The berry industry has shown significant growth in Mexico's agricultural sector, establishing itself as one of the highest-value agricultural export products, primarily to the United States, the Middle East, Southeast Asia, and the European Union (SADER, 2023). By 2024, according to the Agri-Food and Fisheries Information System (SIAP), berry production exceeded one million tonnes, with strawberry (59.9%), blackberry (23.8%), and raspberry (16.3%) standing out as the highest-yielding species. This increase in production has given rise to major phytosanitary challenges. Among the principal phytopathogens responsible for yield and economic losses in strawberry cultivation are Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Rhizoctonia spp., and, more recently, Neopestalotiopsis rosae (Castellanos-González et al., 2020; Rebollar-Alviter et al., 2020; Dholi et al., 2023).

Strawberry plants infected with N. rosae develop symptoms as root rot, as well as the development of lesions on leaves and fruit, which can cause losses of up to 70%. The infection triggers wilting, chlorosis, and foliar necrosis, which may ultimately result in plant death. The disease occurs in strawberry transplants grown both in open fields and under tunnels, at temperatures of 23-27 °C, and its development is favored by rainfall and high leaf wetness (Rebollar-Alviter et al., 2020; Olivares-Rodríguez et al., 2024). As this is a relatively recent disease in strawberry cultivation, available information on its management remains limited.

Currently, the control of N. rosae relies primarily on the preventive and curative application of synthetic chemical fungicides, such as thiram, hymexazol, prochloraz, cyprodinil + fludioxonil, and pydiflumetofen + fludioxonil (Essa et al., 2018; Acosta-González et al., 2024). However, the effectiveness of these fungicides is limited by residuality, environmental impact, and pathogenic resistance (Dukare et al., 2019; Islam et al., 2024). Considering this situation, the implementation of integrated management using environmentally friendly methods is being promoted, such as the use of plant extracts (PEs) as a biorational control strategy (Dėnė and Valiuškaitė, 2021; Cenobio-Galindo et al., 2024).

In this context, plants of the genus Baccharis and the species Fouquieria splendens, distributed across the Chihuahuan and Sonoran deserts, have shown antifungal potential. In particular, F. splendens has demonstrated activity against F. oxysporum, Rhizoctonia solani, and the oomycete Pythium aphanidermatum, an effect attributed primarily to its phytochemical composition, which is rich in polyphenols (Pinto et al., 2022; Ramírez-Méndez et al., 2024; Ramírez-Méndez et al., 2025). However, to date, only antibacterial activity has been reported for Baccharis salicifolia, primarily against the genus Staphylococcus (Escalante-Hernández, 2022), with no studies on its antifungal activity. Therefore, the objective of the present study was to identify phytochemical compounds present in extracts of B. salicifolia and F. splendens, and to evaluate their antifungal activity and in vitro effect on cellular structures of N. rosae.

Collection and processing of plant material. In August 2024, physiologically mature plants of B. salicifolia and F. splendens were collected in Saltillo, Coahuila (25°27'28.6"N, 101°06'44.4"W; 1780 m a. s. l.), and transported to the Mycology and Biotechnology Laboratory of the Universidad Autónoma Agraria Antonio Narro (UAAAN). The simples were washed, oven-dried at 60 °C to constant weight, ground, sieved (0.2 mm), and stored in amber glass vials.

Plant extracts. Plant extracts (PEs) were obtained following the methodology proposed by Jasso de Rodríguez et al. (2015), with some adaptations. Briefly, 42 g of dry powder from

B. salicifolia leaves and F. splendens stems were placed in 500 mL flasks and were extracted with 375 mL of 96% methanol as solvent. The mixtures were kept under continuous agitation for 72 h at 60 °C on a stirring hotplate (Thermo Scientific Cimarec, USA). Subsequently, the solvent was removed by rotary evaporation (IKA RV 10 digital V, USA) operated at 150 rpm and 70 °C. The resulting methanolic fraction was transferred to glass containers and dried in an oven (Arsa AR-130D, México) at 50 °C for 72 h. Finally, the PEs were stored in amber vials at 4 °C under refrigeration until use.

