Ismail, Manal S. ¹ and Hussien, Mennat-Allah N. ²

¹✉ Vegetable and Ornamental Plant Mites Department, Plant Protection Research Institute, Agricultural Research Center (ARC), Giza, Egypt.
²Vegetable Plants Breeding Department, Horticulture Research Institute, ARC, Giza, Egypt.

2024 - Volume: 64 Issue: 2 pages: 370-384

Original research

Keywords

Abstract

Introduction

Eggplant, Solanum melongena L., is a non-tuberous member of the nightshade Solanaceae family. From 2003 until the present, Egypt ranks third in world production. Egypt planted over 0.049 million hectares with a yield of 1.34 million tons, whereas the global yield production from 1.88 million hectares reached 56.62 million tons (Faostat 2020). Several pests hinder eggplant production, with spider mites being the most destructive (Basha et al., 2021).

The two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae) (red form) infestation can cause direct damage to the eggplant leaves, resulting in stippling, chlorosis, and leaf drop, thereby reducing the quality and crop yield (Farouk et al. 2021). TSSM feeding on leaf tissue is the key factor here as it sucks the mesophyll cell contents causing cellular oxidative damage to the whole plant (Foyer and Noctor 2003). This oxidative damage is attributed to the rapid accumulation of reactive oxygen species (ROS) in the form of hydrogen peroxide (H₂O₂) and superoxide (O₂^•‾). Plant cells have a number of ROS scavenging systems that can maintain a relatively low ROS concentration by the relevant protective mechanisms using (enzymatic defense mechanisms [such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO)] and non-enzymatic defense mechanisms [carotenoids and chlorophyll derivatives] (Apel and Hirt 2004; Panda 2012). Both mechanisms contribute to detoxifying ROS and maintaining its levels within a safe range (Gill et al. 2010).

SOD is a crucial endogenous antioxidant enzyme that functions as a component of the first-line defense system against ROS. It catalyzes the dismutation of two molecules of O₂^•‾ to H₂O₂ and O₂ (Fridovich 1995; Ighodaro and Akinloye 2018). The H₂O₂ is then scavenged by CAT, which decomposes it into H₂O and O₂ (Willekens et al. 1995). However, they retain low steady-state levels to maintain redox signaling pathways (Noctor and Foyer 1998).

Some traits of TSSM impede its management, including a short life cycle, arrhenotokous parthenogenesis, and its remarkable adaptability to various hosts and environmental conditions. Currently, chemical and biological control are the most prevalent mite control methods (Khanamani et al. 2012; Agut et al. 2018). To avoid acaricide resistance and contribute to environmental sustainability, new approaches to pest control that emphasize non-chemical methods should be developed (García-Marí and González-Zamora 1999). In recent years, vegetable grafting has gained increased attention as a sustainable agricultural practice (Louws et al. 2010). Grafting in Solanaceae using resistant rootstocks was recommended as an effective tool for controlling numerous pathogens and increasing tolerance to environmental stresses, in addition to improving yield quality and production (Ioannou 2001; Frey et al. 2020; Sudesh et al. 2021).

There has been a growing interest in grafting eggplant due to a prohibition on methyl bromide use and a shortage of stress-resistant eggplant genotypes (Bletsos 2005). Utilizing rootstocks with genetic resistance traits to foliar arthropod pests may improve the success of grafting in IPM. Previous research has shown that grafting using a resistant rootstock decreased the whitefly Bemisia tabaci Gennadius population in tomatoes, which in turn decreased virus infection (Alam et al. 1995; Álvarez-Hernández et al. 2009; Žanić et al. 2017). To date, few studies on eggplant defense responses to TSSM infestation through grafting have been reported. Although grafting may increase pest resistance, the underlying mechanisms are not completely understood. Consequently, the hypothesis being tested in this study is that the grafting plant mitigates the harmful effects of TSSM by enhancing the antioxidant enzyme activities to scavenge H₂O₂.

Our objective was to evaluate (1) the influence of grafted eggplant using Solanum torvum Sw. as a rootstock on the incidence of TSSM. (2) the eggplant defense response and how grafting can alleviate the negative impact of TSSM by monitoring the activity of antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT) as well as hydrogen peroxide (H₂O₂) and photosynthetic pigments contents in leaves at different TSSM infestation levels. (3) the influence of grafted eggplant on vegetative growth and marketable yield.

Material and methods

Plant material and grafting method

The commercial eggplant S. melongena cultivar (cv.) A338 F1 was utilized as a scion, and it was grafted onto the rootstock S. torvum cv. STT3. Seeds were purchased from Vilmorin Seed Company, France.

The rootstock seeds were planted ten days before the scion seeds. Under greenhouse conditions, both seeds were sown in 150- cell styrofoam trays filled with a soil mixture (peat moss and perlite mixes in a 1:1 v/v).

As described by Honma (1977), splice grafting (top grafting) was utilized. When seedlings had four leaves, grafting was performed 30 days after sowing the scion. In order to prevent shoot growth, the cotyledons of rootstock seedlings were severed. The cut may be made at an angle between 45 to 60 degrees with a razor blade. The scion seedling was subsequently trimmed at the same angle as the rootstock. A silicon grafting clip was utilized to affix the scion to the rootstock. The seedlings were kept in a healing chamber for 7 days at temperatures between 24-28 °C and relative humidity (RH) between 90-95%.

Field trial

The field trial was conducted during the winter season on 15 October 2022 at the experimental farm of Suez Canal University (SCU), Ismailia, Egypt (30°37′22″ N, 32°16′30″ E). Four replicates of a randomized complete block design were implemented. Non-grafted eggplants including rootstock (STT3) and scion (A338) were used as a control. Grafted and non-grafted plants were planted in 90 cm rows with 45 cm spacing between plants (50 plants per replication). The cultivation was conducted without chemicals, and all agricultural practices and fertilizer amounts were applied to the plants per the Egyptian Ministry of Agriculture's recommendations. The fertilization was applied at a rate of 320 kg N (ammonium sulfate, 20.5% N), 150 kg P₂O₅ (calcium superphosphate, 15.5% P₂O₅), and 220 kg K (potassium sulfate, 48% K₂O) per hectare.

