Introduction

Muskmelon (Cucumis melo L.) is one of the ten most popular fruits in the world and is a very important horticultural and cash crop worldwide (Song et al., 2022). In recent years, facility cultivation has become the main mode of muskmelon production, the soil salinization has been exacerbated as a result of high multiple cropping index values, crop rotation difficulties, and the excessive application of chemical fertilizer. High concentrations of salt in soil can cause salt damage or induce salt stress and lead to a series of physiological and biochemical changes in plants, such as altered permeability of the plasma membrane and increased osmotic stress, oxidative stress, and ion toxicity, which can diminish the photosynthetic capacity and drive nutrient absorption imbalances. Together, these changes can slow plant growth and development rates and reduce biomass or cause death (Liu et al., 2019). Therefore, soil salinization has become one of the main obstacles hindering sustainable melon production. The application of exogenous additives is widely considered as an efficient, inexpensive, and environmentally friendly means of solving the problem of secondary soil salinization.

Silicon is a beneficial element that can not only improve plant growth and nutrient absorption but also improve resistance to various types of biological and abiotic stress. In recent years, the focus on the mechanism driving the salt stress alleviation association with Si has intensified. Previous work demonstrates that Si deposition in phloem can reduce the extracellular transport of Na⁺ to xylem, thus reducing the upward transfer of Na⁺ (Zhu et al., 2019). Bosnic et al. (2018) showed that Si mediates Na⁺ transport from the xylem to the branches and regulates the accumulation and segregation of Na⁺ by inducing the expression of ZmSOS1, ZmSOS2, ZmHKT1 and ZmNHX genes. Multiple studies have also shown that Si can enhance plant salt tolerance by regulating the expression of hormone and hormone response genes (Yin et al., 2019) and that it alleviates salt-induced osmotic stress by regulating the biosynthesis and metabolism of aquaporins and osmoregulatory substances (Laifa et al., 2021). Salicylic acid (SA) is a natural plant hormone signaling molecule that plays an important role in regulating plant responses to certain types of biological and abiotic stress. Synthetic SA is highly efficient, low-cost, non-toxic and residue-free (Tasgin et al., 2006) and is involved in the regulation of many physiological and biochemical processes (e.g., growth and development, stomatal regulation, enzyme biosynthesis, membrane protection, and flowering) (Sako et al., 2020). Prior work in wheat indicates that 50 mg·L^-1 SA can improve the germination potential, seedling height, and fresh weight under salt stress (Afzal et al., 2006). SA concentrations of 10^-6 and 10^-5 mol·L^-1 can increase the activities of SOD (superoxide dismutase), GST (glutathione S-transferase), and GPOX (glutathione peroxidase) in Arabidopsis under salt stress and reduce the accumulation of H₂O₂ and MDA (malondialdehyde), suggesting that SA can reduce oxidative damage resulting from salt stress by regulating key reactive oxygen species and antioxidant enzymes. This alteration to enzyme activities may be due to the transcriptional regulation of important detoxification genes such as GST (Horvath et al., 2015). Most investigations of the effects and mechanisms of Si or SA in alleviating plant injury are laboratory experiments that induce stress using NaCl or excessive NO^3-. However, there are differences between salt stress produced in laboratory settings and stress experienced by plants growing in salinized agricultural soils. In many commercial agricultural operations, greenhouses have been used for more than ten years, and the soil in such greenhouses contains a number of ion species whose concentrations exceed standard limits, also supporting the contention that crop stress differs from the type induced in laboratory settings. Moreover, it is evident that Si and SA trigger induced resistance in plants and that salinized soils cannot be improved by their use.

