The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil Biology & Biochemistry 38 (2006) 153–163 www.elsevier.com/locate/soilbio

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie J. Kevin Vessey*,1, George N. Chemining’wa2 Department of Plant Science, University of Manitoba, Winnipeg, Canada R3T 2N2 Received 6 October 2004; received in revised form 7 April 2005; accepted 18 April 2005

Abstract Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) R0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of R0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains. q 2005 Elsevier Ltd. All rights reserved. Keywords: Pisum sativum; Rhizobium leguminosarum bv. viciae; Genetic diversity; Inoculation; Microbial ecology; Plasmid profiling; PCR-RFLP

1. Introduction Optimization of the symbiosis between legume crop plants and their respective rhizobial microsymbionts requires the presence in the rhizosphere of competitive,

* Corresponding author. Tel.: C1 902 420 5089; fax: C1 902 496 8772. E-mail address: [email protected] (J. Kevin Vessey). 1 Present address: Faculty of Graduate Studies and Research, Saint Mary’s University, 923 Robie Street, Halifax, NS, Canada B3H 3C3. 2 Present address: Department of Crop Science, University of Nairobi, P.O. Box 30197, Nairobi, Kenya.

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.04.036

infective strains of compatible rhizobia which are highly efficient at fixing N2 and are present in sufficient numbers to maximize nodulation. In agricultural situations, the rhizobial microsymbionts can come from two sources: (1) from commercial inoculant normally placed on, or with the seed at the time of seeding and (2) from resident populations of rhizobia existing in the field soil. Likewise, the long-term resident population of rhizobia may originate from two sources: (1) from compatible rhizobial strains which inoculate wild, uncultivated legumes that are native to the area and (2) from commercial inoculant strains used in previous cropping events in the field or area. The symbiotic efficiency of resident populations can be important to legume production in cases where commercial inoculant is not used, or where the resident population interacts in either

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an additive or competitive fashion with the strain(s) of rhizobia in the applied commercial inoculant. In a companion study (Chemining’wa and Vessey, 2005), it was shown that long-term resident populations containing infective strains of Rhizobium leguminosarum bv. viciae (Rlv) are common in soils of the eastern Canadian prairie in fields which had previously grown a legume crop (pea, lentil or faba bean) treated with a commercial rhizobial inoculant. For the most part, these resident populations contained relatively effective strains, with a only a few sites showing a growth response by P. sativum to inoculation with a commercial rhizobial inoculant. This previous study raised the questions of whether the strains in the resident populations originated from previously used commercial inoculants, and if so, how genetically similar or diverse are they compared to these originating strains. Because symbiosis-related genes of Rlv primarily exist on plasmids (Mazurier and Laguerre, 1997), a study of the genetic diversity of this bacterium must consider genetic variability within both plasmid(s) and the chromosome. Plasmid profiling is a common means of strain identification of Rlv and analysis of restriction fragment length polymorphisms (RFLP) of PCR-amplified sequences from internally transcribed spacer region (ITS) between the 16S and 23S rDNA sequences is useful in characterizing the diversity of the chromosome in this bacterium (Nour et al., 1994; Laguerre et al., 1994, 1996). In this study, these two analysis methods are used to assess the diversity of Rlv isolated from nodules of uninoculated pea plants from 20 sites across southern Manitoba in the eastern region of the Canadian prairie. In addition, the competitiveness of

a commercial inoculant is assessed by using plasmid profiling to identify Rlv isolated from nodules located on the root close to a source of commercial inoculant (proximal nodules) or from nodules located further away from the inoculation point (distal nodules).

2. Materials and methods 2.1. Field sites Twenty field sites (Table 1) were selected across a broad geographical region of southern Manitoba as part of a larger study on the abundance, efficacy, and genetic diversity of Rlv populations in the soils of the eastern Canadian prairie (Chemining’wa and Vessey, 2005). Sites were located within farmers’ fields, on agricultural research stations run by the University of Manitoba, or in uncultivated areas (Table 1). Details on physiochemical characteristics of the soils, cropping histories, and plot establishment are available in Chemining’wa and Vessey (2005). At 8 weeks after planting, root cores (6.5 cm diameter, 15 cm depth) positioned at the center of four randomly chosen plants pea were taken from the four inner rows (one pea plant per row). Root cores were kept cool and transported to the laboratory where six nodules were randomly selected from each root core (for a total of 24 nodules per site, except at Westbourne #2 and Stuartburn where due to limited nodulation only six and two nodules, respectively, were sampled). A total of seven nodules were also selected from

Table 1 Information on the 20 sites in southern Manitoba from which Rlv were isolated from uninoculated pea plants in 1998 Site

Site code

Type of site

Latitude and longitude

Years since last inoculation

Commercial source of last inoculanta

Arborg #1 Arborg #2 Carman #1 Carman #2 Carman #3 Carman #4 Glenlea #1 Glenlea #2 Letellier Morden #1 Morden #2 Morris #1 Morris #2 Souris #1 Souris #2 Stuartburn Teulon Westbourne #1 Westbourne #2 Winnipeg

Ag1 Ag2 Cn1 Cn2 Cn3 Cn4 Ga1 Ga2 Lr Mn1 Mn2 Ms1 Ms2 Ss1 Ss2 Sn Tn Wn1 Wn2 Wg

Farmer’s field Farmer’s field Research station Research station Research station Research station Research station Research station Farmer’s field Farmer’s field Farmer’s field Farmer’s field Farmer’s field Farmer’s field Farmer’s field Uncultivated Farmer’s field Farmer’s field Uncultivated Research station

