- Email: [email protected]
Growth response of the saltbush Atriplex nummularia L. to inoculation with the arbuscular mycorrhizal fungus Glomus intraradices
ARTICLE IN PRESS Journal of Arid Environments
Journal of Arid Environments 61 (2005) 535–540 www.elsevier.com/locate/jnlabr/yjare
Growth response of the saltbush Atriplex nummularia L. to inoculation with the arbuscular mycorrhizal fungus Glomus intraradices C. Plenchettea,, R. Duponnoisb a
INRA, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France IRD, UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2, Laboratoire des Symbioses Tropicales et Me´diterrane´ennes (LSTM), France
b
Received 2 October 2003; received in revised form 8 October 2004; accepted 21 October 2004 Available online 2 December 2004
Abstract Plantlets of Atriplex nummularia were inoculated with the arbuscular mycorrhizal fungi Glomus intraradices in a pot experiment. Plants were grown in a low P soil. Highly significant growth response of a Chenopodiaceae was recorded for the first time. Mycorrhizal colonization of roots was well developed, internal hyphae and vesicles were observed, but not arbuscules. These observations suggest that arbuscules are not necessary to obtain significant growth stimulation from mycorrhizal inoculation. Atriplex nummularia is already used as forage crops, its high mycorrhizal dependency offers possibilities to develop this production and revegetation strategies. r 2004 Elsevier Ltd. All rights reserved. Keywords: Atriplex nummularia; Glomus intraradices; Salt; Growth response; Cover crop
1. Introduction In arid and semi-arid zones the main problem in agricultural systems, which arises concurrently with decreasing availability of fresh water, is the increasing level of Corresponding author. Tel.: +33 3 80 69 30 32; fax: +33 3 80 46 32 62.
E-mail address: [email protected] (C. Plenchette). 0140-1963/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2004.10.003
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salinity of the soils. The rehabilitation of salinized areas for crop production will be a challenge in the next decades since there will be an increasing demand for food in many regions where the population continuously increases. The situation is complicated by the fact that the soil of these regions generally contains little organic matter and bioavailable mineral nutrients. Thus, the establishment of salt tolerant crops is difficult without the use of fertilizers which are usually not available to farmers with low incomes. Some soil-born micro-organisms are thought to be helpful for plants growing in poor soil or stressed environment. Among them, the arbuscular mycorrhizal (AM) fungi (Morton and Benny, 1990), which are ubiquitous in terrestrial ecosystems, are beneficial to plant growth (see Smith and Read, 1997). Mycorrhizae are a symbiotic association between AM fungi and plant roots that constitutes a fundamental link between the soil and the plant since their hyphae are directly involved in nutrient uptake and transport. AM fungi exhibit no specificity and mycorrhizae were found in average on 80–90% of land plants. Nonetheless, in few families, mainly in Chenopodiaceae and Cruciferaceae (Gerdemann, 1968), early studies reported the absence of mycorrhizae. Crop plants show various degrees of tolerance to various types of stresses and it is possible to select salt tolerant species for rehabilitation of arid or semi-arid areas. But the level of salinity encountered in millions of hectares in Asia, Australia and Africa often exceeds the upper limit of salt tolerance of most crops (Choukr-Allah, 1996, pp. 3–16). Therefore more economic approach is to use halophytes. Halophytes can be herbaceous species, shrubs or trees. Many halophytes species have been evaluated for their use as a forage for livestock (Malcom and Pol, 1986; O’Leary, 1988, pp. 773–790). A particular interest was raised for members of Chenopodiaceae, a group that includes in average 25% of the halophytes known world-wide (Le Houe´rou, 1993, pp. 403–422). Early observations fail to find AM fungal structures in members of Chenopodiaceae and it has long been assumed that they were non-mycotrophic (Asai, 1934). Subsequently, however, observations of mycorrhizal structures in several species of this family were reported (Hirrel et al., 1978). The evidence for mycotrophy in Chenopodiaceae is often contradictory. In some cases, AM fungal structures were observed in plants of this family when grown with a mycotrophic companion plant (Hirrel et al., 1978; Plenchette and Trouvelot, 1986, pp. 559–561). It was suggested that in such cases the association was probably not mutual because arbuscules, considered as the site of exchange between the plant and the fungus, were not observed. However, Williams et al. (1974) reported that the presence of vesicles and hyphae in Atriplex canescens enhanced the growth and survival of this Chenopodiaceae. More recently the mycorrhizal status of numerous Chenopodiaceae, particularly the genus Atriplex, was confirmed (Lindsey, 1982; Dhillion et al., 1995; Aguilera et al., 1998; Hildebrandt et al., 2001), and inoculation of Atriplex gardeneri with AM spores was proven efficient to enhance the growth of this species (Allen, 1983). Among Chenopodiaceae the genus Atriplex is probably the most studied, probably because Atriplex species are used for rehabilitation of saline soils.
