In Vitro Antifungal Activity and Chemical Composition of Piper Auritum Kunth Essential Oil Against Fusarium Oxysporum and Fusarium Equiseti

agronomy

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

César Chacón 1 , Emanuel Bojórquez-Quintal 2 , Goretty Caamal-Chan 3,Víctor M. Ruíz-Valdiviezo 1 , Joaquín A. Montes-Molina 1, Eduardo R. Garrido-Ramírez 4 , Luis M. Rojas-Abarca 5 and Nancy Ruiz-Lau 6,*

1 Tecnológico Nacional de México/Instituto Tecnológico de Tuxtla Gutiérrez, Carretera Panamericana km. 1080, 29050 Tuxtla Gutiérrez, Chiapas, Mexico; [email protected] (C.C.); [email protected] (V.M.R.-V.); [email protected] (J.A.M.-M.) 2 CONACyT-Laboratorio de Análisis y Diagnóstico del Patrimonio, El Colegio de Michoacán. A.C., Cerro de Nahuatzen 85, Fracc, Jardines del Cerro Grande, 59370 La Piedad, Michoacán, Mexico; [email protected] 3 CONACyT-Centro de Investigaciones Biológicas del Noroeste, S.C. Instituto Politécnico Nacional 195, Playa Palo de Santa Rita Sur, 23096 La Paz, Baja California Sur, Mexico; [email protected] 4 Campo Experimental Centro de Chiapas-INIFAP, Carretera Ocozocoautla-Cintalapa Km3, 29140 Ocozocoautla, Chiapas, Mexico; [email protected] 5 Laboratorio de Análisis y Diagnóstico del Patrimonio, El Colegio de Michoacán. A.C., Cerro de Nahuatzen 85, Fracc, Jardines del Cerro Grande, 59370 La Piedad, Michoacán, Mexico; [email protected] 6 CONACyT-Tecnológico Nacional de México/Instituto Tecnológico de Tuxtla Gutiérrez, Carretera Panamericana km. 1080, 29050 Tuxtla Gutiérrez, Chiapas, Mexico * Correspondence: [email protected]; Tel.: +52-9993605266 Citation: Chacón, C.; Bojórquez-Quintal, E.; Caamal-Chan, Abstract: The essential oils of plants of the genus Piper have secondary metabolites that have G.; Ruíz-Valdiviezo, V.M.; Montes- Molina, J.A.; Garrido-Ramírez, E.R.; antimicrobial activity related to their chemical composition. The objective of our work was to Rojas-Abarca, L.M.; Ruiz-Lau, N. In determine the chemical composition and evaluate the antifungal activity of the aerial part essential Vitro Antifungal Activity and oil of P. auritum obtained by hydrodistillation on Fusarium oxysporum and Fusarium equiseti isolated Chemical Composition of Piper from Capsicum chinense. The antifungal activity was evaluated by direct contact and poisoned food auritum Kunth Essential Oil against tests, and the minimum inhibitory concentration (MIC50) and maximum radial growth inhibition Fusarium oxysporum and Fusarium (MGI) were determined. The identiﬁcation of oil metabolites was carried out by direct analysis in real equiseti. Agronomy 2021, 11, 1098. time mass spectrometry (DART-MS). By direct contact, the essential oil reached an inhibition of over https://doi.org/10.3390/ 40% on Fusarium spp. The 8.4 mg/mL concentration showed the highest inhibition on F. oxysporum agronomy11061098 (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 Received: 30 April 2021 oil showed [2M-H]− and [M-H]− adducts of high relative intensity that were mainly attributed to Accepted: 25 May 2021 eugenol and thymol/p-cimen-8-ol. The ﬁndings found in this study show a fungistatic effect of the Published: 28 May 2021 essential oil of P. auritum on Fusarium spp.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: aerial part; inhibition; fungistatic; metabolites; safrole; eugenol; thymol published maps and institutional afﬁl- iations.

1. Introduction Diseases caused by pathogenic fungi transmitted by the soil constitute an important Copyright: © 2021 by the authors. limitation for the yield and quality of vegetable crops and fruit in intensive agriculture [1]. Licensee MDPI, Basel, Switzerland. Chili (Capsicum spp.) is a fruit and spice native to South and Central America [2], which This article is an open access article belongs to the Solanaceae family and is known for its ﬂavor and pungency [3]. The genus distributed under the terms and Capsicum is made up of a group of herbaceous plants [4], of which ﬁve species (C. annuum, conditions of the Creative Commons C. baccatum, C. chinense, C. frutescens and C. pubescens) were domesticated and are currently Attribution (CC BY) license (https:// cultivated in different parts of the world [4,5]. Therefore, from an economic point of view, creativecommons.org/licenses/by/ it is one of the most important vegetables worldwide [4]. 4.0/).

Agronomy 2021, 11, 1098. https://doi.org/10.3390/agronomy11061098 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 1098 2 of 13

Fungal wilt, root rot, and charcoal rot are the most damaging diseases for plant crops, including those of the genus Capsicum [6]. Crown and root rots are by far the most widespread disease caused by Fusarium spp., and several species of Fusarium are recognized as causative agents [7]. Recent reports have reported F. oxysporum as a causative agent of chili root and basal stem rot [8], and most shoot vascular wilt is caused by this species [9]. Fusarium equiseti is a pathogenic species associated with leaf spot, root and crown rot, and fruit rot, which cause seed decomposition and seedling infection in different fruit and vegetable crops, as well as cereals and spices [10]. To combat microbial diseases, chemical control methods are applied that are irritating, toxic, mutagenic, teratogenic, and carcinogenic to users, as well as having serious ecological consequences. Synthetic chemicals provided promising results against wilt; however, fungicide application under field conditions involves environmental contamination, in- consistency in efficacy, and high costs [11]. The development of resistance of pathogenic fungi towards synthetic fungicides is of great concern. Therefore, it is important to develop safe, effective, and friendly fungicides with the environment and with the farmers and producers. This generates interest in alternative (green) sources of antifungal compounds that are more environmentally friendly, and that help reduce the intensive use of these products. Plants have, and continue to be, sources of antifungal compounds [12]. Many of these compounds have biological activity, are more degradable than many pesticides, and can significantly reduce the risk of adverse ecological effects [13]. Plant extracts are an alternative method to the use of chemical substances as a green treatment, since they have a wide variety of secondary metabolites that have demonstrated antimicrobial properties [14]. Essential oils produced by aromatic plants are a complex mixture of volatile secondary metabolites [15] known for their natural components, such as monoterpenes, diterpenes, and hydrocarbons with various functional groups, which have been studied for their antifungal activities [16]. Piper is the largest genus of the Piperaceae family, which is distributed throughout the tropics, with significant concentrations in Central and South America, the Caribbean, Africa, Asia, and some Pacific islands. The aroma emitted by its leaves, fruits and roots has caused the species of this genus to be widely used as a flavoring ingredient in cuisine [17]. It is also used for its therapeutic properties in traditional medicine. The secondary metabolites in extracts from various parts of this plant have shown antifungal, insecticidal, antifeedant, bactericidal and cytotoxic activity [18]. Piper’s secondary compounds such as safrole, dilapiol, myristicin, and methylene- dioxyphenyl have been reported to have biological activity [18]. In the analysis of antifungal activity chemical compounds such as safrole and dillapiole obtained from essential oil of P. auritum and P. holtonii, respectively, have been reported to have a direct fungitoxic effect in vitro against Botryodiplodia theobromae and Colletotrichum acutatum, and it was found that the highest values of inhibition of mycelial growth of both fungi were obtained for dillapiole, compared to safrole [19]. There are reports that test different phytoextracts obtained from P. auritum on different phytopathogenic fungi, including the genus Fusarium. The essential oil of P. auritum has been tested against Alternaria solani, inhibiting mycelial growth at seven and fourteen days [20]. In another study, a total inhibition (fungicidal effect) of the mycelial growth of Curvularia lunata, Sarocladium oryzae and Bipolaris oryzae was reported at 96 h [21]. In a previous report, it was observed that the essential oil of P. auritum leaves had an inhibitory effect on the growth of F. oxysporum f. sp. comiteca (75.32%), F. oxysporum f. sp. tequilana (86.57%), and F. solani f. sp. comiteca (63.36%), indicating a fungistatic effect [22]. While in another report, the essential oil of P. auritum caused total inhibition of the growth of F. solani (F2 and F5 isolates) and of F. redolens (F3 isolates) [23]. The increase in food production, the regulations on the use of synthetic fungicides, the development of resistance in phytopathogens justify the search for other control alter- natives [6], as the phytochemical that together with an integrated management of the crop could be successful. Therefore, the objective of the present research was to determine the Agronomy 2021, 11, 1098 3 of 13

