- Email: [email protected]
Rhizobium leguminosarum bv. viciae populations in soils with increasing heavy metal contamination: abundance, plasmid profiles, diversity and metal tolerance
Soil Biology & Biochemistry 34 (2002) 519±529
www.elsevier.com/locate/soilbio
Rhizobium leguminosarum bv. viciae populations in soils with increasing heavy metal contamination: abundance, plasmid pro®les, diversity and metal tolerance Amir Lakzian, Phillip Murphy, Andrew Turner 1, Jim L. Beynon, Ken E. Giller* Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK Received 17 April 2001; received in revised form 19 October 2001; accepted 6 November 2001
Abstract Populations of Rhizobium leguminosarum bv. viciae were investigated from plots of a long-term sewage sludge experiment in Braunschweig, Germany, which represented a gradient of increasing metal contamination. The number of R. leguminosarum bv. viciae decreased from 10 5 cells g 21 soil in uncontaminated plots to between 7 and 10 2 cells g 21 soil with increasing Zn concentration (from 50 to 400 mg kg 21). Rhizobia were isolated from nodules of Vicia hirsuta inoculated with dilutions of soil from seven of the plots (,50 isolates per plot). The rhizobial isolates had between three and nine plasmids which varied in size from approximately 100 to 850 kb. Although a total of 49 plasmid pro®le groups were identi®ed, PCR±RFLP analysis using primers which ampli®ed an intergenic spacer (ITS) region between the 16S and 23S rRNA genes revealed only 20 groups. Ten ITS groups were found among the isolates from the uncontaminated control plot but only two groups were found in the most contaminated plot, and six to eight groups in the plots with intermediate metal contamination. Numbers of plasmid pro®le groups increased with moderate metal-contamination but were strongly reduced when total Zn concentrations exceeded 300 mg kg 21. Isolates from the less contaminated plots had only three to ®ve plasmids whereas isolates with seven to nine plasmids were abundant in the plots with metal concentrations of 200 mg kg 21 or more. Whereas plasmid pro®les indicated considerable changes in strains with increasing metal contamination, one ITS group (group 1) was present in all plots. Isolates from ITS group 1 contained only three to four plasmids in the control plots but those from the most contaminated plots had eight to nine plasmids. There was a marked increase in metal tolerance of isolates belonging to ITS group 1 as metal contamination increased, which was associated with the increase in the number of plasmids carried. However, another ITS group (group 2), which had only three large plasmids was present only in the most contaminated plot. Isolates from this group had less tolerance to Zn than many isolates from the uncontaminated plots. Possible mechanisms for the survival of these isolates in the metal contaminated soils are discussed. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Metal toxicity; Sewage sludge; Pollution; Zinc; Microbial ecology; Rhizobium; Plasmids; Metal tolerance; Diversity
1. Introduction Soil microorganisms are sensitive to long-term exposure to moderate concentrations of heavy metals in soil (Giller et al., 1998). Effective strains of Rhizobium leguminosarum bv. trifolii, the nitrogen-®xing symbiont of white clover (Trifolium repens L.), were eliminated at metal concentrations too small to cause toxicity to plants (McGrath et al., 1988; Giller et al., 1989). Studies of the populations of rhizobia at Woburn, UK, demonstrated that only a single * Corresponding author. Present address: Department of Plant Sciences, Group Plant Production Systems, Wageningen University, P.O. Box 430, 6700 AK Wageningen, The Netherlands. Tel.: 131-317-485818; fax: 131317-484892. E-mail address: [email protected] (K.E. Giller). 1 Present address: School of Natural and Environmental Science, Coventry University, Priory Street, Coventry CV1 5FB, UK.
isolate of Rhizobium survived (Giller et al., 1989; Hirsch et al., 1993) which was heavy metal tolerant (Chaudri et al., 1992a) but ineffective in N2-®xation with white clover (Giller et al., 1989). Further research in long-term experiments at Braunschweig, Germany, demonstrated complete loss of R. leguminosarum bv. trifolii (Chaudri et al., 1993) at metal concentrations within the guidelines for environmental protection in the European Union (CEC, 1986). This led to recommendations for increased stringency in the guidelines for the prevention of Zn accumulation in UK soils receiving sewage sludges (MAFF/DoE, 1993). Evidence from ongoing research on soil microorganisms is contributing to revisions of legislation for environmental protection in Europe. The ®eld experiments at Braunschweig form an ideal basis for the study of microbial ecology, as two series of 20 and 28 plots were established which differ little in soil
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00210-3
520
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
organic matter accumulation (from 0.9 to 1.6%) but range widely in Zn contamination. Concentrations of Zn range from the background of 50 to 400 mg kg 21 (Chaudri et al., 1993), which is slightly above the EU guidelines for environmental protection (150±300 mg Zn kg 21; CEC, 1986). The metal contamination in the plots was caused by additions of metal-contaminated sewage sludges from local sewage works between 1980 and 1991, with some treatments receiving sludges amended with heavy metals. All plots which received sludge are contaminated with a cocktail of metals, but across the range of treatments Cu concentrations reached only 115 mg kg 21 and Cd concentrations were less than 3 mg kg 21 (Chaudri et al., 1993; Knight et al., 1997). We investigated the populations of Rhizobium leguminosarum bv. viciae surviving along the gradient of increasing metal contamination in the long-term Old-Arable ®eld experiment at Braunschweig. R. leguminosarum bv. viciae is the symbiont of pea (Pisum sativum L.) and ®eld bean (Vicia faba L.) which are important arable legume crops in Northern Europe. Earlier work that focused on R. leguminosarum bv. trifolii (e.g. Chaudri et al., 1993) had not addressed aspects of population structure and diversity. Our primary hypothesis was that the abundance and diversity of the rhizobia would progressively decrease with increasing heavy metal stress. Rhizobial numbers were counted and isolates were typed using restriction fragment length polymorphisms (RFLPs) of the variable internally transcribed spacer region (ITS) between the 16S and 23S rDNA genes (Laguerre et al., 1996) and on the basis of their plasmid pro®les (Giller et al., 1989; Laguerre et al., 1992; Hirsch et al., 1993). Experiments were then conducted to examine the Zn tolerance of isolates from soils with different heavy metal loading as Zn is the heavy metal thought to be responsible for causing the deleterious microbial effects in the Braunschweig experiments (Giller et al., 1998).
2. Materials and methods 2.1. Field experiments All of the rhizobia studied were isolated from soils from the Old Arable Soil ®eld experiment at the Federal Research Centre for Agriculture (FAL), Braunschweig, north east Germany. This experiment had been established in 1980 in a uniform area of 0.2 ha of a ®eld which was previously under arable cultivation. Soils from the plots had a pH range of 5.9±7.6, total C contents of 0.91±1.62% and total N of 0.09±0.16%. Further details of the experiment are described by Chaudri et al. (1993). Rhizobial populations were assessed in all four replicate plots of each of the seven treatments, and isolates studied in detail from soils representing the range of metal concentrations from the following treatments (applied between 1980
and 1990): inorganic fertilizer at a rate of 180 kg N ha 21 yr 21 (Plots 2-8 and 2-26), 100 m 3 ha 21 yr 21 sewage sludge (Plot 4-23), 300 m 3 ha 21 yr 21 sewage sludge (Plot 6-2), 100 m 3 ha 21 yr 21 metal-amended sewage sludge (Plot 525), 48 t ha 21 yr 21 ®lter-pressed sewage sludge (Plot 9-30) and 300 m 3 ha 21 yr 21 metal-amended sewage sludge (Plots 7-13 and 7-31). The ®rst digit of the plot codes signi®es the treatment and the second number, the individual plot number. Total Zn concentrations in these soils determined by atomic absorption spectrometry after digestion in Aqua Regia (using the method of McGrath and Cunliffe (1985)) were Plot 2-8, 54 mg kg 21; Plot 2-26, 53 mg kg 21; Plot 4-23, 104 mg kg 21; Plot 5-25, 166 mg kg 21; Plot 6-2, 208 mg kg 21; Plot 9-30, 245 mg kg 21; Plot 7-13, 340 mg kg 21; and Plot 7-31, 399 mg kg 21 (Knight et al., 1997). All plots were 6 m £ 28.5 m separated by 1± 2 m paths and the soil samples were taken from the central 2 m 2 of the plots (to avoid `edge effects') in June 1994. Soils were also sampled from the paths adjacent to Plots 2-8 and 7-13 in May 1996. All soils were sieved (4 mm) and stored moist in loosely tied polyethylene bags at 4 8C prior to rhizobial isolation. 2.2. Rhizobial counts and isolation Numbers of R. leguminosarum bv. viciae were estimated by the most probable number (MPN) plant infection method (Vincent, 1970). Hairy vetch (Vicia hirsuta L.) seeds were surface sterilized with concentrated sulphuric acid for 2 min and rapidly washed 10 times with sterile deionized water. The seeds were germinated on Petri dishes containing 1% agar. Two days later, 10±20 mm long seedlings were transferred to tubes containing 15 ml Hewitt's-N solution (Hewitt, 1952). Tenfold serial dilutions of soil were prepared by shaking 10 g of soil in 90 ml sterilized distilled water, and serial dilutions up to 10 28 prepared (1 ml in 9 ml sterilized water). Triplicate plant infection tubes were inoculated with 1 ml aliquots of each dilution. Tubes were placed in a controlled environment cabinet with 14 h light, and day and night temperatures of 21 and 16 8C, respectively. Plants were examined for nodulation after 3 and 5 weeks. The numbers of R. leguminosarum bv. viciae were estimated using the mpnes computer program (Woomer et al., 1990). Nodules were surface-sterilized by immersing in 3% Na hypochlorite for 3 min and washing ®ve times with sterile deionized water. Nodules were squashed in a drop of sterilized water onto the surface of yeast-extract mannitol (YEM) agar in Petri dishes and incubated at 28 8C. Isolates were streaked out on YEM plates and a single colony picked to avoid problems of mixed isolates. 2.3. DNA isolation and PCR±RFLP analysis of the ITS region Isolates of R. leguminosarum bv. viciae were grown in tryptone-yeast (TY) broth for 2 days at 28 8C with shaking (150 rev min 21). A 3 ml aliquot of the grown cells was
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
