INHIBITORY ACTIVITY OF PROPOLIS EXTRACTS FROM STINGLESS BEES (MELIPONINI) ON THE DEVELOPMENT OF PHYTOPATHOGENIC FUNGI: IN VITRO EVALUATION

Objective: The present study aimed to assess the alcoholic extracts of propolis from Tetragona clavipes , Scaptotrigona bipunctata , Tetragonisca angustula , and Melipona quadrifasciata . Theoretical Framework: There is growing interest in the bioactive metabolites of propolis and honey produced by different species of the group of native stingless bees (NSB) belonging to the Meliponini tribe. Method: The extracts were prepared in a proportion of 30 g of propolis to 70 mL of 96 % v/v ethanol (1:2). Afterwards, its chemical constituents were characterized by HPLC. A PDA medium was used to evaluate the antifungal activity, with the extracts added to the melting medium (55 °C) in different concentrations. The effect of increasing concentrations (1.6 %, 3.2 %, 6.4 %, and 12.0 % v/v) of extracts on the mycelial growth of the fungi Sclerotinia sclerotiorum , Fusarium sp., Colletotrichum gloeosporioides , Botrytis cinerea , and Botryosphaeria sp. was evaluated. Fungal development was determined by measuring mycelial diameter on the 14th day of inoculation


INTRODUCTION
Native stingless bees (NSB) belonging to the Meliponini tribe of the Apidae family (Hymenoptera) are found in all tropical and subtropical regions of the world (Barbieri;Francoy, 2020).In addition to producing honey as a nutritional source for the offspring, NSB produce cerumen and propolis, which provide mechanical and biological protection to the hive (Lavinas et al., 2018).
Propolis is formed by a mixture of plant exudates, mainly resinous, that bees collect from some plants and mix with waxes produced in specialized glands found in these insects.
Typically, propolis results in a viscous material with variable color and flavor that performs several functions for the hive and individuals (Przybyłek & Karpínski, 2019).The composition of propolis can vary, as it results from the combination of different materials that already have a quite varied composition (Pereira et al., 2002;Huang et al., 2014).Propolis extracts, including from native stingless bees, have been investigated regarding their antimicrobial and antiinflammatory potential (Paris et al., 2018) and antifungal activity (Rocha et al., 2024).
NSB propolis has been increasingly investigated in recent years, and studies have demonstrated qualitative and quantitative profiles of the various chemical components present, which may vary according to the producing species and place of origin (Bueno-Silva et al., 2017;Salatino & Salatino, 2021).The complex and diverse chemical composition of different species of stingless bees has also been observed, including phenolic acids, flavonoids, coumarins, benzophenones, terpenes, steroids, alkaloids, fatty acids and sugars (Pereira et al., 2021;Lim et al., 2023).
Given the diversity of bioactive compounds produced by NSB and their recognized biological activity, there is growing interest in studying their properties relative to the biological activity on phytopathogenic fungi, especially considering agricultural environments.
Due to the widespread and often excessive use of synthetic pesticides, which favor the emergence of resistant species via natural selection and have caused environmental contamination of soil, water, and ecosystems (Sangiorgio et al., 2022), the search for products of natural origin as less aggressive alternatives for controlling plant diseases.Therefore, care is taken to maintain the sustainability and resilience of production systems.Among the alternative products, the expansion of research into NSB propolis extracts in controlling phytopathogenic fungi of agricultural interest stands out.
Therefore, the present study aimed to evaluate the chemical composition of alcoholic extracts of propolis from NSB Tetragona clavipes, Scaptotrigona bipunctata, Tetragonisca angustula, and Melipona quadrifasciata, and the effect of these extracts on the mycelial development of the phytopathogenic fungi Sclerotinia sclerotiorum, isolated from lettuce, and Fusarium sp., Colletotrichum gloeosporioides, Botrytis cinerea, and Botryosphaeria sp., isolated from grapevine crops.

COLLECTION OF PROPOLIS AND EXTRACT OBTAINMENT
The NSB propolis samples were collected in February 2023 from hives in small producers located in the Northeast region of the State of Rio Grande do Sul, Southern Brazil.
Information regarding the species, common name, and collection location is shown in Table 1.

