In Vitro Antifungal Activity and Chemical Composition of Piper auritum Kunth Essential Oil against Fusarium oxysporum and Fusarium equiseti

The essential oils of plants of the genus Piper have secondary metabolites that have antimicrobial activity related to their chemical composition. The objective of our work was to determine the chemical composition and evaluate the antifungal activity of the aerial part essential oil of P. auritum obtained by hydrodistillation on Fusarium oxysporum and Fusarium equiseti isolated from Capsicum chinense. The antifungal activity was evaluated by direct contact and poisoned food tests, and the minimum inhibitory concentration (MIC50) and maximum radial growth inhibition (MGI) were determined. The identification of oil metabolites was carried out by direct analysis in real time mass spectrometry (DART-MS). By direct contact, the essential oil reached an inhibition of over 40% on Fusarium spp. The 8.4 mg/mL concentration showed the highest inhibition on F. oxysporum (40–60%) and F. equiseti (>50%). The MIC50 was 6 mg/mL for F. oxysporum FCHA-T7 and 9 mg/mL for F. oxysporum FCHJ-T6 and F. equiseti FCHE-T8. DART-MS chemical analysis of the essential oil showed [2M-H]− and [M-H]− adducts of high relative intensity that were mainly attributed to eugenol and thymol/p-cimen-8-ol. The findings found in this study show a fungistatic effect of the essential oil of P. auritum on Fusarium spp.


Introduction
Diseases caused by pathogenic fungi transmitted by the soil constitute an important limitation for the yield and quality of vegetable crops and fruit in intensive agriculture [1]. Chili (Capsicum spp.) is a fruit and spice native to South and Central America [2], which belongs to the Solanaceae family and is known for its flavor and pungency [3]. The genus Capsicum is made up of a group of herbaceous plants [4], of which five species (C. annuum, C. baccatum, C. chinense, C. frutescens and C. pubescens) were domesticated and are currently cultivated in different parts of the world [4,5]. Therefore, from an economic point of view, it is one of the most important vegetables worldwide [4]. chemical composition and evaluate the antifungal activity of the essential oil of the aerial part of P. auritum on the species of F. oxysporum and F. equiseti causing the wilt in chile.

Plant Material and Essential Oil
Leaves and inflorescences (aerial part) of P. auritum were used, which were collected from plants in flowering stage in the municipality of Ocozocoautla de Espinosa, Chiapas in the community of Ocuilapa de Juárez (290 • W, between 16 • 51 16 N and 93 • 24 40 W, 940 mamsl). The leaves and inflorescences were dried at room temperature for 22 days. The dried and ground plant material was stored in conditions of darkness and room temperature until use. The essential oil extraction was carried out by Clevenger hydrodistillation using 20 g of dry plant material and 300 mL of distilled water, for 1 h at 250 • C and at 1000 rpm. Subsequently, the essential oil was separated from the aqueous phase with ethyl ether, the organic phase was collected and evaporated to dryness [24]. The yield in percentage by weight (%, w/w) of the essential oil was calculated with the following equation: Yield (%) = (W1 × 100)/W2 (1) where W1 is the weight of the essential oil after evaporation of the solvent and W2 is the weight of the dry and ground plant material used for extraction [25]. The density and viscosity parameters of the essential oil were determined with an Anton Paar ® Stabinger model SVM 3000 digital viscometer.

Direct Analysis in Real Time Mass Spectrometry (DART-MS)
For the DART-MS analysis, the essential oil was directly analyzed as a liquid material of organic kind. A capillary tube was immersed in an aliquot of undiluted P. auritum essential oil, then the capillary tube was placed between the helium stream from the DART ionization source and the vacuum interface to obtain mass spectra (MS) [26]. The analysis was carried out on a JMS-T100LP AccuTOF LC-PLUS spectrometer (JEOL, Tokyo, Japan) with a DART SVP100 ion source (Ionsense, Saugus, MA, USA). The DART-MS conditions were in negative mode, the DART ion source was run with helium for analysis and nitrogen for standard mode, the gas temperature (He) was 300 • C, the inlet pressure was 0.55 MPa, and the voltage was ±600 V. The acquisition of the mass spectra was recorded with the Mass Center System Version 1.5.0 k software in a mass range of m/z 50-1000 Da. The analysis was performed between 15 s to 90 s, and the sample was detected at least three times [26].

