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Postharvest Biology and Technology 75 (2013) 24–27

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

journa l h o me pa g e: www.elsev ier .com/ locate /postharvbio

esearch note

ffectiveness of postharvest treatment with chitosan and other resistancenducers in the control of storage decay of strawberry

ianfranco Romanazzi ∗, Erica Feliziani, Marilla Santini, Lucia Landiepartment of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, Via Brecce Bianche, 60131 Ancona, Italy

r t i c l e i n f o

rticle history:eceived 5 June 2012ccepted 22 July 2012

eywords:otrytis cinerearagaria × ananassaenicillium spp.

a b s t r a c t

This study compared the effectiveness of practical grade chitosan when used in solution with acetic, glu-tamic, formic and hydrochloric acids, and a water-soluble commercial chitosan formulation, in controllingpostharvest diseases of strawberry. The commercial chitosan formulation and other resistance inducersbased on benzothiadiazole, oligosaccharides, soybean lecithin, calcium and organic acids, and Abies sibir-ica and Urtica dioica extracts were also tested. The commercial chitosan formulation was as effectiveas the practical grade chitosan solutions in the control of gray mold and Rhizopus rot of strawberriesimmersed in these solutions and kept for 4 days at 20 ± 1 ◦C. Moreover, the treatment with commercial

ostharvest diseasesesistance inducershizopus stolonifer

and experimental resistance inducers reduced gray mold, Rhizopus rot and blue mold of strawberriesstored 7 days at 0 ± 1 ◦C and then exposed to 3 days shelf-life. The highest disease reduction was obtainedwith the commercial chitosan formulation, followed by benzothiadiazole, calcium and organic acids. Thecompounds that provided the best results in postharvest applications to control storage decay of straw-berries, should be tested in further trials through preharvest treatments, applied at flowering and a fewdays before harvest.

. Introduction

Strawberries are a particularly perishable fruit during posthar-est storage, susceptible to drying, mechanical injury, decay andhysiological disorders. Gray mold and Rhizopus rot are causedy Botrytis cinerea (Pers.) and Rhizopus stolonifer (Ehrenb.), respec-ively, and they are the main causes of postharvest decay oftrawberry (Fragaria × ananassa Duch.) (Maas, 1998). The infectionf the fruit by gray mold can be ascribed to an infection on the flow-rs in the field. The B. cinerea fungus remains latent underneath theepals until fruit ripening, and then close to or after harvest it canurn from a saprophyte into a parasite (Powelson, 1960). The dis-ase often starts close to the pedicel, and at times also in woundsn the fruit produced during harvest, resulting in its colonization.. cinerea can also develop at low temperatures (even at 0 ◦C), withhe consequent shortening of the length of storage and marketing.hizopus rot can spread at temperatures greater than 4–6 ◦C, and it

s more common on fruit exposed to rain in the field or grown underlastic tunnels in several rows, when located at their border. Both

f these diseases spread quickly to other fruit, a phenomenon thats known as nesting. Infections from Penicillium spp. (blue mold)nd Mucor spp. (Mucor rot) also occur occasionally (Maas, 1998).

∗ Corresponding author. Tel.: +39 071 2204336; fax: +39 071 2204856.E-mail address: g.romanazzi@univpm.it (G. Romanazzi).

925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.postharvbio.2012.07.007

© 2012 Elsevier B.V. All rights reserved.

In conventional agriculture, these diseases are usually managedby fungicide treatments that are applied around flowering, and arerepeated up to harvest, depending on the disease pressure and thepreharvest interval of the formulation. However, in organic agri-culture and after harvest, the use of synthetic fungicides is notpermitted, so there is a need for alternatives. Among these, theuse of resistance inducers has the potential for large-scale applica-tion. Resistance inducers can increase plant defenses, and at timescan also exploit their antimicrobial properties. Among the naturalcompounds, chitosan has received much interest for application inagriculture and for the food industry. Chitosan can decrease graymold and Rhizopus rot of strawberries through the reduction ofmycelial growth and spore germination, and induction of morpho-logical alterations in the causal organisms (El Ghaouth et al., 1992).Moreover, chitosan acts as a potent elicitor, to enhance plant resis-tance against pathogens (Amborabé et al., 2008). Chitosan needsto be dissolved in dilute acid solution to exploit its properties, andseveral acids can dissolve this biopolymer, the best of which areacetic, hydrochloric, glutamic and formic acids (Romanazzi et al.,2009). So far, there are no data on the effectiveness in the controlof postharvest decay of strawberry of commercial chitosan for-mulations, either alone or compared with practical grade chitosan

dissolved in dilute acids.

