Colonization of wheat {Triticum vulgare L.) by Ng-fixing cyanobacteria: I. A survey of soil cyanobacterial isolates forming associations with roots

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1 Phytol. (1991), 118, Colonization of wheat {Triticum vulgare L.) by Ng-fixing cyanobacteria: I. A survey of soil cyanobacterial isolates forming associations with roots BY M. GANTAR\ N. W. KERBY*, P. ROWELL AND Z. OBREHT^ Department of Biological Sciences, University of Dundee, Dundee DDl 4HN, United Kingdom ^ Institute of Biology, University of Novi Sad, Novi Sad, Yugoslavia (Received 24 October 1990; accepted 26 March 1991) SUMMARY A variety of heterocystous, Nj-fixing cyanobacteria, isolated from soils, were identified as members of the genera, Anabaena and Cylindrospermum. These isolates were tested for their ability to fortn associations with the roots of wheat seedlings grown in liquid culture. Two types of associations were recognized: loose associations with cyanobacteria] growing between root hairs, which were typical of the Anabaena isolates, and tight associations of microcolonies in intimate association with the root surface, which were restricted to certain isolates. Differences in nitrogenase activities of the free-living and associated cyanobacteria, together with the effects of added nitrate, indicate that nitrogenase activity may be influenced by the plant and/or its products. Key words: Anabaena, cyanobacteria, nitrogen-fixation,, Triticum vulgare (wheat). INTRODUCTION Cyanobacteria are photosynthetic prokaryotes, many species of which fix Ng. They are almost exclusively free-living and are ideally suited to an independent existence. Nevertheless, a few species form specific associations with various plant groups and fungi (Stewart, Rowell & Rai, 1983; Rowell, Rai & Stewart, 1985; Rai, 1990). In such symbiotic associations the cyanobacteria (cyanobionts) become modified morphologically, physiologically and biochemically while retaining their ability to fix atmospheric N^ (Stewart & Rowell, 1977). N^ fixed by the cyanobionts is transferred to, and used by, their plant or fungal partners (see Rai, 1990). At present, there is much interest in establishing novel associations between higher plants and a variety of Ng-fixing microorganisms. The long-term objective is to introduce the ability to fix Ng into plants, thereby reducing the demand for nitrogenous fertilizers. The creation of novel symbioses with Ngfixing microorganisms may provide an alternative method to the introduction of isolated nif genes into plants. To date, novel associations between * To whom correspondence should be addressed. Rhizobium and rice (Al-Mallah, Davey & Cocking, 1989) and between Anabaena and tobacco (Gusev et al., 1986) have been reported. The former association was obtained by treating rice roots with cell wall degrading enzymes followed by inoculation with rhizobia. Rhizobia were located between cells and also within degenerating cells. However, nitrogenase activity was barely detectable. The latter association relied on introducing Nj-fixing cyanobacteria into tobacco calli. Subsequently, cyanobacteria showing nitrogenase activity were located on the surfaces and in intercellular spaces of regenerated shoots. In this and the following paper we describe novel associations between cyanobacteria ( and Anabaena spp.) and wheat seedlings, and assess the ability of natural cyanobacterial soil isolates to form such associations. MATERIALS AND METHODS Organisms and grov)th conditions cyanobacteria were isolated from various soil types in Yugoslavia and are listed in Table 1. Cultures were maintained, without shaking, in BGl 1 medium (Rippka et al., 1979) with (BGIIN) or without (BGllfl) nitrate at a photon fiuence rate of 32

2 478 M. Gantar, N. W. Kerby, P. Rowell and Z. Obreht Figure 1. (a), surface view of a wheat root colonized hy Anabaena C5, which forms a loose association; (b), cross section of a wheat root colonized by Anabaena C5; (c), surface view of a wheat root colonized by 2S9B, forming a tight association; {d), cross section of wheat root colonized by 2S9B. CF, cyanobactenal ; CM, cyanobacterial microcolonies: Bar, \00/ira. 20/imol m ^s ^ and at room temperature. The colonization of roots of wheat (winter wheat, Triticum vulgare L., cv. Longbow) was carried out in the following manner: 3 d after germination wheat seedlings were placed in Eppendorf tubes that had their tips removed to permit root growth through the tube. The modified Eppendorf tubes were placed in 20 ml test tubes inoculated with 4 ml (28 fig, chlorophyll a) of 15 d old cyanobacteria] cultures and fresh BG-11 medium was added to cover the roots. Colonization of wheat roots (4 seedlings per treatment) was carried out with 12 different strains in BGll medium with or without nitrate. Cocultivation was carried out in an incubator with continuous light at a photon fluence rate of 10 //mol m"^ s"* at 24 C. During co-cultivation the medium in the test tubes was maintained at constant volume by adding fresh medium. After 15 d of co-culture, the type and degree of cyanobacterial colonization of the roots was determined. Nitrogenase activity Acetylene reduction was used to estimate nitrogenase activity (Stewart, Fitzgerald & Burris, 1967) in freeliving cyanobacterial cultures and for cyanobacteria in association with the roots of wheat seedlings. Tn both cases cyanobacteria and wheat plants were grown in BGl 1 medium with and without nitrate for 15 d. In the case of cyanobacteria associated with wheat roots, whole plants were placed with their roots immersed in 5 ml medium in sealed glass

