Alternatives to synthetic fungicides to control postharvest diseases of

Transcript

UNIVERSITÁ POLITECNICA DELLE MARCHE
Department of Agricultural, Food, and
Environmental Sciences
PhD School in Agricultural Sciences (XI cycle, new series)
curriculum “Crop Production and Environment”
Disciplinary sector: AGR/12 – Plant pathology
Alternatives to synthetic fungicides to
control postharvest diseases of
strawberry, sweet cherry, and table grapes
Academic tutor:
Dr. Gianfranco Romanazzi
PhDstudent:
Coordinator:
Dr. Erica Feliziani
Prof. Bruno Mezzetti
A Viola,
che è il mio pensiero felice
INDEX
1
2
3
4
RIASSUNTO ............................................................................................. 8
ABSTRACT .............................................................................................. 9
INTRODUCTION .................................................................................. 10
PROLONGED STORAGE AND SHELF LIFE EXTENSION OF
FRESH FRUIT AND VEGETABLES BY CHITOSAN
TREATMENT ........................................................................................ 15
Abstract ......................................................................................... 15
2.1
Introduction ................................................................................... 15
2.2
Preharvest application of chitosan for postharvest decay control 19
2.3
Postharvest application of chitosan for storage decay control ...... 26
2.4
Activity of chitosan against postharvest decay causing fungi ....... 34
2.5
Induction of resistance by chitosan on fruits ................................. 38
2.6
Effect of chitosan treatment on retention of fruit quality and
health promoting compounds ........................................................ 57
2.7
Effect of chitosan on foodborne pathogens ................................... 64
2.8
Conclusions and future trends ....................................................... 69
EFFECTIVENESS OF POSTHARVEST TREATMENT WITH
CHITOSAN AND OTHER RESISTANCE INDUCERS IN THE
CONTROL OF STORAGE DECAY OF STRAWBERRY ............... 72
Abstract ......................................................................................... 72
3.1
Introduction................................................................................... 73
3.2
Materials and methods .................................................................. 75
3.2.1
Fruit ...................................................................................... 75
3.2.2
Resistance inducers............................................................... 75
3.2.3
Treatments ............................................................................. 76
3.2.4
Data recording ...................................................................... 76
3.2.5
Experimental design and statistics........................................ 77
3.3
Results and discussion .................................................................. 77
PREHARVEST TREATMENTS WITH ALTERNATIVES TO
SYNTHETIC FUNGICIDES TO PROLONG SHELF LIFE OF
STRAWBERRY FRUIT ........................................................................ 84
Abstract ......................................................................................... 84
4.1
Introduction................................................................................... 85
4.2
Materials and methods .................................................................. 87
4.2.1
Preharvest treatments ........................................................... 87
4.2.2
Decay evaluation .................................................................. 88
4.2.3
Determination of fruit-quality parameters............................ 89
4.2.4
Statistical analysis ................................................................ 90
4.3
Results........................................................................................... 90
4.3.1
First harvest .......................................................................... 90
4.3.2
Second harvest ...................................................................... 93
4.3.3
Strawberry color and firmness after field treatments ........... 96
4.4
Discussion ..................................................................................... 97
5 PRE
AND
POSTHARVEST
TREATMENT
WITH
ALTERNATIVES TO SYNTHETIC FUNGICIDES TO
CONTROL POSTHARVEST DECAY OF SWEET CHERRY ...... 102
Abstract .................................................................................................. 102
5.1
Introduction................................................................................. 103
5.2
Materials and methods ................................................................ 105
5.2.1
Antimicrobial activities of the resistance inducers in vitro . 105
5.2.2
Postharvest treatments ........................................................ 105
5.2.3
Preharvest treatments ......................................................... 106
5.2.4
Data recording for the in vivo trials ................................... 107
5.2.5
Statistical analysis .............................................................. 108
5.3
Results......................................................................................... 108
5.3.1
Antimicrobial activities of resistance inducers in vitro....... 108
5.3.2
Postharvest treatments ........................................................ 109
5.3.3
Preharvest treatments ......................................................... 112
5.4
Discussion ................................................................................... 114
6 PREHARVEST FUNGICIDE, POTASSIUM SORBATE, OR
CHITOSAN USE ON QUALITY AND STORAGE DECAY OF
TABLE GRAPES ................................................................................. 119
Abstract ....................................................................................... 119
6.1
Introduction................................................................................. 119
6.2
Material and methods.................................................................. 121
6.2.1
Vineyard treatments ............................................................. 121
6.2.2
Natural postharvest decay ................................................... 123
6.2.3
Postharvest decay after inoculation with B. cinerea ........... 123
6.2.4
Quality characteristics......................................................... 124
6.2.5
6.2.6
6.2.7
Chitinase activity ................................................................. 125
Hydrogen peroxide content .................................................. 126
Hydrogen peroxide localization by scanning electron
microscope ........................................................................... 127
6.2.8
Phenolic compound analysis ............................................... 128
6.2.9
Effect of residual fungicide content of berries on
postharvest decay................................................................. 129
6.2.10 Statistical analysis ............................................................... 130
6.3
Results......................................................................................... 130
6.4
Discussion ................................................................................... 141
7 OVERALL CONCLUSIONS .............................................................. 147
8 ACKNOWLEDGMENTS .................................................................... 150
9 REFERENCES ..................................................................................... 151
RIASSUNTO
La tesi di dottorato ha riguardato la valutazione dell’efficacia di
applicazioni di composti alternativi ai fungicidi di sintesi, al fine di
prolungare la conservazione di fragole, ciliegie, ed uva da tavola. Prodotti
quali chitosano, benzotiadiazolo, laminarina, oligosaccaridi, calcio ed acidi
organici, lecitina di soia, bicarbonato di potassio, sorbato di potassio, estratti
di abete, e di ortica hanno ridotto marciumi postraccolta, principalmente
causati da Botrytis cinerea su fragola ed uva da tavola, e da Monilinia laxa
su ciliegie, sia quando applicati in prove postraccolta, tramite immersione
della frutta, sia tramite trattamenti prima della raccolta. In particolare,
l’efficacia di una formulazione commerciale a base di chitosano è stata
paragonabile a quella ottenuta tramite applicazioni di fungicidi di sintesi nel
contenimento dei marciumi che naturalmente si sviluppano sulla frutta dopo
la raccolta. I vari prodotti testati hanno agito sia grazie alla loro attività
antimicrobica, testata in vitro, sia tramite il fenomeno dell’induzione di
resistenza nella pianta. In particolare, il trattamento preraccolta con
chitosano su uva da tavola ha aumentato nei tessuti vegetali l’attività di
proteine relazionate alla patogenesi, come la chitinasi, ed il contenuto di
composti fenolici, mentre ha diminuito la quantità di perossido di idrogeno.
Inoltre, le applicazioni dei vari composti non hanno dato effetti negativi su
parametri qualitativi, fondamentali per la commercializzazione della frutta
trattata. In conclusione, i vari prodotti testati potrebbero affiancare, e in
alcuni casi sostituire, l’uso dei fungicidi di sintesi per il contenimento dei
marciumi postraccolta di fragole, ciliegie, ed uva da tavola, considerando
anche i recenti orientamenti delineati della Unione Europea che, con la
Direttiva 128/2009, rende obbligatoria l’applicazione della difesa integrata
sull’intero territorio comunitario a decorrere da gennaio 2014.
8
ABSTRACT
The PhD project dealt with the evaluation of the effectiveness of
alternative compounds to synthetic fungicides for the prolongation of storage
of strawberry, sweet cherry, and table grapes. Postharvest rot was reduced by
chitosan, benzothiadiazole, laminarin, oligosaccharides, calcium and organic
acids, soybean lecithin, potassium bicarbonate, potassium sorbate, and
extract of nettle and of fir. This was seen for rot mainly caused by Botrytis
cinerea on strawberry and table grapes, and by Monilinia laxa on sweet
cherry. These compounds were effective both when applied at the
postharvest stage, by dipping the fruit, or through treatments before the
harvest, with spraying in the experimental fields. In particular, the
effectiveness of a commercial chitosan formulation was comparable with
that obtained by synthetic fungicides for the control of the rot that naturally
occurs on these fruit after harvest. The various products tested were active
both through their antimicrobial activities, which were tested in in vitro
trials, and through resistance induction in the plants. In particular, preharvest
chitosan treatments on table grapes increased the content of phenolic
compounds and the activity of pathogenesis-related proteins in the plant
tissues, such as chitinase, while they decreased the hydrogen peroxide levels.
Moreover, the application of the various compounds tested did not show any
negative effects on any of the quality parameters, which are important for the
commercialization of the fruit. In conclusion, the compounds tested can
complement, and in some cases replace, the use of synthetic fungicides in
the control of postharvest decay of strawberry, sweet cherry, and table
grapes. This needs to be considered as the recent Directive 128/2009 has
made the integrated pest management mandatory in all European Union
countries starting from January 2014.
9
1
INTRODUCTION
In recent decades, agriculture has undergone great changes to adapt
itself to the fast evolution of the market and to the changing requirements of
the consumer. Intensive agriculture has resulted in the development of
particularly dedicated geographical areas, which has in turn created
overproduction in specific zones and at certain times of the year. To be
distributed over time and space, these products need to be subjected to
varying times of transport and storage, in terms of the product characteristics
and the market demands. In the case of fresh fruit and vegetables that are
particularly perishable, this scenario is even more difficult to manage. The
request by consumers for fresh produce that is available all year round
and/or comes from exotic production areas increases the need for storage and
transport of fruit and vegetables.
In addition, the Food and Agriculture Organization (FAO, 2009) has
estimated that by 2050 the world population will reach 9.1 billion. Nearly all
of this population increase will occur in developing countries, and about
70% of the global population will be urban. To feed this larger, more urban,
and richer population, food production must increase by 70% and it will
need to be moved from the areas where it is produced to the areas where it is
consumed (FAO, 2009). This is the current challenge for agriculture. For
fruit and vegetables production, the decrease in postharvest losses of
horticultural perishables can provide an effective way of increasing food
availability and reducing the land needed for its production (Kader, 2005). A
recent study from the FAO (FAO, 2011) estimated that with respect to the
total amounts of fruit and vegetables produced globally, somewhere between
15% and 50% are lost at the postharvest stage, before even reaching the
tables of the consumers. The highest losses were recorded in the developing
countries of Africa and Asia, which lack the necessary technologies to
prolong the storage life of fresh produce.
Fruit and vegetables are highly perishable, and the causes of
postharvest losses can generally be ascribed to physiological deterioration
associated with consumption of the internal water and reserve substances,
10
______________________________________________________ 1 - Introduction
changes in the nutritive values and quality parameters, pathological
breakdown due to fungi and bacteria infections, pathophysiological injury
during storage due to excessive chilling or lighting or to anomalies in the
atmospheric gaseous composition, and physical injury, such as mechanical
damage. In many instances, these causes are interrelated; i.e., mechanical
injury can be associated with postharvest decay from many causes. However,
like any other food, fruit and vegetables are very prone to microbial spoilage
because of their succulent nature. Frequently, infection by microorganisms
that cause postharvest decay can occur before the harvest at the field stage,
such that they can remain latent until storage, when the environmental
conditions are favorable for disease development. Indeed, the rate of
postharvest deterioration depends on several external factors, including
storage temperature, relative humidity, air speed, atmospheric composition
(concentrations of oxygen, carbon dioxide, and ethylene), and sanitation
procedures (Kader et al., 2005). Table 1 summarizes some of the most
common postharvest diseases and pathogens of fruit. Many of these can
develop very rapidly from rotted fruit next to the healthy fruit, causing
extensive breakdown of the commodity, and sometimes spoiling entire lots.
Moreover, aside from direct economic considerations, diseased produce
poses a potential health risk, as some fungal genera are known to produce
mycotoxins under certain conditions, such as Penicillium spp., Aspergillus
spp. and Alternaria spp.
11
1 - Introduction ______________________________________________________
Table 1. Some of pathogens that can cause postharvest decay of fruit.
Fruit
Disease
Causal agent
Berries
Gray mold
Botrytis cinerea
Rhizopus rot
Rhizopus spp. and Mucor spp.
Blue mold
Penicillium spp.
Table grapes
Gray mold
Botrytis cinerea
Blue mold
Penicillium spp.
Rhizopus rot
Rhizopus spp.
Black rot
Aspergillus niger
Stone fruit
Brown rot
Monilinia spp.
Gray mold
Botrytis cinerea
Blue mold
Penicillium spp.
Rhizopus rot
Rhizopus stolonifer
Alternaria rot
Alternaria alternata
Pome fruit
Blue mold
Penicillium spp.
Gray mold
Botrytis cinerea
Brown rot
Monilinia spp.
Alternaria rot
Mucor rot
Mucor piriformis
Citrus fruit
Blue mold
Penicillium italicum
Green mold
Penicillium digitatum
Alternaria rot
Alternaria spp.
Tropical fruit
Anthracnose
Colletotrichum spp.
The correct choice of fruit maturity at harvest, and careful handling
and use of technologies that delay fruit ripening during storage are
fundamental for decay control (Mari et al., 2009). At the same time, the
application of synthetic fungicides remains the most common method to
control postharvest rot of fruit and vegetables. However, for some
commodities, the application of postharvest fungicides is not permitted, due
to the several normative restrictions for fungicide use. Also, the appearance
of pathogens resistant to fungicides has dissuaded their repeated use. In
addition, increasing public concern towards healthy foods has contributed to
12
______________________________________________________ 1 - Introduction
the promotion of interest in the development of alternative methods for
controlling postharvest decay caused by fruit and vegetables plant
pathogens, which need to be integrated into, if not totally replace, the use of
synthetic fungicides. Research efforts have led to the development of novel
control tools, as alternatives to synthetic fungicide treatments. For schematic
reasons, these can be grouped into four main categories: (i) natural
compounds; (ii) compounds generally recognized as safe (GRAS); (iii)
biological control agents (BCAs); and (iv) physical methods alone or the
combination of all four groups (Mari et al., 2009; Romanazzi et al., 2012).
BCAs are mainly bacteria and yeast that are ‘antagonistic’ to
pathogens that can cause postharvest fruit spoilage. These can act through
several mechanisms, including competition for nutrients and space,
antibiosis, parasitism, induction of resistance in the host tissue, and
production of volatile metabolites (Jamalizadeh et al., 2011).
Natural or GRAS compounds are substances that are known not to
be harmful to the environment and to human health, and these are used for
their antimicrobial properties or their induction of plant defenses. Among the
natural compounds, plant extracts and essential oils have been reported to
control postharvest diseases, both in vitro and in vivo, and to prolong the
overall quality and storage life of fresh commodities (Antunes and Cavaco,
2010). Inorganic salts have been shown to be active antimicrobial agents
against a range of phytopathogenic fungi, and among these agents,
bicarbonates have been proposed as safe and effective alternative means to
control postharvest rot of fruit and vegetables. Also, as well as these salts
being nontoxic and having minor environmental impact at effective
concentrations, they are inexpensive (Sanzani et al., 2009). Several sanitizers
classified as GRAS have been applied to extend the postharvest storage of
various produce, including acetic acid, electrolyzed oxidizing water, and
ethanol (Romanazzi et al., 2012). Resistance inducers are plant or pathogens
constituents, or their analogs, that act as plant elicitors, as they can activate
plant defense mechanisms, and thus simulate the presence of pathogens.
Among the resistance inducers, the natural biopolymer chitosan and the
13
1 - Introduction ______________________________________________________
synthetic elicitor benzothiadiazole have been reported to activate systemic
acquired resistance in horticultural produce (Terry and Joyce, 2004).
Physical measures include heat treatment, UV light, treatment at
pressures higher or lower than atmospheric pressure, and exposure to
modified or controlled atmospheres or to ozone, among others measures.
Physical treatments can have dual effects on the fruit, as these are active
against the pathogen and at the same time they can induce host defense
responses (Wilson et al., 1994).
Depending on the characteristics of the commodity and on the
specific situation, one strategy of controlling postharvest decay of fruit might
be more appropriate than another. To overcome the drawbacks that can arise
with the use of a unique strategy, the integration of methods might provide
additive or synergistic effects for disease control (Mari et al., 2007).
According to a recent review (Romanazzi et al., 2012), the ideal alternative
means of controlling postharvest decay should improve on the current
practices, should be affordable and easy to implement, and should not have
any negative influence on the fruit to which it is applied or on the
environment or human health. In particular, one aspect that is important to
consider is the effect of these alternative methods on food safety. Recent
studies have considered fresh fruit and vegetables as vehicles for the
transmission of human pathogens (Berger et al., 2010), and it has been
estimated that foodborne illness has significant global economic and human
cost every year (Bubzy and Roberts, 2009).
14
2
PROLONGED STORAGE AND SHELF LIFE EXTENSION OF
FRESH
FRUIT
AND
VEGETABLES
BY
CHITOSAN
TREATMENT
Abstract
Among alternatives to the use of synthetic fungicides to preserve
fruit and vegetables during storage and shelf life, chitosan has been proposed
for applications either at pre or postharvest, and commercial products based
on the biopolymer are commercially available. Chitosan has a dual action, on
the pathogen and on the vegetal tissues, since it reduces the growth of decay
causing fungi and induces resistance responses in the host. The antimicrobial
activity of chitosan, in addition, could control contamination by foodborne
pathogens eventually occurring on fresh commodities. Chitosan coating
forms a semipermeable film on the vegetables and fruit surface that delays
respiration process and decreases transpiration losses, prolonging the quality
of fresh produce during storage. Moreover, the coating could provide a
substrate for the incorporation of other functional natural food additives that
could improve the chitosan antimicrobial activity or the nutritive properties
of fresh commodities. Chitosan coating has been approved as GRAS
(Generally Recognized As Safe) substance by USFDA (United States Food
and Drug Administration) and its application is safe for the consumer and the
environment. This review summarizes the most relevant and recent
knowledge in the application of chitosan on postharvest decay control and
retention of fruit and vegetables quality.
2.1
Introduction
Susceptibility of fresh produce to postharvest diseases and
15
2 - Prolonged storage and shelf life extension of fresh fruit and vegetable by
chitosan treatment ___________________________________________________
deterioration of quality attributes increase after harvest and prolonged
storage as a result of physiological changes in the commodities that favor
pathogen development. The postharvest losses due to fruit and vegetables
active metabolism can be reduced during the postharvest operations by
harvesting at suitable maturity stages and by adopting appropriate
postharvest handling methods, such as the prevention of mechanical injury,
storage at low temperatures and optimal relative humidity, and correct
transportation during the supply chain (Sugar, 2009; Baloch and Bibi, 2012).
However, in some instances, these practices could be insufficient to maintain
the quality as a result of physiological changes setting in due to prolonged
storage, and a residual protection against postharvest diseases is important
after removal from cold storage at the retailer’s market shelf. On the other
hand, postharvest disease control for horticultural fresh produce begins in the
field and involves cultural practices and, usually, fungicide applications. The
adverse effects of synthetic chemical residues on human health and
environment, and the possibility of the development of fungicide resistant
pathogens have led to intensified world-wide research efforts to develop
alternative control strategies. In addition, consumer trend is towards
experiencing a tread of "green" consumerism, desiring fewer synthetic
additives in food together with increased safety, quality and shelf life.
Furthermore, a potential of foodborne outbreaks exists due to possible
contamination of fruit in the field due to dirty irrigation water or amendment,
or at postharvest for human handling or improper sanitation (Beuchat, 2002).
Application of chitosan treatment at pre or postharvest stage has
been considered as an alternative treatment to the use of synthetic fungicides
to prevent fruit postharvest decay and to extend the storage life while
retaining the overall quality of different fresh commodities (Bautista-Baños
et al., 2006). Chitosan (poly b-(1-4)N-acetyl-d-glucosamine), has been
identified as having the properties of an ideal coating, with antimicrobial
properties itself and that could induce plant defense when applied in vegetal
tissues (Devlieghere et al., 2004). Chitosan coating provides a substrate for
16
2 - Prolonged storage and shelf life extension of fresh fruits and vegetables by
___________________________________________________ chitosan treatment
incorporation of other functional natural food additives that could improve
its antimicrobial property and preventing fruit quality deterioration (Vargas
et al., 2008). The application of chitosan treatment in fresh produce industry
is safe for consumer and environmentally in postharvest or preharvest
application, indeed chitosan has been approved by the United State Food and
Drug Administration (USFDA) as a Generally Retained As Safe (GRAS)
food additive.
Nowadays commercial chitosan formulations are available on the
markets, and among them, some have been tested in experimental trials in
controlling postharvest decay of fruit and vegetables (Table 2) or for plant
protection in general. The latter include Chitogel (Ecobulle, France) (Ait
Barka et al., 2004; Elmer and Reglinski, 2006), Biochikol 020 PC (Gumitex,
Lowics, Poland) (Nawrocki, 2006), Armour-Zen (Botry-Zen Limited,
Dunedin, New Zealand) (Reglinski et al., 2010), Elexa 4 Plant Defense
Booster (Plant Defense Booster Inc., USA) (Elmer and Reglinski, 2006), and
Kendal Cops (Iriti et al., 2011). The main differences between the practical
grade chitosan solutions and the commercial chitosan formulation arise from
the techniques of their preparation and application, which is more immediate
for the commercial formulations (Romanazzi et al., 2013).
17
Table 2. Chitosan based commercial products
postharvest decay in fruit and vegetables.
Product
Company
Formulation a.i.
name
(Country)
(%)
Chito
ChiPro
Powder
99.9
Plant
GmbH
(Bremen,
Germany)
OII-YS
ArmourZen
Biorend
FreshSeal
ChitoClear
Bioshield
Biochikol
020 PC
available for the control of
Fruit
Reference
Table
grapes,
sweet
cherry,
strawberry
Table
grapes
Feliziani et al.,
2013a, 2013b;
Romanazzi et
al., 2013
Venture
Innovations
(Lafayette,
LA, USA)
Botry-Zen
Limited
(Dunedin,
New Zealand)
Bioagro S.A.
(Chile)
Liquid
5.8
Liquid
14.4
Peach,
table
grapes
Casals et al.,
2012; Feliziani
et al., 2013b
Liquid
1.25
Fornes et al.,
2005
BASF
Corporation
(Mount
Olive, NJ,
USA)
Primex ehf
(Siglufjordur,
Iceland)
Seafresh
(Bangkok,
Thailand)
Gumitex
(Lowics,
Poland)
Liquid
-
Clementine
mandarin
fruit
Banana
Powder
100
Rambutan
fruit
Powder
100
Mango
MartínezCastellanos et
al., 2009
Jitarrerat et al.,
2011
Liquid
2
Potato
18
Feliziani et al.,
2013b
Baez-Sañudo et
al., 2009
Kurzawińska
and Mazur, 2007
2 - Prolonged storage and shelf life extension of fresh fruits and vegetables by
The aim of this review is to summarize the most recent and relevant
advances in the application of chitosan on postharvest decay control,
retention of quality, health promoting compounds and food safety issues in
fresh produce industry.
2.2
Preharvest application of chitosan for postharvest decay control
While there is relevant information about the effectiveness of
postharvest chitosan treatments, fewer data are available concerning the
evaluation of preharvest application in the control of postharvest decay of
fruit and vegetables (Tables 3, 4, and 5). However, chitosan applications
before the harvest could be suitable for fruit, such as table grapes and
strawberries that have a bloom on the surface and/or can suffer postharvest
wetting or handling. Moreover, preharvest treatment could have a preventive
effect other than a curative one, since the development of postharvest decay
often arises from an inoculum that survives and accumulates on the fruit in
the field and or in the packaging chain after the harvest.
19
Table 3. Chitosan treatments with other applications on storage decay of temperate
fruit.
Produce
Decay
Integration to
References
chitosan
(moment of
application)
Table grapes
Gray mold
Romanazzi et al., 2002
(pre and postharvest)
Acid solutions
(postharvest)
Ethanol
(postharvest)
Grape seed extract Xu et al., 2007b
(postharvest)
Gray mold and
UV
blue mold
(preharvest)
Decay (in general) Cryptococcus
Meng and Tian, 2009
laurentii
(preharvest); 2010b
(postharvest)
Strawberry
Gray mold
El Ghaouth et al.,
1991a, 1992a
(postharvest); Zhang
and Quantick, 1998
(postharvest);
(pre and postraccolta);
Reddy et al., 2000a
(preharvest); Mazaro et
al., 2008 (preharvest)
Lemon essential
Perdones et al., 2012
oil
(postharvest)
Red thyme,
Vu et al., 2011
oregano extract,
(postharvest)
limonene,
peppermint
20
Produce
Decay
Integration to
chitosan
Rhizopus rot
-
Cladosporium rot
-
Decay (in general)
Calcium lactate,
calcium
gluconate, vitamin
E
Calcium
gluconate
Oleic acid
Raspberry
Decay (in general)
Gray mold and
Rhizopus rot
Calcium lactate,
calcium
gluconate, vitamin
E
-
Blueberry
Decay (in general)
-
Apple
Blue mold
UV-C, Candida
satoiana, harpin
Cryptococcus
21
References
(moment of
application)
El Ghaouth et al., 1992a
(postharvest); Zhang
and Quantick, 1998
(postharvest);
(pre and postharvest);
Park et al., 2005
(postharvest)
Park et al., 2005
(postharvest)
Han et al., 2004
(postharvest)
Hernández-Muñoz et
al., 2006 (postharvest);
2008 (postharvest)
Vargas et al., 2006
(postharvest)
Han et al., 2004
(postharvest)
Zhang and Quantick,
1998 (postharvest)
Duan et al., 2011
(postharvest)
De Capdeville et al.,
2002 (postharvest)
Yu et al., 2007
Produce
Decay
Integration to
chitosan
laurentii
Candida satoiana
Heat treatment
Gray mold
Candida satoiana
Heat treatment
Pear
Blue mold
Peach
Brown rot
Calcium chloride,
Cryptococcus
laurentii
Heat treatment
Sweet cherry
Decay (in general)
Hypobaric
treatment
-
Orange
Blue mold
Black spot disease
Bergamot, thyme,
tea tree essential
oil
-
Decay (in general)
-
Decay (in general)
-
Tankan citrus
fruit
Clementine
mandarin fruit
22
References
(moment of
application)
(postharvest)
El Ghaouth et al., 2000
(postharvest)
Shao et al., 2012
(postharvest)
El Ghaouth et al., 2000
(postharvest)
Shao et al., 2012
(postharvest)
Yu et al., 2012
(postharvest)
Li and Yu, 2000
(postharvest)
Casals et al., 2012
(postharvest)
(pre and postharvest)
(preharvest); Feliziani
et al., 2013a (pre and
postharvest)
Cháfer et al., 2012
(postharvest)
Canale Rappussi et al.,
2009 (postharvest);
2011 (postharvest)
Chien and Chou, 2006
(postharvest)
Fornes et al., 2005 (pre
or postharvest)
Table 4. Chitosan treatments with other applications on storage decay of tropical
fruit.
Produce
Decay
Integration to
References
chitosan
Banana
Anthracnose
Zahid et al., 2012
(postharvest)
Arabic gum
Maqbool et al., 2010a
(postharvest); 2010b
(postharvest)
Crown rot
Cinnamon extract
Win et al., 2007
(postharvest)
Mango
Anthracnose
Zhu et al., 2008
(postharvest); Abd-Alla
and Haggag, 2010
(postharvest)
Irradiation
Abbasi et al., 2009
(postharvest)
Papaya
Anthracnose
Hewajulige et al., 2009
(postharvest); Ali et al.,
2010 (postharvest);
Zahid et al., 2012
(postharvest)
Aqueous extract of
Bautista-Baños et al.,
papaya seeds
2003 (postharvest)
Ammonium
Sivakumar et al., 2005b
carbonate, sodium
(postharvest)
bicarbonate
Dragon
Anthracnose
Zahid et al., 2012
fruit
(postharvest)
Litchi fruit Blue mold and
Potassium
Sivakumar et al., 2005a
Cladosporium rot metabisulphite
(postharvest)
Longan
Decay (in
Jiang and Li, 2001
fruit
general)
(postharvest)
23
Table 5. Chitosan treatments with other applications on storage decay of
vegetables.
Produce
Decay
Integration to
References
chitosan
Tomato
Gray mold
El Ghaouth et al., 1992b
(postharvest); Badawy and
Rabea, 2009 (postharvest)
Gray mold
Liu et al., 2007 (postharvest)
and blue mold
Blackmold rot
Reddy et al., 2000b
(postharvest)
Sweet
Decay (in
Cinnamon oil
Xing et al., 2011a (postharvest)
pepper
general)
Melon
Fusarium rot
Natamycin
Cong et al., 2007 (postharvest)
and black rot
Table grape bunches sprayed in the field with chitosan at three
different concentrations (1%, 0.5% and 0.1%), either once, 21 days before
harvest, or twice, 21 and 5 days before harvest, significantly reduced gray
mold infections after 30 days storage at 0 °C, followed by 4 days of shelf
life. Decay control by chitosan was not different from grapes treated in the
field with procymidone and stored with sulfur dioxide (Romanazzi et al.,
2002). Berries treated at preharvest stage with chitosan showed decreased
incidence and severity of artificially inoculated postharvest gray mold, with
the best results obtained 1-2 days after the application (Romanazzi et al.,
2006). Postharvest decay was reduced by preharvest chitosan treatment or
postharvest UV-C (0.36 J/cm2) irradiation for 5 min, and their combination
resulted in a synergistic action (Romanazzi et al., 2006). Application on the
day before harvest of the antagonistic fungus Cryptococcus laurentii
combined with 1% chitosan significantly reduced natural decay of table
grapes stored 42 days at 0 °C and then exposed to 3 days shelf life at 20 °C
(Meng et al., 2010b). In another work three different commercial
formulations containing chitosan have been compared in a field trial, in
24
which they were applied at four periods during the development of
‘Thompson Seedless’ grapes (berry set, pre-bunch clousure, veraison, and 2
weeks before harvest). The natural incidence of postharvest gray mold after
storage at 2 °C for 5 weeks was reduced by the chitosan applications as well
as the infections of detached berries after artificial inoculation with conidia
of Botrytis cinerea (Feliziani et al., 2013b).
