Accepted Manuscript Short communication Effect of chitosan and its derivatives as antifungal and preservative agents on postharvest green asparagus Miao Qiu, Chu Wu, Gerui Ren, Xinle Liang, Xiangyang Wang, Jianying Huang PII: DOI: Reference:

S0308-8146(14)00047-8 http://dx.doi.org/10.1016/j.foodchem.2014.01.026 FOCH 15251

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

1 July 2013 7 December 2013 13 January 2014

Please cite this article as: Qiu, M., Wu, C., Ren, G., Liang, X., Wang, X., Huang, J., Effect of chitosan and its derivatives as antifungal and preservative agents on postharvest green asparagus, Food Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.foodchem.2014.01.026

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1

Effect of chitosan and its derivatives as antifungal and preservative

2

agents on postharvest green asparagus

3 4

Miao Qiu, Chu Wu, Gerui Ren, Xinle Liang, Xiangyang Wang*, Jianying Huang*

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Key Laboratory of Fruits and Vegetables Postharvest and Processing Technology

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Research of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang

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Gongshang University, Hangzhou, 310018, PR China

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ABSTRACT

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The antifungal activity and effect of high-molecular weight chitosan (H-chitosan), low-

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molecular weight chitosan (L-chitosan) and carboxymethyl chitosan (C-chitosan)

11

coatings on postharvest green asparagus were evaluated. L-chitosan and H-chitosan

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efficiently inhibited the radial growth of Fusarium concentricum separated from

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postharvest green asparagus at 4 mg/ml, which appeared to be more effective in

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inhibiting spore germination and germ tube elongation than that of C-chitosan. Notably,

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spore germination was totally inhibited by L-chitosan and H-chitosan at 0.05 mg/ml.

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Coated asparagus did not show any apparent sign of phytotoxicity and maintained good

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quality over 28 days of cold storage, according to the weight loss and general quality

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aspects. Present results inferred that chitosan could act as an attractive preservative agent

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for postharvest green asparagus owing to its antifungal activity and its ability to stimulate

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some defense responses during storage.

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* *

Corresponding author: Tel: 86-571-28877777, E-mail: [email protected]. Corresponding author: Tel: 86-571-28877777, E-mail: [email protected]

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Keywords: Chitosan; asparagus; Fusarium concentricum; antifungal activity; spore

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germination.

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1. Introduction

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Green asparagus (Asparagus officinalis L.) is eaten worldwide as a popular fresh

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vegetable due to its high nutritional values. However, its respiration rate makes it

28

particularly susceptible to postharvest perish. Moreover, asparagus is easily infected by

29

the Fusarium species causing stem and crown blight (Logrieco, Doko, Moretti, Frisullo

30

& Visconti, 1998), resulting in the decay of postharvest asparagus. Application of

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chemical fungicides, such as benomyl, is controversial in many countries due to health

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risk concerns, although it is by far the most effective method to control postharvest

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diseases.

34

A common method to extend the storage life of asparagus is atmospheric

35

modification and cold storage (McKenzie, Greer, Heyes & Hurst, 2004). Gradually, other

36

reagents including aqueous ozone (An, Zhang & Lu, 2007), 6-benzylaminopurine (Wei &

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Ye, 2011), salicylic acid (Wei, Liu, Su, Liu & Ye, 2011) and silver nanoparticles-PVP

38

(An, Zhang, Wang & Tang, 2008) have been used combined with optimal conditions,

39

such as cold storage or modified atmospheres packaging (MAP), to prevent or lessen

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postharvest asparagus changes in physicochemical and chemical composition.

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At a high relative humidity, most fruit and vegetable can retain good quality.

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However, they are particularly susceptible to postharvest fungal infection under such

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conditions. The common storage method, refrigeration, is uneconomical and removes

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moisture, though it can reduce the respiration rate of asparagus. Thus, there is an ongoing

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challenge to develop efficient and safe approaches for the control of postharvest diseases

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and maintenance of postharvest green asparagus quality.

