In Vitro Sensitivity of Isolates of Neopestalotiopsis rosae, Causal Agent of Strawberry Crown Rot, to Usnic Acid

Abstract

Root and crown rot in strawberries caused by Neopestalotiopsis rosae (N. rosae) results in yield losses of approximately 70%. The main method of control is based on the application of fungicides; however, the excessive use of these products can induce resistance by pathogens to the active ingredients. The use of secondary metabolites is an alternative to disease management. Usnic acid (UA), a secondary metabolite produced by lichens, has shown antimicrobial and antifungal activities that could be useful for the management of phytopathogens, particularly the (+) enantiomer. To provide alternatives to fungicides, the potential of UA as an alternative for N. rosae management was evaluated under in vitro and in vivo conditions. Using the “poisoned medium” technique, concentrations of 0 (UA0), 100 (UA1), 200 (UA2), and 400 (UA4) µg/mL UA at a dose of 2.5 mL/L PDA were evaluated on N. rosae mycelial growth and the number of spores. The UA at 400 µg/mL exhibited a fungistatic effect, reducing the mycelial growth of isolates of N. rosae in 50–60%. In the in vivo assay, sprayed UA (400 µg/mL) reduced hydrogen peroxide (48.59%) and malonaldehyde (77.62%) contents in “Albion” strawberry seedlings inoculated with 466 and FREC2 strains, respectively. These findings suggest that UA could be a potential tool for N. rosae management and could help mitigate the oxidative stress induced by infection. However, field trials are required to evaluate and validate this response.

1. Introduction

Strawberry is a crop with a high demand in the world market because of its high nutritional value and wide use in the food industry [1]. The main strawberry-producing countries are China, United States, Turkey, and Egypt. Mexico is ranked 5th at 641,552 tons, with Michoacán being the leading state in production, accounting for 58.7% of the national total [2].

Fungal diseases are among the main factors limiting strawberry yield and postharvest life [3,4]. Approximately 43 species of fungi have been reported to affect crops, among which Alternaria spp., Botrytis cinerea, Colletotrichum acutatum, Rhizoctonia spp., Verticillium spp., Fusarium oxysporum, and Neopestalotiopsis rosae (N. rosae) are the most important because of their incidence and level of damage [5,6,7,8]. N. rosae, the causal agent of crown and root rot in strawberries, is a recent disease with a significant impact on production, as it can cause losses of up to 70% [6,9]. N. rosae has recently been reported in Taiwan, Egypt, China, the United States, Germany, and Italy [4,10,11,12,13,14]. In Mexico, the presence of this pathogen has been reported in the municipalities of Zamora and Jacona in Michoacán [6].

This fungus affects the vascular tissues of the root and crown, causing rotting, wilting, yellowing, and necrotic spots on the leaves, which eventually intensify and cause plant death. These symptoms have been reported in strawberry seedlings established both in open field conditions and in tunnels, at temperatures between 23 and 27 °C. The incidence and spread of the disease are favored by the presence of rain and high foliar humidity [6].

Because this disease is a recent occurrence in strawberry cultivation, there is little information available on its management or soil fumigation, and applications of preventive and curative fungicides are usually used, such as prochloraz, cyprodinil + fludioxanil, and pidiflumetofen + fludioxanil, which exhibited 99–100% efficacy when applied preventively to strawberry plants under greenhouse conditions [9]. These ingredients inhibit the synthesis of methionine, ergosterol, and glycerol, enzymes involved in fungal metabolism and respiration, and mechanisms related to the germination and growth of the germ tube of conidia [15]. The excessive use of these chemical molecules can induce resistance in pathogens, and the persistence of fungicides can negatively affect human health and the environment [3,16]. Strawberries are a product of fresh consumption destined for export and must meet quality and safety standards, which require the use of products with low levels of toxic residues [1]. Given the scarcity of management alternatives, it is necessary to propose and evaluate new methods to help counteract the damage caused by N. rosae in strawberries. One of these is the use of secondary metabolites, as it has been reported that some of them inhibit mycelial growth in certain fungi and phytopathogenic oomycetes or improve plant resistance to diseases [17].

Several studies have reported the positive effects of the use of secondary metabolites in the control of phytopathogens. Petrovic et al. [18] reported the effectiveness of carvacrol and 0.1% e-cinnamaldehyde (10 μg/10 mL) in inhibiting mycelial growth in 100% of Botryosphaeria dothiodea and Diplodia mutila, Diplodia seriata, Dothiorella iberica, and Neofosicoccum parvum under in vitro conditions.

