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Analysis of rhizobacterial communities in perennial Graminaceae from polluted water meadow soil, and


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FEMS Microbiology Ecology 52 (2005) 153–162 www.fems-microbiology.org

Analysis of rhizobacterial communities in perennial Graminaceae from polluted water meadow soil, and

screening of metal-resistant, potentially plant growth-promoting bacteria
Elena Dell?Amico, Lucia Cavalca, Vincenza Andreoni
*
` degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita Received 23 July 2004; received in revised form 27 September 2004; accepted 2 November 2004 First published online 2 December 2004

Abstract This study investigates the impact of long-term heavy metal contamination on the culturable, heterotrophic, functional and genetic diversity of rhizobacterial communities of perennial grasses in water meadow soil. The culturable heterotrophic diversity was investigated by colony appearance on solid LB medium. Genetic diversity was measured as bands in denaturing gradient gel electrophoresis (DGGE) obtained directly from rhizosphere soil and rhizoplane DNA extracts, and from the corresponding culturable communities. In the two rhizospheric fractions the DGGE pro?les of the direct DNA extracts were similar and stable among replicates, whereas in the enriched cultures the pro?les of the fractions di?ered, but among the replicates they were similar. One hundred isolates were collected into 33 di?erent operational taxonomic units by use of ampli?ed internal transcribed spacers and into 19 heavy metal-resistant phenotypes. The phylogenetic position of strains belonging to 18 operational taxonomic units, representing more than 80% of the isolates, was determined by 16S rRNA gene sequencing. Several heavy metal-resistant strains were isolated from rhizoplane. Finally, metal-resistant rhizobacteria were tested for plant growth-promoting characteristics; some were found to contain 1-aminocyclopropane-1-carboxylic acid deaminase and/or to produce indole acetic acid and siderophores. Two strains resistant to cadmium and zinc, Pseudomonas tolaasii RP23 and Pseudomonas ?uorescens RS9, had all three plant growth-promoting characteristics. Our ?ndings suggest that bacteria can respond to soil metal contamination, and the described methodological approach appears promising for targeting potential plant growth-promoting rhizobacteria. ? 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Heavy metal; Rhizobacterial communities; Metal-resistant plant growth-promoting bacteria

1. Introduction Heavy metals are an important class of pollutants deriving from agriculture (fertilizers and sewage sludge), industrial activity (metal mining and smelting) and disposal of waste matter. In agricultural soils heavy metal pollution can a?ect all types of organisms and ecosystem

Corresponding author. Tel.: +39 02 50316728; fax: +39 02 50316694. E-mail address: vincenza.andreoni@unimi.it (V. Andreoni).

*

processes, including microorganism-mediated processes [1,2]. Long-term exposure to heavy metals can alter the qualitative and quantitative structure of microbial communities, resulting in decreased metabolic activity and diversity [3–5] and in adverse e?ects on various parameters in?uencing quality and yield of plant [1,6]. Microbial response to soil contamination varies due to variations in bioavailability and exposure to harmful metals. Soil bacteria can resist toxicity by transforming metals into less toxic forms, by immobilising metals on the cell surface or in intracellular polymers, and by precipitation or biomethylation [7].

