Tulasnella calospora (UAMH 9824) retains its effectiveness at facilitating orchid symbiotic germination in vitro after two decades of subculturing.

BACKGROUND: The technique of symbiotic germination-using mycorrhizal fungi to propagate orchids from seed in vitro-has been used as one method to cultivate orchids in North America and abroad for > 30 years. A long-held assumption is that mycorrhizal fungi used for this purpose lose their effectiveness at germinating seeds over time with repeated subculturing. RESULTS: We provide evidence for the lingering efficacy of one particular strain of Tulasnella calospora (266; UAMH 9824) to stimulate seed germination exemplified by the North American terrestrial orchid, Spiranthes cernua, as a case study. This fungus was originally acquired from roots from Spiranthes brevilabris in 1999 and sub-cultured during the two decades since. Seeds inoculated with the fungus in vitro developed to an advanced protocorm stage after 16 days, and leaf elongation was pronounced after 42 days. In a pilot study, seedlings co-cultured with Tulasnella calospora 266 were deflasked after 331 days and later transferred to soil under greenhouse conditions where they eventually initiated anthesis. During the course of two decades, seeds of 39 orchid species, cultivars and hybrids spanning 21 genera, germinated in vitro co-cultured with Tulasnella calospora 266. These orchids included temperate terrestrials and tropical epiphytes alike. CONCLUSIONS: The sustained effectiveness of this fungus is noteworthy because it argues against the concept of mycorrhizal fungi losing their symbiotic capability through prolonged subculturing. This study serves as an example of why in situ habitat preservation is essential for the conservation of orchids as a source of potentially useful mycorrhizal fungi.

Projected Status of the Ghost Orchid (Dendrophylax lindenii) in Florida during the Next Decade Based on Temporal Dynamic Studies Spanning Six Years.

The enigmatic ghost orchid, Dendrophylax lindenii (Lindley) Bentham ex Rolfe, is a showy leafless epiphyte restricted to low-lying forests in south Florida and western Cuba. Because of its appeal and reputation for being difficult to cultivate, D. lindenii remains vulnerable to poaching and environmental changes. About 2000 individuals are assumed to remain in Florida, most confined within water-filled cypress domes in the Fakahatchee Strand, but virtually no information exists on current population numbers throughout the region. This paper provides a preliminary summary of the ghost orchid's projected status based on six continuous years of data collected within the Florida Panther National Wildlife Refuge (FPNWR) from 2015-2020. The orchids were clustered in seven different populations, each separated by ca. 5 km. Quantitative data were collected spanning three age classes (seedlings, juveniles, mature plants) for each population, and survival, flowering, and fruiting were noted. To estimate the temporal variability in the demographic rates, size-structured integral projection models (IPMs) were constructed for each annual transition (e.g., 2015-2016, 2016-2017). Results for all seven populations pooled suggest that D. lindenii numbers will decline by 20% during the next decade in the absence of external adverse factors. Seedling recruitment is not expected to keep pace with the projected decline. Only one population, which was also from the wettest location, continuously harbored spontaneous seedlings, suggesting that most populations within the FPNWR lack conditions suitable for reproduction.

Environmental and Management Effects on Demographic Processes in the U.S. Threatened Platanthera leucophaea (Nutt.) Lindl. (Orchidaceae).

Populations of the U.S. threatened orchid, Platanthera leucophaea, are restricted to fragmented grassland and wetland habitats. We address the long-term (1998-2020) interactive effects of habitat (upland prairie vs. wetland), fire management (burned vs. unburned) and climatic variation, as well as pollination crossing effects, on population demography in 42 populations. Our analysis revealed the consistent interactive effects of habitat, dormant season burning, and climatic variation on flowering, reproduction, and survival. Burning increased flowering and population size under normal or greater than normal precipitation but may have a negative effect during drought years apparently if soil moisture stress reduces flowering and increases mortality. Trends in the number of flowering plants in populations also correspond to precipitation cycles. As with flowering and fecundity, survival is significantly affected by the interactive effects of habitat, fire, and climate. This study supports previous studies finding that P. leucophaea relies on a facultative outcrossing breeding system. Demographic modeling indicated that fire, normal precipitation, and outcrossing yielded greater population growth, and that greater fire frequency increased population persistence. It also revealed an ecologically driven demographic switch, with wetlands more dependent upon survivorship than fecundity, and uplands more dependent on fecundity than survivorship. Our results facilitate an understanding of environmental and management effects on the population demography of P. leucophaea in the prairie region of its distribution. Parallel studies are needed in the other habitats such as wetlands, especially in the eastern part of the range of the species, to provide a more complete picture.

Fungal Diversity of Selected Habitat Specific Cynorkis Species (Orchidaceae) in the Central Highlands of Madagascar.