Strawberries treated with chitosan at full bloom, or at green fruit
stage or whitening fruit stage showed a decrease in gray mold and Rhizopus
rot infections from natural inocula after 10 days of storage at 0 °C followed
by 4 days of shelf life; and the decay control with 1% chitosan was in almost
all treatments significantly better than the chemical standards, of
procymidone at the full bloom and green fruit stage, and pyrimethanil at the
whitening fruit stage (Romanazzi et al., 2000). Preharvest treatments with
1% and 2% chitosan decreased postharvest gray mold from natural
inoculum, and after preharvest and postharvest inoculation these applications
performed significantly better than a fungicide. The treatment with 1%
chitosan also performed better than that with 2%, the latter being
occasionally phytotoxic (Mazaro et al., 2008). Preharvest sprays with 0.2,
0.4 and 0.6% chitosan decreased postharvest gray mold and maintained the
keeping quality of strawberries during storage at 3 and 13 °C. The incidence
of decay decreased with increased chitosan concentration (Reddy et al.,
2000a).
Sweet cherries treated 7 days before the harvest with 0.1%, 0.5%
and 1% chitosan decreased gray mold and brown rot after 2 weeks of storage
at 0 °C followed by 7 days of shelf life, as compared to the untreated control.
At the highest chitosan concentration, the decay reduction was not different
with respect to that seen after application of tebuconazole (Romanazzi et al.,
1999). Similar results were obtained when 1% chitosan was applied 3 days
before the harvest, since it reduced sweet cherries postharvest diseased at a
level comparable to the one obtained with the synthetic fungicide
fenhexamid (Feliziani et al., 2013a). Seven days before harvest 1% chitosan
25
application and postharvest hypobaric treatments at 0.25 or 0.50 atm for 4 h
showed a synergistic effect in the control of total rots of sweet cherries
stored at 0 °C for 14 days, and then exposed to 7 day shelf life at 20 °C
(Romanazzi et al., 2003).
In another trials, Clemenules mandarin fruit were treated 86 days
before harvesting or at postharvest with low concentration solutions of
chitosan. In association with antisenescence effects, chitosan reduced the
water spot incidence of the Clemenules mandarins, and this effect increased
with increasing concentration (Fornes et al., 2005).
2.3
Postharvest application of chitosan for storage decay control
Compared to preharvest trials, large amount of data are available on
the effectiveness of chitosan treatment applied to produce after harvest
(Tables 3, 4, and 5), mainly because field trials are carried out only
following positive results obtained in studies where the compound was
applied at postharvest stage.
On temperate fruit the use of chitosan to control postharvest decay
has been tested since many years. On strawberries the effectiveness in
controlling postharvest gray mold and Rhizopus rot of chitosan coating was
comparable to the one obtained with synthetic fungicide applications (El
Ghaouth et al., 1991a; 1992a; Zhang and Quantick, 1998). Cladosporium sp.
and Rhizopus sp. infections decreased in artificially inoculated strawberry
fruit that were coated with chitosan and stored up to 20 days at 4-6 °C (Park
et al., 2005). Similar results were obtained for table grape small bunches
dipped in 0.5% and 1% chitosan solutions, artificially inoculated by spraying
with a B. cinerea conidial suspension, and stored at cool or room
temperatures. The treatment decreased the spread of gray mold infection
from a berry to the closest neighbors (nesting) (Romanazzi et al., 2002). Li
and Yu (2000) reported that 0.5% and 0.1% chitosan reduced significantly
the incidence of brown rot caused by Monilinia fructicola in peach stored at
23 °C and delayed the development of disease compared with the untreated
26
fruits. Similarly, application of 1% chitosan reduced the postharvest disease
of sweet cherry (Feliziani et al., 2013a). Treatments with chitosan and
oligochitosan reduced the disease incidence caused by Alternaria kikuchiana
and Physalospora piricola and inhibited the lesion expansion of the two
fungi in pear fruit stored at 25 °C; the disease control effects of chitosan and
oligochitosan were concentration-dependent and weakened over inoculated
time (Meng et al., 2010a). For vegetables such as tomatoes, lower disease
severity than control treatment was achieved with applications of low
molecular chitosan regardless concentration (Bautista-Baños and BravoLuna, 2004).
On the other hand, the recent advances concerning chitosan
application on postharvest temperate fruit deal with the possibility to
combine the biopolymer with other alternatives to fungicides, such as
decontaminating agents, plant extract or essential oil, biocontrol agents, or
physical mean in order to have a synergic action against fruit decay in
addition to the one already obtained with chitosan alone.
On postharvest control chitosan application was applied in
combination with biocontrol agents, such as Candida satoiana or
Cryptococcus laurentii, microorganisms that show an antagonistic activity
toward postharvest pathogens (El-Ghaouth et al., 2000; De Capdeville et al.,
2002; Yu et al., 2007; Meng et al., 2010b; Yu et al., 2012). Spraying the
yeast, C. laurentii, after postharvest chitosan coating significantly reduced
natural decay of table grapes stored at 0 °C. The chitosan coating enhanced
the effectiveness of the preharvest spray (Meng et al., 2010b). C. laurentii
associated with 0.5% chitosan and calcium chloride was also effective in the
reduction of postharvest blue mold in pear as well. Their combination
resulted in more effective mold control than chitosan or C. laurentii alone,
although chitosan at 0.5% inhibited growth of the biocontrol yeast in vitro
and in vivo. Moreover, after 6 days of incubation, the combined treatment
with C. laurentii, chitosan and calcium chloride inhibited mold decay by
nearly 89%, which was significant higher than that treated with C. laurentii,
27
chitosan and calcium chloride alone, or the combined treatment with C.
laurentii and chitosan, or with C. laurentii and calcium chloride (Yu et al.,
2012). Combination of chitosan and C. laurentii on apple resulted in a
synergistic inhibition of the blue mold rot, being the most effective at the
optimal concentration of 0.1% of chitosan (Yu et al., 2007). In tropical fruit,
the application of the bacterium Lactobacillus plantarum, alone or in
combination with 2% chitosan, preserved quality characteristics of rambutan
fruit (Martínez-Castellanos et al., 2009). Similarly, the combination of C.
saitoana with 0.2% glycolchitosan was more effective in controlling gray
and blue mold of apple and green mold of oranges and lemons than the yeast
or glycolchitosan alone (El-Ghaouth et al., 2000). On contrary, the
combination of chitosan with C. saitoana or with UV-C had no synergistic
effect on the progress of blue mold on apple, although the single treatment
provides significant reductions (De Capdeville et al., 2002).
Extracts obtained from many plants have recently gained popularity
and scientific interest for their antimicrobial properties, and recently their
activity against postharvest fungi of fruit and vegetables has been tested
(Gatto et al., 2011). Chitosan coating could be used as a carrier to
incorporate plant essential oils or extracts that have antifungal activity or
neutraucetical properties. Chitosan incorporated with limonene, a major
component of lemon essential oil, which has gained the GRAS status from
USFDA, preserved strawberry fruit during their shelf life (Vu et al., 2011).
The addition of lemon essential oil enhanced the chitosan antifungal activity
both in in vitro tests and during cold storage of strawberries inoculated with
a spore suspension of B. cinerea (Perdones et al., 2012). Coatings based on
chitosan either combined or not with oleic acid at different percentage
delayed the appearance of fungal infection in comparison to uncoated
strawberries. When oleic acid was added to the chitosan coating, fewer signs
of fungal infection were visible during the strawberry storage, especially
when the coatings contained higher levels of oleic acid that enhanced the
antimicrobial properties of chitosan (Vargas et al., 2006). On table grapes,
28
the combination of 1% chitosan and a grapefruit seed extract improved
decay control respect to chitosan single applications and maintained the
keeping quality of table grapes (Xu et al., 2007b). Similarly, chitosan
coatings containing bergamot oil and cinnamon oil improved the quality of
stored table grapes (Sánchez-González et al., 2011) and sweet pepper (Xing
et al., 2011a). Chitosan coatings, containing or not essential oils (bergamot,
thyme and tea tree oil), were applied to oranges as a preventive or curative
treatments against blue mold. In all cases the addition of essential oil
improved the antimicrobial activity of chitosan, however, preventive and
curative antimicrobial treatments with coatings containing tea tree oil and
thyme respectively were the most effective in the reducing the microbial
growth, as compared to the uncoated samples (Cháfer et al., 2012). On the
other hand, in another study combinations of cinnamon extract and chitosan
resulted not compatible since cinnamon extract reduced the effectiveness of
chitosan in controlling banana crown rot and in delaying fruit senescence
during storage (Win et al., 2007). Treatments of papaya with 0.5% or 1.5%
chitosan or the combination of 1.5% chitosan with aqueous extract of papaya
seed controlled the development of anthracnose disease of fruit inoculated
with Colletotrichum gloeosporioides. However, no synergistic effect was
obtained with the combination of chitosan at 1.5% and aqueous extract of
papaya to control the fungal growth (Bautista-Baños et al., 2003). Similarly,
a limited control of Rhizopus stolonifer was observed on chitosan-coated
tomatoes in combination with beeswax and lime essential oil (Ramos-García
et al., 2012).
Postharvest application of chitosan was combined with physical
mean, such as UV-C irradiation, hypobaric treatment and heat curing in
controlling postharvest decay of fruit and vegetables. Shao et al. (2012)
studied the effects of heat-treatment at 38 °C for 4 days before or after
coating with 1% chitosan on apples. Besides the completely control of blue
mold and gray mold on artificially inoculated apples during storage, chitosan
coating followed by heat treatment improved the quality of the stored fruit.
29
Moreover, the presence of chitosan coating prevented the occurrence of heat
damages on fruit surfaces (Shao et al., 2012). In anotherinvestigation, the
development of postharvest brown rot on peaches and nectarines was
controlled through the heating of fruit at 50 °C for 2 h and 85% RH, which
eradicated the eventual pre-existing Monilinia spp. infections coming from
the field, and the application of 1% chitosan at 20 °C, which protected the
fruit during handling in packinghouses until the consumer usage (Casals et
al., 2012). The combination of immersion in hot water (46.1 °C for 90 min)
and in 2% chitosan was beneficial to the storage qualities of mango
compared to the untreated mangoes or to fruit treated only with hot water or
chitosan (Salvador-Figueroa et al., 2011). Sweet cherries dipped in 1%
chitosan and soon after exposed to a hypobaric treatment (0.50 atm for 4 h)
had a significant reduction of postharvest brown rot, gray mold, and total
rots in comparison with the control and with each treatment applied alone.
This combination produced a synergistic effect in the reduction of brown rot
and total rots of stored sweet cherries (Romanazzi et al., 2003). The
combination of chitosan coating and modified atmosphere packaging was
effective in preventing decay, browning and retaining the pericarp color in
litchi fruit. In this study, chitosan was applied as a technology to improve the
benefices obtained with modified atmosphere packages (De Reuck et al.,
2009).
To improve its efficacy in controlling postharvest decay of fruit and
vegetables, chitosan was combined with decontaminating agents as well. The
combination of 0.5% chitosan with 10 or 20% ethanol, which is commonly
used for its antifungal properties in food industry, improved decay control
with respect to their single treatments in B. cinerea inoculated table grapes
single berries or clusters (Romanazzi et al., 2007). Application of natamycin,
which is a common food additive used against mold and yeast growth, in
combination with a bilayer coating containing chitosan and polyethylene
wax microemulsion extended the shelf life of Hami melon by decreasing its
weight loss and decay (Cong et al., 2007). Chitosan alone or in combination
30
with sodium bicarbonate or ammonium carbonate significantly reduced the
severity of anthracnose in both inoculated and naturally infected papaya
fruit. The effect of chitosan with ammonium carbonate on the incidence and
severity of anthracnose was greater than chitosan alone, or chitosan with
sodium bicarbonate (Sivakumar et al., 2005b). Similarly, the combination of
chitosan with potassium metabisulphite was tested in litchi fruit. Both
chitosan and the combination of chitosan and potassium metabisulphite
decrease postharvest decay of litchi fruit, however no synergistic
effectiveness was recorded (Sivakumar et al., 2005a).
In addition, it is worth of mention the combination of chitosan with
gum arabic, which is a common polysaccharide frequently used in industry
as a food additive, that controlled banana anthracnose caused by
Colletotrichum musae either in vitro or in vivo and enhanced the shelf life of
banana fruit (Maqbool et al., 2010a; 2010b).
Some other studies, tested the most suitable acids to dissolve
chitosan powder, indeed practical grade chitosan must be dissolved in an
acid solution to activate its antimicrobial and eliciting properties. Chitosan
dissolved in 10 different acids was effective in reducing gray mold incidence
on single table grape berries (Romanazzi et al., 2009). However, the greatest
reduction in gray mold decay (about 70% compared with the control) was
observed after immersion of the berries in chitosan dissolved in acetic or
formic acids, whereas intermediate effectiveness was observed with chitosan
dissolved in hydrochloric, lactic, L-glutamic, phosphorous, succinic, or Lascorbic acids. The least effective treatments were chitosan dissolved in
maleic or malic acids (Romanazzi et al., 2009).
Being chitosan potentially applied as a coating to prolong the
postharvest life of fruit and vegetables (Bautista-Baños et al., 2006), its
antimicrobial activity was tested as a wide range of pathogenic fungi causing
postharvest losses (Table 6).
31
Table 6. Growth inhibition by chitosan on decay-causing fungi affecting produce
during storage.
Fungus
Infected species
Reference
Tomato
Sánchez-Dómínguez et al.,
2007; 2011
Alternaria kikuchiana
Pear
Meng et al., 2010a
Aspergillus phoenicus
Pear
Cé et al., 2012
Aspergillus niger
Plascencia-Jatomea et al., 2003
Botrydiplodia lecanidion Tankan citrus fruit
Chien and Chou, 2006
Botrytis cinerea
Tomato, potato, bell
El Ghaouth et al., 1992a;
pepper, cucumber,
1992b; 1997; 2000; Du et al.,
peach, strawberries,
1997; Romanazzi et al., 2002;
table grapes, pear,
Ben-Shalom et al., 2003; Ait
apple, Tankan citrus
Barka et al., 2004; Badawy et
fruit
al., 2004; Chien and Chou,
2006; Lira-Saldivar et al., 2006;
Elmer and Reglinski, 2006; Liu
et al., 2007; Xu et al., 2007a;
Badawy and Rabea, 2009;
Rabea and Badawy, 2012
Cladosporium sp.
Litchi fruit,
Park et al., 2005; Sivakumar et
strawberry
al., 2005a
Colletotrichum
Mango, papaya
Bautista Baños et al., 2003;
gloeosporioides
2005; Sivakumar et al., 2005b;
Jitareerat et al., 2007; Ali and
Mahmud, 2008; Hewajulige et
al., 2009; Abd-Alla and Haggar,
2010; Ali et al, 2010; Zahid et
al., 2012
Colletotrichum musae
Banana
Win et al., 2007; Maqbool et
al., 2010a, 2010b; Zahid et al.,
2012
Colletotrichum spp.
Table grapes and
Muñoz et al., 2009
tomato
Fusarium solani
Eweis et al., 2006
32
Fungus
Fusarium sulphureum
Fusarium spp.
Geotricum candidum
Guignardia citricarpa
Infected species
Potato
Banana
Lasiodiplodia
theobromae
Monilinia fructicola
Monilinia laxa
Penicillium citrinum
Penicillium digitatum
Banana
Penicillium expansum
Litchi fruit,
strawberries, apple,
pear, tomato
Tankan citrus fruit
Penicillium italicum
Orange
Apple, peach
Sweet cherry
Jujube
Orange, lemon,
Tankan citrus fruit
Penicillium stolonifer
Phytophthora cactorum
Physalospora piricola
Rhizopus stolonifer
Pear
Strawberries
Pear
Peach, strawberries,
papaya, tomato
Sclerotinia sclerotiorum
Carrot
33
Reference
Li et al., 2009
Win et al., 2007
El-Mougy et al., 2012
Canale Rappussi et al., 2009;
2011
Win et al., 2007
Yang et al., 2010; 2012a
Feliziani et al., 2013a
Xing et al., 2011b
El Ghaouth et al., 2000;
Bautista-Baños et al., 2004;
Chien and Chou, 2006; ElMougy et al., 2012
El Ghaouth et al., 2000;
Sivakumar et al., 2005a; Liu et
al., 2007; Yu et al., 2007
Chien and Chou, 2006; ElMougy et al., 2012
Cé et al., 2012
Eikemo et al., 2003
Meng et al., 2010a
El Ghaouth et al., 1992b;
Bautista-Baños et al., 2004;
Park et al., 2005; GuerraSánchez et al., 2009; García
Rincón et al., 2010; HernándezLauzardo et al., 2010; Ramos
García et al., 2012
Cheah et al., 1997; Molloy et
al., 2004
2.4
Activity of chitosan against postharvest decay causing fungi
The antimicrobial activity of chitosan seems to rely on electrostatic
interactions between positive chitosan charges and the negatively charged
plasma membrane phospholipids that integrate the fungi membrane.
Chitosan first binds to the target membrane surface and covers it, and in a
second step, after a threshold concentration has been reached, chitosan
causes membrane permeabilization and release of cellular content (PalmaGuerrero et al., 2010). Low levels of Ca2+ are usually kept by the fungi in
their cytosol, due to the barrier forming the plasmatic membrane, which has
hermetic seals that regulate the passage of Ca2+ gradients; this process,
which also involves the homeostatic mechanism, where the Ca2+
concentration regulates itself within the cytosol, sends the Ca2+ excess out of
the cell or stores it in the cell organelles. Thus, as the chitosan is applied, the
homeostatic mechanism becomes drastically transformed, because as it
forms channels in the membrane, it allows the free passage of calcium
gradients, causing deadly instability in the cell (Palma-Guerrero et al., 2009).
In addition, inhibitory effect of chitosan on H+-ATPase in the plasma
membrane of R. stolonifer was reported. The authors suggested that the
decrease in the H+-ATPase activity could induce the accumulation of protons
inside the cell, which would result in the inhibition of the chemiosmotic
driven transport that allows the H+/K+ exchange (García-Rincón et al., 2010).
Moreover, it was reported as an effect of chitosan treatment a rapid efflux of
potassium from cells of R. stolonifer and in increment in pH of the culture
medium that were chitosan concentration dependent. Both phenomena were
related to the leaking of the internal cell metabolites (García-Rincón et al.,
2010). Similarly, when R. stolonifer was grown in media containing
chitosan, the release of proteins by the fungal cell was increased
significantly. And it was proposed that the liberation of proteins from the cell
to the supernatant is due to the fact that there are sites in which the cellular
membrane is damaged by chitosan (Guerra-Sánchez et al., 2009).
Beside its capacity of membrane permeabilization, chitosan was able
34
to penetrate into the fungal cells. Fluorescent labeled chitosan was detected
into fungal conidia and it was hypothesized that chitosan itself permeabilizes
the plasma membrane allowing it to enter the cytoplasm (Palma-Guerrero et
al., 2008; 2009). Another study demonstrated, through fluorescence
visualization, that oligochitosan could penetrate cell membrane of
Phytophthora capsici and that it could bind to intracellular targets such as
DNA and RNA (Xu et al., 2007a). Similarly, observation made on
Aspergillus niger revealed presence of labeled chitosan both outside and
inside the cell, and the permeated chitosan was suggested to block the DNA
transcription and therefore to inhibit the growth of the fungus (Li et al.,
2008).
Several works described the morphological changes induced by
chitosan on fungal hyphae and reproductive structures. Scanning electron
microscopy observations of Fusarium sulphureum treated with chitosan
revealed the effects on the hyphae morphology. The growth of hyphae
treated with chitosan was strongly inhibited, it was tightly twisted and
formed rope-like structures. Spherical or club-shaped abnormally inflated
ends were observed on the twisted hyphae that were swollen and with
excessive branching. Further transmission electron microscopy observation
indicated the ultrastructural alterations by chitosan of the hyphae. These
changes included cellular membrane disorganization, cell wall disruption,
abnormal distribution of cytoplasm, non-membranous inclusion bodies
assembling in cytoplasm, considerable thickening of the hyphal cellular
walls, and very frequent septation with malformed septa (Li et al., 2009).
Examination of ultrasections of the hyphae and conidia of chitosan-treated
Alternaria alternata, revealed marked alterations on the cell wall. The
chitosan-treated mycelium showed predominantly loosened cell walls and in
some areas, lysis was observed. The conidia exposed to chitosan were
intensely damaged, usually eroded and broken cell walls were seen
containing in some cases no cytoplasm (Sánchez-Dómínguez et al., 2007;
2011). R. stolonifer subjected to the formulation with chitosan combined
35
with beeswax and lime essential oil showed no development of the typical
reproductive structures, and its mycelium was distorted and swollen (Ramos
García et al., 2012). In another investigation, chitosan-treated spore of R.
stolonifer showed numerous and deeper ridge formations that were not
observed on not-treated spores (Hernández-Lauzardo et al., 2008). Chitosan
induced morphological changes of mycelium of B. cinerea and R. stolonifer
characterized by excessive hyphal branching as compared to the control (El
Ghaouth et al., 1992a). This was confirmed by another study, in which
induced marked morphological changes and severe structural alterations
were observed in chitosan treated cells of B. cinerea. Microscopic
observations showed coagulation in the fungus cytoplasm characterized by
the appearance of small vesicles in mycelium treated with chitosan. In other
cases, the mycelium contained larger vesicles or even empty cells devoid of
cytoplasm (Ait Barka et al., 2004). The area and the elliptical form of spores
were significantly different when C. gloeosporioides was grown on PDA
(potato dextrose agar) amended with chitosan from the sole PDA (BautistaBaños et al., 2003). Similarly, hyphal and germ tube morphology of C.
gloeosporioides growing on chitosan showed malformed hyphal tips with
thickened walls. Many swellings occurred in the hyphae or at their tips
whereas in controls cell walls and germ tubes were smooth with no swelling
or vacuolation (Ali and Mahmud, 2008; Ali et al., 2010). The scanning
electron micrographs showed normal growth of hyphae in untreated control
for C. gloeosporioides, whereas hyphal agglomeration and formation of
large vesicles in mycelia were observed in samples treated with chitosanloaded nanoemulsions (Zhaid et al., 2012). The fungal mycelium of
Sclerotinia sclerotiorum exposed to chitosan was deformed, twisted and
branched, or dead with no visible cytoplasm into the fungal cells, whereas
untreated mycelium was normal in appearance (Cheah et al., 1997).
Not all the fungi have the same sensitivity to chitosan, which may be
due their intrinsic characteristics. New findings about the permeabilization
of the plasma membrane of different cell types of the fungi Neurospora
36
crassa and the membrane composition among various resistant and
nonresistant-chitosan fungi appear to be important factors (Palma-Guerrero
et al., 2008; 2009; 2010). By imaging fluorescently labeled chitosan using
confocal microscopy, it was seen that chitosan binds to the conidial surfaces
of all species tested, but only consistently permeabilizes the plasma
membranes of some fungi. Some others could form a barrier to chitosan. The
analysis of the main plasma membrane components revealed important
differences in fatty acid composition between chitosan-sensitive and
chitosan-resistant fungi. A higher content of the polyunsaturated fatty acid
linolenic acid, a higher unsaturation index and lower plasma membrane
fluidity were measured in the membranes of chitosan-sensitive fungi.
Chitosan binding should induce an increase in membrane rigidity in the
regions to which it attaches. This interaction will enhance differences in
fluidity between different membrane regions, causing membrane
permeabilization. In a saturated, more rigid membrane, the changes in
rigidity induced by chitosan binding would be much lower and little
permeabilization, even in the presence of negatively charged phospholipids
headgroups, should be induced (Palma-Guerrero et al., 2010).
The antifungal activity of chitosan was reported to vary according to
its molecular weight and concentration. It was also noted that, in general, the
fungal growth inhibition increased as the concentration of chitosan was
increased in the case of B. cinerea (El Ghaouth et al., 1992a; 2000; Ben
Shalom et al., 2003; Chien and Chou, 2006; Liu et al., 2007), R. stolonifer
(El Ghaouth et al., 1992a), Penicillium citrinum (Xing et al., 2011b);
Penicillium digitatum (Chien and Chou, 2006), Penicillium italicum (Chien
and Chou, 2006), Penicillium expansum (El Ghaouth et al., 2000; Liu et al.,
2007; Yu et al., 2007), Monilinia fructicola (Yang et al., 2010; 2012a),
Botrydiplodia lecanidion (Chien and Chou, 2006), C. gloeosporioides
(Bautista-Baños et al., 2005; Jitareerat et al., 2007; Muñoz et al., 2009; Ali
and Mahmud, 2008; Ali et al., 2010; Abd-Alla and Haggar, 2010), Fusarium
solani (Eweis et al., 2006), A. kikuchiana (Meng et al., 2010a) and P.
37
piricola (Meng et al., 2010a), but decreased in the case of A. niger (Li et al.,
2008). In some studies antifungal activity of chitosan decreased with the
increase of molecular weight (Li et al., 2008). The highest inhibitory effect
against the growth of R. stolonifer was observed with low molecular weight
chitosan, while the high molecular weight chitosan affected more the
development of the spores (Hernández-Lauzardo et al., 2010). High
molecular weight chitosan had the lowest inhibitory effect on the B. cinerea
growth compared to low molecular weight chitosan (Rabea and Badawy,
2012). Spore germination and germ tube elongation of A. kikuchiana and P.
piricola were significantly inhibited by chitosan and oligochitosan, but,
compared to chitosan, oligochitosan was more effective on inhibition of
spore germination (Meng et al., 2010a). However, in other investigations, it
was noted fungal growth inhibition by chitosan, regardless of the type of
chitosan (Chien and Chou, 2006), or any fungicidal or fungistatic pattern
among low, medium, and high molecular weight chitosans tested with
different isolates of C. gloeosporioides (Bautista-Baños et al., 2005) and R.
stolonifer (Guerra-Sánchez et al., 2009).
2.5
Induction of resistance by chitosan on fruit
Plant resistance toward pathogens occurs through hypersensitive
response that results in cell death at the penetration site, structural alteration,
accumulation of reactive oxygen species, synthesis of secondary metabolites
and defense molecules, and activation of “pathogenesis related proteins” (PR
proteins) (Van-Loon and Van-Strien, 1999). The application of external
elicitors on vegetal tissue could trigger plant resistance, simulating the
pathogen presence. Several studies reported that chitosan induces a series of
enzyme activities or compound production that are correlated with plant
defense reactions to pathogen attack (Bautista-Baños et al., 2006) (Tables 7,
8, and 9).
38
Table 7. Physiological changes occurring in temperate fruit after chitosan treatment.
