In Vitro and In Planta Evaluation of Trichoderma spp. for the Control of Neopestalotiopsis rosae on Strawberry

Abstract

Neopestalotiopsis rosae is an emerging fungal pathogen that causes leaf blight and fruit rot on strawberry. Due to limited fungicide availability and the small number of substances confirmed to be effective against this pathogen, alternative disease control strategies have become a focus of current research. This study aimed to assess, quantify, and compare the efficacy of extracts and inocula of Trichoderma spp. with the conventional fungicide Switch in controlling N. rosae. In the presence of T. harzianum T16 and T. asperellum T23 extracts, conidia production of N. rosae was reduced by 45.0% and 62.7%, respectively. Extracts of T. koningiopsis T10 strongly inhibited both mycelial growth and conidia production (>92.0%), demonstrating efficacy comparable to that of the reference fungicide. Furthermore, T. koningiopsis T10 extracts were able to inhibit N. rosae conidia viability by 55.6%. Under greenhouse conditions, strawberry plants treated with extracts from T. koningiopsis T10 showed protection from N. rosae leaf spots at levels similar to Switch. These findings highlight T. koningiopsis T10 extracts as a promising alternative to chemical fungicides in the integrated management of N. rosae on strawberry.

1. Introduction

Strawberries are among the most economically important berry crops worldwide [1,2]. Cultivation of this crop is highly challenging, requiring significant investment of resources and labor, resulting in high production costs, particularly with regard to the persistent threat of pathogen infestation [3,4,5]. Multiple plant pathogens can infect strawberry plants and pose a serious threat to strawberry production [6,7]. Leaf spots, fruit, crown, and root rots as well as wilt are particularly deleterious diseases, incited by fungal pathogens such as Ramularia sp., Botrytis sp., Phytophthora sp., Colletotrichum sp., Fusarium sp., and Verticillium sp. [8,9,10,11,12]. While it is crucial to distinguish between these different pathogens for the implementation of targeted control strategies, the emergence of new pathogens further reinforces the need for proactive monitoring systems and dynamic management responses to safeguard strawberry production [5].

Newly emerging pathogens such as Neopestalotiopsis rosae pose a substantial risk to strawberry production [5]. Early-stage infections by N. rosae may be misidentified, and even when correctly diagnosed, effective control measures are rather limited [13,14,15,16]. N. rosae is a fungal pathogen known to cause leaf spot, crown rot, and fruit rot in strawberries [5,17,18,19,20]. Recently, multiple outbreaks of N. rosae have been reported in the United States, where no chemical control strategies are available [13,19], leading to significant crop losses with yield reduction of up to 70% [13,17,19,21]. Furthermore, major strawberry-producing regions worldwide, such as China, India, and Mexico, as well as Taiwan, have reported severe yield losses due to infestation with N. rosae [15,18,20,22]. In Germany, N. rosae has not yet been included in the national list of quarantine plant pathogens, thus no officially approved methods of control have been established yet [23].

A limited number of chemical fungicides have demonstrated partial efficacy against N. rosae, and options for effective management are even more restricted [13,14,15,16]. Cyprodinil and Fludioxonil, either individually or in combination, were able to completely inhibit mycelial growth of N. rosae [13]. Both fungicides are currently approved and widely utilized in Germany for the control of Botrytis cinerea and other fungal pathogens in strawberries [23]. Nevertheless, the application of chemical fungicides is associated with concerns regarding environmental and public health, particularly due to residue accumulation and the subsequent decline in biodiversity [24]. Furthermore, the emergence of fungicide resistance poses a significant risk to the long-term effectiveness of this control measure [25,26]. For example, Amiri et al. (2013) have shown 19.8% of 392 tested Botrytis cinerea isolates to have reduced sensitivity to Switch in strawberry [25].