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 chili.

2. Materials and Methods 2.1. 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◦5101600 N and 93◦2404000 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.

2.2. 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].

2.3. 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].

2.4. 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 Agronomy 2021, 11, 1098 4 of 13

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:

PIRG (%) = [(RC − RT)/RC] × 100 (2)

where RC is the radius of the mycelium of the negative control and RT is the radius of the mycelium of the treatment [21].

2.5. 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 [28–30]. 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 [31–33]. Radial growth inhibition was calculated as a percentage using Equation (2).

2.6. MGI and MIC50 Determination MGI was deﬁned as the concentration of the essential oil that caused the maximum inhibition of radial growth and MIC50 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 MIC50. The radial growth inhibition percentages were calculated using Equation (2).

2.7. Statistical Analysis Statistical analysis was carried out using a completely randomized design of experi- ments 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. Agronomy 2021, 11, 1098 5 of 13

Agronomy 2021, 11, x FOR PEER REVIEW 5 of 14

3. Results 3.1. Aerial Part Essential Oil of P. auritum 3. Results 3.1.Extraction Aerial Part ofEssential the essential Oil of P. oilauritum by hydrodistillation using a Clevenger apparatus showed a yield of 2.20% (w/w, dry basis) (Figure1). The aerial part essential oil extracted consisted Extraction of the essential oil by hydrodistillation using a Clevenger apparatus ofshowed a translucent a yield of yellow 2.20% ( liquid,w/w, dry oily basis) to (Figure the touch, 1). The with aerial a lightpart essential consistency oil extracted and a strong characteristicconsisted of a odortranslucent (Table yellow1). In liquid, the Piper oily togenus, the touch, a yield with ofa light 3.0% consistency (w/w, dry and basis) a has beenstrong reported characteristic for the odor aerial (Table part 1). In essential the Piper oilgenus, of P.a yield divaricatum of 3.0% (wobtained/w, dry basis) by thishas same techniquebeen reported [36], and for the 1.58% aerial (w / partw, dry essential basis) oil for of the P. oil divaricatum essential obtained from P. auritumby this sameleaves [37]. Thus,technique the yield [36], obtained and 1.58% from (w/w leaves, dry basis) and inﬂorescencesfor the oil essential of P. from auritum P. auritumis within leaves the [3 previously7]. reportedThus, the values. yield obtained from leaves and inflorescences of P. auritum is within the previously reported values.

Figure 1. Extraction of essential oil from aerial part of Pipper auritum through hydrodistillation Figureusing 1.a ClevengerExtraction apparatus. of essential oil from aerial part of Pipper auritum through hydrodistillation using a Clevenger apparatus. Table 1. Extraction of aerial part essential oil of P. auritum.

Table 1. ExtractionParameter of aerial part essential oil of P. auritum. Found Value 1 ParameterYield 2.20% Found (w/w) Value Density 0.93 g/mL 1 DynamicYield viscosity 0.812.20% mPa·s (w /w) Density 0.93 g/mL Kinematic viscosity 0.87 mm2/s Dynamic viscosity 0.81 mPa·s 1 Reported value on dry basis. Kinematic viscosity 0.87 mm2/s 1 Reported3.2. Mass value Spectrometry on dry basis. by Direct Analysis in Real-Time (DART-MS) The chemical profile derived from DART-MS for the aerial part essential oil of P. 3.2.auritum Mass is Spectrometry presented in by Figure Direct 2. Analysis Several molecular in Real-Time ions (DART-MS) were observed, the most abun- dantThe being chemical 149.0308 proﬁleand 163.0 derived181 m/z. fromBased DART-MSon previous forreports the, it aerial was possible part essential to make oil of P.assignments auritum is presented for several in of Figure these observed2. Several m/z molecular ions of the ions metabolites were observed, present in the extracts most abun- dantof P. being auritum 149.0308. The ions and 149.0308, 163.0181 163.0181 m/z., and Based 193.0237 on previousm/z corresponded reports, to it [M was-H]− possible and to make[M-H+O assignments2]− adducts forof compounds several of with these formula observed C10H m/z14O, C ions10H12O of2 and the C metabolites10H10O2, which present is in extractsconsistent of P.with auritum the possible. The presence ions 149.0308, of thymol, 163.0181, eugenol and and 193.0237safrole, respectively m/z corresponded (Table to 2). Table 3 shows the metabolites with molecular formula M that are assigned to the ad- [M-H]− and [M-H+O ]− adducts of compounds with formula C H O, C H O and ducts found in the DART2 mass spectrum. Most of the m/z molecular ions10 (adducts)14 10 were12 2 C H O , which is consistent with the possible presence of thymol, eugenol and safrole, 10attributed10 2 to compounds of a terpenic (cymene and thymol/p-cyme-8-ol) and phe- respectively (Table2). Table3 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 Agronomy 2021, 11, 1098 6 of 13 Agronomy 2021, 11, x FOR PEER REVIEW 6 of 14

alcoholnylpropanoid group (Z3, kind Z6, (safrole E8-dodecatrien-1-ol), and eugenol), although aldehyde compounds (2-hexenal), belonging ketone to the (4-hydroxy-4- alcohol methyl-2-pentanone),group (Z3, Z6, ester E8 (methyl-dodecatrien 2,4-decadienoate),-1-ol), aldehyde fatty acid (2- (hexadecanoichexenal), ketone acid), and alkane(4-hydroxy (tetradecane)-4-methyl were-2-pentanone), also detected. ester (methyl 2,4-decadienoate), fatty acid (hexadecanoic acid), and alkane (tetradecane) were also detected.

