the initiation and development of root nodules of the actinorhizal tree species, Elaeagnus angustifolia L. (Elaeagnaceae). Two pure cultured Frankia strains were ...
Protoplasma 128, 107-119 (1985)
PROTOPL6SMA 9 by Springer-Verlag 1985
The Initiation, Development and Structure of Root Nodules in Elaeagnus angustifolia L. (Elaeagnaceae) I. M. MILLER and D. D. BAKER* Battelle---C. F. Kettering Research Laboratory, Yellow Springs, Ohio Received September 17, 1984 Accepted May 14, 1985
Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species, Elaeagnus angustifolia L. (Elaeagnaceae). Two pure cultured Frankiastrains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such as Alnus or Myrica the mode of infection of Elaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomycete Frankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizing Frankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizing Frankia. No random division of root cortical cells, indicative of prenodule formation was observed in Elaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancing Frankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative
* Correspondence and Reprints: Battelle--C. F. Kenering Research Laboratory, 150 East South College Street, Post Office Box 268, Yellow Springs, OH 45387, U.S.A.
developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.
Keywords: Actinorhizal root nodules; Development; N 2 fixation; Elaeagnus," Frankia; Symbiosis; Ultrastructure.
1. Introduction In recent years there have been numerous studies which
describe the initiation and development of nitrogen fixing root nodules induced in certain host plant species by soil-borne actinomycetes of the genus Frankia. Nodule initiation and early development have been studied most extensively in Alnus (Betulaceae) symbiotic associations (PoMMER 1956, TAUBERT1956, ANGUI~O CARMONA 1974, AN6ULO CARMONA et al. 1976, LALONDE1977, BERRYand TORREY 1983). In a general survey of observations on the Alnus system, LALONDE (1977) summarized that in reaction to inoculation the plant root hairs, which are initially straight, become deformed and some are subsequently invaded by the actinomycete. Endophytic hyphae then enter into and proliferate in the cortical cells of the root underlying the root hair, this causing limited division of the cortex and giving rise to a prenodule (CALLAHAMand TORREY 1979). During this period the endophyte induces the formation of a primary nodule meristem from the pericycle of the root vasculature. As the primary nodule tissue grows it becomes infected by penetration of its cortex by endophyte already resident in the prenodule. A similar process of infection and early development has been shown to occur in other actinorhizal symbiotic
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systems. Root hair deformation, invasion and prenodule formation have been shown in Casuarina (TORREY 1976, KANTand NARAYANA1977, CALLAHAM et al. 1979), in Comptonia peregrina (CALLAHAMand TORREY t977, CALLAHAMet al. 1979) and in Myrica (FLETCHER 1955, TORREY and CALLAHAM 1979, CALLAHAMet al. 1979). This considerable quantity of evidence has led to the acceptance of the view that root hair deformation, invasion and prenodule formation is the ubiquitous and exclusive mode of infection in actinorhizal symbiotic systems. However it must be borne in mind that all the evidence comes from studies, albeit extensive and rigorous, on a limited number of species from only three out of the eight families which are known to exhibit actinorhizal symbioses. In the course of studies on nodulation in Elaeagnus spp. (EIaeagnaceae) it was observed that nodulation occurred in regions of the root devoid of root hairs; indeed, nodule formation occurred preferentially in root hair free areas. This present work describes for the first time, using complementary light, scanning (SEM) and transmission electron (TEM) microscopy, direct intercellular penetration as the mode of infection and nodule induction in Elaeagnus angustifolia L. (Elaeagnaceae). 2. Materials and Methods
2.2. Host Plant Material Embryos from the seeds of Elaeagnus angusl~folia L. were excised, sterilized in 30% hydrogen peroxide and germinated for 7 days in the dark under moist sterile conditions. Seedlings were dip inoculated in suspensions of homogenized pure cultured Frankia and then transferred either to glass water culture jars containing 400ml modified Crone's solution (BOND 1951) or to sterile tubes containing 25 grams of turf ace soil material (International Minerals and Chemical Corp.). Sample material was removed from the plants between 7-33 days after inoculation.
2.3. TEM Root nodules at various stages of development were removed from the plants and fixed in 3 % glutaraldehyde in 0.1 M phosphate buffer, pH6.8 at 20~ for 4 to 12hours depending on the size of the specimen. After brief rinsing in distilled water, the nodules were postfixed in 2% buffered osmium tetroxide for 2 to 4hours and then dehydrated in a graded water/ethanol/1,2-epoxypropane series. The dehydrated material was embedded in Poly/Bed 812 (Polysciences, Inc.) and thin sections cut using an LKB Ultratome III. The sections were stained in methanolic uranyl acetate for 45 minutes followed by alkaline lead citrate for 10 minutes and viewed in a Philips 200 TEM.
