Vol. 56, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1990, p. 224-2290099-2240/90/010224-06$02.00/0Copyright © 1990, American Society for Microbiology

Symbiotic Effectiveness of Indigenous Soybean Bradyrhizobia asRelated to Serological, Morphological, Rhizobitoxine, and

Hydrogenase PhenotypestJ. FUHRMANN

Delaware Agricultural Experiment Station, Department ofPlant Science, College ofAgricultural Sciences,University ofDelaware, Newark, Delaware 19717-1303

Received 17 July 1989/Accepted 19 October 1989

A collection of 360 isolates of Bradyrhizobium japonicum was developed from soybean (Glycine max [L.]Merrill) nodules taken from 18 locations in Delaware. The isolates were characterized serologically with anenzyme-linked immunosorbent assay, morphologically by colony type on yeast extract-mannitol agar, and forproduction of rhizobitoxine symptoms with soybean plants. These analyses revealed 12 and 3 groups based onserology and morphology, respectively. The more common identifiable isolates were in serogroups 94, 6, 122,and 76. Nearly 33% of the isolates were rated nonreactive with all of the antisera tested. Overall, 18% of theisolates produced rhizobitoxine symptoms, and these were associated with five known serogroups (31, 46, 76,94, and 130) and the nonreactive grouping, but with only one colony type. A subsample of 92 isolates was ratedfor N2-fixing ability in the greenhouse and for hydrogenase phenotype in the laboratory. The nitrogen contentof plant shoots was strongly and comparably related to both the serological and morphological groupings.Rhizobitoxine and hydrogenase phenotypes were relatively poor predictors of symbiotic effectiveness. Amongthe serologically reactive isolates, those in serogroups 38-115, 122, and 110 fixed the most N2, whereas onecolony type (that containing isolates producing rhizobitoxine) was clearly inferior to the remaining twomorphological groupings. Isolates displaying hydrogenase activity (approximately 15% of the isolates tested)correlated with three serologically reactive groupings (serogroups 110 and 122 and a 122/123 cross-reactivegroup) and two colony types, none of which coincided with groupings containing bradyrhizobia rated positivefor rhizobitoxine production.

The characterization of indigenous populations ofBradyrhizobiumjaponicum, the root-nodulating symbiont ofsoybean plants (Glycine max [L.] Merrill), is important tothe development of strategies to enhance nodulation bysymbiotically superior bradyrhizobia in soils previouslycropped to soybeans. Serological characterizations are mostcommon and have revealed large regional differences inserogroup frequency. Bradyrhizobia serologically related tostrain USDA 123 are dominant in the midwestern UnitedStates (5, 12, 18), whereas serogroups 31 and 76 are commonin the southeastern United States (3, 18). A survey ofbradyrhizobia in the soils of Delaware indicated that themajor identifiable serogroups are 94, 6, 76, and 31 (lOa).The high incidences of isolates in serogroups 94 and 76

may be important because the corresponding serotypestrains produce rhizobitoxine, a phytotoxin that causes adistinctive chlorosis of newly formed leaves of susceptiblesoybean genotypes (7, 21, 25). It is possible that indigenousbradyrhizobia belonging to these serogroups also producethe toxin. Although rhizobitoxine symptoms are often severeunder greenhouse conditions, symptoms in the field aregenerally transitory and, therefore, are thought to have anegligible effect on yields (9). However, definitive studiesregarding yield effects are not in the literature.Other criteria besides serological reaction have been used

to characterize indigenous bradyrhizobia, including the elec-trophoretic mobilities of cellular proteins (16, 24), intrinsicantibiotic resistance (23), and bacteriophage typing (19).Although these and the serological studies have provided

t Miscellaneous paper no. 1280 of the Delaware AgriculturalExperiment Station.

