Effect of essential oils on the development of Colletotrichum sp. fungus in fragments of Feijoa sellowiana fruits

The aim of this work was to evaluate the effect of the essential oils of Syzygium aromaticum, Cymbopogon citratus, Eucalyptus citriodora and Rosmarinus officinalis on the mycelial development of the fungus Colletotrichum sp. in fragments of Feijoa sellowiana fruits. The essential oils were incorporated in the PDA (Potato-Dextrose-Agar) medium in the concentrations of 250, 500 and 1000 ppm, 0 ppm (PDA only) (negative control), and fungicide fluazinam 1% (positive control). The area under the mycelial growth curve (AUMGC) and percent inhibition of mycelial growth (PIMG) were calculated. In the second evaluation, fruits fragments bordering the disease symptom were immersed in essential oils aqueous solution of S. aromaticum, C. citratus, and E. citriodora, at the concentration of 5000 ppm, 0 ppm (water only negative control) and fluazinam 1% (positive control). The immersion times in the treatments were: 2, 4, 8, 12 and 24 hours, with subsequent incubation in Agar-Agar medium at 25°C. This evaluation was performed daily for 15 days, observing the moment of fungal germination through the emission of the mycelium. It was verified from the obtained results that all treatments reduced the fungal growth, and the essential oils of C. citratus and S. aromaticum totally inhibited its growth from the dose 500 and 1000 ppm, respectively. Regarding the test on fruit fragments, the essential oil of S. aromaticum at the immersion times of 12 and 24 hours was effective in inhibiting the fungus until the 15 day of evaluation.


Introduction
In Brazil, the abundance of autochthonous food species is substantial; nevertheless, this biodiversity is scarcely used compared to its potential. For this reason, it is the great relevance the encouragement of strategies aimed to increase the biological diversity, managing native species, as well as, to generate an alternative income. In this scenario, the South region of Brazil is home to several food species, among which Feijoa sellowiana O. Berg (sin. Acca sellowiana) stands out. This species is native to the southern Brazilian plateau and northeast of Uruguay, with dispersions in Argentina and Paraguay (Keller & Tressens, 2007). The interest in the study of this myrtaceae is because this species is adapted to the edaphoclimatic conditions of south Brazil colder regions, and for presenting fruits with significant organoleptic potential (Ducroquet, Hickel, & Nodari, 2000).
However, among the challenges that this species offers, the phytosanitary issues are mentioned, and the most significant disease of F. sellowiana is anthracnose (Andrade & Ducroquet, 1993). This disease is caused by the fungus Colletotrichum sp., reported as one of the most important pathogens that infect the fruit trees (Agrios, 2005). In F. sellowiana, this fungus can cause plantlets tipping, loss of large numbers of seedlings, partial or total drying of branches, even causing the death of adult plants. In addition, in conditions where this disease is more intensified, it may damage 100% of the fruits near maturation and juveniles (Andrade & Ducroquet, 1993;Ducroquet et al., 2000). The control of anthracnose is carried out basically by choosing genotypes and resistant cultivars, management and cultural treatments, sanitization measures and mainly using chemical products (Agrios, 2005).
The large-scale consumption of pesticides to control pests and diseases has increased significantly; nevertheless, its excessive use has been raising concerns for the population due to problems for human health and for the environment. Moreover, the society seeks healthier lifestyles (Ghini & Kimati, 2000;Bettiol & Ghini, 2003;Bettiol, Maffia, & Castro, 2014).
Based on this context, studies with alternative methods in the control of plant diseases have been standing out, since these methods have the objective of controlling diseases without having the same problematic effect of agricultural pesticides. Among them, the use of medicinal plants has been exploited due to its importance in the contribution as a natural source of molecules in the control of diseases and pests in agriculture (Schwan-Estrada & Stangarlin, 2005).
Medicinal plants have secondary compounds that can either present direct activity through crude extracts, hydroalcoholics and plant essential oils on phytopathogens, or indirectly, by activating plant defense mechanisms to pathogens (Hammerschmidt & Dann, 1997). Several crude extracts and essential oils have already been tested on phytopathogens in several studies (Fiori et al., 2000;Itako, Tolentino Júnior, & Schwan-Estrada, 2013;Cruz et al., 2013;Mattos, Povh, Rissato, Schwan, & Schwan-Estrada, 2019). Promising results regarding the fungus of the genus Colletotrichum using plant compounds have been obtained both, in the in vitro conditions on its development, and in postharvest protection in some crops of agricultural importance (Anaruma et al., 2010;Perumal, Sellamuthu, Nambiar, & Sadiku, 2016;Andrade & Vieira, 2016).
Thus, the purpose of this study was to evaluate the effectiveness of essential oils of Syzygium aromaticum, Cymbopogon citratus, Eucalyptus citriodora and Rosmarinus officinalis in the in vitro development of Colletotrichum sp. and in diseased fragments of Feijoa sellowiana fruits.

