The Impact of Biocontrol Bacteria on  Cotton Resistance and Their Effects on Signaling Pathways Related to Defense Against Verticillium Wilt Infection

The Impact of Biocontrol Bacteria on Cotton Resistance and Their Effects on Signaling Pathways Related to Defense Against Verticillium Wilt Infection | 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 The Impact of Biocontrol Bacteria on Cotton Resistance and Their Effects on Signaling Pathways Related to Defense Against Verticillium Wilt Infection Yongbin Fan, Jianwei Cao, Yuanyuan Liu, Chongdie Wu, Jingyi Ye, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4479911/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 This study prepared a mixed fermentation broth using two strains of Bacillus and investigated its inhibitory effects on the cotton Verticillium wilt pathogen, as well as its impact on the signaling pathways related to defense against Verticillium wilt infection in cotton.Biocontrol bacteria can effectively defend against plant diseases by competitively inhibiting pathogens and inducing plant immunity. Through plate confrontation assays, antimicrobial tests using mixed microbial fermentation broth and its dilutions, and their impacts on cotton seed germination, this study explores the defensive potential of the mixed fermentation broth.During the study, it was discovered that The mixed microbial fermentation broth could produce lipopeptide substances. The cotton's immunity against Verticillium wilt, following treatment with this broth, was assessed using DAB and trypan blue histological staining methods. Furthermore, the study involved monitoring the induced expression of resistance-related genes (PR1, PR5, NPR1), as well as the effects on the activities of defense-related enzymes in cotton (SOD, CAT, PPO, POD).The results indicate that The combination of two biocontrol bacterial strains exhibited a certain inhibitory effect on the cotton Verticillium wilt pathogen. Root drenching with the mixed fermentation broth significantly enhanced the transient burst of reactive oxygen species in cotton's defense signaling pathways, inducing an immune response. This response increased the sensitivity of cotton's hypersensitive response (HR), induced the expression of disease resistance-related genes, and heightened the activity of enzymes involved in reactive oxygen species scavenging, thereby enhancing systemic acquired resistance (SAR) in cotton. This study reveals that the mixed fermentation broth improved cotton's resistance to Verticillium wilt, significantly affecting the defense signaling pathways in response to the pathogen, with varying effects on induced resistance in different resistance genotypes of cotton. Mixed microbial fermentation broth Cotton Verticillium wilt Antimicrobial activity Lipopeptides Induced resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Cotton Verticillium wilt, often referred to as ‘cotton cancer,' has been spreading in China since its introduction in 1935 with the import of American cotton varieties(Mokhtari et al., 2023 ). Since the 1990s, this disease has spread to all cotton-growing areas, including Xinjiang. In 2023, the affected area reached 135,000 hectares, primarily in localized regions of Bortala Mongol, Changji Hui Autonomous Prefecture, and Aksu. The pathogen of Verticillium wilt ( Verticillium dahliae Kleb) belongs to the Fungi Imperfecti, class Hyphomycetes, order Hyphomycetales, family Plectosphaerellaceae, and genus Verticillium (Inderbitzin et al., 2013 ). Currently, the strains causing Verticillium wilt in Xinjiang cotton are primarily Verticillium dahliae and its subspecies. This pathogen is characterized by a wide host range, strong infectivity, significant destructiveness, and high variability. The low pollution, low cost, and environmentally friendly nature of biological control have made it an increasingly popular field of study in the control of pathogens in recent years. The mechanisms of biological control of pathogens mainly include competition for nutrients and space, antagonism; it can also induce plants to develop broad-spectrum, systemic, and non-specific systemic resistance, resisting the invasion of pathogens(Borriss et al., 2019 ;Yao et al., 2023 ;Wang et al., 2018 ;Köhl et al., 2019 ;Boulahouat et al., 2023 ). Biocontrol bacteria induce a variety of defense responses in plants. upon pathogen invasion, plants rapidly initiate a hypersensitive response (HR), and local resistance genes in the tissues surrounding the infection site are activated. This leads to the rapid accumulation of various defense-related enzymes and antimicrobial phytoprotectants, affecting the entire plant(Ji et al., 2022 ). The Pathogenesis-Related (PR) protein gene family plays a crucial role in the immune system of plants, particularly in the Systemic Acquired Resistance (SAR) response(Yu et al., 2021 ;Abu-Bakar et al., 2021 ;Han et al., 2022). The PR gene family comprises multiple members, among which PR1 , PR5 , and NPR1 are key genes that have been extensively studied(Li et al., 2021;Yu et al., 2022 ;Köhl et al., 2019 ). The PR1 gene is one of the earliest identified pathogenesis-related genes and is commonly used as a marker gene for SAR response(Brambilla et al., 2023 ).The PR5 gene encodes a protein similar to trypsin inhibitors in animals and exhibits antifungal activity(Ng et al., 2013 ). NPR1 is a key regulatory factor crucial for Systemic Acquired Resistance (SAR) in plants. It is activated upon sensing pathogen attack signals and regulates the expression of downstream PR genes, including PR1 and PR5 (Backer et al., 2019 ). The expression and function of these genes are not isolated but form an internal defense network in plants, collectively participating in the fight against the invasion of external pathogens. Plants are capable of constructing a multi-tiered defense system to effectively resist various pathogens(Li et al., 2021b ;Rezaei et al., 2019 ;Nie et al., 2017 ). In the defense of cotton against Verticillium wilt, the expression of disease resistance-related genes is induced, which helps to enhance the disease resistance of cotton and reduce the impact of the disease. Investigating the antibacterial capability of composite biocontrol bacteria, their role in inducing resistance in cotton, and their impact on the signaling pathways related to defense against Verticillium wilt infection provides a theoretical and practical foundation for the development and practical utilization of microbial agents. 2. Materials and methods 2.1 Materials 2.1.1 Test Strains and Cotton Varieties Pathogen: Verticillium dahliae Vd-278, a non-defoliating physiological race and microsclerotia-producing strain, provided by the Key Laboratory of Biotechnology at Xinjiang Agricultural Reclamation Science Academy. Test Strains: Bacillus tequilensis C-9 and Sphingobacterium A1, isolated and preserved by the Key Laboratory of Agricultural Biotechnology at Shihezi University. Cotton Varieties: Xinluzao 7 (susceptible genotype); Xinluzao 33 (resistant genotype). 2.1.2 Culture Media(Zhao et al., 2016) PDA: potato 200 g, sucrose 20 g, agar 20 g, distilled water 1000 ml, pH 7.0. LB Medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 20 g, distilled water 1000 ml, pH 7.0. 2.2 Antimicrobial Activity Test of Volatile Compounds from Biocontrol Bacterial Strains A 5 mm diameter sterile punch was used to make holes in PDA plates inoculated with Verticillium dahliae Vd-278. The mycelial plugs were then transferred to the center of new PDA plates. On two other new PDA plates, Strains C-9 and A1 were streaked to cover the entire surface. These plates with C-9 and A1 were then inverted over the Verticillium dahliae-inoculated plates and sealed. The plates inoculated with Verticillium dahliae were incubated inverted in an incubator at 25°C for 5–7 days. Post incubation, The colony diameter of Verticillium dahliae was measured using the cross method(Cheng et al., 2020 ). 2.3 Assessment of the Inhibitory Effects of Mixed Microbial Fermentation Filtrate on Verticillium Wilt and Its Spores 2.3.1 Preparation of Mixed Microbial Fermentation Filtrate Separate inoculations of C-9 and A1 were made into 50 ml potato dextrose broth (PDB) in autoclaved flasks at 121°C for 15 minutes. These were then incubated at 160 rpm and 28°C on a shaker for 18 hours to obtain the fermentation seed liquids of C-9 and A1. The seed liquids were then mixed in a 9:1 ratio, and 5% of this mixture was inoculated into new flasks containing 50 ml of sterilized PDB. After incubation at 160 rpm and 28°C for 3 days, the cultures were centrifuged at 5000 rpm for 10 minutes to remove the bacterial cells. The supernatant was filtered through a 0.45 µm pore size filter to remove any remaining bacteria and transferred into sterile Eppendorf tubes for further use(Huang et al., 2022 ). 2.3.2 Preparation of Verticillium dahliae Spore Suspension A 5 mm diameter punch was used to create holes in PDA plates inoculated with Verticillium dahliae . Two mycelial plugs were then inoculated into a flask containing 50 ml PDB and incubated at 160 rpm and 25°C on a shaker for 7–10 days. The culture was filtered through eight layers of sterile gauze to remove the mycelia, and the spore suspension of Verticillium dahliae was diluted to 10^7 cfu/ml before being transferred into sterile Eppendorf tubes for later use(Bandi et al., 2022 ). 2.3.3 Inhibition Test of Mixed Microbial Fermentation Filtrate on Verticillium dahliae and Its Spores 100 µl of 10^7 CFU/ml Verticillium dahliae spore suspension was evenly spread on PDA solid medium. Three equidistant holes were punched 2 cm away from the center of the PDA medium. Mycelial plugs were taken, and the prepared mixed microbial fermentation filtrate was diluted to concentrations of 10%, 25%, and 50% of the original. 100 µl of the original and each diluted fermentation filtrate were added into the three holes on different PDA plates (control group added 100 µl liquid PDB). After incubation at 25°C for 5–7 days, the plates were observed and the diameters of the inhibition zones were measured using the cross method. The mixed fermentation filtrate was mixed 1:1 with Verticillium dahliae spore suspension in Eppendorf tubes (control was a mixture of PDB and spore suspension), and incubated on a shaker at 25°C for 24–48 hours. Once the germination rate of spores in the control exceeded 80%, the germination of spores in the treated group was observed under a microscope(AlSalam A. AlKarem and .Kamran. Hasan, 2023). 