Abstract:Orchid flora of Madagascar is one of most diverse with nearly 1,000 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 40 orchid taxa for isolating the orchid mycorrhizal fungi (OMF), 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 86 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 mycorrhizae from Powered by Editorial Manager® and ProduXion Manager® from Aries Systems CorporationMadagascar. 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 mycorrhizae, density and their distribution within the Itremo orchid hotspot areas will be discussed. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

BackgroundTropical 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.ResultsWe 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.ConclusionsOur study demonstrates that peloton-forming fungi in root samples can retain viability up to 3 weeks after collection.

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