Plant Extracts from the Yucatan Peninsula in the In Vitro Control of Curvularia lunata and Antifungal Effect of Mosannona depressa and Piper neesianum Extracts on Postharvest Fruits of Habanero Pepper

Plant extracts are a valuable alternative for the control of phytopathogenic fungi in horticultural crops. In the present work, the in vitro antifungal effect of ethanol and aqueous extracts from different vegetative parts of 40 native plants of the Yucatan Peninsula on Curvularia lunata ITC26, a pathogen of habanero pepper (Capsicum chinense), and effects of the most active extracts on postharvest fruits were investigated. Among these, the ethanol extracts of Mosannona depressa (bark from stems and roots) and Piper neesianum (leaves) inhibited 100% of the mycelial growth of C. lunata. The three extracts were partitioned between acetonitrile and n-hexane. The acetonitrile fraction from M. depressa stem bark showed the lowest mean inhibitory concentration (IC50) of 188 µg/mL against C. lunata. The application of this extract and its active principle α-asarone in the postharvest fruits of C. chinense (500 µg/mL) was shown to inhibit 100% of the severity of the infection caused by C. lunata after 11 days of contact. Both samples caused the distortion and collapse of the conidia of the phytopathogen when observed using electron microscopy at 96 h. The spectrum of M. depressa enriched antifungal action is a potential candidate to be a botanical fungicide in the control of C. lunata in cultivating habanero pepper.


Introduction
The species of the genus Capsicum spp. (pepper) are among the most appreciated vegetables worldwide with an annual production of 3,112,480 tons in Mexico, meaning that it is considered to rank second [1]. Among the Capsicum species, the habanero pepper (Capsicum chinense Jacq.) is mainly cultivated in the states of Campeche, Yucatan, and Quintana Roo (25,128 tons in 2022), which is appreciated worldwide for its spiciness due to its capsaicin content that has diverse applications in medicine and biotechnology [2,3].

In Vitro Activity of Plant Extracts on Curvularia lunata
The results of the screening of species effective against C. lunata ITC26 are presented in Table 1 In total, 13 extracts were active against C. lunata at 2000 µg/mL, corresponding to eight ethanolic extracts from Alvaradoa amorphoides leaves, Licaria sp. roots., Helicteres baruensis leaves and stems, Mosannona depressa stem bark and root bark, and Piper neesianum leaves and roots. The most active were the ethanolic extracts from Mosannona depressa stem bark and root bark and Piper neesianum leaves with lethal effects on C. lunata. In contrast, the extracts from the leaves of A. amorphoides, Licaria sp., and H. baruensis and the roots of P. neesianum moderately affected (MCI = 75%) the mycelial growth of this pathogen (Table 1). Only five aqueous extracts showed a significant capacity to inhibit the growth of C. lunata (MCI = 25%) at a concentration of 3% (w/v), corresponding to the extracts of Byrsonima bucidifolia leaves, stems, and roots and Morella cerifera leaves and roots.

Determination of Minimum Inhibitory Concentration of Extracts, Fractions, and α-Asarone
The three most active extracts from M. depressa and P. neesianum were partitioned, and their minimum inhibitory concentration (MIC) against C. lunata ITC26 was determined. The results showed that the lowest MIC was 250 µg/mL caused by the ethanolic extracts of stem bark and root bark from M. depressa, both with fungicidal effects. With lower activity and a MIC of 500 µg/mL, the precipitate of M. depressa root bark, the ethanolic extract from P. neesianum leaves, and its acetonitrile fraction were detected as well as the standard commercial α-asarone, all with fungistatic effects. The hexane fractions from the three active ethanolic extracts were inactive against C. lunata ITC26 (Table 2).

Inhibitory Concentration (IC 50 and IC 95 ) of the Most Active Extracts, Fractions, and α-Asarone
The ethanolic extracts from M. depressa stem bark showed the lowest IC 50 and IC 95 of 188 and 218 µg/mL, respectively, against C. lunata (Table 3). The α-asarone showed an IC 50 of 190 µg/mL, which is equivalent to the ethanolic extracts, but its IC 95 of 325 µg/mL was higher. The IC 50 and IC 95 for the precipitate from M. depressa root bark (229 and 265 µg/mL, respectively) and P. neesianum acetonitrile (222 and 256 µg/mL, respectively) fractions were equivalent against C. lunata (Table 3).

