Postharvest Biology and Technology 145 (2018) 134–143

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Wound healing in citrus fruit is promoted by chitosan and Pichia T

membranaefaciens as a resistance mechanism against Colletotrichum
gloeosporioides
⁎
Yijie Zhaoa,b, Lili Denga,b, Yahan Zhoua, Jian Minga,b, Shixiang Yaoa,b, Kaifang Zenga,b,
a
College of Food Science, Southwest University, Chongqing 400715, PR China
b
Chongqing Engineering Research Center of Regional Food, Chongqing 400715, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Colletotrichum gloeosporioides is the most important pathogen of citrus fruit and is a serious concern in rainy,
Chitosan humid regions and seasons. In this study, the effects of chitosan and Pichia membranaefaciens on anthracnose
Pichia membranaefaciens control, wound healing, and the cell wall changes in citrus fruit and the relationship between wound healing and
Colletotrichum gloeosporioides cell wall composition were evaluated. We found that chitosan and P. membranaefaciens increased the wound
Wound healing
healing process via wound tissue appearance changes, lignin accumulation, increases in cell wall compounds
(pectin, cellulose) and reduced activities of cell wall enzymes - polygalacturonase (PG); pectin methylesterase
(PME); cellulase (CEL) - to control anthracnose. During wound healing, acid-soluble pectin (ASP) and cellulose
had strong positive correlations with lignin.

1. Introduction
The pathogen that causes serious anthracnose disease in citrus fruit
is Colletotrichum gloeosporioides, which was recently reported as the
eighth most important group of plant pathogenic fungi that infects at
least 1000 plant species worldwide (Aiello et al., 2015; Bill et al., 2016;
Wu et al., 2016). C. gloeosporioides also causes preharvest symptoms
such as withered twig tips, tear stain, stem-end rot of fruits and citrus
post bloom fruit drop (Huang et al., 2013; Lima et al., 2011; Rhaiem
and Taylor, 2016). Anthracnose particularly infects citrus that grows in
rainy, humid regions and seasons and causes serious decreases in yield.
In China, this pathogen is very common in Chongqing Province and is
severe in some rainy months, such as May and June. Valencia orange is
currently the major variety of orange (Citrus sinensis) grown in China.
Wounding commonly occurs to fresh fruit from harvest to final use and
induces a series of physiological and defensive responses to prevent
further damage, which is the process called wound healing. Wound
healing shows resistance to pathogen invasion (Ayon-Reyna et al.,
2017; Leverentz et al., 2000; Shao et al., 2010; Spotts and Chen, 1987);
thus, the aim of this project was to study anthracnose and the me-
chanism after wounding of Valencia orange.
Wound healing plays an important role in maintaining postharvest
quality and shelf life. Plant responses to wounding have been described
previously (Cheong et al., 2002; De Bruxelles and Roberts, 2001;
Shanker and Venkateswarlu, 2011; Wu et al., 1997) and include (i) ROS
burst, (ii) mechanical barriers to invading organisms, and (iii) defensive
compounds accumulate from the wound, especially the lignin. Fur-
thermore, PAL is an enzyme recognized as highly important in wound
healing, particularly wound-induced suberization. Wounding in apple
promotes the lignification process (Vilanova et al., 2014) and healing of
minor injuries to Valencia orange is also associated with PAL increase
and lignin accumulation (Ismail and Brown, 1979). One study found
that potato tubers develop a wound barrier that is effective against
pathogen infection (Kumar and Knowles, 2003). Robert showed that
healing of wounds decreases the decay in pear caused by B. cinerea, M.
piriformis, P. expansum, and P. solitum (Spotts et al., 1998), and Vilanova
found that the wound response prevents infection by P. expansum at
20 °C (Vilanova et al., 2014). Earlier studies on wound healing suggest
that ABA is involved in the regulation of wound healing in potato tuber
(Espelie and Kolattukudy, 1985; Lulai et al., 2008; Soliday et al., 1978),
Arabidopsis root (Efetova et al., 2007), tomato fruit (Tao et al., 2016),
and kiwifruit (Han et al., 2018, 2017). Moreover, ethylene is relevant to
wound healing in grapevine stems (Sun et al., 2007).
Many studies demonstrate that chitosan and P. membranaefaciens

