Plants have evolved diverse strategies to cope with different environmental stresses, but many studies have shown that most plant responses to biotic and abiotic stress rely on an assortment of common physiological and molecular mechanisms [18, 19]. Particularly, it has been reported that avocado ‘Dusa’ rootstock response to R. necatrix infection involves the impairment of water relations and photosynthesis [68–70] as well as the induction of genes related to water stress and pathogen defense responses [43]. These findings are in agreement with results of the present study on the response of ‘Dusa’ avocado to water stress. This response was dependent upon water stress intensity, since mild-WS and severe-WS treatments affected leaf water status differentially (i.e. decreased values of leaf water potential and RWC) as well as photosynthetic performance, shown by the enhancement of photoprotective mechanisms (i.e. NPQ and qN values) and the decrease in gas exchange parameters (i.e. AN and gs). These physiological changes are consistent to those previously described in response to mild and severe water stress in other woody plants [71, 72] and in avocado trees [73, 74]. ‘Dusa’ rootstock response to either R. necatrix infection or water stress treatments displayed water potential and gs values that dropped below -1.0 MPa and 0.05 mol m-2 s-1, respectively, suggesting an oxidative burst in photosynthetic tissues [75, 76]. This agrees with the higher NPQ and qN values [77, 78] and with a potential vulnerability to cavitation that could limit water flow from roots towards the upperparts of the trees, especially in severe-WS [79, 80]. In the R. necatrix/avocado interaction, this limitation of water flow is consistent with the profuse invasion of root vascular system during pathogen root colonization [70, 81].
Molecular responses at the root level showed the up-regulation of six out of the thirteen tested genes under both water stress treatments (Table 2). These genes, besides being involved in the avocado response to soilborne pathogens (P. cinnamomi and R. necatrix), are also induced in the response of other horticultural and woody species (i.e. Citrus spp., Malus domestica, Populus trichocarpa) to water deficit [45, 82–86]. It is remarkable the increased overexpression of NAC transcription factor accordingly to the intensity of the water stress level, which could be supporting a major accumulation of ROS species under severe-WS since, among other functions, this gene has been associated with the up-regulation of ROS-scavenging genes under abiotic stresses [61]. On the other hand, mild-WS repressed seven out of the thirteen genes, three of which remained down-regulated in the severe-WS (Table 2). NPR1 and PR5 repression is in consonance with the ABA biosynthesis and signalling induced under water stress [87], known to exert an antagonistic effect on the salicylic acid (SA) pathway [88] in which NPR1 functions as a master regulator inducing the expression of pathogenesis related proteins (PR) such as PR5 [89, 90], which are potentially involved in the maintenance of osmotic adjustment in cells [65].
The results stated above indicate that pathways involved in the avocado response
Plants have evolved diverse strategies to cope with different environmental stresses, but many studies have shown that most plant responses to biotic and abiotic stress rely on an assortment of common physiological and molecular mechanisms [18, 19]. Particularly, it has been reported that avocado ‘Dusa’ rootstock response to R. necatrix infection involves the impairment of water relations and photosynthesis [68–70] as well as the induction of genes related to water stress and pathogen defense responses [43]. These findings are in agreement with results of the present study on the response of ‘Dusa’ avocado to water stress. This response was dependent upon water stress intensity, since mild-WS and severe-WS treatments affected leaf water status differentially (i.e. decreased values of leaf water potential and RWC) as well as photosynthetic performance, shown by the enhancement of photoprotective mechanisms (i.e. NPQ and qN values) and the decrease in gas exchange parameters (i.e. AN and gs). These physiological changes are consistent to those previously described in response to mild and severe water stress in other woody plants [71, 72] and in avocado trees [73, 74]. ‘Dusa’ rootstock response to either R. necatrix infection or water stress treatments displayed water potential and gs values that dropped below -1.0 MPa and 0.05 mol m-2 s-1, respectively, suggesting an oxidative burst in photosynthetic tissues [75, 76]. This agrees with the higher NPQ and qN values [77, 78] and with a potential vulnerability to cavitation that could limit water flow from roots towards the upperparts of the trees, especially in severe-WS [79, 80]. In the R. necatrix/avocado interaction, this limitation of water flow is consistent with the profuse invasion of root vascular system during pathogen root colonization [70, 81].
