Introduction

The Brazilian semiarid region covers approximately 1 million km², with high temperatures (~ 30 °C) and limited rainfall (~ 600 mm/year), concentrated over four months1. In response to these challenging environmental conditions, smallholders rely on resilient crops, such as the prickly-pear cactus (Nopalea cochenillifera L.), a drought-tolerant species, showing the crassulacean acid metabolism (CAM), that conserves water through regulated transpiration2,3. Additionally, prickly-pear cactus contains high levels of essential nutrients, including ascorbic acid, vitamin E, carotenoids, and fiber4. These physiological and biochemical characteristics make prickly-pear cactus valuable in semiarid regions, both for conserving the plant and providing water and nutrients to animals5. Particularly, the prickly-pear cactus genotypes ‘Baiana’ and ‘Doce’ are among the most widely cultivated in the Brazilian semiarid region due to their strong adaptation to harsh environmental conditions (Oliveira et al., 2010)6.

Despite its ecological and economical importance to semiarid regions, prickly-pear cactus often has low productivity and quality, mainly due to a lack of inputs such as chemical fertilizers7primarily because of their high cost8. However, the use of plant growth-promoting bacteria (PGPB) can offer a sustainable alternative by enhancing growth via nitrogen fixation, phosphate solubilization, and hormone production9,10,11. In semiarid regions, studies have reported the benefits of PGPB to important crop species, such as cowpea, maize, sorghum, and cassava12,13,14.

While PGPB benefits are well-documented, less is known about their potential effects on soil microbial biomass and enzymatic activity, particularly in the rhizosphere of plant genotypes grown in semiarid conditions. This knowledge gap is critical, as these biological parameters drive soil organic matter (SOM) accumulation and nutrient availability15directly influencing drought resilience. Thus, increased soil organic matter (SOM) content contributes to conservate soil moisture, while higher nutrient availability supports the plant growth. A key impact of PGPB on the rhizosphere is the stimulation of root exudation, which provides carbon and nitrogen sources to support microbial biomass16. Root exudates, composed of sugars, amino acids, and organic acids, act as selective agents that shape the composition and function of rhizosphere microbial communities, including those involved in enzymatic activity and nutrient cycling17,18. Indeed, Anderson et al.17 reported that root exudate can shape rhizosphere microbial communities, with effects on microbial biomass and soil enzyme activities18. In addition, root exudate composition and PGPB effects on microbial biomass and enzymatic activity both vary significantly depending on plant genotype and developmental stage19,20.

Although previous studies have highlighted the positive effects of PGPB inoculation on the growth of plant species in semiarid regions, some gaps remain unanswered. Some key novelties of this study would be is how prickly-pear cactus genotypes modulate PGPB effects, as also affected by early and late growth stage, on soil microbial biomass and enzymatic activity in the rhizosphere. Specifically, we hypothesized that: (a) inoculation with B. subtilis and Paenibacillus sp. would increase microbial biomass and enzymatic activity in the rhizosphere, but the magnitude of these effects would differ between the genotypes ‘Baiana’ and ‘Doce’; and (b) these effects would be stronger at 90 days than at 270 days due to higher microbial activity during early plant development. To test these hypotheses, we inoculated Bacillus subtilis and Paenibacillus sp. in two genotypes of prickly-pear cactus (‘Baiana’ and ‘Doce’) and assessed the effects on microbial biomass C and N and enzymatic activity in the rhizospheric soil. This study is important to support sustainable agriculture in semiarid regions with drought conditions by harnessing plant-microbe interactions to support crop resilience and soil health.

Materials and methods

Study area

This field-study with prickly-pear cactus was conducted at the Campus Prof. Cinobelina Elvas from Federal University of Piauí, located in Bom Jesus, Piauí State (9° 4’ 30’’ S, 44° 21’ 26’’ W). The predominant soil is classified as Yellow Latosol which presents 17% clay, 5% silt, and 78% sand. Soil chemical properties were assessed Teixeira et al.21 at the beginning of the study (Table S1). During the study, the climatic conditions were recorded (Fig. 1).

Fig. 1
figure 1

Climatic conditions at planting and soil collections.

