Nano-selenium enhances the antioxidant capacity, organic acids and cucurbitacin B in melon (Cucumis melo L.) plants

Ecotoxicology and Environmental Safety 241 (2022) 113777

Available online 20 June 2022

0147-6513/© 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).

Nano-selenium enhances the antioxidant capacity, organic acids and cucurbitacin B in melon (Cucumis melo L.) plants

Lu Kang

^a,b

, Yangliu Wu

^a

, Jingbang Zhang

^a

, Quanshun An

^a

, Chunran Zhou

^a

, Dong Li

^a

, Canping Pan

^a,*,1

aInnovation Center of Pesticide Research, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China

bInstitute of Agricultural Quality Standards and Testing Technology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

A R T I C L E I N F O Edited by Dr Muhammad Zia-ur-Rehman Keywords:

Nano-selenium Melon

Antioxidant activity Organic acids Cucurbitacin B Biological resistance

A B S T R A C T

Pesticides are widely used in melon production causing safety issues around the consumption of melon and increasing pathogen and insect tolerance to pesticides. This study investigated whether a nano-selenium (Nano- Se) spray treatment can improve resistance to biological stress in melon plants, reducing the need for pesticides, and how this mechanism is activated. To achieve this, we examine the ultrastructure and physio-biochemical responses of two melon cultivars after foliar spraying with Nano-Se. Nano-Se treatment reduced plastoglobu- lins in leaf mesophyll cells, thylakoid films were left intact, and compound starch granules increased. Nano-Se treatment also increased root mitochondria and left nucleoli intact. Nano-Se treatment enhanced ascorbate peroxidase, peroxidase, phenylalanine ammonia lyase, β-1,3-glucanase, chitinase activities and their mRNA levels in treated melon plants compared to control plants (without Nano-Se treatments). Exogenous application of Nano-Se improved fructose, glucose, galactitol, stachyose, lactic acid, tartaric acid, fumaric acid, malic acid and succinic acid in treated plants compared to control plants. In addition, Nano-Se treatment enhanced cucurbitacin B and up-regulated eight cucurbitacin B synthesis-related genes. We conclude that Nano-Se treat- ment of melon plants triggered antioxidant capacity, photosynthesis, organic acids, and up-regulated cucurbi- tacin B synthesis-related genes, which plays a comprehensive role in stress resistance in melon plants.

1. Introduction

Melon (Cucumis melo L.) is a vital economic crop in many countries for its fabulous flavor and exceptional nutrition value (Jing et al., 2018;

a). In recent years, there has been an increased focus on protected cultivation (modification of the natural environment to achieve opti- mum plant growth) for oriental melon production (Wang et al., 2021).

China is the biggest oriental melon producer and contributes about 51%

of the worldwide production (Wang et al., 2021). Two melon cultivars are commonly grown in China; Bai Ke Kou Qi (BKKQ), a susceptible cultivar, and Yu Meiren (YMR), a resistant cultivar to pathogens and insects (Kesh and Kaushik, 2021). Pathogens and insects such as pow- dery mildew (Glawe, 2008), fusarium wilt disease (Zhao et al., 2011), and aphids and whiteflies (Juarez et al., 2013; Schoeny et al., 2019) are commonly found during melon cultivation. These pests and diseases are usually controlled by pesticides, however there are growing consumer concerns about food safety and environmental pollution from increasing

pesticide residues (Carvalho, 2017; Schoeny et al., 2019), combined with pesticide resistance from long-term pesticide application (Bass et al., 2015; Xu et al., 2019). Therefore, there are many advantages to developing new environmentally-friendly methods to enhance resis- tance by controlling pathogens and insects in melon plants and reduce pesticide use.

Pathogen infections cause a rapid increase of reactive oxygen species (ROS) in plants (Barna et al., 2012; Yao et al., 2019). ROS is scavenged by enhancing the antioxidant enzyme activity of plants (catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxi- dase (POD)) (Mei et al., 2020). Selenium (Se) is also known to have an antioxidant effect (Han et al., 2020; Hu et al., 2020; Joshi et al., 2021) and sodium selenate (Na2SeO3) has been shown to increase APX activity in arsenic-stressed radishes (Hu et al., 2020). The zero-valent oxidation form (Se⁰) has also attracted interest due its high Nano-Se bioavailability to plants (Zhai et al., 2017). Bioaccumulated Se content in Se-treated plants is generally much higher than the original selenate application

* Corresponding author.

E-mail address: canpingp@cau.edu.cn (C. Pan).

1 Address: 2 Yuanmingyuan Western Road, Haidian District, Beijing 100193, China., Fax: +86–10-62733620.

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

https://doi.org/10.1016/j.ecoenv.2022.113777

Received 2 February 2022; Received in revised form 12 June 2022; Accepted 14 June 2022

content, and the Nano-Se form has an even higher bioaugmentation effect than its inorganic form (Neysanian et al., 2020). SOD and CAT induce a decline of harmful substances (Han et al., 2020; Shah et al., 2020), while foliar spraying of selenium has shown increased SOD, POD, CAT, and APX activities in wheat (Wu et al., 2020). Oligochitosan and nanosilica-oligochitosan increased chitinase (CHT) activity and the resistance of dragon fruit plants to brown spot disease (Tuan et al., 2018). In addition, it has been reported that phenylalanine ammonia- lyase (PAL), β-1,3-glucanase (GLU), SOD activities and mRNA levels are improved in tomato plants treated with Nano-Se (Joshi et al., 2021).

Currently, a number of biochemical studies have found that Se en- hances photosynthetic rates (Yin et al., 2019), malic acid, succinic acid (Zahedi et al., 2019), and citric acid (Zahedi et al., 2019; Xu et al., 2020).

The application of Na2SeO3 and sodium selenite (Na2SeO4) was found to promote the formation of chlorophyll in rice plants (Lidon et al., 2018).

It was also reported that spraying Nano-Se on sugarcane seedlings enhanced chlorophyll a and chlorophyll b (Elsheery et al., 2020). Se was also found to enhance photosynthesis of cowpea plants via photosyn- thetic pigments, and antioxidant capacity (Silva et al., 2018; Lanza et al., 2021). Foliar spraying of selenate triggered the transformation of chlo- rophyll a to chlorophyll b in cowpea and enhanced the overall chloro- phyll content (Silva et al., 2020). Furthermore, selenite was also found to increase chlorophyll a and chlorophyll b concentrations in wheat (Wu et al., 2020).

