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Symbiotic characteristics and selection of autochthonous strains of Sinorhizobium meliloti populations in different soils
PERGAMON
Soil Biology and Biochemistry 31 (1999) 1039±1047
Symbiotic characteristics and selection of autochthonous strains of Sinorhizobium meliloti populations in dierent soils Encarna VelaÂzquez a, *, Pedro F. Mateos a, Nieves Velasco a, Fernando Santos b, Pedro A. Burgos c, Pablo Villadas c, NicolaÂs Toro c, Eustoquio Mart|Â nez-Molina a a
Departamento de Microbiolog|Âa y GeneÂtica, Facultad de Farmacia, Edi®cio Departamental, Universidad de Salamanca, Salamanca 37007, Spain b Departamento de Biolog|Âa Animal, Ecolog|Âa, Edafolog|Âa, Parasitolog|Âa y Qu|Âmica Agr|Âcola, Facultad de Farmacia, Universidad de Salamanca, Salamanca 37007, Spain c Departamento de Microbiolog|Âa, EstacioÂn Experimental del ZaidõÂn, C.S.I.C., Profesor Albareda No. 1, Granada, Spain
Abstract We studied the symbiotic characteristics of populations of Sinorhizobium meliloti present in soils with dierent cultivation histories. Usually the most abundant strains of S. meliloti did not bear cryptic plasmids. However, most of the very competitive strains bare other plasmids as well as pSym (the symbiotic plasmid). In a high proportion of these plasmids the presence of nfe genes was detected; these genes are involved in the competitiveness of S. meliloti strains. Studies on eectiveness of the strains bearing this nfe zone in a cryptic plasmid revealed that they form a number of nodules and ®x amounts of nitrogen signi®cantly higher than strains bearing only the symbiotic plasmid, conferring them advantages over other strains coexisting with them in the host plant rhizosphere. These ®ndings suggest the possibility of using the presence of the nfe zone as a criterion for the selection of strains that can be used as inoculants. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction An ecient rhizobia-legume symbiosis passes through a series of steps beginning with the colonization of the plant rhizosphere by the microorganism and ending with the biological ®xation of nitrogen within the nodule. In the root colonization phase, one important factor is the relative abundance of each strain in the soil, which indicates its adaptation to this environment. Although attempts have been made to increase legume production by increasing the number of bacteria surrounding the roots (Hossain and Alexander, 1984), the decisive factor in the ®rst stages of the process of infection is the competitiveness between strains present in the rhizosphere. Hence, the use of highly infective but not very competitive inoculants is inecient * Corresponding author. Fax: +34-923-224876; e-mail: evp@ gugu.usal.es
(Broughton et al., 1987a). It has been reported that nodulation ability and competitiveness may be related to the presence of cryptic plasmids (Brom®eld et al., 1985; Pankhurst et al., 1986; Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Pardo et al., 1994; Hartmann et al., 1998) In S. meliloti GR4 genes involved in competitiveness, have been described called nfe, which are linked to plasmid pRmeGR4b (SanjuaÂn and Olivares, 1989; Toro and Olivares, 1986; Soto et al., 1993; Villadas et al., 1995). In some cases, the eectiveness of nitrogen ®xation has also been related to the presence of certain cryptic plasmids (Thurman et al., 1985; Pankhurst et al., 1986; Barbour and Elkan, 1989; Hynes and MacGregor, 1990; Kuykendall et al., 1994; VelaÂzquez et al., 1995). These cryptic plasmids can be transferred from one strain to others in the rhizosphere (Broughton et al., 1987b; Scho®eld et al., 1987; Rao et al., 1994). The importance of cryptic plasmids is related to their abundance in cells and their great stability (Weaver et al., 1990; Mercado-Blanco and Olivares, 1993). The role of
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 1 8 - 8
0.38 0.00 39.60 5.80 0.06 1.41 9 yr with alfalfa a
S. meliloti count.
loamy sandy
1.47104
6.40
2.86 1.89 12.60 1.80 0.07 1.38 6 yr with alfalfa 4.62106
7.60
2.19 1.14 14.90 5.50 0.08 1.86 3 yr with alfalfa 1.49106
loamy clayey loamy
Planting history
calcic Rhodoxeralf typic Haploxeralf typic Xerochrept
Determination of the most probable number (MPN) of rhizobia was carried out according to Brockwell (1982). For each site, the pooled soil was sieved and mixed thoroughly. A 10 g sample from each soil was emulsi®ed in 90 ml of sterile water (hereafter, lower soil dilution). Serial decimal dilutions were made from
Nuevo Naharros (soil 1) Florida de LieÂbana (soil 2) Parada de Arriba (soil 3)
2.2. Evaluation of abundance of S. meliloti strains
Texture
Soil samples were taken from the sites shown in Table 1. The soils had been cultivated over dierent periods with M. sativa var. AragoÂn (Table 1). Soil samples were taken at a depth of 15±20 cm from each site. They were placed in a cool box for transport and stored at 58C. Samples were used for plant inoculation tests within 2 d of collection. Soil analysis were performed according to the guidelines of the Soil Conservation Service (1972). The soils were classi®ed according to their morphology and the analytical data following the US Soil Taxonomy (Soil Survey Sta, 1994). The characteristics of the three soils are shown in Table 1.
Table 1 Characteristics of three soils used in this study
2.1. Sample sites and collection of soil samples
Type
2. Materials and methods
MNPa (cells g ÿ 1)
pH
Organic matter (%)
Total N (%)
Total P (mg kg ÿ 1)
Total K (mg kg ÿ 1)
CO3Ca (%)
cryptic plasmids in rhizobia has been reviewed by Mercado-Blanco and Toro (1996). On establishing a pluriannual culture for a legume, of which Medicago sativa is perhaps the best example, the bacterial community of the soil may undergo selection pressure by the host (Jenkins and Bottomley, 1985; Brom®eld et al., 1986; Demezas and Bottomley, 1986a,b; Cregan et al., 1989) and therefore the contact between the cells in the rhizosphere increases, thus facilitating horizontal gene transfer among them and variations in the bacterial community of the rhizosphere. This accounts for the great variability observed in the plasmid pro®les of strains isolated in zones habitually cultivated with a single host (Brom®eld et al., 1987). Therefore the factors aecting rhizobia-legume symbiosis are very complex and it is necessary to study the ecology of indigenous populations of rhizobia as a prerequisite for elucidating problems of inoculant establishment and persistence in competitive situations (Brom®eld et al., 1986). The autochthonous populations of S. meliloti in soils cultivated with M. sativa during dierent periods and the symbiotic characteristics of the strains isolated were studied with the aim of selecting strains showing suitable adaptation to the soil, as well as high competitiveness and eectiveness that can be used as inoculants for alfalfa.
6.65
Mg (cmol kg ÿ 1)
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Site
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this suspension up to 1:107. Three 1 ml aliquots were used to inoculate three M. sativa var. AragoÂn plants grown axenically in Rigaud and Puppo (1975) solution. On day 10 after inoculation, the nodulated plants in each of the dilutions were counted and the most probable number (MPN) was calculated following Brockwell's method Brockwell (1982). The MPN for each soil are shown in Table 1. Surface-sterilized seeds of M. sativa var. AragoÂn (Brockwell, 1982) were germinated axenically in Petri dishes. Seedlings were transferred to pots with sterile vermiculite and watered with Rigaud and Puppo (1975) nutrient solution. Aliquots (5 ml) from each dilution of the three soils were added to the plants. The inoculated pots were placed for 30 d in a plant growth chamber with mixed incandescent and ¯uorescent lighting (400 mE m ÿ 2 s ÿ 1; 400±700 nm), programmed for a 16 h photoperiod, day±night cycle, with a constant temperature varying from 25±278C and 50±60% relative humidity. S. meliloti strains were isolated from surface-sterilized root nodules of M. sativa var. AragoÂn plants on YMA dishes (Vincent, 1970). Sixty infective and eective strains were isolated from nodules formed in the roots of M. sativa grown in the pots inoculated with the lower dilution of each soil (20 strains from each soil). Also, 30 infective and eective strains were isolated from nodules formed in the roots of M. sativa grown in the pots inoculated with the higher dilution of each soil in which nodules were formed. 2.3. Plasmid pro®le analysis The strains isolated were subjected to plasmid pro®le analysis using vertical 0.7% agarose gel electrophoresis and following the technique of Eckhardt (1978), modi®ed as follows: electrophoresis was performed in vertical gels (701103 mm) in a MiniProtean II cuvette (BioRad) for 30 min at 10 mA and 90 min at 40 mA. The reference strain for calculating the molecular weights of the cryptic plasmids was S. meliloti GR4, which has a cryptic plasmid of 140 MDa and another of 114 MDa (Toro and Olivares, 1986). 2.4. Preparation of cell lysate DNA, oligonucleotide primers and PCR conditions Bacterial cells grown on TY agar plates (Beringer, 1974) were suspended in 100 mL of water (OD600 = 0.6) containing 0.1% of sarkosyl. Cells were pelleted at 6000 g for 20 min, washed in 1 mL of water and pelleted again in a microcentrifuge tube 2 min at 10000 g. Finally, the pelleted cells were suspended in 100 mL of water. The cell suspension was boiled for 5±10 min and the cell lysate was kept at 48C.
