Scientia Horticulturae 288 (2021) 110317

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Physiological response and evaluation of melon (Cucumis melo L.)

germplasm resources under high temperature and humidity stress at
seedling stage
Jinyang Weng, Pengli Li, Asad Rehman, Likang Wang, Xing Gao, Qingliang Niu *
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China

A R T I C L E I N F O A B S T R A C T

Keywords: Melon is a favourite fruit and is widely planted in China. High temperature and humidity (HTH) stress describes
High temperature and humidity stress the main limiting factors for melon due to the continuous hot and rainy weather in summer. The physiological
Subordinate function analysis response of melon to HTH stress and an evaluation method for identifying varieties resistant to HTH have not
Cluster analysis
been reported. In this study, seedlings of 56 melon germplasm resources were used to measure the growth in­
Correlation analysis
Comprehensive evaluation
dexes and physiological and biological indexes under high temperature and humidity stress and to evaluate the
Melon breeding tolerance of different varieties to HTH. Compared with the control, the plant height (PH) grew rapidly, and stem
diameter (SD), leaf length (LL), and leaf width (LW) were inhibited in all melon varieties. The photosynthetic
parameters Pn and Fv/Fm decreased compared with the control. Most of the varieties showed increases in
chlorophyll (Chl), soluble protein (SP) and MDA. According to the membership function value and cluster
analysis methods, three varieties, M32, M52 and M56, were selected for their better HTH tolerance. Through
correlation analysis, it was determined that Fv/Fm, Pn, Chl, LL, LW, PH, MDA and SD can be used as the
identification indexes of early tolerance to HTH. In conclusion, this work provides basic materials for melon
breeding and basic theories for the molecular mechanism of HTH tolerance in melon.

1. Introduction The adverse effects of abiotic stress may impact the plant growth,
development, metabolism, and productivity of crops, causing severe
Faced with the constant increases in global temperature and specific economic losses (Khan et al., 2015). It has been proven that photosyn­
humidity, heat stress is a relevant stress condition that is projected to thesis is inhibited and that the cell membrane is damaged when plants
intensify through the 21st century, which may threaten agricultural are subjected to HTH stress, leading to cell aging and cell death (Hao
production (Coffel et al., 2017). Moreover, global warming trends are et al., 2019). According to a previous study, specific environmental
irreversible since extremely high temperatures and rainstorms are stresses, such as high temperatures and humidity pressures, contribute
frequent as a result of the deterioration of the greenhouse effect (Liu to the overproduction of ROS, leading to oxidative stress in plants (Liu
et al., 2018). Most crops grow not only under single stress conditions but et al., 2017). When plants are under abiotic stress, a cascade of stress
also under several abiotic stresses, such as unsuitable soil moisture, reactions is activated to resist the harm from the external environment
temperature, sunlight, pathogens and pest infestation (Mittler, 2006). and maintain a normal growth state (Gong et al., 2020). Although
HTH stress are the main abiotic stresses that limit crop growth and extensive research has been carried out in recent years on crop pro­
productivity in summer (Liepert and Previdi, 2009; Deryng et al., 2014). duction when subjected to abiotic stress, little attention has been given
Thus, it is important to identify genetic resources with HTH tolerance to the physiological response of melon (Cucumis melo L.) under an
and to further understand the mechanisms contributing to adaptation to environment with high temperature and humidity.
stress conditions typically co-occurring in the field, such as high tem­ Melon (Cucumas melo L.) is one of the ten most famous fruits in the
perature and humidity (Rollins et al., 2013). word. It is widely cultivated worldwide and is welcomed by people all

Abbreviations: HTH, high temperature and humidity; PH, plant height; SD, stem diameter; LL, leaf length; LW, leaf width; E, transpiration rate; Gs, stomatal
conductance; Pn, net photosynthetic rate; Ci, intracellular CO2 concentration; Chl, chlorophyll; Fv/Fm, maximum quantum efficiency of psll; RC, relative conduc­
tivity; SP, soluble protein; MDA, malondialdehyde; SS, soluble sugar; HTHC, high temperature and humidity tolerance coefficient; ROS, reactive oxygen species.
* Corresponding author.

https://doi.org/10.1016/j.scienta.2021.110317
Received 19 February 2021; Received in revised form 25 April 2021; Accepted 25 May 2021
Available online 10 June 2021
0304-4238/© 2021 Elsevier B.V. All rights reserved.
J. Weng et al. Scientia Horticulturae 288 (2021) 110317

