chitosan disrupts penicillium expansum and controls...
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Food Control 41 (2014) 56e62
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Food Control
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Chitosan disrupts Penicillium expansum and controls postharvest bluemold of jujube fruit
Liting Wang a, Hao Wu a, Guozheng Qin b,**, Xianghong Meng a,*
aCollege of Food Science and Engineering, Ocean University of China, Qingdao 266003, ChinabKey Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
a r t i c l e i n f o
Article history:Received 24 September 2013Received in revised form12 December 2013Accepted 22 December 2013
Keywords:ChitosanJujube fruitPenicillium expansumPostharvest diseasePlasma membrane
* Corresponding author. Tel./fax: þ86 532 8203209** Corresponding author. Tel.: þ86 10 62836900; fa
E-mail addresses: [email protected] (G. Qin), men
0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.12.028
a b s t r a c t
Chitosan has been shown to be effective for control of postharvest diseases on various fruit. However,little is known about the effect of chitosan on blue mold caused by Penicillium expansum on jujube fruit.Here we show that application of chitosan reduced disease development of blue mold caused byP. expansum in wounded and inoculated jujube fruit at 25 �C. Chitosan also provided an inhibitory effecton natural decay of jujube fruit during storage at 0 �C. Application of a chitosan coating to fruit hadhardly any significant effect on the changes of weight loss, soluble solid contents, titratable acidity, andvitamin C, as storage time increased. To investigate the mechanisms underlying the effectiveness ofchitosan against blue mold on jujube fruit, we analyzed the growth of P. expansum after chitosantreatment. Results indicated that spore germination, germ tube length and mycelial growth ofP. expansum were significantly inhibited by chitosan in a concentration-dependent mode. Using thefluorescent probe propidium iodide, we found that the plasma membrane of P. expansum collapsedsignificantly after chitosan treatment. Further observation by electron microscopy revealed that plasmamembrane of P. expansum was gradually disrupted after chitosan application. Our data suggest thatchitosan may be potentially used for controlling postharvest diseases in jujube fruit without negativeeffect on fruit quality.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Winter jujube (Ziziphus jujube Mill. cv. Dongzao) has flourishedin China for over 4000 years (Li, Fan, Ding, & Ding, 2007). Due to itshigh nutritional value and savory taste, winter jujube has beenextensively grown as a commercial crop. Although the fruit can bestored at low temperature for up to two months, they are veryperishable and highly susceptible to postharvest color fading,browning, decay and water loss (Lin, Tian, Wan, Xu, & Yao, 2004).Spoilage fungi such as Aspergillus flavus and Penicillium citrinum cancause the production of mycotoxins and off-flavors, which in turnaffect the quality and shorten shelf-life (Li, Xing, Jiang, Ding, & Li,2009). Alternaria rot, caused by Alternaria alternata, is a majordisease of jujube fruit in the field (Wang et al., 2009), but bluemold,caused by Penicillium expansum, is one of the most serious post-harvest diseases of the fruit in China (Qin & Tian, 2004). Blue moldoccurs during postharvest storage as well during shipping and
3.x: þ86 10 [email protected] (X. Meng).
All rights reserved.
marketing of the fruit, dramatically reducing shelf life and causingserious losses. Synthetic chemical fungicides are still the primarymeans of controlling postharvest diseases of jujube fruit in Chinabut the adverse impact of synthetic chemical fungicides on humanhealth and the environment is a cause for public concern. It istherefore necessary to develop alternatives to synthetic chemicalcontrol to reduce environment risks and raise consumer confidence(Zhang, Zheng, & Yu, 2007).
