Bacillus pumilus - A Potent IAA Producing Plant Growth Promoting Rhizobacteria with  In Vitro PGP Traits and Antagonism Against  Fusarium equiseti

Bacillus pumilus - A Potent IAA Producing Plant Growth Promoting Rhizobacteria with In Vitro PGP Traits and Antagonism Against Fusarium equiseti | 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 Bacillus pumilus - A Potent IAA Producing Plant Growth Promoting Rhizobacteria with In Vitro PGP Traits and Antagonism Against Fusarium equiseti Shambhu Swarnakar, Arka Pratim Chakraborty This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5477377/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Plant growth promotion through microbial mediation has garnered increasing focus in sustainable agriculture because of its capacity to enhance agricultural yield mustard [ Brassica juncea (L.) Czern] as well as resilience. Bacillus pumilus , a PGPR, is known for synthesizing key phytohormones, such as IAA, which play a crucial role in modulating plant development. Tryptophan, a precursor for IAA biosynthesis, is another essential compound linked to the plant-bacteria interaction. HPLC serves as a powerful tool for the precise quantification of these metabolites, offering insights into the microbial mechanisms promoting plant growth. Additionally, antagonism against plant pathogens, such as Fusarium equiseti , further highlights the potential of Bacillus pumilus in crop protection. Results In this study, a bacterial isolate identified as Bacillus pumilus was evaluated for its PGP traits. The isolate demonstrated the production of significant amounts of IAA, as confirmed through HPLC analysis, revealing its metabolic capacity for enhancing plant growth. Alongside IAA production, the isolate exhibited other in vitro PGP activities, such as phosphate solubilization, and siderophore production, which are critical for improving nutrient availability to plants. Furthermore, Bacillus pumilus showed strong antagonistic activity against Fusarium equiseti , a notorious soil-borne pathogen, suggesting its role in biocontrol. Conclusion The study underscores Bacillus pumilus as a potent IAA-producing PGPR with multiple in vitro plant growth-promoting traits and effective antagonism against Fusarium equiseti . The use of HPLC for accurate quantification of IAA and tryptophan provides valuable insights into the microbial mechanisms driving plant growth promotion. These findings emphasize the potential of Bacillus pumilus as a bioinoculant in sustainable agriculture, offering a dual benefit of enhancing crop productivity and providing natural protection against plant pathogens. This research highlights the role of microbial-mediated strategies in advancing environmentally sustainable farming practices. Plant growth-promoting bacteria indole-3-acetic acid tryptophan HPLC microbial interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The intensification of agricultural practices has raised significant challenges regarding soil health, crop productivity, and sustainability within agroecosystems. Consequently, there is an increasing interest in sustainable agricultural practices that enhance soil fertility while minimizing the reliance on chemical fertilizers and pesticides. Among various strategies, the utilization of plant growth-promoting rhizobacteria (PGPR) has emerged as a promising approach to support plant growth, enhance nutrient acquisition, and improve resilience to biotic and abiotic stresses[ 2 ]. PGPR are beneficial soil bacteria that colonize the rhizosphere and have been shown to enhance plant growth through multiple mechanisms, including nutrient uptake, phytohormone production, and biocontrol of plant pathogens. Specifically, these bacteria can facilitate the solubilization of essential nutrients such as phosphorus, improve iron availability, and synthesize phytohormones like indole-3-acetic acid (IAA), which play critical roles in regulating various physiological processes in plants, including cell division, elongation, and differentiation[ 10 ]. B. pumilus is one such PGPR that is recognized for its remarkable ability to produce IAA and other beneficial metabolites. IAA, as a key auxin, significantly influences root development, shoot elongation, and overall plant architecture, thereby enhancing plant vigor and growth potential. Furthermore, B. pumilus can metabolize tryptophan, an essential amino acid that acts as a precursor for IAA biosynthesis, highlighting its importance in plant-microbe interactions[ 15 ]. In addition to its role in hormone production, B. pumilus exhibits critical traits such as phosphate solubilization and siderophore production, which directly contribute to improved nutrient availability for plants. Siderophores are organic compounds that chelate iron, facilitating its absorption by plants and thereby supporting their growth in iron-deficient soils. The capacity of B. pumilus to solubilize phosphorus from insoluble compounds further enhances its value as a biofertilizer, promoting sustainable agricultural practices[ 14 ]. Moreover, B. pumilus has been reported to possess antagonistic properties against various phytopathogens, including Fusarium equiseti , a soil-borne fungus known for causing significant crop diseases. The ability of B. pumilus to inhibit the growth of such pathogens not only protects crops but also reduces the need for chemical fungicides, aligning with the principles of integrated pest management (IPM) in sustainable agriculture[ 6 ]. Given the multifaceted roles of B. pumilus as a PGPR, this study aims to evaluate its plant growth-promoting traits, specifically focusing on IAA and tryptophan production, phosphate solubilization, and antagonistic activity against Fusarium equiseti . By elucidating the mechanisms of action of B. pumilus in promoting plant growth and health, we aim to contribute to the advancement of microbial-based solutions for enhancing agricultural productivity and sustainability[ 11 ]. Materials and Methods Collection of Seeds The seeds of Brassica juncea (L.) Czern. were collected from Debraj Saha, Farm Manager at Dakshin Dinajpur Krishi Vigyan Kendra, Majhian, P.O. Patiram, District Dakshin Dinajpur − 733133, West Bengal, India. The seed material was identified by Dakshin Dinajpur Krishi Vigyan Kendra. After collection, the seeds were grown in the experimental field at Karnajora, Raiganj, Uttar Dinajpur − 733130, West Bengal, India, where the study was conducted. Bacterial Isolation and Culture Sample Collection Soil samples were collected from Khowaspur (Lat. 25.772671 o ; Long. 88.032848 o ), Karandighi, Uttar Dinajpur District, West Bengal, in January 2022, specifically targeting the rhizospheric soil associated with mustard plants to isolate PGPR. The temperature varied between 13.3°C and 24.7°C, with an average humidity of 53%. Sterile polythene bags were used for collection, and the samples were transported to the laboratory. The samples were labeled and stored at 4°C. Soil analysis revealed that the soil type is clay loam (40% silt, 30% clay, 30% sand), pH 5.7 and with a depth range of 0–10 cm. [ 5 ]. Isolation Procedure The serial dilution technique was used to isolate bacteria from the collected soil samples. The soil suspensions were prepared in distilled sterile water and diluted in steps. Diluted samples were plated on nutrient agar media supplemented with tryptophan to promote the growth of IAA-producing bacteria. Incubation The inoculated plates were incubated at 28–30°C for the next 24–48 hours to allow for the growth of bacterial colonies. Pure Culture Selection Distinct bacterial colonies were chosen based on their morphology. The selected colonies were streaked to produce pure cultures on