Symbiosis of Trifolium subterraneum with mycorrhizal fungi and Rhizobium trifolii as affected by ammonium sulphate and nitrification inhibitors

The symbioses between Trifolium subterraneum, mycorrhizal fungi and Rhizohium are affected by (NH₄)₂SO₄ and by the nitrification inhibitors 2-chloro-6 (trichloromethyl) pyridine (N-Serve) and 2-trichloromethyl pyridine (2TMP). At 50 μg · g^?1 soil N-Serve and 2TMP had toxic effects on plant growth, measured as leaf expansion, root length and dry weight. Lower concentrations of N-Serve also produced some toxic symptoms. The addition of (NH₄)₂SO₄ to the soil at 2 and 6 m-equiv NH⁺₄ per pot, resulted in reduced root length and nodulation. Shoot dry weight was reduced at 6 m-equiv NH⁺₄ per pot. In the presence of (NH₄)₂SO₄ the toxic effects of the nitrification inhibitors on plant growth were less.Both nitrification inhibitors reduced development of mycorrhizal entry-points and extent of root colonization (% infection). Percentage infection of the root system was also reduced by (NH₄)₂SO₄. Development of nodules on the lateral roots was increased in the presence of N-Serve at 5 and 15 μ · g^?1. This effect, however, was accompanied by a marked reduction in N₂ase activity. Smaller increases in nodulation were apparent with 2TMP and were associated with variable N₂ase activity.     

Effect of strains of Rhizobium trifolii on the establishment of oversown white clover (Trifolium repens)

Strains of Rhizobium trifolii incorporated into commercial peat inoculants were compared for their effect on the establishment and growth of oversown white clover (Trifolium repens) on soils devoid of infective rhizobia.There were marked differences in numbers of seedlings establishing and clover dry matter production per hectare with the various strains. However, when adjusted to a constant number of established seedlings, dry matter production from all strains, apart from one strain at one site, were similar indicating that the strains did not appear to influence the growth of individual clover plants.The marked differences in establishment of clover inoculated with the various strains could not be accounted for by differences in the number of rhizobia in the peat inoculant.Selecting strains of rhizobia for ability to increase establishment is considered important where clover is oversown onto soils devoid of rhizobia.     

Nodulation of Trifolium subterraneum by introduced rhizobia in competition with naturalized strains

The ability of 4 strains of Rhizobium trifolii to compete with naturalized strains in nodulating Trifolium subterruneum cv. Mt Barker and cv. Woogenellup was assessed at 5 sites in New South Wales. The populations of naturalized rhizobia at these sites ranged from 4 × 10⁶ rhizobia/g to one where no rhizobia were detected. The introduced strains were inoculated singly or as mixed strain inocula onto seed of the host at 2 × 10⁶ rhizobia/seed. There were marked differences in competitive ability between the strains but these differences were modified by the host cultivar and the site.At the R. trifolii-free site the inoculum strain formed 100% of the nodules in the 1st yr; by the second year serologically unrelated strains had invaded the plots and these formed almost all of the nodules in the 3rd yr. At the site where competition was greatest (4 × 10⁶ naturalized rhizobia/g), there were no differences in the competitive abilities of the strains in the first year but at all other sites WU95 was superior whether used as a single strain or in a mixed strain inoculum. In these sites also the proportion of nodules formed by the inoculum strains declined markedly by the 2nd yr.     

Competitive advantage of bacteriocin and phage-producing strains of Rhizobium trifolii in mixed culture

Bacteriocinogenic and lysogenic strains of Rhizobium trifolii were grown with sensitive strains in sterile broth or peat culture. In broth culture the producing strains strongly suppressed growth of sensitive strains. In peat culture a considerable degree of suppression was also observed, the effect being stronger when the peat was “wet” than when it was “damp”. Apparently, conditions which favour growth in peat also favour dominance by bacteriocinogenic or lysogenic bacteria in cultures of mixed strains. The results are discussed in relation to preparation of mixed-strain legume inoculants and to studies of competition between strains in the field environment.     

