Postharvest Biology and Technology 86 (2013) 437446

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Postharvest Biology and Technology

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Effects of hot water treatment on anthracnose disease in papaya fruit

and its possible mechanism
Xueping Li 1 , Xiaoyang Zhu 1 , Nan Zhao, Danwen Fu, Jun Li, Wen Chen, Weixin Chen
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory for Postharvest Science and
Technology of Fruits and Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, PR China

a r t i c l e

i n f o

Article history:
Received 11 May 2013
Accepted 22 July 2013
Keywords:
Hot water treatment
Fruit ripening
Epicuticular wax
Disease resistance
Papaya

a b s t r a c t
Harvested papaya fruit are perishable due to rapid ripening and softening and susceptibility to biotic or
abiotic stresses. Hot water treatment (HWT) can preserve fruit quality by reducing decay. The present
study investigated effects of HWT on controlling fungal pathogens of papaya fruit and the possible mechanism by which HWT induced disease resistance. HWT (54 C, 4 min) of papaya fruit had a pronounced
effect on reducing the carrier rate of Colletotrichum gloeosporioides (C. gloeosporioides) in fruit peel, significantly inhibited the incidence of anthracnose and stem-end rot, effectively delayed fruit softening, but
slightly promoted the rate of fruit coloring. HWT reduced the anthracnose index and fruit ripeness to a
certain extent and induced changes in the wax arrangement on the surface of treated fruit, causing the
wax to melt. The cracks and most stomata appeared to be partially or completely plugged by the melted
wax, thereby providing a mechanical barrier against wound pathogens. HWT induced the expression of
CpPGIP and promptly induced the expression of CpNPR1, and then regulated the expression of the CpPR1
gene, which may enhance the resistance of the fruit to anthracnose disease and reduce the decay rate.
Together, these results conrm that HWT could reduce disease incidence and induce resistance, and thus
maintain postharvest quality during storage and prolong the shelf-life of papaya fruit.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Papaya (Carica papaya L.) is a climacteric fruit and very perishable after harvesting, with rapid ripening and softening, and
susceptibility to biotic or abiotic stresses (Kader, 2002). An import
factor that may affect papaya postharvest quality and limit the
extension of storage life is the occurrence of rots. Anthracnose
is one of the most important postharvest diseases in papaya (Cia
et al., 2007), as well as other fruit (Shi et al., 2011). Postharvest diseases, especially anthracnose, can infect young papaya
fruit and remain latent during fruit growth in the eld Thus it is
hard to prevent disease development, and this may signicantly
reduce quality and commercial value of the fruit during storage
and transport. Anthracnose is usually controlled by application
of postharvest fungicides such as prochloraz or propiconazole.
However, development of fungicide resistance, and public concern over the potential impact of fungicides on human health and
the environment have created interest in seeking new alternative
strategies for disease management (Gamagae et al., 2004; Droby
et al., 2009). Extensive research is imperative to develop advanced

Corresponding author. Tel.: +86 20 38294892; fax: +86 20 85288280.

E-mail address: wxchen@scau.edu.cn (W. Chen).
1
These authors contributed equally to this work.
0925-5214/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.postharvbio.2013.07.037

postharvest treatments in environmentally friendly ways to

maintain high commercial quality during the storage and marketing period. Different postharvest diseases of various fruit have
been effectively controlled using modied atmosphere packaging (Karabulut and Baykal, 2004), sodium chloride (Malakou and
Nanos, 2005), biocontrol (Shi et al., 2010), antagonistic microorganisms (Calvo et al., 2010), salicylic acid (Chan et al., 2008), borate
application (Shi et al., 2011, 2012), etc.
Among various non-chemical approaches, hot water treatment
(HWT) appears to be one of the most effective and promising methods, especially for organically grown crops, to control relatively
high rates of postharvest decay in environmentally friendly ways
(Mari et al., 2007; Jemric et al., 2011; Fruk et al., 2012; Liu et al.,
2012). HWT has been known for a relatively long time as an effective and economic method for controlling plant pathogens (Rodov
et al., 1995, 2000; Schirra and DHallewin, 1997; Hong et al., 2007;
Liu et al., 2012), improving fruit resistance to chilling (Rodov et al.,
1995; Schirra et al., 2004; Lu et al., 2010a; Rui et al., 2010), inhibiting
ripening of many fruit and vegetables, and alleviating some physiological storage disorders (Fallik, 2004; Ciou et al., 2011; Jemric et al.,
2011), thus, maintaining fruit quality and prolonging storage (Klein
and Lurie, 1991; Malakou and Nanos, 2005; Fruk et al., 2012; Liu
et al., 2012). There has therefore, been increasing interest in using
postharvest heat treatments for fruit. Ripening of most climacteric fruit is characterized by softening of the esh, enhanced color