newly prepared nutrient agar plates [ 16 ]. Pure cultures were maintained on agar slants at 4ºC for future analysis. Screening for Indole-3-Acetic Acid (IAA) Production Inoculum Preparation Pure bacterial cultures, including Bacillus pumilus (ON783696), were inoculated into tryptophan-supplemented broth and incubated at 28°C for 48–72 hours. Tryptophan, as a precursor, was added to enhance IAA production by the bacterial isolates. Sample Preparation The dried residue was resuspended in methanol, and the solution was filtered using a 0.45 µm membrane filter. The specimens were kept at -20°C until analyzed by HPLC [ 16 ]. HPLC Analysis for IAA Quantification Instrument Setup Using an HPLC system outfitted with a C18 reverse-phase column and a UV-Vis detector calibrated at 254 nm, the ideal wavelength for IAA detection, the IAA concentration in bacterial extracts was measured. Standard Preparation Pure IAA standard solutions were made at predetermined concentrations, and a calibration curve was generated by plotting peak areas against standard concentrations. Chromatographic Conditions Methanol and water (80:20, v/v) at a flow rate of 1.0 mL/min made up the mobile phase. A 20 µL injection volume of the sample was used. Sample Injection and Analysis Standard solutions and IAA Samples were added to the HPLC device. IAA concentrations were ascertained by comparing the sample peak regions with the standard calibration curve [ 19 ]. Tryptophan Quantification Tryptophan Extraction We used acidified ethanol to extract tryptophan from bacterial cultures. The obtained supernatant was used for solvent extraction after the cultures were centrifuged. Sample Preparation The extracted tryptophan was passed through a membrane filter measuring 0.45 µm and diluted appropriately for HPLC analysis. HPLC Analysis Tryptophan concentrations were quantified using HPLC under the same conditions as the IAA quantification, except for detector adjustments for optimal tryptophan detection. A calibration curve using pure tryptophan standards was generated to calculate sample concentrations [ 8 ]. Screening for In Vitro Plant Growth-Promoting (PGP) Traits Phosphate Solubilization Bacillus pumilus was tested for its ability to solubilize phosphate by growing the bacteria on Pikovskaya’s agar medium. Clear halo zones around the colonies indicated phosphate solubilization. Siderophore Production The bacterial isolates were grown on Chrome Azurol S (CAS) agar plates in order to measure the generation of siderophores. The colonies' orange halo was thought to be a sign that siderophore production was proceeding well [ 4 ]. Antagonism Against Fusarium equiseti (ON783721) Dual Culture Assay The antagonistic activity of Bacillus pumilus (ON783696) against Fusarium equiseti , a common phytopathogen, was tested using a dual culture assay. The bacterial isolate and Fusarium equiseti were co-inoculated onto potato dextrose agar (PDA) plates. After incubating at 28°C for 5–7 days, the fungal growth was shown to be inhibited. Quantification of Inhibition The percentage of fungal growth inhibition was calculated by measuring the radial growth of the pathogen in the presence and absence of the bacterial isolate. The formula used was: $$\:\%Inhibition=\left(\frac{Control\:growth-Treated\:growth}{Control\:growth}\right)\times\:100$$ This allowed for quantitative assessment of Bacillus pumilus (ON783696) antagonistic potential against Fusarium equiseti (ON783721). Statistical Analysis Data Processing Agilent software was used to process data from the HPLC examination of the amounts of tryptophan and IAA. Standard deviations and mean values were computed as part of descriptive statistics. Regression Equation o The equation is followed by the calibration curves: y = ax + b In this case, y stands for the area or height of the instrument response. x is a representation of the compound's quantity. The coefficients a and b are found using the calibration data. Inferential Statistics Student's t-test was used in the statistical analysis to determine the significance of the variations in antagonistic activity and IAA generation amongst the bacterial isolates. P-values less than 0.05 were considered statistically significant [ 7 ]. Quality Control Measures Instrument Calibration Standard IAA and tryptophan solutions were used to calibrate the HPLC system regularly to guarantee the precision and accuracy of the measurement. Control Samples Positive and negative control samples were included in each analytical batch to ensure assay performance and identify any possible systematic errors. Reproducibility To ensure the results could be repeated, all tests, including the antagonism assays and HPLC quantification, were run in triplicate [ 12 ]. Results Isolation and Identification of Bacillus pumilus Isolates (ON783696) To comprehend PGPR's involvement in sustainable agriculture, it is essential to identify and isolate them. The soil samples that were gathered from Uttar Dinajpur's mustard-growing regions were serially diluted and then plated on nutritional agar that contained 50 µg/mL of kanamycin. separate colonies emerged following a 48-hour incubation period at 30°C, showing diverse morphological features, such as color, size, and texture. Colonies exhibited creamy white, yellow, and pink hues [ 13 ]. Initially, Gram staining and motility tests were used to identify the bacterial isolates, revealing that the majority of isolates were Gram-positive, rod-shaped, and motile. Biochemical assays, including catalase activity, IAA production, Siderophore Production and phosphate solubilization tests, were conducted to evaluate their plant growth-promoting traits [Table 1, Fig. 1 & Fig. 2 (a&b)]. Among these, the isolate Bacillus pumilus (ON783696) demonstrated strong PGP potential. For precise molecular identification via 16S rRNA gene sequencing was performed. The sequences were compared with reference sequences in NCBI databases, confirming that the isolate belonged to the species Bacillus pumilus (Fig. 3). This isolate was designated Bacillus pumilus MUSRH-05 (ON783696) and selected for further analysis of its PGP traits and antagonistic activity against Fusarium equiseti . IAA and Tryptophan Production by Bacillus pumilus One of the key plant growth-promoting traits of Bacillus pumilus MUSRH-05 (ON783696) is its ability to synthesize IAA, a vital phytohormone that enhances root development and overall plant growth. The isolate was cultured in tryptophan-supplemented broth to stimulate IAA and tryptophan production. After incubation, the IAA and tryptophan were extracted using ethyl acetate for subsequent quantification [ 3 ]. The extracts' HPLC examination verified that tryptophan and IAA were produced. The retention times for IAA and tryptophan were found to be 3.131 minutes and 2.644 minutes, respectively [Fig. 4 (a & b)]. Quantitative analysis revealed that Bacillus pumilus MUSRH-05 produced IAA at a concentration of 303.333 ng/µL, while tryptophan production was quantified at 151.383 ng/µL, based on peak areas of 5704.8125 and 2027.7877, respectively. The significant production of IAA by Bacillus pumilus (ON783696) highlights its potential to promote plant growth through the synthesis of auxin, which regulates various aspects of plant development. The production of tryptophan, a precursor to IAA, further underscores the metabolic capabilities of this strain in influencing plant-microbe interactions [ 1 ]. Antagonism Against Fusarium equiseti The antagonistic activity of Bacillus pumilus MUSRH-05 against the phytopathogen Fusarium equiseti was evaluated in vitro . The purpose of the dual culture assays was to assess the inhibition of fungal growth by the bacterial isolate. The results