# Rooting for resilience: Arbuscular Mycorrhizal Fungi (AMF) boost citrus tolerance to water scarcity in rangpur lime and carrizo citrange rootstocks

Sajjad Hussain, Mehwish Jan, Muhammad Fasih Khalid, Sakeen Tul-Ain

Haider, Muhammad Arif Ali, Shakeel Ahmad, Samreen Sabir, Mamoudou

Sétamou, Raphaël Morillon

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Sajjad Hussain, Mehwish Jan, Muhammad Fasih Khalid, Sakeen Tul-Ain Haider, Muhammad Arif Ali, et al.. Rooting for resilience: Arbuscular Mycorrhizal Fungi (AMF) boost citrus tolerance to water scarcity in rangpur lime and carrizo citrange rootstocks. Horticulturae, 2025, 11 (1), pp.24. 10.3390/horticulturae11010024 . hal-05246348

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Article

# Rooting for Resilience: Arbuscular Mycorrhizal Fungi (AMF) Boost Citrus Tolerance to Water Scarcity in Rangpur Lime and Carrizo Citrange Rootstocks

Sajjad Hussain <fnref n="1" />,<fnref n="2" />,* ID, Mehwish Jan <fnref n="2" />, Muhammad Fasih Khalid ³ID, Sakeena Tul-Ain Haider ²ID,
Muhammad Arif Ali ⁴ID, Shakeel Ahmad ⁵ID, Samreen Sabir ²ID, Mamoudou Sétamou <fnref n="1" /> and Raphael Morillon ⁶ID

**Citation:** Hussain, S.; Jan, M.; Khalid, M.F.; Haider, S.T.-A.; Ali, M.A.; Ahmad, S.; Sabir, S.; Sétamou, M.; Morillon, R. Rooting for Resilience: Arbuscular Mycorrhizal Fungi (AMF) Boost Citrus Tolerance to Water Scarcity in Rangpur Lime and Carrizo Citrange Rootstocks. *Horticulturae* 2025, 11, 24. https://doi.org/10.3390/horticulturae11010024

**Copyright:** © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/licenses/by/4.0/).

**Abstract:** Fruits and vegetables play a crucial role in addressing food security challenges posed by the growing global population. Citrus fruits are among the most widely cultivated crops worldwide; however, their production is steadily declining due to climate change. Among the various biotic and abiotic stresses affecting citrus production, water scarcity caused by climate change stands out as a significant issue. Interestingly, the rhizosphere of citrus plants is home to beneficial fungi known as arbuscular mycorrhizal fungi (AMF). AMF have been shown to enhance the growth and development of host plants. They also improve the plants' tolerance to various stresses and enhance soil structure. This study aimed to evaluate the response of two different citrus rootstocks—Rangpur lime and Carrizo citrange—when subjected to three mycorrhizal treatments, namely, AMF+ (inoculated with AMF), AMF- (treated with fungicide to eliminate AMF), and a control (naturally occurring AMF), under conditions of water deficit. The results indicated that the AMF+ treatment had a significant positive effect on both rootstock genotypes compared to the AMF- treatment. Physiological attributes such as photosynthesis, stomatal conductance, transpiration, non-photochemical quenching, and both dark and light quantum yield exhibited significantly smaller declines under water deficit conditions in AMF+ plants compared to those in the AMF- and control groups. Conversely, stress indicators—such as malondialdehyde (MDA) and hydrogen peroxide ($H_2O_2$)—increased significantly in the AMF- treatment compared to AMF+. Additionally, the increase in antioxidative enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APx) and osmotic adjustment (proline (PRO))) was more pronounced in the AMF+ treatment in the leaves and roots of both citrus rootstocks. In conclusion, the findings suggest that the presence and application of AMF in citrus roots may enhance the plants' ability to cope with water scarcity more effectively.