over the world because of its unique flavor and nutritional value (Shao In this study, 56 melon germplasm resources were used to analyze
et al., 2020). Melon is a warm seasonal plant that usually matures in the morphological index, photosynthetic index, physiological and
summer and is often affected by extremely high temperature and hu­ biochemical index response to high temperature and humidity stress. To
midity stress; the damage to melon in particular is increasingly serious obtain HTH tolerant varieties and to screen the key indexes to identify
(Laur and Tian, 2011). China is a secondary melon center with many high temperature and tolerance, 11 common melon genotypes were
germplasm resources (Kerje and Grum, 2000). The majority of crop identified at the seedling stage. Moreover, the purpose of this study was
geneticists consider that enrichment of the cultivated gene pool and to create a theoretical foundation to provide a theoretical basis for the
genetic diversity are essential for crop breeding (Tester and Langridge, breeding and cultivation of high temperature and humidity resistant
2010). Thus, a wide understanding of the physiological responses of melon germplasm resources and to provide a theoretical basis for the
melon under HTH conditions and evaluation of germplasm resources are molecular mechanism of melon tolerance to HTH stress.
vital for maintaining suitable productivity.
In recent years, the method of membership function value analysis
has provided a way to comprehensively evaluate the stress tolerance of 2. Material and methods
plants on the basis of multi-index measurements. The results are scien­
tific and reasonable, avoiding the one-sidedness of a single index. This 2.1. Plant materials
method has been widely used in the assessment of many crops in recent
years (Sun et al., 2017; Diao et al., 2019). Several researchers have re­ In this study, 56 main melon cultivars from different provinces of
ported on the assessment of abiotic stress in germplasm resources and China and other countries were selected. These melon germplasms were
the screening out of many tolerance indicators (Zhao et al., 2017; He obtained from the Chinese Melon Genetics Resource center of Zhengz­
et al., 2018). For instance, it has been reported that wheat varieties with hou Fruit Research Institute, Chinese Academy of Agricultural Science.
drought resistance can be identified by measuring plant height (PH), flag The source information and origin of all melon germplasm resources are
leaf area (FLA), ear length (SL) and related antioxidant enzyme activities shown in Table 1 and Fig. 1.
(Song et al., 2017). Photosynthesis and chlorophyll fluorescence have
been used to evaluate tolerance to temperature stress in tomato (Zhou 2.2. Experimental design and treatment
et al., 2015; Zhou et al., 2018). The morphological indexes, total chlo­
rophyll content, photosynthesis, conductivity and MDA in the seedling The experiment was conducted in a greenhouse at the School of
development process can also be used as indexes of the response to high Agriculture and Biology, Shanghai Jiao Tong University. Full melon
temperature stress (Pareek et al., 2019). However, to date, the response seeds were selected and soaked in warm water at 55 ◦ C for 30 min and
of melons to high temperature and humidity and the selection of vari­ then placed in a plant growth chamber at 28 ◦ C for germination in­
eties with HTH tolerance have not yet been reported. duction. After 90% seed germination, sprouts were transferred to a 21-
well plate in the greenhouse for seedling breeding. At the three-leaf

Table 1
Melon germplasm resource materials used in this study.
Code Strain name Origin Code Strain name Origin

M1 3 HAO LU PI CHN-HA M29 TAI YANG HONG CHN-ZJ

M2 XIN SHI JI CHN-HA M30 JIN TAI LANG JP
M3 GUANG ZHOU MI GUA CHN-GD M31 NIU JIAO MI CHN-HA
M4 HONG ZI HUANG MI GUA CHN-HA M32 JIN DI CHN-HN
M5 JIN XIANG LIU CHN-LN M33 YANG SHUI GUA CHN-JS
M6 XI TIAN YI HAO CHN-SN M34 HU HE 3 HAO CHN-IM
M7 BAI XIAN GUA CHN-SN M35 MA TI HUANG CHN-HE
M8 WANG HAI CHN-HA M36 ZHU YE QING CHN-SN
M9 PING HU BAI GUA CHN-ZJ M37 HUANG JIN JIU HAO JP
M10 HUANG HUA ZHUI CHN-HA M38 YI DU BAI YIN CHN-SD
M11 BAI FENG CHN-JL M39 JIU LI XIANG CHN-ZJ
M12 HUANG JIN GUA CHN-SH M40 YUAN HUANG LI CHN-HL
M13 JIN LING GUA CHN-JS M41 HU PI CUI CHN-SX
M14 HANG ZHOU HUANG JIN CHN-ZJ M42 WEN XI BAI GUA CHN-SD
M15 BEI JING MI ZHI GUA CHN-ZJ M43 XIAO CUI GUA CHN-HE
M16 HAI DONG QING CHN-SH M44 BAI TU WA CHN-SN
M17 GUANG XI LI GUA CHN-GX M45 HUANG YU TW
M18 LI ER CUI CHN-SX M46 SHI LENG HUANG JIN GUA CHN-SD
M19 ZHI MA ZI CHN-SD M47 HONG RANG ZI CHN-HL
M20 YANG JIAO MI CHN-SD M48 TIAN SI MI CHN-JL
M21 HUANG MI BAO CHN-HA M49 WANG GU DAN CHN-HL
M22 BAI GUA CHN-LN M50 BAI XIANG JIAO CHN-HL
M23 CHAO YANG BAI CHN-HE M51 YU TIAN YI HAO CHN-HA
M24 HUANG XIANG GUA CHN-JS M52 HEI PI CHN-HL
M25 BAI XIANG TIAN GUA CHN-JS M53 YA MI XIANG GUA CHN-JS
M26 JIN XIANG YU CHN-HA M54 HU HE 4 HAO XUAN CHN-IM
M27 JIN BAO JP M55 LIU JIN GUA CHN-SD
M28 YA DAN QING CHN-JS M56 HE FEI BAI GUA CHN-AH

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J. Weng et al. Scientia Horticulturae 288 (2021) 110317

Fig. 1. The 56 melon varieties were selected with origin from different places as shown in map.

stage, seedlings were transferred to a growth chamber to be pretreated placed into the leaf chamber for parameter determination. Chlorophyll
at 30 ◦ C/20 ◦ C for day and night, with light at 600 μmol m− 2 s− 1 with a fluorescence was determined using a chlorophyll fluorescence imaging
relative humidity of 60% to adapt for three days and then began to adapt system (IMAGING PAM; Heinz Walz, Effeltrich, Germany). To measure
to HTH stress. For high temperature and humidity treatment, the tem­ the maximum photochemical efficiency of PSII (Fv/Fm), melon seed­
perature was 42℃/32℃ for day and night (12 h/12 h) (Zhang et al., lings were dark-adapted for 30 min. The Fv/Fm ratio and the photo­
2014; Liu et al., 2017), with light at 600 μmol m− 2 s− 1, soil moisture at chemical efficiency of PSII (ΦPSII) were determined using the whole
100%, and ambient air humidity at 85%/95% for day and night, area of the second true leaf (Tattini et al., 2015).
respectively (Zhang et al., 2012) . For control treatments, the tempera­
ture was 30℃/20℃ for day and night (12 h/12 h), with light at 600 2.5. Determination of physiological and biochemical indicators
μmol m− 2 s− 1 and ambient humidity at 60% relative humidity all day for
the experimental treatment for five days, after which the second leaf was The total chlorophyll (Chl) content was evaluated according to the
taken and the indicators measured. method defined by Cao et al. (2015). The stability of the cell membrane
was determined by recording the relative conductivity of leaf leakage
2.3. Measurements of agronomic traits (RC) according to Aghaie et al. (2018). The soluble protein (SP) content
determination method, which involves the binding of Coomassie Bril­
After 5 days of HTH stress and control treatments, plant height (PH), liant Blue G-250 to protein, was described by Bradford (1976). Malon­
stem diameter (SD), leaf length (LL), and leaf width (LW) were deter­ dialdehyde (MDA) content was measured as thiobarbituric acid
mined. PH, SD, LL, and LW were measured as the distance between the (TBA)-reactive metabolites using the method described by Shah et al.
soil surface and the top of the flag leaf, and the width of the stem under a (2001). Soluble sugar (SS) contents were measured using the
cotyledon was determined by a Vernier calliper, along with the distance anthrone-sulfuric acid method as described by Zhong et al. (2019).
from tip to petiole on the third leaf and the width of the middle of the
third leaf. 2.6. Data processing and evaluation methods for the indicator