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X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

development, increased sugar/acid ratio, respiratory activity and

ethylene production (Prasanna et al., 2007). Exposing fruit to high
temperatures retards some of these processes while enhancing
others. The physiological and biochemical changes in heat-treated
fruit are more advanced in some ripening characteristics than nontreated fruit, thus maintaining the fruit quality for a longer period
during storage and shelf-life.
Postharvest decay controlled by HWT, however, involves effects
on both plant pathogen and plant host (Pavoncello et al., 2001; Liu
et al., 2012). Several studies have reported that HWT had a pronounced effect on decreasing decay, reducing chilling injury and
maintaining quality of fruit (Lu et al., 2010a; Fruk et al., 2012).
However, improper temperature and duration of HWT can have
a negative impact on fruit quality. On the other hand, other studies
have reported that HWT could directly inhibit fungal germination
and growth, or even kill the fungus (Jemric et al., 2011; Liu et al.,
2012). With regard to the mechanisms of control of postharvest
decay by HWT, several studies have indicated that HWT could
induce a defense response of fruit and vegetables, sequentially
preventing the pathogen spreading throughout the tissues (BenYehoshua et al., 1995; Fallik et al., 1996b). With papaya fruit, while
a few studies have applied heat treatments, including hot air and
hot water, to control postharvest decay and for other commercial
applications (Chan Jr et al., 1981; Nishijima et al., 1992; Lay-Yee
et al., 1998; Kader, 2002; Diczbalis-Deedi et al., 2012), no systematic studies on effects of postharvest HWT on papaya fruit and the
underlying molecular biology mechanisms have been reported.
Expansins are known to participate in several processes during
plant growth and development, particularly where wall extension
and cell expansion are required. They are also suggested to prepare
cell walls for subsequent degradation by cell wall hydrolases during
ripening, particularly in climacteric fruit. As a class of nonenzymatic
cell wall proteins, expansins have been found to play an important
role in cell wall loosening and extension (Cosgrove, 2000). Close
relationships between expansins and postharvest fruit softening
have been shown in a number of fruit (Wang et al., 2006; Yang et al.,
2008). Fruit rmness is related to fruit quality and is negatively
associated with the incidence of preharvest and postharvest rots
(Barritt, 1980).
In the present study, a simple and effective HWT (54 C, 4 min)
for papaya fruit was screened and the physiological basis and
molecular biology mechanism of HWT-induced systemic resistance
studied. The overall objective of this work was to elucidate the
physiological defense reactions and the possible molecular mechanisms of systemically acquired resistance.

2. Materials and methods

2.1. Plant materials and hot water treatment
Papaya fruit (Carica papaya cv. Sunrise) at physiological maturity indicated by a color break stage of 1015% yellow, were
harvested from a local commercial plantation near Guangzhou,
South China, transported to the laboratory and sorted by size, shape
and maturity. Uniform fruit free from visual symptoms of any disease or blemishes were randomly selected and rst cleaned, and
dipped in a 0.3% hypochloride solution for 10 min. Preliminary
investigations revealed that treatment with 54 C for 4 min was the
most effective in controlling diseases and/or delaying the ripening of papaya fruit. The selected papaya fruit were dipped into hot
water (54 C) for 4 min, taken out and then allowed to air-dry at
25 C. The control group was dipped into water at normal temperature water (25 C) for 4 min. Every treatment was repeated three
times and each treatment included 90 fruit. Thereafter, fruit were
placed into unsealed plastic bags (0.02 mm thick) and stored at

