Mechanisms of different cultivars of Cucurbita pepo in resistance to Podosphaera xanthii infection through improvement of antioxidative defense system and gene expression

Background: Powdery mildew is one of the world’s most destructive diseases of cucurbit and the major cause of losses in its production worldwide. A number of strategies have been developed and applied to discover some suitable and alternative safe methods to manage the powdery mildew disease occurrence, but little information is regarding to screen of resistant pumpkins (Cucurbita pepo L.) germplasm and explore the mechanisms of their preventing the disease occurrence at physiological, biochemical, and molecular levels. Therefore, we evaluated and determined the ability and mechanisms of two commercial pumpkin cultivars in resistance to Podosphaera xanthii infection. Results: Compared with mock-inoculated seedlings, small and sparse spots were observed on the cultivar of Sixing F 1 leaves at the 13 th day after inoculation with P. xanthii, whereas a large number of disease spots or a layer of white powdery mildew were observed on the surface of Jin 12 F 1 leaves. Increased the inoculation time (7, 9, 11 and 13 days) signicantly and continuously increased the disease incidence and index of pumpkin seedlings after inoculation. The disease incidence and index of Jin 12 F 1 were signicantly higher than the cultivar of Sixing F 1 . At Day 13, the disease incidence and index of Jin 12 F 1 were 80.0% and 72.6, whereas the cultivar of Sixing F 1 was 22.3% and 17.7, respectively. The contents of H 2 O 2 , MDA, lignin and total phenolics in the leaves of Sixing F 1 and Jin 12 F 1 were markedly accelerated after inoculation with P. xanthii. However, the cultivar of Sixing F 1 exhibited less reactive oxygen species (ROS) accumulation, a lower rate of lipid peroxidation and higher level of lignin and total phenolics contents after inoculation, whereas the cultivar of Jin 12 F 1 exhibited higher level of ROS accumulation and rates of lipid peroxidation, and lower level of lignin and total phenolics contents. Higher activity and transcript level of antioxidant enzymes and gene of PAL expression were observed on different tissues of both cultivars after inoculated with P. xanthii. Compared with pumpkin seedlings that were inoculated with sterile water but not P. xanthii, the level of PAL activity and gene expression in leaves, petioles and stems of Sixing F 1 and Jin 12 F 1 were upregulated and increased signicantly at different time points after inoculation. The enhancement expression of PAL activity and gene in different tissues of Sixing F 1 was signicantly higher than Jin 12 F 1 , and higher in leaves, lower in petioles and stems. Conclusions: Our results indicate that the cultivar of Sixing F 1 exhibited the highest ability in resistance to P. xanthii infection in comparison to Jin 12 F 1 , and one novel possible mechanism is related to the cultivars in resistance to P. xanthii infection by activating and enhancing the antioxidative defense system and gene expression to prevent the pathogens infection.


