Bioactivity of Ethanolic Extracts of Dipteryx punctata on Colletotrichum musae

Abstract

Bioactivity with antifungal properties has already been reported for some species of the genus Dipteryx. However, little is known about Dipteryx punctata. We evaluated the antifungal activity, in vitro and in vivo, of extracts of leaves, branches and fruits of D. punctata on Colletotrichum musae, the causal agent of anthracnose in bananas. The extracts and the coumarin standard were tested in vitro, at concentrations of 10%, 20%, 30%, 40%, and 50% (w/v), added in potato-dextrose-agar (PDA) medium. The experimental design was completely randomized, factorial design, with four replications. The average diameter of the fungal colonies was evaluated daily for eight days, and at the end, the percentage of inhibition and growth rates were calculated. For in vivo tests, the products were tested at concentrations of 40% and 50%, in preventive and curative applications. In these tests, three medium disks (5 mm) containing phytopathogen structures were deposited at opposite points of the fruits, and remained in a humid chamber for 48 h. Anthracnose lesions were measured at an interval of two days, up to eight days after fungus inoculation. In the in vitro essay, the extracts of the branches and residues of the fruits of D. punctata and the coumarin pattern caused the biggest reductions in the average diameter of the colonies of the phytopathogen. D. punctata and coumarin extracts were promising in terms of in vivo antifungal activity, especially in preventive applications, being an important source of investigation for the formulation of natural products as sustainable phytosanitary measures.

1. Introduction

Banana (Musa spp.) is one of the most important fresh fruits in the world food market and a source of income for millions of rural families in developing economies. Data pointed to a record 21,718,000 t of global banana exports in 2019, and this strong supply growth was driven by countries in Latin America and the Caribbean, followed by Asia and Africa [1]. However, the Food and Agriculture Organization of the United Nations (FAO) infers in its reports that during the restrictions arising from the COVID-19 pandemic, there was an increase in the costs of inputs, packaging and transport, production shortages due to adverse weather conditions and the spread of diseases, leading to reductions in banana exports in 2020 (21,542.000 t) and 2021, with approximately 20 million tons [1].

As for the diseases that affect banana trees, Banana Fusarium Wilt Tropical Race 4 (TR4) stands out, caused by the soil fungus Fusarium oxysporum f. sp. cubense (W.C. Snyder & H.N. Hansen), currently confirmed in 23 banana producing countries, predominantly in South and Southeast Asia, but also in the Middle East, Africa and Latin America, with Colombia reporting the first infection in August 2019 and the second in Peru in April 2021. To date, no fungicide or other eradication method available has been effective in controlling it, and the greater the spread of TR4, the greater the economic impacts on global banana production and trade, which would lead to considerable losses of income and employment in the affected countries, as well as significantly higher costs for the consumer on importation [1]. Additionally, anthracnose, caused by fungi of the genus Colletotrichum that attacks several fruit species, such as papaya (Carica papaya L.), mango (Mangifera indica L.), cashew (Anacardium occidentale L.), passion fruit (Passiflora edulis Sims) [2] and guava (Psidium guajava L.) [3].

The species Colletotrichum musae (Berk & Curt.) von Arx is the cause of anthracnose in bananas, being favored in the field by high temperature and relative humidity that contribute to the development of the phytopathogen [4]. Defoliation, drying of branches and the decomposition of fruits occur in the hottest and wettest periods [5], characterized by large rounded, necrotic lesions, containing depressed tissues in the center, where masses of orange-colored conidia are produced, causing soft rot in the fruits [3].

The most commonly employed method for controlling the disease is chemical. However, the application of fungicides, used alone, unsatisfactorily reduces the losses caused by anthracnose [6]. Additionally, the continuous and intensive use of these products can interrupt natural biological control since non-target organisms are affected [7], select resistant pathogens, causing disease outbreaks [8], making production costs unfeasible for some farmers [5] and limiting exports due to increased restrictions on maximum levels of chemical residues in food [1].

Considering that the activities of natural compounds play a crucial role in the maintenance of human health and environmental conservation [9], the search for alternative methods to control anthracnose, such as oils and plant extracts, has indicated the potential of these products to control phytopathogens [10], both by direct fungicide action, through inhibition of mycelial growth and spore germination, and by induction of phytoalexins [11,12].