showed that Bacillus pumilus MUSRH-05 (ON783696) exhibited strong antifungal activity, with a significant inhibition zone around the bacterial colony i.e., 80% [Fig. 5 (a & b]. This antagonism is likely due to the production of secondary metabolites by Bacillus pumilus , which interfere with the growth of Fusarium equiseti (ON783721) [ 18 ]. The antagonistic property of Bacillus pumilus (ON783696) is a valuable trait for biological control, providing a sustainable substitute for chemical fungicides in the control of soil-borne illnesses. The dual action of promoting plant growth through IAA production and controlling pathogens demonstrates the multifaceted benefits of Bacillus pumilus as a PGPR. Comparison of HPLC and Spectrophotometric Methods for IAA Quantification To validate the accuracy of HPLC quantification, the outcomes were contrasted with the spectrophotometric method's findings. While the spectrophotometric approach is commonly used for IAA measurement, it is less specific due to interference from other indolic compounds. In contrast, HPLC offers higher accuracy and precision by separating IAA from additional substances found in the sample. The HPLC method showed a higher correlation coefficient (R² = 0.9987) for IAA quantification compared to the spectrophotometric method (R² = 0.840), emphasizing the superiority of HPLC for accurate IAA analysis [ 9 ]. Reproducibility and Linearity of HPLC Quantification The reproducibility and linearity of the HPLC method were evaluated by analyzing multiple replicates and constructing calibration curves. The relative standard deviation (RSD) values for both IAA and tryptophan quantification were consistently below 2%, demonstrating the high precision of the method. Calibration curves for IAA and tryptophan showed excellent linearity, with R² values of 0.9987 and 0.9991, respectively [ 17 ], confirming the method's reliability for plant growth-promoting studies. Therefore, Bacillus pumilus MUSRH-05 exhibits significant plant growth-promoting traits, including high levels of IAA production and effective antagonism against Fusarium equiseti (ON783721). The accuracy and precision of HPLC analysis, combined with the robust antagonistic properties of this bacterial isolate, suggest its potential as a bioinoculant for enhancing crop productivity and disease resistance. These findings demonstrate the utility of Bacillus pumilus in sustainable agriculture. Discussion The present study highlights the plant growth-promoting potential and biocontrol efficacy of Bacillus pumilus MUSRH-05 (ON783696), isolated from the rhizosphere of mustard crops in Uttar Dinajpur. The successful isolation and identification of this strain underscore its adaptability to the soil environment and its role in sustainable agriculture. The characterization of Bacillus pumilus MUSRH-05 demonstrated its ability to produce indole-3-acetic acid (IAA) and solubilize phosphate, both of which are crucial for plant growth and development. The production of IAA, a key phytohormone, enhances root elongation and facilitates nutrient uptake, thereby improving plant vigor. High-performance liquid chromatography (HPLC) quantification confirmed the production of IAA at a concentration of 303.333 ng/µL with a retention time of 3.131 minutes, while tryptophan, an essential precursor for IAA biosynthesis, was detected at 151.383 ng/µL. These findings indicate that Bacillus pumilus MUSRH-05 possesses a robust metabolic pathway for auxin synthesis, making it a promising candidate for plant growth enhancement. The antagonistic potential of Bacillus pumilus MUSRH-05 against the phytopathogenic fungus Fusarium equiseti was evaluated through dual culture assays, which demonstrated significant fungal growth inhibition (80%). This inhibitory effect is likely attributed to the secretion of antimicrobial compounds that interfere with fungal development. The strong antifungal activity of this strain positions it as a potential biocontrol agent, offering an eco-friendly alternative to synthetic fungicides. To ensure the accuracy and precision of IAA quantification, a comparative analysis between HPLC and spectrophotometric methods was conducted. The HPLC method exhibited a higher correlation coefficient (R² = 0.9987), confirming its superiority over spectrophotometry (R² = 0.840) in detecting and quantifying IAA. The reproducibility and linearity of the HPLC method, with relative standard deviation (RSD) values below 2%, further validate its reliability for future studies on bacterial plant growth promotion. The results of this study highlight the dual role of Bacillus pumilus MUSRH-05 as a plant growth promoter and biocontrol agent, reinforcing its potential as a bioinoculant for sustainable agricultural practices. Its ability to enhance crop productivity while suppressing soil-borne pathogens presents a viable alternative to chemical fertilizers and fungicides. Future investigations should focus on field trials and elucidating the molecular mechanisms underlying its biocontrol and growth-promoting activities. The implementation of Bacillus pumilus MUSRH-05 as a biofertilizer and biopesticide could significantly contribute to environmentally sustainable agricultural practices, reducing chemical inputs while enhancing crop resilience and yield. Conclusion This study highlights Bacillus pumilus MUSRH-05 as a potent PGPR with significant plant growth-promoting and biocontrol traits. The strain demonstrated high IAA and tryptophan production, efficient phosphate solubilization, and siderophore production, contributing to enhanced nutrient availability and plant health. Additionally, its strong antagonistic activity against Fusarium equiseti underscores its potential as a biocontrol agent. The multifunctional properties of Bacillus pumilus position it as a promising biofertilizer and biopesticide candidate for sustainable agriculture. Future research should focus on field trials and microbial consortia to optimize its application in diverse agroecosystems. Implementing Bacillus pumilus -based bioinoculants could reduce dependency on chemical fertilizers and pesticides, promoting environmentally friendly and resilient agricultural practices. Abbreviations PGPR: Plant Growth-Promoting Rhizobacteria, IAA: Indole-3-Acetic Acid, HPLC: High-performance liquid chromatography, PGP: Plant Growth-Promoting. Declarations Acknowledgements Mr. Debraj Saha, Farm Manager at Dakshin Dinajpur Krishi Vigyan Kendra, Majhian, P.O. Patiram, Dist - Dakshin Dinajpur - 733133, West Bengal, India. Is acknowledged for providing the Brassica seeds for experimental purposes. Heredity Life Sciences Pvt Ltd, Bhubaneswar, Odisha and Edison Life Science Laboratory, Kolkata, West Bengal, India are thankfully acknowledged for the molecular identification of bacterial strain. Authors also acknowledged Environcheck for HPLC analysis of IAA, tryptophan. Authors' contributions Mr. Shambhu Swarnakar wrote the manuscript and prepared the figures and tables. Dr. Arka Pratim Chakraborty reviewed the manuscript and finalized it before communication to the journal Conflict of Interest Authors declare that there are no conflict of interest. Compliance with Ethical Standards Disclosure of potential conflicts of interest The authors declare that they have no conflicts of interest. Research involving human participants and/or animals This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent No informed consent is required. Data Availability Statement This research work did not involve the generation or analysis of separate datasets. All relevant data collected during the study are presented in the manuscript. Further enquiries can be directed to the corresponding author. Funding No funding support is availed. Consent of publication I, Shambhu Swarnakar performed the experiment and surveyed the field for soil collection. The photo belongs to me. I have no objection if image is used for publication purposes in the article. References Adil M, Filimban F Z, AmbrinQuddoos A, Sher AA, Naseer M (2024). 