Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long-term

Authors: Webb, Kimberly; Holman, Gregory; Duke, Sara; Greene, Stephanie; MCCLUSKEY, KEVIN - Kansas State University

Submitted to: Annals of Applied Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2018
Publication Date: 7/12/2018
Citation: Webb, K.M., Holman, G.E., Duke, S.E., Greene, S.L., McCluskey, K. 2018. Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long-term. Annals of Applied Biology. 173:133-140. https://doi.org/10.1111/aab.12442.
DOI: https://doi.org/10.1111/aab.12442

Interpretive Summary: Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. Cryopreservation using the vapor phase above liquid nitrogen provides the most stable and long term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage on a diverse collection of Fusarium species. Therefore, we took a collection of 374 Fusarium strains and stored them at -165°C LN2. After five years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells, and measuring the rate of growth of each isolate. We found that all of the isolates stored at -165°C grew at a faster rate than isolates stored at -80°C and that most of the isolates had greater than 80% cell viability. However, there were some isolates that had less than 50% cell viability and that these also generally had fewer conidia germinate. In conclusion, storage at -165°C LN2 provided reliable preservation of a diverse collection of Fusarium spp. over 5 years but some isolates that are known to have low cell viability should be checked for growth more frequently.

Technical Abstract: Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. For most microbial strains, cryopreservation in the vapor phase above liquid nitrogen provides the most stable and long term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage on a diverse collection of Fusarium species. In this work, a collection of 374 Fusarium strains from the Fungal Genetics Stock Center, including 24 unique species, were duplicated and sent to the National Laboratory for Genetic Resource Preservation for storage in the vapor phase above liquid nitrogen. After five years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells, and measuring the rate of growth of each isolate. Additionally, the rate of growth for approximately 10% of the isolates were compared with the same isolates which had been stored at -80°C at the Fungal Genetics Stock Center over the same timeframe to determine if cryopreservation in liquid nitrogen vapor provided a comparable method of storage. All of the NLGRP isolates, grew after being stored at -165°C for 5 years. In general, the isolates that were stored at -165°C grew at a faster rate than the isolates stored at -80°C for the same time period. Most of the isolates had greater than 80% cell viability, however, those isolates that had less than 50% cell viability generally also had fewer conidia germinate. These isolates may be at greater risk for storage over longer times. In conclusion, storage at -165°C LN2 provided reliable preservation of a diverse collection of Fusarium spp. over 5 years, and data indicate that for most isolates, culture viability data indicates that they will remain viable during additional storage for longer periods.