0168-6496/$22.00 ? 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.11.005

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Plant–root interaction with large numbers of di?erent microorganisms, together with soil conditions, are the major determinants of plant growth and proliferation. The best-known mechanisms of plant growth promotion are nitrogen ?xation, phytohormone production, solubilization of phosphorous, and the synthesis of siderophores, antibiotics and enzymes that all modulate plant growth and development [8–10]. It is well documented that plants respond to biological and environmental stresses by synthesizing ‘‘stress’’ ethylene [11–13]. In higher plants ethylene is produced from L -methionine via the intermediates S-adenosyl-L -methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC) [14]. Plant growth-promoting rhizobacteria (PGPR) that produce the enzyme ACC deaminase [15– 17] can cleave ACC and lower the level of ethylene in plants growing in the presence of heavy metals [18]. Instead, PGPR-siderophore producers can help plants acquire su?cient iron for optimal growth in the presence of heavy metals that hinder iron acquisition [19,20]. Whereas microbial communities in metal-polluted bulk soils have been studied [21,22], there is little information on the composition of microbial community of plant rhizospheres growing in soils that are highly polluted with heavy metals. Studies on heavy metal-polluted soils could provide a new insight into bacterial diversity under unfavourable conditions, and into the exploitation of new isolates and genetic information on metal resistance. In this study with culture-dependent and cultureindependent techniques, we examined rhizosphere- and rhizoplane-associated bacterial communities of perennial Graminaceae grasses growing in water meadow with known heavy-metal contamination. Our objectives were to analyse the di?erences in bacterial species composition of rhizosphere and rhizoplane soils, to analyse culturable rhizobacterial diversity and to characterize the isolates, identi?ed by partial 16S rRNA gene sequencing, with regard to their metal resistance and their capability to produce siderophores, indole acetic acid and ACC deaminase.
Table 1 Soil characteristics and bacterial counts Texture Sandy-loam Metal content (mg kg?1dry matter) Total Zn Ni Cd 10325.58 77.49 71.85 pH (KCl) 6.1 Bioavailable 5835 16.9 37.40

2. Materials and methods 2.1. Field site description, sampling and rhizobacterial counts The studied water meadow lies to the south of Milan, Italy. Table 1 shows the physico-chemical characteristics [23] of the soil where the vegetation was one of perennial Graminaceae grasses. Ten plants with their roots were taken from four sampling sites, randomly located in the middle of the ?eld in March 2001. Root samples from each site were put together to form four composite samples (a,b,c,d). The rhizosphere soil (RS), de?ned as the soil released from around the root after gentle shaking, and the rhizoplane fraction (RP), the roots plus very tightly adhering soil, were analyzed separately [24]. The number of heterotrophic bacteria of the RS and RP fractions was based on three replicate experiments using conventional plating techniques from a slurry prepared as follows: 3 g of RS or 3 g of RP were suspended in 27 ml of sodium pyrophosphate solution (0.2%, w/v), shaken on a rotary shaker for 1 h, left to rest for 30 min without shaking and then plated, with 1 ml dilutions of supernatant, on LB (Luria Bertani) solidi?ed medium supplemented, after autoclaving, with cycloheximide (100 lg ml?1) to inhibit fungus growth. To determine the number of Cd-, Zn-, and Ni-tolerant bacteria, three series of plates were supplemented with 1 mmol l?1 Cd (II), 3 mmol l?1 Zn (II) and 0.8 mmol l?1Ni (II). Given the disparity of the results on which metal concentrations are toxic to microorganisms [2], Cd and Ni ion concentrations were chosen at the range of their respective total concentration measured in soil; higher Zn concentration, instead, was not tested as the metal precipitated in LB medium. The respective colonies were counted after incubation at 30 °C for 8 days. The bacterial number of RS fraction was expressed as CFU g?1 soil (dry weight), and the bacterial number of RP fraction as CFU g?1 (root and adhered soil, dry weight).

OMa (g kg?1dry matter) 6.9 CFU g?1 dry matter RP Zn-T Ni-Tc Cd-Td CHBe
b

C/N 11 RS
7 6

7.1 · 10 ± 3 · 10 2.4 · 108 ± 3 · 107 1.4 · 107 ± 1 · 106 3.7 · 108 ± 2 · 107

6.1 · 107 ± 1 · 106 2.2 · 108 ± 3 · 107 1.0 · 107 ± 1 · 106 3.1 · 108 ± 2 · 107

Each CFU value is the average of a triplicate experiment ± standard deviation. a Organic matter. b Zinc-tolerant bacteria grown on LB + 3 mmol L?1 Zn(II). c Nickel-tolerant bacteria grown on LB + 0.2 mmol L?1 Ni(II). d Cadmium-tolerant bacteria grown on LB + 1 mmol L?1 Cd(II). e Culturable heterotrophic bacteria grown on LB.

