Abstract
Introduction
Identifying pathogenic fungi from vegetable is a critical first step in disease management and development of effective plant-based antifungal agent, aligning with environmentally friendly and sustainable agricultural practices. This study aimed to isolate, identify, and characterize the major fungal pathogens of vegetables and to evaluate the antifungal efficacy of selected plant extracts against vegetable pathogenic fungi. Therefore clear identification is crucial for designing effective management protocols against these persistent fungal threats in Western Hararghe, Ethiopia.
Method
Diseased onion samples (leaves, bulbs, and roots) and pepper samples (leaves and fruits) were collected from Gemechis and Boke district, Western Hararghe, Ethiopia, respectively, from July 2023 to January 2025. Data were analyzed using a Completely Randomized Design (CRD) and ANOVA. Three medicinal plants (Allium sativum, Azadirachta indica, and Acokanthera schimperi) were selected based on traditional usage and literature evidence. Crude extracts were prepared using maceration method, while essential oils were obtained through hydro distillation, using Clevenger apparatus. The chemical composition of the crude extracts was assessed using standard phytochemical screening and essential oils was analyzed by using gas chromatography–mass spectrometry (GC–MS). Antifungal activity of both extract was evaluated using the agar well diffusion method and detached leaf assay for obligate biotroph.
Result
A total of six major fungal pathogens (Peronospora destructor, Rhizoctonia solani, and Fusarium oxysporum from onions; and Erysiphe species, Sclerotinia sclerotiorum, and Aspergillus niger from pepper) were identified. Phytochemical screening of these selected plants confirmed the presence of alkaloids, flavonoids, phenolics, tannins, and sulfur compounds in the crude extracts. Antifungal assays results demonstrated that the plant extracts significantly inhibited fungal growth, particularly at higher concentrations (200 µl/ml). Acokanthera schimperi showed the highest antifungal activity, followed by Azadirachta indica, while Allium sativum exhibited moderate effects.
Conclusion
The findings underscored the potential of botanical extracts as environmentally friendly alternatives for managing fungal diseases in vegetable crops.
Keywords: Acokanthera schimperi, Antifungal activity, Essential oil, Erysiphe species, Onion, Pepper, Rhizoctonia solani
Introduction
Background
Vegetables are essential dietary components that supplement staple foods and serve as a consistent income source for small-scale farmers across Ethiopia, particularly in the central Rift Valley region and the present study area [17]. Vegetables also serve as protective foods, offering significant health benefits and contributing to disease prevention [34]. Common vegetables cultivated in Ethiopia include cabbage, carrot, cucumber, garlic, onion, pepper, potato, shallot, and tomato [17].
Onion (Allium cepa L.), a member of Amaryllidaceae family which is one of the most widely cultivated and economically valuable vegetables globally, grown extensively for both domestic consumption and export [30]. Major onion-producing countries include China, India, the United States, Pakistan, and Turkey. In recent years, onion cultivation has expanded significantly in Ethiopia, especially in the central Rift Valley. Pepper (Capsicum species), a member of the Solanaceae family, is another vital vegetable and spice crop cultivated globally [7]. In Ethiopia, it holds economic and cultural significance, with an average adult consumption rate of 15 g/day. Hot pepper alone accounts for approximately 67.98% of the total area under vegetable cultivation in the country (CSA, 2011/2012).
Despite their importance, the production of onion and pepper in Ethiopia is heavily constrained by various pests, including phytophagous insects, plant diseases, and weeds. These constraints are exacerbated in tropical and subtropical climates, where year-round favorable conditions support the spread of pathogens and their hosts. Farmers frequently face total crop failure due to high disease pressure and the inaccessibility of improved crop varieties [2, 6].
Globally, 35–40% of onion yield is lost due to diseases during pre- and post-harvest stages [22]. Fungal pathogens such as Aspergillus species, Fusarium oxysporum, Rhizopus stolonifera, Mucor, and Penicillium digitatum, along with bacterial species like Pseudomonas, Enterobacter, and Escherichia coli, have been identified as major contributors to vegetable spoilage. These pathogens can infect crops at any stage seed, field, harvest, storage, or distribution [4]. In Ethiopia, purple blotch (Alternaria porri) and downy mildew (Peronospora destructor) are significant constraints on onion productivity [33]. Mycotoxins produced by fungi such as Aspergillus, Fusarium, and Penicillium can pose serious health risks, including immune suppression and cancer, particularly aflatoxins produced by Aspergillus species [27].
While chemical fungicides and pesticides are widely used, they present problems such as pathogen resistance, chemical residues, phytotoxicity, and environmental and public health concerns [31]. The World Health Organization estimates that one million pesticide poisoning cases occur annually, causing approximately 20,000 deaths of human. In light of increasing demand for organic produce, there has been a shift toward alternative, eco-friendly disease management practices. Among these, the use of plant extracts and essential oils is gaining attention due to their low toxicity, biodegradability, and rich composition of secondary metabolites [5, 15, 31].
Plants produce secondary metabolites including essential oils as part of their defense mechanisms against pests and pathogens. Highly bioactive essential oils include cinnamon (cinnamaldehyde), clove (eugenol), lemongrass (citronellal, citral), mint (menthol, pulegone), oregano (carvacrol), thyme (thymol), and rosemary (eucalyptol). Certain phenolic compounds in these oils exhibit antifungal activity against pathogens like Botrytis cinerea, Pythium ultimum, and Pythium aphanidermatum [12]. Studies have shown that neem oil inhibits Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii, and Phytophthora species [8]. Eucalyptus extracts have proven effective against Pythium species and other soil-borne fungi [20]. Garlic has demonstrated a range of protective effects, including antifungal, antibacterial, insecticidal, and nematicidal properties.
