PJAR_38_4

Introduction

Chitosan is one of the components of chitin found in the exoskeletons of crustaceans, fungi, and insects. It strengthens cell walls after being extracted from chitin, appearing as a white powder with a slight reddish tint. It is insoluble in water but dissolves in organic acids (Chakraborty et al., 2019). Chitosan is a derivative of chitin, which ranks second after cellulose as the most abundant natural biopolymer (Moran et al., 2018). Chitosan is distinguished from other polysaccharides by possessing amino groups in its structural backbone, granting it unique properties when used in biological applications. Chitosan is composed of two compounds: D-glucosamine and N-acetyl-D-glucosamine (Kheiri et al., 2017). In agricultural applications, chitosan has been studied and used to promote plant growth and stimulate plant defenses under biotic stress conditions. Studies have shown that the use of chitosan enhances plant resistance to pests and pathogenic infections (Fan et al., 2022). Excessive use of insecticides and fungicides can be harmful to humans, animals, and plants (Tsalidis, 2022). To reduce or avoid the use of chemical substances, natural elicitors such as chitosan can serve as alternatives for pest and disease management. Chitosan is used in many agricultural applications as well as an antifungal agent, a biopesticide, and in seed treatment. It is also used in the food industry and in medicine as a drug delivery agent, antibacterial agent, and in pharmaceuticals as a packaging material (Liburdi et al., 2016). Microgel represents one of the most recent applications in converting polymers into gel-like structures capable of absorbing water in three-dimensional networks. Microgel can absorb water up to 400 times its dry weight and has the ability to gradually release water and the compounds loaded onto it, while chitosan’s antifungal effects are well documented, limited studies have addressed its synergistic microgel form combined with bio-chemical inhibitors against R. solani in peppers (Michael et al., 2022). This study aimed to prepare chitosan microgel and use it as an inhibitor, carrier, and aid for the controlled release of loaded inhibitors, thereby reducing dependence on chemical fungicides in suppressing plant pathogens. Moreover, it is an environmentally friendly and biodegradable material.

Materials and Methods

Medium preparation

Preparation of potato dextrose agar (PDA): Potato Dextrose Agar medium was prepared by adding 19.5 g of the nutrient medium to 500 ml of distilled water in a sterilized 1000 ml flask. The mixture was stirred well using a hotplate. The flask opening was sealed with sterile cotton and aluminum foil and then placed in an autoclave at 121°C and 1 bar/cm² pressure for 15 minutes. After sterilization, the medium was cooled, and 500 mg of the antibiotic Amiflux was added and mixed thoroughly. The medium was then poured into 9 cm diameter Petri dishes inside the inoculation room after sterilizing the dishes with alcohol (Al-Jbory et al., 2023).

Preparation of nutrient broth: Nutrient broth medium was prepared by adding 13 g of the nutrient medium to 1000 ml of distilled water in a sterilized 2000 ml flask. The mixture was stirred well using a hotplate. The flask opening was sealed with sterile cotton and aluminum foil and then placed in an autoclave at 121°C and 1 bar/cm² pressure for 15 minutes (Al-Jbory et al., 2025).

Isolation and identification of the causal agents of pepper root rot from different areas of Salah Al-Din Governorate

Fungi associated with the roots of pepper plants (C. annum) showing symptoms of infection, such as yellowing, wilting, plant weakness, and root rot, were isolated. Infected samples were collected from various areas in Salah Al-Din (Al-Alam, Tikrit, Samarra, Al-Duluiya, and Al-Dujail) and transported to the postgraduate laboratory at the College of Agriculture, University of Tikrit for isolation procedures. The plant roots were thoroughly washed with water to remove soil and debris. Then, they were cut into small pieces (1 cm diameter) using scissors sterilized with alcohol. The pieces were surface sterilized with 3% sodium hypochlorite solution (NaClO) for one minute, washed with distilled water, and transferred to the inoculation room. Each piece was plated onto a Petri dish containing the nutrient medium PDA and incubated at 25 ± 2°C for five days. The isolates were then purified by transferring a portion from the colony margin onto new Petri dishes containing PDA medium.