Background
Pumpkin (Cucurbita pepo L.) is one of the most important vegetable crops for human nutrition all over the world [1]. However, powdery mildew is a common and major disease of pumpkin that affects the seedlings and mature plants growth [2], and also is one of the limiting factors that causing severely economic losses in pumpkin by shortening the ripening and harvest intervals, reducing photosynthesis and yields, and decreasing fruit quality in eld and greenhouse [3,4]. Normally, the pumpkin yield losses due to the disease of powdery mildew by 30 to 50% [5]. In addition, Podosphaera xanthii has been considered as one of the most important pathogens that causing the disease of cucurbit powdery mildew and reducing the production worldwide [6]. Thus, suitable control measurements and alternative safe methods, such as effective and friendly environment control methods are needed to overcome these problems.
In past few years, some strategies have been adapted to manage the disease of powdery mildew in agriculture, including the use of chemical, biological fungicides and breeding the resistance varieties [7].
Similar study has reported that the application of fungicides is the most e cient method to control powdery mildew in Iran and elsewhere [8]. However, chemical fungicide that is not healthy and safe due to it hazardous effects on human, animals, plants, and bene cial organisms, as well as developing pathogen resistance [9]. Although biological control agents have been applied to control of powdery mildew, their e cacy mainly depends on climatic conditions [10]. Therefore, screening of resistant pumpkin germplasm will be the best way for developing new cultivars to prevent the disease of powdery mildew occurrence. The resistance to powdery mildew was rst observed in cucumber (Cucumis sativus L. cv. Puerto Rico 37) [11], thereafter, a large number of resistant materials were found in local cucurbit varieties from South and East Asia [12], but little is known about the speci c knowledge for discovering and developing new resistant varieties of pumpkin to prevent powdery mildew occurrence in China, and the mechanisms of pumpkin in resistance to P. xanthii infection is a very complex phenomenon and the nature remains unresolved.
A number of studies have been demonstrated that plants can develop appropriate defense mechanisms to recognize and resist inevitable pathogen attacks, i.e. plants defend themselves against fungal infection through the activation of complex defense responses [13]. One of the earliest these responses is the rapid generation of reactive oxygen species (ROS), which includes superoxide anion (O 2-), hydroxyl radical (OH -) and hydrogen peroxide (H 2 O 2 ) [14]. Meanwhile, phenylpropanoid pathway is another most important secondary metabolism pathways and defense responses in higher plants [15,16]. Phenylalanine ammonia-lyase (PAL) is the rst enzyme in the phenylpropanoid pathway, participating in the formation of a series of structural and defensive lignin and phenolic compounds [17][18][19][20]. Furthermore, PAL gene has been widely studied in participating in plant growth, development and defense systems [21][22][23], such as the upregulated expression of PAL gene in plants that can help plant to develop resistance to phytopathogens infection [24][25][26]. However, to our knowledge, there is little published information regarding the mechanisms of different cultivars of C. pepo in resistance to P. xanthii infection at physiological, biochemical, and molecular levels.
In view of the above background, the aims of our present study were to (i) evaluate the ability and effectiveness of two commercial pumpkin cultivars in resistance to P. xanthii infection, and (ii) determine the defense responses pathway in two commercial pumpkin cultivars after inoculated with the pathogen of P. xanthii at different time points, and (iii) explore the possible mechanisms involved in two different pumpkin cultivars in response to P. xanthii infection at physiological, biochemical, and molecular levels.