Chitosan is a deacetylated derivative of the linear poly-saccharide chitin, which is the second most abundant poly-saccharide found in nature. It consists of b-(1,4)-linked residues ofeb-D-glucosamine(Cruz-Romero, Murphy, Morris, Cummins, &Kerry, 2013). The net positive charge of chitosan confers a varietyof unique physiological and biological properties to this compound.It has broad-spectrum antibacterial activity (Jeon, Park, & Kim,2001; Liu et al., 2006; Zheng & Zhu, 2003) and is also effective ininhibiting spore germination, germ tube elongation, mycelialgrowth, and sporulation of fungal phytopathogens (Liu, Tian, Meng,& Xu, 2007; Meng, Yang, Kennedy, & Tian, 2010; Palma-Guerrero,Jansson, Salinas, & Lopez-Llorca, 2008). The antimicrobial activityof chitosan varies according to its molecular weight, degree ofdeacetylation, pH of the chitosan solution, and the sensitivity of the
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target organism (Xu, Zhao, Han, & Du, 2007). Kushwaha, Rai, andSingh (2010) has proposed that the antibacterial action of chito-san is brought about when positively charged chitosan reacts withnegatively charged molecules at the cell surface, altering cellpermeability and inhibiting the transport of compounds across theplasma membrane. However, the mechanism by which chitosanaffects the development of phytopathogenic fungi has not beenfully elucidated. As a biopolymer, chitosan, as well its derivatives,has excellent film forming properties (Fernández-Saiz, Sánchez,Soler, Lagaron, & Ocio, 2013) and is able to form a semi-permeable film on fruit, which might modify the internal atmo-sphere, as well as decrease weight loss due to transpiration andimprove overall fruit quality (Jiang & Li, 2001; Zhang & Quantick,1997). Chitosan coating could act as a mechanical barrier protect-ing fruits from pathogen infection and also as an exogenous elicitorof host-defense responses (Bautista-Baños et al., 2006; Meng, Li,Liu, & Tian, 2008; Sébastien, Stépahne, Copinet, & Coma, 2006),thus decreasing decay during storage.
Due to its multifunctional activity, chitosan has been proposedas a postharvest fungicide and preservative of fruit (No, Meyers,Prinyawiwatkul, & Xu, 2007; Zhang, Li, & Liu, 2011). Pre- andpost-harvest application of chitosan has been reported to effec-tively control the decay of table grapes (Meng et al., 2008;Romanazzi, Nigro, Ippolito, Di Venere, & Salerno, 2002), andextend the shelf life of tomato (El Ghaouth, Ponnampalam,Castaigne, & Arul, 1992) and strawberry (El Ghaough, Arul,
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Fig. 1. Effect of chitosan on disease incidence (A) and lesion diameter (B) of artificiallywounded jujube fruit inoculated with P. expansum. Bars represent standard deviationsof the means based on three independent experiments. Within each day, columnsfollowed by different letters are statistically different according to the Duncan’s mul-tiple range test (P < 0.05).
Ponnampalam, & Boulet, 1991). The effect of chitosan on controlof blue mold and other types of decay in winter jujube fruit, as wellas the effect on fruit quality has not been explored. The objectives ofthe present study were to investigate the effect of chitosan againstblue mold caused by P. expansum and natural decay on jujube fruitand the detailed mechanisms involved. Influence of chitosancoating on fruit quality was also determined.
2. Materials and methods
2.1. Chitosan
Chitosan, with approximately a 90% deacetylation and averagemolecular weight of 350 kDa, was prepared at starting concentra-tion of 25 g/L in 1% (v/v) HCl by stirring at 25 �C.
2.2. Pathogen inoculum
P. expansum was isolated from jujube fruit with typical bluemold symptoms and cultured on potato dextrose agar (PDA) at25 �C. Spores of P. expansum were removed from 2-week-old cul-tures by adding 5 mL of sterile water containing 0.05% (v/v) Tween-80 to the Petri plates with gentle agitation. The spore suspensionswere filtered through four layers of sterile cheesecloth to removemycelia. Spore concentrationwas adjusted to the desired level withthe aid of a hemocytometer prior to use.
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Fig. 2. Effect of chitosan coating on natural decay incidence (A) and disease index (B)in jujube fruit at different storage stage. Bars represent standard deviations of themeans based on three independent experiments. Within each day, columns followedby different letters are statistically different according to the Duncan’s multiple rangetest (P < 0.05).
Table 1Effect of chitosan coating on quality parameters of jujube fruit stored at 0 �C.
