Mycorrhizal fungi from protocorms, seedlings and mature plants of the Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nutt.) Lindley: a comprehensive list to augment conservation.
The Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nutt.) Lindley (Orchidaceae; Fig. 1), of the tallgrass prairie has experienced long-term decline largely due to habitat loss/ degradation and is currently listed as Federally threatened (U.S. Fish & Wildlife Service, 2007). Formerly widespread across the Midwest, east into Ontario and Maine, the species is limited to prairie remnants and wetlands (fens) north of the Wisconsinan glacial boundary (Bowles et al., 2005). In 2007, 79 extant populations were identified, but only 28% of these sites had full legal protection (U.S. Fish & Wildlife Service, 2007). Well-coordinated efforts to recover this species are currently in place involving a network of scientists, private landowners and volunteers, yet the conservation of P. leucophaea remains a precarious endeavor. Propagation through seed has been a high priority, but progress has been limited by low fruit set and poor seed quality (i.e., low embryo viability) probably linked to genetic inbreeding among closely-related individuals in smaller populations (Bowles et al., 2002). These problems are compounded by the species' reliance on mycorrhizal fungi necessary to propel the orchid life cycle to completion in situ--a requirement of all members of the Orchidaceae (Rasmussen, 1995).
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To address the latter, the technique of symbiotic seed germination (Clements et al., 1986; Dixon, 1987) was applied as a means to acquire laboratory-grown seedlings for reintroduction into suitable habitats. Although Platanthera leucophaea has since been propagated in this manner to a leaf-bearing stage under laboratory conditions (Zettler et al., 2001, 2005), achieving seedling survival ex vitro has been problematic for reasons that remain unclear. Until this dilemma can be resolved, the propagation of this orchid hinges solely on directed seed sowing in situ. This practice, coupled with artificial hand pollination, has been employed for almost a decade and may explain an increase in 20 populations (59 to 79) since 1999 (U.S. Fish & Wildlife Service, 2007). The appearance of spontaneous seedlings suggests that the mycorrhizal fungi specific to the germination and maturation process have persisted in these sites. Unfortunately, the fungi utilized by P. leucophaea have sporadic distributions (Zettler et al., 2005; Piskin et al., 2003), and targeting their whereabouts remains a hit or miss affair. Considering that viable P. leucophaea seeds are sometimes difficult to acquire in sufficient quantity, sowing seeds indiscriminately is not a preferable option.
To improve seed germination in situ, techniques are needed to locate fungal "hotspots" and to artificially amend soil with fungal inoculum. In Australia, several such protocols have surfaced (e.g., Batty et al., 2006a, b; Swarts, 2007) that may have practical merit for Platanthera leucophaea here in North America. In anticipation of the latter possibility, we provide a comprehensive list of the mycorrhizal fungi isolated from P. leucophaea protocorms, seedlings and mature plants during the last 10 y, from sites in Illinois and Michigan. The strains that have prompted seed germination and development in vitro are highlighted, and their accession numbers at the University of Alberta Microfungus Collection and Herbarium in Canada (UAMH) are provided. We also report mycorrhizal fungi from roots of established plants from three additional sites in Illinois (Nachusa, Ogle Co.; Grant Creek, Will Co.; Somme Prairie Grove, Cook Co.), and from protocorms acquired from two prairie remnants that currently lack the species (Anderson Prairie, Christian Co.; Denby Prairie, Macoupin Co.).
