Isolation and characterization of Plant growth-promoting hormones by an endophytic bacterium, Curtobacterium. citreum AVN2 isolated from the rhizome of Curcuma longa L

Isolation and characterization of Plant growth-promoting hormones by an endophytic bacterium, Curtobacterium. citreum AVN2 isolated from the rhizome of Curcuma longa L | 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 Isolation and characterization of Plant growth-promoting hormones by an endophytic bacterium, Curtobacterium. citreum AVN2 isolated from the rhizome of Curcuma longa L Sanneboyina NARMADA, Shaik. Mahekal KOUSAR This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7602699/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Graphical Abstract Abstract Endophytic bacteria have become essential in promoting plant growth and development, and provide resistance against phytopathogens through diverse biochemical mechanisms. This study isolated an endophytic bacterium, AVN2, from Curcuma longa's rhizome and identified it as () using 16S rRNA partial gene sequencing. C. citreum AVN2 was deposited in NCBI with accession number PQ215941. The strain exhibited significant antagonistic activity against . Sp. , a major causative agent of tomato wilt, in a dual culture assay, and is confirmed as a biocontrol agent by GC-MS analysis of its secondary metabolites, Lupeol, 2-Piperidinone, Stigmastan-3,5-diene, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 5-Pyrrolidino-2-pyrrolidone, Diethyl Phthalate, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-. Plant growth-promoting characterization revealed the production of important growth-promoting phytohormones, including indole compounds (6.40 µg/mL), gibberellic acids (29.49 µg/mL), and cytokinins (Kinetin 15.49 µg/mL, and 6-Benzyladenosine 27.78 µg/mL), which were quantified by HPLC analysis. Seed bacterization with . bioformulation enhanced the seed vigour index of tomato seeds compared to the control. Bio-formulated AVN2 significantly enhanced root and shoot growth, and overall biomass under greenhouse conditions, demonstrating biocontrol efficacy and plant growth-promotion (PGP) potential. This study presents the first evidence of as an endophyte in , exhibiting dual biofertilizer and bioprotectant properties. These findings underscore its promise as a sustainable, eco-friendly alternative to chemical fertilizers and fungicides in horticultural crop production. Curtobacterium citreum endophytic bacteria Fusarium. oxysporum plant growth-promoting traits germination percentage germination index liquid bioformulation HPLC analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Infectious Plant diseases caused by phytopathogenic microorganisms contribute to approximately 16% of global agricultural losses, primarily due to their aggressiveness in the soil and the influence on various crops during harvesting and post-harvest practices [ 1 ]. To address this challenge, much focus is needed to alleviate 70% of food security by developing biological biostimulants/ biofertilizers from microorganisms and having synergistic activities with the plant. This problem highlights the growing interest in biological bio-stimulants and biofertilizers sourced from microorganisms residing in the rhizosphere, phyllosphere, and as endophytes, due to their capacity to stimulate plant development and enhance systemic resistance in crops. Endophytes Endophytes are microorganisms that reside in intercellular or intracellular spaces of a host tissue without causing any adverse effects. Like PGPR, Plant growth-promoting endophytes [PGPE] show a range of direct and indirect strategies to boost plant health. PGPE facilitates the synthesis and regulation of Phytohormones, such as auxin, cytokinin, and gibberellin, as well as the uptake of nitrogen, phosphate, and iron from the soil through a direct mechanism [ 2 ]. In recent times, along with Plant Growth Promoting Rhizobacteria (PGPR), endophytic microorganisms have gained much focus due to their many advantages. Application of endophytic microorganisms includes nitrogen fixation, absorbing nutrients, producing phytohormones, withstanding biotic and abiotic challenges, and producing bioactive secondary metabolites, such as phenols, flavonoids, polyketones, peptides, steroids, alkaloids, terpenoids, and quinols. These substances are important in medicines, sustainable agriculture, and industrial biotechnology [ 2 , 3 ]. Curcuma longa Curcuma longa (turmeric) is a medicinal plant used for thousands of years to treat various diseases in the Indian traditional medicine system of Ayurveda. The medicinal properties and numerous applications of C. longa enhance its research potential in biomedicine. Similarly, endophytic microbes that inhabit these host plants significantly promote plant health by generating bioactive secondary metabolites of ecological significance, which confer protection against biotic and abiotic stressors via horizontal gene transfer [ 4 ]. Such endophytes, isolated from medicinal plants, enhance soil structure, augment water retention, improve fertility, serve as biocontrol agents and biofertilizers, detoxify toxic compounds, accelerate seed germination, confer tolerance to abiotic stressors, and promote plant health. As a result, they have attracted considerable attention to sustainable agriculture practices because of their combined functions. PGPE indirectly helps to withstand biotic stress and induce systemic resistance to the plant by producing volatile organic compounds, antibiotics, and antioxidant stress enzymes that neutralize reactive oxygen species [ROS] [ 5 ]. Endophytes have a great deal of promise as biocontrol agents and biofertilizers, and their complex interconnections make them strong friends in sustainable agriculture. This study aims to isolate endophytic bacteria from the rhizome of Curcuma longa , which has plant growth-promoting activities and biocontrol activity against phytopathogens and can be used as a biofertilizer and bio-stimulant. Materials and Methods Collection of plant material Healthy and mature C. longa plants were collected from organic farms of Erode (Turmeric city of India), (11.3410° N, 77.7172° E), Tamil Nadu, bought in a sterile zip-lock bag, and transferred to the lab facility at Acharya Nagarjuna University, Guntur, Andhra Pradesh, India, and processed within 24 hours to preserve the viability of endophytic bacteria. To avoid surface contamination, rhizomes were not washed before transport, and surface sterilisation was done in the lab under sterile conditions. Collection of Fusarium. oxysporum A lyophilized culture of F. oxysporum var. Lycopersici (MTCC10270) was brought from the Microbial Type Culture Collection (MTCC), Chandigarh. It was grown in Sabouraud’s dextrose media (SDA) and Potato dextrose agar (PDA) (Oxoid) plates according to the protocol given by MTCC Chandigarh and stored at 4℃ for later use. Isolation of endophytic bacteria Initially, Rhizomes of C. longa were washed thoroughly with sterile water to remove adhering rhizospheric soil, dust, debris, and other contaminants. Later, the surface sterilization of rhizomes was done with 20% H 2 O 2 for 2 minutes, 70% ethanol for 5 minutes, and repeatedly rinsed with 3% Sodium hypochlorite with 5 minutes of incubation for each rinse, and finally rinsed with sterile saline water (0.85% NaCl). Sterilized rhizome was aseptically cut into 1-inch pieces (weighing 1g) and homogenized with a mortar and pestle in 1% sterilized NaCl. Then the homogenized tissue was made up to 10 mL with 1% NaCl solution, and serially diluted to 10 − 1 to 10 − 10 [ 6 ]. Nutrient agar medium (10 g/L of peptone, 3g/L of beef extract, 5g/L of NaCl, and 20 g/L of agar) is supplemented with 2.0 g/L sucrose for endophytic bacteria isolation. The NAM medium was sterilised in an autoclave at 121℃ for 20 minutes and then cooled to 50℃. Endophytic bacteria were isolated using the spread plate method, and 100 µL of each dilution was taken and spread onto the nutrient agar medium. A 100 µL aliquot of the last rinse from surface disinfection was subjected to a sterility check on nutrient medium to confirm the surface disinfection and verify that the surface was free from biological contamination [ 7 ]. Plates were allowed to be incubated at 30℃ for 48 hours. The isolated bacterial colonies are further sub-cultured by the quadrant streaking method on fresh nutrient agar plates and incubated for 48 hours. A pure single colony in the fourth quadrant of the NAM plate was transferred to agar slants and stored in the refrigerator at 4℃ for further characterization [ 6 ]. Molecular identification and Phylogenetic analysis Genomic DNA from the bacterial isolate AVN2 was subjected to PCR amplification of the 16S rRNA gene sequence using universal primers: 785F 5' (GGA TTA GAT ACC CTG GTA) 3', 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3, 907R 5' (CCG TCA ATT CMT TTR AGT TT) 3', and 1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3' [ 8 ]. Amplification and sequencing were outsourced to Macrogen Inc. (Seoul, Republic of Korea). Phylogenetic analysis was conducted using the neighbour-joining method based on 100% sequence similarity to determine the taxonomic identity of the isolate. The 16S rRNA Partial gene sequence of strain AVN2 was deposited in the NCBI GenBank database (India). Screening of plant growth promotion (PGP) traits Indole-3-acetic acid (IAA) Endophytic bacterial isolate AVN2 was screened qualitatively and quantitatively for Indole-3-acetic acid (IAA) production, a key phytohormone associated with plant growth promotion and development. The qualitative assay was performed using the Salkowski reagent method [ 9 ]. AVN2 was cultured on nutrient agar medium, supplemented with 0.1% (w/v) L-tryptophan (HiMedia) and incubated for 72 hours. Upon addition of Salkowski reagent (1 mL of 0.5 M FeCl 3 in 50 mL of 35% HClO 4 ), development of a pink color confirmed the production of IAA. For quantitative estimation, AVN2 was cultured and grown in nutrient broth supplemented with L-tryptophan (0.1% w/v), and was incubated at 28℃ on a rotary shaker (180 rpm) for 72 hours. After incubation, the culture was harvested by centrifugation at 10,000 × g for 10 minutes, and 1 mL of the supernatant was collected and mixed with two drops of orthophosphoric acid and 2 mL of Salkowski reagent, and then incubated at 28 ± 2℃ for 20 minutes in the dark. Absorbance was measured at 530 nm using a UV-Vis spectrophotometer. This experiment was replicated thrice, and the data presented are the average of the three similar experiments. The absorbance of the samples obtained was plotted against a standard to determine the concentration of IAA produced, and the results are expressed as µg of compound (IAA) per mL of filtrate. Inorganic phosphate solubilization The ability of the bacterial isolate AVN2 to solubilize Inorganic phosphate was assessed, qualitatively and quantitatively, according to [ 10 ]. Using the streak plate method, Pikovskaya’s agar medium was used to determine inorganic phosphate solubilization. Inorganic phosphate solubilization was estimated after 5 days of incubation at room temperature. The appearance of a clear zone around the bacterial colony confirmed the occurrence of Inorganic phosphate solubilization activity. For quantitative estimation, 100 µL of bacterial culture AVN2 was inoculated into 50 mL of nutrient broth supplemented with tricalcium phosphate(1µg/mL) and incubated in an incubator shaker at 180 rpm at 30 ± 0.1℃ for 5 days. An uninoculated broth with tricalcium phosphate served as a control. After incubation, the culture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was estimated using the Vanado-molybdate yellow color method. 2.5 mL of Barton’s reagent was added to 0.5 mL of the supernatant, and the volume was brought up to 50 mL with deionized water. The absorbance of the resultant color complex produced was measured after 10 minutes at 430 nm in a UV/Visible spectrophotometer. The total phosphate solubilized by the bacterial endophyte was calculated from the regression equation using the standard curve, and the values of soluble phosphate were expressed as ppm ml − 1 . Ammonia production Ammonia production by endophytic bacteria AVN2 was determined using peptone water, following the method [ 11 ]. For qualitative analysis, AVN2 was inoculated onto a peptone agar plate and incubated at 36 ± 2°C for 48 hours. The development of a yellow to brown color with 0.5 mL of Nessler's reagent was a positive test for ammonia production. For quantitative estimation, a freshly grown bacterial culture was inoculated in 10 mL of peptone water and incubated at 36 ± 2°C for 72 hours. After incubation, the culture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. To 1 mL supernatant, 0.5 mL Nessler's reagent was added, and the absorbance of the resultant yellow-orange complex was determined by comparison to the standard curve of ammonium sulphate. Antifungal activity In vitro screening of antifungal activity was assayed using the dual culture method [ 12 ]. F. oxysporum Lycopersici was cultured for 7 days on Sabouraud's dextrose agar media (40g/L of dextrose, 10g/L of peptone, 15 g/L of agar). A 5-mm mycelial plug of freshly grown fungal culture was inoculated at the centre of a newly prepared nutrient agar plate for the assay. AVN2 was streaked 3 cm away from the fungal disc toward the periphery of the plate. Plates were incubated at 35°C for 4 to 7 days. Control plates were maintained with fungal inoculation alone, without the bacterial isolate. The formula expressed the antifungal activity as the percentage of radial growth inhibition of the pathogen in the presence of AVN2 compared to the control. a: Distance between the fungus and the endophyte isolated in the centre of the Petri dish. b: The distance between the fungus and the space in the centre of the Petri dish is endophytic-free. This experiment was replicated thrice, and results were expressed as the mean percentage of inhibition of phytopathogen by the bacterial endophyte AVN2. Growth Optimization studies The growth behavior of the bacterial isolate AVN2 was assessed under varying environmental and nutritional conditions by monitoring optical density (O.D.) at 600 nm, following the method [ 13 ]. Growth optimization was carried out across different parameters, including temperature, pH, carbon, and nitrogen sources. Temperature optimisation was performed by incubating cultures at various temperatures (25°C, 30°C, 35°C, 40°C, and 45°C). Similarly, pH optimization was assessed by adjusting the medium to pH values (5.0, 6.0, 7.0, 8.0, and 9.0). For carbon source