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The oligonucleotide primers were synthesized and purchased from Isogen Bioscience B.V. (Amsterdam, The Netherlands). The nucleotide sequences for primers derived from S. meliloti GR4 plasmid pRmeGR4b nfe genes were: P6 (5 0 0 AGCCGCTGGTTCAGACGTAA-3 ) and F2 (3 0 AGACACGAGCTTCTATAGCT-5 0 ) (Soto et al., 1993). PCR reactions were carried out as reported by Villadas et al. (1995) on the DNA obtained. 2.5. Eectiveness studies The eectiveness studies were made in Leonard jars with Rigaud and Puppo (1975) solution adjusted to pH 7, using sterile vermiculite as a support for the plants, in a chamber with controlled photoperiod, temperature and humidity conditions. Each jar contained 10 plants. Five jars were used for each of the strains of S. meliloti studied. The jars were placed in a plant growth chamber with mixed incandescent and ¯uorescent illumination (400 mE m ÿ 2 s ÿ 1; 400±700 nm), programmed for a 16 h photoperiod, with a constant temperature varying from 25 to 278C, day±night cycle and 50±60% relative humidity. 2.6. Experimental design and statistics The plants were cultivated for 40 d in the growth chamber. At harvest the number of nodules was counted and the length and the dry weight of the aerial part of the inoculated plants were determined. Plant nitrogen content was measured according to the A.O.A.C. method (Johnson, 1990). The data obtained were analyzed by one-way analysis of variance. Mean values were compared by the Fisher's protected LSD (least signi®cant dierence).
3. Results 3.1. Plasmid pro®le analysis Fig. 1 and Table 2 show the results of the plasmid pro®le analysis for the strains isolated from the three soils studied. In the three soils studied, 13 plasmid pro®les and 11 dierent cryptic plasmids were identi®ed on the basis of their molecular weights (Fig. 1, lane A), which ranged between 20 and 700 MDa. In the strains isolated from soil 1, with a recent cultivation of alfalfa, 7 dierent plasmid pro®les (Fig. 1, lanes A, B, C, D, H, J and K) including 7 dierent plasmids (molecular weights ranging between 30 and 700 MDa) were observed.
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Fig. 1. Plasmid pro®les of strains included in this study: lane A, pSym; lane B, pSym and cryptic plasmids of 140 and 114 MDa; lane C, pSym and cryptic plasmid of 140 MDa; lane D, pSym and cryptic plasmid of 114 MDa; lane E, pSym and cryptic plasmid of 300 MDa; lane F, pSym and cryptic plasmids of 300 and 20 MDa; lane G, pSym and cryptic plasmid of 30 MDa; lane H, pSym and cryptic plasmids of 200 and 50 MDa; lane I, pSym and cryptic plasmid of 100 MDa; lane J, pSym and cryptic plasmid of 90 MDa; lane K, pSym and cryptic plasmids of 700 and 30 MDa; lane L, pSym and cryptic plasmid of 500 MDa; lane M, pSym and cryptic plasmids of 700 and 90 MDa.
All the plasmid pro®les were found in strains isolated from the lower dilution of soil 1, no new pro®les appearing in the higher soil dilution, in which 4 dierent pro®les were found (Fig. 1, lanes A, B, C and H), with 4 dierent cryptic plasmids on the basis of their molecular weights, which ranged between 50 and 200 MDa. The strains isolated most frequently from the lower soil dilution were those bearing the pSym and two cryptic plasmids of 140 and 114 MDa (Fig. 1, lane B), those bearing one cryptic plasmid of 140 MDa (Fig. 1, lane C), those bearing one cryptic plasmid of 114 MDa (Fig. 1, lane D) and the strains bearing only the pSym plasmid (Fig. 1, lane A). The remaining pro®les were represented by a single strain (Fig. 1, lanes H, J and K). The strains isolated most frequently from the higher soil dilution were those bearing only the pSym plasmid (Fig. 1, lane A), followed by those bearing two cryptic plasmids of 50 and 200 MDa (Fig. 1, lane H). One strain alone bore a cryptic plasmid of 140 MDa (Fig. 1, lane C). In the strains isolated from soil 2, with a well-established alfalfa culture, 7 dierent plasmid pro®les were observed (Fig. 1, lanes A, B, C, D, I, L and M), with 6 dierent plasmids, according to their molecular weights, which ranged from 90 to 700 MDa. All the plasmid pro®les were found in strains isolated from the lower dilution of soil 2, no new pro®les appearing in the higher dilution, in which 5 dierent pro®les were detected (Fig. 1, lanes A, C, I, L and M)
with 5 dierent plasmids, according to their molecular weights, which ranged between 90 and 700 MDa. The strains isolated most frequently from the lower solution of soil 2 were those bearing only pSym (Fig. 1, lane A), followed by those also bearing a cryptic plasmid of 140 MDa (Fig. 1, lane C), two cryptic plasmids of 140 and 114 MDa (Fig. 1, lane B), one cryptic plasmid of 100 MDa (Fig. 1, lane I) or two cryptic plasmids of 700 and 90 MDa (pro®le M). The remaining pro®les were represented by a single strain (Fig. 1, lanes D and L). The strains isolated most frequently in the higher dilution of soil 2 were those bearing a plasmid of 140 MDa (Fig. 1, lane C), followed by those bearing only the pSym (Fig. 1, lane A), those bearing a cryptic plasmid of 500 MDa (Fig. 1, lane L) and those bearing two of 90 and 700 MDa (Fig. 1, lane M). A single strain bore a cryptic plasmid of 100 MDa (Fig. 1, lane I). In the strains isolated from soil 3, with the longest period of alfalfa cultivation, 7 dierent plasmid pro®les were detected (Fig. 1, lanes A, B, C, D, E, F and G), with 5 dierent cryptic plasmids, on the basis of their molecular weights, which ranged between 20 and 300 MDa. All the plasmid pro®les were found in strains isolated from the lower dilution of soil 3, no new pro®les appearing in the higher dilution, in which 2 dierent pro®les were found (Fig. 1, lanes A and G) with a cryptic plasmid of 30 MDa. The strains most frequently isolated from the lower dilution of soil 3 were those bearing only the pSym
a
01, 07 08, 11, 14 15 16 17 18,
Parada de Arriba (3) (PMSA)
Plasmid pro®les are presented in Fig. 1.
