Volatiles of Zanthoxylum limoncello as Antifungal Agents against the Postharvest Rot of Manzano Pepper Triggered by Fusarium temperatum

Abstract

The manzano pepper (Capsicum pubescens) is an exportation product that generates substantial earnings for local producers in Mexico. Herein we report on the most relevant metabolic changes that occur during the postharvest rot of manzano peppers caused by Fusarium temperatum. Simultaneously, we describe the effect of the Zanthoxylum limoncello leaf essential oil (ZlEO) and its major volatiles on the control of this devastating disease. According to our results, ZlEO, 2-undecanone (34%), 2-undecenal (32%), and 2-dodecenal (8%) exerted in vitro fungicide activity on F. temperatum (MIC, 104.6–218.3 mg L⁻¹) and a strong in situ fungistatic effect in manzano peppers previously infected with F. temperatum. A differential fungistatic activity was observed for the natural agents assayed. However, the best results were confirmed with 2-dodecenal, which improved the shelf life of infected peppers up to 16 d post-inoculation. The protective effect of ZlEO and its major volatiles resulted in the conservation of fruit firmness, pH, protein, fat, fiber, ascorbic acid, and nutraceuticals of manzano peppers (carotenoids and capsaicinoids). Our findings endorse the potential use of ZlEO and its major volatiles as natural antifungals to prevent the soft rot triggered by F. temperatum.

1. Introduction

The manzano pepper (Capsicum pubescens) is one of the few hot peppers able to grow between 1700 and 2500 MASL and is also envisioned as a potential source of nutraceuticals with antioxidant, chemoprotective, and selective antimicrobial activity [1]. These properties are linked to the high amount of carotenoids, ascorbic acid, and capsaicinoids contained in this fruit [2,3,4]. The production of manzano pepper under open field conditions (>10 t ha⁻¹) is considered a sustainable activity for local producers from Mexico [3,5]. The introduction of this fruit into Mexico was recorded at the beginning of the 20th century. Since then, manzano pepper has been tagged as an important economic profit in several provinces because of its huge demand for the international market [2,3,4,6]. In the northern highlands of Puebla-Mexico, the state of Mexico, as well as in Michoacán-Mexico, manzano peppers are considered an irreplaceable condiment for local cuisine [2].

On the other hand, the high precipitation rate and relative humidity in which manzano peppers grow make this fruit susceptible to the attack of several fungal pathogens, especially those of the Fusarium genus. Recent investigations confirmed Fusarium sambucinum as a persistent phytopathogen of the manzano pepper rot, triggering substantial losses in fruit quality and economic losses for local producers [7,8]. Interestingly, the manzano pepper rot may be produced by the single or combined action of diverse phytopathogens such as Botrytis spp., Phytium spp., and Fusarium spp. [9]. A recent phytopathological report on manzano peppers described Fusarium temperatum as an emergent fungus able to cause serious losses of this fruit [10]. The infection of F. temperatum on manzano peppers produced rot spots with evident mycelial growth at the center of the spot and signs of necrosis in the periphery with changes in the size of the fruit, loss of turgor, and discoloration. An epidemic caused by this pathogen was documented in plots located in the northern highlands of Puebla-Mexico [10]. Since F. temperatum had been considered an infective agent of maize crops around the world [11,12], the pathogenicity of this fungus may be visualized as a new threat for the producers of manzano peppers in Mexico. Thus, new strategies for controlling this phytopathogen need to be envisioned. As is known, essential oils (complex mixtures of volatiles) and their bioactive compounds exert strong antimicrobial activity against a wide spectrum of phytopathogens [13,14]. In addition, these chemicals have shown negligible side effects on both human health and the environment if compared with synthetic antimicrobials [13,14]. The in vitro antifungal activity of essential oils has revealed the potential use of volatile fractions as natural agents for controlling fungal diseases; however, little is known about the in situ effects of these fractions on specific targets [13,15,16]. Pacheco-Hernández et al. [15] reported the in situ antifungal properties of the essential oil from Lepidium virgnicum and its main volatiles (phenylacetonitrile and alpha-terpineol) in tamarillo fruit previously infected with a wild strain of Colletotrichum acutatum. According to these authors, the essential oil of L. virginicum and phenylacetonitrile improved the shelf life of infected fruit up to 11 d. In addition, these treatments avoided the degradation of the main pigments of tamarillo fruit. Since Zanthoxylum limoncello is tagged as a sustainable condiment for local dishes in southern Mexico and a source of bioactive volatiles [16], this investigation was focused on determining the preservation effect of the Z. limoncello leaf essential oil on manzano peppers infected with Fusarium temperatum.

