Soil microbial response during the phytoremediation of a PAH contaminated soil

The aim of this trial was to quantify and compare the responses of soil microbial communities during the phytoremediation of polycyclic aromatic hydrocarbons (PAHs) in a laboratory trial. The experiment was conducted in 1-kg pots and planted treatments consisted of a mixed ryegrass (Lolium perenne) and white clover (Trifolium repens) sward together with a rhizobial inoculum (Rhizobium leguminosarum bv. trifolii). Throughout the 180-d experiment soil microbial biomass and communities of PAH degraders were monitored. PAH degradation was enhanced in planted treatments that received a rhizobial inoculum. Microbial biomass was enhanced in planted treatments, but there were no significant differences between treatments that had received a rhizobial inoculum and those that had not. However, numbers of PAH degraders were greater in the treatment that had received a rhizobial inoculum.     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.     

The effect of acidity on the distribution and symbiotic efficiency of rhizobia in Lithuanian soils

The distribution and symbiotic efficiency of nodule bacteria Rhizobium leguminosarum_bv. trifolii F., Sinorhizobium meliloti D., Rhizobium galegae L., and Rhizobium leguminosarum bv. viciae F. in Lithuanian soils as dependent on the soil acidity were studied in the long-term field, pot, and laboratory experiments. The critical and optimal pH values controlling the distribution of rhizobia and the symbiotic nitrogen fixation were determined for every bacterial species. The relationship was found between the soil pH and the nitrogen-fixing capacity of rhizobia. A positive effect of liming of acid soils in combination with inoculation of legumes on the efficiency of symbiotic nitrogen fixation was demonstrated.     

Renodulation and characterization of Rhizobium isolates from cicer milkvetch (Astragalus cicer L.)

In 1993 and 1994, 12 bacterial isolates were isolated from root nodules of cicer milkvetch (Astragalus cicer). In the tests for nodulation of A. cicer by these bacterial isolates, five were found to form hypertrophic structures, while only two formed true nodules. These true nodules were formed in a sterilized soil system. This system might be able to act as a DNA donor to provide residual DNA to other microbes in the soil. The rhizobial isolates were thought to have lost genetic material crucial to nodulation during the isolation process. This hypothesis was supported by an experiment in which isolate B2 was able to nodulate A. cicer in vermiculite culture after being mixed with heat-killed rhizobia, Rhizobium leguminosarum bv. trifolii and R. loti. The nodulation would not occur in vermiculite culture system without the heat-killed rhizobia. Based on the biochemical data, the B2 and 9462L, which formed true nodules with A. cicer, were closely related. The rhizobia type cultures that nodulate A. cicer include Bradyrhizobium japonicum, Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viceae, and R. loti. All of these rhizobia were from different cross-inoculation groups. The B2 and 9462L isolates could only nodulate Medicago sativa, Phaseolus vulgaris, and Melilotus officinalis, but not these species within the genus from which they were isolated: Astragalus. The traditional cross-inoculation group concept obviously does not fit well in the classification of rhizobia associated with Astragalus. The rhizobia isolated from A. cicer can be quite different, and the rhizobia able to renodulate A. cicer also quite diverse. Received: 27 June 1996     

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.     

Overproduction of the rhizobial adhesin RapA1 increases competitiveness for nodulation

Competition for nodulation is a complex problem where bacterial adhesins, which are required for root colonization, may play a role. However, the possible influence of adhesins on competitiveness was scarcely studied. In this work, the Rhizobium leguminosarum bv trifolii adhesion protein RapA1 was overproduced from a pHC60-derived plasmid and expressed in R200 strain. When an overproducing strain and a control-carrying empty vector were co-inoculated on clover plants, a positive effect of RapA1 on competition for nodule occupation was observed. Therefore, optimization of RapA1 expression may be considered for improvement of rhizobial competitiveness.     

