Biodegradable Hydrogel for Controlled Delivery of Biocontrol Bacteria and Micronutrients against Macrophomina phaseolina

Biodegradable Hydrogel for Controlled Delivery of Biocontrol Bacteria and Micronutrients against Macrophomina phaseolina | 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 Biodegradable Hydrogel for Controlled Delivery of Biocontrol Bacteria and Micronutrients against Macrophomina phaseolina Fatima Numan, Amna Shoaib, Humera Aslam Awan, Mahrukh Akram, Mahnoor Shahid This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9392239/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plant growth-promoting rhizobacteria (PGPR) represent a sustainable alternative for plant disease management; however, their field application is often limited by poor survival and inefficient delivery. In this study, a multifunctional biopolymer-based hydrogel composed of carboxymethyl cellulose and κ-carrageenan, crosslinked with citric acid, was developed as a carrier system for controlled delivery of PGPR and micronutrients. The engineered hydrogel exhibited high porosity (71.7%), rapid swelling capacity (2.5 g/g), and substantial biodegradability (80.2% within 14 days), following first-order degradation kinetics. Swelling behavior conformed to pseudo-second-order kinetics with Fickian diffusion as the dominant mechanism. The system demonstrated a biphasic release profile, achieving complete bacterial release within 24 h while maintaining ≥ 76% cell viability up to 35 days. Encapsulated PGPR showed strong antifungal activity, inhibiting Macrophomina phaseolina by up to 88%. Additionally, the hydrogel improved soil moisture retention and enhanced seed germination performance. Overall, the developed system integrates material functionality with microbial delivery efficiency, highlighting its potential as a biotechnological platform for improved PGPR-based disease management. Hydrogel micronutrients Controlled release Biopolymer processing Release kinetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Three Key Points Biodegradable hydrogel ensured rapid PGPR release with long-term bacterial viability. Encapsulated PGPR showed up to 88% inhibition of Macrophomina phaseolina . Hydrogel improved soil moisture, seed germination, and sustainable disease management. Introduction Soil-borne plant diseases remain a persistent constraint to global agriculture, particularly under climate stress conditions such as drought and rising temperatures (Sharma et al., 2025 ). Among these, Macrophomina phaseolina (Tassi) Goid is a highly aggressive necrotrophic fungus with an extensive host range exceeding 500 plant species, including cereals, legumes, oilseeds, and vegetables. The pathogen causes charcoal rot, stem rot, and root rot, characterized by tissue maceration, vascular occlusion due to abundant sclerotia formation, and eventual plant wilting and death (Sohaliya et al., 2025 ). Globally, M. phaseolina is responsible for severe yield losses ranging from 60–100% in crops such as soybean, maize, and cotton, with significant economic impact in tropical regions. In Pakistan, maize yield losses of 30–70% have been reported under field conditions (Khan et al., 2018 ; Ahmed & Shoaib, 2024 ). A major challenge in managing M. phaseolina lies in its ability to persist in soil through melanized sclerotia, which can survive for extended periods under adverse environmental conditions. These structures germinate upon encountering a suitable host, making disease control difficult even in well-managed cropping systems (Shirai & Eulgem, 2023 ). Conventional fungicides often provide inconsistent control and pose environmental and health risks. Moreover, their excessive use can disrupt soil microbiota and promote the development of resistant pathogen populations (Hossain et al., 2022 ). These limitations highlight the need for sustainable and biologically integrated disease management strategies. Plant growth-promoting rhizobacteria (PGPR) have emerged as promising biocontrol agents due to their ability to enhance plant growth and suppress pathogens. Certain Bacillus spp. exhibit strong antifungal activity through the production of antibiotics, lytic enzymes, and siderophores, which are effective against pathogens such as M. phaseolina (Bakr et al., 2025 ). In contrast, other PGPR, such as Klebsiella oxytoca , contribute primarily to plant growth through mechanisms including nitrogen fixation, phytohormone production, and improved nutrient availability (Liang et al., 2023 ; Jin et al., 2024 ). The use of functionally diverse microbial consortia can therefore provide a dual benefit by enhancing plant growth while simultaneously suppressing disease (Maciag et al., 2023 ). Micronutrients such as zinc (Zn) and iron (Fe) also play a critical role in plant defense responses, redox regulation, and microbial activity (Verma et al., 2021 ; Jeevanraj et al., 2025 ). However, their direct application in soil is often limited by leaching losses, uneven distribution, and rapid transformation, reducing their bioavailability (Salamat et al., 2021 ). Similarly, the effectiveness of PGPR in field conditions is frequently constrained by poor survival and limited establishment in the rhizosphere. These challenges necessitate the development of efficient carrier systems capable of ensuring sustained and targeted delivery of both beneficial microbes and micronutrients. Hydrogels derived from natural polymers, such as carboxymethyl cellulose (CMC) and κ-carrageenan, have gained considerable attention as potential delivery matrices due to their biodegradability, high water-retention capacity, and porous structure (Kang and Yun, 2022 ). Crosslinking with organic agents such as citric acid further enhances their structural stability while maintaining environmental compatibility (Khan et al., 2018 ). Such systems can provide a protective microenvironment for encapsulated microbes and enable controlled release of active components. Despite increasing interest in hydrogel-based delivery systems, limited studies have explored multifunctional formulations integrating PGPR consortia and micronutrients for the targeted management of M. phaseolina . Therefore, the present study aimed to develop and evaluate a CMC/κ-carrageenan-based hydrogel co-loaded with Bacillus spp., Klebsiella oxytoca , and micronutrients (Zn and Fe) as a sustainable delivery platform for enhanced microbial performance and effective suppression of M. phaseolina . Materials and Methods Microbial cultures Macrophomina phaseolina (Accession no. FCBP-PTF-1156) was procured as a pure culture, which was subsequently subcultured on 2% MEA and refrigerated at 4°C. Klebsiella oxytoca (KO), Bacillus sp. (BS1), and Bacillus velezensis (BV) bacterial strains were obtained from Decqan Biosciences, Lahore, Pakistan and grown on nutrient agar at 30 ± 2 C for 24–48 hours. Bio-compatibility assay The cross-streak procedure was employed to test compatibility among bacterial strains BV, BS1, and KO on nutrient aga (Raja et al., 2006 ). Each strain was streaked on nutrient agar (NA) plates in the center, and the other strains were streaked perpendicular to them after 24 hours of incubation at 28 ± 2°C. Plates were re-incubated for 24–48 hours to view interactions. Compatibility was assessed by simply viewing inhibition or overlap at streak junctions. Antifungal efficacy The bacterial lawn co-culture method was employed to determine the antifungal activity of bacterial isolates against M. phaseolina (Yadav et al., 2023). A uniform lawn was prepared by spreading 1 mL of each bacterial suspension (OD₆₀₀ = 1.0; ~10⁹ CFU/mL) uniformly over the surface of NA plates, incubating for 24 hours at 28°C. The center of every plate was then a 6 mm disc of mycelia from a 7-day-old culture of M. phaseolina grown on malt extract agar. Bacterial lawn-free control plates were maintained in parallel for comparison. Following seven days of incubation at 28 ± 2°C, the antifungal activity was measured by calculating the zone of inhibition and fungal growth inhibition on each plate. Formulation and stability evaluation of hydrogel Six bio-polymeric hydrogels were developed with zinc (Zn) and iron (Fe) supplementation, citric acid as a crosslinker, sodium carboxymethyl cellulose (NaCMC), κ-carrageenan (Car), and a bacterial consortium consisting of BV, BS1, and KO (Kang and Yun, 2022 ). NaCMC and Car were solubilized separately in autoclaved distilled water (60°C) and stirred for 30 minutes to become uniform. Citric acid was added as a crosslinking initiator for increasing the gel strength, and Zn and Fe salts for micronutrient supplementation. Standardized bacterial suspensions (OD₆₀₀ = 1.0, ~ 10⁹ CFU/mL) were aseptically added to the cooled mixture and stirred gently to promote entrapment. The gel was poured (10 mL) into sterile plastic moulds and dried overnight at 45 C to obtain uniform hydrofilms. Stability of the hydrogels was assessed by soaking 0.5 g of each dried film in 10 mL of distilled water at room temperature (25 ± 2°C) and noting the time to complete disintegration. Out of the six formulations screened, the hydrogel (HG 6 ) with maximum stability in water was selected. Hydrogel characterization Gel cross-linked proportion analysis The hydrogel cross-linked proportion was measured using the tea bag method by Uyanga et al. ( 2020 ) and calculated gel fraction percentage to check the extent of crosslinking and structural stability. Swelling behavior and kinetic modeling of hydrogel Swelling dynamics of the best optimized hydrogel formulation (HG 6 ) were gravimetrically investigated by soaking 0.2 g dry film in 10 mL autoclaved distilled water at room temperature (Hüther et al., 2004 ). The swelling percentage was assessed on regular intervals, and by fitting the data to a pseudo-second-order model, the swelling rate and mechanism was determined. The Higuchi model determines Fickian diffusion properties, while the Korsmeyer-Peppas model determines the diffusional exponent and release mechanism (Dal and Onursal, 2023 ). Swelling response of the hydrogel was assessed under ionic conditions and pH changes, hydrogel