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Le Houe´rou (1993, pp. 403–422) reported that in the last 25 five years 100,000 hectares in average were planted with Atriplex spp. in the mediterranean basin. The aim of this work is to assess the responsiveness of Atriplex nummularia, an exotic species from Australia to mycorrhizal inoculation.
2. Materials and methods Seeds of A. nummularia harvested in the area of Kaolack (Senegal) were surface sterilized with 30% hydrogen peroxide for 5 min, rinsed and left to soak for 12 h in sterile distilled water. Seeds were then transferred aseptically to Petri dishes filled with 1% (w:v) water agar medium and plates were incubated at 25 1C in the dark. The germinating seeds were used when rootlets were 1–2 cm long. The arbuscular mycorrhizal fungus Glomus intraradices Schenk & Smith (DAOM 181602, Ottawa Agricultural Herbarium) has been cultivated on leek (Allium porrum L.) for 12 weeks under greenhouse conditions in TerragreenTM substrate. This calcined clay (particule size average 5 mm), Oil-Dri US-special Ty/IIIR (Oil-Dri Company, Chicago, USA) is an attapulgite from Georgia, commonly used for the propagation of mycorrhizal fungi (Plenchette et al., 1996). Leek plants were uprooted, gently washed and cut into pieces 0.5 cm long (around 250 vesicles cm 1). Non-mycorrhizal leek roots, prepared as above, were used for the control treatment. Seedlings were grown in 1 L pots filled with a disinfected sandy soil collected in a millet field located at the east of Ouagadougou (Burkina Faso). After sampling, the soil was crushed, passed through a 2-mm sieve and autoclaved for 40 min at 140 1C to eliminate the native microflora. After autoclaving, the physico-chemical characteristics of this soil were as follows: pH (H2O) 5.6; clay 4.6 (%); fine silt 0.0 (%); coarse silt 0.8 (%); fine sand 25.5 (%); coarse sand 69.1 (%); carbon 0.204 (%); nitrogen 0.04 (%); C/N 52; P (Olsen) 4.3 ppm. A hole (1 cm 3 cm) was made in each pot to receive 1 g (fresh weight) of mycorrhizal leek roots and a germinated seed of Atriplex which was then covered with the disinfected soil. Control treatment received 1 g (fresh weight) of nonmycorrhizal leek roots. Plants were watered twice a week with tap water without fertilizer. The pots were arranged in a randomized complete block design with 10 replicates per treatment. They were placed in a greenhouse during the hot season under natural light (daylight approximately 12 h, daily average temperature 35 1C). After 6 months of growth, Atriplex plants were uprooted and the root systems detached from the shoots and gently washed. The root systems were cut into 1-cmroot pieces and mixed. The internal colonization of arbuscular mycorrhizal fungus was quantified by clearing and staining a sub-sample of the roots according to the method of Phillips and Hayman (1970). The root pieces were placed on a slide for microscopic observation at 250 magnification (Brundrett et al., 1985). About fifty 1-cm-root pieces per plant were observed. The level of mycorrhizal colonization was expressed in terms of fraction of root length with mycorrhizal internal structures (vesicles or hyphae): (length of root fragments colonized/total length of root fragments) 100. Shoots’ and roots’ dry mass was measured after drying for 1 week
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at 65 1C). Ground samples of leaves were ashed (500 1C), digested in 2 ml HCL (6N) and 10 ml HNO3 (N) and then analysed by colorimetry for P (John, 1970). Other sub-samples of leaf tissue were grounded and digested in 15 ml H2SO4 36N containing 50 g l 1 salicylic acid for N (Kjeldhal) determination. All data were subjected to a one-way analysis of variance (Po0:05).
3. Results A. nummularia grown in a P-poor (4.3 ppm Olsen) soil responded greatly to inoculation by G. intraradices (Table 1). Inoculated plants had higher biomass than non-inoculated plants. The growth stimulation was highly significant and the mycorrhizal dependency, calculated accordingly to Plenchette et al. (1983), reached 86%. Shoots’ P-content of inoculated plants was also significantly higher (Table 1), but no difference was observed for shoots’ N-content. The colonization of A. nummularia roots by G. intraradices was well developed, with 77% of root length colonized, but only internal hyphae and vesicles were observed. Arbuscules were not found.
