Effects of Rhizosphere Bacteria on Strawberry Plants (Fragaria × ananassa Duch.) under Water Deficit

Due to the observed climate warming, water deficiency in soil is currently one of the most important stressors limiting the size and quality of plant crops. Drought stress causes a number of morphological, physiological, and biochemical changes in plants, limiting their growth, development, and yield. Innovative methods of inducing resistance and protecting plants against stressors include the inoculation of crops with beneficial microorganisms isolated from the rhizosphere of the plant species to which they are to be applied. The aim of the present study was to evaluate 12 different strains of rhizosphere bacteria of the genera Pantoea, Bacillus, Azotobacter, and Pseudomonas by using them to inoculate strawberry plants and assessing their impact on mitigating the negative effects of drought stress. Bacterial populations were assessed by estimates of their size based on bacterial counts in the growth substrate and with bioassays for plant growth-promoting traits. The physiological condition of strawberry plants was determined based on the parameters of chlorophyll fluorescence. The usefulness of the test methods used to assess the influence of plant inoculation with rhizosphere bacteria on the response of plants growing under water deficit was also evaluated. A two-factor experiment was performed in a complete randomization design. The first experimental factor was the inoculation of plant roots with rhizosphere bacteria. The second experimental factor was the different moisture content of the growth substrate. The water potential was maintained at −10 to −15 kPa under control conditions, and at −40 to −45 kPa under the conditions of water deficit in the substrate. The tests on strawberry plants showed that the highest sensitivity to water deficiency, and thus the greatest usefulness for characterizing water stress, was demonstrated by the following indices of chlorophyll “a” fluorescence: FM, FV, FV/FM, PI, and Area. Based on the assessment of the condition of the photosynthetic apparatus and the analysis of chlorophyll “a” fluorescence indices, including hierarchical cluster analysis, the following strains of rhizosphere bacteria were found to have favorable effects on strawberry plants under water deficit: the Bacillus sp. strains DLGB2 and DKB26 and the Pantoea sp. strains DKB63, DKB70, DKB68, DKB64, and DKB65. In the tests, these strains of Bacillus sp. exhibited a common trait—the ability to produce siderophores, while those of Pantoea sp. were notable for phosphate mobilization and ACCD activity.


Introduction
Due to the observed climate warming, water deficiency in soil is currently one of the most important stressors limiting the size and quality of plant crops. On a global scale, drought affects one-third of soils [1]. In modern agriculture, the unfavorable impact of

Bacterial Counts in Substrate
In the present study, in the case of optimum moisture content, the lowest numbers of bacteria were observed in the control variants, i.e., K0 (3.2 × 10 5 CFU/g of substrate) and KMg with magnesium sulfate (7.2 × 10 5 CFU/g of substrate). Only one strain, Pantoea sp. DKB 63, caused a reduction in the number of bacteria in relation to KMg (to the level of 1.2 × 10 5 CFU/g of substrate), while the remaining strains caused an increase in the number of bacteria in the soil. The highest numbers of bacteria were observed after inoculation with Pseudomonas sp. PJ 1.2 (2.1 × 10 7 CFU/g of substrate) and Azotobacter AJ 1.1 (1.3 × 10 7 CFU/g of substrate). In the case of moisture deficit in the substrate, a negative correlation was observed for all the variants at the level of −0.46, which was manifested in a considerable reduction in the total number of bacteria in the soil. The lowest values of CFU per g of substrate were recorded for the controls K0 (8.5 × 10 3 ) and KMg (1.3 × 10 4 ). After bacterial inoculation, the highest numbers of bacteria were recorded for the strains of Pseudomonas sp. PJ 1.2 (8.2 × 10 5 CFU/g of substrate), Pantoea sp. DKB

Chlorophyll Fluorescence
The study analyzed the frequency of statistically significant differences in the values of the determined parameters of chlorophyll "a" fluorescence in strawberry, between the control variants (K0 and KMg) and the variants differing in the bacterial strains used for inoculation, regardless of the substrate moisture level. The analysis revealed four inoculation strains that accounted for 56% of the variability of chlorophyll "a" fluorescence indices; they were: Bacillus sp. DKB

