Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

Distribution of dry weight between root and shoot in white clover dependent on N2 fixation or utilizing abundant nitrate nitrogen

Summary Plants of white clover var. Blanca were grown singly in pots in controlled environments, or in small swards in a glasshouse, to determine how their distribution of dry weight between root and shoot was influenced when they were dependent on N₂ fixation in their root nodules or when they lacked nodules but utilized an abundant supply of nitrate nitrogen. In single plants and in swards, changes in root/shoot ratio with increasing age and plant development were not influenced by the source of nitrogen, but nodulated plants always displayed a higher root/shoot ratio. When nodulated plants were supplied with nitrate nitrogen, root/shoot ratio declined to values intermediate between those of nodulated and of nitrate plants. The results are discussed in relation to the persistence of white clover, and the general level of productivity, in grass-clover swards. The Grassland Research Institute is financed through the Agricultural Research Council.     

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.     

Studies on the Characterization of Root Morphology of White Clover Infected by Transconjugant of Rhizobium leguminosarum

White clover plants were inoculated with transconjugant strain' 290 which was obtained from introduction of host specific nodulation genes of wild-type Rhizobium trifolii strain ANU 843 to Rhizobium leguminosarum strain 300. The characterization of root morphology of white clover induced by the transconjugant was observed and compared to the plants induced by the parent strains. White clover started tO form a typical root hair curling inoculated with transconjugant strain 290 24h after inoculation, at 48h a part of cell wall of root hair was degradated, infection thread was observed in the infected root hair cell, cortical cell divisions occurred extensively. All these characterizations were similar to that infected by strain ANU 843. Plant inoculation test indicated that no nodule was formed when inoculated by R. leguminosarum strain 300, while plants nodulated when inoculated with transconjugant strain 290 as well as R. trifolii ANU 843. This suggests that introduction of host specific nodulation genes of R. trifolii results in conferring the nodulation ability of R. leguminosarum on white clover.     

Sym plasmid genes of Rhizobium trifolii expressed in Lignobacter and Pseudomonas strains.

A 14-kilobase (kb) fragment of Rhizobium trifolii Sym plasmid containing nodulation (nod) genes or the pSym plasmid of R. trifolii cointegrated with a broad-host-range vector R68.45 (pPN1) were transferred to Lignobacter strain K17 and Pseudomonas aeruginosa strain PAO5 by conjugation. Lignobacter transconjugants carrying Sym plasmid pPN1 formed nodules on white, red, and subterranean clover plants. Lignobacter transconjugants containing a 14-kb fragment of nod genes cloned into a multicopy plasmid nodulated only white and subterranean clover plants, whereas transconjugants carrying the same fragment cloned into a low-copy plasmid vector nodulated only white clover plants. All nodules formed by Lignobacter transconjugants showed bacterial release from the infection threads into the host cytoplasm. Pseudomonas transconjugants with plasmid pPN1 formed nodule-like structures on white clover plants. These structures were not invaded by bacteria; however, a few bacteria were found within the intercellular spaces of the outermost cells of the structures. Pseudomonas transconjugants carrying the 14-kb fragment of R. trifolii nod genes did not form nodules on tested clover plants. All clover plants inoculated with either Pseudomonas or Lignobacter transconjugants containing a 14-kb fragment of nod genes (but not entire Sym plasmid) showed the "thick-and-short-root" response when compared to the control plants inoculated with the R. trifolii wild-type strain.     

THE EFFECTIVENESS OF STRAINS OF THE NODULE ORGANISM WHEN ASSOCIATED WITH DIFFERENT SPECIES OF CLOVER

The effectiveness in nitrogen fixation of fifty-four strains of the clover nodule organism has been tested on red, white and alsike clover in aseptic tube-culture by comparing the nitrogen content of nodulated plants grown on nitrogen-free medium against that of non-nodulated plants grown on media with and without nitrate-nitrogen. The performance of the strains was to a considerable degree consistent on all three hosts. Minor changes were shown but for the most part did not exceed the experimental error. Thus in respect of these three clovers there is no marked host species effect. Seed inoculants or naturally occurring strains effective on one species are likely to be effective on the other species.     