Cucurbitacin-B (Cuc-B) is the main component of cucurbitacin in melons (Hua et al., 2019). Melon bitterness is produced by Cuc-B, which is considered to be a defensive response by the plant to repel insects and herbivores (Hua et al., 2019). Cucurbitacin-B plays a significant role in bacterial resistance and insect attacks (Zhou et al., 2016). Yousaf et al.

(2018) also found that Cuc-B reduced the adult longevity and fecundity of melon aphids. Gene clusters (CmBi, Cm170, Cm180, Cm710, Cm890, Cm490, and acyltransferase (ACT)) were identified to be involved in Cuc-B biosynthesis in melon plants (Luo et al., 2020). However, as far as we know, the ability of Se treatments to increase bitterness and enhance resistance to insects in melon has not yet been reported.

Nano-Se can be considered as a biostimulant, insecticide, or fertilizer when applied through foliar spraying. Although Nano-Se protects melon plants from pathogens and insect infestations by enhancing resistance to biotic stress, the actual mechanism requires further exploration. In the present study, we hypothesize that Nano-Se treatments enhance anti- oxidant capacity, organic acids and up-regulates Cuc-B synthesis-related genes to enhance resistance to powdery mildew in melon plants.

Therefore, the purpose of current study was to (i) investigate the effect of Nano-Se treatments on the antioxidant capacity and photosynthesis of melon plants, and (ii) clarify the molecular mechanism of Nano-Se enhancing organic acids and cucurbitacin B. The outcome of this research will form a framework for future Nano-Se biostimulant or fer- tilizer method developments to improve melon plant resistance under biotic stress.

2. Materials and methods 2.1. Chemical reagents

Chromatography-grade methanol and acetonitrile were obtained from Fisher Scientific (Beijing, China). Chromatography-grade formic acid was purchased from Fuchen Chemical Reagent Co., LTD (Tianjin, China). Nano-Se was supplied by Guilin Jiqi Group Co. Ltd. (Guilin, China). Fructose (purity 99.5%), glucose (purity 99.8%), sucrose (purity 99.8%) and salicylic acid (purity 98.9%) standards were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Galactinol (purity 99.0%), sta- chyose (purity 98.0%) standards were purchased from J&K Scientific (Beijing, China). Lactic acid (purity 90.1%), tartaric acid (purity 99.7%), fumaric acid (purity 99.6%), malic acid (purity 98.9%), citric acid (purity 97.2%), succinic acid (purity 99.5%), and indole-3-acetic acid (purity 99.7%) standards were purchased from BePure (Beijing, China).

A Cucurbitacin-B (purity 98.4%) standard was obtained from ANPEL (Shanghai, China).

2.2. Growing conditions and melon seeding treatments

Two melon cultivars (BKKQ and YMR) were chosen as the experi- mental subjects. BKKQ and YMR are susceptible and resistant to pow- dery mildew, respectively. BKKQ and YMR seeds were purchased from Xinke Seed Co. Ltd. (Xinjiang, China) and Yifeng Seed and Seedling Co.

Ltd. (Xinjiang, China), respectively. Both cultivars were planted at the Institute of Crop Varieties and Resources, Xinjiang Academy of Agri- cultural Sciences in 2020 (Xinjiang, China). Experimental pot trials were conducted indoors under fluorescent lights at 35 ±3 ^◦C and a relative humidity of 50 ±5% to investigate the effect of Nano-Se treatment sprays on the two melon plant cultivars. BKKQ and YMR seeds were planted in 1.5 L flowerpots, with 10 seeds per pot. Three seedlings were selected from each pot after germination. Melon seedlings were watered once every other day. The experiment was conducted with four groups and eight replications per group. When the melon seedlings developed two normal-sized leaves, the leaves were sprayed with Nano-Se (0- control, 2.5, 5.0, and 10.0 mg⋅L⁻¹, respectively) once every 7 days for a period of 3 weeks. At the end of the 3 week treatment period, leaf tissue (approximately 1 mm²) without veins, and roots (approximately 2–3 mm) were collected for transmission electron microscopy (TEM) from each treatment. Leaves were also collected for biochemical and molec- ular analyses.

2.3. Transmission electron microscopy (TEM)

Collected leaves and root tips were immersed in 2.5% (v/v) glutar- aldehyde solution and then washed with a phosphate-buffer solution (pH 7.2–7.4) three times at room temperature for 30 min. The samples were post-fixed with 1% (v/v) OsO4 at room temperature for 2 h and were washed with a phosphate-buffer solution (pH 7.2–7.4) three times for 30 min. Dehydration was performed in a series of ethanol solutions (50%, 70%, 80%, 90% and 100%) where samples were sequentially soaked for 30 min in each solution, and then embedded into epoxy resin.

Thin sections (approximately 100 nm) were prepared using an ultra- micro slicer (EM UC6; Leica, Wetzlar, Germany) and were dyed by soaking in uranyl acetate and lead citrate solutions for about 30 min.

Images were acquired digitally using a transmission electron microscope (JEM1230; JEOL Ltd., Tokyo, Japan). Imaging software was RADIUS (version 2.0; Germany).

2.4. Gene expression analysis using quantitative real time PCR (RT- qPCR)

Total RNA was isolated from the melon leaf samples using a RNA prep pure plant plus kit (Tiangen Biotech Company, Ltd., Beijing, China) according to the manufacturer’s instructions. Total RNA was reverse- transcribed with Transcript® first-stand cDNA synthesis supermix (Transgen Biotech Company, Ltd., Beijing, China) in a total reaction volume of 20 μL containing 50 ng of template RNA, anchored oligo (dT)18, 10 μL of 2 ×TS reaction mix, transcript RT enzyme mix and RNase-free water. RT-qPCR program was conducted according to a previously published method (Li et al., 2020) with the primers listed in Table S1. Relative mRNA levels were calculated according to the 2^–ΔΔCt method (Liu et al., 2021; Zhang et al., 2022a) using actin as an internal control. Three biological replicates were performed for all analyses.

