Ethanolic extract of Copaifera, Croton and Lippia on the control of phytopathogenic fungi

ABSTRACT The search for vegetable extracts for phytosanitary control has been expanded to find new active ingredients to control plant diseases. This study aimed to evaluate the in vitro effect of the fixed constituents of Copaifera luetzelburgii, Croton zehntneri and Lippia lasiocalycina, at the concentrations of 2, 20, 200 and 2,000 μg mL-1, on the percentage of mycelial growth inhibition of Colletotrichum siamense, C. truncatum, Fusarium sacchari, F. udum, Lasiodiplodia theobromae and Thielaviopsis ethacetica, as well as the conidium concentration of C. siamense, F. sacchari and F. udum produced in culture medium with all the extracts. The tested ethanolic extract, especially at the highest concentration, inhibited the percentage of mycelial growth and/or conidium concentration of the evaluated fungi. The other concentrations showed low inhibitory effects or no activity against the fungi. The average values for percentage of mycelial growth inhibition of the ethanolic extract from L. lasiocalycina, C. zehntneri and C. luetzelburgii against the six fungi were 62.5, 53.4 and 51.0 %, respectively. The ethanolic extract of L. lasiocalycina showed the most significant effect on the percentage of mycelial growth inhibition and conidia concentration. The fixed constituents of C. luetzelburgii, C. zehntneri and L. lasiocalycina at 2,000 μg mL-1 showed to be efficient in inhibiting the mycelial growth of C. siamense, C. truncatum, F. sacchari, F. udum, L. theobromae and T. ethacetica, and inhibit the conidia production of C. siamense, F. sacchari and F. udum.

Plants bioactive compounds have been successfully tested and used to control phytopathogenic fungi (Lengai et al. 2020, Pham et al. 2021).Their action can be proven by inhibiting mycelial growth and spore production (Saravanakumar et al. 2015, Mourão et al. 2017).The advantages of these compounds include the low cost and toxicity, ease of use, high efficiency and biodegradability (Kumar et al. 2019).Consequently, farmers are increasingly using products formulated with bioactive compounds extracted from plants due to the growing demand for organic products in the market.
Fixed oils (natural non-volatile oils) are among the bioactive compounds extracted from plant seeds, bark and fruit pulp.These oils are complex mixtures and water-insoluble (hydrophobic), what means they are soluble in nonpolar compounds (organic, predominantly formed by triglycerides) (Saravanakumar et al. 2015).
Several plants present antifungal properties.Therefore, they are excellent sources for oils or extracts with such activity.In this context, Cerrado (Brazilian Savanna) plants stand out (Colli et al. 2020).This biome covers about 23 % of the Brazilian territory and presents approximately 12,000 species of angiosperms (Zappi et al. 2015).Some native species of this biome (e.g.: Copaifera spp., Croton spp.and Lippia spp.) have demonstrated antimicrobial activity and can be used against plant pathogens (Carvalho et al. 2013, Peixoto et al. 2018, Andrade et al. 2020).
Thus, this study aimed to evaluate the in vitro effect of the ethanolic extract of Copaifera luetzelburgii, Croton zehntneri and Lippia lasiocalycina on the mycelial growth of Colletotrichum siamense, Colletotrichum truncatum, Fusarium sacchari, Fusarium udum, Lasiodiplodia theobromae and Thielaviopsis ethacetica, in addition to evaluating the concentration of conidia of C. siamense, F. sacchari and F. udum produced in culture medium with the three extracts.