The species Dipteryx punctata belongs to the Fabaceae family and has relevant chemical compounds, such as coumarin (1,2-benzopyrone) [13,14], to be analyzed for their biological potential. In view of the above, we evaluated, in vitro and in vivo, the antifungal activity of extracts from leaves, branches, residues and seeds of D. punctata fruits, on Colletotrichum musae, the causal agent of anthracnose in bananas.

2. Materials and Methods

2.1. Collection and Obtaining of Plant Extracts from Dipteryx punctata

The plant material to obtain the extracts was collected in five agroforestry systems (AFS) implemented and monitored by the Technical Assistance and Rural Extension Company of the State of Pará (Emater), in the rural communities of Água Fria, Terra de Areia and Boa Fé, located in the municipality of Mojuí dos Campos, Eastern Amazon, State of Pará, Brazil.

Seven trees were sampled in each area (35 trees), and leaves, branches and flowers were collected in the rainy season in the Amazon (February to March), and 1050 fruits (30 fruits per tree; 210 fruits per area) in the dry season (September).

Exsiccates were prepared, botanically identified as Dipteryx punctata and deposited in the Herbarium of the Universidade Federal do Oeste do Pará: HSTM 11897, HSTM 11898, HSTM 11899, HSTM 11900 and HSTM 11901 and, in the Herbarium of the Botanical Garden of Rio de Janeiro: RB 772255, RB 772253, RB 772256, RB 772257 and RB 772254. The research was registered in the National System for the Management of Genetic Resources and Associated Traditional Knowledge (SisGen, Brazil), protocol A1B0150.

The plant materials were separated into leaves, branches and fruits, the latter into residues (epicarp + mesocarp + endocarp) and seeds, dried in an oven at 45 °C, with forced air circulation for 20 days, crushed and weighed, obtaining 70 g of leaves, 35 g of branches, 70 g of residues and 35 g of seeds for each extraction.

Extractions were performed in triplicate via Soxhlet, using 92.8% ethyl alcohol (distilled with sodium hydroxide, 2 g to 2 L, for 4 h). Each procedure lasted 8 h and after the processes, the solvent was removed from the extractive solutions in a rotary evaporator and by drying at room temperature (±27 °C). The dried ethanolic extracts were stored in sterilized amber glass containers [15].

2.2. Isolate Obtention of Colletotrichum musae

The fungus C. musae was obtained from commercialized banana fruits showing symptoms of anthracnose using the direct isolation method [16]. Microscopic slides were prepared to confirm the identification of the fungus through the morphology of the conidia [17], and axenic cultures obtained in PDA medium to perform the pathogenicity test and the in vitro and in vivo essays.

2.3. Antifungal Activity, In Vitro, of Dipteryx punctata and Coumarin Extracts

We evaluated the effect of the ethanolic extracts of D. punctata and the standard of coumarin (1,2-benzopyrone-Dinâmica^®) on the phytopathogen using the means of the addition of these products in PDA culture media, adjusted at concentrations of 10%, 20%, 30%, 40%, and 50%, homogenized and poured into Petri dishes (90 × 15 mm). After solidification of the culture medium, a medium disc (5 mm) containing structures of the phytopathogen [18] was deposited centrally, which was incubated at 25 °C, under a 12 h photoperiod. The control treatment consisted of deposition of the fungus only in PDA. The experimental design was completely randomized, in a factorial scheme (5 × 5 × 6), with five collection areas, five products (four extracts and coumarin) and six concentrations (including the control), with four replications (one plate per repetition).

The evaluations were carried out by measuring the average diameter of the colonies, daily, with the aid of a digital caliper, for eight days. With the data on the average diameter of the colonies, the percentage of inhibition of growth (PIG), the mycelial growth speed index (MGSI) and the growth rate (Gr) were determined.

The PIG was calculated by the difference between the mean diameter of the control colony (C) and the mean diameter of the treatment colony (T), divided by the mean diameter of the control colony multiplied by 100 [19]:

(PIG = (C−T/ C) × 100).

With high anti-fungal activity being considered when the extracts provided inhibition equal to or greater than 50% [20].

