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Long-term viability and infectivity of intraradical forms of Glomus intraradices vesicles encapsulated in alginate beads
Mycol. Res. 107 (5): 614–616 (May 2003). f The British Mycological Society
614
DOI: 10.1017/S0953756203007482 Printed in the United Kingdom.
Long-term viability and infectivity of intraradical forms of Glomus intraradices vesicles encapsulated in alginate beads
Christian PLENCHETTE1 and De´sire´ G. STRULLU2 1
Institut Nationale de Recherche Agronomique (INRA), Unite´ de Malherbologie et Agronomie, 17 rue Sully, 21065 Dijon Cedex, France. 2 Universite´ d’Angers, 2 Bd Lavoisier, 49045 Angers Cedex, France. E-mail : [email protected] Received 19 September 2002; accepted 6 February 2003.
Intraradical vesicles of Glomus intraradices were isolated, entrapped in alginate beads, and stored at 4 xC for periods from 2–74 months. The beads were used to inoculate leeks grown under standard conditions for 6 wk, then development of root colonization by G. intraradices was recorded. Colonization of leeks was high (mean >50 % in length) and did not vary markedly until five years of storage. After six years of storage, the inoculum proved infective and viable.
INTRODUCTION Inoculum production of arbuscular fungi remains a matter for research in spite of the development of numerous pot culture methods using inert substrata (Plenchette, Furlan & Fortin 1982), hydroponic (Mosse & Thompson 1984), aeroponic cultures (Sylvia & Hubbel 1986, Hung & Sylvia 1988), or in vitro culture in association with excised roots, transformed or not by the Ri T-DNA of Agrobacterium rhizogenes (Mugnier & Mosse 1987). Few teams have directed their research on the in vitro culture of AM fungi, but this proved promising (Strullu & Plenchette 1990, 1991, Diop, Plenchette & Strullu 1994, Declerck et al. 1996). Whatever the inoculum production method proposed, questions arise as to the survival of axenic or non-axenic propagules (spores, vesicles or hyphae) and their long-term viability. External hyphae (Jasper, Abbott & Robson 1993, Addy, Miller & Peterson 1997) and spores (Tommerup 1984, Douds & Schenck 1990, Brundett & Abbott 1994) can survive environmental stress due to storage conditions. Alginate beads were proposed for the encapsulation of microorganisms more than 20 years ago (Dommergues, Diem & Dives 1979, Jung 1979). They were used for the inoculation of Rhizobium (Jung et al. 1982), of ectomycorrhizal fungi (Mauperin et al. 1987), or the dual inoculation of phosphate-solubilizing and nitrogen-fixing microorganisms (Vassilev et al. 2001). Immobilization procedures in alginate beads for AM root pieces (Strullu & Plenchette 1990), AM root pieces, spores, and hyphae (Ganry et al. 1985), and isolated vesicles and internal hyphae (Strullu
& Plenchette 1991), or in alginate films for spores (Calvet, Camprubi & Rodriguez-Kabana 1996), can preserve the ability of AM fungi to regrow hyphae. Storage at 4 xC for one month of alginate beads entrapping intraradical forms of Glomus intraradices did not limit hyphal regrowth and formation of mycorrhizas (Strullu & Plenchette 1991). We conducted experiments to determine the viability of such encapsulated material after several years of storage.
