APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1994, p. 427-433 Vol. 60, No. 20099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Symbiotic Characteristics of Rhizobium leguminosarum bv.trifolii Isolates Which Represent Major and Minor Nodule-

Occupying Chromosomal Types of Field-GrownSubclover (Trifolium subterraneum L.)t

K. LEUNG,' F. N. WANJAGE,2 AND P. J. BOTTOMLEYl'2*Department of Microbiology' and Department of Crop and Soil Science,2 Oregon State University,

Corvallis, Oregon 97331-3804

Received 5 August 1993/Accepted 7 November 1993

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soilisolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented bothmajor and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that werehighly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbagedry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupantsof field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants,ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions.ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolateswere mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successfulat nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolatesthat were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered fromnodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with aneffective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodulesoccupied by the former increased as its numerical advantage increased. Although subclover yields declined asnodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which largepercentages of nodules were still occupied by ET31-5.

Over a period of 50 years numerous studies have shown thatsome rhizobial strains can occupy a greater percentage ofnodules than other strains when the organisms are inoculatedonto legumes as a mixture of strains (15, 38, 50). Twohypotheses have been formulated to explain this interstrainvariation in nodulating ability. First, there may be interstrainvariation at the level of the plant-microbe interactions whichoccur during nodule initiation and development (50). Supportfor this hypothesis comes from situations where specific strainsor subpopulations dominate nodules regardless of their rela-tive contributions to an inoculant mixture or to a soil popula-tion (37, 42). In contrast, several studies have shown thatnodulation success by specific strains is influenced by therelative numbers of these strains in a mixture of strains (2, 4,20-22, 33). In these cases, relative nodule occupancy can bechanged if the ratio of strains is changed (2, 14, 21, 22). Thesituation that occurs when legumes grow in soil containingheterogeneous rhizobial populations has not been explained.Although it has been determined that subtypes in heteroge-neous soil rhizobial populations occupy the majority of rootnodules on soil-grown legumes (13, 17, 28, 29, 34, 36, 37), it hasnot been established that nodule-dominant types possess su-perior competitive traits compared with minor occupants.Indeed, there is evidence to the contrary. For example, minor

* Corresponding author. Phone: (503) 737-1844. Fax: (503) 737-0496. Electronic mail address: bottomlp@ucs.orst.edu.

t Oregon Agricultural Experiment Station Technical Paper no.10,286.

nodule-occupying types of Rhizobium meliloti and Rhizobiumleguminosarum bv. viciae were as competitive as nodule-dominant types when the organisms were evaluated undernonsoil conditions (12, 36).

In an accompanying paper we describe serotype AS6 ofRhizobium leguminosarum bv. trifolii which consistently dom-inates nodules of field-grown subclover (32b). In anotherstudy, the majority of the AS6 isolates were found to possessspecific chromosomal types (electrophoretic types [ETs]) andto belong to a genetically distinct group of isolates in the soilpopulation (32a). The objectives of this study were to charac-terize the symbiotic and competitive abilities of isolates be-longing to the nodule-dominant ETs and to compare theseisolates with isolates representing minor nodule-occupyingETs.

MATERIALS AND METHODS

Seed source and germination. Seeds of cultivar Nangeelasubclover were obtained from G. Evers, Texas A&M Univer-sity Agricultural Experiment Station, Angleton. Ten grams ofseeds was sieved through a 2-mm sieve, and abnormally smalland large seeds and seeds with seed coat damage werediscarded. The seeds were surface disinfested by standardprocedures (52), incubated on water agar plates at 4°C for 24h, and germinated overnight at room temperature.

