APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1989, p. 2046-20550099-2240/89/082046-10$02.00/0Copyright © 1989, American Society for Microbiology

Localization of Bacteria and Hemoglobin in Root Nodules ofParasponia andersonii Containing Both Bradyrhizobium

Strains and Rhizobium leguminosarum biovar trifoliiMICHAEL J. TRINICK,* DAVID J. GOODCHILD, AND CELIA MILLER

Division of Plant Industry, Commonwealth Scientific and Industr-ial Researc-h Organization,GPO Box 1600, Canberr-a City, 2601, AuistraliaReceived 21 February 1989/Accepted 22 May 1989

Dual occupancy of Parasponia andersonii nodules with different Bradyrhizobium strains and Rhizobiumleguminosarum biovar trifolii was frequently obtained when two strains were inoculated into plants grownaseptically in tubes. Since reisolates of Bradyrhizobium strains from dually occupied nodules acquired theability to nodulate Trifolium repens, the spatial relationship of the two species of bacteria during noduleinitiation and development was investigated and their proximity was demonstrated. By using light microscopyand electron microscopy and immunogold labeling, R. leguminosarum biovar trifolii NGR66 inoculated aloneonto P. andersonii produced small ineffective nodules, with bacteria embedded in matrix material inintercellular spaces and in a few nonliving host cells rather than in infection threads (CP299). In dualinfections, the two bacterial species were shown to be adjacent to one another in the matrix of noduleintercellular spaces and in some host nodule cells. However, when two different Bradyrhizobium strainsoccupied a single nodule, they were located in different lobes of the same nodule. Immunogold labeling showedthat Parasponia hemoglobin was localized in the cytoplasm of young infected nodule cells. This suggests that thenitrogen-fixing phase of Parasponia nodule cells is short-lived and correlates with previous acetylene reductiondata from nodule slices. Hemoglobin was associated only with areas of nodule tissue infected with the effectivenitrogen-fixing strain CP299 and absent from areas infected with R. leguminosarum biovar trifolii.

Three Parasponia species are nodulated by a group ofstrains of Bradyrhizobium spp. that appear to differ fromstrains that usually nodulate legumes (28). Root nodulebacteria from legumes are divided into groups based on theplants they are able to nodulate. These bacteria belong totwo separate physiological groups and accordingly are

placed into two genera, Rhizobium and Bradyrhizobium.Rhizobium leguminosarlum biovar trifolii nodulates Trifo-lium species, and some members are also able to nodulatePhaseolus vulgaris and Pisium sativum, but they do notnodulate diverse plants usually nodulated by Bradyrhizo-bium spp. (27).M. J. Trinick and P. A. Hadobas (unpublished data) found

that most strains of R. leguminosaruin biovar trifolii nodu-late Parasponia sp. if sufficient time is allowed for infectionto develop. They found that when Bradyrhizobium sp. was

included in low numbers with R. leguminosaumin biovartrifolii in the inoculum, the nodules formed often containedboth organisms but the symbiotic effectiveness of the nod-ules depended on the relative numbers of Bradyrhizobiimnorganisms in the nodule. In addition, Brcadyrhizobiuimstrains, after association with R. legulminosar-ium biovartrifolii in the nodule of Parasponia andersonii, were subse-quently able to form ineffective nodules or nodulelike struc-tures on Trifolium repens, depending on the reisolate used.Thus, in dual infections, Bradyrhizobium sp. appears to haveacquired characteristics from R. legiuminosari-in biovar tri-

folii that enable it to nodulate T. repens (unpublished data).The structure of nodules on Parasponia spp. formed by

effective strains of Bradyrhizobitum sp. is well documented(12, 13, 26), but the location of hemoglobin in Parasponianodules (1) has not been investigated. Also, the structure of

* Corresponding author.

ineffective nodules on Parasponia spp. formed by eitherBradyrhizobiulm sp. or R. legluminosarum biovar trifolii hasreceived only limited attention (3). The gold-labeling tech-niques developed by Craig and Goodchild (5) permits notonly observation of the location of hemoglobin in nodulecells but also identification and location of root nodulebacteria with different serological characteristics. A highincidence of nodules containing more than one Rhizobium or

Bradyr4hizobium strain has been observed in cowpeas (30),soybeans (15), clovers (8), Lens esclulenta (17), and Para-sponia spp. (29). The spatial relationships of two differentstrains of root nodule bacteria in a nodule have not previ-ously been studied in legumes or nonlegumes. The presentstudy examined the location of hemoglobin in and thestructure of nodules on P. andersonii that were formed bytwo effective Bradyr-hizobiuin strains and by Bradyrhizobiumsp. with an ineffective R. leguminosar-uin biovar trifoliistrain and assessed the likelihood of the acquisition byBradyrhizobiiiin sp. of characters from R. leguminosariumbiovar trifolii.