Temperate
Physiological change
Integration to
References
fruit
chitosan
Table grapes Phenilalanine ammonia- Romanazzi et
lyase
al., 2002; Meng
et al., 2008
Cryptococcus
Meng and Tian,
laurentii
2009; Meng et
al., 2010b
Peroxidase
Meng et al.,
2008
Polyphenol oxidase,
Meng et al.,
superoxide dismutase
2008
Cryptococcus
Meng and Tian,
laurentii
2009; Meng et
al., 2010b
Chitinase, myricetin
Feliziani et al.,
2013b
Quercetin
Feliziani et al.,
2013b
Putrescine
Shiri et al.,
2013
Respiration
Bergamot oil
SánchezGonzález et al.,
2011
Resveratrol
UV
Romanazzi et
al., 2006
Feliziani et al.,
2013b
Soluble solids content
Meng et al.,
2008
Bergamot oil
2011
39
Temperate
fruit
Titratable acidity
Integration to
chitosan
Cryptococcus
laurentii
-
Total phenolic content
Cryptococcus
laurentii
Putrescine
Weight loss, color,
texture
Bergamot oil
Putrescine
Strawberry
Shattering and cracking
Grape seed extract
Putrescine
Titratable acidity
Grape seed extract
-
Vitamin E
Calcium gluconate
pH
Calcium gluconate
40
References
Meng et al.,
2010b
Meng et al.,
2008
Meng et al.,
2008
Meng et al.,
2010b
Shiri et al.,
2013
2011
Shiri et al.,
2013
Xu et al., 2007b
Shiri et al.,
2013
Xu et al., 2007b
El Ghaouth et
al., 1991a;
Zhang and
Quantick, 1998;
Reddy et al.,
2000a
Han et al.,
2004; 2005;
HernándezMuñoz et al.,
2008;
Temperate
fruit
Integration to
chitosan
Vitamin E
-
Antocyanin content
Oleic acid
Total polyphenol
-
Soluble solids content
Color
Vitamin E
Calcium gluconate
Vitamin E
Firmness
Calcium gluconate
-
Vitamin C content
-
Glutathione
-
Chitinase, β-1,3
glucanase
-
41
References
2008;
Han et al., 2004
El Ghaouth et
al., 1991a;
Zhang and
Quantick, 1998;
Reddy et al.,
2000a
Vargas et al.,
2006
Kerch et al.,
2011
Han et al., 2005
2008
Han et al.,
2004; 2005
2008
El Ghaouth et
al., 1991a
Zhang and
Quantick, 1998;
Kerch et al.,
2011; Wang and
Gao, 2013
Wang and Gao,
2013
Zhang and
Quantick, 1998
Temperate
fruit
Integration to
chitosan
-
Phenilalanine ammonialyase
Weight loss
Respiration
Raspberry
Apple
Pear
Vitamin E
-
Catalase, glutathioneperoxidase, guaiacol
peroxidase,
dehydroascorbate
reductase,
monodehydroascorbate
reductase
Weight loss, color, pH,
titratable acidity
Ascorbic acid, titratable
acidity, firmness,
antocyanin content
Respiration, firmness,
weight loss, titratable
acidity
Polyphenol oxidase,
chitinase, β-1,3
glucanase,
ROS, catalase,
superoxide dismutase,
ascorbate peroxidase,
glutathione reductase
Peroxidase
-
Respiration,
-
References
Romanazzi et
al., 2000
Han et al., 2004
El Ghaouth et
al., 1991a;
Vargas et al.,
2006
Wang and Gao,
2013
Vitamin E
Han et al., 2004
-
Zhang and
Quantick, 1998
Heat
Shao et al.,
2012
-
Meng et al.,
2010a
Li et al., 2010
-
42
Meng et al.,
2010a; Li et al.,
2010
Zhou et al.,
Temperate
fruit
Apricot
Peach
Sweet cherry
Orange
Integration to
chitosan
permeability of cell
membrane, weight loss
Soluble solid contents,
titratable acidity,
firmness
Total phenolic content,
antioxidant activity,
weight loss
Titratable acidity,
ascorbic acid,
respiration, firmness,
ethylene and
malondialdehyde
production, superoxide
dismutase
Polyphenol oxidase,
peroxidase, ascorbic
acid oxidase,
polygalacturonase,
vitamin C
Titratable acidity,
soluble solid, catalase,
peroxidase, polyphenol
oxidase, phenylalanine
ammonia-lyase,
respiration
Ascorbic acid
Ascorbic acid
Ascorbic acid
Phenols content,
antocyanin content
Water loss, firmness
2008
Lin et al., 2008
Lin et al., 2008
-
Ghasemnezhad
et al., 2010
-
Li and Yu, 2000
CaCl2 coating +
PEpackage +
intermittent warming
Ruoyi et al.,
2005
-
Dang et al.,
2010
-
Dang et al.,
2010; Kerch et
al., 2011
Kerch et al.,
2011
Cháfer et al.,
Bergamot, thyme, tea
43
References
Temperate
fruit
Integration to
chitosan
tree essential oil
-
Color
Tankan
citrus fruit
Jujube
Chitinase, b-1,3glucanase, polyphenol
oxidase
Peroxidase
-
Superoxide dismutase,
catalase, ascorbate
peroxidase, glutathione
reductase, hydrogen
peroxide content,
ascorbate content
Firmness, weight loss,
titratable acidity,
ascorbic acid, soluble
solids
Polyphenol oxidase,
phenolic compounds
-
-
Chien and
Chou, 2006
-
Xing et al.,
2011b
Wu et al., 2010
Xing et al.,
2011b
Qiuping and
Wenshui, 2007
Qiuping and
Wenshui, 2007
Qiuping and
Wenshui, 2007
Wu et al., 2010
1methylcyclopropene
1methylcyclopropene
1methylcyclopropene
Zinc, cerium
Firmness
Weight loss
44
2012
Canale
Rappussi et al.,
2011
Canale
Rappussi et al.,
2009
Canale
Rappussi et al.,
2009; Zeng et
al., 2010
Zeng et al.,
2010
-
Zinc, cerium
-
Ascorbic acid
References
Temperate
fruit
Integration to
chitosan
Zinc, cerium
Respiration, soluble
solids
References
Wu et al., 2010
Table 8. Physiological changes occurring in tropical fruit after chitosan treatment.
Tropical
Physiological
Integration to
References
fruit
changes
chitosan
Banana
Titratable acidity
Kittur et al., 2001
1-methylcyclopropene
Baez-Sañudo et al.,
2009
Arabic gum
Maqbool et al.,
2010a, 2010b
Respiration
Kittur et al., 2001
2009
Arabic gum
Maqbool et al.,
2011
Firmness, soluble
Kittur et al., 2001;
solids content
Win et al., 2007
2009
Arabic gum
Maqbool et al.,
2010a, 2010b; 2011
Color
Kittur et al., 2001;
Win et al., 2007
2009
Arabic gum
Maqbool et al.,
2011
Weight loss
Arabic gum
Maqbool et al.,
2010a, 2010b; 2011
Longan
Respiration, weight
Jiang and Li, 2001
45
Tropical
fruit
fruit
Mango
Physiological
changes
loss, color change,
polyphenol oxidase,
titratable acidity,
total soluble solids,
ascorbic acid
Titratable acidity,
weight loss
Integration to
chitosan
References
-
Jitareerat et al.,
2007; Zhu et al.,
2008
Salvador-Figueroa
et al., 2011
Zhu et al., 2008
Salvador-Figueroa
et al., 2011
Salvador-Figueroa
et al., 2011
Jitareerat et al.,
2007
Jitareerat et al.,
2007; Zhu et al.,
2008
Ali et al., 2010;
2011
Al Eryani et al.,
2008
Ali et al., 2011
Al Eryani et al.,
2008
Ali et al., 2011
Sivakumar et al.,
2005b
Al Eryani et al.,
2008
Ali et al., 2010;
Hydrothermal process
Papaya
Total soluble solids,
firmness, color
change
pH
Chitinase, b-1,3glucanase
Respiration,
ascorbic acid
-
Titratable acidity,
total soluble solids
-
-
Calcium infiltration
Ascorbic acid
Weight loss, color
Ammonium carbonate,
sodium bicarbonate
Firmness
46
Tropical
fruit
Physiological
changes
Chitinase, b-1,3glucanase
Respiration
Litchi fruit
Weight loss
Integration to
chitosan
Ammonium carbonate,
sodium bicarbonate
Aqueous extract of
papaya seeds
-
-
Organic acids
Titratable acidity
-
Organic acids
Total phenolic
content, flavonoid
content
-
Anthocyanin
47
References
2011
Sivakumar et al.,
2005b
Bautista-Baños et
al., 2003
Hewajulige et al.,
2009
Hewajulige et al.,
2009, Ali et al.,
2011
Zhang and
Quantick, 1997;
Jiang and Li, 2001;
Sivakumar et al.,
2005a; Sun et al.,
2010; Lin et al.,
2011
Joas et al., 2005;
Caro and Joas,
2005
Jiang et al., 2005;
Sivakumar et al.,
2005a; Sun et al.,
2010
Joas et al., 2005;
Caro and Joas,
2005
Zhang and
Quantick, 1997;
Sivakumar et al.,
2005a
Zhang and
Tropical
fruit
Physiological
changes
content
Respiration
Color
Integration to
chitosan
Modified atmosphere
packaging
-
Organic acids
Peroxidase
Ascorbic acid
Modified atmosphere
packaging
Ascorbic acid
-
Polyphenol oxidase
Ascorbic acid
Modified atmosphere
packaging
-
Superoxide
Ascorbic acid
Modified atmosphere
packaging
Ascorbic acid
Total soluble solid
48
References
Quantick, 1997;
Jiang et al., 2005;
Sivakumar et al.,
2005a;
De Reuck et al.,
2009
Lin et al., 2011
Zhang and
Quantick, 1997;
Ducamp-Collin et
al., 2008
Caro and Joas,
2005; Joas et al.,
2005
Sun et al., 2010
De Reuck et al.,
2009
Jiang et al., 2005
Sun et al., 2010
Zhang and
Quantick, 1997;
Dong et al., 2004
Sun et al., 2010
De Reuck et al.,
2009
Zhang and
Quantick, 1997;
Jiang et al., 2005;
Lin et al., 2011
Sun et al., 2010
De Reuck et al.,
2009
Sun et al., 2010
Tropical
fruit
Rambutan
Guava
Physiological
changes
dismutase, catalase,
hydrogen peroxide,
malondialdehyde;
ascorbic acid
content
Firmness, soluble
solid, titratable
acidity
Firmness,
peroxidase
superoxide
dismutase, catalase,
inhibition of
superoxide free
radical production,
titratable acidity,
ascorbic acid,
weight loss, soluble
solids, chlorophyll
and
malondialdehyde
content
Integration to
chitosan
References
Lactobacillus
plantatum
MartínezCastellanos et al.,
2009
Hong et al., 2012
-
49
Table 9. Physiological changes occurring in vegetables after chitosan treatment.
Vegetables Physiological changes
Integration
References
to chitosan
Tomato
Respiration, color, ethylene,
El Ghaouth et al.,
firmness, titratable acidity
1992b
Polyphenol oxidase, phenolic
Liu et al., 2007;
content
Badawy and Rabea,
2009
Peroxidase
Liu et al., 2007
Protein content
Badawy and Rabea,
2009
Polygalacturonase, pectate
Reddy et al., 2000b
lyase, cellulose, phytoalexin
production, pH
Potato
Peroxidase, polyphenol
Sun et al., 2008
oxidase, flavonoid content,
lignin content
Phenylalanine ammonia-lyase Gerasimova et al.,
2005
Sweet
Superoxide dismutase,
Cinnamon
Xing et al., 2011a
pepper
peroxidase, catalase
oil
Respiration, weight loss, color El Ghaouth et al.,
1991b;
Cucumber Respiration, weight loss, color El Ghaouth et al.,
1991a
Melon
Weight loss, ascorbic acid, pH Natamycin
Cong et al., 2007
50
Phenylalanine ammonia lyase (PAL) is the key enzyme in phenol
synthesis pathway (Cheng and Breen, 1991) and the accumulation of phenols
that act as phytoalexins has been considered the primary inducible response
of plants against a number of biotic and abiotic stresses (Bhattacharya et al.,
2010; Großkinsy et al., 2012). Chitosan application has been reported to
increase PAL activity in treated fruit tissue. Table grape bunches with a
preharvest spraying with chitosan showed a three-fold increase in PAL
activity in the berry skin 24 h and 48 h after the application (Romanazzi et
al., 2002). PAL elicitation by chitosan was confirmed with table grapes
sprayed in the vineyard with or without C. laurentii and coated at
postharvest, then stored at 0 °C (Meng et al., 2008; 2010b; Meng and Tian,
2009). Chitosan treatments induced activities of PAL in sweet cherry (Dang
et al., 2010) and strawberry (Romanazzi et al., 2000) enhancing the fruit
defense responses during cold storage.
Chitinase and β-1,3-glucanase are two kinds of PR protein that
participate in defense against pathogens, since they are capable of partially
degrading fungal cell wall (Van-Loon and Van-Strien, 1999). The increase in
the activity of chitinase and β-1,3-glucanase was demonstrated as result of
chitosan application in ‘Valencia’ oranges, 24 h after chitosan treatment. It
was proposed that this change in enzyme activity could have contributed for
the reduction of black spots in the orange fruit (Canale Rapussi et al., 2009).
High activity of chitinase and β-1,3-glucanase activities in chitosan treated
strawberries compared to the untreated fruit, reinforced the microbial
defense mechanism of the fruit and accentuated the resistance against fungal
invasion (Zhang and Quantick, 1998; Wang and Gao, 2013). Chitinase and
β-1,3-glucanase activities of papaya and mango subjected to chitosan
treatment, were much higher than in the untreated fruit (Jitareerat et al.,
2007; Hewajulige et al., 2009) and oligochitosan treatment significantly
enhanced the activity of chitinase and β-1,3-glucanase in pear fruit (Meng et
al., 2010a). In table grapes, preharvest chitosan treatments, from three
different commercial formulations, induced activity of endochitinase, while
51
two of the chitosan formulations induced exochitinase activity (Feliziani et
al., 2013b).
In fruit tissue, high activity of pectic enzymes such as
polygalacturonase, cellulase and pectate lyase was shown to be closely
associated with weakening of plant cell wall, which resulted in softening of
the fruits and greater susceptibility to storage rots (Stevens et al., 2004).
Down-regulation of polygalacturonase resulted in firmer fruit (Atkinson et
al., 2012). In peach fruit chitosan treatments inhibited polygalacturonase
activity somewhat throughout the storage period. And in particular, the
combination consisting of the coating of chitosan and calcium chloride, the
polyethylene package, and intermittent warming markedly inhibited
polygalacturonase activity at the end of the refrigerated storage (Ruoyi et al.,
2005). Macerating enzyme activity, such as polygalacturonase, pectate lyase,
and cellulase in tomato tissue in the vicinity of lesions caused by the
pathogen A. alternata was less than half in chitosan-treated fruit compared
with untreated fruit. Chitosan inhibited the development of black mold rot of
tomatoes and reduced the production of pathogenic factors by the fungus
(Reddy et al., 2000b).
Chitosan treatment could induce fruit disease resistance by
regulating the reactive oxygen spicies levels, antioxidant enzyme and the
ascorbate-glutathione cycle (Tables 7, 8, and 9). Reactive oxygen species
(ROS), such as H2O2, O2-, are the earliest events correlated with plant
resistance to pathogens (Baker and Orlandi, 1995) and are involved in the
development of disease resistance (Torres et al., 2003). Although ROS could
contribute to the enhancement of plant defense, high level of ROS may cause
lipid peroxidation and lead to the loss of membrane integrity of plant organs.
To prevent harmful effects of ROS excess, they could be detoxified by an
antioxidant system, consisting of not enzymatic antioxidants, such as
ascorbic acid, glutathione, phenolic compounds, and antioxidant enzymes
such as superoxide dismutase (SOD), peroxidases (POD) and catalases
(CAT). Chitosan application was reported to reduce ROS in tissue of treated
52
fruit, such as pear (Li et al., 2010), or guava (Hong et al., 2012), and to
lowered hydrogen peroxide content in litchi (Sun et al., 2010), pear (Li et al.,
2010), table grapes (Feliziani et al., 2013b) and strawberry (Romanazzi et
al., 2013). This may be due to a direct effect, since chitosan itself has
antioxidant activity and scavenges hydroxyl radicals (Yen et al., 2008), or an
indirect effect, since chitosan induces the plant antioxidant system.
Higher level of glutathione was reported after chitosan treatment in
litchi fruit (Sun et al., 2010), strawberry (Wang and Gao, 2013) and orange
(Zeng et al., 2010). And higher quantity of ascorbic acid was found
subsequently to chitosan application in fruit tissues of strawberry (Wang and
Gao, 2013), peach (Li and Yu, 2000; Ruoyi et al., 2005), sweet cherry (Dang
et al., 2010; Kerch et al., 2011), jujube (Qiuping and Wenshui, 2007; Xing et
al., 2011b); orange (Zeng et al., 2010), citrus (Chien and Chou, 2006),
longan (Jiang and Li, 2001), guava (Hong et al., 2012), mango (Jitareerat et
al., 2007; Zhu et al., 2008) and litchi (Sun et al., 2010). The reduction of
ascorbic acid loss in chitosan coated sweet cherries was proposed to be due
to the low oxygen permeability of the chitosan coating around fruit surface,
which lowered the oxygen level and reduced the activity of the ascorbic acid
oxidase enzymes, preventing oxidation of ascorbic acid (Dang et al., 2010).
The presence of antioxidants, such as phenols, could substantially
reduce the ROS content of plant tissue, since their hydroxyl groups and
unsaturated double bonds make them very susceptible to oxidation (RiceEvans et al., 1997). Chitosan coating was effective in the intensification of
total antioxidant capacity of treated apricot, increasing the phenolic
compounds in fruit tissue (Ghasemnezhad et al., 2010). In tomato, the
content of phenolic compounds increased in chitosan treated fruit compared
to the untreated ones (Liu et al., 2007) and this increase was directly
proportional to the chitosan concentration used (Badawy and Rabea, 2009).
Table grapes treated with chitosan had higher phenolic compounds content
(Shiri et al., 2013; Feliziani et al., 2013b). Anthocyanin, flavonoid and total
phenolics contents of chitosan treated litchi decreased more slowly than in
53
the untreated fruit (Zhang and Quantick, 1997; Jiang et al., 2005; De Reuck
et al., 2009;). Kerch et al. (2011) reported that total phenols and anthocyanin
content increased in chitosan treated sweet cherry after 1 week of cold
storage, while their contents decreased in chitosan treated strawberry stored
with the same conditions. Similarly in strawberry, chitosan coated fruit had
lower anthocyanin content since they were synthesized at a slower rate than
non-treated berries (El Ghaouth et al., 1991a) and the rate of pigment
development was lower with the increase in chitosan concentration (Reddy
et al., 2000a). Anthocyanin contents significantly decreased throughout
storage in strawberries coated with chitosan combined with oleic acid,
whereas no significant changes were observed in uncoated samples, at the
end of the storage (Vargas et al., 2006). On the contrary, Wang and Gao
(2013), reported that strawberries treated with chitosan maintained better
fruit quality with higher levels of phenolics, anthocyanins and flavonoids.
Chitosan treatment has been reported to have an influence on
antioxidant enzyme activities of fruit tissues (Tables 7, 8, and 9).
Strawberries treated with chitosan, compared to untreated, maintained higher
levels of antioxidant enzyme activity such as CAT, glutathione-peroxidase,
guaiacol
peroxidase,
dehydroascorbate
reductase,
and
monodehydroascorbate reductase (Wang and Gao, 2013). Ascorbate
peroxidase or glutathione reductase activities were increased in pear that
were treated with chitosan (Lin et al., 2008; Li et al., 2010). Compared to
uncoated fruit, higher activity of SOD, CAT, and POD was reported after
chitosan application in tissue of pear (Lin et al., 2008; Li et al., 2010), sweet
pepper (Xing et al., 2011a) and tropical fruit, such as guava (Hong et al.,
2012). In addition, POD activity increase after chitosan application was
reported in several other commodities, such as table grapes (Meng et al.,
2008), pear (Meng et al., 2010a), sweet cherry (Dang et al., 2010), orange
(Canale Rappussi et al., 2009), tomato (Liu et al., 2007), and potato (Sun et
al., 2008). On contrary, in other studies decreased POD activity was reported
in litchi fruit after chitosan application combined or not with other
54
treatments (Zhang and Quantick, 1997; De Reuck et al., 2009; Sun et al.,
2010). While, the treatment of litchi fruit with a combination of chitosan and
ascorbic acid increased the activities of SOD and CAT and the contents of
ascorbic acid and glutathione (Sun et al., 2010). Treatments with chitosan
alone or in combination with C. laurentii decreased the SOD activity in table
grape tissues (Meng et al., 2008, 2010b; Meng and Tian, 2009). Treatments
of navel oranges with 2% chitosan effectively enhanced the activities of
POD, SOD and ascorbate peroxidase, but decreased activities of CAT and
the content of ascorbic acid (Zeng et al., 2010).
Physiological changes concerning polyphenol oxidase (PPO)
activity was observed after application of chitosan to fruit (Tables 7, 8, and
9). This has a great impact on fruit quality, indeed PPO is a phenol-related
metabolic enzymes which catalyzes oxidation of phenolic compounds, that
are involved in plant defense against biotic and abiotic stress and in
pigmentation/browning of fruit and vegetables tissues (Lattanzio et al., 2006;
Bhattacharya et al., 2010; Großkinsy et al., 2012). In some investigations
chitosan decreased PPO activity and its inhibitory effect is probably a
consequence of the ability of chitosan positive charges to adsorb suspended
PPO, its substrates, or its products (Badawy and Rabea, 2009). The other
possibility is that the selective permeability to gases due to the chitosan
coating generates low levels of oxygen around the fruit surface, that delays
the deteriorative oxidation reactions, and partially inhibits the activities of
oxidases such as PPO (Ayranci and Tunc, 2003). The chitosan coating of
litchi markedly reduced PPO activity and delayed skin browning during fruit
shelf life. The maintenance of the skin color of the litchi fruit after chitosan
treatment may be accounted for the higher level of anthocyanin content in
the skin resulting from the inhibition of the PPO activity (Zhang and
Quantick, 1997; Jiang et al., 2005; De Reuck et al., 2009). Similarly, by
treating harvested litchi fruit with ascorbic acid and 1% chitosan solution,
activities of PPO and POD and relative parameters of browning index in
pericarp were markedly lowered in treated litchi fruit (Sun et al., 2010). In
55
chitosan treated tomato (Badawy and Rabea, 2009) and jujube (Wu et al.,
2010; Xing et al., 2011b), the decrease of PPO activities was concomitant
with enhanced phenolic content and, in sweet cherry (Dang et al., 2010) with
the reduction in the tissue browning. The combination of chitosan, calcium
chloride and intermittent warming decreased the PPO activity in the tissues
of treated peaches that were cold stored for 50 days (Ruoyi et al., 2005). On
contrary, in other works PPO activities of fruit tissue increased after chitosan
treatment. Chitosan treatment significantly enhanced the activities of PPO in
flesh around wound of pear fruit (Meng et al., 2010a). An increase in the
activity of PPO was demonstrated as result of chitosan application in
‘Valencia’ oranges, 24 h after chitosan treatment (Canale Rapussi et al.,
2009). Chitosan treatment in tomato fruit stored at 25 and 2 °C increased the
content of phenolic compounds and induced the activities of PPO, whose
level was almost 1.5-fold that in wounded control fruit at the same time (Liu
et al., 2007). In this study there was no direct relationship between the PPO
activities and the content of phenolic compounds, although phenolic
compounds could be oxidized by the action of PPO and POD to produce
quinones (Campos-Vargas and Saltveit, 2002). It is likely that regulation of
phenolic metabolism by the action of other enzymes such as PAL, which
participates in the biosynthesis of phenolic compounds, also play a role (Liu
et al., 2007). This could explain even the reason why in some investigations
PPO level of fruit tissue after chitosan application is variable. Preharvest
spray with C. laurentii combined with postharvest chitosan coating increased
the activities of PPO of table grapes in storage, but after 3 days of shelf life,
PPO activities in treated fruit were lower than in untreated (Meng et al.,
2010b). During cold storage PPO activity of litchi fruit coated with chitosan
increased slowly, reached a peak, and then decreased (Zhang and Quantick,
1997).
56
2.6
Effect of chitosan treatment on retention of fruit quality and
health promoting compounds
Chitosan coatings could provide a semipermeable film around the
fruit surface, which modifies the internal atmosphere by reducing oxygen
and/or elevating carbon dioxide levels, that decreases the fruit respiration
level and metabolic activity, hence retards the fruit ripening and senescence
process (Özden and Bayindirli, 2002; Olivas and Barbosa-Cánovas, 2005;
Vargas et al., 2008). A suppressed respiration rate slows down the synthesis
and the use of metabolites, resulting in lower soluble solids due to the slower
hydrolysis of carbohydrates to sugars (Ali et al., 2011; Das et al., 2013).
However, there are numerous confounding factors that could account for
soluble solids concentration in fruit tissues, e.g. the fruit studied, its stage of
ripeness, the storage conditions and the thickness of chitosan coatings (Ali et
al., 2011). On the other hand, since organic acids, such as malic or citric
acid, are substrates for the enzymatic reactions of respiration process, an
increase in acidity and a reduction in pH value are expected in low-respiring
fruit (Yaman and Bayindirli, 2001). Above all, the chitosan coating with its
filmogenic properties has been used as a water barrier to minimize water and
weight loss of fruit during storage (Vargas et al., 2008; Bourlieu et al., 2009).
All these physiological changes were reported in fruits and
vegetables treated with chitosan (Tables 7, 8, and 9). Chitosan coating
minimized weight loss of stored apples, and its combination with heat
treatment showed the lowest respiration rate, significantly reduced pH value,
and increased titratable acidity content (Shao et al., 2012). Chitosan coating
treatments on pears during storage reduced their vital activities, in
particularly respiration rate, maintaining the fruit quality and contributing to
longer shelf life. Coated pears showed a significantly reduced weight loss
(Zhou et al., 2008). In pear, either the chitosan coating alone or the
combination of chitosan with ascorbic acid decreased respiration rate,
delayed the increase of weight loss and retained greater total soluble solids
57
and titratable acidity content (Lin et al., 2008). Chitosan-treated peaches
showed lower respiration rate and higher titratable acidity content than
untreated peaches (Li and Yu, 2000).
Chitosan formed a coating film on the outside surface of the sweet
cherries, that effectively retarded the loss of water and the changes in
titratable acidity and total soluble solids of sweet cherries (Dang et al.,
2010). Strawberries coated with either chitosan or chitosan combined with
calcium gluconate had a reduced weight loss and respiration activity that
delayed the ripening and the progress of fruit decay due to senescence.
Regardless of the addition of calcium gluconate to the chitosan, coated
strawberry had higher titratable acidity, lower pH and soluble solids
(Hernández-Muñoz et al., 2008). Calcium or Vitamin E added or not to
chitosan coatings significantly decreased weight loss, and delayed the
change in pH and titratable acidity of strawberries or red raspberries during
cold storage (Han et al., 2004, 2005). Chitosan coatings combined with
bergamot oil provided a significant water vapor barrier on cold stored table
grapes, reducing fruit weight losses. The addition of bergamot oil, thanks to
its hydrophobic nature, lower further the weight loss (Sánchez-González et
al., 2011). Similarly, weight loss reduction in coated table grapes was
observed combining the chitosan with putrescine (Shiri et al., 2013) or grape
seed extract (Xu et al., 2007b). The complex of zinc (II) and cerium (IV)
with chitosan film-forming material applied to preserve quality of Chinese
jujube fruit, reduced the fruit respiration rate and weight loss, while
increased its total soluble solids, as compared to the uncoated fruit (Wu et
al., 2010). In another study, after 42 days of storage at 13 °C, chitosancoated citrus fruit exhibited less weight loss and showed higher titratable
acidity and total soluble solids. Weight loss of citrus fruit decreased as the
concentration of chitosan was increased (Chien and Chou, 2006). Coating
tomato fruit with chitosan solutions reduced the respiration rate and ethylene
production, with greater effect at 2% than 1% chitosan. Coating increased
the internal CO2, and decreased the internal O2 levels of the tomatoes.
58
Chitosan-coated tomatoes were higher in titratable acidity (El Ghaouth et al.,
1992b).
Similar changes in the respiration, weight loss, pH, titratable acidity
and soluble solids content were reported after chitosan treatment of tropical
fruit (Table 8). Polysaccharide-based coatings, including chitosan coating,
applied on banana fruit displayed reduced carbon dioxide evolution, loss in
weight and titratable acidity. Moreover, the reducing sugar content and total
soluble solids of coated fruit were lower than uncoated, suggesting that the
former synthesized reducing sugars at a slower rate, having slowed down the
metabolism (Kittur et al., 2001). Similarly in bananas, chitosan coating alone
or in combination with 1-methylcyclopropene, reduced by 32% the rate of
respiration compared to untreated banana, decreased titratable acidity and
increased total soluble solids (Baez-Sañudo et al., 2009). The composite
coating consisting of arabic gum and chitosan provided an excellent
semipermeable barrier around the banana fruit, which reduced weight loss,
modified the internal atmosphere and suppressed ethylene evolution,
reducing respiration and delaying ripening process. After 33 days of storage,
soluble solids concentration of treated banana fruit was lowered, whereas
titratable acidity were increased by chitosan and arabic gum coating
(Maqbool et al., 2010a, 2010b, 2011). The application of chitosan delayed
the change in eating quality, reduced respiration rate and weight loss, while
increased total soluble solid and titratable acidity of stored longan (Jiang and
Li, 2001) and guava fruit (Hong et al., 2012). In mango fruit, the decline in
respiration rate, fruit weight, and titratable acidity were all effectively
inhibited by chitosan coating (Jitareerat et al., 2007), while the increase in
total soluble solids was retarded during storage (Zhu et al., 2008). Mango
fruit coated with a chitosan and subjected to hydrothermal process treatment
had less weight loss, lower pH and soluble solids, but higher acidity than
fruits treated or not with the hydrothermal process (Salvador-Figueroa et al.,
2011). The CO2 concentration in the internal cavity of chitosan-treated
papaya was significantly higher than untreated fruit. The formation of a
59
chitosan film on the fruit, as a barrier for O2 uptake, slowed the rate of
respiration and the metabolic activity and consequently the ripening process
(Hewajulige et al., 2009). Similarly in papaya, chitosan provided an effective
control in reducing weight loss, delayed changes in soluble solids
concentration during 5 weeks of storage. The titratable acidity declined
throughout the storage period, though at a slower rate in the chitosan coated
fruit as compared to the untreated papaya (Bautista-Baños et al., 2003; Ali et
al., 2010, 2011). Chitosan coating combined or not with calcium infiltration
markedly slowed the ripening of papaya as shown by their retention of
weight loss, delay in titratable acidity decrease, and in soluble solid and pH
increase (Al Eryani et al., 2008). In litchi fruit during storage, the chitosan
treatment produced an effective coating that reduced the respiration and
transpiration of fruit during storage (Lin et al., 2011), and reduced the
decrease in concentrations of total soluble solids and titratable acidity (Jiang
et al., 2005). Similar results were obtained with the combination of chitosan
with ascorbic acid that significantly increased the titratable acidity and total
soluble solids of stored litchi fruit (Sun et al., 2010).
Fruit firmness is a major attribute that dictates the postharvest
quality of fruit (Barrett et al., 2010). Fruit softening is a biochemical process,
normally attributed to the deterioration in cell wall composition that involves
the hydrolysis of pectin by enzymes, for example, polygalacturonase
(Atkinson et al., 2012). Low levels of oxygen and high levels of carbon
dioxide restricted the activities of these enzymes and allowed retention of the
fruit firmness during storage (Maqbool et al., 2011). Moreover, water
retention due to reduced transpiration gives turgor to the fruit cells. Banana
fruit treated with composite edible coatings of chitosan and arabic gum
presented significantly higher firmness than uncoated bananas at the end of
storage period and that firmness decreased as the coating concentrations
decreased (Maqbool et al., 2011). Chitosan coatings exerted a beneficial
effect on strawberry firmness such that, by the end of the storage period,
treated fruit were with higher flesh firmness values than untreated
60
(Hernández-Muñoz et al., 2008). In several other works chitosan coating
maintained firmness during storage of table grapes (Xu et al., 2007b;
Sánchez-González et al., 2011), apple (Shao et al., 2012), pear (Lin et al.,
2008), peach (Li and Yu, 2000), jujube (Qiuping and Wenshui, 2007); orange
(Chien and Chou, 2006; Cháfer et al., 2012), banana (Kittur et al., 2001; Win
et al., 2007; Baez-Sañudo et al., 2009), mango (Zhu et al., 2008; SalvadorFigueroa et al., 2011), papaya (Bautista-Baños et al., 2003; Sivakumar et al.,
2005b; Ali et al., 2010; 2011), rambutan (Martínez-Castellanos et al., 2009),
guava (Hong et al., 2012) and tomato (El Ghaouth et al., 1992b) (Tables 7, 8,
and 9).
