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The in vitro experiments were conducted at the Laboratory of Agricultural Microbiology and Phytopathology of the Federal University of Grande Dourados (UFGD), Dourados, Mato Grosso do Sul, from September to December 2013.

Mycelial sensitivity and sporulation of C. gloeosporioides in response to natural products and a fungicide

The fungus C. gloeosporioides was isolated from disease lesions in “Formosa” papaya fruits and cultivated in potato-dextrose-agar (PDA) medium for six days at 25ºC, with a 12h photoperiod. After fungal colony growth, consecutive repetitions were made until pure cultures were obtained. The confirmation of fungus identification was based on its morphological characteristics, per ^{TARNOWSKI (2009}) and ^{TORRES-CALZADA et al. (2012}), through slide preparation and microscopic observations.

We used a completely randomized design, with ten treatments and eight replications. For evaluation of mycelial growth, we implemented a 10 × 4 factorial scheme, with ten treatments and four incubation periods (2, 4, 6, and 8 days). The treatments were aqueous extract at concentrations of 5 and 15% of clove, cinnamon and ginger, 1 and 3% chitosan, the fungicide Prochloraz at 100 µg.mL^-1, and a control (no treatment). The concentrations established for the extracts were based on the results of previous tests.

Ginger rhizome, cinnamon bark, and clove bud were used to prepare the aqueous extracts. Thirty grams of plant material were ground in 120 mL of distilled water for 1 min. After grinding, the material was filtered through a sieve, gauze, and cotton, conditioned in an Erlenmeyer flask, and kept in a water bath at 65ºC for 60 min to decontaminate. From this solution, the necessary volume was subsequently removed to obtain a concentration of 5 and 15% of the plant extract in the PDA culture medium.

One and three percent chitosan solutions were prepared and autoclaved at 15 psi for 15 min, per ^{HERNÁNDEZ et al. (2011}).

The extract, chitosan, and fungicide solutions were added to the PDA culture media, to obtain the predetermined concentrations. After homogenization of the media, they were poured into previously identified Petri dishes, where, after solidification, 0.5 cm of C. gloeosporioides mycelium discs were transferred from cultures after 12 days of incubation. Then, the Petri dishes were sealed with plastic film and incubated in a BOD incubator at 25ºC, with a 12h photoperiod.

The evaluations were performed every two days for eight days; we measured mycelial diameter growth in two directions, perpendicularly, with a graduated ruler graduated in millimeters (mm).

At the end of the evaluation of mycelial growth, sporulation of C. gloeosporioides was also evaluated. For each repetition, a suspension of conidia was prepared by placing 10 mL of sterile distilled water plus Tween 20 (0.1 mL.mL^-1) on the surface of the Petri dish with fungal mycelium, smoothly scraping it with Drigalsky’s handle. The suspension was filtered through a double layer of sterile gauze to count conidia in a hemocytometer.

The mycelial growth and sporulation data were submitted to analysis of variance. The average values, when significant, were compared by Tukey’s test and the average of the evaluation periods for mycelial growth was adjusted by the regression analysis, at 5% level of significance.

Sensitivity of C. gloeosporioides conidia to natural products and a fungicide

For the evaluation of inhibition of spore germination, a completely randomized design was used, with six treatments and five replications. The treatments were 7.5% aqueous extract of clove, cinnamon and ginger, 1.5% chitosan, the fungicide Prochloraz at 50 µg.mL^-1, and control (sterilized distilled water).

After 12 days of propagation, the suspension of conidia in the culture was adjusted to 1.4 × 10⁵ conidia.mL^-1, using a hemocytometer. The spore germination test followed the methodology described by ^{TAVARES; SOUZA (2005}).

The conidia germination data were submitted to analysis of variance, and, when significance was found, the averages were compared by Tukey’s test at 5% level of significance.

