Pseudomonas ﬂuorescens SP007 S Formulations in Controlling Soft Rot Disease and Promoting Growth in Kale

: Kale has gained popularity as a healthy food choice due to its rich nutrient profile. However, kale production systems are also affected by various pests and diseases, especially plant pathogenic bacteria, such as Pectobacterium carotovorum . The purpose of this research was to investigate the biocontrol activity of the Pseudomonas fluorescens strain SP007s and develop a formulation that provides stability, long shelf life, and disease control, as well as promoting growth in kale; we expect this formulation to have further commercial applications in the cultivation of kale. The results indicated that a unique mixture of paper sludge, sugar sludge, and glycerol at 40% relative humidity promoted SP007 survival for 6 months at room temperature (30 ◦ C) with measures of 5.92 × 10 6 CFU/g. This treatment maintained the control efficacy of P. carotovorum in infected soil within 28 days of soil drenching. We evaluated the SP007s formula for controlling soft rot disease in greenhouse conditions, and our results revealed that it can reduce the disease in kale by 65.2% and promotes growth in terms of canopy width, shoot height, number of leaves per plant, fresh weight, and dry weight, which were superior to the control. In addition, the formula can induce the accumulation of endogenous salicylic acid and phenolic compounds, as well as a systemic acquired resistance pathway in the plant defense system. Therefore, the formula of SP007s can be a promising novel biocontrol formula for use in kale production, as it promotes plant growth and acts against P. carotovorum causal soft rot disease.


Introduction
Kale (Brassica oleracea var. acephala) is a nutritious food rich in antioxidants, vitamin C, vitamin K, and beta-carotene [1]. This cultivar contains minerals that can contribute to heart health, weight loss, eye health, and other conditions [2]. However, the production of kale still faces problems related to diseases, especially the soft rot disease caused by P. carotovorum in tropical and sub-tropical areas [3]. Biocontrol involves the use of beneficial micro-organisms or natural products to prevent or control plant diseases that have been characterized based on their antagonitic effects against agricultural pathogens [4]. Moreover, such micro-organisms have been exploited as eco-friendly substitutes for pesticides, as they produce antimicrobial compounds that can activate plant immunity to combat a broad range of plant pathogens. Previous studies revealed the general approaches and potential of biocontrol agents that have been explored as a means of controlling soft rot diseases in vegetables, including the use of Trichoderma spp., Bacillus spp., and Pseudomonas spp. [5,6]. The utilization of beneficial microbes or their metabolites is a crucial strategy for preventing and controlling soil-borne diseases. Azaiez et al. [7] reported that the use of Bacillus amyloliquefaciens strain Ar10 suppresses the growth of the soft rot bacterium P. carotovorum by producing glycolipid-like compounds and biofilm, which can act as tion, the wells were made using a 5 mm cork borer. Then, 10 µL of SP007s suspension (10 8 CFU/mL) was dropped in the well and its evolution was compared against a negative control (sterilized water) and the positive controls. Two types of positive control, including amoxicillin (200 g/mL) as an antibiotic and copper hydroxide (77%) as a chemical control, were used in this experiment separately. The Petri dishes were incubated at 28 • C for 72 h. The calculation of the antagonistic activity was determined by observing a distinct area of inhibition after 72 hours.

Preparation of P. fluorescens SP007s Formulations
The SP007s cell suspension was formulated into six different formulations (final concentration as 10 10 CFU/mL), as shown in Table 1. The inert carriers, enrichment, and additive materials were mixed and sterilized by autoclaving at 121 • C, 15 min, twice. The SP007s cells were added to each formulation and mixed well under aseptic conditions; then, the mixtures were air-dried in a laminar flow chamber for 48 h. After drying, a 1 g sample was removed to proceed with the initial population counts. Aliquots of 1 mL from serial dilutions (10 −10 ) of the homogenates were spread on a King's B medium. The colonies were picked up and grown in the King's B medium and validated through polymerase chain reaction (PCR) amplification of their 16S rDNA gene. The genomic DNA was extracted using the CTAB DNA extraction method [17]. The bacterial 16S rDNA primer Ps-for (5 GGTCTGAGAGGATGATCAGT3 ) and Ps-rev (5 TTAGCTCCACCTCGCGGG3 ) were used to amplify the 16S rDNA gene region [18]. The PCR was performed using a DNA Engine DYAD™ Peltier Thermal Cycler. Thermal cycling was performed by applying an initial denaturation at 94 • C for 5 min, followed by 30 cycles at 94 • C for 1 min, 55 • C for 1 min, 72 • C for 1.5 min, and a final extension at 72 • C for 8 min. The obtained fragments were analyzed via gel electrophoresis (24 × 12 cm) with 2% agarose, and configured at 80 V for 2 h. The resulting PCR products were taken for sequencing, which was performed using the BLAST analysis program.

