Materials and methods

This investigation was carried out in the Agricultural Microbiology Laboratory of the Tibaitatá Research Center of the Corporación Colombiana de Investigación Agropecuaria AGROSAVIA.

Plant and pathogenic material. Azúcar mango variety fruits (susceptible to anthracnose) in the stage of physiological maturity (slightly green with a pinkish-yellow tone and a yellow pulp), asymptomatic to any pathology, sized 8 to 10 cm and an average weight of 100 g, from a commercial plantation in the municipal area of Ciénaga were used (department of Magdalena, Colombia). To reduce the expression of quiescent infections of the pathogen, the fruit underwent a hydrothermal treatment at 53 °C for 5 minutes (Memmert WNB 14 waterbath) and tempering by immersion in water at 17 °C for 10 min, followed by producing two lesions, each 2 mm deep, with a sterilized dissection needle on one of the faces of the fruit, with a distance of 5 cm from each other (based on the methodology by ^{Trinidad-Angel et al. (2017}), modified).

To select the pathogen, three isolations were evaluated (Y1, Y2, Y3). Y3 (C. gloeosporioides) was selected from the Yulima variety of mangoes, identified by its morphology and selected due to its pathogenicity when carrying out Koch’s postulates (data not shown). Conidia inoculum was obtained from cultures in Potato Dextrose Agar (PDA, Oxoid^®) previously incubated at 25 °C for 10 days and adjusted to 1x10⁵ conidia mL^-1 in sterile distilled water.

Efficacy of antagonistic microorganisms in the control of anthracnose. The AGROSAVIA Microorganism Germplasm Bank provided the antagonistic microorganisms characterized in previous studies and which displayed control over different pathogens (^{Zapata and Cotes, 2013}; ^{Díaz-García et al., 2015}), which were the bacteria Bacillus amyloliquefaciens Bs006 (^{Gámez et al., 2015}), Bacillus safensis AP280 and Lysinibacillus xylaniticus AP282; and the yeasts Candida oleophila Lv037, Pichia onychis Lv297 and Rhodotorula glutinis Lv316 (^{Moreno et al., 2012}; ^{Zapata et al., 2011}). In order for them to grow, the bacteria were cultivated in a Luria Bertani broth (LB, Oxoid^®), and the yeasts, in a broth composed of malt extract and yeast (YM, Oxoid^®) at 125 rpm, 28 °C for 48 and 72 hours, respectively. For the inoculant, suspensions were made in sterile distilled water at concentrations of 1x10⁸ cells mL^-1 for the bacteria and 1x10⁷ cells mL^-1 for the yeasts. The fruit was inoculated by immersion in the suspensions of each microorganism for 10 min, they were later dried at 18 °C and a 5 μL drop of the pathogen was inoculated on the lesions.

Efficacy of chitinous additives in the control of anthracnose. Chitin (Sigma^® C7170) was used in concentrations of 0.5; 0.8; 10; 100 and 1000 mg L^-1. The fruit was immersed in the suspension of the additive in each defined concentration for 10 min, dried at 18 °C and 5 μL drop of the pathogen was inoculated on the lesions.

Efficacy of the combination of alternatives in the control of anthracnose in mangoes inoculated with C. gloeosporioides. Four treatments were proposed to determine the efficacy in the control of anthracnose in mangoes inoculated with C. gloeosporioides (Table 1). First, the hydrothermal treatment was carried out, followed by the lesions on the fruit and then depositing them in suspensions of controlling microorganisms or the concentration of chitin (10 mg L^-1) for 10 min. They were then inoculated with the pathogen at the concentration described earlier and storage in humid chambers.

Efficacy in quiescent C. gloeosporioides infections: The same treatments mentioned above (Table 1) were evaluated on quiescent C. gloeosporioides infections, without inoculating the pathogen. The hydrothermal treatment was carried out, followed by the placement of the fruits in suspensions of controlling microorganisms for 10 minutes. For the treatment that contained chitin, it was added during the hydrothermal treatment. They were then left to dry and storage in humid chambers.

For the treatments in which the efficacy of the microorganisms and the different concentrations of chitinous substrates were determined, the fruits were places in humid chambers (Relative humidity (RH) of 90%), storage at 23 °C for seven days, under conditions considered optimal for the pathogen and severe for the selection of alternatives. On the other hand, the fruits corresponding to the combination of alternatives were storage in humid chambers at 13 °C for 19 days as an approximation to the storage of the fruit.

Variables evaluated. As a response variable, we calculated the efficacy in the control of the disease by applying the Schneider-Orelli formula: Percentage of efficacy = ((b - k) / (100 - k)) * 100, where b= area under the disease progress curve (AUDPC) in the treatment depending on the diameter of the lesion in fruits and k= AUDPC in the pathogen control (^{Koller et al., 2016}). The irregular distribution of the lesions of the disease were taken into consideration and therefore, in quiescent infections, Brodrick’s disease severity scale (1978, cited by ^{Corkidi et al., 2006}) was used (Table 2, Figure 1).