50890 0 N 97822 0 W 50890 0 N 97822 0 W 49829 0 N 98800 0 W 49829 0 N 98800 0 W 49829 0 N 98800 0 W 49829 0 N 98800 0 W 49864 0 N 97812 0 W 49864 0 N 97812 0 W 49808 0 N 97818 0 W 49811 0 N 98806 0 W 49811 0 N 98806 0 W 49835 0 N 97837 0 W 49835 0 N 97837 0 W 49837 0 N 100815 0 W 49837 0 N 100815 0 W 49813 0 N 96877 0 W 50838 0 N 97827 0 W 50807 0 N 98834 0 W 50807 0 N 98834 0 W 49847 0 N 97808 0 W

O10 3 2 3 1 O10 5 1 3 3 2 3 4 2 3 NA O10 4 NA O10

Unknown Unknown Company #3 Company #3 Company #3 Company #3 Company #3 Company #3 Unknown Company #3 Company #3 Company #3 Company #3 Company #1 Company #1 NA Unknown Company #3 NA Unknown

NA, Not applicable; no record of an inoculated crop grown at the site. a Commercial source is identified by numbers only for proprietary reasons.

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root cores of wild Lathyrus sp. growing at the field edges near the Glenlea, Morden, and Teulon plots. 2.2. Isolation of Rlv strains from nodules Rlv was isolated from nodules from uninoculated ‘trap plants’ from each site. For the nodule occupancy experiment (see below), Rlv was also isolated from plants inoculated with the commercial strain PBC108 at the Morden #2 site only. Although considerable diversity can exits within this genotype of rhizobia isolated from pea nodules (Mutch and Young, 2004), pea is not a promiscuous nodulator like common bean (Martinez-Romero, 2003), which can be nodulated by multiple species of rhizobia. Therefore, one can be relatively confident that rhizobia isolated from pea nodules are strains of Rlv. Isolation of Rlv strains from nodules was carried out according to the protocols by Beattie and Handelsman (1989) and Rice and Olsen (1993) with a modifications. Sampled nodules were stored from 3 to 10 weeks in 20% glycerol at K17 8C before rhizobia were isolated. After crushing the surface-sterilized nodules with the apparatus described by Beattie and Handelsman (1989), the nodule suspensions were spotted onto yeast mannitol agar (YMA; Rice and Olsen, 1993) plates and incubated at 28 8C for at least 2 days. Rhizobia growing on each spot were streaked onto YMA and incubated at 28 8C for 2–3 days. Single colonies were then picked and transferred to new YMA plates. It was assumed that each single colony represented a single strain and that each nodule contained a single strain, or if more than one strain existed per nodule, only the most abundant strain was sampled. It was noted that the recovery of viable rhizobia from nodules was high (above 80%), even after the nodules had been stored for more than 6 months. In contrast, the recovery of viable rhizobial strains from wild Lathyrus sp. plants was relatively low and these nodules were generally small and white in colour. 2.3. Plasmid profiling Plasmid profiling was used as a component of rhizobial strain identification and to assess diversity among strains. Plasmid profiles of 230 strains were examined. The strains included: 207 isolates from field pea root nodules; seven native Lathyrus sp. isolates (four isolates from Teulon, two from Morden, and one from Glenlea); and 16 strains used in commercial Rlv inoculants (Table 2) in the eastern Canadian prairie over approximately the previous 20 years (generously provided by each of the inoculant companies). Plasmid profiles of isolates were examined using a modified Eckhardt (1978) rapid visualization technique as described by Hynes et al. (1986) with modifications. Rlv isolates were grown for at least 3 days on the HP agar medium (2-fold strength and 3.0% agar) at 28 8C. A sample from this culture was then grown for 18 h on a liquid version of

155

Table 2 Commercial Rlv inoculant strains, their plasmid profile identifier, and the sites where the profile was identified in bacteria isolated from nodules of uninoculated pea plants Commercial source

Strain identifier

Plasmid profilea

Site(s) where isolates had a similar profile

Company #1

C1b RGP2b RGP4b RGL4b RGAA1 RGFPb RP212-2 RP212-13 RP212-19b RP212-37b RP213-5b PEA082b 128C56Gb 175G10B 99AA1b PBC108b

5–1 4–3 4–2 4–4 3–1 5–2 4–6 5–3 4–8 4–5 5–1 4–7 5–1 4–9 4–6 4–1

None Cn1, Mn2, Ss1 None None None None Ga2, Mn2 None Mn2, Wn1 Ms1, Ms2 None None None None Ga2, Mn2 Ms1, Ms2, Ss1, Wn1

Company #2

Company #3 Company #4 Company #5 a b

Correspond to profile identifiers in Table 3. Selected for PCR-RFLP of 16S-23S rDNA ITS.

the same medium at 28 8C. The sarkosyl (at 0.6%)/culture mixture was allowed to stand on ice for 15 min prior to centrifugation. Likewise, the lysis of the subsequent centrifugation pellet was carried out on ice for 20 min. The electrophoresis was carried out on 1% agarose (containing 10% SDS) and at 82 V for 4.5 h. Rhizobium etli CE3 (generously provided by Dr David Romero, University of Mexico) with five well-characterized plasmids (630, 510, 390, 270, and 175 kb) was included in each gel as the reference for determining the approximate molecular weights of plasmids of test strains. Bands were imaged using a Fluor-S Multi-Imager (Bio-Rad Laboraotires, Hercules, CA, USA) and Eagle Eye (Stratagene, La Jolla, CA, USA) and visual observations were used to determine plasmid band sizes. Isolates were placed in different plasmid profile groups, based on the number and size of plasmid bands. Isolates that were identical in number and size of plasmid bands were considered to belong to the same plasmid profile group. Plasmid profile groups were assigned an identifier (nKn), with first number indicating the number of plasmids in the profile and the second number being sequentially assigned to indicate a different pattern within isolates with the same number of plasmids. For example, plasmid profile group ‘3–6’ was the sixth identified pattern among isolates with three plasmids in their profile. Diversity in plasmid profiles within each site was expressed in terms of plasmid profile diversity index (PPDI) which we defined as the number of distinct plasmid profiles divided by the total number of isolates typed.

