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.     

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.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Populations ofRhizobium japonicum associated with the surfaces of soil-grown roots

Summary Populations of nativeRhizobium japonicum 123 in the rhizospheres of field and pot grown plants as determined by immunofluorescence were calculated on the basis of root surface area. The density ofR. japonicum 123 on the root fluctuated between a few hundred to over a thousand per square centimeter of root surface. As root volume expanded rapidly, the Rhizobium density fell to less than one hundred per unit area. There was no appreciable effect due to different plant, nitrogen amendment, or addition of another strain ofR. japonicum, on the surface density of the nativeR. japonicum population on roots. Nor did the native population influence the added strain. Direct examination of root surface segments revealed that naturalized rhizobia existed sparsely on root surfaces in the form of short rods. They were observed to be attached sideways or in a polar manner on root hairs, epidermal cells, and at junctions of tap and lateral roots. There was no evidence of specific stimulation of the homologous Rhizobium by the host plant as a prelude to nodulation.     

The role of formate metabolism in nitrogen fixation in Rhizobium Spp.

Formate metabolism supported nitrogen-fixation activity in free-living cultures of Rhizobium japonicum. However, formate0dependent nitrogense activity was observed only in the presence of carbon sources such as glutamate, ribose or aspartate which by themselves were unable to support nitrogenase activity. Formate-dependent nitrogenase activity was not detected in the presence of carbon sources such as malate, gluconate or glycerol which by themselves supported nitrogenase activity. A mutant strain of R. japonicum was isolated that was unable to utilise formate and was shown to lack formate dehydrogenase activity. This mutant strain exhibited no formate-dependent nitrogenase activity. Both the wild-type and mutant strains nodulated soybean plants effectively and there were no significant differences in the plant dry weight or total nitrogen content of the respective plants. Furthermore pea bacteroids lacked formate dehydrogenase activity and exogenously added formate had no stimulatory effect on the endogenous oxygen uptake rate. The role of formate metabolism in symbiotic nitrogen fixation is discussed.Abbreviation FDH formate dehydrogenase     

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.     

Genetic stability in rhizobia in the field

Genetic instability within strains of rhizobia maintained on laboratory media is well recognized, although rarely has the mutation been characterized. Variability within a strain introduced into the field is very difficult to recognise due to poor understanding of naturally-occurring populations of rhizobia. We have examined populations of Rhizobium leguminosarum bv. trifolii from both laboratory cultures and field populations and found significant variation in symbiotic effectiveness within both. In Australia, the only significant introduction of Bradyrhizobium japonicum has been strain CB1809 (=USDA136b). Symbiotic tests on field reisolates obtained by plant entrapment indicate little or no change in symbiotic effectiveness up to nine years after introduction. The RFLP pattern, using the RS probe (Hahn and Hennecke, 1987a) was unchanged but marked differences in serological characters were observed.     

Root nodulation studies inAeschynomene aspera

Summary Fifteen isolates of nodule bacteria were isolated from root and stem nodules ofAeschynomene aspera and they were characterized as Rhizobium by well known laboratory tests. All these isolates together with other efficient strains of known rhizobia belonging to different cross-inoculation groups were evaluated for their nodulation abilities onAeschynomene aspera, Cajanus cajan (pigeon pea),Cicer arietinum (chickpea),Pisum sativum (pea),Trifolium repens (clover),Medicago sativa (lucerne),Lens culinaris (lentil),Glycine max (soybean),Vigna sinensis (cowpea),Vigna radiata (mung bean),Vigna mungo (urd bean) andArachis hypogea (peanut). The results demonstrated that Rhizobium fromAeschynomene could form nodules only on its homologous host (Aeschynomene) but not on other legumes tested. Secondly, none of the rhizobia of other cross-inoculation groups could nodulateA. aspera.     

Detection of genetic variation in Bradyrhizobium japonicum USDA 110 variants using DNA fingerprints generated with GC rich arbitrary PCR primers

Bradyrhizobium japonicum USDA 110 has been shown to contain several genetically similar naturally occurring colony morphology variants. These variants differ in symbiotic nitrogen fixation ability and in the utilization of various carbon substrates. They have been shown to share extensive DNA homology and appear to be derived from a common ancestor. Despite these similarities certain B. japonicum USDA 110 variants have been shown to be devoid of symbiotic nitrogen fixation. One of these variants (L2-110), however, was recently shown to possess significant levels of explanta nitrogen fixation and to synthesize functional dinitrogenase enzyme within bacteroids. In an effort to identify genetic markers which could explain differences in symbiotic nitrogen fixation between B. japonicum variants, DNA fingerprints were generated by PCR using arbitrary primers. Two of these primers with GC rich sequences were able to differentiate between B. japonicum USDA 110 variants I-110, L2-110, and MN-110. Unique markers have now been identified which could be examined further to determine if they explain the differences in symbiotic nitrogen fixation between USDA 110 variants.     

Foliar chlorosis in legumes induced by cowpea rhizobia

Summary While screening cowpea rhizobia from West Africa for ability to nodulate various host species, foliar chlorosis was observed in young mung bean and soybean plants inoculated with certain strains. The chlorosis occurred in the first and sometimes the second trifoliate, but not on subsequent leaves. There was no correlation of symptoms with the presence of nodules. Where extreme chlorosis was induced in soybeans, there was stunting of the primary root. Disease symptoms were obtained with culture-broth supernatants free of rhizobia, indicating an extracellular toxin. In common with rhizobitoxine-producing strains ofR. japonicum, chlorosis-inducing cowpea strains were able to nodulate ‘non-nodulating’ soybeans of the rj₁rj₁ genotype.     

