Preservation of Spores of Vesicular-Arbuscular Endophytes by L-Drying

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1979, p. 831-835 0099-2240/79/05-0831/05$02.00/0 Vol. 37, No. 5 Preservation of Spores of Vesicular-Arbuscular Endophytes by L-Drying INEZ C.

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1979, p /79/ /05$02.00/0 Vol. 37, No. 5 Preservation of Spores of Vesicular-Arbuscular Endophytes by L-Drying INEZ C. TOMMERUP* AND DENIS K. KIDBY Department of Soil Science and Plant Nutrition, Institute of Agriculture, University of Western Australia, Nedlands 6009, Australia Received for publication 22 February 1979 The spores of four species of vesicular-arbuscular endophytes were L-dried at 22 C, and their viability was tested after heating at 80 C for up to 40 min. L- drying of spores in the soil in which they developed was a very effective method of preservation of all spore types examined. Slow L-drying of spores separated from soil and supported on glass fiber filters also gave high viability for spores of some species. A scheme for the long-term preservation of vesicular-arbuscular endophyte spores is proposed. Vesicular-arbuscular (VA) endophytes are biotrophic fungi which form mycorrhizas within plant roots. VA mycorrhizal roots increase the uptake of nutrients, particularly phosphorus, by plants (13). Despite the recent increased interest in VA mycorrhizas, there is almost no information on methods for their long-term preservation. Microbes have inherent genetic instability (11); therefore, development of techniques for preserving genotypes deserves high priority. Although no method ensures complete preservation of genotypes, long-term preservation can be achieved by storage of microbes after lyophilizing, freezing, or L-drying (2, 11, 12). Fungi which grow readily in axenic culture are frequently preserved under mineral oil by air drying, lyophilizing, or freezing (14) after laboratory culture, but VA endophytes have not yet been grown without roots (13). As a method of preservation, the advantages of L-drying in comparison with lyophilization and freezing have been recently discussed (11). Lyophilization of mycorrhizal roots is possible (10, 15), although low viability of the endophytes was observed (5). The present study investigated L-drying procedures (2-4) for the routine preservation of VA endophyte spores. An accelerated storage test (7, 8, 15) was used to give an estimation of the potential for long-term survival of dried spores. MATERIALS AND METHODS Preparation of spores. Spores of Glomus caledonius (Nicol. & Gerd.) Trappe and Gerdemann (6) and Acaulospora laevis Gerdemann and Trappe (6) were isolated from soil under an annual clover and grass pasture. Glomus monosporus Gerdemann and Trappe (6) and a species of Gigaspora which resembled G. calospora (Nicol. & Gerd.) Gerdemann and Trappe (6) were grown in pot culture by L. K. Abbott, using the methods described for G. monosporus (1). All spores used in these experiments formed on plants grown for 6 months. Soil containing spores was air dried to 0.5 to 1.0% moisture content and stored at 22 C. In the experiment described, mature spores which had been stored in soil for 5 to 12 months were used. Spores were L-dried either in the soil in which they had grown or after separation from soil. Spores were separated from soil by wet sieving, sedimentation under kaolinite, and flotation in 50% (wt/vol) sucrose. A sample of 50 g of soil was stirred in 150 ml of water and immediately decanted and wet sieved through a stack of three sieves of mesh sizes 2 mm, 250,im, and 100,um. This procedure of stirring and decanting was repeated 15 to 20 times. The stack of sieves was then washed with a strong jet of water. The fraction retained on the 100-tim sieve was washed into a 50-ml, thick (3 mm)-walled Pyrex centrifuge tube, shaken with 2 g of powdered kaolinite (particle size, <130 tm), and centrifuged at 72 m s-2 for 5 min. The kaolinite compacted the pellet containing spores and soil particles. After removal of the supernatant, the pellet was dispersed in 50% aqueous sucrose (J. C. Sutton, personal communication) and centrifuged at 72 m s-2 for 45 s, and the supernatant containing the spores was filtered under vacuum onto Whatman no. 540 filter paper. Spores were washed and transferred onto a Whatman GF/A glass fiber filter pad, from which they could be readily isolated from adhering debris by using the tip of a fine needle. Spores were transferred to strips of glass fiber filter pads (5 by 2 mm) and placed in ampoules. b-drying. The drying manifold used has been described elsewhere as the vertical manifold (4). Either 0.75 g of soil containing spores or a segment of glass fiber filter pad bearing spores was placed in each 2.5- ml glass ampoule. The ampoules were plugged with absorbent cotton wool half-way down their length, dried at 22 C over P205 by evacuating to 0.01 torr, removed from the manifold and constricted to a volume of about 1 ml, re-evacuated at 22 C over P205 to 0.01 torr, and sealed in vacuo at that pressure. 331

