Genotype-Specific Synergy Between Arbuscular Mycorrhizal Fungi and Olive Cultivars Enhances Drought Resilience in China’s Olive Belt

Abstract

To address severe seasonal drought affecting over 60% of China’s olive-growing regions, this study evaluates whether arbuscular mycorrhizal fungi (AMF) can enhance drought tolerance in elite olive cultivars (Arbequina and Koroneiki) under simulated arid conditions. A controlled pot experiment inoculated seedlings with two AMF strains (Rhizophagus intraradices [AMF1], Funneliformis mosseae [AMF2]) under full irrigation or a 32-day water deficit. Biomass, root colonization, photosynthesis, PSII efficiency, osmolytes, antioxidants, and lipid peroxidation were measured. Data were analyzed via two-way ANOVA, Pearson’s correlation, and principal component analysis (PCA). Under optimal hydration, both AMF strains colonized >60% of roots, significantly increasing Arbequina biomass by 25–35% (p < 0.05) and Koroneiki biomass. Drought reversed benefits in Arbequina but triggered resilience: AMF1 restored photosynthesis (18%), Fv/Fm (37%), and water potential (18%) (p < 0.05) while reducing lipid peroxidation (79%) (p < 0.01). In Koroneiki, AMF2 restored Ψw to 47% of pre-irrigation levels and increased root volume (137%), PSII efficiency (43%), osmolytes (100%), and carotenoids (28%) (p < 0.01). PCA ranked Arbequina–drought–AMF1 as the most resilient combination. Pairing AMF strains with specific cultivars offers a scalable, chemical-free strategy to stabilize olive productivity in southwest China’s aridifying climate, advancing climate-smart agriculture for drought-prone regions.

1. Introduction

Olive (Olea europaea L.), a long-lived oil fruit belonging to the Oleaceae family, is widely cultivated in China’s marginal mountainous zones due to its high yield and proven tolerance to drought, salinity, and infertility, which promise reliable returns [1,2,3]. However, field experiments reveal that even this resilient species becomes highly sensitive to water deficit during two distinct developmental stages [4,5]. First, during late-winter to early-spring vernalization, bud-break, and fruit-set, even mild soil-water shortages can rapidly trigger flower and drupe abscission [4,6], whereas second, from late summer to harvest, drought readily inhibits mesocarp metabolism and abruptly halts oil synthesis [5,7]. Moreover, the high temperatures and infrequent rainfall that characterize these periods intensify the deficits, so growth is curtailed and yields are severely depressed. Consequently, olive orchards have assumed strategic importance for rural development in China’s main producing regions—namely Wudu in the Bailong River basin of Gansu, Xichang in the hot–dry Jinsha River basin of Sichuan, and the Three Gorges Reservoir area. But these regions face prolonged seasonal drought lasting more than six months annually (April–November) [8]. Compounding the problem, shallow infertile hillside soils and limited irrigation infrastructure magnify the stress, while climate change is simultaneously increasing the frequency of extreme events [9]; therefore, the search for olive cultivars capable of sustaining productivity under prolonged water shortage has become urgent.

Drought redirects assimilates from growth to water-saving metabolism and initiates osmotic adjustment, thereby disrupting olive morphogenesis and biomass accumulation [3,10,11]. As predawn leaf water potential declines, specific leaf area and net photosynthetic rate (Pn) fall [12]. Among the Italian cultivars Giarraffa, Leccino, and Maurino, Leccino suffers the steepest reductions in Pn and photochemical efficiency of photosystem II (Fv/Fm) and is the first to respond [13]. Mediterranean germplasm copes through coordinated biochemical, morphological, and physiological adaptations that include osmolyte accumulation [14,15], antioxidant mobilization [16], leaf anatomical remodeling, and precise stomatal control [17,18]. The Liangshan base of the China National Germplasm Collection Center preserves 122 accessions displaying a broad spectrum of drought tolerance. For instance, Arbequina maintains cell viability under −3.2 MPa stress by shrinking leaf area and thickening palisade parenchyma [13,19], while Koroneiki employs leaf rolling, proline accumulation, and elevated superoxide dismutase (SOD) and peroxidase (POD) activities to bolster osmotic and antioxidant defenses [16]. Although these traits support high yields, field observations reveal that growers depend almost exclusively on synthetic fertilizers, which pollute soil and water, erode biodiversity and endanger human and animal health [20,21]. Sustainable olive production therefore demands practical biological strategies, and recent Chinese research confirms that selecting cultivars matched to the local climate can markedly enhance both productivity and sustainability.

Arbuscular mycorrhizal fungi (AMF) are an eco-friendly asset in organic agriculture [22]. They colonize root cortical cells, extend extraradical hyphae that enlarge the soil–root interface, and, via high-affinity P-transporters (GiPT, GmosPT) and ammonium transporters (GintAMT1, GintAMT2), deliver up to 90% of plant P and 25% of plant N [23]; siderophore-mediated chelation and glomalin secretion further solubilize Cu, Zn, Fe, and Mn, acting as a living fertilizer where humus is thin or nutrients are scarce [24]. Termed arbuscular mycorrhizal symbiosis, the union binds >150 AMF species to >80% of vascular plant families [25,26]. Within the subphylum Glomeromycotina, four orders—Glomerales, Paraglomerales, Archaeosporales, and Diversisporales—cluster 25 genera [27]. Investigators have tracked these fungi into the rhizospheres of most angiosperms, bryophytes, pteridophytes, and gymnosperms [28], recording services that span mineral nutrition [29,30], salt-stress alleviation [31], drought resistance via improved water supply [32,33], soil bioremediation [26,34], phytohormone production [35], and ecosystem stability [34,36]. Olive trees, a classic host, profit substantially from this partnership; Rhizophagus intraradices, for instance, reduced lipid peroxidation in ‘Picholine marocaine’ by 35% while boosting proline and soluble sugars (SS) by 42% and 28%, respectively [37,38,39]. Under drought, AMF raised mineral uptake 12–18% across cultivars, with genotype strongly modulating the response—‘Moraiolo’ and ‘Frantoio’ inoculated with Funneliformis mosseae outperformed ‘Leccino’ and local strains surpassed commercial inocula by 20–35% in biomass [37,40]. However, most evidence stems from the Mediterranean littoral, leaving the performance of elite Chinese cultivars such as ‘Arbequina’ and ‘Koroneiki’ under China’s summer-rainfall regime largely unexplored.

This study aimed to clarify the role of arbuscular mycorrhizal fungi (AMF) in promoting seedling growth and enhancing drought resistance in leading Chinese olive cultivars. It also sought to determine whether cultivars exhibit selective preferences for specific AMF under water stress. We hypothesized that Rhizophagus intraradices and Funneliformis mosseae, previously isolated from olive rhizospheres, could improve growth and development of Arbequina and Koroneiki under both well-watered and water-deficit conditions. Therefore, a controlled pot experiment was established with the aim to determine (1) whether AMF inoculation improves morphological and physiological performance across cultivars under drought and (2) whether cultivar–AMF combinations differ in symbiotic efficiency depending on water availability. By integrating morpho-physio-biochemical responses, this work intends to optimize AMF strain selection for elite olive cultivars exposed to seasonal drought in China.

