Drought Resistance of Different Scion Varieties Grafted onto Apple SH40 Interstock

Abstract

Apple production in the arid and semi-arid regions of Northwest China, such as Lingwu in Ningxia, faces severe challenges due to water scarcity, which is exacerbated by climate change. To address this issue, this study aimed to identify superior drought-tolerant apple varieties grafted onto the dwarfing interstock SH40 for cultivation in the Lingwu region. Seven major commercial varieties (‘Yanfu 3’, ‘Yanfu 6’, ‘Yanfu 8’, ‘Huashuo’, ‘Golden Delicious’, ‘Starking Delicious’, and ‘Red General’) were evaluated. Under natural drought stress conditions in Lingwu, we measured physiological and biochemical indices, photosynthetic parameters, leaf anatomical structure, and post-harvest fruit quality and yield. Principal component analysis (PCA) and membership function analysis were then employed for a comprehensive evaluation of drought resistance. The results revealed significant varietal differences. ‘Red General’ exhibited superior antioxidant enzyme activities (peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and higher photosynthetic rates (net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr). ‘Golden Delicious’ showed the highest malondialdehyde (MDA) content but also possessed advantageous leaf anatomical traits, such as a high palisade-to-spongy tissue ratio. PCA extracted five principal components with a cumulative variance contribution rate of 95.492%. Membership function analysis ranked overall drought resistance as follows: ‘Red General’ > ‘Golden Delicious’ > ‘Starking Delicious’> ‘Huashuo’ > ‘Yanfu 6’ > ‘Yanfu 8’ > ‘Yanfu 3’. In conclusion, the mid-season varieties ‘Red General’, ‘Golden Delicious’, and ‘Starking Delicious’ demonstrated excellent comprehensive drought tolerance and are recommended as promising candidates for cultivation in the arid Lingwu region.

1. Introduction

Apple (Malus domestica Borkh.) is one of the important economic fruit trees, and its yield and quality are directly related to the agricultural economy and industrial sustainable development. China is the world’s largest apple producer, but abiotic stress problems such as drought and salinity seriously restrict the production potential of major producing areas [1,2]. Particularly in Lingwu, Ningxia, which is a typical arid and semi-arid region in Northwest China, the annual precipitation is scarce and unevenly distributed, and seasonal droughts occur frequently. According to the meteorological bureau, in 2024, there were 299 rainless days, accounting for 81.9% of the whole year. There are often concentrated drought periods, mainly during the transition from winter to spring, the critical growth period in late spring, the early stage of late summer, and midsummer. The longest precipitation-free period can reach up to 31 days. Drought periods are often accompanied by relatively high temperatures and low relative humidity, and seasonal droughts are prominent, mainly concentrated in summer, which is the critical period for crop water demand. This poses a significant threat to the growth and development, yield formation, and fruit quality of apple trees. Therefore, Apple cultivation has long been faced with the challenges of low water use efficiency and tree premature senescence [3]. In this context, exploring the differences in drought resistance among the main cultivated varieties, such as ‘Yanfu 3’, ‘Yanfu 6’, ‘Yanfu 8’, ‘Huashuo’, ‘Golden Delicious’, ‘Starking Delicious’, and ‘Red General’, grafted on SH40 interstock has important practical significance for optimizing the local variety layout and formulating precise cultivation strategies. Therefore, screening and breeding interstock–scion combinations with strong drought resistance has become an important research direction to improve the stress resistance of the apple industry.

The application of dwarfing interstocks is one of the core technologies in intensive apple cultivation. By regulating tree vigor, promoting early fruiting and high yield, and improving photosynthetic efficiency, they significantly enhance orchard management efficiency and economic benefits [4,5]. As an excellent dwarfing interstock independently bred in China, SH40 possesses multiple traits such as cold resistance, drought resistance, and resistance to ring rot [6,7]. It has been widely promoted on a large scale in major producing areas, including Hebei, Shandong, and Shaanxi provinces, and has shown superior comprehensive performance compared to traditional interstocks M26 and M9-T337 [8,9,10]. However, the drought resistance of interstocks is not only affected by their own genetic characteristics but also closely related to the physiological responses of grafted varieties. Existing studies have mostly focused on the drought resistance evaluation of interstocks alone, while systematic research on the differences in drought resistance and their molecular mechanisms among different varieties grafted onto SH40 remains insufficient. This issue is particularly urgent to be addressed in Lingwu, Ningxia, where ecological conditions are harsh.

The interaction between interstocks and scions can affect drought resistance through physiological metabolic pathways, photosynthesis regulation, and reshaping of leaf anatomical structures. Interstocks can enhance the activities of antioxidant enzymes in scion leaves, such as POD, CAT, and SOD, thereby reducing the damage of reactive oxygen species (ROS) to photosystem II (PS II) and maintaining the stability of light energy conversion efficiency (Fv/Fm) and electron transport rate (ETR). For example, Fuji apples grafted with the Y-1 interstock showed significantly higher chlorophyll content and ΦPS II values than those grafted with the SH1 interstock in the late growth stage, indicating that they adapt to drought environments by delaying the degradation of photosynthetic pigments and enhancing the repair capacity of PS II [11].