Evaluation of mite density

After one month from planting, bi-monthly sampling began to evaluate the abundance of TSSM on grafted and non-grafted eggplants. The leaves (5 leaves × 5 plants × 4 replications) were gathered and stored in polythene bags and then transported to the laboratory for counting. The mobile and egg stages were counted and recorded in a 2.5 cm² area on the underside of the leaf under a Stereo-Binocular microscope (Olympus SZ-PT, Japan).

Evaluation of vegetative growth and crop yield

Plants' height, stem diameter, and leaves' dry weight were recorded every month until the end of the experiments. The plant height of the main stem was measured by using a steel measuring tape (5 m long). A Vernier caliper (0-150 mm, accuracy range of 0.05 mm) was utilized to measure the main stem diameter 5 cm above the ground for rootstock, scion and grafted plants. The leaves dry matter percentage (DM%) was measured by weighing 50 leaves from 10 plants per replicate and then dried in an oven at 70 °C for three days to determine dry weight. DM% was calculated using the following formula: leaf dry weight (g)/leaf fresh weight (g) x 100. Total marketable fruit weight and number were also recorded from ten plants per replicate.

Greenhouse trial

Mite culture

The TSSM was collected from infested leaves of castor bean plants (Ricinus communis L.) at the experimental farm of SCU, Ismailia, Egypt. The TSSM colony was reared on two-week-old beans (Phaseolus vulgaris L.) under controlled ambient conditions (27 ± 2 °C, 60 ± 5% RH and a 16 L:8D h photoperiods). Before being used in the experiment, approximately ten generations of these mites were reared.

Experimental design

Seedlings of grafted, non-grafted rootstock (STT3), and non-grafted scion (A338) plants were individually transplanted into pots (20 cm diameter × 25 cm deep) filled with sandy loam soil. The experiment was conducted in the glass greenhouse of the Faculty of Science, SCU. A complete randomized experimental design with four replicates was used. Each replicate consisted of five plants for each treatment. After one month from transplanting, the plant seedlings were artificially infested with TSSM (10, 30, and 50 adults per plant), and non-infested plants served as a control group. Each seedling was then individually isolated within a net cage (50 cm L × 50 cm W × 100 cm H) to prevent mite dispersal between plants. Each plant was watered using 20-20-20 (N-P-K) at a dose of 1g L⁻¹.

Evaluation of eggplant defense response

After 15 days, fresh leaves were collected from the various seedlings in each treatment group for biochemical analysis. Absorbance measurements were recorded with a UV-visible spectrophotometer (Milton Roy, Spectronic 1201, USA).

Enzyme activities determination

The fresh leaves (0.5 g) were subjected to an extraction assay as described by Hildebrand et al. (1986a).

The determination of superoxide dismutase activity (SOD; EC 1.15.1.1) was based on Beauchamp and Fridovich (1971) and modified by Giannopolitis and Ries (1977). The activity was measured by observing the inhibition of nitro blue tetrazolium's photochemical reduction. The absorbance was measured at 560 nm.

Using the method of Aebi (1984), the activity of catalase (CAT; EC 1.11.1.6) was determined by observing the decrease in absorbance at 240 nm caused by the decomposition of hydrogen peroxide.

Hydrogen peroxide (H₂O₂) determination

The H₂O₂ was extracted from leaves, according to Okuda et al. (1991). The absorbance was determined at 590 nm.

Photosynthetic pigments determination

The content of photosynthetic pigments [chlorophyll (Chl.) a, Chl. b and carotenoids] were measured using the methods provided by Arnon (1949) and Wettstein (1957), respectively. The absorbance was measured at 644 and 662 nm for chlorophyll, and at 440 nm for carotenoids using a spectrophotometer (Unico UV/VIS 2100, USA).

Statistical Analysis

Utilizing the SPSS 23 for Windows statistical software, statistical analysis was conducted. The means were compared using the Tukey multiple comparison test at a significance level of 0.05. Two-tailed paired Student t-test was used to analyze the fruit number, fruit weight per plant, and average fruit weight. The linear regression analysis was carried out to examine the relationship between the density of TSSM infestation and the activity of CAT and SOD, as well as the concentration of H₂O₂, and photosynthetic pigments in both grafted and non-grafted eggplant.

Results

Field Trial

Effect of grafting on the incidence of TSSM

The results indicated that the grafting had a significant influence on the abundance of TSSM (Figure 1). The number of TSSM eggs increased and reached a peak during January and February but decreased during March. The rootstock showed the fewest number of TSSM eggs monthly, and the grafted A338 exhibited fewer monthly TSSM eggs than the non-grafted A338 plants (Figure 1A). Regarding nymphs, their population reached its peak in March. Non-grafted A338 had the highest density of nymphs throughout all months. There was no significant difference between grafted A338 and non-grafted rootstock STT3 in November, December, January, and March (Figure 1B). The number of TSSM adults and eggs followed the same pattern (Figure 3C).

Figure (2) depicts the average total number of TSSM eggs, nymphs, and adults in grafted and non-grafted eggplant during the study period. The non-grafted A338 had the highest total number of eggs, nymphs, and adults, averaging 42.70 ± 7.06, 24.38 ± 4.03, and 10.82 ± 1.69, respectively. In contrast to non-grafted plants, the total number of eggs, nymphs, and adults was lower in the grafted A338 plants, averaging 25.57 ± 4.78, 16.65 ± 3.70, and 7.84 ± 1.43, respectively. Conversely, the lowest total number of TSSM eggs, nymphs, and adults was observed in the non-grafted rootstock, with an average of 15.24 ± 2.64, 8.08 ± 2.26, and 4.49 ± 0.95, respectively, as compared to grafted and non-grafted A338.