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legend.\u003c/p\u003e","description":"","filename":"figure4..jpg","url":"https://assets-eu.researchsquare.com/files/rs-5477377/v1/7deacfd851272f351aa0115c.jpg"},{"id":80338235,"identity":"375c7a83-7f91-449f-b808-25d91c572ec9","added_by":"auto","created_at":"2025-04-10 16:59:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":362796,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure5.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5477377/v1/b21eef302bec2eada68bdd07.jpg"},{"id":80339413,"identity":"63e8ef02-d078-4689-957a-d29f18a8de45","added_by":"auto","created_at":"2025-04-10 17:23:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2742985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5477377/v1/59f7855a-f95b-4db5-8bba-7c89ce4593bd.pdf"},{"id":80337561,"identity":"1f1458ca-60f9-46ab-9271-fe452e644d28","added_by":"auto","created_at":"2025-04-10 16:51:08","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":209785,"visible":true,"origin":"","legend":"","description":"","filename":"Table1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-5477377/v1/83aa34d4db8f446fd99f3ba1.jpg"},{"id":80337563,"identity":"9e332a71-9f9f-4a8b-bc6c-ad968b5f3c0e","added_by":"auto","created_at":"2025-04-10 16:51:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1917398,"visible":true,"origin":"","legend":"","description":"","filename":"Plagiarismreport.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5477377/v1/517dc8e544284ae683141536.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bacillus pumilus - A Potent IAA Producing Plant Growth Promoting Rhizobacteria with In Vitro PGP Traits and Antagonism Against Fusarium equiseti","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe intensification of agricultural practices has raised significant challenges regarding soil health, crop productivity, and sustainability within agroecosystems. Consequently, there is an increasing interest in sustainable agricultural practices that enhance soil fertility while minimizing the reliance on chemical fertilizers and pesticides. Among various strategies, the utilization of plant growth-promoting rhizobacteria (PGPR) has emerged as a promising approach to support plant growth, enhance nutrient acquisition, and improve resilience to biotic and abiotic stresses[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePGPR are beneficial soil bacteria that colonize the rhizosphere and have been shown to enhance plant growth through multiple mechanisms, including nutrient uptake, phytohormone production, and biocontrol of plant pathogens. Specifically, these bacteria can facilitate the solubilization of essential nutrients such as phosphorus, improve iron availability, and synthesize phytohormones like indole-3-acetic acid (IAA), which play critical roles in regulating various physiological processes in plants, including cell division, elongation, and differentiation[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. pumilus\u003c/em\u003e is one such PGPR that is recognized for its remarkable ability to produce IAA and other beneficial metabolites. IAA, as a key auxin, significantly influences root development, shoot elongation, and overall plant architecture, thereby enhancing plant vigor and growth potential. Furthermore, \u003cem\u003eB. pumilus\u003c/em\u003e can metabolize tryptophan, an essential amino acid that acts as a precursor for IAA biosynthesis, highlighting its importance in plant-microbe interactions[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to its role in hormone production, \u003cem\u003eB. pumilus\u003c/em\u003e exhibits critical traits such as phosphate solubilization and siderophore production, which directly contribute to improved nutrient availability for plants. Siderophores are organic compounds that chelate iron, facilitating its absorption by plants and thereby supporting their growth in iron-deficient soils. The capacity of \u003cem\u003eB. pumilus\u003c/em\u003e to solubilize phosphorus from insoluble compounds further enhances its value as a biofertilizer, promoting sustainable agricultural practices[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, \u003cem\u003eB. pumilus\u003c/em\u003e has been reported to possess antagonistic properties against various phytopathogens, including \u003cem\u003eFusarium equiseti\u003c/em\u003e, a soil-borne fungus known for causing significant crop diseases. The ability of \u003cem\u003eB. pumilus\u003c/em\u003e to inhibit the growth of such pathogens not only protects crops but also reduces the need for chemical fungicides, aligning with the principles of integrated pest management (IPM) in sustainable agriculture[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the multifaceted roles of \u003cem\u003eB. pumilus\u003c/em\u003e as a PGPR, this study aims to evaluate its plant growth-promoting traits, specifically focusing on IAA and tryptophan production, phosphate solubilization, and antagonistic activity against \u003cem\u003eFusarium equiseti\u003c/em\u003e. By elucidating the mechanisms of action of \u003cem\u003eB. pumilus\u003c/em\u003e in promoting plant growth and health, we aim to contribute to the advancement of microbial-based solutions for enhancing agricultural productivity and sustainability[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection of Seeds\u003c/h2\u003e \u003cp\u003eThe seeds of \u003cem\u003eBrassica juncea\u003c/em\u003e (L.) Czern. were collected from Debraj Saha, Farm Manager at Dakshin Dinajpur Krishi Vigyan Kendra, Majhian, P.O. Patiram, District Dakshin Dinajpur \u0026minus;\u0026thinsp;733133, West Bengal, India. The seed material was identified by Dakshin Dinajpur Krishi Vigyan Kendra. After collection, the seeds were grown in the experimental field at Karnajora, Raiganj, Uttar Dinajpur \u0026minus;\u0026thinsp;733130, West Bengal, India, where the study was conducted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial Isolation and Culture\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eSample Collection\u003c/b\u003e Soil samples were collected from Khowaspur (Lat. 25.772671\u003csup\u003eo\u003c/sup\u003e; Long. 88.032848\u003csup\u003eo\u003c/sup\u003e), Karandighi, Uttar Dinajpur District, West Bengal, in January 2022, specifically targeting the rhizospheric soil associated with mustard plants to isolate PGPR. The temperature varied between 13.3\u0026deg;C and 24.7\u0026deg;C, with an average humidity of 53%. Sterile polythene bags were used for collection, and the samples were transported to the laboratory. The samples were labeled and stored at 4\u0026deg;C. Soil analysis revealed that the soil type is clay loam (40% silt, 30% clay, 30% sand), pH 5.7 and with a depth range of 0\u0026ndash;10 cm. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation Procedure\u003c/b\u003e The serial dilution technique was used to isolate bacteria from the collected soil samples. The soil suspensions were prepared in distilled sterile water and diluted in steps. Diluted samples were plated on nutrient agar media supplemented with tryptophan to promote the growth of IAA-producing bacteria.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncubation\u003c/b\u003e The inoculated plates were incubated at 28\u0026ndash;30\u0026deg;C for the next 24\u0026ndash;48 hours to allow for the growth of bacterial colonies.