Although onion and pepper are among Ethiopia’s most economically and culturally significant crops (Sadhna et al., 30, their productivity remains below global and African averages. Their increasing cultivation is threatened by biotic stresses especially diseases. Common pathogens affecting vegetables in Ethiopia include those causing black rot in cabbage, bulb rot, downy mildews, garlic rust, pepper wilt, stem blight, powdery mildews, and purple blotch. Notably, onion purple blotch alone causes a 26% loss in yield, while late blight results in 15–67% loss in potatoes and 38–65% in tomatoes. Field surveys in southern Ethiopia have reported the prevalence of purple blotch, downy mildew, damping-off, and iris yellow spot virus in onions [17]. In vitro and in vivo tests in Horo Guduru Wollega identified Phytophthora capsici, Cercospora capsici, and Fusarium oxysporum as dominant pathogens in hot pepper. Moreover, 42% of Ethiopian pepper samples were found to be contaminated with aflatoxins [7].
Effective plant disease control requires ongoing discovery of new antimicrobial agents. Current strategies emphasize rational antimicrobial use and natural products such as plant extracts and essential oils. Plant-derived pesticides are preferable due to their rapid volatilization, low persistence, and minimal toxicity to vertebrates, reducing environmental and human health risks [29].
Although previous studies have examined onion and pepper yield in Western Hararghe Zone [6, 13], there is limited or no documented studies on fungal pathogens affecting vegetables in current study area. Most prior research in Ethiopia has focused on disease surveys and documentation, and little research have made in Ethiopia on the use of plant extracts to control plant disease. Fungal pathogens such as Erysiphe species, Sclerotinia sclerotiorum, Aspergillus niger, Perospora destructor, Rhizoctonia solani, and Fusarium oxysporum are particularly destructive to various host plants. Reliable identification of such pathogens using morphological and biochemical methods is vital for effective antifungal screening, especially using plant-based alternatives aligned with sustainable agriculture. Therefore, the purpose of this study is to identify most common pathogenic fungi of onion and pepper and evaluate the antifungal activity of selected plant extracts. The outcomes of this research will address critical gaps in vegetable disease management, support the production of safe and nutritious vegetables, and enhance the economic sustainability of farming in the study area.
Material and method
Description of the study area
The study was conducted in selected districts of West Hararghe Zone (Gemechis and Boke) from July 2023 to January 2025. West Hararghe Zone is one of the Zones in the Oromia region, Ethiopian. It is bounded on the south by the Shebelle River which separates it from Bale, on the southwest by Arsi, on the northwest by the Afar Region, on the North by the Somali Region and on the East by East Hararghe. It is located at 8°39'59.99"N and 40° 29' 59.99" E (Fig. 1). Based on the 2007 Census conducted by the CSA, this Zone has a total population of 1,871,706 with an increase of 47.16% over the 1994 census, of whom 958,861 are men and 912,845 women: with an area of 15,065.86 square kilometers. West Hararghe has a population density of 124.23, while 160,895 or 9.36% are urban inhabitants, a further 10,567 or 0.56% are pastoralists. A total of 395,127 households were counted in this Zone, which results in an average of 4.74 persons per household, and 380,019 housing units. West Hararghe is characterised by a diversity of agro-climatic conditions which determine the composition of the rural economy. From lowlands (kolla) to midlands (weyna dega) and highlands (dega) the economy reflects patterns which are to a certain extent typical for Ethiopia in general: the lower the altitude the more important becomes the livestock-based economy while, due to climatic constraints, possibilities of crop production are limited.
Fig. 1.
Shows the map of Boke and Gemechis district with their relative location
Study design and sampling techniques
This cross-sectional study employed a purposive sampling technique to select study districts based on their potential for onion and pepper production. To collect the infected plant sample, farms and infected samples were randomly selected, with 80 infected onion samples (leaves, bulbs and roots) with disease symptoms were collected from Gemechis district, and 60 infected pepper samples (leaves and fruits) were taken from Boke district.
Sterilization of materials and media preparation
The inoculating needle and cork borer were sterilized by flaming with a spirit lamp. Glassware, including petri dishes, conical flasks, and test tubes, was washed, dried, and sterilized in a hot air oven at 160 °C for 2 h. The working surface was disinfected using 70% ethanol for 30 min. Culture media used for the isolation and characterization of test organisms such as Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar (PDA) were prepared according to the manufacturer’s instructions.
Isolation and identification of fungal pathogens
Onion and pepper tissues (bulbs, leaves, fruits and roots) showing signs of rotting and discoloration were collected from selected districts of Western Hararghe Zone, Ethiopia during the 2023–2025 growing season. Onion samples were obtained from Gemechis district, while pepper samples were collected from Boke district. The samples were placed in separate polyethylene bags, properly labeled, and transported to the Plant Science Laboratory at Oda Bultum University. Upon arrival, they were stored in a refrigerator at 4 °C for further analysis.
To isolate pathogenic fungi, plant materials (bulbs, leaves, fruits and roots) was washed under running tap water to remove debris and soil (root and onion bulb) then surface sterilized using 2% sodium hypochlorite for 60 s, rinsed three times in sterile distilled water, and dried on sterile filter paper. The infected parts of plants (3–5 cm) were cut from the margins of diseased tissues using a sterile scalpel and macerated in 9 ml of distilled water. Serial dilutions (10⁻1 to 10⁻5) were prepared, and 1 ml from each dilution was plated on potato dextrose agar (PDA) supplemented with chloramphenicol to inhibit bacterial growth. Plates were incubated at room temperature (22–28 °C) for 7 days. Fungal isolates were identified based on morphological characteristics and confirmed through slide culture, microscopic examination, chlamydospore formation, germ tube test, sugar assimilation, motility test, and urease activity [10] and Booth [9]. In addition to microscopic examination the researchers used identified image of the same plants from different literature [19], Haile 17 and Hemida et al., [18]. Identification was verified by using a standard mycological atlas.
