Pseudomonas fluorescens PDS1 and Bacillus subtilis KA9 in terms of chili plant growth and its biocontrol efficiency against Ralstonia solanacearum, experiments were conducted both in vitro and in vivo. A closure assembly was designed using a half-inverted plastic bottle to demonstrate plant–microbial interactions via volatile compounds. The most common volatile organic compounds were identified and reported; they promoted plant development and induced systemic resistance (ISR) against wilt pathogen R. solanacearum. The PDS1 and KA9 VOCs significantly increased defensive enzyme activity and overexpressed the antioxidant genes PAL, POD, SOD, WRKYa, PAL1, DEF-1, CAT-2, WRKY40, HSFC1, LOX2, and NPR1 related to plant defense. The overall gene expression was greater in root tissue as compared to leaf tissue in chili plant. Our findings shed light on the relationship among rhizobacteria, pathogen, and host plants, resulting in plant growth promotion, disease suppression, systemic resistance-inducing potential, and antioxidant response with related gene expression in the leaf and root tissue of chili.Microbial volatiles benefit the agricultural ecological system by promoting plant growth and systemic resistance against diseases without harming the environment. To explore the plant growth-promoting efficiency of VOCs produced by  Pseudomonas and Bacillus, have been identified to release VOCs exhibiting antibacterial action [Bacillus spp., such as benzaldehyde, 1,2-benzisothiazol-3(2H)-one, and 1,3-butadiene, were found to have a substantial inhibitory effect against R. solanacearum, which causes bacterial wilt disease [Bacillus, Pseudomonas, Serratia, and Arthrobacter are linked to plant growth promotion and the induction of systemic resistance [Bacillus subtilis GB03 and B. amyloliquefaciens IN937 [Bacillus VOC 2,3-butanediol was demonstrated to greatly improve resistance in Arabidopsis against Erwinia carotovora subsp. carotovora [Pseudomonas syringae pv. lachrymans, which causes cucumber bacterial angular leaf spot [Bacillus subtilis GBO3 VOCs, a transcriptional study of Arabidopsis revealed that the VOCs can regulate auxin and result in the commencement of growth promotion [Bacillus species are the most effective PGPR bacteria because they can produce spores that survive in harsh environments [Bacillus strain KA9 and Pseudomonas strain PDS-1 on plant growth promotion, induction of systemic resistance, and gene transcriptional expression in chili.Bacterial wilt is a catastrophic soil-borne disease that affects nearly 450 crop species, primarily those belonging to the Solanaceae family ,2. Bactel action ,11,12,17 disease . It was sistance ,14,18,19ns IN937 . In addirotovora . 3-Pentaeaf spot . The effeaf spot ,13,22,23romotion . Bacilluronments . The majR. solanacearum cultures used in this study were taken from the Plant Bacteriology Lab, Division of Plant Pathology, Indian Council of Agricultural Research—Indian Agricultural Research Institute, New Delhi, with accession numbers MT491101 and MN368159. The rhizobacteria cultures utilized in this investigation were collected from diverse agroclimatic zones, characterized in 2019, and kept in the lab in glycerol stock, slants, and active plates. These studies were run since 2019 and repeated three times to ensure a valid and stable hypothesis. These cultures were grown in TSA medium , KB medium , CPG medium  [Rhizobacteria and  0.005%) . A singlR. solanacearum on a separate CPG plate. The agar plate covers were then removed, and the R. solanacearum-inoculated plate was flipped over the rhizobacteria-inoculated plate (R. solanacearum-inoculated plates over a plain agar plate (designated as D1 in this experiment). The growth of R. solanacearum in control was compared against its growth in the presence of the rhizobacteria (designated as D2).A dual-plate assay was first used to determine the potential of rhizobacterial isolates PDS1 and KA9 in producing volatile inhibitory compounds. A colony of each rhizobacterium was smeared on an NA plate, and the same process was repeated for ed plate , wherebyD1 and D2 (Equation (1)).D1 is the diameter growth of R. solanacearum only (control), and D2 is the diameter growth of R. solanacearum co-inoculated with rhizobacteria.The inhibition (%) by the rhizobacteria was calculated using Pseudomonas fluorescens (PDS1) and Bacillus subtillis (KA9) were cultured on a sterile LB agar plate at 28 ± 1 °C for 48 h. They were diluted with sterile distilled water to 108 CFU/mL. The pathogen was grown on CPG agar medium at 28 ± 1 °C for 48 h, and the inoculum load was maintained at 108 CFU/mL. Chili cv. Pusa Jwala seeds were planted in seed starter trays with 96 wells containing a 2:1:1 mixture of peat mass, vermiculite, and sand in a Phytotron under controlled conditions at 28 ± 2 °C, watered daily. As illustrated in R. solanacearum present in the rhizospheric zone of the upper part of the assembly where the chili was transplanted. The plants were simultaneously treated with rhizobacteria and R. solanacearum UTT-25 5 days after transplantation. After 48 h, cultures of wilt pathogen and biocontrol agents were scraped from the Petri plates and mixed in sterile distilled water to maintain the bacterial population at 0.1 OD (600 nm) according to a spectrophotometer. Then, a 2 mL suspension of R. solanacearum was inoculated in the rhizospheric zone after 5 days of transplanting. Subsequently, 5 mL of antagonists were inoculated in the lower chamber of the assembly. The plants treated with pathogen R. solanacearum only and uninoculated plants were also maintained as positive and negative controls, respectively. The observations were recorded at 7 day intervals for 40 days after transplanting. Three replicates were maintained for the experiment.Rhizobacterial isolates B. subtilis KA9), T-3 (P. fluorescens PDS1), T-4 (B. subtilis KA9 + R. solanacearum UTT-25), T-5 (P. fluorescens PDS1 + R. solanacearum UTT-25), T6 (P. fluorescens PDS1 + B. subtilis KA9 + R. solanacearum UTT-25), and T-7 (R. solanacearum UTT-25). The surviving plants were assessed for wilt intensity and fresh and dry weight at the time of harvesting. The degree of wilt was studied at 7 day intervals for 60 days. The average percentage of wilt in each treatment was calculated by following the scale of disease severity, where 1 indicates no symptoms, 2 indicates one wilted leaf, 3 indicates two to three wilted leaves, 4 indicates four or more wilted leaves, and 5 indicates that the entire plant has wilted (dead plant). Wilt incidence was estimated using the 30 days after inoculation according to the following formula [i is the number of plants with a specific disease rating, vi is the disease rating , V is the highest disease rating, and N is the number of plants observed. Biocontrol efficacy was calculated as described in [The seven treatments were as follows: T-1 (control), T-2 , β-mercaptoethanol (0.2 mL), and 1.0 g of insoluble polyvinyl pyrrolidone (PVPP), centrifuged at 12,000 rpm for 30 min at 4 °C. In the supernatant, the concentration of ammonium sulfate was measured. Extraction buffer (3 mL) was used to recover the pellets from the supernatant after 1 h of centrifugation at 12,000 rpm for 30 min at 40 °C, and the pellets were utilized for assays to evaluate defense enzymes such as SOD, POD, PPO, and PAL.The polyphenol oxidase activity was determined according to the method described in . The reaOne unit of enzyme activity was described by a change in absorbance of 0.1 per minute. The specific activity was calculated using the following formula:2O2), 2 mL of potassium phosphate buffer, and 1 mL of pyrogallol were combined. The enzyme extract (0.1 mL) was added to the mixture and left to rest for 3 min at room temperature. Then, 0.5 mL of sulfuric acid was added to terminate the reaction. The OD at 420 nm was measured for 5 min, and one unit of enzyme activity was defined as a change in absorbance of 0.1 per minute. The specific activity was calculated using the following formula:Peroxidase activity was determined using the method described in . Brieflyx is the specific activity per mg protein in the enzyme extract. One unit of enzyme activity was defined as a 50% reduction in the blue color formed by NBT/30 min/mg protein. OD S = OD T − OD C, where OD T is the absorbance of the sample, OD C is the absorbance of the dark control, and OD LS is the absorbance of the light control.Superoxide dismutase activity was determined using the NBT method described by Beauchamp and Fridovich . First, g for 10 min, and the supernatant was used for the enzyme activity assay as described by Sadisivam and Manickam [Chili leaves and roots (0.2 g each) of cv. Pusa Jwala were homogenized in 2 mL of 25 mM borate buffer  containing 2 μL of β-mercaptoethanol and a trace amount of polyvinyl polypyrrolidone (PVP). The homogenate was filtered through a 0.45 μm nylon membrane filter and centrifuged at 12,000× Manickam . One uniR. solanacearum and rhizobacteria in the plant system were assessed. Three chili plants were randomly selected from each treatment, and 1 g of the root was crushed in 5 mL of a 0.85% brine solution, before diluting serially. To determine the growth of R. solanacearum and rhizobacteria, a 100 µL aliquot was disseminated equally over TTC and LA media. The infected Petri plates were incubated at 28 °C for 48 h. The number of colony-forming units (CFU) per gram of plant fresh weight was counted. The trials were carried out in a glass house in triplicate under identical conditions, and the data were pooled for statistical analysis.After 30 days of inoculation, the levels of R. solanacearum present in the rhizospheric zone. The jar assembly was placed at 25 °C in a 12 h light/12 h dark photoperiod for 30 days. The induction of systemic resistance was determined by measuring the difference in regulation of defensive enzymes PAL, PPO, POD, and SOD in the root and shoot tissues of seven different treatments. The seven treatments were as follows: T-1 (uninoculated control treated with sterile water only), T-2 (B. subtilis KA9), T-3 (P. fluorescens PDS1), T-4 (B. subtilis KA9 + R. solanacearum UTT-25), T-5 (P. fluorescens PDS1 + R. solanacearum UTT-25), T6 (P. fluorescens PDS1 + B. subtilis KA9 + R. solanacearum UTT-25), and T-7 (R. solanacearum UTT-25 only).A low-cost closed assembly resembling an Earth system was established using a half-inverted plastic bottle to demonstrate plant–microbial interactions via volatile compounds. This system collects microbial volatile compounds from the interior of an inverted plastic bottle, featuring a downward-pointing spout covered with a muslin cloth. The closure has an open pore through which volatile compounds may flow from the inverted plastic bottle . The lowThe separation and identification of volatile compounds from the