Cryopreservation of spores of vesicular arbuscular mycorrhizal

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1 New Phytol. (1990), 115, Cryopreservation of spores of vesicular arbuscular mycorrhizal BY DAVID D. DOUDS, JR.^ AND N. C. SCHENCK^ ^ US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Philadelphia, PA 19118, USA ^ Plant Pathology Department, University of Florida, Gainesville, FL 32611, USA {Received 21 July 1989; accepted 23 April 1990) SUMMARY Storage of spores of vesicular arbuscular (VA) mycorrhizal fungi in soil at 5 C is a common way of preserving these fungi. This method was satisfactory for Glomus intraradix Schenck & Smith but not for Gigaspora margarita Becker & Hall, Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe, and Acaulospora longula Spain & Schenck. Preservation of spores at 60 to 70 C was examined. Cryoprotectants such as DMSO, glycerol, mannitol, and sucrose were ineffective using the freeze-damage sensitive species G. margarita. Incubation for 47 h in 0-75 to l-o M trehalose conferred a measure of freeze damage protection to the spores such tbat germination rates of previously frozen spores of G. margarita were one tenth to one sixth of controls. The best method of cryoprotection and cryopreservation was found to be slow drying of pot culture soil and the spores in situ. This procedure was satisfactory for tbe five genera of VA mycorrhizal fungi evaluated. Key words: VA mycorrhizal fungi, cryopreservation, germination. INTRODUCTION The most commonly used method of maintaining vesicular-arbuscular (VA) mycorrhizal fungi is continuous culture in a pot of soil with a plant host. This method is labour, time, and space consuming and may not retain the genetic variability of fungi collected from the field. Therefore, a method for long-term storage of pot culture inoculum is essential. Spores of VA mycorrhizal fungi commonly are stored at 4-5 C in dried pot culture soil (Siqueira et al., 1985). Cultures of Glomus fasciculatum have been stored successfully for 4 years in dry soil at 3-5 C (Ferguson & Woodhead, 1982). There are exceptions to the applicability of cool, dry storage, however. Mugnier & Mosse (1987) have stored sporocarps of Glomus mosseae for 4 years at 4 C over saturated salt solutions which maintain high relative humidities. Spores from tropical habitats survived storage better in wet than dry sand or soil (Daft, Spencer & Thomas, 1987). Attempts to preserve these fungi in liquid nitrogen have not been successful (Sylvia, 1984). Single stage lyophilization has been effective only for species with thick-walled spores (Dalpe, 1987). Slow of pot culture soil, soil + kaolinite, or kaolinite to 40 C followed by rapidly to 196 C, has been shown to preserve the infectivity of VA mycorrhizal fungi in colonized root pieces, but cryoprotective solutions were ineffective (Tommerup & Bett, 1985). Tommerup (1988) has successfully preserved spores and hyphae of VA mycorrhizal fungi by L-drying and storage under vacuum. Tommerup (1988) states, however, that 'cryopreservation may be the ultimate choice in methods (but) there are few studies aimed at developing this as a routine procedure for VA fungi.' This study was conducted to quantify the loss in germinability of VA mycorrhizal fungal spores when stored in soil at 5 C. In addition, we explored the use of cryoprotectants to permit storage of spores at 60 to 70 C, and developed a broadly applicable, simple, and effective method of spores in pot culture soil. * Florida Agriculture Experiment Station Journal Series No. R Use of a product name does not constitute an endorsement by the Florida Institute of Food and Agricultural Sciences. t Figures within square brackets represent International Culture Collection of VA Mycorrhizal Fungi isolate number. MATERIALS AND METHODS Storage of spores in soil at 5 C Spores of Acaulospora longula Spain & Schenck [316]t, Gigaspora margarita Becker & Hall [185],