Abbreviations: PAL, L-phenylalanine ammonia-lyase; RH, relative humidity; WSP, water-soluble pectin; ASP, acid-soluble pectin; PG, polygalacturonase; PME,
pectin methylesterase; CEL, cellulase
⁎
Corresponding author at: College of Food Science, Southwest University, Chongqing 400715, PR China.
E-mail address: zengkaifang@hotmail.com (K. Zeng).

https://doi.org/10.1016/j.postharvbio.2018.07.007
Received 12 December 2017; Received in revised form 9 July 2018; Accepted 11 July 2018
0925-5214/ © 2018 Elsevier B.V. All rights reserved.
Y. Zhao et al.
have antimicrobial activity against Vaccinium corymbosum, Penicillium
digitatum, P. expansum, Alternaria alternata, Botrytis cinerea, Rhizopus
stolonifer, and M. fructicola based on different mechanisms (Cao et al.,
2015; Chan and Tian, 2005; Qin et al., 2004; Shao et al., 2015; Vieira
et al., 2016; Wang et al., 2015; Hernandez-Montiel et al., 2018), which
include competition for space and nutrients (Di Francesco et al., 2018,
2017); promotion of plant resistance (Parafati et al., 2016; Yan et al.,
2018); decrease in firmness-related enzymatic activity (Jongsri et al.,
2016; Luo et al., 2013); production of hydrolytic enzymes (Tian et al.,
2018; Zhou et al., 2016); generation of volatile organic compounds
(VOCs) (Hernandez-Montiel et al., 2018; Oro et al., 2018; Parafati et al.,
2017); induction of oxygen radical scavenging enzymes and oxidative
stress (Luo et al., 2013); and induction of lignin accumulation (Chan
et al., 2007; Deng et al., 2015; Luo et al., 2012). Although PAL and
lignin are relevant to wound healing (PAL, lignin), no direct study has
been conducted on the effects of chitosan and P. membranaefaciens on
wound healing. Moreover, wound healing is associated with appear-
ance change, including cell wall changes (Cajuste and Lafuente, 2007;
Carvajal et al., 2015; Vicente et al., 2013), which change during wound
healing (Neubauer et al., 2012; Spotts et al., 1998). However, the link
between wound healing and cell wall structure has not been studied.
Thus, the objectives of this study were as follow: (1) to determine the
effects of chitosan and P. membranaefaciens on wound healing and the
cell wall changes in citrus fruit with and without C. gloeosporioides in-
oculation, and (2) to study the relationship between wound healing and
cell wall components.
2. Materials and methods
2.1. Fruit and pathogen inoculum
Valencia orange [Citrus sinensis (L.) Osbeck] fruits were harvested at
commercial maturity and transported to the laboratory. The fruits
without wounds or rot were selected based on uniform size and color,
disinfected in 2 g L-1 sodium hypochlorite solution for 2 min, and air-
dried.
The fungal pathogen C. gloeosporioides (isolate Cg-7) was isolated
from infected citrus fruit in the laboratory and maintained on a potato
dextrose agar plate at 4 °C. The spore concentration of the suspensions
was determined using a hemocytometer and adjusted to 105 spores
mL−1 using sterile distilled water.
2.2. Treatment preparation
The Pichia membranaefaciens yeast strain was obtained from the
Chinese General Microbiological Culture Collection Center, Beijing,
China. P. membranaefaciens was prepared according to the method of
Zhou (Zhou et al., 2016). According to the method of Luo and Zhou
(Luo et al., 2012; Zhou et al., 2016), the yeast was re-suspended in
sterile distilled water and adjusted to an initial concentration of 108
cells mL−1.
Chitosan (Jinan Haidebei Marine Bioengineering Co., Ltd.,
Shangdong, China) with an intrinsic viscosity of 30 centipoise was
obtained. According to the method of Zhou (Zhou et al., 2016), the
chitosan was dissolved at 0.01 g of chitosan in 100 mL of sterilized
distilled water containing 0.1 % acetic acid and subsequently adjusted
to a pH value of 5.4.
2.3. Postharvest treatment and inoculation
Fruit was randomly divided into 2 treatment groups: group A and
group B, with 300 fruits for each group. For group A, 10 μL of (1)
distilled water was used as a control, (2) 0.01 % chitosan was added, or
(3) 108 cells mL−1 P. membranaefaciens yeast were placed in a 1 mm
wide, 2 mm deep wound made with a sterilized iron nail at 5 points in
the equatorial region of each fruit. For group B, the same treatments
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Postharvest Biology and Technology 145 (2018) 134–143
were applied as those in group A. After 24 h at room temperature, 10 μL
of 105 spores mL−1 pathogen was inoculated in the same wounds of
group B. After air-drying, all fruit were separately packed in plastic bags
and stored at 28 °C and 90 to 95 % room humidity (RH). The disease
incidence and lesion diameter of each fruit were measured at 6, 9, 12,
and 15 d during storage, with the lesion diameter measured without the
width of the hole. Each treatment was performed in three replicates
with ten fruits per replicate, and the experiment was conducted twice.
