Lawrence W. Zettler

Symbiotic germination of three semi-aquatic rein orchids (Habenaria repens, H. quinquiseta, H. macroceratitis) from Florida

The destruction of wetlands in populated areas (e.g. Florida) has prompted interest in habitat restoration. We describe a symbiotic technique to germinate seeds of three semi-aquatic rein orchid species from Florida (Habenaria repens, H. quinquiseta, H. macroceratitis) and to cultivate H. repens seedlings on soil ex vitro. Seeds of all three Habenaria spp. germinated within 21 days of inoculation. Leaf-bearing seedlings of H. repenswere obtained using two fungal isolates (Epulorhiza spp.) recovered from Florida orchids Spiranthes brevilabrisand Epidendrum conopseum . Seedlings infected with the S. brevilabris fungus that were transferred to peat in a greenhouse had the highest (88.9%) survival (>159 days ex vitro). One H. repens seedling initiated anthesis 18 months after seed sowing. The methods outlined by this study have the potential to be adopted by wetland restoration projects seeking to include an orchid ( H. repens) and mycorrhizal fungi as biotic agents.

Water requirements of terrestrial and epiphytic orchid seeds and seedlings, and evidence for water uptake by means of mycotrophy.

The use of endomycorrhizal fungi as an energy source (=mycotrophy) initiates seedling development and supplements or replaces photosynthesis in all orchids in nature. Fungus-infected and non-infected seeds of the monkey face orchid, Platanthera integrilabia, a US Federally-threatened terrestrial species, had a different set of water relations than seeds of the green fly orchid, Epidendrum conopseum, a subtropical epiphyte. Seeds of the terrestrial species had lower water loss rates, smaller activation energies for water loss and absorbed water from lower relative humidities. Thus, the epiphyte lacks the enhanced water retention capacity associated with the terrestrial species, implying that epiphytic orchids are capable of germinating quickly given an adequately moist substrate. After germination, water content of fungus-infected seeds was higher. These results provide first time fundamental information related to habitat preference by analyzing seed. Germination is considerably enhanced with mycorrhizal fungi that facilitate the absorption of free water by their orchid seed hosts.

Mycorrhizal Fungi and Cold-assisted Symbiotic Germination of the Federally Threatened Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nuttall) Lindley

Abstract The 70% decline of the Federally threatened eastern prairie fringed orchid, Platanthera leucophaea (Nuttall) Lindley, has prompted concern for its recovery through artificial propagation. We describe a technique to germinate seeds and cultivate seedlings of P. leucophaea in vitro using cold treatments (=stratification) and mycorrhizal fungi (=symbiotic seed germination). Five fungal isolates were recovered from mature P. leucophaea plants in Illinois and Michigan and were identified as members of the anamorphic genus Ceratorhiza Moore. Stratified seeds inoculated with mycorrhizal fungi germinated within 25 d of sowing. Leaf-bearing seedlings were obtained by chilling young seedlings (protocorms) for 107 d. Our successful culture of leaf-bearing seedlings with a presumed mycotrophic capability may make it possible for this threatened orchid to be propagated in soil ex vitro, followed by reintroduction into suitable habitats.

Symbiotic Seed Germination and Mycorrhizae of Federally Threatened Platanthera praeclara (Orchidaceae)

Abstract In vitro culture of mycotrophic leaf-bearing seedlings of federally threatened Platanthera praeclara Sheviak and Bowles, a terrestrial orchid native to the midwestern prairies, is reported for the first time. Symbiotic germination was evaluated to: (1) determine need for cold moist stratification to enhance seed germination and seedling development and (2) identify the mycobionts that support in vitro germination and development. Germination was improved by exposing seeds to both 4- and 6-mo stratification periods; whereas seeds without stratification failed to germinate in a pilot study. Pretreatment of seeds with 6 mo of stratification combined with inoculation with mycorrhizal fungus derived from a seedling (Ceratorhiza sp., UAMH 9847) supported development of higher stage protocorms and some leaf-bearing seedlings of P. praeclara. Protocorms with developing leaf primordia were also obtained by coinoculation with strains of Epulorhiza and Ceratorhiza. Fungi derived from mature P. praeclara plants failed to promote seedling development to advanced growth stages. Results indicate that in vitro development of P. praeclara is best supported when stratified seeds are cultured with fungi isolated from young seedlings.

Internal and external mycoflora of the American dog tick, Dermacentor variabilis (Acari: Ixodidae), and its ecological implications

ABSTRACT Scopulariopsis brevicaulis, the anamorph of Microascus brevicaulis (Microascaceae, Ascomycota), has been identified in the body contents of the tick Dermacentor variabilis. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of D. variabilis with a novel technique used to recover potential biological control agents.

Mycorrhizal Fungi from Protocorms, Seedlings and Mature Plants of the Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nutt.) Lindley: A Comprehensive List to Augment Conservation

Abstract The Federally threatened Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nutt.) Lindley (Orchidaceae), has experienced long-term decline largely due to habitat loss and degradation. Although this species has been propagated from seed in the laboratory, achieving seedling survival ex vitro has been problematic, forcing conservationists to sow seeds directly into field sites in an attempt to generate seedlings. Given that the mycorrhizal fungi needed for germination in situ have sporadic distributions, sowing seeds of this threatened species indiscriminately is not a preferable option. Thus, locating fungal “hotspots” using seed baits, and amending soil with fungi may have practical merit. In anticipation of the latter possibility, we provide a comprehensive list of the 75 mycorrhizal fungi isolated from P. leucophaea protocorms, seedlings and mature plants during the past 10 y from sites in Illinois and Michigan, including newly acquired strains from five additional sites in Illinois. Collectively, 66 of the 75 isolates (88%) were assignable to the anamorphic form-genus Ceratorhiza, including all of the fungi recovered from the five additional sites. This further supports the hypothesis that P. leucophaea relies primarily on Ceratorhiza to fulfill its initial and long-term mycotrophic needs. Although Ceratorhiza appears to be an ubiquitous associate of P. leucophaea, it should not be assumed that specific strains of this genus are equally widespread. Thus, we advocate that the fungi used in conservation should be limited to strains acquired from the same or nearby populations.

