Liquid Culture Production Research Articles

Inhibition of sporulation, glycopeptide antibiotic production and resistance in Streptomyces toyocaensis NRRL 15009 by protein kinase inhibitors

Production of the glycopeptide antibiotic A47934 by Streptomyces toyocaensis NRRL 15009 begins in the late exponential phase in liquid culture and peaks in the early stationary phase. The pattern of cellular phosphoprotein production changes upon onset of A48934 production with the appearance of several novel phosphoproteins only when an antibiotic is being produced. Phosphoamino acid analysis revealed that S. toyocaensis NRRL 15009 produces proteins phosphorylated on His, Ser, Thr and Tyr, with most being membrane-associated. Addition of the isoflavones genistein or quercetin abolishes A47934 production in liquid culture and sporulation on solid medium. Furthermore, genistein slows the onset of inducible glycopeptide antibiotic resistance in S. toyocaensis NRRL 15009. These results support the participation of protein kinase pathways in A47934 biosynthesis and resistance and cell differentiation in S. toyocaensis NRRL 15009.

Growth of Pseudomonas aureofaciens PGS12 and the Dynamics of HHL and Phenazine Production in Liquid Culture, on Nutrient Agar, and on Plant Roots.

The growth of Pseudomonas aureofaciens PGS12 was followed in nutrient broth (NB), on nutrient agar (NA), and on plant roots by monitoring cell numbers, the production of the autoinducer hexanoyl-homoserine lactone (HHL), and the antibiotic phenazine-1-carboxylic acid (PCA). In NB, as the growth rate declined in transition phase, HHL synthesis increased rapidly, shortly followed by PCA production. During stationary phase, HHL concentration declined rapidly while PCA concentration continued to increase slowly. The luxAB reporter genes were inserted in the phzB gene of the phenazine operon and phenazine transcriptional activity was monitored using measurement of luminescence. Levels and pattern of light output were similar to HHL accumulation and indicated that gene expression was maximal in transition phase and silenced in stationary phase. PCA production continued in stationary phase, suggesting that the protein products of the phenazine operon were maintained in the cell after down regulation. HHL accumulation was 60 times higher on NA than in NB per equivalent volume because of a 60-fold increase in cell density on NA. Higher levels of PCA per cell (6.8 times) and per equivalent volume (360-fold) accumulated in a colony compared to that found in broth. HHL remained at a high concentration in a colony for a longer period compared to a short burst in NB, and this may explain the increased PCA production. In contrast, on wheat seedlings and bean plant roots, bacterial growth was observed, but neither HHL nor PCA was detected; however, transcriptional activity of the phzB::luxAB reporter occurred on the bean plant roots.

Inhibition of sporulation, glycopeptide antibiotic production and resistance in Streptomyces toyocaensis NRRL 15009 by protein kinase inhibitors

Production of the glycopeptide antibiotic A47934 by Streptomyces toyocaensis NRRL 15009 begins in the late exponential phase in liquid culture and peaks in the early stationary phase. The pattern of cellular phosphoprotein production changes upon onset of A48934 production with the appearance of several novel phosphoproteins only when an antibiotic is being produced. Phosphoamino acid analysis revealed that S. toyocaensis NRRL 15009 produces proteins phosphorylated on His, Ser, Thr and Tyr, with most being membrane-associated. Addition of the isoflavones genistein or quercetin abolishes A47934 production in liquid culture and sporulation on solid medium. Furthermore, genistein slows the onset of inducible glycopeptide antibiotic resistance in S. toyocaensis NRRL 15009. These results support the participation of protein kinase pathways in A47934 biosynthesis and resistance and cell differentiation in S. toyocaensis NRRL 15009.