2 668 D. D. Douds and N. C. Schenck Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe [156], and Gl. intraradix Schenck & Smith [208] were isolated from fresh pot culture soil (Arredondo fine sand; loamy, siliceous, hyperthermic Grossarenic Paleudult), or a 14:10 (v/v) mix of Arredondo fine sand and calcined clay ('Emathlite', Mid-Florida Mining Co.^ Lowell, FL) for Gl. intraradix, by wet sieving (Gerdemann & Nicolson, 1963) and centrifugation (Jenkins, 1964). Twenty to 40 spores were pipetted on moist membrane filters (0 45 ^m pore size) which then were folded and placed in tissue specimen bags (Shandon Southern Instruments, Inc., Sewickley, PA). Three membrane filters per VA mycorrhizal fungus, each at five matric potentials, were prepared for four sample periods (i.e. 3x5x4). Filters in specimen bags were then buried in plastic bags full of soil at one of the five matric potentials [^m]: -0-01, -0-05, -0.50, -1.30, and MPa (6-7, 5-0, 4-0, 3-0, and 2-0% water (wt/wt), respectively, for Arredondo fine sand; and 201, 28 5, 16 3, 13-6, and 11 8 % water, respectively, for the soil and calcined clay mix) and incubated at 5 C. Soil water contents at various ^^ were determined using a ceramic plate extractor (no. 1500, Soil Moisture Equipment Co., Santa Barbara, CA). Spores were incubated in the same soil type in which they were produced. Samples were removed after 1, 6, 12, and 20 months of incubation. In addition, three filters per species were prepared to assay germination of spores not subjected to storage. Spore germination was measured by placing the membrane filter in the specimen bag on a layer of moist soil. The bag then was covered with 1 cm of soil and incubated in the dark at 22 C. At the end of 1 month, filters were removed, stained with 0-05 % trypan blue, and scored for germination. Cryopreservation experiments A variety of cryoprotective agents were tested for use in storage of VA mycorrhizal fungal spores at 60 to 70 C. Spores were isolated from soil and pipetted into 1 ml cryotubes (Nunc, Postbox 280, Kamstrup, DK 4000 Roskilde, Denmark) containing deionized water, sucrose (0-1, 0-5, 0-6, 1-2 M), glycerol [10, 12-5, 15, 20, and 30% (w/v)], mannitol (0-1 and 0-5 M), or trehalose (1-0, 0-75, 0-50, 0-25, and M) with or without 9 and 17% (v/v) DMSO. Tubes were incubated at 22 or 5 C and then were transferred to 60 to 70 C. After no less than 3 h, the cryotubes were removed from the freezer and immersed in warm water (35-40 C) for fast thawing. Spores were rinsed, pipetted on to membrane filters, and assayed for germination as outlined above. Gi. margarita [185] was used for tests of the effectiveness of cryoprotective solutions because of its sensitivity to freeze damage. Freshly isolated populations of spores exhibit consistently high rates of germination (80-95 %), and their size and col< _, make them easy to manipulate and good indicators of injury due to. Healthy spores have numerous small lipid droplets. Spores become brownish and these droplets appear to coalesce upon aging or freeze damage. Spores were also frozen in situ, that is in the soil in which they were produced. Freshly watered pot cultures, typically Arredondo fine sand or Wachula sand (sandy, siliceous, hyperthermic Ultic Haplaquods) in 165 cm'' conical plastic pots ('Super-cell,' Stuewe & Sons, Inc., Corvallis, OR 97333, USA), with Paspalum notatum Flugge or Allium cepa L. as host plants, were moved from the greenhouse to the laboratory and were allowed to dry for 4 5 wk. A sampling strategy was devised which allowed for the soil column in the pot to dry in an as undisturbed state as possible. At sampling intervals of 3-14 d, the shoot and root/soil column was removed from the pot and a 25 cm^ section of soil was cut from the bottom of the column. The unused portion of the root/soil column, plus attached shoot, was returned to the pot to continue drying by evapotranspiration. The section removed was mixed and divided into three samples. One sample was used for the determination of soil water content, another was placed in cryotubes and frozen at 60 to 70 C, and