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Inoculation with Pseudomonas fluorescens biocontrol strains does not affect the symbiosis between rhizobia and forage legumes
Soil Biology & Biochemistry 34 (2002) 545±548
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Short communication
Inoculation with Pseudomonas ¯uorescens biocontrol strains does not affect the symbiosis between rhizobia and forage legumes Leonardo De La Fuente a,b,*, Leticia Quagliotto a, Natalia Bajsa a, Elena Fabiano a, Nora Altier c, Alicia Arias a a
Laboratorio de EcologõÂa Microbiana, Dpto. de BioquõÂmica, IIBCE, Av. Italia 3318, Montevideo, CP 11600, Uruguay b Facultad de Ciencias, Unidad Asociada BioquõÂmica, Igua 4225, Montevideo, CP 11400, Uruguay c INIA-Las Brujas, CC 33085, Las Piedras, CP 90200, Uruguay Received 3 November 2000; received in revised form 27 August 2001; accepted 5 September 2001
Abstract Pseudomonas ¯uorescens strains UP61, UP143 and UP148, isolated from Uruguayan soils, have shown the ability to control soil-borne fungal pathogens that cause damping-off in birdsfoot trefoil. In this communication, we study the effect of these strains on the symbiotic ef®ciency of rhizobia from commercial inoculants in birdsfoot trefoil, alfalfa and white clover. Shoot dry weights and the rate of nodulation by rhizobia were not modi®ed by the presence of Pseudomonas strains, despite antagonistic activity against rhizobia in vitro. Survival of P. ¯uorescens UP61 and rhizobia on roots in non-sterile soil were not affected by co-inoculation of the selected forage legumes. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Fluorescent Pseudomonas; Rhizobia; Forage legumes; Co-inoculation; Biocontrol
Forage legumes play an important role in the farming production of Uruguay as a major source of high quality feed for livestock. Their nitrogen ®xing capability in symbiotic association with rhizobia confers a bene®cial residual effect in crop rotations. Important losses in their establishment are caused by soil-borne pathogens, mainly Pythium spp. (Altier, 1996). The application of chemical pesticides for seed treatment may have a detrimental effect on symbiotic nitrogen ®xation (Altier and Pastorini, 1988). The use of rhizosphere bacteria as biocontrol agents has been studied using Pseudomonas ¯uorescens native Uruguayan strains UP61, UP143 and UP148, which are able to control damping-off caused by Pythium ultimum and Rhizoctonia solani in birdsfoot trefoil (Lotus corniculatus L.) (Bagnasco et al., 1998). Biocontrol mechanisms of these strains may involve siderophores, HCN and antibiotics. UP61 produces the antibiotics 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (Plt) and pyrrolnitrin (De La Fuente, unpublished results), and UP148 a phenazine-derivative non-previously described (Bagnasco, unpublished results). * Corresponding author. Address: Laboratorio de EcologõÂa Microbiana, Dpto. de BioquõÂmica, IIBCE, Av. Italia 3318, Montevideo, CP 11600, Uruguay. Tel.: 1598-2-487-1616/145-147; fax: 1598-2-487-5548. E-mail address: [email protected] (L. De La Fuente).
Since for practical applications rhizobia and pseudomonads should be simultaneously inoculated on legume seeds, it is important to be aware of their interactions. The objective of this study was to evaluate the effect of native P. ¯uorescens strains on the symbiosis of rhizobia with birdsfoot trefoil, alfalfa (Medicago sativa L.), and white clover (Trifolium repens L.). Different parameters were considered including in vitro inhibition of rhizobia by pseudomonads, effects on shoot dry weights, the rate of nodulation and root colonisation. P. ¯uorescens strains used were UP61, UP143 and UP148. UP148FS 2 is a mutant of UP148 defective in ¯uorescent siderophore production (kindly provided by Robledo). Mesorhizobium loti B816, Sinorhizobium meliloti MCH3 and Rhizobium leguminosarum bv. trifolii U28 are local commercial inoculant strains for birdsfoot trefoil, alfalfa and white clover, respectively. Spontaneous mutants resistant to rifampicin (Rif) P. ¯uorescens UP61.2R, to kanamycin (Km) M. loti B816.2K and S. meliloti MCH3 (naturally resistant to Km) were used for pot assays. Pseudomonads were routinely grown on King's B medium (KB; King et al., 1954), and rhizobia on Tryptone Yeast Extract (Beringer, 1974) or Yeast Extract Mannitol (Vincent, 1970). The antibiotics Rif (50 mg ml 21), cycloheximide (50 mg ml 21) and Km (50 mg ml 21) were added to the media when necessary.