Fusarium Strains
The F. oxysporum FCHJ-T6, F. oxysporum FCHA-T7, and F. equiseti FCHE-T8 (GenBank number MG020428, MG020429, MG020433, respectively) strains were provided by Dr. Jairo Cristóbal Alejo and belong to the microbial collection of the Instituto Tecnológico de Conkal. The phytopathogens were isolated from the roots of the habanero pepper (C. chinense). The Fusarium strains were seeded onto 50 mm diameter Petri dishes with potato dextrose agar (PDA) and incubated at 30 ± 2 • C for seven days. On the periphery of the plate, sterile 6 mm diameter Whatman ® No. 1 filter paper discs were placed to promote the growth of the phytopathogen on them. Mycelial discs were used as inoculum in all bioassays [27].

Mycelium Disk Microdiffusion Assay
The antifungal effect by direct contact of the essential oil was determined using the disk microdiffusion method with mycelium [23]. The test was carried out using 50 mm diameter Petri dishes with sterile PDA medium. In each plate, a sterile 6 mm diameter Whatman ® No. 1 filter paper disk was deposited on the solidified medium. A volume of 10 µL of essential oil was added to each disc, and then a mycelial disc of Fusarium sp. was placed on them, ensuring direct contact of the mycelium of the fungus with the essential oil. Sterile distilled water was used as a negative control and Sportak ® (prochloraz) prepared at a concentration of 1.5 mL/L according to the manufacturer's instructions as a positive control. The plates were sealed and incubated at 30 ± 2 • C. Three repetitions were used for all the treatments. The mycelial diameter of the fungus was measured with a graduated ruler every 24 h (average of two measurements diametrically opposite) for five days for FCHA-T7 and FCHJ-T6, and six days for FCHE-T8 until the negative control reached the edge of the plate. The evaluation of the percentage of inhibition of radial growth (PIRG) was carried out using the following equation: where RC is the radius of the mycelium of the negative control and RT is the radius of the mycelium of the treatment [21].

Poisoned Food Assay
The effect of the essential oil concentration on the antifungal activity on F. oxysporum FCHJ-T6 and FCHA-T7 and F. equiseti FCHE-T8 was determined using the previously reported poisoned food technique [28][29][30]. The essential oil was incorporated into the sterilized and cooled PDA (~50 • C) at the proportions required to reach 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 3 mg/mL, 5 mg/mL, and 8.4 mg/mL of medium and was poured into sterile 50 mm diameter Petri dishes. Subsequently, a 6 mm diameter mycelial disk of F. oxysporum FCHJ-T6 and FCHA-T7, and F. equiseti FCHE-T8 was placed in the center of the Petri dish with agar. The disk was placed with the mycelium side down [31]. Two types of control and a solvent control were established. The negative control consisted of Petri dishes treated with sterile distilled water (Petri dishes containing PDA without essential oil), the positive control consisted of plates treated with Sportak ® (prochloraz) prepared at a concentration of 1.5 mL/L according to the manufacturer's instructions and the solvent control consisted of Petri dishes containing PDA plus aliquots of ethyl ether used to dissolve the essential oil prior to its incorporation into the sterilized and cooled PDA. The Petri dishes were sealed and incubated in the dark at 30 ± 2 • C. The mycelial diameter was measured every 24 h (average of two measurements diametrically opposite) for six days for FCHA-T7 and FCHJ-T6 and, seven days for FCHE-T8 until the negative control reached the edge of the plate. Three repetitions per treatment were used [31][32][33]. Radial growth inhibition was calculated as a percentage using Equation (2).

