Harnessing a Microbial Consortium and Compost to Control Grapevine Pathogens: A Sustainable Viticulture Strategy for Disease Suppression and Quality Enhancement

Abstract

Beneficial microorganisms are emerging as promising alternatives to conventional pesticides for the biological control of plant diseases. This study evaluated the efficacy of a consortium composed of Pseudomonas yamanorum and Trichoderma longibrachiatum and compost against three grapevine pathogens, Botrytis cinerea, Erysiphe necator, and Plasmopara viticola, in three cultivars: Victoria, Superior Seedless, and Early Sweet. The microbial consortium (P. yamanorum + T. longibrachiatum) combined with compost (treatment T4) significantly outperformed the individual treatments, reducing disease severity indices (DSIs) to 7.72, 5.35, and 3.37% in Victoria; 5.70, 6.95, and 3.32% in Superior Seedless; and 4.98, 2.35, and 2.84% in Early Sweet. The treatment also enhanced physiological traits, such as the chlorophyll content, and defense responses, including ascorbate peroxidase (APX), peroxidase (POX), and catalase (CAT) enzyme activities. Biochemical markers, including the total protein content, phenolic content, and reduced malondialdehyde (MDA) levels, indicated an improved oxidative stress tolerance. The soil analysis confirmed an increased pH, organic matter, nitrogen content, and microbial biomass. T4 further reduced the fruit disease incidence and improved quality attributes, including the sugar content and size, while lowering nitrate accumulation. These findings highlight the synergistic benefits of combining a microbial consortium with compost as a sustainable strategy to promote grapevine health, productivity, and soil resilience.

1. Introduction

The grapevine (Vitis vinifera L.) is one of the most economically important fruit crops worldwide, and is cultivated to produce wine, dried fruits, and fresh table grapes [1]. In 2023, global grapevine cultivation covered an area of 7.2 million hectares, equally divided between pressed and unpressed grapes. The major production regions are concentrated in the European Union, with Spain having the largest vineyards (945 kha in 2023), followed by France (792 kha), Italy (720 kha), Romania (187 kha), and Portugal (182 kha) [2]. This extensive production highlights the global demand and economic relevance of grapevine cultivation.

However, plant diseases continue to pose significant threats to global food security and agricultural productivity. Approximately 30% of global crop yields are lost annually due to plant diseases, which not only reduce the harvest volume but also negatively impact crop quality, farmer livelihoods, and input costs [3]. Among the various threats to grapevines, fungal pathogens such as Botrytis cinerea (gray mold), Erysiphe necator (powdery mildew), and Plasmopara viticola (downy mildew) are particularly destructive. B. cinerea initially behaves as a biotroph before entering a necrotrophic phase, during which it secretes virulence factors, including oxalic acid, cell wall-degrading enzymes, and hormone analogues that impair host metabolism and immunity. These changes lead to tissue softening, browning, and rapid colonization characterized by abundant gray conidia [4,5]. This fungus is estimated to reduce grape yields by 20–30% annually, and in severe cases, up to 50% [6].

E. necator, an obligate biotrophic fungus, depends on photosynthetically active host cells to complete its life cycle. Its conidiospores germinate on the leaf surface, forming lobed appressoria and releasing enzymes such as lipases, esterases, and cutinases to penetrate the cuticle [7]. The infection presents as white powdery patches on leaves, stems, and fruits, causing chlorosis, deformation, and a decreased photosynthetic capacity, ultimately leading to yield and quality losses [8,9,10].

P. viticola, the causal agent of downy mildew, begins as yellow spots on leaves and progresses to necrosis, often resulting in complete crop failure [11]. These pathogens are typically controlled through chemical fungicides; however, they are associated with environmental toxicity, resistance development, and residual contamination, affecting human health and soil microbiomes [12,13,14,15].

As an alternative, biological control using beneficial microorganisms offers a promising solution. Microbial consortium combinations of bacteria and fungi with complementary properties can improve plant resilience through mechanisms such as antagonism, competition, and the induction of systemic resistance [16,17]. Strains with high enzymatic activity (e.g., chitinases) and bioactive metabolites (e.g., siderophores and phytohormones) can enhance soil fertility and disease resistance more effectively than single-strain applications [18].

Among these, Trichoderma spp. and Pseudomonas spp. have demonstrated effective biocontrol properties. For example, T. harzianum T39 induces defense enzymes such as peroxidase, polyphenol oxidase, and β-1,3-glucanase in grapevines, leading to lignin deposition and enhanced cell wall strength [19]. Trichoderma species also produce bioactive compounds that promote growth, nutrient uptake, and disease resistance [20]. Similarly, Pseudomonas spp. produce antibiotics and phytohormones (e.g., gibberellic acid and jasmonic acid) that suppress pathogens and enhance systemic resistance [21]. Combinations of T. harzianum, Pseudomonas fluorescens, Penicillium, and Bacillus strains have shown improved disease control in grapevines compared to individual applications [22,23].

In addition to microbial biocontrol agents, compost serves as an effective organic amendment that improves the soil structure, microbial diversity, and disease suppression [24]. Compost derived from grapevine residues is a valuable source of potassium and can enhance soil fertility and pathogen suppression [25]. When combined with microbial agents, compost improves rhizosphere colonization and systemic plant resistance [26,27,28].

