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Delayed inoculation and competition of nitrogen-fixing strains in Medicago noeana (Boiss.) and Medicago polymorpha (L.)
Applied Soil Ecology 17 (2001) 175–181
Delayed inoculation and competition of nitrogen-fixing strains in Medicago noeana (Boiss.) and Medicago polymorpha (L.) L.A. Materon a,∗ , L. Zibilske b b
a The University of Texas-Pan American, 1201 West University Drive, Edinburg, TX 78539, USA Research Soil Scientist, USDA-Agricultural Research Service, Integrated Farming and Natural Resources Unit, 2413 E. Hwy 83, Weslaco, TX 78596-8344, USA
Received 23 July 2000; accepted 21 November 2000
Abstract Seed inoculation is frequently essential for annual Medicago establishment in dryland farming systems, particularly in Mediterranean-type environments. As post-planting soil inoculation is often practised when seed inoculation fails, the effect of delayed inoculation was investigated. Roots of Medicago noeana ICARDA sel. 1938, and Medicago polymorpha cv. Circle Valley, were pre-exposed to Rhizobium meliloti strains. Subsequently, roots were exposed to a secondary inoculum after 6, 48 and 168 h to simulate delayed inoculation, and subsequent establishment of other strains in the nodules were investigated. Combinations of highly effective and host compatible strains (M29 and M15) and effective–ineffective strains (M29 and M33) were used to evaluate proportional nodulation responses. Plants were harvested after 6 weeks of growth under environmentally controlled conditions. Nodules were assessed for distribution in the root system and for occupancy based on their differential resistance to spectinomycin and streptomycin, and, in the case of M33, on nodule characteristics. The strain M29 was a better competitor than M15 when applied in equal density to M. polymorpha, at zero time. When forming nodules with M. noeana, M15 was equally competitive under the same conditions. With M29 as the primary inoculum, and M15 inoculation delayed for 6, 48 and 168 h, the incidence of M29 nodules increased on M. noeana from 55% (at zero time) to 83, 80, and 95% and, on M. polymorpha, from 72% (at zero time) to 80, 90, and 97% for the three inoculation time delays, respectively. Conversely, strain M15 dominated nodule production at all time intervals when used as primary inoculant on both hosts. The percentage of total nodulation by M33, applied at the three later inoculation times, was markedly lower (21, 2 and 0%, respectively) when M. polymorpha was pre-exposed to M29. This suggested a host preference for M29, even if applied as a late inoculum. Pre-exposure of 2-day-old M. noeana seedlings to the ineffective strain M33 as the primary inoculant resulted in nodule number increases (P ≤ 0.01) as compared with M29. Nonetheless, when M29 was the primary inoculum, M33 was able to produce significantly fewer nodules than its competitor when applied at the 6 and 48 h time delays. Results indicate that the early events in the nodulation process of annual medics coupled with host-specificity factors are perhaps the most critical for competition among R. meliloti strains for nodule formation. Therefore, remedial inoculation after the seed has been planted may be of little benefit. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Delayed inoculation; Rhizobium meliloti; Annual Medicago; Nitrogen fixation
∗ Corresponding author. Tel.: +1-956-381-3537; fax: +1-956-381-3657. E-mail addresses: [email protected] (L.A. Materon), [email protected] (L. Zibilske).
0929-1393/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 ( 0 1 ) 0 0 1 2 1 - 4
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L.A. Materon, L. Zibilske / Applied Soil Ecology 17 (2001) 175–181
1. Introduction Annual ‘medics’ (Medicago spp.) are economically important pasture legumes grown in most regions of the world with Mediterranean-type climate. Exploitation of their nitrogen-fixing potential is often hampered by the need of artificial inoculation of seed. This practice frequently follows planting as a remedial inoculation alternative. Inoculant rhizobial strains have to compete for root infection sites with native strains in the soil. Competition for nodulation is usually measured by comparing the ability of introduced Rhizobium strains to form nodules on the chosen host (Vincent and Waters, 1953; Nutman, 1965; Winarno and Lie, 1979; Dowling and Broughton, 1986). This can be of agronomic concern when the desired inoculum strain fails to establish in nodules because of competition from indigenous and often ineffective rhizobia, which are less efficient in fixing nitrogen (Marques-Pinto et al., 1974). The presence of ineffective rhizobia is a major constraint in cereal rotation systems with annual Medicago species, where inoculation with selected rhizobial strains does not always result in effective root nodulation, significantly reducing herbage yields (Materon, 1993). This is partly because of the nodulation requirements of Medicago spp. with specific rhizobial strains, but is also often due to failure of the inoculum strains to compete with indigenous soil rhizobia (Ham, 1980; Materon and Cocks, 1988; Materon, 1991, 1993, 1994). The number of nodules formed and the effectiveness of the nodules are governed by both host plant and Rhizobium genes, and can result in large differences in nodulation quality and nitrogen fixation efficiency. Specificity among strains of Rhizobium meliloti, particularly with Medicago spp. also plays an important role in establishing an effective nitrogen-fixing symbiosis. The required compatibility is a function of the genetic constitution of both partners, as is the relative nodulating success of competing rhizobial strains (Vincent and Waters, 1953). Moreover, the essential factors or the critical stages of the infection process responsible for the success or failure of a strain have not yet been fully identified. Mechanisms that confer competitive advantage to a strain are still poorly understood; several factors of both the rhizobial symbiont and the host play important roles in determining which strain succeeds in
occupying most nodules (Vincent, 1980; Materon and Vincent, 1980; Dowling and Broughton, 1986). In many situations, appropriate inoculum may not be available in time to treat seeds prior to planting. In such cases, inocula are sometimes applied later, directly onto the soil. The effectiveness of this delayed application is debatable. In contrast to strain competition and host-specificity, there is little information on the effect of delayed inoculation in pasture and forage legumes. Skrdleta (1970) and Kosslak et al. (1983) found that pre-exposure of soybean seedlings to a primary inoculum predisposed the plants to that strain for further nodulation. Materon (1994) using effective and ineffective strains in Medicago rigidula and Medicago rotata reported that nodule formation was dependent on the specificity and intrinsic competitiveness of the strains regardless of delayed inoculation treatments. Pre-exposure to the primary inoculant had a significant competitive advantage over the secondary strain, either effective or ineffective, when applied at different time intervals on both annual medics. Hence, in this study, delayed inoculation with specific strains was used to pinpoint the period of time in the nodulation process most critical to competition among strains. The relative nodulation success of either effective or ineffective strains applied at three time intervals following root exposure to an inoculum was investigated under controlled conditions in two annual Medicago hosts.
