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Article

Differential Effects of Arbuscular Mycorrhizal Fungi on Rooting and Physiology of ‘Summer Black’ Grape Cuttings

by
Yi-Yuan Peng
1,
Chun-Yan Liu
1,* and
Yong Hao
2,*
1
Hubei Key Laboratory of Spices & Horticultural Plant Germplasm Innovation & Utilization, College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China
2
College of Urban Construction, Yangtze University, Jingzhou 434025, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(7), 825; https://doi.org/10.3390/horticulturae11070825
Submission received: 17 June 2025 / Revised: 6 July 2025 / Accepted: 8 July 2025 / Published: 10 July 2025
(This article belongs to the Section Viticulture)
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Abstract

Arbuscular mycorrhizal fungi (AMF) symbiosis has great potential in improving grapevine performance and reducing external input dependency in viticulture. However, the precise, strain-specific impacts of different AMF species on ‘Summer Black’ grapevine cuttings across multiple physiological and morphological dimensions remain underexplored. To address this, we conducted a controlled greenhouse pot experiment, systematically evaluating four different AMF species (Diversispora versiformis, Diversispora spurca, Funneliformis mosseae, and Paraglomus occultum) on ‘Summer Black’ grapevine cuttings. All AMF treatments successfully established root colonization, with F. mosseae achieving the highest infection rate. In detail, F. mosseae notably enhanced total root length, root surface area, and volume, while D. versiformis specifically improved primary adventitious and 2nd-order lateral root numbers. Phosphorus (P) uptake in both leaves and roots was significantly elevated across all AMF treatments, with F. mosseae leading to a 42% increase in leaf P content. Furthermore, AMF inoculation generally enhanced the activities of catalase, superoxide dismutase, and peroxidase, along with soluble protein and soluble sugar contents in leaves and roots. Photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), were dramatically increased in AMF-colonized cutting seedlings. Whereas, P. occultum exhibited inhibitory effects on several growth metrics, such as shoot length, leaf and root biomass, and adventitious lateral root numbers, and decreased the contents of Nitrogen (N), potassium (K), magnesium (Mg), and iron (Fe) in both leaves and roots. These findings conclusively demonstrate that AMF symbiosis optimizes root morphology, enhances nutrient acquisition, and boosts photosynthetic efficiency and stress resilience, thus providing valuable insights for developing targeted bio-fertilization strategies in sustainable viticulture.

1. Introduction

Grape (Vitis vinifera L.), belonging to the Vitaceae family, is globally recognized as one of the most widely cultivated and economically important fruit crops. Its popularity is largely attributed to delicious fruits, which are rich in diverse nutrients, including organic acids, glucose, and various vitamins [1], as well as a wide range of beneficial phytochemicals [2,3]. These compounds contribute to the well-established health benefits of grapes, such as anti-inflammatory, cardioprotective, anti-cancer, and anti-diabetic properties [3]. The rapid expansion of the global grape industry in recent decades has highlighted the urgent need for sustainable agricultural practices, as current intensive cultivation often relies heavily on chemical fertilizers, leading to soil fertility degradation, ecosystem balance disruption, etc. In this context, exploring biological alternatives for enhancing grape cultivation is of paramount importance for fostering ecological and sustainable development.
Root system structure directly determines nutrient and water absorption efficiency, which profoundly influences plant growth performance, stress resilience (e.g., drought and disease resistance), and ultimately, fruit quality [4]. Specific root components, such as root hairs, adventitious roots, and lateral roots, all play crucial roles. Especially lateral roots are particularly prominent in anchoring plants, expanding the absorptive surface, and storing nutrients. The dense network of root hairs is indispensable for the efficient uptake of less mobile nutrients, notably the easily fixed element phosphorus (P) [5].
Arbuscular mycorrhizal fungi (AMF) represent a ubiquitous and ecologically significant group of soil microorganisms that form mutualistic symbioses with the roots of the vast majority of terrestrial plants [6]. This ancient symbiosis is fundamental to ecosystem stability and sustainable productivity in agricultural systems [7]. Upon infecting host roots, they form characteristic structures such as intraradical hyphae, vesicles, and arbuscules, and extensive extraradical hyphal networks in the soil [8]. This network acts as an extension of the host’s root system, significantly increasing water and nutrient uptake, thereby boosting plant growth and vigor [9]. AMF are particularly renowned for their capacity to improve plant acquisition of mineral nutrients, especially P, by mobilizing and transporting it from the soil, thus providing a robust nutritional foundation for plant development [10]. Beyond P, they also enhance the absorption and utilization of other essential nutrients, such as nitrogen, potassium, calcium, iron, copper, zinc, manganese, and boron. Furthermore, AMF inoculation could improve plant photosynthetic efficiency by influencing pigment content and enzyme activities [11]. Meanwhile, AMF contribute significantly to plant stress tolerance by enhancing osmotic regulation [12] and resistance to various abiotic stresses, e.g., drought [13], salinity [14], heavy metals [15], and temperature extremes [16]. They also play a role in improving soil structure [17] and conferring disease resistance [18].
It is well-known that AMF has positive effects on grapevines, which include promoting growth, improving nutrient absorption, and enhancing tolerance to abiotic stresses such as water deficit and high temperatures, as well as biotic stresses [19]. Notably, the effectiveness of AMF inoculation is highly variable and strain-specific, with different fungal species or isolates exhibiting distinct impacts on various plant species and genotypes. For instance, Meng et al. [20] demonstrated that inoculation with different AMF strains (Diversispora versiformis, Funneliformis mosseae, and Rhizophagus intraradices) significantly enhanced multiple growth parameters and root morphological traits in Tinospora sagittata plants. In grape cultivation, Luciani et al. [21] inoculated AMF on one-year-old ‘Sangiovese’ self-rooted seedlings and three-year-old ‘Sangiovese’ grafted seedlings (rootstock 420A) and found that Glomus iranicum significantly increased total root volume and promoted root extension in grapevines. Similarly, inoculation with G. intraradices or G. mosseae enhanced new shoot growth in grapes after five weeks [22]. In addition, Fattahi et al. [23] demonstrated that inoculation with G. mosseae, G. intraradices, G. etunicatum, and G. verruciforme on ‘Asgari’ grapevines significantly increased chlorophyll content, relative leaf water content, and concentrations of P, K+, and Ca2+ in grape tissues. Under water-limited conditions, G. verruciforme and G. etunicatum provided the most significant protection to grapevines. This variability highlights the importance of characterizing specific AMF strains for targeted applications.
While existing research has identified the general benefits of AMF for grape growth and stress tolerance, there are still critical knowledge gaps regarding the specific impacts of AMF on the rooting process and early growth of grape cuttings. Establishment of a robust root system is fundamental for the successful propagation and subsequent field performance of grapevines, but the exact effects of different AMF strains on the development of complex root architecture in cuttings remain underexplored.
Vitis vinifera ‘Summer Black’ is a European-American hybrid triploid variety developed in Japan through the crossbreeding of Vitis vinifera and Vitis labrusca. This variety is characterized by its dark purple to black, seedless berries, which are primarily utilized for fresh consumption and wine production. While ‘Summer Black’ grapes exhibit strong stress resistance and high yield, their root systems tend to be shallower compared to those of other varieties. Hence, we hypothesized that AMF inoculation would significantly enhance the rooting success rate and early growth performance of ‘Summer Black’ grape cuttings. Therefore, the present study was aimed at evaluating the mycorrhizal colonization capabilities of each AMF strain in grape roots. Simultaneously quantify their impact on overall growth performance and detailed root system architecture of grape cuttings, as well as elucidate their impact on the physiological activities of regulation of nutrient absorption and accumulation in grape tissues. Thus, highly efficient AMF strains with great practical application potential in sustainable viticulture could be identified. The findings will provide a crucial theoretical and practical foundation for optimizing AMF technology in grape production, ultimately contributing to establishing more resilient and environmentally friendly cultivation systems.

