Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. En prensa. ISSN (en línea) 1853-8665.

Original article

Essential oils and extracts from Argentinian northwest plants as potential biofungicides for olive and grapevine pathogens: in vitro studies

Aceites esenciales y extractos de plantas del noroeste argentino como potenciales biofungicidas de patógenos de olivo y vid: estudios in vitro

María Sayago¹^,²,

Ivana Ormeño²,

María Teresa Ajmat²,

Natalia Barbieri¹^,² ^*

¹ Universidad Nacional de Chilecito. CONICET. Departamento de Ciencias Básicas y Tecnológicas. 9 de Julio 22, Chilecito F5360CKB. La Rioja. Argentina.

² Universidad Nacional de Chilecito. Instituto de Ambiente de Montaña y Regiones Áridas.

^* nbarbieri@undec.edu.ar

Abstract

This work studies the effect of 12 botanical products from Argentinian northwest plants on spores and mycelium of Verticillium dahliae and Phaeoacremonium parasiticum, two pathogens of agronomic importance for the region. The fungi were exposed to essential oils (EOs) or ethanolic extracts (EEs), determining the percentage of germinated spores and mycelial growth. All tested EOs and EEs showed varying degrees of antifungal activity, dependent on plant species, extract type, pathogen, and targeted fungal structures. V. dahliae germination was completely inhibited by Zuccagnia punctata and Clinopodium gilliesii EOs. In experiments with EEs, Z. punctata EE was the most effective in suppressing spore germination of both fungi. The C. gilliesii EE also controlled V. dahliae germination. The EEs of Z. punctata, C. gilliesii and Lippia turbinata were the most active against mycelial growth. These three EEs had a fungistatic effect on P. parasiticum while Z. punctata and L. turbinata EEs showed a fungicidal effect on V. dahliae. The products obtained from Z. punctata, C. gilliesii and L. turbinata have potential as biocontrollers against V. dahliae and P. parasiticum. This is encouraging since no effective treatments are available for the diseases involving these pathogens.

Keywords: Verticillium dahliae, Phaeoacremonium parasiticum, botanical antifungals, mycelial inhibition, conidial susceptibility

Resumen

Este trabajo estudia el efecto de 12 productos de plantas del noroeste argentino sobre las esporas y micelio de Verticillium dahliae y Phaeoacremonium parasiticum, dos patógenos de importancia agronómica. Los hongos fueron expuestos a los aceites esenciales (AE) o extractos etanólicos (EE), y se determinó el porcentaje de germinación y crecimiento micelial. Todos los AE y EE mostraron actividad antifúngica, la cual dependió de la especie vegetal, del extracto, del patógeno y de las estructuras fúngicas objetivo. La germinación de V. dahliae fue inhibida con los AE de Zuccagnia punctata y Clinopodium gilliesii. El EE de Z. punctata fue el más efectivo para suprimir la germinación de ambos hongos. El EE de C. gilliesii también fue capaz de controlar la germinación de V. dahliae. Mientras que los EE de Z. punctata, C. gilliesii y Lippia turbinata fueron los más activos sobre el micelio. Estos tres EE fueron fungistáticos sobre P. parasiticum mientras que los EE de Z. punctata y L. turbinata fueron fungicidas sobre V. dahliae. Los productos obtenidos de Z. punctata, C. gilliesii y L. turbinata son potenciales biocontroladores de V. dahliae y P. parasiticum. Esto es alentador ya que no se dispone de tratamientos eficaces para las enfermedades en las cuales participan estos patógenos.