**Keywords:** drought stress; stress physiology; citrus rootstocks; photosynthesis; oxidative enzymes; arbuscular mycorrhiza

<footnote n="1">¹ Citrus Center, Texas A&M University-Kingsville, Weslaco, TX 78599, USA; mamoudou.setamou@tamuk.edu</footnote>

<footnote n="2">² Department of Horticulture, Faculty of Agricultural Science & Technology, Bahauddin Zakariya University, Multan 60000, Pakistan; samreensajjad736@gmail.com (S.S.)</footnote>

<footnote n="3">³ Environmental Science Centre, Qatar University, Doha 2713, Qatar; mfasih.khalid@qu.edu.qa</footnote>

<footnote n="4">⁴ Department of Environmental Sciences, Faculty of Science, Bahauddin Zakariya University, Multan 60000, Pakistan; arif1056@bzu.edu.pk</footnote>

<footnote n="5">⁵ Department of Agronomy, Faculty of Agricultural Science & Technology, Bahauddin Zakariya University, Multan 60000, Pakistan; shakeelahmad@bzu.edu.pk</footnote>

<footnote n="6">⁶ Structure Evolutive des Agrumes, Polyploïdie et Amélioration Génétique (SEAPAG), UM AGAP, Département BIOS, CIRAD, Avenue Agropolis, 34398 Montpellier, Cedex 5, France; raphael.morillon@cirad.fr</footnote>

<footnote>* Correspondence: sajjad.hussain@tamuk.edu</footnote>
# 1. Introduction

The ever-increasing global population and changing dietary patterns are driving demand for more food. According to reports from the World Bank, nearly 690 million people worldwide are currently hungry, a number expected to rise by approximately 60 million by 2025 [1]. The challenge of food security is becoming more difficult, particularly due to agriculture's extreme vulnerability to climate change. The negative impacts of climate change are already evident, manifested in rising temperatures, weather variability, shifting agro-ecosystem boundaries, the spread of invasive crops and pests, and more frequent extreme weather events. These factors are reducing the crop yields and nutritional quality of major cereals, as well as lowering livestock productivity, exacerbating food security challenges.

The continuously changing climate imposes numerous biotic and abiotic stresses on plants, hindering their growth, development, and productivity [2]. Among these abiotic stresses, water scarcity is the most critical factor impacting crop health and productivity [3]. Drought stress poses a significant threat to citrus production globally, particularly in regions characterized by arid and semi-arid climates. The physiological and biochemical responses of citrus plants to drought stress are complex, often leading to reduced growth, yield, and fruit quality [4,5]. When plants are exposed to water deficit conditions, they close their stomata to minimize water loss through transpiration. Prolonged water deficit ultimately leads to oxidative stress due to the increased production of reactive oxygen species (ROS). These include superoxide (O²⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (HO⁻), which can act as signaling molecules. However, excessive production of ROS disrupts metabolic processes in the chloroplasts and mitochondria [2,6]. To mitigate the adverse effects of ROS, plants generate various antioxidative enzymes and osmolytes. These enzymes scavenge ROS, helping plants to tolerate drought stress more effectively [4,5,7,8].

Among various strategies to mitigate these adverse effects, the use of mycorrhizal fungi, particularly arbuscular mycorrhizae (AM), has emerged as a promising approach. Mycorrhizae form symbiotic relationships with plant roots, enhancing nutrient uptake, improving soil structure, and increasing plant resilience to abiotic stresses, including drought [9–12]. Jiang et al. reported that 80% of terrestrial plant species form symbiotic relationships with AMF. In this relationship, the host plant provides carbon compounds for the growth of the arbuscular mycorrhizae fungus, while this fungus assists the plant in absorbing water and nutrients from the soil [13].

Research has demonstrated that mycorrhizal fungi can significantly enhance the drought tolerance of citrus plants by modifying root architecture and altering metabolic pathways. For instance, Wu et al. highlighted that AM fungi can influence the fatty acid composition of citrus roots, which is closely associated with drought tolerance [11]. This alteration in root composition may enhance the plant's ability to maintain cellular integrity under water-limited conditions. Furthermore, the presence of mycorrhizae has been shown to improve osmotic regulation and ionic balance in plants, which are critical for sustaining physiological functions during drought stress [14].