Figure 2. DART mass spectra of essential oil of P. auritum with a heated argon gas temperature of 300 °C in negative ion Figure 2. DART mass spectra of essential oil of P. auritum with a heated argon gas temperature of 300 ◦C in negative ion mode. The major molecular ions m/z are represented as adducts [M-H]−, [M-H+O2]−, or [2M-H]−. (a) Intensity vs. m/z. (b) − − − mode.Relative The major intensity molecular vs. m/z. ions The m/zmass areto charge represented ratio (m/z) as adductsof each anion [M-H] is represented, [M-H+O by2] the, or value [2M-H] of the.( peaks.a) Intensity The letter vs. m/z. (b) RelativeM in blue intensity indicates vs. the m/z. molecular The mass weight to of charge the metabolite. ratio (m/z) of each anion is represented by the value of the peaks. The letter M in blue indicates the molecular weight of the metabolite. Table 2. Main metabolite adducts generated in aerial part essential oil of P. auritum by DART-MS and proposed chemical formulas. Table 2. Main metabolite adducts generated in aerial part essential oil of P. auritum by DART-MS and proposed chemicalIdentification formulas. [M-H]− [M-H+O2]− [2M-H]− Thymol 149.0308 1 299.0773 − − − IdentiﬁcationEugenol 163.0181 [M-H] [M-H+O 2] 327.0462[2M-H] CymenoThymol 149.0308 1 165.0216 299.0773 Z3,Z6,E8-Eugenoldodecatrien-1-ol 179.0047 163.0181 327.0462 CymenoSafrole 193.0237 165.0216 Z3,Z6,E8-dodecatrien-1-olPalmitic acid 255.1757 179.0047 1 Most abundantSafrole ion. Adducts observed in the DART-MS spectrum 193.0237 of aerial part essential oil of P. auritum (FigurePalmitic 2). The acid essential oil was analyzed 255.1757 in negative mode with a heated argon gas tem- 1 Mostperature abundant of 300 ion. °C by Adducts DART-MS. observed The major in the molecular DART-MS ions spectrum m/z value of aerialare represented part essential as adducts oil of P. auritum ◦ (Figure[M-H]2).−, The [M- essentialH+O2]−, and oil was[2M- analyzedH]−. The letter in negative M indicates mode the with molecular a heated weight argon gas (MW) temperature of the metabo- of 300 C by − − − DART-MS.lite (identification) The major. molecular ions m/z value are represented as adducts [M-H] , [M-H+O2] , and [2M-H] . The letter M indicates the molecular weight (MW) of the metabolite (identiﬁcation).

3.3. Antifungal Activity of Essential Oil 3.3.1. 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 Table4. The inhibition percentages were greater than 60% in FCHJ-T6 and FCHA-T7 at ﬁve days and Agronomy 2021, 11, 1098 7 of 13

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.

Table 3. List of observed negative molecule and cluster ions detected in aerial part essential oil of P. auritum analyzed in negative mode by DART-MS.

Suggested Metabolite Molecular Formula (M) Adduct Ion m/z Value Terpenes − 1 Thymol/p-cymen-8-ol C10H14O [M-H] 149.0308 MW: 150.1044 g/mol [2M-H]− 299.0694 − Cymeno (o, p, m) C10H14 [M-H+O2] 165.0216 MW: 134.1095 g/mol Phenylpropanoids − 1 Eugenol C10H12O2 [M-H] 163.0181 MW: 164.08373 g/mol [2M-H]− 327.0462 − Safrole C10H10O2 [M+Cl] 179.0004 − MW: 162.0680 g/mol [M-H+O2] 193.0237 Alcohols − Z3,Z6,E8-dodecatrien-1-ol C10H20O [M-H] 179.0047 MW: 180.1514 g/mol Ketones or aldehydes − 4-hydroxy-4-methyl-2-pentanone C6H12O2 [M-H] 115.0495 MW:116.0837 g/mol [2M-H]− 231.0744 − 2-hexenal C6H10O [M-OH] 115.0495 MW:98.0731 g/mol Esters − Methyl 2,4-decadienoate C11H18O2 [M-H] 181.0234 MW:182.1306 [M-OH]− 199.0508 Fatty acids − Palmitic acid/Hexadecanoic acid C16H32O2 [M-H] 255.1757 MW:256.2402 g/mol Alkanes − Tetradecane C14H30 [M-H] 197.0267 MW:198.2347 g/mol [M+Cl]− 233.1018 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).

3.3.2. 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 Table5. 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.

3.3.3. MGI and MIC50 Determination Due to the fungistatic effect of aerial part essential oil of P. auritum on Fusarium spp., the MGI and MIC50 of the aromatic extract were determined. The results are presented in Table6. For FCHJ-T6 and FCHE-T8, the MGI and MIC 50 were found at 9 mg/mL, while FCHA-T7 showed a MIC50 at 6 mg/mL and an MGI at 9 mg/mL (66.7%). AgronomyAgronomy2021Agronomy 20, 1121Agronomy,, 109811 20, x21 FOR, 11 20, PEERx21 FOR, 11 ,REVIEW PEERx FOR REVIEW PEER REVIEW 8 of8 13 of 14 8 of 14 8 of 14 AgronomyAgronomy 2021Agronomy , 11 20, x21 FOR, 11 20, PEERx21 FOR, 11 ,REVIEW PEERx FOR REVIEW PEER REVIEW 8 of 14 8 of 14 8 of 14

Table 4. Antifungal activity of the aerial part essential oil of P. auritum on F. oxysporum FCHA-T7 and FCHJ-T6 and F. equiseti Table Table4. Antifungal Table4. Antifungal 4. activity Antifungal activity of the activity ofaerial the ofaerialpart the essential partaerial essential part oil essentialof P.oil auritum of P.oil auritum of on P. F. auritum oxysporumon F. oxysporumon F. FCHA oxysporum FCHA-T7 and FCHA-T7 FCHJ and-T7 -FCHJT6 and and- T6FCHJ F. and -T6 F. and F. FCHA-T8.Table Table4. Technique: Antifungal Table4. Antifungal 4. mycelium activityAntifungal activity of disc the activity microdiffusion. ofaerial the ofaerialpart the essential aerialpart essential part oil essentialof P.oil auritum of P.oil auritum of on P. F. auritum oxysporumon F. oxysporumon F. FCHA oxysporum FCHA-T7 and FCHA-T7 FCHJ and-T7 -FCHJT6 and and -FCHJT6 F. and -T6 F. and F. equisetiequiseti FCHAequiseti FCHA-T8. Technique: FCHA-T8. Technique:-T8. mycelium Technique: mycelium disc mycelium microdiffusion. disc microdiffusion. disc microdiffusion. equisetiequiseti FCHAequiseti FCHA-T8. Technique: FCHA-T8. Technique:-T8. mycelium Technique: mycelium disc mycelium microdiffusion. disc microdiffusion. disc microdiffusion. TreatmentTreatmentTreatment Treatment FCHJFCHJ-T6 -FCHJ FCHJ-T6T6 -T6 FCHAFCHA FCHA-T7-T7 FCHA-T7 -T7 FCHE FCHE-T8FCHE-T8 FCHE-T8 -T8 TreatmentTreatment Treatment FCHJFCHJ-T6 FCHJ-T6 -T6 FCHAFCHA-T7 FCHA-T7 -T7 FCHEFCHE-T8 FCHE-T8 -T8

