alternatives to synthetic fungicides to control postharvest diseases of

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

RIASSUNTO ............................................................................................. 8 ABSTRACT .............................................................................................. 9 1 INTRODUCTION .................................................................................. 10 2 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

3 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 4 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 Chitinase activity ................................................................. 125 6.2.6 Hydrogen peroxide content .................................................. 126 6.2.7 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

8

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.

9

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.

10

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,

______________________________________________________ 1 - Introduction

11

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.

1 - Introduction ______________________________________________________

12

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


Rhizopus rot Rhizopus spp.

Black rot Aspergillus niger

Stone fruit Brown rot Monilinia spp.

Gray mold Botrytis cinerea


Rhizopus rot Rhizopus stolonifer

Alternaria rot Alternaria alternata

Pome fruit Blue mold Penicillium spp.

Gray mold Botrytis cinerea

Brown rot Monilinia spp.

Alternaria rot Alternaria alternata

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

______________________________________________________ 1 - Introduction

13

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

1 - Introduction ______________________________________________________

14

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).

15

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

2 - Prolonged storage and shelf life extension of fresh fruit and vegetable by

chitosan treatment ___________________________________________________

16

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

2 - Prolonged storage and shelf life extension of fresh fruits and vegetables by

___________________________________________________ chitosan treatment

17

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).



18

Table 2. Chitosan based commercial products available for the control of

postharvest decay in fruit and vegetables.

Product

name

Company

(Country)

Formulation a.i.

(%)

Fruit Reference

Chito

Plant

ChiPro

GmbH

(Bremen,

Germany)

Powder 99.9 Table

grapes,

sweet

cherry,

strawberry

Feliziani et al.,

2013a, 2013b;

Romanazzi et

al., 2013

OII-YS Venture

Innovations

(Lafayette,

LA, USA)

Liquid 5.8 Table

grapes

Feliziani et al.,

2013b

Armour-

Zen

Botry-Zen

Limited

(Dunedin,

New Zealand)

Liquid 14.4 Peach,

table

grapes

Casals et al.,

2012; Feliziani

et al., 2013b

Biorend Bioagro S.A.

(Chile)

Liquid 1.25 Clementine

mandarin

fruit

Fornes et al.,

2005

FreshSeal BASF

Corporation

(Mount

Olive, NJ,

USA)

Liquid - Banana Baez-Sañudo et

al., 2009

ChitoClear Primex ehf

(Siglufjordur,

Iceland)

Powder 100 Rambutan

fruit

Martínez-

Castellanos et

al., 2009

Bioshield Seafresh

(Bangkok,

Thailand)

Powder 100 Mango Jitarrerat et al.,

2011

Biochikol

020 PC

Gumitex

(Lowics,

Poland)

Liquid 2 Potato Kurzawińska

and Mazur, 2007

2 - Prolonged storage and shelf life extension of fresh fruits and vegetables by


19

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.



20

Table 3. Chitosan treatments with other applications on storage decay of temperate

fruit.

Produce Decay Integration to

chitosan

References

(moment of

application)

Table grapes Gray mold - Romanazzi et al., 2002

(pre and postharvest)

Acid solutions Romanazzi et al., 2009

(postharvest)

Ethanol Romanazzi et al., 2007

(postharvest)

Grape seed extract Xu et al., 2007b

(postharvest)

Gray mold and

blue mold

UV Romanazzi et al., 2006

(preharvest)

Decay (in general) Cryptococcus

laurentii

Meng and Tian, 2009

(preharvest); 2010b

(postharvest)

Strawberry Gray mold - El Ghaouth et al.,

1991a, 1992a

(postharvest); Zhang

and Quantick, 1998

(postharvest);

Romanazzi et al., 2000

(pre and postraccolta);

Reddy et al., 2000a

(preharvest); Mazaro et

al., 2008 (preharvest)

Lemon essential

oil

Perdones et al., 2012

(postharvest)

Red thyme,

oregano extract,

limonene,

peppermint

Vu et al., 2011

(postharvest)



21


chitosan

References

(moment of

application)

Rhizopus rot - El Ghaouth et al., 1992a

(postharvest); Zhang

and Quantick, 1998

(postharvest);


(pre and postharvest);

Park et al., 2005

(postharvest)