Soil acidity and the composition of an indigenous population of Rhizobium tripolii in nodules of different cultivars of Trifolium subterraneum L.

Three methods of strain identification were used to determine the composition of the Rhizobium trifolii population in nodules formed on four cultivars of Trifolium subterraneum L. grown in a soil unamended (pH 4.8) or amended with CaCO₃ (pH 6.4). Whole cell somatic tube agglutination placed 68–90% of the nodule occupants from each of the cultivars Mt Barker, Nangeela and Howard into four identifiable serogroups. Only in the case of cultivar Woogeneliup were the majority of the isolates unidentifiable with the antisera at our disposal. In unamended soil different serogroups dominated each cultivar whereas in limed soil serogroup 27 was the dominant identifiable serogroup on all of the cultivars. The latter serogroup was also the most promiscuous nodule occupant being found in seven of eight treatments. Gel-immune-diffusion analysis revealed three serotypes within serogroup 27; serotype 27-A was dominant in the nodules on cultivar Nangeela alone whereas serotype 27-B was dominant on both cultivars Mt Barker and Woogenellup. Separation of cellular proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed four and five different strains within serotypes 27-A and 27-B respectively. Only one strain from serotype 27-B could be considered a common nodule occupant on cultivars Mt Barker and Woogenellup. Two of three strains, representing 82% of the total isolates of serotype 27-B found on cultivar Mt Barker, were nodule occupants of that cultivar in both soil treatments. In contrast, only one of four strains, representing 38.5% of the total isolates of serotype 27-A found on cultivar Nangeela, could be considered a common nodule occupant in both soil treatments.     

Polysaccharides and lipopolysaccharides and infectivity of Rhizobium trifolii

The extracellular polysaccharides obtained from infective and noninfective strains of Rhizobium trifolii were analysed to investigate if infective properties were correlated with polysaccharide composition. The results indicate that soluble extracellular polysaccharides were not a determinant factor in infectivity. However, electrophoresis of bacterial surface lipopolysaccharides showed a relation between the loss of infectivity and changes in electrophoretic mobility.     

Establishment and persistence of Rhizobium trifolii in western australian soils

Large scale experiments with inoculated and drill sown Trifolium subterraneum, T. hirtum, and T. cherleri showed that recent isolates of Rhizobium trifolii from healthy plants in problem pastures were superior to the strains used in commercially available inoculants. The new rhizobia are also shown to persist in the soil longer than the commercial strains. Evidence was obtained of different levels of performance by R. trifolii strains on different soils. Following the inclusion of one of the superior isolates in commercial peat inoculants, a number of farmer-sown pastures were examined for strain persistence. The new isolates showed much improved persistence over the older inoculant strains.     

The nature of non-protein nitrogen accumulation in Trifolium subterraneum root nodules

Non-protein nitrogen accumulated in nodules formed on Trifolium subterraneum cv. Tallarook by Rhizobium trifolii strain NA30, but not in nodules formed by strain TA1. Studies with six R. trifolii strains and four T. subterraneum cultivars indicated that the accumulation of non-protein nitrogen was a characteristic of certain strains and that it was accompanied by a greater development of nodule tissue. With normal symbiotic associations, approximately 6 per cent of the total plant nitrogen was located in the nodule system whereas nodules accumulating non-protein nitrogen contained, on average, 12 per cent of the total nitrogen in the plant.The principal component of the accumulating non-protein nitrogen was identified as “bound” γ-aminobutyric acid. Moderate to high concentrations of γ-aminobutyric acid (0.3–1.7 mmoles/g nodule dry weight) were found in nodules formed by 10 strains (of 36 examined) on Tallarook. With two “accumulating” strains, higher concentrations of γ-aminobutyric acid were found in nodules formed on the cultivar Clare (2.0 mmoles/g nodule dry weight) than in nodules formed on Tallarook or Yarloop (1 1.4 mmoles). No γ-aminobutyric acid was found in cultured cells of either an accumulating strain (NA30) or a nonaccumulating strain (TA1) of R. trifolii.The accumulation of non-protein nitrogen as γ-aminobutyric acid, and the accompanying increase in nodule tissue, each resulting in the export of a lower proportion of the nitrogen fixed to the host, is considered to be a factor causing a lower degree of symbiotic effectiveness.     