Evaluation of symbiotic effectiveness of soil isolates of R.leguminosarum bv. trifolii on subclover. (i) Effectiveness test:experiment 1. The isolates used represented the major nodule-occupying serotype (AS6) and a spectrum of the numerous

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minor nodule-occupying serotypes recovered from subclovergrowing at a field site in western Oregon (32b). We choseseven isolates to represent several of the different chromo-somal types (ETs) which comprise serotype AS6 (32a); theseisolates were strains ET2-1, ET3-3, ET3-8, ET3-18, ET7-3,ET7-14, and ET8-2. We chose five isolates to representdifferent chromosomal types of serotypes that are infrequentnodule occupants of subclover at the Oregon site; theseisolates were serotype AS27 strains ET30-1, ET30-3, andET31-5, serotype AG4 strain ET17-1, and serotype AS21 strainET15-2. The effectiveness potentials of these isolates werecompared with the effectiveness potential of an inoculantquality strain 162X95 (= ET82-1), which was provided by R. S.Smith, LiphaTech Co., Milwaukee, Wis. The isolates weregrown in yeast extract-mannitol broth for 3 days at 28°C beforeseedlings were inoculated.

(ii) Effectiveness test: experiment 2. After initiating thisstudy, we discovered that serotype AS6 is composed of severalsubtypes and that subtype AS6-a is restricted to ET 2 andsubtype AS6-b is restricted almost exclusively to ET 3 (32a,32b). Since subtype AS6-a (ET 2) isolates were poorly repre-sented in the first experiment, a follow-up study was conductedwith subtype AS6-a isolates (strains ET2-1, ET2-2, and ET2-3)and additional representatives of subtype AS6-b (strainsET3-1, ET3-2, and ET3-3). Serotype AR23 isolate ET23-1 wasused as a highly effective strain for yield comparisons.

Plant growth conditions. Seedlings were transplanted intoLeonard jar assemblies (52). Each unit was composed of abottomless 1-quart (0.946-liter) beer bottle sitting neck downin a wide-mouth 800-ml mason jar. Approximately 70 g of aperlite-vermiculite mixture (1:1 vol/vol) was dispensed intoeach beer bottle, and a cheesecloth wick was used to transportnutrient solution from the mason jar to the perlite-vermiculitemixture. A 400-ml portion of an N-free mineral nutrientsolution was dispensed into the perlite-vermiculite mixture,and a 600-ml portion was added directly to the mason jar. TheN-free mineral nutrient solution (pH 6.5) was composed of thefollowing ingredients: 5.98 mM CaSO4 2H20, 0.14 mMCaCl2 . 2H20, 1.99 mM MgSO4- 7H20, 4.99 mM K2SO4, 1.25mM K2HPO4, 0.8 mM KH2PO4, 10 mg of ferric citrate perliter, and 10 ml of a micronutrient solution (18). The tops ofthe assemblies were covered with aluminum foil, and brownpaper bags were placed around the junctions between thebottles and the jars. The units were autoclaved for 60 min andallowed to cool to room temperature for at least 24 h beforeplanting.

After four seedlings were transplanted into each assembly,the preparations were inoculated with individual isolates of R.leguminosarum bv. trifolii which had been grown in yeastextract-mannitol broth for 3 days at 28°C. The assemblies werecovered with petri dish lids and incubated in a growth chamberfor about 5 days. The seedlings were thinned to one seedlingper assembly, and sterile paraffinized sand was applied to thesurface of each jar to a depth of 2 cm. The plants weretransferred to a greenhouse and arranged in a randomizedcomplete block design. Shoots were excised 54 days afterplanting, and three fully expanded leaves were left attached toeach crown. In experiment 1, regrowth was allowed to takeplace for 28 days, and then the herbage was harvested anddried for 14 days at 55°C and the dry weights of the plantmaterial from this second harvest were determined.

Total-nitrogen analysis. Dried herbage was ground in aWiley mill and digested with a combination of Kjeldahl catalystand concentrated H2SO4. The ammonium in the resultingdigests was analyzed by using standard procedures (7).

Statistical analyses. An analysis of variance and separation

of means were performed on the data by using the generallinear model of the SAS software (Statistical Analysis SystemInstitute, Inc.).

Competitive nodulation studies. Three ET 2 isolates (ET2-1,ET2-2, ET2-3) and three ET 3 isolates (ET3-1, ET3-2, andET3-3) were used to examine competitive nodulation poten-tials compared with the competitive nodulation potentials ofrepresentatives of other ETs. The ET 2 and 3 isolates wereused at population ratios of approximately 1:1 with the follow-ing isolates: ET8-1 and ET12-1, representing the most com-mon serotype AS6-c and serotype AS21 ETs, respectively; andET33-2, ET23-1, and ET31-5, representing minor nodule-occupying serotypes AP17, AR23, and AS27, respectively.