Qualitative phytochemical characterization by colorimetry. The PEs were prepared at a concentration of 2 000 mg L⁻¹ and evaluated using specific qualitative tests to detect various groups of secondary metabolites. The following tests were applied: Dragendorff and Sonnenschein for alkaloids; Fehling and Benedict for reducing sugars; Molisch for carbohydrates; H₂SO₄ and FeCl₃ for carotenoids; Ehrlich for coumarins; Liebermann-Burchard and Salkowski for sterols and terpenes; Shinoda and NaOH for flavonoids; Grignard for cyanogenic glycosides; HCl for purines; Bornträger (H₂SO₄, NH₄OH) for quinones; foam test for saponins; and tests with gelatin, FeCl₃, and K₃[Fe(CN)₆] for tannins (Sahgal et al., 2009; Usman et al., 2009).

Isolation of Neopestalotiopsis rosae. The N. rosae isolates were provided by the Centro Universitario Regional Centro Occidente of the Universidad Autónoma Chapingo, and are coded 466, 323, FREC, and 372. The isolates were obtained from strawberry plants showing symptoms in cultivars 'Albion' and 'Festival', from commercial plots in the municipalities of Zamora and Jacona, Michoacán. These isolates were molecularly characterized in the study conducted by Rebollar-Alviter et al. (2020). For preservation, the isolates were maintained on potato dextrose agar (PDA) and refrigerated at 4 °C until use.

Antifungal activity of Baccharis salicifolia and Fouquieria splendens extracts against Neopestalotiopsis rosae. To evaluate the antifungal activity of the PEs against N. rosae, the broth microdilution method was used in 96-well polystyrene microplates following the methodology of Tucuch-Pérez et al. (2020), with some modifications. The first column was designated as the negative control (medium without fungal cells or PEs) and the second column as the positive control (medium with fungal cell suspension without PEs). To each well, 110 μL of liquid Sabouraud medium and 40 μL of indicator solution (2,3,5-triphenyltetrazolium chloride) were added. Starting from the second column, a fungal suspension of N. rosae (1×10⁶ conidia mL⁻¹) was incorporated. The four isolates were evaluated individually in separate microplates, with both PEs (B. salicifolia and F. splendens) used in each case. From columns 3 to 12, PE treatments were added at concentrations ranging from 2 000 to 3.9 mg L⁻¹. Each microplate was considered one replicate with eight experimental units. The microplates were incubated at 28 ± 2 °C for 72 h, after which absorbance was measured at 490 nm using a microplate reader (Thermo Scientific Multiskan Go 51119200, USA). The inhibition percentage was calculated using the following formula:

Inhibition percentage = ((A − B) / A) × 100

Where: A = Absorbance of the positive control; B = Absorbance of the treatment.

Determination of the effect of B. salicifolia and F. splendens extracts on cellular structures of N. rosae. The effects of the PEs on fungal cellular structures were evaluated using the methodology of Ramírez-Méndez et al. (2024), with modifications. The PE doses consisted of 10 two-fold serial dilutions, with concentrations ranging from 2 000 to 3.9 mg L⁻¹. The fungal suspensions contained 1×10⁶ conidia mL⁻¹ of N. rosae. For the bioassays,

1.5 mL Eppendorf tubes were used, adjusting a total volume of 1 000 µL composed of liquid Sabouraud medium and the corresponding dose of each plant extract; subsequently, 10 µL of the fungal suspension was added. The cultures were incubated for seven days at 24 ± 2 °C, and morphological alterations were evaluated using a light microscope (10×, 40×, and 100×) equipped with a Dino-Lite® digital camera. Image capture was performed using DinoCapture 2.0 software. Each concentration was applied in triplicate, and an untreated control consisting solely of Sabouraud medium and the fungal suspension, without PEs, was included.

Statistical analysis. Probit analysis was applied to the in vitro assays to determine the 50% and 90% inhibitory concentrations (IC₅₀-₉₀) for each treatment. In addition, an ANOVA was performed followed by Tukey's mean comparison test (p ≤ 0.05). All procedures were carried out using SAS software version 9.0.

Qualitative phytochemical characterization of plant extracts. Qualitative phytochemical characterization was performed by colorimetric tests (Table 1). The results indicated that both extracts B. salicifolia and F. splendens contained the presence of alkaloids, carbohydrates, flavonoids (xanthones), cyanogenic glycosides, saponins (triterpenoids), tannins (gallic acid and catechol derivatives), and phenols. However, B. salicifolia also contained purines and carotenoids, whereas in F. splendens the presence of quinones (benzoquinones) were detected. These results are consistent with previous studies reporting the presence of some of these phytochemicals in these plant species.