2.5. HPLC-MS/MS for the detection of Cuc-B, salicylic acid, and indole- 3-acetic acid

The HPLC mobile phase was composed by mixing methanol (A) and 5 mM ammonium acetate modified with 0.1% (v/v) formic acid (B). A 50 ×2.1 mm Acquity BEH C18 column (1.7 µm particle size; Waters,

Milford, MA, USA) was equilibrated with methanol for 30 min prior to the injection of 2 μL of sample. The gradient elution steps were as fol- lows: 10%− 90% B for 0–2 min and 90%− 10% B for 2–4 min, then held at 10% B for 1 min, 60% B for 5–7 min, and 90% B for 7–10 min. The flow rate was 0.3 mL/min throughout. Ionization and detection of Cuc-B were performed with electrospray ionization in positive mode under the MRM conditions listed in Table S2. Ionization and detection of salicylic acid and indole-3-acetic acid were performed with electrospray ioniza- tion in negative mode. The ion source temperature was 150 ^◦C and the dissolvent temperature 350 ^◦C. Flow rate of N2 gas and N2 conical gas were set at 650 L/h and 250 L/h, respectively.

2.6. Organic acids and soluble sugars

Fresh melon leaves were ground using a mortar and pestle, with additions of liquid nitrogen to achieve a fine, homogeneous powder.

Powdered samples (1 mg) were placed into plastic centrifuge tubes with 10.0 mL of deionized water, and spun at 1412×g for 2 min. Samples were further centrifuged at 10,000×g for 2 min. The supernatant was collected and filtered using a 0.45 µm microporous filter. Organic acids (lactic acid, tartaric acid, fumaric acid, malic acid, citric acid, and suc- cinic acid contents) were measured on the filtrate using a high perfor- mance liquid chromatography (HPLC) system coupled to an ultraviolet detector (Alliance e2695, Waters, USA). A Platisil ODS column (5 µm particle size and 250 ×4.6 mm) was used for chromatographic sepa- ration. Column temperature was kept at 40 ^◦C. The mobile phase, con- sisting of 0.1% formic acid solution (phase A) and methanol (phase B), was supplied at a flow rate of 2.0 mL/min. Gradient elution of the mobile phase was programmed as follows: 97.5% A for 0–10 min, from 2.5% to 100% B for 10–15 min, 97.5% A for 15–21 min. Injection volume was 20 μL and ultraviolet light wavelength was 210 nm. Soluble sugar contents were detected using a HPLC system equipped with refractive index de- tector. A 250 ×4.6 mm NUCLEODUR 100–5 NH2-RP column (5 µm particle size) was used for chromatographic separation. Column tem- perature was kept at 25 ^◦C. The mobile phase, consisting of acetonitrile (79% phase A) and water (21% phase B) was supplied at a flow rate of 2.0 mL/min.

2.7. Enzyme activity assay

Detection kits were used to determine SOD, CAT, POD, APX, PAL, GLU, CHT, acid invertase (AI), neutral invertase (NI), sucrose synthetase (SS), sucrose phosphate synthetase (SPS), and starch. The main steps were as follows. Melon leaves were ground using mortar containing liquid nitrogen. Obtained powder samples were placed in tubes with extraction solution. Then tubes were homogenized and were centrifuged at 800×g at 4 ^◦C for 10 min for SOD, CAT, and POD, at 10,000×g at 4 ^◦C for 10 min for APX, at 12,000×g at 4 ^◦C for 10 min for PAL and GLU, at 10,000×g at 4 ^◦C for 20 min for CHT, at 12,000×g at 4 ^◦C for 10 min for AI and NI, at 8000×g at 4 ^◦C for 10 min for SS and SPS, and 3000×g at 4

◦C for 10 min for starch. Obtained supernatant was treated with kits for SOD (Kit number: A001–3), CAT (Kit number: A007–1–1), POD (Kit number: A084–3), and starch (Kit number: A148–1–1) using a multiscan spectrum microplate spectrophotometer (Bio Tek Instruments Inc, USA).

APX (Kit number: A123–1–1), PAL (Kit number: A137–1–1), GLU (Kit number: BC0830), CHT (Kit number: A139–1–1), AI (Kit number:

BC0565), NI (Kit number: BC0575), SS (Kit number: BC0585), SPS (Kit number: BC0565) were determined using a UV–vis spectrophotometer (Shanghai Shunyu Hengping Instruments Co., Ltd, China) with kits following the manufacturer’s instructions. The kits for SOD, CAT, POD, APX, PAL, CHT, and starch were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The kits for GLU, AI, NI, SS, and SPS were purchased from Solarbio Science & Technology Company Ltd (Beijing, China). Obtained liquid was used to measure absorbance at 450 nm for SOD, at 405 nm for CAT, at 420 nm for POD, at 290 nm for APX and PAL, at 540 nm for GLU, CHT, AI, and NI, at 480 nm for SS and

SPS, and at 620 nm for starch.

2.8. Photosynthetic pigments

Photosynthetic pigments assay. Leaf tissue (1.0 g) was extracted with 10.0 mL of precooled acetone at 4 ^◦C (80% v-v). The centrifuge tubes were placed in the dark for 24 h at 4 ^◦C before testing. Obtained su- pernatant was centrifuged at 10,000 ×g for 10 min. The concentrations of chlorophyll a (C_a), chlorophyll b (C_b), and total chlorophyll (C_a+b) were measured using a UV-Vis spectrophotometer (Model 752, China) (Cabral Gouveia et al., 2020). The calculation formulas of photosyn- thetic pigments (Lichtenthaler, 1987) are as follows:

C_a =12.25 A₆₆₃ – 2.79 A647

Cb =21.50 A647 – 5.10 A663

C_a+b =7.15 A₆₆₃ +18.71 A647

2.9. Statistical analysis

Two-way Analysis of variance (ANOVA) was performed using a Generalized Linear Model in SPSS (version 26 SPSS Inc., Chicago, IL) to evaluate the effect of different factors on measured parameters. ANOVA was performed to test the significance difference among cultivars, Nano- Se concentrations and their interactions. Paired multiple comparison Student-Neuman-Keuls tests were used to measure significant differ- ences (p < 0.05). Different lowercase letters indicate significance at p <

0.05 between different groups. All data is based on 3 biological repli- cates and statistical data was analyzed using Graph Pad Prism software (version 8.02; San Diego, CA).

