عنوان مقاله [English]
Introduction: Tomato (Solanum lycopersicum) is one of the most important crops worldwide, which suffers from several destructive diseases. Rhizoctonia solani is a necrotrophic fungus which is known as the causal agent of tomato damping-off, seed rot, root and crown rot diseases. Control of this phytopathogenic fungus is very difficult due to its long-term survival in the soil, high level of genetic diversity in R. solani populations and the lake of complete resistance in plants to the pathogen. Although partial genetic resistance to Rhizoctonia diseases has been reported, no major gene responsible for resistance has been found so far. Most of the traditional cultivars, planted in tomato-growing areas, are susceptible to Rhizoctonia diseases. Therefore, an intensive use of other crop protection methods such as an application of chemicals or biological agent which are capable of activating plant defense responses seems to be necessary to suppress the disease damage in tomato fields. The growing concern on negative environmental effects of fungicides and the appearance of fungicide- resistant pathogens led to increased research interest in the alternative protection methods. Among these novel disease management strategies, induced resistance (IR) has emerged as a potential supplement in plant protection trials.
Materials and Methods: The cultivar CH Falat was used as a susceptible tomato genotype to R. solani. The seeds were surface sterilized with 1% sodium hypochlorite for 1min, rinsed 3 times with sterile distilled water and incubated for 5 days on a wet sterile filter paper in Petri dishes. Germinated seeds were each sown in plastic pots filled with autoclaved commercial potting soil and kept in greenhouse conditions. The tomato leaves were treated with various concentrations of thiamine, pyridoxine, and homoserine lactones and inoculated with the pathogen. Disease symptoms were evaluated at 5 days post-inoculation (dpi) and the most effective inducer of resistance at the best concentration was selected for the rest of the experiments. The role of phenolic compounds and peroxidase in thiamine-IR was investigated in our pathosystem. For phenolics measurement, leaf and stem samples (500 mg FW) were crushed into fine powder in liquid N2 with a mortar and pestle and were extracted separately in 8 ml of 80% methanol. The extracts were centrifuged at 4000 g for 5 min. To determine the number of total phenols, 50 ml of the extract was placed in microtubes containing 700 ml of distilled water and 50 ml of Folin. After 5 min, 100 ml of 20% sodium carbonate and 100 ml distilled water were added and the tube contents were mixed. Absorbance at 720 nm was determined after 60 min using a spectrophotometer. Total phenol was expressed as the milligrams per gram of tissue fresh weight. Soluble phenolic contents were calculated using a standard curve obtained with different concentrations of caffeic acid. For determining peroxidase activity, enzyme extracts (containing 30 mg of total protein) were added to 30 ml of 200 mM guaiacol and 25mM citrate phosphate (pH 5.4). To each sample, 30 ml of 30% H2O2 was added and the absorbance was measured at 470 nm using a spectrophotometer.
Results and Discussion: Disease index evaluations after various treatments revealed that thiamine 20 mM had the best effect in inducing resistance to the pathogen. This vitamin was used for treating the tomato leaves in subsequent experiments. For investigation of the role of peroxidase in thiamine- induced resistance, NaN3 was used as a speciﬁc peroxidase inhibitor and phenolic contents were measured in infected samples treated with thiamine and NaN3 at 0, 24, 48 and 72 hours after inoculation. There was a considerable difference between the phenolics level in different samples at various time points investigated in this study. The phenolics level in the samples treated with NaN3 was lower than other treatments. As a result, considering the role of peroxidase in phenolics production, the inhabitation of peroxidase activity leads to a reduction of phenolics and decrease of resistance to the pathogen in the tomato leaves treated with thiamine together with NaN3.
Conclusion: The results obtained in this study revealed the great potential of thiamine, as an environmentally safe vitamin, for induction of defense responses and partial protection of tomato plants against the destructive necrotrophic fungus R. solani AG4 HG I. It can be concluded that peroxidase and phenolics have an important role in thiamine-IR in tomato- R. solani interaction.