The co-inoculation of Bradyrhizobium with other plant growth-promoting microorganisms (arbuscular mycorrhizal fungi, Bacillus, Azospirillum, and Trichoderma) has been increasingly used in agriculture (Kaschuk et al., 2010; 2022; Zeffa et al., 2020; Barbosa et al., 2021, 2022). Co-inoculation relies on the assumption that multifunctional microbial consortia can potentially enhance crops through complementary mechanisms of growth promotion (Pandey et al., 2012; Molina-Romero et al., 2017; Besset-Manzoni et al., 2018; Liu et al., 2023). In this study, soybean plants were subjected to inoculation by individual strains or consortia of four microbial inoculants containing Bradyrhizobium, Azospirillum, Bacillus, and Trichoderma, with the hypothesis that the complementarity effects generated by multifunctional microbial inputs lead to higher productivity that the inoculation of solo strains (Table 1). Overall, our results indicated that inoculating multifunctional microbial consortia may be viable for maximizing crop yields. Still, certain aspects associated with consortia construction need to be carefully considered.
One significant aspect is that inoculation of specific multifunctional microbial consortia resulted in a low number of germinated seeds and negatively impacted plant emergence in both the greenhouse and the field, leading to diminished plant density (Table 3). Seed germination is influenced by several factors, including seed quality and environmental conditions (Tekrony et al., 1980; De Luca and Hungria, 2014; De Luca et al., 2014). Additionally, it can be affected by inputs applied, including inoculants, during sowing (Queiroz-Rego et al., 2018). The reduced germination rates in plants subjected to inoculation by consortia may have been attributed to the larger microbial cell density in the inoculants, potentially overwhelming the rhizosphere with biochemical compounds produced during the establishment of symbiotic or commensal associations (Gadhave et al., 2016). Furthermore, it is plausible that the introduced microorganisms engaged in antibiosis or competition with other beneficial populations (Gadhave et al., 2016; Besset-Manzoni et al., 2018). Usually, a low plant density in the field correlates with decreased agricultural land productivity across various crops (Egli, 1988). However, there are reports that photosynthesis and biological N2 fixation per plant can be augmented, compensating yield (De Luca and Hungria, 2014).
During the early stages of crop development (V4), the inoculation of specific microbial consortia had a negative impact on plant growth. For instance, the inoculation of Bradyrhizobium, Azospirillum, and Trichoderma (T11) resulted in shorter roots and shoots compared to the co-inoculation of Bradyrhizobium and Azospirillum (T6) (Table 4, 5). Remarkably, these results support the effectiveness of co-inoculation of Bradyrhizobium and Azospirillum in maximizing plant growth and yield (Barbosa et al., 2021). However, these findings contradict our initial hypothesis that including a more diverse consortium of microorganisms would lead to higher plant growth. The results involving the consortium, including Trichoderma, align with the decreased soybean shoot biomass observed in plants inoculated with T. asperellum and Azospirillum, along with several other strains, when compared to the non-inoculated control (Silva et al., 2020). Therefore, we propose that the responses are not solely explained by the number of microbial strains but rather by the compatibility of the microorganisms within the consortia. In our experiment, this could potentially be attributed to the antibiosis effect of the Trichoderma strain.
Under greenhouse conditions, the inoculation of Trichoderma alone or in combination with Bacillus (T5, T8, T11, and T12) also led to a significant reduction in nodule mass during the R1 stage, in comparison to the treatment single inoculated with Azospirillum (T3) (Table 5). The reasons behind this inhibitory effect might rely on properties such as the production of hydrogen cyanide (HCN), a highly toxic volatile compound (Rawat and Tewari, 2011; Abdenaceur et al., 2022), or another antibiotic molecule.
Nonetheless, any adverse effects attributed to Trichoderma may have exhibited transient characteristics throughout crop development. Plants subjected to the inoculation of multifunctional microbial consortia (T11 and T12, both including Trichoderma) had higher shoot N concentration compared to those non-inoculated or single inoculated with Bradyrhizobium (Table 6), even though their nodule masses were comparatively lower (Table 5). The variations in nodulation patterns imply that Trichoderma could regulate the allocation of plant photosynthates, thereby influencing nodulation negatively. However, its impact on various activities, such as phytohormone production (Iturralde et al., 2020), could potentially have stimulated bacteroid activity later in crop development. This stimulation, in turn, may have facilitated increased biological N2 fixation, irrespective of the initial plant size.
