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Article

Biotization with Plant Growth-Promoting Bacteria Benefits the Survival and Production of Potato (Solanum tuberosum L.) In Vitro and In Vivo

by
Yulimar Castro Molina
1,2,
Joyce Dória
3,
Ana Milena Gómez Sepúlveda
1,
Luna Queiroz Carvalho
3,
Moacir Pasqual
3 and
Ederson da Conceição Jesus
4,*
1
Biology Department, Federal University of Lavras, Lavras 37200-000, MG, Brazil
2
Biology Department, University of the Andes, Mérida 5101, Venezuela
3
Agriculture Department, Federal University of Lavras, Lavras 37200-000, MG, Brazil
4
Embrapa Agrobiologia, Rodovia BR-465, Km 7, Seropédica 23897-970, RJ, Brazil
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 393; https://doi.org/10.3390/horticulturae11040393
Submission received: 12 February 2025 / Revised: 31 March 2025 / Accepted: 2 April 2025 / Published: 8 April 2025
(This article belongs to the Special Issue The Role of Plant Growth Regulators in Horticulture)
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Abstract

:
Bacterial inoculation stimulates growth and adaptation in micropropagated plants. This study evaluated the effects of biotization on in vitro seedling production and in vivo adaptation in two potato cultivars, Agatha and Duvira. Nine bacterial strains were tested for hormone production and ACC deaminase activity and then inoculated in vitro and re-inoculated in vivo. Growth, adaptation, and tuber production were assessed. Biotization significantly enhanced seedling growth, survival, and tuber yield. Biotized seedlings had a 1.3-fold higher survival rate than the controls. Azospirillum brasilense Ab-V5 and Rhizobium tropici CIAT 899 promoted at least one growth variable in both cultivars under in vitro and in vivo conditions. A. brasilense Ab-V5 consistently improved plant performance across production stages, with re-inoculated plants showing 1.2–1.3-fold increases in stem and root length and a 1.1-fold gain in total dry biomass. Additionally, inoculated plants produced 1.9 times more tubers than the controls. Biotization effects were strain-dependent, with A. brasilense Ab-V5 improving in vitro seedling quality and enhancing plant performance and survivability in vivo.

Graphical Abstract

1. Introduction

Micropropagation has great commercial potential due to the rate of propagation and the ability to produce vigorous disease-free plants [1]. Generally, the micropropagation technique is performed under entirely aseptic conditions: during the establishment of in vitro cultures, the explant is superficially sterilized to eliminate all microorganisms [2]. Therefore, when it comes to plant tissue culture, bacteria have often been described as contaminants [3,4]. However, due to the current knowledge about the performance of endophytic microorganisms and associative bacteria in promoting growth and inducing stress tolerance, there has been an increasing interest in their use in some of the steps of micropropagation, which is known as “biotization” [5]. This term was first introduced by Herman [6] in his article on the benefits of bacteria and fungi co-cultured with plants during micropropagation. The idea of biotization began with the study conducted by Herman [7] on potato (Solanum tuberosum L.), where he observed that contamination in potato micropropagation actually increased seedling growth [8].
Plant growth-promoting bacteria (PGPB) are microorganisms that establish mutualistic interactions with plants, stimulating their development through direct and indirect mechanisms [9,10]. Their key functions include the synthesis of phytohormones, such as indole-3-acetic acid (IAA), gibberellic acid (GA), and salicylic acid (SA), as well as the production of organic metabolites that modulate plant physiological processes, increasing agricultural productivity [11]. Inoculation with PGPB in vitro culture systems has proven to be a relevant biotechnological strategy, optimizing explant multiplication, shoot elongation, rhizogenesis, and transplant survival, thereby enhancing the efficiency of micropropagation protocols [5,8].
According to Sousa [12], the effect of bacterial indole-3-acetic acid (IAA) from the strain identified as N39, isolated from the rhizospheric soil of Axonopus catharinensis Valls, favored the in vitro rhizogenesis and acclimatization stages in apple tree rootstock “Marubakaido” (Malus prunifolia), resulting in rooted seedlings of high quality. In addition to the production of auxins such as IAA, PGPB also produces other compounds such as gibberellic acid (GA) and salicylic acid (SA), considered an important mechanism that stimulates plant growth and productivity [13]. Likewise, explants growing under controlled and very sensitive environmental conditions are more likely to be impaired and therefore need to be strengthened before facing in vivo conditions [8]. To this end, the enzyme ACC deaminase (ACCd) that converts the ethylene precursor 1-aminocyclopropane-1carboxylic acid (ACC) into ammonium and α-ketobutyrate and is produced by PGPB protects plants from excessive ethylene concentrations by decreasing abiotic stress [14].
Research on PGPB has been increasing, and several in vitro and in vivo experiments have been conducted on different crops [15]. Potato (Solanum tuberosum L.), considered the world’s third most important food crop after rice and wheat [16], is one of these crops. It is a crop with high fertilizer demand for optimal yield [17]. Several initiatives have been established to promote efficient potato production, including searching for tolerant genotypes and increasing micropropagation efficiency [8]. Bacteria of the genus Azospirillum, Bacillus, and Bradyrhizobium are models in studies of beneficial plant–microorganism interactions and can stimulate the growth of potato seedlings inoculated in vitro [18]. Kargapolova [19] demonstrated the efficacy of inoculation with Ochrobactrum citisi in potato micro-plants, where a 50% increase in the mitotic index of root meristem cells and a 34% increase in aerial part length were reported under in vivo conditions.
Therefore, this study aimed to evaluate the capacity and growth promotion efficacy of nine bacterial strains on in vitro and in vivo plants of two potato cultivars. We tested the hypothesis that they possess multiple PGPB characteristics and can be used as biostimulants in tissue culture through the biotization technique.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

Table 1 lists the strains used in this study. All strains were provided by the Agricultural Microbiology culture collection of the Federal University of Lavras (CCMA-UFLA), Lavras, Minas Gerais, Brazil, and the Johanna Döbereiner Biological Resource Center, Embrapa Agrobiologia (CRB), Seropédica, Rio de Janeiro, Brazil. The bacteria were preserved in nutrient broth (peptone 5 g L−1, yeast extract 3 g L−1, and sodium chloride 5 g L−1) with glycerol 40% in the freezer (4 °C).
For inoculum preparation, the strains were cultured for 48 h at 25 °C in 7 mL of nutrient broth at 120 rpm. Subsequently, the cells were centrifuged at 4000 rpm for 10 min and washed twice with 1 mL of sterile distilled water. Finally, the cell pellets were resuspended in 10 mL of sterile distilled water, and the solution concentration was adjusted to 1.5 × 108 CFU mL−1 according to the 0.5 tube of the McFarland scale.

2.2. Biochemical Characterization of Bacteria

The analyses of auxin production, gibberellins, salicylic acid, and ACC deaminase activity were performed using as inoculum the bacterial strains initially grown in nutrient broth at 25 °C for 48 h.

