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Control of postharvest fungal rots in grapes through the use of Baccharis trimera and Baccharis dracunculifolia essential oils
Journal Pre-proof Control of postharvest fungal rots in grapes through the use of Baccharis trimera and Baccharis dracunculifolia essential oils Carine Pedrotti, Rute Terezinha da Silva Ribeiro, Joséli Schwambach PII:
S0261-2194(19)30258-3
DOI:
https://doi.org/10.1016/j.cropro.2019.104912
Reference:
JCRP 104912
To appear in:
Crop Protection
Received Date: 7 November 2018 Revised Date:
24 July 2019
Accepted Date: 1 August 2019
Please cite this article as: Pedrotti, C., Silva Ribeiro, R.T.d., Schwambach, José., Control of postharvest fungal rots in grapes through the use of Baccharis trimera and Baccharis dracunculifolia essential oils, Crop Protection (2019), doi: https://doi.org/10.1016/j.cropro.2019.104912. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Control of postharvest fungal rots in grapes through the use of Baccharis trimera and
2
Baccharis dracunculifolia essential oils
3
Carine Pedrotti*, Rute Terezinha da Silva Ribeiro and Joséli Schwambach
4
*
5
of Biotechnology, University of Caxias do Sul, Rua Francisco Getúlio Vargas, 1130 –
6
Petrópolis,
7
[email protected]
8
Keywords
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Alternative control, Essential oil, Grape, Botrytis cinerea, Colletotrichum acutatum
Laboratory of Plant Disease Control and Laboratory of Plant Biotechnology, Institute
95070-560,
Caxias
do
Sul,
RS,
Brazil.
E-mail:
10
Abstract
11
Postharvest diseases cause considerable losses during transportation and storage.
12
Synthetic fungicides are primarily used to control postharvest disease loss, however the
13
development of new and alternative agrochemicals is necessary. In this study, the effect
14
of Baccharis trimera and Baccharis dracunculifolia essential oils (EOs) to control of
15
postharvest fungal rots in grapes was evaluated. The chemical composition and the
16
antifungal activity of B. trimera and B. dracunculifolia EOs and the effect of EOs
17
against Botrytis cinerea and Colletotrichum acutatum was determinate in vitro by
18
mycelial growth (contact and volatile phase) and conidia germination. The in vivo
19
efficacy study consisted of spraying the EO in harvested grapes of Vitis labrusca × Vitis
20
vinifera cv. “Isabela” followed by inoculation with the fungus. The major compound
21
found in B. trimera essential oil (BtEO) was carquejyl acetate and in B. dracunculifolia
22
essencial oil (BdEO) were β-pinene, ledol, spathulenol and limonene. For in vitro tests,
23
BdEO showed a fungistatic action, whereas as BtEO showed fungicidal action. For this
24
reason, the later was selected for in vivo testing. For in vivo test, the all concentrations
25
of EO (200, 400 and 600 ppm (µL mL-1) were efficient, reducing the incidence and
26
severity of disease caused by B. cinerea and C. acutatum, both as preventive and
27
curative treatments. These results are promising and indicate that the BtEO might be
28
further investigated as natural alternative to synthetic fungicides for the control of rots
29
on grapes diseases.
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1.
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Grape is one of the most important fruit crops worldwide. In Brazil, Vitis vinifera and
32
Vitis labrusca are common cultivated species. “Isabela” (Vitis labrusca × Vitis vinifera)
33
is the most widely planted variety in Serra Gaúcha, the southern viticultural region of
34
Brazil. This cultivar is used to make red table wine and juice as well as being
35
commercialized as table grape (Mello, 2014; Silveira et al., 2015). Postharvest decay in
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the supply chain has been identified as a major factor causing fruit loss which could
37
result in significant economic loss, especially in the fruit marketing chain, due to
38
previously established infections, such as latent and quiescent infections and incipient
39
infections occurring through wounds resulting from harvesting operations (Prusky,
40
2011).
41
Botrytis cinerea Pers. Fr. and Colletotrichum acutatum Simmonds cause fungal rot and
42
are considered the main causal agents of postharvest disease in table grapes (Droby and
43
Lichter, 2004; Steel et al., 2007; Whitelaw-Weckert et al., 2007). As a postharvest
44
treatment, grapes are usually fumigated with sulfur dioxide fumigation during storage
45
(Droby and Lichter, 2004). However, the use of synthetic fungicides and sulfur dioxide
46
is not allowed for organic food (Gabler and Smilanick, 2001). In addition, growing
47
public concerns about health and environmental hazards associated with pesticide use
48
have resulted in a considerable interest in developing alternative non-polluting control
49
methods (Youssef and Roberto, 2014). Among the possibilities of alternative control it
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is the use of essential oils (EOs), known for their antimicrobial and biodegradable
Introduction
51
properties and for not leaving any residual effect on fresh produce (Isman, 2000; Burt,
52
2004; Bakkali et al., 2008).
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The genus Baccharis L. (Asterales, Asteraceae, tribe Asterae, sub-tribe Baccharidinae)
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comprises around 500 species, with significant popular use in South America as natural
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medicinal product (Verdi et al., 2005). B. trimera (Less) is widely distributed in Brazil
56
and has been extensively studied for its chemical composition and biological activity
57
including antifungal activity (Caneschi et al., 2015). B. dracunculifolia (D.C.) is also a
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native plant from Brazil, with a variety of chemical compounds and pharmacological
59
activities, including antifungal properties (Oliveira et al., 2015). This study evaluated
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the effectiveness of Baccharis trimera essential oil (BtEO) and Baccharis
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dracunculifolia essential oil (BdEO) on the inhibition in vitro of mycelial growth and
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conidia germination of B. cinerea and C. acutatum and the control to postharvest grape
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rot in vivo.
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2.
Materials and methods
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2.1.
Fungal isolation
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Strains of B. cinerea (A58/09) and C. acutatum (A009/13) used in this work were
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isolated from grapes in Caxias do Sul (Serra Gaúcha, RS, Brazil) and preserved in the
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fungal collection of the Laboratory of Phytopathology, University of Caxias do Sul -
69
Brazil, on PDA (Potato Dextrose Agar) medium. The molecular confirmation of both
70
fungi was conducted using Internal Transcribed Sequence (ITS)-PCR identification.
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The DNA extraction was according to Murray and Thompson (1980) and ITS-PCR
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amplified the region ITS-5.8S rDNA according to White et al. (1990). Sequencing was
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performed at the Human Genome Center – USP. The sequences obtained were edited
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with the software BioEdit Sequence Alignment Editor (1997-2005) and used to search
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for similar sequences using Blastn at NCBI.
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2.2. Plant material
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Leaves of B. trimera and B. dracunculifolia were collected from plants found in Bento
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Gonçalves, RS, Brazil. A voucher specimen of each plant species was deposited in the
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Herbarium of the University of Caxias do Sul accession number 43211 for B. trimera
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and accession number 43210 for B. dracunculifolia.
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2.3. Essential oils extraction and analysis
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EOs were extracted by steam distillation from dried plant leaves for 1 hour according to
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Cassel et al. (2009). Protocol described by Tomazoni et al. (2018) was used for
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identification and quantification of compounds in the EOs, using HP 6890 gas
85
chromatograph (GC) coupled with a Hewlett Packard MSD5973 mass selective (MS)
86
detector equipped with HP Chemstation software and Wiley 275 mass spectra data. The
87
analyses were conducted using a HP-Innowax fused silica capillary column (30 m ×
88
0.25 mm i.d., 0.25 µm film thickness, Hewlett Packard, Palo Alto, USA). The
89
constituents of the oils were identified by comparing their mass spectra with those of
90
the Wiley library (GC / MS) and comparing the practical linear retention index with
91
literature data (Nist). The linear retention index was calculated using the Van den Dool
92
and Krats equation using a standard solution of C8 to C26 hydrocarbons. The relative
93
percentage of each component was obtained from chromatographic peak areas,
94
assuming the sum of all eluted peaks being 100%.
95
2.4.In vitro antifungal assay
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2.4.1. Antifungal activity of essential oil on mycelial growth
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The antifungal properties of EOs were assessed for contact and volatile phase effects.
98
Contact phase effect of EOs was tested according to Pedrotti et al. (2017).
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Concentrations of EOs ranging from 100 to 700 ppm (µL mL-1), with the addition of
100
Tween 20 (1:1), diluted on autoclaved and melting PDA (Potato Dextrose Agar) (40ºC)
101
under sterile conditions were used for both fungi. The control treatment was PDA
102
medium with addition of Tween 20 equal to the highest concentration used to emulsify
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the EO. These emulsions were poured into 9 cm diameter Petri dishes and after medium
104
solidification, inoculated with 5 mm diameter agar disks colonized by B. cinerea or C.
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acutatum from 7 days pre-cultures.