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Chitosan has emerged not only as a promising and economic source for efficient and

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versatile antimicrobial material, but also as a biocompatible and biodegradable polymer

49

with various applications (Dutta, Dutta & Tripathi, 2004; Bansal, Sharma, Sharma, Pal &

50

Malviya, 2011; Zhang, Geng, Jiang, Li & Huang, 2012; Geisberger, Gyenge, Hinger,

51

Kach, Maake & Patzke, 2013). It has been extensively used as edible coatings to preserve

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the quality of many foods (Chien, Sheu & Yang, 2007; Mohan, Ravishankar & Lalitha,

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2012; Huang, Chen, Qiu & Li, 2012; Dutta, Tripathi, Mehrotra & Dutta, 2009) and as a

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bioactive fungicide (EI Ghaouth, Arul, Grenier & Asselin, 1992).

55

Herein, Fusarium concentricum was isolated from postharvest asparagus, which was

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stored at 28 °C, 70% RH, subsequently identified by internal transcribed spacer (ITS)

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sequences in combination with the morphological characters. The antifungal activity of

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three different chitosan derivatives against F. concentricum was evaluated. Moreover, in

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order to assess the potential of chitosan as a natural food preservative, the effect of those

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coatings on postharvest green asparagus quality during cold storage was investigated, to

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determine if chitosan coatings directly affect fungal growth and induce lignification.

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2. Materials and methods

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2.1. Plant material

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Green asparagus spears were harvested from a production site located in Hangzhou

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(China). After harvesting, the samples were rapidly placed in crushed ice and transported

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to the laboratory within 2 hours. Spears selected for the study were straight, undamaged,

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16–20 mm in diameter and ~30 cm in length with closed bracts and no visible signs of

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injury.

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2.2. Fungal isolation

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Diseased spear stems were surface-sterilized in 0.1% potassium permanganate for 3-

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5 min, then excised with a sterile scalpel and plated on antibiotic potato-dextrose agar

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(PDA). The plates were incubated at 28 °C in the dark and examined daily for 7 days

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(Karolewski et al., 2011).

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2.3. Morphological identification

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Identification was achieved by placing a drop of sterile water on a clean slide with

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the aid of a mounting needle, where a small portion of the mycelium from the fungal

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cultures was removed. The mycelium was spread very well on the slide with the aid of

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the needle. A cover slip was gently applied with little pressure to eliminate air bubbles.

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The slide was then mounted and observed with a × 10 eyepiece and × 40, × 100 objective

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lenses, respectively. Cultural characters were assessed by eye, further detailed

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microscopic examinations of the isolate was performed using an Olympus BX51

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polarizing microscope. The species encountered were identified in accordance with the

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reference (Nirenberg & O’ Donnell, 1998).

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2.4. Molecular identification

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Species identification of the collections studied was based on the analysis of the ITS

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sequences in combination with the morphological characters. DNA was extracted and

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purified using the phenol-chloroform-isoamyl alcohol extraction method (Murray &

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Thompson, 1980). ITS was amplified with the primer sets ITS1/ITS4 (Takaiwa, Kiyoharu

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& Masahiro, 1985). PCR was performed in an Applied Bio systems 2620 thermocycler.

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The amplification was confirmed on 1% agarose electrophoresis gels stained with

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ethidium bromide (Stöger, Schaffer & Ruppitsch, 2006). DNA sequencing was performed

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at Beijing Inovogen Tech. Co..

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2.5. Antifungal ability assays

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Antifungal assays were performed based on the method of Jasso de Rodríguez,

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Hernández-Castillo, & Rodríguez-García (2005). The L-chitosan, with a viscosity of 20

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mPa.s and deacetylated degree of 96.1% (Haixin Biological Products Co., Ltd., Zhejiang,

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China), and C-chitosan, with a viscosity of 92.5 mPa.s and deacetylated degree of 91.0%

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(Qingdao Honghai Biotechnology Company, Shandong, China) to be tested was

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dissolved in deionized water, while H-chitosan, with a viscosity of 423 mPa.s and

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deacetylated degree of 95.6%, was dissolved in 0.1% acetic acid, and the pH was then

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adjusted to 5.6 with 1 M NaOH solution. Potato dextrose agar (PDA) media including 0.5,

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1.0, 2.0, 4.0 mg/ml chitosans solutions were sterilized at 121 °C for 20 min and then

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poured onto sterile petri dishes (9-cm diameter). The plates were inoculated with 3-mm-

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diameter plugs taken from the margins of 7 days old colonies on PDA. Control plates

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were also inoculated with the fungus. Subsequently, all plates were incubated in the dark

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at 28 °C. Three replicates for each sample concentration were used. When the mycelium

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of fungi reached the edges of the control plate (without the added samples), the antifungal

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index was calculated as follows:

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Antifungal index (%) = (1 – Da/Db) × 100%

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Where Da is the diameter of the growth zone in the test plates and Db is the diameter of

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growth zone in the control plate.

