The Infection of Trifolium subterraneum Root Hairs by Rhizobium trifolii

Both host cultivar and Rhizobium strain influence the numberof infected root hairs of Trifolium subl-errctneum, seedlings;Yarloop had fewer infections than Cranmore, Mount Barker, orTallarook and Rhizobium trifolii strain 5 infected fewer hairsthan strain TA1. Hybrid lines bred for sparse or abundant nodulationhad similar numbers of infected hairs, but. as in the cultivars,these always greatly exceeded the number of nodules formed.More infection threads aborted early during growth in the roothairs of Cranmore than in other hosts and early abortion wasmore common with strain 5 than strain TA1 In all hosts and with both Rhizobium strains, infection beganon day 3 and was initially restricted to one or two zones alongthe root with later infections extending these zones or initiatingnew ones. The exponential rate of infection (least for Yarloop)slows sharply when nodules appear. Early nodules and lateral roots formed at different places indifferent hosts, and in most cultivars and hybrid lines nodulesand laterals occurred in mutually exclusive zones. Primordiaarising above the first nodule failed to develop.     

Influence of Lime and Phosphate on Nodulation of Soil-Grown Trifolium subterraneum L. by Indigenous Rhizobium trifolii

Previous research had identified four serogroups of Rhizobium trifolii indigenous to the acidic Abiqua soil (fine, mixed, mesic Cumulic Ultic Haploxeroll). Nodulation of subterranean clover (Trifolium subterraneum L.) by two of the serogroups, 6 and 36, was differentially influenced by an application of CaCO₃ which raised the pH of the soil from 5.0 to 6.5. These studies were designed to characterize this phenomenon more comprehensively. Liming the soil with either CaCO₃, Ca(OH)₂, MgO, or K₂CO₃ significantly (P = 0.05) increased the percent nodule occupancy by serogroup 36, whereas the percent nodule occupancy by serogroup 6 was decreased, but the decrease was significant (P = 0.05) only after application of either CaCO₃ or Ca(OH)₂. Application of KH₂PO₄ (25 mg of P kg of soil⁻¹), which did not change soil pH, also significantly (P = 0.05) increased the percent nodule occupancy by serogroup 36. Application of KH₂PO₄ in combination with Ca(OH)₂ produced the same increase in nodule occupancy by serogroup 36 as did individual application of the two materials. Soil populations of serogroup 36 consistently, and in the majority of cases significantly (P = 0.05), outnumbered those of serogroup 6 before planting and after harvest regardless of soil treatment or the outcome of nodulation. Soil chemical and plant analyses provided no evidence that liming was simulating phosphate addition by increasing the availability and subsequent uptake of soil P_i by the subclover plants. Liming did, however, result in a significant transformation (30 to 50 mg of P kg of soil⁻¹) of P_i from the residual soil P_i fraction into an NaOH-extractable organic P fraction during the preplant equilibration period.     

The Effect of Moisture Stress on Infection of Trifolium subterraneum L. by Rhizobium trifolii Dang

Moisture stress and method of inoculation greatly affected thenumber and distribution of infected root hairs and nodules ofyoung seedlings of Trifolium subterraneum. A reduction of soilmoisture from 5·5 to 3·5% (–0·36to –3·6 x 10⁵ Pa) significantly decreased the numberof infection threads and completely inhibited nodulation, althoughthe number of rhizobia in the rhizosphere was unaffected. Atlow soil moisture levels the root hairs were abnormally shortand swollen. Infection and nodulation were little affected between5·5 and 9·5% moisture (–0·36 to –0·089x 10⁵ Pa). Distribution of infected root hairs depended on the initialplacement of the inoculum; with the inoculum mixed evenly throughthe soil, infection threads occurred at discrete foci alongthe root. With seedlings inoculated at planting, infection threadswere restricted to the top 1–2 cm of root, even at thehighest soil moisture tested. Watering increased the number of infections in plants grownat 3·5% moisture; nodules were formed at a rate equivalentto non-stressed plants. Watering also enabled movement of theseedling-borne inocula; new infections were formed along theroot surface bearing mature root hairs.     

nodT,a positively-acting cultivar specificity determinant controlling nodulation of Trifolium subterraneum by Rhizobium leguminosarum biovar trifolii