MGI and MIC 50 Determination
MGI was defined as the concentration of the essential oil that caused the maximum inhibition of radial growth and MIC 50 as the minimum concentration of essential oil that inhibited 50% of the radial growth [34]. The essential oil was added to PDA medium sterilized at 35 • C [32] to reach final concentrations of 5.0 mg/mL, 6.0, 7.0 mg/mL, 8.0 mg/mL, and 9.0 mg/mL. The inoculation was carried out by placing a 6 mm diameter mycelial disk of each fungus in the center of the plate. The negative control contained only PDA. Incubation conditions were in the dark at a temperature of 30 ± 2 • C [31]. All treatments were carried out in triplicate [35]. Measurement of mycelial growth was performed to determine the MGI and MIC 50 . The radial growth inhibition percentages were calculated using Equation (2).

Statistical Analysis
Statistical analysis was carried out using a completely randomized design of experiments and the data obtained were analyzed with one-way ANOVA and Tukey's test at 95% reliability using the STATGRAPHICS Centurion XV.II software, StatPoint Technologies Inc., The Plains, VA, USA.

Aerial Part Essential Oil of P. auritum
Extraction of the essential oil by hydrodistillation using a Clevenger apparatus showed a yield of 2.20% (w/w, dry basis) ( Figure 1). The aerial part essential oil extracted consisted of a translucent yellow liquid, oily to the touch, with a light consistency and a strong characteristic odor (Table 1). In the Piper genus, a yield of 3.0% (w/w, dry basis) has been reported for the aerial part essential oil of P. divaricatum obtained by this same technique [36], and 1.58% (w/w, dry basis) for the oil essential from P. auritum leaves [37]. Thus, the yield obtained from leaves and inflorescences of P. auritum is within the previously reported values.

Aerial Part Essential Oil of P. auritum
Extraction of the essential oil by hydrodistillation using a Clevenger apparatus showed a yield of 2.20% (w/w, dry basis) ( Figure 1). The aerial part essential oil extracted consisted of a translucent yellow liquid, oily to the touch, with a light consistency and a strong characteristic odor (Table 1). In the Piper genus, a yield of 3.0% (w/w, dry basis) has been reported for the aerial part essential oil of P. divaricatum obtained by this same technique [36], and 1.58% (w/w, dry basis) for the oil essential from P. auritum leaves [37]. Thus, the yield obtained from leaves and inflorescences of P. auritum is within the previously reported values.

Mass Spectrometry by Direct Analysis in Real-Time (DART-MS)
The chemical profile derived from DART-MS for the aerial part essential oil of P. auritum is presented in Figure 2. Several molecular ions were observed, the most abundant being 149.0308 and 163.0181 m/z. Based on previous reports, it was possible to make assignments for several of these observed m/z ions of the metabolites present in extracts of P. auritum. The ions 149.0308, 163.0181, and 193.0237 m/z corresponded to [M-H] − and [M-H+O2] − adducts of compounds with formula C10H14O, C10H12O2 and C10H10O2, which is consistent with the possible presence of thymol, eugenol and safrole, respectively ( Table  2). Table 3 shows the metabolites with molecular formula M that are assigned to the adducts found in the DART mass spectrum. Most of the m/z molecular ions (adducts) were attributed to compounds of a terpenic (cymene and thymol/p-cyme-8-ol) and phe-

Mass Spectrometry by Direct Analysis in Real-Time (DART-MS)
The chemical profile derived from DART-MS for the aerial part essential oil of P. auritum is presented in Figure 2. Several molecular ions were observed, the most abundant being 149.0308 and 163.0181 m/z. Based on previous reports, it was possible to make assignments for several of these observed m/z ions of the metabolites present in extracts of P. auritum.  Table 2). Table 3 shows the metabolites with molecular formula M that are assigned to the adducts found in the DART mass spectrum. Most of the m/z molecular ions (adducts) were attributed to compounds of a terpenic (cymene and thymol/p-cyme-8-ol) and phenylpropanoid kind (safrole and eugenol), although compounds belonging to the alcohol group (Z3, Z6, E8-dodecatrien-1-ol), aldehyde (2-hexenal), ketone (4-hydroxy-4methyl-2-pentanone), ester (methyl 2,4-decadienoate), fatty acid (hexadecanoic acid), and alkane (tetradecane) were also detected.