2.4. Light Microscopy Processing was as for routine TEM except that 2gm semi-thin sections were cut using the Ultratome, mounted on glass slides, stained with either 1% methylene blue in 1% sodium tetraborate or l% toluidine blue in 1% sodium tetraborate and examined with a Leitz Ortholux II photomicroscope.
2.1. Frankia Cultures Two pure-cultured Frankia strains were used as inocula in these developmental studies. The first strain, WgCc 1.17, originally isolated from Colletia crueiata, was obtained from Dr. Antoon Akkermans of the Agricultural University, Wageningen, The Netherlands, and propagated on DPM broth (BAKER and O'KEEFE 1984). The second strain, G 2 (syn. ORS 020604), originally isolated from Casuarina equisetifolia was obtained from Dr. Yvon Dommergues of the Laboratory of Microbiology, ORSTOM, Dakar, Senegal and propagated on YCz broth (BAKER and O'KEEFE 1984). Both of these strains are infective and effective for Elaeagnus.
2.5. S E M Sections of root containing nodules were fixed in 3 % glutaraldehyde in 0.1 M phosphate buffer, pH 6.8, for 18 hours at 20 ~ Following postfixation in 2% buffered osmium tetroxide the nodules were dehydrated in a graded water/ethanol/l,2-epoxypropane/Freon I13 series. The nodules were then dried in a Polaron E 3000 critical point drier using CO 2 as the drying solvent, coated with a 30 nm layer of platinum using a Polaron E5100 sputter coater and examined in an ISI DS 130 SEM.
Fig. 1. TS of a small lateral root from a host plant grown in water culture and inoculated with Frankia sp. WgCc 1.17 showing initial infection sites (IS) at the epidermal layer. Although this section shows a developmentally very early stage, infection sites can be readily identified by the presence of a densely staining intercellular material secreted from the epidermal and exodermal cells in response to the invading actinomycete hypha. Cell division in the pericycle, which will give rise to the pl:imary nodule primordium (P), can be seen. CO root cortex, EP epidermis, EX exodermis. x 205 Fig. 2. TS of a small lateral root from a host plant grown in turf ace and inoculated with Frankia sp. WgCc 1.17. Because the infection in this root is at a more developmentally advanced stage than that in Fig. i, a primary nodule primordium (NP) can be seen. x 197 Fig. 3. TEM of an infection site. The Frankia hypha (H) has penetrated the thin cuticle (C) and then penetrates the middle lamella between two epidermal cells. Note the densely stained material in the intercellular spaces (arrow). A number of eubacteria populate the surface of the root (B). x 2,620 Fig. 4. Detail of the point of penetration of the Frankia hypha into the middle lamella (ML) between two epidermal cells (EP). x 22,690
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3. Results
3.1. Root Anatomy When grown in standing water culture the roots of Elaeagnus seedlings develop few root hairs; when grown in turface, certain regions of the root develop root hairs normally although much of the root surface remains root hair free. The root structure of Elaeagnus from hydroponic culture is as shown in Fig. 1. The vascular tissue appears to be a diarch bundle and is surrounded by a tanninised pericycle. The outer region consists of a small-celled epidermis and an underlying layer of somewhat larger exodermal cells; the cortex is composed of large, irregular cells between which are enlarged air-spaces (Fig. 1). The basic structure of roots from turface grown plants is identical to that of hydroponic roots, although differences in hydration levels leads to visible differences in cell shapes (Fig. 2). In general the cells of turface grown roots are more irregular and compressed with less intercellular space between them; the epidermal cells, full and cuboidal in hydroponic roots, are flattened and elongate in turface grown roots.
3.2. Root Penetration by Frankia The infection process in Elaeagnus, in contrast to that reported in other actinorhizal associations is by direct apoplastic penetration, wherein the infecting hyphal strand breaks through the cuticle and enters the intercellular spaces of the root cortex by penetrating and growing through the middle lamella between two adjacent epidermal cells (Figs. 1-4). The same mode of entry was observed in plants grown either in turf ace or water culture inoculated with either Frankia spp. WgCc 1.17 or G 2 . Although nodules were observed more frequently in root hair free areas, in those regions of the root where both nodules and root hairs did occur, infection was always by direct epidermal penetration.
