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Title: Long-term Preservation of a Collection of Rhizoctonia solani, using Cryogenic Storage

Author
item Webb, Kimberly
item Hill, Amy
item LAUFMAN, JULIE - Us Forest Service (FS)
item Hanson, Linda
item Panella, Leonard

Submitted to: Annals of Applied Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2011
Publication Date: 5/1/2011
Citation: Webb, K.M., Hill, A.L., Laufman, J., Hanson, L.E., Panella, L.W. 2011. Long-term Preservation of a Collection of Rhizoctonia solani, using Cryogenic Storage. Annals of Applied Biology. 158(3):297-304.

Interpretive Summary: Rhizoctonia solani is an important plant pathogen on a number of crops and maintaining an extensive collection of reference isolates is important in understanding relationships of this pathogen with multiple hosts. Current long-term storage methods typically call for frequent transfer increasing the risk of changes to morphological or virulence characteristics. Cryopreservation using storage in liquid nitrogen was evaluated to examine the potential for storage of a R. solani culture collection containing 106 isolates. Cultures were stored on autoclaved barley grains at –160C in the vapor phase of liquid nitrogen. After 60d, 5y, and 10y in storage, all isolates were removed, cultured on PDA and examined for fungal growth by calculating the percentage of barley grains from which R. solani mycelia grew. After 5y, only one isolate had a significant decrease in fungal growth when compared to the percent growth 60d after initially being placed in storage, all other isolates had comparable percent growth. After 10y in storage 67 of 106 isolates had no significant decrease in percent fungal growth. Thirty nine of 106 isolates had a significant decrease in percent growth but only nine isolates had less than 10% growth, with four having no growth. A subset of isolates stored for 10y, were tested for pathogenicity on a susceptible (FC901) and resistant (FC703) sugar beet germplasm. All isolates tested maintained approximately the same level of virulence that they had prior to storage on both germplasms. This indicates that cryogenic methods are suited for the preservation or storage of R. solani culture collections, although efficacy may vary with individual isolates.

Technical Abstract: Rhizoctonia solani is an important plant pathogen on a number of crops and maintaining an extensive collection of reference isolates is important in understanding relationships of this pathogen with multiple hosts. Current long-term storage methods typically call for frequent transfer increasing the risk of changes to morphological or virulence characteristics. Cryopreservation using storage in liquid nitrogen was evaluated to examine the potential for storage of a R. solani culture collection containing 106 isolates. Cultures were stored on autoclaved barley grains at –160C in the vapor phase of liquid nitrogen. After 60d, 5y, and 10y in storage, all isolates were removed, cultured on PDA and examined for fungal growth by calculating the percentage of barley grains from which R. solani mycelia grew. After 5y, only one isolate had a significant decrease in fungal growth when compared to the percent growth 60d after initially being placed in storage, all other isolates had comparable percent growth. After 10y in storage 67 of 106 isolates had no significant decrease in percent fungal growth. Thirty nine of 106 isolates had a significant decrease in percent growth but only nine isolates had less than 10% growth, with four having no growth. A subset of isolates stored for 10y, were tested for pathogenicity on a susceptible (FC901) and resistant (FC703) sugar beet germplasm. All isolates tested maintained approximately the same level of virulence that they had prior to storage on both germplasms. This indicates that cryogenic methods are suited for the preservation or storage of R. solani culture collections, although efficacy may vary with individual isolates.