Chlorophyll Fluorescence
The study analyzed the frequency of statistically significant differences in the values of the determined parameters of chlorophyll "a" fluorescence in strawberry, between the control variants (K0 and KMg) and the variants differing in the bacterial strains used for inoculation, regardless of the substrate moisture level. The analysis revealed four inoculation strains that accounted for 56% of the variability of chlorophyll "a" fluorescence indices; they were: Bacillus sp. DKB 58, Pantoea sp. DKB 64, Pantoea sp. DKB 65, and Pantoea sp. DKB 68. It should be noted that after including the next three strains-i.e., Bacillus sp. DKB 84, Pantoea sp. DKB 70, and Bacillus sp. DKB 26-the seven strains together accounted for 85% of the variability of the determined indices of chlorophyll "a" fluorescence, regardless of the substrate moisture level (Figure 2). for 85% of the variability of the determined indices of chlorophyll "a" fluorescence, regardless of the substrate moisture level ( Figure 2).

Figure 2.
Frequency of statistically significant changes in the indices of chlorophyll "a" fluorescence in strawberry as a result of inoculation with rhizosphere bacteria, between optimal soil moisture and water deficit conditions.
The hierarchical cluster analysis revealed three important clusters: cluster I and cluster II represented the similarity of the variability of chlorophyll "a" fluorescence indices under the conditions of water deficit in the substrate, and cluster III was a complex cluster encompassing the variability under optimal moisture levels. Cluster I included strains whose variability coincided with the changes in the control samples K0 and KMg. Cluster II, which was mainly responsible for the variability of these traits caused by different moisture levels, included the strains: Pantoea sp. DKB 64, Pantoea sp. DKB 68, Pantoea sp. DKB 70, Bacillus sp. DKB 58, Pantoea sp. DKB 65, and Bacillus sp. DKB 26. Cluster III showed the dependence of the variability of the effect of the inoculation strains on the magnitude of the determined indices of chlorophyll "a" fluorescence in a manner dependent on K0 and KMg. The most important feature observed in the experiment is the total distinctiveness in terms of the tested fluorescence indices between the optimal soil moisture and water deficit ( Figure 3).  The hierarchical cluster analysis revealed three important clusters: cluster I and cluster II represented the similarity of the variability of chlorophyll "a" fluorescence indices under the conditions of water deficit in the substrate, and cluster III was a complex cluster encompassing the variability under optimal moisture levels. Cluster I included strains whose variability coincided with the changes in the control samples K0 and KMg. Cluster II, which was mainly responsible for the variability of these traits caused by different moisture levels, included the strains: Pantoea sp. DKB 64, Pantoea sp. DKB 68, Pantoea sp. DKB 70, Bacillus sp. DKB 58, Pantoea sp. DKB 65, and Bacillus sp. DKB 26. Cluster III showed the dependence of the variability of the effect of the inoculation strains on the magnitude of the determined indices of chlorophyll "a" fluorescence in a manner dependent on K0 and KMg. The most important feature observed in the experiment is the total distinctiveness in terms of the tested fluorescence indices between the optimal soil moisture and water deficit ( Figure 3).
The results of the descriptive statistics of the conducted experiment are presented in Tables 2 and 3 Table 4. The F 0 coefficient varied in the range from 193 to 376 in the conditions of optimal moisture, reaching a CV of 9.5%, while in the conditions of water deficit, the coefficient was between 159 and 373 and CV = 11%. The lowest values were recorded when inoculated with the Bacillus sp. DLGB 2 (OM) and Pantoea sp. DKB 63 (WD) strains, and the highest when inoculated with the Bacillus sp. DLGB 2 (OM) and Pantoea sp. DKB 63 (WD) strains (Table 2, Figure 4).     The results of the descriptive statistics of the conducted experiment are presented in Tables 2 and 3 Table  4. The F0 coefficient varied in the range from 193 to 376 in the conditions of optimal moisture, reaching a CV of 9.5%, while in the conditions of water deficit, the coefficient was between 159 and 373 and CV = 11%. The lowest values were recorded when inoculated with the Bacillus sp. DLGB 2 (OM) and Pantoea sp. DKB 63 (WD) strains, and the highest when inoculated with the Bacillus sp. DLGB 2 (OM) and Pantoea sp. DKB 63 (WD) strains (Table 2, Figure 4).                    The F M coefficient averaged 1258 and varied in the range from 984 to 1487 under optimal moisture conditions, reaching a CV of 6.5%, while under water deficiency conditions, the coefficient was between 779 and 1464 and CV = 7%. The lowest values were recorded in the control K0 (OM) and when inoculated with the Pantoea sp. DKB 63 (WD) strain, and the highest when inoculated with the Pseudomonas sp. PJ 1.2 (OM) strain and KMg (WD) (Table 4, Figure 5).
The F V coefficient averaged 1032 and varied from 736 to 1259 under optimal moisture conditions, reaching a CV of 7.5%, while under water deficit, the coefficient was between 554 and 1237 and CV = 9.  Figure 10).