The sustainable management of phytopathogens can be achieved through integrated pest management (IPM), including the utilization of biocontrol agents (BCAs) [27,28]. Research on BCAs has been ongoing for several decades [29,30,31,32,33]. Members of the genus Trichoderma are among the most extensively studied, owing to their diverse mechanisms in suppressing fungal pathogens [34,35,36,37,38], including mycoparasitism [39,40,41], induction of plant resistance to pathogens [42,43], and cell wall-degrading capabilities [44,45]. Another notable mode of action is the high competition capability for space and nutrients [46]. Moreover, the production of antifungal secondary metabolites by Trichoderma spp. has been well characterized [47,48,49,50]. Despite numerous studies investigating secondary metabolite production by Trichoderma spp., their efficacy in controlling fungal pathogens remains insufficiently understood [51]. Trichoderma secondary metabolites have shown significant antagonistic effects against Fusarium moniliforme in maize and Phakopsora pachyrihzi on soybean [52,53]. The germination of B. cinerea conidia has been shown to be inhibited by gliotoxin, a metabolite produced by Trichoderma spp. [54]. Additionally, 6-pentyl-alpha-pyrone (6PAP) has been reported not only to reduce mycelial growth of F. oxysporum, B. cinerea, and R. solani, but also to promote plant growth and induce systemic resistance [34,54]. There are currently limited reports describing the control of N. rosae using culture extracts or secondary metabolites of Trichoderma spp. Therefore, this study explored the potential of Trichoderma spp. and their secondary metabolites as a biological control measure for N. rosae.

The specific objectives of this study were: (1) to investigate and quantify the antifungal effects of culture extracts of Trichoderma spp. on N. rosae and to compare their efficacy with that of the conventional fungicide Switch (cyprodinil + fludioxonil); and (2) to compare their effectiveness to the BCA treatments utilizing conidial suspensions as well as to Switch in a greenhouse setting.

2. Materials and Methods

2.1. Micobial and Plant Materials

The fungal strains Trichoderma koningiopsis T10 (GenBank ACNO: OQ822265), T. harzianum T16 (GenBank ACNO: MW520837), T. asperellum T23 (GenBank ACNO: MW509067), and Neopestalotiopsis rosae AETS11 (GenBank ACNO: PQ511123) were obtained from the Institute of Phytomedicine at the University of Hohenheim. All microorganisms were cultivated on glucose-medium-7 (GM7) according to Rieker et al. 2023 [55]. Strawberry plants of the cultivated variety Herzle were provided by Hummel InVitro (Reinhold Hummel GmbH & Co. KG, Stuttgart, Germany).

2.2. Preparation of Conidial Suspensions from Trichoderma spp. and N. rosae

GM7-agar plates (90 mm ⍉) were inoculated with an agar plug (5 mm ⍉) of 14 day-old cultures of Trichoderma spp. or N. rosae. The cultures were sealed airtight with Parafilm (Bemis, Neenah, WI, USA) and incubated for 14 d at 21 ± 2 °C and 14/10 h (light/dark) cycle. After the addition of 4 mL sterile water to each culture, fungal biomass was completely removed with a spatula. To separate conidia from mycelial fragments, the resulting suspension was filtered through two layers of sterile gauze (Hartmann, Neuhausen am Rheinfall, Switzerland) for N. rosae and four layers for Trichoderma spp. The conidial density was determined using a Fuchs-Rosenthal counting chamber (Brand, Wertheim, Germany) and adjusted to 10⁷ and 10⁵ conidia mL⁻¹ for Trichoderma spp. and N. rosae, respectively.

2.3. Extraction of Secondary Metabolites from Trichoderma spp.

Secondary metabolites from cultures of Trichoderma spp. were obtained according to the procedure described by El-Hasan et al. [53]. Briefly, 100 mL of potato dextrose broth (PDB) (Merck, Darmstadt, Germany) in 250 mL Erlenmeyer flasks were inoculated with 15 agar plugs (5 mm ⍉) from fully grown and sporulating T. koningiopsis T10, T. harzianum T16, or T. asperellum T23. As a negative control, agar plugs without fungal growth were used. Each treatment was performed in triplicate. Inoculated Erlenmeyer flasks were incubated at 150 rpm and 21 ± 2 °C in the dark. Based on our previous studies, the incubation time was set to 12 d for T. harzianum T16 and T. asperellum T23 [30,56]. An incubation time of 2 d, previously optimized for T. koningiopsis T10 (unpublished data), was used in this study. Fungal biomass was filtered through sterile filter paper, and the filtrates were extracted twice with 100 mL ethyl acetate (EtOAc; Merck KGaA, Darmstadt, Germany). Organic phases were collected, dehydrated with Na₂SO₄, and subsequently evaporated in a rotary evaporator under reduced pressure (150–170 mbar) at 40 °C. Dried residues were redissolved in 1 mL acetone. Extracts were subsequently sealed airtight and stored at 7 °C in the dark.