films were submerged for 10 minutes at room temperature in buffer solutions with pH values of 4, 6, and 8, as well as in NaCl and CaCl₂ solutions with concentrations ranging from 0.1 to 0.9 M (Azeem et al., 2023 ). For all the experiments, the swelling ratio was calculated by the same gravimetric equation. For measurements of network structure and mechanical strength, polymer volume fraction (V₂) was determined according to Peppas and Merrill et al. (1977) method and crosslink density (Vₑ) was calculated according to Flory-Rehner theory (1943) using χ = 0.49 for water-polymer systems and V₁ as the molar volume of water. Porosity and moisture retention testing Hexane was employed for the liquid displacement procedure to measure porosity (Qazi et al.,2022). Empirical parameters (n and m), to quantify moisture retention and sensitivity of hydrogel in soil conditions, were used to fit the data to the van Genuchten model (1980), including residual and saturated water content, to try and investigate moisture retention behavior. Soil water retention analysis For water retention determination in the soil, 100 g of soil was mixed with 1 g of hydrogel, and 60 mL of water was used to water them; weight loss was measured each day. Cumulative water loss and water retention efficiency were determined, as by Thombare et al. ( 2018 ), compared to untreated controls. Biodegradability assessment Biodegradability was determined using the soil burial technique (Dharmalingam et al.,2016), 1 g films of hydrogel were buried 5 cm deep in soil and weight loss was measured over a period of 14 days. First-order kinetics was simulated, and half-life (t₁ / ₂) was calculated (Kamenova et al., 2024 ). Bacterial release The release of bacteria from 0.5 g hydrogel was tracked in phosphate buffer solution at pH 7.0 under 25 ± 1 C conditions over 24 hours by monitoring OD. Cumulative release (%) was determined from the maximum OD₆₀₀ at regular intervals (Mohamadzadeh et al., 2025 ).. Viability assessment A suspension of 1 g of bacteria loaded hydrogel in 10 mL of sterile saline was vortexed to test for microbial viability. CFUs were measured after serial dilutions (10⁻¹ to 10⁻⁶) were plated on nutrient agar and incubated for 24–48 hours at 28–30°C. Viability was tracked for 60 days at 7-day intervals to determine shelf life and microbial stability (Mohamadzadeh et al., 2025 ). Optical analysis Morphological variations in dried and swollen hydrogels were studied under a compound light microscope (Labomed LX400) with 40× magnification. Samples were imaged prior to and following equilibrium swelling for evaluating structural variations (Gull et al., 2019 ). Antifungal potential of hydrogel against M. phaseolina Antifungal activity of hydrogel formulations at 0.1, 0.5, and 1% (w/v) concentrations was tested against M. phaseolina with a broth macro-dilution assay (Kabiru et al., 2023 ). Mycelial discs (6 mm) of 7-day-old cultures of fungi were inoculated into 10 mL sterile 2% malt extract hydrogel medium. A control was given 1 mL PBS instead of hydrogel. All treatments were incubated under controlled conditions for 7 days (28 ± 2°C). OD₆₀₀ was measured, and fungal biomass was filtered through pre-weighed Whatman No. 1 filter papers. Fresh weight was determined immediately, and dry weight was from drying at 60°C for 48 h. Antifungal activity as a percentage inhibition of fungal biomass compared to the control not treated was computed. Seed germination assays Dk 6321 or Dekalb 6321 maize seed variety was obtained from Bayer, Pakistan. A 1% NaOCl solution was used to surface sterilize the seeds for three to four minutes. Three layers of sterilized blotter were arranged in pre-sterilized Petri dishes, and seven seeds per plate were placed in it. Two treatments with three replicates were implied for this experiment, one was control (without hydrogel) and the other was with hydrogel embedded in the layers of blotter paper. Regular irrigation and observations were made during the six-day experiment. The data for percentage of germination was calculated (Siddiqui et al., 2023 ). The following additional formulas were used to evaluate the germination characteristics of maize seeds: $$\:Hydrogel\:Efficiency\:ratio=\:\frac{Germination\:rate\:with\:hydrogel}{Germination\:rate\:in\:control}\:$$ $$\:Germination\:index\:=\:\frac{\sum\:No.\:of\:seeds\:germinated\:in\:a\:day}{Total\:no.\:of\:days\:for\:germination}\:\times\:100$$ $$\:Mean\:germination\:time\:\left(MGT\right)\:=\:\sum\:\left(\frac{No.of\:seeds\:germinated\:in\:a\:day\:\times\:\:Day\:no.}{Total\:no.\:of\:seeds\:germinated}\right)$$ $$\:Gernimation\:velocity=\:\frac{1}{MGT}$$ $$\:Garmination\:rate=\frac{Total\:no.of\:germinated\:seeds}{Total\:no.\:of\:days\:for\:germination}\:\times\:100$$ Statistical analysis All graphs were created using Microsoft Excel, and the data were modeled using kinetic models and equations. Standard error was calculated to represent variability. A least significant difference (LSD) test was also applied using STATISTIX software to determine the statistical significance among treatment means. Results Bacterial compatibility and antifungal bioassays Each bacterial isolate grew independently in control and the cross streaked bacterial strains showed no visible zones of inhibition at intersection point in interaction plates, showing mutual compatibility and absence of any antagonistic (Fig. S1 ). Bacterial lawn assay on 2% MEA indicated that Bacillus spp. (BV and BS1) restricted the radial growth of fungus by 80% and 83% respectively, with distinct inhibition zones of 6 mm and 4 mm, respectively. On the other hand, KO, showed poor antifungal activity with 7% mycelial inhibition and a 1 mm zone of inhibition (Fig. S2). Hydrogel aqueous stability evaluation Among the six hydrogels (HG 1 -HG 6 ), the maximum structural stability was seen for HG 6 (lasted for 24 h). This one used the optimized amount of polymer (0.75 g/L Car, 1 g/L NaCMC), higher dose of crosslinker (0.75 g/L citric acid), micronutrient supplementation (Zn and Fe at 5 mg/L each), and full microbial consortium (3 × 10⁹ CFU/mL) for further bioassays (Table S1 ). Gel fraction composition The synthesized hydrogel (HG 6 ) had a gel fraction of 84%, determined from its fresh and dry weights (0.2 g and 0.174 g, respectively), which reflects high network integrity after swelling (Fig. S3). Swelling behavior and kinetic modeling of the hydrogel Swelling in water and kinetic modeling The rapidly swelling initial response of the synthesized hydrogel reached almost-equilibrium in 110 minutes and leveled off thereafter, with the highest equilibrium swelling ratio of 2.50 g/g (Fig. 1 A). Pseudo-second-order kinetic modeling showed good correlation with the experimental data (R² = 0.998), validating chemisorption as the prevailing swelling mechanism. The predetermined rate constant (k = 0.0253 g/g min) indicates a mid-range swelling rate appropriate for controlled water release (Fig. 1 B). Additional comparison with the Higuchi model (R² = 0.85) and the Korsmeyer–Peppas model (R² = 0.92; n < 0.5) confirmed Fickian diffusion as a major mechanism of water uptake (Table S2). Swelling behavior in electrolyte solution In NaCl, the maximum swelling (415%) occurred at 0.3 M, while CaCl₂ had a maximum of 390% at 0.5 M (Fig. 2 ). In both cases, higher concentrations led to lower swelling, which reflects ionic crosslinking and compaction of the network at higher salinity. This was measured by calculated crosslinking volume (V c ), with the lowest values at optimal salt concentrations (NaCl: 3032 cm³/mol at 0.3 M; CaCl₂: 2613 cm³/mol at 0.5 M), affirming maximum network relaxation and expansion at intermediate ionic strength (Fig. 5 B). Associated polymer volume fractions (V₂) were also lowest at these concentrations (NaCl: 0.19; CaCl₂: 0.20), indicating a more open hydrogel matrix for water imbibition (Fig. 5 C). These observations indicate that the hydrogel works best at moderate salinity, as in the case of Fickian-controlled release behavior appropriate for saline or dryland soils (Table 3S). Swelling at different pH levels The hydrogel exhibited pH-sensitive swelling, with the highest uptake (390%) at pH 6. The swelling was found to reduce under acidic (305% at pH 4) and alkaline (370% at pH 8) environments (Fig. 3 ). This trend was associated with network structure changes, with lowest crosslink density (Vₑ = 0.035 mol/cm³) and polymer volume fraction (V₂ = 0.20) at pH 6. Greater Vₑ and V₂ at pH 4 and 8 implied denser gel networks, which accounted for the reduced water absorption. Moisture absorption and porosity estimation The hydrogel had a porosity of 71.7% and a swelling ratio of 3.05, with internal pore capacity determined to be 1.3 mL through hexane displacement. The range of pore size estimated to be around 50–200 µm is ideal for water retention and slow release in soil use. The results affirm the gel's ability to store and release moisture slowly, improving soil water-holding potential at the most agronomical appropriate range for plant uptake. The soil-amended hydrogel showed enhanced water retention throughout, with volumetric water content rising steadily by 1.6-2.0% within the 10–100 cm suction range over the untreated soil, and a highest enhancement of 3.18% at 40–50 cm suction as per van Genuchten model analysis (Fig. 4 ; Table S4). At equilibrium (0 cm) moisture content increased slightly from 0.45–0.457 cm³/cm³, whereas effectiveness reduced at suctions ≥ 200 cm, reflecting compromised moisture availability under extreme drought conditions. Soil water retention Hydrogel treatment dramatically decreased moisture loss rate from soil over a 10-day dry-down period (Fig. 5 ). The control and hydrogel-amended soils had practically identical initial weights (150 g and 151 g, respectively), by day 10 the hydrogel-treated soil held more water (116 g) than the control (111 g), corresponding to 77% vs. 74% moisture retention. The water loss was 38.90 g in 10 days in control, whereas for the hydrogel-treated sample, 35.07 g were lost. Retention efficiency was also consistently high in hydrogel-added soil, at greater than 