Potential of Three Plant Extracts in Suppressing Potato Dry Rot Caused by Fusarium incarnatum Under Normal and Cold Storage

Abstract

The potato (Solanum tuberosum L.) is one of the most widely consumed vegetable crops worldwide. During storage, potato tubers are vulnerable to various phytopathogenic fungi. Dry rot, caused by Fusarium incarnatum, is a common and serious disease that affects potato tubers, leading to partial or complete decay during storage. The current study assessed the effectiveness of three ethanolic extracts including cinnamon bark (CIB), clove buds (CLB), and avocado seeds (AVS) in controlling potato dry rot under both normal and cold storage conditions. In vitro bioassay demonstrated that all tested extracts exhibited a dose-dependent fungistatic effect against F. incarnatum, with inhibition percentages of 83.33% for CIB, 72.22% for CLB, and 67.77% for AVS at the highest tested concentration. Moreover, dipping potato tubers in the tested extracts markedly reduced the severity of dry rot disease under both normal and cold storage conditions. Additionally, treated tubers showed increased activities of defense-related enzymes, including catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase. Furthermore, there were higher levels of total soluble phenolics and flavonoids, along with an increase in lignin content and a reduction in the weight loss of stored potato tubers compared to the control group. Moreover, the extracts mitigated infection stress and lowered malondialdehyde levels in the treated potato tubers. These extracts show potential as environmentally friendly alternatives to chemical fungicides for managing potato dry rot caused by F. incarnatum under normal and cold storage.

1. Introduction

The potato (Solanum tuberosum L.) is a significant vegetable crop belonging to the Solanaceae family. It is a cash crop for over a billion people in 150 countries [1]. In 2023, the global production of potato tubers reached 383.08 million tons, cultivated over 16,799,108 hectares [2]. Moreover, Egypt cultivated 235,625 hectares of potato, yielding 29.153 tons per hectare with a total production of 6.86 million tons in the same year [2]. Potato crops are frequently threatened by several phytopathogenic bacteria, fungi, and viruses before harvest, leading to significant economic losses.

Additionally, potato tubers are susceptible to numerous postharvest and storage diseases, primarily caused by phytopathogenic fungi such as Fusarium spp., Helminthosporium solani, Rhizoctonia solani, and Colletotrichum coccodes. These diseases can greatly reduce the quality and marketable yield of the crop. Furthermore, some of these fungi produce mycotoxins that pose health risks to humans [3]. Fusarium, a significant genus of phytopathogenic fungi, is particularly responsible for storage-related dry rot in potato tubers and field wilt [4].

Dry rot, or Fusarium rot, caused by different species of Fusarium, is a significant and destructive disease affecting stored potato tubers [5,6]. It is prevalent worldwide in all potato-growing regions, resulting in yield losses ranging from 10 to 25%, and in extreme cases, the losses can soar to 60% [7,8,9]. Symptoms of dry rot disease in tubers manifest as shrinkage, wilting, and mummification, accompanied by spots and cracks on the dead tissues. These symptoms typically emerge during the storage phase, and cottony growths, which are the mycelium of the pathogen, become visible in the impacted areas. Over time, the affected tubers deplete their reserves of dry matter and nutrients [10].

More than thirteen species of Fusarium are known to cause potato dry rot disease worldwide. The distribution of each species varies depending on the season and geographical location [11]. The most prevalent species globally include F. solani, F. sambucinum, F. oxysporum, F. avenaceum, and F. culmorum [12,13]. However, Egypt’s primary species responsible for potato dry rot are F. sambucinum, F. incarnatum, F. solani, F. oxysporum, and F. verticillioides [14,15]. In our previous study, we isolated F. incarnatum from stored potato tubers, identified it genetically as the F. incarnatum isolate AE 2024, and deposited it in GenBank with the deposit number PP086049 [16].

Synthetic fungicides such as imazalil, flusilazole, thiabendazole (TBZ), and difenoconazole have been commonly used to control Fusarium infections [4]. However, the emergence of resistance in Fusarium species has become a major challenge [17]. Recent studies highlight an alarming rise in resistance to these chemical fungicides, reducing their efficacy in managing Fusarium-related diseases [18,19]. Consequently, there is a critical need to update effective and sustainable technologies for controlling potato dry rot. Some alternatives include plant extracts, organic and inorganic salts, essential oils, natural fumigants, ultraviolet radiation, biocontrol agents, and nanoparticles [20].

Plant extracts have been widely recognized for their effectiveness in controlling potato dry rot, demonstrating significant efficacy in suppressing and managing Fusarium species [8,21]. Among these, cinnamon (Cinnamomum cassia L.), an aromatic plant from the Lauraceae family, is extensively used as a natural fungicide due to its potent antifungal properties against various phytopathogenic fungi [22]. It contains several bioactive compounds, notably cinnamaldehyde and eugenol, which are well-documented for their antifungal and antibacterial activities [22,23,24].

Similarly, cloves (Syzygium aromaticum L.), a member of the Myrtaceae family, are highly valued in traditional medicine for their diverse biological activities. They are rich in phytochemicals such as monoterpenes, sesquiterpenes, phenols, and hydrocarbons which contribute to their antibacterial, antifungal, antioxidant, and anticancer properties [25].

Eugenol, the primary active compound in cloves, exhibits strong antifungal and antimicrobial effects, making it a promising natural alternative for managing plant diseases [25]. Avocado (Persea americana) seeds can also be made into plant extracts for disease management, offering a sustainable and eco-friendly alternative to chemical fungicides [20]. The selection of CIB, CLB, and AVS extracts in this study is based on their demonstrated effectiveness in previous research. For instance, CIB extract has exhibited significant antifungal activity against Botrytis cinerea [26], F. oxysporum, and F. solani [27]. Likewise, CLB extract has shown remarkable efficacy in controlling several postharvest diseases, including Alternaria alternata, A. solani, and B. cinerea [28].