Effect of natural products and fungicide on the control of C. gloeosporioides during post-harvest of “Formosa” papaya “Tainung 1”

“Formosa” papaya “Tainung 1” from a traditional growing area, in the municipality of Ivinhema, Mato Grosso do Sul, were harvested in May 2014. We selected fruits at stage 2 of ripeness (up to 25% yellow surface), per ^{COSTA et al. (2010}).

After being harvested, the fruits were wrapped in newspaper and packed in plastic boxes, and then transported to the Laboratory of Agricultural Microbiology and Phytopathology at UFGD, in Dourados, Mato Grosso do Sul, where they were classified. We discarded those with lesions or inadequate coloration, to standardize maturation stage and fruit quality. The fruits were washed with water and detergent, and kept at room temperature until reaching 50% yellow surface; then, they were submitted to the treatments.

The fruits were immersed in sterilized distilled water (control), aqueous extracts (clove, cinnamon, and ginger) at 15% concentration and 8% chitosan for 5 min, while immersion in the fungicide Prochloraz, at a concentration of 33.75 g a.i./100 L, was carried out for 2 min. In both cases, drying occurred naturally.

The plant extract solutions were prepared using the methodology described in the in vitro experiment for mycelial sensitivity of C. gloeosporioides. The 8% chitosan solution was prepared by dissolving 800 g of chitosan in 10,000 mL of distilled water, then heating at 100ºC with constant stirring for 5h. During the preparation, the solution was adjusted and maintained at pH 5.5 by the addition of acetic acid P.A.

After the treatments, the fruits were packed in polystyrene cups with the stem region facing upwards, inoculated with C. gloeosporioides, per ^{NERY-SILVA et al. (2007}), and stored in a temperature-controlled chamber at 25ºC with a 12h photoperiod.

The evaluations were performed after 2, 4, and 6 days of inoculation, and scores were given based on the severity of stem-end-rot symptoms, as suggested by ^{NERY-SILVA et al. (2007}):

A completely randomized design was used, in a 6 × 3 factorial design, with six treatments and three evaluation periods (2, 4, and 6 days), with four replicates per treatment. The data were submitted to analysis of variance and, when significance was found, the averages between the treatments were compared by Tukey’s test at 5% level of significance.

Mycelial sensitivity and sporulation of C. gloeosporioides in response to natural products and a fungicide

Mycelial growth was completely inhibited by treatments with 5 and 15% clove, 1 and 3% chitosan, and the fungicide Prochloraz, which differed from the other treatments (Fig. 1). In the treatments in which mycelial growth occurred, the highest averages were obtained in the control, 5% ginger, and 5% cinnamon; no significant difference was observed during the evaluation period. However, at the 15% concentration, the two extracts resulted in lower mycelial growth. The effect of 15% ginger on mycelial growth was significantly greater than the treatment with 15% cinnamon from the first evaluation.

Figure 1. Mycelial growth (cm) of Colletotrichum gloeosporioides under the effect of treatments: control (Cont.); 5% ginger (Ging. 5); 15% ginger (Ging. 15); 5% cinnamon (Cin. 5); 15% cinnamon (Cin. 15); 5% clove (Clo. 5); 15% clove (Clo. 15); 1% Chitosan (Ch. 1); 3% Chitosan (Ch. 3) and fungicide Prochloraz (FgP). Dourados, Mato Grosso do Sul, UFGD, 2014. 

Inhibition of mycelial growth by ginger extract was also confirmed by ^{BRITO; NASCIMENTO (2015}): they found that the 5% concentration of ginger extract provided 70% inhibition of mycelial growth of Curvularia eragrostidis. According to ^{DIEMER (2016}), it was not possible to relate the antimicrobial activity of ginger only to the amounts found for the major constituents (α-zingiberene, geranial, β-bisabolene, neral, and β-sesquiphellandrene). Therefore, these may be related to the synergistic effect between the constituents.

Inhibition of mycelial growth by cinnamon extract was also observed by ^{VENTUROSO et al. (2011}). They found higher antifungal activity in the highest concentrations of cinnamon aqueous extract (10 and 20%), in vitro, without total inhibition on the phytopathogenic fungi studied, including Colletotrichum spp. The antimicrobial action of cinnamon extract is related to cinnamaldehyde, its main component (^{BERALDO et al., 2013}).