2. Materials and Methods

2.1. Chemicals

The HPLC solvents were provided by J.T. Baker^® whereas Rezasurin, 2-undecanone, 2-undecenal, 2-dodecenal, capsaicin, and dihydrocapsaicin were from Sigma-Aldrich Co. (St. Louis, MO, USA).

2.2. Obtainment of Zanthoxylum limoncello Leaf Essential Oil

The aerial parts (40 kg) of Zanthoxylum limoncello PLANCH. & OERST. ex TRIANA & PLANCH. (Rutaceae) were harvested in local plots from Yaonáhuac Puebla (19°56′55″ N 97°26′26″ W; 1997 MASL) from March to October 2020. Previously, voucher 158768 was deposited at the FCME-Herbarium-UNAM located in México City. The leaves were extracted by hydrodistillation process using a Clevenger-type apparatus, and the essential oil was gradually accumulated in an amber glass container to get enough for biological assessment. The essential oil was resuspended in n-hexane and dehydrated, as reported by Villa-Ruano et al. [16].

2.3. GC Profiling of Zanthoxylum limoncello Essential Oil (ZlEO)

Gas chromatography-mass spectrometry approaches were made, as previously reported by Villa-Ruano et al. [16], whereas metabolite identity was assigned in accordance with mass spectrum (70 eV) and Kovats index [17]. The identity of 2-undecanone, 2-undecenal, and 2-dodecenal was confirmed by the co-injection of authentic analytical standards. The semi-quantification of the volatiles was performed by integration of FID chromatograms.

2.4. Isolation of F. temperatum from Manzano Peppers

Manzano peppers showing symptoms of soft rot were collected in Yaonáhuac, Puebla, México (19°56′55″ N 97°26′26″ W; 1997 MASL) during the rainy season of 2020 (June–August 2020). The plants of manzano pepper from this geographical location (Capsicum pubescens Ruiz and Pav.) were certified by Ramiro Cruz-Duran at the herbarium of the FCME-UNAM where the voucher 177887 was deposited. The causal agent used for antifungal assays was that previously reported by Pérez-Vázquez et al. [10]. A monosporic culture of Fusarium temperatum (CP4) was recovered from the ceparium of DITCo-BUAP-Mexico and subjected to in vitro and in situ antifungal assays. The nucleotide sequence of the translation elongation factor 1-alpha gene isolated from F. temperatum was previously deposited in the National Center for Biotechnology Information with the accession MW570680.