2.4 Crude Extraction of Lipopeptides and their Antimicrobial Activity Assessment 2.4.1 Lipopeptide Extraction The mixed fermentation broth of C-9 and A1 was centrifuged at 8000 rpm for 20 minutes, and the supernatant was transferred to a sterilized conical flask. 6 mol/L HCl was slowly added to the supernatant with stirring until the pH reached 2.0. The mixture was then left overnight at 4°C. After another centrifugation at 8000 rpm for 20 minutes, the precipitate was collected and allowed to settle for 30–60 minutes. Methanol at pH 7.0 was added to the precipitate and the mixture was left to extract at 4°C for 10 hours (the extraction was repeated twice). The combined extracts were then centrifuged at 4500 rpm for 10 minutes to collect the supernatant, which served as the crude lipopeptide extract. The crude extract was filtered through a 0.45 um sterile filter and aliquoted into sterile Eppendorf tubes for later use(López-Gutiérrez et al., 2022 ;Armbruster and Meredith, 2018 ;Ma et al., 2020 ). 2.4.2 Inhibition Effect of Lipopeptides on Cotton Verticillium Wilt 100 µl of 10^7 CFU/ml Verticillium dahliae spore suspension was evenly spread on a PDA plate. A hole of 5mmdiameter was punched at the center of the plate and the mycelial plug was removed. 100 µl of the aforementioned crude lipopeptide extract was then added to the hole (PDB medium was used for the control). After 3 days, the diameter of the clear zone was measured using the cross method(Nacef et al., 2023 ;Carvalho et al., 2022 ). 2.4.2.1 Effects of Lipopeptides on Spore Yield, Soluble Protein Production, Mycelial Yield, and Cell Membrane Permeability of Cotton Verticillium Wilt The mixed microbial fermentation filtrate and prepared lipopeptides were inoculated at a 5% inoculum rate into new flasks containing 50 ml PDB (control group inoculated with 5% fresh PDB). A 5mmdiameter punch was used on PDA plates of Verticillium dahliae , and mycelial plugs were added to The flasks (2 plugs per flask), which were then incubated at 160 rpm and 25°C for 7 days. The treated Verticillium dahliae spore suspension was filtered through eight layers of sterile gauze to remove the mycelia, and spore yield was counted under a microscope using a hemocytometer. The spore suspension was centrifuged at 5000 rpm for 10 minutes, discarding The pellet and retaining the supernatant, and the soluble protein content was measured using the Coomassie brilliant blue method(Nagata et al., 2022 ). The mycelia collected by filtering through eight layers of sterile gauze were dried at 60°C for 4 hours and then weighed. The collected mycelia were washed five times with sterile water, vacuum-filtered for 15 minutes, and resuspended in 20 ml sterile water. The conductivity of the sterile water was measured every 10 minutes for 2 hours using a conductivity meter, with The final conductivity determined after boiling the resuspended mycelia in sterile water for 5 minutes(Hao et al., 2023 ;Lin et al., 2023 ;Cai et al., 2023 ). The relative electrical conductivity of the Verticillium dahliae mycelium was calculated using the following formula: Relative Electrical Conductivity (%) = Measured Conductivity / Final Conductivity × 100% 2.4.2.2 The Effect of Lipopeptides and Mixed Microbial Fermentation Filtrate on Cotton Seed Germination and Disease Resistance One hundred full-grain Xinluzao 7 cotton seeds were selected and divided into two batches of 50 seeds each. Each batch of 50 seeds was placed in two beakers containing 95 ml of water, with 5 ml of the mixed microbial fermentation filtrate added to one beaker and 5 ml of lipopeptide solution to the other, for a soaking period of 30 minutes (Sterile water was used for the control group). on two layers of filter paper in 5 cm diameter petri dishes, 1 ml of sterile water was applied evenly with a rod to fully wet the filter paper. the first batch of soaked seeds was then placed on the wet filter paper using tweezers (three seeds per Petri dish), and incubated at 25°C for 2 days for germination (1 ml of water was added every 6 hours to keep the filter paper moist). The second batch of seeds was placed on the wet filter paper and an additional 1 ml of the prepared Verticillium dahliae spore suspension was spread evenly with a rod, and incubated at 25°C for 2 days for germination (1 ml of water was added every 6 hours to keep the filter paper moist). The experimental steps for Xinluzao 33 cotton seeds were the same as for Xinluzao 7(Chattopadhyay et al., 2015 ). 2.5 Induction of Resistance in Cotton by Biocontrol Bacteria 2.5.1 Detection of Reactive Oxygen Species (ROS) Burst When cotton plants developed two true leaves, Xinluzao 7 and 33 (three pots each) were root-drenched with 50 ml of mixed microbial fermentation filtrate per pot for 7 days (control groups were treated with an equal volume of PDB liquid). The treated and control cotton plants were then subjected to root drenching with 50 ml of 10^7 CFU/ml Verticillium dahliae spore suspension using the root injury method. After 48 hours, the third leaves from the top of each treatment group were subjected to DAB staining in the dark for 8 hours(Daudi and O’Brien, 2012 ), followed by decolorization in 95% alcohol in a boiling water bath until the green color of the leaves completely faded. The leaves were then cleared of air bubbles in 70% glycerol for microscopic observation and photography. 2.5.2 Detection of the Hypersensitive Response (HR) Third leaves from the top of Xinluzao 7 and 33 cotton plants, 48 hours after treatment with Verticillium dahliae spore suspension, were submerged in trypan blue stain solution (1g phenol, 0.1 mg trypan blue, 1 ml lactic acid, 1 ml glycerol dissolved in 50 ml sterile water) and subjected to a boiling water bath for 15 minutes for staining. After cooling to room temperature, the leaves were decolorized in 2.5 g/ml chloral hydrate for 30 minutes, followed by decolorization in 95% alcohol in a boiling water bath until the green color of the leaves completely faded. The leaves were then cleared of air bubbles in 70% glycerol for microscopic observation and photography(Thordal-Christensen et al., 1997 ). 2.5.3 Detection of Induced Resistance in Cotton Functional leaves of Xinluzao 7 and 33 cotton plants, 48 hours post-treatment with Verticillium dahliae spore suspension, were disinfected with 10% sodium hypochlorite and placed on PDA plates. Using A 5mmdiameter punch, holes were made in plates fully grown with Verticillium dahliae, and mycelial plugs were placed on the surface of the cotton leaves (one plug per leaf). The samples were then incubated at a constant temperature of 25°C for 7 days, after which the disease progression on the leaves was observed and photographed. 2.5.4 Detection of Induced Root Vitality Roots of Xinluzao 7 and 33 cotton plants, 48 hours post-treatment with Verticillium dahliae spore suspension, were rinsed with tap water. A 0.3 g sample of the roots was then weighed and root vitality was determined using the TTC (Triphenyl Tetrazolium Chloride) method(Richter et al., 2007 ). 2.6 The Induction of Defense Enzyme Activities and the Effect on Defense Enzyme Gene Expression in Cotton by Mixed Microbial Fermentation Filtrate 2.6.1 Methods for Detecting Defense-Related Enzyme Activity When cotton plants developed two true leaves, Xinluzao 7 and 33 (three pots each) were treated with 50 ml of mixed microbial fermentation filtrate per pot (control group was treated with an equivalent volume of PDB liquid). leaves were collected every 24 hours for 7 days, rinsed with tap water, and dried with filter paper for the measurement of defense-related enzyme activities in cotton. Seven days post treatment with the fermentation filtrate, the plants were root-drenched with 50 ml of 10^7 CFU/ml Verticillium dahliae spore suspension. leaves were collected every 12 hours, rinsed, and dried as before, for the measurement of defense-related enzyme activities(Alici and Arabaci, 2016 ). 2.6.2 Real-Time Quantitative PCR(Livak and Schmittgen, 2001 ) cotton leaves stored at -80°C were taken out, and RNA was extracted using the Polysaccharide and Polyphenol Plant RNA Extraction Kit (R8611). High-quality RNA was then reverse-transcribed. The RNA reverse transcription was conducted according to the instructions of the TransScript II First-Stand cDNA Synthesis SuperMix Kit by All-In-One. β-Tubulin gene (Actin-8) in cotton was used as an internal reference. The reactions were carried out using the SYBR Green I Master Mix Kit by Roche and amplified using the Light Cycler® 480 II PCR system (Roche). The reaction conditions were 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 seconds, 56°C for 15 seconds, and 72°C for 20 seconds. Each sample was repeated three times, and data analysis was performed using the 2-ΔΔCT method(Michaelidou et al., 2013 ). 3. Result and Discussion 3.1 Antimicrobial Activity Test of Volatile Compounds from Biocontrol Bacteria The volatile compounds from C-9 and A1 effectively inhibited the growth rate of cotton Verticillium wilt and altered the morphology of the colonies (Fig. 1). The diameter of the untreated control Verticillium wilt colonies was 5.66 ± 0.017 cm, which was reduced to 2.90 ± 0.074 cm after treatment with C-9, achieving an inhibition rate of 48.76%; and to 3.32 ± 0.061 cm after Treatment with A1, with an inhibition rate of 38.16%. This indicates that the volatile compounds from both biocontrol strains can inhibit Verticillium wilt, and change the colony morphology from microsclerotia to mycelial form, effectively reducing the production of Verticillium wilt microsclerotia. 3.2 Inhibition Test of Mixed Microbial Fermentation Filtrate on Cotton Verticillium Wilt and its Spores The mixed microbial fermentation filtrate of C-9 and A1 exhibited antimicrobial activity against cotton Verticillium wilt. When diluted to 10% (Fig. 2), the inhibition zone was opaque with a diameter of 1.19 ± 0.026 cm; At 25% dilution, the inhibition zone was opaque with a diameter of 1.41 ± 0.016cm; at 50% dilution, the inhibition zone was clear with a diameter of 2.14 ± 0.099cm; with the undiluted filtrate, the inhibition zone was clear with a diameter of 3.03 ± 0.057cm. The test indicated that the higher the concentration of the fermentation filtrate, the stronger its antimicrobial activity, with The undiluted filtrate showing the strongest activity. The germination rate of Verticillium wilt spores in the control group reached 89.24% (Fig. 2F, G). The number of Verticillium wilt spores treated with the mixed microbial fermentation filtrate was fewer compared to the control group and did not germinate, indicating that the composite biocontrol bacteria formed by the fermentation filtrate strongly inhibited the germination of Verticillium wilt spores. 