Introduction
Postharvest diseases are one of
the most important problems of crops during storage period. If postharvest
diseases are not controlled sufficiently, they may cause huge losses in the
quality and yield of crops. The losses caused by postharvest diseases are estimated
between 20–50% per year (Narayanasamy 2006). B. cinerea, causing pre- and postharvest
losses, is probably one of the most common and widely distributed diseases of
vegetables, fruits, and even field crops throughout the world (Elad
1994).
If grey mold disease is not prevented adequately, it may cause important
losses in yield and quality (Coley-Smith 1980; Baraldi et al. 2002). In Turkey,
grey mold and black rot (Alternaria
alternata) are the most economically important postharvest diseases of
pepper. Grey mold and other postharvest diseases may be controlled by classical
fungicides; however, most of the traditional fungicides embody the risk of
leaving remnants on products and have negative effects on humans, animals and
environment (Agrios 2005; Wightwick and Allinson 2007). Furthermore, B. cinerea may
easily develop races with high resistance against many synthetic fungicides
(Abou-Jawdah and Itani 1995; Archbold et al. 1997).
In this context, recent studies have focused on
alternative chemicals, biological agents and plant extracts for controlling the
pathogens (Skirdal and Eklund 1993; Farooq et al. 2011). Plant extracts, sodium and
potassium salts and some antimicrobial food additives have gained importance in
the control of pathogens. Nowadays, especially, salts, like sodium and
potassium, and antimicrobial food additives have attracted attention in the
control of pathogens. In the recent studies against postharvest diseases with
alternative substances, sodium bicarbonate and eugenol are remarkable. In these
studies, both alternative chemicals have been found effective against various
phytopathogens (Yildirim and Yapici 2007; Wang et
al. 2010; Romanazzi et al. 2012). The objective of the present work
was to determine the effectiveness of sodium bicarbonate and eugenol against
grey mold disease on peppers after harvest.
Materials and Methods
Plant material
Peppers (Capsicum annuum L.) cvs. Charleston F1
and Demre F1 were selected manually from commercial orchards in Çanakkale
(Turkey) after harvest and used as plant material. The study was carried out
between May–July 2014 in Çanakkale Onsekiz Mart
University Agriculture Faculty Plant Protection Department Laboratory and Cold
Storage Facilities.
Origin of Botrytis
cinerea isolate and inoculum
B. cinerea isolate was
obtained from diseased pepper plant in Çanakkale-Turkey. Isolate which was on
Potato Dextrose Agar (PDA), were developed at 21°C in the incubator and to get
the stock cultures, it was transferred to tubes which included PDA. Conidia
were harvested two-weeks-old cultures that were growing on PDA at 21°C under 12
h light/dark periods. Conidia dislodged by rubbing the surface area with glass
rod. Fungal suspension was filtered through two layers of fine sterile muslin
to remove the mycelium and other fragments. Spore suspension was then counted
with a haemocytometer and adjusted to 106 spores mL-1.
For inoculation, the pepper fruits were wounded in the stem and end region by
means of a sterile pipette tip. They were inoculated with 60 µL spore suspension from both the
wounded sides and incubated in dark storage at 20–22°C for 12 h.
Treatments
and storage conditions
Eugenol and sodium bicarbonate (NaHCO3) were
used as nature-friendly alternative chemicals to the use of fungicides for
controlling postharvest disease B.
cinerea in peppers. Some properties of both chemicals are given in Table 1.
After incubation, peppers were treated with different doses of eugenol and
sodium bicarbonate. Eugenol was applied in warm water at 47 ± 1°C. The
treatment doses are given in Table 1. Besides, control fruits were only treated
with warm water. Then all the fruits were kept on air dry at naturally room
temperature for 1 hour. Peppers were stored at 9–10°C and 85–90% relative
humidity conditions after inoculations for 8 days, and the daily improvement of
grey mold was measured both sides of the peppers with digital calipers.