Cladosporium rot - Park et al., 2005

(postharvest)

Decay (in general) Calcium lactate,

calcium

gluconate, vitamin

E

Han et al., 2004

(postharvest)

Calcium

gluconate

Hernández-Muñoz et

al., 2006 (postharvest);

2008 (postharvest)

Oleic acid Vargas et al., 2006

(postharvest)

Raspberry Decay (in general) Calcium lactate,

calcium

gluconate, vitamin

E

Han et al., 2004

(postharvest)

Gray mold and

Rhizopus rot

- Zhang and Quantick,

1998 (postharvest)

Blueberry Decay (in general) - Duan et al., 2011

(postharvest)

Apple Blue mold

UV-C, Candida

satoiana, harpin

De Capdeville et al.,

2002 (postharvest)

Cryptococcus Yu et al., 2007



22


chitosan

References

(moment of

application)

laurentii (postharvest)

Candida satoiana El Ghaouth et al., 2000

(postharvest)

Heat treatment Shao et al., 2012

(postharvest)

Gray mold

Candida satoiana El Ghaouth et al., 2000

(postharvest)

Heat treatment Shao et al., 2012

(postharvest)

Pear Blue mold Calcium chloride,

Cryptococcus

laurentii

Yu et al., 2012

(postharvest)

Peach Brown rot - Li and Yu, 2000

(postharvest)

Heat treatment Casals et al., 2012

(postharvest)

Sweet cherry Decay (in general) Hypobaric

treatment


(pre and postharvest)

- Romanazzi et al., 1999

(preharvest); Feliziani

et al., 2013a (pre and

postharvest)

Orange Blue mold Bergamot, thyme,

tea tree essential

oil

Cháfer et al., 2012

(postharvest)

Black spot disease - Canale Rappussi et al.,

2009 (postharvest);

2011 (postharvest)

Tankan citrus

fruit

Decay (in general) - Chien and Chou, 2006

(postharvest)

Clementine

mandarin fruit

Decay (in general) - Fornes et al., 2005 (pre

or postharvest)



23

Table 4. Chitosan treatments with other applications on storage decay of tropical

fruit.


chitosan

References

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

papaya seeds

Bautista-Baños et al.,

2003 (postharvest)

Ammonium

carbonate, sodium

bicarbonate

Sivakumar et al., 2005b

(postharvest)

Dragon

fruit

Anthracnose - Zahid et al., 2012

(postharvest)

Litchi fruit Blue mold and

Cladosporium rot

Potassium

metabisulphite

Sivakumar et al., 2005a

(postharvest)

Longan

fruit

Decay (in

general)

Jiang and Li, 2001

(postharvest)



24

Table 5. Chitosan treatments with other applications on storage decay of

vegetables.


chitosan

References

Tomato

Gray mold El Ghaouth et al., 1992b

(postharvest); Badawy and

Rabea, 2009 (postharvest)

Gray mold

and blue mold

Liu et al., 2007 (postharvest)

Blackmold rot Reddy et al., 2000b

(postharvest)

Sweet

pepper

Decay (in

general)

Cinnamon oil Xing et al., 2011a (postharvest)

Melon Fusarium rot

and black rot

Natamycin Cong et al., 2007 (postharvest)

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



25

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



26

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



27

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 Bravo-

Luna, 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,



28

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,



29

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.



30

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



31

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 L-

ascorbic 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).



32

Table 6. Growth inhibition by chitosan on decay-causing fungi affecting produce

during storage.

Fungus Infected species Reference

Alternaria alternata 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

pepper, cucumber,

peach, strawberries,

table grapes, pear,

apple, Tankan citrus

fruit

El Ghaouth et al., 1992a;

1992b; 1997; 2000; Du et al.,

1997; Romanazzi et al., 2002;

Ben-Shalom et al., 2003; Ait

Barka et al., 2004; Badawy et

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,

strawberry

Park et al., 2005; Sivakumar et

al., 2005a

Colletotrichum

gloeosporioides

Mango, papaya Bautista Baños et al., 2003;

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

tomato

Muñoz et al., 2009

Fusarium solani - Eweis et al., 2006



33

Fungus Infected species Reference

Fusarium sulphureum Potato Li et al., 2009

Fusarium spp. Banana Win et al., 2007

Geotricum candidum El-Mougy et al., 2012

Guignardia citricarpa Orange Canale Rappussi et al., 2009;