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Volatile phase effect of EOs was conduced according Pedrotti et al. (2017) to determine
107
the action of EOs on the mycelial growth of fungi. Briefly, agar disks (5 mm diameter)
108
colonized by B. cinerea or C. acutatum from 7 days pre-cultures were placed in the
109
center of the Petri dish containing PDA culture medium. EOs concentrations were 12.5,
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25, 50 (with the addition of Tween 20 (0.1%)) and 100% (pure EO, without addition of
111
Tween 20). A 100 µL sample of the EOs was placed onto a cotton ball attached to the
112
inner face of a Petri dish lid, thereby preventing direct contact of the EO with the
113
culture medium and the mycelium disk creating a saturated atmosphere of volatile
114
compounds. The control treatment was PDA medium and 100 µL of Tween 20 (0.1%)
115
applied onto a cotton ball.
116
In both tests, for each concentration, ten replicate plates were used. Plates were
117
incubated performed at 25ºC with a 12 hours photoperiod for 14 days. Fungal growth
118
was recorded at 3, 5, 7, 10 and 14 days by measurement of the orthogonal diameter.
119
2.4.3 Transfer experiments
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Transfer experiments were performed to provide a distinction between the fungistatic
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and fungicidal effects of EOs on the target microorganisms. For this purpose, mycelial
122
plugs that did not grow were transferred to fresh PDA dishes to assess their viability and
123
growth after 5 days at 25°C. The fungal growth was measured.
124
2.4.4. Antifungal activity of essential oil on conidia germination
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Antifungal activity of EOs on conidia germination was tested according to Pedrotti et al.
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(2017). Conidia of B. cinerea and C. acutatum were harvested from a 14-day-old colony
127
growing on PDA at 25ºC with a 12 hour photoperiod. The conidia were gently
128
dislodged by add in 5 ml sterile water and scraping the surface with a sterile glass rod,
129
the suspension was filtered through three layers of cheesecloth to remove mycelia
130
fragments. Conidial concentration was determined using a hemocytometer under a
131
microscope and adjusted to 1 × 106 conidia mL-1. Aliquots of conidia suspension (50
132
µL) were placed in microtubes containing 500 µL of PDB (Potato Dextrose Broth)
133
medium treated with EOs at 100, 200, 300 and 400 ppm, with the addition of Tween 20
134
(1:1). The control treatment was PDB with addition of Tween 20 (similar to the highest
135
concentration used to emulsify the EOs). The tubes were incubated at 25ºC for 16 hours.
136
The samples were placed on a hemocytometer chamber and germination was observed
137
under a microscope at 10 × magnification. All experiments were conducted with 10
138
replicates and 100 conidia were evaluated in each replicate. The conidia were
139
considered germinated when the length of the germ tube equaled or exceeded the length
140
of the conidia.
141
2.5. In vivo antifungal assay
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2.5.1. Inoculum preparation
143
Conidia of B. cinerea and C. acutatum were harvested from a 14-day-old colony
144
growing on PDA at 25ºC with a 12 hour photoperiod as described above.
145
suspension was diluted with sterile water to obtain a suspension of 1 × 106 conidia mL-1.
146
2.5.2. Fruit
147
Grapes (Vitis labrusca × Vitis Vinifera “Isabela”) conventionally grown in Bento
148
Gonçalves, RS, Brazil were used in experiments. The grapes were collected in the
149
morning and the test conducted on the same day.
The
150
2.5.3. Antifungal activity of essential oil in grapes
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The antifungal activity of BtEO on grapes was evaluated as curative and preventive
152
treatments according to the methodology described by Pedrotti et al. (2017). Wounds
153
with as size approximately 2 mm deep were made 10 berries in clusters of grape. After
154
wounding, in the postharvest curative treatment, a conidia suspension of B. cinerea or
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C. acutatum was inoculated (10 µL in each wound). After 4 hours, grape clusters were
156
sprayed with EO concentrations of 200, 400 and 600 ppm. To evaluate the potential
157
preventive effect, after wounding, same concentrations of EO were sprayed in grape
158
clusters and 24 hours later inoculation was made as described above. For both
159
experiments, the grapes were placed in plastic boxes (30 cm wide × 40 cm long × 15 cm
160
high) and, the boxes were incubated at 25 ± 1° C / 80-90% relative humidity with a 16
161
hour photoperiod for 5 days for those inoculated with B. cinerea and 7 days for those
162
inoculated with C. acutatum. After the incubation, disease incidence and severity were
163
assessed. For incidence, 10 inoculated berries from each cluster of grapes were
164
evaluated and disease incidence was calculated. For severity, decayed area on the
165
surface of grape berry was visually evaluated using a scale from 0 to 100% as described
166
previously (Pedrotti et al., 2017).
167
2.6.Statistical analysis
168
Data normality was determined by a Kolmogorov-Smirnov test and homogeneity of
169
variances was determined using Levene’s test. Data were analyzed by ANOVA and the
170
threshold for statistical significance was set at P < 0.05. In the case of statistical
171
significance, Dunnett’s T3 test was applied to separate the means. All statistical analysis
172
was performed using SPSS 22.0 for Windows.
173
3. Results
174
3.1. Chemical composition of the essential oil
175
The number of compounds and the relative amount of each found in EOs varied
176
according to plant species and the particular compound (Table 1). The major compound
177
found in BtEO was carquejyl acetate (67.48%) and 18 other component present in lower
178
amounts. The majority of these compounds (79.29%) correspond to monoterpenes
179
(9.96% hydrocarbons and 69.33% oxygenated) and 10.15% were sesquiterpenes (2.26%
180
hydrocarbons and 7.89% oxygenated). The major compounds found in BdEO were β-
181
pinene (18.01%), ledol (13.55%), spathulenol (13.43%) and limonene (10.11%).
182
Seventeen other components were present in lower amounts, of which 39.61%
183
correspond to monoterpenes (36.44% hydrocarbons and 3.17% oxygenated) and
184
45.47% to sesquiterpenes (8.20% hydrocarbons and 37.27% oxygenated). Dried leaves
185
of B. trimera and B. dracunculifolia yielded 1.08 and 0.11% EO (mL 100g-1 of dried
186
leaves), respectively.
187
3.2. In vitro antifungal effect of B. trimera and B. dracunculifolia essential oils
188
3.2.1. Antifungal activity of essential oils on mycelial growth
189
The in vitro antifungal activity of EOs differed between the fungi, the plant species and
190
among the concentrations tested at contact phase experiments. The effect of BtEO on
191
the mycelial growth of B. cinerea resulted in complete inhibition at concentration 400
192
ppm and above the fungicidal action was confirmed by the transfer experiment, where it
193
was not observed mycelial growth. For the 100 ppm concentration of BtEO there was a
194
significant inhibition until the 7th day and concentrations 200 and 300 ppm there was a
195
significant inhibition until the 14th day, compared to control for B. cinerea (P > 0.05).
196
On the other hand, BtEO there was a fungistatic effect on mycelial growth of C.
197
acutatum which varied according to the concentration tested, at where, to 100 ppm
198
concentration inhibited growth until the 10th day compared the control. For
199
concentrations 300, 500 and 700 ppm, a significant inhibition was observed until the
200
14th day compared to control (Table 2).
201
BdEO had only a fungistatic effect on the mycelial growth of B. cinerea and C.
202
acutatum (Table 3). For B. cinerea, 100 ppm concentration of BdEO there was a
203
significant inhibition until the 7th day. The 200 and 300 ppm concentrations inhibited
204
until the 10th day compared to control. Concentration 400 ppm there was a significant
205
inhibition until the 14th day compared to control. For C. acutatum, all concentrations
206
tested (100, 300, 500 and 700 ppm) a significant inhibition was observed until the 3rd,
207
10th and 14th day compared to control and, no differences were observed in 5th and 7th
208
day compared to control.
209
The effect of volatiles of BtEO on the mycelial growth of B. cinerea at the
210
concentrations of 50% and 100% there was a significant inhibition until 14th day
211
compared to control. For C. acutatum, concentrations 12.5 and 25% inhibited growth
212
until 7th day. Concentrations of 50 and 100% inhibited growth until the 14th day
213
compared to control (Table 4). Volatiles compounds of BdEO reduced the mycelial
214
growth of B. cinerea at the concentration of 100% until the 14th day compared to the
215
control. For C. acutatum, all concentrations presented a significant inhibition until 5rd
216
day compared to control (Table 5).
217
3.2.2. Antifungal activity of essential oils on conidia germination
218
BtEO inhibited completely conidia germination of B. cinerea at the highest
219
concentration 400 ppm. At concentrations 100, 200 and 300 ppm a significant reduction
220
in the germination of conidia was observed. The conidia germination of C. acutatum
221
was completely inhibited at the lowest concentration (100 ppm) (Fig. 1 A). BdEO was
222
unable to completely inhibit conidia germination of both fungi. BdEO reduced the
223
conidia germination of B. cinerea at all concentrations when compared to control.
224
However, BdEO did not reduce the conidia germination of C. acutatum (Fig. 1 B).
