Transposon mutagenesis of Rhizobium japonicum

Summary We report here successful mutagenesis with Transposon Tn5 of three slow-growing strains of Rhizobium japonicum USDA 122, 61A76, USDA 74 and one fast-growing strain, USDA 191. Strains were chosen as representatives of different DNA homology and serogroups of this divergent species, which effectively nodulate North American soybean cultivars. The source of Tn5 was the suicide plasmid pGS9, which possesses broad host range N-type transfer genes in a narrow host range p15A replicon. The selection of Tn5 mutants was facilitated by the expression of the Tn5 encoded streptomycin gene in R. japonicum. Kanamycin and streptomycin resistant colonies appeared from interspecific crosses with E. coli at optimal frequencies of 10^-6 for R. japonicum USDA 61A76 and USDA 191 and 5x10^-7 for R. japonicum USDA 122 and USDA 74. Altogether, 6550 Tn5 mutants were isolated in USDA 122 and 61A76, and a small number from USDA 74 and USDA 191. Colony hybridization showed that all tested mutants of 61A76 and USDA 122 contained Tn5. Physical analysis of total DNAs from representative numbers of USDA 122, 61A76 and USDA 191 mutants revealed that each of them carried one copy of the transposon integrated randomly in the genome. This was also true for most USDA 74 mutants. Screening of mutants for auxotrophy showed frequencies of 0.2% for USDA 122 and 0.08% for 61A76. Several symbiotically defective mutants were identified on plants, Glycine soja and G. max.     

Regulation of nodulation in the soybean-Rhizobium symbiosis : strain and cultivar variability

Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod⁺ on McCall] and USDA257 [Nod⁻ on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall × 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions.     

Conservation of a Symbiotic DNA Region in Soybean Root Nodule Bacteria

Bradyrhizobium japonicum USDA 3I1b110 contains a DNA region in which symbiotic genes and many repeated sequences are closely linked. Hybridization analysis revealed that this region was highly conserved in some B. japonicum strains (USDA 24, USDA 122, USDA 123, ATCC 10324, 61A24) but not in others (USDA 76, 61A76, 61A101). The genomic presence of multiple copies of one of the repeated sequences (RSα) appeared to be specifically characteristic for soybean root nodule bacteria, including the fast-growing Rhizobium fredii, which carries most of these RSα copies on the symbiotic plasmid.     

Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

Symbiotic Potential, Competitiveness, and Serological Properties of Bradyrhizobium japonicum Indigenous to Korean Soils

The symbiotic potential of Bradyrhizobium japonicum isolates indigenous to seven Korean soils was evaluated by inoculating soybeans with 10- and 1,000-fold-diluted soil suspensions (whole-soil inocula). At both levels, significant differences in the symbiotic potential of the indigenous B. japonicum isolates were demonstrated. The relationship between rhizobial numbers in the whole-soil inocula (x) and nitrogen fixation parameters (y) was best predicted by a straight line (y = a + bx) when the numbers in the inocula were 100 to 10,000 ml^-1, while the power curve (y = ax^b) predicted the variation when the numbers were 1 to 100 ml^-1. Thirty isolates from three soils showed wide differences in effectiveness (measured as milligrams of shoot N per plant), and several were of equal or greater effectiveness than reference strain B. japonicum USDA 110 on soybean cultivars Clark and Jangbaekkong. On both of the soybean cultivars grown in a Hawaiian mollisol, the Korean B. japonicum isolate YCK 213 and USDA 110 were of equal effectiveness; USDA 110 was the superior strain in colonization (nodule occupancy). Korean isolates YCK 117 and YCK 141 were superior colonizers compared with USDA 110. However, B. japonicum USDA 123 was the superior colonizer compared with isolates YCK 213, YCK 141, and YCK 117. In an immunoblot analysis of 97 indigenous Korean isolates of B. japonicum, 41% fell into the USDA 110 and USDA 123 serogroups. Serogroups USDA 110 and USDA 123 were represented in six of the seven soils examined. In one Korean soil, 100% of the B. japonicum isolates reacted only with antisera of YCK 117, an isolate from the same soil.     

