Characterization of Phages Virulent for Sarothamnus scoparius Bradyrhizobia

Four virulent phages—ΦDl, ΦTl, ΦCYT21, and ΦOS6, infective on Sarothamnus scoparius rhizobia—were isolated from the soil and characterized for morphology, host range, rate of adsorption to bacterial cells, and genome size. New phages were separated into two morphological families: Siphoviridae with long, noncontractile tails (ΦDl, ΦTl) and Myoviridae with long, contractile tails (ΦCYT21, ΦOS6). They were also classified into two groups by a host specificity. One of them included viruses (ΦDl and ΦTl) that lysed S. scoparius bradyrhizobia and Bradyrhizobium sp. (Lupinus) strain Dl, and the second one comprised phages (ΦCYT21 and ΦOS6) that parasitized only Scotch broom native microsymbionts. Phages specific for S. scoparius rhizobia were differentiated not only by morphology and host range but also by a genome size that was in the range from 47,583 to 60,098 b.p. An erratum to this article is available at .     

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia

Pairwise comparisons of Genista tinctoria (dyer’s weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer’s weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.The GenBank accession numbers for the sequences reported in this paper are as follows: nodC, DQ139776–DQ139781; nodA, DQ135897, Q135898; nodZ, DQ135899–DQ135903.     

Numerical Taxonomy of Sarothamnus scoparius Rhizobia

Twenty nodule isolates from Sarothamnus scoparius (broom) growing in Poland and nine strains from plants growing in Japan were studied for phenotypic properties, plasmid presence, phage sensitivity, and host plant specificity. By numerical analysis of phenotypic properties, it was found that the studied nodule bacteria, originating from geographically different countries, constitute two separate groups affiliated to the bradyrhizobium cluster. The membership of S. scoparius rhizobia in the Bradyrhizobium genus was also supported by their long generation time, alkaline reaction in YEM medium with mannitol, lack of plasmids, and wide host plant range. Received: 12 June 2000 / Accepted: 17 July 2000     

Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure

Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules. The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris. The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes. By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A. cicer microsymbionts to Rhizobium sp. (Oxytropis) was found.     

Nitrogen-fixing potential of micropropagated clones of Acacia mangium inoculated with different Bradyrhizobium spp. strains

Five A. mangium seedlings of different shoot lengths were selected from a 600-seed screening experiment and micropropagated. Two-week-old rooted microcuttings of the 5 micropropagated clones were inoculated with 3 specific Bradyrhizobium spp. strains in 15 combinations. After 5 months of growth, nodule dry weight and shoot dry weight data showed significant effects of clone and Bradyrhizobium spp. strain. Clones RR-G₁ and IR-M₂ and Bradyrhizobium sp. Aust13c resulted in the highest dry-matter production and most efficient nodulation. No interaction was observed between clone and Bradyrhizobium spp. strain, which indicates that the Bradyrhizobium spp. strain and the host plant can be selected separately.     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

The Effect of Variability in the Phenology of the Reproductive Stages of Scotch Broom (Cytisus scoparius) on the Synchronization of the Life Stages of Broom Seed Beetle (Bruchidius villosus) in New Zealand

Variability in timing of the reproductive stages of Scotch broom (Cytisus scoparius) may influence synchronization and establishment of the broom seed beetle (Bruchidius villosus), a biological control agent. A sampling scheme was devised to compare the phenologies of Scotch broom at different sites in the same season and in different seasons at the same site. The synchrony of the broom seed beetle's life stages with those of the host plant was also determined. The phenology of Scotch broom varied only slightly from season to season at Lincoln, but could vary considerably between sites in the same season. At both sites where it is established, the broom seed beetle was synchronized with its host; adult beetles were present throughout the flowering period. Eggs were found on suitable green pods. The broom seed beetle appears capable of adapting to the phenology of its host and has the potential to be an effective agent for Scotch broom. Variability in phenology of the reproductive stages of Scotch broom, even at nearby sites, must be taken into account by practitioners of biological control when releasing broom seed beetles and later when sampling beetles to determine establishment.     