MATERIALS AND METHODS

Bradyrhizobium and Rhizobium strains. R. leguminosaroinbiovar trifolii NGR66, isolated from Trifolium repens grow-

ing in the Wau-Bulolo area of Papua New Guinea, was

selected for study. This strain was representative of a

number of strains found to be capable of forming dualinfections in nodules on P. ander.sonii when inoculated withmixtures of R. legioininosariuin biovar trifolii and Bradyrhizo-biiun sp. R. leguininosaritun biovar trifoii strains formineffective nodules on Parasponia species (1; unpublisheddata). The two Bradyrhizobium strains used, CP299 andCP273, were effective on P. ander-sonii and were isolated

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from P. rigida and P. rugosa in the Lae-Bulolo area of PapuaNew Guinea and Panguna, Bougainville, respectively.

Antibody production and specificity. Antiserum againsteach bacterial strain was prepared in laboratory rabbits as

described by Vincent (34). Each was shown to be specific forits homologous antigen by gel immunodiffusion (22) and tubeagglutination (34) techniques. No cross-reactions betweenthe antigens of the strains used when they were tested withfull-strength antisera were recorded. To avoid nonspecificlabeling problems for immunogold labeling, the immunoglob-ulin G fraction of NGR66 antiserum was prepared by(NH4)2SO4 precipitation (11) and its antiserum was diluted to1:100, which was a considerably lower dilution than the1:1,600 of the antiserum to CP299.Antiserum to hemoglobin from P. andersonii root nodules

was raised in rabbits by the method described by Fleming etal. (10) for Casuarina hemoglobin. Western blot (immuno-blot) analysis of nodule tissue and purified P. andersoniihemoglobin showed that the antibodies were specific (C. A.Appleby, personal communication).

Plant culture. P. andersonii plants having nodules withsingle or dual occupancy were grown in glass tubes (150 by25 mm) of sloped nutrient agar (29) with only a trace ofnitrogen (initially, 1 mM N03-). Pregerminated seeds were

sown high on the slope, and after inoculation, the tubes were

placed in a greenhouse maintained at 28°C by day and 23°Cby night. The tubes were protected from direct sunlight byshade cloth that passed 50% of the incident light. Nodulescontaining Bradyrhizobium sp. or R. leguminosarium biovartrifolii appeared at 11 to 20 or 70 to 100 days, respectively.Plants were harvested 4 months after inoculation.

Production of nodules with dual occupants. Cultures weregrown on yeast extract (Difco Laboratories)-mannitol me-

dium for 7 days at 28°C. The mixed inoculum was suspendedin water, and 1 ml containing approximately 107 to 108 cellsof R. leguminosarum biovar trifolii per ml and 1 to 10 cells ofBradyrhizobium sp. per ml was added to each tube contain-ing a P. andersonii seedling. Two dual-infection experimentswere performed, and up to 50 tubes were inoculuated foreach treatment. In some tests, Bradyrhizobium sp. was

added after a delay of 2 weeks.At harvest, nodules were surface sterilized with 1% mer-

curic chloride (34), and in some experiments, they were cutinto halves. One half of the nodule was squashed in sterilewater and streaked on yeast extract-mannitol plates with orwithout 10 [Lg of chlortetracycline per ml (29), and the otherwas fixed for light microscopy and electron microscopy as

described below. The identity of the nodule isolate(s) was

checked serologically by the tube agglutination (34) andfluorescent-antibody (25) techniques.

Microscopy. Small nodules were carefully sliced from theroot and fixed whole. Larger nodules were cut longitudinallyinto two pieces, and in one experiment, one half of thenodule was used to determine the strains present, while theother half was examined microscopically. Since sectionswere to be used for immunocytochemistry, a fixation-and-embedding procedure was adopted to fulfill the requirementsof both light microscopy and electron microscopy.Specimens were fixed in 25 mM sodium phosphate buffer