2 832 TOMMERUP AND KIDBY APPL. ENVIRON. MICROBIOL. Accelerated storage test. Sealed ampoules were heated in a water bath at 80 C for up to 40 min. Immediately after heating, ampoules were cooled to 20 C, the spores were removed, and their viability was tested. Viability test. Spores were sandwiched between a pair of 0.45-,m membrane filters (Millipore Corp.) which was inserted between 1-cm layers of steamed (1) Lancelin sand with nutrients added (9). The soil was wet to 80% of field capacity. In nutritional studies, this unsterile soil solution was the medium giving the highest percentage of germination and the greatest germ tube elongation (I. C. Tommerup, unpublished data). After 3 weeks of incubation at 22 C in a 200-ml, lidded plastic container, each pair of filters was recovered, and the spores and hyphae on the filters were stained with trypan blue. The percentage of germination and germ tube lengths of spores were recorded. Only spores that had germinated were considered to be viable. Experimental. Spores were dried by one of several regimes, and their viability was tested. Soil containing spores was dried at 22 C in one of two ways. (i) Soil was air dried for 5 to 12 months and then L-dried (Table 1, A). (ii) Soil was air dried for 5 to 12 months and then dried over silica for 21 days to 0.2 to 0.4% water content and L-dried (Table 1, B). After L-drying spores were subjected to the accelerated storage test and separated from soil by suspension in 50% sucrose followed by filtration onto a 0.45-ltm Millipore filter, and their viability was tested. The viability of spores in soil which had not been L-dried was also tested. Spores separated from soil were dried at 22 C on segments of glass fiber filter pads by one of the following regimes. (i) The spores in unplugged ampoules were air dried for 1 day and then L-dried (Table 2, A). (ii) Spores in either plugged ampoules (Table 2, B, i) TABLE 1. or unplugged ampoules (Table 2, B, ii) were dried over silica for 21 days and then L-dried. (iii) Spores in unplugged ampoules were dried over silica for 1 to 14 days (Table 3). (iv) Spores, dried as in (iii), were further dried over P205 by evacuation to 0.01 torr, i.e., the first stage of L-drying (Table 3). (v) Spores, dried as in (iv), were dried again over P205 to 0.01 torr and sealed in vacuo, i.e., L-dried. Only L-dried spores were subjected to the accelerated storage test. After a drying treatment spores were transferred directly from an ampoule to a Millipore filter, and their viability was tested. The viability of spores which had not been dried after separation from soil was also tested. RESULTS Spores of all four species of VA endophytes L- dried in soil which was previously air dried or dried over silica had high viability. The viability of the L-dried spores heated at 800C for up to 40 min was also high (Table 1). L-drying spores which had been separated from soil and air dried on glass fiber filter pads for 1 day resulted in low viability for A. laevis and zero viability for G. caledonius (Table 2, A). However, the viability of L-dried spores of both endophytes dried on glass fiber filter pads over silica for 21 days before L-drying was high (Table 2, B). The viability of spores in the ampoules heated at 800C was also high. To slow the rate of drying, one set of ampoules was plugged during drying over silica. The plugging treatment did not affect the viability of spores when they were L-dried. To determine the period of drying over silica required to give high viability of spores when L-dried, the viability of spores during each phase of the drying process was examined in a timecourse experiment (Table 3). Drying spores on glass fiber filter pads over silica did not affect the viability of G. caledonius, A. laevis, or G. monosporus but reduced the viability of Gigaspora sp. Drying for 1 to 3 days over silica before drying once over P205 or L-drying greatly reduced the viability of spores of G. caledonius but not those of A. laevis. With increased time of drying over silica, up to 11 days, the viability of spores of G. caledonius after L- drying and heating increased. L-dried spores of G. caledonius which had been dried over silica Viability of spores after L-drying in soil which had been air dried or air dried and dried over silica Storage % Germination' of spores Method of drying Endophyte spores Before L After L-drying (time of heating at 80 C) (mo) drying 0 min 5 min 10 min 20 min 40 min (A) Air dried G. caledonius ± 5 75 ± ± 0 87 ± ± 21 -b beforel- A. laevis 5 95±4 95±3 100±0 100±0 100±0 100±0 drying (B) Dried over G. caledonius ± ± ± ± ± ± 0 siica A. laevis ± ± 0 65 ± 6 83 ± ± ± 21 before L- G. monosporus 8 89 ± ± ± 0-67 ± ± 0 drying Gigaspora sp ± ± ± ± 0 Values are means with standard deviations of 5 or more replicates of 5 to 13 spores. b-, Inadequate recovery of spores from each ampoule.