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2.4. PCR-RFLP of 16S-23S rDNA ITS Restriction fragment length polymorphisms (RFLP) of PCR-amplified sequences from internally transcribed spacer (ITS) region between the 16S and 23S rDNA sequences is very useful in strain identification and diversity characterization in Rlv (Nour et al., 1994; Laguerre et al., 1994, 1996). Twenty-eight Rhizobium isolates/strains [12 commercial Rlv strains (Table 2), 15 field Rlv isolates, and one reference R. leguminosarum bv. phaseoli strain (H441, from France)] were selected for PCR-RFLP analysis of the 16S-23S rDNA ITS to relate plasmid profiles to chromosomal backgrounds and to evaluate the diversity of isolates at the chromosomal level. The selection was carried out to include: (1) strains from the same site with identical or different plasmid profiles; (2) strains from different geographical distances exhibiting identical or different plasmid profiles; (3) a field strain with the same plasmid profile as an inoculant strain; (4) commercial strains from different inoculant companies with the same or different plasmid profiles (Table 2). Genomic DNA was isolated from the aforementioned strains following the method described by Laguerre et al. (1992) with minor modifications (i.e. the Tris–HCl was at pH 8.0 and the final DNA pellet was washed twice in ethyl alcohol before drying and dissolving in TE buffer. Purity and concentration of DNA was determined using UVspectrophotometry. All enzymes were purchased from Sigma–Aldrich, St Louis, MO, USA. Oligonucleotides primers used to amplify the ITS sequences between 16S and 23S rDNA genes were the forward primer, 16SR11 (5 0 -GCCCGGCTACTTGCAGAGATGGAAGGTTCCC-3 0 ), and the reverse primer, FGPL132 0 (Laguerre et al., 1996). These oligonucleotides were synthesized by Canadian Life Technologies, Burlington, ON, CDA. DNA amplification was carried out as described by Laguerre et al. (1996) with minor modifications. PCR reactions were performed in a final volume of 150 mL containing approximately 0.5 mg template genomic DNA. All reagents were purchased from Canadian Life Technologies, Burlington, ON, CDA. The amplified DNA fragments were analysed by horizontal gel electrophoresis in 1% agarose gels (containing 0.5 mg LK1 ethidium bromide) in TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0). Electrophoresis was carried out at 90 V for 1 h using 6 cm by 8 cm standard gels. Ethidium bromide (0.5 mg LK1) was added to the electrophoresis buffer before each run. The gels were imaged using the Fluor-S MultiImager. The sizes of the PCR products were estimated by the Eagle-Eye program based on a molecular weight standard (Norgen Biotek, St Catharines, ON, CDA). Seven restriction endonucleases, AluI, CfoI, DdeI, HaeIII, MspI, NdeII, and TaqI (Canadian Life Technologies, Burlington, ON, CDA), were used to digest the amplified DNA. Ten microlitre of the PCR products were digested with 12–15 units of enzyme, following the manufacture’s

instructions. The restriction fragments were analyzed by horizontal gel electrophoresis in TBE buffer on 4% NuSieve 3:1 agarose (FMC Corp., Rockland, ME, USA) gels containing 0.5 mg LK1 ethidium bromide. The gels were run at 82 V for 4 h and imaged as described above. Restriction fragment bands smaller than 100 bp were not included in the analysis because they were generally not well resolved on 4% NuSieve 3:1 agarose gels. Dendogram construction and analysis of the restriction patterns were carried out as described in Laguerre et al. (1996). 2.5. Nodule occupancy Isolates were obtained from plants inoculated with PBC108 strain (with no N-fertilizer) at Morden #2 in 2000 (Chemining’wa and Vessey, 2005). Root cores of four plants (one plant per replicate) were taken as described above and eight nodules were picked from each plant close to the base of the stem (proximal nodules) and bagged separately. An additional 2-cm wide cylinder of soil was taken from the periphery of the hole left by the coring procedure and eight nodules picked from this soil sample (distal nodules) and bagged separately. The nodules were placed in Eppendorf tubes, filled with 20% glycerol, thoroughly vortexed, and stored at K17 8C. Rhizobia were later isolated and cultured as described above. Plasmid profiling (as described above) was used to determine the proportion of the proximal (borne on the tap root near the base of the stem) and distal (borne on lateral roots farther from the base of the stem) nodules occupied by the inoculant strain. Inoculant strain PBC108 was run in every gel as the reference strain. Relative plasmid profile diversity between proximal and distal nodule isolates was determined by calculating the PPDI for each group.

3. Results 3.1. Plasmid profiling Plasmid profiling (Fig. 1) was attempted on 24 isolates from each site, except for Stuartburn and Westbourne #2 where due to limited nodulation only two and six isolates, respectively, were attempted, and Morden #2 where 36 isolates were attempted (due to the high success rate of

Fig. 1. Examples of plasmid profiles of Rhizobium strains segregated in a 1% agarose Eckhardt gel. Reference strain, R. leguminosarum bv. phaseoli CE3, with identified plasmid sizes in Lanes 1 and 6, and R. leguminosarum bv. viciae inoculant strains RGAA1, RGP2, RGP4, and C1 in Lanes 2–5, respectively.

