Promising antifungal activity of two varieties of Capsicum chinense against Sclerotinia sclerotiorum , Rhizopus stolonifer and Colletotrichum goleosporoides

Peppers ( Capsicum sp.) which belong to the Solanaceae family constitute an important segment in the vegetable sector, both in agriculture and in the food industry. This paper aims to investigate in vitro antifungal activity of hexane extracts from Capsicum chinense fruit (unripe bode pepper – ‘HE-UB’ – and ripe little beak pepper – ‘HE-RB’) against Sclerotinia sclerotiorum , Rhizopus stolonifer and Colletotrichum gloeosporioides . Antifungal activity was evaluated by the disk diffusion method (DDM) at doses between 25 µL and 300 µL of both diluted extracts. Chemical analyses revealed that the major constituent in both extracts was E -caryophyllene. HE-RB inhibited 100% of S. sclerotiorum , R. stolonifer and C. gloeosporioides growth at doses of 200 µL, 100 µL and 300 µL, respectively. HE-UB also inhibited 100% of fungal growth at doses of 100 µL ( S. sclerotiorum ), 150 µL ( C. gloeosporioides ) and 200 µL ( R. stolonifer ). HE-RB and HE-UB were active against the fungi under study; thus, screening of medicinal plants provides another alternative to produce chemical fungicides that are relatively non-toxic and cost-effective.


Introduction
Peppers (Capsicum sp.) which belong to the Solanaceae family constitute an important segment in the vegetable sector, both in agriculture and in the food industry.They are special to produce spices due to their characteristics of fruit color and active ingredients which bestow aroma and flavor (Bianchi et al., 2020).
Little beak pepper (Capsicum chinense Jacq.) is a species that has small round fruit -with tails that resemble a bird's beakwhich have low pungency and are characterized as sweet fruit that may be consumed fresh or processed (Diel et al., 2020).Another Capsicum variety that grows in Brazil was evaluated by this study: bode pepper (pimenta-bode in Brazilian Portuguese).It has special aroma and its unripe fruit are sold fresh while the ripe whole ones (yellow or red) are mainly canned (with vinegar or olive oil) and transformed into sauces (Jesus et al., 2020).
Regarding medicinal plants, the literature has broadly described the importance of plant extracts, isolated compounds and essential oils (EOs) to fight against phytopathogens which damage several crops that are economically relevant (Seepe et al., 2021).Some phytopathogens are fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides.The one that causes the disease known as white mold and attacks soybean crops is S. sclerotiorum (Silva et al., 2019).R. stolonifer damages mainly fruit since it causes the post-harvest disease known as soft rot (Rezende et al., 2020).C. gloeosporioides causes anthracnose, the post-harvest disease that leads to fruit rot, which affects several fruit, such as mango, avocado and passion fruit), and prevents commercialization (Gomes et al., 2021).
Taking into consideration the bioactive potential of C. chinense extracts (Morais et al., 2019;Santos et al., 2022), this study aimed at investigating the in vitro antifungal potential of hexane extracts from two Brazilian varieties of C. chinense fruit (unripe bode pepper -'HE-UB' -and ripe little beak pepper -'HE-RB' -Figure 1) and at determining their chemical composition by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).

Plant material
Capsicum chinense fruit (unripe bode pepper -'HE-UB'and ripe little beak pepper -'HE-RB') were bought in fairs in Santa Helena de Goiás and in Rio Verde, two cities in Goiás (GO) state, Brazil.Fruit were identified by the botanist Luzia Francisca de Souza and a voucher specimen of C. chinense (HJ558CCripe little beak pepper) and (HJ559CC -unripe bode pepper) were deposited at the Herbarium Jataiense Professor Germano Guarim Neto.They were then taken to the Laboratory of Natural Product Chemistry at IF Goiano -Campus Rio Verde, located in Rio Verde, GO, where they were washed with distilled water.Afterwards, they were dried with paper towels and had their peduncles removed.Fruit were then weighed and dehydrated in an air circulation oven at 40 °C for 96 h.Finally, they were ground, placed into a sealed container and stored in a refrigerator up to the preparation of crude hexane extracts (HE-UB and HE-RB).

