Nodulation Of Clover Research Articles

Effects of tillage and lime on Rhizobium trifolii populations and survival in wheat-subterranean clover rotation in southeastern Australia

On strongly acidic sandy clay loams in southeastern Australia there can be problems with the growth and persistence of subterranean clover in clover-ley rotations. During a 4 year rotation, which involved wheat crops and then the re-establishment of clover, the effects of direct drilling and cultivation, and agronomic methods on the nodulation of subterranean clover and the survival of Rhizobium leguminosarum biovar trifolii were compared. In the season in which the clover was sown, the plants on the unlimed soil were poorly nodulated, with no nodules at all on the clover that was sown without inoculation and where the soil had been cultivated. Inoculation of the clover seed increased the nodulation of the clover in the absence of lime. The populations of R. trifolii were estimated throughout the wheat-clover sequence in the rotation. Prior to establishing the experiment the site had been in a subterranean clover-based pasture for 12 years, and the populations of rhizobia were about 200–300 g −1 soil. Without lime the rhizobia populations declined to about 10 or less rhizobia g −1 soil over the period the site was cropped, but where the soil was limed there was no change in the rhizobia populations with cropping. When the subterranean clover was re-established the populations of R. trifolii were increased with all soil treatments, with the unlimed soil obtaining populations similar to those present at the start of the experiment and the rhizobia number on the limed soil increasing to about 10 5 g −1 soil. The most favoured soil environment for R. trifolii was where the pH was raised mostly in the soil surface, and the soil was only minimally disturbed with the direct-drill treatment.

Rhizobial siderophore as an iron source for clover

Iron uptake by clover plants (Trifoliuin pratense L. cv. Hruszowska) was studied using radioactive ferric iron (55FeCl2). As shown by autoradiography of non‐infected plants, purified rhizobial siderophore isolated from Rhizobium leguminosarum by, trifolii, stimulated the uptake and shoot transport of iron. Addition of rhizobial siderophore into the growth medium of nodulated clover did not affect the iron transport to the shoots. In the absence of the rhizobial siderophore, clover infected by either nitrogen‐fixing (Nod− Fix+) or nonfixing (Nod+ Fix−) R. L. trifolii strains took up and transported into the shoots more iron than the non‐infected control plants. Nodulated clover reduced Fe(III) more efficiently than the non‐infected control plants.

Seedling establishment characteristics of alternative legume species in tussock grassland environments

Four alternative legume species, Caucasian clover (Trifolium ambiguum), zigzag clover (T. medium), crown vetch (Coronilla varia), and birdsfoot trefoil (Lotus corniculatus), were compared with white clover (T. repens) on four tussock grassland sites. Seed was inoculated and pelleted, and then oversown at a rate equivalent to 5 kg/ha viable seed. Germination, nodulation and survival of white clover, Monaro Caucasian clover, and zigzag clover were similar on the two lowest sites (600 and 830 m). However, low rates of zigzag clover germination occurred on the two higher sites (1090 and 1100 m). Germination of crown vetch appeared to be prevented or delayed by the low temperatures, and no plants established on any site. Low nodulation occurred in Treeline Caucasian clover and birdsfoot trefoil. Further research is required to select more suitable strains of rhizobia for both these legumes. The results provide information for estimating likely plant densities of these new cultivars when oversown in tussock grassland environments. Keywords Coronifla varia, establishment, germination, inoculation, Lotus corniculatus, nodulation, Trifoliumambiguum, Trifolium medium, Trifolium repens

Competition between a native isolate of Rhizobium leguminosarum bv trifolii and two commercial inoculant strains for nodulation of clover

A naturalized rhizobial isolate from nodules obtained from persistent clover pastures was evaluated for its ability to form effective nodules in the presence of two commercial inoculant strains Rhizobium leguminosarum bv trifolii U28 and WU290 recommended for white clover and subterranean clover, respectively. The competition experiment was performed in tubes, and indirect ELISA was used for identification of rhizobia present in the nodules. The ratios between the number of nodules containing only the naturalized isolate (PA8) and the number of nodules containing only the U28 strain were directly proportional to their respective inoculum densities when white clover was used as host plant. However, when the competition assay was done with different mixtures of PA8 and WU290, no WU290 bacteria were identified in subterranean clover nodules. The symbiotic performance of PA8, together with its persistence and competitive ability, suggests that naturalized strains may be useful in attempting to establish and maintain high-yielding pastures in Uruguay.

