Long-term cryopreservation of non-spore-forming fungi in Microbank(TM) beads for plant pathological investigations

Authors: Lakshman, Dilip; SINGH, VIMLA - Icar-Indian Institute Of Maize Research; CAMACHO, MANUEL - North Carolina State University

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2018
Publication Date: 4/13/2018
Citation: Lakshman, D.K., Singh, V., Camacho, M.E. 2018. Long-term cryopreservation of non-spore-forming fungi in Microbank(TM) beads for plant pathological investigations. Journal of Microbiological Methods. 148:120-126. https://doi.org/10.1016/j.mimet.2018.04.007.
DOI: https://doi.org/10.1016/j.mimet.2018.04.007

Interpretive Summary: Long-term preservation of experimental fungi without genetic and pathological changes is critical for plant pathological investigations. Several low temperature preservation methods are available for long-term storage of fungi, but the methods are cumbersome, hazardous, and expensive and are often not suitable for long-term storage of non-spore-forming (sterile) fungi. A method of preservation of spore-forming fungi in commercially available porous bead (MicrobankTM) under low-temperature (-80' C) conditions was successfully utilized for non-spore-forming plant pathogenic fungal isolates belonging to three genera of Rhizoctonia fungi. Viable fungal isolates of all test species were retrieved after five years of storage. The fungal isolates retained morphological, pathological and genetically identifiable characteristics of the parent isolates. Our findings demonstrated the utility of low-temperature preservation in Microbank beads as a convenient alternative to conventional long-term storage of a wide group of fungal cultures for plant pathological investigations, and served as the first report of using porous beads under cryogenic conditions for long-term storage of sterile fungi. This information will be useful to scientists investigating the biology and management of the important soilborne plant pathogen, Rhizoctonia solani.

Technical Abstract: Long-term preservation of experimental fungi without genetic, morphological, and pathogenic changes is of paramount importance in mycological and plant pathological investigations. Several cryogenic and non-cryogenic methods are available for the preservation of fungi, but the methods can be cumbersome, hazardous, expensive, and often not suitable for long-term storage of non-spore-forming (sterile) fungi. A method of preservation of spore-forming fungi in commercially available porous beads (MicrobankTM) under cryogenic conditions was successfully tested for three non-spore-forming basidiomycetes genera: Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) (n = 19), Ceratobasidium species (n = 1), and Waitea circinata (n = 3), and a non-spore forming ascomycete, Sclerotinia sclerotiorum (n = 1). For comparison, spore-forming ascomycetous fungi, Alternaria alternata (n = 1), Bauveria basiana (n = 2), Botrytis cinerea (n = 1), Fusarium oxysporum f.sp. gladiolii (n = 1), Trichoderma spp. (n = 3), and Thielaviopsis basicola (n = 2) were also cryopreserved in Microbank beads. Viable fungal isolates of all test species were retrieved after five years of storage at -80° C, which was longer than the viabilities of the corresponding isolates cryopreserved in agar plugs or colonized wheat seeds. Fungi revived from the Microbank beads maintained identical morphology and cultural characteristics of the parent isolates. Randomly selected Rhizoctonia isolates revived from the Microbank beads maintained respective pathological properties of the parent isolates. Also, no mutation was detected in the internal transcribed spacer (ITS) ribosomal DNA when compared with respective cultures maintained at ambient temperature. This finding demonstrated the utility of cryopreservation in Microbank beads as a convenient alternative to conventional long-term preservation of a wide group of fungal cultures for plant pathological investigations, and serves as the first report of using porous beads under cryogenic conditions for long-term storage of sterile fungi.