Drought-resistant interstocks can also reduce stomatal Gs and Tr to decrease water loss while maintaining a relatively high Ci, thus achieving a balance between stomatal and non-stomatal limitations [7,12]. The introduction of drought-resistant interstocks can significantly alter the leaf morphological and anatomical characteristics of scion varieties, such as increasing leaf thickness, the degree of cuticle development, and the ratio of palisade tissue to spongy tissue, thereby reducing transpiration rate and improving water use efficiency.

Under typical conditions, plants in arid environments exhibit adaptive responses in antioxidant enzyme activity, osmoregulatory substances, and photosynthetic characteristics to mitigate the detrimental effects of drought stress. However, drought stress inevitably impacts fruit external quality and yield, manifesting as reduced fruit weight, lower productivity, uneven coloration, and so on [13].

In summary, this study systematically evaluates the differences in drought resistance among 7 main apple cultivars grafted onto SH40 interstock. By combining physiological indices (photosynthetic characteristics, antioxidant enzyme activities, osmotic adjustment substances) with changes in leaf microstructure, and fruit quality traits including appearance and yield, it aims to reveal the effect of interstock-scion interaction on drought resistance, thereby providing a theoretical basis and technical support for apple interstock selection and stress-resistant cultivation in arid regions of Northwest China. The results of this study will fill the gap in research on the multi-cultivar adaptability of SH40 interstock and lay a scientific foundation for the formulation of regional cultivar layout and efficient water resource utilization strategies.

2. Materials and Methods

2.1. Research Materials

The experiment was conducted at the Germplasm Resource Conservation Bank of Famous and Excellent Economic Forest Tree Species in Lingwu Horticultural Experiment Station, Ningxia, located at 106°22′ E, 38°38′ N, with an altitude of 1185 m. Lingwu City has an annual sunshine duration of 3008 h, averaging 8.3 h per day, with a sunshine percentage of 67.9% and strong solar radiation, and the total annual solar radiation reaches 35.2 kJ/m². The average temperature is 8.8 °C, with an active accumulated temperature ≥ 0.6 °C of approximately 3351 °C. The average annual frost-free period is 172 days, with an annual precipitation of 230.7 mm and an annual evaporation of 2862.2 mm. The specific drought conditions are shown in Figure 1.

Figure 1. The drought situation in Lingwu, Ningxia, in 2024. In 2024, the number of rainless days was 299. (Rainless weather: no rainfall or rainfall < 0.01 mm) The yellow background indicates that there were long-term consecutive rainless periods in that month. Specifically, there were four such periods: from 22 February to 23 March, there was no precipitation for 31 consecutive days; from 4 May to 24 May, there was no precipitation for 21 consecutive days; from 7 June to 19 June, there was no precipitation for 13 consecutive days; from 22 June to 4 July, there was no precipitation for 13 consecutive days.

The 7 apple cultivars grafted onto SH40 interstock included ‘Yanfu 3’, ‘Yanfu 6’, ‘Yanfu 8’, ‘Huashuo’, ‘Golden Delicious’, ‘Starking Delicious’, and ‘Red General’, all of which were planted in 2016. The row and plant spacing were 4.0 m × 1.5 m, and the tree shape was spindle-shaped.

The orchard soil is mainly light gray calcareous soil and aeolian sandy soil. The irrigation method is flood irrigation with Yellow River water, that is, flooding the fields with water from the Yellow River. Approximately 250 cubic meters of water were used per mu. Irrigation was carried out once each in May, June, July, and August, with a total of 4 times of summer-autumn irrigation throughout the year, plus one winter irrigation in November. 1.5 kg of compound fertilizer (primarily nitrogen-based, supplemented with phosphorus and potassium) was applied per plant before flowering to promote germination and fruit setting. During fruit enlargement, 1.5 kg of fertilizer was applied. In autumn, 75 m³/hm² of mature organic fertilizer was applied as base fertilizer [14].

2.2. Research Methods

2.2.1. Collection of Apple Leaf Samples

In the summer of 2024, under the conditions of consecutive rainless and high-temperature weather, before 10:00 a.m., 7 varieties of fruit trees grafted on SH40 interstocks with robust growth and no diseases or pests were selected. For each variety, 3 plants were randomly chosen, and samples were collected randomly from the east, west, south, and north directions of the tree crown. After mixing the samples of each variety, they were divided into 3 parts. One part was used for chlorophyll content determination, one part for physiological and biochemical index determination, and the other part was stored as a backup sample at −80 °C.

2.2.2. Determination of Physiological and Biochemical Index Contents

The activity of POD was determined by the guaiacol method [15]; the activity of SOD was determined by the nitroblue tetrazolium (NBT) method [16]; the activity of CAT was determined by the ultraviolet absorption method; the content of MDA was determined by the thiobarbituric acid method [17]; the content of proline (Pro) was determined by the visible spectrophotometry; the content of soluble protein (SP) was determined by the bicinchoninic acid (BCA) method [18]. All the above kits were produced by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). In addition, the ethanol dark extraction method [19] was used to determine chlorophyll (Chl) and its content. Each experiment was repeated three times.

