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Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6 % minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

Symbiotic nitrogen fixation is the main route for sustainable input of nitrogen into ecosystems. Nitrogen fixation in agriculture can be improved by inoculation of legume crops with suitable rhizobia. Knowledge of the biodiversity of rhizobia and of local populations is important for the design of successful inoculation strategies. Soybeans are major nitrogen-fixing crops in many parts of the world. Bradyrhizobial inoculants for soybean are very diverse, yet classification and characterization of strains have long been difficult. Recent genetic characterization methods permit more reliable identification and will improve our knowledge of local populations. Forage legumes form another group of agronomically important legumes. Research and extension policies valorizing rhizobial germplasm diversity and preservation, farmer training for proper inoculant use and legal enforcement of commercial inoculant quality have proved a successful approach to promoting the use of forage legumes while enhancing biological N 2 fixation. It is worth noting that taxonomically important strains may not necessarily be important reference strains for other uses such as legume inoculation and genomics due to specialization of the different fields. This article points out both current knowledge and gaps remaining to be filled for further interaction and improvement of a rhizobial commons.

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.

Quantification of acidity tolerance in the laboratory may be the first step in rhizobial strain selection for the Amazon region. The present method evaluated rhizobia in Petri dishes with YMA medium at pH 6.5 (control) and 4.5, using scores of 1.0 (sensitive, "no visible" growth) to 4.0 (tolerant, maximum growth). Growth evaluations were done at 6, 9, 12, 15 and 18 day periods. This method permits preliminary selection of root nodule bacteria from Amazonian soils with statistical precision. Among the 31 rhizobia strains initially tested, the INPA strains 048, 078, and 671 presented scores of 4.0 at both pHs after 9 days of growth. Strain analyses using a less rigorous criterion (growth scores higher than 3.0) included in this highly tolerant group the INPA strains 511, 565, 576, 632, 649, and 658, which grew on the most diluted zone (zone 4) after 9 days. Tolerant strains still must be tested for nitrogen fixation effectiveness, competitiveness for nodule sites, and soil persistence before their recommendation as inoculants.

Symbiotic instability occurs in some elite strains of Rhizobium leguminosarum bv. viciae during continued cultivation and preparation as inoculants resulting in loss of nodulation ability. This project aims to understand the symbiotic instability of these strains and identify criteria for the selection of stable elite inoculant strains of pea root nodule bacteria.

A plasmid-cured derivative of Rhizobium loti strain NZP2037, strain PN4010, was significantly more effective in N2 fixation and showed an improved capacity to compete with other root nodule bacteria for nodulation of Lotus pedunculatus, relative to that of strain NZP2037. Reintroduction of the plasmid pRlo2037 into PN4010 resulted in a return to the NZP2037 level of symbiotic effectiveness and competitiveness. The enhanced effectiveness and competitiveness of PN4010 for L. pedunculatus was associated with its increased capacity to form nodules. Strain PN4010 did not differ from NZP2037 in its growth characteristics in pure culture, in its resistance to L. pedunculatus root flavolan or in its ability to multiply in the L. pedunculatus rhizosphere in the presence of a competing strain of R. loti. An examination of 35 R. loti strains (including 25 field isolates) revealed two strains (NZP2014 and NZP2042) that did not contain a single large indigenous plasmid; both were significantly more effective in N2 fixation with L. pedunculatus than was NZP2037 or two other Nod+ Fix+ plasmid-containing strains of R. loti. Transfer of pRlo2037 into NZP2014 and NZP2042 reduced their effectiveness for L. pedunculatus.

Leakage of materials reacting with the fluorescent probe 1-anilino-8-naphthalcne sulphonate (ANS) from root-nodule bacteria subjected to desiccation and rehydration suggests that death of these bacteria is associated with changes in membrane permeability. The slow-growing rhizobia and the fast-growing Rhizobium meliloti differ from other fast-growing bacteria of the R. leguminosarum group in that they exhibit little fluorescence in the presence of ANS. This probably reflects differences between these groups of bacteria in the polar/non-polar nature of the outer cell envelopes. Following desiccation and rehydration, all groups of root-nodule bacteria fluoresced strongly in the presence of ANS. Repair of desiccation-induced damage to the cell envelopes of R. japonicum QA372. R. trifolii SU297/32B and R. meliloti CC131 was not observed using a range of screening agents. However, repair of significant numbers of lysozyme-sensitive bacteria was observed with R. leguminosarum TA101 within an hour of rehydration.

