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.

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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.

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.

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