Table 1
Information regarding the propolis samples studied in the present work.
It is important to note that alcoholic extracts are part of the list of inputs allowed in organic production, both animal and vegetable, and are provided for in the Brazilian Ministry of Agriculture (MAPA) Normative Instruction No. 17, of June 18, 2014(Brasil, 2014;Rivero et al., 2021).

DETERMINATION OF THE LEVELS OF PHENOLIC COMPOUNDS AND FLAVONOIDS IN THE EXTRACTS
The levels of phenolic compounds and total flavonoids were determined by analyzing the extract after dilution with 96 % v/v ethanol, in a proportion of 5 mL of extract to 95 mL of ethanol.The content of phenolic compounds was determined by the Folin-Ciocalteu method, according to the procedure described by Pereira et al. (2017).The total flavonoid content was determined by the aluminum chloride spectrophotometric method, according to the procedure proposed by Matic et al. (2017).

DETERMINATION OF INDIVIDUAL LEVELS OF PHENOLIC COMPOUNDS
The individual levels of phenolic compounds in propolis extracts were analyzed by High-Performance Liquid Chromatography (HPLC) according to the procedure and specifications described by Agostini (2017).Initially, the samples were pre-treated by dilution  in Milli-Q water (5.0 g•L -1 ) and filtered through a nylon membrane with a 0.45 µm pore diameter.An HP model 1100 liquid chromatograph was used, coupled to a Lichrospher RP18 column (5 µm) and a UV detector at a wavelength of 210 nm.The reversed-phase analysis comprised solvent A (Milli-Q water with 1.0 % v/v phosphoric acid) and solvent B (acetonitrile).The mobile phase pumping system was gradient type, with 90 % of solvent A from zero to 5 min, 60 % of A from 5 min to 40 min, and 90 % of A from 45 min to 50 min.
The mobile phase flow rate was maintained at 0.5 mL•min -1 , as proposed by Morelli (2010).
Quantification was carried out using the external standardization method through the correlation of the peak area of the compound to the curve of each standard evaluated.The compounds gallic acid, epigallocatechin, catechin, epicatechin, epigallocatechin gallate, rutin, ferulic acid, naringin, hesperidin, myricetin, resveratrol, quercetin, apigenin and kaempferol were evaluated.The results were expressed in milligrams of each compound per 100 mL of sample (mg•100 mL -1 ).

EXTRACTS
The antioxidant activity of alcoholic propolis extracts was evaluated using the evaluation methods by inhibition of DPPH • and ABTS •+ radicals.To evaluate the inhibition of the DPPH • radical, the methodology proposed by Yamaguchi et al. (1998) was used, where Tris-HCl buffer (100 mM, pH 7.0) containing 250 µM of the DPPH • radical dissolved in ethanol was added to the extracts.The tubes were stored in the dark for 20 min, and absorbance was determined at a wavelength of 517 nm.
The ability to reduce the ABTS •+ radical was determined according to the method proposed by Rufino et al. (2007), with modifications.The ABTS •+ radical was generated by the reaction in an aqueous solution of the ABTS •+ solution (7 mM) with a potassium persulfate solution (140 mM).This solution was kept in the dark at room temperature (20 -25 °C) for 12 -16 h before use.Then, the ABTS •+ solution was diluted with 99 % v/v ethanol to an absorbance of 0.700±0.02at 734 nm.Afterward, 3.0 mL of diluted ABTS •+ solution was added to 30 µL of the extracts, and the absorbance was read every 6 min after the initial mixing.The results for the two antioxidant activity assays were expressed as percentage inhibition of DPPH  For the evaluations, potato-dextrose-agar (BDA) medium was prepared, and after autoclaving, dilutions of the different extracts were made, from zero, 1.6 %, 3.2 %, 6.4 %, and 12.0 % v/v, added to the melting culture medium (55 °C).The same settings were used to prepare the experiments using only 96 % v/v ethanol as a negative control.After the PDA medium solidified, a 4.0 mm diameter disc of the fungus mycelium to be tested was placed in the center of the Petri dish.Optical microscopy was used to identify possible differences between the morphological behavior of fungal hyphae exerted by the alcoholic extract of propolis and 96 % v/v ethanol.
Incubation was carried out in a BOD chamber at 25 ºC and a 12-h photoperiod for 14 days.The mycelial growth of the fungi was evaluated on the 14th day by measuring the orthogonal diameter of the colonies, and the percentage of inhibition was calculated relative to the control (plates without the addition of extracts).

EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS
The HPLC tests and the evaluation of the antioxidant activity of the extracts were carried out in triplicate.The antifungal activity tests followed a bifactorial design (factors: type of extract and concentration) and were carried out in quintuplicates, with each replicate consisting of a Petri dish.Each fungus was isolated and tested separately.
The results obtained were checked for homoscedasticity (Levene's test) and normality of residuals (Shapiro-Wilk's test) and were subsequently subjected to analysis of variance (ANOVA).The means were compared by Tukey's test at a 5 % significance level (α = 0.05) with the aid of the AgroEstat ® software (Brazil).

ANTIFUNGAL ACTIVITY OF PROPOLIS EXTRACTS
The results regarding the levels of phenolic compounds and total flavonoids for the extracts evaluated and the antioxidant activity are compiled in Table 2.
It was possible to verify that the propolis extract from the species T. clavipes presented the highest amount of phenolic compounds, followed by S. bipunctata.However, the flavonoid content was highest for M. quadrifasciata, followed by T. clavipes.Several studies address the profile of chemical compounds in propolis, mainly concerning the richness of phenolic compounds, molecules that have an antimicrobial effect (Popova et al., 2021).Similar studies are also found with Apis propolis extracts, highlighting phenolic compounds and the inhibitory effect of these materials on phytopathogenic fungi (Vicã et al., 2023).It was possible to verify that the propolis extract from the species T. clavipes presented the highest amount of phenolic compounds, followed by S. bipunctata.However, the flavonoid content was highest for M. quadrifasciata, followed by T. clavipes.Several studies address the profile of chemical compounds in propolis, mainly concerning the richness of phenolic compounds, molecules that have an antimicrobial effect (Popova et al., 2021).Similar studies are also found with Apis propolis extracts, highlighting phenolic compounds and the inhibitory effect of these materials on phytopathogenic fungi (Vicã et al., 2023).9 Antioxidant activity was observed in all ANSF propolis extract samples (Table 2).
According to Lim et al. (2023), the antioxidant capacity of propolis is mainly attributed to the presence of flavonoids and phenolic compounds.
Among the chemical compounds observed in the present study, naringin (730.20 mg•L - 1 ), gallic acid (199.04 mg•L -1 ), hesperidin (172.62 mg•L -1 ), and ferulic acid (147.98 mg•L -1 ) were identified at highest contents.The individual levels of phenolic compounds, determined by HPLC, are compiled in Table 3. Flavonoids, such as quercetin, apigenin, naringenin, and aromadendrin, and phenolic acids, ferulic acid, and caffeic acid, among others, have also been identified in propolis from several species of ANSF, mainly when coming from plants of the genus Baccharis (Jasinski et al., 2014;Almeida et al., 2021).According to Zulhendri et al. (2021), the main bioactive compounds in propolis are related to the content of the plant's secondary metabolites, such as phenolic compounds and terpenoids, which are assimilated and used by bees.
The results observed relative to the phenolic and flavonoid compounds identified in the present work (Table 3) help understand propolis's antimicrobial activity and its extracts on phytopathogenic fungi.Naringin was the main compound in the group of flavonoids identified, possibly due to the presence of species from the Citrus sp. group close to the ANSF collection sites.According to Khan et al. (2014) and Zeng et al. (2018), naringin is a flavonoid found mainly in citrus fruits.However, this substance has also been reported in other plant species  et al., 2017).This compound has proven biological activity, especially antioxidant and antimicrobial activity (Veiko et al., 2023).
Ferulic acid, identified in propolis samples in the present work, is one of the most abundant phenolic compounds in plants, widely distributed in several plant species' cell walls.
This substance is known mainly for its antioxidant activity and antimicrobial effects, among other biological properties, with ferulic acid being commonly found in plants from the Poaceae family, among other species (Kumar & Pruthi, 2014;Antonopoulou et al., 2022;Velho et al., 2023).
Gallic acid, identified in propolis extracts from the species Tetragona clavipes, is a phenolic compound widely identified in different parts of various plant species, such as fruits, leaves, roots, branches, and flowers, from different genera (Fidelis et al., 2020;Alaca et al., 2021;Zhang et al., 2023).
