Novel Cucumis enzymes associated with host-specific disease resistance to Phytophthora melonis Katsura

Abstract

The hemibiotrophic oomycetes are significant threats to a wide range of Cucurbitaceae species, causing substantial losses of plant productions. Particularly, Phytophthora melonis Katsura evokes severe symptoms, thus dramatically limiting the yield in cucumber. However, the information about cucumber–P. melonis interaction is still limited. This study explored the changes in the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), superoxide dismutase (SOD) and polyphenol oxidase (PPO) in cucumber roots of two resistant genotypes (Soheil and Ramezz), one moderately resistant genotype (Baby) and three highly susceptible genotypes (Extrem, Mini 6–23 and Yalda), over the time courses of 7, 14 and 21 days after inoculation (DAI). The results indicated that the activities of defense-related enzymes differed between the resistant and highly susceptible genotypes. Although the defense-related enzymatic activities were elevated sharply in the resistant and moderately resistant genotypes after inoculation, no significant correlations were present between the activity trends of PPO, SOD and CAT and resistance characteristics. Moreover, no significant changes in enzyme activities were found in the control plants, non-inoculated plants of the six genotypes during the testing period. Altogether, the resistance of cucumber to P. melonis is related to POX and PAL activities, but does not show relationship with PPO, SOD and CAT activities. Studying the physiological metabolic pathways of POX and PAL appears to be an important direction in research to elucidate the resistance to P. melonis in cucumber genotypes.

Keywords:

• Phenylalanine ammonia-lyase
• peroxidase
• catalase
• superoxide dismutase
• and polyphenol oxidase
• cucumber
• Phytophthora resistance

Introduction

Phytophthora melonis Katsura, a hemibiotrophic organism, belongs to Oomycota phylum and is one of the causal agents of damping-off disease. It is considered a potentially destructive disease of cucumber (Cucumis sativus L.; 2n = 2x = 14) across Iran and other parts of the world, as it causes economic losses [1,2]. Therefore, it is vital to elucidate the mechanisms of plants in response to damping-off. Typical symptoms of the disease are root and root collar rot, stem lesions, foliar blight, fruit rot and plant death [3,4]. Although current control measures for P. melonis are based on the use of fungicides [5], identification of disease-resistant genotypes may be an effective and environmentally friendly strategy in modern crop production [6–9]. To date, analyses of the interaction between P. melonis and C. sativus have been limited on screening resistant genotypes to damping-off [1,6] and no cucumber genotype immune to P. melonis has been reported. In the never ending struggle against pathogens, plants gradually developed a series of complex defense mechanisms involving several factors like defense-related enzymes and inhibitors which lead to prevent infection of pathogens [10]. However, the plant response during the P. melonis–C. sativus interactions or, more specifically, the underlying factors that increase susceptibility and/or resistance of the cucumber plant is not fully understood. So, identification and application of candidate resistance genes and defense enzymes associated with P. melonis responses can be an efficient method to build up the host resistance in cucumber genotypes. In 2019, three defense-related genes of CsWRKY20, CsLecRK6.1 and LOX1 genes were reported to be involved in the resistance to the P. melonis infection in cucumber [6,11]. A complex network of defense-related enzymes, such as peroxidase (POX; EC1.11.1.7), superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and phenylalanine ammonia lyase (PAL; EC4.3.1.5), promote the scavenging of ROS and are related to resistance inducement in plants [12–14]. PAL has a crucial role in flavonoid productions and lignin biosynthesis, which play a key role in the phenylpropanoid biosynthetic pathway [15]. PAL is one of the most extensively studied enzymes with respect to plant responses to environmental stresses, including pathogen infection [16,17]. Polyphenol oxidase (PPO) and peroxidase (POX) are important oxidative enzymes found in most plant species. PPO and POX catalyze the formation of lignin and other oxidative phenols, thereby contributing to the formation of defense barriers by structural reinforcement, to protect against pathogens [18,19]. In cucumber the activities of peroxidase increased following the inoculation with Fusarium oxysporum [20]. [21] reported that high activities of antioxidant enzymes played a major role in both basal and induced resistance of cabbage to black rot. Moreover, regarding the oxidative burst, CAT and SOD are useful biochemical indicators of disease resistance [22]. The activities of different defense-related enzymes vary in different plants after pathogen attack and are highly complex [23,24]. To the best of our knowledge, no comprehensive enzymic analyses have yet been performed on cucumber plants to compare defense-related enzymes in resistant and susceptible cucumber genotypes upon inoculation with P. melonis. Therefore, the present study determined the activities of PAL, POX, CAT, SOD and PPO quantitatively and investigated their roles in response to P. melonis inoculation in the cucumber genotypes. A better insight in cucumber defense responses can help to establish biochemical characteristics for the selection of resistant cucumber sources and provide a theoretical basis for disease control and breeding of cucumber plants with higher resistance toward damping-off.

