Microbial-Enhanced Abiotic Stress Tolerance in Grapevines: Molecular Mechanisms and Synergistic Effects of Arbuscular Mycorrhizal Fungi, Plant Growth-Promoting Rhizobacteria, and Endophytes

Abstract

Grapevines (Vitis vinifera L.) face significant challenges from abiotic stresses caused by climate change, including drought, salinity, and temperature extremes. This comprehensive review examined the role of beneficial microorganisms in enhancing grapevine tolerance to these stresses, focusing on arbuscular mycorrhizal fungi (AMF), plant growth-promoting rhizobacteria (PGPR), and endophytes. The study analyzes species-specific effects and their molecular mechanisms, highlighting how single and consortium inoculations improve plant resilience. AMF species, particularly Funneliformis mosseae and Rhizophagus irregularis, demonstrated significant enhancement in drought and salinity tolerance through improved nutrient uptake and stress response modulation. The PGPRs, Bacillus and Pseudomonas species, show remarkable abilities to mitigate various abiotic stresses through mechanisms including phytohormone production and antioxidant defense enhancement. Endophytic microorganisms such as Pseudomonas fluorescens RG11 and Serendipita indica play crucial roles in stress mitigation through melatonin production and improved water retention, respectively. The synergistic effects of combined AMF, PGPR, and PGPF applications led to a significant increase in grapevine drought and salinity tolerance, improving nutrient uptake, photosynthesis rates, and antioxidant defense mechanisms. Molecular analysis revealed that these microbial consortia regulate the expression of stress-responsive genes, particularly VvNCED and VvP5CS, enhancing grapevine resilience through improved osmotic adjustment, ROS scavenging, and hormonal regulation. These findings provide valuable insights into the molecular pathways underlying stress tolerance, offering promising strategies for sustainable viticulture under climate change.

1. Introduction

Grapes are a highly valued and extensively cultivated horticultural crop [1], currently covering 7.2 million ha of cultivated land and producing 74.7 million tons of grapes globally [2]. They thrive in temperate regions globally, from hot, arid environments to tropical climates, and in very cold areas, producing fruits that most usually are utilized for wine, table grapes, and raisin production [3]. This worldwide grapevine expansion implies the existence of a broad spectrum of physiological mechanisms that provide the necessary adaptation potential to different environments [4]. Multi-century targeted selection of cultivated grapevine (Vitis vinifera L.) and specialized management practices have contributed to its worldwide adaptation [4]. Under these circumstances, grapevines frequently encounter less-than-ideal growth conditions, resulting in environmental or abiotic stress caused by factors such as drought, salinity, temperature extremes, and nutrient imbalances [5]. Global climate change has broad effects on grapevine cultivation by imposing challenges such as an increase in the unpredictability and severity of abiotic stresses [6]. Abiotic stress is defined as an environmental condition that finally reduces growth and yield, affecting most of the globally cultivated area [7], as only a small part of this area can be considered unaffected by some environmental constraints [8]. Meanwhile, modern viticulture faces the challenge of achieving a complex set of management objectives to ensure both financial viability and sustainability. These goals mean always meeting the high grape quality standards needed for wine production or for selling grapes in the fresh grape market. At the same time, grape growers must keep grapevine production profitable. To achieve this, they must solve production management risks that affect market access, farm strength, and sustainability, especially as climate change worsens [9]. Given the worldwide expansion of vineyards, we can conclude that, in the overall context of a highly competitive market environment coupled with climate change challenges and threats, abiotic stresses may have a large impact on grapevine cultivation [10].

The responses of grapevines to abiotic stresses, including drought, salinity, and extreme temperatures, are typically complex and interconnected. Despite differences in the symptomatology of these responses, some common features of the underlying mechanisms exist. For example, a common feature in the response to all stresses is the onset of oxidative stress through the production of reactive oxygen species (ROS) [11]. Adverse abiotic factors perturb the equilibrium between ROS production and ROS scavenging [12]. In general, although each stress condition induces a distinct response, there is some overlap between reactions to abiotic stressors (such as drought, heat, cold, salt, or high light) and biotic stressors (such as pathogens) [13].

The primary physiological responses of grapevines to water deficits include a reduction in water potential (Ψ), diminished growth rates, and stomatal closure, which leads to decreased photosynthesis and reduced evaporative cooling [13]. Initially, vines resist growth reduction through osmotic adjustment, but prolonged drought leads to complete stomatal closure and potential wilting. Increased stress associated with the summer season can lead to dehydration and heat damage, whereas irregular rainfall patterns can stress vines, leading to oxidative damage and compromising grape quality [14]. Continuous water stress can cause xylem embolism, potentially leading to vine death. Notably, grapevines exhibit “vulnerability segmentation”, shedding leaves to protect stems from embolism, allowing survival in extreme drought, and potential recovery in subsequent seasons [15]. Although water shortages typically decrease crop yields, they can enhance red wine quality in all but the most severe cases. Although not typically applied for white wine production, very mild water deficits, when carefully managed, can improve white wine quality, whereas severe water deficits are detrimental to yield and quality [16]. However, as climate change leads to increased water deficits, grape harvests are negatively affected, thereby reducing the economic viability of wine production. Consequently, adapting to drier growing environments is becoming crucial for viticulture worldwide [16].

When cultivated in saline soils, own-rooted grapevine tends to accumulate salt, resulting in diminished yields and altered berry growth and chemical composition. This phenomenon can adversely affect the sensory attributes of wine, potentially compromising its profitability and the reputation of wine production in salt-affected regions. Moreover, the sustainability of viticultural areas may be jeopardized if soil salinity reaches levels at which grapevines produce fruits with salt concentrations exceeding permissible limits [5,17]. Plants subjected to increased soil salinity experience osmotic stress, ion toxicity, and oxidative damage that may intensify and ultimately co-occur as the growing season progresses, making it challenging to attribute plant responses to either adaptation mechanisms or the direct effects of salinity [17]. Owing to these negative effects of salinity, grapevines exposed to high salt levels in the soil have hindered growth, reduced yield, decreased photosynthesis, and degraded fruit quality through reduced sugar content and elevated acidity. The severity of these effects is most severe during the vegetative growth stage rather than the reproductive phase [18].