2. Materials and Methods

2.1. Soil Collection and Fungal and Plant Materials

A pot experiment was conducted at the China West Normal University Experimental Station in Nanchong, Sichuan Province, China (30°35′–31°51′ N, 105°27′–106°58′ E; 603 m a.s.l.). Soil was collected at a depth of ~20 cm after removing surface litter and the top 2 cm of soil. The growth substrate was prepared by blending sieved topsoil (collected via multi-point sampling at 10 m intervals and processed using the quartering method) with sphagnum moss, perlite, and vermiculite in a 5:3:1:1 (v/v) ratio. The mixture was sieved through a 2 mm sieve to ensure homogeneity and exclude gravel and organic debris. Olive seedlings of Arbequina (Arb) and Koroneiki (Kor) cultivars, sourced from the Olive Base in Xichang, Liangshan, Sichuan, in early March, were transplanted individually into 30 cm × 20 cm pots. Two arbuscular mycorrhizal fungi (AMF) strains—Glomus intraradices (AMF1) and Funneliformis mosseae (AMF2)—were obtained from the Institute of Root Biology, Yangtze University (Jingzhou, Hubei). Inocula consisted of pure culture medium, root fragments, spores (15 spores/g), and extraradical mycelium mixed with soil at a 1:3 (fungus-to-soil, v/v) ratio. An uninoculated control (non-AMF) received 20 g of autoclaved inoculum (121 °C, 0.1 MPa, 30 min) to maintain consistent soil volume and microbial neutrality.

2.2. Experimental Design and Treatments

2.2.1. Experimental Design

The experiment used a factorial layout. Two olive cultivars were tested: Arbequina and Koroneiki. Experimental variants are summarized in

Table 1. Experimental design.

Treatment Group	AMF Status	Water Regime	FC (%)
AMF1 + WW	Gl. intraradices	Well-watered (WW)	80%
AMF1 + WS	G. intraradices	Water-stressed (WS)	30%
AMF2 + WW	F. mosseae	Well-watered (WW)	80%
AMF2 + WS	F. mosseae	Water-stressed (WS)	30%
Non-AMF + WW	None	Well-watered (WW)	80%
Non-AMF + WS	None	Water-stressed (WS)	30%

. Two irrigation regimes were imposed: well-watered (WW, WW; soil moisture at 80% field capacity [FC]) and water-stressed (WS, 30% FC). Three arbuscular mycorrhizal fungi (AMF) treatments were applied: non-AMF, AMF1, and AMF2. The design produced four cultivar × water combinations: Arb-WW, Arb-WS, Kor-WW, and Kor-WS. Each combination was subdivided into the three AMF treatments, giving 12 treatment combinations.

2.2.2. Water Treatments and Fungal Inoculation

Drought management employed scheduled, volume-controlled irrigation. Three months post-transplantation, olive seedlings were subjected to drought stress experiments, and treatments began in August 2024 and lasted 32 days. WW plants received 600 cm³ of water every four days, maintaining soil moisture at 80% of FC, while WS plants received 600 cm³ every eight days, corresponding to 30% of FC. FC was defined here as the maximum soil water content retained against gravity drainage (≈15% volumetric water content, VWC), measured following saturation and drainage over 24 h. Total available water capacity (TAWC) was calculated as the difference between FC and wilting point (≈5% VWC), yielding a TAWC of 10% VWC. Thus, WW and WS treatments represented 80% and 30% of TAWC, respectively. Measurements of photosynthetic parameters, chlorophyll fluorescence, and leaf water potential (Ψw) were conducted during the final two weeks of treatment.

For AMF inoculation, 20 g of live inoculum (containing R. intraradices or F. mosseae) was applied per pot at 10 cm root depth. Non-AMF controls received 20 g of autoclaved inoculum. No organic amendments were introduced during inoculation. Each experimental unit consisted of independent olive seedlings (n = 6 per treatment combination), representing biological replicates to account for natural variability. The design included 12 treatment combinations (3 AMF treatments × 4 water/inoculation groups), with each combination replicated six times biologically. Technical replicates were not applied, as measurements (e.g., photosynthetic parameters) were performed once per biological replicate.

2.3. Determination of Plant Biomass and Mycorrhizal Colonization

Root samples were processed for mycorrhizal analysis: segments (1 cm) were cleared in 10% KOH (w/v) (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China) at 90 °C, acidified with 2% HCl (Chengdu Kelong Chemicals Co., Ltd., Chengdu, China), and stained with 0.05% trypan blue [41]. Arbuscular mycorrhizal colonization was quantified using a biological microscope (NE610, Ningbo Yongxin Optical Co., Ltd., Ningbo, China), with colonization rate (%) and mycorrhizal dependency (%) calculated as the proportion of infected root length relative to total root segments.

Prior to harvest, shoot length and basal diameter were measured. At harvest, plants were rinsed with deionized water and dissected into roots, stems, and leaves. Biomass parameters—including aboveground fresh weight (AFW), underground fresh weight (UFW), plant height, and basal diameter—were immediately recorded. Fresh root and leaf tissues were flash-frozen in liquid nitrogen and stored at −80 °C. Subsets of samples were freeze-dried (ALPHA 1-2 LD plus, Marin Christ, Niederbipp, Switzerland) and ground (SCIENTZ-48, Xinzhi, Ningbo, China) for biochemical analysis. Additional samples were oven-dried at 70 °C to a constant weight to determine dry weights (aboveground ADW, underground UDW), with aboveground biomass calculated as the sum of leaf and stem dry weights. Root morphology was assessed by imaging three representative lateral roots via the WinRHIZO root scanning system (Epson, Tokyo, Japan). Quantitative parameters, including total root length, surface area, volume, average diameter, and counts of root tips and intersections, were derived from digital analyses. For total plant biomass, each organ was oven-dried at 70 °C for 48 h, and individual dry weights were summed to calculate overall plant biomass. This standardized protocol ensured precise and reproducible measurement of growth, structural, and compositional traits across treatments.

2.4. Determination of Photosynthetic and, Fluorescence Parameters and Plant Water Status

Photosynthetic measurements were carried out between 9:00 AM and 11:30 AM on clear days. For each treatment group, five seedlings were randomly chosen and assessed using a LI—6400XT portable photosynthetic meter (Li-Cor, Lincoln, NE, USA). Employing a closed-airway system, two to five fully expanded upper functional leaves were uniformly selected from each plant for the determination of the net photosynthetic rate (Pn) concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr). The fluorescence parameters were measured on the identical leaves used for the photosynthetic measurements. Prior to measurement, seedling leaves underwent a 20 min dark treatment. Subsequently, a Handy-FLM handheld chlorophyll fluorescence imaging system (MIN-PAM-II, Qianfeinuo, Shanghai) was utilized to measure the minimal fluorescence (Fo) and maximal fluorescence (Fm) for each treatment. The maximum Fv/Fm was then automatically calculated.

To evaluate plant water status, Ψw was measured in the leaves selected for photosynthetic analysis between 6:00 and 8:30 AM. This measurement was conducted using a dew-point water potential measurement system (Psypro, Wescor, Logan, Utah, MO, USA). On the day prior to harvest, the relative water content (RWC) of the same leaves was also determined. The fresh weight (FW) of these leaves was measured following the method of Barrs and Barrs and Weatherley [42]. Subsequently, the initial fresh weight (W_f) was precisely weighed using an electronic balance with an accuracy of 0.0001 g. The leaves were then placed in the dark at 4 °C to facilitate saturated water absorption over a 24 h period, after which the saturated fresh weight (W_d) was measured. Finally, the leaves were oven-dried at 70 °C until a constant weight was reached (48 h), and the dry weight (W_t) was then recorded. The RWC of the leaves can be calculated using the following formula [42]:

$$\mathrm{RWC} (\%) =(W_f-W_d)/\left(W_t-W_d\right)\times 100$$

(1)

W_f indicates the initial fresh weight; W_t indicates the dry weight; W_d indicates the saturated fresh weight.