104% starting from day 4 and as high as 105% on day 8. The largest differences in percentage for water retention were between days 3 to 6, with the hydrogel treatment holding 2–3% more water compared to the control. Treated soil held 76.8% of initial water compared to 74.1% in the control by day 10. Biodegradability test in soil The burial test in soil showed that the hydrogel was readily biodegradable in natural conditions as there is progressive loss of weight over 14 days (Fig. 6 A). There was an initial 9% degradation in the first day, which reflected early commencement of biodegradation. There then followed a fast stage of degradation on days 3–7 when there was a progressive increase in biodegradability from 26.7% to 53.1%. After day 7, degradation proceeded at a sluggish but linear rate to 80.2% cumulative degradation by day 14. Kinetic modeling validated that degradation was first order, with a rate constant (k) of 0.117 day⁻¹ and a half-life (t₁ / ₂) of 5.93 days. The fit of the model was excellent (R² = 0.99), validating a systematic and exponential trend of decomposition with respect to time (Fig. 6 B). Bacterial release kinetics Release kinetics of bacterial cells from hydrogel was a distinct biphasic kinetic pattern within 24 hours (Fig. S4). There was a lag phase with 8% and 16% cumulative release at 1 and 2 hours, respectively, which was followed by a rapid exponential release to 86% at hour 8. A gradual plateau phase followed with ultimate release (100%) at 24 hours. Microbial viability Viability assays verified that the bacterial populations were well preserved in the hydrogel matrix on storage. From an initial cell count of 3.27 × 10⁹ CFU mL⁻¹, cell counts were relatively constant, declining to a small degree only to 2.61 × 10⁹ CFU mL⁻¹ (79.8%) at 7 days. Between 14–35 days, viable counts varied between 2.48 × 10⁹ and 2.80 × 10⁹ CFU mL⁻¹, corresponding to 76–86% retention. Even after 35 days, viability was good at 2.64 × 10⁹ CFU mL⁻¹ (80.7%), with only a 0.11 log₁₀ unit loss (Fig. 7 ). Optical observations Optical microscopy of swollen and dry hydrogel also supported these observations. Dry hydrogel contained a compact and uniform polymer network (Fig. 8 A). After swelling, the matrix swelled and exhibited well-defined, interconnected pores (Fig. 8 B), giving the structure a porous nature ideal for water uptake and microbial encapsulation. Anti-fungal analysis of the bioformulation Antifungal potential of the hydrogel was tested against M. phaseolina with different concentrations (0.1, 0.5, and 1.0% w/v). Morphologically, increasing distortion and curling of hyphae with increasing concentration of hydrogel were noted which exhibited inhibitory activities towards fungal growth. The fungus biomass reduction and inhibition of corresponding mycelial growth was 11, 34, and 88%, thereby confirming the high antifungal activity of hydrogel at high concentrations (Fig. 9 ). Germination bioassays The germination activity of seeds treated with the hydrogel formulation was compared with untreated control seeds for 6 days. Quantitative data (Table 4S) indicated that hydrogel-treated seeds germinated at 100%, whereas the control germinated to 90% on day 6. Mean germination time (MGT) was significantly lower in the hydrogel treatment (2.431 days) than for the control (3.006 days), reflecting a faster process of germination. As a result, the germination speed, the inverse of MGT, was also increased in the hydrogel-treated group (0.411) over that in the control (0.333), establishing increased germination kinetics. The germination index (GI), a blended parameter of germination rate and synchronicity, was significantly augmented in hydrogel treatment (12.44) compared to the control (9.35), suggesting both quicker and synchronized germination. Moreover, hydrogel efficiency ratio in final germination was 1.11, further highlighting the effectiveness of the hydrogel in triggering early seed emergence. Discussion The present study investigated the physicochemical characteristics, bio-efficacy against M. phaseolina , and maize growth promotion of a multi-functional hydrogel (HG 6 ) made from CMC/κ-carrageenan crosslinked with citric acid with Zn²⁺, Fe³⁺, and a bacterial consortium ( Bacillus velezensis , Bacillus sp., and Klebsiella oxytoca ). Compatibility tests established that the strains of the bacteria were mutually non-antagonistic, a necessary condition for effective co-formulation and synergistic bioactivity (Swiontek et al., 2022). In antifungal tests, Bacillus strains expressed high antagonism (≥ 80% inhibition), which resulted most probably from lipopeptide production (iturins, fengycins, surfactins) and hydrolytic enzymes (Zhou et al., 2022 ; Hong et al., 2022 ). K. oxytoca , although weakly antagonistic, has applications due to its plant growth-promoting features such as nitrogen fixation and phytohormone production (Youseif et al., 2025 ). HG 6 exhibited the maximum structural integrity (24 h) among all formulations. This was a result of synergistic interactions of bicomponent polymers, high citric acid crosslinking, metal ion coordination, and bacterial encapsulation (Trivedi et al., 2023; Zhou et al., 2024 ). Porosity (71.7%) and intense crosslinking (84% gel content) were proposed for a macroporous matrix that can transport microorganisms and retain water in soils (Guilherme et al., 2015 ). Water swelling exhibited pseudo-second-order kinetics (R 2 = 0.998) and reached equilibrium (2.5 g/g) in 2 h, which is typical of hydrogels that are chemisorption dominant. Electrolytes and pH influenced swelling responses: a dose of moderate amount of NaCl and CaCl₂ promoted absorbance, but excessive salinity or pH reduced it, as Flory's theory and ionic crosslinking character of divalent cations would predict (Landsgesell et al., 2023). Swelling was maximal at pH 6, which is the same as the pH of most agricultural soils, with increased availability of nutrition and conditioning of the soil. Soil water retention experiments indicated that hydrogel-modified soils stored 1.6–3.2% more water by volume in the suction range of 0-100 cm. Maximum improvement was noticed at 40–50 cm suction-it is essential for root uptake by plants. Weight-loss measurement indicated 77% water retention in hydrogel-amended soil on day 10 compared to 74% in control, confirming its effectiveness in maintaining soil water (Abdelghafar et al., 2024 ). Capacity for water availability extension justifies its use to counter drought stress in dry environments. Biodegradation tests demonstrated gradual degradation with 80.2% loss in mass after 14 days and a half-life of 5.9 days, following first-order kinetics (R² = 0.99). This confirms the environment-sensitive degradation of the hydrogel without any formation of residues to warrant its application as a safe soil amendment (Kamenova et al., 2024 ). Biphasic release profiles for bacteria were observed, with 86% of the release at 8 hours and complete release at 24 hours. The controlled release is a result of network relaxation due to swelling in the hydrogel, which maximizes the dispersion of microbes (Mo et al., 2022 ). Microbial viability was relatively high (> 76%) up to 35 days, indicating that the hydrogel is effective in protecting bacterial cells and enabling long-term delivery under field conditions (Lima-Tenório et al., 2024 ). Microscopy established an expanded pore structure upon swelling with pores of 50–200 µm diameter to facilitate microbial diffusion and water retention capacity (Montesano et al., 2015 ; Lin et al., 2023 ). The hydrogel also exhibited inherent antifungal activity in inhibiting mycelial biomass by 88% using 1% (w/v) concentration. This may be attributed to acidity derived from citric acid and the inherent antifungal character of Zn²⁺ ions (Korbecka-Glinka et al., 2022 ), by a dual action mechanism. Collectively, these results determine the universal applicability of the Zn/Fe-bacteria enriched hydrogel as a multi-functional, eco-friendly platform for promoting plant growth and disease control. Its biodegradability, water-holding capacity, and site-directed delivery of microbes solve the biggest challenges in sustainable crop management against biotic and abiotic stresses. The hydrogel itself exhibited strong concentration-dependent antifungal activity against M. phaseolina in vitro . Hydrogel treated hyphae became deformed even at 0.1 and 1%, mycelial biomass was reduced by 88% compared to control. This may be attributed to several reasons: the citric acid crosslinker has the potential to acidify the microenvironment, and the released Zn²⁺ is already reported to possess antifungal activity (Korbecka-Glinka et al., 2022 ). Overall, the results show that the developed hydrogel is a stable, slow-release carrier for bacterial biocontrol and micronutrient supply, improving soil water holding capacity and having direct antifungal action. These results validate its potential as a biodegradable soil amendment for integrated disease management and promotion of plant growth. The germination test showed clearly that the maize seeds germinated more effectively when the hydrogel was used compared to the control. All the seeds treated with the hydrogel germinated, in contrast to the control group showed slightly lower 90% germination. Not only germinated more seeds but even quicker-the average time of germination was less, and both vigor and germination percentage were greatly improved. This increase is probably because hydrogel can hold water and seep it out slowly around the seeds, a uniformly wet condition to aid early metabolic activity (Montesano et al., 2015 ; Agbna and Zaidi, 2025 ). This consistent supply of moisture is particularly critical under arid or abnormal conditions, when water stress will inhibit or retard germination. Comparable findings have been reported from other research using superabsorbent hydrogels, which assist seeds by supplying the surrounding soil with the exact amount of water to activate enzymes and promote development (Qureshi et al., 2022 ). Together, these results suggest the potential of the hydrogel as a simple but efficient method of