3 VOL. 37, 1979 TABLE 2. Method of drying L-DRYING OF VA ENDOPHYTE SPORES 833 Viability of spores L-dried after air drying or drying over silica on glass fiber filter pads Endophyte % Germination' of spores Storage Before After L-drying (time of heating at 80 C) of spores air- drying or (mo) drying 0 min 5 min 10 min 20 min 40 min over silica (A) Air dried for 1 day G. caledonius ± before L-drying A. laevis 5 95 ± 4 19 ± ± 2 21 ± ± ± 18 (B) Dried over silica for 21 days before L-drying (i) Ampoules G. caledonius ± ± ± ± ± ± 21 plugged during A. laevis 5 71 ± ± 12 NDb ND ND ND drying over silica (ii) Ampoules not G. caledonius ± ± ± ± ± 14 ND plugged during A. laevis 5 97 ± 6 89 ± ± ± ± ± 8 drying over silica a Values are means with standard deviations of 4 or more replicates of 10 spores. b ND, Not determined. for 11 days or longer had high viability both before and after heating at 80 C for 40 min. Spores of G. monosporus dried on glass fiber filter pads over silica survived each phase of the L-drying process. That few L-dried spores of G. monosporus survived the heating treatment may be due to the low viability of the batch of spores. In some samples, spores of Gigaspora sp. survived L-drying but not the heating treatment. The results suggest that if spores survived drying over silica, then they also survived drying over P205 and L-drying. Spores of A. laevis required 2 days, G. caledonius 11 days, and G. monosporus and Gigaspora sp. probably 8 to 14 days of drying over silica to give high viability after L-drying. In all experiments, when spores germinated after drying and heating treatments the lengths of the germ tubes were the same as those of spores which had not been dried. Therefore, the effect of these treatments was on the ability of the spores to germinate and not on outgrowth. DISCUSSION A recommended scheme for the long-term preservation of spores of VA endophytes is outlined in Fig. 1. This scheme incorporates the protocol used to successfully L-dry spores of VA endophytes, the test used to determine their potential for long-term storage, and the recommendation that L-dried spores be stored at low temperatures. Spores of VA endophytes have shown high viability after L-drying; however, the capacity of the spores to survive was affected by several factors. Both the physical and chemical forms of the medium in which microorganisms are dried influence their viability during and after drying (4, 8, 12, 16). Of the two media used in this study, soil gave high viability for spores of all endophytes after L-drying and after heat treatments. Although preservation of spores in soil having low spore numbers was inefficient, the protective properties of soil make it useful for spores having low viability when L-dried after separation from soil. The age of VA endophyte spores may affect their survival during and after L-drying. Compared to spores stored for 3 months, spores stored for 12 months require additional nutrients for germination and outgrowth (Tommerup, unpublished data). Such differences may be accentuated by L-drying. After L-drying, the variation in viability of spores stored for 5 months was less than for spores stored for 12 months. In the absence of further information, it is recommended that young rather than old spores be preserved. The rate of drying of spores on glass fiber filter pads significantly influenced their viability. Rapid drying over P205 of spores which had been air dried or dried over silica for 1 day gave low viability, and few of the spores which survived L-drying were viable after heating at 80 C. Longer and therefore slower drying of spores over silica before drying over P205 gave high viability. The requirement for a period

4 834 TOMMERUP AND KIDBY APPL. ENVIRON. MICROBIOL. TABLE 3. Effect of three drying treatments on the viability of spores on glass fiber filter padsa % Germinationb of spores Dried over silica After L-drying (time of heating at Endophyte Time (days) Before drying Dried over silica and once 80 C) over silica over P205 0 mm 40mm G. caledonius 0 96 ± ± ±8 21±30 7± NDc 6± ND ND ND ± ND 67 ± A. laevis ±3 56±41 31±40 17± ±17 40±31 42±34 45± ±42 61±15 72±25 52± ±27 55±10 49±31 55± ±3 71±19 85±23 53± ±17 58±44 52±42 40± ±28 63±13 47±25 55±24 G. monosporus 0 32 ± ± ± ±11 10±12 27±22 8± ± 7 49± 23 20± ±12 4±7 42±37 0 Gigaspora sp ±0 4 20± ±15 44± ± 14 8± ±7 0 a Spores of G. caledonius and A. laevis were stored for 12 months, and spores of G. monosporus and Gigaspora sp. were stored for 8 months. b Values are means with standard deviations of 4 or more replicates of 10 spores. c ND, Not determined. FIG. 1. Scheme for the slow L-drying of VA endophyte spores. of slow drying varied among the species of VA endophytes. The survival of microorganisms dried over P205, under vacuum, and stored in vacuo for extended periods has been correlated with their viability after incubation at elevated temperatures (7, 8, 15). When spores of VA endophytes dried slowly in soil or on glass fiber filter pads were L-dried, their viability was high in unheated ampoules and in ampoules heated at 80 C for up to 40 min. The high viability of these slowly L-dried spores after heating indicates a high potential stability of the spores and suggests a high potential for long-term survival. Residual water content of dried microorganisms sealed in vacuo affects their long-term survival and their stability as measured by accelerated storage tests (7, 8, 15, 16). The high heat stability of slowly L-dried endophyte spores suggests that the residual water content of the preparations may be close to the optimum. Storage of spores of VA endophytes for long

