Combinatorial approach in evaluation of biocontrol bacterial species with biochemical formulation mitigating Verticillium wilt of cotton

Combinatorial approach in evaluation of biocontrol bacterial species with biochemical formulation mitigating Verticillium wilt of cotton | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Combinatorial approach in evaluation of biocontrol bacterial species with biochemical formulation mitigating Verticillium wilt of cotton Kamaldeep Kaur, MM Mehdi, Joydeep Dutta, Shalini Pathak, Sharon Nagpal, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8359830/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present study aimed to elucidate the potential of biological control bacterial strains, viz. Pseudomonas aeruginosa, Lysinibacillus macroides , and Bacillus subtilis against V. dahliae causing wilt of Cotton. Out of seven, three strains, viz. Pseudomonas aeruginosa , Lysinibacillus macroides , and Bacillus subtilis have shown significant antagonistic effects in dual culture assay. Antagonistic assays, viz., in vitro inhibition, in vivo field trials, and enzymatic studies revealed significantly superior potential for wilt in Pseudomonas aeruginosa under natural field and pot trials. Pseudomonas aeruginosa bacterized plants also exhibited higher growth metrics and productivity. The current combinatorial study effectively promotes the use of bacterial biocontrol strains for the reduction in disease incidence, promoting crop yield and growth by combining soil solarization and biochemical formulation for sustainability. Cotton biological control biochemical formulation enzymatic activity field trials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Gossypium , commonly known as cotton, is considered white gold grown throughout the world and cultivated since the prehistoric era (Zhang et al. 2018 ). Countries contributing massively tocotton production are the USA, India, Pakistan, Turkey, Argentina and Egypt (Li et al. 2012 ). Verticillium wilt, caused by soil-borne fungi Verticillium dahliae is a common cause of yield loss in cotton (Zhang et al. 2018 ). The disease first reported from Manisa province (Turkey) in 1941 (Pullman and Devay 1981) shows symptoms like wilting in infected leaves with yellowing between veins before showing necrosis. Endophytic bacteria, fungal strains, and actinomycetes have been used for control of cotton wilt (Eljounaidi et al.2016). Biochemical formulations are biologically active products containing one or more beneficial microbial strains in an easy-to-use form with the addition of chemical supplements, promoting plant growth. The present study aims to isolate endophytic bacteria from healthy parts of cotton and characterize their antagonistic, biosurfactant, microbial enzymes for its application as potential biocontrol strains to inhibit Verticillium wilt in cotton under polyhouse and natural field conditions. A combinatorial approach with the use of resistant variety and seed bacterization with endophytic bacterial strains, can find its use as an effective biochemical formulation against Verticillium wilt in cotton. Materials and methods Randomized sample collected from selected sites 20 diversified sites were selected for sample collection from different villages of the Bathinda region. Two sites per village were selected for the collection of non-infested and infested cotton plant samples. Soil samples and plants were collected in a replication of five per sample from the different layers of each segregated plot (Oros sichler et al.2006). The cotton plants and soil were characterized as:(A) healthy plants in non-infested fields (non-infected) (B) healthy plants in infested fields (infected) (C) unhealthy plants in infested fields (infected) (D) rhizosphere soil of healthy plants in non-infested fields (non-infected)(E) rhizosphere soil of healthy plants in infested fields (infected) (F) rhizosphere soil of unhealthy plants in infested fields (infected)(G) bulk soil of non-infested fields (non- infected). (H) bulk soil of infested fields (infected). Cotton plant parts such as healthy leaves, stems, and roots collected from cotton growing fields. Characterization and identification of pathogenic and endophytic bacterial strains Isolation of pathogenic fungi was done by using collected infected and non-infected plant parts and soil samples (Zheng et al., 2011 ). Isolated fungal strains were purified and stored in refrigerator until further experimentation (Zheng et al. 2011 ). Endophytic bacterial strains were isolated and purified from healthy plant parts, viz., roots, stems, and leaves. Isolated bacterial strains were purified by streaking using NA plates with slight modifications (Zheng et al. 2011 ). Preliminary identification of pathogenic fungi was done by examining characteristics and features of colony and aerial hyphae, morphology of resting spores and conidiophores (Xue et al. 2013 ). Potential fungal strain dahlia was sent to Science Farmers, Bangalore, India, for 18s rRNA molecular-based identification and bacterial culture was identified based on 16s rRNA sequencing from Barcode & Biosciences, Bangalore, India. Dual Culture Assay for in vitro evaluation of endophytic bacterial cultures against V. dahliae Verticillium dahliae was inoculated at 2cm apart from the side of the Petri plate and incubated for 48 hours at 26 0 °C. After 48 hours, bacterial isolates from NA medium were inoculated on the other half of the plate by zigzag streaking. A plate with a sole culture of Verticillium dahliae was kept as a control. After incubation at 28 ± 1 0 C for 6 days, each plate was observed for growth. After six days, the growth of both endophytic bacterial strains and Verticillium dahliae was measured to calculate the percentage of growth inhibition (Skidmore and Dickinson, 1976 ), with slight modifications. Calculation of the percentage of growth inhibition was done using: PGI = R1-T1/R1*100 PGI = Percentage of Growth Inhibition, R1 = Growth of Fungus in control, T1 = Growth of Bacterial Culture in Test Preparation of crude cellulase, protease and chitinase from endophytic bacteria Endophytic bacterial strains were grown in liquid Czapek-Mineral salt Medium for 5 days at 26 0 C for cellulase assay, Casein broth for 5 days at 26 0 °C for protease and Chitinase Detection Medium for production of chitinase enzyme. The medium was passed through filter paper followed by centrifugation for 10 min at 10000 ppm at 4°C. The resultant supernatant was separated and stored in 1ml sterilized vials for further screening (Vijayaraghavan et al.2011). Determination of cellulase, protease, and chitinase activity by the well diffusion method Czapek- mineral salt agar medium, casein agar medium, and chitinase agar medium were used for the determination of cellulase, protease, and chitinase activities. Plates were solidified, and a hole was punched in the centre. Supernatant extracted from endophytic bacterial strains was poured into the well. Plates were incubated for 3–4 days at 26 0 C. Congo red solution was poured into petri plates for 15 min to determine cellulase activity. Then, plates were flooded with NaCl (1M) for 1 min. Formation of a yellow opaque area was observed around the well. Diameter of colonies was measured and recorded (Vijayaraghavan et al. 2011 ). Bromocresol green dye was used for protease activity for screening purposes by flooding the plates. Chitinase agar medium was prepared, and pH of the medium was adjusted to 4.7 then autoclaved. Bromocresol green dye was poured onto plates. Purple coloured zone formation around the well was measured and recorded. This experiment was done in triplicates; wherein distilled water was used as a control (Vijayaraghavan et al.2011). Well diffusion method for Biosurfactant Activity Iturin and Fengycin at different concentrations viz. 1ppm, 5ppm and 10ppm were used against V. dahlia. A pathogenic fungal strain was inoculated in the close vicinity of the well. Biosurfactant compounds of different concentrations were poured into the well (Debode et al.2007). Plates were then incubated at 26 0 C for 5 days, and the zone of inhibition was measured (mm). Phytochemical extract formulation Phytochemical extract formulation Different parts of floral plants, viz Clotropis procera, Azadirachta indica, Citrullus colocynthis, Ricinus communis, Tinospora cardifolia, Withania somnifera, Datura stramonium, and Solanum xanthocarpum were selected. Copper sulphate was also added. Selected plant parts were cut into small pieces with a sterile knife and soaked in cow’s urine with fermented acidophilus milk for 30 days in a large bucket. After 30 days, the extract was sieved through muslin cloth. Formulation in the form of a spray with a ratio of 1:9 (biochemical formulation: water) with endophytic bacterial strains was used for seed bacterization. Analysis of biochemical formulation Liquid chromatography Mass Spectrometry technique was used for the analysis of biochemical formulation. One mg of biochemical formulation was sent to Central Drug Research Institute, Lucknow, India for LCMS. In vitro antifungal activity of biochemical formulation against Verticillium dahliae. Antifungal activity of bioformulation was checked at 10ppm, 50ppm, and 100ppm concentration by the Well diffusion method (Debode et al. 2007 ). Control consisting of distilled water was used and plates were incubated for 5 days at 26 0 C. Zones of inhibition were measured in mm. The experiment was done in triplicate. Seed bacterization in polyhouse and field trials against Verticillium dahliae Bacterization of delinted seeds of Verticillium dahliae sensitive local variety Rasi 134 Bt was done for both field and pot experiments with slight modifications (Göre et al. 2008). Nutrient Broth medium was used for antagonistic bacterial growth for 24 hours at 37 