H O(−) H2O (H−)22 O (H−)2 O (−) H2O (H−)2 O (H−)2 O (−)

0.0 ± 00..0c0 ± 00.0.0.0c0 ± 00.0c.0c 0.0 ± 00.0.0.0c0 ± 00.0c0..0c0 ± 0.0c 0.0.00 ± 00.0c0..0c0 ± 00..0c0 ± 0.0c 0.0 ± 00..0c0 ± 00..0c0 ± 0.0c 0.0 ± 00..0c0 ± 00..0c0 ± 0.0c 0.0 ± 00..0c0 ± 00..0c0 ± 0.0c

EOFSEO FS EOFS EOFSEO EOFSFSEO FS

63.4 ±63. 3.2b4 ± 63.3.2b4 ± 3.2b 60.7 ±60. 4.3b7 ± 60.4.3b7 ± 4 .3b 42.1 ±42. 2.8b1 ± 42.2.8b1 ± 2 .8b 63.4 ±63. 3.2b4 ± 63. 63.43.2b4 ±± 33.2b.2b 60.7 ±60. 60.74.3b7 ±± 60. 44.3b.3b7 ± 4.3b 42.42.11 ±42. 22.8b.8b1 ± 42. 2.8b1 ± 2.8b

SPK (+)SPK (+)SPK (+) SPK (+)SPKSPK (+)(+)SPK (+)

99.6 ±99. 0.4a6 ± 99. 0.4a6 ± 0.4a 99.6 ±99. 0.4a6 ± 99. 0.4a6 ± 0.4a 99.2 ±99. 0.4a2 ± 99. 0.4a2 ± 0.4a 99.6 ±99. 0.4a6 ± 99. 99.60.4a6 ± 00.4a.4a 99.6 ±99. 99.60.4a6 ± 99. 00.4a.4a6 ± 0.4a 99.99.22 ±99. 00.4a.4a2 ± 99. 0.4a2 ± 0.4a The valuesThe values areThe presented values are presented are in presented percentage in percentage in of percentage inhibition of inhibition of of inhibition radial of radial growth. of radial growth. FCHA growth. FCHA-T7 and FCHA-T7 FCHJ and-T7 - FCHJT6: and 120 -T6: FCHJ h 120 (five-T6: h 120days); (five h days); (five days); TheThe values valuesThe are presented values The are presented values are in percentage presented are in presented percentage of inhibition in percentage in of of percentage radial inhibition of growth. inhibition of of FCHA-T7 inhibition radial of radial andgrowth. of FCHJ-T6: radial growth. FCHA growth.120 FCHA- hT7 (ﬁve and FCHA- days);T7 FCHJ and FCHA-T8:-T7 - FCHJT6: and 120 - FCHJT6:144 h h 120 (five(six-T6: days).h 120days); (five h days); (five days); AgronomyFCHAFCHA-T8: 2021 144,FCHA -11AgronomyT8: h, x (six144 FOR-T8: days).h PEER 20(six 14421 ,days). Valuesh11REVIEWAgronomy (six, x FOR days).Values are PEER 20 the21 Values are, averageREVIEW11 ,the x FORare average of thePEER three average ofREVIEW repetitions three of repetitionsthree ± repetitionsstandard ± standard error. ± standard Valueserror. Valueserror. with differentValues with different with letters different lettersin the lettersin the9 of in 14 the 9 of 14 9 of 14 AgronomyFCHAFCHA-T8: 2021 144,FCHA -11AgronomyT8: h, x (six 144FOR-T8: days).h PEER 20 (six14421 , days). hValues11REVIEWAgronomy (six, x FOR days).Values ±are PEER20 the21 Values are, average11REVIEW ,the x FORare average of thePEER three average ofREVIEW repetitions three of repetitionsthree ± repetitionsstandard ± standard error. ± standard Valueserror. Valueserror. with Valuesdifferent withp different ≤with letters different lettersin the lettersin the9 of in 14 the 9 of 14 9 of 14 Values are the average of three repetitions standard2 error.2 Values with different letters in the same column are different ( 0.05). H2O same columnsame columnsame are differentcolumn are different are(p ≤ different 0.05). (p ≤ H0.05). 2(Op ≤( − H0.05).): 2Onegative ( −H):2 negativeO (control−): negative control (sterile control (sterile distilled (sterile distilled water); distilled water); EOFS: water); EOFS:essential EOFS:essential oil fromessential oil the from P.oil theau- from P. au-the P. au- − same column are different (p ≤ 0.05).2 H2O (−): negative control (sterile distilled water); EOFS: essential oil from the P. au- ( ):same negative columnsame control columnsame are (sterile differentcolumn are distilleddifferent are (p ≤different water);0.05). (p ≤ H0.05). EOFS: (Op ≤( − H0.05).): essential Onegative ( −H):2 Onegative oil (control− from): negative thecontrol ®(sterileP. auritum control ®(sterile distilledaerial ®(sterile distilled part;water); distilled SPK water); EOFS: (+): water); positive EOFS:essential EOFS:essential control oil fromessential (prochloraz, oil thefrom oilP. the au-from P. theau- P. auritum® aerialritum partaerialritum; SPK partaerial (+):; SPK part positive (+):; SPK positive control(+): positive control (prochloraz, control (prochloraz, (prochloraz,Sportak Sportak® ); and Sportak® )PIRG:; and® PIRG:)percentage; and PIRG:percentage of percentage inhibition of inhibition ofof inhibitionradial of radialgrowth. of radialgrowth. growth. Sportakritum); aerialritum and PIRG: partaerialritum; percentage SPK partaerial (+):; SPK part positive of (+):; inhibition SPK positive control(+): ofpositive radialcontrol (prochloraz, growth.control (prochloraz, (prochloraz,Sportak Sportak® ); and Sportak® )PIRG:; and® )PIRG:percentage; and PIRG:percentage of percentage inhibition of inhibition ofof inhibitionradial of radialgrowth. of radialgrowth. growth.