Neither deformation nor invasion of the root hairs was observed. Sites of infection of the root by the actinomycete can be identified readily; in response to penetration by the hyphae, both the epidermal and exodermal cells are stimulated to secrete into the intercellular spaces a material which stains intensely with methylene blue (Figs. 1 and 2). At the ultrastructural level this material appears electron dense (Fig. 3). Frequently, more than one infection site is found in the same transverse section of the root (Fig. 1).
3.3. Nodule Induction and Development At an early stage in the infection process, when the Frankia hypha has just entered the middle lamella between two epidermal cells, the stimulation of division in the pericycle has occurred and is already visible (Fig. 1). This division, which will result in a primary nodule primordium is periclinal and occurs in the pericycle either at the protoxylem poles or at 90 ~ to the poles. As the infection proceeds, the endophytic hypha completely passes through the middle lamella between the epidermal cells and moves into the material-filled intercellular spaces between the epidermal and exodermal cells (Fig, 5). The endophyte possibly digests the secretory material as a zone of apparent dissolution of this material is evident around the outer surface of the hypha (Fig. 5). As the hypha penetrates deeper into the root tissue, the cortical cells are stimulated to secrete quantities of material which, because of the large size of the intercellular spaces in the cortex, only partially fills these spaces (Figs. 6 and 7). At the light microscope level, the endophytic hyphae cannot be visualized due to the intensity of the staining reaction of the secretory material (Fig. 7). At the ultrastructural level however, the path of the endophyte from the extracellular spaces of the epidermal/exodermal region into the cortical apoplast can be easily followed (Fig. 8).
Fig. 5. The hypha has completelypenetrated the middle lamellabetween two epidermalcells and has passed into the material-filledintercellular spaces between the epidermal and exodermalcells. A zone of dissolution of the secretory material can be seen around the outer surface of the hypha suggesting enzymatic digestion of the material by the endophyte (arrow). EP epidermal cell, EX exodermal cell, H hypha, X external environment, x 10,800 Fig. 6. TS of a small lateral root at an infection site (boxed).denselystaining secretorymaterial fillsthe intercellular spacesbetweenthe epidermal and exodermalcells, but only partly fills the intercellular spaces in the root cortex. The primary nodule primordium (NP) is developingand is surrounded by a tanninised protoperiderm. The epidermis is being disrupted by the developing nodule primordium (arrow). x 143 Fig. 7. Detail of the enclosed area in Fig. 6 showing the distribution of the dense intercellular material, x 597 Fig. 8. TEM of an area similarto that shown in Fig. 7. The path of the endophyte through the epidermaland exodermallayersinto the cortex can be traced (arrows). Note the eubacteria at the root surface. CO cortical cell, EP epidermal cell, EX exodermal cell. x 3,342 Fig. 9. SEM of a portion of a small lateral root where a nodule, at the same developmentalstage as the nodule shown in Fig. 6 is emerging. The cells of the protoperiderm of the young primary nodule primordium are visible through the split in the epidermis (arrow). x 109
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Neither hypertrophy nor hyperplasia of the root cortical cells was found to occur in response to the invading actinomycete hyphae. Occassionally, around the surface of non-sterile roots microcolonies of eubacteria were observed which, although closely associated with the epidermis, never entered the host plant tissue (Figs. 3 and 8). Root nodules however, were formed on axenic plants inoculated only with frankiae. Under these circumstances no eubacteria were observed at the root surface. As the penetration of the root cortex proceeds, the primary nodule primordium enlarges considerably, now possesses a heavily tanninised protoperiderm and is beginning to disrupt and break apart the epidermal layers of the root (Figs. 6 and 9). The cells within the periderm become clearly delimited into a central vascular area and a peripheral nodule cortex region (Fig. 10). By the time the primary nodule primordium has reached this state of differentiation, the endophytic hyphae have traversed the root cortex, penetrated the nodule periderm and are beginning to ramify throughout the nodule cortex. The path of infection from the epidermis to the nodule periderm, as indicated by the occurrence of intercellular secretory material, can be seen in the very low power electron micrograph in Fig. 11. More than one infection path, approaching the same nodule, is commonly seen in the root cortex (Fig. 10). An interesting side feature of the progress of the endophyte through the root cortex is that in every young nodule studied, at least one endophytic septate vesicle can be found embedded in the intercellular secretory material (Figs. 11 and 12). The cells of the nodule periderm are also secretory, the intercellular spaces between them being entirely filled with a densely staining material similar to that secreted by the epidermal and root cortical cells; secretory vesicles containing a fibrous material are found both in the cytoplasm and fusing with the plasmalemma of the periderm cells (Fig. 13). The endophytic hyphae grow through this material to reach the nodule cortex.
