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Revista mexicana de fitopatología

versión On-line ISSN 2007-8080versión impresa ISSN 0185-3309

Rev. mex. fitopatol vol.35 no.3 Texcoco sep. 2017 

Scientific articles

Reduction in the incidence of grey mold in table grapes due to the volatile effect of a garlic extract

Paola Campa-Siqueiros1 

Socorro Vallejo-Cohen1 

Consuelo Corrales-Maldonado1 

Miguel Ángel Martínez-Téllez1 

Irasema Vargas-Arispuro1  * 

Graciela Ávila-Quezada2 

1Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a la Victoria km 0.6, C.P. 83304 Hermosillo, Sonora.

2Universidad Autónoma de Chihuahua. Facultad de Zootecnia y Ecología, Periférico Francisco R. Almada Km 1, Zootecnia, C.P. 31453, Chihuahua, Chihuahua.


Gray mold in grape clusters caused by Botrytis cinerea, is one of the diseases that most affect the table grape market. To control this disease sulphur dioxide releasing pads (SO2) and systemic fungicides are used, currently in regulatory review. In this work, it was proposed to use an extract of garlic (EHA), allicin, dialyldisulfide (DADS) and dialyltrisulfide (DATS) in volatile form to evaluate the incidence of gray mold on clusters of table grapes of the Flame seedless variety. Treatments were impregnated with cellulose, which allowed the release of the volatile compounds on clusters of grape inoculated with 1x106 spores of B. cinerea/mL. The results showed that the volatile emitted by allicin and DADS had a lower effect than that of EHA and DATS. Those compounds similarly inhibited (P≤0.05) disease development in clusters of grape for 14 days at 4 and 25 °C, showing increased effectiveness at low temperature (4 °C). This is a result of particular relevance in the problematic of this disease that occurs during grape cold storage. The effectiveness of garlic-derived compounds applied in their volatile form opens the possibility to use them as an alternative to the traditional fungicides for controlling post-harvest diseases.

Key words: Vitis vinifera; Allium sativum; cluster rot disease; sulphur volatile

Botrytis cinerea Pers.:Fr.[teleomorph: Botryotinia fuckeliana (Bary) Whetz.], the ascomycete that causes gray mold, is one of the most important fungal plant pathogens affecting table grapes. The fungus affects table grapes production both in pre-harvest and post-harvest stages in a wide range of environment conditions and geographic areas (Williamson et al., 2007). Development of gray mold on table grapes affects their market value and also causes them to be rejected upon being inspected at the borders of importing countries. Temperature and moisture conditions during transportation and/or cold storage favor the development of B. cinerea, because during these stages berries achieve their maximum sugar concentration allowing the fungus to grow vigorously and spread easily among grape clusters (Lichter et al., 2006). In northwestern Mexico, 90% of table grape production is exported to markets in the United States, Europe and Asia. To reach those markets, they are sent in refrigerated (4±1 °C) containers over land or sea and have to withstand journeys of 14-25 days (Vázquez, 2011; Lichter et al., 2008).

A common method to control gray mold on table grapes during transportation and cold storage is the use of sulphur dioxide (SO2) releasing pads; SO2 is a gaseous compound that disperses inside the container and prevents B. cinerea spores to germinate (Sanzani et al., 2012). However, SO2 damages the berries by bleaching (Milkota et al., 2010) and may produce hypersensitivity reactions in consumers (Ruiz-Moreno et al., 2015). For this reason, besides the commercial importance of table grape exports, efforts have been made to find alternatives to using SO2. From the various alternatives considered, the most accepted have been those whose active component comes from natural sources (Tripathi et al., 2008). From natural sources, garlic compounds have shown fungicidal effect (Perelló et al., 2013; Wallock-Richards et al., 2014). Such bioactivity has been especially attributed to the allicin compound (Curtis et al., 2004), one of the most abundant components formed when garlic is mechanically damaged. Other sulphur compounds derived from garlic that have also shown antifungal effects are diallyl disulfide (DADS) and dialyltrisulfide (DATS) (Tsao and Yin, 2001; Gándara-Ledezma et al., 2015). A particularly attractive feature of sulphur compounds derived from garlic is that they are volatile. This characteristic allows them to have bioactivity at vapor phase and makes it easier to use them in a gaseous form to control diseases. This is a relevant aspect of table grape marketing because, after being harvested, the berries must not be in contact with liquid substances (Codex Stan 255, 2007). In a previous research, it was found that a hydroalcoholic extract of garlic (EHA) inhibited B. cinerea in vitro spore germination in a magnitude similar to that of allicin and dialyltrisulfide compounds (Gándara-Ledezma et al., 2015).

