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The entrapment of Glomus sp. in alginate beads and their use as root inoculum
Mycol. Res. 95 (10): 1194-1196 (1991)
Printed in Great Britain
1194
The entrapment of Glomus sp. in alginate beads and their use as root inoculum
D. G. STRULLU Universite' d'Angers, Lahoratoire de Phytonique, 2 hd Lavoisier 49045 Angers, France
C. PLENCHETTE 1.N.R.A. Station d'Agronomie, 17 rue Sully, 21034 Dijon Cedex, France
Intraradical forms of Glomus sp. (vesicles and mycelium fragments) were entrapped in alginate and used as inoculum. Isolated intraradical material was found to regenerate in alginate beads and the regenerated mycelium infected roots under controlled conditions. Storage at 4 °C for one month did not limit the hyphal regeneration and the formation of mycorrhizas.
Producing inoculum of vesicular-arbuscular mycorrhizal (VAM) fungi and assessing its use in field and greenhouse conditions is important in view of the well known benefits of such fungi on uptake of mineral nutrients and, possibly, the water relations and photosynthesis levels of plants (Harley & Smith, 1983). Moreover, VAM fungi reduce the susceptibility of their host to certain pathogens (Dehne, 1982). In the past it has been difficult to obtain large amounts of VAM inoculants for agronomic use. These obligate symbionts can be grown from various propagules but their spores produce mycelium with limited independent growth (Hepper, 1984). Mycelia regenerated in pure culture from intraradical vesicles have the ability to infect root cortices (Strullu & Romand, 1987). Ectomycorrhizal fungi, in contrast, can now be produced in a fermentor and by entrapping the mycelium in alginate beads, several workers (Le Tacon ef al., 1985; Mauperin et aI., 1987) have produced inoculum for use in forestry. Recently, entrapment has emerged as a tool for the production of inoculants of VAM fungi. Immobilization procedures can preserve the physiological properties of mycorrhizal roots, promote the regeneration of mycelium and the formation of mycorrhizas (Strullu & PlencheHe, 1990). We conducted experiments to ascertain whether entrapment inside alginate gels can also stabilize the infectivity of a VAM fungus isolated in its intraradical forms i.e. vesicles or interand intracellular mycelial fragments. Glomus sp. 3 (deposited in Canada Department of Agriculture Herbarium, DAOM 181602) was used for the experiments and mycorrhizal roots were recovered from pot cultures of leek (Allium porrum L. cv. Olaf) grown on calcined clay and fed with a nutrient solution (PlencheHe, Furlan & Fortin, 1982). The selected mycorrhizal roots contained about 100 vesicles mm- 1 (Fig. 1). Two forms of inoculum were obtained.
(a) Root pieces: roots were surface sterilized as previously described (Strullu & Romand, 1986) and cut in 1-3 mm pieces by hand under a 10 x microscope. (b) Isolated vesicles: 0'5 g of mycorrhizal roots were lysed overnight at 23° in 5 ml of the following enzymatic solution (pH/H 2 0 = 5'6): 0'2 g 1-1 Macerozyme RIO, 0'5 g 1-1 Driselase (Yakult Honsha Co. Ltd) and 1 g 1-1 cellulase RIO. Lysed root tissues were then washed in distilled water and homogenized for two minutes at 4500 rpm in a blender (Omni mixer, Ivan Sorvall Inc). Intact vesicles and intraradical mycelium were recovered by passage through a 250 I-lm sieve which retained most of the root debris. Material of each of the two types of inoculum was suspended in a 20 g 1-1 solution of sodium alginate (d = 1'0046; viscosity = 14000 centipoises) and the mixture was dropped into a 0'1 M solution of calcium chloride through a needle, using a peristaltic pump. This process entraps vesicles, mycelium or small root fragments in calcium alginate beads averaging 2 mm in diameter (Figs 2, 3, 4). It is possible to obtain about 20000 beads containing 30 vesicles or 500 beads containing 3-5 fragments of 1-3 mm from one gram of selected mycorrhizal roots. Four-week old plantlets of leek (Allium porrum L. cv. Olaf) were prepared in pots containing 200 ml of calcined clay (Plenchette ef aI., 1982) and fed weekly with 20 ml of nutrient solution (Hewitt, 1966). Each experiment consisted of three inoculation treatments: (a) control without beads; (b) plants inoculated with 50 beads each containing 3-5 root pieces; (c) plants inoculated with 50 beads containing 30 isolated vesicles. In experiment 1 the beads were prepared immediately
D. G. Strullu and C. Plenchette
1195
Fig. 1. Mycorrhizal roots showing vesicles of Glomus sp. 3 after staining by fuschin acid. Scale bar = 5000 ~m. Fig. 2 (Dark field microscopy) and 3. Optical micrographs of alginate beads containing vesicles (v), mycelia (my) and root cell fragments (*). Scale bar = 500 ~m. Fig. 4. Vesicles (v) immobilized in alginate gel. Scale bar = 150 ~m.