2.2.3. Determination of Leaf Photosynthetic Parameters

During the consecutive rainless and high-temperature weather, from 9:00 a.m. to 11:00 a.m., leaves at the same leaf position (the 5th functional leaf from top to bottom) were randomly selected. The photosynthetic analyzer GFS-3000 (WALZ Company, Effeltrich, Germany) was used to determine Pn, Ci, Tr, and Gs of the leaves [20]. For each treatment, 2–3 plants were selected, and 6 functional leaves were measured for each plant.

2.2.4. Observation of Leaf Anatomical Structure

Healthy and disease-free mature leaves of each variety were collected and stored in FAA fixative. They were sent to Wuhan Servicebio Technology Co., Ltd. (Wuhan, China) for sectioning, then observed and photographed under a microscope. The leaf thickness (LT), mesophyll thickness (MesT), upper epidermis thickness (TU), lower epidermis thickness (TL), palisade tissue thickness (TP), spongy tissue thickness (TS), and midvein thickness (MV) of each variety were recorded [21]. There were 10 data points in each group, and each group of experiments was repeated 3 times in parallel. The required indices were calculated according to the following formulas:

Palisade tissue and spongy tissue ratio (P/S) = TP/TS

Tightness of leaf palisade tissue (CTR) = TP/TL × 100%

Looseness of leaf spongy tissue (SR) = TS/TL × 100%

2.2.5. Measurement of Fruit External Quality Characteristics Across Seven Apple Cultivars

During the ripening period, 5 trees were randomly selected, and fruit samples were collected from the east, south, west, and north of the crown. 15 fruits of each variety were collected for quality index analysis. The results were repeated three times, and the average value was taken.

The apple’s external morphology was described in accordance with the “Descriptors for apple germplasm resources” [22,23]. Measurements of the fruit’s transverse, longitudinal, and lateral diameters were taken using an electronic vernier caliper [24], with the fruit shape index (longitudinal diameter to transverse diameter ratio) calculated. The mass of individual fruits was determined using an electronic scale (WH-B09, Yicheng International Trade (Shenzhen) Co., Ltd., Shenzhen, China). For each variety, 10 representative trees were randomly selected to survey individual fruit counts, and the yield per tree was calculated as the number of fruits per tree × mass of individual fruit.

2.2.6. Data Analysis

Microsoft Excel 2021 and SPSS 27.0 (IBM Corp., Armonk, NY, USA) were used to analyze the collected data, while the graphical representation was created using Origin 2021(OriginLab Corp., Northampton, MA, USA).

Using SPSS 27.0 software, we conducted principal component analysis (PCA) and membership function analysis on the data. First, we preprocessed the quantitative variables and removed outliers. Data suitability was verified through KMO and Bartlett’s test of sphericity (KMO ≥ 0.6 and p < 0.05). Subsequently, we extracted principal components with eigenvalues ≥ 1 using the PCA method. After maximum variance rotation, a clear factor loading matrix was obtained. We then named the principal components based on the meanings of variables with high loadings. Finally, regression analysis was employed to calculate the principal component scores and composite scores for each sample, achieving data dimensionality reduction and extraction of core information.

The calculation formula for the membership function value (D) is as follows:

D(X_i) = (X_i − X_{i min})/(X_{i max} − X_{i min})

Here, X_i represents the measured value of the indicator, while X_{i max} and X_{i min} denote the maximum and minimum values of this indicator among all samples, respectively [25].

In addition, after evaluating whether there are significant differences among the means of each group through analysis of variance (ANOVA), a least significant difference (LSD) test was employed for post hoc multiple comparisons, with the confidence level set at 95% (α = 0.05).

3. Results

3.1. Analysis of Leaf Physiological and Biochemical Indexes of Different Varieties

Under natural drought stress, there were significant differences in antioxidant capacity among seven apple varieties grafted on SH40. The activities of POD, CAT, and SOD in leaves of ‘Red General’ were significantly higher than those of other varieties, being 3.2, 2.9, and 6.7 times those of ‘Huashuo’, respectively. ‘Yanfu 6’ ranked second, with 2.8, 2.6, and 6.1 times those of ‘Huashuo’ (Figure 2a–c), indicating that both varieties could significantly enhance antioxidant enzyme activities to scavenge ROS and alleviate oxidative damage. The MDA content in leaves of ‘Golden Delicious’ was significantly higher than that of other varieties, while those of ‘Yanfu 8’ and ‘Yanfu 6’ were significantly lower (Figure 2d), suggesting that the former suffered severe oxidative damage, while the latter two had strong antioxidant capacity and slight cell damage. The proline content in leaves of ‘Starking Delicious’ was significantly the highest, and the soluble protein contents in ‘Yanfu 3’, ‘Yanfu 8’, ‘Huashuo’, and ‘Red General’ were relatively high (Figure 2e,f). ‘Yanfu 6’ had the highest total chlorophyll content, followed by ‘Yanfu 3’, and ‘Huashuo’ had the lowest (Figure 2g), indicating that high chlorophyll content is beneficial to enhancing drought resistance.