Under field conditions in Tunisia, the aim of this study was to evaluate the antifungal potential of T. longibrachiatum, P. yamanorum, and compost either separately or in combination. Three grapevine cultivars, Victoria, Superior Seedless, and Early Sweet, were used to assess disease severity resulting from B. cinerea, E. necator, and P. viticola. Different parameters were analyzed, including physiological parameters (chlorophyll content); oxidative stress indicators, e.g., malondialdehyde (MDA) levels; antioxidant enzyme activities (APX, POX, and CAT); biochemical defense responses (total protein and phenolic contents); fruit quality traits; and soil health indicators. T. longibrachiatum and P. yamanorum were selected based on successful isolations in the laboratory of Plant Protection and Biological Sciences at the Regional Center for Agricultural Research (CRRA), Tunisia, and their promising biocontrol activity, particularly the studied efficacy of T. longibrachiatum and Pseudomonas sp. against B. cinerea [29]. This thorough assessment seeks to provide a sustainable, biologically based plan for enhancing grapevine health, yield, and agroecosystem resilience.

2. Materials and Methods

2.1. Study Site

This study was conducted in the Regueb region of Sidi Bouzid (34°51′33.552″ N 9°47′ 11.544″ E), Tunisia, in 2023. According to the climate classification of [30], Regueb exhibits a hot desert climate (BWh). Temperatures generally fluctuate between 11 °C and 30 °C annually, although they may occasionally decrease to 1 °C or ascend to 47 °C. The mean annual precipitation totals approximately 171 mm, with 27 days of rainfall exceeding the 1 mm threshold each year. Regueb receives an annual average of 3949 h of sunshine, with a daylight duration ranging from 9 h and 50 min to 14 h and 26 min daily.

2.2. Detection of Fungal and Fungal-like Pathogens

The phenotypic identification of B. cinerea was determined by the growth rate calculated as the difference between growth at 48 h and growth at 24 h [31], as well as by the morphology and pigmentation of the mycelium, duration until sporulation, time required for sclerotia formation, quantity of sclerotia, and the morphology and pigmentation of sclerotia [32,33,34].

Phenotypic characters of Erysiphe necator, including mycelium on the host; appressoria; the size and shape of conidia, conidiophores and chasmothecia; the shape, size and number of ascospores; and number of asci per chasmothecium, were determined according to [35,36].

For Plasmopara viticola, phenotypic criteria, including the structure and branching pattern of sporangiophores; shape and arrangement of sporangia; type and formation of zoospores; morphology of the intercellular mycelium; shape and presence of intracellular haustoria; and characteristics of oospores, were determined according to [37].

B. cinerea, E. necator, and P. viticola were deposited in the Fungarium of Suez Canal University (https://ccinfo.wdcm.org/collection/by_id/1180; accessed on 14 November 2023), at the Botany and Microbiology Department, Faculty of Science, Ismailia 41,522, Egypt, under the accession numbers SCUF0000711 to SCUF0000713, respectively.

2.3. Experimental Design

This study aimed to evaluate the effectiveness of the microbial consortium and compost in suppressing fungal diseases in three grapevine (Vitis vinifera L.) cultivars: Victoria, Superior Seedless, and Early Sweet. The experimental vineyard trial was conducted using a randomized block design, with each block assigned to one cultivar. Within each block, four different treatments were randomly assigned to individual rows for each cultivar and applied to rows within the cultivar blocks (not on border rows). Each row contained 30 grapevines, with 6 plant replicates per treatment per cultivar; two untreated grapevines were left as buffer plants between each treatment group.

A 15 m buffer zone separated the treated experimental row from the main cultivar blocks to minimize the risk of treatment cross-contamination (Figure 1). The treatments consisted of T1 (untreated control), T2 (the microbial consortium (T. longibrachiatum and P. yamanorum), T3 (compost alone), and T4 (compost combined with the microbial consortium). Drip irrigation was applied uniformly, and no additional fertilizers or chemical treatments were used.

2.4. Biocontrol Strains and Compost Preparation for Field Applications

The compost used in the experiment was produced from vineyard pruning residues, pomace, and manure in a ratio of 30:30:40, which were composted on site at the composting unit in the farm of the Regional Center of Agricultural Research (CRRA) in Sidi Bouzid (35.03824° latitude, 9.484263° longitude). The materials were shredded, mixed, and composted for 3 months, reaching thermophilic temperatures that ranged from 56 to 65 °C. Compost piles were turned regularly to ensure aeration. On average, the compost was characterized by a pH of 7.55 ± 0.28; moisture content of 42.32% ± 6.8; organic matter content of 38.6% ± 8.3% (dry matter); organic carbon content of 23.1% ± 4.2%; total nitrogen content of 1.37% ± 0.64%; C/N ratio 16.87± 3.80; and 6.3 × 10³ CFUs g−1 of total coliforms and was free from pathogenic microbes and weed seeds.

In this work. two microbial agents were used, Pseudomonas yamanorum (GenBank accession number PQ555427) and Trichoderma longibrachiatum strain Tr1Tm (GenBank accession number OP799680), which were obtained from the Plant Protection and Biological Sciences Laboratory at the Regional Center for Agricultural Research (CRRA), Tunisia.

The P. yamanorum strain was cultured in sterile Luria Bertani (LB) broth after an incubation in a rotary shaker incubator (Optic Ivymen System, Spain) at 150 rpm and 28 °C, and the bacterial concentration was adjusted to 10⁸ CFUs/mL using a spectrophotometric measurement at 600 nm. T. longibrachiatum was initially cultured on potato dextrose agar (PDA) at 28 °C for seven days, followed by sub-culturing in potato dextrose liquid (PDL) medium and incubating for five days under the same conditions. The resulting spore suspension was standardized to 1 × 10⁶ spores/mL and stored at 4 °C. Compost was applied at a rate of 4 kg per grapevine plant, based on application rates recommended in previous research [38,39]. For microbial consortium treatments, 0.5 L of each microbial suspension was applied per plant (Figure 2).

