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Revista mexicana de fitopatología

On-line version ISSN 2007-8080Print version ISSN 0185-3309

Rev. mex. fitopatol vol.36 n.3 Texcoco Oct./Dec. 2018

http://dx.doi.org/10.18781/r.mex.fit.1805-3 

Phytopathological notes

Antifungal property of honey on in vitro development of Colletotrichum gloeosporioides

Víctor Albores-Flores1  * 

Ivana Janet Marín-Saenz1 

José Alfonso López-García1 

Adriana Sánchez-Gutiérrez1 

Julieta Grajales-Conesa1 

1 Instituto de Biociencias, Universidad Autónoma de Chiapas. Boulevard Príncipe Akishino s/n. Colonia Solidaridad 2000. Tapachula Chiapas, CP. 30798, México.

Abstract:

In addition to the high toxicity of chemicals for phytosanitary control of fungal diseases in field conditions, the microorganism causing disease is not completely eliminated. Therefore, products of natural origin, both plant and animal, have generated interest for the control of pests and diseases in plants. Thus this study aimed to evaluate the antifungal activity of honey in the in vitro growth of Colletotrichum gloeosporioides. Nine samples of honeys were evaluated, corresponding to three bees species: Melipona solani, M. beecheii and Scaptotrigona mexicana. We registered a decreased colony diameter of C. gloeosporioides at higher concentration of honey, with lower 40% growth of the colony compared to the control treatment within 12 days of incubation. In general, growth rate of the fungus colony in the three types of bee honey was in average, 40% lower than the control treatment. Inhibition rate value observed in bee honeys is 70% higher than that obtained with the fungicide Chlorothalonil®.

Key words: mycelial growth; Melipona solani; Melipona beecheii; Scaptotrigona mexicana; Clorotalonil®

In this century, products used in the control of plant health do not counteract disease development, since they do not eradicate the problem due to the low efficiency of penetration into the cuticle, the activity of the active principle and the accumulation in plant tissue (Campa et al., 2017; Pérez et al., 2017). Therefore, the development of alternative products with antifungal activity and of a microbial, plant or animal nature are currently an option and a reality (Correa et al., 2015; Ramírez et al., 2016). Recent studies have identified honey as an alternative in the control of fungi in vitro, since it has a natural origin, derived from the process of transformation of nectar by bees (Apis mellifera).

Honey presents a variety of neutracetical properties (Ramalivhana et al., 2014) and has also been used as a medicinal alternative for centuries (Vallianou et al., 2014). Recent studies have found a relation between the anti-microbial properties and the concentration and composition of honey (Fangio et al., 2007; Pimentel et al., 2013). Its anti-fungal action has been observed against the fungal genera of Trichophyton, Microsphorum, Aspergillus, Penicillium and Candida (Moussa et al., 2012; Londoño-Orozco et al, 2008; Montenegro et al., 2009; Olaitan et al., 2007). Thus the aim of the present study was to evaluate the anti-fungal activity of honey in the in vitro growth of Colletotrichum gloeosporioides.

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:

µ=(Db-Da)/(tb-ta)

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).

Table 1 Effect of the use of honey on the speed of growth and mycelial inhibition of C. gloeosporioides and its comparison with Clorotalonil® equivalents. 

Tratamiento Concentración
(%)
Velocidad de
crecimiento
(h-1)
Inhibición
micelial
(%)
Equivalentes de
Clorotalonil®
(mg ml-1)
Testigo 0 0.025 a* 0 0
SMCA 25 0.023 bc 40.4 i* 29.2 f*
50 0.020 e 66.1 c 44.8 b
100 0.006 h 95.2 a** 62.5 a**
SMIC 25 0.023 bc 39.0 jkl 28.3 f
50 0.020 e 63.6 d 43.3 c
100 0.004 i 95.2 a** 62.5 a
SMFMB 25 0.023 bc 38.5 kl 28.0 f
50 0.021 de** 59.1 e 40.5 d
100 0.007 g 94.6 ab 62.1 a**
SMID 25 0.023 bc 39.0 ijkl 28.3 f
50 0.021 de** 58.5 ef 40.2 d
100 0.008 f 94.4 ab 62.1 a
MSTA 25 0.024 b 39.8 ijk 28.8 f
50 0.021 de 57.7 fg 39.5 d
100 0.007 g 94.0 ab 61.8 a
MSSJB 25 0.023 bc 40.0 ij 28.9 f
50 0.020 e 63.4 d 43.2 c
100 0.007 g 94.6 ab 62.2 a
MBTB 25 0.024 b 38.1 l 27.8 f
50 0.021 de 58.3 ef 40.1 d
100 0.008 f 94.7 ab 62.2 a
MSLC 25 0.024 b 34.8 ll 25.8 g
50 0.022 cd 56.5 g 38.9 d
100 0.007 g 93.8 b 61.7 a
MBTA 25 0.024 b 33.1 m 24.7 g
50 0.022 cd 54.3 h 37.6 e
100 0.007 g 93.5 b 61.5 a

*p≤ 0.05.

** Same letters indicate no difference between averages

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.

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Received: May 16, 2018; Accepted: July 30, 2018

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