Figure 2. Physiological and biochemical analysis of grafting different varieties of apple SH40: (a) POD activity, (b) CAT activity, (c) SOD activity, (d) MDA content, (e) Pro content, (f) SP content, and (g) chlorophyll content. Lowercase letters in the same column indicate significant differences between varieties at the p = 0.05 level.

3.2. Effects of Different Grafted Varieties on Leaf Photosynthetic Characteristics Under Drought Stress

When water is insufficient, plant photosynthesis is inhibited, leading to a reduction in organic matter synthesis and affecting plant growth. Under natural drought stress, there were significant differences in photosynthetic characteristics among the seven apple varieties grafted on SH40. Among them, the Tr, Gs, and Pn of leaves in ‘Red General’ were significantly higher than those in other varieties. The Ci of leaves in ‘Golden Delicious’ was significantly higher than that in other varieties (Table 1).

Table 1. Comparison of photosynthetic characteristics of different grafted apple varieties under drought stress.

Varieties	Tr/(mmol·m−2·s−1)	Gs/(mmol·m−2·s−1)	Pn/(μmol·m−2·s−1)	Ci/(μmol·mol−1)
Yanfu 3	1.849 ± 0.015 e	93.617 ± 1.615 e	8.409 ± 0.158 e	362.677 ± 1.856 f
Yanfu 6	2.471 ± 0.010 d	135.673 ± 0.435 d	9.612 ± 0.053 d	390.798 ± 0.552 d
Yanfu 8	3.376 ± 0.095 c	199.159 ± 1.551 c	11.388 ± 0.352 b	423.928 ± 1.848 c
Huashuo	1.671 ± 0.071 f	80.088 ± 0.515 f	4.564 ± 0.284 f	439.270 ± 11.741 b
Golden Delicious	3.978 ± 0.099 b	226.383 ± 1.583 b	10.091 ± 0.131 c	464.020 ± 0.723 a
Starking Delicious	1.598 ± 0.074 f	74.043 ± 1.411 g	3.620 ± 0.100 g	424.954 ± 0.789 c
Red General	5.463 ± 0.026 a	343.578 ± 1.397 a	12.589 ± 0.372 a	378.567 ± 0.974 e

Note: Under drought conditions, the measured parameters include transpiration rate (Tr), stomatal conductance (Gs), net photosynthetic rate (Pn), and intercellular CO₂ concentration (Ci). Lowercase letters in the same column indicate significant differences between varieties at the p = 0.05 level.

3.3. Analysis and Comparison of Leaf Anatomical Structure Characteristics of Different Grafted Varieties Under Drought Stress

The leaf anatomical structure characteristics varied among different varieties (Table 2). Under natural drought stress, the thickness of upper epidermis and midvein of leaves in ‘Yanfu 3’ was the largest; the thickness of leaves, mesophyll, and spongy tissue in ‘Huashuo’ was the largest; the palisade–spongy ratio and cell tightness of leaves in ‘Golden Delicious’ were significantly greater than those in other varieties; ‘Red General’ had a large lower epidermis thickness and high cell tightness.

Table 2. Comparison of leaf anatomical structures of different grafted apple varieties under drought stress.

Varieties	TU/μm	TL/μm	TP/μm	TS/μm	LT/μm	MesT/μm
Yanfu 3	21.056 ± 0.536 a	14.944 ± 0.096 b	129.833 ± 1.201 e	142.556 ± 2.009 b	299.278 ± 1.347 c	257.556 ± 2.070 d
Yanfu 6	19.722 ± 0.509 b	14.389 ± 0.096 c	134.500 ± 2.021 d	129.556 ± 1.295 c	291.667 ± 2.404 d	273.167 ± 2.186 b
Yanfu 8	18.444 ± 0.255 c	13.556 ± 0.192 d	143.000 ± 0.167 c	129.889 ± 0.420 c	310.444 ± 1.171 b	266.500 ± 2.682 c
Huashuo	17.500 ± 0.441 d	14.222 ± 0.192 c	133.500 ± 2.048 d	165.500 ± 0.333 a	343.500 ± 1.364 a	299.222 ± 2.084 a
Golden Delicious	20.333 ± 0.289 b	14.222 ± 0.536 c	153.667 ± 2.000 a	120.667 ± 1.302 d	309.500 ± 0.764 b	276.667 ± 1.667 b
Starking Delicious	17.389 ± 0.385 d	13.611 ± 0.255 d	122.778 ± 0.536 f	129.056 ± 1.834 c	276.444 ± 1.072 e	247.778 ± 0.822 e
Red General	17.722 ± 0.347 d	15.500 ± 0.441 a	147.222 ± 0.855 b	130.722 ± 1.619 c	296.889 ± 3.155 c	272.833 ± 2.566 b
Varieties	MV/μm	M/L/μm	P/S	CTR%	SR%
Yanfu 3	1645.500 ± 1.202 a	5.498 ± 0.029 a	0.911 ± 0.014 f	43.383 ± 0.487 d	47.632 ± 0.484 a
Yanfu 6	1413.444 ± 0.948 c	4.846 ± 0.037 c	1.038 ± 0.005 d	46.120 ± 1.075 b	44.424 ± 0.813 c
Yanfu 8	1026.944 ± 0.347 d	3.308 ± 0.014 d	1.101 ± 0.005 c	46.064 ± 0.209 b	41.840 ± 0.123 d
Huashuo	799.667 ± 1.093 f	2.328 ± 0.012 g	0.807 ± 0.013 g	38.865 ± 0.607 e	48.181 ± 0.099 a
Golden Delicious	876.111 ± 3.513 e	2.831 ± 0.018 e	1.274 ± 0.021 a	49.649 ± 0.526 a	38.988 ± 0.407 e
Starking Delicious	1504.278 ± 2.810 b	5.442 ± 0.011 b	0.951 ± 0.011 e	44.414 ± 0.231 c	46.686 ± 0.780 b
Red General	773.500 ± 2.848 g	2.606 ± 0.035 f	1.126 ± 0.007 b	49.590 ± 0.246 a	44.030 ± 0.120 c