stimulating germination and subsequent seedling viability. Conclusion This study successfully designed and explored an eco-friendly hydrogel-based bioformulation for microbial delivery, fortified with Zn and Fe, for management of charcoal rot disease. Bacillus velezensis and Bacillus sp. showed effective antifungal activity against M. phaseolina , with more than 80% inhibition, and all tested strains were mutually compatible for co-formulation. The optimized hydrogel formulation (HG 6 ) had excellent physicochemical stability, facilitating effective encapsulation of microbes and long-term soil delivery. Quick swelling capacity (2.5 g/g equilibrium in 2 hours), high gel content (84%), and pH- and salinity-sensitive water absorbency (up to 390%) established its high-strength structure and water-retention capacity. HG 6 improved soil moisture retention by 1.6–3.2% within the plant-available range and minimized water loss over 10 days, substantiating its function in drought stress mitigation. The hydrogel supported biphasic, controlled release of bacteria with 86% release within 8 hours and complete release in 24 hours, and 76–86% viability of bacteria for 35 days. It is also biodegradable up to 80.2% in 14 days, making it safe to the environment, which supports its use as a comprehensive, dual-purpose bioformulation for suppressing disease. Declarations Acknowledgments University of the Punjab, Lahore, Pakistan is thanked for providing facilities to accomplish the task. Author contribution F. N. Performed experiments, collected data and drafted the manuscript; A. S. Designed and supervised the experiments, Prepared graphs and revised the manuscript; H. A. A . Bacteria provision and cultivation; M. K : Methodology ; M.S : Methodology All authors have substantial contributions to the final manuscript and approved this submission. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval All procedures in this experiment were carried out in accordance with relevant guidelines of the university field of the University of the Punjab, Lahore, Pakistan. 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Front Microbiol 13:983781. https://doi.org/10.3389/fmicb.2022.983781 Zhou Q, Yang W, Lu S, Puglia D, Gao D, Xu P, Ma P (2024) Constructing robust and recyclable self-powered polysaccharide-based hydrogels by adjusting Zn 2+ /Li + bimetallic networks. Green Chem 26(8):4609–4621. https://doi.org/10.1039/D3GC05109A Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTARYFILE.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9392239","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634841514,"identity":"e066a9d5-2350-4641-9e4b-e823054d4d8d","order_by":0,"name":"Fatima Numan","email":"","orcid":"","institution":"University of the Punjab","correspondingAuthor":false,"prefix":"","firstName":"Fatima","middleName":"","lastName":"Numan","suffix":""},{"id":634841515,"identity":"c0e13586-7526-49f1-8a4a-fead1f865050","order_by":1,"name":"Amna Shoaib","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3OsQrCMBCA4ROhLgXXQNW+wklBBKG+SkqgXUpxFBTMVEc3wclX8BEUoS7tXsgzCB0dRLz2AWLdBPPDwS3fJQAm08+GND1Zb10aqy2xz1+ROsZbkv6+uFSw8P3j4Z4xWM4C6aRcf1slggEKcVJJyCCPAjnIzvpnVIxEugKdeMI66TWQLJJa4arYewBuhHvIibxaEFR0HPDqQ2kTkTUJ9R8bE5lyvHHMEzHlWeSlg4xryYg+VlbP1dzdFpeyWs+GOydFLWmiq4EEu1nAYp9B0xxq0tSWmEwm09/0BsaUQoFxnQzsAAAAAElFTkSuQmCC","orcid":"","institution":"University of the Punjab","correspondingAuthor":true,"prefix":"","firstName":"Amna","middleName":"","lastName":"Shoaib","suffix":""},{"id":634841516,"identity":"7dd04a03-3e6b-423f-8a0a-1999b35a7470","order_by":2,"name":"Humera Aslam Awan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Humera","middleName":"Aslam","lastName":"Awan","suffix":""},{"id":634841517,"identity":"32fb3c37-ebc5-4730-aa46-319fc2b51e16","order_by":3,"name":"Mahrukh Akram","email":"","orcid":"","institution":"University of the Punjab","correspondingAuthor":false,"prefix":"","firstName":"Mahrukh","middleName":"","lastName":"Akram","suffix":""},{"id":634841518,"identity":"ff7f50e5-6420-4ad9-a05a-963de40470a4","order_by":4,"name":"Mahnoor Shahid","email":"","orcid":"","institution":"University of the Punjab","correspondingAuthor":false,"prefix":"","firstName":"Mahnoor","middleName":"","lastName":"Shahid","suffix":""}],"badges":[],"createdAt":"2026-04-12 07:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9392239/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9392239/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108805768,"identity":"ec27e7b2-4235-4c6e-ad94-dc2385301aad","added_by":"auto","created_at":"2026-05-08 15:26:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A \u0026amp; B)\u003c/strong\u003e: Swelling behavior and Pseudo-second-order kinetic analysis of hydrogel. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3 × 10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/bd17025e6be92c6b5aa1e0e7.png"},{"id":108633858,"identity":"b6925f13-aed9-427c-87ba-0befcf8fa858","added_by":"auto","created_at":"2026-05-06 17:15:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A-C):\u003c/strong\u003e Swelling behavior and network characteristics of the hydrogel in different electrolyte solutions. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3×10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/a5b4f3589c1ecfcc23586403.png"},{"id":108806207,"identity":"01ff3488-b419-4948-9d46-36fa3e39fe05","added_by":"auto","created_at":"2026-05-08 15:27:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":340647,"visible":true,"origin":"","legend":"\u003cp\u003eSwelling behavior, polymer volume fraction (V\u003csub\u003e₂\u003c/sub\u003e), and crosslink density (V\u003csub\u003eₑ\u003c/sub\u003e) of the hydrogel at different pH levels. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3×10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/c731f36fa34db40d29aff007.png"},{"id":108633860,"identity":"94d582ba-851c-4f27-95d6-46ef06d0d3b6","added_by":"auto","created_at":"2026-05-06 17:15:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":310772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSoil moisture retention\u003c/strong\u003e and predicted architecture of hydrogel. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3×10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/142197e74bcedb1762a105b7.png"},{"id":108806349,"identity":"c43d17a9-6eaf-4572-bd78-bc894bbc7022","added_by":"auto","created_at":"2026-05-08 15:28:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":92616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A \u0026amp; B):\u003c/strong\u003e Soil water retention over 10 days in control and hydrogel-treated conditions. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3×10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/70978aff713fd380fb7a64a9.png"},{"id":108806081,"identity":"7fcc65a2-4baf-42b9-8004-62f070c4318c","added_by":"auto","created_at":"2026-05-08 15:27:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A \u0026amp; B): \u003c/strong\u003eBiodegradation percentage of hydrogel after being placed in soil for 14 days and First-order kinetic model fit to the biodegradation data. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3×10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/6178c663428b8fe7fd0ba371.png"},{"id":108805549,"identity":"3c457a69-74ed-4c8f-b6c0-1a91cee02795","added_by":"auto","created_at":"2026-05-08 15:26:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":22515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMicrobial viability of encapsulated bacteria in hydrogel over 35 days of storage at ambient conditions.\u003c/em\u003e Colony-forming units (CFU mL⁻¹) were recorded at 7-day intervals using the plate count method. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3 × 10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/25a45f89626415ada6df0e97.png"},{"id":108633863,"identity":"cd708f75-7502-4c1e-a517-61dbb8383bf6","added_by":"auto","created_at":"2026-05-06 17:15:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":297228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A-B):\u003c/strong\u003e Microphotographs of hydrogel. Compact cross-linked void microstructures in dried hydrogels (A) and increased pore size in swollen hydrogels (B) under a compound microscope at 40 × resolution. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3 × 10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/fc6091825ec6979bf1553ccf.png"},{"id":108633865,"identity":"df005798-5d46-4409-b094-31dae0cb25ab","added_by":"auto","created_at":"2026-05-06 17:15:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":421596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A-C):\u003c/strong\u003e Antifungal potential of hydrogel against \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e. Colony morphology (A). hyphal structure changes (B), and mycelial growth inhibition (C), after incubation for 7 days at 30 ℃. The formulation included carrageenan (0.75% w/v), citric acid (0.75% w/v), sodium carboxymethyl cellulose (1% w/v), iron (0.005% w/v), zinc (0.005% w/v), and bacteria (3×10\u003csup\u003e9\u003c/sup\u003e CFU).\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/e35cb3547742a03f737ca002.png"},{"id":108809921,"identity":"7d3e2dd2-dde5-4a7a-b589-79f733acf2ae","added_by":"auto","created_at":"2026-05-08 15:56:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1910660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/a0e19454-c973-40d0-8d6b-3d218005446d.pdf"},{"id":108633855,"identity":"b4f1f9f7-80de-4131-9263-a9c8ed99be7d","added_by":"auto","created_at":"2026-05-06 17:15:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":740699,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFILE.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9392239/v1/ce966d5502bb62e3ee7ffb2b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biodegradable Hydrogel for Controlled Delivery of Biocontrol Bacteria and Micronutrients against Macrophomina phaseolina","fulltext":[{"header":"Three Key Points","content":"\u003col\u003e\n \u003cli\u003eBiodegradable hydrogel ensured rapid PGPR release with long-term bacterial viability.