A STUDY OF THE BACTERIA ASSOCIATED WITH THE ROOTS OF SUBTERRANEAN CLOVER AND WIMMERA RYE-GRASS

SUMMARY: A study of the bacteria from the surfaces of roots of subterranean clover ( T. subterraneum L.) and Wimmera rye-grass ( L. rigidum Gaud.) revealed that 21 genera were represented among the isolates from clover and 16 genera among those from rye-grass. Bacteria showing branched forms predominated and accounted for 63% of the 151 clover isolates and 78% of the 167 grass isolates. Most of these were identified as Arthrobacter , but from clover a significant proportion were Nocardia -like types. Members of the genera Mycoplana, Micromonospora, Mycobacterium , and Mycococcus were also identified among the branching forms.
Although the soil had been inoculated with effective rhizobia and the clover plants were effectively nodulated only one of the 318 isolates was capable of nodulating subterranean clover. The majority of the isolates were chromogenic and Gram-negative, produced acid from glucose and ammonia from peptone, were catalase-positive and grew best aerobically. Approximately half the isolates liquefied gelatin and produced hydrogen sulphide from peptone.     

Symbiosis of Leguminous Plants and Nodule Bacteria: V. The Growth of Red Clover at Different Oxygen Tensions

A comparison has been made of the effect of oxygen concentrationon the growth of nodulated red clover plants dependent on nodule-nitrogenand of non-nodulated plants provided with ammonium-nitrogen.Two techniques have been used, the first providing for the exposureof the root system only to adjusted oxygen supply, while inthe second the whole plant was so exposed. Four levels of oxygenwere employed, namely, 21, 12, 5, and 1 per cent. With the first method each step in the reduction of oxygen supplywas attended by a significant curtailment of growth in nodulatedplants. The responses of the ammonium plants were less marked.It is concluded that clover nodules function best when wellsupplied with oxygen. No clear effects of oxygen supply were obtained by the secondmethod, this being due, it is considered, to the severe limitationimposed by the necessary experimental arrangement on the growthof the plants.     

Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables

The identification and conservation of indigenous rhizobia associated with legume plants and their application as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diversity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phenotypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnII and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic composition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance.     

Interaction of Azospirillum and Rhizobium Strains Leading to Inhibition of Nodulation

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac⁻), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.     

Effects of Nitrogen Nutrition on Leaf Expansion and Photosynthesis of Trifolium subterraneum L. II. Comparison between Nodulated Plants and Plants Supplied with Combined Nitrogen

Subterranean clover plants depending on symbiotic nitrogen fixationhad smaller leaf areas than control plants supplied with combinednitrogen in the nutrient solutions. There were no differencesin chlorophyll content per unit fresh weight of leaves or petioles,nor in net rates of carbon dioxide uptake per unit leaf areaat light intensities above 2000 fc and at carbon dioxide concentrationsabove 300 ppm. Dark respiration by the shoots of the nodulatedplants was considerably higher than for the controls. This couldhave been a direct result of nodule activity and is suggestedas a possible factor contributing to the slower growth of theseplants compared with the controls. A comparison of the nitrogen contents of shoots and roots showeda sub-optimal nitrogen status, particularly in the roots, ofthe nodulated plants. This is suggested as another factor contributingto the slower growth of the nodulated plants compared with thecontrols. The response patterns before and after the addition of combinednitrogen differed in a number of important respects from thosefound previously under conditions of a sub-optimal nitrogensupply in the nutrient solution outside the roots. These arebriefly discussed.     

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover: II. COMPARISONS OF THE COST OF NITROGEN FIXATION AND THE UTILIZATION OF COMBINED NITROGEN

Plants of soyabean, cowpea, and white clover were grown singlyin pots in Saxcil growth cabinets at 23/18 °C, 30/24 °C,and 20/15 °C, respectively, until seed maturation or for85 d (white clover). Two populations were produced within eachspecies: one population effectively nodulated and wholly dependentfor nitrogen on fixation in the root nodules, and a second populationcompletely lacking nodules but receiving abundant nitrate nitrogen.In each species, the two populations were compared in termsof rate of gross photosynthesis, rate of shoot respiration,and rate of root respiration. Source of nitrogen had littleor no effect on rate of photosynthesis or shoot respiration.In contrast, the rate of respiration of the nodulated rootsof plants fixing their own nitrogen was greater, sometimes two-foldgreater, than that of equivalent plants lacking nodules andutilizing nitrate nitrogen. This superiority in terms of rateof root respiration was generally confined to the period ofintense nitrogen fixation. An analysis of the magnitude of thisrespiratory burden in terms of daily photosynthesis indicatesthat, in all three legumes, plants fixing their own nitrogenrespire 11–13% more of their fixed carbon each day thanequivalent plants lacking nodules and utilizing nitrate nitrogen.     

Effect of cadmium, chromium and copper on symbiotic and free-living Rhizobium leguminosarum biovar trifolii

Abstract Plasmid-minus derivatives of Rhizobium leguminosarum biovar trifolii have been isolated. Cured strains lacked symbiotic properties, however they showed increased heavy metal resistance. In the presence of 70 ppm chromium the parent strain, unlike cured derivatives, is unable to grow explanta but can nevertheless nodulate clover.
We propose that rhizobia can circumvent exposure to the heavy metal by entering the plant roots.
Acetylene reduction tests showed that nodulated plants, grown in the presence of 10 ppm of chromium, had an increased nitrogenase activity compared to the control plants.     