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The metal tolerance of the heterotrophs was expressed as percentage growth on LB agar without metal addition. 2.2. Enrichment cultures Soil bacterial communities were enriched in the presence of cadmium and zinc to determine their response to metal stress. Each composite RS and RP sample was separately suspended in Tris minimal medium containing gluconate 0.8% (w/v) (TMMG) [25] in the presence of 1 mM Cd or 1 mM Zn. The cultures were incubated at 30 °C for 6 days on a rotary shaker. To verify the presence of bacteria capable of utilising ACC as the sole nitrogen source each RS and RP sample was suspended separately in DF (Dworkin and Faster) [26] minimal medium supplemented with 3 mM ACC. The cultures were adapted to the metals, and to ACC, by re-inoculating 20 ml of the ?fresh? medium with 1 ml of the cell suspensions three times. 2.3. Bacterial isolates, media and growth conditions From sample site ?a?, 50 colonies of RS and 50 colonies of RP, grown on LB plates, were isolated randomly to be identi?ed and characterised for their resistance to heavy metals. Single colonies were streaked to purity on the same LB medium and the strains maintained in glycerol stocks at ?70 °C. Prior to use the strains were grown in LB broth at 30 °C with shaking to mid-exponential phase. 2.4. Resistance to heavy metals

scribed spacers (ITS), sequences between the small (16S) and the large (23S) ribosomal subunit, were ampli?ed using universal 16Sf (5 0 -TGYACACACCGCCCCG-3 0 ) and bacterial-speci?c 23Sr primers (5 0 0 GGGTTBCCCCATTCRG-3 ) according to Ranjard et al. [29]. DGGE analysis of the PCR products was performed in a DCode Universal Mutation Detection System (Biorad, USA) apparatus, essentially as described by Muyzer et al. [30]. The linear denaturing gradient of urea and formamide ranged from 40% to 60%. Gels were run at a constant voltage of 70 V for 16 h at 55 °C. After completion of electrophoresis, the gels were stained in an ethidium bromide containing solution (0.5 mg l?1) and documented with the GelDoc System (Biorad, USA). Sorensen?s index (S) was calculated according to Magurran [31] on the basis of DGGE pro?les (S1,2 = 2a/2a + b + c, where ‘‘a’’ is the number of bands common to both samples, ‘‘b’’ and ‘‘c’’ the bands in samples 1 and 2, respectively), and was used to evaluate the biodiversity of the cultures enriched by Cd, Zn and ACC. (S = 0 indicates that two samples are completely di?erent, whereas S = 0.5 indicates identical samples). The GelDoc software package was used to analyse the DGGE pro?les after assigning bands to the gel tracks. The nucleotide sequences of 16S rRNA genes of the isolates were determined according to the Perkin–Elmer ABI Prism protocol (Applied Biosystems, USA). Primers used in the PCR reaction for sequencing were the same as those in normal 16S rDNA PCR reactions. Forward and reverse samples were run on an Applied Biosystems 310A sequence analyser. 2.6. Statistics

The bacterial level of resistance to Cd, Zn, Ni, and Co was analyzed in liquid TMMG supplemented with the appropriate amount of soluble metals, checking for growth after 4 days incubation at 30 °C. The metals used (Sigma–Aldrich) were Cd as CdCl2, Zn as ZnSO4, Ni as NiCl2 and Co as CoSO4. 2.5. Molecular methods DNA was extracted from soil samples, enrichment cultures and isolated strains. Soil DNA and enrichment culture DNA were extracted by a bead-beating method (MOBIO, USA) and the BIO101 method (Resnova, Italy), respectively, according to manufacturer?s instructions. Proteinase K (1 mg ml?1) was used to extract DNA from strains according to Cavalca et al. [27]. PCR ampli?cation of the 16S rDNA was performed on the extracted DNA using eubacterial universal primer P27f and P1495r referred to the Escherichia coli nucleotide sequence numbering of the 16S rDNA gene. Nested PCR reaction for V3 ampli?cation was carried out according to Muyzer and Smalla [28]. Internal tran-

