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Published in Soil Sci. Soc. Am. J. 67:1746-1754 (2003).
© 2003 Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

DIVISION S-3—SOIL BIOLOGY & BIOCHEMISTRY

Phenotypic and Genetic Diversity of Rhizobia Isolated from Nodules of Clover Grown in a Zinc and Cadmium Contaminated Soil

T. A. Delormea, J. V. Gagliardia, J. S. Angle*,a, P. van Berkumb and R. L. Chaneyb

a Dep. of Natural Resource Sciences, Univ. of Maryland, College Park, MD 20742
b USDA-ARS, Beltsville, MD 20705

* Corresponding author (ja35{at}umail.umd.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Metal contamination can affect the diversity of microbes living in soil. We compared phenotypic and genetic characteristics of Rhizobium leguminosarum bv trifolii isolated from clovers (Trifolium pratense) found at a metal contaminated and a control site, or isolated from nodules of clovers used in most probable number (MPN) determinations of rhizobia in the metal contaminated soil. Analysis of variations in genetic patterns using BOX-PCR was used for genetic characterization. Zinc and Cd tolerance of each isolate was also determined. Rhizobia isolated from the control soil compared with the metal contaminated soil differed both genetically and phenotypically. Genetic diversity, measured by AMOVA, was high and not related to the presence or the absence of high metal concentrations. Isolates originating from the metal contaminated soil were more tolerant to both Cd (minimal inhibitory concentration [MIC50] = 2.5 µM) and Zn (MIC50 = 92.9 µM) compared with those observed for isolates of control soil (Cd MIC50 = 2.0 µM and Zn MIC50 = 41.9 µM). Rhizobia originating from the metal contaminated soil expressed a higher number of metal phenotypes (9) compared with isolates of control soils (6). Slow rates of metal accumulation over the years favored an adaptation of the rhizobia to the metal rather than elimination of metal sensitive organisms and the selection of a few preexisting metal tolerant organisms.

Abbreviations: AMOVA, analysis of molecular variance • DTPA, dimethyltriethyl-pentaacetic acid • HBED, N, N'-di (2-hydroxybenzyl) ethylenediamine-n, n-diacetic dihydrochloride dihydrate • MIC, minimal inhibitory concentration • MPN, most probable number • NTA, nitrotriacetate acid • PCR, polymerase chain reaction • REP, repetitive extragenic palindromic • RFLP, restriction fragment length polymorphism • TBE, Tris-borate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THERE ARE NUMEROUS reported studies showing an adverse effect of heavy metals on rhizobia and N fixation. By studying the long-term effects of biosolids on the growth of Trifolium repens, McGrath et al. (1988) observed a decrease in clover yield in biosolid-amended soil. This decrease was related to lower numbers of rhizobia that formed only ineffective nodules on T. repens. The ineffective rhizobia were shown to contain similar plasmid profiles, which were suggested to indicate that only a single genotype had survived metal exposure (Giller and McGrath, 1989; Giller et al., 1989). Hirsch et al. (1993) later confirmed this from results using restriction fragment length polymorphism (RFLP) analysis.

Obbard and Jones (1993), however, observed the presence of rhizobia capable of forming effective N-fixing symbioses with white clover (Trifolium repens.) in a metal-contaminated soil. They also reported a decrease in the number of rhizobia and low rates of N fixation at low soil pH. Ibekwe et al. (1995) studying the effects of biosolids on nodulation and N fixation of T. repens, found that symbioses were generally ineffective and that nodulation was reduced at low soil pH. They suggested that if pH is maintained at 6.0 or above, heavy metals in soil have no effect on nodulation and N fixation. Studying the diversity of rhizobia isolated from biosolids-amended and control soils using repetitive extragenic palindromic (REP) polymerase chain reaction (PCR), Ibekwe et al. (1997) further reported that isolates from the most contaminated soils were more genetically diverse than isolates from control soils. The authors hypothesized that soil pH and not heavy metal content might be the most important factor in the selection of rhizobia that formed ineffective symbioses. However distinction between metal activity and pH is delicate, since pH is a critical determinant in metal speciation and toxicity.