Significant correlations exist between the ability of root-nodule bacteria to fix molecular nitrogen and some of their properties (accumulation of riboflavin, cobalamine, ATP, cell pyrolysis, effect on plant vegetation characteristics) which can be used for quickly assessing the activity of the process in individual strains of rhizobia.

Rhizobium trifolii organisms associated with eleven species ofTrifolium were isolated and studied by means of antigen-antibody agglutination and immune diffusion techniques. Ten serologic types were differentiated from the eleven species of nativeTrifolium.

Eighteen strains of Rhizobium lupini were shown to form effectively nitrogen-fixing root nodules in Lotus uliginosus whose rhizobia appeared culturally similar to R. lupini. Six strains formed effective or (in one case) semi-effective nodules in Ornithopus sativus.

SUMMARY: The application of computer techniques to study a group of organisms comprising 83 strains of the genus Rhizobium and 38 strains belonging to the genera Agrobacterium, Chromobacterium, Beijerinckia and Bacillus is described. One hundred different features including vitamin, carbohydrate and nitrogen nutrition, antibiotic sensitivities, morphological and colonial characteristics, and infective attributes were used to determine the relationships between these organisms. Members of the genera Chromobacterium, Bacillus and Beijerinckia showed little affinity with the other species studied, or with each other. The results obtained for the genus Rhizobium indicate the need for major taxonomic changes. These changes would include: (1) the consolidation of R. trifolii, R. leguminosarum and R. phaseoli into a single species, R. leguminosarum; (2) The consolidation of A. radiobacter and A. tumefaciens and their inclusion as R. radiobacter in the genus Rhizobium. The creation of a new genus Phytomyxa to contain strains of slow-growing root-nodule bacteria is also proposed.

Trichloro-, dichloro- and monochloroacetic and α-dichloro-, α-monochloro-and β-monochloropropionic acids as sodium salts in 0.10–1.0mm concentration accelerated the growth of a strain of Rhizobium leguminosarum in a liquid synthetic medium with biotin as sole accessory growth factor. The strongest effect was shown by β-monochloropropionate. Concentrations of 10mm and above were inhibitory. α-Monochlorobutyrate did not accelerate growth.

The survival over summer of field-grown root nodule bacteria was studied in the field and the laboratory during the course of an investigation into a nodulation problem of annual clovers. Dry field soils containing Rhizobium trifolii and R. lupini were subjected to a range of temperatures in the laboratory, the bacteria surviving 6 h exposure to temperatures as high as 80°C. Soil temperatures during summer were recorded at different depths. Populations of rhizobia were estimated at these depths from the end of the growing season (October) to early autumn (April) in plots which had carried dense swards of subterranean clover and Serradella. High populations of R. lupini were maintained in the Serradella plots throughout the summer. Populations of R. trifolii in the subterranean clover plots were initially much lower, and declined with both time and depth. The problem known as ‘second-year clover mortality’ is primarily due to low numbers of clover rhizobia in the soil at the end of the growing season. This situation is aggravated over the long hot dry summer, when there is a further decline in numbers.

SUMMARY The growth and survival in peat culture of Rhizobium spp. from 3 inoculation groups was markedly affected by the gas exchange properties of the container. Complete restriction of aeration caused rapid reduction in numbers after only 1 week. Low moisture contents restricted growth but higher levels up to 60% (on a wet weight basis) favoured survival. Within the range tested the size of the inoculum was unimportant in determining the number of rhizobia in sterilized peat provided sufficient time elapsed for multiplication. Storage temperatures between 4° and 26° affected the rate of growth but not survival of rhizobia in pure culture for periods up to 26 weeks provided moisture was not limiting.

SUMMARY: The vitamin requirements of 63 strains of Rhizobium and 18 of Agrobacterium were examined. R. trifolii, R. leguminosarum and R. phaseoli responded in most cases to thiamine, biotin, and calcium pantothenate. Strains of R. meliloti and the slow-growing rhizobia sometimes responded to biotin, but not to the other vitamins. An unknown growth factor, active for the slow-growing rhizobia, was detected. Three strains of Agrobacterium required thiamine.

