A Native Arbuscular Mycorrhizal Fungus, Acaulospora scrobiculata Stimulated Growth of Mongolian Crested Wheatgrass (Agropyron cristatum (L.) Gaertn.)
Burenjargal Otgonsuren1 and Ming-Jen Lee2
1Graduate Institute of Agriculture, National Chiayi University, 300 University Rd., Chiayi 60004, Taiwan,
ROC; Department of Ecology, Mongolian State University of Agriculture, Ulaanbaatar 210153, Mongolia
2Department of Forestry and Natural Resources, National Chiayi University, 300 University Rd., Chiayi 60004, Taiwan
Agropyron cristatum (L.) Gaertn. (crested wheatgrass) is an endemic plant species, which dominates most area of the Mongolian steppe and forest steppe. In the present study, spores of arbuscular mycorrhizal fungi in the rhizosphere soil of crested wheatgrass were isolated with wetsieving/ decanting methods, and the major species was identifi ed as Acaulospora scrobiculata Trappe. For arbuscular-mycorrhizal resynthesis, the spores of A. scrobiculata were propagated with corn pot-culture technique and inoculated onto the roots of crested wheatgrass seedlings. The inoculated crested wheatgrass seedlings exhibited vigor in growth, and examination of the root structure revealed the occurrence of arbuscules and vesicles in the cortical cells. These results demonstrated that A. scrobiculata could effectively form arbuscular mycorrhizas with crested wheatgrass and promote its growth, which can be used to restore Mongolian grassland.
Keyword: Acaulospora scrobiculata,Agropyron cristatum,arbuscular mycorrhizas,biomass,crested wheatgrass,growth, mineral,mycorrhizal dependency
A gropyron cristatum (L.) Gaertn. (crested wheatgrass) is a dominant native plant species in the Mongolian steppe. Available forages in this area consist primarily of Stipa krylovii, crested wheatgrass and Allium polyrrhizum (representing 80% of available phytomass), all of which are regarded as d esirable forage plants (Retzer, 2007). Crested wheatgrass is widely used in the restoration of the Mongolian prairie. Arbuscular mycorrhiza (AM) is important components of virtually all terrestrial ecosystems (Brundrett, 1991; Smith & Read, 2008). Arbuscular mycorrhizal fungi (AMF) are one of the most important soil microbes as they form mutualistic association with more than 80% of land plants (Ulrich et al., 2002). AMF are known to be widespread in semi-arid grasslands plants, and their association with grasses is important in biomes grazed by large ungulates (T rappe, 1981). I t has been well documented that the major benefi ts of plants from these relationships are improvement of uptakes in water and inorganic nutrients, especially phosphorus (S anders & Koide, 1994). Additional benefi ts include increased tolerance to the environmental stresses such as nutrient defi ciency, diseases, drought and salinity (Smith & Read, 2008; Gupta & Kumar, 2000). AMF may further infl uence plant community structure (Marler et al., 1999), biodiversity (Hartnett et al., 1993), primary production (Hedlund, 2002), ecosystem dynamics (Van Der Heijden et al., 1998), and survival of tree seedlings (Hetrick et al., 1988; Smith & Read, 2008; Stinson et al., 2006). Previous research has examined the distribution of AMF in sandy area (Blaszkowski et al., 2002), in agricultural soils (Oehl et al., 2009), and in certain natural ecosystems (Guadarrama & Alvarez-Sanchez, 1999), but few of them have looked into grasslands (Smith & Read, 2008), especially in arid and semiarid areas (Lugo et al., 2002). Mongolian grasslands are facing rapid desertifi cation due to the uncontrolled growth of animal herding, mining and global warming. The grassland has been thinning out in 75 percent of the Mongolian land area, while 7 percent has completely turned into deserts. Some actions must be taken to halt the process to help Mongolia keep its grasslands. Thus, the aims of this study were to isolate, identify and propagate the AMF and to assess the effect of this native AMF on growth of Mongolian crested wheatgrass seedlings through mycorrhizal resynthesis. It is hoped that the fi nding from this study will contribute to the use of mycorrhizal technique in grassland restoration.