2.4. Sampling
The fruit pericarp was collected at 0, 3, 6, 9, 12, and 15 d and
sampled at 5 mm from the edge of the wound after storage. Tissue
samples were mixed and frozen immediately in liquid nitrogen and
stored at −40 °C. Each treatment was replicated three times.
2.5. Water content determination
Tissue samples (5 mm wide × 2 mm deep) were excised, including
the holes, with a sterilized hole puncher. Each fruit had 5 points in the
equatorial region, and 5 fruits were used for each treatment. The
samples were incubated at 65 °C for 12 h, and the results were calcu-
lated as the weight after drying divided by the fresh weight. Each
treatment was replicated three times.
2.6. Determination of pectin, cellulose and lignin
Pectin was extracted according to the methods of Cheng and
McComb and McCready (Cheng et al., 2008; McComb and McCready,
1952). Uronic acid concentrations in water-soluble pectin (WSP) and
acid-soluble pectin (ASP) were measured as previously reported by Bu
(Bu et al., 2013). The pectin concentration is expressed as mg GalA kg-1.
Cellulose was determined according to the method of Bu (Bu et al.,
2013), and the cellulose concentration is expressed as g kg-1. Lignin was
determined according to the method of Deng (Deng et al., 2015), and
the lignin concentration is expressed as ΔOD280 kg-1.
2.7. Enzyme extraction and assay
The pectin methylesterase (PME) activity was measured according
to the method of Carvajal (Carvajal et al., 2015) with some modifica-
tions. One gram of frozen tissue was ground in a mortar and extracted
at 4 °C with 5 mL of 1 M NaCl containing 1 % PVPP. The homogenate
was shaken for 4 h at 4 °C and then centrifuged at 4 °C and 8000 × g for
30 min. The supernatant was collected and adjusted to pH 7.5 with
1 mM NaOH. The reaction mixture contained 1 mL of enzymatic ex-
tract, 6 mL of 0.5 % (w v-1) pectin pH 7.5, 1.5 mL of 0.01 % (w v-1)
bromothymol blue pH 7.5, and 1 mL of H2O pH 7.5. The reduction in
the absorbance at 620 nm was followed for 20 min. The PME activity is
expressed as ΔOD620 s-1 kg-1 protein. The extraction method of poly-
galacturonase and cellulase was adapted from Zhang (Zhang et al.,
2010). The polygalacturonase (PG) activity was assayed according to
Zhang (Zhang et al., 2010), and PG activity is expressed as g GalA h-
1
kg-1 protein. The cellulase (CEL) activity was assayed according to
Carvajal (Carvajal et al., 2015). CEL activities were calculated using a
glucose standard curve and are expressed as g Glc h-1 kg-1 protein. The
PAL activity was measured according to the method of Gao (Gao et al.,
2016) and is expressed as ΔOD290 s-1 kg-1 protein.
2.8. Statistical analyses
The experiments were performed using a completely randomized
design. All statistical analyses for this experiment were performed using
the SPSS statistical software package (SPSS Inc., Chicago, IL, USA). Data
from repeated experiments were statistically analyzed using analysis of
variance (ANOVA) applied to percentages previously subjected to arc-
Y. Zhao et al.
sine transformation to assure homogeneity of variances. Fisher’s least
significant differences (LSD, p < 0.05) were determined to compare
differences between means. Data are presented as the mean and stan-
dard error of the mean (SE).
3. Results
3.1. Effects of chitosan and P. membranaefaciens treatments on controlling
anthracnose in citrus fruit
Chitosan and P. membranaefaciens with pathogen treatment sig-
nificantly reduced disease incidence and lesion diameter of citrus fruit
caused by inoculation with C. gloeosporioides compared with those of
the water control (p < 0.05), particularly the P. membranaefaciens
with pathogen treatment. At 15 d, the disease incidence of chitosan and
P. membranaefaciens with pathogen-treated fruit was significantly lower
than that of the control, with values that were 48 and 20 % lower,
respectively, than that in water with pathogen-treated fruit.
Furthermore, the lesion diameters of chitosan and P. membranaefaciens
with pathogen-treated fruits were 39 and 18 % lower, respectively, than
that of water with pathogen-treated fruit (Fig. 1).
Disinfected fruit were wounded, and 10 μL of each treatment was
injected into the wounds, followed by challenge inoculation with 10 μL
of 1 × 105 spores mL-1 suspension of C. gloeosporioides. Treatments
consisted of (A) sterile distilled water, (B) 0.01 % chitosan, and (C) P.
membranaefaciens (1 × 108 cells mL-1). Vertical bars indicate the stan-
dard error of the mean for three replicates. Values followed by different
letters indicate a significant difference according to Fisher’s least sig-
nificant difference (LSD) test at p < 0.05.
3.2. Effects of chitosan and P. membranaefaciens treatments on wound
appearance in wound tissues
The differences in the wound healing appearance among the dif-