MATERIAL AND METHODS
The experiment was conducted under laboratory conditions at the Universidade Federal do Piauí (UFPI), in Teresina, Piauí state, Brazil, from February 2019 to February 2020.
The plant material and extracts preparation are described as it follows: L. lasiocalycina (Verbenaceae): made available by the Embrapa Meio-Norte (Teresina, Piauí state), with voucher specimen deposited at the Embrapa Recursos Genéticos e Biotecnologia herbarium (number CEN92437); C. zehntneri (Euphorbiaceae): collected in Simões (Piauí state), being deposited at the Graziela Barroso herbarium of the UFPI (number 27.273); C. luetzelburgii (Fabaceae): quilombola community, São Miguel do Tapuio, Piauí state.An exsiccation of the species was deposited at the Graziela Barroso herbarium under the number TEPB 26235.More details about the plant material, extraction process and analysis can be seen in previous publications (Almeida et al. 2018, Fonseca et al. 2019, Almeida et al. 2021, Lima et al. 2021).
The organic geochemistry research group at the UFPI provided the ethanolic extract of C. zehntneri, L. lasiocalycina and C. luetzelburgii.The collected material (C.zehntneri stem bark and C. zehntneri and L. lasiocalycina leaves) was dried in an oven with humid air draught at the temperature of 40 ºC, for 48 h, and stored away from light and moisture.Then, the material was subjected to extraction in ethanol.After the extraction, a simple filtration was performed, and the extracts were concentrated at a rotary evaporator under reduced pressure.
The extracts of C. luetzelburgii, C. zehntneri and L. lasiocalycina were solubilized in P. A. ethanol and homogenized using a vortex mixer.Then, they were filtered on a 0.22 μm PES membrane and stored in sterile 15 mL conical polypropylene tubes.Concentrations of 2, 20, 200 and 2,000 μg mL -1 were used for all the extracts.The extracts were added separately to the PDA culture medium, melted at a maximum temperature of 45 ºC, containing the antibiotic streptomycin at a concentration of 0.2 g L -1 , and then poured into sterile Petri dishes with 90 mm diameter.Each plate was inoculated, in the center, with a 5 mm diameter disk of culture medium containing mycelium from pure cultures of the fungal isolates.The plates were kept at 26 ± 2 ºC, with a photoperiod of 12 h, in a growth chamber.The control comprised Petri dishes containing only PDA culture medium with the addition of streptomycin and inoculated with a 5 mm fungal disc in the center (Cruz et al. 2018).
Assessments were performed daily at the same time, starting at 24 h after the beginning of incubation.The diameters of the colonies were measured on the orthogonal axis (average of two diametrically opposite measurements) until the control treatment reached the total diameter of the Petri dish.After the evaluations, the percentage of mycelial growth inhibition (PMGI) was calculated in comparison to the control sample, without the addition of extract, where: PMGI = [(diameter of the control treatment -diameter of the treatment)/mean diameter of the control] x 100 (Salgado et al. 2003).
The experimental design was completely randomized in a factorial scheme, with five replications.The obtained data were submitted to analysis of variance with the "F" test and regression analysis when significant, evaluating the trend line and "R 2 ".The data analysis was performed using the R software v. 3.5.1.
To each colony's surface were added 10 mL of sterilized distilled water and Tween 20 (1 %), followed by scraping with the aid of a Drigalski loop.The conidia from each plate were filtered with gauze and resuspended in 50 mL of sterilized distilled water.Three 100 μL aliquots of each conidia suspension were transferred separately to a Neubauer chamber.The conidia suspension was shaken to homogenize the sample.Next, the slide was covered with a coverslip, and the suspension was transferred with the aid of a micropipette.A drop was applied to one of the coverslip vertices, and the slide was tilted little by little to fill the compartments with liquid.After preparing the slide with the suspension, a perfect spore distribution was waited for 2 to 3 min before counting.Then, the spores were counted under an optical microscope (Carvalho et al. 2013).
The "C" compartment was used to count the six fungi in all the fixed oils.Individual counts were carried out in the four sub-compartments "c" in the corners and the center.Then, the average of five counts was calculated, and the following formula was used (Alfenas & Mafia 2016): inoculum concentration = (average number of conidia in the "C" compartment) x (2.5 x 10 5 ).
The obtained data were subjected to analysis of variance and the means compared by the Tukey test at 5 % of significance, using the R software v. 3.5.1.The Kruskal-Wallis test was applied in the absence of data normality.