225
3.3. Antifungal activity of essential oil in postharvest grapes
226
From the results obtained in in vitro tests, BtEO was selected for in vivo tests in
227
postharvest grapes. Different concentrations of the EO were efficient, reducing the
228
incidence and severity of disease caused by B. cinerea and incidence of disease caused
229
by C. acutatum, both in preventive and curative treatment. As a preventive treatment,
230
the 400 and 600 ppm concentrations reduce incidence of B. cinerea when compared to
231
the control. The preventive treatment also reduced disease severity but only at the
232
highest concentration (600 ppm) (Fig. 2 A). In a curative treatment all EO
233
concentrations (200, 400 and 600 ppm) reduced incidence of B. cinerea when compared
234
to the control. The curative treatment reduced disease severity starting at the lowest
235
concentration (200 ppm) (Fig. 2 B).
236
As a preventive treatment, the 400 and 600 ppm concentrations reduced the incidence of
237
C. acutatum when compared to control (Fig. 3 A). As a curative treatment, all EO
238
concentrations reduced disease incidence when compared to control (Fig. 3 B). The
239
severity of disease caused by C. acutatum, in preventive and curative treatment, was
240
unaffected by BtEO compared to control (Fig. 3 A and B).
241
Discussion
242
EOs are complex, volatile and plant compounds, known for antiseptic, bactericidal and
243
fungicidal characteristics (Bakkali et al., 2008). Several studies have explored the
244
potential of EOs as antifungal agents (Arici et al., 2011; Badea and Delian, 2014).
245
Simões-Pires et al. (2005) and Besten et al. (2013) found carquejyl acetate (35.5 to 68%
246
and 40.7 to 73.5%, respectively) as major compound of BtEO collected in different
247
places of southern Brazil, similarly to the results here presented. Thus, we can observe
248
that the composition of BtEO is highly specific species, independent of the influence of
249
environmental factors. Parreira et al. (2010) found spathulenol, β-pinene and limonene
250
in BdEO, but not as major compounds. Thus, for BdEO we observed that the
251
composition is dependent of the influence of environmental factors, with geographical
252
origins and harvest seasons (Simões-Pires et al., 2005; Díaz-Maroto et al., 2006).
253
Oliveira et al. (2015) evaluated BdEO activity against Candida albicans and
254
demonstrated that it had antifungal activity at high concentrations. Duarte et al. (2005)
255
also tested BdEO against C. albicans and found that it had a low fungicidal activity. In
256
this study, BdEO had a fungistatic effect at high concentrations on both fungi,
257
demonstrating its low fungicidal activity, when added to the solid media (contact
258
phase). The BtEO tested against Trichophyton rubrum and Microsporum canise
259
exhibited fungicide potential (Caneschi et al., 2015), corroborating with results obtained
260
in this study, where BtEO demonstrated they fungicidal activity against B. cinerea,
261
when added to the solid media (contact phase). Tian et al. (2012) suggested that the
262
effect of different EOs on microbial growth might be the result of phenolic compounds
263
and terpenoids present in the EOs altering microbial cell permeability, causing
264
deformation of the cell structure, functionality and permiting the loss of
265
macromolecules from the cell interior causing inhibition of cell growth.
266
Lombardo et al. (2016) assessed the bioactivity of the volatile compounds of BtEO and
267
BdEO in the control of Phyllosticta citricarpa and demonstrated that BtEO inhibited
268
mycelial growth whereas BdEO had lower inhibition, similarly to the results here
269
presented. Thus, we can conclude that the volatile compounds of the BtEO and BdEO
270
have a low fungicidal effect (volatile phase), but when the EOS were applied in the
271
culture medium (contact phase), they presented has greater capacity to control of fungal
272
mycelial growth. Moreover, we confirmed that the BtEO has greater effect antifungal on
273
mycelial growth control of B. cinerea and C. acutatum than BdEO.
274
Nychas (1995) suggested that besides inhibiting the mycelial growth, compounds of
275
EOs also affect the enzymes responsible for conidia germination and interfere with
276
amino acids that are necessary in the germination processes. Several studies
277
demonstrate the capabilities of different EOs to inhibit conidia germination of B.
278
cinerea and C. acutatum (Alzate et al., 2009; Soylu et al., 2010; Pedrotti et al., 2017). In
279
this study, BtEO inhibited completely conidia germination of B. cinerea and C.
280
acutatum, but BdEO was unable to completely inhibit conidia germination of both
281
fungi, demonstrating that the BtEO has greater effect antifungal on conidia germination
282
control of B. cinerea and C. acutatum than BdEO.
283
The greater inhibition when compared to BdEO, on mycelial growth and conidia
284
germination of B. cinerea and C. acutatum by BtEO is probably due to monoterpenes,
285
such, carquejyla acetate and β-pinene. This class of substances present in the Asteraceae
286
family is related to antifungal activity (Tabassum and Vidyasagar, 2013; Caneschi et al.,
287
2015).
288
EO inhibits postharvest pathogens mainly due to direct effects on the mycelial growth
289
and conidia germination by affecting the cellular metabolism of the pathogens (Serrano
290
et al., 2005; Regnier et al., 2010). In this study, we evaluated the effect of BtEO in
291
postharvest grapes in the curative treatment, when the grapes were inoculated with the
292
pathogens before the application of the BtEO treatment (to simulate pre-existing
293
infections) and in the preventive treatment, when the inoculation was carried out
294
afterwards to treatments with application of the BtEO (to simulate possible re-infections
295
of grapes during handling or storage). From the results obtained, we can observe that
296
BtEO was able to reduced the incidence and severity of disease caused by B. cinerea
297
and incidence of disease caused by C. acutatum, both in preventive and curative
298
treatment. Demonstrating that, BtEO was efficient in control of postharvest fungal rots
299
diseases in grapes and, can be applied in postharvest chain in the storage or packaging
300
process of grapes. According to Tripathi et al. (2008) EOs of Ocimum sanctum, Prunus
301
persica and Zingiber officinale had inhibitory effects on infection caused by B. cinerea
302
in postharvest grapes fruits. Similarly Pedrotti et al. (2017) proved that Foeniculum
303
vulgare EO controlled the incidence of postharvest fungal rots on grapes caused by B.
304
cinerea and C. acutatum, corroborating the results obtained in this study.
305
4. Conclusions
306
These results demonstrate the in vitro and the in vivo antifungal activities of B. trimera
307
EO against B. cinerea and C. acutatum and its potential use as biological fungicide for
308
the control of postharvest fungal rots diseases caused by these micro-organisms on
309
grapes fruits. However, more studies are required before B. trimera EO is recommended
310
as commercial and natural antifungal agent to increase the postharvest storage life of
311
grapes.
312
Acknowledgement
313
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de
314
Nível Superior - Brasil (CAPES) - Finance Code 001.
315
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Tomazoni, E.Z., Griggio, G.S., Broilo, E.P., Ribeiro, R.T.S., Soares, G.L.G., 2018 Screening for inhibitory activity of essential oils on fungal tomato pathogen Stemphylium solani Weber. Biocatalysis and Agricultural Biotechnology 16, 364372. Verdi, L.G., Brighent, I.M.C., Pizzolatti, M.G., 2005. Gênero Baccharis (Asteraceae): aspectos químicos, econômicos e biológicos. Quimica Nova. 28, 85–94. White, T.J., Bruns, T., Lee, S., Taylor, J.W., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: INNIS, M. A. (Eds.). PCR protocols: a guide to methods and applications. San Diego: Academic. 315-322. Whitelaw-Weckert, M.A., Curtin, S.J., Huang, R., Steel, C.C., Blanchard, C.L., Roffey, P.E., 2007. Phylogenetic relationships and pathogenicity of Colletotrichum acutatum isolates from grape in subtropical Australia. Plant Pathology. 56, 448–463. Youssef, K., Roberto, S.R., 2014. Applications of salt solutions before and after harvest affect the quality and incidence of postharvest gray mold of ‘Italia’ table grapes. Postharvest Biology and Technology. 87, 95–102.
453 Table 1 Chemical composition of essential oils from Baccharis trimera and Baccharis dracunculifolia. Compounds
RI ¹
RA ² B. trimera
B. dracunculifolia 36.44 4.02 18.01 0.89 1.65 10.11 1.76
Monoterpene Hydrocarbons α-pinene α-thujene Camphene β-pinene Sabinene Myrcene Limonene Cis-β-ocimene
14.209 14.576 15.768 19.302 21.383 21.895 24.024 26.960
9.96 6.20 0.98 0.31 1.45 0.67 0.38
Oxygenated monoterpenes β-isophorone Camphor Linalool δ-isopulegol Myrtenal Carquejyl acetate
34.169 39.776 40.734 43.144 43.548 47.993
69.33 0.17 1.68 67.48
3.17 1.21 1.54 0.42 -
Sesquiterpene Hydrocarbons α-caryophyllene Germacrene D Byciclogermacrene δ-cadinene α-curcumene
46.126 47.686 48.649 49.432 50.158
2.26 2.26 -
8.20 0.33 2.53 3.88 1.23 0.23
Oxygenated sesquiterpenes Caryophylene oxide Globulol Palustrol Ledol Germacrene-δ-4-ol Viridiflorol Spathulenol Ledene oxide β-eudesmol
56.964 58.238 55.669 57.971 58.092 58.838 59.563 61.586 61.700
7.89 0.18 2.41 3.12 0.67 0.49 0.44 0.58
37.25 2.12 0.84 0.19 13.55 5.39 13.43 1.73 -
Others 0.85 0.00 Trans-pinocarvyl acetate 45.179 0.14 Cryptone 46.205 0.71 ¹ RI, retention index determined relative to n-alkanes (C8–C20). ² RA, Relative amounts of the compounds identified based on the area of each peak in the total chromatogram area.