(i) Preparation of peat inoculants. To minimize the risk ofchanges that might occur in the ratio of inoculant strainsduring the time that the bacteria were harvested, mixed, andinoculated onto plants, the inoculants were prepared in sterilepeat (LiphaTech Co.). Each isolate was grown in 30 ml ofdefined glutamate-mannitol nutrient broth at 28°C for 3 days.Portions (24 ml) of each culture were transferred aseptically tosterile 160-ml milk dilution bottles containing 30-g portions ofsterile peat. The peat-culture mixtures were incubated at 27°Cfor 2 days, thoroughly mixed, and then incubated for another12 days. Samples were taken from each peat inoculant todetermine the rhizobial population density by a plate countassay. Portions (0.5 g) of each peat culture were transferred to500-ml volumes of a sterile mineral salt solution (pH 7)containing 3.67 mM K2HPO4, 0.81 mM MgSO4 - 7H20, and1.71 mM NaCl. The suspensions were diluted through a10-fold dilution series to a final dilution of 10-7. Portions (0.1ml) of the higher dilutions were plated onto yeast extract-mannitol agar plates and incubated at 27°C. The populationdensities of the peat inoculants ranged between 1.7 x 109 and4.4 x 109 cells per g. Each peat inoculant culture wassubdivided into six sterile 20-ml scintillation vials and refriger-ated until it was used.

(ii) Preparation of inoculant mixtures and plant growthconditions. Cultivar Nangeela subclover seeds were surfacedisinfested, germinated, and transplanted into Leonard jarassemblies as described above. A 0.5-g portion of each peatinoculant was diluted 103-fold into 500 ml of a cold (4°C)sterile mineral salt solution to achieve a final cell concentrationof approximately 106 cells per ml. The peat suspensions wereshaken vigorously for 10 min and then were allowed to settlefor 5 min. A 20-ml portion of supernatant representing eitheran ET 2 isolate or an ET 3 isolate was mixed with an equalvolume of supernatant of a competitor, and the mixture waskept in an ice bath until the seedlings were ready for inocula-tion. For the 10:1 ratios, 2 ml of the 10' suspension wasmixed with 18 ml of cold sterile mineral salt solution and thenwith a 20-ml portion of the 10-3-diluted peat inoculant of thecompetitor before inoculation onto subclover seedlings. Eachseedling was inoculated with 1 ml of the appropriate inoculantmixture. Four replicate assemblies were prepared for eachinoculant treatment. The controls included seedlings inocu-lated with each of the individual strains and uninoculatedseedlings supplemented or not supplemented with 8 mMKNO3. The different paired strain combinations used aredescribed in the Results. The actual inoculant strain ratioswere determined later by staining samples with fluorescein-labeled immunoglobulin conjugates (FAs) specific to eachmember of the mixture. Each assembly was covered with asterile petri dish lid and transferred to a growth chamber thatwas programed for a 14-h light-10-h dark cycle. After 5 daysthe petri dish lids were removed, the seedlings were thinned totwo seedlings per assembly, and the perlite-vermiculite mixture

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was covered with sterile paraffinized sand (depth, approxi-mately 2 cm) to prevent aerial contamination and reducesurface evaporation. The assemblies were transferred to agreenhouse and arranged in a randomized complete blockfashion, and the blocks were rotated at 7-day intervals. Day-light illumination was supplemented with high-intensity lampsby using a 14-h light-10-h dark cycle, and the temperature wasmaintained at 25 to 27°C. The assemblies were replenishedwith mineral nutrient solution as needed. The plants wereharvested 6 weeks after inoculation, and the root nodules werecollected for nodule occupancy analysis.