ITS analysis was used to characterise isolated strains. Fragment size was estimated with a linear regression equation between the molecular mass of the DNA ladder and the log of the distance covered by fragments within the same gel run. A distance matrix was built, and the UPGMA method was used to build a similarity tree by the Jaccard coe?cient using the NTSYS software package. 2.7. Qualitative determination of potential plant-growth characteristics of isolates Metal resistant isolates were tested for their ability to grow on ACC as the sole N source, and to produce siderophores and indole acetic acid. 2.7.1. Utilization of ACC as a sole nitrogen source The ability to use ACC as N source is a consequence of enzymatic activity of ACC deaminase. The bacteria were cultured ?rst in rich medium (TSB, Difco) and then transferred into tubes with DF medium containing 3.0

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mM ACC instead of (NH4)2SO4 as N source. Solution of ACC (0.5 M) (Sigma–Aldrich) was ?lter-sterilized (0.2 lm) and the ?ltrate aliquoted and frozen at ?20 °C. Prior to inoculation, the ACC solution was thawn and appropriately added to sterile DF medium. Following inoculation with the appropriate strain, the cultures were incubated at 30 °C on a rotary shaker at 200 rpm for 48 h. Growth was positive when the cultures developed turbidity. The ability of a strain to utilize ACC was veri?ed by inoculating the strain in control tubes containing DF medium without any N source, and incubating the tubes in the mentioned conditions for 10 days. The absence of growth con?rmed the utilization of ACC as N source. 2.7.2. Indole acetic acid production The bacteria were cultured for 4 days in ?asks containing 20 ml of DF medium supplemented with 0.5 mg ml?1 of tryptophane. After incubation, a 1 ml cell suspension was transferred into a tube and mixed vigorously with 2 ml of Salkowski?s reagent (150 ml of concentrated H2SO4, 250 ml of distilled H2O, 7.5 ml of 0.5 M FeCl3 ? 6H2O) [32] and allowed to stand at room temperature for 20 min, after which time a pink colour developed in the cell suspensions. 2.7.3. Synthesis of siderophores Siderophore secretion by strains was detected by the ‘‘universal’’ method of Schwyn and Neilands [33] using blue agar plates containing the dye Chrome azurol S (CAS) (Sigma–Aldrich). Orange halos around the colonies on blue agar were indicative of siderophore excretion.

3.2. Genetic diversity of soil and of enriched bacterial communities by DGGE A comparison of the patterns of DNA directly extracted from RS and RP samples indicated that the bacterial community structure was very similar in all samples, and the DGGE pro?les showed a smear of bands with 11 common dominant bands (Fig. 1). In contrast, the DGGE pro?les of the Cd- and Zn-enriched cultures (Fig. 2) showed composition di?erences in the metal-resistant bacterial species. The DGGE pro?les of the Cd-enriched rhizosphere cultures resembled each other and consisted of 12 bands, while those of the rhizoplane had 10 bands, similar to each other but di?erent from those of RS as the Sorensen index between the RS and RP cultures was 0.29. Analogously, the band pro?les of the Zn-enriched cultures with a Sorensen index of 0.44 were similar within the same fraction but di?ered slightly between RP and RS. The DGGE pro?les of the ACC-enriched cultures of rhizosphere and rhizoplane di?ered (SRP–RS = 0.34), and RP had more bands than RS (Fig. 3). 3.3. Characterization of dominant rhizosphere isolates DGGE analyses showed no di?erences among the four composite samples of soils or enrichment cultures. Fifty isolates were selected from randomly picked colonies grown from each RSa and RPa sample. DNA was extracted from the isolates and ITS analysis was performed. The resulting 100 patterns were compared,

