In Vitro Cell.Dev.Biol.—Plant (2007) 43:178-186 DOI 10.1007/s11627-006-9023-4 SIVB SYMPOSIUM PROCEEDINGS-MICROPROPAGATION Symbiotic seed germination and evidence for in vitro mycobiont specificity in Spiranthes brevilabris (Orchidaceae) and its implications for species-level conservation Scott L. Stewart & Michael E. Kane Received: 26 July 2006 /Accepted: 27 December 2006 / Published online: 13 March 2007 / Editor: B. R. Reed # The Society for In Vitro Biology 2007 Abstract Orchid–mycobiont specificity in the Orchidaceae and water through the action of mycotrophy. The digestion was considered controversial and not well understood for of these mycobionts and subsequent uptake of nutrients by many years. Differences in mycobiont specificity during the immature orchid embryo stimulates seed germination, germination in vitro vs in situ have lead some to consider protocorm development, and seedling growth (Arditti, orchid–mycobiont specificity as being generally low; 1966; Clements, 1988; Rasmussen, 1995). For this reason, however, others have suggested that specificity, especially the survival of orchids in managed or restored habitats may in vitro, is surprisingly high. Mycobiont specificity may be require the presence of appropriate mycobionts to support genus or species specific. An in vitro symbiotic seed plant development and subsequent seedling recruitment germination experiment was designed to examine myco- (Zettler, 1997a). Symbiotic seed germination techniques biont specificity of the endangered Florida terrestrial orchid represent an efficient way to promote the orchid–fungus Spiranthes brevilabris using mycobionts isolated from both association under in vitro conditions and to study in vitro the study species and the endemic congener Spiranthes orchid–mycobiont specificity (Dixon, 1987; Zettler, 1997a, floridana. In a screen of mycobionts, isolates Sflo-305 b; Stewart and Kane, 2006). While a number of symbiotic (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (origi- seed germination techniques exist for terrestrial orchid nating from S. floridana) supported higher initial (stage 1) species, their germination efficiency is often lower than seed germination than isolate Sbrev-266 (32.4%) (originat- expected (Anderson, 1991, 1996; Zettler and McInnis, ing from S. brevilabris) after 3 wk culture. However, only 1992; Zettler and Currah, 1997; Zettler and Hofer, 1998; isolate Sbrev-266 supported advanced germination and Zettler et al., 2001; Stewart and Zettler, 2002; Sharma et al., protocorm development to stage 5 (53.1%) after 12 wk 2003; Stewart et al., 2003; Zettler et al., 2005; Stewart and culture. These findings suggest that S. brevilabris maintains Kane, 2006), especially when compared to asymbiotic a high degree of mycobiont specificity under in vitro germination studies of the same taxa. This low seed germi- symbiotic seed germination conditions. High orchid–myco- nation efficiency is likely because of a degree of specificity biont specificity in S. brevilabris may be indicative of the many terrestrial orchids appear to have for certain myco- rare status of this orchid in Florida. bionts at the time of germination vs later life stages. How- ever, this specificity has apparently been overlooked by Keywords Ceratorhiza . Epulorhiza . Fungal co-culture . previous symbiotic culture practitioners. Mycobiont speci- Mycorrhizae . Native . Protocorm ficity was shown to play an important role in symbiotic orchid propagation and is thought to play a critical role in the establishment of orchids into field sites (Zettler, 1997a, b; Introduction Stewart et al., 2003; Batty et al., 2006a, b). Orchid–mycobiont specificity was considered controver- In nature, orchids utilize naturally occurring endophytic sial for many years. Many researchers have considered the mycorrhizal fungi as sources of carbohydrates, nutrients, orchid–fungus relationship to be happenchance and non- specific both in vitro and in situ (Knudson, 1922;Curtis, * : S. L. Stewart ( ) M. E. Kane 1939;Hadley,1970; Masuhara and Katsuya, 1989; Masuhara Department of Environmental Horticulture, University of Florida, – P.O. Box 110675, Gainesville, FL 32611, USA et al., 1993). Differences in orchid fungal specificity were e-mail: [email protected] identified under in vitro vs in situ conditions (Masuhara and MYCOBIONT SPECIFICITY IN S. BREVILABRIS 179 Katsuya, 1994; Taylor and Bruns, 1999; Taylor et al., 2003; Fig. 1b). The orchid’s typical habitats are grassy roadsides, Bidartondo and Bruns, 2005), and these differences have led cemeteries, and pine flatwoods—all of which are becoming some to consider orchid–mycobiont specificity as generally restricted and degraded in Florida because of habitat loss low (Hadley, 1970; Stewart and Zettler, 