Introduction

Papaya (Carica papaya L.) is a climacteric fruit, grows throughout the year in tropical and subtropical regions, and shows a characteristic flavor and excellent nutritional qualities (Oliveira and Vitoria 2011). Mexico ranks third in the worldwide-production of papaya and is one of the main exporters (FAOSTAT 2020). However, papaya production is compromised by its deterioration rate and susceptibility to mechanical damage and fungal infections (Ayón-Reyna et al. 2017).

Anthracnose is the main disease affecting papaya production and is caused by the fungus Colletotrichum gloeosporioides, one of the most harmful phytopathogens in the world (Siddiqui and Ali 2014). C. gloeosporioides commonly infects papaya on the field; after harvesting, fruit reaches its climacteric peak and the fungus has the adequate growth conditions (temperature, absence of phytoalexins, and high content of simple sugars); then, the fungus induces the anthracnose symptomatology which is characterized by sunken areas and water-soaked spots with a salmon coloration, which rapidly expand and become soft and black (Gullino et al. 2018; Maha-Laksha et al. 2019).

Since the twentieth century, papaya producers have employed many synthetic fungicides (e.g., propiconazole, prochloraz, and thiabendazole) to prevent and control anthracnose (Ayón-Reyna et al. 2017). The effectiveness and mechanism of action of such fungicides are well-known; however, resistant C. gloeosporioides strains have appeared. Besides, the extensive production and use of synthetic fungicides have been questioned by environmental and human health concerns (Elshahawy et al. 2018; Luan and Xo 2018). Consequently, new innocuous and environmental-friendly fungicides are required.

In this regard, plants have been used since ancient times to provide non-toxic compounds with biological activities and Mexico has a great advantage: it comprises between 10 and 12% of the plant world-biodiversity (Villaseñor 2016). Thus, many Mexican plants could be sources of compounds with activity against C. gloeosporioides and reduce the negative impact of the anthracnose in papaya fruit.

Many plant extracts inhibit the anthracnose development, as well as other phytopathological diseases: methanol extracts (ME) of Lantana camara (200 mg/mL) (Maha-Laksha et al. 2019) and ethyl acetate extracts of Echinops sp. (250 mg/mL) (Ademe et al. 2013) reduce the incidence of anthracnose in papaya fruit. Meanwhile, seed aqueous extract of Camellia semiserrata (10 mg/L) inhibits the development of C. gloeosporioides in mango and C. musae in banana fruit (Meng et al. 2015). Besides the use of crude extracts, other authors have employed semi-purified plant extracts to boost their effect; in this regard, Verma et al. (2018) found that a hexane fraction obtained from the Jasminum mesnyi extract exhibits greater antibacterial activity than the original extract. Other studies have also shown improved biological activities (e.g., antimutagenic and parasitocidal) for semi-purified plant fractions (López-Angulo et al. 2016; Montes-Avila et al. 2017).

The selected plant models from Sinaloa, Mexico have antifungal properties; however, such activity has been mainly demonstrated in vitro. For instance, extracts of Bromelia pinguin and Psidium sartorianum are active against Trichophyton spp., which are human pathogens (Camacho-Hernández et al. 2002 and 2004), extracts of Randia echinocarpa (Salinas-Sánchez et al. 2009) and Echeveria spp. (Martínez-Ruiz et al. 2013) were active against Candida albicans; and extracts of Pithecellobium dulce and Vitex mollis reduce the mycelial growth of phytopathogens such as Botrytis cinerea, Penicillium digitatum, and Fusarium spp. (Bautista-Baños et al. 2003; Valencia-Botin et al. 2018). However, scientific evidence about the potential of plant extracts from Sinaloa, Mexico, to inhibit the mycelial growth of C. gloeosporioides and the development of anthracnose disease is scarce. Therefore, this investigation aims to evaluate the in vitro antifungal activity and toxicity against Artemia salina of crude plant extracts from Sinaloa, Mexico, and their semi-purified fractions; as well as to determine the effect of the most effective factors (MEF) on the development of anthracnose and quality parameters of papaya fruit during its commercial storage.