Mycelium Disc Microdiffusion Assay
The mycelium disk microdiffusion test is used to demonstrate the natural antifungal effect of the essential oil by direct contact on the fungal microorganism. The results of the biological activity of the essential oil of P. auritum on F. oxysporum FCHJ-T6 and FCHA-T7, and F. equiseti FCHE-T8 after 120 h and 144 h, respectively, are presented in Table 4. The inhibition percentages were greater than 60% in FCHJ-T6 and FCHA-T7 at five days and less than 50% in FCHE-T8 at six days. The results found in the present study indicate that the antifungal effect of the essential oil on the three phytopathogens was also fungistatic.  1 Most abundant ions in DART-MS spectrum. The essential oil was analyzed in negative mode with a heated argon gas temperature of 300 • C by DART-MS. The analysis was performed between 15 s to 90 s, and the sample was detected at least three times. Metabolites were observed in most of the DART-MS spectra. The mass to charge ratio (m/z) value of adduct is indicated. The letter M indicates the molecular weight (MW) of the metabolite (formula).

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopathogens using PDA added with essential oil at six different concentrations. The results of F. oxysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In general, an increase in mycelial growth inhibition was observed at the highest concentrations. EO-8.4 showed the greatest inhibition on the three isolates, greater than 50% for FCHJ-T6 and FCHE-T8.
The in vitro antifungal activity of essential oils from Piper spp. at different concentrations against fungi have been previously studied, observing in most cases, fungistatic effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum showed a high antifungal activity against F. solani f. sp. piperis (greater than 90%) after seven days at a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part essential oil of P. auritum in poisoned food test corroborates the fungistatic effect, and the inhibition percentage was dependent on the concentration used.

MGI and MIC 50 Determination
Due to the fungistatic effect of aerial part essential oil of P. auritum on Fusarium spp., the MGI and MIC 50 of the aromatic extract were determined. The results are presented in Table 6. For FCHJ-T6 and FCHE-T8, the MGI and MIC 50 were found at 9 mg/mL, while FCHA-T7 showed a MIC 50 at 6 mg/mL and an MGI at 9 mg/mL (66.7%).

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopatho using PDA added with essential oil at six different concentrations. The results of ysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In eral, an increase in mycelial growth inhibition was observed at the highest conce tions.  showed the greatest inhibition on the three isolates, greater than 50 FCHJ-T6 and FCHE-T8.

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopathogens using PDA added with essential oil at six different concentrations. The results of F. oxysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In general, an increase in mycelial growth inhibition was observed at the highest concentrations.  showed the greatest inhibition on the three isolates, greater than 50% for FCHJ-T6 and FCHE-T8.

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopathogens using PDA added with essential oil at six different concentrations. The results of F. oxysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In general, an increase in mycelial growth inhibition was observed at the highest concentrations.  showed the greatest inhibition on the three isolates, greater than 50% for FCHJ-T6 and FCHE-T8.

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopatho using PDA added with essential oil at six different concentrations. The results of ysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In eral, an increase in mycelial growth inhibition was observed at the highest conce tions.  showed the greatest inhibition on the three isolates, greater than 50 FCHJ-T6 and FCHE-T8.

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopathogens using PDA added with essential oil at six different concentrations. The results of F. oxysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In general, an increase in mycelial growth inhibition was observed at the highest concentrations.  showed the greatest inhibition on the three isolates, greater than 50% for FCHJ-T6 and FCHE-T8.