2.5. Antifungal Assays

A monosporic culture of F. temperatum was grown on potato dextrose agar (PDA; Bioxon^®, Franklin Lakes, NJ, USA) at 28 °C. The culture was stressed with sunlight for 3 h during 10 d. Hyphal discs from cultures developed in PDA (7 d old) were extracted using the nozzle connection of a sterile tip (0.2 milliliters capacity) and deposited in 96 well-plates containing 2 × 10⁻⁴ L potato dextrose broth (PDB; Bioxon ^®, Franklin Lakes, NJ, USA). Then, resazurin (1 mM) was mixed with the culture medium as an indicator of cell viability [18]. The minimum inhibitory concentration (MIC) of the essential oil and its main volatiles was determined by dose-response curves (0.01–3 mg L⁻¹) of ZlEO, 2-undecanone, 2-undecenal and 2-dodecenal. The assayed volatiles were commercially acquired, as stated in Section 2.1. Readings were recorded at 630 nm after incubation at 28 °C for 48 h. Each concentration was assayed twenty-five times (n = 25). The conidial germination of F. temperatum (n = 100) was explored by the agar microdilution method performed in contact lenses filled with PDA (100 µL) combined with ZlEO (120 mg L⁻¹), 2-undecanone (150 mg L⁻¹), 2-undecenal (220 mg L⁻¹) and 2-dodecenal (130 mg L⁻¹) during 48 h. These concentrations were evaluated based on the MIC values previously obtained by the broth microdilution method. For in situ experiments, conidial suspensions were adjusted to 1 × 10⁸ conidia L⁻¹ using a Neubauer’s chamber [15]. Then, the asymptomatic manzano pepper fruit were collected in Yaonáhuac-Puebla, sanitized, and 1000 conidia (0.001-mL of 1 × 10⁸ conidia L⁻¹ solution) were inoculated in the exocarp of the fruit by penetration using a micropipette tip (0.1 milliliter capacity). The MIC determined from the in vitro assays was considered for preparing emulsified stock solutions of ZlEO (120 mg L⁻¹), 2-undecanone (150 mg L⁻¹), 2-undecenal (220 mg L⁻¹), and 2-dodecenal (130 mg L⁻¹). The solutions (1 × 10⁻⁴ L) were sprayed in the zone of inoculation using a MicroSprayer (Penn-Century, Inc) every 96 h during 16 d. Each chemical agent (ZlEO, 2-undecanone, 2-undecenal, and 2-dodecenal was assayed in twenty different chili peppers (twenty replicates were considered for each treatment). The control groups were only treated with the same volume of vehicle (EtOH) using the same number of replicates (n = 20). The samples were incubated in translucid plastic containers equipped with an airflow system using the conditions reported by Morales-Rabanales et al. [18]. The emergence of rot symptoms was followed for 16 d. Fruits showing signs of mycelial proliferation and loss of turgor were considered diseased samples.

2.6. Determination of pH and Fruit Firmness

Changes in the pH of pepper fruits (2 g fresh tissue) were determined according to Villa-Ruano et al. [19]. Firmness tests were ran in accordance with Morales-Rabanales et al. [20]. All assays were performed in quintuplicate.

2.7. Proximate Analysis and Ascorbic Acid Determination

The contents of protein, fiber, fat, and ascorbic acid were estimated in accordance with Villa-Ruano et al. [19] using 25 g fresh tissue. All assays were performed in quintuplicate.

2.8. Determination of Carotenoids in Manzano Peppers

The extraction of carotenoids started from 3 g fresh tissue in accordance with Rodríguez-Burruezo et al. [21], using the same equipment and conditions described by Pérez-Vázquez et al. [8]. The standards of violaxanthin, cis-violaxanthin, luteoxanthin, antheraxanthin, lutein, zeaxanthin, and beta-carotene were isolated from continuous HPLC runs to obtain the absolute concentration of these metabolites through calibration curves.

2.9. Determination of Capsaicinoids in Manzano Peppers

The extraction and identification of capsaicin and dihydrocapsaicin by HPLC were done in accordance with Pérez-Vázquez et al. [8]. The identity of these compounds was contrasted with authentic standards.

2.10. Statistical Analysis

Statistically significant differences among treatments were predicted by ANOVA Tukey tests (p < 0.05) using GraphPad 7.0 software.

3. Results

3.1. Essential Oil Composition and In Vitro Antifungal Activity

The yields of ZlEO obtained from Z. limoncello grown in Puebla-México oscillated between 2.3 and 2.8% (w/w). ZlEO mainly contained 2-undecanone (34%), 2-undecenal (32%) and 2-dodecenal (8%) which constituted around 74% of the volatile content (

Table 1. Volatile composition of the essential oil of Zanthoxylum limoncello grown in Puebla-México.

Peak Number	Compound	RI	1TRI	Abundance (%)	+Concentration
1	1-Hexen-3-ol	768	769	1.4	ND
2	Camphene	946	946	1.8	ND
3	1-Octanal	996	998	1.1	ND
4	β-Ocimene, (E)-	1042	1044	2.0	ND
5	2-Nonanone	1088	1087	2.3	ND
6	* Linalool	1095	1095	1.3	23.1
7	Nonanal	1102	1100	1.9	ND
8	2-decanone	1190	1190	1.5	ND
9	* 1-Decanal	1203	1201	2.8	63.6
10	(2E)-Decenal	1258	1260	1.1	ND
11	* 2-Undecanone	1292	1293	34.0	313.9
12	Undecanal	1306	1035	1.3	ND
13	* 2-Undecenal	1362	1357	32.0	276.5
14	* 2-Dodecenal	1454	1454	8.4	128.2
15	2-Tridecanone	1493	1495	2.9	ND
	Total			95.8

¹TIR, retention index reported by Adams [17]. * Identity corroborated with authentic standards. ⁺ Concentration in µg mL⁻¹; ND, non-determined. The peak numbers are those specified in Figure S2.