2.2.1. Indole-3-Acetic Acid (IAA) Biosynthesis

The Salkowski technique was used to determine indoleacetic acid (IAA) synthesis, according to the protocol described by Blanco and Castro [20]. Inocula were prepared in Trypticase Soy Broth (TSB) supplemented with 0.5 mg/mL of L-tryptophan (Sigma-Aldrich, Saint Louis, MO, USA). Readings were made 3, 4, 5, 6, and 9 days after inoculation using a Multiskan™ microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA). IAA concentration was calculated using a standard curve with commercial IAA (Fluka Chemie AG, Buchs, Switzerland) within a range of 10–200 mg mL−1.

2.2.2. Gibberellic Acid (GA3) Biosynthesis

The DNPH (2,4-dinitrophenylhydrazine) method was used to determine the concentration of gibberellic acid (GA3), according to Graham and Thomas [21], modified by Sagar [22]. The strains were incubated in nutrient broth at 25 °C while shaking for 6 days. Measurements were made 2, 4, and 6 days after inoculation. The GA3 concentration was calculated using a regression equation derived from a standard curve with pure gibberellic acid (Sigma-Aldrich, Saint Louis, MO, USA), in a range of 10–100 mg mL−1.

2.2.3. Quantification of Bacterial Salicylic Acid (SA)

SA production was determined according to the methodology described by De Meyer [23], with modifications by Islam [24]. The absorbance was measured at 520 nm using a Multiskan™ microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA), 2, 4, and 6 days after inoculation. SA levels were determined using a standard curve with known concentrations of commercial SA (250, 125, 62.5, and 31.25 µg mL−1).

2.2.4. ACC Deaminase Activity (ACCd)

ACCd activity was assayed according to the method of Penrose and Glick [25], which measures the amount of product α-ketobutyrate released after ACC hydrolysis. The measurements were made 3, 4, 5, 6, and 7 days after inoculation. An aliquot of 250 µL of the bacterial suspension grown in nutrient broth was transferred to tubes with DF minimum salt medium [26] supplemented with 5 mM ACC and incubated at 26 °C for 48 h with orbital shaking. The absorbances were read on a Multiskan™ microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA) in terms of α-ketobutyrate production at 540 nm compared with the standard curve of α-ketobutyrate, which ranged from 0.1 to 20 μmol [27]. Next, the total protein concentration of the toluenized bacterial cells was estimated using bovine serum albumin (BSA) to create the protein calibration curve [28].

2.3. Plant Material, Inoculation, and In Vitro Growth Conditions

The experiment was conducted in the Tissue Culture Laboratory, Department of Agriculture, Federal University of Lavras (UFLA). The plant material was provided by the Genetics and Phytopathology Laboratories of UFLA. To establish in vitro cultures, nodal segments of potato cultivars Duvira and Ágata were used as explants. The size of the nodal segments was standardized at 2 cm in length with one leaf and one lateral bud, and they were placed in test tubes containing 15 mL of Murashige–Skoog nutrient medium [29] free of hormones and added with 20 g L−1 of sucrose and 6 g L−1 of agar. The pH of the medium was adjusted to 5.8. The tubes were then transferred to a rack and kept in a growth room for seven days at a temperature of 25 °C, a 16 h photoperiod, and a light intensity of about 40 μM m−2 s−1. Immediately after this time, the explants of each cultivar were inoculated with different bacteria. For inoculation, a volume of 0.1 mL of the resulting bacterial suspension (108 cells mL−1) was added to the test tubes to obtain a final bacterial concentration of 107 cells mL−1. For the control treatment, 0.1 mL of sterile distilled water was used. Subsequently, the tubes were sealed with plastic film and placed in the growth room under the same conditions for 23 days. An entirely randomized design was established with ten treatments (the nine strains and the control treatment, without inoculation) and four replicates. Each treatment consisted of 24 seedlings (six per replicate) for Duvira and Ágata.
At the end of this period, eight micro-plants of each treatment and each cultivar were removed from the tubes. The root system was washed to remove residues from the medium and dried with paper towels, and the following morphometric variables were evaluated: leaf fresh mass, root fresh mass, leaf dry mass, root dry mass, number of nodes, root length, and shoot length.

2.4. Acclimatization and Re-Inoculation of Potato Seedlings of cv. Duvira and cv. Ágata in a Greenhouse

The experiments for acclimatization and re-inoculation were carried out with the two cultivars, Duvira and Ágata. These cultivars were examined separately; they were not treated as factors in the experiments. The experimental design was completely randomized, with nine treatments and eight replicates (seedlings) per treatment. The nine treatments corresponded to T1: Rhizobium leucaneae (BR 935); T2: Azospirillum brasilense (Ab-V6); T3: Azospirillum brasilense (Ab-V5); T4: Bradyrhizobium japonicum (CCMA0088); T5: Sinorhizobium fredii (CCMA0122); T6: Rhizobium tropici (CIAT899); T7: Bacillus subtilis (CCMA0401); T8: Bacillus megaterium (CCMA0004); and T9: absolute control (not inoculated).
For in vivo adaptation, treatments with 30 days of in vitro growth were transferred to plastic cups with a commercial substrate and were taken to the greenhouse at 25 ± 2 °C during the day and 16 ± 2 °C at night. The substrate was sterilized for three consecutive days for one hour before the transfer. A polypropylene bag with holes was placed in each pot to provide a humid chamber and avoid dehydration by changing the substrate and environment. The bag was removed for 2 h daily for two days.
Next, one group of plants (eight seedlings per treatment for both cv. Duvira and cv. Ágata) was re-inoculated with three milliliters of bacterial inoculum. Three milliliters of sterile distilled water was added to the control plants seven days after transplanting. The remaining group of seedlings was maintained with a single inoculation.
The plants were watered daily by automatic irrigation and fertilized once a week with 10 mL of a half-strength Hoagland solution [30], as Jarstfer and Sylvia [31] reported. The survival rate and morphometric variables of the seedlings were again recorded after 25 days, including tuber production, to determine the performance of the seedlings during the acclimatization and re-inoculation process.

2.5. Statistical Analysis

All statistical analyses were performed using the 2011 version of the INFOSFAT software. Data were analyzed by applying analysis of variance (ANOVA) with Tukey’s test at a significance level of p < 0.05. The Shapiro–Wilk test was applied to verify the normal distribution of the data. Values of morphometric variables were reported as mean values ± standard error (n = 8).

3. Results

3.1. Biosynthesis of Indol-3-Acetic Acid (IAA), Gibberellic Acid (GA3), and Salicylic Acid (SA) in PGPB

All bacteria were able to synthesize at least one phytohormone. In the case of IAA production, which is the main auxin in plants and an indicator of plant growth promotion, the results showed the formation of two major groups of auxin-producing bacteria (Figure 1A). One group was formed by the strains B. japonicum CCMA0088, A. brasilense Ab-V5, Ab-V6, and S. fredii CCMA0122; the other was formed by bacteria of the genus Bacillus and Rhizobium. The highest IAA concentration was reached between the fifth and sixth days of incubation for most of the strains evaluated, with a concentration of 54.1 μg mL−1 for B. japonicum strain CCMA0088, followed by strains Ab-V5 and Ab-V6, with respective concentrations of 48.3 and 46.5 μg mL−1 of IAA. The other strains had an IAA production between 17.8 and 6.72 μg mL−1.
Regarding GA3 production, the nine strains were markedly divided into three groups (Figure 1B). The first group was formed by the A. brasilense Ab-V5 and Ab-V6, which synthesized the highest GA3 concentrations (13 and 10.02 µg mL−1, respectively). A second group was formed by the three strains of the genera Bacillus, R. tropici CIAT 899, and B. japonicum CCMA0088, with values between 5.09 and 2.08 μg mL−1. Finally, the third group was formed by the control and S. fredii CCMA0122. All strains synthesized the highest GA3 amounts after 4 days of incubation.
As for the production of salicylic acid (SA), bacteria of the genera Azospirillum, Bacillus, and the R. tropici strain were identified as the most outstanding producers on the sixth day of incubation. A. brasilense Ab-V5 produced 9.8 μg mL−1, followed by B. megaterium with 9.0 μg mL−1 of SA. B. japonicum CCMA0088, and R. leucaneae BR935 produced the lowest SA amounts (1.3 and 1.72 μg mL−1), respectively (Figure 1C).