spores were isolated from the third as a control. After 3 h or more, cryotubes with soil were removed from the freezer and thawed as above. Control and previously frozen spores were isolated from soil and incubated in the germination assay for 2-3 wk as outlined above. Implicit in these experiments is the assumption that damage to the spores during cryopreservation occurs during the and thawing processes. A quick thaw is generally accepted to be the best way to thaw quickly frozen organisms (Fennel, 1960). Five species were frozen for 3 months to test the assumption that the spores could survive at 60 to 70 C for an indefinite period and germinate after a quick thaw. However, the freezer malfunctioned after three months and they thawed slowly. Spores then were isolated from soil and germinated. Spores in soil also were air dried quickly. Pot cultures were watered, brought into the laboratory, and the soil was spread out and air dried over 2-3 d. Triplicate soil samples were removed each day and spores were frozen and germinated as above. The density of spores in soil was also increased by adding spores, isolated by wet sieving and centrifugation, in 100 /i\ water to moist soil in a cryotube. This soil was dried, frozen, thawed, and spores were incubated in the germination assay outlined above. Vesicular-arbuscular mycorrhizal fungi used in cryopreservation experiments were: A. longula [316]; Acaulospora sp. [776]; A. morrowiae Spain & Schenck [506]; Entrophospora colombiana Spain & Schenck [562]; Entrophospora sp. [283]; Gi. mar-

3 Cryopreservation of VA mycorrhizal fungi 669 garita [105], [185], [328], and [680]; Gi. gigantea Nicolson & Gerdemann [109]; Gl. etunicatum Becker & Gerdemann [236] and [329]; Gl. occultum Walker; Gl. mosseae [322] and [336]; Gl. versiforme (Karsten) Berch [231], Gl. macrocarpum Tul. & Tul. [925], Gl. claroides Schenck & Smith [884]; Gl. sp. [878B]; Gl. clarum Nicolson & Schenck [204]; Gl. intraradix [208]; Scutellispora heterogama (Nicol. & Gerd.) Walker & Sanders [117]; S. pellucida (Nicol. & Schenck) Walker & Sanders [337]; S. dipapillosa (Walker & Koske) Walker & Sanders [400] and [678]; and S. gregaria (Schenck & Nicol.) Walker & Sanders [223B]. Arcsin-transformed germination data were analysed with analysis of variance and linear regression. Significant treatment effects were characterized further using Tukey's method of multiple comparisons. RESULTS Storage of pot culture soil at 5 C Gi. margarita and A. longula failed to germinate after 20 months storage in pot culture soil at 5 C for all \Jf^ studied (Fig. 1 a, c). Germination of spores of G. mosseae was low and variable at 12 months and averaged only 1 % after 20 months (Fig. 1 b). Gl. intraradix was the only species studied which showed substantial germination after 20 months (Fig. \d). Cryopreservation of spores in pot culture soil Spores of VA mycorrhizal fungi in soil were better able to survive to 60 to 70 C when the soil had dried to near air-dry equilibrium moisture content [ % H^O (wt/wt)] (Table 1). Gl. etunicatum was the only species which germinated at rates equal to controls after in moist soil (Table 1) or in water (data not shown). Gl. occultum germinated after being frozen in water-saturated soil, but not as well as controls (12 vs. 87%, respectively). This technique was tested for a wide range of species and was found to be broadly applicable. The only species which did not exhibit germination of previously frozen spores at 50 % or greater of control, were Gl. clarum [204], Gi id) h rf MPa i Storage duration (months) Figure 1. Percent germination of spores of (a) Gigaspora margarita, (b) Glomus mosseae, {c) Acaulospora longula, and {d) Glomus intraradix. Spores were germinated upon isolation from pot culture soil (time zero, shown by cross-hatched bars), or stored at 5 C in soil at five matric potentials for up to 20 months and then incubated at 22 C in soil at field capacity to induce germination. Bars represent the means of three samples of spores+ SEM.