2. Materials and methods All seed and bacterial cultures were obtained from the International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria. All rhizobial cultures for inoculation were derived from single-colony re-isolates. Antibiotic-resistant sub-strains were isolated as single-step spontaneous mutants, which were resistant either to streptomycin or spectinomycin (Obaton, 1971; Materon and Vincent, 1980). Thus, sub-strains of M29 and M15 were spontaneous mutants resistant to final concentrations of 60 g ml−1 of streptomycin sulfate and 100 g ml−1 spectinomycin dihydrochloride, respectively (Obaton, 1971). Strain M33 was also resistant to 100 g ml−1 of spectinomycin sulfate. Both antibiotic mutant strains were previously tested for effectiveness
L.A. Materon, L. Zibilske / Applied Soil Ecology 17 (2001) 175–181
on the medic hosts described in the next paragraph. Following Vincent (1970) and Beck et al. (1993), the rhizobial stock cultures were kept lyophilized to prevent genetic changes. Three strains of R. meliloti, M29, M15, and M33 were used in delayed inoculation studies with Medicago noeana selection 1938 (Siirt, Turkey) and Medicago polymorpha cv. Circle Valley (Coolgardie, Australia). The M29 and M15 strains are equally effective with M. noeana and M. polymorpha, whereas M33 is ineffective on both Medicago hosts (Materon, 1991). The rhizobial strains originated from Mediterranean soils (center of origin of annual medics). The strains have been selected for use as inoculants both in greenhouse and field conditions with several species of annual medics in several Mediterranean-type environments (Materon and Cocks, 1988). Seeds were mechanically scarified, and surfacesterilized by exposure to 95% ethanol for 1 min followed by immersion in a 5.25% sodium hypochlorite (NaOCl) solution for 2 min, then repeatedly rinsed in deionized sterile water as per Vincent (1970). Seeds were then germinated on moist paper in petri-dishes under sterile conditions. The 2-day-old seedlings with 2–3 mm radicles were selected for experimentation. Seedlings of both Medicago species were transferred to 20 cm × 2.4 cm glass tubes containing sterile vermiculite moistened with 1:4 strength Hoagland (Hoagland and Arnon, 1950) nitrogen-free solution (Beck et al., 1993). Each tube contained one seedling and was covered with a cotton-wool plug and placed in a rack. Controls consisted of noninoculated seedlings and seedlings inoculated with only one strain at each time period. Mixture of strains at zero time was based on turbidity to obtain an equal proportion of each Rhizobium strain. For inoculation, 1 ml of 2-day-old yeast mannitol broth culture was applied adjacent to the seedling root. The viable inoculum count was determined from serially diluted suspensions plated on yeast mannitol agar (YMA) plates according to standard procedures (Vincent, 1970). For combining effective rhizobial strains, three different treatments were used. For each Medicago species, at zero time, one group (10 plants) received both effective strains. The second group (30 plants) received only M29, and the third group (30 plants) received only M15. After 6, 48 and 168 h, 10 tubes
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each from groups 2 and 3 received strains M15 and M29 as secondary inoculants, respectively. Similarly, for combining effective and ineffective strains, three different treatments were used. For each Medicago species, at zero time, one group (10 plants) received both effective (M29) and the ineffective strain (M33). The second group (30 plants) received only M29, and the third group (30 plants) received only M33. After 6, 48 and 168 h, 10 tubes each from groups 2 and 3 received strains M33 and M29 as secondary inoculants, respectively. Plants were grown aseptically in a growth chamber at 24◦ C with a light intensity of 168 einstein cm−2 s−1 and a 12 h photoperiod. Four weeks after secondary inoculation, the plants were harvested and nodules separated according to their position on the root (taproot and lateral root). Ten nodules from each plant were used for strain identification. Nodules were surface-sterilized by exposure to 95% ethanol for 30 s, followed by immersion in 5.25% sodium hypochlorite (NaOCl) solution for 90 s, and then vigorously rinsed six times in deionized water. Identification of rhizobia in the nodules was based on resistance or sensitivity to antibiotics. Each nodule was slowly squeezed with the tip of sterile broad-ended forceps; the small volume of nodule suspension thus liberated was gently deposited on wells of ELISA plates containing 1 ml of yeast mannitol broth. After growth of 2 days at 26◦ C, a small volume of culture was taken with a cotton-wool swab from each well and gently deposited on petri-dish quadrants containing YMA or YMA supplemented with 60 g streptomycin sulfate ml−1 or, 100 g spectinomycin sulfate ml−1 . The presence or absence of growth served as the criterion to distinguish strains (Obaton, 1971; Materon and Hagedorn, 1984; Beck et al., 1993). Growth in the presence of both antibiotics indicated dual-strain occupancy in the nodule. Nodule morphology and lack of leghemoglobin were traits also used to distinguish the ineffective strain M33 from the other two mutant strains. Glass tubes, each containing a single plant, were arranged in a completely randomized design with 10 replications per treatment. As data for relative nodulation success were expressed in percentages (p), they were transformed to degrees, to improve the equality of variance according to Bartlett (1947). Values of 0 were substituted by 1/n, and values of 1
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by (4n √ − 1)/4n. Other values were transformed to arcsine( p × 57.297795). Pooled treatment data at 0 time were also subjected to χ 2 to test random distribution of strains in the nodulation of the Medicago hosts. Analysis of variance was used for comparative analysis for each strain combination.
3. Results and discussion Nodule occupancy of either effective strain on both Medicago hosts did not differ with nodule location on primary or secondary roots when the strains were applied in equal proportion at zero time. Materon (1994) obtained similar results on nodule distribution with M. rigidula and M. rotata. In contrast, Skrdleta (1970) found that all taproot nodules were formed by the primary strain. However, our results are consistent with those of Kosslak et al. (1983) who found that nodules produced by the strain that was introduced as the primary inoculant were more abundant and equally distributed along the primary and secondary roots at all delay intervals.