19 and 20
09, 10 12, 13
02, 03, 04, 05, 06
27, 28, 29, 30
21, 22, 23, 24, 25, 26
26 27, 28 29, 30
17
20
23, 24, 25
13 11, 14
01, 09 08 12, 10, 15 16, 18 19,
Florida de LieÂbana (2) (FMSA)
29, 30
28
21, 22, 23, 24, 25 26, 27
21, 22
01 and 02 03, 04, 06, 08, 09 05, 07 10, 12, 13 11 14, 15, 16, 17 18 19 20
Naharros Nuevo (1) (NMSA)
Strains isolated from higher dilution of soil
02, 03, 04, 05, 06, 07
Strains isolated from lower dilution of soil
Soil
Table 2 Characteristics of the strains of S. meliloti isolated in this study
A B B C C D E F G
A B B C C D I L M
A B B C C D H J K
Plasmid pro®lea
and 30
and 50
and 114 and 114
140 and 114 140 and 114 140 140 114 300 300 and 20 30
140 and 114 140 and 114 140 140 114 100 500 700 and 90
140 140 140 140 114 200 90 700
Molecular weight of cryptic plasmids (MDA)
ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ
ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ
ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ
nfe
E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047 1043
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plasmid (Fig. 1, lane A), followed by those also bearing two cryptic plasmids of 140 and 114 MDa (Fig. 1, lane B), one cryptic plasmid of 140 MDa (Fig. 1, lane C), one cryptic plasmid of 114 MDa (Fig. 1, lane D) or one cryptic plasmid of 30 MDa (Fig. 1, lane G). The remaining pro®les were represented by a single strain (Fig. 1, lane F). The strains isolated most frequently from the higher soil dilution bore only the pSym plasmid (Fig. 1, lane A), followed by those bearing a cryptic plasmid of 30 MDa (Fig. 1, lane G). 3.2. PCR ampli®cation with primers derived from GR4 nfe genes The results are shown in Table 1. The nfe zone was never detected in the strains isolated in the higher dilution of the three soils. By contrast, in the lower dilution of soil 1 it was present in 15% of the strains; in soil 2 in 20% of the strains and in soil 3 in 20% of the strains. Considering the total number of strains isolated from each soil (30), in soil 1 the nfe zone was present in 10% of the strains and in soils 2 and 3 in 13% of the strains. In the three soils studied together, the nfe zone was displayed by strains bearing a cryptic plasmid of 140 MDa (Table 2, pro®le C) (38% of strains bearing this cryptic plasmid displayed the nfe zone) or two cryptic plasmids, one of 140 MDa and the other of 114 MDa (Table 2, pro®le B) (46% of strains bearing these cryptic plasmids displayed the nfe zone). 3.3. Eectiveness studies All the strains isolated were infective and eective (data not shown) and 9 strains were chosen for the study of symbiotic characteristics. The selection of strains was also made taking into account the following characteristics: adaptation to the soil (strains PMSA 30, NMSA 29 and FMSA 29 are well adapted
to each of the three soils since they were isolated from the higher dilution of each soil); plasmid pro®les (strains PMSA25, NMSA10, PMSA11, FMSA11, PMSA10 and FMSA09, whose plasmid pro®le was found very frequently in the three soils Ð pro®les A, B and C Ð were selected) and presence of the nfe zone (for example, strains PMSA11 and FMSA11 show the same plasmid pro®le, nevertheless the strain FMSA11 contains the nfe zone which is absent from strain PMSA11). The results of the eectiveness assays are shown in Table 3. No signi®cant dierences were observed between strain PMSA25 (which only bore the symbiotic plasmid) and strain FMSA29 in dry plant weight and total nitrogen contents. By contrast, signi®cant dierences were observed between strain PMSA25 and other strains tested (Table 3). Strains NMSA10, PMSA11, FMSA11, PMSA10, FMSA09, PMSA30 and NMSA29 ®xed signi®cantly more nitrogen than PMSA25 (see Table 3). It was also seen that the strain FMSA11 formed a signi®cantly higher number of nodules in alfalfa compared to strains PMSA25, FMSA29, NMSA10, PMSA11, FMSA09, PMSA30 and NMSA29. By contrast, signi®cant dierences were not found between the FMSA11 and the PMSA10 strain and both strains display the nfe zone.
4. Discussion The results for the strains isolated from the soils we studied point to a high genetic diversity according to the number and molecular weights of the plasmids detected. In each soil, seven plasmid pro®les were found, with a number of cryptic plasmids ranging from 0 to 2 (Fig. 1) and, overall, 13 dierent plasmid pro®les were observed. Eleven dierent cryptic plasmids were detected on the basis of the dierences in
Table 3 Some symbiotic characteristics of S. meliloti strains isolated from soils of Salamanca (Spain) Inoculant strain Nodules per plant Shoot height (cm) Shoot dry weight (mg) Shoot total nitrogen (mg) Cryptic plasmids (MDa) nfe zone PMSA 25 NMSA 10 PMSA 11 FMSA 11 PMSA 10 FMSA 09 PMSA 30 NMSA 29 FMSA 29 a
16abc 22def 18bc 25fg 23defg 15ab 18bc 21def 15ab
27bcde 25abcd 27bcde 30ef 26bcd 30ef 29def 30ef 24ab
83ab 120defg 119defg 131efg 102c 122defg 120defg 120defg 85ab
3a 5bc 5bc 5bc 4bc 5bc 4bc 4bc 3a
± 140 140 140 140 140 30 200 700
and 114 and 114 and 20 and 90
ÿ ÿ ÿ + + ÿ ÿ ÿ ÿ
Values are the means of ®ve jars. In each column, values followed by the same letter are not signi®cantly dierent according to Fisher's protected LSD (P > 0.05).
E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047
their molecular weights, which ranged between 20 and 700 MDa (Table 1). These ®ndings are consistent with those reported by Broughton et al. (1987b), Scho®eld et al. (1987) and Rao et al. (1994) who described strong variability in plasmid pro®les in soils cultivated with alfalfa, due to the transfer of genetic material among microorganisms in the plant rhizosphere. The number and diversity of the cryptic plasmids of the strains isolated decreased as the soil dilution increased, so in the highest soil dilutions we only found the most abundant strains. Also, whereas in the lower soil dilution there were up to seven dierent plasmid pro®les, in the higher dilution the maximum number of plasmid pro®les observed was ®ve. Attempts have been made to relate the presence of cryptic plasmids with adaptation by rhizobia to the soil conditions (Thurman et al., 1985). From our results it may be inferred that, overall, the most abundant strains Ð and hence those best adapted to the soil Ð isolated from the three soils studied lacked cryptic plasmids (Fig. 1, lane A). However, in soil 3 there were strains bearing a cryptic plasmid of 30 MDa that were well adapted to the soil and likewise in soil 2, there were strains having a plasmid of 40 MDa. This suggests a certain involvement of these plasmids in adaptation to the soil. In this study we estimated the competitiveness and abundance of S. meliloti strains present in a soil under natural conditions. In order to do so, we compared strains isolated from alfalfa inoculated with the lower soil dilution (1:10) and those isolated from alfalfa plants inoculated with the higher dilution of the same soil in which nodule production was detected. In pots inoculated with the lower soil dilution, the number and diversity of rhizobia were high and for a strain to be successful in nodulation it must compete with many other strains of rhizobia that coexist with it in the soil. Accordingly, strains with the nfe zone, which confers greater competitiveness (Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995), were isolated in relatively high proportions. By contrast, in pots inoculated with the higher soil dilution the number and diversity of rhizobia were lower; only the most abundant rhizobia were found because they hardly had to compete to be successful in nodulation. At this dilution, no strains showing the nfe zone were isolated, indicating that the strains bearing this zone were not abundant in the soils studied. Comparison of the strains isolated from both soil dilutions suggests the following: the strains isolated very frequently from nodules of pots inoculated with the higher soil dilution were very abundant (the strains bearing the pSym plasmid in the three soils studied and strains bearing a cryptic plasmid of 30 MDa in the soil 3); the less abundant strains which were isolated very frequently from nodules formed in pots
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inoculated with the lower soil dilution were competitive strains (strains that displayed the B or C plasmid pro®les in the three soils studied) and the strains bearing the nfe zone, which confers greater competitiveness, were never isolated from nodules formed in pots inoculated with the higher soil dilution, implying that they were not abundant (strains that displayed the B or C plasmid pro®les in the three soils studied). Therefore we observed that the strains having a cryptic plasmid of 140 MDa (Fig. 1, lane C) or two plasmids of 140 and 114 MDa (Fig. 1, lane B) were very competitive in the three soils studied. This is in agreement with the results of Brom®eld et al. (1985), Pankhurst et al. (1986), Toro and Olivares (1986), SanjuaÂn and Olivares (1989) and Pardo et al. (1994) who related competitiveness to the presence of certain cryptic plasmids. A zone (nfe) present in a 140 MDa cryptic plasmid (pRmeGR4b) directly involved in the competitiveness of the S. meliloti GR4 strain has been described (Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995). In our study, the nfe zone was only detected in highly competitive strains having the B or C plasmid pro®les in the three soils studied (see Table 2). Our results show that the proportion of strains in which the nfe zone was detected was lower than that of strains in which the presence of the pRmeGR4b type plasmid was detected. These results were consistent with those reported by Villadas et al. (1995). From the study of the symbiotic characteristics assayed it may be deduced that strains PMSA10 (soil 3) and FMSA11 (soil 2) bearing the pSym and a cryptic plasmid of 140 MDa (Fig. 1, lane C) and the pSym and two cryptic plasmids of 140 MDa and 114 MDa (Fig. 1, lane B), respectively, are very eective as can be seen by the amount of nitrogen ®xed per plant and show signi®cant dierences with strain PMSA25 bearing only the pSym plasmid (Fig. 1, lane A). As reported above, these strains were also observed to have high competitiveness and their cryptic plasmids display the nfe zone, whose in¯uence in this symbiotic characteristic has been demonstrated (Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995). This suggests that cryptic plasmids were not only involved in competitiveness but also in the eectiveness of S. meliloti strains (VelaÂzquez et al., 1995). In view of the results, strain FMSA 11, with a plasmid pro®le that contains pSym and a 140 MDa plasmid in which the nfe zone was detected, has good symbiotic characteristics as far as both infectivity (number of nodules formed) and eectiveness (plant dry weight and nitrogen ®xed) are concerned. Also, the presence of the nfe zone is important since it has been shown to be involved in competitiveness (Toro
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E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047
and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995). It may thus be concluded that in soil with S. meliloti populations there are autochthonous strains with high competitiveness, infectivity and eectiveness that have advantages over others coexisting with them; hence, they may be very suitable as inoculants. Moreover, this suggests that the nfe zone can be used as a criterion for the selection of strains for this purpose. These `selected local strains' must show advantages in competitiveness, nodulation eectiveness and nitrogen ®xation compare to others isolated in allochthonous ecosystems and their use as inoculants will lead to an increase in the production of the host legume which is, of great interest from the economic point of view.