In several studies, panelists were asked to observe and then rate the
overall appearance, or just the flavor, of the fruit treated or not with chitosan
using hedonic scales (Tables 7, 8, and 9). The results showed that chitosan
could preserve the taste of pear fruit, that after storage was similar to the
taste of fresh fruit (Zhou et al., 2008). Similar results were obtained with the
combination of chitosan and cinnamon oil coating that retained sweet pepper
quality and no off-flavour was developed (Xing et al., 2011a). Consumer
acceptance, which was based on color, flavor, texture, sweetness and acidity
was improved by chitosan coating and/or heat treatment of apple fruit (Shao
et al., 2012). On table grapes, chitosan coatings and the combination of
putrescine treatments significantly maintained sensory quality in comparison
with the untreated bunches (Shiri et al., 2013) and the combination with
grape seed extract delayed rachis browning and dehydration, and maintained
the visual aspect of the berry without detrimental effects on taste, or flavor
(Xu et al., 2007b). In another study, chitosan coating had a strong effect on
the maintenance of quality attributes, such as visual appearance, color, taste
and flavor of the sweet cherries, since it had a protective effects preventing
cherry surface browning, cracking, and the leaking of juice (Dang et al.,
2010). On strawberry, results from consumer testing indicated that chitosan
coatings increased the appearance acceptance of the strawberries
(Devlieghere et al., 2004), whereas coatings containing chitosan and vitamin
61
E developed the waxy-and-white surface of the samples (Han et al., 2005).
In another study, aroma and flavour of coated strawberries was considered
less intense than those of uncoated samples, which were preferred by the
panelists. Likewise, panelists detected an untypical oily aroma in samples
coated with the combination of chitosan and oleic acid (Vargas et al., 2006).
On bananas, Baez-Sañudo et al. (2009) reported that chitosan
coating did not affect the sensory quality of treated banana. However,
sensory evaluation of the bananas for taste, pulp, color, texture, flavor, and
overall acceptability, revealed that fruits treated with Arabic gum and
chitosan attained the highest scores by the panelists in all tested parameters
and that this coating improved banana fruit quality during storage. Whereas
those fruits coated with higher concentration of Arabic gum combined with
chitosan were unable to ripen properly after about 1 month of storage and
developed poor pulp color and inferior texture and were off-flavored
(Maqbool et al., 2011). Similarly, the sensory evaluation of papayas for taste,
peel color, pulp color, texture and flavor revealed that the fruits treated with
1.5% chitosan attained maximum score by the panelists in all tested
parameters. The untreated fruits or those treated with 0.5% chitosan ripened
after 3 weeks of storage and, thereafter began to decompose, while the fruits
treated with 2.0% chitosan were unable to ripen properly after more than 1
month of cold storage because of the thickness of chitosan coating, which
blocked the lenticels and caused fermentation inside, and in both cases the
fruits were discarded from the evaluation due to unacceptable quality. The
flavor of the fruits with 1.5% chitosan coating was rated excellent, because
the pulp was not only sweet and pleasant, but also possessed a characteristic
aroma (Ali et al., 2010; 2011). Litchi fruit subjected to chitosan alone or
combined with carbonate salts also had a good eating quality (Sivakumar et
al., 2005a).
Several other investigations reported the changes after chitosan
application in color fruit peels that were revealed either by technical
instrumentations or only by visual appearance (Tables 7, 8, and 9). The
62
application of chitosan coating in longan fruit delayed the peel fruit
discoloration and this was related to the concomitant inhibition of PPO
activity, which is an enzyme responsible of polyphenol oxidation (Jiang and
Li, 2001). Papaya fruit with higher concentration of chitosan coatings
underwent light changes in their peel color, as indicated by the slower
increase in lightness and chroma values. The delay of color development in
the papaya fruit treated with higher concentrations of chitosan could be
attributed to the slow rate of respiration and reduced ethylene production,
leading to a delayed fruit ripening and senescence (Ali et al., 2011).
Similarly, calcium infiltrated and chitosan coated samples combined
treatment had greatest effect in delaying color surface changes of papaya
fruit as noted from the lower values of lightness and chroma and higher
value of hue angle in treated papaya compared to untreated (Al Eryani et al.,
2008). During storage, chitosan coating delayed color changes in banana
(Kittur et al., 2001; Win et al., 2007; Baez-Sañudo et al., 2009; Maqbool et
al., 2011), litchi fruit (Zhang and Quantick, 1997; Caro and Joas, 2005; Joas
et al., 2005; Ducamp-Collin et al., 2008; De Reuck et al., 2009; Sun et al.,
2010;), mango (Zhu et al., 2008; Salvador-Figueroa et al., 2011), citrus
(Canale Rapussi et al., 2011), strawberry (Han et al., 2004; 2005;
Hernández-Muñoz et al., 2008), and tomato (El Ghaouth et al., 1992b).
Sensory analyses revealed beneficial effects of chitosan coating in terms of
delaying rachis browning and maintenance of the visual aspect of the table
grape berries (Xu et al., 2007b; Sánchez-González et al., 2011).
Fruit and vegetables treated with chitosan could have a nutritional
value added, in fact chitosan could retain ascorbic and phenolic compounds
contents (Tables 7, 8, and 9), which are positively correlated with
antioxidant capability (Rapisarda et al., 1999). Moreover, chitosan coating
can be used as a vehicle for incorporating functional ingredients, such as
antimicrobials, minerals, antioxidants and vitamins. Some of these
combinations could enhance the effects of chitosan coating or reinforce the
nutritional value of commodities (Vargas et al., 2008). Chitosan-based
63
coatings demonstrated their capabilities to carry high concentrations of
calcium or vitamin E, thus significantly increasing the content of these
nutrients in the fresh and frozen strawberry and raspberry. Incorporation of
calcium or vitamin E into chitosan-based coatings did not significantly alter
its antifungal property and enhanced nutritional value of fresh and frozen
strawberry and raspberry (Han et al., 2004). In addition, calcium was
incorporated in chitosan coating since it increased the stability of the cell
wall and middle lamella of strawberry tissue and improved resistance to
enzymes caused by fungal pathogens (Hernández-Muñoz et al., 2006; 2008).
Calcium was added to chitosan coating when used in papaya
(Al Eryani
et al., 2008), pear (Yu et al., 2012) and peach (Ruoyi et al., 2005). Lin et al.
(2008) reported that the combination of chitosan with ascorbic acids not only
controlled the core browning of pear, which is the main problem during
storage, but also increased ascorbic acid content and the antioxidant
capability of pears. The combination of chitosan with ascorbic acid showed
similar results when applied on litchi fruit (Sun et al., 2010).
2.7
Effect of chitosan on foodborne pathogens
Foodborne illnesses are diseases caused by agents that enter the
human body through the ingestion of food. The Centers for Disease Control
and Prevention in 2011 estimated that in the United States each year occur
48 million foodborne illnesses, responsible of 128,000 hospitalizations and
3,000 deaths. The World Health Organization estimates that in 2005 1.5
million people died, worldwide, from diarrheal diseases that in great
proportion of the cases were foodborne. This problem is actual and
worldwide spread. Furthermore in the next future, the growth of population,
in particularly the elderly band, and the movement of goods and people at
global scale make the scenario more complicated and difficult to manage.
Recent investigations have identified fruit and vegetables, and in
particular leafy greens, as important vehicles for transmission of many
disease outbreaks (Berger et al., 2010). Furthermore, nowadays, it is
64
increasing the demand for fresh, minimally processed vegetables, such
“ready to eat” vegetables, which retain much of their indigenous microflora
after minimal processing. All type of produce have potential to harbor
pathogens, but Salmonella spp., Shigella spp., Escherichia coli,
Campylobacter jejuni, Listeria monocytogenes, Yersinia enterocolitica,
Bacillus cereus, Clostridium botulinum, Aeromonas hydrophila, some
viruses and others parasites are of greatest public health interest (Beuchat,
2002). Fruit and vegetables can be contaminated by these microorganisms
during the preharvest stage, mainly by contaminated water or sewage and
faeces, or during the postharvest by handling and storage of the horticultural
products. The growth of microorganisms on fresh-cut produce may also
occur during the cutting and slicing operations (Beuchat, 2002).
Chitosan edible coating, beside its potentiality as mechanical barrier,
could be used for its antimicrobial properties to preserve fresh fruit and
vegetables after harvest (Vargas et al., 2008). Some works reported the
antibacterial activity of chitosan films against foodborne pathogens of fresh
fruit and vegetables (Table 10).
Table 10. Application of chitosan on fruit and vegetables to control foodborne
microorganisms.
Microorganism
Substrate of
Integration to
References
growth
chitosan
Escherichia coli
Tomato
Inatsu et al., 2010
Tomato
Beeswax + lime
Ramos-García et
essential oil
al., 2012
Salmonella spp.
Whole
Allyl
Chen et al., 2012
cantaloupe
isothiocyanate,
nisin
Inatsu et al. (2010) evaluated different sanitizers to prevent growth
of four strains of E. coli on tomato surface and found that chitosan at 0.1%
was effective applied after sodium chloride washing treatment. However, in
65
this case, other combinations of sanitizers (lactic acid 0.1% with sodium
chloride 0.05%) were more effective. Chitosan coating reduced native
microflora on the surface of litchi fruit (Sivakumar et al., 2005a) and
strawberry (Ribeiro et al., 2007), but not in table grapes (Romanazzi et al.,
2002). However, several additives could be incorporated into the chitosan
coating, which can provide more specific functions, such as antimicrobial
activity, aiming to, either prevent, or reduce, the growth of foodborne
microorganisms (Vargas et al., 2008). Coatings consisting of chitosan and
allyl isothiocyanate on cantaloupe reduced the Salmonella presence till the
limit of detection after 2 weeks of storage (Chen et al., 2012). And when it
was simulated a recontamination of cantaloupe with Salmonella, the results
indicated that the chitosan-allyl isothiocyanate coating not only reduced
more Salmonella than the current practice based on acid washing, but also
maintained antibacterial activity for a longer period of time. Furthermore,
the native microflora monitored by microbial counts for total aerobic
bacteria, yeast and mold on cantaloupe surface during storage were reduced
by chitosan and allyl isothiocyanate coating (Chen et al., 2012). Essential
oils are among the antimicrobial agents that could be incorporated into the
chitosan coating (Vargas et al., 2008; Antunes and Cavaco, 2010). Coatings
with chitosan and bergamot oil reduced the counts of moulds, yeasts, and
mesophiles of the table grape berries compared to the untreated fruits. And
the addition of bergamot oil enhanced the antimicrobial activity of the pure
chitosan coatings (Sánchez-González et al., 2011). In another study, growth
of E. coli DH5α did not take place when the bacterium was incubated on
substrates amended with chitosan and beeswax containing or not thyme or
lime essential oils (Ramos-García et al., 2012).
The antimicrobial activity of chitosan seems to be due to its
policationic characteristics, which allow chitosan to interact with the
electronegative charges on the cell surface of the fungi or bacteria, causing,
as a result, microbial cell permeability, internal osmotic disequilibrium, and
cellular leakage (Helander et al., 2001; Rabea et al., 2003; Liu et al., 2004;
66
Raafat et al., 2008; Mellegård et al., 2011). A 12 h exposure period to
chitosan resulted in a higher level of glucose and protein in the supernatant
of cell suspension of Staphylococcus aureus than those observed in the
media without chitosan. The reactive amino groups in chitosan could
conceivably have the ability to interact with a multitude of anionic groups on
the cell surface to alter cell permeability and cause the leakage of
intracellular components, such as glucose and protein, leading to the cell
death (Chung et al., 2011). Furthermore, the possibility of a direct interaction
of chitosan with negatively charged nucleic acids of microorganisms, and
consequently an interference with RNA and protein synthesis was proposed
(Rabea et al., 2003). On the contrary, Raafat et al. (2008) considered the
probabilities of penetration of chitosan into the nuclei of the bacteria rather
low, since the dimension of the molecule of hydrated chitosan is bigger than
the cell wall pores. Raafat et al. (2008) examined the cell damage of
Staphylococcus simulans after exposure to chitosan and found irregular
structures protruding from the cell wall and “vacuole-like” structure possibly
resulting from a disruption of the equilibrium of cell wall dynamics, such as
ion, water effluxes and decreased internal pressure, but, on the other hand,
the cell membrane was intact. These results showed how chitosan could not
interact directly with bacterial internal structures, but just with external cell
wall polymers. Other mechanism proposed for the antimicrobial activity is
the fact that chitosan has a strong affinity with nutritionally essential metal
ions. Rabea et al. (2003) reported that the binding of bacterial trace metals
by chitosan inhibited both microbial growth and production of bacterial
toxins.
The susceptibility of the foodborne microorganisms to chitosan
depends also on the characteristics of the microorganisms themselves. Since
the antimicrobial activity of chitosan relies on electrostatic interactions, the
nature of the bacterial cell wall can influence the capacity of chitosan to
inhibit microorganisms growth. The main important foodborne
microorganisms are gram negative and gram positive bacteria. E. coli,
67
Salmonella spp., Shigella spp., A. hydrophila, C. jejuni and Y. enterocolitica,
belonging to the group of gram-negative, are characterized by an outer
membrane consisting essentially of lipopolysaccharides that contain
phosphate and pyrophosphate groups which cover their surface of negative
charges. The gram-positive bacteria, such as L. monocytogenes, B. cereus,
and C. botulinum have cell wall composed essentially by peptidoglycan
associated to polysaccharides and teichoic acids which are negatively
charged. According to several authors gram positive bacteria are more
susceptible than gram negative to chitosan (No et al., 2002; Takahashi et al.,
2008; Jung et al., 2010; Tayel et al., 2010), according to others it is valid the
opposite (Devlieghere et al., 2004). Furthermore, a recent study reported the
effectiveness of chitosan and its derivatives against well-established biofilms
formed by foodborne bacteria, which are assumed to be very recalcitrant to
cleaning and disinfection practices. The results showed that one hour
exposure to chitosan caused a viable cell reduction on L. monocytogenes
mature biofilms and reduced significantly the attached population of the
other organisms tested, B. cereus, Salmonella enterica and Pseudomonas
fluorescens, except S. aureus (Orgaz et al., 2011).
In the food industry chitosan is frequently used as antioxidant,
clarifying agent and enzymatic browning inhibitor. When applied to food,
the antimicrobial activity of chitosan could be affected by pH or matrix.
Indeed the pKa of chitosan, at which half of its amino group is protonated
and half is not, is around 6.5; therefore it means that at pH lower than 6.5 the
protonated form predominates, resulting in higher positive charge density
that leads to strong and more frequent electrostatic interactions, and to an
higher antimicrobial effectiveness (Helander et al., 2001; Devlieghere et al.,
2004; Jung et al., 2010; Kong et al., 2010). The growth of Candida lambica
was completely inhibited at pH 4.0, while at pH 6.0, the same chitosan
concentration led to a rather small decrease in growth rate (Devlieghere et
al., 2004). Furthermore, this explains why chitosan is less soluble in water
alone than in solution with acetic acid.
68
Chitosan with higher degree of deacetilation, which has higher
numbers of positive charges, would be expected to have stronger
antibacterial activity (Jung et al., 2010; Kong et al., 2010; Tayel et al., 2010).
On the other hand, numerous studies have generated different results
concerning correlation between chitosan bactericidal activity and its
molecular weight. In some studies, chitosans with the lower molecular
weight were more effective against bacteria than those with higher molecular
weights (Liu et al., 2006; Tayler et al., 2010; Kim et al., 2011). In other
works, this trend was observed for gram-negative bacteria, but not for grampositive (No et al., 2002; Zheng and Zhu, 2003). According to Benhabiles et
al. (2012), when the molecular weight of chitosan is low, its polymer chains
have greater flexibility to create more binds, and are better able to interact
with the microbial cells. In other studies, no trends in antibacterial action
related to increasing or decreasing molecular weight were observed (Jung et
al., 2010; Mellegård et al., 2011).
2.8
Conclusions and future trends
This review reports the recent and most relevant works concerning
preharvest sprays and postharvest applications of chitosan showing that this
biopolymer can effectively maintain fruit and vegetables quality during
storage and can control postharvest decay. Studies dealing with chitosan
antimicrobial mechanisms of action against postharvest fungi and foodborne
bacteria were summarized. Film forming properties, antimicrobial activity,
and the ability of induce plant resistance seem to be the key factors of
chitosan success. With its intrinsic properties, and because of the double
activity on the host and on the pathogen, chitosan can be considered the first
of a new class of plant protection products (Bautista-Baños et al., 2006).
Moreover, chitosan has been under considerable investigation for
applications in biomedicine, biotechnology, and in the food industry due to
its biocompatibility, biodegradability, and bioactivity (Synowiecki and Al69
Khateeb, 2003; Tharanathan and Kittur, 2003; Wu et al., 2005). Chitosan is
not toxic for human and its safe use as pharmaceutical excipient was
reported (Baldrick, 2010). FDA recognizes chitosan as GRAS substance.
Available toxicology data indicated that high oral doses in rodents and
rabbits are generally well tolerated. Any chitosan that enters the body by
absorption is not likely to cause any issue of accumulation/retention, due to
conversion to naturally occurring glucosamine derivatives, which are either
excreted or used in the amino sugar pool (Baldrick, 2010). A study carried
out to test the acute toxicity and effects on the blood parameters of rats,
which were treated with high dosage carboxymethyl chitosan (1350 mg/kg)
showed that no acute toxicity was detected and no significant effects were
found on the parameters of coagulation, anticoagulation, fibrinolysis or
hemorheology of rats. This indicated that carboxymethyl chitosan has no
significant toxicity on the blood system of rats since it is firstly absorbed in
the abdominal cavity and then degraded gradually in the blood (Yang et al.,
2012b). Another study reported that, in general, chitosan is a relatively nontoxic and biocompatible material, but care must be taken to ensure that it is
pure, since contaminants could potentially cause many deleterious effects
both in derivative syntheses and in dosage forms (Kean and Thanou, 2010).
Multicomponent edible chitosan coatings may be produced with
suitable ingredients to provide the desired barrier protection, and to be used
as vehicles to incorporate specific additives that enhance functionality, such
as antioxidants or antimicrobials, which can avoid the pathogen or foodborne
microorganism’s growth on the surface of vegetables products (ValenciaChamorro et al., 2011). Combinations of chitosan with minerals, vitamins or
other nutraceuticals compounds could reinforce the nutritional value of
commodities, without reducing taste acceptability. The new generation of
edible coating is being especially designed to allow the incorporation and/or
controlled release of antioxidants, vitamins, nutraceuticals, and natural
antimicrobial agents (Vargas et al., 2008; McClements et al., 2009). Chitosan
coating on food was proposed as carrier for drugs or pharmaceutical
70
compounds (Tharanathan and Kittur, 2003; Baldrick, 2010).
The availability of chitosan commercial products, that can be easily
dissolvable in water, provides a real and prompt alternative to the growers
for the control of diseases of fruit and vegetables. The present review
summarizes application either before and after the harvest. However,
postharvest treatment is not advisable for fruit characterized by thin waxy
pericarp and succulent flesh, which could be easily damaged. Therefore,
preharvest treatment even at 1 day before harvest has been considered as a
promising method to control postharvest decay of fruit (Meng et al., 2009).
Even if a lot of information about the effectiveness of chitosan in preventing
postharvest decay of fruit and vegetables are available, its application in
large-scale tests and integration into commercial agricultural practices are
key points that need to be further investigated. Additional research
concerning its exact mechanisms of action is needed. Indeed, several
mechanisms about its antifungal and antibacterial activity are still unclear.
New knowledge about these aspects will improve information to support
decision regarding how to prepare chitosan, which molecular weight to use,
and the commercial formulation.
71
3
EFFECTIVENESS OF POSTHARVEST TREATMENT WITH
CHITOSAN AND OTHER RESISTANCE INDUCERS IN THE
CONTROL OF STORAGE DECAY OF STRAWBERRY
Abstract
This study compared the effectiveness in controlling postharvest
diseases of strawberry of practical grade chitosan when used as solutions
obtained by dissolving it in acetic, glutamic, formic and hydrochloric acids,
with a water-soluble commercial chitosan formulation. The commercial
chitosan formulation and other resistance inducers based on
benzothiadiazole, oligosaccharides, soybean lecithin, calcium and organic
acids, and fir and nettle extracts were also tested, to evaluate their
effectiveness in the control of postharvest decay of strawberry. The
commercial chitosan formulation was as effective as the practical grade
chitosan solutions in the control of gray mold and Rhizopus rot of
strawberries immersed in these solutions and kept for 4 days at 20 ±1 °C.
Moreover, the treatment with commercial and experimental resistance
inducers reduced gray mold, Rhizopus rot and blue mold of strawberries
stored 7 days at 0 ±1 °C and then exposed to 3 day shelf life. The highest
disease reduction was obtained with the commercial chitosan formulation,
followed by benzothiadiazole, calcium and organic acids. The compounds
that provided the best results in postharvest applications could be tested in
further trials thought preharvest treatments to control storage decay of
strawberries, applied at flowering and a few days before harvest.
72
3 - Effectiveness of postharvest treatment with chitosan and other resistance
_______________________ inducers in the control of storage decay of strawberry
3.1
Introduction
Strawberry is a particularly perishable fruit during postharvest
storage, as it is susceptible to drying, mechanical injury, decay and
physiological disorders. Gray mold and Rhizopus rot are caused by Botrytis
cinerea (Pers.) and Rhizopus stolonifer (Ehrenb.), respectively, and they are
the main causes of postharvest decay of strawberry (Fragaria  ananassa
Duch.) (Maas, 1998). The infection of the fruit by gray mold can be ascribed
to an infection on the flowers in the field. The B. cinerea fungus remains
latent underneath the sepals until fruit ripening, and then close to or after
harvest it can turn from a saprophyte into a parasite (Powelson, 1960). The
disease often starts close to the pedicel, and at times also in wounds on the
fruit produced during harvest, which results in its colonization. B. cinerea
can also develop at low temperatures (even at 0 °C), with the consequent
shortening of the length of storage and marketing. Rhizopus rot can spread at
temperatures greater than 4 °C to 6 °C, and it is more common on fruit
exposed to rain in the field or grown under plastic tunnels in several rows,
when located at their border. Both of these diseases spread quickly to other
fruits, a phenomenon that is known as nesting. Infections from Penicillium
spp. (blue mold) and Mucor spp. (Mucor rot) also occur occasionally (Maas,
1998).
In conventional agriculture, these diseases are usually managed by
fungicide treatments that are applied around flowering, and are repeated up
to harvest, depending on the disease pressure and the preharvest interval of
the formulation. However, in organic agriculture and after harvest, the use of
synthetic fungicides is not permitted, so the exploitation of alternatives is
desirable. Among these, the use of resistance inducers has the potential for
large-scale application. Resistance inducers can increase plant defenses, and
at times they can also exploit their antimicrobial properties. Among the
natural compounds, chitosan has received much interest for application in
agriculture and for the food industry. Chitosan can decrease gray mold and
73
inducers in the control of storage decay of strawberry _______________________
Rhizopus rot of strawberry through the reduction of mycelial growth and
spore germination, and the induction of morphological alterations in the
causal organisms (El Ghaouth et al., 1992a). Moreover, chitosan acts as a
potent elicitor, to enhance plant resistance against pathogens (Amborabé et
al., 2008). Chitosan needs to be dissolved in dilute acid solution to exploit its
properties, and several acids can dissolve this biopolymer, the best of which
are acetic, hydrochloric, glutamic and formic acids (Romanazzi et al., 2009).
So far, there are no data on the effectiveness in the control of postharvest
decay of strawberry of commercial chitosan formulations, either alone or
compared with practical grade chitosan dissolved in dilute acids.
A number of resistance inducers are available on the market today.
Benzothiadiazole (BTH or acibenzolar-S-methyl) is an elicitor of systemic
acquired resistance in plants. It is a photostable analog of salicylic acid, and
it has proven to be effective in the management of gray mold of strawberry
(Terry and Joyce, 2000; Muñoz and Moret, 2010). Oligosaccharides can also
elicit plant defenses, and their presence on host tissue can simulate the
presence of pathogens and activate the plant responses (Shibuya and
Minami, 2001). Fir and nettle extracts are available as commercial and
experimental formulations, respectively, and as with some other plant
extracts, they have recently gained popularity and scientific interest for their
possible antimicrobial activities (Velázquez del Valle et al., 2008; Gatto et
al., 2011).
The objectives of this study were: (i) to compare the effectiveness in
the control of postharvest diseases of strawberry of solutions obtained by
dissolving practical grade chitosan in acetic, glutamic, formic and
hydrochloric acids, and of the water soluble commercial chitosan
formulation; and (ii) to evaluate the effectiveness of a commercial chitosan
formulation, and benzothiadiazole, oligosaccharides, soybean lecithin,
calcium and organic acids, and extracts of fir and nettle in the control of
postharvest decay of strawberry.
74
3.2
3.2.1
Materials and methods
Fruit
Trials were carried out on the strawberry cultivar Camarosa in
commercial orchards located in the Marche region, central-eastern Italy,
grown according to the standards of organic agriculture. The fruit were
selected for the absence of defects, uniformity in size, and degree of ripening
(2/3 red on the surface) (Rosati and Cantoni, 1993), and they were used for
the experiments on the day of harvest.
3.2.2
Resistance inducers
The effectiveness in the control of postharvest strawberry diseases of
chitosan dissolved in different acid solutions, and of the commercial chitosan
formulation was tested. Crab shell chitosan (Sigma Chemical Co. St Louis,
MO, USA) was ground to a fine powder in a mortar, washed with distilled
water, pelleted by low-speed centrifugation, and air dried. For experimental
use, four different 1% solutions (w/v) of chitosan were prepared by
dissolving the chitosan in 1% (v/v) acetic, hydrochloric, glutamic or formic
acids under continuous stirring, to obtain chitosan acetate, chloride,
glutamate and formate (Romanazzi et al., 2009). When dissolved, the pH of
the chitosan solution was adjusted to 5.6 using 1 N NaOH, and 0.05% (w/v)
Triton X-100 surfactant was added to improve the wetting properties of these
solutions. A commercial chitosan-based formulation, known as Chito Plant
(ChiPro GmbH, Bremen, Germany), was prepared by dissolving the powder
(1%, w/v) directly in distilled water 2 h before use. Distilled water was used
as the control.
The effectiveness of different commercial resistance inducers in the
control of postharvest strawberry diseases was compared. These were based
on chitosan (Chito Plant, 1%, w/v), oligosaccharides (Algition, Socoa
75
Trading, Bologna, Italy; 1%, v/v), benzothiadiazole (Bion, Syngenta Crop
Protection, Switzerland; 0.2%, w/v), calcium and organic acids (Fitocalcio,
Agrisystem, Lamezia Terme, CZ, Italy; 1%, v/v), soybean lecithin (Xedabio,
Certis, Saronno, VA, Italy; 1%, v/v), a fir extract from Abies sibirica (Abies,
Agritalia, Villa Saviola di Motteggiana, MN, Italy; 1%, v/v) and an
experimental formulation based on a nettle extract (1%, w/v). This last
compound was obtained by infusion of Urtica dioica leaves in water (10%,
w/v) for one month, with the macerate filtered through a double layer of
cheesecloth, and then diluted 1:10 in deionised water.
3.2.3
Treatments
Strawberries were pooled together and randomized, and then they
were immersed for 10 s in a 5 liter volume of the respective solutions.
Strawberries immersed in deionised water were used as the control. After the
treatments, the fruit were dried in air for 1 h, and then individually arranged
in small plastic boxes. These were then placed in covered plastic boxes and
stored for 7 days at 0 ±1 °C, 95% to 98% RH, and then exposed to 3 day
shelf life at 20 ±1 °C, 95% to 98% RH. Five replicates of 30 strawberries
were used for each of the treatments.
The infections which subsequently developed resulted from
naturally-occurring inoculum.
3.2.4
Data recording
During the storage, the percentage of decayed strawberries was
recorded. Moreover, disease severity was recorded according to an empirical
scale with six degrees: 0, healthy strawberry; 1, 1% to 20% fruit surface
infected; 2, 21% to 40% fruit surface infected; 3, 41% to 60% fruit surface
infected; 4, 61% to 80% fruit surface infected; 5, more than 81% of the
strawberry surface infected and showing sporulation (Romanazzi et al.,
76
2000). The empirical scale allowed the calculation of the McKinney’s index,
expressed as the weighted average of the disease as a percentage of the
maximum possible level (McKinney, 1923). This parameter also includes
information on both disease incidence and disease severity.
3.2.5
Experimental design and statistics
The trials were arranged in a completely randomized design, and
each experiment was repeated at least twice. Data from two or more
experiments were pooled, as the statistical analysis to determine the
homogeneity of variances was tested using Levene’s test (SPSS Inc.,
Chicago, IL, USA). To normalize the data, the appropriate transformations
were determined empirically using normal probability plots. The arcsine of
the square root of the proportion was applied to the decay incidence data.
The values were submitted to analysis of variance and the means were
separated by Duncan’s Multiple Range Test (SuperANOVA, Abacus
Concepts, Inc., Berkeley, CA, USA). Actual values are shown.