2011

Lasiodiplodia

theobromae

Banana Win et al., 2007

Monilinia fructicola Apple, peach Yang et al., 2010; 2012a

Monilinia laxa Sweet cherry Feliziani et al., 2013a

Penicillium citrinum Jujube Xing et al., 2011b

Penicillium digitatum Orange, lemon,

Tankan citrus fruit

El Ghaouth et al., 2000;

Bautista-Baños et al., 2004;

Chien and Chou, 2006; El-

Mougy et al., 2012

Penicillium expansum Litchi fruit,

strawberries, apple,

pear, tomato

El Ghaouth et al., 2000;

Sivakumar et al., 2005a; Liu et

al., 2007; Yu et al., 2007

Penicillium italicum Tankan citrus fruit Chien and Chou, 2006; El-

Mougy et al., 2012

Penicillium stolonifer Pear Cé et al., 2012

Phytophthora cactorum Strawberries Eikemo et al., 2003

Physalospora piricola Pear Meng et al., 2010a

Rhizopus stolonifer Peach, strawberries,

papaya, tomato

El Ghaouth et al., 1992b;

Bautista-Baños et al., 2004;

Park et al., 2005; Guerra-

Sánchez et al., 2009; García

Rincón et al., 2010; Hernández-

Lauzardo et al., 2010; Ramos

García et al., 2012

Sclerotinia sclerotiorum Carrot Cheah et al., 1997; Molloy et

al., 2004



34

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 (Palma-

Guerrero 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



35

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



36

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 (Bautista-

Baños 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 chitosan-

loaded 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



37

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.



38

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).



39

Table 7. Physiological changes occurring in temperate fruit after chitosan treatment.

Temperate

fruit

Physiological change Integration to

chitosan

References

Table grapes Phenilalanine ammonia-

lyase

- Romanazzi et

al., 2002; Meng

et al., 2008

Cryptococcus

laurentii

Meng and Tian,

2009; Meng et

al., 2010b

Peroxidase - Meng et al.,

2008

Polyphenol oxidase,

superoxide dismutase

- Meng et al.,

2008

Cryptococcus

laurentii

Meng and Tian,

2009; Meng et

al., 2010b

Chitinase, myricetin - Feliziani et al.,

2013b

Quercetin - Feliziani et al.,

2013b

Putrescine Shiri et al.,

2013

Respiration Bergamot oil Sánchez-

González et al.,

2011

Resveratrol UV Romanazzi et

al., 2006

- Feliziani et al.,

2013b

Soluble solids content - Meng et al.,

2008

Bergamot oil Sánchez-

González et al.,

2011



40

Temperate

fruit


chitosan

References

Cryptococcus

laurentii

Meng et al.,

2010b

Titratable acidity - Meng et al.,

2008

Total phenolic content - Meng et al.,

2008

Cryptococcus

laurentii

Meng et al.,

2010b


2013

Weight loss, color,

texture

Bergamot oil Sánchez-

González et al.,

2011


2013


Shattering and cracking Putrescine Shiri et al.,

2013


Strawberry Titratable acidity - El Ghaouth et

al., 1991a;

Zhang and

Quantick, 1998;

Reddy et al.,

2000a

Vitamin E Han et al.,

2004; 2005;

Calcium gluconate Hernández-

Muñoz et al.,

2008;

pH Calcium gluconate Hernández-

Muñoz et al.,



41

Temperate

fruit


chitosan

References

2008;

Vitamin E Han et al., 2004

Antocyanin content - El Ghaouth et

al., 1991a;

Zhang and

Quantick, 1998;

Reddy et al.,

2000a

Oleic acid Vargas et al.,

2006

Total polyphenol - Kerch et al.,

2011

Soluble solids content Vitamin E Han et al., 2005

Color Calcium gluconate Hernández-

Muñoz et al.,

2008

Vitamin E Han et al.,

2004; 2005

Firmness Calcium gluconate Hernández-

Muñoz et al.,

2008

- El Ghaouth et

al., 1991a

Vitamin C content - Zhang and

Quantick, 1998;