MATERIALS AMD METHODS
Fungal isolation from orchid tissues.--All mycorrhizal fungus strains were isolated using standard protocols (Currah et al., 1987, 1990; Zettler et al., 2003, 2005). Living orchid tissues from leafless, mycotrophic seedlings (protocorms) and lateral roots from mature plants and seedlings were promptly transported to the laboratory within 24 h of collection. Protocorms were obtained using the seed baiting technique described by Rasmussen and Whigham (1993). Briefly, seeds of Platanthera leucophaea were placed between two sheets of nylon mesh suspended within 35 mm plastic slide mounts (Polaroid Corp., Cambridge, MA) which were then subsequently buried in sod for 1-2 y. The small pore size of the mesh (95 [micro]m) allowed fungal hyphae present in the sod to infiltrate the mesh, infect the seeds and prompt germination to the protocorm stage. Protocorms trapped between the mesh are then recovered, and their associated mycorrhizal fungi are subsequently isolated in the laboratory. Orchid tissues (roots, protocorms) were initially rinsed under tap water to remove surface debris, followed by surface sterilization using a solution of 5% absolute ethanol (EtOH), 5% Clorox[R] bleach (5.25% NaOCI; Clorox Company, Oakland, CA) and 90% sterile DI water. Tissues were immersed in this solution for 1 min under agitation. Alter this time, the solution was decanted off, and tissues were then rinsed twice in sterile DI water. Protocorms were added to a drop of sterile DI water within empty 9 cm diameter Petri plates. The epidermis of roots from mature plants and seedlings was scraped off by scalpel, and clumps of cortical tissues were removed and placed in a drop of sterile DI water within separate Petri dishes. These tissues were then macerated by scalpel, releasing individual cortical cells into the water droplet that were visible to the naked eye. Molten modified Melin-Norkran's agar (MMN; Marx, 1969) or fungal isolation medium (FIM; Hollick, 2004) was poured over the droplet, gently swirled and allowed to cool and solidify. Plates were incubated at ambient temperature (ca. 22 C) for up to 1 wk until individual hyphae could be observed emanating from cortical cells/pelotons suspended in the agar. Using a dissection microscope, pure cultures were obtained by excising hyphal tips with a sterile scalpel, followed by transfer to the surface of potato dextrose agar (PDA; Difco[TM], Becton, Dickinson and Co., Sparks, MD).
Fungal identification and storage.--Genus-level taxonomic treatment of the mycorrhizal fungi reported herein follows Moore (1987). Orchid mycorrhizal strains were distinguished from common molds facilitated by published descriptions, namely Currah et al. (1987, 1990, 1997), Richardson et al. (1993) and Zettler et al. (2003). All strains recovered from Platanthera leucophaea that were assignable to known mycorrhizal genera were retained for further study. Strains that originated from the same tissue source were carefully screened for subtle differences in pure culture. Those that exhibited visual differences (e.g., growth rate, colony coloration, presence/absence of monilioid cells) were retained and assigned an accession number. These cultures were then stored at Illinois College under refrigeration (4 C) in darkness on PDA slants in screw-cap tubes and/or over modified oats medium (Dixon, 1987). Every attempt was made to retain mycorrhizal fungi that appeared unique in culture at the time of isolation. Cultures were sub-cultured to fresh agar slants yearly. To retain culture viability in storage > 1 y between sub-culturing, sterile heavy mineral/paraffin oil (Fisher Scientific, Fair Lawn, NJ) was added over the surface of the culture on agar slants and stored at 4 C. Cultures that either prompted germination and development to the protoconn stage, or were deemed potentially important (e.g., acquired from different protocorms in situ) were deposited into the University of Alberta (Canada) Microfungus Collection and Herbarium (UAMH) for safekeeping and future use through cryopreserration.