Previous studies mostly assessed the impact of heavy metals on rhizobial populations in soils contaminated by rapid amendments of biosolids. Little information is available on metal effects in sites contaminated by very slow and progressive increases in metal concentration over long periods of time such as contamination resulting from air deposition of metal oxides by Zn smelter emissions.

Contrary to sites exposed to very fast acute contamination resulting in the selection of only few extremely resistant organisms (Giller et al., 1998), the slow and progressive contamination may favor the adaptation/acclimation of more rhizobia to cope with the metal toxicity and may lead to the maintenance of high rhizobial diversity in the soil. To assess this hypothesis, we analyzed the diversity of natural populations of R. leguminosarum bv. trifolii in a long-term contaminated site using both phenotypic and genetic approaches. Rhizobial isolates were tested for their capacity to nodulate and for effective association on red clover (Trifolium pratense L.), and their metal tolerances were determined on agar medium. Genetic variation within rhizobia was assessed using BOX-PCR and was shown to a useful method for differentiating closely related rhizobia (De Bruijn, 1992; Martin et al., 1992; Versalovic et al., 1994). This method was used to assess whether the presence of heavy metals in soil led to noticeable changes in rhizobia diversity. BOX-PCR analysis was preferred to other genetic approaches such as (16S-DNA analysis) due to its ability to detect DNA mutations, insertions, and deletions resulting from exposure to high levels of heavy metals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Red clover plants were collected at a Zn/Cd enriched and a relatively unpolluted site in the vicinity of a Zn smelter in Palmerton, PA. The first site, a heavy metal contaminated area (Cd: 10.8 mg kg-1, and Zn: 575 mg kg-1) was located 0.5 km downwind from the Zn smelter. The second site had lower soil metal content (Cd: 1.3 mg kg-1 and Zn: 174 mg kg-1) and was located in Lehighton, PA (14 km northwest and upwind of Palmerton). Both soils were classified as a Klinesville very stoney silt loam (loamy, skeletal, mixed active mesic Lithic Dystrocrept). The vegetation of both sites differed drastically. The low metal soil supported a healthy flora (maple[Acer sp.]-beech [Fagus sp.]-birch forest type [Betula sp.]). The high metal soil supported poorly growing and sparse scrub vegetation. Both soils were collected from the surface horizon (0–15 cm) and were sieved through a 4-mm stainless steel sieve to remove rocks and large organic materials. The Soil Testing Laboratory of the University of Maryland (Table 1) analyzed soils for soil texture, pH, phosphate content, and N content.


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  		Table 1. Chemical and physical characteristics of soils used to assess impacts of heavy metal on rhizobial diversity.

 
Soil Metal Analysis
For total metals, 5 g of air-dried soil was digested in 10 mL of concentrated nitric acid and taken to dryness, and then the residue was digested with 20 mL of 3M HCl. After filtration on Whatman filter paper #40 (Whatman International Ltd., Maidstone, England), the metal concentrations in the filtrate were determined with an atomic absorption spectrophotometer.

Available metals in soils were extracted using 5 mM dimethyltriethyl-pentaacetic acid (DTPA) (Lindsay and Norvell, 1978; Brown et al., 1994, 1995). Soil and extracting solutions were used at a ratio of 1:2 (weight/volume). After shaking for 2 h, the soil extracts were filtered through Whatman filter paper #40, and the metal concentrations in the filtrate were determined on an atomic absorption spectrophotometer (Table 2).


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  		Table 2. Total (hot nitric digestion) and DTPA available metal concentrations of two soils in the vicinity of a Zn smelter in Palmerton, PA.

 
Preparation of Plant Infection Tubes
Seeds of T. pratense were surface sterilized for 10 min in a solution of 70% (v/v) ethanol followed by 10 min in a 0.5% (v/v) solution of Na hypochloride and rinsed three times in sterile distilled water. Sterile seeds were germinated on sterile water-agar Petri plates. After 7 d, seedlings were transferred into sterile tubes containing a slant of N-free agar described by Weaver and Graham (1994). After three more days, plant infection tubes were filled with the same N free but liquid medium, and incubated four more days before inoculation with the adequate soil dilution. Tubes were placed in a controlled environment growth chamber at 28°C (day)/24°C (night) with 12 h photoperiod.

