Cogent Food & Agriculture

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Chemical composition and antifungal activity of essential

oils extracted from Pimenta dioica and Piper auritum
leaves grown in Mexico

Anayancy Lam-Gutiérrez, Teresa Ayora-Talavera, Eduardo Raymundo

Garrido-Ramírez, Víctor Manuel Ruíz-Valdiviezo, Jorge Martín Guzmán-
Albores & Jairo Cristóbal-Alejo

To cite this article: Anayancy Lam-Gutiérrez, Teresa Ayora-Talavera, Eduardo Raymundo

Garrido-Ramírez, Víctor Manuel Ruíz-Valdiviezo, Jorge Martín Guzmán-Albores & Jairo Cristóbal-
Alejo (2024) Chemical composition and antifungal activity of essential oils extracted from
Pimenta dioica and Piper auritum leaves grown in Mexico, Cogent Food & Agriculture, 10:1,
2356935, DOI: 10.1080/23311932.2024.2356935

To link to this article: https://doi.org/10.1080/23311932.2024.2356935

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Cogent Food & Agriculture
2024, VOL. 10, NO. 1, 2356935
https://doi.org/10.1080/23311932.2024.2356935

Soil & Crop Sciences  | Research Article

Chemical composition and antifungal activity of essential oils extracted
from Pimenta dioica and Piper auritum leaves grown in Mexico
Anayancy Lam-Gutiérreza , Teresa Ayora-Talaverab , Eduardo Raymundo Garrido-Ramírezc ,
Víctor Manuel Ruíz-Valdiviezod , Jorge Martín Guzmán-Alborese and Jairo Cristóbal-Alejof
a
Tecnológico Nacional de México/ITS de Cintalapa, Cintalapa de Figueroa, Chiapas, México; bCentro de Investigación y Asistencia en
Tecnología y Diseño del Estado de Jalisco, A.C., Subsede Sureste, Parque Científico Tecnológico de Yucatán, Mérida, Yucatán, Mexico;
c
Universidad Tecnológica de la Selva, entronque Toniná Km. 0.5 Carretera Ocosingo-Altamirano, Ocosingo, Chiapas, México;
d
Tecnológico Nacional de México/IT de Tuxtla Gutiérrez, Tuxtla Gutiérrez, Chiapas, México; eTecnológico Nacional de México, Instituto
Tecnológico de Tapachula, Tapachula, Chiapas, México; fTecnológico Nacional de México/IT de Conkal, Avenida Tecnológico s/n
Conkal, Conkal, Yucatán, México

ABSTRACT ARTICLE HISTORY

Diseases caused by mycotoxin-producing fungi, especially Aspergillus ochraceus and Fusarium Received 15 October
moniliforme, are on the rise, leading to contamination of agricultural products and seeds 2023
during processing and storage, resulting in significant economic losses and posing serious Revised 22 April 2024
Accepted 15 May 2024
health risks to humans and animals. Controlling these diseases primarily relies on synthetic
fungicides, which can leave behind toxic residues harmful to human and animal health, as KEYWORDS
well as the environment. Various strategies have been proposed for controlling these fungal Pimenta dioica; Piper
diseases, including biological control using essential oils (EOs) derived from plants. EOs auritum; antifungal
extracted from plants of the Piper and Pimenta genera contain secondary metabolites with activity; phytopathogens;
antimicrobial properties. This study aims to analyze the effect of EOs from Piper auritum and fungal of crops; Fusarium
moniliforme; Aspergillus
Pimenta dioica on the fungi A. ochraceus and F. moniliforme. The EOs were obtained through ochraceus
steam distillation, and their antifungal activity was assessed using minimum inhibitory
concentration (MIC), minimum fungicidal concentration (MFC), and % sporulation. The REVIEWING EDITOR
phytochemical composition of the EOs was determined by GC-MS. The fungal species Manuel Tejada,
exhibited susceptibility to the EOs, with MIC and MFC values of 0.16 and 1.21 mg/mL, Cristalografía, Mineralogía
respectively, for P. dioica against A. ochraceus, and 0.43 mg/mL against F. moniliforme. y Química Agrícola,
Universidad de Sevilla,
Meanwhile, the MIC and MFC values for P. auritum were 0.16 and 15 mg/mL, respectively, Seville, Spain
against A. ochraceus. The EOs demonstrated effective antifungal activity, likely attributed to
the presence of bioactive compounds. SUBJECTS
Agriculture &
GRAPHICAL ABSTRACT Environmental Sciences;
Microbiology;
Environmental Studies

CONTACT Anayancy Lam-Gutiérrez [email protected] Tecnológico Nacional de México/ITS de Cintalapa, Carretera Panamericana km
995, Cintalapa de Figueroa 30400, México.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/23311932.2024.2356935.
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unre-
stricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the
posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
2 A. LAM-GUTIÉRREZ ET AL.