Mass Production Potential of the Bacto-Helminthic Biocontrol Complex Heterorhabditis indica - Photorhabdus luminescens

Heterorhabditis indica is a potential agent for the biological control of grubs in sugarcane fields in India. The type strain LN 2 was transferred to monoxenic cultures on its symbiont Photorhabdus luminescens and successfully produced on solid media. In liquid cultures, a mean dauer juvenile yield of 457 000 was obtained with a maximum of 648 000 per ml. Comparatively high yields have not been reported before. Therefore, costs related to the liquid culture production of H. indica will be lower than for other entomopathogenic nematodes currently used in biocontrol. Different bacterial clones had no significant influence on the dauer juvenile yields in liquid media. The exit from the dauer juvenile stage (recovery) after inoculation and the number of hermaphrodites significantly decreased when culture temperature was increased from 25-30 ° C; the dauer juvenile yields were not affected. The cell density of P. luminescens in batch cultures was higher at 25 and 30 ° C than at growth temperatures of 35 and 37 ° C. In continuous culture, the bacterial growth was inhibited when the growth temperature reached 38 ° C. After approximately 60 h, the bacteria adapted to higher temperature and the growth rate increased again. When the temperature was further increased to 40 ° C, the bacterial growth was inhibited.

Correlation between oxalic acid production and copper tolerance in Wolfiporia cocos

The increased interest in copper-based wood preservatives has hastened the need for understanding why some fungi are able to attack copper-treated wood. Due in part to accumulation of oxalic acid by brown-rot fungi and visualization of copper oxalate crystals in wood decayed by known copper-tolerant decay fungi, oxalic acid has been implicated in copper tolerance by the formation of copper oxalate crystals. Nineteen isolates of the brown-rot fungus Wolfiporia cocos were evaluated for oxalic acid production and weight loss on wood treated with 1.2% copper citrate. Twelve of 19 isolates that caused moderate to high weight losses in copper citrate-treated wood produced low oxalic acid in liquid culture, whereas isolates with high oxalic acid production had low weight losses in treated wood. Seven W. cocos isolates demonstrated enhanced weight loss in Cu-treated wood. Wood weight loss was unaffected by the presence of copper citrate for two W. cocos isolates and weight loss was lower for 10 isolates compared to weight losses in untreated wood. Citrate did not significantly influence oxalic acid production in liquid culture. Previous hypotheses linking oxalic acid and copper tolerance were based upon observations of single isolates of Postia and Tyromyces. Although most isolates produced more oxalic acid in copper citrate-treated wood than in untreated wood, we found no statistical relationship between the amount of oxalic acid production in liquid culture or wood and copper tolerance in W. cocos. Production of oxalic acid does not seem to be the factor controlling copper tolerance in W. cocos. The diversity seen within W. cocos demonstrates that caution should be used when reporting results, so that generalizations are not based on the behavior of a single isolate.

Potato Cultivar, Pathogen Isolate and Antagonist Cultivation Medium Influence the Efficacy and Ranking of Bacterial Antagonists of Fusarium Dry Rot

The process of selecting biological control agents for further development frequently does not involve conducting bioassays of strain effectiveness on a range of pathogen isolates or host cultivars. Additionally, though previous studies have demonstrated that the medium used to produce biomass of an antagonist can alter its efficacy, this factor is also rarely considered when selecting for the most effective antagonist. Host cultivar, pathogen isolate, and the cultivation medium used to produce the antagonists' biomass were examined as factors of potential importance for assessing the relative effectiveness of bacterial biocontrol strains accurately. Five bacterial antagonists that control Fusarium dry rot on stored potato tubers were assayed for effectiveness against 10 isolates of Gibberella pulicaris . All antagonists reduced disease severity (35-81%) regardless of the specific assays conducted. However, when the antagonists' biomass were produced on two media that differed both in nutrient composition and phase, the efficacy ranking of antagonist Enterobacter sp. S11:P:08 varied from first to fourth most effective. For the antagonists studied, the phase of a nutritionally identical medium had little impact on the efficacy ranking of the five antagonists. Four of the five antagonists had efficacy rankings that ranged from first to last depending on the isolate of the pathogen used to conduct the bioassay. The cultivar of the host also caused variations in the efficacy ranking of the antagonists. These results indicate that bioassays should be conducted using a range of liquid culture production media, pathogen isolates, and host cultivars in order to choose an antagonist that has the highest likelihood for commercial development as an effective biological control product.

