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Legume nodulation by rhizobia can supply crops with nitrogen and reduce environmental impacts caused by chemical fertilization. The soybean crop in Brazil is an impressive example of how biological N2 fixation can be employed with a plant species of high economic value. However, the development of more productive cultivars, along with the increasing global climatic changes demand agricultural practices to become more productive and yet more environmentally friendly. Plant growth-promoting rhizobacteria (PGPR) are highly beneficial to agriculture worldwide, acting in plant nutrition, protection, and growth stimulation. Azospirillum is, certainly, the most employed PGPR in the world, but little is known about its interaction with rhizobia, when both are applied to legume seeds. We have evaluated the co-inoculation of bradyrhizobia and azospirilla on soybean seeds under different soil and climate conditions in Brazil. Our results demonstrated that co-inoculation is efficient and beneficial to the crop, and promotes yield increases without adding any chemical N fertilizers even in soils where established populations of soybean bradyrhizobia exist. The strategy of co-inoculation thus represents a new biotechnological tool to improve soybean yield without adding any chemical N fertilizers, thus contributing to current practices of sustainability in agriculture.

The utilization of inoculants containing Azospirillum is becoming more popular due to increasing reports of expressive gains in grain yields. However, incompatibility with pesticides used in seed treatments represents a main limitation for a successful inoculation. Therefore, in this study we searched for alternatives methods for seed inoculation of maize and wheat, aiming to avoid the direct contact of bacteria with pesticides. Different doses of inoculants containing Azospirillum brasilense were employed to perform inoculation in-furrow, via soil spray at sowing and via leaf spray after seedlings had emerged, in comparison to seed inoculation. Experiments were conducted first under greenhouse controlled conditions and then confirmed in the field at different locations in Brazil. In the greenhouse, most parameters measured responded positively to the largest inoculant dose used in foliar sprays, but benefits could also be observed from both in-furrow and soil spray inoculation. However, our results present evidence that field inoculation with plant-growth promoting bacteria must consider inoculant doses, and point to the need of fine adjustments to avoid crossing the threshold of growth stimulation and inhibition. All inoculation techniques increased the abundance of diazotrophic bacteria in plant tissues, and foliar spray improved colonization of leaves, while soil inoculations favored root and rhizosphere colonization. In field experiments, inoculation with A. brasilense allowed for a 25 % reduction in the need for N fertilizers. Our results have identified alternative methods of inoculation that were as effective as the standard seed inoculation that may represent an important strategy to avoid the incompatibility between inoculant bacteria and pesticides employed for seed treatment.

Biological nitrogen fixation with the soybean crop can be improved by seed inoculation with superior Bradyrhizobium strains, but factors that reducethe population of inoculated bradyrhizobiaon the seedwill directly affect the efficiencyof the process. Seed treatment with fungicides has been broadly practiced as cheap insurance against seed-and soil-borne pathogens, but toxicity of most fungicides to bradyrhizobia has often been underestimated. The compatibility between seed treatment with fungicides in single or mixed applications (including Benomyl, Captan, Carbendazin, Carboxin, Difenoconazole, Thiabendazole, Thiram, Tolylfluanid) and bradyrhizobial inoculants was examined in laboratory, greenhouse and field experiments during five crop seasons in Brazil. Bacterial survival on the seeds was severely affected by all fungicides, resulting in mortalities of up to 62% after only 2 h and of 95% after 24 h. Fungicides also reduced nodule number, total N in grains and decreased yield by up to 17%. The toxic effects of fungicides were more drastic in sandy soils withoutsoybean inoculation and cropping history, reducing nodulation by up to 87%, but were also important in areas with established populations of soybean bradyrhizobia. Therefore, fungicides should be used only when the seeds or soil are contaminated with pathogens,otherwisebiologicalN 2 fixation may be severely affected.