Materials and methods

Plant material

Two cucumber genotypes showing different levels of resistance to damping-off (Soheil and Ramezz), one moderately resistant genotype (Baby) and three susceptible genotypes (Extrem, Mini 6–23 and Yalda) were selected among a collection of thirty-eight commercial genotypes of domestic and exotic hybrids, and inbred lines from different seed companies. They were recently investigated and were screened in another study to identify resistant and susceptible genotypes [6,25,26]. The six genotypes are listed from least to most susceptible according to the disease severity index in greenhouse investigations in Table 1. One hundred seeds per genotype were grown in seedling nursery trays filled with forced-air oven sterilized mix of sand-peat moss in equal parts at 200 °F for 30 min. Cucumber seedlings were grown in a greenhouse (26 ± 2 °C), provided with 16 h photoperiod and 65% relative humidity. The experiment was arranged in a completely randomized factorial design with three replications [27,28].

Table 1. List of Cucumis sativus genotypes, properties, breeding company, method of cultivation, mortality rate and reaction to damping-off at transplanting stage used in this study.

No	Genotype	Properties	Breeding company	Method of cultivation	Mortality rate (%)	Susceptibility to damping-off
1	Soheil	hybrid F1	Nongwoobio	Open field	8.89^c± 2.32	Resistant
2	Ramezz	hybrid F1	Seminis	Open field	7.92^c ± 0.73	Resistant
3	Baby	hybrid F1	Ergon	Green house	14.17^d ± 2.89	Moderately resistant
4	Extrem	hybrid F1	Seminis	Green house	100^a ± 0.0	Highly susceptible
5	Mini 6–23	hybrid F1	NeginBazar	Green house	86.67^b ± 2.03	Highly susceptible
6	Yalda	hybrid F1	Rijk Zwaan	Green house	100^a ± 0.0	Highly susceptible

Note: Values followed by the same letters do not differ statistically among themselves.

Pathogen culture and plant inoculation

An aggressive isolate of P. melonis obtained from naturally infected cucumber plants exhibiting post-emergence damping-off and root rot symptoms and identified as P. melonis (MH924841) was used. Inoculum was obtained by growing the P. melonis isolate in 500-mL flasks containing Water-soaked wheat seeds, which had been autoclaved at 120 °C for 30 min on 2 consecutive days. The flasks were inoculated with 5 cm disks cut from a 10-day-old culture of the P. melonis isolate, and placed in the dark for 2 weeks at 25 ± 2 °C. then, 45-day-old seedlings were inoculated by 10 g of wheat seed (106 sporangia mL⁻¹) and incubated for two days under saturated moist conditions in the greenhouse [6,29–31]. In the seedling stage, inoculated and control root samples were harvested in three biological replicates over the time courses of 7, 14 and 21 days after inoculation (DAI) and then immediately transferred to liquid nitrogen and kept at −80 °C. All experiments were repeated three times.