(pH 7.2) containing 5% acrolein and 1% glutaraldehyde.Preliminary experiments with glutaraldehyde alone showedthat gold labeling of the somatic antigens frequently oc-curred at a distance from the bacterial cell wall. However,by incorporating acrolein with glutaraldehyde, the goldlabeling was restricted mainly to the bacterial cell walls; thissuggests that glutaraldehyde alone does not completely

immobilize these antigens during fixation. Cell wall localiza-tion of the somatic antigens was essential for strain identifi-cation of adjacent bacteria in electron microscope sections.Nodule tissue fixed in this medium was dehydrated inethanol and embedded in LR White resin (London ResinCo., Basingstoke, United Kingdom) (7). In some experi-ments, primary fixation was followed by 2% osmium tetrox-ide in 25 mM sodium phosphate buffer, dehydration inethanol, and embedment in Spurr resin (24).For light microscopy, sections approximately 0.5 ,um thick

were cut on a Reichert Ultracut microtome. The anatomicalfeatures of nodules were highlighted by staining with tolui-dine blue 0 (9) or methylene blue. Sections from any onenodule were cut, stained, and examined in a light microscope(Zeiss) until a region of interest was identified, when approx-imately 80-nm-thick sections were cut for electron micros-copy.Immunocytochemistry was performed on sections mount-

ed on Parlodion-carbon-coated nickel electron microscopegrids. Rabbit antibodies in a high-salt buffer (0.5 M NaCl,0.01 M sodium phosphate with 1% Tween 20 [pH 7.1]) (5)were applied as previously described (2), followed by goatanti-rabbit 20-nm-diameter gold particles (GARG-20; Jans-sen Pharmaceutica) diluted 1:19 with high-salt buffer todetect the location of the primary antibody. ImmunoglobulinG to R. leguminosarum biovar trifolii NGR66 was used at adilution of 1:100, while antiserum to Bradyrhizobium sp.strain CP299 was used at a dilution of 1:1,600. Followingimmunostaining, grids were treated with uranyl acetate andlead citrate to enhance the contrast of cellular features. Inone experiment, the contrast of gold labeling of light micro-scopic sections was increased by the silver enhancementmethod (Intense; Janssen Pharmaceutica). For somatic an-tigens NGR66 and CP299, similar densities of antibody goldlabel were obtained on non-osmium tetroxide-treated, LRWhite resin-embedded tissue and osmium tetroxide-treated,Spurr resin-embedded tissue, provided that the latter wasetched with a saturated solution of sodium periodate, fol-lowed by 0.1 N HCI, before immunolabeling (6). For study-ing the hemoglobin in Parasponia nodules, LR White-em-bedded tissue gave much higher levels of gold label than didosmium tetroxide-treated, Spurr resin-embedded tissue.Immunocytochemical controls were initially performed on

sections of embedded bacteria of pure strains to demonstratelocalization of the antibodies to the bacterial cell periphery.In most subsequent experiments, only internal controls wereused, as mentioned in the Results section (Microscopy, ii),after it was first ascertained that no nonspecific labelingoccurred when GARG-20 was used alone or antibody tobovine serum albumin was used as the primary antibody onnodule tissue.

RESULTS

Bacterial isolations. (i) Dual occupancy with Bradyrhizobiumsp. and Rhizobium sp. The resistance of Bradyrhizobiumstrains to chlortetracycline enabled easy separation from R.leguminosarum biovar trifolii (29) and identification of cul-tures prepared from nodule halves squashed and streaked onyeast extract-mannitol medium with or without this antibi-otic. The same nodules were examined by light microscopyand electron microscopy. Dual occupancy was found in 76%of the nodules examined. Bradyrhizobium sp. and R. legu-minosarlim biovar trifolii were sometimes apparent in nod-ules in equal numbers, but frequently Bradyrhizobium sp.dominated the nodule populations. When nodules were cut

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FIG. 1. Electron micrograph of ineffective R. Iegiimninosarmin FIG. 2. Electron micrograph of adjoining P. andei-soni cellsbiovar tr-ifoiji NGR66 invasion of P. aidersonii root tissue (em- (embedded in LR White resin) invaded by the ineffective strainbedded in LR White resin) showing intercellular matrix material (I) NGR66. Remains of host cell cytoplasm (CR) are evident, and thewith bacterial embedded in it. The adjacent cell was invaded by dense and comparatively featureless appearance is consistent withbacteria in matrix material (M). Many bacteria (SB) appeared to live nonliving cytoplasm. The cell on the left is filled with matrix materialsaprophytically in host cells. (Insert) Single bacterium at higher (M) between the bacterial profiles. No matrix material is evident inmagnification, showing the specificity of immunogold labeling by the the cell on the r-ight. and the bacteria are somewhat larger than thosepresence of 20-nm-diameter gold particles only at the periphery of in the left host cell and have less dense cytoplasmic contents, whichthe bacterial cell. The section was labeled with somatic cell anti-bodies to strain NGR66, followed by GARG-2(0. All of the bacteria may idicate a saprophytic state. Bar, I 1Lm.showed similar cell periphery labeling. Host cell walls, vacuoles,and matrix material were not gold labeled. No cytoplasm wasevident in invaded host cells. Bar, 1 yLm.

into transverse segments, both types of root nodule bacteriawere isolated from nodule tissue from the tips to the bases ofthe nodules. Frequently, when inoculation with BrladvrNiz.o-biiin sp. (CP299) was delayed 14 days after inoculation withR. legiuninosaruiiin biovar trifolii (NGR66), the latter was

present in higher numbers, thus allowing easier detection bymicroscopy.

