Nodulation of Lotus pedunculatus in acid rooting solution by fast- and slow-growing rhizobia

The nodulation of Lotus pedunculatus and the multiplication of three Rhizobium loti (fast-growing, acid-producing) and two Bradyrhizobium (slow-growing, alkali-producing) strains was investigated in acidified rooting solution. R. loti strains multiplied at pH 4.5 but Bradyrhizobium strains failed to multiply. No difference in growth rate between R. loti and Bradyrhizobium strains was apparent in rooting solution at pH 6.7. Similar responses to pH were observed in yeast extract-mannitol broth except that Bradyrhizobium strains multiplied more slowly than R. loti at pH 6.7. All strains nodulated L. pedunculatus growing in acid (pH 4.5) rooting solution when presented as single cultures. Following inoculation with 1:1 mixtures of R. loti and Bradyrhizobium strains, R. loti formed 93% of nodules at pH 4.5 and significantly fewer nodules (66%) at pH 6.7. These results demonstrate a competitive advantage for acid-tolerant strains over acid-sensitive strains in nodulation of their lost legume at pH 4.5.     

Characterization of Rhizobium trifolii isolated from soils of different pH

Rhizobium trifolii were isolated from soils along a transect covering a range of soil pH (3.6–5.6) using two varieties of white clover by either growing seedlings directly in soil or in nutrient solution in tubes inoculated with soil. Rhizobia were present at pH 4.5 but absent at pH 3.9. Neither nodule number nor effectiveness were influenced by the method of isolation and the clover variety on which the strain was isolated. There was no relationship between the pH of the soils and either the number of nodules or the effectiveness of the isolates from those soils. Screening the isolates for tolerance of acidity and Al showed that multiplication was unaffected at pH 5.0 but was slowed for all strains at pH 4.5. Multiplication at pH 5.5 was unaffected by 10 μM Al but was inhibited by 50 μM Al. At pH 4.5 all but 16% of the isolates were inhibited by 10 μM Al; none multiplied with 50 μM Al. The strains which multiplied at pH 4.5 with and without Al were isolated equally from soils in the range pH 4.5–5.6. They were also isolated in almost equal proportions from the two varieties of clover and by the two isolation methods. Overall there was little variation in the effectiveness and acid- and Al-tolerance of isolates from these soils of different pH.     

Rhizobial and bradyrhizobial symbionts of mesquite from the Sonoran Desert: salt tolerance, facultative halophily and nitrate respiration

Rhizobial symbionts were isolated from the surface (0-0.5 M) and phreatic (3.9-5.0 M) root environments of a mature mesquite woodland in the Sonoran Desert of Southern California, and from variable depths (0-12 m) of non-phreatic mesquite ecosystems in the Chihuahuan Desert of New Mexico. They were tested for their ability to tolerate high salinity, and respire NO₃⁻ as mechanisms of free-living survival. Sixteen of 25 isolates were grown in yeast-extract mannitol (YEM) broth at NaCl concentrations of 2 (basal concentration), 100, 300, 500 and 600 mM, and their specific growth rates, cell dry weight and lag times were determined. Twenty of the 25 isolates were also grown in YEM broth under anaerobic conditions with or without 10 mM KNO₃. Three categories of NaCl salinity responses were observed: (1) eight isolates showed decreased specific growth rates at NaCl concentrations of 100, 300 and 500 mM, but they nevertheless remained viable at 500 mM NaCl concentration; (2) the specific growth rate of six isolates increased significantly at 100 and 300 mM NaCl; and (3) specific growth rates of two isolates were significantly greater than the base-rate at all concentrations of NaCl. Five of 11 of the Bradyrhizobium isolates tested respired NO₃⁻, but showed no growth. Seven Rhizobium isolates, three from the deep (3.9-5 m) phreatic rhizobial community, and four from the surface community denitrified NO₃⁻ but only the isolates from the phreatic community displayed anaerobic growth. Long-term interactions between rhizobial and bradyrhizobial communities and the surface and phreatic root environments of the mature Sonoran Desert mesquite woodland appear to have selected for strains of NO₃⁻ respiring rhizobia, general salt tolerance of both rhizobial and bradyrhizobial symbionts, and strains of weak facultative halophilic bradyrhizobia. These survival characteristics of mesquite rhizobia may be important regarding mesquite's establishment and long-term productivity in marginal desert soils, and may provide novel types of rhizobia for food crops growing in harsh environments.     