Symbiotic mutants of USDA191, a fast-growing Rhizobium that nodulates soybeans

Summary Symbiotic and auxotrophic mutants of Rhizobium japonicum strain USDA191 were isolated using Tn5 mutagenesis and techniques that cause plasmid deletions and plasmid curing. Characterization of several mutants that are unable to nodulate (Nod^-) or unable to fix nitrogen (Fix^_) showed that nod and nif genes are located within one regions of a 200 MD plasmid (pSym191). Blot hybridization analysis of plasmids in other fast-growing R. japonicum strains showed that nod as well as nif sequences are located on plasmids in eight strains but are apparently carried in the chromosome in two strains.     

Nodulation and Competition for Nodulation of Selected Soybean Genotypes among Bradyrhizobium japonicum Serogroup 123 Isolates

Twenty recently obtained field isolates of Bradyrhizobium japonicum serogroup 123 were tested for their nodule mass production on the standard commercial soybean (Glycine max (L.) Merr. cv. Williams) and on two soybean plant introduction (PI) genotypes previously determined to restrict nodulation by strain USDA 123. Four of the field isolates showed similar restricted nodulation on the two genotypes, while all 20 isolates produced a normal amount of nodules on G. max cv. Williams. Serological analyses with adsorbed fluorescent antibodies showed that members of the 123 serotype ranked low in nodulation of the two PIs, in contrast to members of serotypes 127 and 129. Competition studies on the PIs indicated that isolates which were restricted were not competitive for nodule occupancy against strain USDA 110. However, unrestricted isolates of serogroup 123 were very competitive against USDA 110. On G. max cv. Williams, all serogroup 123 isolates tested were very competitive against USDA 110.     

Comparison of the host range of fast-growingR. japonicum strains with a fast-growing isolate from Lablab

Summary Fast-growingRhizobium japnicum strains derived from the People's Republic of China were compared with a fast-growingRhizobium isolate from Lablab for their ability to nodulate tropical legumes grown in Leonard-jars and test tube culture. Fast-growingR. japonicum strains were all effective to varying degrees in their symbiosis withVigna unguiculata. Two strains USDA 192 and USDA 201, effectively nodulatedGlycine whightii and one strain, USDA 193, effectively nodulatedMacroptilium atropurpureum. Other nodulation responses in tropical legumes were ineffective. The fast-growing isolate from Lablab was more promiscuous, effectively nodulating with a larger host range. The fast-growing Lablab strain was considered more akin, on a symbiotic basis, to the slow-growing cowpea type rhizobia than the fast-growing China strains ofR. japonicum whilst maintaining physiological characteristics of other fast-growing rhizobia.     

Competition of Rhizobium japonicum Strains in Early Stages of Soybean Nodulation

The effects of preexposure of soybean (Glycine max L. Merrill) roots to Rhizobium japonicum strains and subsequent establishment of other strains in the nodules were investigated by using combinations of effective strains (USDA 110 and USDA 138) and effective-ineffective strains (USDA 110 and SM-5). Strain USDA 110 was a better competitor than either USDA 138 or SM-5 on cultivars Lee and Peking. However, when either of the two less-competitive strains was inoculated into 2-day-old seedlings before USDA 110 was, their nodule occupancy increased significantly on both cultivars. With USDA 138 as the primary inoculum and USDA 110 delayed for 6, 48, and 168 h, the incidence of USDA 138 nodules increased on cultivar Peking from 6% (at zero time) to 28, 70, and 82% and on cultivar Lee from 17% (at zero time) to 32, 88, and 95% for the three time delays, respectively. Preexposure of 2-week-old roots of cultivar Lee to USDA 138 had essentially the same effect: the incidence of USDA 138 nodules increased from 23% at zero time to 89 and 97% when USDA 110 was delayed for 24 and 72 h, respectively. When the ineffective strain SM-5 was used as the primary inoculum, followed by USDA 110 72 h later, the percentage of nodules containing SM-5 increased from 7 to 76%. These results indicate that the early events in the nodulation process of soybeans are perhaps the most critical for competition among R. japonicum strains.     

