Integrated use of Consortia-based Microbial Inoculants and Nutrient Complex Stimulates the Rhizosphere Microbiome and Soybean Productivity

Soybean (Glycine max (L.) Merrill) is among the most important cultivated crops worldwide due to its high agro-economic value and diverse utility in the feed and food, pharmaceutical and other industries. Global consumption of chemical fertilizers and pesticides for agricultural productivity to meet the global demand of the increasing world population continues to rise, despite their harmful impacts on the environment and human health (Devi et al., 2022). In order to reduce the use of agrochemicals, increase organic farming and achieve sustainable soybean production under environmental, climatic and economic challenges, researchers have investigated different alternatives, including the use of microbial inoculants (O’Callaghan et al., 2022).
       
Seed inoculation with nitrogen-fixing Bradyrhizobium strains improves nodulation, plant properties, seed yield and yield quality while satisfying soybean nitrogen demands (Nakei et al., 2022). Besides rhizobia, Bacillus and Azotobacter strains are the most commercially utilized plant growth-promoting bacteria (PGPB) in the production of field and vegetable crops (Gómez-Godínez et al., 2023). Their use as biofertilizers is based on nitrogen fixation, solubilization of nutrients, production of siderophores and synthesis of plant hormones, which have significant effects on soil and crop productivity (Aloo et al., 2022; Virk et al., 2022). Additionally, Bacillus strains can be used as biopesticides and improve the health of plant-soil systems via the production of various antimicrobial compounds involved in the biocontrol of plant pathogens (Miljaković et al., 2020; Kannan et al., 2021).
       
The effectiveness of microbial inoculants depends upon their ability to survive and multiply in soils, colonize plant roots and perform their beneficial actions in the complex plant-soil system (Liu et al., 2022). However, various abiotic and biotic factors, as well as improper management practices, can obstruct the inoculation outcome in the field (Kong et al., 2018). Therefore, it is necessary to develop microbial formulations that will improve the actions of each component and comprehend the issues affecting their application (Herrmann and Lesueur, 2013). In this regard, one of the most promising approaches is the use of consortia-based microbial inoculants (Khan, 2022). Furthermore, the application of nutrient complexes through soil, foliar and seed treatments provides the essential elements for growth, development and metabolism of both microorganisms and plants, ensuring their maximum uptake and a minimum loss to the environment (Farooq et al., 2012). A deficiency of macro- and micronutrients in the soil combined with the inappropriate application of fertilizers may result in micronutrient malnutrition (Arabhanvi et al., 2015). Nevertheless, knowledge on the integrated use of microbial inoculants and nutrients for sustainable agricultural activities and the effects of such formulations is still poorly documented. In particular, the responses of the rhizosphere microbiome and soybean plant to consortia of rhizobia and other PGPB combined with nutrients have not yet been well explored. Understanding such interactions should contribute to improving the effectiveness of inoculants in field conditions through the joint application of microbial consortia and plant nutrients. Therefore, we examined the effect of a microbial consortia consisting of nitrogen-fixing Bradyrhizobium japonicum strains in different combinations with plant growth-promoting strains of Azotobacter chroococcum, Bacillus subtilis and Bacillus megaterium) and a nutrient complex (S, Mg, Mn, Fe, Zn, Cu, B and Mo) on the abundance and activity of microorganisms in the rhizosphere, soybean growth and development, as well as the quantity and quality of seed yield under field conditions.

The experiment was conducted at the Rimski šanèevi experimental field (45°19'N, 19°50'E), Institute of Field and Vegetable Crops (IFVC, Novi Sad, Serbia), during the 2021 growing season. The soil was classified as Haplic Chernozem (FAO, 2015). It belonged to a group of humus soils with a slightly alkaline pH reaction, a low content of available phosphorus and a high content of available potassium (Table 1). The mean temperature and precipitation sum at the experimental field were 19.2°C and 319.1 mm, respectively.
 

       
Bacterial strains from the culture collection of the Section for Microbiological Preparations (IFVC) were used as inoculants. They were cultured in their respective liquid media: yeast extract manitol medium for Bradyrhizobium strains, nutrient medium for Bacillus strains and Burk’s nitrogen-free medium for Azotobacter strains (Hi Media Laboratories Pvt. Limited, Mumbai, India). The chemical composition of the nutrient complex was as follows (% m/m): S- 5.2; Mg- 3; Mn- 1.5; Fe- 1; Zn- 1; Cu- 0.5; B- 0.3; Mo- 0.01.
       