The results obtained with the Prochloraz treatment concur with those obtained by ^{TAVARES; SOUZA (2005}). These authors also verified, in vitro, 100% inhibition of mycelial growth of C. gloeosporioides, with 10 µg.mL^-1 of Prochloraz, Propiconazole, and Tebuconazole.

The effectiveness of in vitro mycelial growth inhibition by clove extract and chitosan resembled the fungicide, demonstrating them to be promising alternatives for the control of phytopathogenic fungi. According to ^{SILVA et al. (2009}), the effective antimicrobial action of clove extract is related to the presence of eugenol (83.6%), its main constituent. However, regarding chitosan, according to ^{MAIA et al. (2010}) and ^{FREDDO (2012}), the control efficacy is not only a function of concentration and pathogen but also of factors such as degree of deacetylation, molecular weight, pH, and temperature.

Although sporulation did not occur in treatments with 5 and 15% clove, 1 and 3% chitosan, and the fungicide Prochloraz, it did not differ from the 15% cinnamon and control treatments (Table 1).

The occurrence of higher production of conidia with the highest concentration of ginger was also found by ^{RODRIGUES et al. (2006}). The in vitro study conducted by this author showed that under an aqueous extract of ginger there was greater Helminthosporium spp. conidia production at concentrations of 20 and 40%, compared to those of 1, 5, and 10%.

Greater inhibition of sporulation with 15% cinnamon, compared to 5%, suggests the possibility of complete inhibition with a higher concentration. However, according to ^{ARAUJO et al. (2009}), the control effectiveness of an extract does not only vary by its concentration but also by the species of fungus to be controlled. This was observed with the aqueous extract of 10% cinnamon, which inhibited the sporulation of Penicillium roqueforti, but had no effect on the production of Aspergillus ochraceus spores.

The results obtained with the clove extract are equivalent to those obtained with the fungicide, showing it to be a promising alternative in the control of phytopathogenic fungi. ^{LORENZETTI et al. (2011}), when evaluating the effect of clove essential oil on Botrytis cinerea, found that, although this pathogen had presented mycelial growth, it did not produce spores during the evaluation. This result supports disease control, due to increasing the time required for reproduction. These authors attributed the fungistatic and fungicidal effect to the presence of eugenol, which is the major component of clove.

The inhibition of sporulation by chitosan was also observed by ^{BARRETO (2016}). It was verified that the incorporation of chitosan at 4 mg.mL^-1 in culture media with Aspergillus niger and Rhizopus stolonifer inhibited sporulation of both to less than 50%, which differed from the control. However, the association of chitosan and oregano oil (Origanum vulgare L.) provided 100% inhibition of sporulation. Similar results were also obtained for spore germination and mycelial growth. ^{SILVA et al. (2015}) found that the in vivo use of 2% chitosan reduced the spore concentration and the production of Aspergillus parasiticus in peanuts, and promoted, in vitro, morphological changes in the spores, such as swelling, larger diameter (20 µm), and absence of spicules. According to ^{COQUEIRO; DI PIERO (2011}), the exact mechanism of action on the pathogen is not yet fully understood. However, it is believed that its cationic nature contributes to the performance of chitosan, since its positive charges can interact with the negatively charged residues of macromolecules exposed on the surface of the pathogen cell, altering cell membrane permeability.

Sensitivity of C. gloeosporioides conidia to natural products and a fungicide

The germination of C. gloeosporioides spores presented high sensitivity to treatments with 1.5% chitosan, 7.5% clove, and the fungicide Prochloraz; these treatments presented significantly lower average values than the other treatments. The germination of conidia exposed to 7.5% cinnamon extract was significantly lower than in the control (Table 2).

