Induction of chlamydospores in pleurotus pulmonarius (grey oyster mushroom) as inoculum and development of lyophilized spawn

Abstract

P. pulmonarius is cultivated by using spawn inoculated with mycelial cultures obtained from spore or tissue culture. However, the application of spore is prone to contamination while tissue culture application may contribute to somaclonal variation. The mushroom grain spawn can be only stored for 30 days. Hence, this study was conducted to improve production and preservation technique for P. pulmonarius spawns by using the combination of induce asexual sporulation and lyophilization techniques respectively. The chlamydospore was induced using various stress media namely DGlucose Soluble Starch, D-Glucose CaCl.2H₂O, D-Glucose NaCl, D-Glucose PEG6000, D-Glucose Glycerol, and D-Glucose Na₂SO₄. Three parameters namely; pH, temperature, and incubation period were selected for optimization of chlamydospore formation. It was found that, D-glucose soluble starch stress medium showed the highest 4.5 x107 chlamydospore production with highest chlamydospore production achieved after 78 hours, at pH 6 and 26˚C of temperature by using 1.5 x 107 inoculum size. Next, the spawn was pre-treated with 20% mixture of trehalose and skimmed milk lyoprotectant and lyophilized by using freeze dryer (Labconco) at -40ºC for 2 to 3 days. The spawn was sealed and stored at room temperature for 1 month. After that, the spawn was treated with 2 hours of rehydration treatment prior to inoculation on PDA medium. The chlamydospore-lyophilized spawn was then compared with the chlamydospore spawn which preserved using cryopreservation, sub-culturing, and 4˚C refrigeration techniques in aspect of growth rate and yield. Growth kinetic analysis found that chlamydospore spawn exhibited higher growth rate; 0.24d-1 compared to tissue culture spawn.