Considering all the aspects above mentioned, this research focused on evaluating the effect of the volatile compounds from a hydroalcoholic extract of garlic, allicin, dialyldisulfide and dialyltrisulfide on the incidence of gray mold on table grapes inoculated with B. cinerea at transportation (4 °C) and marketing temperature (25 °C).


Botrytis cinerea Pears

Botrytis cinerea was isolated from infected grapes of the Flame Seedless variety in a vineyard located in the wine region of Pesqueira, Sonora. The strain was identified by its morphological and molecular characteristics such as conidia size, colony appearance and ITS1-5.8S RNA-ITS2 sequence (access AY568636). For growth, it was sown in agar-potato-dextrose (PDA) medium culture and kept in darkness at 25 °C from 7 to 9 days (Tzortzakis et al., 2007). The fungus was sown again by taking a piece of mycelium and inoculating it into PDA at 25 °C from 7 to 9 days, or until the fungus produced enough spores to prepare a 1x106 spore/mL suspension. To prepare the suspension, 5 mL of distilled water with 0.01% of 0.02% Tween 80 were poured on the surface of the plate containing the fungus; then, it was scratched using a glass rod, the liquid containing fungal spores was collected and filtered through muslin cloth to remove the mycelium, and the mixture was centrifuged at 5000 x g. The collected spores were re-suspended in sterile water until a 1x106 spores/mL concentration was obtained, and quantified in a Neubauer chamber, according to the methodology of Moo-Koh et al. (2014).

Hydroalcoholic extract of garlic (EHA) and its derived sulphur compounds (DADS, DATS and allicin)

The extract was prepared using garlic of the Regional variety purchased at the local market. Ten gram of garlic cloves were blended with 14 mL of distilled water for 1 min at room temperature. The mixture was centrifuged at 15 000 x g at 4 °C for 20 min, then 6 mL of ethanol were added to the supernatant and the mixture was centrifuged again at 8000 x g at 4 °C for 20 min (Jansen et al., 1987). The supernatant represented the hydroalcoholic extract of garlic. Standards with purity above 98% allicin, dialyldisulfide (DADS) and dialyltrisulfide (DTS) were purchased from Neem Biotech (Cardiff, UK).

In vitro evaluation of the effect of EHA, DADS, DATS and allicin on gray mold development caused by Botrytis cinerea on grape clusters

Grape clusters of the Flame Seedless variety were collected at harvest in vineyards in the wine region of Pesqueira, Sonora. The clusters were reduced to 10 berries per cluster, ensuring that they did not show visible symptoms of gray mold caused by B. cinerea. The clusters were disinfected by immersion in a 2% (v/v) sodium hypochlorite solution for 5 min, rinsed with sterile distilled water and dried on paper towels at room temperature. A puncture wound near the peduncle of each berry was made with a sterile scalpel. Each cluster was sprayed with 1 mL of the 1x106 spores/mL suspension of B. cinerea prepared in dextrose sabouraud agar (CDS). The inoculated clusters were placed in 50 cm3 plastic chambers (one cluster per chamber) and exposed to volatile compounds from EHA, DADS, DATS and allicin at the doses per treatment shown in Table 1. The compounds were impregnated in cellulose pads (6x2 cm) that had been previously stuck on the coverlid of the chambers. The control treatment was impregnated with a water-glycerol solution because glycerol was used as a retention agent to control the release of the emitter’s volatiles. This compound was incorporated into the treatments before impregnating the emitter. The disease incidence was quantified every other day during 14 days at 4 and 25 °C. Berries of each cluster with gray mold symptoms were counted. The results are presented as the disease incidence (percentage of infected berries) calculated using the formula proposed by Nally et al. (2012). Three replications per treatment were prepared and the evaluation was performed three times.

Table 1 Treatments used to evaluate in vitro incidence of gray mold on table grape clusters at 4 and 25 °C. 