Material and Methods
Sample collection. Root samples of crested wheatgrass and their rhizosphere soil were collected from grassland at Bogd Mountain in the vicinity of Ulaanbaatar city (E 107008’31’’, N 47045’767’’ 1597 m) of Mongolia in October 2008. Feeder roots, soil and root samples were collected from 0 to 20 cm depth of eight individual plants, put in polyethylene bags and stored at 5-10°C until analyzed. Seeds of crested wheatgrass were also collected from natural grassland at the same site. Extraction, identifi cation, and propagation of spores. Spores of AMF in rhizosphere soils of crested wheatgrass were extracted by wet sieving and decanting method (Gerdermann & Nicolson, 1963; Tommerup, 1992) and sucrose density gradient centrifugation (Daniels & Skipper, 1982), and then identifi ed with reference to the key provided by INVAM (International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi, homepage: www.invam.caf.wvu.edu). After identifi cation, spores were propagated subsequently with corn seeds germinated in sterilized sand pot culture for AM fungal inoculum preparation. Spore sand was quantifi ed for further inoculation test. Seedling raising. After surface cleaning with running tap water, seeds of crested wheatgrass were sterilized with a 10% sodium hypochlorite solution for 15 min, rinsed three times with sterile distilled water, and then germinated in sterilized mixture of peat moss, vermiculite and perlite (1:1:1, v/v). As the seedlings attained 4 cm in height, they were transferred to pots fi lled with sterilized sand for AM resynthesis. Resynthesis. The grass seedlings were inoculated with 10 g sand spore (containing 15±5 spores/g sand). Non-inoculated seedlings treated with the sand-spore fi ltrate served as the control. All seedlings were cultured in greenhouse at 20±30C, and 1000±200 !mole photons m-2 sec-1 photosynthetic photon fl ux density, and watered with deionized water as needed without supplemental fertilization. The seedlings were examined after 6 months of cultivation. Morphology of mycorrhiza. After 6 months, the roots of seedling were sampled and cleaned with water in a supersonic oscillator (Upson et al., 2007). Roots were cut into 1 cm segments, cleared in 10% KOH, treated with 3% H2O2 and 1% HCl, and then stained with 0.05% trypan blue. The morphology of mycorrhiza was observed with a stereomicroscope (Abbott, 1982; Brundrett et al., 1996). The percentage of root colonization was calculated accordingly (Phillips & Hayman, 1970). For ultrastructural study, root samples were fi xed with 2.5% glutaraldehyde and 4% paraformaldehyde fi xative in a phosphatebuffered solution (0.1 M, pH 7.0) for 4 hr at room temperature, then rinsed with the phosphate-buffered solution three times each time for 15 min, followed by serial dehydration in 30, 50, 70, 80, 95, and 100% ethanol and 100% acetone, and fi nally dried in a criticalpoint dryer using liquid carbon dioxide. Dried materials were mounted on an aluminum stub with adhesives, coated with gold, and observed with a scanning electron microscope (Brundrett et al., 1996). Mycorrhizal root colonization was assessed by grid line intersection method (Giovannetti & Mosse, 1980). Mineral content analysis. For mineral content analysis, root, shoot, and leaf samples were oven-dried at 70±2oC and digested with concentrated H2SO4 and H202. Nitrogen contents of root, shoot, and leaf were estimated by microkjeldahl method (MacDonald, 1977). Phosphorus, potassium, calcium, sodium, and magnesium contents were estimated by inductively coupled plasma atomic emission spectrometry. Quantifi cation of mycorrhizal dependency. Mycorrhizal dependency was defi ned as the ratio of the dry weight of seedlings with and without inoculation with AMF (Graham & Syvertsen, 1985). Statistical analysis. After 6 months, noninoculated and inoculated seedlings were harvested. Growth characters, such as plant height, root length, leaf and root fresh weights were recorded. Dry weight was determined after drying in an oven at 70±2°C for 48 hr. Statistical analysis was performed using the software Statistical Package for the Social Science (SPSS 12.0, IL. USA) for windows program. All data represent means of 4 separate experiments ± standard error (n = 4). Differences in growth rates among treatments were analyzed by Tukey’s multiple range test at P<0.05 signifi cant level.