Jenkins and Bottomley, 1985) reported a strong host influence of Medicago sativa L. on the relative ability of R. meliloti strains to compete for nodule sites. Significant interactions among the hosts and inoculant strains for nodule formation and the effect of delayed inoculation were observed in M. rotata and M. rigidula by Materon (1994). Following primary inoculation, strain M15 was significantly out-competed by the primary inoculum (M29) on M. noeana; nodulation due to this strain declined significantly, averaging 42, 14, 16 and 5% after 0, 6, 48 and 168 h intervals, respectively (Fig. 1a). The same trend was observed when M29 was applied as secondary inoculum. M29 was only able to produce 24% of the nodules when applied 6 h following application of the primary inoculum (M15); thereafter, its nodulation was greatly reduced by the primary inoculum (Fig. 1c). Relative nodulation success of M29 on M. polymorpha was affected by a marked host preference
3.1. M29 versus M15 The mix ratio of both effective strains when applied to M. noeana and M. polymorpha was 0.94:1.0 equivalent to 1.31 × 108 rhizobia ml−1 for M29 and 3.76 × 108 rhizobia ml−1 for M15. Therefore, rhizobial presence in the rhizosphere of both Medicago species by the two strains was nearly equal. At zero time, the relative nodulation success of both strains was not significantly different in M. noeana, as each strain produced approximately half of the nodules. When the mixture of effective rhizobial strains was applied to M. polymorpha at zero time, M29 showed superior colonization and competitive ability. Nodule formation was approximately 3.5 times as common as M15 (Fig. 1b). The superior competitiveness of M29 was apparent and highly significant (P ≤ 0.01), whether tested by χ 2 distribution of all nodules, or by an analysis of variance based on identified nodules of replicate plants. There were no significant differences between M15 and M29 for nodule occupancy when applied in equal proportion to M. noeana at zero time. Fifty-five percent of nodulation was due to M29 (Fig. 1a). Other workers (Marques-Pinto et al., 1974;
Fig. 1. Effect of pre-exposure of seedlings of M. noeana and M. polymorpha to an effective strain of Rhizobium meliloti on nodule occupancy by another effective strain (M29 as primary inoculum for (a) and (b) and M15 for (c) and (d)).
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for this strain as compared with M15 only when they were applied in approximately equal quantities. Seventy-two percent of nodules were produced by M29 when applied as mixed inoculum in approximately equal proportions (0.94:1:00) at 0 time. Despite host preference for M29, when this strain was applied at the delay intervals, its nodule occupancy declined to 28, 7 and 5%, at the 6, 48 and 168 h time intervals (Fig. 1d). Similarly, we detected little nodulation by M15 on M. polymorpha when introduced as secondary inoculum as shown in Fig. 1b. Of the total 1600 nodules corresponding to this combination of strains, 8.75% were mixed infected and co-occupied by both strains in both hosts (5% in M. noeana and 3.75% in M. polymorpha) at zero time and delayed time intervals. 3.2. M29 versus M33 It has been observed that rhizobial strain M33 produces parasitic and abundant nodulation in several annual Medicago species including M. noeana and M. polymorpha (Materon and Cocks, 1988; Materon, 1993). On the other hand, strain M29 is an effective competitor and efficient nitrogen-fixing strain. Therefore, this combination of competitors was chosen to determine if the introduction of an ineffective strain (M33) would influence M29 in effectively nodulating both Medicago hosts. At zero time, the mixture of these strains was close to equality with a ratio of 0.95:1.00 which consisted of rhizobial population densities of 1.15 × 108 cells ml−1 and 3.00 × 108 cells ml−1 for M29 and M33, respectively. No significant differences were detected between strains for nodule occupancy on M. noeana at zero time (Fig. 2a and c). This finding suggested no host preference nor specificity for either strain, and no intrinsic competitive advantage between the two strains as reflected by a non-significant χ 2 analysis on nodulation formation. Four percent of nodules had dual-strain occupancy. In contrast, significantly (P ≤ 0.05) more nodules were produced by M29 on M. polymorpha at zero time (Fig. 2b and d). In this host and at this time interval, the superior competitiveness of M29 was significant (P ≤ 0.05), whether tested by χ 2 distribution of all nodules, or by an analysis of variance based on identified nodules of replicate plants. There were about 1.5 times more
Fig. 2. Effect of pre-exposure of seedlings of M. noeana and M. polymorpha to both an effective and ineffective strain of R. meliloti (M29 as primary inoculum for (a) and (b) and M33 for (c) and (d)).