Antioxidant enzyme extraction and activity assays

The pre-weighed infected and non-infected roots were homogenized in 4 mL buffer (50 mmol L⁻¹ Tris pH = 8.5, 14.4 mmol L⁻¹ 2-mercaptoethanol) and 1% (w/w) insoluble polyvinylpolypyrrolidone and centrifuged at 6000 g for 15 min at 4 °C to determine the SOD, CAT, POX, PPO and PAL activities [32]. The total protein content of each enzyme extract was determined using bovine serum albumin as the standard [33]. The CAT activity was determined by measuring the rate of H₂O₂ conversion to O₂ at 240 nm and expressed as U mg⁻¹ protein [34]. The POX activity was determined by the method described by [35]. The photometric intensity of the reaction was measured using a spectrophotometer (470 nm) in a 40 mmol L⁻¹ hydrogen peroxide solution and expressed as units of enzyme activity per milligram of soluble protein per minute (U mg⁻¹ protein). The activity of PPO was determined using the method of Constabel & Ryan [36]. Supernatant was added to substrate consisting of 5 mg mL⁻¹ L-3,4-dihydroxyphenylalanine (L-Dopa). The assay solution consisted of 100 mmol L⁻¹ NaPO₄ (pH 7), 0.015% (w/v) sodium dodecyl sulfate, and catalase (280 U mL⁻¹). The absorbance was measured at 490 nm and the PPO activity was expressed as U mg⁻¹ protein. The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) following the method of [37] and expressed as units of enzyme activity per milligram of soluble protein per minute (U mg⁻¹ protein). The PAL activity was assayed following the method of [38]. The photometric intensity of the reaction was measured using a spectrophotometer (290 nm) with 15 mmol L⁻¹ phenylalanine as the substrate. PAL specific activity was expressed in U mg⁻¹.

Statistical analysis

The data collected from all experiments were analyzed separately for each parameter and subjected to two-way analysis of variance (ANOVA) using SAS (ver. 9.4, SAS Institute, Cary, NC). The means were compared for significance using least significant differences (L.S.D.) at p ≤ 0.05 and the values presented are means of three replicates [39]. The Spearman correlation coefficients were determined between cucumber genotypes susceptibility to damping-off and activity of defense-related enzymes after inoculation by P. melonis.

Results and discussion

Disease response

P. melonis-inoculated plants of all tested genotypes revealed a wide range of symptoms depending on their genetic makeup and none of the inoculated plant was symptomless. The genotypes that responded as resistant showed minor symptoms, moderately resistant ones indicated moderate, while highly susceptible genotypes revealed severe symptoms as earlier reported by [6]. The defense responses in the resistant genotypes were able to stop pathogen growth and infection, whereas in the susceptible genotypes, necrosis continued to progress along the roots and stem until the plant died (Figure 1).

Figure 1. Comparison of damping-off symptom severity among six cucumber genotypes inoculated with P. melonis. (A) Soheil (resistant), (B) Baby (moderately resistant), (C) Ramezz (resistant), Mini 6–23 and Extrem (highly susceptible), (D) Yalda (highly susceptible).

Defense enzymes activities

Early and elevated levels of the expression of various defense enzymes are an important feature of plant disease resistance to different pathogens [10,40]. Although defense-related enzymes constitute an important protective system for plants against pathogen invasion, 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 provide relevant information on damping-off resistance. Data suggest that diverse mechanisms contribute to the specific levels of resistance to P. melonis in different genotypes. Although oxidative stress caused by P. melonis led to increased activities of defense-related enzymes, the overall enzyme activity patterns were distinct by enzyme and genotype. Correlation analysis was performed between cucumber genotypes susceptibility to damping-off and the activity of defense-related enzymes, including PAL, PPO, POX, SOD and CAT, after inoculation with P. melonis (Table 2).

Table 2. Spearman correlations (correlation index) between cucumber genotypes susceptibility to damping-off and activity of defense-related enzymes, including PAL, PPO, POX, SOD and CAT, after inoculation by P. melonis.

Enzymes	7 DAI	14 DAI	21 DAI
PAL	−0.94**	−0.82*	−0.68
PPO	−0.64	−0.74	−0.75
POX	−0.77	−0.92*	−0.64
SOD	−0.56	−0.14	−0.24
CAT	−0.67	−0.55	−0.58

Note: Asterisks represent statistically significant linear relationship (**, p < 0.01 and *, p < 0.05) between the genotypes’ susceptibility to damping-off and the activity of defense-related enzymes according to Spearman correlation. Positive values indicate that the more susceptible genotypes showed increased activity of enzymes. In contrast, negative values indicate that the more susceptible genotypes showed decreased activity of enzymes.