A number of resistance inducers are available on the markettoday. Benzothiadiazole (BTH or acibenzolar-S-methyl) is an elic-itor of systemic-acquired resistance in plants. It is a photostable

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nalog of salicylic acid, and it has proven to be effective in theanagement of gray mold of strawberry (Terry and Joyce, 2000;unoz and Moret, 2010). Oligosaccharides can also elicit plant

efenses, and their presence on host tissue can simulate the pres-nce of pathogens and activate the plant responses (Shibuya andinami, 2001). Abies sibirica and Urtica dioica extracts are available

s commercial and experimental formulations, respectively, and asith some other plant extracts, they have recently gained popular-

ty and scientific interest for their possible antimicrobial activitiesVelázquez del Valle et al., 2008; Gatto et al., 2011).

The objectives of this study were: (i) to compare the effec-iveness in the control of postharvest diseases of strawberry ofolutions obtained by dissolving practical grade chitosan in acetic,lutamic, formic and hydrochloric acids, and of the water-solubleommercial chitosan formulation and (ii) to evaluate the effective-ess of a commercial chitosan formulation, and benzothiadiazole,ligosaccharides, soybean lecithin, calcium and organic acids, andxtracts of A. sibirica and U. dioica in the control of postharvest decayf strawberries.

. Materials and methods

.1. Fruit

Trials were carried out on the strawberry cultivar ‘Camarosa’Fragaria × ananassa Duch) in commercial orchards located in the

arche region, central-eastern Italy, grown according to the stan-ards of organic agriculture. The fruit were selected for the absencef defects, uniformity in size, and degree of ripening (2/3 red on theurface) (Rosati and Cantoni, 1993), and were used for the experi-ents on the day of harvest.

.2. Resistance inducers

The effectiveness in the control of postharvest strawberry dis-ases of chitosan dissolved in different acid solutions, and of theommercial chitosan formulation was tested. Crab shell chitosanSigma Chemical Co., St. Louis, MO, USA) was ground to a fineowder in a mortar, washed with distilled water, pelleted by low-peed centrifugation, and air-dried. For experimental use, fourifferent 1% solutions (w/v) of chitosan were prepared by dis-olving the chitosan in 1% (v/v) acetic, hydrochloric, glutamic orormic acids under continuous stirring, to obtain chitosan acetate,hloride, glutamate and formate (Romanazzi et al., 2009). Whenissolved, the pH of the chitosan solution was adjusted to 5.6 using

N NaOH, and 0.05% (w/v) Triton X-100 surfactant was added tomprove the wetting properties of these solutions. A commercialhitosan-based formulation, known as Chito Plant (ChiPro GmbH,remen, Germany), was prepared by dissolving the powder (1%,/v) directly in distilled water 2 h before use. Distilled water wassed as the control.

The effectiveness of different commercial resistance inducersn the control of postharvest strawberry diseases was com-ared. These were based on chitosan (Chito Plant, 1%, w/v),ligosaccharides (Algition, Socoa Trading, Bologna, Italy; 1%, v/v),enzothiadiazole (Bion, Syngenta Crop Protection, Switzerland;.2%, w/v), calcium and organic acids (Fitocalcio, Agrisystem,amezia Terme, CZ, Italy; 1%, v/v), soybean lecithin (Xedabio, Certis,aronno, VA, Italy; 1%, v/v), an A. sibirica extract (Abies, Agritalia,illa Saviola di Motteggiana, MN, Italy; 1%, v/v) and an experimental

ormulation based on a U. dioica extract (1%, w/v). This last com-ound was obtained by infusion of U. dioica leaves in water (10%,/v) for one month, with the macerate filtered through a double

ayer of cheesecloth, and then diluted 1:10 in deionised water.

and Technology 75 (2013) 24–27 25

2.3. Treatments

Strawberries were pooled together and randomized, and thenimmersed for 10 s in a 5 L volume of the respective solutions. Straw-berries immersed in deionised water were used as the control. Afterthe treatments, the fruit were dried in air for 1 h, and then indi-vidually arranged in small plastic boxes. These were then placedin covered plastic boxes and stored for 7 days at 0 ± 1 ◦C, 95–98%RH, and then exposed to 3 days shelf-life at 20 ± 1 ◦C, 95–98% RH.Five replicates of 30 strawberries were used for each of the treat-ments. The infections which subsequently developed resulted fromnaturally occurring inoculum.

