Survival and persistence of genetically modified Sinorhizobium meliloti in soil

Applied Soil Ecology 22 (2003) 1–14

Survival and persistence of genetically modified Sinorhizobium meliloti in soil H.N. Da, S.P. Deng∗ Department of Plant and Soil Sciences, Oklahoma State University, Stillwater, OK 74078-6028, USA Received 26 February 2002; accepted 6 August 2002

Abstract Studies were conducted to evaluate the survival and persistence of Sinorhizobium meliloti 104A14 and two acid phosphatasenegative mutants in Kirkland (fine, mixed, thermic Udertic Paleustolls) silt loam soils with various fertility levels, and to assess the impact of inoculation on nodule occupancy and soil microbial community structure in the inoculated alfalfa (Medicago sativa L.) rhizosphere. Recovery of the inoculated strains was 100% (in the order of 108 cells g−1 soil) immediately following inoculation to soils, but decreased from 108 cells g−1 soil to undetectable levels in a nutrient-poor soil within 32 days. In a nutrient-rich soil, approximately 2–3% (4.7–7.43×106 cells g−1 soil) of the mutants and 23% (5.84×107 cells g−1 soil) of the wild-type inocula persisted for more than 64 days. Survivability and persistence of the wild-type S. meliloti were significantly greater than that of the genetically modified acid phosphatase negative mutants in all the soils tested. The persistence and nodule occupancy of the introduced S. meliloti in sterile and non-sterile soils were also tested for two repeated alfalfa growth periods in the same plant growth units, with a 1 month interval in between and no additional inoculation for the second period. Nodule occupancy of the introduced S. meliloti in non-sterile soils ranged from 30 to 60% for the first period and 85 to 100% for the second period. Our results suggest that survival and persistence of S. meliloti was enhanced by alfalfa cultivation and increased soil fertility, but impaired by mutation of acid phosphatase genes regardless of phosphorus nutritional levels. Moreover, inoculation with genetically modified S. meliloti strain 104A14 promoted indigenous bacterial growth in soil (increased bacterial population from 1.4 × 106 to 4.3 × 106 cells g−1 soil), but not the growth of fungi and yeast. However, inoculation of the wild-type S. meliloti or genetically modified mutants did not result in significant changes in microbial community structure as indicated by EP indices and ratios of r/K strategists. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Survival and persistence; Sinorhizobium meliloti; Genetically modified microorganisms; Soil microbial community

1. Introduction Symbiotic nitrogen (N) fixation contributes more than 100 million metric tons of N per year to the global N supply (Graham, 1998). Unfortunately, some native soil rhizobia are not effective in N fixation (Segovia ∗ Corresponding author. Tel.: +1-405-744-9591; fax: +1-405-744-5269. E-mail address: [email protected] (S.P. Deng).

et al., 1991). Inoculation with highly effective N-fixing rhizobia, a common practice in agricultural production (Catroux et al., 2001), requires survival and establishment of inoculated rhizobia in the soil environment. Extensive studies have been conducted to evaluate the ability of rhizobia to survive in soil with much of the effort focused on Rhizobium competition, soil pH, moisture, temperature, and predation (e.g. Ramirez and Alexander, 1980; Osa-Afiana and Alexander,

0929-1393/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 ( 0 2 ) 0 0 1 2 7 - 0

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1982; Moawad et al., 1984; Brockwell et al., 1991). Under limiting phosphorus (P) conditions, organic P is an important P source for rhizobia such as Rhizobium huakuii and Bradyhizobium japonicum (Salminen and Streeter, 1987; McGrath et al., 1998). With 20–80% of total soil P in organic forms (Dalal, 1977), phosphatases may play an important role in symbiotic N fixation. Activities of alkaline and acid phosphatases of rhizobia are induced under P-limiting conditions (Samrt et al., 1984; Al-Niemi et al., 1997). Although single mutations of the genes encoding each of the two acid phosphatases found in the periplasm of S. meliloti 104A14 did not affect symbiotic performance significantly in sand-supporting systems regardless of P levels (Deng et al., 1998, 2001), it is not clear whether these acid phosphatases are involved in the survival and persistence of S. meliloti in a P-limiting soil environment. Genetic improvements of rhizobia have made significant contributions to agricultural production (Esperanza and Rosenblueth, 1990; Bosworth et al., 1994). Release of engineered S. meliloti with an extra copy of both nifA and dctABD (a regulatory N fixation gene and C4 -dicarboxylic acid transport gene, respectively) into soil increased alfalfa (Medicago sativa L.) yield by 12.9% (Bosworth et al., 1994). Unfortunately, the use of genetically modified microorganisms (GMMs) on a wide scale is still hampered by a lack of knowledge of the possible ecological impacts that such organisms might have once they are released into the environment. One of the concerns is that GMMs may affect non-target microorganisms (Levin et al., 1987). Diversity indices of soil microbial communities increased upon introduction of a genetically modified Pseudomonas cepacia (Bej et al., 1991). However, the benefits and risks related to the release of GMMs depend on their establishment and persistence in the environment. The objectives of this study were: (1) to assess the effects of mutation of acid phosphatase genes on survival and persistence of S. meliloti 104A14 in soil; (2) to determine the effect of alfalfa growth on establishment and persistence of genetically modified S. meliloti in the soil environment; and (3) to evaluate the impact of genetically modified S. meliloti on soil microbial populations and community structure in the alfalfa rhizosphere.

