Diversity and specificity of orchid mycorrhizal fungi in a leafless epiphytic orchid, Dendrophylax lindenii and the potential role of fungi in shaping its fine-scale distribution

Orchids grow in diverse habitats worldwide with most (approximately 69%) growing on trees as epiphytes. Although orchid mycorrhizal fungi have been identified as potential drivers for terrestrial orchid distribution, the influence of these fungi on the fine-scale distribution of epiphytic orchids is poorly understood. In this study, we investigated the mycorrhizal fungal community and fine-scale distribution of Dendrophylax lindenii, a rare and endangered epiphytic orchid that is leafless when mature. We used amplicon sequencing to investigate the composition of orchid mycorrhizal fungi in the roots of 39 D. lindenii individuals in their natural habitat, the swamps of Florida. We compared the orchid mycorrhizal fungi of D. lindenii to those of co-occurring epiphytic orchids, as well as to the orchid mycorrhizal fungal communities of bark from potential host trees, with and without D. lindenii. Our results show that D. lindenii has a high specificity for a single Ceratobasidium species, which is widely distributed on phorophytes and detected in both wet and dry periods in the orchid’s habitat. This Ceratobasidium species was mostly absent or only recorded in low frequency in the roots of co-occurring epiphytic orchids. Phylogenetic analysis documented that this Ceratobasidium was conspecific with the strain that is used to germinate D. lindenii ex-situ. However, our findings suggest that laboratory germinated adult D. lindenii transplanted into the field had lower read abundances of this Ceratobasidium compared to naturally occurring plants. These findings suggest that this orchid mycorrhizal fungus may play a significant role in the fine-scale distribution of naturally occurring D. lindenii

An expanded role for in vitro symbiotic seed germination as a conservation tool: two case studies in North America (Platanthera leucophaea and Epidendrum nocturnum)

Interest in using mycorrhizal fungi to cultivate orchids from seed in vitro (=symbiotic seed germina- tion) has intensified in recent years and this approach is now an important conservation tool worldwide. In North America, symbiotic germination has been attempted for a growing number of orchid species in peril as a means to acquire seedlings suitable for reintroduction.Interest in using mycorrhizal fungi to cultivate orchids from seed in vitro (=symbiotic seed germina- tion) has intensified in recent years and this approach is now an important conservation tool worldwide. In North America, symbiotic germination has been attempted for a growing number of orchid species in peril as a means to acquire seedlings suitable for reintroduction

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

Abstract 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. repens were obtained using two fungal isolates (Epulorhiza spp.) recovered from Florida orchids Spiranthes brevilabris and 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

Crossing effects on seed viability and experimental germination of the federal threatened Platanthera leucophaea (Orchidaceae)

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Fragrance composition of Dendrophylax lindenii (Orchidaceae) using a novel technique applied in situ

The ghost orchid, Dendrophylax lindenii (Lindley) Bentham ex Rolfe (Orchidaceae), is one of North America’s rarest and well-known orchids. Native to Cuba and SW Florida where it frequents shaded swamps as an epiphyte, the species has experienced steady decline. Little information exists on D. lindenii’s biology in situ, raising conservation concerns. During the summer of 2009 at an undisclosed population in Collier County, FL, a substantial number (ca. 13) of plants initiated anthesis offering a unique opportunity to study this species in situ. We report a new technique aimed at capturing floral headspace of D. lindenii in situ, and identified volatile compounds using gas chromatography mass spectrometry (GC/MS). All components of the floral scent were identified as terpenoids with the exception of methyl salicylate. The most abundant compound was the sesquiterpene (E,E)-α-farnesene (71%) followed by (E)-β-ocimene (9%) and methyl salicylate (8%). Other compounds were: linalool (5%), sabinene (4%), (E)-α-bergamotene (2%), α-pinene (1%), and 3-carene (1%). Interestingly, (E,E)-α-farnesene has previously been associated with pestiferous insects (e.g., Hemiptera). The other compounds are common floral scent constituents in other angiosperms suggesting that our in situ technique was effective. Volatile capture was, therefore, possible without imposing physical harm (e.g., inflorescence detachment) to this rare orchid

A comparison of presettlement and present-day forests in northeastern lower Michigan

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 frommature 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

In Vitro Seed Germination and Seedling Development of <i>Dracula felix</i> (Luer) Luer—An Orchid Native to Ecuador

Effects of daily temperature fluctuations that mimic on-site environmental conditions were tested on seed germination and development in Dracula felix, a native epiphytic orchid from the neotropics. Mature seeds collected from a native population lost their viability from 60% to 37.78% and 0% after 8 and 16 weeks., respectively, under 22 ± 2 °C. Seed viability was completely lost when seeds were maintained at −10 °C in the dark. Less than 50% germination was observed in D. felix seed across all treatments. Seed germinated regardless of the light or temperature treatment. However, significant improvement in germination was observed at 17/22 °C compared to constant temperature treatments. Early seedling development stages were observed only on 1/2XMS and VW media at 17 °C or 17 °C/22 °C under a 12 h light photoperiod. Neither germination nor seedling development were improved by any fungal strain tested using standard symbiotic germination protocols. Information obtained from this study is critical to ensure the ex-situ conservation of this and other rare Dracula species under current and future climate change scenarios

Symbiotic Seed Germination and Seedling Development of <i>Epidendrum geminiflorum</i> Knuth from Ecuador

A greater understanding of the relationship between native orchids and their mycorrhizal symbionts is needed to ensure more effective orchid conservation strategies. A protocol for symbiotic seed germination and seedling development was developed for E. geminiflorum. Mature seeds were collected from a naturally occurring orchid population in Ecuador. Putative mycorrhizal fungi isolated from other native orchid species were used to screen their ability to facilitate germination and seedling development in vitro in either a 0/24 h or 12/12 h light/dark photoperiod at 20 °C. The mycorrhizal fungus Tulasnella calospora (UAMH 9824) isolated from Spiranthes brevilabris in Florida, USA, was also included in this study. Sterilization treatments using 0.3%, 0.5% sodium hypochlorite/ethanol or 2% calcium hypochlorite were tested for their effectiveness as sterilant and their subsequent effects on seed germination percentage. Effective surface seed sterilization was achieved with either 0.5% NaClO/ethanol or 2% calcium hypochlorite. However, significantly higher percentages of germinated embryos developed into protocorms when NaOCl solutions were used compared to the other treatments. Seed germination occurred in both photoperiods tested; however, delayed germination was observed under complete darkness. Seeds of E. geminiflorum germinated without fungal inoculation; however, co-culture with Tulasnella strains improved germination significantly. Seedling development was only observed when seeds were cultured in asymbiotic medium or co-cultured with T. caloscopa (UAMH 9824). Significantly longer seedlings were obtained when T. calospora was present in the culture compared with seedlings cultured in asymbiotic medium. The establishment of mycorrhizal associations was confirmed by the presence of pelotons in the roots of E. geminiflorum seedlings