Effect of temperature on competition amongst strains of Rhizobium trifolii for nodulation of two white clover varieties

The effect of temperature and soil type on the relative success in nodulating cultivars of white clover (Trifolium repens) by mixtures of antibiotic-resistant mutants of Rhizobium trifolii was studied. Under aseptic test-tube culture, 75 str nodulated the plants 5 days earlier than 33 spc at the lowest temperature, but the temperature ×Rhizobium strain interaction was not significant. 33 spc was more effective than 75 str at 25°C and, although no significant difference was found between the two mutant strains at the lower temperatures, die temperature ×Rhizobium strain interaction was highly significant. In soil, when inoculated with mixed inoculum, cv. S100 was more uniformly nodulated by 75 str (81%) than S184 (49%). Success in nodulation could be altered by temperature and the temperature × bacterial strain interaction was significant. In the mixed inoculum treatments, 75 str was most compatible with S100 at 12°C, whereas 33 spc was most compatible with SI84 at 25°C; the bacterial strain × variety × temperature interaction was highly significant. The results are discussed from the point of view of improving symbiotic nitrogen fixation by selecting effective strains of Rhizobium which are compatible with the particular host cultivar, which are competitive with the indigenous population and whose optimum temperatures for nodulation and competitiveness are similar to the soil temperature at the times of inoculation.     

The inheritance of preference for strains of Rhizobium trifolii by white clover (Trifolium repents)

The preference or selection of Rhizobium strains by both parental and F₁ white clover plants was studied using antibiotic resistant mutants of Rhizobium trifolii. Highly significant differences were found between crosses in their preference for 75 str. Progenies from crosses between plants in which the preference for 75 str was 90–100% had 85% of the nodules inhabited by that strain. Progenies of crosses between plants in which the preference for 75 str was less than 50% had only 13% of their nodules inhabited by 75 str. Intermediate values were found for progenies from other crosses. The inheritance for this particular character was clearly additive and without any dominance or maternal effect. The results are discussed from the point of view of improving symbiotic nitrogen fixation by breeding white clover for uniform preference for effective strains of R. trifolii.     

Effects of some fungicides used against cereal pathogens on the growth of Rhizobium trifolii and its capacity to fix nitrogen in white clover

Fungicide residues in soils may affect nitrogen fixation by legumes. Effects of nine systemic fungicides, used against cereal pathogens, on Rhizobium trifolii and white clover were measured. Fenarimol and oxycarboxin inhibited growth of R. trifolii in vitro. The weight of clover plants after 10 weeks' growth in soil containing carboxin, oxycarboxin, benodanil, tridemorph and pyracarbolid was reduced. Symbiotic nitrogen fixation was decreased by carboxin, oxycarboxin and tridemorph present in soil at concentrations somewhat greater than that likely to result from several applications of the fungicide. Only tridemorph reduced nodulation. None of the compounds seems likely to affect nitrogen fixation in the field if applied at the recommended rate.     

Construction of an Acid-Tolerant Rhizobium leguminosarum Biovar Trifolii Strain with Enhanced Capacity for Nitrogen Fixation

Strain ANU1173 is an acid-tolerant Rhizobium leguminosarum biovar trifolii strain that is able to nodulate subterranean clover plants growing in agar culture at pH 4.4 At pH 6.5, its symbiotic effectiveness in association with Trifolium subterraneum cv. Mt. Barker was 80% relative to that of strain ANU794, a Sm^r derivative of the commercial inoculant R. leguminosarum bv. trifolii TA1. Strain ANU1173 contained four indigenous megaplasmids, the smallest of these being the symbiotic (Sym) plasmid. The critical pH requirement for growth of strain ANU1173 in laboratory media was shown not to be associated with this plasmid. When the Sym plasmid of strain ANU1173(pSym-1173) was mobilized into a Nod^- strain of R. leguminosarum bv. viciae, the plasmid conferred to the transconjugant a level of symbiotic effectiveness in association with T. subterraneum that was similar to that observed with ANU1173. The symbiotic effectiveness of strain ANU1173 was improved by first curing pSym-1173 (generating strain ANU1184) and replacing it with another R. leguminosarum bv. trifolii Sym plasmid, pBR1AN. Subterranean clover plants inoculated with strain ANU1184 (pBR1AN) exhibited a 35 or 53% increase in acetylene reduction activity and a 20 or 17% increase in dry weight when grown at pH 6.5 and pH 4.4, respectively, compared with plants inoculated with strain ANU1173 and grown under the same pH conditions. It was further shown that pBR1AN was stably maintained in strain ANU1184 under free-living and symbiotic conditions. These results indicate that it is possible to construct an acid-tolerant strain of R. leguminosarum bv. trifolii with an enhanced capacity for nitrogen fixation.     