\u003c/li\u003e\n \u003cli\u003eEncapsulated PGPR showed up to 88% inhibition of \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eHydrogel improved soil moisture, seed germination, and sustainable disease management.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eSoil-borne plant diseases remain a persistent constraint to global agriculture, particularly under climate stress conditions such as drought and rising temperatures (Sharma et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Among these, \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e (Tassi) Goid is a highly aggressive necrotrophic fungus with an extensive host range exceeding 500 plant species, including cereals, legumes, oilseeds, and vegetables. The pathogen causes charcoal rot, stem rot, and root rot, characterized by tissue maceration, vascular occlusion due to abundant sclerotia formation, and eventual plant wilting and death (Sohaliya et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Globally, \u003cem\u003eM. phaseolina\u003c/em\u003e is responsible for severe yield losses ranging from 60\u0026ndash;100% in crops such as soybean, maize, and cotton, with significant economic impact in tropical regions. In Pakistan, maize yield losses of 30\u0026ndash;70% have been reported under field conditions (Khan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ahmed \u0026amp; Shoaib, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA major challenge in managing \u003cem\u003eM. phaseolina\u003c/em\u003e lies in its ability to persist in soil through melanized sclerotia, which can survive for extended periods under adverse environmental conditions. These structures germinate upon encountering a suitable host, making disease control difficult even in well-managed cropping systems (Shirai \u0026amp; Eulgem, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Conventional fungicides often provide inconsistent control and pose environmental and health risks. Moreover, their excessive use can disrupt soil microbiota and promote the development of resistant pathogen populations (Hossain et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These limitations highlight the need for sustainable and biologically integrated disease management strategies.\u003c/p\u003e \u003cp\u003ePlant growth-promoting rhizobacteria (PGPR) have emerged as promising biocontrol agents due to their ability to enhance plant growth and suppress pathogens. Certain \u003cem\u003eBacillus\u003c/em\u003e spp. exhibit strong antifungal activity through the production of antibiotics, lytic enzymes, and siderophores, which are effective against pathogens such as \u003cem\u003eM. phaseolina\u003c/em\u003e (Bakr et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, other PGPR, such as \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e, contribute primarily to plant growth through mechanisms including nitrogen fixation, phytohormone production, and improved nutrient availability (Liang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jin et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The use of functionally diverse microbial consortia can therefore provide a dual benefit by enhancing plant growth while simultaneously suppressing disease (Maciag et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicronutrients such as zinc (Zn) and iron (Fe) also play a critical role in plant defense responses, redox regulation, and microbial activity (Verma et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jeevanraj et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, their direct application in soil is often limited by leaching losses, uneven distribution, and rapid transformation, reducing their bioavailability (Salamat et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, the effectiveness of PGPR in field conditions is frequently constrained by poor survival and limited establishment in the rhizosphere. These challenges necessitate the development of efficient carrier systems capable of ensuring sustained and targeted delivery of both beneficial microbes and micronutrients.\u003c/p\u003e \u003cp\u003eHydrogels derived from natural polymers, such as carboxymethyl cellulose (CMC) and κ-carrageenan, have gained considerable attention as potential delivery matrices due to their biodegradability, high water-retention capacity, and porous structure (Kang and Yun, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Crosslinking with organic agents such as citric acid further enhances their structural stability while maintaining environmental compatibility (Khan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Such systems can provide a protective microenvironment for encapsulated microbes and enable controlled release of active components.\u003c/p\u003e \u003cp\u003eDespite increasing interest in hydrogel-based delivery systems, limited studies have explored multifunctional formulations integrating PGPR consortia and micronutrients for the targeted management of \u003cem\u003eM. phaseolina\u003c/em\u003e. Therefore, the present study aimed to develop and evaluate a CMC/κ-carrageenan-based hydrogel co-loaded with \u003cem\u003eBacillus\u003c/em\u003e spp., \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e, and micronutrients (Zn and Fe) as a sustainable delivery platform for enhanced microbial performance and effective suppression of \u003cem\u003eM. phaseolina\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial cultures\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e (Accession no. FCBP-PTF-1156) was procured as a pure culture, which was subsequently subcultured on 2% MEA and refrigerated at 4\u0026deg;C. \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e (KO), \u003cem\u003eBacillus\u003c/em\u003e sp. (BS1), and \u003cem\u003eBacillus velezensis\u003c/em\u003e (BV) bacterial strains were obtained from Decqan Biosciences, Lahore, Pakistan and grown on nutrient agar at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2 C for 24\u0026ndash;48 hours.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBio-compatibility assay\u003c/h3\u003e\n\u003cp\u003eThe cross-streak procedure was employed to test compatibility among bacterial strains BV, BS1, and KO on nutrient aga (Raja et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Each strain was streaked on nutrient agar (NA) plates in the center, and the other strains were streaked perpendicular to them after 24 hours of incubation at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Plates were re-incubated for 24\u0026ndash;48 hours to view interactions. Compatibility was assessed by simply viewing inhibition or overlap at streak junctions.\u003c/p\u003e\n\u003ch3\u003eAntifungal efficacy\u003c/h3\u003e\n\u003cp\u003eThe bacterial lawn co-culture method was employed to determine the antifungal activity of bacterial isolates against \u003cem\u003eM. phaseolina\u003c/em\u003e (Yadav et al., 2023). A uniform lawn was prepared by spreading 1 mL of each bacterial suspension (OD₆₀₀ = 1.0; ~10⁹ CFU/mL) uniformly over the surface of NA plates, incubating for 24 hours at 28\u0026deg;C. The center of every plate was then a 6 mm disc of mycelia from a 7-day-old culture of \u003cem\u003eM. phaseolina\u003c/em\u003e grown on malt extract agar. Bacterial lawn-free control plates were maintained in parallel for comparison. Following seven days of incubation at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, the antifungal activity was measured by calculating the zone of inhibition and fungal growth inhibition on each plate.\u003c/p\u003e\n\u003ch3\u003eFormulation and stability evaluation of hydrogel\u003c/h3\u003e\n\u003cp\u003eSix bio-polymeric hydrogels were developed with zinc (Zn) and iron (Fe) supplementation, citric acid as a crosslinker, sodium carboxymethyl cellulose (NaCMC), κ-carrageenan (Car), and a bacterial consortium consisting of BV, BS1, and KO (Kang and Yun, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). NaCMC and Car were solubilized separately in autoclaved distilled water (60\u0026deg;C) and stirred for 30 minutes to become uniform. Citric acid was added as a crosslinking initiator for increasing the gel strength, and Zn and Fe salts for micronutrient supplementation. Standardized bacterial suspensions (OD₆₀₀ = 1.0, ~\u0026thinsp;10⁹ CFU/mL) were aseptically added to the cooled mixture and stirred gently to promote entrapment. The gel was poured (10 mL) into sterile plastic moulds and dried overnight at 45 C to obtain uniform hydrofilms.\u003c/p\u003e \u003cp\u003eStability of the hydrogels was assessed by soaking 0.5 g of each dried film in 10 mL of distilled water at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and noting the time to complete disintegration. Out of the six formulations screened, the hydrogel (HG\u003csub\u003e6\u003c/sub\u003e) with maximum stability in water was selected.\u003c/p\u003e\n\u003ch3\u003eHydrogel characterization\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGel cross-linked proportion analysis\u003c/h2\u003e \u003cp\u003eThe hydrogel cross-linked proportion was measured using the tea bag method by Uyanga et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and calculated gel fraction percentage to check the extent of crosslinking and structural stability.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSwelling behavior and kinetic modeling of hydrogel\u003c/h3\u003e\n\u003cp\u003eSwelling dynamics of the best optimized hydrogel formulation (HG\u003csub\u003e6\u003c/sub\u003e) were gravimetrically investigated by soaking 0.2 g dry film in 10 mL autoclaved distilled water at room temperature (H\u0026uuml;ther et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The swelling percentage was assessed on regular intervals, and by fitting the data to a pseudo-second-order model, the swelling rate and mechanism was determined. The Higuchi model determines Fickian diffusion properties, while the Korsmeyer-Peppas model determines the diffusional exponent and release mechanism (Dal and Onursal, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSwelling response of the hydrogel was assessed under ionic conditions and pH changes, hydrogel films were submerged for 10 minutes at room temperature in buffer solutions with pH values of 4, 6, and 8, as well as in NaCl and CaCl₂ solutions with concentrations ranging from 0.1 to 0.9 M (Azeem et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For all the experiments, the swelling ratio was calculated by the same gravimetric equation. For measurements of network structure and mechanical strength, polymer volume fraction (V₂) was determined according to Peppas and Merrill et al. (1977) method and crosslink density (Vₑ) was calculated according to Flory-Rehner theory (1943) using χ\u0026thinsp;=\u0026thinsp;0.49 for water-polymer systems and V₁ as the molar volume of water.