Enumeration of Rhizobia by the Most Probable Number Method
In both soils, numbers of rhizobia able to nodulate red clover were determined by the MPN method (Weaver and Graham, 1994). Ten grams of soil were blended in sterile distilled water for 1 min at 22000 rpm in a Waring blender (Waring, New Hartford, CT). After allowing sand to settle for 1 min, ten-fold dilutions were made using water. Three plant infection tubes were inoculated with 1-mL aliquots at each dilution step and the tubes were incubated in a controlled environment growth chamber at 28°C (day)/24°C (night) under 12 h of light. Positive (inoculation with R. leguminosarum bv. trifolii USDA 2145 obtained from the USDA Rhizobium Germplasm Collection at Beltsville, MD) and negative controls (inoculation with sterile water) for nodulation were added to each set of tubes. All tubes were examined for nodulation after 3 wk. Numbers of rhizobia were calculated using MPN tables (Woomer, 1994).

Isolation of Rhizobia and Determinations for Effectiveness
Rhizobia were isolated from nodules of plants of both soils and from nodules of plants used for the MPN determination in the high metal soil. Nodules were surface sterilized for 10 min in a solution of 90% (v/v) ethanol followed by 10 min in a solution of Na hydrochloride at 0.5% (v/v). Nodules were then washed three times with sterile distilled water. Nodules were crushed in approximately 0.1 mL of sterile distilled water to release bacteroids. The bacteroid suspensions were plated on Mannitol-Yeast Extract-Congo red agar to obtain single colonies. Colonies were transferred and plated three times on modified arabinose-gluconate medium (van Berkum, 1990) to ensure purity before testing for effectiveness. Isolates were checked for nodule formation on red clover: a single colony was mixed with 1 mL of sterile distilled water. For each isolate, three plant infection tubes without N (Weaver and Graham, 1994) were inoculated with a loop-full of the bacterial suspension. The result was that only one rhizobial isolate was obtained per nodule.

Determination of Cd- or Zn-Tolerance
Rhizobia were tested for metal resistance on a modified medium (1.0 L): Ca (NO3)2 4H2O, 0.75 g; MgSO4 7H2O, 0.246 g; MES (2-(N-Morpholino) ethanesulfonic acid), 4.88 g; Mannitol, 4.50 g; KH2PO4, 13.60 g; Vitamins: 1.0 mL of solution stock; Trace elements, 1 mL of the stock solution. Fe-HBED (N, N'-di (2-hydroxybenzyl) ethylenediamine-n, n-diacetic dihydrochloride dihydrate), 5.53 mg. Vitamin Stock Solution (1.0 L): biotin, 0.1 g; Niacinamide, 0.35 g; Thiamine 2HCl, 0.3 g. Trace Element Stock Solution (1.0 L) CuCl2 2H2O, 426 mg; ZnCl2, 348 mg; MnSO4 H2O, 396 mg, H3BO3, 6.18 mg; MoO3, 28.8 mg, NiCl2 6H2O, 118 mg; CoCl2 6H2O, 119 mg. The pH was fixed at 6.15 to limit metal precipitation occurring at higher pH. Rhizobial colonies were grown three separate times on this medium before metal tolerance testing. Metals (Cd or Zn) were added as an NTA (nitrotriacetate acid) chelate to maintain the same level of free metal during incubation (Angle and Chaney, 1989). The concentration of free metal for each of the metals tested was calculated using Geochem PC (Parker et al., 1995). Metal concentrations varied from 1.8 to 3.3 µM and 15.9 to 484 µM for Cd and Zn, respectively. Rhizobial growth was observed after incubation for 7 d at 28°C. Each isolate was screened three times on the different metal concentrations.