plastic bottle assembly were achieved using a Shimadzu QP 2000 fitted with an Rtx-5ms column (30 m × 0.25 mm) packed with 95% dimethyl poly siloxane for GC–MS analysis. Helium with a flow rate of 1 mL/min was used as the carrier gas. First, 1.0 μL of each sample was injected into the injection port with temperature maintained at 230 °C. The initial temperature of the oven was programmed at 40 °C for 4 min, before it was increased to 220 °C with 5 °C ramping rate and held for 2 min. Finally, the temperature was increased to 270 °C with a ramping rate of 15 °C and held for 1 min. The running time of the sample was 45 min. The ion source was kept at a constant temperature of 200 °C. For GC–MS analysis, electron impact ionization (EII) at 70 eV was utilized, and the data were analyzed using TIC  for compound identification and quantification. The spectra of each molecule were compared to a recognized spectral database in the GC–MS library (NIST14), and the data were then processed by measuring the peak regions using Turbo-Mass-OCPTVS-Demo SPL software.The roots of the chili plants cv. Pusa Jwala were collected. Then, 5 mL of 0.85% brine solution was used to crush 1.0 g of root, before diluting serially. To determine rhizobacteria growth, a 100 µL aliquot was evenly disseminated on Luria agar medium. The inoculated Petri plates were incubated at 28 ± 1 °C for 48 h. The colonies were enumerated to compute the number of colony-forming units (CFU) per gram of plant fresh weight.First, 100 mg of fresh root and leaf samples were carefully gathered from the infected spot (about 10 mm) and homogenized in liquid nitrogen. With an RNA isolation kit, total RNA was extracted from the tissues using the silica spin column technique to obtain pure and undamaged RNA  .Diluting the samples with RNase-free water allowed the RNA content to be quantified. The absorbance of the samples was measured at 260 and 280 nm, where 40 g/mL RNA equates to an absorbance reading of 1.0 at 260 nm. The A260/A280 ratio was used to determine the sample purity. The RNA purity is judged satisfactory for values >1.6.w/v) bromophenol blue, 0.25% (w/v) xylene glycol, and 30% (v/v) glycerol). The RNA was separated on the gels at 100 V for 1 h. The fluorogram was documented photographically using a gel documentation system . The following reagents /buffers were used: 0.8% agarose, 1 mM EDTA, 40 mM Tris acetate, 0.25% bromophenol blue, 0.25% xylene glycol, and 30% glycerol. The 0.8% agarose was melted in 1× TAE buffer  diluted with DEPC-water). Then, 0.5 µg/mL ethidium bromide (EtBr) was added when the mixture cooled to around 60 °C. The gel medium was placed onto horizontal gel trays with combs to generate vertical gel loading pockets for solidification. The polymerized gels were placed into an RNase-free gel tank with 1× TAE buffer. Next, 2 μL of RNA was placed into the wells using 6× loading buffer . The kit’s components were thawed, combined, and briefly centrifuged before being stored on ice. The following reagents were introduced in order to a sterile, nuclease-free tube on ice: 5 g of total RNA, 1 μL of oligo (dT) primer, and 12 μL of nuclease-free water. The reagents were gently mixed, centrifuged at 2500 rpm, and incubated at 65 °C for 5 min. The tube was chilled on ice before being spun down and then returned to the ice. Next, 4 µL of 5× reaction buffer, 1 µL of RNase inhibitor, 2 µL of 10 mM dNTP Mix, and 1 µL of M-MuLV Reverse Transcriptase, made up to a total volume of 20 µL were mixed gently and centrifuged at 2500 rpm for 5 min. The samples were incubated for 60 min at 42 °C for gene-specific primed cDNA synthesis. The process was stopped after 5 min of heating at 70 °C.R. solanacearum and rhizobacteria. The expression levels of Lox2, UBI-3, POD, SOD, WRKYa, PAL1, DEF1, CAT2, HSFC1, WRKY40, PAL, NPRI, and UBI-3 genes were determined at 48 h post inoculation using real-time PCR, by establishing a gradient concentration using 40 nM of primers and 10–100 ng of cDNA. The PCR conditions involved initial activation at 95 °C for 5 min, denaturation at 95 °C for 30 s, and combined annealing/extension at 60 °C for 30 s. The PCR product was melted at 72 °C for 1 min and then ramped from 72 to 98 °C by 1 °C every 5 s.Primer pairs specific to chili defensive genes were designed and used. Primers for chili ubiquitin E3 gene (NCBI accession number XM_016720450.1) cDNA  were used as an internal control. All RT-PCR experiments were conducted in three replicates. Quantitative RT-PCR analysis was conducted to profile the expression of selected defensive genes. The expression of chili genes representing the upstream and downstream pathogenesis response system was profiled by qRT-PCR after infection of chili seedlings with The following formula was used to compute the relative gene expression in terms of fold change: fold change = 2 − ΔΔCt where, ΔCt = Ct, target – Ct, normalizer, and ΔΔCt = ΔCt, stimulated − ΔCt, control.All experiments were performed in a completely randomized design with three replicates. Prior to statistical analysis, all bacterial counts were logarithmically converted. One- and two-way analyses of variance (ANOVAs) using SAS (SAS Institute 1998) were applied to evaluate significant differences between treatments at a significance level of 0.05. Tukey’s post hoc multiple comparison test  was usedR. solonacearum in our previous study [R. solonacearum cells, we examined the pathogen growth under the influence of rhizobacteria producing volatile compounds. It was found after 7 days of observation that the maximum inhibition was achieved by the PDS1 isolate (72.24%), followed by KA9 (68.38%), BDS1 (66.04%), UK2 (64.35%), and UK4 (61.48%)  . Electro strains .Pseudomonas fluorescens PDS1 and Bacillus subtilis KA9 was conducted using the GC–MS method. A total of 63 compounds were identified from the GC−MS analysis. The predominant compounds produced by Bacillus subtilis strain KA9 in terms of peak area were 2-ethyl-3,6-dimethyl pyrazine (22.59%), 3-hydroxy-2-butanone (acetoin) (13.68%), benzaldehyde (0.32%), cycloheptasiloxane (0.73%), 2-methyl pyrazine (15.95%), diethyl phthalate (7.75%), cyclohexanone (0.22%), 3-isobutyl hexahydropyrrolo (3.96%), 4-hydroxy-5-methyl-2-hexanone (0.43%), 2,4-di-tert-butylphenol (0.47%), hexadecane (0.98%), hexahydropyrrolo,  pyrazine-1,4-dione (4.78%), isoamyl alcohol (10.07%), and disulfide dimethyl (2.87%). The predominant compounds produced by Pseudomonas fluorescens strain PDS-1 were 2-ethyl-3,6-dimethyl pyrazine (28.07%), 3-hydroxy-2-butanone (acetoin) (10.02%), benzaldehyde (0.43%), 2-methyl pyrazine (24.40%), 1-undecene (7.10%), diethyl phthalate (7.10%), cyclohexanone (0.20%), 3-isobutyl hexahydropyrrolo (0.91%), 4-hydroxy-5-methyl-2-hexanone (0.87%), 2,4-di-tert-butylphenol (0.46%), hexadecane (0.23%), hexahydropyrrolo,  pyrazine-1,4-dione (3.23%), isoamyl alcohol (20.09%), and disulfide dimethyl (6.34%). Most compounds were benzene derivatives, with pyrazine-1,4-dione, 3-hydroxy-2-butanone (acetoin), 2-ethyl 3, 6-dimethyl pyrazine, 2-methyl pyrazine, 2-ethyl-3,6-dimethyl pyrazine, and diethylphthalate having peak areas between 2.6% and 28.07% , the chemicals potentially produced by rhizobacteria treatments were identified. The relative content of each compound is shown as a mean (n = 3). The peak area of each compound was calculated as a percentage relative to the total peak area of all volatile organic compounds in a particular treatment. Several minor peaks were not included.Leaves−1·mg−1 protein) followed by T5 (0.080 ± 0.002 ΔOD·min−1·mg−1 protein) at 48 hapi in leaf tissue. At 24 h after pathogen inoculation, the maximum PO activity was observed in T6 (0.056 ± 0.003 ΔOD·min−1·mg−1 protein) followed by T5 (0.049 ± 0.002 ΔOD·min−1·mg−1 protein) and T4 (0.046 ± 0.003 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated plant (0.029 ± 0.002 ΔOD·min−1·mg−1 protein). At 72 hapi, the maximum PO activity was observed in T6 (0.064 ± 0.002 ΔOD·min−1·mg−1 protein) followed by T5 (0.058 ± 0.002 ΔOD·min−1·mg−1 protein) and T3 (0.061 ± 0.002 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated plant (0.043 ± 0.002 ΔOD·min−1·mg−1 protein) (Peroxidase activity was measured as the change in OD per minute per milligram of protein. The maximum PO activity was observed in treatment T6 (KA9 + PDS1 + RS) (0.090 ± 0.002 ΔOD·minprotein) b.R. solanacearum-treated plants had considerably higher PO activity at 48 h, i.e., 0.059 ± 0.002, than the untreated plants. However, P. fluorescens PDS1 produced volatile compounds that induced higher PO activity than B. subtilis KA9.The PO activity was increased in all treatments until 48 hapi, after which it declined significantly . Plants Roots−1·mg−1 protein). PO activity in roots expressed similar trends to those seen in leaves, with the maximum activity observed in T6 (0.0092 ± 0.002 ΔOD·min−1·mg−1 protein) followed by T5 (0.0082 ± 0.002 ΔOD·min−1·mg−1 protein). At 24 h after pathogen inoculation, the maximum PO activity was observed in T6 (0.0073 ± 0.021 ΔOD·min−1·mg−1 protein) followed by T4 (0.0060 ± 0.027 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated control (0.0052 ± 0.027 ΔOD·min−1·mg−1 protein). At 48 hapi, the maximum PO activity was observed in T6 (0.0092 ± 0.020 ΔOD·min−1·mg−1 protein) followed by T5 (0.0082 ± 0.020 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated control (0.0077 ± 0.020 ΔOD·min−1·mg−1 protein). At 72 hapi, the maximum PO activity was observed in T3 (0.0050 ± 0.031 ΔOD·min−1·mg−1 protein) followed by T4 (0.0043 ± 0.039 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated control (0.0036 ± 0.021 ΔOD·min−1·mg−1 protein) (PO activity is expressed as the unit change in absorbance per minute per milligram of protein (ΔOD·minprotein) b.R. solanacearum only showed an increase in PO activity after 48 h, i.e., 0.0077 ± 0.020 ΔOD·min−1·mg−1 protein, which was substantially higher than in untreated plants. PO activity was more significant in P. fluorescens PDS1 than in B. subtilis KA9. According to the principal component analysis in glasshouse conditions, the effects of volatile compounds produced by biocontrol agents on the induction of peroxidase (PO) activity in chili leaves and roots against Ralstonia solanacearum were greatest for treatment T6 at 48 h . However, the induction of this defense enzyme was slightly higher in leaves (1.07-fold) than in roots . The maximum PPO activity at 24 hapi was observed in T6 (0.055 ± 0.001 ΔOD·min−1·mg−1 protein) followed by T5 (0.047 ± 0.001ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated plant (0.027 ± 0.001 ΔOD·min−1·mg−1 protein). At 48 hapi, the maximum PPO activity was observed in T6 (0.082 ± 0.001 ΔOD·min−1·mg−1 protein) followed by T5 (0.074 ± 0.001 ΔOD·min−1·mg−1 protein). At 72 hapi, the maximum PPO activity was observed in T6 (0.064 ± 0.003 ΔOD·min−1·mg−1 