White Clover Rhizobia Research Articles

Ecological consequences of urbanization on a legume–rhizobia mutualism

Mutualisms are key determinants of community assembly and composition, but urbanization can alter the dynamics of these interactions and associated effects on ecosystem functions. Legume–rhizobia mutualisms are a model interaction to evaluate the ecological and ecosystem‐level effects of urbanization, particularly urban‐driven eutrophication and nitrogen (N) deposition. Here, we evaluated how urbanization affected the ecology of the mutualism between white cloverTrifolium repensand its rhizobial symbiontRhizobium leguminosarumsymbiovartrifoliialong an urbanization gradient. We found that the abundance of rhizobium nodules on white clover decreased with urbanization. White clover acquired N from mixed sources of N fixation and uptake from the soil for the majority of the urbanization gradient, but white clover primarily acquired N from the soil rather than N fixation by rhizobia at the urban and rural limits of the gradient. Importantly, we identified soil N as a critical nexus for urban‐driven changes in the white clover–rhizobium mutualism. Taken together, our results demonstrate that urbanization alters the ecological consequences of a legume–rhizobium mutualism, with direct and indirect effects of the urban landscape on an ecologically‐important mutualistic interaction.

Effect of rhizobia from caucasian clover (Trifolium ambiguum) on nodulation and nitrogen fixation of white clover (Trifolium repens)

Three glasshouse trials investigated N2fixation in white clover following inoculation with rhizobia from caucasian clover. We grew white clover in agar, vermiculite/perlite and soils low in available‐N and with no detectable rhizobia. Media were subsequently treated with inoculum containing rhizobia effective on either (i) caucasian clover, (ii) white clover, or (iii) mixtures of the two. The percentage of ineffective nodules on white clover increased as the proportion of rhizobia from caucasian clover in the inoculant mix increased. However, the percentages of ineffective nodules on white clover were less than the proportion of ineffective rhizobia in the inoculant, indicating that the ineffective rhizobia were less competitive in nodule formation. Foliage yield and symbiotic N2fixation in white clover were not significantly affected when ineffective rhizobia from caucasian clover made up 67% or less of the inoculant. However, when ineffective rhizobia made up a high proportion of the inoculant mix (>92%), both foliage yield and N2 fixation were significantly reduced compared with plants inoculated with only white clover rhizobia. Extrapolation of the present glasshouse results to the field is difficult and further field trials are required to determine if rhizobia from caucasian clover have any effect on symbiotic N2fixation of white clover in pastoral situations.

A study of the impacts of Zn and Cu on two rhizobial species in soils of a long-term field experiment

Two agriculturally important species of rhizobia, Rhizobium leguminosarum biovar viciae (pea rhizobia) and R. leguminosarum bv. trifolii (white clover rhizobia), were enumerated in soils of a long-term field experiment to which sewage sludges contaminated predominantly with Zn or Cu, or Zn plus Cu, were added in the past. In addition to total soil Zn and Cu concentrations, soil pore water soluble Zn and free Zn2+, and soluble Cu concentrations are reported. Pea and white clover rhizobia were greatly reduced in soils containing ≥200 mg Zn kg-1, and soil pore water soluble Zn and free Zn2+ concentrations ≥7 and ≥3 mg l-1, respectively, in soils of pH 5.9–6. Copper also reduced rhizobial numbers, but only at high total soil concentrations (>250 mg kg-1) and not to the same extent as Zn. Yields of field grown peas decreased significantly as total soil Zn, soil pore water soluble Zn and free Zn+2 increased (R2 = 0.79, 0.75 and 0.75, respectively; P < 0.001). A 50% reduction in seed yield occurred at a total soil Zn concentration of about 290 mg kg-1, in soils of pH 5.9–6. The corresponding soil pore water soluble Zn and free Zn2+ concentrations were about 9 and 4 mg l-1, respectively. Pea seed yields were not significantly correlated with total soil Cu (R2 = 0.33) or soil pore water soluble Cu (R2 = 0.39). Yield reductions were due to a combination of greatly reduced numbers of free-living rhizobia in the soil due to Zn toxicity, thus indirectly affecting N2-fixation, and Zn phytotoxicity. These effects were exacerbated in slightly acidic soils due to increased solubility of Zn, and to some extent Cu, and an increase in the free Zn2+ fraction in soil pore water. The current United Kingdom, German and United States limits for Zn and Cu in soils are discussed in view of the current study. None of these limits are based on toxicity thresholds in soil pore water, which may have wider validity for different soil types and at different pH values than total soil concentrations.

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.

Absence of nitrogen fixation in clover grown on soil subject to long-term contamination with heavy metals is due to survival of only ineffective Rhizobium

All strains of Rhizobium isolated from ineffective nodules on white clover (> 50 separate isolations) formed in metal-contaminated soils from a field experiment were demonstrated to be wholly ineffective in nitrogen fixation in plant infection tests on N-free nutrient agar. The plasmid profiles of these isolates were all very similar indicating a lack of genetic diversity in the population surviving in high concentrations of heavy metals. Isolates from comparable field plots of uncontaminated soil had a wide diversity of plasmid profiles. Inoculation of white clover at sowing with a large inoculum of effective Rhizobium leguminosarum bv. trifolii on metal contaminated soil resulted in effective N 2-fixation. However, if the inoculated soils were held for 2 months in a moist condition before sowing with white clover, N 2-fixation was not detected with inoculum doses of 10 7 cells pot −1 or less but was obvious where a very large inoculum of 10 10 cells pot −1 was added. This indicates that white clover rhizobia are unable to survive (or at least unable to remain effective) in the presence of concentrations of heavy metals close to the current Commission of the European Communities guidelines for environmental protection. A rapid method of assessment of the toxic effects of pollutants on populations of Rhizobium spp is described.