Symptoms of Cucurbita pepo after inoculation with Podosphaera xanthii
Compared with mock-inoculated Jin 12 F 1 (Fig. 1C) and Sixing F 1 seedlings (Fig. 1D), small and sparse spots were observed on the cultivar of Sixing F 1 leaves at the 13 th day after inoculation with P. xanthii ( Fig. 1B), whereas a large number of disease spots were observed on the surface of Jin 12 F 1 leaves, even a layer of white powdery mildew was covered on the leaves (Fig. 1A) in comparison to the mockinoculated Jin 12 F 1 seedlings (Fig. 1C). However, the cultivars of Jin 12 F 1 (Fig. 1C) and Sixing F 1 (Fig. 1D) leaves have no disease spots occurred in the control group.
Disease severity of Cucurbita pepo after inoculation with Podosphaera xanthii Compared with pumpkin leaves that were inoculated with sterile water but not P. xanthii, the cultivars of Jin 12 F 1 and Sixing F 1 were begun to show the disease symptoms after inoculated with the pathogen of P.
xanthii at the 5 th and 7 th days, respectively. However, the disease incidence and index were signi cantly different between the cultivars of Sixing F 1 and Jin 12 F 1 after inoculation with P. xanthii. In contrast, the mock-inoculated seedlings of Sixing F 1 and Jin 12 F 1 were grown normally, and have no disease symptoms at the recorded days after inoculation, respectively. Increased the inoculation time (7,9,11 and 13 days) signi cantly and continuously increased the disease incidence and index of two cultivars pumpkin seedlings. The disease incidence and index of Jin 12 F 1 were signi cantly higher than the cultivar of Sixing F 1 . At Day 13, the disease incidence and index of Jin 12 F 1 were 80.0% and 72.6, respectively, whereas the cultivar of Sixing F 1 was 22.3% and 17.7, respectively.In addition, the cultivar of Jin 12 F 1 began to show symptoms at the 5 th day after inoculation, while the cultivar of Sixing F 1 at the 7 th day after inoculation. Furthermore, the speed of disease spots expansion of the cultivar Jin 12 F 1 was faster than Sixing F 1 with the increase of inoculation time (Table 1 and Table 2). Table 1 The disease incidence of different cultivars of Cucurbita pepo after inoculation with Podosphaera xanthii  Table 1 Hydrogen peroxide (H 2 O 2 ) and lipid peroxidation (MDA) contents in pumpkin seedling The H 2 O 2 and MDA contents of Sixing F 1 and Jin 12 F 1 seedling leaves after inoculated with P. xanthii were increased with the increase of days post inoculation from 1 to 9 or 11 days, peaked at the 9 th and 11 th days, and then declined gradually.    Data are means of twelve replicates. Different letters in the same column denote signi cant differences at the p < 0.05 level by Duncan's new multiple range test (n=12). The treatments are detailed in the footnote of Table 1 Levels of defense enzyme expression The activity of defense enzyme PAL in different cultivars and tissues of pumpkin (Sixing F 1 and Jin 12 F 1 ) was increased signi cantly at the 5 th day after inoculated with the pathogen of P. xanthii, peaked at the 7 th and 9 th days, and then declined gradually ( Fig. 2 and Fig. 3). However, the