2. Materials and methods 2.1. Survival and persistence in soil 2.1.1. Soils Three soils (designated as A, B, and C) with considerable variation in P levels were chosen for this experiment. Surface soil (0–15 cm) samples were taken from a long-term continuous winter wheat (Triticum aestivum L.) experiment located in central OK, USA. The soils are Kirkland (fine, mixed, thermic Udertic Paleustolls) silt loams with a mean particle-size distribution of 37.5% sand, 40% silt and 22.5% clay. Composite soil samples, including 18 cores per plot at 0–10 cm, were taken in January 2000. Descriptions of the experimental site and soils can be found in Parham et al. (2002). Soils were ground, sieved through a 2-mm screen, air-dried and stored at room temperature. The pH value was determined using a combination glass electrode (Thomas, 1996). The organic C and total N were detected by dry combustion using a Carlo-Erba NA 1500 Nitrogen/Carbon/Sulfur Analyzer (Schepers et al., 1989). Phosphorus levels in Mehlich-3 extracts (Mehlich, 1984) were determined by the Murphy and Riley method (MRP) (Murphy and Riley, 1962). Long-term (over 70 years) soil management practices had resulted in considerable variations in soil properties (Table 1). 2.1.2. Bacterial strains and preparation of inoculants Creation and characterization of two acid phosphatase-negative mutants NapD and NapE, derived from S. meliloti 104A14, are described previously Table 1 Properties of soils used Soila

pHb

Organic C (g C kg−1 soil)

Total N (g N kg−1 soil)

MRPc (g P kg−1 soil)

A B C

4.85 5.70 4.70

6.70 9.00 7.80

0.67 0.86 0.77

8 22 57

a Soils used in this experiment belong to Kirkland silt loam series that were under different management practices for over 70 years. Soil A was an untreated control; soil B was treated with cattle manure every 4 years at 269 kg N ha−1 (approximately 89.7 kg P ha−1 ); and soil C was applied 14.6 kg P ha−1 per year. b Soil: 0.1 M CaCl ratio = 1 : 2.5. 2 c MRP: P concentration in Mehlich-3 (Mehlich, 1984) extracts determined by Murphy and Riley method (1962).

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(Deng et al., 1998, 2001). The acid phosphatase genes, NapD and NapE, were inactivated by inserting an 8-kb transposon Tn5-B20, which contains a promoterless lacZ and a kanamycin-resistant gene (Simon et al., 1989). The inserted transposons were in the correct orientation based on the reporter-enzyme studies and the enzymes were constitutively expressed (Deng et al., 1998, 2001). Bacteria were cultured in yeast mannitol broth (YMB) for 48 h at 30 ◦ C on an orbital shaker at 200 rpm. The cells were pelleted by centrifugation for 5 min at 1500×g, washed twice with sterile 0.85% (w/v) NaCl solution to remove P and other nutrients from the medium. Subsequently, the pellets were re-suspended to approximately 109 cells ml−1 based on estimation from culture density (0.5 absorbance at 595 nm ≈ 1010 cells ml−1 ). The actual culturable cell concentrations were determined by plate count on yeast mannitol agar (YMA) plates. 2.1.3. Incubation and enumeration of S. meliloti in soil A 20-g soil sample was placed in a 50-ml flask and adjusted to the desired pH with modified universal buffer (MUB, Tabatabai, 1994), and 60% water holding capacity (WHC). MUB contained 0.02 M of tris (hydroxymethyl)aminomethane, maleic acid, citric acid and boric acid in 1 N NaOH and was adjusted to a desired pH using 0.1 N HCI or NaOH. The flask was then covered with aluminum foil and the total weight was recorded. Soils in flasks were autoclaved at 0.6 MPa and 121 ◦ C for 35 min on each of four consecutive days. Sterile water was added to each flask, based on the weight change during each autoclaving, to maintain 60% WHC. After the fourth autoclaving, soils were inoculated with washed cells prepared as described above at a concentration of approximately 108 CFU g−1 soil, while 60% soil WHC was maintained. In the control soil, an equivalent volume of sterile 0.85% (w/v) NaCl solution was added. Soils were incubated at 28 ◦ C for 0, 1, 2, 4, 8, 16, 32, and 64 days. An open container filled with water was placed inside the incubator to keep the humidity at near saturation level. Soil moisture loss during incubation was replaced with sterilized water every 3 days based on weight changes. Each treatment had three replicates. The Sinorhizobium meliloti population after different incubation times was enumerated using a modification of the procedure described by Zuberer

