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

Extrato etanólico de Copaifera, Croton e Lippia no controle de fungos fitopatogênicos

Lorenna Leal Pires lorennapires@hotmail.com

Universidade Federal do Piauí, Brasil

Bruno Arcanjo Silva bruno.arc.anjo@hotmail.com

Universidade Federal do Piauí, Brasil

Enayra Silva Sousa enayra.sousa@hotmail.com

Universidade Federal do Piauí, Brasil

Enedina Rodrigues da Silva Neta enedinarodriguees@hotmail.com

Universidade Federal do Piauí, Brasil

Sidney Gonçalo de Lima sidney@ ufpi.edu.br

Universidade Federal do Piauí, Brasil

José Evando Aguiar Beserra evando@ufpi.edu.br

Universidade Federal do Piauí, Brasil

Plant diseases caused by fungi can affect the yield and quality of agricultural products, resulting in economic losses to the producer. Colletotrichum, Fusarium, Lasiodiplodia and Thielaviopsis are important pathogens of crops such as Fabaceae, Cucurbitaceae and fruit and palm trees in Brazil, causing severe damage (Cannon et al. 2012, Borges et al. 2019, Nikitin et al. 2023). Several measures can be taken to control the diseases. For example, chemical fungicides usually show a high efficiency (Molina et al. 2019). However, the continued use of these products leads to environmental and human health problems and selection of insensitive pathogens, since many of them are highly toxic and persistent (Carvalho 2017). The inherent disadvantages of chemical fungicides drive the search for alternative products to control fungal diseases (Zubrod et al. 2019).

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.

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.

Six isolates belonging to the phytopathogenic fungi collection of the UFPI (COUFPI) were used. The isolates were previously identified by morphological markers and multilocus phylogenetic analysis (data not shown): Colletotrichum siamense (COUFPI 233), C. truncatum (COUFPI 227), Fusarium sacchari (COUFPI 72), F. udum (COUFPI 34), Lasiodiplodia theobromae (COUFPI 264) and Thielaviopsis ethacetica (COUFPI 01). All isolates were cultivated in potato-dextrose-agar (PDA) medium and kept at 26 ± 2 ºC in a growth chamber, with a photoperiod of 12 h, until the experiments were carried out.

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 “R²”. 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 10⁵).

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.

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).

Figure 1
Effect of Copaifera luetzelburgii, Lippia lasiocalycina and Croton zehntneri ethanolic extracts on the percentage of mycelial growth inhibition (PMGI) of Colletotrichum siamense (A), Colletotrichum truncatum (B), Fusarium sacchari (C), Fusarium udum (D), Thielaviopsis ethacetica (E) and Lasiodiplodia theobromae (F).

The highest percentage of mycelial growth inhibition (100 %) was observed for the L. lasiocalycina extract on T. ethacetica (Figure 1E), while C. zehntneri was the lowest one (34 %) on F. sacchari (Figure 1C). The most significant inhibitions among all the fungal species were observed on T. ethacetica, as the extracts from L. lasiocalycina, C. zehntneri and C. luetzelburgii presented mycelial growth inhibition of 100, 87.3 and 72.4 %, respectively (Figure 1E). The extracts from L. lasiocalycina, C. zehntneri and C. luetzelburgii showed mycelial growth inhibition of 86.1, 66.5 and 45.4 %, respectively, on L. theobromae (Figure 1F).

Thus, the highest concentration of all fixed extracts was the one that exerted the most significant 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.

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 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.

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).

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.

Table 2
Concentration of Fusarium sacchari 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).

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.

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).

The ethanolic extracts yields were 4.9, 8.8 and 11.2 % for C. luetzelburgii, C. zehntneri and L. lasiocalycina, respectively. Phytochemical tests for the fixed extract of C. luetzelburgii stem bark indicated the substantial presence of compounds such as anthocyanins, anthocyanidins, aurones, chalcones, flavanones, flavanonols, flavonols, leucoanthocyanidins and xanthones, all of which have a potential antioxidant activity (Lima et al. 2021). Furthermore, compounds such as trans-docosanil, ferulate, acetyl aleuritolic acid, 3-O-methylquercetin, E-anethole, 2-hydroxy-4,6-dimethoxyacetophenonan, 3-O-methylquercetin, β-sitosterol, stigmasterol (Santos et al. 2017), crototropone (Bracher et al. 2008) and triterpene acetyl aleuritolic acid (Bezerra et al. 2021) have been identified in fixed extracts of C. zehntneri. Moreover, in the ethanolic extract of leaves and stem of L. lasiocalycina, phenylpropanoid (Forsitoside B) and verbacoside were detected (Funari et al. 2012). Overall, volatile constituents have been widely used since ancient times, and their role and action have been discussed regarding their bioactivity as antibacterial, antiviral, antioxidant and antidiabetic by Tanu & Harpreet (2016).

It is known that several species from the Lippia genus have antifungal properties in their oils/extracts (Pandey et al. 2016, 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 can 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 (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.

The fixed constituents of Copaifera luetzelburgii, Croton zehntneri and Lippia lasiocalycina at 2,000 μg mL^-1 are efficient in inhibiting the mycelial growth of Colletotrichum siamense, C. truncatum, Fusarium sacchari, F. udum, Lasiodiplodia theobromae and Thielaviopsis ethacetica. Furthermore, they inhibit the conidia production of C. siamense, F. sacchari and F. udum.