25 C. Samples were collected at 0, 12, 24 and 36 h and 2, 3, 4, 5,

6, 7, 8, 9 and 10 days after treatment. For all samples, peel around
the equatorial fruit, about 2 mm thick, and 2 cm thick esh, were
collected, then frozen in liquid nitrogen and stored at 80 C until
use.
2.2. Color index and disease index evaluation
Fruit peel color and coloring index were calculated as in our
previous study (Zhu et al., 2012). Disease severity of anthracnose in terms of lesion diameter was classied into nine grades
according to Lay-Yee et al. (1998): 0 = without lesion; 1 = 15 mm
lesion diameter = <20; 2 = 615 mm lesion diameter; 3 = lesion proportion = <1/16; 4 = lesion proportion = <1/8; 5 = 1/8 = <lesion proportion = <1/4; 6 = 1/4 = <lesion proportion = <1/2; 7 = 1/2 = < lesion
proportion = <2/3; 8 = decay completely. Use of this empirical
scale made it possible to calculate a disease index (DI) showing the average disease
severity as a proportion of the maximum
disease severity. DI = [ (disease grade number of fruit with disease)/(total number of fruit maximum disease grade)] 100.
Periodic observations were made for incidence of stem-end rot,
the severity of the stem-end rot disease being measured with the
following scale: 0 = no disease; 1 = the proportion of the stem-end
portion surface affected with disease <25%; 2 = the proportion of
the stem-end portion surface affected with disease 2550%; 3 = the
proportion of the stem-end portion surface affected with disease
5075%; 4 = the proportion of the stem-end portion surface affected
with disease
 <75%; 5 = the disease spread to the other part of the
fruit. DI = [ (disease grade number of fruit with disease)/(total
number of fruit maximum disease grade)] 100.
2.3. The determination of carrier rate of Colletotrichum
gloeosporioides in fruit peel
After being stored for 4 days, Colletotrichum gloeosporioides (C.
gloeosporioides) was isolated from fruit peel both in the control
fruit and the HWT-fruit. Fruit peel was cut into 2 mm 2 mm portions, 1 mm thick, soaked in 75% alcohol for 10 s, and then soaked in
0.5% mercuric chloride for 2 min. The treated peel was then washed
with sterile water 3 times and maintained on potato dextrose agar
(PDA) at 28 C for 34 days. The carrier rate of C. gloeosporioides was
statistically analyzed according to the following formula:
The carrier rate of C. gloeosporioides
=

 number of fruit with C. gloeosporioides 

total number of fruit

100.

Thirty fruit were used for each treatment and each treatment was
replicated three times.
During the C. gloeosporioides isolation process, the treated peels
were maintained on potato dextrose agar (PDA) and used to isolate
C. gloeosporioides. Whether or not the fungi were latent or active in
the fruit peel, it could develop into colonies in the PDA after a few
days of incubation.
2.4. Determination of pectin content
The method of Wang et al. (2008) was used to determine pectin
content. A mixture of 5 g of sample powder and 30 mL of hot absolute ethanol was heated in a centrifuge tube for 10 min in a boiling
water bath and centrifuged at 10,000 rpm for 10 min at 4 C. The
residues were dried for 24 h at 35 C, and alcohol insoluble solids
(AIS) were obtained. One milliliter of water was added drop-wise
with stirring, for 35 min to a mixture of 5 mg of AIS and 2 mL of
concentrated sulfuric acid, in a test-tube until the AIS were dissolved. The mixture was transferred to a 25 mL volumetric ask

X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

and made up to volume with distilled water for total pectin examination.
A mixture of 80 mg of AIS and 20 mL of distilled water was
stirred in a centrifuge tube for 5 min at room temperature and the
centrifuged at 10,000 rpm for 10 min at 4 C. The residues were
extracted twice with 2 20 mL of distilled water. All the supernatants were transferred into a 100 mL volumetric ask and made
up to volume with distilled water for water soluble pectin examination.
In an ice bath, 1 mL of the above sample solution was added to
6 mL of 12.5 mM sodium tetraborate (in concentrated sulfuric acid)
and then heated for 5 min in a boiling water bath. Color development followed addition of 0.1 mL of 0.15% m-hydroxydiphenyl and
incubation for 20 min at room temperature. NaOH (0.1 mL) was
added instead of 0.15% m-hydroxydiphenyl to the control. Both
total pectin and water soluble contents were expressed as galacturonic acid equivalents.

439

Hybridization was then performed overnight in the same buffer

containing the gene-specic digoxin (DIG)-labeled probes at 45 C.
Probes were prepared with a DIG probe synthesis kit (Roche Diagnostics) by following the manufacturers instructions. Following
hybridization, membranes were washed twice for 10 min with 2
SSC containing 0.1% SDS at 37 C, followed by washing twice in 0.1
SSC containing 0.1% SDS for 30 min at 63 C. The chemiluminescence signals were detected using CDP-Star TM (Roche Diagnostics,
Gmbh, Mannheim, Germany) as described by the manufacturer.
For each treatment (membrane), three repeats were performed.
The specic primers used for synthesis of DIG-labeled probes were
identical to those used for RT-PCR listed in Supplementary Table 1.