optimization, a minimal broth medium was supplemented with 1% (w/v) of various carbon sources (starch, sucrose, fructose, mannitol, dextrose, lactose, glucose, glycerol, and maltose). For nitrogen source optimisation, 0.5% (w/v) of different nitrogen sources (peptone, beef extract, yeast extract, urea, ammonium sulfate, ammonium chloride, sodium nitrate, and potassium nitrate) was added to minimal broth. In each case, 100 µL of freshly grown bacterial suspension was inoculated into the respective media and incubated for 48 h. Post-incubation, bacterial growth was quantified by measuring O.D. at 600 nm using a UV-Visible spectrophotometer. All experiments were replicated thrice, and the absorbance values were recorded for comparative analysis. Preparation of Liquid Bioformulation Following the optimization of physicochemical parameters—temperature, pH, carbon, and nitrogen sources—using a one-factor-at-a-time (OFAT) approach, a liquid bioformulation of the bacterial isolate AVN2 was prepared. The formulation medium was developed using the optimal conditions identified from growth studies: the most suitable carbon and nitrogen sources and the ideal pH and incubation temperature. Freshly grown culture medium was inoculated into the optimised medium and incubated under a rotary shaker (180 rpm) for 48 h. The resulting liquid culture, containing a high density of viable bacterial cells, was used as the liquid bioformulation for further applications. Extraction of the bioactive metabolites Bioactive secondary metabolites were extracted from the optimised culture broth of the active bacterial strain AVN2 using the ethyl acetate solvent extraction method [ 14 ]. The strain was inoculated into the optimized broth formulated with the previously optimized physicochemical conditions under a rotary shaker (180 rpm) for 48 hours of incubation. After incubation, the culture broth was harvested by centrifuging at 10,000 rpm for 15 minutes. The supernatant was then collected and vigorously shaken with an equal volume of ethyl acetate (1:1, v/v) in a separating funnel and allowed until phase separation occurred. The upper organic layer was collected and subjected to two additional extractions using fresh ethyl acetate. The pooled organic extracts were concentrated to dryness under reduced pressure using a rotary evaporator at 40°C. The crude extract containing bioactive compounds was stored at 4°C and utilized for subsequent bioactivity assays. Quantification of Plant Growth-Promoting Hormones by HPLC The production of important growth-promoting phytohormones like indole-3-acetic acid (IAA), Gibberellins, and cytokinins from the endophytic bacteria AVN2 was assessed using High-performance liquid chromatography (HPLC). Culture supernatants were extracted with ethyl acetate, and the concentrated extracts were subjected to chromatographic analysis to quantify hormones. IAA Ethyl acetate extract of the isolate was concentrated to 10 mL using a Rota evaporator and adjusted to a pH of 2.8. HPLC analysis was conducted by injecting 20 µL aliquots into an Inertsil ODS C-18 (250 mm × 4.6mm; 5 µm id) column connected to an HPLC pump. A ratio of 1:30:70 of acetic acid: methanol: water (v/v) was used as the mobile phase and filtered using 0.2 µm membrane filters. The flow rate was adjusted to 1.0 mL/ min, and the operating pressure was 1200.5 ± 25 psi. Absorbance was measured at 280 nm. The retention times for compounds were compared to those of the standard values of IAA [ 15 ]. Gibberellins Gibberellins (GA) analysis was done using the modified procedure established by [ 16 ]. The ethyl acetate extract of the isolate was concentrated to up to 10 mL using a rotary evaporator and was adjusted to pH 2.5. Concentrated ethyl acetate extract of the culture supernatant was solubilized in 60% methanol (MeOH), with the pH adjusted to 8.0 ± 0.3 using 2 N NH 4 OH. A methanolic suspension of concentrated ethyl acetate extract of isolates was subjected to HPLC by injecting 20 µL aliquots into an Inertsil ODS C-18 (250 mm×4.6mm; 5µ id) column connected to an HPLC pump. Isopropanol, 25% Ammonium Hydroxide, and water in a (10:1:1 v/v) ratio, filtered through 0.2 mm membrane filters, were constructed as the mobile phase. The flow rate was adjusted to 0.5 mL/min with the operating pressure 1240.5 ± 25 psi. Absorbance was monitored at 206 nm. Retention times for compounds were compared to those of standard values for GA. Cytokinins The presence of Cytokinin compounds was assayed according to [ 17 ]. Ethyl acetate extract of the isolate was concentrated up to 10 mL using a rotary evaporator and adjusted to a pH of 2.8. HPLC was performed using an Inertsil ODS C-18 (250 mm×4.6mm; 5µ id) column connected to an HPLC pump. The mobile phase consists of n-butanol, acetic acid, and water in the ratio of (12:3:5 v/v/v) with an injection volume of 20 µL, a flow rate of 0.5 mL/min, a column temperature of 35°C, a walking time of 20 minutes, and a detection wavelength of 254 nm was constructed as the mobile phase. Retention times for compounds were compared to those of standard values for cytokinin. Greenhouse studies Preparation of bacterial cell suspension The selected bacterium AVN2 was inoculated into 200 mL of optimized broth and incubated at 35 ℃ for 48 hours. The bacterial cell suspension was prepared by harvesting cells by centrifugation at 6000 rpm for 15 minutes. The inoculum was re-suspended in sterile distilled water, and the concentration was adjusted to 10 − 8 CFU/ml [ 18 ]. Seed treatment and nursery experiments To determine the effect of AVN2 on the growth of tomato seeds ( Lycopersicon esculentum L .), Royal variety tomato seeds were purchased from a certified nursery in Guntur, Andhra Pradesh, India. Tomato seeds were surface sterilized with 5% sodium hypochlorite for 20 min, and 70% ethanol for 1 minute, and then thoroughly washed five times with double-sterilized distilled water [ 19 ]. Then, tomato seeds were treated with a 48-hour-old (approximately 10 8 CFU/ml concentration) culture of the isolate for 30 minutes and shade-dried at 28℃ for 1 hour, and subsequently sown in 98-well nursery trays (5 seeds/well) using sterilized coco peat. The control seeds were treated with only sterile distilled water under identical conditions. Seedling vigour index or Germination percentage (Gp), Speed of seed germination (S), Germination rate (G R ), and Germination index (G I ) are calculated in the first week. Germination percentage (Gp) = Ni/NX100 Speed of seed germination (S) = ni/di Germination rate (G R ) = no. of germinated seeds/ day of first count +…… + no. of germinated seeds/day of last count Germination index (GI) = Σ G/T Where: N = Total no. of seeds, Ni = germinated seeds, ni = germinated seeds per day, di = counting day, G = percentage of seed germination per day, T = germination period. Pot experiment The bio-efficacy of AVN2-based liquid bioformulation was evaluated under a greenhouse pot experiment (average temperature of 25℃) at Acharya Nagarjuna University, Guntur, Andhra Pradesh, India, in a completely randomized design by replicating each treatment about seven times. Soil Preparation and Transplantation The experimental soil used for the greenhouse trials is sterilized in an autoclave (121°C, 15 psi, 1 h) to eliminate native microbial flora. Two-week-old tomato seedlings were carefully transplanted from the nursery bed to the pots (30 cm diameter) until the final harvest and watered daily. Treatment Application The AVN2 liquid bioformulation (5 mL) was applied weekly to the seedlings through collar application until the final harvest. Control plants received the same volume of the untreated (placebo) formulation prepared identically but without microbial inoculation. Growth and Biochemical Parameters Plants were harvested from each replicate at 2-week intervals to evaluate growth and physiological parameters: root length (cm), Shoot length (cm), Fresh biomass (g), and Primary metabolite content, including Total proteins and total carbohydrates. The primary metabolite content was determined following the protocol of [ 20 ]. Statistical analysis All experimental data were subjected to data analysis using IBM SPSS PASW Statistics 18 software. The significance of the differences between the control and Treated groups was analyzed using one-way ANOVA. Means, standard errors, and percentage increases were calculated from at least three replicates. Statistical significance was determined at p < 0.05. Results In this study, eight morphologically distinct endophytic bacteria were isolated from the rhizome of C. longa . The isolate designated AVN2 was selected for further characterization based on its distinct colony morphology and bioactivity potential (Fig. 1). Identification of AVN2 The 16S rRNA gene sequence analysis of the isolate AVN2 revealed 99.65% similarity with the partial sequence of Curtobacterium oceanosedimentum (GenBank ID: NR-104839.1). Phylogenetic analysis further confirmed that AVN2 clusters closely with C. oceanosedimentum , supporting its taxonomic assignment (Fig. 2). Accordingly, the isolate was identified as Curtobacterium citreum AVN2, and the sequence was deposited in the NCBI GenBank with the accession number PQ215941. Plant growth-promoting (PGP) traits: C. citreum AVN2 exhibited multiple plant growth-promoting attributes. Upon addition of Salkowski reagent, AVN2 showed a positive response to Indole acetic acid production by producing a pink coloured complex. Halo zone formation shows the positive response of AVN2 towards inorganic phosphate solubilization, and the appearance of brick red color confirms the ability of AVN2 in ammonia production. Quantitative assessment revealed that C. citreum AVN2 produced 7.2 µg/mL of IAA, 100 ppm of Inorganic phosphate solubilization, and 0.7 µg/mL of Ammonia production. These results suggest the strain's potential as a bio-inoculant with direct PGP activities. Antifungal activity The antagonistic potential of C. citreum AVN2 was evaluated against Fusarium oxysporum f. sp. L ycopersici using the in vitro technique (Dual culture assay). AVN2 exhibited significant antifungal activity, inhibiting the overall growth of the phytopathogen by 91%. The pronounced inhibition zone indicates the strong biocontrol potential of this endophyte. Growth optimization Optimal growth of C. citreum AVN2 was achieved under the following culture conditions: temperature 35°C, pH 7, with 1% mannitol as the carbon source and 0.5% beef extract as the nitrogen source (Fig. 3). These optimised parameters not only enhanced bacterial proliferation but also significantly increased PGP metabolite production. Compared to the culture media before and after optimisation, the optimised media showed the enhanced production of IAA, Inorganic phosphate solubilisation, and ammonia production by 300%, 140% and 200%, respectively (Table 1). Table 1 Enhancement of PGP traits of C. citreum AVN2 before and after optimisation IAA production (µg/mL) Inorganic Phosphate solubilization (ppm) Ammonia production (µg/mL) Before optimization After optimization Before optimization After optimization Before optimization After optimization Control 0.3 ± 0.26 0.53 ± 0.35 2 ± 0.35 4.4 ± 0.45 0.3 ± 0.32 0.5 ± 0.35 C. citreum AVN2 7.2 ± 0.25 28.8 ± 2.4* (300%) 100 ± 0.35 240 ± 3.0* (140%) 0.7 ± 0.4 2.1 ± 0.35* (200%) Values are the mean of three replicates. ± SE, IAA production, Inorganic phosphate solubilization, and Ammonia production have been increased by 300%, 140%, and 200% after optimisation Liquid bioformulation Based on the growth optimization studies, a liquid bioformulation was developed using the optimised medium and designated AVN2 Liquid Formulation. This formulation was designed as a flowable or aqueous suspension, offering a viable alternative to conventional carrier-based inoculants. Liquid bioformulations are advantageous due to their longer shelf life, higher microbial load, and easier application in field and greenhouse settings. Quantification of Secondary Metabolites by GC-MS In GC MS chromatogram, Metabolic Profile of Bacterial extract of Curtobacterium citreum AVN2 as per the NIST Database, bioactive metabolites such as Phenol,2,4’-isopropylidenedi, Lupeol, N-[2-Hydroxyethyl]succinimide, 2-Piperidinone, Stigmastan-3,5-diene, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 5-Pyrrolidino-2-pyrrolidone, Diethyl Phthalate, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-, Aziridine,2-(1,1-dimethylethy)-3-methyl-1-(2- propenyl)-, trans-, respectively, were observed (Fig. 4; Table 2) Table 2 Metabolic Profile of Curtobacterium citreum AVN2 S. No RT Height % Name of the compound Molecular formula Mol wt Area % occupied Biological activity Reference 1 22.935 51.96 Phenol,2,4’-isopropylidenedi C15H16O2 228 61.72 No activity found 2 28.064 12.81 Lupeol C30H50O 426 21.21 Antimicrobial activity [ 21 ] 3 16.852 7.01 N-[2-Hydroxyethyl] succinimide C6H9NO3 143 2.96 No activity found 4 8.723 6.09 2-Piperidinone C5H9NO 99 3.66 Antimicrobial activity [ 22 ] 26.308 6.60 Stigmastan-3,5-diene C29H48 396 4.35 Antifungal activity [ 23 – 25 ] 5 19.850 2.82 Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- C11H18N2O2 210 0.99 Antifungal activity [ 26 – 29 ] 6 18.770 2.75 5-Pyrrolidino-2-pyrrolidone C8H14N2O 154 1.13 Indole compound (Plant growth compound) [ 30 ] 7 15.225 2.67 Diethyl Phthalate C12H14O4 222 0.76 Antimicrobial activity [ 31 ] 8 17.680 2.30 Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro- C7H10N2O2 154 1.00 Antifungal activity [ 26 – 28 ] 9 17.01 2.25 Aziridine,2-(1,1-dimethylethy)-3-methyl-1-(2–propenyl)-, trans- C10H19N 153 0.85 No activity found HPLC analysis of Plant growth-promoting hormones HPLC analysis was performed on metabolites extracted from AVN2 culture broth using the solvent extraction method to confirm the production of plant growth-promoting hormones. The study validated the presence of indole-3-acetic acid (IAA) and other Phytoactive compounds, supporting the strain’s role in direct plant growth stimulation. Quantification of Plant Growth Hormones by HPLC: IAA HPLC fingerprinting of C. citreum AVN2 revealed a distinct peak at retention time (RT) 0.70 min, with a peak area of 1488%, confirming the presence of an indole compound. The quantified concentration of IAA was 6.40 µg/mL (Fig. 5a) Gibberellins (GA) The HPLC chromatogram of the AVN2 extract showed a prominent peak at RT 5.9 min, with a peak area of 55353%, corresponding to the presence of gibberellic acid (GA). The concentration was quantified as 29.49 µg/mL (Fig. 5b). Cytokinins The HPLC profile of C. citreum AVN2 showed two distinct peaks: one at RT 5.3 min with a peak area of 3579%, confirming