Inoculation with Bacillus cereus DW019 Modulates Growth, Yield and Rhizospheric Microbial Community of Cherry Tomato

: Plant growth-promoting rhizobacteria (PGPR) play an important role in promoting plant growth and increasing crop yield. Bacillus cereus DW019, which was previously isolated from an ion-absorbed rare-earth ore of Ganzhou in Southeastern China, has been considered as a PGPR due to its production of indole-3-acetic acid (IAA), ammonia and siderophore, but its promoting effect on plants remains poorly understood. In this study, autoclaved dead cells and viable cells of Bacillus cereus DW019 at different concentrations were inoculated into pot-cultivated cherry tomato ( Lycopersicon esculentum ) to investigate the promoting effect on plant growth and yield. A total of 70 days after inoculation, the plants and fruits of cherry tomato were harvested, and their growth indicators, yields, and nutrients were measured. The results showed that biomass, stem thickness, plant height and root length were signiﬁcantly promoted and that the vitamin C, soluble sugar and soluble protein were signiﬁcantly increased. Inoculation with Bacillus cereus also modulated the rhizospheric microbial community diversity and structure, especially the proportions of Proteobacteria and Actinobacteriota , which in turn improved the plant height, fresh weight, nutritional quality and rhizosphere soil bacterial diversity of cherry tomato. All the ﬁndings suggest that Bacillus cereus DW019 is beneﬁcial to the growth of crops and improves the yield of cherry tomato, suggesting that Bacillus cereus DW019 could be developed into a potential biofertilizer to be used as an agricultural inoculant to increase crop yield and improve the soil ecosystem.


Introduction
Cherry tomato, also known as grape tomato and small tomato, is characterized by a beautiful appearance and rich nutrition [1]. It contains vitamin C, vitamin A, lycopene and other nutrients; and the water content is 70% and higher [2]. Due to its effects that protect the liver and soften the blood vessels, cherry tomato is one of the most popular fruits and profitable crops worldwide. It can be used for processing jam, fruit juice, beverages and canned food, and its demand continues to expand. It is necessary to develop an environmentally friendly way to fully improve the yield of this crop and its nutritional quality. Various alternatives have been investigated to improve the yield of the crop and to reduce the use of chemical compounds. Plant growth-promoting rhizobacteria (PGPR) are beneficial in agriculture because they are environmentally friendly, cheap and sustainable [3][4][5][6]. PGPR, a kind of microorganism, have a mutual beneficial relationship with plant roots and promote plant growth through unique PGPR substances. PGPR have different growth-promoting mechanisms, so they are also used as biological fertilizers, biostimulants and biological protectants [7,8]. It has been shown that inoculation with Bacillus subtilis is able to effectively control the early blight of tomato and reduce the severity of the plant disease [9]. One study has verified that when the liquid formulation of B. mesonae TP-H20-5 was used to treat vegetables, including tomatoes, cherry tomatoes, strawberries and cucumbers, the final market yield was significantly increased compared with untreated vegetables [10].
Bacillus has been proven to be one of the most promising PGPR strains due to their function in the promotion of plant growth [11][12][13]. For instance, several Bacillus species have been shown to actively colonize the rhizosphere and to promote plant growth by rendering phosphorus, iron and nitrogen bio-available as well as producing beneficial phytohormones, such as indole-3-acetic acid [14,15]. It has been reported in previous studies that bacterial genera, such as Azotobacter, Burkholderia, Erwinia, Flavobacterium, Micrococcous, Pseudomonas, Serratia and Bacillus, etc., have demonstrated plant growth-promoting properties (PGPs) [16]. Additionally, these microorganisms are beneficial in the growth promotion of their host plants through the fixation of atmospheric nitrogen, potassium and zinc; the solubilization of phosphate; the production of siderophore, ammonia, hydrogen cyanide, 1-aminocyclopropane-1-carboxylate (ACC) and phytohormones (auxins, cytokinin and gibberellic acid); and the depletion of ethylene biosynthesis. Thus, host plants increase their nutrient uptake, supply tolerance to biotic and abiotic stresses and obtain biocontrol agents against plant pathogens. Compared with other genera, the Bacillus of the above-mentioned PGPR plays a critical role due to their resilience to harsh environmental conditions through spore formation. Therefore, many Bacillus strains are commercially applied as biocontrols, biofertilizers and biostimulants in agricultural practices today [17][18][19][20].
Plant growth-promoting Bacillus strains have also been isolated from the rhizosphere of phytoremediation plants in an ion-absorbed rare-earth ore in Ganzhou, Jiangxi [21], where ionic rare earths were originally detected [22]. Ion-adsorption rare earths, known as "industrial vitamins", are non-renewable national strategic resources and are considered as strategic resources indispensable for the development of high-tech and cutting-edge national defense purposes all over the world [23][24][25][26]; as such, they are highly valued. However, the previous crude mining methods have caused serious environmental problems [27]. The soil ecosystems of rare earth mining areas have been seriously damaged and polluted through mining, resulting in the low level of comprehensive soil fertility, which has caused considerable impact on crops, especially on cherry tomatoes [28]. To ensure the proper growth and yield of economic crops, the isolation and application of rhizosphere plant-growth promoting microorganisms is a an environmentally friendly and feasible approach.
Our previous study revealed that the Bacillus cereus DW019 strain, isolated from the rhizosphere of the slash pine from an ion-type rare earth ore, is capable of IAA production, iron carrier production, and nitrogen fixation [21]. In this study, the different concentrations of the cells of Bacillus cereus DW019 were inoculated into the potted seedlings of cherry tomato plants to investigate the relationship between DW019 and growth indicators, nutritional quality and the inter-root soil microbial community of cherry tomatoes. Therefore, the aim of this study is to explore the growth-promoting effects on cherry tomato. In addition, the relationships between inoculation treatments and growth indicators, nutritional quality and the inter-root soil microbial community were analyzed to understand how inoculants affect plant growth and rhizosphere microbial diversity.