(iii) Nodule analysis. The Leonard jar assemblies were

dismantled, and the perlite-vermiculite mixture was washedaway from the roots. Regardless of the strain combination,between 100 and 140 nodules were recovered from the com-bined root systems of the two plants in each assembly. Fromthis pool 20 nodules were picked at random, surface disin-fested, and crushed in 50 to 200 [lI of sterile deionized water,and smears were prepared on microscope slides. The smearswere air dried, heat fixed, prestained with 1 drop of rhoda-mine-gelatin conjugate (5), and analyzed by immunofluores-cence with the appropriate FAs. In the treatments in which ET3 representatives competed against ET2-1, duplicate smearswere examined with FA-AS6 adsorbed with a combination ofET2-1 and ET8-1 and with FA-AS20 adsorbed with ET3-3. ET2 smears reacted only with adsorbed FA-AS20, whereas onlyET 3 isolates reacted positively with adsorbed FA-AS6. Nod-ules occupied by ET 3 isolates were distinguished from nodulescontaining ET8-1 since only nodules occupied by the ET 3isolates reacted with FA-AS6 adsorbed with isolate ET8-1;nodules containing ET8-1 were identified by their strongreactions with FA-AS21 (32b).

(iv) Indirect immunofluorescence procedure for identifyingrepresentatives of serotype AS27. Although strains ET30-3 andET31-5 reacted with antiserum AS27 in immunodiffusion tests,they did not react with the fluorescein-labeled conjugateprepared to the same antiserum. Presumably, the cross-reac-tive antibody was lost during preparation of the FA. Anindirect FA method involving fluorescein-labeled goat anti-rabbit immunoglobulin G (Sigma Chemical Co., St. Louis,Mo.) combined with whole AS27 antiserum was used to detectstrains ET30-3 and ET31-5 in nodule smears (46). Nodulesmears were stained with rhodamine-gelatin conjugate andthen incubated for 15 min with AS27 antiserum (diluted 1:100and adsorbed three times with isolate AS6-1 to remove anti-bodies that were cross-reactive with members of serotypeAS6). Excess antiserum was washed off the smears with 0.02 Msodium phosphate buffer (pH 7.2), and the smears wererestained with the manufacturer's recommended dilution(1:80) of the fluorescein-labeled goat anti-rabbit immunoglob-ulin G (in 0.02 M sodium phosphate buffer, pH 7.2). Afterincubation for 15 min, the smears were rinsed, air dried, andexamined by performing immunofluorescence tests as de-scribed above.

RESULTS

Subclover grew vigorously in the Leonard jar assembliesunder greenhouse conditions. A ninefold difference (4.92 to0.56 g) in herbage dry matter accumulation was observedbetween the most and least effective plant-isolate combinationsover the two herbage harvests (Table 1). In general, isolatesthat represented minor nodule-occupying ETs on field-grownplants were significantly more effective than any of the eightisolates that represented the ETs that dominate nodules ofsubclover growing under field conditions. Indeed, two isolates,

TABLE 1. Symbiotic effectiveness potential (shoot dry matter yield)of R. leguminosarum bv. trifolii isolates which represent major and

minor nodule occupants of field-grown subclover

Dry matter yield (g plant- ')Signifi-Isolate Harvest 1 + cancea

Harvest 1 Harvest 2 harvest 2

Minor field nodule-occupying ETs

ET30-1 1.54 3.38 4.92b SET30-3 1.53 3.35 4.88b SET82-1c 1.24 2.46 3.69d SET15-2 1.17 2.77 3.94d SET31-5 1.16 2.59 3.75d SET17-1 1.02 2.62 3.64d S

Major field nodule-occupying ETs

ET3-8 0.76 1.52 2.28e SET8-2 0.60 0.53 1.13f NSET8-1 0.59 0.90 1.49f NSET3-18 0.58 0.97 1.56f sET3-3 0.53 1.62 2.lSe SET7-3 0.40 0.53 0.939 NSET2-1 0.25 0.30 0.569 NSET7-14 0.21 0.37 0.579 S

LSDO.O5h 0.31 0.32 0.52

a S, second-harvest yields were significantly greater (P c 0.05) than first-harvest yields, as determined by a paired t test; NS, first- and second-harvestyields were not significantly different.

be.ef,g Values which are not followed by the same letter are significantlydifferent (P c 0.05) as determined by Duncan's multiple-range test.

c Strain ET82-1 (= 162X95) is an inoculant quality strain.h LSDO05, least significant difference (P c 0.05).

