Production of Agaricus bisporus on substrates pre-colonized by Scytalidium thermophilum and supplemented at casing with protein-rich supplements.

The objective of this study was to evaluate performance of Agaricus bisporus (Ab) on substrates pre-colonized by Scytalidiumthermophilum (St), a thermophilic fungus known to enhance yields of Ab and increase selectivity of the substrate. The radial extension rate (RER) of the mycelium of three strains of St and their influence on the growth of a brown strain of Ab were evaluated. We also determined the time required for colonization of pangola grass by St in a compost pile and the influence of three protein-rich supplements on yield of Ab on pangola grass (Digitaria decumbens) colonized by St. RER of St ranged from 10.1mm/d on grass to 18.9 mm/d on potato dextrose yeast extract agar, with significant differences among substrates and among strains. Ab grew faster on substrate colonized for 1, 2, or 3 days by St (RER of 3.31, 3.29, 3.23 mm/d, respectively) compared to non-colonized substrate (1.85 mm/d). Ab was cultivated on substrate samples selected daily from the St-inoculated pile, with biological efficiencies (BE) ranging from 4% (day 0) to 73.9% (day 2). Protein-rich supplements (soybean, black beans and cowpeas) added at casing significantly stimulated mushroom yield on St-colonized substrate compared to the non-supplemented control. BE varied from 26.1% on substrate non-supplemented to 73.1% on compost supplemented with ground soybean. There were no significant differences in mushroom yield observed among supplements evaluated.

Yield, size and bacterial blotch resistance of Pleurotus eryngii grown on cottonseed hulls/oak sawdust supplemented with manganese, copper and whole ground soybean.

Experiments were performed to determine effects of supplementation of cottonseed hull/sawdust substrate with Mn, Cu, and ground soybean on yield, mushroom size, and bacterial blotch resistance of two commercial strains of Pleurotus eryngii. A basal formulation (d.w.) of cottonseed hulls (62%), aged red oak sawdust (27%), whole ground soybean (6%), corn distiller's waste (4%) and calcium sulfate (1%) was supplemented to 50, 150 or 250 microg/g Mn or Cu and to 4%, 8% and 12% whole ground soybean. The cottonseed hulls content in the basal substrate was adjusted to compensate for the addition of ground soybean. Formulated substrates were mixed, placed in 1050ml bottles, and sterilized at 121 degrees C for 90min. Mushroom yields were significantly higher from substrates containing Mn at 50 microg/g and soybean at 8% and 12% supplementation compared to the basal substrate. As the level of soybean addition to substrate increased, yield also increased. The addition of Mn at levels of 150 and 250 microg/g significantly enhanced yield as well, although less than did the 50 microg/g treatment. To assess the influence of mushroom strain and substrate composition on blotch disease severity, pilei of P. eryngii were inoculated with Pseudomonas tolaasii. Strain WC888 was more resistant to disease than WC846. Disease severity was greater when substrates were amended with Cu to 150 or 250 microg/g. There was a significant difference in inherent levels of Cu in the basidiomata of different strains, but P. eryngii did not accumulate Cu and disease severity was not correlated with Cu content of the basidiomata.

Yield, mushroom size and time to production of Pleurotus cornucopiae (oyster mushroom) grown on switch grass substrate spawned and supplemented at various rates.

To find a cost effective alternative substrate, Pleurotus cornucopiae 608 (yellow basidiomata) was grown on: (1) chopped, pasteurized switch grass (Panicum virgatum, 99%) with 1% ground limestone and (2) a mixture of pasteurized cottonseed hulls (75% dry wt.), 24% chopped wheat straw, and 1% ground limestone (all ingredients wt./wt.). The substrates were spawned at various levels (2.5%, 3.75% or 5% wet wt., crop I) and non-supplemented or supplemented with commercial delayed release nutrient (Campbell's S-41) at various levels (0%, 1.5%, 3%, 4.5%, 6%, 7.5% and 9% dry wt., crop II). Maximum yield (weight of fresh mushrooms harvested at maturity) was obtained on cottonseed hull/wheat straw substrate at a 3.75-5% spawn level and 6% S-41 supplement. On switch grass substrate, increasing spawn levels and supplement levels stimulated yields in a linear fashion. However, maximum yields were only 46% or less for those of similar treatments on cottonseed hull/wheat straw substrate. Yields were three times higher on switch grass that was harvested after the grass had senesced (winter; beige color) compared to material that was harvested when the grass was green (summer; time of flowering). Additional physical processing of the material, such as milling, may improve yield potential of this material.

Resistance of Pre- and Post-epidemic Strains of Agaricus bisporus to Trichoderma aggressivum f. aggressivum.

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.

Influence of spawn rate and commercial delayed release nutrient levels on Pleurotus cornucopiae (oyster mushroom) yield, size, and time to production.