M A T E R I A L S, M E T H O D S A N D R E S U L T S Glomus intraradices (DAOM 181602) was chosen for our experiments, since it forms abundant internal vesicles. The original material was mycorrhizal leek (Allium porrum cv. ‘ Olaf ’) roots recovered from pot cultures on calcined clay (Plenchette & Perrin 1992) fed with a nutrient solution (Hewitt 1966). Selected mycorrhizal roots contained about 100 vesicles mmx1. Root pieces 4–5 cm in length were first surface-sterilized (Strullu & Romand 1986). Then 0.5 g of mycorrhizal roots were lysed overnight at 23 x (Strullu, Romand & Plenchette 1991) in 5 ml of the following enzymatic solution (pH/H2O=5.6) : 0.2 g lx1 Macerozyme R10 (Sigma, St Louis), 0.5 g lx1 Driselase (Yakult Honsha, Tokyo) and 1 g lx1 cellulase R10 (Sigma). Lysed root tissues were then washed in distilled water and homogenized for 2 min at 4500 rpm in a blender (Omni Mixer, Ivan Sorvall, Norwalk, CN). Intact vesicles and intraradical mycelium pieces were recovered by sieving through a 250 mm sieve which
C. Plenchette and D. G. Strullu
615
Table 1. Percentage mycorrhizal colonization of leeks ( % length and intensity), grown for 6 wk on calcined clay inoculated with 50 alginate beads bearing AM fungal vesicles, after different periods of storage at 4 xC. Replicates 1
2
3
4
Meansa
5
Storage (months)
L ( %)
I ( %)
L ( %)
I ( %)
L ( %)
I ( %)
L (%)
I ( %)
L ( %)
I ( %)
L (%)
I ( %)
2 4 41 50 62 74
88 86 96 67 84 9
42 28 62 40 39 40
96 91 99 75 88 21
37 22 52 45 36 20
96 63 98 83 84 5
38 30 62 60 38 20
94 71 88 73 98 25
38 22 56 65 45 28
86 78 97 72 78 6
35 24 49 35 24 26
92.0 a 77.8 bc 95.6 a 74.0 c 86.4 ab 13.2 d
38.0 b 25.2 d 56.2 a 49.0 a 36.4 bc 26.8 cd
a
Column numbers not followed by the same letters are significantly different (LSD, P>0.05).
retained most of the root debris. The vesicles were then suspended in a 20 g lx1 solution of sodium alginate (d=1.0046 ; viscosity=14 000 centipoises) and the mixture was dropped into a 0.1 M solution of calcium chloride through a needle using a peristaltic pump. The alginate beads, averaging 2 mm diam each contained about 30 vesicles. Although the enzymatic procedure leads to the obtaining of isolated vesicles as well as hyphae, we did not attempted to define the contribution of hyphae separately. The batch was separated into plastic vials, each containing 500 beads and 5 ml of autoclaved H2O, and stored at 4 x in the dark until used. Each experiment was conducted under the same conditions. Small plastic pots containing 200 ml of calcined clay (OIL-Dri, Chicago, ILL) were planted with a 4 wk plantlet of the leek cultivar and fed weekly with 20 ml of Long Ashton Solution (Hewitt 1966) diluted 10-fold to reduce the phosphorus concentration. Inoculation was at the time of pot-filling by placing beads 5 cm deep. Pots were placed in a growth chamber (16/ 8 h light/dark ; 22/18 x day/night and an average photon flux density of 220 mmoles sx1 mx2 ; 80 % r.h.). Five replicates were made for each experiment, which took place after 2, 4, 41, 50, 62 and 74 months of storage. After 6 wk, the root systems were collected, washed free of soil, and a sample cleared in 10 % KOH for 1 h at 90 x, before staining for 15 min with acid fuchsin (0.05 % in lactoglycerol). Infected root length percentages were evaluated using the gridline intersect method (Giovannetti & Mosse 1980). At each intersect, the abundance of internal vesicles as well as hyphae was noted, i.e. the intensity of the infection, was estimated visually using different classes, based on the apparent proportions of root volume occupied by the fungus (1–20 ; 20–40; 40–60; 60–80 ; 80–100%). Results were expressed as a percentage as follows : (20x+40y +60z+80t+100v)/(x+y+z+t+v), where x, y, z, t, v are the number of intersects in each class (Plenchette & Morel 1996). In all treatments corresponding to different periods of storage, beads containing isolated intraradical forms of G. intraradices produced new hyphae and initiated new mycorrhizas. The percentage of root length