Note: Under drought conditions, the measured parameters include upper epidermis thickness (TU), lower epidermis thickness (TL), palisade tissue thickness (TP), spongy tissue thickness (TS), leaf thickness (LT), mesophyll thickness (MesT), Midvein thickness (MV), palisade tissue and spongy tissue ratio (P/S), tightness of leaf palisade tissue (CTR) and looseness of leaf spongy tissue (SR). Lowercase letters in the same column indicate significant differences between varieties at the p = 0.05 level.

3.4. The Fruit Shapes of Seven Apple Varieties

The fruit trait characteristics of seven apple varieties grafted with SH40 as the interstock are as follows: The fruit coloring types include three categories, namely streaked red, blotched red, and striped red. The ground colors of the fruits cover a variety of types, such as green, yellowish green, yellowish white, and golden yellow. If the overcolor of the fruit surface is predominantly bright red, it indicates a good overall coloring degree. The specific trait characteristics are presented in Table 3.

Table 3. Description of external morphological traits of 7 apple varieties grafted on SH40 interstock.

Varieties	Fruit Ground Color	Fruit Overcolor	Fruit Coloration Intensity	Fruit Coloration Pattern
Yanfu 3	Yellowish Green	Bright red	Above-Medium Coloring	Blotched Red
Yanfu 6	Yellowish Green	Dark red	Medium Coloring	Streaked Red
Yanfu 8	Yellowish White	Bright red	High Coloring	Blotched Red
Huashuo	Yellowish Green	Bright red	High Coloring	Blotched Red
Golden Delicious	Golden Yellow	No or Slight Red on the Sunny Side	Low Coloring	No Coloring
Starking Delicious	Yellow	Deep red	High Coloring	Streaked Red
Red General	Yellow	Bright red	High Coloring	Blotched Red

3.5. Variation in Fruit Diameter, Shape Index, and Yield per Plant Across 7 Apple Varieties

There is rich diversity in the size, single-fruit mass, and fruit shape of the fruits of different cultivars grafted on SH40 (Table 4), and there are significant differences in most traits among cultivars (p < 0.05). In terms of fruit size, the ‘Red General’ cultivar shows significant advantages. The transverse, longitudinal, and lateral diameters of ‘Red General’ are all the largest, and there are significant differences compared with most cultivars. In contrast, ‘Yanfu 3’, ‘Yanfu 6’, and ‘Yanfu 8’ have significantly smaller values in all size indicators, with overall smaller fruit specifications. In terms of single-fruit mass, ‘Red General’ has the highest value, significantly higher than other cultivars, followed by ‘Huashuo’, and both are representatives of large-fruit types. The analysis of the fruit shape index shows that the fruit shape is mainly round or nearly round. ‘Golden Delicious’ has the highest fruit shape index, and its fruit shape is the closest to a circle; ‘Starking Delicious’, ‘Huashuo’, and ‘Red General’ also have relatively high fruit shape indices (all 0.88), and their fruit shapes are relatively regular; while ‘Yanfu 3’ has the lowest fruit shape index (0.73), and its fruits are obviously flat.

Table 4. Comparison of fruit external quality and yield of 7 apple varieties grafted on SH40 interstock.

