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Characterization of rhizobia isolated from soybean in Mozambique and strategies to maximize the contribution of biological nitrogen fixation

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Soybean inoculation with effective rhizobial strains makes unnecessary the use of nitrogen fertilizers in the tropics. A frequently reported problem is the failure of the inoculant strain to overcome the competition imposed by indigenous rhizobial populations. The screening of indigenous rhizobia, adapted to local conditions, for highly effective strains for use as inoculants represents a promising strategy in overcoming inoculation failure. On the other hand, the strong evidences of inoculation success in areas with high rhizobial populations in Brazil open a window for inoculation research in other geographic regions in the world. Therefore, the objectives of this thesis were: 1) To characterize indigenous rhizobia and to identify strains that hold potential to be included in inoculant formulations for soybean production, with both promiscuous and non-promiscuous varieties, in Mozambican agro-climatic conditions; 2) To compare the performance of four elite strains from Brazil (SEMIAs 587, 5019, 5079 and 5080) and another strain from the US (USDA 110) in trials carried out with non-promiscuous soybean varieties in Brazil and Mozambique. For the first objective, 105 isolates were obtained from nodules of promiscuous soybean grown at 15 sites in Mozambique and screened for N-fixation effectiveness in the greenhouse along with four strains used in inoculants in Brazil, Bradyrhizobium japonicum SEMIA 5079, B. diazoefficiens SEMIA 5080, B. elkanii strains SEMIA 587 and SEMIA 5019, and one strain used in Africa, B. diazoefficiens USDA 110. Eighty-seven isolates confirmed the ability to form effective nodules on soybean and were used for genetic characterization, by rep-PCR (BOX) and DNA sequencing of the 16S rRNA gene, and symbiotic effectiveness. The BOX-PCR fingerprinting revealed remarkable genetic diversity, with 41 clusters formed, considering an arbitrary similarity level of 65%. The 16S rRNA analysis assigned the isolates to the Bradyrhizobium (75%) and Agrobacterium/Rhizobium (25%) clades. Great variability in symbiotic effectiveness was detected among the indigenous rhizobia from Mozambique with ten isolates performing better than USDA 110, the best reference strain, and 51 isolates with inferior performance than all the reference strains. Thirteen of the isolates with the highest symbiotic effectiveness were evaluated, along with the five reference strains, in two promiscuous (TGx 1963-3F and TGx 1835-10E) and one non-promiscuous (BRS 284) soybean varieties in a second greenhouse trial. The 13 isolates were also characterized for tolerance to acidity and alkalinity (pH 3.5 and 9.0, respectively), salinity (0.1, 0.3 and 0.5 mol L−1 of NaCl) and high temperatures (35, 40 and 45 ºC) in vitro. Five isolates, three (Moz 4, Moz 19 and Moz 22) belonging to the sub-group B. elkanii and two (Moz 27 and Moz 61) assigned to the sub-group B. japonicum, consistently showed high symbiotic effectiveness, suggesting that the inoculation with indigenous rhizobia adapted to local conditions represents a possible strategy for increasing yields of soybean in Mozambique. For the second objective, the five reference strains tested in the first study were evaluated in the 2013/2014 and 2014/2015 crop seasons in Brazil (four sites) and Mozambique (five sites). In both countries, the trial sites were in tropical and temperate climate zones and the areas had soybean rhizobial population ranging from < 10 to over 1×103 cells g−1 of soil. The treatments were: (1) NI, non- inoculated control with no N-fertilizer; (2) NI+N, non-inoculated control with 200 kg of N ha−1; and inoculated with (3) Bradyrhizobium japonicum SEMIA 5079; (4) B. diazoefficiens SEMIA 5080; (5) B. elkanii SEMIA 587; (6) B. elkanii SEMIA 5019; (7) B. diazoefficiens USDA 110; (8) SEMIAs 5079 + 5080 (treatment only tested in Brazil). The best inoculation treatments across sites and crop seasons in Brazil were SEMIAs 5079 + 5080, SEMIA 5079 and USDA 110, with average grain yield gains of 4–5% in relation to the NI treatment. Strains SEMIA 5079, SEMIA 5080, SEMIA 5019 and USDA 110 were the best in Mozambique, with average 20–29% grain yield gains over the NI treatment. Moreover, the four best performing strains in Mozambique resulted in similar or better yields than the NI+N treatment, confirming the BNF as a sustainable option to the use of environmentally polluting N-fertilizers. The results also confirm the feasibility of transference of technologies related to BNF with soybean between countries with similar agro-climates.
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