Acknowledgements This work was supported by the Junta de Castilla y LeoÂn and the DGICYT (DireccioÂn General de InvestigacioÂn Cient|Â ®ca y TeÂcnica). The authors thank N. Skinner and Imelda Geldart for revising the English version of the manuscript.
References Barbour, W.M., Elkan, G.H., 1989. Relationship of the presence and copy number of plasmids to exopolysaccharide production and symbiotic eectiveness in Rhizobium fredii USDA 206. Applied and Environmental Microbiology 55, 813±818. Beringer, J.E., 1974. R factor transfer in Rhizobium leguminosarum. Journal of General Microbiology 84, 188±198. Brockwell, J., 1982. Plant-infection counts of rhizobia in soils. In: Vincent, J.M. (Ed.), Nitrogen Fixation in Legumes. Academic Press, New York, pp. 41±57. Brom®eld, E.S.P., Lewis, D.M., Barran, L.R., 1985. Cryptic plasmid and rifampin resistance in Rhizobium meliloti in¯uencing nodulation competitiveness. Journal of Bacteriology 164, 410±413. Brom®eld, E.S.P., Sinha, I.B., Wolynetz, M.S., 1986. In¯uence of location, host cultivar and inoculation on the composition of naturalized populations of R. meliloti in Medicago sativa nodules. Applied and Environmental Microbiology 51, 1077±1084. Brom®eld, E.S.P., Thurman, N.P., Whitwill, S.T., Barran, L.R., 1987. Plasmids and symbiotic eectiveness of representative phage types from two indigenous populations of Rhizobium meliloti. Journal of General Microbiology 133, 3457±3466. Broughton, W.J., Heycke, N., Priefer, U., Schneider, G.M., Stanley, J., 1987a. Ecological genetics of Rhizobium meliloti: diversity and competitive dominance. FEMS Microbiology Letters 40, 245±249. Broughton, W.J., Samrey, U., Stanley, J., 1987b. Ecological genetics of Rhizobium meliloti: symbiotic plasmid transfer in the Medicago sativa rhizosphere. FEMS Microbiology Letters 40, 251±255. Cregan, P.B., Keyser, H.H., Sadowsky, M.J., 1989. Host plants eects on nodulation and competitivenes of the Bradyrhizobium japonicum serotype strains constituting serocluster 123. Applied and Environmental Microbiology 55, 2532±2536. Demezas, D.H., Bottomley, P.J., 1986a. Autoecology in rhizospheres and nodulating behavior of indigenous Rhizobium trifolii. Applied and Environmental Microbiology 52, 1014±1019.
Demezas, D.H., Bottomley, P.J., 1986b. Interstrain competition between representatives of indigenous serotypes of Rhizobium trifolii. Applied and Environmental Microbiology 52, 1020±1025. Eckhardt, T.H., 1978. A rapid method for the identi®cation of plasmid deoxyribonucleic acid in bacteria. Plasmid 1, 584±588. Hartmann, A., Giraud, J.J., Catroux, G., 1998. Genotypic diversity of Sinorhizobium (formely Rhizobium) meliloti strains isolated directly from a soil and from nodules of alfalfa (Medicago sativa) grown in the same soil. FEMS Microbiology Ecology 25, 107±116. Hossain, A.K.M., Alexander, M., 1984. Enhancing soybean rhizosphere colonization by Rhizobium japonicum. Applied and Environmental Microbiology 48, 468±472. Hynes, M.F., MacGregor, N.F., 1990. Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-®xing nodules by Rhizobium leguminosarum. Molecular Microbiology 4, 567±574. Jenkins, M.B., Bottomley, P.J., 1985. Evidence for a strain of Rhizobium meliloti dominating the nodules of alfalfa. Soil Science Society of America Journal 49, 326±328. Johnson, F.J., 1990. Detection method of nitrogen (total) in fertilizers. In: Elrich, K. (Ed.), Ocial Methods of Analysis of the Association of Ocial Analytical Chemists. Association of Ocial Analytical Chemists. Arlington, VA, pp. 17±19. Kuykendall, L.D., Abdel-Wahab, S.M., Hashem, F.M., Van Berkum, P., 1994. Symbiotic competence and genetic diversity of Rhizobium strains used as inoculants for alfalfa and berseem clover. Letters on Applied Microbiology 19, 477±482. Mercado-Blanco, J., Olivares, J., 1993. Stability and transmissibility of the cryptic plasmids of Rhizobium meliloti GR4: their possible use in the construction of cloning vectors for rhizobia. Archives of Microbiology 160, 477±485. Mercado-Blanco, J., Toro, N., 1996. Plasmids in rhizobia: the role of nonsymbiotic plasmids. Molecular Plant Microbe Interaction 9, 535±545. Pankhurst, C.E., MacDonald, P.E., Reeves, J.M., 1986. Enhanced nitrogen ®xation and competitiveness for nodulation of Lotus pedunculatus by a plasmid-cured derivative of Rhizobium loti. Journal of General Microbiology 132, 2321±2328. Pardo, M.A., LaguÂnez, J., Miranda, J., Mart|Â nez, E., 1994. Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase. Molecular Microbiology 11, 315± 321. Rao, J.R., Fenton, M., Jarvis, B.D.W., 1994. Symbiotic plasmid transfer in Rhizobium leguminosarum biovar trifolii and competition between the inoculant strain Icmp2163 and transconjugant soil bacteria. Soil Biology & Biochemistry 26, 339±351. Rigaud, J., Puppo, A., 1975. Indole-3-acetic catabolism by soybean bacteroids. Journal of General Microbiology 88, 223±228. SanjuaÂn, J., Olivares, J., 1989. Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that aect nodulation eciency. Journal of Bacteriology 171, 4154±4161. Scho®eld, P.R., Gibson, A.H., Dudman, W.F., Watson, J.M., 1987. Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population. Applied and Environmental Microbiology 53, 2942±2947. Soil Conservation Service, 1972. In: Soil Survey laboratory methods and procedures for collecting soil samples. USDA, Washington. Soil Survey Sta, 1994. In: Keys to soil taxonomy, 6th ed. Soil Conservation Service, USDA, Washington. Soto, M.J., Zorzano, A., Mercado-Blanco, J., Lepek, V., Olivares, J., Toro, N., 1993. Nucleotide sequence and characterization of Rhizobium meliloti nodulation competitiveness genes nfe. Journal of Molecular Biology 229, 570±576. Thurman, N.P., Lewis, D.M., Gareth Jones, D., 1985. The relationships of plasmid number to growth, acid tolerance and symbiotic eciency in isolates of Rhizobium trifolii. Journal of Applied Bacteriology 58, 1±6.
E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047 Toro, N., Olivares, J., 1986. Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation. Molecular General Genetics 202, 331±335. VelaÂzquez, E., Mateos, P.F., Pedrero, P., Dazzo, F.B., Mart|Â nezMolina, E., 1995. Attenuation of symbiotic eectiveness by Rhizobium meliloti SAF22 related to the presence of a cryptic plasmid. Applied and Environmental Microbiology 61, 2033±2036. Villadas, P.J., VelaÂzquez, E., Mart|Â nez-Molina, E., Toro, N., 1995. Identi®cation of nodule-dominant Rhizobium meliloti strains carrying pRmeGR4-type plasmid within indigenous soil populations by
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PCR using primers derived from speci®c DNA sequences. FEMS Microbiology Ecology 17, 161±168. Vincent, J.M., 1970. The cultivation, isolation and maintenance of rhizobia. In: Vincent, J.M. (Ed.), A Manual for the Practical Study of Root-Nodule Bacteria. Blackwell Scienti®c Publications, Oxford, pp. 1±13. Weaver, R.W., Wei, G.R., Berryhill, D.L., 1990. Stability of plasmids in Rhizobium phaseoli during culture. Soil Biology & Biochemistry 22, 465±469.