Another example is the study by Pham et al. [19], who reported the antifungal activity of rhein obtained from Cassia alata in inhibiting the mycelial growth of Phytophthora spp. by 57.1% at a concentration of 150 μg/mL under in vitro conditions. Additionally, late blight was controlled by 87.9% to 300 μg/mL in tomato seedlings in vivo. Hussain et al. [20] reported the antifungal activity of alkaloids, flavonoids, and terpenoids obtained from Carthamus tinctorius at a concentration of 20 mg/mL by inhibiting the mycelial growth of Aspergillus spp. by 95, 91, and 96%, respectively.

In this context, usnic acid (UA), a secondary metabolite produced by lichens of the genera Cladonia, Alectoria, Usnea, Lecanora, Ramalina, and Evernia [21,22], has been shown to have antimicrobial and antifungal activities that may be useful for agricultural production in disease management [22]. However, most studies have focused on pathogens related to the medical field, with few focusing on agricultural pathogens.

Paguirigan et al. [23] reported the strong antibacterial and antifungal activity of UA by inhibiting the growth of Clavibacter michiganensis subsp. michiganensis, exhibiting a minimum inhibitory concentration (MIC) of 7.8 μg/mL and the mycelial growth of Diaporthe eres and D. actnidiae at MICs of 18.07 and 1.47 μg/mL under in vitro conditions. Similarly, UA reduced the growth of Burkholderia cepacia by 90% at an MIC of 12.5 μg/mL [24]. Similarly, Cintra et al. [25] observed a bactericidal effect of UA on Xanthomonas axonopodis and Xanthomonas campestris, with MIC values of 3.12 μg/mL and 6.23 μg/mL, respectively.

Therefore, the objective of this study was to evaluate the potential of UA to inhibit the mycelial growth of N. rosae under in vitro conditions. The possible results can potentially be used as a strategy in the integrated management of the disease, after the validation of its behavior in open field and protected agricultural conditions.

2. Materials and Methods

2.1. Fungal Isolates

Five fungal isolates (FREC2, FREC, 372, 323, and 466), morphologically and molecularly identified as N. rosae, were provided by Rebollar-Alviter et al. [6] (Regional University Center Universitario Centro Occidente, of the Autonomous University of Chapingo). The isolates were stored in potato dextrose agar (PDA, MCD LAB. S.A. de C.V., Oaxaca, Mexico) medium and refrigerated at 4 °C until use. For in vitro assays, the isolates were reactivated on PDA and placed in an incubator (ECOSHEL 9045, Científica Vela Quin S. de R.L de C.V, México) at 23 °C in the dark for 7 days (d).

2.2. In Vitro Sensitivity Assay of N. rosae Isolates to Usnic Acid

In vitro tests were used to evaluate the sensitivity of N. rosae isolates to different concentrations of UA (400, 200, and 100 µg/mL), coded as UA4, UA2, UA1, and the control UA0 (potato dextrose agar, PDA). The usnic acid used was a (+) enantiomer (Sigma-Aldrich Inc., St. Louis, MO, USA). A chemical control (MiravisTM Prime fungicide of Syngenta^®; pidyflumetofen-fludioxonil + PDA) and a control with solvent (acetone + PDA) were used. Different concentrations of UA (+) were prepared from 400 µg/mL stock solutions. For the preparation, 12 mg of AU was weighed and dissolved in acetone (30 mL). After dilution, the concentrations were determined (C₁V₁ = C₂V₂). For each UA concentration, a dose of 2.5 mL/L was added to the previously sterilized PDA medium and poured into 90 mm Petri dishes. The same dose was used for the fungicide and solvent.

Once the medium was solidified, a 5 mm explant of each N. rosae isolate was placed in each Petri dish with the medium and doses of UA, fungicide, and solvent. The Petri dishes were placed in an incubation oven at 23 °C for 7 d under dark conditions.

The experimental design was a completely randomized factorial with two factors: A (N. rosae isolate) with 5 levels and B (UA concentration) with 4 levels, giving a total of 30 treatments, including a solvent and a chemical control; four replications were carried out and a Petri dish was used as the experimental unit.