Collection of plant materials, extraction, and chemical analysis
Collection of plant materials
Plant species tested for anti-fungal activity (Acokanthera schimperi, Allium sativum, Azadirachta indica) were selected based on their traditional use as control agents of plant disease in the study area and related literature [25]. The authors first identified the plant species at the plants collection sites by comparing the characteristics of specimens with the published books from Ethiopia and Eritrea and then specimens were labelled with the local name, date of collection, voucher collection number, name of collector, description and place of collection and then pressed and taken to Addis Ababa University National Herbarium for further identification following the international code of botanical nomenclature (ICBN) guidelines and finally the identified plant specimens were deposited at Oda Bultum University Department of Biology, Botanical Science Laboratory. The identification of plants was conducted by botanist Muhidin Tahir and the spelling of botanical names where checked using world flora online. (https://www.worldfloraonline.org).
Extraction and analysis of the chemical composition of the plant extracts
The collected plant materials were shade dried at room temperature, chopped, and ground into a coarse powder using an electric blender at the Department of Chemistry, Oda Bultum University. Crude extracts were prepared by mixing 100 g of powdered plant material with 1000 ml of distilled water and allowing extraction for 12 h [1]. The extracts were filtered using Whatman No. 1 filter paper, and the solvents were evaporated at 45 °C using a rotary evaporator. Extracts were stored in sealed vials at 4 °C until further use. Phytochemical analysis was conducted using standard methods like Mayer’s and Dragendorff’s reagents for alkaloids, magnesium and hydrochloric acid for flavonoids, gelatin and sodium chloride (NaCl) for tannins, and frothing tests for saponins [16].
Essential oils were extracted by hydro-distillation methods, using Clevenger type apparatus. About 500 g of shade dried plant materials; A. schimperi (leaves), A. sativum (bulbs), and A. indica (leaves) were mixed with 300 ml of distilled water in a 1 L distillation flask in a Clevenger apparatus for 3 h. The resulting oil was separated with chloroform in a separatory funnel, dried over anhydrous Sodium sulphate (Na₂SO₄), and concentrated under reduced pressure. Oils were stored in amber glass bottles at 4 °C until analysis, and then the oil was analyzed using Gas Chromatography-Mass Spectrometry (GC–MS).
In vitro fungicidal activity of crude extracts and essential oils
Fungicidal activity was evaluated using the agar well diffusion method Sharma and Tripath [32]. A spore suspension (0.5–5 × 104 CFU/ml) was spread on potato dextrose agar (PDA) at 20 ml. After solidification, 8 mm wells were made using a sterile cork borer, and 1 ml of each plant extract (crude extracts and Essential oils) at a concentration of 100 and 200 µl/ml was introduced into the wells. Carbendazim at a concentration of 5 µL/mL was employed as a positive control, and distilled water at concentration of 100 µL/mL was employed as a negative control. Plates were incubated at 26 ± 2 °C for 7 days. Mycelial growth inhibition was calculated using the formula: Inhibition (%) = [(Dc—Dt)/Dc] × 100, where Dc = average diameter of fungal colony in control and Dt = diameter in treatment Pinto et al., [26]. Obligate biotrophs (Peronospora destructor) were assessed using a detached leaf assay [23]. Susceptible host leaves were placed on moistened filter paper in petri dishes. Leaves were treated with plant extracts, allowed to dry, and then inoculated with fungal sporangia. Plates were incubated in a humid chamber at 18–22 °C. Fungicidal activity was measured based on reductions in spore germination, sporulation intensity, and enzyme activity associated with plant defense responses, compared to control treatments Table 1.
Table 1.
Lists of plant species used with their scientific name, family names, and local name
| Scientific name of selected plant | Family name | Local name in Afaan Oromoo | Part used | Voucher number |
|---|---|---|---|---|
| Allium sativum L | Amaryllidaceae | Qullubbii Adii | Leaf | AAOBU0015 |
| Acokanthera schimperi (A.DC.) Benth, and Hook. f. ex Schweinf | Apocynaceae | Qararuu | Leaf | AAOBU0016 |
| Azadirachta indica A.Juss | Meliaceae | Eebicha | Bulb | AAOBU0017 |
Experimental design and statistical analysis
The experiment was conducted using a Completely Randomized Design (CRD) with three replications, following Gomez and Gomez (1984). Data were analyzed using Analysis of Variance (ANOVA), and mean differences were determined using the Least Significant Difference (LSD) test at a significance level of p < 0.05.
Result
Morphological and biochemical characterization of pepper and onion pathogenic fungi
Morphological and biochemical characterization of onion pathogenic fungi
According to current study, three major fungal pathogens were isolated from diseased onion tissues showing foliar, root, and basal rot symptoms. Based on their morphological and biochemical analyses, three most prevalent fungal pathogens of onion (Peronospora destructor, Rhizoctonia solani, and Fusarium oxysporum) were identified. Among identified fungi Peronospora destructor (downy mildews), was characterized by its dark-pigmented conidia, septate hyphae, and branching conidiophores as observed under a light microscope, whereas Rhizoctonia solani (damping-off) was characterized by production of non-septate, right-angled branching hyphae and sclerotia (Table 2). Fusarium oxysporum, responsible for basal rot, produced sickle-shaped macroconidia, pink-pigmented colonies, and tested positive for urease activity and potassium hydroxide (KOH) solubility.
Table 2.