Effect of P. fluorescens SP007s Formulation on Bacterium Survival
The shelf life of the SP007s formulations stored at room temperature was determined for 6-month storage. The viable cells in each formulation were evaluated by counting cfu using the standard dilution plating method on the King's B medium.

Effect of P. fluorescens SP007s Formulation on Inhibiting P. carotovorum in Soil
The soil was inoculated with P. carotovorum by mixing 100 mL of the bacterial suspension into 1 kg of sterile soil, adjusted to 10 8 CFU/g, and then subjected to air-drying for 20 min. The infested soil was treated with the SP007s formulation (1 × 10 10 CFU/g) to be compared with the SP007s cell suspension (1 × 10 10 CFU/mL), the copper hydroxide 77%, and the water-mock. Subsequently, 10 g of the SP007s formulation was added to each soil pot that contained 500 g of infected soil; then, it was mixed well under aseptic conditions (approximately 10 8 CFU/g), and kept at room temperature. The soil samples were collected from each treatment to evaluate the P. carotovorum population every 7, 14, 21, and 28 days after the treatment application using the serial dilution method [19]. Then, 2 g of soil was mixed into 2 mL of sterile phosphate-buffered saline (PBS). Afterwards, the tubes were centrifuged for 5 min at 5000× g, and 1 mL of supernatant was mixed with 9 mL of PBS, yielding a 1:10 dilution. The spread plate technique was used to count the number of P. carotovorum bacterial colonies evenly distributed on the pectobacterium selective agar (PSA) medium. Then, DNA sequencing and BLAST analysis were performed to validate the strain.

Effect of SP007s Formulation on Controlling Soft Rot Disease
The experiment was conducted in a completely randomized design (CRD) with four replications. The selected formulation was evaluated for its ability to control soft rot disease. Kale seedlings were sown in 30 cm pots which contained 500 kg of sterilized soil. The SP007s formulation at a dose of 10 g was mixed in the pot twice at 15 and 30 days post-planting. For positive controls, 10 mL doses of SP007s cell suspension and copper hydroxide were utilized, whereas sterilized water was used as the negative control. The disease incidents of soft rot were recorded using the following equation: p = D/T, where "D" is the total number of infected plants, and "T" is the total number of plants. The experiment was conducted in CRD with four replications. Kale leaf samples (0.5 g) were obtained from each treatment 45 days after planting to evaluate the endogenous SA contents. The leaf samples were homogenized with 1 mL of a buffer solution composed of 90% methanol, 9% glacial acetic acid, and 1% water after being immersed in liquid nitrogen (LN2). The ground samples were centrifuged at 14,000× g for 10 min at 4 • C. The supernatant was transferred into an Eppendorf tube containing 0.5 mL of 0.02 M ferric ammonium sulfate and incubated for 5 min at 30 • C. Then, using a Bio-Tek microplate reader, the absorbance at 530 nm was measured in 200 µL of each sample on a 96-well plate (Winooski, VT, USA). The endogenous SA was calculated through comparison with the standard references.