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(1994). Briefly, the 20-g soil sample in each flask was transferred aseptically to a 500-ml flask containing 180 ml sterile water and 0.18% sodium pyrophosphate. The suspension was then shaken at 200 rpm on an orbital shaker for 15 min. After settling for about 30 s, a 5-ml sample was transferred to a 125-ml flask containing 45 ml of sterile Ringer solution. Serial dilutions were performed and 100 ␮l of each dilution was spread on four separate YMA plates. Kanamycin-containing YMA plates were used for the two acid phosphatase-negative mutants (Deng et al., 1998, 2001). Colony forming units (CFU) were recorded after 3 days incubation at 30 ◦ C. 2.2. Survival and persistence in the alfalfa rhizosphere 2.2.1. Soil Surface (0–15 cm) Renfrow soil (Udertic Paleustolls, silty clay loam) was collected from central OK, USA. This soil was chosen because it has a near neutral pH value (pH 6.53) and was also under continuous winter wheat cultivation. The soil sample was taken, prepared and stored as described above. The soil organic C was 11.4 g C kg−1 soil and total N was 1.16 g N kg−1 soil. 2.2.2. Microcosm and plant growth A plant growth unit consisted of three Magenta boxes (Sigma, St. Louis) arranged as a Leonard jar (McDermott and Kahn, 1992). The lower box contained nutrient solution and the upper box enclosed the shoot. A soil sample (200 g, < 2 mm) was placed in the middle Magenta box of a plant growth unit. Nutrient solution in the lower box was transported to soil in the middle box through a wick made of filter paper. One set of plant growth units was autoclaved at 0.6 MPa and 121 ◦ C on four consecutive days for 60 min each day, while another set remained non-sterile. Each plant growth unit was filled with 200 ml nutrient solution before the fourth autoclaving. The nutrient solution was modified from that described by Wych and Rain (1978). It contained no N but 1.0 mM K2 SO4 , 0.5 mM KH2 PO4 , 0.25 mM K2 HPO4 , 0.5 mM MgSO4 ·7H2 O, 2.0 mM CaSO4 ·2H2 O, 25 ␮M KCl, 13 ␮M H3 BO3 , 1.0 ␮M MnSO4 ·H2 O, 1.0 ␮M ZnSO4 ·H2 O, 0.25 ␮M CuSO4 ·5H2 O, 2.5 ␮M CoCl2 ·6H2 O, 20 ␮M FeCl3 ·6H2 O, and 0.25 ␮M NaMoO4 ·2H2 O, and was adjusted to pH 6.5.

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Alfalfa seed surface-sterilization, germination, and plant growth conditions were performed as described by Al-Niemi et al. (1997). Sterile seedlings were transferred to growth units (four seedlings per unit) and inoculated immediately with washed cells of S. meliloti strains tested. The alfalfa plants were grown in a growth chamber for 6 weeks with a 16-h photoperiod and a 19/24 ◦ C night/day cycle. After 6 weeks of growth, plant tissue dry weight and nodule numbers were recorded. The plant growth units were then kept in the dark at room temperature for 1 month. The experiment was repeated as described above in the same growth units, but without additional inoculation, to evaluate survival and persistence of inoculants under the prevailing conditions. Each treatment was replicated four times. 2.2.3. Isolation of rhizobia from alfalfa nodules Nodules from each treatment were surface sterilized by sequentially immersion in 70% ethanol for 10 s, 1.5% sodium hypochlorite for 1 min, followed by 6 changes of sterile distilled water. The surface-sterilized nodules were homogenized in 200 ␮l 0.85% NaCl solution in microcentrifuge tubes. After serial dilution, the nodule suspensions were spread on YMA plates and incubated at 30 ◦ C for 3 days. Forty to fifty isolated colonies were isolated randomly from each treatment, purified on YMA plates, and stored in 40% glycerol at −80 ◦ C for further studies. 2.2.4. Colony hybridization Approximately 3 ␮l of late log-phase YMB bacterial culture (approximately 24 h) at about 106 CFU ml−1 was placed on YMA plates. The wild-type S. meliloti was used as a positive control and YMB as a negative control. The plates were then incubated overnight at 30 ◦ C for additional growth. Colonies were lifted using a nylon membrane (lmmobilonTM -Ny+, Millipore Corp. Bedford, MA, USA). The cells on the membrane were lysed in a 0.5 N NaOH-1.5 M NaCl solution and the released DNA was fixed to the membrane by UV crosslinking. The membrane with fixed bacterial DNA was immersed in the prewashing solution (five times sodium chloride-sodium citrate (SSC), 0.5% sodium dodecyl sulfate (SDS) and 1 mM EDTA) for 30 min at 50 ◦ C with gentle agitation. The membrane was