Materials and methods

Plant material

The following plant models were selected based on their previously reported antifungal properties: pulp and peel of B. pinguin (aguama) (Camacho-Hernández et al. 2002); pulp and seed of P. sartorianum (arrayan) (Camacho-Hernández et al. 2004); pulp, peel, and seed of P. dulce (guamuchil) (Bautista-Baños et al. 2003); pulp and peel of S. purpurea (mexican plum) (Marisco et al. 2017); pulp and seed of R. echinocarpa (papache) (Salinas-Sánchez et al. 2009); pulp and seed of V. mollis (uvalama) (Valencia-Botin et al. 2018); and leaves of three Echeveria species (E. subrigida, E. craigiana and E. kimnachii) (Martínez-Ruiz et al. 2013). Plant materials were collected in the state of Sinaloa, Mexico, between April and August 2019. Each plant model was stored at -70 °C until freeze-drying and then pulverized and sieved with a 40 mesh (0.425 mm of diameter).

For the in vivo antifungal assays, papaya fruits (Carica papaya L.) with orange skin and some light green areas (ripening stage 4) (Ayón-Reyna et al. 2017) were harvested on April 2020 at a local plantation near to Culiacan, Sinaloa, Mexico. Damage- and pesticide-free fruits with uniform characteristics (size and color) were selected for the experiments.

Pathogen

C. gloeosporioides was previously isolated by our working team from infected papaya and identified by molecular analysis (18S rRNA-accession number HM222960.1) (Ayón-Reyna et al. 2017). Conidia were obtained from a two-week culture and placed in sterilized water. The conidial suspension was adjusted to 1 × 106 conidia/mL using a hemocytometer.

Preparation of plant extracts

Crude methanol extracts

Methanol extracts (ME) were prepared as described by López-Angulo et al. (2014) with minor modifications. Each pulverized plant (20 g) was mixed with methanol (1:10 w/v), sonicated for 20 min at 35 °C, and the supernatant was recovered by centrifugation (5 min, 3000 × g); the residue was extracted two additional times under the same conditions. The supernatants were mixed and centrifuged for 15 min at 3000 × g to eliminate any solid residue, and the supernatant was recovered. The solvent was eliminated in a rotary evaporator (BÜCHI Labortechnick AG, Flawil, Switzerland) at 37 °C and the residue was freeze-dried to obtain the ME.

Preparation of the semi-purified fractions

Fractionation of the ME was carried out by liquid–liquid chromatography as described by López-Angulo et al. (2016). ME (10 g) was dissolved in 90% methanol (1:2 w/v). The solution was partitioned 3 times with hexane (3 × 50 mL) and the hexane phase was recovered; the polar fraction was concentrated, mixed with distilled water, and partitioned 3 times with ethyl acetate (3 × 50 mL); and the ethyl acetate and aqueous fractions were recovered. The hexane and ethyl acetate fractions were concentrated at 37 °C under vacuum and the aqueous fraction by freeze-drying to obtain the hexane (HF), ethyl acetate (EAF), and aqueous (AF) fractions. These fractions were protected from light and stored under a N2 atmosphere at -20 °C.

In vitro antifungal and toxicity assays

Antifungal activity of the crude ME

The antifungal activity was assessed by the methodology of mycelial growth inhibition. The crude ME was dissolved in methanol (400 mg/mL) and filtered through a sterile polytetrafluoroethylene filter (0.45 µm of aperture) (Pall Corporation, New York, USA) to remove airborne spores. The extracts (100 µL) were added on Petri dishes containing potato dextrose agar (PDA) medium, spread using a sterile plastic rod spreader, and air-dried on a laminar flow cabinet. Each Petri dish was inoculated into the center with 1 μL of a C. gloeosporioides suspension (1 × 106 conidia/mL) and incubated at 25 °C for 6 days. Dishes containing methanol instead of the crude ME were used as negative control. Thiabendazole at 0.5 mg/mL was used as positive control following the recommendations of the European Commission (2020). Six Petri dishes were used per treatment. Results were expressed as percentage of inhibition using the formula described by Ayón-Reyna et al. (2017).

$${\%\;Inhibition =}\frac{\text{Rc-R}}{\text{Rc}}\text{ X 100}$$

where,

Rc = Radial growth in the negative control.

