Abstract
Jujube leaf tea is a functional beverage that soothes the nerves. In this study, we evaluated the effects of carbendazim and harpin on disease index, biomass accumulation, H2O2, antioxidant contents, and phenyl alanine ammonia lyase (PAL) activity in young jujube leaves. Compared to harpin, carbendazim decreased the disease index and induced higher H2O2 content. Additionally, the pesticide reduced young leaf biomass accumulation. In contrast, harpin increased vitamin C, glutathione, total phenolics, and total antioxidant capacity in young leaves compared to carbendazim. Compared with the control, harpin enhanced the PAL activity. Carbendazim residues were present in treated leaves for 14 days. Our study findings provide a method for improving jujube leaf tea quality from a pesticide utilization perspective.
Graphical abstract
1 Introduction
Chinese jujube (Ziziphus jujuba Mill.) is an economic crop grown in the Northern Hemisphere that can be traced back 4,000 years [1,2]. Jujube has attracted considerable interest within the scientific community due to its nutritional and medicinal value [3]. In traditional Chinese medicine, jujube leaf is used to improve sleep, nourish the heart, and soothe the nerves [4,5]. Jujube leaf can be processed into green and black tea of fresh smell and pleasant taste through different processing techniques [4,6].
Jujube fruit, which is rich in bioactive compounds (e.g., vitamin C and phenolics), has physiological and pharmacological functions [7,8]. Phenolics are the main bioactive compounds in jujube leaves [4,9]. Polyphenols, which are among the most widely distributed secondary products in the plant kingdom [10], are involved in diverse processes such as resistance to different types of stress and participation in redox reactions [10,11]. However, this new functional tea was made only from the young leaves of wild jujube [4,5]. Environmental stress (e.g., drought and heat) affects the accumulation of bioactive compounds in jujube leaf [12]. Diseases caused by fungi and phytoplasmas reduce fruit yield and nutrient quality of jujube [13]. Pesticides improve yield and visual quality of harvested products. However, the incidence and severity of pests and pathogens depend on growing conditions and changes over the year [13]. Interestingly, both chemical pesticides and pathogens directly influence the phenolic metabolism in plants [10,11]. For example, carbendazim, a common fungicide in China [14], which can be degraded in plants via glutathione-dependent pathway [15], induces phenolic accumulation in tobacco plants at low doses [16]. Similarly, the elicitor harpin, which was initially isolated from Erwinia amylovora, causes multiple effects including hypersensitive reactions and defense responses in plants [17] and stimulates the activity of phenylalanine ammonia lyase (PAL), an enzyme involved in the biosynthesis of polyphenolic compounds in jujube plant [18].
Published data have shown that there are different antioxidant responses between young and old leaves in plants [19,20]. For example, there is a greater salt-induced accumulation of phenolics but lower lipid peroxidation in young leaves than in old leaves of Carthamus tinctorius [19]. Young leaves are required for jujube leaf tea production. In this study, we posed three questions. How do harpin and carbendazim affect disease development and biomass accumulation in young jujube leaves? Do harpin and carbendazim affect total phenolic accumulation in young leaves? What are the possible mechanisms involved? The objective of our study was to develop a method for improving jujube leaf tea quality from a pesticide utilization perspective.
2 Methods
2.1 Plant growth and pesticide application
The experiment was carried out in an artificial orchard chamber located in Luoyang Normal University, China in 2019–2021. The jujube plants (Z. jujuba Mill. var. Spinosa [Bunge] Hu ex H. F. Chow) were 5 months old (75 ± 5 cm height) and grown in plastic buckets (50 L, 50 cm height). The soil (pH 6.4) contained 78.5, 14.3, and 86.4 mg kg−1 of available N, P, and K, respectively. The temperature was set at 15 ± 1°C (dark) and 25 ± 1oC (light). We watered the plants every 2 days. Harpin protein and carbendazim were obtained from Haibos Biotech Co. (Chengdu, China) and Macklin Biochemistry & Technique Co. (Shanghai, China), respectively.