2.5. Soil Sampling After Treatment

Samples were collected at three time points: 30 days (M1), 60 days (M2), and 90 days (M3) after treatment application. At each time point, leaf, fruit, and soil samples were taken from each treatment plot. Soil samples were collected from the topsoil layer using sterile tools and placed in labeled plastic zip-lock bags. These were air-dried at 37 °C, sieved, and homogenized for the laboratory analysis. Leaf samples were selected from fully expanded leaves at similar developmental stages, placed in sterile bags, and transported on ice to the laboratory, where they were stored at 4 °C until analysis. Fruit samples were randomly collected from different vines per treatment to ensure representativeness. Composite fruit samples were labeled and stored at 4 °C.

2.6. Soil Physicochemical and Microbial Analyses

Soil pH was measured according to the AFNOR X 31-103 standard using a calibrated pH meter. Nitrate was extracted from 20 g of soil mixed with 50 mL of distilled water and allowed to settle before measurement using a nitrate meter. The total nitrogen content was determined by the Kjeldahl method [40], and the organic carbon and organic matter contents were analyzed using the Walkley and Black method [41]. The soil bulk density was measured by inserting a 100 cm³ cylinder into the soil, drying the sample, and calculating the dry weight per unit volume.

The soil microbial load was quantified using the serial dilution plate method. Colony-forming units (CFUs) were counted to assess microbial abundance, as described by [42].

2.7. Disease Severity and Incidence Assessments

The disease severity index uses descriptive scales from 0 to 4 to evaluate the symptoms and damage caused to grapevine plants by B. cinerea, E. necator, and P. viticola. The disease severity index (DSI) is calculated using McKinney’s formula [29]:

$$\mathrm{D}\mathrm{S}\mathrm{I}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathsf{\Sigma }(\mathrm{v}\times \mathrm{n})}{\left(\mathrm{N}\times \mathrm{V}\right)}}\times 100$$

(1)

where v represents the value of the disease index scale, n is the number of plants assigned to each disease index scale value, N is the total number of plants evaluated, and V is the maximum scale value.

The fruit disease incidence was assessed based on the percentage of the surface area affected by gray mold and powdery mildew. This was estimated using the following formula:

$$\mathrm{PFA} (\%)=\left({\displaystyle \frac{\mathrm{L}\mathrm{A}}{\mathrm{T}\mathrm{D}}}\right)\times 100$$

(2)

where LA is the lesion area and TD is the total diameter of the fruit [29].

2.8. Physiological Parameters

2.8.1. Chlorophyll Content

Chlorophyll content was used as an indicator of plant health and photosynthetic efficiency. It was measured in situ using a portable SPAD-502Plus chlorophyll meter (Konica Minolta, Tokyo, Japan) on fully expanded leaves from each cultivar and treatment group at each sampling time.

2.8.2. Biochemical and Antioxidant Analyses

Leaf samples were analyzed for several defense-related biochemical markers. Ascorbate peroxidase (APX) activity was determined using the method of [43], catalase (CAT) activity according to [44] and peroxidase (POX) activity by following [45]. The total protein content (TP) was determined by Bradford’s method [46] (1976), while the total phenolic content (TPC) was measured using the Folin–Ciocalteu reagent, as described by [47]. Lipid peroxidation was assessed by quantifying malondialdehyde (MDA) levels using the method of [48]. Leaf or fruit samples (200 mg) were homogenized in 2 mL of 1% trichloroacetic acid (TCA) and centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatant was mixed with thiobarbituric acid (TBA) prepared in 20% TCA, incubated at 90 °C for 20 min, and then cooled on ice. Absorbance was measured at 532 and 600 nm, and the MDA content was calculated using an extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed as µmol/g fresh weight.

2.9. Fruit Quality Analysis

Fruit juice was prepared using a blender to analyze the physicochemical parameters. The pH of the juice was measured using a digital pH meter, and electrical conductivity (EC) was measured using a HI99301 conductivity meter (Hanna Instruments, Virginia, USA). The sugar content was measured in degrees Brix using a digital pocket refractometer (Atago PAL, Tokyo, Japan). The nitrate concentration was determined using a LAQUA-twin nitrate meter (Horiba Ltd, Kyoto, Japan ). The water content (WC) was calculated as follows:

$$\mathrm{W}\mathrm{C}(\mathrm{\%})={\displaystyle \frac{\mathrm{F}\mathrm{F}-\mathrm{F}\mathrm{S}}{\mathrm{F}\mathrm{F}}}\times 100$$

(3)

where FF is the fresh fruit weight and FS is the dry fruit weight. The fruit diameter was measured using digital calipers.

2.10. Mineral Analysis of Leaves

To determine the mineral composition, leaves were washed with sterile water, dried at 60 °C, and ground to a fine powder. One gram of dry sample was ashed at 500 °C for 4 h in a muffle furnace. The resulting ash was dissolved in 1 M HNO₃, evaporated to dryness, and re-dissolved in 0.1 M HCl to a final 80-fold dilution. Phosphorus (P), iron (Fe), magnesium (Mg), chlorine (Cl), calcium (Ca), and zinc (Zn) levels were quantified by Flame Atomic Absorption Spectroscopy.