Evaluation of the synergistic effect among DADS, DATS and allicin on gray mold caused by Botrytis cinerea on grape clusters

To determine a possible synergistic effect among DADS, DATS and allicin compounds, a mixture containing 20 μL of each compound and 90 μL of glycerol was prepared. The mixture was homogenized with gentle agitation and kept in a cold-water bath until it was applied to the emitter. The treatments used are shown in Table 2. The same procedure described for independent compounds was used to determine the effect of the compound mixture on the incidence of gray mold on grape clusters.

Table 2 Treatments used to evaluate the synergy among DADS, DATS and allicin in the 

Experiment design and statistical analysis

Independent compounds were arranged in a completely randomized design with 5 treatments and 3 replications; the mixture of compounds was arranged in a completely randomized design with 3 replications and 3 treatments. The experiment unit in both treatments was a 10-berry cluster, and the response variable the number of berries with gray mold symptoms. The percentage of disease incidence was calculated using the equation of Nally et al., 2012. Data of the percentage of gray mold incidence were converted to y = arsin (sqrt (y/100)) and then subjected to an analysis of variance with a factor and, in case of significance, a media comparison was made using Tukey-Kramer test (P≤0.05) and NCSS statistical program (Number Cruncher Statistical System, 2007).


Effect of EHA, DADS, DATS and allicin on gray mold development caused by Botrytis cinerea on grape clusters

The effect of volatiles emitted by the hydroalcoholic extract of garlic and DADS, DATS and allicin on gray mold development on table grape clusters evaluated during 14 days after treatment application is shown in Figure 1. The figure shows that volatiles emitted by T2 (EHA) controlled gray mold development on grape table clusters both at 4 °C and 25 °C. From T3, T4 and T5 treatments evaluated at 25 °C (Figure 1B), only T4 showed a similar effect (p≤0.05) to EHA on gray mold development. Clusters treated with T3 showed 70% of gray mold incidence, and T5 a similar incidence (p≤0.05) to that of the control, reaching 100% on day 10 of the 14-day evaluation.

The effect of the treatments evaluated at 4 °C showed that T2, T3 and T4 controlled gray mold development (Figure 1A), and although T5 allowed 10% of disease incidence, the statistical analysis (p≤0.05) was the same as that of T2, T4 and T5. Special importance should be given to the effect of the treatments evaluated at 4 °C, because during the first 8 days under treatment, T5 showed only 5% of gray mold incidence, whereas the control presented 30%.

Effectiveness of the treatments evaluated to inhibit gray mold development on table grape clusters is shown in Table 3. Results show that T2 was more effective in controlling the disease during 14 days at 25 and 4 °C, followed by T4 treatment that controlled the disease 8 and 12 more days than the control at 25 and 4 °C, respectively.

Figure 1 Effect of volatiles emitted by EHA, DADS, DATS and allicin during gray mold development on table grape clusters evaluated at 25 and 4 °C during 14 days. Values represent the media of three replications of 10 berries each. Vertical bars represent the standard deviation (n=3). Values with the same letter are not statistically different from each other for P≤0.05, according to Tukey-Kramer. The experiment was conducted three times. 

Table 3 Evaluation in vitro of the effectiveness of treatments to protect clusters of table grape from gray mold. 

z Disease development in EHA started 14 days after.

Synergistic effect of a mixture of DADS, DATS and allicin on gray mold development caused by Botrytis cinerea on table grape clusters

The mixture of DADS, DATS and allicin had a similar effect (P≤0.05) that of EHA on gray mold development on grape clusters (Figure 2). When evaluated at 4 °C, both EHA and the mixture of compounds inhibited disease development during 14 days (Figure 2A), while when evaluated at 25 °C only EHA controlled the disease during 14 days. The mixture of DADS, DATS and allicin resulted in 9% of gray mold incidence at day 10; this value remained until day 14, when the evaluation ended.

Figure 2 Synergistic effect of a mixture of DADS, DATS and allicin on gray mold development evaluated at 25 and 4 °C during 14 days, compared with EHA and its control. Vertical bars represent the standard deviation (n=3). Values with the same letter are not statistically different from each other for P≤0.05, according to Tukey-Kramer. 


