Abundant Allelochemicals and the Inhibitory Mechanism of the Phenolic Acids in Water Dropwort for the Control of Microcystis aeruginosa Blooms

Abstract

In recent years, with the frequent global occurrence of harmful algal blooms, the use of plant allelopathy to control algal blooms has attracted special and wide attention. This study validates the possibility of turning water dropwort into a biological resource to inhibit the growth of harmful Microcystis aeruginosa blooms via allelopathy. The results revealed that there were 33 types of allelopathic compounds in the water dropwort culture water, of which 15 were phenolic acids. Regarding water dropwort itself, 18 phenolic acids were discovered in all the organs of water dropwort via a targeted metabolomics analysis; they were found to be mainly synthesized in the leaves and then transported to the roots and then ultimately released into culture water where they inhibited M. aeruginosa growth. Next, three types of phenolic acids synthesized in water dropwort, i.e., benzoic, salicylic, and ferulic acids, were selected to clarify their inhibitory effects on the growth of M. aeruginosa and their mechanism(s) of action. It was found that the inhibitory effect of phenolic acids on the growth of M. aeruginosa increased with the increase of the exposure concentration, although the algae cells were more sensitive to benzoic acid than to salicylic and ferulic acids. Further study indicated that the inhibitory effects of the three phenolic acids on the growth of M. aeruginosa were largely due to the simultaneous action of reducing the number of cells, damaging the integrity of the cell membrane, inhibiting chlorophyll a (Chl-a) synthesis, decreasing the values of F₀ and F_v/F_m, and increasing the activity of the antioxidant enzymes (SOD, POD, and CAT) of M. aeruginosa. Thus, the results of this study indicate that both culture water including the rich allelochemicals in water dropwort and biological algae inhibitors made from water dropwort could be used to control the growth of noxious algae in the future.

Keywords: Microcystis aeruginosa; allelopathic metabolite; antioxidant enzyme; biological control; cellular structure; phenolic acids; photosynthesis; water dropwort.

Figure 1

The inhibitory effect of the water dropwort culture water on the growth of M. aeruginosa on day 7. (a) 0 mL, (b) 25 mL, and (c) 50 mL of culture water were added to Erlenmeyer flasks containing 100 mL of M. aeruginosa, and the total volume of the culture solution in each group was maintained at 200 mL by replenishing sterile water. The final cell concentration was 2 × 10⁷ cells/mL. Each treatment included three parallel samples.

Figure 2

The heatmap of the contents of allelopathic compounds in the culture water of water dropwort at different growth stages. The bricks represent the log10A abundance of each compound in each sample. The histogram of different colors on the left represents fatty acids (blue), phenolic acids (pink), and terpenes (green). The histogram of different colors in the top-right corner represents samples from different growth stages of water dropwort.

Figure 3

Relative abundances of allelopathic phenols in the culture water. (a) The heatmap of the contents of allelopathic phenols in the culture water of water dropwort at different growth stages. The bricks represent the relative abundance of each compound in each sample. The values are centered and scaled in the row direction to show the content differences of each compound more clearly among different stages. Compound names in red text signify the compounds further investigated in this study. (b) The relative abundances of allelopathy phenols in the juvenile-stage culture water.

Figure 4

The contents of three phenolic acids in the targeted metabolomics analysis of water dropwort. There are three repetitions in each group. The data are reported as the mean ± standard error. Lowercase letters indicate a significant difference between the same phenolic acid in different organs.

Figure 5

The effects of different concentrations of ferulic, salicylic, and benzoic acids on the growth of M. aeruginosa. (a) The change in the cell density of M. aeruginosa with treatment by different phenolic acids. (b) The inhibition rate of M. aeruginosa with treatment by different phenolic acids. There are three repetitions in each group. The data are reported as the mean ± standard error. Lowercase letters of different treatments at the same time indicate significant differences.

Figure 6

SEM images of M. aeruginosa treated with different concentrations of phenolic acid. (A1–A4): 25, 30, 35, and 40 mg/L benzoic acid treatment, respectively; (B1–B4): 25, 30, 35, and 40 mg/L salicylic acid treatment, respectively; (C1–C4): 30, 40, 50, and 60 mg/L ferulic acid treatment, respectively; (D): control of the BG-11 medium.

Figure 7

The effects of different concentrations of phenolic acids on the Chl-a content in M. aeruginosa. There are three repetitions in each group. The data are reported as the mean ± standard error. Lowercase letters at the same time indicate significant differences between different treatments.

Figure 8

The fluorescence parameters F₀ and F_v/F_m of M. aeruginosa under different concentrations of phenolic acids. (A–C): The F₀ of M. aeruginosa under benzoic acid, salicylic acid and ferulic acid treatments, respectively; (D–F): The F_v/F_m of M. aeruginosa under benzoic acid, salicylic acid and ferulic acid treatments, respectively. There are three repetitions in each group. The data are reported as the mean ± standard error. Lowercase letters of different treatments at the same time indicate significant differences.

Figure 9

The effects of different concentrations of phenolic acid on the enzyme activities of M. aeruginosa. (A–C): The SOD activity of M. aeruginosa under benzoic acid, salicylic acid and ferulic acid treatments, respectively; (D–F): The POD activity of M. aeruginosa under benzoic acid, salicylic acid and ferulic acid treatments, respectively; (G–I): The CAT activity of M. aeruginosa under benzoic acid, salicylic acid and ferulic acid treatments, respectively. There are three repetitions in each group. The data are reported as the mean ± standard error. Lowercase letters of different treatments at the same time indicate significant differences.