A comparison of competitiveness and persistence amongst five strains of Rhizobium tripolii

Five strains of Rhizohium trifolii were used to inoculate Trifolium subterraneum cv. Woogenellup sown into two soils with naturally-occurring populations of R. trifolii. In the 1st year all inoculant strains used singly were present at high frequency in the sampled nodule populations from the inoculated plots. Where an inoculant containing a mixture of equal parts of the 5 strains was used. one strain (WU95) predominated at both sites.The persistence of the strains was followed for a further 3 years at one site. Three of the strains WU95, CC2480a and WU290, were maintained at a high frequency (>75% of nodules sampled) for the entire period, but the other two strains showed poor persistence in this environment. Highly effective strains of rhizobia, not identifiable as inoculant strains, nor present in the nodule population at the first sampling, appeared during the course of the study. One strain. WU290, showed a high degree of variation in symbiotic effectiveness between single colony isolates from the stock culture and also between field isolates that were serologically identical with this strain.     

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand-vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.     

Competition for nodule occupancy between introduced and native strains of Rhizobium leguminosarum biovar trifolii

The aim of this work was to evaluate the competitive ability between Rhizobium leguminosarum bv trifolii strain U204 used as commercial inoculants in Uruguay for Trifolium repens L. and Trifolium pratense L. and two native strains isolated from inoculated pastures of T. pratense. T126 is an efficient nitrogen fixer and a melanin producer strain; T70 is inefficient and a melanin non-producer strain; and U204 is very efficient in both hosts but is a melanin non-producer strain. Competitiveness between the strains was determined in experiments in pots and in growth pouches under controlled conditions. In the last experiment, we evaluated pH of plant nutrient solution and inoculum ratios. Plant dry weight was determined, and the identification of nodule bacteria was done using melanin production and DNA fingerprinting (GTG₅-PCR). The U204 symbiotic efficiency was not affected by the co-inoculation with the others two native strains. The T70 strain was a poor competitor when was co-inoculated with one of the effective strains in both experiments. Our results confirmed a “selective nodulation” because an effective symbiosis occurred preferentially over an ineffective one in Trifolium species. The native effective strain competed with U204 for nodule formation in both clovers species, but the nodule occupancy depended on the inoculum ratio. The pH of nutritive solution did not affect competition ability of the studied strains. It may be possible to isolate efficient, competitive, and genetically different native rhizobial strains to be used as inoculant strains for clover pastures in Uruguay. Both (GTG)₅-PCR and melanin production were useful methods to identify nodulating bacteria in competition studies.     

Effect of strains of Rhizobium trifolii on the establishment of oversown white clover (Trifolium repens)

Strains of Rhizobium trifolii incorporated into commercial peat inoculants were compared for their effect on the establishment and growth of oversown white clover (Trifolium repens) on soils devoid of infective rhizobia.There were marked differences in numbers of seedlings establishing and clover dry matter production per hectare with the various strains. However, when adjusted to a constant number of established seedlings, dry matter production from all strains, apart from one strain at one site, were similar indicating that the strains did not appear to influence the growth of individual clover plants.The marked differences in establishment of clover inoculated with the various strains could not be accounted for by differences in the number of rhizobia in the peat inoculant.Selecting strains of rhizobia for ability to increase establishment is considered important where clover is oversown onto soils devoid of rhizobia.     

Competitive advantage of bacteriocin and phage-producing strains of Rhizobium trifolii in mixed culture

Bacteriocinogenic and lysogenic strains of Rhizobium trifolii were grown with sensitive strains in sterile broth or peat culture. In broth culture the producing strains strongly suppressed growth of sensitive strains. In peat culture a considerable degree of suppression was also observed, the effect being stronger when the peat was “wet” than when it was “damp”. Apparently, conditions which favour growth in peat also favour dominance by bacteriocinogenic or lysogenic bacteria in cultures of mixed strains. The results are discussed in relation to preparation of mixed-strain legume inoculants and to studies of competition between strains in the field environment.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

Diversity and phylogeny of rhizobial strains isolated from Lotus uliginosus grown in Uruguayan soils