valuable information regarding the diversity of indigenousbradyrhizobia, their usefulness is limited because they typ-ically have not assessed the N2-fixing abilities of thebradyrhizobia represented by the resulting groupings. It istherefore uncertain whether these classification techniquescan meaningfully predict the symbiotic characteristics ofindigenous soybean bradyrhizobia.One criterion for characterizing indigenous populations

that may directly infer symbiotic effectiveness is the pres-ence of an active hydrogen uptake (Hup) system (hydroge-nase phenotype). Superior rates of N2 fixation have beenreported for strains of B. japonicum exhibiting the Hup+phenotype relative to Hup- strains (2, 13, 31), althoughrecent work has contradicted these findings (8). Surveys ofthe frequency of Hup+ phenotypes in indigenous popula-tions of soybean bradyrhizobia have revealed considerableregional differences (18, 28). Keyser et al. (18) reported thatapproximately 25% of the soybean bradyrhizobia in Dela-ware were Hup+, as estimated from a limited sampling of 28nodules.The studies reported here evaluated various criteria for

characterizing the diversity and symbiotic effectiveness ofindigenous soybean bradyrhizobia. The phenotypes of 360nodule isolates were determined with respect to serologicalreaction, colony morphology, and production of rhizobitox-ine symptoms with soybean plants. Representative isolatesfrom the serological and morphological groupings were ratedfor N2-fixing ability in the greenhouse and for hydrogenasephenotype in the laboratory. The various phenotypes werethen evaluated for their abilities to predict the symbioticeffectiveness of the indigenous population.

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MATERIALS AND METHODS

Field sampling and isolation of bradyrhizobia. Soybeanroot nodules were collected during August 1987 from 18farms located throughout Delaware. Several soybean culti-vars were represented, and plant growth stages at samplingranged from V3 to R5 depending on location (10). Samplesfor each location consisted of the tap roots from a total of atleast 12 plants taken from three or more random locations ina field. Roots were transported to the laboratory in ice-cooled containers and stored at -20°C. For each location, 20tap root nodules were randomly selected for isolation of B.japonicum as previously described (360 isolates total) (10a).

Serological and morphological analyses. Heat-treated,whole-cell antigens from yeast extract-mannitol (YM) (30)broth cultures of each isolate were analyzed serologicallywith an indirect enzyme-linked immunosorbent assay (lOa,11). Each isolate was tested in duplicate with polyclonalantisera produced against the following U.S. Department ofAgriculture (USDA) strains of B. japonicum: 31, 38, 46, 62,76, 94, 110, 122, 123, 130, 135, and 138. Correspondingserogroups were 31, 38-115, 46, 62, 76, 94, 110, 122, 123, 130,135, and 6, respectively (17). Serological reactions weremeasured spectrophotometrically and expressed as a per-centage of that observed for the corresponding USDA anti-gen strain. The criteria employed for assigning isolates toparticular serogroups have already been described (lOa).The colony morphology of each isolate when grown on YMagar for 7 to 10 days at 28°C was also recorded. Isolates wereplaced into groupings according to three classificationschemes based on serological, morphological, and combinedserological-morphological characteristics, respectively.Greenhouse experiments. All 360 isolates were tested for

nodulating ability and rhizobitoxine production with Forrestsoybean plants in the greenhouse (verification study). Iso-lates rated positive and negative for rhizobitoxine produc-tion will be referred to as RT+ and RT-, respectively.Surface-sterilized seeds (95% ethanol for 5 min) were sownto duplicate greenhouse pots (590 ml) containing horticul-tural vermiculite, inoculated with bradyrhizobia from YMbroth cultures (ca. 109 bacteria pot-'), and watered with anN-free nutrient solution (22). Noninoculated plants wereincluded as contamination checks. The plants were rated forthe presence of effective nodulation and rhizobitoxine symp-toms at 44 to 50 days after planting.Randomly selected representatives of each combined se-

rological-morphological grouping were subsequently testedfor N2-fixing ability in the greenhouse (symbiotic effective-ness study). The number of isolates used from each groupingwas approximately proportional to its corresponding fre-quency in the indigenous population. A total of 92 of theoriginal 360 bradyrhizobia was tested. The USDA antigenstrains and noninoculated controls were also included. Theexperimental design was a randomized complete block withfour replicates. Uniform-sized Forrest soybean seeds weresurface sterilized, sown 4 seeds pot-', and watered asdescribed above. Plants were thinned to 2 seedlings pot-' at10 days after planting. Plants were harvested by block at 42to 44 days after planting. The presence and severity ofrhizobitoxine symptoms were recorded. Shoots were clippedat a point just below the cotyledon scar, dried at 65°C for 72h, weighed, and analyzed for total N by a semi-micro-Kjeldahl procedure.Hydrogenase assay. The field isolates included in the

symbiotic effectiveness study were tested for hydrogenaseactivity during heterotrophic growth, using a procedure