2.4. Data recording

During the storage, the percentage of decayed strawberries wasrecorded. Disease severity was also recorded according to an empir-ical scale with six degrees: 0, healthy fruit; 1, 1–20% fruit surfaceinfected; 2, 21–40% fruit surface infected; 3, 41–60% fruit surfaceinfected; 4, 61–80% fruit surface infected; 5, more than 81% of thestrawberry surface infected and showing sporulation (Romanazziet al., 2000). The empirical scale allowed the calculation of the McK-inney’s index, expressed as the weighted average of the disease as apercentage of the maximum possible level (McKinney, 1923). Thisparameter also includes information on both disease incidence anddisease severity.

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 ormore experiments were pooled, as the statistical analysis to deter-mine the homogeneity of variances was tested using Levene’s test(SPSS Inc., Chicago, IL, USA). To normalize the data, the appropriatetransformations were determined empirically using normal prob-ability plots. The arcsin of the square root of the proportion wasapplied to the decay incidence data. The values were submittedto analysis of variance and the means were separated by Duncan’sMultiple Range Test (SuperANOVA, Abacus Concepts, Inc., Berkeley,CA, USA). Actual values are shown.

3. Results and discussion

Research to reduce fungicide applications in agriculture throughthe discovery of new natural antimicrobials is needed to meet thegrowing consumer demand for food without chemical preserva-tives and to respond to the needs of sustainable farming. Due tothe nontoxic and biocompatible properties of chitosan (Wu et al.,2005), it has been considered a candidate for substitution of fungi-cides in horticultural cultivation (Bautista-Banos et al., 2006). Themain difference between the practical grade chitosan solutions andthe commercial chitosan formulation arises from the techniques oftheir preparation. Indeed, to dissolve the chitosan in the variousacids, it is necessary to prepare the solutions 2 days in advance andto monitor and adjust the pH; in contrast, the commercial chitosanformulation can be prepared only 1–2 h before application, just bydissolving the powder in water. Moreover, the resulting solutionwith the commercial chitosan formulation has a lower viscositycompared to chitosan acetate, so it can be applied more easily in thefield using standard sprayers. These details are relevant when thepractical 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 andformate, and in the commercial chitosan formulation, showed sig-nificant reduction of gray mold and Rhizopus rot decay, as well as

26 G. Romanazzi et al. / Postharvest Biology and Technology 75 (2013) 24–27

Table 1Decay, disease severity and McKinney index of gray mold and Rhizopus rot recorded on strawberries treated with solutions obtained by dissolving practical grade chitosanin acetic, glutamic, formic and hydrochloric acids, and with commercial chitosan formulation. The fruit were kept for 4 days at 20 ± 1 ◦C, 95–98% RH.

Treatment Decay (%) Disease severity (1–5) McKinney index (%)

Gray mold Rhizopus rot Gray mold Rhizopus rot Gray mold Rhizopus rot

Control 91.8 A 93.0 A 4.6 A 4.8 A 84.5 A 89.3 AChitosan acetate 37.4 B 14.1 B 3.1 B 3.7 B 23.2 B 10.4 BChitosan chloride 51.3 B 29.9 B 3.2 B 3.4 B 32.8 B 20.3 BChitosan formate 43.1 B 25.9 B 3.4 B 3.4 B 29.3 B 17.6 BChitosan glutamate 44.7 B 25.6 B 3.4 B 3.6 B 30.4 B 18.4 B