2.4. Gas exchange and chlorophyll fluorescence measurements The high temperature and humidity tolerance coefficient (HTHC)
was calculated as the ratio of the data derived from the high temperature
Stomatal exchange parameters [net photosynthetic rate (Pn), tran­ and humidity (HTH) stress and normal control (CK) treatments of the
spiration rate (E), intercellular carbon dioxide concentration (Ci), sto­ same accession for each trait using Eq. (1) (Chen et al., 2012).
matal conductance (Gs)] were determined using the Portable Gas Xij HTH
Exchange Fluorescence System (GFS-3000, WALZ, Effeltrich, Germany) HTHCij = (1)
Xij CK
from 9:00 am to 11:30 am at the laboratory. The instrument was first
warmed up in measuring mode for 30 min. Menu parameters in the Where HTHCij is the high temperature and humidity tolerance co­
photosynthesis system were set as follows: temperature at 25 ◦ C and efficient of trait (j) for cultivar (i); XijHTH and XijCK are the value of trait
humidity at 60%. The CO2 concentration during measurement varied (j) for cultivar (i) evaluated under HTH stress and normal control (CK)
between 420 and 460 ppm, the steady light intensity was 600 μmol m− 2 treatments, respectively.
s− 1 , and the air velocity was 750 μmol s− 1. The seedlings were removed The high temperature and humidity tolerance levels were evaluated
from the growth chamber, and the second leaf of melon was quickly by the membership function value. According to the HTHC, the modified

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J. Weng et al. Scientia Horticulturae 288 (2021) 110317

Table 2
Statistical analysis of 14 characteristics in 56 melon accessions under HTH stress treatment.
Traits Control group Treatment group Growth rate (%)

Mean±SD CV (%) Mean±SD CV (%)

PH (mm) 252.23 ± 66.55 b 26.38 314.26 ± 79.73 a 25.37 24.59%

SD (mm) 3.64 ± 0.65 a 17.86 3.37 ± 0.64 b 18.99 − 7.42%
LL (mm) 78.13 ± 12.14 a 15.54 67.94 ± 12.36 b 18.19 − 13.04%
LW (mm) 79.31 ± 14.21 a 17.92 68.71 ± 12.51 b 18.21 − 13.37%
Pn (µmol m− 2 s − 1) 11.48 ± 2.72 a 23.69 7.37 ± 2.16 b 29.31 − 35.80%
E (mmol m− 2 s− 1) 2.98 ± 1.12b 37.58 5.25 ± 1.34 a 25.52 76.17%
Gs (mmol m− 2 s− 1) 298.82 ± 77.52 b 25.94 511.51 ± 130.18 a 25.45 71.18%
Ci (ppm) 405.51 ± 63.97 b 15.78 460.35 ± 62.95 a 13.67 13.52%
Fv/Fm 0.68 ± 0.01a 1.47 0.64 ± 0.02 b 3.13 − 5.88%
Chl (mg/g FW) 1.30 ± 0.19 b 14.62 1.36 ± 0.28 a 20.59 4.62%
RC (%) 43.44 ± 6.53 a 15.03 36.01 ± 6.35 b 17.63 − 17.10%
SP (mg/g) 7.65 ± 2.40 a 31.37 7.44 ± 2.43 b 32.66 − 2.75%
MDA (nmol/g) 3.27 ± 0.74 b 22.63 3.96 ± 1.16 a 29.29 21.10%
SS (mg/g) 25.77 ± 12.75 a 49.48 22.56 ± 8.71 b 38.61 − 12.46%

membership function value of MHTH tolerance was calculated accord­ experiments.

