Technique for Long-Term Preservation of Phytopathogenic Fungi

Dahmen, H., Staub, Th., and Schwinn, F. J. 1983. Technique for long-term preservation of phytopathogenic fungi in liquid nitrogen. Phytopathology 73:241-246. Survival, pathogenicity, and sporulation of 31 phytopathogenic fungi temperature or in water at 20 or 40 C. For successful recovery, all (including species from the Myxomycetes, Oomycetes, Ascomycetes, Oomycetes and Colletotrichum lagenarium required slow freezing. The Basidiomycetes, and Deuteromycetes) were tested after storage in liquid recovery of all other fungi was equally good after rapid or slow freezing. The nitrogen for up to 9 yr. The protective solutions used and rates of freezing rust fungi required thawing at 40 C. All other fungi survived both quick or and thawing were crucial for successful long-term preservation. Some fungi slow thawing. All but one of the 31 fungal species studied could be preserved survived as well in water as in a protective solution. Skim milk-glycerol for up to 9 yr. We failed to preserve Erysiphe graminis, but we could (8.5% + 10%) and DMSO (15%) were good cryoprotectants for all preserve other powdery mildew fungi such as Podosphaeraleucotrichaand propagules tested except urediospores of rust fungi, which required dry Erysiphe cichoracearum. This study demonstrates that with suitable storage. The samples were frozen either rapidly by direct immersion of the protective solutions and proper handling the viability and pathogenicity of vials in liquid nitrogen or slowly (decreasing 1 C/min to -40 C) followed by a wide range of fungi can be preserved in liquid nitrogen for long periods of transfer into liquid nitrogen. The samples were thawed either in air at room time. Additional key word: cryopreservation. Phytopathogenic fungi are difficult to maintain in pure culture the effectiveness of cryogenic (liquid nitrogen) preservation of a without loss of pathogenicity, virulence, and the ability to wide range of phytopathogenic fungi with DMSO, skim milksporulate. The handling and maintenance of obligate plant glycerol and, for some species, L-proline-glycerol used as pathogens can present several problems, especially when large cryoprotectants. For the rust fungi, these methods were compared quantities of inocula are frequently required. Several methods for to the known procedures for preserving dry urediospores in liquid preserving living cultures are known, such as oil-cover slants, soil nitrogen. or sand cultures, drying (6,8), freezing (1,3), or freeze-drying. Freeze-drying is a useful method for preserving many fungi MATERIALS AND METHODS (11,13,25-27). However, alternatives to freeze-drying are needed for fungal species or isolates that do not sporulate and for others Production of fungal material for storage. The spores of the whose spores do not survive the freeze-drying process (eg, obligate parasites were produced on their host plants under suitable sporangia of the Oomycetes and conidia of the powdery mildew conditions in growth chambers. Spores or mycelia of the facultative pathogens [8,14]). parasites were produced on artificial media (Table 1). Spore Successful storage of a broad range of living material (semen, germination, mycelial growth, and pathogenicity were tested prior blood cells, etc.) in liquid nitrogen at -196 C has led to the to freezing. adaptation of this method for the preservation of fungal cultures. A Preparation of the fungal material for storage. Freshly produced number of microorganisms are routinely stored in liquid nitrogen spores were collected bywashing them from infected leaves in type culture collections (14,16) and in the brewing and (obligate parasites) or agar plates (nonobligate parasites) with fermentation industry to minimize variability of cultures deionized water, or for dry storage, by brushing them from infected (12,23,29). The methods for cryogenic storage of rust fungi, which leaves. require storage in dry conditions, are already well established To obtain a high spore concentration, spores suspended in the (5,7,17,18,25). Methods for the preservation of other fungi, such as washing solution were centrifuged (about 3,000-5,000 g for 10 min) the Oomycetes and powdery mildew fungi, are not well defined and resuspended in a smaller volume of the protective solution. The (2,9,19,24). final spore density varied from 106 to 108 spores per milliliter Several cryoprotectant solutions have been reported in the depending on the fungus. literature. Several authors (2,7,9,15,19) have reported DMSO to be Monilinia fructigena, Phytophthora citrophthora, and P. an excellent cryoprotective agent for various fungi. Skim milk cactorum, all nonsporulating strains, were stored by suspending alone was used as protective solution for freeze-drying bacteria (11) mycelial fragments directly in the protective solution. Rhizoctonia and glycerol alone gave varying results with bacteria (11,22) and solani either was suspended in a protective solution, when present fungi (7,8,10,14-16,19). L-Proline was an excellent cryoprotectant as mycelial mat, or was stored dry, in colonized grains. The resting for cell cultures of higher plants (30,31). Preliminary experiments spores of