The result obtained with 1.5% chitosan was also found by ^{MAIA et al. (2010}). They found that treatment with 160 mg.L^-1 of chitosan reduced the germination of spores of Elsinoe ampelina by 98%, 12 and 24h after incubation. Inhibition of spore germ tubes by chitosan is related to the modification of plasma membrane permeability, making it difficult for nutrients to enter the cells (^{DI PIERO; GARDA, 2008}).

The sensitivity observed with the 7.5% clove treatment was also shown by ^{LUCAS (2012}), who observed complete inhibition of Alternaria solani conidia germination, under the effect of clove essential oil in vitro. ^{CELOTO et al. (2008}) found that aqueous extracts provided a greater percentage of inhibition of C. gloeosporioides spore germination, in relation to hydroethanolic extracts.

The inhibition verified with 7.5% aqueous cinnamon extract was also found by ^{MAMPRIM et al. (2013}) with M. anisopliae. When spraying aqueous extract of 10% cinnamon on the fungus that was previously inoculated in PDA culture medium, they verified that conidia germination was reduced by 24%. ^{LIMA et al. (2012}) observed that with the increase in cinnamon aqueous extract concentration (10, 25, 50, and 100%), there was a reduction in Aspergillus flavus spore germination.

The germination of spores shown with 7.5% ginger extract was also observed by ^{CELOTO et al. (2008}), who found that the 20% aqueous extract of ginger did not inhibit C. gloeosporioides germination in vitro.

The efficacy observed in the fungicide treatment was also obtained by ^{TAVARES; SOUZA (2005}); their study showed that the fungicide Prochloraz, in the concentration of 100 µg.mL^-1, provided 100% inhibition of the C. gloeosporioides spore germination.

Effect of natural products and fungicide on the control of C. gloeosporioides in post-harvest “Formosa” papaya “Tainung 1”

In vivo evaluations showed no significant differences between treatments on the second day of evaluation. However, anthracnose control was verified by treatments with 15% clove, 8% chitosan, and Prochloraz on the fourth and sixth day. The treatment with 15% cinnamon presented intermediate control, differing from the control on the fourth day. The 15% ginger treatment did not differ from the control group on the fourth and sixth day of evaluation, showing the same pattern of lesion growth throughout the evaluations; and the severity of the disease increased between the second and fourth day, without differing between the fourth and sixth day. However, in treatments where there was greater control of the disease, the severity increased significantly after the second evaluation (Table 3).

The lower efficacy of the treatment with 15% ginger extract indicates the control indexes shown in the in vitro experiments, in relation to the other treatments. However, ^{STANGARLIN et al. (1999}), verifying the elicitor effect of phytoalexins in bioassays with sorghum and soybean, found that the raw aqueous extract of ginger had the capacity to activate defense mechanisms in these plants.

The intermediate control obtained with the 15% cinnamon extract indicates the possibility of obtaining a better result with a higher concentration. In the literature, no studies evaluating the phytopathogenicity of cinnamon aqueous extract were found in vivo, but only in vitro. However, there are in vivo studies with cinnamon essential oil that have demonstrated its antimicrobial action. ^{ANDRADE; VIEIRA (2016}) observed that the low efficacy of the cinnamon essential oil in the post-harvest control of C. gloeosporioides in “Golden” papaya was due to low dosage (100 µL), since there was gradual inhibition of this fungus with increased concentrations.

The inhibition of the growth of C. gloeosporioides lesions found in the 8% chitosan treatment is related, according to ^{MAIA et al. (2010}), to the fungistatic effect of chitosan and its potential to activate enzymes and phenolic compounds related to the plant defense mechanism.

Although our study demonstrated anthracnose control with the 15% clove treatment, there are no studies that demonstrate the in vivo behavior of clove in the control of phytopathogens. However, in vitro studies by ^{COSTA et al. (2011}) and ^{LUCAS (2012}) show that eugenol and β-caryophyllene act on the degradation of the cell wall, with morphological changes in fungal mycelium, disorganization of cellular contents, intense fragmentation, and less turgidity of the hyphae.