AMF improve plant tolerance under water deficit conditions through various mechanisms, including maintaining stomatal regulation via hormonal signals [15], aiding in the direct uptake of water and nutrients through mycorrhizal hyphae [16], enhancing the plant's defense mechanisms through osmolytes and antioxidative enzymes [8], improving soil structure through glomalin excreted from the hyphae [17], and upregulating stress-related genes [10,18,19]. In addition to improving physiological traits, mycorrhizal fungi also contribute to the overall health of the soil ecosystem. They promote the formation of a common mycorrhizal network, facilitating nutrient exchange and communication between neighboring plants [9]. This interconnectedness can lead to enhanced resilience not only in
individual plants but also across entire populations, thereby improving the sustainability of citrus cultivation in drought-prone areas. Moreover, mycorrhizal associations have been linked to increased antioxidant enzyme activity, which plays a crucial role in mitigating oxidative stress induced by drought [20].

Citrus is the world's major fruit crop and plays a vital role in the economies of many countries. Different citrus rootstocks exhibit variations in their tolerance mechanisms [21], with differences in photosynthesis and antioxidative defense mechanisms when exposed to water deficit stress [4,22]. It has been observed that rootstocks capable of tolerating greater drought stress tend to possess higher levels of antioxidative enzymes [4,5,20]. In the citrus rhizosphere, many soil microorganisms, such as bacteria, fungi, nematodes, protozoa, and algae, thrive, influenced by root exudates, plant species, plant developmental stages, and soil properties [23]. Studies have shown that AMF impact the photosynthetic attributes of citrus [24–26] and enhance both shoot and root growth. Plants associated with AMF also demonstrate better osmotic adjustment and improved photosynthetic attributes compared to non-AMF plants [26–28].

The integration of mycorrhizal fungi into citrus cultivation practices presents a mul-
tifaceted approach to enhance drought resilience. By fostering these beneficial symbiotic
relationships, growers can potentially improve the productivity and sustainability of cit-
rus crops in the face of increasing water scarcity and climate variability. This research
paper aims to explore the mechanisms by which mycorrhizae contribute to drought stress
mitigation in two citrus rootstocks: rangpur lime (Citrus limonia) and Carrizo citrange
(Poncirus trifoliata L. × Citrus sinensis L.), providing insights into their practical applications
in agricultural systems.

**2. Materials and Methods**

2.1. *Plant Material*

Healthy six-month-old plants of uniform size from two citrus rootstocks, Rangpur
lime (Citrus limonia) and Carrizo citrange (Poncirus trifoliata L. × Citrus sinensis L.), were
sourced from the Horticultural Research Station in Sahiwal, Pakistan. The pots of the same
size (9-inch diameter) and weight were chosen and filled with the same amount of dry
soil (4 kg soil in each pot). Seedlings were transplanted into these pots and kept in the
greenhouse at the Department of Horticulture, Bahauddin Zakariya University, Pakistan,
for the next six months to allow them to grow properly. They were regularly irrigated
with Hoagland solution. After one month, 36 uniform-sized plants of each rootstock were
selected for the experiment. The plants were inoculated with AMF inoculum of Rhizophagus
irregularis, obtained from the Department of Soil Science at Bahauddin Zakariya University,
Multan, Pakistan. Each rootstock was divided into three treatment groups: M₀ (Control, no
inoculum/natural microbiota), M₁ (AMF+, with AMF inoculation of 5 g per 20 kg of soil),
and M₂ (AMF-, with no microbiota). A negative control was established using Topsin-M
(Thiophanate Methyl 70% wettable powder), applied at a rate of 50 mg per kg of soil.

The plants were irrigated with Hoagland solution regularly to ensure optimal growth conditions during the acclimatization phase before the drought treatment. After two months of AMF treatments, half of the plants in each group (M₀, M₁, and M₂) were subjected to water deficit conditions by withholding irrigation for 12 days. The other half were treated as control plants and continued to be irrigated regularly with Hoagland solution. The experiment was replicated three times, and a randomized complete block design with three factors was employed for statistical analysis.
## 2.2. *Studied Attributes*

The data were collected at the end of the experiment after 12 days. All physiological analyses were performed on the last day before harvesting. One plant was used for biochemical analysis of roots and shoots, while the other plant was used to measure the soil colonization of AMF within each block per treatment.