Fungal isolation and extraction of essential oils
The experiment was conducted at the Phytopathology Laboratory of the Universidade Federal de Santa Catarina (UFSC), Curitibanos Campus. The fungus Colletotrichum sp. was isolated from diseased fruits and stored in PDA (Potato-Dextrose-Ágar) culture medium and incubated at 24°C for 12 hours of photoperiod.
Healthy leaves of C. citratus (DC) Stapf, Poaceae family, E. citriodora Hook, family Myrtaceae, and R. officinalis L. Family: Lamiaceae were collected in the University's Medicinal and Aromatic Plants Garden. The essential oils were obtained by steam distillation using a Clevenger apparatus. After the extraction, they were stored in amber glasses at room temperature. Only the essential oil of dried floral of S. aromaticum (L. Merr. & L. M. Perry, Myrtaceae family) was purchased commercially.

Evaluation of mycelial growth on the fungus Colletotrichum sp.
In order to evaluate each essential oil in the mycelial growth of the fungus Colletotrichum sp., the aliquots of oil were incorporated in the PDA melting medium in the doses of 250, 500 and 1000 ppm and distributed in Petri dishes. As negative control the dose used was 0 ppm (PDA only). As a positive control, the chemical fungicide fluazinam at 1% concentration was used. In all treatments 1% (v v -1 ) of Tween20 ® was added to the culture medium to facilitate the emulsification of each of the essential oils.
After the medium solidification, a 6 mm diameter disc of the mycelial fungus, with 7 days growth, was deposited in the center of the Petri dish with the culture medium. The plates were sealed and incubated at 24ºC for a photoperiod of 12 hours. The mycelial growth was evaluated by measuring two opposite diameters of every colony at 24 hours intervals, and these measurements were finished when the control had the fungus colony established on 80% of the growing media surface.
The mycelial growth data obtained by the daily measurements of the colonies were used to calculate the area under the mycelial growth curve (AUMGC). The equation proposed by Campbell and Madden (1990) was used Equation 1: where: y i and y i+1 are the colony growth values observed in two consecutive evaluations, ti+1 and ti are the periods of the evaluations. The AUMGC was used in the calculation of percentage of inhibition of mycelial growth (PIMG) using the formula described by Bastos (1997): PIMG (%) = t*100/T, where T is the AUMGC of the control (dose 0 ppm) and t is the AUMGC of the treatments. Acta Scientiarum. Biological Sciences, v. 43, e53055, 2021 The experimental design was completely randomized (DCR) with five replicates. Each Petri dish was considered a repetition. Data were submitted to analysis of variance and then to the non-linear regression analysis using package 'drc' of software R by the model of Gompertz with 3 parameters,( = • − •(log( )− ) ), where D is the upper limit parameter, B is the relative slope of the curve and E parameter is the logarithm of the inflection point.

Tests in fruits fragments of Feijoa sellowiana fruits
Feijoa sellowiana fruits (cv. Alcântara) with symptoms of anthracnose were collected from plants belonging to the active germplasm bank of Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (Epagri), Experimental Station of São Joaquim, located in São Joaquim, state Santa Catarina.
For the evaluation of the treatments with the essential oils in different immersion times, fragments of 10 mm were removed in the lesioned area of the fruits presenting symptoms of the disease. The fruit fragments were previously disinfested with 70% alcohol (1 min.) and then with 2% sodium hypochlorite (2 min.) and rinsed in sterile distilled water (Punja & Rahe, 1993). After the surface disinfection, the fruit fragments were immersed in aqueous solution of the essential oils of S. aromaticum, C. citratus and E. citriodora at the concentration of 5000 ppm. In addition to the essential oils, distilled water was used as a negative control and as a positive control the chemical fungicide fluazinam at the concentration of 1%. In all treatments 1% (v v -1 ) of Tween20 ® was added to facilitate emulsification.
The immersion times in the previously described treatments were: 2, 4, 8, 12 and 24 hours. After this period four fragments were arranged equidistantly in Petri dishes on the surface of the agar-agar (AA) culture medium. The plates were sealed and incubated at 24°C and for a photoperiod of 12 hours. The evaluations were performed daily, for 15 days, observing the fungal germination time through the emission of mycelium around the fragment of the fruit.
The experimental design was completely randomized with four replicates. Each Petri dish with four fruit fragments was considered a repetition. The data were submitted to survival analysis using the 'survival' package from statistical software R. Survival curves of Kaplan-Meier (survfit function) were calculated. The curves were compared by the log-rank test Mantel-Haenszel (G-rho Family test) (survdiff function) against the control and the fungicide fluazinam (Dudley, Wickham, & Coombs, 2016).