Palabras clave: Verticillium dahliae, Phaeoacremonium parasiticum, antifúngicos botánicos, inhibición micelial, susceptibilidad conidial

Originales: Recepción: 07/05/2024 - Aceptación: 23/12/2024

Introduction

Olive and grapevine cultivation in La Rioja province (northwest Argentina) is economically significant. Fungal diseases affect productivity causing considerable losses ⁽^7,
12⁾. Vascular wilt disease in olives caused by Verticillium dahliae Kleb has acquired great importance worldwide producing tree mortality, fruit yield reduction, and organoleptic defects in virgin olive oil extracted from infected plants ⁽^{18, 19}⁾. Olive verticillium wilt is one major concern for olive growers in the semi-arid regions of Argentina. Rattalino (2023) has recently shown that V. dahliae is widely spread in La Rioja olive-growing regions, estimating 24% disease incidence.

Grapevine trunk diseases are the principal fungal diseases affecting viticulture worldwide ⁽¹⁷⁾. Among these pathologies, hoja de malvón (related to Esca) and young vine decline (Petri disease) are among the most devastating and challenging diseases in many wine regions of Argentina. They are caused by multiple wood fungal pathogens, with Phaeoacremonium parasiticum being mostly prevalent ⁽^9,
10⁾.

Unfortunately, effective treatments against these mycoses are not available, and their management remains difficult. To date, recommendations focus on timely monitoring of these diseases and integrated management strategies including biological control as a potential tool ⁽^{17, 19}⁾.

Plant essential oils (EOs), extracts and related molecules have demonstrated inhibitory efficacy against pathogenic fungi ⁽^3,
26⁾. They represent eco-friendly control alternatives for integrated disease management, contributing to sustainable agricultural production. The antifungal activity (AA) of some EOs and a few plant extracts is reported against V. dahliae ⁽^{6, 8, 11, 14, 24}⁾. However, insufficient studies focus on biological control of P. parasiticum using plant products. This study focused on plant species with previous AA against dermatophytes or molds: Zuccagnia punctata, Clinopodium gilliesii, Lippia turbinata, Lippia integrifolia, Argemone subfusiformis, Erythrostemon gilliesii, and Senecio subulatus var. salsus ⁽¹⁾. We explore their AA against olive and grapevine pathogenic fungi, hypothesizing that plant products from these species could control plant pathogenic fungi in regional crops. We evaluated the effect of 12 botanical products (secondary metabolites) obtained from the mentioned plants on spore viability and mycelial growth of V. dahliae and P. parasiticum.

Material and methods

Plant Material

Z. punctata Cav., C. gilliesii Kuntze, L. turbinata Griseb., L. integrifolia Hieron., A. subfusiformis Ownbey., E. gilliesii (Hook.) Klotzsch and S. subulatus var. salsus (Griseb.) were collected in 2018. Georeferenced specimens were deposited in the herbarium of the Universidad Nacional de Chilecito (UNDEC). Supplementary Table 1 provides data on collection sites, yield and voucher specimens.

Obtaining essential oils (EOs) and ethanolic extracts (EEs)

Air-dried canopies were used. EOs were obtained by hydrodistillation in a Clevenger-type apparatus and stored at -20°C until further use. To obtain EEs, the plant material was macerated in ethanol 96° for 24 h, filtered and the solvent evaporated. Then, waxes were removed by precipitation from an ethanol-water solution. Later, EEs were dissolved in 50% ethanol, shaken using a 40 kHz ultrasonic cleaning bath (1 h) and centrifuged (5000 rpm, 10 min). Finally, the separated supernatants were evaporated and samples were stored until use ⁽²⁾.

Phytopathogenic fungi

We used a native non-defoliating strain of V. dahliae Kleb. previously isolated from an infected olive plant in La Rioja ⁽²¹⁾. The P. parasiticum strain was obtained from the Phytopathology Laboratory of INTA Mendoza, Argentina. First, stock cultures (stored at -80°C) were activated in potato dextrose agar (PDA, Britania, Argentina) and grown in microcultures (PDA block on a microscopic slide) to check morphological traits (Supplementary Figure 1). Secondly, fungi were maintained in PDA for antifungal assays.