before the experiment and in experiment 2, they were stored for 1 month at 4° before use as inoculum. In both experiments beads were stratified 5 em deep in the substrate of each pot. Pots were placed in a growth chamber (22°/18°, 16 h day at 220 \-lmole S-l m- 2, 80% r.h.). Plants were harvested and their root systems were collected after
two months for experiment 1 and one month for experiment 2, for staining and determination of mycorrhizal infection using the method of Giovannetti & Mosse (1980). The results are summarized in Table 1. Non-inoculated plants did not develop mycorrhizas, but plants inoculated with beads containing mycorrhizal root pieces or isolated intra-
VAM inoculum in alginate beads
1196
Table 1. Results of inoculation of leek plantlets (5 replicates) with alginate beads (i) used immediately after their preparation and (ii) after one month storage at 4 0
Treatment
Proportion of mycorrhizal infection of roots (%)
The authors wish to thank Dr J. A. Fortin for critically reading the manuscript and improving the English.
(0
Control Beads with infected root pieces Beads with vesicles
0 57
0 II
0 51
0 7
0 20
12
7
34
15
51
0 82
0 5
0 55
0 64
0 43
2
70
50
82
66
(ii)
Control Beads with infected root pieces Beads with vesicles
mycelium can infect roots under controlled conditions. We can now consider the possibility of producing high quality inoculum for field experiments.
radical material all developed mycorrhizal associations. In both experiments, there was no Significant difference (Student's t-test after arc sin transformation) between the infection obtained with beads containing root fragments and that obtained with beads containing isolated vesicles and mycelium. Furthermore, fungal structures immobilized in alginate capsules did not lose their ability to infect root after storage at 4° for one month. Regeneration is a major physiological process for vesicles; so it is important to know whether the immobilization in alginate permits isolated intraradical forms of Glomus sp. to produce new hyphae and mycorrhizas. Previous results concerning saprophytic growth of VAM fungi have been reviewed by Harley & Smith (1983). Regrowth of hyphae from dead re-wetted roots (Tommerup & Abbott, 1981) and fungal regrowth in sterilized soil (Hepper & Warner, 1983) suggest that under natural conditions VAM fungi have some saprophytic ability. Moreover mycorrhizal root fragments proved to be useful and infective inoculum. Mosse (1988) reported that hyphae of Glomus intraradices, regenerated from roots, grew vigorously in dual culture with carrot roots without infection. In contrast, vesicles of Glomus caledonium yielded negligible fungal growth and had poor infection capacity (Burggraaf & Beringer, 1989). Strullu et al. (1989) suggested that calcium may be important for the regeneration of intraradical mycelium. In our experiments, the final concentration in alginate beads capsules was 10 mM Ca 2 +, similar to amounts found in most culture media used for VAM fungi (Hepper, 1984). These results indicate that isolated intraradical forms of Glomus sp. can grow in alginate beads and that the regenerated (Received for publication 5 December 1990 and in revised form 12 March 1991)
REFERENCES Burggraaf. A. j. P. &; Beringer, j. E. (1989). Absence of nuclear DNA synthesis in vesicular-arbuscular mycorrhizal fungi during in vitro development. New Phytologist 11. 25-33. Dehne, H. W. (1982). Interaction between vesicular arbuscular mycorrhizal fungi and plant pathogens. Phytopathology 72, II 15-II 18. Giovannetti, M. &; Mosse, B. (1980). An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist 84. 489-500. Harley, j. L. &; Smith, S. E. (1983). Mycorrhizal symbiosis. London, U.K.: Academic Press. Hepper, C M. (1984). Isolation and culture of vesicular-arbuscular mycorrhizal (VAM) fungi. In VA mycorrhiza (ed. C L. Powell &; D. j. Bagyaraj), pp. 95-Il2. Boca Raton, Florida, U.s.A.: CRC Press. Hepper, eM. &; Warner, A. (1983). Role of organic matter in growth of a vesicular arbuscular mycorrhizal fungus in soil. Transaction of the British Mycological Society 81, 155-156. Hewitt, E. ]. (1966). Sand and water culture methods used in the study of plant nutrition. In Technical Communication no. 22, 2nd edn revised, pp. 430--434. Commonwealth Agricultural Bureaux, London, U.K. Le Tacon, F., jung, G., Mugnier, )., Michelot, P. &; Mauperin, C (1985). Efficiency in a forest nursery of an ectomycorrhizal inoculum produced in a fermentor and entrapped in polymeric gels. Canadian Journal of Botany 63, 1664-1668. 