Total Phenolic Compounds
A slightly modified Folin-Ciocalteu assay [20] was used to analyze the phenolic compound accumulation. Leaf samples (0.5 g) were soaked in LN2 and then ground with 2.5 mL of 80% methanol. The finely ground samples were centrifuged at 14,000× g for 10 min at 4 • C. Next, 0.2 mL of the supernatant was transferred to an Eppendorf tube containing 0.2 mL of Folin-Ciocalteu reagent and mixed using a vortex mixer. Then, 2 mL of 2% sodium carbonate was added prior to incubation for 20 min at 30 • C under total darkness. Subsequently, 200 µL of each sample was transferred to a 96-well plate, and the absorbance was measured at 760 nm using a Bio-Tek microplate reader (Winooski, VT, USA). Gallic acid was used as the calibration standard, and the total phenolic content was expressed as milligrams of Gallic acid equivalent on a fresh weight basis (mg GAE g −1 FW).

Effect of P. fluorescens SP007s Formulation on Plant Growth
Plant growth parameters, including canopy width, plant height, root length, number of leaves per plant, fresh weight, and dry weight, were recorded 45 days after planting for all treatments.

Statistical Analysis
The experiments were performed under laboratory and greenhouse conditions based on the application of a completely randomized design (CRD) with four replications, and repeated three times. The obtained experimental data were analyzed using one-way ANOVA with SPSS 20 (SPSS Inc., Chicago, IL, USA). Significant differences in means among groups were analyzed using Duncan's multiple range test at p ≤ 0.05.

Antagonistic Activity of P. fluorescens SP007s to Inhibit P. carotovorum
The antagonist testing experiment showed that SP007s produced a strong inhibitory effect on P. carotovorum growth during the co-culture assays on NA ( Figure 1). The diameters of the inhibition areas of the different treatments were measured. The SP007s treatment produced a measured inhibition area of 3.45 ± 0.10 mm in diameter, followed by the amoxicillin treatment with 3.26 ± 0.05 mm and the copper hydroxide treatment with 1.20 ± 00.00 mm ( Table 2).
The experiments were performed under laboratory and greenhouse conditions based on the application of a completely randomized design (CRD) with four replications, and repeated three times. The obtained experimental data were analyzed using one-way ANOVA with SPSS 20 (SPSS Inc., Chicago, IL, USA). Significant differences in means among groups were analyzed using Duncan's multiple range test at p ≤ 0.05.

Antagonistic Activity of P. fluorescens SP007s to Inhibit P. carotovorum
The antagonist testing experiment showed that SP007s produced a strong inhibitory effect on P. carotovorum growth during the co-culture assays on NA ( Figure 1). The diameters of the inhibition areas of the different treatments were measured. The SP007s treatment produced a measured inhibition area of 3.45 ± 0.10 mm in diameter, followed by the amoxicillin treatment with 3.26 ± 0.05 mm and the copper hydroxide treatment with 1.20 ± 00.00 mm ( Table 2).

P. fluorescens SP007s Formulations and Evaluation of the Survival Bacterial Populations
The experiment was based on the development of six microbial formulations in the form of bio-fertilizers for comparing the material efficiency under humidity at 20%, 30%, 40%, and 50%. The six resulting microbial formulations were characterized by a fine texture and crumbly appearance ( Figure S1). The population of the SP007s in each formulation was examined after being kept at room temperature after mixing and then stored for 6 months with varying moisture levels (Tables 3 and 4). The results showed that the moisture content and formula factors influenced the SP007s population. The treatment that included paper sludge, sugar sludge, and 1% glycerol (w/v) at a moisture content of 30% showed the largest SP007s population with 5.92 × 10 6 CFU/g followed by 40%, 50%, and 20% moisture content, with 5.60 × 10 6 , 5.36 × 10 6 , and 4.98 × 10 6 CFU/g, respectively.

P. fluorescens SP007s Formulations and Evaluation of the Survival Bacterial Populations
The experiment was based on the development of six microbial formulations in the form of bio-fertilizers for comparing the material efficiency under humidity at 20%, 30%, 40%, and 50%. The six resulting microbial formulations were characterized by a fine texture and crumbly appearance ( Figure S1). The population of the SP007s in each formulation was examined after being kept at room temperature after mixing and then stored for 6 months with varying moisture levels (Tables 3 and 4). The results showed that the moisture content and formula factors influenced the SP007s population. The treatment that included paper sludge, sugar sludge, and 1% glycerol (w/v) at a moisture content of 30% showed the largest SP007s population with 5.92 × 10 6 CFU/g followed by 40%, 50%, and 20% moisture content, with 5.60 × 10 6 , 5.36 × 10 6 , and 4.98 × 10 6 CFU/g, respectively.