3.3 Crude Extraction and Antimicrobial Effectiveness of Lipopeptides Antimicrobial tests conducted with the crude extract of lipopeptides obtained through methanol extraction showed a clear inhibition zone with a diameter of 2.45 ± 0.047 cm, indicating significant antimicrobial activity (Fig. 2H, I). The impact of lipopeptides on the soluble protein production and mycelial yield of Verticillium dahliae strains was explored (Fig. 3A, B). The mixed microbial fermentation filtrate and lipopeptides significantly reduced the mycelial yield and soluble protein content of Verticillium dahliae. The effect of lipopeptides on the cell membrane permeability of Verticillium dahliae (Fig. 3C) showed that, compared to the control group, the relative electrical conductivity of Verticillium dahliae treated with lipopeptides was higher, indicating that the cell membranes of the mycelia had ruptured and intracellular solutes had leaked, resulting in significant changes in relative electrical conductivity. The results of the group treated with mixed microbial fermentation filtrate were similar to those treated with lipopeptides, suggesting that both the fermentation filtrate and lipopeptides can cause rupture and death of Verticillium dahliae cell membranes, which is one of the mechanisms of their inhibitory effect on Verticillium wilt infection. 3.4 The Effect of Lipopeptides and Mixed Microbial Fermentation Filtrate on Cotton Seed Germination and Disease Resistance In the experiments testing the effect of lipopeptides and mixed microbial fermentation filtrate on the germination and disease resistance of cotton seeds (Fig. 4), compared to the control group, the treatment of Xinluzao 7 and 33 seeds with lipopeptides neither inhibited nor promoted seed germination, and no Verticillium wilt symptoms were observed following infection with Verticillium dahliae spore suspension. the seeds of Xinluzao 7 and 33 treated with mixed microbial fermentation filtrate showed enhanced germination and no Verticillium wilt symptoms post infection. In contrast, seeds of both cotton varieties not treated with fermentation filtrate or lipopeptides displayed disease symptoms in the radicle when infected with Verticillium dahliae spore suspension. The experiment indicates that the mixed microbial fermentation filtrate not only acts in controlling Verticillium wilt but also promotes the germination of cotton seeds. 3.5 Biocontrol Bacteria Induced Resistance in Cotton The DAB staining method was used to detect the burst of reactive oxygen species in cotton leaves (Fig. 5A). In Xinluzao 7 and 33 cotton leaves treated with the mixed microbial fermentation filtrate, a larger amount of dark brown precipitate was observed, while leaves treated with Verticillium dahliae spore suspension showed only a small amount of light brown precipitate in localized areas, indicating that the fermentation filtrate effectively induces the burst of reactive oxygen species in cotton leaves. Trypan blue staining was used to detect the HR response in cotton leaves (Fig. 5B). leaves of Xinluzao 7 and 33 treated with mixed microbial fermentation filtrate showed more blue precipitate, not only around the veins but also around the leaf margins. leaves treated only with Verticillium dahliae spore suspension showed a small amount of blue precipitate around the veins, with almost none at the leaf margins, suggesting that biocontrol bacteria can induce HR response in both types of cotton. Post root-drenching with mixed microbial fermentation filtrate (Fig. 5C), and after 7 days of incubation in a constant temperature incubator, it was observed that Xinluzao 7 leaves treated with the filtrate had fewer Verticillium dahliae mycelia attachments and less necrotic leaf area, while Xinluzao 33 leaves showed no mycelial attachment and were undamaged. In contrast, untreated Xinluzao 7 leaves had a large amount of mycelial attachment and greater necrotic area, and Xinluzao 33 leaves showed milder mycelial attachment and smaller damage area. The difference in leaf damage after infection in the two cotton varieties may be due to different tolerances to Verticillium dahliae based on their resistance genotypes, indicating that treating cotton with mixed microbial fermentation filtrate can enhance the plant's tolerance to Verticillium dahliae , though the degree of induced resistance varies between different genotypes. root vitality was measured (Fig. 5D), and for untreated Xinluzao 7 CK and Xinluzao 33 CK groups, root vitality was 12.86 ± 1.24 mg/(g×h) and 16.07 ± 1.32 mg/(g×h), respectively. In contrast, for Xinluzao 7 and 33 in the TR groups treated with mixed microbial fermentation filtrate, root vitality was 72.93 ± 1.45mg/(g×h) and 55.43 ± 1.12 mg/(g×h), respectively, indicating an increase of 5.67 times for Xinluzao 7 and 3.45 times for Xinluzao 33 compared to the CK, showing that treatment with the fermentation filtrate effectively enhances the root vitality of cotton plants. 3.6 The Induction of Defense Enzyme Activities and the Effect on Defense Enzyme Gene Expression in Cotton by Mixed Microbial Fermentation Filtrate 3.6.1 Detection of Defense-Related Enzyme Activity During the 6-day period of treatment with mixed microbial fermentation filtrate, significant changes were observed in the activity of the defense enzyme superoxide dismutase (SOD) in Xinluzao 7 and 33 cotton, compared to the untreated control group (CK) (see Figure A). Initially, on day 0 of the treatment, there was no significant difference in the activities of SOD and peroxidase (POD). However, as time progressed, particularly on the 2nd and 3rd days post-treatment, the SOD activity in the treated groups was significantly higher than in the CK group. By the 4th to 6th days, however, SOD activity in the treated groups decreased significantly, suggesting that the induction of cotton’s defense enzyme activities by the mixed microbial fermentation filtrate occurred rapidly in the early stages but decreased significantly later, possibly due to the plant's own defense regulatory mechanisms. Similarly, the activity of polyphenol oxidase (PPO) did not change significantly in the initial stages of treatment with the fermentation filtrate (see Figures C, E), but increased significantly from the 1st to the 4th day post-treatment. After the 4th day, the PPO activities in the treated and CK groups were similar, with no apparent differences. In contrast, the activities of catalase (CAT) and peroxidase (POD) in Xinluzao 7 and 33 cotton did not vary as much or as dramatically as SOD and PPO throughout the treatment, showing only slight changes in the later stages of treatment, namely on the 5th and 6th days. In summary, the mixed microbial fermentation filtrate had a significant inducing effect on the SOD and PPO enzyme activities in Xinluzao 7 and 33 cotton. Following the application of mixed microbial fermentation filtrate to Xinluzao 7 and 33 cotton, and subsequent infection with Verticillium dahliae , significant changes were observed in the plant's own defense enzyme activities. In the first 24 hours of treatment, no significant difference in defense enzyme activities was observed between the groups infected with Verticillium dahliae (Vd) and those pre-treated with fermentation filtrate followed by Verticillium dahliae infection (Vd + TR) (see Figures B, D, F). However, 36 to 72 hours post Verticillium dahliae infection, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO) in Xinluzao 7 and 33 cotton were significantly affected, with enzyme activities in the Vd group being significantly higher than in the Vd + TR group. Previous research has shown that cotton's defense enzyme system activity significantly increases after infection with Verticillium dahliae . These results suggest that cotton activates its own defense mechanisms by enhancing the activities of key enzymes when infected with Verticillium wilt. In the treatment where cotton was infected with Verticillium dahliae (Verticillium dahliae) post-application of mixed microbial fermentation filtrate, a decrease in enzyme activities was observed. Under the conditions of composite biocontrol bacteria formed by two biocontrol strains, the biocontrol bacteria might inhibit the pathogenicity and activity of Verticillium dahliae to some extent, thereby weakening the reactive oxygen system response stimulated in cotton by Verticillium dahliae. This suggests that under the influence of biocontrol bacteria, cotton's defense response to Verticillium dahliae may be modulated, leading to changes in its defense enzyme activities. 3.6.2Induction of Defense Gene Expression in Cotton by Mixed Microbial Fermentation Filtrate In Xinluzao 7 and 33 cotton leaves treated with mixed microbial fermentation filtrate (TR), the expression of the PR1 gene increased initially and then decreased within 0 to 48 hours (Figs. 7A, D), reaching its peak at 12 hours. The expression of PR1 in the TR group was higher than in the control group (CK) throughout 0–48 hours. In Xinluzao 7, the expression of PR1 was significantly increased at 12 hours post-treatment with the fermentation filtrate, whereas in Xinluzao 33, it was significantly elevated from 12 to 48 hours under the fermentation filtrate treatment. The expression of the PR5 gene in Xinluzao 7 leaves (TR group) first increased, then decreased, and then increased again within 0 to 48 hours (Fig. 7B), with a significant increase at 48 hours. The expression of PR5 in Xinluzao 33 was higher and significantly enhanced at all time points under the fermentation filtrate treatment. The PR5 gene in both Xinluzao 7 and 33 was significantly induced by the mixed microbial fermentation filtrate between 36–48 hours. The expression of the NPR1 gene in Xinluzao 7 and 33 leaves (TR group) first decreased and then increased within 0 to 48 hours (Figs. 7C, F), reaching its maximum at 48 hours. Compared to the CK group, the expression of the NPR1 gene in the TR group was significantly higher at 36 and 48 hours, indicating a consistent effect on NPR1 expression, with significant increases in the TR group compared to the CK group between 36–48 hours. Overall, in Xinluzao 7 and 33, the induction of PR1 gene expression by the mixed microbial fermentation filtrate mainly occurred in the early phase of treatment, while the induction of PR5 and NPR1 genes primarily occurred in the later phase. Notably, for both Xinluzao 7 and 33, the pattern of defense response induced by the same defense enzyme genes under the fermentation filtrate treatment was consistent. 