Abstract:
Orchid mycorrhiza are symbiotic association between fungi and plants. The role of fungi is to provide nutrients for seed germination and protocorm development of orchids. Thailand is one of the countries in tropic region which has the greatest diversity of orchid mycorrhiza. Presently, Thai native orchids are threatened which extinction due to disturbance of urban sprawl and commercial collection. This research aims to study diversity of mycorrhizal fungi associated with orchid, Doritis pulcherrima Lindl. Symbiotic-seed germination of this orchid was also tested. D. pulcherrima orchids were surveyed and collected from various parts of Thailand such as Chaiyaphum, Chumphon, Krabi, Loei, Pangnga and Phetchabu. Forty-three fungal isolates were isolated from single pelotons within cortex cells of roots on 1/6 Nutrient Dectrose Yeast Extract medium. Fungal identification based on morphological and molecular characteristics revealed that most of isolates were Rhizoctonia-like fungi. They were previously reported as orchid mycorrhiza namely genera Ceratorhiza and Epulorhiza. The other fungi were genera Alternaria, Amanita, Fomes Eutypella and Schizophyum. Phylogenetic relationships based on the internal transcribed spacer sequences of 30 Rhizoctonia-like fungal isolates were analyzed. Phylogenetic tree suggested that two fungal genera, Ceratobasidium (perfect stage of Ceratorhiza) and Epulorhiza associated with Thai D. pulcherrima. There were at least four Epulorhiza species. Test of in vitro symbiotic seed germination of D. pulcherrima with representatives of fungal isolates such as isolate Kr01, Cu02, Cy03, Lo01 and Pb02 demonstrated that all isolates supported seed germination at stage 1 with 50% of viable seeds while control treatment (seed sowing in Oat Meal medium) had the percentage of seed germination at the same stage only 5.17% within 1 month. Only fungi isolate Kr01 promoted germination and protocorm development to stage 5 (24.95%) within 4 months. The seeds sowing in Vacin and Went medium also exhibited germination and protocorm development to stage 4 within 2 months.