Bacterial strain and Inoculum Preparation
Bacillus cereus DW019 was isolated from an ion-absorbed rare-earth mining area in Ganzhou, Jiangxi Province, China (24 • 50 N, 114 • 51 E), and the strain was identified based on 16S rRNA gene sequencing analysis and physiochemical analysis [21]. Bacillus cereus DW019 was kept at −80 • C in our laboratory and grown on L broth agar plate (g·L −1 : tryptone-10 g; yeast extract-5 g; NaCl-8.8 g; agar-15 g) for 24 h at 37 • C, and then one single colony was transferred to LB liquid medium and incubated for 7 h at 37 • C Agronomy 2023, 13, 1458 3 of 15 and 150 r·min −1 to reach the logarithmic growth stage for inoculum. A total of 1% of the inoculum was inoculated into a new LB liquid medium for bacterial cell culture. The bacterial cell culture was collected via centrifugation (8000 r·min −1 , 4 • C, 10 min) and washed three times using sterile saline (0.9%). The colony-forming units (CFU) of inoculant culture were confirmed through LB agar medium and optimal density OD 600 (OD 600 of 1.0 = 2 × 10 9 CFU·mL −1 ). Bacterial cells were adjusted to 10 8 CFU·mL −1 , 10 9 CFU·mL −1 and 2 × 10 9 CFU·mL −1 with sterile water for later use. Meanwhile, bacterial cell suspension at 10 8 CFU·mL −1 was autoclaved at 121 • C for 20 min to prepare dead cells.

Seedling Preparation
The test soil was collected from the around 20 cm-deep topmost layer in Yang Meidu Park, Ganzhou, China (25 • 84 N, 114 • 90 E). The soil samples were dried naturally after removing grass roots, dead leaves, stones and other debris and then sieved through a 100 mm mesh [20]. Cherry tomato seeds (Carlson Pink Jiao F1) of uniform particle size were selected and treated with 75% ethanol for 10 min, and then washed with sterile water 3-4 times to obtain sterile seeds. After surface disinfection, 10 seeds were sown in a pot containing 3.5 kg of the prepared soil after two days of watering. A total of 7 days after germination, one seedling was kept in each pot.

Inoculation and Pot Cultivation of Cherry Tomato
Subsequently, 350 mL of the prepared bacterial cell suspension at different concentrations was inoculated into the soil around the cherry tomato seedling in each pot. Taking the sterile saline inoculation as a control (CK), inoculation treatments included inoculation with autoclaved 10 8 CFU·mL −1 dead cells (DC8), 10 8 CFU·mL −1 viable cells (VC8), 10 9 CFU·mL −1 viable cells (VC9) and 2 × 10 9 CFU·mL −1 viable cells (DVC9). Therefore, the number of inoculated cells per 100 g of soil in the different-treatment pots was 0, 10 8 , 10 9 and 2 × 10 9 CFU, respectively. The planting lasted for 70 days after inoculation, and the soil was watered every day. Five replicates were created for each of the above treatments. Inoculation treatments were performed three times a week from May 2022. After 70 days, cherry tomatoes were harvested and rhizosphere soil was collected for later analysis.

Plant Growth Measurement
Plant height was calculated as the distance between the roots and the highest point of the plant, stem thickness was measured at the point where the above-ground and belowground parts of the plant meet, and both above-ground and below-ground biomass were measured in fresh weight [29].

Plant Yield Measurement
The number of cherry tomatoes was obtained using the counting method. To obtain a yield of cherry tomatoes, ripe fruits were picked and wiped with sterile cotton (adsorbed with 75% ethanol) and then weighed.