Discussion
Members of the Bacillus, Pseudomonas, and Azotobacter genera of bacteria have been extensively reported as plant growth enhancers [41][42][43][44][45]. Those of the genus Pantoea, although usually known as plant pathogens, have been reported in some studies to include strains with plant growth-promoting capabilities [32,46,47]. Indole-3-acetic acid (IAA), a plant hormone, is a natural auxin produced by rhizobacteria; as one of the phytohormones, IAA can act as a reciprocal signaling molecule in microbe-plant interactions [48]. Siderophore-producing PGPRs increased the Fe(III) ion supply to plants in the rhizosphere and are, therefore, known to enhance plant growth and crop productivity [49]. The ability to produce a clear zone around the bacterial colony implies that the bacteria can solubilize mineral phosphorus in the rhizosphere [50]. Plant growth-promoting rhizobacteria which possess the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase facilitate plant growth and development by decreasing ethylene levels, inducing a reduction in drought stress in plants [51]. The production of key traits for PGPR strains was found among many representatives of the bacteria used in this work and they were also tested in the context of drought [52][53][54][55].
There are a number of reports on the mitigation of adverse effects of drought by the addition of PGP microbes [56,57]. The most often used for this purpose are strains of the genus Bacillus [27,58,59], with those of Pantoea [60] and Azotobacter [61] used to a lesser extent.
There are not many reports on the changes in the numbers of microorganisms under drought conditions. For example, culture-based methods (intact grassland monoliths from natural habitats) have indicated that microbial physiological response was modulated by moisture content. The highest numbers of bacteria were observed with wetted treatments consistently being over 10 8 CFU/g of substrate, and there was up to a 40-fold reduction in CFU in dried treatments, compared with the continually wetted treatments [62]. Similar observations were noted by Omar et al. [63], where the decrease in the numbers of bacteria, after they had been subjected to drought stress, varied according to the rice genotype from 9.3% to 20%. When comparing the survival of Azospirillum strains under stressful conditions in maize cultivation, Ilyas et al. [64] observed a decrease in the number of these bacteria by 40%.
In this study, the smallest decrease in the number of bacteria in the case of moisture deficit, compared with optimum moisture content, was observed following inoculations with the strains Pantoea sp. DKB 65 (approx. 5 times), Bacillus sp. DKB 58 (approx. 11 times) and Bacillus sp. DLGB 2 (approx. 13 times).
Chlorophyll fluorescence can be considered the basic indicator for the analysis of the relationship between photosynthesis and plant growth environment. It is used, inter alia, in studies on the response of various plant species to stressors [65][66][67]. Under the influence of water deficiency, the probability of PSII damage increases, manifested by a reduced photosynthetic efficiency and an increase in the dissipation of absorbed energy in the form of non-photochemical quenching [68].
The study did not show any influence of the experimental factors or their interactions on the values of the parameters F 0 and T FM . The F 0 index indicates the amount of loss in the excitation energy during its transmission from the antennas to the PSII active center. T FM is the time to peak fluorescence. In plants growing under the conditions of water deficiency, the study found a decrease in the parameters F M , F V , F V /F M , and also PI and Area, which confirmed the presence of the state of stress. This is indicative of a significant impact of drought on the state of the photosynthetic apparatus of the tested plant species, a lower quantum yield of PSII, the inability to reduce all electron acceptors, and the occurrence of energy losses in the form of heat. According to Xu [69] and Angelini et al. [70], the ratio F V /F M is considered a reliable indicator of the photochemical activity of the photosynthetic apparatus, which determines the potential efficiency of PSII. When this ratio becomes lower, it proves that the plant has been exposed to a stressor. A reduction in the F V /F M value due to drought, which was demonstrated in our study, has also been observed in other species such as: Lycopersicon esculentum Mill. [71], Viburnum tinus L. [72], and Vigna inguiculata L. Walp. [73]; in the case of the latter-as a result of prolonged water deficiency stress. There are also reports of the relatively low sensitivity of the F V /F M ratio to water deficiency, which has been found in various species: Phaseolus vulgaris L. [74], Glycine max. (L.) Merr [75], Secale cereale L. [76], and also Fragaria × ananassa Duch. [77]. The PI index is considered a reliable parameter for assessing the tolerance of plants to abiotic stresses and is an indicator of the efficiency of the PSII system [78]. Our study showed a significant decrease in the value of this index by 10.6% due to water deficiency. Similar results of research on the impact of drought stress on the PI parameter in rye had been reported by Czyczyło-Mysza and Myśków [76].
There is evidence that by inducing various mechanisms, such as the production of phytohormones (IAA, cytokinins, ABA), the production of bacterial exopolysaccharides (EPS), and the synthesis of the ACC deaminase enzyme, rhizospheric microorganisms can promote plant growth under drought stress [79,80]. In the present study, the inoculation with the tested strains of rhizospheric bacteria did not affect the value of the parameters F M , F V , and F V /F M , neither under the conditions of drought nor under optimal substrate moisture ( Figures 5-7). Barnawal et al. [81], however, had shown, under water deficit, a beneficial effect of the inoculation of wheat with the Bacillus subtilis LDR2 strain on the efficiency of the PSII system, which was manifested by an increase in the F V /F M ratio. Similarly, Khan et al. [82] had shown an increase in F V /F M, as a result of using a consortium of the bacteria Bacillus subtilis, Bacillus thuringiensis, and Bacillus megaterium in two varieties of Cicer arietinum L. contrasting for drought tolerance, growing under water deficit. This increase was particularly evident in the drought-sensitive variety.
In the case of the plants growing under water deficit, the present study showed an increase, in comparison with the control, in the PSII vitality index due to inoculation with the Bacillus sp. strains, DLGB2 and DKB26, as well as the Pantoea sp. strains, DKB63, DKB70, DKB68, and DKB64. There was also evidence of a beneficial effect of inoculation with the Pantoea sp. strains, DKB70, DKB68, and DKB65 on the increase, relative to the control, in the value of the Area parameter in the strawberry plants growing under the conditions of water deficit in the substrate. This may indicate an increase in the efficiency of electron transport from the reaction centers to the plastoquinones. A synergistic effect of the Pseudomonas strains in the production of ACC deaminase, auxin synthesis, the ability of mineral phosphate solubilizing, and the production of siderophores, has been found to significantly improve the yield-related traits of sweetcorn under the limited availability of irrigation water. Moreover, there was an increase in the F V /F M index and a decrease in the F 0 index after inoculation with bacteria [83].