Vermicompost is produced by the biodegradation of organic waste by earthworms. It has good porosity (79.9%–82.2%), a favorable water content (54.1%–62.7%), useful shrinkage rates (19.7%–31.6%), a high water holding capacity (166 g–197 g), and a large surface area that supports populations of many beneficial microorganisms (Fornes et al., 2012). Vermicompost can increase the aerobic activity of soil and contains cytokinin, auxin, gibberellin, and other plant hormones that are beneficial for plant growth, production, and quality (Atiyeh et al., 2000; Uma and Malathi, 2009). Earthworm digestion also contributes to the formation of water-stable aggregates (Edwards and Burrows, 1988). In short, vermicompost can enhance soil fertility via its physical, chemical, and biological effects and shows strong potential for ameliorating soil salinity. However, single-measure approaches cannot sufficiently reduce the complex soil salinization associated with long-term continuous cropping; thus, it may be necessary to integrate different management practices (Nazari et al., 2022). Comprehensive treatment strategies that employ both soil and plant management aspects will be crucial to help plants manage biological and abiotic stress in the future. Recent work points to the high potential of an integrated treatment of vermicompost and SA to manage crop disease (Sahni et al., 2021), and we hypothesize that the application of vermicompost combined with SA or Si will mitigate soil salinization and be a promising soil salinization management strategy. Such an approach combines the potential of SA- or Si-induced plant salt resistance with soil amendment using vermicompost.

In this study, we tested the effects of the application of vermicompost manure in conjunction with exogenous Si or SA on salt stress in melon seedlings. We also explored the photosynthetic and resistance physiology associated with this treatment to provide a theoretical basis for the sustainable greenhouse development of Cucumis melo L.

Materials and Methods

Materials

Cucumis melo L., commonly known as muskmelon or cantaloupe, is an annual herb in the gourd family. The cultivar ‘Beitian No.5’ was used in this research. Soil with moderate salinity was collected from a greenhouse operated by the Guangxi Beihai Xingyou Melon and Fruit Farmers Professional Cooperative (Bao, 2000). The basic soil physical and chemical properties of the soil are presented in Table 1. Cow manure and sugarcane filter mud were used as the feeding sources for the earthworms that produced the vermicompost used here. On August 3, 2022, robust melon seedlings of a uniform size were selected and transplanted into pots, each containing 3.5 kg of soil previously collected from the greenhouse. Test seedlings were placed under a canopy (transparent roof, open on all sides) located at the Guangxi Academy of Agricultural Sciences experimental park and received routine weeding and insect control treatments.

Experimental design

Four treatments were selected based on the optimal application concentration determined from preliminary tests: (1) a control (CK) treatment, in which soils were treated with a compound fertilizer containing the same nutrient content as vermicompost used in the other treatment groups (10.8 g/pot); (2) the vermicompost (VC) treatment, in which 10% vermicompost by weight was added to each pot; (3) the vermicompost and Si (VC+Si) treatment, in which 10% vermicompost by weight and exogenous Si was added to each pot; and (4) the vermicompost and SA (VC+SA) treatment, in which 10% vermicompost by weight was added to each pot and SA was applied to the seedling leaves. Each treatment group contained six replicates.

A 0.04% Si solution poured evenly into each pot in the VC+Si group four times each week, and a 100 µmol/L SA solution was sprayed onto the leaf surfaces every other day in the pots in the VC+SA treatment group. When the SA solution was applied, the soil was covered with plastic film to prevent application to the soil surface. The CK, VC, and VC+Si treatment groups were sprayed with the same volume of 0 µmol/L salicylic acid.

The Si solution was prepared by dissolving analytically pure K₂SiO₃·9H₂O in 2000 mL water. The SA solution was prepared using the following protocol: 1) dissolve 0.1 g of SA into 7 mL of anhydrous ethanol; 2) add distilled water to prepare a 1 mmol/L mother solution. The 0 µmol/L salicylic acid solution used here was prepared using the same volumes of ethanol and distilled water.

Index determination and sample collection

On August 28, 2022, the chlorophyll fluorescence and photosynthetic characteristics of the muskmelon seedlings were measured using a LI-6800 portable photosynthetic-fluorometer (LI-COR). Four pots were selected from each treatment, and each plant’s penultimate functional leaf was selected for determination. The instrument was equipped with a multiphase flash, which uses multi-colored LEDs to emit light, along with a detector to measure fluorescence in a 6 cm² leaf chamber. Test plants were acclimated to the dark overnight prior to the LI-COR analysis. The measuring light was set to 50 Hz and the rectangular flash was set to 8000 µmol/(m·s), 100 Hz for 1000 ms according to the manufacturer’s instructions. The activated light was turned off, and the blade was clamped until the fluorescence values stabilized. The initial fluorescence (F0) and maximum fluorescence (Fm) were measured. The manufacturer’s measurement light and rectangle flash settings were retained and the instrument was set to the “Dark Pulse” setting for plant measurements under F0' light. The active light was turned on and set to ambient light intensity (1000 µmol/m·s). When the fluorescence value stabilized, readings were collected for F0', Fm' and Fs. These readings were used to calculate the maximum photochemical efficiency (Fv/Fm), PSII effective quantum yield (Fv'/Fm'), actual photochemical efficiency (ΦPSII), photochemical quenching coefficient (qP) and non-photochemical quenching coefficient (qN). Red and blue light were used to test the photosynthetic characteristic parameters. Based on preliminary experiments, 1200 mol·m^-2·s^-1 was used as the value for photosynthetically active radiation (PAR), 25°C for the leaf chamber temperature, and the atmospheric CO₂ concentration for the CO₂ concentration. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), and transpiration rate (Tr) were measured.