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Table 3 The number of isolates for which plasmid profiling was attempted, the number of isolates for which plasmids was successfully profiled, the number of unique profiles per site, the plasmid profile diversity index (PPDI), and plasmid profiles identifiers of Rlv isolates from uninoculated pea plants and wild Lathyrus plants from 20 sites across southern Manitoba Site

Isolates tested

Profiled isolates

Unique profiles

PPDI

Plasmid profiles identifiersa

Ag1 Ag2 Cn1 Cn2 Cn3 Cn4 Ga1 Ga2

24 24 24 24 24 24 24 24

5 3 5 3 3 3 14 22

5 2 3 3 2 2 10 19

1.00 0.67 0.60 1.00 0.67 0.67 0.71 0.86

Lr Mn1 Mn2

24 24 36

4 9 33

3 3 17

0.75 0.33 0.52

Ms1 Ms2

24 24

14 22

11 15

0.79 0.68

Ss1 Ss2 Sn Tn Wn1 Wn2 Wg Lathyrus

24 24 2 24 24 6 24 7

18 10 1 10 22 4 2 7

13 7 1 2 13 1 1 5

0.72 0.70 0.00 0.20 0.59 0.00 0.00 NA

1–3, 2–1, 2–5, 3–3, 3–7 1–5, 1–6 1–3, 3–2, 4–3 1–7, 3–2, 4–18 3–2, 5–9 1–2, 3–2 2–7, 3–2, 3–8, 3–10, 4–11, 4–18, 4–21, 5–13, 5–17, 6–3 1–1, 1–4, 1–10, 2–1, 2–4, 3–4, 3–6, 4–6, 4–11, 4–14, 4–15, 4–16, 4–17, 5–5, 5–7, 5–10, 6–2, 6–3, 7–2 1–5, 2–7, 5–11 1–3, 4–12, 5–5 1-2, 1-4, 1-5, 1-7, 3-2, 3-3, 3-9, 4-3, 4-6, 4-8, 4-10, 4-13, 5-4, 5-5, 5-6, 6-3, 8-1 1–3, 1–5, 3–2, 3–5, 4–1, 4–5, 4–19, 5–5, 5–7, 5–13, 7–1 1–3, 2–4, 2–6, 2–8, 3–4, 3–5, 4–1, 4–5, 4–11, 5–4, 5–5, 5–6, 5–8, 5–13, 6–1 1–2, 4–1, 4–3, 4–11, 4–12, 4–13, 4–14, 5–5, 5–8, 5–10, 5–13, 5–14, 5–15 1–3, 1–8, 2–4, 3–2, 4–11, 4–20, 5–16 1–4 1–4, 7–1 1–3, 1–6, 1–7, 1–8, 1–9, 2–2, 2–3, 3–2, 3–4, 4–1, 4–8, 4–12, 5–12 1–4 3–4 2–9, 5–18, 5–19, 6–4, 7–3

PPDI, no. of individual profiles/no. of profiled isolates, except where only one profile exists per site in which case the diversity index is considered to be 0. NA, Not applicable; Lathyrus was sampled from different sites; a site specific PPDI can not be calculated. a First number indicates the number of plasmids in the profile; the second number was sequentially assigned to indicate a different pattern within isolates with the same number of plasmids.

profiling attempts at this site (Table 3). Successful profiles were not generated from all isolates, with Winnipeg being the least successful site (2 successful profiles out of 24 isolates) and Morden #2 being the most successful site (33 successful profiles out of 36 isolates). It was noticed that isolates which formed colonies with particularly thick mucous were less likely to be successfully profiled. The inability to profile plasmids from some Rlv strains has been reported by others (Zhang et al., 2001a). Strains varied in number of plasmid from one to eight. Inoculant strains had three to five plasmid bands (Table 2), while most field isolates exhibited one to five plasmid bands (Table 3). Single- and four-plasmid band isolates were the most common, comprising 27.8 and 27.4% of all the isolates. The percentage of isolates with two, three, and five bands was 6.1, 14.3, and 19.1, respectively; isolates with six to eight plasmid bands constituted just about 5% of the total isolates. Strains varied in plasmid size from less than 50 kb to more than 1000 kb. In previous studies, isolates from pea, faba bean, and lentil nodules have been shown to vary in plasmid content from about one to nine and in plasmid size from less than 50–950 kb (Laguerre et al., 1992; van Berkum et al., 1995; Handley et al., 1998; Wilson et al., 1998; Zhang et al., 2001a; Lakzian et al., 2002). Out of 230 field isolates and inoculant strains typed, 77 distinct plasmid profiles were established (Tables 2 and 3).

Most of the distinct profiles were shared by at least two isolates, as only 35 strains had unique plasmid profiles. No single profile was dominant across sites. Profiles 1–3 and 1–4 had single bands and were the most common, each constituting 7% (16 out of 230) of all the isolates typed. Profile 3–2 had three bands and was isolated 15 times. Profile 4–11 with four bands was isolated eight times, while profile 5–5 with five plasmid bands was isolated 11 times. Profile 3–2 was found in mores sites (nine sites) than all the profiles, followed by 1–3 and 5–5 which were isolated from seven and six sites, respectively. Some isolates appeared to be dominant at some sites. Nine out of 10 of the isolates typed at Teulon #2 exhibited profile 1–4, while seven out of nine isolates typed at Morden #1 had profile 1–3. It is interesting that the five plasmid profiles isolated by wild Lathyrus plants (Table 3), sampled adjacent to some of the sites, were unique and not isolated from any of the uninoculated pea plants within the test plots. Considerable diversity in plasmid profiles, as measured by PPDI, was demonstrated in most sites (Table 3). For the 19 sites which had two or more isolates typed, PPDI was approximately 0.6. It is interesting that isolates obtained from pea plants at Teulon and Westbourne #2, sites where pea or lentil was not grown in over 10 yr, registered low PPDIs. Pearson correlation coefficients, relating PPDI to soil pH, soil organic matter, soil electrical conductivity, soil