; Figure S2). Nevertheless, twelve other minor volatiles were also found in ZlEO.

According to the antifungal assays using resazurin as an indicator of redox metabolism, ZlEO had a MIC of 104.6 mg L⁻¹, whereas 2-undecanone, 2-undecenal and 2-dodecenal had MICs of 135.1, 218.3 and 126.8 mg L⁻¹, respectively (Figure 1 and Figure S1). The MIC of each chemical agent was the first value of the asymptotic curve.

The results of the agar microdilution method using a soft lens demonstrated that solid surfaces impregnated with 120 mg L⁻¹ ZlEO, 150 mg L⁻¹ 2-undecanone, 220 mg L⁻¹ 2-undecenal, and 130 mg L⁻¹ 2-dodecenal inhibited conidial germination and mycelial proliferation of F. temperatum (Figure 2).

3.2. In Situ Antifungal Activity of ZlEO and Its Major Volatiles

Manzano peppers infected with F. temperatum showed soft rot symptoms on the third-day post-inoculation. Conversely, the in situ application of ZlEO (120 mg L⁻¹), 2-undecanone (150 mg L⁻¹), 2-undecenal (220 mg L⁻¹), and 2-dodecenal (130 mg L⁻¹) on infected peppers produced a differential delay in the emergence of rot symptoms (Figure 3).

The application of ZlEO and 2-undecenal retarded the emergence of rot symptoms on the tenth and eleventh days, respectively (Figure 4). No substantial differences were observed between these treatments. On the other hand, 2-undecanone delayed the emergence of rot symptoms up to the fifth-day post-inoculation (Figure 4). According to our results, 2-dodecenal produced the best in situ antifungal effects delaying symptom emergence up to the fourteenth-day-inoculation (Figure 4).

3.3. Protective Effect of ZlEO and Its Volatiles on Fruit Firmness and pH

The infection of F. temperatum caused a decrease in the pH of pepper fruit (Figure 5A). This tendency was more evident in samples showing severe signs of infection. Similar results were observed for fruit firmness (Figure 5B). Remarkably, firmness depletion was quite marked at the sixteenth-day post-inoculation (>90%). Conversely, manzano peppers treated with ZlEO and major volatiles maintained the levels of fruit firmness and pH during the same period (Figure 5; Tables S1 and S2). Interestingly, 2-dodecenal and 2-undecenal showed a more potent effect on fruit firmness than 2-undecanone and ZlEO.

3.4. Protective Effect of ZlEO and Its Volatiles on the Nutritional Content of Manzano Pepper

The bromatological composition of manzano pepper was modified by the infection of F. temperatum. The levels of fat, protein, fiber, and vitamin C dramatically decreased on the sixteenth-day post-inoculation (Figure 6, Tables S3–S6). As expected, the application of ZlEO, 2-dodecenal, and 2-undecenal avoided 60–80% degradation of fat, protein, fiber, and vitamin C from the fifth day to the sixteenth-day post-inoculation. On the other hand, the effect of ZlEO on the conservation of fiber was less effective than that of the other volatiles assayed (Figure 6C). Remarkably, the application of 2-dodecenal and 2-undecenal was the most effective in avoiding the loss of fiber in manzano pepper. Nevertheless, infected chili peppers treated with 2-dodecenal conserved the levels of these basic nutrients during 16 d post-inoculation.

3.5. Protective Effect of ZlEO and Its Volatiles on the Nutraceutical Content of Manzano Pepper

The levels of capsaicin and dihydrocapsaicin were markedly reduced in manzano peppers previously inoculated with conidia from F. temperatum (Figure 7, Tables S7 and S8). Unexpectedly, the levels of these alkaloids increased on the fifth-day post-inoculation. This increase was not observed in chili peppers treated with ZlEO and its main volatiles (Figure 7). Nevertheless, on the tenth day, the levels of both alkaloids decreased by approximately 60% compared with those levels observed at day zero. Interestingly, the levels of both compounds were preserved by the application of 2-dodecenal (Figure 7; Tables S7 and S8).