2.3. Mycelial Growth and Percentage of Inhibition (%)

Mycelial growth was measured daily with a digital caliper (Sure Bilt^®, Best Parts, Inc. Memphis, TN, USA), taking north–south and east–west measurements, and the average mycelial diameter developed was determined. Measurements were performed until the mycelial growth of N. rosae isolates from the control (UA0) covered the total diameter of the Petri dish. The experiment was performed in triplicate. The percentage of growth inhibition was determined using Abbott’s formula [26]:

$$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}={\displaystyle \frac{(mycelialgrowthcontrol-mycelialgrowthtreament)}{mycelialgrowthcontrol}}\times 100$$

Using the inhibition percentages, the mean inhibitory concentration (CI₅₀) was determined by probit analysis.

2.4. Final Sporulation

When the control (UA0) showed sporulation (37 d), spore suspensions were prepared. Briefly, 10 mL of sterile distilled water was added to each Petri dish, and the mycelium was scraped and homogenized with water using a sterile glass rod to obtain a suspension [27]. The spore suspension was placed in sterile 15 mL Falcon tubes, and for spore counting, 20 μL of the suspension was collected and deposited in a hemocytometer, which was visualized under a microscope (VELAB^® Model VE-BC3 PLUS, TX, USA) with a 10× objective. The number of spores was determined by averaging six counts, and the data were expressed as the number of spores per mL.

2.5. In Vivo Assay of Usnic Acid in Strawberry Seedlings Inoculated with N. rosae Isolates

Based on the results of the in vitro test, 400 µg/mL was selected to determine its effect on strawberry seedlings inoculated with N. rosae isolates under controlled conditions. Strawberry seedlings of the “Albion” variety were placed in polystyrene containers with peatmoss/perlite substrate with a 1:1, v/v, ratio. Seedlings were placed in a growth chamber at 25 °C and 66% illumination with a 12 h light/12 h dark cycle and a photon flux density of 25 ± 5 µmol m⁻² s⁻¹ (Nambei NRQ250, Henan, China) Fertilization was performed on Steiner’s nutrient solution [28] at 100%, with an electrical conductivity of 1.7 mS m⁻¹ and a pH of 5.8. Two applications of UA were made via foliar application: the first one prior to inoculation at stage BBCH-16, and the second one 7 d after inoculation. For the in vivo assay, UA was prepared according to Lechowski et al. [29] at pH 6.7–6.8 with a dispersant (PenaTrex^® AGROformuladora Delta’ S.A. de C.V., Mexico) and sprayed to the dew point (5 mL/plant).

Inoculation was performed during the BBCH-16 stage (Biologische Bundesanstalt, Bundessortenamt, und Chemische Industrie), six true leaves unfolded (1: leaf development; 6: six true leaves unfolded) [30]. Mycelial disks (5 mm diameter) from the four 19-day old isolates, grown on PDA medium at 23 °C under dark conditions were used. The surface to be inoculated was disinfected with 70% alcohol, and then punctures were made with the help of a sterile syringe needle (3 mL) to cause wounds at the base of the crown. Three disks of mycelia were placed on the wounds and covered with a plastic film to prevent the dehydration of the inoculum. Ten days after inoculation, when the control showed symptoms of the disease, the severity was determined [9], and destructive sampling was carried out on the root and crown, which were placed on ice, frozen at 20 °C, and then freeze-dried (Freeze Dryer, model ECO-FD10PT, Biobase Meihua Trading Co., Ltd. Shandong, China). Hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and phenylalanine ammonia lyase (PAL) contents were determined in the lyophilized tissue.

The experimental design was completely randomized, with 20 treatments, including a chemical control, a control without usnic acid (UA0), and a control with the solvent used, with three replicates and one seedling as the experimental unit, for a total of 60 seedlings (

Table 1. Treatments evaluated on the in vivo activity of (+) usnic acid in strawberry seedlings inoculated with different N. rosae isolates.

Code	Treatment	Code	Treatment
T1	FREC2 + UA 400 µg/mL	T11	323 + NaOH (solvent UA)
T2	FREC2 + Chemical control	T12	323 + distilled water
T3	FREC2 + NaOH (solvent UA)	T13	372 + UA 400 µg/mL
T4	FREC2 + distilled water	T14	372 + Chemical control
T5	FREC + UA 400 µg/mL	T15	372 + NaOH (solvent UA)
T6	FREC + Chemical control	T16	372 + distilled water
T7	FREC + NaOH (solvent UA)	T17	466 + UA 400 µg/mL
T8	FREC + distilled water	T18	466 + Chemical control
T9	323 + UA 400 µg/mL	T19	466 + NaOH (solvent UA)
T10	323 + Chemical control	T20	466 + distilled water

).