Effect of Active Extracts on Curvularia lunata
Exposure of C. lunata hyphae and conidia to the negative control for 96 h confirmed the well-formed hyphae and ovoid-shaped conidia characteristics of the fungal species ( Figure 1A-C). After 96 h exposure to ethanolic extracts from M. depressa bark from the stems and roots at 2000 µg/mL as well as to standard α-asarone at 500 µg/mL, dehydrated and contorted hyphae and fully dry conidia were observed ( Figure 1D-F).

In vivo Effect of Extracts and α-Asarone against C. lunata on Habanero Pepper Fruits
The analysis of variance to estimate the effect of the treatments on the decrease in severity or control of C. lunata isolated from postharvest fruits showed significant statistical differences (p ≤ 0.01) among the treatments (Table 4, Figure 2). Treatments T1, T2, T4, T7, and T10 had a lethal effect (IC = 100%) on the control of C. lunata infection in the post-

In Vivo Effect of Extracts and α-Asarone against C. lunata on Habanero Pepper Fruits
The analysis of variance to estimate the effect of the treatments on the decrease in severity or control of C. lunata isolated from postharvest fruits showed significant statistical differences (p ≤ 0.01) among the treatments (Table 4, Figure 2). Treatments T1, T2, T4, T7, and T10 had a lethal effect (IC = 100%) on the control of C. lunata infection in the postharvest fruits of C. chinense 11 days after inoculation. The most effective treatments corresponded to the ethanolic extract from M. depressa stem bark and the standard α-asarone at 500 µg/mL (Table 5). In the T3, T6, and T9 treatments, a reduction in infection severity (3.4-1.5%) was observed at 250 µg/mL, which is statistically equal to both extracts and α-asarone. In the negative control (T11), 11 days after inoculation, necrosis was observed in the habanero pepper fruits. The 1% Tween 80 solvent (T12) was not toxic to postharvest habanero pepper.