1. Introduction Found in the Myrtaceae family, Piper auritum has

traditionally been used as an antipyretic and diuretic,
Pathogenic fungi pose a significant economic chal-
as well as a material for pest control. It has also been
lenge to crop and food production. Many of these
employed to treat conditions such as angina pecto-
fungi are associated with health risks such as esoph-
ris, erysipelas, common ailments, colic, and head-
ageal cancer, carcinogenesis, mutagenicity, and neu-
aches. Additionally, it has been used as an appetite
ral tube defects, attributed to the production of
suppressant, a wound poultice, and a local anes-
harmful secondary metabolites. This issue is particu-
thetic. It has been recorded that the Chinantec and
larly concerning for the safety of food production.
Mayan tribes used the decoction of the leaves to
Fungi belonging to the Fusarium, Aspergillus, and
facilitate childbirth and to heal wounds, respectively.
Penicillium genera are deemed most significant as
Salleh and Nuzul (2021) reported the latest findings
they are primary producers of mycotoxins such as
for this plant, where aqueous extracts of leaves
aflatoxins, fumonisins, and ochratoxins. Furthermore,
revealed the presence of antifungal activity against
their presence can result in diminished livestock
Candida albicans, Cladosporium, Colletotrichum acu-
production, loss of human and animal life, food con-
tatum, and Botryodiplodia theobromae. However, no
tamination, reduced yields, and heightened food
results were found during the in vitro evaluation of
insecurity and prices (Seepe et al., 2021; Taniwaki
the P. auritum leaf essential oil against Fusarium ver-
et al., 2018; Zabka et al., 2009; Magnoli et al., 2007).
ticillioides and Aspergillus ochraceus.
Additionally, the economic losses incurred in food
Moreover, essential oils extracted from Pimenta
production due to mycotoxins amount to hundreds
dioica (allspice), found in the Piperaceae family, have
of millions to billions of dollars annually (Pinotti
been tested in vitro against Fusarium oxysporum,
et al., 2016).
Fusarium verticillioides, Penicillium expansum,
The adverse effects associated with the use of syn-
Penicillium brevicompactum, Aspergillus flavus, and
thetic pesticides, including environmental and water
Aspergillus fumigatus (Zabka et al., 2009). Extracts
contamination, as well as impacts on human and ani-
from P. dioica leaves are known as a folk remedy for
mal health, have underscored the necessity of seek-
the treatment of obesity, high blood pressure, and
ing control strategies that are both cost-effective and
digestion problems in Central America, and are also
environmentally safe. Research into medicinal plants
used for menstrual and abdominal pain (Zhang &
as control agents for phytopathogens has garnered
Lokeshwar, 2012). Although only in vitro studies exist
attention, given their ready availability and the
regarding the antifungal activity of P. dioica, most
diverse array of biodegradable secondary metabolites
focus on allspice and only a few on leaf extracts and
they contain (Seepe et al., 2021; Perczak et al., 2019;
not EOs.
D’agostino et al., 2019; Durant-Archibold et al., 2018;
Plant-derived natural pesticides, particularly those
Liu et al., 2017). Additionally, they exhibit insecticidal,
derived from essential oils, offer promising alterna-
anti-inflammatory, antioxidant, anti-allergic, and anti-
tives for crop protection (Khaldi et al., 2017; Kumar
cancer properties, alongside antimicrobial characteris-
et al., 2016). Therefore, this study aimed to contrib-
tics (Perczak et al., 2019, Raveau et al., 2020; Gomes
ute to the understanding of their antifungal proper-
da Rocha-Voris et al., 2018). Thus, essential oils from
ties by evaluating the in vitro effects of essential oils
various plants have demonstrated antimicrobial and
extracted from P. dioica and P. auritum on the myce-
biological activity against fungal pathogens that
lial growth and spore germination inhibition of phy-
affect plants, animals, and humans. They are also
topathogens such as A. ochraceus and F. moniliforme.
widely utilized in medical and clinical microbiology,
Additionally, the phytochemical composition of the
pharmaceutical botany, the fragrance industry, and
EOs was estimated by GC-MS.
food flavoring and preservation (Salehi et al., 2019;
Choi, 2018; Borges et al., 2016).
Essential oils obtained by steam distillation 2. Materials and methods
from plants in the Myrtaceae and Piperaceae fami-
2.1. Collection of plant material and extraction
lies have shown a wide variety of antimicrobial
of essential oils
activities. These activities have been intensively
explored in recent years, primarily in response to The leaves were collected in two municipalities from
consumers’ overwhelming concern about the Chiapas, México. Pimenta dioica leaves were obtained
safety of synthetic food additives (Seepe et al., in Copainalá (16° 39’ N/93°44’ W), and Piper auritum
2021; Yi-Xin et al., 2014). in Villaflores (16° 14’ N/93° 16’W). The identity of the
 Cogent Food & Agriculture 3