Morphological and biochemical characterization of pathogenic fungi isolated from infected Onion
| Isolated Fungi | Morphological features | Biochemical tests | Disease |
|---|---|---|---|
| Peronospora destructor | Purplish-gray to violet sporulation mainly on underside leaves, dichotomously branched, slender sporangiophores and smooth walled ellipsoidal sporangia, no zoospores in onion | Not applicable; cannot be cultured on culture media | Downy mildews |
| Rhizoctonia solani | Brown to tan mycelium; right-angle hyphal branching, absence of spores, large sclerotia | Nitrate reduction and oxidase (+), conidia formation (-) | Damping-off |
| Fusarium oxysporum | Cottony white to pinkish mycelium; sickle-shaped macroconidia | Lactophenol cotton blue and urease (+), growth in KOH Medium | Basal rot |
+ positive,— negative
Morphological and biochemical characterization of pepper pathogenic fungi
Based on morphological and biochemical test, three fungal pathogens of pepper exhibiting leaves and fruit infections were identified. Among identified fungal species Erysiphe species, the causal agent of powdery mildews, were characterized by the formation of chain-like conidia and a powdery growth on leaf surfaces, staining distinctly with Lactophenol Cotton Blue (Table 3). Sclerotinia sclerotiorum was identified by its cottony white mycelium and black sclerotia that used as a key identification features along with positive oxidase activity. Aspergillus niger, characterized by producing black, radiating spore heads and was biochemically confirmed by positive urease and citrate utilization tests.
Table 3.
Morphological and biochemical characterization of pathogenic fungi isolated from infected Pepper
| Isolated Fungi | Morphological features | Biochemical tests | Disease |
|---|---|---|---|
| Erysiphe species | White powdery mycelial growth on leaf surface; conidia in chains | Lacto phenol blue and catalase (+) | Powdery Mildews |
| Sclerotinia Sclerotiorum | White, fluffy aerial mycelium; formation of black scerotia | Oxidase test (+); acid production in glucose agar (+); sclerotia formation (+) | White Mold |
| Aspergillus niger | Black, granular spore masses; rough conidiophores; septate hyphae | Citrate utilization; urease test and Lactophenol blue and catalase (+) | Black Mold |
+ positive for respective tests
Phytochemical composition of plant extracts (crude and essential oils)
Phytochemical composition of crude extracts
Table 4 below presents the results of phytochemical screening for crude extracts of Acokanthera schimperi, Azadirachta indica, and Allium sativum. Azardirachta indica showed rich in alkaloids and saponins (+++), moderate levels of flavonoids, tannins, phenolics, and terpenoids (++), but not having sulfur compounds. Key constituents included nimbin and azadirachtin. Acokanthera schimperi was rich in tannins and phenolics (+++), moderate alkaloids and flavonoids (++), and low saponins and terpenoids (+); major bioactive compounds includes geranin and gallic acid. Allium sativum was rich in sulfur compounds (+++), with moderate amount of phenolic (++), and low levels of alkaloids, flavonoids, saponins and tannins (+); allicin and alliin might be the main bioactive compounds of A. sativum.
Table 4.
Chemical composition of crude extracts of A. indica, A. schimperi and A. sativum
| Composition | Azardirachta indica (Neem) | Acokanthera schimperi | Allium sativum |
|---|---|---|---|
| Alkaloids | + + + | + + | + |
| Flavonoids | + + | + + | + |
| Saponins | + + + | + | + |
| Tannins | + + | + + + | + |
| Phenolics | + + | + + + | + + |
| Terpenoids | + + | + | + |
| Sulphur compounds | - | - | + + + |
| Other compounds | Nimbin, Azadirachtin | Glycosides, steroids | Allicin, Alliin |
+ low, + + moderate, + + + abundant, — not detected
Phytochemical composition of essential oils
Essential oils (EOs) from the same plant sources exhibited distinct chemical profiles compared to their crude extracts. Azadirachta indica Eos was rich in Germacrene-B, Palmitic acid, Oleic acid (+ + +), moderate in alpha-Bicyclogermacrene and Phytol (+ +), and low in beta-Caryophyllene gamma-Elemene. Acokanthera schimperi Eos was rich in alpha-pinene, 1, 8-cineole and Oleanolic acid, moderate in Borneol, terpen-4-ol and low in limonene and Beta-caryophyllene (+). The Eos of Allium sativum showed a strong presence of sulfur compounds (+ + +) such as diallyl disulfide and diallyl trisulfide, with moderate level of Allyl methyl trisulfide and Diallyl tetra sulfide (Table 5).
Table 5.
Chemical composition of essential oils A. indica, A. schimperi and A. sativum
| Plant species | Major components | Relative abundance |
|---|---|---|
| Azardirachta indica | Germacrene-B | + + + |
| Beta- Caryophyllene | + | |
| Gamma-Elemene, | + | |
| Alpha-Bicyclogermacrene | + + | |
| Palmitic acid | + + + | |
| Oleic acid | + + + | |
| Phytol | + + | |
| Acokanthera schimperi | Oleanolic acid | + + + |
| Alpha-pinene, | + + + | |
| Borneol | + + | |
| Limonene | + | |
| Tepinen-4-ol | + + | |
| 1, 8-cineole | + + + | |
| Beta-caryophyllene | + | |
| Allium sativum | Diallyl disulfide, | + + + |
| Diallyl trisulfide | + + + | |
| Allyl methyl trisulfide | + + | |
| Diallyl tetra sulfide | + + |
+ low, + + moderate, + + + abundant, — not detected
Antifungal activity of plant extracts against onion and pepper pathogenic fungi
The results reveal that both the crude extracts and essential oil of Acokanthera schimperi exhibited concentration-dependent antifungal activity against all tested fungal pathogens of onion and pepper. The essential oil consistently showed higher inhibition zones than the crude extract across all pathogens, confirming the greater potency of volatile compounds in Eos.