\u003c/p\u003e\n\u003ch3\u003ePorosity and moisture retention testing\u003c/h3\u003e\n\u003cp\u003eHexane was employed for the liquid displacement procedure to measure porosity (Qazi et al.,2022). Empirical parameters (n and m), to quantify moisture retention and sensitivity of hydrogel in soil conditions, were used to fit the data to the van Genuchten model (1980), including residual and saturated water content, to try and investigate moisture retention behavior.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSoil water retention analysis\u003c/h2\u003e \u003cp\u003eFor water retention determination in the soil, 100 g of soil was mixed with 1 g of hydrogel, and 60 mL of water was used to water them; weight loss was measured each day. Cumulative water loss and water retention efficiency were determined, as by Thombare et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), compared to untreated controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiodegradability assessment\u003c/h2\u003e \u003cp\u003eBiodegradability was determined using the soil burial technique (Dharmalingam et al.,2016), 1 g films of hydrogel were buried 5 cm deep in soil and weight loss was measured over a period of 14 days. First-order kinetics was simulated, and half-life (t₁\u003csub\u003e/\u003c/sub\u003e₂) was calculated (Kamenova et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBacterial release\u003c/h2\u003e \u003cp\u003eThe release of bacteria from 0.5 g hydrogel was tracked in phosphate buffer solution at pH 7.0 under 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 C conditions over 24 hours by monitoring OD. Cumulative release (%) was determined from the maximum OD₆₀₀ at regular intervals (Mohamadzadeh et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eViability assessment\u003c/h2\u003e \u003cp\u003eA suspension of 1 g of bacteria loaded hydrogel in 10 mL of sterile saline was vortexed to test for microbial viability. CFUs were measured after serial dilutions (10⁻\u0026sup1; to 10⁻⁶) were plated on nutrient agar and incubated for 24\u0026ndash;48 hours at 28\u0026ndash;30\u0026deg;C. Viability was tracked for 60 days at 7-day intervals to determine shelf life and microbial stability (Mohamadzadeh et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOptical analysis\u003c/h2\u003e \u003cp\u003eMorphological variations in dried and swollen hydrogels were studied under a compound light microscope (Labomed LX400) with 40\u0026times; magnification. Samples were imaged prior to and following equilibrium swelling for evaluating structural variations (Gull et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntifungal potential of hydrogel against\u003c/b\u003e \u003cb\u003eM. phaseolina\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAntifungal activity of hydrogel formulations at 0.1, 0.5, and 1% (w/v) concentrations was tested against \u003cem\u003eM. phaseolina\u003c/em\u003e with a broth macro-dilution assay (Kabiru et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Mycelial discs (6 mm) of 7-day-old cultures of fungi were inoculated into 10 mL sterile 2% malt extract hydrogel medium. A control was given 1 mL PBS instead of hydrogel. All treatments were incubated under controlled conditions for 7 days (28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C). OD₆₀₀ was measured, and fungal biomass was filtered through pre-weighed Whatman No. 1 filter papers. Fresh weight was determined immediately, and dry weight was from drying at 60\u0026deg;C for 48 h. Antifungal activity as a percentage inhibition of fungal biomass compared to the control not treated was computed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSeed germination assays\u003c/h2\u003e \u003cp\u003eDk 6321 or Dekalb 6321 maize seed variety was obtained from Bayer, Pakistan. A 1% NaOCl solution was used to surface sterilize the seeds for three to four minutes. Three layers of sterilized blotter were arranged in pre-sterilized Petri dishes, and seven seeds per plate were placed in it. Two treatments with three replicates were implied for this experiment, one was control (without hydrogel) and the other was with hydrogel embedded in the layers of blotter paper. Regular irrigation and observations were made during the six-day experiment. The data for percentage of germination was calculated (Siddiqui et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The following additional formulas were used to evaluate the germination characteristics of maize seeds:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Hydrogel\\:Efficiency\\:ratio=\\:\\frac{Germination\\:rate\\:with\\:hydrogel}{Germination\\:rate\\:in\\:control}\\:$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Germination\\:index\\:=\\:\\frac{\\sum\\:No.\\:of\\:seeds\\:germinated\\:in\\:a\\:day}{Total\\:no.\\:of\\:days\\:for\\:germination}\\:\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:Mean\\:germination\\:time\\:\\left(MGT\\right)\\:=\\:\\sum\\:\\left(\\frac{No.of\\:seeds\\:germinated\\:in\\:a\\:day\\:\\times\\:\\:Day\\:no.}{Total\\:no.\\:of\\:seeds\\:germinated}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:Gernimation\\:velocity=\\:\\frac{1}{MGT}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:Garmination\\:rate=\\frac{Total\\:no.of\\:germinated\\:seeds}{Total\\:no.\\:of\\:days\\:for\\:germination}\\:\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll graphs were created using Microsoft Excel, and the data were modeled using kinetic models and equations. Standard error was calculated to represent variability. A least significant difference (LSD) test was also applied using STATISTIX software to determine the statistical significance among treatment means.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBacterial compatibility and antifungal bioassays\u003c/h2\u003e \u003cp\u003eEach bacterial isolate grew independently in control and the cross streaked bacterial strains showed no visible zones of inhibition at intersection point in interaction plates, showing mutual compatibility and absence of any antagonistic (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBacterial lawn assay on 2% MEA indicated that \u003cem\u003eBacillus\u003c/em\u003e spp. (BV and BS1) restricted the radial growth of fungus by 80% and 83% respectively, with distinct inhibition zones of 6 mm and 4 mm, respectively. On the other hand, KO, showed poor antifungal activity with 7% mycelial inhibition and a 1 mm zone of inhibition (Fig. S2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHydrogel aqueous stability evaluation\u003c/h2\u003e \u003cp\u003eAmong the six hydrogels (HG\u003csub\u003e1\u003c/sub\u003e-HG\u003csub\u003e6\u003c/sub\u003e), the maximum structural stability was seen for HG\u003csub\u003e6\u003c/sub\u003e (lasted for 24 h). This one used the optimized amount of polymer (0.75 g/L Car, 1 g/L NaCMC), higher dose of crosslinker (0.75 g/L citric acid), micronutrient supplementation (Zn and Fe at 5 mg/L each), and full microbial consortium (3 \u0026times; 10⁹ CFU/mL) for further bioassays (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGel fraction composition\u003c/h2\u003e \u003cp\u003eThe synthesized hydrogel (HG\u003csub\u003e6\u003c/sub\u003e) had a gel fraction of 84%, determined from its fresh and dry weights (0.2 g and 0.174 g, respectively), which reflects high network integrity after swelling (Fig. S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSwelling behavior and kinetic modeling of the hydrogel\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSwelling in water and kinetic modeling\u003c/h2\u003e \u003cp\u003eThe rapidly swelling initial response of the synthesized hydrogel reached almost-equilibrium in 110 minutes and leveled off thereafter, with the highest equilibrium swelling ratio of 2.50 g/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Pseudo-second-order kinetic modeling showed good correlation with the experimental data (R\u0026sup2; = 0.998), validating chemisorption as the prevailing swelling mechanism. The predetermined rate constant (k\u0026thinsp;=\u0026thinsp;0.0253 g/g min) indicates a mid-range swelling rate appropriate for controlled water release (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additional comparison with the Higuchi model (R\u0026sup2; = 0.85) and the Korsmeyer\u0026ndash;Peppas model (R\u0026sup2; = 0.92; n\u0026thinsp;\u0026lt;\u0026thinsp;0.5) confirmed Fickian diffusion as a major mechanism of water uptake (Table S2).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSwelling behavior in electrolyte solution\u003c/h2\u003e \u003cp\u003eIn NaCl, the maximum swelling (415%) occurred at 0.3 M, while CaCl₂ had a maximum of 390% at 0.5 M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In both cases, higher concentrations led to lower swelling, which reflects ionic crosslinking and compaction of the network at higher salinity. This was measured by calculated crosslinking volume (V\u003csub\u003ec\u003c/sub\u003e), with the lowest values at optimal salt concentrations (NaCl: 3032 cm\u0026sup3;/mol at 0.3 M; CaCl₂: 2613 cm\u0026sup3;/mol at 0.5 M), affirming maximum network relaxation and expansion at intermediate ionic strength (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Associated polymer volume fractions (V₂) were also lowest at these concentrations (NaCl: 0.19; CaCl₂: 0.20), indicating a more open hydrogel matrix for water imbibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These observations indicate that the hydrogel works best at moderate salinity, as in the case of Fickian-controlled release behavior appropriate for saline or dryland soils (Table\u0026nbsp;3S).