The MGSI (mm/day) was determined by the formula [21]:

MGSI = Σ (D−Da)/N, where: D = current average colony diameter; Da = mean colony diameter from the previous day; N = number of days after inoculation.

The Gr by the ratio between the final colony diameter (Df) and the number of days of incubation (ND), multiplied by 100 [22]:

(Gr = Df/ND × 100).

2.4. Antifungal Activity, In Vivo, of Dipteryx punctata and Coumarin Extracts

The effects of the products under in vivo conditions were evaluated in preventive and curative trials [23]. Healthy banana fruits were previously surface disinfected with sodium hypochlorite (NaClO) at 2% a.i. for two minutes, washed with sterile distilled water and dried on sterile filter paper. The experimental design was completely randomized, in a factorial scheme (5 × 5 × 3), with five collection areas, five products (four extracts and coumarin) and three concentrations (including the control), with three replications (one banana fruit per replication).

In the preventive application test, the banana fruits were manually sprayed with the extracts and coumarin, at concentrations of 40% and 50%, pre-selected in the in vitro test. For the control treatment, the fruits were sprayed with water. After the application and natural drying of the products on the 225 bananas (a simultaneous process that took place for approximately one hour), three superficial wounds were made on each fruit (opposite points), with sterilized needles, on which a disk of PDA medium (5 mm) containing somatic and reproductive structures of the phytopathogen was deposited. The inoculated fruits were submitted to a humid chamber for 48 h and incubated at 25 °C, under a photoperiod of 12 h. After the onset of symptoms, anthracnose lesions were measured with the aid of a digital caliper, at intervals of two days, up to eight days after inoculation.

For the curative application test, superficial wounds were first made on the disinfected fruits and inoculation of the phytopathogen similar to the preventive test. After 48 h of incubation, the initial symptoms of anthracnose were observed, the lesions present in the fruits were measured and then the extracts, coumarin and water (control) were sprayed. Incubation and evaluations were carried out in the same way as the preventive application test.

2.5. Statistical Analysis

With the data obtained in the tests, analysis of variance and comparison between the means of the treatments were performed using the Tukey test (p ≤ 0.05), using the statistical software SISVAR 5.6 [24]. The MGSI and Gr data were also used for simple linear regression analysis.

3. Results

3.1. Antifungal Activity, In Vitro, of Dipteryx punctata and Coumarin Extracts

In the evaluation of the average diameter of C. musae colonies, there was a significant difference both for the factors alone and for the interaction between them (areas x parts of the plant x concentrations).

All ethanol extracts and coumarin reduced the mycelial growth of the pathogen, regardless of the concentration and origin of the evaluated plant material (

Table 1. Mean diameter of Colletotrichum musae colonies subjected to ethanolic extracts of Dipteryx punctata and to coumarin standard, at different concentrations.