colonized was always high until 62 months of storage (Table 1). Nevertheless, some significant variations occured. After a long period of storage (41 months) the level of colonization recovered was similar to the value at 2 months, when even the intensity of colonization was higher. After a long period of storage the percentage of infection (% length) was reduced, but it remained higher than 10% and the intensity of infection averaged 26 %. Such values are commonly encountered for plants growing in the field and colonized by indigenous fungi. DISCUSSION AND CONCLUSIONS Works on the survival of AM fungi after long-term storage were always conducted with external spores and/or hyphae. It had been shown that spores of AM fungi needed a critical cold-storage period to break their dormancy (Tommerup 1983, Gemma & Koske 1988, Juge et al. 2002). It seems that such physiological phenomenon do not arise with internal vesicles. For a long time and in different studies, we used fresh roots bearing internal vesicles, from different plants and colonized with different AM fungi (Plenchette et al. 1981, 1982, 1983, Duvert, Perrin & Plenchette 1999, Plenchette 2000). We always succeeded and obtained a welldeveloped AM infections on inoculated plants. Our previous results indicated that internal vesicles exhibit no dormancy, and our new results showed that 4 x cold storage does not reduce the percentage germination of internal vesicles. This is apparently not the case with spores, which need a particular cold storage period for germination, and whose viability decreases with the duration of storage (Mugnier & Mosse 1987, Kuszala, Gianinazzi & Gianinazzi-Pearson 2001). It is already a common laboratory practice to store biological materials at 4 x, and moreover the encapsulation of isolated vesicles in alginate beads is easy and does not require sophisticated equipment such as freeze-dryers (Dalpe´ 1987, Tommerup 1988). Our storage technique makes inoculation much easier, and facilitates the quantification and protection of the fungus during the germination period. Encapsulated vesicles of AM fungi are promising approach to developing large-scale field
Viability and infectivity of encapsulated Glomus intraradices inoculation experiment and also for the maintenance of AM fungal strains in genetic resource collections. ACKNOWLEDGEMENT We wish to thank Joseph A. Fortin for revision of the manuscript, and Josiane Mortier for technical assistance.
REFERENCES Addy, H. D., Miller, M. H. & Peterson, R. L. (1997) Infectivity of the propagules associated with extraradical mycelia of two AM fungi following winter freezing. New Phytologist 135 : 745–753. Brundett, M. C. & Abbott, L. K. (1994) Mycorrhizal fungus propagules in the jarrah forest. I. Seasonal study of inoculum levels. New Phytologist 127 : 539–546. Calvet, C., Camprubi, A. & Rodriguez-Kabana, R. (1996) Inclusion of arbuscular mycorrhizal fungi in alginate films for experimental studies and plant inoculation. HortScience 31: 285. Dalpe´, Y. (1987) Spore viability of some Endogonaceae submitted to a single stage lyophilisation. In Proceedings of the Seventh North American Conference on Mycorrhiza (D. M. Sylvia, L. L. Hung & J. H. Graham, eds): 279. University of