Another compound identified was hesperidin, a flavonoid occurring mainly in citrus fruits (Tang et al., 2016;Bodalska et al., 2019).Like the other secondary metabolites found in larger amounts in this work, hesperidin also has antimicrobial activity (Öngün et al., 2021).
From the results of this work, it was possible to observe that the antifungal activity increased with the increase in the concentration of alcoholic extract of propolis from NSB.
However, at 6.4 % and 12.0 % v/v, the results were equal to the treatment control (ethanol 96 % v/v), whose mycelial development was inhibited entirely in most treatments evaluated (Tables 4 to 7).In this sense, the best results for antimicrobial activity were between the concentrations of 1. 11 The phytopathogenic fungus B. cinerea suffered partial inhibition, mainly at a concentration of 1.6 % v/v, when compared to the control (ethanol 96 % v/v).It was also possible to observe that extracts of S. bipunctata, followed by M. quadrifasciata at a concentration of 3.2 % v/v, are also promising in controlling this fungus when compared to the control (ethanol 96 % v/v) (Table 4).Similar results were observed by Costantin et al. (2023) in the evaluation of different NSB propolis extracts in the control of the fungus Botrytis sp.
It is important to note that from a concentration of 6.4 % v/v, all extracts, including the control treatment (ethanol 96 % v/v), completely inhibited the development of this fungus.In this sense, treatment with 96 % v/v ethanol at concentrations of 6.4 % and 12.0 % v/v may be masking the antimicrobial compounds in extracts from native stingless bees, or the high ethanol concentration at 96 % v/v is responsible for the antifungal effect observed here (Table 4).
It can be observed that the mandaçaia and jataí propolis extracts had an inhibition effect, albeit modest, on the development of B. cinerea at a concentration of 1.6 % v/v (Table 4).A decrease in the number of B. cinerea conidia exposed to the alcoholic extract of M.
quadrifasciata propolis (Figure 1B) was also observed compared to the control containing only the BDA medium (Figure 1A) and the control with ethanol (Figure 1C).This reduced number of conidia in the control (Figure 1C) could result from the fungus' defense mechanisms due to ethanol's aggressive effect.It is understood that fungi have mechanisms to combat and mitigate biotic and abiotic factors that impair their development and/or reduce cell viability.As a result, the organism can change its morphology, increase its growth rate, and reduce or increase conidial development and number (Han, 2022).
A similar behavior was observed for the phytopathogenic fungus C. gloeosporioides, according to the results compiled in Table 5. 12 Control of the fungus C. gloeosporioides occurred mainly at a concentration of 1.6 % v/v in all extracts evaluated, except for the alcoholic extract of T. angustula.At a concentration of 6.4 % v/v, all extracts showed total inhibitory activity (100 %) against the fungus C.
gloeosporioides.However, the control treatment (ethanol 96 % v/v) showed a degree of inhibition of 83 %.At a concentration of 12 % v/v, all treatments showed total control, thus leaving this result questionable.According to Meneses et al. (2009) and Abo-Elyousr et al.
(2021), propolis extracts from native bees are capable of delaying the growth of phytopathogenic fungi in vitro, such as Botrytis cinerea, Penicillium expansum, and Colletotrichum sp., among others.However, the possible antifungal activity of stingless bee propolis has not yet been widely explored.
An antifungal effect was also observed by Silva et al. (2019), who reported inhibitory activity of NSB propolis at 0.5 -2.0 mL•L -1 (0.05 -0.2 % v/v) in the control of Colletotrichum spp.isolated from avocado fruit.In the present work, propolis extracts from M. quadrifasciata, S. bipunctata, T. clavipes, and T. angustula at concentrations of 3.2 % and 6.4 % v/v were more effective when compared to the control (ethanol 96 % v/v).
Assessing the control of the phytopathogen Fusarium sp. at a concentration of 3.2 % v/v, the inhibitory effect of propolis extracts from M. quadrifasciata, S. bipunctata, and T.
clavipes were slightly lower when compared to the control (ethanol 96 %v/v).In other concentrations evaluated, no adequate control was observed in any propolis extracts compared to the control (Table 6).

