Abstracts Biological Sciences

Isolating mycorrhizal fungi from Spiranthes vernalis for subsequent in vitro seed germination

by William Grant Morton

The Spring Ladies’ Tresses orchid’s (<i>Spiranthes vernalis </i>) range extends throughout eastern North America. Although the taxon’s distribution is reportedly widespread the species is considered threatened or endangered in many areas throughout its range and is reportedly only found in small populations in old prairie remnants, mainly next to railways. One goal for any conservation program is to successfully increase population numbers, possibly by re-introduction of plants into existing populations. It is commonly known that orchids form unique relationships with mycorrhizal fungi and the association is often essential for orchid seed germination and enhances seedling survival and growth rate. The intent of this study is to isolate and identify mycorrhizal fungi associated with <i>Spiranthes vernalis</i> individuals from a population in Madison County, Illinois. The fungi were isolated by excising fungal pelotons from the roots of an adult orchid because of the growth rate and morphology of the fungal isolates,. Fungi assignable to the ubiquitous genera <i>Ceratorhiza</i> and <i>Epulorhiza</i> were isolated. After ITS sequencing of fungal DNA BioEdit Sequence Alignment Editor an NCBI GenBank BLAST (blastn) search was used to confirm the identities of the mycorrhizal fungi found to be in association with the Spiranthes vernalis were <i>Ceratorhiza (Ceratobasidium</i> sp. UAMH 9847). Seeds from three individuals were plated with four fungal types on specialized media to see if any fungi and seed combinations had greater germination rates than other combinations. The percent of germination was calculated based on seed counts and the stage of seed development. Identification of the superior fungi will allow more seeds to be germinated and seedlings grown for eventual transplantation into their native Illinois populations. One of the fungal isolations did in fact germinate seeds with higher success with one of the seed sets used. Overall, each of the <i>Ceratorhiza</i> spp. isolates germinate well with at least one seed set with higher success than the other two seed sets. In addition, after nine months (±one week) the seed germination experiment was split into two groups for one month; cold stratified at 4°C with no light, and room temperature ±21°C with a photoperiod of 8 hours a day. This was done to investigate if additional cold stratification was beneficial to the germinated seedlings or if that was the right time to expose germinated seedlings to light. This cold stratification and light exposure treatment resulted in having no a distinct effect on the germination rates. To further confirm that the <i>Ceratorhiza</i> spp. used in the germination experiment was truly responsible for success in germination, re-isolation of the fungi was attempted and successful, but yet unconfirmed by ITS sequencing.