Lotus uliginosus is generally nodulated by rhizobia of the genus Bradyrhizobium when used for improvement of Uruguayan pastures. The genetic diversity and phylogenetic relationships of 111 isolates from nodules of L. uliginosus collected from four fields with or without prior inoculation history were analyzed in this study. Genetic diversity estimated by ERIC-PCR revealed 75 different genomic fingerprints, and showed a relatively greater value compared with other methods and varied by soil type. 16S ribosomal RNA gene RFLP analysis revealed three different ribogroups, A, B and C, with 71 isolates in ribogroup A, three isolates in ribogroup B and only one in ribogroup C. Phylogenetic analyses based on 16S RNA gene sequences, ITS, as well as atpD, recA and glnII gene sequences indicated that ribogroup A strains were affiliated with B. japonicum bv. genistearum strains. The three isolates in ribogroup B did not clearly associate with any Bradyrhizobium species described previously and could represent a novel species within this genus. Unlike B. japonicum strains these isolates were able to nodulate and fix nitrogen with other Lotus species as well as with Spartium, a leguminous shrub. The unique isolate in ribogroup C clustered with Mesorhizobium and appeared genetically and phenotypically related to broad host-range Mesorhizobium sp. NZP2037. Our data suggest that Uruguayan soils contain native or naturalized bradyrhizobia that are able to nodulate L. uliginosus as efficiently as the commercial strain NZP2309 but could have adaptive advantages making them more suitable for inoculant purposes.     

Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations

Many of the microbial inoculants all over the world are based on solid peat formulations. This has been mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been assayed employing mannitol or glycerol as C sources. Some media maintained more than 10⁹ cfu ml^?1 of Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The performance of different formulations of two highly effective soybean rhizobia strains was assayed under field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed yields that were not significantly different to those produced by peat-based inoculants.     

Co-introduction of exotic rhizobia to the rhizosphere of the invasive legume Acacia saligna,an intercontinental study

Invasive woody legumes have profound impacts in the nitrogen content and cycling of invaded ecosystems due to the ability to enter into symbiosis with nitrogen-fixing bacteria. In spite of the relevance of this symbiosis, the identity and origin of the symbionts involved in invasion are not well understood. We conducted a study to assess the diversity of symbiotic root-nodulating bacteria associated with the invasive Acacia saligna, in newly colonized areas in Portugal and Australia. BOX-PCR was used to discriminate the isolated bacteria and 16S rRNA and nifD genes were sequenced to identify the different isolates and their geographic origin. Bradyrhizobium and Mesorhizobium nodulated A. saligna in Australia while only Bradyrhizobium spp. were found in Portugal. The dominant strains nodulating A. saligna were related to Bradyrhizobium liaoningense and Bradyrhizobium canariense. Co-occurring Acacia longifolia and A. saligna in Australia harbor different rhizobial communities. As an example, we found Mesorhizobium sp. and Phyllobacterium trifolii in A. saligna and A. longifolia respectively, being this the first report for this association. The analysis of the phylogeographic marker nifD clustered most of the sequences obtained in this study with sequences of Australian origin, indicating that exotic bradyrhizobia might have been co-introduced with A. saligna in Portugal. This result highlights the risks of introducing exotic inoculants that might facilitate the invasion of new areas and alter native soil bacterial communities, hindering the recovery of ecosystems.     

Interactions of the microflora from nodulation problem and non-problem soils towards Rhizobium spp on agar culture

Microorganisms (348 fungi, 388 actinomycetes and 319 bacteria) were isolated from a nodulation problem soil, a non-problem virgin soil, a cultivated problem soil and the rhizosphere of clover plants grown in the problem soil. Rhizobium trifolii TA 1 which failed to establish in problem soils was inhibited on laboratory media by a greater number of these soil microorganisms than the better soil colonizing R. trifolii (WU95 and WU290) and R. lupini (WU425). R. lupini was not inhibited or stimulated on agar by many soil or rhizosphere isolates. R. meliloti showed greater stimulation than either R. trifolii or R. lupini and was inhibited by relatively few soil microorganisms so that its poor soil survival was thought to be due to chemical or physical soil conditions rather than to biotic factors. The greatest incidence of rhizobial inhibitors, mainly associated with TA 1, was found among the isolates from the clover rhizosphere. There was a reduction in the relative numbers of rhizobial inhibitors isolated from the cultivated soil compared with the virgin problem soils, a result possibly due to the changed soil environment changing with cultivation, altered vegetation and the addition of superphosphate. Inhibitors of rhizobia were more frequent amongst the bacteria than fungi or actinomycetes. Strong stimulation was more commonly shown by fungi than by actinomycetes or bacteria. The interaction on agar between rhizobia and the soil microflora is related to soil colonization and persistence.     