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alues with the same letter are not statistically different according to Duncan’s Mu

educed severity and McKinney index, when compared to the con-rol after 4 days shelf-life at 20 ± 1 ◦C (Table 1). The treatments withhitosan acetate, chitosan formate, the commercial chitosan, chi-osan glutammate and chitosan chloride provided McKinney indexeductions in gray mold of 73%, 65%, 64%, 64% and 61% respectively,nd of Rhizopus rot of 88%, 80%, 84%, 79% and 77% respectively, asompared to the control. However, no significant differences in dis-ase control were observed for the solutions obtained starting fromractical grade chitosan compared to the commercial chitosan for-ulation. In these trials, significant infections of blue mold were

ot observed.The treatment of the strawberry slices with chitosan acetate

ignificantly decreased hydrogen peroxide production at 2, 4 and h after treatment, as compared to the untreated control (dataot shown). Chitosan solutions have antioxidant capacity, likeydrogen peroxide scavengers, and the use of chitosan as an antiox-

dant and anti-browning agent is widespread in the food industryDevlieghere et al., 2004). The oxygen radicals scavenging capaci-ies, the levels of phenylpropanoid compounds, and the antioxidantnzyme activity increased in strawberries after the treatment withhitosan (Wang and Gao, in press).

On strawberries cold-stored 7 days (0 ± 1 ◦C) and then exposedo 3 days shelf-life (20 ± 1 ◦C), the reductions, as compared to theontrol, in the McKinney index for gray mold were 79%, 73%,0%, 63%, 60%, 56% and 46% for the fruit treated with com-ercial chitosan, benzothiadiazole, calcium with organic acids,

ligosaccharides, Abies extract, soybean lecithin, and Urtica extract,espectively and for blue mold were 90%, 84%, 71%, 61%, 59% and1% for the fruit treated with commercial chitosan, benzothiadia-ole, calcium with organic acids, Abies extract, Urtica extract andligosaccharides, respectively. Only treatments with chitosan andalcium with organic acids reduced the McKinney Index of Rhizo-us rot, respectively of 84% and 79%, as compared to the control

Table 2).

Chitosan has a dual effect on host–pathogen interactionshrough its antifungal activity and its ability to induce plant defenseesponses (Romanazzi, 2010). Moreover, as chitosan can form an

able 2ecay, severity and McKinney index of gray mold, Rhizopus rot and blue mold recorded o

ruit were stored for 7 days at 0 ± 1 ◦C, 95–98% RH, followed by 3 days of shelf life at 20 ±

Treatment Decay (%) Disease sever

Gray mold Rhizopus rot Blue mold Gray mold

Control 63.5 a 48.9 a 56.9 a 4.2 a

Abies extract 29.8 bc 36.2 ab 28.3 bc 2.2 c

Oligosaccharides 29.0 bc 36.3 ab 40.4 ab 3.4 ab

Benzothiadiazole 25.1 c 20.8 ab 12.6 cd 2.9 bc

Chitosan 20.4 c 8.6 b 4.8 d 2.7 bc

Ca-organic acids 23.5 c 12.7 ab 28.5 bc 3.4 b

Urtica extract 44.6 b 24.2 ab 28.2 bc 2.9 bc

Soybean lecithin 36.8 bc n.d.a n.d.a 3.2 b

alues with the same letter are not statistically different according to Duncan’s Multiple

a Disease not developed in the trials in which the compound was used.

B 3.6 B 30.1 B 13.9 B

Range Test at p < 0.01.

edible film when applied to the surface of fruit and vegetables,it is clearly effective in conferring a physical barrier to moistureloss, delaying dehydration and fruit shriveling. Therefore, its coat-ing can prolong storage life, delay the drop in sensory quality,and control the decay of strawberry fruit (Han et al., 2004; Parket al., 2005; Chaiprasart et al., 2006; Hernández-Munoz et al., 2006;Ribeiro et al., 2007). Chitosan coating can be used as a vehiclefor incorporating functional ingredients, such as antimicrobials ornutraceutical compounds that could enhance the effects of chi-tosan coating or reinforce the nutritional value of the strawberries(Vargas et al., 2006; Vu et al., 2011; Perdones et al., 2012). Pos-itive effects of treatment with practical grade chitosan coatingon the decay of strawberries artificially inoculated with B. cinereaand R. stolonifer and held at 13 ◦C have been shown (El Ghaouthet al., 1992). Preharvest sprays of practical grade chitosan signif-icantly reduced postharvest fungal rot of strawberries stored at3 ◦C and 13 ◦C and maintained the quality of the fruit comparedto the control (Reddy et al., 2000). In the same way, preharvestand postharvest treatments with practical grade chitosan on straw-berries reduced the postharvest gray mold and Rhizopus rot afterstorage at 0 ± 1 ◦C followed by shelf-life at 20 ± 1 ◦C (Romanazziet al., 2000).

