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.     

Breeding of nodulated red clover (Trifolium pratense) for high yield

The symbiotic effectiveness of four cultivars of red clover were compared and breeding programmes undertaken to increase nitrogen fixation and yield of agar- and pot-grown plants. Programme I used the moderately effective Rhizobium trifolii strain 0403 and Programme II the highly effective strain 5. Aggregate scores of plant size (leaf area) were chosen as the criterion of selection. All cultivars, inoculated with strain 0403, differed in time of initial nodulation and in dry matter yield and three differed in leaf area. None differed in nodule number or N-content. Dry matter and leaf area were highly correlated. Diallel crosses among highly effective selections in Programme 1 gave progeny yields that exceeded those of crosses between modal selections by averages of 6% in the first generation, 5% in the second generation and 23% in the third generation. Yields of the high cross category exceeded those of the original cultivar by an average of 9% in the second generation and by 25–101% in the third generation. Effects on leaf area were similar. Highly effective progeny tended to nodulate earlier and have more nodules than the remainder but differences were very small. Crosses between cultivars were generally more effective than those within cultivars, indicating heterotic effects. Similar results for yield were obtained in Programme II. The average increase in yield of crosses of third-generation material between highly effective selections compared with cv. S123 was 63%. The modal crosses were intermediate. In each Programme and generation there were large differences between parents within each cross category.     

Symbiotic and competitive properties of motility mutants of Rhizobium trifolii TA1

Non-motile mutants of Rhizobium trifolii defective in either flagellar synthesis or function were isolated by transposon Tn5 mutagenesis. they were indistinguishable from motile control strains in growth in both laboratory media and in the rhizosphere of clover roots. When each non-motile mutant was grown together with a motile strain in continuous culture, the numbers of motile and non-motile organisms remained in constant proportion, implying that their growth rates were essentially identical. When inoculated separately onto clover roots, the mutants and wildtype did not differ significantly in the number of nodules produced or in nitrogen fixing activity. However, when mixtures of equal numbers of mutant and wild-type cells were inoculated onto clover roots, the motile strain formed approximately five times more nodules than the flagellate or non-flagellate, non-motile mutants, suggesting that motility is a factor in competition for nodule formation.     

Succinate-Induced Morphology of Rhizobium trifolii 0403 Resembles That of Bacteroids in Clover Nodules

Morphological changes which accompany nutrient enrichment of Rhizobium trifolii 0403 were studied. Assays of cell number and size coupled with scanning electron microscopy and immunofluorescence microscopy showed that succinate induces cells to stop dividing in vitro and to swell either in the cell center or at one cell pole. The extent and frequency of in vitro cell swelling were in direct relation to the concentration of succinate added to the enrichment medium. The in vitro swelling of cells in 16.6 mM succinate plus Casamino Acids, glucose, and yeast extract closely resembled that of bacteroids of R. trifolii 0403 in nitrogen-fixing nodules of white clover. We hypothesize that succinate may be involved in the transformation of vegetative bacteria into the bacteroid morphology found in nitrogen-fixing nodules.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Establishment of phage and bacteria in a sterilized compost in a glasshouse

Summary Four strains ofRhizobium trifolii were individually inoculated to pots containing sterilized sand vermiculite mixture, half of which were seeded with red clover and half not. Pots were maintained in an ordinary glasshouse and watered with tap water.Phage was first detected after 4 months, and almost all pots contained one or more phages againstRhizobium trifolii after 9 months. The presence of plants increased the titer of phages in some pots inoculated withR. trifolii, but had no effect on the number of different phages.The pots also contained phages against soil bacteria other than Rhizobium indicating that phages are spread readily and constitute a normal part of the life cycle of soil bacteria.The number of different phages isolated from the pots was affected by the strain of Rhizobium used as inoculum.     