Effect of grafting on some vegetative growth and yield component

Table (1) illustrates that grafting has a significant effect on the length and diameter of stems, as well as the percentage of dry weight in leaves. Non-grafted A338 plants had the shortest stem length (85.33 ± 1.28 cm), whereas non-grafted rootstock had the longest stem length (180.59 ± 2.90 cm), followed by grafted A338 plants (101.42 ± 2.00 cm), which had an intermediate length. The non-grafted rootstock STT3 plants had the largest stem diameter compared to the other treatments. Additionally, the findings revealed no significant difference between the stem diameter of grafted and non-grafted A338 plants. Furthermore, the highest percentage of dry weight in leaves was observed in non-grafted rootstock plants (29.19 ± 0.36%), followed by grafted A338 plants (26.95 ± 0.47%), while non-grafted A338 plants showed the lowest percentage (24.59 ± 0.57%).

The results also indicated a significant variance between grafted and non-grafted A338 plants in the number of fruits per plant (T = -3.191, P < 0.05) and the total weight per plant (T = -3.600, P < 0.05) (Figure 3). However, the two groups demonstrated no significant difference in the average fruit weight (T = -0.620, P > 0.05). Grafted A338 was higher in fruit number (9.50 ± 1.06 No./plant) than non-grafted A338 plants (5.72 ± 0.53 No./plant). The fruit weight was also higher in grafted A338 (2.38 ± 0.09 Kg/plant) than in non-grafted A338 plants (1.40 ± 0.26 Kg/plant).

Greenhouse trial

Effect of TSSM density on the activity of antioxidant enzymes

The results revealed a significant effect of TSSM infestation density on the activity of CAT and SOD (F = 240.63, P < 0.05, and F = 122.84, P < 0.05), respectively. As TSSM density increased the activities of CAT and SOD increased compared to non-infested plants (Figure 4). At the highest density of TSSM (50 adults/plant), non-grafted rootstock STT3 plants showed the highest activity of CAT (531.02 ± 13.01 U/g FW), followed by grafted A338 (367.25 ± 10.02 U/g FW). In contrast, the lowest CAT activity was recorded in non-grafted A338 (114.37 ± 8.95 U/g FW). Moreover, the results indicated no considerable differences between grafted and non-grafted A338 plants in CAT activity when the population density of TSSM was either 10 or 30 adults/plant (Figure 4A).

At the highest density of TSSM, the grafted A338 plants exhibited the most significant elevation in SOD activity (412.84 ± 5.12 U/g FW) as compared to non-infested plants. Whereas, there were no significant differences between the non-grafted A338 (370.71 ± 3.14 U/g FW) and non-grafted STT3 (350.96 ± 2.57 U/g FW) at the highest TSSM density. At all TSSM infestation levels, the grafted eggplants demonstrated high levels of SOD activity compared to non-grafted A338 or STT3 plants (Figure 4B).

Table (2) depicts the correlation coefficient (R) and determination coefficient (R²) for the relationship between the density of TSSM infestation and the activity of CAT and SOD. The results indicate a significant and positive correlation between them in all plant treatments. In addition, this correlation was stronger in the grafted A338 and non-grafted rootstock for CAT and SOD activity, respectively, compared to the non-grafted A338 plants.

Effect of TSSM density on the content of H₂O₂

The results showed that the content of H₂O₂ was significantly affected by the density of TSSM (F = 77.19, P < 0.05), and as the density of infestation increased, H₂O₂ content also increased (Figure 4C). The grafted plant and non-grafted scion had the highest content of H₂O₂ (6.3 ± 0.36 and 5.9 ± 0.33 nM/g FW) when exposed to the highest infestation density (50 adults/plant). At all infestation levels, the non-grafted rootstock exhibited the lowest level of H₂O₂. There was a significantly positive correlation between TSSM density and H₂O₂, as shown in Table (2). The correlation was stronger in the grafted A338 plants (R = 0.940, R² = 0.884) compared to both non-grafted A338 and rootstock.

Effect of TSSM density on the photosynthetic pigment contents

The findings demonstrated that the content of Chl. a+b and carotenoids were notably impacted by the density of TSSM (Figure 5). The statistical analysis revealed a significant effect with F values of 161.60 (P < 0.05) and 29.83 (P < 0.05) for Chl. a+b and carotenoids content, respectively.

The results revealed that the content of chlorophyll a+b increased when the density of TSSM was 10 adults/plant but decreased when the density increased to 30 and 50 adults/plant compared to non-infested plants (Figure 5A). The highest level of Chl. a+b was found in non-grafted rootstock STT3 (234.58 ± 6.57 mg/100g FW) at a TSSM density of 10 adults/plant. At a TSSM density of 50 adults/plant, non-grafted A338 had the lowest Chl. a+b content (62.60 ± 1.76 mg/100g FW). Furthermore, the results indicated a significant difference in Chl. a+b content between grafted and non-grafted A338 plants at TSSM densities of 10 adults/plant (169.61 ± 4.75 and 143.88 ± 4.03 mg/100g FW, respectively) and 50 adults/plant (91.16 ± 2.55 and 62.60 ± 1.76 mg/100g FW, respectively).

Concerning the carotenoids content, the results indicated a decrease in the content at all TSSM densities when compared to non-infested plants (Figure 5B). The carotenoids content was the highest in non-grafted rootstock and grafted A338 plants at all TSSM densities compared to non-grafted A338 plants. At TSSM density of 50 adults/plant, the carotenoids content was the lowest in non-grafted A338 plants (47.84 ± 1.34 mg/100g FW), as compared to non-grafted rootstock STT3 (73.18 ± 2.05 mg/100g FW) and grafted A338 (69.77 ± 1.96 mg/100g FW).