Neighbourhood effect of genotypes of Rhizobium leguminosarum biovar trifolii, Trifolium repens and Lolium perenne

 Ryegrass, white clover and Rhizobium isolated from the corresponding clover nodules, were harvested from a natural pasture in the Massif Central mountains (France). The specificity between Lolium, Trifolium and Rhizobium, and the genetic diversity of Rhizobium were examined. This study showed that: 1) Natural neighbouring combinations of white clover and ryegrass, re-planted together in pots, accumulated a higher biomass than non-neighbouring ones. This increase of mass is higher in the presence of the native strain of Rhizobium. 2) When white clover was inoculated with a mixture of Rhizobium strains, nodules were more often formed by its native strain. 3) The genetical diversity of the Rhizobium leguminosarum biovar trifolii was very high, as revealed by electrophoresis of esterases on seven substrates. These results support the hypothesis that there is a co-adaptation between white clover, ryegrass and Rhizobium Received: 25 March 1996 / Accepted: 13 September 1996     

Water deficit and plant competition effects on growth and water-use efficiency of white clover ( Trifolium repens,L.) and ryegrass (Lolium perenne,L.)

The combined effects of soil water deficit and above and below ground interspecific plant competition on the growth, water-use efficiency (WUE), and measured carbon isotopic composition (δ¹³C) values of white clover and ryegrass were studied. White clover and ryegrass were grown in specially designed crates 1) individually; 2) in shoot competition; or 3) in shoot + root competition and either well-watered or at a moderate or severe soil water deficit. The effects of shoot + root competition on shoot dry matter growth were substantial and benefited both white clover and ryegrass when well-watered or at a moderate soil water deficit, while severely reducing white clover shoot dry matter growth at severe soil water deficit. Plant competition did not affect the WUE of white clover or ryegrass. As soil water deficit increased, the WUE of white clover did not change whereas the WUE of ryegrass increased and was greater than that of white clover. This was attributed to the lower leaf water conductance of ryegrass which conserved water and maintained growth longer compared to white clover. A stronger correlation existed between soil water deficit and measured δ¹³C values for ryegrass at each plant competition level (P<0.001) than existed for white clover (individual: P<0.01; shoot + root: P<0.001; shoot: P<0.10). Unlike white clover, the relationship between measured δ¹³C values and shoot dry matter growth indicated that C assimilation for ryegrass was dependent on type of plant competition. That WUE remained constant for white clover while measured δ¹³C values increased as soil water deficit increased, suggests that the role below ground respiration rate played in determining δ¹³C values increased. The WUE of white clover appears to be independent of the nature of the competition between plants and the soil water deficit level at which it is grown, whereas for ryegrass, the addition of root competition to shoot competition should lead to increases in its WUE. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sitona weevils (Coleoptera: Curculionidae) as agents for rapid transfer of nitrogen from white clover (Trifolium repens L.) to perennial ryegrass (Lolium perenne L.)

The effects of root feeding by larvae of Sitona hispidulus (F.) (a common weevil pest of white clover) on the rate of transfer of nitrogen between plants of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were investigated using a nutrient slant board technique. Clover plants, labelled with ¹⁵N were grown adjacent to ryegrass plants and were either infested with Sitona larvae or not infested. Ryegrass plants associated with the infested clover plants had a significantly higher dry matter yield and nitrogen content (75% and 74% respectively) than the uninvested plants, after 33 days exposure to insect herbivory. It was concluded that root feeding insects could play an important role in the cycling of nitrogen in grass/clover swards.     

The Role of Plant Size and Nutrient Concentrations in Associations between Medicago, and Rhizobium and/or Glomus