R = Radial growth in the treatment.

Toxicity of the crude ME against A. salina

The in vitro toxicity assay was carried out using A. salina and following the protocol described by Solis et al. (1993). Eggs of A. salina (600 mg/L) were hatched in a container with artificial sea medium (ASM), containing NaCl and distilled water (38 g/L), provided with an oxygenation system and low continuous light regime (tungsten light bulb, 72 W). After 48 h at 22–29 °C, the hatched eggs were placed in fresh ASM. The crude extracts were prepared at 20 mg/mL with Tween-80® (60%) and mixed with ASM to obtain a final concentration of 2 mg/mL. The toxicity of TBZ was assessed at 0.2 mg/mL. The negative control consisted of 60% Tween-80®. The evaluations were carried out using 10 A. salina larvae per assay tube and six assay tubes per treatment. The samples were incubated for 24 h at 22–29 °C under continuous light. The number of live larvae in each tube was counted. The mortality rate of each extract was determined by the correction of the Abbott formula:

$$\%\;M=\frac{\text{r}}{{s}^{'}}\;{\times\;100}$$

where:

M = Mortality rate.

r = Dead larvae on each treatment.

s' = Live larvae on the negative control.

Antifungal activity and toxicity of the semi-purified fractions

The crude ME with the highest antifungal activity and the lowest toxicity against A. salina were selected and fractionated to obtain the different fractions. The antifungal activity and toxicity of these fractions were assessed as described in Sects. 2.4.1. and 2.4.2.

Determination of the MIC

Fractions with the highest antifungal activity were prepared at different concentrations (10, 20, 30, 40, 50, 100, and 200 mg/mL) to establish their MIC, which is the minimum concentration to get 100% of mycelial inhibition. The MIC values were used in the in vivo antifungal assays.

In vivo antifungal assays

Papaya fruit was washed and disinfected with a 1% (v/v) sodium hypochlorite solution for 5 min and then rinsed with sterile distilled water. Fruit was inoculated by immersion in a C. gloeosporioides suspension (1 × 106 conidia/mL) and incubated at 25 °C for 16 h to allow the fungus to adhere on the fruit surface. Fractions were mixed with lecithin (60 mg/mL) to facilitate the solubilization. Considering the differences between the in vitro and in vivo assays, extract solutions were adjusted at its MIC and a two-fold concentration, to cover the additional factors (fruit composition, volatility, humidity, and time) involved in the in vivo assay. Fractions (2 mL) were manually rubbed over the fruit surface. To ensure a uniform application, extracts were rubbed all over the fruit for 1 min. Lecithin (60 mg/mL) and TBZ (0.5 mg/mL) were used as negative and positive controls, respectively. TBZ was applied by immersion as reported for synthetic fungicides (The European Commission 2020). Fruit was stored at 13 ºC for 16 d. The relative humidity was maintained at 90–95% during the storage. Nine fruits were used per treatment, and three replicates were carried out.

Anthracnose incidence and severity

Disease incidence (DI) and disease severity (DS) were determinate according to Ayón-Reyna et al. (2017). DI and DS were assessed to determine the in vivo antifungal activity of the MEF. DI was daily evaluated until all fruit presented symptoms and was expressed as the number of fruit showing anthracnose symptoms out of the total fruit number in each treatment. Whereas DS was measured every 4 d and reported as the percentage of fruit surface with anthracnose symptoms.

Quality parameters of papaya fruit treated with the MEF

Papaya quality parameters were carried out according to Ayón-Reyna et al. (2017). Weight loss was measured using a Sartorius balance (Goettingen, Alemania) and reported as percentage of weight loss. Firmness (reported in Newtons, N) was measured on horizontally-cut fruit slices with a Chatillon penetrometer (DFE 100; AMETEK Inc., Largo, Fla., USA) fitted with an 11 mm diameter cylindrical probe, penetration depth of 5 mm, and speed of 50 mm/min. Total soluble solids (TSS) were measured with a handheld refractometer (Atago, Fisher Scientific, GA, USA) and results were expressed as °Brix. pH was measured with a potentiometer (Orion Research Inc., Beverly, Mass., USA), and the titratable acidity (TA) was determined with NaOH (0.1 N) and reported as percentage of citric acid.