Discussion
The present contribution is an addition to the in vitro bioprospecting of native plant extracts against the phytopathogenic fungi of habanero pepper [29]. In particular, the C. lunata ITC26 was isolated for the first time from the postharvest fruits of habanero pepper [9] with the phytopathogen being recognized as the causal agent of leaf spot, leaf curl, and the subsequent defoliation in C. frutescens [7,30], anthracnose in C. annumm [31,32], and C. chinense seeds in conservation (strain ITCC 02) [33]. In Yucatan, C. lunata strains have been isolated from the leaves of Solanum lycopersicum L. [26] and Tridax radiata Lodd., Ex-Schult., and Schult. f. [27]. Therefore, the contribution of alternatives to the control of C. lunata is necessary. The 40 species under study were collected from six different locations and evaluated against Fusarium equiseti strain FCHE and F. oxysporum strain FCHJ as phytopathogens on habanero pepper [29], the root-knot nematode Meloidogyne incognita, M. javanica [34], as well as the repellent and oviposition inhibitory effect against Bemisia tabaci [35].
The in vitro antifungal assay of 184 extracts from the different vegetative parts obtained of the plant species from the Yucatan Peninsula led to the detection of 13 extracts (7% of the total, Table 2) with activity against C. lunata ITC26. The data reveal that C. lunata ITC26 is more sensitive to the ethanolic extracts from A. amorphoides, Helicteres baruensis, Licaria sp., M. depressa, and P. neesianum than the aqueous extracts. This effect is similar to those previously reported with F. equiseti FCHE and F. oxysporum FCHJ, which were more sensitive (MGI = 100%) to ethanolic extracts from M. depressa, Parathesis cubana, and P. neesianum at 2000 µg/mL [29]. The active aqueous extracts (5.4%) showed a low antifungal capacity (MGI = 25%) at the 3% w/v. To date, the extracts of the species studied have not been reported against the pathogen C. lunata except for aqueous extracts from M. depressa stem bark and root bark and P. neesianum leaves with the highest inhibition of sporulation and the germination of conidia (100%) at 3% w/v strain ITC22 isolated from tomato [26]. Other reports of effective aqueous extracts applied in vitro against C. lunata include the leaves of Lawsonia inermis L. (2%, w/v) [36], Ocimum sanctum (10% w/v) [37], and B. flammea stem bark (3%, w/v) [27], which showed 21, 75, and 89%, respectively, effect on the mycelial growth of the pathogen.
In contrast, the ethanolic extracts from M. depressa bark from stems and roots and P. neesianum leaves were lethal against C. lunata ITC26. A previous study with the ethanolic extracts of Calycopteris floribunda leaves and methanolic extract of Tribulus terrestris stem against C. lunata showed MICs of 250 and 300 µg/mL, respectively [38,39]. The hexane and chloroformic extracts of Costus speciosus rhizome and the chloroformic extract of Piper betle presented higher MICs (500-100 µg/mL) [40,41], and the ethanolic extract from Acorus calamus leaves inhibited 57% of C. lunata growth at 1000 µg/mL [42].
The fractions of the ethanolic extract of M. depressa stem bark were less effective against C. lunata, showing higher MIC, IC 50 , and IC 95 , as well as the pure α-asarone compound. Previously, it was documented that α-asarone is the major metabolite of the acetonitrile fraction of M. depressa stem bark. This is contrary to what was observed against F. equiseti and F. oxysporum, where α-asarone showed lower IC 50 (236 and 482 µg/mL, respectively) compared to the ethanolic extract of M. depressa bark (IC 50 = 468 and 944 µg/mL, respectively) and its acetonitrile fraction (IC 50 = 462 and 472 µg/mL, respectively) [29]. The loss of activity of the extract when fractionated may be due to the loss of material during the fractionation, degradation, and evaporation of the more potent components; it may also be attributed to the loss of the synergistic effect between the compounds in the mixture when they are separated [43]. The metabolite α-asarone from M. depressa stem bark could be synergized with other components of the ethanolic extract responsible for the antifungal activity against C. lunata. The synergistic effect among compounds has been previously documented; for example, the combination of eugenol and citral showed synergistic antifungal activity against Penicillium roqueforti, reducing the dose of oil required to damage the fungal cell membrane [44].
Based on the results of the in vitro antifungal bioassays against C. lunata in this study and the most effective and renewable plant part, the ethanolic extracts from M. depressa stem bark, P. neesianum leaves, as well as α-asarone were selected for evaluation on the postharvest fruits of C. chinense. The ethanolic extract from M. depressa stem bark was the most effective followed by P. neesianum. This study is the first in vivo report of the antifungal potential of M. depressa, P. neesianum, and α-asarone to control necrosis caused by C. lunata on C. chinense fruits. The present contribution adds to the few in vivo studies reporting the activity of plant extracts against C. lunata. Other reports include the aqueous extract of B. flammea stem bark (3%, w/v), which showed 100% control of the severity of the C. lunata infection in the postharvest fruits of C. chinense [9], and Azadirachta indica oil (1%, w/v), which controlled 50% of C. lunata infection in Capsicum annuum [8]. The effect of active ethanolic extracts (2000 µg/mL) and α-asarone (500 µg/mL) on C. lunata mycelium and conidia observed with SEM was similar to that reported by Cruz-Cerino et al. [29] against F. equiseti and F. oxysporum. Ethanolic extracts of M. depressa and α-asarone, by contact, deform and dehydrate conidial and fungal cells, tear the cell wall, and rupture the fungal cell membrane, which prevent the synthesis of essential components such as ergosterol [45,46]. The α-asarone is a potent inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that catalyzes ergosterol synthesis in fungi [47]. Their synthetic analogs showed a high effect on recombinant HMGR from Candida glabatra with IC 50 values of 42.65 and 28.77 µM for 2-(2-Methoxy-5-nitro-4-propylphenoxy) acetic acid and 2-(2-Methoxy-4-propylphenoxy) acetic acid, respectively [48]. Additionally, the effect of the compound α-asarone, when evaluated at 500 µg/mL on the hyphal morphology of C. lunata, is reported for the first time.
In addition, the species P. neesianum confirmed its antifungal capacity, whose ethanolic extract was highly effective in vitro and in the postharvest fruits of habanero pepper to prevent the infection of C. lunata ITC26 strain isolated from tomato, while aqueous extract from P. neesianum leaves effectively inhibited the sporulation and germination of the conidia of C. cassiicola ITC22 isolated from tomato [26]. As previously reported, the ethanol extract and its acetonitrile fraction showed an IC 50 of 788 and 462 µg/mL on F. equiseti FCHE isolated from habanero pepper [29] (Cruz-Cerino et al., 2020). Other studies have previously reported the antifungal effect of Piper caninum leaf extract at 3% on Nigrospora orizae and Curvularia verriculosa pathogens on Oryza sativa [60,61]. The essential oils from P. neesianum leaves were identified as bicyclogermacrene, germacrene D, and β-caryopyllene (7.5%) and were major compounds among the 19 detected with gas chromatography coupled to mass spectrometry analysis [62].

