Preservation of in vitro cultures of the mite pathogenic fungus Neozygites tanajoae

Neozygites tanajoae has recently been described as a new fungal pathogen distinct from Neozygites floridana. This pathogen is currently being used as a classical biological control agent against the cassava green mite, Mononychellus tanajoa (Bondar), in Africa. Neozygites tanajoae is a particularly fastidious species, and in vitro cultures of isolates from Brazil and Africa have only recently been established. In this study, the efficacy of several cryoprotectants at different exposure times, cooling rates, and warming rates for protecting hyphal bodies of N. tanajoae during cryopreservation was investigated. A protocol for preservation of cultures of N. tanajoae at ultra-low temperatures of -80 degrees C or -196 degrees C, using 1% trehalose + 2% dimethyl sulfoxide as cryoprotective agents, is described in detail. In this study, we demonstrate that N. tanajoae differs remarkably from N. floridana (isolates ARSEF 662 and ARSEF 5376) in the ability to withstand the stress of cold temperature (4 degrees C) and cryopreservation. In vitro cultures of the 2 N. floridana isolates remained viable at 4 degrees C for up to 47 d; however, cultures of N. tanajoae did not survive this temperature for 4 d. Cryopreservation methods successful for N. tanajoae isolates are not suitable for N. floridana and are unusual in comparison to those for many fungi.