RESULTS AND DISCUSSION
The highest concentration (2,000 μg mL -1 ) of C. luetzelburgii, C. zehntneri and L. lasiocalycina extract inhibited the mycelial growth of all the evaluated fungi.The other concentrations demonstrated low inhibitory effects.Therefore, the following results refer only to the highest concentration of the extracts tested.
The extracts' average percentages of mycelial growth inhibition from L. lasiocalycina, C. zehntneri and C. luetzelburgii were 62.5, 53.4 and 51.0 %, respectively, against the six fungi species.The extract that best inhibited the mycelial growth of the six fungi was L. lasiocalycina, reaching percentages of 100, 86.1 and 43.4 % on T. ethacetica,L. theobromae and C. truncatum,respectively (Figures 1B,1E and 1F).
Thus, the highest concentration of all fixed extracts was the one that exerted the most significant The conidia production of C. siamense (Table 1) was completely inhibited by 2,000 µg mL -1 of C. luetzelburgii and L. lasiocalycina extracts.The other concentrations tested for the three extracts were inefficient in inhibiting the conidia production.The e-ISSN 1983-4063 -www.agro.ufg.br/pat-Pesq.Agropec. Trop., Goiânia, v. 53, e75126, 2023 Ethanolic extract of Copaifera, Croton and Lippia on the control of phytopathogenic fungi extract from C. zehntneri did not differ from the control in any of the concentrations.There is already a report of the volatile composition of C. zehntneri oils, whose main component is trazol, with known bactericidal activity (Andrade et al. 2015).This compound is also present in the fixed constituents.
Apparently, this compound (and others present in the extract) does not show antifungal activity against C. zehntneri.
In their highest concentration, the extracts from C. luetzelburgii, C. zehntneri and L. lasiocalycina were the most efficient in reducing the F. sacchari conidia production (74, 62 and 83 %, respectively) (Table 2).Furthermore, lower concentrations of extracts from C. luetzelburgii and L. lasiocalycina had less pronounced effects on the inhibition of conidia germination.
The most efficient concentration in reducing the F. udum conidia production was 2,000 µg mL -1 of the extracts from C. zehntneri and L. lasiocalycina (67 and 50 %, respectively) (Table 3).The extracts from C. luetzelburgii did not differ from the control.
It is known that several species from the Lippia genus have antifungal properties in their oils/extracts (Pandey et al. 2016al. , Peixoto et al. 2018).However, until the time that this study was carried out, the effects of the ethanolic extract of L. lasiocalycina against phytopathogenic fungi were not known.According to the results, the ethanolic extract of L. lasiocalycina presented fungicide activity against a wide variety of fungi, such as Colletotrichum, Fusarium, Lasiodiplodia and Thielaviopsis, showing antifungal properties at different levels.This activity   (Almeida et al. 2018).
The hydrophobic monoterpenes (e.g., limonene and piperitenone oxide) and other compounds in the L. lasiocalycina essential oil act on the plasma membrane of Candida albicans, which is a yeast capable of causing mycosis in humans.The antifungal property can be attributed to the disruption of the plasma membrane (Almeida et al. 2018).
There are more than 20 Copaifera spp.already described, some of which have had their antimicrobial activity evaluated on bacteria and dermatophyte fungi (Santos et al. 2008, Zimmermmam-Franco et al. 2013).However, this is the first time that the effect of the ethanolic extract of C. luetzelburgii is assessed on the mycelial growth and conidium concentration of phytopathogenic fungi.The main compounds in the essential oils of Copaifera spp.are sesquiterpenes and diterpenes, which seem responsible for their antifungal properties (Veiga-Júnior et al. 2001).
The larvicidal and antifungal properties of Croton spp.oils, including C. zehntneri, have already been demonstrated (Sing et al. 2006, Fontenelle et al. 2008).The main compounds in the C. zehntneri oil are estragole and anethole (Fontenelle et al. 2008), which may be responsible for inhibiting the growth of dermatophyte and saprophyte fungi.However, the inhibitory concentrations against fungi have been high, i.e., between 25,000 and 100,000 µg mL -1 (Pereira et al. 2021).In this study, the concentration of 2,000 µg mL -1 was moderately effective in inhibiting the growth of the fungi evaluated, showing an excellent potential in controlling phytopathogenic fungi.
Only one qualitative study presents the composition of the fixed oil of C. luetzelburgii through chemical tests (Araújo et al. 2021).Its preliminary results indicate the presence of flavanones, flavanonols and flavonols.It also studied its antioxidant effect and toxicity on the reproductive system in rats.The subchronic toxicity was evaluated through the number, morphology and functional viability of spermatozoa and histopathology.
The findings of the present study emphasize the potential of these plant extracts as effective agents for phytosanitary control, suggesting their possible application as environmentally friendly alternatives to synthetic pesticides.However, further research is necessary to explore their full potential and ensure their safe and practical use in agricultural practices.
e-ISSN 1983-4063 -www.agro.ufg.br/pat-Pesq.Agropec.Trop., Goiânia, v. 53, e75126, 2023    percentages of mycelial growth inhibition on the fungi tested.A mycelial growth inhibition above 50 % was exerted by the three extracts on C. siamense and T. ethacetica and by C. zehntneri and L. lasiocalycina on L. theobromae.The conidia production was determined at the end of the mycelial growth assessment.Only the C. siamense, F. sacchari and F. udum fungi produced conidia.The others (i.e., C. truncatum, L. theobromae and T. ethacetica) were not evaluated, since they did not produce conidia in any of the treatments.

Table 1 .
Concentration of Colletotrichum siamense conidia in doses of Copaifera luetzelburgii, Lippia lasiocalycina and Croton zehntneri extracts.Means followed by the same letter do not differ statistically from each other by the Tukey test (p > 0.05).Means followed by the same letter do not differ statistically from each other by the Tukey test (p > 0.05).

Table 3 .
Concentration of Fusarium udum conidia in doses of Copaifera luetzelburgii, Lippia lasiocalycina and Croton zehntneri extracts.Means followed by the same letter do not differ statistically from each other by the Tukey test (p > 0.05).be attributed to the piperitenone oxide, which represents about 58 % of the essential oil composition.Piperitenone oxide has several industrial applications and has shown potential properties against herpes and Aedes aegypti, vasodilatory effects in hypertensive rats and antiparasitic effect *e-ISSN 1983-4063 -www.agro.ufg.br/pat-Pesq.Agropec.Trop., Goiânia, v. 53, e75126, 2023can