丛枝菌根真菌和根瘤菌对白三叶氮同化的影响

为揭示丛枝菌根真菌(AMF)和根瘤菌在白三叶氮(N)同化中的作用,该研究对白三叶进行单一或联合接种隐类球囊霉(Paraglomus occultum)和三叶草根瘤菌(Rhizobium trifolii),分析其对白三叶的生长、光合作用、叶片N和氨基酸含量以及N同化相关酶活性的影响。结果表明:(1)单一接种AMF或根瘤菌以及联合接种AMF和根瘤菌均显著增加了白三叶的株高、匍匐茎长度、叶片数、地上部生物量、总生物量、叶绿素b和总叶绿素含量、稳态光量子效率和叶片N含量,这种增强效应是联合接种>单一AMF>单一根瘤菌>未接种处理。(2)联合接种AMF和根瘤菌显著增加了白三叶叶片中丙氨酸、精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸和组氨酸的含量,显著提升了叶片N同化相关酶如硝酸还原酶、亚硝酸还原酶、谷氨酰胺合成酶、谷氨酸合成酶、谷氨酸脱氢酶、天冬酰胺合成酶和天冬氨酸转氨酶的活性,显著促进AMF对白三叶根系的侵染。综上认为,联合接种AMF和根瘤菌通过激活N同化相关酶活性有效促进N同化,产生更多氨基酸,进一步促进白三叶植株生长; 联合接种AMF和根瘤菌具有协同作用,有效促进了白三叶的N同化。     

Improving N2 fixation from the plant down: Compatibility of Trifolium subterraneum L. cultivars with soil rhizobia can influence symbiotic performance

Plant genotypes of Trifolium subterraneum L. (subterranean clover) were evaluated for differences in symbiotic N₂ fixation with soil rhizobia, with the long-term aim of using plant selection to overcome sub-optimal N₂ fixation associated with poorly effective soil rhizobia. Symbiotic performance (SP) was assessed for 49 genotypes of subterranean clover with each of four pure Rhizobium strains isolated from soil. Plants were grown in N free media in the greenhouse and their shoot dry weights measured and expressed as a percentage of dry weight with R. leguminosarm bv. trifolii WSM1325, the recommended commercial inoculant. Average SP with two Rhizobium strains (H and J) ranged from completely ineffective to 80% of potential for the subterranean clover genotypes. Two clover cultivars with high (cv. Campeda) and low (cv. Clare) SP values were investigated in more detail. Campeda typically fixed more N₂ than Clare when inoculated with 30 soil extracts (4.2 vs 2.4 mg N₂ fixed/shoot) and with 14 pure strains isolated from those soils (4.2 vs 2.2 mg N₂ fixed/shoot). The poor performance of Clare could be attributed to interruptions at multiple stages of the symbiotic association, from nodule initiation (less nodules), nodule development (small, white nodules), through to reduced nodule function (N₂ fixed/mg nodule) depending on the inoculation treatment. Through the careful use of subterranean clover genotypes by plant breeders it should be possible to make significant gains in the SP of future subterranean clover cultivars.     

Competition among Strains of Rhizobium leguminosarum biovar trifolii and Use of a Diallel Analysis in Assessing Competition

Competition between indigenous Rhizobium leguminosarum biovar trifolii strains and inoculant strains or between mixtures of inoculant strains was assessed in field and growth-room studies. Strain effectiveness under competition was compared with strain performance in the absence of competition. Field inoculation trials were conducted at Elora, Ontario, Canada, with soil containing indigenous R. leguminosarum biovar trifolii. The indirect fluorescent-antibody technique was used for the identification of nodule occupants. Treatments consisted of 10 pure strains, a commercial peat inoculant containing a mixture of strains, and an uninoculated control. Inoculant strains occupied 17.5 to 85% of nodules and resulted in increased dry weight and nitrogen content, as compared with the uninoculated control. None of the strains was capable of completely overcoming resident rhizobia, which occupied, on average, 50% of the total nodules tested. In growth-room studies single commercial strains were mixed in all possible two-way combinations and assessed in a diallel mating design. Significant differences in plant dry weight of red clover were observed among strain combinations. Specific combining ability effects were significant at the 10% level, suggesting that the effectiveness of strain mixtures depended on the specific strain combinations. Strains possessing superior effectiveness and competitive abilities were identified by field and growth-room studies. No relationship was detected between strain effectiveness and competitive ability or between strain recovery and host cultivar. The concentration of indigenous populations was not considered to be a limiting factor in the recovery of introduced strains at this site.     