0 C. Centrifugation at 5000g for 5 min at 4 0 C was done to collect the pellets. Pellets were suspended in sterile phosphate buffer with 1.5% CMC solution. For control, seeds were treated with 1.5% CMC without bacteria, and soaking of seeds with antagonistic bacterial suspension of 100mg with 1ml of 1.5% CMC was done. The mean concentration of bacterial strains was 1 X 10 8 cfu/ml + 1.5% CMC for test in both polyhouse and field trials. After soaking, all the seeds were kept separately in sterile conditions in a laminar flow for 12 hours (Bölek et al. 2005). Screening of biological control of Verticillium wilt in poly house and Natural Field conditions with and without biochemical formulation Verticillium dahliae sensitive local cotton variety, was used for screening biochemical formulation along with an endophytic bacterial strain against wilting. A mixture of autoclaved soil-sand-peat-vermiculite in an equal ratio was transferred into plastic pots for sowing of both bacterized and control seeds (Naraghi et al. 2010 ). Seeds treated with each endophytic bacterial strain and bacteria + bioformulation treatments were sown into pots for polyhouse experiments and in the natural field, 10-day-old culture of V. dahliae was used for inoculation after sowing of bacterized seeds, diluted into 3x10 6 conidia/ml. Drops of conidial suspension (5-l0 drops) were inoculated into the stem of a cotton plant through a 22-gauge needle by puncturing the first internode of the stem at six true leaf stages (Hanson 2000 ). Cotton plants were treated with a 16-8-24 ratio of N-P-K liquid fertilizer. The experiment was repeated in triplicate for each bacterial strain. Non-bacterial plants were used as controls with sterile distilled water only. Disease severity index of each plant was checked after fifteen days of inoculation from sowing to harvesting period at 4 to 0 rating scale 4 = (Dead plant), 3 = 67–97% (Wilt or Defoliation), 2 = 34–66% (Necrosis), 1 = 1–33% (Acropetal Chlorosis), 0 = (healthy plant) for both control and test plants (Bejarano-Alcazar et al., 1995). Biocontrol protection % (Li et al. 2012 ) and disease index (Zhu et al. 2013 ) were calculated using the formula mentioned below: Disease Incidence (%) = [(n1 + n2 + n3 + n4)/n] x 100., Disease Severity Index (%) = [(0n0 + 1n1 + 2n2 + 3n3 + 4n4)/4n x 100; Biocontrol Protection% = 100- Vascular discoloration (treatment)/Vascular discoloration (control) x 100 Statistical Analysis All experiments, viz. antagonistic activity, hydrolytic enzymatic activity and in vivo trials were repeated in triplicate. The results were represented as means ± standard error (SE). IBM SPSS Statistics 22 software, USA, was used to statistically analyze all of the experiment data using one-way analysis of variance (ANOVA). Comparison of means was made by Scott- Knott analysis at 5% level of probability. Results and Discussion Isolation of pathogenic fungal strains from collected plant/soil samples Verticillium dahlia was reported from infected cotton plant parts and rhizospheric and bulk soil. The pathogenic fungal strain isolated from infected samples showed whitish mycelium in the initial incubation period on PDA plates. Purification of fungi was performed by using PDA plates. Table 1 depicts the prevalence rate of V. dahliae isolated from samples from different sites V. dahliae was isolated from all samples of different sites. Prevalence rate of Verticillium dahliae from 160 collected samples, of 20 different sites of Bathinda, Punjab, India, was 73%. Percentage of V. dahliae from collected samples-= Prevalence rate of V. dahliae from total 8 sample fields= Total number of fungal strains isolated from 8 sample fields- No of V. dahliae isolated from 8 sample fields/100 Prevalence rate of V. dahliae from total 8 sample fields= Total number of fungal strains isolated from 5 infected sample fields- No of V. dahliae isolated from 5 infected sample fields/100 Isolation of endophytic bacterial strains from healthy plant parts Seven bacterial strains of endophytic nature were isolated from healthy cotton plant parts, viz., roots, stems, and leaves on Nutrient Agar medium (NAM) by streaking method and purified by streaking. The isolates were named as VTS DB-V1/NI/L/S1/B2 from the healthy stem, VG DB-V2/NI/L/S1/B1 from the healthy leaves of a cotton plant, collected from different locations of Bathinda. A similar study was done by Mcinroy and Kloeeper in 1995, in which isolation of endophytic bacterial strains from cotton roots and stem tissues was reported. These seven isolates were further screened through antagonistic tests, and positive bacterial strains were identified through 16S rRNA sequencing. 18s rRNA sequencing for microbial Identification Verticillium dahliae was reported from all infected plant parts of cotton and the rhizospheric and bulk soil of cotton fields. V. dahliae was preliminarily identified by its morphological colony characters, air hyphae, and structure of microsclerotia (Fig. 1 ). Conidiophores were erect, hyaline, verticillately branched, and septate. Phialides frequently arise directly from hyphae. Microsclerotia of V. dahliae was dark brown to black in color, thick-walled, and abundant with almost swollen globular cells (Inderbitzin et al., 2011). Identification of microbes was done by PCR methods through the molecular 18S rRNA gene, which was conducted by outsourcing through Science Farmers, Bangalore, India, by the DNA extraction method. Integrated Transcribed Spacer (ITS1 5՜- 5՜-GTAGTCATATGCTTGTCTC-3՜) and (ITS4 5՜-CTTCCGTCAATTCCTTTAAG-3՜) primers were amplified by the 18S rRNA gene region, sequenced, and analyzed for similarity among species in the NCBI CBI database. A total of 7 endophytic bacterial strains were isolated and purified, out of which 3 strains exhibited positive antagonistic activity during in vitro screening and were hence used for in vivo trials. 16S rRNA gene identification by PCR was conducted by outsourcing through Barcode Bioscience Bangalore, India, by the DNA extraction method. Integrated Transcribed Spacer (ITS1 5՜- 5՜-GTAGTCATATGCTTGTCTC-3՜) and (ITS4 5՜- CTTCCGTCAATTCCTTTAAG-3՜) primers were amplified by the 16S rRNA gene region, sequenced, and analyzed for similarity among species in the NCBI CBI database. Three endophytic bacterial strains were identified through 16S rRNA and named as VTS DB- V1/NI/S/S1/B2 ( Pseudomonas aeruginosa ), VTS DB-V1/NI/R/S1/B2 ( Bacillus subtilis ), and VTS DB-V1/NI/S/S1/B1 ( Lysinibacillus macroides ). Selection of endophytic bacterial strains as potential biocontrol agents Preliminary screening of endophytic bacterial strains by Dual Culture Assay During this study, endophytic bacterial strains were isolated, and in vitro screening was performed to check antagonistic activity against V. dahliae . The dual culture method was the preliminary step to check the antagonistic activity because it eliminates the growth of the host pathogen, and most likely by direct antagonism against the pathogenic strain (Khan, 2013). Primary screening of all culture strains has been done through a dual culture assay for the selection of the best positive endophytic bacterial strains for in vivo evaluation. Different endophytic bacterial strains showed in vitro antagonistic activity towards V. dahliae . The experiment was performed in triplicate for 0 to 144 hours of incubation. Maximum percentage of inhibition in growth was shown by Pseudomonas aeruginosa (VTS DB-V1/NI /S/S1/B2) with 61.12% followed by Lysinibacillus macroides (VTS DB-V1/NI /S/S1/B1), Bacillus subtilis (VTS DB-V1/NI /R/S1/B2), VG DB-V1/NI /L/S1/B2, VTS DB-V1/NI /L/S1/B2, VG DB-V2/NI /L/S1/B1 and VG DB-V1/NI /S/S1/B1 with 59.2%, 57.64%, 56.25%, 48.62%, 45.2% and 40.29% respectively during Dual culture assay. Statistical analysis in Fig. 2 shows the in vitro antagonism against V. dahliae , in which mean values with stars represent a significant difference between compared means at 0- 144 hours of incubation period. Values without a star on means of represent a lack of significant differences at different incubation times (ANOVA, p ≥ 0.05) Assay for cell wall-degrading enzymes in endophytic bacterial strains through in vitro tests Cellulase Enzyme Assay Additional in vitro screening served as a useful secondary parameter for the selection of biocontrol agents, as it facilitated the degradation of pathogenic fungal cell walls through hydrolytic enzymatic activity. Endophytic bacterial strains were further evaluated for their biocontrol potential by assessing their in vitro antagonistic activity during hydrolytic enzyme production. Cellulase enzyme assay was performed by growing the isolates in liquid Czapek Mineral Salt Medium. After five days of incubation, the endophytic bacterial strains produced varying inhibition zones around the wells. Pseudomonas aeruginosa (VTS DB- V1/N1/S/S1/B2) exhibited the maximum inhibition zone (8.56 mm), followed by Lysinibacillus macroides (VTS DB-V1/N1/S/S1/B1) (7.3 mm), VTS DB-V1/N1/L/S1/B2 (6.8 mm), Bacillus subtilis (VTS DB-V1/N1/R/S1/B2) (5.23 mm), VG DB-V2/N1/L/S1/B2 (5.2 mm), VG DB-V2/N1/L/S1/B2 (5.0 mm), and VG DB-V1/N1/S/S1/B1 (4.5 mm) during cellulase activity. Among these, P. aeruginosa (VTS DB-V1/N1/S/S1/B2) demonstrated the most pronounced inhibition, followed by L. macroides . Statistical analysis in Table 2 revealed no significant differences (LSD test) among the mean values of VTS DB- V1/N1/L/S1/B2, VG DB-V1/N1/L/S1/B2, VG DB-V2/N1/L/S1/B1, VG DB-V1/N1/S/S1/B1,and VTS DB-V1/N1/R/S1/B2, as indicated by the same alphabetic grouping. Conversely, the mean values of VTS DB-V1/N1/S/S1/B2 and VTS DB-V1/N1/S/S1/B1 differed significantly, as denoted by distinct alphabetic representations (ANOVA, p ≥ 0.05). Protease Enzyme Assay Protease activity was assessed on Casein Agar medium. During this assay, endophytic bacterial strains