Antifungal Activity of Medicinal Plant Extracts Against Postharvest Fungi of Fruits and Vegetables with in vivo Protection of Tomato Fruits

2.1 Plant sample collection and extract preparation

The plant parts, clove flowers, cinnamon bark, bay leaves, cumin seeds, anise seeds, and wild thyme flowers were collected from local markets (Table 1) and milled into a fine powder to show the important effect on pathogenic fungi. Extraction was performed using the cold maceration method as described by research [7], 5 g of the plant powder were infused in 50 mL of sterile distilled water for 48 hours using a shaker in the lab. temperature (25 ℃) and the extraction was repeated twice. The mixture was filtered using Whatman No. 1 filter paper to obtain the crude extract (100%) from which all other concentrations were prepared: 0, 25, 50, 75, 100% [8]. The extract should be stored in the refrigerator at 4 ℃.

Table 1. Plants used in the study

2.2 Isolation and identification of fungi

Fungi (previously identified) were isolated from infected fruits and vegetables on potato dextrose agar (PDA) medium after surface sterilization of the tissues with 1% sodium hypochlorite solution. The isolates were purified, and the isolated fungi were observed microscopically based on the morphological characteristics of the spores and conidia, and using the taxonomic keys of research [9].

2.3 Effect of extracts on radial growth

The poisoned food technique was used to test the effect of extracts on radial growth. Different concentrations of each extract were added to sterile PDA medium before solidification. The plates were inoculated with a fungal disc (5 mm) and incubated at 27 ± 2 ℃. The adjusted colony diameter (cm) was measured at full growth in the control treatment, and the percentage inhibition was calculated according to the equation:

$\operatorname{Inhibition}(\%)=\frac{d_c-d_t}{d_c} \times 100$

where, d_c = colony diameter in the control; d_t = colony diameter in the treatment.

2.4 Effect of extracts on biomass: Fresh and dry weight

The experiment was performed in potato dextrose broth medium (PDB) following research [10] with some modifications. A total of 600 mL of medium was aliquoted into six 200 mL Erlenmeyer flasks, with 100 mL of PDB per flask. Plant extracts were added to each flask to achieve a final concentration of 100%, after which the flasks were inoculated with one mycelial plug and incubated at 28 ℃ for 14 days (pH 6.7). The experiment was repeated twice. After incubation, the fresh and dry weights were measured by balance:

Fresh weight: Mycelium was harvested by filtration using filter paper No. 1, washed with distilled water, and surface-dried using blotting paper prior to measurement [11].

Dry weight: Mycelium was oven-dried at 60 ℃ for 24 hrs. until a constant weight was reached, and the dry weight was recorded in milligrams [12].

2.5 In vivo tomato fruit protection assay

An in vivo test was performed to evaluate the protective efficacy of the extracts on tomato fruits (Solanum lycopersicum) according to study [13]. Healthy fruits were superficially sterilized and then immersed in the plant extract (optimum concentration) for 5 minutes. After drying, a 2 mm deep wound was made where the fruits were inoculated with a spore suspension (1 × 10⁶ spores/ml). The fruits were incubated in humid chambers at 25 ℃. The severity of infection was assessed by measuring the rot rate after 7 days.

A special scale was employed for the disease index, comprising four degrees of disease severity [14]:

0 = Healthy fruit

1 = Fruits covered by rot at a rate of 1–25% of their surface area

2 = Fruits covered by rot at a rate of 26 – 50% of their surface area

3 = Fruit covered by rot at a rate exceeding 50% of its area

The percentage of infection severity was calculated according to the following equation:

Infection severity $(\%)=\frac{\left(n_0 \times 0\right)+\left(n_1 \times 1\right)+\left(n_2 \times 2\right)+\left(n_3 \times 3\right)}{N \times 3} \times 100$

2.6 Statistical analysis

Data were statistically analyzed using analysis of variance (ANOVA) according to a completely randomized design (CRD). Mean values were compared using the least significant difference (LSD) test at the 0.05 level of significance.

The Statistical Packages of Social Sciences [15] program was used to test the effect of group differences on the study variables. LSD was used to compare the level of significance of the difference between means.

Table 2 shows that increasing the concentration of clove extract significantly reduced the growth diameters of all fungi species examined, indicating a concentration-dependent inhibitory effect. The effect was mostly observed at higher concentrations (75% and 100%), where the lowest fungal growth values were recorded. This aligns with study [16] which explained that the antifungal activity of clove increases with increasing concentration of its active phenolic compounds, particularly eugenol; It has good solubility in organic solvents, and moderate solubility in water. This inhibition is attributed to eugenol ability to disrupt fungal cell membrane integrity and influence the activity of vital enzymes, thereby impairing fungal growth [17]. This study indicates that the substance eugenol affects the cell membrane and thus affects on the fungus viability, and this is confirmed by our current study through its effect on the diameter of the fungus and its biomass.