plants was confirmed by the Flora Department, 2.3. Agar diffusion assay
belonging to the Botanical Garden of the Secretary
The inhibitory effect of the essential oil was prelimi-
of Environmental Protection and Natural History from
narily evaluated using the agar well diffusion method.
the State of Chiapas, México under registration num-
Plates with a diameter of 9 mm were prepared with
ber 49538 for P. auritum and number 27743 for P.
Sabouraud Agar. Wells with a diameter of 6 mm were
dioica. Essential oils were extracted by steam distilla-
cut out of the agar and filled with 100 μL of essential
tion from the fresh leaves of Pimenta dioica (23.12 Kg)
oils previously diluted with dimethyl sulfoxide (Sigma
and Piper auritm (49.25 Kg), at 120 °C and pressure of
Aldrich, USA) at concentrations of 1, 10, 100, and
1 kg/cm2. The performance calculations were based
270 mg/mL. Ketoconazole, serving as the positive
on a wet basis.
control, was also included. Fungal spore concentra-
 Extracted EOs ( g ) 
Yield EOs =   × 100 tion was prepared at 1x105 spores/mL, and 1 mL was
 Fresh leaves ( g )  spread over plates that were incubated at 32 °C. The
measure of the inhibition zone was reported in milli-
meters after 48 hours (Lam-Gutiérrez et al., 2019).
2.2. Fungal species and spore production
Two fungal species, F. moniliforme and A. ochraceus, 2.4. Minimum inhibitory concentration (MIC) and
were obtained from the “Instituto Nacional de minimum fungicide (MFC)
Investigaciones Forestales y Pecuarias” (INIFAP),
México. These fungi were subcultured on Sabouraud The MIC and MFC were assessed. The MIC value of
agar at 30 °C for at least 15 days before being used. the essential oils was tested using a microdilution
The selection of Sabouraud agar as the culture assay. Tubes containing 9 mL of Sabouraud broth
medium was based on its suitability for the growth with essential oil dilutions ranging from 0.09 to
of a wide range of fungi and its ability to support 15 mg/mL were inoculated with 1 mL of a fungal sus-
robust sporulation. Spore suspensions were pre- pension containing 5x104 spores/mL (A. ochraceus
pared by gently washing the surface of the fungal and F. monilifore, respectively). MIC was determined
cultures with 10 mL of sterile distilled water con- by identifying the lowest concentration of essential
taining 0.01% (vol:vol) Tween (R) 80 (Merck, oil at which no fungal growth was observed after
Darmstadt, Germany). The addition of Tween 80 48 hours of incubation at 32 °C. The MFC was deter-
served to reduce surface tension and facilitate the mined by streaking 1 mL aliquots from the treated
release of spores from the fungal hyphae. tubes onto plates. MFC was defined as the lowest
Subsequently, a sterilized spreader was carefully concentration of essential oils that completely inhib-
used to evenly distribute the spore suspension over ited fungal growth (Lam-Gutiérrez et al., 2019).
the agar surface. To ensure the accuracy of spore
quantification, the final spore concentration was 2.5. Effect of essential oils on inhibition of spore
determined using Neubauer chambers under a light germination
microscope (Carl Zeiss, Primo Star iLED). Briefly, the
spore suspension was appropriately diluted to The effect of P. dioica and P. auritum essential oils on
obtain a countable number of spores per grid spore germination of A. ochraceus and F. moniliforme
square. The number of spores present in several was assessed as follows. Fungal spores were collected
grid squares was then counted, and the average from 15 day old cultures growing on Sabouraud agar
spore count per grid square was calculated. This plates (MFC) at 32 °C. To collect the spores, plates
value was used to determine the concentration of were flooded with 10 mL of sterile distilled water
spores per milliliter of the original suspension, containing 0.01% (vol:vol) Tween(R) 80, after which
which was expressed as spores/mL. the spores were gently scraped with an L-shaped
For A. ochraceus, the equation used to calculate: glass spreader. The resulting spore suspension was
counted with a Neubauer chamber.
Spores
= ( Number of spoes )(10 , 000 )( dilution factor )
mL  SGPC − SGEO 
Inh ibition % =   x 100
Similarly, for F. moniliforme, the equation used:  SGPC 
SGPC refers to spores grown in the positive con-
Spores trol, while SGEO refers to spores grown in the pres-
= ( Number of spores )( 250 , 000 )( dilution factor )
mL ence of EOs.
4 A. LAM-GUTIÉRREZ ET AL.