the presence of Kinetin at 15.49 µg/mL, and another at RT 6.2 min with a peak area of 6419%, corresponding to 6-Benzyladenosine, quantified at 27.78 µg/mL (Fig. 5c). Greenhouse studies Seedling vigour index The seedling vigor index exhibited by 48-h bacterized tomato seeds treated with the endophytic bacterium C. citreum AVN2 demonstrated a significant improvement (p ≤ 0.05) by showing 81.6% of Germination percentage (Gp) and 54.5% of Germination index (Gi), respectively when compared to the control (Table 3; Fig. 6). Specifically, C. citreum AVN2 treatment resulted in an 81.6% increase in Gp and a 54.5% increase in GI compared to the untreated control (Table 3; Fig. 6). Table 3 Seedling vigour index of tomato seeds treated with C. citreum AVN2 Growth parameters Control C. citreum AVN2 Germination percentage (G p ) GP Ni/NX100 31.6% 81.6% Speed of seed germination (S) S = ni/di Day1 = 10 Day2 = 50 Day3 = 2.8 Day 4 = 2 Day1 = 10.2 Day2 = 18.75 Day3 = 15 Germination rate (G R ) (G R ) = seed/1st day=……seed/n th day 103.6 192 Germination index (GI) GI/G/T 30.8 54.48 The values are the mean of three replicates. ± SE, Germination percentage = No. The number of seeds germinated per time duration. Vigour index calculated during the first week after germination. The Value in parentheses is the percentage increase of growth parameters compared to the control . Plant growth promotion The C. citreum AVN2 liquid bioformulation application significantly enhanced tomato seedlings' growth parameters under greenhouse conditions. A progressive increase in plant growth was observed from the 4th to the 12th week of treatment (Table 4). Biomass increased by 233% in treated plants compared to the control (p < 0.001). Table 4 Impact of inoculation of C. citreum AVN2 bioformulation on plant growth of tomato seedlings. Biomass (gm) 4th week 6th week 8th week 10th week 12th week Control 0.07 0.07 0.12 0.16 0.18 C. citreum AVN2 0.08 ± 0.03 (14.29%) 0.12 ± 0.04 (71.43%) 0.28 ± 0.03 (133.33%) 0.4 ± 0.03 (150.00%) 0.6 ± 0.03 (233.33%) Plant height (cm) Control 10 10.6 11.4 12.1 12.4 C. citreum AVN2 11.2 ± 0.3 (12.00%) 12.76 ± 0.04 (20.38%) 16 ± 0.4 (40.35%) 17.4 ± 0.4 (43.80%) 19.5 ± 0.4 (57.26%) Shoot height (cm) Control 9 9.2 9.8 10.2 10.4 C. citreum AVN2 10.1 ± 0.4 (12.22%) 11 ± 0.35 (19.57%) 14 ± 0.25 (42.86%) 15 ± 0.30 (47.06%) 16.2 ± 0.3 (55.77%) Root length (cm) Control 1 1.4 1.6 1.9 2 C. citreum AVN2 1.1 ± 0.4 (10.00%) 1.73 ± 0.25 (23.57%) 2 ± 0.35 (25.00%) 2.4 ± 0.4 (26.32%) 3.3 ± 0.25 (65.00%) Protein content (µg/mL) Control 2.3 0.25 3.5 6.7 8.3 C. citreum AVN2 2.6 ± 0.5 (13.04%) 3 ± 0.2 (30.43%) 4.6 ± 0.3 (31.43%) 9.4 ± 0.15 (40.3%) 11.8 ± 0.4 (42.17%) Carbohydrate content (µg/mL) Control 2.5 2.5 3.1 4 4.8 C. citreum AVN2 2.7 ± 0.3 (8.0%) 3.3 ± 0.4 (32.0%) 4.4 ± 0.4 (41.9%) 5.8 ± 0.3 (45.0%) 7.9 ± 0.4 (64.58%) As detailed in Table 4, C. citreum AVN2 treatment resulted in a 55.7% increase in shoot height and a 65% increase in root length relative to untreated controls, demonstrating a substantial effect on morphological growth traits. Furthermore, there was a significant enhancement in primary metabolite content, with protein content increasing by 42.17% and carbohydrate content by 64.58% (p < 0.001) in treated plants. These findings underscore the plant growth-promoting potential of C. citreum AVN2, highlighting its role in improving seed germination, vegetative growth, biomass accumulation, and metabolic productivity in tomato plants under pot experiments. Discussion Endophytic microorganisms, including bacteria and fungi, inhabit various niches within agricultural plants, such as seeds, the phyllosphere, endosphere, and rhizosphere, and have emerged as significant sources of bioactive compounds with medicinal and agricultural relevance [ 32 , 33 ]. These endophytes often colonize the intercellular spaces of host tissues and help in promoting plant growth by enhancing the energy uptake by the production of primary metabolites, especially extracellular enzymes, and improve the host plant’s resilience through the production of secondary metabolites with anti-phytopathogenic properties and plant-beneficial traits. C. longa (turmeric), a medicinal plant, has been traditionally used in biomedicine for thousands of years. The medicinal properties and numerous applications of C. longa enhance its research potential, leading to promising, potent discoveries at the molecular and genetic levels. Similarly, endophytes that inhabit these host plants play a crucial role in promoting plant health by generating bioactive secondary compounds of ecological significance, which confer protection against biotic and abiotic stressors through horizontal gene transfer. In this present study, AVN2 isolated from the rhizome of C. longa is identified as Curtobacterium. citreum (99.65%) by 16s rRNA partial gene sequencing and deposited in the NCBI database with accession number PQ215941 (Fig. 2), validating the isolate's taxonomic classification. C. citreum as an endophyte from C. longa rhizomes has not yet been reported . However, C. citreum has been identified as an endophyte from various plant sources in the past, such as traditional rice cultivars [ 34 ], tea leaves ( Camellia sinensis var. assamica ), and prairie grasses [ 35 , 36 ]. C. citreum AVN2 has multiple plant growth-promoting traits (IAA, ammonia production, and inorganic phosphate solubilization), and antagonistic activity against a phytopathogen, F. oxysporum Lycopersici . And serving as an effective bioformulation for plant growth promotion in tomato seedlings by exhibiting notable improvements in primary metabolite content, biomass accumulation, seedling vigour, and overall plant development, considering that the diversity and potentiality in PGP activity allow the endophytic C. citreum to adapt and establish beneficial interactions with plants rather than the subject of origin [ 37 , 38 ]. The first emerging field of study in sustainable crop protection is the potential of Curtobacterium species as biocontrol agents. Under in vitro circumstances, C. citreum AVN2 demonstrated potent antagonistic activity against F. oxysporum in our investigation, which is consistent with previous studies that reported Curtobacterium strains with comparable antimicrobial properties [ 39 ]. Similarly, A. oryzae AVNF4 isolated from the rhizome of C. longa exhibited antifungal activity against F. oxysporum in the dual culture method [ 20 ]. GC-MS analysis of ethyl acetate extracts of culture supernatant revealed the presence of potential compounds, a pyrrole compound, Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro- produced by Curtobacterium citreum AVN2, is known for its various therapeutic applications, such as antifungal, antibiotic, anti-tumor, and anti-inflammatory activities [ 40 ]. Stigmastan-3,5-diene produced in tissue extracts of Portulacaria afra , and roots of Stellera chamaejasme have antioxidant activity, antimicrobial activity, and antifungal activity [ 23 , 24 , 25 ]. Volatile compounds Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- and Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-, produced by B. subtilis, A. oryzae AVNF4, and Streptomyces Species have shown biocontrol activity against Alternaria Solani , Pyricularia oryzae, F. oxysporum , and Sclerotium bataticola [ 20 , 26 , 27 , 28 , 29 ]. Diethyl phthalate produced by Streptomyces strain has shown antibacterial activity against E. coli [ 31 ]. 2-piperidinone, extracted from dried pomegranate peels, has demonstrated antimicrobial activity against Pseudomonas aeruginosa, Proteus mirabilis , and Candida albicans [ 22 ]. 5-Pyrrolidino-2-pyrrolidone, an indole compound produced by the Bacillus strain, validates in plant growth-promotion in Gossypium hirsutum (cotton) [ 30 ]. These findings validate the production of these secondary metabolites and could serve as both a natural biocontrol and a bio-stimulant agent. Growth optimization studies are of paramount importance in understanding the factors that affect bacterial growth and their metabolism, thereby enhancing the production of plant growth-promoting traits. To improve the production of plant growth-promoting (PGP) features, growth optimization studies are essential for comprehending the environmental and nutritional factors that affect bacterial metabolism and proliferation. The present investigation's optimisation tests demonstrated that the optimal physicochemical conditions for the growth of C. citreum AVN2 were 35°C, pH 7, 1% mannitol as the carbon source, and 0.5% beef extract as the nitrogen source. These conditions also markedly improved the production of important PGP characteristics (Fig. 3; Table 1). The carbon supply had the greatest effect on developing PGP characteristics out of all the variables examined, highlighting its crucial significance above pH, temperature, and nitrogen availability. These conclusions are corroborated by earlier research showing that basic nutritional elements, including temperature, pH, and carbon sources, are important for bacterial metabolism and growth-promoting capacity [ 41 , 42 ]. Significantly affecting the effectiveness of PGP are bacterial strains such as Rhizobium VMA 301, Bacillus, and Streptomyces spp. that have shown a high sensitivity to environmental changes, particularly those related to pH and temperature [ 43 ]. C. citreum AVN2 demonstrated the ability to produce indole-3-acetic acid (IAA), a crucial auxin controlling plant growth and development. A quantitative study using L-tryptophan as a precursor showed that AVN2 generated 7.2 µg/mL of IAA under unoptimized conditions and 28.8 µg/mL following optimization. Furthermore, IAA's presence was verified by HPLC quantification at 6.4 µg/mL and 0.70 m retention time (Fig. 5a). It's interesting to note that C. citreum AVN2 produced IAA even when exogenous L-tryptophan was absent. This suggests that either endogenous tryptophan biosynthesis or a tryptophan-independent IAA synthesis pathway may be involved [ 44 ]. The strain's strong biosynthetic potential and ability to sustain plant development in nutrient-limited environments are highlighted by this metabolic flexibility. IAA is pivotal in regulating plant development, including cell division, elongation, and tissue differentiation [ 45 ]. The root system enhances vascular tissue development, stimulates lateral and adventitious root formation, and improves nutrient and water uptake by reshaping root architecture [ 46 ]. Notably, the influence of IAA is often organ-specific and varies with the plant’s developmental stage [ 47 ], making it a critical regulator in plant–microbe interactions. Although endophytes are not typically recognised for gibberellin (GA) synthesis, several rhizobacteria—including Rhizobium phaseoli , Bacillus pumilus , B. licheniformis , B. cereus , and B. macrolides —have been reported to produce gibberellins and related compounds such as GA₉, GA₂₀, GA₅, GA₈, GA₃₄, GA₄₄, and GA₅₃. In addition, multiple fungal endophytes, including Chrysosporium pseudomerdarium , Fusarium sacchari , F. subglutinans , F. konzum , Gibberella fujikuroi , Phoma herbarum , and Scolecobasidium tshawytschae , have also been identified as GA producers [ 48 – 50 ]. In line with these findings, our study presents the first report of gibberellin production by the endophytic strain C. citreum AVN2, isolated from the rhizome of C. longa . The strain produced a notable concentration of 29.49 µg/mL of gibberellin compound, as determined by HPLC analysis (Fig. 5b), expanding the current understanding of GA-producing endophytes and their potential applications in sustainable agriculture. Curtobacterium citreum AVN2 produced 15.49 µg/mL of kinetin and 27.78 µg/mL of 6-benzyladenosine. Azospirillum species have been reported to synthesise approximately 0.75 µg/mL of cytokinins and 20–40 µg/mL of gibberellic acid [ 51 ]. Similarly, Bacillus sp. strain BPSAC6, isolated from Clerodendrum colebrookianum , was found to produce indole-3-acetic acid (IAA) at 3.61 µg/mL , kinetin at 6.2 µg/mL , and 6-benzyladenine (BA) at 7.9 µg/mL [ 52 ]. Stenotrophomonas maltophilia BE 25, isolated from the roots of Musa spp., demonstrated IAA production at 39.72 µg/mL [ 53 ]. In contrast, Bacillus sp., isolated from the rhizosphere of Cattleya walkeriana , produced 2.3 µg/mL of IAA [ 54 ]. Comparatively, the endophytic bacterium C. citreum AVN2 has been reported to detect and quantify IAA, GA, kinetin, and BA for the first time, highlighting the strain's distinctive characteristics and varied PGP potential. Using these endophytes to create bioformulations or biofertilizers can help control plant health and promote sustainable agriculture by providing environmentally benign substitutes for chemical inputs. Liquid bioformulations, sometimes called aqueous or flowable suspensions, have become powerful substitutes for conventional carrier-based microbial inoculants in recent years. These alternatives provide benefits like increased microbial load, extended shelf life, and simplicity of use. A liquid formulation of C. citreum AVN2 was created and administered to tomato ( Solanum lycopersicum ) seeds to evaluate its bioinoculant potential, with the seedling vigour index, germination percentage (Gp), and germination index (Gi) rising by 81.6% and 54.5% , respectively, in comparison to the untreated control, the treatment resulted in a notable improvement in seedling performance (Table 3; Fig. 6). These results demonstrate how C. citreum AVN2 -based liquid formulations have the potential to improve early seedling development and create strong crop stands. According to previous research [ 55 ], lactic acid bacteria from rhizospheric soil improved tomato seed germination by 6% and raised the seedling vigour index by 259. This highlights the relative efficacy of C. citreum AVN2 as a strong bioinoculant. This highlights the relative efficacy of C. citreum AVN2 as a strong bioinoculant . Several plant growth parameters, such as germination percentage, seedling vigor, germination index, plant height, shoot height, root length, biomass accumulation, and primary metabolite content, were significantly enhanced in tomato seedlings using C. citreum AVN2-based liquid bioformulation inoculation in the current study (Table 4). According to earlier publications, creating various plant growth-promoting (PGP) compounds by AVN2 is responsible for the observed boost in plant growth [ 35 ]. Moreover, releasing a wide range of antagonistic substances, including enzymes and antibiotics, probably suppresses phytopathogens [ 56 ]. According to [ 56 ], members of the Curtobacterium genus are known to produce a variety of PGP