By the time the endophytic hyphae have penetrated the nodule periderm, the basally located cells of the nodule cortex have matured and increased somewhat in size (Fig. 14). The nodule periderm is now well developed, particularly at the apex, and the primary nodule has disrupted and protrudes through the root epidermis considerably into the external environment (Figs. 14 and 15). The path from root epidermis to primary nodule taken by the actinomycete is still visible in the root tissue (Fig. 14). Once entry into the nodule cortex has been attained, the endophytic hypha disseminates rapidly throughout the small intercellular spaces which occur at the junctions of 3 or more of the relatively tightly packed nodule cortical cells (Figs. 16-18). These young cortical cells have numerous Golgi bodies, amyloplasts and mitochondria; strands of rough endoplasmic reticulum are also found located in the cell periphery near the cell wall (Figs. 17 and 18). Large secretory vesicles which contain a material similar in appearance to that which fills the intercellular spaces are found in the cytoplasm either alongside or fused with the plasmalemma (Figs. 17 and 18). The next stage in the process of nodule development is characterized at the light microscope level by hypertrophy of the basal cortical ceils of the developing nodule (Figs. 19 and 20). The hypertrophy leads to an overall increase in nodule size; this results in the root epidermis being disrupted and split for a considerable distance on either side of the nodule (Fig. 21). The cells in the outer layers of the nodule periderm have become enlarged and loosely knit giving the developing nodule a characteristic textured surface appearance (Fig. 21). 3.4. Host Cell Invasion
The endophytic hyphae, already present in the intercellular spaces at multiple cell junctions, proceed to grow along the middle lamellae between adjacent cells. Side branches arise from the hyphae in the middle tamellae which penetrate the host plant cells. At the points of penetration the host cell walls do not appear broken or contorted in any way indicative of a physi-
Fig. 10. LS of a young developingnodule showingthe central vascular tissue, the developingnodule cortex (NC) and the tanninised periderm (PE). Note the presence of two possible infectionpaths (arrows). x 124 Fig. 1I. Very low power TEM of infection path 1 shown in the previous figure. Note the presence of an endophytic septate vesicle in the intercellular material between two cortical cells (arrow). PE periderm cell. x 605 Fig. 12. Detail of the endophytein the root cortex.An endophyticseptate vesicleis present in the intercellularmaterial (arrow). Sectionsof the penetrating hyphae can be seen in the material among the cortical cells (CO). PE periderm cell. x 2,010 Fig. 13. TEM montage of the root cortex/periderm/nodulecortex boundary. Sections of hyphae can be seen in the material surrounding the periderm ceils (arrows). Secretoryvesicles (SV) containing a fibrous material can be seen in the cytoplasm and fusing with periderm cell membranes. CO root cortex, PE periderm cell, NC nodule cortex, x 2,270
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cally forced entry by the hyphae; indeed, the absence of damage along with the tight "fit" of the hyphae in the cell wall of the host is suggestive of a gentler enzyme mediated cell entry (Figs. 22 and 23). The plant cells undergo hypertrophy in response to being entered by the hyphal side branches (Figs. 20-22). Although the cell wall is penetrated by the endophyte, the integrity of the host cell membrane remains intact (Figs. 24 and 25), the endophyte being always separated from the host cell cytoplasm by the cell membrane. As soon as the hyphae penetrate the cell, secretory vesicles containing a fibrous material fuse with the host cell membrane (Figs. 24 and 25). The material in these vesicles encapsulates the bacterial hyphae while their delimiting membranes are added to the host cell plasmalemma, allowing an increase in the surface area of the host membrane in response to the continued growth of the hyphae. Having entered the host plant cell, the hyphae continue to ramify throughout the now considerably enlarged host plant cells (Figs. 26 and 27). Even at this advanced stage in development, with infection of the host plant cells well underway, infection paths from the epidermis to the basal nodule cortex can still be readily identified (Fig. 26). After the hyphae have penetrated the cell the distal end of some of the hyphal strands swell (Fig. 25) and subsequently develop into septate vesicles (Fig. 27), randomly distributed among the hyphae throughout the cell, in which the fixation of nitrogen is presumed to take place.