activity of PAL differed signi cantly between the cultivars of Sixing F 1 and Jin 12 F 1 . Higher level of PAL activity was expressed in the cultivar of Sixing F 1 leaves after inoculation in comparison to the Jin 12 F 1. Compared with control, PAL activity of Sixing F 1 was increased by 39.15% and 32.52% in leaves ( Fig. 2A), as well as 27.19% and 22.31% in petioles (Fig. 2B), and 13.83% and 13.75% in stems (Fig. 2C) at the 7 th and 9 th days, respectively. In contrast, the PAL activity of Jin 12 F 1 was increased by 9.27% and 12.84% in leaves ( Fig.   3A), as well as 11.86% and 11.75% in petioles (Fig. 3B), 7.12% and 6.16%in stems (Fig. 3C) at the 7 th and 9 th days, respectively. In addition, the expression level of PAL activity was higher in leaves, lower in petioles and stems. Overall, the control treatment followed a similar trend as the other treatments in terms of the change of PAL activity during the course of the experimental period, but the control treatment had signi cantly lower PAL activity at a given measurement date starting at the 1 st day after inoculation.

Levels of defense gene expression
Higher transcript level of PAL gene expression was observed on different cultivars and tissues of pumpkin (Sixing F 1 and Jin 12 F 1 ) after inoculated with the pathogen of P. xanthii ( Enhancement expression of PAL gene after inoculation with Podosphaera xanthii The enhancement changes of PAL gene expression were signi cantly different in different cultivars and tissues of pumpkin (Sixing F 1 and Jin 12 F 1 ) after inoculated with P. xanthii (Fig. 6). However, the increased PAL gene expression in Sixing F 1 was signi cantly higher than the cultivar of Jin 12 F 1 , as well as higher in leaves (Fig. 6A) than the petioles (Fig. 6B) and stems (Fig. 6C). The enhancement changes of PAL gene expression in different tissues and cultivars of pumpkin also showed a trend of increased rst, and thereafter it declined with the increase of inoculation time. The enhancement expressionof PAL gene reached its maximum at the 9 th , 7 h and 7 th days in leaves (Fig. 6A), petioles (Fig. 6B) and stems (Fig. 6C) after inoculation and thereafter it declined.
Lignin and total phenolics contents in pumpkin seedlings The cultivars of Sixing F 1 and Jin 12 F 1 with P. xanthii treatment increased the lignin and total phenolics contents in seedling leaves from 1 to 9 or 11 days, and peaked at the 9 th and 11 th days. The level of lignin and total phenolics contents of Sixing F 1 signi cantly higher in comparison to the cultivar of Jin 12 F 1. The average contents of lignin and total phenolics were signi cantly increased by 21.24% and 21.09% in Sixing F 1 seedlings leaves from 9 to 11 days after inoculation in comparison to the control under sterile water treatment, respectively. However, in the cultivars of Jin 12 F 1 , the contents of lignin and total phenolics in the seedling leaves were increased by 12.38% and 18.65% from 9 to 11days after inoculation, compared with the control under sterile water treatment, respectively (Table 4).
Note: F represents forward, R represents reverse