2.5. Scanning electron microscopy (SEM) analysis

SEM analysis was conducted by the method described by Hu
et al. (Hu et al., 2012), with minor modications. A small piece of
tissue (2 mm 2 mm 1 mm) was torn with forceps, xed and preserved in a xative solution (90 mL of 50% ethanol, 5 mL of formalin,
and 5 mL of acetic acid) until further processing. Before scanning,
the slices were dehydrated in a series of ethanol solutions and
dried at a critical point of liquid CO2 in a desiccator. The specimens
were mounted onto aluminum pecimen stubs using conductive
silver glue and sputter-coated with gold. SEM scanning was carried out with an XL-30 scanning electron microscope (Philips, The
Netherlands) at 20 kV.
2.6. RNA extraction and genes clone
Total RNA was extracted using the hot borate method described
by Wan and Wilkins (1994) and then treated with DNAse I digestion
using an RNAse-free kit (TaKaRa, Japan) to eliminate the potential DNA contamination. The RNA concentration and purity were
evaluated. Those RNA samples that had fully lled the criteria
(i.e. OD260/280 > 1.8 and OD260/230 > 2.0) were used for further
analyses. The extracted total RNA was reversed transcribed with
reverse transcription kit into cDNA, which were used as templates
for RT-PCR. The product (the rst-strand cDNA) was subjected to
PCR amplication. The primer pairs of CpEXP1, CpPGIP, CpNPR1,
and CpPR1 were designed based on the sequences obtained from
NCBI and CpEXP2 was cloned by using degenerate primers. All the
primers were listed in Supplementary Table 1. PCR were subjected
to one cycle of 94 C for 3 min, 35 cycles each at 94 C for 1 min, 52 C
for 45 s and 72 C for 2 min, and then one cycle of 72 C for 10 min.
PCR products of the predicted size were puried and cloned into
pGEM-Teasy vector (Promega, Madison, WI, USA) and sequenced
by Beijing Genomics Institute (BGI, China).
2.7. Northern blot analysis
Total RNA (10 g per lane) from each sample was separated
by electrophoresis on 1.0% agarose-formadehyde gel in 1 MOPS
(morpholinopropanesulfonic acid) buffer and capillary blotted onto
positively-charged nylon membrane (Biodyne B, 0.45 m, PALL
Co., Sarasota, FL). The RNA was xed to the membrane by baking for 2 h at 80 C and then cross-linked to the membranes using
an ultraviolet cross linker (Amersham Biosciences, Piscataway, NJ).
The membranes were pre-hybridized in SDS buffer [50% deionized formamide (v/v), 5 SSC, 7% SDS, 2% blocking reagent (Roche
Diagnostics, Mannheim, Germany), 50 mM sodium-phosphate
(pH 7.0) and 0.1% N-lauroylsarcosine (w/v)] for more than 3 h.

Fig. 1. Color index (A), water soluble pectin content (B) and total pectin content (C)
of hot water treated fruit or untreated fruit (control) during ripening at 25 C. Each
data point represents a mean standard error (n = 3).

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X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

2.8. Statistical analysis

All experiments were arranged in completely randomized
design, and each treatment comprised of three replicates. Data
were tested by the analysis of variance using SPSS version 16.0.
The Duncans multiple range tests (P < 0.05) were used to compare
differences among mean values.

Correlation analysis showed that fruit rmness with water

soluble pectin, PME activities and PG activities displayed negative
correlations. Fruit rmness was signicantly and negatively correlated with WSP context (R2 = 0.9407) (Supplementary Fig. 2A) and
PME activities (R2 = 0.9958) (Supplementary Fig. 2D). However, no
obviously close relationship was observed between fruit rmness
and total soluble pectin (Supplementary Fig. 2B).

3. Results

3.2. The effect on carrier rate of C. gloeosporioides in fruit peel

3.1. Fruit evaluation during storage

As shown in Fig. 2A, HWT had a strong impact on carrier rate

of C. gloeosporioides in fruit peel. After 4 days storage, the carrier
rate of C. gloeosporioides in control fruit was about 48%, compared
to 20% in the HWT fruit, HWT effectively reduced the carrier rate
of C. gloeosporioides in peel by 58.3%.

Changes in visual inspection of the peel color, respiration, and

ethylene for at least thirty fruit per treatment group were periodically monitored. As shown in Fig. 1A, fruit color index gradually
increased as a function of storage time and almost turned yellow at
6 days in the HWT-group, but the control fruit fully turned yellow
at 8 days. Clearly the HWT accelerated peel de-greening of the
fruit (Fig. 1A).
Correlation analysis showed that ethylene production, color
change and respiration rate had positive correlations. Signicant
correlation was observed between the ethylene production and
the respiration rate (R2 = 0.8151) (Supplementary Fig. 1).
Pectin degradation plays an important role in fruit softening.
As shown in Fig. 1B, total pectin showed a moderate increase in
the earlier period but decreased rapidly in later stages. During the
later storage times, the level of total pectin in the HWT-fruit was
higher than that in the control fruit. HWT signicantly delayed
and reduced papaya fruit total pectin decrease during the later
storage period. In contrast, water soluble pectin (WSP) content
gradually increased during the entire storage time (Fig. 1C), and
WSP in HWT-fruit was obviously lower than in the control fruit.