modified from that of van Berkum (29). Bradyrhizobia werecultured in a modified medium (4) containing HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 1.3 gliter-'; MES (2-N-morpholinoethanesulfonic acid), 1.1 gliter-'; arabinose, 1.0 g liter-'; sodium gluconate, 1.0 gliter-'; yeast extract, 1.0 g liter-'; and the following salts:NH4Cl, 6.0 mM; KCI, 2.0 mM; Na2SO4, 1.8 mM; Na2HPO4,0.88 mM; MgSO4, 0.73 mM; CaCl2, 88 ,uM; FeCl3, 25 ,uM;and NiCl2, 5 ,uM. The acidity was adjusted to pH 6.6 with 6M NaOH. Starter cultures were grown for 7 days in testtubes containing 1 ml of medium. Serum bottles (160 ml)containing 5 ml of medium were inoculated with ca. 0.1 ml ofstarter culture, sealed with a serum stopper, and purged withN2. H2 and 02 were then added to the headspace, both to aconcentration of 2% (vol/vol). Similarly prepared bottlesinoculated with USDA 110 and USDA 123 served as positiveand negative controls, respectively, while bottles containinguninoculated medium served as reference blanks. Duplicateserum bottles were prepared for all treatments. The cultureswere incubated at 25°C for 84 h on a gyratory shaker set at100 rpm. The concentration of H2 in the headspace wasdetermined by injecting 0.2-ml samples into a Hewlett-Packard gas chromatograph (model 5830A) equipped with amolecular sieve column (0.3 by 182 cm; 55°C) and a thermalconductivity detector (90°C). N2 at a flow rate of 20 ml minm-was used as the carrier gas. Hydrogenase activity wasassessed by comparing the rate of H2 disappearance inbottles inoculated with bradyrhizobia with that for the ref-erence blanks.

Statistical analyses. Data from the serological and morpho-logical analyses were arranged in contingency tables andtested with the chi-square statistic to determine whethersampling location significantly affected the frequency ofoccurrence of the various groupings. Shoot N contents fromthe symbiotic effectiveness study were subjected to analysisof variance procedures to determine the relationship be-tween the abilities of the bradyrhizobia to fix N2 and theirrespective serological, morphological, hydrogenase, andrhizobitoxine phenotypes. Separation of means was accom-plished by least significant difference procedures, employingStudent's t distribution.

RESULTS

Classification of the indigenous bradyrhizobia accordingto serological and morphological criteria indicated the pres-ence of 12 serological groupings, three colony types, and 16combined serological-morphological categories (Table 1).The 12 serological groupings consisted of known serogroupscorresponding to 10 of the 12 antisera used, a 122/123cross-reaction serogroup, and those isolates which did notreact significantly with any of the antisera tested. The122/123 cross-reaction serogroup probably correlates withserogroup 129 in the 123 serocluster (26). Among thoseisolates exhibiting positive reactions, the more commonserogroups were 94, 6, 122, and 76. None of the knownserogroups constituted a majority of the isolates collectedfrom any of the sampling locations, and nearly a third of theisolates could not be identified serologically. Serogroupswhich contained RT+ isolates included 31, 46, 76, 94, and130, plus the nonreactive grouping.Three readily distinguishable colony types were produced

on YM agar: large mucoid (LM), large watery (LW), andsmall dry (SD) (Fig. 1). The large and small colony typeswere >1 mm and s1 mm in diameter after 7 to 10 days

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TABLE 1. Serological and morphological distribution of soybeanbradyrhizobia collected from 18 locations in Delaware andcorresponding frequency of RT+ phenotypes with Forrest

soybean plants grown under greenhouse conditions(verification experiment)

Mean FrequencyColony No. of relative range Frequency

Serogroup type isolates frequency locationsb group (S)

6 LM 37 10.3 0-40 031 LW 18 5.0 0-25 2838-115 LM 18 5.0 0-35 046 LW 11 3.1 0-15 2776 LW 21 5.8 0-35 9094 LW 63 17.5 0-45 37110 LM 10 2.8 0-20 0110 SD 5 1.4 0-10 0110 All 15 4.2 0-20 0122 LM 14 3.9 0-25 0122 SD 17 4.7 0-15 0122 All 31 8.6 0-30 0123 LM 10 2.8 0-10 0122/123C SD 15 4.2 0-15 0130 LW 3 0.8 0-5 67NId LM 73 20.3 0-45 0NI LW 18 5.0 0-25 61NI SD 27 7.5 0-55 0NI All 118 32.8 0-75 9All LM 162 45.0 10-75 0All LW 134 37.2 0-80 48All SD 64 17.8 0-75 0All All 360 18

a Percentage based on 360 isolates.b Percentage based on 20 isolates.c Isolates exhibiting strong cross-reaction with 122 and 123 antisera.d NI, Not identified.