4. Conclusions This study conducted inhibition tests on Verticillium dahliae using volatile compounds and mixed microbial fermentation filtrate produced by two biocontrol bacterial strains. The results demonstrated that the volatile compounds produced by the two biocontrol strains possess antimicrobial activity. The mixed microbial fermentation filtrate significantly inhibited the mycelial growth and spore germination of Verticillium dahliae. It altered the morphological characteristics of Verticillium dahliae colonies, transforming them from a microsclerotia form to a mycelial form. This indicates that the two antagonistic strains can inhibit the growth and microsclerotia production of Verticillium dahliae . This study indicates that lipopeptides not only inhibit the mycelial growth of Verticillium dahliae and reduce the production of spores and soluble proteins, but also cause the rupture and death of Verticillium dahliae cell membranes, demonstrating the ability of lipopeptides to inhibit the infection by Verticillium dahliae. seed germination tests showed that lipopeptides do not promote the germination of cotton seeds but can inhibit the infection of cotton seeds by Verticillium dahliae during germination. In agricultural application, treating cotton seeds with lipopeptides before field sowing can effectively inhibit the impact of Verticillium dahliae on seed germination. Alternatively, spraying lipopeptides in the field before sowing can effectively suppress the growth of Verticillium dahliae in the soil, thus protecting the germinating cotton seeds from infection and ensuring their healthy development. The burst of reactive oxygen species (ROS) and the hypersensitive response (HR) are important pathways for plant stress resistance(Rayees A Ahanger et al., 2014). The root system is a crucial organ for plant growth, and its vitality can enhance the plant's ability to absorb nutrients and increase resistance to external biotic stresses. This study treated two cotton genotypes with different resistance profiles with mixed microbial fermentation filtrate and found that the root drench treatment significantly induced ROS burst, HR reaction, and root vitality in both cotton types, leading to induced immune resistance to Verticillium dahliae . The degree of resistance induction was related to the genetic nature of the cotton. There were clear differences in the changes in enzyme activity levels and response times in different resistant cotton varieties. The defense-related enzyme activities in the resistant genotypes were higher than in the susceptible varieties, and their defense responses were also faster than in the susceptible varieties. In the PR gene family, the PR1 gene is an indicator of the salicylic acid pathway and a marker of systemic acquired resistance in plants, while the PR5 gene is a resistance gene in the salicylic acid pathway, and NPR1 is a regulatory gene of this pathway. After treatment with mixed microbial fermentation filtrate and subsequent Verticillium dahliae infection, the effects on the expression patterns of defense-related genes in Xinluzao 7 and 33 cotton were studied. The results showed that the fermentation filtrate treatment significantly increased the expression of the salicylic acid pathway indicator gene PR1 in cotton, inducing systemic resistance to resist Verticillium dahliae infection. The expression patterns of the PR1 gene were consistent in both Xinluzao 7 and 33, but the upregulation of PR1 gene expression in Xinluzao 33 was significantly higher than in Xinluzao 7, which might be one of the reasons for the higher resistance of Xinluzao 33 to Verticillium dahliae. the fermentation filtrate treatment significantly increased the expression of the PR5 gene in both cotton varieties. The expression patterns of the PR5 gene in the CK groups of Xinluzao 7 and 33 were consistent, while the expression levels in the TR groups were completely different, with the PR5 gene expression first upregulated and then downregulated in Xinluzao 7, while in Xinluzao 33, the expression pattern was consistent and the maximum expression was 2.5 times that of the CK group. This might be another reason for the higher resistance of Xinluzao 33 to Verticillium dahliae . the fermentation filtrate treatment also significantly increased the expression of the systemic acquired resistance indicator gene NPR1 in cotton. The expression patterns of NPR1 gene from 0h to 48h in the CK groups of both Xinluzao 7 and 33 were identical, but the upregulation in the TR group of Xinluzao 7 was larger compared to Xinluzao 33. The experiment shows that the mixed microbial fermentation filtrate treatment can induce the expression of defense-related genes in the salicylic acid pathway in cotton, enhancing the resistance of both cotton varieties to Verticillium dahliae . However, the antagonistic and synergistic actions of the salicylic and jasmonic acid pathways in different resistant cotton varieties differ, which might be the reason for the stronger resistance of Xinluzao 33 compared to Xinluzao 7. This provides a theoretical and practical basis for further research on the induction of disease resistance signaling pathways in cotton by biocontrol bacteria, offering a new approach to control Verticillium wilt in cotton and a theoretical basis for the biological control of cotton Verticillium wilt. Declarations Author Contribution Conflict of interest :The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Author contributionsYongbin Fan: Data curation, Formal Analysis, Methodology, Writing – original draft, Software, Visualization. Jianwei Cao: Data curation, Formal Analysis, Methodology, Writing – original draft. Jingyi Ye: Investigation, Methodology, Writing – original draft. Yuanyuan Liu : Data curation, Investigation, Writing – original draft. Chongdie Wu: Investigation, Writing – original draft. Gaijie Liu: Methodology, Writing – original draft. Aiying Wang: Funding acquisition, Project administration, Writing – review and editing. Yongbin Fan: Investigation, Methodology, Writing – original draft. Jianwei Cao: Data curation, Investigation, Writing – original draft. Aiying Wang: Project administration, Writing – review and editing. 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Rice 14, 91. https://doi.org/10.1186/s12284-021-00532-6 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4479911","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":313223701,"identity":"eba92587-c1ca-4a72-942e-737e0ae9572b","order_by":0,"name":"Yongbin Fan","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Yongbin","middleName":"","lastName":"Fan","suffix":""},{"id":313223702,"identity":"5506aa8b-d66e-4217-b524-aa52b42f5602","order_by":1,"name":"Jianwei Cao","email":"","orcid":"","institution":"Shihezi 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01:16:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12312645,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4479911/v1/f5dbf412-b278-4df0-b6cc-0e6ee4003c2c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Impact of Biocontrol Bacteria on Cotton Resistance and Their Effects on Signaling Pathways Related to Defense Against Verticillium Wilt Infection","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCotton Verticillium wilt, often referred to as \u0026lsquo;cotton cancer,' has been spreading in China since its introduction in 1935 with the import of American cotton varieties(Mokhtari et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since the 1990s, this disease has spread to all cotton-growing areas, including Xinjiang. In 2023, the affected area reached 135,000 hectares, primarily in localized regions of Bortala Mongol, Changji Hui Autonomous Prefecture, and Aksu. The pathogen of Verticillium wilt (\u003cem\u003eVerticillium dahliae\u003c/em\u003e Kleb) belongs to the Fungi Imperfecti, class Hyphomycetes, order Hyphomycetales, family Plectosphaerellaceae, and genus \u003cem\u003eVerticillium\u003c/em\u003e(Inderbitzin et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Currently, the strains causing Verticillium wilt in Xinjiang cotton are primarily Verticillium \u003cem\u003edahliae\u003c/em\u003e and its subspecies. This pathogen is characterized by a wide host range, strong infectivity, significant destructiveness, and high variability.\u003c/p\u003e \u003cp\u003eThe low pollution, low cost, and environmentally friendly nature of biological control have made it an increasingly popular field of study in the control of pathogens in recent years. The mechanisms of biological control of pathogens mainly include competition for nutrients and space, antagonism; it can also induce plants to develop broad-spectrum, systemic, and non-specific systemic resistance, resisting the invasion of pathogens(Borriss et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e;Yao et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Wang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e;K\u0026ouml;hl et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e;Boulahouat et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Biocontrol bacteria induce a variety of defense responses in plants. upon pathogen invasion, plants rapidly initiate a hypersensitive response (HR), and local resistance genes in the tissues surrounding the infection site are activated. This leads to the rapid accumulation of various defense-related enzymes and antimicrobial phytoprotectants, affecting the entire plant(Ji et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Pathogenesis-Related (PR) protein gene family plays a crucial role in the immune system of plants, particularly in the Systemic Acquired Resistance (SAR) response(Yu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e;Abu-Bakar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e;Han et al., 2022). The PR gene family comprises multiple members, among which \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePR5\u003c/em\u003e, and \u003cem\u003eNPR1\u003c/em\u003e are key genes that have been extensively studied(Li et al., 