Items Storage time (days)
0 30 50 70
Firmness (N) Control 5.18 � 0.60 a 5.59 � 0.51 a 5.67 � 0.25 a 5.05 � 0.43 aChitosan 5.18 � 0.60 a 5.42 � 0.51 a 5.45 � 0.39 a 5.36 � 0.56 a
SSC (�B) Control 20.90 � 0.26 a 21.53 � 0.15 a 21.37 � 0.21 a 21.67 � 0.12 aChitosan 20.90 � 0.26 a 21.30 � 0.10 a 20.87 � 0.15 b 21.30 � 0.10 b
TA (�10�6 mol/kg FW) Control 29.33 � 1.33 a 28.89 � 0.77 a 32.67 � 0.67 a 32.67 � 0.67 aChitosan 29.33 � 1.33 a 29.78 � 2.04 a 33.56 � 1.68 a 33.00 � 0.33 a
Vc (g/kg FW) Control 1.704 � 0.13 a 2.709 � 0.02 b 2.892 � 0.06 a 2.821 � 0.00 aChitosan 1.704 � 0.13 a 2.763 � 0.02 a 2.845 � 0.01 a 2.806 � 0.04 a
Weight loss (%) Control 0.00 � 0.00 0.603 � 0.15 a 1.273 � 0.20 a 2.061 � 0.29 aChitosan 0.00 � 0.00 0.522 � 0.21 a 0.885 � 0.28 a 1.330 � 0.19 b
Data are followed by standard deviations of the means. For each fruit quality, values at a defined time followed by a different letter showed significant difference at P < 0.05according to the Duncan’s multiple range test.
Table 2Effect of chitosan on spore germination and germ tube length of P. expansum after12 h of incubation.
Treatment Spore germination (%) Germ tube length (mm)
0 mg/mL chitosan(control)
82.00 � 2.46 a 116.67 � 6.03 a
1 mg/mL chitosan 3.37 � 0.67 b 17.67 � 2.52 b5 mg/mL chitosan 0.00 � 0.00 0.00 � 0.00
Data are followed by standard deviations of the means. Values of each columnfollowed by a different letter showed significant difference at P < 0.05 according tothe Duncan’s multiple range test.
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2.3. Effect of chitosan on blue mold infection of jujube fruit
Jujube fruit were harvested at a pre-climacteric but physiolog-ically mature stage from an orchard located in Dongying city in theShandong Province, China. Fruit were sorted to obtain a subsetsimilar in size, ground color, and freedom from physical injuries orinfections. After disinfection with 1% sodium hypochlorite (v/v) for2 min, jujube fruit were air dried, wounded (3 mm deep and 3 mmwide) with a sterile nail at the equator, and 10 mL of conidial sus-pension of P. expansum (1 � 104 spores/mL) was added to eachwound using a micropipette. After 2 h, 10 mL of a chitosan solution(1, 5, 10, 15 or 20 mg/mL) was added to each wound. Sterile distilledwater was used as the control. The treated fruit were placed in
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Fig. 3. Effect of chitosan on the mycelial growth of P. expansum. The fungi wereincubated at 25 �C on potato dextrose agar containing different concentrations ofchitosan and fungal growth was measured up to 7 days. Bars represent standard de-viations of the means according to three independent experiments. Within each day,columns followed by different letters are statistically different according to the Dun-can’s multiple range test (P < 0.05).
200 mm � 130 mm � 50 mm plastic boxes, overwrapped withplastic bags to maintain a 90e95% relative humidity (RH), andstored at 25 �C. Decay incidence of jujube fruit and the lesiondiameter was measured at 1, 2, 3 and 4 days after inoculation. In-hibition of lesion diameter was calculated by comparing lesiondiameter of treated and untreated fruits. Each treatment containedthree replicates with 30 fruits per replicate and the experiment wasrepeated three times.