Relationships Between Soil Characteristics, Distribution and Restoration Potential of The Federal Threatened Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nutt.) Lindl

Abstract The Federal threatened eastern prairie fringed orchid (Platanthera leucophaea) occupies prairies, sedge meadows, bogs and fens, primarily north of the Wisconsinan glacial boundary. In the Midwest, where restoration is a recovery objective, its southern distribution is thought to be limited by the transition from nutrient-rich Wisconsinan-aged soils to more acidic nutrient poor soils of Illinoian-aged glacial drift. To better understand edaphic factors affecting its distribution and potential for establishment of new populations, we analyzed soil characteristics across the range of habitats occupied by this species, as well as from unoccupied habitats on the Illinoian Till Plain. We found that P. leucophaea occupies a complex edaphic gradient in variation of % organic matter, base content and soil texture. On Wisconsinan-aged substrates, it occurs in circum-neutral base-rich organic prairie soils in Illinois and Wisconsin and in less calcareous soils with slightly higher pH and lower organic matter content in Michigan lake plain prairies. Eastern sand prairie and sedge meadow habitats on Wisconsinan-aged drift and on unglaciated soils are moderately acid and nutrient poor, while bog and fen habitats are more strongly acidic and highly organic, with no evidence for an underlying calcareous substrate. In comparison, unoccupied prairie soils on the Illinoian till plain have lower pH, % organic matter and base concentrations. These soils also have relatively high % silt content which results in comparatively low available soil moisture holding capacity. This combination of soil conditions may exceed the tolerance limits of P. leucophaea and prevent this species from occurring south of the Wisconsin glacial boundary in the Midwest. On the other extreme, calcareous fens have high pH levels as well as extremely high calcium concentrations, which may exceed the tolerance limits of this species. These findings have implications for guiding efforts to establish P. leucophaea into habitats that should be suitable for this species.

Comparative water balance profiles of Orchidaceae seeds for epiphytic and terrestrial taxa endemic to North America

The Orchidaceae have dust-like seeds that use wind currents for long-distance dispersal. Lacking endosperm, orchid embryos consume free-living, mycorrhizal fungi as a carbon source (mycotrophy) after settling on a substrate. Few studies have investigated orchid seed morphology as it relates to ecology, but conceivably variations in seed size and testa characteristics could be linked to water loss rates aimed at maximizing germination in a particular habitat. Seeds of 2 epiphytic, 1 aquatic, and 7 terrestrial orchids native to North America were compared with respect to water balance profiles: Cleistes bifaria, Encyclia tampensis, Epidendrum nocturnum, Habenaria repens, Isotria medeoloides, Liparis elata, L. hawaiensis, Platanthera holochila, P. integrilabia, and P. leucophaea. Water content, water loss rate, activation energy, and equilibrium humidity were assessed for each species. Seeds of epiphytic orchids were smaller, lighter, more porous, and had higher water loss rates compared to terrestrials. No active mechanism for water absorption exists in seeds of either group. Water loss appears to be a species-specific phenomenon that may be linked to the ecological niches these species occupy.

Mycoflora of a Trogloxenic Cave Cricket, Hadenoecus cumberlandicus (Orthoptera: Rhaphidophoridae), from Two Small Caves in Northeastern Kentucky

Abstract External and internal mycoflora of the cave cricket Hadenoecus cumberlandicus Hubbell were examined and consisted of eight deuteromycetes (Aspergillus niger, Beauveria sp., Colletotrichum acutatum, Geotrichum candidum, Penicillium sp., Pestalotia sp., Trichoderma sp., and Mycelia sterilia), five zygomycetes (Absidia corymbifera, Cunninghamella sp., Mucor racemosus, Phycomyces sp., and Rhizopus sp.), and one ascomycete (Thielavia terrestris). Most are soil saprophytes and typical in a cave setting. Internal isolates included Colletotrichum acutatum and Pestalotia sp., plant pathogens that were probably acquired during foraging. Noteworthy among external fungi was Beauveria sp., a genus of entomopathogens, revealing that fungi may have the potential to impact the cave ecosystem by reducing cave cricket populations.

Water balance of a tick-fungus relationship, featuring the life cycle of the fungus Scopulariopsis brevicaulis (sacc.) Bainier (Deuteromycota) in a tick host

Abstract We report isolation of the fungus Scopulariopsis brevicaulis from the lone star tick, Amblyomma americanum (L.), and document its association with hemolymph and integumental glands. The relationship thrives because the water balance of both the fungus and the tick are mutually preserved in spite of enormous fungus burdens, based on moisture requirements and antimycotic fungus removal. The fact that ticks are ideal to perpetuate this fungal life cycle is of public health interest because S. brevicaulis is known to cause infections in humans.