3. Results 3.1. Soil characteristics and rhizobacterial counts Table 1 shows soil characteristics, heavy metal content and CFU of the heterothrophs and Cd-, Zn-, Nitolerant bacteria. The heavy metal content of soil was very high: the Cd and Zn concentrations were one or two orders higher than the typical range of world-wide soils, while Ni concentrations were within it [34]. For the four composite samples, the Fisher?s LSD test (P 6 0.05) revealed no di?erences among the heterotrophs or the metal-tolerant bacteria, nor was there any di?erence between the RS and the corresponding RP samples (data not shown). In Table 1 we report the averages of CFUs of heterotrophs and metal-tolerant bacteria for RP and RS. The proportions of metaltolerant bacteria were 65% and 70% for Ni-tolerant, 19.1% and 19.8% for Zn-tolerant and 3.7% and 3.2% for Cd-tolerant in RP and RS, respectively, cadmium being the most toxic.

Fig. 1. DGGE analysis on a 35–60% denaturant gel of the V3 regions of soil 16S rDNA. Odd lanes: PCR products from DNA extracted from rizhosphere soils (RS); even lanes: PCR products from DNA extracted from rizhoplane (RP); a, b, c, d: site sampled.

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Fig. 2. DGGE analysis on a 35–60% denaturant gel of the V3 regions 16S rDNA of cultures enriched in the presence of metals. Lanes 1–8, PCR products obtained from DNA extracted from cultures enriched by 1 mM Cd; lanes 9–16, PCR products obtained from DNA extracted from cultures enriched by 1 mM Zn. Odd lanes, RP samples; even lanes, RS samples; a,b,c,d: site sampled.

species with the same relative abundance, and a few dominant species were detected. Eighteen OTUs representing 80% of the isolates were identi?ed. The choise of the strains to be identi?ed was mainly performed on the basis of plant growth-promoting (PGP) characteristics and metal resistance: 16 isolates belonging to 16 different OTUs were metal-resistant but only 14 of these had plant growth-promoting characteristics (Table 3). Besides these, two non-metal-resistant strains where chosen: one strain from a dominant group of rhizoplane (I25) and one from a rhizosphere group (I12) (Fig. 4). A total of 14 genera were identi?ed, and used to assign each of the isolates to a broad taxonomic group. Analysis of the phylogenetic tree (Fig. 4) showed that the 33 haplotypes were represented mainly by Gram-negative bacteria (62%). High-G + C Gram-positive bacteria (mainly Rhodococcus, Arthrobacter and Cellulomonas) had a high relative abundance in both rhizoplane and rhizosphere, whereas the Gamma Proteobacteria (Pseudomonas and Serratia) prevailed in rhizosphere. The dominant OTUs belonged to Pseudomonas, Agrobacterium, Serratia and Streptomyces. All isolates showed at least 97% homology to known 16S rRNA genes. 3.4. Resistance to heavy metals We found that 84% of the 100 isolates were metalresistant strains and equally distributed between rhizoplane and rhizosphere fractions. Among these isolates we detected 26 di?erent heavy metal-resistant phenotypes. Table 2 shows the phenotypes of the isolates. Most of the isolates were simultaneously resistant to various metals; 16 isolates were resistant only to Cd, Zn, or Ni and no isolate was resistant only to cobalt. Our data gave no indication of any correlation between the OTU and the metal-resistant phenotype, and each OTU included di?erent metal-resistant phenotypes or susceptible strains. The rhizoplane isolates showed higher Cd- and Zn-resistance levels than the rhizosphere ones. In fact rhizoplane was found to have bacteria resistant to 2 mM Cd and 2 mM Zn, and the number of 1 mM Cd-, and 1 mM Zn-resistant bacteria was twice higher than that in rhizosphere fraction. 3.5. Selection of metal-resistant plant growth-promoting rhizobacteria

