Celery and Spinach Flavonoid-Rich Extracts Enhance Phytoalexin Production in Powdery Mildew-Infected Cucumber Leaves

Abstract

Phytoalexins are antimicrobial compounds of diverse chemical classes whose production is triggered in plants in response to pathogen infection. This study demonstrated that spraying with a celery flavonoid-rich extract (CFRE) or a spinach flavonoid-rich extract (SFRE) enhanced the production of phytoalexins in cucumber leaves artificially infected with powdery mildew incited by Podosphaera fusca. High-performance liquid chromatographic (HPLC) analysis revealed a noticeable increase in the content of phenolic acids, including caffeic acid, ellagic acid, ferulic acid, gallic acid, p-coumaric acid, and syringic acid, as well as the flavonoid rutin in both non-inoculated and inoculated leaves of cucumber seedlings treated with CFRE and SFRE, compared to healthy untreated leaves used as a control. Fluorescence microscopy revealed the accumulation of phenolic acid compounds in chloroplasts and at the periphery of epidermal cells. Overall, results suggest the reduced severity of P. fusca infection following the application of CFRE and SFRE in cucumber leaves could be due, at least in part, to the production of phytoalexins of polyphenolic nature. These findings provide insights into the mechanisms of systemic resistance induced by CFRE and SFRE. Moreover, they confirm these two natural flavonoid-rich products could be promising alternatives to synthetic chemical fungicides for the safe and ecofriendly control of cucumber powdery mildew.

Keywords: Apium graveolens; Cucumis sativus; Spinacia oleracea; flavonoid-rich plant extracts; phenolic compounds; phytoalexins; powdery mildew.

Figure 1

Measured phytoalexin amounts (µg per 100 mg of the dry weight of the extract) (A) caffeic acid; (B) ellagic acid; (C) ferulic acid; (D) gallic acid; (E) p-coumaric acid; (F) syringic acid; and (G) rutin in the inoculated/non-inoculated cucumber leaves at different sampling intervals (0, 1, 2, 4, and 8 d) following treatment with celery flavonoid-rich extract (CFRE). Significant differences between each group and its respective control at the same time interval are indicated by symbols (Tukey’s multiple comparison test): 0 ‘***’ for p ≤ 0.001, ‘**’ for p ≤ 0.01, ‘*’ for p ≤ 0.05, and ns = non-significant.

Figure 2

Clustering heatmap of the measured phytoalexin amounts (caffeic acid, ellagic acid, ferulic acid, gallic acid, p-coumaric acid, rutin, and syringic acid) after foliar application of celery flavonoid-rich extract in the four sets of cucumber seedlings: inoculated untreated seedlings (IN-UT), inoculated treated seedlings (IN-TR), non-inoculated untreated seedlings (NO-UT), and non-inoculated treated seedlings (NO-TR), at various sampling time intervals (0, 1, 2, 4, and 8 d).

Figure 3

Measured phytoalexin amounts (µg per 100 mg of the dry weight of the extract) (A) caffeic acid; (B) ellagic acid; (C) ferulic acid; (D) gallic acid; (E) p-coumaric acid; (F) syringic acid; and (G) rutin in the non-inoculated/inoculated cucumber leaves at different sampling intervals (0, 1, 2, 4, and 8 d) following treatment with spinach flavonoid-rich extract (SFRE). Significant differences between each group and its respective control at the same time interval are indicated by symbols (Tukey’s multiple comparison test): 0 ‘***’ for p ≤ 0.001, ‘**’ for p ≤ 0.01, ‘*’ for p ≤ 0.05, and ns = non-significant.

Figure 4

Heatmap of the measured phytoalexin amounts (caffeic acid, ellagic acid, ferulic acid, gallic acid, p-coumaric acid, rutin, and syringic acid) following foliar application of spinach flavonoid-rich extract in the four sets of cucumber seedlings: inoculated untreated seedlings (IN-UT), inoculated treated seedlings (IN-TR), non-inoculated untreated seedlings (NO-UT), and non-inoculated treated seedlings (NO-TR) at different sampling time intervals (0, 1, 2, 4, and 8 d).

Figure 5

Venn diagrams showing the number and percentage of common phytoalexins (caffeic acid, ellagic acid, ferulic acid, gallic acid, p-coumaric acid, rutin, and syringic acid) present in and shared among (A): non-inoculated untreated seedlings (NO-UT) and non-inoculated treated seedlings (NO-TR); (B): inoculated untreated seedlings (IN-UT) and inoculated treated seedlings (IN-TR); (C): all four sets (non-inoculated untreated seedlings (NO-UT), non-inoculated treated seedlings (NO-TR), inoculated untreated seedlings (IN-UT), and inoculated treated seedlings (IN-TR)) of cucumber seedlings. Seedlings were sprayed with either celery or spinach flavonoid-rich extract, and samples were collected at various sampling time intervals after the treatment (0, 1, 2, 4, and 8 d).

Figure 6

Fluorescence microscopy images showing the distribution patterns of phenolic acid compounds in inoculated cucumber leaves on 1 (A), 2 (B), 4 (C), and 8 (D) days post-treatment with celery flavonoid-rich extract. Black arrows = stomatal guard cells, blue arrows = periphery of epidermal cells, green arrows = chlorophyll. Bar = 10 μm.

Figure 7

Fluorescence microscopy images showing the distribution patterns of phenolic acid compounds in inoculated cucumber leaves on 1 (A), 2 (B), 4 (C), and 8 (D) days post-treatment with spinach flavonoid-rich extract. Black arrows = stomatal guard cells, blue arrows = periphery of epidermal cells, green arrows = chlorophyll. Bar = 10 μm.