2.4. Bioautography Assay

To estimate the antifungal potential of the crude extracts, thin-layer chromatography (TLC) and bioautography procedures described in our previous study [47] were used with modifications. Briefly, 50 µL of the crude extract of Trichoderma spp. T10, T16, and T23 were applied to TLC plates (5 × 5 cm) (Machery-Nagel, Düren, Germany). GM7 extracts and acetone served as controls. Each treatment was carried out in triplicate. TLC plates were sprayed with a total of 30 mL GM7 at approximately 50 °C, followed by a conidial suspension of N. rosae with a concentration of 10⁵ conidia mL⁻¹, using an airbrush compressor (N026, KNF, Freiburg, Germany). Subsequently, TLC plates were incubated in a humidity chamber, at 25 °C, in the dark, for 24 h. Each plate was photographed and fungal growth was determined using ImageJ (v. 1.54d) software (National Institute of Health, Bethesda, MD, USA).

2.5. Poisoned Agar Plug Assay

To further assess the inhibitory effects of crude extracts on growth and sporulation of N. rosae, GM7 petri dishes (35 mm ⍉, Sardstedt, Nümbrecht, Germany) were each inoculated with one centered agar plug (5 mm ⍉) of an actively growing N. rosae culture. Each agar plug was then treated separately with 50 µL crude extract of Trichoderma spp. T10, T16, or T23, respectively. GM7 extract and acetone were used as controls. Switch (Syngenta, Basel, Switzerland) (a.i.: 375 mg g⁻¹ Cyprodinil + 250 mg g⁻¹ Fludioxonil) in the recommended application dosage of 2.5 µg µL⁻¹ was used as a positive control. Each treatment was carried out in triplicate. Inoculated petri dishes were incubated for 5 d at 21 ± 2 °C and 14/10 h (light/dark) cycle. Fungal colonies were then photographed, and the fungal growth and agar coverage rate was determined with the software ImageJ. To assess the effect on sporulation, the initial agar plugs were removed, and conidia formation was determined by adding 2 mL of sterile water to each petri dish. Fungal biomass was scraped off with a sterile spatula and filtered through two layers of sterile gauze, and conidial density was determined using a Fuchs-Rosenthal counting chamber.

2.6. Inhibition of Conidia Viability of N. rosae

To assess the inhibitory effect of crude extracts on the conidial viability of N. rosae, a 2-(3,5-diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole bromide (MTT) assay was utilized. Trichoderma spp. T10, T16, and T23 extracts, and acetone were diluted in liquid GM7 to final concentrations of 1–5% (v/v), in 1% increments. GM7 medium served as the negative control, while Switch (2.5 µg µL⁻¹) in liquid GM7 served as the positive control. Each treatment (100 µL) was pipetted into a flat-bottom 96 well microplate (Greiner, Frickenhausen, Germany). Subsequently, 100 µL of a conidial suspension (3.1 × 10⁵ conidia mL⁻¹) from N. rosae containing 0.2 µL mL⁻¹ Break-Thru S 301 (Evonik Industries, Essen, Germany) was added to each well, resulting in a final volume of 200 µL per well. Three wells were filled with 100 µL of liquid GM7 and 100 µL of conidia suspension from N. rosae without Break-Thru S 301. The remaining wells were filled with 200 µL of liquid GM7 and were used as blanks. The microplate was sealed airtight with Parafilm, placed inside a plastic box with a transparent lid, and incubated for 24 h at 21 ± 2 °C and 14/10 h (light/dark) cycle.

Then, 50 µL MTT solution (2 mg µL⁻¹) prepared in salt buffer was added to each well. The salt buffer consisted of 8 g L⁻¹ NaCl, 0.2 g L⁻¹ KCl, 1.78 g L⁻¹ Na₂PO₄ · 2 H₂O and 0.27 g L⁻¹ KH₂PO₄ in sterile deionized water. After adding the MTT to the salt buffer, the solution was filtered through a 0.2 µm microfilter.