Peroxidase activity (POX)

Analysis of variance and changes in POX activity in the roots of the six cucumber genotypes with different levels of resistance after inoculation with P. melonis was represented in Tables 3 and 4 and Figure 2. In Ramezz and Baby, POX activity indicated a significant increase in 14 DAI, and increased to maximum at 21 DAI (Figure 2(A,B)). Highest POX activity of 80.8 units was recorded in resistant Ramezz after 21 DAI, which was 6 times higher than that of the untreated control, which showed 13.8 units POX activity after 21 DAI (Figure 2(B)). POX activity in Soheil increased at 7 and 14 DAI, decreased at 21 DAI and showed no significant difference between control and inoculated plants at 28 DAI (Figure 2(A)). Pattern study of POX activity in inoculated susceptible samples of Mini 6-23 and Extrem were similar to the uninoculated samples, but, the enzyme activity was significantly higher in inoculated susceptible samples of Yalda at 21 DAI. Interestingly, the POX activity in infected plants of Extrem was lower than that in controls (Figure 2(B)). The increase in POX activity caused by inoculation varied among the genotypes and had a strong negative correlation with damping-off susceptibility at 14 DAI (Table 2). Generally, POX activity in plant tissues is induced by pathogen infection and a greater increase was recorded in resistant plants compared to the susceptible ones [41,42]. The increase in POX activity observed in the resistant genotypes in this study was similar to that previously observed in castor infected with Fusarium oxysporum [41], cucumber infected by Pythium aphanidermatum [43], and zucchini and cucumber infected by mosaic virus [44]. 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. [24] 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 are widely distributed in higher plants and protect cells against the damaging effects of H₂O₂ by catalyzing its decomposition [45].

Figure 2. Trends of changes in peroxidase (POX) activity in the six cucumber genotypes after inoculation with P. melonis. Uninoculated (control) genotypes (A) vs. inoculated genotypes (B) from day 7 to day 21.

Table 3. Analysis of variance in relation to quantification of enzyme activities in inoculated resistant, moderately resistant and susceptible cucumber genotypes as compared to controls (non-inoculated ones).

S.V	D.F	Mean square (M.S)
		Total protein	PPO	POX	SOD	CAT	PAL
			Activity	Activity	Activity	Activity	Activity
Cultivar (A)	5	0.81**	18.0**	40.0**	3.04**	22.2**	12.2**
Time (B)	2	1.45**	11.5**	6.7**	0.15**	6.1**	5.5**
Treatment (C)	1	1.21**	67.4**	104.5**	9.98**	16.5**	54.1**
AB	10	0.81**	17.8**	10.2**	1.25**	9.8**	7.7**
AC	5	0.28**	11.3**	20.0**	1.84**	13.3**	9.4**
BC	2	0.36**	12.1**	16.2**	0.07*	0.8*	5.2**
ABC	10	0.25**	12.7**	88.0**	1.03**	4.5**	6.4**
Error	72	0.01	0.1	0.05	0.02	0.2	0.1
C.V%		7.5	20.0	8.5	14.5	9.4	26.2

^nsnon-significant; * and; ** significant at p < 0.05 and p < 0.01, respectively.

Table 4. Peroxidase (POX) activities in inoculated resistant, moderately resistant and susceptible cucumber genotypes as compared to controls (non-inoculated ones).