2.11. Statistical Analysis

All collected data were analyzed using analysis of variance (ANOVA) in R software (version 4.2.1). The data were checked for normality according to the Shapiro–Wilk test, and homogeneity of variances using Levene’s test. Duncan’s multiple range test was applied to assess significant differences between treatment means at a threshold of p ≤ 0.05. All graphs and visualizations were created using the “ggplot2” package in RStudio (Version 2024.12.0+467).

3. Results

3.1. Antifungal Activity of the Microbial Consortium and Compost Against Fungal Diseases

The antifungal efficiency of the treatments was evaluated using the disease severity index (DSI) on grapevine plants of three cultivars (Victoria, Superior Seedless, and Early Sweet) against B. cinerea, E. necator, and P. viticola (Figure 3). For the Victoria cultivar, the negative control (T1) showed severe DSI rates of 86.69%, 93.47%, and 73.49% for B. cinerea, E. necator, and P. viticola, respectively, confirming the harmful impacts of these fungal pathogens on grapevines. A significant reduction resulted when using Trichoderma and Pseudomonas, which reduced the DSI to 32.05%, 28.43%, and 26.33%, while the application of compost reduced the impacts of diseases to 17.25%, 16.56%, and 12.36%. In this context, treatment T4 was able to reduce the pathogens to 7.72%, 5.35%, and 3.37% (Figure 3a).

Similar trends were observed in other studied cultivars. In Superior Seedless grapevines, the application of the microbial consortium was able to reduce the severity of B. cinerea, E. necator, and P. viticola diseases by 28.05%, 22.72%, and 25.20 (Figure 3b). Remarkably, treatment T4 reduced the DSI to 5.70%, 6.95%, and 3.32%. Additionally, for the Early Sweet cultivar, it was revealed that the untreated grapevine exhibited a DSI of 82.56%, 89.01%, and 90.73% for B. cinerea, E. necator, and P. viticola, respectively (Figure 3c). Meanwhile, the combined effect of compost with the microbial consortium resulted in the lowest DSI values of 4.98%, 2.35%, and 2.84%. Consequently, for all cultivars the combination of the microbial consortium with compost exhibited the most significant reduction in disease severity for B. cinerea, E. necator, and P. viticola as compared to the disease control, which suggests a synergistic effect on the management of fungal infections.

3.2. Effects of the Microbial Consortium and Compost on Plant Growth Promotion

In this study, the chlorophyll content was assessed regarding its efficiency as key indicator of photosynthetic and plant health. Statically significant (p < 0.01) treatment effects were noted when measuring the chlorophyll content across all assessed cultivars compared to the untreated control (

Table 1. Effects of the microbial consortium and compost applications on the chlorophyll content.

Cultivars	Treatments	M1 (30 Days)	M2 (60 Days)	M3 (90 Days)
Victoria	T1	19.5 ± 3.20 c	31.7 ± 0.361 c	31.6 ± 1.70 c
	T2	23.0 ± 0.723 c	32.1 ± 1.54 c	39.9 ± 2.56 b
	T3	23.4 ± 1.25 b	36.0 ± 3.38 b	39.5 ± 4.21 b
	T4	35.1± 1.74 a	47.6 ± 1.72 a	45.4 ± 0.416 a
	p-value	<0.01	<0.01	<0.01
Superior Seedless	T1	22.4 ± 2.89 c	33.9 ± 0.900 b	36.6 ± 2.95 b
	T2	24.1 ± 0.755 c	35.2 ± 6.28 b	34.6 ± 4.80 b
	T3	26.4 ± 0.458 b	38.6 ± 0.458 b	46.3 ± 3.50 a
	T4	34.9 ± 0.954 a	45.4 ± 4.59 a	44.7 ± 3.44 a
	p-value	<0.01	<0.01	<0.01
Early Sweet	T1	33.0 ± 0.153 c	37.4 ± 2.14 c	37.4 ± 2.48 c
	T2	41.7 ± 1.01 b	38.1 ± 0.265 c	40.3 ± 1.47 c
	T3	42.0 ± 0.737 b	41.7 ± 0.802 b	42.4 ± 2.80 b
	T4	43.7 ± 1.32 q	46.1 ± 0.379 a	48.6 ± 1.90 a
	p-value	<0.01	<0.01	<0.01

ANOVA. Means in a column (±standard errors) followed by the same letter are not significantly different according to Duncan’s test (p < 0.05 and p < 0.01). (T1—untreated control, T2—microbial consortium, T3—compost, T4—microbial consortium + compost).

). For the Victoria cultivar, a significant increase was observed in the chlorophyll content with the combined treatment T4. The chlorophyll content increased from 19.50 SPAD (T1) to 35.07 at 30 days, with a continued improvement from 31.7 (T1) to 47.63 at 60 days and from 31.57 to 45.43 at 90 days. Notably, Superior Seedless grapevines exhibited a similar response pattern, with T4 producing the highest chlorophyll levels at all sampling moments compared to the control (T1) (34.90 at 30 days, 45.37 at 60 days, and 44.70 at 90 days). Early Sweet grapevines treated with T4 recorded a strong chlorophyll content as well at the different sampled moments (M1, M2, and M3), which was accounted as 43.70, 46.07, and 48.57, respectively. Furthermore, for the Victoria and Superior Seedless cultivars, significant differences in treatment responses were observed throughout the sampling moments, while Early Sweet demonstrated an early activity after treatment with both T2 and T3, showing statistically equal levels (41.67 and 41.97, respectively).