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00194-8
546
L. De La Fuente et al. / Soil Biology & Biochemistry 34 (2002) 545±548 Table 1 Effect of co-inoculation with rhizobia and P. ¯uorescens strains on shoot dry weights in different forage legumes Inocula a
Plant Rhizobia Birdsfoot trefoil B816 B816 B816 B816 Alfalfa Fig. 1. Rate of nodulation of M. loti B816 on birdsfoot trefoil plants coinoculated with native P. ¯uorescens strains. Plants were inoculated with M. loti B816 alone (A) or together with P. ¯uorescens UP61 (W), P. ¯uorescens UP143 (O) or P. ¯uorescens UP148 (V). The graph shows the results from one of three independent experiments.
Rhizobia inhibition in vitro by pseudomonads strains was tested by bioassays as described by Homma et al. (1989). Assays were independently repeated at least four times. The rate of nodulation and the effect on shoot dry weights were tested in tubes containing Jensen medium (Vincent, 1970) according to Bagnasco et al. (1998). Seedlings were coinoculated with M. loti B816 (5 £ 10 6 CFU seedling 21) and P. ¯uorescens UP61, UP143 or UP148 (5 £ 10 6 CFU seedling 21). Treatments were replicated 15 times and three independent experiments were performed. Each experiment included two controls: one without bacterial inoculation and one inoculated only with M. loti B816. Number of nodulated plants was periodically recorded. Fifty days after sowing, plants were harvested and their aerial dry weights were measured. For pot assays, seeds were bacterised according to Weller and Cook (1983). P. ¯uorescens UP61 density ranged from 8 £ 10 5 to 6 £ 10 7 CFU seed 21, and rhizobia from 1 £ 10 5 to 8 £ 10 6 CFU seed 21 (see Fig. 2). To determine plant response to inoculation, 15 seeds of birdsfoot trefoil, alfalfa or white clover, inoculated separately or co-inoculated with their speci®c rhizobia and UP61, were sown in 300 cm 3 pots containing a mixture of soil and sand (Bagnasco et al., 1998). Treatments were replicated six times and three independent experiments were performed. After 30 d for birdsfoot trefoil, 60 d for white clover and 75 d for alfalfa, aerial dry weight was measured. To study root colonisation, four birdsfoot trefoil or alfalfa seeds, inoculated separately or co-inoculated with their speci®c rhizobia and P. ¯uorescens UP61.2R were sown in 50 cm 3 pots and incubated as described by Bagnasco et al. (1998). Treatments were replicated ®ve times. Plants were harvested periodically and rhizospheric populations were determined by plating in selective media containing antibiotics. Shoot dry weight accumulation in tubes and pots and colonisation experiments were set in a block design and data were analysed by ANOVA (General Lineal Model
MCH3 MCH3
White clover U28 U28
Shoot dry weight (mg plant 21) b
Pseudomonads Tube assay
Pot assay
1 1 1
UP61 UP143 UP148
5.15a 5.58a 5.60a 4.85a
5.37bc 6.28a 5.98ab 4.74c
1
UP61
ND ND
128.25a 114.65a
1
UP61
ND ND
16.51a 14.99a
a Birdsfoot trefoil, alfalfa and white clover plants were inoculated with their speci®c rhizobia (M. loti B816, S. meliloti MCH3 or R. leguminosarum bv. trifolii U28, respectively) and one of the following native biocontrol strains: P. ¯uorescens UP61, UP143 or UP148. b Values represent means corresponding to one of three independent experiments. Data were analysed in a one-way ANOVA. For each plant species and in the same column, different letters mean signi®cant differences P , 0:05: ND, non-determined.
