# Eco-Friendly Suppression of Grapevine Root Rot: Synergistic Action of Biochar and Trichoderma spp. Against Fusarium equiseti

Sabrina Esposito Oliveira da Mota, Jamilly Alves De Barros, Kedma Maria Silva Pinto, José Eduardo Cordeiro Cezar Santos, Alberto dos Passos Vieira, Elisiane Martins De Lima, Diogo Paes Da Costa, Gustavo Pereira Duda, José Romualdo de Sousa Lima, Mairon Moura da Silva, et al.

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Sabrina Esposito Oliveira da Mota, Jamilly Alves De Barros, Kedma Maria Silva Pinto, José Eduardo Cordeiro Cezar Santos, Alberto dos Passos Vieira, et al. Eco-Friendly Suppression of Grapevine Root Rot: Synergistic Action of Biochar and Trichoderma spp. Against Fusarium equiseti. Agriculture, 2025, 15, 10.3390/agriculture15161774. [https://doi.org/10.3390/agriculture151617203]

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Article

# Eco-Friendly Suppression of Grapevine Root Rot: Synergistic Action of Biochar and *Trichoderma* spp. Against *Fusarium equiseti*

Sabrina Esposito Oliveira da Mota <fnref n="1" />, Jamilly Alves de Barros <fnref n="1" /> ID, Kedma Maria Silva Pinto <fnref n="1" />, José Eduardo Cordeiro Cezar Santos <fnref n="1" />, Alberto dos Passos Vieira <fnref n="1" /> ID, Elisiane Martins de Lima <fnref n="1" /> ID, Diogo Paes da Costa <fnref n="1" />, Gustavo Pereira Duda <fnref n="1" />, José Romualdo de Sousa Lima <fnref n="1" /> ID, Mairon Moura da Silva <fnref n="1" />, Carlos Alberto Fragoso de Souza <fnref n="1" />, Rafael José Vilela de Oliveira <fnref n="1" /> ID, Claude Hammecker <fnref n="2" /> ID and Erika Valente de Medeiros <fnref n="1" />,* ID

<figure><img src="image_2.png" /></figure>

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updates

Academic Editor: Giuseppe Lima

Received: 9 June 2025

Revised: 2 August 2025

Accepted: 11 August 2025

Published: 19 August 2025

**Citation:** da Mota, S.E.O.; Barros, J.A.d.; Pinto, K.M.S.; Santos, J.E.C.C.; Vieira, A.d.P.; Lima, E.M.d.; Costa, D.P.d.; Duda, G.P.; Lima, J.R.d.S.; da Silva, M.M.; et al. Eco-Friendly Suppression of Grapevine Root Rot: Synergistic Action of Biochar and *Trichoderma* spp. Against *Fusarium equiseti*. *Agriculture* **2025**, *15*, 1774. https://doi.org/10.3390/agriculture15161774

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

## Abstract

The application of biochar and beneficial microorganisms has gained attention as a sustainable strategy to enhance soil health and plant resistance to pathogens. *Trichoderma* spp. play critical roles in nutrient mobilization, rhizosphere colonization, and suppression of soilborne diseases. However, little is known about the interactive effects of biochar and *Trichoderma* on the suppression of *Fusarium equiseti* (P1I3)-induced root rot in grapevine seedlings. In this study, we investigated the effects of two *Trichoderma aureoviride* strains (URM 6668 and URM 3734), with and without grapevine pruning-derived biochar (BVP), on disease severity, plant growth, and soil properties. Our results revealed that the combination of biochar and *Trichoderma* significantly reduced disease incidence and promoted biomass accumulation. Notably, BVP and *T. aureoviride* URM 3734 were the most effective at reducing leaf disease severity, resulting in a 53% decrease. Conversely, the combination of BVP and *T. aureoviride* URM 6668 led to the greatest reduction in root disease severity, with a 56% decrease. These findings suggest a synergistic relationship between biochar and beneficial fungi, reinforcing the role of organic soil amendments in promoting plant health. The integrated use of biochar and *Trichoderma* strains offers a viable, environmentally sound approach for managing grapevine root rot and enhancing seedling health in sustainable viticulture systems.

**Keywords:** biocarbon; biological control; severity; management practices

## 1. Introduction

Biochar, a carbon-rich material produced through the pyrolysis of organic residues, has garnered increasing interest as a multifunctional amendment in sustainable agriculture. Its

<footnote n="1">¹ Department of Agronomy, Universidade Federal do Agreste de Pernambuco, Campus Garanhuns, Avenida Bom Pastor, s/n Boa Vista, Garanhuns 55292-270, Brazil; sabrina.mota@ufape.edu.br (S.E.O.d.M.); jamillybarros.pnpd@ufape.edu.br (J.A.d.B.); kedma.pinto@ufape.edu.br (K.M.S.P.); eduardo.cezar@ufrpe.br (J.E.C.C.S.); alberto.vieira@ufape.edu.br (A.d.P.V.); elisiane.mlima2@ufrpe.br (E.M.d.L.); diogopaes.pnpd@ufape.edu.br (D.P.d.C.); gustavo.duda@ufape.edu.br (G.P.D.); romualdo.lima@ufape.edu.br (J.R.d.S.L.); mairon.moura@ufape.edu.br (M.M.d.S.); carlosfragoso.pnpd@ufape.edu.br (C.A.F.d.S.); rafael.oliveira@cetene.gov.br (R.J.V.d.O.)</footnote>

<footnote n="2">² Institute de Recherche Pour le Développement (IRD), Place Pierre Viala, 34060 Montpellier, France; claudie.hammecker@ird.fr</footnote>

