNA) and 2.4-kb (S-RNA) and a minor segment of 5.2-kb (M-RNA). L-, M-, and S-RNAs co-purified with spherical fungal vesicles measuring approximately 75 nm in diameter. The three dsRNAs were intimately associated with the vesicles as suggested by their lower buoyant density in cesium sulphate (1.27 g/cc) compared to that of phenol-extracted dsRNAs (1.42 g/cc) and by their resistance to hydrolysis by ribonuclease at low ionic strength. Using a variety of conditions during purification, no virus-like particles were found to be associated with the dsRNAs. In Northern analysis, L-, M-, and S-RNAs failed to cross-hybridize with the genomic dsRNAs of La France isometric virus. We report here the first description of non-encapsidated, vesicle-associated, dsRNA genetic elements in the common cultivated mushroom.

The production of the button mushroom Agaricus bisporus with mycelium-colonized alginate pellets as an inoculant of the growing medium was investigated. Pellets having an irregular surface and porous internal structure were prepared by complexing a mixture of 1% sodium alginate, 2 to 6% vermiculite, 2% hygramer, and various concentrations of Nutrisoy (soy protein) with calcium chloride. The porous structure allowed the pellets to be formed septically and then inoculated and colonized with the fungus following sterilization. By using an enzyme-linked immunosorbent assay (ELISA) to estimate fungal biomass, the matrix components of the pellet were found to be of no nutritive value to A. bisporus. Pellets amended with Nutrisoy at a concentration of 0.5 to 8% supported extensive mycelial growth, as determined by significantly increased ELISA values, with a concentration of 4% being optimal and higher concentrations proving inhibitory. The addition of hydrated, mycelium-invaded pellets to the compost or casing layer supported the thorough colonization of the growing substrate and culminated in the formation of mushrooms that showed normal development and typical morphology. Yields and sizes of mushrooms were comparable from composts seeded with either colonized pellets or cereal grain spawn. Similarly, amending the casing layer with pelletized-mycelium-colonized compost resulted in a 2- to 3-day-earlier and more-synchronous emergence of mushrooms than with untreated casing. This technology shows the greatest potential as a pathogen-free inoculant of the casing layer in the commercial cultivation of mushrooms.

A bacilliform virus from the cultivated mushroom, Agaricus bisporus, has been extensively purified and shown to be a unique mycovirus in that the genome is single-stranded (ss) RNA. The purification procedure entailed extraction at pH 6, followed by polyethylene glycol-NaCl precipitation, and differential, rate-zonal, and equilibrium centrifugation. Mushroom bacilliform virus (MBV) has a buoyant density of 1.317 g/cm(3) in Cs2SO4, A260/A280 nm absorbance ratio of 1.67, and contains approximately 20% nucleic acid by weight. SDS-polyacrylamide gel electrophoretic analyses revealed that MBV contains a single major nucleic acid species with a molecular weight of 1.4 x 108 and a single capsid polypeptide of molecular weight 24,400. The nucleic acid was hydrolyzed by pancreatic ribonuclease A in high ionic strength buffer and by 0.4 M KOH, but not by pancreatic deoxyribonuclease I. Further, the kinetics of melting of MBV RNA upon thermal denaturation closely paralleled that of the ssRNA of tobacco mosaic virus. Based on this evidence, we propose that MBV is the first mycovirus shown to possess a ssRNA genome.

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg(2+) and the four nucleoside triphosphates and was insensitive to actinomycin D, alpha-amanitin, and rifampin. The (3)H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 x 10(6) and 1.4 x 10(6); they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs(2)SO(4) equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 x 10(6) and 1.4 x 10(6). The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.