RESULTS AND DISCUSSION
To date, 75 mycorrhizal fungus strains have been recovered from Platanthera leucophaea protocorms, seedlings and mature plants spanning nine populations in Illinois (Fig. 2) and Michigan (Table 1). The majority (66/75 or 88070) of the fungi were identified as members of the anamorphic genus Ceratorhiza (Fig. 1) and the remaining number were assignable to Epulorhiza (Table 1). A third anamorphic genus associated with orchids worldwide Moniliopsis (teleomorphs = Thanatephorus/Waitea; Currah et al., 1997) - has yet to be isolated from P. leucophaea. Nearly half (35/75) of these strains have been deposited into the UAMH collection for safekeeping and future use (Table 1). This total (75) expands upon the previous number (41) reported by Zettler et al. (2005), in part, because of subsequent isolations that ensued during 2007-2008 involving three new sites in Illinois (Ogle, Will and Cook Cos.; Table 1). Two additional prairie sites (Christian, Macoupin Cos., IL) that lacked P. leucophaea but appeared suitable for the species were also sampled for fungi though the use of seed baits. This resulted in 15 additional strains, 14 of which originated from Denby Prairie in Macoupin Co. (Table 1). Of special significance is that these 15 strains were isolated from protocorms--the earliest growth stage of the orchid (Fig. 3). The acquisition of protocorms from sites lacking P. leucophaea suggests that these sites harbor suitable fungi for germination, but seedling recruitment might be restricted by limited seed dispersal. Protocorms of terrestrial orchids are subterranean and colorless, deriving all of their carbon from fungi during this initial growth stage (Rasmussen, 1995). In a natural setting, orchids like P. leucophaea must make physical contact with this fungus host via seed dissemination, and failure to do so results in an abrupt end to the orchid's life cycle. For ex situ conservation protocols aimed at terrestrial orchids (e.g., Swarts and Dixon, 2009a, b), acquiring fungal strains that prompt germination is of paramount importance and is best achieved by their extraction from orchid tissues (Rasmussen, 1995), preferably protocorms (Zettler et al., 2003). The 15 protocorm-derived strains reported herein, combined with the seven reported previously (Zettler et al., 2005) brings the total number of protocorm isolates to 22 spanning three different prairie sites (Hildy, Anderson, Denby). Of these 22, six have been secured in Canada (UAMH 10217, 10218, 10457, 19498, 10499, 10500). The total deposition of 35 mycorrhizal strains from P. leucophaea into UAMH represents a start, but only six orchid populations have been surveyed for fungi so far. In Illinois alone, >30 additional populations await study (Fig. 1). Studies that utilize molecular techniques (e.g., DNA sequencing of ITS genes) to ascertain genetic variation among these (and newer) strains are also needed.
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Compared to other terrestrial orchids in North America, the prairie fringed orchids (Platanthera leucophaea, P. praeclara, P. lacera) have received modest attention with respect to their mycorrhizal associations (e.g., Bowles et al., 2005; Sharma et al., 2003a, b; Zelmer and Currah, 1995; Zelmer et al., 1996; Zettler et al., 2001, 2005; L.W. Zettler, unpub, data). As a group, fungi assignable to Ceratorhiza have been recovered from protocorms, seedlings and mature plants of both P. leucophaea and P. praeclara alike, and with regularity (Zettler et al., 2001, 2005; Sharma et al., 2003a, b). Strains of Epulorhiza--one of the most common mycorrhizal associates of terrestrial orchids worldwide (Currah et al., 1997)--have also been isolated from these orchids but to a lesser extent. While Ceratorhiza isolates have prompted in vitro seed germination of P. leucophaea (Zettler et al., 2001, 2005), Epulorhiza strains have not been effective. Taken together, the prevalence of Ceratorhiza spanning all three growth stages (protocorms, seedlings, mature plants) combined with in vitro germination results suggests that P. leucophaea relies primarily on Ceratorhiza to fulfill its mycotrophic needs (Zettler et al., 2005). Our recovery of additional Ceratorhiza strains from P. leucophaea at five new sites in Illinois lends further support for this hypothesis. In SW Australia, the abundance and composition of orchid mycorrhizal fungi is influenced by fire (Ramsay et al., 1986), and the same could be true for P. leucophaea of the tallgrass prairie. Thus, additional types of mycorrhizal fungi from frequently-burned areas may await discovery. Curtis (1939) predicted that orchid species restricted to particular habitats would harbor fewer fungal associates. Similarly, Swarts and Dixon (2009a) proposed that rare orchids ("specialists") may be closely tied to specific biotic agents (pollinators, mycorrhizal fungi) that themselves have narrow distributions, whereas common orchids ("generalists") are less discriminate.