Discussion
Previous studies have been demonstrated that the disease resistance screening in introduced germplasm was important to get resistant and tolerant germplasm for breeding new cultivars to control powdery mildew in eld and greenhouse-grown pumpkins [27], but the mechanisms of pumpkin germplasm in resistance to Podosphaera xanthii infection remain unresolved because speci c information about the improvement of antioxidative defense system and gene expression in different cultivars are virtually unknown at the physiological, biochemical and molecular levels. In the present study, we evaluated the ability and unveiled the mechanisms of two commercial pumpkincultivars in resistance to P. xanthii infection. Interestingly, we discovered that the cultivar of Sixing F 1 exhibited higher ability in resistance to P. xanthii infection, and less ROS accumulation, lower rates of lipid peroxidation, and higher level of PAL activity and gene expression, lignin and total phenolics contents than Jin 12 F 1 . Thus, our results indicate that Sixing F 1 and Jin 12 F 1 can be considered as the resistant cultivar and susceptible cultivar, respectively, and the mechanism for their resistance to P. xanthii infection through enhancing the level of PAL activity and gene expression to reduce the ROS accumulation, and increase the lignin and total phenolics contents in their tissues to activate the defense system to prevent the pathogens infection. Our results will provide a vital theoretical basis and new insight for the mechanisms of different cultivars of pumpkinin resistance to P. xanthii infection. To the best of our knowledge, this is the rst report suggesting that the cultivars of pumpkin in resistance to P. xanthii infection through stimulating the defense system response in different tissues.
De Oliveira Rabelo et al. (2017) reported that breeding for resistance is one of the best strategies to decrease powdery mildew damage, and for the selection of cucurbits cultivars in resistance to powdery mildew [28]. Our results showed that the cultivar of Sixing F 1 exhibited higher ability in resistance to P.
xanthii infection, whereas the cultivar of Jin 12 F 1 exhibited lower ability. The disease incidence and index of Jin 12 F 1 were signi cantly higher than Sixing F 1 at different time points after inoculation . Yan et al. [29] showed that the disease incidence of the cultivar of Guangban was signi cantly higher than the cultivars of Sanxing, Erxing, and Hongfu after inoculation with the pathogen of P. xanthii. However, our results indicate that the cultivar of Sixing F 1 presented higher ability in resistance to P. xanthii infection than  [14]. Meanwhile, the excess ROS can lead to the peroxidation of unsaturated lipids of membranes in plants [36], such as the accumulation levels of ROS led to the increased contents of MDA in both the resistant and susceptible cultivars of faba bean during the interaction of B. fabae [37]. Our results indicate that the induction of H 2 O 2 and MDA in inoculated plants may be one of the pumpkin defense mechanisms against the invading pathogen infection, and the lower levels of ROS and MDA correlated with the susceptibility of pumpkin leaf tissues to infection with P. xanthii. El-Komy (2014) demonstrated that the resistant cultivar showed less ROS accumulation, a lower rate of lipid peroxidation and higher activity of the enzymatic ROS scavenging system compared with susceptible cultivar during the interaction of B. fabae [37].
In addition, plants can possess an inherent capacity to eliminate the harmful effects of reactive oxygen species (ROS) through the involvement of an antioxidative system that protects cell constituents from the oxidative damage [38]. Pathogenesis-related protein is one of the most important accumulated biotic components in plants to defend the pathogens after their attack and infection, including PAL, chitinase peroxidase, and other proteins [39]. Among all the pathogenesis-related proteins, PAL is the rst enzyme in the phenylpropanoids pathway, which produces the precursors for lignin and phenolic secondary metabolites that may be enhanced after pathogen infection [40]. Also, PAL is one of the key genes in the phenylpropane synthesis pathway, and is closely related to plant resistance to external stresses [41]; PAL gene is among those most relevantly upregulated in plants that develop resistance to phytopathogens reported that the expression level of PAL activity andgene in cucumber leaves were activated by P. xanthii inoculation [41]. Additionally, we found that the activity of PAL and the expression level of PAL gene in different tissues of resistant cultivar were signi cantly higher than the susceptible cultivar, as well as higher expression in leaves than petioles and stems. Similarly, higher transcript levels of PAL activity and gene expression were found in pathogens inoculated plants than the no-inoculated plants. Several previous studies also reported that the increased levels of the gene expression or the enzymic activity have been observed in plants after inoculation with pathogenic microbe [43][44][45]. Thus, our results indicate that PAL gene plays a signi cant role in coding the PAL activity to induce the ability of pumpkin resist to P. xanthii infection. The expression level of PAL gene in different tissues of pumpkin cultivars reached its maximum at the 7 th and 9 th days after inoculation and thereafter it declined with the increase of inoculation time. Gao et al. [46] revealed that the relative expression level of CsPAL gene in cucumbers exhibited a trend of increased rst after inoculation with powdery mildew, and thereafter decreased with the increase of inoculation time.
PAL is a key and rate-limiting enzyme in catalyzing phenylalanine to trans-cinnamic acid to improve the disease resistance of plants by promoting the synthesis of phenolic substances and lignin [47].
Additionally, phenylalanine ligni cation is one of the physical and biochemical changes of plant cell wall which could be induced by pathogen infection [48], and also phenolic compound plays an important role in participating in the defense mechanism against fungal infection [49,50]. Our results found that a signi cant increase in lignin and total phenolics contents in seedling leaves of Sixing F 1 and Jin 12 F 1 after treatment with P. xanthii, the level of lignin and total phenolics contents in the cultivar of Sixing pathogen of Colletotrichum orbiculare infection [51], and also the level of total phenolics was increased in cucumber seedlings after inoculated with the P. xanthii [41].

