Enhancer trap lines with GFP driven by smad6b and frizzled1 regulatory sequences for the study of epithelial morphogenesis in the developing zebrafish inner ear.

Live imaging in the zebrafish embryo using tissue-specific expression of fluorescent proteins can yield important insights into the mechanisms that drive sensory organ morphogenesis and cell differentiation. Morphogenesis of the semicircular canal ducts of the vertebrate inner ear requires a complex rearrangement of epithelial cells, including outgrowth, adhesion, fusion and perforation of epithelial projections to generate pillars of tissue that form the hubs of each canal. We report the insertion sites and expression patterns of two enhancer trap lines in the developing zebrafish embryo, each of which highlight different aspects of epithelial cell morphogenesis in the inner ear. A membrane-linked EGFP driven by smad6b regulatory sequences is expressed throughout the otic epithelium, most strongly on the lateral side of the ear and in the sensory cristae. A second enhancer trap line, with cytoplasmic EGFP driven by frizzled1 (fzd1) regulatory sequences, specifically marks cells of the ventral projection and pillar in the developing ear, and marginal cells in the sensory cristae, together with variable expression in the retina and epiphysis, and neurons elsewhere in the developing central nervous system. We have used a combination of methods to identify the insertion sites of these two transgenes, which were generated through random insertion, and show that Targeted Locus Amplification is a rapid and reliable method for the identification of insertion sites of randomly inserted transgenes.

Preventing Extinction of a Critically Endangered Dactylorhiza incarnata subsp. ochroleuca in Britain Using Symbiotic Seedlings for Reintroduction.

The yellow early marsh-orchid (Dactylorhiza incarnata subsp. ochroleuca) is critically endangered in the UK. Reintroduction of this threatened orchid to former haunts that have been restored is a long-term objective of this study. Identifying germination-specific mycorrhizal fungus lineages from closely related species is used as a method due to the extremely small number of plants left in the wild. A putative orchid mycorrhizal fungus of the family Tulasnellaceae, isolated from Dactylorhiza praetermissa, supported in vitro seed germination to produce reintroduction-ready seedlings. Reintroduced symbiotic seedlings survived over the winter months in the flooded reintroduction site (RS). The comparative soil analysis for key nutrients before reintroduction showed that phosphorus content in the RS is very low compared to the soil collected from the wild site (WS) where the last viable population exists. On the other hand, C:N ratio in the soil at the WS and RS were not significantly different. To our knowledge, this is the first-ever report on the reintroduction of symbiotic seedlings of a threatened orchid back to the wild in the UK.

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.

Diversity of root-associated culturable fungi of Cephalanthera rubra (Orchidaceae) in relation to soil characteristics.

Cephalanthera rubra (L.) Rich., Red Helleborine, is a widespread orchid in Europe but known only from three very small populations in England. These populations are in decline with no natural seed setting for more than a decade. The species may become extinct in the UK soon unless viable strategies are in place for ex situ conservation, especially the use of symbiotic propagation. Because of the fragile nature of the populations in England mycorrhizal fungal diversity study is not feasible. Therefore, to understand the factors needed for healthy Red Helleborine populations, soil characteristics and diversity of culturable root-derived fungi of the populations from a small area in the Loire Valley in France were studied. The main objectives of the study were: (1) Which culturable mycorrhizal fungi associated with C. rubra roots and (2) To what extent is variation in fungal communities related to variation in soil characteristics? Here, we report a significant difference in diversity of culturable mycorrhizal and non-mycorrhizal fungi depending on soil pH and phosphorus content. Mycorrhizal associations were favoured by plants in locations with low soil nutrient availability and comparatively higher pH. Our study shows that mycorrhizal fungi, both ecto and endo, can be cultured from roots of plants at different maturity stages.

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.

Endophytic fungal diversity of Fragaria vesca, a crop wild relative of strawberry, along environmental gradients within a small geographical area.

BACKGROUND: Fungal endophytes are highly diverse ubiquitous asymptomatic microorganisms, some of which appear to be symbiotic. Depending on abiotic conditions and genotype of the plant, the diversity of endophytes may confer fitness benefits to plant communities. METHODS: We studied a crop wild relative (CWR) of strawberry, along environmental gradients with a view to understand the cultivable root-derived endophytic fungi that can be evaluated for promoting growth and tolerating stress in selected plant groups. The main objectives were to understand whether: (a) suboptimal soil types are drivers for fungal distribution and diversity; (b) high pH and poor nutrient availability lead to fungal-plant associations that help deliver fitness benefits; and (c) novel fungi can be identified for their use in improving plant growth, and alleviate stress in diverse crops. RESULTS: The study revealed that habitats with high pH and low nutrient availability have higher fungal diversity, with more rare fungi isolated from locations with chalky soil. Plants from location G were the healthiest even though soil from this location was the poorest in nutrients. Study of environmental gradients, especially extreme habitat types, may help understand the root zone fungal diversity of different functional classes. Two small in vitro pilot studies conducted with two isolates showed that endophytic fungi from suboptimal habitats can promote plant growth and fitness benefits in selected plant groups. DISCUSSION: Targeting native plants and crop wild relatives for research offers opportunities to unearth diverse functional groups of root-derived endophytic fungi that are beneficial for crops.

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.

Ret signalling integrates a craniofacial muscle module during development.

An appropriate organisation of muscles is crucial for their function, yet it is not known how functionally related muscles are coordinated with each other during development. In this study, we show that the development of a subset of functionally related head muscles in the zebrafish is regulated by Ret tyrosine kinase signalling. Three genes in the Ret pathway (gfra3, artemin2 and ret) are required specifically for the development of muscles attaching to the opercular bone (gill cover), but not other adjacent muscles. In animals lacking Ret or Gfra3 function, myogenic gene expression is reduced in forming opercular muscles, but not in non-opercular muscles derived from the same muscle anlagen. These animals have a normal skeleton with small or missing opercular muscles and tightly closed mouths. Myogenic defects correlate with a highly restricted expression of artn2, gfra3 and ret in mesenchymal cells in and around the forming opercular muscles. ret(+) cells become restricted to the forming opercular muscles and a loss of Ret signalling results in reductions of only these, but not adjacent, muscles, revealing a specific role of Ret in a subset of head muscles. We propose that Ret signalling regulates myogenesis in head muscles in a modular manner and that this is achieved by restricting Ret function to a subset of muscle precursors.