4. Discussion F r o m the ultrastructural details described above it is evident that the mode of entry into the roots of Elaeagnus by symbiotic Frankia spp. and subsequent
nodule development, departs radically from the root hair infection process, presently considered to be the typical mode of infection of actinorhizal plant species (CALLAHAMet al. 1979). The earliest events of root hair deformation and curling (POMMER 1956, ANGbTLO CARMONA et al. 1976, BERRY and TORREY 1983) are not observed in this novel process and indeed root hairs do not seem to be involved at all since root nodules can be initiated in root zones lacking hairs. In this study plants were inoculated under both axenic and non-axenic conditions to determine whether the infection mechanism was influenced by extraneous rhizosphere bacteria. Under no circumstances was a change in the mode of infection observed. Therefore we feel that the influences of other bacteria which have been proposed for root hair deformation and infection of Alnus (KNOWLTONet al. 1980, KNOWLTONand DAWSON 1983) are not responsible for the novel infection mechanism reported here. The formation of what is termed a prenodule (CALLAHAMand TORREY 1977), a limited proliferation of root cortical cells in response to Frankia invasion, is not observed in the infection of Elaeagnus. Rather the plant secretes a material into the intercellular spaces around epidermal and cortical cells where Frankia has penetrated and the hyphae permeate this material instead of invading nearby root cortical cells. Once the endophytic bacterium has grown into the nodule primordium the actual penetration of nodule cortical cells is very similar if not identical to the process observed in Ceanothus integerrimus by STRAND and LAETSCH (1977). In both cases, a hypha penetrates the plant cell wall by apparent degradation of the wall material. The plasmalemma remains intact but secretory vesicles fuse with the wall releasing their fibrous material to form an encapsulation sheath around the actinomycete. The invaded cell does not differentiate
Fig. 14. LS of a young developingnodule. The nodule periderm(PE)is welldevelopedand heavilytanninised. The basal nodule corticalcells(NC) have increased in size.The root epidermalhas been split apart by the developingnodule. Note that an infection path through the root cortex is still visible (arrow). x I44 Fig. 15. SEM of a portion of lateral root containing a nodule at the same stage of development as that in Fig. 14. x 119 Fig. 16. TEM of the cortical region from a young nodule. Hyphal profilescan be seen in many of the intercellular spacesat multiple cellj unctions (arrows). The boxed area is shown in more detail in Fig. I8. x 1,600 Fig. 17. TEM of the boundary between the nodule periderm (PE) and cortex (NC). The young cortical cellscontain numerous amyloplastsand mitochondria and secretory vesicles(arrows) are seen fusing with the host cell membranes. Hyphal profiles (H) can be seen in the intercellular spaces, x 1,942 Fig. 18. Detail of the boxed area in Fig. 16. The endophytichyphae (arrow) can be seen in the intercellularmaterial at the multiple celljunction. The cortical cells contain secretory vesicles(SV), mitochondria (M), Golgi bodies (G) and endoplasmic reticulum (ER). x 21,250
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Fig. 19. LS of a young nodule showing hypertrophy of the basal cortical cells (NC). Remnants of an infection path from root epidermis to nodule cortex are still visible (arrow). x 91 Fig. 20. Detail of the nodule cortex. Hypertrophied cells (HC) contain endophytic hyphae, • 564 Fig. 21. SEM of a nodule at the same stage of development as that in Fig. 19. The root epidermis has been split considerably. The loosely knit cells of the periderm give the emerging nodule a characteristic textured appearance, x 37 Fig. 22. TEM of the basal cortical cells. The endophytic hyphae can be found in the intercellular spaces and material (1), in the middle lamella between adjacent host cells (2) and entering the host cell through the cell wall (3). x 2,000 Fig. 23. Detail of entry into the host plant cell. The hyphae (H) occupies the middle lamella between two host plant cell walls (W1 and W2). A hyphal side branch (SB) has penetrated through one of the host cell walls, x 29,167
Fig. 24. The endophytic hyphae are always separated from the host cell cytoplasm by the host cell plasmalemma (arrows). Secretory (SV) vesicles containing a fibrous material fuse with the host plasmalemma around the hyphae. The material encapsulates the hyphae while the vesicle membranes allow growth of the host cell membranes, x 44,563 Fig. 25. The endophytic hyphae become completely surrounded by the material (SM) secreted by the host plant ceil. This material separates the hyphai wail from the host cell membrane (arrows). The terminal portions of some of the hyphae are swollen (HV). SVsecretory vesicles, x 25,000 Fig. 26. Lateral longitudinal section through a young nodule. The hypertrophied cells of the nodule cortex (NC) now contain much endophytic hyphae. Note that infection paths are still visible in the root cortex (arrows). x 111 Fig. 27. TEM of an infected enlarged nodule cortical cell from a young nodule. An intra-lamellar hypha, a side branch of which probably infected this cell, can be seen (IH). Septate vesicles (HV) have now developed and are found randomly distributed among the hyphae, throughout the cell.