<footnote>* Correspondence: erika.valente@ufape.edu.br; Tel.: +55-87-999454577; Fax: +55-87-3764-5500</footnote>
highly porous structure and alkaline nature improve key soil attributes, including water retention, pH regulation, and the provision of habitats for microorganisms, thereby enhancing both soil health and plant productivity [1–3]. Widely applied as a soil conditioner, biochar contributes to improvements in physical, chemical, and biological properties, particularly through the neutralization of acidity and support for a more resilient soil microbiome [4–6]. Moreover, biochar plays a role in plant defense by inducing systemic resistance, stimulating beneficial microbial activity, and adsorbing phytotoxic compounds [7,8]. It has also been identified as a promising tool for disease suppression across cropping systems, as it promotes the functional diversity of plant-growth-promoting rhizobacteria through its catalytic carbon surface and contribution to the soil organic matter cycle [9,10]. Additionally, biochar enhances the survival and activity of beneficial microorganisms, such as *Trichoderma* spp., which are well-known antagonists of soilborne pathogens [5,11,12].

In this sense, *Trichoderma* spp. play a pivotal role in promoting soil health and are among the most widely used biological control agents in plant disease management due to their multifaceted mechanisms of action [13–15]. Species within this genus act as plant-growth-promoting fungi (PGPF), enhancing root development, producing phytohormones, and facilitating nutrient solubilization [16,17]. Additionally, *Trichoderma* spp. secrete a variety of lytic enzymes capable of degrading pathogen cell walls and employ multiple antagonistic strategies, including mycoparasitism, antibiosis, competition for space and nutrients, and induction of systemic resistance in host plants [11]. Their efficacy against soilborne pathogens is further reinforced by their ability to persist and colonize the rhizosphere under adverse environmental conditions [18,19]. Given these properties, *Trichoderma* spp. represent a viable alternative to synthetic fungicides, which are increasingly limited by issues such as declining efficacy, resistance development, and the need for repeated application factors that elevate production costs and select for fungicide-tolerant pathogen strains [20]. Moreover, synthetic fungicides pose significant environmental risks, including leaching into aquatic ecosystems, long-term soil persistence, bioaccumulation in food webs, and negative impacts on biodiversity [21–25].

Although the combined application of biochar and *Trichoderma* spp. has shown promise in a few cropping systems [8,13], there is still a gap in understanding how this interaction impacts substrates used to produce grapevine seedlings. Grapevine root rot is a devastating disease complex involving over 30 fungal genera [26], leading to substantial yield losses and high mortality rates, particularly in young vines [27,28]. These pathogens compromise the longevity and productivity of mature vineyards and threaten nursery production and vineyard establishment. Many are introduced through infected propagation material [29–31], while others colonize asymptomatic vines latently, residing within the vascular system until favorable conditions promote disease expression [32–34]. Among these, the genus *Fusarium* has been frequently implicated in grapevine decline worldwide due to its high adaptability, soil persistence, and ability to survive on non-host plants, making it difficult to manage [35–37]. Its wide ecological amplitude and long-term survival strategies underscore the urgent need for sustainable and effective disease control approaches in viticulture.

Several studies have demonstrated the plant-growth-promoting potential of *Trichoderma* spp., associated with the production of phytohormones and enzymes that enhance root development, nutrient solubilization, and water uptake, contributing to disease suppression [15,17]. Likewise, the application of biochar has been shown to improve soil chemical and biological properties and serve as a promising tool in managing plant diseases caused by soilborne pathogens [7,8]. Despite these advances, the co-application of biochar and *Trichoderma* spp. has been little explored, particularly in the context of grapevine propagation and root disease management. Considering the potential of inte-
grating biological control agents with sustainable amendments derived from viticultural residues, this study aimed to evaluate whether biochar produced from grapevine pruning, co-applied with *Trichoderma aureoviride* isolates, could reduce the severity of root rot caused by *Fusarium equiseti* and improve soil quality indicators. Specifically, we tested the hypothesis that this combination enhances microbial biomass carbon (MBC) and enzymatic activity in the rhizosphere. To this end, two *T. aureoviride* isolates (URM 3734 and URM 6668) were applied individually or in combination with grape-derived biochar to substrates inoculated with *F. equiseti* (P1I3) in grapevine seedlings.

## 2. Materials and Methods

### 2.1. Biochar Production and Characterization

Biochar was produced via pyrolysis of grapevine pruning residues (BVP), collected from two sites in Pernambuco, Brazil: the experimental vineyard of the Agronomic Institute of Pernambuco (IPA), located in Brejão-PE (9°01'48" S latitude and 36°34'08" W longitude), and a commercial vineyard named Vale das Colinas in Garanhuns-PE (8°56'17.85" S latitude and 36°31'21.45" W longitude) at ~900 m altitude. The residues, composed of fine grapevine branches, were air-dried in a shaded area at ambient temperature and then subjected to pyrolysis. The thermal conversion was carried out using a small-scale kiln adapted from a design widely used by Thai farmers [38], operating under oxygen-limited conditions at 450 °C for 12 h with a chamber capacity of approximately 140 L [39]. After pyrolysis, the resulting biochar was ground using a Willey-type knife mill, yielding 5 kg of material, corresponding to a conversion efficiency of 55.6%.