Reaction to P.melonis	Genotype	POX specific activity U mg^−1
		Day 7		Day 14		Day 21
		Control	Inoculated	Control	Inoculated	Control	Inoculated
Resistant	Soheil	12.3^m–p	41.5^c	11.4^o,p	42.3^c	11.2^o,p	11.5^n,o,p
	Ramezz	14.3^i–l	15.7^i	15.2^i,j	30.3^e	13.8^j–m	80.8^a
Moderately resistant	Baby	14.5^i–l	26.7^f	15.8^i	34.0^d	17.9^h	66.7^b
Susceptible	Extrem	14.7^i,j,k	13.5^j–m	12.8^l–o	8.1^q	13.3^k–n	10.8^p
	Mini 6–23	1.1^r	1.1^r	1.5^r	0.4^r	2.0^r	1.0^r
	Yalda	12.7^l–o	7.5^q	13.4^k,l,m	14.2^i–l	14.4^i–l	23.5^g

Note: Each value represents the mean of three replicates. Means in each column indicated with the same letter are not significantly different according to Fisher’s LSD test (p > 0.05).

Superoxide dismutase (SOD)

Analysis of variance and the activities of SOD in the roots of susceptible and resistant cucumber genotypes subjected to pathogen infection are presented in Tables 3 and 5 and Figure 3. The SOD activity in the control of each genotype showed little change from day 7 to day 21 (Table 5). However, the SOD activity in the inoculated cucumbers changed dramatically. Before inoculation, the SOD activities of all genotypes were relatively low. In resistant and moderately resistant genotypes, the SOD activity increased after inoculation by P. melonis at 14 DAI, remained high in Soheil and Baby and decreased in Ramezz at 24 DAI (Figure 3(A,B)). The level of SOD in inoculated Soheil ranged from 0.42 U mg⁻¹ (7 DAI) to 1.68 U mg⁻¹ (21 DAI); in Ramezz, it ranged from 0.21 U mg⁻¹ (7 DAI) to 0.35 U mg⁻¹ (21 DAI) and in Baby from 2.19 U mg⁻¹ (7 DAI) to 0.79 U mg⁻¹ (21 DAI). The genotype with the highest activities was the moderately resistant Baby. The SOD activity in ‘Baby’ was 4.09 times greater compared to the control at 14 DAI. The induction of SOD in all genotypes found little to no significant differences compared to the control plants at 7 DAI, except Baby and Yalda (Table 5). The SOD activity of the susceptible Mini 6–23 also showed an increase of 1.47 U mg⁻¹ on day 21 after inoculation, but the susceptible Yalda began to decline on day 21 after inoculation. The maximum amount of SOD activity between the three susceptible genotypes in inoculated plants was observed in Mini 6–23 at 21 DAI (Figure 3(B)). The increase in SOD activity caused by inoculation did not correlate with damping-off susceptibility (Table 2, varied between −0.56 and −0.14). 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 [46,47] and other plants in a way similar to our study [22].

Figure 3. Trends of changes in superoxide dismutase (SOD) activity in the six cucumber genotypes after inoculation with P. melonis. Uninoculated (control) genotypes (A) vs. inoculated genotypes (B) from day 7 to day 21.

Table 5. Superoxide dismutase (SOD) activities in inoculated resistant, moderately resistant and susceptible cucumber genotypes as compared to controls (non-inoculated ones).

Reaction to P.melonis	Genotype	SOD specific activity U mg^−1
		Day 7		Day 14		Day 21
		Control	Inoculated	Control	Inoculated	Control	Inoculated
Resistant	Soheil	0.41^p,q,r	0.42^o–r	0.47^n–q	0.86^g,h	0.59^l,m,n	1.67^c
	Ramezz	0.19^t	0.21^t	0.26^s,t	0.91^f,g	0.23^s,t	0.35^q,r,s
Moderately resistant	Baby	0.65^j–m	2.19^a	0.49^n,o,p	2.00^b	0.66^i–m	0.79^g,h,i
Susceptible	Extrem	0.20^t	0.24^s,t	0.30^r,s,t	0.21^t	0.27^s,t	0.27^s,t
	Mini 6–23	0.73^h-k	0.55^l–o	0.49^n,o,p	0.53^m–p	0.67^i–l	1.47^d
	Yalda	0.77^h,i,j	1.19^e	0.73^h-k	1.02^f	0.63^k,l,m	0.56^l,m,n

Note: Each value represents the mean of three replicates. Means in each column indicated with the same letter are not significantly different according to Fisher’s LSD test (p > 0.05).

