Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

Influence of Phosphate on the Growth and Nodulation Characteristics of Rhizobium trifolii

The growth and nodulating characteristics of Rhizobium trifolii 6 and 36 differed under different external phosphate conditions. Under growth conditions designed to deplete the internal phosphate content of the rhizobia, strain 6 maintained a generation time of 5 h during the exponential phase over two cycles of growth in phosphate-depleted medium. In contrast, the generation time of strain 36 was extended from 3.5 to 9.8 h over two cycles of phosphate-depleted growth, although the organism eventually achieved the same cell density and cellular phosphate content as that of strain 6 at stationary phase. Phosphate-depleted strain 6 required 0.51 ± 0.08 μM phosphate to commence proliferation, whereas phosphate-depleted strain 36 required 0.89 ± 0.04 μM phosphate under the same conditions. Phosphate-depleted strain 6 maintained viability when exposed to external phosphate concentrations subcritical for growth to occur, whereas phosphate-depleted strain 36 lost viability within 48 h when exposed to medium containing phosphate at concentrations subcritical for growth. Phosphate-depleted strain 36 was inferior to phosphate-depleted strain 6 at nodulating subterranean clover (Trifolium subterraneum L. cv. Mt. Barker) by taking 2 to 4 days longer to develop nodules in phosphatedepleted plant grown medium at pH 5.5. Nodulation by phosphate-depleted strain 36 was accelerated either by including phosphate in the plant growth medium at pH 5.5 or by raising the solution pH of phosphate-depleted plant growth medium to pH 6.5. External phosphate and pH effects were not observed on the nodulating capabilities of phosphate-depleted strain 6 or on luxury phosphate-grown cells of either strain. Phosphatedepleted strains 6 and 36 proliferated to a similar extent on the rhizoplanes even under stringently low external P_i concentrations. The phosphatase activities of both phosphate-depleted strains were significantly (P = 0.05) higher at pH 6.5 than at pH 5.5, and the activity of strain 6 was significantly higher (P = 0.05) than that of strain 36 at pH 5.5 and 5.0.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Influence of Lime and Phosphate on Nodulation of Soil-Grown Trifolium subterraneum L. by Indigenous Rhizobium trifolii

Previous research had identified four serogroups of Rhizobium trifolii indigenous to the acidic Abiqua soil (fine, mixed, mesic Cumulic Ultic Haploxeroll). Nodulation of subterranean clover (Trifolium subterraneum L.) by two of the serogroups, 6 and 36, was differentially influenced by an application of CaCO₃ which raised the pH of the soil from 5.0 to 6.5. These studies were designed to characterize this phenomenon more comprehensively. Liming the soil with either CaCO₃, Ca(OH)₂, MgO, or K₂CO₃ significantly (P = 0.05) increased the percent nodule occupancy by serogroup 36, whereas the percent nodule occupancy by serogroup 6 was decreased, but the decrease was significant (P = 0.05) only after application of either CaCO₃ or Ca(OH)₂. Application of KH₂PO₄ (25 mg of P kg of soil⁻¹), which did not change soil pH, also significantly (P = 0.05) increased the percent nodule occupancy by serogroup 36. Application of KH₂PO₄ in combination with Ca(OH)₂ produced the same increase in nodule occupancy by serogroup 36 as did individual application of the two materials. Soil populations of serogroup 36 consistently, and in the majority of cases significantly (P = 0.05), outnumbered those of serogroup 6 before planting and after harvest regardless of soil treatment or the outcome of nodulation. Soil chemical and plant analyses provided no evidence that liming was simulating phosphate addition by increasing the availability and subsequent uptake of soil P_i by the subclover plants. Liming did, however, result in a significant transformation (30 to 50 mg of P kg of soil⁻¹) of P_i from the residual soil P_i fraction into an NaOH-extractable organic P fraction during the preplant equilibration period.     

Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

Summary Three distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.     

Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover

Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.     

Nodulation of Trifolium repens at low pH by single and paired strains of Rhizobium leguminosarum biovar trifolii

Detailed individual nodulation profiles were obtained for five strains of Rhizobium leguminosarum biovar trifolii inoculated onto roots of Trifolium repens seedlings growing on an agar medium of pH 4.5. The time of appearance and the location of every nodule were noted for a period of 10 days after inoculation. Using these nodulation frequency profiles, pairings of strains were identified and six mixed-strain inoculation (1:1 ratio) experiments were subsequently performed at pH 4.5. Results from the mixed-inoculum experiments showed that the performance of a Rhizobium strain in single culture could not be reliably used to predict the outcome of a paired-inoculation study and that some seedlings were exclusively nodulated by rhizobia that performed poorly at low pH in single-culture inoculations. Received: 26 November 1996 / Accepted: 18 April 1997     

