Daniel J. Royse

A systematic assessment of Morchella using RFLP analysis of the 28S ribosomal RNA gene

Morels (Morchella spp.) are one of the most highly prized edible mushrooms found in the wild of North America. Because environmental conditions can cause members of the genus to vary morphologically, the number of species has been disputed. Some au? thors classify the genus into three to five species, while others argue for as many as 50 species. DNA from lines of Morchella and a closely related genus (Verpa) was isolated. The large subunit of the ribosomal DNA repeat was amplified using the polymerase chain re? action and then digested with restriction enzymes. Re? striction fragment length polymorphisms were found among the lines investigated and used to separate the lines into genotypic classes. More genetic variation may occur between geographically isolated popula? tions of the same species than between two putatively distinct species. The phylogenetic tree developed from restriction data suggests that the black morels (M. an- gusticeps, M. elata, and M. conica) and the yellow morels (M. esculenta, M. crassipes, and M. deliciosa) are separate taxonomic groups.

Use of Isozyme Variation to Identify Genotypic Classes of Agaricus Brunnescens

Thirty-four commercial and 162 research-maintained lines of Agaricus brunnescens deposited in The Pennsylvania State University Mushroom Culture Collection were examined electrophoretically for isozyme variation. Based on the variability at five structural gene loci (Gpt, Adh, Mpi, Pep-LLL-1, and Pep-LLL-2) they were partitioned into 27 genotypic classes. The allelic variability observed in mycelial extracts of these lines potentially allows the recognition of over 20,000 unique genotypic classes. The fact that only five genotypic classes were actually observed among commercial lines examined is evidence that the common mushroom is a near monoculture. Data from single spore-derived cultures of A. brunnescens revealed that most of these cultures retained heterozygosity at those loci heterozygous in the parental line. A limited number of recombinants were noted, i.e., heterozygous for some loci and homozygous for others. A small fraction (<5%) were homozygous at multiple loci suggesting that they may be homokaryotic. These observations support the contention that low levels of meiotic recombination occur and the chromosomal segregation is nonrandom in A. brunnescens.

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 (Campbells 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.

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.

Effect of spawn run time and substrate nutrition on yield and size of the shiitake mushroom

Sawdust substrate supplemented with millet or wheat bran or both, were inoculated with spawn made from an isolate (PSU 305) of Lentinus edodes. Biological efficiency (%) and size data, on mushrooms harvested from sawdust substrate with spawn run (vegetative growth) periods of 58 and 116 days, were analyzed. Biological efficiencies were two to three times greater for the longer incubation period. Production rate (r), defined as the daily average biological efficiency, was highest (r = 0.79%/da) for a 116 day spawn run with both millet and bran as nutritional supplements. Larger mushrooms generally were produced with longer spawn runs. Practical application of these methods should increase the efficiency of shiitake cultivation on supplemented sawdust.

Phylogenetic relationships of Trichoderma harzianum causing mushroom green mold in Europe and North America to other species of Trichoderma from world-wide sources

Nucleotide sequences of internal transcribed spacers (ITS-1 and ITS-2) and the 5.8 S gene of the ribosomal DNA repeat were examined in 15 lines of Trichoderma spp. Six lines representing four bioty...

Botanical fractions of rice straw colonized by white-rot fungi: changes in chemical composition and structure