2. Materials and Methods

2.1. Plant Material and Growth Substrate

Cuttings of Vitis vinifera L. ‘Summer Black’ were obtained from Jingqiuyuan Company (Jingzhou, Hubei, China), and all the cuttings were from a single mother plant; each branch was approximately 30 cm long with the stem thickness of 1 cm. Initially, mother branches were stored in sand for 8 weeks under controlled conditions (4–7 °C with consistent moisture, maintaining a relative humidity of approximately 80%) to prevent desiccation and maintain bud viability prior to propagation. Subsequently, uniform stem segments approximately 10 cm in length, each containing two plump buds, were prepared for cutting propagation. Before cutting, we checked all maternal branches to ensure that no calluses appeared. These cuttings were planted into plastic pots (upper inner diameter: 20 cm, bottom inner diameter: 15 cm, height: 18 cm, approximately 3 L) with 2.5 kg of growth substance.
The potting substrate consisted of a 1:1 (v/v) mixture of garden soil and sand. The soil had a pH of 6.1, and organic carbon (C), available N, Oslen-phosphorus (P), and available potassium (K) were 9.7 mg/kg, 11.8 mg/kg, 15.3 mg/kg, and 21.5 mg/kg, respectively. To eliminate indigenous arbuscular mycorrhizal fungi (AMF) spores and other soil-borne pathogens, the substrate was sterilized by autoclaving at 121 °C and 0.1 MPa for 2 h with each cycle lasting 1 h and performed twice [20].

2.2. Arbuscular Mycorrhizal Fungal Inocula

Four AMF species were used in this study: All fungal inocula were Diversispora versiformis, Diversispora spurca, Funneliformis mosseae, and Paraglomus occultum, obtained from the Institute of Root Biology at Yangtze University (Jingzhou, Hubei, China). Each inoculum consisted of colonized root segments, AMF spores, and river sand, propagated in maize trap cultures. The approximate spore density in each inoculum was about 3500 spores per 80 g. The control (non-AMF) treatment consisted of 80 g of sterilized mycorrhizal inoculum (autoclaved at 0.11 MPa and 121 °C for 2 h) supplemented with 2.5 mL of a filtrate (through a 25 µm filter) from the inoculum to maintain similar microbial communities, excluding the AMF strain [20].

2.3. Experimental Design and Cultivation

A single-factor completely randomized experimental design was employed, comprising five treatments: inoculation with D. versiformis, D. spurca, F. mosseae, P. occultum, and a non-inoculated control (non-AMF). For AMF treatments, 80 g of the respective AMF inoculum was applied using a “layered inoculation method” during planting [24]. This method involved placing approximately half of the sterilized substrate into each pot, then evenly spreading the 80 g of AMF inoculum, placing the grape cutting on top, and finally covering it with the remaining sterilized substrate. This ensures optimal contact between the inoculum and the developing root system, facilitating early colonization. For the non-AMF control, 80 g of sterilized inoculum (prepared by autoclaving the mixed inoculum at 120 °C, 0.1 MPa for 2 h to inactivate viable spores) was added to each pot to standardize the organic matter input and exclude viable AMF spores while minimizing microbial community differences.
One grape cutting was planted per pot. Each treatment consisted of six replicates, with each pot representing an experimental unit, totaling 30 pots and 30 seedlings. All pots were randomly arranged in a greenhouse. Grape cuttings were cultivated under controlled greenhouse conditions for 12 weeks. The specific environmental parameters maintained were the following: the photosynthetic photon flux density was 982 µmol/m2/s with a day/night temperature regime of 28 °C/20 °C, relative humidity of 65 ± 5%, and a 16-h light/8-h dark photoperiod with a photosynthetically active radiation intensity of approximately 400 µmol m−2·s−1 at plant canopy height, provided by natural sunlight supplemented with artificial grow lights [25]. Plants were regularly watered with distilled water to maintain optimal soil moisture content at approximately 70% of field capacity, determined gravimetrically by weighing representative pots daily to avoid water stress that could confound AMF effects.