Fungi toxic activity of the essential oils
Regarding the percentage of inhibition of mycelial growth (PIMG) ( Table 1), all treatments with oils significantly reduced fungal growth as compared to negative control, including the fungicide treatment (positive control), which inhibited 100% of mycelial growth (data not shown). The response was dosedependent adjusted to the Gompertz model with 3 parameters, except for the essential oil treatment of R. officinalis which did not fit the model. The essential oil of C. citratus completely inhibited the mycelial development of the fungus from the 500 ppm dose (Table 1). Also, S. aromaticum inhibited from the 1000 ppm dose. The E. citriodora essential oil inhibited mycelial development in 70.6% at 1000 ppm dose. The essential oil of R. officinalis at the dose of 250 Acta Scientiarum. Biological Sciences, v. 43, e53055, 2021 ppm had higher mycelial growth than the control. There was only inhibition of mycelial development at doses of 500 to 1000 ppm by 11.2 and 49.1%, respectively.
The efficacy of C. citratus and S. aromaticum oil has been proven in several studies in other pathosystems. Fiori et al. (2000) assessed several essential oils and verified that C. citratrus oil completely inhibited spore germination and growth of the fungus Dydimella bryoniae. Ranasinghe, Jayawardena, and Abeywickrama (2002) verified the fungistatic and fungicidal effect of volatilization of the oils of S. aromaticum and Cinnamomum zeylanicum in the development of the fungi Lasiodiplodia theobromae, Colletotrichum musae and Fusarium proliferatum. Itako et al. (2013) evaluated the effect of increasing doses of C. citratus on the development of Alternaria solani and the induction of pathogenesis-related enzymes. They also observed the fungitoxic effect of the oil and its ability to induce the peroxidase and polyphenoloxidases enzymes in Solanum lycopercisi L. Perumal et al. (2016) investigated the antifungal effect by volatilization of five essential oils on Mangifera indica L. and observed that the oil of T. vulgaris and S. aromaticum, entirely inhibited the development of the fungus C. gloeosporioides. Oliveira, Oliveira, Vieira, Câmara, and Souza (2018) evaluated the effect of C. citratus oil, associated with a chitosan to control five pathogenic species of Colletotrichum in guava (Psidium guajava L.) and in vitro, and observed an inhibition of mycelial growth of all tested fungal species. These results corroborate with those obtained in the present work, since the oil of S. aromaticum and C. citratus completely inhibited the development of the fungus. Salgado et al. (2003), evaluating the oils of different eucalyptus species, verified that the essential oil of Eucalyptus urophylla presented greater fungitoxic action and this was attributed to the presence of the compound globulol, absent in Eucalyptus camaldulensis and E. citriodora. According to Silva (2006), the chemical composition and quantity vary depending on the age of the plant, the type of tissue, its habitat and the type of soil. This partially explains the discrepancy found among the studies conducted at different locations using the same methodology and same plant species.
Regarding the results with essential oil of R. officinalis similar results were obtained by Daferera, Ziogas, and Polissiou (2003) in which evaluated six essential oils including R. officinalis on the development of fungi Botrytis cinerea, Fusarium sp. and observed among the evaluated oils, the R. officinalis showed lower inhibitory activity against fungi.