Biology of theParasponia-Bradyrhizobium symbiosis

Parasponia remains the only non-legume known to nodulate withRhizobium/Bradyrhizobium. It is a pioneer plant that is capable of rapid growth and fixing large quantities of nitrogen. In addition to its high agronomic potential, the symbiosis offers the scientist the unique opportunity of studying differences at the molecular level of both partners, and to investigate any possible extension of the symbiosis to other non-legumes of importance. Haemoglobin has been found in the nodule tissue ofParasponia and other nodulated non-legumes and the gene for it has been found and expressed in non-nodulating plants such asTrema tomentosa andCeltis australis. Bradyrhizobium strains isolated from species ofParasponia growing in Papua New Guinea form a group that are more specific in their host requirements thanBradyrhizobium strains from tropical legumes from the same area. They do not effectively nodulate (except CP283) tropical legumes, andParasponia is not readily nodulated withRhizobium andBradyrhizobium strains from legumes. The effectiveness of the symbiosis is influenced by host species, theBradyrhizobium strain and the environment.Parasponia andersonii forms a more effective symbiosis than the other species tested. In competition studies with strains from legumes, isolates fromParasponia always dominate in nodules onParasponia.     

Phenotypic,genomic and phylogenetic characteristics of rhizobia isolated from root nodules of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98–99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771–961 kb megaplasmids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

Genetic diversity and distribution of Bradyrhizobium and Azorhizobium strains associated with the herb legume Zornia glochidiata sampled from across Senegal

Herb legumes have great potential for rehabilitation of semi-arid degraded soils in Sahelian ecosystems as they establish mutualistic symbiosis with N₂-fixing rhizobia. A phylogenetic analysis was performed for 78 root nodule bacteria associated with the common Sahelian herb legume Zornia glochidiata Reichb ex DC in Senegal. Based on ITS (rDNA16S-23S) and recA sequences, these strains were shown to belong to the two genera Bradyrhizobium and Azorhizobium. Strains of this latter, although frequent, formed small and ineffective nodules and suggested a parasitism rather than a symbiotic association. A potential negative effect of Azorhizobium on Zornia growth was tested for when inoculated alone or in association with a Bradyrhizobium strain. Bradyrhizobium isolates were distributed in four groups. Groups A and B were two sister clades in a larger monophyletic group also including Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, and Bradyrhizobium japonicum. Strains of cluster D fell in a sister clade of the photosynthetic Bradyrhizobium sp. group, including ORS278, whereas group C appeared to be divergent from all known Bradyrhizobium clusters. Amplified fragment length polymorphism (AFLP) clustering was congruent with ITS and recA phylogenies, but displayed much more variability. However, within the main Bradyrhizobium clades, no obvious relationship could be detected between clustering and geographical origin of the strains. Each sub-cluster included strains sampled from different locations. Conversely, Azorhizobium strains showed a tendency in the phylogeny to group together according to the site of sampling. The predominance of ineffective Azorhizobium strains in the nodules of Zornia roots, the large Bradyrhizobium genetic diversity and the geographical genetic diversity pattern are explored.     

花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp.MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B.diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P<0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P<0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P<0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。     

Phage susceptibility and plasmid profile analysis of Sinorhizobium fredii

This is the first report identifying bacteriophages and documenting megaplasmids of Sinorhizobium fredii. Plasmid DNA content and bacteriophage typing of eighteen strains of S. fredii were determined. S. fredii strains fell into ten plasmid profile groups containing 1 to 6 plasmids, some evidently larger than 1000 MDa. Twenty-three S. fredii lytic phages were isolated from soil, and they lysed six different S. fredii strains. The host range and plaque morphology of these phages were studied. Susceptibility to S. fredii phages was examined for S. meliloti; Rhizobium leguminosarum bvs. viceae, trifolii and Phaseoli; R. loti; Bradyrhizobium japonicum; B. elkanii and Bradyrhizobium sp. (Arachis). Several phages that originally lysed S. fredii strain USDA 206 also lysed strains of all three S. fredii serogroups described originally by Sadowsky et al. Phages that infected S. fredii strains USDA 191 and USDA 257 were highly specific and lysed only serogroup 193 strains. S. meliloti strains L5-30 and USDA 1005 were lysed by three of the phages that lysed S. fredii strain USDA 217. No other Rhizobium or Bradyrhizobium strain tested was susceptible to lysis by any of the S. fredii phages. The present investigation indicates that phage susceptibility in conjunction with plasmid profile analysis may provide a rapid method for identification and characterization of strains of S. fredii.     

