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Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations
Applied Soil Ecology 35 (2007) 441–448 www.elsevier.com/locate/apsoil
Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations Matthew D. Denton a,b,c,*, Christopher R. Hill a, William D. Bellotti a, David R. Coventry a b
a The University of Adelaide, Roseworthy Campus, SA 5371, Australia School of Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia c Primary Industries Research Victoria (PIRVic), Department of Primary Industries, Rutherglen Centre, RMB 1145 Chiltern Valley Road, Rutherglen Victoria 3685, Australia
Received 17 August 2005; received in revised form 19 February 2006; accepted 7 April 2006
Abstract The size of the background rhizobial population can often determine the success of field nodulation and persistence of inoculant rhizobia. Field experiments were conducted to determine the nodulation response of annual medics (Medicago spp.) in a pasturewheat-pasture rotation when grown in soils of contrasting pH and rhizobial populations. Medicago truncatula Gaertn. and M. polymorpha L. were inoculated with one of three different strains of Sinorhizobium medicae (WSM540, WSM688) or S. meliloti (NA39) or left uninoculated and sown in two fields of pH (CaCl2) 5.9 and 7.2 of differing soil rhizobial backgrounds (11 and 7.1 104 cells/g soil, respectively). Nodulation was assessed in years 1 and 3 of the rotation. At the site with a small rhizobial background, M. polymorpha nodulated poorly when inoculated with the acid-sensitive strain NA39 but nodulated well when inoculated with acid-tolerant strains WSM688 and WSM540. M. truncatula had a similar extent of nodulation with each of the rhizobial inoculants. At the site with a large rhizobial background all treatments had greater than 85% of plants nodulated. Nodule occupancies, assessed by PCR, provided further insight: at the site with a small rhizobial background both medic species successfully nodulated with the acid-tolerant strains WSM540 and WSM688 and these strains persisted to year 3. However, at the site with large rhizobial background, only one strain, WSM688, was identified from M. truncatula nodules in year 3. This study highlights the importance of edaphic constraints and plant–rhizobia interactions to the successful development of nodulation in a field environment. # 2006 Published by Elsevier B.V. Keywords: Legumes; Medicago truncatula; Medicago polymorpha; Nodule occupancy; Polymerase chain reaction (PCR); Rhizobia; Sinorhizobium meliloti
1. Introduction
* Corresponding author. Tel.: +61 2 6030 4559; fax: +61 2 6030 4600. E-mail address: [email protected] (M.D. Denton). 0929-1393/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.apsoil.2006.08.001
Annual medic (Medicago spp.) pastures contribute substantially to the nitrogen (N) economy of cereal cropping systems in low rainfall (<400 mm) environments of southern Australia (Peoples and Baldock, 2001). In addition to N contributions, the medic component
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enhances soil carbon accumulation and provides opportunities for animal-based enterprises and weed management (Reeves and Ewing, 1993). In southern Australia, annual medics occur on 20 million ha of mainly neutral to alkaline soils (Hill and Donald, 1998). In long-term crop/medic rotations, the pasture legume component typically regenerates during the pasture phase from seed banks in the soil (Ewing, 1999). To maximise N2 fixation, it is essential that elite rhizobia, introduced as legume pasture inoculants, are able to survive in the absence of the legume host (Brockwell et al., 1991). Rhizobia may survive in nodule fragments, by colonising the rhizospheres of non-hosts, or as freeliving components of the soil microflora. Survival of the rhizobia in these circumstances must be sufficient to act as an effective source of inoculation of the regenerating legume (Howieson, 1995). Medicago species vary in their specificity for appropriate rhizobia, Sinorhizobium meliloti and S. medicae. While Medicago truncatula nodulates and fixes N2 with a relatively wide range of rhizobial genotypes (Brockwell and Hely, 1966; Brockwell, 2001), M. polymorpha is more specific (Brockwell and Hely, 1966; Ballard and Charman, 2000; Bena et al., 2005). Sinorhizobium spp. typically have a strong preference for neutral to alkaline soils. Survival, persistence, and nodulation of medic rhizobia are all restricted under acid conditions (Howieson et al., 1991; Watkins et al., 2003). Poor adaptation to acid soils leads to small populations of medic rhizobia in low pH soils (Brockwell et al., 1991) which can be further reduced by tillage (Coventry and Hirth, 1992). Below approximately pH 6.5 rhizobial populations are adversely affected (Brockwell, 2001). The selection of rhizobia from acid soils has resulted in strains with increased acid tolerance (Dilworth et al., 2001). Strain WSM688, for example, was isolated from a Sardinian soil of pH 4.2 (Howieson et al., 1991). The size of the background rhizobial population also influences the success of nodulation and persistence of the inoculant rhizobia (Dowling and Broughton, 1986; Howieson and Ballard, 2004). Survival of inoculant rhizobia in a small background population (<100 cells/ g soil) will depend primarily upon rhizobial tolerance of the edaphic environment. Conversely, survival of rhizobia introduced into soil with a large background population will depend upon the relative abilities of background and introduced strains to compete for the formation of nodules (Vlassak and Vanderleyden, 1997; Denton et al., 2003). Since rhizobial persistence is crucial to the successful establishment, N2 fixation and continuing productivity of