4 670 D. D. Douds and N. C. Schenck Table 1. Germination of spores of VA mycorrhizal fungi dried slowly in situ before and after in soil to -60 to - 70 C for 3-24 h-f Days of drying Regression Regression _ Regression Regression Soil moisture [% H,O (wt/wt)] Percent germination Before Acaulospora longula [316] a a a a * Entrophospora colombiana [562] a a a a a n.s. Glomus etunicatum [236] la a a a Gigaspora margarita [105] a a a 81-6a 72-9 a a n.s. After 18-7b 73-7b 81-5 a Ik* 2-4 b 2-3 b 65-5 a 58-2a * 68-9 a 59-0 a 57-1 a 45-7a n.s. 51-la b 61-3b 65-1 a * f Each number represents the mean of two samples of spores, each with spores. J Numbers for a paired comparison, directly across columns only, followed by tbe same letter are not significantly different (a = 0-05). Results of linear regression, germination as dependent variable and soil moisture as independent variable, *P> 0-05, **P < 0-01, n.s., not significant. margarita [328], 5. pellucida [337], and S. gregaria [223B] (Table 2). All species but Gl. occultum in Table 2 did not survive if water or 10 or 20% (w/v) glycerol were added to the soil before (data not shown). Spores survived 3 months at 60 to 70 ^C followed by a slow thaw (Table 3). There were no significant differences in percentage germination between 3 h and 3 months of. Rapid air-drying of spores and soil yielded successful cryopreservation of two of the three species tested. Spores of Entrophospora sp. [283], in pot culture soil dried slowly to a moisture content of 0-59% over 17 d, exhibited fresh and frozen germination of 45-9 and 32-7%, respectively. Spores from soil quick-dried over 2 d to 0-35 % water exhibited fresh and frozen rates of germination of 89-1 and 64-8%, respectively. Spores of A. longula [316], in pot culture soil dried slowly over 38 d to 0-28 % water, exhibited fresh and frozen germination of 89'8 and 81-9%, respectively. Spores from soil quick-dried over 4 d to 0-26 % water exhibited fresh and frozen rates of germination of 75'2 and 78-2, respectively. Spores of 5. dipapillosa [678], in pot culture soil dried slowly over 17 d to 0-48% water, exhibited fresh and frozen germination of 58*6 and 41-0%, respectively. However, spores from soil quick-dried to 0*34% water over 2 d exhibited fresh and frozen germination of 70-0 and 13-4%, respectively. When spores were isolated from fresh pot culture soil, added to pasteurized soil, and dried over 3 d, two of the four species tested failed to germinate after (Table 4). Indeed, the drying process itself depressed the germination of S. pellucida and Gi. gigantea. These experiments were conducted with other VA mycorrhizal fungi. A. morrowiae [506] exhibited fresh vs. frozen germination of 34-2 vs. 39-9%; Gi. margarita [105], 87-1 vs. 66-3%; Gi. margarita [328], 13-5 vs. 4-6%; S. heterogama [117], 23-3% vs 27-3%; Gl. mosseae [336], 14-3 vs. 0-0%; and S. dipapillosa [400], 24-6 vs. 4-3%. Preservation of spores in cryoprotective solutions Spores of Gi. margarita [185] were incubated in glycerol solutions for 30 and 60 min and frozen. Glycerol treatment did not markedly affect germination of unfrozen spores (84-97 % germination), but no spores germinated after. Similarly, no spores survived after stepwise progression through 10, 12-5, 15, and 20% glycerol. No spores germinated after preceded by a 19 h incubation in 10% glycerol. Another experiment was conducted in which spores were incubated for 1, 19, or 93 h in 0-1 or 05 M mannitol or sucrose. Though incubations did not affect the germination of unfrozen spores of S. heterogama [117], Gl. mosseae [336], and Gi. margarita [105], spores did not germinate after. Incubation of Gi. margarita [185] spores in 0-6 and 1-2 M sucrose for 30 min did not affect germination (74 82%), but spores did not germinate after. Different concentrations of trehalose were evaluated for their cryoprotective properties with Gi. margarita [185]. Incubation for 2 d in 0-5, 0-75, or 1 0 M trehalose yielded germination percentages of frozen spores approximately one sixth those of unfrozen spores (Table 5). Incubation for an additional day before caused a decrease in germination after. Duration of incubation in 0-5 M trehalose affected germination of spores before (r^ = 0-59, P>F= 0-026). In addition, S.