Phytochemical analysis

The presence or absence of phytochemicals in the MEF was determinate by tube assays or TCL as described below: the Salkownski reaction for terpenes; Shonoda’s test for flavonoids; reaction with a gelatin solution (1%) and a quinine sulfate solution with FeCl3 for tannins; foam formation for saponins; yellow fluorescence by reaction with NaOH for coumarins and reaction with the Dragendorff, Mayer and Wagnes reagents for alkaloids (López-Angulo et al. 2014).

Statistical analysis

A completely randomized experimental design with three replicates was carried out for all experiments. A one-way analysis of variance was performed for the in vitro assays and a multivariate analysis of variance for the in vivo assays. The Statgraphics Centurion XVI software was employed (Statpoint Technologies, Inc., Warrenton, Virginia, USA) and the means were compared using the Fisher’s least significant difference (LSD) test (I < 0.05).

Results

In vitro antifungal and toxicity assays

Crude ME

The extraction yields of the crude ME varied from 4.73 to 60.8% (Table 1). Pulp extracts showed the highest values, while seed extracts the lowest. Seven out of the 16 crudes ME inhibited the in vitro mycelial growth of C. gloeosporioides: the best one was obtained from the pulp of V. mollis, being superior to the positive control (TBZ); whereas the other six ME showed moderate activity (19.5–25.7%).

Table 1 Extraction yield, activity against Colletotrichum gloeosporioides, and in vitro toxicity of crude ME of plants from Sinaloa, Mexico
Full size table

Regarding the in vitro toxicity against A. salina, only crude ME with antifungal activity higher than 5% were tested. Only three of the seven evaluated ME were toxic against A. salina (Table 1): the highest values were for the seed (100%) and peel (83.3%) of P. dulce, followed by P. sartorianum seed (53.3%). The toxicity of TBZ (0.2 mg/mL) was statistically higher than the crude ME, except for the toxicities of the methanol extracts of P. dulce. Based on these results, only the ME of P. sartorianum (pulp), E. kimnachii (leaf), and V. mollis (pulp) were further studied. The seed extract from V. mollis was not selected, given that its activity may be caused by either the same compounds found on pulp but in minor concentration or the traces of pulp on this tissue.

Semi-purified fractions

The highest fractionation yields were for the aqueous fractions (32.3–53.0%) (Table 2). On the other hand, the values for the low polarity fractions ranged from 0.4 to 4.4%. On the antifungal evaluation, the highest values (100%) were for the hexane (HF-VM) and ethyl acetate (EAF-VM) fractions of V. mollis, and the ethyl acetate fraction of E. kimnachii (EAF-EK), being better than TBZ (52.3%). Meanwhile, the ethyl acetate fraction of P. sartorianum (EAF-PS) had a moderate activity (53.3%). Compared with their corresponding crude ME, EAF-EK and EAF-PS had an activity boost of about 260% and 430%, respectively, whereas the EAF-VM and HF-VM kept the same value presented on the crude ME. On the in vitro toxicity against A. salina (Table 2), EAF-EK, EAF-VM, and HF-VM were innocuous at 2 mg/mL, showing better results than TBZ. Therefore, these fractions may be useful to inhibit the anthracnose development on papaya fruit. EAF-PS was excluded due to its low antifungal activity.

Table 2 Extraction yield, activity against Colletotrichum gloeosporioides, and in vitro toxicity of fractions from ME of plants from Sinaloa, Mexico
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The antifungal activities of EAF-EK, EAF-VM, and HF-VM were promising; however, the tested concentration (400 mg/mL) is inappropriate for in vivo analysis, given that the fruit surface could be stained and a considerable quantity of plant extract would be required for a postharvest application. Thus, MIC values were calculated for the in vivo assays.