produced clear inhibition zones around the wells compared to the control. All experiments were performed in triplicate. The maximum inhibition zone was observed in Pseudomonas aeruginosa (VTS DB-V1/N1/S/S1/B2) (8.70 mm), followed by Lysinibacillus macroides (VTS DB-V1/N1/S/S1/B1) (7.32 mm), Bacillus subtilis (VTS DB- V1/N1/R/S1/B2) (6.66 mm), VG DB-V1/N1/L/S1/B2 (5.5 mm), VG DB-V2/N1/L/S1/B1 (4.2 mm), VG DB-V1/N1/S/S1/B1 (3.9 mm), and VTS DB-V1/N1/L/S1/B2 (3.2 mm). Chitinase Enzyme Assay Chitinase activity was evaluated on Chitinase Detection Medium containing basal chitin. The growth of purple-coloured zones was measured as an indicator of enzymatic activity, and all assays were carried out in triplicate. Endophytic bacterial strains produced distinct purple inhibition zones around the wells, reflecting significant chitinase activity. The largest zone was observed in Pseudomonas aeruginosa (VTS DB-V1/N1/S/S1/B2) (7.4 mm), followed by Lysinibacillus macroides (VTS DB-V1/N1/S/S1/B1) (7.1 mm), Bacillus subtilis (VTS DB- V1/N1/R/S1/B2) (6.3 mm), VG DB-V1/N1/L/S1/B2 (6.1 mm), VTS DB-V1/N1/L/S1/B2 (5.7 mm), VG DB-V2/N1/L/S1/B1 (4.5 mm), and VG DB-V1/N1/S/S1/B1 (3.9 mm). Biosurfactants antifungal activity against V. dahliae In the present investigation, Fengycin and Iturin have been evaluated as potential biocontrol agent against V. dahliae by checking their in vitro antifungal activity by well diffusion assayas depicted in Fig. 3 . The experiment was performed at different concentration viz., 5ppm and 10 ppm showing 2.3 ± 0.1ab mm and 3.2 ± 0.05b mm inhibition zones respectively and Iturin at the same concentrations showing 2.9 ± 0.15b mm, 3.8 ± 0.2ab mm inhibition zones, respectively as compared to control plate. Both Fengycin and Iturin have shown no inhibition zones against V. dahliae at 1ppm concentration. Statistical test was performed as ANOVA, p ≥ 0.05). LC-MS of biochemical formulation: LCMS has been widely applied in the field of both proteomics and metabolomics research. This spectrometry is considered to highly advanced and analytical technology. LCMS is used for the profiling of different compounds in samples with further structural elucidation. The different compounds in samples can be detected relatively or absolutely through analysing spectral features from LC-MS data. The compounds' identification in the LCMS chromatograph is carried out based on mass-to-charge ratio and retention time. In our study, profiling of phytoconstituents in aqueous extract of different parts of Ricinus communis, Tinospora cardifolia, Withania somnifera, Datura stramonium, Clotropis procera, Azadirachta indica, Citrullus colocynthis, Solanum xanthocarpum was carried out through positive and negative ionization MS-ESI mode of LC-MS method. The LC-MS chromatograms in positive and negative ionization mode are shown in Fig. 4 . In comparison with the previous literature, the bioactive compounds were characterized by their mass spectrum, retention times, and fragmentation profiles. The list of identified phytocompounds with their molecular weight, retention time, monoisotopic mass, and chemical formula is shown in Table 3. Soil solarization of natural field before sowing of seeds The soil solarization technique was followed before sowing of seeds under natural field conditions. The field was prepared according to the experimental field layout as mentioned earlier. Four equal ridges have been prepared with a wooden leveler and properly covered with polythene sheets for 30 days. No growth of V. dahliae was recorded after soil solarization. Hence, soil solarization plays an important role in controlling wilt disease. Soil sterilization and seed bacterization for polyhouse experiments Evaluation of potential antagonistic microbial strains against Verticillium dahliae was performed in polyhouse conditions. Soil has been tested for the non-presence of V. dahliae periodically after sterilization before sowing of seeds. Verticillium dahliae was not observed in any PDA plate after soil sterilization. Bacterization of delinted seeds of Verticillium dahliae sensitive local variety Rasi 134 Bt was done for both field and pot experiments ( Fig. 5 ) . Biological Management of Cotton Verticillium Wilt by Endophytic Bacterial Strains With and Without Biochemical Formulation During the current research, Pseudomonas aeruginosa, Lysinibacillus macroides, and Bacillus subtilis were assessed as biocontrol agents against Verticillium dahliae under natural field conditions after showing strong in vitro antagonistic activity. The tests were carried out under monoculture conditions, and the incidence of wilt was observed fortnightly (Figs. 3 , 4 and 5 ). In control plants without treatment, disease symptoms became apparent following the flowering phase in August, with yellowing and interveinal chlorosis among leaf veins. Bacterized plants, however, manifested significantly lower disease incidence. P. aeruginosa and L. macroides –treated seeds completely suppressed wilt symptoms (0% severity), while B. subtilis –treated seeds displayed mild acropetal chlorosis (28% severity). Following 135 days of planting, control plants had 88% wilt mortality, while the treatments with biocontrol strongly suppressed wilt incidence. The maximum biocontrol activity was on P. aeruginosa (73.6%), followed by L. macroides (68.5%) and B. subtilis (62.5%) (Table 4). To improve further disease control, the same bacterial isolates were compared in combination with a biochemical formulation sprayed over the foliage fortnightly. Wilt suppression under this treatment improved. Seeds bacterized with P. aeruginosa and L. macroides again showed complete protection (0% severity), while B. subtilis recorded 43% severity. After 135 days, mortality in non-bacterized controls reached 82%, whereas biocontrol protection was significantly higher in treated plants: P. aeruginosa (76.6%), L. macroides (69.5%), and B. subtilis (58.3%). In general, seed bacterization and bacterization plus biochemical formulation both lowered wilt severity and improved plant survival. In the strains that were tested, P. aeruginosa always showed the greatest biocontrol activity, followed by L. macroides and B. subtilis . These findings proved the role of endophytic bacteria, specifically P. aeruginosa , as promising biocontrol agents in controlling Verticillium wilt of cotton. Biological management of Verticillium wilt of cotton under polyhouse tests with and without biochemical formulation Polyhouse tests were done to assess the biocontrol potential of endophytic bacterial strains, with and without biochemical formulation (BCF), against Verticillium dahliae. Cotton seeds were bacterized with Pseudomonas aeruginosa, Lysinibacillus macroides , and Bacillus subtilis , whereas non-bacterized seeds were taken as controls. Wilt symptoms were observed on regular intervals after artificial inoculation with V. dahliae. In non-bacterized controls, initial symptoms were light-yellow interveinal chlorosis followed by a rapid development of wilting. The disease severity index reported 89% incidence of wilt in controls. In contrast, seeds bacterized with P. aeruginosa were symptomless (0% severity), whereas seeds treated with L. macroides and B. subtilis were partially protected with disease severities of 19% and 32%, respectively (0–4 rating scale). Corresponding biocontrol protection was observed as 69.6% ( P. aeruginosa ), 65.2% ( L. macroides ), and 62.5% ( B. subtilis ), which is significantly different from that of the controls (36.4%, 29.7%, and 32.2%) (Table 4). At the application of biochemical formulation in combination with seed bacterization, suppression of wilt further increased. Control plants again exhibited high disease infection (86%), whereas P. aeruginosa + BCF and L. macroides + BCF treatments fully suppressed wilt symptoms (0% severity). B. subtilis + BCF decreased the severity to 32%, which showed moderate protection. Protection in biocontrol with these treatments was ascertained as 70.6% ( P. aeruginosa ), 68.6% ( L. macroides ), and 64.5% ( B. subtilis ), whereas the controls varied between 36.5% and 39.2% (Table 4). Generally, all results confirmed that P. aeruginosa always posed the highest protection against wilt, both when used individually as well as in combination with biochemical formulation, followed by L. macroides and B. subtilis . The enhanced activity with biochemical formulation indicated a synergistic action, which augmented the potential of bacterial strains to inhibit V. dahliae . These results are consistent with previous reports that microsclerotia of V. dahliae infect roots to cause infection (El-Zik, 1985; Berg et al., 2001 ; Mercado-Blanco et al., 2004), and that endophytic bacteria can inhibit pathogen activity in the root interface. Assessment of plant growth-promoting potential of endophytic bacterial strains Endophytic bacterial strains were reported to possess potential beneficial effects on the growth on cotton plants. However, their biocontrol efficacy of endophytic bacterial strains had a relationship with their in vitro antagonistic activity against V. dahliae or their cell wall- degrading enzymes. Table 10–11 depicts bacterial strains aiding in increased biomass or cotton yield, and Fig. 10 shows the effect of bacterial strains on the height of treated plants in both polyhouse and field trials as compared to control plants. A study on growth parameters, viz. number of leaves, height, canopy development, and number of bolls under poly house and natural field conditions with statistical analysis was conducted, and means with a star denoted a significant difference, and lack of a star shows no significant difference between the values (ANOVA, p ≥ 0.05). Yield and growth of seed bacterized plants with Pseudomonas aeruginosa under natural field conditions were more than