ferent treatments were obvious (Fig. 2). The change in the control was
not obvious (Fig. 2A1–A3). For chitosan-treated fruit, the pericarp was
collapsed, shrunken, and hardened (Fig. 2A4–A6); we also found that
the color changed to brown in P. membranaefaciens-treated fruit, and
the change was similar in chitosan-treated fruit (Fig. 2A7–A9). After
inoculation with C. gloeosporioides in the water with pathogen-treated
fruit, the color of the pericarp around the wound changed to brown,
and then, the pericarp turned leathery, and the brown area was ex-
panded (Fig. 2B1–B3). Water loss was observed for chitosan with pa-
thogen-treated fruit pericarp at the beginning, and then, the pericarp
collapsed and turned brown (Fig. 2B4–B6). For P. membranaefaciens and
pathogen treated-fruit, the wound appearance did not change obviously
during storage, and the edge of wound showed visible dehydration and
became hardened (Fig. 2B7–B9).
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Postharvest Biology and Technology 145 (2018) 134–143
Fig. 1. Effects of chitosan and P. membranae-
faciens treatments on (A) disease incidence and
(B) lesion diameter of citrus fruit caused by C.
gloeosporioides. Disinfected fruit were wounded
and 10 μL of each treatment was injected into
the wounds, followed by challenge inoculation
with 10 μL of 1 × 105 spores mL-1 suspension
of C. gloeosporioides. Treatments consisted of
(A) sterile distilled water, (B) 0.01 % chitosan,
(C) P. membranaefaciens (1 × 108 cells mL-1).
Vertical bars indicate standard error of means
for three replicates. Values followed by dif-
ferent letters indicate a significant difference
according to Fisher’s least significant differ-
ence (LSD) test at p < 0.05.
3.3. Effects of chitosan and P. membranaefaciens treatments on water
content in wound tissues
As shown in Fig. 3, water content of each group decreased con-
tinuously during storage. The water content in the chitosan and P.
membranaefaciens treatments decreased significantly, and significant
differences were detected among the groups (p < 0.05). At 15 d, the
water content of the chitosan- and P. membranaefaciens-treated fruits
was significantly lower than that of the control (p < 0.05), with these
values 94 and 90 % lower, respectively, than that in the control fruit
(Fig. 3A). After inoculation of C. gloeosporioides, the chitosan and P.
membranaefaciens treatments slowed the speed of water loss, particu-
larly at the end of storage. At 15 d, the water content of the pathogen-
treated fruits in the chitosan and P. membranaefaciens groups was
higher than that of the pathogen-treated fruit in the water group, and
these values were 1.02 and 1.06-fold higher, respectively, than that of
the water group (Fig. 3B).
3.4. Effects of chitosan and P. membranaefaciens treatments on cell wall
components in wound tissues
As shown in Fig. 4A1 and A2, the WSP content showed stable in-
crease, whereas the ASP content showed a decrease after a slight in-
crease. The chitosan and P. membranaefaciens treatments reduced the
WSP content and increased the ASP content compared with those of the
control during the end of storage (p < 0.05), and the values were 0.49
and 0.76-fold lower, respectively, and 1.74 and 1.23-fold higher, re-
spectively, than those of the control at 15 d. As shown in Fig. 4A3, the
cellulose content of the control fluctuated, whereas the contents in the
chitosan and P. membranaefaciens treatments increased significantly
during storage (p < 0.05); the cellulose content was 1.23 and 1.40-fold
higher, respectively, than that in the control at 15 d. Lignin content of
each group increased continuously during storage. The chitosan and P.
membranaefaciens treatments significantly increased the lignin content,
with significant differences among the groups (p < 0.05). At 15 d, the
lignin content of chitosan- and P. membranaefaciens-treated fruit was
significantly higher than that of the controls (p < 0.05), and the con-
tents were 1.10 and 1.13-fold higher, respectively, than that of the
control fruit (Fig. 4A4).
After inoculation of C. gloeosporioides, as shown in Fig. 4B1 and B2,
the WSP content increased, whereas the ASP content decreased sig-
nificantly after a slight increase. The chitosan and P. membranaefaciens
with pathogen treatments significantly reduced the WSP contents
(p < 0.05), which were 0.62 and 0.52-fold lower, respectively, than
that in the water with pathogen-treated fruit at 15 d, and increased the
ASP contents, which increased 5.41 and 7.12-fold, respectively, com-
pared with that in the water with pathogen-treated fruit at 15 d. No-
tably, the ASP content of the water with pathogen treatment was sig-
nificantly higher than that in the other treatments (p < 0.05), with an
approximate 1.40-fold increase. As shown in Fig. 4B3, the contents of
Y. Zhao et al. Postharvest Biology and Technology 145 (2018) 134–143