Pleurotus cornucopiae 608 was grown on a mixture of pasteurized cottonseed hulls (75% dry wt). 24% chopped wheat straw, and 1% ground limestone. The substrate was spawned at various levels (1.25%, 2.5%, 3.75%, or 5% wet wt) and not supplemented or supplemented with commercial delayed release nutrient (Campbell's S-41) at various levels (0%, 3%, 6%, 9%, or 12%). Maximum yield (weight of fresh mushrooms harvested at maturity) was obtained at 3.75-5% spawn level and 6% S-41 supplement. As supplement levels exceeded 6%, yields declined significantly. There was a negative correlation (r=-0.81) between spawn rate and days to production. As the spawn rate increased, the number of days to production decreased. By using a spawn rate of 3.75% of the wet substrate wt, it was possible to reduce the time to production by a mean of 9.2 days compared with a spawn rate of 1.25%.

Effects of genotypes of maitake (Grifola frondosa) on biological efficiency, quality and crop cycle time.

Twenty-three genotypes of maitake (Grifola frondosa) from Asia, North America and Europe were compared for biological efficiency (BE), quality and crop cycle time. Significant differences among lines were found for BE and crop cycle time when mushrooms were produced on nutrient-supplemented (15% millet, 10% wheat bran and 0.2% gypsum) oak sawdust substrate. Four isolates (WC828, M036, M037, and M040) were found to have the most consistent and highest BEs (38.5%, 39.5%, 35.8%, and 38.9%, respectively) and quality ratings (1.2, 1.3, 1.4, and 1.2, respectively, where I is highest quality and 4 is lowest quality). A commercial line (M039) used in China had the shortest crop cycle time (8 weeks). Lines deemed to have the best commercial potential were of Asian origin.

Vacuum-soaking of wood chip shiitake (Lentinula edodes) logs to reduce soak time and log weight variability and to stimulate mushroom yield.

Synthetic logs were vacuum-soaked or regular-soaked to determine the effects of soaking on yield and mushroom size, log weight variability and water distribution within the log. Yields (g/log) from substrates vacuum-soaked were higher by 26.7%, 18.6% and 35.8% (mean = 27.2%) for crops I, II and III, respectively, when compared with regular-soaked. However, mushroom size averaged only 11.2 g for vacuum-soaked logs vs 17 g for regular-soaked logs (51.8% larger for regular-soaked). The time required for vacuum-soaking logs was generally less than 3 min, compared with regular-soaking times ranging over 3-15 h. Water tended to accumulate more in the outside zone in the vacuum-soaked logs, compared with regular-soaked logs. Mean moisture contents for crops I and II for outside, middle and interior zones of vacuum-soaked logs were 66%, 47.5% and 42.2%, respectively, while regular-soaked logs for the same zones were 62.4%, 52.1% and 50.9%, respectively. Vacuum-soaked log weights had lower standard deviations than weights for regular-soaked logs in four out of six soaks, indicating a more uniform soaking process.

Effects of nutrient supplements on biological efficiency, quality and crop cycle time of maitake (Grifola frondosa).

The effects of various combinations of wheat bran, rye and millet (at 20% and 30% of total dry substrate wt) on crop cycle time, biological efficiency (BE) and mushroom quality were evaluated for a commercially used isolate of Grifola frondosa (maitake). Supplements were combined with a basal ingredient of mixed oak (primarily red oak) sawdust, and the resulting mixture was pasteurized, cooled, inoculated and bagged with an autoclaving mixer. Times to mushroom primordial formation and mushroom harvest were recorded, and mushroom quality was rated on a scale of 1-4, where 1 was the highest quality and 4 was the lowest quality. The combinations of 10% wheat bran, 10% millet and 10% rye (BE 47.1%, quality 1.8 and crop cycle 12 weeks) and 10% wheat bran plus 20% rye (BE 44%, quality 1.7 and crop cycle 10 weeks) gave the most consistent yields and best basidiome quality over time.

Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust.

Transcription of laccase and cellulase genes of Lentinula edodes was examined during growth and development under different temperature and moisture levels on a sawdust-based substrate. RNA was extracted from samples of mycelium and fruit bodies at various stages of development and gene expression was determined by competitive RT-PCR. The level of laccase transcripts was maximal during the mycelial growth stage, and then declined rapidly at the fruiting stage. In contrast, the cellulase transcript level peaked at the veil-break stage during fruit body development. Gene expressions for laccase and cellulase were triggered by microclimatic changes, specifically lower temperature and osmotic pressure.

Influence of substrate wood-chip particle size on shiitake (Lentinula edodes) yield.