procedure, SAS Institute). Means were separated using Fisher's protected LSD test P , 0:05: Strains UP61, UP143 and UP148, reference strains P. ¯uorescens Q2-87, CHA0 and P. aureofaciens 30-84 (Thomashow and Weller, 1996) antagonised M. loti B816 on KB plates. Antagonistic activity of UP143, UP148, Q287 and 30-84, were strongly reduced on KB with 100 mM FeCl3 (KB 1 Fe). On the other hand, UP61 and CHA0 were still able to inhibit B816 growth on KB 1 Fe, although to a lesser extent than on KB. In vitro antagonistic activity of all these strains against S. meliloti MCH3 was observed under low-iron conditions, and no activity was detected on KB 1 Fe. UP148FS 2 did not inhibit M. loti B816 and S. meliloti MCH3, both under low- and high-iron conditions (data not shown). All the tested strains produce different active metabolites against a broad range of microorganisms. Production of DAPG and Plt is not affected by iron concentration in CHA0 (Duffy and DeÂfago, 1999). However, the production of phenazine-1-carboxylic acid in P. ¯uorescens 2-79 (Slininger and Jackson, 1992) and HCN in CHA0 (Keel et al., 1989) is stimulated by iron. According to available literature, among the active metabolites produced by the tested strains, siderophores are the only compounds whose synthesis is induced under iron-limited conditions. This, together with the fact that UP148FS 2 was unable to inhibit rhizobial strains, suggests a role for siderophores in the detected in vitro inhibition. The rate of nodulation was evaluated to determine if the presence of pseudomonads affects early steps of nodule formation. Nodulation of birdsfoot trefoil by M. loti B816 was not affected by the presence of P. ¯uorescens UP61 and
L. De La Fuente et al. / Soil Biology & Biochemistry 34 (2002) 545±548
547
Fig. 2. Root colonisation of birdsfoot trefoil (I) and alfalfa (II) by P. ¯uorescens UP61.2R and rhizobia. Strains M. loti B816.2K (I), S. meliloti MCH3 (II) and UP61.2R (I and II) were used to inoculate seeds separately (solid bars) or as a mixture (lined bars) of the speci®c rhizobia with P. ¯uorescens strain. Rhizospheric population values (log CFU (g root) 21) used in the graph correspond to the means of ®ve replicates. Numbers in parentheses represent the initial inocula used. Population sizes were logarithmically transformed before analysis. Data from each graph were independently analysed in a three-factor (treatment, time and block) ANOVA with interactions. Different letters in the same graph indicate signi®cant difference for time factor. Treatment effect was signi®cant for P. ¯uorescens but not for rhizobia. No effect of the block factor and no interactions were found P , 0:05:
548
L. De La Fuente et al. / Soil Biology & Biochemistry 34 (2002) 545±548
UP143 (Fig. 1). About 75% of the plants were nodulated by the 11th day after inoculation, and 100% by the 27th day. When UP148 was co-inoculated on seeds, a delay was observed during the ®rst 13 days. By the eighth day after inoculation only 25% of the plants containing UP148 nodulated, while 60% of the plants inoculated with B816 alone or together with UP61 or UP143, had nodules. However, all treatments reached a similar nodulation level by the 13th day. Similar results were found in three independent experiments. The effect of M. loti B816 on dry weight of birdsfoot trefoil was not negatively affected P , 0:05 by the introduction of native P. ¯uorescens strains. Even more, UP61 showed a moderate plant-growth promoting effect in pot assays (Table 1). To determine if the effect of UP61 depends on the forage legume, assays with alfalfa and white clover were performed in soil. Shoot dry weights of both plant species, inoculated with their speci®c commercial inoculants, were not affected by the presence of UP61 (Table 1). Competitive exclusion among rhizospheric microorganisms is directly associated to the ability of colonising the root surface successfully. Colonisation of birdsfoot trefoil and alfalfa rhizospheres by rhizobia and P. ¯uorescens UP61.2R were not affected by the presence of each other (Fig. 2). In general, population densities of pseudomonads and rhizobia were maintained at the initial levels for the ®rst days, and then a small drop was observed. The exception to this trend was S. meliloti MCH3 