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We have investigated the effectiveness of a new soybean fertilization technique, named crack fertilization, which involves the application of nodule bacteria on biochar to soybean roots through cracks formed between planting rows during midterm tillage. In the present study, the factors and timing of crack fertilization were investigated in an upland field converted from a paddy. The variables investigated were: 1) crack formation without the application of any agricultural materials, and 2) the application of biochar or 3) nodule bacteria on biochar into cracks. The treatment periods were: 1) before sowing, 2) during midterm tillage, and 3) during both periods. The combination of crack fertilization and reduced tillage was also tested. The method of crack fertilization that increased yield was the combination of crack formation and the application of biochar, and the most effective period for the treatment was before sowing in the reduced tillage field. Seed yields in conventional and reduced tillage fields were comparable in the upland field converted from the paddy. These results suggest that the application of biochar into cracks after scratching the soil surface to remove weeds before sowing is a practical method of increasing soybean yield in upland fields converted from paddies.

Strigolactones (SLs), a group of plant secondary metabolites, play an important role as a host recognition signal for symbiotic arbuscular mycorrhizal (AM) fungi in the rhizosphere. SLs promote symbioses with other beneficial microbes, including root nodule bacteria. Root parasitic weeds also take advantage of SLs as a clue to locate living host roots. In plants, SLs function as plant hormones regulating various growth and developmental processes including shoot and root architectures. Plants under nutrient deficiencies, especially that of phosphate, promote SL production and exudation to attract symbionts and to optimize shoot and root architecture.

Waterlogging is the constraint for soybean growth and yield, because soybean is often cultivated in upland fields converted from paddy in Japan. However, efficient cultivation techniques for alleviating the adverse effects have not been developed. We have proposed the new soybean cultivation technique named crack fertilization which enables yield increase due to enhancing new root growth and N acquisition by increasing nodulation. Waterlogging induces N deficiency due to the suppression of nutrient uptake by the inhibition of root growth and nodule activity. Thus, it is hypothesized that crack fertilization would be effective to alleviate the inhibition of soybean growth and yield. The soybean cultivar of Sachiyutaka was planted in 1/5000 a Wagner pots and root boxes. Two separate waterlogging treatments were imposed to soybean plants at different growth stages, V1 and R4, and crack fertilization was done at V3. After these treatments, soybean plants were sampled at R5 in 2012 and 2013 experiments, respectively. Waterlogging at V1 and R4 inhibited the growth and yield of soybean and nodule growth, and the decreases in physiological parameters of soybean such as photosynthesis, chlorophyll content, and xylem sap exudation rate were observed. The adverse effects of waterlogging at V1 were alleviated by crack fertilization at V3, whereas crack fertilization could not alleviate the adverse effects of waterlogging at R4. Thus, crack fertilization after waterlogging at early vegetative stage would be the cultivation technique that enables to alleviate the adverse effects.

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA. Electronic supplementary material The online version of this article (doi: 10.1186/s40793-017-0298-3) contains supplementary material, which is available to authorized users.

Thirty two rhizobial isolates were obtained from different bioclimatic regions of Tunisia using as trap plants, Medicago sativa, Medicago ciliaris, Medicago polymorpha and Medicago minima. To study their diversity and characterize them in relation to Mediterranean conditions, abiotic stress resistance, symbiotic properties and genetic diversity in terms of 16S rRNA and nodA sequences were assessed. Five isolates from M. sativa, three from M. ciliaris and three from M. minima could grow at 45°C. Only two isolates from M. sativa grew at 4% NaCl. The most stress tolerant isolates were obtained from arid soils. A phylogenetic analysis of 16S rRNA genes revealed 29 isolates to be closely related to Ensifer including one (Pl.3-9) that showed a 16S rRNA sequence similar to that of Ensifer meliloti and nodA sequence similar to that of Ensifer medicae. However, three isolates were categorized into Agrobacterium containing the nodA of Ensifer. Furthermore, these isolates developed nodules on original hosts. The results for the four isolates suggest horizontal gene transfer between the species.

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.

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root zone (0–5 mm) and decreased in the root zones susceptible to rhizobia (5–20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.