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. (1988). Some studies relating to independent growth of vesiculararbuscular endophytes. Canadian Journal of Botany 66, 2533-2540. Plenchette, c.. Furlan, V. &; Fortin, j. A. (1982). Effects of different endomycorrhizal fungi on five host plants grown on calcined montmorillonite clay. Journal of American Society for horticultural Science 107, 535-538. Strullu, D. G. &; Plenchette, C (1990). Encapsulation de la forme intraracinaire de Glomus dans l'alginate et utilisation des capsules camme inoculum. Comptes Rendus de I'Academie des Sciences, Ser. III, 310, 447-452. Strullu, D. G. &; Romand, C (1986). Methode d'obtenbon d'endomycorhizes 11 vesicules et arbuscules en conditions axeniques. Comptes Rendus de I'Academie des Sciences 303, 245-250. Strullu, D. G. &; Romand, C (1987) Culture axenique de vesicules isolees 11 partir d'endomycorhizes et re-association in vitro 11 des racines de tomates. Comptes Rendus de [,Academie des Sciences 305, 15-19. Strullu, D. G., Romand, C, Callac, P., Teoule, E. &; Demarly, Y. (1989). Mycorrhizal synthesis in vitro between Glomus spp. and artificial seeds of alfalfa. New Phytologist 113, 545-548. Tommerup, I. C &; Abbott, L. K. (1981). Prolonged survival and viability of VA mycorrhizal hyphae after root death. Soil and Biochemistry 13, 431-433.
Printed in Great Britain
1194
The entrapment of Glomus sp. in alginate beads and their use as root inoculum
D. G. STRULLU Universite' d'Angers, Lahoratoire de Phytonique, 2 hd Lavoisier 49045 Angers, France
C. PLENCHETTE 1.N.R.A. Station d'Agronomie, 17 rue Sully, 21034 Dijon Cedex, France
Intraradical forms of Glomus sp. (vesicles and mycelium fragments) were entrapped in alginate and used as inoculum. Isolated intraradical material was found to regenerate in alginate beads and the regenerated mycelium infected roots under controlled conditions. Storage at 4 °C for one month did not limit the hyphal regeneration and the formation of mycorrhizas.
Producing inoculum of vesicular-arbuscular mycorrhizal (VAM) fungi and assessing its use in field and greenhouse conditions is important in view of the well known benefits of such fungi on uptake of mineral nutrients and, possibly, the water relations and photosynthesis levels of plants (Harley & Smith, 1983). Moreover, VAM fungi reduce the susceptibility of their host to certain pathogens (Dehne, 1982). In the past it has been difficult to obtain large amounts of VAM inoculants for agronomic use. These obligate symbionts can be grown from various propagules but their spores produce mycelium with limited independent growth (Hepper, 1984). Mycelia regenerated in pure culture from intraradical vesicles have the ability to infect root cortices (Strullu & Romand, 1987). Ectomycorrhizal fungi, in contrast, can now be produced in a fermentor and by entrapping the mycelium in alginate beads, several workers (Le Tacon ef al., 1985; Mauperin et aI., 1987) have produced inoculum for use in forestry. Recently, entrapment has emerged as a tool for the production of inoculants of VAM fungi. Immobilization procedures can preserve the physiological properties of mycorrhizal roots, promote the regeneration of mycelium and the formation of mycorrhizas (Strullu & PlencheHe, 1990). We conducted experiments to ascertain whether entrapment inside alginate gels can also stabilize the infectivity of a VAM fungus isolated in its intraradical forms i.e. vesicles or interand intracellular mycelial fragments. Glomus sp. 3 (deposited in Canada Department of Agriculture Herbarium, DAOM 181602) was used for the experiments and mycorrhizal roots were recovered from pot cultures of leek (Allium porrum L. cv. Olaf) grown on calcined clay and fed with a nutrient solution (PlencheHe, Furlan & Fortin, 1982). The selected mycorrhizal roots contained about 100 vesicles mm- 1 (Fig. 1). Two forms of inoculum were obtained.