Inhibition of spore germination

The phytopathogens V. dahliae and P. parasiticum were cultured for 7 and 14 days, respectively, allowing spore development. To obtain spores, 2 mL sterile distilled water were added, and mycelia was gently scraped with a Drigalsky spatula. The suspension was recovered and adjusted to 1 x 10³ spores/mL using a Neubauer counting chamber. For the assays, a 100 uL spore suspension was incubated with 100 uL of different concentrations of EO or EE (1-3 mg/mL) for 1h at 24°C. For plant products with 100% inhibitory activity at 1 mg/mL, lower concentrations (range of 0.2-1 mg/mL) were also evaluated. Following incubation, an aliquot was taken and seeded in PDA. After 48 h for V. dahliae and 72 h for P. parasiticum at 24°C incubation, spores were counted and the percentage of inhibited spores (number of non-germinated spores/total number of spores × 100) was determined ⁽¹⁶⁾. Growth control for each tested phytopathogen (distilled water), solvent control (DMSO or ethanol 96°) and EO and EE sterility controls were included. According to own experimental data, Benomyl (fungicide) constituted the positive control in concentrations ranging from 0.1 to 0.4 mg/mL for V. dahliae and 7 to 10 mg/mL for P. parasiticum. The minimum inhibitory concentration (MIC) was defined as the lowest EO or EE concentration producing 100% inhibition of spore germination.

Synergism (checkerboard test)

The MIC values obtained previously served as a reference and combinations ranging from 0.125xMIC to 1xMIC of EOs, EEs and Benomyl were formulated. Inhibition on spore germination was determined using the methodology described above. To evaluate combination effects, the fractional inhibitory concentration (FIC) index was calculated as FIC index = FICA + FICB, where FICA and FICB are the minimum concentrations inhibiting fungal growth (MIC) for samples A and B, respectively. FICA = (Combination MICA) / (MICA alone), FICB = (Combination MICB)/ (MICB alone). According to the FIC index, results indicated synergism (≤ 0.5), addition (> 0.5 and ≤ 1.0), indifference (> 1.0 and ≤ 2.0), or antagonism (> 2.0) ⁽²⁵⁾.

Inhibition of mycelial growth

EEs were added at different concentrations (0.25-3 mg/mL) on molten PDA. Petri dishes with PDA plus EE were inoculated with a 5-mm diameter mycelial disc obtained from the edge of 7-and 14-day-old cultures of V. dahliae and P. parasiticum, respectively. Growth control for each tested phytopathogen (PDA plate with the mycelial disc), solvent control (PDA plate plus 96° ethanol with the mycelial disc), and positive control (PDA plate plus Benomyl with the mycelial disc) were included. Inoculated plates were incubated at 24°C and growth of V. dahliae and P. parasiticum was evaluated at 7 days by measuring mycelial diameter of each colony. Percentage of growth inhibition was calculated by equation 1:

where

D = colony diameter of growth controls

d = diameter in EE or Benomyl treatments

The MIC was equal to the lowest EE concentration at which mycelial growth was completely inhibited ⁽²⁴⁾. When EEs inhibitory effects were fungicidal or fungistatic, PDA plates with mycelium discs and different EE treatments would be incubated for 2-5 additional days (mycelial inhibition at 9 days for V. dahliae and 12 days for P. parasiticum). When no mycelium re-growth occurred during additional incubation, EE was considered fungicidal. Otherwise, it was considered fungistatic.

Total phenolic and flavonoid content (PC and FC) in the EEs

Total PC was determined by the Folin-Ciocalteu spectrophotometric method. Different volumes of EE solutions were mixed with Folin-Ciocalteu reagent and sodium carbonate. After incubation, absorbance was measured at 765 nm. The PC was determined using a Gallic acid calibration curve and results were expressed as mg Gallic acid equivalents/g of dry extract (mg GAE/g) ⁽²⁾. FC was estimated by a spectrophotometric assay based on aluminum chloride complexes. Serial dilutions from EEs were mixed with aluminum chloride, and incubated for 1 h. Absorbance was measured at 420 nm. FC was calculated using a Quercetin calibration curve and expressed as mg quercetin equivalents/g of dry extract (mg QE/g) ⁽²⁾.