A difference in the sensitivity of the studied fungi was also observed, with species, such as Fusarium and A. niger exhibiting a greater response to higher concentrations. This may be ascribed to variations in cellular structure and defense mechanisms among the fungal species [18]. These results are consistent with recent studies confirming the potential use of clove extracts as natural and relatively safe alternatives to chemical fungicides, especially in the agricultural and food sectors [19].

The results in Table 3 demonstrate that cinnamon concentrations had a clear and significant inhibitory effect on the growth of all fungi isolated from fruits and vegetables. A gradual decrease in colony diameters was observed with increasing cinnamon concentration compared to the control treatment. Results also reveal variation in the fungi response to different cinnamon concentrations, Fusarium was the most sensitive, while Aspergillus niger exhibited relatively higher resistance. This difference may be attributed to variations in the chemical composition of fungal cell walls, as well as differences in physiological defense mechanisms among fungal species, such as the efficiency of their toxicity pumps and their tolerance to oxidative stress [20]. The antifungal activity of cinnamon is attributed to its content of bioactive compounds, most notably cinnamaldehyde and phenolic compounds, which disrupt cell membrane integrity, inhibit the activity of vital enzymes, and disrupt essential metabolic processes within the fungal cell, ultimately leading to growth inhibition or fungal cell death [21, 22]. The results of this study are consistent with several recent studies that have confirmed the effective role of cinnamon extracts and oils in inhibiting the growth of food spoilage fungi, and their promising role as natural and safe alternatives to traditional chemical pesticides, especially in the post-harvest preservation of fruits and vegetables [23].

The results in Table 4 showed that thyme extract has antifungal properties. The aqueous extract of thyme possesses concentration-dependent antifungal activity, attributed to water-soluble polar compounds such as phenols and flavonoids [24]. Aqueous extracts have relatively limited effectiveness due to the low solubility of the active compounds (such as thymol and carvacrol) in water, while essential oils exhibit a much higher inhibition of fungal growth. The antifungal effect of thyme is attributed to the presence of phenolic compounds, flavonoids, and traces of thymol (even in the aqueous extract, but in lower concentrations). The mechanism of action includes: Disruption of the fungal cell membrane, increased cell permeability, inhibition of vital enzymes, and disruption of metabolic processes. Studies have shown that these effects lead to damage to the fungal cell wall and membrane, and leakage of cell components [25]. Recent studies indicate that the aqueous extract mainly contains: phenolic compounds, flavonoids, tannins, and antioxidants [26].

The results in Table 5 clearly demonstrate an inverse relationship between the concentration of bay extract and the mycelial growth diameters of the fungi, with higher concentrations leading to a significant decrease in growth. This effect is attributed to the presence of bioactive compounds in bay, particularly bay leaves (Laurus nobilis L.), such as phenols, and terpenes, which possess antifungal activity. Recent studies have indicated that these compounds can inhibit fungal growth by disrupting cell membrane permeability and affecting essential enzymatic functions within the fungal cell [27, 28]. The results also revealed a difference in the degree of fungal sensitivity to the extract, with Penicillium and Fusarium appearing to exhibit varying levels of sensitivity. They were more affected by a 100% concentration compared to A. niger and Rhizopus. This difference is supported by variations in genetic and cellular structure among the fungal species, as well as differences in cell wall thickness and chitin and beta-glucan content, which affect the ability of plant compounds to penetrate fungal cells [29]. The results are consistent with recent studies showing that natural plant oils and extracts are promising alternatives to chemical pesticides in controlling food spoilage fungi, especially those isolated from stored fruits and vegetables. Several studies have confirmed that extracts of Laurus nobilis exhibit antifungal activity against Aspergillus, Penicillium, and Fusarium genera at relatively high concentrations [30].

The study results in Table 6 showed that anise extract had a significant inhibitory effect on the growth of the studied fungi. The diameter of the fungal colonies decreased gradually with increasing concentration, indicating a dose-dependent effect. Fusarium oxysporum was the most susceptible compared to the other species, while Aspergillus niger exhibited relative resistance. This effect is attributed to the presence of active compounds in aniseed, such as phenols and anethole, which damage the cell membrane and increase its permeability, leading to leakage of cell components and growth inhibition. These results are consistent with previous studies that demonstrated the antifungal activity of aniseed extracts, particularly against Aspergillus and Penicillium species. However, it should be noted that aqueous extracts are less effective than essential oils [31, 32].