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2.6. Spore germination of conidia

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Aliquots of 25 ml of different chitosan solutions (0.01, 0.02, 0.03, 0.05 mg/ml)

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approximately containing 1 × 106 conidia/ml were placed at the centre of multi-well

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microscope slides each containing 10 wells. After being incubated in the dark at 28 °C for

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8 h, the numbers of germinated and non-germinated conidia were recorded and the

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percentage germination was calculated (Tikhonov et al., 2006). The length of spores were

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measured after 8 h of incubation at 400 × with the Image-Pro Plus 6.3 analyzing software

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(Media Cybernetics, Bethesda, MD, USA).

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2.7. Pathogenicity test

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A spore suspension (50 µl) containing a concentration 1 × 106 colony-forming units

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per ml (cfu/ml) was inserted into the 5 mm deep puncture wounds made by a sterile cork

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borer (3 mm diameter) in the top of fresh asparagus. Asparagus inoculated with sterile

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water was used as the control. Then they were placed in polythene bags, tightly sealed,

126

and incubated in the dark at 28 °C for 4 days. After incubation, the crown was cut in half

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longitudinally through the inoculation site with a sterile knife and examined for the extent

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of soft rot. The pathogenicity test was conducted twice.

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2.8. Treatment and storage of asparagus samples

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Coating was carried out at room temperature by dipping the asparagus spears for 10

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min in 1%, 0.25% (w/v) H-chitosan including 0.5% acetic acid suspensions, 1%, 0.25%

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(w/v) C-chitosan and L-chitosan suspensions, and deionized water as a control,

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respectively. After slowly drying at ambient conditions (22 °C, 70% RH) for about 2 h,

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the treatments were stored at 2 °C with 95% RH for subsequent quality assessments at 0,

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7, 14, 21, 28 and 35 days throughout the storage.

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2.9. Visual appearance

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The sensory evaluation of asparagus was determined by a sensory panel of 10

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trained assessors. A four-point score based on a hedonic scale was employed (1, very

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good; 2, good; 3, acceptable; 4, unacceptable). A value of three was considered as the

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commercial acceptability threshold (Villanueva, Tenorio, Sagardoy, Redondo & Saco,

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2005).

142

2.10. Weight loss

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The weight loss was determined by periodical weighing, and the results were

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expressed as the percentage loss from the initial weight (Sothornvit & Kiatchanapaibul,

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2009).

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2.11. Lignin determination

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The lignin content of the different parts of the asparagus spears was determined with

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the thioacidoglycolysis method, according to the literature (Bruce & West, 1989).

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2.12. Statistical analysis

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The data were analyzed using analysis of variance (ANOVA) (P < 0.05). The mean

151

differences were established via the Duncan’s multiple range tests. The data were

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analyzed by SPSS software (Version 18.0, SPSS Inc., Chicago, IL).

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3. Results and discussion

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3.1. Identification of fungi

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The ITS sequence obtained has been deposited into the NCBI GenBank database

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and matched against the nucleotide–nucleotide database through Blast for final

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identification. Representative sequences with high similarity were aligned using ClustalX

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1.81, and then a phylogenetic tree was constructed by mega 5.0 on the basis of a UPGMA

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analysis with 1000 bootstrap replications (Fig. 1). The resultant dendrogram were well-

160

fitted with all the bootstrap values higher than 98. Cluster analysis clearly revealed the

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strain sample belonging to the species of Fusarium. oxysporum or Fusarium.

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concentricum.

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The colony grew very rapidly (0.87–0.94 cm/day) on PDA at the optimum

164

temperature of 28 °C. As shown in Fig. 2, the mycelium on PDA was floccose, whitish

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(Fig. 2A). The pigment on the reverse plate on PDA varied from greyish-rose to carmine

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red (Fig. 2B). The conidia borne in the aerial mycelium were obovoid or oval to allantoid.

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Conidiophores of the mycelium were sparsely branched with only a few polyphialides,

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short and wide (Fig. 2D). Based on the shape of the conidiophore and the characteristics

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described in previous reports (Nirenberg & O’ Donnell, 1998), the strain sample possibly

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attributed to the species of F. concentricum.