whose population density increased with time. Population densities of both rhizobia strains did not differ when they were inoculated alone or co-inoculated with P. ¯uorescens UP61.2R (P 0:73 for M. loti and P 0:64 for S. meliloti). A significant difference in pseudomonads densities on roots was found between treatments inoculated with UP61.2R alone or co-inoculated with rhizobia in both plant species, which was related to the differences found in the initial seed inocula (Fig. 2). A direct linear relationship between bacterial seed and root populations has been previously demonstrated by Bull et al. (1991). In birdsfoot trefoil UP61.2R densities on seeds were larger when co-inoculated with rhizobia (2 £ 10 6 versus 6 £ 10 7CFU seed 21), while in alfalfa were smaller when co-inoculated (2 £ 10 7 versus 8 £ 10 5CFU seed 21). However, for all UP61.2R treatments, the dynamics of populations declined in a similar way in spite of the co-inoculation with rhizobia. Despite the in vitro rhizobia inhibition observed, the presence of Pseudomonas did not signi®cantly affect rhizobia symbiosis (Table 1, Figs. 1 and 2). This suggests that the iron concentration might not be extremely low in the rhizospheric environment of plants growing in natural soil (Loper and Lindow, 1994). The knowledge that native Pseudomonas strains are able to survive on peat for more than 6 months (Bagnasco et al.,
1998) and our results, indicate that a mixed pseudomonads± rhizobia inoculant could be developed in order to improve forage legumes establishment and yield. Acknowledgements This work was partially supported by PEDECIBA, IFS and INCO-DC EU. References Altier, N., 1996. Enfermedades de leguminosas forrajeras: diagnoÂstico, epidemiologõÂa y control. In: DõÂaz, M. (Ed.). Manejo de Enfermedades en Cereales de Invierno y Pasturas. Serie TeÂcnica No. 74 INIA, Montevideo, Uruguay, pp. 87±104. Altier, N., Pastorini, D., 1988. Curasemillas en leguminosas forrajeras: efecto sobre los rizobios. Est. Exp. La Estanzuela. Hoja de divulgacioÂn No. 74. 2p. Bagnasco, P., De La Fuente, L., Gualtieri, G., Noya, F., Arias, A., 1998. Fluorescent Pseudomonas spp. as biocontrol agents against forage legume root pathogenic fungi. Soil Biology and Biochemistry 30, 1317±1322. Beringer, J.E., 1974. R factor transfer in Rhizobium leguminosarum. Journal of General Microbiology 84, 188±198. Bull, C.T., Weller, D.M., Thomashow, L.S., 1991. Relationship between root colonisation and suppression of Gaeumannomyces graminis var. tritici by Pseudomonas ¯uorescens strain 2-79. Phytopathology 81, 954±959. Duffy, B.K., DeÂfago, G., 1999. Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas ¯uorescens biocontrol strains. Applied and Environmental Microbiology 65, 2429±2438. Homma, Y., Sato, Z., Hirayama, F., Konno, K., Shirahama, H., Suzui, T., 1989. Production of antibiotics by Pseudomonas cepacia as an agent for biological control of soilborne plant pathogens. Soil Biology and Biochemistry 21, 723±728. Keel, C., Voisard, C., Berling, C.H., Khar, G., DeÂfago, G., 1989. Iron suf®ciency, a prerequisite for the suppression of tobacco black root rot by Pseudomonas ¯uorescens strain CHA0 under gnotobiotic conditions. Phytopathology 79, 584±589. King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of pyocianin and ¯uorescein. Journal of Laboratory and Clinical Medicine 44, 301±307. Loper, J.E., Lindow, S.W., 1994. A biological sensor for iron available to bacteria in their habitats on plant surfaces. Applied and Environmental Microbiology 60, 1934±1941. Slininger, P.J., Jackson, M.A., 1992. Nutritional factors regulating growth and accumulation of phenazine-1-carboxylic acid by Pseudomonas ¯uorescens 2-79. Applied Microbiology and Biotechnology 37, 388± 392. Thomashow, L.S., Weller, D.M., 1996. Current concepts in the use of introduced bacteria for biological disease control: mechanisms and antifungal metabolites. In: Stacey, G., Keen, N. (Eds.). Plant±Microbe Interactions, vol. 1. Chapman & Hall, New York, pp. 187±235. Vincent, J.M., 1970. A Manual for the Practical Study of the Root Nodule Bacteria. IBP Handbook No. 15. Blackwell, New York. Weller, D.M., Cook, R.J., 1983. Suppression of take-all of wheat by seed treatments with ¯uorescent pseudomonads. Phytopathology 73, 463± 469.