(ii) Dual occupancy with Bradyrhizobium strains. Fluores-cent-antibody and tube agglutination studies of Bradyrhizo-billn strains isolated from nodule halves allowed rapididentification of strains and detection of nodules with dualinfections. Dual infections were detected in two-thirds of thenodules examined, but only nodule halves with high popu-

lations of both strains were used for microscopy. Theeffectiveness of nodulation depended upon the ratio ofBracdyrhizobiuin sp. cells to R. leguiininosarmn biovar triftliicells in the nodules. Ineffective nodules had a dominance ofR. legtininosariuitn biovar trifolii (unpublished data).

Microscopy. (i) Infection by R. leguminosarum biovar trifoliiNGR66 alone. Strain NGR66 produced small nodules on theroots of P. ander-son ii. A cut nodule surface was not visiblypink, and plants grown on medium lacking N, were palegreen and resembled uninoculated controls. This suggestedthat nodulation was ineffective and supported findings pre-

viously reported (unpublished data). Sections of small nod-ules stained with toluidine blue or methylene blue for lightmicroscopy revealed densely staining matrix material inintercellular spaces, frequently with individual bacteria em-

bedded in it (Fig. 1). The characteristic intracellular infectionthreads of normal N,-fixing nodules, in which densely

packed bacteria are surrounded by a dense celluloselikewall, were not encountered. In light micrographs, the con-tents of some cells in the interior of a nodule were stainedalmost uniformly and bacteria were clearly identified.

Close observation of young nodule tissue, at both the lightmicroscope and electron microscope levels, revealed thepresence of matrix material in intercellular spaces from nearthe bases of multicellular root hairs to the nodule interior(data not shown). Electron microscope sections confirmedthat the mode of entry of the bacteria was similar to thatdescribed by Lancelle and Torrey (12). Cell proliferation wasnoticed at the bases of root hairs and was apparently inducedby the extracellular presence of the bacteria. This celldivision produced what was, in effect, a multicellular roothair. Distortions at the bases of the root hair cells caused bycell division produce cleavage at the intercellular wall inter-faces through which bacteria enter the cortical root tissue.By immunogold labeling, it was possible to demonstratevegetative bacteria of strain NGR66 external to the rootsurface in intercellular air spaces and embedded in anintracellular matrix material in internal nodule tissues. Thismatrix material (Fig. 1, I) was often fibrillar and diffuise,unlike infection thread wall material within cells of effectivenodules (see Fig. 3). The gold-labeling pattern on NGR66cells produced by antibodies to their somatic antigen waslocalized near the bacterial cell wall (Fig. 1, insert). Figures1 and 2 show bacteria in matrix material (M) within a hostcell. Many other bacteria were free of the matrix within thesame cell (Fig. 1) and in an adjacent cell (Fig. 2). Theseintracellular bacteria were usually larger than those withinthe matrix material and had distinct cell walls and a lessdense internal matrix, but their gold-labeling pattern wassimilar to that of matrix-contained bacteria. Both bacterium-filled nodule cells in Fig. 2 show remnants of electron-dense

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FIG. 3. Electron micrograph of cells from a nodule of P. ander-sonii (embedded in LR White resin) invaded with the effective strainBradx,/izobi,um sp. strain CP299. Threads (T) are filled with bacte-ria, and many mitochondria (MI) can be seen at the host cellperiphery. Uninfected interstitial cells (IS) adjoin the infected cell.This section was immunolabeled for Pacraisponiia hemoglobin, notreadily apparent at this magnification, and the label appeared onlyover the cytoplasm of the infected cell. Bar, 1 pAm.

host cell cytoplasm (CR). Only a few cells in any nodulestructure were infected in the manner described above. As afurther indication of the ineffectiveness of the infection of P.andersoniii by strain NGR66, the presence of Par-asponiithemoglobin could not be demonstrated in nodule cells byelectron microscope immunocytochemistry.