2.6. Gas Chromatography-Mass Spectrometry Table 1. Yield and physical characteristics of the
(GC-MS) analysis from essential oil essential oil from P. dioica and P. auritum leaves.
EOs
Gas Chromatography-Mass Spectrometry (GC-MS) Characteristics P. dioica P. aritum
analysis of the essential oils was conducted as fol- % Yield 0.77 0.268
lows. The P. dioica and P. auritum essential oils Odor Aromatic
Color Light amber
obtained by steam distillation, were analyzed using Density (g/mL) 0.954 0.95
an Agilent Technologies 7890 A gas chromatogra-
phy/mass spectrometry system equipped with a
each exceeding 5.0%, were eugenol (71.73%), beta-
60 m DB-WAXetr column with an internal diameter
caryophyllene (6.747%), and (-)-beta-pinene (11.447%).
of Ø = 0.25 mm and a thickness of 25 μm. The pure
essential oil was diluted in hexane, and the injected
volume was 1 μL. The injector temperature was set 3.3. Agar diffusion assay
at 70 °C, while the detector temperature was
250 °C. Helium served as the carrier gas at a flow The susceptibility of EOs was confirmed by an inhibi-
rate of 1 mL/min. Fragmentation patterns were tion halo (well diffusion). EOs from P. dioica exhibited
analyzed based on the direct comparison of the a significant inhibition halo (p < 0.05) at 10 mg/mL for
Kovats index and ion fragmentation (Lam-Gutiérrez both A. ochraceous and F. moniliforme. Regarding P.
et al., 2019). auritum EOs, they inhibited growth at concentrations
>100 mg/mL, resulting in inhibition halo sizes rang-
ing from 20 to 25 mm in the well diffusion test for
2.7. Statistical analysis both strains of phytopathogenic fungi.
Statistical analysis was performed using Statgraphic
Centurion version XV software. All data were 3.4. Minimum inhibitory concentration (MIC) and
expressed as the mean ± standard deviation (SD). minimum fungicidal concentration (MFC)
Statistically significant differences among mean val-
ues were determined by one-way ANOVA (p < 0.05). The values of the minimum inhibitory concentration
Pairwise comparisons were conducted using Tukey’s (MIC), and the minimum fungicidal concentration
tests (p < 0.05). (MFC) for the EOs tested on the fungi are presented in
Table 4. The fungal species exhibited susceptibility to
the EOs, with MIC and MFC values for P. dioica at 0.16
3. Results and 1.21 mg/mL for A. ochraceous, respectively, and
0.43 mg/mL for F. moniliforme. Conversely, the MIC and
3.1. Yield of EO
MFC values for P. auritum were 0.16 and 15 mg/mL for
Steam distillation of fresh P. dioica and P. auritum A. ochraceous, respectively (refer to Figures 1 and 2).
leaves yielded a light amber and viscous oil, with
average yields of 0.77% and 0.268%, respectively, as
3.5. Effect of essential oils on inhibition of spore
shown in Table 1.
germination
The effect of EOs on inhibition of spore germination of
3.2. Essential oil composition
fungal strains (as seen in Table 5), revealed that the
Chemical constituents were identified in the essential lowest concentrations of P. auritum (ranging from
oils of P. dioica and P. auritum leaves using gas 0.09 mg/mL to 0.16 mg/mL) inhibited sporulation capa-
chromatography-mass spectrometry (GC-MS). The bilities for F. moniliforme, with inhibition observed in the
main constituents in the P. auritum leaves numbered range of 80.63% to 100%. Similarly, for A. ochraceous,
27, listed according to their elution order (see Table 2). the effect was comparable, resulting in 100% inhibition
The major identified components quantified in at 0.09 mg/mL. Furthermore, P. dioica EOs at a concen-
amounts greater than 5.0% were safrole (51.383%), tration of 0.26 mg/mL showed significant inhibition of
gamma-terpinene (12.62%), terpinolene (9.639%), and 83.37% for F. moniliforme, with inhibition reaching 100%
beta-terpinene (5.94%). at 0.43 mg/mL. Concerning A. ochraceous, P. dioica EOs
The GC-MS analysis pertaining to the P. dioica leaf demonstrated substantial inhibition at concentrations
EOs resulted in the identification of a total of 29 con- ranging from 0.09 to 0.26 mg/mL, with 100% sporula-
stituents (see Table 3). The main identified components, tion inhibition observed at 0.16 mg/mL.
 Cogent Food & Agriculture 5

Table 2. GC/MS analysis of EOs derived from Piper auritum.