454
Table 2 Effect of different concentrations of Baccharis trimera essential oil, added to the solid media, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (contact phase). B. cinerea Essential oil concentrations Day
0
100
200
300
400 (ppm)
3ʳ ͩ
43.71 ± 2.06 a
0.00 ± 0.00 b
0.00 ± 0.00 b
0.00 ± 0.00 b
0.00 ± 0.00 b
5ͭͪ
86.57 ± 3.43 a
10.93 ± 0.20 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
7ͭͪ
87.24 ± 2.76 a
33.78 ± 1.89 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
10 ͭ ͪ
90.00 ± 0.00 a
68.24 ± 4.77 a
13.55 ± 2.06 b
0.00 ± 0.00 c
0.00 ± 0.00 c
14 ͭ ͪ
90.00 ± 0.00 a
89.56 ± 0.19 a
31.19 ± 4.40 b
13.22 ± 3.27 bc 0.00 ± 0.00 c
C. acutatum Essential oil concentrations Day
0
100
300
500
700 (ppm)
3ʳ ͩ
28.02 ± 0.54 a
0.00 ± 0.60 b
0.00 ± 0.00 b
0.00 ± 0.00 b
0.00 ± 0.00 b
5ͭͪ
38.78 ± 0.67 a
10.22 ± 0.40 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
7ͭͪ
73.24 ± 1.19 a
29.56 ± 2.62 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
10 ͭ ͪ
86.47 ± 1.83 a
42.84 ± 3.24 b
10.44 ± 0.27 c
0.00 ± 0.00 d
0.00 ± 0.00 d
14 ͭ ͪ
90.00 ± 0.00 a
66.06 ± 5.81 a
17.81 ± 1.18 b
10.50 ± 0.18 b
8.90 ± 0.54 b
*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).
455 Table 3 Effect of different concentrations of Baccharis dracunculifolia essential oil, added to the solid media, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (contact phase). B. cinerea Essential oil concentrations Day
0
100
200
300
400 (ppm)
3ʳ ͩ
57.81 ± 1.37 a
22.07 ± 1.99 b
12.06 ± 0.32 b
12.97 ± 1.12 b
11.43 ± 0.62 b
5ͭͪ
78.89 ± 2.82 a
32.35 ± 1.05 b
21.88 ± 0.47 c
20.57 ± 0.98 c
17.67 ± 0.42 c
7ͭͪ
90.00 ± 0.00 a
53.95 ± 3.56 b
33.80 ± 0.75 c
32.81 ± 1.48 c
30.99 ± 1.74 c
10 ͭ ͪ
90.00 ± 0.00 a
68.49 ± 4.37 a
45.22 ± 1.25 b
45.49 ± 3.00 b
39.76 ± 1.97 b
14 ͭ ͪ
90.00 ± 0.00 a
80.79 ± 4.25 a
70.51 ± 2.61 ab 64.36 ± 3.23 ab 54.89 ± 3.34 b
C. acutatum Essential oil concentrations Day
0
100
300
500
700 (ppm)
3ʳ ͩ
27.40 ± 0.38 a
18.50 ± 0.48 b
14.75 ± 0.72 b
13.30 ± 0.65 b
12.84 ± 0.49 b
5ͭͪ
42.80 ± 1.48 a
29.05 ± 3.41 a
25.11 ± 2.19 ab 21.82 ± 1.72 ab 20.08 ± 1.43 ab
7ͭͪ
51.30 ± 3.10 a
35.44 ± 4.66 a
29.95 ± 3.13 ab 27.93 ± 1.89 ab 25.42 ± 1.91 ab
10 ͭ ͪ
80.15 ± 0.75 a
44.65 ± 5.64 b
37.38 ± 3.79 b
34.62 ± 1.93 b
33.46 ± 1.44 b
14 ͭ ͪ
90.00 ± 0.00 a
55.91 ± 6.20 b
46.85 ± 4.58 b
46.38 ± 2.93 b
45.30 ± 1.91 b
*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).
456
Table 4 Effect of different concentrations of Baccharis trimera essential oil, applied on the lid, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (volatile phase). B. cinerea Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
62.88 ± 5.04 a
41.13 ± 5.50 a
34.34 ± 2.13 ab
26.77 ± 2.28 ab
17.44 ± 1.50 b
5ͭͪ
90.00 ± 0.00 a
64.02 ± 4.61 b
54.45 ± 2.66 b
40.85 ± 2.94 b
24.95 ± 1.57 c
7ͭͪ
90.00 ± 0.00 a
77.80 ± 3.49 a
70.66 ± 1.54 ab
52.85 ± 2.77 c
35.04 ± 1.63 d
10 ͭ ͪ
90.00 ± 0.00 a
80.63 ± 3.58 a
74.99 ± 2.66 ab
56.79 ± 2.23 c
41.34 ± 1.20 d
14 ͭ ͪ
90.00 ± 0.00 a
81.11 ± 3.49 a
75.49 ± 2.45 ab
57.89 ± 2.19 c
43.86 ± 1.79 d
C. acutatum Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
29.11 ± 0.35 a
19.83 ± 0.47 b
17.42 ± 0.83 b
15.28 ± 0.40 bc
10.61 ± 0.44 d
5ͭͪ
41.97 ± 0.90 a
28.71 ± 0.79 b
23.58 ± 1.04 b
19.90 ± 0.45 bc
15.31 ± 0.58 d
7ͭͪ
66.25 ± 2.41 a
51.49 ± 1.87 b
41.44 ± 1.03 b
32.31 ± 1.76 bc
24.13 ± 1.37 c
10 ͭ ͪ
74.96 ± 2.67 a
64.36 ± 2.48 a
55.81 ± 0.94 ab
38.91 ± 1.64 bc
30.89 ± 1.21 c
14 ͭ ͪ
83.93 ± 2.93 a
76.03 ± 2.91 a
69.45 ± 2.18 a
45.77 ± 1.59 b
42.71 ± 2.03 b
*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).
457 Table 5 Effect of different concentrations of Baccharis dracunculifolia essential oil, applied on the lid, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (volatile phase). B. cinerea Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
46.45 ± 4.66 a
44.12 ± 5.32 a
38.71 ± 5.02 a
37.26 ± 4.46 a
28.53 ± 3.72 b
5ͭͪ
74.17 ± 1.60 a
67.30 ± 2.45 a
63.01 ± 2.69 a
60.01 ± 2.76 a
52.65 ± 2.06 b
7ͭͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
72.90 ± 1.54 b
10 ͭ ͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
73.95 ± 1.68 b
14 ͭ ͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
76.37 ± 2.45 b
C. acutatum Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
27.77 ± 0.29 a
26.85 ± 0.40 a
26.80 ± 0.15 a
26.16 ± 0.34 a
25.23 ± 0.46 ab
5ͭͪ
43.43 ± 0.43 a
38.31 ± 0.50 b
34.41 ± 1.24 b
33.04 ± 0.74 bc
33.51 ± 0.62 bc
7ͭͪ
75.53 ± 1.75 a
65.42 ± 1.98 a
61.88 ± 1.79 ab
59.98 ± 2.83 ab
59.82 ± 2.27 ab
10 ͭ ͪ
84.53 ± 1.13 a
83.84 ± 1.14 a
76.08 ± 1.52 a
73.61 ± 1.21 ab
72.68 ± 1.02 ab
14 ͭ ͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
*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).
458
459
460 461
Fig. 1 Effect of different concentrations of Baccharis trimera (A) and Baccharis dracunculifolia (B)
462
essential oils on conidia germination of Botrytis cinerea (■) and Colletotrichum acutatum (■). Values are
463
the average of ten replicates per treatment ± SE. Means followed by same letter do not differ by Dunnett's
464
T3 test (p < 0.05).
465 466 467 468
469
470 471
Fig. 2 The effects of different concentrations of Baccharis trimera essential oil applied to grapes for
472
disease control. Incidence (●) and severity (■) of disease caused by Botrytis cinerea as to preventive (A)
473
and curative (B) treatment. Values are the average of ten replicates per treatment ± SE. Means followed
474
by same letter do not differ by Dunnett's T3 test (p < 0.05).
475 476 477 478 479
480
481 482
Fig. 3 The effects of different concentrations of Baccharis trimera essential oil applied to grapes for
483
disease control. Incidence (●) and severity (■) of disease caused by Colletotrichum acutatum as to
484
preventive (A) and curative (B) treatment. Values are the average of ten replicates per treatment ± SE.