2.4. Physiological Parameter Measurements

After 12 weeks of inoculation with AMF, seedlings—both AMF-inoculated and non-AMF-inoculated were harvested. Prior to harvest, on a sunny day between 9:00 and 11:00 a.m., photosynthetic parameters were measured. The fifth fully expanded functional leaf below the apex of each cutting was selected for measurement. A portable CO2/H2O analysis system, the LI-6400 (Li-COR, Lincoln, OR, USA) was used to determine net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), and stomatal conductance (Gs). The effective radiation of the light source was PAR 1000 m2 mol/(m2.s), the leaf chamber used was 2 cm × 3 cm, and the gas flow rate was 500 mmol.s−1. Three groups of leaves were determined randomly at each layer of each plant [26].

2.5. Growth and Root Morphological Measurements

At harvest (12 weeks after planting), grape cuttings were carefully removed from their pots, and their root systems were thoroughly washed free of soil. Overall plant growth parameters, including the number of shoots, branch length, branch thickness, and number of leaves, were manually recorded: the number of shoots was counted; branch length was measured from the substrate surface to the tip of the longest shoot using a ruler; branch thickness was measured at 1 cm above the substrate surface using a digital caliper; and the total number of fully expanded leaves was counted.
Intact root systems were then spread in a clear root tray and scanned using an Epson Perfection V700 Photo Dual Lens System (J221A, Seiko Epson Corporation, Jakarta Selatan, Indonesia). Root configuration parameters, including total root length, root surface area, projected area, and root volume, were analyzed using WinRHIZO Version 2007b software (Regent Instruments Inc., Quebec, QC, Canada). Subsequently, adventitious roots and lateral roots at each order were manually counted [25]. Lateral roots were classified based on their origin: primary lateral roots emerged directly from the main adventitious root axis, secondary lateral roots branched from primary ones, and tertiary lateral roots originated from secondary ones.

2.6. Mycorrhizal Infection and Soil Hyphal Length

Approximately 300 g of rhizosphere soil samples were collected from each pot immediately after plant removal and stored at −20 °C for subsequent analysis of soil hyphal length. Soil hyphal length was determined using the method described by Bethlenfalvay and Ames [27].
Remove the root tip (approximately 0–0.5 cm) from the root hair zone, specifically 2–6 cm from the root tip. Sixty 1-cm-long fresh root segments from each whole cutting root system, categorized by class (ten primary adventitious roots, twenty 1st-order lateral adventitious roots, twenty 2nd-order lateral adventitious roots, and ten 3rd-order lateral adventitious roots) per treatment, were randomly selected for mycorrhizal colonization determination. These samples were cleared in a 10% (w/v) KOH (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) solution at 90 °C for 1.5 h in a water bath, rinsed, acidified with 2% HCl (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and then stained with 0.05% (w/v) trypan blue (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in lactic acid [28]. Root segments were observed under a Leica DME binocular light microscope (Leica Microsystem Inc., Wetzlar, Germany). Mycorrhizal infection rate was quantified using the gridline intersect method [29]. Specifically, each stained root segment was placed on a slide, and a minimum of 100 intersections between root segments and gridlines were examined per replicate. Mycorrhizal infection rate was calculated as (number of infected gridline intersections/total number of gridline intersections) × 100%.

2.7. Chemical and Biochemical Analyses

Grape roots and leaves were separately rinsed, heat-inactivated at 105 °C for 30 min, and then oven-dried at 80 °C until constant weight (typically 48 h). Dried samples were ground into a fine powder and sieved through a 4-mm mesh for subsequent chemical analysis.
Nitrogen (N) content in grape roots and leaves was determined using a Smart Chem 200 chemical analyzer (AMS-Alliance, Rome, Italy) after digestion with a mixture of concentrated sulfuric acid and hydrogen peroxide. The contents of other mineral elements, including phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), and boron (B), were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, IRIS Advantage, Thermo Electron Corp., Waltham, MA, USA) after digestion with a mixture of concentrated nitric acid and perchloric acid [25].
The activities of antioxidant enzymes, specifically catalase (CAT) and superoxide dismutase (SOD), were determined spectrophotometrically according to the methods described by Poobathy et al. [30]. Peroxidase (POD) activity was measured using the guaiacol method [31]. Soluble protein content was quantified by the Coomassie Brilliant Blue G-250 method [32], as cited by Qi et al. [33]. Soluble sugar content was determined using the anthrone-sulfuric acid colorimetric method [34].

2.8. Statistical Analysis

All experimental data were subjected to one-way analysis of variance (ANOVA) using IBM SPSS Statistics 27 software (http://www.ibm.com/legal/copytrade.shtml, accessed on 6 July 2025). Significant differences among treatment means were determined by Duncan’s multiple range test, with a significance level of p < 0.05. Data visualization and graph generation were performed using Microsoft Excel 2019.

3. Results

3.1. Mycorrhizal Establishment and Colonization Rate

All four inoculated AMF species successfully colonized the root of ‘Summer Black’ grape cuttings, forming typical mycorrhizal structures including intracellular and extracellular hyphae, vesicles, and arbuscules (Figure 1a–d). F. mosseae inoculation resulted in the highest mycorrhizal infection rate, reaching 93.73% (Figure 2a), but resulted in the lowest soil hyphae density (16.33 cm g−1) (Figure 2b). Conversely, the opposite results appeared in D. versiformis treatment, manifested as the lowest mycorrhizal colonization (Figure 2a) and the greatest soil hyphae density among the inoculated groups (Figure 2b).