Transference of a crystal protein gene from Bacillus thuringiensis and its expression in Bradyrhizobium sp. cells

The plasmid pHT409 that harbours the cryIA(a) gene for the production of a -endotoxin (crystal protein) from Bacillus thuringiensis was transferred into Bradyrhizobium sp. A conjugal transfer system aiming to introduce the plasmid into the Bradyrhizobium sp. host from colonies of an Escherichia coli donor strain (DH5::pHT409) has been developed. As a result exconjugants were obtained in which the transferred plasmid has been detected by both microbiological and electrophoresis techniques. The cryIA(a) gene when inside the new host had a low expression level which was detected by immunoblotting.     

The interplay between host toxins and parasitism by Amoebophrya

The parasitic dinoflagellate Amoebophrya sp. ex Karlodinium veneficum was used to test two hypotheses: (1) infection of cells decreases with increasing host toxicity and (2) parasitism causes the catabolism of host toxin. To test the first hypothesis, host strains differing in toxin content were inoculated with dinospores of Amoebophrya sp. derived from infected cultures of toxic and non-toxic K. veneficum, with resulting infections assessed following 24-h incubations. Contrary to expectations, infection of K. veneficum by Amoebophrya sp. was positively correlated with host toxicity. To examine the second hypothesis, synchronous infection with >80% of cells being parasitized was induced using a toxic strain of K. veneficum, and total toxin concentration (intracellular plus extracellular levels of KmTX1) was followed over the 3-day infection cycle. Toxin content ml⁻¹ increased with growth of K. veneficum in uninfected control cultures, but declined in infected cultures as the parasite completed its life cycle. On a cellular basis, toxin content of infected and uninfected cultures differed little during the experiment, suggesting that the parasite does not actively catabolise host toxin. Rather, infection appears to promote degradation of toxins via death of host cells and subsequent bacterial activity. Results indicate that Amoebophrya sp. ex K. veneficum has greater potential to impact toxic strains relative to non-toxic host strains in natural systems. Thus, Amoebophrya sp. ex. K. veneficum may limit the occurrence of toxic K. veneficum blooms in marine and estuarine environments, while simultaneously functioning as a pathway for dissipation of host toxin.     

Siderophore production byBradyrhizobium spp. strains nodulating groundnut

EighteenBradyrhizobium spp. strains, fourRhizobium spp. strains and oneAzorhizobium caulinodans strain were grown under Fe limitation and assayed for siderophore production. It was further assessed if Fe accumulation in two groundnut cultivars was influenced by inoculant strain or nitrate fertilisation. Growth ofBradyrhizobium spp. strains nodulating groundnut was slow with mean generation times from 11–24 h. All strains, except MAR 967, showed a reduced growth rate when deprived of Fe; none of the strains showed starvation at 1 M Fe. In the CAS (chrome azurol S)-agar assay, all strains, which formed colonies, produced siderophores as visualised by orange halos around the colonies on blue plates.Bradyrhizobium strains produced much smaller halos than the referenceRhizobium meliloti strain. In the CAS-supernatant assay, all strains, except MAR 967, gave positive responses (measured as absorbance at 630 nm) when supernatants of Fe-depleted cultures were assayed with CAS-indicator complex in comparison with Fe-supplemented cultures. Responses of all fourRhizobium spp. strains were large, while responses of allBradyrhizobium strains, exceptB. japonicum MAR 1491 (USDA 110), were small and mostly insignificant. A small response, i.e. a low Fe-scavenging ability, implies either the production of small quantities of siderophores or the production of low affinity siderophores. Among theBradyrhizobium strains, MAR 1574 and MAR 1587 gave the largest responses taken over the two assays. Fe accumulation in groundnut cultivar Falcon was seven times larger than in cultivar Natal Common. No correlation was found between the quantity of nodule tissue and Fe accumulation, making it unlikely that bacteroids are involved in Fe acquisition by groundnuts. Nitrate-fertilised plants accumulated significantly more Fe, suggesting involvement of nitrate reductase in Fe assimilation in groundnut. The two most successful Fe-scavengingBradyrhizobium spp. strains were also the most effective in nodulating groundnut, the reverse also being true. Strain MAR 967, with the lowest Fe requirement, produced the largest nodule dry weight. These data indicate that improved Fe scavenging properties and/or reduced Fe requirement improve rhizospheric growth and with that nodulation effectiveness.     