A comparison of soil tests to predict the growth and nodulation of subterranean clover in aluminium-toxic topsoils

Total Al concentration or pH in 1∶5 10 mM CaCl2 extracts and exchangeable Al in 100 mM BaCl2 extracts cannot always distinguish between Al-toxic and Al-nontoxic topsoils. Our objectives were to compare the abilities of different measures of Al and pH in various extracts to predict the effects of acidity on growth and nodulation of subterranean clover. In a glasshouse experiment,Trifolium subterraneum L. cv. Mt Barker was grown in acidic soils from 3 sites in the Western Australian wheatbelt with different histories of phosphate fertilizer application. The pH was adjusted to give a range of 3.8–7 in the centrifuged soil solution (SS). Total (Al-tot), reactive Al (8-hydroxyquinoline-extractable, Al-HQ) and pH were measured in SS and 1∶5 extracts of KCl, CaCl2 and LaCl3. Another method of estimating reactive Al (Al which reacts with Chelex-100) was also measured in SS only. Other measurements included exchangeable Al and H, Ca in SS, and P in SS and the CaCl2 extracts. Both plant growth and early nodulation decreased with increasing acidity. Plant growth in the acidified and unlimed treatments of all soils was best described by Al-HQ in SS, KCl or CaCl2 (r2=0.68–0.70). Multiple regression of relative yield against Al or pH with the concentration of P in SS increased the percentage variation explained by 10% and 30%, respectively. Early nodulation was well correlated (r2=0.67–0.91) with pH or exch. H, Al-tot or exch. Al and Al-HQ. No improvement in the correlation was gained by including P using multiple regression. At constant ionic strength, increasing the valence of the extracting cation decreased the ability of soil tests to distinguish phytotoxic Al.

P ‐ Mo interactions on the yield and nodulation of white clover grown on limed‐lignite overburden materials

Abstract A greenhouse study using lignite overburden surface soil from a lignite mine in northwest Louisiana was conducted to determine the yield response and nodulation of inoculated and non‐inoculated white clover (Trifolium repens L.) to P (0, 40, and 80 kg/ha) and Mo (0, 90, and 180 g/ha) fertilization. Results manifested a highly significant response of inoculated white clover to a single application of P and Mo. Application of P at the rate of 40 and 80 kg/ha increased the dry matter yield (DMY) of white clover by 164% and 154%, while Mo fertilization at the rate of 90 and 180 g/ha increased the dry matter yield (DMY) by 21% and 37%, respectively, over the control. The yield of white clover was also significantly affected by Rhizobium inoculation. Yield was increased from 2.1 to 2.5 Mg/ha which is equivalent to a 19% yield improvement over the non‐inoculated plants. Nodule formation (NF), likewise, was significantly favored by P and Mo fertilization. Results also revealed that Rhizobium inoculation ...

Influence of herbicides on growth and nodulation of white clover, Trifolium repens

Five herbicides commonly used for suppression of weed growth in white clover seed crops were tested for toxicity to white clover ( Trifolium repens), the nodule-forming bacteria Rhizobium trifolii, and the nitrogen-fixing symbiosis of these organisms. Trials were carried out under bacteriologically-controlled and glasshouse conditions. Overall, paraquat was more toxic to R. trifolii growth than MCPB, followed by bentazone, fusilade and kerb as the least toxic. Bentazone showed the least toxicity to plant growth, nodulation and N 2 fixation under bacteriologically-controlled conditions. All the other herbicides inhibited C 2H 2 reduction, while nodule number and plant fresh weight were lowered by paraquat. MCPB and kerb. However, when plants grown in soil were treated, kerb showed the least toxicity with fusilade, bentazone and MCPB being intermediate in toxicity. This study also showed that changes in growth medium, nitrogen status and soil moisture content can alter the activity of particular herbicides. Possible toxicity of herbicides to plant growth and N 2 fixation should be considered when using herbicides to control weeds in leguminous crops.