washed and pelleted in an Eppendorf tube. Total DNA was isolated by a simple procedure involving lysis with proteinase-K and RNase (10 mg ml 21) and 0.5% sodium dodecyl sulphate at 65 8C, phenol extraction and ethanol precipitation. Precipitated DNA resuspended in water (5 ml) was run on 0.8% agarose and compared with 1 ml l DNA (500 ng) standard cut with HindIII and EcoRI to get an approximate DNA concentration. A dilution to about 10 ng ml 21 of each sample was made for PCR ampli®cation. The populations from ®ve plots were studied in detail by characterizing roughly 50 isolates per plot (except for Plot 7-31 where 33 isolates were studied as the population in this plot was small) giving a total of 231 isolates. Approximately 40 isolates from Plots 7-13, 2-26 and 9-30 were also characterized for comparison. 2.4. PCR ampli®cation and sequence analysis The two primers chosen for ampli®cation of the ITS region of the 16S±23S rRNA were the forward primer C (5 0 -GGCTGGATCACCTCCTTTCT-3 0 ) corresponding to the 3 0 end of 16S gene, and the reverse primer D (5 0 CCGGGTTTCCCCATTCGG-3 0 ) corresponding to the 5 0 region of the 23S gene (Laguerre et al., 1996). The primer sequences are highly conserved and complemented in a wide range of bacteria. Two other primers were used for ampli®cation of the 5 0 region of the 16S gene: the forward primer A (5 0 -GGAGAGTTAGATCTTGGCTTGGCTCAG3 0 ) corresponding to the 5 0 region of the 16S gene, and the reverse primer B (5 0 -CCAGTGTGGCCGGTCGCCCTCTC3 0 ) which is approximately 250±350 bp downstream of primer A (Weisburg et al., 1991). PCR ampli®cation was carried out in a 50 ml reaction volume. DNA was ampli®ed by mixing 2 ml of the template DNA (,10 ng ml 21), 5 ml of the polymerase reaction buffer 10 £ , 2 ml of 50 mM MgCl2, 2 ml of each primer pair (10 mM), 3 ml of dNTPs (2.5 mM), 0.5 units of Taq DNA polymerase per reaction and 34 ml water. DNA was ampli®ed in a Perkin±Elmer 9600 thermal cycler with the following temperature pro®le: an initial denaturation temperature at 94 8C for 5 min; 30 cycles of denaturation (30 s at 94 8C), annealing (30 s at 58 8C) and extension (45 s at 72 8C) and a ®nal extension time at 72 8C for 3 min. Ampli®ed DNA product was examined by horizontal electrophoresis, 100 V for 2 h, in 1% agarose with a 7 ml aliquot of PCR product. The PCR products were then used for RLFP analysis. The sequence of a ,230 bp fragment of the 16S rRNA gene was determined for two selected isolates by direct automatic sequencing of the PCR product of primers A and B. 2.5. DNA digestion and electrophoresis Restriction endonucleases were digested with (10±12 ml) aliquots of the mixture for each PCR reaction. Ten restriction endonucleases MspI, AciI, HhaI, DdeI, MboI, BfaI, HaeI, RsaI, HinfI, HindIII were initially tested. Two
521
restriction enzymes (HhaI and DdeI) had good discriminating power (gave three to ®ve bands of different sizes with distinct strains) and were selected for general RFLP analysis of isolates. Digested products were examined by horizontal gel electrophoresis using 2% agarose gels at 80 V for 3 h. Finally, gels were stained with ethidium bromide and photographed under UV illumination with Polaroid 665 positive/ negative ®lm. 2.6. Strain typing using plasmid pro®les Isolates of R. leguminosarum bv. viciae were grown on TY plates (Beringer, 1974) for 28 h and then in PA broth (0.4% w/v Difco Bacto-peptone and 2 mM MgSO4) for 14± 16 h at 28 8C with shaking (150 rev min 21; Hirsch et al., 1980). The cultures (0.7 ml) were spun down at 13,000g for 3 min and the supernatant discarded. Bacterial cells were resuspended in 32 ml of lysis solution (1 ml resuspension buffer (372.2 mg EDTA, 12 g sucrose and 151 mg Tris base in 50 ml); 50 ml lysozyme (20 mg Sigma L-6876 crystallized lysozyme in 1 ml water); 50 ml RNase stock solution (400 mg ml 21) and 16 ml xylene cyanol) and kept on ice before loading onto gels. Plasmids were separated by running 16±20 ml samples on water cooled Eckhardt (Eckhardt, 1978) agarose gels (0.6% agarose in electrophoresis buffer of 89 mM Tris base, 2.5 mM EDTA and 8.9 mM boric acid; 0.4% agarose with 1% SDS behind the well-former) for about 3.5 h at 210 V. One lane was loaded with the R. leguminosarum bv. viciae strain T83K3 which has six plasmids of 480, 440, 300, 255, 210 and 155 kb (Wang et al., 1986). A total of 345 isolates were typed from seven plots. Analysis of the populations by plasmid pro®les was dif®cult when attempting to classify isolates into groups across plots, as a number of the plasmids were similar in size. This necessitated their examination in adjacent lanes on Eckhardt gels, thus requiring a large number of gels for cross-comparisons. 2.7. Zn tolerance of rhizobial isolates The degree of Zn tolerance in 70 isolates (14 from each of the ®ve plots) was assessed on TY plates to which varying concentrations of analytical grade ZnSO4 were added. Isolates were grown in TY broth at 28 8C for 2 days and washed twice in sterile deionized water and three replicate drops applied to the TY plates. The Zn tolerance of 10 isolates was also compared using a cation-exchange resin (Amberlite IRI 20-plus) method designed to ensure a constant ionic activity in solution (for details see Knight, 1996). In this method, isolates were grown in buffered media at four or ®ve different Zn concentrations (from 0 to 50 mg l 21) and the number of cells surviving over 72 h was measured using a drop-plate counting method (Miles and Misra, 1938). Two R. leguminosarum bv. trifolii isolates were included for comparison: S9 from a heavy metal contaminated soil and isolate F5 from uncontaminated soil (Giller et al., 1989; Chaudri et al., 1992a).
522
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
concentrations found across the metal gradient. All of the isolates gave one or two ITS bands of between 800 and 1300 bp before digestion. In total 20 ITS `strain' groups were identi®ed on the basis of the RFLP patterns after digestion with the restriction enzyme HhaI (Figs. 2(b) and (d) and 3(b)). Groupings using DdeI to digest the ITS±PCR product were exactly the same as those obtained with HhaI. Where isolates from different plots had similar RFLP patterns, their similarity was con®rmed by comparing them on the same gel. The largest number of ITS groups (10 groups) was found in the control plot (Plot 2-8) and the least number of groups (two groups) in the most contaminated plot (Plot 7-31), with no clear pattern to the number found in the other plots (Fig. 3(b)). The most striking result was the occurrence and abundance of ITS group 1 in all plots. The dominance of this group tended to increase with increasing Zn concentration: it comprised 82% of the isolates in the 300 m 3 metalamended sewage sludge treated plot, but only 23±55% of the isolates in the other plots. In the 300 m 3 sewage sludge treated plot, 23% of the isolates were from group 1 and a further 51% of the isolates were from group 3. Isolates from ITS group 3 were present only in Plot 6-2 and were differentiated from ITS group 1 only on the basis of an extra band with both restriction enzymes HhaI (Fig. 2(d)) and DdeI. The population from another plot with high metal loading (Plot 9-30; 48 t ha 21 yr 21 ®lter-pressed sewage sludge) was also dominated by ITS groups 1 and 3. Fig. 1. Numbers of R. leguminosarum bv. viciae estimated by the MPN plant infection method plotted against the total Zn concentration in the soils in plots from the Old Arable Soil, Braunschweig. The vertical lines indicate the current maximum Zn concentrations allowed to accumulate in soils receiving sewage sludge in Germany and in the UK. Arrows indicate the plots sampled for detailed analysis of the populations.
3. Results 3.1. Abundance of R. leguminosarum bv. viciae The number of indigenous R. leguminosarum bv. viciae decreased progressively as heavy metal concentrations increased (Fig. 1). Rhizobial numbers ranged from 1.4 £ 10 4 to 8 £ 10 4 cells g 21 soil in plots which had received 180 kg ha 21 yr 21 inorganic N fertilizer with resulting Zn concentrations around 50 mg Zn kg 21 to between 7 and 2.3 £ 102 cells g 21 soil in plots treated with 300 m 3 ha21 yr 21 metal-amended sewage sludge in which Zn concentrations were 338±399 mg Zn kg 21. 3.2. Diversity of R. leguminosarum bv. viciae based on ITS± RFLP patterns The diversity of 231 isolates from ®ve plots was assessed using the ITS±PCR method. The plots were selected to represent the range of population sizes and metal
3.3. Diversity and complexity of plasmid pro®les Plasmid pro®le groupings were based on visual analysis of the number and size of plasmids seen on Eckhardt gels (Fig. 2). In the three plots with the least metal contamination (Plots 2-8, 4-23 and 5-25), plasmid pro®les of the isolates were fairly simple with three, four or ®ve bands. There was also a great variety of banding patterns (10±14 types) present among the 50 isolates from the plots with less than 250 mg Zn kg 21 (Figs. 2(a) and (c) and 3(a)). Plasmid pro®les varied between plots although a number of pro®les were common to different plots (Fig. 3(a)). With increasing metal concentrations, rhizobia with more complex pro®les of six to nine large plasmids were isolated (Figs. 2(c) and 3(a)). Such isolates formed an increasingly large proportion of the isolates from each plot, although some isolates with only two to ®ve plasmids were present in plots with higher metal loading (Figs. 2(c) and 3(a)). One plasmid pro®le group (group 1) with eight plasmids was particularly dominant, comprising 83±91% of the isolates in the two most contaminated plots (Plots 7-13 and 7-31) which had received the 300 m 3 metal-amended sewage sludge treatment (Figs. 2(c) and 3(a)). Plasmid pro®les of ,50 isolates from each of the four additional plots of the control (180 kg ha 21 yr 21 inorganic N fertilizer) and 100 m 3 ha 21 yr 21 sewage sludge treatments were also examined. All isolates from these additional plots
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
523
Fig. 2. Photographs of (a and c) Eckhardt gels showing plasmid pro®les and (b and d) agarose gels showing the corresponding RFLPs of the PCR products of the ITS region digested using HhaI of R. leguminosarum bv. viciae isolates from plots of the Old Arable Experiment at Braunschweig. Nearly all plasmid pro®le types are represented and for cross-comparison the isolates were ordered so that isolates with similar (but not identical) plasmid pro®les were close together on the gels. The isolates are from a range of plots and treatments: Plot 2-8, control treatment which received 180 kg ha 21 yr 21 inorganic N fertilizer; Plot 4-23, 100 m 3 ha 21 yr 21 sewage sludge treatment; Plot 6-2, 300 m 3 ha 21 yr 21 sewage sludge treatment; Plot 7-31, 300 m 3 ha 21 yr 21 metal-amended sewage sludge treatment; and Plot 9-30, 48 t ha 21 yr 21 ®lter-pressed sewage sludge treatment. The plasmid size marker in (a) is T3K83. A 1 kb ladder is shown in lane 1 of (b) and (d).