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopathogens using PDA added with essential oil at six different concentrations. The results of F. oxysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In general, an increase in mycelial growth inhibition was observed at the highest concentrations.  showed the greatest inhibition on the three isolates, greater than 50% for FCHJ-T6 and FCHE-T8. 42

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopatho using PDA added with essential oil at six different concentrations. The results of ysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In eral, an increase in mycelial growth inhibition was observed at the highest conce tions.  showed the greatest inhibition on the three isolates, greater than 50 FCHJ-T6 and FCHE-T8.

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopathogens using PDA added with essential oil at six different concentrations. The results of F. oxysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In general, an increase in mycelial growth inhibition was observed at the highest concentrations.  showed the greatest inhibition on the three isolates, greater than 50% for FCHJ-T6 and FCHE-T8.

Poisoned Food Assay
The biological activity of the essential oil was tested on the three phytopathogens using PDA added with essential oil at six different concentrations. The results of F. oxysporum and F. equiseti after 144 h and 168 h, respectively, are shown in Table 5. In general, an increase in mycelial growth inhibition was observed at the highest concentrations.  showed the greatest inhibition on the three isolates, greater than 50% for FCHJ-T6 and FCHE-T8.         The values are presented in percentage of inhibition of radial growth and correspond to the average standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): ne distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicat mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper s trations against fungi have been previously studied, observing in effect by the bioextracts evaluated. The aerial part essential oil of high antifungal activity against F. solani f. sp. piperis (greater than a concentration of 5 mg/mL [36]. Therefore, the antifungal effect o of P. auritum in poisoned food test corroborates the fungistatic e percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repet standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentra mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at differe trations against fungi have been previously studied, observing in most cases, effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum high antifungal activity against F. solani f. sp. piperis (greater than 90%) after se a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part e of P. auritum in poisoned food test corroborates the fungistatic effect, and the percentage was dependent on the concentration used. The values are presented in percentage of inhibition of radial growth and correspond to the average of three repetitions ± standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control (sterile distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentration in mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at different concentrations against fungi have been previously studied, observing in most cases, fungistatic effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum showed a high antifungal activity against F. solani f. sp. piperis (greater than 90%) after seven days at a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part essential oil of P. auritum in poisoned food test corroborates the fungistatic effect, and the inhibition percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): ne distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicat mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper s trations against fungi have been previously studied, observing in effect by the bioextracts evaluated. The aerial part essential oil of high antifungal activity against F. solani f. sp. piperis (greater than a concentration of 5 mg/mL [36]. Therefore, the antifungal effect o of P. auritum in poisoned food test corroborates the fungistatic e percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repet standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentra mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at differe trations against fungi have been previously studied, observing in most cases, effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum high antifungal activity against F. solani f. sp. piperis (greater than 90%) after se a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part e of P. auritum in poisoned food test corroborates the fungistatic effect, and the percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repetitions ± standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control (sterile distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentration in mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at different concentrations against fungi have been previously studied, observing in most cases, fungistatic effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum showed a high antifungal activity against F. solani f. sp. piperis (greater than 90%) after seven days at a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part essential oil of P. auritum in poisoned food test corroborates the fungistatic effect, and the inhibition percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): ne distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicat mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper s trations against fungi have been previously studied, observing in effect by the bioextracts evaluated. The aerial part essential oil of high antifungal activity against F. solani f. sp. piperis (greater than a concentration of 5 mg/mL [36]. Therefore, the antifungal effect o of P. auritum in poisoned food test corroborates the fungistatic e percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repeti standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentra mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at differe trations against fungi have been previously studied, observing in most cases, effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum high antifungal activity against F. solani f. sp. piperis (greater than 90%) after sev a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part e of P. auritum in poisoned food test corroborates the fungistatic effect, and the percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repetitions ± standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control (sterile distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentration in mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at different concentrations against fungi have been previously studied, observing in most cases, fungistatic effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum showed a high antifungal activity against F. solani f. sp. piperis (greater than 90%) after seven days at a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part essential oil of P. auritum in poisoned food test corroborates the fungistatic effect, and the inhibition percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): ne distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicat mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper s trations against fungi have been previously studied, observing in effect by the bioextracts evaluated. The aerial part essential oil of high antifungal activity against F. solani f. sp. piperis (greater than a concentration of 5 mg/mL [36]. Therefore, the antifungal effect o of P. auritum in poisoned food test corroborates the fungistatic e percentage was dependent on the concentration used.