From each group [A (low metal soil-indigenous plants), B (high metal soil-indigenous plants) and C (high metal soil, MPN isolates)], 59 rhizobia were tested for Cd or Zn tolerance. For each group of rhizobia, percentages of surviving rhizobia were calculated for each metal concentration, and the Cd and Zn minimal inhibitory concentrations leading to the death of 50% of the isolates (MIC50) were determined.

Bacterial Strains, DNA Preparation, and BOX-PCR Analysis of the Isolates
Rhizobia were examined by BOX-PCR (Versalovic et al., 1991; Martin et al., 1992; Versalovic et al., 1994). Isolates were grown for 16 h on MAG agar medium at 28°C and single colonies from the plates were suspended in 0.1% Tween 20, heated to 95°C for 10 min, and used directly as a template for PCR.

The oligonucleotide primers BOXA1R (5'-CTACGGCAAGGCGACGCTGACG-3') used to amplify the rhizobial DNA were synthesized with an Applied Biosystems DNA synthesizer (Model 380B, Applied Biosystems, Foster City, CA). The PCR mixture contained 0.11 µL of the primer BOXA1R (40pM/100 µL RX), 0.05 µL of each 100mM deoxynucleoside triphosphate, 2.5 µL of 10X Promega buffer (Promega, Madison, WI, USA), 1.5 µL of 25 mM MgCl, 0.13 µL of Taq DNA Polymerase (5 U µL-1) (Perkin Elmer, Norwalk, CT), 0.13 µL of extender and 3 µL rhizobial DNA., DNase and RNase free filter sterile water to a final volume of 25 µL. Each PCR was performed with a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). The PCR was initiated by incubating the reaction mixture at 95°C for 10 min, followed by 30 cycles of 30 s at 94°C; 1 min at 52°C; and 6.5 min at 65°C. The reaction was terminated with an extension step consisting of 16 min incubation at 65°C. All PCR experiments contained a positive control (DNA of R. leguminosarum bv. trifolii USDA 2066 obtained from the USDA Rhizobium Germplasm Collection at Beltsville, MD) and a negative control (no DNA).

Polymerase chain reaction samples (10 µL) were loaded on a 1.0% horizontal agarose together with at least two wells containing molecular size markers of lambda DNA digested with Hind III and Eco RI (0.5 µg well-1), and the positive control. All gels were run in 0.5X TAE (10 mM Tris, 5 mM acetate, 0.1 mM EDTA pH 7.4) for 20 h at room temperature and 30 to 35 V, stained for 20 min in 0.5X TAE buffer containing 0.5 µg mL-1 ethidiumbromide, and immediately photographed on an UV transillumination table using Polaroid Type 57 films (Polaroid Corp., Waltham, MA).

The BOX-PCR fingerprint patterns were scored for each strain by recording the presence or absence of bands to construct a rectangular matrix of data. Differences in migration between gels were corrected by analyzing the migration of the molecular size markers. The genetic structure of the population was investigated with the analysis of molecular variance (AMOVA) (Excoffier et al., 1992) using the Arlequin program (http://lgb.unige.ch/arlequin/) (Schneider et al., 2000). One dendogram based on molecular distances calculated during the AMOVA analysis was constructed to illustrate the genetic relationship between isolates. The dendogram was produced by the minimum spanning tree method (Rohlf, 1973).

Statistical Analyses
Data were analyzed using SAS version 6.12 (SAS Institute, Cary, NC). Means of the different treatments were separated using the Waller-Duncan k-ratio t test after it was determined that there was a significant (P < 0.05) treatment effect using the general linear model procedure.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
As expected from a long-term contamination by a Zn smelter, metal analysis (Table 2) showed a drastic increase in both total and DTPA Cd and Zn concentrations in the high metal soil compared with the low metal soil.