Plant Collection and Processing
The 40 plants from 6 different sites in the Yucatan Peninsula, Mexico, were collected and processed as previously described by Cruz-Cerino et al. [29]. One specimen of each plant species was deposited in the herbarium U Najil Tikin Xiw (the house of dry grass in Mayan) of the Natural Resources Unit of the Centro de Investigación Científica de Yucatán, A.C. (CICY) with their respective collection numbers (Table 5).

Plant Extracts and Partition of Active Extracts
The aqueous and ethanol extracts were obtained as previously reported by Cruz-Cerino et al. [29]. Briefly, plant material (1.5 g) was extracted for 15 min with boiled water, filtered, and diluted with distilled water (25 mL) to a final concentration of 6% (w/v). The aqueous extract was sterilized through a 0.22 µm Millipore filter (Merck-Millipore, Burlington, MA, USA) and frozen at −17.5 ± 0.5 • C until use. In comparison, the ethanolic extracts were obtained using sonication at 20 kHz (Cole-Parmer, Chicago, IL, USA) at room temperature for 20 min each time (three times). The solvent was removed under vacuum in a rotary evaporator (IKA model RV-10, Staufen, DE) until dryness was reached.
The ethanol extracts with a lethal effect on C. lunata were fractionated with hexaneacetonitrile three times (2:1, 1:1, 1:1 v/v), obtaining a hexane fraction (a), acetonitrile fraction (b), and methanol-soluble precipitate (c) of each sample after eliminating the solvents via evaporation as described above.

Fungal Cultures
Curvularia lunata ITC26 was obtained from the fungal collection of the Phytopathology Laboratory, Tecnológico Nacional de México, campus Conkal. The strain was isolated from lesions of the habanero pepper fruit [9], maintained in (a) 20% glycerol (v/v) frozen at −80 • C, (b) sterile distilled water, and (c) potato dextrose agar in slant tubes (PDA, BD, Bioxon, Edo. Mex., MX) at 4 • C in the dark.

Preparation of Conidial Suspension
C. lunata strain was cultured on an oat agar culture medium and incubated as described with slight modifications by Cruz-Cerino et al. [29]. Briefly, a sterile saline solution (5 mL) was added to the surface of the fungal culture (11 days), and the conidia were scraped with a sterile brush. Then, the conidial suspension was filtered through a double layer of sterile cheesecloth and adjusted to a final concentration of 1 × 10 5 conidia/mL using a hemocytometer. The antifungal evaluation of the aqueous and ethanolic extracts against C. lunata was carried out with the microdilution bioassay [29].

Bioassay with Aqueous Extracts
The mycelial growth inhibition (MGI) of the C. lunata strain was determined using a 96-microwell plate, as described by Abou et al. [63] and, with slight modifications, Cruz-Cerino et al. [29]. The aqueous extracts (100 µL of each 6%) were transferred to each microwell. The fungicide prochloraz (5 µL, 450 g a.i./L; Bayer CropScience, Clayton, NC, USA) and 100 µL of the conidial suspension as the negative control were used. Finally, 100 µL of the conidial suspension was added to each well for a final concentration of 3% w/v of the aqueous extracts, 0.112% of prochloraz (w/v), and 5 × 10 4 conidia/mL of C. lunata strain. Each sample was tested in triplicate, and all microdilution plates were maintained at 27 ± 2 • C for 16 h light/8 h dark. The mycelial growth was recorded at 96 h using a 0-4 scale, where 4 is full (0% MGI), and 0 is the absence of MG (MGI = 100%) [64].
The mycelial growth (MG) data were converted to a percentage of MGI using Abbott's formula: [(% MG in the negative control − % MG in the treatment)/% MG in the negative control)] × 100.

Minimum Inhibitory Concentration
Serial dilutions of the selected ethanol extracts (2000, 1000, 500, 250, and 125 µg/mL) and their fractions (1000, 500, 250, and 125 µg/mL) were prepared as described above and tested using a microdilution assay to determine the MIC [28]. The pure α-asarone standard was acquired from Sigma-Aldrich (St. Louis, MO, USA) and was tested at 500, 250, and 125 µg/mL. The samples were performed with four replicates three times. The same controls and incubation conditions (96 h) were used as described above. The lowest extract concentration, at which no mycelial growth was observed in the wells, was registered as the MIC.
Finally, the fungicidal or fungistatic effect was determined for all microwells that did not grow after 96 h. Then, 10 µL from each microwell was transferred to PDA in a Petri dish and maintained at 27 ± 2 • C. The absence of mycelial growth after 72 h indicated the sample's fungicidal effect, and the mycelial growth was fungistatic [65].