Furthermore, avocado peel and seed extracts have demonstrated strong antifungal activity against F. oxysporum, B. cinerea, Rhizoctonia solani, Aspergillus niger, and Penicillium expansum, both in vitro and in vivo [29]. These findings highlight the potential of these natural extracts as effective biocontrol agents for plant disease management, further supporting their selection in this study.

In this study, we explored the in vitro antifungal properties of ethanolic extracts from cinnamon (CIB), clove (CLB), and avocado seeds (AVS) against F. incarnatum. Additionally, we examined the effects of these extracts on potato tubers infected with F. incarnatum under storage conditions using a whole-plant bioassay. We propose that these extracts offer sustainable, alternative, and eco-friendly strategies to partially or completely replace chemical fungicides in managing fungal diseases in potatoes.

2. Materials and Methods

2.1. Plant Materials

Potato tubers (S. tuberosum cv. Spunta) were obtained from a private farm in Tanta, Gharbia Governorate, Egypt (30.7885° N, 31.0019° E). These tubers were carefully selected for their uniform size and absence of diseases and damage. Immediately after harvest, the healthy tubers were transported to the plant pathology laboratory at the Faculty of Agriculture at Tanta University for further experimentation.

2.2. Fusarium incarnatum, the Causal Agent of Potato Dry Rot

F. incarnatum isolate AE 2024 was originally obtained from potato tubers showing typical dry rot symptoms as detailed in our previous study [16]. This isolate was purified using the single-spore method and identified according to morphological and macroscopic characteristics. Subsequently, it was molecularly identified based on sequencing of the internal transcribed spacer (ITS) region, and the sequence was submitted to GenBank under accession number PP086049. The spore suspension was prepared as follows: A 0.5 cm F. incarnatum plug was placed in a potato dextrose agar (PDA) medium and incubated for 10 days at 25 ± 2 °C. After incubation, spores were harvested from the agar surface using a sterile glass rod. The high-density spore suspension was filtered through more than one layer of cheesecloth. The spore concentration was determined using a hemacytometer and diluted with sterile water to achieve a spore inoculum concentration of 1 × 10⁶ spores per mL.

2.3. Preparation of Plant Extracts

Cinnamon bark (CIB), clove buds (CLB), and avocado seeds (AVS) isolated from the fruits were purchased from the local market. The plant materials were separately dried at 40 °C, ground into powder using a household electric grinder (Generic, Ningbo, China), and then sieved through a 1.5 mm mesh to ensure a uniform particle size. Subsequently, 100 g of each powdered plant material was mixed with 1000 mL of 80% ethanol (purchased from Top Medica company, Cairo, Egypt), and agitated at 200 rpm for 48 h [30]. Afterward, the solutions were filtered, and the ethanol was evaporated using a rotary evaporator (purchased from Lap Egypt company, Giza, Egypt), to obtain the crude extracts, which were then kept at 4 °C for additional use.

2.4. In Vitro Antifungal Properties of Plant Ethanolic Extracts

The crude extracts were first dissolved in 2 mL of pure dimethyl sulfoxide (DMSO) and then diluted with distilled water to create a 50 mg/mL stock solution. Specific volumes of this stock solution were mixed with PDA medium to achieve the desired concentrations (2.5, 5, 7.5, and 10 mg/mL), and each concentration was replicated six times. A 0.5 cm plug of F. incarnatum was placed at the center of a 9 cm Petri dish, which was then incubated for one week at 25 ± 2 °C. Control PDA plates contained DMSO (0.2%), without the ethanol extract. The inhibition of mycelial growth was calculated using the following formula:

$$\mathrm{Inhibition} (\%)={\displaystyle \frac{C-T}{C}}\times 100$$

(1)

where ‘C’ represents the mycelial growth in the control (measured in cm), and ‘T’ represents the growth in the treated plates (measured in cm).

2.5. Control of Potato Dry Rot Disease in Tubers Under Storage Conditions

Based on the results obtained from in vitro experiments, we conducted further experiments between March and May of 2023 and 2024 to evaluate the effectiveness of CIB, CLB, and AVS ethanolic extracts at a concentration of 10 mg/mL in controlling F. incarnatum. To determine the practical application of these extracts in controlling dry rot, potato tubers were treated with the same concentration (10 mg/mL) and stored under various conditions, including normal and cold conditions. The storage experiments were conducted at the Faculty of Agriculture, Tanta University, Egypt. Potato tubers were washed with running water, treated with 2% sodium hypochlorite for three minutes, rinsed with distilled water, and air-dried for two hours. Each tuber was then punctured with a sterilized nail (3 mm wide and deep) around the middle, and 20 µL of F. incarnatum conidial suspension (1 × 10⁶ conidia/mL) was applied to each puncture. The tubers were then incubated in darkness at 24 ± 2 °C for 24 h in plastic containers with sterile water in uncovered Petri dishes to maintain high humidity. Subsequently, the tubers were dipped for ten minutes in a 10 mg/mL ethanol extract solution or distilled water (for the control group) and air-dried for two hours at room temperature. The inoculated and treated tubers were then stored under normal (room temperature, approximately 20–25 °C, with 70–80% humidity) or cold conditions (10 ± 1 °C, with 80–90% humidity) for 90 days. The disease severity was assessed after 30, 60, and 90 days by measuring the diameter of the lesions, following the methodology outlined in [21].