Experiment design
and statistical analysis
The experiment was arranged in completely randomized
design with three replications. Ten pepper fruits were assessed for each
replication. Analysis of variance was conducted on the efficacy of the test
chemicals by using ‘Minitab 16 Software’ and means were compared by using
‘Tukey’s method.
Results
According to results, lesion diameter showed an increase
especially on untreated pepper fruits during the storage duration. Infected
area developed significantly after five days after inoculation.
Susceptibilities of artificially inoculated places (tip and middle) of fruits
against B. cinerea were not different,
but both pepper varieties showed different response against pathogen.
‘Charliston’ was more sensitive against fungus than ‘Demre’ (P < 0.01). Meantime, lesion
development was significantly different according to varieties at 6th,
7th and 8th days (P
< 0.05, Fig. 1).
According to last 3th days measurements after
inoculation, the mean of lesion diameters on ‘Charleston’ was found bigger than
on ‘Demre’ (Fig. 1). Besides significant difference between the mean diameters
of lesions developed on ‘Demre’ was not observed, whereas the lesion
development on ‘Charleston’ continued to increase until 8th day (P = 0.05).
Lesion diameter on untreated (control) and treated pepper fruits
significantly showed diversity according to varieties (P < 0.01; Fig. 2). In Fig. 3 (a, b, c), the development of B.
cinerea lesions in untreated (Control), sodium bicarbonate and eugenol
treated pepper fruits (cv. 'Demre') were fixed.
The lesion diameters of control fruits were 43.2 mm on
‘Charleston’ and 26.8 mm on ‘Demre’ peppers, and these were significantly
reduced on fruits treated with test chemicals with the increasing dose levels (Fig.
2). The highest lesion development was observed on pepper varieties treated
with eugenol at 0.75% dosage, 23.7 mm (Charliston), 12.7 mm (Demre), and lesion
diameter of eugenol (1.5%) treated fruits was not significantly different than
that of sodium bicarbonate (0.5%) treated ones (P < 0.01). However, in fruits of both pepper varieties treated
with 1.0% dosage of sodium bicarbonate, no lesion development was observed (Fig.
2).
Chemical applications at postharvest had high efficacy
against B. cinerea, and effectiveness
of chemical applications showed significant differences (P < 0.01) according to treatment dosages eight days after of
inoculation (Fig. 4).
Sodium bicarbonate at 1.0% completely inhibited gray
mold caused by B. cinerea (100%) in
both pepper cultivars and at half dosage (0.5%) exhibited similar effect on
‘Demre’ (Fig. 3). Sodium bicarbonate at 0.5% on ‘Charleston’ and eugenol at
1.5% on ‘Demre’ showed effectiveness close to each other. At half dosage (0.75%),
eugenol on ‘Demre’ showed similar effect at dosage of 1.5% on ‘Charleston’;
however, it had at least efficacy with 45.6% at dosage of 0.75% on ‘Charleston’
(Fig. 4).
Discussion
Table 1: Some properties of the test chemicals
|
Trade Names
of Chemicals |
Active ingredients |
Formulation
and rate of active ingredient (%) |
Dosage (%) |
|
Bioxeda |
Eugenol
(4-allyl-2-methoxyphenol) |
Liquid, 18 |
0.75; 1.5 |
|
Baking
powder |
NaHCO3 |
WP, 99 |
0.5; 1.0 |
%201126-1130_files/image002.png)
Fig. 1: Development of disease on pepper varieties
%201126-1130_files/image004.png)
Fig. 2: Effect of chemicals on the size of lesion on pepper
fruit caused by Botrytis cinerea
Nowadays, the alternative control of plant diseases has
become more important. Especially, plant extracts, sodium and potassium salts
have gained importance in the control of pathogens. In this study, the
effectiveness of sodium bicarbonate and eugenol against B. cinerea (Grey mold disease) on two pepper varieties ‘Demre’ and
‘Charleston’ were studied during at storage conditions. ‘Demre’ and
‘Charleston’ showed different sensitivities against grey mold disease, and this
case reflected also to the effectiveness of alternative chemicals. ‘Charleston’
was found more sensitive to B. cinerea
during eight days of storage period than 'Demre' (Fig. 1 and Fig. 2).