ing to the following equations: The 56 melon varieties come from different places, abbreviated as
follows. CHN-HA, Henan Province of China; CHN-GD, Guangdong
HTHCij − HTHCjmin
Uij = (2) Province of China; CHN-LN, Liaoning Province of China; CHN-SN,
HTHCjmax − HTHCjmin
Shaanxi Province of China; CHN-ZJ, Zhejiang Province of China; CHN-
HTHCij − HTHCjmin JL, Jilin Province of China; CHN-SH, Shanghai of China; CHN-GX,
Uij = 1 − (3) Guangxi Province of China; CHN-SX, Shanxi Province of China; CHN-
HTHCjmax − HTHCjmin
JS, Jiangsu Province of China; CHN-SD, Shandong Province of China;
1∑ n CHN-HE, Hebei Province of China; CHN-HN, Hunan Province of China;
Di = Uij (4) CHN-IM, Inner Mongolia Province of China; CHN-HL, Heilongjiang
n j=1
Province of China; CHN-AH, Anhui Province of China; JP, Japan; TW,
Where Uij is the membership function value of trait (j) for cultivar (i) Taiwan.
for high temperature and humidity tolerance. HTHCjmax is the maximum
value of the high temperature and humidity coefficient for trait (j), and 3. Results
HTHCjmin is the minimum value of HTHCij. The subordinator function
reflected a positive correlation between a particular index variable and 3.1. Changes in the different melon indexes under HTH stress
high temperature and humidity stress expressed by Eq. (2), whereas a
negative correlation was expressed by Eq. (3). Di is the average value of Under high temperature and humidity stress, melon produces a series
the membership function of 11 traits for cultivar (i) by Eq. (4), for high of reactions, mainly in the morphology of growth, photosynthesis, and a
temperature and humidity tolerance. number of stress reactions in cells. Consequently, the response states of
melon against stress can be seen by measuring 14 indicators (Table 2).
The average PH of 56 melon varieties was 24.59% higher than that of the
2.7. Verification of melon varieties resistant to temperature and humidity
control. The average growth rates of SD, LL and LW were significantly
lower than that of the control group. Compared with the control group,
To further verify the high temperature and humidity tolerance of
the LL and LW were significantly reduced by 13.04% and 13.37%,
melon germplasm resources screened by the membership function
respectively. The Pn average was lower than that of the control,
method, we selected varieties that were resistant to HTH, M32, M52,
decreasing by 35.80%, while E and Gs increased by 76.17% and 71.18%,
and M56, and sensitive varieties, M30, M45, and M37, for the experi­
respectively. Ci also slightly increased by 13.52% compared with the
ment. All varieties were grown in a plant growth chamber, soaked at 55
control. Through physiological and biochemical assessments, compared
◦
C for 30 min, and placed at 28 ◦ C for germination induction. Then, the
with the control, melon average Chl, Fv/Fm, and SP increased by 4.62%
seeds were sown in a 21-well plate at 30 ℃/20 ℃ for day and night, with
and decreased by 5.88% and 2.75%, respectively, while RC and SS
light at 600 μmol m− 2 s− 1, with a relative humidity of 60%. When the
decreased by 17.10% and 12.46%, respectively. Under high temperature
seedlings grew to three-leaf stage, HTH stress was initiated for ten days,
and humidity stress, MDA increased by 20.21%. By calculating 14 melon
the stress situations were compared, and the second true leaf of the
stress indexes, the CV coefficient of melon seedlings treated with high
seedling was measured for Fv/Fm determination.
temperature and humidity was between 3.13 and 38.61, and the CV
coefficient of control melon seedlings was between 1.47 and 49.48.
2.8. Statistical analysis Among them, there were 12 indexes with coefficients of variation over
15%. The findings showed that the materials tested have a diverse va­
The experiment was a completely randomized design with three riety of agronomic characteristics, strong genetic variations, and rich
replications (three plants per replicate). To test significance, one-way genetic diversity.
ANOVA was performed. The significance of the data differences Data are presented as the mean ± SD (standard deviation). PH: plant
among treatments (P < 0.05) was determined by the LSD test in SPSS height, SD: stem diameter, LL: leaf length, LW: leaf width, E: transpi­
25.0 (IBM, Chicago, IL, USA). Correlation analysis (*, P < 0.05; **, P < ration rate, Gs: stomatal conductance, Pn: net photosynthetic rate, Ci:
0.01) and cluster analysis were also performed using SPSS 25.0. Sub­ intracellular CO2 concentration, Chl: chlorophyll, Fv/Fm: maximum
ordinate function analysis and coefficient of variation (CV%) calcula­ quantum efficiency of psll, RC: relative conductivity, SP, soluble protein,
tions were performed using Microsoft Office Excel 2010, and the graph MDA: malondialdehyde, SS: soluble sugar, Different letters denote sta­
images were generated using GraphPad Prism software. The data are tistically significant differences by least significant difference (LSD) test
presented as the means ± standard deviation of three independent at P < 0.01.

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J. Weng et al. Scientia Horticulturae 288 (2021) 110317