protein) and T4 (0.060 ± 0.003 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated plant (0.049 ± 0.002 ΔOD·min−1·mg−1 protein) (The polyphenol oxidase activity was measured as the change in absorbance per minute per milligram of protein (ΔOD·minprotein) b.R. solanacearum-treated plants showed a substantial increase in PPO activity at 72 hapi, i.e., 0.049 ± 0.002, compared to untreated plants. PPO activity was more increased by volatile molecules generated by P. fluorescens PDS1 than by B. subtilis KA9.PPO activity increased in all treatments until 48 h after pathogen inoculation, before decreasing significantly, except for the pathogenic treatment T7 b. PlantsRoots−1·mg−1 protein). At 72 h after pathogen inoculation, treatment T6 (0.0075 ± 0.034 ΔOD·min−1·mg−1 protein) had the highest PPO activity in root tissues, followed by T3 (0.0064 ± 0.033 ΔOD·min−1·mg−1 protein). The maximum PPO activity was observed in T6, where volatile compounds produced by two rhizobacteria were inoculated (0.0067 ± 0.030 ΔOD·min−1·mg−1 protein) followed by T5 (0.0058 ± 0.032 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated plant (0.0035 ± 0.026 ΔOD·min−1·mg−1 protein) at 24 hapi. The maximum PPO activity was observed in T6 (0.0043 ± 0.028 ΔOD·min−1·mg−1 protein) followed by T5 (0.0038 ± 0.031 ΔOD·min−1·mg−1 protein) and T4 (0.0038 ± 0.027 ΔOD·min−1·mg−1 protein)  enzyme activities in roots and leaves of chilli against R. solanacearum with 0.0050 and 0.052 respectively. This defense related enzyme was activated 10.40-fold more in the leaves than the roots (Polyphenol oxidase activity was measured as the change in absorbance per minute per milligram of protein (ΔOD·minprotein) b. The PPprotein) b. The PPprotein) . Furtherhe roots a.Leaves−1·mg−1 protein) followed by T5 (0.062 ± 0.003 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated plant (0.051 ± 0.002 ΔOD·min−1·mg−1 protein). At 48 hapi, the maximum SOD activity was observed in T5 (0.082 ± 0.004 ΔOD·min−1·mg−1 protein) followed by T4 (0.072 ± 0.002 ΔOD·min−1·mg−1 protein). At 72 hapi, the maximum SOD activity was observed in T6 (0.063 ± 0.003 ΔOD·min−1·mg−1 protein) followed by T3 (0.062 ± 0.003 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated control (0.056 ± 0.004 ΔOD·min−1·mg−1 protein)  b.R. solanacearum showed an increase in SOD activity after 48 h, 0.063 ± 0.003, which was substantially higher than in untreated plants. The volatile compounds generated by B. subtilis KA9 treatment increased SOD activity more than those generated by P. fluorescens PDS1.SOD activity was enhanced in all treatments until 48 h after pathogen inoculation, except T6, before considerably decreasing b. SOD acRoots−1·mg−1 protein) followed by T5 (0.0085 ± 0.025 ΔOD·min−1·mg−1 protein) in the roots of chili plant. The maximum SOD activity at 48 hapi was observed in T5 (0.0075 ± 0.029 ΔOD·min−1·mg−1 protein) followed by T6 (0.0072 ± 0.031 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated control (0.0056 ± 0.032 ΔOD·min−1·mg−1 protein). At 72 hapi, the maximum SOD activity was observed in T4 (0.0068 ± 0.030 ΔOD·min−1·mg−1 protein) followed by T2 (0.0064 ± 0.031 ΔOD·min−1·mg−1 protein) as compared to the pathogen-treated plant (0.0056 ± 0.031 ΔOD·min−1·mg−1 protein) (Maximum SOD activity at 24 hapi was observed due to the volatile compounds produced by rhizobacteria in T6 (0.0091 ± 0.026 ΔOD·minprotein) b.R. solanacearum showed a substantial increase in SOD activity after 48 h, i.e., 0.0065 ± 0.026 ΔOD·min−1·mg−1 protein, compared to untreated plants. However, there was no significant difference between P. fluorescens PDS1 and B. subtilis KA9 in terms of the induction of SOD enzyme activity in the root tissue of chili plants.The SOD activity in chili roots increased until 28 h after pathogen inoculation in all treatments, before decreasing dramatically . Dual rhR. solanacearum with 0.0057 and 0.052 respectively. This defense related enzyme was activated 9.12-fold -fold more in the leaves than the roots  followed by T5 (265.00 ± 1.64 µmol t-cinnamate·min−1·g−1 FW) at 48 hapi. At 24 hapi, the maximum PAL activity was observed in T6 (88.33 ± 3.24 µmol·min−1·g−1 FW) and T5 (84.333 ± 1.27 µmol·min−1·g−1 FW) as compared to the pathogen-treated control (63.666 ± 2.25 µmol·min−1·g−1 FW). At 72 hapi, the maximum PAL activity was observed in T6 (207.000 ± 3.12 µmol·min−1·g−1 FW) followed by T5 (192.00 ± 1.64 µmol·min−1·g−1 FW) and T4 (191.933 ± 2.34 µmol·min−1·g−1 FW) as compared to the pathogen-treated plant (134.333 ± 2.21 µmol·min−1·g−1 FW) (R. solanacearum-treated plants exhibited a significantly increased in PAL activity (188.667 ± 3.24) in comparison to the untreated plants. However, the volatile produced by P. fluorescens PDS1 enhanced the induction of PAL activity in the leaves of chilli cv. PusaJwala as compared to B. subtilis KA9. Phenyl ammonia lyase activity is expressed in µmol ·g−1 FW) b. The PA·g−1 FW) a. The PA·g−1 FW) . After 4Rootst-cinnamate·min−1·g−1 FW) followed by T5 (254.67 ± 3.16 µmol t-cinnamate·min−1·g−1 FW) at 48 hapi in roots of chili cv. Pusa Jwala. At 24 hapi, the maximum PAL activity was observed in T6 (88.00 ± 2.00 µmol·min−1·g−1 FW) and T5 (80.33 ± 2.17 µmol·min−1·g−1 FW) as compared to the pathogen-treated plant (58.00 ± 2.86 µmol·min−1·g−1 FW). At 72 hapi, the maximum PAL activity was observed in T6 (199.33 ± 3.42 µmol·min−1·g−1 FW), as well as T5 (187.00 ± 2.24 µmol·min−1·g−1 FW) and T4 (185.33 ± 2.84 µmol·min−1·g−1 FW), followed by T3 (141.67 ± 2.73 µmol·min−1·g−1 FW) as compared to the pathogen-treated control (124.33 ± 2.92 µmol·min−1·g−1 FW)  b.R. solanacearum-treated plants also showed an increase in PAL activity (184.67 ± 2.65 µmol·min−1·g−1 FW), which was substantially higher than in untreated plants. Both rhizobacteria produced volatile compounds with potential for inducing PAL enzyme activation. P. fluorescens PDS1, on the other hand, triggered greater PAL activity than B. subtilis KA9. A mean value comparison on the effect of volatile compounds produced by biocontrol agents under glasshouse conditions, treatment T6 recorded the maximum PAL enzyme activities in roots and leaves of chilli against R. solanacearum with 149.80 and 145.80 respectively. This defene related enzyme was activated 1.027-fold more in the root tissues than the leaves  in the RS treatment, which was significantly greater than in the uninoculated control. A significant low population of R. solanacearum was observed in the treatments where rhizobacteria were applied with R. solanacearum. As the time period increased, there was decline in the microbial population , T3 (P. fluorescens PDS1), T4 (B. subtilis KA9 + R. solanacearum), T5 (P. fluorescens + R. solanacearum), and T7 (R. solanacearum). Samples were collected from these treatments for GC–MS analysis, which revealed the presence of 257 compounds among all samples. A total of 137 molecules were specifically present in certain treatments, whereas the remaining 120 molecules were common to multiple samples. Furthermore, 13 compounds were common to all treatments (The separation and identification of volatile compounds from the plastic bottle assembly were achieved using a Shimadzu QP 2000 fitted with an Rtx-5 ms column (30 m × 0.25 mm) packed with 95% dimethyl poly siloxane for GC–MS analysis. The mass spectra of the unknown compounds were compared with those of known compounds stored in the NIST-011 library. The active constituents, along with their retention time (RT), molecular weight, molecular formulae, and concentration (peak area%), were determined from the five different rhizobacteria treatments, namely, T2 , 2-methyl-1-butanol (14.3), 4-ethylbenzoic acid, 2-bromo-4-fluorophenyl (17.49), anisole (12), benzaldehyde, 3,5-dimethyl- (13.51), benzothiazole (15.00), butylthiophenol (11.9), methyl butanal (8.2), octanol (12.5), and oxime-, methoxy-phenyl- (8.195) were identified , the chemicals likely produced by various rhizobacterial treatments of entified .R. Solanacearum revealed benzene derivatives, different hydrocarbons in the form of alkanes and esters, and carboxylic acids. Most compounds were aromatic in nature, emphasizing the basic metabolic process of R. solanacearum (The gas chromatography–mass spectrometry (GC–MS) profile of common volatile organic compounds (VOCs) produced by the treatment (T7) with nacearum c.Bacillus subtilis and Pseudomonas fluorescens) from the bottom of the apparatus designed to produce volatile compounds that can induce ISR through the root tissues of plants against pathogenic microbe R. solanacearum , peroxidase (POD), phenyl ammonia lyase (PAL), superoxide dismutase (SOD), catalase (CAT), and defensin gene (DEF) is known to directly reflect ISR at both the transcript and the protein levels. Other genes such as WRKY  and HSF (HSFC) are transcription factors that act as activators or repressors of plant immunity. These genes are also implicated in pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity by influencing gene expression. Furthermore, NPR1 (a nonexpressor of pathogenesis-related genes) contributes to overall plant health when the immune response of the plant is increased. Its expression does not change dramatically upon pathogen infection; hence, its post-translational modifications are essential. It acts as a transcription cofactor, whose expression changes in leaves because of root-associated bacteria.R. solanacearum), KA9 + RS (B. subtilis KA9 + R. solanacearum), PDS1 + RS (P. fluorescens PDS1 + R. solanacearum), KA9 + PDS1 + RS (B. subtilis KA9 + P. fluorescens PDS1 + R. solanacearum), BABA , and mock inoculated treatments) on susceptible chili cv. Pusa Jwala is represented graphically. In the susceptible cv. Pusa Jwala, the mock treatment was used for calibration, revealing that the BABA treatment at 1000 µg/mL resulted in the highest relative expression of PAL (4.48-fold) at 48 h, followed by KA9 + PDS1 + RS (3.75-fold), KA9 + RS (3.25-fold), PDS1 + RS (3.09-fold), and RS (2.54-fold). These was no significant variation observed in relative expression in response to PDS1 + RS and KA9 + RS treatments. Moreover, the dual rhizobacterial inoculation treatment, i.e., KA9 + PDS1 + RS, led to a significant variation compared to other treatments. Upregulation of defensive gene PAL was found in all treatments as compared to UBI3 , followed by KA9 + PDS1 + RS (3.25-fold), KA9 + RS (2.75-fold), PDS1 + RS (2.59-fold), and RS (2.04-fold) as compared to UBI3. A significant variation was observed in the dual KA9 + PDS1 + RS treatment compared to the mock inoculated control, as well as individual rhizobacterial treatments. Upregulation of defensive gene PAL in leaves was found in all treatments as compared to UBI3 . The relIn the susceptible cv. Pusa Jwala, the mock treatment was used to calibrate the expression of defense-related genes. The