Effect of P. fluorescens SP007s Formulation on Inhibiting P. carotovorum in Infested Soil
The application of the SP007s formulation at 10 g per 500 g of infected soil showed the highest reduction in P. carotovorum population, followed by the SP007s cell suspension and the copper hydroxide; whereas the water-mock did not inhibit the pathogen (Figure 2).

Effect of P. fluorescens SP007s Formulation on Controlling soft Rot Disease
The use of the SP007s formulation twice at 10 g per pot decreased the incide soft rot disease at 45 days after sowing. Soft rot disease reductions observed in th plants that were treated with the SP007s formulation, SP007s cell suspension, c hydroxide, and the untreated control were 65.2%, 60.0%, 91.3%, and 0.0%, respec Nevertheless, the protective efficacy of the SP007s formulation and the SP007s ce pension indicated a non-significant disease incidence of the soft rot disease when paring among these treatments (Table 5, Figure 3).

Effect of P. fluorescens SP007s Formulation on Controlling soft Rot Disease
The use of the SP007s formulation twice at 10 g per pot decreased the incidence of soft rot disease at 45 days after sowing. Soft rot disease reductions observed in the kale plants that were treated with the SP007s formulation, SP007s cell suspension, copper hydroxide, and the untreated control were 65.2%, 60.0%, 91.3%, and 0.0%, respectively. Nevertheless, the protective efficacy of the SP007s formulation and the SP007s cell suspension indicated a non-significant disease incidence of the soft rot disease when comparing among these treatments (Table 5, Figure 3).

Effect of P. fluorescens SP007s Formulation on the Simulation of Plant Defense
The effect of the SP007s formulation on endogenous SA content in kale was evaluated. The results showed that the chemical-fertilizer-treated sample revealed the highest content of endogenous SA at 45 days after sowing. This is considered a non-significant difference when compared with the SP007s formulation, the SP007s cell suspension, and the organic fertilizer treatment. Overall, the endogenous SA content of the water-mock sample showed a significantly lower value when compared with all evaluated treatments ( Table 6).

Effect of P. fluorescens SP007s Formulation on the Simulation of Plant Defense
The effect of the SP007s formulation on endogenous SA content in kale was evaluated. The results showed that the chemical-fertilizer-treated sample revealed the highest content of endogenous SA at 45 days after sowing. This is considered a non-significant difference when compared with the SP007s formulation, the SP007s cell suspension, and the organic fertilizer treatment. Overall, the endogenous SA content of the water-mock sample showed a significantly lower value when compared with all evaluated treatments (Table 6). The total phenolic compound contents of the kale plants for each treatment are shown in Table 7. In general, the use of the chemical fertilizer showed the highest total phenolic compound content of 121 mg GAE g −1 FW, followed by the SP007s formulation (120 mg GAE g −1 FW), organic fertilizer (119 mg GAE g −1 FW), SP007s cell suspension (117 mg GAE g −1 FW), and untreated (113 mg GAE g −1 FW). The samples treated with organic fertilizer and with the SP007s cell suspension were significantly lower than the chemical fertilizer treated samples. The samples treated with the SP007s formulation were non-significantly different when compared with the chemical fertilizer.

Effect of P. fluorescens SP007s Formulation on the Growth Parameter
The plant growth parameters, including plant width, plant height, root length, fresh weight, and dry weight, were significantly increased by the chemical fertilizer treatment followed by the organic fertilizer treatment, the SP007s cell suspension treatment, and the SP007s formulation treatment, as shown in Table 8 and Figure 4. The data showed that the kale plant samples treated with the SP007s formulation had significantly higher values in plant height and dry weight: 7.6 cm and 0.73 g, respectively, compared with the untreated samples, 5.40 cm and 0.55 g, respectively.