2.5. Determination of Chlorophylls and Carbon and Nitrogen Detection

Photosynthetic pigment levels (chlorophyll and carotenoid) were analyzed following the method of Arnon [43]. Circular leaf samples, each with a diameter of 6 mm, were collected from the center of the photosynthetic functional leaf. The fresh weight (W, g) of these samples was recorded using an electronic balance with an accuracy of 0.0001 g. Subsequently, the leaf samples were transferred into 5 cm³ of 80% acetone (Chengdu Kelong Chemicals Co., Ltd.) extract, which was stored in a brown centrifuge tube and kept away from light until the samples were completely decolorized. Then, 200 μL of the supernatant was pipetted onto an ELISA plate (V, cm³). The term “absorbance (OD)” refers to Optical Density, and this clarification has been added to the manuscript to ensure terminological precision. Regarding wavelength specifications, the protocol follows Arnon [43], where 663 nm quantifies chlorophyll a absorption, 645 nm quantifies chlorophyll b, and 470 nm measures carotenoids (which absorb strongly in the blue spectrum). The absorbance values (OD) at wavelengths of 663, 645, and 470 nm were measured using an Epoch full-wavelength scanner (BioTek, Windsor, VT, USA). The calculation formulas are as follows [43].

$$\mathrm{Chla} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FW}) = (12.7{\mathrm{OD}}_{663}- 2.69{\mathrm{OD}}_{645}) \times (1000 \times \mathrm{W})$$

(2)

$$\mathrm{Chlb} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FW}) = (22.9{\mathrm{OD}}_{645} - 4.86{\mathrm{OD}}_{663}) \times \mathrm{V}/(1000 \times \mathrm{W})$$

(3)

$$\mathrm{Total} \mathrm{Chl} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FW}) = (20.21{\mathrm{OD}}_{645} + 7.84{\mathrm{OD}}_{663}) \times \mathrm{V}/(1000 \times \mathrm{W})$$

(4)

$$\mathrm{Carotenoid} (\mathrm{mg} {\mathrm{g}}^{-1}\mathrm{FW})={\displaystyle \frac{1000{\mathrm{OD}}_{470}-3.27\mathrm{Chla}-104\mathrm{Chlb}}{299}}\times \mathrm{V}/\left(1000\times \mathrm{W}\right)$$

(5)

Chla indicates chlorophyll a; Chlb indicates chlorophyll b; Total Chl indicates total chlorophyll.

The Vario MACRO cube elemental analyzer measures carbon (C) and nitrogen (N) content via high-temperature combustion. Samples are combusted in an oxygen-rich environment at 1050 °C, converting organic C to CO₂ and N to NO_x;. Gas-phase products are purified and separated using reduction/oxidation columns and a thermal conductivity detector, enabling precise quantification of elemental concentrations. This method aligns with protocols described in Gao et al. [44], ensuring accurate determination of C/N ratios, critical for assessing plant nutrient dynamics under stress conditions. Initially, the samples were dried at 105 °C for 0.5 h, followed by further drying at 80 °C until a constant weight was achieved. Subsequently, the dried samples were ground using an electric grinder and sieved through a 100-mesh sieve. Next, precisely 40 mg of the processed sample was weighed using an electronic balance with an accuracy of 0.0001 g. This weighed sample was then placed into a Vario MACRO cube elemental analysis system (Elementar, Shanghai, China) for quantitative analysis of C and N.

2.6. Determination of Physiological Characteristics

Fresh roots and leaves (0.5 g) from each treatment group were homogenized in 1 cm³ of pre-chilled phosphate buffer (pH 7.8, 4°C, Shanghai Shangbao Biotechnology Co., Ltd., Shanghai, China). After homogenization, the volume was adjusted to 5 cm³, and 2 cm³ of the supernatant was collected. This supernatant was then centrifuged at 12,000× g for 20 min to obtain the enzyme solution. Superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium (NBT, Nanjing Dulai Biology Co., Ltd., Nanjing, China) photochemical reduction method [45] and read at 560 nm with a microplate reader (Thermo Multiskan GO, East Lyme, CT, USA). Peroxidase (POD) activity was measured with guaiacol (Shanghai Aladdin Biochemical Technology Co., Ltd.) at 470 nm (Multiskan FC) [46]. A dark reaction mixture served as a blank. Additionally, malondialdehyde (MDA) content was quantified by the thiobarbituric-acid method [47], and absorbances were recorded at 532 and 450 nm (Multiskan FC).

To explore the drought-resistance mechanisms of two olive varieties inoculated with AMF under drought stress, changes in osmotic regulatory substances in their roots and leaves were examined. Soluble sugar content (SS) was determined using the anthrone colorimetric method [48]. Absorbance measurements were taken at 630 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA), and SS concentrations were calculated via a sucrose standard curve. Soluble protein content (SP) was assessed via the Coomassie Brilliant Blue G-250 method [49], with absorbance readings at 595 nm obtained using the same spectrophotometer. Protein quantification relied on a bovine serum albumin standard curve. Free proline content was measured using the acid ninhydrin colorimetric method [50], where absorbance at 520 nm was recorded (SpectraMax M5 microplate reader, Molecular Devices, Sunnyvale, CA, USA) with pure toluene (Chengdu United Chemical Reagent Research Institute) as the blank control.

2.7. Statistical Analyses

Prior to statistical analysis, we assessed each experimental dataset for normality and homogeneity of variance. Data that failed to meet these assumptions underwent log-transformation. Subsequently, statistical analysis was conducted using SPSS Statistics 24.0 (SPSS Inc., Chicago, IL, USA). To analyze differences between treatment groups, we employed one-way analysis of variance (ANOVA) followed by Duncan’s multiple comparison test. Additionally, LSD tests were carried out, and results were presented as mean ± standard deviation (SD). A two-way analysis was performed to evaluate the interaction between inoculation and water treatment on malondialdehyde and membrane conductivity. For examining the interaction among water treatment, inoculation treatment, and variety on growth, photosynthetic parameters, as well as physiological and biochemical parameters, a three-way analysis was conducted. Finally, Pearson’s correlation analysis and principal component analysis (PCA) were performed using Origin 2024 (Systat Software, Richmond, CA, USA), with a significance level set at $\alpha$ = 0.05.

3. Results

3.1. AMF Colonization and Mycorrhizal Dependency in Roots

After harvest, AMF colonization was observed in all inoculated roots; none were found in non-inoculated controls. Under WW conditions, both Arbequina and Koroneiki formed effective AM associations (Figure 1A,B). AMF2 colonization was consistently higher than AMF1 in both cultivars (p < 0.05). Koroneiki with AMF2 performed best, reaching 67% colonization and a 35% mycorrhizal dependency (Figure 1A,B). Drought did not disrupt the symbiosis; AMF2-inoculated plants maintained colonization >60%. Koroneiki under WS showed higher AMF1 colonization and a sharp rise in AMF2 dependency (62%), whereas Arbequina under WS kept AMF2 dependency lower (37%) (Figure 1B). Two-way ANOVA indicated that the water × fungus interaction significantly affected both mycorrhizal colonization (W × A, p < 0.001) and dependency (W × A, p < 0.01).