± 2.9c
(mg/mL). The values are presented in percentage of inhibition of radial growth and correspond to the average of three repet standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentra mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at differe trations against fungi have been previously studied, observing in most cases, effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum high antifungal activity against F. solani f. sp. piperis (greater than 90%) after se a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part e of P. auritum in poisoned food test corroborates the fungistatic effect, and the percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repetitions ± standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control (sterile distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentration in mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at different concentrations against fungi have been previously studied, observing in most cases, fungistatic effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum showed a high antifungal activity against F. solani f. sp. piperis (greater than 90%) after seven days at a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part essential oil of P. auritum in poisoned food test corroborates the fungistatic effect, and the inhibition percentage was dependent on the concentration used.
46.4 ± 3.3bc  (mg/mL). The values are presented in percentage of inhibition of radial growth and correspond to the average standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): ne distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicat mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper s trations against fungi have been previously studied, observing in effect by the bioextracts evaluated. The aerial part essential oil of high antifungal activity against F. solani f. sp. piperis (greater than a concentration of 5 mg/mL [36]. Therefore, the antifungal effect o of P. auritum in poisoned food test corroborates the fungistatic e percentage was dependent on the concentration used. 57.8 ± 7.4b (mg/mL). The values are presented in percentage of inhibition of radial growth and correspond to the average of three repeti standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentra mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at differe trations against fungi have been previously studied, observing in most cases, effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum high antifungal activity against F. solani f. sp. piperis (greater than 90%) after se a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part e of P. auritum in poisoned food test corroborates the fungistatic effect, and the percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repetitions ± standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control (sterile distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentration in mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at different concentrations against fungi have been previously studied, observing in most cases, fungistatic effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum showed a high antifungal activity against F. solani f. sp. piperis (greater than 90%) after seven days at a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part essential oil of P. auritum in poisoned food test corroborates the fungistatic effect, and the inhibition percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): ne distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicat mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper s trations against fungi have been previously studied, observing in effect by the bioextracts evaluated. The aerial part essential oil of high antifungal activity against F. solani f. sp. piperis (greater than a concentration of 5 mg/mL [36]. Therefore, the antifungal effect o of P. auritum in poisoned food test corroborates the fungistatic e percentage was dependent on the concentration used.  The values are presented in percentage of inhibition of radial growth and correspond to the average of three repeti standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentra mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at differe trations against fungi have been previously studied, observing in most cases, effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum high antifungal activity against F. solani f. sp. piperis (greater than 90%) after se a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part e of P. auritum in poisoned food test corroborates the fungistatic effect, and the percentage was dependent on the concentration used.
100.0 ± 0.0a (mg/mL). The values are presented in percentage of inhibition of radial growth and correspond to the average of three repetitions ± standard error. Values with different letters in the same column are different (p ≤ 0.05). H2O (−): negative control (sterile distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentration in mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ).
The in vitro antifungal activity of essential oils from Piper spp. at different concentrations against fungi have been previously studied, observing in most cases, fungistatic effect by the bioextracts evaluated. The aerial part essential oil of P. divaricatum showed a high antifungal activity against F. solani f. sp. piperis (greater than 90%) after seven days at a concentration of 5 mg/mL [36]. Therefore, the antifungal effect of aerial part essential oil of P. auritum in poisoned food test corroborates the fungistatic effect, and the inhibition percentage was dependent on the concentration used.
The values are presented in percentage of inhibition of radial growth and correspond to the average of three repetitions ± standard error. Values with different letters in the same column are different (p ≤ 0.05). H 2 O (−): negative control (sterile distilled water); EO: essential oil from the P. auritum aerial part, the numbers after the dash indicate the concentration in mg/mL at which it was tested; and SPK (+): positive control (prochloraz, Sportak ® ). Table 6. Fungitoxicity of aerial part essential oil of P. auritum on Fusarium spp.