annual medic pastures, the aim of this study was to track the success of inoculant rhizobia in forming nodules on annual medics over three seasons. The experiments comprised pasture-wheat-pasture rotations at two field sites with contrasting rhizobial population sizes. We investigated the extent to which (i) the legume species and (ii) the size of the background rhizobial population affected the success of inoculants in the pasture phases. M. truncatula and M. polymorpha were inoculated with either an acid-sensitive (NA39) or acid-tolerant rhizobia (WSM688, WSM540) and nodule occupancy of the medics was assessed in years 1 and 3 of the experiment. 2. Materials and methods 2.1. Site characteristics and management Two sites with red-brown earths (chromosols) were chosen in the Upper-North region of South Australia, near Pekina (32846.593S, 138833.728E) and Black Rock (32849.323S, 138842.789E). The Pekina site had a clay soil that contained 39 mg/kg (Colwell) P, 0.65% organic C, 332 mg/kg K. The Black Rock had a clay soil that contained 27 mg/kg P, 1.15% organic C, 771 mg/kg K prior to sowing. The previous crop was field peas at Pekina and pasture at Black Rock. The sites were sown with annual Medicago pastures in the first year of the experiment. The experimental design consisted of two cultivars: Medicago truncatula Gaertn. cv. Caliph and M. polymorpha L. cv. Santiago and four inoculation treatments: inoculation with either S. meliloti strain NA39, S. medicae WSM540 or S. medicae WSM688 or no inoculation. Each of these strains has been used in Australian commercial inoculants at one time or another. NA39 is intolerant of acid soils; strains WSM540 and WSM688 are tolerant of soils with a pH of 4.7 (Howieson et al., 1991). The rhizobia were applied as peat inoculants and seed was lime-pelleted with Plastaid1 in order to enhance rhizobial survival. Following preparation, the pelleted seed was sown at 20 kg/ha by broadcasting and covered by light raking. Triple super phosphate was applied at 20 kg/ha of P prior to sowing. The plot design was a randomised complete block design with 5 m 1.5 m plots separated by 1.1 m spaces from other plots and blocks separated by 5 m. The sites were sprayed as necessary to control insect and weed pests. In the second year wheat (Triticum aestivum cv. Barunga), was sown at 75 kg/ha with 20 kg P/ha as triple super phosphate. In the third year pastures were allowed to regenerate from the soil seed bank. Annual rainfall at Pekina was 455, 348 and
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456 mm for years 1, 2 and 3 and April–October growing season rainfall was 326, 272 and 194 mm. At Black Rock, the annual rainfall was 375, 277 and 343 mm with April–October rainfall 271, 227, 155 mm over the 3 years of the experiment. 2.2. Soil and plant sampling Soil cores were taken at three locations across the field sites at depths of 0–10, 10–20 and 20–50 cm to determine pH (1:5 in H2O and CaCl2; Rayment and Higginson, 1992). Estimations of most probable numbers (MPN) of Sinorhizobium spp. in the soil were made at 0–10 cm depth, using a plant infection method (Brockwell, 1963) with Medicago sativa L. as the test species. At Pekina the soil pH (CaCl2) was slightly acidic but increased with soil depth while the Black Rock soil had a neutral pH (Table 1). Pekina had a small rhizobial population while Black Rock had a large soil surface population (Table 1). In year 1 medic plants with entire root systems were harvested at 15 weeks (early spring), to ensure nodulation had occurred and that nodules were still fresh enough to isolate rhizobia effectively. The percentage of plants nodulated was assessed on 100 plants (20 samples/plot) and nodule occupancy was assessed in 20 nodules from each medic strain treatment using nodules selected from all five replicate plots. In year 3, plants with entire root systems were excavated in early spring. Rhizobia were isolated from nodules (Vincent, 1970) sampled from field plots in years 1 and 3 of the experiments. Isolates were obtained from 20 nodules from each medic strain treatment using nodules from all five replicate plots. Insufficient rhizobia were successfully isolated from M. polymorpha nodulated with strain NA39 in year 1 of the experiment for PCR analysis. One nodule per plant was taken from the top 5 cm of root that was on, or close to,
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the taproot as this nodule usually forms early and is thought to provide a large proportion of nitrogen fixation (Corbin et al., 1977). In addition, some plants had just a single very large nodule on the excavated root system. Nodules were surface sterilised in 95% ethanol for 30 s; then 3% sodium hypochlorite for 1.5–3 min (depending upon nodule size) and washed eight times in sterile water. Nodules were then crushed, streaked on YEM (Vincent, 1970) and incubated at 28 8C for 4 days. To obtain a pure colony, a single isolate from each nodule was re-streaked and used for subsequent PCR analysis. A PCR amplification method (Richardson et al., 1995) using the RP05 primer, was chosen for the identification of rhizobia. A number of nodule isolates were confirmed to be rhizobia by re-inoculating M. sativa under aseptic conditions (Vincent, 1970). 2.3. Statistical analyses Statistical analyses (ANOVA) were performed using Genstat 7.1. Where significant differences were observed in ANOVA, means were tested using a Tukey test. 3. Results 3.1. Nodulation The medics responded differently at each field site (Fig. 1). At the site with a small rhizobial population both medic species had poor nodulation when sown uninoculated (Fig. 1a). The nodules that did form on inoculated plants at this site were typically large. Only half or less of the M. truncatula plants at the site with the small rhizobial background had nodules (Fig. 1a). In contrast, M. polymorpha nodulated well with WSM540 or WSM688 and poorly with the acid-sensitive strain NA39. At the site with a large rhizobial background
Table 1 Soil pH (n = 3) and Sinorhizobium meliloti population sizes (derived from most probable number estimates) at the field sites prior to sowing medic species Site
Soil depth (cm)
Soil pHa (H2O)
Soil pH (CaCl2)
Rhizobial population (rhizobia/g soil)b
Pekina
0–10 10–20 20–50
6.4 0.14 a 6.6 0.06 a 7.7 0.07 b
5.9 0.09 a 6.3 0.17 b 7.2 0.05 c
11
Black Rock
0–10 10–20 20–50
7.7 0.03 a 7.8 0.02 a 8.1 0.09 b
7.2 0.02 a 7.4 0.08 b 7.4 0.06 b
a b
Values within a column group that are followed by different letters are significantly different at P < 0.05 (Tukey). Rhizobial population size estimated using the most probable number technique (Brockwell, 1963).