Whole plant samples were harvested on August 28, 2022. After rinsing with tap water, intact samples were washed with distilled water and dried with paper towels and stored at ‒80°C. The penultimate third unfolded leaf of each plant was mixed to prepare the sample for the determination of the physiological characteristics. Six pots of plants were sampled and separated into above- and below-ground portions, after which they were oven-dried at 90°C for 30 min. The oven’s temperature was reduced to 60°C and the samples were dried to a constant weight. The aboveground biomass and root biomass were weighed using an electronic balance, and the root-shoot ratio was calculated at the same time. The equation used is shown below.

Root shoot ratio = Root biomass / shoot biomass

Malondialdehyde (MAD), superoxide dismutase (SOD), peroxidase (POD), proline (PRO), superoxide anion (OFR), catalase (CAT), soluble sugar and soluble protein were determined using the biochemical colorimetric method outlined by Chongqing Bonuo Hundsun Chemical Technology Co., Ltd. Three replicates from each treatment group were used for these analyses.

Statistical analysis

SPSS 22.0 software was used to conduct a one-way ANOVA to reveal the significance of the influence of different exogenous substances on each index. Duncan’s multiple-range test was used to test the significance of differences between each treatment group. Origin 8.5 software was used for mapping.

Results

Growth characteristics

In the experiment, 100% of the seedlings survived until the end. Leaf number, plant height, stem diameter, and shoot and root biomass were higher in the VC treatment than in CK, but these differences were not significant (p > 0.05; Table 2), indicating that the vermicompost treatment promoted seedling growth but that the magnitude of this effect was limited. The addition of exogenous Si or SA amplified this effect. Plant height and shoot biomass increased significantly with the application of vermicompost combined with SA or Si compared to the CK treatment. Here, the VC+SA treatment exhibited the best results, with leaf number, plant height, and biomass outcomes correspondingly 32.92%, 56.38% and 63.29% higher than those of CK.

Chlorophyll fluorescence

The Fv/Fm, leaf PSII, Fv'/Fm', actual ΦPSII and qP of PSII values from the VC, VC+Si and VC+SA treatment groups were higher than those in CK, but there were no significant differences between Fv/Fm, Fv'/Fm' and qP from the VC treatment compared to CK. Fv/Fm, ΦPSII, and qP increased significantly in the VC+SA treatment group compared to CK (Table 3), and Fv/Fm, Fv'/Fm' and ΦPSII increased significantly in the VC+Si treatment group compared to CK. Fv'/Fm' was highest in the VC+Si treatment group, showing an increase of 3.32% over CK. The non-photochemical quenching coefficient (qN) was lower in the VC, VC+Si, and VC+SA treatment groups relative to CK, but there was no significant difference among these treatments (p > 0.05). Together, these results suggest that the VC, VC+Si, and VC+SA treatments reduce damage to the PSII reaction center under salt stress and reduce the non-photochemical heat dissipation of excitation energy, with VC+SA conferring the most protection.

Photosynthetic characteristics

The photosynthetic parameters were significantly affected depending on the treatment type (p < 0.05; Table 4). All parameters were higher with the addition of vermicompost, but there were no significant differences in the stomatal conductance, intercellular CO₂, or transpiration rate under VC relative to CK. These improvements were magnified with the addition of Si or SA. The VC+Si treatment increased the net photosynthetic rate, stomatal conductance, and intercellular CO₂ significantly, with the VC+SA treatment also significantly increasing the net photosynthetic rate, intercellular CO₂, and transpiration rate compared to CK.