Experimental design and data analysis. Bioassays were established under a completely random block design. The experimental unit consisted of three fruits, with three repetitions and two replicates, including an untreated control and one pathogenic control. An analysis of variance was carried out, along with Tukey’s test (p≤0.05), using the statistics software Statistix 10.0.

Figure 1 Scale of severity for the evaluation of anthracnose in mango cv. Azúcar, appearance of the fruits in each level of severity. A. level 0, B. level 1, C. level 2, D. level 3, E. level 4. 

Results

Efficacy of antagonistic microorganisms in the control of anthracnose. A difference was noted in the performance of the microorganisms between the two replicates carried out in different times, possibly under the influence of the quiescent infections. However, in replicate one the treatments AP282, Bs006 and Lv316 displayed the highest efficacy in the control of the disease, with values between 62% and 65%, whereas in replicate two, the highest efficacy was obtained with AP282 (49%), which was also the most consistent treatment in accordance with the first replicate (Figure 2).

Figure 2 Efficacy of microbial antagonists in the reduction of anthracnose in mango cv. Azúcar fruits inoculated with C. gloeosporioides Y3 after storage for 7 days at 23 °C. Columns with the same letter are not significantly different, according to Tukey’s test (*= P>0.05). 

Efficacy of chitinous additives in the control of anthracnose. The different concentrations of chitin evaluated displayed some percentage of control, although the concentration of 10 mg L^-1 with an efficacy of 42% in replicate one and 49% in replicate two presented the greatest protection against the growth of C. gloeosporioides, and was also the most consistent treatment (Figure 3).

Efficacy of the combination of alternatives in the control of anthracnose in mangoes inoculated with C. gloeosporioides. All treatments displayed some level of control on the development of the disease in mangoes inoculated with C. gloeosporioides. However, the combination of the hydrothermal process and the tempering of the fruits in the L. xylaniticus AP282 suspension presented the greatest control, with an efficacy of 84%, whereas an evaluation of only the hydrothermal treatment presented the lowest performance, with an efficacy of 19% (Figure 4).

Figure 3 Efficacy of chitin in different concentrations on the reduction of anthracnose in mango cv. Azúcar fruits inoculated with C. gloeosporioides Y3 after storage for seven days at 23 °C. Columns with the same letter are not significantly different, according to Tukey’s test (*= P>0.05). 

Efficacy of the combination of alternatives in the control of anthracnose from quiescent C. gloeosporioides infections. Regarding the reduction in the expression of quiescent infections, combining the hydrothermal treatment with the addition of chitin or the later tempered of the fruits in the L. xylaniticus AP282 or R. glutinis Lv316 suspensions reduced the incidence of infections with a greater efficacy in the control of the disease in comparison with the hydrothermal treatment alone, and although these treatments surpassed an efficacy of 80%, the combination of hydrotherapy and the immersion in Lv316 was the most outstanding, with 89% (Figures 5 and 6).

Figure 4 Efficacy of the combination of treatments in the reduction of anthracnose in mango cv. Azúcar fruits inoculated with C. gloeosporioides Y3 after storage for 15 days at 13 °C. Columns with the same letter are not significantly different, according to Tukey’s test (*= P>0.05). 

Figure 5 Efficacy of the combination of treatments in the reduction of anthracnose from quiescent infections of C. gloeospo rioides in mango cv. Azúcar fruits after storage for 19 days at 13 °C. Columns with the same letter are not signifi cantly different, according to Tukey’s test (*= P>0.05). 

Figure 6 Symptoms of anthracnose in mango cv. Azúcar fruits treated with the combination of treatments storage at 13 °C. Top: Fruits inoculated with C. gloeosporioides Y3 after 15 days of storage. Bottom: Quiescent infections of C. gloeosporioides in treated fruits after 19 days of storage. 

Discussion

Lysinibacillus xylaniticus AP282, R. glutinis Lv316 and chitin (10 mg L^-1) proved to control the anthracnose in the fruits inoculated with the pathogen under storage conditions that favors their development (RH of 90% at 23 °C) and afterwards, when evaluating under a condition nearer to commercial storage (13 °C), they maintained or increased their protection and also reduced the expression of quiescent infections. Meanwhile, the single application of the hydrothermal treatment displayed an efficacy of 19%, but when combining it with tempering in the suspension of antagonists or adding chitin, its efficacy increased, particularly in the case of quiescent infections.