Pathogenicity test of the isolated pathogens on pepper seeds

The pathogenicity of the fungi isolated from the roots of infected pepper plants (Rhizoctonia solani, Alternaria sp., Fusarium solani, Macrophomina phaseolina, Fusarium oxysporum) was tested on pepper seeds. The seeds were surface sterilized with 3% sodium hypochlorite solution (NaOCl) for one minute, as described in section (1.3), and then transferred to the inoculation room. The seeds were evenly sown near the edge of Petri dishes containing the nutrient medium BDA, with 10 seeds per dish. A 1.5 cm diameter mycelial disc was placed in the center of the dish, cut from the margin of a 7-days old pure fungal colony grown on BDA medium. Three replicates were used for each fungal isolate, in addition to a control treatment where sterilized pepper seeds were planted in dishes without fungal inoculation, using the same number of replicates. The plates were incubated at 25 ± 2°C for five days. The germination percentage was calculated after the seeds in the control treatment had fully germinated, as follows:

Preparation of chitosan microgel

The hydrogel compound was prepared by utilizing various polymeric materials capable of forming a three-dimensional structure, including chitosan. This was achieved by adding 2 ml of 6% acetic acid to 120 mg of chitosan, then mixing the solution using a connecting tube between two syringes, as illustrated below, for 30 seconds. After mixing, 100 microliters of genipin solution were added to the prepared mixture, followed by a second round of mixing using the dual-syringe system for another 30 seconds. The mixture was then poured into a Petri dish, covered with its lid, and incubated at 30°C for 24 hours. After incubation, the hydrogel was cut into the desired size or converted into microgel particles by passing it through a syringe equipped with a filter of known pore size, creating the required particle sizes by pushing the mixture through the filter. The resulting chitosan microgel particles were collected in a 50 ml Erlenmeyer flask, to which 20 ml of distilled water were added, and the mixture was stirred to obtain a chitosan microgel suspension. The chitosan microgel was resuspended in 10 ml of 70% ethanol and stirred for 5 minutes. Centrifugation was performed at 1000 rpm for 5 minutes, the supernatant was discarded, and the pellet was retained. The washing process was repeated three times with distilled water to remove residual ethanol. Finally, the compound was sterilized by exposure to ultraviolet light for one hour to make it ready for biological evaluation and the addition of other inhibitors (Michael et al., 2022).

Testing the efficacy of biological agents in inhibiting the most pathogenic causal agent

This test was conducted by culturing Bacillus subtilis and Pseudomonas fluorescens in the nutrient broth (N.B) medium, prepared as described in section (1.1.2). The cultures were incubated in a shaking incubator at 37°C for 24 hours. The bacterial filtrate was prepared using 9 cm diameter filter paper. Ten milliliters of the liquid N.B medium containing the bacteria were placed onto the filter paper fixed on the opening of a sterile 100 ml glass flask. The filtrate was then collected using a syringe equipped with a Mollipore filter to obtain a sterile filtrate. Petri dishes containing BDA medium were inoculated with 1 ml of the bacterial filtrate, and the plates were swirled gently to evenly distribute the bacterial filtrate. A 0.5 cm diameter disc was then taken from the margin of a 7-days old pure fungal colony grown on BDA medium and placed at the center of each plate. Three replicates were used for each bacterial type, in addition to three replicates for the control treatment. The plates were incubated at 25 ± 2°C for five days until the fungal growth in the control treatment reached the edge of the plate. The colony diameter was measured by recording the dimensions of the fungal colony.

Testing the efficacy of chemical agents in inhibiting the most pathogenic causal agent

Three types of chemical elements (organic copper, boron, and zinc) were tested. For each element, two concentrations were used: (0.025) and (0.05). Each concentration was dissolved in 100 ml of pre-prepared PDA medium in a 250 ml flask, with a total of 7 flasks (100 ml for each concentration of each element). The media containing the above concentrations were poured into Petri dishes inside a sterile laminar flow hood. A 0.5 cm diameter disc was taken from the margin of a pure R. solani colony grown on PDA medium and placed in the center of each plate. Three replicates were prepared for each concentration. The seventh flask was left without any additive to serve as a control treatment. The plates were labeled with codes and numbers to identify the element and concentration, sealed with parafilm, and incubated at 25 ± 2°C for 5 days until the fungal colony in the control treatment reached the edge of the plate. The colony diameter was measured by recording the dimensions of the growing colony.