Table 3 increasing the permeability of the membrane. Fv/Fm is often used as an

HTHC for physiological indexes in 56 melon lines. indicator of stress severity. In the experiment, the average relative value
Index Mean SD CV (%) Range was less than 1, and the range was 0.86–0.98, indicating that all vari­
eties were under different degrees of stress. MDA, as an essential index of
PH 1.25 0.09 7.20 1.10–1.55
SD 0.92 0.04 4.35 0.80–0.99 membrane peroxidation, had an average relative value of more than 1
LL 0.87 0.07 8.05 0.74–0.96 and a range of 1.01–1.78, indicating that all varieties were damaged by
LW 0.87 0.06 6.90 0.71–0.95 membrane oxidation. Among the relative indexes, the range of coeffi­
Pn 0.64 0.11 17.19 0.41–0.86 cient of variation was 3.19–50.99, which shows that the variety range
E 2.02 1.03 50.99 1.10–4.88
Gs 1.82 0.64 35.16 1.12–4.08
before and after treatment was large, and there were obvious differences
Ci 1.15 0.14 12.17 1.01–1.83 in the high temperature and humidity tolerance of the tested materials.
Fv/Fm 0.94 0.03 3.19 0.86–0.98 The selection of variety types was rich, which was conducive to the
Chl 1.05 0.19 18.10 0.74–1.44 evaluation of high temperature and humidity tolerance of melon and the
RC 0.84 0.14 16.67 0.51–1.23
screening of identification indexes.
SP 1.01 0.29 28.71 0.22–1.55
MDA 1.21 0.19 15.70 1.01–1.78 The mean is the average value of the high-temperature and humidity
SS 0.94 0.25 26.60 0.46–1.54 tolerance coefficients of different indexes of all tested varieties; SD is the
standard deviation of the high-temperature and humidity tolerance co­
efficient. The CV (%) is the coefficient of variation of the high temper­
3.2. Changes in the HTHC in response to high temperature and humidity ature and humidity tolerance coefficient. The range is the value from
stress minimum to maximum of the high-temperature and humidity tolerance
coefficient.
Using the high temperature and humidity stress coefficients of
melon, the difference in each index can be further evaluated. The high
temperature and humidity stress coefficients calculated from the relative 3.3. Correlation analysis of the membership function values of HTH
values of each index are shown in Table 3. The results show that the tolerance characteristics
changes in the relative values of the 14 indicators are different after high
temperature and humidity treatments. In the growth index, the relative The relative values of 14 indexes under high temperature and hu­
value of PH was higher than 1, and the range of change was 1.10–1.55, midity stress were analysed (Table 4). In terms of the growth index, PH
while the relative values of SD, LL, LW were less than 1, and the range of was negatively correlated with SD (r = − 0.331*) and MDA (r =
variation was 0.71–0.99, indicating that under high temperature and − 0.310*) and was positively correlated with LL (r = 0.263*) and LW (r
humidity stress, experimental varieties can grow rapidly by increasing = 0.310*). In terms of photosynthetic indicators, E was significantly
PH and reducing SD, LL, LW. In the photosynthetic index, the mean positively correlated with Gs and Ci and had correlation coefficients of
relative value of Pn was 0.64, while the range of Pn was 0.41 to 0.86. 0.643** and 0.436**, respectively. The correlation coefficient between
The corresponding values of E, Gs, and Ci were all greater than 1, and Gs and Ci was 0.439**. In terms of physiology and biochemistry, SP was
the range of Pn was also between 1.01 and 4.88, which indicated that Pn positively correlated with SS, the correlation coefficient was 0.430**,
decreased and E, Gs, and Ci increased under the conditions of high and Fv/Fm was positively correlated with Chl and negatively correlated
temperature and humidity to resist the stress caused by those conditions. with MDA. Moreover, Fv/Fm was positively correlated with SD, LL, LW,
In the physiological and biochemical indexes, the average relative and Pn. The above results show that there are different degrees of cor­
values of Chl and SP were greater than 1. The range of change was be­ relation among the individual indicators, which makes the indicators
tween 0.22 and 1.55, indicating that the indexes of each variety to cope overlap. Therefore, in the identification of melon varieties with high
with high temperature and humidity are different, but most varieties temperature and humidity tolerance, only using a single index cannot
deal with high temperature and humidity stress by increasing chloro­ reflect the difference in HTH tolerance between different genotypes, it is
phyll and synthetic protein contents. The average relative values of RC necessary to evaluate these indexes comprehensively.
and SS were less than 1.The range value of RC and SS are 0.51–1.23 and
0.46–1.54 respectively, which shows that the trend of the indexes of 3.4. Comprehensive evaluation of HTH tolerance
each variety is inconsistent, most of the varieties’ soluble sugar is
reduced, and most of the varieties improve the response to stress by Then, 56 melon germplasm resource membership values were

Table 4
Pearson correlation coefficients between the subordination function values of 14 traits from 56 varieties of melon under HTH stress.
Index PH SD LL LW Pn E Gs Ci Fv/Fm Chl RC SP MDA SS

PH 1
SD − 0.331* 1
LL 0.263* 0.054 1
LW 0.310* 0.065 0.861** 1
Pn 0.125 0.127 0.217 0.262 1
E 0.091 − 0.077 − 0.124 − 0.040 − 0.037 1
Gs 0.129 − 0.097 − 0.041 0.045 0.234 0.643** 1
Ci 0.089 − 0.155 − 0.010 0.048 0.060 0.436** 0.439** 1
Fv/Fm 0.222 0.283* 0.331* 0.281* 0.453** 0.027 0.031 − 0.073 1
Chl 0.135 0.254 0.123 0.155 0.228 − 0.262 − 0.114 − 0.218 0.332* 1
RC 0.090 0.011 0.128 0.184 − 0.003 0.004 0.218 0.135 0.193 0.022 1
SP 0.252 − 0.046 − 0.023 0.005 0.130 0.078 0.089 − 0.038 0.184 0.201 0.227 1
MDA − 0.310* 0.088 − 0.144 − 0.194 − 0.206 0.063 0.195 0.049 − 0.402** − 0.078 0.013 − 0.044 1
SS − 0.042 0.027 − 0.064 − 0.174 0.007 − 0.020 − 0.137 − 0.005 0.103 0.197 0.179 0.430** 0.244 1
*
Significant difference at the 0.05 probability level

**
Significant difference at the 0.01 probability level.

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Table 5
Different values of the membership function and the comprehensive identification of 56 melon varieties at the seedling stage under HTH stress.
Code PH SD LL LW Pn Fv/Fm Chl RC SP MDA SS D Rank