2021;Yu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;K\u0026ouml;hl et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003ePR1\u003c/em\u003e gene is one of the earliest identified pathogenesis-related genes and is commonly used as a marker gene for SAR response(Brambilla et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).The \u003cem\u003ePR5\u003c/em\u003e gene encodes a protein similar to trypsin inhibitors in animals and exhibits antifungal activity(Ng et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eNPR1\u003c/em\u003e is a key regulatory factor crucial for Systemic Acquired Resistance (SAR) in plants. It is activated upon sensing pathogen attack signals and regulates the expression of downstream PR genes, including \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePR5\u003c/em\u003e(Backer et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The expression and function of these genes are not isolated but form an internal defense network in plants, collectively participating in the fight against the invasion of external pathogens. Plants are capable of constructing a multi-tiered defense system to effectively resist various pathogens(Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e;Rezaei et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e;Nie et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the defense of cotton against Verticillium wilt, the expression of disease resistance-related genes is induced, which helps to enhance the disease resistance of cotton and reduce the impact of the disease. Investigating the antibacterial capability of composite biocontrol bacteria, their role in inducing resistance in cotton, and their impact on the signaling pathways related to defense against Verticillium wilt infection provides a theoretical and practical foundation for the development and practical utilization of microbial agents.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Test Strains and Cotton Varieties\u003c/h2\u003e \u003cp\u003ePathogen: \u003cem\u003eVerticillium dahliae\u003c/em\u003e Vd-278, a non-defoliating physiological race and microsclerotia-producing strain, provided by the Key Laboratory of Biotechnology at Xinjiang Agricultural Reclamation Science Academy.\u003c/p\u003e \u003cp\u003eTest Strains: \u003cem\u003eBacillus tequilensis\u003c/em\u003e C-9 and \u003cem\u003eSphingobacterium\u003c/em\u003e A1, isolated and preserved by the Key Laboratory of Agricultural Biotechnology at Shihezi University.\u003c/p\u003e \u003cp\u003eCotton Varieties: Xinluzao 7 (susceptible genotype); Xinluzao 33 (resistant genotype).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Culture Media(Zhao et al., 2016)\u003c/h2\u003e \u003cp\u003ePDA: potato 200 g, sucrose 20 g, agar 20 g, distilled water 1000 ml, pH 7.0.\u003c/p\u003e \u003cp\u003eLB Medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 20 g, distilled water 1000 ml, pH 7.0.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Antimicrobial Activity Test of Volatile Compounds from Biocontrol Bacterial Strains\u003c/h2\u003e \u003cp\u003eA 5 mm diameter sterile punch was used to make holes in PDA plates inoculated with \u003cem\u003eVerticillium dahliae\u003c/em\u003e Vd-278. The mycelial plugs were then transferred to the center of new PDA plates. On two other new PDA plates, Strains C-9 and A1 were streaked to cover the entire surface. These plates with C-9 and A1 were then inverted over the Verticillium dahliae-inoculated plates and sealed. The plates inoculated with Verticillium \u003cem\u003edahliae\u003c/em\u003e were incubated inverted in an incubator at 25\u0026deg;C for 5\u0026ndash;7 days. Post incubation, The colony diameter of Verticillium \u003cem\u003edahliae\u003c/em\u003e was measured using the cross method(Cheng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e2.3 Assessment of the Inhibitory Effects of Mixed Microbial Fermentation Filtrate on Verticillium Wilt and Its Spores\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Preparation of Mixed Microbial Fermentation Filtrate\u003c/h2\u003e \u003cp\u003eSeparate inoculations of C-9 and A1 were made into 50 ml potato dextrose broth (PDB) in autoclaved flasks at 121\u0026deg;C for 15 minutes. These were then incubated at 160 rpm and 28\u0026deg;C on a shaker for 18 hours to obtain the fermentation seed liquids of C-9 and A1. The seed liquids were then mixed in a 9:1 ratio, and 5% of this mixture was inoculated into new flasks containing 50 ml of sterilized PDB. After incubation at 160 rpm and 28\u0026deg;C for 3 days, the cultures were centrifuged at 5000 rpm for 10 minutes to remove the bacterial cells. The supernatant was filtered through a 0.45 \u0026micro;m pore size filter to remove any remaining bacteria and transferred into sterile Eppendorf tubes for further use(Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Preparation of \u003cem\u003eVerticillium dahliae\u003c/em\u003e Spore Suspension\u003c/h2\u003e \u003cp\u003eA 5 mm diameter punch was used to create holes in PDA plates inoculated with Verticillium \u003cem\u003edahliae\u003c/em\u003e. Two mycelial plugs were then inoculated into a flask containing 50 ml PDB and incubated at 160 rpm and 25\u0026deg;C on a shaker for 7\u0026ndash;10 days. The culture was filtered through eight layers of sterile gauze to remove the mycelia, and the spore suspension of Verticillium \u003cem\u003edahliae\u003c/em\u003e was diluted to 10^7 cfu/ml before being transferred into sterile Eppendorf tubes for later use(Bandi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Inhibition Test of Mixed Microbial Fermentation Filtrate on \u003cem\u003eVerticillium dahliae\u003c/em\u003e and Its Spores\u003c/h2\u003e \u003cp\u003e100 \u0026micro;l of 10^7 CFU/ml Verticillium \u003cem\u003edahliae\u003c/em\u003e spore suspension was evenly spread on PDA solid medium. Three equidistant holes were punched 2 cm away from the center of the PDA medium. Mycelial plugs were taken, and the prepared mixed microbial fermentation filtrate was diluted to concentrations of 10%, 25%, and 50% of the original. 100 \u0026micro;l of the original and each diluted fermentation filtrate were added into the three holes on different PDA plates (control group added 100 \u0026micro;l liquid PDB). After incubation at 25\u0026deg;C for 5\u0026ndash;7 days, the plates were observed and the diameters of the inhibition zones were measured using the cross method. The mixed fermentation filtrate was mixed 1:1 with Verticillium dahliae spore suspension in Eppendorf tubes (control was a mixture of PDB and spore suspension), and incubated on a shaker at 25\u0026deg;C for 24\u0026ndash;48 hours. Once the germination rate of spores in the control exceeded 80%, the germination of spores in the treated group was observed under a microscope(AlSalam A. AlKarem and .Kamran. Hasan, 2023).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Crude Extraction of Lipopeptides and their Antimicrobial Activity Assessment\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Lipopeptide Extraction\u003c/h2\u003e \u003cp\u003eThe mixed fermentation broth of C-9 and A1 was centrifuged at 8000 rpm for 20 minutes, and the supernatant was transferred to a sterilized conical flask. 6 mol/L HCl was slowly added to the supernatant with stirring until the pH reached 2.0. The mixture was then left overnight at 4\u0026deg;C. After another centrifugation at 8000 rpm for 20 minutes, the precipitate was collected and allowed to settle for 30\u0026ndash;60 minutes. Methanol at pH 7.0 was added to the precipitate and the mixture was left to extract at 4\u0026deg;C for 10 hours (the extraction was repeated twice). The combined extracts were then centrifuged at 4500 rpm for 10 minutes to collect the supernatant, which served as the crude lipopeptide extract. The crude extract was filtered through a 0.45 um sterile filter and aliquoted into sterile Eppendorf tubes for later use(L\u0026oacute;pez-Guti\u0026eacute;rrez et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;Armbruster and Meredith, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e;Ma et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Inhibition Effect of Lipopeptides on Cotton Verticillium Wilt\u003c/h2\u003e \u003cp\u003e100 \u0026micro;l of 10^7 CFU/ml Verticillium dahliae spore suspension was evenly spread on a PDA plate. A hole of 5mmdiameter was punched at the center of the plate and the mycelial plug was removed. 100 \u0026micro;l of the aforementioned crude lipopeptide extract was then added to the hole (PDB medium was used for the control). After 3 days, the diameter of the clear zone was measured using the cross method(Nacef et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Carvalho et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e2.4.2.1 Effects of Lipopeptides on Spore Yield, Soluble Protein Production, Mycelial Yield, and Cell Membrane Permeability of Cotton Verticillium Wilt\u003c/p\u003e \u003cp\u003eThe mixed microbial fermentation filtrate and prepared lipopeptides were inoculated at a 5% inoculum rate into new flasks containing 50 ml PDB (control group inoculated with 5% fresh PDB). A 5mmdiameter punch was used on PDA plates of Verticillium \u003cem\u003edahliae\u003c/em\u003e, and mycelial plugs were added to The flasks (2 plugs per flask), which were then incubated at 160 rpm and 25\u0026deg;C for 7 days. The treated Verticillium dahliae spore suspension was filtered through eight layers of sterile gauze to remove the mycelia, and spore yield was counted under a microscope using a hemocytometer. The spore suspension was centrifuged at 5000 rpm for 10 minutes, discarding The pellet and retaining the supernatant, and the soluble protein content was measured using the Coomassie brilliant blue method(Nagata et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The mycelia collected by filtering through eight layers of sterile gauze were dried at 60\u0026deg;C for 4 hours and then weighed. The collected mycelia were washed five times with sterile water, vacuum-filtered for 15 minutes, and resuspended in 20 ml sterile water. The conductivity of the sterile water was measured every 10 minutes for 2 hours using a conductivity meter, with The final conductivity determined after boiling the resuspended mycelia in sterile water for 5 minutes(Hao et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Lin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e;Cai et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relative