Isolation of <i>Tulasnella</i> spp. from Cultivated <i>Paphiopedilum</i> Orchids and Screening of Germination-Enhancing Fungi

Ex situ conservation, an important way to increase the survival and sustainability of endangered species, is widely used in the conservation of endangered orchids. However, long-term ex situ conservation might affect the dominant group of orchid symbiotic fungi, which are crucial for orchid growth and reintroduction. This study investigated the culturable <i>Tulasnella</i> spp. associated with <i>Paphiopedilum</i> orchids after long-term greenhouse cultivation, and identified germination-enhancing isolates. A total of 44 <i>Tulasnella</i> isolates were obtained from the roots of 14 <i>Paphiopedilum</i> spp., and 29 of them were selected for phylogenetic analysis. They clustered mainly with <i>Tulasnella deliquescens</i>, <i>Tulasnella calospora</i>, <i>Tulasnella bifrons,</i> and <i>Tulasnella irregularis</i>, but included two potential new groups. Compared with published uncultured data, most of the isolates were grouped together with the reported types, and the dominant <i>Tulasnella</i> associated with <i>P. armeniacum</i> and <i>P. micranthum</i> could still be isolated after ten years of cultivation, most of which were the first isolation. In vitro symbiotic germination showed that certain root isolates could promote seed germination (e.g., parm152 isolated from <i>P. armeniacum</i>, Php12 from <i>P. hirsutissimum</i>, and prhi68 from <i>P. rhizomatosum</i>). These data indicated that the dominant <i>Tulasnella</i> types colonizing the roots of cultivated <i>Paphiopedilum</i> are stable over time, and germination-enhancing fungi colonizing the roots would benefit for seed reproduction after population reintroduction into the wild.