The most probable number of R. leguminosarum bv. trifolii in both high metal and low metal soils were 2.9 x 103 and 2.0 x 103 rhizobia per gram of soil, respectively. Rhizobia were isolated from nodules present on indigenous clover growing in both low and high metal soil (Groups A and B, respectively). To assess the presence in the metal contaminated soil of rhizobia unable to form nodules on indigenous plants, rhizobia were also isolated from nodules of the plants used to determine the MPN of rhizobia in the high metal soil (Group C). All isolates were regularly checked for nodulation on red clover grown aseptically in a N-free medium (Weaver and Graham, 1994). Within 4 wk, uninoculated plants were stunted and showed typical signs of N deficiency; this was not seen with the inoculation of effective rhizobia. All the isolates from both soils were found to be effective and supported normal growth of red clover on N-free medium.

To determine if the presence of high concentrations of Cd and Zn in the high metal soil affected tolerance to Cd and Zn, isolates were screened on bacterial growth media containing increasing concentrations of metals. For both curves (Fig. 1 and 2) , each point represented the average of three different measures of Zn or Cd tolerance for each individual isolate. Identical results were observed for the three metal tolerance determinations, explaining the lack of error bars on both figures.



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  		Fig. 1. Effect of Cd on the growth of rhizobia isolated from nodules present on indigenous clover growing in both low and high metal soil, or from nodules of the plants used to determine the most probable number (MPN) of rhizobia in the high metal soil. Each point represents the average of three different measures of metal tolerance for each individual isolates. Identical results were observed for the three metal tolerance determinations, explaining the lack of error bars on both figures.

 


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  		Fig. 2. Effect of Zn on the growth of rhizobia isolated from nodules present on indigenous clover growing in both low and high metal soil, or from nodules of the plants used to determine the most probable number (MPN) of rhizobia in the high metal soil. Each point represents the average of three different measures of metal tolerance for each individual isolates. Identical results were observed for the three metal tolerance determinations, explaining the lack of error bars on both figures.

 
Rhizobia from Group B showed higher tolerance to both metals as shown by higher survival curves (Fig. 1 and 2). Their Cd MIC50 and Zn MIC50 (values obtained by linear regression) were higher (2.5 and 92.9 µM, respectively) compared with those observed for isolates of Group A (Cd MIC50 = 2.0 µM and Zn MIC50 = 41.9 µM), or Group C (Cd MIC50 of 2.0 µM and Zn MIC50 of 49.4 µM).

Each rhizobial isolate was characterized and artificially grouped according to their Cd and Zn MICs. Isolates with a Cd MIC between 2.05 and 2.49 µM, or a Zn MIC of 56.5 µM were assumed to exhibit low tolerance for the appropriate metal(s). Low Cd tolerant isolates represented 61% of the total isolates compared with 58% for Zn low tolerant species. Ninety-seven percent of the isolates of Group A are considered as either Cd or Zn low tolerant species, compared with 42% for isolates of Group B. These isolates are mainly thought to express a basic tolerance to Zn or Cd conferred by their intrinsic cellular properties. Rhizobia, which showed lower Zn or Cd MIC's, were characterized as sensitive. For both metals, sensitive isolates represented <10% of the total isolates. Rhizobia with a Cd MIC or Zn MIC higher than 2.49 or 56.5 µM, respectively, were characterized as highly tolerant organisms. The 10% most tolerant organisms were grouped and qualified as extremely tolerant. For each metal, highly tolerant strains represented about 25% of the isolates.

The presence of high levels of Cd and Zn did not lead to the selection of only one particular metal phenotype. Thirteen different metal phenotypes were observed ranging from sensitive to both metals to extreme tolerance to both Cd and Zn (Table 3). Metal phenotypes varied within and between each group of isolates. Some organisms showed a specific tolerance to one metal but not the other (Cd tolerant but Zn sensitive: phenotype 4, and Zn tolerant but Cd sensitive: phenotypes: 2 and 3).


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  		Table 3. Influence of rhizobial origins (indigenous plants or most probable number [MPN] plants) on their metal phenotypes based on Cd and Zn minimum inhibitory concentrations (MIC).