Soil Biology and Biochemistry 31 (1999) 1039±1047
Symbiotic characteristics and selection of autochthonous strains of Sinorhizobium meliloti populations in dierent soils Encarna VelaÂzquez a, *, Pedro F. Mateos a, Nieves Velasco a, Fernando Santos b, Pedro A. Burgos c, Pablo Villadas c, NicolaÂs Toro c, Eustoquio Mart|Â nez-Molina a a
Departamento de Microbiolog|Âa y GeneÂtica, Facultad de Farmacia, Edi®cio Departamental, Universidad de Salamanca, Salamanca 37007, Spain b Departamento de Biolog|Âa Animal, Ecolog|Âa, Edafolog|Âa, Parasitolog|Âa y Qu|Âmica Agr|Âcola, Facultad de Farmacia, Universidad de Salamanca, Salamanca 37007, Spain c Departamento de Microbiolog|Âa, EstacioÂn Experimental del ZaidõÂn, C.S.I.C., Profesor Albareda No. 1, Granada, Spain
Abstract We studied the symbiotic characteristics of populations of Sinorhizobium meliloti present in soils with dierent cultivation histories. Usually the most abundant strains of S. meliloti did not bear cryptic plasmids. However, most of the very competitive strains bare other plasmids as well as pSym (the symbiotic plasmid). In a high proportion of these plasmids the presence of nfe genes was detected; these genes are involved in the competitiveness of S. meliloti strains. Studies on eectiveness of the strains bearing this nfe zone in a cryptic plasmid revealed that they form a number of nodules and ®x amounts of nitrogen signi®cantly higher than strains bearing only the symbiotic plasmid, conferring them advantages over other strains coexisting with them in the host plant rhizosphere. These ®ndings suggest the possibility of using the presence of the nfe zone as a criterion for the selection of strains that can be used as inoculants. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction An ecient rhizobia-legume symbiosis passes through a series of steps beginning with the colonization of the plant rhizosphere by the microorganism and ending with the biological ®xation of nitrogen within the nodule. In the root colonization phase, one important factor is the relative abundance of each strain in the soil, which indicates its adaptation to this environment. Although attempts have been made to increase legume production by increasing the number of bacteria surrounding the roots (Hossain and Alexander, 1984), the decisive factor in the ®rst stages of the process of infection is the competitiveness between strains present in the rhizosphere. Hence, the use of highly infective but not very competitive inoculants is inecient * Corresponding author. Fax: +34-923-224876; e-mail: evp@ gugu.usal.es
(Broughton et al., 1987a). It has been reported that nodulation ability and competitiveness may be related to the presence of cryptic plasmids (Brom®eld et al., 1985; Pankhurst et al., 1986; Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Pardo et al., 1994; Hartmann et al., 1998) In S. meliloti GR4 genes involved in competitiveness, have been described called nfe, which are linked to plasmid pRmeGR4b (SanjuaÂn and Olivares, 1989; Toro and Olivares, 1986; Soto et al., 1993; Villadas et al., 1995). In some cases, the eectiveness of nitrogen ®xation has also been related to the presence of certain cryptic plasmids (Thurman et al., 1985; Pankhurst et al., 1986; Barbour and Elkan, 1989; Hynes and MacGregor, 1990; Kuykendall et al., 1994; VelaÂzquez et al., 1995). These cryptic plasmids can be transferred from one strain to others in the rhizosphere (Broughton et al., 1987b; Scho®eld et al., 1987; Rao et al., 1994). The importance of cryptic plasmids is related to their abundance in cells and their great stability (Weaver et al., 1990; Mercado-Blanco and Olivares, 1993). The role of
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 1 8 - 8
0.38 0.00 39.60 5.80 0.06 1.41 9 yr with alfalfa a
S. meliloti count.
loamy sandy
1.47104
6.40
2.86 1.89 12.60 1.80 0.07 1.38 6 yr with alfalfa 4.62106
7.60
2.19 1.14 14.90 5.50 0.08 1.86 3 yr with alfalfa 1.49106
loamy clayey loamy
Planting history
calcic Rhodoxeralf typic Haploxeralf typic Xerochrept
Determination of the most probable number (MPN) of rhizobia was carried out according to Brockwell (1982). For each site, the pooled soil was sieved and mixed thoroughly. A 10 g sample from each soil was emulsi®ed in 90 ml of sterile water (hereafter, lower soil dilution). Serial decimal dilutions were made from
Nuevo Naharros (soil 1) Florida de LieÂbana (soil 2) Parada de Arriba (soil 3)
2.2. Evaluation of abundance of S. meliloti strains
Texture
Soil samples were taken from the sites shown in Table 1. The soils had been cultivated over dierent periods with M. sativa var. AragoÂn (Table 1). Soil samples were taken at a depth of 15±20 cm from each site. They were placed in a cool box for transport and stored at 58C. Samples were used for plant inoculation tests within 2 d of collection. Soil analysis were performed according to the guidelines of the Soil Conservation Service (1972). The soils were classi®ed according to their morphology and the analytical data following the US Soil Taxonomy (Soil Survey Sta, 1994). The characteristics of the three soils are shown in Table 1.
Table 1 Characteristics of three soils used in this study
2.1. Sample sites and collection of soil samples
Type
2. Materials and methods
MNPa (cells g ÿ 1)
pH
Organic matter (%)
Total N (%)
Total P (mg kg ÿ 1)
Total K (mg kg ÿ 1)
CO3Ca (%)
cryptic plasmids in rhizobia has been reviewed by Mercado-Blanco and Toro (1996). On establishing a pluriannual culture for a legume, of which Medicago sativa is perhaps the best example, the bacterial community of the soil may undergo selection pressure by the host (Jenkins and Bottomley, 1985; Brom®eld et al., 1986; Demezas and Bottomley, 1986a,b; Cregan et al., 1989) and therefore the contact between the cells in the rhizosphere increases, thus facilitating horizontal gene transfer among them and variations in the bacterial community of the rhizosphere. This accounts for the great variability observed in the plasmid pro®les of strains isolated in zones habitually cultivated with a single host (Brom®eld et al., 1987). Therefore the factors aecting rhizobia-legume symbiosis are very complex and it is necessary to study the ecology of indigenous populations of rhizobia as a prerequisite for elucidating problems of inoculant establishment and persistence in competitive situations (Brom®eld et al., 1986). The autochthonous populations of S. meliloti in soils cultivated with M. sativa during dierent periods and the symbiotic characteristics of the strains isolated were studied with the aim of selecting strains showing suitable adaptation to the soil, as well as high competitiveness and eectiveness that can be used as inoculants for alfalfa.
6.65
Mg (cmol kg ÿ 1)
E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047
Site
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E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047
this suspension up to 1:107. Three 1 ml aliquots were used to inoculate three M. sativa var. AragoÂn plants grown axenically in Rigaud and Puppo (1975) solution. On day 10 after inoculation, the nodulated plants in each of the dilutions were counted and the most probable number (MPN) was calculated following Brockwell's method Brockwell (1982). The MPN for each soil are shown in Table 1. Surface-sterilized seeds of M. sativa var. AragoÂn (Brockwell, 1982) were germinated axenically in Petri dishes. Seedlings were transferred to pots with sterile vermiculite and watered with Rigaud and Puppo (1975) nutrient solution. Aliquots (5 ml) from each dilution of the three soils were added to the plants. The inoculated pots were placed for 30 d in a plant growth chamber with mixed incandescent and ¯uorescent lighting (400 mE m ÿ 2 s ÿ 1; 400±700 nm), programmed for a 16 h photoperiod, day±night cycle, with a constant temperature varying from 25±278C and 50±60% relative humidity. S. meliloti strains were isolated from surface-sterilized root nodules of M. sativa var. AragoÂn plants on YMA dishes (Vincent, 1970). Sixty infective and eective strains were isolated from nodules formed in the roots of M. sativa grown in the pots inoculated with the lower dilution of each soil (20 strains from each soil). Also, 30 infective and eective strains were isolated from nodules formed in the roots of M. sativa grown in the pots inoculated with the higher dilution of each soil in which nodules were formed. 2.3. Plasmid pro®le analysis The strains isolated were subjected to plasmid pro®le analysis using vertical 0.7% agarose gel electrophoresis and following the technique of Eckhardt (1978), modi®ed as follows: electrophoresis was performed in vertical gels (701103 mm) in a MiniProtean II cuvette (BioRad) for 30 min at 10 mA and 90 min at 40 mA. The reference strain for calculating the molecular weights of the cryptic plasmids was S. meliloti GR4, which has a cryptic plasmid of 140 MDa and another of 114 MDa (Toro and Olivares, 1986). 2.4. Preparation of cell lysate DNA, oligonucleotide primers and PCR conditions Bacterial cells grown on TY agar plates (Beringer, 1974) were suspended in 100 mL of water (OD600 = 0.6) containing 0.1% of sarkosyl. Cells were pelleted at 6000 g for 20 min, washed in 1 mL of water and pelleted again in a microcentrifuge tube 2 min at 10000 g. Finally, the pelleted cells were suspended in 100 mL of water. The cell suspension was boiled for 5±10 min and the cell lysate was kept at 48C.