3.2. Determination of ACC Deaminase Activity (ACCd) in PGPB

We found that there were differences in ACCd activity among the nine bacteria. Bacillus megaterium CCMA0004, Bacillus subtilis CCMA0401, and Bacillus amyloliquefaciens strains formed the first group exhibiting the highest ACCd activities in the range of 8.51–7.16 µmol α-ketobutyrate per mg cell protein per hour, significantly outperforming the others. ACCd activity increased after 24 h of incubation (1.6-, 1.2-, and 1.1-fold) in cultures of B. amyloliquefaciens CCMA0112, B. megaterium CCMA0004, and B. subtilis CCMA0401, respectively. A second group of strains with low ACCd production consisted of Sinorhizobium fredii CCMA 0122 and Bradyrhizobium japonicum CCMA0088, and they reached their peak activity at day 6 with values of 3.75 and 2.04 μmol α-ketobutyrate−1 mg−1 protein hr−1, respectively, but did not show noticeable increases over time. The third group formed by strains without detectable ACCd activity included the bacteria Azospirillum brasilense Ab-V5, Azospirillum brasilense Ab-V6, and Rhizobium tropici CIAT 899. Regarding R. tropici CIAT 899, Ormeño [32] demonstrated the presence of the acdS gene in the genome of the strain; however, R. tropici CIAT 899 did not show ACC deaminase activity by the colorimetric method used in this work (Figure 2).

3.3. Effect of Biotization on Growth Parameters of Potato cv. Duvira and cv. Ágata Propagated In Vitro

The response to inoculation differed between the two potato cultivars. Two bacterial strains, Azospirillum brasilense Ab-V5 and Rhizobium tropici CIAT 899, significantly promoted at least one growth variable in both cultivars.
In Duvira cultivar, four strains promoted a significant increase in shoot length of 41.3% (A. brasilense Ab-V5), 33.2% (S. fredii CCMA 0122), 21.09% (A. brasilense Ab-V6), and 17.2% (R. tropici CIAT 899) compared to un-inoculated micro-plants. Root length increased significantly with S. fredii CCMA 0122, A. brasilense Ab-V5, and A. brasilense Ab-V6 by 29.6%, 25.4%, and 24.8%, respectively. Dry biomass was increased 3.7, 3.4, and 2.28 times upon biotization with A. brasilense Ab-V5, S. fredii CCMA 0122, and R. tropici CIAT 899 compared to the control (Table 2).
In the case of the Ágata cultivar, the micro-plants biotized with A. brasilense Ab-V5 and B. megaterium CCMA0004 showed the best performance, as they showed a significant increase in all morphometric variables compared to the un-inoculated control. The plants inoculated with A. brasilense Ab-V5 showed the highest values in all growth variables by approximately 25, 48, 78.5, 72.7, and 76% for stem length, root length, stem dry weight, root dry weight, and total biomass, respectively. Potato micro-plants showed significant increases of 16.6% in stem height and 43.7% in root length when inoculated with B. megaterium CCMA0004. Also, the total dry biomass was significantly higher than that of un-inoculated plants, showing an increase of 70% (Table 3).
Inoculation with R. leucaenae BR935 had a negative effect, decreasing microplant growth in both cultivars (Table 2 and Table 3). Regarding B. amyloliquefaciens CCMA0112, bacterial growth increased considerably in the culture medium (SM), resulting in the death of microplants.
Except for the strain A. brasilense Ab-V5, which was effective for both cultivars, differences were observed between the strains of bacteria that promoted the growth of potato micro-plants in the two cultivars. The strains S. fredii CCMA 0122 and A. brasilense Ab-V6, which positively increased some growth parameters in Duvira, decreased growth in Ágata. In turn, the strains belonging to the genus Bacillus, such as B. subtilis CCMA0401 and B. megaterium CCMA0004, were those that promoted the growth of the micro-plants of cv. Ágata but with a negative effect on the micro-plants of cultivar Duvira.

3.3.1. Influence of Biotization on the Development of the Number of Nodes in Potato Micro-Plants cv. Duvira and cv. Ágata

The number of nodes per micro-plant was recorded after four weeks under in vitro conditions. Significant differences were observed between genotypes and inoculation treatments. Plants inoculated with A. brasilense Ab-V5 developed 1.7 times more nodes than control micro-plants in cultivar Duvira. In addition, S. fredii CCMA 0122, A. brasilense Ab-V6, and R. tropici CIAT 899 increased the number of nodes 1.6, 1.4, and 1.3 times, respectively (Figure 3A), producing significant positive effects on the development of micro-plants compared to the control (Figure 3B). In the case of the cultivar Ágata, the highest number of nodes was stimulated by inoculation with A. brasilense Ab-V5 and B. megaterium CCMA 0004, with an increase of 1.4 times, followed by B. subtilis CCMA 0401, which favored an increase in the number of nodes of 1.3 times over the control micro-plants (Figure 3C), promoting shoot and root growth (Figure 3D).

3.3.2. Effect of Biotization on Survival of Seedlings of cv. Duvira and cv. Ágata Under In Vivo Conditions

In general, the seedlings were successfully acclimatized in the greenhouse. Seedling survival during this phase resulted better in those treatments where bacteria promoted in vitro rooting. Biotization affected the survival of potato seedlings ranging from 50% to 100%, depending on the cultivar, and also contrasting with 75% and 87.5% survival of un-inoculated seedlings. The survival percentage in both cultivars was 100% for seedlings biotized with A. brasilense Ab-V5, R. tropici CIAT 899, and S. fredii CCMA 0122, in addition to recovering turgor in the leaves five days after transplanting. In the cultivar Duvira, biotized with A. brasilense Ab-V6, B. subtilis CCMA 0401, and B. megaterium CCMA 0004, the survival rate for the seedlings was 100, 75, and 87.5%, respectively.
As for the cultivar Ágata, the seedling survival rate was 87.5% with A. brasilense Ab-V6 and B. subtilis CCMA 0401 and 100% with B. megaterium CCMA 0004. The treatment inoculated with R. leucaenae BR935 showed a reduction in survival rate in the Duvira cultivar of 50% and 62.5% in the Ágata cultivar (Figure 4).