## 2.3. Physiological Variables

The leaf photosynthetic rate, leaf stomatal conductance, and leaf transpiration rate were measured with an Infra-Red Gas Analyser (LCi-SD, ADC BioScientific Ltd., Hoddesdon, Herts EN11 0NT, United Kingdom). Quantum yield in light-adapted (Fv'/Fm') and in dark-adapted (Fv/Fm) leaves and non-photochemical quenching (NPQ) in dark-acclimated leaves were measured with a FluorPen (FP-100) Průmyslová 470 664 24 Drásov, Czech Republic. Chlorophyll content or leaf greenness was measured with a SPAD meter (SPAD-502, Konica Minolta, Sakai, Osaka, Japan). All light-adapted attributes were measured between 9 am and 11 am, while dark-adapted, leaves were covered for 1 h, and then measurements were taken. Leaf surface area was measured with a leaf area meter (CI-202) and relative water content was measured by using the fresh, dry, and turgid weight of the leaves and applying the formula as described by [5].

## 2.4. *Mycorrhizal Colonization*

The fine roots were washed with potassium hydroxide and then stained with trypan blue solution [28]. The gridline intersect method was used to search for the presence of an AM structure in roots [29].

## 2.5. Stress Indicators

The stress indicators, i.e., hydrogen peroxide ($H_2O_2$) and malondialdehyde (MDA), were measured by homogenizing the fresh samples of leaves and roots in trichloroacetic acid [5]. For $H_2O_2$ measurement, the method described in [30] was followed using potassium iodide and potassium phosphate buffer. For MDA analysis, the method described in [31] was used by using thiobutyric acid and trichloroacetic acid, and absorbance was measured at 600 and 532 nm using a spectrophotometer (UV-1900, BMS, Toronto, ON, Canada).

## 2.6. Antioxidative Enzymes

The fresh leaf and root samples were harvested and stored at -80 °C after crushing in liquid nitrogen for further analysis. The measurements of superoxide dismutase (SOD EC 1.15.1.1) [32] absorbance, measured at 560 nm, catalase (CAT EC: 1.11.1.6) [33] at 240 nm, and peroxidase (POD EC: 1.11.1.7) [32] at 470 nm, were carried out using sodium phosphate buffer pH 7.8 and then centrifuged at 15,000 rpm for 5 min. The method described in [34] was used for further analysis. For ascorbate peroxidase (APx EC: 1.11.1.11), the sample was homogenized in potassium phosphate buffer pH 7.0 by the method explained by [35] and absorbance was measured at 290 nm using a spectrophotometer (UV-1900, BMS, Toronto, ON, Canada).

## 2.7. Total Proline and Protein Content

The total proline content was measured using fresh leaf and root samples homogenized in sulfosalicylic acid. For further analysis, ninhydrin reagent, glacial acetic acid, and toluene were used as described by [36] and absorbance was measured at wavelength 520 nm using a spectrophotometer (UV-1900, BMS, Toronto, ON, Canada). To estimate the total soluble protein content, fresh samples of leaves and roots were homogenized in saline phosphate buffer at 7.2 pH. The solution for further analysis contained deionized water and Coomassie blue dye [37] and absorbance was measured at 595 nm.
## 2.8. Chlorophyll Content, Antioxidant Capacity, and Total Phenolic Content

Chlorophyll a and b were measured by homogenizing the fresh leaf samples in acetone as described by [38]. The absorbance was taken at 645 nm by using a spectrophotometer (UV-1900, BMS, Toronto, ON, Canada), and the following equation was applied:

$$ \text{Chlorophyll 'a' (mg g}^{-1}\text{)} = 100 \times [(0.0127 \times \text{A663} - \text{A645} \times 0.00269)]/0.5 $$

$$ \text{Chlorophyll 'b' (mg g}^{-1}\text{)} = 100 \times [(0.0229 \times \text{A645} - 0.00468 \times \text{A663})]/0.5 $$

The antioxidant capacity and total phenolic content were extracted by homogenizing leaves and roots in a solution containing ethanol, distilled water, and acetic acid (70:29:1 v/v) [39]. For antioxidant capacity, [40] 515 nm and [41] 750 nm wavelength was used to measure the absorbance.