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Short communication
Inoculation with Pseudomonas ¯uorescens biocontrol strains does not affect the symbiosis between rhizobia and forage legumes Leonardo De La Fuente a,b,*, Leticia Quagliotto a, Natalia Bajsa a, Elena Fabiano a, Nora Altier c, Alicia Arias a a
Laboratorio de EcologõÂa Microbiana, Dpto. de BioquõÂmica, IIBCE, Av. Italia 3318, Montevideo, CP 11600, Uruguay b Facultad de Ciencias, Unidad Asociada BioquõÂmica, Igua 4225, Montevideo, CP 11400, Uruguay c INIA-Las Brujas, CC 33085, Las Piedras, CP 90200, Uruguay Received 3 November 2000; received in revised form 27 August 2001; accepted 5 September 2001
Abstract Pseudomonas ¯uorescens strains UP61, UP143 and UP148, isolated from Uruguayan soils, have shown the ability to control soil-borne fungal pathogens that cause damping-off in birdsfoot trefoil. In this communication, we study the effect of these strains on the symbiotic ef®ciency of rhizobia from commercial inoculants in birdsfoot trefoil, alfalfa and white clover. Shoot dry weights and the rate of nodulation by rhizobia were not modi®ed by the presence of Pseudomonas strains, despite antagonistic activity against rhizobia in vitro. Survival of P. ¯uorescens UP61 and rhizobia on roots in non-sterile soil were not affected by co-inoculation of the selected forage legumes. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Fluorescent Pseudomonas; Rhizobia; Forage legumes; Co-inoculation; Biocontrol
Forage legumes play an important role in the farming production of Uruguay as a major source of high quality feed for livestock. Their nitrogen ®xing capability in symbiotic association with rhizobia confers a bene®cial residual effect in crop rotations. Important losses in their establishment are caused by soil-borne pathogens, mainly Pythium spp. (Altier, 1996). The application of chemical pesticides for seed treatment may have a detrimental effect on symbiotic nitrogen ®xation (Altier and Pastorini, 1988). The use of rhizosphere bacteria as biocontrol agents has been studied using Pseudomonas ¯uorescens native Uruguayan strains UP61, UP143 and UP148, which are able to control damping-off caused by Pythium ultimum and Rhizoctonia solani in birdsfoot trefoil (Lotus corniculatus L.) (Bagnasco et al., 1998). Biocontrol mechanisms of these strains may involve siderophores, HCN and antibiotics. UP61 produces the antibiotics 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (Plt) and pyrrolnitrin (De La Fuente, unpublished results), and UP148 a phenazine-derivative non-previously described (Bagnasco, unpublished results). * Corresponding author. Address: Laboratorio de EcologõÂa Microbiana, Dpto. de BioquõÂmica, IIBCE, Av. Italia 3318, Montevideo, CP 11600, Uruguay. Tel.: 1598-2-487-1616/145-147; fax: 1598-2-487-5548. E-mail address: [email protected] (L. De La Fuente).
Since for practical applications rhizobia and pseudomonads should be simultaneously inoculated on legume seeds, it is important to be aware of their interactions. The objective of this study was to evaluate the effect of native P. ¯uorescens strains on the symbiosis of rhizobia with birdsfoot trefoil, alfalfa (Medicago sativa L.), and white clover (Trifolium repens L.). Different parameters were considered including in vitro inhibition of rhizobia by pseudomonads, effects on shoot dry weights, the rate of nodulation and root colonisation. P. ¯uorescens strains used were UP61, UP143 and UP148. UP148FS 2 is a mutant of UP148 defective in ¯uorescent siderophore production (kindly provided by Robledo). Mesorhizobium loti B816, Sinorhizobium meliloti MCH3 and Rhizobium leguminosarum bv. trifolii U28 are local commercial inoculant strains for birdsfoot trefoil, alfalfa and white clover, respectively. Spontaneous mutants resistant to rifampicin (Rif) P. ¯uorescens UP61.2R, to kanamycin (Km) M. loti B816.2K and S. meliloti MCH3 (naturally resistant to Km) were used for pot assays. Pseudomonads were routinely grown on King's B medium (KB; King et al., 1954), and rhizobia on Tryptone Yeast Extract (Beringer, 1974) or Yeast Extract Mannitol (Vincent, 1970). The antibiotics Rif (50 mg ml 21), cycloheximide (50 mg ml 21) and Km (50 mg ml 21) were added to the media when necessary.