7.1 104
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Fig. 1. The percentage of M. truncatula and M. polymorpha nodulated at sites with a low (a) and high (b) background soil rhizobial population in year 1 of the experiments. The frequency of nodulation (nodulated vs. non-nodulated plants) was assessed using 20 plants per replicate (100 plants/ treatment). Columns that do not share the same letter are significantly different (P 0.05).
more than 85% of plants were nodulated regardless of inoculation (Fig. 1b). Across all treatments, M. truncatula had significantly (P < 0.05) greater nodulation than M. polymorpha. 3.2. Nodule occupancy Rhizobial isolates were identified from nodules harvested in years 1 and 3 of the field experiments using PCR with the random primer RP05 (Fig. 2). Banding patterns of some rhizobial isolates clearly matched the banding patterns of the commercial inoculants (lanes 3, 4, 6 and 10–15 in Fig. 2) and were considered to be inoculant strains. Other isolates produced PCR banding patterns that were clearly dissimilar to the commercial strains (lanes 1, 2, 5, 7–9; Fig. 2). All nodule isolates that were re-inoculated onto M. sativa did form nodules, which indicated that banding profiles were from rhizobia. Inoculant strains were not recovered from plots that were not inoculated, indicating that cross contamination of strains was not a factor. Successful nodule occupancy, based on PCR profiling, was determined for all inoculant strains in M. truncatula nodules in year 1 at the low rhizobia site and for M. polymorpha inoculated with WSM540 and
Fig. 2. An example of PCR profiles used to identify rhizobial isolates from the two field sites, amplified using the RP05 primer. Nodule isolates were collected from M. truncatula inoculated with strain WSM688 (lanes 1–9; isolates collected in year 3 of the trial) and from M. polymorpha inoculated with strain WSM540 (lanes 10–15; isolates were collected in year 1 of the trial). Amplification profiles for commercial strains Sinorhizobium spp. strains WSM688 and WSM540 are indicated in lanes on the left of the gel. Matching amplification profiles for inoculant strain WSM688 are shown in lanes 3, 4 and 6; matching amplifications for strain WSM540 are shown in lanes 10–15. SPP1 bacteriophage digested with EcoRI was used as the molecular weight marker.
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Fig. 3. Recovery of inoculant rhizobia from nodules sampled in the field at sites with a small (a and b) and large (c and d) background rhizobial population. Nodule occupancies of M. truncatula and M. polymorpha are shown for years 1 (a and c) and 3 (b and d) of the experiment. Insufficient nodules prohibited a determination for M. polymorpha nodulated with strain NA39 in year 1 at the site with a small rhizobial population.
WSM688 (Fig. 3a and b). In year 3 of the trial, nodule occupancy was only confirmed in M. truncatula and M. polymorpha for the two acid-tolerant strains, WSM540 and WSM688 (Fig. 3b). At the site with a high rhizobial background, nodule occupancies of inoculant rhizobia in year 1 of the trial were typically very low than for the site with a low background rhizobial population (Fig. 3c and d). By year 3 of the trial, M. truncatula inoculated with WSM688 was the only treatment in which nodule occupancy was confirmed (Fig. 3d). 4. Discussion This study demonstrates the importance of understanding both nodulation and nodule occupancy to understand the outcome of competition for nodulation in a field environment. Inoculation success was demonstrated to be highly dependent upon host–strain interactions in field sites with contrasting soil rhizobial populations. 4.1. Plant nodulation The medics at the two locations differed substantially in their nodulation response at the two sites (Fig. 1). The differences in rhizobial populations at these sites is likely to be due to edaphic and cultural attributes influencing the presence or absence of annual
medics, in turn affecting the establishment of rhizobia populations (Denton et al., 2000). When acid-tolerant rhizobia (WSM688, WSM540) were used to inoculate M. polymorpha in a mildly acidic soil, nodulation was increased above that achieved by no inoculation or inoculation with an acid-sensitive strain (NA39). This indicates the value of using strains that are well matched to edaphic conditions. Uninoculated controls produced very few nodulated plants, reinforcing the estimate of a small rhizobial population at the time of sowing the experiment. M. polymorpha had variable nodulation with the three rhizobial strains (10–85% of plants) while M. truncatula nodulated to a similar extent with each of the three strains (40–50% of plants) which indicates the likelihood that host–strain interactions influenced nodulation. In a previous study M. polymorpha also had a variable nodulation response when inoculated with strain CC169 (15%) or strain WSM688 (50%) (Howieson et al., 1991). Plants at the site with a low rhizobial background produced very large crown nodules. Plants at the site with high background rhizobia contained many small nodules. At this site each medic was well nodulated regardless of inoculation, indicating that this background population of rhizobia was sufficiently large and diverse to nodulate both hosts. Surprisingly, 100% nodulation was not observed for all plants, even with the large background of rhizobia. While much care was taken in excavating plant roots,
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some nodules may have been lost in the process. M. truncatula showed a small but significantly greater preference to nodulate than did M. polymorpha, indicating slightly greater compatibility with the background rhizobial population. 