The manzano peppers cultivated in Puebla-México contained seven carotenoids which were identified in accordance with their mass and UV spectra (Figure 8A; Table S9). These compounds were violaxanthin, cis-violaxanthin, luteoxanthin, antheraxanthin, lutein, zeaxanthin, and beta-carotene. From these antioxidants, violaxanthin, cis-violaxanthin, and beta-carotene were the most abundant carotenoids found in the analyzed samples (>2 mg 100 mg⁻¹). As observed for capsaicinoids, the infection of F. temperatum produced a clear depletion in the endogenous levels of carotenoids on the tenth-day post-inoculation (Figure 8; Tables S10–S16). The degradation of these compounds started on the fifth day and finished on the sixteenth-day post-inoculation, where no levels of carotenoids were detected. Exceptionally, the levels of lutein were undetectable on the fifth-day post-inoculation (Table S14). Our results suggested that the levels of carotenoids in manzano peppers present high variability (Figure 8). This trend was clearly observed in the endogenous levels of all carotenoids at day zero. Nevertheless, the comparative measurement of carotenoid levels at day zero and those of the sixteenth day revealed that ZlEO and its main volatiles avoided around 30% degradation of these pigments (Figure 8B,C). Remarkably, 2-dodecenal was the most effective inhibitor of carotenoid degradation.

4. Discussion

A recent report states that F. temperatum must be considered an emergent threat to the production of manzano peppers in Mexico [10]. This fungus causes a more aggressive soft rot than that produced by F. sambucinum in manzano peppers [8]. Consequently, possible damages in the production chain of manzano peppers cannot be discarded. Essential oils and their volatiles have shown reliable results as natural antifungals against postharvest diseases in fruit and as natural preservatives to improve the shelf life of vegetables [8,15,21]. The yields of ZlEO obtained from plants grown in Puebla-Mexico (2.3–2.8% w/w) were slightly higher than those previously reported for the same species grown in Oaxaca-Mexico [16]. The observed yields suggested that the plant material obtained in this investigation is a good source for the extraction of bioactive ZlEO containing 2-undecanone, 2-undecenal, and 2-dodecenal as the main oxygenated hydrocarbons derived from fatty acid metabolism [16]. Interestingly, slight differences were observed in the chemical composition of ZlEO in comparison with that obtained from plants grown in Oaxaca-México. However, our data strongly suggest that the main volatiles of Z. limoncello remain constant independently of the geographical region in which the plant grows (Table 1). Conserved volatile profiles have been observed in other aromatic plants, such as those of Brickellia verononicifolia, which is able to grow in different locations within Mexico [22].