prehybridized with the hybridization buffer (0.5 M sodium phosphate, 2 mM EDTA, 7% SDS, and 0.1% sodium pyrophosphate, pH 7.1) for 2 h at 65 ◦ C. Then, it was hybridized overnight at 65 ◦ C with NapD or nifH probe and gentle agitation. Nonradioactive DIG-labeled probes were prepared by polymerase chain reaction (PCR) using primers specific for NapD or nifH (Table 2), the genomic DNA of wild-type S. meliloti as a template, and the probe synthesis kit from Roche Molecular Biochemicals (Mannheim, Germany). Hybridization between the labeled probes and bacterial DNA was detected by color development. 2.2.5. PCR and DNA sequencing Unless specified, PCR for DNA amplification was performed on an automated thermal cycler (PTC-100, MJ Research Inc., Watertown, MA, USA) with an initial denaturation (94 ◦ C for 120 s), followed by 30 cycles of denaturation (94 ◦ C for 45 s), annealing (65 ◦ C for 30 s) and extension (72 ◦ C for 120 s), and a single final extension (72 ◦ C for 10 min). DNA sequencing was performed using an ABI PRISM 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA). Sequence similarity searches were conducted using the BLAST network service (Altschul et al., 1990), and sequence alignments used the GAP program (Devereux et al., 1984). 2.2.6. Amplified ribosomal DNA restriction analysis (ARDRA) Bacterial 16S rDNA (about 1.6 kb) were generated by PCR using universal primers (Table 2). Approximately 1.0–1.5 ␮g 16S rDNA was digested at 37 ◦ C for at least 3 h using Alul, Haelll, Hpall, and Rsal, respectively (GIBCOBRL® , Life Technologies, Grand Island, NY, USA). The digested DNA was separated on an 8% polyacrylamide gel in Tris-Borate EDTA (TBE) buffer and the banding patterns were visualized by staining in ethidium bromide solution (0.5 ␮g ml−1 ). PCR and enzyme restriction digestions were repeated to confirm the results. 2.2.7. Culturable microbial community After 6 weeks of alfalfa growth, plant tissue and roots were removed aseptically from the growth units. Soil in the unit was mixed thoroughly and 10 g of the soil (fresh weight) was taken for enumeration of bacterial populations, dilution plating onto

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Table 2 Nucleotide sequences of primers used for polymerase chain reactions (PCR) in this study Traget gene

Sequence

16S rDNA

5 -AGA

GTT TGA TCC TGG CTC 5’-GGT TAC CTT GTT ACG ACT T-3

Toyota et al. (1999)

NapD

5 -GCA AAG AAT ACG GCC AGG AG-3 5 -ACG CGC CCT ATC CTG TCT CA-3

Deng et al. (1998)

nifH

5 -GTT CGG CAA GCA TCT GCT CG-3 5 -AAG TGC GTG GAG TCC GGT GG-3

Eardly et al. (1992)

0.1 strength Tryptone soy agar (TSA) plates. TSA has been reported to support the growth of a wide range of bacteria (Martin, 1975) and was used for total culturable bacteria counts (Lawley et al., 1983). Bacteria appearing within 24 h were designated as r-strategists, and the remaining as K-strategists (De Leij et al., 1993). R-strategists are fast growers that proliferate in uncrowded, nutrient-rich environments, while K-strategists are slow growers that are more efficient at breaking down recalcitrant substrates (De Leij et al., 1993). Unless specified, colonies were enumerated on a daily basis for five consecutive days and on day 10. Plates were examined at low magnification (1.5X), and colonies that were visible on each day were marked and enumerated. Thus, 6 counts (classes) were generated per plate. Plates with 20 to 200 colonies were selected for enumeration. When plates became too crowded, the next dilutions were used for enumeration. Distribution of bacteria in each class as a percentage of the total counts gave insight into the distribution of r- and K-strategists in each sample. The distribution of the 6 classes in each sample was also expressed as Eco-Physiological (EP) Index (H ) (De Leij et al., 1993), which was calculated using the equation: H = −