After 1 h of incubation at 37 °C with shaking in double-orbital mode at 100 rpm inside a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany), the absorbance was measured at 570 nm using the orbital (6 mm) mode, with 106 reading points per well.

2.7. Greenhouse Trial

To assess the efficacy of crude extracts of Trichoderma spp. towards N. rosae on strawberry plants and compare their efficacy with the conventional-used live spores biocontrol, two-month-old strawberry plants cv. Herzle were planted in pots (0.34 L) containing soil (SP Topf; Einheitserde Werkverband, Sinntal-Altengronau, Germany) with pH 5.8, 70% organic matter content and 340 mg L⁻¹ N, 260 mg L⁻¹ P₂O₅, 330 mg L⁻¹ K₂O and 100 mg L⁻¹ Mg. Plants were fertilized biweekly with 0.2% Wuxal^® Super (Aglukon, Duesseldorf, Germany).

Crude extracts of Trichoderma spp. T10, T16, and T23 as well as acetone were diluted to 3% (v/v) in sterile water. Five healthy plants were placed in a tray and 2 mL of the respective treatment were pipetted onto the rhizome of each plant. Sterile water and Switch (2.5 µg µL⁻¹) served as the negative and positive controls, respectively. Conidial suspensions of Trichoderma spp. T10, T16, and T23 (10⁷ conidia mL⁻¹) and N. rosae (10⁵ conidia mL⁻¹) were prepared as described in Section 2.3. All conidial suspensions contained 0.02% Break-Thru S 301. To maintain appropriate relative humidity, trays were placed on fleece saturated with water and covered with translucent plastic boxes (l × w × h; 1.8 m × 0.6 m × 0.6 m). Trays were arranged randomly inside the greenhouse cabin. At 24 h intervals, boxes were removed for 8 h, to simulate a daily cycle of humidity. The relative humidity (RH) in the greenhouse cabin varied from 70 to 85%, whereas 100% RH was achieved under the plastic boxes. After seven days, each plant was sprayed with 2 mL of their respective BCA-treatment using an airbrush. Two days after the second BCA application, every plant was sprayed with 2 mL of the N. rosae conidial suspension. The experiment was carried out in triplicate. Plants were incubated for 37 days at 21 ± 2 °C under 14/10 h (light/dark) conditions.

Leaves showing visible signs of infection were scored and the total number of infected and intact leaves of each plant was counted starting two weeks after the inoculation with N. rosae. Scoring was repeated at 7-day intervals for 21 d. Disease symptoms were examined using a digital microscope (VHX-X1; Keyence Deutschland GmbH, Leinfelden-Echterdingen, Germany). The area under disease progress curve (AUDPC) for the developed leaf lesions was calculated using the formula:

$$AUDPC=\sum _{i=1}^{n-1}\left({\displaystyle \frac{y_i+y_{i+1}}{2}}\right){\left(t_{i+1}-t\right)}_1$$

where t is the time of each assessment, y is the percentage of infested plant parts in each assessment, and n is the number of readings. The variable t stands for days [57].

Subsequently, the relative AUDPC (rAUDPC) was derived from the AUDPC using the following formula:

$$rAUDPC={\displaystyle \frac{AUDPC}{\left(t_{max}-t_1\right)\times 100}}$$

2.8. Statistical Analysis

The inhibition of fungal growth, conidia production, and conidia viability were calculated using Abbott’s formula [58]:

$$Inhibition[\%]=\left({\displaystyle \frac{Control-Treatment}{Control}}\right)\times 100$$

Statistical analyses were performed using the GLM procedure in SAS software (version 9.4). A one-factorial analysis of variance (ANOVA) followed by a least-significant difference (LSD) test were used to compare treatment means. Differences between treatments were considered significant at a probability level of p = 0.05.

3. Results

3.1. Mycelial Growth Inhibition of N. rosae on TLC Plates Amended with Trichoderma Extracts

The control and acetone treatments showed no fungal growth inhibition and were statistically similar, confirming that the solvent itself had no antifungal effect within this set up (Figure 1).

Among the Trichoderma extracts, T. asperellum T23 exhibited a pronounced inhibitory effect, producing a small but statistically significant inhibition area ranging between 171.1 ± 13.6 mm². This was followed by the fungicide Switch, which showed a moderate inhibition of about 367.0 ± 59.7 mm². A stronger inhibitory effect was observed with T. harzianum T16, with an inhibition area of 484.4 ± 5.5 mm², while T. koningiopsis T10 produced the highest inhibition of mycelial growth reaching 860.4 ± 68.0 mm².