3.2. Effects of AMF Inoculation on Grape Cuttings Growth Parameters

AMF inoculation significantly influenced the growth parameters of ‘Summer Black’ grape cuttings, with distinct variations among the different fungal species (Table 1).
Compared to the non-AMF control, D. versiformis and F. mosseae inoculation significantly increased shoot length and leaf and stem fresh weight, but they all did not affect the shoot diameter. D. spurca inoculation only specifically increased stem fresh weight and significantly reduced shoot diameter. In contrast, P. occultum inoculation significantly inhibited the plant growth of ‘Summer Black’ cuttings (Table 1).

3.3. Effects of AMF Inoculation on Grape Root Architecture

Significant and varied effects on root architecture were observed among grape cuttings inoculated with different AMF species (Figure 3, Table 2). F. mosseae inoculation uniquely and significantly improved root system architecture, manifested as increased total root length by 8.84%, root surface area by 3.33%, and root volume by 48.56%, and adventitious root by 111.43% and 1st-order lateral adventitious root by 35.00%, as compared to the non-AMF control (Table 2). Conversely, treatments with D. versiformis, D. spurca, and P. occultum did not significantly affect these macroscopic root morphological indicators (total length, surface area, volume), and the effects on adventitious root development were also inconsistent, with varying manifestations (Table 2).

3.4. Effects of AMF Inoculation on Mineral Element Contents in Grapes

AMF inoculation significantly altered the mineral element profiles in both grape leaves and roots (Table 3). A consistent and prominent effect across all AMF treatments was the significant increase in P content in both grape leaves and roots, ranging from 95.29 to 131.76% in leaves and 60.66–104.92% in roots, as compared to the non-AMF control. F. mosseae inoculation resulted in the highest root P content, 1.97 g/Kg, representing an increase of 131.8%, and D. spurca also showed a strong positive effect on leaf P content, with an increase of 104.92%. Furthermore, F. mosseae yielded the highest root P content, 1.97, an increase of 131.76%.
In addition, D. versiformis colonization only significantly increased root K, Mg, Cu, and Fe content, while dramatically decreasing all the leaf mineral element content (except N). D. spurca inoculation markedly increased leaf N, Zn, and root K, Fe, and Mn content but notably decreased the other mineral content except root Mg content. In P. occultum colonized cuttings, higher leaf B, Cu, and Zn and root Mg, Cu, and Mn appeared, and the other elements were all markedly decreased, except root K, as compared with non-AMF colonized cuttings (Table 3).

3.5. Effects of AMF Inoculation on Antioxidant Enzyme Activities in Grapes

AMF inoculation notably influenced the activities of antioxidant enzymes (SOD, POD, CAT) in both grape roots and leaves (Figure 4). In grape leaves (Figure 4a), all four AMF species significantly enhanced leaf CAT activity, with increases ranging from 15.77 to 98.63%. D. versiformis, F. mosseae, and P. occultum significantly increased leaf SOD activity, with D. versiformis demonstrating the most potent effect, an increase of 102.39%. Meanwhile, D. versiformis, D. spurca, and P. occultum also significantly enhanced leaf POD activity, with D. spurca resulting in the highest increase, by 91.07%.
In root, D. spurca significantly increased SOD by 139.86%, POD by 43.18%, and CAT by 20.66%. D. versiformis notably enhanced POD and SOD by 42.55% and 135.7%, respectively. F. mosseae significantly increased SOD and CAT by 30.27% and 78.58%, respectively. Furthermore, inoculation with P. occultum significantly increased the activities of SOD by 214.94%, POD by 64.52%, and CAT by 41.16%, respectively, as compared to the non-AMF control (Figure 4b).

3.6. Effects of AMF Inoculation on Soluble Protein and Soluble Sugar Contents in Grape Tissues

Across grape cuttings, the contents of soluble protein and soluble sugar were consistently higher in leaves than in roots (Table 4). Following different AMF inoculations, leaf soluble protein increased by 10.5–17.86%, and root soluble protein increased by 13.95–24.85%, in which D. versiformis exhibited the highest promoting effects.
Soluble sugar content also differed significantly after AMF inoculation compared to the non-AMF control; the highest soluble sugar content in roots was observed under D. spurca treatment, with an increase of 19.03%. In leaves, P. occultum treatment resulted in the highest soluble sugar content, with an increase of 42.14%.

3.7. Effects of AMF Inoculation on Grape Photosynthesis

All four AMF species, D. versiformis, D. spurca, F. mosseae, and P. occultum, significantly increased Pn, Gs, and Tr in grape leaves compared to the non-AMF control (Figure 5). Among the four different AMF fungi, F. mosseae exhibited the best promotion effects, with Pn, Tr, and Gs reasonably increased by 258.98%, 96.58%, and 86.21%, respectively. Moreover, D. versiformis and D. spurca treatments increased Ci by 14.48% and 16.86%, respectively. In contrast, P. occultum dramatically reduced Ci by 7.84%, and F. mosseae had no significant effect on Ci (Figure 5).

3.8. Pearson Correlations

The Pearson correlation analysis conducted on the dataset aimed to identify correlations unique to each treatment group concerning mycorrhizal development and root growth parameters, variables, and Physiological index (Figure 6). On plant growth and root morphology, mycorrhizal colonization was significantly and positively correlated with total root length and average diameter, and soil hyphae density was positively correlated with 2nd-order lateral root. Meanwhile, root morphology parameters, especially the number of adventitious roots, are closely related to most growth indicators (p ≤ 0.05 or p ≤ 0.01).
In terms of plant physiology, mycorrhizal colonization was significantly positively correlated with Gs but negatively correlated with leaf SOD. Meanwhile, there was a positive relationship between soil hyphae density and Ci and leaf SOD; similarly, a negative relationship also appeared between soil hyphae density and leaf soluble sugar content, Pn, and CAT (p ≤ 0.05 or p ≤ 0.01).
For nutrient absorption, mycorrhizal development may have a better effect on the absorption of large amounts of nutrients than on the promotion of trace elements, although there is a negative correlation between soil hyphae density and the content of some elements, mainly because mycorrhizal infection has maintained the absorption of host plant nutrients at a stable level.