Table 6
Percentage (%) of inhibition of mycelial growth of Fusarium sp.exposed to increasing concentrations of NSB propolis extracts, evaluated 14 days after inoculation.13 from the group of flavonoids.Therefore, the difference in mycelial growth inhibition capacity found in the present work could be attributed to the more efficient extraction of bioactive substances from propolis using ethanol (Pobiega et al., 2019).
Regarding the antimicrobial activity of Botryosphaeria sp.(Table 7), none of the extracts evaluated were effective against this phytopathogen.However, observing the presence or absence of conidia via optical microscopy (Figure 4), a significant reduction in conidia in the 96 % v/v ethanol treatment (Figure 4C) was observed in the different treatments, followed by a decrease in conidia also in the treatment containing alcoholic extract of M. quadrifasciata propolis at a concentration of 3.2 % v/v (Figure 4B), compared to the control (only BDA culture medium), where a large number of conidia of were observed.Therefore, this suggests that propolis extracts may act decreasing the production of fungal spores.Culturato (2011) evaluated using plant extracts and ethanol to control Botryosphaeria fungus.Ethanol caused total inhibition of mycelium growth from a concentration of 10 % v/v.
On the other hand, plant extracts only had an effect at a concentration of 15 % v/v.Given the ineffectiveness of other alcoholic extracts of propolis on the fungus Botryosphaeria, it is understandable the enormous variety of substrates that fungi can transform, including natural polymers such as not only cellulose, lignin, chitin, and starch, but also many anthropogenic products, such as pesticides, explosives and other xenobiotics (Harms et al., 2011;Gadd, 2013).It can be noted that there was partial inhibition of the growth of S. sclerotiorum with exposure from concentrations of 3.2 % v/v when compared to the control (ethanol 96 % v/v).
Research using control with Apis propolis extracts on the development of Sclerotinia sclerotiorum was evaluated by Matny et al. (2014), who demonstrated effective control of the propolis, as mentioned earlier, against this phytopathogenic fungus.
In this work, propolis extracts from M. quadrifasciata, S. bipunctata, and T. angustula were partially effective at a concentration of 3.2 % v/v compared to the control (ethanol 96 % v/v).
In this work, the antimicrobial capacity of propolis at a concentration of 1.6 % v/v was particularly demonstrated in the control of B. cinerea and C. gloeosporioides.Rodrigues-Canales et al. (2023) demonstrated that propolis extract affects the integrity of the fungal cell membrane, resulting in increased permeability, possibly due to the interaction of phenols and flavonoids contained in propolis extract.
In the present work, propolis extracts, when used from a concentration of 3.2 % v/v, showed a more potent inhibitory activity, especially against B. cinerea and C. gloeosporioides.
However, the cytotoxic effect of ethanol and its impact on the antifungal activity of the extracts must be considered, especially at 6.4 % and 12.0 % v/v, which had very similar percentages of mycelial growth inhibition, both for alcoholic extracts of propolis from different species of NSB as in the tests using only 96 % v/v ethanol.According to Visconti et al. (2021), concentration and contact time are important factors that help understand the antifungal activity of ethanol.At concentrations greater than 10 % v/v, the toxicity of ethanol solutions to spores of several fungi, including Penicillium expansum and Penicillium implicatum, has been reported (Geiges & Kuchen, 1981).Yang et al. (2016) evaluated the use of propolis as an agent for the alternative control of the phytopathogens B. cinerea and Rhizopus stolonifer in strawberries.Mycelial growth of both pathogens was completely inhibited in the presence of ethanolic propolis extract at a concentration of 800 mg•L -1 .
According to the data presented in this work, the antimicrobial activity is consistent with the evolutionary heritage of these insects and with the characteristic of propolis in inhibiting undesirable microorganisms in the hive, producing substances rich in polyphenols, with bactericidal and fungicidal functions (Fernández-Calderón et al., 2020;Tran et al., 2020).
However, some phytopathogens presented different mechanisms to the alcoholic extract of propolis, being more or less susceptible to its use.