3. Results

3.1. Ultrastructure of mesophyll cells in melon leaves after Nano-Se treatment

Ultrastructure of mesophyll cells from the control and Nano-Se treatment groups of the two melon cultivars are shown in Fig. 1. The ultrastructure of leaf mesophyll cells from the control group shows plastoglobulin, thylakoid membranes, starch granules, and cell walls (Fig. 1A, E). After Nano-Se treatment, the number of plastoglobulin of mesophyll cells decreased in BKKQ (Fig. 1B) and YMR (Fig. 1F) compared to the control group. Thylakoid membranes (Fig. 1D, H) were left intact, compound SG (Fig. 1D, F) increased, and cell walls thickened (Fig. 1C, G).

3.2. Ultrastructure of root cells of melons treated with Nano-Se

Root cell ultrastructure of the two melon cultivars for the control and Nano-Se treatment groups are displayed in Fig. 2. The ultrastructure of the melon roots in the control and Nano-Se treated group clearly shows the nuclear membranes, vacuole, mitochondria, cell walls, and nucleoli (Fig. 2E, F). In the control group (Fig. 2A, E), nuclear membranes were blurred and vacuole was arranged in a regular pattern (Fig. 2E). In the Nano-Se treated group, mitochondria increased (Fig. 2F), cell walls thickened (Fig. 2B, C), and nucleoli was intact (Fig. 2F). In addition, Nano-Se particles (Fig. 2G) and dictyosome (Fig. 2H) were found after 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments, respectively.

3.3. Antioxidant enzymes and genes

The antioxidant activities of four enzymes (SOD, CAT, APX, and POD), and mRNA levels of three antioxidant genes (SOD, CAT, and APX) were compared between the control group and Nano-Se treated melon

leaves of both cultivars (Fig. 3). SOD activity (Fig. 3A1) and SOD mRNA level (Fig. 3A2) increased significantly (p<0.05) in the three Nano-Se treatments compared to the control group in BKKQ. SOD mRNA levels in the 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treated samples increased signifi- cantly (p<0.05) compared to the 2.5 mg⋅L⁻¹ Nano-Se treated samples.

A significant increase (p<0.05) of SOD mRNA level was found for all concentrations of Nano-Se treatments in YMR. CAT activity (Fig. 3B1) in the three Nano-Se treatment groups and CAT mRNA level (Fig. 3B2) in the 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatment groups increased signifi- cantly (p<0.05) compared to the control group in BKKQ. CAT mRNA

level in the 5.0 mg⋅L⁻¹ Nano-Se treatment group had the highest sig- nificance (p<0.05) among all treatment groups in BKKQ. CAT mRNA levels in the 2.5 and 10.0 mg⋅L⁻¹ Nano-Se treated groups increased significantly (p<0.05) compared to the control group in YMR. APX activity (Fig. 3C1) and APX mRNA levels (Fig. 3C2) were up-regulated significantly (p<0.05) after all three Nano-Se treatments with 2.5, 5.0, and 10.0 mg⋅L⁻¹ in both BKKQ and YMR except for APX mRNA levels for the 2.5 mg⋅L⁻¹ Nano-Se treated group and the control group for YMR. POD activity (Fig. 3D1) showed a significant increase (p<0.05) for all three Nano-Se treatment concentrations, and POD Fig. 1. TEM micrographs of mesophyll cells of BKKQ and YMR melon leaves. A: BKKQ control group; B, C, and D: BKKQ treated with Nano-Se at 2.5, 5.0, and 10.0 mg⋅L⁻¹, respectively; E: YMR control group; F, G, and H: YMR treated with Nano-Se at 2.5, 5.0, and 10.0 mg⋅L⁻¹, respectively. Key: SG: starch granules; Thy:

thylakoid films; PG: plastoglobule; CW: cell wall.

activity in the 5.0 mg⋅L⁻¹ Nano-Se group had the highest significance (p<0.05) among the treatment groups in YMR.

3.4. The mRNA levels of PAL, GLU, CHT and activities

The activities of PAL (Fig. 4A1), GLU (Fig. 4B1), and CHT (Fig. 4C1), as well as and mRNA levels of PAL (Fig. 4A2), GLU (Fig. 4B2), CHT1 (Fig. 4C2), CHT2 (Fig. 4C3) after Nano-Se treatment were investigated in BKKQ and YMR cultivars. BKKQ with 5.0 mg⋅L⁻¹ Nano-Se treatment significantly increased PAL activity (p<0.05) compared with the con- trol group (Fig. 4A1), and 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments increased PAL mRNA levels significantly (p<0.05) (Fig. 4 A2). For

YMR, all the three concentrations of Nano-Se treatments increased PAL activity and PAL mRNA level significantly (p<0.05). Nano-Se treat- ments of 5.0 and 10.0 mg⋅L⁻¹ significantly increased GLU activity (p<0.05) (Fig. 4B1), GLU mRNA levels from the three Nano-Se treat- ment concentrations (Fig. 4B2) increased significantly (p<0.05) rela- tive to the control groups for BKKQ and YMR, respectively. Compared to the control group, the three Nano-Se treatments significantly increased CHT activity (Fig. 4C1), CHT1 mRNA levels (Fig. 4C2), and CHT2 mRNA levels (Fig. 4C3) in both melon cultivars (p<0.05). Moreover, the 5.0 mg⋅L⁻¹ Nano-Se treatment had the highest effect of all three Nano-Se treatments.

Fig. 2.TEM micrographs of BKKQ and YMR melon root tip cells. A: BKKQ control group; B, C, and D: BKKQ treated with Nano-Se at 2.5, 5.0, and 10.0 mg⋅L⁻¹, respectively; E: YMR control group; F, G, and H: YMR treated with Nano-Se at 2.5, 5.0, and 10.0 mg⋅L⁻¹, respectively. Key: M: mitochondria; NM: nuclear membrane;

CW: cell wall; Va:vacuole; Nue: nucleoli; D: dictyosome.