3.2. Biomass Accumulation and Root Growth

Water availability exerted the strongest influence on every biomass parameter (p < 0.001). When irrigation was non-limiting, AMF1 drove a general upsurge in Arb-WW, elevating AFW, UFW, and ADW by 36%, 25%, and 25%, respectively, while the root–shoot ratio fell by two thirds relative to non-mycorrhizal controls; except for the aboveground fresh weight (AFW), none of the other changes reached a significant level. (

Table 2. AMF effects on biomass accumulation of two olive cultivars under water treatments.

Treatments		Height (cm)	Diameter (mm)	AFW (g)	UFW (g)	ADW (g)	UDW (g)	Root–Shoot Ratio
non-AMF	Arb-WW	90.03 ± 3.19 abc	9.00 ± 0.37 b	90.11 ± 2.16 b	16.59 ± 1.85 abc	32.76 ± 0.45 ab	9.62 ± 0.82 ab	0.35 ± 0.03 abc
	Kor-WW	95.65 ± 5.03 ab	7.92 ± 0.29 bcd	69.40 ± 2.70 cd	9.91 ± 1.08 cd	26.70 ± 1.43 bc	5.12 ± 0.32 b	0.27 ± 0.02 bc
	Arb-WS	51.35 ± 3.19 f	6.92 ± 0.33 d	40.56 ± 2.90 e	9.97 ± 1.76 cd	18.71 ± 2.65 cd	7.20 ± 1.73 ab	0.52 ± 0.13 a
	Kor-WS	71.60 ± 3.85 cde	7.18 ± 0.60 cd	39.44 ± 8.85 e	6.38 ± 0.11 d	14.89 ± 3.41 d	6.25 ± 1.47 b	0.43 ± 0.09 ab
AMF1	Arb-WW	77.4 ± 3.21 bcde	8.81 ± 0.83 bc	122.00 ± 9.56 a	20.80 ± 4.01 a	40.84 ± 3.63 a	10.05 ± 1.07 ab	0.21 ± 0.01 c
	Kor-WW	71.77 ± 5.45 cde	8.53 ± 0.55 bcd	72.54 ± 10.41 bc	20.04 ± 6.31 ab	32.23 ± 7.08 ab	11.72 ± 4.69 a	0.26 ± 0.05 bc
	Arb-WS	64.48 ± 4.26 def	7.88 ± 0.28 bcd	52.62 ± 3.18 cde	12.09 ± 1.84 bcd	23.21 ± 2.70 bcd	6.76 ± 0.65 b	0.43 ± 0.04 ab
	Kor-WS	66.83 ± 8.31 def	7.01 ± 0.52 d	42.05 ± 1.57 e	9.34 ± 1.37 cd	20.67 ± 0.96 cd	5.94 ± 0.99 b	0.43 ± 0.04 ab
AMF2	Arb-WW	82.93 ± 3.95 abcd	9.18 ± 0.89 ab	70.01 ± 2.61 bc	13.84 ± 0.36 abcd	26.48 ± 2.05 bc	6.97 ± 0.58 b	0.21 ± 0.02 c
	Kor-WW	98.40 ± 11.04 a	10.56 ± 0.61 a	94.60 ± 9.48 b	16.98 ± 1.79 abc	32.86 ± 4.82 ab	8.39 ± 1.67 ab	0.33 ± 0.07 bc
	Arb-WS	62.33 ± 11.17 ef	7.27 ± 0.30 cd	44.65 ± 6.15 e	10.21 ± 0.85 cd	20.81 ± 3.04 cd	6.50 ± 0.61 b	0.39 ± 0.06 abc
	Kor-WS	82.45 ± 4.00 abcde	7.93 ± 0.27 bcd	50.62 ± 2.92 de	12.21 ± 1.69 bcd	24.83 ± 1.61 bcd	8.71 ± 0.81 ab	0.45 ± 0.01 ab
V		**	ns	**	ns	ns	ns	ns
A		**	**	**	*	*	*	ns
W		**	**	**	**	**	**	**
V × A		**	ns	**	*	ns	**	ns
V × W		ns	ns	**	*	ns	ns	ns
A × W		**	ns	**	*	*	**	ns
V × A × W		ns	ns	*	ns	ns	ns	ns

Explanations: Data are mean ± SE (n = 5). Different lowercase letters within columns indicate significant differences (Duncan, p < 0.05). *, ** denote p ≤ 0.05, p ≤ 0.01; ns, not significant. Arb, Arbequina; Kor, Koroneiki; WW, 80% FC; WS, 30% FC; AMF1, R. intraradices; AMF2, F. mosseae. V, variety; A, AMF; W, water regime. AFW, aboveground fresh weight; UFW, underground fresh weight; ADW, aboveground dry weight; UDW, underground dry weight.

; p > 0.05). Under the same conditions, AMF2 failed to surpass the controls in any biomass variable (p > 0.05), confirming that the growth benefit is fungus-specific. Drought stress significantly reduced the biomass of Arb cultivars (p < 0.01). Although AMF1 or AMF2 inoculation did not significantly enhance plant height, stem diameter, AFW, UFW, or ADW in Arb-WS (p > 0.05), AMF1 still significantly promoted overall growth compared to the drought control (p < 0.05). UDW remained unaffected by cultivar, and the root–shoot ratio exhibited a non-significant downward trend (p > 0.05). In Kor cultivars, response patterns diverged (Table 2). Under WW conditions, AMF1 doubled UFW and UDW, while AMF2 significantly increased basal diameter in Kor-WW (p < 0.01). During drought, both isolates stimulated growth: AMF1 increased plant height, basal diameter, and fresh and dry weights of Kor-WS by 25–30%, though not significantly (p > 0.05); similarly, AMF2 increased the plant height of Kor-WS, which also did not reach a significant level (p > 0.05), while the root–shoot ratio again declined non-significantly (p > 0.05). These results underscore the cultivar- and strain-specific nature of AMF-mediated growth promotion under water stress, with AMF1 exerting a stronger promotive effect on Arb under drought and AMF2 delivering the most pronounced benefit to Kor under water-limited conditions.

Table 3. AMF effects on root growth of two olive cultivars under water treatments.

Treatments		Surface Area of Root (dm2)	Total Root Length (dm)	Roots Volume (cm3)	Number of Root Tips (×1000)	Average Diameter (mm)
non-AMF	Arb-WW	2.52 ± 0.20 cd	12.82 ± 1.02 b	11.40 ± 1.39 b	1.93 ± 0.09 cd	0.50 ± 0.03 bc
	Kor-WW	3.30 ± 0.22 ab	16.40 ± 0.20 a	15.69 ± 1.02 a	2.25 ± 0.17 c	0.63 ± 0.03 ab
	Arb-WS	1.69 ± 0.09 de	7.83 ± 0.54 d	4.35 ± 1.55 c	1.72 ± 0.16 d	0.63 ± 0.09 ab
	Kor-WS	1.62 ± 0.23 e	9.03 ± 1.08 cd	4.45 ± 0.60 c	1.61 ± 0.14 d	0.48 ± 0.05 c
AMF1	Arb-WW	2.89 ± 0.40 bc	16.47 ± 0.80 a	14.16 ± 0.96 a	3.56 ± 0.14 a	0.45 ± 0.03 c
	Kor-WW	2.76 ± 0.36 bcd	12.21 ± 1.59 b	11.42 ± 2.23 b	1.76 ± 0.31 d	0.58 ± 0.03 abc
	Arb-WS	2.31 ± 0.26 cde	11.36 ± 0.79 bc	9.80 ± 0.93 b	1.57 ± 0.04 d	0.54 ± 0.03 bc
	Kor-WS	2.12 ± 0.10 cde	11.42 ± 1.22 bc	9.48 ± 0.12 b	1.92 ± 0.18 cd	0.51 ± 0.02 bc
AMF2	Arb-WW	1.91 ± 0.10 cde	11.76 ± 0.74 bc	10.50 ± 0.49 b	2.22 ± 0.20 c	0.45 ± 0.03 c
	Kor-WW	3.66 ± 0.07 a	16.86 ± 0.34 a	16.81 ± 0.17 a	2.73 ± 0.11 b	0.69 ± 0.01 a
	Arb-WS	1.94 ± 0.08 cde	11.63 ± 0.34 bc	9.00 ± 0.45 b	2.00 ± 0.14 cd	0.53 ± 0.03 bc
	Kor-WS	2.51 ± 0.09 cd	12.69 ± 0.59 b	10.56 ± 0.43 b	2.38 ± 0.14 b	0.54 ± 0.01 bc
V		ns	ns	ns	ns	ns
A		**	**	**	**	*
W		**	**	**	**	ns
V × A		ns	*	ns	ns	**
V × W		*	ns	**	*	*
A × W		ns	ns	*	*	ns
V × A × W		ns	ns	**	**	ns