4. Discussion and conclusion We found that A. nummularia is a highly mycorrhizae-dependent plant (Plenchette et al., 1983). As regularly observed in mycorrhizal inoculation experiments (see Plenchette, 1991), the growth stimulation observed was mainly attributed to a phosphorus nutrition enhancement. In the field, native populations of Atriplex spp. have been more often found colonized by dark septate fungi than by mycorrhizal fungi (Barrow and Osuna, 2002). Our results demonstrate clearly that A. nummularia could benefit from mycorrhizal inoculation, but also that the growth stimulation could be attributed to a better P-nutrition. To our knowledge this is the first report of an AM fungi induced growth stimulation in the Chenopodiaceae. Moreover, this result was obtained without the presence of a mycorrhizal companion plant. Gallaud (1905) described Arum- and Paris-type endomycorrhizae. In this study typical
Table 1 Effect of G. intraradices inoculation on the plant growth and on the leaf mineral content (P and N) after 6 months culture in greenhouse conditions Treatments
Shoot biomass (mg dry weight)
Root biomass (mg dry weight)
Total biomass (mg dry weight)
P (mg/g)
N (mg/g)
Control G. intraradices
191.0aa 2137.1b
104.1a 259.4b
295.1a 2396.5b
2.7a 3.5b
4.5a 4.1a
a
Numbers in the same column are not significantly different according to the one-way analysis of variance (Po0:05) when followed by the same letter.
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Arum-type arbuscules or Paris-type hyphal coils were not observed, so it is not possible to classify A. nummularia mycorrhizae type. It was found that G. mosseae and G. viscosum which are known to form Arum-type mycorrhizae produced Paristype when inoculated to Smilax aspera, a Liliaceae (Bedini et al., 2000). Similarly, G. coronatum inoculated on Asphodelus fistulosus, another Liliaceae, forms Paris-type mycorrhizae (Cavagnaro et al., 2003). These results tend to confirm that arbuscular mycorrhizal infection type is governed by the plant host genotype, and it could be hypothesized that a third type, without arbuscules, could exist when Chenopodiaceae are associated with AM fungi. Salinity is a major concern all over the world (Szabolcs, 1976, 1994). Millions of hectares of land are affected by salinity. Planting halophytes is often the only opportunity to produce forage for livestock. A. nummularia is already used as a forage crop in several countries (Watson, 1990). The yield of such plantation could be enhanced by mycorrhizal inoculation, considering the mycorrhizal dependency of this plant. Inoculation of A. nummularia with mycorhizal fungi may also facilitate revegetation and remediation of brine-contaminated soil in oil-producing regions (Keiffer and Ungar, 2002). Further experiments should be undertaken to confirm the mycorrhizal dependency of A. nummularia under field conditions, and to evaluate the mycorrhizal dependency of different Atriplex species.
References Aguilera, L.E., Gutierrez, J.R., Moreno, R.J., 1998. Vesiculo arbuscular mycorrhizae associated with saltbushes Atriplex spp. (Chenopodiaceae) in the Chilean arid Zone. Revista Chilena de Historia Natural 71, 291–302. Allen, M.F., 1983. Formation of vesicular-arbuscular mycorrhizae in Atriplex gardneri (Chenopodiaceae): seasonal response in a cold desert. Mycologia 75, 773–776. Asai, T., 1934. U¨ber das vorkommen und die bedeutung der wurzelpize in den Landpflanzen. Japanese Journal of Botany 5, 107–150. Barrow, J.R., Osuna, P., 2002. Phosphorus solubilization and uptake by dark septate fungi in fourwing saltbush, Atriplex canescens (Pursh) Nutt. Journal of Arid Environment 51, 449–459. Bedini, S., Maremmani, A., Giovannetti, M., 2000. Paris-type mycorrhizas in Smilax aspera L. growing in a Mediterranean scherophyllous wood. Mycorhiza 10, 9–13. Brundrett, M.C., Piche, Y., Peterson, R.L., 1985. A developmental study of the early stages in vesiculararbuscular mycorrhizal formation. Canadian Journal of Botany 63, 184–194. Cavagnaro, T.R., Smith, F.A., Ayling, S.M., Smith, S.E., 2003. Growth and phosphorus nutrition of a Paris-type arbuscular mycorrhizal symbiosis. New Phytologist 157, 127–134. Choukr-Allah, R., 1996. The potential of halophytes in the development and rehabilitation of arid and semiarid zones. In: Choukr-Allah, R., Malcolm, V.V., Hamdy, A. (Eds.), Halophytes and Biosaline Agriculture. Marcel Dekker, New York, NY 424pp. Dhillion, S.S., Vidiella, P.E., Aguilera, L.E., Friesse, S.E., De Leon, E., Zak, J.C., 1995. Mycorrhizal plant and fungi in the fog free Pacific coastal desert of Chile. Mycorrhiza 5, 381–386. Gallaud, I., 1905. Etudes sur les mycorhizes endotrophes. Revue Ge´ne´rale de Botanique, 17, 5–48, 66–83, 123–135, 223–239, 313–325, 425–433, 479–500. Gerdemann, J.W., 1968. Vesicular-arbuscular mycorrhizae and plant growth. Annual Review of Phytopathology 6, 397–418. Hildebrandt, U., Janetta, K., Ouziad, F., Renne, B., Nawrath, K., Bothe, H., 2001. Arbuscular mycorrhizal colonization of halophytes in Central European salt marshes. Mycorrhiza 10, 175–183.