Wood chips from four commercial hardwood sawmills were screened with 10 US standard sieves (4-0.21 mm) to assess particle size distributions. 96-98% of wood chips were < 4 mm while 95-99% of particles were > 0.21 mm. The majority (mean = 64.5%) of wood chips passed through US standard sieve size 14 (< 1.4 mm). Shiitake (Lentinula edodes) was grown in three crops to determine the effect of four particle size classes (1 = 2.8-4 mm; 2 = 1.7-2.8 mm; 3 = 0.85-1.7 mm; 4 = < 0.85 mm) on mushroom yield. Yields from substrates prepared with wood chips from class 4 (< 0.85 mm) were lower by 27.7%, 12.4% and 2% (mean = 14.9%) for Crops I, II, and III, respectively, when compared to controls. Profiling of wood chips may help growers optimize their production media and reduce production costs.

Adapting substrate formulas used for shiitake for production of brown Agaricus bisporus.

A pasteurized, non-composted substrate (basal mixture) consisting of oak sawdust (28%), millet (29%), rye (8%), peat (8%), alfalfa meal (4%), soybean flour (4%), wheat bran (9%), and CaCO3 (10%) was adapted from shiitake culture to produce the common cultivated mushroom (brown; portabello), Agaricus bisporus. Percentage biological efficiency (ratio of fresh mushroom harvested/oven-dry substrate weight, %BE) ranged from a low of 30.1% (when wheat straw was substituted for sawdust) to 77.1% for the basal mixture. Special, high gas-exchange bags were required to optimize mycelial growth during spawn run. Our formula may allow specialty mushroom growers to produce portabello mushrooms on a modified, pasteurized (110 degrees C for 20 min) substrate commonly used for shiitake production without the added expense of compost preparation.

Molecular Phylogenetic Analyses of Verticillium fungicola and Related Species Causing Dry Bubble Disease of the Cultivated Button Mushroom, Agaricus bisporus.

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.

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

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

Homelessness and gender in out-of-treatment drug users.

This study examines 5225 out-of-treatment crack users and drug injectors drawn from five different geographic areas to examine selected factors associated with homelessness. Of these crack users, 27% considered themselves undomiciled, and 60% had previously entered some type of drug treatment. Logistic regression found that substance abusers who were married, female, and persons of color were less likely to be without a home when other variables were controlled. Trading sex for money and perceived chance of getting acquired immunodeficiency syndrome (AIDS) were associated positively with homelessness, while participating in methadone detoxification and methadone maintenance programs seemed to offer some protection from homelessness.

PCR-based genotyping of epidemic and preepidemic Trichoderma isolates associated with green mold of Agaricus bisporus.

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.

Attitudes toward assisted suicide: a survey of hospice volunteers.

Technological advances have lengthened our years and, often, the dying process as well. While studies have been conducted of physicians and dying patients concerning their views on assisted suicide, no prior studies have examined the attitudes of hospice volunteers. This survey of 277 hospice volunteers found that overall their attitudes were more supportive of assisted suicide than that of a convenience sample of the public. Thirty-seven percent of the volunteers endorsed the view that there are situations when assisting death may be morally acceptable; 4% had been asked to provide assistance to help a patient end his or her life.

Evidence for two independent lineages of shiitake of the americas (Lentinula boryana) based on rDNA and beta-tubulin gene sequences.

Portions of ribosomal RNA genes (rDNA) were sequenced from members of the genus Lentinula and were used, along with partial beta-tubulin gene sequences, for phylogenetic reconstructions. The rDNA sequences of L. boryana were separated into two well-defined lineages. Lineage 1 was composed of isolates from Mexico and Costa Rica while lineage 2 encompassed isolates from the United States, Venezuela, and Brazil. The two South American isolates of L. boryana had nearly identical ITS sequences and very closely related beta-tubulin sequences. This high level of similarity may indicate that sexual reproduction occurs among the sampled populations, although this is difficult to reconcile with the large geographic distances (over 4000 km) that separate some of the collecting locations. An alternative explanation may be that the isolates sampled are the product of a rapid population expansion over a large geographic area. Analyses of partial beta-tubulin gene sequences that were rooted using Pleurotus spp. support the hypothesis that L. boryana is monophyletic.

Molecular Phylogenetic Analyses of Biological Control Strains of Trichoderma harzianum and Other Biotypes of Trichoderma spp. Associated with Mushroom Green Mold.

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.

Spatial Distribution of Green Mold Foci in 30 Commercial Mushroom Crops.

Statistical analyses were performed on spatial distributions of mushroom green mold foci caused by Trichoderma spp. in 30 standard Pennsylvania doubles (743 m2 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 m2. 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.

Mentoring high-risk minority youth: evaluation of the Brothers project.

Mentoring as an intervention for at-risk teens is becoming increasingly popular despite sparse evidence of its effectiveness. This research, part of a larger evaluation effort, reports on a four-year mentoring project developed specifically for African-American adolescents. Self-esteem, attitudes toward drugs and alcohol, grades, school attendance, and disciplinary infractions were examined using an experimental design. No significant differences were found between the control and intervention groups. However, multiple explanations are offered to account for why it is so difficult to document the positive benefits of mentoring.