Bacterial Community in Sugarcane Rhizosphere Under Bacillus subtilis Inoculation and Straw Return

Abstract

Straw return enhances soil biological properties by increasing carbon and energy availability, thereby improving conditions for microbial communities. However, the introduction of beneficial bacteria, such as Bacillus subtilis, can further shape the rhizosphere bacterial composition. In this study, we combined sugarcane straw return with B. subtilis inoculation to test whether this synergy reduces microbial specialization in the sugarcane rhizosphere. Three treatments were evaluated: (I) bulk soil (bulk), (II) rhizosphere soil with straw return but no B. subtilis inoculation (straw), and (III) rhizosphere soil with straw return and B. subtilis inoculation (straw + Bacillus). The bacterial community, including plant-growth-promoting bacteria (PGPB), was analyzed via 16S rRNA amplicon sequencing. Neither straw return nor B. subtilis inoculation significantly altered bacterial richness, diversity, or phylum-level abundance in the rhizosphere. Actinobacteria, Firmicutes, and Proteobacteria dominated the community, with Bacillus, Bradyrhizobium, and Paenibacillus as the predominant PGPB genera. Notably, only Bradyrhizobium abundance increased in the rhizosphere when straw was co-applied with B. subtilis. A co-occurrence network analysis revealed stronger microbial interactions under straw return, while B. subtilis enhanced connectivity among the PGPB. Although niche occupancy remained stable, PGPB specialization was higher with straw alone, suggesting that B. subtilis fosters a more generalist community. In conclusion, while straw return and B. subtilis inoculation did not affect overall bacterial diversity, B. subtilis increased PGPB interactions and reduced functional specialization, promoting a more generalized microbial community.

1. Introduction

Sustainable agricultural practices have been widely encouraged to achieve optimal crop yields while maintaining and improving soil properties. These practices are essential for ensuring long-term agricultural productivity, mitigating environmental degradation, and promoting ecosystem stability. Among these practices, straw return stands out as a key strategy, offering significant ecological benefits such as enhanced soil nutrient supply and sustainable crop productivity [1,2,3]. For example, studies demonstrate that straw return increases the availability of essential nutrients, such as nitrogen (N), phosphorus (P), and potassium (K), by approximately 14%, 10%, and 18%, respectively [4]. Additionally, it boosts total organic carbon (TOC) levels by 12% [2], a critical factor in improving soil fertility and carbon sequestration. The decomposition of straw serves as a vital source of carbon and energy, further stimulating microbial activity [5].

Interestingly, straw return can also reshape microbial communities in the rhizosphere [6]. This is important for plant growth, as the rhizosphere is recognized as a hotspot for root–microbe interactions, which are associated with plant nutrient acquisition [7]. Thus, any changes in the microbial community in the rhizosphere can significantly affect plant growth. Specifically, the bacterial community in the rhizosphere influences plant growth through multiple mechanisms, including enhancing nutrient availability, releasing phytohormones, and controlling pathogens [8]. Previous studies have reported that straw return drives the abundance, diversity, and richness of bacterial communities in the rhizosphere [9,10]. According to Chen et al. [9], straw return alters soil properties, particularly biological properties and TOC, which effectively influence the bacterial community.

In addition to straw return, other crop management practices, such as microbial inoculation, have been employed to enhance plant growth and soil health. As an important example, Bacillus subtilis, a well-known plant-growth-promoting bacterium (PGPB), has been widely recognized for its ability to enhance plant growth through multiple mechanisms, including nutrient solubilization, phytohormone production, and disease suppression [11]. In addition, B. subtilis can modify the bacterial community and affect enzyme activities, such as urease and phosphatase [11]. For instance, Sui et al. [12] inoculated B. subtilis in Populus sp. and found changes in bacterial structure and abundance in the rhizosphere, while the bacterial diversity did not change. The authors concluded that B. subtilis has the potential to enhance the structure of the rhizosphere microbial community as well as promote plant growth. Therefore, the combined application of straw return and B. subtilis inoculation presents a promising strategy for improving soil biological properties and plant growth.

However, the application of B. subtilis in conjunction with straw return could alter soil biological properties and shape the bacterial community in the rhizosphere. Indeed, little is known about the combined effect of B. subtilis in conjunction with straw on the bacterial community in the rhizosphere of sugarcane. In this study, we applied sugarcane straw return combined with B. subtilis inoculation to test the hypothesis that this combination could reduce microbial specialization in the rhizosphere of sugarcane. In Brazil, sugarcane straw return has been applied, as sugarcane is harvested without burning and the straw returns to soil. Specifically, this study assessed the effects of returned sugarcane straw alone and in combination with B. subtilis on the composition, diversity, and interactions of the bacterial community in the sugarcane rhizosphere.