Varieties	Horizontal Diameter /mm	Vertical Diameter /mm	Lateral Diameter /mm	Single Fruit Weight/g	Fruit Shape Index	Yield per Plant (kg/Plant)
Yanfu 3	77.35 ± 5.35 bc	56.89 ± 5.8 e	57.47 ± 5.68 d	192.15 ± 28.41 d	0.73 ± 0.04 d	22.67 ± 1.04 ab
Yanfu 6	74.27 ± 4.01 cd	58.65 ± 5.37 de	59.76 ± 6.28 d	202.60 ± 38.78 bcd	0.79 ± 0.06 c	24.08 ± 1.42 a
Yanfu 8	73.13 ± 3.42 d	57.38 ± 4.29 de	56.64 ± 4.64 d	197.00 ± 25.01 cd	0.78 ± 0.04 c	21.67 ± 1.26 abc
Huashuo	79.75 ± 3.81 b	69.71 ± 2.63 b	68.56 ± 3.16 b	220.30 ± 15.43 bc	0.88 ± 0.06 a	19.50 ± 1.32 c
Golden Delicious	79.36 ± 3.91 b	71.48 ± 2.41 ab	69.71 ± 1.74 ab	190.05 ± 8.37 d	0.90 ± 0.06 a	19.83 ± 2.02 bc
Starking Delicious	80.50 ± 3.91 b	70.68 ± 2.41 b	70.60 ± 1.74 ab	196.20 ± 8.37 d	0.88 ± 0.06 a	18.67 ± 2.02 c
Red General	84.58 ± 2.44 a	74.81 ± 3.9 a	73.22 ± 4.35 a	222.26 ± 22.64 b	0.88 ± 0.04 a	21.00 ± 2.00 bc

Note: Lowercase letters in the same column indicate significant differences between varieties at the p = 0.05 level.

Overall, ‘Red General’ shows a balanced performance in terms of fruit size, single-fruit mass, and fruit shape, with the best appearance quality; ‘Golden Delicious’ has an elegant fruit shape but average single-fruit mass; the Yanfu series cultivars generally perform poorly in terms of appearance quality. The per-plant yield ranges from 18.67 to 24.08 kg, with ‘Starking Delicious’ having the lowest yield and ‘Yanfu 6’ having the highest.

3.6. Principal Component Analysis of Seven Apple Cultivars Grafted on SH40 Interstock

PCA was performed on 28 indicators, and the results showed that the cumulative variance contribution rate of the five principal components was 95.492%, indicating that these five principal components could represent most of the information of drought resistance indicators (Table 5).

Table 5. Principal component load matrix and contribution rate.

Index	The First Principal Component	The Second Principal Component	The Third Principal Component	The Fourth Principal Component	The Fifth Principal Component
POD	0.930	−0.265	0.197	0.002	0.064
CAT	0.722	−0.312	0.018	0.553	−0.096
SOD	0.553	−0.392	0.578	0.381	−0.238
Concentration of soluble protein	−0.261	0.279	−0.373	0.393	0.299
Pro	0.551	−0.054	0.807	−0.024	−0.188
MDA	0.204	0.279	0.06	−0.621	0.701
Total chlorophyll content	0.567	−0.805	0.089	0.022	0.021
TU	0.114	−0.555	−0.49	−0.259	0.478
TL	0.368	0.042	−0.151	0.73	0.503
TP	0.689	0.422	−0.564	−0.165	0.026
TS	−0.814	0.394	−0.089	0.382	0.111
LT	−0.462	0.688	−0.538	−0.001	0.006
MesT	−0.19	0.713	−0.427	0.162	−0.184
MV	−0.296	−0.889	0.301	−0.052	0.165
M/L	−0.213	−0.883	0.392	−0.053	0.133
P/S	0.889	0.023	−0.254	−0.378	−0.048
CTR%	0.978	−0.117	−0.091	−0.125	0.008
SR%	−0.727	−0.099	0.384	0.536	0.163
Tr	0.868	0.308	−0.282	0.201	0.008
Gs	0.856	0.297	−0.283	0.243	−0.005
Pn	0.717	−0.107	−0.639	0.204	−0.039
Ci	−0.037	0.582	0.055	−0.771	−0.231
Horizontal Diameter	0.328	0.561	0.523	0.341	0.433
Vertical Diameter	0.377	0.741	0.529	0.035	0.142
Lateral Diameter	0.361	0.684	0.602	0.065	0.133
Single Fruit Weight	−0.036	0.61	0.058	0.754	−0.226
Fruit Shape Index	0.312	0.775	0.485	−0.183	−0.075
Yield per Plant	0.086	−0.685	−0.553	0.374	−0.157
Eigenvalue	8.861	7.654	4.732	3.841	1.650
Variance contribution rate	31.645	27.335	16.901	13.718	5.893
Accumulation rate	31.645	58.981	75.881	89.599	95.492

3.7. Comprehensive Evaluation of Seven Apple Cultivars Grafted on SH40 Interstock

Through the subordinate function analysis method, the comprehensive drought resistance scores of the seven apple cultivars grafted on SH40 interstock ranged from 0.281 to 0.779. The order of drought resistance from high to low was as follows: ‘Red General’, ‘Golden Delicious’, ‘Starking Delicious’, ‘Huashuo’, ‘Yanfu 6’, ‘Yanfu 8’, and ‘Yanfu 3’. The top-ranked is ‘Red General’, indicating that it had higher drought resistance than the other cultivars in Lingwu City, with better field performance, and was suitable for large-scale promotion and application in the Lingwu area. However, the variety scores of the Yanfu series are not very high, and therefore this variety is not suitable for the arid conditions in this area (Table 6).