There was a significant and negative correlation between TSSM density and the content of Chl. a+b and carotenoids, as shown in Table (2). The correlation between Chl. a+b and TSSM density was stronger in non-grafted A338 plants (R = -0.894, R² = 0.798) compared to non-grafted rootstock (R = -0.878, R² = 0.770) and grafted A338 (R = -0.872, R² = 0.761). Similarly, the correlation between carotenoids and TSSM density was the highest in non-grafted A338 plants (R = -0.796, R² = 0.634), followed by grafted A338 (R = -0.722, R² = 0.521), and the lowest in non-grafted rootstock STT3 (R = -0.683, R² = 0.466).

Discussion

Grafting is a technique commonly used in vegetable production to improve plant growth, yield (Miceli et al. 2014; Sabatino et al. 2018), and disease resistance (Murata et al. 2022). One of its potential benefits is increasing resistance against pests. In a study by Žanić et al. (2018), grafted tomato on different rootstocks cv. Emperador, Arnold, Maxifort, and Buffon showed a lower number of B. tabaci as compared to non-grafted plants. The adults number on grafted plants was significantly decreased by about 26.59% than on non-grafted tomato. A similar finding was reported by Mandušić et al. (2019), who found that the tomato grafted onto the same previous rootstocks exhibited lower attractiveness to the greenhouse whitefly Trialeurodes vaporariorum Westw. than non-grafted plants. Furthermore, Álvarez-Hernández et al. (2009) found that grafted tomatoes onto six wild relative rootstocks exhibited resistance to phloem feeders such as Bemisia tabaci Gen., Bactericera cockerelli (Sulc), and Aphis gossypii Glov. The incidence of these insects was 1.7 to 3 times lower in grafted compared to non-grafted tomato plants. Likewise, Cortez-Madrigal (2010) reported that leaf miner Liriomyza spp. and aphids incidence was significantly lower in grafted tomatoes (2.18 ± 2.16 and 0.32 ± 0.35, respectively) than in non-grafted plants (3.9 ± 3.18 and 0.76 ± 0.98, respectively). In contrast, there was no significant difference in the incidence of B. tabaci, Frankliniella occidentalis (Perg.), and A. gossypii between the grafted tomato on Onyx rootstock and non-grafted ones (Waiganjo et al. 2013).

In general, the influence of grafted eggplant on the population dynamics of TSSM has received scant attention. The results presented herein showed that the abundance of TSSM egg, nymph and adult stages was significantly lower in the non-grafted rootstock STT3 and the grafted A338 as compared to the non-grafted A338. This result is consistent with the previous study of Edelstein et al. (2000), who indicated that the plant resistance to mite infestations can be transferred by grafting from rootstock to scion.

Tetranychus urticae feeding causes physical damage to plant cells, which can lead to the destruction of chloroplasts and cause a reduction in the rate of photosynthesis resulting in a decrease of chlorophyll content in the plant which can lead to reduced growth and productivity (Sances et al. 1981). The Chl. a+b and carotenoids contents in eggplant leaves were negatively impacted by TSSM infestation level. At a low mite density of 10 adults/plant, Chl. a+b contents increased compared to non-infested plants. However, at moderate to high mite densities of 30 and 50 adults/plant, Chl. a+b and carotenoids contents were significantly lower than non-infested plants. The grafting A338 plants exhibited the lowest Chl. a+b (38%) and carotenoids (13%) reductions at the highest mite density compared to non-infested plants. In addition, the non-grafted rootstock showed moderate reductions in Chl. a+b (49%) and carotenoids (14%). In contrast, the non-grafted A338 showed the highest reduction in Chl. a+b (55%) and carotenoids (27%).

The findings of this study are in agreement with those of a previous study that investigated the effects of TSSM density and duration of feeding on the chlorophyll fluorescence parameters of P. vulgaris. They showed that with higher densities of TSSM and longer feeding periods the results are greater chlorophyll content reductions of up to 50% (Iatrou et al., 1995). In addition, Hildebrand et al. (1986b) indicated that as TSSM populations increased, leaf chlorophyll and carotenoids contents decreased significantly by 55.26% and 79.3%, respectively. Similarly, another study on the effect of TSSM feeding on cucumber plants revealed that the total Chl. content was reduced by 55% and 80%, respectively, due to feeding by immature and adult TSSM (Park and Lee 2002). They suggest that mite infestation can negatively impact plant growth and development, especially at high densities. In contrast, Bounfour et al. (2002) investigated the feeding effect of T. urticae and Eotetranychus carpini borealis. In response to both spider mite feeding, the Chl. a, b, and total Chl. levels decreased slightly. However, the present study's findings indicate that grafting can provide some protection against these negative effects, and the rootstock utilized can also impact the level of reduction in chlorophyll and carotenoid content.

It was demonstrated that the TSSM infestation could trigger the production of ROS, including H₂O₂, in the plant tissues as a defense mechanism against this infestation (Santamaría et al. 2017, 2018; Golan et al. 2021). Excessive ROS production can cause oxidative stress in the plant cells, damaging lipids, proteins, and DNA, ultimately affecting plant growth and development (Apel and Hirt 2004). To combat the damaging effects of ROS, plants have developed a combination of many secondary compounds and antioxidant defense enzymes for inhibiting or quenching free radicals. The activation of these defensive enzymes directly affects the level of herbivore resistance shown by the plant (War et al. 2012).

In response to pest infestations, antioxidant defense enzymes and non-enzymatic antioxidants can be induced to enhance plant defense against oxidative stress. Previous research has demonstrated that POD activity increased in response to TSSM infestation (Trevisan 2003). Moreover, TSSM-infested plants exhibited greater PPO and POD activity than uninfested plants. Nevertheless, the resistant genotypes exhibited even more significant increases in these enzyme activities than the susceptible ones (Shoorooei et al. 2013). In addition, TSSM damage to soybean foliage increased POD but did not affect the levels of CAT and SOD (Hildebrand et al. 1986a). On the other hand, SOD activity was the highest after 24 hours, with a 10% increase upon B. tabaci infestation. However, CAT activity showed no significant difference compared to the control (Zhang et al. 2008).