characteristics, including phytohormones, which greatly enhance the vigor and growth of inoculated agricultural plants. The present investigation has effectively isolated and identified C. citreum AVN2, indicating that it can function as both a biocontrol agent and an endophyte that promotes plant growth. C. citreum AVN2 showed strong antagonistic action against Fusarium oxysporum f. sp. Lycopersici , a serious pathogenic fungus that affects tomato crops. Greenhouse experiments also demonstrated its practical utility by confirming its effectiveness in boosting seed germination, seedling vigour, and inhibiting fungal infection. This early data shows the significance of AVN2 in combined crop improvement and disease management techniques. The results of this study are of great importance to the study of the mechanism of action and antagonism, which will lay the foundation for future applications in the biological control of plant growth and diseases. Conclusion C. citreum AVN2 exhibits a wide range of plant growth-promoting (PGP) characteristics and strong antifungal activity against wilt-causing phytopathogens in vitro. This study is the first to report on the isolation of endophytic bacteria C. citreum from the rhizome of C. longa . Significant promise was shown in improving tomato seed germination, seedling vigor, and overall plant growth using the liquid bioformulation made with C. citreum AVN2. These findings represent a promising step towards facilitating the development of non-toxic and ecologically friendly biofertilizers. When used as a liquid bioinoculant, C. citreum AVN2 could be an excellent substitute for chemical inputs, supporting environmentally friendly and sustainable farming methods, especially when growing vegetable crops. Such bioformulations' economic and agronomic significance should be further enhanced by future studies concentrating on their mechanistic comprehension and field validation. Declarations Funding This study was supported by a University Research Fellowship via reference number ANU/UCS/URF/Research Fellowship-2021–2022 and 2022-23 Ethics statement This article does not contain any studies on Human participants or animals performed by any authors. Conflict of Interest: The authors declare no conflict of interest. Author Contribution Sanneboyina NARMADA: Conceptualization, Methodology, Investigation, Supervision, Writing – Original draft, Correspondence.Shaik. 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Revista Brasileira de Ciência do Solo 35(3):729–737 Konappa NM, Maria M, Uzma F, Krishnamurthy S, Nayaka SC, Niranjana SR, Chowdapp S (2016) Lactic acid bacteria mediated induction of defence enzymes to enhance the resistance in tomato against Ralstonia solanacearum causing bacterial wilt. Scientia Hortic 207:183–192. 10.1016/j.scienta.2016.05.029 Christina A, Christapher V, Bhore SJ (2013) Endophytic bacteria as a source of novel antibiotics: An overview. Pharmacogn Rev 7(13):11–16. https://doi.org/10.4103/0973-7847.112833 Additional Declarations No competing interests reported. Supplementary Files Supplementarydocument.docx 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. 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1","display":"","copyAsset":false,"role":"figure","size":74953,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation of endophytic bacteria from \u003cem\u003eC. longa\u003c/em\u003e rhizome. a: Control plate, b: Endophytic bacteria designated as AVN2\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/ec0f020a0b9624f43fdd5fdc.jpg"},{"id":92416243,"identity":"20bf2749-68f0-4805-b3ea-05682624c985","added_by":"auto","created_at":"2025-09-29 13:25:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67343,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of AVN2 (\u003cem\u003eCurtobacterium citreum\u003c/em\u003e AVN2 PQ215941)\u003c/p\u003e\n\u003cp\u003eNeighbor-joining tree based on partial 16s rRNA gene sequence showing relationship between \u003cem\u003eCurtobacterium \u003c/em\u003eisolate \u003cem\u003eCurtobacterium citreum \u003c/em\u003estrain AVN2 and related members of the genus \u003cem\u003eCurtobacterium\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/e6b92b905994a50118e56777.jpg"},{"id":92414840,"identity":"19022fa1-c0be-4803-b2e7-560644996b5b","added_by":"auto","created_at":"2025-09-29 13:17:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1420342,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth optimization of \u003cem\u003eC. citreum\u003c/em\u003e AVN2. a. Effect of Temperature, b: Effect of pH, c: Effect of 1% carbon sources, d: Effect of 0.5% nitrogen sources\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/fc5827ce7ec0687132f7c216.jpg"},{"id":92414844,"identity":"05f6a4ad-b2ca-48dc-989f-9b212d015690","added_by":"auto","created_at":"2025-09-29 13:17:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":322368,"visible":true,"origin":"","legend":"\u003cp\u003eGC-MS analysis of \u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/affc3e04229563ac8958aebf.jpg"},{"id":92414846,"identity":"c09173a1-27e6-4dd2-972b-78980c7c68f5","added_by":"auto","created_at":"2025-09-29 13:17:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136470,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of Plant growth-promoting Hormones by HPLC analysis, a: Indole compounds, b: Gibberellic acids, c: Cytokinins (kinetin, 6-Benzyladenosine) compounds\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/acfe17b80f940c7a67e79402.jpg"},{"id":92416247,"identity":"40c7add9-40cc-4386-b263-06c8925ab6fe","added_by":"auto","created_at":"2025-09-29 13:25:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":359993,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 liquid bioformulation on plant growth of Tomato seedlings under Greenhouse conditions, a: Nursery experiment under greenhouse conditions, b: Control tray, c: \u003cem\u003eC. citreum\u003c/em\u003e AVN2 bacterized tomato seedlings tray, d: Post harvest, e: Root system, f: Effect of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 liquid bioformulation on tomato seedlings\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/0e0513f24ed07e304e600df2.jpg"},{"id":92416249,"identity":"0e2aa26d-7127-4d85-83cf-06cad0fc1a82","added_by":"auto","created_at":"2025-09-29 13:25:30","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"graphical-abstract","size":119175,"visible":true,"origin":"","legend":"Endophytic bacteria have become essential in promoting plant growth and development, and provide resistance against phytopathogens through diverse biochemical mechanisms. This study isolated an endophytic bacterium, AVN2, from Curcuma longa's rhizome and identified it as () using 16S rRNA partial gene sequencing. C. citreum AVN2 was deposited in NCBI with accession number PQ215941. The strain exhibited significant antagonistic activity against . Sp. , a major causative agent of tomato wilt, in a dual culture assay, and is confirmed as a biocontrol agent by GC-MS analysis of its secondary metabolites, Lupeol, 2-Piperidinone, Stigmastan-3,5-diene, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 5-Pyrrolidino-2-pyrrolidone, Diethyl Phthalate, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-. Plant growth-promoting characterization revealed the production of important growth-promoting phytohormones, including indole compounds (6.40 \u0026micro;g/mL), gibberellic acids (29.49 \u0026micro;g/mL), and cytokinins (Kinetin 15.49 \u0026micro;g/mL, and 6-Benzyladenosine 27.78 \u0026micro;g/mL), which were quantified by HPLC analysis. Seed bacterization with . bioformulation enhanced the seed vigour index of tomato seeds compared to the control. Bio-formulated AVN2 significantly enhanced root and shoot growth, and overall biomass under greenhouse conditions, demonstrating biocontrol efficacy and plant growth-promotion (PGP) potential. This study presents the first evidence of as an endophyte in , exhibiting dual biofertilizer and bioprotectant properties. These findings underscore its promise as a sustainable, eco-friendly alternative to chemical fertilizers and fungicides in horticultural crop production.","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/9fdcea5eef32bf7f8339123c.png"},{"id":96248914,"identity":"e7bcd3a8-4024-4dd0-8901-e53d8ea56dbc","added_by":"auto","created_at":"2025-11-19 07:29:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4061224,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/0ef579c0-45a5-4226-96ae-90b679ec045b.pdf"},{"id":92414850,"identity":"75f3142b-0f46-4bb7-bfea-96d57a7473d9","added_by":"auto","created_at":"2025-09-29 13:17:30","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1080742,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydocument.docx","url":"https://assets-eu.researchsquare.com/files/rs-7602699/v1/b61e1de492bc4924c6f5a9eb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation and characterization of Plant growth-promoting hormones by an endophytic bacterium, Curtobacterium. citreum AVN2 isolated from the rhizome of Curcuma longa L","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfectious Plant diseases caused by phytopathogenic microorganisms contribute to approximately 16% of global agricultural losses, primarily due to their aggressiveness in the soil and the influence on various crops during harvesting and post-harvest practices [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To address this challenge, much focus is needed to alleviate 70% of food security by developing biological biostimulants/ biofertilizers from microorganisms and having synergistic activities with the plant. This problem highlights the growing interest in biological bio-stimulants and biofertilizers sourced from microorganisms residing in the rhizosphere, phyllosphere, and as endophytes, due to their capacity to stimulate plant development and enhance systemic resistance in crops.\u003c/p\u003e\n\u003ch3\u003eEndophytes\u003c/h3\u003e\n\u003cp\u003eEndophytes are microorganisms that reside in intercellular or intracellular spaces of a host tissue without causing any adverse effects. Like PGPR, Plant growth-promoting endophytes [PGPE] show a range of direct and indirect strategies to boost plant health. PGPE facilitates the synthesis and regulation of Phytohormones, such as auxin, cytokinin, and gibberellin, as well as the uptake of nitrogen, phosphate, and iron from the soil through a direct mechanism [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent times, along with Plant Growth Promoting Rhizobacteria (PGPR), endophytic microorganisms have gained much focus due to their many advantages. Application of endophytic microorganisms includes nitrogen fixation, absorbing nutrients, producing phytohormones, withstanding biotic and abiotic challenges, and producing bioactive secondary metabolites, such as phenols, flavonoids, polyketones, peptides, steroids, alkaloids, terpenoids, and quinols. These substances are important in medicines, sustainable agriculture, and industrial biotechnology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCurcuma longa\u003c/h2\u003e\u003cp\u003e\u003cem\u003eCurcuma longa\u003c/em\u003e (turmeric) is a medicinal plant used for thousands of years to treat various diseases in the Indian traditional medicine system of Ayurveda. The medicinal properties and numerous applications of \u003cem\u003eC. longa\u003c/em\u003e enhance its research potential in biomedicine. Similarly, endophytic microbes that inhabit these host plants significantly promote plant health by generating bioactive secondary metabolites of ecological significance, which confer protection against biotic and abiotic stressors via horizontal gene transfer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Such endophytes, isolated from medicinal plants, enhance soil structure, augment water retention, improve fertility, serve as biocontrol agents and biofertilizers, detoxify toxic compounds, accelerate seed germination, confer tolerance to abiotic stressors, and promote plant health. As a result, they have attracted considerable attention to sustainable agriculture practices because of their combined functions.\u003c/p\u003e\u003cp\u003ePGPE indirectly helps to withstand biotic stress and induce systemic resistance to the plant by producing volatile organic compounds, antibiotics, and antioxidant stress enzymes that neutralize reactive oxygen species [ROS] [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Endophytes have a great deal of promise as biocontrol agents and biofertilizers, and their complex interconnections make them strong friends in sustainable agriculture. This study aims to isolate endophytic bacteria from the rhizome of \u003cem\u003eCurcuma longa\u003c/em\u003e, which has plant growth-promoting activities and biocontrol activity against phytopathogens and can be used as a biofertilizer and bio-stimulant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eCollection of plant material\u003c/h2\u003e\u003cp\u003eHealthy and mature \u003cem\u003eC. longa\u003c/em\u003e plants were collected from organic farms of Erode (Turmeric city of India), (11.3410\u0026deg; N, 77.7172\u0026deg; E), Tamil Nadu, bought in a sterile zip-lock bag, and transferred to the lab facility at Acharya Nagarjuna University, Guntur, Andhra Pradesh, India, and processed within 24 hours to preserve the viability of endophytic bacteria. To avoid surface contamination, rhizomes were not washed before transport, and surface sterilisation was done in the lab under sterile conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCollection of\u003c/b\u003e \u003cb\u003eFusarium. oxysporum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA lyophilized culture of \u003cem\u003eF. oxysporum var.