Whether or not P. leucophaea should be regarded as a "specialist" or a "generalist" remains unclear; however, the degree that P. leucophaea relies on Ceratorhiza, combined with its requirement for moth pollinators (e.g., Sphinx erremitus; Pollack, 2009), suggests the balance to be tilted in favor of the former.
New approaches to conservation with mycorrhizal fungi.--In Australia, several protocols within the framework of integrated conservation (Falk, 1990; Ramsay and Dixon, 2003; Stewart, 2007; Swarts, 2007) have already been advanced that may have an application for rare species in North America (e.g., Platanthera leucophaea). Of particular interest are in situ and ex situ methods used to detect the presence of mycorrhizal fungi in suitable habitats and methods that involve inoculation of soil. Quay et al. (1995) acquired seedlings of two Caladenia species from seed in 10 wk on pre-inoculated potting mix (ex vitro germination), and noted that seed germination was absent in containers lacking mycorrhizal fungi and in potting mix that was not previously sterilized. Batty et al. (2006a) inoculated potting media with mycorrhizal fungi to promote the survival of symbiotically-grown seedlings in a greenhouse setting but concluded that the additional dose of inoculum was unnecessary.
Brundrett et al. (2003) devised an innovative fungal baiting technique that could detect the presence of mycorrhizal fungi ex situ. They concluded that fungal inoculum was most prevalent in the layer of coarse organic matter (>2 mm litter) and topsoil. The same study also yielded a novel in situ method for detecting mycorrhizal fungi using multi-chambered seed packets that permitted simultaneous assessment of seed germination spanning multiple orchid genera. Swarts (2007) used ex situ baiting to locate "fungal hotspots" in field sites and proceeded to reintroduce seedlings of critically rare Caladenia huegelii into these areas. Our recovery of mycorrhizal fungi from P. leucophaea protocorms using Rasmussen and Whigham's (1993) initial seed baiting protocol can also be used to locate "fungal hotspots," but these newer Australian techniques may prove more effective.
The availability of the 35 Platanthera leucophaea isolates combined with protocols applied in Australia should provide conservationists with an opportunity to amend soil with mycorrhizal fungi in situ. Doing so could conceivably lead to the establishment of additional fungus "hotspots" for seed germination and the establishment of "safe sites" that improve survival rates of re-introduced seedlings (Swarts, 2007). Developing a "safe site" protocol for P. leucophaea seems warranted given the difficulties associated with the establishment of laboratory-grown seedlings to date. To our knowledge, amending soil in situ has yet to be attempted in North America. In Australia, Swarts (2007) utilized sterilized millet seed colonized by mycorrhizal fungi to amend soil prior to reintroducing Caladenia huegelii seedlings. However, he reported that mice seeking the millet seed as a food source destroyed the out-planted seedlings by digging under wire cage barriers. Given the likelihood of small mammals in the prairie ecosystem, a more distasteful substitution for millet seed might be considered. Batty et at (2006b) inoculated field sites with fungi using small (3 mm) cubes of oat meal agar placed adjacent to translocated tubers of two species grown symbiotically (Thelymitra manginiorum, Diuris micrantha) but concluded that there was no benefit to this practice on orchid survival rates possibly because fungi were already present at the site. Additional studies are needed to determine if amending soil imparts a benefit on seedling survival in sites that lack suitable mycorrhizal fungi determined through seed baiting (Batty et al., 2006b). We also urge caution when selecting specific fungal strains for this purpose, at least until more is known about fungal invasion ecology. Desprez-Loustau et al. (2007) warned that the introduction and spread of exotic fungi is one of the most important problems facing conservation biology exacerbated by limited baseline data on fungal communities. Although Ceratorhiza appears to be a ubiquitous associate of P. leucophaea, it should not be assumed that specific strains of that genus are equally widespread. Wright et al. (20t0) determined that mycorrhizal associates differ between populations of Caladenia tenlaculata and that local fungus diversity exists within individual orchid populations. Thus, we advocate that the fungi used in amending soil should be limited to strains acquired from the same or nearby populations until more information is gathered.
