2. RESULTS
2.1 Root zone warming enhances tomato resistance to Bc induced disease
To determine the role of mild heat acclimation in tomato disease response, we examined the effect of warming tomato roots to 28ºC on pathogenesis of the necrotrophic fungus B. cinerea, the causative agent of gray mold disease. Bc infects more than 1400 plant species, infecting various organs of many important crops (Elad et al., 2016b). Tomato plants of wild type (WT) cv. Brigade, a susceptible tomato cultivar, and WT cv. M82, a less susceptible tomato cultivar, were grown at 21ºC. Heat-induced disease reduction was previously reported in tomato for Bc in the Brigade cultivar (Elad, 2018). For experiments, treated plants were placed on a hot plate device which warmed the root zone to 28ºC, with constant temperature monitoring, on a ”long-day” cycle, for 7 days. Further experimental information is provided in the materials section. Mock plants were mounted on a similar device which was not activated. After 7 days, plants were removed from the heating device, and infected with B. cinerea . Disease progression was monitored for 7 days. Throughout the experiment, the plant shoot remained at 21ºC, as ensured by constant temperature measurement. RZW decreased the severity of Bc induced disease by about 60% in cv. Brigade (Figure 1a,b) and 40% in cv. M82 (Figure 1c,d). In both cultivars, disease was significantly lower at all time-points in the root-warmed plants when compared with the mock plants. The differences in the level of disease reduction are likely attributable to the initial difference in the level of disease susceptibility among the two cultivars.
Since warming the roots resulted in disease resistance in the shoots, which remained at 21ºC during the entire experiment, the disease protectant effect generated by the treatments is systemic, as was previously suggested (Elad, 2018; Elad et al., 2016a).
Previous reports have indicated that there are several different types of heat acclimation in plants. The treatment we applied most resembles thermo-tolerance to moderate – high temperatures; TMHT (Yeh et al., 2012). To examine activation of the plant thermo-tolerance machinery as a result of the RZW we applied, we assayed the expression of classical heat shock genes in these plants in comparison with the mock plants. The heat-stress machinery was activated in the plants that received the RZW treatment (Figure 2), though it appears to be activated to a lesser degree than reported in the literature in connection with more classical ”heat shock” experiments conducted, where typically, more extreme heat treatments are applied (Yang et al., 2016; Snyman and Cronjé, 2008; Fragkostefanakis et al., 2016). The induction levels of HSP and Hsf genes in tomato following our RZW treatment as compared with the induction achieved in a heat-shock experiment conducted in tomato (Fragkostefanakis et al., 2016), where higher induction values were observed (Supplemental Figure S1 ).
To examine the robustness and timing of disease protection in the plants which received the RZW treatments, we employed a second treatment protocol, where we applied the warming treatment for 48 h and subsequently infected the plants with Bc at different time-points after the warming treatment was applied. Three different time-points were selected, ranging from plants that received the heat treatment 5 days prior to Bc inoculation, all the way up to plants that received the heat treatment immediately prior to Bc inoculation. Plants which received the warming treatment 3 days prior to Bcinoculation, spending 72h in 21ºC recovery after the heat treatment and prior to Bc application, showed the greatest reduction in disease levels, in both Brigade (Figure 3a,b ) and M82 cultivars (Figure 3c,d ). This indicates that the acclimation processes occurring within the plant after RZW are amplified, in the context of immune system activation, after spending some time back in optimal temperatures.