M1 0.44 0.11 0.73 1.00 0.44 0.75 0.16 0.39 0.41 0.91 0.07 0.49 39
M2 0.40 0.84 0.41 0.71 0.69 0.75 0.01 0.49 0.77 0.77 0.35 0.56 26
M3 0.64 0.00 0.86 1.00 0.38 0.25 0.00 0.65 0.72 0.94 0.26 0.52 32
M4 1.00 0.32 0.59 0.58 0.18 0.67 0.53 0.74 0.87 0.97 0.77 0.66 13
M5 0.38 0.63 0.86 1.00 0.09 0.67 0.76 0.28 0.77 0.81 0.67 0.63 15
M6 0.09 0.58 0.77 0.75 0.42 0.33 0.19 0.71 0.56 0.35 0.86 0.51 36
M7 0.64 0.42 0.95 0.88 0.27 0.42 0.44 0.58 0.41 0.90 0.07 0.54 29
M8 0.20 0.84 0.45 0.42 0.07 0.58 0.29 0.53 0.55 0.60 0.50 0.46 43
M9 0.13 0.53 0.73 0.71 0.53 0.25 0.29 0.79 0.28 0.40 0.22 0.44 49
M10 0.56 0.26 0.41 0.29 0.13 0.25 0.07 0.43 0.89 0.48 0.80 0.42 53
M11 0.47 0.89 0.59 0.83 0.00 0.33 0.10 0.32 0.62 0.75 0.31 0.47 42
M12 0.33 0.63 0.41 0.38 0.51 0.42 0.37 1.00 0.66 0.52 0.08 0.48 41
M13 0.49 0.84 0.64 0.83 0.60 0.58 0.40 0.56 0.54 0.96 0.02 0.59 24
M14 0.27 0.79 0.05 0.29 0.33 0.50 0.50 0.60 0.52 0.83 0.25 0.45 47
M15 0.18 0.89 0.23 0.38 0.27 0.42 0.80 0.67 0.69 0.21 0.65 0.49 40
M16 0.56 0.79 0.86 0.88 0.91 0.75 0.60 0.14 0.87 0.31 1.00 0.70 7
M17 0.36 0.16 0.00 0.33 0.78 0.25 0.26 0.74 0.59 0.57 0.61 0.42 50
M18 0.22 0.95 0.59 0.75 0.27 0.00 0.54 0.60 0.43 0.00 0.63 0.45 45
M19 0.09 0.79 0.64 0.79 0.47 0.75 0.53 0.88 0.69 0.66 0.25 0.59 20
M20 0.56 0.74 0.82 0.83 0.69 0.75 0.71 0.47 0.60 0.96 0.16 0.66 11
M21 0.38 0.74 0.09 0.29 0.29 0.75 0.30 0.60 0.77 0.96 0.52 0.52 34
M22 0.44 0.47 0.55 0.83 0.73 0.42 0.96 0.42 0.44 1.00 0.28 0.59 21
M23 0.60 0.58 0.86 0.79 0.47 0.83 0.66 0.47 0.63 0.23 0.34 0.59 25
M24 0.51 0.63 0.64 0.75 0.62 0.67 0.61 0.69 0.50 0.97 0.09 0.61 18
M25 0.16 0.84 0.27 0.33 0.58 0.83 0.39 0.26 0.74 0.87 0.72 0.54 28
M26 0.49 0.84 0.73 0.79 0.64 0.92 0.39 0.00 0.74 0.57 0.39 0.59 22
M27 0.09 0.74 0.41 0.58 0.31 0.42 0.17 0.81 0.40 0.77 0.26 0.45 46
M28 0.27 0.42 0.45 0.50 0.96 0.92 0.31 0.42 0.79 0.56 0.49 0.55 27
M29 0.38 0.58 0.77 0.88 0.44 0.50 0.01 0.44 0.75 0.83 0.24 0.53 31
M30 0.13 0.47 0.55 0.71 0.47 0.00 0.13 0.60 0.47 0.66 0.19 0.40 56
M31 0.02 0.84 1.00 0.88 0.40 0.75 0.49 0.33 0.35 0.43 0.18 0.52 35
M32 0.56 0.84 0.82 0.96 0.91 0.92 0.89 0.68 0.88 0.96 0.54 0.81 1
M33 0.24 0.84 0.91 0.83 0.69 0.92 0.61 0.64 0.71 1.00 0.57 0.72 4
M34 0.36 1.00 0.86 0.88 0.96 0.75 0.70 0.54 0.50 0.82 0.47 0.71 5
M35 0.31 0.79 0.68 0.88 0.69 0.83 0.84 0.61 0.64 0.92 0.56 0.70 6
M36 0.36 0.68 0.36 0.63 0.40 0.67 0.11 0.49 0.35 0.97 0.00 0.46 44
M37 0.00 0.84 0.00 0.17 0.64 0.67 0.50 0.71 0.36 0.40 0.27 0.41 54
M38 0.04 0.79 0.95 0.92 0.87 1.00 0.13 0.58 0.50 0.92 0.68 0.67 10
M39 0.60 0.89 0.41 0.58 0.87 0.83 0.24 0.60 0.53 0.87 0.29 0.61 17
M40 0.07 0.42 0.27 0.33 0.22 0.33 0.31 0.63 0.65 0.70 0.67 0.42 51
M41 0.22 0.74 0.05 0.29 0.44 0.67 0.19 0.74 0.26 0.86 0.42 0.44 48
M42 0.60 0.47 0.95 0.79 0.82 0.83 0.20 0.29 0.44 0.96 0.52 0.62 16
M43 0.22 0.68 0.95 0.83 0.27 1.00 0.30 0.42 0.92 0.62 0.74 0.63 14
M44 0.47 0.47 0.09 0.29 0.33 0.75 0.74 0.61 0.38 0.64 0.73 0.50 37
M45 0.09 0.74 0.32 0.54 0.42 0.25 0.30 0.51 0.28 0.40 0.63 0.41 55
M46 0.22 0.21 0.00 0.00 0.53 0.33 0.49 0.31 1.00 0.84 0.66 0.42 52
M47 0.16 0.79 0.68 0.71 0.24 0.75 0.81 0.28 0.26 0.91 0.35 0.54 30
M48 0.04 0.68 0.59 0.75 0.56 0.58 0.29 0.31 0.49 0.92 0.49 0.52 33
M49 0.49 0.42 0.91 0.96 0.53 0.92 0.81 0.53 0.56 0.74 0.56 0.68 8
M50 0.11 0.84 0.41 0.50 0.42 0.50 1.00 0.49 0.98 0.83 0.60 0.61 19
M51 0.47 0.84 0.64 0.71 0.42 0.92 0.59 0.69 0.40 0.96 0.64 0.66 12
M52 0.51 0.84 0.82 0.88 0.93 0.92 0.90 0.65 0.87 0.97 0.64 0.81 2
M53 0.67 0.26 0.86 0.92 0.76 0.92 0.70 0.58 0.45 0.94 0.34 0.67 9
M54 0.33 0.63 0.32 0.42 0.49 0.92 0.66 0.46 0.87 0.92 0.48 0.59 23
M55 0.20 0.68 0.95 0.38 0.36 0.58 0.16 0.88 0.00 0.92 0.33 0.49 38
M56 0.38 0.89 1.00 0.92 1.00 0.92 0.70 0.76 0.86 0.96 0.51 0.81 3