Table 6. The membership function values and comprehensive evaluation values of drought resistance in 7 apple varieties.

Varieties	U1	U2	U3	U4	U5	D value	Ranking
Yanfu 3	0.278	0.000	0.209	0.626	1.000	0.281	7
Yanfu 6	0.590	0.004	0.302	0.643	0.000	0.342	5
Yanfu 8	0.479	0.371	0.000	0.334	0.092	0.319	6
Huashuo	0.000	1.000	0.323	0.620	0.364	0.455	4
Golden Delicious	0.876	0.663	0.241	0.000	0.649	0.563	2
Starking Delicious	0.440	0.309	1.000	0.320	0.366	0.480	3
Red General	1.000	0.695	0.415	1.000	0.507	0.779	1

4. Discussion

Lingwu, Ningxia, is located in the arid region of northwestern China, where climate change has intensified the frequency of seasonal droughts. In this context, the SH40 interstock has become an important choice for local dwarf and dense planting due to its strong cold resistance and shoot-drying resistance [9,26].

In this study, PCA and membership function method were used to comprehensively evaluate the drought resistance of seven apple cultivars grafted on SH40 interstock. The cumulative variance contribution rate reached 95.492%, indicating that this method can effectively integrate multi-dimensional physiological, biochemical, and structural indicators (such as antioxidant enzyme activity, photosynthetic parameters, and leaf anatomical characteristics) and fruit appearance quality and yield per plant, thus avoiding the limitations of a single indicator. This is consistent with the theory proposed that “crop drought resistance needs to be evaluated through multi-dimensional synergy” [27]. The final ranking (‘Red General’ > ‘Golden Delicious’ > ‘Starking Delicious’ > others) not only provides a direct basis for cultivar selection in the arid region of Lingwu but also reveals the diversity of drought resistance strategies among different cultivars: ‘Red General’ is primarily characterized by physiological activity, ‘Golden Delicious’ relies on structural adaptation, and ‘Starking Delicious’ demonstrates a stable drought resistance capacity across various aspects.

Under drought stress, ‘Red General’ showed excellent antioxidant capacity: its SOD, POD, and CAT activities were significantly higher than those of other cultivars (p < 0.05). As the first line of defense against superoxide free radicals [28], SOD, together with POD and CAT, synergistically reduces the damage of ROS to the membrane system, which explains its low accumulation of MDA (a marker of membrane lipid peroxidation). In contrast, ‘Golden Delicious’ had the highest MDA content, suggesting insufficient membrane repair capacity. However, it is worth noting that it maintained cellular osmotic balance by significantly increasing Pro content (the highest among cultivars, with ‘Starking Delicious’ being the highest) [29], and this osmotic adjustment compensation mechanism is an important basis for its second place in the membership function ranking. In addition, ‘Starking Delicious’, which ranks third, exhibits relatively strong antioxidant capacity. This indicates that under drought conditions, while maintaining a relatively stable state, it can also mitigate the threats posed by drought stress, thereby effectively regulating itself.

There is a certain synergistic adaptation between photosynthetic performance and anatomical structure. Drought significantly inhibited photosynthesis, but there were significant differences among cultivars: ‘Red General’ maintained the highest Pn, Gs, and Tr, indicating its excellent stomatal regulation ability, which can maintain carbon assimilation efficiency when water is limited. This phenomenon is consistent with the “stomatal limitation priority” theory proposed by Flexas et al. [30]: stomatal closure in the early stage of drought reduces water loss, but excessive closure leads to a decrease in Pn, while ‘Red General’ achieves a balance between the two.

Leaf anatomical structure further reveals the adaptation mechanism: ‘Golden Delicious’ had the significantly largest palisade–sponge ratio (ratio of palisade tissue to sponge tissue thickness) and cell compactness, which is consistent with the evolutionary strategy of “leaf compaction” in plants in arid regions [31]. A high palisade–sponge ratio enhances light energy capture efficiency, and the tight cell arrangement reduces the water diffusion path, thereby reducing transpiration loss. In contrast, the thickening of the upper epidermis and midrib in ‘Yanfu 3’ (physical barriers to reduce water loss) and the thickening of the mesophyll in ‘Huashuo’ (expanding photosynthetic area) represent different directions of structural adaptation.

‘Golden Delicious’ showed a unique contradiction of “high damage-high adaptation”: it had the highest MDA content (severe membrane damage), but ranked second in the membership function, and had a significantly optimal palisade–sponge ratio and cell compactness. This indicates that structural adaptability can partially offset physiological damage [32]; the osmotic protection of Pro alleviated oxidative damage; and the weight of the “structural factor” in principal component analysis highlights its value. However, in extreme drought years, the vulnerability of the membrane system of ‘Golden Delicious’ may become a limiting factor, so it is recommended to complement their use by promoting water-saving irrigation technologies.

