Larry W. Richardson

Protocorms of an epiphytic orchid (Epidendrum amphistomum A. Richard) recovered in situ, and subsequent identification of associated mycorrhizal fungi using molecular markers

Epiphytic orchids have received considerable study, yet little has been published on their germination requirements in situ involving mycorrhizal fungi. Such research has been hampered by the small, dust-like size of seeds and leafless seedlings (protocorms) which are difficult to pinpoint on natural substrates, especially those on arboreal substrates (tree limbs). We report a novel seed sowing and retrieval method, modified from one applied to terrestrial orchids, used in the acquisition of epiphytic orchid protocorms from the Florida Panther National Wildlife Refuge. Seeds from two epiphytic orchid species (Epidendrum amphistomum A. Richard, E. nocturnum Jacquin) were placed in separate nylon mesh packets secured within 35 mm plastic slide mounts, and affixed to tree bark using gutter mesh and a staple gun. To confirm that the embryos were viable, some seeds were also sown on asymbiotic media in the laboratory which subsequently germinated after 52 days incubation. Of 60 packets distributed among 18 tree limb sites, one packet - harboring seeds of E. amphistomum affixed to pop ash (Fraxinus caroliniana Mill.) on a moss substrate - harbored protocorms after 267 days. Using molecular markers, a fungus assignable to the Ceratobasidiaceae, appears to be the mycorrhizal associate of these protocorms suggesting that this fungus may be associated with the germination process in situ.

Comparative seed germination and seedling development of the ghost orchid, Dendrophylax lindenii (Orchidaceae), and molecular identification of its mycorrhizal fungus from South Florida

Background and Aims The endangered leafless ghost orchid, Dendrophylax lindenii, one of the most renowned orchids in the world, is difficult to grow under artificial conditions. Published information on asymbiotic and symbiotic (co-culture with a mycobiont) seed germination, seedling anatomy and developmental morphology of this leafless orchid is completely lacking. This information is critical for the development of efficient procedures for ghost orchid production for successful reintroduction. Methods Ghost orchid seedling early development stages were morphologically and anatomically defined to compare germination, embryo and protocorm maturation and seedling development during asymbiotic and symbiotic culture with one of two mycorrhizal strains (Dlin-379 and Dlin-394) isolated from ghost orchid roots in situ. Key Results Seeds symbiotically germinated at higher rates when cultured with fungal strain Dlin-394 than with strain Dlin-379 or asymbiotically on P723 medium during a 10-week culture period. Fungal pelotons were observed in protocorm cells co-cultured with strain Dlin-394 but not Dlin-379. Some 2-year-old seedlings produced multinode inflorescences in vitro. Production of keikis from inflorescence nodes indicated the capacity for clonal production in the ghost orchid. Conclusions Ghost orchid embryo and seedling development were characterized into seven stages. Fungal strain Dlin-394 was confirmed as a possible ghost orchid germination mycobiont, which significantly promoted seed germination and seedling development. Internal transcribed spacer sequencing data confirmed that Dlin-394 belongs within the genus Ceratobasidium. These results offer the opportunity to examine the benefits of using a mycobiont to enhance in vitro germination and possibly ex vitro acclimatization and sustainability following outplanting.

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.

Pestiferous scale insects on native epiphytic orchids in south Florida: a new threat posed by exotic species

Abstract Scale insects belong to the superfamily Coccoidea, which includes serious plant pests such as soft scales, armored scales, and mealybugs. In 2009, in a natural habitat surrounded by urban development in Naples, FL, pestiferous scale insects (Coccidae) and mealybugs (Pseudococcidae) were discovered on inflorescences of Dendrophylax lindenii (Ghost Orchid). The next year, a follow-up survey was initiated to also include plants in two orchid-rich habitats within the Florida Panther National Wildlife Refuge. There, exotic scales were found on six different epiphytic orchid taxa. Of the scales encountered, Coccus hesperidum (Brown Soft Scale) and Diaspis boisduvalii (Boisduval Scale) are both well-known and serious pests of cultivated plants. Of particular concern is the possibility that these invasive insects have thoroughly infiltrated isolated, natural habitats of rare native orchids.

An Update on Mealybugs and Scale Insects (Hemiptera) on Native Epiphytic Orchids in South Florida, Including a New record for Pseudococcus microcirculus (Pseudococcidae)

Abstract We provide the first published record that populations of the orchid mealybug, Pseudococcus microcirculus McKenzie (Hemiptera: Pseudococcidae), occur on epiphytic orchids in both the Florida Panther National Wildlife Refuge and the Fakahatchee Swamp in the Big Cypress Basin eco-region of South Florida. In California, this mealybug has been a pest of orchids in greenhouses and was the target of state quarantine and eradication efforts in 1962. To date, no published records have documented P. microcirculus on orchids in either Californias or Floridas natural habitats. In 2013, 322 epiphytic orchids were surveyed to document scale and mealybug levels. Pseudococcus microcirculus was found on 5 endangered epiphytic orchid taxa, and 8 (2.5%) individual plants harbored another greenhouse pest, Boisduval scale Diaspis boisduvalii Signoret (Hemiptera: Diaspididae), a species sometimes associated with orchids. How these invasive species were able to establish in these State-protected regions is unknown. With active orchid restoration efforts currently underway in these and other important orchid habitats, efforts should be made to prevent the further spread of these plant parasites.