Diversity of rhizobia isolated from an agricultural soil in Argentina based on carbon utilization and effects of herbicides on growth

Seventy-six rhizobial isolates belonging to four different genera were obtained from the root nodules of several legumes (Vicia sativa, Vicia faba, Medicago sativa, Melilotus sp., Glycine max and Lotus corniculatus). The action of five commonly used herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate (GF), dicamba, atrazine and metsulfuron-methyl] on the growth of rhizobial strains was assessed. Subsequently, GF and 2,4-D were tested in a minimum broth as C and energy sources for 20 tolerant strains. The ability of these strains to metabolize different carbon sources was studied in order to detect further differences among them. Tolerance of the bacteria to agrochemicals varied; 2,4-D and GF in solid medium inhibited and diminished growth, respectively, in slow-growing rhizobial strains. Among slow-growing strains we detected Bradyrhizobium sp. SJ140 that grew well in broth + GF as the sole C and energy source. No strain was found which could use 2,4-D as sole C source. The 20 strains studied exhibited different patterns of C sources utilization. Cluster analysis revealed three groups, corresponding to four genera of rhizobia: Rhizobium (group I), Sinorhizobium (group II) and Mesorhizobium–Bradyrhizobium (group III). On the basis of the results obtained on responses to herbicides and C sources utilization by the isolates investigated, it was possible to differentiate them at the level of strains. These results evidenced a considerable diversity in rhizobial populations that had not been previously described for Argentinean soils, and suggested a physiological potential to use natural and xenobiotic C sources.     

Inhibition of growth of Bradyrhizobium japonicum bacteroid by spermidine and spermine in yeast extract

Abstract

Bacteroids are defined as the symbiotic forms of Rhizobium or Bradyrhizobium cells in the root nodules of their legume host. The differentiation to bacteroids involves various physiological changes and may be associated with some genetical changes. Single-colony isolates from a nodule formed by a Bradyrhizobium strain often differed in their effectiveness and intrinsic antibiotic resistance (Weaver and Wright 1987; Ozawa unpublished data). Detailed analysis of the changes in the gene structure requires the isolation of individual bacteroids from a nodule. However it has been reported that the viability of bacteroids in culture is very low and only a small fraction of a bacteroid population could produce colonies on yeast extract-mannitol (YEM) agar (Sutton et al. 1977). Bergersen (1974) concluded that the reversion of bacteroids to the vegetative, growing form is very rare.     

Inhibitory effects of acidic minesoil on the sericea lespedeza/Bradyrhizobium symbiotic relationship 1

Effects of acidic minesoil on sericea lespedeza [Lespedeza juncea (L.F.) var. sericea (Mig.)] and its nitrogen (N₂)‐fixing symbiotic relationship with Bradyrhizobium spp. were examined. Sericea lespedeza was grown in pots with N fertilization, without N fertilization, or with commercial Bradyrhizobium as a seed inoculant. Minesoil (pH 5.2) was fertilized with calcium (Ca), phosphorus (P), molybdenum (Mo), and potassium (K), and the pH level was adjusted to 4.8 or 4.5 with aluminum or iron sulfate [Al₂(SO₄)₃; Fe₂(SO₄)₃]. Minesoil was also limed to pH 6.1. Shoot dry weights, shoot N concentrations, nodule dry weights, and nodule numbers were significantly lower (P < 0.05) when inoculated plants were grown in soil at pH 4.5 and 4.8 compared to limed soil. Thus, the N₂ fixation process was adversely affected below pH 5.0. Nitrogen‐fertilized plants grew well in acidified soil, and there were no significant differences in shoot dry weights of such plants among the soil acidification treatments including limed soil. Thus, the N₂‐fixing symbiosis appeared to be more sensitive to acidified soil than the plant host. The effects of Al toxicity versus other factors could not be determined because Al₂(SO₄)₃‐ and Fe₂(SO₄)₃‐amended soils contained similar levels of toxic Al at the highest pH (4.8) that prevented N₂ fixation.