Cherry Tomato Nutrients Measurement
The contents of soluble sugar, vitamin C and soluble protein were determined using an enzyme kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

DNA Extraction, PCR Amplification and Sequencing of 16S rRNA Genes
The soil microbial sequencing was completed by Shanghai OE Biotech Co., Ltd. The total DNA of soil microorganisms was extracted using the CTAB (Hexadecyl Trimethyl Ammonium Bromide) method [30]. The V3-V4 region of the 16S rRNA was amplified with bacterial specific primers 343F (5 -TACGGRAGGCAGCAG-3 ) and 798R (5 -AGGGTATCTAATCCT-3 ) [31]. ReadyMix high-fidelity enzymes were used in the PCR to ensure the efficiency and accuracy of amplification. Each group of the samples was subjected to two PCR amplifications [32]. When completed, equal amounts of samples were mixed according to the concentration of PCR products, and the primer regions were sequenced. Sequencing samples were harvested in cherry tomatoes' rhizosphere soil. The original image data files obtained via high-throughput sequencing were converted into original sequencing sequences using base calling analysis, and the results were stored in the FASTQ (referred to as fq) file format, which contained the sequence information of the sequencing sequences (reads) and their corresponding sequencing quality information [33][34][35]. Cutadapt software was used to cut away the primer sequences from the raw data sequences. Based on Qiime2 default parameters, the QC analysis of the qualified double-ended raw data was performed to obtain the presentative sequences and ASV abundance tables through DADA2. QC analysis included quality filtering, noise reduction, splicing and chimera removal. Based on sequence similarity, they were divided into multiple operational taxonomic units (OTUs) via comparison with the database. Gene sequences with a similarity level of 0.03 (97% of sequence similarity, approximately equaling the species level) were selected for subsequent analysis, and OTU representative sequences were annotated.

Statistical Analysis
Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA) was used to process data and plot above-ground and below-ground fresh weight, plant height, stem thickness, fruit number, yield and fruit nutritional quality. The values in the graph are the means of the number of replicates ± standard error (SE). Statistical analyses were performed using IBM SPSS Statistical Version 20.0 (IBM Corporation Armonk, New York, NY, USA). Tukey's test and one-way analysis of variance (ANOVA) were performed on all samples to determine the significances of any differences (p < 0.05).
Data related to the 16S rRNA gene sequences were computed in R, version 3.5.1. The R package was used for graphical operations. Alpha indexes (Chao and Shannon) were calculated by Mothur (version 1.31.2). Beta diversities were calculated using the weighted UniFrac method in QIIME. Principal Coordinate Analysis (PCoA) was conducted based on weighted UniFrac (WUF) distance and taxon abundances, and alpha (α-) and beta (β-) diversity were calculated.

Effect of B. cereus DW019 on the Growth of Cherry Tomato
The plant growth of cherry tomato after inoculation with B. cereus DW019 was better than that of the control without inoculation ( Figure 1, Table 1). B. cereus DW019 was able to promote the biomass, stem thickness, plant height and root length of cherry tomato (Table 1), but there was no significant difference between the control and the plants inoculated with dead cells. Inoculation with viable cells positively increased all the above growth indexes of cherry tomato. However, although the promotion effect of DVC9 on plant growth was the highest, VC9 and DVC9 presented no significant differences in the below-ground biomass, plant height and root length of cherry tomato, indicating the inoculation of viable cells per 100 g of soil was appropriate within 10 8~2 × 10 9 CFU. All these results showed that the inoculation with viable cells of B. cereus DW019 can promote the growth of cherry tomato.

Effect of B. cereus DW019 on the Yield of Cherry Tomato
The average number of cherry tomato fruit in the inoculation treatments was remarkably larger than the control (Figure 2a), as was the yield (Figure 2b). Compared with the control, the fruit number of DC8, VC8, VC9 and DVC9 treatment increased by 45.83%, 87.50%, 400.00% and 462.50%, respectively, while the weights increased by 42.58%, 242.96%, 291.43% and 425.18%, respectively. The results showed that the number and weights of cherry tomato was positively related to the inoculation concentration of DW019, but only the VC9 and DVC9 treatments increased the yield significantly, which indicated that the inoculation of viable cells could also promote the yield of cherry tomato. * Data are shown as mean ± standard deviation. Different letters indicate significant differences at p < 0.05. Control without inoculation, CK; inoculation with dead cells at 10 8 CFU mL −1 , DVC8; inoculation with viable cells at 10 8 CFU mL −1 , VC8; inoculation with viable cells at 10 9 CFU mL −1 , VC9; inoculation with viable cells at 2 × 10 9 CFU mL −1 , DVC9.

Effect of B. cereus DW019 on the Yield of Cherry Tomato
The average number of cherry tomato fruit in the inoculation treatments was remarkably larger than the control (Figure 2a), as was the yield (Figure 2b). Compared with the control, the fruit number of DC8, VC8, VC9 and DVC9 treatment increased by 45.83%, 87.50%, 400.00% and 462.50%, respectively, while the weights increased by 42.58%, 242.96%, 291.43% and 425.18%, respectively. The results showed that the number and weights of cherry tomato was positively related to the inoculation concentration of DW019, but only the VC9 and DVC9 treatments increased the yield significantly, which indicated that the inoculation of viable cells could also promote the yield of cherry tomato.

