4 │ Discussion
Early and elevated levels of the expression of various defense enzymes are an important feature of plant disease resistance to different pathogens (Andersen et al., 2018). Although defense-related enzymes constitute an important protective system for plants against pathogen invasion but the underlying mechanism of defense reactions and their relationships with damping-off diseases in cucumber remain unclear. Using uninoculated cucumber genotypes and time points before inoculation as controls, this study analyzed the changes in the activities of PPO, PAL, SOD, POX, and CAT in the roots of six cucumber genotypes with different susceptibilities to damping-off to identify indices that allow relevant information on damping-off resistance, which suggests that diverse mechanisms contribute to the specific levels of resistance toP. melonis in genotypes. Although oxidative stress caused byP. melonis led to increased activities of defence‐related enzymes, the overall enzyme activity patterns were distinct by enzyme and genotype.
Generally, POX activity in plant tissues are induced by pathogen infection and a greater increase was recorded in resistant plants compared to the susceptible ones (Mydlarz & Harvell 2006; Bharathi et al., 2019). Similar increase in POX activity, as observed in the resistant genotypes in this study was observed in castor infected withFusarium oxysporum (Bharathi et al. 2019), cucumber infected byPythium aphanidermatum (Sabbaghi et al., 2018), and zucchini and cucumber infected by mosaic virus (Riedle-Bauer, 2000). The increased activity of POX in the resistant genotypes after inoculation at 14 DAI was correlated with damping-off susceptibility in the cucumber genotypes and related to plant disease resistance. Su et al. (2016) found that the POX activity levels could potentially be used as a genetic marker for resistant evaluation during the early phase of infection. Our results support this indication that POX may also play an important role in cucumber resistance to damping-off. POX and POD is widely distributed in higher plants and protects cells against damaging effects of H2O2 by catalyzing its decomposition (Fernandes et al., 2006).
After inoculation with P. melonis , the SOD activity increased in the resistant genotypes. Although the SOD activity also increased in the highly susceptible genotypes Mini 6-23 and Yalda, the response time and range of increase were far lower than those in the resistant genotypes. Therefore, the differences in the activity of SOD between the six genotypes may be due to the complex activity patterns of SOD and appeared to be an important physiological basis for resistance to disease. However, the SOD activities did not correlate with damping-off susceptibility. Other researchers have indicated an increase in the activity of SOD after inoculation in cucumber (Nostar et al., 2013; Moradi et al., 2016) and other plants in a way similar to our study (Su et al., 2019).
The CAT enzyme activity displayed different patterns in different genotypes under stress and mainly affects plant disease resistance through two physiological pathways (Jiang et al., 2019). This seems to be an important H202-scavenging enzyme in plants and be localized in the peroxisomes. Catalase can be induced and then be consumed as a result of oxidative stress (Garg & Manchanda, 2009). In this study, there was a difference in the CAT activity between the resistant and susceptible genotypes, which means the alternative of CAT showed intraspecific genotypic variation in response to P. melonis inoculation and suggested that altered CAT activity also had an important effect on damping-off resistance. However, the physiological pathway by which CAT affects resistance to P. melonis remains unclear and further investigations are needed. This result is consistent with other studies for Lilium hybrids (Su et al., 2019), walnut (Jiang et al., 2019) andBrassica juncea (Pandey et al., 2017).
Due to the importance of PAL in the phenylpropanoid pathway and the relationship between PAL and plant disease resistance, it has always been a hot research topic. Several previous studies using different plant species have shown that the PAL activity is increased after fungal infection and played an important role in plant defense against pathogenic fungi (Saunders and O’neill, 2004; Zhang et al., 2017). In this study, significant changes in the PAL activity occurred after inoculation with P. melonis in resistant and moderately resistant genotypes. There were also some differences among the genotypes. Numerous studies also revealed that the activation of PAL and subsequent increase in phenolic content in plants is a general response associated with disease resistance. In black rice, PAL contributes to the resistance mechanism against Xanthomonas oryzae pv. oryzae(Solekha et al., 2019). In tomato, PAL activity was enhanced in roots by a biotic elicitor Fusarium mycelium extract (Mandal and Mitra, 2007). In cucumber roots, high levels of PAL were induced after inoculation with P. aphanidermatum (Chen et al., 2000). In transgenic soybean, overexpression of GmPAL2.1 increases resistance toPhytophthora sojae (Zhang et al., 2017). Therefore, increase in level of PAL in the infected root tissue of cucumber genotype showed that phenyl propanoid pathway accumulate phenolics might have prevented the pathogen invasion, and thus, the activity maintained at higher levels during the infection period.
Our findings show that, although the PPO activity increased in roots of infected plants in comparison to the control in the resistant and moderately resistant genotypes, but the activities varied among the three genotypes. Several studies have indicated induction of PPO in plants in response to infection by different pathogens (Vanitha et al., 2009; Khodadadi et al., 2020). In present study, systemic induction of PPO expression in the resistant and moderately resistant genotypes in response to P. melonis might provide an additional line of defense to protect cucumber plants against further attack by pathogens. The PPO appear to play a role in resistance to P. melonis , since this compound was present in considerably higher levels in roots of inoculated plants of resistant genotypes which is consistent with observations made for cucumber, walnut and common bean plants in which PPO was demonstrated to be induced by Fusarium oxysporum,Xanthomonas arboricola and arbuscular mycorrhizal fungi, respectively (Moghbeli et al., 2017; Jiang et al., 2019; Abdel-Fattah et al., 2011). Additionally, a significant correlation between the antioxidant enzymes indicate the role of antioxidant enzymes system under P. melonis infection. The role of antioxidant enzymes in eliciting systemic resistance against Fusarium oxysporum ,Pyricularia oryzae , Alternaria sp. and Sclerotiumsp. has been reported earlier (Rais et al., 2017). These findings indicated the complex interaction between both oxidative-burst and response to damping- off. Tt seems that the oxidativeburst and H2O2 accumulation also happened in resistant genotypes as an adaptive response to P. melonis infection, and resistant genotypes may handle stress better than susceptible genotypes during the infection due to other mechanisms. However, the results of the changes in defensive enzyme activity are far from sufficient to explain damping-off resistance in cucumber. To understand cucumber resistance physiology more clearly, other physiological indicators should be considered.
The results of this study indicated that the disease defense response is more pronounced in the resistance genotypes as demonstrated by an earlier defense response through increasing the content of defense-related enzymes. In addition, the results suggest the PAL and POX have active roles in disease resistance against damping-off; however, there was no direct connection between damping-off resistance and PPO, SOD and CAT activities. Meanwhile, the varied response of defense-related enzymes suggests that the oxidative-burst response after inoculation by P. melonis , and the response may have differed depending on the genotype and inoculation period. These findings can help us to understand the resistance physiology of damping-off and provide indicators for cucumber breeding. However, diverse mechanisms contribute to the specific levels of resistance to P. melonis in genotypes and the mechanisms by which defense-related enzymes accumulation contribute to resistance in cucumber remain to be explored in future studies.