Time periods required for cells of Bradyrhizobium strains to multiply by a factor of 10⁴ were significantly longer (P ≤ 0.05) in extracts of Al₂(SO₄)₃‐amended soil (pH 4.8 and 4.5) than in extracts of calcium carbonate [CaCO₃]‐amended soil (pH 6.1). These increases suggested that reduced multiplication of Bradyrhizobium in acidified minesoils may have been at least partially responsible for the large decreases in nodulation and N₂ fixation observed in these soils. It was also reasoned that the inability of existing bacteria to infect and nodulate plant roots may also have been a factor, based on the high inoculation rates used and the abilities of Bradyrhizobium cells to survive and multiply (albeit at a reduced rate) in extracts of acidified soil. Sericea lespedeza is known to tolerate soils of pH 4.5. However, results of this study suggested sericea lespedeza may not fix appreciable N₂ in acidic soil below pH 5 when inoculated with commercial Bradyrhizobium, even after the establishment of lespedeza plants tolerant of such conditions.     

Laboratory prescreening of Bradyrhizobium japonicum for low pH,Al and Mn tolerance can be used to predict their survival in acid soils

We examined whether strains of Bradyrhizobium japonicum selected for growth on acid media in vitro would also survive and grow better in acid soils. Four agar screening media for acid-tolerant rhizobia, which differed in the number of acid soil stresses imposed (pH, low calcium (Ca) and phosphorus (P), high aluminum (Al) and manganese (Mn)), were assessed for their effects on the survival of 14 Indonesian strains and two commercial strains of B. japonicum. Survival of B. japonicum in the agar media was compared with that in two acid soils. A repeat stab inoculation method which provided a declining range of inoculum cell number to 10³ cells per stab was used to assess the daily growth of the strains on the screening media at 5 pH levels (3.8, 4.2, 4.5, 5.0, and 6.8). The growth and survival of the 16 strains were then measured at days 1, 8, 18, and 28 after inoculation in two acid soils (pH 4.24 and 4.35) sterilized using γ-irradiation at 5.0 Mrad. Selectivity of the agar media improved as more acid stress factors were incorporated in the media. Those strains of Bradyrhizobium identified as acid, Al and Mn-tolerant in acidic agar media, also had better survival in the low pH soils. There was no relationship between acid or alkali production on agar media and acid tolerance on agar or in soil. There was no apparent relationship between symbiotic performance and acid tolerance, and one acid-tolerant strain was as effective as the commercial inoculant strain CB1809. The most acid-tolerant strain was also the most ineffective.     

Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan

Abstract

Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S?23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj ₂ Rj ₃) and Fukuyutaka (Rj ₄), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.     

Nodulation and nitrogen fixation of Lupinus species with Bradyrhizobium (lupin) strains in iron-deficient soil