Peak # Retention time (min) CAS# Compound Class %
1 6.735 007785-70-8 Pinene Monoterpene (Terpenes) 4.669
2 7.556 79–92-5 Camphene Monoterpene (Terpenes) 0.805
3 8.396 127-91-3 (-) Z beta-pinene Monoterpene (Terpenes) 4.423
4 8.613 555-10-2 Beta –phellandrene Monoterpene (Terpenes) 0.986
5 9.247 007785-70-8 3-carene Bicyclic Monoterpene (Terpenes) 1.416
6 9.636 0099-83-2 Alpha-phellandrene Monoterpene (Terpenes) 0.349
7 10.015 000099-86-5 Beta-terpinene Monoterpene (Terpenes) 5.940
8 10.843 – Unrecognized – 0.336
9 11.961 000099-85-4 Gamma-terpinene Monoterpene (Terpenes) 12.626
10 12.860 000535-77-3 O-cymene Aromatic hydrocarbon 3.351
11 13.318 000586-62 Terpinolene Monoterpene (Terpenes) 9.639
12 17.673 00821-55-6 2-nonanone Symmetrical ketone 0.071
13 22.059 (+) – cyclozativene 0.108
14 22.460 3856-25-5 Copaene Sesquiterpene (Terpenes) 0.319
15 23.668 0076-22-2 Camphor Bicyclic monoterpene ketone (Terpenes) 0.146
16 26.299 5655-61-8 Bornyl acetate Esters 0.173
17 26.835 Beta-cubebene Tricyclic sesquiterpene (Terpenes) 0.114
18 27.105 0087-44-5 Caryophyllene Polycyclic Sesquiterpenes (Terpenes) 0.206
19 32.319 10208-80-7 Muurolene Sesquiterpene and a carbobicyclic compound (Terpenes) 0.239
20 32.799 3242-08-8 Elixene Sesquiterpenes (Terpenes) 0.086
21 33.841 00483-76-1 Delta-cadinene Sesquiterpenes (Terpenes) 0.118
22 38.598 00512-44-5 P-cymenol Alkylbenzene, sesquiterpene (Terpenes) sesquiterpene 0.074
23 40.154 000094-59-7 Safrole Phenylpropanoid 51.383
24 47.055 0093-15-2 Methyl eugenol Phenylpropanoid 0.063
25 52.607 6859-40-9 Spatulenol Tricyclic sesquiterpene alcohol (Terpenes) 0.076
26 54.635 80-56-8 Pinene Unsaturated bicyclic monoterpenes (Terpenes) 0.104
27 59.115 79–92-5 Camphene Bicyclic organic compound, monoterpene (Terpenes) 0.150

Table 3. GC/MS analysis of EOs derived from Pimenta dioica.

Retention time
Peak # (min) CAS# Compound Class %
1 6.304 2867-05-2 3-thujene Bicyclic Monoterpenes (Terpenes) 0.156
2 7.657 499-97-8 Pseudolimonene Terpenes 0.87
3 7.826 99-83-2 Phellandrene Monoterpene (Terpenes) 0.194
4 8.519 127-91-3 (-)-Beta-pinene Monoterpene (Terpenes) 11.447
5 9.059 99-86-5 Alpha-terpinene Monoterpene (Terpenes) 0.195
6 9.524 5989-27-5 D-limonene Monoterpene (Terpenes) 0.474
7 10.255 3779-61-1 Trans-beta-ocimene Monoterpene (Terpenes) 0.101
8 10.806 3338-55-4 (Z)-beta-Ocimene Monoterpene 2.723
9 11.664 202–796-7 P-cymene Monoterpene (Terpene) 0.162
10 12.073 586-62-9 Terpinolene Monoterpene (Terpene) 0.344
11 21.212 3856-25-5 Copaene tricyclic sesquiterpenes (Terpene) 0.365
12 23.026 78-70-6 Linalool Monoterpene (Terpene) 0.419
13 25.058 87-44-5 Beta-caryophyllene Monoterpene (Terpene) 6.747
14 26.693 25246-27-9 Alpha-aromadendrene Sesquiterpenoids (Terpene) 0.084
15 27.592 6753-98-6 α-Humulene Monocyclic sesquiterpene (Terpene) 1.110
16 28.230 – Unrecognized – 0.107
17 28.529 21747-46-6 Viridiflorene Carbotricyclic sesquiterpene (Terpene) 0.167
18 29.107 13744-15-5 Beta-cubebene Tricyclic sesquiterpene (Terpene) 0.346
19 29.632 483-75-0 Alpha-amorphene Sesquiterpenes (Terpene) 0.061
20 30.138 29873-99-2 Gamma-elemene Sesquiterpene (Terpene) 0.359
21 31.086 523-47-7 Beta-cadinene Sesquiterpenoids (Terpene) 0.336
22 34.186 – Unrecognized – 0.068
23 41.657 1139-30-6 Caryophyllene oxide Sesquiterpenoid (Terpene) 0.059
24 42.647 93-15-2 Eugenol methyl ether Phenylpropene (a type of 0.309
phenylpropanoid)
25 44.701 87-44-5 (-)-Caryophyllene-(L1) Bicyclic sesquiterpene (Terpene) 0.055
26 45.016 552-02-3 Viridiflorol Sesquiterpenoid (Terpene) 0.088
27 46.238 6750-60-3 Spathulenol Tricyclic sesquiterpenoid 0.08
28 47.520 97-53-0 Eugenol Allylbenzene (Terpene) 71.73
29 48.424 1898-13-1 Cembrene Monocyclic diterpene (Terpene) 0.838