Determination of MIC

The antifungal activity of the crude ME and their fractions at different concentrations (10–400 mg/mL) is shown in Table 3. Fractions were more active than their corresponding ME, and the best fractions were those obtained from the V. mollis pulp, showing the following MIC values (mg/mL): ME (400) > EAF (30) > HF (20). The ME and EAF of E. kimnachii had high MIC values (400 mg/mL) and were not considered for further analysis. Thus, the HF and EAF of V. mollis were selected as the MEF and used in the in vivo antifungal assay.

Table 3 Mycelial growth inhibition (%) of Colletotrichum gloeosporioides by different concentrations of crude ME and selected fractions obtained from leaves of Echeveria kimnachii and pulp of Vitex mollis
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In vivo antifungal assays

Considering the differences between the in vitro and in vivo assays, evaluated concentrations were at their MIC and an additional two-fold concentration: HF, 20 (HF20) and 40 mg/mL (HF40); and EAF, 30 (EAF30) and 60 mg/mL (EAF60). TBZ was used as positive control.

Anthracnose incidence and severity

The HF40 treatment showed better protection against the infection of papaya with C. gloeosporioides. This treatment decelerated the development of symptoms such as circular sunken areas, and salmon-colored conidial masses (Figs. 1 and 2). Analyzing the anthracnose incidence on papaya fruit (Fig. 1A), disease symptoms appeared in all treatments after 3 d. On the next day, values from the HF40 and EAF60 treatments were lower than those from control treatment (P < 0.05). After 5 d of storage, HF40 showed the lowest values (P < 0.05); whereas after 6 d, the effects of HF40 and TBZ treatments were similar. Considering the anthracnose severity on papaya fruit (Fig. 1B), both HF and EAF treatments protected the fruit, and protection at the highest evaluated concentrations (HF40 and EAF60) was similar to TBZ.

Fig. 1
figure 1

Effect of the fractions of Vitex mollis pulp (hexane fraction, HF; ethyl acetate fraction, EAF) on anthracnose incidence (a) and severity (b) of papaya infected with Colletotrichum gloeosporioides and stored for 16 d at 13 °C. TBZ was the positive control. Evaluated concentrations were as follows: HF at 20 (HF20) and 40 mg/mL (HF40) and EAF at 30 (EAF30) and 60 mg/mL (EAF60). Values are means of three replicates. Vertical bars indicate standard deviation (SD). Values with the same letter within the same evaluation day are not significantly (P > 0.05) different according to the Fisher’s least significant difference (LSD) test

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Fig. 2
figure 2

Representative images of papaya fruit infected with Colletotrichum gloeosporioides and treated with fractions of Vitex mollis pulp (hexane fraction, HF; ethyl acetate fraction, EAF). TBZ was the positive control. Fruit was stored for 16 d at 13 °C. Evaluated concentrations were as follows: HF at 20 (HF20) and 40 mg/mL (HF40) and EAF at 30 (EAF30) and 60 mg/mL (EAF60). The experiment had three replicates

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Quality parameters of papaya fruit treated with the MEF

The fruit quality parameters were not negatively affected by the application of the V. mollis fractions dissolved in lecithin (60 mg/mL). Weight loss showed significant differences among treatments after 16 d of storage (Table 4). The values obtained for the HF and EAF treatments were lower compared with those from the TBZ treatment. For the other quality parameters, notable changes were observed as the fruits reached maturity; however, there were no differences among treatments.

Table 4 Quality parameters of papaya fruit infected with Colletotrichum gloeosporioides and treated with TBZ and fractions of Vitex mollis pulp. Fruit was stored for 16 d at 13 °C
Full size table

Phytochemical analysis

The HF and EAF from V. mollis were positive in five out of the six phytochemicals evaluated, where only saponins evaluation was negative (Table 5). A greater diversity of phytochemicals was observed in the EAF-VM, showing the presence of tannins, flavonoids, coumarins, and terpenes; meanwhile alkaloids, coumarins, and terpenes were observed in the HF-VM.

