Control of Postharvest Fungal Rots on Grapes Using Essential Oil of Foeniculum vulgare Mill .

Fungal rots, caused by Botrytis cinerea and Colletotrichum acutatum, are the main causes of postharvest decay of table grapes in Brazil. The aim of this study was to assess the chemical composition and the fungicidal effect of Foeniculum vulgare essential oil in vitro, on mycelial growth (contact and volatile phase) and conidia germination, and in vivo on postharvest grapes against both fungi. The major compounds found in F. vulgare essential oil were trans-anethole (79.14%), fenchone (11.94%) and estragole (5.76%). The mycelial growth (contact phase) and conidia germination of B. cinerea were inhibited completely at concentrations of 50 and 100 ppm (μL mL), respectively. For C. acutatum, mycelial growth (contact phase) and conidia germination were inhibited completely at concentrations of 100 and 200 ppm, respectively. The volatile phase had a fungistatic effect on mycelial growth of both fungi at different concentrations tested, and the pure essential oil (100%) presented fungicidal effect against B. cinerea. In vivo tests were carried out using grapes of Vitis spp. cv. “Isabella” and the concentrations of essential oil tested were efficient, reducing the incidence of disease caused by B. cinerea and C. acutatum at postharvest, both in preventive and curative treatment. The concentration 200 ppm completely inhibited the incidence of both fungi. In conclusion, F. vulgare essential oil presented fungicidal action against postharvest fungal rots on grapes.


Introduction
Grape is one of the most important fruit crops worldwide.In Brazil, grape production destined for processing (wine, juice and derivatives) was 673.422 million kilos in 2014, representing 46.89% of the national production.The remaining production (53.11%) was intended for consumption in natura (Mello, 2014)."Isabella" grape is one of the most important varieties of Vitis spp.and it is the most diffused variety in the Serra Gaúcha, the southern viticultural region of Brazil, where it represents near 57% of the total production."Isabella" grape is used to make red table wine and juice and it is also commercialized as table grape (Mello, 2014;Silveira, Hoffmann, & Garrido, 2015).
The economic losses due to fungal infection in fruit and vegetables within the postharvest chain are variable and not well documented and they usually reach any where from 30 to 50% and on some occasions rots can lead to total loss of the produce (Smilanick, Brown, & Eckert, 2006;Youssef & Roberto, 2014).Botrytis cinerea Pers.Fr. and Colletotrichum acutatum Simmonds cause fungal rot on a large number of economically important agricultural crops and they are considered the main cause of great losses of postharvest in table grapes (Pearson & Goheen, 1988;Peres, Kuramae, Dias, & Souza, 2002;Steel, Greer, & Savocchia, 2007;Whitelaw-Weckert et al., 2007).
By over efficient that it is the phytosanitary treatment made in the field, it is not enough to dismiss it in postharvest (Lichter et al., 2002).As a postharvest treatment, grapes are usually stored with sulfur dioxide fumigation (Droby & Lichter, 2004).However, the use of synthetic fungicides and sulfur dioxide is not allowed on organic grapes (Mlikota Gabler & Smilanick, 2001).In addition, the growing public concerns about health Several studies have proven the effect of extracted compounds isolated from essential oils of plants, which act as natural fungicides inhibiting fungal activity (Chao & Young, 2000).Moreover, the majority of essential oils are classified by the FDA (Food and Drug Administration) as GRAS (Generally Recognized as Safe), recognized as safe for use in foods, so there has been a growing interest in using them in the treatment of fruits and vegetables (Gonzáles-Aguilar et al., 2008).
The aim of this study was to identify the chemical composition and evaluate the fungicidal effect of Foeniculum vulgare essential oil in vitro, on mycelial growth (contact and volatile phase) and conidia germination, and in vivo on postharvest grapes against B. cinerea and C. acutatum.

Isolated Fungi
Strains of B. cinerea (A58/09) and C. acutatum (A009/13) used in this work were isolated from grapes of Caxias do Sul (Serra Gaúcha, RS, Brazil) and preserved in the fungal collection of the Laboratory of Phytopathology, University of Caxias do Sul, Brazil, on PDA (Potato Dextrose Agar) medium.The molecular confirmation of both fungi was done using Internal Transcribed Sequence (ITS)-PCR identification.The DNA extraction was according to Murray and Thompson (1980) and ITS-PCR amplified the region ITS-5.8SrDNA according to White, Bruns, Lee, and Taylor (1990).Sequencing was proceed at the Human Genome Center-USP and the sequences obtained were edited with the software BioEdit Sequence Alignment Editor (1997Editor ( -2005) ) and used to search for similar sequences using Blastn at NCBI.