Kerch et al.,

2011; Wang and

Gao, 2013

Glutathione - Wang and Gao,

2013

Chitinase, β-1,3

glucanase

- Zhang and

Quantick, 1998



42

Temperate

fruit


chitosan

References

Phenilalanine ammonia-

lyase

- Romanazzi et

al., 2000

Weight loss Vitamin E Han et al., 2004

Respiration - El Ghaouth et

al., 1991a;

Vargas et al.,

2006

Catalase, glutathione-

peroxidase, guaiacol

peroxidase,

dehydroascorbate

reductase,

monodehydroascorbate

reductase

- Wang and Gao,

2013

Raspberry Weight loss, color, pH,

titratable acidity

Vitamin E Han et al., 2004

Ascorbic acid, titratable

acidity, firmness,

antocyanin content

- Zhang and

Quantick, 1998

Apple Respiration, firmness,

weight loss, titratable

acidity

Heat Shao et al.,

2012

Pear Polyphenol oxidase,

chitinase, β-1,3

glucanase,

- Meng et al.,

2010a

ROS, catalase,

superoxide dismutase,

ascorbate peroxidase,

glutathione reductase

Li et al., 2010

Peroxidase - Meng et al.,

2010a; Li et al.,

2010

Respiration, - Zhou et al.,



43

Temperate

fruit


chitosan

References

permeability of cell

membrane, weight loss

2008

Ascorbic acid Lin et al., 2008

Soluble solid contents,

titratable acidity,

firmness

Ascorbic acid Lin et al., 2008

Apricot Total phenolic content,

antioxidant activity,

weight loss

- Ghasemnezhad

et al., 2010

Peach Titratable acidity,

ascorbic acid,

respiration, firmness,

ethylene and

malondialdehyde

production, superoxide

dismutase

- Li and Yu, 2000

Polyphenol oxidase,

peroxidase, ascorbic

acid oxidase,

polygalacturonase,

vitamin C

CaCl2 coating +

PEpackage +

intermittent warming

Ruoyi et al.,

2005

Sweet cherry Titratable acidity,

soluble solid, catalase,

peroxidase, polyphenol

oxidase, phenylalanine

ammonia-lyase,

respiration

- Dang et al.,

2010

Ascorbic acid - Dang et al.,

2010; Kerch et

al., 2011

Phenols content,

antocyanin content

- Kerch et al.,

2011

Orange Water loss, firmness Bergamot, thyme, tea Cháfer et al.,



44

Temperate

fruit


chitosan

References

tree essential oil 2012

Color - Canale

Rappussi et al.,

2011

Chitinase, b-1,3-

glucanase, polyphenol

oxidase

- Canale

Rappussi et al.,

2009

Peroxidase - Canale

Rappussi et al.,

2009; Zeng et

al., 2010

Superoxide dismutase,

catalase, ascorbate

peroxidase, glutathione

reductase, hydrogen

peroxide content,

ascorbate content

- Zeng et al.,

2010

Tankan

citrus fruit

Firmness, weight loss,

titratable acidity,

ascorbic acid, soluble

solids

- Chien and

Chou, 2006

Jujube Polyphenol oxidase,

phenolic compounds

- Xing et al.,

2011b

Zinc, cerium Wu et al., 2010

Ascorbic acid - Xing et al.,

2011b

1-

methylcyclopropene

Qiuping and

Wenshui, 2007

Firmness 1-

methylcyclopropene

Qiuping and

Wenshui, 2007

Weight loss 1-

methylcyclopropene

Qiuping and

Wenshui, 2007




45

Temperate

fruit


chitosan

References

Respiration, soluble

solids


Table 8. Physiological changes occurring in tropical fruit after chitosan treatment.

Tropical

fruit

Physiological

changes

Integration to

chitosan

References

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


2011

Firmness, soluble

solids content

- Kittur et al., 2001;

Win et al., 2007


2009


2010a, 2010b; 2011

Color - Kittur et al., 2001;