The aim of this research was to carry out a critical study of the method of obtaining size equivalence between non-symbiotic alfalfa and alfalfa associated with Glomus and/or Rhizobium by applying fixed addition rates of nutrients to the non-symbiotic controls. The experimental design included three nutrient response curves in which the levels of added phosphorus and/or nitrogen were constant during the whole plant growth process: 1) a phosphorus response curve, in order to compare the growth of double symbiotic plants with that of only-Rhizobium inoculated ones; 2) a nitrogen response curve, that consisted of a comparison between the growth of double symbiotic alfalfa and four treatments associated only with Glomus; 3) a phosphorus and nitrogen response curve, to compare the growth of non-inoculated alfalfa with that of double symbiotic plants. Although similar size was achieved among some treatments at harvest, shoot growth over time and nutrient concentrations in tissues differed, indicating that growth equivalence did not mean functional equivalence. A second experimental design was performed taking into account the establishment of microsymbionts for determining the adequate moment to add supplemental phosphorus and/or nitrogen. It included four treatments: a) double symbiotic plants (MR); b) plants inoculated with Rhizobium only (R); c) plants inoculated with Glomus only (M), and d) non-inoculated plants (N). Great similarity in terms of plant growth and nutrient contents in tissues were obtained. Moreover, symbiotic plants were able to produce similar dry matter than non-symbiotic ones under P and N limitations.     

Variation within and between white clover varieties in their preference for strains of Rhizobium trifolii

Variation within as well as between varieties of white clover (Trifolium repens L.) in their selection or preference for rhizobial strains in nodulation was studied using antibiotic-resistant mutant strains of Rhizobium trifolii. Significant differences between varieties were found. Cv. S100 was nodulated almost exclusively by 75 str but there was more variation within S184 and, in particular, Pajbjerg smalbladet. In this experiment 93% of the total variance was genotypic, of which 68% was due to the difference between varieties and 25% due to variance between genotypes within varieties. Significant correlations were found between the preference for rhizobial strains of plants grown from seeds and of stolon lines vegetatively propagated from the former plants, indicating that the preference for rhizobial strains is genetically controlled by the host. A considerable drift in preference towards selection of more compatible rhizobial strains by the clonally propagated stolons compared with the original white clover plants was observed. The results are discussed from the point of view of improving symbiotic nitrogen fixation by combining highly effective and competitive Rhizobium strains with a host variety with uniform preference for those strains.     

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.     

Nitrogen fixation by white clover when competing with grasses at moderately low temperatures

It was the aim of this study to determine the way in which low temperature modifies the effect of a competing grass on nitrogen fixation of a forage legume. White clover (Trifolium repens L.) was grown in monoculture or in different planting ratios with timothy (Phleum pratense L.) or perennial ryegress (Lolium perenne L.) in growth chambers at either 7.5/5°C (LoT) or 15/10°C (HiT) average day/night temperatures, and with 2.5 or 7.5 mM ¹⁵N-labelled nitrate in the nutrient solution.Competition with grass led to a marked increase in the proportion of clover nitrogen derived from symbiosis (% N_sym). This increase was slower at LoT where % N_sym was reduced considerably; it was closely related to the reduction in the amount of available nitrate as a result of its being utilized by the grass.Nitrogen concentration in white clover herbage and dry matter yield per clover plant were reduced, for the most part, when a competing grass was present. The amount of nitrogen fixed per plant of white clover decreased markedly with temperature. Low temperature consequently accentuated competition for nitrate. The capacity of white clover to compete successfully was limited by its slower growth and nitrogen accumulation.     

Subnanomolar concentrations of membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii are fully capable of eliciting symbiosis-related responses on white clover

Axenic seedling bioassays were performed on white clover, vetch, and alfalfa to assess the variety and dose responses of biological activities exhibited by membrane chitolipooligosaccharides (CLOSs) from wild type Rhizobium leguminosarum bv. trifolii ANU843. Subnanomolar concentrations of CLOSs induced deformation of root hairs (Had) and increased the number of foci of cortical cell divisions (Ccd) in white clover, some of which developed into nodule meristems. In contrast, ANU843 CLOSs were unable to induce Had in alfalfa and required a 10⁴-fold higher threshold concentration to induce this response in vetch. Also, ANU843 CLOSs were not mitogenic on either of these non-host legumes. In addition, CLOS action also increased chitinase activity in white clover root exudate. Thus, the membrane CLOSs from wild type R. leguminosarum bv. trifolii are fully capable of eliciting various symbiosis-related responses in white clover in the same concentration range as extracellular CLOSs of other rhizobia on their respective legume hosts. These results and our earlier studies indicate that membrane CLOSs represent one of many different classes of bioactive metabolites made by R. leguminosarum bv. trifolii which elicit more intense symbiosis-related responses in white clover than in other legumes. Therefore, CLOSs evidently play an important role in symbiotic development, but they may not be the sole determinant of host-range in the Rhizobium-clover symbiosis.Abbreviations Ccd cortical cell division - CLOS chitolipooligosaccharide - Had root hair deformation     