Soil temperature,mycorrhizal infection and nodulation of Medicago truncatula and Trifolium subterraneum

Establishment of vesicular-arbuscular mycorrhizal fungi in plant roots involves a pre-infection phase of propagule germination, hyphal growth and appressorium formation, followed by growth of the fungus within the root. The effect of soil temperature on the pre-infection stage was examined by counting the numbers of fungal “entry-points” on the main roots of Medicago truncatula and Trifolium subterraneum, grown at soil temperatures of 12°, 16°, 20° and 25°C for periods up to 12 days. Increased root temperature was positively associated with increased numbers of “entry-points”. This effect was more marked between 12° and 16°C than at higher temperatures, as shown by comparing plants at the same stage of development (emergence of spade leaf) and by calculating the results as entry points per cm root.The first root nodules appeared sooner at higher temperatures (20° and 25°), but subsequent development of nodules (measured as nodule number and aggregate volume of nodules per plant, up to 21 days) was best at 16°C for both host Rhizobium combinations in non-sterile and autoclaved soil. There was no evidence that competition between mycorrhizal fungi and Rhizobium for infection sites occurred.A method of obtaining numbers of infective propagules of vesicular-arbuscular mycorrhizal fungi in soil is described.     

Studies of field populations of rhizobium: Effectiveness of strains of rhizobium trifolii associated with trifolium subterraneum L. pastures in South-Eastern Australia

Field populations of Rhizobiuin trifollii from eight regions in south-eastern Australia were sampled over a period of 5 years from 1966 to 1970. The R. trifolii isolates were tested under bacteriologically-and environmentally-controlled conditions for effectiveness of nitrogen fixation in combination with Trifalium subterraneum L. cv. Bacchus Marsh; the effective strain TA1 was used as a standard of comparison.Mean effectiveness of the R. trifolii populations for any region at any sampling varied between 62 and 93 percent of the effectiveness of the standard strain. The principal feature was the large variability within sampling sites, between sites within paddocks, and between paddocks within regions. In addition there was some variability with time in the range of effectiveness values of isolates within a site and in the absolute values for both sites and paddocks. Effectiveness values were not related to soil pH, size of population of R. trifolii, inoculation procedure at sowing, age of pasture, annual rate of fertilizer application, or mean annual rainfall.     

Aluminium toxicity and multiplication of Rhizobium trifolii in a defined growth medium

The responses of six strains of R. trifolii to pH, Ca, Al and phosphate were studied in the defined medium of Munns and Keyser (1981) supplemented with vitamins. Wild-type strains responded in the same way as rifampicin-resistant mutant strains. Multiplication was inhibited by acidity alone at pH4.3, and by 50 μM Al at pH 5.5 (despite the precipitation of Al). This inhibitory effect of Al was overcome by increasing the pH above 6.0, or by increasing the phosphate concentration from 10 to 100 μm. Ca concentration had no effect, nor did holding the medium at 22°C for 36 weeks before inoculation. There was variation between strains in their responses to concentrations of A1 < 50 μM. These responses indicate that the defined medium is a satisfactory model for the white clover rhizosphere.Results from these studies and from chemical analyses indicate that the inhibitory effect of Al under partially-neutralized conditions is not due to a direct effect of monomeric A1, nor to an indirect effect of polymeric A1 in reducing the concentration of phosphate, but is due to a direct effect of polymeric A1.     

