Viability of Metarhizium majus UICC 295 after preservation by freezing at -80°C

Reliable preservation method is required to ensure the integrity of entomopathogenic features of fungus entomopathogen. This study evaluated the viability of entomopathogenic fungus Metarhizium majus UICC 295 maintained in freezing preservation at -80°C by using two different cryoprotectants. Metarhizium majus UICC 295 was cultivated in Saboraud Dextrose with Yeast Extract Agar (SDAY) and SDAY without 10% (w/v) cricket powder. Two different cryoprotectants were used, 10% (v/v) glycerol and 10% (v/v) glycerol with 5% (w/v) trehalose. Sterile water was used as control. The initial cell number was (5.04x10⁶) CFU/mL in SDAY, and (2.5x10⁷) CFU/mL in SDAY with cricket powder. The spore and hyphal suspensions were pre-incubated at 4°C for one hour to allow the cryoprotectants to diffuse into the cell, before storing at -80°C. Viability was evaluated by enumeration using total plate count (TPC) on SDAY and quantifying the percentage of viable cell number at 1, 14, and 30 days of preservation. Each treatment was replicated three times. The results showed that viability of spores and hyphae of M. majus was maintained in this study. The fungal strain was able to withstand the extreme temperature. Adequate recovery of spores and hyphae could be obtained without using a cryoprotectant. Highest percentage of viability (62.10%) with the cell number (3.13x10⁶CFU/mL) was obtained from the sample prepared from SDAY, which was maintained in sterile water. The present study showed that the preservation method at -80°C in sterile water allowed the recovery of the fungus, while preservation at - 80°C in cryoprotectants was not effective and resulted in low recovery of the fungus.