In vitro evaluation of chitosan, β-glucan and chitosan-copper-β-glucan nanoconjugate against Sclerotium rolfsii causing tuberose stem rot

Introduction

Medicinal plants have greatly gained importance in the management and treatment of human diseases, globally [1] . It constitutes the main source of primary healthcare in most rural populations, especially in many African countries, due to its affordability and availability. However, as the usage of these medicinal plants becomes more widespread around the world, there are questions and misgivings about the quality, efficacy, and safety of the products utilized in the health sector [2,3,4] . These concerns have hampered the sensible use and validation of Tuberose (Agave amica) is an important commercial ornamental crop valued for its fragrant white flowers and its use in loose flowers, garlands, floral arrangements and perfume production [1,2] . However, its production is affected by several fungal diseases, among which stem rot caused by S. rolfsii is an important constraint, particularly under warm and humid conditions [3,4] . The pathogen produces persistent sclerotia that serve as important survival structures and contribute to disease recurrence [5,6] . Management of S. rolfsii mainly relies on chemical fungicides and biological control agents, but limitations associated with repeated chemical application and variable biocontrol performance have encouraged the search for alternative approaches [7,8] . Chitosan and βglucans are biologically active polysaccharides capable of interacting with plant defence systems and chitosan can function both as a direct antimicrobial compound and as an elicitor of plant defence responses. Chitin-and β-glucan-derived molecular patterns can participate in plant immune recognition, while chitosan has been associated with activation of defencerelated responses and secondary metabolism [9,10] . Incorporation of copper into chitosanbased formulations may further enhance antimicrobial activity [11,12] .

Materials and Methods

The present investigation entitled "In vitro evaluation of chitosan, β-glucan and chitosan-copper-β-glucan nanoconjugate against tuberose stem rot (Sclerotium rolfsii)" was carried out during 2025-2026 in the Department of Plant Pathology, Govt. College of Agriculture, Pune, Maharashtra, India. The wellcharacterized nanomaterials used in the study were obtained from Vasantdada Sugar Institute (VSI), Manjari (Bk.), Pune, Maharashtra, India [15] . The stock concentrations provided were 3000 ppm each for chitosan and β-glucan and 2000 ppm for CN-Cu-BG nanoconjugate.

Characterization of chitosan, β-glucan and chitosancopper-β-glucan nanoconjugate

The physicochemical characteristics of these nanomaterials have been previously reported by Chavanke and Dalvi [10,15] . Chitosan and β-glucan nanoparticles showed nanoscale particle sizes, with average zeta-sizer sizes of 64.43 nm for chitosan and 60.16-73.66 nm for β-glucan nanoparticles [10] . The CN-Cu-BG nanoconjugate exhibited a uniformly spherical morphology with an average particle size of 41.97 nm and spectroscopic and elemental analyses confirmed the incorporation of copper within the chitosan-β-glucan matrix [15] . These previously reported characteristics confirm the nanoscale nature of the nanomaterials used in the present investigation.

Isolation and identification of the pathogen

Diseased samples of tuberose showing typical stem rot symptoms were collected from AICRP on Floriculture, ZARS, Ganeshkhind, Pune (18°33'44.7"N 73°49'30.4"E) and brought to the laboratory for isolation of the pathogen. The pathogen was isolated from infected stem tissues using the tissue isolation technique on potato dextrose agar (PDA). The emerging fungal growth was purified by hyphal-tip isolation and maintained on PDA. The isolate was identified based on cultural and morphological characteristics, including mycelial colour, texture, growth pattern and sclerotial characteristics and identified as S. rolfsii.

in vitro evaluation of antifungal activity

The antifungal activity of chitosan, β-glucan and chitosancopper-β-glucan nanoconjugate against S. rolfsii was evaluated using the poisoned food technique. The respective concentrations were incorporated into sterilized PDA medium and 20 mL of the amended medium was poured into sterile 90-mm Petri plates. PDA without nanomaterial served as the untreated control. After solidification, a 5-mm mycelial disc from an actively growing culture of S. rolfsii was placed at the centre of each plate. The plates were incubated at 28 °C. Mycelial growth was observed for approximately 5-7 days and observations were recorded when the untreated control plate was completely covered by fungal growth. Colony diameter was measured in centimetres. Chitosan and β-glucan were evaluated at 50, 100, 200, 300, 400, 500, 600 and 700 ppm, whereas the nanoconjugate was evaluated at 50, 100, 150, 200, 250 and 300 ppm, along with an untreated control. Each treatment was maintained in three replications.