3.3
Results and discussion
Research to reduce fungicide applications in agriculture through the
discovery of new natural antimicrobials is needed to meet the growing
consumer demand for food without chemical preservatives and to respond to
the needs of sustainable farming. Due to the nontoxic and biocompatible
properties of chitosan (Wu et al., 2005), it has been considered a candidate
for substitution of fungicides in horticultural cultivation (Bautista-Baños et
al., 2006). The main difference between the practical grade chitosan
solutions and the commercial chitosan formulation arises from the
techniques of their preparation. Indeed, to dissolve the chitosan in the
various acids, it is necessary to prepare the solutions two days in advance
and to monitor and adjust the pH; in contrast, the commercial chitosan
77
formulation can be prepared only 1-2 h before application, just by dissolving
the powder in water. Moreover, the resulting solution with the commercial
chitosan formulation has a lower viscosity compared to the chitosan acetate,
so it can be applied more easily in the field using standard sprayers. These
details are relevant when the practical application is proposed, as farmers
can easily and quickly prepare and apply the compound at the field scale. In
our work, strawberries immersed in chitosan acetate, chloride, glutamate and
formate, and in the commercial chitosan formulation, showed significant
reduction of gray mold and Rhizopus rot decay, as well as reduced severity
and McKinney index, when compared to the control after 4 day shelf life at
20 ±1 °C (Table 11).
Table 11. Decay, disease severity and McKinney index of gray mold and Rhizopus
rot recorded on strawberries treated with solutions obtained by dissolving practical
grade chitosan in acetic, glutamic, formic and hydrochloric acids, and with
commercial chitosan formulation. The fruit were kept for 4 days at 20 ±1 °C, 95% to
98% RH.
Decay
Disease severity McKinney Index
(%)
(1-5)
(%)
Gray
Rhizopus Gray Rhizopus Gray Rhizopus
mold
rot
mold
rot
mold
rot
Control
91.8 Aa
93.0 A 4.6 A
4.8 A
84.5 A 89.3 A
Chitosan acetate
37.4 B
14.1 B 3.1 B
3.7 B
23.2 B 10.4 B
Chitosan chloride
51.3 B
29.9 B 3.2 B
3.4 B
32.8 B 20.3 B
Chitosan formate
43.1 B
25.9 B 3.4 B
3.4 B
29.3 B 17.6 B
Chitosan glutamate
44.7 B
25.6 B 3.4 B
3.6 B
30.4 B 18.4 B
Commercial chitosan 43.0 B
19.3 B 3.5 B
3.6 B
30.1 B 13.9 B
a
Values with the same letter are not statistically different according to Duncan’s
Multiple Range Test at p <0.01.
The treatments with chitosan acetate, chitosan formate, the
commercial chitosan, chitosan glutammate and chitosan chloride provided
McKinney index reductions in gray mold of 73%, 65%, 64%, 64% and 61%
78
respectively, and of Rhizopus rot of 88%, 80%, 84%, 79% and 77%
respectively, as compared to the control. However, no significant differences
in disease control were observed for the solutions obtained starting from
practical grade chitosan compared to the commercial chitosan formulation.
In these trials, significant infections of blue mold were not observed.
The treatment of the strawberry slices with chitosan acetate
significantly decreased the hydrogen peroxide production at 2, 4 and 6 h
after treatment, as compared to the untreated control (data not shown).
Chitosan solutions have antioxidant capacity, like hydrogen peroxide
scavengers, and the use of chitosan as an antioxidant and anti-browning
agent is widespread in the food industry (Devlieghere et al., 2004). The
oxygen radicals scavenging capacities, the levels of phenylpropanoid
compounds, and the antioxidant enzyme activity increased in strawberries
after the treatment with chitosan (Wang and Gao, 2013).
On strawberries cold-stored 7 days (0 ±1 °C) and then exposed to 3
day shelf life (20 ±1 °C), the reductions, as compared to the control, of
McKinney index for gray mold were 79%, 73%, 70%, 63%, 60%, 56% and
46% for the fruit treated with commercial chitosan, benzothiadiazole,
calcium with organic acids, oligosaccharides, fir extract, soybean lecithin,
and nettle extract, respectively and for blue mold were 90%, 84%, 71%,
61%, 59% and 31% for the fruit treated with commercial chitosan,
benzothiadiazole, calcium with organic acids, fir extract, nettle extract and
oligosaccharides, respectively. Only treatments with chitosan and calcium
with organic acids reduced the McKinney Index of Rhizopus rot,
respectively of 84% and 79%, as compared to the control (Table 12).
79
Table 12. Decay, severity and McKinney index of gray mold, Rhizopus rot and blue
mold recorded on strawberries treated with commercial and experimental resistance
inducers. The fruit were stored for 7 days at 0 ±1 °C, 95% to 98% RH, followed by
3 days of shelf life at 20 ±1 °C, 95% to 98% RH.
Decay (%)
Severity (1-5)
McKinney index (%)
Gray Rhizopus Blue Gray Rhizopus Blue Gray Rhizopus Blue
mold
rot
mold mold
rot
mold mold
rot
mold
63.5aa 48.9a 56.9a 4.2a
3.8a
3.8a 53.3a 44.8a 40.8a
Control
29.8bc 36.2ab 28.3bc 2.2c
3.8a 2.9abc 21.2bc 29.6ab 16.0bc
Fir extract
2.5a
3.4ab 19.7bc 32.8ab 28.0b
Oligosaccharides 29.0bc 36.3ab 40.4ab 3.4ab
2.2a
1.6bc 14.6bc 15.2ab 6.4c
Benzothiadiazole 25.1c 20.8ab 12.6cd 2.9bc
20.4c
8.6b
4.8d 2.7bc
1.8a
1.0c 11.1c
7.2b
4.0c
Chitosan
1.6a 2.2abc 16.0bc 9.6b
12.0c
Ca-organic acids 23.5c 12.7ab 28.5bc 3.4b
44.6b 24.2ab 28.2bc 2.9bc
2.3a 1.9abc 27.9b 13.6ab 16.8bc
Nettle extract
n.d.* 3.2b
n.d.*
n.d.* 23.6bc n.d.*
n.d.*
Soybean lecithin 36.8bc n.d.*
a
Values with the same letter are not statistically different according to Duncan’s
Multiple Range Test at p <0.05.
*Disease not developed in the trials in which the compound was used.
Chitosan has a dual effect on host–pathogen interactions through its
antifungal activity and its ability to induce plant defense responses
(Romanazzi, 2010). Moreover, as chitosan can form an edible film when
applied to the surface of fruit and vegetables, it is clearly effective in
conferring a physical barrier to moisture loss, delaying dehydration and fruit
shriveling. Therefore, its coating can prolong storage life, delay the drop in
sensory quality, and control the decay of strawberry fruit (Han et al., 2004;
Park et al., 2005; Chaiprasart et al., 2006; Hernández-Muñoz et al., 2006;
Ribeiro et al., 2007). Chitosan coating can be used as a vehicle for
incorporating functional ingredients, such as antimicrobials or nutraceutical
compounds that could enhance the effects of chitosan coating or reinforce
the nutritional value of the strawberries (Vargas et al., 2006; Vu et al., 2011;
Perdones et al., 2012). Positive effects of treatment with practical grade
chitosan coating on the decay of strawberries artificially inoculated with B.
80
cinerea and R. stolonifer and held at 13 °C have been shown (El Ghaouth et
al., 1992a). Preharvest sprays of practical grade chitosan significantly
reduced postharvest fungal rot of strawberries stored at 3 °C and 13 °C and
maintained the quality of the fruit compared to the control (Reddy et al.,
2000a). In the same way, preharvest and postharvest treatments with
practical grade chitosan on strawberries reduced the postharvest gray mold
and Rhizopus rot after storage at 0 ±1 °C followed by a shelf life at 20 ±1 °C
Benzothiadiazole is a functional analog of salicylic acid and an
acquired systemic resistance activator that can elicit activation of genes
involved in plant defense and pathogenesis-related proteins (Lawton et al.,
1996; Vallad and Goodman, 2004). Our results are in agreement with Terry
and Joyce (2000), who reported the possibility to delay the development of
gray mold on strawberry fruit held at 5 °C by about 1.2-fold, through single
or multiple preharvest foliar treatments at anthesis with benzothiadiazole,
with no phytotoxic effects seen for either fruit or plant. Postharvest treatment
of strawberries with benzothiadiazole induced disease resistance by
enhancing fruit antioxidant systems and free radical-scavenging capabilities
(Cao et al., 2011).
In the formulation whose composition is based on calcium and
organic acids, the calcium reinforces the structural composition of the plant
cell wall through the binding of pectins with salts, and therefore provides
more resistance during the manipulation and transport of fruit. Calcium is
one of the most widely used treatment alternatives to fungicides with table
grapes, with the aim to protect the berries from preharvest and postharvest
gray mold, and it is used in both organic and conventional agriculture
When oligosaccharides are applied to plants, these can simulate the
presence of a pathogen and thus induce plant defense responses (Chisholm et
al., 2006). These compounds derived from the degradation of plant cell-wall
polysaccharides are one class of well characterized elicitors that, in some
81
cases, can induce defense responses at very low concentrations (Shibuya and
Minami, 2001).
In the present study, the application of soybean lecithin was tested as
a resistance inducer. In a previous study, Hoa and Ducamp (2008) reported
that treatments with soybean lecithin delayed mango ripening during storage
at ambient temperatures, thus slowing the changes of the biochemical
ripening indicators. Lecithin could also work as an antioxidant, since
hydrogen peroxide content in strawberry tissues treated with lecithin was
reduced compared to the control (data not shown). In the food industry,
soybean lecithin is normally used as a natural and non-toxic compound with
antioxidant properties, and it is approved by the United States Food and
Drug Administration for human consumption, with the status of “Generally
Recognized As Safe”.
Over the last few years, there has been increasing interest for
scientific research into plant extracts for their antimicrobial actions and their
safe application (Gatto et al., 2011). The activity of the fir extract could be
due to its triterpene acids, which act as plant-growth regulators and facilitate
cell division and shoot regeneration (Korolev et al., 2003). From the present
study, the fir extract appears to have good antimicrobial activity. The nettle
extract has also been shown to have antimicrobial and antioxidant effects
(Gülçin et al., 2004), and here we show its antimicrobial properties, as it
reduced gray and blue molds. Moreover, this nettle extract is used by organic
farmers, who claim that they can achieve a reduction in aphid numbers.
Among these formulations tested, the commercial chitosan
formulation and benzothiadiazole provided the highest disease reduction,
which indicates their possible application in IPM. Resistance inducers also
have the advantage of triggering wide-spectrum resistance, for activity
against several classes of plant pathogen and pest (Inbar et al., 1998).
Chitosan treatment showed an antioxidant activity on strawberry tissue.
However, further studies are needed to better understand the mechanisms of
action of these resistance inducers. Moreover, the appreciation from the
82
consumers of fruits treated with these resistance inducers needs to be
investigated.
83
4
PREHARVEST TREATMENTS WITH ALTERNATIVES TO
SYNTHETIC FUNGICIDES TO PROLONG SHELF LIFE OF
STRAWBERRY FRUIT
Abstract
The effectiveness of the control of postharvest gray mold on
strawberry (Fragaria × ananassa Duch, cv. Alba) fruit following field
applications of chitosan (0.5%, 1%), laminarin, fir extract, and
benzothiadiazole were compared with a fungicide strategy based on
cyprodinil, fludioxonil and pyrimethanil. Four or five field treatments with
these compounds were applied every five days throughout the season, from
strawberry flowering to maturity, with two consecutive harvests carried out.
For both harvests, all of the treatments reduced postharvest decay and
McKinney index of strawberry fruit after cold-storage for at least one week
and exposure to shelf life conditions for 3 or 4 days. The mean postharvest
decay reductions, compared to the untreated control, across the first and
second harvests, were 60%, 74%, 41%, 61%, 45% and 94%, for chitosan
0.5%, chitosan 1%, laminarin, fir extract, benzothiadiazole, and the
fungicides, respectively. After the second harvest, the effectiveness in
reducing the McKinney index for gray mold of 1% chitosan was not
significantly different from that of the fungicide strategy. Chitosan 1% and
the fungicide strategy decreased the area under disease progress curve
(AUDPC) of strawberry gray mold during shelf life both after the first and
second harvest. The treatments with these alternative compounds did not
have any negative effects on strawberry quality parameters, including for
color and firmness; only benzothiadiazole application reduced the red tone
of strawberry fruit, although this was not detrimental to the strawberry
84
4 - Preharvest treatments with alternatives to synthetic fungicides to prolong shelf
_________________________________________________ life of strawberry fruit
appearance. Laminarin at 1% gave phytotoxic signs on strawberry leaves,
but not on the fruit.
4.1
Introduction
Strawberry (Fragaria × ananassa Duch) is a perishable fruit that
can easily undergo fungal spoilage after harvesting. The main pathogen that
affects strawberry during storage is Botrytis cinerea, a saprophytic fungus
that is the causal agent of gray mold (Snowdon, 1990). Pathogen infection
occurs during strawberry cultivation, while symptoms develop mainly after
harvesting, and the infection can easily move to the nearby fruit, a
phenomenon known as nesting (Maas, 1998). Usually, to prevent this
postharvest rot, fungicides are repeatedly sprayed on the strawberry plants
through the season, from flowering to harvest. However, the normative
restrictions and the growing concern of consumers regarding the healthiness
of food have led to the search for alternatives to the use of synthetic
fungicides. Furthermore, fungicide resistance has been detected in B. cinerea
isolates exposed to fungicides that were constantly applied in the field to
control gray mold on small fruit (Weber et al., 2011).
Ideally, alternative compounds to fungicides will be nontoxic for
human health and the environment, will not have negative effects on the
quality of the fruit, and will complement or improve current productive
practices (Romanazzi et al., 2012). Alternative compounds to synthetic
fungicides are characterized by antimicrobial activity against the main
postharvest pathogens that cause fruit rot, or they are resistance inducers that
activate plant defenses, to simulate the present of a pathogen. Indeed,
resistance inducers are analogs of pathogen or plant constituents. Among
these, there are two particular resistance inducers that have been reported to
stimulate plant defenses and to prevent disease development (Aziz et al.,
2003; Bautista-Baños et al., 2006): chitosan, which is a natural biopolymer
in the cell wall of many pathogenic fungi (Synowiecki and Al-Khateeb,
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4 - Preharvest treatments with alternatives to synthetic fungicides to prolong
shelf life of strawberry fruit _________________________________________
2003), and laminarin, which is an oligosaccharide that is one of main
constituents of algal tissue (Rioux et al., 2007). Alternatively,
benzothiadiazole is an analog of salicylic acid that has been applied to plant
tissues as an activator of systemic acquired resistance (Lawton et al., 1996).
Furthermore, plant extracts can be considered as useful alternatives to the
use of synthetic fungicides in the management of postharvest rot of fruit and
vegetables (Gatto et al., 2011).
The effectiveness of such alternative compounds to control
strawberry gray mold have been tested in preliminary studies carried out at
the postharvest stage, by dipping the strawberries in solutions of these
compounds (Cao et al., 2011; Romanazzi et al., 2013), or at the preharvest
stage under controlled conditions in plastic tunnels (Reddy et al., 2000a;
Terry and Joyce, 2000; Mazaro et al., 2008). Romanazzi et al. (2000) applied
practical grade chitosan either at postharvest or under field conditions,
spraying 0.1%, 0.5% or 1% chitosan on strawberries at the growth stages of
full bloom, green fruit and whitening fruit. Chitosan reduced the postharvest
rot caused by B. cinerea and R. stolonifer, with the greatest reductions
observed for 1% chitosan applied to strawberry fruit at the whitening stage.
For the present study, we selected some of the most promising
compounds and tested them under field conditions, with repeated treatments
from strawberry flowering to fruit maturity. The aim of this study was to
determine the effectiveness in the control of postharvest decay of strawberry
of field applications of: chitosan (0.5% and 1%), laminarin, of a fir extract,
and benzothiadiazole. The effectiveness of these compounds was compared
to the control treated with water and to the spraying of a fungicide strategy
that is currently used in conventional agriculture, as a combination of
cyprodinil, fludioxonil, and pyrimethanil.
86
4.2
4.2.1
Preharvest treatments
The trials were carried out in an experimental strawberry field in a
flat area of central-eastern Italy (Agugliano; 43°31′60″N, 13°22′60″E). The
strawberry cv. Alba was planted under field conditions using the plastic hill
culture production system, with twin rows at intervals of 30 cm × 30 cm, and
where each of the twin rows was separated by 1 m. Through the season the
plants were irrigated using a drip system.
For the trial, different treatments of the strawberry plants were
compared. The strawberries were treated with chitosan at two different
concentrations (0.5% and 1%; Chito Plant, ChiPro GmbH, Bremen,
Germany), laminarin (1%; K&A Frontiere, BioAtlantis, Tralee, Ireland), a
fir extract from Abies sibirica (1%; Abies, Agritalia, Villa Saviola di
Motteggiana, Italy), benzothiadiazole (0.2%; Bion, Syngenta, Basilea,
Switzerland), or a fungicide strategy of cyprodinil and fludioxonil (0.08% of
Switch, Syngenta, Basilea, Switzerland) for 2 initial applications, followed
by pyrimethanil (0.15% of Scala, Bayer Crop Science, Monheim am Rhein,
Germany) for 3 applications. Strawberry plants treated with water were used
as the control.
A randomized block design with 4 replicates was used, and the
treatments were assigned to plots using a random-number generator (Excel;
Microsoft Corp., Redmond, WA, USA). Along the twin rows, each plot was
6.5 m in length, which corresponded to ca. 45 plants per plot. The plots were
divided from each other by 0.5 m of untreated plants. The treatments were
distributed by spraying a volume equivalent to 1000 l/ha using a motorized
backpack sprayer (GX 25, 25cc, 0.81 kW; Honda, Tokyo, Japan). To
indicate the flowers that were completely opened and with 5 petals, just
before the first treatment a tag was put on their stems. The first treatments
were carried out on April 17, 2012, at flowering, and further treatments
87
followed every 5 days, for a total of 5 treatments. The first harvest was
carried out at the beginning of May, 5 days after the plants had received 4
treatments, and just before they received the fifth treatment. The second
harvest was carried out 5 days after the plants had received the full 5
treatments. At both harvest times, only the ripe strawberry fruit in each plot
that had the tags on the stems and were red over ≥2/3 of their surface were
picked (Rosati and Cantoni, 1993), to be sure that they had received 4 and 5
treatments from flowering to maturity at the first and second harvests,
respectively. After each harvesting, the strawberry fruit were selected for
absence of defects and uniformity of color and shape. The strawberry fruit
harvest from each plot was randomly divided in groups of 6 fruits that were
placed into small boxes, which were then placed into large covered boxes.
To create the humid condition of storage, a layer of wet paper was placed in
the bottom of the large boxes. Approximately, 6 small boxes (ca. 0.7 kg
fruit) of strawberry fruit were obtained per plot at the first harvest, and 9
small boxes (ca. 1 kg fruit) per plot at the second harvest. The strawberry
fruit from the first harvest were stored for 7 days at 0.5 ±1 °C, and then
exposed to a shelf life at 20 ±1 °C and 95% to 98% relative humidity for 4
days. Similarly, the strawberry fruit from the second harvest were stored for
10 days at 0.5 ±1 °C, and then exposed to a shelf life at 20 ±1 °C and 95% to
98% relative humidity for 3 days.
4.2.2
Decay evaluation
During the shelf life period, the percentages of decayed strawberry
fruit were recorded. Disease severity was also recorded according to an
empirical scale with six degrees: 0, healthy fruit; 1, 1%-20% fruit surface
infected; 2, 21%-40% fruit surface infected; 3, 41%-60% fruit surface
infected; 4, 61%-80% fruit surface infected; 5, ≥81% of the fruit surface
infected and showing sporulation (Romanazzi et al., 2000). The infection
index (or McKinney index), which incorporates both the incidence and
88
severity of the disease, was expressed as the weighted means of the disease
as a percentage of the maximum possible level (McKinney, 1923). This was
calculated by the formula: I = [Σ(d×f)/(N×D)] ×100, where d is the category
of rot intensity scored on the strawberry fruit and f is its frequency, N is the
total number of examined strawberry fruit (healthy and rotted), and D is the
highest category of disease intensity that occurred on the empirical scale
(Romanazzi et al., 2001). Moreover, the area under the disease progress
curve (AUDPC), which represents a quantitative summary of the disease
intensity over time (Jeger and Viljanen-Rollinson, 2001), was calculated
through the formula: AUDPC = Σ[(Yi+n+Yi)/2][Xi+n-Xi], where Yi+n and Yi
are the decay percentages recorded for two consecutive decay evaluations,
and Xi+n and Xi are the days when these two decay evaluations were carried
out. As the decay evaluations were always carried out daily, [Xi+n-Xi] was
always 1.
4.2.3
Determination of fruit-quality parameters
One week after the last treatment, a third harvest was carried out
with the aim of determining the fruit-quality parameters, as the strawberry
fruit color and firmness. For each plot, 10 strawberry fruits were randomly
selected according to the absence of deformity and uniformity of size and
degree of maturation. The fruit were transported to the laboratory and their
color and firmness were determined.
The fruit color was measured on both sides of each fruit, using a
colorimeter (Chroma Meter CR 400; Konica Minolta, Tokyo, Japan). The
instrument provided the parameters of L*, a* and b*, which relate to the
luminescence, red tone and yellow tone, respectively, of the fruit color. In
addition, the chroma (C*) and hue (h*) parameters were obtained
mathematically from the a* and b* values, calculated according to the
formula C* = [(a*2+b*2)½] and h* = tan−1(b∗/a∗) (Nunes et al., 2006). The
fruit firmness was measured on the same strawberries used for the color
89
analysis, using a penetrometer (Fruit Pressure Tester 327, Effegì, Ravenna,
Italy), with the data expressed in g.
4.2.4
Statistical analysis
The data were analyzed statistically by two-way ANOVA, followed
by Tukey’s Honestly Significant Difference (HSD) test, at P = 0.05 (Statsoft,
Tulsa, OK, USA). In the statistical analysis of the randomized complete
block design at the first and second harvest, the block was considered as a
second factor. When the range of percentages was greater than 40, the
percentage data were arcsine transformed before analysis, to improve the
homogeneity of the variance. The actual values are shown.
4.3
4.3.1
Results
First harvest
After the first harvest, the four treatments with the alternative
compounds to fungicide use reduced the development of strawberry rot after
4 days of shelf life, which was mainly through gray mold. In particular, and
as given in Table 13, compared to the untreated control, the treatments with
0.5% chitosan, 1% chitosan, laminarin, fir extract, benzothiadiazole and the
fungicide strategy significantly decreased the decay by 70%, 81%, 47%,
74%, 36% and 94%, respectively. Similarly, the McKinney index of
strawberry disease was significantly decreased compared to the control, by
82%, 90%, 56%, 77%, 48%, and 97%, respectively (Table 13). The disease
severity of the postharvest gray mold was also significantly reduced by both
chitosan treatments (0.5%, 45%; 1%, 48%), and by the fir extract (31%) and
the fungicides (51%), but not by laminarin and benzothiadiazole (Table 13).
90
Table 13. Decay, severity, and McKinney index of postharvest gray mold on cv.
Alba strawberry fruit developed after 4 days of shelf life following 4 treatments of
the indicated compounds through the season, from flowering to maturity. After
harvest the strawberries were stored for 7 d at 0.5±1 °C, and then exposed to shelf
life at 20±1 °C and 95% to 98% relative humidity.
Decay
Disease severity
McKinney index
(%)
(1-5)
(%)
Control
38.33 ± 10.81 aa
2.04 ± 0.43 a
16.67 ± 6.95 a
Chitosan 0.5%
11.54 ± 6.43 cd
1.12 ± 0.17 bc
2.95 ± 2.41 cd
Chitosan 1%
7.41 ± 5.82 cd
1.06 ± 0.08 bc
1.67 ± 1.42 cd
Laminarin
20.37 ± 12.77 bc
1.51 ± 0.37 abc
7.41 ± 4.51 bc
Fir extract
9.85 ± 14.33 cd
1.41 ± 0.68 bc
3.79 ± 5.84 cd
Benzothiadiazole 24.40 ± 12.32 b
1.64 ± 0.56 ab
8.69 ± 5.56 b
Fungicides
2.27 ± 2.27 d
1.00 ± 0.00 c
0.45 ± 0.45 d
a
Values followed by unlike letters are significantly different according to Tukey’s
HSD (P = 0.05).
Figure 1 illustrates these first harvest data for the evolution over time
of the strawberry decay during the shelf life. Overall, as a quantitative
summary of the disease intensity over time, the AUDPC over these 4 days of
shelf life was significantly reduced again by both chitosan treatments (0.5%,
78%; 1%, 85%) and by the fungicides treatments (96%), but not by
laminarin, fir extract, and benzothiadiazole (Figure 2). It was also noted
however, that the laminarin treatment resulted in phytotoxic signs, which
included red spots on the strawberry leaves (data not shown).
91
Decay (%)
45
40
35
30
25
20
15
10
5
0
Control
Chitosan 0.5 %
Chitosan 1%
Laminarin
Fir extract
Benzothiadiazole
Fungicides
1
2
3
Days of shelf life
4
Figure 1. Decay evolution during shelf life (20 ±1 °C; 95%-98% relative humidity)
of gray mold that developed on the cv. Alba strawberry fruit from the first harvests,
according to the various treatments (as indicated) used for four times during the
season, from flowering to maturity.
92
60
50
a
AUDPC
40
ab
30
ab
20
b
10
b
ab
b
0
Figure 2. AUDPC during shelf life (20 ±1 °C; 95%-98% relative humidity) of gray
mold that developed on the cv. Alba strawberry fruit from the first harvests,
according to the various treatments (as indicated) used for four times during the
season, from flowering to maturity. Different letters show significantly different
values according to Tukey’s HSD (P = 0.05).
4.3.2
Second harvest
As with the first harvest, the five treatments with the alternative
compounds up to the second harvest reduced the development of postharvest
strawberry fruit gray mold after 3 days of shelf life. Compared to the
untreated control, the treatments with 0.5% chitosan, 1% chitosan, laminarin,
fir extract, benzothiadiazole and the fungicide strategy significantly
decreased the decay by 51%, 68%, 35%, 48%, 54%, and 93% (Table 14).
Thus, again, the treatments with the fungicides provided the greatest
protection of the strawberry fruit from postharvest decay during the shelf
93
life. Similarly, the McKinney index of strawberry disease was significantly
decreased compared to the control, by 61%, 77%, 38%, 66%, 67%, and 96%,
respectively (Table 14). The effectiveness of 1% chitosan in the control of
the postharvest gray mold was also not significantly different to that for the
fungicide strategy. The disease severity of this strawberry fruit postharvest
gray mold was significantly reduced by both chitosan treatments (0.5%,
31%; 1%, 40%), and by the fir extract (34%), benzothiadiazole (32%), and
the fungicides (49%), but not by laminarin (Table 14).
Table 14. Decay, severity, and McKinney index of postharvest gray mold on cv.
Alba strawberry fruit developed after 3 days of shelf life following 5 treatments of
the indicated compounds through the season, from flowering to maturity. After
harvest the strawberries were stored for 10 d at 0.5±1 °C, and then exposed to shelf
life at 20±1 °C and 95% to 98% relative humidity.
Decay
Disease severity
McKinney index
(%)
(1-5)
(%)
Control
61.85 ± 7.43 aa
2.03 ± 0.76 a
26.17 ± 9.80 a
Chitosan 0.5 %
30.46 ± 21.62 bc
1.39 ± 0.46 bc
10.23 ± 9.33 bc
Chitosan 1%
20.06 ± 12.39 c
1.22 ± 0.08 bc
5.92 ± 3.90 cd
Laminarin
40.39 ± 22.19 b
1.64 ± 0.52 ab
16.35 ± 12.62 b
Fir extract
32.32 ± 11.84 bc
1.35 ± 0.21 bc
8.99 ± 2.26 bc
Benzothiadiazole
28.33 ± 10.80 bc
1.38 ± 0.34 bc
8.67 ± 4.37 bc
Fungicides
4.17 ± 7.28 d
1.03 ± 0.04 c
1.00 ± 1.59 d
a
HSD (P = 0.05).
Figure 3 shows the evolution over time of the strawberry fruit decay
during the shelf life after the second harvest. Here, after 3 days of shelf life,
the AUDPC was significantly reduced by chitosan treatments at 1%
concentration (66%) and the fungicides (93%), but not by the other
compounds. As for the first harvest, the laminarin treatment resulted in
phytotoxic signs, which included red spots on the strawberry leaves (data not
shown).
94
Decay (%)
70
60
50
40
30
20
10
0
Control
Chitosan 0.5 %
Chitosan 1%
Laminarin
Fir extract
Benzothiadiazole
1
2
Days of shelf life
3
Fungicides
Figure 3. Decay evolution during shelf life (20 ±1 °C; 95%-98% relative humidity)
of gray mold that developed on the cv. Alba strawberry fruit from the second
harvests, according to the various treatments (as indicated) used for five times
during the season, from flowering to maturity.
95
AUDPC
45
40
35
30
25
20
15
10
5
0
a
ab
abc
abc
bc
abc
c
Figure 4. AUDPC during shelf life (20 ±1 °C; 95%-98% relative humidity) of gray
mold that developed on the cv. Alba strawberry fruit from the second harvests,
according to the various treatments (as indicated) used for five times during the
season, from flowering to maturity. Different letters show significantly different
values according to Tukey’s HSD (P = 0.05).
4.3.3
Strawberry color and firmness after field treatments
Almost all of the strawberry fruit color parameters showed no
significant changes from the control for all of these field treatments, with the
measurements of the luminescence (L*), red tone (a*), yellow tone (b*),
Chroma (C*), and hue (h*). However, the treatments with benzothiadiazole
significantly decreased the a* values of the strawberry fruit skins compared
to the control, and also compared to 1% chitosan, the fir extract and
fungicides strategy (Table 15). Similarly, although not significantly, the C*
96
value after the benzothiadiazole treatments was slightly lower than for the
control; again, this reduction did reach significance compared to the
treatments with 1% chitosan and the fir extract and fungicides strategy
(Table 15). The only further significant change seen was with the laminarin
treatment, which saw a slight reduction in L* for these strawberry fruit,
although this only reached significance when compared to the fungicides
treatments (Table 15).