2.2 Experimental design
Before treatment, we watered all plants with tap water. We randomly divided the plants into three groups (in triplicate): group 1 (foliar spraying with water, control), group 2 (foliar spraying with harpin), and group 3 (foliar spraying with carbendazim). Based on the product recommendations, we used 30 mg L−1 harpin and 1,000 mg L−1 carbendazim. For spraying, we used a 1 L plastic sprayer until the shoots become complete wet. After treatment for 0, 3, 7, and 14 days, we harvested the second fully expanded leaves from the apex. Moreover, young leaves were hand-harvested in the early morning and transported to the laboratory within 2 h. We selected leaves of similar shape and appearance and devoid of visible defects. Each replicate contained 10 g of leaves. We stored the leaves at −70°C for analyses of pesticide residues, hydrogen peroxide (H2O2), vitamin C, glutathione, total phenolics, total antioxidant capacity, PAL, and polyphenol oxidase.
2.3 Pesticide residue assay
We obtained pure carbendazim from the Institute for the Control of Agrochemicals, Ministry of Agriculture (Beijing, China) and extracted pesticide residues from 5 g of chopped leaf tissues. We homogenized the leaves with petroleum ether (PE) and anhydrous sodium sulfate (ASS) in a high-speed homogenizer (12,000×g for 5 min). We used a Büchner funnel (7 cm) containing 10 g of ASS to filter the leaf mixture and 50 mL of redistilled PE to wash the filter cake three times. The filtrates were mixed in a flat-bottomed flask (0.5 L) and dried under a N2 stream. We collected 5 mL of carbendazim dissolved in redistilled PE for analysis using gas chromatography. Quantitative analysis was performed using a gas chromatograph equipped with a phosphorus filter and a flame photometric detector.
2.4 Hydrogen peroxide determination
We determined the leaf H2O2 as previously reported [21]. After homogenizing leaf tissues (0.1 g) on ice in 0.1% (w/v) TCA, we centrifuged the homogenate at 10,000×g for 15 min at 4°C. An aliquot of the supernatant (0.5 mL) was combined with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI. We measured the absorbance at 390 nm and calculated H2O2 based on a standard curve of known H2O2 concentrations.
2.5 Antioxidant measurements
We measured the vitamin C content by the titrimetric method with 2,6-dichloro-phenol-indophenol (2,6-DPI). Briefly, 1 g of homogenized fresh sample was mixed with 20 mL of 2% oxalic acid. The mixture was homogenized, diluted to 0.1 L with 2% oxalic acid, and filtered. We titrated 10 mL of the filtered solution with 0.01% of 2,6-DPI. The final point was when the solution retained the pink color for 15 s. The calibration of 2,6-DPI was performed using 0.05% ascorbic acid. The results were expressed as µmol of ascorbic acid equivalents per gram of dry weight (µmol g−1 DW). The assays were finished within 10–15 min.
We measured the reduced glutathione (GSH) levels fluorometrically [22] using o-phthaldialdehyde as the fluorophore. Fluorescence intensity was recorded at 420 nm after excitation at 350 nm on a Perkin-Elmer LS 55 fluorescence spectrophotometer (Perkin-Elmer, Waltham, MA, USA).
We measured the total phenolic content of the leaf extracts spectrophotometrically by the Folin–Ciocalteu procedure of Singleton and Rossi using Folin–Ciocalteu as reactive reagent and gallic acid as standard [23]. We prepared the homogenates as reported for antioxidant activity determination, and the clear supernatant was used for the determination of total phenolic content. The results were expressed as mg gallic acid equivalents g−1 dry weight.
2.6 Total antioxidant capacity assay
We measured the total antioxidant capacity as reported by Benzie and Strain [24]. Leaves were ground in liquid N2 using a mortar and pestle. We transferred 5 g of leaf powder to 1 L of 80% (w/v) aqueous methanol and incubated at room temperature for 2 h in the dark. Filtered extracts were pooled, and the solvent was discarded under vacuum at 45°C using a rotary evaporator. We stored the crude extracts in a desiccator at 4°C for total antioxidant capacity analysis using the ferric reducing ability of plasma assay.
2.7 Phenolic-related enzyme activity assay
We extracted 5 g of frozen leaf tissue using extraction buffer (50 mL) consisting of potassium phosphate (50 mM, pH 7.5), KCl (10 mM), EDTA (1 mM), dithiothreitol (5 mM), and polyvinylpolypyrrolidone (1:4, w/w). The homogenates were centrifuged at 12,000×g for 30 min, and the supernatant was used for enzyme assays.