3.4. Evaluation of the Effect of Biotization on the Performance of Potato Seedlings cv. Duvira and Ágata in the Acclimatization and Re-Inoculation Phase

When the micro-plants were transferred to soil, a positive influence of biotization on in vivo adaptation was observed. A. brasilense Ab-V5, R. tropici CIAT 899, S. fredii CCMA 0122, and A. brasilense Ab-V6 stimulated a significant increase in all physiological variables of Duvira cultivars during the acclimatization phase ((A) in Table 4). The highest values for shoot and root length were stimulated by A. brasilense Ab-V5, with increases of 50 and 65.63%, respectively, followed by the R. tropicii strain, with increases of 43.61% in the shoot and 33.39% in the root. The strains S. fredii CCMA 0122 and A. brasilense Ab-V6 also showed significant differences compared to the control. Regarding shoot and root dry weight, the A. brasilense Ab-V6 strain significantly increased these variables (3.67- and 4.16-fold), respectively. The values obtained by A. brasilense Ab-V5, R. tropici CIAT 899, and S. fredii CCMA 0122 were significantly higher than those of the control. Plant dry matter accumulation is directly related to its growth rate; therefore, the same group of strains A. brasilense Ab-V5, R. tropici CIAT 899, S. fredii CCMA 0122, and A. brasilense Ab-V6 accumulated greater amounts of dry biomass, with A. brasilense Ab-V6 increasing this variable 3.85-fold compared to the control.
A second dose (re-inoculation) with A. brasilense Ab-V5, R. tropici CIAT 899, S. fredii CCMA 0122, and A. brasilense Ab-V6 significantly influenced the increase in all variables ((B) in Table 4). The highest values for shoot and root length were obtained with the A. brasilense Ab-V5 strain, with increases of 1.7- and 1.8-fold, respectively. Re-inoculation with the A. brasilense Ab-V5 and A. brasilense Ab-V6 strains favored increases in total biomass (4- and 5-fold, respectively) compared to the control.
It should be noted that plants subjected to a second inoculation manifested better development than those with a single inoculation. The average gains in stem length with A. brasilense Ab-V5 and A. brasilense Ab-V6 were 1.4 times. In root length, the increase was 1.3-fold with R. tropici CIAT 899, followed by A. brasilense Ab-V5 with 1.2-fold and S. fredii CCMA 0122 with 1.1-fold. Similarly, inoculations with A. brasilense Ab-V5 and A. brasilense Ab-V6 in the total dry biomass variable increased 1.1 and 1.02 times, respectively.
In the case of cv. Ágata, A. brasilense Ab-V5, B. japonicum CCMA0088, B. megaterium CCMA0004, and R. tropici CIAT 899 provided a significant increase in stem length compared to the control during the acclimatization phase. This increase ranged from 27 to 13%. The increase in root growth of plants inoculated with A. brasilense Ab-V5, B. japonicum CCMA0088, and B. megaterium CCMA0004 was of 37, 33, and 28%, respectively. On the other hand, stem and root dry mass were higher in treatments with A. brasilense Ab-V5 (65 and 82%), R. tropici CIAT 899 (54 and 61%), B. japonicum CCMA0088 (43 and 63%), and B. megaterium CCMA0004 (40 and 64%) ((A) in Table 5).
Likewise, re-inoculation in plants of cv. Ágata promoted the increase in different variables. Inoculation with A. brasilense Ab-V5, R. tropici CIAT 899, B. megaterium CCMA0004, and B. japonicum CCMA0088 increased stem and root length, with values between 39 and 21%. The plants with significantly higher dry biomass were those re-inoculated with A. brasilense Ab-V5, R. tropici CIAT 899, B. megaterium CCMA0004, B. japonicum CCMA0088, and B. subtilis CCMA0401. Moreover, the average gain between the first and second inoculation in the treatment with A. brasilense Ab-V5 was 1.1-fold in stem length and 1.2-fold in root length. In dry biomass, there was an increase from 4.15 to 5.08 g in this treatment. B. subtilis CCMA0401 promoted plant growth after re-inoculation, with significant differences in all morphometric parameters. In this treatment, the average gain between the first and second inoculation was 1.2 and 1.3 times in stem and root length, and for stem and root dry biomass it was 1.7 and 3.1 times, respectively ((B) in Table 5).

3.4.1. Effect of Biotization with PGPB on Tuber Formation and Weight in cv. Duvira and cv. Ágata

After 25 days of acclimatization, only inoculation with A. brasilense Ab-V5 promoted significant differences in the number and weight of tubers in plants with a single inoculation. Specifically, this treatment showed increases of 1.6 times in cv. Duvira and 2.3 times in cv. Ágata, for the number of tubers (Figure 5A). Likewise, the treatment with A. brasilense Ab-V5 increased the tuber fresh weight 3.8 times in cv. Duvira and 2.3 times in cv. Ágata (Figure 5B). Inoculations with R. tropici CIAT 899 and S. fredii CCMA 0122 in the cultivar Duvira promoted high values in these variables but without significant differences.
Regarding cv. Ágata, the treatments R. tropici CIAT 899 and B. megaterium CCMA0004 presented a higher number and weight of tubers compared to non-inoculated seedlings, although this difference was not statistically significant.
Biotization in cv. Duvira significantly improved tuber formation with A. brasilense Ab-V5 (Figure 5C,D). Similarly, in the cultivar Ágata, biotization with A. brasilense Ab-V5 favored tuber growth and formation (Figure 5E) compared to un-inoculated plants (Figure 5F).

3.4.2. Effect of PGPB Re-Inoculation on Tuber Formation and Weight in cv. Duvira and cv. Ágata

Re-inoculation assays with plant growth-promoting bacteria (PGPB) demonstrated a differential impact on tuber formation and weight between Duvira and Ágata cultivars, highlighting the key role of Azospirillum brasilense Ab-V5.
Plants of cultivar Duvira re-inoculated with A. brasilense strain Ab-V5 showed a higher number of tubers, with a significant 1.9-fold increase compared to the control. For the cultivar Ágata, two strains increased the number of tubers, A. brasilense Ab-V5 by 2.2 times and B. japonicum CCMA0088 by 1.6 times (Figure 6A). If we compare the number of tubers in singly inoculated and the re-inoculated treatments, we observe that A. brasilense Ab-V5 increased 1.2 times in cv. Duvira and 1.2 times when co-inoculated with B. japonicum CCMA0088 in cv. Ágata. Regarding tuber fresh weight, only A. brasilense Ab-V5 strain statistically outperformed the controls, both in Duvira and Ágata, with 2.6- and 2.4-fold increases, respectively (Figure 6B). The inoculation with A. brasilense Ab-V5 promoted good plant physiological development in cv. Duvira (Figure 6C) compared to the control (Figure 6D), maintaining the same effect in Ágata (Figure 6E,F).