2. Materials and Methods

2.1. Experimental Site Characterization

The experiment was conducted during the 2023/2024 crop season in a sugarcane (Saccharum spp.) field planted with the RB306066 cultivar, located in the rural community of Cabeceiras, Palmeira, Piauí, Brazil (8°40′42″ S, 44°15′12″ W). The climate is classified as tropical (Aw, Köppen-Geiger), with an average annual rainfall of 900 mm and a mean annual temperature of 30 °C. The soil is classified as a Yellow Latosol (Oxisol, USDA Soil Taxonomy). The chemical properties of the soil before the experiment are detailed in Supplementary Table S1. Sugarcane was planted in an area characterized by high soil moisture levels.

2.2. Experimental Design and Treatments

The experiment was conducted using a randomized block design with three treatments: (I) bulk soil (bulk), (II) rhizosphere soil with straw return without B. subtilis inoculation (straw), and (III) rhizosphere soil with straw return with B. subtilis inoculation (straw + Bacillus). The design included 12 plots, with six plots receiving straw return without B. subtilis and six plots receiving straw return with B. subtilis inoculation. For the straw treatments, sugarcane straw was applied at a rate of 15 tons per hectare two months before sampling. Soil samples were collected from the rhizosphere (soil adhering to sugarcane roots) within each plot. For bulk soil sampling, soil was collected 20 cm away from the plant roots.

2.3. Experimental Condition

The planting and topdressing fertilization were carried out according to specific recommendations for sugarcane [13]. The inoculum of Bacillus subtilis (strain AP-3) was prepared by culturing the bacteria in a nutrient-rich medium under controlled conditions until it reached a concentration of 1.0 × 10⁸ cells per mL. After the application of straw return, B. subtilis was inoculated using a suspension containing 2 L of inoculum in 100 L of water per hectare. This suspension was adjusted to each plot size and applied via spraying at the base of the plants. The application was homogeneous to ensure suitable distribution and proper fixation of the microorganism in both the soil and the plant.

The inoculation was conducted during the tillering stage of sugarcane, a period characterized by enhanced root activity and an increased demand for plant-growth-promoting microorganisms. Each experimental plot had a total area of 84 m², comprising six rows of 10 m each, spaced 1.4 m apart. The effective sampling area within each plot was 56 m², designated for plant collection and analysis.

Rhizospheric soil sampling took place 60 days after the application of straw and B. subtilis. Before sampling, the equipment was sterilized with 70% alcohol to prevent cross-contamination. In each plot, three plants per plot were selected for the collection of soil adhered to the roots and put together to form a composite sample per plot. Additionally, bulk soil samples (non-rhizospheric soil) were collected 20 cm from the roots. The samples were stored in refrigerated containers and transported to the Sugarcane Genetic Improvement Program at the Federal University of Piauí (UFPI), where they were kept at −18 °C until analysis.

2.4. DNA Extraction and Sequencing

Microbial DNA was extracted from rhizosphere and bulk soil samples using 0.5 g of each sample with a PowerLyzer PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) following the manufacturer’s instructions. The DNA extraction was performed in triplicate for each sample. The quality and concentration of the extracted DNA were determined by using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).

The V4 region of the 16S rRNA gene was amplified with region-specific primers (515F/806R) [14]. Each 25 μL PCR reaction contained the following: 12.25 µL of nuclease-free water (Certified Nuclease-free, Promega, Madison, WI, USA), 5.0 µL of buffer solution 5x (MgCl2 2Mm), 0.75 μL of solution of dNTPs (10 mM), 0.75 μL of each primer (515 F 40 μM e 806 R 10 μM), 1.0 unit of Platinum Taq polymerase High Fidelity in a concentration of 0.5 μL (Invitrogen, Carlsbad, CA, USA), and 2.0 µL of template DNA. Moreover, a control reaction was performed by adding water instead of DNA. The conditions for PCR were as follows: 95 °C for 3 min to denature the DNA, 35 cycles at 98 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, and a final extension of 3 min at 72 °C to ensure complete elongation.

After indexing, the PCR products were cleaned up using Agencourt AMPure XP—PCR purification beads (Beckman Coulter, Brea, CA, USA) according to the manufacturer’s manual and quantified using a dsDNA BR assay Kit (Invitrogen, Carlsbad, CA, USA) on a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Once quantified, equimolar concentrations of each library were pooled into a single tube. After quantification, the molarity of the pool was determined and diluted to 2 nM, denatured, and then diluted to a final concentration of 8.0 pM with a 20% PhiX (Illumina, San Diego, CA, USA) spike for loading into the Illumina MiSeq sequencing machine (Illumina, San Diego, CA, USA).