Antifungal activity of crude extracts and Eos of A. schimperi on fungal pathogen of onion and pepper
The essential oil showed strongest inhibition against P. destructor (9.00 mm at 200 µl/ml), followed by R. solani (8.30 mm), and F. oxysporum (7.60 mm). These pathogens are major constraints to onion productivity in Ethiopia, and their control using plant-based solutions like A. schimperi EOs provides a sustainable alternative to synthetic fungicides. The activity may be attributed to the presence of α-pinene, limonene, and β-caryophyllene, which disrupt fungal membranes and inhibit mycelial growth. Crude extracts also showed significant inhibition, particularly against P. destructor (7.17 mm at 200 µl/ml), indicating that non-volatile phytochemicals such as tannins, flavonoids, and saponins play a role in fungal suppression. Pepper pathogens were equally susceptible, with the essential oil showing the strongest inhibition against Erysiphe species (8.90 mm at 200 µl/ml) and Sclerotinia sclerotiorum (8.40 mm) (Table 6). Garlic-like sulfur compounds in A. schimperi, though not yet fully characterized, may contribute to its broad-spectrum action. Crude extracts were also effective, particularly at higher concentrations, with up to 7.40 mm inhibition against Erysiphe species.
Table 6.
Antifungal activity of A. schemperi against isolated fungal pathogen of onion and pepper
| Isolated fungi | Antifungal activity of A. indica (Mean SD) |
|||||
|---|---|---|---|---|---|---|
| Crude extract | Essential oil | CD (5%) | ||||
| 100 µl/ml) | 200(µl/ml) | 100 µl/ml) | 200(µl/ml) | |||
| Onion | Peronospora destructor | 5.30
|
7.20
|
4.33
|
6.67
|
0.75 |
| Rhizoctonia solani | 4.90
|
6.80 0.28 |
3.33
|
4.01 0.22 |
0.55 | |
| Fusarium oxysporum | 4.40
|
6.30
|
2.33
|
3.67 0.28 |
0.64 | |
| Pepper | Erysiphe species | 6.10
|
7.17
|
5.33
|
7.67 0.33 |
0.75 |
| Sclerotinia sclerotiorum | 4.30
|
5.90
|
4.65
|
4.01 0.67 |
1.52 | |
| Aspergillus niger | 4.00
|
5.60
|
3.65
|
5.67 0.02 |
0.047 | |
CD critical difference value greater than 0.68 considered significant at a P-value of 0.05, SD standard deviation
Antifungal activity of the crude extracts and Eos of A. indica against onion and pepper pathogenic fungi
Azadirachta indica showed moderate but broad-spectrum antifungal activity. In onion pathogens, its inhibition zones ranged from 2.33 mm to 6.67 mm, with highest inhibition against Peronospora destructor (6.67 mm at 200 µl/ml) and lowest against Fusarium oxysporum (2.33 mm at 100 µl/ml). In pepper, it showed good activity against Erysiphe species (7.67 mm at 200 µl/ml), moderate effect on Sclerotinia sclerotiorum (4.01 mm), and 5.67 mm against Aspergillus niger. Azadirachta indica (neem) EOs exhibited moderate antifungal activities, with inhibition zones reaching 7.20 mm against Peronospora destructor, 6.80 mm against Rhizoctonia solani, and 6.30 mm against Fusarium oxysporum at 200 µl/ml. Similarly, it showed notable activity against pepper pathogens, including 7.67 mm against Erysiphe species and 5.90 mm against Sclerotinia sclerotiorum (Table 7).
Table 7.
Antifungal activity of A. indica against isolated fungal pathogen of onion and pepper
| Isolated fungi | Antifungal activity of A. indica (Mean  SD) |
|||||
|---|---|---|---|---|---|---|
| Crude extract | Essential oil | CD (5%) | ||||
| 100 µl/ml) | 200(µl/ml) | 100 µl/ml) | 200(µl/ml) | |||
| Onion | Peronospora destructor | 5.30 
|
7.20 
|
4.33 
|
6.67 
|
0.75 |
| Rhizoctonia solani | 4.90 
|
6.80  0.28 |
3.33 
|
4.01  0.22 |
0.55 | |
| Fusarium oxysporum | 4.40 
|
6.30 
|
2.33 
|
3.67  0.28 |
0.64 | |
| Pepper | Erysiphe species | 6.10 
|
7.17 
|
5.33 
|
7.67  0.33 |
0.75 |
| Sclerotinia sclerotiorum | 4.30 
|
5.90 
|
4.65 
|
4.01  0.67 |
1.52 | |
| Aspergillus niger | 4.00 
|
5.60 
|
3.65 
|
5.67  0.02 |
0.047 | |
CD critical difference value greater than 0.68 considered significant at a P-value of 0.05, SD standard deviation
Antifungal activity of A. sativum crude extracts and Eos against onion and pepper pathogenic fungi
Allium sativum, while being the least effective overall, still showed measurable antifungal activity, especially at higher concentrations. In onion pathogens, inhibition zones ranged from 2.65 mm (F. oxysporum at 100 µl/ml) to 5.00 mm (Peronospora destructor at 200 µl/ml) (Table 8). In pepper, the extract demonstrated up to 6.00 mm inhibition against Sclerotinia sclerotiorum and 5.01 mm against Aspergillus niger at 200 µl/ml. Allium sativum (garlic) EOs, while less potent than the other two, still showed measurable antifungal activity. It was most effective against Sclerotinia sclerotiorum (6.00 mm at 200 µl/ml) and Erysiphe species. (5.90 mm), pathogens that are significant constraints in pepper production. Against onion pathogens, the inhibition ranged from 3.60 mm to 5.80 mm.