3 Plant-cyanobacterial associations 479 Table 1. List of cyanobacterial isolates, morphological features and habitat Taxonomic assignment Isolate designation Morphological features Isolated from (soil type) Anabaena Anabaena Anabaena Cylindrospermum 2S3B 2S9B 2S6B LC2 2C1 SI 2S10 S8 C2 C3 C5 2S7 Hormogonia, Hormogonia, Hormogonia, Hormogonia Hormogonia, Hormogonia Hormogonia Black meadow Chernozem Chernozem Chernozem Chernozem vessels containing 17 ml of 10"i, (v/v) acetylene in air and were incubated on an orbital shaker at a photon fluence rate of 20/*mol m~^ s~^ for 60 min. Free-living cultures were assayed as described above except that 1 ml of culture was incubated in a 7 ml sealed glass bottle. Chlorophyll a determination Chlorophyll a (chl a) was extracted into methanol in the dark at 4 C. The absorbance at 663 nm was measured and chl a concentration calculated according to MacKinney (1941). Light and electron microscopy The surfaces of fresh roots and freshly cut transverse sections were examined with an Olympus BH-2 microscope. Cross sections of roots colonized by cyanobacteria were prepared for scanning electron microscopy by freezing in liquid nitrogen and sectioning with a razor blade. Pieces of roots were freeze-dried, mounted on stubs, coated with gold and examined with a Jeol-JSM-35 scanning electron microscope. RESULTS Certain cyanobacteria, isolated from different soil types, were able to colonize the roots of wheat seedlings grown in liquid culture (Figs la,\c). Table 1 lists the cyanobacterial isolates that were tested for wheat root colonization. Some isolates did not form associations with roots but continued to grow, free-living, in the grow^th media (Table 2). Two different kinds of association between roots and cyanobacteria were observed. Loose attachment of cyanobacteria was characterized by long loosely packed together, growing between root hairs (Fig. la). In the case of Anabaena C5, individual were observed on the surface of roots and around the root in freshly cut transverse sections (Fig. 1 b)\ tbey were not intimately associated witb root epidermis. Scanning electron microscopy (Fig. 2b) revealed tbat the cyanobacterial were entrapped in a mucilaginous layer. Tbis form of association occurred among both Anabaena and strains (Table 2). Two isolates of (2S3B, and 2S9B) formed tight associations. Figure 1 c sbow^s tightly packed of the isolate 2S9B forming elongated microcolonies/ packages on a root surface. Individual w ithin packages were not discernible although single were observed prior to microcolony formation. The cyanobacterial packages made an intimate contact with the root surface and followed the contours of the root epidermis (Figs. Id, 2b). In contrast to strains forming a loose attachment, these strains were not characterized by abundant mucilage production (Fig. 26) and were very difficult to remove by water washmg. Tbe two isolates (2S3B, 2S9B) tbat formed tight associations produced bormogonia and aseriate stages during tbeir developmental cycles (Table 1). The first stage of colonization of wheat roots by these isolates was probably the migration of hormogonia, with the subsequent development of aseriate pack-