Fig. 3. DGGE analysis on a 35–60% denaturant gel of the V3 regions 16S rDNA of cultures enriched by ACC as N source. Odd lanes, RP samples; even lanes, RS samples; a,b,c,d: site sampled.

giving 33 di?erent haplotypes, i.e., operational taxonomic units (OTUs) (Fig. 4). Twelve haplotypes were exclusive to rhizosphere, another 12 to rhizoplane and 9 were in both fractions. Sharing OTUs were well represented (60% of the total isolates); there were numerous

In stressed environment rhizosphere bacteria with PGP characteristics may play an important role on plant growth. To identify potential PGP rhizobacteria, all 84 metal-resistant isolates were tested for their ability to produce indole acetic acid, to utilize ACC as the sole N source and to secrete siderophores into the growth medium. Table 3 shows that 25 isolates from rhizoplane and 13 from rhizosphere had at least one PGP trait. Approximately 31% and 5% of the metal-resistant

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Fig. 4. Distribution of 100 di?erent isolates among 33 OTUs from RS (closed squares) and RP (open squares) samples. The phylogenetic tree was constructed using the NTSYS software pakage with Jaccard coe?cient. Distance are marked at junctions: the branch lengths are the percentage mismatch between two nodes. Species identi?cations were obtained by 16S rDNA nucleotide sequence analysis of representing strains.

strains had, as phenotypic traits, the capacity to produce IAA and to grow on ACC, respectively, and 26% could secrete siderophores. Two strains, P. tolaasii RP23 and P. ?uorescens RS9 had all three traits. The PGP rhizobacteria belonged to di?erent phylogenetic groups and the majority were Gram-negative bacteria.

4. Discussion In the present study we used PCR-DGGE in conjunction with cultivation-based methods to analyse the diversity of the rhizosphere-associated bacterial communities of heavy metal-contaminated soil. To achieve our goal we used perennial species of Graminaceae grasses

grown in a water meadow with a long history of metal pollution (more than 25 years). The e?ect of heavy metals on the number of culturable bacteria is uncertain as the ?ndings of studies di?er [2]. In the present investigation, the numbers of heterotrophs were comparable to those reported for non-polluted soils that often have more than 107–108 CFU per g of soil [35,21], but higher than those reported for highly-polluted soils [36,37]. The high percentage of heterotrophs tolerant to cadmium, zinc and nickel indicated that these populations had been under constant metal stress for a long time. The highly bioavailable metal content of the soil (Table 1) could have exerted a selective pressure upon rhizospheric bacteria that should have an inherent way of dealing with the metals.

E. Dell’Amico et al. / FEMS Microbiology Ecology 52 (2005) 153–162 Table 2 Heavy metal-resistant phenotypes Phenotype OTU Metal (mM) Cd 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 I2, I4 I2, I4, I9, I23 I3, I17, I19 I3, I5, I6 I5, I6 I2 I4, I26 I5, I29 I1, I3, I5 I5 I2, I9 I1, I6, I8, I14 I1 I9, I27 I15 I20, I30 I7, I32 I2 I6, I7, I18, I31, I33 I9 I3, I14 I15 I3, I22 I7, I11, I16, I24 I6, I13, I22 I6, I7 + + + 1 + + 2 Zn 0.5 1 2 Ni 0.5 1 Co 0.5 1 2 8 0 8 4 0 4 4 0 0 4 6 2 6 0 2 4 0 10 6 2 0 2 6 6 0

159

Percentage of isolates RS RP

+ + + + + + + + +

+ + + + +

+ + + +

+

+ + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + +

+ + +

+ + + + + +

+ + + + + + + + +

+ + + + + + +

+ + + + + + + + + + + + + + +

+

+

+

+

0 0 6 0 0 2 0 4 8 6 6 4 0 6 4 2 4 4 4 4 2 2 0 6 2 6

The percentage of isolates calculated on the 50 isolates is reported.