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3.2. In vitro antifungal effect

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In the case of F. concentricum, considerable differences were observed in the

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treatments of L-chitosan and H-chitosan with respect to the C-chitosan. As shown in

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Fig. 3, the antifungal activity of chitosan was concentration-dependent. When using a

175

concentration of 4 mg/ml, there was a marked fungus radial growth inhibition in

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evaluations of L-chitosan and H-chitosan. The antifungal indexes for L-chitosan and H-

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chitosan were 89% and 74%, respectively. Whereas, the antifungal index of C-chitosan

178

was only 10%.

179

It has been well speculated that the negatively charged residues of chitosan can

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interfere with the fungal cell surface to change the permeability of the plasma membrane,

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thereby inhibiting the growth of several phytopathogenic fungi (Leuba & Stossel, 1986).

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In the present study, the interactions between different chitosan derivatives and the fungal

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cell surface are different. L-Chitosan can easily approach the fungal cells to give a target

184

inhibition by disturbing fungi metabolism due to its relatively small and flexible structure

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compared to H-Chitosan. Previous studies have reported that chitosan possessing proper

186

molecular weight at proper lower concentration had preferable inhibition ability against

187

Aspergillus niger (Li, Feng, Yang, Wang & Su, 2008). Our study also showed that L-

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chitosan exhibited significantly (P < 0.05) better inhibition ability against F.

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concentricum than that of H-chitosan. However, C-chitosan was observed to have lower

190

antifungal activity, probably due to its amphipathic structure hampering the interaction of

191

the negatively charged residues with the phospholipid groups present in the fungal cells.

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3.3. Spore germination

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Spores were clearly more sensitive to chitosan than hyphae. The effect of the

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chitosans on the germination of conidia of F. concentricum was studied at a concentration

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range from 0.01 to 0.05 mg/ml (Table 1). As shown in Table 1, L-chitosan and H-chitosan

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markedly reduced the spore germination and germ tube elongation of F. concentricum. At

197

the concentration of 0.05 mg/ml, spore germination was almost totally inhibited by L-

198

chitosan and H-chitosan (P < 0.05). However, C-chitosan showed no effect on the

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germination of F. concentricum even exposed at a concentration of 0.05 mg/ml. In this

200

study, the inhibitory effect of L-chitosan and H-chitosan was dose-dependent, and L-

201

chitosan exhibited a slightly higher inhibitory activity than H-chitosan. Since the pattern

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associated with the molecular weight of chitosan and its effect on spore germination is

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not clear (Bautista-Banos, Hernandez-Lopez, Hernandez-Lauzardo, Trejo-Espino,

204

Bautista-Ceron & Melo-Giorgana, 2005), it is necessary to carry out more studies about

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the mode of action of the physicochemical properties of chitosan on F. concentricum.

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The inhibitory effects of the three chitosans on germ tube elongation were also

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different. Germ tube elongation of F. concentricum was significantly inhibited when the

208

H-chitosan and L-chitosan concentration were higher than 0.02 mg/ml (P < 0.05). The

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shortest germ tubes of F. concentricum were observed with H-chitosan and L-chitosan at

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0.05 mg/ml. The longest germ tubes were observed for C-chitosan.

211

3.4. Pathogenicity test

212

Symptoms are the visible reactions including wilt, rot, vascular discoloration, as

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well as brown lesions on root and stem surfaces, when asparagus are infected and

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colonized by Fusarium species (Corpas-Hervias, Melero-Vara, Zurera-Muñoz &

215

Basallote-Ureba, 2006), ultimately resulting in disease.

216

The brown lesions on spears were obviously observed, and the growth of mycelia

217

was visible from the 4th day of storage at 28 °C. This causal pathogen was re-isolated

218

from the infected asparagus, and the diagnosis was confirmed by the morphological

219

changes and molecular identification previously developed for F. concentricum. This

220

study has fulfilled Koch’s postulates for F. concentricum as a causative pathogen fungus

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of green asparagus.