2.6. Biochemical Changes in Treated Potato Tubers

All biochemical measurements were performed using a UV-160 spectrophotometer (Model SM1200 UV-Vis, AZZOTA Corporation, Claymont, DE, USA). Biochemical parameters were assessed in both normal and cold storage experiments. Samples from the treated potato tubers were collected at 1, 2, 3, 4, and 5 days after plant extract treatment.

2.6.1. Activities of Defense-Related Enzymes

Samples were taken at intervals of 24, 48, 72, 96, and 120 h post-treatment (hpt), with the tested extracts being used to assay defense-related enzymes, such as catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL). For the assays of CAT, POD, and PPO, 500 mg of potato tissue was homogenized with 3 mL of 50 mM Tris buffer (pH 7.8) containing polyvinylpyrrolidone (PVP; 7.5%) and ethylenediaminetetraacetic acid (EDTA-Na₂; 1 mM). The homogenate was then centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was harvested for enzyme assays, following the methods described by [31].

The Catalase Enzyme Activity

The activity of the CAT enzyme was measured following a modified protocol from references [31,32]. This method consisted of mixing 0.075 mL of crude enzyme extract with 0.150 mL of 269 mM hydrogen peroxide and 3 mL of 0.1 mM sodium phosphate buffer (pH 6.5). The extinction coefficient for hydrogen peroxide was 0.040 mM⁻¹ cm⁻¹. The CAT activity was determined by tracking the reduction in hydrogen peroxide at 240 nm using a quartz cuvette.

The Peroxidase Activity

The activity of POD was evaluated using a method based on [33], with some modifications as described in [34]. The reaction mixture included 2.2 mL of 100 mM sodium phosphate buffer (pH 6.0), 0.1 mL of 12 mM hydrogen peroxide, 0.1 mL of guaiacol, and 0.1 mL of crude enzyme extract. The formation of the conjugate was monitored by measuring the increase in absorbance at 436 nm, using an extinction coefficient of 26.6 mM⁻¹ cm⁻¹.

The Polyphenol Oxidase Activity

The PPO enzyme activity was evaluated following the method outlined by Malik and Singh [35]. In this assay, 3 mL of a freshly prepared catechol solution (0.01 M) in 0.1 M phosphate buffer (pH 6.0) was mixed with 0.1 mL of crude enzyme extract. The PPO activity was measured by recording the absorbance changes at 495 nm every 30 s over three minutes.

Phenylalanine Ammonia-Lyase

Phenylalanine ammonia-lyase (PAL) was extracted using 5 mL of a 100 mM boric acid buffer (pH 8.8) containing 4% (w/v) PVP, 1 mM EDTA, and 50 mM β-mercaptoethanol. The mixture was centrifuged at 12,000 rpm for 30 min at 4 °C [36], and the supernatant was used for enzyme assays. The enzyme activity was measured using a modified version of the method described by [37]. In this assay, 0.5 mL of the enzyme extract was mixed with 0.5 mL of 20 mM L-phenylalanine and 2 mL of 50 mM borate buffer (pH 8.8) and incubated at 37 °C for 1 h. The reaction was terminated by adding 0.1 mL of 6M HCl. The increase in absorbance at 290 nm, indicating trans-cinnamate formation, was measured with a spectrophotometer. PAL activity was expressed as 0.01 A290 mg⁻¹ protein h⁻¹ [37].

2.6.2. Total Soluble Phenolic Compounds

To determine the total soluble phenolic compounds, the Folin–Ciocalteu reagent was used, following the method outlined by Kähkönen [38]. Potato tubers were sampled 24, 48, 72, 96, and 120 h post-treatment (hpt). A 100 mg sample of potato tissue was homogenized and extracted with 20 mL of 80% methanol for 24 h. The reaction mixture consisted of 0.2 mL of the methanolic extract, 1 mL of 10% Folin–Ciocalteu reagent, and 0.8 mL of 7.5% sodium carbonate (w/v). After vortexing for 30 s, the mixture was incubated at room temperature for 30 min. Absorbance was measured at 765 nm using a spectrophotometer. A standard gallic acid (GA) curve was used to quantify the total soluble phenolics, expressed as mg of gallic acid equivalents per gram of fresh weight (mg GAE g⁻¹ FW).

2.6.3. Total Soluble Flavonoid Compounds

The total soluble flavonoid content was measured using the method specified in [39]. In summary, 1 mL of methanolic extract from potato tubers was mixed with 1 mL of a 2% aluminum chloride solution in methanol. The mixture was thoroughly combined and incubated at room temperature for 15 min. Absorbance was then recorded at 430 nm using a spectrophotometer. The total soluble flavonoids were quantified and expressed as milligrams of rutin equivalents per gram of fresh weight (mg RE g⁻¹ FW).

2.6.4. Assessment of Malondialdehyde as an Indication of Lipid Peroxidation

Malondialdehyde (MDA) levels were determined with slight modifications to the method described in [40]. Briefly, 1 g of fresh potato tissue was homogenized and mixed with 20% trichloroacetic acid (TCA) containing 0.01% butylated hydroxytoluene (BHT). The mixture was then heated to 95 °C and centrifuged at 12,000 rpm for 10 min. The absorbance of the supernatant was recorded at 532 nm and 600 nm using a spectrophotometer to determine the MDA concentration. The results were expressed in nanomoles per gram of fresh weight (FW).

2.7. Determination of Lignin Content (%)

The percentage of lignin content was determined according to the methods outlined by [41]. First, 10 g of potato slices (m) were immersed in 12 mL of a 70% sulfuric acid solution for 60 min at a temperature of 40 °C. After this period, the mixture was diluted to 200 mL of 4% sulfuric acid and boiled for 60 min. The resulting sediment was thoroughly rinsed with distilled water and dried overnight at 60 °C. Once dried, the sediment was weighed (m1). The lignin content (LC) was then calculated using the following formula:

$$\mathrm{LC} (\%)={\displaystyle \frac{m1}{m}}\times 100$$

(2)

2.8. The Weight Loss (%) of Potato Tubers

The weight loss (%) of potato tubers was determined by calculating the percentage reduction from their initial weight after being stored under both normal and cold conditions for three months. This involved weighing the tubers before and after the storage period to assess the extent of weight loss.