In the firmness analysis of these strawberry fruit, none of these
treatments showed any significant effects (Table 15).
Table 15. Color and firmness parameters recorded following the third harvest of cv.
Alba strawberry fruit following five treatments of the plants with the indicated
compounds through the season, from flowering to maturity.
L*
a*
b*
C*
h
Firmnessa
b
Control
37.3±0.4 ab 39.0±1.0 a 22.4±0.9 a 45.1±1.3 ab 29.8±0.5 a 462±62 a
Chit. 1%c 37.7±0.5 ab 39.1±0.5 a 23.2±0.9 a 45.5±0.7 a 30.6±0.9 a 468±85 a
Chit. 0.5%d 37.8±1.6 ab 38.6±1.5 ab 22.8±2.8 a 44.9±2.6 ab 30.4±2.2 a 473±45 a
Laminarin 36.7±0.9 b 38.2±0.7 ab 22.5±1.3 a 44.4±1.2 ab 30.4±1.2 a 438±78 a
Fir extract 37.7±1.3 ab 39.1±0.7 a 23.6±1.7 a 45.8±1.3 a 30.9±1.5 a 468±129 a
BTH
37.4±1.6 ab 37.5±1.6 b 22.2±1.9 a 43.7±2.3 b 30.5±1.1 a 484±68 a
Fungicides 38.1±1.3 a 39.1±0.9 a 23.6±1.5 a 45.7±1.5 a 31.0±1.2 a 453±116 a
a
The unit of measurement of firmness is g.
b
HSD (P = 0.05).
c
Chitosan 1%.
d
Chitosan 0.5%.
4.4
Discussion
The present study has revealed how preharvest treatments with a
range of alternative compounds to fungicides can reduce the development of
postharvest rot of strawberry fruit. In particular, the chitosan treatments at
both concentrations were effective in the control of gray mold decay. And
97
for the second harvest, in particular, according to the McKinney index, the
higher concentration of chitosan (1%) was the only treatment that showed
disease protection that was not significantly different from the effectiveness
of the fungicides treatments. These data confirm the results obtained in some
previous studies, where treatments with chitosan also reduced the
postharvest decay of strawberry fruit, when chitosan was applied at the
postharvest stage either alone (El Ghaouth et al., 1991a; Reddy et al., 2000a;
Romanazzi et al., 2013) or when it was mixed with other compounds
(Vargas et al., 2006; Hernández-Muñoz et al., 2008; Perdones et al., 2012),
or when it was applied at the preharvest stage under controlled conditions in
plastic tunnels (Mazaro et al., 2008; Reddy et al., 2000a) or under field
conditions (Romanazzi et al., 2000).
With respect to previous preharvest trials, in the present study, the
chitosan commercial formulation was used, which can easily be dissolved in
water, and the experimental conditions carried out here were a close
simulation of the scenario of strawberry fruit production for commercial
purposes. The commercial chitosan formulation used here has been reported
to be as effective as the practical grade chitosan solutions for the control of
postharvest gray mold and Rhizopus rot of strawberry fruit kept for 4 days at
20 ±1 °C (Romanazzi et al., 2013). Chitosan at 1% also performed better
than 0.5% chitosan. Similarly, Reddy et al. (2000a) reported that the
incidence of strawberry fruit decay decreased with increased chitosan
concentration.
The effectiveness of chitosan appears to be ascribed to its antifungal
activity against B. cinerea (Muñoz and Moret, 2010) and to its triggering of
plant defenses, such as enzymes or compounds related to pathogenesis in
strawberry tissue (Wang and Gao, 2013) and in other fruit (Yan et al., 2011;
Feliziani et al., 2013b). Furthermore, the formation of a semi-permeable film
by chitosan around the fruit surface has been reported to decrease
postharvest strawberry fruit weight loss and to slow the gaseous exchange,
which helps to slow the fruit metabolism, and hence to delay senescence
98
(Hernández-Muñoz et al., 2008). Chitosan has also been shown to be
effective for the reduction of the microbial load that fruit can harbor,
including the microorganisms that are responsible for food-borne illnesses
(Tsai et al., 2004; Friedman and Juneja, 2010).
Benzothiadiazole is a molecule that has been defined as a functional
analog of salicylic acid; indeed, activation of systemic-acquired resistance
was seen in Arabidopsis mutants for salicylic acid when they were treated
with benzothiadiazole (Lawton et al., 1996). Application of benzothiadiazole
to strawberry fruit has been reported to decrease the development of decay
compared to control fruit, by enhancing the strawberry antioxidant system
and free-radical-scavenging capability (Cao et al., 2011). As well as its
action as a resistance inducer, benzothiadiazole also has antimicrobial
properties against some of the fungi that can cause postharvest decay,
including B. cinerea (Terry and Joyce, 2000; Feliziani et al., 2013a). In the
present study, benzothiadiazole indeed reduced the postharvest decay of the
strawberry fruit. Similar results have been obtained in other studies carried
out at the postharvest (Romanazzi et al., 2013) and preharvest (Terry and
Joyce, 2000; Mazaro et al., 2008) stages. Likewise, the treatments with the
fir extract reduced the postharvest rot of the strawberry fruit, compared to
the untreated control. The antimicrobial activity of these fir extracts appear
to be ascribable to certain of its constituents, which include flavonoids,
lignans and phenols (Yang et al., 2008), and which have been reported to be
active in plant defenses (Lattanzio et al., 2006). In a similar way, in other
studies, the immersion of strawberries on a fir extract solution reduced
postharvest gray mold and blue mold (Romanazzi et al., 2013). Also,
postharvest dipping and preharvest spraying with a fir extract can reduce
sweet cherry postharvest rot (Feliziani et al., 2013a). However, on the
present study, the effectiveness of benzothiadiazole or of fir extract was not
sufficient to decrease the AUDPC of gray mold developed during strawberry
shelf life, while the treatments with chitosan at 1% or fungicides lowered
significantly the AUDPC both in the first and second harvest.
99
Treatments with the oligosaccharide laminarin reduced the
postharvest strawberry fruit gray mold, although the AUDPC relative to
these laminarin treatments after both the first and second harvests did not
differ from the control. Also, for both of the harvests, phytotoxic signs were
observed on the strawberry leaves. The high concentration of laminarin used
here could have induced an overreaction in the plant tissues that showed as
red spots on the leaf surfaces. Indeed, laminarin is a resistance inducer that
can activate plant defense systems (Klarzynski et al., 2000; Jayaraj et al.,
2008), and here, massive programmed cell death might have occurred. In
other studies, laminarin was shown to reduce postharvest decay of sweet
cherry (Feliziani et al., 2013a), and to elicit defense responses in grapevine,
where it can induce protection against B. cinerea and P. viticola (Aziz et al.,
2003).
In the monitoring of the fruit-quality parameters after the third
harvest, the color of the strawberry fruit was affected by the
benzothiadiazole treatments, while none of the other compounds that were
applied altered the external appearance of the strawberry fruit compared to
the untreated control. In particular, the a* value, which is a measure of
redness, was reduced by the benzothiadiazole spraying. These data appear to
be in contrast with previous studies that have reported that benzothiadiazole
treatment of strawberry plants increased the fruit content of anthocyanin, a
pigment that contributes to the red color of the strawberry fruit (Nunes et al.,
1995), and induced enzyme activities related to anthocyanin and phenol
metabolism in the strawberry fruit after harvesting (Hukkanen et al., 2007;
Cao et al., 2010). However, Ayala-Zavala and coworkers (2004) reported
that although they did not find differences in skin color among strawberries
stored at different low temperatures, they observed differences in the total
anthocyanin content of the fruit pulp. These authors hypothesized that an
increase in the internal anthocyanin content in the strawberry fruit flesh
might not necessarily be the same in the external strawberry tissue, which
might maintain the same anthocyanin concentrations after treatment, and
100
therefore the same color appearance (Ayala-Zavala et al., 2004). Some
studies have asserted that there is no direct relationship between the external
color and the flesh color of the strawberry fruit (Hernanz et al., 2008), and
contrary to what might be generally believed, the surface color is not
necessarily a reliable indicator of the anthocyanin content of these fruit
(Ordidge et al., 2012). However, the slight reduction of a* by
benzothiadiazole in the present study did not have any detrimental effects on
the strawberry fruit appearance, as the color parameters were in line with
previous analyses (Capocasa et al., 2008) and are within the range of 6 units
to 7 units, which is the usual color tolerance that takes into account the
natural variability of this fruit (Perdones et al., 2012).
Color and firmness are the quality parameters that are fundamental
for consumer acceptability (Hernández-Muñoz al., 2008; Hernanz et al.,
2008), and in the present study, none of these treatments negatively affected
the external appearance of the strawberry fruit, which is of primary
importance. In previous studies, and as reported here, benzothiadiazole did
not alter the firmness of the fruit compared to the control (Mazaro et al.,
2008), while, chitosan treatments of strawberry have been reported to
increase the pulp consistency (Reddy et al., 2000; Vargas et al., 2006;
Hernández-Muñoz et al., 2008; Mazaro et al., 2008). However, the field
conditions adopted in the present study compared to laboratory trials might
have diluted out the effects of the chitosan treatments. Moreover, the harvest
for the analysis of the quality parameters was carried out almost one week
after the final treatments, and this could have further weakened the effects of
chitosan on the strawberry fruit. As indicated above, the laminarin
treatments produced phytotoxic signs that were visible on the leaves of these
strawberry plants at the end of the season. However, the quality parameters
of the strawberry fruit were not affected by these oligosaccharides. Indeed,
the effects of laminarin on the strawberry leaves might be ascribed to the
relatively high concentration of laminarin used here.
101
5
PRE
AND
ALTERNATIVES
POSTHARVEST
TO
TREATMENT
SYNTHETIC
WITH
FUNGICIDES
TO
CONTROL POSTHARVEST DECAY OF SWEET CHERRY
Abstract
The effectiveness of alternatives to synthetic fungicides for the
control of pathogens causing postharvest diseases of sweet cherry was tested
in vitro and in vivo. When amended to potato dextrose-agar,
oligosaccharides, benzothiadiazole, chitosan, calcium plus organic acids, and
nettle macerate reduced the growth of Monilinia laxa, Botrytis cinerea and
Rhizopus stolonifer. Treatment of sweet cherries three days before harvest or
soon after harvest with oligosaccharides, benzothiadiazole, chitosan, calcium
plus organic acids, nettle extract, fir extract, laminarin, or potassium
bicarbonate reduced brown rot, gray mold, Rhizopus rot, Alternaria rot, blue
mold and green rot of cherries kept 10 d at 20±1 °C, or 14 d at 0.5±1 °C and
then exposed to 7 d of shelf life at 20±1 °C. Among these resistance
inducers, when applied either preharvest or postharvest, chitosan was one of
the most effective in reducing storage decay of sweet cherry, and its
antimicrobial activity in vitro and in field trials was comparable to that of the
fungicide fenhexamid. Benzothiadiazole was more effective when applied
postharvest than with preharvest spraying. These resistance inducers could
represent good options for organic growers and food companies, or they can
complement the use of synthetic fungicides in an integrated disease
management strategy.
102
5 - Pre and postharvest treatment with alternatives to synthetic fungicides to control
_______________________________________ postharvest decay of sweet cherry
5.1
Introduction
Sweet cherry (Prunus avium) is a perishable fruit, and during storage
it can undergo postharvest decay. This is mainly caused by Monilinia spp.
and Botrytis cinerea, and occasionally by Rhizopus stolonifer, Alternaria
alternata, Penicillium expansum, and Cladosporium spp. (Romanazzi et al.,
2001). At present, preharvest treatments with synthetic fungicides are the
main means for postharvest disease control in stone fruit in general.
However, alternatives to the use of synthetic fungicides are needed for the
sweet cherry market, where no fungicides are registered for postharvest
applications and none are allowed in organic agriculture. Compared to
synthetic fungicides, alternative methods might also have the benefits of
lower risk of the development of fungal resistance, lower cost, and
application close to the harvest. Moreover, they have the potential to reduce
the impact of agriculture on the environment and on human health (Elmer
and Reglinski, 2006; Mari et al., 2010).
Natural compounds with antimicrobial activity and eliciting
properties might represent alternatives to synthetic fungicides in the control
of postharvest disease of fruit (Bautista-Baños et al., 2006). Resistance
inducers are compounds that have a composition based on pathogen or plant
constituents, or their analogs, such that they can react with plant receptors
and can activate plant defenses; this can then prevent pathogen infection
(Terry and Joyce, 2004; Elmer and Reglinski, 2006). Benzothiadiazole is a
synthetic analog of salicylic acid, and it has been reported to induce systemic
acquired resistance in plants. Furthermore, it has been shown to be effective
in the control of gray mold on strawberry (Terry and Joyce, 2000;
Romanazzi et al., 2013). In the same way, some oligosaccharides that are
derived from the degradation of fungal and plant cell-wall polysaccharides
represent a class of well-characterized elicitors that in some cases can induce
plant defense responses at very low concentrations (Shibuya and Minami,
2001). Also, the natural polysaccharide chitosan has been reported to have
103
postharvest decay of sweet cherry _______________________________________
antimicrobial activity against a long list of postharvest fungi and to be
effective in inducing an array of responses in plant tissue (Bautista-Baños et
al., 2006; Romanazzi et al., 2009; Reglinski et al., 2010; Feliziani et al.,
2013a). Chitosan treatment can elicit plant defenses through the stimulation
of enzymes related to pathogenesis and prolonged fruit and vegetables
storage (Li and Yu, 2000; Meng et al., 2010; Romanazzi et al., 2012; Wang
and Gao, 2013). Additionally, chitosan treatment can form a coating on the
surface of the fruit that slows down the respiration and ripening processes
(Romanazzi et al., 2009; Dang et al., 2010).
Recently, interest in the use of plant extracts and essential oils for
their antimicrobial activity increased, because these are considered to be safe
for both the environment and human health. Indeed, some such preparations
have shown a broad spectrum of activity against plant pathogens, and
particularly those responsible for postharvest diseases of fruit and vegetables
(Tripathi and Dubey, 2004; Gatto et al., 2011). Furthermore, inhibitory
effects of inorganic salts against postharvest diseases have been reported on
different commodities (Sanzani et al., 2009; Mari et al., 2010), among these,
the control of postharvest rots on the sweet cherry by sodium bicarbonate
and potassium sorbate have been demonstrated (Ippolito et al., 2005;
Karabulut et al., 2001; 2005a).
The aims of the present study were to: (i) evaluate the in vitro ability
of oligosaccharides, benzothiadiazole, chitosan, calcium plus organic acids,
nettle extract, as alternatives to fungicides, to inhibit the growth of Monilinia
laxa, B. cinerea, R. stolonifer and A. alternata; and (ii) evaluate the
effectiveness of preharvest and postharvest applications of these and other
resistance inducers, such as laminarin, potassium bicarbonate, fir extract for
the control of brown rot, gray mold, Rhizopus rot, Alternaria rot, blue mold
and green rot during the storage of sweet cherries at room temperature (20±1
°C) and at cold temperature (0.5±1 °C).
104
5.2
5.2.1
Antimicrobial activities of the resistance inducers in vitro
The antimicrobial activities of a range of resistance inducers were
tested for their ability to reduce mycelial growth of fungal colonies. Agar
plugs, with a diameter of 6 mm, from M. laxa, B. cinerea, A. alternata or R.
stolonifer colonies in active growth were placed in the centers of Petri dishes
containing 10 mL potato dextrose-agar in water without (control) or with
additions, after autoclaving, of oligosaccharides (1%, Algition, Socoa
Trading, Bologna, Italy), benzothiadiazole (0.2%, Bion, Syngenta, Basilea,
Switzerland), chitosan (1%, Chito Plant, ChiPro GmbH, Bremen, Germany),
calcium plus organic acids (COA) (1%, Fitocalcio, Agrisystem Srl, Lamezia
Terme, Italy), extract from Urtica dioica (1%), or fenhexamid (0.05%,
Teldor, Bayer CropScience, Monheim am Rhein, Germany). The U. dioica
extract was prepared by macerating 10 kg of green leaves and stems of the
nettle in 100 L water and leaving this for 1 month. The suspension thus
obtained was filtered through a double layer of cheesecloth, and diluted 10fold. To determine the antimicrobial activities of the formulations used, the
radial growth of the fungal colonies was measured daily, until one of the
treatments reached the edge of the Petri dish. Seven replicates were used for
each fungus and each treatment. This period corresponded approximately to
3-4 days for R. stolonifer colonies and to 1 week for B. cinerea, M. laxa and
A. alternata.
5.2.2
Postharvest treatments
Commercial sweet cherry ‘Sweet Heart’ and ‘Burlat’ were harvested
from an organic orchard in Ancona, central-eastern Italy. The fruit were
selected for uniformity in size and color, and absence of deformity or
disease. The ‘Sweet Heart’ cherries were treated with nettle extract (1%),
105
benzothiadiazole (0.2%), chitosan (1%), oligosaccharides (1%), or COA
(1%). The ‘Burlat’ cherries were treated with benzothiadiazole (0.2%),
chitosan (1%), fir extract from Abies sibirica (1%, Abies, Agritalia, Villa
Saviola di Motteggiana, Italy), laminarin (1%, K&A Frontiere, BioAtlantis,
Tralee, Ireland) or potassium bicarbonate at different concentrations (0.4,
0.9, 1.7, 2.6, 3.4 or 4.3% ) (Karma, Certis Europe, Saronno, Italy). Distilled
water was used as the control. The cherries were randomized and immersed
for 1 min in the tested solutions. Three replicates of thirty cherries per
treatment were placed into small plastic boxes that were then placed into
large boxes. To create humid condition of storage, a layer of wet paper was
placed in the bottom of the large boxes. The ‘Sweet Heart’ cherries were
kept 10 d at 20±1 °C, 95% to 98% relative humidity (RH), while the ‘Burlat’
cherries were stored for 14 d at 0.5±1 °C, and then exposed to 7 d of shelf
life at 20±1 °C, 95% to 98% RH.
5.2.3
The trials were carried out in an experimental orchard located in a
hilly area of the Ancona Province (43° 31’ 60’’ N, 13° 22’ 60’’ E; 203 m
a.s.l.) in central-eastern Italy in 2009 and 2011. The trees were selected for
uniformity of production and ripening. In 2009, the canopy of ‘Sweet Heart’
trees was sprayed with a solution of chitosan (1%), nettle extract (1%), or
fenhexamid (0.05%), 3 days before the harvest. In 2011, ‘Blaze Star’ trees
were sprayed with a solution of chitosan (1%), fir extract (1%),
benzothiadiazole (0.2%), or fenhexamid (0.05%), 3 days before the harvest.
The spraying used a back pump (model WJR2525, Honda, Tokyo, Japan) to
deliver the equivalent volume of 1000 L/ha. Untreated trees were used as
controls for both years. On the day of the harvest, the cherries were selected
for uniformity in size and color, and absence of deformity or disease. Ten
replicates of 750 g cherries per treatment were collected in plastic boxes that
were then placed into large boxes. To create humid condition of storage, a
106
layer of wet paper was placed in the bottom of the large boxes. The ‘Sweet
Heart’ and ‘Blaze Star’ cherries were stored for 14 d at 0.5±1 °C, and then
exposed to 7 d of shelf life at 20±1 °C, 95% to 98% RH. In the present trials,
we simulated real agricultural practices using preharvest applications of a
commercial chitosan formulation. Commercial formulations for chitosan
have the advantage of more practical use, as its viscosity is lower than that of
the biopolymer dissolved in acid solution, and it has the same effectiveness
as chitosan dissolved in acetic acid (Romanazzi et al., 2009; 2013).
5.2.4
Data recording for the in vivo trials
In the in vivo trials, at the end of the storage, the levels of decay due
to each of the pathogens were assessed separately according to the
symptoms. In any cases of doubt, isolations from rotted tissues were carried
out on potato dextrose-agar, and the causal agent was identified according to
the morphological properties. The diseases incidence was expressed as the
percentage of infected fruit. The severity was assigned to five classes
according to the percentage of cherry surface covered by fungal mycelia: 0,
uninfected cherry; 1, surface mycelia just visible to 25% of the cherry
surface; 2, 26% to 50% of the cherry surface covered with mycelia; 3, 51%
to 75% of the cherry surface covered with mycelia; and 4, >75% of the
cherry surface covered with mycelia (Romanazzi et al., 2001). The infection
index (or McKinney index), which incorporates both the incidence and
severity of the disease, was expressed as the weighted means of the disease
as a percentage of the maximum possible level (McKinney, 1923). In
particularly, it was calculated by the formula: I=[Σ(d×f)/(N×D)]×100, where
d is the category of rot intensity scored on the sweet cherry and f its
frequency; N the total number of sweet cherries examined (healthy and
rotted) and D is the highest category of disease intensity occurring on the
empirical scale (Romanazzi et al., 2001).
107
5.2.5
Statistical analysis
The data were analyzed statistically by one-way ANOVA, followed
by Tukey’s HSD test, at P = 0.05 (Statsoft, USA). Percentage data were
arcsine transformed before analysis to improve homogeneity of variance
when the range of percentages was greater than 40. Actual values are shown.
5.3
5.3.1
Results
Antimicrobial activities of resistance inducers in vitro
When added to potato dextrose-agar, all of the tested resistance
inducers reduced the radial growth of M. laxa, B. cinerea and R. stolonifer,
as compared to the controls. A. alternata growth was also inhibited except
for the oligosaccharides and COA. Fenhexamid and chitosan had the highest
ability of reducing the mycelial growth of all of the tested fungi. In
particular, growth of M. laxa and A. alternata was completely inhibited with
fenhexamid and chitosan, and B. cinerea did not grow with fenhexamid. R.
stolonifer growth was inhibited by all of the resistance inducers, although
none of them completely stopped its growth (Table 16).
108
Table 16. Radial mycelial growth (mm) of fungal colonies (Monilinia laxa, Botrytis
cinerea, Alternaria alternata, Rhizopus stolonifer) on PDA amended with water
(control), oligosaccharides, benzothiadiazole, chitosan, calcium plus organic acids,
nettle extract, and fenhexamid.
Radial growth (mm)
Monilinia Botrytis Rhizopus Alternaria
Treatment
laxa
cinerea stolonifer alternata
Control
29 aa
70 a
80 a
25 a
Oligosaccharides
23 b
55 b
70 b
21 ab
Benzothiadiazole
10 d
32 d
37 d
12 c
Chitosan
0e
9e
31 e
0d
Calcium plus organic acids
11 d
49 c
71 b
22 ab
Nettle extract
18 c
32 d
57 c
18 b
Fenhexamid
0e
0f
18 f
0d
a
Values followed by different letters are significantly different within columns,
according to Tukey’s HSD (P = 0.05).
5.3.2
Postharvest treatments
The postharvest treatments with oligosaccharides, benzothiadiazole,
chitosan, COA, and nettle extract all reduced the postharvest decay of the
‘Sweet Heart’ cherries kept 10 d at 20±1 °C, 95% to 98% RH (Table 17).
109
Table 17. Effects of postharvest treatment with water (control), oligosaccharides,
benzothiadiazole, chitosan, calcium plus organic acids, and nettle extract on
McKinney infection index of postharvest diseases (brown rot, gray mold, Rhizopus
rot, Alternaria rot, and total rot) of sweet cherries cv. ‘Sweet Heart’ kept 10 d at
20±1 °C, 95% to 98% RH.
McKinney infection index (%)
Brown
Gray
Rhizopus Alternaria
Total
Treatment
rot
mold
rot
rot
rota
b
Control
24.6 a
21.3 a
32.8 a
49.2 a
67.2 a
Oligosaccharides
11.5 b
12.2 b
6.6 b
26.2 b
36.1 b
Benzothiadiazole
9.8 b
13.9 b
12.3 b
24.6 b
44.2 b
Chitosan
6.6 b
11.5 b
7.4 b
22.9 b
29.5 b
Calcium + organic acids
9.8 b
16.4 b
20.5 b
27.7 b
49.3 b
Nettle extract
4.9 b
6.5 b
4.9 b
16.4 b
23.0 b
a
Total rot includes brown rot, gray mold, Rhizopus rot, Alternaria rot, blue mold and
green rot.
b
There were no statistical differences among these treatments.
Compared to the control, the application of nettle extract, chitosan,
oligosaccharides, benzothiadiazole, and COA reduced the sweet cherry total
rots of 66%, 56%, 46%, 34% and 27%, respectively. The infection index of
the total rot, that included gray mold, brown rot, Rhizopus rot, Alternaria rot,
blue mold and green rot, was lower than the sum of the single infection
indices as multiple infections can occur on the same cherry.
The postharvest treatment with benzothiadiazole, chitosan, fir
extract, the algal oligosaccharide laminarin, and potassium bicarbonate at
different concentrations decreased the total rot of the ‘Burlat’ cherries coldstored for 14 d (0.5±1 °C) and then exposed to 7 d of shelf life (20±1 °C,
95% to 98% RH; Table 18).
110
Table 18. Effects of postharvest treatment with water (control), benzothiadiazole,
chitosan, fir extract, laminarin, and potassium bicarbonate at different concentrations
on McKinney infection index of postharvest diseases (brown rot, gray mold, and
total rots) of sweet cherries cv. ‘Burlat’ stored 14 d at 0.5±1 °C and then exposed to
7 d of shelf life at 20±1 °C, 95% to 98% RH.
Brown
Gray
Total
Treatment (%)
rot
mold
rota
b
Control
44.9 a
61.3 a
70.4 a
Benzothiadiazole
8.7 b
33.8 b
37.6 bc
Chitosan
14.9 b
7.6 c
17.3 de
Fir extract
14.4 b
30.7 b
35.6 bc
Laminarin
13.1 b
25.6 bc
34.2 bcd
Potassium bicarbonate (0.4)
12.7 b
18.9 bc
27.8 bcde
15.8 b
14.4 bc
25.1 cde
12.4 b
13.6 bc
22.7 cde
11.1 b
5.1 c
16.7 e
23.6 ab
14.9 bc
36.9 bc
28.4 ab
18.0 bc
44.2 b
a
green rot.
b
The most effective treatments in controlling postharvest total rots of
sweet cherry were chitosan and potassium bicarbonate at concentration
ranging from 0.4 to 2.6%. For brown rot, gray mold and total rot, chitosan
reduced the infection indices by 67%, 88% and 75%, respectively, and 2.6%
potassium bicarbonate by 75%, 92% and 76%, respectively. Gray mold
infections were reduced by all the tested resistance inducers. While infection
indices for brown rot were not decreased by potassium bicarbonate at 3.4
and 4.3%, but when it was used at lower concentrations. The treatment with
111
potassium bicarbonate induced phytotoxic effects that were visible from
concentrations >0.9%, and that increased further with concentration (data not
shown). These phytotoxic signs consisted of pedicel browning and the
formation of dark spots on the cherry surface. Moreover, the pedicels of the
sweet cherries treated with potassium bicarbonate dried earlier.
5.3.3
Preharvest treatments with chitosan, nettle macerate, and
fenhexamid significantly reduced brown rot, gray mold, and Rhizopus rot of
the ‘Sweet Heart’ cold-stored for 14 d (0.5 ±1 °C) and then exposed to 7 d of
shelf life (20±1 °C, 95% to 98% RH, Table 19), and among these treatments,
there were no statistical differences. Compared to the control, for brown rot,
gray mold, Rhizopus rot and Alternaria rot, chitosan treatment reduced
infection indices by 63%, 28%, 31% and 47%, respectively, while
fenhexamid reduced them by 79%, 59%, 42% and 57%, respectively.
Alternaria rot was not affected by the nettle extract, however it reduced the
infection indices for brown rot, gray mold and Rhizopus rot by 63%, 31%
and 27%, respectively. Fenhexamid provided the greatest reduction of the
total rot, at 58%, a level significantly greater than all of the other treatments.
112
Table 19. Effect of treatments applied 3 days before the harvest with water (control),
chitosan, nettle extract, and fenhexamid on McKinney infection index of postharvest
diseases (brown rot, gray mold, Rhizopus rot, Alternaria rot, and total rots) of sweet
cherries cv. ‘Sweet Heart’ stored 14 d at 0.5±1 °C and then exposed to 7 d of shelf
life at 20±1 °C, 95% to 98% RH.
Brown
Gray
Rhizopus Alternaria
Total
Treatment
rot
mold
rot
rot
rota
b
Control
13.6 a
19.3 a
18.6 a
33.6 a
74.3 a
Chitosan
5.0 b
13.9 b
12.9 b
17.9 b
42.9 b
Nettle extract
5.0 b
13.4 b
13.6 b
28.6 ab
48.6 b
Fenhexamid
2.9 b
7.9 b
10.7 b
14.3 b
31.4 c
a
green rot.
b
The preharvest treatments with chitosan, fir extract, and fenhexamid
reduced brown rot on the ‘Blaze Star’ cherries cold-stored for 14 d (0.5 ±1
°C) and then exposed to 7 d of shelf life (20±1 °C, 95% to 98% RH; Table
20). Compared to the control, the infection indices for brown rot (the most
predominant decay that occurred in these trials) was reduced by 91%, 62%
and 57% after treatments with fenhexamid, chitosan and fir extract,
respectively, and the effects of these treatments were not statistically
different from each other.
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Table 20. Effect of treatments applied 3 days before the harvest with water (control),
chitosan, fir extract, benzothiadiazole, and fenhexamid on McKinney infection index
of postharvest diseases (brown rot, Alternaria rot, and total rots) of sweet cherries cv.
‘Blaze Star’ stored 14 d at 0.5±1 °C and then exposed to 7 d of shelf life at 20±1 °C,
95% to 98% RH.