Pesticide screening experiment

Three types of reputable and widely available fungicides (Beltanol, Moncut, and Topsin) were tested against the most pathogenic causal agent. For each fungicide, two concentrations were used: (0.025) and (0.05). Each concentration was dissolved in 100 ml of pre-prepared PDA medium in a 250 ml flask, with a total of 7 flasks (100 ml for each concentration of each fungicide). The media containing the above concentrations were poured into Petri dishes inside a sterile laminar flow hood. A 0.5 cm diameter disc was taken from the margin of a pure R. solani colony grown on PDA medium and placed in the center of each plate. Three replicates were prepared for each concentration. The seventh flask was left without any additive to serve as a control treatment.

The plates were labeled with codes and numbers to identify the type of fungicide and concentration, sealed with parafilm, and incubated at 25 ± 2°C for 5 days until the fungal colony in the control treatment reached the edge of the plate. The colony diameter was measured by recording the dimensions of the growing colony.

Enhancement of chitosan microgel with biological and chemical inhibitors and its use as a controlled-release agent for inhibitors, and evaluation of its single and combined inhibitory efficiency

The PDA medium was prepared as described in section (1.1) in 5 flasks of 250 ml capacity, with 100 ml per flask. The Chitosan Microgel compound was fortified with three treatments as follows, leaving the fourth treatment with Chitosan Microgel alone, while the fifth treatment was left without any addition:

(0.025) ml Chitosan Microgel + (0.025) ml of Pseudomonas fluorescens bacterial filtrate
(0.025) ml Chitosan Microgel + (0.025) ml Moncut fungicide
(0.025) ml Chitosan Microgel + (0.025) ml Boron (B)
(0.05) ml Chitosan Microgel
Control treatment

The above treatments were added separately to each flask. Then, the culture media containing the inhibitors were poured into Petri dishes. After the medium solidified, a 0.5 cm disc was taken from the margin of a pure Rhizoctonia solani colony grown on PDA medium and placed at the center of the plate. Three replicates were made for each treatment, while ensuring a control plate without any additives was included. The plates were labeled with codes and numbers to identify the type of compound and concentration, sealed with parafilm, and incubated at 25 ± 2°C for 5 days until the colony in the control treatment reached the edge of the plate. The colony diameter was measured by recording the dimensions of the growing colony.

Statistical analysis

Experimental data resulting from the complete random design (C.R.D) were analysed using the SAS programme and The least significant difference test was used to compare the means at a probability level of (0.05%).

Results and Discussion

Effect of different fungi on pepper seed germination percentage in petri dishes

The results of testing the effect of fungi on pepper seed germination percentage in Petri dishes Figure 2 showed that most fungal isolates caused a significant reduction in the germination percentage of pepper seeds, which ranged between 6.66 % and 96.6% compared to the control treatment, which reached 96.6 %. The Rhizoctonia solani isolate was superior, preventing pepper seed germination with a rate of 6.66 %, followed by Macrophomina phaseolina, Fusarium solani and Fusarium oxysporum, with germination rates of 63.33%, 50 %, and 10 %, respectively. The highest germination rate (76.66%) was recorded with the Alternaria alternata isolate.

The variation in the effect of fungal isolates on seed germination is attributed to differences in their pathogenicity, toxin production, the quantity and type of these toxins, their genetic makeup, and their ability to secrete enzymes such as cellulase, pectinase, and protease, which contribute to seed decay and prevent germination. In contrast, non-pathogenic fungal isolates lack these enzymes (Hashi, 2020; Avan et al., 2021). El-Kazzaz et al. (2022) reported that R. solani secretes metabolites and cellulase and pectinase enzymes during the initial infection stage, playing a vital role in degrading the cell wall to facilitate host penetration. The more enzymes and toxins an isolate produce, the more pathogenic it is to the plant host.