Effect of B. cereus DW019 on the Yield of Cherry Tomato
The average number of cherry tomato fruit in the inoculation treatments was remarkably larger than the control (Figure 2a), as was the yield (Figure 2b). Compared with the control, the fruit number of DC8, VC8, VC9 and DVC9 treatment increased by 45.83%, 87.50%, 400.00% and 462.50%, respectively, while the weights increased by 42.58%, 242.96%, 291.43% and 425.18%, respectively. The results showed that the number and weights of cherry tomato was positively related to the inoculation concentration of DW019, but only the VC9 and DVC9 treatments increased the yield significantly, which indicated that the inoculation of viable cells could also promote the yield of cherry tomato.  Control without inoculation, CK; inoculation with dead cells at 10 8 CFU mL −1 , DVC8; inoculation with viable cells at 10 8 CFU mL −1 , VC8; inoculation with viable cells at 10 9 CFU mL −1 , VC9; inoculation with viable cells at 2 × 10 9 CFU mL −1 , DVC9. Different lowercase letters above columns indicate significant differences at p < 0.05.

Effect of B. cereus DW019 on the Fruit Nutrients of Cherry Tomato
As shown in Figure 3a, the vitamin C content of cherry tomato in inoculation treatments increased with higher inoculation concentration, being 8.27 mg·100 g −1 , 11.20 mg·100 g −1 , 14.44 mg·100 g −1 and 14.66 mg·100 g −1 , respectively. The inoculation of the viable cells of DW019 was able to significantly increase the content of vitamin C in cherry tomato fruits compared with the control. It can be seen from Figure 3b that the soluble sugar content in all different treatments increased with rates of 6.21%, 21.56%, 83.93% and 91.83%, respectively. However, only VC9 and DVC9 treatments enhanced the content significantly. The protein contents of the inoculation treatments were 0.17 mg·g −1 , 0.27 mg·g −1 , 0.29 mg·g −1 and 0.33 mg·g −1 , respectively (Figure 3c). Compared with the control, VC8, VC9 and DVC9 treatments increased by 34.12%, 41.39% and 63.18%, respectively, except for a slight decrease in the DC8 treatment.

Effect of B. cereus DW019 on the Fruit Nutrients of Cherry Tomato
As shown in Figure 3a, the vitamin C content of cherry tomato in inoculation treatments increased with higher inoculation concentration, being 8.27 mg·100 g −1 , 11.20 mg·100 g −1 , 14.44 mg·100 g −1 and 14.66 mg·100 g −1 , respectively. The inoculation of the viable cells of DW019 was able to significantly increase the content of vitamin C in cherry tomato fruits compared with the control. It can be seen from Figure 3b that the soluble sugar content in all different treatments increased with rates of 6.21%, 21.56%, 83.93% and 91.83%, respectively. However, only VC9 and DVC9 treatments enhanced the content significantly. The protein contents of the inoculation treatments were 0.17 mg·g −1 , 0.27 mg·g −1 , 0.29 mg·g −1 and 0.33 mg·g −1 , respectively (Figure 3c). Compared with the control, VC8, VC9 and DVC9 treatments increased by 34.12%, 41.39% and 63.18%, respectively, except for a slight decrease in the DC8 treatment. Control without inoculation, CK; inoculation with dead cells at 10 8 CFU mL −1 , DVC8; inoculation with viable cells at 10 8 CFU mL −1 , VC8; inoculation with viable cells at 10 9 CFU mL −1 , VC9; inoculation with viable cells at 2 × 10 9 CFU mL −1 , DVC9. Different lowercase letters above columns indicate significant differences at p < 0.05.
The above results were comprehensively analyzed, and it was concluded that inoculation with viable cells of B. cereus DW019 was able to significantly improve fruit nutrients, including vitamin C, soluble sugar and protein content in cherry tomato. However, similarly to the case of effectiveness of growth, no significant difference was found between VC9 and DVC9, indicating that inoculation concentration probably has limited effects on the promotion of plant growth and yield.

Impact of Inoculation on Rhizospheric Bacterial Community Diversity and Composition
After harvesting, rhizospheric soil samples of cherry tomato were collected and analyzed via bacterial 16S rDNA gene sequencing, yielding high-quality sequences between 78,047 and 81,403 from the various treatments. By comparison, 48,837-54,346 sequences (72.04% of all sequences) were identified as bacterial sequences, containing 16,727 to 35,531 bacterial sequences per sample. Based on the 97% sequence similarity clustering, 2462 OTUs were obtained, containing 1207 to 1724 OTUs per sample ( Table 2). The samples were analyzed for alpha diversity and beta diversity at the OTU level, community composition and clustering analysis.  Control without inoculation, CK; inoculation with dead cells at 10 8 CFU mL −1 , DVC8; inoculation with viable cells at 10 8 CFU mL −1 , VC8; inoculation with viable cells at 10 9 CFU mL −1 , VC9; inoculation with viable cells at 2 × 10 9 CFU mL −1 , DVC9. Different lowercase letters above columns indicate significant differences at p < 0.05.
The above results were comprehensively analyzed, and it was concluded that inoculation with viable cells of B. cereus DW019 was able to significantly improve fruit nutrients, including vitamin C, soluble sugar and protein content in cherry tomato. However, similarly to the case of effectiveness of growth, no significant difference was found between VC9 and DVC9, indicating that inoculation concentration probably has limited effects on the promotion of plant growth and yield.