Resistant physiology characteristics

Antioxidant enzyme activities (SOD, CAT, POD) were similar across the treatments, with activities of VC+SA > VC > VC+Si > CK (Fig. 1A), suggesting that the addition of vermicompost improved the antioxidant enzyme activity in muskmelon seedlings under salt stress. Compared to CK, the CAT and POD activities increased significantly in the groups treated with exogenous substances. Enzyme activities in the VC+Si treatment group were lower than in the VC group (Fig. 1A). Higher activity was most obvious under the VC+SA treatment where the SOD, CAT and POD activities were respectively 49.52%, 22.96%, and 73.46% higher than in the CK case (Fig. 1A). Moreover, compared to the VC-only group, the VC+SA treatment significantly increased SOD, CAT, and POD activities, whereas SOD and POD levels were decreased significantly in the VC+Si treatment group (Fig. 1A).

Under the VC, VC+Si, and VC+SA treatments, seedling MDA and superoxide anion contents were significantly lower than under CK (Fig. 1B and 1C). Moreover, superoxide anion and MDA contents were significantly lower under the VC+Si and VC+SA treatments compared to the VC-only group (Fig. 1B and 1C).

Proline, soluble sugar, and soluble protein are important physiological osmoregulators, and their accumulation can improve water absorption and retention in cells under stress. The application of exogenous substances significantly increased proline, soluble sugar, and soluble protein contents (Fig. 1D, 1E and 1F). Compared to the VC treatment group, plants treated with VC+Si and VC+SA had significantly higher soluble sugar (Fig. 1E) and protein contents (Fig. 1F).

Fig. 1.

Muskmelon seedling resistance physiology characteristics.

Discussion

Salt stress inhibits the growth and development of plants, resulting in leaf wilting, yellowing, and reduced root growth and plant height (Ali et al., 2021), indicating that salt stress strongly influences plant growth. Applying vermicompost has been widely reported to promote plant growth, possibly due to its association with increased nutrient uptake levels and other beneficial effects, such as its stimulation of phytohormone production (Li et al., 2021). In this study, a single application of vermicompost enhanced plant growth under salt stress, but the effect was limited, similar to other findings (Mohammed et al., 2018). The improved soil nutrient content and the addition of other growth-promoting materials resulting from the added vermicompost may not be enough to mitigate intense salt stress. Thus, it is necessary to supplement vermicompost with exogenous boosters such as phytohormones or biostimulants that improve plants’ tolerance of salt stress. Compared to CK, the combination of vermicompost and SA or Si resulted in significant increases in plant height and shoot biomass. Previous work has documented that Si and SA can trigger salt resistance in plants, which may be the reason for this effect (Jini and Joseph, 2017; Zhu et al., 2020).

Chlorophyll fluorescence parameters reflect electron transfer and the operation of the PSII reaction center during plant photosynthesis and can be influenced by abiotic stressors (Zhang et al., 2022), with higher chlorophyll fluorescence (Fv/Fm, Fv'/Fm', ΦPSII and qP) suggestive of an increased photosynthetic capacity. The ionic and osmotic stress induced by soil salinity damages the PSII reaction center, reducing the system’s electron transport efficiency and photosynthetic activity (Kalaji et al., 2016). In this study, a single application of vermicompost increased the photosynthetic capacity in muskmelon seedlings under salt stress relative to CK, but Fv/Fm, Fv'/Fm' and qP outcomes did not change significantly. When treated with vermicompost combined with SA or Si, PSII activity and growth characteristics both increased significantly, suggesting that the addition of either Si or SA can reduce salt-stress-induced damage to the PSII reaction center and mitigate photoinhibition. The photosynthetic and fluorescence parameters exhibited similar trends. Under salt stress, the effects of vermicompost combined with Si or SA on muskmelon seedling photosynthesis were better than those from vermicompost alone. It has been widely reported that the application of either Si or SA can help plants adapt to salinity stress by improving photosynthesis (Nazar et al., 2011; Gurmani et al., 2013; Rizwan et al., 2015; Souana et al., 2020). Treatment with Si and SA mediates these improvements by enhancing photosynthetic pigments, the antioxidant capacity, and the abundance of proteins related to photosynthesis and energy metabolism (Wang et al., 2010; Gadallah and Sayed, 2014; Prabhakaran et al., 2017; Haghighi et al., 2023). Moreover, SA can activate the protective components of PSII, thereby reducing chlorophyll degradation and PSII excess excitation energy (Gupta and Gupta, 2016; Li et al., 2020). This study underscores the ability of the Si and SA treatment to increase the antioxidant capacity.