4. Discussion

This study comprehensively investigated the diverse impacts of four distinct AMF species, D. versiformis, D. spurca, F. mosseae, and P. occultum, on the growth, root architecture, mineral nutrition, and physiological functions of ‘Summer Black’ grapevine cuttings. The results robustly demonstrate that inoculation with different AMF strains elicits distinct and measurable responses in the host plant.
The foundation of AMF-induced benefits lies in the successful establishment of the symbiotic relationship. Our results confirmed that all four inoculated AMF species successfully colonized the roots of ‘Summer Black’ grapevine cuttings (Figure 1); this initial compatibility is a prerequisite for any subsequent beneficial effects. Among the tested strains, F. mosseae exhibited the highest mycorrhizal colonization, which was obviously higher than other strains, suggesting a high degree of compatibility with the ‘Summer Black’ grapevine under the experimental conditions. In contrast, D. versiformis, despite its comparatively lower intraradical colonization, developed the most extensive soil extraradical mycelial density, 24.87 cm g−1. This divergence highlights that the functional effectiveness of an AMF strain is not solely dictated by its internal root colonization percentage but also by its capacity for prolific external hyphal growth, which is critical for soil exploration and nutrient uptake beyond the root depletion zone [35]. This differential emphasis on internal vs. external fungal biomass development likely contributes to the varied plant responses observed across the strains.
The most conspicuous outcome of successful AMF symbiosis is often enhanced plant growth [36]. Our results demonstrated that F. mosseae and D. versiformis consistently promoted the above-ground growth of ‘Summer Black’ grape cuttings, significantly increasing shoot length and biomass (Table 1). This generalized growth enhancement is a direct consequence of improved resource acquisition and optimized plant physiological processes by AMF [37]. The plant root system is the main organ for obtaining nutrient resources from soil. In this study, the superior total root length, root surface area, root volume, and primary and 1st-order adventitious root numbers were observed in F. mosseae-inoculated cuttings, which was very beneficial for promoting robust above-ground growth as it provided a greater absorptive surface. In addition, D. versiformis and D. spurca showed inconsistent effects on root system architecture of grape cuttings, indicating that different AMF strains can “engineer” distinct root architectures tailored to different soil resource foraging strategies. Conversely, P. occultum intensely exhibited the inhibitory effects on several growth parameters and adventitious root development, suggesting that while symbiosis was established, it may impose a metabolic cost or be less functionally compatible with the ‘Summer Black’ grapevine under these specific conditions, highlighting the inherent variability in AMF-host interactions [38].
Enhanced nutrient uptake, particularly of P, is a cornerstone of AMF benefits. Our study consistently revealed a significant increase in P content in both grape leaves and roots across all AMF treatments (Table 3), with F. mosseae and D. spurca exhibiting the most pronounced effects in leaves and F. mosseae in roots. This confirms the well-established role of AMF in facilitating P acquisition by extending the effective absorption zone beyond the root depletion zone and potentially accessing less available P forms [35]. The impact on other mineral elements, however, was highly variable and strain-dependent, reflecting the complex interplay between host demand, fungal transport capabilities, and soil nutrient dynamics. For instance, while D. spurca, F. mosseae, and P. occultum generally increased leaf Zn and Cu, D. versiformis led to their reduction. Similarly, root Fe content generally increased, but P. occultum treatment resulted in a decrease. This nuanced selectivity in nutrient absorption and translocation among different AMF species underscores that AMF do not simply enhance general nutrient uptake but actively modulate the plant’s mineral profile in a highly specific manner [39]. Such differential nutrient foraging strategies could be linked to the varied requirements of the fungal strains or their specific transporter systems, providing a competitive advantage for certain elements [40,41]. In addition, in this study, we observed different responses of AMF inoculation to various mineral elements, which may be related to the nutritional level of the cultivation substrate itself, as additional N, P, and K fertilization may inhibit AMF infection and thus affect nutrient absorption under field conditions [42,43]. Furthermore, a previous study also showed that field application of AMF (Acauloapora scrobiculata, Diversispora spurca, D. versiformis) was able to improve the fruit’s external quality of Citrus sinensis via increasing soil ammonium N, nitrate N, Olsen P, and available K content [44]. Subsequently, similar results were obtained on Camellia oleifera after application of the endophytic fungus Serendipita indica in field conditions, as well as the content of P, K, and Ca significantly increasing in Camellia oleifera leaves and roots [45]. These results indicated that microorganisms in the soil, especially endophytic fungi, can interact with mineral elements in the soil, thereby promoting the absorption and utilization of nutrients by host plants.
Previous studies have demonstrated that AMF inoculation significantly elevated soluble sugars and soluble protein levels, thereby enhancing host resilience to drought stress and bolstering defense against biotic stressors, including grape [46]. In this study, the four different AMF strains tested in the present study all dramatically altered the content of soluble protein in varying degrees, but we all know that this is mainly attributed to the improvement of nutrient conditions, which enhances the resistance of grape cuttings. We observed differential responses of AMF inoculation to various mineral elements, potentially due to the interplay between N, P, K fertilization and AMF colonization, especially the content of N, P, and K. Previous field experiments have demonstrated that some biostimulants, such as Ausuma, Bioilsa, and BF-Ecomix, can enhance the abundance and species richness of mycorrhizal fungi in the grapevine rhizosphere [42,43].
Beyond macronutrients, AMF symbiosis also significantly modulated plant physiological resilience. Our study revealed notable increases in the activities of key antioxidant enzymes, SOD, POD, and CAT, in both grape roots and leaves following AMF inoculation (Figure 4). These enzymes are vital components of the plant’s defense system against reactive oxygen species (ROS), which are naturally produced during metabolism and amplified under stress conditions [12]. The elevation in antioxidant enzyme activities across multiple AMF treatments suggests an enhanced capacity for oxidative stress mitigation, even under the relatively optimal conditions of this experiment. This pre-priming or strengthened basal defense mechanism could confer greater intrinsic resilience to future environmental challenges such as drought, salinity, or pathogen attack encountered in field settings [34,47].
The benefits of AMF symbiosis also extended to the photosynthetic machinery of ‘Summer Black’ grape cuttings (Figure 5). All four AMF species significantly enhanced Pn, Gs, and Tr, indicating improved carbon assimilation efficiency and water flux through the plant. F. mosseae demonstrated the most pronounced positive effects on Pn, Gs, and Tr, consistent with its superior root colonization and overall growth promotion. The increased Gs facilitates greater CO2 influx into the leaves, supporting higher photosynthetic rates. Changes in Ci were more variable: D. versiformis and D. spurca increased Ci, suggesting that photosynthetic carboxylation capacity might have become more limiting than stomatal opening. Conversely, P. occultum reduced Ci, indicating potential stomatal limitations. The overall enhancement of photosynthetic efficiency, combined with improved nutrient availability, directly contributes to increased biomass production by providing more photosynthates for plant growth and for supporting the fungal symbiont. This is further corroborated by the elevated soluble protein and soluble sugar contents in both leaves and roots of AMF-inoculated plants (Table 4), signifying enhanced metabolic activity and efficient carbon partitioning. These soluble compounds also serve as crucial osmolytes, energy reserves, and building blocks for various physiological processes and stress responses [34].
In conclusion, our study provides robust evidence for the significant and highly species-specific benefits of AMF symbiosis for ‘Summer Black’ grapevine cuttings. We confirm that different AMF strains vary markedly in their colonization patterns (intraradical vs. extraradical development), their capacity to promote specific aspects of plant growth and root architecture, their selective influence on mineral nutrient profiles, and their differential enhancement of antioxidant defenses and photosynthetic efficiency. F. mosseae and D. versiformis emerged as particularly effective strains for promoting general growth and physiological vigor in grapevines, largely attributed to their strong capacities for nutrient uptake and enhanced photosynthetic performance. The complex and nuanced responses observed across different AMF strains underscore that selecting the appropriate AMF strain is crucial for maximizing the benefits in viticulture and achieving desired outcomes under specific cultivation conditions.