Difference of sensitivity of both pepper varieties to disease maybe related
with ethylene production of plants in different concentration during ripening
postharvest (Woltering and Doom 1988). Ethylene
causes increase of the tissue sensitivity to diseases in plants, at the same
time it stimulates the conidial germination and hyphal growth of B. cinerea (Elad 1988; Elad and Evensen
1995). In addition, pepper varieties as well as other plant varieties
may have difference defense system against B.
cinerea. Dix and Webster (1995) reported that some workers determined that
the waxes extracts obtained from various plant leaves (broad bean, sugar beet,
red beet, birch, lettuce, tomato, black current and chrysanthemum) had
inhibitory effect on spore germination of B.
cinerea, and this was caused by the inhibitory substances in waxes
(Blakeman and Sztejnberg 1973).
%201126-1130_files/image006.png)
Fig. 3: In 'Demre' pepper variety,
lesion development of Botrytis cinerea
in untreated pepper fruits (a) and
peppers treated with (b) sodium
bicarbonate at 1% dosage and (c)
eugenol 1.5% dosage
%201126-1130_files/image008.png)
Fig. 4: Effectiveness of chemicals to grey mold caused by Botrytis
cinerea on pepper fruit
Both treatment doses (0.5 and 1%) of sodium bicarbonate
showed strong inhibition on the lesion development of B. cinerea in both the pepper varieties. However, its effectiveness
against grey mold disease at the dosage of 0.5% on ‘Charleston’ was a little lower
than that of on ‘Demre’. Disease severity on the pepper fruits especially on
'Charleston’ treated with eugenol was higher than that of sodium bicarbonate.
Thus, sodium bicarbonate was more successful for the control of B. cinerea according to eugenol. In
contrast to eugenol, sodium bicarbonate at all doses had a higher effectiveness
against grey mold on both the pepper varieties. It completely inhibited the
disease at 1% dose and showed similar effect at ‘Demre’ at 0.5% treatment dose.
However, this effect was observed only at high dose (1%) of the chemicals on
‘Charleston’. That could be considered as a collective effect of the host
resistance and effectiveness of the chemical. Sodium bicarbonate is one of the
best-known alternative substances, and previously research works conducted with
alternative chemicals had shown that sodium bicarbonate alone or in combination
with other antifungal agents could be highly effective against numerous pre-
and postharvest diseases (Palmer et al. 1997; Zaker 2014; Mlikota and
Smilanick 2001).
Contrary to
results of this study, it is reported that salts also including sodium bicarbonate
applied to table grape after harvest did not show suppressive effect to B. cinerea and the preharvest-treatment
on grape was more effective to mold than postharvest immersion (Youssefa
and Roberto 2014). However, this study on
pepper fruits artificially inoculated with B.
cinerea after harvest demonstrated that sodium
bicarbonate had strong inhibitory effect to grey mold at 0.5 and 1.0% doses on
both pepper varieties ‘Demre’ and ‘Charleston’. This difference between two
studies may be due to the different sensitivities of the plants to the pathogen
or due to the buffering response of B. cinerea to high pH caused by sodium bicarbonate.
Bicarbonate ion
(HCO3-) having a buffering property in aqueous solution
causes alkalinization by elevating the pH of environment, and this results in
inhibition of the mycelial growth and conidia germination of B. cinerea as well as the
polygalacturonase activity (Palmer et al. 1997; Fagundes et al.
2013). However, high pH may not be sufficient to explain the mode of action of
salts in all circumstances; thus, it is reported that high pH of salt solution
was not enough for the inhibition of B. cinerea on grape berries (Nigro et al. 2006). Thus, the
production of extracellular enzymes (polygalacturonases, pectin methylesterases, proteases
and laccases) playing
important role on pathogenicity show diversity according to host and
environmental factors. However, high pH may not be sufficient to explain the
mode of action of salts in all circumstances (Wang et al. 2010; Shah et
al. 2012; Fagundes et al. 2013). It is reported that the hydrogen
ion concentration existing in environment controlled the ionization of salts
and hence the availability of ions to the fungus, and the permeability of the
plasmalemma (Dix and Webster 1995). Thus, ion uptake, enzyme activity and
extracellular enzyme activity is influenced depending on pH values. Besides,
one of the mode actions of salts is the reduction of the turgor pressures of
fungal structures, and this results in the collapse and shrinkage of fungal
hyphae as well (Fallik et al. 1997).