4 480 M. Gantar, N. W. Kerhy, P. Rowell and Z. Ohreht Table 2. Colonization of wheat roots by cyanobacterial isolates and their rates of acetylene reduction when grown in medium with () or without ( N) nitrate Isolate 2S3B 2S9B 2S6B LC2 2C1 SI 2S10 S8 Anabaena C2 Anabaena C3 Anabaena C5 Cylindrospermum Association -hn -hn 2S7 T.A. T.A. T.A. T.A. Colonization (fig chl a plant"^) Acetylene reduction (nmol Cjtt /ig'^ chl a Associated '12 Free O'OO O'OO T.A., tight association;, loose attachment;, no attachment;, not detected; associated, cyanobacteria associated with roots; free, free-living cyanobacteria. ages. However, other isolates (e.g. 2S6B, S8) with a similar developmental cycle (Table 1) formed only a loose association (Table 2). One of the isolates (SI), which formed hormogonia and an in liquid culture, was not capable of forming an association with roots (Table 2); another strain { S8) formed an association only in the presence of nitrate. This suggests that factors other than these distinct developmental stages are involved in establishing associations with wheat roots. The extent of cyanobacterial colonization of roots was estimated by measuring the amount of chl a extracted from the colonized roots. Chlorophyll a was not detected in roots grown either in the absence of cyanobacteria or in the presence of cyanobacteria unable to form associations. The two tight associations with isolates had high chl a concentrations as did the loose association with Cylindrospermum. Nitrate stimulated the growth of cyanobacteria in association with roots (Table 2) and the chl a concentration was increased up to 45 times. In the absence of nitrate, nitrogenase activity was always higher in free-living cultures than in associations. Highest nitrogenase activity was found when roots were colonized by Anabaena C5 grown in nitrogen-free medium. With the exception of Anabaena C5, the nitrogenase activity of free-living isolates was suppressed by nitrate (Table 2). However, addition of nitrate restored the nitrogenase activity of strains in tight association with wheat roots to a level similar to that found in freeliving cultures incubated without nitrate. In contrast, nitrate inhibited the nitrogenase activity oi Anabaena associations. Growth of wheat was not significantly affected during 15 d in the absence of nitrate. In addition to forming a tight association, one strain { 2S9B) also appeared to penetrate some root cells (Fig. 3). Cyanobacteria were found in both epidermal and cortical cells and were present either as single or as packages of cells. DISCUSSION Several isolates of soil cyanobacteria, mainly of the genera and Anabaena, are capable of forming novel associations with wheat roots grown in liquid culture. To our knowledge this is the first report of such an association. Although Anabaena was capable of forming loose associations with wheat roots, tight associations were

$Plant-cyanobacterial associations 481 (a) Figure 2. Scanning electron microscopy of root cross sections, (a), loose association with Anabaena 05; (b). tight association with 2S9B. Bar. \00 /im.$

5 Plant-cyanobacterial associations 481 (a) Figure 2. Scanning electron microscopy of root cross sections, (a), loose association with Anabaena 05; (b). tight association with 2S9B. Bar. \00 /im. found only with certain strains. The ability of strains to form tight associations may be related, in part at least, to the characteristic developmental Hfe cycle of : beterocystous, hormogonia and an that consists of heterocystous packaged tightly together (LazarofT, 1973). However, since only two of the isolates tested formed tight associations, other factors must be important. These could involve recognition of specific molecules including lectins (Mellor et al., 1981; Ladha & Watanabe, 1984; McCowen, Mac- Arthur & Gates, 1987) and components of root exudates. Polysaccharides of plant and cyanobacterial origin may play an important role in the attachment of cyanobacteria to roots, as has been shown for the attachment of cyanobacteria to plant

6 482 M. Gantar, N. W. Kerby, P. Rowell and Z. Obreht Figure 3. Intracellular associations of 2S9B with epidermal and cortical cells of wheat roots. Arrows indicate cells containing associated cyanobacteria. Bar, 100/(m. cells and inert surfaces (Robins et al., 1986). Specificity of associations between cyanobacteria and other organisms has been reported; for example, associations between the higher plant Gunnera and members of the genus (Bonnett & Silvester, 1981). Additionally, studies on reconstitution of symbioses with Anthoceros and Blasia have shown that only certain species of form successful associations (Rodgers & Stewart, 1977; Enderlin & Meeks, 1983). Addition of ammonia to Ng-fixing cultures of cyanobacteria almost invariably suppresses nitrogenase activity and heterocyst development, whereas nitrate addition may do so either completely or partially (Meeks e/a/., 1983; Rowell & Kerby, 1991). The nitrogenase activity of all except one of the isolates was severely inhibited by addition of nitrate to free-living cultures. The insensitivity oi Anabaena C5 nitrogenase to nitrate is possibly due to an inability either to take up or to metabolize nitrate. In association with wheat roots, nitrogenase activity in Anahaena C5 was suppressed in the presence of nitrate. This may have been a consequence of the liberation of products of nitrate assimilation by the plant. Nitrogenase activity of other Anabaena isolates was suppressed by nitrate, whether the cyanobacterium was free-living or in association. However, in those isolates forming tight associations, nitrogenase activity, which was inhibited by nitrate in free-living culture, was stimulated by nitrate in association. This observation strongly suggests that plant products, which may be dependent on the composition of the growth medium, can infiuence nitrogenase activity in the associated cyanobacteria. In addition to demonstrating associations of cyanobacteria with the root surfaces of wheat we present preliminary evidence that cyanobacteria may penetrate root tissues. The mode of cyanobacterial penetration into the root cells and the benefit (if any) to the plant of such an intracellular association is, as yet, unclear. An ultrastructural study of the tight association between 2S9B and wheat roots, together with information on the distribution of cyanobacteria within the plant, is presented in the accompanying paper (Gantar, Kerby & Rowell, 1991). ACKNOWLEDGEMENTS This work was supported by the British Council, the Scientific Fund of the Province of Vojvodina and the University of Dundee. REFERENCES AL-MAU-AH, M, K., DAVEY, M. R. & COCKING, E. C- (1989). Formation of nodular structures on rice seedlings by rhizobia. Journal of Experimental Botany 40, BONNETT, H. & SILVESTER, W. B. (1981). Specificity in the Gunneraostoc endasymhiosis. New Phytologist 89, ENDERLIN, S. & MEEKS, J. C. (1983). Pure culture and reconstitution of the Anthocerosostoc symbiotic association. Planta 158, GANTAR, M., KERBY, N. W. & ROWELI., P. (1991). Colonization of wheat by N^-fixing cyanobacteria II, An ultrastructural study of the association. Nev; Pfiytologist 118, GusEv, M. V., KoRZHENEvsKAYA, T. G., PYVOVAROVA, L. V., BALLINA, O. I. & BuTENKO, R. G. (1986). Introduction of nitrogen-fixing cyanobacterium into shoot regenerates. Planta 167, 1-8. LADHA, J. K. & WATANABE, 1. (1984). Antigenic similarities among Anabaena azollae separated from different species of Azolla. Biochemical and Biophysical Research Communications 109, LAZAROFF, N. (1973). Photomorphogenesis and nostocacean development. In; The Biology of Blue-Green Algae (Ed. by N. G. Carr & B. A. Whitton), pp Blackweli Scientific Publications, Oxford. MACKINNEY, G. (1941). Absorbtion of light by chlorophyll solutions. Journal of Biological Chemistry 140, MEEKS, J. C, WYCOFF, K. L., CHAPMAN, J. S. & ENDERLIN, C. S.