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eSwelling at different pH levels\u003c/h2\u003e \u003cp\u003eThe hydrogel exhibited pH-sensitive swelling, with the highest uptake (390%) at pH 6. The swelling was found to reduce under acidic (305% at pH 4) and alkaline (370% at pH 8) environments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This trend was associated with network structure changes, with lowest crosslink density (Vₑ = 0.035 mol/cm\u0026sup3;) and polymer volume fraction (V₂ = 0.20) at pH 6. Greater Vₑ and V₂ at pH 4 and 8 implied denser gel networks, which accounted for the reduced water absorption.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eMoisture absorption and porosity estimation\u003c/h2\u003e \u003cp\u003eThe hydrogel had a porosity of 71.7% and a swelling ratio of 3.05, with internal pore capacity determined to be 1.3 mL through hexane displacement. The range of pore size estimated to be around 50\u0026ndash;200 \u0026micro;m is ideal for water retention and slow release in soil use. The results affirm the gel's ability to store and release moisture slowly, improving soil water-holding potential at the most agronomical appropriate range for plant uptake.\u003c/p\u003e \u003cp\u003eThe soil-amended hydrogel showed enhanced water retention throughout, with volumetric water content rising steadily by 1.6-2.0% within the 10\u0026ndash;100 cm suction range over the untreated soil, and a highest enhancement of 3.18% at 40\u0026ndash;50 cm suction as per van Genuchten model analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Table S4). At equilibrium (0 cm) moisture content increased slightly from 0.45\u0026ndash;0.457 cm\u0026sup3;/cm\u0026sup3;, whereas effectiveness reduced at suctions\u0026thinsp;\u0026ge;\u0026thinsp;200 cm, reflecting compromised moisture availability under extreme drought conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSoil water retention\u003c/h2\u003e \u003cp\u003eHydrogel treatment dramatically decreased moisture loss rate from soil over a 10-day dry-down period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The control and hydrogel-amended soils had practically identical initial weights (150 g and 151 g, respectively), by day 10 the hydrogel-treated soil held more water (116 g) than the control (111 g), corresponding to 77% vs. 74% moisture retention. The water loss was 38.90 g in 10 days in control, whereas for the hydrogel-treated sample, 35.07 g were lost. Retention efficiency was also consistently high in hydrogel-added soil, at greater than 104% starting from day 4 and as high as 105% on day 8. The largest differences in percentage for water retention were between days 3 to 6, with the hydrogel treatment holding 2\u0026ndash;3% more water compared to the control. Treated soil held 76.8% of initial water compared to 74.1% in the control by day 10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eBiodegradability test in soil\u003c/h2\u003e \u003cp\u003eThe burial test in soil showed that the hydrogel was readily biodegradable in natural conditions as there is progressive loss of weight over 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). There was an initial 9% degradation in the first day, which reflected early commencement of biodegradation. There then followed a fast stage of degradation on days 3\u0026ndash;7 when there was a progressive increase in biodegradability from 26.7% to 53.1%. After day 7, degradation proceeded at a sluggish but linear rate to 80.2% cumulative degradation by day 14. Kinetic modeling validated that degradation was first order, with a rate constant (k) of 0.117 day⁻\u0026sup1; and a half-life (t₁\u003csub\u003e/\u003c/sub\u003e₂) of 5.93 days. The fit of the model was excellent (R\u0026sup2; = 0.99), validating a systematic and exponential trend of decomposition with respect to time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eBacterial release kinetics\u003c/h2\u003e \u003cp\u003eRelease kinetics of bacterial cells from hydrogel was a distinct biphasic kinetic pattern within 24 hours (Fig. S4). There was a lag phase with 8% and 16% cumulative release at 1 and 2 hours, respectively, which was followed by a rapid exponential release to 86% at hour 8. A gradual plateau phase followed with ultimate release (100%) at 24 hours.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicrobial viability\u003c/h3\u003e\n\u003cp\u003eViability assays verified that the bacterial populations were well preserved in the hydrogel matrix on storage. From an initial cell count of 3.27 \u0026times; 10⁹ CFU mL⁻\u0026sup1;, cell counts were relatively constant, declining to a small degree only to 2.61 \u0026times; 10⁹ CFU mL⁻\u0026sup1; (79.8%) at 7 days. Between 14\u0026ndash;35 days, viable counts varied between 2.48 \u0026times; 10⁹ and 2.80 \u0026times; 10⁹ CFU mL⁻\u0026sup1;, corresponding to 76\u0026ndash;86% retention. Even after 35 days, viability was good at 2.64 \u0026times; 10⁹ CFU mL⁻\u0026sup1; (80.7%), with only a 0.11 log₁₀ unit loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eOptical observations\u003c/h2\u003e \u003cp\u003eOptical microscopy of swollen and dry hydrogel also supported these observations. Dry hydrogel contained a compact and uniform polymer network (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). After swelling, the matrix swelled and exhibited well-defined, interconnected pores (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), giving the structure a porous nature ideal for water uptake and microbial encapsulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eAnti-fungal analysis of the bioformulation\u003c/h2\u003e \u003cp\u003eAntifungal potential of the hydrogel was tested against \u003cem\u003eM. phaseolina\u003c/em\u003e with different concentrations (0.1, 0.5, and 1.0% w/v). Morphologically, increasing distortion and curling of hyphae with increasing concentration of hydrogel were noted which exhibited inhibitory activities towards fungal growth. The fungus biomass reduction and inhibition of corresponding mycelial growth was 11, 34, and 88%, thereby confirming the high antifungal activity of hydrogel at high concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eGermination bioassays\u003c/h2\u003e \u003cp\u003eThe germination activity of seeds treated with the hydrogel formulation was compared with untreated control seeds for 6 days. Quantitative data (Table\u0026nbsp;4S) indicated that hydrogel-treated seeds germinated at 100%, whereas the control germinated to 90% on day 6. Mean germination time (MGT) was significantly lower in the hydrogel treatment (2.431 days) than for the control (3.006 days), reflecting a faster process of germination. As a result, the germination speed, the inverse of MGT, was also increased in the hydrogel-treated group (0.411) over that in the control (0.333), establishing increased germination kinetics. The germination index (GI), a blended parameter of germination rate and synchronicity, was significantly augmented in hydrogel treatment (12.44) compared to the control (9.35), suggesting both quicker and synchronized germination. Moreover, hydrogel efficiency ratio in final germination was 1.11, further highlighting the effectiveness of the hydrogel in triggering early seed emergence.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study investigated the physicochemical characteristics, bio-efficacy against \u003cem\u003eM. phaseolina\u003c/em\u003e, and maize growth promotion of a multi-functional hydrogel (HG\u003csub\u003e6\u003c/sub\u003e) made from CMC/κ-carrageenan crosslinked with citric acid with Zn\u0026sup2;⁺, Fe\u0026sup3;⁺, and a bacterial consortium (\u003cem\u003eBacillus velezensis\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e sp., and \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e). Compatibility tests established that the strains of the bacteria were mutually non-antagonistic, a necessary condition for effective co-formulation and synergistic bioactivity (Swiontek et al., 2022). In antifungal tests, \u003cem\u003eBacillus\u003c/em\u003e strains expressed high antagonism (\u0026ge;\u0026thinsp;80% inhibition), which resulted most probably from lipopeptide production (iturins, fengycins, surfactins) and hydrolytic enzymes (Zhou et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hong et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eK. oxytoca\u003c/em\u003e, although weakly antagonistic, has applications due to its plant growth-promoting features such as nitrogen fixation and phytohormone production (Youseif et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHG\u003csub\u003e6\u003c/sub\u003e exhibited the maximum structural integrity (24 h) among all formulations. This was a result of synergistic interactions of bicomponent polymers, high citric acid crosslinking, metal ion coordination, and bacterial encapsulation (Trivedi et al., 2023; Zhou et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Porosity (71.7%) and intense crosslinking (84% gel content) were proposed for a macroporous matrix that can transport microorganisms and retain water in soils (Guilherme et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Water swelling exhibited pseudo-second-order kinetics (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.998) and reached equilibrium (2.5 g/g) in 2 h, which is typical of hydrogels that are chemisorption dominant. Electrolytes and pH influenced swelling responses: a dose of moderate amount of NaCl and CaCl₂ promoted absorbance, but excessive salinity or pH reduced it, as Flory's theory and ionic crosslinking character of divalent cations would predict (Landsgesell et al., 2023). Swelling was maximal at pH 6, which is the same as the pH of most agricultural soils, with increased availability of nutrition and conditioning of the soil.