Table 5 Phytochemical analysis of Vitex mollis fractions
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Discussion

The higher extraction yields on pulp are caused by the extensive presence of polar compounds in this tissue, which are easily dissolved in methanol. Additionally, it is reported that methanol is useful to extract a wide variety of metabolites with different polarities (e.g. carbohydrates, flavonoids, and fatty acids) (López-Angulo et al. 2014). On the other hand, tissues such as peels, seeds, and leaves contain polymers (e.g., cellulose, hemicellulose, and lignin), which show low solubility in methanol (González-Centeno et al. 2010). Regarding the extract concentration for the in vitro assays, it must be mentioned that these were evaluated at a high concentration (400 mg/mL) to assess their antifungal potential and rapidly discard those without antifungal activity.

All plant models with activity against C. gloeosporioides found in this study (P. sartorianum, E. kimnachii, P. dulce, and V. mollis); exhibit antifungal activity against yeast or other phytopathogen fungi of importance in agriculture such as B. cinerea and P. digitatum (Bautista-Baños et al. 2003; Camacho-Hernández et al. 2004; Martínez-Ruiz et al. 2013). Leaf and stem extracts from V. mollis have shown important antifungal properties (Valencia-Botin et al. 2018); however, this is the first study where pulp and seed are evaluated to inhibit a phytopathogen. It is important to mention that even though the antifungal properties of these plant models are reported, the identity of their antifungal compounds and mechanism involved are still unknown. For V. mollis pulp, the elevated activity exhibited may be caused by the presence of numerous monoterpenes, characteristic components among Vitex spp. fruits, which possess a strong activity against many microorganisms (Miron et al. 2014). Eucalyptol, sabinene, α-pinene, and limonene are among the main monoterpenes in some Vitex spp. (Pío-León et al. 2014).

For the in vitro toxicity against A. salina, extracts were evaluated at a low concentration (2 mg/mL) to discard the most toxic extracts. Of all evaluated plant parts of this study, only the pulp of P. dulce has been studied regarding its in vitro and in vivo toxicity (Megala and Getha 2012; Toudji et al. 2017). There are many toxicity studies; however, most of them are focused on other Psidium and Vitex species (Rani and Sharma 2013; Manekeng et al. 2019).

As expected, pulp extracts were not toxic given their common consumption. For the case of leaf extract toxicities, it varied among plant models. Psidium guajava extracts have low in vivo toxicity and are used to treat colitis (Etuk and Francis 2003), whereas the Catharanthus roseus and Azadirachta indica extracts show acute toxicity in mice, and their consumption is not recommended (Kevin et al. 2012; Deng et al. 2013). Seed and peel extracts commonly contained toxic compounds such as arsenic, cyanide, phytic acid, protease inhibitors, and tannins (Popova and Mihaylova, 2019). Cavalcante-Fonseca et al. (2013) evaluated the toxicity against A. salina of extracts (2 mg/mL) of several fruit seeds, and half of them induced 100% mortality due to their high cyanide content.

When the extracts of P. sartorianum (pulp), E. kimnachii (leaf), and V. mollis were fractioned, the aqueous fractions had the highest yields (32.3–53.0%) (Table 2). This is a consequence of the elevated content of polar compounds in the ME. Compared with the aqueous and hexane fractions that were inactive against C. gloeosporioides, the activity boost on the EAF-PS and EAF-EK suggested that the active compounds against C. gloeosporioides are in the EAF fraction. EAF-VM and HF-VM exhibited a strong antifungal activity. Even though these fractions share some compounds, both showed the maximum inhibition, suggesting that different compounds are involved in the antifungal activity. Similar results have been found in other plant models, showing higher activity for fractions of low and medium polarity (López-Angulo et al. 2016; Verma et al. 2018).

On the in vivo antifungal assay, the anthracnose symptomatology on papaya fruit was consistent with previous reports, this is, the presence of circular sunken areas and as the disease spread, appearance of salmon-colored conidial masses and white cottony areas (Fig. 2) (Ademe et al. 2013; Maha-Laksha et al. 2019).