4. Discussion

4.1. Growth-Promoting Functions of Bacteria

Beneficial bacteria play essential roles that directly or indirectly positively affect plant growth and development [19]. In this study, 50% of the bacterial strains evaluated revealed a remarkable ability to synthesize phytohormones known for regulating numerous biological processes in the plant. The formation of two main groups in IAA biosynthesis reflects intrinsic differences in the efficiency of the biosynthetic pathways of the bacteria evaluated, such as tryptophan dependence and concentration, as well as incubation time. The strain B. japonicum CCMA0088, with the highest IAA production, coincides with previous studies that highlight Bradyrhizobium as efficient producers of auxins, essential for nodulation in legumes and which favors the delay in nodule senescence [33]. On the other hand, A. brasilense Ab-V5 and A. brasilense Ab-V6, along with S. fredii CCMA 0122, produced significant amounts of IAA, highlighting the importance of this group of bacteria as plant growth promoters, not only in nitrogen fixation but also in phytohormone biosynthesis [34]. Interestingly, the strains of the Bacillus genus evaluated did not produce large amounts of IAA, which differs from other studies reporting that different species of the Bacillus genus produced optimal levels of IAA [35,36]. Indole-3-acetic acid (IAA), recognized as one of the most relevant phytohormones, presents variable production among bacterial species. This variability is conditioned by factors such as culture conditions, microbial growth phase, and substrate source [37]. Furthermore, IAA also promotes responses to abiotic stress and interactions with pathogens [38,39] through its intricate interconnection with other phytohormones [40].
Similarly, the bacteria tested produced gibberellins at concentrations ranging from 13 to 2.08 µg mL−1. A. brasilense Ab-V5 and A. brasilense Ab-V6 stood out as the main producers of GA, reinforcing their role as plant growth-promoting bacteria. Azospirillum brasilense has been reported to promote plant growth by increasing the production of phytohormones such as gibberellins, auxins, and cytokinins [41].
Strains of the Bacillus and Rhizobium genera produced smaller but significant amounts of GA. Several studies have reported that the bacterial genera Sinorhizobium, Rhizobium, and Bacillus exhibit physiologically desirable traits such as the production of IAA, cytokinins, gibberellins, riboflavin, and Nod factors, which play diverse roles in improving plant growth and productivity [42,43,44].
Other phytohormones, such as salicylic acid, also play an essential role in plant stress relief by modulating the activity of antioxidant enzymes [45,46]. Furthermore, it has been shown to play a crucial role as a regulator of systemic acquired resistance (SAR) against abiotic stress [47]. In our study, the genera Azospirillum and Bacillus synthesized significant amounts of salicylic acid. The high synthesis in A. brasilense Ab-V5 (9.8 μg mL−1) is consistent with studies linking this species to the modulation of plant defense responses, as reported by Kasim [48] where they evaluated the interaction between the SA produced by Azospirillum and the response of wheat plants under water stress. Egamberdieva [49] reported SA production by root-associated bacteria such as B. licheniformis MML2501 (18 µg mL−1) and Pseudomonas sp. PRGB06 (6.8 µg mL−1). Furthermore, SA production was reported for Pseudomonas tremae with values of 57.05 µg mL−1 and Curtobacterium herbarum 46.22 µg mL−1 [24], demonstrating that phytohormone biosynthesis differs depending on the bacterial strain [50]. The differential production of SA in Azospirillum, Bacillus, and R. tropici, as the most active in our study, highlights its potential to induce systemic resistance in plants against stress.
As a strategy to improve plant adaptation to stress conditions, ACC deaminase-producing bacteria can influence plant growth directly or indirectly [51]. The biochemical evaluation of bacteria by quantifying the amount of α-ketobutyrate produced is necessary and feasible to confirm the positive character of ACCd production, so the method used in this study is widely accepted for the identification of ACCd-producing rhizobacteria [52]. In our experiments, five strains represented by the genera Bacillus, Sinorhizobium, and Bradyrhizobium have ACCd activities. No strain of the genus Azospirillum was positive for ACCd. According to Joe [53], despite possessing all plant growth-promoting traits, most Azospirillum spp. are considered negative for ACC deaminase (acdS) activity except a few strains of Azospirillum lipoferum (AZm5 4B, CRT1, CN1, N4, and TW3).

4.2. Influence of Biotization on Potato In Vitro Growth

Biotized plants benefit from microbial presence through increased photosynthetic efficiency and biomass production. Generally, PGPB improve growth by releasing necessary compounds that promote micropropagation and rooting [18]. In our study, we demonstrated that biotization with strains of the genera Azospirillum, Bacillus, Rhizobium, Bradyrhizobium, and Sinorhizobium, identified as producers of phytohormones (IAA, GA, and SA), improved the development of potato micro-plants. A. brasilense Ab-V5 was the strain that benefited the growth and rooting in vitro of micro-plants and potato seedlings in both cultivars. Stem length was the most stimulated variable under in vitro conditions, with an increase of up to 41% with Azospirillum brasilense Ab-V5 strain. This is probably due to the biosynthesis of gibberellins and auxin, which acts as a growth promoter and regulates various development processes, such as stem elongation, seed germination, sexual expression, and fruit formation [41]. Similarly, the total dry biomass increased up to 82% with Azospirillum brasilense AbV5. The S. fredii CCMA 0122 strain improved the development of the Duvira cv., while B. japonicum CCMA0088, B. megaterium CCMA0004, and B. subtilis CCMA0401 promoted the growth of the Ágata cultivar, both in the aerial parts and in the root system. This group of bacteria produced significant amounts of at least two phytohormones, which could have had a positive impact on plant growth in vitro.
Micro-plants inoculated with the Bacillus amyloliquefaciens CCMA0112 strain adversely affected the development of potato explants, impairing micro-plant growth. Therefore, this treatment was not evaluated on potato seedlings under in vitro and in vivo conditions. We believe that the rapid growth of B. amyloliquefaciens may have generated nutritional stress for the micro-plants due to the formation of biofilms on the roots, which may have physically interfered with water and nutrient absorption.
ACC deaminase activity is another critical characteristic of bacteria that promotes plant growth. Bacteria of the genera Agrobacterium, Bacillus, Burkholderia, Methylobacterium, Pseudomonas, and Rhizobium have been reported to produce ACC deaminase with the ability to increase plant growth parameters, as well as decrease biotic and abiotic stress [54]. In this study, the strains of the genera Bacillus, Bradyrhizobium, and Sinorhizobium with ACC deaminase activity significantly increased the vegetative parameters of length and biomass weight in potato micro-plants compared to un-inoculated micro-plants.
However, the effects of biotization with different bacterial strains on the growth of potato micro-plants in vitro were very different, indicating the specificity of the bacterium’s interaction with the plant genotype. A study by Weinert [55] determined that potato genotype is one of the factors shaping the rhizosphere-associated microbial community. Five potato cultivars and two strains were evaluated. Differences in bacterial communities between plant genotypes were significant in some cultivars, suggesting a rather subtle influence of plant genotype on tuber-associated bacteria.