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nitrate-N, and soil P content, resulted in non-significant correlations (data not shown). A small, but significant, negative correlation (rZK0.57, PZ0.05) was demonstrated between PPDI and number of years since previous pea or lentil cultivation (data not shown) However, this correlation was non-significant when Westbourne #2, which had not been grown to pea or lentil in the previous 25 yr, was excluded in the analysis. 3.2. PCR-RFLP analysis of 16-23S rDNA ITS Single PCR bands were generated for all the strains tested (data not shown), with the exception of one (the inoculant strain RGL4). Estimates showed that the length of the 16S23S rDNA ITS regions of the strains ranged from 1700 to 2000 bp, confirming that the ITS for rhizobia is much longer than 400 bp which is the average ITS length for most other bacteria (Navarro et al., 1992). The PCR products generated in the current study are slightly larger than the 1160–1400 bp 16S-23S ITS products generated in Laguerre et al. (1996), but can be explained by the use of a different forward primer in the current study which corresponds to an oligonucleotide closer to the 3 0 end of the 16S rDNA. The number of distinct RFLP patterns produced by the seven individual restriction endonucleases ranged from 14 to 23 (Fig. 2; Table 4). Alu1 was the least discriminating endonuclease, while DdeI was the most discriminating. Enzymes HaeIII, NdeII, MspI, Cfo1, and TaqI each produced 16–17 different profiles. Two to eight bands per restriction pattern were obtained with each restriction enzyme. A total of 148 polymorphic bands were scored for all the enzymes, resulting in a 28!148 matrix. The range in band numbers per endonuclease varied from 24 for TaqI to 17

Table 4 Restriction patterns of 12 commercial Rlv inoculant strains, 15 isolates from nodules of uninoculated pea plants collected from 10 sites across southern Manitoba, and one R. leguminosarum bv. phaseoli strain (H441) of the amplified 16S-23S rDNA intergenic space obtained with seven restriction enzymes Strain/ isolatea

Restriction patterns AluI

CfoI

DdeI

HaeIII

MspI

NdeII

TaqI

RP212-19 PEA082 Ms1-B RGP4 RGFP 99AA1 128C56G C1 RGP2 Cn1-A Ms1-A Ms1-C Ss1 Cn1-B PBC108 Ga1-A Ga1-B Tn Mn2 Wg-A Wg-B Wn1 Ss2 RP213-5 RGL4 H441 RP212-37 Ms2 No. of patterns Bands per profile

A1 A2 A2 A2 A3 A4 A4 A4 A4 A4 A4 A4 A4 A5 A6 A7 A8 A9 A10 A10 A10 A6 A10 A7 A11 A12 A13 A14 14

C1 C2 C2 C2 C3 C4 C4 C4 C5 C6 C5 C5 C5 C7 C8 C9 C10 C11 C12 C12 C12 C13 C14 C9 C15 C16 C17 C13 17

D1 D2 D3 D3 D4 D5 D6 D7 D8 D5 D9 D10 D5 D11 D12 D13 D14 D15 D16 D17 D17 D18 D19 D13 D20 D21 D22 D23 23

H1 H2 H2 H2 H3 H4 H4 H4 H4 H4 H4 H4 H4 H5 H6 H7 H8 H9 H10 H10 H10 H6 H11 H12 H13 H14 H15 H16 16

M1 M2 M2 M2 M3 M4 M4 M4 M5 M5 M5 M5 M5 M6 M7 M8 M9 M10 M11 M11 M11 M12 M13 M8 M14 M15 M16 M17 17

N1 N2 N3 N2 N4 N1 N1 N1 N1 N1 N1 N1 N1 N5 N6 N3 N7 N8 N9 N9 N9 N10 N11 N12 N13 N14 N15 N16 16

T1 T2 T3 T3 T4 T5 T5 T5 T5 T5 T5 T5 T5 T6 T7 T8 T9 T10 T11 T11 T11 T12 T13 T13 T14 T15 T16 T17 17

5–8

2–6

4–6

3–6

3–7

4–8

3–6

a

Strains isolated from pea nodules from the field are designated by the site code from Table 1 and are followed by a capital A, B or C if the site was represented by two or more isolates; the rest (in italics) are commercial isolates of R. leguminosarum bv. viciae, except for H441 which is a reference strain of R. leguminosarum bv. phaseoli.

Fig. 2. Examples of restriction patterns of PCR-amplified 16S-23S rDNA ITS regions of R. leguminosarum bv. viciae inoculant strains (RP212-19, FGFP, PEA082, 99AA1, PBC108) digested with AluI, HaeIII, and MspI in a 4% agarose gel.

from CfoI (data not shown). The 28!148 matrix was used to generate a SMC matrix (28!28). SMCs ranged from 0.58 to 1 (data not shown). The SMC matrix was used to generate a phenogram (Fig. 3), depicting similarity in the rDNA ITS regions of Rhizobium strains, using UPGMA. Clustering of the SMCs using UPGMA placed the isolates in different groupings. The different clusters mirrored the similarity in restriction patterns among the isolates demonstrated in Table 4. The tree had three major groupings that had SMCs of 0.73 or greater: the first grouping had had 12 strains (from RP212-19 to PEA082 in Fig. 3); the second had eight strains (from Cn1-B to Ss); and the third had four strains (Wn1, PBC108, Ms2, and RGL4). Two inoculant strains (RGFP and RP212-37), plus the isolate from Teulon 1, and, as expected, R. leguminosarum bv. phaseoli strain (H441) were categorized in their own