3.5. Photosynthetic products, chlorophyll, sucrose, and sucrose- metabolized enzyme activities

Fructose, glucose, galactinol, stachyose, and starch (Table 1) were measured in melon plants after foliar treatments of 2.5, 5.0, and

10.0 mg⋅L⁻¹ Nano-Se. The results of ANOVA revealed significant effects of foliar Nano-Se treatments (Se), two cultivars (C, BKKQ and YMR) and their interaction (C ×Se) on photosynthetic products, chlorophyll, su- crose, and sucrose-metabolized enzyme activities (Table 2). The three Nano-Se treatments and the 2.5 mg⋅L⁻¹ Nano-Se treatment significantly Fig. 3.Antioxidant enzymes activity and antioxidant genes expression from BKKQ and YMR. A1: SOD activity, A2: SOD mRNA level, B1: CAT activity, B2: CAT mRNA level, C1: APX activity, C2: APX mRNA level, D1: POD activity. Different letters indicate a significant difference between treatments (p<0.05) and same letters indicate an insignificant difference between treatments (p≥0.05). The error bars represent standard deviations (n=3). CK, 2.5, 5.0, and 10.0 represent the control group and different spray concentrations of 2.5, 5.0, and 10.0 mg⋅L⁻¹ Nano-Se, respectively. (*) indicates that the cultivar effect (C), Nano-Se treatments (Se), and C ×Se are significant at *p<0.05 and **p<0.01.

increased (p<0.05) fructose, glucose levels for the BKKQ and YMR cultivars, respectively, compared to the control group. All three Nano-Se treatment groups significantly increased (p<0.05) galactinol and sta- chyose in both melon cultivars compared to the control group. All three Nano-Se treatment groups significantly increased (p<0.05) starch in BKKQ. Nano-Se treatments of 5.0 and 10.0 mg⋅L⁻¹ significantly increased (p<0.05) chlorophyll a, chlorophyll b, and total chlorophyll

in BKKQ. Treatments of 5.0 and 10.0 mg⋅L⁻¹ Nano-Se also significantly increased (p<0.05) chlorophyll a, while the three Nano-Se treatments significantly increased (p<0.05) chlorophyll b and total chlorophyll in YMR. The three Nano-Se treatments significantly increased (p<0.05) sucrose and four sucrose-related metabolic enzyme activities (SS, SPS, AI, and NI) in both melon cultivars (Table S3, S4), but not SS activity in YMR. Treatments of 5.0 and 10.0 mg⋅L⁻¹ Nano-Se significantly Fig. 4. PAL, GLU, CHT activities and PAL, GLU, CHT1, CHT2 mRNA levels in BKKQ and YMR. A1: PAL activity, A2: PAL mRNA level, B1: GLU activity, B2: GLU mRNA level, C1: CHT activity, C2: CHT1 mRNA level, C3: CHT2 mRNA level. The different letters above each bar and CK, 2.5, 5.0, and 10.0 are as in Fig. 3. (*) indicates that the cultivar effect (C), Nano-Se treatments (Se), and C ×Se are significant at *p<0.05 and * *p<0.01.

increased (p<0.05) SS activity in YMR.

3.6. Salicylic acid, indole-3-acetic acid, and organic acids

Salicylic acid, indole-3-acetic acid, and organic acid levels are shown in Table 1 and are related to melon pathogen resistance. The ANOVA results revealed significant differences for foliar Nano-Se treatments (Se), two cultivars (C, BKKQ and YMR) and their interaction (C ×Se) with salicylic acid, indole-3-acetic acid, and organic acids (Table 2). The three Nano-Se treatments significantly increased (p<0.05) salicylic acid and indole-3-acetic acid in BKKQ and YMR, respectively. The 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) lactic acid in BKKQ, and all three Nano-Se treatments significantly increased (p<0.05) lactic acid in YMR. Conversely, tartaric acid in both BKKQ and YMR was reduced after spraying with Nano-Se. The three Nano-Se treatments significantly increased (p<0.05) fumaric acid and succinic acid in both melon cultivars. The 2.5 and 5.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) malic acid in BKKQ, and all three Nano-Se treatments significantly increased (p<0.05) malic

acid in YMR. The three Nano-Se treatment groups significantly increased (p<0.05) citric acid in BKKQ, and 10.0 mg⋅L⁻¹ Nano-Se significantly increased (p<0.05) citric acid in YMR.

3.7. Cuc-B and Cuc-B biosynthesis-related genes

Cuc-B and eight Cuc-B (CmBt, CmBi, Cm170, Cm180, Cm490, Cm710, Cm890, and ACT) biosynthesis-related gene expressions related to melon insect resistance are shown in Fig. 5. The three Nano-Se concentrations significantly increased (p<0.05) Cuc-B (Fig. 5A1) in BKKQ and YMR.

The 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) CmBt mRNA level (Fig. 5A2) in both cultivars. The 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) CmBi mRNA levels in BKKQ, while the three Nano-Se treatment groups significantly increased (p<0.05) transcription levels of CmBi in YMR (Fig. 5A3). The three Nano-Se treatment groups significantly increased (p<0.05) Cm170 mRNA levels in BKKQ, while the 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) Cm170 mRNA levels in YMR (Fig. 5A4). The 10.0 mg⋅L⁻¹ Nano-Se treatment signifi- cantly increased (p<0.05) transcription levels of Cm180 in BKKQ, and the three Nano-Se concentrations significantly increased (p<0.05) Cm180 mRNA levels in YMR (Fig. 5A5). The three Nano-Se treatment groups significantly increased (p<0.05) Cm490 mRNA levels in YMR (Fig. 5A6). The three Nano-Se treatment groups significantly increased (p<0.05) transcription levels of Cm710 in BKKQ, while the 10.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) Cm710 mRNA levels in YMR (Fig. 5A7). The three Nano-Se treatment groups significantly increased (p<0.05) Cm890 mRNA levels in BKKQ, and the 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) Cm890 mRNA levels in YMR melon plants (Fig. 5A8). The 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments significantly increased (p<0.05) ACT mRNA levels in BKKQ, while three Nano-Se treatment groups significantly increased (p<0.05) ACT transcription levels in YMR (Fig. 5A9).

4. Discussion

Selenite, selenate, and Nano-Se are three main Se forms that occur (Li et al., 2020). During leaf senescence, the thylakoid membrane de- teriorates and generates abundant fatty acids (Liu, 2016), which are a temporary reserve for plastioglobulin, resulting in a dramatic increase of plastioglobulin within the leaf (Fan et al., 2013). All Nano-Se treatments Table 1

Effects of Nano-Se foliar sprays on photosynthetic pigments, organic acids, and plant hormones in two melon cultivars. Presented data are the mean of three replicates with standard error. Different letters in the same line represent a significant difference between treatments (p<0.05) and the same letters in the same line represent no significant difference between treatments (p≥0.05).