Conclusions
In summary, the results of our study suggest that Sixing F 1 can be considered as the resistant cultivar, and Jin 12 F 1 can be considered as the susceptible cultivar. One novel possible mechanism is related to the pumpkin cultivars in resistance to P. xanthii infection through enhancing the level of PAL activity and gene expression to promote the synthesis of phenolic substances and lignin, and reduce the ROS accumulation to activate the defense system in different tissues to prevent the pathogens infection nally. However, more research is needed to determine other defense genes in pumpkins that coding the pathogenesis-related protein expression in resistance to P. xanthii infection in the future.

Materials And Methods
Experiments were carried out at the Laboratory of Plant Pathology, College of Plant Protection; Gansu Provincial Key Laboratory of Arid Land Crop Science, Gansu Agricultural University. All treatments in the experiments described below had twelve replicates.

Seeds treatment
Seeds of two commercial pumpkin cultivars (Sixing F 1 and Jin 12 F 1 ) were selected and kindly provided by the company of Wuwei Golden Apple co. LTD. Seeds with a uniform size were surface-sterilized with 5% NaOCl (v/v) for 3 min. After disinfection, all the seeds were rinsed with sterile water for 5 times, and then were soaked in sterile water for 12 hours for germination.

Greenhouse experiments
The experiments were carried out in the greenhouse of Gansu Agricultural University in August, 2013. Seeds of two pumpkin commercial cultivars were germinated in 9-cm diameters Petri dishes, and covered with two layers of absorbent cotton and blotter papers in a constant temperature at 25°C for germination.
Thereafter, the germinated seeds with a uniform size were planted in 12-cm diameters pots that contained 500 g of sterilized soil. Each pot was planted with 8 seeds and each cultivar with 12 pots (a total of 96 plants) after germination. The experiment was arranged in a completely randomized design with twelve replications, took place in a greenhouse with the inside temperature maintained between 25°C and 20°C (day and night), the supplemental photoperiod was 16/8 hours light/dark, and the relative humidity at 60%. Irrigation was done twice weekly at each treatment and control.

Podosphaera xanthii identi cation and inoculum preparation
Pumpkin leaves infected with the pathogen of powdery mildew were collected from the eld (Wuwei, China) in July 15, 2013 for microscopic observation of the pathogen. The conidia observed under light microscope (E200, Germany) were oidium type and conidiospore with cylindrical brosin bodies of conidia which was identi ed to be Podosphaera xanthii according to the earlier published papers [52,53]. Thereafter, arti cial inoculation of host plants seedlings were performed by manually through dusting the sporulated leaves and P. xanthii isolate in host plants were kept for 20 days in greenhouse. The tested plants were inoculated at the 4-true leaf stage with the suspension of powdery mildew fungal pathogen P. xanthii spores by smear method, and 5 plants with relatively consistent growth were selected for each pot, and each plant was inoculated with 3 leaves. After that, the inoculated seedlings were placed in a greenhouse at 25°C and 20°C (day and night), relative humidity of 60% and light of 16/8 hours for development of powdery mildew. Control plants (mock-inoculated) were inoculated with the same volume of sterile water and maintained separately from the inoculated plants in the same greenhouse. The seedlings disease incidence, disease index H 2 O 2 ,MDA, lignin and total phenolics contents, and the level of 1: the infected areas less than 30% in the front of leaves, and no symptoms in the reverse of leaves; 3: the infected areas greater than 30% in the front of leaves, and less than 10% in the reverse of leaves; 5: the infected areas greater than 30% and 10% in the front and reverse of leaves, respectively, and a few lesions appeared on the petioles; 7: the powdery mildew covered in the front of leaves and the infected areas greater than 10% in the reverse of leaves, and more lesions appeared on the petioles and a few on the main stems; 9: the powdery mildew covered in the front of leaves, petioles and main stems, and the infected areas greater than 10% in the reverse of leaves.
Disease index = (ΣNDL GL) / (TNIL HGL) 100…………………………………………. (2) where NDL is the number of diseased leaves in each lever; GL is grade levels; TNIL is total number of Thereafter, the reaction mixture was centrifuged at 10, 000 g for 10 min, and the contents of H 2 O 2 in extracts were determined and calculated according to the method described by Willekens et al. (1997) [55]. The contents of H 2 O 2 were expressed as µmol g -1 FW.
The level of lipid peroxidation was determined by quantifying the MDA contents in different cultivars of pumpkin seedlings according to the method described by Hodges et al. (1999) [56] and Tian et al. (2015) [57] with some modi cations. For the determination of the accumulation of MDA in pumpkin seedling leaves, 0.5 g fresh leaf samples were homogenized in 2.5 ml of 0.1% trichloroacetic acid and the homogenate. Afterwards, the reaction mixture was centrifuged at 10, 000 g for 15 min and the absorbance of supernatant was recorded at 532 nm wavelength. The content of MDA was expressed as nmol g -1 FW.