The results for anthracnose incidence and severity showed that EAF60 treatment delayed the spread of symptoms, but only had a significant effect on anthracnose incidence after 4 d of storage. On the other hand, HF40 treatment had a consistent effect on both criteria. The HF40 treatment reduced the anthracnose symptoms, showing similar results throughout the storage with the fruit treated with TBZ. Comparing with the EAF60 treatment, a lower concentration of HF (HF40) was required to obtain better results; besides, the extraction yield from HF was slightly higher. Consequently, the HF of V. mollis could be considered a better treatment against anthracnose disease on papaya.

The in vitro assays showed that HF-VM at 20 mg/mL and EAF-VM at 30 mg/mL completely inhibited the mycelial growth of C. gloeosporioides; nevertheless, the in vivo results showed that papaya fruit from all treatments exhibited symptomatology. This could be by the multiple variables involved in the in vivo analysis. For instance, in vitro analysis involves nutrient intake limitations, but different micronutrients in papaya fruit could boost the fungi pathogenicity (Iskandarov et al. 2006). Additionally, a large number of spores were already germinated when treatments were applied to the fruit (16 h later); therefore, germinated spores could have less sensitivity to the antifungal compounds on the fractions.

Different plant extracts have been employed to inhibit anthracnose. As observed in our study, Ademe et al. (2013) applied an Echinops sp. extract on papaya fruit and reduced the incidence and severity of anthracnose, showing a similar reduction with a synthetic fungicide (carbendazim). Similar results were obtained on mango and banana fruit by the application of Camellia semiserrata extracts (Meng et al. 2015).

Concerning the chemical compounds involved, on the phytochemical analysis metabolites such as polyphenols, alkaloids, and terpenes were detected on the MEF, and these are important natural antimicrobials (Pío-León et al. 2014). On a preliminary assay (data not shown), HF-VM and EAF-VM were obtained by Soxhlet extraction (90 °C, 5 h). The results showed that the extraction conditions affected the composition of EAF-VM, causing a decrease in its activity, whereas the activity of the HF-VM was unchanged. This suggests that the activity of EAF-VM is caused by thermolabile compounds such as polyphenols, and the activity of the HF-VM by more resistant compounds such as terpenes. (Ali et al. 2015 and 2016) found that terpenes from ginger and lemongrass (α-pinene, 1,8-cineole, borneol, geranial, and neral) inhibit the spore germination, mycelial growth, and development of anthracnose in papaya fruit stored at 12 and 25 °C. The mechanism of these compounds involves the breakdown of the fungi mycelium as observed by optical microscopy on C. gloeosporioides and C. musae hyphae treated with natural terpenes (Garcia et al. 2008). This breakdown may be caused by the interaction of such compounds with ergosterol, forming a membrane channel that debilitates many cellular structures (Miron et al. 2014).

Regarding quality parameters, when fungicidal agents are applied on fruits and vegetables, producers look for antifungals with no effects on the ripening process and their postharvest quality parameters. In this sense, any of the measured quality parameters was negatively affected, only weight loss had a significant reduction on fruit treated with lecithin (mixed or not with the V. mollis fractions) after 16 d of storage. This is caused by the lecithin coating, which has hydrophobic properties, and limits water exchange (Pérez-Gago et al. 2003).

It is important to mention that even though moisture retention was observed; the respiration rate may not be reduced by the lecithin coating, given that the maturation process was not decelerated as shown on the quality parameters. Similar results have been obtained with the application of other natural extracts, showing positive results on reducing the incidence and severity without negative effects on fruit quality parameters (Ali et al. 2016; Maha-Laksha et al. 2019). On the other hand, Ali et al. (2015) observed a delay in the maturation process when a ginger extract or oil was applied on papaya; however, this was achieved due to the addition of gum Arabic, which formed a more efficient coating.

Conclusions

The best crude ME to inhibit the mycelial growth of C. gloeosporioides were obtained from Psidium sartorianum (pulp), Echeveria kimnachii (leaves), and V. mollis (pulp), resulting better for V. mollis. The hexane fraction obtained from the crude ME of V. mollis showed the lowest MIC value in vitro and the best effect in vivo without negative effects on fruit quality parameters; thus, this fraction is a good alternative to the synthetic antifungal TBZ against anthracnose development on papaya fruit.