Location of the Experiment, Plant Material, and Growth Conditions
The experiment was conducted in a greenhouse, located at the West Pomeranian University of Technology in Szczecin (53 • 25 N, 14 • 32 E, 25 m a.s.l., sub-zone 7a USDA). On 5 October 2020, plantlets of the strawberry cultivar Polka (Strawberry plant nursery J.G. Mendyk, Koronowo, Poland) were planted. Strawberry cv. Polka is one of the tastiest medium-late varieties of strawberries. The fruits are medium size, spherical, heart-shaped, or broad-conical-shaped. They have uniformly red to dark red skin with a slight gloss. The plants are moderately strongly growing. 'Polka' is resistant to frost, leaf scorch disease, as well as powdery mildew, and are susceptible to common gray mold. Plantlets (BBCH13) were planted individually into black round PVC pots with a diameter of 19 cm, and a capacity of 3.0 dm 3  conditions of water deficit in the substrate. This may indicate an increase in the efficiency of electron transport from the reaction centers to the plastoquinones. A synergistic effect of the Pseudomonas strains in the production of ACC deaminase, auxin synthesis, the ability of mineral phosphate solubilizing, and the production of siderophores, has been found to significantly improve the yield-related traits of sweetcorn under the limited availability of irrigation water. Moreover, there was an increase in the FV/FM index and a decrease in the F0 index after inoculation with bacteria [83].