Three species of white-rot fungi (Cyathus stercoreus (Cs) ATCC-36910, Phanerochaete chrysosporium (Pc) BKM, and Pleurotus sajor-caju (Ps) 537) were grown on leaf blade (leaf) or stem plus leaf sheath (stem) of rice straw for 30 d by solid state fermentation (SSF). Physical and chemical methods were employed to evaluate substrate specificity, substrate composition and histology. Changes in histology of decayed material were evaluated before and after ruminal digestion by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Control leaf and stem were similar in IVDMD (38%), although leaf was higher in crude protein and lower in cell wall compared to stem (3.7 vs. 2.8%; 73.9 vs. 80.7%, respectively). The changes were due mostly to a higher concentration of silica in leaf compared to stem (17.0 vs. 13.1%). After 30 d of SSF, Cs and Ps increased the IVDMD of leaf from 38.1 to 49 and 46.3%, respectively, by selective degradation of hemicellulose as opposed to cellulose. In contrast, Pc degraded cellulose and hemicellulose indiscriminately in leaf and lowered the IVDMD of leaf to 30.1%. Partially degraded lignin, silica and hemicellulose of leaf were negatively correlated (r) with IVDMD in contrast to cellulose (r = −0.49, −0.54, −0.16 and 0.85, respectively). Prediction of IVDMD of fungal-decayed leaf was primarily a function of hemicellulose and cellulose with a coefficient of IVDMD = −0.155 + 2.14 (cellulose) −0.87 (hemicellulose); R2 = 0.98. Stem decayed by Pc and Cs became less digestible compared to the control (18.5 and 20.3% vs. 39.7%, respectively), although hemicellulose and cellulose of stem were poorly degraded after SSF. Only Ps improved the IVDMD of stem compared to the control (44.1 vs. 39.7%). SEM sections of leaf decayed by Pc showed complete degradation of mesophyll but the more recalcitrant vascular and epidermal tissues resisted rumen degradation and resulted in lower IVDMD. Leaf tissues colonized by Cs and Ps showed presence of all tissues but after 72 h rumen microorganisms completely degraded mesophyll tissue which resulted in a higher IVDMD. Observation of TEM sections showed that fungal treatment facilitated rumen microbial penetration of lignified tissues. Improvement of digestibility of decayed straw depends upon the fungal species, the plant substrates and the botanical fractions.

Molecular evolution of Agaricus species based on ITS and LSU rDNA sequences

Phylogenetic analyses of 62 isolates of 42 Agaricus and related secotioid species (A. inapertus, Gyrophragmium dunalii and Longula texensis) were conducted based on sequence data of the internal transcribed spacers (ITS) and partial large subunit (LSU) of ribosomal DNA. Bayesian, maximum likelihood and maximum parsimony analyses were used to reveal evolutionary groups within the genus Agaricus, while molecular clock analyses were carried out to obtain more information about the divergence times during the evolution of Agaricus within the Basidiomycota. Six major distinct clades were found within the genus with varying levels of support. These monophyletic groups suggested interspecific relationships both confirming and challenging previous morphological sections in several cases. Our results show that most morphological features likely have evolved in apparently similar ways multiple times independently during evolution, and that the secotioid A. inapertus, Gyrophragmium dunalii and Longula texensis evolved from Agaricus species in Clade I. A new name Agaricus aridicola, and a new combination Agaricus texensis are suggested to replace the names Gyrophragmium dunalii and Longula texensis, respectively. Molecular clock estimates for minimal age of separation of the genus Agaricus from its closest relatives were 32.63 ± 8.06 and 15.45 ± 3.82 Ma, respectively, using calibrations based on other molecular clock studies on fungi and fossil data. However, Agaricus likely diverged much earlier (73.30 ± 18.12 Ma), as suggested by the estimate based on the most robust calibration.

Genetic relatedness and its application in selective breeding of Agaricus brunnescens

Thirty-four commercial and 162 research-maintained lines of Agaricus brunnescens were assigned to 27 genotypic classes based on allelic variability at five biochemical loci. Coefficients of genetic similarity between genotypic classes were determined. Three comparisons of genetic relatedness were made to determine the best lines from which to generate homokaryons (breeding stock) for crossing. Comparisons of relatedness included mean, highest, and lowest coefficients of genetic similarity between genotypic classes. A dendrogram was constructed from similarity coefficients to provide further insights into the relationships of these classes. A comprehensive and practical approach to the development of improved cultivars of the commercial mushroom by increasing variation within and among lines is discussed.

Confirmation of crosses between lines of Agaricus brunnescens by isozyme analysis

Abstract In this paper are presented the results of isozyme analysis which confirmed intraspecific crosses between putative homokaryotic lines of Agaricus brunnescens . Putative homokaryotic lines were recognized by electrophoresis of single-spore-derived cultures and used as breeding stock. These breeding stock lines were grown together in dual culture on agar and selections were made from the interaction zone. These selections were then transferred to make spawn. Alternatively, spawns from each of two selected lines were mixed together and inoculated directly into compost. Intraspecific crosses were confirmed by the presence of heteromeric isozymes. Many of these newly developed lines were characterized by unique isozymic phenotypes reflecting unique genotypic classes.