3.3. Effect of hot water treatment on disease inhibition

HWT signicantly reduced the incidence of decay. As shown in
Fig. 2B, HWT signicantly prevented anthracnose. The DI was 13.12
in the control fruit at 6 d but was only 4.37 in the HWT-fruit, with
a maximum inhibition of 66.6%. At 7 d after treatment, the control fruit were nearly decayed with a DI of 23.35, whereas the DI
for the HWT-fruit was 10.37. The controlling effect reached a level
of 55.4%. At 8 d after treatment, the control fruit were over-ripen
and anthracnose expanded rapidly, with a DI of 43.34. However,
anthracnose expanded at a relatively low rate in the treated fruit,
with a DI of 27.12. The controlling effect reached a level of 37.42%.
HWT signicantly controlled stem-end rot as well. As shown in
Fig. 2C, HWT signicantly reduced stem-end rot incidence and DI
(P < 0.05). At 7 d after treatment, the disease incidence was 35.3%
that of the control fruit, compare with a disease incidence of 11.8%

Fig. 2. The effects of HWT on the carrier rate of Colletotrichum and disease control. (A) The carrier rate of Colletotrichum gloeosporioides in fruit peel. (B) The effect of HWT
on control of anthracnose. (C and D) The effect of hot water treatment on control of stem end rot. Each data point represents a mean standard error (n = 3). *Signicantly
different at P 0.05.

X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

441

wax melted to oil drop dimensions and unevenly covered the fruit
surface in the treated fruit (Fig. 4B1). There were several natural
openings on the untreated fruit surface (Fig. 4A2 and A3), but the
openings or stomas were sealed with molten wax material caused
by HWT (Fig. 4B2 and B3). It is supposed that the molten wax caused
by HWT partially or entirely sealed natural openings in the epidermis, and this could physically prevent the invasion of the pathogen,
and thus reduce disease incidence.
3.6. Differential expression of CpEXP1 and CpEXP2

Fig. 3. Relative correlation coefcient of fruit ripeness with disease index.

in HWT-fruit, which was reduced by almost 3-fold as compared to

that of the control fruit. The DI in HWT-fruit was 9.88 at 7 d, which
was 50% that of the control (Fig. 2D).

Two expansin genes, CpEXP1 and CpEXP2, were cloned and their
expression patterns were investigated in papaya fruit peel and pulp.
As shown in Figs. 5 and 6, the stable levels of CpEXP1 and CpEXP2
mRNA gradually increased throughout the entire storage period,
both in peel and pulp with fruit ripening. CpEXP1 expression in the
HWH-fruit of peel was signicantly lower than that in the control
fruit (Fig. 5). However, the CpEXP2 mRNA levels in HWT-fruit were
slightly higher than those in the control fruit during the rst 2 days,
but no noticeable differences were observed thereafter (Fig. 5). The
results showed that HWT inhibited CpEXP1 expression but slightly
induced CpEXP2 expression during the early period in the peel. Similar changes were observed in the pulp (Fig. 6), but the inhibition of
CpEXP1 expression by HWT in pulp was more severe than in peel.

3.4. Correlation of anthracnose index and fruit ripeness

The relationship between anthracnose index and fruit ripeness
is shown in Fig. 3. The DI was signicantly and positively correlated with fruit ripeness (Fig. 3). In the control fruit, the correlation
was highly signicant (P < 0.01), with a correlation coefcient of
0.9796. Signicant correlation was also observed between anthracnose index and fruit ripeness in the treated fruit (P < 0.05), with
a relatively low correlation coefcient of 0.8535. HWT reduced
the correlation coefcient between anthracnose index and fruit
ripeness to some extent. These results indicated that HWT signicantly delayed the development of anthracnose.
3.5. The effects of hot water treatment on structural changes of
epicuticular wax
As shown in Fig. 4, the wax material on the untreated fruit was
regularly arrayed as ovuliferous scales (Fig. 4A1). However, the

