Characterization of pseudomonads isolated from decaying sporocarps of oyster mushroom.

Pleurotus ostreatus is one of the most extensively cultivated mushrooms in the world; however, the success of cultivation often depends on the proliferation of different bacterial pathogens. Pseudomonas tolaasii is thought as the major cause of brown blotch disease of Agaricus bisporus and yellowing of Pleurotus ostreatus. In this study we examined the pathogenicity and assessed the industrial damage causing effect of 41 Pseudomonas strains isolated from deformed, yellowing oyster mushroom (P. ostreatus) sporocarps. Identification of the isolates at species level by the partial sequence analysis of the hypervariable region of the rpoB gene revealed nine Pseudomonas sps. We analyzed the presence of the tolaasin gene-cluster, the production of fluorescent pigments, the oxidase- and nitrite reductase activities, the growth at restrictive temperatures and the carbon source utilizing abilities of each strain. Complex lipopeptide production (including tolaasin) was examined with thin layer chromatography and a novel in vitro necrosis-test was developed and evaluated for the investigation of the pathogenic effect of Pseudomonas strains. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process. Strong correlations were found between the secretion of trypsin-like proteases and lipases and the necrotic effect of these bacteria. All the results clearly established that besides Ps. tolaasii, Ps. fluorescens biovar V strains were pathogenic to P. ostreatus and cause serious losses during mushroom production. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process, especially the trypsin-like proteases and lipases.

Microbial community structure changes during oyster mushroom substrate preparation.

Although oyster mushroom (Pleurotus spp.) is a valuable food, cultivated worldwide on an industrial scale, still very little is known about the microbial dynamics during oyster mushroom substrate preparation. Therefore, the characterization of the microbial dynamics by chemical and biological tools was the objective of this study. During substrate preparation, enzymatic digestibility of the substrate improved by 77%, whereas the cellulose and hemicellulose to lignin ratios decreased by 9% and 19%, respectively. Fluorescein diacetate hydrolysis reached its minimum value at the temperature maximum of the process during the composting phase and exceeded the initial level at the end of the process. Fungal species played part in the initial mesophilic phase of the substrate preparation process, but they disappeared after pasteurization in tunnels at constant elevated temperatures. Changes in the microbiota showed a marked bacterial community succession during substrate preparation investigated by 16S ribosomal deoxyribonucleic acid-based terminal restriction fragment length polymorphism (T-RFLP). Mature samples represented the least variance, which indicated the effect of the standardized preparation protocol. The relation between mushroom yield and the bacterial community T-RFLP fingerprints was investigated, but the uniformity of mushroom yields did not support any significant correlation.

Molecular identification of Trichoderma species associated with Pleurotus ostreatus and natural substrates of the oyster mushroom.

Green mold of Pleurotus ostreatus, caused by Trichoderma species, has recently resulted in crop losses worldwide. Therefore, there is an emerging need for rapid means of diagnosing the causal agents. A PCR assay was developed for rapid detection of Trichoderma pleurotum and Trichoderma pleuroticola, the two pathogens causing green mold of P. ostreatus. Three oligonucleotide primers were designed for identifying these species in a multiplex PCR assay based on DNA sequences within the fourth and fifth introns in the translation elongation factor 1alpha gene. The primers detected the presence of T. pleurotum and/or T. pleuroticola directly in the growing substrates of oyster mushrooms, without the need for isolating the pathogens. The assay was used to assess the presence of the two species in natural environments in which P. ostreatus can be found in Hungary, and demonstrated that T. pleuroticola was present in the growing substrates and on the surface of the basidiomes of wild oyster mushrooms. Other Trichoderma species detected in these substrates and habitats were Trichoderma harzianum, Trichoderma longibrachiatum and Trichoderma atroviride. Trichoderma pleurotum was not found in any of the samples from the forested areas tested in this study.