Molecular responses at the root level showed the up-regulation of six out of the thirteen tested genes under both water stress treatments (Table 2). These genes, besides being involved in the avocado response to soilborne pathogens (P. cinnamomi and R. necatrix), are also induced in the response of other horticultural and woody species (i.e. Citrus spp., Malus domestica, Populus trichocarpa) to water deficit [45, 82–86]. It is remarkable the increased overexpression of NAC transcription factor accordingly to the intensity of the water stress level, which could be supporting a major accumulation of ROS species under severe-WS since, among other functions, this gene has been associated with the up-regulation of ROS-scavenging genes under abiotic stresses [61]. On the other hand, mild-WS repressed seven out of the thirteen genes, three of which remained down-regulated in the severe-WS (Table 2). NPR1 and PR5 repression is in consonance with the ABA biosynthesis and signalling induced under water stress [87], known to exert an antagonistic effect on the salicylic acid (SA) pathway [88] in which NPR1 functions as a master regulator inducing the expression of pathogenesis related proteins (PR) such as PR5 [89, 90], which are potentially involved in the maintenance of osmotic adjustment in cells [65].
The results stated above indicate that pathways involved in the avocado response to gradually imposed water stress lead to the induction of genes expressed in incompatible interactions against fungal pathogens [43, 90]. In this regard, co-occurrence of water stress and soilborne pathogens could have a positive effect in achieving tolerance against the pathogen (i.e. cross-tolerance, [20, 21]) or a negative additive effect, making plants more susceptible [16, 91–94]. Additional studies on avocado are required to clarify this point.
Previous studies have suggested the use of ‘priming’ [10–12] with drought stress to achieve tolerance to forthcoming diseases [16]. This acquired tolerance is based on sustained changes on the basal levels of cellular and molecular defense in primed plants after cessation of stimuli compared to non-primed ones [11, 12, 16]. In the present study, water status and photosynthetic performance was completely restored in drought-primed plants one week after re-watering regardless of the pre-drought intensity. This fast recovery suggests that impairment of whole plant transpirational flow and photosynthesis did not lead to irreversible changes on avocado and can be indicative of some degree of drought adaptation [78].
However, at the root level, re-watering induced the upregulation of defense related genes, suggesting a ‘primed state’ of the previously water stressed avocado plants. Gene overexpression, which could be associated with crosstalk between the different signaling pathways underlying plant tolerance/resistance to biotic and abiotic stress such as the abscisic (ABA), jasmonic (JA) and salicylic (SA) acids [95–98], was more remarkable in mild-WS compared to severe-WS. Particularly, the induction of NPR1 transcription factor in mild-WS primed plants suggests the activation of salicylic acid-mediated defense responses [52, 89, 90] and the deactivation of ABA-related responses after water stress [99]. In addition, this ‘primed state’ is accompanied by the significant accumulation of PR proteins (i.e. PR4 and PR5) which have been correlated with the development of systemic acquired resistance [48] and are considered the most promising candidates for developing multiple stress tolerance [89]. It is also remarkable that the expression of genes related with fungal cell wall degradation, such as endochitinase, was only up-regulated in plants recovered after mild-WS. Genes encoding metallothionein, universal stress protein, protease inhibitor and NAC domain containing protein 72 remained overexpressed in mild-WS primed plants. These genes are involved in the general plant response to stress [51, 58, 60, 62, 64, 67, 82, 100–103], playing the last two a fundamental role in avocado defense to R. necatrix [43]. It should be highlighted the marked overexpression of the gene encoding the NAC domain containing protein 72 (24 fold over mild-WS) in roots recovered from mild-WS compared to severe-WS, suggesting a higher promotion of root development [104, 105], although further studies are necessary to clarify its importance on the water stress recovery response.