growth at 28°C, respectively. The LM colonies were mucoid,convex, entire, and uniformly translucent to opaque,whereas the LW colonies were flat and translucent, formedextensive areas of confluent watery growth, and tended tohave a mottled or granular appearance and irregular margins.The SD colonies were raised to convex, entire, and translu-cent to nearly opaque. Bradyrhizobia producing LM colo-nies were most prevalent in the indigenous population,whereas those exhibiting the SD type were least common(Table 1). Each colony type was dominant (.75% of theisolates recovered) at one or more of the sampling locations.Only isolates producing LW colonies induced rhizobitoxinesymptoms in Forrest soybean plants, although not all LWisolates were rated RT+. Overall, 18% of the isolates wereRT+ in the verification experiment.

Multiple colony types were observed for three of theserological groupings (Table 1). Bradyrhizobia in serogroups110 and 122 produced LM and SD colonies, and those fromthe nonreactive grouping exhibited all three colony types.Most of the nonreactive isolates produced LM colonies. Allserogroups having the LW colony type, including the non-reactive grouping, contained some RT+ isolates.The frequency of occurrence of the serological and mor-

phological groupings differed greatly among locations (Table1). Chi-square analysis of the data revealed that thesedifferences were highly significant (P c 0.001) regardless ofthe classification system employed (results not shown).Similarly, the frequency of RT+ isolates ranged from 0 to65% among sampling locations (results not shown).

Large differences in mean symbiotic effectiveness were

observed among the phenotypes composing each classifica-tion system examined (Table 2). Among the serologicallyreactive groupings, isolates in serogroups 38-115, 122, and 110fixed the most N2, while those in serogroups 94 and 76 were

least effective. Isolates producing LM colonies tended to fixmore N2 than did those of the SD type, whereas LW isolateswere clearly inferior. The range in mean N contents ofsoybean shoots produced by the LM, SD, and LW colonytypes were 49.1 to 86.1, 52.2 to 71.4, and 37.4 to 59.4 mg ofNplant-', respectively (data not shown). The mean N contentsof plants nodulated by the indigenous serogroups were incon-sistently related to those for the corresponding USDA strains.

Isolates displaying the Hup+ phenotype were restricted tofour serological groupings (110, 122, 122/123, and the nonre-active isolates) and two colony types (LM and SD) (Table 2).Only the 122-LM and 122/123-SD combined groupings con-

tained solely Hup+ isolates. Overall, approximately 15% ofthe isolates tested were Hup+. Nodulation by isolates havingHup+ phenotypes resulted in plants having greater N con-

tents, biomass, and N concentrations than those nodulatedby Hup- phenotypes when compared within the LM colonytype but not within the SD colony type (Table 3). Availablecomparisons within serogroups produced similar results(data not shown). Of those isolates producing high shoot Ncontents (>75 mg ofN plant-'), five were Hup- (77.5 to 86.1mg of N plant-') and four were Hup+ (75.8 to 83.8 mg of Nplant-') (data not shown).As in the verification study, all isolates rated RT+ in the

effectiveness study produced LW colonies, although rhizo-bitoxine symptoms occurred at a lower frequency (Table 2).Comparisons within the LW colony type between RT+ andRT- isolates revealed that the former yielded lower N con-tents and biomass but produced greater shoot N concentra-tions (Table 3). Isolates producing LW colonies fixed signifi-cantly less N than the other isolates, even when RT+ isolateswere excluded from consideration (results not shown).