Growth of Lotus nodule bacteria (Strain LC 296) was temporarily suppressed in a yeast-extract mineral-salts medium containing 2,4-DB at 50 and 100 µg/ml. However, the 2,4-DB concentration needed to completely suppress the growth of the bacteria appeared to be above 500 µg/ml. When added during the logarithmic phase of growth 2,4-DB at 50 and 100 µg/ml did not inhibit the growth of the cells, so that the growth-inhibiting effect of the herbicide apparently was directed primarily against lag phase cells. Pre-incubation of rhizobia in a medium containing 2,4-DB at concentrations up to 10 µg/ml did not affect the capacity of the bacteria to effectively nodulate their host plant. Attempts to induce the rhizobia to utilize 2,4-DB as a sole carbon source failed. Nevertheless, when theLotus nodule bacteria (LC 296) were grown in a medium containing 2,4-DB-1-C14, C14O2 was released. Similar results were obtained with other strains of the Lotus rhizobia and other species of Rhizobium. C14O2 evolution was also demonstrated when radioactive 2,4-DB was added to both intact (excised) and crushed trefoil nodules. The results indicate that the nodule bacteria are capable of degrading 2,4-DB; although the mechanism is still obscure it does not appear to be via cleavage of the ether link.

Bacteriophage for clover nodule bacteria can be found on roots and nodules of all naturally grown clover plants and also in the soil surrounding the roots, but not in soil without clover plants. Alternative hosts for the phage of clover bacteria are pea bacteria, and vice versa. The bacteria and the phage are heterogeneous in the sense that only a proportion of strains of clover bacteria and of pea bacteria are susceptible to lysis by a given race of phage, and only a proportion of races of phage can lyse a given bacterial strain. There does not seem to be any association between the susceptibility of bacterial strains to lysis by phage and any other features such as antigenic structure and effectiveness in nitrogen fixation. There may be an association with avirulence, i.e. inability to infect the host plant. The behavior of phage–bacterial mixtures depends on the surrounding medium. The longevity of phage in soil or in a soil-like medium such as a vermiculite mixture is relatively short, and the effect of phage can be localized so that phage-susceptible bacteria and the phage can exist close to each other without any apparent interaction. However, as long as the phage is present, phage-resistant bacterial mutants are usually present also. The phage-resistant mutants may also be mutants in other respects such as effectiveness in nitrogen fixation. In the presence of weakened phage, bacterial mutants were found to occur that differ from the parent form in effectiveness but resemble it in susceptibility to the phage.

A new species, Rhizobium fredii, is proposed for fast-growing root nodule bacteria isolated from soybeans. The type strain was isolated from a root nodule of Glycine max growing in Honan Province, China, and is designated strain USDA 205 (= ATCC 35423 = PRC 205). This new species is differentiated from currently recognized Rhizobium and Bradyrhizobium species by deoxyribonucleic acid hybridization comparisons, plant specificity, generation times, antibiotic resistance, and serology. The strains of R. fredii are differentiated into two proposed chemovars, R. fredii chemovar fredii chemovar nov. and R. fredii chemovar siensis chemovar nov., by deoxyribonucleic acid hybridization tests, growth in the presence of erythromycin (20 μg/ml), final pH of yeast extract-mannitol medium, and serology.

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Antisera prepared against 58 strains of root-nodule bacteria and against 16 strains belonging to the genusAgrobacterium were tested against 113 strains ofRhizobium, 20 strains ofAgrobacterium and 20 strains of other, possibly related, bacteria.

MR. VINCENT, who courteously sent me a copy of this letter before publication, suggests that the juice from roots bearing effective nodules is more stimulating to the growth of Rhizobium as a result of effective association. This is in agreement with our views. We state in our paper1 (p. 489) that there is evidence of “an increased stimulating effect from juices of roots bearing effective nodules, possibly connected with the products of nitrogen fixation”.

Variations in the susceptibility of rhizobia to antibiotic substances have been previously reported. Fogle and Allen (1948) considered lupin and soybean rhizobia to be more susceptible to various 'streptomycete antibiotics than other species of rhizobia. In contrast, Landerkin and Lochhead (1948) noted that strains of Rhizobium japonicum were less susceptible to actinomycete antibiotics than were other soil organisms tested.