ET30-1 and ET30-3, were significantly more effective thanisolate 162X95 (a commercial subclover inoculant strain)under our growth conditions.

While most of the effective plant-isolate combinations pro-duced at least twofold more dry matter during the secondgrowth period than during the first growth period, the yields offour of the six least effective plant-isolate combinations did notincrease significantly between the first harvest and the secondharvest. Indeed, only one isolate, ET3-3, produced more thantwofold more herbage dry weight. Although the overall effec-tiveness rankings of most isolates did not change betweenharvests, there was one notable exception. The yield of 162X95was equivalent to the yields of the two superior strains (ET30-1and ET30-3) at the first harvest, but this strain declined in itsoverall ranking during the second growth interval.The total N2 fixed by the plant-isolate combinations followed

the trends observed with dry weights (Table 2). In addition, theN concentrations in the subclover herbage produced by ET 2,3, and 7 isolates were substantially lower (26.0 to 32.2 mg of Nper g) than the N concentrations in the herbage produced byisolates representing the minor nodule-occupying ETs (35.4 to38.6 mg of N per g). Nevertheless, the amount of N2 fixed bysome plant-isolate combinations during the interval betweenthe first harvest and the second harvest was up to threefoldgreater than the amount of N2 fixed before the first harvest. Asa result, the concentrations of N in second-harvest herbage ofplants nodulated by ET 3, 7, and 8 isolates were not signifi-cantly less than the concentrations in high-yielding plant-isolate combinations.

Since isolates belonging to one of the major nodule-occupy-ing lineages, ET 2, were not adequately represented in the

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TABLE 2. Symbiotic effectiveness potential (amount of N2 fixed and herbage N concentration) of R. leguminosarum bv. trifolii isolates whichrepresent major and minor nodule occupants of field-grown subclover

Kjeldahl digestible nitrogen in herbage

Isolate Harvest 1 Harvest 2 Total N2 fixed(mg plant )

mg plant- mg g-i mg plant- mg g-l

Minor field nodule-occupying ETsET30-1 60 38.6 153a 45.2 213*ET30-3 54 35.4 149a 44.6 203"ET82-1 44 36.3 114a 46.2 158CET15-2 43 36.4 128a 46.2 171CET31-5 44 37.3 117a 45.3 161CET17-1 38 38.0 118a 45.0 156'

Major field nodule-occupying ETsET3-8 22 28.6 67a 43.2 89"ET8-2 21 34.2 23e 43.0 44f'ET3-18 18 31.6 42a 42.7 60fET3-3 17 31.3 71a 43.6 88dET7-3 13 32.2 21e 42.1 349ET2-1 8 30.0 lOe 33.0 18'ET7-14 5 26.0 14a 37.7 19q

LSDO.05h 13 3.6 15 2.7 25

a Second-harvest values for the amount of nitrogen per plant were significantly greater (P c 0.05) than first-harvest values, as determined by a paired t test.b,c,dfg Values which are not followed by the same letter are significantly different (P c 0.05) as determined by Duncan's multiple-range test.e First- and second-harvest values for the amount of nitrogen per plant were not significantly different.h LSDO05, least significant difference (P c 0.05).

two-harvest experiment, another less extensive experiment wasperformed to assess the symbiotic potential of these organisms(Table 3). The data obtained confirmed that both ET 2 and ET3 isolates are suboptimally effective on subclover despite theirhigh occupancy of nodules on field-grown plants.

Competition experiments were performed in Leonard jarassemblies which gave the plants an opportunity to growvigorously. Despite the fact that the three ET 3 isolates werenot significantly different in symbiotic effectiveness, theseorganisms were different in competitive ability (Table 4).Although isolates ET3-1 and ET3-2 occupied significantlymore nodules than all of their competitors except ET23-1, theresults obtained with isolate ET3-3 were less consistent. Al-though ET23-1 was recovered from the field on only oneoccasion, it was equally competitive with each of the three ET3 isolates under nonsoil conditions.

