Elicitors have been proposed as a harmless tool for integrated pest management in agriculture, among which MeJA, and SA are the most widely used. In avocado, the effect of the exogenous application of MeJA and SA in the molecular response of young avocado ‘Dusa®’ plants, tolerant to the hemibiotrophic oomycete Phytophthora cinnamomi, revealed the involvement of these compounds in the molecular pathways associated with a successful defense response against P. cinnamomi [34]. This study also revealed that, among others, plant-defense-related genes were overexpressed after 6, 18 and 24 h of the application of both elicitors, suggesting the induction of a “primed-state” that could confer some protection against further pathogen attacks. Therefore, it is reasonable to suggest the use of these elicitors for reducing susceptibility of avocado ‘DusaⓇ’ to the necrotrophic ascomycete R. necatrix, in the same way as the primed-state induced by mild water stress slowed disease progression (i.e., cross-factor priming; [28]). In the present work, we have studied the effect of those elicitors on the physiological and molecular response of avocado ‘DusaⓇ’ plants before and after inoculation with the necrotoph, R. necatrix, as well as their impact on the disease progression.
Physiological and molecular response of avocado ‘Dusa ® ’ rootstocks to MeJA and SA application.
Exogenous application of MeJA and SA (at the concentrations used in this study), induced a differential response in ‘Dusa®’ avocado plants at the morpho-physiological and molecular levels. Thus, in comparison to non-elicited control plants, MeJA- and SA-treated plants increased photoprotective mechanisms (i.e., NPQ values), consistently with the observed decrease in the intrinsic photochemical efficiency of the open reaction centers of PSII (Fv’/Fm′) [36], supporting the triggering of energy dissipation mechanisms related to the alleviation of oxidative stress, in line with their role in the plant defense response against pathogens and other abiotic stress [11, 14, 37]. However, although both elicitors induced a similar response at the photochemical level, gas exchange photosynthetic parameters were only improved by SA, which agrees with previous work reporting the enhancement of net photosynthetic rates (AN), transpiration rates (E), stomatal conductance (gs) and intercellular CO2 concentration (Ci) by exogenous application of SA [38–43]. This enhancement of the photosynthetic capacity was not associated with higher chlorophyll content (i.e., SPAD index was similar in all treatments; [39]) nor to a differentially higher stomatal density [44–46], but with higher rubisco instantaneous carboxylation efficiency (AN/Ci), suggesting that SA stimulated rubisco activity rather than an increase in pigment content [40, 47]. However, the increased stomatal opening of SA-treated plants resulted in higher transpiration rates, involving higher water loss consistent with the lower RWC values, as well as lower intrinsic water use efficiency (AN/gs). These results would imply a lower ability of SA-treated plants to withstand water stress [28, 39], and contrast with the involvement of SA in plant tolerance to water stress [48, 49] and with the SA-induced stomatal closure described in previous studies [50–52]. It is noteworthy, that the higher photosynthetic rates of SA-treated plants did not translate into higher biomass (i.e., it was similar to that of non-treated and MeJA-treated plants), contrasting with the role of SA as a growth promoter [53–56]. These discrepancies, may be due to the fact that the mode of action of exogenous SA application is highly dependent on several factors [16, 57], such as plant species [38], concentration applied [58], environmental conditions [40] and the time lapsed after elicitor application [38, 55].
Conversely to SA, photosynthetic performance of avocado ‘Dusa®’ plants was not affected by MeJA, despite the observed increase in leaf mass area (LMA) in MeJA-treated avocado plants. This increase of LMA is consistent with the MeJA-induced enhancement in leaf thickness and/or leaf density previously reported [59, 60], and would entail lower water and CO2 diffusion through mesophyll cells and lower photosynthetic rates [61, 62]. Likewise, it has also been reported that exogenous MeJA application reduced assimilation and transpiration rates and induced stomatal closure in other plant species [63–66], associated to a water conservative strategy under water stress [63]. Consequently, the higher stomatal density (of similar size) of the MeJA-treated avocado leaves, could counteract the diffusional limitations of photosynthesis by increasing the amount of CO2 entering the mesophyll cells and thus enhancing photosynthetic capacity [45]. Anyhow, it should be kept in mind that the effect of MeJA on photosynthesis is dose- and plant species-dependent [13, 66, 67].
At the plant level, although elicitor treatments did not significantly affect most biomass related parameters, MeJA showed a tendency to reduce leaf dry weight and significantly increase root dry weight, resulting in a significantly higher root/shoot ratio compared to control and SA-treated plants. This result suggests that MeJA induces greater carbon partitioning to roots in avocado, in agreement with previous reports describing increased adventitious root biomass and root length by the exogenous application of MeJA in other crops [68, 69]. Consistently, MeJA-treated plants showed lower total plant leaf surface than in the other treatments, suggesting a smaller leaf size that may be associated with lower leaf water losses, and, consequently, lower plant transpiration [70]. These characteristics together with high root/shoot ratios, have been related to plant adaptability to water stress [71–74] but also to avocado tolerance to R. necatrix [31, 75] and P. cinnamomi [76, 77].