Discussion
In general, essential oils account for less than 5% of dry plant material [37] and among them there is a wide diversity of yield and chemical composition [38]. This variation is under the effect of factors such as species and genotype, ecological conditions, growth stage, and extraction methods [39]. In the present study, we obtained a yield of 2.20%, which is within the range reported for this species.
The Piperaceae family is widely used in cuisine and traditional medicine, and one of its best known genera is Piper. The essential oils of plants of this genus have a wide metabolic diversity [36]. There are more than 270 compounds identified in essential oils from Piper species, and more than 80 of these compounds belonged to the mono and sesquiterpene hydrocarbon classes, followed by aldehydes, alcohols, acids, ketones, esters, and phenols. Some species have a simple profile, while others, such as P. auritum, contain very diverse groups of secondary metabolites. Depending on the organ, it is possible to find differences in the chemical composition of the essential oils of fruits, leaves, and aerial parts [40].
The presence of compounds of a terpenic and phenylpropanoid kind in aerial part essential oil of P. auritum tested in this study could be related to the fungal effect on Fusarium spp. observed in tests by direct contact (Table 4) and poisoned food (Table 5). However, the role played by the other components detected in the mechanism of inhibition of fungal cell growth is not ruled out.
The bioactivity of essential oils has been reported to result from the interaction between structural components, particularly the main components, although the other compounds in the oil may also have a vital function due to a synergistic effect [44,45] and the direct fungitoxic action of these compounds [31]. Research on the antimicrobial effects of monoterpenes suggests that they diffuse into cells and damage the cell membrane [46]. In addition, other substances present in the essential oil could inhibit mycelial growth or spore germination [31]. For its part, safrole is a methylenedioxy-type compound and its toxic properties have been associated with its structure as a derivative of benzene [45]. This type of compound can reduce the mycelial growth of some plant pathogens, acting as phytoanticipins [19].
Compounds of botanical origin that exhibit resistance to attack by phytopathogenic fungi, such as the essential oil of P. auritum, can serve as agrochemicals taking advantage of their antifungal activity [13]. Therefore, essential oils are one of the most promising groups of natural compounds as an alternative for the development of antifungal agents that are harmless to the environment in the management of diseases in the field and postharvest, due to the effect they exhibit on phytopathogenic fungi [38].
The control of phytopathogenic microorganisms using essential oils can be improved by nanomaterials since nanoparticles are considered as another control alternative [47]. Lipid nanoparticles obtained by encapsulating essential oil have proven to be more efficient and are currently being studied for various purposes [47,48]. Based on the percentages of inhibition obtained with the aerial part essential oil of P. auritum, this could be a good candidate to be used together with this new technology.

Conclusions
The results in this study show that the aerial part essential oil of P. auritum had a fungistatic effect on the three Fusarium phytopathogenic isolates tested, finding a MIC50 of 9 mg/mL for F. oxysporum (FCHJ-T6) and F. equiseti (FCHE-T8). F. oxysporum (FCHA-T7) was more sensitive to the bioactivity of the oil, with a MIC50 of 6 mg/mL and MGI of 9 mg/mL, with a percentage of mycelial inhibition greater than 60%. On the other hand, the compounds identified by DART-MS presented chemical diversity, pointing out those of a terpenic and phenylpropanoid kind.