Biological control of fusarium dry rot of potato tubers under commercial storage conditions

Lack of effective chemicals impedes control of Fusarium dry rot of stored potato tubers destined for processed and table stock use. Biological control of dry rot incited byGibberella pulicaris (anamorph=Fusarium sambucinum) has been demonstrated in laboratory studies but not in commercial storage environments. Several Gram-negative bacterial strains that were efficacious and amenable to production in liquid culture in laboratory studies were selected for pilot studies in Idaho and for bin trials at commercial storage houses in Idaho and North Dakota. In the first year of pilot studies,Pseudomonas fluorescens S22:T:04 (≈1 x 108 cfu/ml) decreased dry rot caused byG. pulicaris by 19% when coinoculated with the pathogen compared to controls and to the fungicide thiabendazole (TBZ) applied at label rates. In second year pilot studies,P. fluorescens P22:Y:05 andEnterobacter cloacae S11:T:07 (≈4 x 108 cfu/ml) reduced the severity of disease incited byG. pulicaris (25% and 17% average disease decrease, respectively) when antagonists were applied after pathogen inoculum. Treatment effects on dry rot that developed from inoculation withNectria haematococca (anamorph=Fusariumsolani var.coeruleum) were variable and influenced by interactions between antagonists, a wetting agent, and TBZ. In commercial storage bin trials,E. cloacae S11:T:07 reduced naturally occurring levels of dry rot by an average of 21% compared to 14% for TBZ, demonstrating that this antagonist was effective when produced using a liquid culture medium and methodology that approximates commercial practices for producing biomass.

Lipids of in Vivo and in Vitro Cultured Heterorhabditis bacteriophora

The lipid, sterol, and fatty acid compositions of Heterorhabditis bacteriophora infective juveniles reared by in vivo and in vitro methods (Galleria mellonella, Popillia japonica, solid and liquid culture) were compared. The greatest accumulation of lipids per dry weight was achieved by growing nematodes in P. japonica and solid culture (54.7 and 60.3%, respectively). Nematodes produced by liquid fermentation accumulated lower levels of sterols (2%) per total lipids compared with other production methods. However, the level of polar lipids was higher in nematodes produced in artificial media. Fractionation of the polar lipids revealed that an increased level of phosphatidylglycerol accompanied production in liquid culture. This was not observed for nematodes produced in solid culture in which the major polar lipid fractions were phosphatidylcholine and phosphatidylethanolamine. Lipids from nematodes produced in artificial media had more linoleic (C18: 2) fatty acids, whereas lipids from in vivo cultures had more stearic (C18: 0) fatty acids. The ratio of saturated to unsaturated fatty acid composition decreased in cultures produced in vitro. Artificial media composition should be adjusted to meet the nutritional composition of a natural host. Media for H. bacteriophora culture should be modified to boost deficiencies in sterols (e.g., liver extract) and oleic acid (e.g., olive oil).

POLYGALACTURONASE PRODUCTION BY ASPERGILLUS NIGER: EXPRESSION IN ONION SEEDS AND POSSIBLE INVOLVEMENT IN VIRULENCE

SUMMARY Nine Aspergillus niger isolates differing in virulence were assayed for polygalacturonase (PG) content and isoenzyme pattern in spores, liquid cultures and inoculated onion seeds of cultivar ‘Bianca Agostana’. The isolates differed in PG production quantitatively and in terms of isoenzyme pattern. Isolates An1 and An5, characterised by low PG production in liquid culture and low virulence, lacked detectable levels of three PG isoforms (PG3, PG4 and PG5). One acidic PG band (PG6) was detected only during growth in vitro by most of the isolates. Pectin lyase activity was not detected from spores, liquid cultures, or infected seeds. The ability of three isolates differing in virulence, An6, An7 and An14 to colonise five onion cultivars was also tested. The isolates differed in virulence and in quantitative and qualitative PG production. The results suggest that particular PGs from A. niger may contribute to virulence during onion seed colonisation.