5 VOL. 37, 1979 L-DRYING OF VA ENDOPHYTE SPORES 835 periods has not yet been investigated. The capacity of a great range of dried microorganisms to remain viable is considerably higher at 0 to 4 C than at ambient temperatures (17 to 300C), and storage at 37 C causes a rapid reduction in viability (4, 8, 15, 16). The survival of L-dried spores of VA endophytes may be enhanced by storage at 0 to 40C. ACKNOWLEDGMENTS The technical help of Susan Birch, Teresa Hayes, and Nyet Fah Eagleton is warmly acknowledged. We thank A. D. Robson, J. P. Beilby, and L. K. Abbott for comments on the draft. This work was supported by the Rural Credits Development Fund of the Reserve Bank of Australia, the Australian Meat Research Committee, and a Postdoctoral Research Fellowship from the University of Western Australia (I.C.T.). LITERATURE CITED 1. Abbott, L. K., and A. D. Robson Growth stimulation of subterranean clover with vesicular-arbuscular mycorrhizas. Aust. J. Agric. Res. 28: Annear, D. I The preservation of bacteria by drying in peptone plugs. J. Hyg. 54: Annear, D. I Observations on drying bacteria from the frozen and from the liquid state. Aust. J. Exp. Biol. 36: Annear, D. I Recoveries of bacteria after drying on cellulose fibres. Aust. J. Exp. Biol. 40: Crush, J. R., and A. C. Pattison Preliminary results on the production of vesicular-arbuscular mycorrhizal inoculum by freeze drying, p In F. E. Sanders, B. Mosse, and P. B. Tinker (ed.), Endomycorrhizas. 6. Gerdemann, J. W., and J. M. Trappe The Endogonaceae in the Pacific North West. Mycol. Mem. 5: Greaves, R. I. N Some factors which influence the stability of freeze-dried cultures, p In A. S. Parkes and A. U. Smith (ed.), Recent research in freezing and drying. Blackwell Scientific Publications, Oxford. 8. Grieff, D., and W. A. Rightsel Stability of suspensions of influenza virus dried to different contents of residual moisture by sublimation in vacuo. Appl. Microbiol. 16: Hill, J., A. D. Robson, and J. F. Loneragan The effects of copper and nitrogen supply on the retranslocation of copper in four cultivars of wheat. Aust. J. Agric. Res. 29: Jackson, N. E., R. H. Miller, and R. E. Franklin The influence of vesicular-arbuscular mycorrhizae on uptake of 90Sr from soil by soybeans. Soil Biol. Biochem. 5: Kidby, D. K Culture maintenance and productivity, p In P. A. Sandford and A. Laskin (ed.), Extracellular microbial polysaccharides. American Chemical Society, Washington, D.C. 12. Lampage, S. P., J. E. Shelton, T. G. Mitchell, and A. R. MacKenzie Culture collections and the preservation of bacteria, p In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 3A. 13. Mosse, B Advances in the study of vesicular-arbuscular mycorrhiza. Annu. Rev. Phytopathol. 11: Onions, A. H. S Preservation of fungi, p In C. Booth (ed.), Methods in microbiology, vol Proom, H., and L. M. Hemmons The drying and preservation of bacterial cultures. J. Gen. Microbiol. 3: Scott, N. J A mechanism causing death during storage of dried micro-organisms, p In A. S. Parkes and A. U. Smith (ed.), Recent research in freezing and drying. Blackwell Scientific Publications, Oxford.

COMPONENTS OF VA MYCORRHIZAL INOCULUM AND THEIR EFFECTS ON GROWTH OF ONION

COMPONENTS OF VA MYCORRHIZAL INOCULUM AND THEIR EFFECTS ON GROWTH OF ONION New Phytol. (1981) 87, 3 5 5.161 355 OMPONENTS OF VA MYORRHIZAL INOULUM AND THEIR EFFETS ON GROWTH OF ONION BY A. MANJUNATH AND D. J. BAGYARAJ Depart?nent of Agricultural Microbiology, University of Agricultural