Catalase activity (CAT)

The changes in CAT activity of the six cucumber genotypes after inoculation with P. melonis are shown in Tables 3 and 6 and Figure 4. Before inoculation, the CAT activities in resistant and moderately resistant genotypes (Soheil and Baby), were significantly higher than in the other resistant genotype (Ramezz) (Table 6). After inoculation, the CAT activity in Soheil increased sharply to 20.3 U mg⁻¹ at 14 DAI and then decreased at 21 DAI (Figure 4(A)). A similar pattern was also observed in Ramezz. However, the CAT activity in another moderately resistant genotype (Baby) showed a different reaction, with a non-significant decrease after inoculation at 14 DAI and a significant increase at 21 DAI. The maximum CAT activity in roots of inoculated plants was observed in Ramezz and Soheil (Table 6). No significant difference was found in two susceptible genotypes, Extrem and Mini 6–23 (Table 6 and Figure 4(A,B)). In another susceptible genotype (Yalda), CAT activity never exceeded the activity before inoculation at 14 and 21 DAI (Figure 4(B)). However, no correlation was observed between damping-off susceptibility and CAT activity changes (Table 2). The CAT enzyme activity displayed different patterns in different genotypes under stress and mainly affects plant disease resistance through two physiological pathways [18]. This is an important H₂O₂-scavenging enzyme in plants and is localized in the peroxisomes. Catalase can be induced and then be consumed as a result of oxidative stress [48]. In this study, there was a difference in the CAT activity between the resistant and susceptible genotypes, suggesting that CAT showed intraspecific genotypic variation in response to P. melonis inoculation and that altered CAT activity also had an important effect on damping-off resistance. However, the physiological pathway by which CAT affects the resistance to P. melonis remains unclear and further investigations are needed. This result is consistent with other studies for Lilium hybrids [22], Juglans [18] and Brassica juncea [49].

Figure 4. Trends of changes in catalase (CAT) activity in the six cucumber genotypes after inoculation with P. melonis. Uninoculated (control) genotypes (A) vs. inoculated genotypes (B) from day 7 to day 21.

Table 6. Catalase (CAT) activities in inoculated resistant, moderately resistant and susceptible cucumber genotypes as compared to controls (non-inoculated ones).

Reaction to P.melonis	Genotype	CAT specific activity U mg^−1
		Day 7		Day 14		Day 21
		Control	Inoculated	Control	Inoculated	Control	Inoculated
Resistant	Soheil	10.5^i,j	15.6^b	12.8^c–h	20.3^a	13.7^c	12.5^c–h
	Ramezz	5.3^n,o,p	9.5^j,k	6.6^m,n	11.6^e–i	6.6^m,n	8.3^k,l
Moderately resistant	Baby	11.9^d–i	11.5^f–i	10.6^i,j	10.3^i,j	10.5^i,j	15.6^b
Susceptible	Extrem	4.7^o,p	5.2^n,o,p	5.3^n,o,p	4.0^p	5.2^n,o,p	4.1^p
	Mini 6–23	13.1^c–f	11.2^h,i	13.3^c,d	14.0^b,c	12.7^c–h	13.1^c–f
	Yalda	12.8^c–h	7.9^l,m	11.4^g,h,i	10.3^i,j	12.4^c–h	5.9^n,o

Note: Each value represents the mean of three replicates. Means in each column indicated with the same letter are not significantly different according to Fisher’s LSD test (p > 0.05).