524
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
Fig. 3. Histograms showing the distribution of the groups: (a) based on plasmid pro®les; and (b) based on RFLP analysis using the ITS±PCR method in the different plots (approximately 50 isolates per plot) of R. leguminosarum bv. viciae isolated from the Old Arable Soil, Braunschweig. Numbers above each bar in (a) indicate the number of plasmid bands present in isolates belonging to that group.
had ®ve or less plasmids with many plasmid pro®les, similar to those of other control plots. Overall, mean plasmid number increased markedly with metal concentration (Fig. 4). Additional soil samples were taken in May 1996 from the paths adjacent to the heavily contaminated plots 7-13 and 7-31. These soil samples represented adjacent uncontaminated soil which had not been subject to any treatment effects. All 60 isolates of R. leguminosarum bv. viciae made from these samples had a range of plasmid banding patterns with two to six plasmids (data not shown), adding further
evidence that the dominance of isolates with complex plasmid pro®les in these plots resulted from the heavy metal stress. 3.4. Species af®nity of the isolates Two isolates, one from the most contaminated plot (isolate 7-31-1 from ITS group 1 which contained 55% of the isolates overall) and one from the control plot (isolate 28-1 from ITS group 5) were characterized by partial
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
525
Fig. 4. The relationships between mean numbers of plasmids (X) in ,50 isolates per plot of R. leguminosarum bv. viciae, and diversity of rhizobial types based on both plasmid pro®les and ITS±RFLPs (W) from plots on the Old Arable Soil, Braunschweig against soil Zn concentration.
sequence analysis of ,260 bp from the 5 0 end of the 16S rRNA gene. In both cases, the nucleotide sequence was 100% homologous with that of the R. leguminosarum bv. viciae type strain (National Center for Biotechnology Information Accession number U29386), which con®rmed their expected species af®nity. 3.5. Zn tolerance of the isolates As a major difference between the soils from the plots was the elevated Zn concentration where sewage sludges had been applied, a simple method of screening isolates for growth on agar plates was used to evaluate their relative tolerance to Zn. With some exceptions the tests indicated that there was a general, though not particularly marked increase in Zn tolerance with increasing metal loading in the soil (data not shown; A. Lakzian, unpublished PhD Thesis, Wye College, University of London, 1998). Within a given plot, different isolates from the same ITS group had similar Zn tolerance. In the two most contaminated plots isolates from ITS group 1 grew at higher concentrations of Zn than isolates of the same group from the less contaminated plots. Isolates of ITS group 1 that had eight or nine plasmids were clearly more Zn tolerant than isolates from ITS group 2 from the same plot which had only three plasmids. Similar tests of tolerance to Cu and Cd on agar plates revealed no consistent differences between the isolates. Metal toxicity of only a limited number of isolates was tested using the buffered solution method due to the laborious nature of this assay. The two test strains F6 and S9 were clearly differentiated using this method; the metal tolerant strain S9 grew at all metal concentrations, whereas F6 did
not grow at the higher concentrations (Fig. 6). Isolates from ITS groups 1, 2, 6, 12, 14 and 17 with only three plasmids were all killed after 72 h with the 30 and 50 mg Zn l 21 treatments (results not shown). Isolates which both belonged to ITS group 1 and had eight plasmids (e.g. Fig. 6(b)) were clearly the most tolerant of all the isolates from the Braunschweig soils tested as they grew at 30 mg Zn l 21, though these isolates did not possess the same degree of metal tolerance as the test strain S9 (Fig. 6(d)). Isolate 731-15 (ITS±RFLP group 2) with only three plasmids from the most contaminated plot was as sensitive as all other isolates tested using this method (Fig. 6(a)).
4. Discussion 4.1. Effects of heavy metal pollution on rhizobial abundance The progressive reductions in numbers of R. leguminosarum bv. viciae with increasing heavy-metal contamination in the soil (Fig. 1) were similar to the results with R. leguminosarum bv. trifolii reported by Chaudri et al. (1993). However, their results indicated that the numbers of R. leguminosarum bv. trifolii were more strongly reduced in plots which received 300 m 3 ha 21 yr 21 sewage sludge and no clover rhizobia were detected in some of the most contaminated plots. Rhizobia are known to be highly competent heterotrophs which can survive for long periods in the absence of a compatible legume host (Giller, 2001). As no legumes have been grown on these plots since before 1970 (although some compatible legumes could have occurred sporadically as weeds) these changes in rhizobial populations have occurred in the absence of the in¯uence of
526
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
Fig. 5. Characterization of 10 isolates of R. leguminosarum bv. viciae from a plot of the 300 m 3 ha 21 yr 21 metal-amended sewage sludge treatment (Plot 7-13) and 10 isolates from a plot of the control treatment which received 180 kg ha 21 yr 21 inorganic N fertilizer (Plot 2-26). Photographs of (a) Eckhardt gels showing plasmid pro®les; (b) gel showing the PCR product of the ITS region. A 1 kb ladder is shown in the two central lanes; and (c) gel showing RFLPs generated by digesting the PCR product of the ITS region using HhaI. The central lane is a 50 bp±2 kb marker.
the host legume. The metal concentrations in these plots are also suf®ciently small that growth of plants is unaffected, so it is unlikely that the changes in population sizes are due to indirect effects of plant rhizospheres, or inputs of C through root exudates. 4.2. Comparison of rhizobial diversity based on ITS±RFLP typing and plasmid pro®le groups In total, 49 plasmid pro®le groups were identi®ed among isolates from the Old Arable Soil but only 20 ITS±RFLP groups were distinguished (Fig. 3). Cross-comparison of the isolate groupings using the two methods revealed that many isolates belonging to the same RFLP group had distinct plasmid pro®les. There was a clear trend of increasing plasmid number with increasing metal concentration in the soil (Fig. 4). Due to the wide differences in population size, the sample of isolates studied is equivalent to the whole population per gram soil in the most contaminated plot, but it represents only 0.001% of the culturable population in the control plot. However, we have no reason to assume that there was any bias in the sampling of isolates and typing of greater numbers was not feasible. Isolates which belonged to ITS group 1 were abundant in all of the plots, yet none of the plasmid pro®le groups were common to all of these plots (Fig. 3). Comparison of the plasmid pro®le and ITS±RFLP groups revealed that a wide range of plasmid pro®les were associated with some of the ITS groups (Fig. 2). The ITS groups which were represented
by only single isolates, of course, contained only a single plasmid pro®le type but generally when isolates of the same ITS group had been isolated from different plots, the plasmid pro®les differed (e.g. see plasmid pro®le groups associated with ITS groups 1 and 4; Fig. 2). The widest diversity of plasmid pro®les was associated with ITS group 1 which was the most abundant group in almost all of the plots. Further evidence for the similarity of ITS groups 1 and 3 is the occurrence of isolates from plasmid pro®le group 1 in both of these ITS groups. Plasmid pro®le group 1 had eight plasmids and was the dominant type in the most contaminated plot, and another very similar plasmid pro®le group (group 9) which had seven plasmids was abundant in Plot 6-2 (Fig. 3). Isolates from other plots with high metal loading (Plot 9-30; 48 t ha 21 yr 21 ®lter-pressed sewage sludge and Plot 7-13; 300 m 3 ha 21 yr 21 metal-amended sewage sludge) also had many isolates with large numbers of plasmids which belonged to two similar ITS groups 1 and 3 (Fig. 2(c) and (d)). A comparison of 10 isolates from each of a control plot (Plot 2-26) and a contaminated plot (Plot 7-13) clearly demonstrated the dominance of ITS group 1 in both plots (Fig. 5). Several different plasmid groups were represented in the control plot, all of which had only two to four plasmids, whereas the 10 isolates from the contaminated plot all belonged to ITS group 1 with eight plasmids. Certain plasmids are often associated with particular chromosomal types (Young and Wexler, 1988; Souza and Eguiarte, 1997), and many plasmid incompatibility groups
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
527
Fig. 6. Metal tolerance of two isolates of R. leguminosarum bv. viciae from the most contaminated plot (Plot 7-31; 399 mg Zn kg 21) of the Old Arable Experiment, Braunschweig based on growth/survival of cells in buffered Zn solutions of varying concentration. Two isolates of R. leguminosarum bv. trifolii (F6 and S9) from Woburn, UK known to differ in metal tolerance were included for comparison.
occur so that certain plasmids can or cannot coexist in the same bacterium (Shaw, 1987). Our study clearly demonstrates that certain chromosomal types maintain larger numbers of plasmids under conditions of heavy metal stress in the soil. These strains with many plasmids are almost exclusively dominant in the small populations in the contaminated plots, suggesting that the extra plasmids confer increased ®tness under stressed conditions. At variance to our initial hypothesis, that numbers and diversity of rhizobia would progressively decrease with increasing metal concentrations, a strong reduction in diversity (in both numbers of plasmid pro®le groups (Fig. 3(a)) and ITS±RFLP groups (Fig. 3(b)) was only found at the highest metal concentrations. At intermediate metal concentrations, the diversity of plasmid pro®le groups tended to increase, and the diversity of groups clearly increased when isolates are grouped on the basis of both their PCR±RFLPs and their plasmid pro®les (Fig. 4). This is consistent with ecological models which suggest that moderate environmental stresses may reduce exclusion due to particular competitive genotypes, thus reducing dominance and giving apparent increases in diversity with intermediate stress (Austin and Smith, 1989).