Rhizobium leguminosarum biovar trifolii strain TA1 nodulates a range of Trifolium plants including red, white and subterranean clovers. Nitrogen-fixing nodules are promptly initiated on the tap roots of these plants at the site of inoculation. In contrast to these associations, strain TA1 has a Nod^- phenotype on a particular cultivar of subterranean clover called Woogenellup (A.H. Gibson, Aust J Agric Sci 19: (1968) 907–918) where it induces rare, poorly developed, slow-to-appear and ineffective lateral root nodules. By comparing the nodulation gene region of strain TA1 with that of another R. leguminosarum bv. trifolii strain ANU843, which is capable of efficiently nodulating cv. Woogenellup, we have shown that the nodT gene (B.P. Surin et al., Mol Microbiol 4: (1990) 245–252) is essential for nodulation on cv. Woogenellup. The nodT gene is naturally absent in strain TA1. A cosmid clone spanning the entire nodulation gene region of strain TA1 was capable of conferring nodulation ability to R.l. bv. trifolii strains deleted for nodulation genes, but only on cultivars of subterranean clovers nodulated by strain TA1. This shows that cultivar recognition events are, in part, determined by genes in the nodulation region of strain TA1. Complementation studies also indicated that strain TA1 contains negatively-acting genes located on the Sym plasmid and elsewhere, which specifically block nodulation of cv. Woogenellup.     

Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.     

Effect of Acidity on the Composition of an Indigenous Soil Population of Rhizobium trifolii Found in Nodules of Trifolium subterraneum L

Acidity affected which members of an indigenous soil population of Rhizobium trifolii nodulated Trifolium subterraneum L. cv. Mt. Barker. In three experiments involving plants grown either in mineral salts agar adjusted to pH 4.8 or 6.8 and inoculated with a soil suspension or grown directly in samples of unamended soil (pH 4.8) or soil amended with CaCO(3) (pH 6.4), 121 of 151 isolates of R. trifolii were placed into four serogroups. Seventy-nine of these isolates were placed into two serogroups (6 and 36) whose nodulating ability was affected by the pH of the plant root environment. Representatives of serogroup 6 occupied the greatest percentage of the nodules at the low pH in both mineral salts agar (77%) and in unlimed soil (47 and 57%). The same serogroup was a minor nodule occupant at the higher pH in mineral salts agar (0%) and in limed soil (0 and 10%). In contrast, serogroup 36 was virtually absent in nodules formed at the low pH, whereas it was the dominant serogroup at the higher pH in both mineral salts agar (32%) and in limed soil (35 and 49%). Despite the isolates from within each serogroup being antigenically identical, separation of cellular proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed four and six different gel types within serogroups 6 and 36, respectively. Isolates represented by one or two gel types dominated the contribution of each serogroup to the nodule population. Further evidence for differences between isolates within each gel type were revealed from measurements of symbiotic effectiveness.     

Effect of Acidity on the Composition of an Indigenous Soil Population of Rhizobium trifolii Found in Nodules of Trifolium subterraneum L.

Acidity affected which members of an indigenous soil population of Rhizobium trifolii nodulated Trifolium subterraneum L. cv. Mt. Barker. In three experiments involving plants grown either in mineral salts agar adjusted to pH 4.8 or 6.8 and inoculated with a soil suspension or grown directly in samples of unamended soil (pH 4.8) or soil amended with CaCO₃ (pH 6.4), 121 of 151 isolates of R. trifolii were placed into four serogroups. Seventy-nine of these isolates were placed into two serogroups (6 and 36) whose nodulating ability was affected by the pH of the plant root environment. Representatives of serogroup 6 occupied the greatest percentage of the nodules at the low pH in both mineral salts agar (77%) and in unlimed soil (47 and 57%). The same serogroup was a minor nodule occupant at the higher pH in mineral salts agar (0%) and in limed soil (0 and 10%). In contrast, serogroup 36 was virtually absent in nodules formed at the low pH, whereas it was the dominant serogroup at the higher pH in both mineral salts agar (32%) and in limed soil (35 and 49%). Despite the isolates from within each serogroup being antigenically identical, separation of cellular proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed four and six different gel types within serogroups 6 and 36, respectively. Isolates represented by one or two gel types dominated the contribution of each serogroup to the nodule population. Further evidence for differences between isolates within each gel type were revealed from measurements of symbiotic effectiveness.     

The intracytoplasmic membrane system of the bacteroids of subterraneum clover nodules (Trifolium subterraneum L.)