4. Discussion et al. (2012). However, it is known that variations in

In general, EO yields from P. dioica were higher than the proportion of each component and composition
those of P. auritum. The yield of P. auritum was dou- are determined by factors such as the extraction
ble that reported in the study conducted by Pineda method, geographical origin, plant type, vegetative
6 A. LAM-GUTIÉRREZ ET AL.

stage, plant part, environmental conditions, storage much as 100%. In this study, the agar diffusion
conditions, among others (Salehi et al., 2019). method was used as a simple and quick test to
Safrole (phenylpropanoids) is the main compo- measure the susceptibility of fungi to essential oils,
nent in the EO from P. auritum leaves, consistent serving as a pre-screening step for determining anti-
with previous reports. However, it is interesting to fungal activity of EOs (Yi-Xin et al., 2014). An effec-
note that EOs from China and Ecuador reported cin- tive EO can inhibit the growth of microorganisms,
namaldehyde and γ-terpinene as the main compo- such as fungi, resulting in the formation of a zone
nents (Salleh & Nuzul, 2021). EOs from P. auritum of inhibition.
characterized in Panama contained 70% safrole, while Although there are few reports about EOs from P.
those originating in Cuba contained 64.5% (Da Silva dioica leaves and their respective MIC and MFC val-
et al., 2017). ues against A. ochraceous and F. moniliforme, it has
On the other hand, the constituents in EOs from been revealed that biological activity occurs against
P. dioica leaves, are consistent with Zhang and other genera of microorganisms, as well as within
Lokeshwar (2012), who revealed that the main com- the same genus but different species. MIC values for
ponents found were lignins, terpenoids and polyphe- EOs from P. dioica fruit have been reported against
nols with eugenol being the most common Acinetobacter (8 mg/mL), Klebsiella pneumonia (4 mg/
component in fruits and leaves. mL), Proteus vulgaris (4 mg/mL), Enterococcus fecalis
Seepe et al. (2021), reported antifungal activity (4 mg/mL), Staphylococcus aureus (1 mg/mL), and
for EOs obtained from P. dioica fruit when using Staphylococcus epidermidis (1 mg/mL) (Chouhan et al.,
1 µL/mL in the agar dilution method, resulting in 2017). Eugenol, the most important component iso-
inhibition of F. oxysporum and F. verticillioides by as lated from P. dioica, has been shown to inhibit the
growth of many phytopathogens, such as A. flavus
(2% eugenol v/v), A. terreus, and Fusarium spp. (4%
Table 4. Minimal inhibitory concentration (MIC), and mini-
v/v) (Olea et al., 2019; Rodrigues et al., 2019; Yi-Xin
mum fungicidal concentration (MFC) of EOs obtained from P.
dioica and P. auritum on A. ochraceous. and F. moniliforme. et al., 2014).
Pimenta dioica Piper auritum It has been confirmed via static water toxicity
Plant pathogenic MIC (mg/ MFC (mg/ MIC (mg/ MFC (mg/ tests in animal models that eugenol and other con-
fungi mL) mL) mL) mL) stituents from EOs are non-persistent in fresh water
Aspergillus 0.16 1.21 0.16 15 and lack of noticeable resistance development
ochraceus
Fusarium 0.43 0.43 0.09 0.16 (Isman, 2000; Raveau et al., 2020). In our study, EOs
moniliforme from P. dioica leaves contained 71.73% eugenol.