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00194-8
546
L. De La Fuente et al. / Soil Biology & Biochemistry 34 (2002) 545±548 Table 1 Effect of co-inoculation with rhizobia and P. ¯uorescens strains on shoot dry weights in different forage legumes Inocula a
Plant Rhizobia Birdsfoot trefoil B816 B816 B816 B816 Alfalfa Fig. 1. Rate of nodulation of M. loti B816 on birdsfoot trefoil plants coinoculated with native P. ¯uorescens strains. Plants were inoculated with M. loti B816 alone (A) or together with P. ¯uorescens UP61 (W), P. ¯uorescens UP143 (O) or P. ¯uorescens UP148 (V). The graph shows the results from one of three independent experiments.
Rhizobia inhibition in vitro by pseudomonads strains was tested by bioassays as described by Homma et al. (1989). Assays were independently repeated at least four times. The rate of nodulation and the effect on shoot dry weights were tested in tubes containing Jensen medium (Vincent, 1970) according to Bagnasco et al. (1998). Seedlings were coinoculated with M. loti B816 (5 £ 10 6 CFU seedling 21) and P. ¯uorescens UP61, UP143 or UP148 (5 £ 10 6 CFU seedling 21). Treatments were replicated 15 times and three independent experiments were performed. Each experiment included two controls: one without bacterial inoculation and one inoculated only with M. loti B816. Number of nodulated plants was periodically recorded. Fifty days after sowing, plants were harvested and their aerial dry weights were measured. For pot assays, seeds were bacterised according to Weller and Cook (1983). P. ¯uorescens UP61 density ranged from 8 £ 10 5 to 6 £ 10 7 CFU seed 21, and rhizobia from 1 £ 10 5 to 8 £ 10 6 CFU seed 21 (see Fig. 2). To determine plant response to inoculation, 15 seeds of birdsfoot trefoil, alfalfa or white clover, inoculated separately or co-inoculated with their speci®c rhizobia and UP61, were sown in 300 cm 3 pots containing a mixture of soil and sand (Bagnasco et al., 1998). Treatments were replicated six times and three independent experiments were performed. After 30 d for birdsfoot trefoil, 60 d for white clover and 75 d for alfalfa, aerial dry weight was measured. To study root colonisation, four birdsfoot trefoil or alfalfa seeds, inoculated separately or co-inoculated with their speci®c rhizobia and P. ¯uorescens UP61.2R were sown in 50 cm 3 pots and incubated as described by Bagnasco et al. (1998). Treatments were replicated ®ve times. Plants were harvested periodically and rhizospheric populations were determined by plating in selective media containing antibiotics. Shoot dry weight accumulation in tubes and pots and colonisation experiments were set in a block design and data were analysed by ANOVA (General Lineal Model
MCH3 MCH3
White clover U28 U28
Shoot dry weight (mg plant 21) b
Pseudomonads Tube assay
Pot assay
1 1 1
UP61 UP143 UP148
5.15a 5.58a 5.60a 4.85a
5.37bc 6.28a 5.98ab 4.74c
1
UP61
ND ND
128.25a 114.65a
1
UP61
ND ND
16.51a 14.99a
a Birdsfoot trefoil, alfalfa and white clover plants were inoculated with their speci®c rhizobia (M. loti B816, S. meliloti MCH3 or R. leguminosarum bv. trifolii U28, respectively) and one of the following native biocontrol strains: P. ¯uorescens UP61, UP143 or UP148. b Values represent means corresponding to one of three independent experiments. Data were analysed in a one-way ANOVA. For each plant species and in the same column, different letters mean signi®cant differences P , 0:05: ND, non-determined.