3.7. Regulation of expression of CpPGIP, CpNPR1, CpPR1 genes by

hot water treatment
CpPGIP expression decreased during storage, as the fruit ripened
and with severe levels of disease. However, CpPGIP expression
transiently increased at 36 h in HWT-fruit and was higher than that
in the control fruit in peel (Fig. 7). A similar scenario was shown
in the papaya fruit pulp (Fig. 8), but the expression of CpPGIP was
lower in peel later than in pulp. These results indicated that CpPGIP
expression in both the peel and pulp were enhanced by HWT
(Figs. 7 and 8).
Both CpNPR1 and CpPR1 were cloned in papaya fruit. As shown
in Figs. 7 and 8, CpNPR1 was expressed more strongly in peel than
in pulp. In peel, the steady state levels of CpNPR1 mRNA remained
almost unchanged throughout the rst 4 d period in the control
fruit, but they gradually decreased from 5 d to 9 d (Fig. 7). However,
CpNPR1 was profoundly induced by HWT, increasing signicantly

Fig. 4. Structural changes of epicuticular wax induced by hot water treatment.

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X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

Fig. 5. Differential expression patterns of CpEXP1 and CpEXP2 in the pericarp during ripening at 25 C. Total RNA (10 g per lane) was separated by electrophoresis on
1.0% agarose-formadehyde gel and hybridized with DIG-labeled probes. The top section showed the RNA gel blot analysis while the bottom section indicated the ethidium
bromide-stained rRNA bands as a loading control of the gel. The graphics below indicated the ratio between the signal detected in each lane and the corresponding upper
band of the ethidium bromide stained gel (integrating optical density: IOD) using GelPro Analyzer 4.0 software.

Fig. 6. Differential expression patterns of CpEXP1 and CpEXP2 in the peel and pulp during ripening at 25 C. Total RNA (10 g per lane) was separated by electrophoresis on
1.0% agarose-formadehyde gel and hybridized with DIG-labeled probes. The top section showed the RNA gel blot analysis while the bottom section indicated the ethidium
bromide-stained rRNA bands as a loading control of the gel. The graphics below indicated the ratio between the signal detected in each lane and the corresponding upper
band of the ethidium bromide stained gel (IOD) using GelPro Analyzer 4.0 software.

X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

443

Fig. 7. Regulation of CpPGIP, CpNPR1 and CpPR1 in HWT-fruit in pericarp. Total RNA (10 g per lane) was separated by electrophoresis on 1.0% agarose-formadehyde gel and
hybridized with DIG-labeled probes. Ethidium bromide-stained rRNA was used as the loading control. The graphics below indicate the ratio between the signal detected in
each lane and the corresponding upper band of the ethidium bromide stained gel (IOD) using GelPro Analyzer 4.0 software.

from 12 h to 48 h in treated fruit and then gradually decreasing to

a low level. The level in HWT fruit was still higher than that in the
control fruit during the entire storage period (Fig. 7). CpNPR1 was
expressed weakly in the pulp both in control and treated fruit and
no signicant difference was found between both groups except
at 2 d (Fig. 8). These results indicated that HWT could induce the
expression of CpNPR1 in peel of papaya fruit.
The expression of CpPR1 gene showed a close relationship with
pathogen infection. As shown in Fig. 7, CpPR1 accumulation could
only be detected after 3 d and was dramatically increased from 5 d
to 9 d with the occurrence of pathogen, which severely infected
peel in control fruit (Fig. 7). In HWT-fruit, CpPR1 accumulation
was delayed and was only detected after 5 d, then dramatically
increased. However, its expression was much lower than that in
control fruit (Fig. 7). In contrast to expression in peel, CpPR1 was
undetectable in the pulp tissues during the whole storage period
(Fig. 8). These results showed that CpPR1 expression was profoundly induced by the pathogen and inhibited by HWT.
4. Discussion
Papaya is a typical climacteric fruit. Ethylene production and
respiration rate increase as fruit ripen. HWT has drawn increasing
attention due to the growing demand of reducing environmental pollution caused by postharvest use of chemical fungicides.