Figure 1. Minimum Fungicidal Concentration (MFC). The lowest concentration of essential oil from P. dioica that completely
eliminate A. ochraceus. Treatment: A) Negative control and B) 1.21 mg/mL of essential oil from P. dioica.
 Cogent Food & Agriculture 7

Figure 2. Minimum Fungicidal Concentration (MFC). The lowest concentration of essential oil from P. auritum that completely
eliminate F. moniliforme. Treatment: A) Negative control and B) 0.16 mg/mL of essential oil from P. auritum.

Table 5. Inhibition of spore germination: Effect of EOs from P. dioica and P. auritum. Values were
expressed as mean ± SD; Different letters (p < 0.05) indicated significant differences according to
Tukey test. K = ketoconazole.
EO
P. dioica P. auritum

Concentrations % sporulation
Treatment (mg/mL) F. moniliforme A. ochraceous F. moniliforme A. ochraceous
1 0.09 83.296 ± 3.318 a 19.509 ± 1.713 a 19.365 ± 0.677 a 100 ± 0 a
2 0.16 51.363 ± 2.918 b 0 0 100 ± 0 a
3 0.26 16.663 ± 0.380 c 0 0 100 ± 0 a
4 0.43 0 0 0 100 ± 0 a
5 0.73 0 0 0 100 ± 0 a
6 1.21 0 0 0 100 ± 0 a
7 2 0 0 0 100 ± 0 a
8 3.4 0 0 0 100 ± 0 a
9 5.6 0 0 0 38.604 ± 3.100 b
10 8 0 0 0 22.656 ± 2.616 c
11 15 0 0 0 0
12 Ketoconazol 0 0 0 0

Piper species have also been used in traditional mL) (Valarezo et al., 2021; Van-Vuuren & Holl, 2017).
medicine, as it has been reported that EOs from P. Based on these categories, EOs from P. auritum and
auritum leaves have activity against the leishmania P. dioica presented very high MIC values, which may
parasite (Da Silva et al., 2017). Additionally, MIC val- be due to the presence of eugenol and safrole.
ues have been reported for Colletotrichum acutatum Eugenol has been reported to cause morphological
(364.70 µg·mL−1) and Botryodiplodia theobromae changes in hyphae and conidiophores, to reduce
(254.40 µg·mL−1) (Salleh & Nuzul, 2021), as well as an ergosterol, and to inhibit ochratoxin A production by
MIC50 of 9 mg/mL for F. oxisporum and F. equiseti iso- A. ochraceus (Hua et al., 2014). Furthermore, this ter-
lates (Chacón et al., 2021). pene inhibits the H + ATPase system, causing intracel-
In an attempt to define in vitro antimicrobial activ- lular acidification and cell death (Ahmad et al., 2011).
ity, different investigations have suggested the devel- On the other hand, safrole is a methylenedioxy com-
opment of a scale for EOs and extracts, where MIC pound whose toxicity is related to the structure of its
values are classified as having moderate antifungal benzene derivative, capable of reducing mycelial
activity (between 500–1000 mg/mL), strong activity growth in some plant pathogens and can also act as
(101–500 mg/mL), and very strong activity (≤100 mg/ an phytoantipeptide (Chacón et al., 2021).
8 A. LAM-GUTIÉRREZ ET AL.