5. Conclusions

This comprehensive study confirms the successful symbiotic establishment of all four inoculated AMF strains with ‘Summer Black’ grapevine roots. F. mosseae consistently demonstrated superior efficacy, leading to the highest root colonization, significant improvements in overall root system development (total root length, surface area, and volume), and the most pronounced increases in P absorption by both leaves and roots. This strain also significantly enhanced above-ground growth parameters and photosynthetic rate (Pn, Gs, and Tr). Furthermore, all tested AMF species to varying degrees increased antioxidant enzyme activities (SOD, POD, CAT) in both roots and leaves, as well as soluble protein and soluble sugar contents. This multifaceted physiological enhancement collectively improved grapevine resilience and promoted robust growth and development. Conversely, P. occultum, despite forming symbiosis, exhibited inhibitory effects on several growth metrics and specific root architectural traits, underscoring the potential for less compatible or even detrimental interactions depending on the specific AMF-host combination. Therefore, F. mosseae is a recommended candidate for AMF-based inoculation in grapevine propagation.

Author Contributions

Conceptualization, Conceptualization, C.-Y.L. and Y.H.; methodology, Y.-Y.P., C.-Y.L. and Y.H.; data curation and statistical analysis, Y.-Y.P. and C.-Y.L.; writing—original draft preparation, Y.-Y.P. and C.-Y.L.; writing—review and editing, C.-Y.L. and Y.H.; supervision, C.-Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data is contained within the article.