\u003c/em\u003e Lycopersici (MTCC10270) was brought from the Microbial Type Culture Collection (MTCC), Chandigarh. It was grown in Sabouraud\u0026rsquo;s dextrose media (SDA) and Potato dextrose agar (PDA) (Oxoid) plates according to the protocol given by MTCC Chandigarh and stored at 4℃ for later use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIsolation of endophytic bacteria\u003c/h3\u003e\n\u003cp\u003eInitially, Rhizomes of \u003cem\u003eC. longa\u003c/em\u003e were washed thoroughly with sterile water to remove adhering rhizospheric soil, dust, debris, and other contaminants. Later, the surface sterilization of rhizomes was done with 20% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 2 minutes, 70% ethanol for 5 minutes, and repeatedly rinsed with 3% Sodium hypochlorite with 5 minutes of incubation for each rinse, and finally rinsed with sterile saline water (0.85% NaCl). Sterilized rhizome was aseptically cut into 1-inch pieces (weighing 1g) and homogenized with a mortar and pestle in 1% sterilized NaCl. Then the homogenized tissue was made up to 10 mL with 1% NaCl solution, and serially diluted to 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nutrient agar medium (10 g/L of peptone, 3g/L of beef extract, 5g/L of NaCl, and 20 g/L of agar) is supplemented with 2.0 g/L sucrose for endophytic bacteria isolation. The NAM medium was sterilised in an autoclave at 121℃ for 20 minutes and then cooled to 50℃. Endophytic bacteria were isolated using the spread plate method, and 100 \u0026micro;L of each dilution was taken and spread onto the nutrient agar medium. A 100 \u0026micro;L aliquot of the last rinse from surface disinfection was subjected to a sterility check on nutrient medium to confirm the surface disinfection and verify that the surface was free from biological contamination [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Plates were allowed to be incubated at 30℃ for 48 hours. The isolated bacterial colonies are further sub-cultured by the quadrant streaking method on fresh nutrient agar plates and incubated for 48 hours. A pure single colony in the fourth quadrant of the NAM plate was transferred to agar slants and stored in the refrigerator at 4℃ for further characterization [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMolecular identification and Phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA from the bacterial isolate AVN2 was subjected to PCR amplification of the 16S rRNA gene sequence using universal primers: 785F 5' (GGA TTA GAT ACC CTG GTA) 3', 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3, 907R 5' (CCG TCA ATT CMT TTR AGT TT) 3', and 1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3' [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Amplification and sequencing were outsourced to Macrogen Inc. (Seoul, Republic of Korea). Phylogenetic analysis was conducted using the neighbour-joining method based on 100% sequence similarity to determine the taxonomic identity of the isolate. The 16S rRNA Partial gene sequence of strain AVN2 was deposited in the NCBI GenBank database (India).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eScreening of plant growth promotion (PGP) traits\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eIndole-3-acetic acid (IAA)\u003c/h2\u003e\u003cp\u003eEndophytic bacterial isolate AVN2 was screened qualitatively and quantitatively for Indole-3-acetic acid (IAA) production, a key phytohormone associated with plant growth promotion and development. The qualitative assay was performed using the Salkowski reagent method [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. AVN2 was cultured on nutrient agar medium, supplemented with 0.1% (w/v) L-tryptophan (HiMedia) and incubated for 72 hours. Upon addition of Salkowski reagent (1 mL of 0.5 M FeCl\u003csub\u003e3\u003c/sub\u003e in 50 mL of 35% HClO\u003csub\u003e4\u003c/sub\u003e), development of a pink color confirmed the production of IAA. For quantitative estimation, AVN2 was cultured and grown in nutrient broth supplemented with L-tryptophan (0.1% w/v), and was incubated at 28℃ on a rotary shaker (180 rpm) for 72 hours. After incubation, the culture was harvested by centrifugation at 10,000 \u0026times; g for 10 minutes, and 1 mL of the supernatant was collected and mixed with two drops of orthophosphoric acid and 2 mL of Salkowski reagent, and then incubated at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃ for 20 minutes in the dark. Absorbance was measured at 530 nm using a UV-Vis spectrophotometer. This experiment was replicated thrice, and the data presented are the average of the three similar experiments. The absorbance of the samples obtained was plotted against a standard to determine the concentration of IAA produced, and the results are expressed as \u0026micro;g of compound (IAA) per mL of filtrate.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eInorganic phosphate solubilization\u003c/h3\u003e\n\u003cp\u003eThe ability of the bacterial isolate AVN2 to solubilize Inorganic phosphate was assessed, qualitatively and quantitatively, according to [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Using the streak plate method, Pikovskaya\u0026rsquo;s agar medium was used to determine inorganic phosphate solubilization. Inorganic phosphate solubilization was estimated after 5 days of incubation at room temperature. The appearance of a clear zone around the bacterial colony confirmed the occurrence of Inorganic phosphate solubilization activity. For quantitative estimation, 100 \u0026micro;L of bacterial culture AVN2 was inoculated into 50 mL of nutrient broth supplemented with tricalcium phosphate(1\u0026micro;g/mL) and incubated in an incubator shaker at 180 rpm at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1℃ for 5 days. An uninoculated broth with tricalcium phosphate served as a control. After incubation, the culture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was estimated using the Vanado-molybdate yellow color method. 2.5 mL of Barton\u0026rsquo;s reagent was added to 0.5 mL of the supernatant, and the volume was brought up to 50 mL with deionized water. The absorbance of the resultant color complex produced was measured after 10 minutes at 430 nm in a UV/Visible spectrophotometer. The total phosphate solubilized by the bacterial endophyte was calculated from the regression equation using the standard curve, and the values of soluble phosphate were expressed as ppm ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAmmonia production\u003c/h2\u003e\u003cp\u003eAmmonia production by endophytic bacteria AVN2 was determined using peptone water, following the method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For qualitative analysis, AVN2 was inoculated onto a peptone agar plate and incubated at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 48 hours. The development of a yellow to brown color with 0.5 mL of Nessler's reagent was a positive test for ammonia production. For quantitative estimation, a freshly grown bacterial culture was inoculated in 10 mL of peptone water and incubated at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 72 hours. After incubation, the culture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. To 1 mL supernatant, 0.5 mL Nessler's reagent was added, and the absorbance of the resultant yellow-orange complex was determined by comparison to the standard curve of ammonium sulphate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAntifungal activity\u003c/h2\u003e\u003cp\u003eIn vitro screening of antifungal activity was assayed using the dual culture method [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eF. oxysporum Lycopersici\u003c/em\u003e was cultured for 7 days on Sabouraud's dextrose agar media (40g/L of dextrose, 10g/L of peptone, 15 g/L of agar). A 5-mm mycelial plug of freshly grown fungal culture was inoculated at the centre of a newly prepared nutrient agar plate for the assay. AVN2 was streaked 3 cm away from the fungal disc toward the periphery of the plate. Plates were incubated at 35\u0026deg;C for 4 to 7 days. Control plates were maintained with fungal inoculation alone, without the bacterial isolate. The formula expressed the antifungal activity as the percentage of radial growth inhibition of the pathogen in the presence of AVN2 compared to the control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ea: Distance between the fungus and the endophyte isolated in the centre of the Petri dish.\u003c/p\u003e\u003cp\u003eb: The distance between the fungus and the space in the centre of the Petri dish is endophytic-free.\u003c/p\u003e\u003cp\u003eThis experiment was replicated thrice, and results were expressed as the mean percentage of inhibition of phytopathogen by the bacterial endophyte AVN2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eGrowth Optimization studies\u003c/h2\u003e\u003cp\u003eThe growth behavior of the bacterial isolate AVN2 was assessed under varying environmental and nutritional conditions by monitoring optical density (O.D.) at 600 nm, following the method [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Growth optimization was carried out across different parameters, including temperature, pH, carbon, and nitrogen sources. Temperature optimisation was performed by incubating cultures at various temperatures (25\u0026deg;C, 30\u0026deg;C, 35\u0026deg;C, 40\u0026deg;C, and 45\u0026deg;C). Similarly, pH optimization was assessed by adjusting the medium to pH values (5.0, 6.0, 7.0, 8.0, and 9.0). For carbon source optimization, a minimal broth medium was supplemented with 1% (w/v) of various carbon sources (starch, sucrose, fructose, mannitol, dextrose, lactose, glucose, glycerol, and maltose). For nitrogen source optimisation, 0.5% (w/v) of different nitrogen sources (peptone, beef extract, yeast extract, urea, ammonium sulfate, ammonium chloride, sodium nitrate, and potassium nitrate) was added to minimal broth. In each case, 100 \u0026micro;L of freshly grown bacterial suspension was inoculated into the respective media and incubated for 48 h. Post-incubation, bacterial growth was quantified by measuring O.D. at 600 nm using a UV-Visible spectrophotometer. All experiments were replicated thrice, and the absorbance values were recorded for comparative analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Liquid Bioformulation\u003c/h2\u003e\u003cp\u003eFollowing the optimization of physicochemical parameters\u0026mdash;temperature, pH, carbon, and nitrogen sources\u0026mdash;using a one-factor-at-a-time (OFAT) approach, a liquid bioformulation of the bacterial isolate AVN2 was prepared. The formulation medium was developed using the optimal conditions identified from growth studies: the most suitable carbon and nitrogen sources and the ideal pH and incubation temperature.\u003c/p\u003e\u003cp\u003eFreshly grown culture medium was inoculated into the optimised medium and incubated under a rotary shaker (180 rpm) for 48 h. The resulting liquid culture, containing a high density of viable bacterial cells, was used as the liquid bioformulation for further applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eExtraction of the bioactive metabolites\u003c/h2\u003e\u003cp\u003eBioactive secondary metabolites were extracted from the optimised culture broth of the active bacterial strain AVN2 using the ethyl acetate solvent extraction method [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The strain was inoculated into the optimized broth formulated with the previously optimized physicochemical conditions under a rotary shaker (180 rpm) for 48 hours of incubation. After incubation, the culture broth was harvested by centrifuging at 10,000 rpm for 15 minutes. The supernatant was then collected and vigorously shaken with an equal volume of ethyl acetate (1:1, v/v) in a separating funnel and allowed until phase separation occurred. The upper organic layer was collected and subjected to two additional extractions using fresh ethyl acetate. The pooled organic extracts were concentrated to dryness under reduced pressure using a rotary evaporator at 40\u0026deg;C. The crude extract containing bioactive compounds was stored at 4\u0026deg;C and utilized for subsequent bioactivity assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eQuantification of Plant Growth-Promoting Hormones by HPLC\u003c/h2\u003e\u003cp\u003eThe production of important growth-promoting phytohormones like indole-3-acetic acid (IAA), Gibberellins, and cytokinins from the endophytic bacteria AVN2 was assessed using High-performance liquid chromatography (HPLC). Culture supernatants were extracted with ethyl acetate, and the concentrated extracts were subjected to chromatographic analysis to quantify hormones.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eIAA\u003c/h2\u003e\u003cp\u003eEthyl acetate extract of the isolate was concentrated to 10 mL using a Rota evaporator and adjusted to a pH of 2.8. HPLC analysis was conducted by injecting 20 \u0026micro;L aliquots into an Inertsil ODS C-18 (250 mm \u0026times; 4.6mm; 5 \u0026micro;m id) column connected to an HPLC pump. A ratio of 1:30:70 of acetic acid: methanol: water (v/v) was used as the mobile phase and filtered using 0.2 \u0026micro;m membrane filters. The flow rate was adjusted to 1.0 mL/ min, and the operating pressure was 1200.5\u0026thinsp;\u0026plusmn;\u0026thinsp;25 psi. Absorbance was measured at 280 nm. The retention times for compounds were compared to those of the standard values of IAA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eGibberellins\u003c/h2\u003e\u003cp\u003eGibberellins (GA) analysis was done using the modified procedure established by [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The ethyl acetate extract of the isolate was concentrated to up to 10 mL using a rotary evaporator and was adjusted to pH 2.5. Concentrated ethyl acetate extract of the culture supernatant was solubilized in 60% methanol (MeOH), with the pH adjusted to 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 using 2 N NH\u003csub\u003e4\u003c/sub\u003eOH. A methanolic suspension of concentrated ethyl acetate extract of isolates was subjected to HPLC by injecting 20 \u0026micro;L aliquots into an Inertsil ODS C-18 (250 mm\u0026times;4.6mm; 5\u0026micro; id) column connected to an HPLC pump. Isopropanol, 25% Ammonium Hydroxide, and water in a (10:1:1 v/v) ratio, filtered through 0.2 mm membrane filters, were constructed as the mobile phase. The flow rate was adjusted to 0.5 mL/min with the operating pressure 1240.5\u0026thinsp;\u0026plusmn;\u0026thinsp;25 psi. Absorbance was monitored at 206 nm. Retention times for compounds were compared to those of standard values for GA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCytokinins\u003c/h2\u003e\u003cp\u003eThe presence of Cytokinin compounds was assayed according to [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ethyl acetate extract of the isolate was concentrated up to 10 mL using a rotary evaporator and adjusted to a pH of 2.8. HPLC was performed using an Inertsil ODS C-18 (250 mm\u0026times;4.6mm; 5\u0026micro; id) column connected to an HPLC pump. The mobile phase consists of n-butanol, acetic acid, and water in the ratio of (12:3:5 v/v/v) with an injection volume of 20 \u0026micro;L, a flow rate of 0.5 mL/min, a column temperature of 35\u0026deg;C, a walking time of 20 minutes, and a detection wavelength of 254 nm was constructed as the mobile phase. Retention times for compounds were compared to those of standard values for cytokinin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eGreenhouse studies\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003ePreparation of bacterial cell suspension\u003c/h2\u003e\u003cp\u003eThe selected bacterium AVN2 was inoculated into 200 mL of optimized broth and incubated at 35 ℃ for 48 hours. The bacterial cell suspension was prepared by harvesting cells by centrifugation at 6000 rpm for 15 minutes. The inoculum was re-suspended in sterile distilled water, and the concentration was adjusted to 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e CFU/ml [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eSeed treatment and nursery experiments\u003c/h2\u003e\u003cp\u003eTo determine the effect of AVN2 on the growth of tomato seeds (\u003cem\u003eLycopersicon esculentum L\u003c/em\u003e.), Royal variety tomato seeds were purchased from a certified nursery in Guntur, Andhra Pradesh, India. Tomato seeds were surface sterilized with 5% sodium hypochlorite for 20 min, and 70% ethanol for 1 minute, and then thoroughly washed five times with double-sterilized distilled water [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Then, tomato seeds were treated with a 48-hour-old (approximately 10\u003csup\u003e8\u003c/sup\u003e CFU/ml concentration) culture of the isolate for 30 minutes and shade-dried at 28℃ for 1 hour, and subsequently sown in 98-well nursery trays (5 seeds/well) using sterilized coco peat. The control seeds were treated with only sterile distilled water under identical conditions. Seedling vigour index or Germination percentage (Gp), Speed of seed germination (S), Germination rate (G\u003csub\u003eR\u003c/sub\u003e), and Germination index (G\u003csub\u003eI\u003c/sub\u003e) are calculated in the first week.