electrical conductivity of the Verticillium dahliae mycelium was calculated using the following formula:\u003c/p\u003e \u003cp\u003eRelative Electrical Conductivity (%)\u0026thinsp;=\u0026thinsp;Measured Conductivity / Final Conductivity \u0026times; 100%\u003c/p\u003e \u003cp\u003e2.4.2.2 The Effect of Lipopeptides and Mixed Microbial Fermentation Filtrate on Cotton Seed Germination and Disease Resistance\u003c/p\u003e \u003cp\u003eOne hundred full-grain Xinluzao 7 cotton seeds were selected and divided into two batches of 50 seeds each. Each batch of 50 seeds was placed in two beakers containing 95 ml of water, with 5 ml of the mixed microbial fermentation filtrate added to one beaker and 5 ml of lipopeptide solution to the other, for a soaking period of 30 minutes (Sterile water was used for the control group). on two layers of filter paper in 5 cm diameter petri dishes, 1 ml of sterile water was applied evenly with a rod to fully wet the filter paper. the first batch of soaked seeds was then placed on the wet filter paper using tweezers (three seeds per Petri dish), and incubated at 25\u0026deg;C for 2 days for germination (1 ml of water was added every 6 hours to keep the filter paper moist). The second batch of seeds was placed on the wet filter paper and an additional 1 ml of the prepared Verticillium dahliae spore suspension was spread evenly with a rod, and incubated at 25\u0026deg;C for 2 days for germination (1 ml of water was added every 6 hours to keep the filter paper moist). The experimental steps for Xinluzao 33 cotton seeds were the same as for Xinluzao 7(Chattopadhyay et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Induction of Resistance in Cotton by Biocontrol Bacteria\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Detection of Reactive Oxygen Species (ROS) Burst\u003c/h2\u003e \u003cp\u003eWhen cotton plants developed two true leaves, Xinluzao 7 and 33 (three pots each) were root-drenched with 50 ml of mixed microbial fermentation filtrate per pot for 7 days (control groups were treated with an equal volume of PDB liquid). The treated and control cotton plants were then subjected to root drenching with 50 ml of 10^7 CFU/ml Verticillium dahliae spore suspension using the root injury method. After 48 hours, the third leaves from the top of each treatment group were subjected to DAB staining in the dark for 8 hours(Daudi and O\u0026rsquo;Brien, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), followed by decolorization in 95% alcohol in a boiling water bath until the green color of the leaves completely faded. The leaves were then cleared of air bubbles in 70% glycerol for microscopic observation and photography.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Detection of the Hypersensitive Response (HR)\u003c/h2\u003e \u003cp\u003eThird leaves from the top of Xinluzao 7 and 33 cotton plants, 48 hours after treatment with Verticillium dahliae spore suspension, were submerged in trypan blue stain solution (1g phenol, 0.1 mg trypan blue, 1 ml lactic acid, 1 ml glycerol dissolved in 50 ml sterile water) and subjected to a boiling water bath for 15 minutes for staining. After cooling to room temperature, the leaves were decolorized in 2.5 g/ml chloral hydrate for 30 minutes, followed by decolorization in 95% alcohol in a boiling water bath until the green color of the leaves completely faded. The leaves were then cleared of air bubbles in 70% glycerol for microscopic observation and photography(Thordal-Christensen et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Detection of Induced Resistance in Cotton\u003c/h2\u003e \u003cp\u003eFunctional leaves of Xinluzao 7 and 33 cotton plants, 48 hours post-treatment with Verticillium dahliae spore suspension, were disinfected with 10% sodium hypochlorite and placed on PDA plates. Using A 5mmdiameter punch, holes were made in plates fully grown with Verticillium dahliae, and mycelial plugs were placed on the surface of the cotton leaves (one plug per leaf). The samples were then incubated at a constant temperature of 25\u0026deg;C for 7 days, after which the disease progression on the leaves was observed and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Detection of Induced Root Vitality\u003c/h2\u003e \u003cp\u003eRoots of Xinluzao 7 and 33 cotton plants, 48 hours post-treatment with Verticillium dahliae spore suspension, were rinsed with tap water. A 0.3 g sample of the roots was then weighed and root vitality was determined using the TTC (Triphenyl Tetrazolium Chloride) method(Richter et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e2.6 The Induction of Defense Enzyme Activities and the Effect on Defense Enzyme Gene Expression in Cotton by Mixed Microbial Fermentation Filtrate\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Methods for Detecting Defense-Related Enzyme Activity\u003c/h2\u003e \u003cp\u003eWhen cotton plants developed two true leaves, Xinluzao 7 and 33 (three pots each) were treated with 50 ml of mixed microbial fermentation filtrate per pot (control group was treated with an equivalent volume of PDB liquid). leaves were collected every 24 hours for 7 days, rinsed with tap water, and dried with filter paper for the measurement of defense-related enzyme activities in cotton. Seven days post treatment with the fermentation filtrate, the plants were root-drenched with 50 ml of 10^7 CFU/ml Verticillium dahliae spore suspension. leaves were collected every 12 hours, rinsed, and dried as before, for the measurement of defense-related enzyme activities(Alici and Arabaci, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Real-Time Quantitative PCR(Livak and Schmittgen, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e)\u003c/h2\u003e \u003cp\u003ecotton leaves stored at -80\u0026deg;C were taken out, and RNA was extracted using the Polysaccharide and Polyphenol Plant RNA Extraction Kit (R8611). High-quality RNA was then reverse-transcribed. The RNA reverse transcription was conducted according to the instructions of the TransScript II First-Stand cDNA Synthesis SuperMix Kit by All-In-One. \u003cem\u003eβ-Tubulin\u003c/em\u003e gene (Actin-8) in cotton was used as an internal reference. The reactions were carried out using the SYBR Green I Master Mix Kit by Roche and amplified using the Light Cycler\u0026reg; 480 II PCR system (Roche). The reaction conditions were 95\u0026deg;C for 2 minutes, followed by 45 cycles of 95\u0026deg;C for 15 seconds, 56\u0026deg;C for 15 seconds, and 72\u0026deg;C for 20 seconds. Each sample was repeated three times, and data analysis was performed using the 2-ΔΔCT method(Michaelidou et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Result and Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Antimicrobial Activity Test of Volatile Compounds from Biocontrol Bacteria\u003c/h2\u003e \u003cp\u003eThe volatile compounds from C-9 and A1 effectively inhibited the growth rate of cotton Verticillium wilt and altered the morphology of the colonies (Fig.\u0026nbsp;1). The diameter of the untreated control Verticillium wilt colonies was 5.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 cm, which was reduced to 2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.074 cm after treatment with C-9, achieving an inhibition rate of 48.76%; and to 3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.061 cm after Treatment with A1, with an inhibition rate of 38.16%. This indicates that the volatile compounds from both biocontrol strains can inhibit Verticillium wilt, and change the colony morphology from microsclerotia to mycelial form, effectively reducing the production of Verticillium wilt microsclerotia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Inhibition Test of Mixed Microbial Fermentation Filtrate on Cotton \u003cem\u003eVerticillium\u003c/em\u003e Wilt and its Spores\u003c/h2\u003e \u003cp\u003eThe mixed microbial fermentation filtrate of C-9 and A1 exhibited antimicrobial activity against cotton Verticillium wilt. When diluted to 10% (Fig.\u0026nbsp;2), the inhibition zone was opaque with a diameter of 1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026 cm; At 25% dilution, the inhibition zone was opaque with a diameter of 1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016cm; at 50% dilution, the inhibition zone was clear with a diameter of 2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.099cm; with the undiluted filtrate, the inhibition zone was clear with a diameter of 3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057cm. The test indicated that the higher the concentration of the fermentation filtrate, the stronger its antimicrobial activity, with The undiluted filtrate showing the strongest activity. The germination rate of Verticillium wilt spores in the control group reached 89.24% (Fig.\u0026nbsp;2F, G). The number of Verticillium wilt spores treated with the mixed microbial fermentation filtrate was fewer compared to the control group and did not germinate, indicating that the composite biocontrol bacteria formed by the fermentation filtrate strongly inhibited the germination of Verticillium wilt spores.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Crude Extraction and Antimicrobial Effectiveness of Lipopeptides\u003c/h2\u003e \u003cp\u003eAntimicrobial tests conducted with the crude extract of lipopeptides obtained through methanol extraction showed a clear inhibition zone with a diameter of 2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047 cm, indicating significant antimicrobial activity (Fig.\u0026nbsp;2H, I). The impact of lipopeptides on the soluble protein production and mycelial yield of Verticillium \u003cem\u003edahliae\u003c/em\u003e strains was explored (Fig.\u0026nbsp;3A, B). The mixed microbial fermentation filtrate and lipopeptides significantly reduced the mycelial yield and soluble protein content of Verticillium dahliae. The effect of lipopeptides on the cell membrane permeability of Verticillium dahliae (Fig.\u0026nbsp;3C) showed that, compared to the control group, the relative electrical conductivity of Verticillium dahliae treated with lipopeptides was higher, indicating that the cell membranes of the mycelia had ruptured and intracellular solutes had leaked, resulting in significant changes in relative electrical conductivity. The results of the group treated with mixed microbial fermentation filtrate were similar to those treated with lipopeptides, suggesting that both the fermentation filtrate and lipopeptides can cause rupture and death of Verticillium dahliae cell membranes, which is one of the mechanisms of their inhibitory effect on Verticillium wilt infection.