Lysinibacillus macroides followed by Bacillus subtilis when compared with control (non-bacterized). Endophytic bacteria such as Pseudomonas aeruginosa is considered as most significant bacteria and act as a biocontrol agent to help in growth of plant (Spiers et al. 2020). Disease severity and symptoms were significantly reduced after seed bacterization with Pseudomonas aeruginosa followed by Lysinibacillus macroides and Bacillus subtilis in both poly house and field trials. Conclusion In the present study, soil-borne pathogenic fungus Verticillium dahliae was obtained from infected samples of unhealthy cotton fields, and potential biocontrol bacterial strains were isolated from healthy cotton plant parts. Results of growth parameters clearly indicate a significant increase with bacterial strains when compared to control plants. Suppression of wilt symptoms was also recorded during pot and field trials in bacterized cotton plants. The major effect of V. dahliae was defoliation, and the poor growth and development of the cotton plant were also significantly suppressed by Pseudomonas aeruginosa, Lysinibacillus macroides , and Bacillus subtilis . Wilt symptoms were recorded after three months of non- bacterized seeds sowing under controlled and natural field conditions as compared to bacterized seeds. Endophytic bacterial strains under polyhouse and natural conditions field trials of Bathinda, Punjab in combination with biochemical formulation, have shown positive biocontrol efficacy in integrated management of wilt disease. This approach also affects on growth of plants and increases the yield of cotton in the Bathinda region. Declarations Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare no conflict of interest. Funding sources This work is partially supported from the grant of Lovely Professional University. Author Contribution Author contribution Kamaldeep Kaur: Investigation, Formal analysis, Writing– original draft. MM Mehdi: review & editing. JoyDeep Dutta: review & editing. Shalini Pathak, SharonNagpal, Faisal Mushtaq: review & editing. Ashish Vyas: Conceptualization, Methodology, Resources, Supervision Acknowledgement Author acknowledged Lovely Professional University (LPU), Punjab, India for providing facility in lab. Data Availability The original contributions presented in the study are included in the article, and further inquiries can be directed to the corresponding author. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8359830","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":574483662,"identity":"0bb3ef21-a87d-4690-90a1-b3b1d7c7aa07","order_by":0,"name":"Kamaldeep Kaur","email":"","orcid":"","institution":"Akal University","correspondingAuthor":false,"prefix":"","firstName":"Kamaldeep","middleName":"","lastName":"Kaur","suffix":""},{"id":574483663,"identity":"7704d36a-b5a7-4909-8f9a-364b60aeb0c6","order_by":1,"name":"MM Mehdi","email":"","orcid":"","institution":"Lovely Professional 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1","display":"","copyAsset":false,"role":"figure","size":1222966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eColony of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eVerticillium dahliae\u003c/em\u003e isolated from unhealthy soil after 72 hours of incubation \u003cstrong\u003e(b) \u003c/strong\u003eColony of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eV. dahliae\u003c/em\u003e isolated from roots of infected cotton field after 96 hours of incubation period\u003cstrong\u003e (c) \u003c/strong\u003e\u003cem\u003eV. dahliae\u003c/em\u003e after 14 days of post inoculation under lab conditions\u003cstrong\u003e (d) \u003c/strong\u003eMature microsclerotia of \u003cem\u003eV. dahliae\u003c/em\u003e under light microscope\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8359830/v1/9c24ffea56d7c54902dda060.png"},{"id":100426290,"identity":"700d8474-c332-49c3-8831-15228d08e064","added_by":"auto","created_at":"2026-01-16 14:19:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e screening of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBacillus subtilis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVertcillium dahliae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after every 24 hours of incubation\u003c/strong\u003e \u003cstrong\u003e(b)\u003c/strong\u003e \u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e screening of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas aeruginosa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eagainst \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVertcillium dahliae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after every 24 hours of incubation (c) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e screening of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLysinibacillus macroides \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eagainst \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVertcillium dahliae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein Dual Culture Assay after every 24 hours of incubation \u003c/strong\u003e(Standard error bars are indicated. Mean with star denote a significant difference between compared producers; without star denote a lack of significant differences between compared means (ANOVA. p≥0.05))\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8359830/v1/b669366bb8f91e9e4aaaf578.png"},{"id":100426295,"identity":"494d6ad2-a023-4b7a-ac27-38f0d905db5f","added_by":"auto","created_at":"2026-01-16 14:19:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e screening of antifungal activity of Fengycin and Iturin at 1ppm, 5ppm and 10 ppm concentrations against pathogenic fungus\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e V. dahliae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eduring “Well diffusion assay”\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8359830/v1/e02130f3326b99027232f7e9.png"},{"id":100426250,"identity":"a0a912e3-5d54-492d-b52c-245291a133b1","added_by":"auto","created_at":"2026-01-16 14:19:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":197487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a,b) \u003c/strong\u003eLCMS chromatogram of biochemical formulation in positive mode showing peaks with identified compounds\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8359830/v1/7e221b13df9c9485e39091db.png"},{"id":100426347,"identity":"f087b0d9-17d1-476f-b94f-6ccc79750925","added_by":"auto","created_at":"2026-01-16 14:19:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eProcess of Seed bacterization with endophytic bacterial strains (a) Surface sterilization of seeds (b) Washing with 0.5 M phophate buffer (c,d) Soaking of seeds in 1.5% CMC solution with endophytic bacterial strains (e) Bacterized seeds were kept separately in sterile condition in laminar flow for 12 hours\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-8359830/v1/f4bd42232b9fc65a50c48a35.png"},{"id":100553989,"identity":"fd3880d1-6454-4a19-ae1f-c7cb51b184cf","added_by":"auto","created_at":"2026-01-19 08:38:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3901500,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8359830/v1/c186244b-123e-4a76-8e98-e4d4c486bc5c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCombinatorial approach in evaluation of biocontrol bacterial species with biochemical formulation mitigating\u003cem\u003e Verticillium wilt\u003c/em\u003e of cotton\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eGossypium\u003c/em\u003e, commonly known as cotton, is considered white gold grown throughout the world and cultivated since the prehistoric era (Zhang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Countries contributing massively tocotton production are the USA, India, Pakistan, Turkey, Argentina and Egypt (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eVerticillium\u003c/em\u003e wilt, caused by soil-borne fungi \u003cem\u003eVerticillium dahliae\u003c/em\u003e is a common cause of yield loss in cotton (Zhang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The disease first reported from Manisa province (Turkey) in 1941 (Pullman and Devay 1981) shows symptoms like wilting in infected leaves with yellowing between veins before showing necrosis. Endophytic bacteria, fungal strains, and actinomycetes have been used for control of cotton wilt (Eljounaidi et al.2016). Biochemical formulations are biologically active products containing one or more beneficial microbial strains in an easy-to-use form with the addition of chemical supplements, promoting plant growth. The present study aims to isolate endophytic bacteria from healthy parts of cotton and characterize their antagonistic, biosurfactant, microbial enzymes for its application as potential biocontrol strains to inhibit Verticillium wilt in cotton under polyhouse and natural field conditions. A combinatorial approach with the use of resistant variety and seed bacterization with endophytic bacterial strains, can find its use as an effective biochemical formulation against \u003cem\u003eVerticillium\u003c/em\u003e wilt in cotton.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRandomized sample collected from selected sites\u003c/h2\u003e \u003cp\u003e20 diversified sites were selected for sample collection from different villages of the Bathinda region. Two sites per village were selected for the collection of non-infested and infested cotton plant samples. Soil samples and plants were collected in a replication of five per sample from the different layers of each segregated plot (Oros sichler et al.2006). The cotton plants and soil were characterized as:(A) healthy plants in non-infested fields (non-infected)\u003c/p\u003e \u003cp\u003e(B) healthy plants in infested fields (infected) (C) unhealthy plants in infested fields (infected) (D) rhizosphere soil of healthy plants in non-infested fields (non-infected)(E) rhizosphere soil of healthy plants in infested fields (infected) (F) rhizosphere soil of unhealthy plants in infested fields (infected)(G) bulk soil of non-infested fields (non- infected). (H) bulk soil of infested fields (infected). Cotton plant parts such as healthy leaves, stems, and roots collected from cotton growing fields.