3.3. Impacts of the Microbial Consortium and Compost on the Grapevine Plant Defense System

3.3.1. Antioxidant Enzymatic Activities in Grapevine Leaves

The application (T4) of compost in combination with P. yamanorum and T. longibrachiatum significantly enhanced antioxidant enzyme activities in grapevine leaves across all cultivars. An analysis of ascorbate peroxidase activity (APX) revealed an enhancement of the antioxidant defense system with the combined treatment of compost and the microbial consortium (T4). In all cultivars, the combined approach regularly resulted in higher enzyme activities compared to the control (T1) and the separate treatments (T2 and T3). In the Victoria cultivar, T4 resulted in 7.67 units·mg protein⁻¹·min⁻¹ at 30 days (M1), peaked at 19.89 at 60 days (M2), and reached 9.67 at 90 days (M3), which were notably higher than the control values of 1.11, 3.67, and 6.33, respectively. Meanwhile, similar improvements were observed in Superior Seedless and Early Sweet vines (Figure 4a). Peroxidase activity (POX) showed a remarkable increase in the T4-treated plants, particularly in the Victoria cultivar, where it rose from 0.46 units·mg protein⁻¹·min⁻¹ at M1 to 6.74 at M3. Similar trends were observed in Early Sweet, and in Superior Seedless, which, despite their lower overall activities, still demonstrated a consistent increase (Figure 4b). Catalase (CAT) activity also responded positively to treatments, with T4-treated Victoria leaves showing an increase from 9.02 at M1 to 12.66 at M3, and Early Sweet reaching a peak of 30.82 at M3. Superior Seedless leaves receiving T4 showed a steady increase from 6.76 at M1 to 12.00 at M3, while T2 and T3 provided only modest improvements (Figure 4c). For the three cultivars, it was noted that the effect of T4 was characterized by a long-term effect until M3, as shown in Figure 4c, where values of catalase increased through the sampling period.

3.3.2. Stress Markers and Defense-Related Compounds in Leaves

The levels of malondialdehyde (MDA), a marker of lipid peroxidation, decreased significantly after the T4 treatment, confirming a reduction in oxidative stress. In the Victoria cultivar, the MDA content decreased by 53% at M1, 51% at M2, and 62% at M3 compared to the untreated control (Figure 4d). Similar patterns were observed in Superior Seedless and Early Sweet, though with a slight rebound at M3 in the latter, suggesting that a second application may be beneficial. In addition, T4-treated plants showed a significant accumulation of protein and phenolic compounds (Figure 4e, f). In Victoria, total protein content increased from 29.61 at M1 to 76.74 at M3 with T4, whereas the control was maintained at only 7.83. The total phenolic content in the same cultivar increased from 7.14 to 8.64. Similar enhancements were recorded in Early Sweet and Superior Seedless, indicating the activation of both enzymatic and non-enzymatic defense mechanisms.

3.4. Soil Physicochemical Responses

The application of compost and microbial inoculants resulted in significant changes in the soil physicochemical properties (p < 0.01; Figure 5). Soil pH values ranged from 7.53 to 8.00, with untreated plots displaying the highest values, suggesting more alkaline conditions. Treatments with compost, particularly T4, slightly reduced the soil pH, facilitating better nutrient solubility and uptake (Figure 5a). The nitrate content was also significantly affected, with the Victoria cultivar exhibiting the highest nitrate level (836.67 mg/kg) at 30 days with T4. This was followed by a gradual decline over time, indicating nutrient uptake by the plant. Early Sweet recorded the lowest nitrate values but still responded positively to the T4 treatment (Figure 5b).

The soil organic carbon and organic matter contents increased markedly with T4 across all cultivars and time points. In Victoria, the organic carbon content rose to 0.34% and organic matter content to 0.59% with T4 at M3, compared to just 0.11% and 0.19% in the control. Similar improvements were observed in Early Sweet (0.44% and 0.76%) and Superior Seedless (0.47% and 0.81%) with T4 at M3 (Figure 5c, d). A concurrent improvement in the soil structure was noted, as reflected by the reduction in bulk density values. In Early Sweet, the bulk density decreased from 1.60 at M1 to 1.42 at M3 with T4, while Superior Seedless and Victoria recorded values of 1.55 and 1.46, respectively (Figure 5e). Moreover, the total nitrogen content increased significantly across all treated plots, with the highest values again observed with T4. At M3, the nitrogen content reached 1.97% in Victoria, 2.17% in Superior Seedless, and 2.07% in Early Sweet (Figure 5f), indicating improved nitrogen availability and retention due to the treatments.

3.5. Soil Microbial Abundance

The effects of treatments on the soil microbial community revealed that T4 significantly increased bacterial populations in all cultivars compared to the control (p < 0.01). In Victoria, the bacterial count with T4 was 3.00 × 10⁵ CFUs/g soil, whereas T1, T2, and T3 recorded much lower levels at 0.60, 0.68, and 0.88 × 10⁵ CFUs/g soil, respectively. Similarly, Superior Seedless and Early Sweet showed microbial counts of 4.73 × 10⁵ and 3.82 × 10⁵ CFUs/g soil with T4, far surpassing other treatments (Figure 6). These findings emphasize the synergistic roles of compost and the microbial consortium in enriching the soil microbiota.

3.6. Antioxidant Enzyme Activities and Stress Markers in Grapevine Fruits

Significant variations were observed in fruit antioxidant enzyme activities and stress markers across treatments (p < 0.01;

Table 2. Effects of Trichoderma longibrachiatum, Pseudomonas yamanorum, and compost applications on antioxidant enzymatic activities and stress markers in grapevine fruits.