Spore morphology and identifi cation. After extraction, spores of AMF were collected. The dominant species was identifi ed as Acaulospora scrobiculata. Spores were formed singly in soil and distributed abundantly in sampling sites. Spores of A. scrobiculata were orange yellow to orange brown, and globose to subglobose (Figs. 1, 2). The morphology of the spore clearly showed that tits cell wall is consisted of 3 layers (L1, L2 and L3). Also, germinal wall, hypha remnant and cicatrix were found (Fig. 2 a-c). The ultrastructure showed that the spore surface is smooth with little pits (Fig. 2b-d). Morphology of arbuscular mycorrhiza. Vesicles, intracellular and intercellular hyphae were observed in the root cortical cells of crested wheatgrass collected from the sampling sites in Mongolian grassland (Fig. 3). Root colonization rate in crested wheatgrass collected from its natural habitat was 25.5%. The crested wheatgrass seedlings inoculated with A. scrobiculata were found to produce more leaves and roots than the control seedlings (Fig. 4a-b). After six months of cultivation, AM fungal colonization was as high as 100 percent in crested wheatgrass seedlings inoculated with A. scrobiculata (Fig. 5). Roots of seedlings inoculated with A. scrobiculata produced a network of external hyphae (Fig. 5a-b). Staining of root samples rev ealed that AM developed well in the roots of inoculated seedlings. Arbuscules and vesicles were present abundantly in root tissues of inoculated seedlings (Fig. 5c, e-f). In roots of inoculated seedl ings, hyphae extended intercellularly and formed arbuscules, which showed Arum-type morphology (Fig. 5e-f). The number of vesicles was very high in roots, where an average of 12-15 vesicles per cm root was observed (Fig. 5c). However, no hypha e, arbuscules, and vesicles were found in the roots of the non-inoculated seedlings (Fig. 6). Seedling growth. In the fi rst two weeks, relatively uniform growth of inoculated and noninoculated seedlings was observed. However, after three weeks, the infl uence of inoculation on seedling growth was evident as the growth of inoculated seedlings increased sharply. After eight weeks, there were signifi cant differences in growth between inoculated and noninoculated seedlings (Fig. 4). After six months, seedling height and root length of inoculated seedlings were signifi cantly higher (175% and 191%, respectively) than the control (Fig. 4ab; Table 1). Meanwhile, fresh and dry weights of leaves and roots of inoculated seedlings were also signifi cantly higher than the control (Table 2).
After six months of cultivation, the nitrogen, phosphorus, potassium, calcium, sodium, and magnesium concentrations of roots, stems, and leaves of inoculated crested were also higher than those of the control (Tables 3-6). Mycorrhizal dependency of the crested wheatgrass on arbuscular mycorrhiza with A. scrobiculata was estimated to be 839.5% based on biomass accumulation.
In nature, more than 90% of all higher plants are associated with mycorrhiza and more than 80% of these plants form AM relationships (Smith & Read, 2008). In this study, the dominant spore isolated from rhizosphere soil of Mongolian crested wheatgrass was identifi ed as AMF Acaulospora scrobiculata (Fig. 1-2). AM characteristics were observed in root cortical cells of crested wheatgrass (Fig. 3). Other fungi, such as Glomus macrocarpum, G. macrocarpum var. macrocarpum were also found to form AM with Agropyron smithii in Colorado, USA (Singh, 2004). Generally, the soils of Mongolian grassland are nutrient defi cient. In this study, we found that percent root colonization was as low as 25.5% in crested wheatgrass collected from the grassland at Bogd Mountain near Ulaanbaatar, Mongolia. AMF effi ciency can be measured in terms of host plant growth under different environmental conditions (Ruiz-Lozano et al., 1996). Our study showed that crested wheatgrass seedlings inoculated with A. scrobiculata increased the growth of leaves and roots signifi cantly under greenhouse condition (Fig. 4, Table 1). And, six months after inoculation, 100% root colonization and high spore production were observed in crested wheatgrass seedlings (Fig. 5). Based on the frequency of occurrence, A. scrobiculata was clearly the dominant AM fungal species. The mycorrhizal infl uence was more pronounced in aerial biomass than in root biomass (Table 2) which may be because of a proportionally greater allocation of carbohydrates to the shoot than to the root tissues after AMF colonization (Schwab et al., 1982). Previous works reported that mycorrhizal fungi are able to increase the shoot height, fruit number, shoot and root biomass of plants (Andrade et al., 1998; Mendeiros et al., 1994; Utkhede, 2006). Smith and Read (2008) reported that AMF increase plant growth mainly by increasing nutrient acquisition and thus enhanced the plant’s resistance to biotic and abiotic stresses. A possible mechanism by which AM fungi increase root size could be that AM fungi modify root function and change root architecture (Andrade et al., 1998). The Arum-type structures were found in roots of crested wheatgrass seedlings inoculated with A. scrobiculata (Fig. 5e). Arum-type structures have been reported to associate with cultivated plants, which are usually fast-growing with plenty of sunlight (Smith & Smith, 1997). O’Connor et al. (2001) also reported that the Arum-type structures are present in all of the 21 species of herbaceous AM plants growing in Australian desert. AM inoculation signifi cantly increased the nitrogen and mineral (P, K, Ca, Mg, and Na) contents in roots, stems and leaves of crested wheatgrass seedlings inoculated with A. scrobiculata (Table 3-6). A previous study has shown that growth and mineral nutrition of plants are commonly enhanced by AMF inoculation (Clark & Zeto, 2000). Our results confi rm signifi cant effects of A. scrobiculata inoculation on growth of crested wheatgrass. The 839.5% mycorrhizal dependency of crested wheatgrass with A. scrobiculata indicated a high degree of responsiveness of crested wheatgrass growth to mycorrhizal colonization.