Testing the effect of different fungicides on the radial growth of R. solani (cm²)

The results of testing the fungicides on the fungal growth of Rhizoctonia solani Figure 3 showed inhibition of fungal growth, with varying effects among the fungicides Beltanol, Moncut, and Topsin. The chemical fungicide Moncut was the most effective, achieving complete inhibition of the pathogenic fungal colony, with a radial growth of 0 cm² at a concentration of 0.05, which was significantly different from the radial growth of 1.83 cm² at a concentration of 0.025, compared to the control treatment where the inhibition percentage was 0.0%. Beltanol followed, with a radial growth of 0.33 cm² at 0.05 concentration compared to 0.66 cm² at 0.025 concentration. The fungicide Topsin recorded the highest radial growth among the fungicides, with values of 3 cm² and 1.5 cm² at 0.025 and 0.05 concentrations, respectively. The superiority of the fungicide Moncut in inhibiting the pathogenic fungus is attributed to its ability to inhibit respiration by affecting the fungal mitochondria, leading to the inhibition of NADH oxidase activity and causing the fungal cell death. It is considered a protective and curative fungicide effective against basidiomycete fungi (Yang and Li, 2012). This study agrees with Al-Jbory et al. (2019) and Abedalred et al. (2019) regarding the inhibitory capacity of fungicides against various fungal pathogens, including R. solani.

Testing the effect of different chemical elements on the fungal growth of R. solani (cm²)

The results in Figure 4 showed the effect of different chemical elements on the fungal growth of R. solani. All treatments caused a significant reduction in the colony diameter. The boron treatment at a concentration of 0.05 resulted in the lowest radial growth, reaching 2 cm², which was significantly different compared to the 0.025 concentration that resulted in a radial growth of 3.83 cm², compared to the control treatment which recorded a radial growth of 9 cm².

Next was the copper treatment, which achieved a radial growth of 2.2 cm² at 0.05 concentration compared to 4.16 cm² at 0.025 concentration. The zinc treatment recorded the highest radial growth among the treatments, with 2.33 cm² at 0.05 concentration compared to 4.66 cm² at 0.025 concentration. These results are in agreement with the study by Erper et al. (2019), who used a 1% concentration of boron against R. solani isolates, leading to a reduction in fungal growth by (95.07%–97.61%). The results indicate that boron may serve as an alternative to synthetic fungicides against root rot. Madusanka et al. (2024) also reported, in their study on several elements against R. solani, the ability of copper to inhibit the fungus, achieving the highest inhibition rate among the tested elements at 73.43% at a concentration of 1000 mg/L. Mahmoud (2024) similarly noted, in his study on root and stem rot caused by R. solani in faba bean, the inhibitory effect of zinc on the fungus, showing a significant reduction in fungal growth.

Testing the effect of P. fluorescens and B. subtilis bacterial isolates on the radial growth of R. solani (cm²)

The experiment results in Figure 5 illustrate the effect of the two bacterial isolates on the radial growth of R. solani. The bacterium P. fluorescens gave the highest inhibition of the fungal radial growth, reaching 3.1 cm² and 6.7 cm² at concentrations of 0.05 and 0.025 respectively, compared to the control treatment, which recorded an inhibition rate of 0.0%. This was followed by B. subtilis, which gave a lower inhibition rate of fungal colony growth, reaching 3.7 cm² and 7.1 cm² at concentrations of 0.05 and 0.025, respectively.

P. fluorescens produces several secondary metabolites such as antibiotics like pyrrolnitrin, phenazine, and DAPG (Raaijmakers and Mazzola, 2012). Antagonistic bacteria act by producing antifungal substances that inhibit the hyphal growth of pathogenic fungi; P. fluorescens produces many antifungal compounds such as pyrrolnitrin, pyoluteorin, phenazines, siderophores, cyanide, and 2,4-diacetylphloroglucinol (Al-Jbory et al., 2025).