Florida, Gainsville, FL. Declerck, S., Strullu, D., Plenchette, C. & Guillemette, T. (1996) Entrapment of in vitro produced spores of Glomus versiforme in alginate beads: in vitro and in vivo inoculum potentials. Journal of Biotechnology 48 : 51–57. Diem, H. G., Kalifa, B., Neyra, M. & Dommergues, Y. (1989) Recent advances in the inoculant technology with special emphasis on plant microorganisms. In Advanced Technologies for Increased Agricultural Production (U. Leone, G. Riadli & R. Vanore´, eds): 196–209. Universita` degli studi di Genova, Roma. Diop, T. A., Plenchette, C. & Strullu, D. G. (1994) In vitro culture of sheared mycorrhizal roots. Symbiosis 17: 217–227. Dommergues, Y., Diem, H. G. & Dives, C. (1979) Microbiological process for controlling the productivity of cultivated plants. [US patent 4, 153, 737.] Douds, D. D. & Schenck, N. C. (1990) Cryopreservation of spores of vesicular-arbuscular mycorrhizal fungi. New Phytologist 115: 667–674. Duvert, R., Perrin, R. & Plenchette, C. (1990) Soil receptiveness to V.A. mycorrhizal association: concept and method. Plant and Soil 124: 1–6. Ganry, F., Diem, H. G., Wey, J. & Dommergues, Y. R. (1985) Inoculation with Glomus mosseae improves N2 fixation by fieldgrown soybeans. Biology and Fertility of Soils 1 : 15–23. Gemma, J. & Koske, R. (1988) Seasonal variation in spores abundance and dormancy of Gigaspora gigantea and in mycorrhizal inoculum potential of a dune soil. Mycologia 80: 211–216. Giovannetti, M. & Mosse, B. (1980) An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist 84: 489–500. Hewitt, E. J. (1966) Sand and Water Culture Methods used in the Study of Plant Nutrition. [Technical communication No. 22] 2nd edn (revised). Commonwealth Agricultural Bureaux, Farnham Royal. Hung, L. L. & Sylvia, D. M. (1988) Production of vesicular-arbuscular mycorrhizal fungus inoculum in aeroponic culture. Applied and Environmental Microbiology 54: 353–357. Kuszala, C., Gianinazzi, S. & Gianinazzi-Pearson, V. (2001) Storage conditions for the long-term survival of AM fungal propagules in wet sieved soil fractions. Symbiosis 30 : 287–299. Jasper, D. A., Abbott, L. K. & Robson, A. D. (1993) The survival of infective hyphae of vesicular-arbuscular mycorrhizal fungi in dry soil: an interaction with sporulation. New Phytologist 124: 473– 479. Juge, C., Samson, J., Bastien, C., Vierheilig, H., Coughlan, A. & Piche´, Y. (2002) Breaking dormancy of spores of the arbuscular mycorrhizal fungus Glomus intraradices : a critical cold-storage period. Mycorrhiza 12 : 37– 42.
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Jung, G. (1979) Proce´de´ d’inclusion de microorganismes dans une matrice de polyme`re. [French patent 7, 908, 597.] Jung, G., Mugnier, J., Diem, H. G. & Dommergues, Y. (1982) Polymer-entrapped Rhizobium as an inoculant for legumes. Plant and Soil 65: 219–231. Mauperin, C., Mortier, F., Garbaye, J., Le Tacon, F. & Carr, G. (1987) Viability of an ectomycorrhizal inoculum produced in a liquid medium and entrapped in calcium alginate gels. Canadian Journal of Botany 65 : 2326–2329. Mosse, B. & Thompson, J. P. (1984) Vesicular-arbuscular endomycorrhizal inoculum production. I. Exploratory experiment with beans (Phaseolus vulgaris) in nutrient flow culture. Canadian Journal of Botany 62 : 1523–1530. Mugnier, J. & Mosse, B. (1987) Vesicular-arbuscular mycorrhizal infection in transformed root-inducing T-DNA grown axenically. Phytopathology 77: 1045–1050. Plenchette, C. (2000) Receptiveness of some tropical soils from banana fields of Martinique to the arbuscular fungus Glomus intraradices. Applied Soil Ecology 15: 35–36. Plenchette, C., Furlan, V. & Fortin, J. A. (1981) Growth