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159

RP212-19 99AA1 128C56G

C1 Ms1-A

Ss1 Ms1-C RGP2 Cn1-A Ms1-B RGP4 PEA082 Cn1-B Ga1-A RP213 -5 Ga1-B Mn2 Wg-A Wg-B Ss2

Group 1

Group 2

Tn Wn1 PBC108 Ms2 RGL4 RGFP H441 RP212-37 0.64

0.73

0.82

0.91

Group 3

1.00

Simple matching coefficient Fig. 3. Phenogram depicting genetic similarities among 15 field isolates and 12 commercial strains of R. leguminosarum bv. viciae, and one R. leguminosarum bv. phaseoli strain (H441) based upon UPGMA analysis of PCR-RFLP patterns of the 16S-23S rDNA ITS regions.

groups, each with SMCs of 0.70 or less relative to all other strains/isolates. 3.3. Nodule occupancy Plasmid profiles of 64 Rlv isolates obtained from nodules occupying proximal and distal root regions of four inoculated pea plants grown at Morden 2 in 2000 were examined. Nine different plasmid profiles that occurred

more than once were identified among these 64 isolates (including the profile of PBC108, the inoculant strain) (Table 5). The profiles of 22% of the proximal isolates (seven out of 32 isolates) were identical to that of the inoculant strain, while only 6% of the distal isolates (2 out of 32 isolates) had the same profile as the inoculant strain. There was no single profile that dominated pea nodules. The most common profile (M7) was found in 18.8% of all the 64 isolates, representing 34.4% of all the isolates obtained from

Table 5 Distribution of plasmid profiles that were identified more than once among isolates from proximal and distal root regions of pea plants inoculated with Rlv strain PBC108 at Morden 2 site in 2000 Profile

Nodule source

Plant 1

Plant 2

Plant 3

Plant 4

Total

% of isolates

M1 M1 M2 M2 M7 M7 M10 M10 M12 M12 M13 M13 M14 M14 M15 M15 PBC108 PBC108

Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal

0 0 0 2 1 2 1 0 0 0 0 0 0 3 0 0 3 0

0 1 0 3 0 1 0 0 0 3 0 1 0 1 1 0 1 0

0 2 0 1 0 4 3 0 0 0 1 0 1 0 1 0 2 2

0 0 0 1 0 4 0 0 0 0 0 2 0 0 3 0 1 0

0 3 0 7 1 11 4 0 0 3 1 3 1 4 5 0 7 2

0 9.4 0 21.9 3.1 34.4 12.5 0 0 9.4 3.1 9.4 3.1 12.5 15.6 0 21.9 6.3

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distal root nodules. Isolates obtained from proximal and distal root regions of pea plants appeared to have different profile types. For example, profiles M1 and M2 were isolated three and seven times, respectively, from distal root nodules but none were isolated from proximal root nodules. In contrast, profile M10 was absent in distal isolates but it was isolated four times from proximal isolates. Some profiles appeared to be associated more with some plants than with others. For example, profile M12 was isolated three times from Plant 2 but none in the other plants, while profile PBC108 was isolated three times from Plant 1, once in Plant 2, four times from Plant 3 and once in Plant 4. The diversity amongst plasmid profiles of isolates from within the proximal and distal nodule sources was characterized by calculating PPDI for each of these groups. Isolates from proximal nodules had a higher PPDI than that for distal nodules. The PPDI for proximal isolates was 0.53, approximately double that of isolates from distal nodules (PPDIZ0.25).

4. Discussion 4.1. Genetic diversity among field isolates—plasmid profiling Plasmid profiling of Rlv isolates from nodules collected across southern Manitoba indicate a relatively high degree of variability. Of the 214 field isolates analyzed, 67 different plasmid profiles were identified. These results are in agreement with some studies demonstrating that rhizobial isolates originating from pea, lentil, and faba bean are diverse in terms of their plasmid composition. Kucey and Hynes (1989) observed a total of 10 plasmid profile types out of 24 pea isolates from the western Canadian prairie, while van Berkum et al. (1995) found 13 distinct plasmid profiles out of 22 isolates from V. faba, but from disparate geographical locations around the world. In contrast, Zhang et al. (2001a,b) identified only 41 different plasmid profiles among 360 V. hirsutu and pea isolates from restored browncoal mining fields in Germany, and Brockman and Bezdicek (1989) found only 18 profiles among 192 isolates originating from pea root nodules harvested from eastern Washington state. The diversity of Rlv strains varied within and between sites and was considerably high in the majority of the sites. Among sites with 10 or more isolates, the PPDIs ranged from 0.2 at Teulon 1 to 0.83 at both Glenlea sites. The PPDIs for all the arable sites, with the exception of Teulon 1, were above 0.5. Variations in diversity among and within sites have been reported previously for nodule isolates from pea and other legumes. On the basis of plasmid profile and RAPD-PCR analyses, Handley et al. (1998) characterized numerous isolates of R. leguminosarum from multiple sites in Britain and observed large variations in diversity between the sites. The diversity index (number of RAPD profiles/total number