4.2. Nodule occupancy Nodule occupancies were particularly informative: occupancy of inoculant rhizobia differed markedly at the two field sites and for each host legume. At the site with a small background of rhizobia, assessment of nodule occupancy indicated that both medics nodulated with the acid-tolerant strains WSM540 and WSM688 in the year of inoculation far more successfully than the acid-sensitive strain NA39. The acid tolerant strains persisted through a year with a non-host (wheat) and two periods of hot, dry summers to nodulate both medics in year 3. This result indicates the importance of using an acid-tolerant strain of rhizobia to survive saprophytically in this mildly acidic soil. The particular attributes that allowed these acid-tolerant strains to persist in the soil and nodulate medics in the third year remain to be elucidated. Acid tolerance in rhizobia has been shown to involve maintenance of intracellular pH (O’Hara et al., 1989) which may be maintained by increased concentrations of potassium or phosphorus (Watkins et al., 2003). At the site with a large rhizobial background, establishment of inoculant strains was generally poor in the first year, with a maximum of 20% colonisation of M. truncatula nodules by WSM688. This is not surprising, given the large background of rhizobia (7.1 104 rhizobia/g soil) into which inoculant rhizobia were placed. Large populations of rhizobia can have a significant impact on inoculation and are likely to be of concern when the background strains of rhizobia have a poor capacity to fix N2, when inoculation becomes futile (Denton et al., 2002, 2003). At the site with a large background rhizobial population, inoculation with different strains provided 0–20% nodulation in year 1 and inoculant strains occupied very few nodules in year 3. This compared poorly with the site that had a small rhizobial background in which nodule occupancy typically varied between 50 and 100%. An interesting result was that strain WSM688 occupied 45% of M. truncatula nodules assessed in year 3 of the experiment at the site with a large background rhizobial population. Strain WSM688 occupied nodules of M. truncatula successfully at the site with a small rhizobial population where there was very little competition for nodulation from indigenous
strains. However, in the presence of such a large background population, it is difficult to understand why strain WSM688 was so successful in colonising nodules of M. truncatula. Strain WSM688 had not been previously inoculated at this site and the density of plants and nodules was not higher in this treatment in year 1 of the experiment (data not presented), indicating that there was no initial competitive advantage for strain WSM688. The success of WSM688 in year 3 of the experiment is notable given that only 20% of nodules were colonised with this strain in the first year of the trial. Brockwell et al. (1995) have outlined the difficulty in assuring successful nodulation by inoculant strains under the pressure of competing with large numbers of background rhizobia. The successful nodulation between M. truncatula with WSM688 indicates that the specific interactions between the legume and rhizobia and are likely to have determined the outcome of nodulation. This finding indicates that under certain conditions, particular Medicago–Sinorhizobium combinations favour the nodulation of particular strains of rhizobia, despite the presence of large competing background strains of rhizobia. Host species have recently been observed to express a degree of selectivity in nodulation with inoculants in a field environment (Yates et al., 2005). The present results differ from other inoculation experiments that show that the rhizobial population is the primary factor influencing the outcome of nodulation (Brockwell et al., 1982; Thies et al., 1991). Given that M. polymorpha is considered more selective for rhizobia genotypes than M. truncatula (Brockwell, 2001), M. polymorpha was considered to be a more likely candidate to nodulate with WSM688. However, some naturalised M. polymorpha were observed at the Black Rock field site and are likely to have maintained a population of soil rhizobia wellmatched with M. polymorpha. In this case, efficient nodulation of M. polymorpha with the soil rhizobial population may have restricted nodulation of with inoculant strains. While the reason for this particular result is unknown, it is instructive to note the importance that legume–rhizobia relationships may play in environments with large populations of soil rhizobia. By selecting (or genetically enhancing) legumes and rhizobia to achieve highly specific plant-rhizobial signaling, it may be possible to manipulate the outcomes of inoculation, despite the size of the background rhizobial population into which the legume is sown. It is likely, however, that the genetic structure
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and physiology of the rhizobial population present at a site may be as important to the successful outcomes of inoculation, as is the size of the population. Further studies would be required to understand the mechanisms that allow the successful nodulation outcome for M. truncatula with WSM688 on these soil types. These results highlight the importance of (i) using acid-tolerant strains and (ii) selecting rhizobia that are well matched to the legume genotypes of interest for successful nodulation in a field environment. These data indicate that, if legumes are suitably matched with rhizobia, the possibility exists that effective inoculation can proceed, whether the nodulation constraint is a large soil population of rhizobia or an acid soil. Acknowledgements The authors wish to thank Mr. John Cozens, Black Rock and Mr. Kym Fromm, Pekina for the use of land for these experiments. Dr. Annette Anderson and Mrs. Irnayuli Sitepu are thanked for assisting with running PCR and isolating rhizobia from nodules. Dr. Alan Richardson, CSIRO, Plant Industry, Canberra is thanked for providing the RP05 primer. The input of Dr. Alan H. Gibson early in this project was much appreciated. This work was supported by the Grains Research and Development Corporation. References Ballard, R.A., Charman, N., 2000. Nodulation and growth of pasture legumes with naturalised soil rhizobia. 1. Annual Medicago spp. Aust. J. Exp. Agric. 40, 939–948. Bena, G., Lyet, A., Huguet, T., Olivieri, I., 2005. Medicago–Sinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago. J. Evol. Biol. 18, 1547–1558. Brockwell, J., 1963. Accuracy of a plant-infection technique for counting populations of Rhizobium trifolii. Appl. Microb. 11, 377–383. Brockwell, J., 2001. Sinorhizobium meliloti in Australian soils: population studies of the root-nodule bacteria for species of Medicago in soils of the Eyre Peninsula, South Australia. Aust. J. Exp. Agric. 41, 753–762. Brockwell, J., Bottomley, P.J., Thies, J.E., 1995. Manipulation of rhizobia microflora for improving legume productivity and soil fertility: a critical assessment. Plant Soil 174, 143–180. Brockwell, J., Gault, R.R., Zorin, M., Roberts, M.J., 1982. Effects of environmental variables on the competition between inoculum strains and naturalized populations of Rhizobium trifolii for nodulation of Trifolium subterraneum L. and on rhizobia persistence in the soil. Aust. J. Agric. Res. 33, 803–815. Brockwell, J., Hely, F.W., 1966. Symbiotic characteristics of Rhizobium meliloti: an appraisal of the systematic treatment of nodulation and nitrogen fixation interactions between hosts and rhizobia of diverse origins. Aust. J. Agric. Res. 17, 885–899.