3.2. Poisoned Agar Plug Assay

The results show clear differences among treatments in their inhibitory effects on mycelial growth and conidia production of N. rosae (Figure 2, Appendix A). Acetone insignificantly inhibited mycelial growth by 2.8 ± 20.9%. When treated with T. asperellum T23 or T. harzianum T16, N. rosae showed low growth inhibition with no significant differences compared to the acetone treatment. On the other hand, in the presence of T. koningiopsis T10 or Switch, N. rosae showed the highest growth inhibition measured by 92.3 ± 4.7% and 96.1 ± 0.9%, respectively. Both latter treatments did not differ significantly from each other.

Concentrations of conidia of N. rosae were highest in the GM7 control and acetone treatments at 4.2 × 10⁶ conidia mL⁻¹ and 3.9 × 10⁶ conidia mL⁻¹, respectively. While T. harzianum T16 and T. asperellum T23 led to a moderate spore formation inhibition (45.0 ± 38.3% and 62.7 ± 20.6%, respectively), N. rosae sporulation was almost completely inhibited when treated with T. koningiopsis T10 (96.5 ± 4.8%) or Switch (99.9 ± 0.0%).

In general, conidia production appeared to be more sensitive than mycelial growth to T. harzianum T16 and T. asperellum T23 extracts, while T. koningiopsis T10 extract and Switch were highly effective against both parameters.

3.3. Inhibition of Conidia Viability of N. rosae by Trichoderma Crude Extracts

The effect of Trichoderma spp. extracts on conidia viability of N. rosae in liquid culture was evaluated using the MTT assay at concentrations ranging from 1 to 5% and compared with the acetone control at equivalent concentrations. Conidia viability in the acetone control remained consistently high across all tested concentrations, with values above 90% at 1–3% acetone and only a slight decrease observed at higher concentrations (85.1 ± 4.1% at 5%, Figure 3). This indicates that acetone, even at increasing concentrations, had minimal inhibitory effects on conidia viability of N. rosae. In contrast, all Trichoderma spp. extracts significantly reduced conidia viability relative to the acetone control in a concentration-dependent manner. Extracts of T. asperellum T23 caused a moderate but consistent inhibition, with viability decreasing from 85.1 ± 1.7% at 1% to 73.5 ± 3.9% at 5%. T. harzianum T16 extracts proved to be significantly more effective in inhibiting conidia viability, showing a progressive reduction in viability, decreasing from 83.3 ± 3.4% at 1% to 58.3 ± 2.5% at 5%. However, extracts of T. koningiopsis T10 exhibited the strongest inhibitory effect, ranging between 44.4 ± 0.8% and 37.5 ± 3.6% depending on their concentration.

3.4. In Planta Assesment of the Effects of Crude Extracts and Conidia of Trichoderma spp. Against N. rosae

Leaves infected with N. rosae exhibited characteristic disease symptoms, including brown leaf spots, discoloration of leaf tissue, leaf curling (Figure 4A–C), and formation of characteristic black acervuli (Figure 4D,E) and conidia (Figure 4E,F), confirming the efficacy of the inoculation method and the identity of N. rosae.

Leaf infestation by N. rosae, expressed as rAUDPC (%) at 28 days after inoculation (dpi), was significantly affected by the different treatments. The infected control showed a high level of disease development (31.1 ± 22.0%), whereas the healthy control exhibited the lowest rAUDPC values (16.3 ± 15.0%; Figure 5).

Conidial (C) treatments of Trichoderma spp. (T10C, T16C, and T23C), as well as the extract (E) treatment T16E, produced intermediate rAUDPC values ranging between 23.8% and 27.5%. These treatments insignificantly reduced leaf infestation and remained significantly higher than the healthy control.

Among the extract treatments, clear differences were observed. The application of acetone and T23E resulted in relatively high rAUDPC values (35.8 ± 16.0% and 37.0 ± 25.0%, respectively) and did not differ significantly from the infected control, indicating no suppressive effect on disease development. In contrast, treatment with T10E significantly (20.5 ± 11.0%) reduced disease severity and was among the most effective extract treatments. The fungicide treatment (Switch) resulted in a strong reduction of disease development (17.0 ± 16.0%) and differed insignificantly from the healthy control, indicating high efficacy under the experimental conditions.