\u003c/p\u003e \u003cp\u003e3.4 The Effect of Lipopeptides and Mixed Microbial Fermentation Filtrate on Cotton Seed Germination and Disease Resistance\u003c/p\u003e \u003cp\u003eIn the experiments testing the effect of lipopeptides and mixed microbial fermentation filtrate on the germination and disease resistance of cotton seeds (Fig.\u0026nbsp;4), compared to the control group, the treatment of Xinluzao 7 and 33 seeds with lipopeptides neither inhibited nor promoted seed germination, and no Verticillium wilt symptoms were observed following infection with Verticillium dahliae spore suspension. the seeds of Xinluzao 7 and 33 treated with mixed microbial fermentation filtrate showed enhanced germination and no Verticillium wilt symptoms post infection. In contrast, seeds of both cotton varieties not treated with fermentation filtrate or lipopeptides displayed disease symptoms in the radicle when infected with Verticillium dahliae spore suspension. The experiment indicates that the mixed microbial fermentation filtrate not only acts in controlling Verticillium wilt but also promotes the germination of cotton seeds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Biocontrol Bacteria Induced Resistance in Cotton\u003c/h2\u003e \u003cp\u003eThe DAB staining method was used to detect the burst of reactive oxygen species in cotton leaves (Fig.\u0026nbsp;5A). In Xinluzao 7 and 33 cotton leaves treated with the mixed microbial fermentation filtrate, a larger amount of dark brown precipitate was observed, while leaves treated with Verticillium dahliae spore suspension showed only a small amount of light brown precipitate in localized areas, indicating that the fermentation filtrate effectively induces the burst of reactive oxygen species in cotton leaves. Trypan blue staining was used to detect the HR response in cotton leaves (Fig.\u0026nbsp;5B). leaves of Xinluzao 7 and 33 treated with mixed microbial fermentation filtrate showed more blue precipitate, not only around the veins but also around the leaf margins. leaves treated only with Verticillium dahliae spore suspension showed a small amount of blue precipitate around the veins, with almost none at the leaf margins, suggesting that biocontrol bacteria can induce HR response in both types of cotton. Post root-drenching with mixed microbial fermentation filtrate (Fig.\u0026nbsp;5C), and after 7 days of incubation in a constant temperature incubator, it was observed that Xinluzao 7 leaves treated with the filtrate had fewer Verticillium dahliae mycelia attachments and less necrotic leaf area, while Xinluzao 33 leaves showed no mycelial attachment and were undamaged. In contrast, untreated Xinluzao 7 leaves had a large amount of mycelial attachment and greater necrotic area, and Xinluzao 33 leaves showed milder mycelial attachment and smaller damage area. The difference in leaf damage after infection in the two cotton varieties may be due to different tolerances to Verticillium \u003cem\u003edahliae\u003c/em\u003e based on their resistance genotypes, indicating that treating cotton with mixed microbial fermentation filtrate can enhance the plant's tolerance to Verticillium \u003cem\u003edahliae\u003c/em\u003e, though the degree of induced resistance varies between different genotypes. root vitality was measured (Fig.\u0026nbsp;5D), and for untreated Xinluzao 7 CK and Xinluzao 33 CK groups, root vitality was 12.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 mg/(g\u0026times;h) and 16.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32 mg/(g\u0026times;h), respectively. In contrast, for Xinluzao 7 and 33 in the TR groups treated with mixed microbial fermentation filtrate, root vitality was 72.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45mg/(g\u0026times;h) and 55.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12 mg/(g\u0026times;h), respectively, indicating an increase of 5.67 times for Xinluzao 7 and 3.45 times for Xinluzao 33 compared to the CK, showing that treatment with the fermentation filtrate effectively enhances the root vitality of cotton plants.\u003c/p\u003e \u003cp\u003e3.6 The Induction of Defense Enzyme Activities and the Effect on Defense Enzyme Gene Expression in Cotton by Mixed Microbial Fermentation Filtrate\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1 Detection of Defense-Related Enzyme Activity\u003c/h2\u003e \u003cp\u003eDuring the 6-day period of treatment with mixed microbial fermentation filtrate, significant changes were observed in the activity of the defense enzyme superoxide dismutase (SOD) in Xinluzao 7 and 33 cotton, compared to the untreated control group (CK) (see Figure A). Initially, on day 0 of the treatment, there was no significant difference in the activities of SOD and peroxidase (POD). However, as time progressed, particularly on the 2nd and 3rd days post-treatment, the SOD activity in the treated groups was significantly higher than in the CK group. By the 4th to 6th days, however, SOD activity in the treated groups decreased significantly, suggesting that the induction of cotton\u0026rsquo;s defense enzyme activities by the mixed microbial fermentation filtrate occurred rapidly in the early stages but decreased significantly later, possibly due to the plant's own defense regulatory mechanisms. Similarly, the activity of polyphenol oxidase (PPO) did not change significantly in the initial stages of treatment with the fermentation filtrate (see Figures C, E), but increased significantly from the 1st to the 4th day post-treatment. After the 4th day, the PPO activities in the treated and CK groups were similar, with no apparent differences. In contrast, the activities of catalase (CAT) and peroxidase (POD) in Xinluzao 7 and 33 cotton did not vary as much or as dramatically as SOD and PPO throughout the treatment, showing only slight changes in the later stages of treatment, namely on the 5th and 6th days. In summary, the mixed microbial fermentation filtrate had a significant inducing effect on the SOD and PPO enzyme activities in Xinluzao 7 and 33 cotton.\u003c/p\u003e \u003cp\u003eFollowing the application of mixed microbial fermentation filtrate to Xinluzao 7 and 33 cotton, and subsequent infection with Verticillium \u003cem\u003edahliae\u003c/em\u003e, significant changes were observed in the plant's own defense enzyme activities. In the first 24 hours of treatment, no significant difference in defense enzyme activities was observed between the groups infected with Verticillium \u003cem\u003edahliae\u003c/em\u003e (Vd) and those pre-treated with fermentation filtrate followed by Verticillium \u003cem\u003edahliae\u003c/em\u003e infection (Vd\u0026thinsp;+\u0026thinsp;TR) (see Figures B, D, F). However, 36 to 72 hours post Verticillium dahliae infection, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO) in Xinluzao 7 and 33 cotton were significantly affected, with enzyme activities in the Vd group being significantly higher than in the Vd\u0026thinsp;+\u0026thinsp;TR group. Previous research has shown that cotton's defense enzyme system activity significantly increases after infection with Verticillium \u003cem\u003edahliae\u003c/em\u003e. These results suggest that cotton activates its own defense mechanisms by enhancing the activities of key enzymes when infected with Verticillium wilt. In the treatment where cotton was infected with Verticillium dahliae (Verticillium dahliae) post-application of mixed microbial fermentation filtrate, a decrease in enzyme activities was observed. Under the conditions of composite biocontrol bacteria formed by two biocontrol strains, the biocontrol bacteria might inhibit the pathogenicity and activity of Verticillium \u003cem\u003edahliae\u003c/em\u003e to some extent, thereby weakening the reactive oxygen system response stimulated in cotton by Verticillium dahliae. This suggests that under the influence of biocontrol bacteria, cotton's defense response to Verticillium \u003cem\u003edahliae\u003c/em\u003e may be modulated, leading to changes in its defense enzyme activities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2Induction of Defense Gene Expression in Cotton by Mixed Microbial Fermentation Filtrate\u003c/h2\u003e \u003cp\u003eIn Xinluzao 7 and 33 cotton leaves treated with mixed microbial fermentation filtrate (TR), the expression of the \u003cem\u003ePR1\u003c/em\u003e gene increased initially and then decreased within 0 to 48 hours (Figs.\u0026nbsp;7A, D), reaching its peak at 12 hours. The expression of \u003cem\u003ePR1\u003c/em\u003e in the TR group was higher than in the control group (CK) throughout 0\u0026ndash;48 hours. In Xinluzao 7, the expression of \u003cem\u003ePR1\u003c/em\u003e was significantly increased at 12 hours post-treatment with the fermentation filtrate, whereas in Xinluzao 33, it was significantly elevated from 12 to 48 hours under the fermentation filtrate treatment. The expression of the \u003cem\u003ePR5\u003c/em\u003e gene in Xinluzao 7 leaves (TR group) first increased, then decreased, and then increased again within 0 to 48 hours (Fig.\u0026nbsp;7B), with a significant increase at 48 hours. The expression of \u003cem\u003ePR5\u003c/em\u003e in Xinluzao 33 was higher and significantly enhanced at all time points under the fermentation filtrate treatment. The PR5 gene in both Xinluzao 7 and 33 was significantly induced by the mixed microbial fermentation filtrate between 36\u0026ndash;48 hours. The expression of the \u003cem\u003eNPR1\u003c/em\u003e gene in Xinluzao 7 and 33 leaves (TR group) first decreased and then increased within 0 to 48 hours (Figs.\u0026nbsp;7C, F), reaching its maximum at 48 hours. Compared to the CK group, the expression of the \u003cem\u003eNPR1\u003c/em\u003e gene in the TR group was significantly higher at 36 and 48 hours, indicating a consistent effect on NPR1 expression, with significant increases in the TR group compared to the CK group between 36\u0026ndash;48 hours. Overall, in Xinluzao 7 and 33, the induction of \u003cem\u003ePR1\u003c/em\u003e gene expression by the mixed microbial fermentation filtrate mainly occurred in the early phase of treatment, while the induction of \u003cem\u003ePR5\u003c/em\u003e and \u003cem\u003eNPR1\u003c/em\u003e genes primarily occurred in the later phase. Notably, for both Xinluzao 7 and 33, the pattern of defense response induced by the same defense enzyme genes under the fermentation filtrate treatment was consistent.