There are few studies on the defensive mechanisms conferred by grafting in eggplant plants against TSSM infestation. The present study could provide new insights into the defense mechanisms activated in grafted eggplants upon mite infestation and determine if grafting modulates those mechanisms to boost resistance against this pest. Our results showed that the grafted plants exhibited higher SOD activity and presumably greater TSSM tolerance than the non-grafted plant A338. The present study is in conformity with other studies of Lu et al. (2017) and Zhang et al. (2023) illustrated that plants with higher SOD activity in leaves tend to be more tolerant to TSSM infestation.

The findings of this study also showed that CAT activity rises as the density of TSSM increases, with the most significant rise occurring in the non-grafted rootstock followed by the grafted A338 plant compared to the non-grafted A338. Plants tend to increase CAT activity to eliminate excess H₂O₂ generated and reduce oxidative damage (Mai et al. 2013). This suggests that CAT activity was inversely correlated with H₂O₂ content (Kaur et al. 2014). In this study, the non-grafted rootstock with the highest CAT activity was able to break down the most H₂O₂. On the other hand, the non-grafted A338 plants with the lowest CAT activity accumulated the most H₂O₂. The grafted plants, with moderate CAT activity, also had moderate H₂O₂ content. These results indicate that higher CAT activity could maintain lower H₂O₂ content and vice versa.

Host plant resistance is a crucial tactic in the management of TSSM infestation. There are numerous ways in which host plants can exhibit resistance to pest attack. One of these is through antibiosis, where the plant produces allelochemicals and secondary metabolites that are toxic or repellent to TSSM. Another way is through antixenosis, where the plant has morphological and chemical traits such as glandular trichomes, thick cuticles, and waxy surface that deter TSSM feeding and oviposition. Lastly, tolerance is the plant's ability to withstand or recover from TSSM damage without significant yield loss (Painter 1951; Kogan and Ortman 1978). Grafting can be an effective tool for mitigating the detrimental effects of pests on crops (Louws et al. 2010). However, the exact defense mechanisms associated with this technique are not fully understood. It has been suggested that grafting improves plant defense by transferring defense signals from the rootstock that prime the scion's defense system (Edelstein et al. 2000; Cortez-Madrigal 2010). In addition, the changes in leaf characteristics (i.e., trichomes density, lamina, and mesophyll thickness) caused by the rootstock may influence the incidence of pests on plants (Žanić et al. 2018; Nord et al. 2020). Moreover, in the current study, grafting activates the antioxidant defense system in plants by upregulating the antioxidant enzymes like SOD and CAT, which detoxify the excessive ROS and safeguard cells from oxidative stress. This result agrees with previous studies showing that the grafted plant enhanced the antioxidant defense response under different stresses (Goreta et al. 2008; Zhang et al. 2010; Sánchez-Rodríguez et al. 2012; Liu et al. 2014).

Grafting has been widely used in horticulture to improve plant growth, increase yield, and enhance resistance to abiotic and biotic stresses. The current study showed that grafted A338 on S. torvum had a higher fruit number per plant (9.50 ± 1.06) than non-grafted plants (5.72 ± 0.53). Additionally, the total fruit weight of grafted A338 plants was found to be two times higher than non-grafted plants. However, the result showed that there was no significant difference in the average fruit weight between the two groups. The obtained results are consistent with Nord et al. (2020), who stated that the yield of grafted eggplant was as much as six times greater than the non-grafted variety. Other studies, including those by Moncada et al. (2013), Miceli et al. (2014) and Sabatino et al. (2018), established that grafting could influence yield and fruit quality in eggplant. The rootstock has the ability to influence the scion growth in several ways, as suggested by Lee (1994). It can enhance the minerals uptake through the roots. The rootstock also influences the production of hormones within the plant which play an important role in growth processes. Therefore, grafting can improve the growth and development of plant such as stem height, biomass, and leaf number of plant, as well as yield increase even under TSSM infestation.

In conclusion, our study highlights the potential benefits of grafting as an effective tool in IPM programs for managing pests. Plants with higher antioxidant enzyme activity may have better tolerance to mite infestation and can alleviate the negative impact of TSSM infestation. However, it is noteworthy that grafting alone may not be sufficient for effective pest management and should be used in combination with other IPM strategies. The choice of rootstock and scion should be carefully considered to ensure compatibility and maximize the potential benefits of grafting in IPM programs. Further studies are needed to understand the specific defense pathways triggered by different rootstocks to enhance scion resistance to mites. This study provides valuable insights into the role of antioxidant enzymes in plant defense against TSSM and the potential benefits of grafting for enhancing plant resistance to pests and diseases.