The aim of these studies was to investigate whether residual toxic effects of exposing soybean root nodule bacteria to Al in a solid defined media (SDM) alter tolerance to Al, survival, sensitivity to antibiotics, N2 fixation effectiveness and genetic diversity of Bradyrhizobium strains. After being exposed four times to Al, strains showed variation in Al tolerance but there was no evidence of change in their original Al tolerance, sensitivity to the antibiotics or genetic diversity. Exposure of Bradyrhizobium strains to SDM plus Al did not alter biological N2 fixation effectiveness of five strains. Strain SEMIA 587 showed a reduction in its N2 fixation effectiveness but it seems that it was just a superficial toxic effect because one single passage through the plant eliminated this effect. Residual Al did not cause increases in Al tolerance and reductions in the survival and N2 fixation effectiveness of Bradyrhizobium strains USDA 143, SEMIA 586, SEMIA 5019, SEMIA 5039 and SEMIA 5073. It also did not alter the resistance to antibiotics of strains USDA 143, SEMIA 5039 and SEMIA 5073, and the genetic diversity of the strains SEMIA 587 and SEMIA 5019. O objetivo deste trabalho foi investigar se os efeitos tóxicos residuais do Al afetam a tolerância Al, sobrevivência, sensibilidade a antibióticos, eficiência de fixação de nitrogênio e diversidade genética de estirpes de Bradyrhizobium. Quando os nódulos de bactérias da raiz de soja foram expostos ao Al, em meio sólido por quatro vezes consecutivas, as estirpes de Bradyrhizobium mostraram variações na tolerância ao Al. Não houve alterações nos níveis originais de tolerância ao Al, sobrevivência, sensibilidade aos antibióticos testados e diversidade genética. Cinco das estirpes expostas ao Al não alteraram a eficiência de fixação de nitrogênio. A estirpe SEMIA 587, por sua vez, mostrou uma redução da sua eficiência de fixação de N2, mas apenas superficial, porque uma simples passagem através da planta eliminou esse efeito tóxico. Sucessivas exposições de estirpes de Bradyrhizobium ao Al não aumentam a tolerância ao Al, não reduzem a sobrevivência e a eficiência de fixação simbiótica do N2 das estirpes USDA 143, SEMIA 586, SEMIA 5019, SEMIA 5039 e SEMIA 5073, não alteram a tolerância a antibióticos das estirpes USDA 143, SEMIA 5039 e SEMIA 5073 e não alteram a diversidade genética das estirpes SEMIA 587 e SEMIA 5019.

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.

The Sinorhizobium-legume interaction is sensitive to a number of environmental factors: one of the important stresses is soil acidity. Low pH imposes severe constraints on legume productivity. In the Sinorhizobium-Medicago partnership, prokaryotic partner is the most acid-sensitive. The isolation of acid-tolerant Sinorhizobium strains from the eastern and southern Mediterranean rim have allowed the successful development of medic pastures on over I million ha of land in Western Australia, posing two important questions: • How do acid-tolerant strains differ from acid-sensitive counterparts? • How do agriculturally important root nodule bacteria respond to low pH? Knowledge of the genetic circuits required and activated by the cell in acidic conditions is vital to understand the response and survival of bacteria in these conditions. The molecular basis of acid tolerance has been investigated with several approaches (Glenn et al. 1999).

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.

Root nodule bacteria infect and form nodules in the roots on various leguminous plant hosts. The bacteria undergo morphological changes in the cells of the nodules; from free-living to bacteroidal form. The bacteroidal form is classified to two types: Rhizobium and Bradyrhizobium. The bacteroid morphology is determined by rhizobial genera, the host plant and some unknown mechanisms.

One of the most interesting aspects of Rhizobium and Bradyrhizobium ecology is the competitivity for nodulation between strains. Native rhizobial populations, often poorly effective for symbiotic nitrogen fixation, are well adapted to soil conditions and are able to occupy most of the nodules formed in the presence of bacterial strains applied to seeds. This is a cause of the common fail of attempts carried out to improve productivity in agricultural fields by inoculation with superior strains of root nodule bacteria.

Summary Cross-inoculation group specificity in Cicer arietinum L. (chickpea) was studied. Seventy-one strains of root nodule bacteria of C. arietinum were examined for nodulation on 87 species of legumes. These species represent all the known cross-inoculation groups and were selected from various tribes and genera of family Fabaceae and Mimosaceae. In a reciprocal cross-inoculation study, 287 strains of root nodule bacteria from 52 of the 87 species, were examined on various genotypes of C. arietinum. C. arietinum and its root nodule bacteria did not show cross inoculation affinity with any of the members of the known cross-inoculation groups: alfalfa, clover, pea, bean, soybean, lupin-lotus and cowpea miscellany; except for some loose non-reciprocal kinship with Sesbania, which in its turn has strong affinity with the cowpea miscellany. However, under natural conditions in soil, even the loose non-reciprocal kinship between Cicer and Sesbania was rare. For practical reasons, it is suggested that C. arietinum and its root nodule bacteria should be considered in a separate cross-inoculation group. ‘Cross-inoculation host specificity’ appears to be of some help in the revision of the taxonomy of order Leguminosales and supports the placement of Cicer in a monogeneric tribe Cicereae.