This study is consistent with Mohamed et al. (2021) regarding the effect of some bacterial isolates in inhibiting fungal growth in vitro and studying their antagonistic activity, where B. subtilis gave the highest reduction in pathogen radial growth, with an inhibition rate of 74.76%.

Testing the effect of different concentrations of chitosan and chitosan microgel on the radial growth (cm²) of R. solani

The results in Figure 6 showed the effect of different concentrations of Chitosan and Chitosan Microgels on the radial growth of R. solani. Both Chitosan and Chitosan Microgels resulted in the lowest radial growth of the fungal isolate, reaching 4.16 cm² and 2.83 cm², respectively, at concentrations of 100% and 0.08 each, compared to the control treatment, which showed a growth of 9 cm². This was followed by Microgel concentrations of 75% and 50%, with inhibition values of 5.33 cm² and 4 cm², and Chitosan at concentrations of 0.04 and 0.06, with inhibition values of 7 cm² and 5.33 cm², respectively. The lowest reduction in fungal growth was recorded at a concentration of 25% for Microgels (6.33 cm²) and 0.02 for Chitosan (7.83 cm²).

This study agrees with Freddo et al. (2014), who evaluated the effectiveness of Chitosan in reducing the growth of R. solani, using four different concentrations of Chitosan (0, 0.25, 0.5, 2%), showing a significant effect on fungal growth in vitro, with inhibition increasing as the concentration increased. Fungi tend to produce acids at varying levels, making the growth medium acidic, which stimulates protonation of the amino group in Chitosan, leading to the destruction of biomolecules. Chitosan activity against fungi is higher at low pH values due to its increased solubility compared to other media (Sathiyabama and Muthukumar, 2020; Ahmed et al., 2025).

Results of the effect of several treatments with chitosan microgel on the radial growth of R. solani (cm²)

The results in Figure 7 showed a significant reduction in the radial growth of the fungal colony in all treatments, with values ranging from 0 to 2.66 cm², compared to the control treatment, which showed a radial growth of 9 cm².The treatment Chitosan Microgel + pesticide gave the lowest radial growth of the pathogenic fungus, reaching 0 cm², followed by Chitosan Microgel + Boron and Chitosan Microgel + Bacteria with radial growths of 1.83 cm² and 2.41 cm², respectively.

The Microgel pure treatment recorded the highest radial growth compared to the other treatments, reaching 2.66 cm². This is consistent with Mahdi et al. (2024), who indicated that the use of hydrogels reduces the impact of plant pathogens. These results also align with several studies that concluded that using multiple control agents has a greater effect in inhibiting the pathogen through a synergistic interaction of these agents. Additionally, they contribute to making the environment unsuitable for the pathogen’s growth by limiting its access to nutritional requirements, as well as inhibiting and restricting its growth (Al-Mashhadani, 2022).

Conclusions

From the above, it can be concluded that the Chitosan Microgel compound has a high inhibitory efficacy against the pathogen R. solani, and that its inhibitory efficiency can be enhanced by loading it with chemical inhibitors and biological filtrates. This makes it an inhibitory agent and a carrier material that aids in the controlled release of inhibitors, in addition to being environmentally friendly and biodegradable and This approach may reduce synthetic fungicide dependency in greenhouse and field conditions.

Acknowledgment

We are greatly thankful to our staff of Plant Protection Department, College of Agriculture, Tikrit University for their help and support.

Novelty Statement

This manuscript represents a new aspect in the development of natural compounds and their conversion into gel form for use in combating plant diseases.

Author’s Contribution

Sara H. Aftean: The first author did the work Conceived the idea, Wrote abstract, Methodology.

Awf A. Ahmed Al-Jbory: The second author did the work SPSS analysis, Conclusion, Technical Input at every step, Overall Management of the article, Data collection, Result and discussion, introduction, References.

Funding

This study is funded by the researchers’ own account.

Generative AI and AI-assisted technology statement

The authors declare that no Genrative AI was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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