Benzothiadiazole is a functional analog of salicylic acid and anacquired systemic resistance activator that can elicit activation ofgenes involved in plant defense and pathogenesis-related proteins(Lawton et al., 1996; Vallad and Goodman, 2004). Our results arein agreement with Terry and Joyce (2000), who reported the pos-sibility to delay the development of gray mold on strawberry fruitheld at 5 ◦C by about 1.2-fold, through single or multiple preharvestfoliar treatments at anthesis with benzothiadiazole, with no phy-totoxic effects seen for either fruit or plant. Postharvest treatmentof strawberries with benzothiadiazole induced disease resistanceby enhancing fruit antioxidant systems and free radical-scavenging

capabilities (Cao et al., 2011).

In the formulation where composition is based on calcium andorganic acids, the calcium reinforces the structural composition ofthe plant cell wall through the binding of pectins with salts, and

n strawberries treated with commercial and experimental resistance inducers. The1 ◦C, 95–98% RH.

ity (1–5) McKinney index (%)

Rhizopus rot Blue mold Gray mold Rhizopus rot Blue mold

3.8 a 3.8 a 53.3 a 44.8 a 40.8 a3.8 a 2.9 abc 21.2 bc 29.6 ab 16.0 bc2.5 a 3.4 ab 19.7 bc 32.8 ab 28.0 b2.2 a 1.6 bc 14.6 bc 15.2 ab 6.4 c1.8 a 1.0 c 11.1 c 7.2 b 4.0 c1.6 a 2.2 abc 16.0 bc 9.6 b 12.0 c2.3 a 1.9 abc 27.9 b 13.6 ab 16.8 bcn.d.a n.d.a 23.6 bc n.d.a n.d.a

Range Test at p < 0.05.

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herefore provides more resistance during the manipulation andransport of fruit. Calcium is one of the most widely used treatmentlternatives to fungicides with table grapes, with the aim to protecthe berries from preharvest and postharvest gray mold, and it issed in both organic and conventional agriculture (Romanazzi et al.,012).

When oligosaccharides are applied to plants, these can sim-late the presence of a pathogen and thus induce plant defenseesponses (Chisholm et al., 2006). These compounds derived fromhe degradation of plant cell-wall polysaccharides are one class ofell characterized elicitors that, in some cases, can induce defense

esponses at very low concentrations (Shibuya and Minami, 2001).In the present study, the application of soybean lecithin was

ested as a resistance inducer. Hoa and Ducamp (2008) reportedhat treatments with soybean lecithin delayed mango ripening dur-ng storage at ambient temperatures, thus slowing the changes ofhe biochemical ripening indicators. Lecithin could also work as anntioxidant, since hydrogen peroxide content in strawberry tissuesreated with lecithin was reduced compared to the control (data nothown). In the food industry, soybean lecithin is normally used as aatural and non-toxic compound with antioxidant properties, and

t is approved by the United States Food and Drug Administrationor human consumption, with the status of “Generally Recognizeds Safe”.

Over the last few years, there has been increasing interest foresearch into plant extracts for their antimicrobial actions and theirafe application (Gatto et al., 2011). The activity of the A. sibir-ca extract could be due to its triterpene acids, which act as plantrowth regulators and facilitate cell division and shoot regenera-ion (Korolev et al., 2003). From the present study, the A. sibiricaxtract appears to have good antimicrobial activity. The U. dioicaxtract has also been shown to have antimicrobial and antioxidantffects (Gülc in et al., 2004), and here we show its antimicrobialroperties, as it reduced gray and blue molds. Moreover, this U.ioica extract is used by organic farmers, who claim that they canchieve a reduction in aphid numbers.

Among these formulations tested, the commercial chitosanormulation and benzothiadiazole provided the highest diseaseeduction, which indicates their possible application in IPM.esistance inducers also have the advantage of triggering wide-pectrum resistance, for activity against several classes of plantathogen and pest (Inbar et al., 1998). However, further studiesre needed to better understand the mechanisms of action of theseesistance inducers, and the appreciation from the consumers ofreated fruit.

cknowledgement

The research was granted by EUBerry Project: EU FP7 KBBE010-4, Grant Agreement No. 265942.

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