4. Discussion

The control of Neopestalotiopsis species, particularly N. rosae and N. clavispora, has become a critical priority due to their emergence as destructive pathogens causing root, crown, and fruit rot in strawberries and flower blight in ornamental plants [59]. Conventional management of these pathogens relies largely on chemical fungicides, which often fail to provide complete control and are associated with resistance development and potential health and environmental risks [60]. Consequently, research is shifting toward more effective and sustainable alternatives for integrated disease management [31,38,61,62,63].

In this study, we assessed the antifungal activity of Trichoderma-derived metabolites in vitro and evaluated their performance in planta.

In the direct-contact TLC assay, extracts of T. koningiopsis T10, T. harzianum T16, and T. asperellum T23 all inhibited mycelial growth of N. rosae, confirming antibiosis as an important mode of action (Figure 1). Among these, T. koningiopsis T10 produced inhibition zones that were 2.5-fold larger than those of the reference fungicide Switch, indicating a particularly strong antifungal effect. Bacterial BCAs including Bacillus cereus, B. amyloliquefaciens, and B. methylotrophicus inhibited N. clavispora growth by about 60–79% in vitro [64], which is lower than the very strong inhibition observed for T. koningiopsis T10 extracts in our experiments. Similarly, cell-free culture filtrates of three Bacillus strains (two B. velezensis and one B. subtilis) significantly suppressed N. clavispora in vitro, with inhibition ranging from 63.3% to 73.6%. In planta, application of these antagonists markedly reduced root rot severity (up to 67%) and enhanced superoxide dismutase, peroxidase, and catalase activities in leaves, indicating that their cell-free metabolites act in combination with induced host resistance [65].

Comparable patterns of strong in vitro inhibition have been observed with botanical extracts: clove (Syzygium aromaticum) and turmeric (Curcuma longa) extracts completely suppressed mycelial growth of Neopestalotiopsis sp. and Pseudopestalotiopsis sp., whereas ginger, lemongrass, and roselle caused only partial inhibition and mangosteen extract only had a minimal effect [66].

When mycelial growth was quantified following the amendment of the agar plugs of the pathogen with Trichoderma extracts, T. koningiopsis T10 achieved 92.3% inhibition of N. rosae after five days, approaching the 96.2% inhibition obtained with the reference fungicide (Figure 2, Appendix A). In contrast, T. harzianum T16 and T. asperellum T23 extracts caused only moderate growth suppression. In vitro, volatile and non-volatile metabolites produced by T. asperellum CMT10 exhibited strong antifungal activity against N. clavispora, inhibiting mycelial growth by 79.7% and 69.8%, respectively [67]. Comparable strain-dependent variability has been reported for Trichoderma isolates antagonizing Fusarium sp., Phytophthora sp., and Botrytis spp. [47,56].

Using botanicals, high in vitro efficacy has been observed: cinnamon essential oil nanoemulsions inhibited N. rosae growth at high dilutions and completely suppressed leaf spot severity [68].

We also quantified the effect of the Trichoderma extracts on N. rosae sporulation. T. harzianum T16 and T. asperellum T23 extracts reduced conidia production by 45% and 62.7%, respectively, whereas T. koningiopsis T10 extracts suppressed conidiation by 96.5%, close to the 99.9% reduction achieved by the reference fungicide (Figure 2). Since conidia are the primary propagules driving Neopestalotiopsis epidemics [42], such strong inhibition of conidia formation is highly relevant for disease management. Similar effects on sporulation have been documented for other Trichoderma-based BCAs: extracts of Trichoderma spp., for example, markedly inhibited sporulation of Fusarium moniliforme and Phakopsora pachyrhizi, resulting in reduced lesion development and lower disease severity [52,53]. Although explicit quantification of conidia reduction is less commonly reported, B. cereus reduced sporulation indirectly by impairing mycelial growth [7]. Compared with botanical agents, our data are similar in magnitude to clove and turmeric extracts, which fully suppressed Neopestalotiopsis and Pseudopestalotiopsis growth at high concentrations [66].