procedure, SAS Institute). Means were separated using Fisher's protected LSD test P , 0:05: Strains UP61, UP143 and UP148, reference strains P. ¯uorescens Q2-87, CHA0 and P. aureofaciens 30-84 (Thomashow and Weller, 1996) antagonised M. loti B816 on KB plates. Antagonistic activity of UP143, UP148, Q287 and 30-84, were strongly reduced on KB with 100 mM FeCl3 (KB 1 Fe). On the other hand, UP61 and CHA0 were still able to inhibit B816 growth on KB 1 Fe, although to a lesser extent than on KB. In vitro antagonistic activity of all these strains against S. meliloti MCH3 was observed under low-iron conditions, and no activity was detected on KB 1 Fe. UP148FS 2 did not inhibit M. loti B816 and S. meliloti MCH3, both under low- and high-iron conditions (data not shown). All the tested strains produce different active metabolites against a broad range of microorganisms. Production of DAPG and Plt is not affected by iron concentration in CHA0 (Duffy and DeÂfago, 1999). However, the production of phenazine-1-carboxylic acid in P. ¯uorescens 2-79 (Slininger and Jackson, 1992) and HCN in CHA0 (Keel et al., 1989) is stimulated by iron. According to available literature, among the active metabolites produced by the tested strains, siderophores are the only compounds whose synthesis is induced under iron-limited conditions. This, together with the fact that UP148FS 2 was unable to inhibit rhizobial strains, suggests a role for siderophores in the detected in vitro inhibition. The rate of nodulation was evaluated to determine if the presence of pseudomonads affects early steps of nodule formation. Nodulation of birdsfoot trefoil by M. loti B816 was not affected by the presence of P. ¯uorescens UP61 and
L. De La Fuente et al. / Soil Biology & Biochemistry 34 (2002) 545±548
547
Fig. 2. Root colonisation of birdsfoot trefoil (I) and alfalfa (II) by P. ¯uorescens UP61.2R and rhizobia. Strains M. loti B816.2K (I), S. meliloti MCH3 (II) and UP61.2R (I and II) were used to inoculate seeds separately (solid bars) or as a mixture (lined bars) of the speci®c rhizobia with P. ¯uorescens strain. Rhizospheric population values (log CFU (g root) 21) used in the graph correspond to the means of ®ve replicates. Numbers in parentheses represent the initial inocula used. Population sizes were logarithmically transformed before analysis. Data from each graph were independently analysed in a three-factor (treatment, time and block) ANOVA with interactions. Different letters in the same graph indicate signi®cant difference for time factor. Treatment effect was signi®cant for P. ¯uorescens but not for rhizobia. No effect of the block factor and no interactions were found P , 0:05:
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L. De La Fuente et al. / Soil Biology & Biochemistry 34 (2002) 545±548
UP143 (Fig. 1). About 75% of the plants were nodulated by the 11th day after inoculation, and 100% by the 27th day. When UP148 was co-inoculated on seeds, a delay was observed during the ®rst 13 days. By the eighth day after inoculation only 25% of the plants containing UP148 nodulated, while 60% of the plants inoculated with B816 alone or together with UP61 or UP143, had nodules. However, all treatments reached a similar nodulation level by the 13th day. Similar results were found in three independent experiments. The effect of M. loti B816 on dry weight of birdsfoot trefoil was not negatively affected P , 0:05 by the introduction of native P. ¯uorescens strains. Even more, UP61 showed a moderate plant-growth promoting effect in pot assays (Table 1). To determine if the effect of UP61 depends on the forage legume, assays with alfalfa and white clover were performed in soil. Shoot dry weights of both plant species, inoculated with their speci®c commercial inoculants, were not affected by the presence of UP61 (Table 1). Competitive exclusion among rhizospheric microorganisms is directly associated to the ability of colonising the root surface successfully. Colonisation of birdsfoot trefoil and alfalfa rhizospheres by rhizobia and P. ¯uorescens UP61.2R were not affected by the presence of each other (Fig. 2). In general, population densities of pseudomonads and rhizobia were maintained at the initial levels for the ®rst days, and then a small drop was observed. The exception to this trend was