Seeds of the soybean cultivar Apolo (maturity group I) were obtained from the Legume Department (IFVC). The following seed treatments were tested: 1. control (without bacterial mixture and nutrient compex); 2. Bradyrhizobium japonicum (BJ1, BJ2, BJ4, BJ6, BJ7, BJ8); 3. Bacillus subtilis (B5, B7, B13, B32); 4. Bacillus megaterium (B8, B12, B15, B17); 5. Br. japonicum + B. subtilis; 6. Br. japonicum + B. megaterium; 7. B. subtilis + Azotobacter chroococcum; 8. B. megaterium + A. chroococcum; 9. Br. japonicum + B. subtilis + A. chroococcum; 10. Br. japonicum + B. megaterium + A. chroococcum; 11. Br. japonicum + nutrient complex; 12. Br. japonicum + B. subtilis + nutrient complex; 13. Br. japonicum + B. megaterium + nutrient complex; 14. Br. japonicum + B. subtilis + A. chroococcum + nutrient complex; 15. Br. japonicum + B. megaterium + A. chroococcum + nutrient complex. Seeds were treated just before sowing by applying a liquid bacterial inoculum (109 CFU/ml) and nutrient complex to the seeds using sterilized peat as a carrier. A total of 40 ml of mixture (bacterial inoculum with or without nutrient complex) on peat was applied per treatment, while the volume of individual components in each mixture was equal. Agrochemicals were not applied. The experiment was carried out in three replications in a split-plot design and the plot size was 5 × 3 m.
       
The effect of seed treatments on microbial abundance and dehydrogenase activity in soybean rhizosphere was determined at full flowering (R2) and full maturity (R8), using the indirect dilution plate method (Trolldenier, 1996) on appropriate nutrient media (Hi Media Laboratories Pvt. Limited, Mumbai, India). Microbial abundance included: total bacteria (10^–7; soil agar), free-living N₂-fixers (10^–6; nitrogen-free medium), actinomycetes (10^–4; synthetic agar) and fungi (10^–4; Czapek-Dox agar). Dehydrogenase activity (DHA) was determined spectrophotometrically according to the method with triphenyltetrazolium chloride (TTC) (EN ISO 23753-1:2019). All microbial analyses were performed in three replicates and the values from two samplings were averaged.
       
The effect of seed treatments on plant height, plant weight, pod number, pod weight, seed number, seed weight and seed yield was determined at full maturity (R8) of soybean. Ten plants were randomly collected from each plot for biometric measurements and yield structure components and the average value for all samplings per plant was calculated. The seed yield from the plots per 1 ha was calculated (based on the 14% moisture content). The content of protein and oil in seeds was determined with the near-infrared spectroscopy (NIRS) method using an Antaris II FT NIR device (Thermo Fisher Scientific, Waltham, MA, USA), while protein and oil yields were calculated from seed yield and the percentage of a given component in seeds.
       
The data were statistically processed (StatSoft Inc., Tulsa, USA) using the analysis of variance (ANOVA) statistical method, followed by mean separation according to the Fisher’s LSD test (P<0.05).

Soil microbial communities have a crucial role in nutrient cycling, organic matter decomposition and many other processes that are necessary for the growth and development of plants and ecosystem functioning (Bender et al., 2016). The results of this study indicate a significant influence of seed treatments on the total bacteria, free nitrogen-fixing bacteria, actinomycetes and dehydrogenase activity (Table 2). Applied treatments mostly had a positive effect on the microbial parameters and the increase compared to control ranged as follows: total bacteria (6-111%), N₂-fixers (15-181%), fungi (7-117%), actinomycetes (15-186%) and dehydrogenase (2-110%). The greatest proliferation of total bacteria, N₂-fixers, actinomycetes and dehydrogenase was obtained by applying consortia-based inoculants with nutrient complex, while the highest abundance of fungi was recorded after Br. japonicum + nutrients treatment. Numerous reports suggest that microbial inoculants can influence indigenous microbial communities, thus promoting free-living nitrogen-fixing bacteria as well as other beneficial bacteria or fungi (Trabelsi and Mhamdi, 2013). Furthermore, Bana et al., (2022) obtained significant improvements in microbial enzyme activity with micronutrient-supplemented fertilizer (N, P, K, Fe, Zn and B). Similarly, Egamberdieva et al., (2018) indicated the positive effect of nutrients (N, P and Mg) on rhizosphere colonization, bacterial proliferation and symbiotic performance of rhizobia inoculated soybean.
 