2.4. Effect of chitosan coating on the natural decay of jujube fruit
A chitosan coating was prepared using 10 mg/mL chitosan and0.1% Tween 80. Disinfected jujube fruit were dipped in the chitosansolution orwater (as control) for 1min and air-dried at 25 �C. Treatedfruit were put in plastic boxes, overwrapped with plastic bags andstored at 0 �C. Each treatment contained three replicates with 5 kg(about 350 fruit) per replicate and the experiment was repeatedthree times. The fruit were examined at 0, 30, 50 and 70 days at 0 �Cand then 3 days at room temperature (70 þ 3 d) to evaluate thenatural decay incidence. A disease index for natural decay wasdetermined according toMeng et al. (2008). Disease severity of eachjujube fruit was rated according to an empirical scale where:
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Fig. 4. Effect of chitosan on plasma membrane integrity of P. expansum. Spores werecultured in potato dextrose broth medium containing 1 or 5 mg/mL of chitosan at25 �C. Culture mediumwithout chitosan was used as control. Spores were stained withpropidium iodide (PI) and observed with a fluorescence microscope. Bars represent thestandard deviation of the mean of three independent experiments. Within each day,columns followed by different letters are statistically different according to the Dun-can’s multiple range test (P < 0.05).
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0 ¼ healthy fruit; 1 ¼ one lesion smaller than 5 mm in diameter;2¼ one lesion smaller than 10mmindiameter; 3¼ several lesions ormore than one-quarter of fruit infected; 4¼more than one-half fruitinfected. The decay index was calculated by the formula,DI ¼ Pðdf Þ=ðNDÞ, where d is the degrees of rot severity scored on ajujube fruit and f represents the quantity of jujube fruit related to thecorrespondingdegrees;N is the total numberof fruit examinedandDis the highest degree of disease severity occurring on the scale.
2.5. Quality assessment of chitosan-coating jujube fruit
Weight loss of jujube fruit was measured by monitoring weightchanges of fruit stored for 0, 30, 50 and 70 days at 0 �C. Weight losswas calculated as the percentage loss from the initial weight.
Ten fruit were used to determine the firmness on oppositepeeled cheeks of the fruits using a Fruit Firmness Tester (FT-327,Italy), equipped with an 8 mm plunger tip. Soluble solids content(SSC) of mesocarp tissue was measured with an Abbe Mark Grefractometer (AO Scientific Instrument, USA) and expressed as�Brix. Titratable acidity (TA) was assayed by titrationwith 0.01 N ofNaOH to pH 8.2 and expressed as malic acid content, grams per1000 g fresh sample weight (FW).
Vitamin C (Vc) was determined by titration with 2, 6-dichlorophenolindophenol. Fruit tissue (20 g) was homogenizedwith 20 mL of 0.25 mM oxalic acid and centrifuged at 13,000 � g at4 �C for 15 min. The supernatant (4 mL) was mixed with 16 mL of0.25 mM oxalic acid and titrated with 1.86 mM 2, 6-dichlor-ophenolindophenol. An equal volume of oxalic acid was alsotitrated as the blank. Titration was repeated three times and the Vccontent was calculated using a standard curve developed from di-lutions of Vc. Results were expressed as grams of ascorbic acid per1000 g of fresh sample weight (FW).
2.6. Effect of chitosan on spore germination and germ tubeelongation
Based on the method of Liu et al. (2007), a stock solution ofchitosan was diluted with sterile, molten PDA to achieve concen-trations of 1 or 5 mg/mL, and the pH of mediumwas adjusted to 5.6by 0.1 N of HCl. Ten milliliters of the chitosan mediumwas added toeach Petri dish and the medium was allowed to solidify. Fiftymicroliter aliquots of conidial suspensions at 1 � 106 spores/mLwere plated in each Petri dish (60 mm in diameter) and the inoc-ulum spread across the surface. PDAwithout chitosan served as thecontrol. The cultures were incubated at 25 �C for 12 h. More than200 spores per treatment were examined for germination andgerm tube length. Each treatment contained three replicates andthe experiment was repeated three times.