calculated based on the fuzzy mathematics membership function comprehensive membership function, the differences in the high tem­
method (Zadeh, 1965) and were measured, and the D value of the perature and humidity tolerances of 56 different germplasms, were
average membership function of each indicator was calculated and evaluated by 11 common indexes of the melon varieties (Table 5). The
ranked. The D value of the average membership function represents the membership values of the different melon varieties ranged from 0.40 to
tolerance of melon to HTH. As it can be seen from Table 3, when melon 0.81, among which 19 varieties were below 0.5, 18 varieties were be­
seedlings were subjected to HTH stress, all Pn decreased, while Gs and Ci tween 0.5–0.6, and 19 varieties were above 0.6, among which 3 varieties
increased. Therefore, it can be seen that the decrease of photosynthesis were above 0.8. It can be inferred from the above data that the response
caused by HTH stress in melon was mainly caused by nonstomatal re­ of each index is quite different, and using a single index cannot fully
striction (Bunce, 1988). In addition, in terms of E, on one hand, it ac­ reflect the HTH tolerance of the varieties. To achieve a comprehensive
celerates the evaporation of water in plants; on the other hand, it ranking system, the evaluation and analysis of the average value of each
reduces leaf temperature through transpiration. Therefore, it is difficult index membership function were used. In this case, the temperature and
to use E to evaluate the tolerance to HTH (Bogeat-Triboulot et al., 2019) humidity tolerance of M32, M52, and M56 were high (D > 0.8), but
. In conclusion, E, Gs and Ci are not suitable for the following mem­ M30, M45, M37, M10, M46, M40, M17, M9, and M41 (D < 0.45) were
bership function evaluation indexes. The average values of the less resistant to high temperature and humidity.

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Fig. 3. Image of melon of different varieties (A), chlorophyll fluorescence im­

aging of maximal quantum yield of PSII photochemistry (B), and Fv/Fm value
(C) for ten days under HTH stress. Bars represent mean ± SD(n = 6). ** in­
dicates a significant difference at P < 0.01.

value was 0.66–0.72, and 10 different varieties represented 17.86% of

the materials tested. Group III was of the weak tolerance category, with
an average membership value of 0.59–0.63, and 12 varieties account for
21.42% of the materials tested. Group V was a nonresistant type, with an
average membership value of 0.44–0.56, with 24 varieties accounting
Fig. 2. Cluster analysis of the 56 different melon varieties based on the D value. for 42.86% of the materials tested. Group IV was a sensitive type, and
The numbers of the Y-axis present the code of the melon accessions; the X-axis the average membership function was lower (D < 0.44), with 7 varieties
presents the Euclidean distance. accounting for 12.5% of the materials tested. Based on the average
membership value for each character and the synthesis of each char­
3.5. Cluster analysis acter, we preliminarily screened three high temperature and humidity
melon germplasm resources.
To comprehensively evaluate the high temperature and humidity
tolerance of different varieties of melon, the mean value of the mem­ 3.6. Screening of identification indexes of HTH tolertance
bership function (D) clustering analysis of each index was performed, in
which the Euclidean distance was 7, and the high temperature and hu­ The physiological phenotypes of HTH-sensitive and HTH-tolerant
midity tolerance of 56 germplasms were divided into 5 groups (Fig. 2). varieties were significantly different after 10 days of stress. The leaves
Group I was a highly resistant type with 3 varieties, accounting for of M30, M45, and M37, which are sensitive to HTH, were almost all
5.36% of the tested materials. The average value of the membership yellow, and the values of Fv/Fm were relatively small. Photosynthetic
function of HTH tolerance was higher (D > 0.8). Group II was of medium function was lost, and the seedlings gradually died out. However, the
type, and the tolerance to HTH was medium. The average membership leaves of M32, M52 and M56 of the HTH-resistant varieties were slightly

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Table 6
Pearson correlation coefficients between D values and 11 traits from 56 accessions of melon under HTH stress.
Index PH SD LL LW Pn Fv/Fm Chl RC SP MDA SS D

PH 1
SD − 0.328* 1
LL 0.260 0.055 1
LW 0.305* 0.068 0.864** 1
Pn 0.125 0.127 0.220 0.265* 1
Fv/Fm 0.225 0.281* 0.330* 0.283* 0.454** 1
Chl 0.135 0.253 0.125 0.155 0.226 0.334* 1
RC − 0.096 − 0.010 − 0.133 − 0.189 0.001 − 0.194 − 0.025 1
SP 0.253 − 0.048 − 0.021 0.004 0.129 0.184 0.201 − 0.229 1
MDA 0.309* − 0.091 0.145 0.193 0.205 0.402** 0.077 0.012 0.041 1
SS − 0.038 0.029 − 0.069 − 0.176 0.008 0.102 0.200 − 0.178 0.433** − 0.244 1
D 0.428** 0.285* 0.620** 0.616** 0.579** 0.721** 0.567** − 0.052 0.377** 0.422** 0.208 1
*
Significant difference at the 0.05 probability level.