Raveau et al. (2020), reported different mecha- aspect of using EOs or their constituents as an
nisms of action related to EOs of vegetal species alternative in crops is their low, favorable mamma-
(even extracts) against microorganisms, including lian toxicity. The new challenges in the use of
the inhibition of cell division, fungi cell wall forma- essential oils lie in the application of new technol-
tion, efflux pumps, protein synthesis, mitochondrial ogies, namely nanoemulsion, encapsulation, and
electron transport and ergosterol synthesis, as well nanoparticles, as an attempt to ensure a more con-
as disruptions in the cell membrane (Mazu et al., trolled release of constituents while also enhancing
2016; Pascual-Villalobos et al., 2019; Estrada-Cano their stability.
et al., 2017).
Fungi have the capacity to develop resistance mech-
Acknowledgments
anisms against specific chemical compounds over time,
a phenomenon exhibiting significant variability across The authors wish to thank biologist Manuel de Jesús
different genera. Certain fungi may harbor enzymes Gutiérrez Morales and Francisco Hernández Najarro in
charge of the office of the ‘Herbarium: Dr. Faustino Miranda
proficient in metabolizing or neutralizing toxic com-
Botanical Garden, in Chiapas, Mexico’ for the identification
pounds, whereas others may possess efflux pump sys- of the plant and tree.
tems facilitating the expulsion of said chemicals from
the cellular milieu (Del Sorbo et al., 2000).
Moreover, the susceptibility of fungi to chemical Disclosure statement
constituents can be intricately linked to their cellu- No potential conflict of interest was reported by the
lar architecture and physiological makeup. author(s).
Disparities in cell membrane composition or the
expression profiles of cellular receptors can pro-
foundly influence a fungus’s ability to either absorb About the author
or withstand particular chemical agents (Adebo
et al., 2020). Lastly, the genetic diversity prevalent
within genera such as Fusarium and Aspergillus
assumes a pivotal role in dictating the sensitivity of
individual species to chemical compounds. These
inherent genetic variations can significantly impact
fungi’s capacity to either endure or metabolize the
toxic constituents found within essential oils (Lee &
Taylor, 2019).
However, despite our results being in agreement
with other studies, further research must be con-
ducted to elucidate the mechanisms of action of
their components. Additionally, it is necessary to
establish whether the synergy between phytochemi- Dr. Lam brings a diverse academic background to her
cals could be partly responsible for the fungicidal research endeavors. With a Ph.D. in Food Sciences and
Biotechnology and expertise in Biochemical Engineering,
effect, or whether it is attributed to a specific she delves into various facets of plant biotechnology and
compound. food science. Her work focuses on Phytochemistry, explor-
ing the potential of agro-industrial waste, understanding
plant biology, and investigating the molecular makeup of
5. Conclusions microorganisms for biotechnological applications. Beyond
her research, Dr. Lam actively contributes to the scientific
EOs from P. auritum and P. dioica leaves exhibited
community as a recognized member of the National and
fungicidal action against phytopathogens belong- State Researchers Systems. Her collaborations extend inter-
ing to the Fusarium and Aspergillus species. Both nationally, including research stints at esteemed institu-
are notorious for causing severe damage to plants tions. She is also engaged in academia through the
and for producing mycotoxins that pose risks to investigation group "Innovation and Technological Transfer
human and animal health. An alternative of added for the Use of Agricultural Products and Waste." Additionally,
she guides students through thesis projects funded by
value is proposed, as P. dioica crops are often dis- TecNM, contributing to the advancement of the field as a
carded in tree prunings or used as compost with faculty researcher at the Technological Institute of
minimal processing. Perhaps the most promising Cintalapa.
 Cogent Food & Agriculture 9

Dra. Lam, in an experimental greenhouse inspecting seed- Molecules (Basel, Switzerland), 24(20), 3713. PMID:
lings of economically significant plant species in the 31619024; PMCID: PMC6832927 https://doi.org/10.3390/
Valle-Zoque region of Chiapas, Mexico. molecules24203713
Del Sorbo, G., Schoonbeek, H. J., & De Waard, M. A. (2000).
Fungal transporters involved in efflux of natural tox-
ORCID ic compounds and fungicides. Fungal Genetics and
Biology: FG & B, 30(1), 1–15. DOI: https://doi.org/10.1006/
Anayancy Lam-Gutiérrez http://orcid. fgbi.2000.1218
org/0000-0001-9124-5721 Durant-Archibold, A. A., Santana, A. I., & Gupta, M. P. (2018).
Teresa Ayora-Talavera http://orcid. Ethnomedical uses and pharmacological activities of
org/0000-0003-2469-3238 most prevalent species of genus Piper in Panama: A re-
Eduardo Raymundo Garrido-Ramírez http://orcid. view. Journal of Ethnopharmacology, 217, 63–82. https://
org/0000-0002-3618-5014 doi.org/10.1016/j.jep.2018.02.008
Víctor Manuel Ruíz-Valdiviezo http://orcid. Estrada-Cano, C., Anaya-Castro, M. A., Muñoz-Castellanos,
org/0000-0003-0572-8845 L., Amaya- Olivas, N., García-Triana, A., & Hernández-Ochoa,
Jorge Martín Guzmán-Albores http://orcid. L. (2017). Antifungal activity of microcapsulated clove
org/0000-0002-3383-4605 (Eugenia caryophyllata) and Mexican Oregano (Lippia ber-
Jairo Cristóbal-Alejo http://orcid. landieri) essential oils against Fusarium oxysporum.
org/0000-0001-9354-1129 Journal of Microbial & Biochemical Technology, 9(1), 567–
571. https://doi.org/10.4172/1948-5948.1000342
Gomes da Rocha-Voris, D., Dos Santos-Dias, L., Alencar-
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