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Hirrel, M.C., Mehravaran, H., Gerdemann, J.W., 1978. Vesicular-arbuscular mycorrhizae in the Chenopodiaceae and Cruciferae: do they occur? Canadian Journal of Botany 56, 2813–2817. John, M.K., 1970. Colorimetric determination in soil and plant material with ascorbic acid. Soil Science 68, 171–177. Keiffer, C.H., Ungar, I.A., 2002. Germination and establishment of halophytes on brine-affected soils. Journal of Applied Ecology 39, 402–415. Lindsey, D.L., 1982. The role of vesicular-arbuscular mycorrhizae in shrub establishment. In: Williams, S.E., Allen, M.F. (Eds.), VA Mycorrhizae and Reclamation of Arid and Semiarid Land. University of Wyoming: Agricultural Experimental Station, pp. 53–68. Le Houe´rou, H.N., 1993. Salt tolerant plants for the arid regions of the Mediterranean isoclimatic zone. In: Lieth, H., Masoom, El. (Eds.), Towards the Rational Use of High Salinity-Tolerant Plants. Kluwer Academic Publication, Dordrecht, The Nederlands 440pp. Malcom, C.V., Pol, J.E., 1986. Grazing and management of saltland shrubs. Journal of Agriculture Western Australia 27, 59–63. Morton, J.B., Benny, G.L., 1990. Revised classification of arbuscular mycorrhizal fungi (Zygomycetes): a new order, glomales, two new suborders, glomineae and gigasporineae, and two new families, acaulopsoraceae and gigasporaceae, with an emendation of glomaceae. Mycotaxon 37, 471–491. O’Leary, J.W., 1988. Saline environments and halophytic crops. In: Whitehead, E.E., Hutchinson, C.F., Timmerman, B.N., Varady, R.G. (Eds.), Arid lands, today and tomorrow. Wetview Press, Boulder, Colorado 1435pp. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society 55, 158–161. Plenchette, C., 1991. Utilisation des mycorhizes en agriculture et horticulture. In: Strullu, D.G., Garbaye, J., Perrin, R., Plenchette, C. (Eds.), Les mycorhizes des arbres et des plantes cultive´es. Technique et Documentation, Lavoisier, Paris, pp. 131–196. Plenchette, C., Trouvelot, A., 1986. Influence du ge´nome de la betterave a` sucre (Beta vulgaris L.) sur son aptitude a` la mycorhization. In: Gianinazzi-Pearson, V., Gianinazzi, S. (Eds.), Physiological and Genetical Aspects of Mycorrhizae. INRA, Paris 832pp. Plenchette, C., Fortin, J.A., Furlan, V., 1983. Growth responses to several plant species to mycorrhizae in a soil of moderate P-fertility. I. Mycorrhizal dependency under field conditions. Plant Soil 70, 199–209. Plenchette, C., Declerck, S., Diop, T., Stullu, D.G., 1996. Infectivity of monoaxenic subcultures of the AM fungus Glomus versiforme associated with Ri-TDNA transformed root. Applied Microbiology and Biotechnology 46, 545–548. Szabolcs, I., 1976. Present and potential salt affected soils, prognosis of salinity and alkalinity. Soils Bulletin 31, 9–13. Szabolcs, I., 1994. Soils and salinization. In: Pessarakli, M. (Ed.), Handbook of Plant Crop Stress. University of Arizona, Tucson, AZ, pp. 3–21. Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis, second Ed. Academic Press Inc., San Diego, CA 605pp. Watson, M.C., 1990. Atriplex species as irrigated forage crops. Agriculture, Ecosystems and Environment 32, 107–118. Williams, S.E., Wollum II., A.G., Aldon, F.E., 1974. Growth of Atriplex canescens (Pursh.) Nutt. improved by formation of vesicular-arbuscular mycorrhizae. Soil Science Society of America Proceedings 38, 962–965.