The performed pathogenicity test shed light on whether this water-stress induced ‘primed state’ was effective for enhancing avocado tolerance to this necrotrophic pathogen. In this sense, the disease progression delay, observed in mild-WS primed plants in comparison with control and severe-WS primed plants, suggests an enhancement of plant ability to cope with R. necatrix infection after priming with mild water stress. This ability could be attributable to differential expression of key genes involved in the tolerance of avocado to soilborne pathogens such as NPR1 and NAC domain containing protein 72, as well as with a lower energy investment for overcoming a moderate water stress compared with severely stressed plants [72]. Moreover, although all of the overexpressed genes in mild-WS primed plants are involved in plant defense against fungi, not all have been described to be related with avocado tolerance to R. necatrix (i.e. NPR1, PR4, PR5 and endochitinase). However, their enhanced expression after drought-priming (i.e. abiotic factor) could also represent a benefit for avocado plants to overcome forthcoming fungal infection (i.e. biotic stressor).
to gradually imposed water stress lead to the induction of genes expressed in incompatible interactions against fungal pathogens [43, 90]. In this regard, co-occurrence of water stress and soilborne pathogens could have a positive effect in achieving tolerance against the pathogen (i.e. cross-tolerance, [20, 21]) or a negative additive effect, making plants more susceptible [16, 91–94]. Additional studies on avocado are required to clarify this point.
Previous studies have suggested the use of ‘priming’ [10–12] with drought stress to achieve tolerance to forthcoming diseases [16]. This acquired tolerance is based on sustained changes on the basal levels of cellular and molecular defense in primed plants after cessation of stimuli compared to non-primed ones [11, 12, 16]. In the present study, water status and photosynthetic performance was completely restored in drought-primed plants one week after re-watering regardless of the pre-drought intensity. This fast recovery suggests that impairment of whole plant transpirational flow and photosynthesis did not lead to irreversible changes on avocado and can be indicative of some degree of drought adaptation [78].
However, at the root level, re-watering induced the upregulation of defense related genes, suggesting a ‘primed state’ of the previously water stressed avocado plants. Gene overexpression, which could be associated with crosstalk between the different signaling pathways underlying plant tolerance/resistance to biotic and abiotic stress such as the abscisic (ABA), jasmonic (JA) and salicylic (SA) acids [95–98], was more remarkable in mild-WS compared to severe-WS. Particularly, the induction of NPR1 transcription factor in mild-WS primed plants suggests the activation of salicylic acid-mediated defense responses [52, 89, 90] and the deactivation of ABA-related responses after water stress [99]. In addition, this ‘primed state’ is accompanied by the significant accumulation of PR proteins (i.e. PR4 and PR5) which have been correlated with the development of systemic acquired resistance [48] and are considered the most promising candidates for developing multiple stress tolerance [89]. It is also remarkable that the expression of genes related with fungal cell wall degradation, such as endochitinase, was only up-regulated in plants recovered after mild-WS. Genes encoding metallothionein, universal stress protein, protease inhibitor and NAC domain containing protein 72 remained overexpressed in mild-WS primed plants. These genes are involved in the general plant response to stress [51, 58, 60, 62, 64, 67, 82, 100–103], playing the last two a fundamental role in avocado defense to R. necatrix [43]. It should be highlighted the marked overexpression of the gene encoding the NAC domain containing protein 72 (24 fold over mild-WS) in roots recovered from mild-WS compared to severe-WS, suggesting a higher promotion of root development [104, 105], although further studies are necessary to clarify its importance on the water stress recovery response.
The performed pathogenicity test shed light on whether this water-stress induced ‘primed state’ was effective for enhancing avocado tolerance to this necrotrophic pathogen. In this sense, the disease progression delay, observed in mild-WS primed plants in comparison with control and severe-WS primed plants, suggests an enhancement of plant ability to cope with R. necatrix infection after priming with mild water stress. This ability could be attributable to differential expression of key genes involved in the tolerance of avocado to soilborne pathogens such as NPR1 and NAC domain containing protein 72, as well as with a lower energy investment for overcoming a moderate water stress compared with severely stressed plants [72]. Moreover, although all of the overexpressed genes in mild-WS primed plants are involved in plant defense against fungi, not all have been described to be related with avocado tolerance to R. necatrix (i.e. NPR1, PR4, PR5 and endochitinase). However, their enhanced expression after drought-priming (i.e. abiotic factor) could also represent a benefit for avocado plants to overcome forthcoming fungal infection (i.e. biotic stressor).