Table3.3.2. 5. 3.3.2.PoisonedInhibition 3.3.2.Poisoned Food of Poisoned radial FoodAssay growth FoodAssay of Assay Fusarium spp. by aerial part essential oil of P. auritum at Table 5. InhibitionTable of 5. radial 3.3.2.Inhibition growthTable 3.3.2.Poisoned of 5.3.3.2.Poisoned radialof Inhibition Fusarium Food Poisoned growth FoodAssay ofspp. ofradial FoodAssay Fusariumby aerialgrowth Assay pspp.art of essentialbyFusarium aerial oilpspp.art of essential byP. auritumaerial oilp artat of different essentialP. auritum concentrationsoil at of different P. auritum concentrations at different concentrations differentThe concentrations biologThe biologicalThe (mg/mL). activitybiological activityical of theactivity of essential the of essential the oil essential was oil tested was oil tested wason the tested on three the on threephytopathogens the threephytopathogens phytopathogens (mg/mL). (mg/mL). The(mg/mL). biologThe biologicalThe activitybiological activityical of theactivity of essential the of essential the oil essential was oil testedwas oil tested wason the tested on three the on threephytopathogens the threephytopathogens phytopathogens using usingPDA usingPDAadded addedPDA with added essentialwith essentialwith oil essential at oilsix atdifferent oilsix atdifferent six concentrations. different concentrations. concentrations. The results The results Theof F. results ox-of F. ox-of F. ox- usingTreatment usingPDA usingPDAadded PDAadded with added essentialwith FCHJ-T6 essentialwith oil essential at oilsix atdifferent oilsix at FCHA-T7different six concentrations. different concentrations. concentrations. The FCHE-T8 results The results The of F. results ox-of F. ox-of F. ox- Treatment Treatment ysporumFCHJTreatmentysporum-T6 and ysporum FCHJF. and equiseti -F. T6and equiseti after F.FCHJ equiseti 144after- T6h 144afterandFCHA h 168 144and-T7 h,h 168 respectively,andFCHA h, 168 respectively, -h,T7 respectively, areFCHA shown are- T7shown FCHEinare Table shown in-T8 Table5. IninFCHE Tablegen-5. In- gen-T85. In gen-FCHE-T8 ysporumysporum andysporum F. and equiseti F. and equiseti after F. equiseti 144after h 144afterand h 168 144and h,h 168 andrespectively, h, 168 respectively, h, respectively, are shown are shown arein Table shown in Table5. inIn Tablegen-5. In gen-5. In general, aneral, increase aneral, increase an in increase mycelial in mycelial in growth mycelial growth inhibition growth inhibition was inhibition observed was observed was at observed the at highest the at highest the concentra- highest concentra- concentra- eral, aneral, increase aneral, increase an in increase mycelial in mycelial in growth mycelial growth inhibition growth inhibition was inhibition observed was observed was at observed the at highest the at highest the concentra- highest concentra- concentra- tions. tions.EO-8.4 tions.EO showed-8.4 EO showed-8.4 the showed greatest the greatest the inhibition greatest inhibition oninhibition the on three the on threeisolates, the threeisolates, greater isolates, greater than greater 50%than for 50%than for50% for tions. tions.EO-−8.4 tions.EO showed-8.4 EO showed-8.4 the showed greatest the greatest the inhibition greatest inhibition oninhibition the on three the on threeisolates, the threeisolates, greater isolates, greater than greater 50%than for 50%than for50% for H2O (−) H2O (−) HH2O2 O((−) ) FCHJ-FCHJT6 and-FCHJT6 FCHE and-T6 FCHE- andT8. FCHE-T8. -T8. FCHJ-FCHJT6 and -FCHJT6 FCHE and-T6 FCHE- andT8. FCHE-T8. -T8. 0.0 ± 0.0e 0.0 ± 0.0e 0.0 ± 0.0e0.0e0.0 ± 0.0d 0.00.0 ± 0.0d0.0d 0.0 ± 0.0d 0.0 ± 0.0f0.0f 0.0 ± 0.0f 0.0 ± 0.0f

EO-0.1 EO-0.1 EOEO-0.1-0.1

1.2 ± 1.4e 1.2 ± 1.4e 1.2 ±± 1.4e1.4e1.1 ± 0.7d 1.11.1 ± 0.7d0.7d 1.1 ± 0.7d29.729.7 ± 0.8de0.8de 29.7 ± 0.8de 29.7 ± 0.8de

EO-0.2 EO-0.2 EO-0.2

4.4 ± 1.1e 4.4 ± 1.1e 4.4 ± 1.1e1.1 ± 0.0d 1.1 ± 0.0d 1.1 ± 0.0d37.8 ± 2.8cd 37.8 ± 2.8cd 37.8 ± 2.8cd

EO-0.4 EO-0.4 EO-0.4

5.2 ± 0.4e 5.2 ± 0.4e 5.2 ± 0.4e2.3 ± 0.7d 2.3 ± 0.7d 2.3 ± 0.7d20.3 ± 0.8e 20.3 ± 0.8e 20.3 ± 0.8e

EO-3.0 EO-3.0 EO-3.0

23.3 ± 0.4d 23.3 ± 0.4d 23.3 ± 0.4d17.1 ± 1.1c 17.1 ± 1.1c 17.1 ± 1.1c34.7 ± 2.0d 34.7 ± 2.0d 34.7 ± 2.0d

EO-5.0 EO-5.0 EO-5.0

41.4 ± 2.9c 41.4 ± 2.9c 41.4 ± 2.9c22.7 ± 3.3c 22.7 ± 3.3c 22.7 ± 3.3c46.4 ± 3.3bc 46.4 ± 3.3bc 46.4 ± 3.3bc

EO-8.4 EO-8.4 EO-8.4

57.8 ± 7.4b 57.8 ± 7.4b 57.8 ± 7.4b42.4 ± 3.6b 42.4 ± 3.6b 42.4 ± 3.6b55.9 ± 2.7b 55.9 ± 2.7b 55.9 ± 2.7b

SPK (+) SPK (+) SPK (+)

98.8 ± 0.0a 98.8 ± 0.0a 98.8 ± 0.0a100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a98.2 ± 1.2a 98.2 ± 1.2a 98.2 ± 1.2a The values areThe presented values inare percentage Thepresented values of inare inhibitionpercentage presented of of inradial inhibitionpercentage growth of of andradial inhibition correspond growth of andradial to correspondthe growth average and toof thecorrespondthree average repetitions toof thethree ± average repetitions of three ± repetitions ± standard error.standard Values with error. differentstandard Values withletters error. different inValues the same letterswith column different in the aresame letters different column in the ( p aresame ≤ 0.05). different column H2O ( p (−):are ≤ 0.05). negativedifferent H2O control ( (−):p ≤ 0.05).negative (sterile H2O control (−): negative (sterile control (sterile distilled water);distilled EO: essential water);distilled oil EO: from essential water); the P. oil auritumEO: from essential theaerial P. oilpart,auritum from the aerialthe numbers P. part,auritum after the aerial numbersthe dash part, indicateafter the numbersthe thedash concentration indicateafter the thedash inconcentration indicate the inconcentration in mg/mL at whichmg/mL it was at tested whichmg/mL; and it was SPK at tested which(+): positive; and it was SPK controltested (+): positive; and(prochloraz, SPK control (+): Sportakpositive (prochloraz,® control). Sportak (prochloraz,® ). Sportak® ).