The total concentrations of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), and boron (B) were quantified by Nair et al. [40]. For each sample, approximately 30 mg of biochar was ashed in a muffle furnace at 500 °C for at least 8 h. After cooling to room temperature, 1.5 mL of nitric acid (HNO₃), diluted in ultrapure water (1:3, v/v), was added to each sample, followed by the addition of 13.5 mL of ultrapure water. The mixtures were homogenized and subsequently filtered through 0.45 µm pore-size membranes. Elemental concentrations were determined using inductively coupled plasma optical emission spectrometry (ICP-OES). The cation exchange capacity (CEC) of the biochar was assessed using a modified ammonium acetate compulsory displacement method, adapted specifically for biochar materials [41]. Total nitrogen (N) content was determined via sulfuric acid digestion following the Kjeldahl method, while organic carbon was quantified by dry combustion in a muffle furnace, as described by Teixeira [42]. The chemical characterization of the two biochar types is shown in Table 1.

<table><caption>**Table 1.** Chemical characterization of biochar from grapevine pruning residues (BVP).</caption><thead><tr><th>Attributes</th><th>Grapevine Pruning Biochar (BVP) *</th></tr></thead><tbody><tr><td>pH</td><td>9.64</td></tr><tr><td>CEC</td><td>17.3</td></tr><tr><td>C (g kg<sup>-1</sup>)</td><td>381.7</td></tr><tr><td>N<sup>1</sup> (g kg<sup>-1</sup>)</td><td>14.7</td></tr><tr><td>C/N</td><td>26</td></tr><tr><td>P (g kg<sup>-1</sup>)</td><td>8.9</td></tr><tr><td>K (g kg<sup>-1</sup>)</td><td>14.5</td></tr><tr><td>Ca (g kg<sup>-1</sup>)</td><td>12.8</td></tr><tr><td>Mg (g kg<sup>-1</sup>)</td><td>4.2</td></tr><tr><td>Na (g kg<sup>-1</sup>)</td><td>4.8</td></tr><tr><td>Sulfur (g kg<sup>-1</sup>)</td><td>3209.4</td></tr><tr><td>Fe (mg kg<sup>-1</sup>)</td><td>3631.9</td></tr><tr><td>Cu (mg kg<sup>-1</sup>)</td><td>36.8</td></tr><tr><td>Mn (mg kg<sup>-1</sup>)</td><td>362.9</td></tr></tbody></table>
<table><caption>**Table 1.** *Cont.*</caption><thead><tr><td>Attributes</td><td>Grapevine Pruning Biochar (BVP) *</td></tr></thead><tbody><tr><td>Zn (mg kg<sup>-1</sup>)</td><td>3205.5</td></tr><tr><td>B<sup>2</sup> (g kg<sup>-1</sup>)</td><td>23.8</td></tr><tr><td>EC (dS m<sup>-1</sup>)</td><td>1.01</td></tr></tbody></table>

## 2.2. *Trichoderma aureoviride* Inoculum Production

We used two strains that were considered the most effective in the in vitro antagonism assays—*Trichoderma aureoviride* URM 6668 (+T1) and *T. aureoviride* URM 3734 (+T2)—which inhibited the growth of *F. equiseti* in pure culture (PDA) medium and in biochar-supplemented medium, in addition to presenting high sporulation (fundamental for application in the in vivo test). Previous studies have also demonstrated that both strains could produce chitinase [43] and were the most effective in antagonistic activity against the pathogen *Fusarium solani*. Both strains were obtained from the URM Culture Collection https://www.ufpe.br/micoteca/ (accessed on 4 January 2025). The strain *Trichoderma aureoviride* (accession number: URM 6668) was isolated in soil contaminated with textile effluent in the city of Toritama, Pernambuco, Brazil, and the strain *T. aureoviride* (accession number: URM 3734) was isolated in a preserved forest area in the city of Recife, Pernambuco, Brazil. Each strain was cultivated in Erlenmeyer flasks (five replicates per isolate) containing PDA (potato dextrose agar) medium. Three mycelial disks (5 mm in diameter) were transferred to each flask and incubated for 5 days in a BOD chamber at 25 °C with a 12 h photoperiod. After the incubation period, the conidial suspensions were prepared by adding sterile saline solution with Tween 80, followed by spore counting using a Neubauer chamber under light microscopy. The concentration of conidia was calculated using the average count from five quadrants, according to the following formula: Conidia mL⁻<fnref n="1" /> = [(E1 + E2 + E3 + E4 + E5)/5] × 10⁴]. Based on this quantification, inoculum suspensions were adjusted to a final concentration of 10⁶ conidia mL⁻<fnref n="1" />, and a volume of 100 mL per pot was applied in the in vivo experiment.

## 2.3. *Fusarium equiseti* Inoculum Production

*F. equiseti* was isolated from diseased wine grape plants by the phytopathology team at a winery in the region. This isolate (P1I3) was identified and stored in the culture collection of the phytopathology laboratory of UFAPE. The multiplication of *F. equiseti* (P1I3) followed the methodology of Steffen and Maldaner [44], using 100 g of parboiled rice moistened with 50 mL of distilled water as substrate. After autoclaving at 121 °C for 25 min, the substrate was inoculated with five 5 mm mycelial disks from pure cultures grown on PDA. The material was incubated at 25 ± 1 °C under a 12 h photoperiod for five days, with daily manual agitation to promote aeration and stimulate sporulation.

## 2.4. Microcosm Experiment

The experiment was arranged in a completely randomized design with seven treatments and four replicates per treatment. The treatments were structured based on two main factors, the first (i), *Trichoderma* strain, with three levels—*T. aureoviride* URM 6668 (T1), *T. aureoviride* URM 3734 (T2), and a non-inoculated (-T)—and the second factor (ii), biochar application, with two levels: absence (−BVP) and presence (+BVP) of grapevine pruning biochar. In addition, two control treatments were included: a positive control (−BVP-T: no *Trichoderma*, no biochar, with pathogen) and a negative control (AD: no *Trichoderma*, no biochar, and no pathogen). All treatments, except the negative control, were inoculated with *Fusarium equiseti* (P1I3).