stimulation of apple trees in unsterilized soil under filed conditions by endomycorrhizal inoculation. Canadian Journal of Botany 59 : 2003–2008. Plenchette, C., Furlan, V. & Fortin, J. A. (1982) Comparative effects of different endomycorrhizal fungi on five host plants grown on calcined montmorillonite clay. Journal of American Society for Horticultural Science 107: 535–538. Plenchette, C., Furlan, V. & Fortin, J. A. (1983) Responses of endomycorrhizal plants grown in a calcined montmorillonite clay to different levels of soluble phosphorus. I. Effect on growth and mycorrhizal development. Canadian Journal of Botany 61: 1377–1383. Plenchette, C. & Perrin, R. (1992) Evaluation in the greenhouse of the effects of fungicides on the development of mycorrhiza on leek and wheat. Mycorrhiza 1: 59–62. Plenchette, C. & Morel, C. (1996) External phosphorus requirement of mycorrhizal and non-mycorrhizal barley and soybean plants. Biology and Fertility of Soil 21 : 303–308. Strullu, D. G. & Plenchette, C. (1990) Encapsulation de la forme intraracinaire de Glomus dans l’alginate et utilisation des capsules comme inoculum. Comptes Rendus de l’Academie des Sciences, Series III 310: 447– 452. Strullu, D. G. & Romand, C. (1986) Me´thode d’obtention, d’endomycorhizes a` ve´sicules et arbuscules en conditions asce´niques. Comptes Rendus de l’Acade´mic des Sciences Se´rie 3. 303: 245– 250. Strullu, D. G., Romand, C. & Plenchette, C. (1991) Axenic culture and encapsulation of the intraradical forms of Glomus spp. World Journal of Microbiology and Biotechnology 7: 292–297. Strullu, D. G. & Plenchette, C. (1991) The entrapment of Glomus sp in alginate beads and their use as root inoculum. Mycological Research 95: 1194 –1196. Sylvia, D. M. & Hubbel, D. H. (1986) Growth and sporulation of vesicular-arbuscular mycorrhizal fungi in aeroponic and membrane systems. Symbiosis 1 : 259–267. Tommerup, I. (1983) Spore dormancy in vesicular-arbuscular mycorrhizal fungi. Transactions of the British Mycological Society 81: 37–45. Tommerup, I. (1984) Effect of soil water potential on spore germination by vesicular-arbuscular mycorrhizal fungi. New Phytologist 83: 193–202. Tommerup, I. (1988) Long term preservation by L-drying and storage of vesicular-arbuscular mycorrhizal fungi. Transactions of the British Mycological Society 90: 585–591. Vassilev, N., Vassileva, M., Azcon, R. & Medina, A. (2001) Interaction of an arbuscular mycorrhizal fungus with free or co-encapsulated cells of Rhizobium trifoli and Yarrowia lipolytica inoculated into a soil–plant system. Biotechnology Letters 23: 149–151.
Corresponding Editor: P. Bonfante
614
DOI: 10.1017/S0953756203007482 Printed in the United Kingdom.
Long-term viability and infectivity of intraradical forms of Glomus intraradices vesicles encapsulated in alginate beads
Christian PLENCHETTE1 and De´sire´ G. STRULLU2 1
Institut Nationale de Recherche Agronomique (INRA), Unite´ de Malherbologie et Agronomie, 17 rue Sully, 21065 Dijon Cedex, France. 2 Universite´ d’Angers, 2 Bd Lavoisier, 49045 Angers Cedex, France. E-mail : [email protected] Received 19 September 2002; accepted 6 February 2003.
Intraradical vesicles of Glomus intraradices were isolated, entrapped in alginate beads, and stored at 4 xC for periods from 2–74 months. The beads were used to inoculate leeks grown under standard conditions for 6 wk, then development of root colonization by G. intraradices was recorded. Colonization of leeks was high (mean >50 % in length) and did not vary markedly until five years of storage. After six years of storage, the inoculum proved infective and viable.