Salt accumulation in plant tissues destroys the antioxidant enzyme system, driving large increases in oxygen free radicals and a continuous decrease in growth, eventually resulting plant growth cessation or even death (Zelm et al., 2020). MDA is the product of membrane lipid peroxidation, and its concentration is an important indicator of lipid peroxidation (Wang et al., 2014). SOD, POD, and CAT are important components of the active oxygen scavenging system in plants. These enzymes cooperate with each other to regulate the balance of active oxygen in plants. Our results indicate that the superoxide anion and MDA contents were higher in CK than in any other treatment group. The inhibition of lipid peroxidation by exogenous substances may be due to better regulation of the antioxidant defense under salt stress. In this study, CAT and POD activities were higher in muskmelon seedlings that were treated with exogenous substances. Moreover, compared to the VC treatment group, superoxide anion and MDA contents decreased significantly in the VC+Si or VC+SA treatments, indicating that the application of Si or SA improves the removal ability of the plants. These results demonstrate that SA improves peroxidation inhibition mainly by stimulating SOD, CAT, and POD activities. Similarly, Chao et al. (2010) found that under high salinity, SA can improve the antioxidant oxidase activity in rice leaves and reduce oxidative damage. Similar to our results, previous studies have also shown that Si can improve antioxidant defenses in plants under salt stress and reduce oxidative damage, thus maintaining the cell structure and membrane integrity (Liang et al., 2006; Zhu et al., 2016). However, compared to VC, the activity of antioxidant enzymes decreased in the VC+Si treatment groups, possibly because an exogenous Si application reduced the cell peroxide content via other mechanisms, such as by modulating the non-enzymatic antioxidant defense system and/or improving the efficiency of the ascorbate-glutathione cycle (Pan et al., 2023; Souza et al., 2023). Thus, the higher antioxidant enzyme activity may not have been necessary for the plants to cope with salt stress.

Proline, soluble sugar and soluble protein are important plant osmoregulators, and their accumulation can regulate the cell osmotic potential and mitigate the effects of salt damage related to osmotic protection (Hatzig et al., 2014; Rajabi et al., 2022). In this study, proline, soluble sugar and soluble protein contents were significantly higher in the VC, VC+Si, and VC+SA treatment groups relative to CK, indicating that the addition of exogenous substances can help muskmelon seedlings cope with soil salinity by increasing the osmoregulator content. Moreover, soluble sugar was significantly higher in the VC+Si and VC+SA treatment groups relative to VC. Thus, the addition of SA and Si strengthened the stability of the protoplasmic colloid of muskmelon seedlings under salt stress. The increase in soluble sugar suggests that Si and SA have positive effects on enzymes related to carbohydrate metabolism (Gou et al., 2023). Moreover, a treatment with SA may alter the respiratory kinetics in petal tissues by uncoupling the mitochondrial electron transport system, which may strongly influence sugar utilization (Norman et al., 2004). Additionally, compared to VC, the VC+SA treatment enhanced the soluble protein content, likely due to the induction of stress proteins that occurs under abiotic stress (Srivastava et al., 2005). Other work suggests that a SA treatment increases the osmoregulator content in plants (Shemi et al., 2021). Our results indicated that the addition of vermicompost can promote intense osmoregulator synthesis in muskmelon seedlings, aiding them in dealing with salt stress. Combining vermicompost with Si or SA improved performance outcomes mostly because Si and SA are better triggers of plant salt resistance.

Taken together, this study demonstrated that a VC, VC+SA, or VC+Si treatment can alleviate the damage to the PSII reaction center, improve the photosynthetic capacity, increase osmotic substance content levels, enhance the antioxidant enzyme activity, and reduce cellular membrane lipid peroxidation to alleviate damage caused by salt stress and improve salt tolerance in muskmelon seedlings. Moreover, compared to the VC-only treatment, VC combined with SA or Si profoundly strengthened salt tolerance and plant growth. Therefore, an approach that combines a vermicompost treatment with the application of SA or Si represents a more promising strategy for the management of saline soil.