Cabernet Sauvignon grapevine seedlings grafted onto the susceptible SO4 rootstock were sourced from Sociedade Vitácea de Desenvolvimento Vitícola LTDA (Caldas,

<footnote n="1">¹ Sulfuric digestion; ² solubilization method: dry digestion. * Total contents (except carbon).</footnote>
MG, Brazil), a certified nursery specializing in high-quality grapevine propagation. The seedlings were transplanted into 4 L pots containing a sterilized soil and organic matter mixture (3:1, v/v). For sterilization, high-temperature-resistant bags were filled with 1 kg of the material and autoclaved three times for 20 min at 121 °C in a vertical autoclave.

The sandy soil utilized in this study was collected from a site undergoing ecological regeneration within a tropical dry forest ecosystem located in Garanhuns, Pernambuco, Brazil (8°56'18.6" S, 36°28'57.9" W; Figure 1), at an elevation of 705 m above sea level. Formerly degraded by low-yield pastureland, the area has been left to regenerate naturally for the past 15 years and is now predominantly colonized by native vegetation typical of the Caatinga biome—the largest expanse of seasonally dry tropical forest globally. According to the World Reference Base for Soil Resources [45], the soil is classified as Regosol. The regional climate is characterized as mesothermal tropical highland (Cwa) under the Köppen climate classification system, with a mean annual temperature of approximately 20 °C and an average annual precipitation of 1300 mm.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** A map of the geographic location of the soil collection site for the experiment.</figcaption></figure>

The soil was collected from the 0–20 cm layer, sieved through a 2 mm mesh to remove coarse fragments, and used immediately to preserve native microbial communities. Chemical characterization of the soil (Table 2) was carried out by Teixeira et al. [42].

<table><caption>**Table 2.** Chemical characterization of sandy soil from tropical dry forest (Regosol) collected at Garanhuns, Pernambuco, Brazil.</caption><thead><tr><th>pH</th><th>EC</th><th>OM</th><th>P</th><th>S-SO<sub>4</sub><sup>2-</sup></th><th>Ca<sup>2+</sup></th><th>Mg<sup>2+</sup></th><th>K<sup>+</sup></th><th>Na<sup>+</sup></th><th>Al<sup>3+</sup></th></tr></thead><tbody><tr><td>(H<sub>2</sub>O)</td><td>(dS m<sup>-1</sup>)</td><td>(g kg<sup>-1</sup>)</td><td></td><td>(mg kg<sup>-1</sup>)</td><td></td><td></td><td>(cmolc kg<sup>-1</sup>)</td><td></td><td></td></tr><tr><td>5.37</td><td>0.42</td><td>5.5</td><td>15.5</td><td>1.8</td><td>1.04</td><td>0.6</td><td>0.14</td><td>0.02</td><td>0.08</td></tr><tr><th>H+Al</th><th>SB<br>(cmolc kg<sup>-1</sup>)</th><th>CEC</th><th>V<br>(%)</th><th>Fe<sup>2+</sup></th><th>Mn<sup>2+</sup></th><th>Cu<sup>2+</sup><br>(cmolc kg<sup>-1</sup>)</th><th>Zn<sup>2+</sup></th><th>B</th><td></td></tr><tr><td>1.16</td><td>1.8</td><td>2.96</td><td>60.69</td><td>50.1</td><td>15.9</td><td>0.34</td><td>1.6</td><td>0.29</td><td></td></tr></tbody></table>

EC = electrical conductivity; OM = organic matter; SB = sum of bases; CEC = cation exchange capacity; V = percentage by base saturation.
The substrate used (registration number: RS 003636-6.000087) consisted of peat, vermiculite, and limestone, with the following physicochemical properties: a pH of 5.5, dry bulk density of 130 kg m⁻³, water holding capacity of 300% (w/w), and electrical conductivity of 0.7 mS cm⁻¹.

Treatments were initiated three months after transplanting. Biochar was first applied as a single surface dose (10 g per pot), followed 15 days later by the application of *Trichoderma* strains (100 mL per pot at a concentration of 10⁶ colony-forming units—CFU—per mL⁻¹). The *Trichoderma* suspensions were applied twice: once prior to pathogen inoculation and again 30 days later. Inoculation with *F. equiseti* (P1I3) was performed 15 days after the first *Trichoderma* application by incorporating 20 g of colonized rice substrate into the soil of each pot. The experiment was maintained for an additional three months post-inoculation. Irrigation was managed based on the available water capacity (AWC) of the substrate, calculated as the difference between soil moisture at field capacity (FC) and at the permanent wilting point (PWP) (AWC = FC – PWP). Distilled water was applied when soil moisture measurements indicated that 30% of the AWC had been depleted.

Disease severity was evaluated using a visual rating scale adapted from Tokeshi and Galli [46], with the following scores: 0 = healthy plant; 1 = up to 25% foliar yellowing; 2 = up to 50% yellowing with marginal necrosis; 3 = up to 75% yellowing with marginal necrosis; and 4 = complete plant death (sudden wilting). Likewise, a visual scale of disease severity was applied to root symptoms, observing the intensity of the lesions and the internal color of the plant collar and roots. Using these data, the percentage of disease severity on leaves (DSL) and disease severity on roots (DSR) was estimated in relation to the total number of plants evaluated per treatment (4 repetitions). To confirm disease incidence, a pathogenicity test was conducted to verify the presence of the pathogen in symptomatic tissues. For each sampled plant, the following parameters were measured: plant height (PH), stem diameter (SD), shoot fresh and dry biomass (SFB and SDB), and root fresh and dry biomass (RFB and RDB). Plant height was measured with a graduated ruler, and stem diameter was assessed using a digital caliper. Fresh biomass was determined immediately after harvest using an analytical balance, while dry biomass was obtained after drying the plant material at 65 °C for 72 h and then weighing.