Mycelial growth inhibition

The percentage inhibition of mycelial growth over the untreated control was calculated using the formula of Vincent [20] :

I = [(C-T)/C] × 100

Where I = percent inhibition, C = mycelial growth in control (cm) and T = mycelial growth in treatment (cm). The effect of the treatments on mycelial morphology was also recorded based on growth pattern and mycelial colour in comparison with the untreated control.

Sclerotial germination assay

The effect of the chitosan-copper-β-glucan nanoconjugate on sclerotial germination of S. rolfsii was evaluated at 50, 100, 150, 200, 250 and 300 ppm along with an untreated control. Five sclerotia were used per replicate and placed on the respective poisoned PDA medium. The plates were incubated at 28 °C for 72 h, after which sclerotial germination was recorded based on visible emergence of mycelium from the sclerotia. The percentage of sclerotial germination was calculated as: Sclerotial germination (%) = (Number of germinated sclerotia/Total number of sclerotia) × 100

Experimental design and statistical analysis

The data recorded for the different observations were analyzed using Completely Randomized Design (CRD). Statistical analysis was carried out following the standard procedures described by Panse and Sukhatme [16] . The significance of treatment differences was tested using analysis of variance and treatment means were compared using the critical difference (C.D.) at the appropriate level of significance. The analysis was performed using the OPSTAT online statistical application.

Results

During the present investigation on the "In vitro evaluation of chitosan, β-glucan and chitosan-copper-β-glucan nanoconjugate against S. rolfsii causing tuberose stem rot", the results obtained are presented below.

Morphological studies of S. rolfsii

The isolated pathogen produced cottony, creamy white to dull white mycelial growth on PDA. The mycelium showed a characteristic radial growth pattern with fan-shaped mycelial strands. Brown to dark brown, round to spherical sclerotia developed after 10-14 days, measuring 0.5-2.05 mm in diameter. Sclerotia were predominantly formed at the periphery of the colony, with a few scattered towards the centre. Microscopic examination of the mycelium revealed septate hyphae with clamp connections. Based on these cultural and morphological characteristics, the isolate was identified as S. rolfsii (Table 1, Plate 1).

in vitro antifungal activity of chitosan nanoparticles against S. rolfsii

Chitosan exhibited a concentration-dependent inhibitory effect on the mycelial growth of S. rolfsii. The untreated control recorded the maximum mycelial diameter of 9.0 cm. With increasing concentration of chitosan, mycelial growth progressively decreased from 4.7 cm at 50 ppm to 0.2 cm at 700 ppm. The corresponding inhibition of mycelial growth increased from 48.15% at 50 ppm to 97.41% at 700 ppm. The maximum inhibition was recorded at 700 ppm (97.41%), followed by 600 ppm (95.93%) and 500 ppm (92.59%). The growth pattern changed from fluffy growth at lower concentrations to compact and subsequently pin-point growth at higher concentrations, while the mycelial colour changed from cottony white to dull white (Table 2, Plate 2).