Desiccation tolerance of four strains of Rhizobium japonicum

The survival of different strains of Rhizobium japonicum was evaluated in three soils with two matric- and three osmotic-induced moisture potentials. Both drying and added NaCl significantly decreased populations. Strains CC 709 and USDA 110 were less affected by the matric- or osmotic-induced desiccation than strains CB 1809 and USDA 123. The survival of CC 709 and USDA 110 at 2 weeks with 0.7% added NaCI was 33 and 46% of initial counts in soils undergoing drying, and 70 and 69% in soils maintained at the 30kPa (0.3 bar) potential. Comparable survivals of CB 1809 and USDA 123 were 15 and 18% and 56 and 59%, respectively. The soil with the greatest clay and organic C contents maintained the highest populations during desiccation. Turbidity measurements indicated similar rates of growth of the four strains at a water activity (A_w) of 0.999 in yeast mannitol broth (YMB). When the YMB was adjusted with glycerol to lower water activities, strains CC 709 and USDA 110 consistently showed greater growth than did strains CB 1809 and USDA 123. No growth of CB 1809 and USDA 123 was observed at an (A_w) of 0.975. Water adsorption isotherms of freeze-dried cells showed that the more desiccation-susceptible strains (CC 709 and USDA 110) retained greater amounts of water at a given relative vapor pressure than did the two more tolerant strains.     

Variation in the selection of Rhizobium trifolii by varieties of red and white clover

The selection of Rhizobium trifolii by six varieties of red and white clover was examined under laboratory conditions. The varieties differed in their selection of strains and there was considerable variation within any one variety. The implications of such variation are discussed with particular reference to clover seed inoculation.     

A comparison of competitiveness and persistence amongst five strains of Rhizobium tripolii

Five strains of Rhizohium trifolii were used to inoculate Trifolium subterraneum cv. Woogenellup sown into two soils with naturally-occurring populations of R. trifolii. In the 1st year all inoculant strains used singly were present at high frequency in the sampled nodule populations from the inoculated plots. Where an inoculant containing a mixture of equal parts of the 5 strains was used. one strain (WU95) predominated at both sites.The persistence of the strains was followed for a further 3 years at one site. Three of the strains WU95, CC2480a and WU290, were maintained at a high frequency (>75% of nodules sampled) for the entire period, but the other two strains showed poor persistence in this environment. Highly effective strains of rhizobia, not identifiable as inoculant strains, nor present in the nodule population at the first sampling, appeared during the course of the study. One strain. WU290, showed a high degree of variation in symbiotic effectiveness between single colony isolates from the stock culture and also between field isolates that were serologically identical with this strain.     

Competition for nodule formation between effective and ineffective strains of Rhizobium meliloti

Ineffective mutants of four effective strains of Rhizobium meliloti were isolated and tested for their ability to compete with effective parents or antibiotic resistant mutants in the formation of nodules on Medicago sativa. In 5 out of the 6 cases studied, ineffective mutants were no different to effective strains of the same origin in their competitive ability. A difference in selection for infection by the host plant was observed between equally effective strains as well as between ineffective strains. Except for one pair of strains, the more-competitive effective strains (resistant or not to antibiotics) had the same origin as the more-competitive ineffective strains. For such strains the ability to compete with other strains to form nodules was a characteristic of each parent strain. Competitiveness was independent of effectiveness and had been retained during mutation.     