Trifolin: A Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 μg protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with M_r ≈ 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-d-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.     

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.     

Influence of Azospirillum Strains on the Nodulation of Clovers by Rhizobium Strains

Mixed cultures of several Azospirillum and Rhizobium trifolii strains caused either an inhibition or stimulation of nodule formation on plant hosts as compared with nodulation of plants inoculated with R. trifolii alone. Azospirillum strains affected the nodulation process at a precise cell ratio (R. trifolii/Azospirillum cells) and time of inoculation. All Azospirillum strains used showed a variation in their ability to inhibit or enhance nodulation by R. trifolii strains. When nonviable cell preparations of Azospirillum strains were used for mixing experiments, no effect on nodulation was observed. A decrease in the effectiveness of normally Nod⁺ Fix⁺R. trifolii strains was observed when an Azospirillum strain caused an increase in nodule number.     

Specific and general effectiveness ofRhizobium trifolii populations from different agricultural locations

Summary The effectiveness ofRhizobium trifolii isolates from five locations in southern Britain representing contrasting soil types has been examined with five white clover varieties. The average effectiveness of Rhizobium isolates varied considerably as did the average productivity of plant varieties. The largest differences were, however, associated with Rhizobium population × plant variety interactions. These were often large enough to reverse relative yield differences between white clover varieties. The implications of these results for improving clover productivity in nitrogen fixation are discussed.     

The effects of shading and defoliation on nodulation and nitrogen fixation by white clover

In a glasshouse experiment, single plants of ten-weeks old white clover (Trifolium repens L.) were subjected to two levels of shading and two levels of defoliation. Nodulation and nitrogen fixation parameters were measured at six sequential harvest over four weeks. Changes in nodule number and hence nodule dry weight per plant were due to nodule decay, sloughing off and non-production and were closely related to losses in root dry weight. Severe defoliation caused degradation of leghaemoglobin, an effect which was seen in less than three days from treatment. It led also to a temporary but marked decrease in the nitrogen fixation capacity of the nodules as measured by the acetylene reduction assay. Recovery of normal activity by the nitrogenase system took about ten days. The effects of shading and defoliation on the pattern of nodulation have been described briefly.     

Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis

Either NO₃⁻ (16 millimolar) or NH₄⁺ (1 millimolar) completely inhibited infection and nodulation of white clover seedlings (Trifoliin repens) inoculated with Rhizobium trifolii. The binding of R. trifolii to root hairs and the immunologically detectable levels of the plant lectin, trifoliin, on the root hair surface had parallel declining slopes as the concentration of either NO₃⁻ or NH₄⁺ was increased in the rooting medium. This supports the role of trifoliin in binding R. trifolii to clover root hairs. Agglutination of R. trifolii by trifoliin from seeds was not inhibited by these levels of NO₃⁻ or NH₄⁺. The results suggest that these fixed N ions may play important roles in regulating an early recognition process in the Rhizobium-clover symbiosis, namely the accumulation of high numbers of infective R. trifolii cells on clover root hairs.     

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.     

Fine Structure of Succinate-Swollen Rhizobium trifolii 0403

Transmission electron micrographs of glutaraldehyde- OsO₄-fixed Rhizobium trifolii 0403 before and after cells were treated with 16.6 mM succinate showed that treated cells increased in mass by increasing cytoplasmic volume. The morphology of succinate-treated cells was identical to that of bacteroids, and the appearances of the envelope and periplasmic space were similar. The primary difference was in inclusion number and type.     

Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover

White clover root hairs which were inoculated with Rhizobium trifolii 4S (infectious strain) contained infection threads which were observed by light microscopy and scanning electron microscopy. Three morphological types of root hairs retaining infection threads were recognized. The bacteria were strongly attached between the surfaces of two plant cell walls as follows: between surfaces of a root hair tip curled back on itself, between a protuberance from a root hair and its cell surface, or between two root hair tips clinging together. An anatomical analysis documented the attachment site of the infection thread sheath from the inside of the root hair cell.