Acknowledgments.--We express gratitude to Dr. Lynne Sigler for deposition of mycorrhizal fungi into UAMH and to Marlin L. Bowles (The Morton Arboretum) for the photograph of P. leucophaea depicted in Fig. 1. Special thanks are extended to Kelly Neal (Illinois Nature Preserves Commission) and to Dave Nance (Jacksonville, IL) for permission to sow seed baits into Denby and Anderson Prairies, respectively. We also thank Stephen Packard and Bill Kleiman for access to material at Somme Prairie and Nachusa Grasslands, respectively. Valuable feedback from Dr. Nevin Aspinwall (Saint Louis University), Dr. Scott L. Stewart (Kankakee Community College, IL), and Cathy Pollack (U.S. Fish & Wildlife Service) is also appreciated. The technical support of the following Illinois College students is graciously acknowledged: Jenifer Fortney, Jared Hartsock, Erin Wood.
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SUBMITTED 2 SEPTEMBER 2010
ACCEPTED 13 JANUARY 2011
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LAWRENCE W. ZETTLER (1)
Department of Biology, Illinois College, 1101 West College Avenue, Jacksonville 62650
KURT A. PISKIN (2)
Department of Biology, ,Saint Louis University, 3507 Laclede Avenue, ,Saint Louis, Missouri 63103
(1) Corresponding author: e-mail: firstname.lastname@example.org
(2) Current Address: Division of Natural Sciences, University of Illinois - Springfield, 1 University Plaza, Springfield 62703
TABLE 1.--Summary of mycorrhizal fungi isolated from Platanthera leurophaea in Illinois and Michigan prairies (1998-2008). Cultures marked by an asterisk (*) were acquired from protocorms. UAMH accession numbers listed in bold represent cultures that prompted seed germination in vitro (Zettler et al., 2001, 2005). All UAMH cultures listed may be obtained at: www.devonian2.ualberta.ca/uamh/ # Epulo- # Ceratorhiza strains rhiza strains (mature Proto- Seed- State Year Population plant) corm ling Mature MI 1998 Monroe Co. (1) -- -- -- 4 IL 1998 Lake Co. Wrigley/ 4 Abbott IL 1999 Lake Co. 5 -- 4 14 Wadsworth IL 2002 Grundy Co. Hildy 4 7 -- -- IL 2004 Christian Co. Anderson -- 1 -- -- IL 2004 Macoupin Co. -- 14 -- -- Denby IL 2007 Ogle Co. Nachusa -- -- -- 6 IL 2007 Will Co. Grant Creek -- -- -- 2 IL 2008 Cook Co. Somme -- -- -- 10 Prairie Grove Total 9 populations 9 22 4 40 (8 Illinois, 1 Michigan) UAMH accession State Year Population number MI 1998 Monroe Co. (1) 9611# IL 1998 Lake Co. Wrigley/ 9610# Abbott IL 1999 Lake Co. 9855, 9856, 9857, Wadsworth 9858, 9859, 9860, 9861# IL 2002 Grundy Co. Hildy 10219,10220,10217 *, 10218 * IL 2004 Christian Co. Anderson 10457 * IL 2004 Macoupin Co. 10498 *, 10499 *, Denby 10500 * IL 2007 Ogle Co. Nachusa 10974, 10975, 10976, 10977, 10978, 11011 IL 2007 Will Co. Grant Creek 10979, 10980 IL 2008 Cook Co. Somme 10981, 10982, 10983, Prairie Grove 10984, 10985, 10986, 10987, 10988,10989, 10990 Total 9 populations 35 deposited into (8 Illinois, UAMH# 1 Michigan) (1) The identity of the Michigan population has been withheld at the request of Michigan DNR Note: UAMH accession numbers listed in bold indicated with #.
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|Author:||Zettler, Lawrence W.; Piskin, Kurt A.|
|Publication:||The American Midland Naturalist|
|Date:||Jul 1, 2011|
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