Effect of the
sensitivity difference between pepper varieties was clearly observed in application
of eugenol. At the low dosage (0.75%) of eugenol, the effectiveness against the
disease was found more about at 50% ratio on Demre than that of on Charleston (Fig.
2). Eugenol at high dose (1.5%) was as effective as NaHCO3 at low
dose (0.5%). It showed high effectiveness against for eight days. It may be
accepted to be successful for the control of gray mold on peppers.
Eugenol has
antimicrobial activity against various fungal pathogens (Rhayour et al.
2003). In some studies, it has been observed that eugenol could inhibit hyphal
growth of B. cinerea but had no biological activity on conidial
germination. Eugenol triggers H2O2 production in the
cytoplasm, leading to increasing free Ca2+, that lead to membrane
binding and permeability changes and consequently destabilization and
disruption of the plasma membrane (Wang et al. 2010).
As a result, it was determined that each chemical,
eugenol and NaHCO3 had strong postharvest effectiveness against B. cinerea on peppers. In addition, the
effectiveness of applied alternative chemicals was variable depending on the
pepper variety of dose of the chemicals. Eugenol applied at higher dosage to B. cinerea on ‘Demre’ was found as
effective as that of NaHCO3 applied at lower dosage to the pathogen
on ‘Charleston’. These findings mean that the resistance of plants against
pathogens is an important aspect regarding the effectiveness of chemicals.
Conclusion
The eugenol and sodium bicarbonate can be used against
post-harvest diseases in organic and traditional agriculture. The results of
Eugenol and sodium bicarbonate against lead mold disease show difference
according to sensitivities of plant varieties to pathogen. In application
doses, it is necessary to pay attention to the susceptibility of plant
varieties. In general, sodium bicarbonate at 0.5% dose and eugenol 1.5% can be
applied by spraying at the surface of the fruit or by dipping the fruits into
water containing chemical.
Author Contributions
Yildirim made the innoculum,
materialized the innoculation and found literatures, Sakaldas made the
measurements and assesed the statistical datas and found the plant material.
Conflicts of Interes
The authors declare that they have no
conflict of interest.
Data Availability
The data will be made avaialble on
acceptable requests to the corresponding author.
Ethics Approval
Not applicable.
References
Abou-Jawdah Y,
H Itani (1995). Sensitivity of Botrytis
cinerea isolates to fungicides used in Lebanon. In: Proceedings of 9th Congress of the Mediterranean
Phytopathology Union, Vol. 34, pp:100‒108. Kuşadası-Aydin, Türkiye
Agrios GN (2005). Plant Pathology, 5th
edn., p:952. Elsevier-Academic Press, San Diego,
California, USA
Archbold DD, TR Hamiltonkemp, MM Barth, BE Langlois (1997).
Identifying natural volatile compounds that control gray mold (Botrytis cinerea) during postharvest
storage of strawberry, blackberry and grape. J Agric Food Chem
45:4032‒4037
Baraldi E, P Bertolini, E Chierici, B
Trufelli, D Luiselli (2002). Genetic diversity between Botrytis cinerea isolates from unstored and cold stored kiwi fruit.
J Phytopathol 150:629‒635
Blakeman JP,
A Sztejnberg (1973). Effect of surface wax on inhibition of germination of Botrytis cinerea spores on beetroot
leaves. Physiol Plant Pathol 3:269‒278
Coley-Smith
JR (1980). Sclerotia and other structures in survival. In: The Biology of
Botrytis, pp:85‒114.