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization and identification of pathogenic and endophytic bacterial strains\u003c/b\u003e Isolation of pathogenic fungi was done by using collected infected and non-infected plant parts and soil samples (Zheng et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Isolated fungal strains were purified and stored in refrigerator until further experimentation (Zheng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Endophytic bacterial strains were isolated and purified from healthy plant parts, viz., roots, stems, and leaves. Isolated bacterial strains were purified by streaking using NA plates with slight modifications (Zheng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Preliminary identification of pathogenic fungi was done by examining characteristics and features of colony and aerial hyphae, morphology of resting spores and conidiophores (Xue et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Potential fungal strain dahlia was sent to Science Farmers, Bangalore, India, for 18s rRNA molecular-based identification and bacterial culture was identified based on 16s rRNA sequencing from Barcode \u0026amp; Biosciences, Bangalore, India.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual Culture Assay for\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eevaluation of endophytic bacterial cultures against\u003c/b\u003e \u003cb\u003eV. dahliae\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eVerticillium dahliae\u003c/em\u003e was inoculated at 2cm apart from the side of the Petri plate and incubated for 48 hours at 26\u003csup\u003e0\u003c/sup\u003e \u0026deg;C. After 48 hours, bacterial isolates from NA medium were inoculated on the other half of the plate by zigzag streaking. A plate with a sole culture of \u003cem\u003eVerticillium dahliae\u003c/em\u003e was kept as a control. After incubation at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003e0\u003c/sup\u003e C for 6 days, each plate was observed for growth. After six days, the growth of both endophytic bacterial strains and\u003c/p\u003e \u003cp\u003e \u003cem\u003eVerticillium dahliae\u003c/em\u003e was measured to calculate the percentage of growth inhibition (Skidmore and Dickinson, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), with slight modifications.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCalculation of the percentage of growth inhibition was done using:\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePGI\u0026thinsp;=\u0026thinsp;R1-T1/R1*100\u003c/h2\u003e \u003cp\u003ePGI\u0026thinsp;=\u0026thinsp;Percentage of Growth Inhibition, R1\u0026thinsp;=\u0026thinsp;Growth of Fungus in control, T1\u0026thinsp;=\u0026thinsp;Growth of Bacterial Culture in Test\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of crude cellulase, protease and chitinase from endophytic bacteria\u003c/h3\u003e\n\u003cp\u003eEndophytic bacterial strains were grown in liquid Czapek-Mineral salt Medium for 5 days at 26\u003csup\u003e0\u003c/sup\u003e C for cellulase assay, Casein broth for 5 days at 26\u003csup\u003e0\u003c/sup\u003e \u0026deg;C for protease and Chitinase Detection Medium for production of chitinase enzyme. The medium was passed through filter paper followed by centrifugation for 10 min at 10000 ppm at 4\u0026deg;C. The resultant supernatant was separated and stored in 1ml sterilized vials for further screening (Vijayaraghavan et al.2011).\u003c/p\u003e\n\u003ch3\u003eDetermination of cellulase, protease, and chitinase activity by the well diffusion method\u003c/h3\u003e\n\u003cp\u003eCzapek- mineral salt agar medium, casein agar medium, and chitinase agar medium were used for the determination of cellulase, protease, and chitinase activities. Plates were solidified, and a hole was punched in the centre. Supernatant extracted from endophytic bacterial strains was poured into the well. Plates were incubated for 3\u0026ndash;4 days at 26\u003csup\u003e0\u003c/sup\u003e C. Congo red solution was poured into petri plates for 15 min to determine cellulase activity. Then, plates were flooded with NaCl (1M) for 1 min. Formation of a yellow opaque area was observed around the well. Diameter of colonies was measured and recorded (Vijayaraghavan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Bromocresol green dye was used for protease activity for screening purposes by flooding the plates. Chitinase agar medium was prepared, and pH of the medium was adjusted to 4.7 then autoclaved. Bromocresol green dye was poured onto plates. Purple coloured zone formation around the well was measured and recorded. This experiment was done in triplicates; wherein distilled water was used as a control (Vijayaraghavan et al.2011).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWell diffusion method for Biosurfactant Activity\u003c/h2\u003e \u003cp\u003eIturin and Fengycin at different concentrations viz. 1ppm, 5ppm and 10ppm were used against \u003cem\u003eV. dahlia.\u003c/em\u003e A pathogenic fungal strain was inoculated in the close vicinity of the well. Biosurfactant compounds of different concentrations were poured into the well (Debode et al.2007). Plates were then incubated at 26\u003csup\u003e0\u003c/sup\u003e C for 5 days, and the zone of inhibition was measured (mm).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhytochemical extract formulation\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003ePhytochemical extract formulation\u003c/div\u003e \u003cp\u003eDifferent parts of floral plants, viz \u003cem\u003eClotropis procera, Azadirachta indica, Citrullus colocynthis, Ricinus communis, Tinospora cardifolia, Withania somnifera, Datura stramonium, and Solanum xanthocarpum\u003c/em\u003e were selected. Copper sulphate was also added. Selected plant parts were cut into small pieces with a sterile knife and soaked in cow\u0026rsquo;s urine with fermented acidophilus milk for 30 days in a large bucket. After 30 days, the extract was sieved through muslin cloth. Formulation in the form of a spray with a ratio of 1:9 (biochemical formulation: water) with endophytic bacterial strains was used for seed bacterization.\u003c/p\u003e\n\u003ch3\u003eAnalysis of biochemical formulation\u003c/h3\u003e\n\u003cp\u003eLiquid chromatography Mass Spectrometry technique was used for the analysis of biochemical formulation. One mg of biochemical formulation was sent to Central Drug Research Institute, Lucknow, India for LCMS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantifungal activity of biochemical formulation against\u003c/b\u003e \u003cb\u003eVerticillium dahliae.\u003c/b\u003e Antifungal activity of bioformulation was checked at 10ppm, 50ppm, and 100ppm concentration by the Well diffusion method (Debode et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Control consisting of distilled water was used and plates were incubated for 5 days at 26\u003csup\u003e0\u003c/sup\u003e C. Zones of inhibition were measured in mm. The experiment was done in triplicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSeed bacterization in polyhouse and field trials against\u003c/b\u003e \u003cem\u003eVerticillium dahliae\u003c/em\u003e\u003c/p\u003e \u003cp\u003eBacterization of delinted seeds of \u003cem\u003eVerticillium dahliae\u003c/em\u003e sensitive local variety Rasi 134 Bt was done for both field and pot experiments with slight modifications (G\u0026ouml;re et al. 2008). Nutrient Broth medium was used for antagonistic bacterial growth for 24 hours at 37\u003csup\u003e0\u003c/sup\u003e C. Centrifugation at 5000g for 5 min at 4\u003csup\u003e0\u003c/sup\u003eC was done to collect the pellets. Pellets were suspended in sterile phosphate buffer with 1.5% CMC solution. For control, seeds were\u003c/p\u003e \u003cp\u003etreated with 1.5% CMC without bacteria, and soaking of seeds with antagonistic bacterial suspension of 100mg with 1ml of 1.5% CMC was done. The mean concentration of bacterial strains was 1 X 10\u003csup\u003e8\u003c/sup\u003e cfu/ml\u0026thinsp;+\u0026thinsp;1.5% CMC for test in both polyhouse and field trials. After soaking, all the seeds were kept separately in sterile conditions in a laminar flow for 12 hours (B\u0026ouml;lek et al. 2005).\u003c/p\u003e \u003cp\u003e \u003cb\u003eScreening of biological control of\u003c/b\u003e \u003cb\u003eVerticillium\u003c/b\u003e \u003cb\u003ewilt in poly house and Natural Field conditions with and without biochemical formulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eVerticillium dahliae\u003c/em\u003e sensitive local cotton variety, was used for screening biochemical formulation along with an endophytic bacterial strain against wilting. A mixture of autoclaved soil-sand-peat-vermiculite in an equal ratio was transferred into plastic pots for sowing of both bacterized and control seeds (Naraghi et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Seeds treated with each endophytic bacterial strain and bacteria\u0026thinsp;+\u0026thinsp;bioformulation treatments were sown into pots for polyhouse experiments and in the natural field, 10-day-old culture of \u003cem\u003eV. dahliae\u003c/em\u003e was used for inoculation after sowing of bacterized seeds, diluted into 3x10\u003csup\u003e6\u003c/sup\u003e conidia/ml. Drops of conidial suspension (5-l0 drops) were inoculated into the stem of a cotton plant through a 22-gauge needle by puncturing the first internode of the stem at six true leaf stages (Hanson \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Cotton plants were treated with a 16-8-24 ratio of N-P-K liquid fertilizer. The experiment was repeated in triplicate for each bacterial strain. Non-bacterial plants were used as controls with sterile distilled water only. Disease severity index of each plant was checked after fifteen days of inoculation from sowing to harvesting period at 4 to 0 rating scale 4 = (Dead plant), 3\u0026thinsp;=\u0026thinsp;67\u0026ndash;97% (Wilt or Defoliation), 2\u0026thinsp;=\u0026thinsp;34\u0026ndash;66% (Necrosis), 1\u0026thinsp;=\u0026thinsp;1\u0026ndash;33% (Acropetal Chlorosis), 0 = (healthy plant) for both control and test plants (Bejarano-Alcazar et al., 1995).