In Baccharis species, the main phytochemicals reported are phenolic compounds, diterpenoids, and triterpenoids, as well as volatile constituents and coumarins (Abad and Bermejo, 2007). For B. salicifolia in particular, several studies support this composition, such as that of Corral et al. (2012), who isolated halimane-type diterpenoids (salicifolic acid and 5-hydroxy-6-hydrosalicifolic acid) from the ethanolic extract of B. salicifolia, as well as the compounds sakuranetin and apigenin, belonging to the flavonoid group, and scopoletin, belonging to the coumarin family. Valdez-Palomino (2014), in turn, reported that hydroalcoholic extracts of B. salicifolia presented phenols and/or tannins, flavonoids, lactones and/or coumarins, catechins, and alkaloids. These results were obtained through colorimetric tests, which are consistent with the findings of the present study.

On the other hand, the phytochemicals of F. splendens have been extensively studied. For example, in the study conducted by Ramírez-Méndez et al. (2023), the methanolic extract of F. splendens stems presented alkaloids, carbohydrates, flavonoids (flavanones, flavones, flavonols, and chalcones), reducing sugars, triterpenoid-type saponins, tannins (gallic acid and catechol derivatives), as well as quinones (anthraquinones and benzoquinones). Finally, Román-Casiano et al. (2025) identified phenolic compounds in the ethanolic extract of F. splendens leaves, including flavonoids, phenolic acids, tannins, and quinic acid derivatives.

Table 1

	A	C	F				GC	AZ	S		T				Q			CU	P	Ca
			F1	F2	F3	F4		AZ1	S1	S2	T1	T2	T3	T4	Q1	Q2	Q3
B. salicifolia	-	+	-	-	-	+	+	-	+	+	+	+	+	+	-	-	-	-	+	+
F. splendens	+	+	-	-	-	+	+	-	+	+	+	+	+	+	-	+	-	-	-	-

+= Phytochemical present, -=Phytochemical absent, A = Alkaloids; C = Carbohydrates; F = Flavonoids; F1 = Aurones or chalcones; F2 = Flavanones; F3 = Flavones; F4 = Xanthones; CG = Cyanogenic glycosides; RS = Reducing sugars; S = Saponins; S1 = Triterpenoid saponins; S2 = Sterols and terpenes; T = Tannins; T1 = Tannins; T2 = Gallic acid derivatives; T3 = Catechol derivatives; T4 = Phenolic compounds; Q = Quinones; Q1 = Anthraquinones; Q2 = Benzoquinones; Q3 = Anthrone derivatives; CU = Coumarins; P = Purines; Ca = Carotenoids.

Antifungal activity of B. salicifolia and F. splendens extracts against N. rosae (Microdilution method). Regarding the inhibition of the different N. rosae isolates with the B. salicifolia extract (Figure 1), isolates 323 and FREC completely inhibited fungal growth at 250 mg L⁻¹, while isolates 466 and 372 required a higher concentration (1 000 mg L⁻¹). As for the IC₅₀₋₉₀ results, isolates 323 (IC₅₀ = 39.91 mg L⁻¹, IC₉₀ = 188.79 mg L⁻¹) and FREC (IC₅₀ = 44.69 mg L⁻¹, IC₉₀ = 211.82 mg L⁻¹) were statistically similar (Table 2). On the other hand, isolates 466 (IC₅₀ = 130.01 mg L⁻¹, IC₉₀ = 608.34 mg L⁻¹) and 372 (IC₅₀

= 101.59 mg L⁻¹, IC₉₀ = 480.21 mg L⁻¹) formed distinct groups, showing higher inhibitory concentrations (Tukey, p < 0.05).