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Experimental design and planting

The study was conducted in a completely randomized design under split-split plot arrangement (2 × 3 × 2). The main plot factor was cactus growth stage (90 and 270 days after cactus planting), the subplot factor was PGPB inoculation (non-inoculation and inoculation with B. subtilis and Paenibacillus sp.), and the sub-subplot factor was two genotypes of prickly-pear cactus (‘baiana’ and ‘doce’). The strains of both B. subtilis and Paenibacillus sp. were IPA-CC29 and IPA-CC38, respectively. These strains were used in previous experiments13and their growth-promoting characteristics include the production of Indole-3-acetic acid (IPA-CC29: 1.41 mg L⁻¹; IPA-CC38: 1.13 mg L⁻¹) and Acetylene-reducing activity (IPA-CC29: 5.03 nmol C₂H₄ h⁻¹; IPA-CC38: 7.10 nmol C₂H₄ h⁻¹).

To obtain the PGPB inoculants, B. subtilis and Paenibacillus sp. were grown in Erlenmeyer flasks containing 50 mL of liquid medium (Tryptone Soy Broth) and incubated in a rotatory shaker (200 rpm; 31 °C) for 72 h. Bacterial growth was assessed by optical density (absorbance) at a wavelength of 540 nm using a spectrophotometer and was approximately 108 CFU (colony-forming unit) mL1. The planting was done using one cladode per hole, with a spacing of 0.75 × 0.50 m. After planting, the inoculation of B. subtilis and Paenibacillus sp. was performed by spraying the liquid inoculant diluted in water at a 5:1 ratio directly in the root area. (Fig. 1).

Soil sampling and analysis

Soil samples were collected from rhizosphere by removing soils adhered to the roots, using a spade while preserving the plant structure as much as possible. In each treatment, four soil samples were collected, totaling 48 soil samples, which were sieved through a 2 mm mesh sieve, and stored at 6 °C.

Soil microbial biomass C22N23and P24 were assessed by irradiation-extraction. Briefly, 10 g of soil from each sample was weighed in duplicate, with one sample being irradiated and the other non-irradiated. The irradiated samples were subjected to a microwave oven in five cycles of 30 s each. An extracting solution was then added to both irradiated and non-irradiated soil samples, which were shaken for 30 min at 150 rpm. After shaking, the samples were filtered. Potassium sulfate (K₂SO₄) was used as the extracting solution for microbial biomass C and N, while sodium bicarbonate (NaHCO₃) was used for microbial biomass P.

To measure the biomass C, potassium dichromate (K₂Cr₂O₇) and sulfuric acid (H₂SO₄) were added to the samples. The solution was heated, then cooled, and diluted with distilled water. The residual dichromate was measured by titration with an ammonium ferrous sulfate solution ((NH₄)₂Fe(SO₄)₂·6 H₂O) in concentrated sulfuric acid (H₂SO₄), using ferroin ([Fe(C₁₂H₈N₂)₃]SO₄) as an indicator.

To measure biomass N, the samples were transferred to test tubes, and citric acid (C₆H₈O₇) and ninhydrin (C₉H₆O₄) were added. The samples were then incubated in a water bath at 100 °C for 25 min. After cooling, an ethanol: water solution (C₂H₆O: H₂O) in a 1:1 ratio was added, and the absorbance was measured with a spectrophotometer at 570 nm.

To measure microbial P, HCl (10 mol L⁻¹) was slowly added to the samples, which were then left to stand for 1 h. After this, the samples were filtered, and reagent A (containing ammonium molybdate - (NH₄)₆Mo₇O₂₄·4 H₂O, potassium antimony tartrate - C₈H₄K₂O₁₂Sb₂·3 H₂O, and sulfuric acid - H₂SO₄), reagent B (containing ascorbic acid - C₆H₈O₆ and sulfuric acid - H₂SO₄), and distilled water were added. The samples were left to stand for 30 min, after which the absorbance was measured with a spectrophotometer at 882 nm.

The quantification of microbial biomass C, N, and P was determined by the difference between irradiated and non-irradiated soils. The extracted C, N, and P were converted into microbial C, N, and P using correction factors Kc = 0.38, 0.48, and 0.40, respectively.

The determination of β-glucosidase25 and acid phosphatase26 activities was carried out by measuring p-nitrophenol, released when 0.5 g of soil was incubated for 1 h at 37 °C with buffered solutions containing the substrates p-nitrophenyl-β-D-glucoside (PNG) and p-nitrophenyl phosphate (PNPP) to β-glucosidase and acid phosphatase, respectively. The p-nitrophenol released by the reaction with the enzymes was extracted from the soil by filtration after the addition of CaCl₂ and NaOH solutions. Readings were taken using a spectrophotometer at a wavelength of 400 nm for both enzymes.