Extracts	Concentrations (%)	Mean Diameter of the Pathogen Colonies (mm)
		Area 1	Area 2	Area 3	Area 4	Area 5
Control	0	65.2 aA	65.2 aA	65.2 aA	65.2 aA	65.2 aA
Leaves	10	41.7 bB	49.0 bA	42.8 cB	42.7 cB	40.5 cB
	20	39.2 dB	44.4 bA	38.1 dB	37.2 eB	37.5 dB
	30	37.4 eB	40.4 cA	35.4 eB	34.8 eB	35.7 eB
	40	35.9 eB	38.0 dA	32.0 fC	31.2 gC	33.8 fB
	50	33.5 fA	34.8 fA	29.0 hB	28.5 hB	29.4 hB
Branches	10	38.9 dB	37.0 eC	40.8 cB	43.2 bA	43.0 bA
	20	37.4 eB	31.3 gD	36.3 eC	40.6 dA	39.8 cA
	30	33.1 fB	29.6 gC	32.2 fB	37.0 eA	36.1 eA
	40	32.1 gB	24.0 hD	29.7 hC	34.3 fA	35.2 eA
	50	27.9 hB	22.2 iD	26.4 hC	32.8 fA	29.7 hB
Residues	10	36.1 eC	41.9 cB	45.5 bA	47.5 bA	47.7 bA
	20	35.0 eB	41.7 cA	41.2 cA	41.2 cA	40.6 cA
	30	30.7 gC	38.3 dB	41.1 cA	38.4 dB	37.7 dB
	40	22.9 hC	31.0 gB	38.4 dA	36.3 eA	36.7 dA
	50	18.6 iD	24.0 hC	32.2 fB	34.5 fA	30.1 gB
Seeds	10	39.0 dC	43.0 bA	44.9 bA	44.2 bA	41.5 cB
	20	36.8 eB	39.6 cA	40.9 cA	38.7 dA	39.8 cA
	30	34.9 eB	37.3 dA	38.2 dA	37.8 dA	37.6 dA
	40	34.4 fB	35.8 eB	37.8 dA	33.0 fC	36.3 eB
	50	32.4 fA	33.0 fA	31.2 gA	26.5 hB	31.8 gA
Coumarin	10	41.0 cA	41.0 cA	41.0 cA	41.0 cA	41.0 cA
	20	34.9 eA	34.9 eA	34.9 eA	34.9 eA	34.9 eA
	30	31.9 gA	31.9 gA	31.9 gA	31.9 gA	31.9 gA
	40	26.8 hA	26.8 hA	26.8 hA	26.8 hA	26.8 hA
	50	25.0 hA	25.0 hA	25.0 hA	25.0 hA	25.0 hA
Coefficient of Variation%						2.2

Means followed by the same lowercase letters in the columns and by the same uppercase letters in the rows do not differ from each other by Tukey’s test (p ≤ 0.05).

and Figure 1). When considering the extracts and coumarin, at different concentrations, within each collection area, it was observed that the greatest reductions in the average diameter of C. musae colonies were caused by the extracts from the residues (area 1; concentration 50%), the extract of branches (area 2; concentration 50%), varying in the other collection areas, with more than one product and concentration showing similarities between them (Table 1).

For the leaves extracts, the best results were found for the material collected in areas 3, 4, and 5, with reductions of 36.2 mm, 36.7 mm and 35.8 mm, respectively (concentration of 50%), in relation to the control (Table 1).

When the pathogen was subjected to branch extracts, the reductions in the average diameter of its colonies, in relation to the control, ranged from 26.3 mm to 37.3 mm (area 1) and from 28.2 mm to 43 mm (area 2).

The extracts obtained from D. punctata residues reduced the growth of C. musae (Table 1), with reductions ranging from 30.7 mm (area 4) to 46.6 mm (area 1), at a concentration of 50%, in relation to the control.

The reductions in the mean diameter of the colonies caused by the seed extract varied from 21 mm to 38.7 mm in relation to the control, in area 4 (Table 1). The smallest mean diameters of C. musae colonies caused by coumarin were 24.2 mm at the 10% concentration and 40.2 mm at the 50% concentration, in relation to the control (Table 1).

It was also verified the change in color and irregularity in the growth of colonies in the presence of D. punctata extracts, both in the open plate and in the reversed surface of the plate (Figure 1).

In determining the percentage of inhibition of growth (PIG), the greatest reductions were a PIG of 71.4% (residues extracts; concentration 50%; area 1) and 66% (extracts from branches; concentration 50%; area 2) (

Table 2. Percentage of inhibition of growth (PIG) of Colletotrichum musae submitted to different concentrations of Dipteryx punctata ethanol extracts and coumarin standard.