About 90% of Cynorkis species are endemic to the biodiversity hotspot of Madagascar. This terrestrial habitat-specific genus received little study for fungal diversity to support conservation. We evaluated the diversity of culturable fungi of 11 species and soil characteristics from six sites spanning a >40 km radius in and along the region's inselbergs. Peloton-forming fungi were grown in vitro from root/protocorm slices and positively identified using DNA sequencing. The fungal diversity was then correlated with soil pH, NO3-N, P, and K. All species harbored either putative mycorrhizal associates in the Rhizoctonia complex or Hypocreales fungi. Tulasnella Operational Taxonomic Units (OTUs) were most prevalent in all soil types while Serendipita OTUs were found in species inhabiting granite/rock outcrops in moist soil (seepage areas). Most Cynorkis species were present in soil with low NO3-N and P levels with diversity of mycorrhizal fungi inversely correlated to NO3-N levels. Of the different orchid life stages sampled, only one species (Cynorkis fastigiata) yielded putative mycorrhizal fungi from juvenile stages. As diversity of mycorrhizal fungi of Cynorkis spp. was negatively correlated with NO3-N, and majority of the studied taxa were found in soils with low NO3-N and P contents, reintroduction studies must include analysis of N and P in detail. For the first time, we showed that the assemblage of culturable fungi in the roots of habitat-specific species of Cynorkis (Orchidaceae) are intimately tied to specific soil characteristics.

Recovery of mycorrhizal fungi from wild collected protocorms of Madagascan endemic orchid Aerangis ellisii (B.S. Williams) Schltr. and their use in seed germination in vitro.

Orchid mycorrhizal fungi (OMF) are critical for seed germination and maintaining natural populations of orchids, yet the degree of specificity of most orchids to their mycorrhizal associates remains unknown. Many orchids are at risk of extinction, whether generalists or specialists, but orchid species of narrow fungal specificity are arguably under increased threat due to their requirement for specific fungal symbionts. This study characterises the fungi associated with Aerangis ellisii, a lithophytic orchid from a site in the Central Highlands of Madagascar. Culturable OMF isolated from spontaneous protocorms of this species from the wild were used for seed germination. In vitro germination and seedling development of A. ellisii were achieved with fungi derived from A. ellisii and an isolate from a different Aerangis species 30 km away. The significance of these findings and their importance to conservation strategies for this species and other Aerangis spp. is discussed. These results have important implications for the conservation of A. ellisii populations in Madagascar.

Further evidence of Ceratobasidium D.P. Rogers (Basidiomycota) serving as the ubiquitous fungal associate of Platanthera leucophaea (Orchidaceae) in the North American tallgrass prairie.

BACKGROUND: In the United States and Canada, ca. one-half of native orchid species are now threatened with extinction. A number of these species are restricted to tallgrass prairies of central North America, such as the Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nutt.) Lindl.-a U.S. Federally threatened species. RESULTS: We provide new records of fungi recovered from roots of P. leucophaea and five other orchid species inhabiting prairie sites in Illinois and neighboring states during a 10-year period (2008-2017). A total of 39 fungal endophytes were isolated from Cypripedium candidum (1), Platanthera lacera (1), P. leucophaea (32), P. peramoena (3), Spiranthes lacera (1), and S. magnicamporum (1), 31 (79%) of which were assignable to Ceratobasidium and the remainder to Tulasnella. These fungi were acquired from 16 different sites, 13 of which are new records including two new state records (Iowa, Wisconsin). Molecular analysis revealed that some Ceratobasidium strains were virtually identical despite being geographically isolated by > 300 km. CONCLUSIONS: This study, encompassing a decade of work, confirms that Platanthera leucophaea is a mycorrhizal specialist with heavy reliance on Ceratobasidium with the tallgrass prairie ecosystem of North America. Our isolation of Ceratobasidium from P. leucophaea spanning additional sites suggests that the association is widespread. Such information should provide conservationists and land managers with more confidence in developing protocols that facilitate the long-term conservation of this prairie orchid.

Techniques for the collection, transportation, and isolation of orchid endophytes from afar: a case study from Madagascar.

BACKGROUND: Tropical orchids need more study with respect to their mycorrhizal associations. For researchers in distant countries who aspire to study these orchids augmenting their conservation, the great distances involved, coupled with limited funds, pose formidable challenges. These challenges are sometimes exacerbated by political unrest, delays in securing permits, unexpected hardships, and the risk that the biological samples collected (e.g., roots harboring mycorrhizal fungi) will not survive long-distance transport. RESULTS: We describe a protocol for the collection and transport of root samples from Madagascar orchids to labs in the United Kingdom (Kew) and the United States (Illinois) where Rhizoctonia-like fungi were subsequently isolated. Three separate trips were made spanning 4 years (2012-2015), with emphasis on the collection of roots from epiphytic, lithophytic, and terrestrial orchids inhabiting the Itremo Massif of the Central Highlands. Collectively, the trips to Madagascar resulted in the isolation of all major groups of Rhizoctonia-like fungi (Ceratobasidium, Tulasnella, Sebacina) from all three orchid growth forms (terrestrials, epiphytes and lithophytes). Sampling of terrestrial and epiphytes during the rainy season (January) yielded best results. CONCLUSIONS: Our study demonstrates that peloton-forming fungi in root samples can retain viability up to 3 weeks after collection.