Photosynthesis, respiration and nitrogen fixation in white clover

AbstractAn apparatus in which shoot and root CO2 exchange and acetylene reduction can be simultaneously measured in specific white cloverrhizobium associations is described. In mature stolonating clover there was a fairly constant ratio between net photosynthesis, root respiration and acetylene reduction. Of the net carbon fixed daily (12 h light of 80 W m‐2, 400–700 nm; day/night temperature 15°C) 12% was lost during the dark period by the shoot and 17% by the nodulated root. Changes in the rate of acetylene reduction by nodulated root systems occurred more slowly than those in rates of root respiration in response to reduced irradiance. In 21·5 h continuous darkness the rate of acetylene reduction remained constant. Responses to increased irradiance were more immediate in both root respiration and acetylene reduction. In plants maintained at 15°C in a 12‐h, 80‐W m‐2 photoperiod there was no significant diurnal variation in acetylene reduction or root respiration. Acetylene depressed root respiration by 20%. Assuming the energy requirement of the nitrogenase system to be the same when reducing acetylene and N the depression can be used as an indication of the energy requirement of fixed N assimilation, metabolism and export in the nodulated root. Of the net carbon fixed daily 3·4% was utilized in this way in plants growing in a 12‐h photoperiod (80 W m‐2, 400–700 nm) at 15°C.

Some problems and potentialities in breeding for improved white clover‐rhizobium (TrifoZium repens‐Rhizobium trifolli) symbiosis

Some problems and potentialities in breeding for improved white clover‐rhizobium (TrifoZium repens‐Rhizobium trifolli) symbiosis

Correction - Studies in the nodulation of Kenya white clover (Trifolium semipilosum) under field conditions in south-east Queensland

The nodulation of Kenya white clover (Trifolium semipilosum) was examined under field conditions to evaluate reported establishment problems attributed to poor nodulation. Two isolates of Rhizobium identified as strain CB782 were obtained from nodules of T. semipilosum located in separate field sowings each of which was considered an establishment failure but which had recovered to healthy stands at the time of sampling. The two isolates were compared with a laboratory maintained culture of CB782 for nodulation and nitrogen fixation. Both isolates and the original strain were equally effective in N-fixation and all three increased plant dry weight, per cent plants with effective (pink) nodules and amount of nitrogen fixed compared with an uninoculated control. Increasing the level of seed application of inoculum up to the normal rate increased effective nodulation, but excess inoculum levels resulted only in greater numbers of smaller effective (pink) nodules without any increase in nodule dry weight. High inoculum levels decreased the number of white and green nodules compared with the control. Over 50 per cent effective nodulation was obtained with less than 50 Rhizobium seed-1 at sowing. Serological identification and effectiveness tests of strains in nodules confirmed that most white and green nodules on T. semipilosum resulted from infection by naturally occurring white clover rhizobia. These were ineffective in nitrogen fixation with T. semipilosum but fully effective on T. repens. Serological typing of nodules also indicated that normal levels of inoculation resulted in more than 80 per cent of nodules being formed by the specific inoculum strain. We conclude that field sowings of T. semipilosum can be effectively nodulated by the specific inoculum strain CB782 which is both effective and persistent under south-east Queensland conditions. We suggest that most reported nitrogen deficiency symptoms of establishing T. semipilosum are due to infection by Rugose Leaf Curl Virus.

Studies in the nodulation of Kenya white clover (Trifolium semipilosum) under field conditions in south-east Queensland

The nodulation of Kenya white clover (Trifolium semipilosum) was examined under field conditions to evaluate reported establishment problems attributed to poor nodulation. Two isolates of Rhizobium identified as strain CB782 were obtained from nodules of T. semipilosum located in separate field sowings each of which was considered an establishment failure but which had recovered to healthy stands at the time of sampling. The two isolates were compared with a laboratory maintained culture of CB782 for nodulation and nitrogen fixation. Both isolates and the original strain were equally effective in N-fixation and all three increased plant dry weight, per cent plants with effective (pink) nodules and amount of nitrogen fixed compared with an uninoculated control. Increasing the level of seed application of inoculum up to the normal rate increased effective nodulation, but excess inoculum levels resulted only in greater numbers of smaller effective (pink) nodules without any increase in nodule dry weight. High inoculum levels decreased the number of white and green nodules compared with the control. Over 50 per cent effective nodulation was obtained with less than 50 Rhizobium seed-1 at sowing. Serological identification and effectiveness tests of strains in nodules confirmed that most white and green nodules on T. semipilosum resulted from infection by naturally occurring white clover rhizobia. These were ineffective in nitrogen fixation with T. semipilosum but fully effective on T. repens. Serological typing of nodules also indicated that normal levels of inoculation resulted in more than 80 per cent of nodules being formed by the specific inoculum strain. We conclude that field sowings of T. semipilosum can be effectively nodulated by the specific inoculum strain CB782 which is both effective and persistent under south-east Queensland conditions. We suggest that most reported nitrogen deficiency symptoms of establishing T. semipilosum are due to infection by Rugose Leaf Curl Virus.