Short-term Changes in CO2 Evolution Associated with Nitrogenase Activity in White Clover in Response to Defoliation and Photosynthesis

Direct, continuous measurements of the CO₂ evolution of rootnodules, calibrated by direct measurements of rate of ethyleneproduction, were utilized to determine the short-term responseof nitrogenase activity to defoliation and photosynthesis inwhite clover. Defoliation (removal of all expanded leaflets) generally resultedin a fall in nodulated root respiration within 10 min; mostrespiration associated with nitrogenase activity ceased within1–2 h. Darkening of the shoot also reduced nodulated root respirationwithin 10 min, but the subsequent fall in respiration, althoughof the same magnitude, was slower. The re-illumination of shootslargely reversed these effects. The inhibition of photosynthesisby DCMU largely simulated the effects of darkening the shoots. It is concluded that, in these white clover plants of 100 mgto 2.0 g total weight, current photosynthate provides the primarysource of energy for N₂ fixation. The mobilization of reserveenergy substrate appeared to play only a small role. The minimumtime interval of 10 min between onset of treatment and fallin nodule respiration probably reflects the time taken to exhaustthe assimilate in transit between leaf and nodule. Key words: White clover, N₂ fixation, Defoliation, Photosynthesis     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

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     

Comparisons of the Respiratory Effluxes of Nodules and Roots in Six Temperate Legumes

The specific respiration rates of nodulated root systems, ofnodules and of roots were determined during active nitrogenfixation in soya bean, navy bean, pea, lucerne, red clover andwhite clover, by measurements on whole plants before and afterthe removal of nodule populations. Similar measurements weremade on comparable populations of the six legumes, lacking nodulesbut receiving abundant nitrate-nitrogen, to determine the specificrespiration of their roots. All plants were grown in a controlled-environmentclimate which fostered rapid growth. The specific respiration rates of nodulated root systems ofthe three grain and three forage legumes during a 7–14-dayperiod of vegetative growth varied between 10 and 17 mg CO₂g^–1 (dry weight) h^–1. This mean value consistedof two components: a specific root respiration rate of 6–9mg CO₂ g^–1 h^–1 and a specific nodule respirationrate of 22–46 mg CO₂ g^–1 h^–1. Nodule respirationaccounted for 42–70 per cent of nodulated root respiration;nodule weight accounted for 12–40 per cent of nodulatedroot weight. The specific respiration rates of roots lackingnodules and utilizing nitrate nitrogen were generally 20–30per cent greater than the equivalent rates of roots from nodulatedplants. The measured respiratory effluxes are discussed in thecontext of nitrogen nitrogen fixation, nitrate assimilation. Glycine max, Phaseolus vulgaris, Pisum sativum, Medicago sativa, Trifolium pratense, Trifolium repens, soya bean, navy bean, pea, lucerne, red clover, white clover, nodule respiration, root respiration, fixation, nitrate assimilation     

Enhancement of symbiotic nitrogen fixation by vitamin-secreting fluorescent Pseudomonas

Fluorescent Pseudomonas sp. strain 267 promotes growth of nodulated clover plants under gnotobiotic conditions. In the growth conditions (60 M FeCl₃), the production of siderophores of the pseudobactin-pyoverdin group was repressed. Plant growth enhancement results from secretion of B vitamins by Pseudomonas sp. strain 267. This was proven by stimulation of clover growth by naturally auxotrophic strains of Rhizobium leguminosarum bv. trifolii and marker strains E. coli thi^- and R. meliloti pan^- in the presence of the supernatant of Pseudomonas sp. strain 267. The addition of vitamins to the plant medium increased symbiotic nitrogen fixation by the clover plants.     

The effect of seed weight on the early growth and nodulation of white clover

The results of growing, in agar culture, nodulated and un-nodulated white clover seedlings from three seed-weight categories, and the subsequent growth of selected plants in soil in pots, are reported. Seed-weight variations greatly influenced plant growth, made substantial differences in nodulation parameters and nitrogen fixation, and contributed appreciably to the observed variability. The relationship of such effects to nitrogen fixation and the relevance to breeding for superior symbiosis are discussed. The growth of plants, as determined by agar culture technique, was not predictive of their subsequent performance in the soil in pots, and restricting seed weight variations did not improve the prediction. The importance of such effects and the relevance of tube culture to breeding work are discussed.     

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.