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N₂ than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N₂ than did the USDA 110 during the 20-day period. The superior N₂ fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N₂-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N₂-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N₂ in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N₂-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N₂ fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N₂-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.     

Stability of Bradyrhizobium japonicum Inoculants after Introduction into Soil

Bradyrhizobium japonicum USDA 125-Sp, USDA 138, and USDA 138-Sm had been used as inoculants for soybean (Glycine max (L.) Merr.) in soils previously free of B. japonicum. At 8 to 13 years after their release, these strains were reisolated from soil samples. A total of 115 isolates were obtained through nodules, and seven colonies were obtained directly by a serological method. The stability of the inoculants was confirmed by comparing the reisolated cultures with their respective parental strains which had been preserved by being lyophilized or stored on a yeast extract-mannitol agar slant at 4°C. Comparisons were made on morphological and serological characters, carbon compound utilization (8 tested), intrinsic antibiotic resistance (9 tested), and enzymatic activity (19 tested). Mucous and nonmucous isolates of serogroup 125 were analyzed for symbiotic effectiveness and restriction fragment hybridization with a DNA probe. Our data suggest that the B. japonicum inoculants have survived for up to 13 years in the soils without significant mutation except for two reisolates with a slightly increased kanamycin resistance level.     

Characterization ofR. fredii QB1130, a strain effective on commercial soybean cultivars

Summary Two rhizobial strains (QB1130 and C3A) from northeast China were identified asRhizobium fredii on the basis of growth rate, media acidification and growth on a wide range of carbon substrates. The strains were shown to be distinct from USDA 191 on the basis of plasmid number and size. Bothnif and commonnod genes were located on the 295 kb plasmid of strains QB1130 and USDA 191, while onlynif genes were identified on this plasmid in C3A. When used to inoculate four commercial soybean (Glycine max) cultivars, one of the strains (C3A) was found to be ineffective, while the other (QB1130) was at least as effective as USDA 191, a strain ofR. fredii reported to be widely effective on North American cultivars of soybean. Further, QB1130 was capable of more effective nodulation of cowpea or the uncultivated soybean line, Peking, than either USDA 191 or the slow-growingBradyrhizobium japonicum USDA 16. Strain QB1130 should be useful for studies directed at improving symbiotic performance in soybean, or for studies of the comparative physiology and genetics of FG and SG strains on a single host.     

Microscopic characterization of early nodulation events in a non-nodulating soybean mutant (NN5) and lack of response to high inoculum dose

The microscopic events leading to nodulation in normally nodulatingsoybean [Glycine max (L.) Merr.] genotypes, and the effectsof Bradyrhizobium strain and inoculum dose on nodulation, wereexamined in the NN5 non-nodulating mutant derived from cv. Williams.The NN5 mutant possesses the recessive genes rj₅ and ,rj₆. BradyrhizoblumJaponicum strain USDA 110 cells attached normally to the rootsurface of NN5, many in a polar manner as in its wild-type parent,but failed to induce root hair curling and sub-epidermal celldivision in the root. Co-culturing NN5 and Williams did notmodify nodulation of either genotype. Hydroponically-grown NN5seedlings did not nodulate at a high inoculum dose (1 x 10¹⁰cells seedling^–1) of any B. japonicum strain tested (USDA110, USDA 26, USDA 136, and the tryptophan metabolic variantsB-14075 and ta 11 Nod⁺). A higher inoculum dose of 3 x 10 USDA136 cells seedling also failed to induce nodulation in NN5 andnod139 (a non-nodulating mutant of cv. Bragg). The lack of nodulationof NN5 at any inoculum dose is contrary to previous observationsof sparse nodulation of other non-nodulating mutants at highinoculum dose. Genetic control of non-nodulation in NN5 is probablysimilar to nodl39. Key words: Nodulation events, non-nodulating mutant, soybean     