nodules due to M29 infection as compared to the ineffective strain at zero time (Fig. 2b and d). M33 produced about 40% of the total nodules on M. noeana when applied 6 h after pre-exposure to M29. Nodulation declined significantly when applied 48 and 168 h after primary inoculation with M29 (Fig. 2a). Plants did not show any nitrogen deficiency symptoms. When the roots of this host were pre-exposed to M33, most of the nodules, regardless of delayed inoculation treatment, were produced by the primary ineffective strain. Nodulation was abundant and parasitic. All plants of this treatment showed typical nitrogen deficiency symptoms because of reduced N2 -fixation capacity. When M 29 was inoculated alone into the root zone of M. polymorpha, nodulation response indicated that this strain prevented the secondary inoculant from infecting this host. Only 21, 2, and 0% of the nodules were formed when M33 was introduced at the 6, 48,
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and 168 h time intervals (Fig. 2b). On the other hand, the situation was dramatically different when M29 was the secondary inoculant. At 6 and 48 h the number of nodules due to M29 were fewer than 2% of the total (P ≤ 0.01). Therefore, strain M33 was dominant over M29 in the rhizosphere, and produced virtually all nodules at 6, 48 and 168 h interval (Fig. 2d). Periods for delayed inoculation used by other investigators in related studies include 72 h (Skrdleta, 1970), and 0 and 6 h (Kosslak et al., 1983). In this investigation, it was evident that a short pre-exposure (6 h or less) was adequate to alter significantly the competition pattern of the strains for nodule formation. Though the technique of studying strains in pairs for competitive ability is laborious, it provides additional and relevant information on the strains studied. It also revealed dual occupancy of nodules, particularly at the earlier time intervals. With this strain combination, dual occupancy did not reach more than 5.25% of total nodules at the 0 or 6 h interval and at the 48 and 168 h intervals in both hosts. Control plants inoculated with the effective strains M15 and M29 produced 16 (±4), 19 (±5) nodules per plant, respectively. A mean nodule number of 32 (±7) for the ineffective strain M33 was recorded. Nodules of the latter, present in chlorotic plants, were always smaller and lacked leghemoglobin. This trait was also used to identify strain M33. Control plants that received no inoculation had no nodules and were chlorotic and stunted. Results from this investigation suggest that interactions which occur during the early period of infection between the Medicago host and its rhizobial partner, are perhaps the most critical for competition among R. meliloti strains. The role of the host in determining the outcome of competition among strains was highlighted by the observation that seedlings with root lengths ranging from 2 to 4 mm at the time of primary exposure to one strain became predisposed to that strain for nodulation. These studies illustrate host preference for a strain, and, in the case of M33, the potential hazard of a strain that is both ineffective and an aggressive colonizer, when it is given an opportunity to infect Medicago seedlings. The presence of such a strain in a mixed inoculum, or if naturally present in the soil, could be a barrier to the establishment of annual species of Medicago. As reported by Materon (1994), R.
meliloti naturally occurring in soils, either effective or ineffective, become strong competitors for inoculant strains if inoculation is delayed or postponed. Thus, it can be expected that most nodules will be initiated by the strain to which the host is pre-exposed. Remedial inoculation after the seed has been planted may be of little benefit. Inoculated strains may have little chance of success in competing with indigenous soil rhizobia for nodulation on some Medicago species. References Bartlett, M.S., 1947. The use of transformations. Biometrics 3, 39–52. Beck, D.P., Materon, L.A., Afandi, F., 1993. Practical Rhizobium-Legume Technology Manual. International Center for Agricultural Research in the Dry Areas (ICARDA), Technical Manual No. 19, Aleppo, Syria, p. 394. Dowling, D.N., Broughton, W.J., 1986. Competition for nodulation of legumes. Ann. Rev. Microbiol. 40, 131–157. Ham, G.E., 1980. Inoculation of legumes with Rhizobium in competition with naturalized strains. In: Newton, W.E., Orme-Johnson, W.H. (Eds.), Nitrogen Fixation, Vol. II. Park Press, Baltimore, MD, pp. 131–138. Hoagland, D.R., Arnon, D.I., 1950. The Water-culture Method for Growing Plants without Soil, Vol. 347. California Agr. Exp. Sta. Circ., CA, USA. Jenkins, M.B., Bottomley, P.J., 1985. Evidence for a strain of Rhizobium meliloti dominating the nodules of alfalfa. Soil Sci. Soc. Am. J. 49, 326–328. Kosslak, R.M., Bohlool, B.B., Dowdle, S., Sadowsky, M.J., 1983. Competition of Rhizobium japonicum strains in early stages of soybean nodulation. Appl. Environ. Microbiol. 46, 870–873. Marques-Pinto, C., Yao, P.K., Vincent, J.M., 1974. Nodulating competitiveness amongst strains of Rhizobium meliloti and Rhizobium trifolii. Aust. J. Agric. Res. 25, 317–329. Materon, L.A., 1991. Symbiotic characteristics of Rhizobium meliloti in west Asian soils. Soil Biol. Biochem. 23, 429–434. Materon, L.A., 1993. Constraints to nodulation of annual medics by indigenous populations of Rhizobium meliloti in west Asian soils. In: Christiansen, S., Materon, L.A., Falcinelli, M., Cocks, P.S. (Eds.), Introducing Ley Farming to the Mediterranean Basin. International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria, pp. 192–202. Materon, L.A., 1994. Delayed inoculation and competition of Rhizobium meliloti in annual Medicago species. Appl. Soil Ecol. 1, 255–260. Materon, L.A., Cocks, P.S., 1988. Constraints to biological nitrogen fixation in ley-farming systems designed for west Asia. In: Murrell, W.G., Kennedy, I.R. (Eds.), Microbiology in Action. Wiley, New York, pp. 93–106. Materon, L.A., Hagedorn, C.H., 1984. Responses of crimson clover to inoculation with genetically-marked strains of Rhizobium trifolii. Comm. Soil Sci. Plant Anal. 15, 33–47.
L.A. Materon, L. Zibilske / Applied Soil Ecology 17 (2001) 175–181 Materon, L.A., Vincent, J.M., 1980. Host specificity and interstrain competition with soybean rhizobia. Field Crops Res. 3, 215–224. Nutman, P.S., 1965. The relation between the nodule bacteria and the legume host in the rhizosphere and in the process of infection. In: Baker, K.F., Snyder, W.C. (Eds.), The Ecology of Soil-borne Plant Pathogens. University of California Press, Berkeley, CA, pp. 231–247. Obaton, M., 1971. Utilisation des mutants spontanés résistants aux antibiotiques pour l’étude écologique de Rhizobium. Compte Rendu Academie de Science (Paris), Vol. 272D, pp. 2630–2633. Skrdleta, V., 1970. Competition for nodule sites between two inoculum strains of Rhizobium japonicum as affected by delayed inoculation. Soil Biol. Biochem. 2, 167–171.
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Vincent, J.M., 1970. A Manual for the Practical Study of Root-Nodule Bacteria. IBP Handbook No. 15. Blackwell Scientific Publications, Oxford, UK, p. 164. Vincent, J.M., 1980. Factors controlling the legume-Rhizobium symbiosis. In: Newton, W.E., Orme-Johnson, W.H. (Eds.), Nitrogen Fixation, Vol. II. University Park Press, Baltimore, MD, pp. 103–129. Vincent, J.M., Waters, L.M., 1953. The influence of the host on competition amongst clover root-nodule bacteria. J. Gen. Microbiol. 9, 357–370. Winarno, R., Lie, T.A., 1979. Competition between Rhizobium strains in nodule formation: interaction between nodulating and non-nodulating strains. Plant Soil 51, 135–142.