Evaluation of Ethanolic Extracts on Hyphal Morphology of Curvularia lunata ITC26
The effect of the root and stem bark of M. depressa (2000 µg/mL) and α-asarone (500 µg/mL) on the mycelium and conidia of C. lunata was observed in a JSM 6360 SEM (Jeol, Tokyo, Japan) at 20 kV. The samples were prepared as previously described by Cruz-Cerino et al. [29]. Briefly, a disk (5 mm) of C. lunata grown on oat agar culture medium for 11 days was exposed to 200 µL of ethanol extract. After 96 h, the sample was filtered through a nylon membrane and fixed in a mixture of 2.5% v/v glutaraldehyde and 0.2 M sodium phosphate (pH 7.2 at 4 • C). After 48 h, the sample was washed (2×, 1 h each time) with phosphate buffer and dehydrated in an ethanol series (1 h each: 30-100%, 2× absolute ethanol). The samples were dried with CO 2 , attached to a sample holder, and coated with gold for 10 min in an ionizing chamber (Dentom Vacuum-Desk II, Moorestown, NJ, USA).

In Vivo Evaluation of Ethanolic Extracts and α-Asarona against C. lunata on Habanero Pepper Fruits
The habanero pepper fruits used in the bioassay were from cv. Jaguar orange when ripe (Seminis Vegetable Seeds ® , Sant Louis, MO, USA). The habanero pepper fruits were selected according to color and size, discarding those which were wrinkled and damaged. These were washed with tap water and superficially disinfected via immersion in a 70% alcohol solution (1 min) and 2% sodium hypochlorite (1 min), and then double rinsed in sterile distilled water (2 min). Immediately, small lesions were made on the surface of the fruits with the help of a sterile needle to promote infection. All ethanolic extracts were diluted in 1% Tween 80 (Sigma-Aldrich). The treatments (T) evaluated corresponded to the stem bark of M. depressa at concentrations of 750 (T1), 500 (T2), and 250 (T3) µg/mL; the leaves of P. neesianum at 750 (T4), 500 (T5), and 250 (T6) µg/mL; the α-asarone standard at 500 (T7), 250 (T8), and 125 (T9) µg/mL; the fungicide Mirage ® CE45 prochloraz (T10) at a concentration of 450 µg/mL as a positive control; and fruits treated with water (T11) and with 1% Tween 80 (T12) as negative controls. The habanero pepper fruits were submerged individually for 5 min in the ethanolic extracts at the indicated concentrations for each treatment. The fruits were left to dry in a laminar flow hood for 40 min and were inoculated via spraying with a suspension of C. lunata spores (1 × 10 5 spores/mL). At 48 h, it was inoculated for the second time [9]. Each treatment was carried out with five replicates, and they were incubated in plastic trays at a temperature of 27 ± 2 • C until the negative controls showed symptoms of the disease.
At 11 days after inoculation, the severity of the damage in the fruits was estimated, and the percentage of the effectiveness of the extracts was evaluated with the use of a scale of four classes, no visible damage (0), low severity (1), medium severity (2), and severe damage (3), as well as reporting the percentage of the average values (% severity) [9]. The experiment was repeated in 3 different events with 15 replicates per treatment (n = 15).

Statistical Analyses
A one-way analysis of variance was performed with the prior transformation of the original % MGI and severity fruits data using the formula y = arsin [sqrt (y/100)]. The treatment means were compared using Tukey's multiple range test (p = 0.05). Variance analyses were performed using SAS ver. 9.4 for Windows (SAS Institute, Cary, NC, USA). Using probit analysis, the IC 50 and IC 95 values for the extracts and effective fractions were calculated (95% confidence intervals).

Conclusions
This research is the first contribution to the in vitro and in vivo evaluations of native plant extracts against the pathogen C. lunata associated with the habanero pepper fruit in the Yucatan Peninsula. Our knowledge about antifungal extracts from native Yucatan species was enriched, particularly the spectrum of action of M. depressa and P. neesianum. The phytopathogen C. lunata ITC26 shows greater sensitivity to ethanol extracts of M depressa and P. neesianum than pure α-asarone. Our native species M. depressa and P. neesianum are viable alternatives in developing a natural antifungal agent to reduce the severity of C. lunata on the postharvest fruits of habanero pepper.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12162908/s1, Table S1: Percentage of inhibition of mycelial growth of Curvularia lunata ITC26 by plant extracts from 40 native species of the Yucatán Peninsula in the microdilution assay.