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study conducted inhibition tests on \u003cem\u003eVerticillium dahliae\u003c/em\u003e using volatile compounds and mixed microbial fermentation filtrate produced by two biocontrol bacterial strains. The results demonstrated that the volatile compounds produced by the two biocontrol strains possess antimicrobial activity. The mixed microbial fermentation filtrate significantly inhibited the mycelial growth and spore germination of Verticillium dahliae. It altered the morphological characteristics of Verticillium dahliae colonies, transforming them from a microsclerotia form to a mycelial form. This indicates that the two antagonistic strains can inhibit the growth and microsclerotia production of Verticillium \u003cem\u003edahliae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis study indicates that lipopeptides not only inhibit the mycelial growth of Verticillium \u003cem\u003edahliae\u003c/em\u003e and reduce the production of spores and soluble proteins, but also cause the rupture and death of Verticillium dahliae cell membranes, demonstrating the ability of lipopeptides to inhibit the infection by Verticillium dahliae. seed germination tests showed that lipopeptides do not promote the germination of cotton seeds but can inhibit the infection of cotton seeds by Verticillium dahliae during germination. In agricultural application, treating cotton seeds with lipopeptides before field sowing can effectively inhibit the impact of Verticillium \u003cem\u003edahliae\u003c/em\u003e on seed germination. Alternatively, spraying lipopeptides in the field before sowing can effectively suppress the growth of Verticillium dahliae in the soil, thus protecting the germinating cotton seeds from infection and ensuring their healthy development.\u003c/p\u003e \u003cp\u003eThe burst of reactive oxygen species (ROS) and the hypersensitive response (HR) are important pathways for plant stress resistance(Rayees A Ahanger et al., 2014). The root system is a crucial organ for plant growth, and its vitality can enhance the plant's ability to absorb nutrients and increase resistance to external biotic stresses. This study treated two cotton genotypes with different resistance profiles with mixed microbial fermentation filtrate and found that the root drench treatment significantly induced ROS burst, HR reaction, and root vitality in both cotton types, leading to induced immune resistance to Verticillium \u003cem\u003edahliae\u003c/em\u003e. The degree of resistance induction was related to the genetic nature of the cotton. There were clear differences in the changes in enzyme activity levels and response times in different resistant cotton varieties. The defense-related enzyme activities in the resistant genotypes were higher than in the susceptible varieties, and their defense responses were also faster than in the susceptible varieties.\u003c/p\u003e \u003cp\u003eIn the PR gene family, the \u003cem\u003ePR1\u003c/em\u003e gene is an indicator of the salicylic acid pathway and a marker of systemic acquired resistance in plants, while the \u003cem\u003ePR5\u003c/em\u003e gene is a resistance gene in the salicylic acid pathway, and \u003cem\u003eNPR1\u003c/em\u003e is a regulatory gene of this pathway. After treatment with mixed microbial fermentation filtrate and subsequent Verticillium dahliae infection, the effects on the expression patterns of defense-related genes in Xinluzao 7 and 33 cotton were studied. The results showed that the fermentation filtrate treatment significantly increased the expression of the salicylic acid pathway indicator gene \u003cem\u003ePR1\u003c/em\u003e in cotton, inducing systemic resistance to resist Verticillium dahliae infection. The expression patterns of the \u003cem\u003ePR1\u003c/em\u003e gene were consistent in both Xinluzao 7 and 33, but the upregulation of \u003cem\u003ePR1\u003c/em\u003e gene expression in Xinluzao 33 was significantly higher than in Xinluzao 7, which might be one of the reasons for the higher resistance of Xinluzao 33 to Verticillium dahliae. the fermentation filtrate treatment significantly increased the expression of the \u003cem\u003ePR5\u003c/em\u003e gene in both cotton varieties. The expression patterns of the \u003cem\u003ePR5\u003c/em\u003e gene in the CK groups of Xinluzao 7 and 33 were consistent, while the expression levels in the TR groups were completely different, with the \u003cem\u003ePR5\u003c/em\u003e gene expression first upregulated and then downregulated in Xinluzao 7, while in Xinluzao 33, the expression pattern was consistent and the maximum expression was 2.5 times that of the CK group. This might be another reason for the higher resistance of Xinluzao 33 to Verticillium \u003cem\u003edahliae\u003c/em\u003e. the fermentation filtrate treatment also significantly increased the expression of the systemic acquired resistance indicator gene \u003cem\u003eNPR1\u003c/em\u003e in cotton. The expression patterns of \u003cem\u003eNPR1\u003c/em\u003e gene from 0h to 48h in the CK groups of both Xinluzao 7 and 33 were identical, but the upregulation in the TR group of Xinluzao 7 was larger compared to Xinluzao 33. The experiment shows that the mixed microbial fermentation filtrate treatment can induce the expression of defense-related genes in the salicylic acid pathway in cotton, enhancing the resistance of both cotton varieties to Verticillium \u003cem\u003edahliae\u003c/em\u003e. However, the antagonistic and synergistic actions of the salicylic and jasmonic acid pathways in different resistant cotton varieties differ, which might be the reason for the stronger resistance of Xinluzao 33 compared to Xinluzao 7. This provides a theoretical and practical basis for further research on the induction of disease resistance signaling pathways in cotton by biocontrol bacteria, offering a new approach to control Verticillium wilt in cotton and a theoretical basis for the biological control of cotton Verticillium wilt.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConflict of interest :The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Author contributionsYongbin Fan: Data curation, Formal Analysis, Methodology, Writing \u0026ndash; original draft, Software, Visualization. Jianwei Cao: Data curation, Formal Analysis, Methodology, Writing \u0026ndash; original draft. Jingyi Ye: Investigation, Methodology, Writing \u0026ndash; original draft. Yuanyuan Liu : Data curation, Investigation, Writing \u0026ndash; original draft. Chongdie Wu: Investigation, Writing \u0026ndash; original draft. Gaijie Liu: Methodology, Writing \u0026ndash; original draft. Aiying Wang: Funding acquisition, Project administration, Writing \u0026ndash; review and editing. Yongbin Fan: Investigation, Methodology, Writing \u0026ndash; original draft. Jianwei Cao: Data curation, Investigation, Writing \u0026ndash; original draft. Aiying Wang: Project administration, Writing \u0026ndash; review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbu-Bakar, N., Juri, N.M., Abu-Bakar, R.A.H., Sohaime, M.Z., Badrun, R., Sarip, J., Hassan, M.A., Ahmad, K., 2021. 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Rice 14, 91. https://doi.org/10.1186/s12284-021-00532-6\u003c/li\u003e\n\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":"Mixed microbial fermentation broth, Cotton Verticillium wilt, Antimicrobial activity, Lipopeptides, Induced resistance","lastPublishedDoi":"10.21203/rs.3.rs-4479911/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4479911/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study prepared a mixed fermentation broth using two strains of Bacillus and investigated its inhibitory effects on the cotton Verticillium wilt pathogen, as well as its impact on the signaling pathways related to defense against Verticillium wilt infection in cotton.Biocontrol bacteria can effectively defend against plant diseases by competitively inhibiting pathogens and inducing plant immunity. Through plate confrontation assays, antimicrobial tests using mixed microbial fermentation broth and its dilutions, and their impacts on cotton seed germination, this study explores the defensive potential of the mixed fermentation broth.During the study, it was discovered that The mixed microbial fermentation broth could produce lipopeptide substances. The cotton's immunity against Verticillium wilt, following treatment with this broth, was assessed using DAB and trypan blue histological staining methods. Furthermore, the study involved monitoring the induced expression of resistance-related genes (PR1, PR5, NPR1), as well as the effects on the activities of defense-related enzymes in cotton (SOD, CAT, PPO, POD).The results indicate that The combination of two biocontrol bacterial strains exhibited a certain inhibitory effect on the cotton Verticillium wilt pathogen. Root drenching with the mixed fermentation broth significantly enhanced the transient burst of reactive oxygen species in cotton's defense signaling pathways, inducing an immune response. This response increased the sensitivity of cotton's hypersensitive response (HR), induced the expression of disease resistance-related genes, and heightened the activity of enzymes involved in reactive oxygen species scavenging, thereby enhancing systemic acquired resistance (SAR) in cotton. This study reveals that the mixed fermentation broth improved cotton's resistance to Verticillium wilt, significantly affecting the defense signaling pathways in response to the pathogen, with varying effects on induced resistance in different resistance genotypes of cotton.\u003c/p\u003e","manuscriptTitle":"The Impact of Biocontrol Bacteria on Cotton Resistance and Their Effects on Signaling Pathways Related to Defense Against Verticillium Wilt Infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-12 15:16:51","doi":"10.21203/rs.3.rs-4479911/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":"50a7bde8-b48d-4151-9693-9601e9aeb7ed","owner":[],"postedDate":"June 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-30T01:08:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-12 15:16:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4479911","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4479911","identity":"rs-4479911","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}