Plant Material
Fruits in the final stage of maturation of F. vulgare were collected from plants localized in the city of Caxias do Sul, Brazil.A voucher specimen of the plant species was deposited in the Herbarium of the University of Caxias do Sul with number 44057.

Essential Oils Extraction and Analysis
The essential oil was extracted by hydrodistillation from dried fruits for 1 hour in a Clevenger-type apparatus according to Agostini et al. (2009).For identification and quantification of compounds of the essential oil, it was used the protocol described in Tomazoni et al. (2016) using a gas chromatograph HP 6890, coupled with a mass selective detector Hewlett Packard MSD5973, equipped with HP Chemstation software and Wiley 275 spectra data.The analyses were conducted using a fused silica capillary column HP-Innowax (30 m × 0.25 mm i.d., 0.25 µm film thickness, Hewlett Packard, Palo Alto, USA).The components were identified by a combination of mass spectrum of the Wiley library and by comparison with data from literature (Adams, 2005).The relative percentage of each component was obtained from chromatographic peak areas, assuming the sum of all eluted peaks being 100%.

Antifungal Activity of Essential Oil on Mycelial Growth
The antifungal properties of essential oil were assessed for its contact and volatile phase effects towards mycelial growth of phytopathogens.Contact phase effect of essential oil was tested according to Feng and Zheng (2007) with minor modifications.Essential oils concentrations used were 10, 50, 100 and 200 ppm (µL mL -1 ), with the addition of Tween 20 (1:1), diluted on autoclaved and melting PDA (Potato Dextrose Agar) (40 °C) under aseptic conditions.The control treatment was just PDA medium with addition of Tween 20 at concentration 200 ppm (similar to the highest concentration used to emulsify the essential oil).These emulsions were poured into 9 cm (Ø) Petri dishes and, after medium solidification, inoculated with 5 mm (Ø) agar disks colonized by B. cinerea or C. acutatum mycelium with seven days of development.
To assess fungicidal action of the volatile phase of essential oil on the mycelial growth of fungi it was utilized the methodology according to Silva (2012) with minor modifications.Agar disks with 5 mm (Ø) colonized by B. cinerea or C. acutatum mycelium with seven days of development were placed in the center of the Petri dish containing PDA culture medium.The concentrations of essential oils used were 12.5, 25, 50 with the addition of Tween 20 (0.1%) and 100% (pure essential oil, without addition of Tween 20).A 100 μL sample of pure essential oil and the solutions were applied onto a cotton ball attached to the inner face of a Petri dish lid, thereby preventing direct contact of the oil with the culture medium and the mycelium disk, creating a saturated atmosphere of volatile compounds.The control treatment was just PDA medium and 100 μL of Tween 20 (0.1%) applied onto a cotton ball.
In both tests, for each concentration, ten replicate plates were used.Incubation was performed at 25 °C temperature and 12 hours photoperiod, during fourteen days.Fungal growth was recorded on the 3 rd , 5 th , 7 th , 10 th and 14 th day by measuring the orthogonal diameter of the fungi.Transfer experiments were performed to provide a distinction between the fungistatic and fungicidal effects of essential oil on the target microorganisms.For this purpose, plugs that did not grow were transferred to fresh PDA dishes to assess their viability and growth after five days of inoculation at 25 °C temperature and 12 hours photoperiod.The residual fungal growth was monitored by measuring the orthogonal diameter of the fungi.

Antifungal Activity of Essential Oil on Conidia Germination
Antifungal activity of essential oil on conidia germination was tested according to Badawy and Rabea (2013), with minor modifications.Conidia of B. cinerea and C. acutatum were harvested from a colony of the fungi 14 days grown in PDA at 25 °C temperature and 12 hours photoperiod.Five milliliter of sterile water was added to a Petri plate culture.The conidia were gently dislodged from the surface with a sterile glass rod and the suspension was filtered through three layers of cheesecloth to remove mycelia fragments.The suspension was diluted with sterile water to obtain a suspension of 1  10 6 conidia mL -1 .Aliquots of conidia suspension (50 µL) were placed in microtubes containing 500 µL of PDB (Potato Dextrose Broth) medium treated with essential oils, at the concentrations of 10, 50, 100 and 200 ppm, with the additon of Tween 20 (1:1).The control treatment was just PDB with addition of Tween 20 at concentration 200 ppm (similar to the highest concentration used to emulsify the essential oil).The tubes were incubated at 25 °C for 16 hours.The samples were placed on a Neubauer chamber and observed under the microscope for conidia germination.The counting of conidia was done using a light microscopy at 10 magnification.All experiments were conducted in ten replicates and in each replicate were evaluated hundred conidia.The conidia were considered germinated when the length of the germ tube equaled or exceeded the length of the conidia.