2.9. Statistical Analysis

The in vitro bioassay was analyzed using a split-plot design, where the extracts (treatments) were assigned to the main plots, while concentrations were assigned to the sub-plots. Significant differences in the main effects of the extracts (p _Treatments), concentrations (p _{Concentrations}), and their interaction (p _{Treatment × Concentrations}) were determined using analysis of variance (ANOVA). Tukey’s honestly significant difference (HSD) test was used for post hoc analysis, based on the interaction p-value (p _{Treatment × Concentrations} ≤ 0.05). Subsequent experiments followed a completely randomized design (CRD). The data were analyzed with ANOVA, and the HSD test (p < 0.05) was used for post hoc analysis and pairwise comparisons. Each experiment was repeated twice, with six replicates per treatment. The results presented are the averages from these two experiments.

3. Results

3.1. In Vitro Antifungal Activity of Tested Ethanolic Extracts Against F. incarnatum

The antifungal properties of three plant extracts, CIB, CLB, and AVS, against F. incarnatum were evaluated in vitro. All extracts demonstrated dose-dependent antifungal activities, significantly reducing mycelial radial growth on potato dextrose agar (PDA) plates (Figure 1A). CIB was the most potent, achieving an 83.33 ± 1.11% inhibition of mycelial growth at 10 mg/mL, significantly more effective than both CLB (72.22 ± 1.11%) and AVS (67.77 ± 1.11%) at the same concentration (p < 0.05). Interestingly, the CIB extract at 7.5 mg/mL had a similar inhibitory effect to the AVS extract at 10 mg/mL, with no significant difference between these treatments (p > 0.05) (Figure 1B). This demonstrates the strong antifungal properties of the CIB extract, even at lower concentrations, highlighting its potential as an effective means of controlling F. incarnatum.

3.2. Control of Potato Dry Rot Using Plant Extracts Under Normal and Cold Conditions

Generally, all tested plant extracts significantly reduced the progression of dry rot disease in potato tubers under both normal and cold storage conditions (Figure 2A,B). Over 30, 60, and 90 days post-treatment (dpt), the CIB extract was the most effective, resulting in the smallest lesion diameters. Under normal storage conditions, the lesion diameters were 6.56 ± 0.84 mm, 15.22 ± 1.17 mm, and 18.11 ± 1.50 mm at 30, 60, and 90 days, respectively. In cold storage, the lesion diameters were 3.32 ± 0.58 mm, 8.02 ± 0.31 mm, and 10.42 ± 0.52 mm at the same intervals. The CLB and AVS extracts also effectively reduced lesion sizes, with no significant differences. In contrast, the control group consistently showed the largest lesion diameters at all time points, measuring 20.18 ± 1.61 mm, 33.00 ± 2.33 mm, and 47.50 ± 0.47 mm under normal conditions, and 8.34 ± 0.20 mm, 14.17 ± 0.55 mm, and 24.73 ± 0.99 mm under cold conditions. Importantly, the statistical analysis indicated no significant interaction between extract type and storage conditions for lesion diameter measurements.

3.3. Tested Extracts Enhanced the Defense-Related Enzymes

3.3.1. Catalase Activity

The influence of dipping potato tubers in ethanolic extracts of CIB, CLB, and AVS compared to the control on CAT activity under normal and cold storage conditions is shown in Figure 3A,B. Generally, all tested extracts markedly increased CAT activity compared to control in both normal and cold conditions. Under normal conditions, CAT activity sharply increased at 24 h post-treatment (hpt) with CLB extract (88.47 ± 5.11 µm H₂O₂ g⁻¹ FW min⁻¹), while CIB and AVS extracts showed peak activity at 72 hpt (142.62 ± 1.78 and 80.98 ± 2.27 µm H₂O₂ g⁻¹ FW min⁻¹, respectively, Figure 3A). Under cold storage conditions, CAT activity exhibited a more gradual increase. CLB and AVS extracts reached their highest activity at 48 hpt, whereas CIB treatment led to a steady rise in CAT activity, peaking at 96 hpt (Figure 3B). These results indicate that storage temperature influences the timing and intensity of the enzymatic response, with cold conditions delaying the peak CAT activity compared to normal storage.

3.3.2. Peroxidase Activity

Similarly, peroxidase (POD) activity peaked at 24 h post-treatment with AVS extract under normal conditions, recording 3.921 ± 0.278 × 10⁻² μM Tetraguaiacol g⁻¹ FW min⁻¹. However, CIB and CLB extracts showed their maximum activity at 48 hpt, with values of 4.383 ± 0.154 and 3.864 ± 0.102 × 10⁻² μM Tetraguaiacol g⁻¹ FW min⁻¹, respectively, as indicated in Figure 4A, followed by a gradual decline. Furthermore, Figure 4B illustrates that the highest peaks of POD enzymatic activity for all tested extracts occurred at 72 hpt under cold conditions, with the CIB extract displaying the highest activity at 6.113 ± 0.219 × 10⁻² μM Tetraguaiacol g⁻¹ FW min⁻¹.

