Antifungal Activity of Propyl-Propane-Thiosulfinate (PTS) and Propyl-Propane-Thiosulfonate (PTSO) from Allium cepa against Verticillium dahliae: In Vitro and in Planta Assays

Abstract

Verticillium wilt, caused by Verticillium dahliae, is the most devastating soil-borne fungal disease of olive trees worldwide. Currently, there is no effective measure available to control the pathogen in diseased plants in open field conditions. Searching more effective and sustainable solutions are a priority for the olive sector. The existing alternatives for disease control include the use of biological control microorganisms and compounds of natural origin from plants, such as Alliaceae. Propyl propane thiosulfinate (PTS) and propyl propane thiosulfonate (PTSO) are two organosulfur compounds derived from Allium cepa with a widely documented antimicrobial activity. The aim of this study was to evaluate the antifungal activity of PTS and PTSO against the defoliating and non-defoliating V. dahliae pathotypes. Firstly, several in vitro tests were performed (Minimum Antifungal Concentration, susceptibility studies according to the Kirby-Bauer disk-diffusion method, antifungal activity through aerial diffusion and effect on mycelial growth). The ability of both compounds to sanitize soil was evaluated using a sterile substrate inoculated with V. dahliae. Finally, challenges in growth chambers were carried out. PTS and PTSO generated growth inhibition zones in agar diffusion and the gas phase, and the mycelial growth of all the V. dahliae strains was significantly altered. The V. dahliae population in soil was considerably reduced after the sanitization. Finally, in planta assays demonstrated the ability of these compounds to reduce disease related parameters and their contribution to control the phytopathogen. In conclusion, the results showed that the PTS and PTSO from Allium cepa display in vitro and in vivo antifungal activity against V. dahliae and suggested that both compounds could be used as natural and environmentally friendly tools for Verticillium wilt management.

Keywords: Verticillium wilt; olive trees; onion; organosulfur compounds; pest management.

Figure 1

Antifungal activity of PTS and PTSO against Verticillium dahliae V136I 1A, V1235I 2B and V1266I 4B isolates using the disk-diffusion method. The image shows inhibition zones at doses of 2.5, 5, 10, 25 and 50 µg/µL from left to right and from top to bottom.

Figure 2

Antifungal activity of PTS and PTSO against Verticillium dahliae V136I 1A, V1235I 2B and V1266I 4B strains via the gas phase. The image shows inhibition zones at doses of 2.5, 5, 10, 25 and 50 µg/µL from left to right and from top to bottom.

Figure 3

Mycelium Growth Curves of Verticillium dahliae isolates at different concentration of PTS and PTSO: control group; 25; 50; 75 and 100 µg/mL. Values are means with SD in bars. ^T p < 0.1; * p < 0.05; ** p < 0.01 respect to control. EC50 values have been calculated for each V. dahliae isolate and each organosulfur compound.

Figure 4

Concentration of Verticillium dahliae in soil after treatment with the blend of PTS and PTSO in 1:1 proportion at different concentrations (100, 500 and 1000 µg/mL) compared to a positive control group (C +) that consists of sterilized soil inoculated with V. dahliae but without treatment. Values are means with standard deviation in bars. For each sampling day, bars with a different letter (a–d) indicate significant differences according to Tukey HSD test at p < 0.05.

Figure 5

Symptoms of Verticillium wilt in olive plantlets of cv. Picual that grew for 2 months in soil infested with the defoliating pathotype V136I 1A of Verticillium dahliae treated with the blend of PTS and PTSO in 1:1 proportion at different concentrations: (a) Plants that were not further treated with AP; (b) Plants that were treated once with 100 mL one week later via irrigation. In both panels (a,b), the following can be observed: (1) Negative control group without fungal inoculation or treatment, (2) Positive control group that grew in soil infested with V. dahliae and not treated. (3) Plants that grew in V. dahliae infected soil treated with the blend of PTS/PTSO at 250 µg/mL and (4) Infected test group treated with the blend of PTS/PTSO at 500 µg/mL.

Figure 6

Disease reaction of olive trees of cv. Picual that grew in soil infested with the defoliating Verticillium dahliae (NT) pathotype treated with the blend of PTS and PTSO in 1:1 proportion at 250 and 500 µg/mL, 92 days after transplanting to the infested soil: (A) Disease incidence expressed as percentage, (B) Disease severity expressed in scale from 0 to 4, (C) Intensity of vascular colonization expressed as percentage, (D) Density of V. dahliae in soil expressed as Log₁₀ CFU/g of soil. All panels include results from plants previously treated with the blend of PTS/PTSO or treated once with 100 mL of their respective dose one week later (Soil + Irrigation). For each panel, bars with a different letter indicate significant differences according to Tukey HSD test at p < 0.05.