Test on fruits fragments
In preliminary tests, doses of 1000, 2000 and 4000 ppm were tested, and these did not show results in inhibiting the fungus in the fruits fragments. Thus, only 5000 ppm was used in the experiment.
Regarding the germination of the fungus in the treatments by immersion of F. sellowiana fruits fragments in solution of the essential oils, the 2 and 4 hours immersion times were not efficient in inhibiting fungus development for any of the essential oils tested. However, the other times presented variable responses of 50 to 90% inhibition (Table 2). Due to the low efficiency in inhibition of mycelial growth of the essential oil of R. officinalis, this oil was not used in the assay with the fruit fragments. In relation to the treatment of the negative control ( Figure 1A), there was no inhibition of fungal germination in all fruit fragments from the 4 th day of evaluation. Yet, the positive control treatment ( Figure 1B) totally inhibited germination until the 15 th day. In the 8 hour immersion time ( Figure 1C, D and E), the fruit fragments treated with the essential oils of S. aromaticum and E. citriodora remained without hyphae formation until the 4 th day, and for the oil of C. citratus until the 5 th day, similar to the negative control treatment. The percentage of fruit fragments without hyphae formation, from 6 th day until 15 th day, was gradually reduced. All treatments with essential oils presented percentages lower than 40% in the inhibition of germination, observing the lowest percentage of 10% in the treatment with E. citriodora oil.
In the case of 12 hours immersion time ( Figure 1F, G and H), the fruit fragments treated with the essential oils of C. citratus and E. citriodora maintained inhibition close to the negative control, and from the 6 to the 12 th day, they had inhibitory effect of 73 and 58%, respectively. The treatment using S. aromaticum oil ( Figure 1G) maintained 100% inhibition until the 8 th day, and from that period to 15 th day the inhibition was 90%. And subsequently, in the 24 hours immersion time ( Figure 1I, J and K) of the fruit fragments, the treatments with the oils of S. aromaticum, C. citratus and E. citriodora inhibited the formation of hyphae for 8, 6 and 5 days respectively. The C. citratus oil inhibited up to 70% germination of the fungus until the last day of evaluation. The oil of S. aromaticum showed to be efficient in the inhibition of the fungus until the 15 th day, inhibiting in up to 90% of the fungus in the fruit fragments.
Regarding to the comparison test between the survival curves (Table 3), it was verified that all treatments with essential oils at immersion times of 8, 12 and 24 hours were higher than the negative control, indicating better efficiency in controlling of the growth by the essential oils. Compared with the positive control, most treatments were lower, except for the treatment with essential oil of cloves in the immersion times of 12 and 24 hours. In this time, the treatment using the S. aromaticum essential oil, the efficacy observed was similar to the positive control.
These results reaffirm the potential of C. citratus and S. aromaticum to possess antifungal and antimicrobial compounds. Barrera-Necha, Bautista-Banos, Flores-Moctezuma, and Estudillo, (2008) evaluated the effectiveness of nine essential oils, including S. aromaticum oil on the in vitro development of C. gloeosporioides isolated from Carica papaya L. and observed that increasing doses (200, 250 and 300 μg mL -1 ) efficiently inhibited mycelial development and spore germination. From the in vitro results, it a test in papaya fruits was performed, and its efficacy was verified through the smaller percentage of infection. However, the treatments did not exceed the activity of the synthetic fungicide. Anaruma et al. (2010), evaluated 28 essential oils of medicinal plants on the development of C. gloeosporioides and observed that the oil of C. citratus, Coriandrum sativum, Cymbopogon flexuosus and Lippia alba inhibited mycelial growth of the fungus, and the oil of C. citratus reduced the rate of anthracnose disease in fruit of Passiflora edulis.
The essential oil of C. citratus has antifungal and antibacterial therapeutic properties, and the main component is citral (Nascimento, Innecco, Marco, Mattos, & Nagao, 2003). The main constituent of S. aromaticum oil is eugenol, a very efficient aromatic compound, with nematicidal, insecticidal, bactericidal and fungicidal activity (Lorenzi & Matos, 2002). Partly, one of the explanations of the action on fungal inhibition in both mycelial growth and sporulation of the fungus Colletotrichum sp. it may be due to the major presence of these components in the essential oil.
Analyzing the results, it was verified the highest efficiency of S. aromaticum oil compared to the other oils tested to inhibit germination of the fungus from the fruits fragments. According to Costa et al. (2011) the antifungal effect of the essential oil of S. aromaticum is related to its hydrophobicity, which allows them to interact with the cell wall constituents (lipids), altering the permeability, causing disturbances in these structures. The same authors studied the action of the S. aromaticum oil in the in vitro mycelial growth of phytopathogenic fungi and evidenced several morphological alterations (disorganization of the cellular contents, less turgidity of the hyphae, decrease in cell wall sharpness) on fungi F. oxysporum, F. solani and R. solani.

Conclusion
The essential oil of S. aromaticum was effective in inhibiting total mycelial growth and germination (hypha formation) in the F. sellowiana fruit fragments in 12 and 24 hours immersion time.
The other essential oils tested showed partial inhibition in the development of the fungus. The results demonstrate the potential of essential oils in the control of plant diseases in fruits.