## 2.9. Statistical Analysis

The collected data were statistically analyzed using Statitix 8.1 (Tallahassee, FL, USA). Mean LSD tests were used to compare at the 5% probability level. All figures were illustrated in SigmaPlot software version 12.5 (Systat Software, San Jose, CA, USA).

# 3. Results

## 3.1. Physiological Variables

Under water deficit conditions, significant effects of mycorrhizal treatments were observed on photosynthetic attributes, including photosynthesis, transpiration, stomatal conductance, and quantum yield, both day and night (Figure 1). Physiological attributes decreased under water deficit conditions compared to the untreated control, but the decrease was more pronounced in M₂ (AMF-). Conversely, the lowest decreases were observed in M₁ (AMF+). Control plants also showed reductions in all physiological attributes, but the reductions were more significant than those in M₁ plants (Figure 1).

Regarding non-photochemical quenching, all stress-treated plants exhibited increased values, with M₂ showing the highest value, followed by M₀. In contrast, M₁ exhibited the lowest value for non-photochemical quenching. Moreover, no significant differences were observed between the genotypes (Figure 1). Leaf greenness was not significantly affected by water deficit conditions, although the lowest values were recorded in M₂ (AMF-) under these circumstances. This indicates a significant interaction between water availability and mycorrhizal treatment (Figure 2A). Leaf greenness was also unaffected by genotype.

The relative water content (%) and leaf surface area were significantly impacted by water deficit (Figure 2B,C). Rangpur lime showed higher relative water content compared to Carrizo citrange under both control and water deficit conditions. A decrease in relative water content and leaf surface area was noted in the plants treated with M₂ (AMF-) in Carrizo citrange (Figure 2B,C). The mycorrhizal colonization percentage was highest in M₁ (AMF+) for both genotypes, while the lowest was found in M₂ (AMF-) (Figure 2D).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Gas exchange and chlorophyll fluorescence in the leaves of Rangpur lime (RL) and Carrizo citrange (CC) rootstock seedlings under control and water deficit conditions: (A) photosynthesis; (B) transpiration rate; (C) stomatal conductance; (D) NPQ; (E) Fv/Fm; (F) Fv'/Fm'. Values are mean ± S.E. (n = 3). The bar graphs correspond to = control, = water deficit. M₀ = control (no inoculum/natural microbiota); M₁ = AMF+ (AMF inoculation 5 g/20 kg soil); M₂ = AMF- (no microbiota). The different letters above bars, it means that those points are statistically significantly different from each other.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

**Figure 2.** (A) Leaf greenness SPAD; (B) relative water content%; (C) leaf surface area; (D) mycorrhizal colonization % in Rangpur lime (RL) and Carrizo citrange (CC) rootstock seedlings under control and water deficit conditions. Values are mean ± S.E. (n = 3). The bar graphs correspond to **= control**, □= water deficit. M₀ = control (no inoculum/natural microbiota); M₁ = AMF+ (AMF inoculation 5 g/20 kg soil); M₂ = AMF- (no microbiota). The different letters above bars, it means that those points are statistically significantly different from each other.