Grapevine responses to different abiotic stresses are not uniform across genotypes. Numerous studies have demonstrated that different grapevine cultivars exhibit distinct physiological and biochemical responses to abiotic stresses. For instance, ‘Cabernet Sauvignon’ is recognized for its moderate drought tolerance, maintaining acceptable yield and berry composition under water-deficit conditions [13]. In contrast, ‘Chardonnay’ vines showed significant sensitivity to salinity, exhibiting reduced shoot growth and photosynthesis at relatively low NaCl concentrations [17]. ‘Grenache Noir’ has been reported to exhibit higher thermotolerance, maintaining stomatal conductance and transpiration rates under high-temperature stress [19]. In contrast, ‘Tempranillo’ and ‘Pinot Noir’ are more heat-sensitive, with studies showing reductions in berry sugar accumulation and overall fruit quality under elevated temperatures [20,21]. Therefore, these differences are crucial for the selection of varieties in stress-prone regions.

In addition to grape cultivars, rootstocks are crucial components of viticultural adaptation strategies. They play a central role in modulating grapevine responses to drought, salinity, and nutrient availability by influencing root architecture, hydraulic conductivity, ion exclusion, and hormonal signaling between roots and scions. A meta-analysis conducted by Marín et al. [22] on Spanish viticulture revealed that rootstock selection significantly affects scion physiological performance, berry sugar accumulation, and berry acidity levels under environmental stress. Rootstocks such as “110 Richter”, “1103 Paulsen”, and “140 Ruggeri” are associated with improved drought tolerance through enhanced water-use efficiency and deeper rooting profiles [22]. In a long-term field study, El-Salhy et al. [23] demonstrated that rootstocks such as ‘Black Balady’ and ‘White Khalili’ showed superior vegetative growth, reduced Na⁺ and Cl⁻ translocation to shoots, and higher photosynthetic activity under saline irrigation conditions. These findings demonstrate the importance of ion exclusion capacity and membrane stability conferred by certain rootstocks in salinity-stressed environments. In addition to physiological responses, rootstocks also modulate the position and function of the grapevine rhizosphere microbiome. Zarraonaindia et al. [24] showed that different rootstocks influence microbial community gathering in the root zone, thereby enhancing nutrient cycling and stress resilience through microbial-mediated mechanisms. Overall, these studies emphasize the importance of rootstock choice as a fundamental element in vineyard stress management.

A diverse array of strategies for adapting grapevines to adverse environmental conditions and mitigating the effects of various abiotic stresses has been implemented over the centuries in the global vineyard, especially in the last two decades under the pressure of climate change. These strategies include the utilization of appropriate rootstocks [25,26] and varieties [11,16] as well as vineyard design options and management practices [16]. In numerous instances, these strategies have been formulated as traditional knowledge, which has ensured the remarkable adaptation of grapevines to highly challenging environmental conditions [16]. Within the context of this wealth of empirical and scientific knowledge on grapevine cultivation, the plant-associated microbiome may contribute to adaptation to adverse conditions as it can enhance tolerance to abiotic stresses [27]. Furthermore, both scions and rootstocks can interact with microorganisms in the root space of grapevines [28]. The plant-associated microbiome represents an additional factor within the environmental interactions occurring in the vineyard, which not only increases the level of complexity but also expands the range of adaptive processes, considering the extended phenotype and the microbiome’s contribution to it [4]. Therefore, considering the challenges posed to grapevine cultivation by abiotic stresses, which are exacerbated under climate change conditions, understanding and leveraging plant-microbe interactions presents a promising avenue for sustainable viticulture. In response to these challenges, plants have developed a ‘crying-for-help’ strategy whereby they recruit beneficial microbes to their holobiont to mitigate stresses and facilitate adaptation to changing environments [29]. This strategy underscores the complex interactions between plants and their associated microbiota, which can be utilized to enhance crop resistance to abiotic stresses. Harnessing these microbial interactions could potentially reduce reliance on chemical fertilizers and pesticides, contributing to more sustainable and environment-friendly viticulture practices.

In this context, beneficial microorganisms have emerged as potential tools for ensuring crop stability under adverse environmental and soil stress conditions [30]. Pacifico et al. [31] stated that these microorganisms contribute to plant growth through diverse mechanisms, both directly and indirectly. These mechanisms include improvement in nutrient uptake and assimilation (through increased root biomass, length, volume, and branching), increase in photosynthetic rates and osmolyte production, and enhancement of leaf water relations and antioxidant defense systems. These enhancements lead to reduced transpiration and increased stomatal resistance, along with regulation of key genes involved in reactive oxygen species (ROS) detoxification. To illustrate, endophytes, such as P. fluorescens, P. phytofirmans, and Pseudomonas migulae, have the ability to synthesize ACC deaminase, which avoids ethylene accumulation, hence alleviating growth inhibition caused by excessive ethylene [31]. In addition, the most frequent mechanisms by which endophytic fungi contribute to plants are siderophore production, phosphate solubilization, and nitrogen (N) fixation. Backer et al. [32] found that PGPR can regulate phytohormone levels, such as auxin, gibberellin, cytokinins, ethylene, abscisic acid, and brassinosteroids, which play pivotal roles in plant development. To demonstrate, the plant-associated bacterial endophyte Burkholderia phytofirmans PsJN modulates carbohydrate metabolism that limits chilling damage to grapevine plantlets exposed to low-temperature stress. Additionally, Habib et al. [33] stated that ACC production by PGPR enhances salt tolerance in okra, increases antioxidant enzyme activities (superoxide dismutase (SOD), ascorbate peroxidase (APX), and CAT (catalase)), and upregulates ROS pathway genes (CAT, APX, and GR). Chen et al. [34] reported that Bacillus amyloliquefaciens (strain SQR9) enhanced salt stress tolerance in maize seedlings by enhancing chlorophyll content, total soluble sugar content, peroxidase/catalase activity, and glutathione content and reducing Na⁺ levels. Furthermore, beneficial strains of mycorrhizal fungi have been applied in combination with PGPR to improve efficiency and promote plant growth, as AM hyphae can reach soil pores and assimilate water and nutrients [35].