The soil chemical parameters analyzed included pH in water (1:2.5), available phosphorus (P), potassium (K), and sodium (Na), and total organic carbon (TOC), following the procedures described by Teixeira and Donagemma [42]. TOC was also quantified using the method of Yeomans and Bremner [47], based on dry combustion in a muffle furnace. Microbial biomass carbon (MBC) was determined using the irradiation-extraction method as outlined by Mendonça and Matos [48] and Bartlett and Ross [49].

Enzymatic activities of β-glucosidase (EC 3.2.1.21), acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1), arylsulfatase (EC 3.1.6.1), and urease (EC 3.5.1.5) were assessed using standard colorimetric methods. β-glucosidase activity was determined using p-nitrophenyl β-D-glucopyranoside as a substrate, incubated at 37 °C for 1 h, with p-nitrophenol (PNP) release quantified spectrophotometrically at 410 nm [50]. Acid and alkaline phosphatase activities were measured using p-nitrophenyl disodium phosphate under the same incubation conditions, with absorbance also read at 410 nm [51]. Arylsulfatase levels were determined with potassium p-nitrophenyl sulfate (PNS) as the substrate, incubated at 37 °C for 1 h, with the release of p-nitrophenol quantified spectrophotometrically at 410 nm [52]. Urease activity was evaluated using urea as the substrate, incubated for 2 h at 37 °C, and the released ammonium was quantified at 690 nm [53].
## 2.5. Statistical Analysis

All analyses were carried out using R v.4.3.1 [54] via the RStudio interface v 2023.06.1 (Build 524) [55]. The assumptions of normality and homogeneity of variances were analyzed using the Shapiro-Wilk and Levene tests, respectively. The statistical approach adopted was a completely randomized design, followed by analysis of variance and the Skott-Knott post hoc test in cases of significant differences, both at a 5% significance level (Supplementary Materials Table S1). Non-parametric data was subjected to the Kruskal-Wallis test followed by Dunn's post hoc test at a 5% significance level. Principal component analysis (PCA) was conducted to study the variance explained by the soil and plant variables simultaneously, identifying the grouping related to the factorial treatments. This modeling was performed using the resources of the R package 'factoextra' [56]. All graphs were created using R's "ggplot2" library.

# 3. Results

The application of biochar and *Trichoderma* significantly affected the disease severity on leaves (DSL) and on roots (DSR) caused by *Fusarium equiseti* (Figure 2). The treatment with biochar and *T. aureoviride* URM 3734 (+BVP+T2) was the most effective in reducing disease severity on leaves (DSL), with a 53% decrease. In contrast, plants treated with biochar and *T. aureoviride* URM 6668 (+BVP+T1) exhibited the greatest reduction in root disease severity, achieving a 56% decrease.

<figure><img src="image_4.png" /></figure>

**Figure 2.** Root rot severity index in grapevine seedling leaves and roots in soil treated with grapevine pruning biochar (BVP) and *Trichoderma aureoviridae* strains. AD = additional control (no *Trichoderma*, no biochar, and no pathogen); -T = no *Trichoderma*; +T1 = *Trichoderma aureoviride* URM 6668; +T2 = *T. aureoviride* URM 3734; -BVP = without biochar; +BVP = with grapevine pruning biochar. DSL = disease severity on leaves; DSR = disease severity on roots. Means followed by the same lowercase letter do not differ significantly for the DSL variable, while means followed by the same uppercase letter do not differ significantly for the DSR variable. Statistical differences were determined using Tukey's test at a 5% significance level (*p* < 0.05). The black (DSL) and green (DSR) dashed horizontal lines correspond to the highest observed severity percentages.

The development of grapevine seedlings inoculated with the pathogen *Fusarium equiseti* (P113) was influenced by the application of biochar and *Trichoderma* strains (Figure 3). Notably, plants treated with the combination of biochar and *T. aureoviride* URM 3734 (+BVP+T2) exhibited the highest number of leaves, a trend that was consistently observed across most growth parameters evaluated (NL = number of leaves; FRM = fresh root mass; DRM = dry root mass; and DMB = dry mass of branches), when compared to the control (no biochar, no *Trichoderma*, and with the pathogen). This improved plant performance was also associated with a significant reduction in foliar disease severity (Figure 2), suggesting a
synergistic effect between biochar and *Trichoderma* in enhancing plant vigor and suppressing pathogen impact.

<figure><img src="image_5.png" /></figure>

**Figure 3.** Grapevine development in soil treated with grapevine pruning biochar (BVP) and *Trichoderma aureoviridae* strains and inoculated with *Fusarium equiseti* (P1I3). AD = additional control (no *Trichoderma*, no biochar, and no pathogen); -T = no *Trichoderma*; +T1 = *Trichoderma aureoviride* URM 6668; +T2 = *T. aureoviride* URM 3734; -BVP = without biochar; +BVP = with grapevine pruning biochar. NL = number of leaves (a); DMB = dry mass of branches (b); FRM = fresh root matter (c); LB = length of branches (d); DRM = dry root mass (e). Means followed by the same lowercase or uppercase letters within each level of *Trichoderma* (Factor 1) and biochar (Factor 2) do not differ significantly, as determined by Tukey's test at the 5% significance level (p < 0.05). Means marked with asterisks differ significantly from the additional control treatment (dashed line), based on Dunnett's test at the 5% significance level.