Statistical analysis

Each treatment had two replicates, and experiments were conducted at least three times using a randomized design. Results are expressed as mean ± standard deviation/standard error. Statistical significance of the data was determined by ANOVA followed by Tukey’s test (MINITAB software version 15 for Windows, SPSS Inc., Chicago, IL), p ≥0.05. Pearson’s correlation coefficient was calculated between AA and phenols or flavonoid content of EEs using InfoStat software ⁽⁵⁾.

Results

Effect of EOs and EEs on V. dahliae and P. parasiticum spore germination

The AA of five EOs and seven EEs obtained from plants in northwest Argentina was evaluated against spore germination of V. dahliae and P. parasiticum. Inhibition of conidia germination varied among treatments and increased with increasing EO or EE concentration.

Only the EOs from Z. punctata and C. gilliesii exhibited 100% inhibitory activity on V. dahliae spores, with MIC values of 3 mg/mL each (figures 1a and b). At the highest concentration tested, the EOs from L. turbinata and L. integrifolia showed remarkable activity against V. dahliae spores, with inhibition values of 96.4 and 96% respectively (figure 1c and d).

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) S. subulatus var. salsus, (f) Benomyl (Fungicide).

Data are expressed as mean ± standard error, n=6-8. Different letters indicate significant differences among concentrations of the same EO. (p˂ 0.05).

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) S. subulatus var. salsus, (f) Benomil (fungicida).

Los datos se expresan como la media ± error estándar, n=6-8. Letras distintas indican diferencias significativas entre concentraciones del mismo AE. (p˂ 0,05).

Figure 1. Effect of essential oils (EOs) on V. dahliae spore germination.

Figura 1. Efecto de los aceites esenciales (AE) sobre la germinación de las esporas de V. dahliae.

Concerning spore germination of P. parasiticum, no EO had a 100% inhibitory effect. C. gilliesii EO inhibited 85.8% of spores at 3 mg/mL, while the remaining oils showed low activity, with 30-54 % inhibition at the highest concentration evaluated (Supplementary Figure 2).

On the other hand, in assays with EEs, only Z. punctata EE effectively controlled spore germination of both pathogenic fungi (figure 2a and 3a). The effective concentration (MIC) of this extract on V. dahliae was 0.4 mg/mL, similar to the MIC obtained with the synthetic antifungal Benomyl (MIC=0.3 mg/mL) (figure 2a and h).

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) A. subfusiformis, (f) E. gilliesii, (g) S. subulatus var. salsus, (h) Benomyl (Fungicide). Data are expressed as mean ± standard error, n=6- 8. Different letters indicate significant differences among concentrations of the same EE. (p˂ 0.05).

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) A. subfusiformis, (f) E. gilliesii, (g) S. subulatus var. salsus, (h) Benomyl (Fungicida). Los datos se expresan como la media ± error estándar, n=6-8. Letras distintas indican diferencias significativas entre concentraciones del mismo EE. (p˂ 0,05).

Figure 2. Effect of ethanolic extracts (EEs) on V. dahliae spore germination.

Figura 2. Efecto de los extractos etanólicos (EEs) sobre la germinación de esporas de V. dahliae.

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) A. subfusiformis, (f) E. gilliesii, (g) S. subulatus var. salsus, (h) Benomyl (Fungicide). Data are expressed as mean ± standard error, n=6-8. Different letters indicate significant differences among concentrations of the same EE. (p˂ 0.05).

Figure 3. Effect of ethanolic extracts (EEs) on P. parasiticum spore germination.

Figura 3. Efecto de los extractos etanólicos (EEs) sobre la germinación de esporas de P. parasiticum.