Research into new products aimed at the agricultural sector is necessary.Studies with extracts from beekeeping products such as NSB propolis, which have a broad and complex chemical composition, are necessary.Most research has focused on the use and applications of propolis produced by Apis mellifera.At the same time, few studies have been conducted on biological compounds and applications of NSB propolis extracts, as well as strategies for the maintenance and/or recovery of forest areas to preserve these essential pollinators.Therefore, efforts are needed to understand better the potential biological activity of these extracts in managing agricultural crops.The results regarding the microscopic evaluation of the morphology of the fungus Botrytis cinerea are shown in Figure 1.In all the images analyzed, it was possible to observe that the hyphae from the control treatment (only BDA) presented normal, uniform morphology, and the number of conidia was much higher when compared to other treatments.
Regarding the morphology of the hyphae and quantity of conidia of the fungus Botrytis cinerea, a significant decrease in the number of conidia was observed when grown in the medium with ethanol 96% v/v, control (Figure 1C) compared to the control containing only the PDA medium (Figure 1A) and the M. quadrifasciata propolis extract (Figure 1B).
Here, a significant decrease in the number of conidia is also observed in the medium with 96 % v/v ethanol (Figure 2C) compared to the control containing only the PDA medium (Figure 2A) and the S. bipunctata propolis extract (Figure 2B).It is assumed that the propolis extract of S. bipunctata at a concentration of 3.2 % v/v, as well as the other propolis extracts evaluated in this work at this same concentration, have antifungal activity as an inhibitor of the development of conidia.(Ali et al., 2014).
Regarding the in vitro activity, it was found that stingless bee propolis is capable of delaying the growth of phytopathogenic fungi such as Botrytis cinerea, Penicillium expansum, Penicillium digitatum, and Colletotrichum spp.(Tripathi & Dubey, 2004;Meneses et al., 2009;Abo-Elyousr et al., 2021).These data align with the results of the present work and indicate the antifungal activity of the different NSB propolis extracts, especially at a concentration of 3.2 % v/v.
Figure 3 shows the optical microscopy of the fungus Fusarium sp. after exposure to the ethanolic extract of M. quadrifasciata propolis and 96 % v/v ethanol.A smaller number of Fusarium sp.conidia can be seen in the medium containing the alcoholic extract of M. quadrifasciata propolis (Figure 3B) and the control with ethanol (Figure 3C), compared to the control containing only the PDA medium (Figure 3A).This reduced number of conidia in the control (Figure 3C) could possibly be the result of the fungus's defense mechanisms due to ethanol's aggressive effect.According to Han (2022), microorganisms can change the morphology or increase the growth rate, as well as the reduction or increase of conidia in the face of abiotic factors.
Figure 4 shows optical microscopy images of the Botryosphaeria fungus after exposure to the alcoholic extract of M. quadrifasciata propolis and 96 % v/v ethanol.When evaluating the fungus Botryosphaeria, it was also possible to verify a significant reduction in conidia in the control treatment with ethanol (Figure 4C), followed by the alcoholic extract treatment of M. quadrifasciata propolis (Figure 4B), compared to the PDA culture medium alone (Figure 4A), where a large number of conidia of this fungus can be observed.
Figure 5 shows optical microscopy images of the fungus S. sclerotiorum after exposure to the alcoholic extract of T. angustula propolis and 96 % v/v ethanol.From this marker, substantial decreases in conidia number were identified in colonies exposed to propolis extracts and ethanol 96 % v/v compared to colonies grown only with the PDA culture medium.
It is important to highlight here that the diverse and complex chemical composition of the different ethanolic propolis extracts can act synergistically, highlighting phenolic compounds as the main constituents of biological activity.According to Costa et al. (2022), exposure to propolis impacts fungal cells, mainly causing a decrease in the number of CFU and a visible reduction in hyphae, observations that align with the results obtained in this work.