Among the soil chemical and biological attributes evaluated, significant differences were observed exclusively for potassium (K), sodium (Na), and microbial biomass carbon (MBC) (Figure 4). Notably, MBC was influenced by the application of biochar in the absence of *Trichoderma* strains (+BVP-T). This treatment led to a 38.77% increase in MBC compared to the positive control (soil inoculated with *Fusarium equiseti*, but without biochar or *Trichoderma*), indicating the favorable impact of biochar alone on microbial biomass accumulation. In contrast, the co-application of biochar with *T. aureoviride* URM 6668 (+T1) resulted in a pronounced suppression of MBC, with a 53.76% reduction relative to the control.

Biochar significantly enhanced K and Na concentrations when applied without *Trichoderma*. In this condition, K and Na levels increased by 53.43% and 45.86%, respectively, compared to the control. However, the simultaneous application of biochar and *T. aureoviride* URM 3734 (+BVP+T2) led to reductions of 94.20% in K and 69.04% in Na, underscoring a potential biochar-mediated modulation of fungal activity and its downstream effects on nutrient mobilization or retention.

The enzymatic activity of the soil was significantly influenced by the combined application of *Trichoderma* strains and biochar, mainly in arylsulfatase, alkaline phosphatase, and urease (Figure 5). The application of biochar with *Trichoderma* (+BVP+T2) enhanced arylsulfatase activity, promoting a 40.64% increase compared to the equivalent treatment without biochar.
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Changes in the attributes of soil treated with biochar and *Trichoderma* strains, cultivated with grape plants, and inoculated with *Fusarium equiseti* (P1I3). K = potassium contente (a); Na = sodium contente (b); MBC = microbial biomass carbon (c). AD = additional control (no *Trichoderma*, no biochar); -T = no *Trichoderma*; +T1 = *Trichoderma aureoviride* URM 6668; +T2 = *T. aureoviride* URM 3734; -BVP = without biochar; +BVP = with grapevine pruning biochar. Means followed by the same lowercase or uppercase letters within each level of *Trichoderma* (Factor 1) and biochar (Factor 2) do not differ significantly, as determined by Tukey's test at the 5% significance level (*p* < 0.05). Means marked with asterisks differ significantly from the additional control treatment (dashed line), based on Dunnett's test at the 5% significance level.</figcaption></figure>

Conversely, alkaline phosphatase activity was markedly enhanced by the application of biochar, even in the absence of *Trichoderma* spp. (+BVP-T), suggesting that the pyrolyzed material alone exerts a positive influence on phosphorus mineralization processes in the soil. In contrast, urease activity was negatively affected by biochar application in the absence of *Trichoderma*, exhibiting a significant reduction compared to the control. However, the inclusion of *T. aureoviride* URM 3734 in the biochar treatment reversed this trend, leading to a 23.03% increase in urease activity compared to the same treatment without biochar.

Principal component analysis (PCA) was employed to investigate the effects of *Trichoderma* strains and biochar on the suppression of grapevine root rot, as well as on plant development and soil biochemical properties (Figure 6). The first two principal components (Dim1 and Dim2) accounted for 24.5% and 15.9% of the total variance, respectively, explaining 40.4% of the variability among treatments.

Overall, treatments clustered distinctly according to the presence or absence of *Trichoderma* strains and biochar amendment. Treatments without *Trichoderma* (–T) and with biochar (+BVP) were primarily associated with elevated MBC, urease activity, and β-glucosidase, suggesting that microbial activity and nitrogen cycling were favored by biochar alone, in the absence of fungal inoculation.

In contrast, co-application of *T. aureoviride* URM 3734 (+T2) with biochar resulted in a unique clustering pattern, with plant-growth-promoting variables such as number of
leaves (NL), fresh and dry branch mass (FMB and DMB), fresh and dry root mass (FRM and DRM), and total organic carbon (TOC). This association suggests a synergistic effect between this specific *Trichoderma* isolate and biochar in stimulating both plant development and soil fertility parameters related to carbon and phosphorus cycling.

<figure><img src="image_7.png" /></figure>

**Figure 5.** Enzymatic activities of soil treated with biochar and *Trichoderma* strains, cultivated with grape plants, and inoculated with *Fusarium equiseti* (P113). Aryl = arylsulfatase activity (a); Alkaline Phosp. = alkaline phosphatase activity (b); Urease = urease activity (c). AD = additional control (no *Trichoderma*, no biochar); -T = no *Trichoderma*; +T1 = *Trichoderma aureoviride* URM 6668; +T2 = *T. aureoviride* URM 3734; -BVP = without biochar; +BVP = with grapevine pruning biochar. Means followed by the same lowercase or uppercase letters within each level of *Trichoderma* (Factor 1) and biochar (Factor 2) do not differ significantly, as determined by Tukey's test at the 5% significance level (*p* < 0.05). Means marked with asterisks differ significantly from the additional control treatment (dashed line), based on Dunnett's test at the 5% significance level.

Interestingly, the combination of *T. aureoviride* URM 6668 (+T1) with biochar showed an intermediate response. This treatment was positioned near the center of the biplot, indicating a more balanced contribution of microbial and plant-related variables, but without the strong positive associations observed for the +T2 treatment.