Journal of Arid Environments 61 (2005) 535–540 www.elsevier.com/locate/jnlabr/yjare
Growth response of the saltbush Atriplex nummularia L. to inoculation with the arbuscular mycorrhizal fungus Glomus intraradices C. Plenchettea,, R. Duponnoisb a
INRA, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France IRD, UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2, Laboratoire des Symbioses Tropicales et Me´diterrane´ennes (LSTM), France
b
Received 2 October 2003; received in revised form 8 October 2004; accepted 21 October 2004 Available online 2 December 2004
Abstract Plantlets of Atriplex nummularia were inoculated with the arbuscular mycorrhizal fungi Glomus intraradices in a pot experiment. Plants were grown in a low P soil. Highly significant growth response of a Chenopodiaceae was recorded for the first time. Mycorrhizal colonization of roots was well developed, internal hyphae and vesicles were observed, but not arbuscules. These observations suggest that arbuscules are not necessary to obtain significant growth stimulation from mycorrhizal inoculation. Atriplex nummularia is already used as forage crops, its high mycorrhizal dependency offers possibilities to develop this production and revegetation strategies. r 2004 Elsevier Ltd. All rights reserved. Keywords: Atriplex nummularia; Glomus intraradices; Salt; Growth response; Cover crop
1. Introduction In arid and semi-arid zones the main problem in agricultural systems, which arises concurrently with decreasing availability of fresh water, is the increasing level of Corresponding author. Tel.: +33 3 80 69 30 32; fax: +33 3 80 46 32 62.
E-mail address: [email protected] (C. Plenchette). 0140-1963/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2004.10.003
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salinity of the soils. The rehabilitation of salinized areas for crop production will be a challenge in the next decades since there will be an increasing demand for food in many regions where the population continuously increases. The situation is complicated by the fact that the soil of these regions generally contains little organic matter and bioavailable mineral nutrients. Thus, the establishment of salt tolerant crops is difficult without the use of fertilizers which are usually not available to farmers with low incomes. Some soil-born micro-organisms are thought to be helpful for plants growing in poor soil or stressed environment. Among them, the arbuscular mycorrhizal (AM) fungi (Morton and Benny, 1990), which are ubiquitous in terrestrial ecosystems, are beneficial to plant growth (see Smith and Read, 1997). Mycorrhizae are a symbiotic association between AM fungi and plant roots that constitutes a fundamental link between the soil and the plant since their hyphae are directly involved in nutrient uptake and transport. AM fungi exhibit no specificity and mycorrhizae were found in average on 80–90% of land plants. Nonetheless, in few families, mainly in Chenopodiaceae and Cruciferaceae (Gerdemann, 1968), early studies reported the absence of mycorrhizae. Crop plants show various degrees of tolerance to various types of stresses and it is possible to select salt tolerant species for rehabilitation of arid or semi-arid areas. But the level of salinity encountered in millions of hectares in Asia, Australia and Africa often exceeds the upper limit of salt tolerance of most crops (Choukr-Allah, 1996, pp. 3–16). Therefore more economic approach is to use halophytes. Halophytes can be herbaceous species, shrubs or trees. Many halophytes species have been evaluated for their use as a forage for livestock (Malcom and Pol, 1986; O’Leary, 1988, pp. 773–790). A particular interest was raised for members of Chenopodiaceae, a group that includes in average 25% of the halophytes known world-wide (Le Houe´rou, 1993, pp. 403–422). Early observations fail to find AM fungal structures in members of Chenopodiaceae and it has long been assumed that they were non-mycotrophic (Asai, 1934). Subsequently, however, observations of mycorrhizal structures in several species of this family were reported (Hirrel et al., 1978). The evidence for mycotrophy in Chenopodiaceae is often contradictory. In some cases, AM fungal structures were observed in plants of this family when grown with a mycotrophic companion plant (Hirrel et al., 1978; Plenchette and Trouvelot, 1986, pp. 559–561). It was suggested that in such cases the association was probably not mutual because arbuscules, considered as the site of exchange between the plant and the fungus, were not observed. However, Williams et al. (1974) reported that the presence of vesicles and hyphae in Atriplex canescens enhanced the growth and survival of this Chenopodiaceae. More recently the mycorrhizal status of numerous Chenopodiaceae, particularly the genus Atriplex, was confirmed (Lindsey, 1982; Dhillion et al., 1995; Aguilera et al., 1998; Hildebrandt et al., 2001), and inoculation of Atriplex gardeneri with AM spores was proven efficient to enhance the growth of this species (Allen, 1983). Among Chenopodiaceae the genus Atriplex is probably the most studied, probably because Atriplex species are used for rehabilitation of saline soils.