of isolates typed) for the sites varied from less than 0.1 to above 0.65. High diversity within a region however is not universal. Zhang et al. (2001a) found 50% of the 41 different profiles they identified among Rlv 360 isolates belonged to just two profiles. High genetic diversity appears to indicate the occurrence of greater disturbance, distinct microsites or unique environmental niches to which different rhizobial strains are adapted (Handley et al., 1998; Palmer and Young, 2000). No correlations could be identified between PPDI of sites and soil characteristics (pH, OM, EC, NO3–N, and P), however, the weak negative correlation between PPDI and number of years since previous pea or lentil cultivation may indicate that the occurrence of a compatible legume host helps to increase a sites rhizobial diversity. Kinkle and Schmidt (1991) found that plasmid transfer frequencies among Rlv strains were higher in the presence of the pea rhizosphere or by additions of plant material, than is soil absent of the plants. Each site had plasmid profiles common with at least one other site, but the number of plasmid profiles common to any two sites was not dependent on the proximity between them. For example, Souris #1 and Souris #2 (1 km apart) had only one plasmid profile in common, while Glenlea and Souris #1, which were about 250 km apart, shared five plasmid profiles. Similarly, in Alberta in the western Canadian prairie, Rlv strains with identical plasmid profiles were obtained from pea fields located more than 40 km apart (Kucey and Hynes, 1989; Hynes and O’Connell, 1990). Isolates of native Lathyrus sp. plants from Teulon, Morden and Glenlea sites did not have any plasmid profiles in common with field pea isolates, including those from adjacent experimental sites. 4.2. Genetic diversity among field isolates—PCR-RFLP Analysis by PCR-RFLP of the 16S-23S rDNA ITS regions of 15 selected isolates from 10 of the 20 sites across southern Manitoba, indicated a relatively high degree of genetic variability. There were only two cases of clusters among field isolates with SMCs of 0.95 or higher (Fig. 3). Those were a cluster including Ms1-A, Ss1, and Ms1-C within SMC Group 1 and a second including Mn2, Wg-A and Wg-B in SMC Group 2. In contrast, to group all field isolates together, SMC had to be reduced to 0.70. Given that the relatively genetically distant R. leguminosarum bv. phaseoli reference strain H441 had a SMC of 0.66 relative to all the field isolates as a group, the 0.70 SMC shared by all the field isolates as a group, indicates a relatively high degree of genetic variability among the field isolates. As was observed with the plasmid profiles, PCR-RFLP analysis indicated that the similarity of 16S-23S ITS regions was not dependent on the proximity of the sites from which they were obtained. There was only one case of identical PCR-RFLP patterns among isolates (Wg-A and Wg-B), and these did come from the same site (Winnipeg). Likewise, two of the three isolates from the Morris #1 site clustered

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together. However, isolate Ss1 from the Souris #1 site also clustered with these isolates from Morris #1 (approximately 220 km between sites), while isolate Ss2, harvested just 1 km away, had a low SMC relative to isolate Ss1. Isolates Cn1-A and Cn1-B, both from the Carman #1 site, also did not have a high SMC. As was seen with the diversity of plasmid profiles in the current study, and with other studies utilizing PCR-RFLP techniques, high genetic diversity among Rlv isolates is not unexpected in arable, cultivated soils (Mutch and Young, 2004; Palmer and Young, 2000). When a strong stress or selection pressure is applied to an area, such as heavy metal loading (Lakzian et al., 2002), the level of diversity among Rlv genotypes can decline. 4.3. Relationships between plasmid profiles and PCR-RFLP patterns among field isolates The only two isolates with identical PCR-RFLP patterns (Wg-A and Wg-B) also had identical plasmid profiles (profile 3–4), indicating that they were the same strain. However, isolate Mn2 which, based on PCR-RFLP, clustered closely to the Wg-A/B strain (SMCZ0.99) had a different plasmid profile. Isolates Ms1-A and Ms1-B, both from the Morris #1 site, clustered together according to PCR-RFLP patterns (SMCZ0.95) but had distinct plasmid profiles (data not shown). Such differences in plasmid profiles amongst isolates with similar chromosomal PCRRFLP patterns is not unexpected because, although plasmid profiles in rhizobia can be considered as a comparatively stable character, strains can lose or have partial deletion of a plasmid (Weaver et al., 1990), have recombination or rearrangement between plasmids (Zhang et al., 2001b), or plasmid exchange with other bacteria (Louvrier et al., 1996). Observations of Rlv field isolates with similar chromosomal genetic composition, but varying plasmid profiles have been made in previous studies (Laguerre et al., 1993). The rates of conjugation between rhizobial genotypes appears to be higher in arable, cultivated soils than in natural, undisturbed sites (Wernegreen et al., 1997). Isolates Ms1-B and Cn1-B were selected for PCR-RFLP analysis because, despite the fact that they were harvested from locations approximately 50 km apart (Morris and Carman), they shared the same plasmid profile (profile 3–2). Nonetheless, their PCR-RFLP patterns were highly dissimilar. Previous reports have indicated that Rlv strains with identical plasmid profiles tended to be very close chromosomally (Handley et al., 1998; Laguerre et al., 1993). It should be kept in mind that similarity in plasmid profiles does not mean similarity in genetic makeup of those plasmids. Plasmid profiles are defined simply by the number and size of plasmids. Assessment of the genetic similarity of plasmids requires genetic analysis of the plasmids themselves, such as PCR-RFLP techniques based upon nod genes on the symbiosis plasmid (pSym) (e.g. Laguerre et al., 1996). Such techniques were not applied in the current study.

