2.5 Seed symbiotic and asymbiotic cultures
Seed symbiotic and asymbiotic cultures were conducted according to the methods described by Zhou and Gao (Zhou & Gao, 2016). Mixed seeds of each treatment were removed from storage at -20℃ and kept at ambient temperature for 10 h. Appropriate amount of seeds of each treatment were surface-sterilized using sodium hypochlorite solution containing 0.1% available chlorine for 3 min followed by washing with ddH2O for three times. Each sterilized circular nylon cloth with a diameter of 2.6 cm and spores of 45 µm was inoculated with 50-100 surface-sterilized seeds using a pipette. Then it was transferred individually to a cylindrical glass bottle (height 9 cm, diameter 6.5 cm) containing 35 mL of AGS (MS 0.9 L/L, natural mature coconut juice 0.1 L/L, carbon powder 1 g/L, sucrose 20 g/L, and agar 6 g/L) or OMA+Epa-01 medium (OMA: oat 4 g/L, agar 8 g/L, pH = 5.8; Epa-01: a highly compatible fungus, which promotes seed germination and protocorm development of P. teres ) such that its surface was completely covered with the Epa-01 strain colony. Each treatment had at least 60 replicates.
Thereafter, all glass bottles containing OMA+Epa-01and AGS medium were incubated at 26.0 ± 0.5℃ and 14 / 10-h light/dark cycle for 60 and 70 days, respectively. Developmental stages of P. teres seeds were determined according to the methods described by Stewart and Kane (Stewart, 2008). P. teres seeds that reached developmental stage ≥2 were considered as germinated. P. teresseedlings with roots were considered as developmental stage 6. Seed states, including developmental stage, germination rate, and fresh weight of germinated seeds, were recorded after incubation. Electronic balance with high accuracy (one-millionth) was used to measure fresh weight of germinated seeds. During incubation, glass bottles contaminated with other fungi were discarded. The calculation of average developmental stage for each treatment was based on the methods described by Nontachaiyapoom et al (Nontachaiyapoom et al., 2011).