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePure Culture Selection\u003c/b\u003e Distinct bacterial colonies were chosen based on their morphology. The selected colonies were streaked to produce pure cultures on newly prepared nutrient agar plates [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Pure cultures were maintained on agar slants at 4\u0026ordm;C for future analysis.\u003c/p\u003e\n\u003ch3\u003eScreening for Indole-3-Acetic Acid (IAA) Production\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eInoculum Preparation\u003c/b\u003e Pure bacterial cultures, including \u003cem\u003eBacillus pumilus\u003c/em\u003e (ON783696), were inoculated into tryptophan-supplemented broth and incubated at 28\u0026deg;C for 48\u0026ndash;72 hours. Tryptophan, as a precursor, was added to enhance IAA production by the bacterial isolates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSample Preparation\u003c/b\u003e The dried residue was resuspended in methanol, and the solution was filtered using a 0.45 \u0026micro;m membrane filter. The specimens were kept at -20\u0026deg;C until analyzed by HPLC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eHPLC Analysis for IAA Quantification\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eInstrument Setup\u003c/b\u003e Using an HPLC system outfitted with a C18 reverse-phase column and a UV-Vis detector calibrated at 254 nm, the ideal wavelength for IAA detection, the IAA concentration in bacterial extracts was measured.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStandard Preparation\u003c/b\u003e Pure IAA standard solutions were made at predetermined concentrations, and a calibration curve was generated by plotting peak areas against standard concentrations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromatographic Conditions\u003c/b\u003e Methanol and water (80:20, v/v) at a flow rate of 1.0 mL/min made up the mobile phase. A 20 \u0026micro;L injection volume of the sample was used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSample Injection and Analysis\u003c/b\u003e Standard solutions and IAA Samples were added to the HPLC device. IAA concentrations were ascertained by comparing the sample peak regions with the standard calibration curve [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTryptophan Quantification\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eTryptophan Extraction\u003c/b\u003e We used acidified ethanol to extract tryptophan from bacterial cultures. The obtained supernatant was used for solvent extraction after the cultures were centrifuged.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSample Preparation\u003c/b\u003e The extracted tryptophan was passed through a membrane filter measuring 0.45 \u0026micro;m and diluted appropriately for HPLC analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHPLC Analysis\u003c/b\u003e Tryptophan concentrations were quantified using HPLC under the same conditions as the IAA quantification, except for detector adjustments for optimal tryptophan detection. A calibration curve using pure tryptophan standards was generated to calculate sample concentrations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eScreening for\u003c/b\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003ePlant Growth-Promoting (PGP) Traits\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhosphate Solubilization\u003c/b\u003e \u003cem\u003eBacillus pumilus\u003c/em\u003e was tested for its ability to solubilize phosphate by growing the bacteria on Pikovskaya\u0026rsquo;s agar medium. Clear halo zones around the colonies indicated phosphate solubilization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSiderophore Production\u003c/b\u003e The bacterial isolates were grown on Chrome Azurol S (CAS) agar plates in order to measure the generation of siderophores. The colonies' orange halo was thought to be a sign that siderophore production was proceeding well [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntagonism Against Fusarium equiseti\u003c/b\u003e (ON783721)\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual Culture Assay\u003c/b\u003e The antagonistic activity of \u003cem\u003eBacillus pumilus\u003c/em\u003e (ON783696) against \u003cem\u003eFusarium equiseti\u003c/em\u003e, a common phytopathogen, was tested using a dual culture assay. The bacterial isolate and \u003cem\u003eFusarium equiseti\u003c/em\u003e were co-inoculated onto potato dextrose agar (PDA) plates. After incubating at 28\u0026deg;C for 5\u0026ndash;7 days, the fungal growth was shown to be inhibited.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantification of Inhibition\u003c/b\u003e The percentage of fungal growth inhibition was calculated by measuring the radial growth of the pathogen in the presence and absence of the bacterial isolate. The formula used was:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\%Inhibition=\\left(\\frac{Control\\:growth-Treated\\:growth}{Control\\:growth}\\right)\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThis allowed for quantitative assessment of \u003cem\u003eBacillus pumilus\u003c/em\u003e (ON783696) antagonistic potential against \u003cem\u003eFusarium equiseti\u003c/em\u003e (ON783721).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cb\u003eData Processing\u003c/b\u003e Agilent software was used to process data from the HPLC examination of the amounts of tryptophan and IAA. Standard deviations and mean values were computed as part of descriptive statistics.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRegression Equation\u003c/h3\u003e\n\u003cp\u003eo The equation is followed by the calibration curves:\u003c/p\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;ax\u0026thinsp;+\u0026thinsp;b\u003c/p\u003e \u003cp\u003eIn this case, y stands for the area or height of the instrument response.\u003c/p\u003e \u003cp\u003ex is a representation of the compound's quantity.\u003c/p\u003e \u003cp\u003eThe coefficients a and b are found using the calibration data.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInferential Statistics\u003c/b\u003e Student's t-test was used in the statistical analysis to determine the significance of the variations in antagonistic activity and IAA generation amongst the bacterial isolates. P-values less than 0.05 were considered statistically significant [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eQuality Control Measures\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eInstrument Calibration\u003c/b\u003e Standard IAA and tryptophan solutions were used to calibrate the HPLC system regularly to guarantee the precision and accuracy of the measurement.\u003c/p\u003e \u003cp\u003e \u003cb\u003eControl Samples\u003c/b\u003e Positive and negative control samples were included in each analytical batch to ensure assay performance and identify any possible systematic errors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReproducibility\u003c/b\u003e To ensure the results could be repeated, all tests, including the antagonism assays and HPLC quantification, were run in triplicate [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIsolation and Identification of\u003c/b\u003e \u003cb\u003eBacillus pumilus\u003c/b\u003e \u003cb\u003eIsolates\u003c/b\u003e(ON783696)\u003c/p\u003e \u003cp\u003eTo comprehend PGPR's involvement in sustainable agriculture, it is essential to identify and isolate them. The soil samples that were gathered from Uttar Dinajpur's mustard-growing regions were serially diluted and then plated on nutritional agar that contained 50 \u0026micro;g/mL of kanamycin. separate colonies emerged following a 48-hour incubation period at 30\u0026deg;C, showing diverse morphological features, such as color, size, and texture. Colonies exhibited creamy white, yellow, and pink hues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInitially, Gram staining and motility tests were used to identify the bacterial isolates, revealing that the majority of isolates were Gram-positive, rod-shaped, and motile. Biochemical assays, including catalase activity, IAA production, Siderophore Production and phosphate solubilization tests, were conducted to evaluate their plant growth-promoting traits [Table\u0026nbsp;1, Fig.