3.3.3. Polyphenol Oxidase Activity

The CLB extract significantly enhanced the enzymatic activity of polyphenol oxidase more than the CIB and AVS extracts, peaking at 24 h of treatment (1.20 ± 0.053 × 10⁻¹ arbitrary units) before declining. Conversely, the CIB and AVS extracts reached their maximum activity at 72 h during the storage experiment under normal conditions (1.45 ± 0.0006 and 1.06 ± 0.128 × 10⁻¹ arbitrary units, respectively, as shown in Figure 5A). However, in refrigerated storage, all extracts achieved their highest activity at 48 h, with the CIB extract exhibiting the greatest polyphenol oxidase activity (Figure 5B).

3.3.4. Phenylalanine Ammonia-Lyase Activity

Additionally, we investigated the activity profile of the enzyme phenylalanine ammonia-lyase (PAL). The results indicated that all tested extracts led to a gradual increase in PAL activity, peaking at 72 h post-treatment. This was followed by a rapid decline in activity at 96 and 120 h post-treatment under both normal and refrigerated conditions, as illustrated in Figure 6A and Figure 6B, respectively. Briefly, in treated potato tubers, PAL activity consistently increased until the third day, reaching levels approximately three times higher than the control. This was followed by a significant decline during the final two days of the study, as shown in Figure 6. Despite this decrease, enzyme activity in the treated samples remained higher than in the control throughout the study period.

3.4. Tested Extracts Enhanced Total Soluble Phenolic Content

In both normal (

Table 1. Effect of tested extracts on total soluble phenolics of F. incarnatum-infected potato tubers under normal conditions.

Treatment	Total Soluble Phenolics (mg GAE g−1 FW)
	Hours Post-Treatment (hpt)
	24	48	72	96	120
Control	12.47 ± 0.57 d	24.81 ± 0.14 d	22.26 ± 0.96 c	6.97 ± 0.36 d	3.56 ± 0.39 d
CIB	15.22 ± 0.02 c	33.30 ± 0.05 b	35.54 ± 0.04 a	23.07 ± 0.04 b	4.72 ± 0.12 c
CLB	27.46 ± 0.03 a	35.95 ± 0.17 a	19.48 ± 0.05 d	16.23 ± 0.04 c	14.13 ± 0.02 a
AVS	16.12 ± 0.13 b	27.23 ± 0.10 c	33.80 ± 0.14 b	25.21 ± 0.08 a	8.08 ± 0.18 b
	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001

Values are presented as the mean ± standard deviation (SD). Different letters within each column indicate significant differences, as determined by Tukey’s HSD test at p < 0.05.

) and cold (

Table 2. Effect of tested extracts on total soluble phenolics of F. incarnatum-infected potato tubers under cold conditions.

Treatment	Total Soluble Phenolics (mg GAE g−1 FW)
	Hours Post-Treatment (hpt)
	24	48	72	96	120
Control	5.38 ± 0.10 d	12.17 ± 0.08 d	8.63 ± 0.22 d	3.14 ± 0.31 c	1.19 ± 0.12 d
CIB	13.38 ± 0.04 b	21.28 ± 0.06 b	23.11 ± 0.03 b	33.23 ± 0.15 a	21.47 ± 0.04 a
CLB	24.45 ± 0.11 a	34.14 ± 0.71 a	26.55 ± 0.71 a	23.52 ± 0.08 b	19.33 ± 0.06 b
AVS	8.59 ± 0.18 c	15.77 ± 0.07 c	17.83 ± 0.10 c	34.39 ± 0.96 a	12.46 ± 0.36 c
	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001

Values are presented as the mean ± standard deviation (SD). Different letters within each column indicate significant differences, as determined by Tukey’s HSD test at p < 0.05.

) conditions, all tested extracts increased the total soluble phenolic content of infected potato tubers similarly to the effects observed with the studied enzymes. Under normal conditions, dipping tubers in ethanolic extracts of CIB, CLB, and AVS significantly boosted the total soluble phenolic content compared to untreated tubers. Initially, the CLB extract exhibited the highest total soluble phenolic content during the first two days, compared to the CIB and AVS extracts (Table 1). However, this trend reversed on the third and fourth days post-treatment, with the CIB and AVS extracts surpassing the CLB extract in total soluble phenolic content (Table 1). Similarly, under cold conditions, the CLB extract was the most effective at enhancing the total soluble phenolic content in potato tubers, showing the highest value after 48 h of treatment. However, after 96 h, both the CIB and AVS extracts surpassed the CLB extract, achieving the highest levels of total soluble phenolic compounds, with no significant difference between them (Table 2).

3.5. Tested Extracts Enhanced Total Soluble Flavonoids

During the first 24 h of storage under normal conditions, there were no significant differences in the total soluble flavonoid content between the control and all tested extracts. However, after 48 h of treatment, a notable increase was observed in the extract treatments (

Table 3. Effect of tested extracts on total soluble flavonoids of F. incarnatum-infected potato tubers under normal conditions.

Treatment	Total Soluble Flavonoids (mg RE g−1 FW)
	Hours Post-Treatment (hpt)
	24	48	72	96	120
Control	1.08 ± 0.49 a	2.76 ± 0.14 c	0.84 ± 0.01 a	0.70 ± 0.21 a	0.39 ± 0.01 a
CIB	3.95 ± 0.01 a	8.08 ± 0.96 a	2.41 ± 0.78 a	1.27 ± 0.07 a	0.55 ± 0.23 a
CLB	2.04 ± 0.01 a	6.93 ± 0.01 ab	1.49 ± 0.01 a	1.11 ± 0.02 a	0.79 ± 0.01 a
AVS	1.75 ± 0.24 a	5.79 ± 0.28 b	2.31 ± 1.30 a	1.47 ± 0.34 a	0.61 ± 0.22 a
	p = 0.0862	p < 0.0001	p = 0.164	p = 0.0091	p = 0.0771

Values are presented as the mean ± standard deviation (SD). Different letters within each column indicate significant differences, as determined by Tukey’s HSD test at p < 0.05.