(pi × log10 pi )

where pi represents CFU on each day as a proportion of the total CFU in that sample in 10 days incubation. Total culturable fungal populations in soils were determined by culturing fungi on 0.1 strength malt extract agar (MEA) plates at 23 ◦ C for 3 days. Each sample was plated onto four separate plates. The countable plates of the highest dilution were

References AG-3

enumerated (Naseby and Lynch, 1998). All plate counts were conducted with four treatment replicates. 2.3. Data analysis All plant and soil measurements were expressed on a dry weight basis. Significant differences between treatments were determined using one-way analysis of variance (ANOVA). Treatment means were compared using the least significant difference (LSD) test at P ≤ 0.05.

3. Results and discussion 3.1. Survival and persistence in soil Survival and persistence of S. meliloti 104A14 and its two acid phosphatase-negative mutants in soil A increased with increasing soil pH from 4.8 to 6.5 (Fig. 1). The inoculants survived less than 1 week at pH 4.8, but more than 30 days when soil pH was increased to 6.5, supporting previous findings that S. meliloti strains are acid sensitive (Rice et al., 1977; Brockwell et al., 1991). A significant positive relationship was observed between soil pH and the population of S. meliloti in soils from 84 field sites with varied pH values (Brockwell et al., 1991). Survival and persistence of S. meliloti were evaluated after soils were adjusted to pH 5.7 (the highest pH value of the three soils used in this study, Table 1). The three soils chosen for this experiment had P levels ranging from 8 to 57 mg P kg−1 soil, resulting from over 70 years of soil management practices (Table 1). The wild-type S. meliloti demonstrated

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Fig. 1. Effect of soil pH on survival and persistence of introduced wild-type Sinorhizobium meliloti and its acid phosphatase-negative mutants in sterilized Kirkland soil A. Bars represent standard errors (n = 3).

significantly higher survivability than the acid phosphatase-negative mutants in all the soils tested regardless of the soil P and nutrient levels (Fig. 2). Thus, the observed difference was not likely due to limited P nutrition, but the presence of additional genes in the genome. It has been proposed that GMMs carry an extra metabolic load for the additional genes, which affect their ability to survive and grow (Da Silva and Bailey, 1986; Davis, 1987; van Elsas et al., 1991). Findings such as this are important in guiding the safe release of GMMs in the environment. GMMs have been developed for pest control, pollution abatement, frost protection, and the stimulation of N fixation (e.g. Keeler, 1988; Lindow and Panopoulos, 1988; Bosworth et al., 1994). Attempts at practical applications of GMMs often fail due to poor understanding

of their survival and persistence in the environment (Hirsch and Spokes, 1994). Persistence of the two mutants in the same soil tested was not significantly different (Fig. 2), which is in agreement with previous findings in that mutation of either of the acid phosphatase genes did not result in significantly different symbiotic phenotypes regardless of the P concentrations in nutrient solutions (Deng et al., 1998, 2001). However, soil properties appeared to be more dominant factors dictating establishment and persistence of S. meliloti than mutation of acid phosphatase genes (Fig. 2). Although 100% of the introduced S. meliloti was recovered immediately following inoculation, none of the inoculants persisted for 32 days or more in soil A (Fig. 2). 4.7 × 106 and 7.43 × 106 out of 2.3 × 108 cells g−1 soil (2–3%) of

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Fig. 2. Survival and persistence of inoculated wild-type Sinorhizobium meliloti and its acid phosphatase-negative mutants in sterilized Kirkland soils at pH 5.7. The circles, triangles, and rectangles indicate inoculation with the wild-type, NapD and NapE, respectively. Bars represent standard errors (n = 3).

the NapD and NapE mutants and 5.84 × 107 out of 2.47×108 cells g−1 soil (23%) of the wild-type inocula persisted in soil B for more than 64 days (Fig. 2). Poor survivability of inoculants in soil A may be related to unsatisfactory P supplies. Growth of S. meliloti was limited when P concentration was 0.06 ␮M P (equivalent to 10 mg P kg−1 soil) or less (Beck and Munns, 1984). MRP level in soil A was only 8 mg P kg−1 soil, 1-seventh of that in soil C (Table 1). It was expected that inoculants persisted the longest in soil B. After over 70 years of cattle manure application, soil B not only exhibited significantly higher soil organic C content, but also total N and MRP were higher compared with soil A (Table 1). It is well accepted that organic matter not only provides nutrients (C, N, P, etc.), but also is the major agent stimulating the formation and stability of soil aggregates that compartment the space and nutrient supplies (Brady and Weil, 1999). As a result, inoculated strains could survive longer in the micro-niches created in this soil escaping predation by such as protozoa and Bdellovibrio (Danso et al., 1975; Keya and Alexander, 1975).