\u003c/p\u003e\u003cp\u003eGermination percentage (Gp)\u0026thinsp;=\u0026thinsp;Ni/NX100\u003c/p\u003e\u003cp\u003eSpeed of seed germination (S)\u0026thinsp;=\u0026thinsp;ni/di\u003c/p\u003e\u003cp\u003eGermination rate (G\u003csub\u003eR\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;no. of germinated seeds/ day of first count +\u0026hellip;\u0026hellip; + no. of germinated seeds/day of last count\u003c/p\u003e\u003cp\u003eGermination index (GI) = Σ G/T\u003c/p\u003e\u003cp\u003eWhere: N\u0026thinsp;=\u0026thinsp;Total no. of seeds, Ni\u0026thinsp;=\u0026thinsp;germinated seeds, ni\u0026thinsp;=\u0026thinsp;germinated seeds per day, di\u0026thinsp;=\u0026thinsp;counting day, G\u0026thinsp;=\u0026thinsp;percentage of seed germination per day, T\u0026thinsp;=\u0026thinsp;germination period.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003ePot experiment\u003c/h2\u003e\u003cp\u003eThe bio-efficacy of AVN2-based liquid bioformulation was evaluated under a greenhouse pot experiment (average temperature of 25℃) at Acharya Nagarjuna University, Guntur, Andhra Pradesh, India, in a completely randomized design by replicating each treatment about seven times.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eSoil Preparation and Transplantation\u003c/h2\u003e\u003cp\u003eThe experimental soil used for the greenhouse trials is sterilized in an autoclave (121\u0026deg;C, 15 psi, 1 h) to eliminate native microbial flora. Two-week-old tomato seedlings were carefully transplanted from the nursery bed to the pots (30 cm diameter) until the final harvest and watered daily.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eTreatment Application\u003c/h2\u003e\u003cp\u003eThe AVN2 liquid bioformulation (5 mL) was applied weekly to the seedlings through collar application until the final harvest. Control plants received the same volume of the untreated (placebo) formulation prepared identically but without microbial inoculation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eGrowth and Biochemical Parameters\u003c/h2\u003e\u003cp\u003ePlants were harvested from each replicate at 2-week intervals to evaluate growth and physiological parameters: root length (cm), Shoot length (cm), Fresh biomass (g), and Primary metabolite content, including Total proteins and total carbohydrates. The primary metabolite content was determined following the protocol of [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experimental data were subjected to data analysis using IBM SPSS PASW Statistics 18 software. The significance of the differences between the control and Treated groups was analyzed using one-way ANOVA. Means, standard errors, and percentage increases were calculated from at least three replicates. Statistical significance was determined at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn this study, eight morphologically distinct endophytic bacteria were isolated from the rhizome of \u003cem\u003eC. longa\u003c/em\u003e. The isolate designated AVN2 was selected for further characterization based on its distinct colony morphology and bioactivity potential (Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of AVN2\u003c/h2\u003e\u003cp\u003eThe 16S rRNA gene sequence analysis of the isolate AVN2 revealed 99.65% similarity with the partial sequence of \u003cem\u003eCurtobacterium oceanosedimentum\u003c/em\u003e (GenBank ID: NR-104839.1). Phylogenetic analysis further confirmed that AVN2 clusters closely with \u003cem\u003eC. oceanosedimentum\u003c/em\u003e, supporting its taxonomic assignment (Fig.\u0026nbsp;2). Accordingly, the isolate was identified as \u003cem\u003eCurtobacterium citreum\u003c/em\u003e AVN2, and the sequence was deposited in the NCBI GenBank with the accession number PQ215941.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlant growth-promoting (PGP) traits:\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2 exhibited multiple plant growth-promoting attributes. Upon addition of Salkowski reagent, AVN2 showed a positive response to Indole acetic acid production by producing a pink coloured complex. Halo zone formation shows the positive response of AVN2 towards inorganic phosphate solubilization, and the appearance of brick red color confirms the ability of AVN2 in ammonia production. Quantitative assessment revealed that \u003cem\u003eC. citreum\u003c/em\u003e AVN2 produced 7.2 \u0026micro;g/mL of IAA, 100 ppm of Inorganic phosphate solubilization, and 0.7 \u0026micro;g/mL of Ammonia production. These results suggest the strain's potential as a bio-inoculant with direct PGP activities.\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eAntifungal activity\u003c/h2\u003e\u003cp\u003eThe antagonistic potential of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 was evaluated against \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. L\u003cem\u003eycopersici\u003c/em\u003e using the in vitro technique (Dual culture assay). AVN2 exhibited significant antifungal activity, inhibiting the overall growth of the phytopathogen by 91%. The pronounced inhibition zone indicates the strong biocontrol potential of this endophyte.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eGrowth optimization\u003c/h2\u003e\u003cp\u003eOptimal growth of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 was achieved under the following culture conditions: temperature 35\u0026deg;C, pH 7, with 1% mannitol as the carbon source and 0.5% beef extract as the nitrogen source (Fig.\u0026nbsp;3). These optimised parameters not only enhanced bacterial proliferation but also significantly increased PGP metabolite production. Compared to the culture media before and after optimisation, the optimised media showed the enhanced production of IAA, Inorganic phosphate solubilisation, and ammonia production by 300%, 140% and 200%, respectively (Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEnhancement of PGP traits of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 before and after optimisation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eIAA production (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eInorganic Phosphate solubilization (ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eAmmonia production (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore optimization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAfter optimization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBefore optimization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAfter optimization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBefore optimization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAfter optimization\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAVN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4*\u003c/p\u003e\u003cp\u003e(300%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e240\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0*\u003c/p\u003e\u003cp\u003e(140%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35*\u003c/p\u003e\u003cp\u003e(200%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003eValues are the mean of three replicates. \u0026plusmn; SE, IAA production, Inorganic phosphate solubilization, and Ammonia production have been increased by 300%, 140%, and 200% after optimisation\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003eLiquid bioformulation\u003c/h2\u003e\u003cp\u003eBased on the growth optimization studies, a liquid bioformulation was developed using the optimised medium and designated AVN2 Liquid Formulation. This formulation was designed as a flowable or aqueous suspension, offering a viable alternative to conventional carrier-based inoculants. Liquid bioformulations are advantageous due to their longer shelf life, higher microbial load, and easier application in field and greenhouse settings.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003eQuantification of Secondary Metabolites by GC-MS\u003c/h2\u003e\u003cp\u003eIn GC MS chromatogram, Metabolic Profile of Bacterial extract of \u003cem\u003eCurtobacterium citreum\u003c/em\u003e AVN2 as per the NIST Database, bioactive metabolites such as Phenol,2,4\u0026rsquo;-isopropylidenedi, Lupeol, N-[2-Hydroxyethyl]succinimide, 2-Piperidinone, Stigmastan-3,5-diene, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 5-Pyrrolidino-2-pyrrolidone, Diethyl Phthalate, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-, Aziridine,2-(1,1-dimethylethy)-3-methyl-1-(2- propenyl)-, trans-, respectively, were observed (Fig.\u0026nbsp;4; Table\u0026nbsp;2)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMetabolic Profile of \u003cem\u003eCurtobacterium citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS. No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHeight %\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eName of the compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMolecular formula\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMol wt\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eArea % occupied\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eBiological activity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22.935\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e51.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePhenol,2,4\u0026rsquo;-isopropylidenedi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC15H16O2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e61.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo activity found\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28.064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLupeol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC30H50O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e426\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e21.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAntimicrobial activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16.852\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN-[2-Hydroxyethyl] succinimide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC6H9NO3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo activity found\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2-Piperidinone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC5H9NO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAntimicrobial activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26.308\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStigmastan-3,5-diene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC29H48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e4.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAntifungal activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19.850\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC11H18N2O2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e210\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAntifungal activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.770\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5-Pyrrolidino-2-pyrrolidone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC8H14N2O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e154\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eIndole compound\u003c/p\u003e\u003cp\u003e(Plant growth compound)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.225\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiethyl Phthalate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC12H14O4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e222\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAntimicrobial activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17.680\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC7H10N2O2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e154\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eAntifungal activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAziridine,2-(1,1-dimethylethy)-3-methyl-1-(2\u0026ndash;propenyl)-, trans-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC10H19N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e153\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo activity found\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eHPLC analysis of Plant growth-promoting hormones\u003c/h3\u003e\n\u003cp\u003eHPLC analysis was performed on metabolites extracted from AVN2 culture broth using the solvent extraction method to confirm the production of plant growth-promoting hormones. The study validated the presence of indole-3-acetic acid (IAA) and other Phytoactive compounds, supporting the strain\u0026rsquo;s role in direct plant growth stimulation.\u003c/p\u003e\n\u003ch3\u003eQuantification of Plant Growth Hormones by HPLC:\u003c/h3\u003e\n\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003eIAA\u003c/h2\u003e\u003cp\u003eHPLC fingerprinting of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 revealed a distinct peak at retention time (RT) 0.70 min, with a peak area of 1488%, confirming the presence of an indole compound. The quantified concentration of IAA was 6.40 \u0026micro;g/mL (Fig.