Survival and plasmid stability of rhizobia introduced into a contaminated soil

The behaviour of Rhizobium strains introduced separately into soil from a contaminated site with high concentrations of heavy metals (mainly Zn and Hg), and the role of plasmids in the ecology of these rhizobia strains were studied. Six Rhizobium leguminosarum biovar trifolii strains, from different sources and with different plasmid contents, were selected. Two of them were isolated from nodules of subterranean clover plants (Trifolium subterraneum) grown in the contaminated soil and four were from an uncontaminated soil. After inoculation with approximately 10⁷ cells g⁻¹ soil, of each strain, survival and plasmid stability were assessed over a period of 12-18 months. Differences in survival of Rhizobium strains were only detected more than 12 months after inoculation. After 18 months it was clear that survival in contaminated soil was greatest in the two strains originally isolated from that contaminated soil, and also by two of the strains originally isolated from uncontaminated soil. The latter two strains were also the only ones that showed changes in their plasmid profiles. The remaining isolates had the lowest populations, and their plasmid profiles were unchanged and similar to the parent strains.     

Isolation and symbiotic characteristics of Mexican Frankia strains associated with Casuarina

In the absence of available symbiotic nitrogen-fixing Frankia strains associated with Casuarina trees in Mexico for reforestation purposes, isolation was undertaken using root nodules from trees growing in different habitats in Mexico, from the coast of the Gulf of Mexico up to 2550 m above the sea level. A total of 24 strains were isolated and clonal cultures were obtained from one filament of each strain. The use of acetate as the sole carbon source was essential for the isolation of the endosymbiont from the nodules due to the fact that other contaminant actinomycetes utilize propionate. Clonal cultures were obtained, and cultural and symbiotic characteristics of pure cultures were assessed. All strains grew well in stirred DPM (defined propionate medium) with no mineral nitrogen. Isolates showed hyphae, multilocular sporangia and characteristic vesicles. The presence of the gene nifH was also demonstrated, with all strains being able to nodulate Casuarina equisetifolia. Nitrogenase activity (acetylene reduction) of the formed root nodules varied among the different associations depending on the isolate used to inoculate the plants. Several of the isolates can be used as inoculants for the propagation of Casuarina trees.     

Competitiveness and persistence of introduced rhizobia on oversown clover: Influence of strain,inoculation rate and lime pelleting

Five strains of R. trifolii were evaluated, at two inoculation levels in the presence or absence of lime pelleting, for their ability to compete and persist in a tussock grassland soil containing a naturalized population of rhizobia. Effects on the growth of the oversown white clover (Trifolium repens) were also investigated.Strains showed marked differences in their ability to form nodules on the host in competition with the naturalized population of rhizobia and also differed in their persistence over 15 months. The most competitive and persistent strain was PDDCC 2163 followed by 2153, 2666 and 2668 and the least competitive was 4144. Lime pelleting or increasing the rate of inoculation increased the competitive ability of strains. Strains that were highly competitive increased clover dry matter and N uptake.     

Commercial rhizobial inoculants significantly enhance growth and nitrogen fixation of a promiscuous soybean variety in Kenyan soils

Low effectiveness of native strains remains a limitation to soybean productivity in sub-Saharan Africa; while in other countries commercial inoculants are produced that provide effective strains that stimulate N fixation and growth. An experiment was set up to evaluate the response of a dual purpose promiscuous soybean variety (TGx1740-2F) and a non-promiscuous variety (Nyala) to commercial rhizobium inoculants in soils from central and coastal Kenya. Highest nodulation was observed in some of the treatments with commercial inoculants applied with nodule weights of 4.5 and 1.0 g plant⁻¹ for TGx1740-2F and Nyala, respectively. Average biomass yields of TGx1740-2F (16 g plant⁻¹) were twice as large as of Nyala (7.5 g plant⁻¹) at the podding stage. Nitrogen fixation was higher in TGx1740-2F than in Nyala, and positively affected by a number of commercial inoculants with more than 50% N derived from the atmosphere. Nodule occupancy was 100% on both soybean varieties, indicating that the commercial strains were extremely infective in both of the tested soils. These results showed that commercial strains can be used to inoculate promiscuous soybean and enhance N fixation and yield.     