Classification schemes based on serology or morphologyalone were essentially identical in their abilities to charac-terize the symbiotic effectiveness of the indigenous brady-rhizobia (Table 4). Combining these criteria produced asignificant improvement in predictive ability relative to thesingle-factor models. Classifications based on hydrogenase orrhizobitoxine phenotypes alone were clearly inferior to theother models, although the latter accounted for more varia-tion in shoot N contents than did the former. Incorporation ofhydrogenase or rhizobitoxine phenotypes into the combinedserology-morphology classification scheme did not apprecia-bly increase the precision of the model (results not shown).Averaged across all phenotypes and sampling locations,

soybean bradyrhizobia indigenous to Delaware producedshoot N contents that were approximately 83 and 70% ofthose produced by USDA 110 and USDA 122, respectively(Table 2). Comparisons among the sampling locations, basedon extrapolated values calculated from the observed fre-quencies and symbiotic performances of the combined sero-logical-morphological groupings, revealed that isolates fromthe sampling location possessing the least effective indige-nous population would be expected to yield an overall shootN content 68% of that for the most effective indigenouspopulation sampled (results not shown). In comparison withinoculant quality strains, the indigenous populations wouldbe predicted to produce relative shoot N contents rangingfrom 65 to 96% and 55 to 81% of those observed for USDA110 and USDA 122, respectively, depending on samplinglocation.

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FIG. 1. Photographs of colonies of soybean bradyrhizobia illustrating the morphological differences between (A) LM and SD colonies and(B) LW and SD colonies. The bacteria were grown on yeast extract-mannitol agar at 28°C for 7 to 10 days. Illumination is by combinedincident and transmitted light. The marker bar represents 1 mm in both photographs.

DISCUSSION

Previous research with recognized strains of soybeanbradyrhizobia has shown that bacteria in the genusBradyrhizobium compose at least two genetically and phe-notypically distinct subgroups (1, 7, 14, 15, 20, 27). Theindigenous soybean bradyrhizobia included in the presentstudy could also be separated into two divergent groups.

One group, identified by the LW colony type, appeared tocorrelate with DNA homology group II of Hollis et al. (14)based on both serological evidence and the presence of RT+isolates (7). Isolates in the second group (LM and SD colonytypes) aligned with DNA homology groups I and Ia (14),although the serological analyses suggested that these col-ony types and homology subgroups did not correlate indi-

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TABLE 2. Mean nitrogen contents of Forrest soybean plantsinoculated with USDA strains and indigenous soybean

bradyrhizobia from the serological and morphological groupings(symbiotic effectiveness study) and corresponding

numbers of Hup+ and RT+ phenotypes

Mean shoot NNo. of content (mg of N

Srgop Colony isltsNo. of No. of plant-') for":Serogroup CtleYisolates Hu+ R+type studied Hup~ RT+a1Indigenous USDAisolatesc straind

38-115 LMe 4 0 0/0 76.7 a 69.8122 LMe 3 3 0/0 70.8 ab 83.4*Nlf LM 21 4 0/0 69.8 bc110 LMe 3 2 0/0 68.7 bc 70.4122 All 7 4 0/0 (67.6)NI All 30 4 0/2 (65.9)122 SD 4 1 0/0 65.1 bcd 83.4*110 All 5 2 0/0 (64.6)NI SD 6 0 0/0 64.0 cde6 LMe 9 0 0/0 61.8 de 67.7122/1239 SD 4 4 0/0 60.7 def123 LMe 2 0 0/0 58.7 def 61.4110 SD 2 0 0/0 58.5 ef 70.4*31 LWe 5 0 0/1 55.0 f 52.646 LWe 3 0 0/1 48.2 g 43.3130 LWe 2 0 1/2 44.6 gh 42.8NI LW 3 0 0/2 42.7 gh94 LWe 16 0 6/7 40.0 h 17.6*76 LWe 5 0 3/4 39.9 h 25.2*All LM 42 9 0/0 (68.2)All SD 16 5 0/0 (62.8)All LW 34 0 10/17 (43.4)All All 92 14 10/17 (58.1)

a The first and second numbers indicate the number of RT+ isolatesobserved for the symbiotic effectiveness study and the verification study,respectively.

b Mean N content of noninoculated controls was 4.5 mg of N plant-'.Combined serological-morphological groupings followed by the same

letter are not significantly different at P 50.05. Values for single factorgroupings are given in parentheses.

d Means followed by asterisks are significantly different from those forcorresponding indigenous isolates at P c 0.05.

e Same colony type as corresponding USDA antigen strain.f NI, Not identified.g Isolates cross-reacting with 122 and 123 antisera.