Brown
Total
Treatment
rot
rota
b
Control
25.0 a
25.2 a
Chitosan
9.4 bc
9.5 bc
Fir extract
10.8 bc
11.0 bc
Benzothiadiazole
17.0 ab
17.3 ab
Fenhexamid
2.2 c
2.2 c
a
green rot.
b
5.4
Discussion
The in vitro ability of chitosan to reduce mycelial growth of M. laxa,
B. cinerea, R. stolonifer and A. alternata were comparable to those obtained
with the synthetic fungicide fenhexamid. These data support previous studies
that have reported that chitosan formulations reduced germination and radial
growth of a list of decay-causing fungi, such as B. cinerea (El Ghaouth et al.,
1992a; Badawy and Rabea, 2009), A. alternata (Sánchez-Dómínguez et al.,
2011), Rhizopus spp. (El Ghaouth et al., 1992a; García-Rincón et al., 2010),
and M. fructicola (Casals et al., 2012; Yang et al., 2012b). Similarly, in the
present study, benzothiadiazole and nettle extract had in vitro ability of
reducing the mycelial growth of the tested fungi. A concentration of 0.2%
benzothiadiazole was sufficient to inhibit B. cinerea radial growth on potato
dextrose-agar media, and the fungus was progressively inhibited with
increasing benzothiadiazole doses (Terry and Joyce, 2000; Muñoz and
114
Moret, 2010). For the nettle extracts, there are no data in the literature on its
effectiveness in the control of postharvest pathogens, although phenolic
compounds derived from herb extracts are known to be effective against
decay causing fungi of fruit and vegetables, such as B. cinerea, M. laxa,
Penicillium spp. and Aspergillus spp. (Gatto et al., 2011).
In the in vivo trials, the present study showed that preharvest and
postharvest treatments with some of these tested resistance inducers can
reduce the development of postharvest decay of sweet cherries. As
previously reported, on sweet cherry, postharvest application of chitosan
delayed their loss of water, maintained the quality attributes during storage,
and induced peroxidase and catalase activity in the fruit (Dang et al., 2010).
Indeed, the combination of hypobaric treatments and the practical grade
chitosan coating applied either preharvest or postharvest had synergistic
effects on the control of postharvest decay of sweet cherries cold-stored for
14 d (0±1 °C) and then exposed to 7 d of shelf life (Romanazzi et al., 2003).
However, little information is available on the effects of preharvest or
postharvest applications of the commercial chitosan formulation, which is
easy for the growers to dissolve in water, on sweet cherry postharvest decay
and the growth of M. laxa, which is one of the main cherry postharvest
pathogens.
Benzothiadiazole reduced the postharvest decay of sweet cherry
when applied postharvest, although preharvest treatment with
benzothiadiazole was not sufficient to control brown rot. In previous studies,
benzothiadiazole treatments induced plant defense systems and protected
strawberry from gray mold (Terry and Joyce, 2000; Romanazzi et al., 2013).
Benzothiadiazole mimics the effects of salicylic acid, which is involved in
plant signal transduction systems and is needed to activate the formation of
defense compounds, such as polyphenols and pathogenesis-related proteins
(Durang and Dong, 2004). On sweet cherry, preharvest treatments with
salicylic acid significantly reduced lesion diameters caused by M. fructicola,
and induced phenylalanine ammonia-lyase, glucanase, and peroxidase
115
activities during early storage of the fruit (Yao and Tian, 2005). In the
present study, benzothiadiazole reduced the disease incidence when applied
postharvest, and showed in vitro ability of reducing the mycelial growth of
the tested fungi. This suggests that beside the well-known induced resistance
of benzothiadiazole, it can also have a direct antimicrobial effect on several
postharvest pathogens. These protective effects against plant pathogens can
thus be ascribed to the combination of its defense-inducing activity on plants
and its adverse effects on the growth and vigor of these pathogens.
Postharvest application of potassium bicarbonate was effective in
reducing postharvest brown rot and gray mold of these ‘Burlat’ sweet
cherries. Potassium bicarbonate has been shown to control Geotrichum
candidum and P. expansum postharvest infections on peaches, nectarines and
plums (Palou et al., 2009). In the present study, signs of potassium
bicarbonate phytotoxicity were recorded only after applications at
concentrations >0.9%. Similarly, in a prior work, slight injury was seen to
the stems of sweet cherries treated with 0.24 mol/L sodium bicarbonate
(Karabulut et al., 2005a). As this was tested on just one cultivar here, it is not
known whether this potassium bicarbonate phytotoxicity is cultivar
dependent, and therefore further studies to understand the optimal dose and
time of application of potassium bicarbonate are needed. We did not rinse
the fruits after the potassium bicarbonate treatments here, and this might be
the reason for this phytotoxicity. In some packinghouses rinsing is common
practice, because the salt solutions must be washed off the fruit surface after
treatment to prevent phytotoxic effects (Palou et al., 2009).
Preharvest or postharvest applications of nettle and fir extracts
reduced these postharvest diseases of sweet cherry. Similarly, treatment with
1% nettle or 1% fir extracts reduced postharvest decay of strawberries stored
for 7 d at 0 °C and then exposed to 3 d shelf life at 20 °C (Romanazzi et al.,
2013). Applications of extracts from wild herbs reduced postharvest brown
rot, gray mold and green mold on table grapes, apricots, nectarines and
oranges (Gatto et al., 2011), and the application of a A. sibirica extract
116
significantly decreased the disease severity of downy mildew on grapevines
under semi-controlled conditions, and its efficacy was equal to copper
treatment (Dagostin et al., 2011).
Postharvest applications of the experimental products containing
oligosaccharides, or of the algal oligosaccharide laminarin, controlled these
postharvest sweet cherry diseases. Oligosaccharides are signaling molecules
that have been applied experimentally to activate plant defense responses
(Esquerré-Tugayé et al., 2000). The application of laminarin elicited defense
responses and reduced disease infections of gray mold and powdery mildew
on grapevine (Aziz et al., 2003). Thus, by simulating the presence of a
pathogen, these oligosaccharides applied to the cherry tissue might have
activated defense responses in advance, and avoided disease development.
The commercial product based on COA decreased the development
of sweet cherry decay, thus prolonging the shelf life of the fruit. Indeed
calcium ions strengthen the plant cell wall, to provide more resistance for the
fruit during postharvest handling. Calcium improves fruit firmness by
binding to the carboxyl groups of the pectic homogalacturonan backbones,
and the reinforced cell wall would be a further barrier to pathogen
penetration and infection, thus delaying disease development on fruit during
storage (Ippolito et al., 2005). Immersion of strawberry fruits on a solution
based on COA or on oligosaccharides reduced the infection index of
postharvest gray mold and blue mold (Romanazzi et al., 2013).
These alternative resistance inducers have here generally shown in
vitro ability of reducing the mycelial growth of the tested fungi and the
ability to reduce postharvest decay of sweet cherries, when applied either
preharvest or postharvest. Compounds such as chitosan, algal extracts or
potassium bicarbonate are substances defined as ‘GRAS’, Generally
Recognized as Safe, by the United States Food and Drug Administration, and
when applied to fruit, they are not expected to be harmful for humans or the
environment. These therefore offer safer alternatives to synthetic fungicides,
and have most of requirements of an ideal mean to control postharvest decay
117
of fruit (Romanazzi et al., 2012). These resistance inducers could represent
good options for organic growers and food companies, or they can
complement the use of synthetic fungicides in an integrated disease
management strategy. However, further studies on the impact of the
treatments with these resistance inducers on the flavor and quality
characteristics of sweet cherries are needed.
118
6
PREHARVEST FUNGICIDE, POTASSIUM SORBATE, OR
CHITOSAN USE ON QUALITY AND STORAGE DECAY OF
TABLE GRAPES
Abstract
Potassium sorbate, a program of four fungicides, or one of three
chitosan formulations were applied to clusters of ‘Thompson Seedless’
grapes at berry set, pre-bunch closure, veraison, and 2 or 3 weeks before
harvest. After storage at 2 °C for 6 weeks, the natural incidence of
postharvest gray mold was reduced by potassium sorbate, the fungicide
program, or both together in a tank mixture, in 2009 and 2010. In 2011, the
experiment was repeated with three chitosan products (OII-YS, Chito Plant,
and Armour-Zen) added. Chitosan or fungicide treatments significantly
reduced the natural incidence of postharvest gray mold among grapes.
Berries harvested from vines treated by two of the chitosan treatments or the
fungicide program had fewer infections after inoculation with B. cinerea
conidia. None harmed berry quality, and all increased endochitinase activity.
Chitosan decreased berry hydrogen peroxide content. One of the chitosan
formulations increased quercetin, myricetin, and resveratrol content of the
berry skin. In another experiment, ‘Princess Seedless’ grapes treated with
one of several fungicides before 4 or 6 weeks of cold storage, had less decay
than the control. Fenhexamid was markedly superior to the other fungicides
for control of both the incidence and spread of gray mold during storage.
6.1
Introduction
After harvest, grapes are particularly susceptible to severe losses by
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6 - Preharvest fungicide, potassium sorbate, or chitosan use on quality and storage
decay of table grapes _________________________________________________
gray mold, caused by Botrytis cinerea, because this pathogen grows under
cold storage temperatures and spreads rapidly from one berry to others
(nesting) by aerial mycelial growth (Snowdon, 1990). Methods to control
postharvest diseases are of interest to both growers who use conventional
fungicides and those who chose to avoid their use, for example, to produce
grapes in compliance with “organic” production rules (USDA, 2011).
Furthermore, fungicide resistance has been frequently detected in B. cinerea
populations exposed to fungicides applied to control bunch rot in grape
vineyards (Jacometti et al., 2010.). The use of chitosan, a natural substance,
has been considered as a valid alternative. Chitosan has been proven to
control numerous pre and postharvest diseases on various horticultural
commodities and fruit (Bautista-Baños et al., 2006; Romanazzi, 2010) and,
among them, to be effective in controlling B. cinerea and elicit plant defense
in table grapes through pre and postharvest applications (Romanazzi et al.,
2002; Trotel-Aziz et al., 2006; Meng et al., 2008; Reglinski et al., 2010;
Romanazzi et al., 2012). However, the influence of preharvest commercial
chitosan treatments on chitinase activity and its nature (endo- or exochitinase
activity), on the composition of phenolic compounds, or on hydrogen
peroxide content of grapes has not been reported. Little is known about the
influence of repeated chitosan applications on many aspects of berry quality,
such as size, texture, and maturation rate.
Potassium applications begun after the onset of veraison are known
to increase the soluble solids contents of grapes and increase berry firmness
(Strydum and Loubser, 2008; Mlikota Gabler et al., 2010; Kelany et al.,
2011). Potassium sorbate inhibits the growth and sporulation of B. cinerea,
and many other fungi, in vitro (Mills et al., 2004). Karabulut and coworkers
(2005b) reported a single application of potassium sorbate applied to
harvested grapes partially controlled subsequent gray mold during cold
storage. Therefore, potassium sorbate could influence both grape quality and
postharvest decay, and be a commercially feasible treatment. It has a low
order of toxicity to workers and the environment, it is inexpensive and
120
_________________________________________________ decay of table grapes
readily available, exempt from residue tolerances (EPA, 2011), and the risk
of resistance in the pathogen population is probably low. Unlike chitosan,
however, it is not approved for use in “organic” products in the United States
(USDA, 2011).
Although there are a number of fungicides approved for use on table
grapes produced under conventional practices, reports about their
effectiveness to control postharvest decay are few (Franck et al., 2005;
Smilanick et al., 2010). No reports describe the influence of residual
fungicide content in the fruit on the incidence and spread of B. cinerea
among grapes during storage, and this information would be valuable in the
selection and timing of fungicides to use in vineyard fungicide programs.
The aim of our work was to compare the effectiveness of several
approaches available to grape growers to control gray mold that could be
applied in vineyards, including three chitosan-containing products,
potassium sorbate, and a program of four conventional fungicide
formulations to control postharvest decay of ‘Thompson Seedless’ grapes.
The size, texture, and appearance of table grapes are of particular importance
compared to grapes grown for wine production or raisins. Therefore, the
influence of materials applied in the vineyard on these aspects is of critical
practical importance. We also determined their effect on berry size, weight,
pH, titratable acidity, soluble solids content, firmness, chitinase activity, and
the contents of potassium, phenolic compounds, and hydrogen peroxide.
6.2
6.2.1
Material and methods
Vineyard treatments
A single vineyard of ‘Thompson Seedless’ grapevines,
approximately 50 years in age, drip irrigated, and located at the San Joaquin
Valley Agricultural Sciences Center in Parlier, CA was used in 2009, 2010,
and 2011. Elemental sulfur dust was applied repeatedly to control powdery
121
mildew. There were 4 treatments in 2009 and 2010 and 6 treatments in 2011,
each repeated in 6 blocks that corresponded to 6 rows, each of them
separated by a two non-treated rows. Each vineyard plot (a total of 24 in
2009 and 2010 and 36 in 2011) consisted of 5 vines spaced 1.7 m apart in
rows with 3.5 m between rows. In all the years a randomized block design
was used, and the treatments were re-randomized each year. Treatments were
assigned to plots using a random number generator (Excel; Microsoft Corp.,
Redmond, WA). The vines were not girdled or treated with gibberellic acid
to increase berry size. The treatments were applied four times; at berry set,
bunch closure, veraison, and 2 weeks before harvest in 2009 and 2010; and
at berry set, bunch closure, veraison, and 3 weeks before harvest in 2011. In
all years, treatments were applied from a powered sprayer and the clusters
were wetted until run-off. All treatments contained 0.3 ml liter-1 of surfactant
(Latron B1956, BFR Products, Five Points, CA). Potassium sorbate (Fruit
Growers Supply, Exeter, CA) was applied 3.33 g per vine from a solution
containing 0.5% (wt/vol) potassium sorbate. The fungicide program
consisted of an initial application of pyrimethanil (Scala SC, Bayer Crop
Science, Research Triangle Park, NC, 1.1 ml per vine) at berry set,
cyprodinil + fludioxonil (Switch 62.5 WG, Syngenta, Wilmington, DE, 0.4 g
per vine) at bunch closure, pyraclostrobin + boscalid (Pristine WG, BASF,
Florham Park, NJ, 0.6 g per vine) at the onset of veraison, and lastly
fenhexamid (Elevate 50WDG, Arysta LifeScience, Cary, NC, 0.5 g per vine)
at two (2009 and 2010) or three (2011) weeks before harvest. These are the
approximate common commercial rates at the time these tests were
conducted (Smilanick, 2010). Potassium sorbate alone, the fungicide
program, and the fungicide program plus potassium sorbate were applied in
2009 and 2010. The chitosan containing products, applied in the 2011 tests
only, were applied at 1% chitosan concentration. The treatment of fungicide
program plus potassium sorbate was omitted in 2011. Chitosan-A (OII-YS;
Venture Innovations, Lafayette, LA), chitosan-B (Chito Plant; ChiPro
GmbH, Bremen, D), and chitosan-C (Armour-Zen; Botry-Zen Limited,
122
Dunedin, NZ) were applied at 112 ml, 6.7 g, and 45 ml, respectively, per
vine. Control plots were treated with water.
6.2.2
Natural postharvest decay
Ten kilos (five or six clusters from each vine of the five vines in
each plot, a total of 27 grape clusters) per each plot were harvested and
placed in plastic bags and placed in expanded polystyrene boxes containing
9 bags each. The grapes selected were free of defective or decayed berries.
The boxes were stored at 2 °C under humid conditions (90-99% RH) in
darkness for 6 weeks when the natural incidence of decay and shatter was
counted and the rachis appearance was rated. Gray mold infected grapes
were identified by the characteristic slip-skin symptom and sporulation. The
slip skin condition is a consequence of the growth of B. cinerea under the
berry skin that causes it to easily separate from the underlying tissues when
touched. The incidence of decay by other fungi was also recorded.
Percentages were calculated by dividing the number of infected berries by
the average total number of berries within each polyethylene bag. The rachis
appearance rating employed a scale of 0 to 5, where 0 = fresh and green; 1 =
pedicels only are brown; 2 = all pedicels and less than 50% of the laterals are
brown; 3 = pedicels and more than 50% of the laterals are brown; and 4 =
pedicels and laterals brown, main rachis stem green; and 5 = rachis entirely
brown. The experiment was conducted three times with the fungicide
program and potassium sorbate (2009, 2010, and 2011) and once with the
chitosan-containing products (2011).
6.2.3
Postharvest decay after inoculation with B. cinerea
B. cinerea isolate 1440 was grown on PDA in Petri dishes and
incubated at 25 ± 1 °C and in dark for 2 weeks. The pathogen was isolated in
1992 from an infected kiwi fruit in California. It was selected because
123
sporulated readily, was virulent, and was sensitive to the fungicides
evaluated in this study. Sterile water containing 0.1% Triton X-100 (wt/vol)
was added to the dishes and conidia were rubbed from the agar surface with
a sterile glass rod. This high-density conidial suspension was passed through
two layers of cheesecloth and the number of conidia counted using a
hemacytometer. The conidial suspension was diluted with sterile water to
contain 104 conidia ml-1. Three repetitions of 30 berries from each plot were
selected from 10 to 20 clusters by clipping the terminal berries from the
second lateral branch located from the top of the rachis. The detached berries
were placed above a grid, sprayed (Spray Gun; Harbor Freight Tools,
Camarillo CA) to run-off with the conidial suspension and stored at 15 °C
under humid conditions (90-99% RH) in darkness. Three weeks after
inoculation the incidence and severity of berry decay were recorded. The
incidence was expressed as the percentage of infected berries. The severity
was assigned into classes according the berry surface percentage covered by
the fungal mycelium. The classes were: 0 = uninfected berry; 1 = infected
and discolored, but no surface mycelium present; 2 = surface mycelium just
visible to 25% of the berry surface; 3 = 26 to 50% of the berry surface
covered with mycelium; and 4 = more than 50% of the berry surface covered
with mycelium. The proportion of infected berries per replicate and
McKinney Index values, which incorporated both incidence and severity,
were calculated as the weighted average of the disease as a percentage of the
maximum possible level (McKinney, 1923). Berries were collected from the
plots three times on consecutive days before the quality harvest and the
experiment repeated with each collection.
6.2.4
Quality characteristics
Berries were selected from clusters by clipping the terminal berries
from the second lateral branch located at the top of the rachis. Soluble solids
were determined repeatedly at biweekly intervals among the treatments
124
before harvest during 2009, 2010, and 2011. In 2009 and 2010, the soluble
solids contents were determined from a 20 berry sample collected from each
of the treatment plots and were pooled and macerated before the soluble
solids were determined, so the variance in these measurements could not be
calculated. In 2011, thorough quality evaluations of the berries were
conducted. One hundred berries were collected from clusters by clipping one
or two terminal berries from the second lateral branch located at the top of
the rachis of mature clusters from each plot. These were weighed to
determine the mean weight per berry and their firmness and diameter were
measured (FirmTech 2; BioWorks, Wamego, KS). The berries were blended
(Blender 5011; Waring, New Hartford, CT) at high speed for 30 s and the
homogenate was centrifuged for 10 min at 9,000 x g. The supernatant pH
(pH Meter 320; Corning, Corning, NY), total acidity (TIM850 Titration
Manager; Radiometer Analytical, Villeurbanne Cedex, F), potassium content
(C-131 Compact Potassium Ion Meter; Horiba, Irvine, CA), and soluble
solids content (Pocket Refractometer PAL-1; ATAGO, Bellevue, WA) were
recorded.
6.2.5
Chitinase activity
Chitinase activity was assessed in berry skin and flesh in
preparations by the method of Byrne et al. (2001) with some modifications.
One hundred berries were collected from clusters by clipping one or two
terminal berries from the second lateral branch located at the top of the
rachis of mature clusters from each plot. Frozen berries, in which seed traces
were manually removed, were blended, 4 g of the homogenate was added to
10 ml of ice-cold 50 mM Na-acetate pH 5.0 containing 0.25% Triton X-100
(wt/vol), and then centrifuged at 9,000 x g for 10 min. Chitinase activity in
the extract supernatant were determined using three different substrates
(Chitinase Assay Kit; Sigma-Aldrich, Saint Louis, MO): i) 4Methylumbelliferyl
β-D-N,N′,N′′-triacetylchitotriose which is an
125
endochitinase
substrate;
ii)
4-Methylumbelliferyl
N-acetyl-β-Dglucosaminide; and iii) 4-methylumbelliferyl-N,N’-diacetyl-β-D-chitobiose.
The last two are exochitinase substrates. From these substrates, chitinase
hydrolysis liberates 4-methylumbelliferone whose fluorescence was
measured using a fluorometer (Spectramax M2; Molecular Device,
Sunnyvale, CA) with excitation at 360 nm and emission at 450 nm. One unit
of chitinase activity released 1 µmole of 4-methylumbelliferone from the
appropriate substrate per minute at pH 5.0 at 37 °C. Chitinase activity was
expressed as units per gram of proteins contained in the extract supernatant.
Protein content was measured through bicinchoninic acid (BCA Protein
Assay Reagent; ThermoFisher Scientific, Rockford, IL), using bovine serum
albumin as standard protein.
6.2.6
Hydrogen peroxide content
Hydrogen peroxide content of the berries was determined with 2’,7’dichlorodihydrofluorescein
diacetate
(H2DCF-DA;
Sigma-Aldrich)
according to the method of Macarisin and coworkers (2007). H2DCF-DA
was dissolved in anhydrous dimethyl sulfoxide (Sigma-Aldrich) to make a
10mM stock solution, which was frozen (-20 °C) in aliquots and thawed just
before the analysis. From each plot a sample of 50 berries per treatment,
selected from clusters by clipping the terminal berries from the second
lateral branch located at the top of the rachis, were frozen in liquid nitrogen
after harvest and stored at -80 °C. The berries were reduced to powder in
liquid nitrogen with a mortar and pestle, and 0.5 g of the powder was placed
in a microcentrifuge tube and diluted in 1 ml of 50 mM 2-(N-morpholine)
ethanesulfonic acid (Sigma-Aldrich) buffer, pH 6.5. Three replicates per
each plot were prepared. The microcentrifuge tubes were vortexed briefly
and centrifuged at 18,000 x g for 5 min at 4 °C. The supernatants were
collected and 150 µl was pipetted in a 96 well plate containing 150 µl 0.02
mM H2DCF-DA in each well. H2O2 content was determined after 24 h of
126
incubation at room temperature and in darkness by measuring fluorescence
intensity (Spectramax M2; Molecular Device, Sunnyvale, CA) with an
excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Hydrogen peroxide content was expressed as a percentage of the hydrogen
peroxide content of the control grapes.
6.2.7
Hydrogen peroxide localization by scanning electron microscope
Grapes were rapidly frozen by plunging them in liquid nitrogen then
they were stored at -80 °C until use. One berry from each treatment was
fractured with a chilled scalpel. Pieces that included the berry epidermis with
approximate dimensions of 6 x 3 mm and 2 mm thick were placed in a 6well holder and the holder was then placed sequentially into a container with
5 µM CeCl3 in 0.1M 1,4-piperazinediethanesulfonic acid buffer (PIPES)
(Sigma-Aldrich), 4% (vol/vol) glutaraldehyde in 0.1M PIPES buffer, and
water purified by reverse osmosis (RO). The grape pieces were processed in
a vacuum capable microwave oven (BioWave Pro 36500; Ted Pella, Inc.,
Redding, CA), while in the 6-well holder they treated using the following
protocol: i) they were placed in the 5 µM CeCl3 solution and treated under
vacuum for 60 s at 150 watts and for 60 s at 0 watts; this cycle was repeated
4 times; ii) they were placed in the 4% glutaraldehyde solution for 60 s at
150 watts and 60 s at 0 watts; this cycle was repeated 4 times; and iii) they
were placed in RO water for 120 s at 100 watts and 120 s at 0 watts; this
cycle was repeated for 3 times and, after a change of water, it was repeated
another 3 times. The grape pieces were transferred through a dehydration
series of 25, 50, 75, 95, and 100% ethanol and finally a second refreshed
100% ethanol solution. With each dehydration step, the grape pieces were
microwaved for 40 s at 150 watts and 40 s at 0 watts and each step of this
cycle was repeated twice. The dehydrated grapes were transferred to
specimen holders and critical point dried (Autosamidri-815B supercritical
drier; Tousimis Research Corporation, Rockville, MD). Dried specimens
127
were mounted on carbon coated aluminum stubs and examined with an
scanning electron microscope (model S-3500N, Hitachi High-Technology
America Corp., Pleasanton, CA). Cerium was detected by energy dispersive
x-ray detector (EVEX, Princeton, NJ).
6.2.8
Phenolic compound analysis
Fifty berries were collected from clusters by clipping one or two
terminal berries from the second lateral branch located at the top of the
rachis of mature clusters from each plot. They were washed with water,
frozen at -20 °C, peeled by hand, and their skins were placed in (1 ml per
berry) 70% acetone and 30% distilled deionized water containing 0.1%
ascorbate (wt/vol) and agitated on an orbital shaker for 24 h in darkness at
room temperature. Extracts were then filtered through Whatman No.1 filter
paper and evaporated (Multivapor P-12; Buchi Corporation, New Castle,
DE) at 35 °C with partial vacuum (400 mm Hg) to remove the acetone. The
evaporated samples were adjusted to 50 ml with distilled deionized water.
Approximately 20 ml of each sample were stored at -20 °C in glass vials. To
determine the composition of phenolic compounds, an HPLC (High
Performance Liquid Chromatograph) (Prominence; Shimadzu Corporation,
Japan) with two pressure pumps and a diode array UV-visible detector (SPDM10 AVP) coupled and connected to LC (Liquid Chromatography) real time
program was used. Samples were thawed and, after adding 1 ml of solvent B
to 250 µl, were centrifuged at 14000 rpm for 10 min. Supernatants were
drawn into auto-sampling vials via a syringe attached to 13 mm filter
(Acrodisc Syringe Filter; Pall Scientific, NY). The samples were loaded into
Novapak RP C18 column 3.9 x 300 mm, 4 µm particle size (Waters, Milford,
MA), was used for the stationary phase. The column was connected to
Novapak guard column with the same material. The flow rate of mobile
phase was 0.5 ml min-1 which separated the individual phenolics. The
solvents, concentration gradient used for phenolic compound separation, and
128
preparation of gallic acid, resveratrol, quercetin, catechin and epicatechin
(Sigma Aldrich) standards were the same as described by Lamuela-Raventós
and Waterhouse (1994). Standard curves were developed by comparing the
concentrations of each standard to its peak area. Individual phenolics were
identified and calculated by comparing the retention time and the absorption
spectrum from 280 to 365 nm on chromatogram plot to those of the
standards.
6.2.9
Effect of residual fungicide content of berries on postharvest decay
A second experiment was conducted to evaluate the influence of the
residual fungicide content deposited on berries on their subsequent
postharvest decay. Clusters of mature ‘Princess Seedless’ table grapes were
placed immediately after harvest on plastic racks and sprayed (Spray Gun;
Harbor Freight Tools, Camarillo CA) to run-off with fungicides at
concentrations that approximate those used commercially in vineyards where
the maximum fungicide application rates indicated on the USEPA-approved
product label in a water volume of 1900 liter ha-1 were used (Smilanick et al.,
2010). They were: i) pyraclostrobin and boscalid, 59 g liter-1 and 116 g
liter-1, respectively; ii) cyprodinil 270 g liter-1 (Vangard; Syngenta,
Wilmington, DE); iii) pyrimethanil 370 l liter-1; and iv) fenhexamid 290 g
liter-1. After the clusters dried in air (about 2 hours), a single berry infected
just before placement by the injection of 20 µl of a suspension containing
106 conidia ml-1 of B. cinerea, isolate 1440, was placed in the center of each
cluster. Two boxes that contained 10 clusters each were prepared for each
fungicide treatment; one was examined after 4 weeks and the other after 6
weeks at 1 °C under humid condition (90 to 99% RH) in darkness.
Observations included: i) the spread of gray mold from a single, untreated
berry previously inoculated with B. cinerea conidia and placed within the
cluster after fungicide treatment. Spread was determined by counting the
number of new infected berries that were adjacent to the inoculated berry; ii)
129
the natural incidence of gray mold infected berries; iii) the incidence of
berries infected by other fungi; and iv) the residues of the applied fungicide
after 6 weeks. A single sample of 50 healthy berries was collected from each
treatment for residue analysis. Fungicides residues were determined by the
method of Karaca et al. (2011). The experiment was done once.
6.2.10 Statistical analysis
Data were analyzed by a one or two-way ANOVA followed by
Fisher’s protected LSD or Tukey’s HSD test at P = 0.05 (SPSS Statistics
17.0 Inc., Chicago, IL). In the statistical analysis of the randomized complete
block design, the block (row) is considered as a second factor. Percentage
data were arcsine transformed before analysis to improve homogeneity of
variance when the range of percentages was greater than 40. Actual values
are shown.
6.3
Results
In 2009 and 2010, the natural incidence of postharvest decay among
the treatments was mostly caused by B. cinerea (Table 21). The most
effective treatment was the fungicide program, alone or with potassium
sorbate, in 2009, while in 2010, the fungicide program and potassium
sorbate were similarly effective. The incidence of decay caused by other
fungi was low in 2009 (1 to 2%) and somewhat (4 to 5%) higher in 2010. In
both years, control of other fungi by the treatments was poor. Among all of
the treatments, the rate of soluble solids increase was slightly higher than the
control treatments among potassium sorbate treated grapes in all years,
although not significantly so in 2011, when the variability among the
treatments was high. The soluble solids contents on the day of harvest of the
control grapes in 2009, 2010, and 2011 was 16.6, 16.9, and 18.2 (± 0.9),
respectively, while among those treated with potassium sorbate it was 17.7,
130
18.1, and 18.7 (± 1.0), respectively. In 2011, there were no statistical
differences in soluble solids of control grapes and those treated with
potassium sorbate.