Explanations: Data are mean ± SE (n = 5). Different lowercase letters within columns indicate significant differences (Duncan, p < 0.05). *, ** denote p ≤ 0.05, p ≤ 0.01; ns, not significant. Arb, Arbequina; Kor, Koroneiki; WW, 80% FC; WS, 30% FC; AMF1, R. intraradices; AMF2, F. mosseae. V, variety; A, AMF; W, water regime. AFW, aboveground fresh weight; UFW, underground fresh weight; ADW, aboveground dry weight; UDW, underground dry weight.

confirms that fungal strain and water treatment governed root growth (p < 0.001), whereas olive cultivar had no influence, and cultivar–strain interactions remained non-significant for surface area of root (SAR), roots volume (RV), and number of root tips (NRT) (p > 0.05). Strain identity therefore dictated the root response. In Arb-WW, AMF1 raised TRL, RV, and NRT by 28%, 24%, and 84% relative to non-mycorrhizal controls (p < 0.05), whereas AMF2 was ineffective. Drought collapsed TRL, RV, and NRT in Arb-WS; both fungi then fully resurrected them, lifting TRL and RV to 45–49% and 107–125% of the control level, respectively (Table 3; p < 0.05). SAR inched up 15–37% yet never crossed the significance threshold. Kor already wielded a larger root frame. In Kor-WW, AMF1 shrank TRL, RV, and NRT (p < 0.05), while AMF2 expanded NRT (p < 0.05). Drought strangled root growth in Kor-WS, yet both strains clawed back biomass. Relative to the control (drought non-AMF), AMF1 increased the RV of Kor-WS by 113%, AMF2 elevated the SAR by 55%, TRL 41%, RV 137%, and NRT 48% (p < 0.05), whereas AD stayed uniform across mycorrhizal treatments (p > 0.05). These results demonstrate that mycorrhizal inoculation markedly enhances root architecture under drought, primarily by proliferating root volume that improves water acquisition and mitigates stress.

3.3. Plant Photosynthetic and Fluorescence Parameters and Water Status

AMF inoculation significantly altered Pn, Gs, Tr, and Ci (p < 0.01), and the identity of both cultivar and watering regime further influenced Pn and Gs (p < 0.01) (Figure 2A–D, Table S1). The three-way interaction among AMF, cultivar, and water was also significant (p < 0.01), whereas Tr and Ci were unaffected by water regime or cultivar, and their two-way interaction remained non-significant (W × V, p > 0.05). Under WW conditions, AMF strongly improved photosynthesis in both cultivars. In Arb-WW, AMF1 raised Gs and Tr (p < 0.01), while AMF2 pushed Pn up by 19%, Gs by 143%, and Tr by 152% (p < 0.01) compared with the non-AMF control. Kor-WW responded even more strongly to AMF2, increasing Pn, Gs, and Tr by 67%, 82%, and 74% (Figure 2A,C,D; p < 0.01), respectively, while Ci remained unchanged (Figure 2B; p > 0.05). Drought suppressed every gas-exchange trait in both cultivars (p < 0.01), yet, under WS and relative to uninoculated drought controls, AMF1 and AMF2 lifted Pn by 18% and 21% in Arb-WS and by 58% and 43% in Kor-WS (p < 0.05), respectively (Figure 2A). In Arb-WS, AMF1 simultaneously slashed Tr by 83% and Gs by 50% compared to the control while elevating Ci by 12% (p < 0.01). Conversely, in Kor-WS, AMF2 raised Gs by 68% without altering Tr or Ci (p > 0.05). These strain-specific shifts reveal distinct stomatal and non-stomatal tactics that enhance photosynthesis under drought, with AMF1 delivering the strongest mitigation in Arb-WS.

Figure 3A–C first reveal that, whereas the water regime clearly reshaped Fv/Fm, Ψw, and RWC, AMF inoculation subsequently exerted a strong, independent effect on Fv/Fm and Ψw (Table S2, p < 0.001); conversely, the olive cultivar itself remained inconsequential (p > 0.05). Although no cultivar × water interaction emerged, a significant three-way interplay (V × A × W, p < 0.001) nevertheless modulated both Fv/Fm and Ψw, thereby underscoring the contextual nature of the response. Mycorrhizal effects on Fv/Fm were positive and strong. Arb-WW rose 12% under AMF1 and 15% under AMF2; Arb-WS rose 37% and 44%, both at p < 0.01. Kor-WW did not react to AMF1 but gained 9% with AMF2 (p < 0.05). Kor-WS climbed 26% with AMF1 and 43% with AMF2 (p < 0.01). Ψw of WW plants stayed flat, whereas WS plants responded sharply: AMF1 lifted Arb-WS by 18% and Kor-WS by 63% (p < 0.01); AMF2 raised Arb-WS by 94% and Kor-WS by 47% (p < 0.01). RWC did not shift in any group. Collectively, these findings indicate that both AMF1 and AMF2 markedly enhance Fv/Fm and Ψw in drought-tolerant olive cultivars, and—more importantly—that AMF inoculation systematically strengthens drought tolerance in both cultivars, with AMF2 delivering the greatest improvements in Fv/Fm and Ψw under Arb-WS.

3.4. Photosynthetic Pigments and Nutrient Elements

Table 4. AMF effects on photosynthetic pigments of two olive varieties under water treatments.