Given the growing interest to find natural alternatives to the use of SO2 to control gray mold on table grape (Parafati et al., 2015), it was proposed to evaluate volatiles emitted by a garlic extract (EHA) and three of its main components (DADS, DATS and allicin) (Iciek et al., 2009) to control the disease. Both EHA and DADS, DATS and allicin have showed to become bioactive at vapor phase (Curtis et al., 2004; Gándara-Ledezma et al., 2015), a characteristic that makes it easier to use them in a gaseous form to control B. cinerea, a pathogen causing gray mold on grape clusters. Due to marketing norms, table grape berries should not come into contact with liquids after being harvested (Codex Stan 255, 2007). Several studies have been published on the antibacterial and antifungal effect of garlic extracts evaluated in liquid media (Harris et al., 2001; Durairaj et al, 2010). However, few studies have been published on volatile evaluations of garlic compounds. Studies published by Curtis et al. (2004) and Gándara-Ledezma et al. (2015) showed the antimicrobial potential of garlic derivatives applied in a volatile manner. In this study, those results were extended to the control of gray mold by demonstrating that volatile compounds emitted by EHA, DADS and DTS resulted in a significant reduction in the incidence of gray mold on table grape clusters (Table 3, Figure 1). EHA and sulphur compounds were more effective in reducing gray mold at low temperatures (4 °C) (Figure 1A), a relevant fact for the purpose of this research since B. cinerea develops vigorously at low temperatures (-0.5 °C) (Lichter et al., 2006) and spreads easily throughout grape clusters during transportation and cold storage (Crisosto et al., 2012). The effect of allicin on the incidence of gray mold showed a contrasting result at both temperatures, showed a similar effect on the control (p≤0.05) at 25 °C, as well as a similar effect on the more effective treatment (EHA) (P≤0.05) at 4 °C. Results from allicin may be associated with thermal instability given that, when it is not in liquid media, at higher temperatures it volatilizes quickly (Chong et al., 2015), which causes a loss of antifungal activity. This was demonstrated by Curtis et al. (2004), who evaluated the effect of allicin from a garlic extract applied in a volatile manner on bacteria and fungi; they observed that the antimicrobial activity of allicin decreased as temperature increased, while at 4 °C activity remained constant. Antifungal activity of garlic extracts has been correlated with allicin content (Singh et al., 2001). However, in this research, DATS volatiles were better than allicin in controlling gray mold, since DATS delayed the onset of the disease by 12 days at 4 °C and 8 days at 25 °C (Table 3), and only EHA controlled the disease during 14 days at both temperatures (Table 3). EHA effectiveness is attributed to its content of sulphur compounds (allicin, DADS and DATS) (Fujisawa et al., 2008), which may be acting in a synergistic way, as it was demonstrated in this research (Figure 2) by evaluating a mixture of DADS, DATS and allicin that controlled gray mold development on grape clusters at 4 °C, while at 25 °C the disease decreased by 94% compared with its control. The mixture of DADS, DATS and allicin produced a better effect on disease incidence than the individual components evaluated under the same temperature conditions and concentration (Figures 1 and 2).

Although in the literature there are reports on active agents for controlling gray mold, such as those by Muñoz and Moret (2010) and Tripathi et al. (2008), who used chitosan and acibenzolar-S-methyl, essential oils, respectively, those compounds were evaluated in liquid media and in direct contact with the fungus. The strength of this research is based on the effectiveness of garlic derived-compounds to control gray mold when they are applied in a gaseous form. According to Martinez et al. (2007), when antifungal agents are applied in a gaseous form they are less effective than when applied in liquid form at the same doses. In spite of the good results to reduce the incidence of gray mold on table grape, sulphur compounds still require further evaluations to be proposed as fungicide agents, especially studies including different dose ranges in large-scale tests. Organoleptic tests should also be conducted to ensure the absence of touches of flavor in fruit conferred by sulphur compounds.


Dialyltrisulfide and hydroalcoholic extract of garlic emitted volatiles that protected grape clusters against gray mold caused by B. cinerea during 14 days at 4 and 25 °C. This potential offers the possibility to use them as an alternative to traditional fungicides to control postharvest diseases.


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The authors wish to thank to the Consejo Nacional de Ciencia y Tecnología (CONACyT) for the support scholarship granted to the first author of this study for her postgraduate studies at Centro de Investigación en Alimentación y Desarrollo, A.C.

Received: July 12, 2017; Accepted: August 21, 2017

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