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The oligonucleotide primers were synthesized and purchased from Isogen Bioscience B.V. (Amsterdam, The Netherlands). The nucleotide sequences for primers derived from S. meliloti GR4 plasmid pRmeGR4b nfe genes were: P6 (5 0 0 AGCCGCTGGTTCAGACGTAA-3 ) and F2 (3 0 AGACACGAGCTTCTATAGCT-5 0 ) (Soto et al., 1993). PCR reactions were carried out as reported by Villadas et al. (1995) on the DNA obtained. 2.5. Eectiveness studies The eectiveness studies were made in Leonard jars with Rigaud and Puppo (1975) solution adjusted to pH 7, using sterile vermiculite as a support for the plants, in a chamber with controlled photoperiod, temperature and humidity conditions. Each jar contained 10 plants. Five jars were used for each of the strains of S. meliloti studied. The jars were placed in a plant growth chamber with mixed incandescent and ¯uorescent illumination (400 mE m ÿ 2 s ÿ 1; 400±700 nm), programmed for a 16 h photoperiod, with a constant temperature varying from 25 to 278C, day±night cycle and 50±60% relative humidity. 2.6. Experimental design and statistics The plants were cultivated for 40 d in the growth chamber. At harvest the number of nodules was counted and the length and the dry weight of the aerial part of the inoculated plants were determined. Plant nitrogen content was measured according to the A.O.A.C. method (Johnson, 1990). The data obtained were analyzed by one-way analysis of variance. Mean values were compared by the Fisher's protected LSD (least signi®cant dierence).
3. Results 3.1. Plasmid pro®le analysis Fig. 1 and Table 2 show the results of the plasmid pro®le analysis for the strains isolated from the three soils studied. In the three soils studied, 13 plasmid pro®les and 11 dierent cryptic plasmids were identi®ed on the basis of their molecular weights (Fig. 1, lane A), which ranged between 20 and 700 MDa. In the strains isolated from soil 1, with a recent cultivation of alfalfa, 7 dierent plasmid pro®les (Fig. 1, lanes A, B, C, D, H, J and K) including 7 dierent plasmids (molecular weights ranging between 30 and 700 MDa) were observed.
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Fig. 1. Plasmid pro®les of strains included in this study: lane A, pSym; lane B, pSym and cryptic plasmids of 140 and 114 MDa; lane C, pSym and cryptic plasmid of 140 MDa; lane D, pSym and cryptic plasmid of 114 MDa; lane E, pSym and cryptic plasmid of 300 MDa; lane F, pSym and cryptic plasmids of 300 and 20 MDa; lane G, pSym and cryptic plasmid of 30 MDa; lane H, pSym and cryptic plasmids of 200 and 50 MDa; lane I, pSym and cryptic plasmid of 100 MDa; lane J, pSym and cryptic plasmid of 90 MDa; lane K, pSym and cryptic plasmids of 700 and 30 MDa; lane L, pSym and cryptic plasmid of 500 MDa; lane M, pSym and cryptic plasmids of 700 and 90 MDa.
All the plasmid pro®les were found in strains isolated from the lower dilution of soil 1, no new pro®les appearing in the higher soil dilution, in which 4 dierent pro®les were found (Fig. 1, lanes A, B, C and H), with 4 dierent cryptic plasmids on the basis of their molecular weights, which ranged between 50 and 200 MDa. The strains isolated most frequently from the lower soil dilution were those bearing the pSym and two cryptic plasmids of 140 and 114 MDa (Fig. 1, lane B), those bearing one cryptic plasmid of 140 MDa (Fig. 1, lane C), those bearing one cryptic plasmid of 114 MDa (Fig. 1, lane D) and the strains bearing only the pSym plasmid (Fig. 1, lane A). The remaining pro®les were represented by a single strain (Fig. 1, lanes H, J and K). The strains isolated most frequently from the higher soil dilution were those bearing only the pSym plasmid (Fig. 1, lane A), followed by those bearing two cryptic plasmids of 50 and 200 MDa (Fig. 1, lane H). One strain alone bore a cryptic plasmid of 140 MDa (Fig. 1, lane C). In the strains isolated from soil 2, with a well-established alfalfa culture, 7 dierent plasmid pro®les were observed (Fig. 1, lanes A, B, C, D, I, L and M), with 6 dierent plasmids, according to their molecular weights, which ranged from 90 to 700 MDa. All the plasmid pro®les were found in strains isolated from the lower dilution of soil 2, no new pro®les appearing in the higher dilution, in which 5 dierent pro®les were detected (Fig. 1, lanes A, C, I, L and M)
with 5 dierent plasmids, according to their molecular weights, which ranged between 90 and 700 MDa. The strains isolated most frequently from the lower solution of soil 2 were those bearing only pSym (Fig. 1, lane A), followed by those also bearing a cryptic plasmid of 140 MDa (Fig. 1, lane C), two cryptic plasmids of 140 and 114 MDa (Fig. 1, lane B), one cryptic plasmid of 100 MDa (Fig. 1, lane I) or two cryptic plasmids of 700 and 90 MDa (pro®le M). The remaining pro®les were represented by a single strain (Fig. 1, lanes D and L). The strains isolated most frequently in the higher dilution of soil 2 were those bearing a plasmid of 140 MDa (Fig. 1, lane C), followed by those bearing only the pSym (Fig. 1, lane A), those bearing a cryptic plasmid of 500 MDa (Fig. 1, lane L) and those bearing two of 90 and 700 MDa (Fig. 1, lane M). A single strain bore a cryptic plasmid of 100 MDa (Fig. 1, lane I). In the strains isolated from soil 3, with the longest period of alfalfa cultivation, 7 dierent plasmid pro®les were detected (Fig. 1, lanes A, B, C, D, E, F and G), with 5 dierent cryptic plasmids, on the basis of their molecular weights, which ranged between 20 and 300 MDa. All the plasmid pro®les were found in strains isolated from the lower dilution of soil 3, no new pro®les appearing in the higher dilution, in which 2 dierent pro®les were found (Fig. 1, lanes A and G) with a cryptic plasmid of 30 MDa. The strains most frequently isolated from the lower dilution of soil 3 were those bearing only the pSym
a
01, 07 08, 11, 14 15 16 17 18,
Parada de Arriba (3) (PMSA)
Plasmid pro®les are presented in Fig. 1.