INTRODUCTION Inoculum production of arbuscular fungi remains a matter for research in spite of the development of numerous pot culture methods using inert substrata (Plenchette, Furlan & Fortin 1982), hydroponic (Mosse & Thompson 1984), aeroponic cultures (Sylvia & Hubbel 1986, Hung & Sylvia 1988), or in vitro culture in association with excised roots, transformed or not by the Ri T-DNA of Agrobacterium rhizogenes (Mugnier & Mosse 1987). Few teams have directed their research on the in vitro culture of AM fungi, but this proved promising (Strullu & Plenchette 1990, 1991, Diop, Plenchette & Strullu 1994, Declerck et al. 1996). Whatever the inoculum production method proposed, questions arise as to the survival of axenic or non-axenic propagules (spores, vesicles or hyphae) and their long-term viability. External hyphae (Jasper, Abbott & Robson 1993, Addy, Miller & Peterson 1997) and spores (Tommerup 1984, Douds & Schenck 1990, Brundett & Abbott 1994) can survive environmental stress due to storage conditions. Alginate beads were proposed for the encapsulation of microorganisms more than 20 years ago (Dommergues, Diem & Dives 1979, Jung 1979). They were used for the inoculation of Rhizobium (Jung et al. 1982), of ectomycorrhizal fungi (Mauperin et al. 1987), or the dual inoculation of phosphate-solubilizing and nitrogen-fixing microorganisms (Vassilev et al. 2001). Immobilization procedures in alginate beads for AM root pieces (Strullu & Plenchette 1990), AM root pieces, spores, and hyphae (Ganry et al. 1985), and isolated vesicles and internal hyphae (Strullu
& Plenchette 1991), or in alginate films for spores (Calvet, Camprubi & Rodriguez-Kabana 1996), can preserve the ability of AM fungi to regrow hyphae. Storage at 4 xC for one month of alginate beads entrapping intraradical forms of Glomus intraradices did not limit hyphal regrowth and formation of mycorrhizas (Strullu & Plenchette 1991). We conducted experiments to determine the viability of such encapsulated material after several years of storage.
M A T E R I A L S, M E T H O D S A N D R E S U L T S Glomus intraradices (DAOM 181602) was chosen for our experiments, since it forms abundant internal vesicles. The original material was mycorrhizal leek (Allium porrum cv. ‘ Olaf ’) roots recovered from pot cultures on calcined clay (Plenchette & Perrin 1992) fed with a nutrient solution (Hewitt 1966). Selected mycorrhizal roots contained about 100 vesicles mmx1. Root pieces 4–5 cm in length were first surface-sterilized (Strullu & Romand 1986). Then 0.5 g of mycorrhizal roots were lysed overnight at 23 x (Strullu, Romand & Plenchette 1991) in 5 ml of the following enzymatic solution (pH/H2O=5.6) : 0.2 g lx1 Macerozyme R10 (Sigma, St Louis), 0.5 g lx1 Driselase (Yakult Honsha, Tokyo) and 1 g lx1 cellulase R10 (Sigma). Lysed root tissues were then washed in distilled water and homogenized for 2 min at 4500 rpm in a blender (Omni Mixer, Ivan Sorvall, Norwalk, CN). Intact vesicles and intraradical mycelium pieces were recovered by sieving through a 250 mm sieve which
C. Plenchette and D. G. Strullu
615
Table 1. Percentage mycorrhizal colonization of leeks ( % length and intensity), grown for 6 wk on calcined clay inoculated with 50 alginate beads bearing AM fungal vesicles, after different periods of storage at 4 xC. Replicates 1
2
3
4
Meansa
5
Storage (months)
L ( %)
I ( %)
L ( %)
I ( %)
L ( %)
I ( %)
L (%)
I ( %)
L ( %)
I ( %)
L (%)
I ( %)
2 4 41 50 62 74
88 86 96 67 84 9
42 28 62 40 39 40
96 91 99 75 88 21
37 22 52 45 36 20
96 63 98 83 84 5
38 30 62 60 38 20
94 71 88 73 98 25
38 22 56 65 45 28
86 78 97 72 78 6
35 24 49 35 24 26
92.0 a 77.8 bc 95.6 a 74.0 c 86.4 ab 13.2 d
38.0 b 25.2 d 56.2 a 49.0 a 36.4 bc 26.8 cd
a
Column numbers not followed by the same letters are significantly different (LSD, P>0.05).