In terms of fruit appearance quality and yield per plant, whether it is the fruit shape index, single-fruit mass, or yield per plant, ‘Red General’ shows excellent performance. This indicates that ‘Red General’ can effectively regulate itself under drought stress and produce high-quality fruits. In contrast, ‘Starking Delicious’ has a relatively light single-fruit mass, and its yield per plant is even the lowest, suggesting that its reproductive growth has been inhibited. However, it has good antioxidant capacity, which implies that drought induces oxidative stress and activates the antioxidant defense system, triggering the allocation strategy of ‘survival before reproduction’ [33]. That is, limited resources are preferentially allocated to defensive and maintenance functions such as the leaf antioxidant system and root water absorption, rather than reproductive processes like fruit development [34]. As a result, the fruits receive insufficient photosynthetic products and nutrients, leading to a decrease in fruit-setting rate and single-fruit mass, and ultimately a reduction in yield per plant. Meanwhile, due to the increased resource input, the antioxidant system related to defense shows stronger activity, which also indicates its good drought resistance. This is the main reason why it ranks third in the final ranking.

Based on the drought-resistance ranking and maturity differences, cultivation strategies for the arid area of Lingwu are proposed. The recommended varieties are as follows: ‘Red General’ (mid-maturing): It has comprehensive physiological resistance, high photosynthetic efficiency, and shows a balanced performance in terms of fruit size, single-fruit mass, and fruit shape, with the best appearance quality. It is suitable for moderately and slightly arid areas. ‘Golden Delicious’ (mid-maturing): It requires supporting water management, and its structural advantages may be more significant in sandy soil areas [35]. ‘Starking Delicious’: It needs optimized water management, with a focus on precise water replenishment during critical growth periods. It is suitable for semi-arid areas and semi-humid drought-prone areas.

This study is based on natural drought stress, which is closer to actual production, but has the limitation that precipitation fluctuations are uncontrollable. It is recommended that, in the future, additional gradient water control experiments be set up to quantify the water requirement thresholds of cultivars; combined with fruit quality analysis (such as sugar–acid ratio, ardness, brittleness), to avoid improving drought resistance at the expense of commodity quality, and to analyze the molecular mechanism of SH40 interstock regulating the drought resistance of scions (such as ABA signaling pathway), so as to provide targets for breeding.

In addition, three replicate samples are still insufficient for a representative sample size. Increasing the number of biological replicate samples remains the optimal choice. However, for the mature fruit trees used in this study, the experimental scale is inherently limited by multiple restrictive factors in practical operations, mainly in two aspects. The first is resource intensity: each combination of tree species or different stand configurations requires a large area of land, and it takes several years of growth for the trees to reach the mature fruiting stage before evaluation can be carried out. The second factor is uniformity of plant materials: to ensure the reliability of comparison results, trees with consistent age, size, and health status must be selected, which further narrows the scope of experimental samples. It should be specifically noted that the experimental unit in our study is individual trees. The specific sampling method (collecting and mixing leaves from four directions) was implemented for three independent trees of each variety, respectively. This protocol aims to obtain representative samples from each biological replicate and minimize intra-tree variation, rather than preparing technical subsamples from a single source. Subsequently, the mixed samples were aliquoted for different analysis items such as chlorophyll detection, biochemical analysis, and reserve testing. The significant and consistent differences observed among different varieties in multiple independent physiological and anatomical indicators are sufficient to demonstrate the robustness and biological significance of the core trends and final rankings. Naturally, the above-mentioned limitations need to be verified through larger-scale subsequent experiments.

In conclusion, by integrating multi-dimensional evidence from physiological and biochemical responses, photosynthetic performance, anatomical structure, as well as fruit appearance quality and yield, the adaptive differentiation mechanism of apple varieties grafted on SH40 in the arid region of Lingwu has been elucidated. The coordinated regulation of antioxidation and stomata in ‘Red General’, the structural optimization in ‘Golden Delicious’, and the overall stability in ‘Starking Delicious’ jointly constitute the scientific basis for regional variety selection. In the future, in-depth exploration is required in the fields of interstock–scion interaction mechanisms and the coordinated optimization of quality and resistance.

5. Conclusions

In conclusion, under natural drought conditions, the drought resistance of seven apple varieties grafted on SH40 in Lingwu City, Ningxia, in descending order is as follows: ‘Red General’, ‘Golden Delicious’, ‘Starking Delicious’, ‘Huashuo’, ‘Yanfu 6’, ‘Yanfu 8’, and ‘Yanfu 3’. Among them, ‘Red General’ exhibits favorable antioxidant capacity and excellent fruit appearance quality, while ‘Golden Delicious’ has a relatively high intercellular CO₂ concentration, palisade-to-spongy tissue ratio, and cell compactness. Therefore, under natural drought conditions, Lingwu City can give priority to the mid-ripening varieties ‘Red General’, ‘Golden Delicious’, and ‘Starking Delicious’ grafted on SH40.