4.3. Impact of Biotization on In Vivo Adaptation and Tuber Formation

When micro-plants are transplanted into the greenhouse, they face obstacles in the growth and development stages due to various factors such as decreased gas exchange, low humidity, reduced photosynthetic capacity, and a lack of root hairs [8]. A critical factor for the adaptation of micro-plants to the soil is good root development [56].
The results obtained in our work demonstrate that biotization with the strains A. brasilense Ab-V5 and A. brasilense Ab-V6 has a significant impact on the adaptation and performance of potato seedlings of both the Duvira and Agata cultivars during the acclimatization and re-inoculation phases. Strains that promoted plant growth in vitro contributed to the production of high-quality rooted micro-plants and a high survival rate of up to 100% during acclimatization, improving their performance under in vivo conditions. The effect of A. brasilense Ab-V5 and Ab-V6 in stimulating root (65.63% and 33.39%, respectively) and shoot (50% and 43.61%) growth suggests that their multifunctional capacity to synthesize phytohormones plays a central role in the modulation of root architecture and leaf development in plants. Previous studies have associated Azospirillum with the induction of lateral roots through the production of phytohormones, especially IAA, which increases the surface area for nutrient and water absorption, a critical factor during transplant stress [48]. Similarly, the marked increase in total dry biomass (up to 4.16-fold with A. brasilense Ab-V6) reflects an improvement in photoassimilation efficiency, likely mediated by the optimization of the seedling nutritional status. Recently, Vishnupradeep [57] observed that inoculation with PGPB was effective in regulating stomatal functions and reducing structural damage to the photosynthetic apparatus in maize plants by inducing the accumulation of osmolytes, including abscisic acid and IAA, in the plant.
Regarding tuber production, Azospirillum brasilense Ab-V5 increased the number of tubers compared to other treatments in both Duvira and Ágata cultivars. A significant effect was also observed after re-inoculation with A. brasilense Ab-V5, which significantly increased the number of tubers in Duvira cultivars (1.9-fold) and Ágata cultivars (2.2-fold). This could be due to the fact that auxin stimulates root development and stolon elongation, improving nutrient uptake and the formation of potential sites for tuberization. However, this process is influenced by an integral hormonal interaction (such as the balance with cytokinins, abscisic acid, and ethylene) and environmental conditions.
The consistency of Azospirillum brasilense Ab-V5 in both cultivars positions this strain as a candidate for use at different stages of potato production: in tissue culture, acclimatization, and growth and production under controlled conditions. These results highlight the importance of studying PGPB inoculation at different stages of crop production.

5. Conclusions

This study demonstrates the multifunctional effect of bacterial strains producing IAA, GA, SA, and ACC deaminase in the biostimulation of potato micro-plants cv. Duvira and cultivar Ágata. Biotization with strains of the genera Azospirillum, Bacillus, and Sinorhizobium (through mechanisms such as phytohormone synthesis and stress regulation via ACC deaminase) induced optimal morphological development in the micro-plants, optimizing critical parameters such as survival rate and in vivo adaptation. Under greenhouse conditions, inoculation with Azospirillum brasilense Ab-V5 improved plant growth and increased tuber formation, demonstrating a significant impact on the agronomic quality of potato plants in both cultivars. Re-inoculation (bacterial application at key phenological stages) appears to be essential to sustaining rhizosphere colonization and maximizing the production of growth-promoting metabolites.
A significant increase in tuber production through inoculation with this strain can significantly increase farmers’ economic benefits and also reduce the environmental pressure caused by excessive fertilizer use.

Author Contributions

Conceptualization, Y.C.M., J.D. and E.d.C.J.; methodology, Y.C.M. and A.M.G.S.; formal analysis, Y.C.M.; investigation, Y.C.M., A.M.G.S. and L.Q.C.; resources, M.P.; data curation, Y.C.M.; writing—original draft, Y.C.M.; writing—review and editing, J.D., A.M.G.S., M.P. and E.d.C.J.; supervision, J.D. and E.d.C.J.; project administration, J.D. and E.d.C.J.; funding acquisition, J.D., M.P. and E.d.C.J. All authors have read and agreed to the published version of the manuscript.

