The experiment was conducted in a greenhouse located at the Citrus Research and Education Center (CREC), Lake Alfred, Florida (28.1026° N, 81.7120° W). ‘Duncan’ grapefruit (Citrus Paradisi) seedlings were propagated from disease-free seeds collected directly from the fruit grown in the CREC CUPS (Citrus Under Protective Screens; Schumann et al., 2023) facility. Seeds were rinsed three times with distilled water to remove any juice vesicles, and then soaked in distilled water to discard floating seeds. The selected seeds were dried with paper towels and the seed coats were carefully removed with a previously sanitized blade (GEM 62-0161 carbon steel extra sharp single-edge blade). Two seeds were planted into each growth media plug in germination trays of the Double Bio Dome Seed-Starting System (Park Seed, Greenwood, SC) and allowed to germinate in the greenhouse. After three weeks of completed germination, the tallest vigorous seedlings with true leaves were transferred individually into 1.67-L plastic pots filled with unsterilized substrate media of 50% Canadian peat moss with the addition of pine bark, perlite, and vermiculite. All plants were fertilized every week until the start of the experiment 18 weeks after transplanting, then fertilization was stopped and only treatments based on fertilizer (T1) and B. velezensis plus fertilizer (T6) continued receiving reduced fertilization every four weeks (25% of the full rate) ( Table 1 ). Monitoring and treating for any potential pest incidence were assessed weekly.

A randomized complete block design with seven treatments and five replications was used, with each plot (experimental unit) composed of four potted grapefruit seedlings. The treatments ( Table 2 ) were applied as a 0.129 L pot^-1 root drench and correspond to: (1) fertilization ( Table 1 ), (2) B. velezensis (LALRISE VITA^®; Lallemand Plant Care, Montreal, Canada), (3) fresh inoculum of B. amyloliquefaciens (commercial product Double Nickel 55^®; Certis, Columbia, MD) where dormant spores were activated in a bioreactor 24 hours before application, (4) fresh inoculum of a consortium of B. subtilis, B. megaterium, and Bacillus sp. (citrus endophytic isolates) (Li et al., 2021), (5) a combination of fresh inoculum of B. amyloliquefaciens and the consortium of three B. subtilis isolates to test for additional synergy with bacterial combinations, (6) B. velezensis with the addition of fertilization and (7) water (control) with no additional application. The fertilizer treatment consisted of three different sources described in Table 1 . The fresh inoculum of Bacillus spp. consortia was prepared by culturing the bacterium in a nutrient broth medium with agitation at 30°C for 24 hours and adjusted to 10⁹ cells per milliliter with water.

Tree seedling heights and diameters in two perpendicular directions were measured every month to obtain canopy volume based on the formula of Thorne et al. (2002), as well as trunk diameter measured 5 cm above the potting media. SPAD chlorophyll index (Konika Minolta, Ramsey, NJ) was collected monthly from three leaves per tree and averaged for each 4-tree plot. At the end of the experiment (three months after treatments began), every plot of four trees was photographed and four leaves per tree were collected and sent for N, P, K, Ca, Mg, S, Zn, B, Mn, Fe, and Cu concentration analysis at the Waters Agricultural Laboratory, Camilla, GA. Tree above-ground biomass and root biomass were obtained by separating leaves, shoots, and roots for each 4-tree plot. Bagged samples were placed in a drying oven (Fisher Scientific, Pittsburgh, PA) for 48 hours at 70°C, and were then weighed to obtain dry mass per plot.

We hypothesized that microbial respiration from the most effective rhizobacterial treatments would increase compared to the controls. Growing media samples in the rhizosphere were obtained from the excavated root systems by gentle shaking into a plastic tray, to collect samples, mix and transfer them into plastic Ziploc bags. A mixed media sample from 4 trees was collected, representing the treatment plot and processed for microbial respiration in the lab by implementing the substrate-induced respiration method (Anderson and Domsch, 1978). Empty Mason jars were weighed, then 50 grams of media from the samples was weighed into the jars. To measure the CO₂ respired, a 20 mL plastic scintillation vial was placed inside the jar with 4 mL of 1M NaOH. After 10 days of incubation in a growth chamber in dark- conditions at 35 degrees Celsius, vials were removed from the jars and capped. Jars were placed in the drying oven at 105 degrees Celsius for 48 hours to dry the media. Dry media was then weighed to calculate the percentage of gravimetric water content. The determination of CO₂ produced by microbial respiration processes was based on the titration of 1M NaOH in vials with standardized 0.1M HCl and the use of phenolphthalein as a color indicator to determine the endpoint. An additional blank jar with a vial inside and no media was included in the incubation to calculate the titration amount with zero respiration.