Fig. 2. Effects of chitosan and P. membranaefaciens treat-

ments on wound appearance in wound tissues. (A) Effects
of chitosan and P. membranaefaciens treatment on wound
appearance after 1, 3 and 15 d. Disinfected fruit were
wounded and 10 μL of each treatment was injected into the
wounds. (B) Effects of chitosan and P. membranaefaciens
treatment interaction with C. gloeosporioides on wound
appearance after 1, 3, and 15 d. Disinfected fruit were
wounded and 10 μL of each treatment was injected into the
wounds, followed by challenge inoculation with 10 μL of
1 × 105 spores mL-1 suspension of C. gloeosporioides.
Treatments consisted of (A) sterile distilled water, (B) 0.01
% chitosan, (C) P. membranaefaciens (1 × 108 cells mL-1). 1,
2, and 3 indicate 1, 3 and 15 d, respectively.

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Y. Zhao et al.
cellulose decreased in each treatment, after a sharp increase from 0 to 6
d. The chitosan and P. membranaefaciens with pathogen treatments
significantly increased the cellulose content, particularly in the P.
membranaefaciens with pathogen treatment group (p < 0.05), and the
levels were 1.71 and 2.11-fold higher, respectively, than that in the
water with pathogen-treated fruit at 6 d. For the lignin content, the
three treatments all promoted lignin accumulation, and the water with
pathogen treatment increased the lignin content significantly
(p < 0.05), with values 1.19 and 1.12-fold higher than the values of
the chitosan and P. membranaefaciens with pathogen-treated fruits, re-
spectively (Fig. 4B4).
3.5. Effects of chitosan and P. membranaefaciens treatments on cell wall-
modifying enzymes in wound tissues
As shown in Fig. 5A1 and A2, PME and PG activities of control fruit
showed a trend of rapid increase. Clearly, the chitosan and P. mem-
branefaciens treatments significantly reduced the PME and PG activities
compared with those of the control (p < 0.05), which were 33.33 and
48.33 % lower, respectively, in the chitosan treatment and 39.46 and
68.86 % lower, respectively, in the P. membranefaciens treatment than
those of the control fruit at 12 d. For the CEL activity shown in Fig. 5A3,
the control fruit first showed an increase and then a decrease after
which the activity increased again and then decreased during storage.
CEL activity of chitosan- and P. membranaefaciens-treated fruits showed
a slight increase during the first 3 d and then fluctuated during storage;
the activity was 61.73 and 58.70 % lower, respectively, than that in the
control fruit at 12 d (p < 0.05). As shown in Fig. 5A4, the PAL activity
of each treatment showed a gradual increase, and the chitosan and P.
membranaefaciens treatments resulted in significantly higher values
than that in control fruit at 15 d, which were 1.07 and 1.11-fold higher
than that of the control, respectively (p < 0.05). Moreover, significant
differences in PME, PG and PAL activities were observed between
chitosan and P. membranaefaciens-treated fruits (p < 0.05).
After inoculation of C. gloeosporioides, PG activity of each treatment
showed a trend of increase (Fig. 5B1). Clearly, PG activity was reduced
by the chitosan and P. membranaefaciens with pathogen treatments;
with activity 78.28 and 73.33 % lower, respectively, than that of the
water with pathogen treatment group at 15 d (p < 0.05). PME activity
of each treatment showed a decreasing trend after a continuous in-
crease (Fig. 5B2). The chitosan and P. membranaefaciens with pathogen
treatments decreased PME activity (64.76 and 44.76 % lower, respec-
tively, at 15 d than that of the water with pathogen treatment), parti-
cularly the P. membranaefaciens with pathogen treatment (p < 0.05).
CEL activity is shown in Fig. 5B3, and the CEL activity trend was similar
to that of PG activity. The chitosan and P. membranaefaciens with pa-