TABLE 3. Herbage dry matter yields of subclover plants nodulatedby several ET 2 and 3 isolates, yield of plants nodulated by a strainthat exhibited superior effectiveness (ET23-1), and yield of plants

that were nitrate fed

Isolate Herbage dry wtor prepn (g plant')

Nitrate fed............................................................................ET23-1...................................................................................ET3-1.....................................................................................ET3-3.....................................................................................ET3-2.....................................................................................ET2-1.....................................................................................ET2-2.....................................................................................ET2-3.....................................................................................Uninoculated ........................................................................

1.08a0.91"0.66c0.64c0.55,0.45c'd0.47c,d0.44cd0.03

LSD0O05e ......................................... 0.16

A somewhat different situation was observed with three ET2 isolates (Table 5). Although ET 2 isolates occupied a highproportion of nodules when they were mixed 1:1 with somecompetitors (ET33-2 and ET12-1), they could not prevent the

TABLE 4. Competitive nodulating abilities of three ET 3 isolateswhen they were tested with representatives of other ETs on cultivar

Nangeela subclover

% of nodules occupied by":Paired strain Signifi-combination' ET 3 Competitor cance'

isolate

ET3-1 + ET2-1 81(67-91) 25 (14-38) SET3-2 + ET2-1 95 (95) 32 (19-43) SET3-3 + ET2-1 58 (48-67) 42 (33-52) NS

ET3-1 + ET8-1 80 (71-86) 20 (9-14) SET3-2 + ET8-1 98 (95-100) 2 (0-5) SET3-3 + ET8-1 86 (71-90) 24 (15-29) S

ET3-1 + ET33-2 81(67-91) 19 (10-33) SET3-2 + ET33-2 84 (81-86) 17 (14-19) SET3-3 + ET33-2 79 (57-95) 25 (14-43) S

ET3-1 + ET12-1 84 (74-91) 16 (10-29) SET3-2 + ET12-1 86 (81-91) 14 (10-19) SET3-3 + ET12-1 62 (43-81) 38 (19-57) NS

ET3-1 + ET23-1 38 (24-57) 62 (16-43) NSET3-2 + ET23-1 36 (24-43) 68 (57-81) NSET3-3 + ET23-1 30 (19-48) 70 (52-81) NS

" The inoculant ratios ranged from 0.98:1 to 1:1.3.b The nodule occupancy values are the means of four replicates and include

the values for both singly occupied nodules and cooccupied nodules. The valuesin parentheses are the ranges of occupancy values.

' S, nodule occupancy value of the ET 3 isolate was significantly (P s 0.05)greater than nodule occupancy value of the competitor as determined by a pairedt test; NS, values were not significantly different.

a.b.C.d Values which are not followed by the same letter are significantlydifferent (P s 0.05) as determined by the Duncan's multiple-range test.

' LSD(.05, least significant difference (P s 0.05).

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TABLE 5. Competitive nodulating abilities of three ET 2 isolateswhen they were tested with representatives of other ETs on cultivar

Nangeela subclover

Paired strain % of nodules occupied by":combination' ~~~~~~~~~~Significancecombination" ET 2 isolate Competitor

ET2-1 + ET33-2 68 (48-95) 44 (33-52) NScET2-1 + ET33-2d 95 (91-100) 5 (0-10) e

ET2-2 + ET33-2 86 (76-91) 26 (24-29) e

ET2-3 + ET33-2 66 (57-76) 39 (24-48) NS

ET2-1 + ET12-1 64 (57-71) 40 (29-48) NSET2-1 + ET12-Id 94 (86-100) 6 (0-14) -'

ET2-2 + ET12-1 63 (52-71) 37 (29-48) NSET2-3 + ET12-1 71(62-81) 29 (19-38) e

ET2-1 + ET23-1 12 (0-24) 93 (81-100) -fET2-1 + ET23-ld 55 (38-76) 49 (24-62) NS

ET2-1 + ET31-5 26 (14-31) 80 (76-90)ET2-1 + ET30-3 6 (5-11) 95 (90-100)

a Inoculant ratios were approximately 1:1 except as indicated below.b The nodule occupancy values are the means of four replicates and include

the values for both singly occupied nodules and cooccupied nodules. The valuesin parentheses are the ranges of occupancy values.