Table 8.
Antifungal activity of A. sativum against isolated fungal pathogen of onion and pepper
| Isolated fungi | Antifungal activity A. sativum (Mean  SD) |
|||||
|---|---|---|---|---|---|---|
| Crude extract | Essential oil | CD (5%) | ||||
| 100 µl/ml | 200 µl/ml | 100 µl/ml | 200 µl/ml | |||
| Onion | Peronospora destructor | 3.65  0.01 |
5.0  0.22 |
4.10  0.24 |
5.80  0.03 |
0.28 |
| Rhizoctonia solani | 3.65  0.24 |
4.0  0.33 |
4.80  0.24 |
5.20 
|
0.65 | |
| Fusarium oxysporum | 2.65  0.33 |
4.0  0.24 |
3.60  0.33 |
4.90  0.01 |
0.38 | |
| Pepper | Erysiphe species | 3.0  0.33 |
4.0 
|
4.30  0.67 |
5.90  0.02 |
0.8 |
| Sclerotinia sclerotiorum | 5.65  0.34 |
6.0  0.66 |
4.00  0.22 |
6.00  0.67 |
1.01 | |
| Aspergillus niger | 4.65  0.24 |
5.01  0.02 |
3.50  0.24 |
4.70  0.22 |
0.63 | |
CD critical difference value greater than 0.68 considered significant at a P-value of 0.05, SD standard deviation
Discussion
In the present investigation, three most prevalent fungal pathogens; Peronospora destructor, Rhizoctonia solani, and Fusarium oxysporum were isolated from diseased onion tissues exhibiting foliar, root, and basal rot, similarly from red pepper three most prevalent pathogenic fungi (Erysiphe species, Sclerotinia sclerotiorum and Aspergillus niger) were isolated and identified from the pepper leaves and fruits with a typical symptoms of disease. Identification utilized an integrated standard approach combining cultural traits, rigorous morphological characteristics, biochemical tests (macroconidia, hyphal septation, pigmentation test, urease test, catalase test, nitirate utilization test and KOH reactions) and literature supported comparisons, in accordance with accepted mycological protocols [23], and [18]. This integrated approach enhances diagnostic reliability and supports targeted disease management strategies in onion and pepper crops.
The current findings reveal that both the crude extracts and essential oils (EOs) of all tested plants (Acokanthera schimperi, Azadirachta indica, and Allium sativum) showed promising antifungal results, with the highest activity exhibited by Acokanthera schimperi against all tested fungi. The essential oils consistently showed higher inhibition zones than the crude extracts across all pathogens, confirming the greater potency of volatile compound. Among all tested plant-derived products, A. schimperi EOs showed the strongest inhibition zones, particularly against Peronospora destructor and Erysiphe species. The superior performance of essential oils over crude extracts can be attributed to the concentration and volatility of active phytochemicals, including α-pinene, limonene, and β-caryophyllene, which have been previously reported for their antifungal mechanisms. These compounds are believed to cause membrane disruption, leakage of cellular contents, inhibition of sporulation, and interference with fungal enzyme systems Mwangi (2020). Specifically, α-pinene and limonene can penetrate the fungal cell wall and alter membrane permeability, leading to cytoplasmic leakage and cell death 14. In addition to direct antifungal effects, essential oils of A. schimperi may also inhibit fungal growth by inducing oxidative stress in fungal cells through the generation of reactive oxygen species (ROS), leading to apoptosis-like responses in pathogens [26].
These findings support the incorporation of A. schimperi into integrated disease management systems for horticultural crops. Particularly for smallholder farmers in Ethiopia, where access to synthetic fungicides is limited due to cost and availability, the use of local plants like A. schimperi offers a low-cost, eco-friendly alternative with minimal environmental impact. Additionally, as resistance to commercial fungicides continues to rise, the deployment of phytochemicals with novel mechanisms of action could help delay resistance development in fungal pathogens [24].
The results also reveal that both the crude extracts and essential oil of Azadirachta indica (neem) exhibited moderate but broad-spectrum antifungal activity. Among onion pathogens, the highest effect was observed against P. destructor, while in pepper pathogens, neem revealed remarkable inhibition, particularly against Erysiphe species. The essential oil also demonstrated moderate antifungal activity, with inhibition zones evident against both P. destructor and Erysiphe species, suggesting its potential utility in managing foliar and systemic fungal infections in vegetable crops. The antifungal efficacy of neem is largely credited to its rich content of limonoids, especially azadirachtin, nimbin, and salannin, which interfere with fungal growth, spore germination, and cell membrane integrity [28]. Furthermore, neem has been found to inhibit ergosterol biosynthesis in fungal cells [11].
Previous studies have also demonstrated neem’s broad antifungal spectrum. For example, [11] reported significant inhibition of Fusarium oxysporum and Alternaria species, using neem oil, highlighting its potential in integrated pest and disease management systems. Although the antifungal activity observed in this study is not as strong as A. schimperi, neem’s availability, traditional acceptance, and multifunctional pesticidal properties make it a practical option for smallholder farmers.