Inoculum Preparation
Conidia of B. cinerea and C. acutatum were harvested from a colony of the fungi 14 days grown in PDA at 25 °C temperature and 12 hours photoperiod as described above.The suspension was diluted with sterile water to obtain a suspension of 1  10 6 conidia mL -1 .

Fruit
Traditionally grown and freshly harvested grapes of cultivar "Isabella" from Bento Gonçalves, RS, Brazil were used in in vivo experiments.

Antifungal Activity of Essential Oil on Grapes
To evaluate the antifungal activity of the essential oil on grapes it was carried out experiments with curative and preventive treatments.The postharvest curative treatment consisted of inoculation of 10 berries for each cluster (10 fruit/treatment) of grape, through wounds, approximately 2 mm deep, with the aid of a syringe (Zahavi et al., 2000).After the injury, the clusters were inoculated by spraying the conidia suspension of B. cinerea or C. acutatum, according to the methodology described by Romanazzi, Nigro, Ippolito, Di Venere, and Salermo (2002) and Thomas, Marois, and English (1988) with modifications.After 4 hours, the application of essential oil was carried out with the concentrations based on the in vitro test (50, 100 and 200 ppm).Subsequently, in order to evaluate the potential of oil in preventing disease, the grape clusters were sprinkled with essential oil in the same concentrations of the previous test and inoculated after 24 hours with the fungi.For both experiments, the clusters were placed in plastic boxes and kept at 25 ± 1 °C/80-90% relative humidity for a period of five days for those inoculated with B. cinerea and seven days for those inoculated with C. acutatum.At the end of this period, assessment of the incidence and severity of disease was performed.To evaluate the incidence, ten berries for each bunch of grapes that were inoculated were evaluated and it was used the mean number of berries with symptoms of the disease.For assessing the severity, a scale from 0 to 100% was created in accordance with the berry area affected by the disease.

Statistical Analysis
Data normality was determined by Kolmogorov-Smirnov test and the homogeneity of variances was determined using Levene's test.Data were analyzed by ANOVA and the threshold for statistical significance was set at p < 0.05.In the case of statistical significance Dunnett's T3 test was applied to separate the means.All statistics analysis was performed using SPSS 22.0 for Windows.

Antifungal Activity of Essential Oil on Mycelial Growth
The in vitro antifungal activity of essential oil differed for each fungi and concentration tested at contact phase experiments (Table 2).The effect of essential oil on the mycelial growth of B. cinerea resulted in completely inhibition at concentration 50 ppm and the fungicidal action was observed by the transfer experiment.For the concentration 10 ppm there was a significant inhibition until the 5 th day compared to control, also the mycelial growth presented a different morphology.On the other hand, the completely inhibition of the mycelial growth of C. acutatum occurred at a higher concentrations (100 and 200 ppm) with fungicidal action proven by the transfer experiment.The concentration 10 ppm presented a fungistatic action until the 3 rd day, and the same was observed for concentration 50 ppm until the 10 th day, being significantly different from control.Both concentrations presented a modified morphology for the mycelial growth.B. cinerea 3 rd day 47.75 ± 2.07 a 14.80 ± 1.26 b 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 5 th day 90.00 ± 0.00 a 73.53 ± 2.37 b 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 7 th day 90.00 ± 0.00 a 81.88 ± 3.58 a 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 b 10 th day 90.00 ± 0.00 a 82.53 ± 3.33 a 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 b 14 th day 90.00 ± 0.00 a 82.75 ± 3.28 a 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 b C. acutatum 3 rd day 17.11 ± 0.39 a 13.23 ± 0.60 b 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00 ± 0.00 c 5 th day 39.99 ± 1.38 a 34.50 ± 0.90 a 6.34 ± 2.25 b 0.00 ± 0.00 b 0.00 ± 0.00 b 7 th day 55.08 ± 3.17 The effect of the volatiles compounds in the mycelial growth of B. cinerea showed a total inhibition at 100% concentration (fungicidal action was confirmed by transfer experiment) and for C. acutatum the same concentration reduced the mycelial growth but do not complete inhibited it (Table 3).At the concentration of 25%, growth was complete inhibited until 7 th day, and the concentration of 50% inhibited the growth until 10 th day of B. cinerea, showing that maybe a reapplication of the essential oil could control the growth of the fungus.

