Efficiency of nodule initiation in cowpea and soybean

When serial dilutions of a suspension of Bradyrhizobium japonicum strain 138 were inoculated onto both soybean and cowpea roots, the formation of nodules in the initially susceptible region of the roots of both hosts was found to be linearly dependent on the log of the inoculum dosage until an optimum dosage was reached. Approximately 30- to 100-fold higher dosages were required to elicit half-maximal nodulation on cowpea than on soybean in the initially susceptible zone of the root. However, at optimal dosages, about six times as many nodules formed in this region on cowpea roots than on soybean roots. There was no appreciable difference in the apparent rate of nodule initiation on these two hosts nor in the number of inoculum bacteria in contact with the root. These results are consistent with the possibility that cowpea roots have a substantially higher threshold of response to symbiotic signals from the bacteria than do soybean roots. Storage of B. japonicum cells in distilled water for several weeks did not affect their viability or efficiency of nodule initiation on soybean. However, the nodulation efficiency of these same cells on cowpea diminished markedly over a 2 week period. These differential effects of water storage indicate that at least some aspects of signal production by the bacteria during nodule initiation are different on the two hosts. Mutants of B. japonicum 138 defective in synthesis of soybean lectin binding polysaccharide were defective in their efficiency of nodule initiation on soybean but not on cowpea. These results also suggest that B. japonicum may produce different substances to initiate nodules on these two hosts.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

Host-Symbiont Specificity Expressed during Early Adsorption of Rhizobium meliloti to the Root Surface of Alfalfa

Early (4 h) adsorption of Rhizobium meliloti L5-30 in low numbers to alfalfa roots in mineral solution was examined for competition with other bacterial strains. All tested competitor strains decreased the adsorption of L5-30 by extents which depended on the strain and its concentration. The decrease of adsorption by R. meliloti competitors (all of them infective in alfalfa) was nearly complete at saturation (97 to 99% decrease). All other heterologous rhizobia and Agrobacterium tumefaciens at saturating concentrations (10⁶ to 10⁷ per ml) decreased adsorption of L5-30 only partially, less than 60%. The differential effects of homologous and heterologous competitors indicate that initial adsorption of R. meliloti to the root surface of its host occurs in symbiont-specific as well as nonspecific modes and suggest the existence of binding sites on roots which are highly selective for the specific microsymbiont in the presence of other heterologous bacteria even in very unfavorable (less than 10⁻⁴) symbiont-competitor concentration ratios.     

Role of Lectins in Plant-Microorganism Interactions: III. Influence of Rhizosphere/Rhizoplane Culture Conditions on the Soybean Lectin-binding Properties of Rhizobia

The influence of rhizosphere/rhizoplane culture conditions on the ability of various rhizobia to bind soybean seed lectin (SBL) was examined. Eleven strains of the soybean symbiont, Rhizobium japonicum, and six strains of various heterologous Rhizobium species were cultured in root exudate of soybean (Glycine max [L.] Merr.) and in association with roots of soybean seedlings which were growing either hydroponically or in montmorillonite clay soil amendment (Turface). All 11 of the R. japonicum strains developed biochemically specific receptors for the lectin when cultured under these conditions, whereas six of the 11 did not develop such receptors when cultured in synthetic salts medium. Two cowpea strains also developed receptors for SBL. The other four heterologous strains of rhizobia gave no evidence of biochemically specific SBL binding in either synthetic salts media or rhizosphere/rhizoplane cultures. These results demonstrate that the environment provided by plant roots is an important factor in the development of specific lectin receptors on the cell surface of R. japonicum.     

