Honey samples

Honey samples were collected during February and March 2016, in meliponaries from the “Asociación de Meliponicultores del Soconusco S. C. del R. L.” in the municipal areas of the Soconusco, Chiapas: Tapachula, Tuxtla-Chico and Cacahoatán.

Nine honey samples were used from three bee species: Melipona solani (n=3, from Trinidad (MsTa), San Jerónimo (MsSjb) and Izapa (Mslc)), M. beecheii (n=2, from Tapachula (MbTa) and (MbTb) and Scaptotrigona mexicana (n=4, from Cacahoatán (Smca), Francisco y Madero (SmFMb), Izapa I (Smic), Izapa II (Smid)). The honey was collected from three different boxes in the meliponaries in order to obtain a mixed honey sample per site, which was taken using sterile 5 ml syringed, and it was stored in properly labelled jars. They were stored at -4 °C until their analysis.

Anti-fungal activity of honey

The anti-fungal activity of honey was evaluated by antagonizing it in the laboratory with a C. gloeosporioides strain from the collection of the Bioscience Institute of the Autonomous University of Chiapas. The strain was reactivated in a potato-dextrose-agar (PDA) medium without honey and a pH of 6.0. The biotrial was carried out with a PDA medium mixed with a honey solution, and for this, we prepared dishes with a volume of solution (in concentrations of 25, 50 and 100 % honey) of 100 µl for every 20 mL of medium. After the strain inoculation (0.5 cm culture discs), the Petri dishes were incubated at 32 °C for 12 days.

Mycelial growth was measured every third day using a Vernier (Stainless Hardebed^®) with a capacity of 0-150 mm (minus the size of the inoculant disc).

The growth rate of every culture was determined with the following equation:

where µ was growth rate of the culture, Db was the diameter of the culture (mm) in time “b”, Da was the diameter of the culture in time “a”; and “tb” and “ta” were the time in which the evaluation was carried out.

Determination of anti-fungal activity was queivalents comparable to Clorotalonil^®

The anti-fungal activity of the honey was converted to equivalents of Chlorothalonil^® (2, 4, 5, 6-tetrachloroisophthalonitrile), which is a chemical fungicide, commonly used in the control of C. gloeosporioides. For this, we determined the growth inhibition (%) of this fungus mediated by the Chlorothalonil^® by prepating solutions with the concentrations 0, 0.9, 1.8, 3.6, 5.4 y 7.2 mg ml^-1. Using the data obtained, we created a callibration curve (Figure 1).

Figure 1 Relation between the concentration of Clorotalonil^® and the degree of inhibition of the development of C. gloeosporioides. 

Analysis of results

To analyze the diameter of the culture, its growth rate and inhibition, and equivalents of Chlorothalonil, an analysis of variance was carried out, followed by a mean comparison using Tukey’s test (p<0.05). The statistical program used was Infostat 2015. All treatments had nine repetitions.

In the treatments with honey, a relation was found between the honey concentration and the size reached by the fungal culture (r=-0.87, p<0.05)i.e., the diameter of the culture decreased as the concentration of honey increased. Starting on day 9 (Figure 2), in the concentrations of 25 and 50% of honey, significant differences were observed (p<0.05) in the diameter of the culture amongst the different types and concentration of honey applied. On average, the maximum diameters reached by the fungal cultures, regardless of the honey type, were 31.1, 20.4 and 3.2 mm for concentrations 25, 50 and 100%, respectively, unlike the control, which was 50 mm (Figure 2).

Figure 2 Growth kinetics of C. gloesporioides in different concentrations of honey (honey concentrations: 25%, 50% and 100%) 

Typical growth rate (µ) was in the range of 0.020 to 0.023 h^-1, with honey concentrations of 25 and 50%, unlike in the control, where it was 0.025 h^-1. At a concentration of 100% of honey, the range was between 0.004 h^-1 and 0.008 h^-1. In general, the growth rate of the culture of C. gloeosporioides in honey was 40% less than in the control (Cuadro 1).

For inhibition activity of C. gloeosporioides, highly significant differences were found (p<0.0001) between the honey from different bees, and it was also found that regardless of the concentration of honey, those coming from S. mexicana (Smca and Smic) inhibited the development of the C. gloeosporioides by 5.2%, while in honey from M. solani (Mssjb and Msta), M. becheii (Mbtb) and from S. mexicana (Smid and Smfmb) inhibition action was between 94% and 95%. The honey with the lowest inhibition values were M. solani (Mslc) and M. becheii (Mbta), with valued of 93.87 and 93.53%, respectively. However, inhibition was still high.

Finally the anti-fungal activity of the related honey in equivalents of Chlorothalonil^® (eC) was also related to the concentration of honey (r=0.98, p<0.05). The highest concentration related to eC with inhibition action on C. gloeosporioides was displayed by honey from S. mexicana (Smca and Smic) with values of 62.5 mg ml^-1, 72.7 mg ml^-1 y 62.5 mg ml^-1, 72.6 mg ml^-1, respectively.

The time taken for the growth of the C. gloeosporioides culture without honey to reach the inner diameter of the Petri dish was 12 days (Figure 2). Unlike the treatments with honey, the growth of the control displayed a sigmoidal shape.

The reduction in mycelial growth of the fungus could be related to the bioactive components of honey, since similar results were reported in the development of Aspergillus spp and Penicillium spp fungal cultures when incubated in the presence of Apis honey (^{Olaitan et al., 2007}). ^{Lira (2003)} and ^{Jasso et al. (2007}, ²⁰¹¹⁾ coincided in that the inhibiting effect on the growth of the mycelium of pathogenic fungi for plants lies in the bioactive compounds of the biological products used, as in the case of plants.

Scaptotrigona or Melipona honey showed potential for use as a product with antifungal action when using a concentration of 500 µL of honey for every 100 mL of solution, reaching a level of inhibition of 95.0%, in comparison with commercial products that can be applied across a wide range of concentrations that range from 0.5 mg kg^-1 to 500 mg kg^-1 (^{Rodríguez et al., 2008}; ^{Warnke et al., 2009}).

The anti-fungal action observed on C. gloeosporioides growth in the presence of honey at different concentrations could be due to the concentration of phenols, flavonoids and pH value, which have been reported by Marín et al. (2017). The structural similarity of these compounds found in the honey with that shown by the commercial chemical molecules used for the same purpose (^{Pérez-Cárdenas et al., 2013}; ^{Gregorí, 2005}) suggests that the antifungal mechanism observed on the honey studied in this investigation could be associated with the alteration of cell division, the permeability of cell membranes and the intracellular transportation in the fungal mycelium.

Scaptotrigona and Melipona honey showed a potential for the control of the pathogenic fungus C. gloeosporioides and also reduces the mycelial growth rate by 95%, which makes it 40% more efficient than the commercial fungicide Clorotalonil^®. The use of honey in the control of plant pathogenic fungi could be a solution alternative within a comprehensive management.