Win et al., 2007


2009


2011

Weight loss Arabic gum Maqbool et al.,

2010a, 2010b; 2011

Longan Respiration, weight - Jiang and Li, 2001



46

Tropical

fruit

Physiological

changes

Integration to

chitosan

References

fruit loss, color change,

polyphenol oxidase,

titratable acidity,

total soluble solids,

ascorbic acid

Mango Titratable acidity,

weight loss

- Jitareerat et al.,

2007; Zhu et al.,

2008

Hydrothermal process Salvador-Figueroa

et al., 2011

Total soluble solids,

firmness, color

change

- Zhu et al., 2008

Hydrothermal process Salvador-Figueroa

et al., 2011

pH Hydrothermal process Salvador-Figueroa

et al., 2011

Chitinase, b-1,3-

glucanase


2007

Respiration,

ascorbic acid


2007; Zhu et al.,

2008

Papaya Titratable acidity,

total soluble solids

- Ali et al., 2010;

2011

Calcium infiltration Al Eryani et al.,

2008

Ascorbic acid - Ali et al., 2011


2008

Weight loss, color - Ali et al., 2011

Ammonium carbonate,

sodium bicarbonate

Sivakumar et al.,

2005b


2008

Firmness - Ali et al., 2010;



47

Tropical

fruit

Physiological

changes

Integration to

chitosan

References

2011

Ammonium carbonate,

sodium bicarbonate

Sivakumar et al.,

2005b

Aqueous extract of

papaya seeds

Bautista-Baños et

al., 2003

Chitinase, b-1,3-

glucanase

- Hewajulige et al.,

2009

Respiration Hewajulige et al.,

2009, Ali et al.,

2011

Litchi fruit Weight loss - Zhang and

Quantick, 1997;

Jiang and Li, 2001;

Sivakumar et al.,

2005a; Sun et al.,

2010; Lin et al.,

2011

Organic acids Joas et al., 2005;

Caro and Joas,

2005

Titratable acidity - Jiang et al., 2005;

Sivakumar et al.,

2005a; Sun et al.,

2010

Organic acids Joas et al., 2005;

Caro and Joas,

2005

Total phenolic

content, flavonoid

content

- Zhang and

Quantick, 1997;

Sivakumar et al.,

2005a

Anthocyanin - Zhang and



48

Tropical

fruit

Physiological

changes

Integration to

chitosan

References

content Quantick, 1997;

Jiang et al., 2005;

Sivakumar et al.,

2005a;

Modified atmosphere

packaging

De Reuck et al.,

2009

Respiration - Lin et al., 2011

Color - Zhang and

Quantick, 1997;

Ducamp-Collin et

al., 2008

Organic acids Caro and Joas,

2005; Joas et al.,

2005

Ascorbic acid Sun et al., 2010

Modified atmosphere

packaging

De Reuck et al.,

2009

Total soluble solid - Jiang et al., 2005


Peroxidase - Zhang and

Quantick, 1997;

Dong et al., 2004


Modified atmosphere

packaging

De Reuck et al.,

2009

Polyphenol oxidase - Zhang and

Quantick, 1997;

Jiang et al., 2005;

Lin et al., 2011


Modified atmosphere

packaging

De Reuck et al.,

2009

Superoxide Ascorbic acid Sun et al., 2010



49

Tropical

fruit

Physiological

changes

Integration to

chitosan

References

dismutase, catalase,

hydrogen peroxide,

malondialdehyde;

ascorbic acid

content

Rambutan Firmness, soluble

solid, titratable

acidity

Lactobacillus

plantatum

Martínez-

Castellanos et al.,

2009

Guava Firmness,

peroxidase

superoxide

dismutase, catalase,

inhibition of

superoxide free

radical production,

titratable acidity,

ascorbic acid,

weight loss, soluble

solids, chlorophyll

and

malondialdehyde

content

- Hong et al., 2012



50

Table 9. Physiological changes occurring in vegetables after chitosan treatment.

Vegetables Physiological changes Integration

to chitosan

References

Tomato Respiration, color, ethylene,

firmness, titratable acidity

- El Ghaouth et al.,

1992b

Polyphenol oxidase, phenolic

content

- Liu et al., 2007;

Badawy and Rabea,

2009

Peroxidase - Liu et al., 2007

Protein content - Badawy and Rabea,

2009

Polygalacturonase, pectate

lyase, cellulose, phytoalexin

production, pH

- Reddy et al., 2000b

Potato Peroxidase, polyphenol

oxidase, flavonoid content,

lignin content

- Sun et al., 2008

Phenylalanine ammonia-lyase - Gerasimova et al.,

2005

Sweet

pepper

Superoxide dismutase,

peroxidase, catalase

Cinnamon

oil

Xing et al., 2011a

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



51

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



52

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



53

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 (Rice-

Evans 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



54

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



55

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



56

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).