Acknowledgments

I extend my sincerest gratitude to Jia-Dong He, for his patient guidance, invaluable advice, and unwavering support throughout the research design, experimental implementation, and manuscript preparation.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Mycorrhizal structures observed in roots of Vitis vinifera L. ‘Summer Black’ cuttings inoculated with different arbuscular mycorrhizal fungi species ((a), D. versiformis; (b), D. spurca; (c), F. mosseae; (d), P. occultum) under controlled greenhouse conditions. (A) Intraradical hyphae; (B) Extraradical hyphae; (C) Spore; (D) Vesicle.
Figure 1. Mycorrhizal structures observed in roots of Vitis vinifera L. ‘Summer Black’ cuttings inoculated with different arbuscular mycorrhizal fungi species ((a), D. versiformis; (b), D. spurca; (c), F. mosseae; (d), P. occultum) under controlled greenhouse conditions. (A) Intraradical hyphae; (B) Extraradical hyphae; (C) Spore; (D) Vesicle.
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Figure 2. (a) Mycorrhizal colonization and (b) soil hyphae density of Vitis vinifera L. ‘Summer Black’ cuttings inoculated with different arbuscular mycorrhizal fungi species under controlled greenhouse conditions. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test.
Figure 2. (a) Mycorrhizal colonization and (b) soil hyphae density of Vitis vinifera L. ‘Summer Black’ cuttings inoculated with different arbuscular mycorrhizal fungi species under controlled greenhouse conditions. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test.
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Figure 3. Growth status of Vitis vinifera L. ‘Summer Black’ cuttings under different arbuscular mycorrhizal fungi. inoculation treatments. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test.
Figure 3. Growth status of Vitis vinifera L. ‘Summer Black’ cuttings under different arbuscular mycorrhizal fungi. inoculation treatments. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test.
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Figure 4. Comparative effects of four arbuscular mycorrhizal fungi species on antioxidant enzyme (SOD, POD, and CAT) activities in (a) leaf and (b) root of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test. SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.
Figure 4. Comparative effects of four arbuscular mycorrhizal fungi species on antioxidant enzyme (SOD, POD, and CAT) activities in (a) leaf and (b) root of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test. SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.
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Figure 5. Comparative effects of four arbuscular mycorrhizal fungi species on (a) Pn, (b) Gs, (c) Ci, and (d) Tr of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test. Pn, net photosynthetic rate; Gs, stomatal conductance; Ci, intercellular CO2 concentration; Tr, transpiration rate.
Figure 5. Comparative effects of four arbuscular mycorrhizal fungi species on (a) Pn, (b) Gs, (c) Ci, and (d) Tr of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions. Data are presented as means ± SD (n = 6). Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05) determined by LSD test. Pn, net photosynthetic rate; Gs, stomatal conductance; Ci, intercellular CO2 concentration; Tr, transpiration rate.
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Figure 6. Pearson correlations between mycorrhizal development and root morphology traits (a), Physiological index (b), and mineral element contents (c) of Vitis vinifera L. ‘Summer Black’ cuttings inoculated with different AMF species. Red and blue colors indicate positive and negative relationships, respectively. Circle sizes indicate the correlation strength. *, p < 0.05, means significant; **, p < 0.01, means extremely significant, ***, p < 0.001, means high extremely significant.
Figure 6. Pearson correlations between mycorrhizal development and root morphology traits (a), Physiological index (b), and mineral element contents (c) of Vitis vinifera L. ‘Summer Black’ cuttings inoculated with different AMF species. Red and blue colors indicate positive and negative relationships, respectively. Circle sizes indicate the correlation strength. *, p < 0.05, means significant; **, p < 0.01, means extremely significant, ***, p < 0.001, means high extremely significant.
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Table 1. Comparative effects of four arbuscular mycorrhizal fungi species on the growth parameters of Vitis.vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
Table 1. Comparative effects of four arbuscular mycorrhizal fungi species on the growth parameters of Vitis.vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
TreatmentPlant Growth StatusBiomass (g FW/plant)
Number of Shoots (#/plant)Shoot Length (cm)Shoot
Diameter (cm)		Leaf Number (#/plant)LeafShootRoot
non-AMF2.6 ± 0.89 a25.3 ± 2.1 c4.41 ± 0.37 a25 ± 2 a21.29 ± 1.35 b21.03 ± 1.52 b5.74 ± 0.40 a
D. versiformis1.8 ± 0.00 c31.1 ± 2.8 a4.18 ± 0.31 a23 ± 2 ab22.34 ± 0.99 a23.35 ± 1.01 a5.90 ± 0.43 a
D. spurca2.4 ± 0.89 a22.5 ± 2.5 c3.78 ± 0.35 b25 ± 2 a21.73 ± 1.43 ab23.77 ± 1.00 a5.03 ± 0.31 ab
F. mosseae2.0 ± 0.00 b28.8 ± 2.7 b4.07 ± 0.32 a21 ± 2 b23.10 ± 1.24 a23.10 ± 0.76 a4.70 ± 0.30 b
P. occultum2.4 ± 0.55 a16.8 ± 1.7 d3.66 ± 0.33 b19 ± 1 b18.24 ± 1.11 c21.12 ± 0.72 b3.51 ± 0.14 c
Note: data (means ± SD, n = 6) followed by different letters above the bars indicate significant differences among treatments (p < 0.05).