(ii) Infection by Bradyrhizobium sp. strain CP299 and dis-tribution of hemoglobin. Establishment of infection by strainCP299 was similar to that described previously for infectionof Parasponia spp. by Bradyihizobilun sp. (12, 13, 26).Intercellular infection threads gave rise to intracellularthreads that were filled with bacteria. In the early stages ofintracellular infection, these threads were surrounded byhost cytoplasm (Fig. 3), but as the threads increased inapparent number, the cytoplasm was confined mainly to thecell periphery, where mitochondria and plastids were alsopresent. This stage is often defined as the mature symbioticstate, and in P. ande)rsoniii it is accompanied by the presenceof a unique hemoglobin (1). In contrast to infection by R.legii;ninosaruim biovar t) ifolii NGR66, the bacteria con-tained deposits of poly-3-hydroxybutyrate and some ap-peared to be longer and more pleomorphic.Immunocytochemical localization of Para-asponii( hemo-

globin in nodule sections was most intense in the cytoplasmof invaded cells (Fig. 4), as evidenced by the pattern of goldlabeling. Antibody to bovine serum albumin, when used asthe primary antibody, did not result in the occurrence of anygold label on sections, apart from an occasional nonspecificparticle. Gold label was also seen over the nuclei of invadedP. ander.sonii cells, in common with the localization ofsoybean leghemoglobin (Goodchild, unpublished data) andpea leghemoglobin (21). No label, other than a very low levelsimilar to the intensity obtained in controls (nonspecificlabeling), was observed over the threads containing the

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FIG. 4. Electron micrograph of the central portion of a nodulecell from P. (tidersonii (embedded in LR White resin) infected withCP299 and immunolabeled for Par-aspotniai hemoglobin. Dense goldlabeling is apparent over the comparatively sparse host cell cyto-plasm but not over threads or in the bacteria contained within them.where only gold particles characteristic of nonspecific labeling arepresent. Bar, 0.5 p.m.

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F1G. 5. Electron micrograph of nodule cells from P. anddersonii(embedded in LR White resin) invaded by strain CP299 and immu-nogold labeled for Paraispotiia hemoglobin. Dense gold labeling canbe seen over the cytoplasm of the invaded cell but not over amitochondrion (MI), the cell wall (CW), or the resin-filled air space(AS). A few gold particles (nonspecific labeling) can be seen over theuninfected interstitial cell (IS) on the right and over the threadscontaining bacteria. Bar. 0.5 pLm.

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FIG. 6. Electron micrograph of dual invasion of P. andersoniiroot tissue (embedded in LR White resin) by strains NGR66 andCP299. NGR66 is present in matrix material in what would normallybe intercellular spaces (AS) in an effective nodule. The matrixmaterial (M) may expand the intercellular space and distort corticalroot cells. Strain specificity was verified by immunogold labelingthat is not evident at this magnification. Bar, 5 p.m.

bacteria in infected host cells or over intercellular infectionthreads. No label was evident over mitochondria or plastidsin infected cell cytoplasm. The level of label over uninvadedinterstitial cells adjacent to infected cells was low andvariable (Fig. 5) and is attributed to nonspecific labeling.This latter finding serves as an internal control for thelocalization of Parasponia hemoglobin.At the mature symbiotic state, immunogold labeling of

hemoglobin was most intense in the cytoplasm. The threadwalls were also lightly stained (Fig. 3). At a later stage ofinfection, the thread walls thickened and became denselystained with electron-dense stains as previously describedby Trinick (26) and Lancelle and Torrey (13). In such cells,the host cytoplasm appears to be absent, leaving the cellsfilled with threads, and this may be a stage in the senescenceof nodule tissue. At this stage, no hemoglobin was detectedby immunocytochemistry. The infected nodule cell typesdescribed often occurred side by side.

(iii) Dual infections by R. leguminosarum biovar trifoliiNGR66 and Bradyrhizobium sp. strain CP299. Nodule devel-opment can take several months, and plants were periodi-cally examined and sampled so that nodules at several stagesof development were used for microscopy. Since the pres-ence of strain NGR66 was accompanied by a densely stain-ing matrix in sections stained for light microscopy, thisfeature was used to screen sections for the proximity of dualinfections in individual nodules. For this purpose, approxi-mately 0.5-p.m-thick serial sections were cut through indi-vidual nodules and stained with methylene blue or toluidineblue.Many of the light microscopic sections revealed that strain

NGR66 was present in intercellular matrix material, and insome sections, NGR66 was present in host cells filled withmatrix material. In nodules with dual infections, many

FIG. 7. Electron micrograph of dual invasion of a single cell(embedded in LR White resin) by strains CP299 and NGR66. Thebacteria in threads (CP299) are evident to the left of the invaded cell,while the NGR66 in matrix material is present to the right of theinvaded cell. The boundary is shown by the arrowheads. Bar, 5 p.m.

intercellular air spaces were filled with matrix material,usually containing NGR66 bacteria, and it was difficult todistinguish these structures from cells filled with matrix andNGR66 bacteria (Fig. 6). The intercellular air spaces wereenlarged, and adjacent cells were frequently distorted, pos-sibly by pressure produced by the increased volume ofmatrix material. By sectioning through a nodule, cells couldbe found in which bacteria in symbiotic-state threads

FIG. 8. Electron micrograph of a portion of a cell (embedded inLR White resin) from a nodule invaded by strains CP299 andNGR66. This cell was invaded only by NGR66 bacteria contained inmatrix material formed into tubes with a definite perimeter. Somebacteria are separated by electron-lucent areas that may contain theremains of host cytoplasm. Bar, 1 p.m.