CONCLUSION
It was possible to verify that ethanolic propolis extracts from different NSB species showed inhibitory activity and promising potential as an alternative to the indiscriminate use of synthetic pesticides.However, other studies are necessary to test the stability of the process due to the uncertainty regarding the safe concentrations of alcoholic propolis extract and its possible phytotoxic effects in future in vivo evaluations.Therefore, the use of propolis-based compounds and new in vivo tests need to be encouraged and further explored.

7 2 . 5
EVALUATION OF THE ANTIFUNGAL ACTIVITY OF PROPOLIS EXTRACTSThe antifungal activity of the extracts was evaluated on the mycelial growth of the phytopathogenic fungi Sclerotinia sclerotiorum, isolated from lettuce, and Fusarium sp., Colletotrichum gloeosporioides, Botrytis cinerea, and Botryosphaeria sp., isolated from grapevine cultivation.
Means followed by the same letter, uppercase in a row and lowercase in a column, do not differ statistically according to the Tukey test at a 5 % error probability.Coefficient of variation: 8.39 %.Source: authors (2024)According toOzan et al. (2007), the most prominent inhibition of the growth of Fusarium spp.species was at a concentration of 1.0 mg•L -1 of the ethanolic extract of Apis mellifera propolis, whose identified antifungal properties were mainly attributed to compounds Inhibitory Activity of Propolis Extracts From Stingless Bees (Meliponini) on the Development of Phytopathogenic Fungi: in Vitro Evaluation ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.7 | p.1-25 | e07446 | 2024.
Means followed by the same letter, uppercase in a row and lowercase in a column, do not differ statistically according to the Tukey test at a 5 % error probability.Coefficient of variation: 14.11 %.Source: authors (2024)

3. 2
MICROSCOPIC EVALUATION OF THE MORPHOLOGY OF FUNGI EXPOSED TO DIFFERENT PROPOLIS EXTRACTS Optical microscopy tests were carried out to verify possible morphological changes in the fungal hyphae and the presence and absence of conidia, evaluated with different alcoholic extracts of propolis and 96% v/v ethanol (control).After cultivation in an incubator at 26 °C for 14 days, the hyphae were removed from the edge of the fungal colony and observed under an optical microscope to observe the morphology and structure of the hyphae and conidia.A concentration of 3.2 % v/v was chosen for the tests as it presented an inhibition factor in all different tests but with the possibility of performing optical microscopy.Alcoholic propolis Inhibitory Activity of Propolis Extracts From Stingless Bees (Meliponini) on the Development of Phytopathogenic Fungi: in Vitro Evaluation ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.7 | p.1-25 | e07446 | 2024.16 extracts were chosen according to the highest degree of effectiveness shown in each test.The other images can be found in the supplementary material.

Figure 1
Figure 1Optical microscopy images (100 µm) to visualize changes in the structure and morphology of the Botrytis fungus cinerea after exposure to the alcoholic extract of M. quadrifasciata propolis and 96 % v/v ethanol, both at a concentration of 3.2 % v/v.A -growth of the fungus in PDA medium; B -exposure to M. quadrifasciata propolis extract; C -exposure to ethanol96 % v/v   (control)

Figure 2
Figure 2 Optical microscopy images (100 µm) to visualize changes in the structure and morphology of the fungus C. gloeosporioides after exposure to the alcoholic extract of S. bipunctata propolis and ethanol 96 % v/v, both at a concentration of 3.2 % v/v.A -growth of the fungus in PDA medium; B -exposure to S. bipunctata propolis extract; C -exposure to ethanol 96 % v/v (control).