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Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations Matthew D. Denton a,b,c,*, Christopher R. Hill a, William D. Bellotti a, David R. Coventry a b
a The University of Adelaide, Roseworthy Campus, SA 5371, Australia School of Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia c Primary Industries Research Victoria (PIRVic), Department of Primary Industries, Rutherglen Centre, RMB 1145 Chiltern Valley Road, Rutherglen Victoria 3685, Australia
Received 17 August 2005; received in revised form 19 February 2006; accepted 7 April 2006
Abstract The size of the background rhizobial population can often determine the success of field nodulation and persistence of inoculant rhizobia. Field experiments were conducted to determine the nodulation response of annual medics (Medicago spp.) in a pasturewheat-pasture rotation when grown in soils of contrasting pH and rhizobial populations. Medicago truncatula Gaertn. and M. polymorpha L. were inoculated with one of three different strains of Sinorhizobium medicae (WSM540, WSM688) or S. meliloti (NA39) or left uninoculated and sown in two fields of pH (CaCl2) 5.9 and 7.2 of differing soil rhizobial backgrounds (11 and 7.1 104 cells/g soil, respectively). Nodulation was assessed in years 1 and 3 of the rotation. At the site with a small rhizobial background, M. polymorpha nodulated poorly when inoculated with the acid-sensitive strain NA39 but nodulated well when inoculated with acid-tolerant strains WSM688 and WSM540. M. truncatula had a similar extent of nodulation with each of the rhizobial inoculants. At the site with a large rhizobial background all treatments had greater than 85% of plants nodulated. Nodule occupancies, assessed by PCR, provided further insight: at the site with a small rhizobial background both medic species successfully nodulated with the acid-tolerant strains WSM540 and WSM688 and these strains persisted to year 3. However, at the site with large rhizobial background, only one strain, WSM688, was identified from M. truncatula nodules in year 3. This study highlights the importance of edaphic constraints and plant–rhizobia interactions to the successful development of nodulation in a field environment. # 2006 Published by Elsevier B.V. Keywords: Legumes; Medicago truncatula; Medicago polymorpha; Nodule occupancy; Polymerase chain reaction (PCR); Rhizobia; Sinorhizobium meliloti
1. Introduction
* Corresponding author. Tel.: +61 2 6030 4559; fax: +61 2 6030 4600. E-mail address: [email protected] (M.D. Denton). 0929-1393/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.apsoil.2006.08.001
Annual medic (Medicago spp.) pastures contribute substantially to the nitrogen (N) economy of cereal cropping systems in low rainfall (<400 mm) environments of southern Australia (Peoples and Baldock, 2001). In addition to N contributions, the medic component
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enhances soil carbon accumulation and provides opportunities for animal-based enterprises and weed management (Reeves and Ewing, 1993). In southern Australia, annual medics occur on 20 million ha of mainly neutral to alkaline soils (Hill and Donald, 1998). In long-term crop/medic rotations, the pasture legume component typically regenerates during the pasture phase from seed banks in the soil (Ewing, 1999). To maximise N2 fixation, it is essential that elite rhizobia, introduced as legume pasture inoculants, are able to survive in the absence of the legume host (Brockwell et al., 1991). Rhizobia may survive in nodule fragments, by colonising the rhizospheres of non-hosts, or as freeliving components of the soil microflora. Survival of the rhizobia in these circumstances must be sufficient to act as an effective source of inoculation of the regenerating legume (Howieson, 1995). Medicago species vary in their specificity for appropriate rhizobia, Sinorhizobium meliloti and S. medicae. While Medicago truncatula nodulates and fixes N2 with a relatively wide range of rhizobial genotypes (Brockwell and Hely, 1966; Brockwell, 2001), M. polymorpha is more specific (Brockwell and Hely, 1966; Ballard and Charman, 2000; Bena et al., 2005). Sinorhizobium spp. typically have a strong preference for neutral to alkaline soils. Survival, persistence, and nodulation of medic rhizobia are all restricted under acid conditions (Howieson et al., 1991; Watkins et al., 2003). Poor adaptation to acid soils leads to small populations of medic rhizobia in low pH soils (Brockwell et al., 1991) which can be further reduced by tillage (Coventry and Hirth, 1992). Below approximately pH 6.5 rhizobial populations are adversely affected (Brockwell, 2001). The selection of rhizobia from acid soils has resulted in strains with increased acid tolerance (Dilworth et al., 2001). Strain WSM688, for example, was isolated from a Sardinian soil of pH 4.2 (Howieson et al., 1991). The size of the background rhizobial population also influences the success of nodulation and persistence of the inoculant rhizobia (Dowling and Broughton, 1986; Howieson and Ballard, 2004). Survival of inoculant rhizobia in a small background population (<100 cells/ g soil) will depend primarily upon rhizobial tolerance of the edaphic environment. Conversely, survival of rhizobia introduced into soil with a large background population will depend upon the relative abilities of background and introduced strains to compete for the formation of nodules (Vlassak and Vanderleyden, 1997; Denton et al., 2003). Since rhizobial persistence is crucial to the successful establishment, N2 fixation and continuing productivity of