c NS, no significant difference between the percentage of nodules occupied byET2-1 and the percentage of nodules occupied by the competitor.

d Inoculant ratios were approximately 10:1 in favor of ET2-1.e The ET 2 isolate occupied significantly more nodules than the competitor (P

c 0.05).fThe competitor occupied significantly more nodules than the ET 2 isolate (P

c 0.05).

competitors from also occupying large proportions of rootnodules. When ET2-1 was given a modest (6:1 to 10:1)numerical advantage over isolates ET33-2 and ET12-1, it madesignificant gains in occupancy at the expense of the twocompetitors. In contrast, although occupancy by isolate ET2-1increased when this organism was given a numerical advantageover ET23-1, the latter isolate still occupied a large percentageof nodules.Although ET2-1 could not outcompete either of the more

symbiotically effective competitors (ET23-1 and ET31-5), weobserved a suppression of plant yield despite the large per-centages of nodules occupied by the highly effective isolates.This yield-suppressing effect was examined in a more detailedexperiment in which we used different inoculant ratios ofET2-1 and ET31-5 (Tables 6 and 7). When the inoculant ratioreached 10:1 in favor of ET2-1, the plant yield and amount of

TABLE 6. Influence of different inoculum ratios on noduleoccupancy by isolates ET2-1 and ET31-5

Ratio of ET2-1 % of nodules occupied by": btoET31-5 ~~~~~~~~~~~Significanceto ETsl-5 ET2-1 ET31-5 Both isolates

25:1 73 (58-86) 43 (20-68) 20 NS10:1 32(20-45) 78(60-95) 13 S1:1 10(0-20) 100 10 S1:10 0 100 0 S

a Nodule occupancy values for ET2-1 and ET31-5 are the means of fourreplicates and include the values for both singly occupied nodules and cooccu-pied nodules. The values in parentheses are the ranges of occupancy values.

b ET31-5 occupied significantly more nodules than ET2-1 (P c 0.05), asdetermined by a paired t test. NS, occupancy values were not significantlydifferent.

TABLE 7. Influence of nodule occupancy by isolates ET2-1 andET31-5 of R. leguminosarum bv. trifolii on subclover

yield parameters

Herbage yield parametersRatio of ET2-1 Total N

to ET31-5 Dry wt N concna (mg plant-)(g plant-') (mg g- l)

ET2-1 0.12 21.1 2.525:1 0.22 32.0 7.010:1 0.61 37.0 22.61:1 0.74 37.5 27.81:10 0.95 34.9 33.2ET31-5 0.98 40.0 39.2

LSD.05, 0.15

aN concentrations were determined by using a composite sample from eachtreatment.

b LSD(,05, least significant difference (P - 0.05).

N2 fixed values were between 30 and 50% less than the valuesobserved with plants nodulated by ET31-5 alone, despite thefact that only 20 to 45% of the nodules were occupied byET2-1. With an inoculant ratio of 25:1, both the yield and theamount of N2 fixed values were similar to values for plantsnodulated by ET2-1 alone, even though 43% of the noduleswere occupied by ET31-5.

DISCUSSION

Our findings have bearing on a number of unresolved issuesconcerning nitrogen fixation in subclover in particular andinterstrain competition for nodulation in general. Despite theimportance of subclover to the productivity of a large area ofpastures on hill and range lands in the far western UnitedStates, there have been several previous reports which haveshown that field-grown subclover in this region is nodulatedprimarily by rhizobia that exhibit suboptimal symbiotic effec-tiveness (13, 25, 27, 30). Furthermore, agronomists havereported that the N content of subclover herbage grownthroughout Oregon and California is erratic and can rangefrom 2 to 4.5% (1, 16, 31, 40, 45). Our observations that thenodule-dominant isolates on the Abiqua soil site fix 2- to10-fold less N2 on subclover than minor occupants fix andproduce first-harvest herbage that has suboptimal proteinconcentrations complement the observations described above.Since suboptimally effective nodule-dominant isolates fromanother site (13) also belong to the same chromosomal lineageas the isolates described in this study (32a), it will be importantto determine how widespread the occurrence of subclovernodulated primarily by strains belonging to this group is.