vidually. The present study complements previous researchon genetic divergence in B. japonicum by extending theseobservations to a large collection of indigenous soybeanbradyrhizobia.The results of this study support the contention that

soybean bradyrhizobia in DNA homology group II (LW

TABLE 3. Effect of Hup and RT phenotype of indigenoussoybean bradyrhizobia on mean N content, biomass,

and N concentration of soybean shoots

Colony Phenotype No. of N content Biomass N concntype Hup RT isolates (mg of N plant-') (g plant-') (%)

LM + - 9 71.7a 1.95a 3.82a- - 33 67.3 1.76 3.67

SD + - 5 61.7 1.64 3.78- - 11 63.2 1.66 3.80

LW - + 10 30.8b 0.75b 4.15b- - 24 48.7 1.21 4.01

a Means for Hup+ and Hup- phenotypes significantly different (P s 0.05)." Means for RT+ and RT- phenotypes significantly different (P s 0.05).

TABLE 4. Summary of statistical models relating nitrogencontent of soybean shoots to groupings of indigenous soybean

bradyrhizobia according to their respective serological,morphological, combined serological-morphological,

hydrogenase, and rhizobitoxine phenotypes

Basis of Coefficient of Coefficient of SDbgrouping determination (r2Y' variation (%)

Serology 0.5913 17.7 10.3Morphology 0.5974 17.4 10.1Serology-morphology 0.6847 15.7 9.1Hydrogenase 0.1478 25.2 14.7Rhizobitoxine 0.4402 20.5 11.9

a All models were significant at P c 0.0001.b SD, Standard deviation.

colony type) are symbiotically defective with at least somesoybean genotypes (6, 7). This conclusion is based on theobservation that rhizobitoxine production was limited to LWisolates and that the most effective LW isolate fixed onlymoderate amounts of N2 when compared with isolates fromthe remaining colony types. Particularly significant is theobservation that LW isolates fixed less N2 than the otherisolates even when RT+ isolates were excluded from analy-sis, suggesting the presence of a more fundamental incom-patibility between the host and group II bradyrhizobia thanthat attributable to possible production of foliar chlorosis.Alternatively, it is conceivable that some of the isolatesrated RT- may have interfered with photosynthesis (andthereby N2 fixation) via production of rhizobitoxine at levelsinsufficient to cause foliar chlorosis. The significant increasein tissue N concentration produced by RT+ relative to RT-isolates probably reflects a reduced capacity for biomassproduction due to foliar chlorosis and stunting of mer-istematic tissues rather than any superiority in symbioticeffectiveness on the part of the RT+ isolates. It should benoted that soybean genotypes differ in rhizobitoxine suscep-tibility (9, 25), and it is not known whether studies withresistant genotypes would yield results similar to thosereported here for Forrest soybeans which exhibit moderatesensitivity to rhizobitoxine when compared with other cul-tivars (J. Fuhrmann, unpublished results).No evidence for the presence of Hup+ isolates in DNA

homology group 11 (14) is reported here, yet Keyser et al.(18) reported significant frequencies of Hup+ bradyrhizobiain serogroups 31, 46, 76, and 94 whose serotype strains alignwith this homology group (7). Similarly, this study did notconfirm the presence of Hup+ isolates in serogroups 6 or 123(18). Reasons for these discrepancies are unclear but mayinclude sampling variations, differences in the proceduresused for detection of hydrogenase activity, or differences inthe protocols used for the accompanying serological charac-terizations. The overall frequency of Hup+ phenotypes notedin the present study (15%) is somewhat lower than thatreported previously for soybean bradyrhizobia in Delaware(18). Although comparisons between Hup+ and Hup- isolatesin the present study are confounded by other genetic dissim-ilarities among the bacteria, the results indicate that hydrog-enase phenotype is not a useful predictor of the symbioticeffectiveness of indigenous soybean bradyrhizobia.