4.3. Interrelationships between ITS groups, plasmid pro®les and Zn tolerance of rhizobial isolates How the isolates of ITS group 2, which were among the most sensitive to Zn in arti®cial media (Fig. 6), are able to survive in the most contaminated plots is unclear. Other studies have shown that exposure to moderate metal concentrations (similar to those in the Braunschweig ®eld experiment) for more than a year is required to signi®cantly reduce rhizobial populations (e.g. Chaudri et al., 1992b; Giller et al., 1993). The soils in this study had been subject to these concentrations of heavy metal contamination for at least 4 yr since sludge applications ceased. The bacteria may be avoiding the heavy metal stress in some way, for instance by physical protection by close adherence to clay particles or within micropores of soil aggregates where the active metal concentrations may be smaller. The metal tolerance tests showed that in spite of the reduction in the number of groups and few types of isolates remaining in the most contaminated plots, there was only a slight increase in the tolerance to Zn (Fig. 6). The Zn concentrations tested in these laboratory assays cannot be extrapolated to concentrations to which the bacteria are
528
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
exposed in soil where the concentrations of the free Zn 21 ion have been measured as less than 600 mg l 21 in all of the plots (B. Knight, unpublished). Nevertheless, when the results were compared for different isolates belonging to a particular ITS group, a pattern of tolerance could be seen and the overall tolerance tended to increase with heavy metal loading in the soil. None of the isolates was as tolerant as the metal-tolerant isolate (S9) which was dominant in a heavy metal contaminated plot at Woburn, England (Giller et al., 1989; Hirsch et al., 1993) and was tolerant to Zn, Cd, Cu and Ni (Chaudri et al., 1992a). Increases in the number of plasmids carried by isolates were associated with increased metal tolerance. This was observed both between isolates from ITS group 1 from the same plot and between isolates from this group from different plots. The dominance of the more tolerant types with large numbers of plasmids in the plots with more metal contamination (Plots 6-2, 9-30, 7-13 and 7-31) is therefore likely to be due to increased metal tolerance associated with the large number of plasmids. A plasmid of approximately 70 kb appeared to be common to all of the isolates which had enhanced metal tolerance and this plasmid may be the source of metal tolerance genes. Genes for heavy metal tolerance in a wide range of bacteria are often carried on plasmids (Silver and Misra, 1988). If acquisition of the plasmids was related to increased metal tolerance, the extra energetic costs of maintaining a large number of plasmids could be partly counterbalanced by the selective pressure of heavy metal toxicity. However, it is highly unlikely that all of the extra plasmids carried by the isolates in the most contaminated plots are involved in metal tolerance. As yet, we have no evidence of any speci®c functions for these plasmids, though it seems unlikely that a rhizobial strain would carry so many plasmids unless they confer some selective advantage. A mixture of organic compounds as well as a number of different metals will have been added to the soils in the sewage sludge treatments. As a period of 4 yr had elapsed since sewage sludge was last added to the plots and the time of sampling, most labile compounds would have already been degraded. It is possible that some of the plasmids actually originated from microorganisms added in the sewage sludge, though we cannot test this suggestion as samples of the sludges added to the soils are not available. Comparison of the rhizobial populations present in the 300 m 3 ha21 yr 21 sewage sludge treatment (Plot 6-2) and the 300 m 3 ha21 yr21 metalamended sewage sludge treatment (Plots 7-13 and 7-31) allows the effects of sludge loading to be separated from effects of metals. Isolates with eight plasmids of the same ITS and plasmid pro®le group as in Plots 7-13 and 7-31 were present in Plot 6-2, but this type was dominant only in the plots with greater heavy metal concentrations, again suggesting that the large number of plasmids in the rhizobial isolates was related to heavy metal stress. Other studies have also failed to reveal a simple relationship between heavy metal toxicity and microbial diversity
(Sandaa et al., 1999, 2001). The complex relationship between diversity of rhizobia and heavy metal stress observed here suggests that the common assumption that increasing stress leads to simple reductions in microbial diversity should be revisited. Acknowledgements This research was conducted with ®nancial support from NERC and the EU-Environment Programme of DGXII. Amir Lakzian is grateful to the Government of Iran for a scholarship. References Austin, M.P., Smith, T.M., 1989. A new model for the continuum concept. Vegetatio 83, 35±47. Beringer, J.E., 1974. R Factor transfer in Rhizobium leguminosarum. Soil Biology and Biochemistry 84, 188±198. CEC, 1986. Council Directive on the protection of the environment, and in particular of soil, when sewage sludge is used in agriculture. Of®cial Journal of the European Communities Directive, 86/278/EEC, No. L181/6-12. Chaudri, A.M., McGrath, S.P., Giller, K.E., 1992a. Metal tolerance of isolates of Rhizobium leguminosarum biovar trifolii from soil contaminated by past applications of sewage sludge. Soil Biology and Biochemistry 24, 83±88. Chaudri, A.M., McGrath, S.P., Giller, K.E., 1992b. Survival of the indigenous population of Rhizobium leguminosarum biovar trifolii in soil spiked with Cd, Zn, Cu and Ni salts. Soil Biology and Biochemistry 24, 625±632. Chaudri, A.M., McGrath, S.P., Giller, K.E., Rietz, E., Sauerbeck, D., 1993. Enumeration of indigenous Rhizobium leguminosarum biovar trifolii in soils previously treated with metal-contaminated sewage sludge. Soil Biology and Biochemistry 25, 301±309. Eckhardt, T., 1978. A rapid method for the identi®cation of plasmid deoxyribonucleic acid in bacteria. Plasmid 1, 584±588. Giller, K.E., 2001. Nitrogen Fixation in Tropical Cropping Systems. 2nd ed. CAB International, Wallingford. Giller, K.E., McGrath, S.P., Hirsch, P.R., 1989. Absence of nitrogen ®xation in clover grown on soil subject to long term contamination with heavy metals is due to survival of only ineffective Rhizobium. Soil Biology and Biochemistry 21, 841±848. Giller, K.E., Nussbaum, R., Chaudri, A.M., McGrath, S.P., 1993. Rhizobium meliloti is less sensitive to heavy-metal contamination in soil than R. leguminosarum bv. trifolii or R. loti. Soil Biology and Biochemistry 25, 273±278. Giller, K.E., Witter, E., McGrath, S.P., 1998. Toxicity of heavy metals to microorganisms and microbial processes in agricultural soilsÐa review. Soil Biology and Biochemistry 30, 1389±1414. Hewitt, E.J., 1952. Sand and Water Culture Methods used in the Study of Plant Nutrition. Commonwealth Agricultural Bureaux, Farnham Royal. Hirsch, P.R., Van Montagu, M., Johnston, A.W.B., Brewin, N.J., Schell, J., 1980. Physical identi®cation of bacteriocinogenic, nodulation and other plasmids in strains of Rhizobium leguminosarum. Journal of General Microbiology 120, 403±412. Hirsch, P.R., Jones, M.J., McGrath, S.P., Giller, K.E., 1993. Heavy-metals from past applications of sewage sludge decrease the genetic diversity of Rhizobium leguminosarum biovar trifolii populations. Soil Biology and Biochemistry 25, 1485±1490. Knight, B., 1996. Unpublished PhD Thesis, Wye College, University of London. Knight, B., McGrath, S.P., Chaudri, A.M., 1997. Biomass carbon
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529 measurements and substrate utilization patterns of microbial populations from soils amended with cadmium, copper, or zinc. Applied and Environmental Microbiology 63, 39±43. Laguerre, G., Mazurier, I.S., Armarger, N., 1992. Plasmid pro®les and restriction fragment length polymorphism of Rhizobium leguminosarum bv. viciae in ®eld populations. FEMS Microbiology Ecology 101, 17±26. Laguerre, G., Mavingui, P., Allard, M.-R., Charnay, M.-P., Louvrier, P., Mazurier, S.-I., Rigottier-Gois, L., Amarger, N., 1996. Typing rhizobia by PCR DNA ®ngerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars. Applied and Environmental Microbiology 62, 2029±2036. MAFF/DoE, 1993. Review of the Rules for Sewage Sludge Application to Agricultural Land: Soil Fertility Aspects of Potentially Toxic Elements. Report of the Independent Scienti®c Committee, PB 1561, MAFF Publications, London. McGrath, S.P., Cunliffe, C.H., 1985. A simpli®ed method for the extraction of metals Fe, Zn, Cu, Ni, Cd, Pb, Cr, Co and Mn from soils and sewage sludges. Journal of the Science of Food and Agriculture 36, 794±798. McGrath, S.P., Brookes, P.C., Giller, K.E., 1988. Effects of potentially toxic metals in soil derived from past applications of sewage sludge on nitrogen ®xation by Trifolium repens L. Soil Biology and Biochemistry 20, 415±424. Miles, A.A., Misra, S.S., 1938. The estimation of the bactericidal power of blood. Journal of Hygiene, Cambridge 38, 732±748.
529
Sandaa, R.-A., Torsvik, V., Enger, é., Daae, F.L., Castberg, T., Hahn, D., 1999. Analysis of bacterial communities in heavy metal contaminated soil at different levels of resolution. FEMS Microbiology Ecology 30, 237±251. Sandaa, R.-A., Torsvik, V., Enger, é., 2001. In¯uence of long-term heavy metal contamination on microbial communities in soil. Soil Biology and Biochemistry 33, 287±295. Shaw, P.D., 1987. Plasmid ecology. In: Kosuge, T., Nester, E. (Eds.). Plant±Microbe Ineractions. Macmillan, New York, pp. 3±39. Silver, S., Misra, T.K., 1988. Plasmid-mediated heavy metal reistances. Annual Reviews of Microbiology 42, 717±743. Souza, V., Eguiarte, L.E., 1997. Bacteria gone native vs. bacteria gone awry: plasmidic transfer and bacterial evolution. Proceedings of the National Academy of Sciences, USA 94, 5501±5503. Vincent, J.M., 1970. A Manual for the Practical Study of Root-Nodule Bacteria. Blackwell, Oxford. Wang, C.L., Beringer, J.E., Hirsch, P.R., 1986. Host plant effects on hybrids of Rhizobium leguminosarum biovars viciae and trifolii. Journal of General Microbiology 132, 2063±2070. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal ampli®cation for phylogenetic study. Journal of Bacteriology 173, 697±703. Woomer, P., Bennet, J., Yost, R., 1990. Overcoming the in¯exibility of most-probable number procedures. Agronomy Journal 82, 349±353. Young, J.P.W., Wexler, M., 1988. Sym plasmid and chromosomal genotypes are correlated in ®eld populations of Rhizobium leguminosarum. Journal of General Microbiology 134, 2731±2739.