3.2. Nodule occupancy Nodule occupancy by an introduced strain is directly related to survival and persistence of the inoculant (Vlassak et al., 1996). Bacteria isolated from nodules are usually composed of a mixture of rhizobia (Evans et al., 1996). Numerous phenotypic, biochemical and molecular methods have been employed to identify rhizobia that were re-isolated from plant nodules (e.g. Noel and Brill, 1980; Bjourson and Cooper, 1988; Fettell et al., 1997; Hebb et al., 1998). In this study, colony hybridization, PCR detection, ARDRA, and DNA sequencing were used to identify the inoculated strains. For colony hybridization, NapD and nifH of S. meliloti were used as probes. NapD encodes one of the acid phosphatases in S. meliloti 104A14 (Deng et al., 1998). Sequence of NapD did not match any known sequences in the GenBank (Deng et al., 1998) and preliminary study indicated negative hybridization signal with bacteria isolated from alfalfa nodules growing in the soil without inoculation. Thus, NapD may be unique to the strain tested. If so, a large num-

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ber of bacterial isolates can be verified quickly and precisely using techniques such as PCR detection. The nifH gene encodes nitrogenase reductase, and has been found to be the most conserved nif gene among N-fixing organisms (Torok and Kondorosi, 1981). The nifH gene has been used to differentiate S. meliloti from other rhizobia (Eardly et al., 1992). Bacteria isolated from alfalfa nodules exhibited two major growth strategies. The fast growers (colony formation within 24 h) did not appear to be the inoculated strain, which was later confirmed based on DNA analyses. All strains isolated from nodules contained nifH as evidenced by colony hybridization and PCR detection (data not shown). However, 16S rDNA from bacterial isolates with +/− NapD-hybridization signals demonstrated different ARDRA patterns (Fig. 3) and sequences (data not shown). 16S rDNA sequences of NapD-positive bacterial isolates belonged to S. meliloti based on the BLAST search (Altschul et al., 1990) and matched with those of the inoculated strain according to sequence alignments (Devereux et al., 1984). Results obtained suggested that NapD is specific for the introduced S. meliloti and the isolates with positive NapD hybridization signal belonged to the inoculated strains. The persistence and nodule occupancy of introduced S. meliloti in autoclaved and non-autoclaved

soils were tested for two repeated alfalfa growth periods in the same plant growth units as described earlier. After the first period of alfalfa growth, 88–98% of bacteria isolated from nodules grown in sterile soils and 30–60% from non-sterile soils were the inoculated strains (Table 3). A small percentage of bacteria isolated from nodules growing in sterilized soils did not hybridize with NapD, suggesting that the soils used in the study were not sterile after being autoclaved four times in four consecutive days for 1 h each day at 121 ◦ C and 0.6 MPa. However, no nodules were observed in the four controls without inoculation. Nodule occupancy by the introduced strains in

Table 3 Persistence and nodule occupancy of the introduced wild-type and genetically modified Sinorhizobium meliloti in sterile and non-sterile soils were tested for two repeated alfalfa growth periods, with 6 weeks each and a 1 month interval in between, and no additional inoculation for the second growth period Growth period

Treatment

First

Second

Fig. 3. Amplified ribosomal DNA restriction analysis (ARDRA) of bacteria isolates with NapD-positive and negative hybridization. 16S rDNA was amplified with primers shown in Table 2. Approximately 1.0 ␮g was loaded in each lane. The template DNA for lanes 1, 4, 7 and 10 were from the wild type Sinorhizobium meliloti 104A14, those of lanes 2, 5, 8 and 11 were from bacteria isolates that hybridized with the NapD probe, and those of lanes 3, 6, 9 and 12 from bacteria isolates that did not hybridize with the NapD probe.

Bacteria isolates from nodule

Soil

Strainsa

Tested (total)b

NapD+ (%)c

Sterile

Control W.T. NapD NapE

0 42 42 40

(0) (194) (175) (179)

0 37 41 36

(0) (88) (98) (90)

Non-sterile

Control W.T. NapD NapE

40 50 40 50

(157) (187) (201) (216)

0 15 24 21

(0) (30) (60) (41)

Sterile

Control W.T. NapD NapE

0 40 40 40

(0) (156) (204) (179)

0 39 40 38

(0) (97) (100) (95)

Non-sterile

Control W.T. NapD NapE

40 40 40 40

(119) (224) (166) (114)

0 38 40 34

(0) (95) (100) (85)

The isolates were verified by colony hybridization using an acid phosphatases gene (NapD) as a probe. a W.T.: NapD and NapE refer to the wild type S. meliloti 104A14 and two acid phosphatase negative mutants, respectively (Deng et al., 1998). Soils were inoculated with wild-type, NapD, and NapE at 1.18 × 108 , 3 × 108 and 1.77 × 108 (CFU g−1 soil), respectively. b Figures in parentheses indicate the total isolates obtained from each treatment. c NapD+ refers to those that hybridized with the NapD probe. Figures in parentheses are number of NapD+ expressed as a percentage of total isolates tested.