It is noteworthy that colony morphology was found to beas useful a predictor of symbiotic effectiveness as wasserological characterization, the latter being a much morecommon, expensive, and laborious procedure than the for-mer. However, these two classification schemes appearsomewhat complementary, as evidenced by the greater

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predictive power of the combined model relative to eithersingle-factor model. Morphological characterization has theadvantage of not being dependent on the availability ofantisera appropriate to the indigenous population underexamination. Additional serological studies to differentiatethe isolates within the nonreactive serological grouping mayproduce classification schemes having greater predictivevalue than those presented here.The current study confirms a previous serological charac-

terization of soybean bradyrhizobia in Delaware by notingboth the relatively high frequency of indigenous isolates inserogroups 94 and 6 and the large effect of sampling locationon serogroup frequency (lOa). Results presented here alsocomplement this previous study by documenting the pres-ence and frequency of RT+ and Hup+ phenotypes within theserological and morphological groupings and by determiningthe symbiotic effectiveness of the corresponding isolates. Ofparticular relevance to future studies are the very highincidences at some sampling sites of apparently symbioti-cally inferior isolates, i.e., those aligning with group II ofHollis et al. (14) as recognized by the LW colony morphol-ogy or RT+ phenotype. The prominence of such indigenouspopulations clearly justifies a critical examination of theirpotential impact on soybean growth and yield.

ACKNOWLEDGMENTS

This research was supported by a grant from the DelawareSoybean Board, Dover.

Antisera and cultures of USDA strains were kindly provided byHarold H. Keyser (USDA, Beltsville, Md.). Appreciation is ex-tended to P. van Berkum (USDA, Beltsville, Md.) for helpfuldiscussions regarding the hydrogenase assay.

LITERATURE CITED1. Agarwal, A. K., and D. L. Keister. 1983. Physiology of ex planta

nitrogenase activity in Rhizobium japonicum. Appl. Environ.Microbiol. 45:1592-1601.

2. Albrecht, S. L., R. J. Maier, F. J. Hanus, S. A. Russell, D. W.Emerich, and H. J. Evans. 1979. Hydrogenase in Rhizobiumjaponicum increases nitrogen fixation by nodulated soybeans.Science 203:1255-1257.

3. Caldwell, B. E., and E. E. Hartwig. 1970. Serological distribu-tion of soybean root nodule bacteria in soils of southeasternUSA. Agron. J. 62:621-622.

4. Cole, M. A., and G. H. Elkan. 1973. Transmissible resistance topenicillin G, neomycin, and chloramphenicol in Rhizobiumjaponicum. Antimicrob. Agents Chemother. 4:248-253.

5. Damirgi, S. M., L. R. Frederick, and I. C. Anderson. 1967.Serogroups of Rhizobium japonicum in soybean nodules asaffected by soil types. Agron. J. 59:10-12.

6. Devine, T. E., L. D. Kuykendall, and B. H. Breithaupt. 1983.Nodule-like structures induced on peanut by chlorosis produc-ing strains of Rhizobium classified as R. japonicum. Crop Sci.23:394-397.

7. Devine, T. E., L. D. Kuykendall, and J. J. O'Neill. 1988. DNAhomology group and the identity of bradyrhizobial strainsproducing rhizobitoxine-induced foliar chlorosis on soybean.Crop Sci. 28:938-941.

8. Drevon, J. J., V. C. Kalia, M. 0. Heckmann, and L. Salsac.1987. Influence of the Bradyrhizobium japonicum hydrogenaseon the growth of Glycine and Vigna species. Appl. Environ.Microbiol. 53:610-612.

9. Erdman, L. W., H. W. Johnson, and F. Clark. 1957. Varietalresponses of soybeans to a bacterial-induced chlorosis. Agron.J. 49:267-271.

10. Fehr, W. R., C. E. Caviness, D. T. Burmood, and J. S.Pennington. 1971. Stage of development descriptions for soy-beans, Glycine max (L.) Merrill. Crop Sci. 11:929-931.

10a.Fuhrmann, J. 1989. Serological distribution of Bradyrhizobiumjaponicum as influenced by soybean cultivar and samplinglocation. Soil Biol. Biochem. 21:1079-1081.

11. Fuhrmann, J., and A. G. Wollum II. 1985. Simplified enzyme-linked immunosorbent assay for routine identification of Rhizo-bium japonicum antigens. Appl. Environ. Microbiol. 49:1010-1013.

12. Ham, G. E., L. R. Frederick, and I. C. Anderson. 1971.Serogroups of Rhizobium japonicum in soybean nodules sam-pled in Iowa. Agron. J. 63:69-72.

13. Hanus, F. J., S. L. Albrecht, R. M. Zablotowicz, D. W. Emerich,S. A. Russell, and H. J. Evans. 1981. Yield and N content ofsoybean seed as influenced by Rhizobium japonicum inoculantspossessing the hydrogenase characteristic. Agron. J. 73:368-372.