## 3.2. *Stress Indicators and Antioxidative Enzymes*

The stress indicators, MDA and H₂O₂, showed significant differences in the leaves and roots of both genotypes (Figures 3 and 4). The highest levels of MDA and H₂O₂ were observed in the leaves of M₂ (AMF-) for both genotypes, Rangpur lime and Carrizo citrange, under water deficit conditions. Conversely, the lowest levels of MDA and H₂O₂ under water deficit conditions were recorded in M₁ (AMF+) for both genotypes (Figure 3). In terms of stress indicators, the maximum levels of MDA and H₂O₂ were also found in the roots of M₂ (AMF-) for both genotypes (Figure 4). Interestingly, the levels of MDA and H₂O₂ were higher in well-irrigated plants of M₂ (AMF-) compared to those under water deficit conditions (Figure 4). The antioxidative enzymes, including SOD, POD, CAT, and APx, demonstrated significant differences between control and water deficit conditions in the leaves and roots of both citrus genotypes (Figures 3 and 4). The highest activities of SOD, POD, CAT, and APx were observed in the leaves and roots of both genotypes treated with M₁ (AMF+) under water deficit conditions. In contrast, the lowest levels of antioxidative enzymes were found in M₂ (AMF-) for both genotypes under drought stress conditions (Figures 3 and 4).
<figure><img src="image_4.png" /><figcaption>**Figure 3.** (A) Superoxide dismutase; (B) peroxidase; (C) catalase; (D) ascorbate peroxidase; (E) malondialdehyde; (F) hydrogen peroxide in the leaves of Rangpur lime (RL) and Carrizo citrange (CC) rootstock seedlings under control and water deficit conditions. Values are mean ± S.E. (n = 3). The bar graphs correspond to = control, = water deficit. M₀ = control (no inoculum/natural microbiota); M₁ = AMF+ (AMF inoculation 5 g/20 kg soil); M₂ = AMF- (no microbiota). The different letters above bars, it means that those points are statistically significantly different from each other.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 4.** (A) Superoxide dismutase; (B) peroxidase; (C) catalase; (D) ascorbate peroxidase; (E) malondialdehyde; (F) hydrogen peroxide in the roots of Rangpur lime (RL) and Carrizo citrange (CC) rootstock seedlings under control and water deficit conditions. Values are mean ± S.E. (n = 3). The bar graphs correspond to **= control**, □ = water deficit. M₀ = control (no inoculum/natural microbiota); M₁ = AMF+ (AMF inoculation 5 g/20 kg soil); M₂ = AMF- (no microbiota). The different letters above bars, it means that those points are statistically significantly different from each other.</figcaption></figure>

### 3.3. Osmolytes and Other Biochemical Compounds

The total proline content in leaves and roots was significantly higher under water deficit conditions compared to the control group (see Figures 5 and 6). The results indicated that the maximum proline was observed in the leaves of both genotypes treated with M₁ (AMF+) during water deficit conditions (Figure 5). Conversely, the minimum proline content was noted in the roots of Rangpur lime rootstock treated with M₂ (AMF-). In the roots, the minimum proline value was also found in the Rangpur lime rootstock with M₂ (AMF-), while the maximum proline content was observed in the M₁ (AMF+)-treated
Carrizo citrange under the same water deficit conditions (Figure 6). Protein content was significantly higher in the M₁ (AMF+) leaves and roots of Carrizo citrange (Figures 5 and 6). In the leaves, higher protein content was recorded under control conditions for Carrizo citrange treated with M₁ (AMF+). In contrast, in the roots, higher protein levels were seen in water deficit plants of Carrizo citrange treated with M₁ (AMF+). The lowest protein values were found in the leaves and roots of Rangpur lime treated with M₂ (AMF-) (Figures 5 and 6).

<figure><img src="image_6.png" /></figure>

**Figure 5.** Measurements of the biochemical parameters along the stress in leaves: (A) total proline content; (B) total soluble proteins; (C) chlorophyll 'a'; (D) chlorophyll 'b'; (E) antioxidant capacity; (F) total phenolic content in the leaves of Rangpur lime (RL) and Carrizo citrange (CC) rootstock seedlings under control and water deficit conditions. Values are mean ± S.E. (n = 3). The bar graphs correspond to = control, = water deficit. M₀ = control (no inoculum/natural microbiota); M₁ = AMF+ (AMF inoculation 5 g/20 kg soil); M₂ = AMF- (no microbiota). The different letters above bars, it means that those points are statistically significantly different from each other.
<figure><img src="image_7.png" /><figcaption>**Figure 6.** Measurements of the biochemical parameters along the stress in roots: (A) total proline content; (B) total soluble proteins; (C) antioxidant capacity; (D) total phenolic content in the roots of Rangpur lime (RL) and Carrizo citrange (CC) rootstock seedlings under control and water deficit conditions. Values are mean ± S.E. (n = 3). The bar graphs correspond to **= control**, □= water deficit. M₀ = control (no inoculum/natural microbiota); M₁ = AMF+ (AMF inoculation 5 g/20 kg soil); M₂ = AMF- (no microbiota). The different letters above bars, it means that those points are statistically significantly different from each other.</figcaption></figure>