Impact of Inoculation on Rhizospheric Bacterial Community Diversity and Composition
After harvesting, rhizospheric soil samples of cherry tomato were collected and analyzed via bacterial 16S rDNA gene sequencing, yielding high-quality sequences between 78,047 and 81,403 from the various treatments. By comparison, 48,837-54,346 sequences (72.04% of all sequences) were identified as bacterial sequences, containing 16,727 to 35,531 bacterial sequences per sample. Based on the 97% sequence similarity clustering, 2462 OTUs were obtained, containing 1207 to 1724 OTUs per sample ( Table 2). The samples were analyzed for alpha diversity and beta diversity at the OTU level, community composition and clustering analysis. The Shannon and Chao indexes of rhizosphere soil bacteria of cherry tomato with various inoculation treatments were analyzed in this study (Figure 4). Compared with CK, the Shannon index of DC8, VC8, VC9 and DVC9 decreased by 0.84%, 1.05%, 4.10% and 3.89%, respectively, while the Chao index decreased by 2.48%, 4.05%, 13.46% and 12.87%, respectively. The results indicated that adding PGPR DW019 to cherry tomato would significantly reduce the total number of species and community diversity in the soil. In general, inoculation with Bacillus cereus DW019 reduced the alpha diversity of the rhizosphere soil bacterial community.
* Data are shown as mean ± standard deviation. Different letters indicate significant differences at p < 0.05.
The Shannon and Chao indexes of rhizosphere soil bacteria of cherry tomato with various inoculation treatments were analyzed in this study (Figure 4). Compared with CK, the Shannon index of DC8, VC8, VC9 and DVC9 decreased by 0.84%, 1.05%, 4.10% and 3.89%, respectively, while the Chao index decreased by 2.48%, 4.05%, 13.46% and 12.87%, respectively. The results indicated that adding PGPR DW019 to cherry tomato would significantly reduce the total number of species and community diversity in the soil. In general, inoculation with Bacillus cereus DW019 reduced the alpha diversity of the rhizosphere soil bacterial community. The PCoA showed that all the soil samples were integrated into five groups despite some overlaps between DC8 and VC8 ( Figure 2). The first principal axis revealed that the closest distance lay between CK and DC8 samples, while the VC8 and DC8 groups were close, and so were the DVC9 and VC9 samples ( Figure 5). The first principal-coordinate axis (PCoA1) contributed 21.32% of the total variation, indicating that the largest separation factor was the inoculation concentration, which clearly separated the rhizosphere soil bacterial community of cherry tomato (p < 0.001). Therefore, inoculation with DW019 in cherry tomato had a significant effect on the rhizosphere bacterial β diversity. The PCoA showed that all the soil samples were integrated into five groups despite some overlaps between DC8 and VC8 ( Figure 2). The first principal axis revealed that the closest distance lay between CK and DC8 samples, while the VC8 and DC8 groups were close, and so were the DVC9 and VC9 samples ( Figure 5). The first principal-coordinate axis (PCoA1) contributed 21.32% of the total variation, indicating that the largest separation factor was the inoculation concentration, which clearly separated the rhizosphere soil bacterial community of cherry tomato (p < 0.001). Therefore, inoculation with DW019 in cherry tomato had a significant effect on the rhizosphere bacterial β diversity.
All bacterial OTUs were further assigned into 36 phylum, 101 classes, 246 orders, 387 families and 709 genus, including some unclassified groups. At the phylum level ( Figure 6), Proteobacteria, Actinobacteria, Bacteroidetes, Gemmatimonadota and Acidobacteria were predominant in the soil, accounting for a larger proportion of the relative abundance in the rhizosphere soil (Table 3). Proteobacteria was the most dominant in all samples, accounting for more than 50% of the total bacteria, followed by Actinobacteria. Compared with CK, Proteobacteria showed an upward trend in each inoculation treatment with an increase of 3.28%, 3.87%, 6.77% and 8.93%, respectively. Compared with CK, Actinobacteria changed significantly in the inoculation treatments, increasing by 2.7%, 14.08%, 31.99% and 25.96%, respectively. However, the Bacteroidetes in each treatment showed a decrease of 9.76%, 9.83%, 11.19% and 13.63%, respectively. The changes of Gemmatimonadota, Acidobacteria and Myxococcota were consistent, presenting a downward tendency with an increase in inoculation concentration.
At the class level (Table 4), a total of 10 predominant classes were observed (>1% of total effective sequences), among which Alphaproteobacteria was the highest, followed by Gammaproteobacteria and Bacteroidia (Figure 7). Compared with CK, inoculation with DW019 had a significant effect on the content of Proteobacteria in the rhizosphere soil of cherry tomato. There was an increase of 1.89%, 5.95%, 16.83% and 6.92% of Alphaproteobacteria in DC8, VC8, VC9 and DVC9 treatments, respectively, and there might be a quadratic function relationship with the effects of inoculation on Alphaproteobacteria. Gammaproteobacteria decreased by 5.11% in the VC9 treatment, but it increased in the other treatments. The Gemmatimonadetes increased by 6.15% in the DC8 treatment and decreases were seen in the VC8, VC9 and DVC9 treatments of 7.28%, 11.42% and 20.08%, respectively. We speculated that some components of the living cells themselves or their metabolites had some influence on the community structure of Bacillus in the rhizosphere soil. Actinobacteria levels in DC8 treatment did not change as much as in other inoculation treatments, increasing by 11.45%, 46.39% and 41.77% with increasing inoculation concentrations. The statistics showed that viable DW019 cells modulated the proportion of Actinobacteria. In particular, compared with CK, the inoculation of viable cells significantly reduced the level of Acidobacteriae by 10.79%, 11.27%, 34.53% and 41.25% in the VC8, VC9 and DVC9 treatments, respectively. However, there were no significant differences in Bacteroidia, Thermoleophilia, Acidimicrobiia and Holophagae among all the treatments. All bacterial OTUs were further assigned into 36 phylum, 101 classes, 246 orders, 387 families and 709 genus, including some unclassified groups. At the phylum level ( Figure 6), Proteobacteria, Actinobacteria, Bacteroidetes, Gemmatimonadota and Acidobacteria were predominant in the soil, accounting for a larger proportion of the relative abundance in the rhizosphere soil (Table 3). Proteobacteria was the most dominant in all samples, accounting for more than 50% of the total bacteria, followed by Actinobacteria. Compared with CK, Proteobacteria showed an upward trend in each inoculation treatment with an increase of 3.28%, 3.87%, 6.77% and 8.93%, respectively. Compared with CK, Actinobacteria changed significantly in the inoculation treatments, increasing by 2.7%, 14.08%, 31.99% and 25.96%, respectively. However, the Bacteroidetes in each treatment showed a decrease of 9.76%, 9.83%, 11.19% and 13.63%, respectively. The changes of Gemmatimonadota, Acidobacteria and Myxococcota were consistent, presenting a downward tendency with an increase in inoculation concentration. Figure 5. Microbial β-diversity weighted UniFrac distances for bacteria in rhizosphere soil after various inoculation treatments: control without inoculation, CK; inoculation with dead cells at 10 8 CFU mL −1 , DVC8; inoculation with viable cells at 10 8 CFU mL −1 , VC8; inoculation with viable cells at 10 9 CFU mL −1 , VC9; inoculation with viable cells at 2 × 10 9 CFU mL −1 , DVC9.   At the class level (Table 4), a total of 10 predominant classes were observed (>1% o total effective sequences), among which Alphaproteobacteria was the highest, followed by Gammaproteobacteria and Bacteroidia (Figure 7). Compared with CK, inoculation with DW019 had a significant effect on the content of Proteobacteria in the rhizosphere soil o cherry tomato. There was an increase of 1.89%, 5.95%, 16.83% and 6.92% of Alphaproteobac teria in DC8, VC8, VC9 and DVC9 treatments, respectively, and there might be a quadratic function relationship with the effects of inoculation on Alphaproteobacteria. Gammaproteobac teria decreased by 5.11% in the VC9 treatment, but it increased in the other treatments. The Gemmatimonadetes increased by 6.15% in the DC8 treatment and decreases were seen in the VC8, VC9 and DVC9 treatments of 7.28%, 11.42% and 20.08%, respectively. We speculated that some components of the living cells themselves or their metabolites had some influence Figure 6. Changes in the relative abundance of major phylum levels of soil bacteria: control without inoculation, CK; inoculation with dead cells at 10 8 CFU mL −1 , DVC8; inoculation with viable cells at 10 8 CFU mL −1 , VC8; inoculation with viable cells at 10 9 CFU mL −1 , VC9; inoculation with viable cells at 2 × 10 9 CFU mL −1 , DVC9.