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Le Houe´rou (1993, pp. 403–422) reported that in the last 25 five years 100,000 hectares in average were planted with Atriplex spp. in the mediterranean basin. The aim of this work is to assess the responsiveness of Atriplex nummularia, an exotic species from Australia to mycorrhizal inoculation.
2. Materials and methods Seeds of A. nummularia harvested in the area of Kaolack (Senegal) were surface sterilized with 30% hydrogen peroxide for 5 min, rinsed and left to soak for 12 h in sterile distilled water. Seeds were then transferred aseptically to Petri dishes filled with 1% (w:v) water agar medium and plates were incubated at 25 1C in the dark. The germinating seeds were used when rootlets were 1–2 cm long. The arbuscular mycorrhizal fungus Glomus intraradices Schenk & Smith (DAOM 181602, Ottawa Agricultural Herbarium) has been cultivated on leek (Allium porrum L.) for 12 weeks under greenhouse conditions in TerragreenTM substrate. This calcined clay (particule size average 5 mm), Oil-Dri US-special Ty/IIIR (Oil-Dri Company, Chicago, USA) is an attapulgite from Georgia, commonly used for the propagation of mycorrhizal fungi (Plenchette et al., 1996). Leek plants were uprooted, gently washed and cut into pieces 0.5 cm long (around 250 vesicles cm 1). Non-mycorrhizal leek roots, prepared as above, were used for the control treatment. Seedlings were grown in 1 L pots filled with a disinfected sandy soil collected in a millet field located at the east of Ouagadougou (Burkina Faso). After sampling, the soil was crushed, passed through a 2-mm sieve and autoclaved for 40 min at 140 1C to eliminate the native microflora. After autoclaving, the physico-chemical characteristics of this soil were as follows: pH (H2O) 5.6; clay 4.6 (%); fine silt 0.0 (%); coarse silt 0.8 (%); fine sand 25.5 (%); coarse sand 69.1 (%); carbon 0.204 (%); nitrogen 0.04 (%); C/N 52; P (Olsen) 4.3 ppm. A hole (1 cm 3 cm) was made in each pot to receive 1 g (fresh weight) of mycorrhizal leek roots and a germinated seed of Atriplex which was then covered with the disinfected soil. Control treatment received 1 g (fresh weight) of nonmycorrhizal leek roots. Plants were watered twice a week with tap water without fertilizer. The pots were arranged in a randomized complete block design with 10 replicates per treatment. They were placed in a greenhouse during the hot season under natural light (daylight approximately 12 h, daily average temperature 35 1C). After 6 months of growth, Atriplex plants were uprooted and the root systems detached from the shoots and gently washed. The root systems were cut into 1-cmroot pieces and mixed. The internal colonization of arbuscular mycorrhizal fungus was quantified by clearing and staining a sub-sample of the roots according to the method of Phillips and Hayman (1970). The root pieces were placed on a slide for microscopic observation at 250 magnification (Brundrett et al., 1985). About fifty 1-cm-root pieces per plant were observed. The level of mycorrhizal colonization was expressed in terms of fraction of root length with mycorrhizal internal structures (vesicles or hyphae): (length of root fragments colonized/total length of root fragments) 100. Shoots’ and roots’ dry mass was measured after drying for 1 week
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at 65 1C). Ground samples of leaves were ashed (500 1C), digested in 2 ml HCL (6N) and 10 ml HNO3 (N) and then analysed by colorimetry for P (John, 1970). Other sub-samples of leaf tissue were grounded and digested in 15 ml H2SO4 36N containing 50 g l 1 salicylic acid for N (Kjeldhal) determination. All data were subjected to a one-way analysis of variance (Po0:05).
3. Results A. nummularia grown in a P-poor (4.3 ppm Olsen) soil responded greatly to inoculation by G. intraradices (Table 1). Inoculated plants had higher biomass than non-inoculated plants. The growth stimulation was highly significant and the mycorrhizal dependency, calculated accordingly to Plenchette et al. (1983), reached 86%. Shoots’ P-content of inoculated plants was also significantly higher (Table 1), but no difference was observed for shoots’ N-content. The colonization of A. nummularia roots by G. intraradices was well developed, with 77% of root length colonized, but only internal hyphae and vesicles were observed. Arbuscules were not found.