**
Significant difference at the 0.01 probability level.

stressed, and the value of Fv/Fm was above 0.6. Thus, Fv/Fm could be by HTH stress (Wang et al., 2018). The measurement of the photo­
used to better identify the HTH tolerance of different melon germplasms chemical efficiency of PSII with chlorophyll fluorescence is an effective
(Fig. 3). The 11 single-index and D value correlation coefficients were method of plant evaluation in PSII. In this work, the Fv/Fm values of
different. Fv/Fm, LL, LW, Pn, Chl, PH, MDA, and SP were highly posi­ leaves of all varieties were lower than that of the control and showed a
tively correlated with the D value (P < 0.01), SD was positively corre­ significant positive correlation with the D value (r = 0.721**). We
lated with the D value (P < 0.05), and the RC, SS, and D values did not selected three HTH-sensitive varieties and three HTH-resistant varieties
reach significance (Table 6). The results show that, with the exception of for determination after 10 days of HTH stress, and significant differences
RC and SS, the remaining 9 indexes can be used as high temperature and were found in their Fv/Fm (Fig. 3). The results indicated that Fv/Fm can
humidity tolerance screening indexes in the seedling stage; among them, be used as an effective screening index of HTH tolerance, which has also
Fv/Fm can be used as the key indicator of screening. been reported in other crops (Banks, 2018; Reis et al., 2020).
The plant response to HTH stress involves numerous regulations
4. Discussion regarding physiological processes caused by changes in organelles
(Wahid et al., 2007). The chlorophyll content is one of the indexes used
Melon is an important vegetable crop worldwide that is cultivated in to assess the degree of stress on plants. In this study, the chlorophyll
a wide area in China (Wang et al., 2014). Under environmental condi­ content of most melon varieties was slightly higher than that of the
tions to cultivate in the summer, melon is often subjected to the abiotic control after 5 days of stress, and the chlorophyll content of a few va­
stresses of high temperature and humidity, which may seriously affect rieties was reduced (Table 3). The results showed that different melon
its growth and quality. The evaluation of germplasm resources, espe­ varieties had different adaptation mechanisms of chlorophyll content to
cially in the selection of resistant species, is an essential fundamental HTH stress and that heat- and humidity-tolerant genotypes could syn­
work in breeding. The photosynthetic, physiological and biochemical thesize more chlorophyll content in the early stages of stress to resist
characteristics of melon respond to high temperature and humidity HTH stress conditions. The content of soluble protein is normally used to
stress, but the sensitivity of different indexes to high-temperature and evaluate the tolerance of plants. In this study, the relative value of sol­
humidity stress is different. In this study, the plant heights of all melon uble protein of melon varieties was between 0.22 and 1.55, with an
varieties were higher than that of the control, but the growth levels of average value of 1.01 (Table 3), suggesting that different genotypes of
stem diameter, leaf length, and leaf width were inhibited under HTH melon have different responses to the indicators of soluble protein
stress after 5 days. These results indicated that under HTH stress, the SD content of melon and that the HTH resistant melon varieties will syn­
and LL and LW of melon grow slowly, and the PH extends continuously thesize more proteins to maintain the normal state of cells. The principal
to speed up its growth to cope with HTH stress by shortening its life cycle product of membrane lipid peroxidation is malondialdehyde (MDA),
(Challinor et al., 2005). Similar conclusions have been drawn from which often acts as an index to measure the degree of lipid peroxidation
previous studies on heat stress in heat-sensitive and heat-tolerant ge­ under abiotic stress (Hamurcu et al., 2020; Hu et al., 2020). In this study,
notypes of wucai (Brassica campestris L.) (Zou et al., 2016). both HTH stresses caused increases in the MDA content in melon, with a
In most plants, the ability to tolerate HTH stress is directly associated significant correlation between MDA and the D value (r = 0.422**). The
with their ability to sustain leaf gas exchange characteristics by main­ results showed that HTH stress could damage the cell membrane of
taining the net photosynthetic rate and integrity of cellular membranes, melon and that MDA was an important evaluation index. Electrolyte
as well as their pigment systems (Nagarajan et al., 2010). Photosynthetic leakage is an important indicator of plant membrane damage levels
parameters are good indicators for detecting the effects of stress on under stress and is widely used to estimate the tolerance of plants (Sun
plants because photosynthesis and growth/yield are closely related (Xu et al., 2020). Cellular osmotic regulation is mainly supplemented by
et al., 2016). In this study, we concluded that the Pn of all varieties of soluble sugar (SS). Plants accumulate soluble sugars, withstand harmful
melons decreased under humidity stress to various degrees, which was environments, maintain their integrity, and exhibit normal structural
consistent with the results of the study (LÜ et al., 2013). Most studies growth. In the present work, the responses of RC and SS of different
have reported that when crops are under stress, E, Gs, and Ci decrease, varieties under stress conditions were different, and there was no
resulting in a reduction in Pn (Zhong et al., 2018). In this study, the E, obvious correlation between the two indexes and the D value (Table 6),
Gs, and Ci of melon comparatively increased under HTH stress, while Pn suggesting that these two indexes are not suitable for the evaluation of
decreased, indicating that the decrease in the photosynthesis of melon melon varieties under HTH stress.
leaves had no significant relationship with stomatal conductance. This Plant stress tolerance is usually a quantitative trait controlled by
may be caused by the degradation of thylakoid membrane proteins in multiple genes. The responses of the identifying index to stress were
leaves and damage to the light system, reducing the photosynthetic rate different for plants with different genotypes, and there were obvious

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J. Weng et al. Scientia Horticulturae 288 (2021) 110317

interactions between the multiple indexes (Table 4). In recent years, the Bogeat-Triboulot, M.B., Buré, C., Gerardin, T., Chuste, P.A., Le Thiec, D., et al., 2019.
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Liu, X., Tang, Q., Zhang, X., Sun, S., 2018. Projected Changes in Extreme High
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interests or personal relationships that could have appeared to influence LÜ, G., WU, Y., BAI, W., MA, B., WANG, C., et al., 2013. Influence of high temperature
stress on net photosynthesis, dry matter partitioning and rice grain yield at flowering
the work reported in this paper.
and grain filling stages. J. Integr. Agric. 12 (4), 603–609. https://doi.org/10.1016/
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Funding Mittler, R., 2006. Abiotic stress, the field environment and stress combination. Trends
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Nagarajan, S., Jagadish, S.V.K., Prasad, A.S.H., Thomar, A.K., Anand, A., et al., 2010.
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Technical System and the Shanghai Agriculture Applied Technology rice in northwestern India. Agriculture Ecosyst. Environ. 138, 274–281. https://doi.
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Development Program, China (Grant NO. T20180304).
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plasticity to high temperature stress in chickpea: adaptive responses and variable
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