thogen treatments resulted in lower CEL activity than that of the water
with pathogen treatment; the values were 82.27 and 74.57 % lower,
respectively, than the control values at 9 d (p < 0.05). We observed the
same trends in Fig. 5B4; the PAL activity of each treatment showed an
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Postharvest Biology and Technology 145 (2018) 134–143
Fig. 3. Effects of chitosan and P. membranaefa-
ciens treatments on water content in wound tis-
sues. (A) Effects of chitosan and P. membranaefa-
ciens treatment on water content in wound tissues.
(B) Effects of chitosan and P. membranaefaciens
treatment interaction with C. gloeosporioides on
water content in wound tissues.Vertical bars in-
dicate standard error of means for three re-
plicates. Vertical bars indicate standard error of
means for three replicates.
increase. Additionally, the chitosan and P. membranaefaciens with pa-
thogen treatments significantly decreased PAL activity before 9 d
(p < 0.05), which was 0.92 and 0.93-fold lower, respectively, than the
control value; then, the PAL activity in these treatments significantly
increased (p < 0.05), with increases of 1.08 and 1.11-fold, respec-
tively, compared with that of the control.
3.6. Correlation analysis between the cell wall components and water
content in citrus fruit treated by an antagonist
To elucidate the relationship between the cell wall and wound
healing before and after inoculation of C. gloeosporioides, we performed
correlation analyses between the cell wall components, the water
content, and the lignin content. The correlation plots for the control,
chitosan treatment, and P. membranaefaciens treatment, with the asso-
ciated p-values, are shown in Fig. 6A and B. ASP and cellulose were
strongly positively correlated with lignin; whereas water was strongly
negatively correlated with lignin. After inoculation of C. gloeosporioides,
the correlation plots for the water with pathogen and chitosan and P.
membranaefaciens with pathogen treatments, with the associated p-va-
lues, are shown in Fig. 6C and D. ASP, cellulose and lignin were strongly
negatively correlated with lignin.
4. Discussion
In our study, chitosan and P. membranaefaciens treatments sig-
nificantly decreased disease incidence and lesion diameter when C.
gloeosporioides was inoculated after incubation for 24 h at 30 °C.
Chitosan and P. membranaefaciens can directly control anthracnose,
producing hydrolytic enzymes (Zhou et al., 2016), but the wound
healing process can also resist anthracnose, as shown in previous stu-
dies. Vilanova et al. (2013) found that 10 d after wounding, P. digitatum
was effectively controlled on orange, and Kim et al. (2008) obtained
similar results with green peppers inoculated with C. acutatum 1 h after
wounding. These findings support the theory that the wound healing
process provides a necessary barrier to resist pathogen invasion
(Neubauer et al., 2012).
We found that wound appearance changed with different treat-
ments, regardless of the presence of the pathogen. Wound healing,
followed by dehydration, peel pitting and browning, was activated by
chitosan and P. membranaefaciens (Fig. 2). In tomato, Tao found that the
wound healing progress could control weight loss (Tao et al., 2016).
However, in our study, we found that wound healing was linked with
water loss of wound tissue (Fig. 3A). The explanation for this dis-
crepancy may be that Tao used tomato, whereas we used citrus fruit;
additionally, Tao measured the whole fruit weight, whereas we only
measured the wound tissue and wound-induced suberization processes:
the closing layer formation and wound periderm formation may cause
wound tissue water loss (Lulai and Neubauer, 2014). Furthermore, the
lesion caused by the pathogen induced serious browning and water loss,
Y. Zhao et al. Postharvest Biology and Technology 145 (2018) 134–143