4. Discussion and conclusion We found that A. nummularia is a highly mycorrhizae-dependent plant (Plenchette et al., 1983). As regularly observed in mycorrhizal inoculation experiments (see Plenchette, 1991), the growth stimulation observed was mainly attributed to a phosphorus nutrition enhancement. In the field, native populations of Atriplex spp. have been more often found colonized by dark septate fungi than by mycorrhizal fungi (Barrow and Osuna, 2002). Our results demonstrate clearly that A. nummularia could benefit from mycorrhizal inoculation, but also that the growth stimulation could be attributed to a better P-nutrition. To our knowledge this is the first report of an AM fungi induced growth stimulation in the Chenopodiaceae. Moreover, this result was obtained without the presence of a mycorrhizal companion plant. Gallaud (1905) described Arum- and Paris-type endomycorrhizae. In this study typical
Table 1 Effect of G. intraradices inoculation on the plant growth and on the leaf mineral content (P and N) after 6 months culture in greenhouse conditions Treatments
Shoot biomass (mg dry weight)
Root biomass (mg dry weight)
Total biomass (mg dry weight)
P (mg/g)
N (mg/g)
Control G. intraradices
191.0aa 2137.1b
104.1a 259.4b
295.1a 2396.5b
2.7a 3.5b
4.5a 4.1a
a
Numbers in the same column are not significantly different according to the one-way analysis of variance (Po0:05) when followed by the same letter.
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Arum-type arbuscules or Paris-type hyphal coils were not observed, so it is not possible to classify A. nummularia mycorrhizae type. It was found that G. mosseae and G. viscosum which are known to form Arum-type mycorrhizae produced Paristype when inoculated to Smilax aspera, a Liliaceae (Bedini et al., 2000). Similarly, G. coronatum inoculated on Asphodelus fistulosus, another Liliaceae, forms Paris-type mycorrhizae (Cavagnaro et al., 2003). These results tend to confirm that arbuscular mycorrhizal infection type is governed by the plant host genotype, and it could be hypothesized that a third type, without arbuscules, could exist when Chenopodiaceae are associated with AM fungi. Salinity is a major concern all over the world (Szabolcs, 1976, 1994). Millions of hectares of land are affected by salinity. Planting halophytes is often the only opportunity to produce forage for livestock. A. nummularia is already used as a forage crop in several countries (Watson, 1990). The yield of such plantation could be enhanced by mycorrhizal inoculation, considering the mycorrhizal dependency of this plant. Inoculation of A. nummularia with mycorhizal fungi may also facilitate revegetation and remediation of brine-contaminated soil in oil-producing regions (Keiffer and Ungar, 2002). Further experiments should be undertaken to confirm the mycorrhizal dependency of A. nummularia under field conditions, and to evaluate the mycorrhizal dependency of different Atriplex species.
References Aguilera, L.E., Gutierrez, J.R., Moreno, R.J., 1998. Vesiculo arbuscular mycorrhizae associated with saltbushes Atriplex spp. (Chenopodiaceae) in the Chilean arid Zone. Revista Chilena de Historia Natural 71, 291–302. Allen, M.F., 1983. Formation of vesicular-arbuscular mycorrhizae in Atriplex gardneri (Chenopodiaceae): seasonal response in a cold desert. Mycologia 75, 773–776. Asai, T., 1934. U¨ber das vorkommen und die bedeutung der wurzelpize in den Landpflanzen. Japanese Journal of Botany 5, 107–150. Barrow, J.R., Osuna, P., 2002. Phosphorus solubilization and uptake by dark septate fungi in fourwing saltbush, Atriplex canescens (Pursh) Nutt. Journal of Arid Environment 51, 449–459. Bedini, S., Maremmani, A., Giovannetti, M., 2000. Paris-type mycorrhizas in Smilax aspera L. growing in a Mediterranean scherophyllous wood. Mycorhiza 10, 9–13. Brundrett, M.C., Piche, Y., Peterson, R.L., 1985. A developmental study of the early stages in vesiculararbuscular mycorrhizal formation. Canadian Journal of Botany 63, 184–194. Cavagnaro, T.R., Smith, F.A., Ayling, S.M., Smith, S.E., 2003. Growth and phosphorus nutrition of a Paris-type arbuscular mycorrhizal symbiosis. New Phytologist 157, 127–134. Choukr-Allah, R., 1996. The potential of halophytes in the development and rehabilitation of arid and semiarid zones. In: Choukr-Allah, R., Malcolm, V.V., Hamdy, A. (Eds.), Halophytes and Biosaline Agriculture. Marcel Dekker, New York, NY 424pp. Dhillion, S.S., Vidiella, P.E., Aguilera, L.E., Friesse, S.E., De Leon, E., Zak, J.C., 1995. Mycorrhizal plant and fungi in the fog free Pacific coastal desert of Chile. Mycorrhiza 5, 381–386. Gallaud, I., 1905. Etudes sur les mycorhizes endotrophes. Revue Ge´ne´rale de Botanique, 17, 5–48, 66–83, 123–135, 223–239, 313–325, 425–433, 479–500. Gerdemann, J.W., 1968. Vesicular-arbuscular mycorrhizae and plant growth. Annual Review of Phytopathology 6, 397–418. Hildebrandt, U., Janetta, K., Ouziad, F., Renne, B., Nawrath, K., Bothe, H., 2001. Arbuscular mycorrhizal colonization of halophytes in Central European salt marshes. Mycorrhiza 10, 175–183.