Considering the importance of abiotic stresses for grapevine cultivation on a global scale in the context of climate change, this review aimed to summarize the inoculation effects of key beneficial microorganisms on grapevine performance under abiotic stress conditions and elucidate the potential of utilizing these microorganisms as an additional strategy for vineyard management under challenging environmental conditions. Unlike prior studies that primarily focused on single-strain inoculations, this review highlights the emerging potential of microbial consortia in creating robust, stress-resilient systems for grapevines. Our main objectives are (1) to investigate the effectiveness of using single versus multiple microbial species in promoting grapevine health under abiotic stress conditions. (2) Explore the synergistic effects of combined microbial species by investigating their roles in phytohormonal regulation, enzymatic activity, and metabolite production. (3) To analyze the stress tolerance pathways activated through gene expression in grapevines and the effects of specific species on plant molecular pathways and gene expression.

2. Materials and Methods

Topic Analysis and Categorization of Studies

A comprehensive bibliometric analysis of the literature from 2017 to 2024, conducted through Scopus, Web of Science, and Google Scholar, revealed a scarcity of studies investigating grapevine responses to abiotic stresses, highlighting an opportunity for further research in this area compared to the more extensively studied biotic stressors. The keywords used in our search included “grapevine abiotic stress”, “Vitis vinifera stress tolerance”, “drought stress grapevine”, “salinity stress grapevine”, “temperature stress viticulture”, “beneficial microorganisms grapevine”, “AMF grapevine”, “PGPR viticulture”, “stress response mechanisms grapevine”, “stress-responsive genes grapevine”, “MYB transcription factors”, “CBL-CIPK signaling”, “heat shock proteins”, “antioxidant enzymes”, “hormone signaling pathways”, and “stress-induced metabolites”.

The literature review identified several distinct research patterns focusing on three primary categories. First, physiological response studies have examined plant-water relations, drought tolerance mechanisms, heat stress responses, salinity tolerance, and oxidative stress responses. Second, beneficial microorganism studies have explored the effects of arbuscular mycorrhizal fungi, plant growth-promoting rhizobacteria, endophytic fungi input, and microbial consortium applications. Third, molecular and genetic studies have investigated gene expression patterns, stress-responsive transcription factors, hormone signaling pathways (ABA-mediated responses), and epigenetic regulation mechanisms. The research has focused on temperature-related stress, water deficit, and salinity. Contemporary studies have demonstrated a significant shift toward integrative approaches combining multiple research methodologies, including high-throughput genomics, metabolomics, and field-based physiological studies, representing a more comprehensive understanding of grapevine stress responses than earlier research periods.

To further enhance our understanding of the overall structure and evolution of relevant research conducted on grapevine responses to abiotic stresses and how they are affected by the application of beneficial microorganisms, we conducted a keyword co-occurrence analysis in a social network analysis [36]. Before the network analysis, keyword cleansing and standardization were conducted using OpenRefine (Version 3.8.7 with Java) software [37]. The obtained keyword network was visualized in network, overlay, and cluster density maps using the VOSViewer (Version 1.6.20, with Java) software. The cluster density map (Figure 1) reveals the overall structure of the topic and specific thematic areas related to the three categories of beneficial organisms.

The overlay visualization map (Figure 2) represents the average years of relevant publications and their trends by thematic area over time. Figures S1–S3 depict the keyword co-occurrence network for each of the beneficial microorganism categories. In general, this analysis revealed ongoing research trends in beneficial microorganism studies within the overall context of grapevine responses to abiotic stress towards sustainable grapevine cultivation directions.

3. AMF and Abiotic Stress Tolerance in Grapevines

Most experimental studies have focused on the beneficial effects of arbuscular mycorrhizal fungal root inoculation, whether through single or multiple species, on the morphophysiological parameters that define the growth, development, and health of grapevine plants under various stress conditions (

Table 1. AMF stress-mitigation mechanisms in the grapevine.

Microbial Inoculant	Product	Stress	Mechanism of Action	Inoculant amount	Form of Inoculum	Age	Location	Reference
Glomus mosseae	-	drought	Enhanced water and nutrient uptake	100 g	powder	1 year	greenhouse	[39]
Glomus intraradices	-	salinity	Increased photosynthesis rate	25 spores g−1	powder	1 year	greenhouse	[40]
Funneliformis mosseae	-	heat	Increased photosynthesis rates	10 g	powder	young	field	[41]
Funneliformis mosseae IMA 1	-	drought	Volatile organic compounds of different metabolic pathways; hormonal balance	2 mL	liquid	4 weeks	greenhouse	[42]
Funneliformis mosseae, Glomus aggregatum, Claroideoglomus etunicatum	MycoApply®	drought	Increased photosynthesis rate increased osmolyte accumulation and antioxidant activities	5 g	powder	2 years	greenhouse	[43]
Rhizophagus intraradices, Funneliformis mosseae, Glomus aggregatum, Glomus etunicatum	Myco Apply Endo Maxx	drought	Regulation of anthocyanin and flavanol metabolisms Improvement of photosynthetic activity	10 g	powder	2 years	field	[19]
Rhizophagus clarus UFSC-14, Rhizophagus intraradices UFSC-32, Dentisculata heterogama UFSC-08	-	copper toxicity	Lower leaf Cu accumulation; activation of chelation mechanisms	-	liquid	young	field	[44]

). Funneliformis mosseae, formerly known as Glomus mosseae and Rhizophagus irregularis, also referred to as Glomus intraradices, are the most commonly investigated species in mutual symbiosis with young grapevine plants subjected to drought, salinity, and temperature extremes in controlled environments (greenhouses). Young grapevines exhibit heightened sensitivity to environmental changes and treatments, making them particularly responsive to water stress. This sensitivity allows researchers to observe the effects of various interventions within a shorter timeframe and with fewer resources [38].