485
Means followed by same letter do not differ by Dunnett's T3 test (p < 0.05).
Highlights •
Essential oils of Baccharis trimera and Baccharis dracunculifolia proved an effective antifungal agent against Botrytis cinerea and Colletotrichum acutatum in vitro.
•
The essential oil of Baccharis trimera proved effective against postharvest fungal rots in grapes.
•
Protection of grapes during storage is possible using essential oil as a natural fungicide.
S0261-2194(19)30258-3
DOI:
https://doi.org/10.1016/j.cropro.2019.104912
Reference:
JCRP 104912
To appear in:
Crop Protection
Received Date: 7 November 2018 Revised Date:
24 July 2019
Accepted Date: 1 August 2019
Please cite this article as: Pedrotti, C., Silva Ribeiro, R.T.d., Schwambach, José., Control of postharvest fungal rots in grapes through the use of Baccharis trimera and Baccharis dracunculifolia essential oils, Crop Protection (2019), doi: https://doi.org/10.1016/j.cropro.2019.104912. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Control of postharvest fungal rots in grapes through the use of Baccharis trimera and
2
Baccharis dracunculifolia essential oils
3
Carine Pedrotti*, Rute Terezinha da Silva Ribeiro and Joséli Schwambach
4
*
5
of Biotechnology, University of Caxias do Sul, Rua Francisco Getúlio Vargas, 1130 –
6
Petrópolis,
7
[email protected]
8
Keywords
9
Alternative control, Essential oil, Grape, Botrytis cinerea, Colletotrichum acutatum
Laboratory of Plant Disease Control and Laboratory of Plant Biotechnology, Institute
95070-560,
Caxias
do
Sul,
RS,
Brazil.
E-mail:
10
Abstract
11
Postharvest diseases cause considerable losses during transportation and storage.
12
Synthetic fungicides are primarily used to control postharvest disease loss, however the
13
development of new and alternative agrochemicals is necessary. In this study, the effect
14
of Baccharis trimera and Baccharis dracunculifolia essential oils (EOs) to control of
15
postharvest fungal rots in grapes was evaluated. The chemical composition and the
16
antifungal activity of B. trimera and B. dracunculifolia EOs and the effect of EOs
17
against Botrytis cinerea and Colletotrichum acutatum was determinate in vitro by
18
mycelial growth (contact and volatile phase) and conidia germination. The in vivo
19
efficacy study consisted of spraying the EO in harvested grapes of Vitis labrusca × Vitis
20
vinifera cv. “Isabela” followed by inoculation with the fungus. The major compound
21
found in B. trimera essential oil (BtEO) was carquejyl acetate and in B. dracunculifolia
22
essencial oil (BdEO) were β-pinene, ledol, spathulenol and limonene. For in vitro tests,
23
BdEO showed a fungistatic action, whereas as BtEO showed fungicidal action. For this
24
reason, the later was selected for in vivo testing. For in vivo test, the all concentrations
25
of EO (200, 400 and 600 ppm (µL mL-1) were efficient, reducing the incidence and
26
severity of disease caused by B. cinerea and C. acutatum, both as preventive and
27
curative treatments. These results are promising and indicate that the BtEO might be
28
further investigated as natural alternative to synthetic fungicides for the control of rots
29
on grapes diseases.
30
1.
31
Grape is one of the most important fruit crops worldwide. In Brazil, Vitis vinifera and
32
Vitis labrusca are common cultivated species. “Isabela” (Vitis labrusca × Vitis vinifera)
33
is the most widely planted variety in Serra Gaúcha, the southern viticultural region of
34
Brazil. This cultivar is used to make red table wine and juice as well as being
35
commercialized as table grape (Mello, 2014; Silveira et al., 2015). Postharvest decay in
36
the supply chain has been identified as a major factor causing fruit loss which could
37
result in significant economic loss, especially in the fruit marketing chain, due to
38
previously established infections, such as latent and quiescent infections and incipient
39
infections occurring through wounds resulting from harvesting operations (Prusky,
40
2011).
41
Botrytis cinerea Pers. Fr. and Colletotrichum acutatum Simmonds cause fungal rot and
42
are considered the main causal agents of postharvest disease in table grapes (Droby and
43
Lichter, 2004; Steel et al., 2007; Whitelaw-Weckert et al., 2007). As a postharvest
44
treatment, grapes are usually fumigated with sulfur dioxide fumigation during storage
45
(Droby and Lichter, 2004). However, the use of synthetic fungicides and sulfur dioxide
46
is not allowed for organic food (Gabler and Smilanick, 2001). In addition, growing
47
public concerns about health and environmental hazards associated with pesticide use
48
have resulted in a considerable interest in developing alternative non-polluting control
49
methods (Youssef and Roberto, 2014). Among the possibilities of alternative control it
50
is the use of essential oils (EOs), known for their antimicrobial and biodegradable
Introduction
51
properties and for not leaving any residual effect on fresh produce (Isman, 2000; Burt,
52
2004; Bakkali et al., 2008).
53
The genus Baccharis L. (Asterales, Asteraceae, tribe Asterae, sub-tribe Baccharidinae)
54
comprises around 500 species, with significant popular use in South America as natural
55
medicinal product (Verdi et al., 2005). B. trimera (Less) is widely distributed in Brazil
56
and has been extensively studied for its chemical composition and biological activity
57
including antifungal activity (Caneschi et al., 2015). B. dracunculifolia (D.C.) is also a
58
native plant from Brazil, with a variety of chemical compounds and pharmacological
59
activities, including antifungal properties (Oliveira et al., 2015). This study evaluated
60
the effectiveness of Baccharis trimera essential oil (BtEO) and Baccharis
61
dracunculifolia essential oil (BdEO) on the inhibition in vitro of mycelial growth and
62
conidia germination of B. cinerea and C. acutatum and the control to postharvest grape
63
rot in vivo.
64
2.
Materials and methods
65
2.1.
Fungal isolation
66
Strains of B. cinerea (A58/09) and C. acutatum (A009/13) used in this work were
67
isolated from grapes in Caxias do Sul (Serra Gaúcha, RS, Brazil) and preserved in the
68
fungal collection of the Laboratory of Phytopathology, University of Caxias do Sul -
69
Brazil, on PDA (Potato Dextrose Agar) medium. The molecular confirmation of both
70
fungi was conducted using Internal Transcribed Sequence (ITS)-PCR identification.
71
The DNA extraction was according to Murray and Thompson (1980) and ITS-PCR
72
amplified the region ITS-5.8S rDNA according to White et al. (1990). Sequencing was
73
performed at the Human Genome Center – USP. The sequences obtained were edited
74
with the software BioEdit Sequence Alignment Editor (1997-2005) and used to search
75
for similar sequences using Blastn at NCBI.
76
2.2. Plant material
77
Leaves of B. trimera and B. dracunculifolia were collected from plants found in Bento
78
Gonçalves, RS, Brazil. A voucher specimen of each plant species was deposited in the
79
Herbarium of the University of Caxias do Sul accession number 43211 for B. trimera
80
and accession number 43210 for B. dracunculifolia.
81
2.3. Essential oils extraction and analysis
82
EOs were extracted by steam distillation from dried plant leaves for 1 hour according to
83
Cassel et al. (2009). Protocol described by Tomazoni et al. (2018) was used for
84
identification and quantification of compounds in the EOs, using HP 6890 gas
85
chromatograph (GC) coupled with a Hewlett Packard MSD5973 mass selective (MS)
86
detector equipped with HP Chemstation software and Wiley 275 mass spectra data. The
87
analyses were conducted using a HP-Innowax fused silica capillary column (30 m ×
88
0.25 mm i.d., 0.25 µm film thickness, Hewlett Packard, Palo Alto, USA). The
89
constituents of the oils were identified by comparing their mass spectra with those of
90
the Wiley library (GC / MS) and comparing the practical linear retention index with
91
literature data (Nist). The linear retention index was calculated using the Van den Dool
92
and Krats equation using a standard solution of C8 to C26 hydrocarbons. The relative
93
percentage of each component was obtained from chromatographic peak areas,
94
assuming the sum of all eluted peaks being 100%.
95
2.4.In vitro antifungal assay
96
2.4.1. Antifungal activity of essential oil on mycelial growth
97
The antifungal properties of EOs were assessed for contact and volatile phase effects.
98
Contact phase effect of EOs was tested according to Pedrotti et al. (2017).
99
Concentrations of EOs ranging from 100 to 700 ppm (µL mL-1), with the addition of
100
Tween 20 (1:1), diluted on autoclaved and melting PDA (Potato Dextrose Agar) (40ºC)
101
under sterile conditions were used for both fungi. The control treatment was PDA
102
medium with addition of Tween 20 equal to the highest concentration used to emulsify
103
the EO. These emulsions were poured into 9 cm diameter Petri dishes and after medium
104
solidification, inoculated with 5 mm diameter agar disks colonized by B. cinerea or C.