2.6. H₂O₂ Determination

H₂O₂ content was determined according to the methodology described by Velikova et al. [31]. Briefly, 1 mL of 0.1% cold trichloroacetic acid (TCA) was added to 10 mg of lyophilized sample and centrifuged (Microcentrifuge Frontier™ FC5515R, Ohaus Corporation, Parsippany, NJ, USA) for 15 min at 12,000× g at 4 °C. Then, 125 μL of the supernatant was recovered and placed in an Eppendorf tube, and 375 μL of cold potassium phosphate buffer (pH 7) and 500 μL of potassium iodide (KI; 1 M) were added. The absorbance of the samples was read using a spectrophotometer (ME-UV1800; MesuLab Instruments Co., Ltd., Guangzhou City, China) at 390 nanometers (nm).

2.7. MDA Determination

The MDA content (nmol g⁻¹ DW) was determined using 50 mg of lyophilized tissue to which 1.25 mL of TCA (0.1%) was added. The samples were centrifuged for 15 min at 10,000 rpm at 4 °C. The supernatant (250 μL) was recovered and placed in an Eppendorf tube by adding 500 μL of a 0.5% TCA/20% thiobarbituric acid (TBA, Cayman Chemical, Michigan, USA) mixture and placed in a water bath (100 °C) for 20 min. Samples were then passed through a cold bath and centrifuged at 6000 rpm for 10 min at 4 °C. The samples were read using a spectrophotometer at 532 and 600 nm [31]. The results were recorded as thiobarbituric acid-reactive substances (TBARS), which represent the MDA equivalents. The MDA concentration was evaluated using the following formula:

$$MDA=(A_{532}-A_{600})\times FW\times V/\epsilon \times 1000$$

A₅₃₂ is the absorbance at 532 nm;

A₆₀₀ is the absorbance at 600 nm;

FW is the sample weight (g);

V is the reaction volume (mL);

ε = extinction coefficient of the complex MDA-TBA = 155 mM⁻¹ cm⁻¹.

2.8. Phenylalanine Ammonia Lyase Activity (EC. 4.3.1.5)

PAL activity was evaluated according to the method of Syklowska-Baranek et al. [32] with some modifications. The enzymatic extract was obtained by homogenizing 50 mg of lyophilized tissue with 5 mg of polyvinyl pyrrolidone (PVP) and 1.5 mL of phosphate buffer pH 7–7.2 (0.1 M) and sonicating (sonicator Baku BK 2000, Guangzhou, Guangdong, China) for 5 min. After centrifugation at 12,500 rpm for 10 min at 4 °C, the supernatant was collected, filtered, diluted at 1:20 in phosphate buffer, and stored at 4 °C. To determine enzyme activity, 25 μL of enzyme extract was taken and 225 μL of phenylalanine (6 mM prepared in phosphate buffer) was added. The samples were incubated in a water bath (40 °C) for 30 min, after which 62.5 μL of HCl (5 N prepared in phosphate buffer) was added. Finally, the samples were placed in a cold bath for 2 min and 1.25 mL of distilled water was added. Readings were recorded at 260 nm. PAL activity was determined using a trans-cinnamic acid straight-line equation (trans-cinnamic acid, ε = 9000 M mL⁻¹).

2.9. Statistical Analysis

The data obtained from the in vitro mycelial growth inhibition test were subjected to a probit analysis using the Minitab 2019 software. Likewise, the data on in vitro mycelial growth inhibition and severity of N. rosae on strawberry seedlings were transformed (Asin) [33] and subjected to a two-way analysis of variance and Tukey’s test of means, as well as data on MDA, H₂O₂, and PAL variables. Finally, mycelial growth data over time were analyzed using Friedman’s test. Analyses were performed at a confidence level of 0.05 using the InfoStat program v. 2020I.

3. Results

3.1. Mycelial Growth and Percentage of Inhibition (%)

The effect of UA concentration on mycelial growth of the N. rosae isolates FREC2, FREC, 372, 323, and 466 is shown in Figure 1. In general, UA4 showed the highest percentage of inhibition (58, 52, 61, 58, and 50%) in all isolates showing significant differences with respect to the control (UA0). The chemical control was the most effective treatment, with inhibition percentages above 80% in all isolates, showing significant differences with respect to the rest of the treatments (p < 0.05). The differences in the percentage inhibition between treatments were influenced by the fungal isolate. For example, in isolate FREC2, treatment with UA4 achieved a mycelial growth inhibition of 58%, while in isolate 466, that same concentration only reached about 50%. This shows that isolates respond differently to the same treatment. The results indicate that the efficacy of UA depends on both the treatment applied and the type of isolate, and that the combination of both factors influences the level of inhibition observed. Therefore, it is important to consider these variations when evaluating the antifungal potential of the compound.