The results in Table 7 are consistent with scientific literature confirming that cumin contains a range of bioactive compounds, including phenols, flavonoids, and terpenoids, as well as cuminaldehyde, which is one of the most important compounds responsible for antifungal activity. These compounds work by disrupting cell membrane integrity and increasing its permeability, leading to leakage of cell components and disruption of vital metabolic processes within the fungal cell. Cumin contains active compounds such as cuminaldehyde and terpenoids and exhibits broad antifungal activity [33].

Table 8 shows that the plant extracts examined exhibited varying degrees of effectiveness in inhibiting fungal growth, reflected in the reduced fresh and dry weight of the fungi compared to the control treatment. Because the results of fungal diameter measurements indicated that a 100% concentration of plant extracts was the most efficient in reducing fungal diameters for all fungi and significantly more so than other concentrations, the experiment was limited to select the most efficient concentration (100%) to determine its effect on the fungal biomass. Cinnamon and clove extract demonstrated the highest inhibitory effect, indicating their broad-spectrum antifungal activity. This effectiveness is attributed to the presence of potent phenolic and terpenoid compounds in these plants, such as thymol, carvacrol, and cinnamaldehyde, which disrupt fungal cell membrane integrity, inhibit cell wall synthesis, and disable enzyme systems responsible for cellular growth [34]. These results go in line with recent studies confirming that plant extracts can reduce the biomass of foodborne pathogenic fungi such as Aspergillus and Fusarium, both in culture media and food applications [35]. The variation in response among different fungal species may be attributed to differences in cell wall structure, chitin and beta-glucan content, and varying resistance to oxidative stress.

Table 9 clearly shows that cinnamon and cloves produced the lowest dry weight values, indicating the strongest inhibitory effect on biomass accumulation. This can be explained through several biochemical and physiological mechanisms:

1. Contain highly active phenolic compounds (eugenol, cinnamaldehyde)
2. Disrupt cell membranes, causing leakage and death
3. Inhibit metabolic and enzymatic pathways
4. Induce oxidative damage in the cells
5. Exhibit broad-spectrum antimicrobial activity. All these mechanisms lead to a decrease in Cell growth, biomass accumulation, and then a decrease in Dry weight

The results in Table 9 show that the aqueous extracts of medicinal plants had a clear effect in reducing the severity of fungal infection of tomato fruits contaminated with A. niger, F. oxysporum, R. stolonifer, and P. expansum. As the infection rates decreased after treatment with plant extracts compared to the condition before treatment, the control treatment recorded the highest severity of infection for all tested fungi, which agrees with research [36].

Cinnamon extract recorded the highest efficiency in reducing the severity of post-treatment infection, as infection rates decreased significantly compared to pre-treatment, especially against A. niger and R. stolonifer. This effect is attributed to cinnamon containing cinnamaldehyde, which has antifungal activity by inhibiting fungal hyphae growth and disrupting cell membrane permeability, thus reducing the fungus's ability to spread within fruit tissues [37].

Clove extract also reflected high efficacy, namely against P. expansum, with decreasing post-treatment infection severity compared to the pre-treatment state. This is attributed to the presence of eugenol, a compound that damages the fungal cell wall and interferes with essential metabolic processes within the fungal cell, leading to inhibited fungal growth or death. This aligns with the findings of studies [38, 39] with regard to the effective role of plant phenolic compounds in fungal resistance.

The results also show that all the studied plant extracts led to a relative decrease in pre-treatment infection severity compared to post-treatment, despite the fact that the degree of this decrease varied depending on the plant species and the fungus species used. This variation is attributed to differences in the chemical composition of the plant extracts and the varying concentrations of their active compounds, as well as differences in the sensitivity of the studied fungi to these compounds. This goes in line with Tripathi and Dubey [40], who observed that R. stolonifer showed resistance to some extracts compared to Aspergillus niger and Penicillium spp. fungi, as infection rates remained relatively higher after treatment. This may be ascribed to the rapid growth of this fungus and its high ability to form spores, in addition to the nature of its cell wall, which reduces the effect of some plant compounds on it, which is consistent with studies [37, 41].

In general, the results confirm that treatment with aqueous extracts of medicinal plants reduced the severity of fungal infection in tomato fruits compared to the pre-treatment condition. This highlights the possibility of using them as natural alternatives or support to chemical fungicides in post-harvest disease management programs, given that they are relatively safe and environmentally friendly.