Effect of Nitrogen Supply on the Grass and Clover Components of Simulated Mixed Swards Grown under Favourable Environmental Conditions I. Carbon Assimilation and Utilization

Simulated mixed swards of Perennial Ryegrass (Lolium perenneL.) cv. S23 and White clover (Trifolium repens L.) cv. S100were grown from seed under a constant 20 °C day/15 °Cnight temperature regime and their growth and carbon economyexamined. The swards received a nutrient solution daily, whichcontained either High (220 mg l^–1) or Low (10 mg l^–1)nitrate N. Rates of canopy photosynthesis and respiration, and final drymatter yields were similar in the two treatments although theproportions of grass and clover differed greatly. The Low-Nswards were made up largely of clover. The grass plants in theseswards had high root: shoot ratios and low relative photosyntheticrates – both signs of N deficiency – and were clearlyunable to compete with the vigorously growing Low-N clover plants.These had higher relative growth rates and dry matter yieldsthan their High-N counterparts. In the High-N swards clovercontributed around 50 per cent to the sward dry weight throughoutthe measurement period despite having a smaller proportion ofits dry weight in photosynthetic tissue (laminae) than grassover much of it. The latter was compensated for, initially bya higher specific leaf area than grass, and later by a higherphotosynthetic rate per unit leaf weight. The results are discussedin relation to observed declines in the clover content of swardsafter the addition of nitrogen fertilizer in the field. Trifolium repens, white clover, Lolium perenne, perennial ryegrass, nitrogen, photosynthesis, carbon balance     

Frost tolerance and biochemical changes during hardening and dehardening in contrasting white clover populations

Successful winter survival of perennial plants, like white clover, is dependent on proper timing of both hardening and dehardening. The purpose of this study was to investigate the regulation of these processes in two cultivars (AberCrest and AberHerald) and two Norwegian ecotypes (Særheim collected at 58°46′N lat. and Bodø at 67°20′N lat.) of white clover (Trifolium repens L.). For hardening and dehardening, plants were exposed to controlled temperature conditions and frost hardiness of stolons was tested by programmed freezing at the rate of 3°C per hour. In addition, stolons were analysed for starch, soluble sugars and soluble amino acids. Cultivars AberCrest and AberHerald, selected for growth at low temperature and winter hardiness in the United Kingdom, were significantly less hardy than the Norwegian populations. After six weeks of hardening (2 weeks at 6°C and 4 weeks at 0.5°C), estimated LT₅₀ values were ?13.8, ?13.0, ?17.8 and ?20.3°C for AberCrest, AberHerald, Saerheim and Bodø, respectively. The rate of dehardening increased with increasing temperature. At low temperature (6°C), the northern ecotype from Bodø was more resistant to dehardening than AberHerald. However, at 18°C the absolute rate of dehardening (°C day^?1) was twice as high in Bodø as in AberHerald plants. Stolon elongation during dehardening was initiated at lower temperatures in AberHerald than in plants of the Bodø ecotype. The content of total soluble sugars, sucrose and the amino acids proline and arginine were significantly higher in hardy plants of Bodø than in those of AberHerald. Sucrose levels decreased during dehardening and correlations between sucrose content and LT₅₀ during this process were statistically highly significant for both Bodø and AberHerald. The least hardy populations of white clover were characterized by thick stolons, long internodes and large leaves.     

Studies on symbiotic nitrogen fixation by a new strain of tetraploid red clover (UO36)

Summary Some characteristics of the symbiotic nitrogen fixation by tetraploid red clover (UO36) have been studied in laboratory experiments. Cross-inoculations were made on tetraploid and diploid clover plants usingRhizobium isolates from both types of clover. Nodulation activity and nitrogen fixation were determined. No sign of specific strains necessary for optimal fixation of nitrogen by theUO36-symbiosis was observed in comparison to the diploid clover strain.Rhizobium cultures effective in connection with cultivation of diploid clover will probably also be effective when cultivating tetraploid clover strains.     