In addition, it is important to mention that the % inhibition at all UA concentrations decreased over time, so that the isolates eventually filled the Petri dish, suggesting that UA slowed the growth rate.

3.2. Mean Inhibitory Concentration

The mean inhibitory concentrations (IC₅₀) determined for N. rosae isolates are listed in

Table 2. Mean inhibitory concentrations of N. rosae isolates exposed to different concentrations of usnic acid.

Isolate	* IC50 (μg/mL)	Fiducial Limits (Lower-Upper)	Probit Equation	p-Value
FREC2	247.112	217.842–277.984	Y= −0.350276 + 0.0014175 X	0.000
FREC	354.327	300.423–449.182	Y= −0.305939 + 0.0008634 X	0.000
372	255.439	231.684–280.885	Y= −0.444146 + 0.0017388 X	0.000
323	254.278	227.583–282.972	Y= −0.393141 + 0.0015461 X	0.000
466	385.798	350.936–433.877	Y= −0.623589 + 0.0016164 X	0.000

* Mean inhibitory concentration.

. The isolate most susceptible to UA activity was FREC2, with an IC₅₀ of 247.112 μg/mL, followed by 323 and 372. In contrast, isolates 466 and FREC exhibited higher IC₅₀ values of 385.798 and 354.327 μg/mL, respectively.

3.3. Mycelial Growth of N. rosae Exposed to Different Concentrations of Usnic Acid

Figure 2a–e shows the mycelial growth of the isolates during the 7 d of incubation with different UA concentrations. The mycelial growth of the isolates decreased as a function of UA concentration and remained below that of the control (UA0) (p < 0.05). The chemical control presented the lowest mycelial growth in the first 5 d of the experiment; however, at the end of this period, the growth was similar to that observed in UA4 (400 µg/mL).

Figure 3 shows the final growth of the isolates with the different treatments (7 d). Figure 4 shows the microphotographs of possible damage to cell structures of N. rosae under usnic acid applications. In general, in the UA treatments, the pigmentation and deformation of the conidia and their appendages were affected, as well as alterations in the morphology of the hyphae (Figure 4a,b,e). Chemical control also affected the morphology of hyphae and conidia (Figure 4d,g), which is in contrast to UA0 (Figure 4c,f), where the structures do not show visible alterations at the light microscopic level.

3.4. Sporulation

The number of spores varied according to the isolate and UA concentration (

Table 3. Number of spores of isolated N. rosae exposed to different concentrations of usnic acid.

Isolated	FREC2	FREC	372	323	466
Treatment (μg/mL)	No. Spores mL−1
400	2.4 × 104 a	3.0 × 103 a	1.2 × 105 a	1.1 × 103 a	1.3 × 105 a
200	1.0 × 104 a	6.2 × 103 a	2.5 × 105 a	9.6 × 104 a	6.7 × 104 a
100	5.7 × 104 a	5.0 × 104 a	6.5 × 104 a	7.5 × 102 a	2.7 × 104 a
0	1.6 × 104 a	2.3 × 105 a	7.3 × 104 a	7.4 × 104 a	8.5 × 104 a
Solvent	5.4 × 104 a	1.5 × 105 a	6.4 × 104 a	9.6 × 104 a	3.2 × 105 a
Chemical control	1.3 × 104 a	4.5 × 104 a	5.5 × 104 a	4.6 × 105 a	2.6 × 103 a

Isolate p = 0.3795; treatment p = 0.4961; isolate × treatment p = 0.0497. Equal letters in columns are not significantly different.

). For isolate FREC2, the 200 µg/mL concentration presented the lowest number of spores (1 × 10⁴) and was statistically equal to the UA0 treatment (1.6 × 10⁴) and chemical control (1.3 × 10⁴). In the case of FREC, the number of spores was 98.6% lower at 400 µg/mL compared to the UA0 but without statistical significance. In isolates 372 and 323, there was no statistically significant difference between treatments. Finally, for isolate 466, the chemical control showed a lower number of spores compared to the other treatments but was statistically equal at concentrations of 200 and 100 µg/mL.