Coley-Smith JR, K Verhoeff, WR Jarvis (Eds.). Academic Press, London
Dix NJ, J Webster (1995). Fungal Ecology. Published
by Chapman and Hall, London
Elad Y (1994). Biological control of grape grey mould by
Trichoderma harzianum. Crop Prot
13:35–38
Elad Y (1988). Involvement of ethylene in the disease
caused by Botrytis cinerea on the
rose and carnation flower sand the possibility of control. Ann Appl Biol
113:589‒598
Elad Y, K Evensen (1995). Physiological
aspect of resistance to Botrytis cinerea.
Phytopathology 85:635‒643
Fagundes C,
MB Pérez-Gago, AR Monteiro, L Palou (2013). Antifungal activity of food
additives in vitro and as ingredients
of hydroxypropyl methylcellulose-lipid edible coatings against Botrytis cinerea and Alternaria alternata on cherry tomato
fruit. Intl J Food Microbiol 166:391‒398
Fallik E, S
Grinberg, O Ziu (1997). Potassium bicarbonate reduces postharvest decay
development on bell pepper fruit. J Hortic Sci 72:35‒41
Farooq M, K
Jabran, ZA Cheema, A Wahid, KH Siddique (2011). The role of allelopathy in
agricultural pest management. Pest Manage Sci 67:493‒506
Mlikota GF, JL Smilanick (2001).
Postharvest control of table grape gray mold on detached berries with carbonate
and bicarbonate salts and disinfectants. Amer J Enol Viticult 52:12‒20
Narayanasamy P (2006). Postharvest Pathogens and
Disease Management, p:578. John Wiley and Sons, Inc., New Jersey, USA
Nigro F, L
Schena, A Ligorio, I Pentimone, A Ippolito, MG Salerno (2006). Control of table
grape storage rots by pre-harvest applications of salts. Postharv
Biol Technol 42:142‒149
Palmer CL, RK Horst, RW Langhans (1997). Use of
bicarbonates to inhibit in vitro growth of Botrytis
cinerea. Plant Dis 81:1432‒1438
Rhayour K, T
Bouchikhi, A Tantaoui-Elaraki, K Sendide, A Remmal (2003). The mechanism of
bactericidal action of oregano and clove essential oils and of their phenolic
major components. J Essent Oil Res 15:286‒292
Romanazzi A,
G Lichter, FG Mlikota, J Smilanick (2012). Recent advances on the use of
natural and safe alternatives to conventional methods to control postharvest
gray mold of table grapes. Postharv Biol Technol 63:141‒147
Shah P, AL Powell,
R Orlando, C Bergmann, G Gutiérrez-Sánchez (2012). Proteomic analysis of ripening
tomato fruit infected by Botrytis cinerea.
J Proteom Res 11:2178‒2192
Skirdal IM, T Eklund (1993). Microculture model studies
on the effect of sorbic acid on Penicillium
chrysogenum, Cladosporium
cladosporioides and Ulocladium atrum
at different pH levels. J Appl Bacteriol 74:191‒195
Wang C, J Zhang-Chen, HY Fan, J Shi (2010). Antifungal
Activity of Eugenol against Botrytis
cinerea. Trop Plant Pathol 35:137‒143
Wightwick A, G Allinson (2007). Pesticide residues in victorian waterways: A review (online). Aust J Ecotoxicol 13:91‒112
Woltering ED, WGV Doom (1988). Role of ethylene in senescence of
petals: Morphological and taxonomical relationships. J Exp Bot 39:1605‒1616
Yildirim I, BM Yapici (2007). Inhibition of conidia
germination and mycelial growth of Botrytis
cinerea by some alternative chemicals. Pak J Biol Sci 10:1294‒1300
Youssefa K,
SR Roberto (2014). Salt strategies to control Botrytis mold of ‘Benitaka’ table
grapes and to maintain fruit quality during storage. Postharv Biol Technol
95:95‒102
Zaker M (2014). Antifungal evaluation of some inorganic
salts against three phytopathogenic fungi. Intl J Agric Crop Sci 7:1352‒1358