ARTICLE IN PRESS 540
C. Plenchette, R. Duponnois / Journal of Arid Environments 61 (2005) 535–540
Hirrel, M.C., Mehravaran, H., Gerdemann, J.W., 1978. Vesicular-arbuscular mycorrhizae in the Chenopodiaceae and Cruciferae: do they occur? Canadian Journal of Botany 56, 2813–2817. John, M.K., 1970. Colorimetric determination in soil and plant material with ascorbic acid. Soil Science 68, 171–177. Keiffer, C.H., Ungar, I.A., 2002. Germination and establishment of halophytes on brine-affected soils. Journal of Applied Ecology 39, 402–415. Lindsey, D.L., 1982. The role of vesicular-arbuscular mycorrhizae in shrub establishment. In: Williams, S.E., Allen, M.F. (Eds.), VA Mycorrhizae and Reclamation of Arid and Semiarid Land. University of Wyoming: Agricultural Experimental Station, pp. 53–68. Le Houe´rou, H.N., 1993. Salt tolerant plants for the arid regions of the Mediterranean isoclimatic zone. In: Lieth, H., Masoom, El. (Eds.), Towards the Rational Use of High Salinity-Tolerant Plants. Kluwer Academic Publication, Dordrecht, The Nederlands 440pp. Malcom, C.V., Pol, J.E., 1986. Grazing and management of saltland shrubs. Journal of Agriculture Western Australia 27, 59–63. Morton, J.B., Benny, G.L., 1990. Revised classification of arbuscular mycorrhizal fungi (Zygomycetes): a new order, glomales, two new suborders, glomineae and gigasporineae, and two new families, acaulopsoraceae and gigasporaceae, with an emendation of glomaceae. Mycotaxon 37, 471–491. O’Leary, J.W., 1988. Saline environments and halophytic crops. In: Whitehead, E.E., Hutchinson, C.F., Timmerman, B.N., Varady, R.G. (Eds.), Arid lands, today and tomorrow. Wetview Press, Boulder, Colorado 1435pp. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society 55, 158–161. Plenchette, C., 1991. Utilisation des mycorhizes en agriculture et horticulture. In: Strullu, D.G., Garbaye, J., Perrin, R., Plenchette, C. (Eds.), Les mycorhizes des arbres et des plantes cultive´es. Technique et Documentation, Lavoisier, Paris, pp. 131–196. Plenchette, C., Trouvelot, A., 1986. Influence du ge´nome de la betterave a` sucre (Beta vulgaris L.) sur son aptitude a` la mycorhization. In: Gianinazzi-Pearson, V., Gianinazzi, S. (Eds.), Physiological and Genetical Aspects of Mycorrhizae. INRA, Paris 832pp. Plenchette, C., Fortin, J.A., Furlan, V., 1983. Growth responses to several plant species to mycorrhizae in a soil of moderate P-fertility. I. Mycorrhizal dependency under field conditions. Plant Soil 70, 199–209. Plenchette, C., Declerck, S., Diop, T., Stullu, D.G., 1996. Infectivity of monoaxenic subcultures of the AM fungus Glomus versiforme associated with Ri-TDNA transformed root. Applied Microbiology and Biotechnology 46, 545–548. Szabolcs, I., 1976. Present and potential salt affected soils, prognosis of salinity and alkalinity. Soils Bulletin 31, 9–13. Szabolcs, I., 1994. Soils and salinization. In: Pessarakli, M. (Ed.), Handbook of Plant Crop Stress. University of Arizona, Tucson, AZ, pp. 3–21. Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis, second Ed. Academic Press Inc., San Diego, CA 605pp. Watson, M.C., 1990. Atriplex species as irrigated forage crops. Agriculture, Ecosystems and Environment 32, 107–118. Williams, S.E., Wollum II., A.G., Aldon, F.E., 1974. Growth of Atriplex canescens (Pursh.) Nutt. improved by formation of vesicular-arbuscular mycorrhizae. Soil Science Society of America Proceedings 38, 962–965.