5 Cryopreservation of VA mycorrhizal fungi 671 Table 2. Germination of spores of VA mycorrhizal fungi dried slowly in situ before and after in soil to 60 to 70 C for 3-24 h* Percent germination Species Soil moisture [% H,O (wt/wt)] Before After Glomus occultum Glomus mosseae [322] Glomus mosseae [336] Glomus versiforme [231] Glomus macrocarpum [925] Glomus claroides [884] Glomus clarum [204] Glomus intraradix [208] Glomus sp. [878B] Entrophospora sp. [283] Gigaspora margarita [680] Gigaspora margarita [185] Gigaspora margarita [328] Scutellispora heterogama [117] Scutellispora pellucida [337] Scutellispora dipapillosa [400] Scutellispora gregaria [223B] 0-46 M at 18-2b 14-3 a 72-3 a 49 7 a 611a 55-4a 55-9a 87-9 a 80-0 a 95-5 a 54-5 a 77-8 a 46-8 a 67-4a 24 6 a 46-3 a 85-1 a 26-8 a 16 8 a 70 9 a 52-0 a 72-Oa 0 9b 529a 85 2 a 52-4a 81-8a 49-8 a 26 3 b 28 6 b 4-2 b 28-6 a 15-Ob * Each number represents the mean of two samples of spores, each with spores. f Numbers for a paired comparison, directly across columns only, followed by the same letter are not significantly different (a = 0-05). X Spores were isolated from roots after drying in soil and. Table 3. Germination of spores of VA mycorrhizal fungi dried in situ, before and after for 3 h and 3 months in soil at 60 to 70 C* Percent germination Species Before After 3h for 3 months Glomus etunicatum [329] Glomus occultum Gigaspora margarita [105] Scutellispora heterogama [117] Acaulospora longula [316] 92-7 a 86 0 a 45-1 a 46-8 a 88-8 a 90-7 a 85-1 a 29-5 a 28-6 b 81-5a 96-0 a 947 a 69-7 a 25-Ob 84-2 a * Each number represents the mean of two samples, each with spores, t Numbers for a comparison, directly across columns only, followed by the same letter are not significantly different (a = 0-05). heterogama [117] exhibited unfrozen vs. frozen rates of germination of 77-3 vs. 0-0 % after incubation in M trehalose for 18 h. Gl. occultum and Gl. etunicatum [236] exhibited unfrozen vs. frozen rates of germination of 60-1 vs and 85-7 vs. 72-7%, respectively (differences not significant at P < 0-05), after incubation in M trehalose for 18 h. Addition of DMSO to trehalose solutions decreased spore germination and this effect increased at the higher DMSO concentration. Nine and 17% DMSO (v/v) in 0-5 M trehalose yielded 76-7 and 37-2% germination of Gi. margarita [185] spores, respectively, for spores incubated 47 h, but no germination after. Three days of incubation of spores in 1-0 M trehalose with 9 and 17 % DMSO yielded 30-9 and 27% germination, respectively, before but no germination after.

6 672 D. D. Douds and N. C. Schenck Table 4. Germination of spores of VA mycorrhizal fungi after isolation from soil {fresh), readdition to soil and drying, and in soil to -60 C Percent germination After drying Before After Species Fresh Acaulospora longula [nb] 48-7 af 335 a b 14-9b Scutellispora pellucida [337] 84-1 a 26-4b 0-0a Gigaspora gigantea [109] 19-3a 0-Ob 0-Ob Entrophospora colombiana [562] 51-1 a 64-Oa 52-2a * Acaulospora longula, S. pellucida and Gi. gigantea were cultured in Arredondo fine sand soil. Initial moisture content of pasteurized soil, 8-61 %; after drying for 3 d, 0-80% (wt/wt). Entrophospora colombiana was cultured in Wachula sand. Initial moisture content of pasteurized soil, 11-30% ; after drying for 3 d, 0-92 %. Each number represents the mean of two samples of spores, each with spores. t Numbers for a comparison, directly across columns only, followed by the same letter are not significantly different (a = 0-05). Table 5. Germination of spores of Gigaspora margarita [185], after incubation in trehalose solution for different times, and before and after at -60 to - 70 C for 3 h* Percent germination After incubation Trehalose Duration of Before After- (M) incubation (h) Fresh at 89-2a 0-Ob b 82-9a 0-Oc a 78-7 a 3-3 b a 87-1 a 0-Ob a 86-6 a 0-Ob a 74-8a 13-1 b a 72-7a 2-9b Oa 89-2a 0-Ob a 75-Ob 0-Oc a 79-9 a 14-5b a 72-8a 3-7b a 64-5 a 0-Ob a 40-9 a 00 c a 73-9a 13-5b a 70-6a 0-Ob * Spores were isolated from soil and incubated in the indicated solutions at 22 C. Each number represents the mean of two samples of spores, each with spores. t Numbered for comparisons, directly across columns