3.5. H₂O₂ Content in Strawberry Plants cv. Albion Inoculated with N. rosae and Exposed to Different Concentrations of Usnic Acid

The H₂O₂ content is shown in Figure 5. The behavior of the isolates showed that 466, 372, and 323 in the control appear to be more aggressive by presenting 39.28%, 39.88%, and 23.80% of H₂O₂ with respect to FREC and FREC2 (mean 1.68 µmol g⁻¹ DW in the control). In general, the application of UA significantly reduced the accumulation of H₂O₂ in isolates 323 and 466, in contrast to the chemical control, which showed the highest values. In isolates (FREC2, FREC, and 372), no significant differences were observed between treatments, although the trend of lower H₂O₂ content with UA was maintained. In particular, it was found that the solvent used induced high levels of H₂O₂ because of the high concentration of NaOH (40%). These results indicate that H₂O₂ content varies according to isolate and treatment applied, suggesting a significant interaction between treatment and isolates (p < 0.05).

3.6. MDA Content in Strawberry Plants cv. Albion Inoculated with N. rosae and Exposed to Different Concentrations of Usnic Acid

The MDA content varied significantly as a function of the isolate and the treatment applied (p < 0.05) (Figure 6). In general, plants inoculated with 466 plants under the four treatments exhibited lower MDA accumulation. The UA treatment and chemical control showed no statistically significant difference between them, except for the FREC2, FREC, and 323 isolates (p < 0.05). The FREC isolate, in combination with the chemical control, showed a lower MDA content.

3.7. Phenylalanine Ammonia-Lyase Activity (PAL) in Strawberry cv. Albion Inoculated with N. rosae and Exposed to Different Concentrations of Usnic Acid

PAL activity varied according to the isolate and treatment (p < 0.05) (Figure 7). In plants inoculated with FREC2 in the fungicide treatment, PAL activity increased by 11.96% compared to the control, whereas AU reduced it by 17.95%. At 372, both fungicide and UA promoted notable increases in PAL compared to the control. In contrast, in FREC and 323, UA reduced PAL activity by 39.46% compared with that in the controls (p > 0.05). For plants inoculated with 466, the reduction was more pronounced (82.72%) than that in the control.

3.8. Severity Percentage in Strawberry Plants cv. Albion Inoculated with N. rosae and Exposed to Different Concentrations of Usnic Acid

The severity percentages are shown in Figure 8 (Figures S1–S5), where significant differences were observed according to the combination of isolate and treatment (p < 0.05). In general, the chemical control showed a notable reduction in severity in most isolates, specifically in FREC2, where the lowest percentage (20%) was observed (p < 0.05). In contrast, in AU, the severity reached levels of up to 80%. NaOH also promoted high severity percentages (100%), particularly for isolate 466 (Figure 8).

4. Discussion

4.1. Mycelial Growth and Percentage of Inhibition (%)

Fungal diseases, the emergence of new pathogens and resistance to fungicides are a limiting factor in agricultural production. Therefore, the search for new antifungal substances that inhibit the growth and sporulation of phytopathogenic fungi has increased in recent years [23]. In this context, the antifungal effects of some metabolites of lichenic origin, such as UA, have recently been evaluated. However, most reports have focused on fungi of clinical importance, thus limiting studies on agricultural pathogens. The present study shows the potential of UA in the inhibition of mycelial growth of different isolates of N. rosae, the causal agent of root and crown rot in strawberries. As shown in Figure 1, the highest inhibition percentage was obtained at a concentration of 400 μg/mL (UA4) at a dose of 2.5 mL/L. In this case, UA decreased the growth rate of the isolates but did not inhibit it by 100%; therefore, its effect can be considered fungistatic.

Although the mode of action of UA as a fungicide and fungistatic is not entirely clear, it could be due to (i) inhibition of nucleic acid synthesis, as observed in bacteria [34], and/or (ii) the inhibition of 4-hydroxyl-phenyl pyruvate dioxygenase (HPPD) [35,36]. The inhibition of nucleic acid synthesis (RNA and DNA) interrupts the processes of replication, transcription, and gene translation. Consequently, protein synthesis and cell division processes are affected, which negatively affects fungal mycelial growth and development [37].