Bacillus strains were incorporated into the experiment due to their role in solubilizing phosphate in the rhizosphere soil, enhancing plant P nutrition (Saxena et al. 2020; Sousa et al. 2020). Interestingly, the analysis of P content in plants grown in the greenhouse and inoculated solely with Bacillus did not reveal significant differences compared to their non-inoculated counterparts (Table 5). However, at the R1 stage in the greenhouse, plants inoculated with microbial consortia containing Bacillus, Bradyrhizobium, and Trichoderma exhibited higher P contents than those inoculated solely with Bacillus (T11; Table 6) despite having shorter root length (Table 5). This suggests that plants inoculated with microbial consortia displayed greater efficiency in absorbing soil P.
In essence, for both N and P, greenhouse-grown plants inoculated with multifunctional microbial consortia demonstrated a superior nutritional status than plants inoculated with fewer microbial strains. The absence of differences in nutritional status observed in the field could potentially be attributed to its favorable soil fertility levels (Table 2), wherein nutrient absorption is not a limiting factor.
Inoculations of multifunctional microbial consortia may have altered the physiological functioning of soybean plants. First, the inoculation of consortia negatively affected the vegetative growth of the crop in the greenhouse (reduced root length and mass, and number of nodules; Table 4) and in the field (reduced height and number of nodules; Table 5). Later, these presumed negative results disappeared, as the soybean cultivar (indeterminate cycle) exhibited high phenotypic plasticity to compensate for initial adversities and recovered its productive capacity to satisfactory agronomic levels. Single inoculation with Bradyrhizobium resulted in the highest plant growth in both experiments (Table 4, 5), and despite showing differences in the number of plants per plot (Table 3), no statistical differences were verified in grain yield (Table 7). Conversely, the inoculation with multifunctional microbial consortia containing Bradyrhizobium, Azospirillum, Bacillus, and Trichoderma produced smaller plants in the vegetative stage but later resulted in higher grain yield. Similarly, Moretti et al. (2020) demonstrated that inoculation of microbial consortia (i.e., Bradyrhizobium spp., Rhizobium spp., B. subtilis, and A. brasilense) increased grain yield by 485 kg ha-1 in comparison to the single inoculation with Bradyrhizobium. Given this, we hypothesize that soybean inoculation with multifunctional microbial consortia consisting of Bradyrhizobium and another plant growth-promoting microorganism can promote yield gain despite the reduced initial growth due to the crop's phenotypic plasticity (Kaschuk et al., 2012; De Luca and Hungria, 2014; De Luca et al., 2014).
The grain composition analyses showed that multiple inoculations altered the redistribution of photosynthates during the reproductive period of the crop. In the case of T11 (responsible for the highest grain mass), it produced grains with lower lipid values compared to the non-inoculated T1 (Table 7), indicating a dilution effect on the composition. Moreover, it is possible that the high number of microorganisms decreased grain lipid concentration due to the degradation of plant photosynthates by rhizosphere respiration (Marschner, 1995). However, Kaschuk et al. (2010; 2012) demonstrated that photosynthetic rates can be compensated by the activity of rhizobia and mycorrhizal fungi in the rhizosphere, resulting in no differences in grain lipid concentration due to Bradyrhizobium inoculation. Furthermore, Marra et al. (2019) showed that soybean inoculation exclusively with Trichoderma increased lipid concentration in the grains. In any case, the results highlight that inoculations of multifunctional microbial consortia strongly depend on the combination of the strains used when designing the consortia.
In summary, despite the potentially favorable perspective provided by the results of this study regarding the recommendation of multifunctional microbial consortia inoculations containing different plant growth-promoting microorganisms in soybean cultivation, several aspects remain to be comprehensively understood. There is a significant research interest in exploring various microbial consortia to ensure more sustainable agriculture with minimal environmental impact through microorganism utilization (Pandey et al. 2012, Liu et al. 2023). Another beneficial aspect of using consortia lies in the potentially higher stability and survival of consortia rather than solo strains when introduced in the soils. Although testing this functional response trait was beyond the scope of this project, it highlights the need to carefully design microbial consortia according to the intended aim. Moreover, given that the responses of microbial inoculation may vary as a function of several factors related to the functional traits of the microbes themselves, but also the native soil microbiome, plant genotype, and environmental conditions, it is imperative to carefully test any consortia before field applications.