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.

A comparison was made between 65 strains of root nodule bacteria from indigenous New Zealand legumes and 45 reference strains including: Rhizobium trifolii, R. phaseoli, R. leguminosarum, R. meliloti, and both “acid-producing” and “non-acid-producing” strains from the Lotus-Lupinus-Ornithopus cross-inoculation group. The strains were classified into 10 clusters on the basis of 37 morphological, cultural, and physiological tests. Relationships disclosed among the reference strains were in accordance with current ideas on Rhizobium taxonomy. The indigenous strains were well separated from both the trifolii-leguminosarumphaseoli complex and R. meliloti but acid-producing strains from the Lotus-Lupinus-Ornithopus cross-inoculation group segregated with the indigenous strains. The principal characters differentiating the clusters are discussed. The base composition of DNA from representative indigenous strains was determined and base sequence homology studied. The relative reassociation at 65°c in 0.2...

Rhizobium trifolii isolates were obtained from nodules of white clover (Trifolium repens L.) growing within a grazing trial in steep, North Island hill country. The white clover was from each of 6 experimental treatments involving 2 fertiliser application regimes and 3 grazing managements. ‘Grasslands Huia’ and the resident Kentish-type white clover were present in pastures in variable proportions. Effectiveness of isolates, measured using Huia as the host plant and compared with a standard strain, ranged from 3 to 170%. Of 285 isolates tested, 9% were at least 20% more effective than the standard strain. Influence of experimental treatments on effectiveness of isolates appeared to be indirect, acting through effects on proportion of Huia and the resident white clover present at sample sites.

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.

Summary The study on cultural and biochemical characteristics of 115 strains of root nodule bacteria of chickpea (henceforth named Cicer-Rhizobium ), suggest that only few characteristics, like the absence of growth on yeast extract mannitol agar (YEMA) in 24 h, no or little growth on glucose peptone agar without change of p H of the medium in 48 h, inability to produce 3-ketolactose, to precipitate calcium glycero-phosphate, to form serum zone coupled with acid production in litmus milk, to tolerate 20 meq Mn ++ /1 lactose medium and p H 10.0 in Hofer's alkaline broth, could be of diagnostic value in the identification of Rhizobium . In other characteristics, the strains were found to vary. The Congo-red colour differentiation was better obtained by pour plate method than by streaking the culture on YEMA plates. The production of acid or alkali on mannitol was found to be independent of the properties of soil from where the strains were isolated; and this did not substantiate Norris's hypothesis that Rhizobium , associated with tropical legumes, produces alkali. The position of Cicer-Rhizobium as a link between the group of fast growing Rhizobium from clover, pea, and beans and the slow growing group of Rhizobium from soybean, lupin, and cowpea miscellany has been indicated.

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.

The effect of the air content of the medium on the development and function of the nodule was studied with sterile cultures of peas inoculated with their appropriate strain of nodule bacteria.In water cultures the plant grows well without added nitrogen, even though all nodules are submerged, if air is bubbled through the medium during the period of growth. Abundant nodulation occurs also in nonaerated liquid cultures; the nodules, however, are much smaller than in aerated cultures, and the plant grows poorly. Subsequent aeration of such cultures results in a distinct increase in the size of the nodules and improvement in the growth of the plant.Nodulation is completely prevented by passing a stream of gaseous N through the medium. That oxygen is indispensable for the function of the nodules is shown also by the fact that the peas grow excellently on added nitrogen in non-aerated cultures.

The failure of pea (Pisum sativum) to grow in ‘goradu’ soil of Central Gujarat without artificial inoculation of the seed with the specific root nodule bacteria has been investigated. The study of a large number of soil fungi isolated from ‘goradu’ soils failed to show that they had any selective antibiotic activity against the pea rhizobia. It has been explained that the high sand content of this soil and the lower thermal death point of the pea rhizobia are the probable causes for the failure. The high sand content does not give any protection to the pea rhizobia against the high temperatures of the summer months with the result that their number gets so reduced and are not able to nodulate plants sufficiently.