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(CP299) with thin walls were present with areas of matrixmaterial within the same cell (Fig. 7). In a few cells contain-ing NGR66, the usually diffuse matrix material was confinedto tubelike structures (Fig. 8). When areas such as thatshown in Fig. 7 were found, thin sections were cut forelectron microscope immunocytochemistry and treated withantibodies to strains NGR66 and CP299 separately to iden-tify the bacterial strains and their localizations. Doublelabeling of sections with both antibodies produced artifactsand was not definitive (4).

Figure 9 shows complementary labeling of adjacent sec-tions. In Fig. 9A, bacteria were labeled for CP299, and inFig. 9B they were labeled for NGR66. Bacteria labeled withone antibody were not labeled with the other. Figures 9 and10 show examples of the close proximity attained by bacte-rial cells of the two different species of root nodule bacteria.In Fig. 10, R. leguminosarum biovar trifolii NGR66, but notBradyrhizobium sp. strain CP299, was labeled. Bradyrhizo-bium sp. strain CP299 bacteria were contained withinthreads in host cells, but the nature of the thread wall variedand appeared to be diffuse and even lacking near the R.leguminosarum biovar trifolii NGR66 contained in matrixmaterial.

In some experiments, strain CP299 was inoculated at thesame time as strain NGR66, and in others, CP299 wasinoculated 14 days after strain NGR66. While both treat-ments produced dual infections, from the nodules examinedmicroscopically there was a greater incidence of dual infec-tions when the Bradyrhizobium strain was inoculated afterthe R. leguminosarum biovar trifolii strain. Dual occupancyof a single nodule by both bacterial species usually resultedin occupation of many cells by R. leguminosarum biovartrifolii NGR66 in matrix material, whereas in nodules withsingle NGR66 infections very few such cells were seen. Thepresence of hemoglobin was noted by immunocytochemistryin the cytoplasm of cells that contained only the Bradyrhizo-bium strain. When host cells contained both Bradyrhizobiumsp. and R. leguminosarum biovar trifolii in matrix material,cytoplasm and hemoglobin were not observed in any of thecells examined.

(iv) Dual infection with two Bradyrhizobium strains. Whentwo Bradyrhizobium strains (CP299 and CP273) that nor-mally nodulate Parasponia sp. effectively were inoculatedtogether on P. andersonii, the bacteria colonized separatelobes of each of five nodules sections. Figure 11A and Bshows light micrographs of adjacent sections that wereimmunogold labeled and then enhanced for light microscopyby the silver enhancement technique. The sections clearlyshow one lobe labeled for strain CP299 (Fig. 11A) and onelabeled for strain CP273 (Fig. 11B). The two lobes areseparated by several cortical cell layers. Additional sectionswere examined in the electron microscope, but no immuno-cytochemical evidence of dual occupancy of lobes was foundin these mature nodules. However, immunochemistry de-tected the presence of hemoglobin in each lobe of thenodules formed due to the final infection by two Bradyrhizo-bium strains.

DISCUSSION

The ineffective association with R. legirninosarum biovartrifolii NGR66 produced nodules whose internal structurediffered greatly from that of effective nodules. However, asR. leguminosarum biovar trifolii had the ability to stimulateroot hair division, entry was possible in a manner whichappeared to be similar to that which resulted in effective

nodules produced by Bradyrhizobium sp. strain CP299. Thepresence of bacteria was associated with unusual quantitiesof intercellular matrix material which, together with themultiplying bacteria, may have caused host cell distortiondue to its expansion. Since no true infection threads wereseen in the few host cells that were invaded, it is possiblethat the presence of bacteria in matrix in these cells is due tophysical pressure forcing the matrix through the host cellwall.The R. leguminosarum biovar trifolii strain thus appears

to have more difficulty in establishing itself in ineffectivehost nodules than does Bradyrhizobium sp. in effectivenodules. Invasion of Parasponia sp. by R. leguminosarumbiovar trifolii has some characteristics in common withinvasion by Agrobacterium tumefaciens after host wounding(31), in which matrix material is also present with bacteria inintercellular spaces (16) but invasion of unwounded cellsdoes not take place (4).