Delayed inoculation and competition of nitrogen-fixing strains in Medicago noeana (Boiss.) and Medicago polymorpha (L.) L.A. Materon a,∗ , L. Zibilske b b
a The University of Texas-Pan American, 1201 West University Drive, Edinburg, TX 78539, USA Research Soil Scientist, USDA-Agricultural Research Service, Integrated Farming and Natural Resources Unit, 2413 E. Hwy 83, Weslaco, TX 78596-8344, USA
Received 23 July 2000; accepted 21 November 2000
Abstract Seed inoculation is frequently essential for annual Medicago establishment in dryland farming systems, particularly in Mediterranean-type environments. As post-planting soil inoculation is often practised when seed inoculation fails, the effect of delayed inoculation was investigated. Roots of Medicago noeana ICARDA sel. 1938, and Medicago polymorpha cv. Circle Valley, were pre-exposed to Rhizobium meliloti strains. Subsequently, roots were exposed to a secondary inoculum after 6, 48 and 168 h to simulate delayed inoculation, and subsequent establishment of other strains in the nodules were investigated. Combinations of highly effective and host compatible strains (M29 and M15) and effective–ineffective strains (M29 and M33) were used to evaluate proportional nodulation responses. Plants were harvested after 6 weeks of growth under environmentally controlled conditions. Nodules were assessed for distribution in the root system and for occupancy based on their differential resistance to spectinomycin and streptomycin, and, in the case of M33, on nodule characteristics. The strain M29 was a better competitor than M15 when applied in equal density to M. polymorpha, at zero time. When forming nodules with M. noeana, M15 was equally competitive under the same conditions. With M29 as the primary inoculum, and M15 inoculation delayed for 6, 48 and 168 h, the incidence of M29 nodules increased on M. noeana from 55% (at zero time) to 83, 80, and 95% and, on M. polymorpha, from 72% (at zero time) to 80, 90, and 97% for the three inoculation time delays, respectively. Conversely, strain M15 dominated nodule production at all time intervals when used as primary inoculant on both hosts. The percentage of total nodulation by M33, applied at the three later inoculation times, was markedly lower (21, 2 and 0%, respectively) when M. polymorpha was pre-exposed to M29. This suggested a host preference for M29, even if applied as a late inoculum. Pre-exposure of 2-day-old M. noeana seedlings to the ineffective strain M33 as the primary inoculant resulted in nodule number increases (P ≤ 0.01) as compared with M29. Nonetheless, when M29 was the primary inoculum, M33 was able to produce significantly fewer nodules than its competitor when applied at the 6 and 48 h time delays. Results indicate that the early events in the nodulation process of annual medics coupled with host-specificity factors are perhaps the most critical for competition among R. meliloti strains for nodule formation. Therefore, remedial inoculation after the seed has been planted may be of little benefit. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Delayed inoculation; Rhizobium meliloti; Annual Medicago; Nitrogen fixation
∗ Corresponding author. Tel.: +1-956-381-3537; fax: +1-956-381-3657. E-mail addresses: [email protected] (L.A. Materon), [email protected] (L. Zibilske).
0929-1393/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 ( 0 1 ) 0 0 1 2 1 - 4
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1. Introduction Annual ‘medics’ (Medicago spp.) are economically important pasture legumes grown in most regions of the world with Mediterranean-type climate. Exploitation of their nitrogen-fixing potential is often hampered by the need of artificial inoculation of seed. This practice frequently follows planting as a remedial inoculation alternative. Inoculant rhizobial strains have to compete for root infection sites with native strains in the soil. Competition for nodulation is usually measured by comparing the ability of introduced Rhizobium strains to form nodules on the chosen host (Vincent and Waters, 1953; Nutman, 1965; Winarno and Lie, 1979; Dowling and Broughton, 1986). This can be of agronomic concern when the desired inoculum strain fails to establish in nodules because of competition from indigenous and often ineffective rhizobia, which are less efficient in fixing nitrogen (Marques-Pinto et al., 1974). The presence of ineffective rhizobia is a major constraint in cereal rotation systems with annual Medicago species, where inoculation with selected rhizobial strains does not always result in effective root nodulation, significantly reducing herbage yields (Materon, 1993). This is partly because of the nodulation requirements of Medicago spp. with specific rhizobial strains, but is also often due to failure of the inoculum strains to compete with indigenous soil rhizobia (Ham, 1980; Materon and Cocks, 1988; Materon, 1991, 1993, 1994). The number of nodules formed and the effectiveness of the nodules are governed by both host plant and Rhizobium genes, and can result in large differences in nodulation quality and nitrogen fixation efficiency. Specificity among strains of Rhizobium meliloti, particularly with Medicago spp. also plays an important role in establishing an effective nitrogen-fixing symbiosis. The required compatibility is a function of the genetic constitution of both partners, as is the relative nodulating success of competing rhizobial strains (Vincent and Waters, 1953). Moreover, the essential factors or the critical stages of the infection process responsible for the success or failure of a strain have not yet been fully identified. Mechanisms that confer competitive advantage to a strain are still poorly understood; several factors of both the rhizobial symbiont and the host play important roles in determining which strain succeeds in
occupying most nodules (Vincent, 1980; Materon and Vincent, 1980; Dowling and Broughton, 1986). In many situations, appropriate inoculum may not be available in time to treat seeds prior to planting. In such cases, inocula are sometimes applied later, directly onto the soil. The effectiveness of this delayed application is debatable. In contrast to strain competition and host-specificity, there is little information on the effect of delayed inoculation in pasture and forage legumes. Skrdleta (1970) and Kosslak et al. (1983) found that pre-exposure of soybean seedlings to a primary inoculum predisposed the plants to that strain for further nodulation. Materon (1994) using effective and ineffective strains in Medicago rigidula and Medicago rotata reported that nodule formation was dependent on the specificity and intrinsic competitiveness of the strains regardless of delayed inoculation treatments. Pre-exposure to the primary inoculant had a significant competitive advantage over the secondary strain, either effective or ineffective, when applied at different time intervals on both annual medics. Hence, in this study, delayed inoculation with specific strains was used to pinpoint the period of time in the nodulation process most critical to competition among strains. The relative nodulation success of either effective or ineffective strains applied at three time intervals following root exposure to an inoculum was investigated under controlled conditions in two annual Medicago hosts.