       
The use of microbial inoculants, especially with nutrients, increases microbial abundance and activity, suggesting their potential to enhance soil fertility. Microbial inoculants have the ability to improve nutrient availability and nutrient uptake, promote the health of soil and crops and contribute to high yields in sustainable ways. Beneficial microorganisms improve the biological, chemical and physical properties of soil and, subsequently, the productivity of agricultural crops. In this study, soybean parameters, namely plant height and weight, pod number and weight, seed number and seed weight, as well as protein yield, were significantly influenced by seed treatments (Table 3 and 4).
 

 

       
A higher effectiveness versus control was observed for most applied treatments, which resulted in the following improvements: plant height (1-23%), plant weight (1-51%), pod number (1-96%), pod weight (2-100%), seed number (5-79%), seed weight (7-102%), seed yield (6-25%), protein content (1-4%), protein yield (0.3-29%), oil content (0.4-3%) and oil yield (2-26%). The highest improvements in plant weight, pod number and weight, seed number and weight in relation to the control were observed with Br. japonicum + nutrients and B. megaterium + A. chroococcum. Seed treatments with inoculants and nutrient complex had a primacy over single and combined inoculants in the case of soybean seed yield, as well as protein and oil yield, while Br. japonicum + B. megaterium + A. chroococcum + nutrients had the best effect. The individual application of Br. japonicum strains, as well as their combination with B. subtilis, A. chroococcum and nutrients, had the equally best effect on the protein content. The highest and significant increase, compared to the control, in the oil content was obtained from inoculation with B. megaterium. Similarly, Moretti et al., (2020) described that the inoculation with bacterial consortia (Br. japonicum, Br. diazoefficiens, B. subtilis and Azospirillum brasilense) increased grain yield and quality of soybean under field conditions when compared to the single inoculation with Bradyrhizobium. In a meta-analysis of studies from 1987 to 2018, Zeffa et al., (2020) reported that co-inoculation of soybean with Bradyrhizobium and PGPB resulted in a significant increase in nodule number and biomass, root and shoot biomass, whereas no significant increase was observed in shoot nitrogen content and grain yield. The significant effects of nutrients on soybean yield and its components were also proven (Kobraee and Shamsi, 2013). Among their broad structural and functional roles, nutrients may affect nitrogen fixation in both legumes and non-legumes at various stages, including infection, nodule formation and function and plant growth (Weisany et al., 2013). Co-inoculation of soybean with Bradyrhizobium japonicum and Azospirillum brasilense in combination with foliar application of Co and Mo increased pod number, grain number per pod, weight of 100 grains and grain yield (Barbosa et al., 2023). Moreover, Jarecki et al., (2016) found that combined application of Bradyrhizobium and a complex of nutrients (Mg, S, B, Cu, Mn, Mo and Zn) increased plant height, pod number and thousand seed weight, while soybean seed yield was significantly higher both in individual and combined treatments as compared to the control. The current study clearly demonstrated that nutrients and their interactions have an important effect on the performance of the inoculation strains on soybean plants. It has been reported that the uptake of main nutrients by plants depends on their available forms and their interactions with other nutrients (Leidi and Rodríguez-Navarro, 2000). Similar findings were reported by Chen et al., (2017), who observed that a higher supply of micronutrients significantly increased biomass and uptake of macronutrients in soybean plants through the improved interactions of rhizobia and arbuscular mycorrhizal fungi (AMF).

The use of consortia-based microbial strains in combination with a nutrient complex could help avoid rhizosphere competence, improve the survival and performance of microbial inoculants, enhance nutrient uptake, increase plant growth and yield and mitigate plant responses to abiotic and biotic stresses. The combination of these two approaches could be integrated to achieve maximum effectiveness and significantly improve crop yields, especially in organic production. Further studies are needed to confirm the efficacy of such formulations in different environmental conditions and for different cultivars.

The research leading to these results has received funding from the European Union‘s Horizon 2020 research and innovation funding program under grant agreement number 771367, ECOBREED. Dataset supporting reported results are available at ECOBREED Zenodo community: https://doi.org/10.5281/zenodo.8210775. The support from Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number: 451-03-47/2023-01/200032) is also acknowledged.
 

All authors declare that they have no known competing financial interests or personal relationship that could have appeared to influence the work reported in this article.