Summary A vesicular intracytoplasmic membrane system is demonstrated in bacteroids from the leghaemoglobin filled zone of effective Trifolium subterraneum nodules after KMnO₄ and OsO₄ fixation. The system appears to be present in all mature bacteroids from this zone, and is derived from tubular invaginations of the plasma membrane of the bacteriod. A granular substance similar to the bacteroid cytoplasm is found in the vesicles which are bounded by a tripartite membrane approximately 80 Å wide, while the interspace between the vesicles is filled with a material of similar appearance to that in the interspace between bacteroid plasma membrane and cell wall.     

Competition between soil- and seed-borneRhizobium trifolii in nodulation of introducedTrifolium subterraneum

Summary Native rhizobia associated withTrifolium albopurpureum, T. bifidum, T. ciliolatum, T. depauperatum, T. dichotomum, T. flavulum, T. melanthum, T. microcephalum, T. microdon, T. oliganthum andT. tridentatum were found in Northern California range soils. These rhizobia nodulate subterranean clover but are ineffective in nitrogen fixation with this host. Native rhizobia compet with those in commercial inoculants to form nodules. To ensure effective nodulation by nitrogen fixing rhizobia, commercial inoculants should be applied at rates greater than those recommended by the manufacturerse Effective nodulation was achieved by an application of 7.5×10⁴ rhizobia per seed, four times the recommended rate.     

Root Rot of Trifolium subterraneum Induced by Fusarium solani

A fungus disease of Trifolium subterraneum, previously unknown in South Africa, was identified as Fusarium root rot caused by F. solani (Mart.) Sacc. Diseased plants were severely stunted. The characteristic internal root discolouration observed in the field, is described. The disease occurred mainly between Swellendam and Humansdorp in the southern and south-eastern Cape Province. The subterranean clover fields inspected were between 95 and 100 % infected with the root rot disease. T. repens and T. pratense were also susceptible.     

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.)

We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait.     

Relationship between Colour, Phenolic Content and Impermeability in the Seed Coat of various Trifolium subterraneum L. genotypes

The relationship between seed colour, phenol content of thetesta and water impermeability in dark and light seeded genotypesof Trifolium subterraneum L. was investigated. The developmentof water impermeability and catechol oxidase activity in expandingseeds of two genotypes was monitored. The results show catecholoxidase activity decreases as seed colour changes from greento purple, but the potential to become impermeable with dehydrationis not acquired until later in seed development. Both waterpermeable and impermeable seeds of dark coloured genotypes containsubstantial amounts of phenol in the lumen of the Malpighiancells; light coloured testas do not. It is concluded that the darkening of the testa of Trifoliumsubterraneum is associated with oxidation of phenol by catecholoxidase but that development of impermeability is independentof this process. Trifolium subterraneum L., subterranean clover, seed coat impermeability, catechol oxidase, phenol, testa colour     

Herbicide effects on the growth and nodulation potential ofRhizobium trifolii withTrifolium subterraneum L.

A study was made of the effect of the herbicides 2,4-D, amitrole, atrazine, chlorsulfuron, diclofop-methyl, diquat, glyphosate, paraquat and trifluralin on the nodulation of sub-clover (Trifolium subterraneum L. ‘Clare’), the growth ofR. trifolii TA1 in liquid nutrient medium and the ability of herbicide-treated inoculum to successfully nodulate sub-clover plants. As concentrations of amitrole, diclofop-methyl and glyphosate in the rooting environment increased from 0 to 20 mg ai L⁻¹, nodulation decreased linearly. The other herbicides at these concentrations caused more severe decreases in nodulation. Growth ofR. trifolii TA1 in nutrient broth was significantly retarded by all concentrations of diquat, 2 mg ai L⁻¹ of paraquat, 10 mg ai L⁻¹ of glyphosate and 2 mg ai L⁻¹ of chlorsulfuron. Other herbicides did not suppress rhizobial growth. Inoculation with TA1 that had been grown in the presence of amitrole, atrazine or glyphosate and then washed free of the herbicide decreased nodulation of sub-clover, indicating that these herbicides may physiologically influence the nodulating potential of certain strains of Rhizobium. The remaining herbicides showed no indications of this effect.     

Symbiotic Characteristics of Rhizobium leguminosarum bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover (Trifolium subterraneum L.)

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.     

Calcium and Nodulation in Subterranean Clover (Trifolium subterraneum L.)

From a study of the effects of Ca ions on the nodulation of subterranean clover in flowing culture solutions it is concluded that root infection or nodule initiation has a higher Ca requirement than either nodule development or host plant growth in the presence of fixed nitrogen.