Figure 3
Figure 3 Optical microscopy images (100 µm) to visualize changes in the structure and morphology of the fungus Fusarium sp. after exposure to the alcoholic extract of M. quadrifasciata propolis and ethanol 96 % v/v, both at a concentration of 3.2 % v/v.A -growth of the fungus in PDA medium; B -exposure to M. quadrifasciata propolis extract; C -exposure to ethanol 96 % v/v (control).

Figure 4
Figure 4Optical microscopy images (100 µm) to visualize changes in the structure and morphology of the Botryosphaeria fungus after exposure to the alcoholic extract of M. quadrifasciata propolis and ethanol 96 % v/v, both at a concentration of 3.2 % v/v.A -growth of the fungus in PDA medium; B -exposure to M. quadrifasciata propolis extract; C -exposure to ethanol96 % v/v   (control)

Figure 5
Figure 5Optical microscopy images (100 µm) to visualize changes in the structure and morphology of the fungus S. sclerotiorum after exposure to the alcoholic extract of T. angustula propolis and ethanol 96 % v/v, both at a concentration of 1.6 % v/v.A -growth of the fungus in PDA medium; B -exposure to T. angustula propolis extract; C -exposure to ethanol 96 % v/v (control).
the S. sclerotiorum hyphae, it is possible to observe a normal and uniform morphology in the control treatment with BDA (Figure4).However, when evaluated in the Inhibitory Activity of Propolis Extracts From Stingless Bees (Meliponini) on the Development of Phytopathogenic Fungi: in Vitro Evaluation ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.7 | p.1-25 | e07446 | 2024.20 presence of T. angustula propolis extract at a concentration of 1.6 % v/v, it is possible to verify changes such as thickened hyphae.When observed in the control treatment with ethanol 96 % v/v, a reduced number of hyphae in the hyphal network could be identified.Optical microscopy images are important markers for understanding the effectiveness of propolis extracts on the morphology and reproductive structures of phytopathogenic fungi.

Table 2
Contents equivalents of gallic acid; EQ -quercetin gram equivalents.Column means followed by the same letter do not differ statistically using the Tukey test at a 5 % error probability.
of total phenolic compounds, total flavonoids, and antioxidant activity of alcoholic extracts of stingless bee propolisSource: authors (2024)

Table 4
Percentage (%) inhibition of Botrytis mycelial growth cinerea exposed to increasing concentrations of NSB propolis extracts evaluated 14 days after inoculation.AA 100.0±0.0AA 100.0±0.0AA 100.0±0.0AA Means followed by the same letter, uppercase in a row and lowercase in a column, do not differ statistically according to the Tukey test at a 5 % error probability.Coefficient of variation: 12.83 %.Source: authors (2024) Inhibitory Activity of Propolis Extracts From Stingless Bees (Meliponini) on the Development of Phytopathogenic Fungi: in Vitro Evaluation 6 % and 3.2 % v/v of the different propolis extracts assessed in this study.The results of inhibition of B. cinerea by propolis extracts, evaluated at increasing concentrations, are presented in Table 4. ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.7 | p.1-25 | e07446 | 2024.

Table 5
Percentage (%) of inhibition of mycelial growth of Colletotrichum gloeosporioides exposed toincreasing concentrations of NSB propolis extracts evaluated after 14 days of inoculation.AA Means followed by the same letter, uppercase in a row and lowercase in a column, do not differ statistically according to the Tukey test at a 5 % error probability.Coefficient of variation: 12.60 %.Source: authors (2024) Inhibitory Activity of Propolis Extracts From Stingless Bees (Meliponini) on the Development of Phytopathogenic Fungi: in Vitro Evaluation ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.7 | p.1-25 | e07446 | 2024.

Table 7
Percentage (%) of inhibition of mycelial growth of Botryosphaeria sp.exposed to increasing concentrations of NSB propolis extracts evaluated 14 days after inoculation.AA Means followed by the same letter, uppercase in a row and lowercase in a column, do not differ statistically according to the Tukey test at a 5% error probability.Coefficient of variation: 7.68 %.

Table 8
Percentage (%) of inhibition of mycelial growth of Sclerotinia sclerotiorum exposed to increasing concentrations of NSB propolis extracts evaluated after 14 days of inoculation.