Extracts	Concentrations (%)	Percentage of Inhibition of Growth (PIG%)
		Area 1	Area 2	Area 3	Area 4	Area 5
Control	0,0	0 aA	0 aA	0 aA	0 aA	0 aA
Leaves	10	36.0 bB	24.8 bA	34.3 cB	34.5 cB	37.9 cB
	20	39.8 dB	31.9 bA	41.6 dB	42.9 eB	42.4 dB
	30	42.6 eB	38.1 cA	45.7 eB	46.6 eB	45.2 eB
	40	45.0 eB	41.8 dA	50.9 fC	52.2 gC	48.2 fB
	50	48.6 fA	46.6 fA	55.6 hB	56.3 hB	54.9 hB
Branches	10	40.3 dB	43.2 eC	37.3 cB	33.8 bA	34.1 bA
	20	42.6 eB	51.9 gD	44.3 eC	37.7 A	38.9 cA
	30	49.3 fB	54.6 gC	50.7 fB	43.2 eA	44.6 eA
	40	50.7 gB	63.1 hD	54.4 hC	47.3 fA	46.0 eA
	50	57.3 hB	66.0 iD	59.5 hC	49.7 fA	54.5 hB
Residues	10	44.6 eC	35.8 cB	30.2 bA	27.1 bA	26.8 bA
	20	46.4 eB	36.0 cA	36.9 cA	36.8 cA	37.7 cA
	30	52.9 gC	41.2 dB	37.0 cA	41.2 dB	42.2 dB
	40	64.9 hC	52.4 gB	41.1 dA	44.3 eA	43.7 dA
	50	71.4 iD	63.3 hC	50.7 fB	47.0 fA	53.8 gB
Seeds	10	40.2 dC	34.1 bA	31.2 bA	32.3 bA	36.4 cB
	20	43.6 eB	39.2 cA	37.3 cA	40.6 dA	39.0 cA
	30	46.4 eB	42.7 dA	41.3 dA	42.0 dA	42.3 dA
	40	47.3 fB	45.1 eB	42.0 dA	49.4 fC	44.4 eB
	50	50.3 fA	49.4 fA	52.2 gA	59.4 hB	51.2 gA
Coumarin	10	37.1 cA	37.1 cA	37.1 cA	37.1 cA	37.1 cA
	20	46.5 eA	46.5 eA	46.5 eA	46.5 eA	46.5 eA
	30	51.1 gA	51.1 gA	51.1 gA	51.1 gA	51.1 gA
	40	58.8 hA	58.8 hA	58.8 hA	58.8 hA	58.8 hA
	50	61.6 hA	61.6 hA	61.6 hA	61.6 hA	61.6 hA
Coefficient of Variation %						4.5

Means followed by the same lowercase letters in the columns and by the same uppercase letters in the rows do not differ from each other by Tukey’s test (p ≤ 0.05).

).

The percentage of inhibition of C. musae growth in the other treatments ranged from 46.6% to 63.3% at the highest concentration and from 24.8% to 44.6% at the lowest tested concentrations of the different products. Only at the concentration of 10% for the extract of leaves (area 2) and extract of residues (areas 3, 4 and 5) was the PIG not higher than 30% (Table 2).

In relation to the mycelial growth speed index (MGSI), all products evaluated reduced the growth velocity of C. musae. The smallest MGSI was caused by the branches extracts. The reduction caused by this product meant a decrease of 0.6 of the control MGSI. The MGSI observed when C. musae was subjected to cumaru extracts were 1.8 times lower (seeds), 1.7 times (leaves), 1.7 times (residues) and 1.7 times (coumarin) than the growth of fungus in the absence of any extract.

For the extract of leaves and branches, the greatest reductions in MGSI occurred from a concentration of 30%. In the residues, the lowest speed indexes were obtained from the concentration of 20% and, for the seed extracts and for the coumarin pattern, the concentration of 50% was highlighted in the reduction of the growth speed of the pathogen (Figure 2).

The averages of the speed indexes in the best concentrations of the extracts of leaves and branches were 61.4% and 81.6% lower than the growth speed of the control. For the extracts of residues, seeds and coumarin, these indices represented 53.4%, 72.7%, and 64.8% of reduction in the speed of growth of the fungus in relation to the control, respectively.

For each product used in the control of C. musae, first-degree functions (y = ax ± b) were plotted, which describe the behavior of the mycelial growth speed index (MGSI), dependent variable (y), in as a function of the concentrations under test (%), independent variable (x), and the speed index of the control (b), with a decreasing trend in the MGSI for C. musae with increasing concentrations, with the lowest speed indexes of daily growth obtained at concentrations of 40% and 50%, in all ethanolic extracts of D. punctata (Figure 3).

The average MGSI, subjected to leaves extracts, residues and coumarin, was two times slower than the control; for the seeds, it was 2.2 times lower, and for the branches the average index was 3.9 times lower than the growth of the fungus only in PDA medium.