Enzyme treatment, PEG, biotin and mannitol, stimulate nodulation of white clover by Rhizobium trifolii

Summary The number of nodules produced on white clover seedlings inoculated with Rhizobium trifolii was increased by more than 64% by treating seedling roots with an enzyme mixture consisting of 1% w/v Cellulase YC and 0.1 % w/v Pectolyase Y23 immediately prior to inoculation with Rhizobium , or by inoculating untreated or enzyme treated seedlings with Rhizobium in the presence of PEG. Nodulation of clover seedlings was also promoted and acetylene reduction was stimulated following addition of biotin to the nitrogen-free medium used to grow the seedlings under axenic conditions. Washing of the roots of inoculated clover seedlings in mannitol solution also increased nodule development. Addition of sucrose to the nitrogen-free medium inhibited nodule development, but promoted acetylene reduction.

Subclover Early Growth Responses to Levels and Placements of Superphosphate and Ammonium Nitrate

AbstractSingle superphosophate (SSP) commonly has been used to correct soil deficiencies of P and S in rangeland seedings of annual legumes. This study evaluated rates and soil placement of P and S as SSP and N as ammonium nitrate on germination (emergence), seedling growth, and nodulation of subterranean clover (Trifolium subterraneum L.). The soil was a Sobrante‐Las Posas association (fineloamy or fine mixed thermic family of mollic haploxeralfs). Plants were grown from seed in three separate greenhouse experiments. The SSP levels varied from 31 to 4000 kg ha−1 (equivalent to 2.4 and 313 kg ha−1 P and 3.7 and 480 kg ha−1 S). Nitrogen varied from 40 to 160 kg ha−1. Placements were: 5 cm below the seed, 1.25 cm side‐banded, broadcast, and seed placed. Responses measured included emergence, growth rate, shoot weight, nodulation, and P toxicity symptoms. Negative effects of SSP mainly reduced emergence and increased plant toxicity symptoms and occurred at levels ≥1000 to 4000 kg ha−1. Negative effects of N (at 160 kg ha−1) were mainly on nodulation. Positive effects on most growth parameters and nodulation were largely confined to SSP levels ≤500 kg ha−1 and were best expressed by an asymptotic regression function. Placement effects generally were small except for seed‐placed reduction of emergence above 1000 kg ha−1 SSP. Soil‐banded placement treatments usually were more beneficial to growth than either broadcast or seed placed.

Expression of Rhizobium leguminosarum CFN42 genes for lipopolysaccharide in strains derived from different R. leguminosarum soil isolates

Two mutant derivatives of Rhizobium leguminosarum ANU843 defective in lipopolysaccharide (LPS) were isolated. The LPS of both mutants lacked O antigen and some sugar residues of the LPS core oligosaccharides. Genetic regions previously cloned from another Rhizobium leguminosarum wild-type isolate, strain CFN42, were used to complement these mutants. One mutant was complemented to give LPS that was apparently identical to the LPS of strain ANU843 in antigenicity, electrophoretic mobility, and sugar composition. The other mutant was complemented by a second CFN42 lps genetic region. In this case the resulting LPS contained O-antigen sugars characteristic of donor strain CFN42 and reacted weakly with antiserum against CFN42 cells, but did not react detectably with antiserum against ANU843 cells. Therefore, one of the CFN42 lps genetic regions specifies a function that is conserved between the two R. leguminosarum wild-type isolates, whereas the other region, at least in part, specifies a strain-specific LPS structure. Transfer of these two genetic regions into wild-type strains derived from R. leguminosarum ANU843 and 128C53 gave results consistent with this conclusion. The mutants derived from strain ANU843 elicited incompletely developed clover nodules that exhibited low bacterial populations and very low nitrogenase activity. Both mutants elicited normally developed, nitrogen-fixing clover nodules when they carried CFN42 lps DNA that permitted synthesis of O-antigen-containing LPS, regardless of whether the O antigen was the one originally made by strain ANU843.