Preparation of hexane extracts (HE-UB and HE-RB)
Fruit (300 g) were air-dried and milled by a Wiley mill.Subsequently, they were exhaustively cold-extracted with hexane.Every resulting extract was filtered and concentrated under reduced pressure.Finally, 6.0 g crude hexane extract from ripe little beak peppers (HE-RB) and 4.3 g crude hexane extract from unripe bode peppers (HE-UB) were collected.

Chemical identification of HE-UB and HE-RB constituents
HE-RB and HE-UB were dissolved in ethyl ether and analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) with the use of Shimadzu QP5000 Plus and GCMS2010 Plus (Shimadzu Corporation, Kyoto, Japan) systems.The temperature of the column in GC-FID was programmed to rise from 60 to 240 °C at 3 °C/min and was held at 240 °C for 5 min; the carrier gas was H 2 at the flow rate of 1.0 mL/min.The equipment was set to operate in the injection mode; the injection volume was 0.1 µL (split ratio of 1:10) while injector and detector temperatures were 240 and 280 °C, respectively.Relative concentrations of components were obtained by normalizing peak areas (%).Relative areas consisted of the average of triplicate GC-FID analyses.GC-MS conditions and the identification have been previously reported (Cabral et al., 2022).Identification of volatile components of hexane extracts (Table 1) was based on their retention indices on an Rtx-5MS (30 m X 0.25 mm; 0.250 µm) capillary column under the same operating conditions used for GC relative to a homologous series of n-alkanes (C 8 -C 20 ).Structures were computer-matched with Wiley 7, NIST 08 and FFNSC 1.2 and their fragmentation patterns were compared with literature data (Adams, 2007).

Antifungal activity of HE-RB and HE-UB
In this study, two methodologies were used for evaluating antifungal activity in vitro (Dias et al., 2022;Cabral et al., 2022).R. stolonifer and C. gloeosporioides strains were isolated from moldy whole papaya in natural conditions and identified.S. sclerotiorum isolates were collected in an area that has been naturally infested by this pathogen in Rio Verde, GO, Brazil.Sclerotia were produced by chopping fungal mycelia and placing them in Erlenmeyer flasks containing previously autoclaved carrot discs.Flasks were incubated at 25 ºC in the dark for 30 days.Afterwards, resulting sclerotia were removed from the flasks, washed under running water and stored at 5 ºC up to their use in experiments.The plate scribing method was used for isolation and purification.Isolated fungal colonies (R. stolonifer and C. gloeosporioides) selected in naturally contaminated papaya were dissolved in sterile saline to make a fungal suspension which was spread on Petri dishes containing potato dextrose agar (PDA) medium and incubated at 28 °C for 3-5 days until complete fungus growth.Grown colonies were re-cultured to obtain pure cultures, transferred to PDA slant medium and stored at 4 °C for further studies.Fungal strains were cultured at 28 °C for 3-5 days and fungal spores on plates were dissolved in sterile saline solution and diluted to the approximate proportion of 10 6 CFU/mL.HE-RB and HE-UB were dissolved in 0.1% Tween 80 to render doses between 25-300 µL.Diluted extracts were filtered by a 0.45 μM microporous filter.Then, 100 μL of every fungal suspension was spread onto PDA plate medium and the sterile filter paper (6.0 mm diameter, 1.0 mm thick) was impregnated with 10 μL of every extract and placed on the surface of seeded Petri plates.Filter paper loaded with solvent was used as the control.Plates were placed in an incubator at 28 °C for 3-5 days.The diameter of the inhibition zone was measured and recorded as an indicator of antifungal activity.Frowncide 500SC was used as the positive control (dose of 5 µL).Pure Tween 80 was also evaluated at the lowest dose under investigation (25 µL) in all steps of the experiment in order to find out whether it would interfere with the assays.Agar diffusion assays applied to every extracts against the three fungi were performed in triplicate.They were incubated at 28 °C and mycelial growth was measured daily up to full growth of the fungus on control dishes.The treatment was carried out in quadruplicate and the experimental design was thoroughly randomized.Data were submitted to the analysis of variance (ANOVA) and means of treatments were evaluated by the Scott-Knott test at 5% significance level by the ASSISTAT software program.The percentage of inhibition of mycelial growth was calculated by the following formula (Equation 1): (1)