\u003c/p\u003e \u003cp\u003eBiocontrol protection % (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and disease index (Zhu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) were calculated using the formula mentioned below:\u003c/p\u003e \u003cp\u003e \u003cb\u003eDisease Incidence (%)\u003c/b\u003e = [(n1\u0026thinsp;+\u0026thinsp;n2\u0026thinsp;+\u0026thinsp;n3\u0026thinsp;+\u0026thinsp;n4)/n] x 100.,\u003c/p\u003e \u003cp\u003e \u003cb\u003eDisease Severity Index (%) =\u003c/b\u003e [(0n0\u0026thinsp;+\u0026thinsp;1n1\u0026thinsp;+\u0026thinsp;2n2\u0026thinsp;+\u0026thinsp;3n3\u0026thinsp;+\u0026thinsp;4n4)/4n x 100;\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiocontrol Protection%\u003c/b\u003e = 100- Vascular discoloration (treatment)/Vascular discoloration (control) x 100\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments, viz. antagonistic activity, hydrolytic enzymatic activity and \u003cem\u003ein vivo\u003c/em\u003e trials were repeated in triplicate. The results were represented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). IBM SPSS Statistics 22 software, USA, was used to statistically analyze all of the experiment data using one-way analysis of variance (ANOVA). Comparison of means was made by Scott- Knott analysis at 5% level of probability.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of pathogenic fungal strains from collected plant/soil samples\u003c/h2\u003e \u003cp\u003e \u003cem\u003eVerticillium dahlia\u003c/em\u003e was reported from infected cotton plant parts and rhizospheric and bulk soil. The pathogenic fungal strain isolated from infected samples showed whitish mycelium in the initial incubation period on PDA plates. Purification of fungi was performed by using PDA plates. \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e depicts the prevalence rate of \u003cem\u003eV. dahliae\u003c/em\u003e isolated from samples from different sites \u003cem\u003eV. dahliae\u003c/em\u003e was isolated from all samples of different sites. Prevalence rate of \u003cem\u003eVerticillium dahliae\u003c/em\u003e from 160 collected samples, of 20 different sites of Bathinda, Punjab, India, was 73%.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003ePercentage of\u003c/b\u003e \u003cb\u003eV. dahliae\u003c/b\u003e \u003cb\u003efrom collected samples-= Prevalence rate of\u003c/b\u003e \u003cb\u003eV. dahliae\u003c/b\u003e \u003cb\u003efrom total 8 sample fields=\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTotal number of fungal strains isolated from 8 sample fields- No of \u003cem\u003eV. dahliae\u003c/em\u003e isolated from 8 sample fields/100\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrevalence rate of\u003c/b\u003e \u003cb\u003eV. dahliae\u003c/b\u003e \u003cb\u003efrom total 8 sample fields=\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal number of fungal strains isolated from 5 infected sample fields- No of \u003cem\u003eV. dahliae\u003c/em\u003e\u003c/p\u003e \u003cp\u003eisolated from 5 infected sample fields/100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of endophytic bacterial strains from healthy plant parts\u003c/h2\u003e \u003cp\u003eSeven bacterial strains of endophytic nature were isolated from healthy cotton plant parts, viz., roots, stems, and leaves on Nutrient Agar medium (NAM) by streaking method and purified by streaking. The isolates were named as VTS DB-V1/NI/L/S1/B2 from the healthy stem, VG DB-V2/NI/L/S1/B1 from the healthy leaves of a cotton plant, collected from\u003c/p\u003e \u003cp\u003edifferent locations of Bathinda. A similar study was done by Mcinroy and Kloeeper in 1995, in which isolation of endophytic bacterial strains from cotton roots and stem tissues was reported. These seven isolates were further screened through antagonistic tests, and positive bacterial strains were identified through 16S rRNA sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003e18s rRNA sequencing for microbial Identification\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eVerticillium dahliae\u003c/em\u003e was reported from all infected plant parts of cotton and the rhizospheric and bulk soil of cotton fields. \u003cem\u003eV. dahliae\u003c/em\u003e was preliminarily identified by its morphological colony characters, air hyphae, and structure of microsclerotia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Conidiophores were erect, hyaline, verticillately branched, and septate. Phialides frequently arise directly from hyphae. Microsclerotia of \u003cem\u003eV. dahliae\u003c/em\u003e was dark brown to black in color, thick-walled, and abundant with almost swollen globular cells (Inderbitzin et al., 2011). Identification of microbes was done by PCR methods through the molecular 18S rRNA gene, which was conducted by outsourcing through Science Farmers, Bangalore, India, by the DNA extraction method. Integrated Transcribed Spacer (ITS1 5՜- 5՜-GTAGTCATATGCTTGTCTC-3՜) and (ITS4 5՜-CTTCCGTCAATTCCTTTAAG-3՜) primers were amplified by the 18S rRNA gene region, sequenced, and analyzed for similarity among species in the NCBI CBI database. A total of 7 endophytic bacterial strains were isolated and purified, out of which 3 strains exhibited positive antagonistic activity during \u003cem\u003ein vitro\u003c/em\u003e screening and were hence used for \u003cem\u003ein vivo\u003c/em\u003e trials. 16S rRNA gene identification by PCR was conducted by outsourcing through Barcode Bioscience Bangalore, India, by the DNA extraction method. Integrated Transcribed Spacer (ITS1 5՜- 5՜-GTAGTCATATGCTTGTCTC-3՜) and (ITS4 5՜-\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCTTCCGTCAATTCCTTTAAG-3՜) primers were amplified by the 16S rRNA gene region, sequenced, and analyzed for similarity among species in the NCBI CBI database. Three endophytic bacterial strains were identified through 16S rRNA and named as VTS DB- V1/NI/S/S1/B2 (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e), VTS DB-V1/NI/R/S1/B2 (\u003cem\u003eBacillus subtilis\u003c/em\u003e), and VTS DB-V1/NI/S/S1/B1 (\u003cem\u003eLysinibacillus macroides\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelection of endophytic bacterial strains as potential biocontrol agents Preliminary screening of endophytic bacterial strains by Dual Culture Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDuring this study, endophytic bacterial strains were isolated, and \u003cem\u003ein vitro\u003c/em\u003e screening was performed to check antagonistic activity against \u003cem\u003eV. dahliae\u003c/em\u003e. The dual culture method was the preliminary step to check the antagonistic activity because it eliminates the growth of the host\u003c/p\u003e \u003cp\u003epathogen, and most likely by direct antagonism against the pathogenic strain (Khan, 2013). Primary screening of all culture strains has been done through a dual culture assay for the selection of the best positive endophytic bacterial strains for \u003cem\u003ein vivo\u003c/em\u003e evaluation. Different endophytic bacterial strains showed \u003cem\u003ein vitro\u003c/em\u003e antagonistic activity towards \u003cem\u003eV. dahliae\u003c/em\u003e. The experiment was performed in triplicate for 0 to 144 hours of incubation. Maximum percentage of inhibition in growth was shown by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (VTS DB-V1/NI\u003c/p\u003e \u003cp\u003e/S/S1/B2) with 61.12% followed by \u003cem\u003eLysinibacillus macroides\u003c/em\u003e (VTS DB-V1/NI /S/S1/B1),\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus subtilis\u003c/em\u003e (VTS DB-V1/NI /R/S1/B2), VG DB-V1/NI /L/S1/B2, VTS DB-V1/NI\u003c/p\u003e \u003cp\u003e/L/S1/B2, VG DB-V2/NI /L/S1/B1 and VG DB-V1/NI /S/S1/B1 with 59.2%, 57.64%,\u003c/p\u003e \u003cp\u003e56.25%, 48.62%, 45.2% and 40.29% respectively during Dual culture assay. Statistical analysis in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the \u003cem\u003ein vitro\u003c/em\u003e antagonism against \u003cem\u003eV. dahliae\u003c/em\u003e, in which mean values with stars represent a significant difference between compared means at 0- 144 hours of incubation period. Values without a star on means of represent a lack of significant differences at different incubation times (ANOVA, p\u0026thinsp;\u0026ge;\u0026thinsp;0.05)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAssay for cell wall-degrading enzymes in endophytic bacterial strains through\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003etests\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eCellulase Enzyme Assay\u003c/h2\u003e \u003cp\u003eAdditional \u003cem\u003ein vitro\u003c/em\u003e screening served as a useful secondary parameter for the selection of biocontrol agents, as it facilitated the degradation of pathogenic fungal cell walls through hydrolytic enzymatic activity. Endophytic bacterial strains were further evaluated for their biocontrol potential by assessing their \u003cem\u003ein vitro\u003c/em\u003e antagonistic activity during hydrolytic enzyme production. Cellulase enzyme assay was performed by growing the isolates in liquid Czapek Mineral Salt Medium. After five days of incubation, the endophytic bacterial strains produced varying inhibition zones around the wells. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (VTS DB- V1/N1/S/S1/B2) exhibited the maximum inhibition zone (8.56 mm), followed by \u003cem\u003eLysinibacillus macroides\u003c/em\u003e (VTS DB-V1/N1/S/S1/B1) (7.3 mm), VTS DB-V1/N1/L/S1/B2 (6.8 mm), \u003cem\u003eBacillus subtilis\u003c/em\u003e (VTS DB-V1/N1/R/S1/B2) (5.23 mm), VG DB-V2/N1/L/S1/B2 (5.2 mm), VG DB-V2/N1/L/S1/B2 (5.0 mm), and VG DB-V1/N1/S/S1/B1 (4.5 mm) during cellulase activity. Among these, \u003cem\u003eP. aeruginosa\u003c/em\u003e (VTS DB-V1/N1/S/S1/B2) demonstrated the most pronounced inhibition, followed by \u003cem\u003eL. macroides\u003c/em\u003e. Statistical analysis in Table\u0026nbsp;2 revealed no significant differences (LSD test) among the mean values of VTS DB- V1/N1/L/S1/B2, VG DB-V1/N1/L/S1/B2, VG DB-V2/N1/L/S1/B1, VG DB-V1/N1/S/S1/B1,and VTS DB-V1/N1/R/S1/B2, as indicated by the same alphabetic grouping. Conversely, the mean values of VTS DB-V1/N1/S/S1/B2 and VTS DB-V1/N1/S/S1/B1 differed significantly, as denoted by distinct alphabetic representations (ANOVA, p\u0026thinsp;\u0026ge;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProtease Enzyme Assay\u003c/h2\u003e \u003cp\u003eProtease activity was assessed on Casein Agar medium. During this assay, endophytic bacterial strains produced clear inhibition zones around the wells compared to the control. All experiments were performed in triplicate. The maximum inhibition zone was observed in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (VTS DB-V1/N1/S/S1/B2) (8.70 mm), followed by \u003cem\u003eLysinibacillus macroides\u003c/em\u003e (VTS DB-V1/N1/S/S1/B1) (7.32 mm), \u003cem\u003eBacillus subtilis\u003c/em\u003e (VTS DB- V1/N1/R/S1/B2) (6.66 mm), VG DB-V1/N1/L/S1/B2 (5.5 mm), VG DB-V2/N1/L/S1/B1 (4.2 mm), VG DB-V1/N1/S/S1/B1 (3.9 mm), and VTS DB-V1/N1/L/S1/B2 (3.2 mm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eChitinase Enzyme Assay\u003c/h2\u003e \u003cp\u003eChitinase activity was evaluated on Chitinase Detection Medium containing basal chitin. The growth of purple-coloured zones was measured as an indicator of enzymatic activity, and all assays were carried out in triplicate. Endophytic bacterial strains produced distinct purple inhibition zones around the wells, reflecting significant chitinase activity. The largest zone was observed in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (VTS DB-V1/N1/S/S1/B2) (7.4 mm), followed by \u003cem\u003eLysinibacillus macroides\u003c/em\u003e (VTS DB-V1/N1/S/S1/B1) (7.1 mm), \u003cem\u003eBacillus subtilis\u003c/em\u003e (VTS DB- V1/N1/R/S1/B2) (6.3 mm), VG DB-V1/N1/L/S1/B2 (6.1 mm), VTS DB-V1/N1/L/S1/B2 (5.7 mm), VG DB-V2/N1/L/S1/B1 (4.5 mm), and VG DB-V1/N1/S/S1/B1 (3.9 mm).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiosurfactants antifungal activity against\u003c/b\u003e \u003cb\u003eV. dahliae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the present investigation, Fengycin and Iturin have been evaluated as potential biocontrol agent against \u003cem\u003eV. dahliae\u003c/em\u003e by checking their \u003cem\u003ein vitro\u003c/em\u003e antifungal activity by well diffusion assayas depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The experiment was performed at different concentration viz., 5ppm and 10 ppm showing 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1ab mm and 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05b mm inhibition zones respectively and Iturin at the same concentrations showing 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15b mm, 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2ab mm inhibition zones, respectively as compared to control plate. Both Fengycin and Iturin have shown no inhibition zones against \u003cem\u003eV. dahliae\u003c/em\u003e at 1ppm concentration. Statistical test was performed as ANOVA, p\u0026thinsp;\u0026ge;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS of biochemical formulation:\u003c/h2\u003e \u003cp\u003eLCMS has been widely applied in the field of both proteomics and metabolomics research. This spectrometry is considered to highly advanced and analytical technology. LCMS is used for the profiling of different compounds in samples with further structural elucidation. The different compounds in samples can be detected relatively or absolutely through analysing spectral features from LC-MS data. The compounds' identification in the LCMS chromatograph is carried out based on mass-to-charge ratio and retention time. In our study, profiling of phytoconstituents in aqueous extract of different parts of \u003cem\u003eRicinus communis, Tinospora cardifolia, Withania somnifera, Datura stramonium, Clotropis procera, Azadirachta indica, Citrullus colocynthis, Solanum xanthocarpum\u003c/em\u003e was carried out through positive and negative ionization MS-ESI mode of LC-MS method. The LC-MS chromatograms in positive and negative ionization mode are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In comparison with the previous literature, the bioactive compounds were characterized by their mass spectrum, retention times, and fragmentation profiles. The list of identified phytocompounds with their molecular weight, retention time, monoisotopic mass, and chemical formula is shown in \u003cb\u003eTable\u0026nbsp;3.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSoil solarization of natural field before sowing of seeds\u003c/h2\u003e \u003cp\u003eThe soil solarization technique was followed before sowing of seeds under natural field conditions. The field was prepared according to the experimental field layout as mentioned earlier. Four equal ridges have been prepared with a wooden leveler and properly covered with polythene sheets for 30 days. No growth of \u003cem\u003eV. dahliae\u003c/em\u003e was recorded after soil solarization. Hence, soil solarization plays an important role in controlling wilt disease.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSoil sterilization and seed bacterization for polyhouse experiments\u003c/h2\u003e \u003cp\u003eEvaluation of potential antagonistic microbial strains against \u003cem\u003eVerticillium dahliae\u003c/em\u003e was performed in polyhouse conditions. Soil has been tested for the non-presence of \u003cem\u003eV. dahliae\u003c/em\u003e periodically after sterilization before sowing of seeds. \u003cem\u003eVerticillium dahliae\u003c/em\u003e was not observed in any PDA plate after soil sterilization. Bacterization of delinted seeds of \u003cem\u003eVerticillium dahliae\u003c/em\u003e sensitive local variety Rasi 134 Bt was done for both field and pot experiments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eBiological Management of Cotton Verticillium Wilt by Endophytic Bacterial Strains With and Without Biochemical Formulation\u003c/h2\u003e \u003cp\u003eDuring the current research, \u003cem\u003ePseudomonas aeruginosa, Lysinibacillus macroides, and Bacillus subtilis\u003c/em\u003e were assessed as biocontrol agents against \u003cem\u003eVerticillium dahliae\u003c/em\u003e under natural field conditions after showing strong \u003cem\u003ein vitro\u003c/em\u003e antagonistic activity. The tests were carried out under monoculture conditions, and the incidence of wilt was observed fortnightly (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn control plants without treatment, disease symptoms became apparent following the flowering phase in August, with yellowing and interveinal chlorosis among leaf veins. Bacterized plants, however, manifested significantly lower disease incidence. \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eL. macroides\u003c/em\u003e\u0026ndash;treated seeds completely suppressed wilt symptoms (0% severity), while \u003cem\u003eB. subtilis\u003c/em\u003e\u0026ndash;treated seeds displayed mild acropetal chlorosis (28% severity). Following 135 days of planting, control plants had 88% wilt mortality, while the treatments with biocontrol strongly suppressed wilt incidence. The maximum biocontrol activity was on \u003cem\u003eP. aeruginosa\u003c/em\u003e (73.6%), followed by \u003cem\u003eL. macroides\u003c/em\u003e (68.5%) and \u003cem\u003eB. subtilis\u003c/em\u003e (62.5%) (Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eTo improve further disease control, the same bacterial isolates were compared in combination with a biochemical formulation sprayed over the foliage fortnightly. Wilt suppression under this treatment improved. Seeds bacterized with \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eL. macroides\u003c/em\u003e again showed complete protection (0% severity), while \u003cem\u003eB. subtilis\u003c/em\u003e recorded 43% severity. After 135 days, mortality in non-bacterized controls reached 82%, whereas biocontrol protection was significantly higher in treated plants: \u003cem\u003eP. aeruginosa\u003c/em\u003e (76.6%), \u003cem\u003eL. macroides\u003c/em\u003e (69.5%), and \u003cem\u003eB. subtilis\u003c/em\u003e (58.3%).