Discussion
Pseudomonas sp. has emerged as a potential biocontrol agent in various studies. In our experiments, we investigated the effectiveness of the integrated control agent Pseudomonas fluorescens SP007s in different formulations that were combined with sugar sludge, paper sludge, and glycerol. The survival of inoculated Pseudomonas sp. in the soil and plant rhizosphere is dependent on many factors, such as the inoculate formulation, soil conditions, and the physiological status of the plant. In our study, the results showed that the formulation based on sugar sludge at 49.5%, paper sludge at 49.5%, and glycerol at 1% (w/v) had larger SP007s populations than the other proposed formulations after being kept at room temperature for 6 months. Paper sludge is a potential source of organic matter and rich in nutrients, as well as a carbon source with cellulose [21,22]. Mohammadi et al. [23] reported that the application of paper mill sludge to pots increased the dry matter yield and the potassium, phosphorus, and micronutrient uptake for sorghum, with a rate of 2% being more effective. In addition, micro-organisms can survive in paper sludge and produce a specific enzyme that is partially dependent on the type and structure of the sludge composition [24]. P. fluorescens is a species of bacteria known to produce ligninolytic enzymes that play a crucial role in the breakdown and recycling of lignin in nature, such as paper mill sludge [25]. The key ligninolytic enzymes include laccases, peroxidases, dyP-type peroxidases, and auxiliary enzymes [26]. These provide bacteria with access to lignocellulosic biomass as a carbon and energy source, contributing to their survival [27]. Sugar sludge is the carbon source for microbial cell culture that a micro-organism uses to build biomass. Micro-organisms utilize the sugars present in the sugar sludge for growth and fermentation by converting the sugars into various products of interest, such as organic acids, alcohols, enzymes, and biofuels