Survival and multiplication ofRhizobium japonicum strains in silt loam

Summary Antibiotic resistant mutants 8-0 Str^R, 110 Tet^R and 138 Kan^R derived from wild typeRhizobium japonicum strains were inoculated into silt loam soil to cell concentrations greater than 2×10⁸/g of soil. Population changes were monitored using antibiotic media and strain identification was done using immunodiffusion assay on microcores of soil. Immunodiffusion bands formed by the mutant strains with homologous antisera essentially duplicated bands formed by the parent strain. Strains 110 Tet^R and 8-0 Str^R had cross reacting antigens whereas antigens of strain 138 Kan^R reacted only with the homologous antiserum. Populations ofR. japonicum strains introduced into sterile soil increased over a period of four weeks under both single and mixed culture inoculations. All populations decreased by the end of six weeks and thereafter remained constant. When theseR. japonicum strains were introduced into non-sterile soil, the population did not increase over the initial population added. Population decreased gradually for two weeks and then maintained thereafter. It was possible to recover very low populations of antibiotic resistantR. japonicum strains from both sterile and unsterile soils using media containing specific antibiotics. Detection ofR. japonicum strains by immunodiffusion was accomplished only when the population was 10⁹ cells/g of soil. The method using antibiotic resistant mutants permitted an evaluation of the interactions of variousR. japonicum strains in soil with respect to their survival and multiplication.     

Degradation of aromatic compounds byRhizobium spp.

Summary The ability of rhizobia to utilize catechol, protocatechuic acid, salicylic acid, p-hydroxybenzoic acid and catechin was investigated. The degradation pathway of p-hydroxybenzoate byRhizobium japonicum, R. phaseoli, R. leguminosarum, R. trifolii andRhizobium sp. isolated from bean was also studied.R. leguminosarum, R. phaseoli andR. trifolii metabolized p-hydroxybenzoate to protocatechuate which was cleaved by protocatechuate 3,4-dioxygenasevia ortho pathway.R. japonicum degraded p-hydroxybenzoate to catechol which was cleaved by catechol 1,2-dioxygenase.Rhizobium sp., a bean isolate, dissimilatedp-hydroxybenzoate to salicylate. Salicylate was converted to gentisic acid prior to ring cleavage. The rhizobia convertedp-hydroxybenzoate to Rothera positive substance. Catechol and protocatechuic acid were directly cleaved by the species.R. japonicum converted catechin to protocatechuic acid.     

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⁺.     

Symbiotic efficiency of Hup+ and Hup− strains ofRhizobium japonicum

Summary Relative efficiency of Hup⁺ and Hup^– R. japonicum strains with Pusa 16 cultivar of soybean was studied. Inoculation with the Hup⁺ strain (A 1014) reduced the protein content in grain as compared to uninoculated control.     

Regulation of the β-ketoadipate pathway in Rhizobium japonicum and bacteroids by succinate

Rhizobium japonicum 61-A-101 and its bacteroids catabolize phenol and p-hydroxybenzoate. With phenol as a carbon source, utilization started only after a prolonged lag phase while p-hydroxybenzoate was almost instantancously metabolized. Succinate, which supports rapid growth of Rhizobium japonicum, completely repressed respication of phenol; the oxidation of p-hydroxybenzoate was partially inhibited. Pyruvate, supporting slower growth than succinate, retarded the onset of phenol consumption but did not affect its maximum rate.Catabolite repression of phenol utilization by succinate appears to be a characteristic feature of rhizobia. In Pseudomonas putida which also actively metabolizes phenol, succinate had no effect on phenol utilization.     

Agglutination ofRhizobium japonicum 3I1b110 by soybean lectin

Summary Conditions leading to agglutination ofRhizobium japonicum 3I1b110 with soybean seed lectin were examined. Ability of cells to be agglutinated was transient and was optimal for cultures grown for 4–5 days on yeast extract mannitol plates. Similar lectin-binding results were obtained with cells from the same cultures using fluorescence microscopy with fluorescein isothiocyanate-labelled lectin. These results revise the previous model for soybean lectin-R. japonicum interactions, since it was based on the inability of soybean lectin to agglutinate these bacteria.     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Lack of specific binding of labeledRhizobium japonicum lipopolysaccharides to wild soybean (Glycine soja) roots

Summary Lipopolysaccharides (LPS) were extracted from two strains ofRhizobium japonicum (61A76NS and 3I1b110-I). The extracted LPS was purified by gel filtration column chromatography and the amount of 2-keto-3-deoxyoctonate (KDO) was determined. Column purified LPS from both strains were conjugated to rhodamine isothiocyanate on celite to examine binding of this purified, labeled surface component to aseptically grownGlycine soja (wild soybean) seedlings as a basis for symbiotic specificity using fluorescent microscopy. Rhodamine conjugated LPS from both strains ofRhizobium japonicum did not exhibit specific binding to wild soybean seedling roots.Paper no. 8130 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

The Influence of Lipopolysaccharides and Glucans from Two Rhizobium leguminosarum bv. viciae Strains on the Formation and Efficiency of Their Symbioses with Pea Plants

The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation tests. Two Rhizobium leguminosarum bv. viciae strains which differed in their symbiotic properties were used: strain 31 (fix⁺, efficient, moderately virulent, and moderately competitive) and strain 248b (fix^–, inefficient, highly virulent, and highly competitive). Preparations of LPS–glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10–26%. Analogous preparations from a less virulent strain 31 did not have this ability. Unseparated LPS–glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18–23% but did not change it in plants inoculated with the other, inefficient strain. No significant influence of LPS preparations on the symbiosis productivity was observed. Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36–56%. In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31. In general, the formation and efficiency of the symbiosis of R. leguminosarum bv. viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation–proficient but less virulent strain 31.