Melon (Cucumis melo L.) is a crop of Cucurbitaceae, Cucumis, as the world’s top ten best-selling fruits, has the characteristics of delicious, sweet, juicy and refreshing. Powdery mildew is one of the most important diseases that harm melon production. It not only reduces the yield of melon, but also affects the maturity and sugar content of melon, thereby reducing the quality and commodity of melon and causing serious economic losses (Chen et al., 2006, Jiangsu Agricultural Sciences, (6): 224-225). Previous studies have proved that under stress, the contents of photosynthetic indexes, malondialdehyde (MDA) and osmoregulation substances (soluble sugar and soluble protein), and the defense enzyme activity changes of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbic acid peroxidase (APX), polyphenol oxidase (PPO), and phenylalanine ammonialyase (PAL) in plants can be used as important index of plant stress resistance (Li et al., 2011). After plants are infected by pathogens, a series of complex physiological and biochemical changes will occur (Ryals et al., 1996; Sticher et al., 1997; Hong et al., 2005; Cavalcanti et al., 2006). The results showed that the relative chlorophyll content and photosynthetic rate of the two wheat varieties decreased when they were infected by powdery mildew (Han et al., 2016). The content of MDA in rose leaves of three resistant varieties increased significantly, and the increase of MDA content of susceptible varieties was the largest (Yan et al., 2010). The activities of SOD, POD, CAT, APX, PAL and PPO were significantly up-regulated (Jiang and Si, 2010; Tian et al., 2015; Zhang et al., 2015). Mo et al. (2016, Jiangsu Agricultural Sciences, 44(6): 229-232) found that after inoculation with powdery mildew, the defense enzyme activity in pepper leaves were improved, and the changes of defense enzyme activities of different pepper varieties were different.

Although there are some reports on the research of melon powdery mildew, there are some differences among the research results. On the other hand, different melon varieties have different mechanisms of stress resistance. Therefore, in order to provide more theoretical basis for resistance identification and genetic breeding of melon powdery mildew, three melon varieties with different resistance to powdery mildew were selected as experimental materials in this experiment. We analyzed the changes of antioxidant defense enzyme activity, MDA and soluble protein content, chlorophyll content and photosynthetic index in different periods after inoculation with powdery mildew.

1 Results and Analysis 

1.1 Changes of antioxidant enzyme activities in different resistant melon varieties

After inoculation with powdery mildew, POD activities of ‘16-6’, ‘04-38’, and ‘15b-42’ were all induced to increase, reached the peak 5 d after inoculation, and then began to decline rapidly. The POD activity of ‘16-6’ and ‘04-38’ was always higher than that of ‘15b-42’ during this period of time. The POD activities of ‘16-6’ and ‘15b-42’ were increased 9 d after inoculation (Figure 1A). With the prolongation of inoculation time of powdery mildew, the activities of CAT and SOD in ‘16-6’, ‘04-38’, and ‘15b-42’ showed a trend of first increase and then decrease. Among them, the CAT activity of ‘16-6’ and ‘04-38’ reached the peak 3 d after inoculation, while the CAT activity of ‘15b-42’ increased slowly and reached the peak 7 d after inoculation, with the increase range of ‘16-6’ >‘04-38’>‘15b-42’(Figure 1B). The SOD activity of ‘16-6’ reached the peak 5 d after inoculation, while the enzyme activities of ‘04-38’ and ‘15b-42’ did not change significantly in the first five days after inoculation, and then began to increase rapidly. 7 d after inoculation, the SOD activity of the three varieties almost reached the same level. During this period, SOD activities of ‘04-38’ and ‘15b-42’ were significantly higher than those of ‘16-6’ (Figure 1C). APX activity of ‘16-6’, ‘04-38’ and ‘15b-42’ increased significantly after inoculation with powdery mildew, peaked 3 d after inoculation, and then began to decrease significantly. The increase range of enzyme activity was ‘16-6’>‘04-38’>‘15b-42’. APX activity of ‘04-38’ and ‘15b-42’ began to increase 5 d after inoculation, reached the second peak after 7 d, and then began to decline rapidly (Figure 1D). 

1.2 Changes of PAL and PPO activities in different resistant melon varieties

There was no significant difference in PAL activity among ‘16-6’, ‘04-38’ and ‘15b-42’ 1 d after inoculation, and then they all began to increase rapidly. After 7 d, PAL activity reached the peak, and the change range of enzyme activity showed ‘16-6’ >‘04-38’>‘15b-42’. After that, PAL activity of the three varieties began to decrease significantly (Figure 2A). PPO activities of ‘16-6’, ‘04-38’ and ‘15b-42’ increased significantly after inoculation with powdery mildew, peaked 5 d after inoculation, and then began to decrease. After 7 d, PPO activities began to increase rapidly. Increase range showed ‘16-6’>‘04-38’>‘15b-42’ (Figure 2B). 