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

Quantitative Study of Nodulation Competitiveness in Rhizobium Strains

We compared the nodulation competitiveness of three strains of Rhizobium leguminosarum by counting the number of nodules formed on faba bean plants after the application at sowing time of different concentrations of the strains to soils already containing Rhizobium strains of the same species. A relationship of type y = axⁿ was found to exist between the ratio of the nodules formed by the applied inoculum strain to the nodules formed by the soil strains and the ratio of Rhizobium cells in the inoculum to the cells in the soil. This relationship was also confirmed in another competition experiment in which two R. meliloti strains of identical competitiveness were mixed in various proportions. The relationship can also be applied to the majority of results reported in the literature. Should it prove to be more widely applicable, it could be used to estimate the relative competitiveness of Rhizobium strains and thus predict the performance of an inoculum in a given soil.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

The uptake and hydrolysis of disaccharides by fast-and slow-growing species of Rhizobium

Slow growing strains of rhizobia appear to lack both uptake systems and catabolic enzymes for disaccharides. In the fast-growing strains of rhizobia there are uptake mechanisms and catabolic enzymes for disaccharide metabolism. In Rhizobium leguminosarum WU 163 and WU235 and R. trifolii WU290, sucrose and maltose uptake appears to be constitutive whereas in R. meliloti WU60 and in cowpea Rhizobium NGR234 uptake of these disaccharides is inducible. There is evidence that there are at least two distinct disaccharide uptake systems in fast-growing rhizobia, one transporting sucrose, maltose and trehalose and the other, lactose. Disaccharide uptake is via an active process since uptake is inhibited by azide, dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone but not by arsenate. Bacteroids of R. leguminosarum WU235 and R. lupini WU8 are unable to accumulate disaccharides.     

Congo Red Absorption by Rhizobium leguminosarum

Congo red absorption is generally considered a contraindication of Rhizobium. However, R. leguminosarum takes up the dye on yeast extract-mannitol agar. The uptake of congo red varies among strains of R. leguminosarum, as shown elsewhere with strains of R. trifolii and R. meliloti. Congo red absorption does not distinguish rhizobia from other bacteria, but may be useful as a strain marker.     

An Ouchterlony double diffusion study on the interaction between legume lectins and rhizobial cell surface antigens

Acidic exopolysaccharides and O-antigen containing lipopolysaccharides were isolated from Rhizobium japonicum, R. leguminosarum, R. lupini, R. meliloti, R. phaseoli, cowpea Rhizobium sp. and a non-nodulating soil bacterium. Lectins from seeds of soybean (Glycine max), garden pea (Pisum sativum), lentil (Lens culinaris), alfalfa (Medicago sativa), field bean (Phaseolus vulgaris), jackbean (Canavalia ensiformis) and from wheat germ were tested for their capacity to precipitate rhizobial exopolysaccharides and lipopolysaccharides in the Ouchterlony double diffusion test. Soybean lectin precipitated exclusively with the exopolysaccharide of R. japonicum, whereas the lectins from pea and lentil precipitated exopolysaccharides from all the fast growing strains of Rhizobium. Host range specific interactions between lipopolysaccharides and lectins were observed in the pea/lentil-R. leguminosarum and in the alfalfa-R. meliloti systems. Concanavalin A precipitated the exopolysaccharides of all fast growing strains of Rhizobium, the exopolysaccharide of the cowpea strain and several lipopolysaccharides of different Rhizobium species and thus did not show any correlation between polysaccharide binding and symbiotic specificity. Non-leguminous wheat germ agglutinin did not precipitate any of the rhizobial polysaccharides tested and the lipopolysaccharide of the soil bacterium did not precipitate with any of the lectins examined.Abbreviations Con A Concanavalin A - CPC cetylpyridinium chioride - EPS exopolysaccharide - FITC fluorescein isothiocyanate - KDO 2-keto-3-deoxyoctonic acid - LPS lipopolysaccharide - PBS phosphate-buffered saline - PS polysaccharide     

Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia

A study was conducted to determine whether colonization of legume roots and nodulation byRhizobium meliloti andBradyrhizobium japonicum could be enhanced by using inocula containing microorganisms that produce antibiotics suppressing soil or rhizosphere inhabitants but not the root-nodule bacteria. An antibiotic-producing strain of Pseudomonas and one of Bacillus were isolated, and mutants ofR. meliloti andB. japonicum sp. resistant to the antibiotics were used. The colonization of the alfalfa rhizosphere and nodulation byR. meliloti were enhanced by inoculation of soil withPseudomonas sp. in soil initially containing 2.7×10⁵ R. meliloti per g. The colonization of soybean roots byB. japonicum was enhanced by inoculating soil with three cell densities ofBacillus sp., and nodulation was stimulated byBacillus sp. added at two cell densities. In some tests, the dry weights of soybeans and seed yield increased as a result of these treatments, and co-inoculation with Bacillus also increased pod formation. Inoculation of seeds withBacillus sp. and the root-nodule bacterium enhanced nodulation of soybeans and alfalfa, but colonization byB. japonicum andR. meliloti was stimulated only during the early period of plant growth. Studies were also conducted withStreptomyces griseus and isolates ofR. meliloti andB. japonicum resistant to products of the actinomycete. Nodulation of alfalfa byR. meliloti was little or not affected by the actinomycete alone; however, both nodulation and colonization were enhanced if the soil was initially amended with chitin andS. griseus was also added. Chitin itself did not affectR. meliloti. Treatments of seeds with chitin orS. griseus alone did not enhance colonization of alfalfa roots byR. meliloti or soybean roots byB. japonicum, but the early colonization of the roots by both bacterial species was promoted if the seeds received both chitin andS. griseus; this treatment also increased nodulation and dry weights of alfalfa and soybeans and the N content of alfalfa. It is suggested that co-inoculation of legumes with antibiotic-producing microorganisms and root-nodule bacteria resistant to those antibiotics is a promising means of promoting nodulation and possibly nitrogen fixation.     

Growth and Movement of Spot Inoculated Rhizobium meliloti on the Root Surface of Alfalfa

Inoculum droplets of approximately 10 nanoliter volume and containing about 10 Rhizobium meliloti cells were placed onto the root surface of alfalfa seedlings in plastic growth pouches at either the root tip, the position of the smallest emergent root hairs, or at a site midway between these points. The droplets were initially confined to an area of about 0.2 square millimeter at the point of application. By 48 and 96 hours after inoculation, the inoculum bacteria and their progeny were distributed over several centimeters of the root between the initial site of deposition and the growing root tip, reaching densities of 10³ to 10⁴ bacteria per centimeter near the site of initial deposition and decreasing exponentially from that point toward the root tip. Graphite particles deposited on the root surface close to the growing tip were similarly distributed along the root length by 48 and 96 hours, suggesting that passive displacement by root cell elongation was primarily responsible for the spread of bacteria. A nonmotile mutant of R. meliloti colonized alfalfa roots to the same extent as the wild type and was usually distributed in the same manner, indicating that bacterial motility contributed little under these conditions to long distance spread of the bacteria. However, when applied in low numbers, R. meliloti mutants defective in motility or chemotaxis were considerably less efficient in initiating nodules near the point of inoculation than the wild type. This implies that motility and/or chemotaxis contribute significantly to local exploration for suitable infection sites. Almost all nodules on the primary root formed within a few millimeters of the spot-inoculation site, indicating that, under our experimental conditions, movement and multiplication of R. meliloti on the root surface were not sufficient to maintain an adequate population in the infectible region of the root during root growth.     