We wish to thank Professor Maurice S. B. Ku for reviewing the manuscript. Financial support from National Chiayi University is gratefully acknowledged.
- Abbott, L. K. 1982. Comparative anatomy of vesicular-arbuscular mycorrhizas formed on subterranean clover. Aust . J. Bot. 30: 485-499.
- Andrade, G., Mihara, K. L. & Liderman, R. G. 1998. So il aggregation status and rhizobacteria in the mycorrhizosphere. Plant Soil, 202: 89-96.
- Blas zkowski, J., Tadych, M. & Madej, T. 2002. Arbuscular mycorrhizal fungi (Glomales, Zygomycota) of the Bledowska Desert Poland. Acta Societatis Botanicorum Poloniae, 71: 71-85.
- Brundrett, M. C. 1991. Mycorrhizas in natural ecosystems. Advances in Ecological Research, 21: 171-313.
- Brun drett, M. C., Bougher, N., Dell, B., Grove, T. & Malajczuk, N. 1996. Working with mycorrhizas in forestry and agriculture. Australian Centre for International Agricultural Research Monograph, 32: 1-374.
- Clark, R. B. & Zeto, S. K. 2000. Mineral acquisition by arbuscular mycorrhizal plants. J. Plant Nutr., 23: 867-902.
- Dani els, B. A. & Skipper, H. D. 1982. Methods for the recovery and quantitative estimation of propagules from soil. In N. C. Schenck (ed.): Methods and Principles of Mycorrhizal Research. The American Phytopathological Society, pp. 29-36.
- Gerdemann, J. W. & Nicolson T. H. 1963. Spores of mycorrhizal Endogone species extracted from soils by wet sieving and decanting methods. Trans. Br. Mycol. Soc., 46: 235-244.
- Giovannetti, M. & Mosse, B. 1980. An evaluation technique for measuring vesicular and arbuscular mycorrhizal infection in roots. New Phytol., 84: 489-500.
- Graham, J. H. & Syvertsen, J. P. 1985 Host determinants of mycorrhizal dependency of citrus rootstock seedlings. New Phytol., 101: 667-676.
- Guadarrama, P. & Alvarez-Sanchez, F. J. 1999. Abundance of arbuscular mycorrhizal fungi spores in different environments in a tropical rain forest Veracruz Mexico. Mycorrhiza, 8: 267-270.
- Gupt a, R. & Kumar, P. 2000. Mycorrhizal plants in response to adverse environmental conditions. In: Mycorrhizal Biology, Plenum Publisher, India, 67-84 pp.
- Hartnett, D. C., Hetrick, B. A. D., Wilson, G. W. T. & Gibson, D. J. 1993. Mycorrhizal infl uence on intra- and interspecifi c neighbour interactions among co-occurring prairie grasses. J. Ecology, 81: 787-795.
- Hedlund, K. 2002. Soil microbial community structure in relation to vegetation management on former agricultural land. Soil Biology and Biochemistry, 34: 1299-1307.
- Hetrick, B. A. D., Kitt, D. G. & Wilson, G. T. 1988. Mycorrhizal dependence and growth habit of warm-season and cool-season tallgrass prairie species. Can . J. Bot., 66: 1376-1380.