\u0026nbsp;5a)\u003c/p\u003e\u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\u003ch2\u003eGibberellins (GA)\u003c/h2\u003e\u003cp\u003eThe HPLC chromatogram of the AVN2 extract showed a prominent peak at RT 5.9 min, with a peak area of 55353%, corresponding to the presence of gibberellic acid (GA). The concentration was quantified as 29.49 \u0026micro;g/mL (Fig.\u0026nbsp;5b).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003eCytokinins\u003c/h2\u003e\u003cp\u003eThe HPLC profile of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 showed two distinct peaks: one at RT 5.3 min with a peak area of 3579%, confirming the presence of Kinetin at 15.49 \u0026micro;g/mL, and another at RT 6.2 min with a peak area of 6419%, corresponding to 6-Benzyladenosine, quantified at 27.78 \u0026micro;g/mL (Fig.\u0026nbsp;5c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\u003ch2\u003eGreenhouse studies\u003c/h2\u003e\u003cp\u003e\u003cb\u003eSeedling vigour index\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe seedling vigor index exhibited by 48-h bacterized tomato seeds treated with the endophytic bacterium \u003cem\u003eC. citreum\u003c/em\u003e AVN2 demonstrated a significant improvement (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) by showing 81.6% of Germination percentage (Gp) and 54.5% of Germination index (Gi), respectively when compared to the control (Table\u0026nbsp;3; Fig.\u0026nbsp;6). Specifically, \u003cem\u003eC. citreum\u003c/em\u003e AVN2 treatment resulted in an 81.6% increase in Gp and a 54.5% increase in GI compared to the untreated control (Table\u0026nbsp;3; Fig.\u0026nbsp;6).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSeedling vigour index of tomato seeds treated with \u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eGrowth parameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAVN2\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGermination percentage (G\u003csub\u003ep\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGP Ni/NX100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e81.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpeed of seed germination (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eS\u0026thinsp;=\u0026thinsp;ni/di\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDay1\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e\u003cp\u003eDay2\u0026thinsp;=\u0026thinsp;50\u003c/p\u003e\u003cp\u003eDay3\u0026thinsp;=\u0026thinsp;2.8\u003c/p\u003e\u003cp\u003eDay 4\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDay1\u0026thinsp;=\u0026thinsp;10.2\u003c/p\u003e\u003cp\u003eDay2\u0026thinsp;=\u0026thinsp;18.75\u003c/p\u003e\u003cp\u003eDay3\u0026thinsp;=\u0026thinsp;15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGermination rate (G\u003csub\u003eR\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(G\u003csub\u003eR\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;seed/1st day=\u0026hellip;\u0026hellip;seed/n\u003csup\u003eth\u003c/sup\u003e day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e103.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e192\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGermination index (GI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGI/G/T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003eThe values are the mean of three replicates. \u0026plusmn; SE, Germination percentage = No. The number of seeds germinated per time duration. Vigour index calculated during the first week after germination. The Value in parentheses is the percentage increase of growth parameters compared to the control\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003ePlant growth promotion\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eC. citreum\u003c/em\u003e AVN2 liquid bioformulation application significantly enhanced tomato seedlings' growth parameters under greenhouse conditions. A progressive increase in plant growth was observed from the 4th to the 12th week of treatment (Table\u0026nbsp;4). Biomass increased by 233% in treated plants compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eImpact of inoculation of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 bioformulation on plant growth of tomato seedlings.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eBiomass (gm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4th week\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6th week\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8th week\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10th week\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12th week\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003cp\u003e(14.29%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003cp\u003e(71.43%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003cp\u003e(133.33%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003cp\u003e(150.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003cp\u003e(233.33%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePlant height (cm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003cp\u003e(12.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003cp\u003e(20.38%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(40.35%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(43.80%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(57.26%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eShoot height (cm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(12.22%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003cp\u003e(19.57%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003cp\u003e(42.86%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\u003cp\u003e(47.06%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003cp\u003e(55.77%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRoot length (cm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(10.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003cp\u003e(23.57%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003cp\u003e(25.00%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(26.32%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003cp\u003e(65.00%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eProtein content (\u0026micro;g/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003cp\u003e(13.04%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003cp\u003e(30.43%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003cp\u003e(31.43%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003cp\u003e(40.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(42.17%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCarbohydrate content (\u0026micro;g/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003cp\u003e(8.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(32.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(41.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003cp\u003e(45.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003cp\u003e(64.58%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAs detailed in Table\u0026nbsp;4, \u003cem\u003eC. citreum\u003c/em\u003e AVN2 treatment resulted in a 55.7% increase in shoot height and a 65% increase in root length relative to untreated controls, demonstrating a substantial effect on morphological growth traits. Furthermore, there was a significant enhancement in primary metabolite content, with protein content increasing by 42.17% and carbohydrate content by 64.58% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in treated plants.\u003c/p\u003e\u003cp\u003eThese findings underscore the plant growth-promoting potential of \u003cem\u003eC. citreum\u003c/em\u003e AVN2, highlighting its role in improving seed germination, vegetative growth, biomass accumulation, and metabolic productivity in tomato plants under pot experiments.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndophytic microorganisms, including bacteria and fungi, inhabit various niches within agricultural plants, such as seeds, the phyllosphere, endosphere, and rhizosphere, and have emerged as significant sources of bioactive compounds with medicinal and agricultural relevance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These endophytes often colonize the intercellular spaces of host tissues and help in promoting plant growth by enhancing the energy uptake by the production of primary metabolites, especially extracellular enzymes, and improve the host plant\u0026rsquo;s resilience through the production of secondary metabolites with anti-phytopathogenic properties and plant-beneficial traits. \u003cem\u003eC. longa\u003c/em\u003e (turmeric), a medicinal plant, has been traditionally used in biomedicine for thousands of years. The medicinal properties and numerous applications of \u003cem\u003eC. longa\u003c/em\u003e enhance its research potential, leading to promising, potent discoveries at the molecular and genetic levels. Similarly, endophytes that inhabit these host plants play a crucial role in promoting plant health by generating bioactive secondary compounds of ecological significance, which confer protection against biotic and abiotic stressors through horizontal gene transfer.\u003c/p\u003e\u003cp\u003eIn this present study, AVN2 isolated from the rhizome of \u003cem\u003eC. longa\u003c/em\u003e is identified as \u003cb\u003eCurtobacterium. citreum\u003c/b\u003e (99.65%) by 16s rRNA partial gene sequencing and deposited in the NCBI database with accession number \u003cb\u003ePQ215941\u003c/b\u003e (Fig.\u0026nbsp;2), validating the isolate's taxonomic classification. \u003cb\u003eC. citreum\u003c/b\u003e \u003cb\u003eas an endophyte from\u003c/b\u003e \u003cb\u003eC. longa\u003c/b\u003e \u003cb\u003erhizomes has not yet been reported\u003c/b\u003e. However, \u003cem\u003eC. citreum\u003c/em\u003e has been identified as an endophyte from various plant sources in the past, such as traditional rice cultivars [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], tea leaves (\u003cem\u003eCamellia sinensis var. assamica\u003c/em\u003e), and prairie grasses [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eC. citreum\u003c/b\u003e \u003cb\u003eAVN2\u003c/b\u003e has multiple plant growth-promoting traits (IAA, ammonia production, and inorganic phosphate solubilization), and antagonistic activity against a phytopathogen, \u003cb\u003eF. oxysporum Lycopersici\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAnd serving as an effective bioformulation for plant growth promotion in tomato seedlings by exhibiting notable improvements in primary metabolite content, biomass accumulation, seedling vigour, and overall plant development, considering that the diversity and potentiality in PGP activity allow the endophytic \u003cem\u003eC. citreum\u003c/em\u003e to adapt and establish beneficial interactions with plants rather than the subject of origin [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe first emerging field of study in sustainable crop protection is the potential of Curtobacterium species as biocontrol agents. Under in vitro circumstances, \u003cem\u003eC. citreum\u003c/em\u003e AVN2 demonstrated potent antagonistic activity against \u003cem\u003eF. oxysporum\u003c/em\u003e in our investigation, which is consistent with previous studies that reported Curtobacterium strains with comparable antimicrobial properties [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Similarly, \u003cem\u003eA. oryzae\u003c/em\u003e AVNF4 isolated from the rhizome of \u003cem\u003eC. longa\u003c/em\u003e exhibited antifungal activity against \u003cem\u003eF. oxysporum\u003c/em\u003e in the dual culture method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. GC-MS analysis of ethyl acetate extracts of culture supernatant revealed the presence of potential compounds, a pyrrole compound, Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro- produced by \u003cem\u003eCurtobacterium citreum\u003c/em\u003e AVN2, is known for its various therapeutic applications, such as antifungal, antibiotic, anti-tumor, and anti-inflammatory activities [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Stigmastan-3,5-diene produced in tissue extracts of \u003cem\u003ePortulacaria afra\u003c/em\u003e, and roots of \u003cem\u003eStellera chamaejasme\u003c/em\u003e have antioxidant activity, antimicrobial activity, and antifungal activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Volatile compounds Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- and Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-, produced by \u003cem\u003eB. subtilis, A. oryzae\u003c/em\u003e AVNF4, and \u003cem\u003eStreptomyces\u003c/em\u003e Species have shown biocontrol activity against \u003cem\u003eAlternaria Solani\u003c/em\u003e, \u003cem\u003ePyricularia oryzae, F. oxysporum\u003c/em\u003e, \u003cem\u003eand Sclerotium bataticola\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Diethyl phthalate produced by \u003cem\u003eStreptomyces\u003c/em\u003e strain has shown antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. 2-piperidinone, extracted from dried pomegranate peels, has demonstrated antimicrobial activity against \u003cem\u003ePseudomonas aeruginosa, Proteus mirabilis\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. 5-Pyrrolidino-2-pyrrolidone, an indole compound produced by the \u003cem\u003eBacillus\u003c/em\u003e strain, validates in plant growth-promotion in \u003cem\u003eGossypium hirsutum\u003c/em\u003e (cotton) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings validate the production of these secondary metabolites and could serve as both a natural biocontrol and a bio-stimulant agent.\u003c/p\u003e\u003cp\u003eGrowth optimization studies are of paramount importance in understanding the factors that affect bacterial growth and their metabolism, thereby enhancing the production of plant growth-promoting traits. To improve the production of plant growth-promoting (PGP) features, growth optimization studies are essential for comprehending the environmental and nutritional factors that affect bacterial metabolism and proliferation. The present investigation's optimisation tests demonstrated that the optimal physicochemical conditions for the growth of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 were 35\u0026deg;C, pH 7, 1% mannitol as the carbon source, and 0.5% beef extract as the nitrogen source. These conditions also markedly improved the production of important PGP characteristics (Fig.