Spore germination of V. dahliae was also completely inhibited at 3 mg/ml of C. gilliesii EE (MIC), while other EEs showed inhibitions ranging between 54% and 89% (figure 2). P. parasiticum spore germination was controlled at 0.75 mg/mL of Z. punctata EE (MIC), a much lower value than the obtained with the antifungal Benomyl (MIC=10 mg/mL) (figure 3a and h). In addition, significant inhibition of P. parasiticum spore germination (94%) was obtained at 3 mg/mL of E. gilliesii EE (figure 3f).

Evaluation of synergistic antifungal effect

The results demonstrated no synergistic effect against V. dahliae and P. parasiticum spores for any of the evaluated combinations. Antifungal interaction was additive or indifferent (Supplementary Table 2).

Effect of EEs on mycelial growth of V. dahliae and P. parasiticum

Since no EOs could completely inhibit P. parasiticum spore germination, and their activity on V. dahliae spore germination was weaker than the extracts, assays considering mycelial growth inhibition were performed with EEs only.

Mycelial growth inhibition increased with EEs concentration. All seven EEs tested showed growth inhibition of over 25% for both phytopathogens (figure 4 and figure 5).

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) A. subfusiformis, (f) E. gilliesii, (g) S. subulatus var. salsus, (h) Benomyl (Fungicide). The percentage of inhibition was determined at 7 and 9 days of incubation at 24°C. Data are expressed as mean ± standard error, n=6- 8. Different letters indicate significant differences among the concentrations tested (p< 0.05).

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) A. subfusiformis, (f) E. gilliesii, (g) S. subulatus var. salsus, (h) Benomyl (Fungicida). El porcentaje de inhibición se determinó a los 7 y 9 días de incubación a 24°C. Los datos se expresan como media ± error estándar, n=6-8. Letras diferentes indican diferencias significativas entre las concentraciones ensayadas. (p< 0.05).

Figure 4. Effect of ethanolic extracts (EEs) on V. dahliae mycelial growth.

Figura 4. Efecto de los extractos etanólicos (EE) sobre el crecimiento micelial de V. dahliae.

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) A. subfusiformis, (f) E. gilliesii, (g) S. subulatus var. salsus, (h) Benomyl (Fungicide). The percentage of inhibition was determined at 7, 9 and 12 days of incubation at 24°C. Data are expressed as mean ± standard error, n=6-8. Different letters indicate significant differences among concentrations. (p< 0.05).

(a) Z. punctata, (b) C. gilliesii, (c) L. turbinata, (d) L. integrifolia, (e) A. subfusiformis, (f) E. gilliesii, (g) S. subulatus var. salsus, (h) Benomyl (Fungicida). El porcentaje de inhibición se determinó a los 7, 9 y 12 días de incubación a 24°C. Los datos se expresan como media ± error estándar, n=6-8. Letras diferentes indican diferencias significativas entre concentraciones. (p< 0,05).

Figure 5. Effect of ethanolic extracts (EEs) on P. parasiticum mycelial growth.

Figura 5. Efecto de los extractos etanólicos (EE) sobre el crecimiento micelial de P. parasiticum.

Considering EEs inhibitory effect on V. dahliae, three treatments (EEs from Z. punctata, C. gilliesii and L. turbinata) completely inhibited mycelial growth (figure 4a-c). Z. punctata EE was the most effective, obtaining the lowest MIC value (MIC=1.5 mg/mL for Z. punctata EE, MIC=2.5 mg/mL for C. gilliesii EE and MIC=3 mg/mL for L. turbinata EE; (figure 4a-c). The EEs of L. integrifolia and E. gilliesii reached inhibition values of 93.7% and 89.5% against V. dahliae at 3 mg/mL (figure 4d and f). The two remaining EE treatments (A. subfusiformis and S. subulatus) achieved 60-70% inhibition figure 4e and g. Given that no mycelium re-growth occurred during additional incubation time (day 9), Z. punctata, L. turbinata, L. integrifolia, A. subfusiformis and S. subulatus EEs resulted fungicidal against V. dahliae (figure 4). In the case of C. gilliesii and E. gilliesii, mycelial recovery was observed at 9 days. The C. gilliesii EE MIC value changed from 2.5 to 3 mg/mL while inhibition percentage of E. gilliesii EE at 3 mg/mL decreased significantly (figure 4b and f). Thus, AA of these EEs on V. dahliae was considered fungistatic.