Fig. 4. Effects of chitosan and P. membranaefaciens

treatments on cell wall components in wound tissues.
(A1) Effects of chitosan and P. membranaefaciens
treatment on water-soluble pectin in wound tissues.
(A2) Effects of chitosan and P. membranaefaciens
treatment on acid-soluble pectin in wound tissues. (A3)
Effects of chitosan and P. membranaefaciens treatment
on cellulose in wound tissues. (A4) Effects of chitosan
and P. membranaefaciens treatment on lignin in wound
tissues. (B1) Effects of chitosan and P. membranaefa-
ciens treatment interaction with C. gloeosporioides on
water-soluble pectin in wound tissues. (B2) Effects of
chitosan and P. membranaefaciens treatment interac-
tion with C. gloeosporioides on acid-soluble pectin in
wound tissues. (B3) Effects of chitosan and P. mem-
branaefaciens treatment interaction with C. gloeospor-
ioides on cellulose in wound tissues. (B4) Effects of
chitosan and P. membranaefaciens treatment interac-
tion with C. gloeosporioides on lignin in wound tissues.
Vertical bars indicate standard error of means for three
replicates.

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Y. Zhao et al. Postharvest Biology and Technology 145 (2018) 134–143

Fig. 5. Effects of chitosan and P. membranaefaciens

treatments on cell wall-modifying enzymes in wound
tissues. (A1) Effects of chitosan and P. membranae-
faciens treatment on PME activity in wound tissues.
(A2) Effects of chitosan and P. membranaefaciens
treatment on PG activity in wound tissues. (A3)
Effects of chitosan and P. membranaefaciens treat-
ment on CEL activity in wound tissues. (A4) Effects
of chitosan and P. membranaefaciens treatment on
PAL activity in wound tissues. (B1) Effects of chit-
osan and P. membranaefaciens treatment interaction
with C. gloeosporioides on PME activity in wound
tissues. (B2) Effects of chitosan and P. membranae-
faciens treatment interaction with C. gloeosporioides
on PG activity in wound tissues. (B3) Effects of
chitosan and P. membranaefaciens treatment inter-
action with C. gloeosporioides on CEL activity in
wound tissues. (B4) Effects of chitosan and P. mem-
branaefaciens treatment interaction with C. gloeos-
porioides on PAL activity in wound tissues. Vertical
bars indicate standard error of means for three re-
plicates.