(a) Root pieces: roots were surface sterilized as previously described (Strullu & Romand, 1986) and cut in 1-3 mm pieces by hand under a 10 x microscope. (b) Isolated vesicles: 0'5 g of mycorrhizal roots were lysed overnight at 23° in 5 ml of the following enzymatic solution (pH/H 2 0 = 5'6): 0'2 g 1-1 Macerozyme RIO, 0'5 g 1-1 Driselase (Yakult Honsha Co. Ltd) and 1 g 1-1 cellulase RIO. Lysed root tissues were then washed in distilled water and homogenized for two minutes at 4500 rpm in a blender (Omni mixer, Ivan Sorvall Inc). Intact vesicles and intraradical mycelium were recovered by passage through a 250 I-lm sieve which retained most of the root debris. Material of each of the two types of inoculum was suspended in a 20 g 1-1 solution of sodium alginate (d = 1'0046; viscosity = 14000 centipoises) and the mixture was dropped into a 0'1 M solution of calcium chloride through a needle, using a peristaltic pump. This process entraps vesicles, mycelium or small root fragments in calcium alginate beads averaging 2 mm in diameter (Figs 2, 3, 4). It is possible to obtain about 20000 beads containing 30 vesicles or 500 beads containing 3-5 fragments of 1-3 mm from one gram of selected mycorrhizal roots. Four-week old plantlets of leek (Allium porrum L. cv. Olaf) were prepared in pots containing 200 ml of calcined clay (Plenchette ef aI., 1982) and fed weekly with 20 ml of nutrient solution (Hewitt, 1966). Each experiment consisted of three inoculation treatments: (a) control without beads; (b) plants inoculated with 50 beads each containing 3-5 root pieces; (c) plants inoculated with 50 beads containing 30 isolated vesicles. In experiment 1 the beads were prepared immediately
D. G. Strullu and C. Plenchette
1195
Fig. 1. Mycorrhizal roots showing vesicles of Glomus sp. 3 after staining by fuschin acid. Scale bar = 5000 ~m. Fig. 2 (Dark field microscopy) and 3. Optical micrographs of alginate beads containing vesicles (v), mycelia (my) and root cell fragments (*). Scale bar = 500 ~m. Fig. 4. Vesicles (v) immobilized in alginate gel. Scale bar = 150 ~m.
before the experiment and in experiment 2, they were stored for 1 month at 4° before use as inoculum. In both experiments beads were stratified 5 em deep in the substrate of each pot. Pots were placed in a growth chamber (22°/18°, 16 h day at 220 \-lmole S-l m- 2, 80% r.h.). Plants were harvested and their root systems were collected after
two months for experiment 1 and one month for experiment 2, for staining and determination of mycorrhizal infection using the method of Giovannetti & Mosse (1980). The results are summarized in Table 1. Non-inoculated plants did not develop mycorrhizas, but plants inoculated with beads containing mycorrhizal root pieces or isolated intra-
VAM inoculum in alginate beads
1196
Table 1. Results of inoculation of leek plantlets (5 replicates) with alginate beads (i) used immediately after their preparation and (ii) after one month storage at 4 0
Treatment
Proportion of mycorrhizal infection of roots (%)
The authors wish to thank Dr J. A. Fortin for critically reading the manuscript and improving the English.
(0
Control Beads with infected root pieces Beads with vesicles
0 57
0 II
0 51
0 7
0 20
12
7
34
15
51
0 82
0 5
0 55
0 64
0 43
2
70
50
82
66
(ii)
Control Beads with infected root pieces Beads with vesicles
mycelium can infect roots under controlled conditions. We can now consider the possibility of producing high quality inoculum for field experiments.