Items BKKQ (ug⋅g⁻¹) YMR (ug⋅g⁻¹)

CK 2.5 mg⋅L⁻¹ 5.0 mg⋅L⁻¹ 10.0 mg⋅L⁻¹ CK 2.5 mg⋅L⁻¹ 5.0 mg⋅L⁻¹ 10.0 mg⋅L⁻¹

Fructose 58±2 d 72±2c 88±3 a 82±2 b 68±6 b 87±2 a 74±3 b 68±7 b

Glucose 92±3c 135±2 b 163±3 a 174±10 a 278±4 b 393±16 a 222±11c 239±9c

Galactinol 349±4 d 362±8c 426±5 b 436±3 a 292±3 d 349±8c 362±5 b 376±5 a

Stachyose 361±21 d 404±4c 461±11 b 511±7 a 407±4c 449±2 b 517±11 a 530±12 a

Starch 10,503±288c 12,288±222 b 13,350±150 a 12,766±172 b 15,766±153 b 15,978±82 b 16,289±339 b 18,246±668 a

Chlorophyll a 245±11 b 254±5 b 334±19 a 362±24 a 269±64 b 309±1 b 327±2 a 345±24 a

Chlorophyll b 276±13 b 314±26 ab 348±27 a 357±20 a 270±19 b 363±5 a 369±8 a 332±24 a

Total chlorophyll 521±4 b 568±30 b 682±30 a 719±28 a 539±46 b 672±5 a 697±10 a 677±30 a

Sucrose 128±9 d 153±4c 164±4 b 219±2 a 154±5c 186±3 b 199±3 b 279±15 a

Salicylic acid 0.243±0.020

b 0.300±0.009

a 0.304±0.028 a 0.313±0.010 a 0.234±0.003

b 0.420±0.023

a 0.386±0.012

a 0.406±0.019 a

Indole-3-acetic

acid 0.479±0.029

d 0.544±0.030c 0.634±0.029

b 0.951±0.010 a 0.510±0.014c 0.821±0.036

a 0.808±0.010

a 0.611±0.025 b

Lactic acid 2517±256c 2871±89c 3798±173 b 8930±549 a 3373±284c 3875±82 b 3889±174 b 6253±106 a

Tartaric acid 485±12 d 558±34c 730±12 b 919±13 a 442±31c 451±30c 698±27 b 866±11 a

Fumaric acid 36±4c 56±5 b 114±12 a 100±10 a 37±8c 69±3 b 96±1 a 92±5 a

Succinic acid 175±5 d 192±7c 225±6 b 321±6 a 155±13c 236±14 b 286±15 a 306±15 a

Malic acid 3871±50 b 4126±115 a 4208±51 a 3051±6c 1809±79c 2258±50 b 2210±53 b 4035±154 a

Citric acid 950±1 d 1392±47c 1618±62 b 2051±63 a 1192±37 b 1085±7 b 1159±127 b 1899±72 a

Table 2

Probability values of different foliar Nano-Se treatments (Se), two cultivars (C, BKKQ and YMR) and their interactions (C ×Se) on photosynthetic pigments, organic acids, and plant hormones.

Variables

Traits Cultivars (C) Foliar Nano-Se treatments (Se) C ×Se

Fructose 0.726 ＜0.01 ＜0.01

Glucose ＜0.01 ＜0.01 ＜0.01

Galactinol ＜0.01 ＜0.01 ＜0.01

Stachyose ＜0.01 ＜0.01 ＜0.05

Starch ＜0.01 ＜0.01 ＜0.01

Chlorophyll a 0.232 ＜0.01 0.129

Chlorophyll b 0.241 ＜0.01 ＜0.05

Total chlorophyll ＜0.05 ＜0.01 ＜0.01

Sucrose ＜0.01 ＜0.01 ＜0.01

Salicylic acid ＜0.01 ＜0.01 ＜0.01

Indole-3-acetic acid ＜0.01 ＜0.01 ＜0.01

Lactic acid 0.105 ＜0.01 ＜0.01

Tartaric acid ＜0.01 ＜0.01 0.059

Fumaric acid 0.312 ＜0.01 ＜0.01

Succinic acid ＜0.01 ＜0.01 ＜0.01

Malic acid ＜0.01 ＜0.01 ＜0.01

Citric acid ＜0.01 ＜0.01 ＜0.01

were observed to reduce plastioglobulin and delay the onset of senes- cence in YMR leaf cells (Fig. 1 F, G, H). Na2SeO3 treatments reduced damage to root cells (Zhao et al., 2019) while thylakoid membranes were observed in Nano-Se treated samples (Zsiros et al., 2019). Previous research reported that mesophyll cells in wheat leaves developed thylakoid membranes after Na₂SeO₄ treatment (Semenova et al., 2017) suggesting that Se improves photosynthetic efficiency. Light energy is collected and absorbed onto the thylakoid membrane, PSI and PSII, as the electron transport chain is among the most significant transfer pathways in thylakoid membranes (Liu et al., 2018). Two reports also support our findings and found that mesophyll cells and leaves devel- oped thylakoid membranes after applied with Na2SeO4 in wheat (Semenova et al., 2017) and that thylakoid membranes were observed in Nicotiana tabacum after Nano-Se supplements were applied (Zsiros et al., 2019). Our results show that Nano-Se treatments delay the onset of mesophyll cells in melon leaves, enhance photosynthesis, and resist the invasion of pathogens by reducing plastioglobulin numbers and increasing electron transport chain efficiency.

The presence of Nano-Se in root cells (Fig. 2 G) may be caused by the following reasons, Nano-Se may transfer to root cells as part of a growth and metabolic effect in melon plants. Foliar sprayed Nano-Se is also transferred to the soil and may be taken up by root cells. Alternatively, Nano-Se may translocate from leaf mesophyll cells through the stalks to the root cells (Valdez Barillas et al., 2012). However, this mechanism is yet to be confirmed. The applications of 5 and 10 mg⋅L⁻¹ Nano-Se foliar sprays could slow down root senescence, while previous studies have also suggested that Na2SeO3 promotes cell wall integrity of Chinese

cabbage (Zhao et al., 2019). Stomata opening allows carbon dioxide to flow into leaves and evapotranspiration to occur into the surrounding environment (Hetherington and Woodward, 2003). Mitochondrial metabolism is associated with stomatal opening (Medeiros et al., 2018).