Influence of Azospirillum Strains on the Nodulation of Clovers by Rhizobium Strains

Mixed cultures of several Azospirillum and Rhizobium trifolii strains caused either an inhibition or stimulation of nodule formation on plant hosts as compared with nodulation of plants inoculated with R. trifolii alone. Azospirillum strains affected the nodulation process at a precise cell ratio (R. trifolii/Azospirillum cells) and time of inoculation. All Azospirillum strains used showed a variation in their ability to inhibit or enhance nodulation by R. trifolii strains. When nonviable cell preparations of Azospirillum strains were used for mixing experiments, no effect on nodulation was observed. A decrease in the effectiveness of normally Nod⁺ Fix⁺R. trifolii strains was observed when an Azospirillum strain caused an increase in nodule number.     

Studies on Acid Tolerance of Rhizobium trifolii in Culture and Soil

Several experiments were carried out in an attempt to isolate and adapt strains of Rhizobium trifolii to growth at low pH on citrate-phosphate buffered yeast-extract mannitol agar (CPYEM). In another experiment, two streptomycin resistant strains (7A str^r and 16 str^r ) with contrasting tolerances to acidity in culture were evaluated for survival in acid upland soils.
No acid tolerant clones were isolated from three strains on CPYEM suggesting that there were no cells with greater tolerance of low pH present. The stepwise subculture of several strains on media of decreasing pH also failed to increase their acid tolerance. Growth initiation on CPYEM at pH 4–4 was a function of the number of viable cells in the inoculum and was accompanied by a rise in pH.
In three acid soils, strain 7A str^r was more persistant than 16 str^r , whereas in culture the converse was true. Survival of these strains in the acid soils was inversely related to acid production on the medium of Norris (1965) in agreement with Norris's hypothesis. Ameliorating soil pH with lime improved the persistency of both strains. Prior exposure of 7A str^r and 16 str^r to the acid soils failed to improve subsequent survival in these soils.     

Polyol metabolism by Rhizobium trifolii.

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.     

Nodulation of Trifolium repens with modified Bradyrhizobium and the nodulation of Parasponia with Rhizobium leguminosarum biovar trifolii

Thirty one strains of Rhizobium leguminosarum biovar trifolii isolated from the North and South American continents, New Guinea, USSR, Turkey and Australia, nodulated P. andersonii ineffectively when grown in plant growth tubes and in Leonard jars. Nodules were slow to form, sometimes taking over 100 days. Reisolates of R. leguminosarum biovar trifolii from P. andersonii nodulated Trifolium repens and their identity was confirmed using serological techniques. Dual occupation of nodules by Rhizobium and Bradyrhizobium in P. andersonii is reported. The reduced effectiveness of the Bradyrhizobium symbiosis depended on the relative numbers of Rhizobium occupants in this dual system. R. leguminosarum biovar trifolii and Bradyrhizobium strains from Parasponia were able to co-exist in nodules on P. andersonii and maintain similar populations in the rhizosphere and on culture media. Bradyrhizobium strains, separated from R. leguminosarum biovar trifolii, were able to initiate and form nodule-like structures on T. repens. Bradyrhizobium bacteria were identified as the sole occupants of the cells of the nodule-like structures on Trifolium repens using an immunogold labelling technique applied to ultrathin sectins. The re-isolates of Bradyrhizobium obtained from these nodule-like structures on T. repens were able to effectively nodulate P. andersonii.     

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.     

Production of Antimicrobial and Bacteriocin-Like Substances by Rhizobium trifolii

Rhizobium trifolii strains IARI and Rel-1 produced substances with broad and narrow activity spectra, respectively. Reproducible inhibitory zones of various sizes produced by R. trifolii IARI (2 to 14 mm) and R. trifolii Rel-1 (2 to 6 mm) were detected, depending upon the indicator organism used. The maximum production of these substances by both strains of R. trifolii was observed on l-arabinose agar. A preliminary characterization of the antimicrobial substance produced by strain IARI showed resistance to heat (75 to 80 degrees C for 45 min), trypsin, lysozyme, DNase I, and RNase A. On the other hand, the substance produced by strain Rel-1 showed sensitivity to heat (75 to 80 degrees C for 45 min) and trypsin, but resistance to lysozyme, RNase A, and DNase I.     

Bacteriocinogeny of Rhizobium trifolii.

Rhizobium trifolii strains differing in cell and colony morphology, streptomycin resistance, phage sensitivity pattern and infectivity to clover plants produced bacteriocins sensitive to proteases. Elimination of bacteriocin production ability wtih SDS and rifampicin treatment indicates that this feature is plasmid controlled. Elimination of bacteriocinogenic plasmid did not influence other features of R. trifolii.