). Among these, the CIB extract showed the highest value (8.07 ± 0.96 mg RE g⁻¹ FW). Under cold storage conditions, the effectiveness of the extracts was further enhanced, significantly boosting the total soluble flavonoid content, as the highest flavonoid content was observed in the AVS treatment, followed by the CIB extract (

Table 4. Effect of tested extracts on total soluble flavonoids of F. incarnatum-infected potato tubers under cold conditions.

Treatment	Total Soluble Flavonoids (mg RE g−1 FW)
	Hours Post-Treatment (hpt)
	24	48	72	96	120
Control	1.79 ± 0.04 a	2.53 ± 0.02 d	1.89 ± 0.13 c	1.40 ± 0.03 c	1.19 ± 0.06 c
CIB	2.16 ± 0.46 a	5.06 ± 0.19 b	3.21 ± 0.20 a	2.53 ± 0.19 a	2.21 ± 0.12 b
CLB	2.28 ± 0.05 a	3.93 ± 0.03 c	2.50 ± 0.04 b	2.22 ± 0.13 b	2.36 ± 0.13 ab
AVS	2.24 ± 0.32 a	8.89 ± 0.18 a	3.42 ± 0.02 a	2.71 ± 0.02 a	2.59 ± 0.03 a
	p = 0.2089	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001

Values are presented as the mean ± standard deviation (SD). Different letters within each column indicate significant differences, as determined by Tukey’s HSD test at p < 0.05.

).

3.6. Tested Extracts Alleviate Lipid Peroxidation

Infection with F. incarnatum markedly increased oxidative stress in potato tubers, as shown by higher malondialdehyde (MDA) levels (Figure 7). However, all tested extracts (CIB, CLB, and AVS) effectively lowered MDA levels, indicating a reduction in oxidative stress. Dipping potato tubers in CIB, CLB, and AVS extracts significantly reduced MDA accumulation in treated infected tubers in both normal and cold conditions (Figure 7A,B). Notably, the CIB extract had the lowest MDA level (17.91 ± 0.211 nmol.g⁻¹ FW) on the fourth day of storage under normal conditions (Figure 7A). Under cold conditions, the AVS extract performed better than CIB and CLB, recording the lowest MDA level throughout the experiment (Figure 7B), with CIB following. These results imply that dipping treatments with these extracts can alleviate oxidative stress in potato tubers infected with F. incarnatum.

3.7. Tested Extracts Elevated Lignin Content in Potato Tubers

Overall, the lignin content in potato tubers remained relatively stable during the first two days of treatment with the tested extracts, regardless of whether they were stored under normal or cold conditions (Figure 8). Briefly, under normal storage, a significant change was observed on the third day post-treatment; the plant extracts reached their highest lignin content values, with the CIB extract showing the highest percentage (26.55 ± 0.37%), followed by the AVS and CLB extracts (21.99 ± 0.84 and 16.37 ± 1.11%, respectively). Notably, the AVS extract recorded the highest value on the fourth day before decreasing (Figure 8A). Under cold storage conditions, a slight change was noted on the third day, with the AVS extract (21.68 ± 1.16%) surpassing the CIB extract to achieve its highest value (25.23 ± 0.89%). Conversely, the cinnamon extract peaked on the fourth day of treatment (Figure 8B).

3.8. Tested Extracts Effectively Minimized the Weight Loss of Stored Potato Tubers

Potato tubers generally experience weight loss under both normal and refrigerated storage conditions. However, the application of plant extracts significantly reduces this weight loss compared to untreated controls. The untreated control exhibited the highest weight loss, reaching 25.56 ± 1.89%. In contrast, the CIB extract was particularly effective, reducing weight loss to 9.93 ± 1.98% under normal storage conditions (Figure 9A). Under refrigerated conditions, the effectiveness of the plant extracts varied. The CLB extract demonstrated remarkable efficacy, reducing weight loss to 8.44 ± 0.57%, while the AVS extract achieved an even lower weight loss of 6.01 ± 1.44%. Additionally, the cinnamon extract showed significant effectiveness, reducing weight loss to 18.12 ± 1.02% compared to the untreated tubers (Figure 9B).

4. Discussion

Potato dry rot, caused by F. incarnatum, is a serious postharvest disease that threatens potato tubers during storage and leads to significant economic losses worldwide. Addressing this issue is vital for ensuring the quality and yield of potato crops. Synthetic fungicides are commonly used to control dry rot. However, their frequent and extensive application can harm human health, the environment, and various organisms. Moreover, these practices have contributed to the development of Fusarium strains that are resistant to these chemicals [42].

Given these challenges, it is essential to explore sustainable and cost-effective strategies. Consequently, researchers are investigating alternatives such as plant extracts [43]. The current study highlights the potent antifungal properties of extracts from cinnamon bark (CIB), clove buds (CLB), and avocado seeds (AVS) against F. incarnatum. The findings demonstrate that these plant extracts inhibit fungal growth in a concentration-dependent manner, with higher concentrations exhibiting more pronounced antifungal effects. This inhibition is likely due to the bioactive compounds present in the extracts, such as cinnamaldehyde in cinnamon, eugenol in cloves [44,45], and persin in avocado seeds [46]. Additionally, avocado extracts contain other bioactive compounds, including phenolic compounds (e.g., hydroxycinnamic acids, hydroxybenzoic acids, flavonoids, and proanthocyanins), acetogenins, phytosterols, carotenoids, and alkaloids, which may also contribute to their antifungal activity [47]. Generally, plant extracts eliminate pathogens through their antimicrobial properties or biologically active compounds [48].