Determination of competitive abilities of Bradyrhizobium japonicum strains in soils from soybean production regions in South Africa

Bradyrhizobium japonicum strain CB 1809 was recently chosen to replace strain WB 1 in commercial soybean [Glycine max (L.) Merr.] inoculants in South Africa, the selection criterion being N₂-fixing effectiveness. Nodulation competitiveness is an additional characteristic required of inoculants and was determined for CB 1809 and WB 1 as well as two other strains, USDA 110 and a Brazilian strain 965, using the gusA marker gene to identify strains. Initial experiments with plants grown in sterile sand showed that the competitive index of strain WB 1 was less than that of the other strains. Further comparisons used plants grown in five soils containing established populations of B. japonicum. When strains were applied in peat inoculum to seed at a rate of 1,000 cells per seed in a soil containing 300 rhizobia g^–1, significant differences in nodule occupancy were detected and strains ranked in the order 965>CB 1809>USDA 110>WB 1. The remaining four soils each contained about 10⁶ rhizobia g^–1 and 5×10⁶ cells were applied per seed. Nodule occupancy by inoculant strains ranged from 22% to 81% between soils. In this experiment, WB 1 was consistently the poorest performer and its competitiveness was significantly less than CB 1809. The competition results supported the recent decision to replace WB 1 with CB 1809 in commercial inoculants. Although WB 1 had been used in inoculants over a period of 19 years, this strain was detected in only one soil, where it comprised 8% of isolates. In contrast, a substantial proportion (32–78%) of isolates from the soils corresponded serologically to a former inoculant strain WB 66, which had been discontinued in 1966. This illustrates the difficulty of replacing a resident population with an introduced strain. The effect of naturalized populations on the establishment of CB 1809 in South African soils will need monitoring Received: 23 November 1999     

Experimental study of sequences of ectomycorrhizal fungi on birch (Betula SP.) seedling root systems

Birch seedlings on which mycorrhizas of different fungi (primary inoculants) were established in aseptic conditions were transplanted into pots of brown earth supplemented with inocula of other mycorrhizal fungi (secondary inoculants) in a glasshouse study. Leccinum scabrum and Amanita muscaria did not persist as primary inoculants after transplanting seedlings to soil, and did not colonize as secondary inoculants, irrespective of the presence of other mycorrhizal fungi. Lactarius pubescens persisted poorly as a primary inoculant after transplanting and did not colonize seedlings as a secondary inoculant in soil; however, Lactarius-type mycorrhizas sometimes developed from naturally occurring inoculum in soil, especially after seedlings had been subjected to dormancy. Hebeloma sacchariolens and Thelephora terrestris persisted and spread as primary inoculants after transplanting and also colonized seedlings as secondary inoculants. These fungi apparently competed with one another, H. sacchariolens being dominant in a brown earth; but H. sacchariolens was ineffective as either primary or secondary inoculant in sphagnum peat, whereas T. terrestris formed abundant mycorrhizas in peat. Two isolates of Paxillus involutus behaved differently from one another: one isolate did not persist as a primary inoculant and did not colonize as a secondary inoculant whereas the other isolate did not persist as a primary inoculant but colonized seedlings extensively as a secondary inoculant in soil.The results demonstrate important and predictable behavioural differences between mycorrhizal fungi that have been termed “early-stage” and “late-stage” in mycorrhizal sequences on birch. Only early-stage mycorrhizal types were suitable for artificial inoculation of seedlings; they influenced subsequent colonization by some other mycorrhizal fungi but did not facilitate colonization by late-stage types typical of mature tree stands.     

Nodulation of Trifolium subterraneum by introduced rhizobia in competition with naturalized strains

The ability of 4 strains of Rhizobium trifolii to compete with naturalized strains in nodulating Trifolium subterruneum cv. Mt Barker and cv. Woogenellup was assessed at 5 sites in New South Wales. The populations of naturalized rhizobia at these sites ranged from 4 × 10⁶ rhizobia/g to one where no rhizobia were detected. The introduced strains were inoculated singly or as mixed strain inocula onto seed of the host at 2 × 10⁶ rhizobia/seed. There were marked differences in competitive ability between the strains but these differences were modified by the host cultivar and the site.At the R. trifolii-free site the inoculum strain formed 100% of the nodules in the 1st yr; by the second year serologically unrelated strains had invaded the plots and these formed almost all of the nodules in the 3rd yr. At the site where competition was greatest (4 × 10⁶ naturalized rhizobia/g), there were no differences in the competitive abilities of the strains in the first year but at all other sites WU95 was superior whether used as a single strain or in a mixed strain inoculum. In these sites also the proportion of nodules formed by the inoculum strains declined markedly by the 2nd yr.     