highest relative expression of POD gene was recorded at 48 h after treatment with BABA (3.5-fold), followed by KA9 + PDS1 + RS (3.22-fold), PDS1 + RS (2.6-fold), KA9 + RS (2.29-fold), and RS (2.26-fold). The relative expression observed in response to RS and KA9 + RS treatments was not significantly different during the study. However, the dual rhizobacterial treatment KA9 + PDS1 + RS was found to vary significantly from individual rhizobacterial inoculated treatments. Upregulation of defensive gene POD was found in all treatments as compared to UBI3 in the plant roots a,b Supp. In leavThe highest relative expression of SOD gene was recorded at 48 h in roots of cv. Pusa Jwala after treatment with BABA (3.68-fold), followed by KA9 + PDS1 + RS (3.57-fold), KA9 + RS (2.46-fold), PDS1 + RS (2.12-fold), and RS (1.03-fold). The relative expression observed in response to PDS1 + RS and KA9 + RS treatments was not significantly different; however, there was a significant variation between dual rhizobacterial treatment (KA9 + PDS1 + RS) and individual rhizobacterial treatments after 48 h. Upregulation of defensive gene SOD was found in all treatments compared to UBI3. In leaves of cv. Pusa Jwala, the highest relative expression of SOD was recorded at 48 h after treatment with BABA (3.60-fold), followed by KA9 + PDS1 + RS (2.70-fold), PDS1 + RS (1.62-fold), KA9 + RS (1.00-fold), and RS (2.03-fold). The relative expression observed in the dual rhizobacterial inoculation treatment (KA9 + PDS1 + RS) was significantly different from that in individual rhizobacterial treatments. The relative expression observed in response to control and KA9 + RS treatments was not significantly different after 48 h inoculation of rhizobacteria. Upregulation of defensive gene SOD in leaves was found in all treatments compared to UBI3 a,b Supp. The relThe highest relative expression of WRKYa gene in roots was recorded at 48 h after treatment with BABA (2.66-fold), followed by KA9 + PDS1 + RS (1.43-fold), KA9 + RS (1.36-fold), PDS1 + RS (0.82-fold), and RS (0.64-fold). No significant variation in the relative expression of WRKYa gene was recorded in roots of cv. Pusa Jwala between dual rhizobacterial treatment (KA9 + PDS1 + RS) and individual rhizobacterial treatments. Upregulation of defensive gene WRKYa was found in all treatments compared to UBI3. In leaves, the highest relative expression of WRKYa was recorded at 48 h after treatment with BABA (1.90-fold), followed by KA9 + PDS1 + RS (1.20-fold), PDS1 + RS (0.82-fold), KA9 + RS (0.80-fold), and RS (0.32-fold). A significant variation in relative expression between dual rhizobacterial treatment (KA9 + PDS1 + RS) and individual rhizobacterial treatments was recorded in leaf samples after 48 h of treatment. However, the relative expression observed in response to KA9 + RS and PDS 1 + RS treatments was not significantly different. Upregulation of defensive gene WRKYa in leaves was found in all treatments compared to UBI3 a,b Supp.The highest relative expression of PAL1 gene was recorded in the roots of cv. Pusa Jwala at 48 h after treatment with BABA (5.04-fold), followed by PDS1 + RS (1.58-fold), RS (1.51-fold), KA9 + PDS1 + RS (1.17-fold), and KA9 + RS (0.99-fold). The relative expression observed after PDS1 + RS treatment was significantly different from the control and other treatments. Upregulation of defensive gene PAL1 was found in all treatments compared to UBI3. In leaves, a similar pattern of PAL1 gene expression was recorded. The highest relative expression of PAL1 was recorded at 48 h after treatment with BABA (2.00-fold), followed by PDS1 + RS (1.90-fold), KA9 + PDS1 + RS (1.57-fold), KA9 + RS (1.50-fold), and RS (0.86-fold). The relative expression observed after the PDS1 + RS treatment differed significantly from the mock control and other treatments. Upregulation of defensive gene PAL1 was found in all treatments except for the uninoculated mock, which presented a downregulation compared to UBI3. Rhizobacteria induced PAL1 gene expression more in leaf tissue than in root tissue, whereas chemical BABA treatment induced PAL1 gene expression more in root tissue than in leaf tissue a,b Supp.The highest relative expression of DEF1 gene was recorded at 48 h after treatment with BABA (4.03-fold), followed by KA9 + PDS1 + RS (2.11-fold), KA9 + RS (1.11-fold), PDS1 + RS (0.48-fold), and RS (1.62-fold). The combined KA9 + PDS1 + RS treatment showed a significant variation in relative expression compared to other rhizobacterial treatments. Upregulation of defensive gene DEF1 was found in all treatments compared to UBI3. In leaves, the highest relative expression of DEF1 was recorded in cv. Pusa Jwala at 48 h after treatment with BABA (3.00-fold), followed by KA9 + PDS1 + RS (1.61-fold), KA9 + RS (1.70-fold), PDS1 + RS (0.90-fold), and RS (1.20-fold). The dual rhizobacterial treatment (KA9 + PDS1 + RS) differed significantly in terms of the relative expression of DEF1 from individual rhizobacterial treatments. Upregulation of defensive gene DEF1 in leaves was recorded in all cases, except for the uninoculated mock which exhibited a downregulation compared to UBI3. It was also noted that, after RS, KA9 + PDS1 + RS, and BABA treatments, DEF1 gene was expressed more in root tissue than in leaf tissue, whereas the opposite was found for KA9 + RS and PDS1 + RS treatments a,b Supp.The highest relative expression of CAT2 gene was recorded at 48 h in root tissue of cv. Pusa Jwala after treatment with BABA (3.75-fold), followed by KA9 + PDS1 + RS (0.91-fold) and KA9 + RS (2.6-fold). There was no significant variation between PDS1 + RS (−0.34-fold) and RS (−0.34-fold) treatments; however, both cases exhibited downregulation of CAT2. The relative expression observed in the dual rhizobacterial inoculation (KA9 + PDS1 + RS) differed significantly from individual rhizobacterial treatments. Upregulation of defensive gene CAT2 was found after treatment with BABA, PDS1 + KA9 + RS, and KA9 + RS, whereas downregulation was observed for PDS1 + RS and RS, compared to UBI3. In leaves, the highest relative expression of CAT2 gene was recorded at 48 h after treatment with BABA (2.70-fold), followed by KA9 + PDS1 + RS (1.91-fold), PDS1 + RS (1.34-fold), KA9 + RS (1.20-fold), and RS (0.60-fold). The relative expression of CAT2 gene observed after dual rhizobacterial treatment with KA9 + PDS1 + RS differed significantly from individual rhizobacterial treatments. Upregulation of defensive gene CAT2 was recorded in leaves, except for the uninoculated mock which exhibited downregulation compared to UBI3. KA9 + RS, KA9 + PDS1 + RS, and BABA treatments induced more CAT2 activity in the roots of cv. Pusa Jwala, whereas the opposite was found for RS and PDS1 + RS treatments a,b Supp.R. solanacearum, which exhibited downregulation compared to UBI3. The WRKY40 gene was relatively expressed more in leaf tissue as compared to root tissue after all treatments , followed by KA9 + PDS1 + RS (0.91-fold), and PDS1 + RS (0.24-fold). The relative expression observed in the dual rhizobacterial inoculation (KA9 + PDS1 + RS) differed significantly from individual rhizobacterial treatments. Upregulation of defensive gene WRKY40 was observed after BABA, PDS1 + KA9 + RS, and PDS1 + RS treatment, whereas downregulation of WRKY40 was observed after KA9 + RS and RS treatment, compared to UBI3. BABA treatment differed significantly from rhizobacterial treatments. In leaves, the highest relative expression of WRKY40 was recorded at 48 h after treatment with BABA (1.50-fold), followed by PDS1 + RS (1.40-fold), KA9 + RS (1.30-fold), KA9 + PDS1 + RS (0.90-fold), and RS (−0.65-fold). The relative expression observed after PDS1 + RS (1.40-fold) and KA9 + RS (1.30-fold) treatments was not significantly different, unlike individual rhizobacterial treatments compared to the dual inoculation (KA9 + PDS1 + RS). Upregulation of defensive gene WRKY40 was recorded in leaves, except for the pathogenic treatment of eatments a,b , followed by KA9 + PDS1 + RS (0.90-fold) and BABA (0.87-fold), KA9 + RS (0.24-fold), and PDS1 + RS (−0.25-fold). Plants treated with RS only showed a significantly higher relative expression of HFSC1 gene in root tissues than in rhizobacterial treatments. Upregulation of defensive gene HFSC1 was found in treatments BABA, PDS1 + KA9 + RS, and KA9 + RS, whereas downregulation was observed in treatment PDS1 + RS. In leaves, the highest relative expression of HFSC1 was recorded after treatment with KA9 + PDS1 + RS (1.5-fold), followed by KA9 + RS (0.90-fold) and RS (0.90-fold), BABA (0.87-fold), and PDS1 + RS (0.80-fold). The dual culture of rhizobacteria KA9 + PDS1 + RS exhibited a higher relative expression of HFSC1 gene than single rhizobacterial treatments. Upregulation of defensive gene HFSC1 in leaf tissue was observed in all samples compared to UBI3 a,b Supp. With thThe highest relative expression of LOX2 gene was recorded in root tissue of cv. Pusa Jwala at 48 h after treatment with KA9 + PDS1 + RS (0.84-fold), followed by PDS1 + RS (0.04-fold), CC (−0.63-fold), KA9 + RS (−0.93-fold), and RS (−0.98-fold). There was no significant difference in expression of LOX2 between uninoculated mock and PDS + RS treatments. The relative expression observed in the dual microbial inoculation was found significantly higher compared to the mock control and other treatments. Upregulation of defensive gene LOX2 was found after PDS1 + KA9 + RS and PDS1 + RS treatments, whereas downregulation was observed after KA9 + RS, RS, and BABA treatments, compared to UBI3. In leaves, the highest relative expression of LOX2 was recorded after treatment with RS (1.98-fold), followed by KA9 + PDS1 + RS (1.84-fold) KA9 + RS (1.07-fold), and PDS1 + RS (1.04). The lowest relative expression of LOX2 gene was recorded after treatment with BABA. Dual rhizobacterial treatment exhibited a significantly higher relative expression of LOX2 gene compared to single rhizobacterial treatments. Upregulation of defensive gene LOX2 in leaves was observed in all samples except for the chemical control, compared to UBI3. Except for the chemical treatment, all treatments induced greater LOX1 gene expression in leaf tissue than in root tissue after 48 h of inoculation in glasshouse conditions a,b Supp.The highest relative expression of NPR1 gene was recorded at 48 h in the tissue of cv. Pusa Jwala after treatment with BABA (3.13-fold), followed by KA9 + PDS1 + RS (0.96-fold), KA9 + RS (0.94-fold), PDS1 + RS (0.82-fold), and RS (0.52-fold). The relative expressional of NPR1 gene in plants treated with KA9 + PDS1 + RS and KA9 + RS did not differ significantly, whereas treatment with the chemical control BABA differed significantly from the mock control and other treatments. Upregulation of defensive gene NPR1 was found in all treatments compared to UBI3. In