The order Sebacinales belongs to a taxonomically, ecologically, and physiologically diverse group of fungi within the Phylum Basidiomycota. Using several molecular techniques they were detected all over the world. Few isolates, classified into the clade B, are available at the moment: the root colonizing mutualistic fungus Piriformospora indica, various Sebacina vermifera isolates from autotrophic orchids, as well as Piriformospora glomeralium (ex multinucleate rhizoctonia DAR29830, Warcup). All of them were described as growth promoting and resistance inducing fungi. In the present work, seven Sebacinales isolates of the clade B were characterized molecularly and phenotypically. In addition, the presence in Germany of fungi closely related to P. indica was proven.Phylogenetic analyses conducted using DNA sequences from the 28S and the translation elongation factor 1 &
#945; gene (TEF) showed that analyzed Sebacinales isolates represent at least 3 distinct groups of isolates. Further, three independent environmental samples, collected from two different areas in Germany were examined. The analysis demonstrated that the sample respresented organisms closely related to P. indica. The analyzed introns of their TEF gene had the same sequence. This finding suggests that one genotype is present in different area of Germany which is associated with roots of taxonomically diverse plants, including Anthyllis, Medicago and Lolium. Moreover, the fungal genome sizes of five sebacinoid isolates were estimated using Pulsed Field Gel Electrophoresis (PFGE) and real time PCR based on the absolute quantification of a single copy gene (TEF). The fungi have at least 4 to 7 chromosomes and a genome size ranging from 21 to 26 Mb.Morphological, physiological and molecular studies of the multinucleate rhizoctonia DAR29830 isolate proved that this strain is the closest related to P. indica, the most investigated member of the Sebacinales. Therefore it is designed in this study as a provisional new species named Piriformospora glomeralium sp. nov.Seven Sebacinales strains were tested for extracellular enzyme production including peroxidase, laccase, protease, pectinase, and cellulase. In general, the enzymatic test demonstrated strong protease activity in cultures of all the analyzed fungi. The orchid mycorrhiza isolates showed stark laccase activity and lack of cellulase production in axenic culture whereas the Piriformospora strains were able to digest this plant cell wall component. Only one isolate proved to secrete pectinase. All Sebacinales isolates except P. indica and P. glomeralium that were co cultured with a fungal competitor (Rhizoctonia solani) generated significant amounts of laccase. The presence of a plant host, life or dead, had noticeable impact on laccase secretion by P. indica but no influence on P. glomeralium enzyme exudation. In addition, production of laccase, peroxidase, esterase, and lipase by P. indica in response to plant host was spectrophotometrically investigated. Diversity in laccase and lipase activity was observed mostly in presence of decay plant material. Differences in the enzyme profile for the analyzed Sebacinales strains agreed mostly with their phylogenetic position. In addition, genome estimation as well as karyotype analysis clearly confirmed the phylogenetic study and proved that Sebacina vermifera should be considered as complex of isolates. Moreover, laccase secretion by P. indica can be induced only by the presence of the plant symbiotic partner.
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