The in vitro antifungalThe in vitro activity antifungalThe in of vitro essential activity antifungal oils of essentialfrom activity Piper oils of spp. essentialfrom at Piperdifferent oils spp. from concen- at Piperdifferent spp. concen- at different concentrations againsttrations fungi against havetrations been fungi previously against have been fungi studied, previously have observingbeen studied, previously in observingmost studied, cases, in fungistatic observingmost cases, in fungistatic most cases, fungistatic effect by the bioextractseffect by the evaluated. bioextractseffect by theThe evaluated. bioextracts aerial part The evaluated.essential aerial part oil The of essential P.aerial divaricat part oil ofum essential P. showed divaricat oil a umof P. showed divaricat aum showed a high antifungalhigh activity antifungal againsthigh activity antifungalF. solani against f. sp.activity F.piperis solani against (greater f. sp. F.piperis solanithan (greater90%) f. sp. after piperis than seven (greater90%) days after than at seven 90%) days after at seven days at a concentrationa concentration of 5 mg/mLa concentration[3 of6 ]5. Therefore,mg/mL [3 of6 the] 5. Therefore,mg/mL antifungal [36 the] .effect Therefore, antifungal of aerial the effect part antifungal ofessential aerial effect part oil ofessential aerial part oil essential oil of P. auritum ofin P.poisoned auritum offoodin P.poisoned auritumtest corroborates foodin poisoned test corroboratesthe foodfungistatic test corroboratesthe effect fungistatic, and thethe effect fungistaticinhibition, and the effect inhibition, and the inhibition percentage waspercentage dependent waspercentage on depe the ndentconcentration was on depe the ndentconcentration used. on the concentration used. used.

Agronomy 2021, 11Agronomy, x FOR PEER2021, 11REVIEWAgronomy, x FOR PEER2021, 11REVIEW, x FOR PEER REVIEW 9 of 14 9 of 14 9 of 14 Agronomy 2021, 11Agronomy, x FOR PEER2021,, 11REVIEWAgronomy,, xx FORFOR PEERPEER2021, 11REVIEWREVIEW, x FOR PEER REVIEW 9 of 14 9 of 14 9 of 14 Agronomy 2021, 11Agronomy , x FOR PEER2021, 11REVIEW Agronomy, x FOR PEER2021, 11REVIEW, x FOR PEER REVIEW 9 of 14 9 of 14 9 of 14 Agronomy 2021, 11 Agronomy , x FOR PEER2021, 11REVIEWAgronomy , x FOR PEER2021, 11REVIEW, x FOR PEER REVIEW 9 of 14 9 of 14 9 of 14

Table 5. InhibitionTableTable of 5.5. radial InhibitionInhibition growthTable ofof 5. radialradialof Inhibition Fusarium growthgrowth ofspp. ofradialof FusariumbyFusarium aerialgrowth spp.pspp.art of essentialbyFusariumby aerialaerial oilpspp.partart of essential essentialbyP. auritumaerial oiloilp artat ofof different essential P.P. auritumauritum concentrationsoil atat of differentdifferent P. auritum concentrationsconcentrations at different concentrations Table 5. InhibitionTable of 5. radial Inhibition growthTable of 5. radialof Inhibition Fusarium growth ofspp. radialof Fusariumby aerialgrowth pspp.art of essentialFusariumby aerial oilpspp.art of essential byP. auritumaerial oilp artat of different essentialP. auritum concentrationsoil at of different P. auritum concentrations at different concentrations (mg/mL). (mg/mL).(mg/mL). (mg/mL). (mg/mL). (mg/mL). (mg/mL). Treatment TreatmentTreatment FCHJTreatment-T6 FCHJFCHJ--T6T6 FCHJ-T6FCHA -T7 FCHAFCHA--T7T7 FCHA-T7FCHE -T8 FCHEFCHE--T8T8 FCHE-T8 Treatment Treatment FCHJTreatment-T6 FCHJ-T6 FCHJ-T6FCHA -T7 FCHA-T7 FCHA-T7FCHE -T8 FCHE-T8 FCHE-T8

H2O (−) H2O (−) H2O (−) Agronomy 2021H2O, 11 (−), 1098 H22O (−) H2O (−) 9 of 13 H2O (−) H2O (−) H2O (−) H2O (−) H2O (−) H2O (−)

0.0 ± 0.0e 0.00.0 ±± 0.0e0.0e 0.0 ± 0.0e 0.0 ± 0.0d 0.00.0 ±± 0.0d0.0d 0.0 ± 0.0d 0.0 ± 0.0f 0.00.0 ±± 0.0f0.0f 0.0 ± 0.0f 0.0 ± 0.0e 0.0 ± 0.0e 0.0 ± 0.0e0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0f 0.0 ± 0.0f 0.0 ± 0.0f

Table 5. Cont. EO-0.1 EO-0.1 EO-0.1 EO-0.1 EO--0.1 EO-0.1 EO-0.1 EO-0.1 EO-0.1 Treatment FCHJ-T6 FCHA-T7 FCHE-T8 1.2 ± 1.4e 1.21.2 ±± 1.4e1.4e 1.2 ± 1.4e 1.1 ± 0.7d 1.11.1 ±± 0.7d0.7d 1.1 ± 0.7d29.7 ± 0.8de 29.729.7 ±± 0.8de0.8de 29.7 ± 0.8de 1.2 ± 1.4e 1.2 ± 1.4e 1.2 ± 1.4e1.1 ± 0.7d 1.1 ± 0.7d 1.1 ± 0.7d29.7 ± 0.8de 29.7 ± 0.8de 29.7 ± 0.8de

EO-0.2 EO-0.2 EOEO-0.2-0.2 EO-0.2 EO--0.2 EO-0.2 EO-0.2 EO-0.2 EO-0.2

4.4 ± 1.1e 4.44.4 ±± 1.1e1.1e 4.4 ± 1.1e1.1e 1.1 ± 0.0d 1.11.1 ±± 0.0d0.0d 1.1 ± 0.0d37.837.8 ± 2.8cd2.8cd 37.837.8 ±± 2.8cd2.8cd 37.8 ± 2.8cd 4.4 ± 1.1e 4.4 ± 1.1e 4.4 ± 1.1e1.1 ± 0.0d 1.1 ± 0.0d 1.1 ± 0.0d37.8 ± 2.8cd 37.8 ± 2.8cd 37.8 ± 2.8cd

EO-0.4 EO-0.4 EOEO-0.4-0.4 EO-0.4 EO--0.4 EO-0.4 EO-0.4 EO-0.4 EO-0.4

5.2 ± 0.4e 5.25.2 ±± 0.4e0.4e 5.2 ± 0.4e0.4e 2.3 ± 0.7d 2.32.3 ±± 0.7d0.7d 2.3 ± 0.7d20.3 ±± 0.8e0.8e 20.320.3 ±± 0.8e0.8e 20.3 ± 0.8e 5.2 ± 0.4e 5.2 ± 0.4e 5.2 ± 0.4e2.3 ± 0.7d 2.3 ± 0.7d 2.3 ± 0.7d20.3 ± 0.8e 20.3 ± 0.8e 20.3 ± 0.8e

EO-3.0 EOEO--3.03.0 EOEO-3.0-3.0 EO-3.0 EO-3.0 EO-3.0

23.3 ± 0.4d 23.3 ± 0.4d 23.3 ± 0.4d17.1 ± 1.1c 17.1 ± 1.1c 17.1 ± 1.1c34.7 ± 2.0d 34.7 ± 2.0d 34.7 ± 2.0d 23.3 ± 0.4d 23.3 ± 0.4d 23.323.3 ±± 0.4d0.4d17.1 ± 1.1c 17.117.1 ±± 1.1c1.1c 17.1 ± 1.1c34.734.7 ±± 2.0d2.0d 34.7 ± 2.0d 34.7 ± 2.0d 23.3 ± 0.4d 23.3 ± 0.4d 23.3 ± 0.4d17.1 ± 1.1c 17.1 ± 1.1c 17.1 ± 1.1c34.7 ± 2.0d 34.7 ± 2.0d 34.7 ± 2.0d