2. Materials and methods All seed and bacterial cultures were obtained from the International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria. All rhizobial cultures for inoculation were derived from single-colony re-isolates. Antibiotic-resistant sub-strains were isolated as single-step spontaneous mutants, which were resistant either to streptomycin or spectinomycin (Obaton, 1971; Materon and Vincent, 1980). Thus, sub-strains of M29 and M15 were spontaneous mutants resistant to final concentrations of 60 g ml−1 of streptomycin sulfate and 100 g ml−1 spectinomycin dihydrochloride, respectively (Obaton, 1971). Strain M33 was also resistant to 100 g ml−1 of spectinomycin sulfate. Both antibiotic mutant strains were previously tested for effectiveness
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on the medic hosts described in the next paragraph. Following Vincent (1970) and Beck et al. (1993), the rhizobial stock cultures were kept lyophilized to prevent genetic changes. Three strains of R. meliloti, M29, M15, and M33 were used in delayed inoculation studies with Medicago noeana selection 1938 (Siirt, Turkey) and Medicago polymorpha cv. Circle Valley (Coolgardie, Australia). The M29 and M15 strains are equally effective with M. noeana and M. polymorpha, whereas M33 is ineffective on both Medicago hosts (Materon, 1991). The rhizobial strains originated from Mediterranean soils (center of origin of annual medics). The strains have been selected for use as inoculants both in greenhouse and field conditions with several species of annual medics in several Mediterranean-type environments (Materon and Cocks, 1988). Seeds were mechanically scarified, and surfacesterilized by exposure to 95% ethanol for 1 min followed by immersion in a 5.25% sodium hypochlorite (NaOCl) solution for 2 min, then repeatedly rinsed in deionized sterile water as per Vincent (1970). Seeds were then germinated on moist paper in petri-dishes under sterile conditions. The 2-day-old seedlings with 2–3 mm radicles were selected for experimentation. Seedlings of both Medicago species were transferred to 20 cm × 2.4 cm glass tubes containing sterile vermiculite moistened with 1:4 strength Hoagland (Hoagland and Arnon, 1950) nitrogen-free solution (Beck et al., 1993). Each tube contained one seedling and was covered with a cotton-wool plug and placed in a rack. Controls consisted of noninoculated seedlings and seedlings inoculated with only one strain at each time period. Mixture of strains at zero time was based on turbidity to obtain an equal proportion of each Rhizobium strain. For inoculation, 1 ml of 2-day-old yeast mannitol broth culture was applied adjacent to the seedling root. The viable inoculum count was determined from serially diluted suspensions plated on yeast mannitol agar (YMA) plates according to standard procedures (Vincent, 1970). For combining effective rhizobial strains, three different treatments were used. For each Medicago species, at zero time, one group (10 plants) received both effective strains. The second group (30 plants) received only M29, and the third group (30 plants) received only M15. After 6, 48 and 168 h, 10 tubes
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each from groups 2 and 3 received strains M15 and M29 as secondary inoculants, respectively. Similarly, for combining effective and ineffective strains, three different treatments were used. For each Medicago species, at zero time, one group (10 plants) received both effective (M29) and the ineffective strain (M33). The second group (30 plants) received only M29, and the third group (30 plants) received only M33. After 6, 48 and 168 h, 10 tubes each from groups 2 and 3 received strains M33 and M29 as secondary inoculants, respectively. Plants were grown aseptically in a growth chamber at 24◦ C with a light intensity of 168 einstein cm−2 s−1 and a 12 h photoperiod. Four weeks after secondary inoculation, the plants were harvested and nodules separated according to their position on the root (taproot and lateral root). Ten nodules from each plant were used for strain identification. Nodules were surface-sterilized by exposure to 95% ethanol for 30 s, followed by immersion in 5.25% sodium hypochlorite (NaOCl) solution for 90 s, and then vigorously rinsed six times in deionized water. Identification of rhizobia in the nodules was based on resistance or sensitivity to antibiotics. Each nodule was slowly squeezed with the tip of sterile broad-ended forceps; the small volume of nodule suspension thus liberated was gently deposited on wells of ELISA plates containing 1 ml of yeast mannitol broth. After growth of 2 days at 26◦ C, a small volume of culture was taken with a cotton-wool swab from each well and gently deposited on petri-dish quadrants containing YMA or YMA supplemented with 60 g streptomycin sulfate ml−1 or, 100 g spectinomycin sulfate ml−1 . The presence or absence of growth served as the criterion to distinguish strains (Obaton, 1971; Materon and Hagedorn, 1984; Beck et al., 1993). Growth in the presence of both antibiotics indicated dual-strain occupancy in the nodule. Nodule morphology and lack of leghemoglobin were traits also used to distinguish the ineffective strain M33 from the other two mutant strains. Glass tubes, each containing a single plant, were arranged in a completely randomized design with 10 replications per treatment. As data for relative nodulation success were expressed in percentages (p), they were transformed to degrees, to improve the equality of variance according to Bartlett (1947). Values of 0 were substituted by 1/n, and values of 1
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by (4n √ − 1)/4n. Other values were transformed to arcsine( p × 57.297795). Pooled treatment data at 0 time were also subjected to χ 2 to test random distribution of strains in the nodulation of the Medicago hosts. Analysis of variance was used for comparative analysis for each strain combination.
3. Results and discussion Nodule occupancy of either effective strain on both Medicago hosts did not differ with nodule location on primary or secondary roots when the strains were applied in equal proportion at zero time. Materon (1994) obtained similar results on nodule distribution with M. rigidula and M. rotata. In contrast, Skrdleta (1970) found that all taproot nodules were formed by the primary strain. However, our results are consistent with those of Kosslak et al. (1983) who found that nodules produced by the strain that was introduced as the primary inoculant were more abundant and equally distributed along the primary and secondary roots at all delay intervals.