To obtain shoot biomass we summed the leaf and stem biomass and then conducted an ANOVA to find the significance among treatments. Similarly, the shoot-to-root ratio was calculated by dividing shoot biomass by root biomass. Finally, the N content in leaves was obtained by multiplying the leaf N concentration and the leaf biomass. In order to test the N fertilizer equivalent saved as a result of using PGPRs, a linear regression line of mg N applied per pot versus mg N/pot accumulated in leaves was calculated for treatments T1 (fertilizer) and T7 (control). The regression equation was then used to interpolate the mg N fertilizer equivalent contributed by each PGPR treatment according to its N accumulation in the leaf biomass.

To identify significant differences between treatments, Analysis of Variance (ANOVA) was performed using Rstudio (Rstudio Team (2020). Rstudio: Integrated Development for R. Rstudio, PBC, Boston, MA URL http://www.rstudio.com/.) and conducting Post hoc comparisons with the Tukey HSD test to find statistical differences between means using the ‘agricolae’ package (De Mendiburu, 2022). The data was tested to be normally and independently distributed, from the seven treatment combinations with 5 replications. Differences were considered statistically significant when P ≤ 0.05.

Results of grapefruit seedling growth measurements included height, canopy volume, trunk diameter, root, stem, and leaf biomass, and SPAD chlorophyll index. Tree height, canopy volume, trunk diameter, shoot biomass, root biomass, and chlorophyll index were significantly greater for plants inoculated with B. velezensis that also received fertilizer ( Figures 1 – 6 ). The B. subtilis + B. amyloliquefaciens consortium treatment (T5) had a significantly higher shoot biomass than the untreated negative control (T7), despite the lack of any added fertilizer. The growth promotion ability may have an effect on roots (Shen et al., 2023) and can lead to an increase in water and nutrient uptake for the overall plant growth (Wang et al., 2020). In our results, Supplementary Figure 3 shows a very highly significant correlation (p<0.001) between leaf N accumulated and root biomass per tree to illustrate the linkage between enhanced root biomass and N uptake. The B. velezensis inoculation + fertilizer treatment (T6) produced significantly more shoot biomass than any other treatment, even including T1, the fertilized positive control ( Figure 4 ), and the B. velezensis inoculation (T2), suggesting synergy between the B. velezensis and fertilizer. The ratio of shoot/root biomass is an estimate of response to physical, chemical, and biological factors related to above-ground architecture (Lopez et al., 2023). Supplementary Figure 1 shows no significant differences among treatments suggesting that the relative changes in root and shoot growth according to treatments were approximately proportional. The B. subtilis + B. amyloliquefaciens treatment produced a significantly higher (p<0.001) SPAD chlorophyll index than the control ( Figure 6 ), but the other B. subtilis and B. amyloliquefaciens treatments did not show evidence of promoting tree growth ( Figures 1 – 6 ).

Soil inoculation with bacteria that promote plant growth is a promising technique for sustainable agricultural management and an effort to increase the efficiency of crops to uptake nutrient via symbiosis with microbes. In the present study, we anticipated that the applied bacteria would successfully establish around the rhizosphere in the potting media, as demonstrated in this study. However, when conducting microbial inoculation in open field soil conditions, the response of soil bacterial communities may be less consistent due to environmental variability such as tillage (Sergaki et al., 2018), pesticides, fertilizers, and sewage sludge amendments applied in raw form (Omotayo and Babalola, 2021). These environmental factors could disrupt the establishment and proliferation of the bacteria. Bacillus species are well known for their plant growth-promoting potential (Gohil et al., 2022), and based on our results, we could see a marked increase in above and below-ground plant biomass with PGPR inoculation. Although the specific mechanisms by which PGPR promotes plant growth are not fully understood (Backer et al., 2018), they are thought to include auxin secretion by B. velezensis, systemic induced resistance, phosphate solubilization (Wang et al., 2020; Wang et al., 2022) pathogen control, and secondary metabolites (Grobelak et al., 2015; Ghazy & El-Nahrawy, 2021) to protect and promote plant growth (Wang et al., 2020).

We hypothesized that microbial respiration from the most effective bacterial treatments would vary compared to the controls, however there were no significant differences among the microbial respiration of the treatments ( Supplementary Figure 2 ). The positive and negative controls (untreated and fertilized) showed respiratory activity even though they were not inoculated with bacteria, and this indicates that unsterilized potting media can harbor a wide variety of microorganisms, whether they are beneficial (Huang et al., 2012; Al-Mazroui and Al-Sadi, 2015) or pathogenic species (Al-Sa’di et al., 2008; Al-Sadi et al., 2011). The fact that in the present work all the treatments showed microbial respiration activity suggests that it could have masked the activity of added PGPR treatments. From previous studies, we can identify the lack of analysis for bacterial communities’ characterization in potting media to assess changes in the activity of inoculated soil microbial communities (Al-Sadi et al., 2016; Mawarda et al., 2022).