\u003c/p\u003e \u003cp\u003eSoil water retention experiments indicated that hydrogel-modified soils stored 1.6\u0026ndash;3.2% more water by volume in the suction range of 0-100 cm. Maximum improvement was noticed at 40\u0026ndash;50 cm suction-it is essential for root uptake by plants. Weight-loss measurement indicated 77% water retention in hydrogel-amended soil on day 10 compared to 74% in control, confirming its effectiveness in maintaining soil water (Abdelghafar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Capacity for water availability extension justifies its use to counter drought stress in dry environments. Biodegradation tests demonstrated gradual degradation with 80.2% loss in mass after 14 days and a half-life of 5.9 days, following first-order kinetics (R\u0026sup2; = 0.99). This confirms the environment-sensitive degradation of the hydrogel without any formation of residues to warrant its application as a safe soil amendment (Kamenova et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Biphasic release profiles for bacteria were observed, with 86% of the release at 8 hours and complete release at 24 hours. The controlled release is a result of network relaxation due to swelling in the hydrogel, which maximizes the dispersion of microbes (Mo et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Microbial viability was relatively high (\u0026gt;\u0026thinsp;76%) up to 35 days, indicating that the hydrogel is effective in protecting bacterial cells and enabling long-term delivery under field conditions (Lima-Ten\u0026oacute;rio et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroscopy established an expanded pore structure upon swelling with pores of 50\u0026ndash;200 \u0026micro;m diameter to facilitate microbial diffusion and water retention capacity (Montesano et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The hydrogel also exhibited inherent antifungal activity in inhibiting mycelial biomass by 88% using 1% (w/v) concentration. This may be attributed to acidity derived from citric acid and the inherent antifungal character of Zn\u0026sup2;⁺ ions (Korbecka-Glinka et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), by a dual action mechanism. Collectively, these results determine the universal applicability of the Zn/Fe-bacteria enriched hydrogel as a multi-functional, eco-friendly platform for promoting plant growth and disease control. Its biodegradability, water-holding capacity, and site-directed delivery of microbes solve the biggest challenges in sustainable crop management against biotic and abiotic stresses.\u003c/p\u003e \u003cp\u003eThe hydrogel itself exhibited strong concentration-dependent antifungal activity against \u003cem\u003eM. phaseolina in vitro\u003c/em\u003e. Hydrogel treated hyphae became deformed even at 0.1 and 1%, mycelial biomass was reduced by 88% compared to control. This may be attributed to several reasons: the citric acid crosslinker has the potential to acidify the microenvironment, and the released Zn\u0026sup2;⁺ is already reported to possess antifungal activity (Korbecka-Glinka et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overall, the results show that the developed hydrogel is a stable, slow-release carrier for bacterial biocontrol and micronutrient supply, improving soil water holding capacity and having direct antifungal action. These results validate its potential as a biodegradable soil amendment for integrated disease management and promotion of plant growth.\u003c/p\u003e \u003cp\u003eThe germination test showed clearly that the maize seeds germinated more effectively when the hydrogel was used compared to the control. All the seeds treated with the hydrogel germinated, in contrast to the control group showed slightly lower 90% germination. Not only germinated more seeds but even quicker-the average time of germination was less, and both vigor and germination percentage were greatly improved. This increase is probably because hydrogel can hold water and seep it out slowly around the seeds, a uniformly wet condition to aid early metabolic activity (Montesano et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Agbna and Zaidi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This consistent supply of moisture is particularly critical under arid or abnormal conditions, when water stress will inhibit or retard germination. Comparable findings have been reported from other research using superabsorbent hydrogels, which assist seeds by supplying the surrounding soil with the exact amount of water to activate enzymes and promote development (Qureshi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Together, these results suggest the potential of the hydrogel as a simple but efficient method of stimulating germination and subsequent seedling viability.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully designed and explored an eco-friendly hydrogel-based bioformulation for microbial delivery, fortified with Zn and Fe, for management of charcoal rot disease. \u003cem\u003eBacillus velezensis\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e sp. showed effective antifungal activity against \u003cem\u003eM. phaseolina\u003c/em\u003e, with more than 80% inhibition, and all tested strains were mutually compatible for co-formulation. The optimized hydrogel formulation (HG\u003csub\u003e6\u003c/sub\u003e) had excellent physicochemical stability, facilitating effective encapsulation of microbes and long-term soil delivery. Quick swelling capacity (2.5 g/g equilibrium in 2 hours), high gel content (84%), and pH- and salinity-sensitive water absorbency (up to 390%) established its high-strength structure and water-retention capacity. HG\u003csub\u003e6\u003c/sub\u003e improved soil moisture retention by 1.6\u0026ndash;3.2% within the plant-available range and minimized water loss over 10 days, substantiating its function in drought stress mitigation. The hydrogel supported biphasic, controlled release of bacteria with 86% release within 8 hours and complete release in 24 hours, and 76\u0026ndash;86% viability of bacteria for 35 days. It is also biodegradable up to 80.2% in 14 days, making it safe to the environment, which supports its use as a comprehensive, dual-purpose bioformulation for suppressing disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUniversity of the Punjab, Lahore, Pakistan is thanked for providing facilities to accomplish the task.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. N.\u003c/strong\u003e Performed experiments, collected data and drafted the manuscript; \u003cstrong\u003eA. S.\u003c/strong\u003e Designed and supervised the experiments, Prepared graphs and revised the manuscript; \u003cstrong\u003eH. A.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e. Bacteria provision and cultivation; \u003cstrong\u003eM. K\u003c/strong\u003e: Methodology\u003cstrong\u003e; M.S\u003c/strong\u003e: Methodology All authors have substantial contributions to the final manuscript and approved this submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures in this experiment were carried out in accordance with relevant guidelines of the university field of the University of the Punjab, Lahore, Pakistan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelghafar R, Abdelfattah A, Mostafa H (2024) Effect of super absorbent hydrogel on hydro-physical properties of soil under deficit irrigation. 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Green Chem 26(8):4609\u0026ndash;4621. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3GC05109A\u003c/span\u003e\u003cspan address=\"10.1039/D3GC05109A\" 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":"Hydrogel, micronutrients, Controlled release, Biopolymer processing, Release kinetics","lastPublishedDoi":"10.21203/rs.3.rs-9392239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9392239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant growth-promoting rhizobacteria (PGPR) represent a sustainable alternative for plant disease management; however, their field application is often limited by poor survival and inefficient delivery. In this study, a multifunctional biopolymer-based hydrogel composed of carboxymethyl cellulose and κ-carrageenan, crosslinked with citric acid, was developed as a carrier system for controlled delivery of PGPR and micronutrients. The engineered hydrogel exhibited high porosity (71.7%), rapid swelling capacity (2.5 g/g), and substantial biodegradability (80.2% within 14 days), following first-order degradation kinetics. Swelling behavior conformed to pseudo-second-order kinetics with Fickian diffusion as the dominant mechanism. The system demonstrated a biphasic release profile, achieving complete bacterial release within 24 h while maintaining\u0026thinsp;\u0026ge;\u0026thinsp;76% cell viability up to 35 days. Encapsulated PGPR showed strong antifungal activity, inhibiting \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e by up to 88%. Additionally, the hydrogel improved soil moisture retention and enhanced seed germination performance. Overall, the developed system integrates material functionality with microbial delivery efficiency, highlighting its potential as a biotechnological platform for improved PGPR-based disease management.\u003c/p\u003e","manuscriptTitle":"Biodegradable Hydrogel for Controlled Delivery of Biocontrol Bacteria and Micronutrients against Macrophomina phaseolina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 17:15:19","doi":"10.21203/rs.3.rs-9392239/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":"1cc0f552-8efb-42e1-b6d9-18b74c26ba16","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T16:55:21+00:00","index":23,"fulltext":""},{"type":"reviewerAgreed","content":"157653536481352016699707838368904044802","date":"2026-05-05T13:14:21+00:00","index":22,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T17:15:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 17:15:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9392239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9392239","identity":"rs-9392239","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}