Table 2. Comparative effects of four arbuscular mycorrhizal fungi species on the root system architectural parameter of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
Table 2. Comparative effects of four arbuscular mycorrhizal fungi species on the root system architectural parameter of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
TreatmentTotal Root Length/cmSurface Area/cm2Volume/cm3Primary
Adventitious Root		Adventitious Lateral Root (#/Plant)
1st-Order2nd-Order3rd-Order
non-AMF283.55 ± 25.33 b13.81 ± 0.45 b4.18 ± 1.75 b35 ± 2 c1030 ± 72 b839 ± 20 b77 ± 3 b
D. versiformis298.40 ± 25.85 ab14.07 ± 0.43 ab4.76 ± 1.90 ab24 ± 2 d724 ± 23 d806 ± 39 b158 ± 13 a
D. spurca301.40 ± 21.00 ab14.17 ± 0.33 ab6.03 ± 1.95 ab61 ± 4 b940 ± 63 c1072 ± 73 a36 ± 2 c
F. mosseae308.61 ± 12.78 a14.27 ± 0.23 a6.21 ± 1.15 a74 ± 6 a1390 ± 34 a684 ± 19 c78 ± 6 b
P. occultum301.80 ± 15.36 ab13.94 ± 0.43 ab5.19 ± 1.39 ab31 ± 2 c726 ± 20 d556 ± 25 d14 ± 1 d
Note: data (means ± SD, n = 6) followed by different letters above the bars indicate significant differences among treatments (p < 0.05).
Table 3. Comparative effects of four arbuscular mycorrhizal fungi species on mineral element contents in leaves and roots (per gram DW) of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
Table 3. Comparative effects of four arbuscular mycorrhizal fungi species on mineral element contents in leaves and roots (per gram DW) of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
OrganMineral ElementNon-AMFD. versiformisD. spurcaF. mosseaeP. occultum
LeafMacronutrients
N (g/kg)10.62 ± 0.40 b10.39 ± 0.14 b13.62 ± 0.16 a9.45 ± 0.21 c8.37 ± 0.15 d
P (g/kg)0.61 ± 0.07 c1.00 ± 0.03 b1.25 ± 0.04 a1.24 ± 0.01 a0.98 ± 0.03 b
K (g/kg)8.26 ± 0.17 a5.14 ± 0.02 c7.36 ± 0.02 b8.40 ± 0.03 a7.25 ± 0.04 b
Ca (g/kg)15.28 ± 0.33 a9.72 ± 0.15 d11.25 ± 0.06 c13.30 ± 0.36 b13.10 ± 0.31 b
Mg (mg/kg)2.23 ± 0.03 a1.51 ± 0.02 d1.72 ± 0.05 c2.10 ± 0.03 b1.99 ± 0.03 b
Micronutrients
B (mg/kg)24.40 ± 0.50 c18.87 ± 0.37 d25.40 ± 0.54 c46.96 ± 0.71 a42.00 ± 1.68 b
Cu (mg/kg)161.04 ± 3.37 d82.58 ± 1.77 e290.79 ± 3.38 c321.39 ± 4.89 b349.16 ± 4.06 a
Fe (mg/kg)353.00 ± 9.43 a166.38 ± 1.93 e307.50 ± 3.50 c194.85 ± 3.34 d338.23 ± 3.93 b
Mn (mg/kg)47.44 ± 0.47 a26.15 ± 0.61 e30.09 ± 0.47 d33.63 ± 0.59 c37.75 ± 0.79 b
Zn (mg/kg)40.27 ± 1.02 d31.96 ± 0.50 e49.61 ± 0.75 c86.61 ± 3.03 a73.44 ± 1.44 b
RootMacronutrients
N (g/kg)5.37 ± 0.04 a4.50 ± 0.13 b4.12 ± 0.02 d4.21 ± 0.03 c4.25 ± 0.04 c
P (g/kg)0.85 ± 0.04 d1.86 ± 0.01 b1.70 ± 0.04 c1.97 ± 0.03 a1.66 ± 0.02 c
K (g/kg)8.36 ± 0.19 b9.97 ± 0.19 a7.51 ± 0.26 c9.41 ± 0.08 a8.44 ± 0.13 b
Ca (g/kg)10.23 ± 0.31 a9.38 ± 0.11 b8.55 ± 0.17 c7.84 ± 0.16 d7.71 ± 0.16 d
Mg (mg/kg)1.51 ± 0.04 b1.72 ± 0.01 a1.60 ± 0.06 b1.45 ± 0.04 bc1.34 ± 0.02 c
Micronutrients
B (mg/kg)18.38 ± 0.64 a12.21 ± 0.29 c16.05 ± 0.18 b12.95 ± 0.27 c15.35 ± 0.31 b
Cu (mg/kg)75.27 ± 0.86 c198.88 ± 7.01 a39.99 ± 0.85 d72.16 ± 1.62 c94.86 ± 3.34 b
Fe (mg/kg)442.66 ± 4.43 d582.81 ± 9.05 c890.79 ± 18.99 a767.25 ± 19.37 b347.81 ± 3.48 e
Mn (mg/kg)14.00 ± 0.24 b14.14 ± 0.28 b18.41 ± 0.21 a13.59 ± 0.28 bc13.37 ± 0.21 c
Zn (mg/kg)39.38 ± 1.20 a37.37 ± 0.73 b26.55 ± 0.31 c38.49 ± 0.65 a37.14 ± 0.88 b
Note: data (means ± SD, n = 6) followed by different letters above the bars indicate significant differences among treatments (p < 0.05).
Table 4. Comparative effects of four arbuscular mycorrhizal fungi species on soluble protein and soluble sugar contents in leaves and roots of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
Table 4. Comparative effects of four arbuscular mycorrhizal fungi species on soluble protein and soluble sugar contents in leaves and roots of Vitis vinifera L. ‘Summer Black’ cuttings under controlled greenhouse conditions.
TreatmentSolube Protein Content (mg/g)Soluble Sugar Content (mg/g)
RootLeafRootLeaf
non-AMF0.0889 ± 0.0009 c0.1114 ± 0.0079 c0.0725 ± 0.0051 c0.1450 ± 0.0091 d
D. versiformis0.1198 ± 0.0093 a0.1580 ± 0.0139 a0.0823 ± 0.0081 a0.1622 ± 0.0054 c
D. spurca0.0902 ± 0.0070 bc0.1142 ± 0.0104 bc0.0863 ± 0.0069 a0.1766 ± 0.0085 b
F. mosseae0.1013 ± 0.0048 b0.1313 ± 0.0122 b0.0767 ± 0.0052 b0.1960 ± 0.0073 a
P. occultum0.1183 ± 0.0036 a0.1231 ± 0.0085 b0.0740 ± 0.0024 bc0.2061 ± 0.0096 a
Note: data (means ± SD, n = 6) followed by different letters above the bars indicate significant differences among treatments (p < 0.05).
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Peng, Y.-Y.; Liu, C.-Y.; Hao, Y. Differential Effects of Arbuscular Mycorrhizal Fungi on Rooting and Physiology of ‘Summer Black’ Grape Cuttings. Horticulturae 2025, 11, 825. https://doi.org/10.3390/horticulturae11070825

AMA Style

Peng Y-Y, Liu C-Y, Hao Y. Differential Effects of Arbuscular Mycorrhizal Fungi on Rooting and Physiology of ‘Summer Black’ Grape Cuttings. Horticulturae. 2025; 11(7):825. https://doi.org/10.3390/horticulturae11070825

Chicago/Turabian Style

Peng, Yi-Yuan, Chun-Yan Liu, and Yong Hao. 2025. "Differential Effects of Arbuscular Mycorrhizal Fungi on Rooting and Physiology of ‘Summer Black’ Grape Cuttings" Horticulturae 11, no. 7: 825. https://doi.org/10.3390/horticulturae11070825

APA Style

Peng, Y.-Y., Liu, C.-Y., & Hao, Y. (2025). Differential Effects of Arbuscular Mycorrhizal Fungi on Rooting and Physiology of ‘Summer Black’ Grape Cuttings. Horticulturae, 11(7), 825. https://doi.org/10.3390/horticulturae11070825

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