retained most of the root debris. The vesicles were then suspended in a 20 g lx1 solution of sodium alginate (d=1.0046 ; viscosity=14 000 centipoises) and the mixture was dropped into a 0.1 M solution of calcium chloride through a needle using a peristaltic pump. The alginate beads, averaging 2 mm diam each contained about 30 vesicles. Although the enzymatic procedure leads to the obtaining of isolated vesicles as well as hyphae, we did not attempted to define the contribution of hyphae separately. The batch was separated into plastic vials, each containing 500 beads and 5 ml of autoclaved H2O, and stored at 4 x in the dark until used. Each experiment was conducted under the same conditions. Small plastic pots containing 200 ml of calcined clay (OIL-Dri, Chicago, ILL) were planted with a 4 wk plantlet of the leek cultivar and fed weekly with 20 ml of Long Ashton Solution (Hewitt 1966) diluted 10-fold to reduce the phosphorus concentration. Inoculation was at the time of pot-filling by placing beads 5 cm deep. Pots were placed in a growth chamber (16/ 8 h light/dark ; 22/18 x day/night and an average photon flux density of 220 mmoles sx1 mx2 ; 80 % r.h.). Five replicates were made for each experiment, which took place after 2, 4, 41, 50, 62 and 74 months of storage. After 6 wk, the root systems were collected, washed free of soil, and a sample cleared in 10 % KOH for 1 h at 90 x, before staining for 15 min with acid fuchsin (0.05 % in lactoglycerol). Infected root length percentages were evaluated using the gridline intersect method (Giovannetti & Mosse 1980). At each intersect, the abundance of internal vesicles as well as hyphae was noted, i.e. the intensity of the infection, was estimated visually using different classes, based on the apparent proportions of root volume occupied by the fungus (1–20 ; 20–40; 40–60; 60–80 ; 80–100%). Results were expressed as a percentage as follows : (20x+40y +60z+80t+100v)/(x+y+z+t+v), where x, y, z, t, v are the number of intersects in each class (Plenchette & Morel 1996). In all treatments corresponding to different periods of storage, beads containing isolated intraradical forms of G. intraradices produced new hyphae and initiated new mycorrhizas. The percentage of root length
colonized was always high until 62 months of storage (Table 1). Nevertheless, some significant variations occured. After a long period of storage (41 months) the level of colonization recovered was similar to the value at 2 months, when even the intensity of colonization was higher. After a long period of storage the percentage of infection (% length) was reduced, but it remained higher than 10% and the intensity of infection averaged 26 %. Such values are commonly encountered for plants growing in the field and colonized by indigenous fungi. DISCUSSION AND CONCLUSIONS Works on the survival of AM fungi after long-term storage were always conducted with external spores and/or hyphae. It had been shown that spores of AM fungi needed a critical cold-storage period to break their dormancy (Tommerup 1983, Gemma & Koske 1988, Juge et al. 2002). It seems that such physiological phenomenon do not arise with internal vesicles. For a long time and in different studies, we used fresh roots bearing internal vesicles, from different plants and colonized with different AM fungi (Plenchette et al. 1981, 1982, 1983, Duvert, Perrin & Plenchette 1999, Plenchette 2000). We always succeeded and obtained a welldeveloped AM infections on inoculated plants. Our previous results indicated that internal vesicles exhibit no dormancy, and our new results showed that 4 x cold storage does not reduce the percentage germination of internal vesicles. This is apparently not the case with spores, which need a particular cold storage period for germination, and whose viability decreases with the duration of storage (Mugnier & Mosse 1987, Kuszala, Gianinazzi & Gianinazzi-Pearson 2001). It is already a common laboratory practice to store biological materials at 4 x, and moreover the encapsulation of isolated vesicles in alginate beads is easy and does not require sophisticated equipment such as freeze-dryers (Dalpe´ 1987, Tommerup 1988). Our storage technique makes inoculation much easier, and facilitates the quantification and protection of the fungus during the germination period. Encapsulated vesicles of AM fungi are promising approach to developing large-scale field
Viability and infectivity of encapsulated Glomus intraradices inoculation experiment and also for the maintenance of AM fungal strains in genetic resource collections. ACKNOWLEDGEMENT We wish to thank Joseph A. Fortin for revision of the manuscript, and Josiane Mortier for technical assistance.
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