Treatments		Chla (mg g−1)	Chlb (mg g−1)	Total Chl (mg g−1)	Carotenoids (mg g−1)
non-AMF	Arb-WW	1.31 ± 0.09 abc	0.55 ± 0.04 bc	1.86 ± 0.11 abc	0.38 ± 0.03 bcd
	Kor-WW	1.31 ± 0.03 abc	0.44 ± 0.02 cd	1.75 ± 0.01 bcd	0.43 ± 0.02 ab
	Arb-WS	1.06 ± 0.09 cd	0.34 ± 0.02 d	1.43 ± 0.11 de	0.25 ± 0.01 e
	Kor-WS	0.96 ± 0.02 d	0.35 ± 0.07 d	1.31 ± 0.08 e	0.29 ± 0.04 de
AMF1	Arb-WW	1.44 ± 0.12 a	0.73 ± 0.04 a	2.17 ± 0.14 a	0.47 ± 0.05 ab
	Kor-WW	1.34 ± 0.07 ab	0.53 ± 0.02 c	1.87 ± 0.08 abc	0.49 ± 0.02 a
	Arb-WS	1.05 ± 0.15 cd	0.34 ± 0.04 d	1.40 ± 0.19 de	0.29 ± 0.02 de
	Kor-WS	1.17 ± 0.08 abcd	0.40 ± 0.03 cd	1.50 ± 0.11 cde	0.31 ± 0.03 d
AMF2	Arb-WW	1.42 ± 0.14 a	0.67 ± 0.07 ab	2.09 ± 0.19 ab	0.40 ± 0.03 bc
	Kor-WW	1.33 ± 0.05 ab	0.53 ± 0.04 c	1.87 ± 0.03 abc	0.43 ± 0.02 ab
	Arb-WS	1.08 ± 0.07 bcd	0.39 ± 0.05 d	1.47 ± 0.12 de	0.32 ± 0.01 cde
	Kor-WS	1.06 ± 0.13 cd	0.30 ± 0.02 d	1.44 ± 0.14 de	0.29 ± 0.03 de
V		ns	**	ns	ns
A		ns	*	ns	ns
W		**	**	**	**
V × A		ns	ns	ns	ns
V × W		ns	**	ns	ns
A × W		ns	ns	ns	ns
V × A × W		ns	ns	ns	ns

Explanations: Chla, chlorophyll a content; Chlb, chlorophyll b content; Total Chl, total chlorophyll content. Data are mean ± SE (n = 5). Different lowercase letters within columns indicate significant differences (Duncan, p < 0.05). *, ** denote p ≤ 0.05, p ≤ 0.01; ns, not significant. Arb, Arbequina; Kor, Koroneiki; WW, 80% FC; WS, 30% FC; AMF1, R. intraradices; AMF2, F. mosseae. V, variety; A, AMF; W, water regime. AFW, aboveground fresh weight; UFW, underground fresh weight; ADW, aboveground dry weight; UDW, underground dry weight.

first underscores that, whereas the water regime acted as the sole main driver of leaf chlorophyll under drought (p < 0.001), chlorophyll b simultaneously responded to the combined influence of cultivar, fungal strain, and water as well as to the cultivar × water interaction (p < 0.001); furthermore, strain identity alone proved significant (p < 0.001). Table 4 shows clear upward trends in well-watered plants. Chlorophyll a and b and total chlorophyll all rose with AMF (p < 0.01); Arb-WW gained 33% in chlorophyll b and 17% in total chlorophyll under AMF1 (p < 0.01). Drought dropped every pigment (p < 0.01), yet AMF1 and AMF2 slowed a loss of chlorophyll a, chlorophyll b, and total chlorophyll in Arb-WS and Kor-WS. Carotenoids followed the same pattern: higher in Arb-WW and Kor-WW, peaking at 0.49 mg g⁻¹ in Kor-WW, but drought lowered them, and mycorrhizae did not lift them back. Thus, AMF boosts chlorophyll b and total chlorophyll only when water is ample; under drought, the benefit disappears.

Figure 4A–D show that water treatment governed leaf C and N after AMF inoculation under drought, whereas cultivar and strain jointly determined leaf C (Table S3, p < 0.001). Root N differed between cultivars, and leaf C responded to cultivar × water and cultivar × strain interactions (V × W and V × A, p < 0.001), while root C and N responded to strain × water interactions (A × W, p < 0.001). Figure 4 shows flat trends for leaf nitrogen: no change in either cultivar, water regime, or fungus (Figure 4C; p > 0.05). Leaf carbon stayed unchanged in Arbequina (p > 0.05) yet rose 2% with AMF1 and 2% with AMF2 in Kor-WS (Figure 4A; p < 0.05). Root N did not shift in Koroneiki or in Arb-WS; Arb-WW gained 36% under AMF2 (Figure 4D; p < 0.05). Root C edged up 2% in Arb-WS and 1% in Kor-WS with AMF2 (Figure 4B; p < 0.05), while AMF1 had no effect on root C or N content (Figure 4B,D). Thus, AMF2 selectively lifts carbon and nitrogen, and the direction and magnitude of the responses depended on cultivar and water availability.

3.5. Antioxidant Enzymes and Osmoregulatory Substances

Figure 5A–F show that SOD (superoxide dismutase), POD (peroxidase), and MDA (malondialdehyde) activities in roots and leaves responded to water regime and AMF strain (Table S4, p < 0.001), and POD differed between cultivars. The three-way interaction affected leaf MDA, leaf SOD, and POD (p < 0.001) but not root enzymes actives (Table S4). Under full irrigation, leaf SOD in Arb-WW and Kor-WW stayed unchanged across fungi (p > 0.05), and root SOD in both cultivars was unaffected by AMF1 (Figure 5A). In Arb-WW roots, SOD responded differently to AMF1 and AMF2 depending on water status. AMF2 lowered leaf SOD in Arb-WS and Kor-WS by 33% and 28% yet raised root SOD in Kor-WS by 60% (p < 0.01). Leaf POD in Arb-WW and Kor-WW rose 97% and 74% under AMF1 (p < 0.01); AMF2 gave smaller, variable increases in droughted plants. Root POD reacted strongly to fungal identity: Arb-WW gained 34% with AMF2 but lost 32% with AMF1; the same 46% drop occurred in Arb-WS (p < 0.01). Kor-WW root POD was unaltered, whereas Kor-WS root POD fell 26% with AMF1 (p < 0.01). Leaf MDA varied with fungus and water: well-watered plants showed cultivar-specific changes, but droughted leaves declined 44%/48% in Arb-WS and 37%/33% in Kor-WS with AMF1/AMF2 (p < 0.01). Root MDA in Arb-WW stayed stable (p > 0.05); Kor-WW root MDA rose with AMF2, yet Kor-WS root MDA dropped 31% under AMF1 and 18% under AMF2 (p < 0.01). Thus, ample water gives variable, fungus-specific responses, whereas drought consistently lowers leaf SOD and root POD, raises leaf POD and root SOD, and strongly reduces MDA in both leaves and roots.

Figure 6A–F reveal that root and leaf SS varied with treatment, whereas both soluble sugar (SS) and proline responded strongly to water status and AMF (Table S5, p < 0.01); in contrast, leaf and root soluble protein (SP) remained unchanged (p < 0.01). Under full water, leaf SS and SP in Arb-WW and leaf SS in Kor-WW stayed flat across fungi (Figure 6A,C, Table S5); Kor-WW root SS and SP varied with strain. Drought reversed the pattern: AMF1 and AMF2 raised Arb-WS leaf SS by 60% and 72%, while AMF2 lifted Kor-WS leaf SS and SP by 28% and 54% (Figure 6A; p < 0.01); Arb-WS leaf SP and root SS rose 29% and 100% (p < 0.01; Kor-WS root SS was unchanged, yet root SP surged 46%/51% in Arb-WS and 136%/95% in Kor-WS under AMF1/AMF2 (Figure 6B,D; p < 0.01). Leaf proline stayed stable for both cultivars and treatments (Figure 6E; p > 0.05); root proline reacted strongly. AMF1/AMF2 raised root proline in Arb-WW by 31%/19% and in Arb-WS by 75%/52%, and in Kor-WW by 23%/40% and in Kor-WS by 100%/94% (Figure 6F; p < 0.01). Thus, SS, SP, and proline in roots are more drought-sensitive than in leaves and may play a more crucial role in regulating plant resistance to water limitation.