222

3.4. Visual appearance

223

Although sensory attributes are an objective parameter, it is a fundamental criterion

224

for determining the shelf life of the product. The changes of visual characteristics of all

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asparagus samples were presented in Table S1. The control batches showed longitudinal

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striation (from no striation up to strong striation), dryness, and colour changes (from

227

bright green to dull olive green) earlier than those chitosan treated samples. The visual

228

characteristics changes were easily noticeable after 28 days for the control, and the

229

asparagus was no longer acceptable for selling. In the case of the chitosan treated batches,

230

the overall shelf life was relatively prolonged compared to the control by the above

231

sensory quality. At the 35th day, 0.25% of the C-chitosan and 0.25% of the L-chitosan

232

treated batches could be acceptable for the public, and a shelf-life extension of 7 days

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was achieved (Table S1).

234

3.5. Weight loss

235

Weight loss gives direct information about the quality of asparagus, which is crucial

236

and valuable, due to the fact that every loss in weight leads to an economical loss. As

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shown in Fig. 4, the losses of all chitosan-treated batches were relatively lower than that

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of control during the entire storage time. At the 21st day, the losses of weight were 4.57%

239

(1% C-chitosan treated), 3.73% (0.25% C-chitosan treated), 5.93% (1% L-chitosan

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treated), 6.52% (0.25% L-chitosan treated), 6.83% (1% H-chitosan treated) and 6.78%

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(0.25% H-chitosan treated) respectively, whereas, that of the control was 8.68%. The

242

chitosan-treated batches significantly (P < 0.05) reduced the weight loss of asparagus

243

during storage. Among those chitosan-treated batches, asparagus treated with 0.25% C-

244

chitosan apparently exhibited the best moisture maintenance ability and contributed to a

245

better quality of asparagus during the cold storage. Chitosan can be used as a protective

246

barrier due to its film-forming property, thus allowing control of the biochemical changes

247

in the metabolism of asparagus by preventing water loss or creating a modified

248

atmosphere surrounding the asparagus. However, it also has the properties of moisture

249

absorption, and will somewhat absorb water from the asparagus texture in case of high

250

viscosity and high concentration conditions, leading to a lesser effect on the weight loss

251

of asparagus. It was further inferred that chitosan edible coatings could create a physical

252

barrier to moisture loss comparable to MAP in terms of retarding dehydration and

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shriveling, while different concentrations and different chitosan derivatives exhibited

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different effects on postharvest asparagus.

255

3.6. Lignin content

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The extent of lignification is another important factor in the quality of asparagus

257

because it can also decrease the fresh market value. As shown in Fig. 5, the lignin

258

concentration of all asparagus samples increased rapidly over the first 7 days, and there

259

was no significant (P > 0.05) difference in the lignin content of the control and those

260

treated. Augmented lignification was observed in the H-chitosan-treated batches till the

261

14th day of storage. After that, the lignification deposition of all chitosan-treated batches

262

increased gradually during the later storage excluding the control batches. At the 35th day

263

of storage, the lignin content of 0.25% treated C-chitosan and 0.25% treated L-chitosan

264

sample were 4.81 mg/100 g FW and 4.74 mg/100 g FW, respectively, while that of the

265

control was 5.74 mg/100 g FW. Previous studies have reported that the lignin deposition

266

in postharvest was correlated with different enzymes, in which phenylalanine ammonia-

267

lyase (PAL) was supposed to be a key enzyme for the biosynthetic pathway of lignin

268

(Flores, Oosterhaven, Martínez-Madrid & Romojaro, 2005; Tzoumaki, Biliaderis &

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Vasilakakis, 2009; Chen & McClure, 2000). These data suggested that the application of

270

exogenous chitosan, especially H-chitosan, can potentiate responses, enhancing PAL

271

activity and lignin biosynthesis in the initial storage period. This may result in the

272

reinforcement of the cell wall and the formation of an efficient physical barrier to restrict

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subsequent fungal penetration and infection. Later, the irritable effect of chitosan on

274

PAL turned to inhibition, thereby reducing the biosynthesis of lignin and maintaining the

275

asparagus quality.

276 277

4. Conclusions

278

Fusarium concentricum isolated from postharvest green asparagus was identified,

279

and the fungal inhibition of chitosan derivatives, as well as quality effect of chitosan

280

derivatives coatings on postharvest green asparagus was evaluated. The chitosan

281

derivative coating was effective in reducing the decay of green asparagus caused by F.