Among the three tested plants, Allium sativum (garlic) demonstrated least antifungal activity, particularly at all concentrations. In higher inhibition zones were reported against the pepper pathogen (Sclerotinia sclerotiorum). The antifungal potential of garlic is primarily linked to allicin, a sulfur-containing volatile compound formed enzymatically when garlic tissue is damaged. Allicin is known to react with thiol groups in fungal enzymes and proteins, impairing essential metabolic processes and ultimately leading to fungal death [3]. Other organosulfur compounds like diallyl disulfide and diallyl trisulfide also contribute to garlic’s antifungal effects, particularly against molds and yeasts 21. Despite being the least effective in this study, garlic still provides a viable supplementary tool for managing fungal pathogens, especially in resource-limited settings where its affordability, safety, and traditional use make it a sustainable disease control agent. Moreover, its wide availability across Ethiopia enhances its applicability in integrated pest and disease management programs, particularly in organic and home gardening contexts.
However, this study has several limitations. First, the findings are based solely on in vitro assays, which do not account for environmental variables present under field conditions. Second, only three plant species were tested, limiting the generalizability of the results. Additionally, the specific mechanisms of antifungal action were not investigated, leaving a gap in understanding how individual compounds exert their effects on fungal cells. Future studies should include greenhouse and field trials, expand the range of plant species evaluated, and apply molecular and microscopic techniques to elucidate the precise modes of action.
Conclusion
The present study successfully identified and characterized major fungal pathogens of onion such as P. destructor, R. solani, and F. oxysporum and pepper Erysiphe species, S. sclerotiorum, and A. niger) through detailed morphological and biochemical analyses. Further this study evaluated phytochemical profiles of three medicinal plants i.e. Azadirachta indica (neem), Acokanthera schimperi (African Wintersweet), and Allium sativum (garlic) revealed the presence of diverse bioactive compounds such as alkaloids, phenolics, and sulfur compounds, and essential oil constituents active component such as α-pinene and diallyl disulfide.
Among the tested plant extracts and essential oils, the extract of A. schimperi exhibited the strongest and broadest antifungal activity against both onion and pepper pathogens. Its essential oil significantly inhibited the growth of all tested fungi, particularly P. destructor and Erysiphe species, likely due to its rich content of monoterpenes and phenolic compounds. A. indica also showed moderate yet consistent antifungal activity, suggesting its usefulness in integrated disease management strategies. Allium sativum, though less potent, demonstrated measurable inhibitory effects, especially against Sclerotinia sclerotiorum and Aspergillus niger.
Overall, the results highlight the potential of plant-derived extracts and essential oils, particularly from A. schimperi, as environmentally friendly alternatives to synthetic fungicides for managing fungal diseases vegetable crops. Further studies on formulation, application methods, and field efficacy are recommended to promote the practical use of these botanical fungicides in sustainable agriculture.
Acknowledgements
We thank Oda Bultum University for funding and their support throughout the study. We would like to thank the local people of the study district (Gemechis, and Boke), and all farmers for providing as with a sample and their hospitality. We also acknowledge Western Hararghe and Gemechis, and Boke districts agricultural Office for providing us the support letter to visit the collect the samples.
Authors’ contributions
A.A. manage the project, Investigation, Conceptualization, writing original manuscript. M.T. Write, review & edit the manuscript and formal analysis. S.A. and G.D. Conceptualization, review & analysis.
Funding
Not applicable.
Data availability
Data will be made available up on reason able request from corresponding author.
Declarations
Ethics approval and consent to participate
Prior sample collection the permission of collecting samples were obtained first from Oda Bultum University directorate of Research, ethics and publication office and further permission were obtained from the Zones and districts agricultural office and from farmers to get the sample. All samples were collected after permission was obtained from the farm owners, after the objective of the study was mentioned.
Consent for publication
Not applicable.
Competing interest
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Abdel-Hafez SII, Abo-Elyousr KAM, Abdel-Rahim IR. Effect of certain plant extracts to control purple blotch disease of onion plants (Allium cepa L). J Plant Physiol Pathol. 2013;1:1000111. 10.4172/2329-955X.1000111. [Google Scholar]
- 2.Abay G, Getu A. Assessments of distribution and status of major disease of hot pepper (Capsicum annuum L.) in West Shoa and East Wollega Zones, Ethiopia. J Plant Sci. 2022; 10(2):57–63. 10.11648/j.jps.20221002.12.
- 3.Ankri S, Mirelman D. Antimicrobial properties of allicin from garlic. Microbes Infect. 1999;1(2):125–9. 10.1016/S1286-4579(99)80003-3. [DOI] [PubMed] [Google Scholar]
- 4.Barth M, Hankinson TR, Zhuang H, Breidt F. Microbiological spoilage of fruits and vegetables. In: Sperber WH, Doyle MP, editors. Compendium of the microbiological spoilage of foods and beverages. New York: Springer; 2009. p. 135–83. [Google Scholar]
- 5.Bekele H. Short report on yellow rust and stem rust of wheat. In: Proceedings of Agronomy Workshop. Melkassa: BADE; 2020. p. 67–78.
- 6.Begna T, Sirba HY, Gojam M. Evaluating the performance of recently released onion (Allium cepa L.) varieties at highland areas of West Hararghe, Ethiopia. Adv Crop Sci Tech. 2022;10:544. [Google Scholar]
- 7.Berke T. The AVRDC Pepper Project. In: Central Statistical Agency Agricultural Sample Survey. Addis Ababa: FDRE Ethiopia; 2002.
- 8.Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of neem (Azadirachta indica). Curr Sci. 2002;82(11):1336–45. [Google Scholar]
- 9.Booth C. The genus Fusarium. Kew, Surrey, England: Commonwealth Mycological Institute; 1971. p. 237.
- 10.Charles RL, Beales PA, Hughes KJD. Fungal plant pathogens. 2nd ed. Applied Techniques; 2024. ISBN 978‑1‑80062‑055‑1328.