Our aim in this study was to obtain a comprehensive picture of the total rhizobacterial community of these grasses. Although we did not characterize the DGGE bands by sequencing, we analysed, in great detail, the population of culturable rhizobacteria with regard to their metal resistance and their potential ability to promote plant growth; to do this we isolated metal-resistant bacteria and potentially plant growth-promoting rhizobacteria. The DGGE pro?les obtained directly from the DNA of rhizosphere and rhizoplane fraction were very similar, indicating similarity in bacterial species composition and that several dominant groups were relatively stable in both rhizospere fractions. Instead, the analysis of culturable bacteria resulted in a di?erent resolution level, revealing some biodiversity within the RP and RS isolate populations. In fact the Sorensen similarity values calculated from the DGGE pro?les revealed a signi?cant di?erence in species composition of RP and RS bacteria (Figs. 2 and 3). This suggests the presence of di?erent subsets of metal-resistant and ACC-utilizing bacteria in the two fractions, probably re?ecting di?erent metal availability and/or nutritional status. The implication is that the metals exert a negative e?ect on certain bacteria by a?ecting their ability to replicate on

laboratory media. This assumption is tenable as there is a relation between culture-forming ability and physiological status, while metal stress could reduce catabolic diversity [22]. Our data indicate that the rhizoplane and rhizosphere e?ects did not signi?cantly a?ect the total bacterial community diversity but they did a?ect the physiological status so that the distribution of bacteria capable of responding to laboratory culture was altered, as found by Kozdroj and van Elsas [5]. RS as well as RP hosted many metal-resistant culturable bacteria, indicating a marked adaptation to the heavy metal content. High phenotypic diversity was found among the 100 isolates that included 19 di?erent heavy metal-resistant phenotypes, indicating the existence of di?erent combinations of genetic determinants for metal resistance, including co-resistance [38,39]. The more common phenotype showed a simultaneous resistance to cadmium, zinc, nickel and cobalt both in rhizosphere and rhizoplane fraction. The strains with single resistance were few, and most of these came from rhizosphere fraction. An e?ect on the diversity of RS and RP metal-resistant bacteria could also be expected as a result of the solubilization of heavy metals by plant roots.

160

Table 3 Metal-resistant plant growth promoting rhizobacteria OTU Strain Closest relative % Homology Phenotypic traits ACC-deaminase activity I1 I6 Rp19 Rp18 Rp16 Rs23 Rp24 Rs2 Rs5 Rp6 Rp9 Rp3 Rp21 Rp17 Rp23 Rp31 Rs8 Rs9 Rs43 Rs11 Rs4 Rs6 Rs Rp2 Rp1 Rp4 Rp15 Rp36 Rp41 Rs28 Rs21 Rp13 Rp14 Rp29 Rs14 Rp5 Rp12 Rp27 Rs3 Rp7 Microbacterium sp. (AY561630) Serratia liquefaciens (AJ306725) 98.6 99.4 ? ? ? ? ? + ? ? ? ? ? ? ? ? ? + ? ? ? ? ? ? ? + ? ? ? ? ? ? + ? ? ? ? ? ? ? IAA production + + + + + + + + + ? + ? ? + + + + + + + + + + ? + ? ? + + ? ? + + ? + + + + Siderophore production + + + + + + ? ? ? + + + + + ? + + + + ? ? + ? ? ? + + ? ? + ? ? ? + ? ? ? ? Metal resistance Cd Zn Zn Ni Zn Ni Zn Zn Ni Co Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd Zn Zn Zn Zn Ni Zn Zn Zn Zn Ni Zn Zn Zn Zn Co Ni

I9

Pseudomonas tolaasii (AF348507)

100

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Co Ni Co Co Ni

I22

Pseudomonas ?uorescens (AF134704)