- Lugo, M. A. & Cabello, M. N. 2002. Native arbuscular mycorrhizal fungi (AMF) from mountain grassland (C#rdoba Argentina) I. Seasonal variation of fungal spore diversity. Mycologia, 94: 579-586.
- MacDonald, D. C. 1977 Methods of soil and tissue analysis used in the analytical laboratory. Canadian Forestry Service Information Report MM-X-78.
- Marler ,M. J., Zabinski, C. A. & Callaway, R. M. 1999. Mycorrhizae indirectly enhance competitive effects of an invasive forb on a native bunchgrass. Ecology, 80: 1180-1186.
- O’Connor, P.J., Smith, S.E. & Smith, F.A. 2001. Arbuscular mycorrhizal associations in the southern Simpson Desert. Aust. J. Bot., 49: 493-9.
- Oehl, F., Sieverding, E., Ineichen, K., Mader, P., Wiemken, A. & Boller, T. 2009. Distinct sporulation dynamics of arbuscular mycorrhizal fungal communities from different agroecosystems in long-term microcosms. Agriculture, Ecosystems and Environment, 134: 257-268.
- Phillips, J. M. & Hayman, D. S. 1970 Improved procedures for clearing and staining parasitic and arbuscular mycorrhizal fungi for rapid assessment of infection.
- Trans. Br. Mycol. Soc., 55: 158-161. Retzer, V. 2007. Forage competition between livestock and Mongolian Pika (Ochotona pallasi) in southern Mongolian mountain steppes. Basic and Applied Ecology, 8: 147- 157
- Ruiz-Lozano, J. M., Azcon, R. & Gomez, M. 1996. Allev iation of salt stress by arbuscular mycorrhizal Glomus species in Lactuca sativa plants. Physiol. Plant., 98: 767-772.
- Sanders, I. R. & Koide, R. T. 1994. Nutrient acquisition and community structure in cooccurring mycotrophic and nonmycotrophic old-fi eld annuals. Func. Ecol., 8: 77-84.
- Schwab, S. M., Johnson, E. L. V. & Meng, J. A. 1982. The i nfl uence of Simazine on formation of vesicular-arbuscular mycorrhizae in Chenopodium qunona Willd. Plant Soil, 64: 283-287.
- Singh , S. 2004. Effect of soil moisture on arbuscular mycorrhizal development in plants. Part 1. In grasses, cereals, and fodder crops. Mycorrhiza News, 15(4): 2-19.
- Smith, S. E. & Read, D. J. 2008. Mycorrhizal symbiosis. 3rd ed. Academic Press, London. Smith, F. A. & Smith, S. E. 1997. Structural diversity in (vesicular) arbuscular mycorrhizal symbioses. J. New Phytol., 137: 373-388.
- Stinson, K. A., Campbell, S. A., Powell, J. R., Wolfe, B. E., Callaway, R. M., Thelen, G. C. & Hallett, S. G. 2006. Invasive plant suppresses the growth of native tree seedlings by disrupting belowground mutualisms. Biology, 4(5): 140.
- Tomme rup, I. C. 1992. Methods for the study of the population biology of vesicular-arbuscular mycorrhizal fungi. Methods in Microbiology, 24: 23-51.
- Trapp e, J. M. 1981. Mycorrhizae and productivity of arid and semiarid rangelands. In J. T. Manasah & E. J. Briskey (eds): Advances in Food Producing Systems for Arid and Semiarid Lands. Academic Press, New York. pp. 581-599.
- Ulrich, H., Katharina, J. & Hermann, B. 2002. Towards growth of arbuscular mycorrhizal fungi independent of a plant host. Applied Environment Microbiology, 68: 1919-1924.
- Upson, R., Read, D. J. & Newsham, K. K. 2007. Microscopy analyses of fi eld-collected Cephaloziella varians. New Phytol., 176: 460- 471.
- Utkhe de, R. 2006. Inc reased growth and yield of hydroponcally grown green house tomato plants inoculated with arbuscular mycorrhizal fungi and Fusarium oxysporum f. sp. radicislycopersici. Biological Control, 51: 393-400.
- Van Der Heijden, M. G. A., Klironomos, J. N., Ursic, M., Moutoglis, P., Streitwolf-Engel, R., Boller, T., Wiemken, A. & Sanders, I. R. 1998. Mycorrhizal fungal diversity determines plant biodiversity ecosystem variability and productivity. Nature, 396: 69-72.