 
More metal phenotypes were observed in rhizobia isolated from the high metal soil in both Group B (9) and Group C (9) compared with isolates from Group A (6). Isolates sensitive to one or both metals represented 37% of the isolates of Group A compared with 5% for Groups B and C. The vast majority of the sensitive isolates of Group A were Zn rather than Cd sensitive. The majority of the isolates of Group A (58%) expressed low tolerance to both Cd and Zn, this phenotype was less common in Group B (17%). Thirty-seven strains of Group B expressed a high tolerance to Cd, compared with only one strain of Group A.

Metal phenotypes expressed by isolates from Group B also differed from those of Group C. Some metal sensitive phenotypes were only found in one group: Phenotype 2 was found only in Group C, and metal Phenotypes 4 and 7 were only observed in Group B. Among isolates of Group C, 63% had the phenotype of low tolerance to Cd and Zn. This percentage was higher than observed in Group A (17%). While absent from Group B, Phenotype 13 having extreme tolerance to both Cd and Zn was found in isolates of Group C.

To determine if differences in metal tolerance were reflected in changes in the genetic diversity of the rhizobia, the DNA of each isolate was amplified by BOX-PCR and compared with rhizobial DNA from the low metal soil. BOX-PCR patterns were found to be highly reproducible from gel to gel and effective in comparing rhizobial isolates.

Polymerase chain reaction with BOX primers yielded multiple DNA products of different sizes ranging from approximately 300 to 4000 BP. To measure all the possible discrepancies between the isolates, all band sizes were used in the analysis. To improve readability of the gels, the different gels were scanned and imported into Adobe PageMaker (Adobe Systems Inc., San Jose, CA). Presence/absence of bands was then visually determined. To compare and minimize DNA profile variations within and between different gels, band migrations were normalized by comparing the migration of the DNA molecular size markers, and also by comparing the migration of identical band patterns. Bands on the same or different gels varying by 1.0 mm relative to each other or more were considered to be different bands. A total of 177 isolates (59 for each group) were studied resulting in 82 different bands.

The gel in Fig. 3 is representative of the results obtained with this analysis. The fingerprint pattern in each of the lanes was used in determining the genetic diversity among isolates. Gel analysis resulted in multiple fingerprint patterns demonstrating that there was no selection for a single genotype. No single rhizobial genotype appears to have established dominance over the other genotypes.



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  		Fig. 3. Examples of BOX polymerase chain reaction (PCR) fingerprint patterns of rhizobial isolates. Molecular size markers of lambda DNA digested with Hind III and Eco RI are: Lanes 1, 12, and 42 in Gel A, and 1 and 25 in Gel B. All the other lanes are BOX-PCR fingerprint patterns of clover rhizobia isolated from low metal polluted soil (Gel A) or high polluted soil (Gel B).

 
The differences in population genetic structure were assessed by AMOVA (Excoffier et al., 1992; Excoffier, 2000; Rousset, 2000). The three populations appeared not to be significantly different; the variance between isolates within each population was much greater (9.695) than the variance observed between populations (1.821). This indicates greater diversity within each group than between them. However, the visual analysis of the dendrogram (Fig. 4) based on the molecular distances between all the isolates, showed that the distribution of the isolates of the different groups was not homogenous. Several isolates were unique and clustered far from those of other groups (or sites). Eighty percent of Group A belong to two clusters (1 and 4) that contained no strain (1) or only a few strains (4) from the other groups (86% A, 12% B, and 2% C). Clusters 2 and 5a contained exclusively (2) or principally (5a [92%]) isolates of Group B. Isolates of Group C were mostly found in Clusters 3 and 5b.



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  		Fig. 4. Dendogram derived from the analysis of fingerprint patterns showing the diversity among clover-nodulating Rhizobium recovered from nodules present on indigenous plants in the low metal soil (Group A), in the high metal soil (Group B) or from nodules present on plants used to determine the most probable number of Rhizobium in the high metal soil (Group C). The dendogram, based on the molecular distances between isolates, was produced by the minimum spanning tree method (Rohlf, 1973).

