Acclimatization with endophytic fungi and reintroduction of Guarianthe skinneri (Bateman) Dressler & W.E. Higgins, a threatened, native orchid of cultural value in southern Mexico

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The acclimatization of vitroplants into natural conditions represents one of the greatest challenges for the conservation of wild orchids. For more than a century, a wide range of orchid species have been propagated in the laboratory (in vitro), with, when attempted, a low percentage of plants surviving the acclimatization process. We designed a rustic protocol for acclimatization using fungal endophytes of vitroplants for the reintroduction of Guarianthe skinneri into semi-natural habitat. In Phase I (21 days), a total of 640 vitroplants measuring 1–2.8 cm were transplanted onto substrates made from pieces of each of: clay tiles; Cedrela odorata bark, Agave fourcroydes string, and the exocarp of Theobroma bicolor fruits, and then placed into trays with transparent lids. These transplanted vitroplants were inoculated with three selected strains of endophytic fungi previously isolated from mature G. skinneri plants (Nigrospora sp., Coprinellus sp., and Fusarium sp.). In Phase II the growing vitroplants were transplanted onto larger, individual pieces of the same substrates and were hung from the metal frames of galleries open to natural climatic conditions, for 120 days. In Phase III, surviving, acclimatized seedlings were reintroduced onto the branches of cocoa and coffee bushes as phorophytes, in two sites and evaluated at seven months. We observed 59.1 % (378/640 plants) survival at the end of Phase I, decreasing to 15 % (96/640 plants) at the end of Phase II. The survival percentages were higher when the vitroplants came from a previous subculture using Dalla Rosa and Laneri (DRL) culture medium and were then established on pieces of clay tiles or Cedrela odorata bark and inoculated with endophytic fungi. For Phase III, survival at seven months in the site at 80 m was 21.9 % (21 plants), with no surviving plants in the site at 1,100 m. Only 3.3 % of the original 640 vitroplants survived to one year. This is the first rustic acclimatization protocol of G. skinneri vitroplants, using fungal endophytes, aiming towards achieving reintroduction under natural conditions. The protocol requires improvement, specifically to be initiated with larger, more developed vitroplants, and continued study to improve the selection and management of symbiotic endophytic fungi, and will be applied in reintroduction and restoration programs for G. skinneri and other endangered wild epiphytic orchids. We report for the first time the genera Nigrospora sp. and Coprinellus sp. as endophytic fungi of G. skinneri. eng