Effect of heterologous bacteria and combined nitrogen on the adsorption ofRhizobium meliloti to lucerne seedling roots

Summary The binding ofRhizobium meliloti strains A₂ (effective) and V₆ (ineffective),Agrobacterium tumefaciens strain B₆S₃ andR. trifolii strain TL₅ to lucerne seedling roots was studied by using¹⁴C or³H-labelled bacteria. When added singly or in combination with the heterologous bacteria, the number of A₂ cells attached to the roots was significantly less than the number of B₆S₃ or TL₅ cells. However, the presence of the heterologous bacteria did not decrease the proportion of A₂ cells added in the inoculum that bind to the roots, suggesting thatR. meliloti is attached to specific sites. In fact, the same number of A₂ or V₆ cells bind to the roots and in mixed inoculation the 2 strains share equally the binding sites. When added to the seedlings growth medium NO ₃ ^– at 5 or 16 mM significantly decreased the number of A₂ cells adhering to lucerne seedling roots. The results suggest that the lectin-recognition hypothesis is probably involved in the attachment ofR. meliloti to lucerne seedling roots.Contribution No. 252 Station de recherches, Agriculture Canada     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation

Co-inoculation with antibiotic-producing bacteria and rhizobia resistant to those antibiotics has been proposed as a means of promoting colonization and nodulation of legumes by root-nodule bacteria. A study was conducted to establish some of the factors affecting co-inoculation with antibiotic-producing strains of Bacillus and Streptomyces griseus. The stimulation of Rhizobium meliloti and yield and N uptake by alfalfa was enhanced with increasing inoculum size of Bacillus sp. S. griseus and chitin added to soil increased nodulation of soybeans by Bradyrhizobium japonicum and increased nodulation, yield, and number of pods on a second crop grown in the same soil. Bacillus sp. persisted in soil in sufficient numbers for at least 51 days to increase colonization of soybean roots by B. japonicum. The populations of S. griseus, Bacillus sp., and antibiotic-resistant isolates of R. meliloti and B. japonicum fell after their addition to seeds. Nevertheless, a benefical effect by the antibiotic-producing bacteria was evident on R. meliloti colonization of the rhizosphere, nodulation, and yield of alfalfa grown from seeds stored 94 days and on B. japonicum colonization, nodule number, yield, and seed weight of soybeans grown from seeds stored 90 days. Because non-antibiotic-producing derivatives of Bacillus sp. and S. griseus did not promote colonization or nodulation of alfalfa roots by R. meliloti, the benefit of this co-inoculation is a result of antibiotic formation.     

Survival of Rhizobium in Acid Soils

A Rhizobium strain nodulating cowpeas did not decline in abundance after it was added to sterile soils at pH 6.9 and 4.4, and the numbers fell slowly in nonsterile soils at pH 5.5 and 4.1. A strain of R. phaseoli grew when added to sterile soils at pH 6.7 and 6.9; it maintained large, stable populations in soils of pH 4.4, 5.5, and 6.0, but the numbers fell markedly and then reached a stable population size in sterile soils at pH 4.3 and 4.4. The abundance of R. phaseoli added to nonsterile soils with pH values of 4.3 to 6.7 decreased similarly with time regardless of soil acidity, and the final numbers were less than in the comparable sterile soils. The minimum pH values for the growth of strains of R. meliloti in liquid media ranged from 5.3 to 5.9. Two R. meliloti strains, which differed in acid tolerance for growth in culture, did not differ in numbers or decline when added to sterile soils at pH 4.8, 5.2, and 6.3. The population size of these two strains was reduced after they were introduced into nonsterile soils at pH 4.8, 5.4, and 6.4, and the number of survivors was related to the soil pH. The R. meliloti strain that was more acid sensitive in culture declined more readily in sterile soil at pH 4.6 than did the less sensitive strain, and only the former strain was eliminated from nonsterile soil at pH 4.8; however, the less sensitive strain also survived better in limed soil. The cell density of the two R. meliloti strains was increased in pH 6.4 soil in the presence of growing alfalfa. The decline and elimination of the tolerant, but not the sensitive, strain was delayed in soil at pH 4.6 by roots of growing alfalfa.     

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 M 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 M 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 M added iron) during the stationary phase of growth.Abbreviations CAS chrome azurol S - HDTMA hexadecyltrime-thylammonium bromide