Antifungal activity
Assays of antifungal activity were divided into two parts and results were shown by graphs in Figures 3-8.The first part consisted in testing all pre-selected doses of HE-RB (25-300 µL) and Tween 80 (25 µL) against S. sclerotiorum, R. stolonifer and C. goleosporoides to find the dose of HE-RB which would be capable of inhibiting 100% of the three fungi (Figures 3-5).In the case of S. sclerotiorum, maximum inhibition was reached at 200 µL of HE-RB while 100 µL was enough to inhibit 100% of R. stolonifer growth and 300 µL inhibited 100% of C. goleosporoides growth.The second part of the analyses aimed at evaluating antifungal potential of HE-UB against the fungi.Results showed that 100 µL, 200 µL and 150 µL inhibited 100% of S. sclerotiorum, R. stolonifer and C. goleosporoides growth, respectively.The positive control was the commercial fungicide Frowncide 500SC at 5 µL (100% inhibition).
Regarding antifungal activity, excellent in vitro results were found.Both HE-RB and HE-UB exhibited high inhibition of mycelial growth of three phytopathogens, i. e., S. sclerotiorum, R. stolonifer and C. goleosporoides.It should be reinforced that they cause incalculable economic losses to important crops worldwide, such as soybeans, and prevent commercialization of several types of fruit due to precocious rot (Wang et al., 2019;Nunes et al., 2020).
In the scenario in which fungi are responsible for severe economic losses and damage in the food sector, natural products with antifungal activity are considered promising alternatives to replace highly toxic synthetic fungicides.Secondary metabolites from plants have already proven to be as active as commercial fungicides used in agriculture (Jiménez-Reyes et al., 2019).
Recent data published by several researchers have shown that C. chinense exhibits relevant antifungal activity, a fact that has been confirmed and corroborated by this short communication.For instance, Anaya-López et al. (2006) showed that C. chinense exhibits activity against the fungus Candida albicans.Dias et al. (2013) reported that C. chinense is active against C. albicans, P. membranifaciens, S. cerevisiae, C. tropicalis and K. marxiannus yeasts.Ethanolic extract based on C. chinense fruit showed its capacity to inhibit Aspergillus parasiticus growth (Buitimea-Cantúa et al., 2020).Besides, some researchers have stated that peptides found in C. chinense fruit have high antimicrobial potential against phytopathogenic fungi, which is strong evidence of the fact that the species is promising in agriculture (Santos et al., 2020;Moguel-Salazar et al., 2011).In addition, a recent study carried out by Aguieiras et al. (2021) showed that C. chinense fruit have bioactive metabolites which are capable of fighting multi-resistant pathogens.
Studies of C. chinense and their active constituents -that have already been published in the literature -may explain the satisfactory results of HE-RB and HE-UB.This study highlights the total inhibition of fungal growth when different doses of hexane extracts were evaluated.Santos et al. (2024) reported that 200 µL ethyl acetate extracted from C. chinense fruit was capable of inhibiting S. sclerotiorum (96.2%),R. stolonifer (87.3%) and C. goleosporoides (98.3%) growth.The methanolic extract was poorly active since it inhibited only around 50% of mycelial growth of the three fungi (Santos et al., 2024).In sum, this study suggests that antifungal activity exhibited by HE-RB and HE-UB may be explained by their high concentrations of E-caryophyllene, since this sesquiterpene has well-known antifungal activity (Nogueira et al., 2020;Hilgers et al., 2021).Another possibility that must also be mentioned is the synergic effect of all constituents of the extracts which act to result in satisfactory antifungal activity (Rueangrit et al., 2019).

Conclusion
Pepper extracts under evaluation have an inhibitory effect on mycelial growth of Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides.Another observation is that the more extract doses, the more the antifungal activity increases.

Figure 3 .
Figure 3. Percentages of inhibition of S. sclerotiorum mycelial growth at different HE-RB doses.

Figure 4 .
Figure 4. Percentages of inhibition of R. stolonifer mycelial growth at different HE-RB doses.

Figure 5 .
Figure 5. Percentages of inhibition of C. goleosporoides mycelial growth at different HE-RB doses.

Figure 6 .
Figure 6.Percentages of inhibition of S. sclerotiorum mycelial growth at different HE-UB doses.

Figure 7 .
Figure 7. Percentages of inhibition of R. stolonifer mycelial growth at different HE-UB doses.

Figure 8 .
Figure 8. Percentages of inhibition of C. goleosporoides mycelial growth at different HE-UB doses.

Table 1 .
Volatile constituents of hexane extracts from ripe little beak peppers (HE-RB) and unripe bode peppers (HE-UB).