Involvement of reactive oxygen species in resistance of cucumber (Cucumis sativus L.) plants to cucumber scab induced by photodynamically active substances

Induced disease resistance of plants is promising for practical use, since it involves internal plant resources and allows reducing the load on the environment. substances affecting the pro/antioxidant balance are shown to participate in the mechanism of systemic acquired resistance. Photodynamically active substances also have a similar ability. This work demonstrates for the first time the ability of photodynamically active substances (methylene blue, Bengal rose and 2-mercapto-pyridine-N-oxide) to protect cucumber plants like the known inducers of systemic resistance (K2HPO4 and β-aminobutyric acid). The objective is to study the protective action of photodynamically active substances on cucumber scab, compare it with that of the known disease resistance inducers and investigate the participation of active oxygen species in the implementation of the induction mechanism. Known and suspected disease resistance inducers were tested: K2HPO4, BABA (β-aminobutyric acid), photodynamic dyes Bengal rose (BR) and methylene blue (MB), light-dependent source of hydroxyl radical 2-MPHO (2-mercapto-pyridine-N-oxide). Cucumber plants ( Cucumis sativus L.) of the Phoenix variety susceptible to cucumber scab were grown in natural soil in a Conviron EF7 climate chamber (Conviron, Canada). At the stage of the 1st true leaf, the plants were treated with solutions of the studied substances (10 drops per leaf, water as control). After approximately 2 weeks, the proportion of drops that caused burns was calculated. 6-8 days after treatment, in half of the plants in all variants, the next, 2nd leaf was infected with a suspension of Cladosporium cucumerinum Ell. et Arth. strain C5 spores at a concentration of 105 spores/ml. In the other half of the plants, drops of water (10 drops of 20 µl) were applied to the 2nd leaf. The plants were placed in a dark thermostat (Smolensk SKTB SPU, Russia) at 18 °С for 22-24 h. Symptoms were counted 6-8 days later. To obtain diffusates of the 1st leaf, drops of water or the studied solutions (10 drops of 20 μl each) were applied to it. They were removed after 24 h. The same drops of distilled water were put in the same points. After another 24 h, they were collected, centrifuged for 10 min at 8000 g, and the supernatant was used. To obtain diffusates of uninfected leaves, water was applied to them. The spore suspension was applied on leaves to obtain diffusates of infected ones. Spore diffusates were obtained from the spore suspension. The formation of superoxide radical was analyzed in diffusates by the oxidation of exogenous adrenaline.To assess spore germination, water, diffusates, or test solutions (40 μl each) were mixed with 5 μl of suspension (3.5×104 spores/ml) and 5 μl of water. The material was incubated in the dark for 24 h in a climatic chamber, then fixed with ethanol (1-2 drops per well), and germinated spores were counted. To assess the involvement of ROS in the action of diffusates on spores, antioxidants were added instead of 5 μl of water, the 50 μg/ml of SOD or 50 μg/ml of catalase, or hydroxyl radical scavengers, the 10 mM mannitol or 1 mM sodium formate. Then, germination was counted in the same way. To study effect of light on germination, a mixture of diffusates with spores was illuminated for 1 h at 90 μmolłm-2·s-1 and then incubated for 23 h in the dark. For a preliminary assessment of the nature of substances secreted by the leaves, we checked how boiling the diffusate (1 h in a water bath) affects spore germination. The effect of putative inducers on spore germination also was studied. The preparation solutions (45 μl) were mixed with 5 μl of suspension (3.5×104 spores/ml) and germinated under the same conditions, in the light and in the dark. It was found that the tested substances locally damaged the 1st leaf. The degree of disease development on the 2nd leaf in the variants with treatment was less, which indicates the induction of systemic resistance. The degree of disease suppression during treatment with photosensitizers was approximately the same as during treatment with known inducers. On the 1st leaf, superoxide formation during treatment with photosensitizers and known inducers was higher than during treatment with water. The same treatments stimulated superoxide formation in both uninfected and infected second leaves. Diffusates of uninfected leaves of untreated plants stimulated spore germination relative to germination in water. BR, MS, K2HPO4 and BAMK reduced stimulation, 2-MPNO imparted fungitoxicity. Diffusates of infected leaves of untreated plants did not stimulate germination. When treated with the studied substances, germination in diffusates decreased (except for K2HPO4), which indicated the release of fungitoxic substances by the infected plants. Since exogenous antioxidants did not affect spore germination in leaf diffusates, the action of exometabolites is not realized through ROS.