Cultivars	Treatments	APX (Units·mg Protein−1·min−1)	POX (Units·mg Protein−1·min−1)	CAT (Units·mg Protein−1·min−1)	MDA (µmol/g)	TPC (mg/g)	TP (mg/g)
Victoria	T1	3.51 ± 0.81 d	1.48 ± 0.27 b	17.50 ± 1.74 c	1.03 ± 0.95 b	0.47 ± 0.01 d	1.09 ± 0.51 c
	T2	9.40 ± 0.59 c	1.55 ± 0.45 b	19.52 ± 1.52 bc	2.71 ± 0.27 a	0.98 ± 0.57 c	8.06 ± 0.92 b
	T3	9.83 ± 0.66 b	1.78 ± 0.59 a	23 ± 0.97 ab	0.39 ± 0.02 c	1.83 ± 0.48 a	13.41 ± 0.87 a
	T4	9.92 ± 0.48 a	1.91 ± 0.63 a	24.64 ± 1.22 a	0.65 ± 0.08 c	1.15 ± 0.92 b	13.64 ± 1.23 a
	p-value	<0.01	<0.05	<0.01	<0.01	<0.01	<0.01
Superior Seedless	T1	3.80 ± 0.44 d	2.21 ± 0.21 b	4.22 ± 0.91 d	10.19 ± 0.45 a	1.07 ± 0.33 c	10.31 ± 1.08 b
	T2	7.52 ± 0.34 c	2.24 ± 0.13 b	4.86 ± 0.67 c	7.57 ± 0.69 b	0.42 ± 0.64 d	11.86 ± 0.97 a
	T3	8.16 ± 0.17 b	2.30 ± 0.44 b	6.10 ± 0.58 b	2.82 ± 0.34 c	1.71 ± 0.28 b	10.54 ± 0.84 b
	T4	9.51 ± 0.75 a	2.49 ± 0.98 a	7.06 ± 0.04 a	0.47 ± 0.05 d	2.50 ± 0.49 a	12.40 ± 0.66 a
	p-value	<0.01	<0.05	<0.01	<0.01	<0.01	<0.01
Early Sweet	T1	1.09 ± 0.18 d	2.05 ± 0.18 a	5.06 ± 0.19 c	4.24 ± 0.08 a	0.92 ± 0.15 d	0.85 ± 0.02 c
	T2	4.99 ± 0.29 c	2.16 ± 0.65 a	5.64 ± 0.36 b	2.47 ± 0.23 b	1.13 ± 0.69 c	7.36 ± 0.31 b
	T3	7.81 ± 0.96 b	2.32 ± 0.32 a	6.14 ± 0.48 b	0.99 ± 0.19 c	1.43 ± 0.09 b	11.01 ± 0.45 a
	T4	15.38 ± 0.33 a	2.42 ± 0.46 a	6.98 ± 0.53 a	0.97 ± 0.24 c	1.80 ± 0.37 a	11.16 ± 0.75 a
	p-value	<0.01	≥0.05	<0.01	<0.01	<0.01	<0.01

ANOVA. Means in a column (±standard errors) followed by the same letter are not significantly different according to Duncan’s test (p < 0.05 and p < 0.01). T1—untreated control, T2—microbial consortium, T3—compost, T4—microbial consortium + compost.

). In Early Sweet, ascorbate peroxidase activity reached 15.38 with T4, while the control recorded only 1.09. Victoria fruits treated with T4 showed the highest catalase activity (24.64) and protein content (13.64), whereas Superior Seedless demonstrated the highest catalase activity (7.06) and phenolic content (2.50) with the same treatment. Importantly, T4 also led to the lowest malondialdehyde content in Superior Seedless (0.47), confirming reduced oxidative stress and enhanced cellular integrity in grapevine fruits.

3.7. Mineral Composition of Grapevine Fruits

The mineral analysis indicated that T4 influenced the nutrient composition of grapevine fruits (

Table 3. Impacts of Trichoderma longibrachiatum, Pseudomonas yamanorum, and compost applications on the mineral profile.

Cultivars	Treatments	Fe (µmol/g)	Mg (µmol/g)	Cl (µmol/g)	Ca (µmol/g)	Zn (µmol/g)	P (µmol/g)
Victoria	T1	2.42	183.75	919.53	5.57	0.33	0.48
	T2	1.67	73.4	699.52	2.23	0.24	0.47
	T3	2.01	68.87	801.06	2.09	0.15	0.57
	T4	2.31	141.37	648.75	4.29	0.2	0.87
Superior Seedless	T1	2.09	28.22	603.62	0.86	0.38	0.61
	T2	2.65	16.05	614.9	0.49	0.52	0.62
	T3	2.31	75.79	535.92	2.3	0.76	0.65
	T4	2.44	76.94	660.03	2.33	0.81	0.79
Early Sweet	T1	1.27	144.99	784.14	4.4	0.04	0.5
	T2	1.75	16.13	693.88	0.49	0.06	0.52
	T3	1.87	34.89	880.04	1.06	0.12	0.56
	T4	1.84	4.2	547.2	0.13	0.08	0.64

T1—untreated control, T2—microbial consortium, T3—compost, T4—microbial consortium + compost.

). In Victoria, iron levels with T4 reached 2.31 µmol/g DW and phosphorus levels were 0.87 µmol/g DW, surpassing other treatments and the control. Superior Seedless showed notable improvements in the zinc (0.81 µmol/g DW) and phosphorus (0.79 µmol/g DW) contents. In Early Sweet, although the changes were less pronounced, T4 effectively reduced the chloride content and moderately increased the phosphorus content, contributing to a more balanced mineral profile and better fruit nutritional quality. Overall, the T4 treatment contributed to a more equitable mineral profile, supporting better fruit quality and potential nutritional value.