The levels of chlorophyll 'a' and 'b' declined under water deficit conditions (Figure 4C,D). In well-irrigated (control) plants, chlorophyll content varied according to the mycorrhizal treatment. The maximum chlorophyll was found in M₁ (AMF+), while the minimum was in M₂ (AMF-) among the control plants (Figure 4C,D). Under water deficit conditions, the highest chlorophyll content was observed in M₁ (AMF+) in Carrizo citrange, whereas the minimum levels of chlorophyll 'a' and 'b' were found in M₂ (AMF-) for both the Rangpur lime and Carrizo citrange cultivars (Figure 4C,D). The antioxidant capacity of plants decreased under water deficit conditions. The maximum antioxidant capacity was recorded in the leaves of Carrizo citrange treated with M₁ (AMF+), while the minimum was observed in Rangpur lime treated with M₂ (AMF-) under water deficit conditions (Figure 4E). Although there was a trend of increasing antioxidant capacity in the roots under water deficit conditions, it was not statistically significant (Figure 6C).

Similarly, in the roots, the highest antioxidant capacity was noted in M₁ (AMF+) and the lowest in M₂ (AMF-). The total phenolic content increased in the leaves and roots of both rootstocks under water deficit conditions (Figures 4F and 6D). The maximum phenolic content was observed in the leaves and roots of plants treated with M₁ (AMF+), while the minimum was seen in those treated with M₂ (AMF-). Carrizo citrange exhibited higher
phenolic content compared to Rangpur lime in leaves, whereas Rangpur lime had more phenolics than Carrizo citrange in roots.

**4. Discussion**

In this study, two citrus rootstocks, i.e., Rangpur lime and Carrizo citrange, were exposed to water deficit conditions after arbuscular mycorrhizal fungi (AMF) treatments (control, AMF+, and AMF-) (Figure 7). Our results showed that AMF+ maintain a better growth under water deficit conditions than control and AMF- (Figure 8). The results of this study highlight the significant role of mycorrhizal treatments in enhancing the physiological responses of citrus plants under drought stress. The observed physiological variables, including photosynthesis, transpiration, stomatal conductance, and quantum yield, were markedly influenced by the presence of arbuscular mycorrhizal fungi (AMF). Specifically, plants treated with M₁ (AMF+) exhibited the least decline in these physiological attributes compared to untreated controls and those without mycorrhiza M₂ (AMF-). The AMF+ application in water deficit citrus rootstocks maintained the physiological attributes (photosynthesis, stomatal conductance, transpiration, non-photochemical quenching, and quantum yield (dark and light)) more efficiently as compared to AMF-. The decrease in photosynthesis under water deficit conditions was observed due to stomatal closure which resulted in limited diffusion of carbon [42]. When the quantum yield ratio decreases, it suggests that the energy transfer processes are disrupted, which can be caused by environmental stressors such as lack of water. In this case, the quantum yield (dark and light) ratios decreased, which is a direct indicator that the plants were suffering from photoinhibition. This aligns with previous findings that mycorrhizal fungi can enhance photosynthetic efficiency and water use efficiency in plants under stress conditions [14,43]. The enhanced physiological performance in M₁-treated plants suggests that AMF may improve the plant's ability to maintain photosynthetic activity and gas exchange, critical for sustaining growth during periods of water deficit.

<figure><img src="image_8.png" /><figcaption>**Figure 7.** Root microscopic analysis: (A) natural; (B) AMF inoculated; (C) no mycorrhiza of the roots of Rangpur lime (RL) and Carrizo citrange (CC) rootstock seedlings.</figcaption></figure>
Rangpur lime

<figure><img src="image_9.png" /></figure>

Carrizo citrange

<figure><img src="image_10.png" /><figcaption>**Figure 8.** Comparison of Rangpur lime and Carrizo citrange. M₀ = control (no inoculum/natural microbiota); M₁ = AMF+ (AMF inoculation 5 g/20 kg soil); M₂ = AMF- (no microbiota).</figcaption></figure>

Moreover, the increase in non-photochemical quenching (NPQ) values in stress-treated plants indicates an adaptive mechanism to dissipate excess energy and protect against photoinhibition under drought conditions. Notably, the highest NPQ was recorded in M₂ (AMF-), suggesting that these plants were less efficient in managing excess light energy, which could lead to oxidative stress [14,43]. The lower NPQ in M₁ (AMF+) indicates a more effective energy dissipation mechanism, which is crucial for maintaining photosynthetic integrity under stress [44]. The lack of significant differences in leaf greenness across treatments suggests that while mycorrhizal inoculation can enhance physiological traits, it may not directly influence chlorophyll content under drought stress, as supported by findings in other studies [45,46].