References

1. Aebi H. 1984. [13] Catalase in vitro. In: Parker L. (Ed). Methods in enzymology. New York: Academic Press. p. 121-126. https://doi.org/10.1016/S0076-6879(84)05016-3
2. Agut B., Pastor V., Jaques J.A., Flors V. 2018. Can Plant Defence Mechanisms Provide New Approaches for the Sustainable Control of the Two-Spotted Spider Mite Tetranychus urticae? Int. J. Mol. Sci., 19(2): 614. https://doi.org/10.3390/ijms19020614
3. Alam M.Z., Hossain M.M., Choudhury D.A.M., Uddin M.J., Haque N.M.M. 1995. Effect of Grafting Technology on Suppression of Whitefly (Bemisia tabaci Gennadius) Disseminating Virus Diseases in Tomato. Ann. Bangladesh Agric., 5(2): 91-98.
4. Álvarez-Hernández J.C., Cortez-Madrigal H., García-Ruiz I., Ceja-Torres L.F., Pétrez-Domínguez, J. F. 2009. Incidence of pests in grafts of tomato (Solanum lycopersicum) on wild relatives. Rev. Col. Entomol., 35: 150-155. https://doi.org/10.25100/socolen.v35i2.9206
5. Apel K., Hirt H. 2004. Reactive Oxygen Species: Metabolism, Oxidative Stress, and Signal Transduction. Annu. Rev. Plant Biol., 55: 373-399. https://doi.org/10.1146/annurev.arplant.55.031903.141701
6. Arnon D.I. 1949. Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24(1): 1-15. https://doi.org/10.1104/pp.24.1.1
7. Basha H.A., Mostafa E.M., Eldeeb A.M. 2021. Mite pests and their predators on seven vegetable crops (Arachnida: Acari). Saudi J. Biol. Sci., 28(6): 3414-3417. https://doi.org/10.1016/j.sjbs.2021.03.004
8. Beauchamp C., Fridovich I. 1971. Superoxide Dismutase: Improved Assays and an Assay Applicable to Acrylamide Gels. Anal. Biochem., 44(1): 276-287. https://doi.org/10.1016/0003-2697(71)90370-8
9. Bletsos F.A. 2005. Use of Grafting and Calcium Cyanamide as Alternatives to Methyl Bromide Soil Fumigation and their Effects on Growth, Yield, Quality and Fusarium Wilt Control in Melon. J. Phytopathol., 153(3): 155-161. https://doi.org/10.1111/j.1439-0434.2005.00945.x
10. Bounfour M., Tanigoshi L.K., Chen C., Cameron S.J., Klauer S. 2002. Chlorophyll Content and Chlorophyll Fluorescence in Red Raspberry Leaves infested with Tetranychus urticae and Eotetranychus carpini borealis (Acari: Tetranychidae). Environ. Entomol., 31(2): 215-220. https://doi.org/10.1603/0046-225X-31.2.215
11. Cortez-Madrigal H. 2010. Resistance to insects of tomato grafted on wild relatives, with emphasis in Bactericera cockerelli (Sulc.) (Hemiptera: Psylidae). Bioagro, 22(1): 11-16.
12. Edelstein M., Tadmor Y., Abo-Moch F., Karchi Z., Mansour F. 2000. The potential of Lagenaria rootstock to confer resistance to the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychidae) in Cucurbitaceae. Bull. Entomol. Res., 90(2): 113-117. https://doi.org/10.1017/S0007485300000213
13. FAOSTAT 2020. Food and Agriculture Organization of the United Nations. FAOSTAT Statistical Database. Rome. [8 April 2023]. Available from: https://www.fao.org/faostat/en/#data/QCL
14. Farouk S., Almutairi A.B., Alharbi Y.O., Al-Bassam W.I. 2021. Acaricidal Efficacy of Jasmine and Lavender Essential Oil or Mustard Fixed Oil Against Two-Spotted Spider Mite and Their Impact on Growth and Yield of Eggplants. Biol. (Basel), 10(5): 410. https://doi.org/10.3390/biology10050410
15. Foyer C.H., Noctor G. 2003. Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol. Plant., 119(3): 355-364. https://doi.org/10.1034/j.1399-3054.2003.00223.x
16. Frey C.J., Zhao X., Brecht J.K., Huff D.M., Black Z.E. 2020. High Tunnel and Grafting Effects on Organic Tomato Plant Disease Severity and Root-Knot Nematode Infestation in a Subtropical Climate with Sandy Soils. HortScience, 55(1): 46-54. https://doi.org/10.21273/HORTSCI14166-19
17. Fridovich I. 1995. Superoxide radical and superoxide dismutases. Annu. Rev. Biochem., 64(1): 97-112. https://doi.org/10.1146/annurev.bi.64.070195.000525
18. García-Marí F., González-Zamora J.E. 1999. Biological Control of Tetranychus urticae (Acari: Tetranychidae) with Naturally Occurring Predators in Strawberry Plantings in Valencia, Spain. Exp. Appl. Acarol., 23:487-495. https://doi.org/10.1023/A:1006191519560
19. Giannopolitis C.N., Ries S.K. 1977. Superoxide Dismutases: I. Occurrence in Higher Plants. Plant Physiol., 59(2): 309-314. https://doi.org/10.1104/pp.59.2.309
20. Gill R., Gupta A., Taggar G., Taggar M. 2010. Role of oxidative enzymes in plant defenses against insect herbivory. Acta Phytopathol. Entomol. Hung., 45(2): 277-290. https://doi.org/10.1556/APhyt.45.2010.2.4
21. Golan K., Jurado I.G., Kot I., Górska-Drabik E., Kmieć K., Łagowska, B., Skwaryło-Bednarz B., Kopacki M., Jamiołkowska A. 2021. Defense Responses in the Interactions between Medicinal Plants from Lamiaceae Family and the Two-Spotted Spider Mite Tetranychus urticae Koch (Acari: Tetranychidae). Agron., 11(3): 438. https://doi.org/10.3390/agronomy11030438
22. Goreta S., Bucevic-Popovic V., Selak G.V., Pavela-Vrancic M., Perica S. 2008. Vegetative growth, superoxide dismutase activity and ion concentration of salt-stressed watermelon as influenced by rootstock. J. Agric. Sci., 146(6): 695-704. https://doi.org/10.1017/S0021859608007855