3.8. Antifungal Activity Against Grapevine Pathogens

An evaluation of the disease incidence revealed that T4 significantly reduced the percentage of grape surface infected by B. cinerea and E. necator (p < 0.01; Figure 7). In Victoria, T4 reduced B. cinerea and E. necator infection to 9.29% and 8.18%, respectively, compared to 72.61% and 89.41% in the control. In Superior Seedless, the infection levels were reduced to 6.53% and 4.93%, while in Early Sweet, the respective values were 9.15% and 7.26%. These data confirm the strong antifungal effect of the combined compost–microbial treatment under field conditions.

3.9. Fruit Quality Attributes

An analysis of the physicochemical and morphometric fruit parameters showed significant (p < 0.05 and p < 0.01) improvement with T4 across all cultivars (

Table 4. Effects of Trichoderma longibrachiatum, Pseudomonas yamanorum, and compost applications on the morphometric and physiochemical characteristics of grape fruits.

Cultivars	Treatments	pH	EC (mS cm−1)	Sugar Content (Brix)	Nitrate Content (mg/kg))	WC (%)	Fruits Caliber Size (mm)
Victoria	T1	3.54 ± 0.51 d	3.44 ± 0.70	12.67 ± 0.53 b	100 ± 3.05 a	59.50 ± 0.64	16.95 ± 0.81 b
	T2	3.80± 0.68 b	3.71 ± 0.24	12.70 ± 0.84 b	94.67 ± 2.67 b	51.17 ± 0.57	19.03 ± 0.75 ab
	T3	3.71 ± 0.77 c	3.41 ± 0.71	14.33 ± 0.99 a	94 ± 1.94 b	58.83 ± 0.92	19.29 ± 0.49 ab
	T4	3.82 ± 0.92 a	3.26 ± 0.37	14.80 ± 0.68 a	77 ± 1.88 c	72.17 ± 0.38	21.20 ± 0.93 a
	p-value	<0.01	<0.01	<0.01	<0.01	≥0.05	<0.05
Superior Seedless	T1	3.85 ± 0.36 d	4.33 ± 0.09	13.10 ± 0.42 c	87 ± 1.28 b	27.83 ± 0.45 c	17.55 ± 1.02 b
	T2	3.91 ± 0.75 c	4.16 ± 0.65	13.60 ± 0.37 b	98.33 ± 1.36 a	38.83 ± 0.69 bc	18.37 ± 0.36 ab
	T3	3.94 ± 0.16 b	4.50 ± 0.44	14.07 ± 0.91 a	81.67 ± 1.45 c	57.67 ± 0.88 a	20.24 ± 0.69 ab
	T4	3.99 ± 0.22 a	3.99 ± 0.18	14.40 ± 0.64 a	83.33 ± 1.89 c	46.83 ± 0.32 ab	20.55 ± 0.55 a
	p-value	<0.01	<0.01	<0.01	<0.01	<0.05	<0.05
Early Sweet	T1	3.73 ± 0.69 d	4.26 ± 0.74 a	11.37 ± 0.86 c	97 ± 1.69 a	68.50 ± 0.22 b	15.76 ± 0.23 a
	T2	3.87 ± 0.82 c	4.01 ± 0.98 a	12.30 ± 0.75 b	65.67 ± 2.09 b	79.17 ± 0.75 a	15.88 ± 1.14 a
	T3	3.91 ± 0.55 b	3.92 ± 0.80 a	12.33 ± 0.67 ab	86.67 ± 1.08 b	79.33 ± 0.64 a	17.03 ± 0.42 a
	T4	3.96 ± 0.45 a	3.34 ± 0.62 a	12.53 ± 0.52 a	84 ± 0.98 b	75.33 ± 0.80 ab	17.81 ± 0.89 a
	p-value	<0.01	≥0.05	<0.01	<0.01	<0.05	≥0.05

ANOVA. Means in a column (±standard errors) followed by the same letter are not significantly different according to Duncan’s test (p < 0.05 and p < 0.01). T1—untreated control, T2—microbial consortium, T3—compost, T4—microbial consortium + compost.

). In Victoria, T4 yielded the highest sugar content (14.80 °Brix) and fruit caliber (21.20 mm), along with the lowest nitrate content (77 mg/kg), a favorable pH (3.82), and reduced electrical conductivity (3.26). In Superior Seedless, T4-treated fruits had the highest sugar level (14.40 °Brix), optimal caliber (20.55 mm), favorable pH (3.99), and improved EC (3.99). Although the EC in Early Sweet was not significantly different (p ≥ 0.05), the T4 treatment resulted in the highest pH (3.96), largest fruit caliber (17.81 mm), highest sugar content (12.53 °Brix), and a reduced nitrate level (84 mg/kg). These outcomes underline the effectiveness of T4 in enhancing fruit quality and market value.

4. Discussion

The present study proposed an integrated strategy that combines a beneficial bacterium (P. yamanorum), fungus (T. longibrachiatum), and compost amendment as a microbial consortium (T4 treatment) to combat major fungal pathogens affecting vineyards in the Ruegeb region of Tunisia. These pathogens include B. cinerea, E. necator, and P. viticola, which pose significant threats to grapevine cultivation. The findings demonstrate that this microbial and organic consortium significantly suppressed disease severity and incidence, offering a sustainable alternative to chemical fungicides. These results align with previous research indicating the effectiveness of a microbial consortium in plant disease control [49,50]. According to previous research conducted in Tunisia, bacterial strains of Bacillus subtilis B27 and B29 decreased grapevine diseases caused by Botrytis cinerea and Uncinula necator. The present study supports these findings by demonstrating that the application of a microbial consortium is effective at suppressing a range of diseases in vineyards, in particular when combined with compost [51].