Activity of phenylalanine ammonia-lyase (PAL)

In each of the six cucumber genotypes, the PAL activities of the control plants indicated no significant changes from day 7 to day 21 (Table 7). In the resistant and moderately resistant genotypes, PAL activities showed significant difference from the control at the same time point after inoculation. After inoculation, PAL activity in the resistant genotype Soheil increased, reaching its first peak on day 7, then it decreased on day 14 and increased to 1.86 U mg⁻¹ on day 21 after inoculation (Figure 5(A)). The CAT activity in resistant Ramezz increased sharply to 2.17 U mg⁻¹ at 7 DAI and then decreased at 14 and 21 DAI (Table 7). The moderately resistant genotype, Baby, showed behavior similar to that of Soheil, but it increased sharply and reached its maximum on day 21 (5.20 U mg⁻¹). The two highly susceptible genotypes Extrem and Mini 6–23 showed increased PAL activity on day 7 and 14 and then decreased on day 21, which was different from the patterns of PAL activity in the other genotypes (Figure 5(A,B)). In contrast, in the other genotype, Yalda, PAL activity increased on day 7 and then decreased on day 14 and 21 (Figure 5(B)). Therefore, the changes in PAL activity showed differences among different genotypes and it was significantly higher in inoculated plants of both moderately resistant and resistant genotypes, as compared to susceptible genotypes. Significant negative correlations between the activity of PAL and damping-off susceptibility were only observed at 7 and 14 DAI (Table 2, r = −0.94 and r = −0.82, respectively); in the highly susceptible genotypes, the increase of PAL activity was lower than in the resistant genotypes. 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 [50,51]. 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 [52]. In tomato, PAL activity was enhanced in roots by a biotic elicitor Fusarium mycelium extract [53]. In cucumber roots, high levels of PAL were induced after inoculation with P. aphanidermatum [54]. In transgenic soybean, overexpression of GmPAL2.1 increases resistance to Phytophthora sojae [51]. Therefore, an increase in the level of PAL in the infected root tissue of cucumber genotypes showed that the phenyl propanoid pathway accumulated phenolics might have prevented the pathogen invasion, and thus, the activity maintained at higher levels during the infection period.

Figure 5. Trends of changes in phenylalanine ammonia-lyase (PAL) activity in the six cucumber genotypes after inoculation with P. melonis Uninoculated (control) genotypes (A) vs. inoculated genotypes (B) from day 7 to day 21.

Table 7. Phenylalanine ammonia-lyase (PAL) activities in inoculated resistant, moderately resistant and susceptible cucumber genotypes as compared to controls (non-inoculated ones).

Reaction to P.melonis	Genotype	PAL specific activity U mg^−1
		Day 7		Day 14		Day 21
		Control	Inoculated	Control	Inoculated	Control	Inoculated
Resistant	Soheil	0.26^m,n	0.12^n	0.60^g–m	1.21^d	0.71^e–j	1.86^b,c
	Ramezz	0.28^m,n	2.17^b	0.18^n	1.84^b,c	0.36^k–n	1.57^c
Moderately resistant	Baby	0.92^d–g	1.70^c	0.94^d,e,f	0.95^d,e,f	1.03^d,e	5.20^a
Susceptible	Extrem	0.32^l,m,n	0.58^g–m	0.33^l,m,n	0.55^h–m	0.26^m,n	0.12^n
	Mini 6–23	0.45^i–n	0.56^h–m	0.33^l,m,n	0.80^e–h	0.39^j–n	0.36^k–m
	Yalda	0.75^e–i	1.04^d,e	0.70^e–k	0.29^m,n	0.92^d–g	0.70^e–k

Note: Each value represents the mean of three replicates. Means in each column indicated with the same letter are not significantly different according to Fisher’s LSD test (p > 0.05).

Activity of polyphenol oxidase (PPO)