Mycelial growth and production of oxalic acid by virulent and hypovirulent isolates of Sclerotinia sclerotiorum

Hypovirulent dsRNA-containing, and virulent dsRNA-free, isolates of Sclerotinia sclerotiorum were compared for mycelial growth and oxalic acid production in liquid culture. When harvested at 14 days post-inoculation, hypovirulenr isolates 91 and 215 produced less dry mycelium than virulent isolates 191 and 275C in potato-dextrose broth (PDB) and PDB amended with sodium succinate (PDBSS). All isolates produced more oxalic acid per gram dry mycelium When grown in PDBSS than in PDB. In a time course study, hypovirulent isolates 91 and 275 produced less mycelium (0.23 and 0.03 g dry wt.) and oxalic acid (0.22 and 0.01 mg/mL) than virulent isolates 191 and 275C (0.81 and 0.91 g dry wt., and 0.50 and 0.63 mg oxalic acid per millilitre, respectively) at 15 days post-inoculation. The amount of oxalic acid produced per gram dry mycelium reached a maximum for virulent isolates 191 and 275C at 3 and 5 days post-inoculation. Hypovirulent isolate 91 reached a maximum at 9 days post-inoculation, and hypovirulent isolate 275 was still increasing at 15 days post-inoculation. The mycelial dry weight of individual isolates was positively correlated with production of oxalic acid and inversely correlated with pH of culture filtrates at harvest. Three near-isogenic pairs of hypovirulent and virulent isolates were compared for mycelial growth and oxalic acid production in PDB and PDBSS. All hypovirulent isolates produced considerably less dry mycelium and oxalic acid than virulent isolates at 2 weeks post-inoculation. The mycelial growth of hypovirulent isolates was still less than the virulent isolates at 4 weeks post-inoculation, but the production of oxalic acid by two hypovirulent isolates increased to levels comparable to that of the virulent isolates at 2 weeks post-inoculation. The results are consistent with the hypothesis that dsRNA-associated hypovirulence in isolates of S. sclerotiorum was associated with reduced or delayed production of oxalic acid, or both, in comparison to virulent isolates, particularly during the first 3 to 7 days of growth.

Production of extracellular enzymes by two Pleurotus species using banana pseudostem biomass

AbstractThe degradation of lignocellulosic biomass of banana pseudoste was investigated during solid state fermentation (SSF) by P. ostreatus and P. sajor‐caju. Both organisms proved to be efficient degraders of banana pseudostem biomass. P.ostreatus degraded hemicellulose (40% of dry weight, d.w.) better than cellulose (17.5% of d.w.) and lignin (10% of d.w.). P. sajor‐caju also degraded hemicellulose (31% of d.w.) better than cellulose (12.4% of d.w.) and lignin (6% of d.w.). In both cases, a preferential removal of hemicellulose during the initial growth period and a delayed degradation of lignin were observed. The kinetics of cellulolytic, hemicellulolytic and lignolytic enzyme production in liquid culture were also examined. The activities of CMCase and β‐glucosidase were highest at 16 days of growth and avicelase activity was at its maximum after 24 days (CMCase ‐ 1.1 IU/ml, β‐glucosidase ‐ 0.09 IU/ml in the case of P. ostreatus; CMCase ‐ 1.0 IU/ml, β‐glucosidase ‐ 0.087 ‐ IU/ml in the case of P. sajor‐caju.). Xylanase and laccase activity reached their maximum after day 16 and day 24 of incubation, respectively. (Xylanase ‐ 1.1 IU/ml and laccase 3.0 IU/ml in the case of P. ostreatus; xylanase ‐ 1.0 IU/ml and laccase ‐ 3.6 IU/ml in the case of P. sajor‐caju.). The efficient degrading capacity of test fungi demonstrated their potential use in the conversion of banana pseudostem biomass into mycelial protein‐rich fermented animal feed.

Optimizing nutritional conditions for the liquid culture production of effective fungal biological control agents

Spores of fungal pathogens of weeds and insects are unique in their ability to actively infect and kill their pest host. While these capabilities are advantageous in terms of their use as a contact biological control agent, or biopesticide, they also require special consideration during spore production. Directed approaches to medium optimization must consider not only spore yield but also spore qualities such as desiccation tolerance, stability as a dry preparation, and biocontrol efficacy. Nutritional conditions during culture growth and sporulation should direct the accumulation of appropriate endogenous reserves so that newly formed spores possess these advantageous qualities. Studies with the bioherbicidal fungus Colletotrichum truncatum and with the bioinsecticidal fungus Paecilomyces fumosoroseus have demonstrated the impact of nutrition on spore ‘fitness’ for use as a biological control agent. The optimization strategy used in these nutritional studies as well as a comparison of the results are presented.