Single Strains vs. Multi-Species

Kamayestani et al. [39] reported that inoculation with Glomus mosseae could increase plant tolerance to drought by improving water and nutrient supply. Inoculation increased the number of leaves in the ‘Pikani’ and ‘Shahroudi’ grape cultivars. Notably, the most substantial fresh and dry stem weights, the highest number of internodes, and the greatest root length were recorded in a water deficit of 60% with AMF. The highest root dry weight was observed under a water deficit of 40%. Funneliformis mosseae IMA1 increased specific volatile compounds (VOCs) in grapevine plants by 85% [39]. Nogales et al. [40] demonstrated that introducing Funneliformis mosseae via cover crops mitigated heat wave effects by improving phosphorus uptake, chlorophyll content, photosynthesis, and stomatal conductance, emphasizing its role in drought and heat stress resilience. In addition, Sensoy et al. [41] reported that Rhizophagus irregularis showed significant improvements in growth parameters such as shoot diameter and height, root width and length, and number of leaves, while also enhancing phosphorus and water uptake. On the other hand, in mitigating copper toxicity, Rhizophagus clarus and Rhizophagus irregularis have been shown to play a vital role by reducing the translocation of copper to the shoots while promoting its accumulation in the roots through chelation mechanisms involving organic compounds released by the fungi and their associated bacteria [44].

Ye et al. [43] reported that combined species of AMF enhanced osmotic adjustment by increasing compatible solutes and photosynthesis and improved carbon assimilation and gas exchange. The combined effects of Funneliformis mosseae, Glomus aggregatum, and Claroideoglomus etunicatum significantly enhanced the production of sucrose and proline, which improved osmotic adjustment capacity, thereby maintaining photosynthetic activity under drought stress conditions. These fungi also increased the activity of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD), which collectively enhanced the grapevine’s ability to scavenge ROS and mitigate oxidative damage [43]. Torres et al. [19] demonstrated that Rhizophagus intraradices, Funneliformis mosseae, Glomus aggregatum, and Glomus etunicatum improved carbon assimilation rates and enhanced gas exchange parameters, including stomatal conductance, as well as increased chlorophyll concentration and overall photosynthetic capacity.

4. PGPR and Abiotic Stress Tolerance in Grapevines

Several studies have explored the efficacy of PGPR in enhancing grapevine resilience to diverse environmental challenges. Bacillus and Pseudomonas were the most commonly used genera (

Table 2. PGPR stress-mitigation mechanisms in grapevines.

Microbial Inoculant	Product	Stress	Mechanism of Action	Inoculant Amount	Form of Inoculum	Age	Location	Reference
Bacillus licheniformis	-	frost	Increase antioxidant and enzyme activity	108 CFU ml−1	Liquid	1–2 years	field	[45]
Arthrobacter	-	lead toxicity		5 mL	Liquid	young	greenhouse	[46]
Agrobacterium rubi A18, Bacillus subtilis OSU 142	-	high pH	Improvement of vegetative growth, leaf physiology and nutrient acquisition	109 CFU mL−1	Liquid	2 years	greenhouse	[47]
Pseudomonas, Enterobacter, Achromobacter	-	drought	Improvement of nutrient uptake, hormonal balance, and antioxidant capacity	150 mL	Liquid	1 year	greenhouse	[48]
Bacillus licheniformis, Micrococcus luteus, Pseudomonas fluorescens	-	arsenic toxicity	Increased antioxidant enzyme activity Reduced peroxidation of membrane lipids	50 mL	Liquid	2 years	field	[49]
Pantoea anthophila, Pantoea agglomerans, Pantoea sp.	-	salinity	Increase in antioxidant enzyme activities Improved photosynthesis performance; production of auxins and siderophores, nitrogen fixation, and phosphate solubilization	150 mL	Liquid	seedlings	greenhouse	[50]
Bacillus amyloliquefaciens QST 713, Sinorhizobium meliloti B2352	HYDROMAAT, FUTURECO BIOSCIENCE®	drought	Regulation of plant hormonal balance	2% w/w	Liquid	seedlings	greenhouse	[51]
Pseudomonas composti Bacillus zhangzhouensis Pseudarthrobacter Aeromonas aquariorum Bacillus methylotrophicus Bacillus aryabhattai	-	heat	Improvement of nutrient acquisition, modulation of hormonal responses, induction of systemic resistance, and enhanced antioxidant activity	107 CFU mL−1	Liquid	seedlings	greenhouse	[52]

).

Single Strains vs. Multi-Species

Köse et al. [45] demonstrated that Bacillus licheniformis decreased the levels of abscisic acid while increasing growth-promoting hormones such as auxins and cytokinins, thereby promoting root and shoot development. According to Papantzikos et al. [51], Bacillus amyloliquefaciens (strain QST 713), under both full and deficit irrigation conditions, enhanced nutrient availability by solubilizing phosphorus and producing phytohormones, which promoted the growth of the plant’s root system. In grapevines, inoculation with Bacillus subtilis has been shown to induce chitinase activity, which contributes to enhanced plant defense by degrading fungal cell wall components and activating systemic resistance pathways, ultimately improving tolerance to environmental stressors [53]. Sinorhizobium meliloti (strain cepa B2352) improved nitrogen availability and exhibited significantly greater leaf area and dry biomass of the roots, shoots, and leaves. This was linked to increased total chlorophyll, leaf relative water content, total phenolic compounds, and proline accumulation. Duan et al. [48] stated that PGPR with ACC deaminase activity, such as Pseudomonas corrugata and Erwinia coli, effectively mitigated drought stress in grapevines by converting 1-aminocyclopropane-1-carboxylic acid into ammonia and α-ketobutyrate, reducing ethylene levels and enhancing root growth. In addition, inoculation with these strains stimulated the production of phytohormones, such as indole-3-acetic acid (IAA), which influences shoot growth, stem elongation, root development, and the uptake of essential nutrients and water.