We measured the polyphenoloxidase (PPO, EC1.14.18.1) activity as reported by Mayer et al. [25]. The reaction mixture consisted of 1.5 mL of 0.1 M sodium phosphate buffer (pH 6.5) and 100 μL of enzyme extract. To start the reaction, we added 0.2 mL of 0.01 M catechol. We expressed the activity as changes in absorbance at 495 nm min−1 mg−1 protein. We determined the PAL (EC4.3.1.5) activity from the rate of conversion of l-phenyl alanine to trans-cinnamic acid at 290 nm [26]. Sample containing 0.1 mL of enzyme extract was incubated with 1.2 mL of 0.1 M borate buffer, pH 8.8 and 1.5 mL of 12 M l-phenyl alanine in the same buffer for 30 min at 30°C. The reaction was stopped with the addition of 1 M trichloroacetic acid. Following incubation for 5 min at 37°C, we measured the absorbance at 290 nm. We expressed the enzyme activity as mmol trans-cimmamic acid min−1 mg−1 protein.
Soluble protein was determined by the method of Bradford [27] using bovine serum albumin as standard.
2.8 Disease index (DI) assay
We assessed the disease symptoms 7 days after treatment. We evaluated the DI according to the method by Leath et al. [28] with some modification: 0 (no symptoms), 1 (slight yellow of lower leaves), 2 (moderate yellow leaves), 3 (yellow halo around brown spots), and 4 (concentric rings of raised and depressed dead tissue). DI was calculated using the following formula:
where n 1–n 4r represents the number of plants in the indicated classes and n t is the total number of plants tested.
2.9 Data analysis
We used a completely randomized design, with three replicates per treatment. We analyzed the data by Duncan’s multiple range test using SPSS 13.0 software (IBM Cop., Armonk, NY, USA) at p < 0.05.
3 Results
3.1 DI, biomass accumulation, and H2O2 content
Both harpin and carbendazim reduce pathogen development by decreasing the DI. For example, application with harpin decreased the DI by approximately 6, 21, 36, and 35% in jujube leaves after 3, 7, 14, and 21 days, respectively, compared to the control (Figure 1a; p < 0.05). Similarly, carbendazim decreased DI by approximately 73, 83, 80, and 72% in young leaves after 3, 7, 14, and 21 days, respectively, over the control (Figure 1a; p < 0.05).
Effects of pesticides on DI, leaf growth, and H2O2 content. Effects of harpin (30 mg L−1) and carbendazim (1,000 mg L−1) on DI (a), biomass accumulation (b), and H2O2 content (c) of jujube leaves after treatment for 0, 3, 7, 14, and 21 days. Bars represent the standard deviations of the means (n = 3). Numbers with the same letter were not significantly different (p < 0.05).
Compared with the control, harpin promoted biomass accumulation in young leaves (Figure 1b; p < 0.05). For example, foliar spraying with harpin enhanced leaf biomass accumulation by approximately 50, 36, 32, and 22% after 3, 7, 14, and 21 days, respectively, over the water control (Figure 1b; p < 0.05). In contrast, carbendazim slightly but significantly inhibited leaf biomass accumulation (Figure 1b; p < 0.05). Foliar spraying with carbendazim reduced leaf biomass accumulation by approximately 10, 8, 16, and 21% after treatment for 3, 7, 14, and 21 days, respectively, compared to the water control (Figure 1b; p < 0.05).
Both harpin and carbendazim induced H2O2 accumulation in young leaves (Figure 1c). Compared with the water control, harpin increased H2O2 content by approximately 76, 42, 19, and 7% in young leaves after 3, 7, 14, and 21 days, respectively (Figure 1c; p < 0.05). However, the H2O2 content increased by approximately 232, 126, 56, and 21% in carbendazim-treated leaves over the control after pesticide application for 3, 7, 14, and 21 days, respectively (Figure 1c; p < 0.05).
3.2 Antioxidants in young leaves
Harpin and carbendazim affected vitamin C, GSH, total phenolic accumulation, and total antioxidant capacity in young jujube leaves after treatment for 7 days (Figure 2). For example, foliar spraying with 30 mg L−1 harpin enhanced vitamin C accumulation by approximately 47, 85, 78, and 66% in young jujube leaves after treatment for 3, 7, 14 and 21 days, respectively, compared to the water control (Figure 2a; p < 0.05). However, vitamin C content decreased by approximately 27, 16, 25, and 27% in young leaves after treatment with carbendazim for 3, 7, 14, and 21 days, respectively, compared with the water control (Figure 2a; p < 0.05). We obtained similar patterns with GSH, total phenolics, and total antioxidant capacity (Figure 2b–d; p < 0.05). For example, application of harpin enhanced the GSH, total phenolic content, and total antioxidant capacity by approximately 87, 79, and 62%, respectively, in young jujube leaves after 7 days (Figure 2b–d; p < 0.05). In contrast, carbendazim application reduced the GSH, total phenolic content, and total antioxidant capacity by approximately 46, 19, and 26%, respectively, in young leaves after 3 days (Figure 2b–d; p < 0.05).