Regarding the inhibition levels of the F. splendens extract (Figure 2), isolate 323 was the most sensitive, reaching 100% inhibition at 250 mg L⁻¹, followed by FREC and 372, which were inhibited 100% at doses of 1 000 mg L⁻¹. Isolate 466 showed the lowest sensitivity, achieving 100% inhibition only at 2 000 mg L⁻¹. These differences reflect marked heterogeneity in the isolates’ responses to the evaluated extract. Regarding the IC₅₀₋₉₀, the F. splendens extract showed a heterogeneous response among the N. rosae isolates, with significant statistical differences (Tukey, p < 0.05) (Table 2). Isolate 323 was the most sensitive (IC₅₀ = 65.76 mg L⁻¹, IC₉₀ = 231.11 mg L⁻¹), followed by FREC (IC₅₀ = 118.08 mg L⁻¹, IC₉₀ = 506.69 mg L⁻¹), 372 (IC₅₀ = 185.48, IC₉₀ = 821.08 mg L⁻¹), while 466 was the most resistant (IC₅₀ = 307.37, IC₉₀ = 1,940 mg L⁻¹).

Table 2

Isolate	B. salicifolia		F. splendens
	IC50	IC90	IC50	IC90
466	130.01a	608.34a	130.01b	608.34b
323	39.91c	188.79c	65.76d	231.11d
FREC	44.69c	211.82c	118.08c	506.69c
372	101.59b	480.21b	185.48a	821.08a

Figure 1

Figure 2

Regarding the comparison of isolate sensitivity to the different doses evaluated between the PEs, as well as the IC₅₀₋₉₀ values, isolates 323 and FREC were the most susceptible to both PEs, followed by isolate 372, which showed a moderate response in both cases. In contrast, isolate 466 exhibited lower sensitivity, evident only against the F. splendens extract. The antifungal activity observed for the PEs against N. rosae isolates is attributed primarily to the presence of phytochemicals such as phenols, tannins, and flavonoids, identified through qualitative tests, as the involvement of these compounds in antifungal activity has been widely reported in various studies (Tucuch-Pérez et al., 2020; Kengne et al., 2023).

Likewise, various studies have demonstrated the efficacy of phytochemicals in controlling Neopestalotiopsis species. For example, Darapanit et al. (2021) evaluated crude PEs against Neopestalotiopsis spp. and Pseudopestalotiopsis sp., observing that PEs of clove (Syzygium aromaticum) and turmeric (Curcuma longa) completely inhibited the growth of all isolates after 14 days of incubation at a concentration of 10 000 mg L⁻¹, values higher than those obtained in the present study. In another study, Tran et al. (2023) reported that the encapsulated formulation with cinnamon essential oil (Cinnamomum verum) showed the greatest antifungal activity against N. rosae, reaching 95.6% inhibition in vitro at a dilution of 1:500. Ye et al. (2023) evaluated the in vitro effect of thymol on Neopestalotiopsis clavispora, and the results showed that it limited mycelial growth with a minimum inhibitory concentration (MIC) of 100 mg L⁻¹. Zheng et al. (2025) demonstrated that carvacrol exhibited the greatest in vitro antifungal activity (EC₅₀ = 19.72 mg L⁻¹) against Neopestalotiopsis saprophytica.

Taken together, these results highlight the potential of phytochemicals as alternatives for managing Neopestalotiopsis species. In this regard, additional studies are needed to identify the bioactive compounds responsible for the antifungal activity, evaluate their efficacy under in vivo conditions and asses their potential integration into sustainable disease management strategies for agriculturally important crops.

Evaluation of the effect of extracts on cellular structures of Neopestalotiopsis rosae. Both the B. salicifolia extract (Figure 3) and the F. splendens extract (Figure 4) induced structural alterations in N. rosae. However, this effect varied between the PEs and the isolates, being observed primarily at high concentrations (2 000, 1 000, or 500 mg L⁻¹) and, in some cases, down to 250 mg L⁻¹. At a concentration of 2 000 mg L⁻¹, all isolates treated with both PEs exhibited inhibition of N. rosae conidial germination, as well as evident structural alterations. Loss of appendages was observed in all isolates treated with the B. salicifolia extract (Figure 3: B1, C1, D1, and E1). Additionally, signs of dehydration and cell wall alterations were recorded, particularly in isolates 323, FREC, and 372 treated with the F. splendens extract (Figure 4: C1, D1, and E1). At a dose of 1 000 mg L⁻¹, the B. salicifolia extract induced vacuolization in the conidia of all N. rosae isolates (Figure 3: B2, C2, D2, and E2). The F. splendens extract also showed structural damage in the conidia, likewise in isolates 323, FREC, and 372 (Figure 4: B2, C2, D2, and E2).