 The effect of six Bradyrhizobium sp. (lupin) strains (WPBS 3201D, WPBS 3211D, USDA 3040, USDA 3041, USDA 3042 and CB 2272) and Fe supply on nodulation, N₂-fixation and growth of three lupin species (Lupinus termis, L. albus and L. triticale) grown under Fe deficiency in an alkaline soil, were examined in sterilized and non-sterilized pot experiments. When inoculated with USDA 3040, 3041, 3042 and CB2272 without Fe addition, the three lupin species had a very low nodule number and mass, low shoot and root dry matter accumulation and lower N yield. However, inoculation with WPBS 3201D and 3211D without Fe treatments increased all these parameters substantially. The ability of WPBS 3201D and 3211D to form nodules on the three lupin species under conditions of Fe stress could be attributed to their ability to scavenge Fe from Fe-deficient environments through their siderophore production. Addition of Fe to the other four strains significantly increased nodulation and N₂-fixation of the three lupin species, indicating that the poorer nodulation and N₂-fixation of these strains in the absence of Fe, resulted from a low ability to obtain Fe from alkaline soils. Bradyrhizobium strains WPBS 3201D and 3211D were superior to the other four strains in terms of promoting greater nodulation, N₂-fixation, plant growth and N accumulation of L. termis and L. albus. However, the other four strains were more efficient in symbiotic association with L. triticale. The greater variations in nodule efficiencies (specific nitrogenase activity) under different levels of Fe supply could be attributed to the quantities of bacteroid protein and leghaemoglobin in the nodules. The results suggested that Bradyrhizobium (lupin) strains differ greatly in their ability to obtain Fe from alkaline soils, and that the selection of bradyrhizobial strains which are tolerant of Fe deficient soils could complement plant breeding for the selection of legume crops for Fe-deficient soils. Received: 5 January 1998     

Bradyrhizobium sp. (Lupinus) in the winter rainfall region of South Africa

Widespread cultivation of lupin has resulted in the establishment of effective populations of Bradyrhizobium sp. (Lupinus) in the winter rainfall region of the Western Cape, South Africa. To determine whether inoculation increased yields of Lupinus angustifolius L., field trials were carried out at five sites in this region. Populations ranged from 380 rhizobia g^-1 in a moderately alkaline (pH 7.6) soil to >5,000 rhizobia g^-1 in four moderately acid (pH 5.5-5.8) soils. Soil isolates were generally similar to the inoculant strain WU425 in nitrogen-fixing effectiveness but several were significantly less effective. Average effectiveness of isolates from certain soils differed significantly. Although inoculation failed to appreciably increase nodule occupancy by WU425 in acid soils containing high populations of rhizobia, nodule occupancy was increased to 98% in the low population alkaline soil. The latter site was later abandoned because of disease. At the other sites, analysis of seed dry mass and protein content showed that yields were not significantly increased by either inoculation, nitrogen fertiliser (45 kg N ha^-1) or molybdenum applications. Analysis of genomic DNA by PCR fingerprinting showed that WU425 (isolated in Western Australia) and serologically related strains from other cultures clustered separately from the soil isolates. Isolates from the four acid soils were genomically diverse, whereas isolates from the alkaline soil formed a homogeneous cluster. Further investigation is required to determine the benefit of inoculation in alkaline soils of the winter rainfall region of the Western Cape.     

The Effect of Soil pH on Nodulation and N2-Fixation of Winged Bean Psophocarpus tetragonolobus (L) D.C. Rhizobium Strains

Nine effective Rhizobium strains of winged bean were tested for their symbiotic performance under different soil pH levels. In general, they performed best at soil pH 5.5. The performance of all the strains was poor when soil pH was either extremely low as 4.5 or high as 7.5. The strains RRIM 56, KUL-JN, KUL-Z₃ were suitable for soils with a pH range of 5.5 to 6.0. The strains KUL-Z₁, KUL-Z₂ and KUL-GP could be only used in soils with a pH of 5.5. The strains KUL-BH, KUL-6.2Z and KUL-6.9Z could be used for inoculation of soils with high pH. Isolating strains of Rhizobium by altering the original pH of the soil of Rhizobium isolation would be a good practice to obtain strains with desirable pH tolerance.     