www.elsevier.com/locate/soilbio
Rhizobium leguminosarum bv. viciae populations in soils with increasing heavy metal contamination: abundance, plasmid pro®les, diversity and metal tolerance Amir Lakzian, Phillip Murphy, Andrew Turner 1, Jim L. Beynon, Ken E. Giller* Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK Received 17 April 2001; received in revised form 19 October 2001; accepted 6 November 2001
Abstract Populations of Rhizobium leguminosarum bv. viciae were investigated from plots of a long-term sewage sludge experiment in Braunschweig, Germany, which represented a gradient of increasing metal contamination. The number of R. leguminosarum bv. viciae decreased from 10 5 cells g 21 soil in uncontaminated plots to between 7 and 10 2 cells g 21 soil with increasing Zn concentration (from 50 to 400 mg kg 21). Rhizobia were isolated from nodules of Vicia hirsuta inoculated with dilutions of soil from seven of the plots (,50 isolates per plot). The rhizobial isolates had between three and nine plasmids which varied in size from approximately 100 to 850 kb. Although a total of 49 plasmid pro®le groups were identi®ed, PCR±RFLP analysis using primers which ampli®ed an intergenic spacer (ITS) region between the 16S and 23S rRNA genes revealed only 20 groups. Ten ITS groups were found among the isolates from the uncontaminated control plot but only two groups were found in the most contaminated plot, and six to eight groups in the plots with intermediate metal contamination. Numbers of plasmid pro®le groups increased with moderate metal-contamination but were strongly reduced when total Zn concentrations exceeded 300 mg kg 21. Isolates from the less contaminated plots had only three to ®ve plasmids whereas isolates with seven to nine plasmids were abundant in the plots with metal concentrations of 200 mg kg 21 or more. Whereas plasmid pro®les indicated considerable changes in strains with increasing metal contamination, one ITS group (group 1) was present in all plots. Isolates from ITS group 1 contained only three to four plasmids in the control plots but those from the most contaminated plots had eight to nine plasmids. There was a marked increase in metal tolerance of isolates belonging to ITS group 1 as metal contamination increased, which was associated with the increase in the number of plasmids carried. However, another ITS group (group 2), which had only three large plasmids was present only in the most contaminated plot. Isolates from this group had less tolerance to Zn than many isolates from the uncontaminated plots. Possible mechanisms for the survival of these isolates in the metal contaminated soils are discussed. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Metal toxicity; Sewage sludge; Pollution; Zinc; Microbial ecology; Rhizobium; Plasmids; Metal tolerance; Diversity
1. Introduction Soil microorganisms are sensitive to long-term exposure to moderate concentrations of heavy metals in soil (Giller et al., 1998). Effective strains of Rhizobium leguminosarum bv. trifolii, the nitrogen-®xing symbiont of white clover (Trifolium repens L.), were eliminated at metal concentrations too small to cause toxicity to plants (McGrath et al., 1988; Giller et al., 1989). Studies of the populations of rhizobia at Woburn, UK, demonstrated that only a single * Corresponding author. Present address: Department of Plant Sciences, Group Plant Production Systems, Wageningen University, P.O. Box 430, 6700 AK Wageningen, The Netherlands. Tel.: 131-317-485818; fax: 131317-484892. E-mail address: [email protected] (K.E. Giller). 1 Present address: School of Natural and Environmental Science, Coventry University, Priory Street, Coventry CV1 5FB, UK.
isolate of Rhizobium survived (Giller et al., 1989; Hirsch et al., 1993) which was heavy metal tolerant (Chaudri et al., 1992a) but ineffective in N2-®xation with white clover (Giller et al., 1989). Further research in long-term experiments at Braunschweig, Germany, demonstrated complete loss of R. leguminosarum bv. trifolii (Chaudri et al., 1993) at metal concentrations within the guidelines for environmental protection in the European Union (CEC, 1986). This led to recommendations for increased stringency in the guidelines for the prevention of Zn accumulation in UK soils receiving sewage sludges (MAFF/DoE, 1993). Evidence from ongoing research on soil microorganisms is contributing to revisions of legislation for environmental protection in Europe. The ®eld experiments at Braunschweig form an ideal basis for the study of microbial ecology, as two series of 20 and 28 plots were established which differ little in soil
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00210-3
520
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
organic matter accumulation (from 0.9 to 1.6%) but range widely in Zn contamination. Concentrations of Zn range from the background of 50 to 400 mg kg 21 (Chaudri et al., 1993), which is slightly above the EU guidelines for environmental protection (150±300 mg Zn kg 21; CEC, 1986). The metal contamination in the plots was caused by additions of metal-contaminated sewage sludges from local sewage works between 1980 and 1991, with some treatments receiving sludges amended with heavy metals. All plots which received sludge are contaminated with a cocktail of metals, but across the range of treatments Cu concentrations reached only 115 mg kg 21 and Cd concentrations were less than 3 mg kg 21 (Chaudri et al., 1993; Knight et al., 1997). We investigated the populations of Rhizobium leguminosarum bv. viciae surviving along the gradient of increasing metal contamination in the long-term Old-Arable ®eld experiment at Braunschweig. R. leguminosarum bv. viciae is the symbiont of pea (Pisum sativum L.) and ®eld bean (Vicia faba L.) which are important arable legume crops in Northern Europe. Earlier work that focused on R. leguminosarum bv. trifolii (e.g. Chaudri et al., 1993) had not addressed aspects of population structure and diversity. Our primary hypothesis was that the abundance and diversity of the rhizobia would progressively decrease with increasing heavy metal stress. Rhizobial numbers were counted and isolates were typed using restriction fragment length polymorphisms (RFLPs) of the variable internally transcribed spacer region (ITS) between the 16S and 23S rDNA genes (Laguerre et al., 1996) and on the basis of their plasmid pro®les (Giller et al., 1989; Laguerre et al., 1992; Hirsch et al., 1993). Experiments were then conducted to examine the Zn tolerance of isolates from soils with different heavy metal loading as Zn is the heavy metal thought to be responsible for causing the deleterious microbial effects in the Braunschweig experiments (Giller et al., 1998).
2. Materials and methods 2.1. Field experiments All of the rhizobia studied were isolated from soils from the Old Arable Soil ®eld experiment at the Federal Research Centre for Agriculture (FAL), Braunschweig, north east Germany. This experiment had been established in 1980 in a uniform area of 0.2 ha of a ®eld which was previously under arable cultivation. Soils from the plots had a pH range of 5.9±7.6, total C contents of 0.91±1.62% and total N of 0.09±0.16%. Further details of the experiment are described by Chaudri et al. (1993). Rhizobial populations were assessed in all four replicate plots of each of the seven treatments, and isolates studied in detail from soils representing the range of metal concentrations from the following treatments (applied between 1980
and 1990): inorganic fertilizer at a rate of 180 kg N ha 21 yr 21 (Plots 2-8 and 2-26), 100 m 3 ha 21 yr 21 sewage sludge (Plot 4-23), 300 m 3 ha 21 yr 21 sewage sludge (Plot 6-2), 100 m 3 ha 21 yr 21 metal-amended sewage sludge (Plot 525), 48 t ha 21 yr 21 ®lter-pressed sewage sludge (Plot 9-30) and 300 m 3 ha 21 yr 21 metal-amended sewage sludge (Plots 7-13 and 7-31). The ®rst digit of the plot codes signi®es the treatment and the second number, the individual plot number. Total Zn concentrations in these soils determined by atomic absorption spectrometry after digestion in Aqua Regia (using the method of McGrath and Cunliffe (1985)) were Plot 2-8, 54 mg kg 21; Plot 2-26, 53 mg kg 21; Plot 4-23, 104 mg kg 21; Plot 5-25, 166 mg kg 21; Plot 6-2, 208 mg kg 21; Plot 9-30, 245 mg kg 21; Plot 7-13, 340 mg kg 21; and Plot 7-31, 399 mg kg 21 (Knight et al., 1997). All plots were 6 m £ 28.5 m separated by 1± 2 m paths and the soil samples were taken from the central 2 m 2 of the plots (to avoid `edge effects') in June 1994. Soils were also sampled from the paths adjacent to Plots 2-8 and 7-13 in May 1996. All soils were sieved (4 mm) and stored moist in loosely tied polyethylene bags at 4 8C prior to rhizobial isolation. 2.2. Rhizobial counts and isolation Numbers of R. leguminosarum bv. viciae were estimated by the most probable number (MPN) plant infection method (Vincent, 1970). Hairy vetch (Vicia hirsuta L.) seeds were surface sterilized with concentrated sulphuric acid for 2 min and rapidly washed 10 times with sterile deionized water. The seeds were germinated on Petri dishes containing 1% agar. Two days later, 10±20 mm long seedlings were transferred to tubes containing 15 ml Hewitt's-N solution (Hewitt, 1952). Tenfold serial dilutions of soil were prepared by shaking 10 g of soil in 90 ml sterilized distilled water, and serial dilutions up to 10 28 prepared (1 ml in 9 ml sterilized water). Triplicate plant infection tubes were inoculated with 1 ml aliquots of each dilution. Tubes were placed in a controlled environment cabinet with 14 h light, and day and night temperatures of 21 and 16 8C, respectively. Plants were examined for nodulation after 3 and 5 weeks. The numbers of R. leguminosarum bv. viciae were estimated using the mpnes computer program (Woomer et al., 1990). Nodules were surface-sterilized by immersing in 3% Na hypochlorite for 3 min and washing ®ve times with sterile deionized water. Nodules were squashed in a drop of sterilized water onto the surface of yeast-extract mannitol (YEM) agar in Petri dishes and incubated at 28 8C. Isolates were streaked out on YEM plates and a single colony picked to avoid problems of mixed isolates. 2.3. DNA isolation and PCR±RFLP analysis of the ITS region Isolates of R. leguminosarum bv. viciae were grown in tryptone-yeast (TY) broth for 2 days at 28 8C with shaking (150 rev min 21). A 3 ml aliquot of the grown cells was
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