1.3 Changes of MDA and soluble protein in different resistant melon varieties

With the prolonged time of inoculation with powdery mildew, the content of MDA in ‘16-6’, ‘04-38’ and ‘15b-42’ showed a trend of first increase and then decrease. One day after inoculation with powdery mildew, the difference of MDA content among the three varieties was not very significant, and then it was induced to increase. After 5 d, MDA content of ‘16-6’ and ‘15b-42’ reached the peak. While, MDA content of ‘04-38’ reached the peak after 7 d. During this period, MDA increased in the order of ‘15b-42’>‘16-6’>‘04-38’ (Figure 3A). The content of soluble protein in three varieties decreased after inoculation with powdery mildew. Among them, ‘15b-42’ decreased faster than ‘16-6’ and ‘04-38’ (Figure 3B).

1.4 Changes of chlorophyll content in different resistant melon varieties

By analyzing the changes of chlorophyll content, it was found that the contents of chlorophyll a, chlorophyll b, carotenoids and total chlorophyll of the three melon varieties after inoculation with powdery mildew showed an overall downward trend. Compared with 1d and 9 d after inoculation, the contents of chlorophyll a, chlorophyll b, carotenoids and total chlorophyll in ‘16-6’ decreased by 39.1%, 22.9%, 27.9% and 34.5%, respectively. In ‘04-38’, the contents decreased by 56.9%, 52.5%, 56.4%, and 55.8%, respectively. And in ‘15B-42’, the contents decreased by 63.4%, 55.4%, 45.6%, and 60.6%, respectively. The above results showed that except for carotenoids, which have the largest decline among the ‘04-38’ variety, the decline of the other indexes was shown as ‘15b-42’>‘04-38’>‘16-6’ (Table 1).

1.5 Changes of photosynthetic efficiency in different resistant melon varieties

With the prolonged time of inoculation with powdery mildew, net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr) and stomatal limitation value (Ls) in ‘16-6’, ‘04-38’, and ‘15b-42’ showed an overall downward trend (Table 2), of which the net photosynthetic rate of ‘16-6’ decreased significantly less than ‘04-38’ and ‘15b-42’, and the transpiration rate of ‘15b-42’ decreased significantly more than ‘04-38’ and ‘16-6’. Water use efficiency (WUE) in ‘16-6’ showed an overall trend of first increase and then decrease. There was no significant change in ‘04-38’ variety, but it showed an overall upward trend in ‘15b-42’.

2 Discussion

Antioxidant enzymes play an important defense role when plants are under stress, it can remove excessive active oxygen and maintain its metabolic balance, protect membrane structure, so that plants can reduce or resist stress damage to a certain extent (Mao et al., 2005; Zhao et al., 2009). PPO can promote the oxidation of phenols to quinones or form lignin. Quinones can inhibit the propagation and spread of pathogenic bacteria by inactivating the respiratory enzymes of pathogenic bacteria, and inhibit or kill pathogenic microorganisms. Lignin can be synthesized and accumulated in a large number in the infected parts, thereby inhibiting the reproduction of pathogenic bacteria (He and Bin, 2001, Plant Physiology Communication, 37(4): 340-345; Ma and Wu, 2006). PAL is a key enzyme and speed limiting enzyme in the synthesis of plant disease resistant substances. It can catalyze the deamination of phenylalanine to produce cinnamic acid. And the improvement of PAL activity can increase the content of the resistant substance lignin (Xu et al., 2000). The results showed that after inoculation with powdery mildew, the activities of POD, CAT, APX, PAL and PPO of melon varieties with different resistance increased significantly within a certain period of time, and the stronger the resistance, the more the enzyme activity and enzyme activity increased. This is the same as that Hu (2007) found that the activities of PDD, CAT and APX in cucumber were significantly increased after inoculation with powdery mildew. Chen (2000) found that the activities of POD, CAT, PAL and PPO in maize increased with the improvement of resistance. And Tian et al. (2015) found that the activities of POD, PAL and PPO in balsam pear were positively correlated with the resistance to powdery mildew. In this experiment, there was no obvious change rule between SOD activity and powdery mildew resistance of melon, which was consistent with the research results of Zhang et al. (2015).