Inoculation with multiple species, including Pantoea anthophila and Pantoea agglomerans (isolated from halophytes), contributed to the activation of antioxidant systems and synthesis of phytohormones, leading to improved growth parameters and enhanced photosynthetic rates, including increased levels of chlorophyll a, chlorophyll b, and total chlorophyll [50]. According to Pinter et al. [49], Bacillus licheniformis, Micrococcus luteus, and Pseudomonas fluorescens increased the activity of antioxidant enzymes, including ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POX), which scavenged reactive oxygen species (ROS) and protected plant cells from oxidative damage. On the other hand, Carreiras et al. [52] found that the application of the marine PGPR consortium of A. aquariorum, B. methylotrophicus, and B. aryabhattai improved photoprotection and cell membrane stability, resulting in better photosynthetic efficiency compared to non-inoculated plants. Inoculated grapevines maintained a higher chlorophyll content and overall plant fitness, which are crucial for productivity under adverse conditions.

5. Endophytes and Abiotic Stress Tolerance in Grapevines

The endophytic communities in grapevines play an important role in enhancing stress tolerance through various mechanisms [30]. The endophytic bacteria Pseudomonas fluorescens (strain RG11) and endophytic fungus Serendipita indica were investigated for their role in antioxidant enzyme production and stilbene enhancement (

Table 3. Endophyte and PGPF stress mitigation in grapevines.

Microbial Inoculant	Type	Stress	Mechanism of Action	Inoculant Amount	Form of Inoculum	Age	Location	Reference
Serendipita indica (Piriformospora indica)	Endophytic fungus	drought	Enhanced plant growth, antioxidant enzyme and plant hormone production.	1% w/v	Liquid	-	greenhouse	[54]
Pseudomonas fluorescens RG11	Endophytic bacteria	salinity	Enhanced endogenous melatonin in plants. Regulated melatonin-related genes: TDC1 (putative tryptophan decarboxylase-1) and SNAT (serotonin N-acetyltransferase).	1 mL	Liquid	young	greenhouse	[55]
Aspergillus niger, Microdochium bolleyi, Westerdikeya centenaria	PGPF	drought	enhancing root growth. increasing nutrient availability. Improved gas exchange variables and mesophyll conductance.	10 mL	Liquid	2 years	field	[56]
Erwinia sp., Pseudomonas sp. Xanthomonas sp. Pantoea sp. And Alternaria sp. Cladosporium sp. Biscogniauxia sp. Didymella sp. Fusarium sp.	Endophytic bacteria and fungi	drought	Stimulation of stilbene and secondary metabolite production; modulation of abscisic acid (ABA) metabolism.	100 μL of bacterial suspension; 100 mg of fungal material	Liquid	young	field	[57]

).

Several of the microbial inoculants used in this study—such as Serendipita indica, Pseudomonas fluorescens RG11, Microdochium bolleyi, and Funneliformis spp.—were originally isolated as endophytes and have been experimentally confirmed to recolonize internal plant tissues following inoculation, including in grapevine (S. indica: [54]; P. fluorescens RG11: [55]; M. bolleyi: [56]; Funneliformis spp.: [42]. For other taxa such as Pantoea agglomerans, Rahnella aquatilis, Aspergillus niger, Cladosporium, and Aureobasidium, endophytic behavior has been reported in specific strains or under certain environmental conditions, though their consistent re-establishment as endophytes post-inoculation is less frequently documented and remains strain-dependent [57].

A study conducted by Ma Yaner et al. [55] showed that inoculation of grapevine roots with the endophytic bacterium Pseudomonas fluorescens (strain RG11) resulted in increased activity of antioxidant enzymes, including SOD, CAT, and POX, which were correlated with increased endogenous melatonin levels that acted as potent antioxidants.

Espareh et al. [54] reported that inoculation of grapevine plants with the endophytic fungus Serendipita indica (formerly known as Piriformospora indica) under drought stress conditions increased the production of cytokinins, which are vital for promoting cell division and improving nutrient transport through the vascular system. Additionally, S. indica promoted higher levels of carbohydrates, proteins, and proline through hexose transporters, thereby influencing plant hormone synthesis. According to Fuentes-Quiroz et al. [56], fungal species isolated from the Atacama Desert, including Aspergillus niger, Microdochium bolleyi, and Westerdikeya centenaria, enhanced root growth and improved the capacity of plants to absorb water and nutrients by producing phytohormones that enhanced root development but also promoted overall plant vigor under challenging conditions.

The study by Aleynova et al. [57] was the first to investigate the effects of key endophytic bacteria (Erwinia sp., Pantoea sp., Pseudomonas sp., and Xanthomonas sp.) and fungi (Alternaria sp., Biscogniauxia sp., Cladosporium sp., Didymella sp., and Fusarium sp.) on cell growth and the production of resveratrol and its derivatives. Each endophytic microorganism was tested individually, rather than as a consortium, allowing for evaluation of the distinct effects of single strains on resveratrol and stilbene accumulation in grapevine cells. They found that bacteria Erwinia sp., Pantoea sp., Pseudomonas sp., and Xanthomonas sp. enhanced the total stilbene content by 2.2 to 5.3 times, while endophytic fungi Alternaria sp., Biscogniauxia sp., Cladosporium sp., Didymella sp. and Fusarium sp. were even more effective, increasing stilbene accumulation by 2.6 to 16.3 times. This increase was associated with the activation of key genes involved in stilbene biosynthesis, specifically phenylalanine ammonia-lyase (PAL) and stilbene synthase (responsible for stilbene biosynthesis, particularly resveratrol). Although the highest stilbene production levels were recorded for fungal endophytes such as Biscogniauxia sp. and Didymella sp., the combination of Didymella sp. and Trichoderma sp. caused cell death and restricted cell culture biomass build-up [57].

6. Synergistic Effects of Combined AMF, PGPR and PGPF

“Diversity is strength”; a diverse community of microbial inoculants can provide a broader range of benefits compared to a single species, optimizing plant performance under various environmental conditions [58]. Liu et al. [59] conducted a global meta-analysis of 51 live-soil studies and revealed that consortium inoculation significantly outperformed single-species treatments in both biofertilization and bioremediation. The diversity of inoculants and synergistic interactions between strains increased plant growth by 48% and population remediation by 80%, whereas inoculations with a single species increased plant growth by 29% and population remediation by 48%.