EO-5.0 EO-5.0 EO-5.0 EO-5.0 EO--5.0 EOEO-5.0-5.0 EO-5.0 EO-5.0 EO-5.0

41.4 ± 2.9c 41.441.4 ±± 2.9c2.9c 41.4 ± 2.9c22.7 ± 3.3c 22.722.7 ±± 3.3c3.3c 22.7 ± 3.3c46.4 ± 3.3bc 46.446.4 ±± 3.3bc3.3bc 46.4 ± 3.3bc 41.4 ± 2.9c 41.4 ± 2.9c 41.4 ±± 2.9c2.9c22.7 ± 3.3c 22.722.7 ±± 3.3c3.3c 22.7 ± 3.3c46.446.4 ± 3.3bc3.3bc 46.4 ± 3.3bc 46.4 ± 3.3bc

EO-8.4 EO-8.4 EO-8.4 EO-8.4 EO--8.4 EOEO-8.4-8.4 EO-8.4 EO-8.4 EO-8.4

57.8 ± 7.4b 57.857.8 ±± 7.4b7.4b 57.8 ± 7.4b42.4 ± 3.6b 42.442.4 ±± 3.6b3.6b 42.4 ± 3.6b55.9 ± 2.7b 55.955.9 ±± 2.7b2.7b 55.9 ± 2.7b 57.8 ± 7.4b 57.8 ± 7.4b 57.8 ±± 7.4b7.4b42.4 ± 3.6b 42.442.4 ±± 3.6b3.6b 42.4 ± 3.6b55.955.9 ± 2.7b2.7b 55.9 ± 2.7b 55.9 ± 2.7b

SPK (+) SPK (+) SPK (+) SPK (+) SPK (+) SPK (+) SPK (+) SPK (+) SPKSPK (+) (+)

98.8 ± 0.0a 98.8 ± 0.0a 98.8 ± 0.0a100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a98.2 ± 1.2a 98.2 ± 1.2a 98.2 ± 1.2a 98.8 ± 0.0a 98.8 ± 0.0a 98.8 ± 0.0a100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a98.2 ± 1.2a 98.2 ± 1.2a 98.2 ± 1.2a The values are Thepresented values98.8 inare ±percentageThe presented0.0a values 98.8 ofinare inhibition±percentage presented0.0a 98.8 of ofin radial ±inhibition±percentage 0.0a 100.0growth of± of and0.0aradial inhibition correspond 100.0100.0growth of±± and0.0aradial to thecorrespond 100.0growth average ± and0.0a toof98.298.2 correspondthe three ± average 1.2a 1.2arepetitions toof98.2 thethree ± ±average 1.2a repetitions of98.2 three ± ± 1.2arepetitions ± The values areThe presented values inare percentage Thepresented values of inare inhibitionpercentage presented of of inradial inhibition percentage growth of of andradial inhibition correspond growth of andradial to correspondthe growth average and toof thecorrespondthree average repetitions ofto thethree ± average repetitions of three ± repetitions ± Thestandard values error. areThe standardpresented Values valuesThe with error. values inare percentagedifferentstandard Thepresented Values are values presented letterswith error. of inare inhibition percentagedifferent inpresented inValues percentage the same withlettersof of inradial ofinhibition columnpercentagedifferent inhibitionin growththe aresame lettersof of differentandradial radial inhibitioncolumn incorrespond growththe growth (p aresame ≤ of 0.05). different and radial tocolumn correspondcorrespondtheH 2growthO average ( (−):pare ≤ 0.05). negative differentand to toof the thecorrespondthreeH average2O average control( p (−):repetitions ≤ 0.05).negative of (sterile threetoof thethreeH2 ±O average control (−): repetitions negative (sterileof three ± control repetitions (sterile ± standard error.standard Values witherror. differentstandard Values withletters error. different inValues the same letterswith columndifferent in the aresame letters different column in the ( p aresame ≤ 0.05). different column H2O ( p (−):are ≤ 0.05). negativedifferent H22O control( (−):p ≤ 0.05).negative (sterile H2O control (−): negative (sterile control (sterile standard error.standard Values with error. differentstandard Values± withletters error. different inValues the same withletters column different in the aresame letters different column in the ( p aresame ≤ 0.05). different column H2O ( (−):pare ≤ 0.05). negativedifferentp H≤2O control ( (−):p ≤ 0.05).negative (sterile− H2O control (−): negative (sterile control (sterile standarddistilled water); error.standarddistilled Values EO: repetitionsessential withwater); error. differentdistilledstandard oilValues EO: standardfrom essential withwater);letters error.the error. P.different oilinValues auritumEO: Values thefrom essential same withletters withtheaerial column P.different different oilin part,auritum thefrom the aresame letters letters theaerial numbersdifferent columnP. in inpart,auritum the the sameafter( pthe aresame ≤ aerial 0.05). columnnumbersthedifferent column dash part,H are2O indicateafter( differentp(−):theare ≤ 0.05). negativenumbersthedifferent the (dash H 2concentrationO 0.05).control indicateafter( (−):p ≤ H0.05). negativethe2 O((sterile thedash H): inconcentration2O control indicate (−): negative (sterile the inconcentration control (sterile in distilled water);distilled EO: negativeessential water); controldistilled oil EO: from (sterileessential water); the distilled P. oil auritumEO: from water); essential theaerial EO: P. oil essentialpart,auritum from the oil aerialthe numbers from P. part,auritum the P.after the auritum aerial numberstheaerial dash part, part, afterindicate the the numbersthe numbers thedash concentration indicateafter after thethe dashthe dash inconcentration indicate the inconcentration in distilledmg/mL at water); whichdistilledmg/mL EO: it was essential at water);tested whichmg/mLdistilled ;oil andEO: it fromwas SPKessential at water); testedthe which (+): P. positive ; oil auritumandEO: it fromwas SPKessential control testedtheaerial (+): P. positive ; oil part,auritumand(prochloraz, from SPKthe control aerialthe numbers(+): P. positiveSportak part, auritum(prochloraz, after the® control). aerial numbersthe dashSportak part,(prochloraz, indicateafter the® ). numbersthe the dashSportak concentration indicateafter® ). the thedash inconcentration indicate the inconcentration in mg/mL at whichmg/mL it wasindicate at tested which themg/mL; and concentrationit was SPK at tested which (+): inpositive;; and mg/mLit was SPK controltested at (+): which positive; and(prochloraz, it was SPK control tested; (+): positiveSportak and (prochloraz, SPK® control (+):). positive Sportak (prochloraz, control®®).). (prochloraz, Sportak® Sportak). ®). mg/mL at whichmg/mL it was at tested whichmg/mL; and it was SPK at tested which(+): positive; and it was SPK controltested (+): positive; and(prochloraz, SPK control (+): Sportakpositive (prochloraz,® control). Sportak (prochloraz,® ). Sportak® ). Table 6. Fungitoxicity of aerial part essential oil of P. auritum on Fusarium spp. The in vitro antifungalThThee inin vitrovitro activity antifungalantifungalThe in of vitro essential activityactivity antifungal oils ofof essentialessentialfrom activity Piper oilsoils of spp. essentialfromfrom at PiperPiperdifferent oils spp.spp. from concen- atat Piperdifferentdifferent spp. concen-concen- at different concen- The in vitro antifungalThe in vitro activity antifungalThe in of vitro essential activity antifungal oils of essentialfrom activity Piper oils of spp. essentialfrom at Piperdifferent oils spp. from concen- at Piperdifferent spp. concen- at different concentrations againsttrationstrations fungi againstagainst havetrations been fungifungi previously against havehave beenbeen fungi studied, previouslypreviously have observingbeen studied,studied, previously in observingobservingmost studied, cases, inin fungistatic observingmostmost cases,cases, in fungistaticfungistatic most cases, fungistatic trationsFungal against Strainstrations fungi against havetrations been fungi previously against MIChave50 been fungi studied, previously have observingbeen studied, previously inMGI observingmost studied, cases, in fungistatic observingmost cases, in fungistatic most cases, fungistatic effect by the bioextractseffecteffect byby thethe evaluated. bioextractsbioextractseffect by theThe evaluated.evaluated. bioextracts aerial part TheThe evaluated.essential aerialaerial partpart oil The of essentialessential P.aerial divaricat part oiloil ofumof essential P.P. showed divaricatdivaricat oil a umofum P. showedshowed divaricat aaum showed a effectFCHJ-T6 by the bioextractseffect by the evaluated. bioextractseffect by theThe evaluated. bioextracts aerial part The 9 mg/mLevaluated.essential aerial part oil The of essential P.aerial divaricat part oil ofum essential P. showed divaricat oil a ofum P. showed divaricat aum showed a high antifungalhighhigh activity antifungalantifungal againsthigh activityactivity antifungalF. solani againstagainst f. sp.activity F. F.piperis solanisolani against (greater f.f. sp.sp. F.piperispiperis solanithan (greater(greater90%) f. sp. after piperis thanthan seven (greater90%)90%) days afterafter than at sevenseven 90%) daysdays after atat seven days at highFCHA-T7 antifungalhigh activity antifungal againsthigh activity antifungalF. 6solani mg/mL against f. sp.activity F.piperis solani against (greater f. sp. F.piperis solanithan 9 (greater mg/mL90%) f. sp. after piperis than seven (greater90%) days after than at seven 90%) days after at seven days at a concentrationaa concentrationconcentration of 5 mg/mLa concentration[3 ofof6 ] 55. Therefore,mg/mLmg/mL [3[3 of66 the] ]5.. Therefore,Therefore,mg/mL antifungal [36 thethe] .effect Therefore, antifungalantifungal of aerial the effecteffect part antifungal ofofessential aerialaerial effect partpart oil ofessentialessential aerial part oiloil essential oil a concentrationFCHE-T8a concentration of 5 mg/mLa concentration[3 of6 ]5. Therefore,mg/mL [3 of6 the ]5. Therefore,mg/mL antifungal 9 mg/mL [36 the] .effect Therefore, antifungal of aerial the effect part antifungal ofessential aerial effect part oil ofessential aerial part oil essential oil of P. auritum ofofin P.P.poisoned auritumauritum offoodinin P.poisonedpoisoned auritumtest corroborates foodfoodin poisoned testtest corroboratescorroboratesthe foodfungistatic test corroboratesthethe effect fungistaticfungistatic, and thethe effecteffect fungistaticinhibition,, andand thethe effect inhibitioninhibition, and the inhibition MIC50: minimumof P. auritum concentration ofin P.poisoned auritum of essential offoodin P.poisoned oil auritumtest that corroborates inhibited foodin poisoned 50% test of corroboratesthe radial foodfungistatic growth test and corroboratesthe effect MGI: fungistatic concentration, and thethe effect fungistaticinhibition of, and the effect inhibition, and the inhibition essentialpercentage oil that caused waspercentage maximumpercentage dependent inhibition waswaspercentage on depedepe ofthe radial ndentndentconcentration growth.was onon depe thethe ndentconcentrationconcentration used. on the concentration used.used. used. percentage waspercentage dependent waspercentage on depe the ndentconcentration was on depe the ndentconcentration used. on the concentration used. used. 4. 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 Agronomy 2021, 11, 1098 10 of 13