Location of the Experiment, Plant Material, and Growth Conditions
The experiment was conducted in a greenhouse, located at the West Pomeranian University of Technology in Szczecin (53°25′ N, 14°32′ E, 25 m a.s.l., sub-zone 7a USDA). On 5 October 2020, plantlets of the strawberry cultivar Polka (Strawberry plant nursery J.G. Mendyk, Koronowo, Poland) were planted. Strawberry cv. Polka is one of the tastiest medium-late varieties of strawberries. The fruits are medium size, spherical, heart-shaped, or broad-conical-shaped. They have uniformly red to dark red skin with a slight gloss. The plants are moderately strongly growing. 'Polka' is resistant to frost, leaf scorch disease, as well as powdery mildew, and are susceptible to common gray mold. Plantlets

Experimental Factors
A two-factor experiment was performed in a complete randomization design with four replications, each represented by a single plant. The first experimental factor was the inoculation of plant roots with rhizospheric bacteria. The inoculum was applied to the growth substrate near the root system, in the amount of 40 cm 3 /plant, with a minimum bacterial density of 10 7 CFU/g, within 7 weeks from planting the plants (BBCH16, Figure  12). The following variants of the first factor were applied: Figure 11. The plantlet of the strawberry cv. Polka.

Experimental Factors
A two-factor experiment was performed in a complete randomization design with four replications, each represented by a single plant. The first experimental factor was the inoculation of plant roots with rhizospheric bacteria. The inoculum was applied to the growth substrate near the root system, in the amount of 40 cm 3 /plant, with a minimum bacterial density of 10 7 CFU/g, within 7 weeks from planting the plants (BBCH16, Figure 12). The following variants of the first factor were applied:  The second experimental factor was the different moisture content of the growth substrate. The water potential was maintained at −10 to −15 kPa under control conditions (optimal soil moisture-variant OM), and at −40 to −45 kPa under conditions of water deficit in the substrate (variant WD). The substrate moisture levels were varied from 6 weeks after inoculation. The substrate moisture was determined using soil contact tensiometers.

Bioassays for Plant Growth Promoting Traits
The mobilization of P from insoluble phosphate was detected by the formation of a transparent halo zone surrounding bacterial colonies on the Pikovskaya medium containing tricalcium phosphate, after 5 days at 28 °C [84]. Siderophore production was detected by the production of an orange halo zone on a standard Chrome Azurol-S (CAS) agar plate after 5 days at 28 °C [85,86].
The quantification of indole-3-acetic acid production was performed with Salkowski's reagent [87]. Bacterial cultures were grown on minimal DF solid medium [88] supplemented with tryptophan (500 µ g·mL −1 ) as the precursor of IAA. The plates were The second experimental factor was the different moisture content of the growth substrate. The water potential was maintained at −10 to −15 kPa under control conditions (optimal soil moisture-variant OM), and at −40 to −45 kPa under conditions of water deficit in the substrate (variant WD). The substrate moisture levels were varied from 6 weeks after inoculation. The substrate moisture was determined using soil contact tensiometers.

Bioassays for Plant Growth Promoting Traits
The mobilization of P from insoluble phosphate was detected by the formation of a transparent halo zone surrounding bacterial colonies on the Pikovskaya medium containing tricalcium phosphate, after 5 days at 28 • C [84]. Siderophore production was detected by the production of an orange halo zone on a standard Chrome Azurol-S (CAS) agar plate after 5 days at 28 • C [85,86].
The quantification of indole-3-acetic acid production was performed with Salkowski's reagent [87]. Bacterial cultures were grown on minimal DF solid medium [88] supplemented with tryptophan (500 µg·mL −1 ) as the precursor of IAA. The plates were covered with Whatman No. 1 filter paper saturated with Salkowski's reagent for 30 min. at 28 • C. A pink zone appeared around the IAA-producing colonies.
ACC deaminase activity was determined by a modified method that measures the amount of α-ketobutyrate (α-KB) when the ACC deaminase enzyme cleaves ACC. The bacterial strains were propagated in a minimal DF medium with 5 mM ACC. The calibration curve was formed on α-ketobutyrate. The ACCD activity was expressed as nM of α-KB·mg protein −1 ·h −1 [89,90].