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.

Preliminary findings on identification of mycorrhizal fungi from diverse orchids in the Central Highlands of Madagascar.

The Orchid flora of Madagascar is one of the most diverse with nearly 1000 orchid taxa, of which about 90% are endemic to this biodiversity hotspot. The Itremo Massif in the Central Highlands of Madagascar with a Highland Subtropical climate range encompasses montane grassland, igneous and metamorphic rock outcrops, and gallery and tapia forests. Our study focused on identifying culturable mycorrhizae from epiphytic, lithophytic, and terrestrial orchid taxa to understand their diversity and density in a spatial matrix that is within the protected areas. We have collected both juvenile and mature roots from 41 orchid taxa for isolating their orchid mycorrhizal fungi (OMF), and to culture, identify, and store in liquid nitrogen for future studies. Twelve operational taxonomic units (OTUs), of three known orchid mycorrhizal genera, were recognized by analysis of internal transcribed spacer (ITS) sequences of 85 isolates, and, by comparing with GenBank database entries, each OTU was shown to have closely related fungi that were also found as orchid associates. Orchid and fungal diversity were greater in gallery forests and open grasslands, which is very significant for future studies and orchid conservation. As far as we know, this is the first ever report of detailed identification of mycorrhizal fungi from Madagascar. This study will help start to develop a programme for identifying fungal symbionts from this unique biodiversity hotspot, which is undergoing rapid ecosystem damage and species loss. The diversity of culturable fungal associates, their density, and distribution within the Itremo orchid hotspot areas will be discussed.

The giant Madagascar hissing-cockroach (Gromphadorhina portentosa) as a source of antagonistic moulds: concerns arising from its use in a public setting.

Cockroaches and their excreta have been linked to allergies and childhood asthma. The giant Madagascar hissing-cockroach (Gromphadorhina portentosa), heralded as an educational tool in classrooms, museums, zoos, is now available to the public as a children's pet raising health concerns. We provide a catalogue of potentially antagonistic moulds associated with this insect. Specimens were obtained from laboratories, classrooms, pet stores and private homes. Three different agar media were used to culture moulds from both internal and external cockroach surfaces. Pure cultures were obtained from hyphal tips excised by scalpel. A total of 14 mould taxa were obtained, mostly from external surfaces. The mycoflora was dominated by species of Rhizopus, Penicillium, Mucor, Trichoderma and Alternaria, and differed little among nymphs, adults, cast skins (exuviae) and faeces. A two-fold increase of Aspergillus niger isolates, however, was detected in exuviae and faeces. The mycoflora appeared to be equally distributed on the body regions in nymphs and adults alike. Most of the moulds recovered are common, well-known saprophytes with a prolific ability to produce asexual spores (e.g. conidia) when supplied with adequate moisture and an organic substrate (e.g. vegetable matter, pet food and exuviae). Cockroach rearing conditions thus serve as an ideal environment for mould growth and proliferation, and the subsequent use (handling) of these insects in a public forum increases the risk of inducing mould-related allergies in humans. Of special concern are moulds also capable of causing secondary infections (e.g. Rhizopus, Mucor, Aspergillus), gaining entry via open wounds and inhalation. This is mainly a point of public awareness aimed at individuals (especially children) prone to infections and allergies that might be exposed to this insect and/or its rearing conditions.

An endosymbiotic conidial fungus, Scopulariopsis brevicaulis, protects the American dog tick, Dermacentor variabilis, from desiccation imposed by an entomopathogenic fungus.

The functional role of an endosymbiotic conidial fungus (Scopulariopsis brevicaulis) prevalent within the integumental glands and hemocoel of the American dog tick (Dermacentor variabilis) was investigated to explore the nature of this tick/fungus association. D. variabilis is normally highly resistant to Metarhizium anisopliae, a widely-distributed entomopathogenic fungus, but when mature female ticks harboring S. brevicaulis were fed a solution containing a mycotoxin (Amphotericin B) to purge this mycobiont internally, the ticks inoculated with M. anisopliae displayed classic signs of pathogenicity, as evidenced by recovery of M. anisopliae from ticks by internal fungus culture, greatly accelerated net transpiration water loss rates (nearly 3x faster than ticks containing S. brevicaulis naturally) and elevation of critical equilibrium humidity (CEH) closer to saturation, implying a reduced capacity to absorb water vapor and disruption of water balance (water gain not equal water loss) that resulted in tick death. The presence of S. brevicaulis within the tick was previously puzzling: the fungus is transmitted maternally and there is no apparent harm inflicted to either generation. This study suggests that S. brevicaulis provides protection to D. variabilis ticks against M. anisopliae. Thus, the S. brevicaulis/tick association appears to be mutualistic symbiosis. Given that both organisms are of medical-veterinary importance, disruption of this symbiosis has potential for generating novel tools for disease control.

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

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.