- 11.Chaturvedi P, Shrivastava R, Upreti DK. Neem (Azadirachta indica): a natural source of eco-friendly pest control agent. Plant Arch. 2014;14(1):1–6. [Google Scholar]
- 12.Daayf F, Ongena M, Boulanger R, El-Hadrami I, Belanger RR. Induction of phenolic compounds in two cultivars of cucumber by treatment with extracts of Reynoutria sachalinensis. J Chem Ecol. 2000;26:1579–93. [Google Scholar]
- 13.Dereje D, Sintayehu G, Gezahegn A. Genetic variability, heritability, genetic advance, and association of characters in small pod hot pepper (Capsicum annuum L.) landraces in West Hararghe, Eastern Ethiopia. Res Square [Preprint]. 2023.
- 14.Da Silva Bomfim N, Nakassugi LP, de Oliveira RC & Kohiyama CY. Antifungal potential of essential oils and phytochemicals against plant pathogenic fungi. J Plant Protect Res. 2020; 60(3), 239–250. 10.24425/jppr.2020.133954
- 15.El Rasheed S, El Rasheed AS. Vegetable diseases control by using essential oils to access organic production in Sudan. Agri Res Tech Open Access J. 2017;6(4):555694. 10.19080/ARTOAJ.2017.06.555694. [Google Scholar]
- 16.Evans WC. Trease and Evans Pharmacognosy. 14th ed. London: WB Saunders Company Ltd; 1998. p. 15–6. [Google Scholar]
- 17.Haile B, Babege T, Hailu A. Diseases and insect pests of onion (Allium cepa L.) in Masha District of Sheka Zone, South-West Ethiopia. Acad J Agric Res. 2016;4(10):629–63. [Google Scholar]
- 18.Hemida KA, Ziedan EH, El-Samman MG, Khattab AN, Mohamed MH. Morphological, molecular biology and pathological characterization of fungi causing root rot on grapevine. Egypt Pharm J. 2024;23(2):368–81. 10.4103/epj.epj_286_23. [Google Scholar]
- 19.Howard RJ, Garland JA, Seaman WL. Diseases and pests of vegetable crops in Canada. Ottawa: Canadian Phytopathological Society; 1994. [Google Scholar]
- 20.Hur J, Ahn SY, Koh JY, Lee C. Antimicrobial properties of cold-tolerant Eucalyptus species against phytopathogenic fungi and food-borne bacterial pathogens. Korean J Plant Pathol. 2000;16(2):150–6. [Google Scholar]
- 21.Kotan R, Kordali S & Cakir A. (2007). Screening of antibacterial activities of twenty-one oxygenated monoterpenes. Zeitschrift für Naturforschung C, 62(7–8), 507–513.Iqbal, M., & Naz, R. Evaluation of the antifungal efficacy of neem (Azadirachta indica) oil against pathogenic fungi. J Med Plants Res. 2021; 15(2), 66–72. [DOI] [PubMed]
- 22.Kumar V, Neeraj. Isolation and identification of fungi from post‑harvest stored onion varieties and their control measures with organic formulations. Eur J Mol Clin Med. 2020; 7(7):2664.
- 23.Lee IS, Kim W, Jo G, Yang KY. Rapid detection of a downy mildew pathogen, Peronospora destructor, in infected onion tissues and soils by loop-mediated isothermal amplification. Phytopathology. 2024;114(6):1237–43. 10.1094/PHYTO-11-23-0440-R. [DOI] [PubMed] [Google Scholar]
- 24.Lucas JA, Hawkins NJ, Fraaije BA. The evolution of fungicide resistance. Adv Appl Microbiol. 2015;90:29–92. 10.1016/bs.aambs.2014.09.001. [DOI] [PubMed] [Google Scholar]
- 25.Mishra RK, Gupta RP. In vitro evaluation of plant extracts, bio-agents and fungicides against Purple blotch and Stemphylium blight of onion. Seeds. 2012;10(26):2842.
- 26.Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus, and dermatophyte species. J Med Microbiol. 2021;68(3):324–31. 10.1099/jmm.0.000928. [DOI] [PubMed] [Google Scholar]
- 27.Pitt JI, Hocking AD. Fungi and food spoilage. 2nd ed. London: Blackie Academic and Professional; 1997. [Google Scholar]
- 28.Siddiqui BS, Afshan F, Faizi S, Naqvi SNH. Two insecticidal tetranortriterpenoids from Azadirachta indica. Phytochemistry. 2003;63(5):695–8. 10.1016/S0031-9422(03)00175-6. [DOI] [PubMed] [Google Scholar]
- 29.Pralle SB. Timing and sequence in agenda-setting and policy change: a comparative study of lawn care pesticide politics in Canada and the US. J Eur Public Policy. 2006;13(7):987–1005. [Google Scholar]
- 30.Sadhna P, Pandey SN, Pandey PH. Post‑harvest management of horticulture crops. India: Kalyani Publishers; 2011. p. 325–8.
- 31.Sajad UN, Wasim RH, Dar MS, Kirmani SN, Mudasir M. New generation fungicides in disease management of horticultural crops. Hortic J. 2017;7(1):1–7. [Google Scholar]
- 32.Sharma N, Tripathi A. Fungi toxicity of the essential oil of Cymbopogon citratus against storage fungi. J Food Prot. 2006;71(7):1334–9. [Google Scholar]
- 33.Wondirad M, Eshetu A, Mohammed Y, et al. Review of vegetable diseases research in Ethiopia. Ethiop J Agric Sci. 2009;19(1):45–67. [Google Scholar]
- 34.Yaradua S, Kankara SL, Yusuf M. Isolation and identification of fungi from some selected vegetables in kankara local government area, katsina state, Nigeria. Ujmr. 2018;3(1). issn: 2616–0668.
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Data Availability Statement
Data will be made available up on reason able request from corresponding author.