The content of MDA can reflect the level of cell lipid peroxidation and the degree of membrane damage. It is an important index to judge the effect of membrane lipid peroxidation (Yoshimura et al., 2004; Liang et al., 2018). This study found that after inoculation with powdery mildew, the increase of MDA content in susceptible variety ‘15b-42’was significantly higher than that in resistant variety ‘16-6’ and medium resistant variety ‘04-38’, which indicated that more free radicals were accumulated in leaves of susceptible varieties, and the damage degree of cell membrane system in infected parts was relatively higher. The change of MDA content has a certain relationship with the resistance of the plant to pathogenic bacteria. This is consistent with the research results of Yan et al. (2010) and Wang et al. (2013) on rose. Soluble protein is an important index in plant physiology research. Studies have shown that the soluble protein content of wheat infected with powdery mildew and tobacco infected with mosaic virus decreased significantly (Mao et al., 2001; Han et al., 2016). In this experiment, the soluble protein content of melon infected with powdery mildew was significantly reduced, which was consistent with the aforementioned scholars’ results. Wang et al. (2013) found that there was no correlation between rose soluble protein content and powdery mildew resistance. However, the results of this experiment showed that the soluble protein content was positively correlated with the resistance of melon to powdery mildew. Therefore, the relationship between soluble protein content and plant disease resistance needs further study. Studies have shown that when plants are infected by pathogenic bacteria, their chlorophyll content and photosynthetic activity are significantly reduced (Li and Shang, 2001; Gu et al., 2004). Yu et al. (2002) found that the contents of chlorophyll a, chlorophyll b and carotenoids decreased significantly after tobacco was infected with CMV. Other studies also showed that when alfalfa or pea were infected with powdery mildew, the content of chlorophyll decreased significantly, and the decline in susceptible varieties was much greater than that in resistant varieties (Xu et al., 2005; Lu et al., 2018). In this experiment, the contents of chlorophyll a, chlorophyll b, carotenoids and total chlorophyll in resistant variety ‘16-6’, medium resistant variety ‘04-38’ and susceptible variety ‘15b-42’ were significantly decreased after inoculation with powdery mildew, and the decrease extent in susceptible variety was significantly greater than that in resistant variety, which was consistent with the aforementioned scholars. It was found that with the aggravation of Cotton Verticillium wilt disease, the net photosynthetic rate, transpiration rate, and stomatal conductance of plant leaves gradually decreased (Chen et al., 2017). Wu et al. (2011) found that the above three parameters also showed a downward trend after tomato was infected with powdery mildew, and the decline range in susceptible varieties was greater than that in resistant varieties. In this study, except stomatal conductance had no significant difference among the three melon varieties, the net photosynthetic rate and transpiration rate were the smallest among the resistant varieties, which was basically consistent with the aforementioned scholars’ results.

The results showed that the resistance of melon to powdery mildew was positively correlated with the enzyme activities of POD, CAT, APX, PAL, PPO, and the soluble protein content, and negatively correlated with the decline rate of MDA content, chlorophyll content, net photosynthetic rate and transpiration rate, but no significant correlation with SOD activity.

3 Materials and Methods

3.1 Experimental melon varieties and strains

The tested melon varieties are high-resistance variety ‘16-6’, medium-resistance variety ‘04-38’ and susceptible variety ‘15b-42’, determined by screening in early cultivation. Its seeds are provided by Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, and the powdery mildew was collected from the Baihe Test Base of Shanghai Academy of Agricultural Sciences.

3.2 Experimental design

Full melon seeds were selected for soaking and accelerating germination treatment, and then put them in a special plastic nutrition bowl for seedling after sprouting, and move to the bioartificial climate room for cultivation. The day and night temperature was 30℃/22℃, and the light cycle was 16 h/8 h (light / dark). When the seedlings grew to 3 leaves and 1 heart, the powdery mildew spores on the infected leaves were brushed in sterile water with a brush. Tween-20 (the final concentration is 0.05%) was added to the spore suspension with a concentration of 1.0×10⁶/mL for spray inoculation, and then placed in a bioartificial climate box for cultivation. During the day, the temperature was controlled at about 25℃, and the relative humidity was 70%~80%. At night, the temperature was controlled at about 16℃, and the relative humidity was 85%~95%, alternating 16 h/8 h (light / dark). At 1 d, 3 d, 5 d, 7 d, and 9 d after inoculation, the second leaf of 15 seedlings were randomly selected to determine the physiological and biochemical indexes. Each index was repeated three times.

3.3 Determination of physiological and biochemical indexes

The enzyme activities of SOD, POD, CAT, APX, PPO, and PAL were determined with reference to the kit instructions of Suzhou Keming Biotechnology Co., Ltd., and the contents of chlorophyll, MDA, and soluble protein were determined by the method of Li (2000). Plant net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), water use efficiency (WUE) and stomatal limitation (Ls) were all determined by portable photosynthesis instrument LI-6400 (made by LI-COR company, USA) from 9:00 am to 11:00 am. The calculation formula is: WUE= Pn/Tr, (Ls)=1-Ci/Co(CO is the CO₂ concentration in the air outside the leaf). During determination, the light intensity was about 320 000 lx, the temperature was (30±1)℃, and the CO₂concentration was about (400±10) μmoL/moL (Li, 2000). Repeat the readings 3 times for each blade.

3.4 Data statistics and analysis

Each index was determined 3 times, and the average value of the results was taken as the experimental data. Excel and SPSS data processing software were used to process and statistically analyze the results, and Excel was used for drawing.

Authors’ contributions

DQN designed the experiment. DQN and CYY performed the statistical analysis and drafted the manuscript. JXJ, CYY, and CYY participated in the design of the experiment, and performed the results analysis. ZYP revised the final manuscript. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by Shanghai Minhang District Program (2019MHC051), Shanghai Fruit Industrial Technology System (Hunongke [2019] No.1), and Excellent Team of Shanghai Academy of Agricultural Sciences (Nongkechuang2017(B-06)).

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