P. parasiticum mycelial growth was completely inhibited by Z. punctata and L. turbinata EEs (MIC=1 mg/mL for Z. punctata EE and MIC=2 mg/mL for L. turbinata EE) (figure 5a and c). In addition, the EEs of C. gilliesii, L. integrifolia, and E. gilliesii showed strong inhibitory effects on P. parasiticum mycelial growth reaching 92.6, 81.4 and 84.9% at 3 mg/mL, respectively (figure 5b, d and f). The EE treatments A. subfusiformis and S. subulatus also inhibited 60-70% of mycelial growth (figure 5e and g). The EE treatments produced reversible inhibition of P. parasiticum mycelium growth. During additional incubation time, the MIC value of Z. punctata and L. turbinata EEs increased to 2 mg/mL and 3 mg/mL, respectively. A significant reduction in inhibition percentage of the other EEs was also observed on day 12 (figure 5). Therefore, these EEs were fungistatic against P. parasiticum.

Phenolic and flavonoid contents (PC and FC) in EEs

Both PC and FC of the studied EEs were significantly different (Supplementary Table 3). The EE of Z. punctata showed the highest PC, followed by C. gilliesii EE, L. turbinata EE, L. integrifolia EE = A. subfusiformis EE = E. gilliesii EE, and S. subulatus EE. Regarding FC, Z. punctata EE presented the highest value (327.6 mg QE/g) and the other EEs ranged between 13 and 73 mg QE/g.

Pearson’s correlation coefficient between PC and spore germination inhibition was r=0.63 (p < 0.0001) for V. dahliae and r=0.39 (p < 0.0001) for P. parasiticum. Additionally, a significant correlation was observed between PC and mycelial growth inhibition, with values of r=0.79 (p < 0.0001) for V. dahliae and r=0.78 (p < 0.0001) for P. parasiticum. The FC and inhibition of spore germination showed correlation coefficients of r=0.73 (p < 0.0001) for V. dahliae and r=0.72 (p < 0.0001) for P. parasiticum, while for FC and mycelial growth inhibition, r=0.60 (p < 0.0001) was observed for V. dahliae and r=0.67 (p < 0.0001) for P. parasiticum.

Discussion

V. dahliae and P. parasiticum are important phytopathogens in La Rioja province, involved in Verticillium wilt of olive and grapevine trunk diseases, respectively ⁽^10,
20⁾. Given the lack of control treatments, searching for antifungal agents is strategic ⁽^{17, 19}⁾. We tested EOs and EEs from seven Argentinian northwest plants as natural alternatives against V. dahliae and P. parasiticum.

Three mg/mL of our EOs had remarkable activity against V. dahliae spore germination (100% inhibitory activity for Z. punctata and C. gilliesii EOs, and 96% inhibitory activity for L. turbinata and L. integrifolia EOs). Similarly, other EOs (oregano, thyme, laurel, and lavender) block V. dahliae conidia germination at concentrations ranging from 0.2 to 3 mg/mL ⁽^{11, 14}⁾.

On the other hand, only the C. gilliesii EO was able to significantly inhibit P. parasiticum conidia germination, suggesting V. dahliae spores are more susceptible to the tested EOs than P. parasiticum. In addition, although previous reports demonstrated the activity of C. gilliesii and L. turbinata EOs against other phytopathogenic fungi ⁽^{15,
22, 27}⁾, this is the first report on AA of Z. punctata and L. integrifolia EOs against this type of pathogens.