in vitro antifungal activity of β-glucan nanoparticles against S. rolfsii

β-glucan exhibited a concentration-dependent inhibitory effect on the mycelial growth of S. rolfsii. The untreated control recorded the maximum mycelial diameter of 9.0 cm. With increasing concentration of β-glucan, mycelial growth progressively decreased from 5.1 cm at 50 ppm to 0.3 cm at 700 ppm. The corresponding inhibition of mycelial growth increased from 43.33% at 50 ppm to 97.04% at 700 ppm. The maximum inhibition was recorded at 700 ppm (97.04%), followed by 600 ppm (93.70%) and 500 ppm (90.37%). The growth pattern changed from fluffy growth at lower concentrations to compact and subsequently pin-point growth at higher concentrations, while the mycelial colour changed from cottony white to dull white (Table 3, Plate 3).

in vitro antifungal activity of chitosan-copper-βglucan nanoconjugate against S. rolfsii

The chitosan-copper-β-glucan nanoconjugate showed a pronounced concentration-dependent inhibition of S. rolfsii mycelial growth. The untreated control recorded 9.0 cm mycelial growth, whereas the growth decreased progressively from 3.9 cm at 50 ppm to 0.2 cm at 300 ppm. The corresponding inhibition increased from 56.30% at 50 ppm to 98.15% at 300 ppm. The highest inhibition was recorded at 300 ppm (98.15%), followed by 250 ppm (94.81%) and 200 ppm (88.15%). At higher concentrations, the mycelial growth pattern changed from fluffy to compact and finally pinpoint, with the mycelial colour changing from cottony white to dull white (Table 4, Plate 4).

Effect of chitosan-copper-β-glucan nanoconjugate on sclerotial germination

The chitosan-copper-β-glucan nanoconjugate markedly reduced the germination of S. rolfsii sclerotia in a concentration-dependent manner. Complete germination (100%) was recorded in the untreated control. Sclerotial germination decreased progressively with increasing nanoconjugate concentration, from 73.33% at 50 ppm to 0% at 300 ppm.

The lowest germination was recorded at 300 ppm, where complete inhibition of sclerotial germination was observed (Table 5, Plate 5).