2002). However, through urban development and habitat mismanagement others have suggested that specificity, especially under in (Main et al., 1999; Marshall and Pielke, 2004). No infor- vitro conditions, is surprisingly high (Clements, 1988; mation exists concerning the symbiotic seed germination, Smreciu and Currah, 1989;TaylorandBruns,1997; mycobiont specificity, or mycobiont diversity of this McKendrick et al., 2002; Selosse et al., 2002; McCormick species. S. brevilabris and S. floridana were shown to be et al., 2006; Stewart and Kane, 2006). In the most general two independent species with a high degree of relatedness sense, it appears that orchid–mycobiont specificity may be based on genetic evidence (L. Dueck, unpublished data), genus or even species specific. and are considered congeners for this reason. Spiranthes brevilabris Lindley, the short-lipped ladies’ The present study investigates the in vitro mycobiont spe- tresses, is an endangered terrestrial orchid historically found cificity of the rare Florida terrestrial orchid S. brevilabris throughout the southeastern United States coastal plain, but is using mycobionts originating from both study species and the now restricted to one roadside population in Levy County in endemic congener S. floridana through the use of symbiotic west central Florida (Brown, 2002; Fig. 1a). The orchid’s seed germination techniques. A concise description and current habitat is a grassy roadside, although little is known tentative identification of all mycobionts are provided. The about the species’ existence in its more natural sunny pine role of fungal specificity in the distribution, current status, flatwood habitat (S. L. Stewart, personal observation). and conservation of S. brevilabris is also given consideration. Information exists on the symbiotic seed germination of S. brevilabris. Stewart et al., (2003) provided a symbiotic seed germination protocol using mycobionts originating from S. Materials and Methods brevilabris and the Florida epiphytic orchid Epidendrum magnoliae Mühlenberg var. magnoliae (syn.=Epidendrum Fungal isolation and identification. Four mycobionts were conopseum R. Brown ex Aiton). However, the mycobiont chosen for in vitro symbiotic seed germination and myco- specificity in S. brevilabris using mycobionts originating biont specificity trials of S. brevilabris (Table 1). Mycobiont from within the genus was not explored. No further research Sbrev-266 was previously isolated by Stewart et al. (2003). exists concerning the symbiotic germination or mycobiont S. floridana mycobionts were isolated on 28 April 2004 specificity in this species. following the procedure outlined by Stewart et al. (2003), Spiranthes floridana (Wherry) Cory emend. P.M. Brown, modified by taking only root sections and not entire the Florida ladies’ tresses, is an endemic terrestrial orchid flowering plants because of the rarity of the species. Root historically ranging throughout north–central Florida, but is systems of five adult flowering plants at the Bradford currently restricted to two populations in Bradford and County (Florida) population of S. floridana were carefully Duval Counties in northeastern Florida (Brown, 2002; excavated and root sections (<10 cm) were removed. Only five plants, representing 20% of the total population, were sampled because of the small size of this population. The Duval County population was not sampled because, at the time of sampling, only two plants were known from this site. The root sections were wrapped in paper towels moistened with sterile deionized water, placed in plastic bags, stored in darkness at ca 10°C, and transported to the laboratory (<4 h). Root sections were rinsed with cold tap water to remove surface debris and were surface cleansed 1 min in a solution containing absolute ethanol:6.00% NaOCl:sterile deionized distilled (dd) water (5:5:90 v/v/v). Clumps of cortical cells containing fungal pelotons were removed, placed on corn meal agar (CMA; Sigma-Aldrich, St. Louis, MO) supple- mented with 50 mg l−1 novobiocin sodium salt, and incubated at 25°C for 4 d. Hyphal tips were excised from actively growing pelotons and subcultured onto one-fifth- strength potato dextrose agar (1/5-PDA): 6.8 g PDA (BD Figure 1. (a) Spiranthes brevilabris inflorescence in situ.(b) S. Company, Sparks, MD), 6.0 g granulated agar (BD floridana inflorescence in situ. Scale bars=5 mm. Company), 1 l dd water. The pH of all previously mentioned 180 STEWART AND KANE Table 1. Sources of fungal mycobionts used in the Isolate Host Collection Identification inoculation of Spiranthes information brevilabris seed Sflo-305 S. floridana Collected 28 April 2003; Bradford Co., FL Ceratorhiza sp. All mycobionts hosted within Sflo-306 S. floridana Collected 28 April 2003;