S. meliloti MCH3 whose population density increased with time. Population densities of both rhizobia strains did not differ when they were inoculated alone or co-inoculated with P. ¯uorescens UP61.2R (P 0:73 for M. loti and P 0:64 for S. meliloti). A significant difference in pseudomonads densities on roots was found between treatments inoculated with UP61.2R alone or co-inoculated with rhizobia in both plant species, which was related to the differences found in the initial seed inocula (Fig. 2). A direct linear relationship between bacterial seed and root populations has been previously demonstrated by Bull et al. (1991). In birdsfoot trefoil UP61.2R densities on seeds were larger when co-inoculated with rhizobia (2 £ 10 6 versus 6 £ 10 7CFU seed 21), while in alfalfa were smaller when co-inoculated (2 £ 10 7 versus 8 £ 10 5CFU seed 21). However, for all UP61.2R treatments, the dynamics of populations declined in a similar way in spite of the co-inoculation with rhizobia. Despite the in vitro rhizobia inhibition observed, the presence of Pseudomonas did not signi®cantly affect rhizobia symbiosis (Table 1, Figs. 1 and 2). This suggests that the iron concentration might not be extremely low in the rhizospheric environment of plants growing in natural soil (Loper and Lindow, 1994). The knowledge that native Pseudomonas strains are able to survive on peat for more than 6 months (Bagnasco et al.,
1998) and our results, indicate that a mixed pseudomonads± rhizobia inoculant could be developed in order to improve forage legumes establishment and yield. Acknowledgements This work was partially supported by PEDECIBA, IFS and INCO-DC EU. References Altier, N., 1996. Enfermedades de leguminosas forrajeras: diagnoÂstico, epidemiologõÂa y control. In: DõÂaz, M. (Ed.). Manejo de Enfermedades en Cereales de Invierno y Pasturas. Serie TeÂcnica No. 74 INIA, Montevideo, Uruguay, pp. 87±104. Altier, N., Pastorini, D., 1988. Curasemillas en leguminosas forrajeras: efecto sobre los rizobios. Est. Exp. La Estanzuela. Hoja de divulgacioÂn No. 74. 2p. Bagnasco, P., De La Fuente, L., Gualtieri, G., Noya, F., Arias, A., 1998. Fluorescent Pseudomonas spp. as biocontrol agents against forage legume root pathogenic fungi. Soil Biology and Biochemistry 30, 1317±1322. Beringer, J.E., 1974. R factor transfer in Rhizobium leguminosarum. Journal of General Microbiology 84, 188±198. Bull, C.T., Weller, D.M., Thomashow, L.S., 1991. Relationship between root colonisation and suppression of Gaeumannomyces graminis var. tritici by Pseudomonas ¯uorescens strain 2-79. Phytopathology 81, 954±959. Duffy, B.K., DeÂfago, G., 1999. Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas ¯uorescens biocontrol strains. Applied and Environmental Microbiology 65, 2429±2438. Homma, Y., Sato, Z., Hirayama, F., Konno, K., Shirahama, H., Suzui, T., 1989. Production of antibiotics by Pseudomonas cepacia as an agent for biological control of soilborne plant pathogens. Soil Biology and Biochemistry 21, 723±728. Keel, C., Voisard, C., Berling, C.H., Khar, G., DeÂfago, G., 1989. Iron suf®ciency, a prerequisite for the suppression of tobacco black root rot by Pseudomonas ¯uorescens strain CHA0 under gnotobiotic conditions. Phytopathology 79, 584±589. King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for the demonstration of pyocianin and ¯uorescein. Journal of Laboratory and Clinical Medicine 44, 301±307. Loper, J.E., Lindow, S.W., 1994. A biological sensor for iron available to bacteria in their habitats on plant surfaces. Applied and Environmental Microbiology 60, 1934±1941. Slininger, P.J., Jackson, M.A., 1992. Nutritional factors regulating growth and accumulation of phenazine-1-carboxylic acid by Pseudomonas ¯uorescens 2-79. Applied Microbiology and Biotechnology 37, 388± 392. Thomashow, L.S., Weller, D.M., 1996. Current concepts in the use of introduced bacteria for biological disease control: mechanisms and antifungal metabolites. In: Stacey, G., Keen, N. (Eds.). Plant±Microbe Interactions, vol. 1. Chapman & Hall, New York, pp. 187±235. Vincent, J.M., 1970. A Manual for the Practical Study of the Root Nodule Bacteria. IBP Handbook No. 15. Blackwell, New York. Weller, D.M., Cook, R.J., 1983. Suppression of take-all of wheat by seed treatments with ¯uorescent pseudomonads. Phytopathology 73, 463± 469.