105
acutatum from 7 days pre-cultures.
106
Volatile phase effect of EOs was conduced according Pedrotti et al. (2017) to determine
107
the action of EOs on the mycelial growth of fungi. Briefly, agar disks (5 mm diameter)
108
colonized by B. cinerea or C. acutatum from 7 days pre-cultures were placed in the
109
center of the Petri dish containing PDA culture medium. EOs concentrations were 12.5,
110
25, 50 (with the addition of Tween 20 (0.1%)) and 100% (pure EO, without addition of
111
Tween 20). A 100 µL sample of the EOs was placed onto a cotton ball attached to the
112
inner face of a Petri dish lid, thereby preventing direct contact of the EO with the
113
culture medium and the mycelium disk creating a saturated atmosphere of volatile
114
compounds. The control treatment was PDA medium and 100 µL of Tween 20 (0.1%)
115
applied onto a cotton ball.
116
In both tests, for each concentration, ten replicate plates were used. Plates were
117
incubated performed at 25ºC with a 12 hours photoperiod for 14 days. Fungal growth
118
was recorded at 3, 5, 7, 10 and 14 days by measurement of the orthogonal diameter.
119
2.4.3 Transfer experiments
120
Transfer experiments were performed to provide a distinction between the fungistatic
121
and fungicidal effects of EOs on the target microorganisms. For this purpose, mycelial
122
plugs that did not grow were transferred to fresh PDA dishes to assess their viability and
123
growth after 5 days at 25°C. The fungal growth was measured.
124
2.4.4. Antifungal activity of essential oil on conidia germination
125
Antifungal activity of EOs on conidia germination was tested according to Pedrotti et al.
126
(2017). Conidia of B. cinerea and C. acutatum were harvested from a 14-day-old colony
127
growing on PDA at 25ºC with a 12 hour photoperiod. The conidia were gently
128
dislodged by add in 5 ml sterile water and scraping the surface with a sterile glass rod,
129
the suspension was filtered through three layers of cheesecloth to remove mycelia
130
fragments. Conidial concentration was determined using a hemocytometer under a
131
microscope and adjusted to 1 × 106 conidia mL-1. Aliquots of conidia suspension (50
132
µL) were placed in microtubes containing 500 µL of PDB (Potato Dextrose Broth)
133
medium treated with EOs at 100, 200, 300 and 400 ppm, with the addition of Tween 20
134
(1:1). The control treatment was PDB with addition of Tween 20 (similar to the highest
135
concentration used to emulsify the EOs). The tubes were incubated at 25ºC for 16 hours.
136
The samples were placed on a hemocytometer chamber and germination was observed
137
under a microscope at 10 × magnification. All experiments were conducted with 10
138
replicates and 100 conidia were evaluated in each replicate. The conidia were
139
considered germinated when the length of the germ tube equaled or exceeded the length
140
of the conidia.
141
2.5. In vivo antifungal assay
142
2.5.1. Inoculum preparation
143
Conidia of B. cinerea and C. acutatum were harvested from a 14-day-old colony
144
growing on PDA at 25ºC with a 12 hour photoperiod as described above.
145
suspension was diluted with sterile water to obtain a suspension of 1 × 106 conidia mL-1.
146
2.5.2. Fruit
147
Grapes (Vitis labrusca × Vitis Vinifera “Isabela”) conventionally grown in Bento
148
Gonçalves, RS, Brazil were used in experiments. The grapes were collected in the
149
morning and the test conducted on the same day.
The
150
2.5.3. Antifungal activity of essential oil in grapes
151
The antifungal activity of BtEO on grapes was evaluated as curative and preventive
152
treatments according to the methodology described by Pedrotti et al. (2017). Wounds
153
with as size approximately 2 mm deep were made 10 berries in clusters of grape. After
154
wounding, in the postharvest curative treatment, a conidia suspension of B. cinerea or
155
C. acutatum was inoculated (10 µL in each wound). After 4 hours, grape clusters were
156
sprayed with EO concentrations of 200, 400 and 600 ppm. To evaluate the potential
157
preventive effect, after wounding, same concentrations of EO were sprayed in grape
158
clusters and 24 hours later inoculation was made as described above. For both
159
experiments, the grapes were placed in plastic boxes (30 cm wide × 40 cm long × 15 cm
160
high) and, the boxes were incubated at 25 ± 1° C / 80-90% relative humidity with a 16
161
hour photoperiod for 5 days for those inoculated with B. cinerea and 7 days for those
162
inoculated with C. acutatum. After the incubation, disease incidence and severity were
163
assessed. For incidence, 10 inoculated berries from each cluster of grapes were
164
evaluated and disease incidence was calculated. For severity, decayed area on the
165
surface of grape berry was visually evaluated using a scale from 0 to 100% as described
166
previously (Pedrotti et al., 2017).
167
2.6.Statistical analysis
168
Data normality was determined by a Kolmogorov-Smirnov test and homogeneity of
169
variances was determined using Levene’s test. Data were analyzed by ANOVA and the
170
threshold for statistical significance was set at P < 0.05. In the case of statistical
171
significance, Dunnett’s T3 test was applied to separate the means. All statistical analysis
172
was performed using SPSS 22.0 for Windows.
173
3. Results
174
3.1. Chemical composition of the essential oil
175
The number of compounds and the relative amount of each found in EOs varied
176
according to plant species and the particular compound (Table 1). The major compound
177
found in BtEO was carquejyl acetate (67.48%) and 18 other component present in lower
178
amounts. The majority of these compounds (79.29%) correspond to monoterpenes
179
(9.96% hydrocarbons and 69.33% oxygenated) and 10.15% were sesquiterpenes (2.26%
180
hydrocarbons and 7.89% oxygenated). The major compounds found in BdEO were β-
181
pinene (18.01%), ledol (13.55%), spathulenol (13.43%) and limonene (10.11%).
182
Seventeen other components were present in lower amounts, of which 39.61%
183
correspond to monoterpenes (36.44% hydrocarbons and 3.17% oxygenated) and
184
45.47% to sesquiterpenes (8.20% hydrocarbons and 37.27% oxygenated). Dried leaves
185
of B. trimera and B. dracunculifolia yielded 1.08 and 0.11% EO (mL 100g-1 of dried
186
leaves), respectively.
187
3.2. In vitro antifungal effect of B. trimera and B. dracunculifolia essential oils
188
3.2.1. Antifungal activity of essential oils on mycelial growth
189
The in vitro antifungal activity of EOs differed between the fungi, the plant species and
190
among the concentrations tested at contact phase experiments. The effect of BtEO on
191
the mycelial growth of B. cinerea resulted in complete inhibition at concentration 400
192
ppm and above the fungicidal action was confirmed by the transfer experiment, where it
193
was not observed mycelial growth. For the 100 ppm concentration of BtEO there was a
194
significant inhibition until the 7th day and concentrations 200 and 300 ppm there was a
195
significant inhibition until the 14th day, compared to control for B. cinerea (P > 0.05).
196
On the other hand, BtEO there was a fungistatic effect on mycelial growth of C.
197
acutatum which varied according to the concentration tested, at where, to 100 ppm
198
concentration inhibited growth until the 10th day compared the control. For
199
concentrations 300, 500 and 700 ppm, a significant inhibition was observed until the
200
14th day compared to control (Table 2).
201
BdEO had only a fungistatic effect on the mycelial growth of B. cinerea and C.
202
acutatum (Table 3). For B. cinerea, 100 ppm concentration of BdEO there was a
203
significant inhibition until the 7th day. The 200 and 300 ppm concentrations inhibited
204
until the 10th day compared to control. Concentration 400 ppm there was a significant
205
inhibition until the 14th day compared to control. For C. acutatum, all concentrations
206
tested (100, 300, 500 and 700 ppm) a significant inhibition was observed until the 3rd,
207
10th and 14th day compared to control and, no differences were observed in 5th and 7th
208
day compared to control.
209
The effect of volatiles of BtEO on the mycelial growth of B. cinerea at the
210
concentrations of 50% and 100% there was a significant inhibition until 14th day
211
compared to control. For C. acutatum, concentrations 12.5 and 25% inhibited growth
212
until 7th day. Concentrations of 50 and 100% inhibited growth until the 14th day
213
compared to control (Table 4). Volatiles compounds of BdEO reduced the mycelial
214
growth of B. cinerea at the concentration of 100% until the 14th day compared to the
215
control. For C. acutatum, all concentrations presented a significant inhibition until 5rd
216
day compared to control (Table 5).
217
3.2.2. Antifungal activity of essential oils on conidia germination
218
BtEO inhibited completely conidia germination of B. cinerea at the highest
219
concentration 400 ppm. At concentrations 100, 200 and 300 ppm a significant reduction
220
in the germination of conidia was observed. The conidia germination of C. acutatum
221
was completely inhibited at the lowest concentration (100 ppm) (Fig. 1 A). BdEO was
222
unable to completely inhibit conidia germination of both fungi. BdEO reduced the
223
conidia germination of B. cinerea at all concentrations when compared to control.