14. Hollis, A. B., W. E. Kloos, and G. H. Elkan. 1981. DNA:DNAhybridization studies of Rhizobium japonicum and relatedRhizobiaceae. J. Gen. Microbiol. 123:215-222.

15. Huber, T. A., A. K. Agarwal, and D. K. Keister. 1984. Extra-cellular polysaccharide composition, ex planta nitrogenase ac-tivity, and DNA homology in Rhizobium japonicum. J. Bacte-riol. 158:1168-1171.

16. Kamicker, B. J., and W. J. Brill. 1986. Identification ofBradyrhizobiumjaponicum nodule isolates from Wisconsin soy-bean farms. Appl. Environ. Microbiol. 51:487-492.

17. Keyser, H. H., and R. F. Griffin. 1987. Beltsville Rhizobiumculture collection catalog. Agricultural Research Service publi-cation no. 60. U.S. Department of Agriculture, Washington, D.C.

18. Keyser, H. H., D. F. Weber, and S. L. Uratsu. 1984. Rhizobiumjaponicum serogroup and hydrogenase phenotype distributionin 12 states. Appl. Environ. Microbiol. 47:613-615.

19. Kowalski, M., G. E. Ham, L. R. Frederick, and I. E. Anderson.1974. Relationship between strains of Rhizobiumjaponicum andtheir bacteriophages from soil and nodules of field-grown soy-beans. Soil Sci. 118:221-228.

20. Kuykendall, L. D., M. A. Roy, J. J. O'Neill, and T. E. Devine.1988. Fatty acids, antibiotic resistance, and deoxyribonucleicacid homology groups of Bradyrhizobium japonicum. Int. J.Syst. Bacteriol. 38:358-361.

21. La Favre, J. S., and A. R. J. Eaglesham. 1986. Rhizobitoxine: aphytotoxin of unknown function which is commonly producedby bradyrhizobia. Plant Soil 92:443-452.

22. McClure, P. R., and D. W. Israel. 1979. Transport of nitrogen inthe xylem of soybean plants. Plant Physiol. 64:411-416.

23. Mueller, J. G., H. D. Skipper, E. R. Shipe, L. W. Grimes, andS. C. Wagner. 1988. Intrinsic antibiotic resistance in Brady-rhizobium japonicum. Soil Biol. Biochem. 20:879-882.

24. Noel, K. D., and W. J. Brill. 1980. Diversity and dynamics ofindigenous Rhizobium japonicum populations. Appl. Environ.Microbiol. 40:931-938.

25. Owens, L. D., and D. A. Wright. 1965. Rhizobial-inducedchlorosis in soybeans: isolation, production in nodules, andvarietal specificity of the toxin. Plant Physiol. 40:927-930.

26. Schmidt, E. L., M. J. Zidwick, and H. M. Abebe. 1986.Bradyrhizobiumjaponicum serocluster 123 and diversity amongmember isolates. Appl. Environ. Microbiol. 51:1212-1215.

27. Stanley, J., G. G. Brown, and D. P. S. Verma. 1985. Slow-growing Rhizobium japonicum comprises two highly divergentsymbiotic types. J. Bacteriol. 163:148-154.

28. Uratsu, S. L., H. H. Keyser, D. F. Weber, and S. T. Lim. 1982.Hydrogen uptake (HUP) activity of Rhizobiumjaponicum frommajor U.S. soybean production areas. Crop Sci. 22:600-602.

29. van Berkum, P. 1987. Expression of uptake hydrogenase andhydrogen oxidation during heterotrophic growth of Bradyrhizo-bium japonicum. J. Bacteriol. 169:4565-4569.

30. Weaver, R. W., and L. R. Frederick. 1982. Rhizobium, p.1043-1070. In A. L. Page, R. H. Miller, and D. R. Keeney(ed.), Methods of soil analysis. Part 2. Chemical and microbio-logical properties, 2nd ed. American Society of Agronomy andSoil Science Society of America, Madison, Wis.

31. Zablotowicz, R. M., S. A. Russell, and H. J. Evans. 1980. Effectof the hydrogenase system in Rhizobium japonicum on thenitrogen fixation and growth of soybeans at different stages ofdevelopment. Agron. J. 72:555-559.

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