\u0026nbsp;1 \u0026amp; Fig.\u0026nbsp;2 (a\u0026amp;b)]. Among these, the isolate \u003cem\u003eBacillus pumilus\u003c/em\u003e (ON783696) demonstrated strong PGP potential.\u003c/p\u003e \u003cp\u003eFor precise molecular identification via 16S rRNA gene sequencing was performed. The sequences were compared with reference sequences in NCBI databases, confirming that the isolate belonged to the species \u003cem\u003eBacillus pumilus\u003c/em\u003e (Fig.\u0026nbsp;3). This isolate was designated \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 (ON783696) and selected for further analysis of its PGP traits and antagonistic activity against \u003cem\u003eFusarium equiseti\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIAA and Tryptophan Production by\u003c/b\u003e \u003cb\u003eBacillus pumilus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne of the key plant growth-promoting traits of \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 (ON783696) is its ability to synthesize IAA, a vital phytohormone that enhances root development and overall plant growth. The isolate was cultured in tryptophan-supplemented broth to stimulate IAA and tryptophan production. After incubation, the IAA and tryptophan were extracted using ethyl acetate for subsequent quantification [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe extracts' HPLC examination verified that tryptophan and IAA were produced. The retention times for IAA and tryptophan were found to be 3.131 minutes and 2.644 minutes, respectively [Fig.\u0026nbsp;4 (a \u0026amp; b)]. Quantitative analysis revealed that \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 produced IAA at a concentration of 303.333 ng/\u0026micro;L, while tryptophan production was quantified at 151.383 ng/\u0026micro;L, based on peak areas of 5704.8125 and 2027.7877, respectively.\u003c/p\u003e \u003cp\u003eThe significant production of IAA by \u003cem\u003eBacillus pumilus\u003c/em\u003e (ON783696) highlights its potential to promote plant growth through the synthesis of auxin, which regulates various aspects of plant development. The production of tryptophan, a precursor to IAA, further underscores the metabolic capabilities of this strain in influencing plant-microbe interactions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntagonism Against\u003c/b\u003e \u003cb\u003eFusarium equiseti\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe antagonistic activity of \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 against the phytopathogen \u003cem\u003eFusarium equiseti\u003c/em\u003e was evaluated \u003cem\u003ein vitro\u003c/em\u003e. The purpose of the dual culture assays was to assess the inhibition of fungal growth by the bacterial isolate. The results showed that \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 (ON783696) exhibited strong antifungal activity, with a significant inhibition zone around the bacterial colony i.e., 80% [Fig.\u0026nbsp;5 (a \u0026amp; b]. This antagonism is likely due to the production of secondary metabolites by \u003cem\u003eBacillus pumilus\u003c/em\u003e, which interfere with the growth of \u003cem\u003eFusarium equiseti\u003c/em\u003e (ON783721) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe antagonistic property of \u003cem\u003eBacillus pumilus\u003c/em\u003e (ON783696) is a valuable trait for biological control, providing a sustainable substitute for chemical fungicides in the control of soil-borne illnesses. The dual action of promoting plant growth through IAA production and controlling pathogens demonstrates the multifaceted benefits of \u003cem\u003eBacillus pumilus\u003c/em\u003e as a PGPR.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparison of HPLC and Spectrophotometric Methods for IAA Quantification\u003c/h2\u003e \u003cp\u003eTo validate the accuracy of HPLC quantification, the outcomes were contrasted with the spectrophotometric method's findings. While the spectrophotometric approach is commonly used for IAA measurement, it is less specific due to interference from other indolic compounds. In contrast, HPLC offers higher accuracy and precision by separating IAA from additional substances found in the sample. The HPLC method showed a higher correlation coefficient (R\u0026sup2; = 0.9987) for IAA quantification compared to the spectrophotometric method (R\u0026sup2; = 0.840), emphasizing the superiority of HPLC for accurate IAA analysis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReproducibility and Linearity of HPLC Quantification\u003c/h2\u003e \u003cp\u003eThe reproducibility and linearity of the HPLC method were evaluated by analyzing multiple replicates and constructing calibration curves. The relative standard deviation (RSD) values for both IAA and tryptophan quantification were consistently below 2%, demonstrating the high precision of the method. Calibration curves for IAA and tryptophan showed excellent linearity, with R\u0026sup2; values of 0.9987 and 0.9991, respectively [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], confirming the method's reliability for plant growth-promoting studies.\u003c/p\u003e \u003cp\u003eTherefore, \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 exhibits significant plant growth-promoting traits, including high levels of IAA production and effective antagonism against \u003cem\u003eFusarium equiseti\u003c/em\u003e (ON783721). The accuracy and precision of HPLC analysis, combined with the robust antagonistic properties of this bacterial isolate, suggest its potential as a bioinoculant for enhancing crop productivity and disease resistance. These findings demonstrate the utility of \u003cem\u003eBacillus pumilus\u003c/em\u003e in sustainable agriculture.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study highlights the plant growth-promoting potential and biocontrol efficacy of \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 (ON783696), isolated from the rhizosphere of mustard crops in Uttar Dinajpur. The successful isolation and identification of this strain underscore its adaptability to the soil environment and its role in sustainable agriculture.\u003c/p\u003e \u003cp\u003eThe characterization of \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 demonstrated its ability to produce indole-3-acetic acid (IAA) and solubilize phosphate, both of which are crucial for plant growth and development. The production of IAA, a key phytohormone, enhances root elongation and facilitates nutrient uptake, thereby improving plant vigor. High-performance liquid chromatography (HPLC) quantification confirmed the production of IAA at a concentration of 303.333 ng/\u0026micro;L with a retention time of 3.131 minutes, while tryptophan, an essential precursor for IAA biosynthesis, was detected at 151.383 ng/\u0026micro;L. These findings indicate that \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 possesses a robust metabolic pathway for auxin synthesis, making it a promising candidate for plant growth enhancement.