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron.

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.

Stimulation of early eosinophil progenitors by a heat stable alveolar macrophage product from ovalbumin‐sensitized and non‐sensitized guinea pigs

Alveolar macrophages (AM) may participate in brochopulmonary hyper-reactivity by secreting cytokines that recruit mature eosinophils, or induce eosinophil production from recruited circulating progenitors. To define whether AM products can contribute to lung eosinophil production in immunized guinea pigs (GP), by analysing the effect of AM culture supernatants (AM-SN) on in vitro eosinophilopoiesis. Liquid and semi-solid bone marrow (BM) cultures were seeded with SN from 95% pure AM exposed to LPS. AM-SN increased very significantly the long-term viability, cell proliferation and eosinophil production in liquid culture and supported formation of eosinophil-bearing mixed colonies, by acting on progenitors depleted of mature eosinophils. The effect on eosinophil production was not duplicated by natural or recombinant sources of GM-CSF (which nevertheless supported GM colony formation by GP BM), not by rhIL-8 (which was active on GP cells) and was not due to residual LPS. FPLC separation of active AM SN yielded a peak of apparent m.w. 43 kDa, active on both liquid and semi-solid cultures. The active moiety was heat- and trypsin-resistant. Neutralizing monoclonal antibodies to hGM-CSF, mGM-CSF, hIL-3 and mIL-3 failed to deplete the activity in AM-SN. Ovalbumin immunization induced its production by AM even without LPS challenge. The lack of T lymphocytes among factor-producing AM, the properties of the active material, the inability of GM-CSF to reproduce these effects, and the failure of MoAbs to GM-CSF and to IL-3 to neutralize the activity indicate it is not due to the major eosinopoietic factors GM-CSF, IL-3 or IL-5.

Liquid culture production of microsclerotia of Colletotrichum truncatum for use as bioherbicidal propagules

Colletotrichum truncatum NRRL 18434 is a host-specific pathogen of hemp sesbania (Sesbania exaltata), a weed in cotton, soybeans and rice. The bioherbicidal potential of C. truncatum microsclerotia (MS) was assessed based on the feasibility of producing and storing microsclerotia and the efficacy of microsclerotia in controlling hemp sesbania. Microsclerotia were produced in highly aerated liquid cultures grown at 28 °C in a basal salts medium with 80 g glucose and 13·2 g Casamino acids l−1. Hyphal aggregation to form microsclerotial initials began on day 4 with maximum concentrations (6 × 106 MS l−1) being attained by day 6. Melanization started on day 6 and was completed by day 10. After air-drying at 22°, microsclerotia: diatomaceous earth preparations were stored at 4°. After storage for 7 months 90% of the dried C. truncatum MS germinated. Efficacy studies showed that the incorporation of either fresh or 1-month-old microsclerotia into potting soil (156 microsclerotia cm−3 soil) killed more than 95% of hemp sesbania seedlings after emergence. Microsclerotia-infested potting mix incited disease of a similar magnitude in a second crop of hemp sesbania seedlings planted 28 d after the first. These studies demonstrate that C. truncatum microsclerotia may have potential for use as an alternative to conidia for biocontrol since they can be produced in submerged culture, remain viable as a dry formulation and effectively control hemp sesbania seedlings when incorporated into potting medium prior to seeding.

Non-fragmenting variants of Streptomyces hyphae have enhanced activity of an enzyme (phospho- N-acetylmuramyl pentapeptide translocase) in peptidoglycan biosynthesis

The repeated use of an enrichment technique for establishment of antibiotic production in liquid culture, where previously production had only been seen on agar, resulted in the selection of stable strain variants with enhanced resistance to fragmentation. The variant was more resistant to tunicamycin, an inhibitor of peptidoglycan biosynthesis, than the wild-type. When peptidoglycan synthesis was compared in the variant and the wild-type strains of one of the isolates, enhanced activity of the enzyme: phospho- N-acetylmuramyl pentapeptide translocase (which catalyses the first membrane bound reaction of cell wall synthesis) was found in the fragmentation-resistant form.