3.6. Principal Component and Correlation Analyses

Principal component analysis (PCA) linked root MDA, Ψw, leaf Pro, SOD, and POD across olive cultivars under well-watered (WW) and water-stressed (WS) regimes with AMF1 or non-AMF inoculation, revealing that PC1 (63.2% variance) and PC2 (14.3% variance) clearly separated treatments by correlating PC1 with root–shoot ratio, leaf/root SOD/POD, Pro, and root MDA, while PC2 tracked RWC, Ψw, Fv/Fm, and leaf MDA; samples clustered into WW (red) and WS (black) groups (Figure 7A), with loadings showing PC1 driven by root–shoot ratio, Fv/Fm, osmoregulation (Pro), and antioxidant enzymes (SOD, POD) and PC2 influenced by water status (RWC, Ψw) and membrane damage (MDA) (Figure 7B). Under WW, Kor + AMF1 aligned on positive PC1 with drought-resistance traits, whereas non-AMF controls and Arb + AMF1 fell on the negative side, indicating cultivar-specific strategies, with AMF1 enhancing drought tolerance in Arbequina (e.g., Arb-WS + AMF1 scored highest on both components) but reducing some indicators in Koroneiki, ultimately positioning Arb + AMF1 as the most resilient combination, Arbequina as the hardier cultivar, and AMF1 as a promising symbiont for olive seedlings under water limitation, thus providing a framework for targeted cultivation.

Pearson’s correlation coefficient analysis under drought stress showed that root POD activity had positive and significant correlations with RSA, leaf MDA, root MDA, and root Pro (p < 0.05), whereas Ψw displayed a negative and significant correlation with root POD (Figure 8; p < 0.05). In contrast, RWC and Fv/Fm exhibited negative but non-significant relationships with root POD, while leaf SOD and root SOD showed positive but non-significant associations (p > 0.05). Leaf Pro and leaf POD were not correlated with root POD (Figure 8; p > 0.05). It may be concluded that seedlings trade off water status maintenance for antioxidant and osmotic defense; genotypes with larger root systems sustain stronger osmotic adjustment yet weaker leaf antioxidant capacity, and lower Ψw is linked to higher drought tolerance. AMF shaped these links in a cultivar-specific manner, guiding the selection of genotype–symbiont combinations that balance canopy water status and underground allocation for maximal resilience.

4. Discussion

4.1. AMF Establishes a Symbiotic Relationship with Two Olive Varieties Under Different Water Conditions

Mycorrhizal colonization reflects both the extent and the affinity of AMF with the host [51]. Our results integrate quantitatively with the literature to refine the ranking of olive cultivars for AMF responsiveness. We confirm that Arbequina sustains the high infection capacity previously reported (100% frequency, up to 66% intensity) [52], because even under drought its AMF2 colonization remained ≥60%, and intensity values were comparable to those earlier data. The new observation is that Koroneiki can equal this performance: under well-watered conditions, Koroneiki–AMF2 reached 68% colonization, i.e., within the upper range recorded for Arbequina, and its dependency (35%) was half of the 61.6% index formerly cited [53], indicating that the cultivar is responsive but not obligate. Water deficit did not break symbiosis in either cultivar, echoing reports on Astragalus adsurgens and Malus hupehensis, where colonization rates were likewise drought-stable [54,55]. However, the sharp rise in Koroneiki AMF2 dependency to 62% under drought while Arbequina–WS stayed at 37% demonstrates—via the significant water × fungus interaction (p < 0.01 for dependency)—that the two cultivars partition carbon to the fungus differently when stressed. Because our non-inoculated roots remained AMF-free, the observed colonization is strictly inoculum-dependent, reinforcing the conclusion that intrinsic cultivar × fungus traits, rather than external moisture alone, govern the final colonization level [56].

4.2. Effects of AMF on Biomass Allocation and Root Growth in Two Olive Varieties Under Different Water Conditions

Under WW, AMF symbiosis promptly enlarged olive growth and biomass: AMF1 drove a general upsurge in Arb-WW, elevating AFW, UFW, and ADW by 35%, 25%, and 25%, respectively, while the root–shoot ratio fell by two-thirds relative to non-mycorrhizal controls (ns); this trait correlated positively with Fv/Fm and Ψw but negatively with osmolytes and antioxidant enzymes. AMF2 failed to surpass controls in any biomass variable in Arb-WW, confirming that the growth benefit is fungus-specific. Under the same conditions, AMF1 doubled UFW, ADW, and UDW, whereas AMF2 significantly increased basal diameter in Kor-WW. These gains mirror reports on Catalpa bungei [57], Sophora davidii [58], black locust [59], Cyclobalanopsis glauca [60], and Arizona cypress (Cupressus arizonica G.), which are credited to extraradical hyphae that expand water–nutrient delivery [61], as under WW conditions, AMF1 doubled UFW, ADW, and UDW, while AMF2 significantly increased basal diameter in Kor-WW, underscoring a species-specific interplay. WS suppressed height, stem diameter, and biomass in both cultivars and slashed total root length, volume, and tips in Arb-WS, yet AMF1 and AMF2 almost reversed these losses: AMF1 increased plant height, basal diameter, and fresh and dry weights of Kor-WS by 25–30% (ns), whereas AMF2 significantly increased plant height in Kor-WS (p < 0.05), paralleling earlier olive biomass declines under drought [10,62] and mycorrhizal restoration in other cultivars [63]. The magnitude and direction of the response hinged on fungal isolate, as AMF1 strongly promoted root growth in Arbequina while AMF2 enhanced specific absorption and raised root length, volume, and tips in Kor-WS, confirming that plant–fungus compatibility steers morphological adjustments and nutrient uptake [64] and indicating that R. intraradices (AMF1) favors Arb whereas F. mosseae (AMF2) favors Kor, each extending hyphal networks that enlarge effective root surface, improve water–nutrient acquisition, and boost biomass accumulation under drought [32].

4.3. Effects of AMF on Photosynthesis, Fluorescence, and Water Status in Two Olive Varieties Under Different Water Conditions

When water was plentiful, AMF consistently raised carbon fixation in both cultivars. In Arb-WW, AMF1 raised Gs and Tr (p < 0.01), while AMF2 increased Pn by 19%, Gs by 143%, and Tr by 152% (p < 0.01) relative to the non-AMF control. Kor-WW responded even more strongly to AMF2, with Pn, Gs, and Tr rising by 63%, 82%, and 74%, respectively; Fv/Fm remained near 0.8 in both cultivars, indicating intact PSII and correlating positively with Ψw [33]. Lower hydraulic resistance for a given Tr kept Gs wide and leaf water status high, mirroring Ye [65], who found no WW difference in Fv/Fm, Fv/F₀, ΦPSII, or NPQ between mycorrhizal and non-mycorrhizal grapevines. Under drought, photosynthesis declined (p < 0.001) and Pn, Gs, Tr, and Ci dropped, in line with Sbbar [18], Boughalleb [12] and Parri [13], yet RWC remained stable in Arb-WS and Kor-WS, confirming olive drought tolerance [66]. AMF1 and AMF2 lifted Pn by 18% and 21% in Arb-WS and by 58% and 43% in Kor-WS (p < 0.05), respectively. In Arb-WS, AMF1 simultaneously reduced Tr by 83% and Gs by 50% compared with non-AMF plants while elevating Ci by 12% (p < 0.01); in Kor-WS, AMF2 raised Gs by 68% without altering Tr or Ci (p > 0.05). Both strains thus alleviated drought limitation, agreeing with Bonetto [67], who reported higher stem water potential and Gs in mycorrhizal ‘Arbequina’ and ‘Barnea’. AMF plants combined lower Gs with higher Tr, showing that partial stomatal closure curbed water loss while transpiration continued under high temperature and low humidity, and because RWC did not differ, the Pn gain was not mediated by leaf water content. Mycorrhizal inoculation also prevented drought-induced loss of PSII efficiency, raised Fv/Fm, and sustained light–energy conversion while elevating leaf Ψw and preserving turgor, with AMF2 giving the largest Fv/Fm and Ψw increase in stressed Arb-WS, echoing Ouledali [38], who recorded positive turgor in one-year-old mycorrhizal olives under drought but a decline in non-mycorrhizal plants. Taken together, the data indicate that AMF enhance electron transport, improve light-use efficiency, and strengthen drought tolerance in olive seedlings.