Regarding HPPD, it is a widely researched target in the development of pesticides, particularly herbicides, and recently, in fungicides [38]. This enzyme participates in tyrosine metabolism by converting 4-hydroxy-phenyl pyruvic acid (HPPA) to homogentisic acid (HGA) for melanin synthesis in fungi [38,39]. Melanin is a pigment that confers protection and resistance to fungi against factors such as UV radiation, drought, extreme temperatures, and oxidative stress, thereby maintaining cell stability and integrity [40]. In addition, it facilitates the penetration, colonization, establishment, and dissemination of the pathogen in the host tissues [41] and is also associated with fungicide resistance because of its antioxidant capacity and the structural reinforcement of cell walls [42]. Therefore, the inhibition of HPPD could affect melanin synthesis and increase the susceptibility of fungi to antifungal substances by negatively affecting mycelial growth.

Wang et al. [43] reported that the compound 9-phenanthrol disrupting the expression of HPPD of Magnaporthe oryzae. Similarly, nitisinone [2-(2-nitro-4-trifluorometilbenzoil)-ciclohexano-1,3-diona], a specific inhibitor of HPPD, has been found to inhibit the growth and differentiation of Paracoccidioides brasiliensis and Cocccidioidies immitis mammalian-associated pathogens [44,45]. This could explain the possible mechanism of action of UA in reducing mycelial growth in N. rosae isolates in this study, although further in-depth studies are required.

4.2. Effect of Usnic Acid on Structures of N. rosae

The changes observed in the conidia and hyphae of N. rosae (Figure 4) suggest possible UA-induced damage, evidenced by the loss of pigmentation in some conidia and alterations in hyphal morphology. These effects could be related to the previously described inhibition of melanin synthesis. However, further investigation is required to confirm the impact of UA on fungal structures and to elucidate its possible mechanism of action.

4.3. Mean Inhibitory Concentration (IC₅₀)

The IC₅₀ values obtained in this study showed the ability of UA to limit mycelial growth as a function of concentration. However, these results differ from those reported by Paguirigan et al. [23], who reported mean effective concentrations (EC₅₀) of 18.07 and 1.47 μg/mL for Diaporthe eres and D. actnidiae, respectively, values below those found in this study. This discrepancy provides evidence that the efficacy of UA varies depending on the pathogen evaluated. According to the IC₅₀ observed in this study and the scale reported by Holetz et al. [46] and Cintra et al. [25], it has been suggested that UA has a moderate antimicrobial capacity.

4.4. Final Sporulation

Sporulation is a key process in the reproduction, survival, and dispersal of phytopathogenic fungi [27,47]. This study showed the possible negative effects of UA on the sporulation process of different N. rosae isolates. It should be noted that this is the first study to evaluate the effect of UA on spore production by this phytopathogen. The FREC isolate was the most affected, with 98.6% fewer spores than its control, but it had one of the lowest percentages of mycelial growth inhibition (52%). In isolates 372 and 323, UA had no significant effect on sporulation. Considering that the N. rosae infection process begins with spore germination [14,48], the decrease in their concentrations could be related to the lower capacity of the fungus to spread and establish infections. Additionally, this reduction could compromise their long-term survival by limiting the availability of the inoculum needed to initiate new infection cycles. It has been reported that N. rosae spores can survive in soil and infected plant tissues, even after several seasons [48,49]. However, UA efficacy varies depending on the isolate, possibly due to genetic differences between isolates that affect their sensitivity to this metabolite.

4.5. H₂O₂ Content in Strawberry Plants Inoculated with N. rosae Strains Exposed to Different Concentrations of Usnic Acid

H₂O₂ content is an indicator of oxidative stress in plants and is closely related to defense during plant–pathogen interactions. In addition to their antimicrobial properties, its accumulation at infection sites promotes lignification and cell wall thickening, the activation of reactive oxygen species (ROS)-dependent signaling pathways, the expression of pathogenesis-related (PR) genes, and programmed cell death, with the aim of limiting the spread of pathogens within plants [50]. However, it is important to mention that high H₂O₂ content can also cause negative effects on plant cells; therefore, it is important to maintain a balance between H₂O₂ content and the antioxidant system.

In this study, plants inoculated with isolate 466 and treated with the chemical control showed the highest H₂O₂ levels, suggesting an intense response to stress, possibly induced by the high virulence of the pathogen and the low effectiveness of the fungicide. This type of product targets the pathogen but does not exert any beneficial effects that stimulate defense pathways in plants. Thus, UA appears to have a positive effect by maintaining stable H₂O₂ levels.