Jenkins and Bottomley, 1985) reported a strong host influence of Medicago sativa L. on the relative ability of R. meliloti strains to compete for nodule sites. Significant interactions among the hosts and inoculant strains for nodule formation and the effect of delayed inoculation were observed in M. rotata and M. rigidula by Materon (1994). Following primary inoculation, strain M15 was significantly out-competed by the primary inoculum (M29) on M. noeana; nodulation due to this strain declined significantly, averaging 42, 14, 16 and 5% after 0, 6, 48 and 168 h intervals, respectively (Fig. 1a). The same trend was observed when M29 was applied as secondary inoculum. M29 was only able to produce 24% of the nodules when applied 6 h following application of the primary inoculum (M15); thereafter, its nodulation was greatly reduced by the primary inoculum (Fig. 1c). Relative nodulation success of M29 on M. polymorpha was affected by a marked host preference
3.1. M29 versus M15 The mix ratio of both effective strains when applied to M. noeana and M. polymorpha was 0.94:1.0 equivalent to 1.31 × 108 rhizobia ml−1 for M29 and 3.76 × 108 rhizobia ml−1 for M15. Therefore, rhizobial presence in the rhizosphere of both Medicago species by the two strains was nearly equal. At zero time, the relative nodulation success of both strains was not significantly different in M. noeana, as each strain produced approximately half of the nodules. When the mixture of effective rhizobial strains was applied to M. polymorpha at zero time, M29 showed superior colonization and competitive ability. Nodule formation was approximately 3.5 times as common as M15 (Fig. 1b). The superior competitiveness of M29 was apparent and highly significant (P ≤ 0.01), whether tested by χ 2 distribution of all nodules, or by an analysis of variance based on identified nodules of replicate plants. There were no significant differences between M15 and M29 for nodule occupancy when applied in equal proportion to M. noeana at zero time. Fifty-five percent of nodulation was due to M29 (Fig. 1a). Other workers (Marques-Pinto et al., 1974;
Fig. 1. Effect of pre-exposure of seedlings of M. noeana and M. polymorpha to an effective strain of Rhizobium meliloti on nodule occupancy by another effective strain (M29 as primary inoculum for (a) and (b) and M15 for (c) and (d)).
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for this strain as compared with M15 only when they were applied in approximately equal quantities. Seventy-two percent of nodules were produced by M29 when applied as mixed inoculum in approximately equal proportions (0.94:1:00) at 0 time. Despite host preference for M29, when this strain was applied at the delay intervals, its nodule occupancy declined to 28, 7 and 5%, at the 6, 48 and 168 h time intervals (Fig. 1d). Similarly, we detected little nodulation by M15 on M. polymorpha when introduced as secondary inoculum as shown in Fig. 1b. Of the total 1600 nodules corresponding to this combination of strains, 8.75% were mixed infected and co-occupied by both strains in both hosts (5% in M. noeana and 3.75% in M. polymorpha) at zero time and delayed time intervals. 3.2. M29 versus M33 It has been observed that rhizobial strain M33 produces parasitic and abundant nodulation in several annual Medicago species including M. noeana and M. polymorpha (Materon and Cocks, 1988; Materon, 1993). On the other hand, strain M29 is an effective competitor and efficient nitrogen-fixing strain. Therefore, this combination of competitors was chosen to determine if the introduction of an ineffective strain (M33) would influence M29 in effectively nodulating both Medicago hosts. At zero time, the mixture of these strains was close to equality with a ratio of 0.95:1.00 which consisted of rhizobial population densities of 1.15 × 108 cells ml−1 and 3.00 × 108 cells ml−1 for M29 and M33, respectively. No significant differences were detected between strains for nodule occupancy on M. noeana at zero time (Fig. 2a and c). This finding suggested no host preference nor specificity for either strain, and no intrinsic competitive advantage between the two strains as reflected by a non-significant χ 2 analysis on nodulation formation. Four percent of nodules had dual-strain occupancy. In contrast, significantly (P ≤ 0.05) more nodules were produced by M29 on M. polymorpha at zero time (Fig. 2b and d). In this host and at this time interval, the superior competitiveness of M29 was significant (P ≤ 0.05), whether tested by χ 2 distribution of all nodules, or by an analysis of variance based on identified nodules of replicate plants. There were about 1.5 times more
Fig. 2. Effect of pre-exposure of seedlings of M. noeana and M. polymorpha to both an effective and ineffective strain of R. meliloti (M29 as primary inoculum for (a) and (b) and M33 for (c) and (d)).
nodules due to M29 infection as compared to the ineffective strain at zero time (Fig. 2b and d). M33 produced about 40% of the total nodules on M. noeana when applied 6 h after pre-exposure to M29. Nodulation declined significantly when applied 48 and 168 h after primary inoculation with M29 (Fig. 2a). Plants did not show any nitrogen deficiency symptoms. When the roots of this host were pre-exposed to M33, most of the nodules, regardless of delayed inoculation treatment, were produced by the primary ineffective strain. Nodulation was abundant and parasitic. All plants of this treatment showed typical nitrogen deficiency symptoms because of reduced N2 -fixation capacity. When M 29 was inoculated alone into the root zone of M. polymorpha, nodulation response indicated that this strain prevented the secondary inoculant from infecting this host. Only 21, 2, and 0% of the nodules were formed when M33 was introduced at the 6, 48,
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and 168 h time intervals (Fig. 2b). On the other hand, the situation was dramatically different when M29 was the secondary inoculant. At 6 and 48 h the number of nodules due to M29 were fewer than 2% of the total (P ≤ 0.01). Therefore, strain M33 was dominant over M29 in the rhizosphere, and produced virtually all nodules at 6, 48 and 168 h interval (Fig. 2d). Periods for delayed inoculation used by other investigators in related studies include 72 h (Skrdleta, 1970), and 0 and 6 h (Kosslak et al., 1983). In this investigation, it was evident that a short pre-exposure (6 h or less) was adequate to alter significantly the competition pattern of the strains for nodule formation. Though the technique of studying strains in pairs for competitive ability is laborious, it provides additional and relevant information on the strains studied. It also revealed dual occupancy of nodules, particularly at the earlier time intervals. With this strain combination, dual occupancy did not reach more than 5.25% of total nodules at the 0 or 6 h interval and at the 48 and 168 h intervals in both hosts. Control plants inoculated with the effective strains M15 and M29 produced 16 (±4), 19 (±5) nodules per plant, respectively. A mean nodule number of 32 (±7) for the ineffective strain M33 was recorded. Nodules of the latter, present in chlorotic plants, were always smaller and lacked leghemoglobin. This trait was also used to identify strain M33. Control plants that received no inoculation had no nodules and were chlorotic and stunted. Results from this investigation suggest that interactions which occur during the early period of infection between the Medicago host and its rhizobial partner, are perhaps the most critical for competition among R. meliloti strains. The role of the host in determining the outcome of competition among strains was highlighted by the observation that seedlings with root lengths ranging from 2 to 4 mm at the time of primary exposure to one strain became predisposed to that strain for nodulation. These studies illustrate host preference for a strain, and, in the case of M33, the potential hazard of a strain that is both ineffective and an aggressive colonizer, when it is given an opportunity to infect Medicago seedlings. The presence of such a strain in a mixed inoculum, or if naturally present in the soil, could be a barrier to the establishment of annual species of Medicago. As reported by Materon (1994), R.