The methodology used for the determination of urease enzyme in soil consisted of measuring ammonia release after incubating the soil in urea solution for 2 h at 37 °C27. Two grams of soil were weighed and added to the urea solution, followed by borate buffer with pH 10. The samples were then incubated in a water bath for 2 h at 37 °C. After the incubation period, potassium chloride (KCl) was added, followed by filtration. After filtering the material, H₂O, salicylate/NaOH, and dichloroisocyanurate (0.1%) were added, and the mixture was incubated for 30 min. The intensity of the color was measured with a spectrophotometer at 690 nm.

Statistical analysis

The data was subjected to the Shapiro-Wilk normality test and analysis of variance (ANOVA). When the treatments differed significantly based on the F test, the means were compared using Tukey’s test at a probability level of 5%, and the analysis was done in RStudio. To reduce the dimensionality of the data and understand which factors have influence on the biological variables, a principal component analysis (PCA) was performed using a correlation matrix with the R software (stats, factoextra, and vegan packages), version 4.1.3 28.

Results

The interactions between growth stage, PGPB, and genotypes

The ANOVA results indicated that PGPB species, prickly-pear cactus genotypes, growth stage, and their interactions had significant effects on microbial biomass and enzymatic activity in the rhizosphere (Table S2). Specifically, PGPB inoculation influenced microbial biomass P, β-glucosidase, and acid phosphatase, whereas cactus genotypes affected microbial biomass C and N. Growth stage also significantly impacted microbial biomass C and N, as well as acid phosphatase. Interactions between PGPB species and cactus genotypes led to notable changes in microbial biomass C, N, β-glucosidase, and acid phosphatase. Additionally, three-way interactions among PGPB species, cactus genotypes, and growth stage influenced microbial biomass C, N, P, and acid phosphatase. The highest levels of microbial biomass C, P, and acid phosphatase were recorded at 90 days, while microbial biomass N peaked at 270 days. In contrast, β-glucosidase and urease activities showed no significant variations across these time periods (Fig. 2).

Fig. 2
figure 2

Microbial biomass and enzymatic activity at 90 and 270 days of growth of prickly-pear cactus. Different lowercase letters indicate significant differences based on Tukey’s HSD test (p < 0.05).

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Microbial biomass and enzymatic activity in response to PGPB and genotypes of prickly-pear cactus

At 90 days, non-inoculated plants and those inoculated with B. subtilis had higher microbial biomass C in the rhizosphere of genotype ‘Doce’ compared to ‘Baiana’ (Fig. 3A). By 270 days, microbial biomass C remained higher in the rhizosphere of non-inoculated genotype ‘Doce’ than in ‘Baiana’ (Fig. 3B). PGPB inoculation did not affect microbial biomass N at 90 days compared to non-inoculated plants (Fig. 3C). However, non-inoculated genotype ‘Baiana’ had higher microbial biomass N in the rhizosphere than genotype ‘Doce’ at this stage. At 270 days, B. subtilis inoculation increased microbial biomass N in the rhizosphere of genotype ‘Doce’ (Fig. 3D). Microbial biomass P increased at 90 days in both genotypes with PGPB inoculation compared to non-inoculated plants (Fig. 3E). By 270 days, PGPB inoculation increased microbial biomass P in the rhizosphere of genotype ‘Doce’, while ‘Baiana’ showed higher microbial biomass P only with B. subtilis inoculation (Fig. 3F).

Fig. 3
figure 3

Carbon (MBC), nitrogen (MBN), and phosphorus (MBP) of microbial biomass in the rhizosphere of ‘Baiana’ and ‘Doce’ prickly-pear cactus, including both non-inoculated and inoculated with Bacillus and Paenibacillus, at 90 and 270 days of plant growth. Lowercase letters indicate differences between inoculants within the same genotype, while uppercase letters indicate differences between genotype within the same inoculant, based on Tukey’s HSD test (p < 0.05).