Table 21. The influence of 4 preharvest applications (at berry set, bunch closure,
veraison, and 2 weeks before harvest) of potassium sorbate alone, or a fungicide
program alone, or a combination of both, on the postharvest decay of ‘Thompson
Seedless’ grapes. The fungicide program consisted of applications of pyrimethanil,
cyprodinil + fludioxonil, pyraclostrobin + boscalid, or fenhexamid at first, second,
third, and final applications, respectively. The grapes were examined after 6 weeks
of storage at 2 °C.
Decay after storage (%)
2009
2010
Treatments
Gray mold Other rotsa Gray mold Other rots
Control
15.1 ab
1.9 a
24.2 a
5.0a
K sorbate
4.9 b
1.4 a
6.9 b
4.2a
Fungicide program
1.1 c
1.1 a
4.7 bc
4.1a
Fungicide program+K sorbate
1.8 bc
1.4 a
3.7 c
4.4a
a
Alternaria spp. and Penicillium spp.
b
Values within columns followed by unlike letters are significantly different
according to Tukey’s HSD (P = 0.05). Statistical analysis employed arcsine
transformed values, actual values are shown.
In 2011, the natural incidence of postharvest gray mold was
markedly reduced by the fungicide regime, and moderately reduced by the
chitosan treatments (Table 22). Decay by other pathogens was most reduced
by the chitosan-A and the fungicide regime. All of the treatments reduced
berry shatter, and some slightly but significantly reduced berry shrivel or
improved rachis appearance, compared to the control. Berry shrivel or
“water berry” disorder (Morrison and Iodi, 1990; Hall et al., 2011) was
present in the vineyard (Table 22).
131
Table 22. Incidence of decay, shatter, shrivel, and rachis appearance of ‘Thompson
Seedless’ table grapes after 6 weeks of storage at 2 °C that had been treated 4 times
before harvest (at berry set, bunch closure, veraison, and 3 weeks before harvest)
with water (control), one of three chitosan containing products (all applied at 1%
chitosan), potassium sorbate (applied at 0.5% wt/vol), or a fungicide program that
consisted of applications of pyrimethanil, cyprodinil + fludioxonil, pyraclostrobin +
boscalid, or fenhexamid at the first, second, third, and final applications,
respectively. Each value is the mean of six replicate 10 kg boxes containing nine
grape cluster bags with three clusters each. Decay, shatter, and berry shrivel values
are the percentage of affected berries. The rachis rating is a scale of 0 to 5, where 0
= fresh and green in appearance to 5 = rachis entirely brown.
Decay (%)
Treatment
Gray mold Other rotsa Shatter (%) Berry shrivel (%) Rachis
rating
Control
3.9 ab
4.8 a
11.3 a
5.6 a
1.6 a
Chitosan-A
2.1 bc
1.0 d
5.7 b
4.9 ab
1.4 ab
Chitosan-B
2.3 bc
3.3 bc
6.8 b
3.1 b
1.3 ab
Chitosan-C
2.0 c
3.3 bc
5.9 b
3.6 ab
1.2 b
K sorbate
2.8 ab
4.3 ab
8.1 b
4.8 ab
1.4 ab
Fungicide
0.7 d
2.5 c
5.3 b
3.0 b
1.0 b
a
b
HSD (P = 0.05).
The number of infections and severity of gray mold infections that
occurred after artificial inoculation with B. cinerea of berries collected from
these treatments was reduced by two of the chitosan formulations and the
fungicide regime (Figure 5). Visible and objectionable brown-colored
deposits were present on the berries where chitosan-C formulation had been
applied.
132
McKinney Index (% ± SD)
35
a
30
25
c
A
bc
20
15
d
10
5
0
35
a
a
ab
30
Decay (% ± SD)
ab
ab
B
bc
cd
25
20
d
15
10
5
0
Control
Fungicides
K sorbate
Chitosan-A
Chitosan-B
Chitosan-C
Figure 5. McKinney Index (A) and incidence of decay (B) (%±SD) after
inoculations with a suspension containing 104 B. cinerea conidia ml-1 of ‘Thompson
Seedless’ grapes that had been treated four times (at berry set, bunch closure,
veraison, and 3 weeks before harvest) with water (control), one of three chitosan
containing products (all applied at 1% chitosan), potassium sorbate (applied at 0.5%
wt/vol), or a fungicide program that consisted of applications of pyrimethanil,
cyprodinil + fludioxonil, pyraclostrobin + boscalid, or fenhexamid at the first,
second, third, and final applications, respectively. The grapes were stored 3 weeks at
15 °C at 90-99% RH in darkness. Unlike letters are significantly different according
to Tukey’s HSD (P = 0.05).
133
In 2011, vineyard treatments significantly but modestly influenced
titratable acidity, firmness, berry weight, and berry diameter, and did not
alter soluble solids, juice pH, and potassium content significantly (Table 23).
None of the treatments were significantly different from the control in
titratable acid content; the lowest values (5.08 g tartaric acid liter -1) were
among the fungicide treated grapes and highest (5.68 g tartaric acid liter -1)
among those treated with potassium sorbate. Firmness was significantly
higher than the control after chitosan-C treatment and significantly lower
after fungicide, chitosan-A or chitosan-B treatments. Berry weight was
lowest (1.52 g) after potassium sorbate treatment and highest (1.75 g) after
fungicide treatment. Berry diameter was largest (14.33 mm) after the
fungicide treatments, followed by the chitosan treatments.
134
Table 23. Characteristics of ‘Thompson Seedless’ grapes at harvest that had been
treated four times (at berry set, bunch closure, veraison, and 3 weeks before harvest)
with water (control), one of three chitosan containing products (all applied at 1%
chitosan), potassium sorbate (applied at 0.5% wt/vol), or a fungicide program that
consisted of applications of pyrimethanil, cyprodinil + fludioxonil, pyraclostrobin +
boscalid, or fenhexamid at the first, second, third, and final applications,
respectively. Value of firmness, weight, and diameter were the mean of 6 replicates
of 100 berries each. Soluble solids, acidity, pH, and potassium content were the
means of 6 replicates of a filtered macerate prepared from 100 berries per replicate.
Sol. Titratable
Berry
size
Potassium
Treatment solidsa
acidity Firmness
Weight Diameter pH
content
Control
18.2
5.28 abcb 2.64 b
1.71 ab 13.93 c
3.4
1026
Chitosan-A 17.8
5.36 abc 2.47 d
1.59 ab 14.08 bc
3.4
1013
Chitosan-B
18.0
5.57 ab
2.59 c
1.58 ab 14.19 ab
3.3
996
Chitosan-C 19.5
5.19 bc
2.82 a
1.65 ab 14.20 ab
3.4
1016
K sorbate
18.8
5.68 a
2.70 b
1.52 b
13.91 c
3.4
1033
Fungicide
18.5
5.08 c
2.50 cd
1.75 a
14.33 a
3.4
1050
a
Units of measurement: soluble solids (% Brix), titratable acidity (g tartaric acid
liter-1), firmness (N), berry weight (g), berry diameter (mm), potassium content
(ppm).
b
HSD (P = 0.05).
All of the treatments increased endochitinase activity, with the larger
increase caused by chitosan formulation or potassium sorbate (Table 24).
Exochitinase activity, as determined using 4-methylumbelliferyl-N,N’diacetyl-β-D-chitobiose as a substrate, was significantly increased only on
those grapes previously treated with chitosan-A or chitosan-C, while all of
the treatments were similar to the control for exochitinase activity, as
determined using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide as a
substrate.
135
Table 24. Chitinase activity (U g-1 protein) at harvest of ‘Thompson Seedless’ grapes
that had been treated four times (at berry set, bunch closure, veraison, and 3 weeks
before harvest) with water (control), one of three chitosan containing products (all
applied at 1% chitosan), potassium sorbate (applied at 0.5% wt/vol), or a fungicide
program that consisted of applications of pyrimethanil, cyprodinil + fludioxonil,
pyraclostrobin + boscalid, or fenhexamid at the first, second, third, and final
applications, respectively.
Treatment
Endochitinasea
Exochitinaseb
Exochitinasec
d
Control
13.7 e
36.2 ab
4.7 b
Chitosan-A
16.9 b
39.7 a
5.3 a
Chitosan-B
15.6 c
33.2 b
4.6 bc
Chitosan-C
17.1 b
38.8 a
5.1 a
Fungicide
14.8 d
35.8 ab
4.5 bc
K sorbate
18.5 a
39.7 a
4.4 c
a
Endochitinase activity determined using 4-methylumbelliferyl β-D-N,N′,N′′triacetylchitotriose
b
Exochitinase activity determined using 4-methylumbelliferyl N-acetyl-β-Dglucosaminide
c
Exochitinase activity determined using 4-methylumbelliferyl-N,N’-diacetyl-β-Dchitobiose
d
Values followed by unlike letters are significantly different by Fisher’s protected
LSD (P = 0.05).
Vineyard applications of the chitosan-B formulation significantly
increased the resveratrol, quercetin and myricetin contents of berry skin
(Table 25). The content of quercetin and myricetin after treatments with the
other two chitosan formulations was higher, but not statistically different
from the control. The sole effect of potassium sorbate application was to
increase resveratrol content of the berry skin. None of the treatments
modified the gallic acid content of the berry skin.
136
Table 25. Gallic acid, quercetin, myricetin, and resveratrol contents (mg kg -1 berry
weight) at harvest of ‘Thompson Seedless’ grapes that had been treated four times
(at berry set, bunch closure, veraison, and 3 weeks before harvest) with water
(control), one of three chitosan containing products (all applied at 1% chitosan),
potassium sorbate (applied at 0.5% wt/vol), or a fungicide program that consisted of
applications of pyrimethanil, cyprodinil + fludioxonil, pyraclostrobin + boscalid, or
fenhexamid at the first, second, third, and final applications, respectively.
Treatment
Gallic acid
Quercetin Myricetin
Resveratrol
a
Control
6.5
17.1 bc
1.8 b
0.36 c
Chitosan-A
6.9
19.2 b
2.0 b
0.37 bc
Chitosan-B
7.2
23.7 a
2.9 a
0.41 ab
Chitosan-C
6.5
17.8 bc
2.1 b
0.35 c
Fungicide
6.3
14.4 c
1.8 b
0.34 c
K sorbate
7.0
14.5 c
1.8 b
0.42 a
a
Values followed by unlike letters are significantly different by Fisher’s protected
LSD (P = 0.05).
Chitosan-A and chitosan-B formulations significantly decreased
hydrogen peroxide content of berries, with the greatest reduction of 70%
from chitosan-A (Figure 6). The location and content of hydrogen peroxide
observed in mature ‘Thompson Seedless’ grape berry tissue as shown by Xray energy dispersive analysis of cerium hydroxide, a reaction product of
hydrogen peroxide and cerium chloride (Figure 7). The carbon coating did
not completely eliminate charging on the specimens so some distortions
were observed. Images indicated relatively high levels of hydrogen peroxide
among berries treated with potassium sorbate, the fungicide program, and
the control, with lower levels among the grapes treated with the chitosan
formulations.
137
Chitosan-A Chitosan-B Chitosan-C
Control
K sorbate
Fungicides
a
a
H2O2 content (% of the control)
120
110
ab
100
b
90
80
70
c
c
60
Figure 6. Relative hydrogen peroxide content immediately at harvest of ‘Thompson
Seedless’ grapes that had been treated four times (at berry set, bunch closure,
veraison, and 3 weeks before harvest) with water (control), one of three chitosan
containing products (all applied at 1% chitosan), potassium sorbate (applied at 0.5%
wt/vol), or a fungicide program that consisted of applications of pyrimethanil,
cyprodinil + fludioxonil, pyraclostrobin + boscalid, or fenhexamid at the first,
second, third, and final applications, respectively. Unlike letters are significantly
different according to Tukey’s HSD (P = 0.05).
138
Figure 7. Location and content of hydrogen peroxide in mature ‘Thompson
Seedless’ grape berry tissue as shown by X-ray energy dispersive analysis of cerium
hydroxide (pink pixels), a reaction product of hydrogen peroxide and cerium
chloride. The epidermis appears at the uppermost portion of each panel with
approximately ten cell layers shown. The grapes were treated four times (at berry
set, bunch closure, veraison, and 3 weeks before harvest) with water (A), potassium
sorbate (B), a fungicide program (C), chitosan-A formulation (D), chitosan-B
formulation (E), or chitosan-C formulation (F). Bar = 100 µm.
139
The residual fungicide content remaining on ‘Princess Seedless’
grapes greatly influenced the spread and incidence of gray mold and
incidence of other decay pathogens, such as Alternaria spp. and Penicillium
spp., during cold storage (Table 26). Fenhexamid was the most effective for
the control of gray mold, but did not influence the incidence of decay by
other fungi. Pyrimethanil and cyprodinil were similar in effectiveness to
each other for the control of gray mold, while pyraclostrobin + boscalid did
not significantly reduce gray mold, but did reduce the incidence of decay by
other fungi. The US EPA maximum residual fungicide content of the berries
of fenhexamid, pyrimethanil, cyprodinil, pyraclostrobin, and boscalid are 4,
5,
2,
2,
and
3.5
mg-kg-1,
respectively
(http://www.epa.gov/opp00001/food/viewtols.htm). Only those of cyprodinil
exceeded the tolerance in our work.
140
Table 26. Effect of postharvest treatments with fenhexamid (FENH), pyrimethanil
(PYRI), cyprodinil (CYPR), or pyraclostrobin + boscalid (PYRA+BOSC) on the
spread of gray mold from a single infected berry placed within ‘Princess Seedless’
grape clusters after fungicide treatment and on the natural incidence of berries
infected by gray mold or other fungi. The decay incidence was evaluated after 4 and
6 weeks of storage at 1 °C. The residual fungicide contents of the berries of the
applied fungicide after 6 weeks of storage were determined.
Gray mold spread
Natural
Natural
Fungicide
from infected berry
gray mold (%)
other
rotsa
(%)
content
Treatments
4 wk
6 wk
4 wk
6 wk
4 wk
6 wk
(mgkg-1)
Control
10.3 a
23.0 a
1.4 ab 4.4 a
5.6
9.7 a
…
FENH
0.1 c
0.9 c
0.7 c
0.8 c
3.7
10.3 a
2.1
PYRI
2.1 b
5.0 b
1.1 b
1.1 b
5.3
8.6 ab
3.8
CYPR
5.0 b
8.9 b
1.6 b
1.4 b
3.7
5.7 b
4.2
PYRA + BOSC
9.3 a
22.8 a
2.6 a
4.0 a
3.1
4.2 b 1.5/1.5
a
b
Values within columns followed by unlike letters are significantly different by
Fisher’s protected LSD (P = 0.05).
6.4
Discussion
Potassium sorbate reduced natural gray mold incidence in two of
three study years, demonstrating that repeated applications are effective, as
has been shown for postharvest applications (Karabulut et al., 2005b). The
inefficacy of potassium sorbate in the third year of the study may have
several possible explanations. The detached berries from these same vines
became infected when inoculated with B. cinerea conidia, perhaps because
the residual potassium sorbate content in grape berries was low.
Investigation of the potassium sorbate content and persistence in the berries
141
would help interpret how vineyard sorbate applications controlled gray
mold. It declines rapidly in fresh citrus fruit (Montesinos-Herrero, 2009).
However, potassium sorbate may control gray mold by other than its
antimicrobial properties. The modest increases in endochitinase activity and
resveratrol content we observed in 2011 indicate induction of resistance by
potassium sorbate could have had some role. It is conceivable potassium
sorbate induced significant resistance to infection in 2009 and 2010, but not
in 2011.
In 2011, a high prevalence of “water berry” caused variability in
maturity among the grapes. This disorder causes phloem death in the rachis
followed by cessation of sugar and water accumulation in berries (Morrison
and Iodi, 1990; Hall et al., 2011). Although we avoided visibly shriveled and
symptomatic berries when sampling, some were probably included and their
lower soluble solids content and softer texture contributed variation in berry
quality measurements and made differences among treatments more difficult
to resolve.
Foliar applications of potassium sorbate (Mlikota Gabler et al.,
2010) or other sources of potassium (Strydum and Loubser, 2008; Mlikota
Gabler et al., 2010; Kelany et al., 2011) to grapes were reported to accelerate
accumulation of soluble solids in grapes, reduce berry size, and increase
titratable acidity. The modest increase in resveratrol content of the berry skin
we observed is somewhat paradoxical because an increase in soluble solids
content, an indication of maturity, was observed after potassium sorbate
applications in prior work (Mlikota Gabler et al., 2010), and advanced
maturity is negatively correlated with the capacity for resveratrol synthesis
(Jeandet et al., 1991; Bais et al., 2000). However, sorbate also reduced berry
size and increased titratable acidity, both characteristic of less mature
berries, which is associated with increased resveratrol content (Jeandet et al.,
1991; Bais et al., 2000). It is conceivable the reduction in berry size alone
could influence the concentration of the sugars and other components within
the berries.
142
Preharvest fungicide regimes in this and prior reports were shown to
significantly reduce subsequent postharvest decay (Franck et al., 2005;
Smilanick et al., 2010). Potassium sorbate could be used alone or in a
mixture with conventional fungicides to provide partial control of
postharvest decay. As a component in a conventional fungicide program, it
may retard the development of fungicide resistant populations of B. cinerea
in vineyards. Of the conventional fungicides we evaluated, the residual
fungicide content that remained after fenhexamid application was markedly
superior for the control of both the natural incidence of gray mold and spread
of the aerial mycelium of B. cinerea among stored grapes. The residual
fungicide content within the berries was below regulatory tolerances (EPA,
2006). Applied after rainfall or immediately before harvest, fenhexamid
would be a good choice for use in San Joaquin Valley vineyards, although
resistance to this fungicide develops rapidly among B. cinerea populations
(Franck et al., 2005; Smilanick et al., 2010). During the hot, dry periods of
the growing season, summer bunch rot is prevalent in this area, while gray
mold is not. Summer bunch rot, caused by a complex of fungi and bacteria,
is not controlled by fenhexamid, which is primarily a botricide, while it can
be partially controlled by other fungicides (Tjamos et al., 2004) and cultural
practices that open the vineyard canopy, such as leaf removal (Schilder et al.,
2010).
This is one of few studies where commercial chitosan formulations
were evaluated and compared to fungicides in effectiveness in a regime that
closely simulated commercial vineyard practices. Relatively non-toxic and
environmentally benign, risk of the development of resistance to them in the
pathogen population is low. Treatment with chitosan-C caused visible and
objectionable brown-colored deposits on berries while the other formulations
did not. Among the three chitosan formulations, control of natural
postharvest gray mold was similar, but chitosan-A most effectively
controlled natural decay by pathogens other than B. cinerea (mainly
Alternaria spp. and Penicillium spp.) and the chitosan-C formulation did not
143
retard the spread of B. cinerea after inoculation. Since the three formulations
were all applied with a chitosan content of 1%, differences in effectiveness
could be ascribed to the chemical characteristics of chitosan and/or to the
other components in the formulations. Chitosan forms films on products
(Romanazzi et al., 2009), and the characteristics of the films created by the
chitosan formulations we used merit study, particularly since they could
constitute a physical barrier to inhibit B. cinerea infections. Cuticle and cell
thickness in the skin are natural barriers associated with resistance in grape
berries to B. cinerea (Mlikota Gabler et al., 2003; Deytieux-Belleau et al.,
2009).
The chitosan formulations increased chitinase activity. This enzyme
is a pathogenesis-related protein with antimicrobial activity that participates
in defense against pathogens (Van Loon and Van Strien, 1999). Previous
work indicated that in addition to antimicrobial activity (Rabea et al., 2003;
Muñoz and Moret, 2010), chitosan induced a series of defensive reactions in
grape against B. cinerea. Phenylalanine ammonia-lyase (PAL), a key enzyme
involved in the synthesis of phytoalexins and phenolic compounds with
antifungal activity, was induced by chitosan both in grape leaves (TrotelAziz et al., 2006; Reglinski et al., 2010;) and berries (Romanazzi et al.,
2002; Meng et al., 2008). In our work, preharvest all chitosan treatments
induced activity of endochitinase, and chitosan-A and chitosan-C
formulations induced exochitinase (from one of the substrates). This result
corroborates findings by Trotel-Aziz et al. (2006) that chitosan applications
induced chitinase activity in detached grape leaves.
Chitosan treatments reduced hydrogen peroxide content, which
confirms work by Romanazzi et al. (2013). It may have been a direct effect,
since chitosan itself has antioxidant activity and scavenges hydroxyl radicals
(Xing et al., 2005; Yen et al., 2008), or an indirect effect, since chitosan was
reported to increase peroxidase activity in table grapes (Meng et al., 2008;
Reglinski et al., 2010), which would reduce their hydrogen peroxide content.
Peroxidase participates in various physiological processes, such as
144
lignification, suberization, wound healing, and defense mechanisms against
pathogen infection (Hiraga et al., 2001). The presence of other antioxidants,
such as phenols, could have reduced the content of hydrogen peroxide as
well since the hydroxyl group and unsaturated double bonds of phenols
make them very susceptible to oxidation (Rice-Evans et al., 1997; Yilmaz
and Toledo, 2004; Iacopini et al., 2008).
The accumulation of phytoalexins trans-resveratrol and other
phenols is considered the primary inducible response of grapevine against a
number of biotic and abiotic stresses (Jeandet et al. 2002; Kretschmer et al.,
2007). We found preharvest treatments with the chitosan-B formulation
induced the production of phenolic compounds, such as resveratrol, which is
a stilbene, and myricetin and quercetin, which are flavonols. The other two
chitosan formulations showed a trend to increase all of these phenolic
compounds except resveratrol, but these were not significantly higher than
the control. In previous work, preharvest chitosan application enhanced the
total phenolic compounds in table grape berries (Meng et al., 2008) and
induction of resveratrol and derivatives was observed following treatment of
grapevine leaves with chitosan alone or in combination with copper sulfate
(Aziz et al., 2006). Our result corroborates the findings of Iriti et al. (2011),
in which weekly vineyard chitosan applications increased total polyphenols
in grape berries. Furthermore, the induction of phenolic compounds by
chitosan is consistent with their induction of PAL activity, a key enzyme in
phenol synthesis (Romanazzi et al., 2002; Trotel-Aziz et al., 2006; Meng et
al., 2008; Reglinski et al., 2010). Moreover, in addition to resveratrol,
myricetin and quercetin were also reported to be involved in grape defense
against pathogens such as B. cinerea (Adrian et al., 1997; Goetz et al., 1999;
Iriti et al., 2004) and Erysiphe necator (Taware et al., 2010). Taware et al.
(2010) showed myricetin and quercetin increased in grapevine leaves and
berries of grape after E. necator infection compared with the asymptomatic
organs. Iriti et al. (2004) reported benzothiadiazole improved resistance to
infection by B. cinerea and enhanced trans-resveratrol content in ‘Merlot’
145
berries from 0.4 to 0.5 mg/kg and cis-resveratrol from 0.1 to 0.2 mg/kg,
respectively. In our work, the concentration of resveratrol in berry skins was
similar and may have been sufficient to inhibit B. cinerea infections.
146
7
OVERALL CONCLUSIONS
Treatments with these compounds as alternatives to synthetic
fungicides can reduce the postharvest decay of strawberry, sweet cherry, and
table grapes. In particular, chitosan was the most promising compound, as its
effectiveness after preharvest applications was comparable to that of the
synthetic fungicides used in the control of postharvest rot of strawberry,
sweet cherry, and table grapes. Similarly, chitosan was effective when
applied at a postharvest stage, to control rot of strawberry and sweet cherry,
and it showed in vitro antimicrobial activity against the main postharvest
pathogens. The effectiveness of the commercial chitosan formulation tested
was the same as the practical grade chitosan dissolved in different acids.
However, the commercial formulation is easy to dissolve in water, and its
introduction into current agronomic practices is realistic. With table grape
berries, chitosan induced plant defense reactions, which triggered chitinase
activity, increased the concentration of phenolic compounds, and lowered the
content of hydrogen peroxide. The application of this natural biopolymer
was not detrimental for the external appearance of the treated fruit.
Benzothiadiazole showed in vitro antimicrobial activity against the
main postharvest pathogens. It was effective in reducing postharvest decay
of strawberry when applied at postharvest or at preharvest. The application
of benzothiadiazole on strawberry reduced the red tone of the fruit skin;
however, this was a relatively slight change, which did not have any
detrimental effects on the external appeal of the fruit. On sweet cherry,
benzothiadiazole was more effective when applied at the postharvest stage
than at preharvest.
Plant extracts derived from nettle and fir showed good performances
in the control of postharvest rot of strawberry and sweet cherry. In particular,
the nettle extract showed antimicrobial activity against postharvest
pathogens in vitro, and when it was applied in vivo at postharvest, it
promoted the reduction of gray and blue mold of strawberry and total rot of
sweet cherry. The fir extract was applied to strawberry and sweet cherry at
147
7 - Overall conclusions _______________________________________________
the postharvest and preharvest stages, and in all cases it reduced fruit decay.
The salts that were tested here were potassium sorbate for table
grapes and potassium bicarbonate for sweet cherry. The former was active in
reducing the gray mold that naturally develops on table grapes, but not on
inoculated B. cinerea, maybe because of the low persistence of the salt. The
resveratrol concentration of the berry skin was increased by potassium
sorbate, and there appeared to be an induction of resistance as well as
antimicrobial activity. Potassium bicarbonate was effective in reducing the
postharvest decay of sweet cherry when it was applied at concentrations
ranging from 0.4% to 2.6%, while at higher doses it produced phytotoxic
signs on the sweet cherry skins and pedicels.
Laminarin and the oligosaccharides applied at the postharvest stage
in sweet cherry reduced the postharvest rot. On strawberry, oligosaccharides
lowered the decay incidence of gray mold, but not of Rhizopus rot or blue
mold, even if they had some antimicrobial activity in vitro. Preharvest
applications of laminarin for strawberry reduced the decay of gray mold, but
not the progress of the disease over time. Phytotoxic signs were seen on
strawberry laminarin-treated leaves, maybe because of the high
concentration used, while fruit showed no negative effects.
The other resistance inducers tested were soybean lecithin and
calcium with organic acids. The former was applied with positive results at
the postharvest stage to reduce strawberry decay. The latter had in vitro
antimicrobial activity, and in vivo it was effective when applied at the
postharvest stage to control the decay of strawberry and sweet cherry.
These results can be considered preliminary for some of these tested
compounds, while they are more convincing for others, although in all cases
they are promising for the introduction of alternative compounds into current
agronomic practices. Further studies to understand their mechanisms of
action are needed, and this new knowledge should provide valuable
information to detail their more practical aspects, such as the time, frequency
and dose of their application, and their most effective formulations. Some of
these compounds are already commercially available, and so their
148
________________________________________________ 7 - Overall conclusions
application alone or in combination with synthetic fungicides is already
feasible.
These results are particularly important considering the new
directions of the European Union policies, which with Directive 128/2009
have made integrated pest management mandatory from January 1, 2014.
This Directive states that the European Member States need to take all of the
necessary measures to promote the use of products with low risk to human
health and the environment from among those available for the same pest
problem. In addition, this Directive considers that where there is the risk of
the development of pathogen resistance against a plant protection measure,
the available anti-resistance strategies should be applied to maintain the
effectiveness of the products. This can include the use of multiple pesticides
with different modes of action or the integration of several means that might
even be nonchemical.
The principles supported by the European Union are in agreement
with the Millennium Development Goals defined by the United Nations and
the FAO. Their eight targets to be reached by 2015 include the achievement
of environmental sustainability and the eradication of hunger. A reduction in
the waste of agricultural products through the use of integrated pest
management with measures that have low risk for the environment and
human health might be one of the effective ways of increasing future food
availability.
149
8
ACKNOWLEDGMENTS
First of all I would like to thank the professor Gianfranco
Romanazzi, tutor of the PhD project, for his constant availability to help me.
I am also very thankful to Dr. Lucia Landi, Dr. Sergio Murolo, and Dr.
Valeria Mancini, that helped me during the experimental trials. The professor
Romanazzi together with the all Plant Pathology research group, through
their models, transmitted me the passion for the research.
Part of the PhD project research was carried out at the USDA
station, located in Parlier, California. To me this was a great opportunity.
Above all, I would like to thanks the Dr. Joe Smilanick for his continuous
help. I am also very thankful to Mr. Kent Fjeld, Dr. Luciana Cerioni, Dr.
Dennis Margosan, Ms. Zilfina Rubio Ames, and Mr. Gabriel Verduzco for
their support with the experimental trials.
The table grapes experimentations were financed by the California
Table Grape Commission and BARD Project No. IS-4476-11. Thanks go to
Dr. Robin Borden and Dr. Lawrence Marais for the donation of OII-YS and
Armour-Zen, and to Dr. Hemant Gohil, Dr. Brodie McCarthy, Dr. Parminder
Sahota and Prof. San Liang Gu for their technical assistance. The sweet
cherry experimentations was carried out within the project “Pre and
postharvest treatments to control storage decay of sweet cherries” granted by
Marche Polytechnic University. Thanks go to Dr. Alberto Belleggia for the
assistance in the first year field trials, and to Dr. Giorgio Murri of the
Experimental farm “Pasquale Rosati”, Marche Polytechnic University for the
help in the second year field trials. The strawberry experimentations were
granted by EUBerry Project: EU FP7 KBBE 2010-4, Grant Agreement No.
265942. Thanks go to the Prof. Bruno Mezzetti, Dr. Franco Capocasa, Dr.
Jacopo Diamanti for the help in the setting of the experimental trials and to
Dr. Massimo Bastianelli, Mr. Piergiorgio Ciarlantini, Dr. Francesca Balducci,
Dr. Roberto Cappelletti, and the workers of the Marche Polytechnic
University experimental farm for their technical assistance.
150
9
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