washed and pelleted in an Eppendorf tube. Total DNA was isolated by a simple procedure involving lysis with proteinase-K and RNase (10 mg ml 21) and 0.5% sodium dodecyl sulphate at 65 8C, phenol extraction and ethanol precipitation. Precipitated DNA resuspended in water (5 ml) was run on 0.8% agarose and compared with 1 ml l DNA (500 ng) standard cut with HindIII and EcoRI to get an approximate DNA concentration. A dilution to about 10 ng ml 21 of each sample was made for PCR ampli®cation. The populations from ®ve plots were studied in detail by characterizing roughly 50 isolates per plot (except for Plot 7-31 where 33 isolates were studied as the population in this plot was small) giving a total of 231 isolates. Approximately 40 isolates from Plots 7-13, 2-26 and 9-30 were also characterized for comparison. 2.4. PCR ampli®cation and sequence analysis The two primers chosen for ampli®cation of the ITS region of the 16S±23S rRNA were the forward primer C (5 0 -GGCTGGATCACCTCCTTTCT-3 0 ) corresponding to the 3 0 end of 16S gene, and the reverse primer D (5 0 CCGGGTTTCCCCATTCGG-3 0 ) corresponding to the 5 0 region of the 23S gene (Laguerre et al., 1996). The primer sequences are highly conserved and complemented in a wide range of bacteria. Two other primers were used for ampli®cation of the 5 0 region of the 16S gene: the forward primer A (5 0 -GGAGAGTTAGATCTTGGCTTGGCTCAG3 0 ) corresponding to the 5 0 region of the 16S gene, and the reverse primer B (5 0 -CCAGTGTGGCCGGTCGCCCTCTC3 0 ) which is approximately 250±350 bp downstream of primer A (Weisburg et al., 1991). PCR ampli®cation was carried out in a 50 ml reaction volume. DNA was ampli®ed by mixing 2 ml of the template DNA (,10 ng ml 21), 5 ml of the polymerase reaction buffer 10 £ , 2 ml of 50 mM MgCl2, 2 ml of each primer pair (10 mM), 3 ml of dNTPs (2.5 mM), 0.5 units of Taq DNA polymerase per reaction and 34 ml water. DNA was ampli®ed in a Perkin±Elmer 9600 thermal cycler with the following temperature pro®le: an initial denaturation temperature at 94 8C for 5 min; 30 cycles of denaturation (30 s at 94 8C), annealing (30 s at 58 8C) and extension (45 s at 72 8C) and a ®nal extension time at 72 8C for 3 min. Ampli®ed DNA product was examined by horizontal electrophoresis, 100 V for 2 h, in 1% agarose with a 7 ml aliquot of PCR product. The PCR products were then used for RLFP analysis. The sequence of a ,230 bp fragment of the 16S rRNA gene was determined for two selected isolates by direct automatic sequencing of the PCR product of primers A and B. 2.5. DNA digestion and electrophoresis Restriction endonucleases were digested with (10±12 ml) aliquots of the mixture for each PCR reaction. Ten restriction endonucleases MspI, AciI, HhaI, DdeI, MboI, BfaI, HaeI, RsaI, HinfI, HindIII were initially tested. Two
521
restriction enzymes (HhaI and DdeI) had good discriminating power (gave three to ®ve bands of different sizes with distinct strains) and were selected for general RFLP analysis of isolates. Digested products were examined by horizontal gel electrophoresis using 2% agarose gels at 80 V for 3 h. Finally, gels were stained with ethidium bromide and photographed under UV illumination with Polaroid 665 positive/ negative ®lm. 2.6. Strain typing using plasmid pro®les Isolates of R. leguminosarum bv. viciae were grown on TY plates (Beringer, 1974) for 28 h and then in PA broth (0.4% w/v Difco Bacto-peptone and 2 mM MgSO4) for 14± 16 h at 28 8C with shaking (150 rev min 21; Hirsch et al., 1980). The cultures (0.7 ml) were spun down at 13,000g for 3 min and the supernatant discarded. Bacterial cells were resuspended in 32 ml of lysis solution (1 ml resuspension buffer (372.2 mg EDTA, 12 g sucrose and 151 mg Tris base in 50 ml); 50 ml lysozyme (20 mg Sigma L-6876 crystallized lysozyme in 1 ml water); 50 ml RNase stock solution (400 mg ml 21) and 16 ml xylene cyanol) and kept on ice before loading onto gels. Plasmids were separated by running 16±20 ml samples on water cooled Eckhardt (Eckhardt, 1978) agarose gels (0.6% agarose in electrophoresis buffer of 89 mM Tris base, 2.5 mM EDTA and 8.9 mM boric acid; 0.4% agarose with 1% SDS behind the well-former) for about 3.5 h at 210 V. One lane was loaded with the R. leguminosarum bv. viciae strain T83K3 which has six plasmids of 480, 440, 300, 255, 210 and 155 kb (Wang et al., 1986). A total of 345 isolates were typed from seven plots. Analysis of the populations by plasmid pro®les was dif®cult when attempting to classify isolates into groups across plots, as a number of the plasmids were similar in size. This necessitated their examination in adjacent lanes on Eckhardt gels, thus requiring a large number of gels for cross-comparisons. 2.7. Zn tolerance of rhizobial isolates The degree of Zn tolerance in 70 isolates (14 from each of the ®ve plots) was assessed on TY plates to which varying concentrations of analytical grade ZnSO4 were added. Isolates were grown in TY broth at 28 8C for 2 days and washed twice in sterile deionized water and three replicate drops applied to the TY plates. The Zn tolerance of 10 isolates was also compared using a cation-exchange resin (Amberlite IRI 20-plus) method designed to ensure a constant ionic activity in solution (for details see Knight, 1996). In this method, isolates were grown in buffered media at four or ®ve different Zn concentrations (from 0 to 50 mg l 21) and the number of cells surviving over 72 h was measured using a drop-plate counting method (Miles and Misra, 1938). Two R. leguminosarum bv. trifolii isolates were included for comparison: S9 from a heavy metal contaminated soil and isolate F5 from uncontaminated soil (Giller et al., 1989; Chaudri et al., 1992a).
522
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
concentrations found across the metal gradient. All of the isolates gave one or two ITS bands of between 800 and 1300 bp before digestion. In total 20 ITS `strain' groups were identi®ed on the basis of the RFLP patterns after digestion with the restriction enzyme HhaI (Figs. 2(b) and (d) and 3(b)). Groupings using DdeI to digest the ITS±PCR product were exactly the same as those obtained with HhaI. Where isolates from different plots had similar RFLP patterns, their similarity was con®rmed by comparing them on the same gel. The largest number of ITS groups (10 groups) was found in the control plot (Plot 2-8) and the least number of groups (two groups) in the most contaminated plot (Plot 7-31), with no clear pattern to the number found in the other plots (Fig. 3(b)). The most striking result was the occurrence and abundance of ITS group 1 in all plots. The dominance of this group tended to increase with increasing Zn concentration: it comprised 82% of the isolates in the 300 m 3 metalamended sewage sludge treated plot, but only 23±55% of the isolates in the other plots. In the 300 m 3 sewage sludge treated plot, 23% of the isolates were from group 1 and a further 51% of the isolates were from group 3. Isolates from ITS group 3 were present only in Plot 6-2 and were differentiated from ITS group 1 only on the basis of an extra band with both restriction enzymes HhaI (Fig. 2(d)) and DdeI. The population from another plot with high metal loading (Plot 9-30; 48 t ha 21 yr 21 ®lter-pressed sewage sludge) was also dominated by ITS groups 1 and 3. Fig. 1. Numbers of R. leguminosarum bv. viciae estimated by the MPN plant infection method plotted against the total Zn concentration in the soils in plots from the Old Arable Soil, Braunschweig. The vertical lines indicate the current maximum Zn concentrations allowed to accumulate in soils receiving sewage sludge in Germany and in the UK. Arrows indicate the plots sampled for detailed analysis of the populations.
3. Results 3.1. Abundance of R. leguminosarum bv. viciae The number of indigenous R. leguminosarum bv. viciae decreased progressively as heavy metal concentrations increased (Fig. 1). Rhizobial numbers ranged from 1.4 £ 10 4 to 8 £ 10 4 cells g 21 soil in plots which had received 180 kg ha 21 yr 21 inorganic N fertilizer with resulting Zn concentrations around 50 mg Zn kg 21 to between 7 and 2.3 £ 102 cells g 21 soil in plots treated with 300 m 3 ha21 yr 21 metal-amended sewage sludge in which Zn concentrations were 338±399 mg Zn kg 21. 3.2. Diversity of R. leguminosarum bv. viciae based on ITS± RFLP patterns The diversity of 231 isolates from ®ve plots was assessed using the ITS±PCR method. The plots were selected to represent the range of population sizes and metal
3.3. Diversity and complexity of plasmid pro®les Plasmid pro®le groupings were based on visual analysis of the number and size of plasmids seen on Eckhardt gels (Fig. 2). In the three plots with the least metal contamination (Plots 2-8, 4-23 and 5-25), plasmid pro®les of the isolates were fairly simple with three, four or ®ve bands. There was also a great variety of banding patterns (10±14 types) present among the 50 isolates from the plots with less than 250 mg Zn kg 21 (Figs. 2(a) and (c) and 3(a)). Plasmid pro®les varied between plots although a number of pro®les were common to different plots (Fig. 3(a)). With increasing metal concentrations, rhizobia with more complex pro®les of six to nine large plasmids were isolated (Figs. 2(c) and 3(a)). Such isolates formed an increasingly large proportion of the isolates from each plot, although some isolates with only two to ®ve plasmids were present in plots with higher metal loading (Figs. 2(c) and 3(a)). One plasmid pro®le group (group 1) with eight plasmids was particularly dominant, comprising 83±91% of the isolates in the two most contaminated plots (Plots 7-13 and 7-31) which had received the 300 m 3 metal-amended sewage sludge treatment (Figs. 2(c) and 3(a)). Plasmid pro®les of ,50 isolates from each of the four additional plots of the control (180 kg ha 21 yr 21 inorganic N fertilizer) and 100 m 3 ha 21 yr 21 sewage sludge treatments were also examined. All isolates from these additional plots
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
523
Fig. 2. Photographs of (a and c) Eckhardt gels showing plasmid pro®les and (b and d) agarose gels showing the corresponding RFLPs of the PCR products of the ITS region digested using HhaI of R. leguminosarum bv. viciae isolates from plots of the Old Arable Experiment at Braunschweig. Nearly all plasmid pro®le types are represented and for cross-comparison the isolates were ordered so that isolates with similar (but not identical) plasmid pro®les were close together on the gels. The isolates are from a range of plots and treatments: Plot 2-8, control treatment which received 180 kg ha 21 yr 21 inorganic N fertilizer; Plot 4-23, 100 m 3 ha 21 yr 21 sewage sludge treatment; Plot 6-2, 300 m 3 ha 21 yr 21 sewage sludge treatment; Plot 7-31, 300 m 3 ha 21 yr 21 metal-amended sewage sludge treatment; and Plot 9-30, 48 t ha 21 yr 21 ®lter-pressed sewage sludge treatment. The plasmid size marker in (a) is T3K83. A 1 kb ladder is shown in lane 1 of (b) and (d).