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 extracts from leaves and inflorescence of P. auritum are essentially a mixture of alka- loids, safrole, amines, butenolides, flavonoids, and terpenes, among other compounds [41], while the essential oil of this same species is composed of more than 40 compounds, some of them mono and sesquiterpenes [37,42]. The biological activity of the essential oils of P. auritum is due to the different types of secondary metabolites present in each genus of the Piperaceae family, among which the lignans, sesquiterpenes, diterpenes, monoterpenes and phenolic compounds stand out [43]. Previously, the essential oil extracted from P. auritum leaves showed a clear fungistatic effect on three phytopathogenic agave fungi, since it had a significant inhibitory effect (96 h) on F. oxysporum f. sp. comiteca (75.32%), F. oxysporum f. sp. tequilana (86.57%), and F. solani f. sp. comiteca (63.36%) [23] Chemical analysis by DART-MS of aerial part essential oil of P. auritum showed adducts possibly attributed to terpenes (cymene and thymol/p-cymene-8-ol) and phenylpropanoids (safrole and eugenol), the most intense signals in the mass spectrum being those corresponding to thymol and eugenol (Figure2, Table2), although compounds belonging to the alcohol group (Z3, Z6, E8-dodecatrien-1-ol), aldehyde (2-hexenal), ketone (4-hydroxy- 4-methyl-2-pentanone), ester (methyl 2,4-decadienoate), fatty acid (hexadecanoic acid), and alkane (tetradecane) were also detected (Table3). 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 (Table4) and poisoned food (Table5). 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.

5. 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, Agronomy 2021, 11, 1098 11 of 13

the compounds identiﬁed by DART-MS presented chemical diversity, pointing out those of a terpenic and phenylpropanoid kind.

Author Contributions: Designed this study, N.R.-L. and C.C.; analyzed the results, N.R.-L., C.C. and E.B.-Q.; DART-MS, analysis and technical support, L.M.R.-A. and E.B.-Q.; statistically analyzed the date, C.C. and J.A.M.-M.; supervision, N.R.-L., E.R.G.-R., G.C.-C., V.M.R.-V. and J.A.M.-M.; wrote the manuscript, N.R.-L. and C.C.; revised and edited the manuscript, N.R.-L., C.C., E.B.-Q. and G.C.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: To the Consejo Nacional de Ciencia y Tecnología (CONACyT)-Mexico for the scholarship awarded to César Chacón (support number 715167) and to Jairo Cristóbal Alejo for providing the fungal strains used in this work. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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