Funding

The authors extend thanks to the Research Support Foundation of the State of Minas Gerais (FAPEMIG) for providing the necessary equipment, financial, and technical support for the experiments, the National Council for Scientific and Technological Development (CNPq) for the grant n° 311796/2019-2 provided to E.d.C.J., and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for students fellowships (financial code 001).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 00393 g001
Figure 1. Quantitative determination of phytohormone biosynthesis in PGPB expressed in μg mL−1. (A) IAA production at 3, 4, 5, 6, and 9 days of incubation in medium supplemented with 0.5 mg mL L-tryptophan; (B) GA3 production at 2, 4, and 6 days of incubation; and (C) SA production at 2, 4, and 6 days of incubation. Bars represent the standard error (SE).
Figure 1. Quantitative determination of phytohormone biosynthesis in PGPB expressed in μg mL−1. (A) IAA production at 3, 4, 5, 6, and 9 days of incubation in medium supplemented with 0.5 mg mL L-tryptophan; (B) GA3 production at 2, 4, and 6 days of incubation; and (C) SA production at 2, 4, and 6 days of incubation. Bars represent the standard error (SE).
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Figure 2. Quantitative determination of ACCd activity in PGPB, expressed in terms of concentration of μmol α-ketobutyrate−1 mg−1 protein hr−1 during seven days of incubation. Bars represent the standard error (SE).
Figure 2. Quantitative determination of ACCd activity in PGPB, expressed in terms of concentration of μmol α-ketobutyrate−1 mg−1 protein hr−1 during seven days of incubation. Bars represent the standard error (SE).
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Figure 3. Effect of biotization on the number of nodes in potato micro-plants. (A) Number of nodes for cultivar Duvira, (B) graphical representation of the development of biotized micro-plants of cv. Duvira, (C) number of nodes for cultivar Ágata, and (D) graphical representation of the development of biotized micro-plants of cv. Ágata. Means followed by the same letter are not significantly different by Tukey’s test (p < 0.05). Bars represent the standard error (SE).
Figure 3. Effect of biotization on the number of nodes in potato micro-plants. (A) Number of nodes for cultivar Duvira, (B) graphical representation of the development of biotized micro-plants of cv. Duvira, (C) number of nodes for cultivar Ágata, and (D) graphical representation of the development of biotized micro-plants of cv. Ágata. Means followed by the same letter are not significantly different by Tukey’s test (p < 0.05). Bars represent the standard error (SE).
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Figure 4. Survival percentage of biotized and non-biotized micro-plants of potato cultivars Duvira and Ágata under in vivo conditions. Bars represent the standard error (SE).
Figure 4. Survival percentage of biotized and non-biotized micro-plants of potato cultivars Duvira and Ágata under in vivo conditions. Bars represent the standard error (SE).
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Figure 5. Effect of biotization with PGPB on tuber formation in cv. Duvira and Ágata. (A) Number and (B) fresh weight of tubers in cv. Duvira and Ágata. (C) Duvira inoculated with A. brasilense Ab-V5 and (D) its uninoculated control. (E) Ágata inoculated with A. brasilense Ab-V5, and (F) its uninoculated control. Means followed by the same letter are not significantly different by Tukey’s test (p < 0.05). Bars represent the standard error (SE).
Figure 5. Effect of biotization with PGPB on tuber formation in cv. Duvira and Ágata. (A) Number and (B) fresh weight of tubers in cv. Duvira and Ágata. (C) Duvira inoculated with A. brasilense Ab-V5 and (D) its uninoculated control. (E) Ágata inoculated with A. brasilense Ab-V5, and (F) its uninoculated control. Means followed by the same letter are not significantly different by Tukey’s test (p < 0.05). Bars represent the standard error (SE).
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Figure 6. Effect of re-inoculation with PGPB on cv. Duvira and Ágata tuber formation. (A) Number of tubers, (B) fresh weight of tubers, (C) cv. Duvira inoculated with A. brasilense Ab-V5 and (D) its un-inoculated control, (E) cv. Ágata inoculated with A. brasilense Ab-V5 and (F) its un-inoculated control. Means followed by the same letter are not significantly different by Tukey’s test (p < 0.05). Bars represent the standard error (SE).
Figure 6. Effect of re-inoculation with PGPB on cv. Duvira and Ágata tuber formation. (A) Number of tubers, (B) fresh weight of tubers, (C) cv. Duvira inoculated with A. brasilense Ab-V5 and (D) its un-inoculated control, (E) cv. Ágata inoculated with A. brasilense Ab-V5 and (F) its un-inoculated control. Means followed by the same letter are not significantly different by Tukey’s test (p < 0.05). Bars represent the standard error (SE).
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Table 1. The bacterial strains used in the experiments.
Table 1. The bacterial strains used in the experiments.
StrainSpeciesGeographic Origin
BR 935Rhizobium leucaneaeCipó-BA, Brazil
Ab-V6 (= CNPSo2084)Azospirillum brasilenseCuritiba-PR, Brazil
Ab-V5 (= CCMA1291)Azospirillum brasilenseCuritiba-PR, Brazil
CCMA0088Bradyrhizobium japonicumArcos-MG, Brazil
CCMA0122Sinorhizobium frediiLuminárias-MG, Brazil
CIAT899Rhizobium tropiciColombia
CCMA0401Bacillus subtilisAlto Garças-MT, Brazil
CCMA0004Bacillus megateriumConfresa-MT, Brazil
CCMA0112Bacillus amyloliquefaciensLuminárias-MG, Brazil
Table 2. Effects of biotization with PGPB on growth variables of potato cv. Duvira after 30 days under in vitro conditions.
Table 2. Effects of biotization with PGPB on growth variables of potato cv. Duvira after 30 days under in vitro conditions.
In Vitro
Duvira Cultivar		Length (cm)Dry Weight (mg)
TreatmentsShootRootShootRootTotal Biomass
R. leucaneae3.6 ± 0.38 e1.3 ± 0.36 ef10.0 ± 1.22 d0.00 ± 0.00 d10.00 d
Ab-V68.3 ± 0.43 c4.1 ± 0.24 a30.0 ± 2.25 c10.00 ± 1.66 de40.00 cd
R. tropici7.7 ± 0.60 c3.3 ± 0.22 bc110.0 ± 3.24 b50.10 ± 2.94 b160.10 b
S. fredii10.8 ± 0.34 b4.3 ± 0.31 a130.0 ± 3.42 ab110.0 ± 4.10 a240.00 ab
B. japonicum5.7 ± 0.12 d3.3 ± 0.20 bc20.0 ± 1.53 c10.0 ± 0.33 d30.00 c
Ab-V512.3 ± 0.28 a4.1 ± 0.22 a150.0 ± 2.19 a110.0 ± 3.51 a260.00 a
B. subtilis3.8 ± 0.16 e2.0 ± 0.26 de10.0 ± 0.86 d10.0 ± 0.70 d20.00 d
B. megaterium3.3 ± 0.20 e2.1 ± 0.25 d30.0 ± 2.68 c0.00 ± 0.00 d30.00 c
Control7.2 ± 0.41 d3.1 ± 0.30 c40.0 ± 2.34 c30.1 ± 1.52 c70.10 c
Data represent mean ± standard error (SE). Means followed by the same letter do not differ significantly by Tukey’s test (p < 0.05).
Table 3. Effects of biotization with PGPB on growth variables in potato cv. Ágata, evaluated after 30 days under in vitro conditions.
Table 3. Effects of biotization with PGPB on growth variables in potato cv. Ágata, evaluated after 30 days under in vitro conditions.
In Vitro
Ágata Cultivar		Length (cm)Dry Weight (mg)
TreatmentsShootRootShootRootTotal Biomass
R. leucaneae3.8 ± 0.26 f1.5 ± 0.23 e20.0 ± 1.58 c0.0 ± 0.00 de20.0 c
Ab-V63.3 ± 0.24 f1.9 ± 0.21 e10.0 ± 0.71 c0.0 ± 0.00 cde10.0 c
R. tropici6.8 ± 0.36 d3.9 ± 0.30 c90.0 ± 1.66 b50.00 ± 1.01 b140.0 b
S. fredii6.5 ± 0.28 d3.9 ± 0.32 c10.0 ± 0.59 c0.0 ± 0.00 e10.0 c
B. japonicum10.6 ± 0.40 c5.1 ± 0.25 b110.0 ± 4.23 b50.0 ± 2.29 b160.0 b
Ab-V512.5 ± 0.32 a6.9 ± 0.18 a140.0 ± 3.85 a110.0 ± 3.55 a250.0 a
B. subtilis11.8 ± 0.37 b3.6 ± 0.16 c110.0 ± 2.45 ab50.0 ± 2.82 b160.0 ab
B. megaterium12.0 ± 0.34 ab6.4 ± 0.16 a120.0 ± 3.04 ab80.0 ± 3.07 ab200.0 ab
Control10.1 ± 0.69 c3.6 ± 0.23 c30.0 ± 1.85 c30.0 ± 1.76 cde60 c
Data represent mean ± standard error (SE). Means followed by the same letter do not differ significantly by Tukey’s test (p < 0.05).
Table 4. Effects of biotization with PGPB on growth variables in potato cv. Duvira evaluated after 25 days in a greenhouse. Acclimatization phase (A) and after re-inoculation (B).
Table 4. Effects of biotization with PGPB on growth variables in potato cv. Duvira evaluated after 25 days in a greenhouse. Acclimatization phase (A) and after re-inoculation (B).
(A) In VivoDuvira Cultivar
AcclimatizationTreatmentsLength (cm)Dry Weight (g)
ShootRootShootRootTotal Biomass
R. leucaenae3.8 ± 0.37 g2.0 ± 0.40 fg0.3 ± 0.03 c0.08 ± 0.10 c0.3 ± 0.10 c
Ab-V613.7 ± 0.39 c14.5 ± 0.32 c1.9 ± 0.14 a1.2 ± 0.08 a3.1 ± 0.15 a
R. tropici16.8 ± 0.41 ab13.7 ± 0.26 bc1.2 ± 0.20 b1.0 ± 0.26 a2.2 ± 0.21 b
S. fredii14.7 ± 0.28 bc12.4 ± 0.35 c1.3 ± 0.24 b1.2 ± 0.11 a2.5 ± 0.11 ab
B. japonicum9.5 ± 0.51 c8.8 ± 0.22 de0.4 ± 0.07 c0.3 ± 0.08 bc0.7 ± 0.20 c
Ab-V517.6 ± 0.38 a17.0 ± 0.40 a1.3 ± 0.26 b0.8 ± 0.21 ab2.1 ± 0.13 b
B. subtilis8.4 ± 0.38 ef7.0 ± 0.19 ef0.3 ± 0.03 c0.2 ± 0.15 c0.5 ± 0.02 c
B. megaterium8.5 ± 0.39 ef8.3 ± 0.26 de0.4 ± 0.03 c0.2 ± 0.13 c0.6 ± 0.17 c
Control11.7 ± 0.46 d10.3 ± 0.38 d0.5 ± 0.02 c0.3 ± 0.15 c0.8 ± 0.19 c
(B) Length (cm)Dry Weight (g)
Re-inoculationTreatmentsShootRootShootRootTotal Biomass
R. leucaenae4.0 ± 0.05 f2.2 ± 0.03 ef0.4 ± 0.20 c0.1 ± 0.10 c0.5 ± 0.03 c
Ab-V615.2 ± 0.16 b15.1 ± 0.05 b2.1 ± 0.16 a2.0 ± 0.26 a4.1 ± 0.11 a
R. tropici17.7 ± 0.20 a15.0 ± 0.02 b1.5 ± 0.33 b1.1 ± 0.37 b2.6 ± 0.05 b
S. fredii15.3 ± 0.11 b13.6 ± 0.11 bc1.2 ± 0.11 b1.0 ± 0.21 b2.2 ± 0.05 b
B. japonicum10.2 ± 0.31 cd8.2 ± 0.25 d0.3 ± 0.10 c0.2 ± 0.27 c0.5 ± 0.14 c
Ab-V519.0 ± 0.35 a18.3 ± 0.21 a2.1 ± 0.14 a1.1 ± 0.15 b3.2 ± 0.15 ab
B. subtilis10.0 ± 0.12 cd8.1 ± 0.22 d0.5± 0.25 c0.3 ± 0.12 c0.8 ± 0.22 c
B. megaterium7.1 ± 0.10 e8.1 ± 0.14 d0.3 ± 0.17 c0.2 ± 0.16 c0.5 ± 0.18 c
Control11.1 ± 0.22 c9.8 ± 0.26 d0.4 ± 0.19 c0.3 ± 0.10 c0.7 ± 0.20 c
Data represent mean ± standard error (SE). Means followed by the same letter do not differ significantly by Tukey’s test (p < 0.05).
Table 5. Effects of biotization with PGPB on growth variables in potato cv. Ágata evaluated after 25 days in the greenhouse. Acclimatization phase (A) and after re-inoculation (B).
Table 5. Effects of biotization with PGPB on growth variables in potato cv. Ágata evaluated after 25 days in the greenhouse. Acclimatization phase (A) and after re-inoculation (B).
(A) In VivoÁgata Cultivar
AcclimatizationTreatmentsLength (cm)Dry Weight (g)
ShootRootShootRootTotal Biomass
R. leucaenae4.0 ± 0.24 e2.1 ± 0.31 c0.1 ± 0.03 e0.1 ± 0.03 d0.2 ± 0.15 ed
Ab-V67.0 ± 0.25 d5.0 ± 0.22 cd0.4 ± 0.12 ce0.2 ± 0.03 cd0.6 ± 0.05 cd
R. tropici14.4 ± 0.30 b5.7 ± 0.16 bc1.6 ± 0.20 b0.9 ± 0.05 b2.5 ± 0.22 b
S. fredii6.9 ± 0.38 d3.2± 0.30 e0.2 ± 0.08 de0.1 ± 0.05 d0.3 ± 0.04 de
B. japonicum15.3 ± 0.49 b7.6 ± 0.15 a1.3 ± 0.10 b0.9 ± 0.16 b2.2 ± 0.18 b
Ab-V517.0 ± 0.27 a8.2 ± 0.15 a2.1 ± 0.14 a2.0 ± 0.29 a4.1 ± 0.26 a
B. subtilis12.3 ± 0.35 c5.8 ± 0.20 bc0.6 ± 0.11 cd0.2 ± 0.11 cd0.8 ± 0.10 cd
B. megaterium14.6 ± 0.39 b7.2 ± 0.33 ab1.2 ± 0.20 b1.0 ± 0.17 b2.2 ± 0.08 b
Control12.5 ± 0.36 c5.1 ± 0.30 cd0.7 ± 0.11 c0.3 ± 0.08 c1.0 ± 0.11 c
(B) Length (cm)Dry Weight (g)
Re-inoculationTreatmentsShootRootShootRootTotal Biomass
R. leucaenae3.8 ± 0.16 e3.1± 0.21 de0.17± 0.05 e0.1 ± 0.02 de0.2 ± 0.06 de
Ab-V68.0 ± 0.11 d5.3 ± 0.24 c0.5 ± 0.01 c0.3 ± 0.08 cd0.8 ± 0.10 cd
R. tropici16.0 ± 0.20 b6.4± 0.24 c1.2 ± 0.07 b0.8 ± 0.05 b2.0 ± 0.15 b
S. fredii5.1 ± 0.04 e4.0 ± 0.31 cd0.3± 0.11 d0.2 ± 0.10 de0.5 ± 0.02 de
B. japonicum14.8 ± 0.21 b6.9 ± 0.31 b1.0 ± 0.14 b0.7 ± 0.12 b1.7 ± 0.21 b
Ab-V519.1 ± 0.18 a10.3± 0.27 a2.4 ± 0.20 a2.6± 0.15 a5.0 ± 0.26 a
B. subtilis14.3 ± 0.11 c8.0 ± 0.33 ab1.0± 0.08 b0.7 ± 0.04 b1.7 ± 0.11 b
B. megaterium15.1 ± 0.20 b7.8 ± 0.30 ab1.1 ± 0.10 b0.8 ± 0.10 b1.9 ± 0.11 b
Control11.7 ± 0.13 cd5.2 ± 0.35 c0.5 ± 0.12 c0.4± 0.08 cd0.9 ± 0.08 cd
Data represent mean ± standard error (SE). Means followed by the same letter do not differ significantly by Tukey’s test (p < 0.05).
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Castro Molina, Y.; Dória, J.; Gómez Sepúlveda, A.M.; Carvalho, L.Q.; Pasqual, M.; Jesus, E.d.C. Biotization with Plant Growth-Promoting Bacteria Benefits the Survival and Production of Potato (Solanum tuberosum L.) In Vitro and In Vivo. Horticulturae 2025, 11, 393. https://doi.org/10.3390/horticulturae11040393