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and wound healing slowed this trend (Fig. 1), because the disease spot
is linked with dead cells (Carvajal et al., 2015); thus, we found wound
healing decreased the cell death caused by C. gloeosporioides, as shown
in Fig. 4B, similar to blue mold control on wild apple (Janisiewicz et al.,
2016).
To study the possible connection between cell wall metabolism and
wound healing during storage, we assessed the cell wall components.
We found that chitosan and P. membranaefaciens treatments increased
the pectin content and delayed ASP dissolving to WSP (Fig. 4A1 and
A2). Chitosan and P. membranaefaciens treatments prevented the solu-
bilization of loosely bound pectin, and the treated fruits showed high
levels of tightly bound pectin, similar to benzylaminopurine-treated
squash (Massolo et al., 2014). We hypothesized that during wound
healing, the ASP will accumulate more quickly and limit its transfer to
WSP. Benhamou found that citrus fruit treated by P. digitatum showed
cellulose accumulation on the cell wall (Benhamou, 2004). In this
study, we found that the C. gloeosporioides significantly increased the
ASP content, indicating that pathogens can induce structural resistance,
and that different pathogens can induce different substances. We also
found that wound healing delayed the pectin degradation caused by the
pathogen (Fig. 4B1and B2). Cellulose is the major constituent of the cell
wall and is as important as pectin to maintain the structural integrity
(Bennett and Labavitch, 2008). The chitosan and P. membranaefaciens
treatments significantly increased the cellulose content (Fig. 4A3). After
inoculation of C. gloeosporioides, the cellulose content decreased after a
small increase during storage (Fig. 4B3). However, the decrease was
prevented by the chitosan and P. membranaefaciens treatments. Typi-
cally, during fruit storage and pathogen infection, the cellulose levels
decrease (Chassot et al., 2007; Cheng et al., 2009; Zhou et al., 2015).
Therefore, the results demonstrated that wound healing can result in
cellulose accumulation and delay cellulose degradation.
The wound response resulted in the accumulation of lignin around
the wounds. In many studies, the wound healing process is correlated
with lignin accumulation in oranges (Vilanova et al., 2013, 2012). In
this paper, we found that chitosan or P. membranaefaciens treatment
significantly increased the lignin content (Fig. 4A4), similar to results
141
Postharvest Biology and Technology 145 (2018) 134–143
Fig. 6. Correlation analysis between the cell
wall components and water content in citrus.
(A, B) Correlation plot between the lignin,
WSP, ASP, cellulose, water in citrus treated
with antagonist. (C, D) Correlation plot be-
tween the lignin, WSP, ASP, cellulose, water in
citrus treated with antagonist and C. gloeos-
porioides. The heat map is described as positive
values set to red color and negative values set
to blue color. Correlations with p < 0.05 are
indicated by *, and p < 0.01 by **.
previously reported by Liu and Deng (Deng et al., 2015; Liu et al.,
2010). Furthermore, after inoculation of C. gloeosporioides, the contents
in all treatments groups were higher than those in the non-inoculated
group (Fig. 4B4). Additionally, the contents in the pathogen treatment
groups were the highest among all treatments. The results are con-
sistent with those of Vilanova, Angel Torres, and Hueckelhoven, in-
dicating that lignin accumulation plays an important role against pa-
thogens and may indicate a healing process that retards damage caused
by C. gloeosporioides. However, C. gloeosporioides is able to overcome the
defense and result in disease.
PME, PG, and CEL are crucial enzymes for cell wall degradation. In
this paper, chitosan or P. membranaefaciens treatment decreased the
activities of PME, PG, and CEL regardless of the presence of C. gloeos-
porioides. After inoculation of C. gloeosporioides, the activities of PME,
PG, and CEL all increased compared with those of the non-inoculated
group, particularly CEL, indicating that pathogen invasion could pro-
duce and induce plant accumulation of cell wall-degrading enzymes
(CWDEs) (Bellincampi et al., 2013). PAL is the key enzyme related to
induction of lignin. The results showed that chitosan or P. membra-
naefaciens treatment could increase PAL activity. After inoculation of C.
gloeosporioides, the PAL activity increased significantly, possibly be-
cause PAL is important for induction of disease resistance in plants after
inoculation of a pathogen (Casals et al., 2010). Therefore, we found that
wound healing could decrease the CWDEs and eventually increase PAL
enzyme activity.
Based on relevant analyses, we found that the ASP and cellulose
were positively correlated with lignin, and lignin was confirmed as an
index of wound healing (Ismail and Brown, 1979; Vilanova et al.,
2014), which indicated that during the wound healing process, the ASP
and cellulose accumulation was accompanied by lignin accumulation.
Therefore, we propose the ASP and cellulose contents as candidate in-
dexes to assess the wound healing process. We found that after in-
oculation of C. gloeosporioides, the relations between ASP and cellulose
and lignin changed to strongly negative correlations, because lignin
quickly accumulates in the wound, which may be because the pathogen
reduces the host defense (Benhamou, 2004); thus, this effect is much
Y. Zhao et al. Postharvest Biology and Technology 145 (2018) 134–143

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