annual medic pastures, the aim of this study was to track the success of inoculant rhizobia in forming nodules on annual medics over three seasons. The experiments comprised pasture-wheat-pasture rotations at two field sites with contrasting rhizobial population sizes. We investigated the extent to which (i) the legume species and (ii) the size of the background rhizobial population affected the success of inoculants in the pasture phases. M. truncatula and M. polymorpha were inoculated with either an acid-sensitive (NA39) or acid-tolerant rhizobia (WSM688, WSM540) and nodule occupancy of the medics was assessed in years 1 and 3 of the experiment. 2. Materials and methods 2.1. Site characteristics and management Two sites with red-brown earths (chromosols) were chosen in the Upper-North region of South Australia, near Pekina (32846.593S, 138833.728E) and Black Rock (32849.323S, 138842.789E). The Pekina site had a clay soil that contained 39 mg/kg (Colwell) P, 0.65% organic C, 332 mg/kg K. The Black Rock had a clay soil that contained 27 mg/kg P, 1.15% organic C, 771 mg/kg K prior to sowing. The previous crop was field peas at Pekina and pasture at Black Rock. The sites were sown with annual Medicago pastures in the first year of the experiment. The experimental design consisted of two cultivars: Medicago truncatula Gaertn. cv. Caliph and M. polymorpha L. cv. Santiago and four inoculation treatments: inoculation with either S. meliloti strain NA39, S. medicae WSM540 or S. medicae WSM688 or no inoculation. Each of these strains has been used in Australian commercial inoculants at one time or another. NA39 is intolerant of acid soils; strains WSM540 and WSM688 are tolerant of soils with a pH of 4.7 (Howieson et al., 1991). The rhizobia were applied as peat inoculants and seed was lime-pelleted with Plastaid1 in order to enhance rhizobial survival. Following preparation, the pelleted seed was sown at 20 kg/ha by broadcasting and covered by light raking. Triple super phosphate was applied at 20 kg/ha of P prior to sowing. The plot design was a randomised complete block design with 5 m 1.5 m plots separated by 1.1 m spaces from other plots and blocks separated by 5 m. The sites were sprayed as necessary to control insect and weed pests. In the second year wheat (Triticum aestivum cv. Barunga), was sown at 75 kg/ha with 20 kg P/ha as triple super phosphate. In the third year pastures were allowed to regenerate from the soil seed bank. Annual rainfall at Pekina was 455, 348 and
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456 mm for years 1, 2 and 3 and April–October growing season rainfall was 326, 272 and 194 mm. At Black Rock, the annual rainfall was 375, 277 and 343 mm with April–October rainfall 271, 227, 155 mm over the 3 years of the experiment. 2.2. Soil and plant sampling Soil cores were taken at three locations across the field sites at depths of 0–10, 10–20 and 20–50 cm to determine pH (1:5 in H2O and CaCl2; Rayment and Higginson, 1992). Estimations of most probable numbers (MPN) of Sinorhizobium spp. in the soil were made at 0–10 cm depth, using a plant infection method (Brockwell, 1963) with Medicago sativa L. as the test species. At Pekina the soil pH (CaCl2) was slightly acidic but increased with soil depth while the Black Rock soil had a neutral pH (Table 1). Pekina had a small rhizobial population while Black Rock had a large soil surface population (Table 1). In year 1 medic plants with entire root systems were harvested at 15 weeks (early spring), to ensure nodulation had occurred and that nodules were still fresh enough to isolate rhizobia effectively. The percentage of plants nodulated was assessed on 100 plants (20 samples/plot) and nodule occupancy was assessed in 20 nodules from each medic strain treatment using nodules selected from all five replicate plots. In year 3, plants with entire root systems were excavated in early spring. Rhizobia were isolated from nodules (Vincent, 1970) sampled from field plots in years 1 and 3 of the experiments. Isolates were obtained from 20 nodules from each medic strain treatment using nodules from all five replicate plots. Insufficient rhizobia were successfully isolated from M. polymorpha nodulated with strain NA39 in year 1 of the experiment for PCR analysis. One nodule per plant was taken from the top 5 cm of root that was on, or close to,
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the taproot as this nodule usually forms early and is thought to provide a large proportion of nitrogen fixation (Corbin et al., 1977). In addition, some plants had just a single very large nodule on the excavated root system. Nodules were surface sterilised in 95% ethanol for 30 s; then 3% sodium hypochlorite for 1.5–3 min (depending upon nodule size) and washed eight times in sterile water. Nodules were then crushed, streaked on YEM (Vincent, 1970) and incubated at 28 8C for 4 days. To obtain a pure colony, a single isolate from each nodule was re-streaked and used for subsequent PCR analysis. A PCR amplification method (Richardson et al., 1995) using the RP05 primer, was chosen for the identification of rhizobia. A number of nodule isolates were confirmed to be rhizobia by re-inoculating M. sativa under aseptic conditions (Vincent, 1970). 2.3. Statistical analyses Statistical analyses (ANOVA) were performed using Genstat 7.1. Where significant differences were observed in ANOVA, means were tested using a Tukey test. 3. Results 3.1. Nodulation The medics responded differently at each field site (Fig. 1). At the site with a small rhizobial population both medic species had poor nodulation when sown uninoculated (Fig. 1a). The nodules that did form on inoculated plants at this site were typically large. Only half or less of the M. truncatula plants at the site with the small rhizobial background had nodules (Fig. 1a). In contrast, M. polymorpha nodulated well with WSM540 or WSM688 and poorly with the acid-sensitive strain NA39. At the site with a large rhizobial background
Table 1 Soil pH (n = 3) and Sinorhizobium meliloti population sizes (derived from most probable number estimates) at the field sites prior to sowing medic species Site
Soil depth (cm)
Soil pHa (H2O)
Soil pH (CaCl2)
Rhizobial population (rhizobia/g soil)b
Pekina
0–10 10–20 20–50
6.4 0.14 a 6.6 0.06 a 7.7 0.07 b
5.9 0.09 a 6.3 0.17 b 7.2 0.05 c
11
Black Rock
0–10 10–20 20–50
7.7 0.03 a 7.8 0.02 a 8.1 0.09 b
7.2 0.02 a 7.4 0.08 b 7.4 0.06 b
a b
Values within a column group that are followed by different letters are significantly different at P < 0.05 (Tukey). Rhizobial population size estimated using the most probable number technique (Brockwell, 1963).