AMA Style

Castro Molina Y, Dória J, Gómez Sepúlveda AM, Carvalho LQ, Pasqual M, Jesus EdC. Biotization with Plant Growth-Promoting Bacteria Benefits the Survival and Production of Potato (Solanum tuberosum L.) In Vitro and In Vivo. Horticulturae. 2025; 11(4):393. https://doi.org/10.3390/horticulturae11040393

Chicago/Turabian Style

Castro Molina, Yulimar, Joyce Dória, Ana Milena Gómez Sepúlveda, Luna Queiroz Carvalho, Moacir Pasqual, and Ederson da Conceição Jesus. 2025. "Biotization with Plant Growth-Promoting Bacteria Benefits the Survival and Production of Potato (Solanum tuberosum L.) In Vitro and In Vivo" Horticulturae 11, no. 4: 393. https://doi.org/10.3390/horticulturae11040393

APA Style

Castro Molina, Y., Dória, J., Gómez Sepúlveda, A. M., Carvalho, L. Q., Pasqual, M., & Jesus, E. d. C. (2025). Biotization with Plant Growth-Promoting Bacteria Benefits the Survival and Production of Potato (Solanum tuberosum L.) In Vitro and In Vivo. Horticulturae, 11(4), 393. https://doi.org/10.3390/horticulturae11040393

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