Identification and Characterization of High Acid Tolerant and Aluminum Resistant Yeasts Isolated from Tea Soils

From acidic tea soils of Kagoshima Prefecture in Japan, some soil properties were determined and 38 strains of acid tolerant microorganisms were isolated. Different Al³⁺ concentrations were applied to YG media to estimate Al resistance. Selected microbial strains could grow strongly in the liquid media in the presence of 100 mM Al³⁺ and survive even in 300 mM Al³⁺ at pH 3.0. Their base sequences of 28S rDNA-D1/D2 were determined and sequence data were searched using the Basic Local Alignment Search Tool (BLAST) system. The results of sequencing revealed that the isolates belong to two different species, Cryptococcus sp. and Candida palmioleophila. When cultivated with various Al³⁺ concentrations, the yeast growth was inhibited at a concentration of 200 mM. Pre-cultivation of these strains with 0–30 mM Al³⁺ did not promote the growth response caused by Al³⁺. Inductively-Coupled Plasma-Mass Spectrometry (ICP-MS) was used to assess the elimination of Al. The amount of Al remaining in culture media was decreased considerably after cultivation. Due to a capacity for resistance to significant Al concentrations as well as high Al elimination, these acid tolerant and Al resistant yeasts may have potential applications in the bio- and phyto-remediation of Al and acid-contaminated soils.     

Phylogeny and nitrogen fixation potential of Bradyrhizobium species isolated from the legume cover crop Pueraria phaseoloides (Roxb.) Benth. in Eastern Cameroon

Pueraria is an herbaceous, perennial legume crop originating in Asia. Pueraria phaseoloides (Roxb.) Benth. (tropical kudzu) is frequently introduced into production systems and is used as green manure, a cover crop and a forage plant, making it important economically. We used P. phaseoloides as a trap crop to study and characterize soil rhizobia in Eastern Cameroon. Bacteria were isolated from fresh nodules collected from field-grown P. phaseoloides roots. The 16S-23S rRNA internal transcribed spacer (ITS) sequences from 30 bacterial isolates were amplified by polymerase chain reaction (PCR) and the reaction products were sequenced. Phylogenetic analysis revealed that all isolates were ascribed to the genus Bradyrhizobium and were grouped into three clusters of Bradyrhizobium sp. strains, one cluster of B. yuanmingense strains, and one cluster of B. elkanii strains. Acetylene reduction assay (ARA) results indicated that the B. yuanmingense strains had significantly higher nitrogen fixation potential and that they could be used as inoculants to enhance nitrogen fixation in Pueraria grown in Eastern Cameroon.     

Comparative efficieny of Rhizobium/Bradyrhizobium spp. strains in nodulating Cajanus cajan in relation to characteristic metabolic enzyme activities

The comparative symbiotic properties of Rhizobium spp. and Bradyrhizobium spp. strains infecting pigeon pea were evaluated. Bradyrhizobium strains (Cajanus) were found to be superior to Rhizobium strains (Cajanus) and the superiority was ascertained to be due to the higher enzyme activity of the tricarboxylic acid (TCA) cycle in comparison to Rhizobium spp. strains. Moreover, metabolic superiority or rapid growth rate does not necessarily correlate with symbiotic effectiveness. The symbiotic performance of isolates varied with the host cultivar. The dry matter accumulation could be correlated with the total acetylene reduction activities rather than nodule number or nodule fresh weight per plant. Received: 3 March 1993     

Antagonistic effect of Pseudomonas spp. on pathogenic fungi and enhancement of growth of green gram (Vigna radiata)