Four distinct types of colonial mutation were obtained by X-ray irradiation of a parasitic strain of alfalfa Rhizobium. Two types were procured from an effective strain. Colonial morphology remained stable throughout serial transfer on artificial medium, but changed considerably after one plant passage. Three variants derived from the parasitic parent were found to be efficient in nitrogen fixation when tested by sterile plant growth procedures and the Virtanen technique. This efficiency increased during the course of two plant passages. Colonial variation and effectivity changes occurred independently of one another.

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.

The Virtanen technique was used to detect effective and ineffective rhizobia. The rhizobia studied varied widely in their action towards different varieties of the same host species. Maximum leghemoglobin concentrations occurred just prior to blossoming in field legumes, but much earlier in greenhouse legumes cultivated under conditions of this experiment. The nodules of the ineffective strains of legume bacteria differed from those produced by effective strains in color, size, distribution, rate of destruction of the bacteroid area, and presence or absence of rodlike bacterial forms. Experimental evidence is presented to show that a supposedly parasitic strain of alfalfa organism can exist.

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 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.

The associated growth of peas and different non-legumes (barley, oats, wheat, and potato) was investigated both under sterile conditions and in ordinary pot cultures. In the former case the medium consisted of quartz sand, and in the latter of clay or sandy loam soil, poor in soluble nitrogen. It is shown that nitrogen was transferred from the pea nodules to the non-legumes.In sterile cultures, where the excreted amino-acids cannot undergo decomposition, oats, wheat and barley utilized only a part of the excreted nitrogen, generally less than 50 per cent. In some instances, however, the barley took up considerably over 50 per cent of the excreted nitrogen. There was an apparent accumulation of aspartic acid in the medium of the associated cultures. In pot cultures, where the amino-acids may have been broken down by contaminating micro-organisms, the transfer of the excreted nitrogen to the potato exceeded 90 per cent.All experiments show that with increasing ratio of non-legumes to legumes the growth of the peas suffers, obviously from lack of nitrogen. Under these conditions, the excretion from the nodules increases to such an extent that the peas receive only a small fraction of the nitrogen fixed by the nodules. The major part of the carbohydrate material furnished by the pea for the amino-acid synthesis in the nodules is excreted into the medium. This naturally causes disturbances in the growth of the pea.

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.

The possibilities of the Rhlzobium active strains selection in the pure culture and on the early stages of the legume- Rhizobium symbiosis formation are discussed.

Fixation of nitrogen by Rhizobium in pure cultures in the presence of nitrate under anaerobic conditions has been established. Both slowly-growing and fast-growing rhizobia were investigated.

THE root nodule bacteria of leguminous plants (Rhizobium spp.) have repeatedly been observed to survive as long as 15–16 yr. in various media1,2. Bisset3 has explained this remarkable longevity as due to formation of endospores in a complicated life-cycle.

Use of the plant-dilution method for the determination of the number of root-nodule bacteria in nonsterile carrier materials has been described for both small and large-seeded legume hosts. Calculation of the most probable number of rhizobia is described using values based on the appropriate tables of Fisher and Yates, and approximate factors of error for 95 per cent fiducial limits are stated. It was found that the plant-dilution method was likely to underestimate the number of rhizobia by a factor not greater than 2, when comparisons were made with pure cultures. However for non-sterile peat the tendency was for the plate count to give the lower estimate. This latter effect was no doubt due to the presence of other organisms and therefore the plant-dilution method is to be preferred in such cases.

IT has been suggested by Thimann1, Link, Wilcox and Link2, and Link3 that β-indole-acetic acid (heteroauxin) is the agent partially or entirely responsible for the formation of nodules on the roots of leguminous plants. Thimann1 advances the hypothesis that the root nodule bacteria convert tryptophane into heteroauxin, (a) by stimulating elongation of a lateral root and (b) by arresting root elongation through continued auxin production, resulting in the formation of a nodule. If this condition exists, there may be differences in the rate of formation and in total amounts of heteroauxin derived from tryptophane, between efficient and inefficient strains of Rhizobia. (Efficient: Rhizobia which infect the host plant and benefit the latter by the symbiosis; inefficient: Rhizobia which infect the host plant but fail to aid in fixing atmospheric nitrogen.) Nodules produced by efficient strains are generally characterized as being large and usually located on the crown of the tap-root, indicating early invasion, while inefficient strains produce very small nodules which are distributed over the secondary root system, indicative of relatively late invasion.

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.