Recently, we observed that HWT delayed ripening and reduced

endogenous ethylene production and the respiration rate in
postharvest papaya fruit (Zhao et al., 2008). In present study, we
observed that HWT also controlled the incidence of postharvest
disease (Fig. 2BD). The inhibition of ripening by HWT may be
mediated by its effect on ethylene (Luo et al., 2010). It has been
reported that hot air treatments of 3540 C inhibit ethylene
synthesis within hours in mangoes, apples and tomatoes (Biggs
et al., 1988; Klein, 1989; Ketsa et al., 1999). In the present study,
the decreased ethylene production caused by HWT contributed to
the delayed ripening of the fruit. Some studies have reported that
reduced ethylene production in HWT-fruit might be attributed to
the reduced activities of 1-aminocyclopropane-1-carboxylic acid
synthase (ACS) and ACC oxidase (Atta-Aly, 1992; Ketsa et al., 1999).
However, the mechanism of the inhibition of ethylene synthesis by
HWT needs further investigation. Ethylene is also associated with
disease incidence, as change in ethylene production is a response
to many pathogen infections in plants. Pathogens induce ethylene
biosynthesis and produce ethylene themselves as well. Our work
has revealed that HWT played a role in inhibiting ethylene production and respiration rate, which may be responsible for its effect on
controlling fruit pathogens, as well on inducing fruit physiological
change.
Many studies have reported that appropriate heat treatment
delayed fruit softening, and extended postharvest life of fruit such

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X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

Fig. 8. Regulation of CpPGIP, CpNPR1 and CpPR1 in HWT-fruit in pulp. Total RNA (10 g per lane) was separated by electrophoresis on 1.0% agarose-formadehyde gel and
hybridized with DIG-labeled probes. Ethidium bromide-stained rRNA was used as the loading control. The graphics below indicate the ratio between the signal detected in
each lane and the corresponding upper band of the ethidium bromide stained gel (IOD) using GelPro Analyzer 4.0 software.

as nectarine (Fruk et al., 2012) and tomatoes (Lurie and Sabehat,

1997), whereas some reduced rmness of treated tomatoes (Lu
et al., 2010a,b). Our previous study has shown that HWT (54 C,
4 min) delayed fruit softening and maintained postharvest quality
(Zhao et al., 2008). The decrease in softening rate may be due to
inhibition of the synthesis of cell wall hydrolytic enzymes such as
PG and PME (Zhao et al., 2008). Pectin degradation plays an important role in fruit softening, which leads to disassembly of cellulose
and the hemicellulose network (Liu et al., 2009). In this work, we
observed that an increase of water soluble pectin (WSP) content
in association with decreased total pectin (TP) were delayed in
HWT-fruit (Fig. 1), which may account for inhibition of fruit softening. Studies on cell walls of apple fruit found less soluble pectin
and more insoluble pectin after exposure to hot-air treatment than
in untreated fruit, indicating inhibition of uronic acid degradation
(Shalom et al., 1996).
Expansins, cell wall-associated proteins, have been proposed to
play an important role in fruit softening (Brummell et al., 1999).
Northern blot analysis in the present work showed that HWT differently affected expression of two expansin genes. This difference
in the earlier postharvest period may be due to a stress response
caused by HWT. In addition, CpEXP1 was expressed more strongly in
pulp than in peel. These results indicate that CpEXP1 may contribute
to delay of softening after HWT. With regard to CpEXP2, there were
no signicant differences during fruit softening at the later storage
stage, indicating that CpEXP2 has a less signicant relationship with
fruit ripening. Similar results have been obtained in strawberry
(Dotto et al., 2011) and Creeping Bentgrass (Zhu et al., 2011).
Anthracnose is one of the most important postharvest diseases
in papaya. Papaya anthracnose has a latent stage in its development
and results in considerable loss in papaya production. The infection

progress of latent pathogens includes preinfection, infection, incubation and pathogenic periods. Fruit are infected by the fungus at
early stages of maturity in the eld and the fungus remains quiescent until the fruit reach the climacteric phase and symptoms
emerge as anthracnose or chocolate spot lesions (Dickman and
Alvarez, 1983; Gamagae et al., 2003). The present study showed
that HWT effectively reduced the carrier rate of C. gloeosporioides
in peel and had a pronounced effect on decreasing decay caused by
anthracnose (Fig. 2B). Gamma and UV-C irradiation have been used
in the postharvest control of papaya anthracnose. It has been found
that gamma irradiation could reduce postharvest losses caused by
anthracnose whereas UV-C irradiation was ineffective in reducing
the occurrence of C. gloeosporioides lesions, but caused fruit browning (Cia et al., 2007). Similar to gamma irradiation, HWT also play a
role in reducing another important postharvest disease in stem-end
rot (Fig. 2C and D).
In the present work, we found that the wax arrangement on
the surface of HWT-fruit had been changed. HWT caused the wax
to melt, and most stomata appeared to be partially or completely
plugged by melted wax (Fig. 4), thereby providing a mechanical
barrier against wound pathogens. Heat treatment can cause the
disappearance of wax platelets normally present in untreated
fruit and make the fruit surface relatively homogeneous (Schirra
et al., 2000). Thus, cuticular fractures, microwounds, and most
stomata are partially or completely lled by the melted wax, and
early-germinated spores may be encapsulated and inactivated by
molten wax as well (Fallik et al., 1999, 2000; Schirra et al., 2000).
The occlusion of possible gaps for wound pathogens as well as
the encapsulation and inactivation of early-germinated spores,
have been considered as additional factors in protection of fruit
against decay. In apple, it has been found that the epicuticular wax