In this sense, the development of C. gloeosporioides may be limited by the absence of nutrients such as organic nitrogen and microelements such as iron, needed in the germination of conidia and later tissue infection processes, as well as by the exposure to metabolites produced by antagonistic microorganisms that may take action on the pathogen from quiescent infections (^{Liu et al., 2013}).

Bacteria and yeasts display action mechanisms that may inhibit the development of different pathogens, namely, competition for space and nutrients, the production of antibiotics, siderophores, parasitism and the induction of defense responses in plants (^{Rungjindamai, 2016}). Although little is known on the action mechanisms of L. xylaniticus, other species of this genus, such as Lysinibacillus sphaericus ZA9, are known to have the ability to inhibit the growth of Aspergillussp., A. alternata, Bipolaris spicifera, Curvularia lunata and Sclerotiniasp. in vitro, by secreting siderophores, hydrocyanic acid (HCN), chitinases, proteases and lypases, cycloalkane and quinoline type compounds (^{Naureen et al., 2017}). However, in order to clarify how L. xylaniticus AP282 controls C. gloeosporioides Y3, its action mechanisms should be stablished.

On the other hand, R. glutinis may compete for space and nutrients with different pathogens. Studies have shown the ability of this yeast to colonize lesions in strawberry and pear fruits, observing a rapid colonization on lesions and control over B. cinerea at 20 °C without affecting quality parameters, such as the loss of mass, firmness and acidity (^{Zhang et al., 2008}, ²⁰¹⁰). In addition, R. glutinis produces rhodotorulic acid, a siderophore that chelates the iron in the substrate, giving it a competitive advantage (^{Calvente et al., 1999}). For R. glutinis Lv316, a study carried out by ^{Zapata and Cotes, (2013}) obtained a control of 60% for B. cinerea in commercial berry crops, higher than that obtained with Prochloraz (58%) and Carbendazim (27%) applications, relating to the reduction of quiescent infections in the flowers.

Regarding chitin, its cationic character displays antifungal activity, mediated by the interaction of its free amino groups (positively charged in an acidic medium) with the negative residues in the molecules of the fungal walls modifying the permeability of the plasmatic membrane, causing the ionic homeostasis of K⁺and Ca²⁺causing the loss of small molecules such as phosphates, nucleotides and substrates of enzyme reactions that will eventually affect the fungal metabolism (^{Ban et al., 2015}; ^{Bautista-Baños et al., 2006}). ^{Lucas-Bautista et al. (2019}) found a greater content of chitinases in mature lyophilized papaya skins (29.000 units of chitinases (UC) g^-1) than those contained in mature frozen skins (1.500 UC g^-1), and they also determined that the chitin contained in C. gloeosporioides as a pathogen present in the papaya postharvest may contain up to 20.6%.

Despite the tendency of hydrothermal treatments to seal or cure lesions, limiting the areas of penetration for the pathogens (^{Schirra et al., 2000}), with the action of chitin (10 mg L^-1) or the tempering of the fruit in the suspensions of L. xylaniticus AP282 or R. glutinis Lv316, it is possible to reduce secondary infections by reinforcing control. However, studies must be carried out to develop biopesticides based on the selected microorganisms to allow their implementation on a commercial scale. On the other hand, considering the efficacy of chitin, it is necessary to have a commercial substitute, considering that this study used reactive grade chitin.

The inoculation of antagonistic microorganisms during the tempering after the hydrothermal treatment has shown a high potential for the reduction of damages by pathogens in postharvest. ^{Karabulut and Baykal (2004}) observed that the incidence of B. cinereaandPenicillium expansum in peaches decreased with a hydrothermal treatment at 55 °C for 10 seconds followed by inoculation with C. oleophila. On the other hand, it is possible that with the combined application of the antagonists, the control may be greater, as proven by ^{Carrillo-Fasio et al. (2005}), who evaluated isolations of the genus Bacillus and Rhodotorula on their own and combined, finding a greater control of anthracnose in Kent mango fruits when applying the mixture of biocontrol agents.

In this sense, controlling quiescent infections with the hydrothermal treatments with the addition of chitin at 10 mg L^-1 or the later tempering of the fruit in the suspension of R. glutinis Lv316 or L. xylaniticus AP282 may be a strategy to treat the mango cv. Azúcar immediately after its harvest, allowing the microorganisms in the fruit to establish and therefore, increase their protective ability during storage.

Conclusions

The combination of the hydrothermal treatment at 53 °C for 5 minutes with the addition of chitin at 10 mg L^-1 or the later tempering of the mangoes in the suspensions of R. glutinis Lv316 or L. xylaniticus AP282 presented an efficacy between 83 and 89% in the control of anthracnose originated from the quiescent C. gloeosporioides infections, alternatives which may be considered in the postharvest treatment of the mango cv. Azúcar.