The cell wall is a protective barrier to prevent pathogen infection (Lahlali et al., 2016). In this study, Nano-Se treatments increased the quantity of mitochondria (Fig. 2F) and thickened cell walls (Fig. 2B, C).

These results indicate that Nano-Se regulates stomatal opening through mitochondrial metabolism and increases the thickness of cell walls to increase photosynthesis and defense mechanisms against pathogen infections.

Pathogen infections cause a rapid increase in reactive oxygen species (ROS) in plants (Barna et al., 2012; Yao et al., 2019). Extensive ROS destroy the plant’s metabolism, which results in oxidative damage to lipids, proteins, and nucleic acids (JM, 2000). ROS is scavenged by enhancing the antioxidant enzyme activity of a plant (CAT, SOD, APX, and POD) (Mei et al., 2020). Exogenous Nano-Se applications have been reported to increase CAT, POD, and PAL activities (Neysanian et al., 2020). Similar results were found for rapeseed antioxidant enzyme ac- tivities (SOD, POD, and APX) and their transcript levels (Ulhassan et al., 2018), and for SOD, POD activities of strawberry after foliar application of Nano-Se (Zahedi et al., 2019). Higher transcript levels of CAT occurred with Se treatments (Luo et al., 2021). Results from the current study indicate that Nano-Se treatments increased SOD, CAT activities, as well as SOD and CAT mRNA levels in BKKQ melons. In YMR melon cultivars, SOD mRNA levels were increased. In addition, Nano-Se ap- plications increased APX and POD activities and transcript levels of APX Fig. 5. Cuc-B and Cuc-B synthesis-related genes expression in BKKQ and YMR. A1: Cuc-B content, A2: CmBt mRNA level, A3: CmBi mRNA level, A4: Cm170 mRNA level, A5: Cm180, mRNA level, A6: Cm490 mRNA level, A7: Cm710 mRNA level, A8: Cm890 mRNA level, A9: ACT mRNA level. The different letters above each bar and CK, 2.5, 5.0, and 10.0 are as in Fig. 3. (*) indicates that the cultivar effect (C), Nano-Se treatments (Se), and C ×Se are significant at *p<0.05 and * *p<0.01.

in both cultivars. Our findings suggest that Nano-Se treatments improve antioxidant capacity of BKKQ and YMR melon plants. Nano-Se foliar application improves resistance to biotic stress in melon plants by enhancing antioxidant capacity and scavenging ROS, which plays a significant role to increase biological stress tolerance (Barna et al., 2012).

PAL and GLU activities neutralize pathogen invasions by producing phytoalexins, and hydrolysis of beta glucans in cell walls (Joshi et al., 2021). The substrate of chitinase is chitin, which is a familiar constituent in pathogen cell walls and insect cuticles, and results in pathogen de- fense by preventing spore germination, germ tube elongation and decomposing mycelial tips (Zhu et al., 2022). β-1,3-glucanase catalyzes the hydrolytic cleavage of 1,3-b-D-glycosidic bonds in b-1,3-glucan, a component of mycelial cell walls (Wang et al., 2003). Nano-Se treatment showed a significant regulatory effect on tomato late blight pathogens, while transcription levels of PAL and GLU also supported reinforcement of the Nano-Se biochemical defense system (Joshi et al., 2021). CHT and GLU mRNA levels increased the pathogen defense response of peach trees (Ji et al., 2021). Compared to the control group, PAL, GLU, CHT activities and PAL, GLU, CHT1, CHT2 mRNA levels of the two melon cultivars increased in response to the 5.0 mg⋅L⁻¹ Nano-Se treatment, implying that Nano-Se applications increased pathogen resistance in melon plants. Although expression levels of CHT1 and CHT2 increased significantly after the 5.0 mg⋅L⁻¹ Nano-Se treatment, expression levels of CHT1 and CHT2 decreased when the Nano-Se concentration increased to 10.0 mg⋅L⁻¹, indicating that pathogen resistance was higher after the 5.0 mg⋅L⁻¹ Nano-Se treatment than the 10.0 mg⋅L⁻¹ Nano-Se treatment of melon plants.

Photosynthetic pigments chlorophyll a and chlorophyll b levels directly relate to photosynthesis rate, and can absorb light energy during photosynthesis (Zhang et al., 2022). As a significant component of chlorophyll, chlorophyll a is the major PSII light collection pigment that assimilates and converts light energy while assimilating long-wave light, while chlorophyll b mainly absorbs short-wave light (Ji et al., 2022).

Sodium selenate treatment increased rice total chlorophyll and soluble sugar (Mostofa et al., 2020). Spraying sugarcane seedlings with nano particles had been reported to enhance chlorophyll and reduce the ef- fects of chilling (lower environmental temperature) stress in sugarcane (Elsheery et al., 2020). The 5.0 and 10.0 mg⋅L⁻¹ Nano-Se treatments increased total chlorophyll in BKKQ, and all three Nano-Se treatment groups increased total chlorophyll in YMR. Nano-Se increased photo- synthesis via PSII, the absorption of short-wave light and influenced carbohydrate metabolism. Se may also influence glycine metabolism to improve carbohydrate metabolism (Silva et al., 2018). SS disintegrates sucrose in cytoplasm and supplies crude materials and precursors for starch and cellulose synthesis (Lan et al., 2020). SPS catalyzes fructose-6-phosphate and UDPG to synthesize sucrose-6-phosphates, which supplies a substrate for sucrose phosphatase to catalyze sucrose synthesis (Hashida et al., 2016). Increased activities of AI and NI are beneficial for sucrose decomposition and enhance glucose and fructose levels (Ji et al., 2022). Spray application of Se dramatically enhanced total soluble sugars (Nawaz et al., 2015). Nano-Se treatments increased sucrose and metabolic enzyme activities (SS, SPS, AI, and NI) (Table S3) in the two cultivars. Sucrose is the main product of photosynthesis that drives plant growth, signal transduction, and adjustment to different stressors (Mostofa et al., 2017). Na2SeO4 supplements influenced starch and sucrose in an active way (Cipriano et al., 2022). Lidon et al. (2018) found that applications of Na2SeO3 and Na2SeO4 increased the contents of sugar (including sucrose, glucose, and fructose) in rice grains.