Potential Effects of Bacterial Diversity and Its Function on the Growth of Cherry Tomato
According to their relative abundances, the top 10 phyla of bacterial communities were subjected to Spearman's correlation analysis to investigate the relationship between their functions and the growth of cherry tomato plants, further uncovering the reasons for the differences in cherry tomato growth and yield (Figure 8).
At the phylum level, there was a significant negative correlation between plant height and Myxococcota. Stem thickness was positively correlated with Proteobacteria and negatively correlated with Acidobacteriota and Myxococcota. There was a significant positive correlation between above-ground biomass and Proteobacteria and Actinobacteriota and a significant negative correlation with Bacteroidota, Acidobacteriota, Myxococcota and Desulfobacterota. Below-ground biomass was positively correlated with Actinobacteriota and negatively correlated with Acidobacteriota, Myxococcota and Desulfobacterota. There was a significant negative correlation between root length and Myxococcota. There was a significant negative correlation between cherry tomato number and Myxococcota. The cherry tomato weight was the same as the above-ground biomass and was positively correlated with Proteobacteria and Actinobacteriota. The difference between number and weight was negatively correlated with Bacteroidota, Acidobacteriota and Myxococcota. Soluble sugar appeared to have a significant negative correlation with Myxococcota. A significant positive correlation was revealed between vitamin C and Bdellovibrionota. However, no significant correlation was found between fruit proteins and the relative abundance of microorganisms.
Agronomy 2023, 13, x FOR PEER REVIEW 10 of 16 46.39% and 41.77% with increasing inoculation concentrations. The statistics showed that viable DW019 cells modulated the proportion of Actinobacteria. In particular, compared with CK, the inoculation of viable cells significantly reduced the level of Acidobacteriae by 10.79%, 11.27%, 34.53% and 41.25% in the VC8, VC9 and DVC9 treatments, respectively. However, there were no significant differences in Bacteroidia, Thermoleophilia, Acidimicrobiia and Holophagae among all the treatments.   At the genus level (Figure 9), plant height was significantly negatively correlated with uncultured bacteria, Ellin6067, Subgroup_7 and Bryobacter and significantly positively correlated with Sphingomonas and Lysobacter. Stem thickness, above-ground biomass, below-ground biomass and fruit number were significantly and positively correlated with Sphingomonas, Lysobacter and SC-I-84 but negatively correlated with uncultured bacteria, Ellin6067, Subgroup_7 and Bryobacter. Root length was significantly positively correlated with Sphingomonas and negatively correlated with the other genera. Fruit yield was significantly negatively correlated with uncultured bacteria and Bryobacter and positively correlated with Sphingomonas, Lysobacter and SC-I-84. Soluble sugars were significantly positively correlated with Sphingomonas and SC-I-84 and negatively correlated with Ellin6067, Subgroup_7 and Bryobacter. Vitamin C content was significantly positively correlated with stem thickness only, rather than with bacterial abundance. There was no significant correlation between fruit protein content and the relative abundance of microorganisms. The correlation results suggested that the application of PGPR can promote crop growth and improve the yield of cherry tomato by changing microbial community composition and the function of root dominant bacteria, especially of rhizosphere bacteria.