Chitinase and laminarinase production in liquid culture by Trichoderma spp. and their role in biocontrol of wood decay fungi

This paper describes macro- and microassay methods to assess the production of the lytic enzymes laminarinase and chitinase, by a range of Trichoderma isolates, and investigates the effect of nutrient composition, glucose amendment and the addition of basidiomycete cell wall material on the production of these lytic enzymes. Large interstrain and interspecies differences exist in the levels of production of both enzymes. Total activities of the enzymes are greater when isolates are cultured in malt medium, but specific chitinase and laminarinase activities are higher in low nutrient conditions. Glucose repressed the production of both laminarinase and chitinase, although this effect was not common to all Trichoderma isolates for the latter enzyme. Cell walls of both N. lepideus and T. versicolor induced increased chitinase activity but this effect was Trichoderma species specific. Mycoparasitism involving lytic enzymes has been described as the mechanism of action of Trichoderma species in the biological control of commercially important plant pathogens. However, little information is available on the significance of this mechanism for the biological control of wood decay fungi. The importance of mycoparasitism as a mechanism in the biological control of wood decay fungi is briefly discussed.

Strain Variation in Tolerance of Water Stress by Idriella(Microdochium)bolleyi, a Biocontrol Agent of Cereal Root and Stem Base Pathogens

Three randomly chosen isolates of Idriella bolleyi differed markedly in tolerance of water stress down to—5 MPa in vitro. The differences were seen with respect to osmotic potential of media adjusted with KCl or matric potential of media adjusted with polyethylene glycol 8000. They were consistent when assessed by linear extension of colonies, spore germination, biomass production in liquid culture and sporulation in liquid culture. In comparative tests, one strain of I. bolleyi showed similar stress tolerance to that oF Fusarium culmorum, noted as a pathogen of cereals in dry conditions, whereas another strain of I. bolleyi showed low tolerance equivalent to that of the take‐all fungus Gaeumannomyces graminis and a Phialophora sp. from maize. The extreme variation in water stress tolerance of I. bolleyi might be used to select strains for biocontrol of different cereal root‐ and foot‐rot pathogens or strains might be combined in seed‐applied inocula for consistency of biocontrol in different site or seasonal conditions.

A method for increasing the success rate of duplicating antibiotic activity in agar and liquid cultures of Streptomyces isolates in new antibiotic screens

During screens for novel secondary metabolite natural products, actinomycete isolates are often encountered which show antibiotic activity on agar but not in liquid culture. In this study, it was found that the liquid culture morphology of Streptomyces species, producing antibiotic on solid media only, was fragmented compared with that of the filamentous mycelia of antibiotic-producing Streptomyces. An enrichment culture method which selected for a more filamentous liquid culture morphology was successful in providing active liquid cultures of the isolates which had previously been active only in agar culture. After repeating the selection procedure five times with several cultures, two stable phenotypes with antibiotic activity in liquid culture and a filamentous morphology were obtained. The correlation of a filamentous morphology with antibiotic production in liquid culture is discussed.

Physiological characteristics ofRhizobium meliloti1021 Tn5 mutants with altered rhizobactin activities

Abstract Rhizobium meliloti 1021 and several Tn5 generated mutants were examined for physiological activities which would reflect the capacity of these strains for efficient iron metabolism. Rhizobactin production in liquid culture, as measured by CAS reactivity, was greatest in R. meliloti 1021 with lesser amounts formed by strains PRR 63 and PRR 62. Examination of the various strains of R meliloti grown under different levels of iron revealed that all strains tested were constitutive for ferric reductase. Evaluation of Medicago saliva L. cultivated in peat pots and inoculated with R meliloti revealed that greatest levels of growth was with R meliloti 1021, the only strain in this study with an efficient rhizobactin system. Evaluation of dinitrogen fixed by nodulated plants grown in a hydroponic system indicated that the amount of nitrogen fixed per bacteroid in the nodule was not constant but was correlated with the rhizobactin producing capability of the R. meliloti strains. Levels of dinitrogen fixed ...