The relative water content (RWC) and leaf surface area were significantly affected by drought, with Rangpur lime displaying higher RWC compared to Carrizo citrange. This observation is consistent with previous research indicating that different citrus rootstocks exhibit varying degrees of drought tolerance, which can be partially attributed to their physiological responses to water stress [47,48]. The decline in RWC and leaf area in M₂ (AMF-)-treated Carrizo citrange further emphasizes the detrimental effects of insufficient mycorrhizal colonization on plant water status and overall health [49,50]. The mycorrhizal colonization percentage was highest in M₁ (AMF+), reinforcing the notion that effective mycorrhizal associations can enhance drought resilience by improving water uptake and nutrient acquisition [51,52].

The stress indicators, malondialdehyde (MDA) and hydrogen peroxide ($H_2O_2$), serve as markers of oxidative stress in plants. The elevated levels of these compounds in M₂ (AMF-) indicate a higher degree of oxidative damage under drought conditions, which cor-
relates with the lower activities of antioxidative enzymes observed in these plants [53,54]. Conversely, the lowest levels of MDA and H₂O₂ in M₁ (AMF+) suggest that mycorrhizal inoculation effectively mitigates oxidative stress, likely through enhanced antioxidant enzyme activity [55,56]. This is further supported by the significant increases in the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APx) in M₁-treated plants, which are crucial for scavenging reactive oxygen species (ROS) generated during drought stress [57,58].

In terms of osmolytes, the total proline content was significantly higher in M₁ (AMF+)-treated plants, indicating a robust osmotic adjustment mechanism under drought stress. Proline accumulation is a well-documented response to osmotic stress, serving both as an osmoprotectant and a signaling molecule [59,60]. The findings that M₁ (AMF+) plants had higher protein content under both control and drought conditions further support the notion that mycorrhizal fungi enhance the overall metabolic capacity of the host plant, enabling better stress management [61,62]. The observed decline in chlorophyll content under drought conditions across all treatments aligns with previous studies that report reduced chlorophyll levels because of water stress [63,64]. However, the maintenance of higher chlorophyll levels in M₁ (AMF+) suggests a protective effect of mycorrhizal inoculation on photosynthetic pigments, which is vital for sustaining photosynthetic activity during drought [65].

Overall, the results underscore the importance of mycorrhizal associations in enhancing the physiological and biochemical responses of citrus plants to drought stress. The differential responses observed between mycorrhizal treatments highlight the potential of AMF as a sustainable strategy for improving drought tolerance in citrus cultivation, thereby contributing to the resilience of agricultural systems in the face of climate variability.

**5. Conclusions**

The application of AMF in two citrus rootstocks, i.e., Rangpur lime and Carrizo citrange, significantly induced the tolerance against water deficit conditions, while the removal of AMF from these rootstocks makes the plants more susceptible to drought stress by decreasing their tolerance. The addition of AMF showed more stability in physiological attributes (photosynthesis, stomatal conductance, transpiration, non-photochemical quenching, and quantum yield) with less increase in stress indicators (MDA, H₂O₂) while increasing antioxidative enzymes (SOD, POD, CAT, APx) and osmotic adjustment (PRO) in the leaves and roots of both citrus rootstocks.

**Author Contributions:** Conceptualization, S.H., M.F.K. and R.M.; Methodology, M.F.K., M.A.A. and S.A.; Software, S.S.; Investigation, M.J. and S.S.; Resources, S.T.-A.H., M.A.A. and S.A.; Data curation, M.J. and M.A.A.; Writing—review & editing, S.H., M.F.K., S.T.-A.H., S.A., S.S., M.S. and R.M.; Supervision, S.H.; Project administration, S.H.; Funding acquisition, S.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.

**Conflicts of Interest:** The authors declare no conflict of interest.

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