Previous studies reported that UA promotes the activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) [51]. The main antioxidant enzymes are responsible for reducing and regulating the content of active oxygen and free radicals, thereby reducing their toxic effects on cells [52]. Kikowska et al. [53] reported an increase in the concentrations of flavonoids and phenolic acids in Eryngium alpinum seedlings following UA application. These compounds are classified as non-enzymatic antioxidants that participate in the reduction and neutralization of ROS [54,55]. This could explain the behavior of UA-treated seedlings in showing a lower H₂O₂ content than the chemical control.

However, it is necessary to mention that the solvent used (40% NaOH) also induced the production of H₂O₂ in response to excess sodium stress, since strawberries are highly susceptible to excess salt. Therefore, new studies are suggested with concentrations lower than 40% for this crop to maintain the solubility and pH of UA.

4.6. MDA Content in Strawberry Plants Inoculated with N. rosae Strains and Exposed to Different Concentrations of Usnic Acid

MDA is an indicator of cell damage in plants under stress conditions, as it reflects the degree of membrane lipid peroxidation [56]. In the present study, the MDA content showed a significant interaction between the aggressiveness of N. rosae isolates and the ability of treatments to counteract seedling damage. Isolate FREC2 was the most aggressive in generating the highest MDA content, suggesting strong lipid peroxidation and oxidative damage in seedlings. In contrast, FREC generated lower levels of MDA, even in the control; therefore, it can be considered the least aggressive and with less capacity to induce oxidative stress. Notably, UA reduced the levels of MDA by 77.4% in plants inoculated with FREC2, indicating a possible protective effect, either by the direct inhibition of the pathogen or by the activation of the antioxidant mechanisms of the plants. This behavior could be related to that observed in the H₂O₂ content, which supports the hypothesis that UA promotes the activity of enzymatic and non-enzymatic antioxidant systems [51,53] that help to neutralize ROS generated during the N. rosae infection process. However, their behavior varies depending on isolate and plant–isolate interactions.

4.7. Phenylalanine Ammonia Lyase Activity in Strawberry Plants Inoculated with N. rosae Strains and Exposed to Different Concentrations of Usnic Acid

The PAL enzyme is directly associated with the defense response in plants, as it acts as a key precursor in the synthesis of antioxidant compounds [57]. PAL activity usually increases in response to pathogen infections [58]. In this study, an increase in PAL activity was observed in the roots of plants inoculated with the FREC2 isolate and treated with the chemical control, while UA reduced its activity by 17.95%. These results suggest that UA can efficiently regulate the antioxidant responses of plants without requiring intense PAL activation. In contrast, plants with fungicide applications appear to require higher PAL production, possibly because their ingredients do not stimulate antioxidant defense systems. Regarding the behavior of the isolates, FREC2 and 466 promoted higher PAL activity, which could indicate higher aggressiveness. In contrast, FRECs 323 and 372 induced lower PAL activity, which could be related to a lower capacity to induce oxidative stress.

4.8. Severity Percentage in Strawberry Plants Inoculated with N. rosae Strains and Exposed to Different Concentrations of Usnic Acid

According to the results, the chemical control had a positive impact on reducing the severity of infection in all N. rosae isolates. This confirms its effectiveness as part of traditional chemical management [9]. Although UA failed to reduce severity, it induced positive biochemical responses in H₂O₂ and MDA content in the treated plants. This suggests that the mode of action of UA may be related more to the antioxidant defense system of plants than to a direct antifungal effect. This also agrees with the results obtained in the in vitro test, as UA exhibited fungistatic properties on N. rosae isolates by reducing their growth rate. However, the high percentage of severity in solvent-treated plants may be associated with the high concentration of NaOH (40%), since plants are highly sensitive to sodium, which may have made them more susceptible to infection by the isolates.

5. Conclusions

UA exerts a fungistatic effect at a concentration of 400 μg/mL on the mycelial growth of N. rosae isolates FREC2, FREC, 372, 323, and 466 under in vitro conditions. The results of the in vivo test showed a significant interaction between the aggressiveness of the isolates, the physiological response of the plants, and the efficacy of the treatments. The FREC2 isolate was the most aggressive. It has been suggested that UA acts as a regulator of the plant antioxidant system, with the capacity to mitigate the oxidative stress generated by N. rosae isolates. These findings suggest that UA is an alternative to conventional fungicides, with physiological benefits to plants. However, it is necessary to evaluate and validate its performance in open fields and protected agricultural conditions. It is also important to carry out studies on other phytopathogens to understand the mode of action of UA as an antifungal and fungistatic agent or as a biostimulant in the plant defense system.