Defoliation-Induced Stress in Nodules of White Clover

Defoliation-Induced Stress in Nodules of White Clover

The Effects of Salt Stress on Nodulation of White Clover

The Effects of Salt Stress on Nodulation of White Clover

Soil solution P concentrations necessary for nodulation and nitrogen fixation in mycorrhizal and non-mycorrhizal red clover ( Trifolium pratense L.)

Soil solution P concentrations necessary for nodulation and nitrogen fixation in mycorrhizal and non-mycorrhizal red clover ( Trifolium pratense L.)

Defoliation-Induced Stress in Nodules of White Clover

Defoliation-Induced Stress in Nodules of White Clover

Phage Induction of Lysogenic Rhizobium leguminosarum biovar trifolii in both the Free-living and the Symbiotic Form

A lysogenic strain, Rhizobium leguminosarum biovar trifolii UK-1: φU, was isolated from a wild white clover nodule. It was symbiotically effective on white clover. A lysogenic phage (U-mole) was induced from this strain by treatment with either UV irradiation or mitomycin C. Phage U-mole had an icosahedral head (40 nm wide), a short tail (9 nm long) and tail fibres (15 nm long). Phage U-mole was induced with mitomycin C both from bacterial cells in infection threads and from bacteroids in nodules. Southern hybridization using EcoRI fragments of phage U-mole as probe indicated that phage U-mole DNA was integrated into the chromosome of strain UK-1: øU at a site involving the 60 kb EcoRI fragment of phage U-mole. Phage U-mole also lysogenized the wild-type strain R. leguminosarum biovar trifolii 4S. The resulting lysogenized strain, 4S:øU, had lost its 315 kb Sym plasmid and its nodulation ability.

Specificity of Arrowleaf Clover for Rhizobia and Its Establishment on Soil From Crimson Clover Pastures

AbstractSuccessful establishment of clover (Trifolium spp.) on soil requires that the appropriate rhizobia are present to establish an N2 fixing association. Experiments were undertaken in the laboratory to determine if the rhizobial requirements of arrowleaf clover (Trifolium vesiculosum Savi) were different from those of crimson clover (Trifolium incarnatum L.) because arrowleaf clover is often being sown in pastures that have previously grown crimson clover. Generally, rhizobia capable of nodulating crimson clover were also able to nodulate arrowleaf clover but fixed little N2. Soil collected from nine pastures that were growing crimson clover and had never been planted to arrowleaf clover contained rhizobia capable of effectively (nodules fixed N2) nodulating arrowleaf clover. Protein profiles of rhizobia isolated from nodules of arrowleaf and crimson clover grown on soil from one of the nine locations indicated that at least 12 rhizobial strains were present, and those that selectively nodulated crimson clover generally were not able to fix N2 or showed delayed nodulation on arrowleaf clover. When arrowleaf clover was grown on the same soil, four of the five strains that formed nodules on its roots were effective. It appears that arrowleaf clover was able to select effective rhizobia from a mixed population and that it may be successfully established on fields that have grown crimson clover, even though the rhizobial requirements for the two hosts are different.

Nodulation and growth of subterranean clover ( Trifolium subterraneum L.) in a drying acid soil

Subterranean clover (cv. Mt Barker) was grown in undisturbed acid soil (pH 4.3), in cultivated acid soil (pH 4.3) and in lime-amended (pH 5.5) soil and after 12 weeks of growth was either watered to field capacity or basally irrigated with the equivalent of half the amount of water transpired by the control plants. Basally-irrigated plants were able to exploit subsoil moisture and maintain adequate plant-water relations whilst the soil at the top of the soil profile dried slowly. Most nodules were confined to the less acidic uppermost and basal regions of the undisturbed acid soil profile and no new nodules were produced on the root system once basal irrigation commenced. The permanent wilting point of the compact, undisturbed acid soil (−1.5 MPa) corresponded with a soil moisture content of 7% w/w and was attained 3 weeks after commencement of basal irrigation. Nitrogen accumulation by the subterranean clover plants grown in this treatment ceased when the top 9 cm of the acid soil profile reached the permanent wilting point. The permanent wilting point of the top 9 cm of the limed and unlimed cultivated soil occurred at a soil moisture content of 5% w/w and was reached 5 weeks after commencement of basal irrigation. Nodules occurred on roots at depth in the limed and unlimed cultivated soil and nodulation continued after commencement of basal irrigation compensating for lower nodule numbers on the upper crown roots that were in contact with the drying soil. Nitrogen continued to be accumulated by these plants throughout the experiment. Nodule size varied in accordance with soil cultivation treatment. In particular, larger nodules were produced on the roots of plants grown in undisturbed acid soil whereas smaller, more numerous nodules occurred on the roots of plants grown in lime-amended soil and to a lesser extent on the roots of plants grown in cultivated acid soil. However, total nodule mass was equivalent on plants grown in each of the soil treatments. Continued nodulation at depth on the roots of subterranean clover due to cultivation and incorporation of lime enabled nitrogen fixation to continue when nodules on roots near the soil surface became desiccated and senescent due to the localized moisture deficit.