7.1 104
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Fig. 1. The percentage of M. truncatula and M. polymorpha nodulated at sites with a low (a) and high (b) background soil rhizobial population in year 1 of the experiments. The frequency of nodulation (nodulated vs. non-nodulated plants) was assessed using 20 plants per replicate (100 plants/ treatment). Columns that do not share the same letter are significantly different (P 0.05).
more than 85% of plants were nodulated regardless of inoculation (Fig. 1b). Across all treatments, M. truncatula had significantly (P < 0.05) greater nodulation than M. polymorpha. 3.2. Nodule occupancy Rhizobial isolates were identified from nodules harvested in years 1 and 3 of the field experiments using PCR with the random primer RP05 (Fig. 2). Banding patterns of some rhizobial isolates clearly matched the banding patterns of the commercial inoculants (lanes 3, 4, 6 and 10–15 in Fig. 2) and were considered to be inoculant strains. Other isolates produced PCR banding patterns that were clearly dissimilar to the commercial strains (lanes 1, 2, 5, 7–9; Fig. 2). All nodule isolates that were re-inoculated onto M. sativa did form nodules, which indicated that banding profiles were from rhizobia. Inoculant strains were not recovered from plots that were not inoculated, indicating that cross contamination of strains was not a factor. Successful nodule occupancy, based on PCR profiling, was determined for all inoculant strains in M. truncatula nodules in year 1 at the low rhizobia site and for M. polymorpha inoculated with WSM540 and
Fig. 2. An example of PCR profiles used to identify rhizobial isolates from the two field sites, amplified using the RP05 primer. Nodule isolates were collected from M. truncatula inoculated with strain WSM688 (lanes 1–9; isolates collected in year 3 of the trial) and from M. polymorpha inoculated with strain WSM540 (lanes 10–15; isolates were collected in year 1 of the trial). Amplification profiles for commercial strains Sinorhizobium spp. strains WSM688 and WSM540 are indicated in lanes on the left of the gel. Matching amplification profiles for inoculant strain WSM688 are shown in lanes 3, 4 and 6; matching amplifications for strain WSM540 are shown in lanes 10–15. SPP1 bacteriophage digested with EcoRI was used as the molecular weight marker.
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Fig. 3. Recovery of inoculant rhizobia from nodules sampled in the field at sites with a small (a and b) and large (c and d) background rhizobial population. Nodule occupancies of M. truncatula and M. polymorpha are shown for years 1 (a and c) and 3 (b and d) of the experiment. Insufficient nodules prohibited a determination for M. polymorpha nodulated with strain NA39 in year 1 at the site with a small rhizobial population.
WSM688 (Fig. 3a and b). In year 3 of the trial, nodule occupancy was only confirmed in M. truncatula and M. polymorpha for the two acid-tolerant strains, WSM540 and WSM688 (Fig. 3b). At the site with a high rhizobial background, nodule occupancies of inoculant rhizobia in year 1 of the trial were typically very low than for the site with a low background rhizobial population (Fig. 3c and d). By year 3 of the trial, M. truncatula inoculated with WSM688 was the only treatment in which nodule occupancy was confirmed (Fig. 3d). 4. Discussion This study demonstrates the importance of understanding both nodulation and nodule occupancy to understand the outcome of competition for nodulation in a field environment. Inoculation success was demonstrated to be highly dependent upon host–strain interactions in field sites with contrasting soil rhizobial populations. 4.1. Plant nodulation The medics at the two locations differed substantially in their nodulation response at the two sites (Fig. 1). The differences in rhizobial populations at these sites is likely to be due to edaphic and cultural attributes influencing the presence or absence of annual
medics, in turn affecting the establishment of rhizobia populations (Denton et al., 2000). When acid-tolerant rhizobia (WSM688, WSM540) were used to inoculate M. polymorpha in a mildly acidic soil, nodulation was increased above that achieved by no inoculation or inoculation with an acid-sensitive strain (NA39). This indicates the value of using strains that are well matched to edaphic conditions. Uninoculated controls produced very few nodulated plants, reinforcing the estimate of a small rhizobial population at the time of sowing the experiment. M. polymorpha had variable nodulation with the three rhizobial strains (10–85% of plants) while M. truncatula nodulated to a similar extent with each of the three strains (40–50% of plants) which indicates the likelihood that host–strain interactions influenced nodulation. In a previous study M. polymorpha also had a variable nodulation response when inoculated with strain CC169 (15%) or strain WSM688 (50%) (Howieson et al., 1991). Plants at the site with a low rhizobial background produced very large crown nodules. Plants at the site with high background rhizobia contained many small nodules. At this site each medic was well nodulated regardless of inoculation, indicating that this background population of rhizobia was sufficiently large and diverse to nodulate both hosts. Surprisingly, 100% nodulation was not observed for all plants, even with the large background of rhizobia. While much care was taken in excavating plant roots,
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some nodules may have been lost in the process. M. truncatula showed a small but significantly greater preference to nodulate than did M. polymorpha, indicating slightly greater compatibility with the background rhizobial population. 