In the few host cells in which R. leguminosarum biovartrifolii in a matrix had penetrated, the host cytoplasm wasreduced to vestiges, and in some host cells the bacteriaappeared to grow saprophytically, possibly at the expense ofthe host cells. In these cells, the bacteria were not sur-rounded by membranes or walls of host origin, and matrixmaterial was frequently absent. The appearance of thebacteria was unlike that of degrading bacteroids in otherindeterminate nodules of legumes (18, 19), and they mayactively grow. Indeed, although NGR66-caused noduleslacked the ability to fix nitrogen, as measured by the absenceof hemoglobin and acetylene reduction, large numbers ofviable R. leguminosarum biovar trifolii organisms wererecovered from these ineffective nodules (unpublished data).

Riboflavin-requiring mutants of R. leguminosarum biovartrifolii have been reported to produce nodules on red cloverin which bacteria surrounded by a matrix, possibly ofpolysaccharide origin, occur in the host cells (20). Anineffective strain of R. meliloti produced a similar effect,suggesting that cellular recognition could be impaired by thepresence of the matrix and thus prevent bacteroid develop-ment (32, 33). Sprent and de Faria (23) have discussed theway in which cell walls may be crossed by infection threads,and it is possible that the presence of matrix material inhibitsthis process so that R. leguminosarum biovar trifolii cannotreadily enter Parasponia root cortical cells. Certainly, theabsence of hemoglobin from these ineffective Parasponianodules is consistent wi.th the data that they do not fixnitrogen. It is also interesting that the host genotype canresult in production by a Rhizobium strain of ineffectivenodules with cells filled with matrix and bacteria (33).The composition of the matrix material is outside the

scope of the present study, but the electron microscopicimages suggest that it is unlike that of cellulose cell wall.Furthermore, when sections were treated with colloidalthorium, the matrix material stained strongly, especiallycompared with effective nodule tissue (data not shown),suggesting the presence of acid polysaccharide (14).The localization of Parasponia hemoglobin to the host

cytoplasm in nodules follows a pattern similar to that re-

ported for leghemoglobin in pea nodules (21). Additionalquestions regarding the efficiency of the Parasponia-Bradyrhizobiuim (CP299) symbiosis are raised, however,because infected cells adjacent to those with hemoglobin-containing cytoplasm lacked any evidence of both hemoglo-bin and cytoplasm and their bacterium-containing threadshad thickened walls. This suggests that the symbiotic statewhich requires hemoglobin is transitory. Evidence from

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nodule dissections and acetylene reduction analyses (i)shows that the nitrogen-fixing nodule tissue is located closeto the nodule apex (26) and (ii) favors the view that thesymbiotic state in Parasponia sp. is transitory comparedwith that of other nodules. For this reason, the term symbi-otic-state threads is proposed for the thin-walled threadstage of nodule cell development in Pawrasponia sp., in whichcytoplasm and hemoglobin are still present. Host cells withno cytoplasm and thick-walled threads probably represent astage in nodule senescence that is not so clearly restricted toa particular tissue zone as it is in other nodules. The findingthat hemoglobin antigen appears to be present in the nucleiof both soybean and Parasponia host cells remains unex-plained.The Bradyrhizobium strains used in these experiments

produced nodule structures that have previously been de-

scribed for effective associations on Parasponia sp. (12, 26).In dual infections, both Bradyrhizobilini sp. and R. legiumi-nosarirn biovar trifjiii were found in close proximity to eachother in intercellular spaces and in the same nodule host cellsof Parasponia sp. Their entrance into roots and their distri-bution within nodules demonstrated that if close proximitywas a factor in the apparent acquisition by Bradyrhizobiumsp. of the ability to produce nodules or nodulelike structureson T. repens, then these conditions existed in ineffective andpartially effective nodules with dual infections. Ninety per-cent of the nodule reisolates of Bradyrhizobium sp. fromdually infected nodules had acquired this characteristic(unpublished data).