\u0026nbsp;3; Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eThe carbon supply had the greatest effect on developing PGP characteristics out of all the variables examined, highlighting its crucial significance above pH, temperature, and nitrogen availability. These conclusions are corroborated by earlier research showing that basic nutritional elements, including temperature, pH, and carbon sources, are important for bacterial metabolism and growth-promoting capacity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Significantly affecting the effectiveness of PGP are bacterial strains such as Rhizobium VMA 301, Bacillus, and \u003cem\u003eStreptomyces\u003c/em\u003e spp. that have shown a high sensitivity to environmental changes, particularly those related to pH and temperature [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2 demonstrated the ability to produce indole-3-acetic acid (IAA), a crucial auxin controlling plant growth and development. A quantitative study using L-tryptophan as a precursor showed that AVN2 generated 7.2 \u0026micro;g/mL of IAA under unoptimized conditions and 28.8 \u0026micro;g/mL following optimization. Furthermore, IAA's presence was verified by \u003cb\u003eHPLC quantification at 6.4 \u0026micro;g/mL and 0.70 m retention time\u003c/b\u003e (Fig.\u0026nbsp;5a). It's interesting to note that \u003cem\u003eC. citreum\u003c/em\u003e AVN2 produced IAA even when exogenous L-tryptophan was absent. This suggests that either endogenous tryptophan biosynthesis or a tryptophan-independent IAA synthesis pathway may be involved [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The strain's strong biosynthetic potential and ability to sustain plant development in nutrient-limited environments are highlighted by this metabolic flexibility. IAA is pivotal in regulating plant development, including cell division, elongation, and tissue differentiation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The root system enhances vascular tissue development, stimulates lateral and adventitious root formation, and improves nutrient and water uptake by reshaping root architecture [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Notably, the influence of IAA is often organ-specific and varies with the plant\u0026rsquo;s developmental stage [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], making it a critical regulator in plant\u0026ndash;microbe interactions.\u003c/p\u003e\u003cp\u003eAlthough endophytes are not typically recognised for gibberellin (GA) synthesis, several rhizobacteria\u0026mdash;including \u003cem\u003eRhizobium phaseoli\u003c/em\u003e, \u003cem\u003eBacillus pumilus\u003c/em\u003e, \u003cem\u003eB. licheniformis\u003c/em\u003e, \u003cem\u003eB. cereus\u003c/em\u003e, and \u003cem\u003eB. macrolides\u003c/em\u003e\u0026mdash;have been reported to produce gibberellins and related compounds such as GA₉, GA₂₀, GA₅, GA₈, GA₃₄, GA₄₄, and GA₅₃. In addition, multiple fungal endophytes, including \u003cem\u003eChrysosporium pseudomerdarium\u003c/em\u003e, \u003cem\u003eFusarium sacchari\u003c/em\u003e, \u003cem\u003eF. subglutinans\u003c/em\u003e, \u003cem\u003eF. konzum\u003c/em\u003e, \u003cem\u003eGibberella fujikuroi\u003c/em\u003e, \u003cem\u003ePhoma herbarum\u003c/em\u003e, and \u003cem\u003eScolecobasidium tshawytschae\u003c/em\u003e, have also been identified as GA producers [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn line with these findings, our study presents the \u003cb\u003efirst report of gibberellin production\u003c/b\u003e by the endophytic strain \u003cem\u003eC. citreum\u003c/em\u003e AVN2, isolated from the rhizome of \u003cem\u003eC. longa\u003c/em\u003e. The strain produced a \u003cb\u003enotable concentration of 29.49 \u0026micro;g/mL\u003c/b\u003e of gibberellin compound, as determined by HPLC analysis (Fig.\u0026nbsp;5b), expanding the current understanding of GA-producing endophytes and their potential applications in sustainable agriculture.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCurtobacterium citreum\u003c/em\u003e AVN2 produced 15.49 \u0026micro;g/mL of kinetin and 27.78 \u0026micro;g/mL of 6-benzyladenosine. \u003cem\u003eAzospirillum\u003c/em\u003e species have been reported to synthesise approximately \u003cb\u003e0.75 \u0026micro;g/mL of cytokinins\u003c/b\u003e and \u003cb\u003e20\u0026ndash;40 \u0026micro;g/mL of gibberellic acid\u003c/b\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Similarly, \u003cem\u003eBacillus\u003c/em\u003e sp. strain BPSAC6, isolated from \u003cem\u003eClerodendrum colebrookianum\u003c/em\u003e, was found to produce \u003cb\u003eindole-3-acetic acid (IAA) at 3.61 \u0026micro;g/mL\u003c/b\u003e, \u003cb\u003ekinetin at 6.2 \u0026micro;g/mL\u003c/b\u003e, and \u003cb\u003e6-benzyladenine (BA) at 7.9 \u0026micro;g/mL\u003c/b\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e BE 25, isolated from the roots of \u003cem\u003eMusa\u003c/em\u003e spp., demonstrated IAA production at \u003cb\u003e39.72 \u0026micro;g/mL\u003c/b\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In contrast, \u003cem\u003eBacillus\u003c/em\u003e sp., isolated from the rhizosphere of \u003cem\u003eCattleya walkeriana\u003c/em\u003e, produced \u003cb\u003e2.3 \u0026micro;g/mL of IAA\u003c/b\u003e [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Comparatively, the endophytic bacterium \u003cb\u003eC. citreum\u003c/b\u003e \u003cb\u003eAVN2\u003c/b\u003e has been reported to detect and quantify \u003cb\u003eIAA, GA, kinetin, and BA\u003c/b\u003e for the first time, highlighting the strain's distinctive characteristics and varied PGP potential.\u003c/p\u003e\u003cp\u003eUsing these endophytes to create bioformulations or biofertilizers can help control plant health and promote sustainable agriculture by providing environmentally benign substitutes for chemical inputs. Liquid bioformulations, sometimes called aqueous or flowable suspensions, have become powerful substitutes for conventional carrier-based microbial inoculants in recent years. These alternatives provide benefits like increased microbial load, extended shelf life, and simplicity of use. A liquid formulation of \u003cb\u003eC. citreum\u003c/b\u003e\u003cb\u003eAVN2\u003c/b\u003e was created and administered to tomato (\u003cb\u003eSolanum lycopersicum\u003c/b\u003e) seeds to evaluate its bioinoculant potential, with the \u003cb\u003eseedling vigour index, germination percentage (Gp), and germination index (Gi) rising by 81.6% and 54.5%\u003c/b\u003e, respectively, in comparison to the untreated control, the treatment resulted in a notable improvement in seedling performance (Table\u0026nbsp;3; Fig.\u0026nbsp;6). These results demonstrate how \u003cb\u003eC. citreum\u003c/b\u003e\u003cb\u003eAVN2\u003c/b\u003e-based liquid formulations have the potential to improve early seedling development and create strong crop stands. According to previous research [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], lactic acid bacteria from rhizospheric soil improved tomato seed germination by 6% and raised the seedling vigour index by 259. This highlights the relative efficacy of \u003cem\u003eC. citreum\u003c/em\u003e AVN2 as a strong bioinoculant. This highlights the relative efficacy of \u003cb\u003eC. citreum\u003c/b\u003e\u003cb\u003eAVN2 as a strong bioinoculant\u003c/b\u003e. Several plant growth parameters, such as germination percentage, seedling vigor, germination index, plant height, shoot height, root length, biomass accumulation, and primary metabolite content, were significantly enhanced in tomato seedlings using \u003cem\u003eC. citreum\u003c/em\u003e AVN2-based liquid bioformulation inoculation in the current study (Table\u0026nbsp;4). According to earlier publications, creating various plant growth-promoting (PGP) compounds by AVN2 is responsible for the observed boost in plant growth [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, releasing a wide range of antagonistic substances, including enzymes and antibiotics, probably suppresses phytopathogens [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAccording to [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], members of the Curtobacterium genus are known to produce a variety of PGP characteristics, including phytohormones, which greatly enhance the vigor and growth of inoculated agricultural plants. The present investigation has effectively isolated and identified \u003cem\u003eC. citreum\u003c/em\u003e AVN2, indicating that it can function as both a biocontrol agent and an endophyte that promotes plant growth. \u003cem\u003eC. citreum\u003c/em\u003e AVN2 showed strong antagonistic action against \u003cem\u003eFusarium oxysporum f. sp. Lycopersici\u003c/em\u003e, a serious pathogenic fungus that affects tomato crops. Greenhouse experiments also demonstrated its practical utility by confirming its effectiveness in boosting seed germination, seedling vigour, and inhibiting fungal infection. This early data shows the significance of AVN2 in combined crop improvement and disease management techniques. The results of this study are of great importance to the study of the mechanism of action and antagonism, which will lay the foundation for future applications in the biological control of plant growth and diseases.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eC. citreum\u003c/em\u003e AVN2 exhibits a wide range of plant growth-promoting (PGP) characteristics and strong antifungal activity against wilt-causing phytopathogens in vitro. This study is the first to report on the isolation of endophytic bacteria \u003cem\u003eC. citreum\u003c/em\u003e from the rhizome of \u003cem\u003eC. longa\u003c/em\u003e. Significant promise was shown in improving tomato seed germination, seedling vigor, and overall plant growth using the liquid bioformulation made with \u003cem\u003eC. citreum\u003c/em\u003e AVN2. These findings represent a promising step towards facilitating the development of non-toxic and ecologically friendly biofertilizers. When used as a liquid bioinoculant, \u003cem\u003eC. citreum\u003c/em\u003e AVN2 could be an excellent substitute for chemical inputs, supporting environmentally friendly and sustainable farming methods, especially when growing vegetable crops. Such bioformulations' economic and agronomic significance should be further enhanced by future studies concentrating on their mechanistic comprehension and field validation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis study was supported by a University Research Fellowship via reference number ANU/UCS/URF/Research Fellowship-2021\u0026ndash;2022 and 2022-23\u003c/p\u003e\n\u003ch2\u003eEthics statement\u003c/h2\u003e\n\u003cp\u003eThis article does not contain any studies on Human participants or animals performed by any authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSanneboyina NARMADA: Conceptualization, Methodology, Investigation, Supervision, Writing \u0026ndash; Original draft, Correspondence.Shaik. Mahekal KOUSAR: Formal analysis, data curation, funding acquisition, project administration, visualization, writing \u0026ndash; Original draft, writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\n\u003cp\u003eThe authors are thankful to the University Research Fellowship for Providing Financial support. Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll datasets generated or analysed during this study are included in the manuscript and supplementary information file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSavary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A (2019) The global burden of pathogens and pests on major food crops. 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Pharmacogn Rev 7(13):11\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4103/0973-7847.112833\u003c/span\u003e\u003cspan address=\"10.4103/0973-7847.112833\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Curtobacterium citreum, endophytic bacteria, Fusarium. oxysporum, plant growth-promoting traits, germination percentage, germination index, liquid bioformulation, HPLC analysis","lastPublishedDoi":"10.21203/rs.3.rs-7602699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7602699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Endophytic bacteria have become essential in promoting plant growth and development, and provide resistance against phytopathogens through diverse biochemical mechanisms. This study isolated an endophytic bacterium, AVN2, from Curcuma longa's rhizome and identified it as () using 16S rRNA partial gene sequencing. C. citreum AVN2 was deposited in NCBI with accession number PQ215941. The strain exhibited significant antagonistic activity against . Sp. , a major causative agent of tomato wilt, in a dual culture assay, and is confirmed as a biocontrol agent by GC-MS analysis of its secondary metabolites, Lupeol, 2-Piperidinone, Stigmastan-3,5-diene, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, 5-Pyrrolidino-2-pyrrolidone, Diethyl Phthalate, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-. Plant growth-promoting characterization revealed the production of important growth-promoting phytohormones, including indole compounds (6.40 \u0026micro;g/mL), gibberellic acids (29.49 \u0026micro;g/mL), and cytokinins (Kinetin 15.49 \u0026micro;g/mL, and 6-Benzyladenosine 27.78 \u0026micro;g/mL), which were quantified by HPLC analysis. Seed bacterization with . bioformulation enhanced the seed vigour index of tomato seeds compared to the control. Bio-formulated AVN2 significantly enhanced root and shoot growth, and overall biomass under greenhouse conditions, demonstrating biocontrol efficacy and plant growth-promotion (PGP) potential. This study presents the first evidence of as an endophyte in , exhibiting dual biofertilizer and bioprotectant properties. These findings underscore its promise as a sustainable, eco-friendly alternative to chemical fertilizers and fungicides in horticultural crop production.","manuscriptTitle":"Isolation and characterization of Plant growth-promoting hormones by an endophytic bacterium, Curtobacterium. citreum AVN2 isolated from the rhizome of Curcuma longa L","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 13:17:25","doi":"10.21203/rs.3.rs-7602699/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":"aafa90be-9948-450e-ba2d-a8d434d9f14e","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T14:39:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 13:17:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7602699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7602699","identity":"rs-7602699","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}