All plant products obtained from D. punctata caused reductions in the mycelial growth rates (Gr) of C. musae that differed from the control. The growth rate of the fungus in the presence of coumarin and submitted to the extracts of the branches was 0.047 mm/day and 0.049 mm/day, respectively. For coumarin, this growth rate was 1.7 times lower than the growth of the fungus only in PDA (control). The extracts from residues, leaves and seeds did not differ significantly from each other, presenting an average rate of 0.052 mm/day.

The mycelial growth rate, evaluated as a function of concentrations, demonstrated that the extracts and coumarin reduced the daily growth of C. musae at all concentrations tested (Figure 4). The higher concentration of products caused reductions above 50% in the growth rate, with emphasis on extracts from branches with 57.4%, residues with 57.2% and for coumarin with 61.6%, when compared to the control.

In the regression analysis of the mycelial growth rate (Gr) of the phytopathogen as a function of the concentrations in test, the extracts and coumarin provided reductions in the rate (Gr) linearly with increasing concentrations (Figure 5), thus emphasizing the inhibitory effect of the products during the days of phytopathogen incubation. The concentrations provided an average growth rate for coumarin 2.0 times lower than the control; for branches, this rate was 1.9 times lower and, for extracts from leaves, residues and seeds, the rates were 1.8 times lower when compared to the control.

3.2. Antifungal Activity, In Vivo, of Dipteryx punctata and Coumarin Extracts

There was a significant difference for the factors alone, as well as the interaction between the plant products and the concentrations evaluated, in the two in vivo essays. The ethanolic extracts of D. punctata and coumarin reduced the mean diameter of the lesions, in relation to the control, both in preventive and curative applications (

Table 3. Mean diameter of anthracnose lesions caused by Colletotrichum musae in banana fruits with preventive application of ethanolic extracts of Dipteryx punctata and the coumarin standard.

Treatments	Concentrations (%)	Mean Diameter of Lesions (mm)
		Leaves	Branches	Residues	Seeds	Coumarin
Control	0	14.8 aA	14.8 aA	14.8 aA	14.8 aA	14.8 aA
Preventive	40	9.2 bA	9.7 bA	9.5 bA	7.7 bA	8.0 bA
	50	8.6 bA	8.7 bA	8.2 bA	7.0 bA	6.6 bA
Coefficient of Variation: 21.47%

Means followed by the same lowercase letters in the columns do not differ from each other by Tukey’s test (≤0.05).

). The origin of the collection material did not influence the effect of the products.

In the preventive application, there was no significant difference in the mean diameter of the lesions between the products and concentrations tested, but there was a difference in these factors in relation to the control treatment (Table 3). Generally, the extracts caused reductions in the size of the lesions on banana fruits, which varied from 34.4% to 48% at a concentration of 40% and from 41.2% to 52.7% at a concentration of 50%, when compared with the control. For coumarin, the variations were 45.9% and 55.4% compared to the control, showing promise in reducing fruit lesions (Figure 6).

In relation to the four evaluations carried out in the preventive trial, the protective effect of the extracts and coumarin (Figure 6) was verified as of the second evaluation, with the treated fruits showing less development of lesions, which is a promising result in terms of the protective action of the fruits (Figure 7).

Similar to what was observed in the preventive application, the evaluated products and concentrations also differed from the control treatment, in the curative application. All extracts, except branches, at a concentration of 50%, caused a greater reduction in the mean diameter of anthracnose lesions, in relation to the concentration of 40% and the control (

Table 4. Mean diameter of anthracnose lesions caused by Colletotrichum musae in banana fruits with curative application of ethanolic extracts of Dipteryx punctata and coumarin pattern.

Treatments	Concentrations (%)	Mean Diameter of Lesions (mm)
		Leaves	Branches	Residues	Seeds	Coumarin
Control	0	31.5 aA	31.5 aA	31.5 aA	31.5 aA	31.5 aA
Curative	40	25.9 bA	26.3 bA	26.3 bA	25.2 bA	19.3 bB
	50	23.5 cAB	25.4 bA	24.5 cAB	23.1 cB	15.7 cC
Coefficient of Variation: 7.87%

Means followed by the same lowercase letters in the columns do not differ from each other by Tukey’s test (≤0.05).