Antioxidant content in young leaves. Effects of harpin (30 mg L−1) and carbendazim (1,000 mg L−1) on vitamin C (a), GSH (b), total phenolics (c), and total antioxidant capacity (d) in young leaves after treatment for 0, 3, 7, 14, and 21 days. Bars represent the standard deviations of the means (n = 3). Numbers with the same letter were not significantly different (p < 0.05). GSH, reduced glutathione.
3.3 Phenolic-related enzyme activity assay
We measured the activities of phenolic-related enzymes such as PAL and PPO in young leaves after treatment with harpin and carbendazim. Application with harpin increased the PAL activity by approximately 71, 90, 65, and 24% in young leaves over the water control after treatment for 3, 7, 14, and 21 days, respectively (Figure 3; p < 0.05). Similarly, PPO activity increased by approximately 34, 19, 11, and 5% in harpin-treated jujube leaves after 3, 7, 14, and 21 days, respectively (Figure 3; p < 0.05). In contrast, carbendazim reduced the PAL activity by approximately 57, 36, 26, and 20%, respectively, in young leaves over the water control after pesticide application for 3, 7, 14, and 21 days, respectively (Figure 3; p < 0.05). However, carbendazim application enhanced the PPO activity by approximately 123, 81, 41, and 21%, respectively, in young leaves over the water control after pesticide application for 3, 7, 14, and 21 days, respectively (Figure 3; p < 0.05).
Phenolic-related enzyme activities. Effects of harpin (30 mg L−1) and carbendazim (1,000 mg L−1) on PAL (a) and PPO (b) activities in young leaves after treatment for 0, 3, 7, 14, and 21 days. Bars represent the standard deviations of the means (n = 3). Numbers with the same letter were not significantly different (p < 0.05). PAL, phenylalanine ammonia-lyase; PPO, polyphenol oxidase.
3.4 Carbendazim residues in young leaves
Plant growth decreased carbendazim residues in young leaves during the first 21 days after foliar spraying (Table 1). Table 1 shows that pesticide residues cannot be detected in carbendazim-treated leaves after 21 days. Compared with the zero day, the residues decreased by approximately 15, 57, and 95% after treatment with carbendazim for 3, 7, and 14 days, respectively (Table 1; p < 0.05).
Pesticide residues in jujube leaves
| 0 day | 3 day | 7 day | 14 day | 21 day | |
|---|---|---|---|---|---|
| Content | 565 ± 19a | 482 ± 11b | 245 ± 13c | 31 ± 4d | nd |
Carbendazim residue content (mg kg−1 dry weight) in jujube leaves following treatment (1,000 mg L−1) for 0, 3, 7, 14, and 21 days. Numbers with the same letter in superscript were not significantly different (n = 3; p < 0.05).
4 Discussion
Plant disease impairs crop yield and quality [29]. Therefore, pesticides play a key role in improving yield and quality of grains, vegetables, and fruits [30]. Pathogens and insects impact yield and quality of jujube fruit [13]. However, young leaves with high levels of bioactive components and few pesticide residues are required in the production of high-quality jujube leaf tea. In this study, we investigated the effects of harpin and carbendazim on disease development and antioxidant accumulation in young jujube leaves.
Figure 1a shows that harpin and carbenzine significantly decreased the DI in jujube leaves. Harpin slightly inhibited disease development in jujube plant during the first 7 days (Figure 1a; p < 0.05). Therefore, harpin fails to quickly reduce pathogen development in diseased plant compared to carbendazim. Moreover, carbendazim residues can be detected in jujube leaves after treatment for 14 days at the recommended concentration (Table 1). In contrast, harpin is a protein elicitor that is an eco-friendly biopesticide in plants without any hazardous residues [31]. Published data have shown that harpin induces disease resistance in muskmelon [32]. Therefore, harpin can be used as a preventive but not “therapeutic” pesticide in jujube plant.