Despite the fact that there is an extensive literature on thesubject of competitive nodulation, we have little understandingof the factors which influence this phenomenon under soilconditions (6, 24). One major problem has been the lack oflaboratory studies performed with isolates that have beenproven to be nodule-dominant types on field-grown plants. Inthis study we showed that such isolates (ET 2 and 3 isolates)are not necessarily more competitive than isolates which areminor nodule occupants on field-grown plants at the samelocation. Our findings agree with those of other workers whohave shown that strains which dominate field nodules are notnecessarily more competitive than minor nodule occupantswhen the organisms are evaluated under environmentallycontrolled nonsoil conditions (12, 36). Nevertheless, we cannotexclude the possibility that enhanced competitiveness is an

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unstable trait which is easily lost during subculturing afterisolation from nodules. It is well documented that symbioticeffectiveness is unstable in some rhizobial strains (23), andboth genomic instability and plasmid instability have beenobserved in R. leguminosarum bv. phaseoli (10, 19, 43). Inaddition, since plasmids play a role in nodulation competitive-ness (8, 9, 11, 35) and plasmid diversity occurs in R. legumino-sarum isolates having the same chromosomal type (32, 44, 55),the possibility that there is intra-ET variation in competitive-ness seems likely.

It is intriguing to speculate about reasons why there areisolates which exhibit enhanced competitiveness and yet areminor occupants of field-grown plants. It is possible that fieldconditions suppress the inherent competitive potential of thesebacteria. Several reports have shown that soil temperature andsoil pH can modify the nodulating success of strains of variousRhizobium species on soil-grown plants (17, 24, 26, 41, 47, 51,53, 54). Equally plausible, however, is the possibility that thehighly effective competitive strains are nonrandomly distrib-uted in the soil at a site because they are recent immigrants orbecause they represent the remnants of a previously well-distributed population that is now in decline. Alternatively,these strains may represent rare intrasite recombinants thathave not had the opportunity to disperse. In an accompanyingpaper (32b), Leung et al. show that some serotypes were rarenodule occupants in most areas of a field but occupiedsubstantially more nodules in one or two localized sites withinthe field. More work is needed to discriminate between soilfactors and nonrandom distribution as explanations for thelack of competitive types in field nodules.

Finally, our observations on yield suppression by isolateET2-1 could provide insight into how suboptimally effectivestrains might exert a negative effect on legume growth in thefield. Previous studies have described antagonistic effects onclover growth when plant nodules were occupied by more thanone rhizobial strain (3, 14, 30). Demezas and Bottomley (14)noted that subclover plants grew suboptimally when theirnodules were occupied by suboptimally effective strain WS2-01despite the fact that 50% of the nodules on the same plantswere occupied by strain WS1-01 which exhibited superioreffectiveness. Additional studies will be needed to determinewhether the lower yield obtained was simply the result of thefact that a high proportion of nodules were occupied by a strainwith poor N2-fixing ability or whether it was due to anantagonistic property of ET2-1 directed either at plant metab-olism or at the metabolism of the other nodule-occupyingstrain. There have been reports of strains of Bradyrhizobiumjaponicum (48) and Rhizobium tropici (39) that have negativeeffects on legume growth, as well as a report of a strain of R.leguminosarum bv. trifolii that inhibits the growth and nodu-lating ability of other strains of the same biovar (49).

ACKNOWLEDGMENTS

This research was supported by grant 87-37120-4738 from theCompetitive Grants Office of the U.S. Department of Agriculture, bythe Oregon Agricultural Experiment Station, and by the N. L. TartarFoundation. F. N. Wanjage was supported by US-AID.We thank C. Pelroy for final processing of the manuscript. W.

Dougherty is recognized for his generous loan of greenhouse facilitieswith high-intensity lighting.

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