At concentrations of 500 and 250 mg L⁻¹ of the B. salicifolia extract, isolate 323 remained the most sensitive (Figure 3: C3-C4), as no conidial germination was observed and the conidia exhibited morphological damage. Isolates FREC and 372 showed evident thickening (Figure 3: D3-D4 and E3-E4). In contrast, isolate 466 exhibited germination without evident structural damage (Figure 3: B3-B4). In the case of F. splendens at doses of 500 and 250 mg L⁻¹, isolate 323 was the most sensitive (Figure 4: C3-C4), showing inhibition of N. rosae conidial germination. In the conidia of isolates 372 and FREC, germinating conidia showed evidence of mycelial thickening (Figure 4: D3-D4 and E3-E4), while in isolate 466 the mycelium showed no evident damage (Figure 4: B3-B4). Finally, at a dose of 125 mg L⁻¹, germination was evident and more developed in all isolates, with conidia showing no evidence of structural malformations with either the B. salicifolia extract (Figure 3: B5, C5, D5, and E5) or the F. splendens extract (Figure 4: B5, C5, D5, and E5). These effects suggest direct antifungal action of the PEs on the integrity and development of the fungus.

Figure 3

The effect observed of the PEs on the structures of the different N. rosae isolates can be attributed primarily to the identified phytochemicals, as evidence exists for the mechanisms by which these compounds act on fungal cells. Various studies have demonstrated that secondary metabolites of plant origin produce similar effects in other phytopathogenic fungi. Polyphenols induce ROS production and cause lipid peroxidation damage in the fungal cell membrane, leading to increased membrane permeability, leakage of cellular components, and alterations in hyphal structure (Jiang et al., 2015; Davidova et al., 2025).

In this regard, the thickening observed in the fungal structures could be associated primarily with processes of cytoplasmic vacuolization induced by these compounds (Agredo-Hoyos et al., 2025). Although various studies have reported the antifungal activity of isolated phytochemical compounds, such as carvacrol and thymol, against Neopestalotiopsis species, the available evidence has focused primarily on individual molecules (Ye et al., 2023; Zheng et al., 2025). To date, no studies have described structural effects induced by complex plant extracts, containing multiple phytochemicals, on the structures of N. rosae.

Figure 4

Both PEs contained alkaloids, flavonoids, saponins, tannins, and phenols; additionally, B. salicifolia contained purines and carotenoids, while F. splendens contained quinones. In the evaluation of in vitro antifungal activity against N. rosae isolates, a clearly differential sensitivity was observed among the isolates. Isolates 323 and FREC were highly sensitive, showing total inhibition and structural damage at low concentrations (250 mg L⁻¹), while isolates 372 and 466 showed lower sensitivity and required higher doses (1 000 and 2 000 mg L⁻¹), with B. salicifolia being the most effective extract. Taken together, these results highlight the potential of both extracts as biorational alternatives for the management of N. rosae, whose efficacy should be confirmed in studies under greenhouse and field conditions.

Limitations. One of the most relevant limitations of the study is the insufficient chemical analysis, as no quantitative characterization of the plant extracts was performed. Given the characteristics of the experiment conducted, it would be advisable to evaluate a greater number of fungal isolates and to incorporate the use of stains that would allow more precise visualization of damage to the structures of the phytopathogenic fungus.

Conflict of interest. The authors declare that they have no conflicts of interest regarding the research, authorship, and/or publication of this Note.

Funding. This study was funded by the Universidad Autónoma Agraria Antonio Narro.

Acknowledgements. To the Universidad Autónoma Agraria Antonio Narro for the financial support and facilities provided for the conduct of this research.

Authors' Contributions. Jesús Eduardo Ramírez-Méndez: conception and development of the research, experimental design, and coordination of the study. Meralida Bartolón-Pérez: data analysis and literature review. Francisco Daniel Hernández-Castillo: methodological guidance and critical review of the manuscript. Yisa María Ochoa-Fuentes: support in the interpretation of results and review of the manuscript. Laura Castro-Rosales: support in the analysis of results and scientific review of the document. Marco Antonio Tucuch-Pérez: experimental execution, data collection, and manuscript writing. Corresponding author