5TH International Congress on Technology - Engineering & Science - Kuala Lumpur - Malaysia (2018-02-01)

Potassium Phosphite Enhanced Defense Responses In Downy Mildew- Infected Cucumber (cucumis Sativus L.) Plants

Potassium phosphite (KPhi) is a widely used chemical resistance inducer to enhance plants tolerance against fungal diseases [1]. In the present study the effect of KPhi application on physiological and molecular defense responses of downy mildew-challenged cucumber plants was investigated. Cucumber plants grown under controlled conditions were treated with KPhi followed by inoculation with Pseudoperonospora cubensis and leaf samples were collected at different time courses for assessments. The plants were treated as following: 1. Leaves were sprayed with 3 μL sterile distilled water (Control), 2. Leaves were treated solely with 2 gL-1 KPhi (KPhi treatment), 3. Each leaf was inoculated with 3 μL drops of 1×105 mL-1 zoospores sporangia on the abaxial surface, 4. Four days after treatment of the leaves with 2 g L-1 KPhi, inoculation with sporangia of P. cubensis was performed (Pre-inoculation), 5. Eight days after inoculation of the leaves with sporangia of P. cubensis, 2 gL-1 KPhi treatment were applied (Post-inoculation). Different parameters including chlorophyll content [2], phytoalexins accumulation [3], alterations in phenylammonialyase (PAL) activity [4] were investigated. Furthermore, molecular analysis of the variations in defense-related genes were conducted through qPCR. All assessments were arranged as factorial experiments and performed in three biological replications in each treatment. The mean data of all other experiments were subjected to analysis of variance (ANOVA) and significant effects of treatments were determined by two-way ANOVA and least significant difference (LSD) methods. The overall results of this study revealed that KPhi treatment led enhanced physiological, biochemical and molecular defense responses in KPhi and P. cubensis-inoculated cucumber plants. The inducing effects of KPhi on plant immune system have been reported by several researchers [5, 6, 7]. Significant difference was observed between treatments and time courses compared to control plants (P≤ 0.05). Results showed that the control plants exhibited a significant increase in chlorophyll (Chl) content (about 10%) during the experiment. By contrast, compared to control plants, in KPhi, pre-inoculated, post-inoculated and P. cubensis inoculated plants a remarkable decrease in Chl a (31%, 23%, 62% and 82%) was observed at 96 h after inoculation. In case of Chl b, alteration was just similar to Chl a and compared to samples collected 24 hours after inoculation, a 12 % increase was recorded for samples collected at 96 hours after inoculation. But in KPhi, pre-inoculated, post-inoculated and P. cubensis inoculated plants a decrease of Chl b (23%, 36%, 92% and 87%) at 96 h was observed compared to control plants (Figure 1 A, B). It was revealed that the highest amount of phytoalexin was recorded in KPhi-treated and pre-inoculated plants (30% higher than the control) at 96 h (0.048 µg per 1 g FW), with a gradual increase over time. The lowest amounts of phytoalexin were observed in control and P. cubensis-inoculated plants at all-time points (from 0.02 µg per 1 g FW at 24 h to 0.038 µg per 1 g FW at 96 h). Higher contents were detected in the pre-inoculated (34%) than post-inoculated plants (21%) compared with the control (Figure 2). KPhi application significantly increased PAL activity during the experiment. However, only in pre-inoculated plants did the enzymatic activity increase at 48 h (0.95 mg per 1 g FW) and then decrease at the end of experiment (0.76 mg per 1 g FW). The highest enzymatic activity was recorded in KPhi at 96 h and in pre-inoculated plants at 48 h, with an approximately 41% increase compared with the control plants (0.95 mg per 1 g FW). The lowest enzyme activity was recorded in the control and P. cubensis-inoculated plants at 24 h (0.5 mg per 1 g FW). The pre-inoculated plants showed more effectiveness (40%) compared with the post-inoculated plants (23%) in terms of efficiency at all-time points (Figure 3). Gene expression analysis indicated that the highest gene rate was observed in plants pre-inoculated with KPhi for 96 h for thaumatin-like protein (TLP) (74%), 72 h for Ribosome-inactivating protein (RIP) (95%) and 48 h for defensin (95%) after P. cubensis inoculation (Figure 4). The findings of the present study indicate that KPhi potassium phosphite primes plants for a rapid and robust response to infection that involves the heightened activation of a range of defense responses.
Ali Dehestani Kolagar, Moazzameh Ramezani, Fatemeh Rahmani, Samira Shabani