Acid-tolerance and symbiotic effectiveness of Rhizobium trifolii associated with a Trifolium subterraneum L.-based pasture growing in an acid soil

The critical range of pH, below which growth in solid media of several strains of Rhizobium trifolii was inhibited, was between pH 4.55 and 4.85. This was consistent with growth of the strains over a range of pH in liquid culture. Concentrations of Al in excess of 150 μM were necessary to inhibit growth of R. trifolii in agar-culture at pH 5.10. Sensitivity of growth to Al was also influenced by the concentration of P in the medium. Acid-tolerance of R. trifolii isolates collected from a very acid soil was considered to be poor and was similar to that observed for R. trifolii strains TA1 and WU95. Only 96 of 481 isolates were capable of growth in solid media at pH 4.70, whereas all isolates made growth at pH 5.10. The proportion of isolates capable of growth at pH 4.70 was not related to the pH of the soil from which they were isolated, but was influenced by the method of isolation. A higher proportion of more acid-tolerant isolates were obtained from clover nodules formed in dilute soil suspensions than from single nodules removed from plants in the field. The symbiotic eflectiveness of 127 isolates of R. trifolii from the acid soil, relative to the effectiveness of R. trifolii strain TA1 in association with Trifolium subterraneum, ranged between 27 and 112% with a mean of 74% over all isolates. Significant negative correlations were found between the symbiotic eflectiveness and the growth of individual isolates in solid media at low pH.

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.

Antigenic and symbiotic characterization of indigenous Rhizobium leguminosarum bv. tripolii recovered from root nodules of Trifolium pratense L. sown into subterranean clover pasture soils

Isolates of Rhizobium leguminosarum bv. trifolii were recovered from root nodules of red clover ( Trifolium pratense L.) cullivar Florie sown umnoculated into two acidic Oregon soils of the Whobrey and Abiqua series which had been in subclover ( Trifolium subterraneum L.) pasture for 30–40 yr. Gel-immune-diffusion studies showed that none of the Whobrey isolates were antigenically identical to serogroups WS 1-01 or WS 2-01, previously shown to dominate nodules of field-grown 7'. subterraneum L. in this soil. Twenty-seven and 50% of the Whobrey isolates were placed into two previously unidentified serogroups. WR 26 and WR 27 respectively. Only two of the Abiqua isolates were antigenically identical to any of five serogroups, AS 6, AS 16, AS 17, AS 27 or AS 36, previously recovered from T. subterraneum L. growing in this soil; a previously unidentified serogroup, AR 21 constituted 50% of the isolates. An antigenic relationship between serogroups WR 27 and AS 16 was confirmed by whole-cell somatic tube agglutination, gel immune-diffusion and fluorescent antibody reactions. An incomplete antigenic relationship between serogroups WR 26 and AR 21 was also established. Immunofluorescence counts confirmed the presence of organisms antigenically related to four of the AS serogroups in samples of Whobrey soil recovered from the field. The population densities of some of the indigenous serogroups observed in Whobrey soil were significantly different ( P = 0.05) from each other. Only 5% of Whobrey isolates were ineffective on red clover, whereas 48% of the isolates were incapable of fixing N on subclover. With few exceptions, the Abiqua red clover isolates were ineffective or suboptimally effective on either host. Most isolates which displayed the same N-fixing trait on red clover expressed at least two effectiveness traits when evaluated on subclover.