4.4. Effects of AMF on Photosynthetic Pigments and Nutrient Elements in Two Olive Varieties Under Different Water Conditions

Under WW conditions, AMF increased olive leaf pigments and nutrients in the same way as reported for lettuce [68]. Chlorophyll a, chlorophyll b, and total chlorophyll all rose with AMF (p < 0.01); Arb-WW gained 33% in chlorophyll b and 17% in total chlorophyll (p < 0.01), implying that hyphal extension enlarges the absorptive surface and that the two isolates differ in C/N uptake efficiency [48,69]. WS depressed Chla, Chlb, total Chl, and carotenoids in Arb-WS and Kor-WS, yet AMF1 and AMF2 slowed the loss of chlorophyll a in Arb-WS and Kor-WS, whereas chlorophyll b and total chlorophyll continued to decline. Carotenoids followed the same pattern: concentrations were higher in Arb-WW and Kor-WW, peaking at 0.49 mg g⁻¹ in Kor-WW. Elevated carotenoids not only harvest light but also detoxify ROS and feed abscisic-acid and strigolactone synthesis, while greater total chlorophyll supports sustained Pn [70]. WS also reduced leaf C, root C, and root N (p < 0.01), yet leaf C rose by 2% with AMF1 and by 2% with AMF2 in Kor-WS (p < 0.05). Root N did not change in Koroneiki or in Arb-WS, whereas Arb-WW gained 36% under AMF2 (p < 0.05). Root C edged up by 2% in Arb-WS and by 1% in Kor-WS with AMF2 (p < 0.05), whereas AMF1 had no effect on root C or N. These results parallel gains recorded in ‘Picholine’ and ‘Arbequina’ inoculated with R. irregularis [71,72]. Thus, AMF reshape C and N uptake and allocation in a cultivar- and water-dependent manner, enhancing pigment synthesis, protein production, and ultimately growth [72].

4.5. Effects of AMF on Antioxidant Enzymes and Osmoregulatory Substances in Two Olive Varieties Under Different Water Conditions

Under full irrigation, neither cultivar displayed water stress, yet subtle AMF effects emerged: soluble sugar and proline responded strongly to both water status and AMF (p < 0.01). In Arb-WW roots, SOD responded differently to AMF1 and AMF2 depending on water status, implying that balanced SOD-POD crosstalk maintains ROS homeostasis in both organs [73]. This protection was confirmed by lower malondialdehyde values: root MDA in Arb-WW remained stable (p > 0.05), whereas Kor-WW root MDA increased with AMF2, collectively demonstrating negligible oxidative damage under ample water. After water withholding, osmotic adjustment became critical and soluble sugars together with soluble proteins served as osmoprotectants and ROS scavengers [14,15,16]. AMF1 and AMF2 increased Arb-WS leaf soluble sugar by 60% and 72%, respectively, while AMF2 elevated Kor-WS leaf soluble sugar by 28% and soluble protein by 54% (p < 0.01). Arb-WS leaf soluble proteins rose by 29% under AMF2 (p < 0.05), and Kor-WS root soluble sugar was unchanged, yet root soluble protein surged by 46%/51% in Arb-WS and 136%/95% in Kor-WS under AMF1/AMF2 (p < 0.01). Leaf proline remained stable for both cultivars and treatments (p > 0.05), trends that point to AMF-driven sugar accumulation. Proline, acting as a membrane stabilizer and radical scavenger [74], was unchanged in leaves under WS, yet AMF1/AMF2 increased root proline in Arb-WW by 31%/19%, in Arb-WS by 68%/52%, in Kor-WW by 23%/40% and in Kor-WS by 100%/94% (p < 0.01). Both strains lowered MDA in leaves and roots of both cultivars, in line with reports that AMF curb lipid peroxidation [75]. Although AMF1 and AMF2 also modulated leaf and root SOD and POD, occasional declines in root POD or leaf SOD occurred, and earlier studies show that R. intraradices colonizes roots more extensively than Glomus spp. and promotes olive biomass more effectively [40,76,77]. Overall, the data reveal that Kor-WS antioxidant machinery responds more sensitively to AMF2, whereas Arb-WS counters oxidative stress more efficiently through AMF1 and thereby achieves greater drought resistance, a conclusion reinforced by principal component analysis that mirrors the findings of Salimonti et al. [78], who used the same multivariate approach to classify ‘Frantoio’ as drought-susceptible and ‘Arbequina’ as tolerant and demonstrated that AMF inoculum alleviates water-deficit damage mainly in the susceptible cultivar by targeting the most pivotal physiological responses.

5. Conclusions

Under well-watered (WW) conditions, both AMF strains colonized over 60% of root systems, significantly enhancing plant growth. AMF1 increased aboveground fresh weight (AFW), underground fresh weight (UFW), and aerial dry weight (ADW) by 25–35% in Arbequina–WW, chlorophyll b content by 33%, and total chlorophyll by 17%. It also elevated above- and underground biomass in Koroneiki–WW. AMF2 further boosted net photosynthesis (Pn) by 18%, stomatal conductance (Gs) by 143%, transpiration rate (Tr) by 152%, and maximum photochemical efficiency (Fv/Fm) by 15%, alongside a 36% rise in root nitrogen (N) content (p < 0.05) in Arbequina–WW. Under water-stressed (WS) conditions, AMF mitigated biomass loss and restored physiological functions. AMF1 restored Arbequina–WS Pn by 18%, Fv/Fm by 37%, and water potential (Ψw) by 18% while reducing leaf malondialdehyde (MDA) content by 44%. AMF2 enhanced Koroneiki–WS plant Pn by 43%, Fv/Fm by 43%, Ψw by 47%, root volume (RV) by 137%, and nitrogen resorption efficiency (NRT) by 48% alongside a 18% reduction in root MDA (p < 0.05). Both strains increased soluble sugars (SS), starch (SP), and proline content by 19–100% in both cultivars. Principal component analysis (PCA; PC1 explaining 63% variance) grouped Arbequina–WS + AMF1 as the most stress-resilient cluster. Significant correlations emerged between root peroxidase (POD) activity, osmoregulation, and lower Ψw, coupled with enhanced root allocation (p < 0.05). While AMF conferred shared benefits under well-watered conditions, drought responses were cultivar-specific: AMF1 provided superior protection to Arbequina, whereas AMF2 maximized Koroneiki’s performance. Both strains improved photosynthesis, water status, pigments, nutrient uptake, and antioxidant capacity, imparting robust drought resistance. This study underscores the critical role of AMF strain specificity and cultivar adaptability in modulating plant responses to water regimes, providing a clear blueprint for strain–cultivar pairing to enhance olive drought resilience and sustainable production under climate change.