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non-sterile soils was considerably higher than those reported by Evans et al. (1996). They showed that inoculant strains on Pisum sativum were found in <15% of the nodules. Moreover, the percentage of nodule occupancy of the introduced strain increased from 85 to 100% in non-sterile soils in the second growth period, suggesting that inoculated strains out-competed indigenous S. meliloti in nodule formation. The reverse situation was reported in a similar study conducted in the field on nodule occupancy by inoculated R. tropici (Vlassak et al., 1996). Nodule occupancy by inoculated R. tropici was dominant (80%) in the first year but not in second year (only 15%) without re-inoculation (Vlassak et al., 1996). The difference might be due to differences in strains and soils tested, and the length of interval between planting in these two studies. 3.3. Total microbial population Survival and persistence of an introduced microorganism is closely related to soil microbial community structure and activity (Moawad et al., 1984). The total culturable bacterial population in the soil tested was 6.14 (log CFU g−1 soil) and was not significantly different (P > 0.05) with or without alfalfa growth (Table 4). Total culturable bacterial populations were Table 4 Total culturable microbial population in soil as affected by 6 weeks of alfalfa growth and inoculation of wild-type and genetically modified Sinorhizobium meliloti 104A14 Treatmenta

Control − alfalfa Control + alfalfa Alfalfa + W.T. Alfalfa + NapD Alfalfa + NapE

Populationsb Bacteria

Fungi and yeast

(Log CFU g−1 soil) 6.15 ± 0.15 B 6.13 ± 0.08 B 6.27 ± 0.13 B 6.69 ± 0.24 A 6.58 ± 0.25 A

3.17 3.30 3.31 3.13 3.26

± ± ± ± ±

0.37 0.13 0.30 0.22 0.23

A A A A A

a Control − alfalfa, no plant and no inoculation. Control + alfalfa: treated with 0.85% NaCl; W.T.: NapD and NapE, inoculated alfalfa seedlings with the wild-type S. meliloti 104A14 or its acid phosphatase negative mutants in 0.85% NaCl solution (Deng et al., 1998, 2001). b Means ± S.D. Different letters indicate significantly different means at P < 0.05 according to least significant difference test (n = 4).

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not affected significantly by inoculation with the wild-type S. meliloti, but increased two-fold [(from 6.14 log units (1.4 × 106 ) to about 6.64 log units (4.3 × 106 ) cells g−1 soil)] due to introduction of the genetically modified S. meliloti strains (Table 4). Reports in the literature on the subject are not consistent. The total bacterial population was not affected by inoculation with the wild-type or genetically modified P. fluorescens (De Leij et al., 1995), but increased with inoculation of a genetically modified P. cepacia (Bej et al., 1991). Bej et al. (1991) suggested that the introduced GMM not only displaced a proportion of the indigenous population, but they also occupied a wider niche or colonized more intensely in the rhizosphere. In this study, the mutants were less persistent than the wild type in soils without alfalfa cultivation. Possibly, a portion of the introduced inoculants died, releasing nutrients that could be utilized by indigenous microorganisms and promoted bacterial growth in that environment. However, this still does not explain the undetected difference in total bacterial population with and without inoculation of the wild-type S. meliloti at 8 log unit cells g−1 soil (Table 4). In fact, Sinorhizobia are not known to be competitive in the rhizosphere. Data from this study indicated that the introduced populations decreased over time and were probably not multiplying significantly. Thus direct competition for nutrients and space is less likely. It is possible that genetic modification stimulated the interaction of alfalfa roots and Sinorhizobium, which resulted in changes in root

Table 5 Mean dry weight of plant tissue and nodule numbers of alfalfa growing for 6 weeks in soils inoculated with the wild-type and genetically modified Sinorhizobium meliloti 104A14 Treatmenta

Plant dry weightb (mg per plant)

Control W.T. NapD NapE

8.7 17.8 18.9 20.6

± ± ± ±

2.1 B 4.62 A 3.86 A 4.65 A

Number of nodules (per plant) 1.4 5.5 6.1 6.5

± ± ± ±

0.2 1.1 0.6 0.7

B A A A

a Control: treated with 0.85% NaCl; W.T.: NapD and NapE were inoculated with the wild-type S. meliloti 104A14 or its acid phosphatase negative mutants in 0.85% NaCl solution (Deng et al., 1998, 2001). b Means ± S.D. Different letters indicate significantly different means at P < 0.05 according to least significant difference test.