2.7. Inhibitory effect of chitosan on mycelial growth of P. expansum
Using the method of Meng et al. (2010), mycelial discs were cut(7mm in diameter) from the edge of actively growing two-week-oldfungal cultures. One mycelial disk was placed in the center of eachPetri dish (60mm)containing10mLof a chitosan (1, 5 and10mg/mL)PDAmedium and incubated at 25 �C. PDAwithout chitosan served asthe control. Mycelial growth was determined by measuring colonydiameter at 2, 3, 4, 5, 6 and 7 days after inoculation. Inhibition ofmycelial growth was determined by comparing colony diameter oftreated and untreated cultures. Each treatment was replicated threetimes and the experiment was repeated three times.
2.8. The integrity and morphological structure of plasma membraneof P. expansum
Aliquots of 100 mL of a conidial suspension were transferred toconical flasks (50 mL) containing 20 mL of potato dextrose broth(PDB) with 1 or 5 mg chitosan/mL to obtain a final concentration of5 � 105 spores/mL, respectively. Spore concentrations were deter-mined with a hemocytometer, and adjusted as required with sterilewater. PDB without chitosan served as the control. All flasks wereincubated at 25 �C and 200 rpm. Spores were collected by centri-fugation at 8000 � g for 10 min at 25 �C from PDB after incubationfor 0, 2 and 4 h. Spores were washed three times with 50 mM so-dium phosphate buffer (pH 7.0) to remove any residual medium.
The integrity of spores was assessed using propidium iodide (PI)as a probe according to the method of Liu et al. (2007). Spore sus-pensions of the control and the only 5 mg/mL chitosan treatmentwere stained with 10 mg/mL of PI for 5 min at 30 �C. Spores werethen collected by centrifugation and washed three times withbuffer to remove residual dye. Spores were observed with a NikonE800 (Nikon Instrument Inc., Tokyo, Japan) equipped with a fluo-rescein filter set. Three fields of view were chosen randomly, andthe number of spores observed in the bright-field was defined asthe total number. The destruction of spores was expressed as thepercentage of spores stained with PI.
Ultrastructural observation of spores was conducted using themethod of Li (2009) with somemodification. After incubation timesof 0, 8, and 12 h, control and treated (1 and 5 mg/mL chitosan)spores werewashedwith 50mM sodium phosphate buffer (pH 7.0),and fixed with 2.5% (v/v) glutaraldehyde and 4% (v/v) para-formaldehyde in 50 mM sodium phosphate buffer for 1 h at roomtemperature. Samples were thenwashed three times with the samebuffer, postfixed in 1% (w/v) OsO4, and then dehydrated in a gradedseries of acetone. The samples were embedded in Spurr’s resin.Ultrathin sections (70 nm) were mounted on Formvar-coated grids,stained with uranyl acetate and lead citrate, and observed with anelectron microscope (JEOL 1210, Japan) at 80 kV.
2.9. Statistical analysis
Statistical analyses for fruit quality, disease development,pathogen growth, and integrity of plasma membrane were per-formed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The data weregrouped at a defined time for analysis by analysis of variance(ANOVA). Mean separations were determined using a Duncan’smultiple range test. Differences at P < 0.05 were considered to besignificant. Data presented in this paper were pooled across threeindependent repeated experiments, as the interaction betweentreatment and experiment as a variable was not significant.
3. Results
3.1. Effect of chitosan on blue mold in wounded and inoculated fruit
Bluemold infection of jujube fruit was observed as early as 1 dayafter inoculation in the control. Similar results were obtained forwounds treated with either 1 or 5 mg/mL of chitosan (Fig. 1A).Chitosan at higher concentrations (10, 15 and 20 mg/mL), however,significantly reduced disease incidence. Although chitosan at con-centration of 15 or 20 mg/mL provided the best control at 2 daysafter inoculation, chitosan at 10 mg/mL showed relatively lastingand stable efficacy. As with the effect of chitosan on mycelialgrowth, the inhibitory effect of chitosan on disease incidencedecreased over time. Lesion diameter was significantly decreasedby chitosan at all concentrations in a concentration-dependent
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manner (Fig. 1B), although the rate of lesion growth was similar infruit treated with chitosan at 10 and 15 mg/mL.
