Role of Nodulation-Enhancing Rhizobacteria in the Promotion of Medicago sativa Development in Nutrient-Poor Soils

Abstract

Legumes are usually used as cover crops to improve soil quality due to the biological nitrogen fixation that occurs due to the interaction of legumes and rhizobia. This symbiosis can be used to recover degraded soils using legumes as pioneer plants. In this work, we screened for bacteria that improve the legume-rhizobia interaction in nutrient-poor soils. Fourteen phosphate solubilizer-strains were isolated, showing at least three out of the five tested plant growth promoting properties. Furthermore, cellulase, protease, pectinase, and chitinase activities were detected in three of the isolated strains. Pseudomonas sp. L1, Chryseobacterium soli L2, and Priestia megaterium L3 were selected to inoculate seeds and plants of Medicago sativa using a nutrient-poor soil as substrate under greenhouse conditions. The effects of the three bacteria individually and in consortium showed more vigorous plants with increased numbers of nodules and a higher nitrogen content than non-inoculated plants. Moreover, bacterial inoculation increased plants' antioxidant activities and improved their development in nutrient-poor soils, suggesting an important role in the stress mechanisms of plants. In conclusion, the selected strains are nodulation-enhancing rhizobacteria that improve leguminous plants growth and nodulation in nutrient-poor soils and could be used by sustainable agriculture to promote plants' development in degraded soils.

Keywords: Medicago; abiotic stress; biofertilizers; degraded soils; legumes; nodulation-enhancing rhizobacteria (NER); nutrient poverty; plant growth-promoting rhizobacteria (PGPR).

Figure 1

Characterization of isolated bacteria. (A) Number of PGP properties in each strain; X-axis indicated the different isolated strains. (B) Percentage of enzymatic activities in the isolates.

Figure 2

In vitro effects of selected PGPR in M. sativa. (A) Number of germinated seeds in 7 days with different inoculums. Values are mean ± S.D. (n = 50). (B) Dry weight of shoots and roots of M. sativa plants after 30 days in BNM plates. Values are mean ± S.D. (n = 5). Different letters indicate means that are significantly different from each other. Lowercase and uppercase letters are used to qualify different variables and are not comparable among them (one-way ANOVA; LSD test, p < 0.001). (C) Number of nodules in plants of M. sativa after 30 days in BNM plates with different inoculums. Values are mean ± S.D. (n = 5). Different letters indicate means that are significantly different from each other (one-way ANOVA; LSD test, p < 0.001). C-: without inoculation; MA11: inoculation with E. medicae MA11; L1: inoculation with strain Pseudomonas sp. L1; L2: inoculation with Chryseobacterium soli L2; L3: inoculation with Priestia megaterium L3; L1+MA11: inoculation with strains L1 and MA11; L2+MA1: inoculation with strains L2 and MA11; L3+MA11: inoculation with strains L3 and MA11; CSL: inoculation with a consortium of strains L1, L2, L3, and MA11.

Figure 3

Effects of selected PGPR in M. sativa under greenhouse conditions in nutrient-poor soils. (A) Length of shoots and roots of M. sativa plants after 60 days. Values are mean ± S.D. (n = 16). Different letters indicate means that are significantly different from each other. Lowercase and uppercase letters are used to qualify different variables and are not comparable among them (one-way ANOVA; LSD test, p < 0.001). (B) Number and size of leaves in plants of M. sativa after 60 days from germination. Values are mean ± S.D. (n = 16). The size of the circles indicates the mean of the diameter of the leaves. Different letters indicate means that are significantly different from each other. Lowercase letters are used to qualify the number of leaves per plant and uppercase letters are used to qualify the diameter of leaves and are not comparable among them (one-way ANOVA; LSD test, p < 0.0001). (C) Dry weight of shoots and roots of M. sativa plants after 60 days from germination. Values are mean ± S.D. (n = 16). Different letters indicate means that are significantly different from each other. Lowercase and uppercase letters are used to qualify different variables and are not comparable among them (one-way ANOVA; LSD test, p < 0.001). (D) Nitrogen content in plants of M. sativa after 60 days from germination. Values are mean ± S.D. (n = 16). Different letters indicate means that are significantly different from each other. Lowercase and uppercase letters are used to qualify different variables and are not comparable among them (one-way ANOVA; LSD test, p < 0.001). (E) Number of nodules in plants of M. sativa after 60 days from germination. Values are mean ± S.D. (n = 16). Different letters indicate means that are significantly different from each other (one-way ANOVA; LSD test, p < 0.0001). C-: without inoculation; MA11: inoculation with strain MA11; L1+MA11: inoculation with strains L1 and MA11; L2+MA1: inoculation with strains L2 and MA11; L3+MA11: inoculation with strains L3 and MA11; CSL: inoculation with a consortium of strains L1, L2, L3, and MA11.

Figure 4

Photosynthetic parameters. (A) Net photosynthetic rate, (B) stomatal conductance, (C) maximum quantum efficiency of PSII photochemistry, and (D) total chlorophyll content in plants of M. sativa after 60 days from germination under greenhouse conditions with a nutrient-poor soil as substrate and different inoculation treatments. Values are mean ± S.D. (n = 16). Different letters indicate means that are significantly different from each other (one-way ANOVA; LSD test, p < 0.005). C-: without inoculation; MA11: inoculation with strain MA11; L1+MA11: inoculation with strains L1 and MA11; L2+MA1: inoculation with strains L2 and MA11; L3+MA11: inoculation with strains L3 and MA11; CSL: inoculation with a consortium of strains L1, L2, L3, and MA11.

Figure 5

Level of antioxidant activities. (A) Catalase, (B) ascorbate peroxidase, (C) guaiacol peroxidase, (D) superoxide dismutase activities in leaves of M. sativa 60 days after germination under greenhouse conditions with a nutrient-poor soil as substrate and different inoculation treatments. Values are mean ± S.D. (n = 16). Different letters indicate means that are significantly different from each other (one-way ANOVA; LSD test, p < 0.001). C-: without inoculation; MA11: inoculation with strain MA11; L1+MA11: inoculation with strains L1 and MA11; L2+MA1: inoculation with strains L2 and MA11; L3+MA11: inoculation with strains L3 and MA11; CSL: inoculation with a consortium of strains L1, L2, L3, and MA11.