7 Plant-cyanobacterial associations 483 (1983). Regulation of expression of nitrate and dinitrogen assimiialion by Anahaena species. Applied and Environmental Microbiology is. 13S1-13.S9. MELLOR, R. B., GADD. G. M., ROWEI.L. P- & STEWART, W, D. P. (1981). A phytohaemaggluiinin from the Azolla-Anabaena symbiosis. Biochemical and Biophysical Research Communications^, McCowEN, S. M.. MACARTHUR, L- & GATES, J. E. (1987). A^olla fern lectins that specifically recognize endosymbiotic cyanobacteria. Current Microbiology 14, RAI, A. N. (1990). CRC Handbook of Symbiotic Cyanobacteria. CRC Press, Boca Raton. RipPKA, R., DERUELI.ES, J., WATERBURY, J. B.. HERDMAN, M. &L STAKIER, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111, ROBINS, R- J., HALL, D. O., SHI, D.-J., TURNER, R. J, & RHODES, M. J.C. (1986). Mucilage acts to adhere cyanobacteria and cultured plant cells to biological and inert surfaces. FEMS Microbiology Letters 34, RoDGERS, G- A. & STEWART, W. D. P. (1977). The cyanophytahepatic symbiosis. I Morpbology and physiology. New Phytologist 78, ROWELL, P. & KERRY, N. W. (1991). Cyanobacteria and tbeir symbionts. In: Biology and Biochemistry of Nitrogen Fixation (Ed. by M, Dilworth & A- Glenn ), pp. 373^07. Elsevier Science Publications, Amsterdam. ROWELL, P., RAI, A. N. & STEWART, W. D. P. (1985). Studies on the nitrogen metabolism of the lichens Peltigera aphthosa and Peltigera canina. In ; Lichen Physiology and Cell Biology (Ed. b\' D. H. Brown), pp Plenum Press, New York, London. STEWART, W. D. P., FITZGERALD, G. P. & BURRIS, R. H. (1967). Jn situ studies on N^-fixation using tbe acetylene reduction technique. Proceedings of the National Academy of Sciences. USA 58, STEWART, W. D. P. & ROWELL, P. (1977). Modifications of nitrogen-fixing algae in lichen symbioses. Nature 265, STEWART, W. D. P., ROWELL, P. & RAI, A. N. (1983)- Cyanobacteria-eukaryotic plant symbioses. Annales de Microbiologie {Institut Pasteur] 145B,

Colonization of wheat {Triticum vulgare L.) by Ng-fixing cyanobacteria: II. An ultrastructural study

Phytol. (199!), 118, 485-492 Colonization of wheat {Triticum vulgare L.) by Ng-fixing cyanobacteria: II. An ultrastructural study BY M. GANTAR^ N. W. KERBY* AND P. ROWELL Department of Biological Sciences,