Effects of resident rhizobial communities and soil type on the effective nodulation of pulse legumes

Communities of resident rhizobia capable of effective nodulation of pulse crops were found to vary considerably over a range of soil environments. These populations from soils at 50 sites in Southern Australia were evaluated for nitrogen fixing effectiveness in association with Pisum sativum, Vicia faba, Lens culinaris, Vicia sativa, Cicer arietinum and Lupinus angustifolius. The values for nitrogen fixing effectiveness could be related to soil pH as determined by soil type and location. It was found that 33% of paddocks had sufficient resident populations of Rhizobium leguminosarum bv viciae for effective nodulation of faba bean, 54% for lentils, 55% for field pea and 66% for the effective nodulation of the vetch host plant. Mesorhizobium cicer populations were very low with only 7% of paddocks surveyed having sufficient resident populations for effective nodulation. Low resident rhizobial populations (<10 rhizobia g⁻¹ soil) of R. leguminosarum bv viciae and M. cicer were found in acid soil conditions. In contrast, Bradyrhizobium populations increased as soil pH decreased. Inoculation increased faba bean yields from 0.34 to 4.4 t ha⁻¹ and from 0.47 to 2.37 t ha⁻¹ for chickpeas on acid soils. On alkaline soils, where resident populations were large there was no consistent response to inoculation. Observations at experimental field sites confirmed the findings from the survey data, stressing the importance of rhizobial inoculation, especially on the acid soils in south-eastern Australia.     

Population densities of clover rhizobia in Texas pastures and response to liming

Summary Clovers are widely used forage legumes on acidic soils in Texas and need inoculation with appropriate rhizobia when first introduced. Acidic soils are not conducive to survival of clover rhizobia. A survey of pastures was undertaken to determine the number of rhizobia present. The effect of liming acidic soils on the survival of clover rhizobia was also evaluated in the laboratory. The number of clover rhizobia was more than 100 cells g^-1 soil in 70% of the pastures surveyed but populations within pastures varied by more than two orders of magnitude. The number of years of clover production beyond 1 year did not affect the rhizobial population density. The soil pH of twelve samples was below 5.0 and six samples had populations of rhizobial lower than 100 g^-1 soil. Eleven out of sixteen samples from fields that had grown clover and had pH values above 6.0 had populations exceeding 1000 g^-1 soil and only three samples had populations lower than 100 g^-1 soil. Incubating indigenous or inoculated rhizobia in well-mixed soils having pH values of 5.1 or below resulted in populations declining to below 10 g^-1 soil in 6 weeks. Mixing of soils with pH values of up to 5.4 induced reduction of rhizobial numbers, possibly by destroying microsites. Liming of soils to increase pH values above 5.5 improved survival of native or inoculated rhizobia in most cases.     

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.     

Numbers of clover rhizobia needed for crown nodulation and early growth of clover in soil

Low soil populations of Rhizobium leguminosarum biovar trifolii indicate a need for inoculating clovers (Trifolium sp.) at planting. The number of rhizobia in soil varies considerably from field to field and the number needed for nodulation on the upper taproot and for vigorous seedling development is not known. Two experiments were undertaken using arrowleaf clover (T. vesiculosum Savi) and crimson clover (T. incarnatum L.) grown in pots filled with soil. Two soils were used; one contained 10 indigenous rhizobia g^-1 and the other contained fewer than three. The treatments consisted of amending each soil with two strains of inoculant rhizobia to contain from 10 to approximately 1×10⁶ rhizobia g^-1 followed by planting to clover. The number of nodules near the top of the root increased as the number of rhizobia in the soil increased to the highest inoculum level. A low number (approximately 1×10³ to 1×10⁴) of rhizobia was sufficient for maximal N content of seedlings. It seems that soil containing 100 or fewer rhizobia g^-1 may respond to inoculation with increased crown nodulation and seedling vigor.     

Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth

The aim of this study was to identifiy the endophytic bacteria recovered from the foliage, tap roots and nodules of red clover plants (Trifolium pratense L.); and to assess the effects of the nodule bacteria, alone and in combination with Rhizobium spp., on the growth and development of red clover seedlings. Thirty-one bacteria species from 14 different genera were recovered from within the foliage, roots and nodules of red clover plants cv. AC Charlie. Genera diversity and species number were greatest in foliage tissues. Pantoea agglomerans (59.6%) was the most frequent species recovered in foliage tissues, Agrobacterium rhizogenes A in the tap root (49.2%) and Rhizobium leguminosarum BV phaseoli and R. loti B in the nodules (27.2% each). Recovery of Rhizobium species was not restricted to the nodules, and species of this genus were systemic throughout the plant. Clover root nodules were host to 12 bacteria species other than rhizobia, of which 8 were specific to this tissue. Using non-selective media, R. leguminosarum BV trifolii constituted only 8.8% of all the root nodule bacteria recovered. In root bacterization experiments, species of nodule bacteria promoted growth of red clover more often when applied in combination with R. leguminosarum BV trifolii than when applied singly. However, Bacillus megaterium, Bordetella avium and Curtobacterium luteum consistently promoted growth either individually or in combination with R. leguminosarum BV trifolii. Nodulation was promoted when R. leguminosarum BV trifolii was coinoculated with Bacillus insolitus, B. brevis or A. rhizogenes A. Single isolate applications of Rhizobium species to roots always led to the depression of clover growth, but mixtures of R. leguminosarum BV trifolii and R. leguminosarum BV phaseoli resulted in growth promotion. The latter is considered further evidence of the beneficial allelopathic side effect of strain competition for the same ecological niche. Received: 27 June 1996     