Inoculation with B. velezensis together with the addition of a low (25%) fertilizer rate significantly increased leaf nitrogen (N), magnesium (Mg), and sulfur (S) concentrations compared to the other treatments of Bacillus species along with the positive and negative control ( Table 3 ). Salvagiotti et al. (2009) reported a synergism in the uptake of N and S by plants, and a synergism of P and Mg uptake by plants was reported by Weih et al., 2021. However, in our study P concentration differences were not significant ( Table 3 ) but increased levels of P uptake were observed from treatments inoculated with B. velezensis, B. amyloliquefaciens, and B. subtilis. Leaf concentrations of K, Ca ( Table 3 ), Mn, and Cu ( Table 4 ) were also not significantly different among treatments.

Nitrogen is one of the essential nutrients for crop growth and has been widely studied (Wang et al., 2014). In our study, due to a low rate of fertilization, leaf N concentration for all treatments was below the optimum rate required for citrus crops (Obreza et al., 2020), however, leaf N concentration for the B. velezensis plus fertilizer treatment, significantly outperformed the rest of the treatments ( Table 3 ). A similar pattern was observed in the SPAD chlorophyll index ( Figure 6 ), where fertilizer treatment B. velezensis plus fertilizer had the highest average chlorophyll content correlated to N status in the leaf.

In the absence of fertilizer, there were non-significant differences in leaf nutrient concentrations among treatments inoculated only with bacteria ( Tables 3 , 4 ), emphasizing the importance of synergistically including fertilizer with PGPR inoculation as some previous studies found, increasing yield in tomato (Solanum lycopersicum L.) (Clagnan et al., 2023) and cowpea (Vigna unguiculata L. Walp) (Stamford et al., 2020). Surprisingly, leaf iron (Fe) concentrations were higher for the control treatment (T7; Table 4 ), although Fe concentration was at the optimum level for all the treatments. For the negative control (T7), it is possible that other nutrients were more limiting than Fe, resulting in a lack of biomass growth and a relative concentration of Fe in the leaves when compared to the more thrifty treatments.

Stamford et al. (2020); Clagnan et al. (2023) and Angulo et al. (2020) have examined the potential beneficial effects of microbial-based fertilizers on the rhizosphere in a competitive nutrient-limited environment. After soil inoculation, these microorganisms contribute to nitrogen fixation, solubilization/mobilization of soil nutrients, and secretion of substances that enhance plant growth (Shahwar et al., 2023). The B. amyloliquefaciens + B subtilis consortia (T5) treatment accumulated the most leaf N per tree for any unfertilized treatment, and significantly more than B. amyloliquefaciens (T3; Supplementary Figure 4 ). PGPRs play a key role when unavailable nutrients are present in the soil or nearby the rhizosphere (Prasad et al., 2019). From the regression line of N fertilizer applied versus leaf N accumulated (y = 0.4015x - 169.19), we estimated the amount of N contributed by PGPRs that was not derived by chemical N fertilizer. T6 was the highest (105 mg N/pot), followed by T5 (47 mg N/pot), T2 (16 mg N/pot), T4 (14 mg N/pot), and T3 (-23 mg N/pot). The negative value for T3 suggests microbial N immobilization. For T6, the amount of fertilizer N applied (82 mg N/pot) was subtracted from the interpolated result, leaving only the equivalent portion derived from PGPR activity (105 mg N/pot). The highest N fertilizer equivalent provided only by PGPRs was T5 (B. amyloliquefaciens + triple B. subtilis consortia), which was 57% of the amount of chemical N fertilizer supplied in treatments 1 and 6. Gohil et al., 2022 reported that Bacillus sp. PG-8 was able to solubilize insoluble nutrients to help the plant growth. Phosphate solubilization is most frequently attributed to Bacillus isolates by 80% compared to Azotobacter, Pseudomonas, and least by Mesorhizobium (Ahmad et al., 2008). The promotion of plant growth can be ascribed to various mechanisms, including the synthesis of plant growth-promoting hormones within the rhizosphere and the involvement in other plant growth-promoting activities (Ahmad et al., 2008). In recent years, numerous strains of B. velezensis have been discovered and extensively examined for their impact on plant growth, disease, and pest resistance, and the induction of systemic plant defenses, while some strains have proven to be pivotal in enhancing crop yield.