Zhang et al. [60] demonstrated that synergistic inoculation of AMF and PGPR could increase the intricacy of co-occurrence networks and structural stability of microbial communities. Additionally, the metabolomic results in their study also indicated that combined microorganisms can upregulate beneficial secondary metabolic pathways (i.e., glycerophospholipid metabolism pathway, sphingolipid signaling pathway, cAMP signaling pathway, cutin, suberin, and wax biosynthesis pathways). Furthermore, integrated inoculation enhanced the expression of beneficial metabolites (i.e., lipids and lipid-like molecules), which regulate plant growth and stress response. The abundance of beneficial bacterial taxa (i.e., Actinobacteria, Chloroflexi and Paenarthrobacter) was significantly increased, as well as the symbiosis between mycorrhizal taxa (i.e., Funneliformis and Rhizophagus) and plants.

Samri et al. [61] found that combined inoculation of Glomus iranicum var. tenuihypharum and Pseudomonas putida significantly improved drought tolerance in table grapes by increasing the root surface area, facilitating better water uptake and nutrient absorption. The enhanced root surface area and water uptake were attributed to the G. iranicum var. tenuihypharum, whereas P. putida contributed to osmolyte accumulation and cellular osmotic balance. Balestrini et al. [62] reported that Funneliformis and Rhizophagus species worked synergistically to colonize grapevine roots, affecting the proteome and upregulating nitrogen metabolism genes, thereby enhancing drought resilience and berry quality by boosting nitrogen metabolism and improving water uptake efficiency. According to Goicoechea et al. [20], Rhizophagus irregularis significantly improved nutrient acquisition and water uptake efficiency and maintained a favorable sugar-to-organic acids ratio. Karimi et al. [63] stated that the combination of Glomus mosseae and Streptomyces rimosus aided the uptake of higher amounts of nutrients in the host plant and improved the physical characteristics of the shoots and roots. Furthermore, chlorophyll molecules strongly maintained their function and structure, resulting in better growth. Additionally, Alimam and Al-A’areji [64] showed that co-inoculation with Bacillus subtilis and Trichoderma harzianum presented the optimum value for total chlorophyll content in leaves (

Table 4. Microbial consortia for stress mitigation in grapevine.

Microbial Inoculant	Type	Product	Stress	Mechanism of Action	Inoculant Amount	Form of Inoculum	Age	Location	Reference
Glomus iranicum var. tenuihypharum, Pseudomonas putida	PGPR + AMF	-	salinity	increase in photosynthesis and growth	1.2 × 104 CFU 100 g−1 and 1 × 108 CFU·100 g−1	liquid	12 years	field	[61]
Funneliformis mosseae, Trichoderma viride T. harzianum Pochonia chlamidosporia Streptomyces spp. (ST60, SB14, SA51) Bacillus subtilis BA41 Pseudomonas fluorescens PN53 Glomus spp. GB67 G. mosseae GP11 Glomus viscosum GC41	AMF + PGPR + PGPF	Micosat F	drought	transcriptional regulation; modification of nutrient transport, hormonal balance and cell wall metabolism	30 g	powder	young	greenhouse	[62]
Septoglomus deserticola, Funneliformis mosseae, Rhizoglomus intraradices, Rhizoglomus clarum Glomus aggregatum Bacillus paenibacillus	AMF + PGPR	Bioradis Gel	heat	increase in photosynthetic rate; increased proline and amino acids	1 g	liquid	1 year	greenhouse	[20]
Glomus mosseae, Streptomyces rimosus	AMF + PGPR	-	salinity	increase in photosynthesis rate and regulation of hormonal balance	100 g and 106 CFU mL−1	liquid	seedlings	greenhouse	[63]
Bacillus subtilis, Trichoderma harzianum	PGPR+ PGPF	-	drought	increase in photosynthesis rate	40 g−1	powder or granular	seedlings	field	[64]

). In grapevine, co-inoculation with B. subtilis and T. harzianum has been shown to increase total chlorophyll content and improve overall plant vigor under water-limited conditions [64]. Beyond grapevine, B. subtilis has been demonstrated to enhance salinity tolerance in maize by boosting antioxidant enzyme activity and stabilizing ion homeostasis [25]. Similarly, T. harzianum has been shown to improve drought tolerance in tomatoes by regulating osmolyte accumulation and increasing water-use efficiency. These findings highlight the versatile roles these organisms play not only through direct interaction with the plant but also potentially by modulating rhizosphere microbial dynamics and triggering systemic physiological responses.

An unresolved question in current research is whether the observed benefits of microbial consortia are due to the direct action of the applied strains or their ability to modulate the existing vineyard microbiome. Recent studies suggest that both mechanisms may play roles, with some consortia enhancing native microbial network complexity and stability [59,60]. Clarifying that these interactions are an important area for future research, particularly through metagenomic and functional assays under field conditions.

7. Grapevine Molecular Pathways and Gene Expression Under Stress Conditions

The grapevine response to stress implicates complex transcriptional reprogramming and the activation of various genes. Heat and drought stress trigger distinct molecular pathways along with sophisticated hormonal crosstalk that leads to antioxidant accumulation and defense responses [65]. The molecular response of grapevines to environmental stresses involves a complex network of gene expressions and interconnected pathways that work together to enhance plant resilience (Figure 3).

Environmental signals trigger a complex signal transduction network involving hormone signaling through the ABA (via PPZC proteins), JA/ET (through JAZ proteins), and SA pathways, in addition to calcium signaling cascades through calmodulin proteins, CBL proteins, and MAPK cascade activation [65]. The WRKY and GRAS family transcription factors organize these responses, resulting in defense mechanisms that include metabolic adjustments (proline synthesis, sugar metabolism, and phenylpropanoid pathway activation) and protective mechanisms (secondary metabolite accumulation, antioxidant enzyme production, and stress response proteins). This integrated network enables grapevines to assemble a coordinated defense response against different environmental stresses through precise molecular regulation and metabolic adaptation [65].