leaves, the highest relative expression of NPR1 gene was recorded at 48 h after treatment with BABA (2.15-fold), followed by KA9 + RS (0.67-fold), PDS1 + RS (−0.22-fold), KA9 + PDS1 + RS (−0.74-fold), and RS (−0.93-fold). The relative expression of NPR1 gene observed in BABA-treated plants significantly differed from other treatments. Upregulation of defensive gene NPR1 in leaves was found in BABA and KA9 + RS treatments, whereas downregulation occurred in KA9 + PDS1+ RS (−0.22-fold) and PDS1 + RS (−0.22-fold) treatments, compared to UBI3. The root tissue of cv. Pusa Jwala showed a greater relative expression of NPR2 gene at 48 h than the leaf tissue after all treatments a,b Supp.B. subtilis KA9 and P. fluorescens PDS1 treatments. In contrast, NPR1 does not show an increase after treatment with individual rhizobacteria, but showed a 1.5 log fold increase after all combined treatments except for Pseudomonas and Ralstonia . It was observed that most genes showed an enhancement of their transcription levels ranging from 0.5–4.0 log fold change. Maximum expression modulation was observed for PAL gene, i.e., ~4.0 log fold increase in both root and leaf tissues. POD, SOD, and CAT2 showed a 3.0 log fold increase after alstonia .B. subtilis KA9 and P. fluorescens PDS1 were selected on the basis of their antagonistic and plant-growth promoting abilities under in vitro conditions. In glasshouse conditions at the National Phytotron Facility, IARI, New Delhi, these rhizobacteria isolates (KA9 and PDS1) were investigated for their bio-efficacy in managing bacterial wilt disease and growth promotion activities in chili cv. Pusa Jwala (susceptible cv.). Individual and dual inoculation of isolates PDS1 and KA9 significantly reduced the bacterial wilt incidence in chili and enhanced the growth of the plants. Minimum disease intensity (21.80%) was recorded in BABA-treated plants followed by treatment with KA9 + PDS1 + RS (22.40%), KA9 + RS (26.50), and PDS1 + RS (26.80) after 40 days of pathogen inoculation.In the present study, two rhizobacteria R. solanacearum-infected plants after 16 days of inoculation, whereas in rhizobacteria-treated plants, appearance of wilt disease was delayed by 10–14 days. Moreover, a significant variation in reduction in wilt disease in chili was recorded among these isolates in glasshouse conditions (p > 0.05) (Maximum biocontrol efficacy (72.05%) was recorded in BABA-treated plants followed by treatment with KA9 + PDS1 + RS (71.28%), KA9 + RS (66.02%), and PDS1 + RS (65.64%). The bacterial wilt disease symptoms were initiated only in  > 0.05) . After 4 > 0.05) a.KA9 + PDS1 + RS treatment resulted in the highest shoot dry weight of 1.23 g, followed by KA-9 + RS (1.18 g), PDS1 + RS (1.14 g), BABA (1.14 g), mock (0.98 g), and RS (0.68 g). The growth promotion efficiency (GPE) of shoots on a dry weight basis was calculated in comparison to the RS treatment, revealing the maximum value for KA9 + PDS1 + RS (80.87%) followed by KA-9 + RS (73.35%), BABA (69.11%), PDS1 + RS (67.64%), and mock (44.11%) . The maxR. solanacearum were investigated in this study. The VOCs produced by these isolates significantly inhibited the growth of R. solanacearum under in vitro conditions. The highest inhibition was recorded by strain PDS-1 (P. fluorescens) (72.24%) compared to the control. The results were concordant with the findings of Raza et al. [P. fluorescens WR-1 restricted the growth and virulence traits of R. solanacearum. The effect of VOCs produced by different biocontrol strains on R. solanacearum has not been recorded; nevertheless, VOCs produced by the fungal pathogen Aspergillus flavus resulted in a fourfold decrease in R. solanacearum growth [P. fluorescens strains produced antimicrobial VOCs and suppressed the growth of the phytopathogen Botrytis cinerea [P. fluorescens B-4117 produced VOCs that inhibited the growth of Agrobacterium tumefaciens and Agrobacterium vitis [Bacillus genera showed significant inhibition activity against R. solanacearum, confirming the findings of Tahir et al. [Bacillus volatile compounds against R. solanacearum in tobacco plant. Similar findings were also reported by Raza et al. [R. solanacearum to volatile organic compounds produced by biocontrol strain B. amyloliquefaciens SQR-9. The inhibitory activity of biocontrol agents in our study was due to presence of 2-ethyl-3,6-dimethyl pyrazine, 3-hydroxy-2-butanone (acetoin), benzothiazole, cycloheptasiloxane, and 2-methyl pyrazine compounds, in line with the results of Goa et al. [B. subtilis CF-3.The effects of volatile compounds (VOCs) produced by five rhizobacterial strains, PDS-1, UK-2, UK-4, BDS-1, and KA-9, on the growth and virulence features of the chili wilt pathogen a et al. , who repm growth . Other i cinerea . P. fluoum vitis . In thisr et al. , who repa et al. , who shoa et al. , who ideB. subtilis KA9 and P. fluorescens PDS1 trigger a cascade of defense enzymes against R. solanacearum. To counter soil-borne diseases, rhizobacteria can induce systemic resistance in a range of plants, according to several studies. Plant growth-promoting bacteria (PGPB) use ISR to prime the entire plant body for disease resistance. Activation of latent defensive responses occurs not just at the induction site, but also systemically across plant tissues [Bacillus species that induce systemic resistance [This repeatedly showed that biocontrol agents such as  tissues . Plant dsistance . PPO andsistance .B. subtilis KA9 and P. fluorescens PDS1 against R. solanacearum resulted in higher polyphenol oxidase, peroxidase, superoxide dismutase, and phenyl ammonia lyase activity, which increased up to 48 hapi, before gradually decreasing in all treatments. There was no increase in enzyme activity in the untreated control group. This result is supported by Jayapala et al. [Bacillus sp. boosted defense-related enzymes such as PAL, peroxidase, and polyphenol oxidase at 48 hapi against Colletotrichum capsici. Chunyu et al. [B. amyloliquefaciens SQRT3 and R. solanacearum boosted PO and PPO activities; their findings were similar to the results of the current investigation. Pretreating tomato seedlings with P. fluorescens conferred systemic resistance against Fusarium oxysporum f.sp. lycopersici, according to Ramamoorthy et al. [R. solanacearum by increasing peroxidase and phenyl ammonia lyase activities. According to Ho et al. [R. solanacearum. The findings of this study are backed up by observations of tomato plants treated with B. subtilis against the pathogen Erwinia carotovora subsp. carotovora, which boosted the activities of phenyl ammonia lyase and superoxide dismutase in seedlings [2O2 to be deposited, which is required for plant disease resistance [P. fluorescens enhanced the defense related enzyme activities like PAL, PO, Lipoxygenase PPO in tomato seedlings post inoculated with R. solanacearum.According to the results of this study, in chili cv. Pusa Jwala, the combined treatment of a et al. , who fouu et al.  found thy et al. . Accordiy et al. , bioprimo et al. , the jaseedlings . Increassistance . The majsistance . Our finsistance , who fouB. subtilis KA9 and P. fluorescens PDS-1), a plant pathogenic bacterium (R. solanacearum), and the host chili plant was demonstrated in this study. Beneficial and pathogenic bacteria coexist in soil, and their efficacy as a PGPR or pathogen is influenced by the rhizosphere population, soil nutrition, and environmental variables. In soil, several relationships influence biological activity. In this work, the interaction effects of volatile compounds (VOCs) produced by both PGPR strains on the elicitation of defense reactions in the host chili were investigated. A significant difference was observed in all treatments of rhizobacteria with respect to defensive enzyme elicitation  when chili seedlings were exposed to volatile compounds produced by these rhizobacterial combinations in all experimental systems. During exposure to the pathogenic bacteria R. solanacearum VOCs, no huge variations were seen. In earlier studies, the inverted plate technique was utilized to determine whether VOCs can promote plant growth or induce systemic resistance. Generally, artificial growth medium is utilized in an inverted plate setup for plant growth studies; however, VOCs can only reach the leaves due to their solid or semisolid nature, leaving the roots unharmed. Another factor to consider is that, when pathogens are inoculated to test for induced systemic resistance, the pathogen grows on the growth medium surface and contaminates the inverted plate setup. To avoid this issue, we modified the method in this study by utilizing a half-inverted bottle system, in which soil mixed with vermiculite was employed as a plant development medium. VOCs could reach seedling roots through small pores in the soil–vermiculite combination in this transparent system. PGPR colonized soil organically, and the VOCs it produced could interact with the plant roots rather than leaves in natural settings. To investigate the growth-stimulating action of rhizobacteria VOCs in soil exposed to roots, the experiment was relocated from in vitro conditions to pots in a growth chamber with an RH of 80% and a temperature of 28 °C. The findings demonstrated that rhizobacterial VOCs encouraged development under soil conditions, and the results are supported by an earlier study by Park et al. [Bacillus-treated tomato plants, which led to a decrease in wilt disease [Microorganism-produced volatiles have been found to play an essential environmental role in the induction of systemic resistance to biotic elements, plant growth promotion, and bacterial pathogen suppression through beneficial interactions between PGPRs and plants . Studiesk et al. . Volatilk et al. . Followik et al. ,49. PPO k et al. . In resp disease .R. solanacearum in chili to understand the biology of systemic resistance induction.Defense-related genes such as lipoxygenase (LOX), peroxidase (POD), phenyl ammonia lyase (PAL), superoxide dismutase (SOD), catalase (CAT), and defensin gene (DEF) were chosen to study their relative expression in the root and leaf tissue of chili induced by rhizobacteria and a chemical known to be directly associated with inducing systemic resistance through expression at both transcript and protein levels ,56,57,58R. solanacearum), KA9 + RS (B. subtilis KA9+ R. solanacearum), PDS1 + RS (P. fluorescens PDS1 + R. solanacearum), KA9 + PDS1 + RS (B. subtilis KA9 + P. fluorescens PDS1 + R. solanacearum), BABA , and mock inoculated treatments) on the Pusa Jwala cultivar of chili was studied. The BABA-treated leaf and root showed the highest relative expression of PAL gene at 48 h, which indicates that the priming of ISR maintains plant fitness when pathogens attack. In addition to chemical-induced priming of ISR, the priming effects can be