224
However, BdEO did not reduce the conidia germination of C. acutatum (Fig. 1 B).
225
3.3. Antifungal activity of essential oil in postharvest grapes
226
From the results obtained in in vitro tests, BtEO was selected for in vivo tests in
227
postharvest grapes. Different concentrations of the EO were efficient, reducing the
228
incidence and severity of disease caused by B. cinerea and incidence of disease caused
229
by C. acutatum, both in preventive and curative treatment. As a preventive treatment,
230
the 400 and 600 ppm concentrations reduce incidence of B. cinerea when compared to
231
the control. The preventive treatment also reduced disease severity but only at the
232
highest concentration (600 ppm) (Fig. 2 A). In a curative treatment all EO
233
concentrations (200, 400 and 600 ppm) reduced incidence of B. cinerea when compared
234
to the control. The curative treatment reduced disease severity starting at the lowest
235
concentration (200 ppm) (Fig. 2 B).
236
As a preventive treatment, the 400 and 600 ppm concentrations reduced the incidence of
237
C. acutatum when compared to control (Fig. 3 A). As a curative treatment, all EO
238
concentrations reduced disease incidence when compared to control (Fig. 3 B). The
239
severity of disease caused by C. acutatum, in preventive and curative treatment, was
240
unaffected by BtEO compared to control (Fig. 3 A and B).
241
Discussion
242
EOs are complex, volatile and plant compounds, known for antiseptic, bactericidal and
243
fungicidal characteristics (Bakkali et al., 2008). Several studies have explored the
244
potential of EOs as antifungal agents (Arici et al., 2011; Badea and Delian, 2014).
245
Simões-Pires et al. (2005) and Besten et al. (2013) found carquejyl acetate (35.5 to 68%
246
and 40.7 to 73.5%, respectively) as major compound of BtEO collected in different
247
places of southern Brazil, similarly to the results here presented. Thus, we can observe
248
that the composition of BtEO is highly specific species, independent of the influence of
249
environmental factors. Parreira et al. (2010) found spathulenol, β-pinene and limonene
250
in BdEO, but not as major compounds. Thus, for BdEO we observed that the
251
composition is dependent of the influence of environmental factors, with geographical
252
origins and harvest seasons (Simões-Pires et al., 2005; Díaz-Maroto et al., 2006).
253
Oliveira et al. (2015) evaluated BdEO activity against Candida albicans and
254
demonstrated that it had antifungal activity at high concentrations. Duarte et al. (2005)
255
also tested BdEO against C. albicans and found that it had a low fungicidal activity. In
256
this study, BdEO had a fungistatic effect at high concentrations on both fungi,
257
demonstrating its low fungicidal activity, when added to the solid media (contact
258
phase). The BtEO tested against Trichophyton rubrum and Microsporum canise
259
exhibited fungicide potential (Caneschi et al., 2015), corroborating with results obtained
260
in this study, where BtEO demonstrated they fungicidal activity against B. cinerea,
261
when added to the solid media (contact phase). Tian et al. (2012) suggested that the
262
effect of different EOs on microbial growth might be the result of phenolic compounds
263
and terpenoids present in the EOs altering microbial cell permeability, causing
264
deformation of the cell structure, functionality and permiting the loss of
265
macromolecules from the cell interior causing inhibition of cell growth.
266
Lombardo et al. (2016) assessed the bioactivity of the volatile compounds of BtEO and
267
BdEO in the control of Phyllosticta citricarpa and demonstrated that BtEO inhibited
268
mycelial growth whereas BdEO had lower inhibition, similarly to the results here
269
presented. Thus, we can conclude that the volatile compounds of the BtEO and BdEO
270
have a low fungicidal effect (volatile phase), but when the EOS were applied in the
271
culture medium (contact phase), they presented has greater capacity to control of fungal
272
mycelial growth. Moreover, we confirmed that the BtEO has greater effect antifungal on
273
mycelial growth control of B. cinerea and C. acutatum than BdEO.
274
Nychas (1995) suggested that besides inhibiting the mycelial growth, compounds of
275
EOs also affect the enzymes responsible for conidia germination and interfere with
276
amino acids that are necessary in the germination processes. Several studies
277
demonstrate the capabilities of different EOs to inhibit conidia germination of B.
278
cinerea and C. acutatum (Alzate et al., 2009; Soylu et al., 2010; Pedrotti et al., 2017). In
279
this study, BtEO inhibited completely conidia germination of B. cinerea and C.
280
acutatum, but BdEO was unable to completely inhibit conidia germination of both
281
fungi, demonstrating that the BtEO has greater effect antifungal on conidia germination
282
control of B. cinerea and C. acutatum than BdEO.
283
The greater inhibition when compared to BdEO, on mycelial growth and conidia
284
germination of B. cinerea and C. acutatum by BtEO is probably due to monoterpenes,
285
such, carquejyla acetate and β-pinene. This class of substances present in the Asteraceae
286
family is related to antifungal activity (Tabassum and Vidyasagar, 2013; Caneschi et al.,
287
2015).
288
EO inhibits postharvest pathogens mainly due to direct effects on the mycelial growth
289
and conidia germination by affecting the cellular metabolism of the pathogens (Serrano
290
et al., 2005; Regnier et al., 2010). In this study, we evaluated the effect of BtEO in
291
postharvest grapes in the curative treatment, when the grapes were inoculated with the
292
pathogens before the application of the BtEO treatment (to simulate pre-existing
293
infections) and in the preventive treatment, when the inoculation was carried out
294
afterwards to treatments with application of the BtEO (to simulate possible re-infections
295
of grapes during handling or storage). From the results obtained, we can observe that
296
BtEO was able to reduced the incidence and severity of disease caused by B. cinerea
297
and incidence of disease caused by C. acutatum, both in preventive and curative
298
treatment. Demonstrating that, BtEO was efficient in control of postharvest fungal rots
299
diseases in grapes and, can be applied in postharvest chain in the storage or packaging
300
process of grapes. According to Tripathi et al. (2008) EOs of Ocimum sanctum, Prunus
301
persica and Zingiber officinale had inhibitory effects on infection caused by B. cinerea
302
in postharvest grapes fruits. Similarly Pedrotti et al. (2017) proved that Foeniculum
303
vulgare EO controlled the incidence of postharvest fungal rots on grapes caused by B.
304
cinerea and C. acutatum, corroborating the results obtained in this study.
305
4. Conclusions
306
These results demonstrate the in vitro and the in vivo antifungal activities of B. trimera
307
EO against B. cinerea and C. acutatum and its potential use as biological fungicide for
308
the control of postharvest fungal rots diseases caused by these micro-organisms on
309
grapes fruits. However, more studies are required before B. trimera EO is recommended
310
as commercial and natural antifungal agent to increase the postharvest storage life of
311
grapes.
312
Acknowledgement
313
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de
314
Nível Superior - Brasil (CAPES) - Finance Code 001.
315
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453 Table 1 Chemical composition of essential oils from Baccharis trimera and Baccharis dracunculifolia. Compounds
RI ¹
RA ² B. trimera
B. dracunculifolia 36.44 4.02 18.01 0.89 1.65 10.11 1.76
Monoterpene Hydrocarbons α-pinene α-thujene Camphene β-pinene Sabinene Myrcene Limonene Cis-β-ocimene
14.209 14.576 15.768 19.302 21.383 21.895 24.024 26.960
9.96 6.20 0.98 0.31 1.45 0.67 0.38
Oxygenated monoterpenes β-isophorone Camphor Linalool δ-isopulegol Myrtenal Carquejyl acetate
34.169 39.776 40.734 43.144 43.548 47.993
69.33 0.17 1.68 67.48
3.17 1.21 1.54 0.42 -
Sesquiterpene Hydrocarbons α-caryophyllene Germacrene D Byciclogermacrene δ-cadinene α-curcumene
46.126 47.686 48.649 49.432 50.158
2.26 2.26 -
8.20 0.33 2.53 3.88 1.23 0.23
Oxygenated sesquiterpenes Caryophylene oxide Globulol Palustrol Ledol Germacrene-δ-4-ol Viridiflorol Spathulenol Ledene oxide β-eudesmol
56.964 58.238 55.669 57.971 58.092 58.838 59.563 61.586 61.700
7.89 0.18 2.41 3.12 0.67 0.49 0.44 0.58
37.25 2.12 0.84 0.19 13.55 5.39 13.43 1.73 -
Others 0.85 0.00 Trans-pinocarvyl acetate 45.179 0.14 Cryptone 46.205 0.71 ¹ RI, retention index determined relative to n-alkanes (C8–C20). ² RA, Relative amounts of the compounds identified based on the area of each peak in the total chromatogram area.