\u003c/p\u003e \u003cp\u003eThe antagonistic potential of \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 against the phytopathogenic fungus \u003cem\u003eFusarium equiseti\u003c/em\u003e was evaluated through dual culture assays, which demonstrated significant fungal growth inhibition (80%). This inhibitory effect is likely attributed to the secretion of antimicrobial compounds that interfere with fungal development. The strong antifungal activity of this strain positions it as a potential biocontrol agent, offering an eco-friendly alternative to synthetic fungicides.\u003c/p\u003e \u003cp\u003eTo ensure the accuracy and precision of IAA quantification, a comparative analysis between HPLC and spectrophotometric methods was conducted. The HPLC method exhibited a higher correlation coefficient (R\u0026sup2; = 0.9987), confirming its superiority over spectrophotometry (R\u0026sup2; = 0.840) in detecting and quantifying IAA. The reproducibility and linearity of the HPLC method, with relative standard deviation (RSD) values below 2%, further validate its reliability for future studies on bacterial plant growth promotion.\u003c/p\u003e \u003cp\u003eThe results of this study highlight the dual role of \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 as a plant growth promoter and biocontrol agent, reinforcing its potential as a bioinoculant for sustainable agricultural practices. Its ability to enhance crop productivity while suppressing soil-borne pathogens presents a viable alternative to chemical fertilizers and fungicides. Future investigations should focus on field trials and elucidating the molecular mechanisms underlying its biocontrol and growth-promoting activities. The implementation of \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 as a biofertilizer and biopesticide could significantly contribute to environmentally sustainable agricultural practices, reducing chemical inputs while enhancing crop resilience and yield.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights \u003cem\u003eBacillus pumilus\u003c/em\u003e MUSRH-05 as a potent PGPR with significant plant growth-promoting and biocontrol traits. The strain demonstrated high IAA and tryptophan production, efficient phosphate solubilization, and siderophore production, contributing to enhanced nutrient availability and plant health. Additionally, its strong antagonistic activity against Fusarium equiseti underscores its potential as a biocontrol agent.\u003c/p\u003e \u003cp\u003eThe multifunctional properties of \u003cem\u003eBacillus pumilus\u003c/em\u003e position it as a promising biofertilizer and biopesticide candidate for sustainable agriculture. Future research should focus on field trials and microbial consortia to optimize its application in diverse agroecosystems. Implementing \u003cem\u003eBacillus pumilus\u003c/em\u003e-based bioinoculants could reduce dependency on chemical fertilizers and pesticides, promoting environmentally friendly and resilient agricultural practices.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePGPR: Plant Growth-Promoting Rhizobacteria, IAA: \u0026nbsp;Indole-3-Acetic Acid, HPLC: High-performance liquid chromatography, PGP: Plant Growth-Promoting.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMr. Debraj Saha, Farm Manager at Dakshin Dinajpur Krishi Vigyan Kendra, Majhian, P.O. Patiram, Dist - Dakshin Dinajpur - 733133, West Bengal, India. Is acknowledged for providing the Brassica seeds for experimental purposes.\u003c/p\u003e\n\u003cp\u003eHeredity Life Sciences Pvt Ltd, Bhubaneswar, Odisha and Edison Life Science Laboratory, Kolkata, West Bengal, India are thankfully acknowledged for the molecular identification of bacterial strain.\u003c/p\u003e\n\u003cp\u003eAuthors also acknowledged Environcheck for HPLC analysis of IAA, tryptophan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMr. Shambhu Swarnakar wrote the manuscript and prepared the figures and tables. Dr. Arka Pratim Chakraborty reviewed the manuscript and finalized it before communication to the journal\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that there are no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDisclosure of potential conflicts of interest\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eResearch involving human participants and/or animals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInformed consent\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo informed consent is required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability Statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work did not involve the generation or analysis of separate datasets. All relevant data collected during the study are presented in the manuscript. Further enquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding support is availed. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent of publication\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI, Shambhu Swarnakar performed the experiment and surveyed the field for soil collection. The photo belongs to me. I have no objection if image is used for publication purposes in the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdil M, Filimban F Z, AmbrinQuddoos A, Sher AA, Naseer M (2024). Phytochemical screening, HPLC analysis, antimicrobial and antioxidant effect of Euphorbia parviflora L.(Euphorbiaceae Juss.). Scientific Reports, \u003cem\u003e14\u003c/em\u003e(1), 5627.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAubier TG, Kopp M, Linn IJ, Puebla O, Rafajlović M, Servedio MR (2024). Negative coupling: the coincidence of premating isolating barriers can reduce reproductive isolation. Cold Spring Harbor Perspectives in Biology, a041435.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalatrava V, Hom EF, Guan Q, Llamas A, Fern\u0026aacute;ndez E, Galv\u0026aacute;n A (2024). Genetic evidence for algal auxin production in Chlamydomonas and its role in algal-bacterial mutualism. Iscience, \u003cem\u003e27\u003c/em\u003e(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Ye X, Yu X, Zhao J, Song M, Yin D, Yu J (2024). Analysis of the regulatory mechanism of exogenous IAA-mediated tryptophan accumulation and synthesis of endogenous IAA in Chlorococcumhumicola. Chemosphere, 141633.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClare EL, Economou CK, Bennett FJ, Dyer CE, Adams K, McRobie B, Littlefair JE (2022). Measuring biodiversity from DNA in the air. Current Biology, \u003cem\u003e32\u003c/em\u003e(3), 693\u0026ndash;700.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFathy WA, AbdElgawad H, Hashem AH, Essawy E, Tawfik E, Al-Askar AA, Elsayed KN (2023). Exploring Exogenous Indole-3-acetic Acid\u0026rsquo;s Effect on the Growth and Biochemical Profiles of \u003cem\u003eSynechocystis\u003c/em\u003e sp. PAK13 and Chlorella variabilis. Molecules, \u003cem\u003e28\u003c/em\u003e(14), 5501.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGharsallah F, Ulusoy HI,Ghorbel N(2024). Highly selective analysis of glipizide as an antidiabetic drug in urine samples using HPLC-DAD technique via magnetic nanocomposites (Fe3O4@ SiO2@ tryptophan). Microchemical Journal, \u003cem\u003e197\u003c/em\u003e, 109861.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGirgin G, Sanajou S, Meric-Deliveli S, Baydar T (2024). Verification studies of tryptophan and kynurenine determination using HPLC and evaluation of the kynurenine pathway and neopterin levels in human colostrum samples. Biomedical Chromatography, \u003cem\u003e38\u003c/em\u003e(2), e5791.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang CL, Wu SW, Hsu TC, Yang CY, Chung WH, Lin XJ, Yu FY (2023). Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for sensitive detecting aristolochic acid I in herbal remedies. Microchemical Journal, \u003cem\u003e191\u003c/em\u003e, 108884.