Cadmium Accumulation and Tolerance in <Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp. (Peanut Microsymbionts)

In this study, the effect of cadmium (Cd) on cell viability and its accumulation in Bradyrhizobium spp. (peanut microsymbionts) as well as the role of glutathione (GSH) in the tolerance to this metal were investigated. A reference strain recommended as peanut inoculant (Bradyrhizobium sp. SEMIA6144) grew up to 10 μM Cd meanwhile a GSH-deficient mutant strain (Bradyrhizobium sp. SEMIA6144-S7Z) was unable to grow at this concentration. Two native peanut isolates obtained from Córdoba soils (Bradyrhizobium sp. NLH25 and Bradyrhizobium sp. NOD31) tolerated up to 30 μM Cd. The analysis of Cd content showed that Bradyrhizobium sp. SEMIA6144 accumulated a high amount of this metal, but a considerable inhibition of growth was observed compared to tolerant strains at 10 μM Cd. At this concentration, the intracellular GSH content of all the Bradyrhizobium sp. strains was not modified in comparison to control conditions. However, at 30 μM Cd, the intracellular GSH content significantly increased in Bradyrhizobium sp. strains NLH25 and NOD31. Thus, the distinct response of each Bradyrhizobium sp. strain to Cd reveals that, even in closely related lineages, there are strain-specific variations influencing the levels of tolerance to this metal. Indeed, the native peanut isolates tolerated higher Cd concentration than the reference strain, possibly due to an increase in GSH levels which could act as a detoxifying agent.     

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N₂-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O₂ flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N₂-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.     

Localization of Bacteria and Hemoglobin in Root Nodules of Parasponia andersonii Containing Both Bradyrhizobium Strains and Rhizobium leguminosarum biovar trifolii

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

Effects of Bradyrhizobium strain and host genotype,nodule dry weight and leaf area on groundnut (Arachis hypogaea L. ssp. fastigiata) yield

Effects of inoculating four Arachis hypogaea ssp. fastigiata cultivars with 17 Bradyrhizobium spp. strains were studied in a glasshouse experiment using a sandy soil devoid of an indigenous Bradyrhizobium population. Firstly, a wide range of parameters, indicative of symbiotic performance, were assessed for their influence on seed yield, by correlation and statistical analyses. It was found that nodule dry weight and leaf area were relevant parameters concerning seed yield. Secondly, the effects of host and strain genotype on those parameters were described.Variations in nodule dry weight did not have an effect on seed yield, except for cultivar Natal Common at lower nodule dry weight values. Therefore, it was concluded that the quantity of nitrogen fixing tissue met the demand for combined nitrogen and did not limit seed yield. This conclusion was further supported by the observation that at low nodule numbers per plant the nodule size increased to generate sufficient nitrogen fixing tissue.Leaf area, which comprises components for both photosynthetic capacity and plant development, was found to correlate well with seed yield. An increase in leaf area resulted in significant seed yield increases for all three spanish-type cultivars, but not for the valencia-type cultivar. Leaf area, thus, appeared as a factor limiting seed yield of spanish-type groundnuts.Cultivar performance concerning seed yield was significantly better for Natal Common compared to the other three cultivars, while Natal Common had a significantly lower plant (biomass excluding seed) dry weight value.Inoculation with different strains of Bradyrhizobium resulted in significantly different nodule dry weight values, but hardly led to significant differences in seed yield. This agreed with the finding that the amount of nitrogen fixing tissue appeared not to limit the availability of combined nitrogen.A large quantity of nitrogen was partitioned to the groundnut seeds: 62% to 76% of total accumulated nitrogen was located in the seeds.This study showed that testing for symbiotic effectiveness in the groundnut Bradyrhizobium symbiosis should include assessment of final (seed and biomass) yield, because parameters measured at stages prior to maturity, like nodulation parameters, may lead to flawed effectiveness ratings.