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The inoculation of B. subtilis increased β-glucosidase activity in the rhizosphere of genotype ‘Doce’ at 90 days, whereas Paenibacillus sp. enhanced β-glucosidase activity in the rhizosphere of genotype ‘Baiana’ (Fig. 4A). At 270 days, PGPB inoculation significantly increased β-glucosidase activity in both genotypes (Fig. 4B). Urease activity in the rhizosphere was unaffected by PGPB or genotype treatments at both time points (Figs. 4C, D). At 90 days, acid phosphatase activity increased in the rhizosphere of genotype ‘Baiana’ with Paenibacillus sp. inoculation (Fig. 4E). However, by 270 days, no significant differences were observed between PGPB and genotype treatments for acid phosphatase (Fig. 4F).

Fig. 4
figure 4

The enzymatic activity of β-glucosidase, urease, and acid phosphatase in the rhizosphere of the ‘Baiana’ and ‘Doce’ prickly-pear cactus, both non-inoculated and inoculated with Bacillus and Paenibacillus, at 90 and 270 days of plant growth. Lowercase letters indicate differences between inoculants within the same genotype, while uppercase letters indicate differences between genotype within the same inoculant, based on Tukey’s HSD test (p < 0.05).

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Multivariate analysis of microbial biomass and enzymatic activity in responses to PGPB and genotypes prickly-pear cactus

Principal component analysis (PCA) explained 68.6% and 58.3% of the total variance at 90 and 270 days, respectively (Fig. 5). At 90 days, the inoculation of B. subtilis in the genotype ‘Baiana’ formed a distinct cluster characterized by increased acid phosphatase activity. The grouping of B. subtilis in the genotype ‘Doce’ with Paenibacillus sp. in the genotype ‘Baiana’ reflected their shared enhancement of microbial biomass P and urease activity. The non-inoculated genotype ‘Doce’ and Paenibacillus sp.-inoculated genotype ‘Doce’ clustered together due to their similar patterns in microbial biomass C and β-glucosidase, while non-inoculated genotype ‘Baiana’ showed only microbial biomass C responses.

Fig. 5
figure 5

The principal component analysis (PCA) of the combination of prickly-pear cactus genotypes, both inoculated and non-inoculated, assessed their effects on microbial biomass (MBC, MBN, MBP), and enzymatic activity (β-glucosidase, urease, and acid phosphatase) at 90 and 270 days of plant growth. PCA a = Permanova < 0.05 and R²: 0.634, PCA b = Permanova < 0.05 and R²: 0.800.

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At 270 days, treatments drove more differentiated responses. Thus, the non-inoculated genotype ‘Doce’ formed a separate cluster defined by higher microbial biomass C. The grouping of non-inoculated genotypes ‘Baiana’ with Paenibacillus sp.-inoculated genotype ‘Baiana’ resulted from their comparable effects on β-glucosidase activity and microbial biomass N and P. The inoculation of B. subtilis in the genotype ‘Baiana’ showed significant association with high acid phosphatase, while the genotype ‘Doce’ primarily enhanced urease activity.

Discussion

This study is the first to assess the effects of inoculation with plant growth-promoting bacteria (PGPB) on prickly-pear cactus genotypes in semiarid regions, specifically assessing their influence on microbial biomass and enzymatic activity in the rhizosphere. Both hypotheses were supported, demonstrating that: (a) microbial biomass and enzymatic activity responses depended on the inoculated PGPB (B. subtilis and Paenibacillus sp.) and prickly-pear cactus genotypes (‘Doce’ and ‘Baiana’), and (b) growth stage (juvenile versus mature plants) differentially influenced microbial biomass and enzymatic activity responses.

The growth stage influences microbial biomass and enzymatic activity in response to PGPB inoculation in prickly-pear cactus

Our study found distinct effects of growth stage (90 and 270 days) on biological parameters in the rhizosphere, with greater microbial biomass and activity during the early growth (90 days) compared to later stage (270 days). This occurs due to more rhizosphere activity found during the early stage than later one27. We observed higher microbial biomass C and P, along with increased acid phosphatase activity, at the early growth stage (juvenile), while microbial biomass N was higher at later stage (maturity). These patterns reflect fundamental shifts in plant-microbe interactions across growth stages. Root exudation, known to vary by plant growth stage29serves as the primary driver of microbial communities in the rhizosphere30. During early growth (90 days), abundant carbon-rich exudates31,32 enhance microbial biomass C.