Our results showed that ZlEO and its major volatiles (2-undecanone, 2-undecenal, and 2-dodecenal) exerted a differential antifungal effect on F. temperatum (Figure 1 and Figure 2). The proven activity of these volatiles strongly suggested their involvement as bioactive compounds dissolved in ZlEO, and probably, these compounds may act synergistically to produce an antifungal effect on F. temperatum. On the other hand, the results of the in vitro antifungal assays using resazurin as an indicator of cell viability revealed that ZlEO had the lowest MIC on F. temperatum. These data endorsed that all agents assayed exerted fungicide activity by blocking the redox activity of fungal cells [17]. In the same context, the results of agar microdilution tests carried out in soft lenses confirmed inhibition of conidial germination, which is translated into the fungistatic activity of ZlEO and its major volatiles (Figure 2). The latter results predicted that the application of MIC on solid surfaces might exert a growth inhibitory activity on F. temperatum under in situ conditions. Our records stated that the application of ZlEO, 2-undecanone, 2-undecenal, and 2-dodecenal on the exocarp of manzano peppers previously infected with F. temperatum, resulted in a significant delay of rot symptoms from the fifth day to tenth-day post-inoculation (Figure 3 and Figure 4). The in vitro antifungal effect of the three main volatiles dissolved in ZlEO has recently been demonstrated in F. oxysporum [23]. Nonetheless, our investigation is the first one demonstrating the in situ antifungal effects of 2-dodecenal, 2-undecenal, and 2-undecanone on an edible fruit infected with a wild strain of F. temperatum. Unexpectedly, 2-dodecenal, and 2-undecenal caused a more prolonged fungistatic effect than that observed for ZlEO and 2-undecanone under in situ conditions (Figure 3 and Figure 4). The amphipathic ability of these oxygenated hydrocarbons to cross fruit cuticle and cell membranes could probably be considered as the mode of action to exert their antifungal activity under in situ conditions [23]. The present investigation demonstrates the individual bioactivity and effectiveness of 2-dodecenal and 2-undecenal for the control of F. temperatum in manzano peppers. Considering this evidence, the escalation of 2-dodecenal and 2-undecenal through chemical or biotechnological platforms should reduce the continuous extraction of ZlEO from monocultures of Z. limoncello. This strategy would also promote the conservation of this plant. It should be considered that the ZlEO and its main volatiles had a similar smell to that produced by coriander [16]. Zanthoxylum limoncello has been used for centuries as a condiment of traditional dishes in southern Mexico. Then, the impregnation of these volatiles in the pericarp of these fruits should be envisioned as a new flavor for manzano peppers. Nevertheless, further sensory analyses need to be performed to determine the acceptance of this additive for use by consumers.

Fruit firmness and pH are key parameters in fruit quality because it defines customers’ fruit acceptability. Some variables, such as the natural ripening stage and phytopathogenic activity, dramatically affect these parameters [24]. The infection caused by F. temperatum reduced these parameters, as well as the bromatological and nutraceutical contents of manzano peppers (Figure 5 and Figure 6). The depletion of these parameters should be related to the ability of filamentous fungi to exploit the external nutrient environment, which is reflected in the consumption of protein, fat, and carbohydrates (reducing sugars and fiber) of their respective hosts [25]. As expected, the in situ application of ZlEO and its major volatiles produced evident conservation of these parameters compared with non-treated peppers on the sixteenth day (Figure 5 and Figure 6). This effect should be a consequence of the fugal growth inhibition induced by these components [24].

As secondary metabolites, capsaicinoids are part of the chemical defense of chili peppers [26]. Previous reports state that the levels of capsaicinoids are triggered by the colonization of Verticillium dahliae and Botrytis cinerea [26]. Our results showed an evident accumulation of capsaicin and dihydrocapsaicin in non-infected chili peppers on the fifth day (Figure 7). This finding should probably be linked to the systemic response of manzano peppers against the colonization of F. temperatum [8]. Similarly, the degradation of the seven carotenoids contained in manzano peppers from the fifth day to the sixteenth day indicates that the infection caused by this fungus may modify the yellow color of the fruit. Due to their capacity to retard the proliferation of F. temperatum, ZlEO and its main volatiles avoided carotenoid degradation (Figure 8). Analogous results were observed by the in situ evaluation of peppermint essential oil, menthone, and menthol on manzano peppers infected with F. sambucinum [8]. Unexpectedly, the levels of lutein were undetectable in manzano peppers infected with both fugal phytopathogens on the tenth-day post-inoculation. This finding may suggest that lutein is used by the fungus as an inductor of α-1,3-glucan accumulation, as already proved in Colletotrichum gloeosporioides and Cochliobolus miyabeanus [27,28]. This effect was recently confirmed in manzano peppers infected with F. sambucinum [8].

5. Conclusions

The essential oil from Z. limoncello collected in Puebla-Mexico and its major volatiles inhibited the in vitro growth of F. temperatum and delayed the emergence of soft rot symptoms in manzano pepper up to 16 d after inoculation with viable conidia from this fungus. The in situ evaluation of these natural components resulted in a protective effect on the nutritional parameters of manzano peppers, such as protein, fat, fiber, and ascorbic acid. Simultaneously, these substances delayed the degradation of valuable nutraceuticals of manzano pepper, such as capsaicinoids and carotenoids. Our results suggest the possible use of the essential oil from Z. limoncello and its natural volatiles for controlling the soft rot caused by F. temperatum.