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Title: PRESERVATION OF IN VITRO CULTURES OF THE MITE PATHOGENIC FUNGUS NEOZYGITES TANAJOAE

Author
item DELALIBERA, ITALO - CORNELL UNIVERSITY
item Humber, Richard
item HAJEK, ANN - CORNELL UNIVERSITY

Submitted to: Canadian Journal of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/2004
Publication Date: 9/30/2004
Citation: Delalibera, I., Humber, R.A., Hajek, A.E. 2004. Preservation of in vitro cultures of the mite pathogenic fungus neozygites tanajoae. Canadian Journal of Microbiology. 50:579-586.

Interpretive Summary: Cassava green mite (CGM), MONONYCHELLUS TANAJOA, is a significant pest of cassava in Brazil and West Africa where this host plant is the main subsistence crop. CGM is often affected in Brazil by a fungal pathogen newly described as NEOZYGITES TANAJOAE because of key differences in the nutrition, physiology, host specificity, and genetics between the CGM fungus and a related fungus, N. FLORIDANA. These differences became apparent when trying to culture and to preserve cultures of N. TANAJOAE. New culture media and techniques for the preservation of N. TANAJOAE were successfully developed and proved to be surprisingly unlike those commonly used for other fungi affecting invertebrates. This study enables future studies in culture with South American and African isolates of N. TANAJOAE, and will also be useful to produce this fungus for practical use as a biocontrol agent. These results also underscore the need to recognize that fungal pathogens of invertebrates that show very high host specificities (as does N. TANAJOAE), may require very special conditions to allow their isolation, maintenance in culture, and long-term preservation to enable substantial laboratory research on such organisms.

Technical Abstract: NEOZYGITES TANAJOAE has recently been described as a new fungal pathogen distinct from NEOZYGITES FLORIDANA. This pathogen is currently being used as a classical biocontrol agent against cassava green mite, MONONYCHELLUS TANAJOA (Bondar), in Africa. NEOZYGITES TANAJOAE is a particularly fastidious species, and in vitro cultures of isolates from Brazil and has 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 (-80C or -196C) 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 (4C) and cryopreservation. In vitro cultures of the two N. FLORIDANA isolates remained viable at 4C 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 useful for N. FLORIDANA and are unusual in comparison to those for many fungi.