57

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



58

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, chitosan-

coated 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.



59

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



60

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



61

(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; Salvador-

Figueroa 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



62

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



63

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



64

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



65

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

growth

Integration to

chitosan

References

Escherichia coli Tomato - Inatsu et al., 2010

Tomato Beeswax + lime

essential oil

Ramos-García et

al., 2012

Salmonella spp. Whole

cantaloupe

Allyl

isothiocyanate,

nisin

Chen et al., 2012

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



66

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;



67

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,



68

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.



69

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 gram-

positive (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 Al-



70

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 non-

toxic 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 (Valencia-

Chamorro 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



71

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.

72

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.

3 - Effectiveness of postharvest treatment with chitosan and other resistance

_______________________ inducers in the control of storage decay of strawberry

73

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


inducers in the control of storage decay of strawberry _______________________

74

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.



75

3.2 Materials and methods

3.2.1 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



76

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.,



77

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



78

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

(1-5)

McKinney Index

(%)

Gray

mold

Rhizopus

rot

Gray

mold

Rhizopus

rot

Gray

mold

Rhizopus

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 aValues 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%



79

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).



80

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

mold

Rhizopus

rot

Blue

mold

Gray

mold

Rhizopus

rot

Blue

mold

Gray

mold

Rhizopus

rot

Blue

mold

Control 63.5aa 48.9a 56.9a 4.2a 3.8a 3.8a 53.3a 44.8a 40.8a

Fir extract 29.8bc 36.2ab 28.3bc 2.2c 3.8a 2.9abc 21.2bc 29.6ab 16.0bc

Oligosaccharides 29.0bc 36.3ab 40.4ab 3.4ab 2.5a 3.4ab 19.7bc 32.8ab 28.0b

Benzothiadiazole 25.1c 20.8ab 12.6cd 2.9bc 2.2a 1.6bc 14.6bc 15.2ab 6.4c

Chitosan 20.4c 8.6b 4.8d 2.7bc 1.8a 1.0c 11.1c 7.2b 4.0c

Ca-organic acids 23.5c 12.7ab 28.5bc 3.4b 1.6a 2.2abc 16.0bc 9.6b 12.0c

Nettle extract 44.6b 24.2ab 28.2bc 2.9bc 2.3a 1.9abc 27.9b 13.6ab 16.8bc

Soybean lecithin 36.8bc n.d.* n.d.* 3.2b n.d.* n.d.* 23.6bc n.d.* n.d.* aValues 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.



81

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



82

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



83

consumers of fruits treated with these resistance inducers needs to be

investigated.

84

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

4 - Preharvest treatments with alternatives to synthetic fungicides to prolong shelf

_________________________________________________ life of strawberry fruit

85

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,

4 - Preharvest treatments with alternatives to synthetic fungicides to prolong

shelf life of strawberry fruit _________________________________________

86

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.



87


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



88

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



89

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



90

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 Results

4.3.1 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).



91

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 aValues 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).



92

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.

0

5

10

15

20

25

30

35

40

45

1 2 3 4

Dec

ay

(%

)

Days of shelf life

Control

Chitosan 0.5 %

Chitosan 1%

Laminarin

Fir extract

Benzothiadiazole

Fungicides



93

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

a

b b

ab

ab

ab

b

0

10

20

30

40

50

60

AU

DP

C



94

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 aValues followed by unlike letters are significantly different according to Tukey’s

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).



95

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.

0

10

20

30

40

50

60

70

1 2 3

Dec

ay

(%

)

Days of shelf life

Control

Chitosan 0.5 %

Chitosan 1%

Laminarin

Fir extract

Benzothiadiazole

Fungicides



96

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*

a

abc

bc

ab

abc abc

c

0

5

10

15

20

25

30

35

40

45

AU

DP

C



97

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 Firmness

a

Control 37.3±0.4 abb 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 Values followed by unlike letters are significantly different according to Tukey’s

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



98

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



99

(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.



100

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



101

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.

102

5 PRE AND POSTHARVEST TREATMENT WITH

ALTERNATIVES TO SYNTHETIC 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.