X. Li et al. / Postharvest Biology and Technology 86 (2013) 437446

of non-heated fruit showed a number of deep surface cracks that

formed an interconnected network on the peel surface and the
cracks became wider and deeper during long-term storage (Roy
et al., 1999). The circular cracks disappeared after heat treatment,
probably as a result of the melting of the wax platelets that
had occurred in the cracks (Roy et al., 1994; Cajuste et al., 2010).
Germinated spores, conidia and hyphae appeared to be covered
and mummied by molten wax after heat treatment in cactus
pears (Schirra et al., 1999). Hot treatment was also found to have
inhibitory effects on spore germination and germ tube elongation
of Monilinia fructicola in peach fruit (Liu et al., 2012).
Polygalacturonase-inhibiting protein (PGIP), a cell wall protein
that can inhibit fungal endopolygalacturonases, is considered to be
an important factor for plant resistance to phytopathogenic fungi
(Toubart et al., 1992). CpPGIP expression was low during fruit ripening and was induced by HWT both in peel and pulp in the present
study (Figs. 7 and 8). In plants, systemically acquired resistance
(SAR) is established as a result of non-expressor of PR gene (NPR1)regulated expression of pathogenesis-related (PR) genes (Wang
et al., 2005). NPR1 gene has been shown to be a key regulator of gene
expression during the onset of a plant SAR (Dong, 2004; Mukhtar
et al., 2009). Expression of NPR1 is induced by pathogen infection
in Arabidopsis (Zhang et al., 1999; Yu et al., 2001). In the present
study, CpNPR1 expression decreased along with the occurrence of
pathogens in the later storage time, and its expression was induced
by HWT in peel. CpNPR1 was mainly expressed in the peel tissue,
indicating that disease resistance might be mainly maintained in
the peel of papaya. Plant PR proteins are induced and accumulated
in plants when they are infected by microbial pathogens or insects.
Several studies have suggested that the induced resistance could
result in accumulation of pathogenesis related protein (PR). However, other reports have revealed that PR was accumulated as fruit
disease developed. Therefore, we investigated the expression of
CpPR1 in the present work and found that the transcripts of CpPR1
increased as the disease increased. All these results demonstrated
that HWT induced disease resistance by inducing the expression of
defense-related genes in papaya fruit and that the papaya fruit peel
played an important defense role rather than the fruit pulp. HWT
of red sweet pepper also signicantly reduced decay caused by B.
cinerea and A. alternata (Fallik et al., 1996a). HWT has been known
to induce the expression of defense-related genes and increase the
activities of their corresponding enzymes in peach fruit, without
any impairment of fruit quality (Liu et al., 2012). Several other studies also have shown that HWT had a favorable effect on the control
of postharvest disease (Jemric et al., 2011; Fruk et al., 2012).
The results of present work and those of other studies have
revealed a possible mechanism of HWT on preservation of papaya
fruit, i.e.: (1) HWT decreased ethylene production and respiration
rates of papaya fruit, and thus reduced the effect of ethylene on fruit
ripening; (2) HWT repressed the expression of CpEXP1, delayed the
activation of PG and PME, and so delayed fruit softening, maintained a relative high rmness and reduced pathogen damage; (3)
HWT caused the melting of wax, which partially or entirely sealed
natural openings in the epidermis, prevented the invasion of the
pathogen and inactivated quiescent/latent infections of pathogen,
and then reduced disease incidence; (4) HWT induced the expression of CpPGIP and CpNPR1, and regulated the expression of CpPR1,
which may result in the gain of systemically acquired resistance
and enhanced disease resistance. However, while this could be the
possible mechanism involved in HWT-induced disease resistance,
further work is required for a better understanding.
In summary, the results showed that HWT at 54 C for 4 min
could maintain the quality of climacteric papaya fruit and induce
resistance to postharvest disease. HWT could be applied to papaya
as an effective pretreatment to maintain postharvest quality during
storage and marketing.

445

Acknowledgments
The research work was supported by the National Key Technology R&D Program of China (2011BAD24B02-4) and National Natural
Science Foundation of China (U0631004).

Appendix A. Supplementary data

Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.postharvbio.
2013.07.037.

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