Based on EEs activity on conidia germination, only Z. punctata EE was able to control both V. dahliae (MIC= 0.4 mg/mL) and P. parasiticum (MIC=0.75 mg/mL). These results coincide with previous research showing the Z. punctata EE effectiveness against soybean pathogenic and brown rot fungi spore development at concentrations between 0.25-0.5 mg/mL ⁽^4,
23⁾. Our results also showed that C. gilliesii EE controlled germination of V. dahliae spores, apparently never studied before against phytopathogenic fungi.

Although no synergistic antifungal effect was found for the mixtures of EO and EE tested, antagonistic absence and additive effects of the combinations of Z. punctata EO/Z. punctata EE and C. gilliesii EO/Z. punctata EE, constitute encouraging outcomes. This suggests that the botanical effective antifungal concentration (MIC) could be halved when combined.

On the other hand, the most effective inhibitors of mycelial growth of both phytopathogens were Z. punctata, C. gilliesii, and L. turbinata EEs. All three extracts behaved as fungistatic on P. parasiticum, while Z. punctata and L. turbinata EEs killed V. dahliae mycelium (fungicidal effect), evidencing that V. dahliae vegetative growth was more susceptible to our EEs than P. parasiticum.

In vitro studies with Z. punctata EE at 1.6 mg/mL could not completely inhibit hyphal growth of Fusarium species associated with Ear Rot in cereals ⁽¹³⁾. In contrast, our findings showed that the AA of Z. punctata EE, ranging from 1-1.5 mg/mL could completely inhibit mycelial growth of V. dahliae and P. parasiticum. Results also showed that Z. punctata EE MIC values were 2-3 times lower on the spores than on the mycelium of both phytopathogens, consistent with previous findings ⁽¹³⁾. Considering C. gilliesii EE, MIC was similar for conidia germination and mycelial growth of V. dahliae. Surprisingly, a complete reduction of V. dahliae and P. parasiticum mycelial growth was observed with L. turbinata EE. However, it did not provide complete control over conidia germination, suggesting a differential effect of extract components on each fungal structure.

Considering all the evaluated EEs, Z. punctata EE was the most effective at suppressing spore germination and mycelial growth. Previous research has corroborated the AA of Z. punctata EE against other phytopathogenic fungi, attributing this property to polyphenolic compounds, especially chalcone type ⁽^{4,
13, 23}⁾. Considering the difference in the AA observed among the different EEs evaluated, phenols and flavonoid content were quantified, showing that Z. punctata EE had the highest content of phenols and flavonoids likely responsible for its potent AA.

Finally, we found that total phenols had the best correlation with mycelial growth inhibition, while flavonoid levels best correlated with inhibition of spore germination. Thus, the AA of studied EEs on conidial germination could be mainly attributed to flavonoid content, while phenols would be responsible for inhibitory effects on mycelial growth.

Conclusions

This work searched for antifungals of plant origin against pathogenic fungi involved in grapevine trunk diseases and Verticillium wilt of olive. We explored the in vitro antifungal properties of five EOs and seven EEs obtained from Argentinian northwest plants. All tested EOs and EEs showed varying AA degrees against both phytopathogenic fungi. This activity depended on plant species, extract type (EO or EE), pathogen identity, and targeted fungal structures. According to our findings, the products obtained from Z. punctata, C gilliesii and L. turbinata were the most effective against V. dahliae and P. parasiticum, suggesting their potential as biofungicides for integrated disease control. This is particularly encouraging considering absent effective treatments against these two pathogens. Further research should determine antifungal effectiveness of these botanical products in plants and identify their specific antifungal compounds.

Acknowledgments

This research was funded by Secretaría de Políticas Universitarias and Universidad Nacional de Chilecito, Argentina (grants PAFCyT I+D 35/18 and FICyT-2022).

We thank Translator A. López López for improving English in the manuscript and M.J. Loyola from UNDEC Herbarium for assisting with plant taxonomic identification.

M.S is a fellow and N.B is a researcher, both at CONICET, Argentina.

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