282

concentricum. As tested in vitro, L-chitosan and H-chitosan inhibited the radial growth of

283

F. concentricum, with a remarkable effect at a concentration of 4 mg/ml, and totally

284

inhibited spore germination at a concentration of 0.05 mg/ml, indicating that chitosan

285

derivatives were either fungistatic or fungicidal. The observed inhibition of decay and

286

augmented lignification was attributed to the fungistatic activity of chitosan and its ability

287

to induce a defense reaction.

288

It was concluded that chitosan was worthy of further study as a natural preservative

289

for green asparagus and other foods prone to fungal spoilage. However, the successful

290

application of any novel antimicrobial agent in food preservation depends on a number of

291

factors. Adequate control of microbial growth in foods using chitosan requires extensive

292

knowledge of the factors that determine chitosan performance.

293 294

Acknowledgments

295

This study was supported by the Natural Science Foundation of the Technology

296

Planning Project of Zhejiang Province of China (2011C12031), and the National Science

297

Foundation of China (21102129). We thank to Beijing Inovogen Tech. Co. for DNA

298

sequencing.

299 300

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401

of

chitin

and

chitosan

with

the

[(2-Hydroxy-3-

402 403 404

Fig. 1. UPGMA tree based on sequence analysis of internal transcribed spacer sequences. Values on

405

the branches indicate percent bootstrap condidence values.

406

407 408

A

B

C

D

409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424

Fig. 2. Asparagus with symptoms of rot caused by F.concentricum. A, observe colony

425

cultures on PDA after incubation at 28 °C for 7 days; B, reverse colony cultures on PDA

426

after incubation at 28 °C for 7 days; C, F. concentricum mold (× 40); D, Detail of

427

conidiophores (× 100).

428

429

100

C-chitosan

L-chitosan

H-chitosan

90

Antifungal index (%)

80 70 60 50 40 30 20 10 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Concentration (mg/mL)

430 431

Fig. 3. Effect of C-chitosan, L-chitosan and H-chitosan on antifungal index of

432

F. concentricum. Vertical bars represent standard deviation of means.

433

434

16

C-chitosan 1% C-chitosan 0.25% L-chitosan 1% L-chitosan 0.25% H-chitosan 1% H-chitosan 0.25% Control

14

Weight loss (%)

12 10

a b

a

8

a

6

b b bb

4 2

a b

c

a a b

c

b c b

ccc

b

c

c c dd

d e

d

c c cb d

0 7

14

21

28

35

Storage time (days)

435 436

Fig. 4. Changes in weight loss of asparagus stored at 2 °C for 35 days. Each data point is

437

the mean of three replicate samples. Vertical bars represent standard deviation of means.

438

439 440

6.0

5.5

Lignin (mg/100 g FW)

5.0

4.5

4.0

3.5

a C-chitosan 1% C-chitosan 0.25% a L-chitosan 1% b bb b L-chitosan 0.25% a cd c H-chitosan 1% b dd H-chitosan 0.25% c cd de a Control e c ab c f bc dd cd de a de ab e abc abc bc cd d jj jj jj j

3.0

2.5 0

7

14

21

28

35

Storage time (days)

441 442

Fig. 5. Changes in lignin content of asparagus stored at 2 °C for 35 days. Each data point

443

is the mean of three replicate samples. Vertical bars represent standard deviation of means.

444 445 446

447

Table 1

448

Effect of C-chitosan, L-chitosan and H-chitosan on spore germination and germ tube

449

length of F.concentricum.

450 451 452

0

Germination,% (mean±standard error) 100

Germ tube length, µm 23.6

C-chitosan

0.05 0.03 0.02 0.01

100 100 100 100

21.5 21.6 22.2 22.9

L-chitosan

0.05 0.03 0.02 0.01

0 42.96±2.15 63.22±0.43 84.44±2.36

7.70 9.27 15.8 16.1

H-chitosan

0.05 0.03 0.02 0.01

0 47.57±4.17 71.23±3.48 95.92±3.76

7.69 9.67 13.9 23.2

Type of chitosan

Concentration, mg/ml

Control

453

The potential for using chitosan as a natural food preservative was assessed.

454

Antifungal ability and spore germination inhibition of chitosan were investigated.

455

Effect of chitosan derivatives coatings on postharvest green asparagus was evaluated.

456 457

Effect of chitosan and its derivatives as antifungal and preservative agents on postharvest green asparagus.

The antifungal activity and effect of high-molecular weight chitosan (H-chitosan), low-molecular weight chitosan (L-chitosan) and carboxymethyl chitos...
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