Our findings are consistent with previous studies that have documented the antifungal properties of cinnamon extracts against various pathogens, including C. longissima [49], B. cinerea [26], F. sambucinum [50], Aspergillus sp., Penicillium sp., C. kikuchii, Colletotrichum sp., F. solani, and Phomopsis sp. [51]. Furthermore, the antifungal activities of clove extract were tested against eight species of phytopathogenic fungi, including R. cerealis, F. graminearum, G. graminis, F. oxysporum f. sp. vasinfectum, Valsa mali, C. gloeosporioides, F. oxysporum f. sp. cucumerinum, and C. lagenarium [52]. Additionally, a study assessed the antifungal effects of avocado peel and seed extract against B. cinerea, R. solani, A. niger, and P. expansum [29].

Cinnamon and clove extracts are renowned for their antifungal properties, attributed to their rich content of bioactive compounds such as flavonoids, phenolics, and terpenoids, which effectively combat various pathogenic fungi and other pathogens [53]. Clove extracts, in particular, contain glucuronides, glucosides, and acids like chlorogenic acid [54], in addition to flavonoids including myricetin, quercetin, kaempferol, and ellagic acid [55], many of which exhibit antifungal properties [56,57].

Similarly, cinnamon extracts, which have a phytochemical profile comparable to clove extracts, have also been validated as antifungal agents [53]. Although both extracts contain similar types of compounds, differences in their composition may explain their varying antifungal effectiveness. Using these natural extracts as antifungal agents aligns with eco-friendly strategies by offering a biodegradable, sustainable, and low-toxicity alternative to synthetic fungicides, thereby reducing chemical residues in food and minimizing environmental risks. Moreover, they can be sourced sustainably, providing a cost-effective solution for managing fungal diseases in crops. The shift towards using plant-based biofungicides aligns with the growing demand for organic farming practices and the need to reduce chemical residues in food products [26].

Additionally, avocado seed extract has demonstrated antifungal properties in various studies, attributed to its rich composition of biologically active compounds. Phytochemical screenings have identified 14 distinct phenolic compounds responsible for antimicrobial activity against a range of 15 microorganisms, including three Gram-negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa, and P. fluorescens), three Gram-positive bacterial strains (Bacillus cereus, Staphylococcus aureus, and Streptococcus pyogenes), and the fungus Candida albicans [58].

Previous research has highlighted that avocado seeds are abundant in biologically active compounds, including phenols, flavonoids, and condensed tannins. These compounds have been examined for their various bioactivities, such as antimicrobial properties [59]. Flavonoids, in particular, have been recognized for their capacity to disrupt microbial DNA synthesis and function, enhancing their antimicrobial effectiveness [60].

Evidence suggests that avocado seed extracts may serve as a promising source of natural antifungal compounds, potentially replacing synthetic fungicides in agriculture. The rich phytochemical profile of these extracts, including phenols, flavonoids, and condensed tannins, underpins their potent antimicrobial and antifungal properties. Continued research into these bioactive compounds could lead to the development of effective natural fungicides and antimicrobial agents. This study suggests that CIB, CLB, and AVS extracts inhibit the growth of F. incarnatum, likely due to their bioactive compounds. However, further research is required to identify these compounds and evaluate their efficacy.

Moreover, in the present study, CIB, CLB, and AVS extracts significantly reduced the progression of dry rot disease in treated potato tubers compared to untreated ones under normal and cold conditions. This reduction is likely due to the stimulation of the antioxidant defense system in the treated tubers. The antioxidant defense system in plants is a complex network designed to protect cells from oxidative damage caused by reactive oxygen species (ROS). This system includes various enzymatic components, which serve as the first line of defense against ROS, and non-enzymatic components, which form the second line of defense. Together, these components work to neutralize ROS and repair oxidative damage [61].

Enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase, and peroxidase play crucial roles in detoxifying ROS [62]. Catalase is particularly important for eliminating ROS, especially hydrogen peroxide (H₂O₂), by converting it into water (H₂O) and oxygen (O₂), thus preventing oxidative damage [63,64]. Additionally, peroxidase neutralizes H₂O₂, helping to maintain redox balance [62]. Polyphenol oxidase plays a crucial role in plant defense, particularly when plants are injured or attacked by pathogens. This enzyme converts phenolic compounds into quinones, which are more toxic and help to deter pathogens [65]. While polyphenol oxidase is not a primary component of the antioxidant defense system that directly neutralizes reactive oxygen species (ROS), it significantly contributes to the plant’s overall defense mechanisms. Additionally, phenylalanine ammonia-lyase (PAL) is an important enzyme involved in the phenylpropanoid and shikimate pathways [66]. It aids in the production of various compounds, such as phytoalexins, lignins, tannins, phenolic compounds, and salicylic acid, all of which enhance plant resistance to diseases [66].

Our study revealed a notable increase in the enzymatic activities of catalase, peroxidase, and polyphenol oxidase in potato tubers treated with the examined extracts, underscoring their antioxidant properties [58,67]. This surge in enzymatic activity is attributable to the bioactive compounds in the extracts, which bolster the plant’s defense mechanisms against oxidative stress and pathogen invasion.

Previous research has consistently highlighted the antioxidant properties of cinnamon, clove, and avocado seed extracts. For example, ref. [53] examined the antioxidant activities of aqueous and methanolic extracts from cinnamon and cloves, discovering that they inhibited the growth of F. oxysporum. Their study confirmed that these extracts enhance the activities of key antioxidant enzymes, thereby strengthening the plant’s defenses against oxidative stress and pathogen invasion.