meliloti naturally occurring in soils, either effective or ineffective, become strong competitors for inoculant strains if inoculation is delayed or postponed. Thus, it can be expected that most nodules will be initiated by the strain to which the host is pre-exposed. Remedial inoculation after the seed has been planted may be of little benefit. Inoculated strains may have little chance of success in competing with indigenous soil rhizobia for nodulation on some Medicago species. References Bartlett, M.S., 1947. The use of transformations. Biometrics 3, 39–52. Beck, D.P., Materon, L.A., Afandi, F., 1993. Practical Rhizobium-Legume Technology Manual. International Center for Agricultural Research in the Dry Areas (ICARDA), Technical Manual No. 19, Aleppo, Syria, p. 394. Dowling, D.N., Broughton, W.J., 1986. Competition for nodulation of legumes. Ann. Rev. Microbiol. 40, 131–157. Ham, G.E., 1980. Inoculation of legumes with Rhizobium in competition with naturalized strains. In: Newton, W.E., Orme-Johnson, W.H. (Eds.), Nitrogen Fixation, Vol. II. Park Press, Baltimore, MD, pp. 131–138. Hoagland, D.R., Arnon, D.I., 1950. The Water-culture Method for Growing Plants without Soil, Vol. 347. California Agr. Exp. Sta. Circ., CA, USA. Jenkins, M.B., Bottomley, P.J., 1985. Evidence for a strain of Rhizobium meliloti dominating the nodules of alfalfa. Soil Sci. Soc. Am. J. 49, 326–328. Kosslak, R.M., Bohlool, B.B., Dowdle, S., Sadowsky, M.J., 1983. Competition of Rhizobium japonicum strains in early stages of soybean nodulation. Appl. Environ. Microbiol. 46, 870–873. Marques-Pinto, C., Yao, P.K., Vincent, J.M., 1974. Nodulating competitiveness amongst strains of Rhizobium meliloti and Rhizobium trifolii. Aust. J. Agric. Res. 25, 317–329. Materon, L.A., 1991. Symbiotic characteristics of Rhizobium meliloti in west Asian soils. Soil Biol. Biochem. 23, 429–434. Materon, L.A., 1993. Constraints to nodulation of annual medics by indigenous populations of Rhizobium meliloti in west Asian soils. In: Christiansen, S., Materon, L.A., Falcinelli, M., Cocks, P.S. (Eds.), Introducing Ley Farming to the Mediterranean Basin. International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria, pp. 192–202. Materon, L.A., 1994. Delayed inoculation and competition of Rhizobium meliloti in annual Medicago species. Appl. Soil Ecol. 1, 255–260. Materon, L.A., Cocks, P.S., 1988. Constraints to biological nitrogen fixation in ley-farming systems designed for west Asia. In: Murrell, W.G., Kennedy, I.R. (Eds.), Microbiology in Action. Wiley, New York, pp. 93–106. Materon, L.A., Hagedorn, C.H., 1984. Responses of crimson clover to inoculation with genetically-marked strains of Rhizobium trifolii. Comm. Soil Sci. Plant Anal. 15, 33–47.
L.A. Materon, L. Zibilske / Applied Soil Ecology 17 (2001) 175–181 Materon, L.A., Vincent, J.M., 1980. Host specificity and interstrain competition with soybean rhizobia. Field Crops Res. 3, 215–224. Nutman, P.S., 1965. The relation between the nodule bacteria and the legume host in the rhizosphere and in the process of infection. In: Baker, K.F., Snyder, W.C. (Eds.), The Ecology of Soil-borne Plant Pathogens. University of California Press, Berkeley, CA, pp. 231–247. Obaton, M., 1971. Utilisation des mutants spontanés résistants aux antibiotiques pour l’étude écologique de Rhizobium. Compte Rendu Academie de Science (Paris), Vol. 272D, pp. 2630–2633. Skrdleta, V., 1970. Competition for nodule sites between two inoculum strains of Rhizobium japonicum as affected by delayed inoculation. Soil Biol. Biochem. 2, 167–171.
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Vincent, J.M., 1970. A Manual for the Practical Study of Root-Nodule Bacteria. IBP Handbook No. 15. Blackwell Scientific Publications, Oxford, UK, p. 164. Vincent, J.M., 1980. Factors controlling the legume-Rhizobium symbiosis. In: Newton, W.E., Orme-Johnson, W.H. (Eds.), Nitrogen Fixation, Vol. II. University Park Press, Baltimore, MD, pp. 103–129. Vincent, J.M., Waters, L.M., 1953. The influence of the host on competition amongst clover root-nodule bacteria. J. Gen. Microbiol. 9, 357–370. Winarno, R., Lie, T.A., 1979. Competition between Rhizobium strains in nodule formation: interaction between nodulating and non-nodulating strains. Plant Soil 51, 135–142.