Competition studies withRhizobium trifolii in laboratory experiments

Summary Competition studies were carried out in soil cores by comparing the nodule forming ability of antibiotic resistance marked strains, inoculated at three inoculum levels onTrifolium repens versus an effective indigenous Rhizobium population of 1.5×10⁵/gm. It was seen that the indigenous marked isolate G1067 formed a high proportion of nodules sampled (>90%) at all three inoculum levels (10⁵, 10⁷ and 10⁹ cells/seed) wherein the introduced foreign strain G1032 formed >50% of the nodules at the highest inoculum level.In a test tube experiment, competition for nodule sites was examined by inoculating mixtures of twoR. trifolii strains at different input levels on two cultivars ofTrifolium repens var. Huia and Titan. It was seen that G1032 was less competitive than G1006 and G1067 on cv. Huia but was more competitive on cv. Titan than the other two strains.     

Interstrain Competition between Representatives of Indigenous Serotypes of Rhizobium trifolii

The symbiotic characteristics of Rhizobium trifolii strains 1-01 and 2-01 were evaluated both individually and in various combinations on two cultivars (Mt. Barker and Woogenellup) of subterranean clover (Trifolium subterraneum L.). Nodules were observed on day 8 independent of cultivar or strain. Cultivar differences were measured in nodulating efficiency by 1-01 since 54% of the primary nodules were formed on cv. Mt. Barker and only 15% were formed on cv. Woogenellup in the zone above, or 1 cm below, the root tip location at the time of inoculation. The percentage of nodules formed in this zone by 2-01 was similar on both cultivars (31 to 32%). When mixtures of strains 1-01 and 2-01 (230:1 and 1:20) were used to inoculate plants, >90% of the nodules on both cultivars were occupied by the more abundant strain in the inoculum regardless of sampling date (4 or 8 weeks). In contrast, large percentages of nodules on 4-week-old plants of both cultivars exposed to a 5:1 inoculum mixture were doubly occupied (64 and 74%). By week 8 these values had decreased significantly (P ≤ 0.01) and were accompanied by large increases in the percentage of nodules occupied by either strain 1-01 alone (1 to 65%) on cv. Mt. Barker or 2-01 alone (4 to 49%) on cv. Woogenellup. The superior (cv. Mt. Barker) and inferior (cv. Woogenellup) symbiotic performance of plants inoculated with the 5:1 mixture correlated more closely with the 8-week than the 4-week nodule occupancy data. Primary nodule occupancy by 1-01 and 2-01 was significantly influenced by changes in the inoculum ratios of 1-01/2-01 from 5.7:1 to 0.67:1 on cv. Mt. Barker and from 1.9:1 to 0.67:1 on cv. Woogenellup. Despite evidence for extensive proliferation of the inoculant strains on the rhizoplanes, no evidence was obtained for either interstrain antagonism or selective proliferation as a valid reason to explain the outcome of primary nodulation.     

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.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Genetic transformation in Rhizobium trifolii.

Markers controlling the synthesis of amino acids and organic bases as well as streptomycin resistance and sensitivity to acriflavine were transformed in Rhizobium trifolii. The results indicate that the str marker was transformed independently of leu, his, ade and trp markers. Co-transformation of leu and utra markers ranged from 3 to 7%, whereas that of thi and acr was 10%.     

Bacteriocinogeny of Rhizobium trifolii.

Rhizobium trifolii strains differing in cell and colony morphology, streptomycin resistance, phage sensitivity pattern and infectivity to clover plants produced bacteriocins sensitive to proteases. Elimination of bacteriocin production ability wtih SDS and rifampicin treatment indicates that this feature is plasmid controlled. Elimination of bacteriocinogenic plasmid did not influence other features of R. trifolii.     

Role of Microniches in Protecting Introduced Rhizobium leguminosarum biovar trifolii against Competition and Predation in Soil

The importance of microniches for the survival of introduced Rhizobium leguminosarum biovar trifolii cells was studied in sterilized and recolonized sterilized loamy sand and silt loam. The recolonized soils contained several species of soil microorganisms but were free of protozoa. Part of these soil samples was inoculated with the flagellate Bodo saltans, precultured on rhizobial cells. The introduced organisms were enumerated in different soil fractions by washing the soil, using a standardized washing procedure. With this method, free organisms and organisms associated with soil particles or aggregates >50 μm were separated. The total number of rhizobia was influenced slightly (silt loam) or not at all (loamy sand) by the recolonization with microorganisms or by the addition of flagellates alone. However, when both flagellates and microorganisms were present, numbers of rhizobia decreased drastically. This decrease was more than the sum of both effects separately. Nevertheless, populations of rhizobia were still higher than in natural soil. In the presence of flagellates, higher percentages of rhizobia and other microorganisms were associated with soil particles or aggregates >50 μm than in the absence of flagellates. In recolonized soils, however, the percentages of particle-associated rhizobia were lower than in soils not recolonized previous to inoculation. Thus, the presence of other microorganisms hindered rhizobial colonization of sites where they are normally associated with soil particles or aggregates.     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate     

Rhizobium trifolii mutants deficient in exopolysaccharide production

Spontaneous mutants of Rhizobium trifolii 24AR5 which did not produce exopoly-saccharide were isolated. The non-mucoid mutants formed small white and ineffective nodules on both red and white clover. These nodules contained infection threads, but only a small number of bacteria were released into nodule cells, and bacteroids were rarely observed. The non-mucoid phenotype was not complemented by the symbiotic plasmid (pJB5JI) of Rhizobium leguminosarum.     

Plasmid deoxyribonucleic acid in Rhizobium trifolii.

In deoxyribonucleic acid of Rhizobium trifolii centrifuged in cesium chloride-ethidium bromide equilibrium was found a sattelite peak containing covalently closed circular deoxyribonucleic acid. The plasmid had a molecular weight of about 64 x 10(6) shown by sedimentation in sucrose gradients and electron microscopy.     

Polyol metabolism by Rhizobium trifolii.

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.