Another study confirmed the antioxidant properties of avocado seed extract, emphasizing its ability to enhance the activity of both enzymatic and non-enzymatic antioxidants in plants. The research demonstrated that avocado seed extract is rich in bioactive compounds, including phenolic acids, flavonoids, and tannins, contributing to its strong antioxidant activity [68]. These properties are crucial for boosting the plant’s defense mechanisms against oxidative damage and pathogen invasion [68]. This aligns with our findings, where increased activities of CAT, POX, PPO, and PAL were observed in treated potato tubers. The significant increase in the activities of these enzymes in potato tubers treated with plant extracts underscores the potential of these natural compounds in enhancing plant defense mechanisms. Further research into the specific bioactive compounds responsible for these effects could lead to the development of effective natural fungicides and antioxidant treatments for agricultural use.

The phenylpropanoid pathway, also known as the phenolic metabolism pathway, is a vital source of secondary metabolites in plants. This pathway produces a diverse range of compounds, including total phenolic compounds, flavonoids, lignin, coumarins, anthocyanins, and volatile compounds. These metabolites play an important role in plant disease management by enhancing plant resistance and suppressing pathogen development [63,69]. Total phenolic compounds, in particular, are a significant group of bioactive molecules that are essential for activating plant defense responses. These compounds possess strong antioxidant properties, allowing them to scavenge reactive oxygen species (ROS) and protect plant cells from oxidative stress [61].

Phenolic compounds enhance the structural integrity of the cell wall, making it more difficult for pathogens to penetrate and establish infections. Flavonoids, a type of phytoalexin, play a dual role in plant defense. They can directly inhibit the growth and development of plant pathogens or indirectly contribute to the induction of host resistance mechanisms [60]. The biosynthesis of lignin via the phenylpropanoid pathway is a complex process that relies on specific enzymes, including phenylalanine ammonia-lyase, peroxidase, cinnamate-4-hydroxylase, and 4-coumarate-CoA ligase. Hydrogen peroxide (H₂O₂) also plays a crucial role in this process. Lignin is a complex polymer that strengthens the cell wall, providing rigidity and acting as a physical barrier against pathogen invasion [64,70].

Our study demonstrated a significant increase in total phenolic, flavonoid, and lignin levels in potato tubers treated with ethanol extracts of CIB, CLB, and AVS throughout the experiment. This biochemical enhancement was associated with a notable reduction in lesion diameters, indicating that these compounds play a crucial role in strengthening tuber resistance to dry rot disease. Phenolic compounds and flavonoids contribute to plant defense by exhibiting antimicrobial properties and reinforcing cell walls, while lignin deposition acts as a structural barrier that limits pathogen invasion [71,72]. Previous studies have reported a direct link between increased lignification and enhanced resistance to fungal infections. Lignin accumulation has been associated with reduced susceptibility to Fusarium spp. Similarly, lignin deposition in potato tubers has been shown to limit the spread of Fusarium infections, contributing significantly to disease suppression [21,73].

The observed rise in these defense-related compounds is likely mediated by the activation of key enzymes involved in the phenylpropanoid pathway, such as PAL, POD, and PPO. These enzymes are critical for cell wall reinforcement, antimicrobial compound synthesis, and overall pathogen suppression [21]. Our findings highlight the potential of natural plant extracts as an effective strategy for enhancing plant defense mechanisms and mitigating fungal infections. However, further investigations are needed to elucidate the precise biochemical pathways underlying these responses and to establish a definitive causal relationship between metabolite accumulation and disease resistance.

Malondialdehyde (MDA) is a low-molecular-weight end-product of membrane unsaturated fatty acid peroxidation and serves as a direct indicator of membrane lipid peroxidation. MDA is formed by the oxidation of lipid molecules, such as enzymes, phospholipids, and polyunsaturated fatty acids, by reactive oxygen species (ROS) [74]. An increase in MDA levels indicates cell membrane damage; however, our results showed a gradual decrease in MDA levels in potato tubers treated with CIB, CLB, and AVS ethanol extracts compared to the control.

The significant decrease in MDA levels is attributed to the increased enzymatic activity of antioxidant enzymes in the treated potato tubers. This enhancement in enzymatic activity likely reduces oxidative damage caused by oxidative stress. The increased activity of these enzymes stimulates the antioxidant defense system, which plays a crucial role in protecting plants from oxidative damage and pathogen invasion. Additionally, activation of the PAL enzyme promotes the biosynthesis of antifungal metabolites, including phenolics, flavonoids, lignin, and phytoalexins, by activating the phenylpropanoid metabolism pathway. This in turn enhances the ability of potato tubers to reduce oxidative stress and lower MDA levels.

Additionally, the extracts examined in this study significantly reduced the weight loss of potato tubers during storage, both under normal and refrigerated conditions. This indicates a beneficial impact of these extracts on the preservation of potato quality. The observed effect can be attributed to two main factors: the extracts’ ability to mitigate the severity of dry rot in potato tubers during storage and the enhancement of defense-related enzyme activities. These findings highlight the potential of CIB, CLB, and AVS extracts as natural preservatives, offering a sustainable alternative to synthetic chemicals for maintaining potato quality during storage.

5. Conclusions

Our study demonstrates the potential of cinnamon bark (CIB), clove bud (CLB), and avocado seed (AVS) extracts in controlling Fusarium incarnatum, the cause of potato dry rot. These extracts showed strong antifungal properties in vitro, significantly inhibiting fungal growth and limiting disease spread in tubers. This is likely due to their direct antifungal effects and the activation of plant defense mechanisms, helping maintain tuber quality during storage. These findings indicate that CIB, CLB, and AVS extracts could be sustainable alternatives to chemical fungicides, supporting organic farming and food safety. Further research should focus on field validation, optimizing application methods, and investigating potential synergistic interactions with other biocontrol strategies for improving postharvest disease management in potatoes.