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.