Plasmodiophora brassicae were stored within cabbage with a mixture of skim milk and glycerol in our laboratory showed root fragments without cryoprotectant solution. Urediospores of promising cryoprotective effects (unpublished). rust fungi were air-dried for 24 hr at room temperature prior to The purpose of this study was to examine more systematically storage. The spore and mycelial suspensions and dry material were dispensed in cryogenic storage vials. For storage of nonsterile The publication costs of this article were defrayed in part by page charge payment. This material, the vials were polypropylene cryogenic screw-cap serum article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § tubes (A/S Nunc, Roskilde, Denmark) with 2or 5-ml sample 1734 solely to indicate this fact. tbs(/ uc okle emr)wt -o -lsml 1734 __solelytoindicatethisfact._ capacity. For sterile storage of nonobligate parasites, 01983 The American Phytopathological Society polypropylene vials for heat-sealing (Steriline, Teddington, UK) Vol. 73, No. 2,1983 241 with a sample capacity of 1.5 ml were used. All vials with screw-cap d. Water control. closures were stored in the vapor phase of nitrogen and all sealed Freezing and thawing procedures. The samples were frozen vials were stored in the liquid phase. The vials were stored in a either slowly at a controlled rate of 1 C/ min from ambient model CG 140 liquid nitrogen refrigerator (Cryodiffusion, LUry, temperature to -40 C followed by quick immersion in liquid France). nitrogen, or rapidly by direct immersion in liquid nitrogen. The Cryoprotective solutions. The following cryoprotective controlled rate of decrease in temperature was achieved with the solutions were used: controlled temperature capabilities of a model L2 Ismatec freezea. Skim milk, 8.5% glycerol, 10% (SG). Stock solutions of 17% dryer (Ismatec, ZUrich, Switzerland). skim milk (Difco) and 20% glycerol (C. Roth KG, Karlsruhe, Three rates of thawing were tested: Germany) were mixed 1:1 after autoclaving. a. Slow thawing-20-30 min stationary in air at room b. Dimethyl sulfoxide (DMSO) (Merck) 15%. Because DMSO temperature; can be toxic to several biological systems (12), it was washed b. Intermediate thawing-the storage vials containing the frozen from the fungal samples after they were removed from material were swirled for 5-7 min (depending on vial size) in a storage. 20 C waterbath; and c. L-Proline, 5%-glycerol, 5%. Stock solutions of 10% L-proline c. Rapid thawing-the storage vials containing the frozen (Senn Chemicals, Dielsdorf, Switzerland) and 10% glycerol material were swirled for 2-4 min in a 40 C waterbath. were mixed 1:1 after autoclaving. The solution was washed These methods do not distinguish between the effects of the from the fungal samples after they were removed from thawing conditions on viability and on cold-induced dormancy, storage. which is important for the rust fungi (17). TABLE 1. Media and techniques used to cultivate various phytopathogenic fungi and to increase their inoculum Media and their ingredients Incubation conditions Incubation Fungal species or references and special techniques time in weeks Phytophthora Rye decoction agar medium: 200 g rye grain, 5 g D-glucose, 2 weeks at 18 C, 1 week at 12 C, in the dark, 3 infestans 20 g agar, I liter deionized water. The rye is boiled for 1.50 Roux-bottles hr, the grains are discarded, the remaining ingredient added; pH 5.6 (C. A. Drandarevski, personal communication) P. cactorum and Rye decoction broth medium: same ingredients as 22 C, in the dark, Roux-bottles 2 P. citrophthora above without agar Pythium ultimum Carrot medium: 150 g grated carrots in I liter of 18 C, in the dark, Roux-bottles 2 deionized water Rhizoctonia Millet medium: 25 g millet suspended in 50 ml 24 C, in the dark, 300 ml flasks 3 solani deionized water Botrytis cinerea Pea agar medium: 250 g canned, peas are blended, 20 g 24 C, cool white fluorescent lighta, Roux-bottles 2 saccharose, and 15 g agar added, and made up with deionized water to I liter; pH 6.5 Pyricularia Oat decoction agar medium: 30 g rolled oats are boiled for 24-26 C, I week in the dark, I week under cool 2 oryzae 60 min in 1 liter of deionized water. The rolled oats are white fluorescent lighta, petri plates discarded, 20 g saccharose and 18 g agar added and made up with water to I liter. Cercospora Czapek-Dox V-8 medium (4) 24 C, cool white fluorescent lighta, 2 arachidicola circular cellulose filterb on the agar, petri plates Alternaria Potato-carrot-agar medium (20) (28) (28) solani Helminthosporium Lactose-casein-hydrolysate medium (28) (28) (28) gramineum and H. oryzae Septoria nodorum Czapek-Dox V-8 medium (4) Colletotrichum V-8 agar medium (28) 24 C, in the dark, in Roux-bottles 2 lagenarium Fusarium oxysporum V-8 agar medium (28) 24 C, in the dark, in Roux-bottles 2 f. sp. lycopersici Gloeosporium V-8 agar medium (28) 24 C, in the dark, in Roux-bottles 2 musarum Penicillium V-8 agar medium (28) 24 C, in the dark, in Roux-bottles 2 digitatum Verticillium V-8 agar medium (28) 24 C, in the dark, in Roux-bottles 2 albo-atrum 'Fluorescent light: Philips TLMF 40W/33 RS tubes. bCellulose filter: manufactured by Macherey-Nagel, Diiren, West Germany.