Defense Mechanisms Induced by Celery Seed Essential Oil against Powdery Mildew Incited by Podosphaera fusca in Cucumber

Abstract

This study aimed to evaluate the effectiveness of essential oil extracted from celery (Apium graveolens) seeds (CSEO) for the control of powdery mildew of cucumber (Cucumis sativus) incited by Podosphaera fusca and to investigate the metabolic and genetic defense mechanisms triggered by the treatment with this essential oil in cucumber seedlings. The main compounds in the CSEO as determined by gas chromatography-mass spectrometry (GC-MS) analysis were d-limonene, 3-butyl phthalide, β-selinene, and mandelic acid. The treatment with CSEO led to an increase in the content of both chlorophyll and phenolic/flavonoid compounds in cucumber leaves. In greenhouse tests, the application of CSEO reduced by 60% the disease severity on leaves of cucumber plants and stimulated the activity of defense-related enzymes such as β-1,3-glucanase, chitinase, phenylalanine ammonia-lyase, peroxidase, and polyphenol oxidase. Moreover, treatment with CSEO induced overexpression of β-1,3-glucanase, chitinase, and phenylalanine ammonia-lyase genes. A highly significant correlation was found between the β-1,3-glucanase, chitinase, and phenylalanine ammonia-lyase enzymatic activities and the relative expression of the corresponding encoding genes in both inoculated and non-inoculated cucumber seedlings treated with the essential oil. Overall, this study showed that CSEO is a promising eco-friendly candidate fungicide that can be exploited to control cucumber powdery mildew.

Keywords: Apium graveolens; Cucumis sativus; chitinase; chlorophyll; d-limonene; flavonoids; gene expression; phenols; phenylalanine ammonia-lyase; β-1,3-glucanase.

Figure 1

Severity of powdery mildew in cucumber seedlings sprayed with different concentrations of celery seed essential oil (100, 200, and 400 μg mL⁻¹) one day after inoculation, compared to inoculated control seedlings treated with dw. Different letters indicate a significant difference for p ≤ 0.01 (LSD test).

Figure 2

Effect of treatment with celery seed essential oil (400 μg mL⁻¹) on the (A) chlorophyll content, (B) phenolic compounds, and (C) flavonoids of leaves of cucumber seedlings inoculated with powdery mildew compared to non-inoculated seedlings, at five different time intervals after the treatment. Different letters indicate significant differences for p ≤ 0.001 (Tukey’s test).

Figure 3

Effect of treatment with celery seed essential oil (400 μg mL⁻¹) on (A) β-1,3-glucanase, (B) chitinase, (C) phenylalanine ammonia-lyase, (D) peroxidase, and (E) polyphenol oxidase activity in cucumber leaves either inoculated or non-inoculated with powdery mildew, compared to untreated seedlings, at five different time intervals after the treatment. Different letters indicate significant differences for p ≤ 0.001 (Tukey’s test).

Figure 4

Effect of celery essential oil (400 μg mL⁻¹) on the relative expression (fold change) of β-1,3-glucanase (A1,A2), chitinase (B1,B2), and phenylalanine ammonia-lyase (C1,C2) in the cucumber leaves inoculated and non-inoculated with powdery mildew at different sampling intervals after the treatment with essential oil. The treatment was carried out 10 d after inoculation soon after the onset of symptoms, and the zero point was within one hour after the treatment. Different symbols indicate significant differences between each set and the corresponding control set at the same time interval according to Tukey’s multiple comparison test. Significance symbols: 0 ***; 0.001 **; 0.01 * 0.05; ns non-significant.

Figure 5

Correlation between the activities of β-1,3-glucanase (A1,A2), chitinase (B1,B2), and phenylalanine ammonia-lyase (C1,C2) and their corresponding gene expressions in cucumber leaves. The leaves were either inoculated (A1,B1,C1) or non-inoculated (A2,B2,C2) with cucumber powdery mildew. The correlation was determined at different sampling intervals after the treatment with celery seed essential oil at a concentration of 400 μg mL⁻¹.

Figure 6

Heatmap of β-1,3-glucanase, chitinase, and phenylalanine ammonia-lyase gene expressions as a result of the treatment with celery essential oil (400 μg mL⁻¹) in cucumber leaves inoculated with cucumber powdery mildew at different sampling intervals after the treatment with essential oil. The red color represents the maximum relative expression (fold change) of each gene, while the blue color represents the minimum relative gene expression compared to untreated healthy cucumber seedlings.