3.2. Effect of chitosan coating on natural decay
Coating jujube fruit with chitosan at 10 mg/mL significantlyreduced natural decay during storage at 0 �C. Natural decay inci-dence in untreated fruit was about 23% at 50 days and increased to40% at 70 days (Fig. 2A). Natural decay incidence of chitosan-coatedfruit was only 10% at 50 days and 18% at 70 days. After 70 days ofstorage at 0 �C and 3 days at room temperature (70þ 3 days), 90% ofthe control fruit were infected while decay incidence was only 38%in the chitosan-coated fruit. The decay index in chitosan-coatedfruit, compared to untreated fruit, was reduced from 0.15 to 0.05at 50 days and 0.27 to 0.08 at 70 days (Fig. 2B).
3.3. Effect of chitosan coating on fruit quality of jujube
Weight loss of jujube fruit increased with time of storage at 0 �C.Coating of fruit with chitosan no significant effect on the weightloss in comparison to the control fruit except that stored 70 d(Table 1). No significant effect of the chitosan coating on fruitfirmness was observed during storage. SSC of all fruit increasedduring storage with no significant effect being observed from thechitosan coating. Results on TA and Vc were similar to those ob-tained for SSC, namely, an increase in both parameters over storagetime with no significant difference between the control andchitosan-coated fruit.
3.4. Effect of chitosan treatment on growth of P. expansum
Spore germination and germ-tube growth were significantlyinhibited in PDB medium containing chitosan. No spore germina-tion was detected in the culture medium containing 5 mg/mL ofchitosan after 12 h of incubation. Spore germinationwas only 3.37%in the medium supplemented with 1 mg/mL of chitosan, while agermination rate of 82% was detected in the medium withoutchitosan (Table 2). The average germ-tube length of 17.67 mm and116.67 mm was observed, respectively, in the medium containing1 mg/mL of chitosan and the control.
Mycelial growth of P. expansum was significantly inhibited in aconcentration-dependent manner by the chitosan at concentra-tions ranging from 1 to 10 mg/mL (Fig. 3). The inhibitory effect ofchitosan gradually diminished with cultivation time increasingfrom 2 to 7 days.
3.5. Chitosan damaged plasma membrane of P. expansumdetermined by fluorescent staining
The integrity of plasmamembrane of P. expansumwas evaluatedby PI staining. Using this probe, red fluorescent staining of thenucleus would be observed if the plasma membrane of a spore wasdisrupted. As shown in Fig. 4, 95% of the spores incubated for 6 h inthe control medium were unstained, indicating that they hadmaintained the integrity of the plasma membrane. In contrast,spores incubated in themedium containing 5 mg/mL chitosanweregradually disrupted over time and more than 95% of the sporeswere stained after 6 h of incubation.
Fig. 5. Ultrastructural changes of P. expansum in response to chitosan treatments. Normal aculture medium without chitosan at 0 h (A, B), 8 h (C, D) and 12 h (E, F). Vacuolation of cytoH) and 12 h (I, J) of incubation in potato dextrose broth medium containing 1 mg/mL chitosaplasma membrane were found at 8 h (K, L), with plasmolysis (arrow) and anamorphic spo
3.6. Chitosan application led to the destruction of plasmamembrane of P. expansum by ultrastructural observation
Ultrastructure of P. expansum after chitosan treatment wasobserved in spores and germ tubes. The morphology of P. expansumincubated in PDB medium for 0, 8 and 12 h appeared normal(Fig. 5A, C, E) with an intact plasma membrane (Fig. 5B, D, F). Incontrast, the cytoplasmic degradation and an increase in vacuolarcontent of P. expansum were observed in culture medium con-taining 1 mg/mL of chitosan (Fig. 5GeJ). Additionally, the invagi-nation and disruption of the plasma membrane was evident afterchitosan treatment (Fig. 5GeJ). When the concentration of chitosanwas increased to 5 mg/mL in the medium, plasma membraneinvagination and disruption were readily apparent after 8 h of in-cubation (Fig. 5K, L), and plasmolysis occurred after 12 h (Fig. 5M).An increase in the number of pleomorphic and anamorphic sporeswas observed after chitosan treatment (Fig. 5I, N).