only, followed by the same letter are not significantly different (a = 0-05). This method is convenient for the sandy soils used at DISCUSSION, T 1/--.1 / ". I I - ritatl/t the International Culture Collection ot VA ivlycor- The poor storage of VA mycorrhizal fungus spores rhizal Fungi (Schenck, 1987) because the soil in pot culture soil at 5 C demonstrates the need for moisture reached at equilibrium with the atmosphere an effective method of cryopreservation. Drying is conducive to cryopreservation of the spores, so the spores in pot culture soil effectively permits them to water content of the soil need not be monitored, withstand the stress of to 60 to 70 C. The method of cryopreservation described re-

7 Cryopreservation of VA mycorrhizal fungi 673 quires slow drying of the soil, in situ, and a density of spores in the soil great enough to make storage in small cryotubes practical. Rapid air drying led to satisfactory survival of for two of the three species tested. The addition to soil of spores freshly isolated from pot culture, in order to increase spore density, did not yield satisfactory germination after subsequent. Spores may not withstand rapid desiccation so soon after hydration caused by wet sieving, centrifugation, and collection, the most reliable method, therefore, is slow drying of spores plus soil in situ, at the original population density. Some VA mycorrhizal fungus spores withstand in aqueous solutions or moist soil, e.g. Gl. occultum and Gl. etunicatum. Cryopreservation of these spores does not require special handling. Common cryoprotective agents utilized in the preservation of other organisms (Tuite, 1969) were ineffective for VA mycorrhizal fungi in this and other studies (Tommerup & Bett, 1985). Cryoprotective agents work by several means. Some can permeate cells and may act as 'noninjurious solutes', increasing bound water and decreasing freeze dehydration at a given temperature (Levitt, 1980, p. 236; Yelenosky & Guy, 1989). Also, cryoprotectants may be impermeable compounds and may act as hypertonic solutions, drawing water out of cells and thereby providing a measure of freeze protection via dehydration. Other cryoprotectants, such as bovine serum albumin and skim milk may serve to absorb wastes produced by the organism which would become toxic as their concentrations increased as water froze. There is evidence that disaccharides such as trehalose, sucrose, and maltose stabilize membranes during dehydration stress (Crowe, Crowe & Chapman, 1984; Crowe et al., 1984, 1986). Much freeze injury is due to freeze-induced dehydration (Levitt, 1980, p. 94). As water becomes less available within the cell during, membranes lose the water which had formed bipolar bridges between phosphate residues of phospholipids (Crowe et al., 1984). The hydroxyl moieties of trehalose and sucrose are believed to take the place of water in membranes during dehydration, maintaining the integrity of the membranes and, therefore, proper compartmentation of enzymes and solutes within the cell. The most common injury observed in Gi. margarita spores which did not survive was the loss of internal integrity of the spore. The many small lipid droplets of the spores coalesced into what appeared to be a large vacuole, suggesting injury to membranes. Indeed, trehalose solutions yielded the greatest survival of spores of Gi. margarita of all cryoprotectants studied. Arredondo fine sand soil reached equilibrium moisture content with the air at % H2O, a xjf^ of approximately 41 MPa. A solution of glycerol at 13 % (w/v) would produce that same water potential, yet did not serve as a cryoprotectant. Evidently, more than dehydration is occurring as spores are exposed to slowly drying soil which prepares them for. Perhaps metabolic processes which result in the maintenance of the fluidity of membranes, such as increased unsaturation of fatty acids (Levitt, 1980, p. 196), increase in phospholipids (Vigh et al., 1986; Borochov et al., 1987; Lynch & Steponkus, 1987), the maintenance of permeability to water (Mazur, 1969; Levitt, 1980, p. 126), and lipid-sugar interactions (Caflfrey, Fonseca & Leopold, 1988) occur as the soil slowly dries, but not in osmotically