\u003c/p\u003e \u003cp\u003eIn general, seed bacterization and bacterization plus biochemical formulation both lowered wilt severity and improved plant survival. In the strains that were tested, \u003cem\u003eP. aeruginosa\u003c/em\u003e always showed the greatest biocontrol activity, followed by \u003cem\u003eL. macroides\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e. These findings proved the role of endophytic bacteria, specifically \u003cem\u003eP. aeruginosa\u003c/em\u003e, as promising biocontrol agents in controlling Verticillium wilt of cotton. Biological management of Verticillium wilt of cotton under polyhouse tests with and without biochemical formulation\u003c/p\u003e \u003cp\u003ePolyhouse tests were done to assess the biocontrol potential of endophytic bacterial strains, with and without biochemical formulation (BCF), against Verticillium dahliae. Cotton seeds were bacterized with \u003cem\u003ePseudomonas aeruginosa, Lysinibacillus macroides\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003esubtilis\u003c/em\u003e, whereas non-bacterized seeds were taken as controls. Wilt symptoms were observed on regular intervals after artificial inoculation with \u003cem\u003eV. dahliae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn non-bacterized controls, initial symptoms were light-yellow interveinal chlorosis followed by a rapid development of wilting. The disease severity index reported 89% incidence of wilt in controls. In contrast, seeds bacterized with \u003cem\u003eP. aeruginosa\u003c/em\u003e were symptomless (0% severity), whereas seeds treated with \u003cem\u003eL. macroides\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e were partially protected with disease severities of 19% and 32%, respectively (0\u0026ndash;4 rating scale). Corresponding biocontrol protection was observed as 69.6% (\u003cem\u003eP. aeruginosa\u003c/em\u003e), 65.2% (\u003cem\u003eL. macroides\u003c/em\u003e), and 62.5% (\u003cem\u003eB. subtilis\u003c/em\u003e), which is significantly different from that of the controls (36.4%, 29.7%, and 32.2%) (Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eAt the application of biochemical formulation in combination with seed bacterization, suppression of wilt further increased. Control plants again exhibited high disease infection (86%), whereas \u003cem\u003eP. aeruginosa\u003c/em\u003e\u0026thinsp;+\u0026thinsp;BCF and \u003cem\u003eL. macroides\u003c/em\u003e\u0026thinsp;+\u0026thinsp;BCF treatments fully suppressed wilt symptoms (0% severity). \u003cem\u003eB. subtilis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;BCF decreased the severity to 32%, which showed moderate protection. Protection in biocontrol with these treatments was ascertained as 70.6% (\u003cem\u003eP. aeruginosa\u003c/em\u003e), 68.6% (\u003cem\u003eL. macroides\u003c/em\u003e), and 64.5% (\u003cem\u003eB. subtilis\u003c/em\u003e), whereas the controls varied between 36.5% and 39.2% (Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eGenerally, all results confirmed that \u003cem\u003eP. aeruginosa\u003c/em\u003e always posed the highest protection against wilt, both when used individually as well as in combination with biochemical formulation, followed by \u003cem\u003eL. macroides\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e. The enhanced activity with biochemical formulation indicated a synergistic action, which augmented the potential of bacterial strains to inhibit \u003cem\u003eV. dahliae\u003c/em\u003e. These results are consistent with previous reports that microsclerotia of \u003cem\u003eV. dahliae\u003c/em\u003e infect roots to cause infection (El-Zik, 1985; Berg et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Mercado-Blanco et al., 2004), and that endophytic bacteria can inhibit pathogen activity in the root interface.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eAssessment of plant growth-promoting potential of endophytic bacterial strains\u003c/h2\u003e \u003cp\u003eEndophytic bacterial strains were reported to possess potential beneficial effects on the growth on cotton plants. However, their biocontrol efficacy of endophytic bacterial strains had a relationship with their \u003cem\u003ein vitro\u003c/em\u003e antagonistic activity against \u003cem\u003eV. dahliae\u003c/em\u003e or their cell wall- degrading enzymes. Table\u0026nbsp;10\u0026ndash;11 depicts bacterial strains aiding in increased biomass or cotton yield, and Fig.\u0026nbsp;10 shows the effect of bacterial strains on the height of treated plants in\u003c/p\u003e \u003cp\u003eboth polyhouse and field trials as compared to control plants. A study on growth parameters, viz. number of leaves, height, canopy development, and number of bolls under poly house and natural field conditions with statistical analysis was conducted, and means with a star denoted a significant difference, and lack of a star shows no significant difference between the values (ANOVA, p\u0026thinsp;\u0026ge;\u0026thinsp;0.05). Yield and growth of seed bacterized plants with \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e under natural field conditions were more than \u003cem\u003eLysinibacillus macroides\u003c/em\u003e followed by \u003cem\u003eBacillus subtilis\u003c/em\u003e when compared with control (non-bacterized). Endophytic bacteria such as \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is considered as most significant bacteria and act as a biocontrol agent to help in growth of plant (Spiers et al. 2020). Disease severity and symptoms were significantly reduced after seed bacterization with \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e followed by \u003cem\u003eLysinibacillus macroides\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e in both poly house and field trials.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, soil-borne pathogenic fungus \u003cem\u003eVerticillium dahliae\u003c/em\u003e was obtained from infected samples of unhealthy cotton fields, and potential biocontrol bacterial strains were isolated from healthy cotton plant parts. Results of growth parameters clearly indicate a significant increase with bacterial strains when compared to control plants. Suppression of wilt symptoms was also recorded during pot and field trials in bacterized cotton plants. The major effect of \u003cem\u003eV. dahliae\u003c/em\u003e was defoliation, and the poor growth and development of the cotton plant were also significantly suppressed by \u003cem\u003ePseudomonas aeruginosa, Lysinibacillus macroides\u003c/em\u003e, and \u003cem\u003eBacillus subtilis\u003c/em\u003e. Wilt symptoms were recorded after three months of non- bacterized seeds sowing under controlled and natural field conditions as compared to bacterized seeds. Endophytic bacterial strains under polyhouse and natural conditions field trials of Bathinda, Punjab in combination with biochemical formulation, have shown positive biocontrol efficacy in integrated management of wilt disease. This approach also affects on growth of plants and increases the yield of cotton in the Bathinda region.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding sources\u003c/h2\u003e \u003cp\u003eThis work is partially supported from the grant of Lovely Professional University.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contribution Kamaldeep Kaur: Investigation, Formal analysis, Writing\u0026ndash; original draft. MM Mehdi: review \u0026amp; editing. JoyDeep Dutta: review \u0026amp; editing. Shalini Pathak, SharonNagpal, Faisal Mushtaq: review \u0026amp; editing. Ashish Vyas: Conceptualization, Methodology, Resources, Supervision\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthor acknowledged Lovely Professional University (LPU), Punjab, India for providing facility in lab.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe original contributions presented in the study are included in the article, and further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAyele A G, Wheeler T A and Dever J K (2020) Impacts of Verticillium Wilt on Photosynthesis Rate, Lint Production, and Fiber Quality of Greenhouse-Grown Cotton (Gossypium hirsutum) Plants 9(7):857\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerg G, Fritze A, Roskot N and Smalla K (2001) Evaluation of potential biocontrol rhizobacteria from different host plants of Verticillium dahliae Kleb. 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Biol Con 56(3):209\u0026ndash;216\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cotton, biological control, biochemical formulation, enzymatic activity, field trials","lastPublishedDoi":"10.21203/rs.3.rs-8359830/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8359830/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study aimed to elucidate the potential of biological control bacterial strains, viz. \u003cem\u003ePseudomonas aeruginosa, Lysinibacillus macroides\u003c/em\u003e, and \u003cem\u003eBacillus subtilis\u003c/em\u003e against \u003cem\u003eV. dahliae\u003c/em\u003e causing wilt of Cotton. Out of seven, three strains, viz. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eLysinibacillus macroides\u003c/em\u003e, and \u003cem\u003eBacillus subtilis\u003c/em\u003e have shown significant antagonistic effects in dual culture assay. Antagonistic assays, viz., \u003cem\u003ein vitro\u003c/em\u003e inhibition, \u003cem\u003ein vivo\u003c/em\u003e field trials, and enzymatic studies revealed significantly superior potential for wilt in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e under natural field and pot trials. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e bacterized plants also exhibited higher growth metrics and productivity. The current combinatorial study effectively promotes the use of bacterial biocontrol strains for the reduction in disease incidence, promoting crop yield and growth by combining soil solarization and biochemical formulation for sustainability.\u003c/p\u003e","manuscriptTitle":"Combinatorial approach in evaluation of biocontrol bacterial species with biochemical formulation mitigating Verticillium wilt of cotton","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 14:10:40","doi":"10.21203/rs.3.rs-8359830/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"77b4b88a-709b-428b-bfcb-6786b34a1a04","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T17:23:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 14:10:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8359830","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8359830","identity":"rs-8359830","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}