454
Table 2 Effect of different concentrations of Baccharis trimera essential oil, added to the solid media, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (contact phase). B. cinerea Essential oil concentrations Day
0
100
200
300
400 (ppm)
3ʳ ͩ
43.71 ± 2.06 a
0.00 ± 0.00 b
0.00 ± 0.00 b
0.00 ± 0.00 b
0.00 ± 0.00 b
5ͭͪ
86.57 ± 3.43 a
10.93 ± 0.20 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
7ͭͪ
87.24 ± 2.76 a
33.78 ± 1.89 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
10 ͭ ͪ
90.00 ± 0.00 a
68.24 ± 4.77 a
13.55 ± 2.06 b
0.00 ± 0.00 c
0.00 ± 0.00 c
14 ͭ ͪ
90.00 ± 0.00 a
89.56 ± 0.19 a
31.19 ± 4.40 b
13.22 ± 3.27 bc 0.00 ± 0.00 c
C. acutatum Essential oil concentrations Day
0
100
300
500
700 (ppm)
3ʳ ͩ
28.02 ± 0.54 a
0.00 ± 0.60 b
0.00 ± 0.00 b
0.00 ± 0.00 b
0.00 ± 0.00 b
5ͭͪ
38.78 ± 0.67 a
10.22 ± 0.40 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
7ͭͪ
73.24 ± 1.19 a
29.56 ± 2.62 b
0.00 ± 0.00 c
0.00 ± 0.00 c
0.00 ± 0.00 c
10 ͭ ͪ
86.47 ± 1.83 a
42.84 ± 3.24 b
10.44 ± 0.27 c
0.00 ± 0.00 d
0.00 ± 0.00 d
14 ͭ ͪ
90.00 ± 0.00 a
66.06 ± 5.81 a
17.81 ± 1.18 b
10.50 ± 0.18 b
8.90 ± 0.54 b
*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).
455 Table 3 Effect of different concentrations of Baccharis dracunculifolia essential oil, added to the solid media, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (contact phase). B. cinerea Essential oil concentrations Day
0
100
200
300
400 (ppm)
3ʳ ͩ
57.81 ± 1.37 a
22.07 ± 1.99 b
12.06 ± 0.32 b
12.97 ± 1.12 b
11.43 ± 0.62 b
5ͭͪ
78.89 ± 2.82 a
32.35 ± 1.05 b
21.88 ± 0.47 c
20.57 ± 0.98 c
17.67 ± 0.42 c
7ͭͪ
90.00 ± 0.00 a
53.95 ± 3.56 b
33.80 ± 0.75 c
32.81 ± 1.48 c
30.99 ± 1.74 c
10 ͭ ͪ
90.00 ± 0.00 a
68.49 ± 4.37 a
45.22 ± 1.25 b
45.49 ± 3.00 b
39.76 ± 1.97 b
14 ͭ ͪ
90.00 ± 0.00 a
80.79 ± 4.25 a
70.51 ± 2.61 ab 64.36 ± 3.23 ab 54.89 ± 3.34 b
C. acutatum Essential oil concentrations Day
0
100
300
500
700 (ppm)
3ʳ ͩ
27.40 ± 0.38 a
18.50 ± 0.48 b
14.75 ± 0.72 b
13.30 ± 0.65 b
12.84 ± 0.49 b
5ͭͪ
42.80 ± 1.48 a
29.05 ± 3.41 a
25.11 ± 2.19 ab 21.82 ± 1.72 ab 20.08 ± 1.43 ab
7ͭͪ
51.30 ± 3.10 a
35.44 ± 4.66 a
29.95 ± 3.13 ab 27.93 ± 1.89 ab 25.42 ± 1.91 ab
10 ͭ ͪ
80.15 ± 0.75 a
44.65 ± 5.64 b
37.38 ± 3.79 b
34.62 ± 1.93 b
33.46 ± 1.44 b
14 ͭ ͪ
90.00 ± 0.00 a
55.91 ± 6.20 b
46.85 ± 4.58 b
46.38 ± 2.93 b
45.30 ± 1.91 b
*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).
456
Table 4 Effect of different concentrations of Baccharis trimera essential oil, applied on the lid, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (volatile phase). B. cinerea Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
62.88 ± 5.04 a
41.13 ± 5.50 a
34.34 ± 2.13 ab
26.77 ± 2.28 ab
17.44 ± 1.50 b
5ͭͪ
90.00 ± 0.00 a
64.02 ± 4.61 b
54.45 ± 2.66 b
40.85 ± 2.94 b
24.95 ± 1.57 c
7ͭͪ
90.00 ± 0.00 a
77.80 ± 3.49 a
70.66 ± 1.54 ab
52.85 ± 2.77 c
35.04 ± 1.63 d
10 ͭ ͪ
90.00 ± 0.00 a
80.63 ± 3.58 a
74.99 ± 2.66 ab
56.79 ± 2.23 c
41.34 ± 1.20 d
14 ͭ ͪ
90.00 ± 0.00 a
81.11 ± 3.49 a
75.49 ± 2.45 ab
57.89 ± 2.19 c
43.86 ± 1.79 d
C. acutatum Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
29.11 ± 0.35 a
19.83 ± 0.47 b
17.42 ± 0.83 b
15.28 ± 0.40 bc
10.61 ± 0.44 d
5ͭͪ
41.97 ± 0.90 a
28.71 ± 0.79 b
23.58 ± 1.04 b
19.90 ± 0.45 bc
15.31 ± 0.58 d
7ͭͪ
66.25 ± 2.41 a
51.49 ± 1.87 b
41.44 ± 1.03 b
32.31 ± 1.76 bc
24.13 ± 1.37 c
10 ͭ ͪ
74.96 ± 2.67 a
64.36 ± 2.48 a
55.81 ± 0.94 ab
38.91 ± 1.64 bc
30.89 ± 1.21 c
14 ͭ ͪ
83.93 ± 2.93 a
76.03 ± 2.91 a
69.45 ± 2.18 a
45.77 ± 1.59 b
42.71 ± 2.03 b
*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).
457 Table 5 Effect of different concentrations of Baccharis dracunculifolia essential oil, applied on the lid, on the mycelial growth of Botrytis cinerea and Colletotrichum acutatum (volatile phase). B. cinerea Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
46.45 ± 4.66 a
44.12 ± 5.32 a
38.71 ± 5.02 a
37.26 ± 4.46 a
28.53 ± 3.72 b
5ͭͪ
74.17 ± 1.60 a
67.30 ± 2.45 a
63.01 ± 2.69 a
60.01 ± 2.76 a
52.65 ± 2.06 b
7ͭͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
72.90 ± 1.54 b
10 ͭ ͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
73.95 ± 1.68 b
14 ͭ ͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
76.37 ± 2.45 b
C. acutatum Essential oil concentrations Day
0.0
12.5
25
50
100 (%)
3ʳ ͩ
27.77 ± 0.29 a
26.85 ± 0.40 a
26.80 ± 0.15 a
26.16 ± 0.34 a
25.23 ± 0.46 ab
5ͭͪ
43.43 ± 0.43 a
38.31 ± 0.50 b
34.41 ± 1.24 b
33.04 ± 0.74 bc
33.51 ± 0.62 bc
7ͭͪ
75.53 ± 1.75 a
65.42 ± 1.98 a
61.88 ± 1.79 ab
59.98 ± 2.83 ab
59.82 ± 2.27 ab
10 ͭ ͪ
84.53 ± 1.13 a
83.84 ± 1.14 a
76.08 ± 1.52 a
73.61 ± 1.21 ab
72.68 ± 1.02 ab
14 ͭ ͪ
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
90.00 ± 0.00 a
*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).
458
459
460 461
Fig. 1 Effect of different concentrations of Baccharis trimera (A) and Baccharis dracunculifolia (B)
462
essential oils on conidia germination of Botrytis cinerea (■) and Colletotrichum acutatum (■). Values are
463
the average of ten replicates per treatment ± SE. Means followed by same letter do not differ by Dunnett's
464
T3 test (p < 0.05).
465 466 467 468
469
470 471
Fig. 2 The effects of different concentrations of Baccharis trimera essential oil applied to grapes for
472
disease control. Incidence (●) and severity (■) of disease caused by Botrytis cinerea as to preventive (A)
473
and curative (B) treatment. Values are the average of ten replicates per treatment ± SE. Means followed
474
by same letter do not differ by Dunnett's T3 test (p < 0.05).
475 476 477 478 479
480
481 482
Fig. 3 The effects of different concentrations of Baccharis trimera essential oil applied to grapes for
483
disease control. Incidence (●) and severity (■) of disease caused by Colletotrichum acutatum as to
484
preventive (A) and curative (B) treatment. Values are the average of ten replicates per treatment ± SE.
485
Means followed by same letter do not differ by Dunnett's T3 test (p < 0.05).
Highlights •
Essential oils of Baccharis trimera and Baccharis dracunculifolia proved an effective antifungal agent against Botrytis cinerea and Colletotrichum acutatum in vitro.
•
The essential oil of Baccharis trimera proved effective against postharvest fungal rots in grapes.
•
Protection of grapes during storage is possible using essential oil as a natural fungicide.