| Abstract
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The Pleurotus eryngii species complex comprises at least five varieties: eryngii, ferulae, elaeoselini, nebrodensis and tingitanus . This species is unique among the genus Pleurotus because in nature it is found in association with specific members of the Umbelliferae and Compositae families. Geographic distribution of Pleurotus eryngii is limited to subtropical regions of the Mediterranean, Central and Southern Europe, Ukraine, North Africa, East and Central Asia and Iran. Pleurotus eryngii var. eryngii and nebrodensis were domesticated in 1970 and 1987, respectively, and are now cultivated in some countries of Asia and Europe. In the United States, the var. eryngii was recently introduced with cultivation beginning in 2000. The varietal status of members of P. eryngii has been widely questioned and the evolutionary relationships among them are unknown. In this research, four regions of the genome were analyzed to establish phylogenetic relationships among isolates of var. eryngii, ferulae, elaeoselini and nebrodensis . No nucleotide variation in the Internal Transcribed Spacer (ITS) region was found among the varieties eryngii, ferulae and elaeoselini although intra-isolate polymorphisms were observed in some isolates. On the other hand, allelic polymorphisms in the partial β-tubulin gene were problematical in phylogenetic studies but allowed delimitation of genetic pools. Informative nucleotide variation in partial sequences of the genes coding for translation elongation factor ( tef1 ) and RNA polymerase II (RPB2 ) were useful for phylogenetic analyses among the varieties. Combined data sets of tef1 and RPB2 indicated that P. eryngii is a monophyletic group. Varieties eryngii, elaeoselini and ferulae are closely related sharing a common ancestor. In all phylogenetic analyses, Pleurotus eryngii var. nebrodensis was placed in a distinct clade, clearly differentiated from the other varieties indicating that this group should be considered a distinct species. Limited nucleotide variation in the genomic regions of the varieties eryngii, ferulae and elaeoselini was indicative that P. eryngii is a taxon that recently diverged and that the speciation mechanism is a result of host adaptations rather than geographical isolation. Since distribution of var. nebrodensis is restricted to elevations of 1,200-2000 m, altitude might also be important in its speciation. A second objective of this research was to elucidate cultural practices that might be used to enhance the concentration of two important antioxidants found in mushrooms: selenium (Se) and ergothioneine (ERGO). In order to enhance Se content in basidiomata, substrates were supplemented with sodium selenite (Na 2 SeO3 ) at two levels (5 and 10 μg/g). Basidiomata of one commercial isolate of P. eryngii var. eryngii linearly accumulated Se up to 4.6 and 9.3 μg/g. On the other hand, ERGO concentration was enhanced in mushrooms produced on a substrate with 55% moisture content compared to the commonly used 60% in commercial cultivation. Mushrooms produced on low-moisture content substrate had ERGO concentrations up to 3.0 mg/g, while mushrooms produced on high-moisture content substrate had less than 2.3 mg/g. Commercial cultivation of P. eryngii in controlled environments usually involves a single harvest; after that, the substrate is discarded. However, on some Italian and Chinese farms, growers use a casing layer to obtain more than one break of mushrooms. A third objective of this research, therefore, was to determine yield, biological efficiency (BE) and number of mushrooms as influenced by casing and substrate supplementation. Application of a casing overlay increased total yield by 141% compared to a non-cased substrate. Supplementation of substrate with delayed release nutrient (Remo's at 4% d.w.) added at substrate fragmentation increased yield by 14% over non-supplemented substrate. When a casing overlay and nutrient supplement were used together, yields increased by 176%. These results may offer growers an opportunity to increase productivity and improve the nutritional and medicinal qualities of P. eryngii .
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