As shown in Table 8, comprehensible difference in PPO activity was not observed in the control of each genotype from day 7 to day 21. After inoculation with P. melonis, the PPO activity in the cucumber plants changed notably. Changes in the PPO activity of the roots of the six cucumber genotypes with different levels of resistance after inoculation with P. melonis are shown in Table 8 and Figure 6. The PPO activities in the resistant and moderately resistant genotypes were significantly higher than those in the three highly susceptible genotypes. In Soheil and Baby, the activity of PPO continuously displayed high activity until 21 DAI (Figure 6(A,B)). In the resistant genotype Ramezz, the significant increase in the activity of POX occurred at 7 DAI and then began to decrease at 14 and 21 DAI after inoculation (Figure 6(B)). The maximum amount of PPO activity was found to be 6-fold higher in the roots of inoculated Ramezz at 7 DAI than the respective control plants. The CAT activity of the three highly susceptible genotypes Extrem, Mini 6–23 and Yalda revealed a similar trend after inoculation: a slow increase at 7 DAI followed by a decrease at 14 and 21 DAI (Table 8). In the present study, PPO activity showed no significant differences in inoculated susceptible genotypes, as compared to non-inoculated plants. There was no correlation between damping-off susceptibility and PPO activity in the cucumber genotypes (Table 2). Furthermore, the activity of PPO was correlated significantly with the POX (r = 0.23) and PAL (r = 0.53) activities in the six genotypes of cucumber with different susceptibilities to damping-off (Table 2). Our findings show that, although the PPO activity increased in the roots of infected plants in comparison to the control in the resistant and moderately resistant genotypes, the activities varied among the three genotypes. Several studies have indicated induction of PPO in plants in response to infection by different pathogens [55,56]. In the 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. PPOs appear to play a role in the resistance to P. melonis, since the PPO activity was considerably higher in the 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 [18,57,58]. Additionally, a significant correlation between the antioxidant enzymes indicates the role of the antioxidant enzymes system under P. melonis infection. The role of antioxidant enzymes in eliciting systemic resistance against Fusarium oxysporum, Pyricularia oryzae, Alternaria sp. and Sclerotium sp. has been reported earlier [59]. These findings indicated the complex interaction between both oxidative-burst and response to damping-off. It can be speculated that oxidative burst and H₂O₂ accumulation also happened in resistant genotypes as an adaptive response to P. melonis infection, and that resistant genotypes may handle stress better than susceptible genotypes during the infection due to other mechanisms. However, the results focused on the changes in defensive enzyme activities are far from sufficient to explain damping-off resistance in cucumber. To understand cucumber resistance physiology more clearly, other physiological indicators should be considered.

Figure 6. Trends of changes in polyphenol oxidase (PPO) activity in the six cucumber genotypes after inoculation with P. melonis. Uninoculated (control) genotypes (A) vs. inoculated genotypes (B) from day 7 to day 21.

Table 8. Polyphenol oxidase (PPO) activities in inoculated resistant, moderately resistant and susceptible cucumber genotypes as compared to controls (non-inoculated ones).

Reaction to P.melonis	Genotype	PPO specific activity U mg^−1
		Day 7		Day 14		Day 21
		Control	Inoculated	Control	Inoculated	Control	Inoculated
Resistant	Soheil	15.1^m,n,o	26.0^i,j,k	16.9^m,n	37.9^e,f	16.8^m,n	96.8^b
	Ramezz	27.4^h,i,j	188.4^a	31.4^g,h	54.2^d	31.7^g,h	33.8^f,g
Moderately resistant	Baby	25.6^j,k	42.5^e	23.9^j,k,l	41.1^e	33.2^f,g	81.4^c
Susceptible	Extrem	30.7^g,h,i	51.0^d	34.9^f,g	34.4^f,g	24.8^j,k	31.2^g,h
	Mini 6–23	16.0^m,n,o	31.3^g,h	14.4^m,n,o	11.7^o	13.1^n,o	16.2^m,n,o
	Yalda	19.2^l,m	22.2^k,l	30.5^g,h,i	24.4^j,k	31.5^g,h	30.6^g,h,i

Note: Each value represents the mean of three replicates. Means in each column indicated with the same letter are not significantly different according to Fisher’s LSD test (p > 0.05).

Conclusions

The results of this study indicated that the disease defense response is more pronounced in the resistant genotypes as demonstrated by an earlier defense response through increasing the content of defense-related enzymes. In addition, the results suggest that 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 oxidative-burst response occurred 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 contributes to resistance in cucumber remain to be explored in future studies.

Acknowledgments

The authors are thankful to Plant Protection Research Division, Isfahan Center for Agricultural and Natural Resources Research and Education (AREEO), Isfahan, Iran and also, Plant Protection Research Institute, Tehran, Iran, for providing facilities to run the project.

Disclosure statement

There is no conflict of interest relating to this article.