Regarding microbial biomass P and acid phosphatase activity, the increases in both biological parameters correspond with phosphorus demand by the plant during its active growth stage33. At later maturity (270 days), reduced plant nitrogen demand contributes to greater microbial N acquisition, increasing soil microbial biomass N34. This effect is associated with elevated soil moisture that facilitates organic matter decomposition and nitrogen release35. Interestingly, β-glucosidase activity remained stable across both growth stages, indicating consistent rates of organic carbon decomposition36. These findings collectively demonstrate how growth-stage dependent changes in plant physiology and environmental conditions shape rhizosphere microbial communities and their functioning. In addition, non-inoculated plants of genotype ‘Doce’ (90 days) and ‘Baiana’ (270 days) showed stronger reliance on microbial biomass N, suggesting a shift toward nitrogen immobilization in the absence of PGPB.

The interaction between PGPB and genotypes of prickly-pear cactus

While there are currently no specific studies comparing the effects of different PGPB species on microbial biomass and enzymatic activity across various plant genotypes in semiarid regions, our findings indicate that plant genotype influences these microbial responses to PGPB inoculation. This agrees with previous studies reporting genotype-dependent effects of PGPB on rhizosphere microbial communities37,38. Our results further suggest that some specific genotypes may better optimize interactions with specific PGPB taxa, thereby shaping rhizosphere microbial biomass and its functional dynamics39.

Interestingly, B. subtilis inoculation differentially affected microbial biomass C, N, and P in the rhizosphere of each genotype, with the most pronounced effects observed in the genotype ‘Doce’. This enhancement can be due the ability of B. subtilis to efficiently colonize roots, form biofilms, and secrete exudates40thereby fostering a favorable microenvironment for soil microbes. Specifically, B. subtilis contributes to phosphorus solubilization, which could explain the increase in microbial biomass P41. Specifically, B. subtilis stimulated phosphorus cycling processes in both genotypes, though through different mechanisms: a direct enzymatic action in the genotype ‘Baiana’ (via increased phosphatase activity during early growth) and microbial biomass accumulation in the genotype ‘Doce’.

Additionally, both B. subtilis and Paenibacillus sp. promoted the activity of β-glucosidase and acid phosphatase. This is interesting since these enzymes play key roles in organic matter decomposition, such as breaking down cellulose (via β-glucosidase) and organic phosphate (via acid phosphatase). Thus, the PGPB strains enhanced microbial biomass C and P, associated with increased β-glucosidase and acid phosphatase activities, suggesting improved microbial access to C and P sources, possibly mediated by increased root-derived substrates43,44. At later growth stages, Paenibacillus sp. in the genotype ‘Baiana’ and B. subtilis in the genotype ‘Doce’ were associated with elevated β-glucosidase activity and microbial biomass P, highlighting their roles in both phosphorus mobilization45 and carbon cycling46. The inoculation of Paenibacillus sp. also increased microbial biomass P in the genotype ‘Baiana’, indicating genotype-specific effects of PGPB strains.

Implications for inoculating B. subtilis and Paenibacillus sp. in genotypes of prickly-pear cactus

This study showed that inoculation with B. subtilis and Paenibacillus sp. significantly influenced microbial biomass C and P and their related enzymatic activities, while had no significant effect on microbial biomass N or associated N-cycling enzymes. While both PGPB taxa are known to participate in N cycling, particularly nitrogen fixation45,47our findings suggest that in the prickly-pear cactus rhizosphere, their primary role involves organic matter decomposition and phosphorus (P) solubilization, rather than N mineralization or fixation. This suggests that prickly-pear cactus genotypes may associate with native soil microorganisms to meet their N requirements, while inoculated PGPB (B. subtilis and Paenibacillus sp.) predominantly enhance C and P availability through microbial biomass turnover and enzymatic processes.

Conclusions

The interactions among growth stage, PGPB taxa, and genotypes significantly shape microbial biomass and enzymatic activity in the rhizosphere of prickly-pear cactus. These dynamics suggest that specific combinations of prickly-pear cactus genotypes with PGPB taxa under distinct plant growth stage shape rhizosphere microbial biomass and enzymatic activity. Particularly, B. subtilis has been proved to be broadly effective for both genotypes of prickly-pear cactus, particularly in microbial biomass and enzymes associated to phosphorus. This mean that carefully pairing prickly-pear cactus genotypes with specific PGPB taxa, mainly B. subtilis, can enhance microbial biomass and enzymatic activity in the rhizosphere. Markedly, this can lead to improved P availability to support cactus growth, mainly in nutrient-poor arid soils.