Further insight was gained from the variable contribution plot (Figure 6c), which identified fresh mass of branches (FMB), fresh root mass, length of branches and dry root mass (FRM, LB, DRM), and number of leaves (NL) as the most influential variables driving the observed clustering. Enzymatic activities such as acid phosphatase and β-glucosidase also contributed moderately to the overall separation, while urease and alkaline phosphatase had lower relative contributions.
<figure><img src="image_8.png" /><figcaption>**Figure 6.** An exploratory analysis of treatments with biochar and *Trichoderma* strains applied to Cabernet Sauvignon grape seedlings (rootstock SO4) inoculated with *Fusarium equiseti* (P1I3). (a) The contributions of the principal components. (b) A biplot of the principal component analysis. The levels of the *Trichoderma* factor are classified in different colors, while the levels of biochar are distinguished by different shapes. (c) The average contribution of the variables to the variance in the multivariate model. AD = additional control (no *Trichoderma*, no biochar); -T = no *Trichoderma*; +T1 = *Trichoderma aureoviride* URM 6668; +T2 = *T. aureoviride* URM 3734; -BVP = without biochar; +BVP = with grapevine pruning biochar. Aryl = arylsulfatase activity; Alkaline Phosp. = alkaline phosphatase activity; Urease = urease activity. NL = number of leaves; DMB= dry mass of branches; DRM = dry root mass; LB = length of branches; DRM = dry root mass; FMB= fresh mass of branches; FRM = fresh root mass; DS = disease severity; SD = stem diameter; K = potassium content; Na = sodium content; MBC = microbial biomass carbon; Aryl = arylsulfatase activity; Alkaline Phosp. = alkaline phosphatase activity; Urease = urease activity.</figcaption></figure>

**4. Discussion**

In recent years, the application of biological control agents has emerged as a promising and environmentally sustainable alternative to synthetic agrochemicals in the management of soilborne pathogens. Among these agents, fungal species of the genus *Trichoderma* have received attention due to their multifaceted mechanisms of action, including mycoparasitism, competition for nutrients and space, induction of systemic resistance, and production of lytic enzymes and secondary metabolites [11,57]. In parallel, biochar has gained recognition for its ability to improve soil physicochemical properties and its function as a potential carrier and enhancer of microbial inoculants [13].

In this study, we evaluated the effects of *T. aureoviridae* strains, applied singly and in co-inoculation with BVP, on the suppression of grapevine root rot caused by *Fusarium equiseti* (P1I3). Consistent with our first hypothesis, the co-application of *Trichoderma*
strains with BVP significantly reduced disease severity in both leaves and roots, indicating that these strains negatively affect the establishment of the pathogen. *T. aureoviridae*, which is well known for its ability to compete for nutrients, cause antibiosis, and activate plant defense pathways, confirming its important role in controlling root rot diseases [58]. Moreover, *Trichoderma* produces antibiotic compounds, including non-ribosomal peptides, terpenoids, pyrones, and indolic-derived compounds that inhibit the growth of plant pathogens through direct antagonism [59]. The secretion of lytic enzymes by *Trichoderma* strains, such as chitinases, also plays a pivotal role in the degradation of fungal cell walls, thereby contributing to the suppression of soilborne phytopathogens [60]. These hydrolytic enzymes disrupt the structural integrity of pathogenic fungi, weakening their defenses and enhancing the efficacy of biological control [61,62].

Interestingly, the *T. aureoviride* URM 3734 with biochar (+BVP+T2) treatment was more effective in reducing leaf symptoms of grapevine root rot, with a 53% decrease compared to the control. In contrast, the combination of biochar with *T. aureoviride* URM 6668 (+BVP+T1) was more effective in reducing disease severity at the root, with a 56% decrease. The differences in response between strains indicate that, while both strains possess biocontrol potential, their mechanisms of action and interactions with the plant and soil microbiome may differ. The *T. aureoviridae* URM 6668 (T1) may exert more localized effects in the rhizosphere, such as direct antagonism through mycoparasitism or competition [43,60,61]. Supporting this hypothesis, a previous in vitro screening for antagonistic potential against *Fusarium equiseti* revealed that T1 exhibited a higher inhibition rate (78.16%) compared to *T. aureoviride* URM 3734 (T2), which achieved 75.71% inhibition.

While the effects of *Trichoderma* strains with biochar were evaluated against grapevine root rot caused by *F. equiseti* (P1I3), we also hypothesized that these inoculations could modify plant development and soil properties. Our results partially supported this hypothesis: notably, the co-application of biochar and *T. aureoviride* URM 3734 (+BVP+T2) resulted in the highest NL, with improvements in shoot and root biomass. This enhanced plant development was closely associated with a substantial reduction in foliar disease severity observed in the same treatment. This suggests that improved plant health may be a direct outcome of effective pathogen suppression in the rhizosphere, which is crucial for plant resilience [13], including in seedlings [63]. In addition, while the biochar likely contributed to better soil aeration, water retention, and nutrient dynamics [39], *T. aureoviride* may have enhanced root colonization and activated plant defense responses, collectively minimizing pathogen impact [59]. This combination may have alleviated stress conditions caused by *F. equisetai* (P1I3), allowing the plant to allocate more energy toward growth and development.