active cryoprotectants. ACKNOWLEDGEMENTS This research was supported by National Science Foundation Grant No. BSR We would like to thank David M. Hubbell and James W. Kimbrough for their review of this manuscript. REFERENCES BOROCHOV, A., WALKER, M. A., KENDALL, E. J., PAULS, K. P. & MCKERSIE, B. D. (1987). Effect of a freeze-thaw cycle on properties of microsomal membranes of wheat. Plant Physiology 84, CAFFREY, M., FONSECA, V. & LEOPOLD, A. C. (1988). Lipid-sugar interactions. Relevance to anhydrous biology. Plant Physiology 86, CROWE, J. H., CROWE, L. M. & CHAPMAN, D. (1984). Infrared spectroscopic studies on interactions of water and carbohydrates with a biological membrane. Archives of Biochemistry and Biophysics 232, 400^07. CROWE, L. M., MOURADIAN, R., CROWE, J. H., JACKSON, S. A. & WoMERSLEY, C. (1984). Effects of carbohydrates on membrane stability at low water activities. Biochimica Biophysica Acta 769, CROWE, L. M., WOMERSLEY, C, CROWE, J. H., REID, D., APPEL, L. & RUDOLPH, A. (1986). Prevention of fusion and leakage in freeze-dried liposomes by carbohydrates. Biochimica Biophysica Acta 861, DAFT, M. J., SPENCER, D. & THOMAS, G. E. (1987). Infectivity of vesicular-arbuscular mycorrhizal inocula after storage under various environmental conditions. Transactions of the British Mycological Society 88, DALPE, Y. (1987). Spore viability of some Endogonaceae submitted to a single stage lyophilisation. In: Proceedings of the Seventh North American Conference on Mycorrhiza (Ed. by D. M. Sylvia. L. L. Hung & J. H. Graham), p May Gainesville, FL, USA. FENNELL, D. I. (1960). Conservation of fungous cultures. Botanical Review 26, FERGUSON, J. J. & WOODHEAD, S. H. (1982). Production of endomycorrhizal inoculum. A. Increase and maintenance of vesicular arbuscular mycorrhizal fungi. In: Methods and Principles of Mycorrhizal Research (Ed. by N. C. Schenck), pp American Phytopathological Society, St Paul, MN, USA. GERDEMANN, J. W. & NICHOLSON, T. H. (1963). Spores of mycorrhizal Endogone species extracted by wet sieving and decanting. Transactions of the British Mycological Society 46, JENKINS, W. R. (1964). A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48, 692. LEVITT, J. (1980). Responses of Plants to Environmental Stresses, vol. I, Chilling, Freezing, and High Temperature Stresses. Academic Press, New York, NY USA.

8 674 D. D. Douds and N. C. Schenck LYNCH, D. V. & STEPONKUS, P. L. (1987). Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secate cereale L. cv. Puma). Plant Physiology 83, MAZUR P. (1969). Freezing Injury in Plants. Annual Revievi of Plant Physiology 20, 419^M5. MUGNIER, J. & MOSSE, B. (1987). Spore germination and viability of a vesicular-arbuscular mycorrhizal fungus, Glomus mosseae. Transactions of the British Mycological Society 88, SCHENCK, N. C. (1987). The International Culture Collection of VA Mycorrhizal Fungi (INVAM). Angewandte Botanik 61, SIQUEIRA, J. D., SYLVIA, D. M., GIBSON, J. & HUBELL, D. H. (1985). Spores, germination, and germ tubes of vesiculararbuscular mycorrhizal fungi. Canadian Journal of Microbiology 31, SYLVIA, D. M. (1984). Production of inocula of VA mycorrhizal fungi. In: Applications of Mycorrhizal Fungi in Crop Production (Ed. by J. J. Ferguson), pp University of Florida, Gainesville, FL. TOMMERUP, I. C. (1988). Long-term preservation by L-drying and storage of vesicular-arbuscular mycorrhizal fungi. Transactions of the British Mycological Society 90, ToMMEHUP, I. C. & BETT, K. B. (1985). Cryopreservation of genotypes of VA mycorrhizal fungi. In: Proceedings of the Sixth North American Conference on Mycorrhiza. (Ed. by R. Molina), p. 235, June, Bend, OR, USA. TuiTE, J. (1969). Plant Pathological Methods : Fungi and Bacteria. Burgess Publishing Co., Minneapolis, MN, USA. ViGH, L., HUITEMA, H., WOLTJES, J. & VAN HASSELT, P. R. (1986). Drought stress-induced changes in the composition and physical state of phospholipids in wheat. Plant Physiology 77, YELENOSKY, G. & GUY, C. L. (1989). Freezing tolerance of citrus, spinach, and petunia leaf tissue. Osmotic adjustment and sensitivity to freeze induced cellular dehydration. Plant Physiology 89,

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