98.5

Co Ni Co Ni Co Co

I27 I13 I3

Ralstonia sp. (AB051684) Ralstonia taiwanenses (AF300324) Alcaligenes sp. (AF511432)

99.7 100 100

Zn Co Zn Co Cd Zn Co Ni Cd Zn Cd Zn Cd Cd Ni Co Cd Zn Co Ni Cd Zn Co Ni Zn Ni Cd Cd Zn Co Ni Cd Zn Cd Cd Ni Zn Ni Zn Ni Zn Ni Cd Zn Ni

I7

Agrobacterium tumefaciens (AT16SRD5)

99.1

I8 I17 I16 I29 I2 I15

Sinorhizobium adhaerenses (AJ420775) Paracoccus sp. (AY616160) Mycobacterium sp. (AY646435) Rhodococcus sp. (AF420422) Cellulomonas sp. (AJ292035) Arthrobacter aurescens (AF501335)

99.9 98.2 97.9 99.8 97.2 100

The strains are designed as follow: Rs strains isolated from rhizosphere soil; Rp strains from rhizoplane.

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The dominant isolates belonged to common soil genera found in soils based on the identi?cation of culturable bacteria [40,41], and the high relative abundance of Gamma Proteobacteria in the molecular (rDNA) isolates is in accordance with other studies [42,21]. The high relative abundance of Arthrobacter, Rhodococcus and other Actinobacteria agrees with the detection of Arthrobacter-like strains in soil contaminated with zinc [40] and in highly lead-contaminated sites [41]. Di?erently from Ellis [22], Bacillus spp. were not isolated. Most of the metal-resistant strains with determined PGP characteristics, i.e., production of siderophores and indole acetic acid, and ACC deaminase, were detected in the rhizoplane fraction (Table 3). The presence of more than one PGP trait in many of these bacteria, as in the case of the two metal-resistant Pseudomonas tolaasii RP23 and P. ?uorescens RS9, facilitates plant growth by the possibility to utilise one or more of these mechanisms at various times during the life cycle of a plant. Our results con?rm the wide distribution of ACC deaminase activity in di?erent bacterial genera and in di?erent species of Pseudomonas [43]. However, it is evident that the ability to produce ACC deaminase is strain-dependent. Heavy metals in soils could stimulate the production of bacterial siderophores [44] that, in turn, alleviate metal toxicity by increased supply of iron to the plant [18]. An increase in root growth and root length, generally promoted by IAA-producing rhizobacteria [10], was also observed under toxic Cr concentration in wheat inoculation experiments using Pseudomonas sp. IAA producers; it was found that plant growth was accompanied by a reduction in the Cr uptake and an increase in the plant auxin content [45]. In conclusion, our work has demonstrated that di?erent microbial communities live in association with rhizosphere soils and rhizoplane, and are able to withstand high heavy metal concentrations. Both Gram-positive and Gram-negative rhizobacteria belonging to di?erent genera such as Pseudomonas, Mycobacterium, Agrobacterium and Arthrobacter were found to have plant growth-promoting characteristics that can potentially support heavy metal uptake and reduce stress symptoms in plants. Several studies have evidenced that heavy metal-resistant Proteobacteria can protect plants from the toxic e?ects of metals, or even enhance metal uptake by hyperaccumulator plants [18,43,46]. The metal-resistant PGP rhizobacteria isolated in this study will be used in experiments of seed inoculation to verify their ability to protect seedlings from the e?ects of heavy metals. Their metal resistance and possibly metaltransforming capacities will be studied as well. The selection of microorganisms that are both metal-resistant and e?cient in producing plant growth-promoting

compounds could prove useful as inocula for processes of re-vegetation and phyto-remediation.

Acknowledgements This work was supported by FIRST and by the University of Milan. E.D.A. is the recipient of a ‘‘Dottorato di Ricerca- Ecologia Agraria’’ research fellowship from the Milan University.

References
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