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exudates pattern. As a result, stimulated growth of the indigenious microorganisms was observed. This is evidenced by higher nodule numbers from NapD and NapE inoculation when compared with the wild-type, though not statistically significant (Table 5). The populations of fungi and yeast ranged from 3.13 to 3.31 (log CFU g−1 soil) (Table 1). Fungal and yeast populations in NapD-inoculated soil were the lowest, but little significant differences were detected among treatments (P > 0.05). This is consistent with results obtained by Naseby and Lynch (1998), who reported that inoculation with wild-type or genetically modified P. fluorescens strains did not affect fungal

population significantly. On the contrary, fungal population was suppressed by inoculation with Pseudomonas spp. (Shanahan et al., 1992) and genetically modified P. cepacea (Doyle et al., 1991). These results demonstrated that a GMM could differentially affect microbial populations in soil. Thus, evaluation of the potential of GMMs to induce unanticipated effects on the microbial ecology and activity of soil should include a broad range of ecological end points (Doyle et al., 1991). However, results obtained from this study suggested that survival and persistence of inoculated S. meliloti were not closely related to fungal and yeast population.

Fig. 4. Culturable bacteria population in soil: (A) in the presence or absence of alfalfa plant; (B) the alfalfa seedlings were inoculated with the wild-type Sinorhizobium meliloti 104A14, NapD, or NapE mutant. Data derived from colony forming units (CFU) appearing on 0.1 strength TSA plates over a period of 10 days incubation. Bars represent standard errors (n = 4).

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3.4. Soil bacterial community structure Although alfalfa cultivation did not result in an increase of total culturable bacteria population, it did affect community structure considerably (Fig. 4A). In the absence of alfalfa, over 60% of the total bacterial population was recovered on day 2. Presence of alfalfa plants resulted in an increase of r-strategists from 17 to 40%, because K-strategists reduced their fitness in a nutrient-rich environment that is more suitable for r-strategists, leading to the dominance of r-strategists in those environments (Mueller and Ayala, 1981). This is further supported by studies of De Leij et al. (1993). In the presence of alfalfa, bacterial growth pattern and distribution of r/K strategists were not significantly different among the three S. meliloti strains tested (Fig. 4B), suggesting that inoculation with genetically modified S. meliloti increased total bacterial population, but did not alter the microbial community structure under the conditions evaluated. This is further evidenced by the EP index, which was 0.4 in the absence of alfalfa and 0.6 in the presence of alfalfa with or without inoculation (Fig. 5). This index was not affected significantly by inoculation of S. meliloti,

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suggesting that the evenness of population distribution and richness of the microbial community were not affected by inoculation. This is contrary to findings reported by Bej et al. (1991) that the evenness of population distribution within a community increased following inoculation with a genetically modified P. cepacia However, their evaluation did not include plant growth. Furthermore, impact on microbial community structure can also be reflected in changes of ecosystem functions, such as plant growth and health. Plant dry matter in the control soil was 8.7 mg per plant with an average of 1.4 nodules per plant, while plants in S. meliloti-inoculated soils ranged from 17.8 to 20.6 mg dry matter and 5.5 to 6.5 nodules per plant. Alfalfa growth was significantly enhanced by inoculation with S. meliloti. However, there were no significant differences (P > 0.05) in plant dry weight and nodule numbers between inoculation of the wild-type and acid phosphatase-negative mutants (Table 5). This is consistent with analyses on soil microbial community structure. Results from studies conducted both in soil (this study) and in sand-supporting systems (Deng et al., 1998, 2001) showed that mutation of acid phosphatase genes resulted in little change in N-fixation symbiosis.

Fig. 5. EP-indices of soils. Controls were performed in the presence or absence of alfalfa plants. Alfalfa seedlings were inoculated with the wild-type Sinorhizobium meliloti 104A14, NapD or NapE mutant, respectively. Different letters indicate significantly different means at P < 0.05 according to least significant difference test. Bars represent standard errors (n = 4).

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4. Conclusion Survival and persistence of inoculated S. meliloti 104A14 was enhanced in soils with adequate nutrient supply, high organic matter, and optimal pH. Presence of host plants favored the establishment and persistence of inoculated strains. Genetic modification, but not specifically by mutation of the acid phosphatase genes in relation to P nutrition, weakened the bacteria’s ability to survive in a soil environment. This impact, however, was not as prominent as the impact resulting from the presence of host plants or variation in soil properties.

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