The Growth and Photosynthesis of Seedling Plants of White Clover at Low Temperature

The growth of white clover (Trifolium repens L.) in conditionstypical of April in Southern England (8 °C day/4 °Cnight, 12 h photoperiod of 90 J m^–2 s^–1 visibleradiation) was extremely slow, whether the plants were dependentfor nitrogen on fixation by their root nodules or were suppliedwith abundant nitrate; although growth was slower in the nodulatedplants. The reasons for slow growth were a large root: shootratio and a small leaf area, particularly in the nodulated plants,and a low photosynthetic rate in all plants. The probable effectsof these characteristics on the growth of white clover withgrasses in mixed pastures are discussed. Trifolium repens L, white clover, low temperature, leaf area, photosynthetic rate, nitrogen supply, growth     

Alfalfa water status and cold hardiness as influenced by cold acclimation and water stress

Abstract Water stress at a nonacclimating temperature (18–20°C) increased the cold hardiness of Medicagosativa L. (alfalfa) plants. This increased cold hardiness was retained when the previously water-stressed plants were cold acclimated (2–9°C) in the absence of water stress. Water stress during cold acclimation also increased cold hardiness. Alfalfa was demonstrated to suffer injury, measured as decreased growth following freezing, at sub-lethal temperatures. During cold acclimation the turgor potential (ψ) of watered plants increased, whereas the solute potential and the water content per unit dry weight decreased. The large positive psgrdap of acclimated plants indicates that the decreased water content per unit dry weight is related to an increased proportion of tissue dry matter rather than to tissue dehydration.     

Effect of Low Temperature and Rh izospheric Application of Naringenin on Pea-Rhizobium leguminosarum biovar viciae Symbiosis

The production of Tsr factor by Rhizobium leguminosarum bv. viciae was influenced by low temperature (10°C) In the presence of seed exudate collected at 10°C and 25°C or naringenin (10fuM). Root exudate collected at 25°C and naringenin induced Tsr factor in R. leguminosarum causing thick and short root phenotype and root hair curling and deformation of host root. Root exudate collected at 10°C also induced root hair curling but Tsr activity was low. low temperature grown plants had poor nodulation, nitrogen fixation, nitrogen content and total blomass as compared to plants grown at 25°C. Rhizospheric application of naringenin partially alleviated the deleterious effect of low temperature on nodulation status and nodule efficiency.     

The response of white clover (Trifolium repens L.) seedlings to spring root temperatures: The relative roles of the plant and the Rhizobium bacteria

Three experiments are reported which examine the relative roles of host and Rhizobium genotypes as factors limiting clover (Trifolium repens L.) growth at low soil temperatures.In the first experiment un-nodulated clover and perennial ryegrass (Lolium perenne L.) were grown with non-limiting nitrate at root temperatures of 8, 10 and 12°C. The ryegrass had substantially better relative growth rates (RGR) than the clover with the biggest difference occurring at 8°C. Alterations in growth rate with temperature were more marked in clover than in ryegrass but the latter still produced several times more dry matter than clover at each temperature.In the subsequent experiments clover nodulated with different strains of rhizobia was grown with and without non-limiting additions of nitrate at root temperatures of 9, 12 and 15°C. Plants receiving nitrate generally produced more dry matter than those dependent upon Rhizobium for nitrogen but differences in yield between these treatments did not alter with temperature. This suggests that limitations imposed by nitrogen fixation are similar at both high and low temperatures. Indeed, there was some evidence that nitrogen limitations were rather more pronounced at the highest temperature. The first experiment clearly demonstrated that the clover genotype makes particularly poor use of nitrate at low root temperatures when compared to its common companion perennial ryegrass.It can be concluded that improvements in spring growth of clover will rest largely with alterations to the plant genotype and its ability to use combined nitrogen for growth at lower temperatures rather than with changes in rhizobia or any symbiotic characters.     

Effects of Salt and pH Stress on Temperature-Tolerant Rhizobium sp. NBRI330 Nodulating Prosopis juliflora

A study was conducted to examine the growth response of a rhizobial strain Rhizobium sp. NBRI330 isolated from root nodules of Prosopis juliflora growing in alkaline soil. The strain had the ability to nodulate P. juliflora. Nursery grown plants inoculated with Rhizobium sp. NBRI330 had 60.6% higher plant dry weight, as compared with uninoculated plants. The individual stress survival limit of a rhizobial strain Rhizobium sp. NBRI330 isolated from alkaline soil in a medium containing 32% (wt/vol) salt was 8 h, and at 55°C up to 3 h. The length of Rhizobium sp. NBRI330 in salt-stressed cells increased significantly to 3.04 μm from 1.75 μm of non-stressed control cells. On the contrary, the length of pH-stressed cells declined to 1.40 μm. Compared with non-stressed control rod-shaped cells, the shape of temperature-stressed cells changed to spherical, of 0.42 μm diameter. High temperature (45°C) was tolerated efficiently by Rhizobium sp. NBRI330 in the presence of salt at pH 12, as compared with pH 7. Received: 13 September 1999 / Accepted: 14 October 1999