5 - Pre and postharvest treatment with alternatives to synthetic fungicides to control

_______________________________________ postharvest decay of sweet cherry

103

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


postharvest decay of sweet cherry _______________________________________

104

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).



105


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 10-

fold. 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%),



106

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.


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



107

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).



108


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 Results

5.3.1 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).



109

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)

Treatment Monilinia

laxa

Botrytis

cinerea

Rhizopus

stolonifer

Alternaria

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 0 e 9 e 31 e 0 d

Calcium plus organic acids 11 d 49 c 71 b 22 ab

Nettle extract 18 c 32 d 57 c 18 b

Fenhexamid 0 e 0 f 18 f 0 d aValues 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).



110

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 (%)

Treatment Brown

rot

Gray

mold

Rhizopus

rot

Alternaria

rot

Total

rota

Control 24.6 ab 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 aTotal rot includes brown rot, gray mold, Rhizopus rot, Alternaria rot, blue mold and

green rot. bValues followed by different letters are significantly different within columns,


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 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 18).



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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. McKinney infection index (%)

Treatment (%) Brown

rot

Gray

mold

Total

rota

Control 44.9 ab 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

Potassium bicarbonate (0.9) 15.8 b 14.4 bc 25.1 cde

Potassium bicarbonate (1.7) 12.4 b 13.6 bc 22.7 cde

Potassium bicarbonate (2.6) 11.1 b 5.1 c 16.7 e

Potassium bicarbonate (3.4) 23.6 ab 14.9 bc 36.9 bc

Potassium bicarbonate (4.3) 28.4 ab 18.0 bc 44.2 b aTotal rot includes brown rot, gray mold, Rhizopus rot, Alternaria rot, blue mold and



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



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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.


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.



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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.


Treatment Brown

rot

Gray

mold

Rhizopus

rot

Alternaria

rot

Total

rota

Control 13.6 ab 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 aTotal rot includes brown rot, gray mold, Rhizopus rot, Alternaria rot, blue mold and



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.


Treatment Brown

rot

Total

rota

Control 25.0 ab 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 aTotal rot includes brown rot, gray mold, Rhizopus rot, Alternaria rot, blue mold and



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



115

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



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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



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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



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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.

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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

6 - Preharvest fungicide, potassium sorbate, or chitosan use on quality and storage

decay of table grapes _________________________________________________

120

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


_________________________________________________ decay of table grapes

121

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 Material and methods

6.2.1 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



122

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,



123

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



124

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



125

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) 4-

Methylumbelliferyl β-D-N,N′,N′′-triacetylchitotriose which is an



126

endochitinase substrate; ii) 4-Methylumbelliferyl N-acetyl-β-D-

glucosaminide; 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



127

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 6-

well 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



128

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 (SPD-

M10 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



129

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)



130

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.


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,



131

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).



132

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 Alternaria spp. and Penicillium spp.

b Values followed by unlike letters are significantly different according to Tukey’s

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.



133

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).

Control Fungicides K sorbate Chitosan-A Chitosan-B Chitosan-C0

5

10

15

20

25

30

35

d

aab

bca B

Dec

ay

(%

± S

D)

0

5

10

15

20

25

30

35

c

ab

cd

bc

ab

d

aA

McK

inn

ey I

nd

ex (

% ±

SD

)



134

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.



135

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 Values followed by unlike letters are significantly different according to Tukey’s

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.



136

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 Exochitinase

b Exochitinase

c

Control 13.7 ed 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-D-

glucosaminide c Exochitinase activity determined using 4-methylumbelliferyl-N,N’-diacetyl-β-D-

chitobiose 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.



137

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

Control 6.5 17.1 bca 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 X-

ray 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.



138

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).

60

70

80

90

100

110

120

aa

ab

b

c

c

FungicidesK sorbateControlChitosan-CChitosan-BChitosan-A

H2O

2 c

on

ten

t (%

of

the

con

trol)



139

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.



140

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.



141

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 (mg-

kg-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 Alternaria spp. and Penicillium spp.

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



142

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.



143

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



144

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 (Trotel-

Aziz 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



145

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’



146

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.

147

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

7 - Overall conclusions _______________________________________________

148

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

________________________________________________ 7 - Overall conclusions

149

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.

150

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.

151

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