Sequence data were processed using QIIME 2 version 2023.7.0. Firstly, the sequences were demultiplexed, and quality control was carried out using DADA2 [15] using the consensus method to remove any remaining chimeric and low-quality sequences. After filtering, approximately 1,395,00 high-quality sequences were obtained, with an average of approximately 70,000 sequences per sample. Singletons and doubletons were removed, and the samples were rarefied to 47,800 sequences, following the number of the lowest sample. The taxonomic affiliation was performed at 97% similarity using the Silva database v. 138 [16], and the generated matrix was further used for statistical analyses. The complete pre-filtered 16 S rRNA data are available at NCBI SRA under the identifier PRJNA1250553.

2.5. Statistical Analysis

Statistical analyses were conducted to compare bulk soil and rhizosphere samples. Data normality and homogeneity of variances were assessed using the Shapiro–Wilk and Levene tests, respectively. Alpha diversity metrics, including richness and Shannon diversity, were calculated for both prokaryotic and plant-growth-promoting bacteria (PGPB) communities based on amplicon sequence variants (ASVs). Relative abundances were compared, and statistical significance was determined using one-way analysis of variance (ANOVA), followed by Dunn’s test (p < 0.05). Co-occurrence network analysis was conducted to examine microbial community interactions. Significant Spearman correlations (positive > 0.7 and negative < −0.7, p < 0.01) were selected for network construction. Networks were visualized using Gephi [17], with nodes, edges, and positive and negative correlations highlighted. Niche occupancy was assessed using the CLAM test, a multinomial classification method for species assignment [18]. This approach categorizes microbial taxa based on their abundance across two habitats, identifying generalists, specialists, and rare taxa. The analysis was performed using the ‘clamtest’ function in the R package ‘vegan,’ applying the supermajority rule (K = 2/3, p < 0.005).

3. Results

The richness and diversity of the bacterial community did not change with straw return with and without B. subtilis (Figure 1).

Similarly, the PGPB community showed no differences in richness or diversity between straw application and B. subtilis treatment. Neither straw return with nor without B. subtilis changed the relative abundance of bacterial phyla (Figure 2A). In both bulk soil and rhizosphere, the most abundant bacterial phyla were Actinobacteria, Firmicutes, and Proteobacteria. At the PGPB level, Bacillus, Bradyrhizobium, and Paenibacillus were the most abundant genera (Figure 2B). Among treatments, only Bradyrhizobium exhibited differences, with lower abundance in bulk soil and higher abundance in the rhizosphere when straw was inoculated with B. subtilis.

The co-occurrence analysis showed a higher number of nodes and edges in the bacterial community within the rhizosphere under straw application (Figure 3A). The topological properties of the bacterial and PGPB co-occurrence networks are detailed in Supplementary Table S2. For the PGPB community, the number of nodes was higher in the rhizosphere with straw application, while the number of edges increased when straw was inoculated with B. subtilis. Niche occupancy analysis of the core bacterial community showed a similar number of specialists between straw with and without B. subtilis (Figure 3B). However, the PGPB community had more specialists with straw application alone compared to straw inoculated with B. subtilis.

4. Discussion

In this study, we have addressed the hypothesis that sugarcane straw return inoculated with B. subtilis could influence the bacterial community in the rhizosphere of sugarcane. The results showed that the straw return and inoculation of B. subtilis did not significantly change the bacterial richness, diversity, and relative abundance of dominant bacterial phyla. These results suggest that the bacterial community, mainly in the rhizosphere, remains stable when B. subtilis is inoculated. Although we did not assess the growth of sugarcane, this result is interesting since the inoculation of B. subtilis, as an ecological strategy to promote plant growth, does not bring any effect on the bacterial community in the rhizosphere of sugarcane [19].

Regarding the composition of the bacterial community (Figure 2), the relative dominance of Actinobacteria, Firmicutes, and Proteobacteria agrees with previous studies that found these three main phyla dominant in soil and the rhizosphere [20,21]. As straw return was applied, it favored the high abundance of Actinobacteria, which is a well-known phylum acting on organic residues [22]. In addition, the increase in organic residue contributes to the increase in the abundance of Proteobacteria, which is favored in conditions of high organic matter [23]. Regarding Firmicutes, the inoculation of B. subtilis (Firmicutes) contributed to an increase in the abundance of Firmicutes. Ecologically, the higher abundance of these phyla is important to soil health and plant growth [24]. Interestingly, Bacillus, Bradyrhizobium, and Paenibacillus were the most abundant PGPB genera found mainly in the rhizosphere, and this contributes to plant growth through nitrogen fixation, phosphate solubilization, and stress resistance mechanisms [25,26]. Our results highlight the higher abundance of Bradyrhizobium in the rhizosphere of sugarcane when straw was inoculated with B. subtilis (Figure 2C). This suggests a positive effect of B. subtilis favoring Bradyrhizobium colonization in the rhizosphere [27]. Functionally, the increased presence of Bacillus, Bradyrhizobium, and Paenibacillus in the rhizosphere could benefit sugarcane by improving plant vigor, increasing biomass accumulation, and potentially resulting in higher yields.