In the conidia viability assay, T. koningiopsis T10 extracts inhibited N. rosae germination by 62.5%, whereas T. harzianum T16 and T. asperellum T23 showed a different efficacy ranking compared to in the sporulation assay, indicating that conidia production and viability are differentially sensitive targets. At the same time, we observed that acetone concentrations of 4% or higher significantly suppressed viability in the MTT assay, meaning that Trichoderma extract efficacy could be reliably evaluated only up to a 3% extract concentration. By comparison, Switch was the only treatment in our study that completely inhibited N. rosae conidial viability, which is consistent with its known multi-stage activity against several strawberry pathogens [1,13,14,59].

Considering the limitations of in vitro assays in capturing the complexity of pathogen–host interactions, both live inocula and crude extracts of Trichoderma spp. were evaluated in planta under greenhouse conditions. Our results show that conidial suspensions only moderately and not significantly reduced N. rosae leaf spot. This outcome suggests that under our specific conditions (Trichoderma strain, inoculum density, timing, environmental parameters, and host cultivar), live inocula did not establish or act sufficiently to provide robust protection. In contrast, T. koningiopsis T10 crude extracts significantly suppressed N. rosae leaf spot severity, reaching control levels comparable to the fungicide Switch (Figure 5). This finding is particularly notable because Switch is regarded as one of the most effective fungicides against Neopestalotiopsis and other strawberry pathogens, with proven efficacy in reducing mycelial growth, sporulation, and infection in field and greenhouse settings [15,69]. Our results show that crude extracts from T. koningiopsis T10 can translate their pronounced in vitro activity into effective in planta disease control. This stands in line with several reports where Trichoderma spp. have shown strong in planta effects. For example, T. asperellum inhibited N. clavispora by 66.7% in vitro and reduced disease severity by 75.8% on strawberry, performing similarly to chemical fungicides [70]. Acosta-González et al. [69] also demonstrated that Trichoderma spp. significantly suppressed N. rosae incidence in strawberry. Similarly, CMT10 of T. asperellum achieved 63.1% biocontrol efficacy against strawberry root rot caused by N. clavispora and concurrently promoted plant growth under greenhouse conditions [67].

Similar extract-based in planta efficacy has also been reported for botanicals: cinnamon essential oil nanoemulsions reduced leaf spot severity by nearly 80% without phytotoxicity [68], and clove and turmeric extracts have shown strong protection of fruits and foliage against Pestalotiopsis-like fungi at high concentrations [66]. Moreover, bacterial BCAs such as B. cereus and B. amyloliquefaciens also reduced disease incidence and severity, e.g. B. cereus Bce-2 lowered N. clavispora leaf spot while strongly activating host defense enzymes, and B. amyloliquefaciens isolates reduced strawberry root rot by >57% and promoted plant growth, but their suppression levels are often slightly lower than those achieved by the synthetic fungicides [7].

Taken together, our results show that crude extract of T. koningiopsis T10 exhibits very strong in vitro activity against N. rosae (on mycelial growth, sporulation, and, to a lesser extent, conidial germination) and can provide in planta disease control equivalent to a commercial fungicide, whereas Trichoderma inocula were not significantly effective under the conditions tested. The effective control of N. rosae by fungicides such as Switch may contribute to its currently low incidence in German strawberry cultivation. However, problems could arise in the future if fungicide performance declines, or N. rosae develops resistance [25,26]. Nevertheless, comparable effectiveness in suppressing N. rosae was shown by T. koningiopsis T10 extracts, suggesting their potential as a suitable alternative to conventional chemical fungicides.

5. Conclusions

The emergence of novel pathogens poses a significant threat to the cultivation of strawberries. Continuous reliance on chemical fungicides has detrimental environmental consequences and is not a sustainable practice over an extended period. This study thus focused on the identification of biological control agents against N. rosae. The results of this study demonstrate that extracts from Trichoderma spp. can effectively inhibit growth and spore formation of N. rosae. Among the isolates tested, T. koningiopsis T10 exhibited efficacy comparable to the reference fungicide (Switch) in terms of its impact on mycelial growth, spore formation, and conidia viability of N. rosae. In greenhouse trials with strawberry plants, T. koningiopsis T10 was likewise as effective as Switch in preventing strawberry leaf infestation.