Bacterial Counts in Substrate
Two grams of each substrate sample was added to 18 mL of 0.9% (w/v) solution of sodium chloride. After homogenization for 1h, this solution was decimally diluted (10 −2 to 10 −6 ), and 100 µL aliquots of the resulting solutions were plated on Tryptone Soya Agar (TSA, Oxoid). After incubation at 28 • C for 3 days, the colony forming units (CFU) were counted. The bacterial counts were performed on substrate samples taken from each pot on 12 April 2021, 18 weeks after the inoculation of the plants.

Chlorophyll "a" Fluorescence
The measurements of direct chlorophyll fluorescence were recorded using a Handy PEA (Handy Plant Efficiency Analyzer) spectrofluorometer (Hansatech Instruments Ltd., King's Lynn, Norfolk, UK), based on the standard apparatus procedure (3 × 650 nm LEDs, maximum actinic light intensity 3500 µmol·m −2 ·s −1 , duration of the light pulse 1s). The leaves were shaded for 20 min. prior to the measurement with a leaf clip (4 mm in diameter). The following parameters of chlorophyll fluorescence induction were measured and calculated using the spectrofluorometer: the index of initial fluorescence excitation energy loss in power antennas (F 0 ); maximum fluorescence after the reduction in acceptors in PSII and after dark adaptation (F M ); variable fluorescence, determined after dark adaptation, a parameter dependent on the maximum quantum yield of PSII (F V = F M − F 0 ); maximum potential photochemical reaction efficiency in PSII determined after dark adaptation and after the reduction in acceptors in PS II (F V /F M ); the time of fluorescence increase to the value of F M (T FM ); PSII vitality index for the overall viability of this system (PI); the surface area above the chlorophyll fluorescence curve and between the F 0 and F M points proportional to the size of the reduced plastoquinone acceptors in PS II (Area) [91]. The measurements of chlorophyll fluorescence parameters were taken 18 weeks after the inoculation of the plants, on healthy, fully grown leaves of each plant.

Statistical Methods
The results of the tests were subjected to bivariate analysis of variance (ANOVA), and Tukey's HDS post hoc test was performed. Additionally, in order to determine the occurrence of the variability of the determined traits depending on the experimental factors used, a cluster analysis was carried out using Ward's method for linkage and the square of the Euclidean distance as a measure of distance [92]. The significance of the clusters was determined using the Sneath graded criterion [93]. The above statistical calculations were performed using Statistica 13.1PL (Cracow, Poland, StatSoft Poland). A Monte Carlo simulation with R Studio software (Boston, USA, RStudio PBA) was performed to confirm the obtained results [94].

Conclusions
The presented studies in this paper on strawberry plants showed that the greatest usefulness for characterizing water stress was demonstrated by F M , F V , F V /F M , PI, and Area. Based on the assessment of the condition of the photosynthetic apparatus and the analysis of chlorophyll "a" fluorescence indices, the most favorable effects on strawberry plants under water deficit had the Bacillus sp. strains, DLGB2 and DKB26 and the Pantoea sp. strains, DKB63, DKB70, DKB68, DKB64, and DKB65. In the case of inoculation with the Pantoea sp. strain, DKB65 and Bacillus sp. strain, DLGB 2, the tests demonstrated the lowest decrease, among those recorded under water deficit, in the numbers of bacteria in the soil. In contrast, the Pantoea sp. strain, DKB64, was one of those that produced, after inoculation under water deficit, the highest number of bacteria. The recently ongoing climatic changes force us to look for sustainable and effective solutions to alleviate the water deficit stress in cultivated plants. Therefore, it is justified and necessary to conduct further, much wider research on the most promising beneficial microorganisms that favorably affect plants under stress conditions and alleviate the negative effects of water stress.

Conflicts of Interest:
The authors declare no conflict of interest.