524
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
Fig. 3. Histograms showing the distribution of the groups: (a) based on plasmid pro®les; and (b) based on RFLP analysis using the ITS±PCR method in the different plots (approximately 50 isolates per plot) of R. leguminosarum bv. viciae isolated from the Old Arable Soil, Braunschweig. Numbers above each bar in (a) indicate the number of plasmid bands present in isolates belonging to that group.
had ®ve or less plasmids with many plasmid pro®les, similar to those of other control plots. Overall, mean plasmid number increased markedly with metal concentration (Fig. 4). Additional soil samples were taken in May 1996 from the paths adjacent to the heavily contaminated plots 7-13 and 7-31. These soil samples represented adjacent uncontaminated soil which had not been subject to any treatment effects. All 60 isolates of R. leguminosarum bv. viciae made from these samples had a range of plasmid banding patterns with two to six plasmids (data not shown), adding further
evidence that the dominance of isolates with complex plasmid pro®les in these plots resulted from the heavy metal stress. 3.4. Species af®nity of the isolates Two isolates, one from the most contaminated plot (isolate 7-31-1 from ITS group 1 which contained 55% of the isolates overall) and one from the control plot (isolate 28-1 from ITS group 5) were characterized by partial
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
525
Fig. 4. The relationships between mean numbers of plasmids (X) in ,50 isolates per plot of R. leguminosarum bv. viciae, and diversity of rhizobial types based on both plasmid pro®les and ITS±RFLPs (W) from plots on the Old Arable Soil, Braunschweig against soil Zn concentration.
sequence analysis of ,260 bp from the 5 0 end of the 16S rRNA gene. In both cases, the nucleotide sequence was 100% homologous with that of the R. leguminosarum bv. viciae type strain (National Center for Biotechnology Information Accession number U29386), which con®rmed their expected species af®nity. 3.5. Zn tolerance of the isolates As a major difference between the soils from the plots was the elevated Zn concentration where sewage sludges had been applied, a simple method of screening isolates for growth on agar plates was used to evaluate their relative tolerance to Zn. With some exceptions the tests indicated that there was a general, though not particularly marked increase in Zn tolerance with increasing metal loading in the soil (data not shown; A. Lakzian, unpublished PhD Thesis, Wye College, University of London, 1998). Within a given plot, different isolates from the same ITS group had similar Zn tolerance. In the two most contaminated plots isolates from ITS group 1 grew at higher concentrations of Zn than isolates of the same group from the less contaminated plots. Isolates of ITS group 1 that had eight or nine plasmids were clearly more Zn tolerant than isolates from ITS group 2 from the same plot which had only three plasmids. Similar tests of tolerance to Cu and Cd on agar plates revealed no consistent differences between the isolates. Metal toxicity of only a limited number of isolates was tested using the buffered solution method due to the laborious nature of this assay. The two test strains F6 and S9 were clearly differentiated using this method; the metal tolerant strain S9 grew at all metal concentrations, whereas F6 did
not grow at the higher concentrations (Fig. 6). Isolates from ITS groups 1, 2, 6, 12, 14 and 17 with only three plasmids were all killed after 72 h with the 30 and 50 mg Zn l 21 treatments (results not shown). Isolates which both belonged to ITS group 1 and had eight plasmids (e.g. Fig. 6(b)) were clearly the most tolerant of all the isolates from the Braunschweig soils tested as they grew at 30 mg Zn l 21, though these isolates did not possess the same degree of metal tolerance as the test strain S9 (Fig. 6(d)). Isolate 731-15 (ITS±RFLP group 2) with only three plasmids from the most contaminated plot was as sensitive as all other isolates tested using this method (Fig. 6(a)).
4. Discussion 4.1. Effects of heavy metal pollution on rhizobial abundance The progressive reductions in numbers of R. leguminosarum bv. viciae with increasing heavy-metal contamination in the soil (Fig. 1) were similar to the results with R. leguminosarum bv. trifolii reported by Chaudri et al. (1993). However, their results indicated that the numbers of R. leguminosarum bv. trifolii were more strongly reduced in plots which received 300 m 3 ha 21 yr 21 sewage sludge and no clover rhizobia were detected in some of the most contaminated plots. Rhizobia are known to be highly competent heterotrophs which can survive for long periods in the absence of a compatible legume host (Giller, 2001). As no legumes have been grown on these plots since before 1970 (although some compatible legumes could have occurred sporadically as weeds) these changes in rhizobial populations have occurred in the absence of the in¯uence of
526
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
Fig. 5. Characterization of 10 isolates of R. leguminosarum bv. viciae from a plot of the 300 m 3 ha 21 yr 21 metal-amended sewage sludge treatment (Plot 7-13) and 10 isolates from a plot of the control treatment which received 180 kg ha 21 yr 21 inorganic N fertilizer (Plot 2-26). Photographs of (a) Eckhardt gels showing plasmid pro®les; (b) gel showing the PCR product of the ITS region. A 1 kb ladder is shown in the two central lanes; and (c) gel showing RFLPs generated by digesting the PCR product of the ITS region using HhaI. The central lane is a 50 bp±2 kb marker.
the host legume. The metal concentrations in these plots are also suf®ciently small that growth of plants is unaffected, so it is unlikely that the changes in population sizes are due to indirect effects of plant rhizospheres, or inputs of C through root exudates. 4.2. Comparison of rhizobial diversity based on ITS±RFLP typing and plasmid pro®le groups In total, 49 plasmid pro®le groups were identi®ed among isolates from the Old Arable Soil but only 20 ITS±RFLP groups were distinguished (Fig. 3). Cross-comparison of the isolate groupings using the two methods revealed that many isolates belonging to the same RFLP group had distinct plasmid pro®les. There was a clear trend of increasing plasmid number with increasing metal concentration in the soil (Fig. 4). Due to the wide differences in population size, the sample of isolates studied is equivalent to the whole population per gram soil in the most contaminated plot, but it represents only 0.001% of the culturable population in the control plot. However, we have no reason to assume that there was any bias in the sampling of isolates and typing of greater numbers was not feasible. Isolates which belonged to ITS group 1 were abundant in all of the plots, yet none of the plasmid pro®le groups were common to all of these plots (Fig. 3). Comparison of the plasmid pro®le and ITS±RFLP groups revealed that a wide range of plasmid pro®les were associated with some of the ITS groups (Fig. 2). The ITS groups which were represented
by only single isolates, of course, contained only a single plasmid pro®le type but generally when isolates of the same ITS group had been isolated from different plots, the plasmid pro®les differed (e.g. see plasmid pro®le groups associated with ITS groups 1 and 4; Fig. 2). The widest diversity of plasmid pro®les was associated with ITS group 1 which was the most abundant group in almost all of the plots. Further evidence for the similarity of ITS groups 1 and 3 is the occurrence of isolates from plasmid pro®le group 1 in both of these ITS groups. Plasmid pro®le group 1 had eight plasmids and was the dominant type in the most contaminated plot, and another very similar plasmid pro®le group (group 9) which had seven plasmids was abundant in Plot 6-2 (Fig. 3). Isolates from other plots with high metal loading (Plot 9-30; 48 t ha 21 yr 21 ®lter-pressed sewage sludge and Plot 7-13; 300 m 3 ha 21 yr 21 metal-amended sewage sludge) also had many isolates with large numbers of plasmids which belonged to two similar ITS groups 1 and 3 (Fig. 2(c) and (d)). A comparison of 10 isolates from each of a control plot (Plot 2-26) and a contaminated plot (Plot 7-13) clearly demonstrated the dominance of ITS group 1 in both plots (Fig. 5). Several different plasmid groups were represented in the control plot, all of which had only two to four plasmids, whereas the 10 isolates from the contaminated plot all belonged to ITS group 1 with eight plasmids. Certain plasmids are often associated with particular chromosomal types (Young and Wexler, 1988; Souza and Eguiarte, 1997), and many plasmid incompatibility groups
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
527
Fig. 6. Metal tolerance of two isolates of R. leguminosarum bv. viciae from the most contaminated plot (Plot 7-31; 399 mg Zn kg 21) of the Old Arable Experiment, Braunschweig based on growth/survival of cells in buffered Zn solutions of varying concentration. Two isolates of R. leguminosarum bv. trifolii (F6 and S9) from Woburn, UK known to differ in metal tolerance were included for comparison.
occur so that certain plasmids can or cannot coexist in the same bacterium (Shaw, 1987). Our study clearly demonstrates that certain chromosomal types maintain larger numbers of plasmids under conditions of heavy metal stress in the soil. These strains with many plasmids are almost exclusively dominant in the small populations in the contaminated plots, suggesting that the extra plasmids confer increased ®tness under stressed conditions. At variance to our initial hypothesis, that numbers and diversity of rhizobia would progressively decrease with increasing metal concentrations, a strong reduction in diversity (in both numbers of plasmid pro®le groups (Fig. 3(a)) and ITS±RFLP groups (Fig. 3(b)) was only found at the highest metal concentrations. At intermediate metal concentrations, the diversity of plasmid pro®le groups tended to increase, and the diversity of groups clearly increased when isolates are grouped on the basis of both their PCR±RFLPs and their plasmid pro®les (Fig. 4). This is consistent with ecological models which suggest that moderate environmental stresses may reduce exclusion due to particular competitive genotypes, thus reducing dominance and giving apparent increases in diversity with intermediate stress (Austin and Smith, 1989).