The study further addressed the efficacy of the treatment on three commercially important grapevine cultivars grown in Tunisia, Victoria, Superior Seedless, and Early Sweet, making the findings directly relevant to regional agricultural practices. Fungicide resistance, often driven by genetic mutations at fungicide target sites, has diminished the effectiveness of chemical treatments [52], further supporting the need for alternative methods.

The integrated T4 treatment reduced disease severity indices, achieving reductions as low as 3–8%, equivalent to a 90% decrease compared to untreated controls. This was significantly more effective than the individual microbial treatment (T2), which yielded only a 63–76% reduction depending on the cultivar and pathogen. These results are consistent with previous findings: Trichoderma harzianum T39 alone reduced P. viticola severity by 67.3% in greenhouse trials [53], and Pseudomonas fluorescens achieved an approximately 35% suppression of grapevine root-rot pathogens [22]. Similarly, ref. [54] found that compost applications suppressed over 50% of 2400 soilborne disease cases. The synergistic effect observed for T4 suggests that compost enhances the efficacy of microbial antagonists, improving disease suppression in grapevines. To ensure long-term consistency across various soil types and climates, multi-season experiments are recommended, as field conditions can change over time.

Plant growth promotion was evidenced through increased leaf chlorophyll content, measured as an indicator of the photosynthetic capacity (Table 1). Thirty days after application, all cultivars showed significant increases. Victoria increased from 19.5 SPAD to 35.07 (+80%), Superior Seedless from 23.37 to 34.90 (+49%), and Early Sweet from 33.03 to 43.70 (+32%). These outcomes are consistent with earlier studies, where compost combined with microbial inoculants increased the chlorophyll content in maize, lettuce, and date palm [55,56]. Such improvements in photosynthesis reflect better plant health and vigor, which are often linked to microbial–root interactions [57].

The consortium treatment (T4) also enhanced the plant’s antioxidant defense system and biochemical response, as shown by increased activities of catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX), and elevated total protein and phenolic contents (Figure 3; Table 2). These defense mechanisms may be activated systemically upon microbial colonization due to a synergistic relationship in which microbial colonization primes the plant’s systemic resistance by triggering defense-related enzymes and pathways that improve pathogen tolerance, as reported in previous studies [58,59,60]. Ref. [61] emphasized that antioxidants regulate stress-responsive gene expression, which may explain the superior disease suppression and growth observed in the T4-treated plants. In order to identify the precise signaling pathways behind the T4 consortium’s actions, more molecular research is required.

Fruit quality parameters were significantly enhanced under the T4 treatment. Fruit pH increased consistently across all cultivars from 3.54 to 3.82 (Victoria), 3.85 to 3.99 (Superior Seedless), and 3.73 to 3.96 (Early Sweet), suggesting microbial-driven metabolic shifts that were not achieved by compost alone. Electrical conductivity (EC) values also decreased with T4, from 3.44 to 3.26 mS/cm in Victoria, 4.33 to 3.99 in Superior Seedless, and 4.26 to 3.34 in Early Sweet, showing 5–8% reductions. Compost’s known ability to improve the soil structure and reduce EC likely plays a role here, alongside the microbial regulation of ion uptake [62].

The sugar content (Brix value) and nitrate levels were also positively affected. Previous studies have shown that a Pseudomonas spp. and Trichoderma-based consortium can raise Brix levels [63,64,65]. In this study, T4 treatment reduced nitrate accumulation in Victoria fruits by 23% (from 100 to 77 mg/kg), consistent with the ability of compost to modulate nitrogen assimilation [66,67]. The fruit water content in Victoria increased from 59.5% to 72.2% (+21%), and similarly in other cultivars, likely due to an improved water retention capacity and enhanced soil structure following the microbial and compost treatment [63]. Lastly, the increases in soil microbial biomass and diversity with T4 suggests that this integrated strategy not only improves plant health and productivity but also contributes to long-term soil fertility and sustainability. The improvements in fruit quality and soil microbial diversity with the T4 treatment demonstrate the promise of this integrated microbial approach for increasing grapevine yield and nutritional value in addition to suppressing diseases. Future research should investigate into the metabolic and physiological processes, as well as assess whether widespread adoption is feasible in a variety of commercial vineyard and environmental settings.

5. Conclusions

This study demonstrated that the integrated application of Pseudomonas yamanorum, Trichoderma longibrachiatum, and compost (T4) significantly enhanced grapevine resistance to the key fungal pathogens B. cinerea, E. necator, and P. viticola in three major cultivars: Victoria, Superior Seedless, and Early Sweet. The consortium reduced the disease severity index and fruit disease incidence while simultaneously boosting plant physiological responses such as the chlorophyll content, enzymatic antioxidant defenses (CAT, POX, and APX), and biochemical markers (protein and phenolic contents).

Additionally, this approach improved key fruit quality traits, including pH, sugar content, water retention, and size, while reducing undesirable components such as nitrate levels and electrical conductivity. Soil health indicators also benefited through an improved organic matter content, microbial activity, and nutrient availability.

Collectively, these findings present a compelling case for replacing or supplementing chemical fungicides with a microbial organic consortium in vineyard management. This eco-friendly strategy not only promotes plant and soil health but also aligns with sustainable agriculture and environmental conservation goals. The application of a microbial consortium integrated with compost thus offers a robust, sustainable path forward for viticulture in Tunisia and comparable agroecosystems.