7.1. Stress Response Mechanisms: Multiple Case Studies

Numerous stress-responsive genes have been identified in grapevine, each providing tolerance through distinct hormonal, signaling, and metabolic pathways. These genes that are involved in ABA biosynthesis, osmotic adjustment, and calcium signaling form a complex molecular network activated under abiotic stress [66].

Several studies have shown that the responses of grapevines to abiotic stresses vary considerably depending on the cultivar, stress duration, and intensity. To illustrate, under a 15-day drought at 30% field capacity, ‘Cabernet Sauvignon’ showed higher proline and ABA accumulation due to the upregulation of VvNCED1 and VvP5CS, while ‘Chardonnay’ exhibited a milder response under similar conditions, indicating cultivar-specific sensitivity [67,68]. While under salt stress (100 mM NaCl, 14 days), ‘Thompson Seedless’ upregulated antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), along with ion transporters like VvNHX1 and VvHKT1, but showed reduced photosynthetic efficiency [69]. In heat–drought combination stress, field trials on ‘Tempranillo’ showed a significant increase in flavonoid and stilbene content [19], while ‘Sangiovese’, when inoculated with Funneliformis mosseae, exhibited enhanced enzymatic antioxidant activity and improved physiological responses [42]. These examples show how stress response and metabolite regulation change not only by stress type and intensity but also by grapevine genotype, emphasizing the need for cultivar-specific management strategies.

Under drought stress, VvNCED genes triggered ABA synthesis for stress signaling, whereas VvP5CS activated proline accumulation for osmotic adjustment [70]. The ABA pathway is activated through PPZC proteins and the JA/ET pathway, which regulates specific gene arrays under abiotic stress. These involve JAZ repressors and ERF transcription factors. Furthermore, stress response genes are triggered by the SA pathway. To maintain water balance, aquaporins (VvSIP, VvPIP1,2) regulate water transport, and the R2R3-MYB family controls phenylpropanoid pathways for stress tolerance. Elevated accumulation of ABA, SA, JA, and hormonal signaling stimulated stomatal closure and protected the cells. During the drought defense response, metabolic changes, such as sugar metabolism and the phenylpropanoid pathway for secondary metabolite production, were enhanced. Another important element in stress signaling is Ca²⁺. Calcium signaling plays a central role in coordinating signaling waves that interact with hormonal MAPK pathways; examples include calcium-dependent protein kinases (CDPKs), calmodulin (CALM), and calcineurin B-like protein/CBL-interacting PKs (CBL/CIPKs). The upregulation of CBL proteins regulates the expression of the MAPK pathway, which counteracts abiotic stress responses [70].

When grapevines encountered salt stress, major pathways were activated, including primary and secondary metabolism, photosynthesis, cell regulation, cellular responses, hormone metabolism and signaling [69]. Additionally, a decrease in stomatal conductance resulted in reduced internal CO₂, which affected the net photosynthesis rate. Notably, the grapevine genes VvTDC1 (tryptophan decarboxylase) and VvSNAT (serotonin N-acetyltransferase) genes initiated serotonin synthesis pathways. Serotonin acts as a multifunctional molecule that contributes to antioxidant activities, regulating stress-related signaling pathways and ion homeostasis, therefore enhancing salt tolerance. Meanwhile, ROS scavenging enzymes were heightened for protection from oxidative stress caused by salinity [69].

Carvalho et al. [67] found that various gene networks have been associated with grapevine plants in heat stress management, interacting with physiological and metabolic pathways. Heat shock transcription factors (HSFs), heat shock proteins (HSPs), proteins and enzymes play major roles in enhancing thermotolerance. The upregulation of the HSP17.9A gene under such stress has been documented in plants, and its significant response to induced heat shock has been confirmed. Furthermore, Carvalho et al. [67] demonstrated that the reference genes UBC and VAG, used for expression profiling of HSP17.9A, are associated with key biological processes, including plant growth and development, carbon fixation, and cell elongation. Heat stress induces the miR156/miR5 superfamily and heat shock proteins while downregulating miR3640-5p, leading to protection of the cell. In addition, upregulation of HSF30 and HSFA2B (heat shock proteins) was found to play an important role in heat and combined stress [67]. Under both heat, drought, and combined stresses, photosynthesis-related and hormone signaling pathways were significantly regulated. Ju et al. [20] showed that combined stresses activated the signal transduction pathway, which affected the metabolism, synthesis, and transport of macromolecular substances in cells.

7.2. Molecular Pathways and Gene Expression Patterns Associated with Beneficial Interactions

Goddard et al. [71] and Campos et al. [72] studied distinct molecular reactions between Rhizophagus irregularis and Funneliformis mosseae. Their findings revealed that grapevines inoculated with R. irregularis exhibited a greater number of differentially expressed microRNAs (DEmiRNAs) when subjected to heat stress than those inoculated with F. mosseae. Notably, members of the miR156/miR529/miR535 superfamily, which are known to play roles in regulating plant growth and development, were significantly overexpressed in R. irregularis-inoculated plants [71]. In contrast, F. mosseae-inoculated plants showed downregulation of specific grapevine miRNAs, such as miR3640-5p. The downregulated miRNAs were associated with processes, such as lignin catabolism, indicating potential differences in cell wall composition and structural integrity under heat stress [72]. Gene ontology analysis revealed that the targets of these downregulated miRNAs were enriched for terms related to lignin metabolism and oxidative stress responses, suggesting that these pathways might be less effectively regulated under heat stress than those in R. irregularis-inoculated plants [71]. The miR156-SPL module was highlighted as crucial for heat stress tolerance and was more robustly activated in grapevines colonized by R. irregularis, suggesting enhanced protective mechanisms against stress [72]. Thus, although both mycorrhizal species contribute positively to grapevine growth under heat stress, Rhizophagus irregularis appears to be more efficient than Funneliformis mosseae. This efficiency is reflected in the greater modulation of specific miRNAs associated with stress tolerance and metabolic pathways that enhance acclimation to high temperatures [71,72]. Furthermore, Duret et al. [73] quantified AMF colonization in grapevines using an RT-qPCR-based approach to assess both mycorrhization intensity and symbiotic vitality. The authors reported that colonization by R. irregularis leads to the upregulation of genes involved in polyphenol biosynthesis, including key enzymes such as cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia-lyase (PAL). These enzymes are crucial for the production of flavonoids and other phenolic compounds, which play vital roles in plant defense mechanisms and stress responses. In addition, these compounds are important contributors to grape and wine sensory qualities, including bitterness and aroma.