4. Discussion
Chitosan is a biodegradable substance which shows great po-tential as a fungicide and preservative due to its antimicrobial ac-tivity. Chitosan has been proven to effectively inhibit postharvestfungal diseases of fruit, such as tomato (Liu et al., 2007; Reddy,Angers, Castaigne, & Arul, 2000), table grapes (Romanazzi et al.,2002), as well as strawberry and raspberry (Zhang & Quantick,1998), but little is known about the effectiveness of chitosanagainst postharvest diseased in jujube fruit.
In the present study, we found that chitosan was effective forcontrol of blue mold caused by P. expansum and natural decay onjujube fruit. Both disease incidence and lesion size of blue moldwere significantly inhibited by chitosan in a concentration-dependent manner. Meanwhile, chitosan application significantlydecreased the overall incidence of natural decay of jujube fruitstored at low temperature. Chitosan may form a stable barrier onthe fruit surface and prevent the evaporation of water and othervolatile compounds (Elsabee & Abdou, 2013). However, effect ofchitosan coating on weight loss, fruit firmness, SSC, TA, and Vcduring storage was not observed in this study.
To investigate the mechanisms by which chitosan decreasesfruit decay, we first detected the growth of P. expansum after chi-tosan treatment. The results indicated that chitosan inhibited sporegermination, germ tube elongation, and mycelial growth in aconcentration-dependent manner. These observations are consis-tent with those reported by Liu et al. (2007). The antifungal activityof chitosan has been connected to its effect on fungal cell walls andcell membranes (El Ghaouth, Arul, Asselin, & Benhamou, 1992). Theintegrity of the plasma membrane in P. expansum as measured byits permeability and the ultrastructure was assessed in order tobetter understand the antifungal action of chitosan. Results of PIstaining indicated an increase in spore staining as incubation timeincreased, indicating that the plasma membrane of spores wasgradually disrupted. Degradation of the cytoplasma and disruptionof the plasma membrane of spores was further verified by the ul-trastructural observations of spores and germ tubes. Chitosantreated spores exhibited a high level of invagination and/ordisruption of the plasmamembrane and even plasmolysis. Previousstudies have suggested that the plasma membrane damage may bedue to the interaction of the positive amino groups of chitosanwith
ppearance and intact plasma membrane (arrowhead) of P. expansum was observed inplasm, invagination and disruption of the plasma membrane was observed after 8 h (G,n. In culture medium containing 5 mg/mL chitosan, the invagination and disruption ofres being observed at 12 h (M, N). The bar was indicated as Figure.
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the negative charges of plasma membrane phospholipids whichresults in a change in the permeability of the plasma membrane(Benhamou, 1992; Laflamme, Benhamou, Bussières, & Dessureault,1999). However, results of Palma-Guerrero et al. (2008, 2009)demonstrated that the uptake of chitosan into fungal cells wasenergy-dependent rather than by endocytosis or passive diffusion.It has also been proposed that chitosan, once inside the cytoplasm,enters the nuclei of fungi and interferes with RNA and proteinsynthesis (Rabea, Badawy, Stevens, Smagghe, & Steurbaut, 2003).The mechanisms by which chitosan inhibits fungal pathogens needfurther investigation.
In summary, as a natural biodegradable substance, chitosancould directly inhibit the growth of P. expansum and control post-harvest blue mold of jujube fruit. A potential mode of actionappeared to be the degradation of the cytoplasma and the damageof the plasma membrane as indicated by the uptake of PI and ul-trastructural observations of plasma membrane invagination anddisruption. Chitosan coating was able to delay natural decaywithout impacting fruit quality. These results indicate that chitosancould be used as both a fungicide and a preservative coating forfresh winter jujube fruit.
Acknowledgments
The research was supported by the Public Benefit ResearchFoundation from State Forestry Administration (201004041), theNational Natural Science Foundation of China (Grant No. 31171762),the National High Technology Research and Development Programof China (863 Program, Grant No. 2012AA101607), and Program forChangjiang Scholars and Innovative Research Team in University.
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