Competitiveness and effectiveness of strains of Rhizobium phaseoli isolated from the sonoran desert

Four strains of Rhizobium phaseoli were examined for N₂ fixation effectiveness and for competitiveness for nodule occupancy by utilizing strain-specific fluorescent antibodies. Competition studies in Leonard jars held in a growth chamber showed strain KIM-5 (a cool season isolate from Kimberly, Idaho) consistently occupied the majority of nodules on bean plants (Phaseolus vulgaris L.) cv. Kentucky Wonder, when applied as a mixed inoculant with desert strains (K-1, 36 or 90). Competitiveness of KIM-5 was relatively independent of cell numbers as shown by the high recovery of KIM-5 from nodules, even when extensively outnumbered in the inoculant. KIM-5 out-competed the desert strains regardless of whether they were ineffective (strains 36 and 90) or highly effective (K-1). Although KIM-5 was more competitive than K-l, no difference in infectiveness (as shown by nodule mass) or effectiveness (as shown by % N, total plant N, C₂H₂ reduction and total plant weight) was observed.In YEM broth, strain K-l showed increasing growth rates when the temperature was increased from 27° to 35°C, and was viable at 40°C. These data indicate K-1 to be an unusually heat-tolerant strain. Growth rates of KIM-5 were constant from 27° to 35°C and the organism was not viable at 40°C. Both strains produced acid in a defined broth medium.The effectiveness of KIM-5 and K-l was also evaluated under field conditions using single strain inoculants with two cultivars of pinto beans (P. vulgaris L.) ev. Mexicali 80 and Delicias 71. Inoculation with K-1 resulted in yield increases with both cultivars over uninoculated plants, whereas there was little difference between KIM-5 inoculated and uninoculated plants.     

Soil microorganisms antagonistic towards Rhizobium japonicum

Success in introducing Rhizobium japonicum strains into soil is related to their interaction with native microorganisms including some that are antagonistic. Actinomycetes, bacteria, fungi and rhizobiophages antagonistic towards strains of R. japonicum were counted directly using soil samples from field plots under different crop and soil management systems. The antagonistic actinomycete population varied from 1.3 × 10³ to 4.5 × 10⁵ g^?1 dry soil and ranged up to 90% of total actinomycetes. Soybean rhizosphere soil samples included antagonistic actinomycetes ranging up to 70% of total actinomycetes. The antagonistic bacterial population was less than 10% of total bacteria and the proportion did not vary significantly with crop or soil management practices. Antagonistic fungi were observed for many of the soils examined but they could not be counted. There were few rhizobiophages and they were found most frequently in soybean rhizospheres. Occasional bacterial and actinomycete colonies that stimulated growth of R. japonicum were randomly observed among the soil samples tested.     

Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N₂ fixation.     

Survival of Rhizobium japonicum in soil-sludge environment

The usage of sewage sludge on agricultural lands is an effective and inexpensive practice that provides nutrients for crops. A successful legume crop also depends on the survival of Rhizobium in the soil environment. The number of R. japonicum (USDA 110) in treatment groups containing various soil-to-sludge ratios (control, 13:1, 9:1 and 5:1) during incubation for 1, 21 and 42 days was investigated. The control group contained soil without sludge. Mecklenburg clay and Enon sandy loam soils (both are fine, mixed, thermic, ultic Hapludalfs) were used. All treatments were adjusted to pH 6.7 and brought to 75% of field capacity with 1 ml inoculum (9 × 10⁸ cells ml^? 1) and distilled water. Samples were incubated at 25 C and monitored periodically for the number of surviving R. japonicum (USDA 110) organism by the plant infcction-MPN method. Strains were identified by gel-immunodiffusion. Recovery of rhizobia from both soils was < 1% in all treatment groups after 42 days. However, for control, 13:1, 9:1 and 5:1 groups, the percentage recovery was higher in Enon sandy loams (7.9, 2.3, 2.3 and 2.3%, respectively) at 21 days. Recovery of rhizobia in the 5:1 group from both soils was 7.9% after 1 day, whereas control values were 92%. A decline in rhizobial populations in higher sludge soils may be due to the heavy metals present and available during mineralization of sludge in soils. However, the number of R. japonicum that survived to 21 days was 1.7 × 10₅g^?1 and 1.7 x 10⁶g^?1 for Mecklenburg clay and Enon sandy loam soils with highest sludge, respectively.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.