Growth of perennial forage legumes in acidic soils of the Appalachian Hill‐Lands after liming

A major constraint to the renovation of forage legume‐based pastures on acidic soils of the Appalachian hill‐lands is thought to be the absence of effective rhizobia. A growth chamber experiment was done with aluminum (Al) toxic, low pH (≥ 4.2) soils from four series (Berks, Lily, Tate, and Westmoreland) that were planted with alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), white clover (Trifolium repens L.), or birdsfoot trefoil (Lotus corniculatus L.). These soils, without lime addition, were previously shown not to contain effective, naturalized populations of rhizobia for these plant species. However, a non‐toxic, pH 6.8, Watauga soil was shown to have such rhizobia but only for alfalfa. In the present study, these five soils were reexamined after liming to pH ≥ 5.5 for effective, naturalized populations of rhizobia and the efficacy of soil inoculation with commercially available rhizobia. In addition to effective, naturalized R. meliloti for alfalfa in the Watauga soil, similar populations of R. trifolii for red clover, and R. lotus for birdsfoot trefoil, were now found. Such rhizobia were also found for alfalfa in the Lily soil and for red clover in the Lily and Tate soil. Thus, liming allowed the expression of effectiveness of natural rhizobia that otherwise would not have been detected in soil pot experiments without lime. Inoculation of the toxic soils after lime addition with commercial rhizobia was effective in about half of the soil‐plant combinations that did not contain populations of effective, naturalized rhizobia. Asymbiotic shoot growth of all the plant species was significantly (P ≤ 0.05) correlated with soil pH over a range of 5.5–6.6. These results indicate that, in the absence of effective, naturalized populations of rhizobia, improvement of rhizobial inocula could increase forage production by ～34% for some species on some of the toxic soils, even after the pH of the soils is increased to ≥ 5.5.     

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.     

Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

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.     

Population size of indigenous Rhizobium leguminosarum biovar trifolii in long-term field experiments with sewage sludge cake,metal-amended liquid sludge or metal salts: Effects of zinc,copper and cadmium

There is conflicting evidence, and therefore continuing concern, as to whether metals in sewage sludge are deleterious to soil microbial processes and long-term agricultural productivity. Nine field experiments with sewage sludge cakes, three with metal-amended liquid sludges and three with inorganic metal salts were set up across Britain in 1994 to give individual metal dose–response treatments to try to answer this question. This study reports on the effects of Zn, Cu and Cd on the population size of Rhizobium leguminosarum biovar trifolii, a nitrogen fixing symbiont of white clover (Trifolium repens), in soils from these experiments over 11 years. Significant (P < 0.05) reductions in indigenous rhizobial numbers occurred on the Zn metal dose–response treatments at eight of the sludge cake sites in 2005, but few consistent effects were evident on the Cu or Cd metal dose–response treatments during the 11-year monitoring period. The soil total Zn concentrations where effects occurred were near to the UK statutory limit of 300 mg kg^?1 for soils receiving sewage sludge. No significant reductions occurred in any treatments on the metal-amended liquid sludge or inorganic metal salt experiments in which the metals would be expected to be in a more bioavailable form, even after 11 years. The effects in the sludge cake experiments were related consistently with soil total Zn, with no recovery to date. The reductions in clover rhizobial numbers in the sludge cake experiments were due to Zn effects on free-living rhizobia in the soil, with gradual die-off over a long time with increasing soil total Zn concentrations. Currently, no consistent adverse effects on rhizobia have been seen at the UK limits for Cu and Cd of 135 and 3 mg kg^?1, respectively.     