While the recent work of Ma et al. [55] suggests a potential role for endophyte-induced melatonin in stress tolerance, current evidence is limited, and further studies are required to confirm these effects in diverse grapevine genotypes and under field conditions.

7.3. Laboratory and Field Trial Evidence

A substantial body of research on microbial biostimulants, particularly arbuscular mycorrhizal fungi, is from controlled laboratory and greenhouse experiments. These studies have been vital in revealing the fundamental mechanisms of plant-microbe interactions, such as nutrient uptake enhancement and stress mitigation. However, the projection of these findings to field conditions requires caution because of the complexity and variability of native vineyard ecosystems [74].

Laboratory studies often utilize sterilized soils and controlled environments, which do not fully convey the interactions between diverse soil microbiota, management practices, and unstable environmental conditions. For example, the efficacy of AMF in enhancing grapevine nutrient uptake and stress tolerance observed in pot experiments may not directly translate to field scenarios in which soil structure, microbial diversity, and abiotic factors are significantly different.

Field trials have provided critical insights into the practical applicability of microbial inoculants. For instance, Aguilera et al. [74] demonstrated that AMF inoculation in Chilean vineyards improved water uptake and nutrient absorption in grapevines under drought conditions, underscoring the potential of AMF to enhance vineyard resilience to climate-induced stresses. Schreiner [75] emphasized the role of AMF in grapevine mineral acquisition under field conditions, highlighting the importance of these symbiotic relationships in vineyard nutrient management.

These findings emphasize the importance of validating laboratory insights by using field-based evidence. Thus, the practical relevance of microbial biostimulants can be confirmed, reinforcing their role in sustainable vineyard management strategies [74,75].

8. Conclusions

A significant discrepancy exists between laboratory findings and field applications, necessitating more comprehensive research to effectively translate microbiome knowledge into practical agricultural solutions in the context of climate change. To address this discrepancy, the effects of beneficial microorganism applications should be evaluated following a holistic approach spanning across all aspects of grapevine cultivation in terms of yield, grape quality, and economic viability. However, the efficacy of microbial inoculants often varies significantly between controlled experiments and field conditions. Despite microbial application, the existing microbiota in vineyards often resist compositional shifts, therefore limiting the establishment of introduced strains. This ecological resilience can also reduce the reliability of achieving the same beneficial outcomes under field conditions. Alternatively, our research showed that combined AMF, PGPR, or endophyte inoculant consortia showed promising results and should be considered for further production by the biofertilizer industry. Microorganisms associated with vines play an essential role in sustainable viticulture and are gaining increasing attention as the demand for sustainable and environmentally friendly farming practices increases. In this context, it is important to measure grapevine responses from the physiological level to integrated indicators of grapevine performance, such as yield, growth, and berry composition, at the whole-vine level, rather than being limited to the organ level. In this pursuit, it is essential to consider several limitations of the existing research that constrain the applicability of the findings to established commercial vineyards. Notably, a significant limitation is the predominant focus on young grapevines and seedlings, whereas there is a relative scarcity of research on mature grapevines with a wider range of grape varieties and rootstocks. Additionally, most experiments have been conducted in controlled greenhouse environments for relatively short periods, and various inoculation methods and microbial concentrations make it challenging to draw definitive conclusions or make standardized recommendations.

To enhance the resilience of grapevines under adverse environmental conditions, it is imperative to develop targeted strategies by comprehending strain-specific effects and their underlying molecular mechanisms. Understanding the complex interactions between different microbial species and how they collectively influence grapevine stress tolerance is also important. The utilization of beneficial microorganisms can diminish reliance on chemical inputs, foster healthier ecosystems, and align with consumer demand for organic and natural wines, which represent significant market trends. There is an increasing movement towards organic viticulture to safeguard local biodiversity and cultivate organic vine crops. For example, Chile has established a sustainability code to integrate sustainable practices within wine companies [69]. Furthermore, soil health emerges from the interaction of physical, chemical, and biological properties that influence soil function, thereby promoting healthy vines and high-quality wine grapes. Growers and industry personnel typically express concern regarding salinity, soil structure, organic matter, biological stress and water stress. Some microbial stimulants or formulations may unintentionally increase soil electrical conductivity (EC), especially in systems with limited leaching capacity. Enhancing soil health in vineyards requires focused research on soil tests to identify reliable indicators and establish techniques for assessment and benchmarking.

To conclude, we have presented a structured overview of the contribution of beneficial microorganisms in improving plant tolerance to abiotic stresses in grapevine cultivars, with an emphasis on multi-species consortia and identifying the molecular mechanisms involved. Based on a literature review of the last seven years, it was observed that AMF, particularly Rhizophagus irregularis and Funneliformis mosseae, have shown high efficacy in improving drought, salt, and heat stress tolerance through the modulation of VvNCED and VvP5CS genes, along with the expression of R2R3-MYB family transcription factors. Mycorrhizal symbiosis has emerged as a critical factor that not only influences miRNA expression but also enhances physiological adaptations through the modulation of key metabolic pathways related to osmotic balance, antioxidant defenses, and energy metabolism. PGPR, Bacillus, and Pseudomonas species have shown significant capabilities in enhancing nutrient uptake and stress tolerance through hormone modulation and activation of the antioxidant system. For PGPF and endophytes, particularly Serendipita indica and Pseudomonas fluorescens RG11, melatonin synthesis and enhanced antioxidant responses facilitated the natural mechanisms of grapevines to withstand stress. Consortium applications of multiple beneficial microorganisms have shown superior results compared to single-species treatments, highlighting the synergistic interactions that enhance plant growth, biocontrol, and environmental sustainability.