PAL activity determination
Fresh pumpkin seedling leaf, petiole and stem samples of 0.5 g were homogenized in 6 ml ice-cold borate buffer (5 mM, pH 8.8) using pre-chilled mortar and pestle, respectively. The supernatant of homogenates were collected and used as crude extracts after centrifuging at 8, 000 g for 20 min at 4°C. Thereafter, the supernatant was mixed with 0.02 M phenylalanine and distilled water to obtain the extractions. The reaction mixtures were placed in a thermostatic water bath at 30°C for 30 min and then measured at 290 nm. For the determination of PAL activity, the crude extraction was measured according to the method described by Hu et al. (2009) [61] and Ruiz et al. (1999) [62]. The activity of PAL was expressed as U min -1 g -1 FW.
Total RNA extraction and rst strand cDNA synthesis The samples of leaves, petioles and stems were collected every 2 days post inoculation (dpi) for both the inoculated and mock-inoculated seedlings, and the collections continued for 1, 3, 5, 7, 9, 11 and 13 dpi from both cultivars of Sixing F 1 and Jin 12  Quantitative real-time PCR (qRT-PCR) analysis The level of PAL gene expression was determined in pumpkin leaves, petioles and stems at different time points after inoculation with P. xanthii or sterile water in each treatment and control. qRT-PCR was performed using a SYBR Premix Ex Taq kit (Takara Biotechnology, Dalian, China) following the manufacturer's instructions. The sequences of the forward and reverse primer pairs used for qRT-PCR analysis were designed according to the EST sequences of pumpkin in NCBI using Primer Express 5.0 software that ampli es the target genes. The actin gene of pumpkin was used as an internal control. The level of PAL gene expression was determined using the method of 2 -ΔΔCt [63]. The gene speci c primers used for this analysis are shown in Table 5.

Leaf cell wall isolation and lignin content determination
The leaf cell wall of different cultivars of pumpkin seedlings were isolated according the method described by Eskandari et al. (2018) [58]. A 0.5 g of fresh leaf samples were frozen and ground to powder using the liquid nitrogen. The powder samples were homogenized in distilled water and then centrifuged at 10, 000 g for 10 min. The precipitation was washed with absolute ethanol, and rinsed in chloroform and methanol (v/v = 1: 2) and then washed with acetone for three times. The cell wall pellet was ltered and nally dried overnight at 35°C. The residue (cell wall) was collected and kept in a desiccator at room temperature until use.
The content of lignin was determined and assayed by following the procedure of Iiyama and Wallis (1990) [59] with a minor modi cation. A 5 mg of cell wall preparation was treated with a mixture (2.5 ml) that consisted of 5% (w/w) acetyl bromide and AcHO, and 0.1 ml of 70% HClO at 70°C for 30 min. Finally, the reaction mixture was treated with 50 ml that contained 2 M NaOH and AcHO after cooling. The lignin content was determined by measuring the absorbance at 280 nm using a speci c absorption coe cient of 20.0 g -1 l cm -1 .

Total phenolics content determination
The contents of total phenolics were measured according to the method described by Singleton and Rossi (1965) with a minor modi cation [60]. A 0.5 g fresh leaf samples were ground with a small amount of quartz sand, and thereafter extracted in 70% ethanol (10 ml) for 10 min. The reaction mixture was centrifuged at 12, 000 g for 20 min. The supernatant of the reaction mixture was collected and used to measure the total phenolic content, and expressed as mg g -1 FW.

Statistical analysis
The data were subjected to variance analysis (ANOVA) using SPSS Version 16.0 (SPSS Inc., Chicago, IL).
Each experiment had twelve replications. Duncan's multiple range test was computed using standard error and T values of adjusted degrees of freedom. The signi cant differences between the treatments were considered at the level of p <0.05. The data of disease incidence, index and gene expression levels of two commercial pumpkin cultivars (Sixing F 1 and Jin 12 F 1 ) were calculated according the formulas as described previously. Figure 1 The symptoms of different cultivars of Cucurbita pepo after inoculation with the pathogen of Podosphaera xanthii at the 13th day. Where A the cultivar of Jin12 F1 after inoculation with P. xanthii; B the cultivar of Sixing F1 after inoculation with P. xanthii; C the cultivar of Jin12 F1 after inoculation with sterile water but not P. xanthii; D the cultivar of Sixing F1 after inoculation with sterile water but not P.