). These reductions corresponded to 25.4% for leaves extracts, 22.2% for residues and 26.7% for seeds. Coumarin was the treatment that resulted in the smallest lesions, with a 50.2% reduction in relation to the control, at a concentration of 50% (Figure 8).

For the curative test, the effect of the concentrations of the treatments in relation to the control was obtained only in the third evaluation, in the concentration of 50%, and in the two concentrations tested in the fourth evaluation performed (Figure 9).

4. Discussion

Evaluating the potential of extracts obtained from different parts of D. punctata plants against anthracnose in banana fruits, it was found that the average diameter of colonies and percentage of inhibition of mycelial growth (PIG%), considered as high antifungal activity when the percentage was greater than 50% [20], were affected by increasing concentrations of plant extracts and the coumarin standard in the in vitro tests. Studies evaluating antifungal activity found this trend of effect in inhibiting the development of phytopathogens with increasing concentrations of plant products in the culture medium [25].

Dipteryx punctata leaves branch extracts contain lup-20(29)-en-3-one and lupeol, which are part of the terpene class. Pharmacologically, this class is a rich source of bioactives, with proven anti-inflammatory, antibacterial, antifungal, and antioxidant activities [26,27]. The activity found in the residues of the fruits of D. punctata is very promising, considering that the residues are discarded after the removal of the seeds for commercialization, being, therefore, an innovative source of destination for this by-product, in addition to contributing to the reduction of environmental contamination.

The variable behavior of the phenotypic characteristics observed in the plates of the same species of Colletotrichum is caused by several factors such as the environmental and temporal conditions of culture in which it was placed [28]. The substances present in the composition of plant products can act synergistically and present broad fungicidal or fungistatic actions [29,30].

The antimicrobial activity of plant extracts on fungal development in vitro and in vivo has been widely studied, and the results demonstrate that both mycelial growth in vitro and banana fruit rot development in vivo can be stimulated or reduced by plant extracts [31]. In vitro, extracts such as those obtained from the leaves of Piper marginatum Jacq. showed an inhibitory effect on C. musae from a concentration of 10% [32], and aqueous extracts of garlic (Allium sativum L.) and cinnamon (Cinnamomum zeylanicum Blume) evaluated in the control of Colletotrichum sp., also provided the smallest colony diameter at the highest concentrations [20]. The aqueous extract of cinnamon was also evaluated in the control of Cercospora kikuchii (T. Matsumoto & Tomoyasu) Gardner, Fusarium solani (Mart.) Sacc., Colletotrichum sp. Corda and Phomopsis sp. (Sacc.) Bubák, linearly reduced the diameter of colonies increasing concentrations [25]. Oil fractions obtained from cumaru seeds (Dipteryx sp.) evaluated on C. musae reduced the mycelial growth of the fungus, especially the dichloromethane fraction, which presented, in addition to coumarin, the compound called 3,4-dihydrocoumarin [33].

For the mycelial growth speed index (MGSI) and the mycelial growth rate (Gr), the reductions found were important, because the slower the development of the pathogen on banana fruits, the smaller the lesions and, consequently, the disease will be less aggressive, which directly implies lower product and economic losses since anthracnose can depreciate the fruit for commercialization. Clove (Eugenia caryophyllata Thunb.), palmarosa (Cymbopogon martini (Roxb.) Will. Watson) and melaleuca or tea tree (Melaleuca alternifolia Cheel) oils were evaluated on mycelial growth and growth rate indices of C. musae, which where efficient in the control, employing a dose of 50 µL/L [34].

In in vivo tests, both preventive and curative applications showed promising results, and in the preventive application, the differences in the reduction of lesions in relation to the control occurred from the second evaluation of the test.

When in an adequate degree of maturation, the banana peel presents ideal protection that guarantees safety and conservation against external agents; however, as well as the hydrolysis of starch that is converted into soluble sugars, which characterizes the sweet taste, reduction of astringency and skin softening, the phenolic compounds are present in bananas, which help in this protection decrease [35]. The action of cumaru and coumarin extracts in the reduction of lesions observed by the preventive application may be linked to the interaction between the compounds present in the extracts and the natural composition of the banana, considering that the studied plant products are rich in phenolic compounds, offering the protection that the bark loses during the ripening process.