Potential Effects of Bacterial Diversity and Its Function on the Growth of Cherry Tomato
According to their relative abundances, the top 10 phyla of bacterial communities were subjected to Spearman's correlation analysis to investigate the relationship between their functions and the growth of cherry tomato plants, further uncovering the reasons for the differences in cherry tomato growth and yield (Figure 8). At the phylum level, there was a significant negative correlation between plant height and Myxococcota. Stem thickness was positively correlated with Proteobacteria and negatively correlated with Acidobacteriota and Myxococcota. There was a significant positive correlation between above-ground biomass and Proteobacteria and Actinobacteriota and a significant negative correlation with Bacteroidota, Acidobacteriota, Myxococcota and Desulfobacterota. Belowground biomass was positively correlated with Actinobacteriota and negatively correlated with Acidobacteriota, Myxococcota and Desulfobacterota. There was a significant negative correlation between root length and Myxococcota. There was a significant negative correlation between cherry tomato number and Myxococcota. The cherry tomato weight was the same as the above-ground biomass and was positively correlated with Proteobacteria and Actinobacteriota. The difference between number and weight was negatively correlated with Bacteroidota, Acidobacteriota and Myxococcota. Soluble sugar appeared to have a significant negative correlation with Myxococcota. A significant positive correlation was revealed between vitamin C and Bdellovibrionota. However, no significant correlation was found between fruit proteins and the relative abundance of microorganisms. At the genus level (Figure 9), plant height was significantly negatively correlated with uncultured bacteria, Ellin6067, Subgroup_7 and Bryobacter and significantly positively correlated with Sphingomonas and Lysobacter. Stem thickness, above-ground biomass, belowground biomass and fruit number were significantly and positively correlated with Sphingomonas, Lysobacter and SC-I-84 but negatively correlated with uncultured bacteria, Ellin6067, Subgroup_7 and Bryobacter. Root length was significantly positively correlated with Sphingomonas and negatively correlated with the other genera. Fruit yield was significantly negatively correlated with uncultured bacteria and Bryobacter and positively correlated with Sphingomonas, Lysobacter and SC-I-84. Soluble sugars were significantly positively correlated with Sphingomonas and SC-I-84 and negatively correlated with Ellin6067, Subgroup_7 and Bryobacter. Vitamin C content was significantly positively correlated with stem thickness only, rather than with bacterial abundance. There was no significant correlation between fruit protein content and the relative abundance of microorganisms. The correlation results suggested that the application of PGPR can promote crop growth and improve the yield of cherry tomato by changing microbial community composition and the function of root dominant bacteria, especially of rhizosphere bacteria.