 
Metal tolerance was not associated with one particular cluster of isolates, but was found spread among all the clusters. Clusters richer in isolates of Group B and C showed higher metal tolerance. Small variations in the distribution of the metal tolerance within isolates of Groups B or C could be noticed: Cluster 2 appeared enriched in rhizobia sensitive or less tolerant to Cd, whereas those closely related to isolates of Group A (Cluster 4b) were highly tolerant to both metals. Isolates of Group C with a low metal tolerance to both metals were preferentially observed in Cluster 3 where they represented 91% of the isolates compared with 48% in Cluster 5.

The presence of 23 siblings with identical BOX-PCR and metal phenotype patterns in Group C might indicate a possible selection of this particular genotype in the soil and/or a preference for infection by this Rhizobium genotype under laboratory conditions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The rhizobial genotypes present in the high metal soil were not significantly different from those in the low metal soil. Populations of both sites appeared to be very diverse. The presence of heavy metals did not lead to a decrease in diversity as observed by Hirsch et al. (1993). To our knowledge, this is the first time that a high level of genetic diversity was observed in a long-term metal contaminated site.

Unlike observations of Giller et al. (1989) and Hirsch et al. (1993), the presence of high levels of metals in soil did not lead to the selection for only one or a few ineffective rhizobia. All isolates in our study were effective in nodulating red clover and fixing N2. Our results demonstrate that effective rhizobia can survive in long-term metal contaminated soil as it was previously reporting in biosolids amended metal-contaminated soils (Mårtensson and Witter, 1990; Obbard and Jones, 1993; Smith, 1997).

In fact, the presence of genetic clusters containing only isolates of one Group A or B might indicate an evolution in the rhizobial population, with the emergence and/or selection of particular subpopulations at the expense of the established ones. This might be seen as an increase in the diversity as it was reported by Ibekwe et al. (1997). The presence of high levels of metals did lead to changes within the rhizobial populations but did not alter the overall diversity of R. leguminosarum bv trifolii.

The high genetic diversity among our isolates also was apparent from the results of the analysis of metal tolerance and metal phenotypes expressed by each of the isolates. An overall increase in Cd and Zn tolerance corresponded with higher diversity in metal phenotypes in the high metal soil compared with the low metal soil. Rhizobia sensitive to one or both metals and rhizobia expressing low tolerance to one or both metals represented up to 98% of the total isolates in the low metal soil, but accounted for only 44% of the isolates of the high metal soil (Group B). Such an increase in metal tolerance is commonly observed in metal contaminated soils (Kozdroj, 1995; Diaz-Ravina and Baath, 1996; Wuertz and Mergeay, 1997; Baath et al., 1998). Chaudri et al. (1992) studying rhizobia isolated from long-term sewage sludge amended soil found higher Cu, Ni, Cd, and Zn tolerances among these isolates compared with those from a control soil. Purchase et al. (1997) also reported greater metal tolerance in rhizobia isolated from soils that had been previously amended with biosolids.

Metal tolerance in highly contaminated soil may result from genetic mutations and/or selection and proliferation of individuals within a population that are intrinsically more resistant to heavy metals (Giller et al., 1998). This may explain why levels of Cd and Zn in the high metal soil led to greater diversity in metal phenotypes.

The increase in the number of metal phenotypes seems in agreement with the hypothesis that the development/emergence of resistance among metal-exposed rhizobia is responsible for this increase rather than the selection of few particular high metal resistances mechanisms. High levels of metals did not select for one (or a few) high metal resistance mechanisms that would be expected with the selection of only one type of rhizobia and/or the lateral dissemination of selected metal resistance mechanisms via genetic transfer. In the contaminated soil, rhizobial metal tolerance varied greatly from sensitivity to both metals to extreme tolerance to both metals. Some rhizobia had high tolerance to only Cd or Zn, not both metals, suggesting that in these organisms, each metal tolerance may be independent and controlled by specific mechanisms of resistance. Genes conferring resistance to toxic concentrations of heavy metals are often encountered on plasmids (Ji and Silver, 1995; Wuertz and Mergeay, 1997). The exchange, acquisition, or modification of plasmids during rhizobial adaptation to increased metal concentration may account for some of the apparent genetic diversity as measured by BOX-PCR and may explain some of the observed discrepancy between genotypes and phenotypes since more than one metal resistances can be found on each plasmid.