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4.4. Relationships between field isolates and inoculant strains Despite the fact that inoculant from Company #3 was the most widely and recently used inoculant in fields at the test sites (Table 1), the plasmid profiles of the two strains used by this company were not isolated from any of the nodules in the study. In fact, across the whole study, there was only one occurrence where the plasmid profile isolated from nodules from a site corresponded to a plasmid profile of an inoculant strain that may have been recently used on that site (profile 4–3, corresponding to inoculant strain RGP2, used 2 years previous at Souris #1). Given that the use of rhizobial inoculants has the potential to add plasmids into a soil’s rhizobial population (Geniaux and Amarger, 1993), the infrequency of plasmid profiles of inoculant strains appearing in field isolates suggests that complete plasmid complements of the recently used inoculant strains either were not widely incorporated, or did not remain conserved, in the resident Rlv populations in the soil. Likewise, Kucey and Hynes (1989) noted that the plasmid profiles of R. leguminosarum bv. phaseoli isolated from bean nodules collected in the western Canadian prairie region were distinct from those of the inoculant strains used 3 years earlier in the same fields. The lack of similarity in plasmid profiles of nodules isolates with previously used inoculant strains is supportive of the concept of a high frequency of plasmid exchange in Rlv (Hynes and O’Connell, 1990; Kinkle and Schmidt, 1991; Mutch and Young, 2004). Despite the lack of similarity in plasmid profiles between inoculant strains and nodule isolates, there is evidence to support an influence of chromosomal material from inoculant strains becoming established in the resident Rlv populations. Firstly, it needs to be recognized that the inoculant strains C1, 128C56G, and 99AA1 were very similar in terms of ITS PCR-RFLP patterns (SMC R0.98). Strains C1 and 128C56G also had the same plasmid profile (profile 5–1), suggesting that the two genotypes are essentially the same, or were recently derived from the same genotype. This cluster of inoculant strains had a relatively high degree of similarity to field isolates from Morris #1, Souris #1, and Carman #1 (SMC R0.92) suggesting that their chromosomes originated from these inoculant strains. Isolate Ms1-B also had a high degree of similarity to inoculant strain RGP4 (SMCZ0.93). However, similarity in PCR-RFLP of ITS regions between field isolates and inoculant strains was not universal. For example, isolates Cn1-B and Tn showed relatively little similarity in ITS PCR-RFLP patterns to any inoculant strains (SMC %0.74). The remaining field isolates show intermediate levels of similarity with one or more inoculant strains (SMCs ranging from 0.75 to 0.88). Overall these results suggest an influence, but not a predominance, of chromosomal genetic material from inoculant strains on the populations of resident Rlv strains found across southern Manitoba. Such influences of current- or formerly used

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commercial inoculants were not observed in the genotype of Rlv isolates ‘trapped’ by pea or faba bean from soils of South Australia based upon analysis of RAPD-PCR banding patterns (Ballard et al., 2004). 4.5. Relationships between isolates from proximal and distal nodules from the same plants The proportion of the nodules occupied by the inoculant strain, PBC108, at Morden 2 was 6% (2 of 32 isolates) for distal root nodules compared to 22% (7 of 32 isolates) for proximal root nodules. Thus, the inoculant strain was more successful in nodulating the proximal root regions than the distal root regions. This finding supports other studies that have demonstrated that inoculant rhizobia applied to the seed produce nodules primarily on the upper portion of the tap root, with little nodulation on the more distal portion of the taproot and lateral roots (McDermott and Graham, 1989; Rice et al., 2000). The failure to achieve effective dispersion of bacteria in the bulk soil with seed inoculation may be attributed to the limited mobility of rhizobia in the soil relative to the rate of root exploration of the soil (Streeter, 1994). The movement of rhizobia in soil varies with water movement, soil texture, soil slope, and rhizobial motility (Issa et al., 1993; Parco et al., 1994), but it is generally poor in most soil conditions, especially in the lateral direction (Date, 1991). Aside from the competitiveness of the resident Rlv population for nodulation of pea plants, the profiling of isolates from within the same pea plants also indicated a greater PPDI in proximal nodules versus distal nodules. The reason for the increased diversity in proximal nodules is not clear, but may simply be indicative of greater disturbance and mixing of soil when the seed was planted, and hence a greater variety of microniches in the area leading to promotion of growth of a wider diversity of genotypes (Postma et al., 1989; Palmer and Young, 2000). Alternatively, the greater diversity nearer the site of inoculant/ seed placement may indicate that that seed inoculation stimulated growth and activity of the resident Rlv population, possibly through increased flavonoid signals from the host or higher nod factor signals in the immediate area.

5. Conclusion The present study indicates that indigenous populations of Rlv in southern Manitoba soils are genetically diverse. It must also be recognized that only a subset of strains from the total soil population of Rlv strains at these sites were sampled in this study. All isolates analyzed were infective in P. sativum and were ‘trapped’ using the same cultivar of pea at all sites. Given that not all rhizobia in a soil will be infective (Bromfield et al., 1995), and host genotype can affect the spectrum of rhizobial genotypes selected from a soil (Handley et al., 1998; Laguerre et al., 2003;

Mutch and Young, 2004), it should be kept in mind that genetic diversity of the complete Rlv populations at the test sites is no doubt greater than that identified in this study. A companion study (Chemining’wa and Vessey, 2005) assessed the symbiotic efficiency of the resident Rlv populations analyzed in the current study. They found infective Rlv populations at all cultivated sites, and that these resident populations were generally quite symbiotically efficient. Hence, despite a relative high degree of genetic variability in the resident infective Rlv populations across southern Manitoba, effective strains are quite prevalent. However, the evidence for non-infective Rlv strains at non-cultivated sites (Stuartburn and Westbourne #2) (Chemining’wa and Vessey, 2005) and for Rlv strains infecting wild Lathyrus plants having plasmid profiles distinct from isolates from pea nodules, suggest that infective/ effective strains of Rlv are not natural to this region of the eastern Canadian prairie. These findings, in combination with the PCR-RFLP evidence of similarities between inoculant strains and some endemic strains, strongly suggest that the infective strains of the resident Rlv populations are naturalized from previously used inoculant strains.

Acknowledgements This work was carried out with financial support from the Canadian Commonwealth Fellowship Plan, the Manitoban Agricultural Research and Development Initiative, the Manitoba Pulse Growers Association, and Philom Bios Inc., J.K. Vessey research program is largely supported by NSERC Canada. The authors are indebted to Mr B. Luit for his technical assistance, the many undergraduate assistants who aided in the project, and especially the farmer cooperators who allowed us to establish research plots on their land.

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