radical material all developed mycorrhizal associations. In both experiments, there was no Significant difference (Student's t-test after arc sin transformation) between the infection obtained with beads containing root fragments and that obtained with beads containing isolated vesicles and mycelium. Furthermore, fungal structures immobilized in alginate capsules did not lose their ability to infect root after storage at 4° for one month. Regeneration is a major physiological process for vesicles; so it is important to know whether the immobilization in alginate permits isolated intraradical forms of Glomus sp. to produce new hyphae and mycorrhizas. Previous results concerning saprophytic growth of VAM fungi have been reviewed by Harley & Smith (1983). Regrowth of hyphae from dead re-wetted roots (Tommerup & Abbott, 1981) and fungal regrowth in sterilized soil (Hepper & Warner, 1983) suggest that under natural conditions VAM fungi have some saprophytic ability. Moreover mycorrhizal root fragments proved to be useful and infective inoculum. Mosse (1988) reported that hyphae of Glomus intraradices, regenerated from roots, grew vigorously in dual culture with carrot roots without infection. In contrast, vesicles of Glomus caledonium yielded negligible fungal growth and had poor infection capacity (Burggraaf & Beringer, 1989). Strullu et al. (1989) suggested that calcium may be important for the regeneration of intraradical mycelium. In our experiments, the final concentration in alginate beads capsules was 10 mM Ca 2 +, similar to amounts found in most culture media used for VAM fungi (Hepper, 1984). These results indicate that isolated intraradical forms of Glomus sp. can grow in alginate beads and that the regenerated (Received for publication 5 December 1990 and in revised form 12 March 1991)
REFERENCES Burggraaf. A. j. P. &; Beringer, j. E. (1989). Absence of nuclear DNA synthesis in vesicular-arbuscular mycorrhizal fungi during in vitro development. New Phytologist 11. 25-33. Dehne, H. W. (1982). Interaction between vesicular arbuscular mycorrhizal fungi and plant pathogens. Phytopathology 72, II 15-II 18. Giovannetti, M. &; Mosse, B. (1980). An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist 84. 489-500. Harley, j. L. &; Smith, S. E. (1983). Mycorrhizal symbiosis. London, U.K.: Academic Press. Hepper, C M. (1984). Isolation and culture of vesicular-arbuscular mycorrhizal (VAM) fungi. In VA mycorrhiza (ed. C L. Powell &; D. j. Bagyaraj), pp. 95-Il2. Boca Raton, Florida, U.s.A.: CRC Press. Hepper, eM. &; Warner, A. (1983). Role of organic matter in growth of a vesicular arbuscular mycorrhizal fungus in soil. Transaction of the British Mycological Society 81, 155-156. Hewitt, E. ]. (1966). Sand and water culture methods used in the study of plant nutrition. In Technical Communication no. 22, 2nd edn revised, pp. 430--434. Commonwealth Agricultural Bureaux, London, U.K. Le Tacon, F., jung, G., Mugnier, )., Michelot, P. &; Mauperin, C (1985). Efficiency in a forest nursery of an ectomycorrhizal inoculum produced in a fermentor and entrapped in polymeric gels. Canadian Journal of Botany 63, 1664-1668. 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. (1988). Some studies relating to independent growth of vesiculararbuscular endophytes. Canadian Journal of Botany 66, 2533-2540. Plenchette, c.. Furlan, V. &; Fortin, j. A. (1982). Effects of different endomycorrhizal fungi on five host plants grown on calcined montmorillonite clay. Journal of American Society for horticultural Science 107, 535-538. Strullu, D. G. &; Plenchette, C (1990). Encapsulation de la forme intraracinaire de Glomus dans l'alginate et utilisation des capsules camme inoculum. Comptes Rendus de I'Academie des Sciences, Ser. III, 310, 447-452. Strullu, D. G. &; Romand, C (1986). Methode d'obtenbon d'endomycorhizes 11 vesicules et arbuscules en conditions axeniques. Comptes Rendus de I'Academie des Sciences 303, 245-250. Strullu, D. G. &; Romand, C (1987) Culture axenique de vesicules isolees 11 partir d'endomycorhizes et re-association in vitro 11 des racines de tomates. Comptes Rendus de [,Academie des Sciences 305, 15-19. Strullu, D. G., Romand, C, Callac, P., Teoule, E. &; Demarly, Y. (1989). Mycorrhizal synthesis in vitro between Glomus spp. and artificial seeds of alfalfa. New Phytologist 113, 545-548. Tommerup, I. C &; Abbott, L. K. (1981). Prolonged survival and viability of VA mycorrhizal hyphae after root death. Soil and Biochemistry 13, 431-433.