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into a transfer cell as has been shown for invaded root hair cells of Alnus and Myrica (BERRY 1984, CALLAHAM et al. 1979). The similarity in the nodule cell invasion mechanisms between Elaeagnus and Ceanothus suggests that the mode of infection might also be identical, and that a second genus in a different actinorhizal family may be infected by intercellular epidermal penetration. It is however, even more probable that the genera HippophaO"and Shepherdia, both in the Elaeagnaceae are also infected by this mechanism since these genera are nodulated by the same bacteria which nodulate Elae~ agnus e t al. 1980). It is of interest to note that in some rhizobial symbioses, infection mechanisms other than root hair infection have recently been demonstrated also. Infection of host plant roots by the intercellular entry of rhizobia between the epidermal cells and the base of root hairs has been shown in the legumes Arachis and Stylosanthes (CHANDLER1978, CHANDLER et al. 1982) and in the ulmaceous species Parasponia rigida (LANCELLE and TORREY 1984). DUHOUX (1984) has shown that in the stem nodules of Sesbania rostrata, the rhizobia infect the host plant by entering dead epidermal cells and subsequently colonizing the intercellular spaces of the cortex prior to invading the nodule primordium. Intercellular pathways of infection may be more common than once thought. In Elaeagnus, more than one infection site may be observed in proximity to a single nodule primordium, and thus it is quite possible that two distinct Frankia strains are able to enter and colonize the same nodule, i.e., multiple infections. Evidence for double infections has only recently been provided by undertaking protein analysis of several isolates from the same nodule (BENSON and HANNA 1983). However this report was for isolated. Frankiae from alder, a plant known to be infected by root hair infection. CALLAHAMand TORREY (1977) state that infections in Comptonia and Casuarina, both root hair infected, are rare events. Therefore it may be more appropriate to study the possibility of multiple infections in Elaeagnus due to the larger number of infections observed per nodule.
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BOND, O., 1951: The fixation of nitrogen associated with the root nodules of Myrica gale L., with special reference to its pH relation and ecological significance. Ann. Dot. 15, 447-459. CALLAHAM, D., NEWCOMB, W., TORREY, J. G., PETERSON, R. L., 1979: Root hair infection in actinomycete-induced root nodule initiation in Casuarina, Myrica and Comptonia. Dot. Gaz. 140, S I-$9. - TORREY, J. G,, 1977: Prenodule formation and primary nodule development in roots of Comptonia (Myricaceae). Can. J. Dot. 55, 2306-2318.
CHANDLER, M. R., 1978: Some observations on infection of Arachis hypogaea by Rhizobiurn. J. exp. Dot. 29, 749-755. DATE, R. A., ROUGHLEY,R. J., 1982: Infection and root-nodule development in Stylosanthes species by Rhizobium. J. exp. Dot. 33, 47-57.
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DLnOUX, E., 1984: Ontog6nase des nodules caulinaires du Sesbania rostrata (16gumineuses). Can. J. Dot. 62, 982-994. FLETCHER, W. W., 1955: The development and structure of rootnodules of Myrica gale L. Ann. Dot. 19, 501 513. KANT, S., NARAYANA, H. S., 1977: Preliminary studies on the development and structure of root nodules in Casuarina equisetifolia L. Proc. Indian Acad. Sci. B. 85, 34-41. KNOWLTON, S., BERRY, A., TORREY, J. G., I980: Evidence that associated soil bacteria may influence root hair infection of actinorhizal plants by Frankia. Can. J. Dot. 26, 971-977. DAWSON,J. O., 1983: Effects ofPseudomonas cepacia and cultural factors on the nodulation ofAlnus rubra roots by Frankia. Can. J. Dot. 61, 2877-2882.
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Acknowledgements The authors would like to thank Dr. YVONNE BARNET for useful discussions during this study; M. DANIELS,D. H1VELY,M. PENCEand S. PERKINSfor technical assistance; S. DUNBARand M. TOOTLE for photographic assistance and Dr. T. V. BHUVANESWAR/for critically reading the manuscript. Contribution no. 868 from the Battelle--C. F. Kettering Research Laboratory.
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