Discussion
PGPR have been widely used as plant growth enhancers or bioremediation agents. For decades, exploiting diverse microbes as PGPR in safe and ecofriendly methods for increas-

Discussion
PGPR have been widely used as plant growth enhancers or bioremediation agents. For decades, exploiting diverse microbes as PGPR in safe and ecofriendly methods for increasing growth and yield has become increasingly popular in sustainable agriculture [36]. Based on taxonomy, PGPR, isolated from the rhizosphere, belong to different bacterial families, including Rhizobium, Pseudomonas, Burkholderia, Micrococcus, Azotobacter, Erwinia, Flavobacterium, Serratia and Bacillus, among others [37][38][39]. Generally, PGPR have the abilities to produce phytohormones (auxins, cytokinins and gibberellins), organic acid, siderophore and ACC deaminase, dissolving phosphorus and potassium, as well as fixing nitrogen [39].
In our previous research, the Bacillus cereus strain DW019 was isolated from the rhizosphere of Pinus elliottii, which is one of the phytoremediation plants found in an abandoned ionic rare earth mine in Ganzhou, China [21]. The strain has growth-promoting abilities, including IAA production, iron carrier production and nitrogen fixation. As we know, IAA is the most important plant hormone, which directly promotes the growth of plants and microorganisms. Endophytic bacteria with IAA-producing abilities can promote root growth and root length, thus increasing root surface area and enabling plants to obtain more nutrients from the soil [36]. Our results support previous research results, that is, that IAA produced by Bacillus is beneficial to plant growth. It has been reported that Bacillus treatment increased the yield of vegetables. For example, Nuntavun et al. (2021) used Bacillus licheniformis BDS31 and Pseudomonas azoicus C2-114 to treat M. capillaris with the aim of promoting plant height and leaf number [40]. Agake et al. (2021) found that the high concentration of Bacillus subtilis TUAT1 on the spermosphere via coating with molybdenum promoted the growth of rice [41]. PGPR have been used as alternatives in sustainable agriculture to improve the germination and development of cucumber shoots, as well as for greater production of phytochemical compounds, which allowed the early establishment in the field and the subsequent growth of the plants [42]. B. cereus inoculation dramatically promoted the growth of ground diameter and the plant height of J. regia [43]. Our results were consistent with those from the applications of Bacillus TP-H20-5 to cherry tomato, mature tomatoes, strawberries and cucumbers, all of which had their fruit yields significantly increased [44]. The results of our study showed that the number and weights of cherry tomato plants can be effectively increased via the inoculation of viable cells into the soil of cherry tomato.
Our study showed that B. cereus DW019 can not only increase the fruit yield of cherry tomato but also promote the accumulation of phytochemicals. The inoculation enhanced the content of vitamin C, soluble sugar and soluble protein in cherry tomato, which was similar to the finding that environmental conditions, mature stage and nutritional status had an impact on the contents of these substances in fruits [45]. The strain also induced plant salt tolerance and alleviated stress, which further affected the content of vitamin C, soluble sugar and soluble protein. It was found that after treatment with B. velezensis, protein and sugar in wheat ears was increased by 56% and 30%, respectively [46]. In this study, the increase in protein and sugar in both VC9 and DVC9 groups was much higher than that of the above results. Taken together, B. cereus DW019 is able to effectively improve fruit quality and yield.
The diversity of soil bacteria is an important biological indicator for determining soil health [47]. Traditional culturing methods, such as the plate-counting method, usually cultivate rapidly growing microorganisms but only 1~2% of bacterial diversity can be estimated in this manner [45]. To evaluate the diversity and ecological characteristics of non-culturable microorganisms, high-throughput sequencing technology was conducted in this study. Compared with the control, the bacterial alpha diversity of rhizosphere soil decreased after inoculation. The UniFrac-weighted PCA analysis, based on OTU composition, showed that the effect of inoculation on bacterial diversity also varied among differently treated soil samples, and beta diversity index of the treated soil was higher than the control. Similarly, the Chao index and Shannon index of the soil in the treatments were also higher than those in the blank control group. At the phylum level, the bacterial community composition of different treatments was also different, of which Proteobacteria and Actinomycetes were the dominant bacteria in the rhizosphere. They played an important role in the ecological and metabolic functions of soil due to their participation in nitrogen fixation, decomposition and humus formation [48,49]. P. vancouverii M1 and E. ludwigensis E15 showed the best growth promotion effect under the dual stress of low light and high sediment organic matter load, with a 36% and 46% increase in height, respectively [50]. PGPR agents were able to regulate the relative abundance of certain rhizosphere bacteria, modulate the alpha diversity and beta diversity of inter-root bacterial communities, and thus promote the growth of and yield of crops.

Conclusions
In this study, the treatment of cherry tomato seedlings with Bacillus cereus DW019 increased the yield of cherry tomato fruit, improved their nutritional quality and changed the diversity of rhizosphere soil microorganisms. The yield, vitamin C content, soluble sugar and soluble protein of cherry tomatoes underwent an increasing trend with increasing inoculation concentration. Moreover, the addition of Bacillus cereus altered the bacterial diversity and increased the species abundance in rhizosphere soil. Hence, as a plant-growthpromoting bacterium, B. cereus DW019 is able to promote the plant growth and the yield of nutritional contents of cherry tomato, which indicates that it would be a suitable alternative to chemical fertilizers for improving plant growth, nutrient quality and even pathogen inhibition. Therefore, we propose that B. cereus DW019 could be applied to other economic crops in the future and highlight that, in the pursuit of sustainable agriculture, it could have great potential in the development of agricultural inoculants to promote crop yield and improve cropland ecosystems.