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang Y, Wu Y, Hu N, Li H, Jiao J (2020). Interactions of bacterial-feeding nematodes and indole-3-acetic acid (IAA)-producing bacteria promotes growth of Arabidopsis thaliana by regulating soil auxin status. Applied Soil Ecology, \u003cem\u003e147\u003c/em\u003e, 103447.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLata DL, Abdie O, Rezene Y (2024). IAA-producing bacteria from the rhizosphere of chickpea (Cicer arietinum L.): Isolation, characterization, and their effects on plant growth performance. Heliyon, \u003cem\u003e10\u003c/em\u003e(21).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammadi F, Roushani M, Valipour A (2024). Development of a label-free impedimetric aptasensor based on Zr-MOF and titaniom carbide nanosheets for detection of L-tryptophan. Bioelectrochemistry, \u003cem\u003e155\u003c/em\u003e, 108584.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustafa YF, Ismael RN, Jebir RM (2024). Natural coumarins from two cultivars of watermelon seeds as biosafe anticancer agents, an algorithm for their isolation and evaluation. Journal of Molecular Structure, \u003cem\u003e1295\u003c/em\u003e, 136644.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaes MDCRP (2023). \u003cem\u003eStudy of melatonin defence priming effects against fungal development in Vitis vinifera grapes during pre and postharvest\u003c/em\u003e (Doctoral dissertation).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatnaningsih HR, Noviana Z, Dewi TK, Loekito S, Wiyon S, Gafur A, Antonius S (2023). IAA and ACC deaminase producing-bacteria isolated from the rhizosphere of pineapple plants grown under different abiotic and biotic stresses. Heliyon, \u003cem\u003e9\u003c/em\u003e(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRychshanova R, Mendybayeva A, Miciński B, Mamiyev N, Shevchenko P, Bermukhametov Z Miciński J (2022). Antibiotic resistance and biofilm formation in Staphylococcus aureus isolated from dairy cows at the stage of subclinical mastitis in northern Kazakhstan. Archives Animal Breeding, \u003cem\u003e65\u003c/em\u003e(4), 439\u0026ndash;448.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrieder MM, Sanches VL, Rostagno MA (2024). Simultaneous extraction, separation, and analysis of 5-caffeoylquinic acid and caffeine from coffee co-product by PLE-SPE\u0026times; HPLC-PDA two-dimensional system. Food Research International, \u003cem\u003e175\u003c/em\u003e, 113690.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerzi M, Theodorou M, Louloudi E, Manousi N, Tzanavaras PD, Zacharis CK (2024). Salicylic acid as a switchable hydrophilicity solvent for the microextraction of the antibiotic amphotericin B from human urine followed by HPLC-UV analysis. Microchemical Journal, \u003cem\u003e199\u003c/em\u003e, 110025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Rong Z, Li Y, Yin Z, Lu C, Zhao H, Ding X (2024). NIT24 and NIT29-mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice. Molecular Plant Pathology, \u003cem\u003e25\u003c/em\u003e(1), e13409.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-plants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Plants](https://link.springer.com/journal/44372)","snPcode":"44372","submissionUrl":"https://submission.springernature.com/new-submission/44372/3","title":"Discover Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Plant growth-promoting bacteria, indole-3-acetic acid, tryptophan, HPLC, microbial interactions","lastPublishedDoi":"10.21203/rs.3.rs-5477377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5477377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePlant growth promotion through microbial mediation has garnered increasing focus in sustainable agriculture because of its capacity to enhance agricultural yield mustard [\u003cem\u003eBrassica juncea\u003c/em\u003e (L.) Czern] as well as resilience. \u003cem\u003eBacillus pumilus\u003c/em\u003e, a PGPR, is known for synthesizing key phytohormones, such as IAA, which play a crucial role in modulating plant development. Tryptophan, a precursor for IAA biosynthesis, is another essential compound linked to the plant-bacteria interaction. HPLC serves as a powerful tool for the precise quantification of these metabolites, offering insights into the microbial mechanisms promoting plant growth. Additionally, antagonism against plant pathogens, such as \u003cem\u003eFusarium equiseti\u003c/em\u003e, further highlights the potential of \u003cem\u003eBacillus pumilus\u003c/em\u003e in crop protection.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, a bacterial isolate identified as \u003cem\u003eBacillus pumilus\u003c/em\u003e was evaluated for its PGP traits. The isolate demonstrated the production of significant amounts of IAA, as confirmed through HPLC analysis, revealing its metabolic capacity for enhancing plant growth. Alongside IAA production, the isolate exhibited other \u003cem\u003ein vitro\u003c/em\u003e PGP activities, such as phosphate solubilization, and siderophore production, which are critical for improving nutrient availability to plants. Furthermore, \u003cem\u003eBacillus pumilus\u003c/em\u003e showed strong antagonistic activity against \u003cem\u003eFusarium equiseti\u003c/em\u003e, a notorious soil-borne pathogen, suggesting its role in biocontrol.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe study underscores \u003cem\u003eBacillus pumilus\u003c/em\u003e as a potent IAA-producing PGPR with multiple \u003cem\u003ein vitro\u003c/em\u003e plant growth-promoting traits and effective antagonism against \u003cem\u003eFusarium equiseti\u003c/em\u003e. The use of HPLC for accurate quantification of IAA and tryptophan provides valuable insights into the microbial mechanisms driving plant growth promotion. These findings emphasize the potential of \u003cem\u003eBacillus pumilus\u003c/em\u003e as a bioinoculant in sustainable agriculture, offering a dual benefit of enhancing crop productivity and providing natural protection against plant pathogens. This research highlights the role of microbial-mediated strategies in advancing environmentally sustainable farming practices.\u003c/p\u003e","manuscriptTitle":"Bacillus pumilus - A Potent IAA Producing Plant Growth Promoting Rhizobacteria with In Vitro PGP Traits and Antagonism Against Fusarium equiseti","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 16:51:03","doi":"10.21203/rs.3.rs-5477377/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-05T07:20:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-26T11:01:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-23T08:53:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335308509127592861433340114527936393928","date":"2025-04-11T09:42:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300631417740214567450016159705599487648","date":"2025-04-10T06:23:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-09T09:37:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-08T12:31:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Plants","date":"2025-03-24T03:07:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-plants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Plants](https://link.springer.com/journal/44372)","snPcode":"44372","submissionUrl":"https://submission.springernature.com/new-submission/44372/3","title":"Discover Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0fe9dbd5-2b44-484d-880a-928a7d36b2da","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-31T09:38:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-10 16:51:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5477377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5477377","identity":"rs-5477377","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}