Selection of Rhizobium strains on their competitive ability for nodulation

The shoot dry weight of alfalfa inoculated with an effective strain of Rhizobium meliloti mixed with an ineffective strain in different ratios was found to be directly proportional to the log of the number of effective nodules. Consequently the comparison of the shoot dry weight of plants inoculated with a mixture of effective and ineffective strains with the shoot dry weight of plants inoculated with the effective strain should allow the estimation of the relative competitiveness of the effective strains. To check this. the competitiveness of 14 antibiotic-resistant strains of R. leguminosarum was evaluated in this way and compared with the ability of the strains to form nodules when inoculated to seeds of Vicia faba planted in a soil containing indigenous R. leguminosarum. The percentage of recovery of the inoculum strains in the nodules of field-grown fababeans was positively correlated with the competitiveness of the strains as estimated by the greenhouse test. This simple way of evaluating the nodulating competitiveness of strains of rhizobia being indicative of their competitive behaviour with indigenous rhizobia in the field could therefore be useful for screening a large number of strains for competitiveness.     

Host–strain mediated selection for an effective nitrogen-fixing symbiosis between Trifolium spp. and Rhizobium leguminosarum biovar trifolii

We have previously reported significant strain–host incompatibility between the microsymbiont Rhizobium leguminosarum biovar trifolii and Trifolium spp. related to geographic and phenological barriers. Additionally, we have shown that an effective symbiosis between strains of R. l. trifolii and clover was established despite the soil harbouring ineffective R. l. trifolii capable of nodulating the host. We termed this “selection” for effective symbiosis. This paper reports glasshouse-based experiments to validate and further explore this in situ selection phenomenon. The effect of cell density and strain ratio at the time of inoculation, as well as soil pH, were investigated on two hosts (Trifolium purpureum and Trifolium polymorphum) that were each exposed to one microsymbiont capable and one incapable of N₂ fixation. In co-inoculation experiments at a cell density of 10⁴ cells/mL, each host nodulated solely with its effective strain, even when this strain was outnumbered 100-fold by the ineffective strain. However, the selection process ceased when the effective strain was outnumbered 1000-fold. At higher basal cell concentrations of 10⁵–10⁸ cells/mL, selection for WSM1325 to form effective nodulation on T. purpureum was evident, but was significantly reduced as the ratio of ineffective cells in the inoculum increased above 4-fold. The results indicate that the selection mechanism is highly dependent on the basal rhizobial cell density. Soil pH did not significantly alter the process, and both strains grew at similar rates and formed nodules at similar rates. A preliminary investigation into the genetic backgrounds of WSM1325 and WSM2304 revealed that although their 16S rRNA sequences were identical, they have considerable differences in their symbiotic and chromosomal replicons through examination of atpD, GSII and nodD sequences.     

The location and distribution in soil of rhizobia under senesced annual legume pastures

Soil cores from under senesced legume swards were partitioned into (1) soils, (2) legume roots, (3) nodules and (4) debris. The viable population of Rhizobium trifolii under subterranean clover was found to be predominantly in the nodule fraction; but with R. lupini under Serradella the distribution was more even. In commercially sown stands the rhizobia often failed to migrate from the original sown rows into the soil between the rows. The relative importance of the various fractions as contributors to the “pool” of rhizobia available for nodulation of subsequent generations of host-plants is discussed.     

Ecological studies of root-nodule bacteria introduced into field environments—V.A critical examination of the stability of antigenic and streptomycin-resistance markers for identification of strains of Rhizobium tripolii

Marked strains of Rhizobium trifolii, distinguishable from other strains antigenically and by streptomycin resistance, were introduced by seed inoculation of subterranean clover (Trifolium subterraneum L.) into a field environment having a natural population of R. trifolii. Isolates from nodules obtained periodically during the following 41 months were classified using both methods of identification in parallel. This procedure made it possible to determine the reliability of each method independently.There was a gradual disappearance of the inoculum strains which occurred more rapidly in plots of cv. Woogenellup than in plots seeded with cv. Mount Barker. At five harvests, there was 95% (or greater) correspondence between inoculum survival using either method of identification. There was evidence that a small proportion of the progeny of the inocula sustained independent loss of antigenic character and/or streptomycin resistance in the field or, alternatively, that strains occurring naturally acquired these characteristics. A few nodules contained more than one strain of rhizobia. These exceptions occurred at low frequency and did not interfere substantially with identification results. It is concluded that gel immune diffusion serology and the use of streptomycin-resistant mutants are both reliable methods for identifying strains of rhizobia reisolated from field environments.