Sinorhizobium fredii USDA257, a cultivar-specific soybean symbiont, carries two copies of y4yA and y4yB, two open reading frames that are located in a region that encodes the type III protein secretion system

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on primitive soybean (Glycine max) cultivar Peking but fails to nodulate the improved cultivar McCall. Cultivar specificity is governed by a plasmid-borne locus, nolXBTUV. By DNA sequence analysis, we have identified two open reading frames, y4yA and y4yB, immediately downstream of nolX. Northern (RNA) blot analysis indicated that the expression of both y4yA and y4yB is inducible by isoflavonoids, and an intact copy of nolX is required. Two copies each of y4yA and y4yB are present in S. fredii USDA257, one on the sym plasmid (y4yAsp and y4yBsp), and the other on the chromosome (y4yAc and y4yBc). The cultivar-nonspecific strain USDA191 lacks y4yAc and y4yBc. Introduction of y4yAc plus y4yBc from USDA257 into USDA191 did not influence the ability of the latter strain to nodulate McCall soybean plants. Unlike nolX, the inactivation of y4yAsp and y4yBsp of USDA257 did not extend the host range of this strain. A double mutant, in which both the plasmid and chromosomal copies of y4yA and y4yB were mutated, had no observable effect on symbiotic ability of USDA257. The y4yAsp and y4yBsp mutants did not influence flavonoid-dependent extracellular protein production. Rhizobium sp. strain NGR234 and S. saheli USDA4893 both contain sequences similar to S. fredii USDA257 y4yAsp and y4yBsp; however, Bradyrhizobium spp., the traditional soybean symbionts, lack these genes.     

A Comparative Study of the Physiology of Symbioses Formed by Rhizobium japonicum with Glycine max, Vigna unguiculata, and Macroptilium atropurpurem

Although Rhizobium japonicum nodulates Vigna unguiculata and Macroptilium atropurpurem, little is known about the physiology of these symbioses. In this study, strains of R. japonicum of varying effectiveness on soybean were examined. The nonhomologous hosts were nodulated by all the strains tested, but effectiveness was not related to that of the homologous host. On siratro, compared to soybean, many strains reversed their relative effectiveness ranking. Both siratro and cowpea produced more dry matter with standard cowpea rhizobia CB756 and 176A22 than with the strains of R. japonicum. Strains USDA33 and USDA74 were more effective with siratro and cowpea than with soybean. The strain USDA122 expressed high rates of hydrogenase activity in symbiosis with the cowpea as well as the soybean host. The strains USDA61 and USDA74 expressed low levels of hydrogenase activity in symbiosis with cowpea, but no activity was found with soybean. Our results indicate host influence for the expression of hydrogenase activity, and suggest the possibility of host influence of nitrogenase for the allocation of electrons to N₂ and H⁺.     

The Type III Secretion System of Bradyrhizobium japonicum USDA122 Mediates Symbiotic Incompatibility with Rj2 Soybean Plants

The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners.     

Case of Localized Recombination in 23S rRNA Genes from Divergent Bradyrhizobium Lineages Associated with Neotropical Legumes

Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii.     