Nano-Se treatments were also shown to improve glucose, fructose, and sucrose in strawberry fruits subjected to saline stress (Zahedi et al., 2019). Our results suggest that applications of Nano-Se increases su- crose, fructose, glucose, galactinol, and stachyose in melon leaves (Table 1). The main carbohydrate transport in melon phloem was sta- chyose, with a small amount of sucrose. Nano-Se treatments of 5.0 and 10.0 mg⋅L⁻¹ were most effective to regulate fructose, glucose,

galactinol, and stachyose. Nano-Se application had the best effect on sucrose, SS, SPS, AI, and NI at a treatment level of 5.0 mg⋅L⁻¹.

Organic acids are significant intermediate products of the central metabolism in plants and contribute to tricarboxylic acid cycles and photosynthesis metabolic pathways (Medeiros et al., 2021). Organic acids are regarded as driving forces of electron and proton transfer, supplying energy via proton gradients, which are conducive to redox transport among cellular compartments (Medeiros et al., 2021). In addition, spraying citric acid can enhance the thickness of plant leaves, palisade and spongy thickness (Mekawi et al., 2019). Salicylic acid plays a major role in plant defense and immune responses (Zhou and Zhang, 2020). Selenite treatments altered plant hormone signal pathways in tea plants (Cao et al., 2018). Salicylic acid signal transduction triggers de- fense nutritional pathogens (Barna et al., 2012). Na2SeO3 supplement increased the endogenous salicylic acid in tomato leaves under drought stress conditions (Fan et al., 2022). Nano-Se supplements showed an accumulation of indole-3-acetic acid, and improved salinity tolerance in strawberry plants (Zahedi et al., 2019). Salicylic acid was found to induce systemic acquired resistance in plants, while jasmonic acid and ethylene contributed to plant induced systemic resistance (Zhao et al., 2020). In this current study, Nano-Se treatments increased salicylic acid in melon plants. We speculate that Nano-Se treatments may increase resistance to biotic stress by systemic acquired resistance, however, this hypothesis needs further verification.

Our findings suggest that Nano-Se treatments increased salicylic acid and indole-3-acetic acid in melon plants compared to the control group, and concurred with research that showed Nano-Se treatments promoted tomato and eggplant growth (Gudkov et al., 2020). Recent research indicates that fumaric acid and malic acid contents of banana root ex- udates play a crucial role in attracting, initiating and promoting rhizo- bacteria colonization during plant growth (Yuan et al., 2015). Organic acids also contribute to the pathogen resistance of faba beans (Lv et al., 2020), and tartaric acid and malic acid have a significant inhibitory effect on Fusarium wilt (Lv et al., 2021). It was also shown that malic acid, citric acid, and succinic acid contents in strawberry fruits increased with exogenous Nano-Se application after saline stress (Zahedi et al., 2019). Organic acids represent the plant’s metabolic state, and reflect the ability of plants to survive and maintain basal metabolism (Samanta et al., 2020). Foliar spraying with Nano-Se increased organic acids, particularly lactic acid, malic acid, and citric acid, and further proved that Nano-Se treatments can improve pathogen resistance in melon plants. Nano-Se treatments improved biotic stress resistance by pro- moting tricarboxylic acid cycle, electron and proton transfer, and increasing thickness of leaf cell wall.

Cuc-B reduced adult longevity and fecundity of melon aphids (Yousaf et al., 2018). Cuc-B is the main component of cucurbitacin in melons (Hua et al., 2019). Bitterness is produced by Cuc-B, which is considered to be a defensive plant response to insects and herbivores (Hua et al., 2019). Cucurbitacins consist of a group of tetracyclic triterpenoids (Kim et al., 2020). It has been reported that Cuc-B has a biosynthetic pathway.

Cuc-B accumulation in melon fruit is regulated by the transcription factor CmBt, which regulates several key biosynthetic genes, including the final acetylation step for cucurbitacin D acetylation by acyl- transferase (ACT) to generate Cuc-B (Zhou et al., 2016). Nano-Se application was noted to increase Cuc-B and Cuc-B bio- synthesis-related mRNA levels. Cuc-B increased by 74%, when bitter melon was treated with carbon-based nanoparticles, fullerol C₆₀(OH)₂₀ (Kole et al., 2013). Transcription factors CmBt may increase Cuc-B (Luo et al., 2020). Our findings reveal that Cuc-B in BKKQ was higher than in YMR plants under control conditions, while Nano-Se application increased Cuc-B in BKKQ and YMR. Overall, Nano-Se applications increased insect resistance by increasing Cuc-B and up-regulating cucurbitacin B synthesis-related genes.

5. Conclusion

Foliar application of Nano-Se was found to improve powdery mildew resistance in melon plants. Nano-Se improved antioxidant capacity in both melon cultivars and Nano-Se foliar treatment of 5.0 mg⋅L⁻¹ had the best pathogen resistance effect. In conclusion, the present study found that Nano-Se treatment increases biotic stress resistance by enhancing antioxidant capacity, photosynthesis and preserving a steady-state bal- ance between scavenging and production of reactive oxygen species.

Nano-Se acts as a biostimulant, insecticide, and fertilizer to enhance resistance to biotic stress in melon plants.

CRediT authorship contribution statement

Lu Kang: Writing − original draft, Methodology, Data curation, Investigation. Yangliu Wu: Conceptualization, Writing − review &

editing. Jingbang Zhang: Investigation, Validation. Quanshun An:

Investigation, Validation. Chunran Zhou: Resources, Validation. Dong Li: Validation. Canping Pan: Conceptualization, Methodology, Writing

− review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Agricultural Science and Technology Innovation Platform Capacity Improvement Construction Project of Xinjiang Academy of Agricultural Sciences, China (XNYPT 2021–003);

Tianshan Talent Plan of Xinjiang Uygur Autonomous Region Phase III, China (2021–2023) and Key Cultivation Project of Scientific and Tech- nological Innovation of Xinjiang Academy of Agricultural Sciences, China (xjkcpy-2022002).

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2022.113777.

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