Induction of phytoalexins and proteins related to pathogenesis in plants treated with extracts of cutaneous secretions of southern Amazonian Bufonidae amphibians

Abstract

Cutaneous secretions produced by amphibians of the family Bufonidae are rich sources of bioactive compounds that can be useful as new chemical templates for agrochemicals. In crop protection, the use of elicitors to induce responses offers the prospect of durable, broad-spectrum disease control using the plant's own resistance. Therefore, we evaluated the potential of methanolic extracts of cutaneous secretions of two species of amphibians of the family Bufonidae found in the Amazon biome-Rhaebo guttatus (species 1) and Rhinella marina (species 2)-in the synthesis of phytoalexins in soybean cotyledons, bean hypocotyls, and sorghum mesocotyls. Additionally, changes in the enzyme activity of β-1,3-glucanase, peroxidase (POX), and polyphenol oxidase (PPO) and in the total protein content of soybean cotyledons were determined. In the soybean cultivar 'TMG 132 RR', our results indicated that the methanolic extract of R. guttatus cutaneous secretions suppressed glyceollin synthesis and β-1,3-glucanase activity and increased POX and PPO activities at higher concentrations and total protein content at a concentration of 0.2 mg/mL. On the other hand, the methanolic extract of R. marina cutaneous secretions induced glyceollin synthesis in the soybean cultivars 'TMG 132 RR' and 'Monsoy 8372 IPRO' at 0.1-0.2 mg/mL and 0.2 mg/mL, respectively. The methanolic extract of R. marina cutaneous secretions also increased the specific activity of POX and PPO in 'Monsoy 8372 IPRO' and 'TMG 132 RR', respectively, and decreased the activity of β-1,3-glucanases in 'Monsoy 8372 IPRO'. At 0.3 mg/mL, it stimulated phaseolin synthesis. The extracts did not express bioactivity in the synthesis of deoxyanthocyanidins in sorghum mesocotyls. The study in soybean suggests that the bioactivity in defense responses is influenced by cultivar genotypes. Therefore, these results provide evidence that extracts of cutaneous secretions of these amphibians species may contribute to the bioactivity of defense metabolites in plants.

Fig 1. Synthesis of glyceollins in soybean cotyledons treated with different concentrations of R. guttatus and R. marina (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL), sterile water (0), and S. cerevisiae (SC).

Cultivars treated with R. guttatus cutaneous secretions (species 1): (A) ‘Monsoy 8372 IPRO’; (B) ‘TMG 132 RR’; and (C) nontransgenic ‘TMG 4182’. Cultivars treated with R. marina cutaneous secretions (species 2): (D) ‘Monsoy 8372 IPRO’; (E) ‘TMG 132 RR’; and (F) nontransgenic ‘TMG 4182’. The experiments were performed three times for each treatment. The same letters indicate absence of significant differences by the Scott–Knott test at P ≤ 0.05. Metric bars indicate the standard error of the mean (SE). Data were transformed as follows: (x+1)^0.5.

Fig 2. Synthesis of phaseolins in bean hypocotyls and deoxyanthocyanidins in sorghum mesocotyls treated with different concentrations of R. guttatus and R. marina (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL), sterile water (0), and S. cerevisiae (SC).

(A) Bean hypocotyls treated with R. guttatus cutaneous secretions; (B) Bean hypocotyls treated with R. marina cutaneous secretions; (C) Sorghum mesocotyls treated with R. guttatus cutaneous secretions; and (D) Sorghum mesocotyls treated with R. marina cutaneous secretions. The experiments were performed three times for each treatment. The same letters indicate absence of significant differences by the Scott–Knott test at P ≤ 0.05. Metric bars indicate the standard error of the mean (SE). Data were transformed as follows: (x+1)^0.5.

Fig 3. Specific activity of peroxidases in ‘Monsoy 8372 IPRO,’ ‘TMG 132 RR,’ and nontransgenic ‘TMG 4182’ treated with different concentrations of R. guttatus and R. marina (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL), sterile water (0), and S. cerevisiae (SC).

Cultivars treated with R. guttatus cutaneous secretions (species 1): (A) ‘Monsoy 8372 IPRO’; (B) ‘TMG 132 RR’; and (C) nontransgenic ‘TMG 4182’. Cultivars treated with R. marina cutaneous secretions (species 2): (D) ‘Monsoy 8372 IPRO’; (E) ‘TMG 132 RR’; and (F) nontransgenic ‘TMG 4182’. Data are the mean of the three independent experiments. Data when significant were subjected to regression analysis (P ≤ 0.05), adjusting the regression equations (R²). Treatment with S. cerevisiae was used as an additional control. Metric bars indicate the standard error of the mean (SE).

Fig 4. Specific activity of polyphenol oxidases in ‘Monsoy 8372 IPRO,’ ‘TMG 132 RR,’ and nontransgenic ‘TMG 4182’ treated with different concentrations of R. guttatus and R. marina (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL), sterile water (0), and S. cerevisiae (SC).

Cultivars treated with R. guttatus cutaneous secretions (species 1): (A) ‘Monsoy 8372 IPRO’; (B) ‘TMG 132 RR’; and (C) nontransgenic ‘TMG 4182’. Cultivars treated with R. marina cutaneous secretions (species 2): (D) ‘Monsoy 8372 IPRO’; (E) ‘TMG 132 RR’; and (F) nontransgenic ‘TMG 4182’. Data were the mean of three independent experiments. Data when significant were subjected to regression analysis (P ≤ 0.05), adjusting the regression equations (R²). Treatment with S. cerevisiae was used as an additional control. Metric bars indicate the standard error of the mean (SE).

Fig 5. Specific activity of β-1,3-glucanases in ‘Monsoy 8372 IPRO,’ ‘TMG 132 RR,’ and conventional ‘TMG 4182’ exposed to different concentrations of R. guttatus and R. marina (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL), sterile water (0), and S. cerevisiae (SC).

Cultivars treated with the extract of R. guttatus cutaneous secretions (species 1): (A) ‘Monsoy 8372 IPRO’; (B) ‘TMG 132 RR’; and (C) nontransgenic ‘TMG 4182’. Cultivars treated with the extract of R. marina cutaneous secretions (species 2): (D) ‘Monsoy 8372 IPRO’; (E) ‘TMG 132 RR’; and (F) nontransgenic ‘TMG 4182’. Data are the mean of three independent experiments. Data when significant were subjected to regression analysis (P ≤ 0.05), adjusting the regression equations (R²). Treatment with S. cerevisiae was used as an additional control. Metric bars indicate the standard error of the mean (SE).

Fig 6. Total protein content in ‘Monsoy 8372 IPRO,’ ‘TMG 132 RR,’ and nontransgenic ‘TMG 4182’ treated with different concentrations of R. guttatus and R. marina (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL), sterile water (0), and S. cerevisiae (SC).

Cultivars treated with R. guttatus cutaneous secretions (species 1): (A) ‘Monsoy 8372 IPRO’; (B) ‘TMG 132 RR’; and (C) nontransgenic ‘TMG 4182’. Cultivars treated with R. marina cutaneous secretions (species 2): (D) ‘Monsoy 8372 IPRO’; (E) ‘TMG 132 RR’; and (F) nontransgenic ‘TMG 4182’. Data are the mean of three independent experiments. Data when significant were subjected to regression analysis (P ≤ 0.05), adjusting the regression equations (R²). Treatment with S. cerevisiae was used as an additional control. Metric bars indicate the standard error of the mean (SE).