Nodulation, Nitrogen Fixation, and Hydrogen Oxidation by Pigeon Pea Bradyrhizobium spp. in Symbiotic Association with Pigeon Pea, Cowpea, and Soybean

The pigeon pea strains of Bradyrhizobium CC-1, CC-8, UASGR(S), and F4 were evaluated for nodulation, effectiveness for N₂ fixation, and H₂ oxidation with homologous and nonhomologous host plants. Strain CC-1 nodulated Macroptilium atropurpureum, Vigna unguiculata, Glycine max, and G. soja but did not nodulate Pisum sativum, Phaseolus vulgaris, Trigonella foenum-graecum, and Trifolium repens. Strain F4 nodulated G. max cv. Peking and PI 434937 (Malayan), but the symbioses formed were poor. Similarly, G. max cv. Peking, cv. Bragg, PI 434937, PR 13-28-2-8-7, and HM-1 were nodulated by strain CC-1, and symbioses were also poor. G. max cv. Williams and cv. Clark were not nodulated. H₂ uptake activity was expressed with pigeon pea and cowpea, but not with soybean. G. max cv. Bragg grown in Bangalore, India, in local soil not previously exposed to Bradyrhizobium japonicum formed nodules with indigenous Bradyrhizobium spp. Six randomly chosen isolates, each originating from a different nodule, formed effective symbioses with pigeon pea host ICPL-407, nodulated PR 13-28-2-8-7 soybean forming moderately effective symbioses, and did not nodulate Williams soybean. These results indicate the six isolates to be pigeon pea strains although they originated from soybean nodules. Host-determined nodulation of soybean by pigeon pea Bradyrhizobium spp. may depend upon the ancestral backgrounds of the cultivars. The poor symbioses formed by the pigeon pea strains with soybean indicate that this crop should be inoculated with B. japonicum for its cultivation in soils containing only pigeon pea Bradyrhizobium spp.     

Long-Term Effects of Metal-Rich Sewage Sludge Application on Soil Populations of Bradyrhizobium japonicum

The application of sewage sludge to land may increase the concentration of heavy metals in soil. Of considerable concern is the effect of heavy metals on soil microorganisms, especially those involved in the biocycling of elements important to soil productivity. Bradyrhizobium japonicum is a soil bacterium involved in symbiotic nitrogen fixation with Glycine max, the common soybean. To examine the effect of metal-rich sludge application on B. japonicum, the MICs for Pb, Cu, Al, Fe, Ni, Zn, Cd, and Hg were determined in minimal media by using laboratory reference strains representing 11 common serogroups of B. japonicum. Marked differences were found among the B. japonicum strains for sensitivity to Cu, Cd, Zn, and Ni. Strain USDA 123 was most sensitive to these metals, whereas strain USDA 122 was most resistant. In field studies, a silt loam soil amended 11 years ago with 0, 56, or 112 Mg of digested sludge per ha was examined for total numbers of B. japonicum by using the most probable number method. Nodule isolates from soybean nodules grown on this soil were serologically typed, and their metal sensitivity was determined. The number of soybean rhizobia in the sludge-amended soils was found to increase with increasing rates of sludge. Soybean rhizobia strains from 11 serogroups were identified in the soils; however, no differences in serogroup distribution or proportion of resistant strains were found between the soils. Thus, the application of heavy metal-containing sewage sludge did not have a long-term detrimental effect on soil rhizobial numbers, nor did it result in a shift in nodule serogroup distribution.     

Strain-Specific Inhibition of nod Gene Induction in Bradyrhizobium japonicum by Flavonoid Compounds

A broad-host-range plasmid, pEA2-21, containing a Bradyrhizobium japonicum nodABC'-'lacZ translational fusion was used to identify strain-specific inhibitors of the genes required for soybean nodulation, the common nod genes. The responses of type strains of B. japonicum serogroups USDA 110, USDA 123, USDA 127, USDA 129, USDA 122, and USDA 138 to nod gene inhibitors were compared. Few compounds inhibited nod gene expression in B. japonicum USDA 110. In contrast, nod gene expression in strains belonging to several other serogroups was inhibited by most of the flavonoids tested. However, the application of two of these strain-specific compounds, chrysin and naringenin, had little effect on the pattern of competition between indigenous and inoculum strains of B. japonicum in greenhouse and field trials. Preliminary studies with radiolabeled chrysin and naringenin suggest that the different responses to nod gene inhibitors may be partly due to the degree to which plant flavonoids can be metabolized by each strain.