Likewise, a differential molecular response of ‘Dusa®’ avocado roots was observed after treatment with MeJA or SA. Hence, the expression of nine of the 10 defense-related genes, selected by their involvement in tolerance to soil-borne pathogens [34, 35], was deregulated depending on the type of elicitor applied.
Thus, while the major effect of MeJA treatment was the upregulation of five defense related genes (i.e., endochitinase, PR5, PR4, glu protease inhibitor and glutathione-S-transferase), SA treatment repressed three out of the five differentially expressed genes (trypsin inhibitor, metallothionein-like protein, NAC domain-containing protein 72) and upregulated two (glutathione-S-transferase, universal stress protein).
MeJA treatment significantly upregulated glu protease inhibitor. This gene encodes proteins linked to different aspects of plant defense such as oxidative stress [78], which may encompass an enhanced ability of plants to withstand R. necatrix infection, consistently with its important role in avocado tolerance to this pathogen [35, 79]. Similarly, the enhanced expression of endochitinases, PR4 and PR5 induced by MeJA could also represent a benefit to overcome WRR disease [28].
Overall, the overexpression of the above-mentioned genes [80, 81], has been described in the response of tolerant avocado plant material to P. cinnamomi and R. necatrix [34, 35, 79, 82], as well as in susceptible rootstocks after priming with mild water stress [28]. The common overexpression of these genes under diverse stress conditions points to their involvement in a broad range of plant responses to biotic [83, 84] and abiotic stress [85, 86]. This agrees with the role of MeJA in triggering plant responses against necrotrophic pathogen attack and other stresses [11, 87–89].
Both MeJA and SA, induced the upregulation of glutathione S-transferase, which has been associated with plant response to oxidative stress and inactivation of toxic compounds [90–93], which is consistent with the higher levels of NPQ observed in treated plants compared to control plants. This gene is induced by several stresses [94, 95] and has been linked mainly to the SA regulation pathway [34, 91], but its overexpression also in MeJA-treated plants suggests a concomitant crosstalk between both regulatory pathways despite their commonly reported antagonistic interaction [10, 96]. This interconnection could also explain the expression pattern of PR5 in MeJA-treated plants [11].
Thus, our results show that the effects of MeJA treatment of avocado are evidenced by the expression of defense-related genes and through changes at the morpho-anatomical level rather than by modifications at the photosynthetic level. Morpho-anatomical changes include functional traits, such as high LMA, high stomatal density, high root/shoot ratios, and ROS-scavenging mechanisms, closely related to strategies for coping with harsh environments, such as water scarcity [97–100] and soil-borne pathogen infections [31, 75, 77].
Effect of MeJA and SA on the response of avocado ‘Dusa ® ’ rootstocks to inoculation with Roselinia necatrix.
Inoculation of ‘Dusa®’ avocado plants with R. necatrix affected the physiological performance of treated and non-treated plants prior to the appearance of any visible symptoms, compared to non-inoculated control plants. Thus, regardless of elicitor treatment, all inoculated plants showed increased levels of energy dissipating mechanisms (NPQ) and decreased efficiency of PSII open reaction centers (Fv’/Fm′), consistent with commonly observed responses following pathogen attacks [75, 101], and with the enhanced generation of ROS by phytotoxic metabolites produced by R. necatrix that could disrupt photosynthetic electron transport [75, 102, 103].
Conversely, R. necatrix inoculation induced a differential response between treated and non-treated plants in gas exchange related parameters and water potential. Thus, AN, E, gs, Ci, and AN/Ci were decreased in inoculated-control plants compared to non-inoculated control and MeJA- and SA-treated plants. The same applies to water potential, suggesting some degree of water stress [28] that could be linked to R. necatrix colonization of the root vascular system [104]. This response supports previous results of the effect of R. necatrix on avocado photosynthesis [35, 75, 104]. These effects were to some extent mitigated in treated plants, which showed similar values to non-inoculated control plants for most parameters (i.e., E, gs, Ci, and AN/gs), except for AN and AN/Ci, which were lowered. These results suggest that MeJA and SA could be counteracting the stomatal and/or diffusional limitations of photosynthesis associated with the effect that R. necatrix infection has on water relations [35, 75], but are also pointing out the impairment of rubisco carboxylation activity in the early stages of disease progression.
Despite the similar effect on photosynthetic performance of both elicitors, a delay in the disease progression was observed in MeJA-treated plants in comparison with control and SA treatments. This suggests that the improved ability of avocado plants to cope with R. necatrix infection after MeJA treatment could be associated with the MeJA-induced morpho-physiological changes and with the activation of different pathways [11] involving the differential expression of specific defense-related genes mentioned above. The increase of leaf thickness/density, stomatal density and root dry weight (%) together with the lower plant leaf surface in MeJA-treated plants could be counteracting the impairment of water relations produced by the collapse of the plant’s vascular system after R. necatrix root invasion [28, 75, 104, 105]. These features in combination with the greater upregulation of key defense-related genes associated with tolerance to R. necatrix in MeJA-treated plants compared to SA-treated plants (i.e., glu protease inhibitor), might be playing an important role in the achievement of better performance of MeJA-treated plants against R. necatrix. This study suggests the use of MeJA to increase avocado tolerance to R. necatrix, as observed against other necrotrophic pathogenic fungi in different crops [106, 107].