In effective or partially effective nodules with dual occu-pancy, the Bradyvhizobiu,n strains in close association withR. legumin1vosaruim biovar tiJolii appeared to be morpholog-

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^b;L, !,i. I\s;; v'K77FIG. 10. Electron micrograph of a section of a P. andersonii host nodule cell (embedded in Spurr resin) invaded by both NGR66 and CP299

strains which was immunogold labeled for NGR66. The close association between the two strains is shown, especially where the thread wall(TW) appears diffuse or even lacking (arrowheads). CP299 bacteria, in threads, usually contain more poly-p-hydroxybutyrate (PB) than doNGR66 bacteria. Bar, 1 p.m.

ically similar to those in single-occupancy cells. Host cyto-plasm was never present in these cells, and CP299 sendsthreads across an NGR66 matrix but not vice versa. Therewas no apparent morphological change in the bacteroid cellsdue to the presence of the two species within the samenodule cells or in the intercellular spaces. Dual occupancy,however, did encourage more variation in the morphology ofhost cells and intercellular spaces invaded by R. leguminosa-rum biovar trifolii. In general, more cells were invaded by

NGR66 in dually occupied nodules than in single infections,and more intercellular spaces were filled with matrix mate-rial, although some of these contained dual infections. Nod-ule cell distortion was increased by expansion of matrixmaterial into intercellular spaces. Often this material was inthe form of thick-walled tubes sometimes separated byelectron-lucent spaces. Since no cytoplasmic profiles consis-tent with living cytoplasm were observed in dually infectedcells and no hemoglobin could be detected, it is likely that

FIG. 11. Complementary light micrographs from adjacent sections of a P. andersonii nodule (embedded in LR White resin) infected withtwo Bradyrhizobium strains, CP299 and CP277. The section in panel A was immunogold stained, with silver enhancement, for strain CP299,and that in panel B was stained for strain CP273. The strains are each confined to separate lobes of the nodule separated by uninfected corticalroot cells. Magnification, x73.

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2054 TRINICK ET AL.

only nonliving cells were invaded by R. legurninosaruirinbiovar trifolii whether Bradyr-hizobiiirn sp. was present ornot. However, when Bradyrhizobillm sp. was present to-gether with R. leguminosarurn biovar trifolii, the threadwalls in host cells were not thickened as they are in singlyinfected nodules, suggesting that the presence of R. leguimi-nosarum biovar tirifolii may modify the senescent stages ofParasponia infection by Bradyrhizobiiim sp.

Bacterial isolation on yeast extract-mannitol medium withchlortetracycline showed that 76% of the nodules had dualoccupancy. Fewer nodules showed dual occupancy by lightmicroscopy or electron microscopic immunocytochemistry.Squashing half of a nodule permitted any available Rhizo-bium cell intra- or intercellularly placed to be detected,whereas microscopic examination revealed only a smallproportion of the dual infections.Dual occupancy of Parasponia nodules with two different

Bradyrhizobium strains originally isolated from Parasponiasp. produced an unexpected result. Although both organismsinfected the root tissue to form nodules, they remained indifferent lobes of the nodules. Neither individual nodule cellsnor adjacent cells with the two Bradyrhizobium strains werefound, despite extensive examination of the material avail-able. It appears that the two Bradyrhizobium strains weremutually excluded from adjcent nodule cells, which resultedin the formation of different lobes for the two strains.The work reported here established morphological differ-

ences in dually infected nodules, depending upon the infec-tive strains, and showed that close associations betweenbacterial species within the same nodule are possible. Inaddition, it demonstrated the ability of immunocytochemicalmethods to specifically localize antigens by electron micro-scope with high spatial resolution. Adjoining bacteria withdiffering somatic antigens can be readily resolved, providedthat there is no immunological cross-reaction. Such methodsand that used to determine the presence and localization ofhemoglobin should prove of increasing use in studyingrhizosphere and symbiotic associations.

ACKNOWLEDGMENTS

We thank, from the Division of Plant Industry, CommonwealthScientific and Industrial Research Organization, P. Hadobas forvaluable technical assistance and F. J. Bergersen, S. Craig, and A.Gibson for valuable discussions during preparation of the manu-script.

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19. Paau, A. S., C. B. Bloch, and W. J. Brill. 1980. Developmentalfate of Rhizobiion teliloti bacteroids in alfalfa nodules. J.Bacteriol. 143:1480-1490.

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21. Robertson, J. G., B. Wells, T. Bisseling, K. J. F. Farnden, andA. W. B. Johnson. 1984. Immuno-gold localization of leghaemo-globin in cytoplasm in nitrogen-fixing root nodules of pea.Nature (London) 311:254-256.

22. Schwinghamer, E. A., and W. F. Dudman. 1980. Methods foridentifying strains of diazotrophs, p. 337-365. In F. J. Bergersen(ed.), Methods for evaluating biological nitrogen fixation. JohnWiley & Sons, Inc., New York.

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33. Vance, C. P., L. E. B. Johnson, and G. Hardarson. 1980.Histological comparisons of plant and Rhizobiium induced inef-fective nodules in alfalfa. Physiol. Plant Pathol. 17:167-173.

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