species were isolated from the rhizosphere of green gram [Vigna radiata (L.) Wilczek] and some of the rhizobacterial isolates were found to have a wide range of antifungal activity inhibiting growth of the phytopathogenic fungi Aspergillus sp., Curvularia sp., Fusarium oxysporum and Rhizoctonia solani in culture. These isolates also showed slight inhibition of the growth of a Bradyrhizobium strain (Vigna) in a spot test which was mainly a result of nutrient competition as culture supernatants of the Pseudomonas isolates did not inhibit the growth of bradyrhizobia but inhibited the growth of fungi. The rhizobacterial isolates produced siderophores in Fe-deficient succinate medium. However, the inhibition of fungal growth by different Pseudomonas isolates in Luria Bertani and King's medium B which were not limiting in Fe³⁺ ions suggested that, besides siderophores, other antifungal compounds (antibiotics) produced by these rhizobacteria were involved in antagonism. On coinoculation of green gram with Pseudomonas strains MRS13 and MRS16 and Bradyrhizobium sp. (Vigna) strain S24, there was a significant increase in nodule weight, plant dry weight and total plant N as compared to inoculation with Bradyrhizobium strain S24 alone, suggesting that the nodule-promoting effects of Pseudomonas sp. lead to an increase in symbiotic N fixation and plant growth. Received: 27 October 1997     

Diversity and phylogeny of rhizobial strains isolated from Lotus uliginosus grown in Uruguayan soils

Lotus uliginosus is generally nodulated by rhizobia of the genus Bradyrhizobium when used for improvement of Uruguayan pastures. The genetic diversity and phylogenetic relationships of 111 isolates from nodules of L. uliginosus collected from four fields with or without prior inoculation history were analyzed in this study. Genetic diversity estimated by ERIC-PCR revealed 75 different genomic fingerprints, and showed a relatively greater value compared with other methods and varied by soil type. 16S ribosomal RNA gene RFLP analysis revealed three different ribogroups, A, B and C, with 71 isolates in ribogroup A, three isolates in ribogroup B and only one in ribogroup C. Phylogenetic analyses based on 16S RNA gene sequences, ITS, as well as atpD, recA and glnII gene sequences indicated that ribogroup A strains were affiliated with B. japonicum bv. genistearum strains. The three isolates in ribogroup B did not clearly associate with any Bradyrhizobium species described previously and could represent a novel species within this genus. Unlike B. japonicum strains these isolates were able to nodulate and fix nitrogen with other Lotus species as well as with Spartium, a leguminous shrub. The unique isolate in ribogroup C clustered with Mesorhizobium and appeared genetically and phenotypically related to broad host-range Mesorhizobium sp. NZP2037. Our data suggest that Uruguayan soils contain native or naturalized bradyrhizobia that are able to nodulate L. uliginosus as efficiently as the commercial strain NZP2309 but could have adaptive advantages making them more suitable for inoculant purposes.     

Isolation and partial characterization of endophytic diazotrophs associated with Japanese sugarcane cultivar

Endophytic bacteria were isolated from the juice of a 6 month-old sugarcane cv. NiF-8, which was collected from Miyako, Okinawa and planted in a field lysimeter in Tsukuba, Ibaraki, Japan. The most probable number of N₂-fixing endophytes was 4.5 × 10⁵ cells per gram of fresh weight sample and 21 isolates have a positive reaction for ARA in an N₂-free semi-solid medium with 10% crystallized sugarcane sugar and 0.5% sugarcane juice. Analyses of some of the biochemical properties of the N₂-fixing isolates indicated that 13 isolates were putative strains of Acetobacter diazotrophicus, 4 isolates showed similar characteristics to those of Herbaspirillum seropedicae, and 4 isolates consisted of Herbaspirillum rubribalbicans-like strains. This study confirmed the existence of N₂-fixing endophytic bacteria in the Japanese sugarcane cv. NiF-8.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Nodulation of African yam bean (Sphenostylis stenocarpa) by Bradyrhizobium sp. isolated from Erythrina brucei

African yam bean (Sphenostylis stenocarpa), which is widely cultivated in Africa because of its growth capability on marginal soils, was nodulated by an endosymbiont (characterized and designed Bradyrhizobium sp. AUEB20) isolated from the Ethiopian tree Erythrina brucei with the formation of a small number of large, indeterminate N₂-fixing nodules. In contrast, 24 other isolates from Ethiopian woody legumes were ineffective. Strain AUEB20 promiscuously nodulated a number of tropical legumes, but none out of five European crop plants tested. Received: 17 September 1996