23. Hildebrand D.F., Rodriguez J.G., Brown G.C., Luu K.T., Volden C.S. 1986a. Peroxidative Responses of Leaves in Two Soybean Genotypes Injured by Two-Spotted Spider Mites (Acari: Tetranychidae). J. Econ. Entomol., 79(6):1459-1465. https://doi.org/10.1093/jee/79.6.1459
24. Hildebrand D.F., Rodriguez J.G., Brown G.C., Volden C.S. 1986b. Twospotted Spider Mite (Acari: Tetranychidae) Infestations on Soybeans: Effect on Composition and Growth of Susceptible and Resistant Cultivars. J. Econ. Entomol., 79(4): 915-921. https://doi.org/10.1093/jee/79.4.915
25. Honma S. 1977. Grafting Eggplants. Sci. Hortic., 7(3): 207-211. https://doi.org/10.1016/0304-4238(77)90017-6
26. Iatrou G., Cook C.M., Stamou G., Lanaras T. 1995. Chlorophyll fluorescence and leaf chlorophyll content of bean leaves injured by spider mites (Acari: Tetranychidae). Exp. Appl. Acarol., 19: 581-591. https://doi.org/10.1007/BF00048813
27. Ighodaro O.M., Akinloye O.A. 2018. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alexandria J. Med., 54(4): 287-293. https://doi.org/10.1016/j.ajme.2017.09.001
28. Ioannou N. 2001. Integrating soil solarization with grafting on resistant rootstocks for management of soil-borne pathogens of eggplant. J. Hortic. Sci. Biotechnol., 76(4): 396-401. https://doi.org/10.1080/14620316.2001.11511383
29. Kaur R., Gupta A.K., Taggar G.K. 2014. Role of catalase, H₂O₂ and phenolics in resistance of pigeonpea towards Helicoverpa armigera (Hubner). Acta Physiol. Plant., 36: 1513-1527. https://doi.org/10.1007/s11738-014-1528-6
30. Khanamani M., Fathipour Y., Hajiqanbar H., Sedaratian A. 2012. Reproductive performance and life expectancy of Tetranychus urticae (Acari: Tetranychidae) on seven eggplant cultivars. J. Crop Prot., 1(1): 57-66.
31. Kogan M., Ortman E.F. 1978. Antixenosis - A New Term Proposed to Define Painter's ''Nonpreference'' Modality of Resistance. Bull. ESA., 24(2): 175-176. https://doi.org/10.1093/besa/24.2.175
32. Lee J.M. 1994. Cultivation of Grafted Vegetables I. Current Status, Grafting Methods, and Benefits. HortScience, 29(4): 235-239. https://doi.org/10.21273/HORTSCI.29.4.235
33. Liu J., Li, J., Su X., Xia Z. 2014. Grafting improves drought tolerance by regulating antioxidant enzyme activities and stress-responsive gene expression in tobacco. Environ. Exp. Bot., 107: 173-179. https://doi.org/10.1016/j.envexpbot.2014.06.012
34. Louws F.J., Rivard C.L., Kubota C. 2010. Grafting fruiting vegetables to manage soilborne pathogens, foliar pathogens, arthropods and weeds. Sci. Hortic., 127(2): 127-146. https://doi.org/10.1016/j.scienta.2010.09.023
35. Lu F., Liang X., Lu H., Li Q., Chen Q., Zhang P., Li k., Liu G., Yan W., Song J., Duan C. Zhang, L. 2017. Overproduction of superoxide dismutase and catalase confers cassava resistance to Tetranychus cinnabarinus. Sci. Rep., 7(1): 40179. https://doi.org/10.1038/srep40179
36. Mai V.C., Bednarski W., Borowiak-Sobkowiak B., Wilkaniec B., Samardakiewicz S., Morkunas I. 2013. Oxidative stress in pea seedling leaves in response to Acyrthosiphon pisum infestation. Phytochem., 93: 49-62. https://doi.org/10.1016/j.phytochem.2013.02.011
37. Mandušić M., Dumičić G., Ban S.G., Selak G.V., Žnidarčič D., Špika M.J., Urlić B. Žanić K. 2019. The potential of tomato rootstocks in the management of Trialeurodes vaporariorum (Westwood). Sci. Hortic., 256: 108566. https://doi.org/10.1016/j.scienta.2019.108566
38. Miceli A., Sabatino L., Moncada A., Vetrano F., D'Anna F. 2014. Nursery and field evaluation of eggplant grafted onto unrooted cuttings of Solanum torvum Sw. Sci. Hortic., 178: 203-210. https://doi.org/10.1016/j.scienta.2014.08.025
39. Moncada A., Miceli A., Vetrano F., Mineo V., Planeta D., D'Anna F. 2013. Effect of grafting on yield and quality of eggplant (Solanum melongena L.). Sci. Hortic., 149: 108-114. https://doi.org/10.1016/j.scienta.2012.06.015
40. Murata G., Uehara T., Lee H.J., Mizutani M., Kadota Y., Shinmura Y., Saito T., Uesugi K. 2022. Solanum palinacanthum Dunal as a potential eggplant rootstock resistant to root‐knot nematodes. J. Phytopathol., 170(3): 185-193. https://doi.org/10.1111/jph.13067
41. Noctor G., Foyer C.H. 1998. Ascorbate and Glutathione: Keeping Active Oxygen under Control. Annu. Rev. Plant Physiol. Plant Mol. Biol., 49(1): 249-279. https://doi.org/10.1146/annurev.arplant.49.1.249
42. Nord R., Cortez-Madrigal H., Rodríguez-Guzmán E., Villar-Luna E. and Gutiérrez-Cárdenas O.G. 2020. Grafting Wild Tomato Genotypes and Mexican Landraces Increases Trichome Density and Resistance Against Pests. Southwest. Entomol., 45(3): 649-662. https://doi.org/10.3958/059.045.0308
43. Okuda T., Matsuda Y., Yamanaka A., Sagisaka S. 1991. Abrupt Increase in the level of Hydrogen Peroxide in Leaves of Winter Wheat is caused by Cold Treatment. Plant Physiol., 97(3): 1265-1267. https://doi.org/10.1104/pp.97.3.1265
44. Painter R.H. 1951. Insect Resistance in Crop Plants, Soil Science 72(6): p. 481. https://doi.org/10.1097/00010694-195112000-00015