Antifungal Activity of Essential Oil on Conidia Germination
Germination of conidia of B. cinerea was inhibited completely at concentration 100 ppm, while the complete inhibition for C. acutatum was at concentration 200 ppm (Figure 1).The concentrations of 10 and 50 ppm

Discuss
The increa to produce volatiles a ( Being lipophilic in nature, essential oil accumulates in plasma membrane, causes swelling of the membrane and makes the membrane proteins inefficient due to increased disorder.This ultimately causes leakage of cell contents and inhibition of cell growth (Helal, Sarhan, Abu Shahla, & Abou El-Khair, 2007).Moreover, each essential oil component makes its own contribution to the biological activity of the oil.The volatile phase of essential oil showed a fungistatic action, partially inhibiting the mycelial growth of fungi.Volatile phase of artemisia, peppermint, basil and thyme essential oils were also reported to posses antimicrobial activity against plant pathogenic fungi (Edris & Farrag, 2003;Soylu et al., 2005).Investigators suggested that the antifungal activity resulted from a direct effect of essential oil vapours on fungal mycelium and postulated that the lipophilic nature of essential oils would make possible for them being absorbed by fungal mycelia (Edris & Farrag, 2003;Inouye et al., 2000).In this work, both fungi hyphae grown on media with essential oils revealed alterations in the morphology.Such modifications may be related to the effect of the essential oil on enzymatic reactions regulating wall synthesis for example (Rasooli, Rezaei, & Allameh, 2013).Soylu et al. (2007) reported the reduction of germination of Sclerotinia sclerotiorum and Aminifard and Mohammadi (2013) reported that conidia germination and germ tube elongation of B. cinerea were inhibited by F. vulgare essential oil.Our results corroborated with that as the essential oil of F. vulgare also inhibited conidia germination of B. cinerea and C. acutatum.Besides inhibiting the mycelial growth, phenolic compounds also affect the enzymes responsible for conidia germination and interfere with amino acids that were necessary in germination processes (Nychas, 1995).
The in vivo test showed that the essential oil of fennel had a positive effect in controlling the incidence of postharvest fungal rots on grapes caused by B. cinerea and C. acutatum.Essential oil inhibits postharvest pathogens mainly due to their direct effect on the mycelial growth of the pathogens and conidia germination by affecting the cellular metabolism of the pathogens (Serrano, Martínez-Romero, Castillo, Guillen, & Valero, 2005;Tzortzakis, 2007aTzortzakis, , 2007b;;Regnier, Combrinck, Du Ploov, & Botha, 2010).Prior studies of fennel essential oil has shown better efficacy against postharvest fungi including Aspergillus species in different test methods (Singh et al., 2006;Barkat & Bouguerra, 2012;Gemeda, Woldeamanuel, Asrat, & Debella, 2014).According to Abdolari, Hassani, Ghosta, Javadi, and Meshkatalsadat (2010), fennel essential oil had good inhibitory effects on infection caused by Alternaria alternata and Penicillium digitatum in postharvest tomato fruits.According to Aminifard and Mohammadi (2013), fennel essential oil inhibited B. cinerea growth on plum fruits compared with the control.Lopez-Reyes, Spadaro, Gullinoa, and Garibaldua (2010) proved that the essential oil of fennel has antifungal activity as postharvest treatments against B. cinerea and Penicillium expansum on apples.
Results showed that antifungal activities of essential oils were different under in vitro and in vivo conditions and these activities were higher under in vitro conditions, requiring a higher concentration of the essential oil in vivo.Dikbas, Kotan, Dadasoglu, and Sahin (2008) noticed that these differences could be attributed to the alternation of site of action of essential oils or alternation in membranes of fungi under in vivo condition.

Conclusions
Considering the inhibition in mycelial growth and germination of conidia of B. cinerea and C. acutatum in vitro, and the reduced incidence of disease symptoms on essential oil treated grapes fruits, we can conclude that F. vulgare (fennel) essential oil could be used as possible biofungicide.However, more studies are required before this essential oil can be recommended as commercial and natural antifungal agent to increase the postharvest storage life of grapes.
Note.Values are the average of ten replicates per treatment ± SE.The letters indicate the comparison among the different essential oil concentrations evaluated in each day (per line).Means followed by same letter do not differ by Dunnett's T3 test (p < 0.05).

Note.
Values are the average of ten replicates per treatment ± SE.The letters indicate the comparison among the different essential oil concentrations evaluated in each day (per line).Means followed by same letter do not differ by Dunnett's T3 test (p < 0.05). Figure

Table 1 .
Chemical composition of essential oil from Foeniculum vulgare fruits

Table 2 .
Effect of different concentrations of Foeniculum vulgare essential oil, added on the solid media, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (contact phase)

Table 3 .
Effect of different concentrations of Foeniculum vulgare essential oil, applied on the lid, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (volatile phase)