The apparent lack of selection for one organism or one resistance mechanism may be linked to the slow and progressive increase (over a century) in metal concentration in the Palmerton soil. Contrary to rapid metal loading and chronic long-term effects, which results in strong selection for organisms with greater metal tolerance (Giller et al., 1998), the slow metal increase favored the adaptation of more rhizobia to cope with the metal toxicity without drastically affecting the overall rhizobial diversity.

The numbers of rhizobia in metal contaminated soil were also similar to those in soil with low metal content. This lack of difference was unexpected especially since plant available metals as determined by DTPA extraction was about six times higher in the high metal soil than in the low metal soil, and that total metal concentrations were also higher than those observed in the biosolids amended Woburn soils where drastic changes in populations were observed (McGrath et al., 1988). A gradual increase over a long period of time in metal concentration may prevent the loss of sensitive organisms often observed in rapid/acute contamination (Kozdroj, 1995) by allowing organisms to progressively acclimate to the change in metal concentrations or find metal protected niches in the soil.

In this study, the presence of indigenous clover may have helped to maintain the observed high levels of rhizobia in both soils by providing these bacteria with protective niches. Legumes may protect rhizobia from toxic metals in soil when they colonize the rhizosphere. The rhizobia surviving in microniches low in metals are further protected by their ability to invade the roots and form a symbiotic relationship with the plant (Giller et al., 1989, 1993, and 1998). The lack of change in rhizobial numbers could also be related to the observations made by Ibekwe et al. (1995) that when soil pH remained above 6.0, heavy metals have little effect on nodulation and nitrogen fixation.

Metal sensitive rhizobia were also found in the high metal soil as was previously described by Angle and Chaney (1991) and Ibekwe et al. (1997). Metal sensitive or low metal tolerant rhizobia able to form nodules on indigenous plants accounted for 22% of the isolates of Group B. This percentage drastically increased and reached 68% of the isolates of Group C (High metal soil-MPN plants). During MPN quantification, soils were blended and diluted with water, breaking apart soil aggregates and freeing metal-protected organisms in a dilute soil solution. Freed rhizobia were then capable of interacting and forming nodules on the trap plants. Rhizobia that were unable to form nodules in the soil due to a lack of competitiveness or a sensitivity to heavy metals were then able to nodulate red clover. The higher percentage of metal sensitive and low metal tolerance phenotypes and the presence of new metal phenotypes in Group C (not present in Group B) demonstrates that metal sensitive rhizobia were able to persist in the high metal soil at a much higher rate than initially suggested by the sole analysis of the rhizobia of Group B. These metal sensitive rhizobia were probably able to survive in the high metal soil by hiding within low metal microniches. Our hypothesis is based on the reports of Wilcke et al. (1996) and Wilcke and Kaupenjohann (1997) who showed a lower reactivity or concentration of the metals inside the aggregates compared with the outside. They attributed these differences in reactivity and concentrations to a preferential weathering of aggregate surfaces, causing a shift from strongly bound to other metal forms, and preferential sorption of deposited metals in the aggregate exteriors, particularly in easily extractable forms.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
High concentrations of Cd and Zn greatly influenced populations of R. leguminosarum bv trifolii. Contrary to previous studies in biosolids amended soils, the overall genetic diversity of rhizobia in the high metal soil did not decrease and diversity of metal phenotypes was higher, suggesting the development of new metal resistance rather than the selection and amplification of few metal resistant phenotypes. Slow rates of metal accumulation over the years may have favored an adaptation of the rhizobia to the metal rather than elimination of metal sensitive organisms, plus selection of a few existing metal tolerant organisms. Sensitive organisms unable to actively nodulate indigenous clover were present in the soil, and were thought to be confined in soil aggregates where exposure to metal is likely lower. This study suggests that metal-contaminated soils may harbor a higher diversity of rhizobia than previously thought especially if the metal contamination was slow and progressive over a long period of time.

Received for publication November 5, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 





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