The co-occurrence analysis showed a higher number of nodes and edges in the bacterial community within the rhizosphere under straw application (Figure 3A), which suggests that straw return increases the bacterial interactions in the rhizosphere. For instance, ref. [28] found that the bacterial co-occurrence network in straw-amended soil showed increased complexity and connectivity. This could be explained by the increased availability of organic matter and root exudates, which support diverse microbial interactions [6]. When assessing the PGPB community, the increase in nodes under a straw application without inoculation indicates a greater diversity of plant-growth-promoting bacteria, suggesting that organic residues stimulate a more interconnected PGPB community [29]. However, the increase in edges when straw was inoculated with B. subtilis suggests that while bacterial diversity did not increase, the interactions among PGPB genera did. This could suggest that B. subtilis plays a role in structuring PGPB interactions in the rhizosphere of sugarcane.

The niche occupancy analysis of the core bacterial community (Figure 3B) revealed a similar number of specialists between straw with and without B. subtilis. This indicates that the inoculation of B. subtilis did not change the ecological niches occupied by dominant bacterial taxa. This confirms the bacterial community stability as found regarding richness and diversity. However, within the PGPB community, more specialists were present in the straw without B. subtilis, which suggests that while straw application promotes niche differentiation among PGPB, the inoculation of B. subtilis reduces specialization, possibly by promoting a more generalist microbial assemblage [27]. This could be due to competition, facilitation, or niche overlap between B. subtilis and native PGPB taxa. It is known that B. subtilis is highly effective in colonizing the rhizosphere [30] and this may have limited the ecological space available for other specialized PGPB.

As a strength, this study shows the stability of the bacterial community in the rhizosphere despite B. subtilis inoculation, which potentially suggests the importance of straw return in enhancing key bacterial phyla and microbial interactions. It also reveals that B. subtilis influences PGPB interactions rather than increasing diversity, providing ecological insights into niche specialization shifts among PGPB. As limitations, the non-significant change in bacterial diversity limits broader conclusions, and the short-term assessment leaves long-term effects unclear. Additionally, the potential competition between B. subtilis and native PGPB was not fully explored.

This study provides novel insights into how B. subtilis inoculation, in combination with straw, shapes the sugarcane rhizosphere microbiome by maintaining community stability while enhancing beneficial microbial interactions. Our findings show that B. subtilis promotes Bradyrhizobium colonization and increases connectivity among the PGPB community. We also demonstrated that B. subtilis reduces PGPB specialization, indicating subtle competitive or synergistic dynamics within a stable microbial niche. These results suggest the potential of B. subtilis in enhancing functionally beneficial microbial networks in the rhizosphere. The strategy of inoculating B. subtilis can be interesting for sustainable agriculture without negatively altering microbial diversity. On the other hand, straw return enhances microbial interactions and key bacterial phyla, reinforcing its role in improving soil health and nutrient cycling. The observed increase in PGPB interactions suggests that B. subtilis could support plant growth. However, potential competition with native PGPB should be considered in the strategy to inoculate B. subtilis in sugarcane.

On the other hand, a limitation of this study is that it focuses primarily on bacterial community structure and interactions, without direct measurements of functional outcomes such as plant growth, nutrient uptake, or crop yield. Furthermore, while B. subtilis enhanced specific PGPB, its potential competition with native PGPB and long-term ecological effects were not fully explored. Future studies should integrate functional assays and field-scale evaluations to validate the agronomic benefits and ecological impacts of B. subtilis inoculation under different environmental conditions.

5. Conclusions

Our study shows that straw return and B. subtilis inoculation maintain bacterial community stability in the rhizosphere of sugarcane, with no significant changes in bacterial richness, diversity, and composition. However, B. subtilis favored Bradyrhizobium colonization in the rhizosphere and increased microbial interactions among PGPB. While niche occupancy remained stable, B. subtilis reduced PGPB specialization due to competition or niche overlap. These findings highlight the resilience of sugarcane-associated microbiomes and suggest potential microbial interactions that could benefit plant growth.