Discussion
Pseudomonas sp. has emerged as a potential biocontrol agent in various studies. In our experiments, we investigated the effectiveness of the integrated control agent Pseudomonas fluorescens SP007s in different formulations that were combined with sugar sludge, paper sludge, and glycerol. The survival of inoculated Pseudomonas sp. in the soil and plant rhizosphere is dependent on many factors, such as the inoculate formulation, soil conditions, and the physiological status of the plant. In our study, the results showed that the formulation based on sugar sludge at 49.5%, paper sludge at 49.5%, and glycerol at 1% (w/v) had larger SP007s populations than the other proposed formulations after being kept at room temperature for 6 months. Paper sludge is a potential source of organic matter and rich in nutrients, as well as a carbon source with cellulose [21,22]. Mohammadi et al. [23] reported that the application of paper mill sludge to pots increased the dry matter yield and the potassium, phosphorus, and micronutrient uptake for sorghum, with a rate of 2% being more effective. In addition, micro-organisms can survive in paper sludge and produce a specific enzyme that is partially dependent on the type and structure of the sludge composition [24]. P. fluorescens is a species of bacteria known to produce ligninolytic enzymes that play a crucial role in the breakdown and recycling of lignin in nature, such as paper mill sludge [25]. The key ligninolytic enzymes include laccases, peroxidases, dyP-type peroxidases, and auxiliary enzymes [26]. These provide bacteria with access to lignocellulosic biomass as a carbon and energy source, contributing to their survival [27]. Sugar sludge is the carbon source for microbial cell culture that a micro-organism uses to build biomass. Micro-organisms utilize the sugars present in the sugar sludge for growth and fermentation by converting the sugars into various products of interest, such as or-ganic acids, alcohols, enzymes, and biofuels [28,29]. This suggestion is consistent with Liu et al. [30], who state that the addition of molasses combined with microbial inoculants can improve the structure of the bacteria community. This can provide a favorable carbon source for the microbial inoculants, helping them to establish and thrive in silage or other systems. Glycerol can serve as a carbon and energy source for bacteria, preventing damage to the cell membranes and contributing to keep the cells alive.
The SP007s formulation exhibited potent antagonistic activity against P. carotovorum (>60%), as our results determined, based on the soil application technique twice every 15 days. The SP007s formulation can effectively suppress P. carotovorum populations in infected soil, which reduces the pathogen population by producing pyoluteorin, 2,4-diacetylphloroglucinol (DAPG), and phenazine as antimicrobial properties that are important in disease suppression [31]. These findings are consistent with Suresh et al. [32], who reported that P. fluorescens VSMKU3054 produced 2,4-DAPG to control Ral-stonia solanacearum, the cause of tomato bacterial wilt, and promoted the growth of tomato seedlings. DAPG belongs to a large group of phenolic antimicrobial substances which possess a well-documented role in triggering systemic acquired resistance (SAR) and induced systemic resistance (ISR) in plants such as arabidopsis, maize, tomato, and pea [33][34][35].
P. fluorescens strains have the potential to produce antimicrobial compounds such as lipase and protease [36,37]. These compounds are known to be associated with the degradation of the fungal cell wall or cell membrane to protect the plants against soil borne and foliar pathogens [38]. Similarly, Chauhan et al. [39] reported that P. fluorescens can produce lipopeptides such as viscosinamide, pseudofactin II tensin, and others as antibacterial agents. Furthermore, lipopeptides are a general intermediate for stimulating the plant immune system [40,41]. Thus, the SP007s formulation in plant immune interactions was further investigated. Our results showed that the observed plant defense parameters were increased, including the endogenous SA and the phenolic compound contents (p < 0.05) when compared with the control.
The SA pathway is the dominant signaling cascade under pathogen attacks due to the presence of communication channels that can promote the generation of energy and the synthesis of potential intermediates and enzymes to inhibit pathogens [42]. According to the scientific literature on the defense mechanisms of various plant species, such as rice, soybeans, and tobacco, it has been observed that immune plants enhance the accumulation of salicylic acid in response to pathogen infection [41,43,44]. Likewise, in their report, Lakkis et al. [45] state that Pseudomonas sp. can be an inducer of ISR in grapevine, which activates both the immune response and the priming phenomenon under Botrytis cinerea invasion. This event is related to the constitutively enhanced expression of SA-related genes and to the increase in the total phenolic content [45]. Phenolic compounds have been noted as the most influential secondary products in the determination of plant-disease resistance [46]. These compounds are involved in the activities of polyphenol oxidase (PPO), POX, catalase (CAT), and IAA oxidase (IAAO) that play important roles in various physiological processes and provide protection against multiple biotic stressors [47]. In addition, we found that the use of the SP007s formulation can promote the growth of kale plants. In our previous experiments, P. fluorescens SP007S was studied on PGP traits using high-performance liquid chromatography (HPLC). The results revealed that SP007s secreted high levels of IAA and GA 3 in nutrient glucose broth: 47.5 and 54.7 g mL −1 , respectively [14]. These results are consistent with numerous reports indicating that P. fluorescens has been recognized as a beneficial agent for promoting plant growth [48][49][50]. They produce different substrates such as phytohormones and solubilizing minerals in soil that positively influence plant growth [51]. Furthermore, P. fluorescens are known for their ability to mediate nutritional and for their growth promotion effects in plants by enhancing nutrient availability, including phosphate solubilization and nitrogen fixation [52,53]. Therefore, the SP007s formulation can be used as an environmentally friendly alternative to combat P. carotovorum and emerge as a promising strategy to assist crops to reduce pathogen populations.

Conclusions
The formulation of Pseudomonas fluorescens SP007s reduced plant disease by up to 65% and enhanced the plant growth of kale. The concentration of the endogenous salicylic acid and phenolic compounds of the harvested kale remained affected by the use of SP007s, regardless of the soil application technique. More specifically, the results showed that the SP007s had positive effects, with statistically significant differences, on the plant height and dry weight when compared with the untreated samples. The canopy width, plant height, plant length, fresh weight, and dry weight parameters were significantly increased by the chemical and the organic fertilizer treatments on soil application. On the other hand, the number of leaves per plant of kale plants was not significantly increased in all the treated samples when compared with the untreated samples. The results from our experiments show that the evaluated SP007s formulation can be used as a bio-stimulant in order to enhance kale production.
Author Contributions: A.N., W.C. and D.A., conceptualization and design. A.N., implementation, investigation, and analyzed the data. A.N., W.T. and D.A., arranged the results, discussion, and wrote the original draft. W.T. and D.A., revised the manuscript. All authors have read and agreed to the published version of the manuscript.