Interference in culture among Rhizobium strains

Growth interference in culture was shown by 57 strains of Rhizobium belonging to different inoculation groups. The assays were conducted in vitro by spot tests on agar plates and using 1007 paired combinations, 240 cases of interference were observed.A frequency of interference of about 30% was found among strains of R. meliloti and R. meliloti, R. lupini, R. trifolii and R. leguminosarum. Only one case out of 168 showed interference between R. meliloti and R. japonicum.     

Isolation and identification of potassium-solubilizing bacteria from tobacco rhizospheric soil and their effect on tobacco plants

Soil potassium supplementation relies heavily on the use of chemical fertilizer, which has a considerable negative impact on the environment. Potassium-solubilizing bacteria (KSB) could serve as inoculants. They convert insoluble potassium in the soil into a form that plants can access. This is a promising strategy for the improvement of plant absorption of potassium and so reducing the use of chemical fertilizer. The objectives of this study were to isolate and characterize tobacco KSB and to evaluate the effects of inoculation with selected KSB strains on tobacco seedlings. Twenty-seven KSB strains were isolated and identified through the comparison of the 16S ribosomal DNA. Among them, 17 strains belonged to Klebsiella variicola, 2 strains belonged to Enterobacter cloacae, 2 strains belonged to Enterobacter asburiae, and the remaining 6 strains belonged to Enterobacter aerogenes, Pantoea agglomerans, Agrobacterium tumefaciens, Microbacterium foliorum, Myroides odoratimimus, and Burkholderia cepacia, respectively. All isolated KSB strains were capable of solubilizing K-feldspar powder in solid and liquid media. K. variicola occurred at the highest frequency with 18 strains. Four isolates, GL7, JM3, XF4, and XF11, were selected for a greenhouse pot experiment because of their pronounced K-solubilizing capabilities. After being treated with the four KSB strains, plant dry weight and uptake of both K and nitrogen (N) by tobacco seedlings increased significantly. These increases were higher with the combination of KSB inoculation and K-feldspar powder addition. Isolate XF11 showed the most pronounced beneficial effect on plant growth and nutrient uptake by tobacco seedlings. Combining the inoculation of KSB and the addition of K-feldspar powder could be a promising alternative to commercial K fertilizer and may help maintain the availability of soil nutrients. Further studies are necessary to determine the effects of these bacterial strains on mobilization of potassium-bearing minerals under field conditions.     

Enhanced dissipation of chrysene in planted soil: the impact of a rhizobial inoculum

Results from an innovative approach to improve remediation in the rhizosphere by encouraging healthy plant growth and thus enhancing microbial activity are reported. Mixed grass-legume systems, together with microbial inoculants, were used to remediate a polycyclic aromatic hydrocarbon (chrysene) spiked agricultural soil. Inoculants were symbiotic rhizobia, which may play an important role in rhizoremediation by increasing plant and root growth. An inoculum of an isolate of Rhizobium leguminosarum bv. trifolii, selected for PAH tolerance, was produced using a peat carrier. The inoculum and white clover (Trifolium repens L.), were planted into soils with ryegrass (Lolium perenne L.). The soils spiked with chrysene (500 mg kg⁻¹) then aged for 4 weeks. Shoot- and root-biomass of plants, and the amount of root nodulation, were determined. Rhizobial populations, soil pH and soil nitrogen were also monitored throughout the trial. In addition, the ability of the inoculated rhizobial strain to utilise chrysene as a sole carbon source was assessed. Direct uptake and/or degradation of chrysene by the clover and ryegrass did not occur to a significant degree. Enhanced losses of chrysene were seen in planted, non-sterile soils that contained a rhizobial inoculum. No direct degradation of chrysene by R. leguminosarum bv. trifolii was observed and no enhanced losses of PAHs were detected in sterile soils after inoculation with rhizobia. Results suggest that the enhanced dissipation of chrysene, observed in the non-sterile planted inoculated pots, was not a result of degradation of chrysene by R. leguminosarum bv. trifolii. The symbiotic association with R. leguminosarum bv. trifolii improved plant vigour and growth in inoculated planted treatments. This may have stimulated the rhizospheric microflora to degrade chrysene.