19 and 20
09, 10 12, 13
02, 03, 04, 05, 06
27, 28, 29, 30
21, 22, 23, 24, 25, 26
26 27, 28 29, 30
17
20
23, 24, 25
13 11, 14
01, 09 08 12, 10, 15 16, 18 19,
Florida de LieÂbana (2) (FMSA)
29, 30
28
21, 22, 23, 24, 25 26, 27
21, 22
01 and 02 03, 04, 06, 08, 09 05, 07 10, 12, 13 11 14, 15, 16, 17 18 19 20
Naharros Nuevo (1) (NMSA)
Strains isolated from higher dilution of soil
02, 03, 04, 05, 06, 07
Strains isolated from lower dilution of soil
Soil
Table 2 Characteristics of the strains of S. meliloti isolated in this study
A B B C C D E F G
A B B C C D I L M
A B B C C D H J K
Plasmid pro®lea
and 30
and 50
and 114 and 114
140 and 114 140 and 114 140 140 114 300 300 and 20 30
140 and 114 140 and 114 140 140 114 100 500 700 and 90
140 140 140 140 114 200 90 700
Molecular weight of cryptic plasmids (MDA)
ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ
ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ
ÿ ÿ + ÿ + ÿ ÿ ÿ ÿ
nfe
E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047 1043
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E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047
plasmid (Fig. 1, lane A), followed by those also bearing two cryptic plasmids of 140 and 114 MDa (Fig. 1, lane B), one cryptic plasmid of 140 MDa (Fig. 1, lane C), one cryptic plasmid of 114 MDa (Fig. 1, lane D) or one cryptic plasmid of 30 MDa (Fig. 1, lane G). The remaining pro®les were represented by a single strain (Fig. 1, lane F). The strains isolated most frequently from the higher soil dilution bore only the pSym plasmid (Fig. 1, lane A), followed by those bearing a cryptic plasmid of 30 MDa (Fig. 1, lane G). 3.2. PCR ampli®cation with primers derived from GR4 nfe genes The results are shown in Table 1. The nfe zone was never detected in the strains isolated in the higher dilution of the three soils. By contrast, in the lower dilution of soil 1 it was present in 15% of the strains; in soil 2 in 20% of the strains and in soil 3 in 20% of the strains. Considering the total number of strains isolated from each soil (30), in soil 1 the nfe zone was present in 10% of the strains and in soils 2 and 3 in 13% of the strains. In the three soils studied together, the nfe zone was displayed by strains bearing a cryptic plasmid of 140 MDa (Table 2, pro®le C) (38% of strains bearing this cryptic plasmid displayed the nfe zone) or two cryptic plasmids, one of 140 MDa and the other of 114 MDa (Table 2, pro®le B) (46% of strains bearing these cryptic plasmids displayed the nfe zone). 3.3. Eectiveness studies All the strains isolated were infective and eective (data not shown) and 9 strains were chosen for the study of symbiotic characteristics. The selection of strains was also made taking into account the following characteristics: adaptation to the soil (strains PMSA 30, NMSA 29 and FMSA 29 are well adapted
to each of the three soils since they were isolated from the higher dilution of each soil); plasmid pro®les (strains PMSA25, NMSA10, PMSA11, FMSA11, PMSA10 and FMSA09, whose plasmid pro®le was found very frequently in the three soils Ð pro®les A, B and C Ð were selected) and presence of the nfe zone (for example, strains PMSA11 and FMSA11 show the same plasmid pro®le, nevertheless the strain FMSA11 contains the nfe zone which is absent from strain PMSA11). The results of the eectiveness assays are shown in Table 3. No signi®cant dierences were observed between strain PMSA25 (which only bore the symbiotic plasmid) and strain FMSA29 in dry plant weight and total nitrogen contents. By contrast, signi®cant dierences were observed between strain PMSA25 and other strains tested (Table 3). Strains NMSA10, PMSA11, FMSA11, PMSA10, FMSA09, PMSA30 and NMSA29 ®xed signi®cantly more nitrogen than PMSA25 (see Table 3). It was also seen that the strain FMSA11 formed a signi®cantly higher number of nodules in alfalfa compared to strains PMSA25, FMSA29, NMSA10, PMSA11, FMSA09, PMSA30 and NMSA29. By contrast, signi®cant dierences were not found between the FMSA11 and the PMSA10 strain and both strains display the nfe zone.
4. Discussion The results for the strains isolated from the soils we studied point to a high genetic diversity according to the number and molecular weights of the plasmids detected. In each soil, seven plasmid pro®les were found, with a number of cryptic plasmids ranging from 0 to 2 (Fig. 1) and, overall, 13 dierent plasmid pro®les were observed. Eleven dierent cryptic plasmids were detected on the basis of the dierences in
Table 3 Some symbiotic characteristics of S. meliloti strains isolated from soils of Salamanca (Spain) Inoculant strain Nodules per plant Shoot height (cm) Shoot dry weight (mg) Shoot total nitrogen (mg) Cryptic plasmids (MDa) nfe zone PMSA 25 NMSA 10 PMSA 11 FMSA 11 PMSA 10 FMSA 09 PMSA 30 NMSA 29 FMSA 29 a
16abc 22def 18bc 25fg 23defg 15ab 18bc 21def 15ab
27bcde 25abcd 27bcde 30ef 26bcd 30ef 29def 30ef 24ab
83ab 120defg 119defg 131efg 102c 122defg 120defg 120defg 85ab
3a 5bc 5bc 5bc 4bc 5bc 4bc 4bc 3a
± 140 140 140 140 140 30 200 700
and 114 and 114 and 20 and 90
ÿ ÿ ÿ + + ÿ ÿ ÿ ÿ
Values are the means of ®ve jars. In each column, values followed by the same letter are not signi®cantly dierent according to Fisher's protected LSD (P > 0.05).
E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047
their molecular weights, which ranged between 20 and 700 MDa (Table 1). These ®ndings are consistent with those reported by Broughton et al. (1987b), Scho®eld et al. (1987) and Rao et al. (1994) who described strong variability in plasmid pro®les in soils cultivated with alfalfa, due to the transfer of genetic material among microorganisms in the plant rhizosphere. The number and diversity of the cryptic plasmids of the strains isolated decreased as the soil dilution increased, so in the highest soil dilutions we only found the most abundant strains. Also, whereas in the lower soil dilution there were up to seven dierent plasmid pro®les, in the higher dilution the maximum number of plasmid pro®les observed was ®ve. Attempts have been made to relate the presence of cryptic plasmids with adaptation by rhizobia to the soil conditions (Thurman et al., 1985). From our results it may be inferred that, overall, the most abundant strains Ð and hence those best adapted to the soil Ð isolated from the three soils studied lacked cryptic plasmids (Fig. 1, lane A). However, in soil 3 there were strains bearing a cryptic plasmid of 30 MDa that were well adapted to the soil and likewise in soil 2, there were strains having a plasmid of 40 MDa. This suggests a certain involvement of these plasmids in adaptation to the soil. In this study we estimated the competitiveness and abundance of S. meliloti strains present in a soil under natural conditions. In order to do so, we compared strains isolated from alfalfa inoculated with the lower soil dilution (1:10) and those isolated from alfalfa plants inoculated with the higher dilution of the same soil in which nodule production was detected. In pots inoculated with the lower soil dilution, the number and diversity of rhizobia were high and for a strain to be successful in nodulation it must compete with many other strains of rhizobia that coexist with it in the soil. Accordingly, strains with the nfe zone, which confers greater competitiveness (Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995), were isolated in relatively high proportions. By contrast, in pots inoculated with the higher soil dilution the number and diversity of rhizobia were lower; only the most abundant rhizobia were found because they hardly had to compete to be successful in nodulation. At this dilution, no strains showing the nfe zone were isolated, indicating that the strains bearing this zone were not abundant in the soils studied. Comparison of the strains isolated from both soil dilutions suggests the following: the strains isolated very frequently from nodules of pots inoculated with the higher soil dilution were very abundant (the strains bearing the pSym plasmid in the three soils studied and strains bearing a cryptic plasmid of 30 MDa in the soil 3); the less abundant strains which were isolated very frequently from nodules formed in pots
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inoculated with the lower soil dilution were competitive strains (strains that displayed the B or C plasmid pro®les in the three soils studied) and the strains bearing the nfe zone, which confers greater competitiveness, were never isolated from nodules formed in pots inoculated with the higher soil dilution, implying that they were not abundant (strains that displayed the B or C plasmid pro®les in the three soils studied). Therefore we observed that the strains having a cryptic plasmid of 140 MDa (Fig. 1, lane C) or two plasmids of 140 and 114 MDa (Fig. 1, lane B) were very competitive in the three soils studied. This is in agreement with the results of Brom®eld et al. (1985), Pankhurst et al. (1986), Toro and Olivares (1986), SanjuaÂn and Olivares (1989) and Pardo et al. (1994) who related competitiveness to the presence of certain cryptic plasmids. A zone (nfe) present in a 140 MDa cryptic plasmid (pRmeGR4b) directly involved in the competitiveness of the S. meliloti GR4 strain has been described (Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995). In our study, the nfe zone was only detected in highly competitive strains having the B or C plasmid pro®les in the three soils studied (see Table 2). Our results show that the proportion of strains in which the nfe zone was detected was lower than that of strains in which the presence of the pRmeGR4b type plasmid was detected. These results were consistent with those reported by Villadas et al. (1995). From the study of the symbiotic characteristics assayed it may be deduced that strains PMSA10 (soil 3) and FMSA11 (soil 2) bearing the pSym and a cryptic plasmid of 140 MDa (Fig. 1, lane C) and the pSym and two cryptic plasmids of 140 MDa and 114 MDa (Fig. 1, lane B), respectively, are very eective as can be seen by the amount of nitrogen ®xed per plant and show signi®cant dierences with strain PMSA25 bearing only the pSym plasmid (Fig. 1, lane A). As reported above, these strains were also observed to have high competitiveness and their cryptic plasmids display the nfe zone, whose in¯uence in this symbiotic characteristic has been demonstrated (Toro and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995). This suggests that cryptic plasmids were not only involved in competitiveness but also in the eectiveness of S. meliloti strains (VelaÂzquez et al., 1995). In view of the results, strain FMSA 11, with a plasmid pro®le that contains pSym and a 140 MDa plasmid in which the nfe zone was detected, has good symbiotic characteristics as far as both infectivity (number of nodules formed) and eectiveness (plant dry weight and nitrogen ®xed) are concerned. Also, the presence of the nfe zone is important since it has been shown to be involved in competitiveness (Toro
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and Olivares, 1986; SanjuaÂn and Olivares, 1989; Villadas et al., 1995). It may thus be concluded that in soil with S. meliloti populations there are autochthonous strains with high competitiveness, infectivity and eectiveness that have advantages over others coexisting with them; hence, they may be very suitable as inoculants. Moreover, this suggests that the nfe zone can be used as a criterion for the selection of strains for this purpose. These `selected local strains' must show advantages in competitiveness, nodulation eectiveness and nitrogen ®xation compare to others isolated in allochthonous ecosystems and their use as inoculants will lead to an increase in the production of the host legume which is, of great interest from the economic point of view.