45. Panda S.K. 2012. Assay Guided Comparison for Enzymatic and Non-Enzymatic Antioxidant Activities with Special Reference to Medicinal Plants. Antioxidant Enzyme. InTech. 14: p. 382-400. https://doi.org/10.5772/50782
46. Park Y.L., Lee, J.H. 2002. Leaf cell and tissue damage of cucumber caused by two-spotted spider mite (Acari: Tetranychidae). J. Econ. Entomol., 95(5): 952-957. https://doi.org/10.1093/jee/95.5.952
47. Sabatino L., Iapichino G., D'Anna F., Palazzolo E., Mennella G., Rotino G.L. 2018. Hybrids and allied species as potential rootstocks for eggplant: Effect of grafting on vigour, yield and overall fruit quality traits. Sci. Hortic., 228: 81-90. https://doi.org/10.1016/j.scienta.2017.10.020
48. Sances F.V., Wyman J.A., Ting I.P., Van Steenwyk R.A., Oatman, E.R. 1981. Spider Mite Interactions with Photosynthesis, Transpiration and Productivity of Strawberry. Environ. Entomol., 10(4): 442-448. https://doi.org/10.1093/ee/10.4.442
49. Sánchez-Rodríguez E., del Mar Rubio-Wilhelmi M., Blasco B., Leyva R., Romero L., Ruiz J. M. 2012. Antioxidant response resides in the shoot in reciprocal grafts of drought-tolerant and drought-sensitive cultivars in tomato under water stress. Plant Sci., 188: 89-96. https://doi.org/10.1016/j.plantsci.2011.12.019
50. Santamaría M.E., Martinez M., Arnaiz A., Ortego F., Grbic V., Diaz I. 2017. MATI, a Novel Protein Involved in the Regulation of Herbivore-Associated Signaling Pathways. Front. Plant Sci., 8: 975. https://doi.org/10.3389/fpls.2017.00975
51. Santamaría M.E., Arnaiz A., Velasco-Arroyo B., Grbic V., Diaz I., Martinez, M. 2018. Arabidopsis response to the spider mite Tetranychus urticae depends on the regulation of reactive oxygen species homeostasis. Sci. Rep., 8: 9432. https://doi.org/10.1038/s41598-018-27904-1
52. Shoorooei M., Lotfi M., Nabipour A., Mansouri A.I., Kheradmand K., Zalom F.G., Madadkhah E., Parsafar A. 2013. Antixenosis and antibiosis of some melon (Cucumis melo) genotypes to the two-spotted spider mite (Tetranychus urticae) and a possible mechanism for resistance. J. Hortic. Sci. Biotechnol., 88(1): 73-78. https://doi.org/10.1080/14620316.2013.11512938
53. Sudesh K.S., Anjanappa M., Manjunathagowda D.C., Shilpashree N., Bharathkumar A., Praveenkumar N.R. 2021. Grafting in brinjal (Solanum melongena L.): a sustainable way of increasing the yield. Vegetos, 34(1): 263-269. https://doi.org/10.1007/s42535-020-00171-0
54. Trevisan M.T.S., Scheffer J.J.C, Verpoorte R. 2003. Peroxidase activity in hop plants after infestation by red spider mites. Crop Prot., 22(2): 423-424. https://doi.org/10.1016/S0261-2194(02)00151-5
55. Wettstein V.D. 1957. Chlorophyll-letale und der submikroskopische Formwechsel der Plastiden. Exp. Cell Res., 12(3): 427-506. https://doi.org/10.1016/0014-4827(57)90165-9
56. Waiganjo M., Omaiyo D., Gathambiri C., Kuria S., Njeru C., Kleinhenz M., Kovach J., Miller S., Erbaugh M. 2013. Effects of grafting and high tunnel tomato production on pest incidence, yield and fruit quality in smallholder farms in central Kenya. East African Agric. For. J., 79(2): 107-111.
57. War A.R., Paulraj M.G., Ahmad T., Buhroo A.A., Hussain B., Ignacimuthu S., Sharma H.C. 2012. Mechanisms of plant defense against insect herbivores. Plant Signal. Behav., 7(10): 1306-1320. https://doi.org/10.4161/psb.21663
58. Willekens H., Inzé D., Van Montagu M., Van Camp, W. 1995. Catalases in Plants. Mol. Breed., 1: 207-228. https://doi.org/10.1007/BF02277422
59. Žanić K., Dumičić G., Urlić B., Selak V.G., Goreta Ban, S. 2017. Bemisia tabaci (Gennadius) population density and pupal size are dependent on rootstock and nitrogen in hydroponic tomato crop. Agric. Forest Entomol., 19(1): 42-51. https://doi.org/10.1111/afe.12179
60. Žanić K., Dumičić G., Mandušić M., Selak V.G., Bočina I., Urlić B., Ljubenkov I., Popović V.B., Goreta Ban S. 2018. Bemisia tabaci MED Population Density as Affected by Rootstock-Modified Leaf Anatomy and Amino Acid Profiles in Hydroponically Grown Tomato. Front. Plant Sci, 9: 86. https://doi.org/10.3389/fpls.2018.00086
61. Zhang S.Z., Hua B.Z., Zhang, F. 2008. Induction of the activities of antioxidative enzymes and the levels of malondialdehyde in cucumber seedlings as a consequence of Bemisia tabaci (Hemiptera: Aleyrodidae) infestation. Arthropod-Plant Interact., 2: 209-213. https://doi.org/10.1007/s11829-008-9044-5
62. Zhang Z.K., Liu S.Q., Hao S.Q., Liu S.H. 2010. Grafting increases the copper tolerance of cucumber seedlings by improvement of polyamine contents and enhancement of antioxidant enzymes activity. Agric. Sci. China, 9(7): 985-994. https://doi.org/10.1016/S1671-2927(09)60181-4
63. Zhang Y., Liu Y., Liang X., Wu C., Liu X., Wu M., Yao X., Qiao Y., Zhan X., Chen, Q. 2023. Exogenous methyl jasmonate induced cassava defense response and enhanced resistance to Tetranychus urticae. Exp. Appl. Acarol., 89: 45-60. https://doi.org/10.1007/s10493-022-00773-0

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