Discussion

The present study demonstrated a clear concentrationdependent antifungal activity of chitosan nanoparticles, βglucan nanoparticles and chitosan-copper-β-glucan nanoconjugate against S. rolfsii. Chitosan nanoparticles reduced mycelial growth from 9.0 cm in the untreated control to 0.2 cm at 700 ppm, corresponding to 97.41% inhibition. The inhibition increased progressively from 48.15% at 50 ppm to 97.41% at 700 ppm. This concentration-dependent response agrees with Kheiri et al. [17] , who reported significant inhibition of Fusarium graminearum by chitosan and chitosan nanoparticles, with maximum inhibition obtained at higher concentrations. Their study also observed dehydration, deformation and collapse of fungal hyphae following treatment with chitosan nanoparticles. The antifungal activity of chitosan against S. rolfsii is further supported by Eweis et al. [18] , who evaluated chitosan and its thiourea derivative against several sugar-beet pathogens, including S. rolfsii. They reported inhibitory effects on mycelial growth and on the germination of sclerotia, with S. rolfsii sclerotia being particularly sensitive to the modified chitosan treatment. Chitosan-mediated disruption of fungal membrane integrity and cellular homeostasis is considered an important basis of this activity [10] . β-Glucan nanoparticles also exhibited a concentrationdependent inhibitory effect, reducing mycelial growth from 9.0 cm in the control to 0.3 cm at 700 ppm and producing 97.04% inhibition. Importantly, Chavanke and Dalvi [10] reported complete inhibition of sugarbeet sclerotium root rot causing S. rolfsii at 160 ppm using β-glucan nanoparticles derived from S. rolfsii and yeast. Their study specifically noted that in vitro investigations of β-glucan nanoparticles against S. rolfsii were limited, making their finding directly relevant to the present investigation. The chitosan-copper-β-glucan nanoconjugate exhibited the highest activity in the present study, reducing mycelial growth from 9.0 cm in the control to 0.2 cm at 300 ppm, corresponding to 98.15% inhibition. Thus, near-complete inhibition was achieved at less than half the concentration required for the individual chitosan nanoparticles and βglucan nanoparticles, indicating that integration of copper within the chitosan-β-glucan nanosystem substantially enhanced antifungal potency. A plausible explanation is that the integrated nanostructure provides multiple antifungal interfaces and mechanisms. Chitosan can facilitate interaction with the fungal surface, whereas coppercontaining nanostructures can perturb cellular integrity and redox homeostasis. Nanocomposite systems may additionally improve the effective presentation, dispersion or delivery of active components relative to their individual forms [15] . This interpretation is strongly supported by independent evidence. Rubina et al. [19] demonstrated that chitosancopper nanocomposites exerted substantial fungicidal activity against the sclerotia-forming pathogens S. rolfsii and Rhizoctonia solani. Their microscopic analyses showed loss of cytoplasmic contents, cytoplasmic coagulation, irregular hyphal morphology and destruction of fungal hyphae following treatment, providing direct evidence that chitosan-copper nanocomposites can cause severe structural damage to fungal cells. More recently, an independently reported chitosan-copperβ-glucan nanocomposite also demonstrated complete inhibition of S. rolfsii mycelial growth at 200 ppm, supporting the particularly strong antifungal potential of integrating chitosan, copper and β-glucan within a single nanosystem [15] . The convergence of these findings with the present results strengthens the proposition that biopolymermetal nanocomposites can provide an efficient platform for suppressing S. rolfsii at comparatively low concentrations. The complete inhibition of S. rolfsii sclerotial germination by the chitosan-copper-β-glucan nanocomposite at 300 ppm is particularly important from a disease-management perspective. Unlike transient inhibition of vegetative mycelial growth, suppression of sclerotial germination directly targets the pathogen's principal survival and inoculum-producing phase. Sclerotia enable S. rolfsii to persist in soil and serve as a major source of primary inoculum; consequently, materials capable of simultaneously restricting mycelial development and preventing sclerotial germination may have greater epidemiological value than treatments acting only on actively growing hyphae. The present observation is consistent with Eweis et al. [18] , who reported pronounced inhibition of S. rolfsii sclerotial germination by chitosan-derived compounds and with Rubina et al. [19] , who demonstrated that chitosan-copper nanocomposites adversely affected both fungal hyphae and sclerotial development. Thus, the activity observed here is relevant not merely as an in vitro growth-inhibition phenomenon but as an indication that the nanocomposite may interfere with two biologically distinct stages of the S. rolfsii life cycle. Overall, the findings establish a clear hierarchy of antifungal performance-chitosan nanoparticles and β-glucan nanoparticles showing strong but concentration-dependent inhibition, whereas the chitosan-copper-β-glucan nanoconjugate achieved near-complete inhibition at a considerably lower concentration and completely suppressed sclerotial germination. The results therefore identify the chitosan-copper-β-glucan nanoconjugate as the most promising candidate among the tested materials for subsequent greenhouse and field evaluation against S. rolfsii.

Conclusion

The present study demonstrated that chitosan nanoparticles, β-glucan nanoparticles and chitosan-copper-β-glucan nanoconjugate exhibited significant, concentrationdependent antifungal activity against S. rolfsii under in vitro conditions. Chitosan and β-glucan nanoparticles recorded maximum mycelial growth inhibition of 97.41 and 97.04%, respectively, at 700 ppm. The chitosan-copper-β-glucan nanoconjugate showed the highest inhibition of 98.15% at 300 ppm and completely inhibited sclerotial germination at the same concentration. This substantially lower effective concentration, together with inhibition of both vegetative growth and the pathogen's persistent survival structure, identifies chitosan-copper-β-glucan nanoconjugate as the most promising antifungal material evaluated. The findings are consistent with previous evidence that chitosan-copper nanocomposites can strongly suppress S. rolfsii growth and sclerotial development.

Overall, the results provide compelling in vitro evidence for the potential of chitosan-copper-β-glucan nanoconjugate as a nano-enabled strategy for S. rolfsii management and provide a strong basis for its further validation under greenhouse and field conditions.

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