Acknowledgements This work was supported by the Junta de Castilla y LeoÂn and the DGICYT (DireccioÂn General de InvestigacioÂn Cient|Â ®ca y TeÂcnica). The authors thank N. Skinner and Imelda Geldart for revising the English version of the manuscript.
References Barbour, W.M., Elkan, G.H., 1989. Relationship of the presence and copy number of plasmids to exopolysaccharide production and symbiotic eectiveness in Rhizobium fredii USDA 206. Applied and Environmental Microbiology 55, 813±818. Beringer, J.E., 1974. R factor transfer in Rhizobium leguminosarum. Journal of General Microbiology 84, 188±198. Brockwell, J., 1982. Plant-infection counts of rhizobia in soils. In: Vincent, J.M. (Ed.), Nitrogen Fixation in Legumes. Academic Press, New York, pp. 41±57. Brom®eld, E.S.P., Lewis, D.M., Barran, L.R., 1985. Cryptic plasmid and rifampin resistance in Rhizobium meliloti in¯uencing nodulation competitiveness. Journal of Bacteriology 164, 410±413. Brom®eld, E.S.P., Sinha, I.B., Wolynetz, M.S., 1986. In¯uence of location, host cultivar and inoculation on the composition of naturalized populations of R. meliloti in Medicago sativa nodules. Applied and Environmental Microbiology 51, 1077±1084. Brom®eld, E.S.P., Thurman, N.P., Whitwill, S.T., Barran, L.R., 1987. Plasmids and symbiotic eectiveness of representative phage types from two indigenous populations of Rhizobium meliloti. Journal of General Microbiology 133, 3457±3466. Broughton, W.J., Heycke, N., Priefer, U., Schneider, G.M., Stanley, J., 1987a. Ecological genetics of Rhizobium meliloti: diversity and competitive dominance. FEMS Microbiology Letters 40, 245±249. Broughton, W.J., Samrey, U., Stanley, J., 1987b. Ecological genetics of Rhizobium meliloti: symbiotic plasmid transfer in the Medicago sativa rhizosphere. FEMS Microbiology Letters 40, 251±255. Cregan, P.B., Keyser, H.H., Sadowsky, M.J., 1989. Host plants eects on nodulation and competitivenes of the Bradyrhizobium japonicum serotype strains constituting serocluster 123. Applied and Environmental Microbiology 55, 2532±2536. Demezas, D.H., Bottomley, P.J., 1986a. Autoecology in rhizospheres and nodulating behavior of indigenous Rhizobium trifolii. Applied and Environmental Microbiology 52, 1014±1019.
Demezas, D.H., Bottomley, P.J., 1986b. Interstrain competition between representatives of indigenous serotypes of Rhizobium trifolii. Applied and Environmental Microbiology 52, 1020±1025. Eckhardt, T.H., 1978. A rapid method for the identi®cation of plasmid deoxyribonucleic acid in bacteria. Plasmid 1, 584±588. Hartmann, A., Giraud, J.J., Catroux, G., 1998. Genotypic diversity of Sinorhizobium (formely Rhizobium) meliloti strains isolated directly from a soil and from nodules of alfalfa (Medicago sativa) grown in the same soil. FEMS Microbiology Ecology 25, 107±116. Hossain, A.K.M., Alexander, M., 1984. Enhancing soybean rhizosphere colonization by Rhizobium japonicum. Applied and Environmental Microbiology 48, 468±472. Hynes, M.F., MacGregor, N.F., 1990. Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-®xing nodules by Rhizobium leguminosarum. Molecular Microbiology 4, 567±574. Jenkins, M.B., Bottomley, P.J., 1985. Evidence for a strain of Rhizobium meliloti dominating the nodules of alfalfa. Soil Science Society of America Journal 49, 326±328. Johnson, F.J., 1990. Detection method of nitrogen (total) in fertilizers. In: Elrich, K. (Ed.), Ocial Methods of Analysis of the Association of Ocial Analytical Chemists. Association of Ocial Analytical Chemists. Arlington, VA, pp. 17±19. Kuykendall, L.D., Abdel-Wahab, S.M., Hashem, F.M., Van Berkum, P., 1994. Symbiotic competence and genetic diversity of Rhizobium strains used as inoculants for alfalfa and berseem clover. Letters on Applied Microbiology 19, 477±482. Mercado-Blanco, J., Olivares, J., 1993. Stability and transmissibility of the cryptic plasmids of Rhizobium meliloti GR4: their possible use in the construction of cloning vectors for rhizobia. Archives of Microbiology 160, 477±485. Mercado-Blanco, J., Toro, N., 1996. Plasmids in rhizobia: the role of nonsymbiotic plasmids. Molecular Plant Microbe Interaction 9, 535±545. Pankhurst, C.E., MacDonald, P.E., Reeves, J.M., 1986. Enhanced nitrogen ®xation and competitiveness for nodulation of Lotus pedunculatus by a plasmid-cured derivative of Rhizobium loti. Journal of General Microbiology 132, 2321±2328. Pardo, M.A., LaguÂnez, J., Miranda, J., Mart|Â nez, E., 1994. Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase. Molecular Microbiology 11, 315± 321. Rao, J.R., Fenton, M., Jarvis, B.D.W., 1994. Symbiotic plasmid transfer in Rhizobium leguminosarum biovar trifolii and competition between the inoculant strain Icmp2163 and transconjugant soil bacteria. Soil Biology & Biochemistry 26, 339±351. Rigaud, J., Puppo, A., 1975. Indole-3-acetic catabolism by soybean bacteroids. Journal of General Microbiology 88, 223±228. SanjuaÂn, J., Olivares, J., 1989. Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that aect nodulation eciency. Journal of Bacteriology 171, 4154±4161. Scho®eld, P.R., Gibson, A.H., Dudman, W.F., Watson, J.M., 1987. Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population. Applied and Environmental Microbiology 53, 2942±2947. Soil Conservation Service, 1972. In: Soil Survey laboratory methods and procedures for collecting soil samples. USDA, Washington. Soil Survey Sta, 1994. In: Keys to soil taxonomy, 6th ed. Soil Conservation Service, USDA, Washington. Soto, M.J., Zorzano, A., Mercado-Blanco, J., Lepek, V., Olivares, J., Toro, N., 1993. Nucleotide sequence and characterization of Rhizobium meliloti nodulation competitiveness genes nfe. Journal of Molecular Biology 229, 570±576. Thurman, N.P., Lewis, D.M., Gareth Jones, D., 1985. The relationships of plasmid number to growth, acid tolerance and symbiotic eciency in isolates of Rhizobium trifolii. Journal of Applied Bacteriology 58, 1±6.
E. VelaÂzquez et al. / Soil Biology and Biochemistry 31 (1999) 1039±1047 Toro, N., Olivares, J., 1986. Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation. Molecular General Genetics 202, 331±335. VelaÂzquez, E., Mateos, P.F., Pedrero, P., Dazzo, F.B., Mart|Â nezMolina, E., 1995. Attenuation of symbiotic eectiveness by Rhizobium meliloti SAF22 related to the presence of a cryptic plasmid. Applied and Environmental Microbiology 61, 2033±2036. Villadas, P.J., VelaÂzquez, E., Mart|Â nez-Molina, E., Toro, N., 1995. Identi®cation of nodule-dominant Rhizobium meliloti strains carrying pRmeGR4-type plasmid within indigenous soil populations by
1047
PCR using primers derived from speci®c DNA sequences. FEMS Microbiology Ecology 17, 161±168. Vincent, J.M., 1970. The cultivation, isolation and maintenance of rhizobia. In: Vincent, J.M. (Ed.), A Manual for the Practical Study of Root-Nodule Bacteria. Blackwell Scienti®c Publications, Oxford, pp. 1±13. Weaver, R.W., Wei, G.R., Berryhill, D.L., 1990. Stability of plasmids in Rhizobium phaseoli during culture. Soil Biology & Biochemistry 22, 465±469.