4.3. Interrelationships between ITS groups, plasmid pro®les and Zn tolerance of rhizobial isolates How the isolates of ITS group 2, which were among the most sensitive to Zn in arti®cial media (Fig. 6), are able to survive in the most contaminated plots is unclear. Other studies have shown that exposure to moderate metal concentrations (similar to those in the Braunschweig ®eld experiment) for more than a year is required to signi®cantly reduce rhizobial populations (e.g. Chaudri et al., 1992b; Giller et al., 1993). The soils in this study had been subject to these concentrations of heavy metal contamination for at least 4 yr since sludge applications ceased. The bacteria may be avoiding the heavy metal stress in some way, for instance by physical protection by close adherence to clay particles or within micropores of soil aggregates where the active metal concentrations may be smaller. The metal tolerance tests showed that in spite of the reduction in the number of groups and few types of isolates remaining in the most contaminated plots, there was only a slight increase in the tolerance to Zn (Fig. 6). The Zn concentrations tested in these laboratory assays cannot be extrapolated to concentrations to which the bacteria are
528
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529
exposed in soil where the concentrations of the free Zn 21 ion have been measured as less than 600 mg l 21 in all of the plots (B. Knight, unpublished). Nevertheless, when the results were compared for different isolates belonging to a particular ITS group, a pattern of tolerance could be seen and the overall tolerance tended to increase with heavy metal loading in the soil. None of the isolates was as tolerant as the metal-tolerant isolate (S9) which was dominant in a heavy metal contaminated plot at Woburn, England (Giller et al., 1989; Hirsch et al., 1993) and was tolerant to Zn, Cd, Cu and Ni (Chaudri et al., 1992a). Increases in the number of plasmids carried by isolates were associated with increased metal tolerance. This was observed both between isolates from ITS group 1 from the same plot and between isolates from this group from different plots. The dominance of the more tolerant types with large numbers of plasmids in the plots with more metal contamination (Plots 6-2, 9-30, 7-13 and 7-31) is therefore likely to be due to increased metal tolerance associated with the large number of plasmids. A plasmid of approximately 70 kb appeared to be common to all of the isolates which had enhanced metal tolerance and this plasmid may be the source of metal tolerance genes. Genes for heavy metal tolerance in a wide range of bacteria are often carried on plasmids (Silver and Misra, 1988). If acquisition of the plasmids was related to increased metal tolerance, the extra energetic costs of maintaining a large number of plasmids could be partly counterbalanced by the selective pressure of heavy metal toxicity. However, it is highly unlikely that all of the extra plasmids carried by the isolates in the most contaminated plots are involved in metal tolerance. As yet, we have no evidence of any speci®c functions for these plasmids, though it seems unlikely that a rhizobial strain would carry so many plasmids unless they confer some selective advantage. A mixture of organic compounds as well as a number of different metals will have been added to the soils in the sewage sludge treatments. As a period of 4 yr had elapsed since sewage sludge was last added to the plots and the time of sampling, most labile compounds would have already been degraded. It is possible that some of the plasmids actually originated from microorganisms added in the sewage sludge, though we cannot test this suggestion as samples of the sludges added to the soils are not available. Comparison of the rhizobial populations present in the 300 m 3 ha21 yr 21 sewage sludge treatment (Plot 6-2) and the 300 m 3 ha21 yr21 metalamended sewage sludge treatment (Plots 7-13 and 7-31) allows the effects of sludge loading to be separated from effects of metals. Isolates with eight plasmids of the same ITS and plasmid pro®le group as in Plots 7-13 and 7-31 were present in Plot 6-2, but this type was dominant only in the plots with greater heavy metal concentrations, again suggesting that the large number of plasmids in the rhizobial isolates was related to heavy metal stress. Other studies have also failed to reveal a simple relationship between heavy metal toxicity and microbial diversity
(Sandaa et al., 1999, 2001). The complex relationship between diversity of rhizobia and heavy metal stress observed here suggests that the common assumption that increasing stress leads to simple reductions in microbial diversity should be revisited. Acknowledgements This research was conducted with ®nancial support from NERC and the EU-Environment Programme of DGXII. Amir Lakzian is grateful to the Government of Iran for a scholarship. References Austin, M.P., Smith, T.M., 1989. A new model for the continuum concept. Vegetatio 83, 35±47. Beringer, J.E., 1974. R Factor transfer in Rhizobium leguminosarum. Soil Biology and Biochemistry 84, 188±198. CEC, 1986. Council Directive on the protection of the environment, and in particular of soil, when sewage sludge is used in agriculture. Of®cial Journal of the European Communities Directive, 86/278/EEC, No. L181/6-12. Chaudri, A.M., McGrath, S.P., Giller, K.E., 1992a. Metal tolerance of isolates of Rhizobium leguminosarum biovar trifolii from soil contaminated by past applications of sewage sludge. Soil Biology and Biochemistry 24, 83±88. Chaudri, A.M., McGrath, S.P., Giller, K.E., 1992b. Survival of the indigenous population of Rhizobium leguminosarum biovar trifolii in soil spiked with Cd, Zn, Cu and Ni salts. Soil Biology and Biochemistry 24, 625±632. Chaudri, A.M., McGrath, S.P., Giller, K.E., Rietz, E., Sauerbeck, D., 1993. Enumeration of indigenous Rhizobium leguminosarum biovar trifolii in soils previously treated with metal-contaminated sewage sludge. Soil Biology and Biochemistry 25, 301±309. Eckhardt, T., 1978. A rapid method for the identi®cation of plasmid deoxyribonucleic acid in bacteria. Plasmid 1, 584±588. Giller, K.E., 2001. Nitrogen Fixation in Tropical Cropping Systems. 2nd ed. CAB International, Wallingford. Giller, K.E., McGrath, S.P., Hirsch, P.R., 1989. Absence of nitrogen ®xation in clover grown on soil subject to long term contamination with heavy metals is due to survival of only ineffective Rhizobium. Soil Biology and Biochemistry 21, 841±848. Giller, K.E., Nussbaum, R., Chaudri, A.M., McGrath, S.P., 1993. Rhizobium meliloti is less sensitive to heavy-metal contamination in soil than R. leguminosarum bv. trifolii or R. loti. Soil Biology and Biochemistry 25, 273±278. Giller, K.E., Witter, E., McGrath, S.P., 1998. Toxicity of heavy metals to microorganisms and microbial processes in agricultural soilsÐa review. Soil Biology and Biochemistry 30, 1389±1414. Hewitt, E.J., 1952. Sand and Water Culture Methods used in the Study of Plant Nutrition. Commonwealth Agricultural Bureaux, Farnham Royal. Hirsch, P.R., Van Montagu, M., Johnston, A.W.B., Brewin, N.J., Schell, J., 1980. Physical identi®cation of bacteriocinogenic, nodulation and other plasmids in strains of Rhizobium leguminosarum. Journal of General Microbiology 120, 403±412. Hirsch, P.R., Jones, M.J., McGrath, S.P., Giller, K.E., 1993. Heavy-metals from past applications of sewage sludge decrease the genetic diversity of Rhizobium leguminosarum biovar trifolii populations. Soil Biology and Biochemistry 25, 1485±1490. Knight, B., 1996. Unpublished PhD Thesis, Wye College, University of London. Knight, B., McGrath, S.P., Chaudri, A.M., 1997. Biomass carbon
A. Lakzian et al. / Soil Biology & Biochemistry 34 (2002) 519±529 measurements and substrate utilization patterns of microbial populations from soils amended with cadmium, copper, or zinc. Applied and Environmental Microbiology 63, 39±43. Laguerre, G., Mazurier, I.S., Armarger, N., 1992. Plasmid pro®les and restriction fragment length polymorphism of Rhizobium leguminosarum bv. viciae in ®eld populations. FEMS Microbiology Ecology 101, 17±26. Laguerre, G., Mavingui, P., Allard, M.-R., Charnay, M.-P., Louvrier, P., Mazurier, S.-I., Rigottier-Gois, L., Amarger, N., 1996. Typing rhizobia by PCR DNA ®ngerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars. Applied and Environmental Microbiology 62, 2029±2036. MAFF/DoE, 1993. Review of the Rules for Sewage Sludge Application to Agricultural Land: Soil Fertility Aspects of Potentially Toxic Elements. Report of the Independent Scienti®c Committee, PB 1561, MAFF Publications, London. McGrath, S.P., Cunliffe, C.H., 1985. A simpli®ed method for the extraction of metals Fe, Zn, Cu, Ni, Cd, Pb, Cr, Co and Mn from soils and sewage sludges. Journal of the Science of Food and Agriculture 36, 794±798. McGrath, S.P., Brookes, P.C., Giller, K.E., 1988. Effects of potentially toxic metals in soil derived from past applications of sewage sludge on nitrogen ®xation by Trifolium repens L. Soil Biology and Biochemistry 20, 415±424. Miles, A.A., Misra, S.S., 1938. The estimation of the bactericidal power of blood. Journal of Hygiene, Cambridge 38, 732±748.
529
Sandaa, R.-A., Torsvik, V., Enger, é., Daae, F.L., Castberg, T., Hahn, D., 1999. Analysis of bacterial communities in heavy metal contaminated soil at different levels of resolution. FEMS Microbiology Ecology 30, 237±251. Sandaa, R.-A., Torsvik, V., Enger, é., 2001. In¯uence of long-term heavy metal contamination on microbial communities in soil. Soil Biology and Biochemistry 33, 287±295. Shaw, P.D., 1987. Plasmid ecology. In: Kosuge, T., Nester, E. (Eds.). Plant±Microbe Ineractions. Macmillan, New York, pp. 3±39. Silver, S., Misra, T.K., 1988. Plasmid-mediated heavy metal reistances. Annual Reviews of Microbiology 42, 717±743. Souza, V., Eguiarte, L.E., 1997. Bacteria gone native vs. bacteria gone awry: plasmidic transfer and bacterial evolution. Proceedings of the National Academy of Sciences, USA 94, 5501±5503. Vincent, J.M., 1970. A Manual for the Practical Study of Root-Nodule Bacteria. Blackwell, Oxford. Wang, C.L., Beringer, J.E., Hirsch, P.R., 1986. Host plant effects on hybrids of Rhizobium leguminosarum biovars viciae and trifolii. Journal of General Microbiology 132, 2063±2070. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal ampli®cation for phylogenetic study. Journal of Bacteriology 173, 697±703. Woomer, P., Bennet, J., Yost, R., 1990. Overcoming the in¯exibility of most-probable number procedures. Agronomy Journal 82, 349±353. Young, J.P.W., Wexler, M., 1988. Sym plasmid and chromosomal genotypes are correlated in ®eld populations of Rhizobium leguminosarum. Journal of General Microbiology 134, 2731±2739.