elicited by rhizobacteria [B. subtilis CBR05 as a biocontrol agent for soft rot disease (Erwinia carotovora subsp. carotovora) in tomato and found that the PAL gene exhibited the most significant upregulation in the transcript profile. The general conclusion was that increases in PAL activity were caused by the increased expression of PAL gene. Previous studies [Colletotrichum acutatum, Wang et al. [B. cereus AR156 treatment stimulated the expression of three defense-related genes, resulting in higher ISR and reduced anthracnose rot.The relative expression of PAL gene in response to different treatments . Upregulation of defensive gene PAL in leaves was found in all treatments compared to UBI3. The relative expression of PAL gene was greater in root tissue than in leaf tissue after all treatments. Kolahi and coworkers  reported2O2 and works in tandem with PAL [Burkholderia anthina MYSP113 in sugarcane crops. In this study, POD expression was higher in root tissue, which agrees with the studies of Bharti et al. [P. fluorescens elicited defensive gene expression in tomato plant against Alternaria blight disease.In the oxidation of phenolic compounds and protection against pathogens, POD is a key scavenger of Hwith PAL ,78. In twith PAL , who shoi et al. , who repi et al. , wherebySl-SOD1 gene sustained high expression in all tested tomato tissues during growth and development. They also recorded a higher expression in root tissue, in line with the results of this study. Corpas et al. [Superoxide dismutase (SOD) activity in plants is due to the expression of the corresponding SOD gene. SODs are important antioxidant enzymes that safeguard organisms against reactive oxygen species (ROS) produced under stress conditions . In thiss et al.  used qRTs et al.  also repB. subtilis KA9 and P. fluorescens PDS1 was found to be significantly better than other individual rhizobacterial treatments. Upregulation of defensive gene WRKYa was found after all treatments compared to UBI3 in root and leaf tissue. Upregulation of defensive gene WRKY40 was observed following BABA, PDS1 + KA9 + RS, and PDS1 + RS treatments, whereas downregulation was observed following KA9 + RS and RS treatments. This may be because WRKY transcription factors are interconnected with plant hormones and stress response pathways [WRKY genes are involved in hormone and mechanic injury stresses. Nan et al. [Phytophthora capsici, ABA, and MeJA). Stress treatment increased the expression of several CaWRKY genes. According to Liu et al. [R. solanacearum. For transcriptional control and production of the defense gene, WRKY40 binds directly to the W4-box element of the ChiIV3 promoter region. ChiIV3 is influenced by WRKY40 gene regulation in a variety of ways. This may be the reason for the lower disease incidence in our treatments where WRKY gene expression was high.WRKY is a conserved protein family implicated in a variety of biological processes in plants ,86,87. Hpathways . Nan andpathways  also anan et al.  characten et al.  also stuu et al. , the WRK2O2 produced in the peroxisomes by photorespiration [Catalase is essential for the removal of Hpiration . As a repiration ,94. Planpiration ,96. In tpiration . Greaterpiration , whereby2O2 in response to stress, thereby playing an important role in adapting to environmental stresses. A greater incidence of wilting disease was recorded in chili plants with low CAT expression. A previous report indicated that deficiency of CAT2 reduced resistance in Arabidopsis plants [Many studies have shown that stress activates catalase expression and activity in plants ,104,105.s plants .B. subtilis KA9 and P. fluorescens PDS1 may reduce the heat stress of chili plants and improve their growth, as indicated by the highest plant growth parameter. Ali et al. [B. cereus was particularly successful in alleviating heat stress. For example, when subjected to diverse abiotic stimuli such as heat, salt, and heavy metals, Tang et al. [Chili is mainly grown by farmers in kharif season in India, with the summer temperature commonly exceeding 35 °C. Therefore, to determine the effect of heat stress on chili growth and development, we evaluated HFSC gene expression. The relative expression of HFSC1 gene was lower in roots than in leaf tissue. This may be because of the direct exposure of leaves to heat in glasshouse conditions ,108,109.i et al.  corrobori et al.  conducteg et al.  found thLOX expression patterns differed significantly between wildtype peanut and cultivated peanut infected with Aspergillus flavus. Upregulation of defensive gene LOX2 in leaves was observed in all cases except after BABA treatment, compared to UBI3. A similar finding was also observed by Mariutto et al. [P. putida BTP1. Downregulation of the LOX gene was recorded by Zhang et al. [Lipoxygenase (LOX) genes are abundant in plants and play an important role in biotic and abiotic stress resistance ,113. Theo et al.  with CaLg et al.  in vegetg et al. .Arabidopsis mutant OCP3 in SA- and JA-dependent induced defense and discovered that OCP3 plants were impaired by P. fluorescens WCS417-triggered induced systemic resistance (ISR) against both P. syrinagae DC3000 and Hyaloperonospora arabidopsidis. Thus, rhizobacteria may affect NPR1’s nucleocytosolic activity in the regulation of JA-dependent defensive responses. NPR1 facilitates the immunological transcriptome not only by activating SA-responsive genes but also by functioning as a corepressor of JA-responsive MYC2 [NPR1 (nonexpresser of PR genes) is a master regulator of salicylic acid (SA) signaling, which is important for plant immunity. NPR1 interacts with transcription factors in the nucleus to boost defense responses by inducing the expression of PR (pathogenesis-related) genes . In thisive MYC2 .Pseudomonas fluorescens PDS1 and Bacillus subtilis KA9 on chili against Ralstonia solanacearum was evaluated in a transparent closed assembly featuring a half-inverted plastic bottle setup, demonstrating the plant–microbial interactions via volatile compounds. The PDS1 and KA9 VOCs significantly increased defensive enzyme activity and overexpressed the antioxidant genes. The overall gene expression was higher in root tissue than in leaf tissue. Our findings shed light on the relationship among rhizobacteria, pathogens, and host plants, which promotes plant growth promotion, suppresses disease, induces systemic resistance, and triggers an antioxidant response in the leaves and roots of chili.Microbes can benefit agricultural systems by promoting plant growth and systemic resistance to diseases in plants without harming the environment. The plant growth-promoting efficiency of VOCs produced by


Streptococcus gallolyticus subsp. gallolyticus is an emerging opportunistic pathogen responsible for septicemia and endocarditis in the elderly. Invasive infections by S. gallolyticus subsp. gallolyticus are strongly linked to the occurrence of colorectal cancer (CRC). It was previously shown that increased secondary bile salts under CRC conditions enhance the bactericidal activity of gallocin, a bacteriocin produced by S. gallolyticus subsp. gallolyticus, enabling it to colonize the mouse colon by outcompeting resident enterococci . In a separate study, we showed that S. gallolyticus subsp. gallolyticus produces and secretes a 21-mer peptide that activates bacteriocin production . This peptide was named CSP because of its sequence similarity with competence-stimulating peptides found in other streptococci. Here, we demonstrate that CSP is a bona fide quorum sensing peptide involved in activation of gallocin gene transcription. We therefore refer to CSP as GSP . GSP displays some unique features, since its N-terminal amino acid lies three residues after the double glycine leader sequence. Here, we set out to investigate the processing and export pathway that leads to mature GSP. Heterologous expression in Lactococcus lactis of the genes encoding GSP and the BlpAB transporter is sufficient to produce the 21-mer form of GSP in the supernatant, indicating that S. gallolyticus subsp. gallolyticus BlpAB displays an atypical cleavage site. We also conducted the first comprehensive structure-activity relationship (SAR) analysis of S. gallolyticus subsp. gallolyticus GSP to identify its key structural features and found that unlike many other similar streptococci signaling peptides (such as CSPs), nearly half of the mature GSP sequence can be removed (residues 1 to 9) without significantly impacting the peptide activity. Streptococcus gallolyticus subsp. gallolyticus, previously known as Streptococcus bovis biotype I, is an emerging opportunistic human pathogen belonging to the highly diverse Streptococcus bovis/Streptococcus equinus complex (SBSEC)   –3. S. gaer (CRC) , 5. Receelopment –8. Usingolyticus . Indeed,S. gallolyticus subsp. gallolyticus strain TX20005 secretes a 21-mer peptide that induces the production of unknown bacteriocins that are active against various oral streptococci  circuitry has been found to regulate competence in 12 streptococcus species belonging to the mitis and anginosus groups (–sigX/comX-inducing peptide)  , 20.comABCDE circuitry in Streptococcus mutans regulates the production of bacteriocins called mutacins directly and competence through activation of the comRS circuitry (22–S. mutans CSP be renamed mutacin-inducing peptide (MIP)  , 26, 27.de (MIP) , 29.S. gallolyticus subsp. gallolyticus possesses both the comABCDE and comRS loci in its genome  and renaming its putative transporter associated genes, currently called comAB and comDE, as blpAB and blpHR . Since GSP is predicted to be a 24-mer peptide while the isolated GSP was found to be a 21-mer peptide starting three residues after the double glycine leader sequence of the precursor peptide, we first tested the activity of GSP 24-mer versus the GSP 21-mer on gallocin transcription using a green fluorescent protein (GFP) reporter construct described in the accompanying paper  is dispensable and can be removed without significantly affecting GSP’s ability to activate its cognate histidine kinase receptor, providing a minimal structure for the development of GSP-based QS inhibitors that could affect S. gallolyticus subsp. gallolyticus fitness during competition with the gut microflora.s genome –32. In t genome –, it is dwith CSP , 10, 33.S. gallolyticus subsp. gallolyticus strain UCN34. GSP is derived from a 45-residue precursor encoded by gsp and is predicted to be processed to a 24-mer peptide by cleavage of the amino-terminal leader sequence after a double glycine motif and then exported to the extracellular environment by the BlpAB transporter. However, GSP was previously isolated as a 21-mer peptide from S. gallolyticus subsp. gallolyticus TX20005   of both peptides . Indeed, the GSP 21-mer peptide EC50 (2.96 nM) was around 100 times lower than GSP 24-mer EC50 (287 nM), indicating that the 21-mer is significantly more active than the 24-mer (We first aimed at determining the active form of GSP in  TX20005 . We firs TX20005 . The pre TX20005 . Therefo (PgllA) . No expre 24-mer .10.1128/mBio.03189-20.1FIG S1S. gallolyticus subsp. gallolyticus GSP expected: H2+2/2 (1246.6855 Da) and H3+3/3 (831.4594 Da). Download FIG S1, PDF file, 0.3 MB.LC-MS of UCN34 supernatant after 16 h incubation. Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. gallolyticus subsp. gallolyticus UCN34. In S. mutans, the equivalent 21-mer MIP peptide encoded by comC is secreted by a specific ABC transporter composed of ComA and ComB proteins  encoding a putative ABC transporter homologous to ComAB were found in the vicinity of the GSP and gallocin genes  following homologous recombination. These bWT strains should display the WT phenotype and are isogenic to their mutant counterparts. Basic phenotypic characterization of several clones for a given mutation was carried out systematically  to rule out any major secondary mutations that might have occurred during the engineering process. We then determined gallocin activity against a highly sensitive bacterium, Streptococcus gallolyticus subsp. macedonicus, using the supernatants of the ΔblpAB mutant compared to its bWT strain. As shown in blpAB mutant supernatant but was present in the bWT strain. Three possibilities can explain these results: (i) absence of GSP in the supernatant of the ΔblpAB mutant, (ii) absence of the two structural peptides (GllA1 and GllA2) constituting the active gallocin in the supernatant of the ΔblpAB mutant, or (iii) absence of both GSP and gallocin peptides in the supernatant of the ΔblpAB mutant.We next aimed at determining how GSP was secreted and processed into a 21-mer peptide in proteins . Two genin genes . To testΔblpAB supernatant to activate gfp transcription in the UCN34 Δgsp pTCVΩPgllA-gfp reporter strain mentioned above. To do so, we resuspended the reporter strain in different supernatants. If GSP is present in the supernatant, the gallocin promoter will be activated, turning on GFP and allowing the bacteria to fluoresce. As shown in gsp strain supernatant) and became fluorescent in the presence of GSP (THY plus GSP and WT supernatant). No GFP fluorescence could be detected using the ΔblpAB strain supernatant, while a strong signal was detected in the bWT strain supernatant, demonstrating that GSP is not secreted in the ΔblpAB mutant, an observation that was verified by liquid chromatography-mass spectrometry (LC-MS) (gllA-gfp) into the ΔblpAB mutant and measuring GFP fluorescence. The gallocin promoter was inactive in the ΔblpAB pTCVΩPgllA-gfp strain, while addition of synthetic GSP in the culture medium restored full gallocin promoter activity  . These ractivity .10.1128/mBio.03189-20.2FIG S2blpAB supernatant after 16 h incubation. No S. gallolyticus subsp. gallolyticus GSP was detected. Download FIG S2, PDF file, 0.4 MB.LC-MS of UCN34 ΔCopyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the blpAB mutant in the presence of synthetic GSP. As shown in gsp mutant. However, no gallocin activity could be detected in the ΔblpAB mutant even in the presence of synthetic GSP, although the gallocin promoter was fully active under these conditions, as mentioned above.We next investigated whether the same ABC transporter could also secrete the gallocin GllA1 and GllA2 peptides, and therefore, we analyzed gallocin activity in the ΔS. mutans, an extracellular protease, SepM, is responsible for CSP/MIP maturation by cleaving the three C-terminal amino acids of ComC after its secretion. BLAST analysis revealed that S. gallolyticus subsp. gallolyticus UCN34 possesses a close SepM homolog  and H3+3/3 (831.4594 Da). Download FIG S3, PDF file, 0.4 MB.LC-MS of UCN34 ΔCopyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gsp or ΔsepM cells in sterile saline solution for 30 min and analyzed the filtrates using LC-MS. Degradation of KNK-GSP to GSP was not observed following incubation with either UCN34 ΔsepM or UCN34 Δgsp . Indeed, the supernatant of the recombinant L. lactis strain induced with anhydrotetracycline was able to activate GFP production, while the control noninduced supernatant could not , and the GSP 21-mer with washed UCN34 ΔN34 Δgsp  and S5. olyticus . We wondould not . Further10.1128/mBio.03189-20.4FIG S4S. gallolyticus subsp. gallolyticus KNK-GSP incubated with UCN34 ΔsepM cells in saline solution for 30 min. KNK-GSP (P1) expected: P1H4+4/4 (716.4046 Da) and P1H4+5/5 (573.3251 Da). S. gallolyticus subsp. gallolyticus GSP-des-D1-L3 (P3) expected: P3H3+3/3 (706.3996 Da) and P1H4+4/4 (530.0515 Da). Download FIG S4, PDF file, 0.4 MB.LC-MS of Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03189-20.5FIG S5S. gallolyticus subsp. gallolyticus KNK-GSP incubated with UCN34 Δgsp cells in saline solution for 30 min. KNK-GSP expected: P1H4+4/4 (716.4046 Da) and P1H5+5/5 (573.3251 Da). GSP-des-D1-L3 expected: P3H3+3/2 (706.3996 Da) and P3H4+/4 (530.0515 Da). Download FIG S5, PDF file, 0.4 MB.LC-MS of Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. gallolyticus subsp. gallolyticus SepM is a functional protease; therefore, we repeated the washed-cell processing assays described above, but this time we added the S. mutans 21-mer CSP to washed UCN34 Δgsp or UCN34 ΔsepM cells. The 18-mer CSP was found in filtrates treated with UCN34 Δgsp but not in filtrates treated with UCN34 ΔsepM, indicating that S. gallolyticus subsp. gallolyticus SepM is functional and capable of processing inactive S. mutans 21-mer CSP into active 18-mer CSP  expected: P2H3+3/3 (831.4594 Da), P2H4+4/4 (623.8464 Da) and P2H5+/5 (499.2786 Da). GSP-des-D1-L3 (P3) expected: P3H3+3/3 (706.3996 Da) and P3H4+4/4 (530.0515 Da). Download FIG S6, PDF file, 0.4 MB.LC-MS of Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03189-20.7FIG S7S. mutans 21-mer CSP incubated with UCN34 Δgsp cells in saline solution for 30 min. S. mutans 21-mer (P4) expected: P4H2+2/2 (1183.128 Da), P4H3+3/3 (789.0878 Da) and P4H4+4/4 (592.0676 Da). S. mutans 18-mer CSP (P5) expected: P5H2+2/2 (1034.0278 Da) and P4H3+3/3 (689.6876 Da). Download FIG S7, PDF file, 0.4 MB.LC-MS of Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03189-20.8FIG S8S. mutans 21-mer CSP incubated with UCN34 ΔsepM cells in saline solution for 30 min. S. mutans 21-mer (P4) expected: P4H2+2/2 (1183.128 Da), P4H3+3/3 (789.0878 Da), and P4H4+4/4 (592.0676 Da). Download FIG S8, PDF file, 0.4 MB.LC-MS of Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03189-20.9FIG S9S. gallolyticus subsp. gallolyticus GSP incubated with S. mutans ΔcomC cells in saline solution for 30 min. GSP (P2) expected: P2H3+3/3 (831.4594 Da). GSP-des-D1-L3 (P3) expected: P3H3+3/3 (706.3996 Da) and P3H4+4/4 (530.0515 Da). Download FIG S9, PDF file, 0.4 MB.LC-MS of Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Our second aim in this study was to evaluate and determine the key structural motifs that drive GSP binding to its cognate histidine kinase, BlpH, thus activating gallocin gene transcription.We first set out to perform a full alanine scan of the 21-mer GSP signal. Peptides were synthesized using standard solid-phase peptide synthesis (SPPS) conditions using a CEM Liberty1 microwave synthesizer on Cl-MPA Protide resin (LL). The peptides were then purified to homogeneity (>95%) using reverse-phase high-performance LC (RP-HPLC) and validated using mass spectrometry . The alanine scan revealed several residues that are important for receptor binding, as mutating them to alanine resulted in significant reduction in activity, yet no one residue was found to be critical for receptor activation, leading to the identification of a competitive inhibitor . SpecifiS. gallolyticus subsp. gallolyticus GSP signal from both ends. We also wanted to identify the residues in S. gallolyticus subsp. gallolyticus KNK-GSP that led to an ∼100-fold reduction in potency compared to GSP; therefore, we included in our analysis the sequential truncation of KNK-GSP to GSP. Starting with KNK-GSP, removal of a single residue (K) from the N terminus was sufficient to increase the potency of the peptide by 20-fold, resulting in an analog, NK-GSP, that was only ∼5-fold less potent than GSP   . RemovalS. gallolyticus subsp. gallolyticus is an emerging human pathogen that utilizes quorum sensing (QS) to regulate the production of gallocin, a class IIb bacteriocin, to gain a competitive advantage over other commensals and colonize the colon under tumoral conditions .S. mutans  similarly to the S. pneumoniae and S. mitis CSPs; however, in these two signaling molecules, the negatively charged N-terminal residue (glutamate) is responsible for receptor activation, and its modification leads to the formation of analogs capable of binding the receptor but not activating it (competitive inhibitors) , since activity was reduced but not abolished in all the variants tested. This activity profile matches that of the CSP/MIP signal in . mutans  and contognition –37. Furtgnition –, 37. Theibitors) –37.Interestingly, it appears that removal of residues is more tolerated than replacement of the side chains of these residues with alanine, suggesting that although the N terminus does not play a significant role in receptor binding, modifications of this region may lead to conformational changes that result in steric clashes with the BlpH receptor and thus significantly lowered potency. Most striking, though, is the fact that nearly half of the GSP sequence (residues 1 to 9) can be removed without significantly affecting the peptide activity (9-fold reduction in potency compared to GSP). As the C-terminal region of GSP is more hydrophilic, whereas the N-terminal region is more hydrophobic, it is tempting to speculate that in the native environment the role of the N terminus is to increase the affinity of GSP to the bacterial cell membrane to prevent the peptide from diffusing away and allowing the C terminus to interact with the BlpH receptor. Overall, our findings revealed the minimal pheromone scaffold needed for gallocin activation and provide the groundwork for rationally designing peptide inhibitors targeting gallocin production.2O) was purified using a Millipore analyzer feed system. Solid-phase Cl-MPA Protide resin was purchased from CEM Corporation. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected l-α-amino acids were purchased from Advanced ChemTech.All chemical reagents and solvents were purchased from Sigma-Aldrich or Chem-Impex and used without further purification. Double-distilled water (ddH2O and HPLC-grade acetonitrile [ACN]) contained 0.1% trifluoroacetic acid (TFA). Preparative RP-HPLC was performed using a Phenomenex Kinetex 5-μm, 100-Å C18 column (250 by 10 mm), while analytical RP-HPLC was performed using a Phenomenex Kinetex 5-μm, 100-Å C18 column (250 by 4.6 mm). Fmoc-based solid-phase peptide synthesis was performed on a Discover Microwave and Liberty1 automated peptide synthesizer (CEM Corp.). Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) data were obtained by mixing 0.75 μl of sample with 0.75 μl of matrix solution (α-cyano-4-hydroxycinnamic acid dissolved in ddH2O-ACN [1:1] with 0.1% TFA) on an MSP 96 polished steel target plate  and allowing it to air dry. Data were obtained using a Bruker Microflex spectrometer equipped with a 60 Hz (337-nm wavelength) nitrogen laser and a reflectron. MALDI-TOF MS data were obtained using reflectron positive ion mode with the following settings: ion source 1, 19 kV; ion source 2, 15.9 kV; lens, 8.75 kV; reflector, 20 kV; up to 300 Da matrix suppression; 200 laser shots per sample; and detector gain, 1,594 V. Exact mass data were obtained on an Agilent Technologies 6230 time-of-flight mass spectrometer (TOF-MS) with the following settings for positive electrospray ionization mode (ESI+): capillary voltage, 3,500 V; fragmentor voltage, 175 V; skimmer voltage, 65 V; octopole radio frequency voltage, peak power point (Oct 1 RF Vpp), 750 V; gas temperature, 325°C; drying gas flow rate, 3 liters/min; and nebulizer, 25 lb/in2.Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using a Shimadzu UFLC system equipped with a CBM-20A communications bus module, two LC-20AT pumps, a SIL-20A auto sampler, a SPD-20A UV–visible-light (UV/Vis) detector, a CTO-20A column oven, and a FRC-10A fraction collector. All RP-HPLC solvents , UCN34 Δgallo_rs10340 (Δgsp), UCN34 Δgsp pTCVΩPgllA-gfp, UCN34 Δgallo_rs03005 (ΔsepM), UCN34 ΔsepM pTCVΩPgllA-gfp, UCN34 Δgallo_rs10390/10395 (ΔblpAB), UCN34 ΔblpAB pTCVΩPgllA-gfp; L. lactis NZ9000 pTCVΩPtetO-blpAB-gsp was also used. The following procedure was followed for each S. gallolyticus subsp. gallolyticus isolate: a freezer stock was streaked onto a plate of Todd-Hewitt agar supplemented with 0.5% yeast extract (THY plate). Strains containing the pTCVΩPgllA-gfp plasmid were grown in the presence of erythromycin (EM) at a final concentration of 10 μg/μl. The plates were incubated for 12 to 24 h in a CO2 incubator (37°C with 5% CO2). Fresh colonies were picked, inoculated into a sterilized culture tube containing 2 ml of sterile THY broth, and incubated statically in a CO2 incubator overnight. The overnight culture was used for the experiments described below. L. lactis strains were grown under similar conditions, with the exception of the absence of CO2 and a growth temperature of 30°C.The Peptide synthesis and purification of analogs was conducted using previously established methods . All pep10.1128/mBio.03189-20.10TABLE S1S. gallolyticus subsp. gallolyticus GSP analogs. Download Table S1, PDF file, 0.1 MB.MS and HPLC data for Copyright © 2021 Harrington et al.2021Harrington et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50s from three trials were calculated using GraphPad Prism software (v. 7.0) using a sigmoidal curve fit.Fluorescence and absorbance measurements were recorded using a Biotek Synergy H1 microplate reader using Gen5 data analysis software (v. 3.03). Biological assays were performed in triplicate per trial. ECStock solutions of peptides (1 mM) were prepared in dimethyl sulfoxide (DMSO) and stored at 4°C in sealed glass vials. The maximum concentration of DMSO used in all biological assays did not exceed 2% (vol/vol). Black polystyrene 96-well microtiter plates (Costar) were used for the GFP cell-based reporter assays.S. gallolyticus subsp. gallolyticus UCN34 Δgsp pTCVΩPgllA-gfp strain. An overnight culture was used to make a fresh 2-ml culture without EM using a 1:10 dilution on the day of the experiment and incubated in a CO2 incubator (37°C with 5% CO2) for 3 to 4 h to reach mid-log to late log phase of growth (OD600 = 0.6 to 0.9). A final 1:10 dilution was made on a larger scale (20 ml) without EM and used for the assay, where 198 μl diluted bacterial culture was added to each well of the microtiter plate containing peptides. Plates were incubated at 37°C with shaking at 200 rpm. Fluorescence  and optical density at 600 nm (OD600) readings were recorded for each well using a plate reader. Measurements were taken 1 h after inoculation and were recorded every 20 to 30 min for up to 3 h to capture the maximum fluorescence signal. The maximum fluorescence signal was normalized with the OD600 value and used to construct dose curves to determine the EC50.Peptide stock solutions were serially diluted with DMSO in either 1:2, 1:3, or 1:5 dilutions, and 2 μl of the diluted solution was added to each of the wells in a black 96-well microtiter plate. Each concentration was tested in triplicate, with DMSO only used as a negative control. Screening of peptide analogs was conducted using the S. gallolyticus subsp. gallolyticus strains (Δgsp and ΔsepM) and S. mutans SMCC3 (ΔcomC) were grown in 60 ml THY overnight at 37°C with 5% CO2 and centrifuged at 4,000 × g for 15 min. The supernatant was discarded, and the cell pellets were washed with 30 ml of sterile saline solution (0.9% [wt/vol] NaCl). The cell suspensions were centrifuged at 4,000 × g for 15 min, and the supernatant was discarded. The cell pellet was resuspended in 6 ml sterile saline solution and used for the SepM function experiment. The peptides tested  were resuspended in sterile H2O to a final concentration of 1 mM. For each strain tested, around 900 μl of the cell suspension was mixed with 100 μl of each peptide separately to give a 100 μM concentration. The cell-only control for each strain was made with 900 μl of each cell suspension with 100 μl of sterile saline solution. The peptide-only control for each peptide was made in sterile saline solution at a 100 μM concentration. All variables were tested in sterile 1.5-ml microcentrifuge tubes. All samples were incubated at 37°C with shaking at 200 rpm for 30 min. After 30 min, the samples were centrifuged at 5,000 × g for 1 min. A 200-μl aliquot from each sample was filtered through a sterile 0.45-μm syringe filter (Phenomenex Phenex-RC 4-mm syringe filter) into a sterile 1.5-ml microcentrifuge tube. LC-MS analysis of each sample was performed using XBridge C18 columns  on an Agilent Technology 1200 series LC connected to Agilent Technologies 6230 TOF-MS. The solvents used for LC-MS were as follows: mobile phase A, ddH2O plus 0.1% formic acid, and mobile phase B, ACN plus 0.1% formic acid. The LC-MS method used to analyze components in the filtrates had an injection volume of 100 μl and a linear gradient of 5% to 95% B over 25 min, with a flow rate of 0.5 ml/min.S. gallolyticus subsp. gallolyticus strains  were grown in 40 ml of sterile THY at 37°C with 5% CO2 for 12 h. The cultures were centrifuged at 4,000 × g for 15 min, the supernatant was discarded, and the cell pellets were resuspended in 5 ml of sterile THY. The resuspended cells were incubated for 16 h and centrifuged at 4,000 × g for 15 min. The supernatant was filter sterilized using a 0.22-μm syringe filter (CELLTREAT PES 30-mm diameter syringe filter) in a sterile 1.5-ml microcentrifuge tube. LC-MS analysis of each sample was performed using an XBridge C18 column  on an Agilent Technology 1200 series LC connected to an Agilent Technologies 6230 TOF-MS. The solvents used for LC-MS were as follows: mobile phase A, ddH2O plus 0.1% formic acid, and mobile phase B, ACN plus 0.1% formic acid. The LC-MS method used to analyze components in the filtrates had an injection volume of 50 μl and a linear gradient of 5% to 95% B over 25 min, with a flow rate of 0.5 ml/min. L. lactis pTCVΩPtetO-blpAB-gsp was inoculated from a fresh plate into 40 ml of sterile THY containing 200 ng/ml anhydrotetracycline to an initial OD600 of 0.1, and grown at 30°C for 1 h with shaking at 200 rpm. Cultures were then centrifuged at 5,000 × g for 15 min, after which the supernatant was filter sterilized using a 0.22-μm syringe filter (CELLTREAT PES 30-mm diameter syringe filter) in a sterile 50-ml centrifuge tube. Ammonium sulfate (60% [wt/vol]) was added to the filtered supernatants to facilitate precipitation of the peptide. Supernatants were incubated with ammonium sulfate for 1 h at 4°C and centrifuged at 5,000 × g for 15 min. The supernatant was discarded, and pelleted proteins were resuspended in 10 ml 50:50 acetonitrile-water, frozen, and lyophilized for 24 h. Following lyophilization, LC-MS analysis was performed on precipitated components of the L. lactis supernatant, using an injection volume of 50 μl and a linear gradient of 5% to 95% B over 50 min, with a flow rate of 0.25 ml/min.S. gallolyticus subsp. macedonicus; OD600 ≈ 0.5) were poured on a THY agar plate. After removal of excess liquid, the plate was dried for 15 min. Using sterile tips, 5-mm-diameter wells were dug into the agar. Wells were filled with 80 μl of S. gallolyticus subsp. gallolyticus filtered supernatant supplemented with 0.1% Tween 20. When the wells were dry, plates were incubated inverted overnight at 37°C, and the inhibition rings were observable the next day.For well diffusion assays, 2 ml of prey bacteria  and ΔsepM  mutants were constructed as reported previously in S. gallolyticus subsp. gallolyticus strain UCN34  at 38°C, a nonpermissive temperature for plasmid replication. Then, excision of the plasmid from the chromosome by a second event of homologous recombination was obtained by successive cultures at 30°C in THY broth without erythromycin, resulting in either gene deletion or reversion back to the wild type (bWT). Mutants were systematically tested for erythromycin sensitivity to confirm the loss of the plasmid and confirmed by PCR and sequencing of the chromosomal locus flanking the deletion region.The Δin UCN34 . The priblpAB and gsp genes were amplified by PCR using the primers listed in tetO vector to obtain pTCVΩPtetO-blpAB-gsp. This vector was transformed and amplified in competent E.coli TOP10 cells (Thermo Fisher), verified by sequencing and finally transformed by electroporation in L. lactis NZ9000.Plasmid construction was performed as described in the accompanying paper . BrieflyS. gallolyticus subsp. gallolyticus strains or L. lactis pTCVΩPtetO-blpAB-gsp) were inoculated at an OD600 of 0.1 in THY and grown at 37°C (30°C for L. lactis). At an OD600 of 1, the cells were pelleted and the supernatants were harvested and filtered in order to test for the presence of GSP. After overnight incubation, 100 μl of the reporter strain S. gallolyticus subsp. gallolyticus Δgsp pTCVΩPgllA-gfp was pelleted and resuspended in 1 ml of the tested supernatant. After 3 h of incubation at 37°C, the fluorescence of the reporter strain was measured by flow cytometry using a MACSQuant VYB flow cytometer.Strains to be tested for GSP production (