Furthermore, the absence of significant differences in some soil attributes, mainly in soil chemicals, indicates that the quantity of inputs and the duration of the experiment were not sufficient to induce measurable changes in soil chemistry. This is consistent with previous findings indicating that chemical transformations in soil often require longer periods of exposure and higher amendment rates to become evident [64,65]. In contrast, biological attributes responded more rapidly to the treatments. The co-application of biochar and *Trichoderma* strains significantly influenced microbial biomass and most soil enzymatic activities. These findings highlight the sensitivity of biological indicators as early markers of soil response to management practices and reinforce the role of biochar–microbe interactions in enhancing microbial activity and functional capacity, even in the short term [8,13]. For example, the application of biochar with *Trichoderma* (+BVP+T2) enhanced arylsulfatase activity, which suggests a synergistic interaction that may stem from improved microbial habitat conditions, the stabilization of enzyme substrates, or biochar-induced shifts in microbial community composition favoring sulfur-mineralizing organisms [66]. In
contrast, urease activity was negatively affected by biochar application in the absence of *Trichoderma*, exhibiting a significant reduction compared to the control. This suppression may result from the adsorption of urease or its substrate (urea) onto the porous structure of the biochar, thereby limiting enzyme–substrate interaction or enzyme stability [67]. Biochar’s high surface area and porosity enable it to adsorb both urease enzymes and urea, reducing their availability for the hydrolysis reaction and thus decreasing urease activity [68].

The PCA revealed a clear separation of treatments based on the presence or absence of *Trichoderma* strains and BVP, indicating that these factors distinctly modulated soil and plant responses. Notably, treatments without *Trichoderma* (–T) but with biochar (+BVP) were clustered together and associated with elevated microbial biomass carbon (MBC), urease activity, and β-glucosidase levels. This pattern suggests that biochar alone was effective in stimulating microbial activity, particularly those related to carbon and nitrogen cycling, even in the absence of introduced fungal strains [69,70]. In contrast, co-application of *T. aureoviride* URM 3734 (+T2) with biochar resulted in a unique clustering pattern, with plant-growth-promoting variables such as the number of leaves (NL), fresh and dry shoot biomass (FMB and DMB), root biomass (FRM and DRM), and total organic carbon (TOC). This association suggests a synergistic effect between this specific Trichoderma isolate and biochar in stimulating both plant development and soil fertility parameters related to carbon and phosphorus cycling [71].

These results underscore the potential of integrating biochar and beneficial fungi to enhance soil health and plant resilience in pathogen-challenged systems. However, they also highlight the critical importance of selecting compatible microbial strains, as the outcome of biochar–microbe interactions is highly specific and can substantially alter both biochemical and agronomic outcomes.

**5. Conclusions**

This study demonstrated that *T. aureoviride* URM 3734, co-applied with grapevine pruning biochar, was more effective than *T. aureoviride* URM 6668 in promoting grapevine growth and reducing foliar disease severity, while *T. aureoviride* URM 6668 was more effective in suppressing root rot caused by *Fusarium equiseti* (P113). Despite limited changes in soil chemical properties, both strains significantly enhanced microbial biomass and soil enzymatic activities, highlighting the responsiveness of biological indicators to biochar–microbe interactions. Future studies should investigate the mechanisms behind these strain-specific effects, including pathogen antagonism, microbial competition, and induced plant resistance, as well as evaluating the long-term field performance of these bioformulations. Overall, this study provides promising evidence for the targeted use of *Trichoderma* strains and biochar as sustainable tools to manage soilborne diseases and support soil biological function in viticulture systems.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15161774/s1, Table S1. Statistical analysis.

**Author Contributions:** Conceptualization, S.E.O.d.M., K.M.S.P., C.H. and E.V.d.M.; Data Curation, D.P.d.C. and E.M.d.L.; Formal Analysis, J.E.C.C.S. and R.J.V.d.O.; Funding Acquisition, E.V.d.M.; Investigation, J.A.d.B., A.d.P.V., M.M.d.S. and C.A.F.d.S.; Methodology, K.M.S.P., G.P.D. and J.R.d.S.L.; Project Administration, E.V.d.M.; Resources, J.A.d.B. and A.d.P.V.; Software, D.P.d.C.; Supervision, E.V.d.M.; Validation, M.M.d.S. and G.P.D.; Visualization, S.E.O.d.M. and J.E.C.C.S.; Writing—Original Draft, S.E.O.d.M. and E.M.d.L.; Writing—Review and Editing, K.M.S.P., C.H. and E.V.d.M. All authors have read and agreed to the published version of the manuscript.
**Funding:** The research received financial support from the National Council for Scientific and Technological Development (CNPq), as well as from the Pernambuco State Foundation for Science and Technology (FACEPE). Additional funding was partially provided by the Coordination for the Improvement of Higher Education Personnel (CAPES-88887.736369/2017-00 and Finance Code 001).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data is not publicly available due to privacy.

**Acknowledgments:** The authors thank the Vale das Colinas winery in Garanhuns-PE-Brazil for providing the material and all the infrastructure for the research.

**Conflicts of Interest:** The authors declare no competing interests.

## Abbreviations

The following abbreviations are used in this manuscript:

<table><tr><td>+BVP</td><td>with grapevine pruning biochar</td></tr><tr><td>-BVP</td><td>without biochar</td></tr><tr><td>T1</td><td><i>Trichoderma aureoviride</i> URM 6668</td></tr><tr><td>T2</td><td><i>Trichoderma aureoviride</i> URM 3734</td></tr><tr><td>NL</td><td>number of leaves</td></tr><tr><td>DMB</td><td>dry mass of branches</td></tr><tr><td>FRM</td><td>fresh root mass</td></tr><tr><td>LB</td><td>length of branches</td></tr><tr><td>DRM</td><td>dry root mass</td></tr><tr><td>MBC</td><td>microbial biomass carbon</td></tr><tr><td>Acid Phosp.</td><td>acid phosphatase</td></tr><tr><td>Alkaline Phos.</td><td>alkaline phosphatase</td></tr><tr><td>Beta</td><td>beta-glucosidase</td></tr><tr><td>Aryl</td><td>arylsulfatase</td></tr><tr><td>Ure</td><td>urease</td></tr></table>

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