4.2. Nodule occupancy Nodule occupancies were particularly informative: occupancy of inoculant rhizobia differed markedly at the two field sites and for each host legume. At the site with a small background of rhizobia, assessment of nodule occupancy indicated that both medics nodulated with the acid-tolerant strains WSM540 and WSM688 in the year of inoculation far more successfully than the acid-sensitive strain NA39. The acid tolerant strains persisted through a year with a non-host (wheat) and two periods of hot, dry summers to nodulate both medics in year 3. This result indicates the importance of using an acid-tolerant strain of rhizobia to survive saprophytically in this mildly acidic soil. The particular attributes that allowed these acid-tolerant strains to persist in the soil and nodulate medics in the third year remain to be elucidated. Acid tolerance in rhizobia has been shown to involve maintenance of intracellular pH (O’Hara et al., 1989) which may be maintained by increased concentrations of potassium or phosphorus (Watkins et al., 2003). At the site with a large rhizobial background, establishment of inoculant strains was generally poor in the first year, with a maximum of 20% colonisation of M. truncatula nodules by WSM688. This is not surprising, given the large background of rhizobia (7.1 104 rhizobia/g soil) into which inoculant rhizobia were placed. Large populations of rhizobia can have a significant impact on inoculation and are likely to be of concern when the background strains of rhizobia have a poor capacity to fix N2, when inoculation becomes futile (Denton et al., 2002, 2003). At the site with a large background rhizobial population, inoculation with different strains provided 0–20% nodulation in year 1 and inoculant strains occupied very few nodules in year 3. This compared poorly with the site that had a small rhizobial background in which nodule occupancy typically varied between 50 and 100%. An interesting result was that strain WSM688 occupied 45% of M. truncatula nodules assessed in year 3 of the experiment at the site with a large background rhizobial population. Strain WSM688 occupied nodules of M. truncatula successfully at the site with a small rhizobial population where there was very little competition for nodulation from indigenous
strains. However, in the presence of such a large background population, it is difficult to understand why strain WSM688 was so successful in colonising nodules of M. truncatula. Strain WSM688 had not been previously inoculated at this site and the density of plants and nodules was not higher in this treatment in year 1 of the experiment (data not presented), indicating that there was no initial competitive advantage for strain WSM688. The success of WSM688 in year 3 of the experiment is notable given that only 20% of nodules were colonised with this strain in the first year of the trial. Brockwell et al. (1995) have outlined the difficulty in assuring successful nodulation by inoculant strains under the pressure of competing with large numbers of background rhizobia. The successful nodulation between M. truncatula with WSM688 indicates that the specific interactions between the legume and rhizobia and are likely to have determined the outcome of nodulation. This finding indicates that under certain conditions, particular Medicago–Sinorhizobium combinations favour the nodulation of particular strains of rhizobia, despite the presence of large competing background strains of rhizobia. Host species have recently been observed to express a degree of selectivity in nodulation with inoculants in a field environment (Yates et al., 2005). The present results differ from other inoculation experiments that show that the rhizobial population is the primary factor influencing the outcome of nodulation (Brockwell et al., 1982; Thies et al., 1991). Given that M. polymorpha is considered more selective for rhizobia genotypes than M. truncatula (Brockwell, 2001), M. polymorpha was considered to be a more likely candidate to nodulate with WSM688. However, some naturalised M. polymorpha were observed at the Black Rock field site and are likely to have maintained a population of soil rhizobia wellmatched with M. polymorpha. In this case, efficient nodulation of M. polymorpha with the soil rhizobial population may have restricted nodulation of with inoculant strains. While the reason for this particular result is unknown, it is instructive to note the importance that legume–rhizobia relationships may play in environments with large populations of soil rhizobia. By selecting (or genetically enhancing) legumes and rhizobia to achieve highly specific plant-rhizobial signaling, it may be possible to manipulate the outcomes of inoculation, despite the size of the background rhizobial population into which the legume is sown. It is likely, however, that the genetic structure
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and physiology of the rhizobial population present at a site may be as important to the successful outcomes of inoculation, as is the size of the population. Further studies would be required to understand the mechanisms that allow the successful nodulation outcome for M. truncatula with WSM688 on these soil types. These results highlight the importance of (i) using acid-tolerant strains and (ii) selecting rhizobia that are well matched to the legume genotypes of interest for successful nodulation in a field environment. These data indicate that, if legumes are suitably matched with rhizobia, the possibility exists that effective inoculation can proceed, whether the nodulation constraint is a large soil population of rhizobia or an acid soil. Acknowledgements The authors wish to thank Mr. John Cozens, Black Rock and Mr. Kym Fromm, Pekina for the use of land for these experiments. Dr. Annette Anderson and Mrs. Irnayuli Sitepu are thanked for assisting with running PCR and isolating rhizobia from nodules. Dr. Alan Richardson, CSIRO, Plant Industry, Canberra is thanked for providing the RP05 primer. The input of Dr. Alan H. Gibson early in this project was much appreciated. This work was supported by the Grains Research and Development Corporation. References Ballard, R.A., Charman, N., 2000. Nodulation and growth of pasture legumes with naturalised soil rhizobia. 1. Annual Medicago spp. Aust. J. Exp. Agric. 40, 939–948. Bena, G., Lyet, A., Huguet, T., Olivieri, I., 2005. Medicago–Sinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago. J. Evol. Biol. 18, 1547–1558. Brockwell, J., 1963. Accuracy of a plant-infection technique for counting populations of Rhizobium trifolii. Appl. Microb. 11, 377–383. Brockwell, J., 2001. Sinorhizobium meliloti in Australian soils: population studies of the root-nodule bacteria for species of Medicago in soils of the Eyre Peninsula, South Australia. Aust. J. Exp. Agric. 41, 753–762. Brockwell, J., Bottomley, P.J., Thies, J.E., 1995. Manipulation of rhizobia microflora for improving legume productivity and soil fertility: a critical assessment. Plant Soil 174, 143–180. Brockwell, J., Gault, R.R., Zorin, M., Roberts, M.J., 1982. Effects of environmental variables on the competition between inoculum strains and naturalized populations of Rhizobium trifolii for nodulation of Trifolium subterraneum L. and on rhizobia persistence in the soil. Aust. J. Agric. Res. 33, 803–815. Brockwell, J., Hely, F.W., 1966. Symbiotic characteristics of Rhizobium meliloti: an appraisal of the systematic treatment of nodulation and nitrogen fixation interactions between hosts and rhizobia of diverse origins. Aust. J. Agric. Res. 17, 885–899.
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