This reduction in the intensity of anthracnose conferred by alternative products may also be linked to the favoring of plant resistance, through the production of secondary metabolites and more efficient structural defense mechanisms, such as the presence of bioflavonoids and polymers, precursors of secondary phytoalexins and chitinases compounds, respectively, responsible for plant defense; and by the germicidal action through the rupture of fungal cell membranes [36].

The antifungal action was analyzed in vitro and in vivo simultaneously in many studies, such as the application of extracts of São Caetano melon (Momordica charantia L.), for the control of anthracnose in banana, in which in vitro a reduction in the diameter of colonies with increasing concentrations and in vivo the treatments differed significantly from the control, indicating the presence of antifungal substances, mainly when applied 1 and 24 h before the fungus inoculation, confirming the protective action of the extracts and reduction of the diameter of anthracnose lesions [37]. The antifungal action, in vitro and in vivo, of thyme (Thymus vulgaris L.), lemongrass (Cymbopogon citratus (DC.) Stapf) and oregano (Origanum vulgare L.) oils at 0.1% associated or not with cassava starch skin at 2 and 3%, in the control of anthracnose in banana, showed the best results for oregano oil in the in vitro test, and for the skins isolated and combined with the oils of lemongrass and thyme in the in vivo evaluations; there was also a reduction of lesions in banana fruits with the application of oils alone [38].

In vitro, clove (Eugenia caryophyllus (Spreng.) Bullock & S.G. Harrison) and thyme (Thymus vulgaris) oils were efficient at all concentrations tested in inhibition of mycelial growth, germination and sporulation of C. musae, and under in vivo conditions, the concentration of 160 μL of ginger oil (Zingiber officinale Roscoe) and the concentrations of 160 and 240 μL of tea tree oil (Melaleuca alternifolia) reduced the severity of anthracnose in fruits [39]. Extracts and oils of rosemary pepper (Lippia sidoides Cham.), flower buds of clove (Caryophyllus aromaticus L.) and eucalyptus (Eucalyptus citriodora Hook.) were tested in vitro and in vivo on C. musae, and the results were significant for rosemary pepper and buds of clove, 100% inhibiting in vitro the growth of the pathogen; however, in in vivo tests, the results showed little efficiency, which can be attributed to the low concentrations of the tested products, as well as the volatility of the products of plant origin [40].

The ethanolic extracts of D. punctata and coumarin need to be tested under field conditions, so that the adequacy of extract production and formulation for large-scale use can be carried out; in addition to the toxicological determination to the environment and to humans [37]. Recent research with D. punctata seed extracts showed the greatest decrease in mycelial growth of the evaluated phytopathogens, which is promising as an alternative control [40]. New techniques, times, doses and application intervals also make it possible to identify a safe and effective method for the control of anthracnose in bananas, since only with the adoption of these sustainable measures, which must start in the field up to the final consumer, quality parameters required by the market could be reached [41].

The data obtained are of fundamental importance both for the knowledge about the species D. punctata, and for the consolidation of the effectiveness of natural products as sustainable alternatives for the control of anthracnose in bananas. Additionally, cheaper and less harmful control methods will help reduce production costs and increase the possibility of revenue from sales of ecologically based products.

5. Conclusions

The extracts showed high antifungal activity against Colletotrichum musae, highlighting the extracts from branches, residues and the coumarin pattern.

The results obtained on growth inhibition, mycelial growth speed and mycelial growth rate (Gr), demonstrate the potential of the products evaluated in the control of C. musae, under the conditions used in this research.

The antifungal activity found in the fruit residues has great potential, allowing the reuse and valorization of a plant by-product, rich in bioactive compounds, contributing to the reduction of solid waste disposal in the environment.

The preventive application of the extracts, at the highest concentrations, was more effective in reducing anthracnose lesions in banana fruits.

More in-depth tests are important to confirm the effectiveness of extracts and coumarin under field conditions, as well as to adjust the production and dosage of extracts and determine the toxicity of these products for both the environment and human health. In addition, studies were carried out to evaluate the conservation of the quality of post-harvest bananas on different days of storage, with a view to the best strategy for marketing the fruits.