Figure 1b shows that harpin application significantly promoted jujube leaf growth by enhancing biomass accumulation (p < 0.05). Similar results were reported in Arabidopsis [33]. However, carbendazim inhibited young leaf growth at the recommended concentration (Figure 1b; p < 0.05). H2O2 promotes plant leaf growth at low concentrations [34]. In contrast, high H2O2-induced oxidative stress inhibits plant growth [35,36]. Therefore, we compared the effects of harpin and carbendazim on H2O2 accumulation in young leaves. Compared to harpin, carbendazim induced a greater accumulation of H2O2 in jujube leaves (Figure 1c; p < 0.05). For example, H2O2 increased approximately by 2.3-fold in young leaves of jujube after treatment with carbendazim for 3 days over the control (Figure 1c; p < 0.05). In contrast, harpin enhanced H2O2 content by approximately 76% relative to the control (Figure 1c; p < 0.05). This result partly explains why carbendazim significantly inhibited young leaf growth at the recommended concentration [35,36]. Oxidative stress reduces antioxidant (e.g., glutathione) accumulation in plant [37].
We investigated the effects of harpin and carbendazim on vitamin C, GSH, total phenolics, and total antioxidant capacity in young leaves of jujube (Figure 2). Compared with the control, harpin promoted antioxidant accumulation and enhanced total antioxidant capacity in young leaves of jujube (Figure 2; p < 0.05). Studies have reported that harpin promotes the accumulation of phenolics and vitamin C in postharvest fruits [38,39]. However, carbendazim significantly decreased antioxidant accumulation and total antioxidant capacity in young leaves of jujube at the recommended concentration, especially during the first 7 days (Figure 2; p < 0.05). High concentrations of carbendazim decreases phenolic content in tobacco plant [16]. Carbendazim induced GSH accumulation over the control during the last stage (Figure 2b; p < 0.05). Low concentrations of carbendazim residues are degraded by GSH and GST in plants [40].
Phenolics are the major bioactive components in jujube leaves [4,9]. Therefore, we measured the activities of two key enzymes (PAL and PPO) involved in the metabolism of phenolics (Figure 3). Figure 3a shows that PAL activity increased with harpin but decreased with carbendazim. This result agrees with previous reports, which showed that high concentrations of carbendazim inhibit PAL activity in tobacco plant [16]. In contrast, harpin significantly enhances PAL activity in jujube plant [18]. Both harpin and carbendazim enhanced the PPO activity in the young leaves (Figure 3b; p < 0.05). Past studies have reported that harpin and carbendazim enhance PPO activity [16,18]. Therefore, the high antioxidant accumulation can be partly attributed to the high PAL activity and the low H2O2 accumulation induced by harpin in jujube leaves. In contrast, the carbendazim-induced overproduction of H2O2 and inhibition of PAL may contribute to the low antioxidant (e.g., phenolics) accumulation in jujube leaves. Why is the recommended concentration of carbendazim harmful to the young leaves of jujube? One plausible explanation can be partly attributed to the high sensitivity of young leaves to chemical pesticides, which may have an inadequate antioxidant defense system compared with mature leaves [41].
5 Conclusions
Some interesting conclusions can be drawn from our study findings. First, harpin contributes to a greater antioxidant accumulation in young leaves of jujube than carbendazim. Second, PAL plays a key role in regulating phenolic accumulation in jujube leaves. Third, harpin promotes biomass accumulation in young leaves of jujube. Finally, high residues of carbendazim can be detected in young leaves after treatment for 14 days. Comparatively, harpin improves jujube leaf quality. Our study findings provide a method for protecting young edible tissues of plant from pathogens and pests.
Acknowledgments
We are grateful to Dr Benliang Deng for his valuable advice.
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Funding information: This work was supported by a grant from the Opening Topic Foundation of “China-Loess Plateau Water Loss and Soil Erosion” Process and Control Key Laboratory in Ministry of Water Conservancy (grant number 190412).
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Author contributions: Shan Tian: conceptualisation, funding acquisition, writing, reviewing, and editing; Ying Chen, Shan Tian, and Qianjin Wang: investigation, project administration, validation, formal analysis, resources; Zhien Cai, Jiarui Zhang, and Zhilan Liu: investigation, reviewing, and editing; Yueyue Li and Xusheng Zhao: supervision. All authors have read and approved the final version of the manuscript.
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Conflict of interest: The authors declare no conflict of interest in the current study.
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Ethical approval: This manuscript does not report on or involve the use of any animal or human data or tissues, and therefore ethics problems are not applicable.
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Data availability statement: All data analyzed during the current study are included in this published article. The detailed data can be provided on reasonable request.
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