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\begin{document}

\title{In situ sampling revealed that 2,4 DTBP contributes to continuous
cropping obstacles in pepper}



\author[1]{Ning Wang}%
\author[1]{Hongyan Yang}%
\author[1]{Chunchun Wang}%
\author[1]{Min  Zhao}%
\affil[1]{Northeast Forestry University}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Continuous cropping (CC) obstacles resulting from the increase in
planting frequency restrict the sustainable development of the vegetable
industry. To understand the mechanisms causing continuous cropping
obstacles in-depth, we analyzed pepper (Capsicum annuum L.) rhizosphere
soils. Samples were collected using a new type of in situ sampler
composed of microporous ceramic tubes. After elution, the samples were
qualitatively and quantitatively analyzed via gas chromatography-mass
spectrometry (GC-MS) and high-performance liquid chromatography (HPLC).
Then, the effects of the main possible allelochemicals on sensitive
plants were investigated. Fourteen chemicals, including
2,4-di-tert-butyl-phenol (2,4-DTBP), diisobutyl adipate (DIBA), dibutyl
phthalate (DBP) ,and limonene, were detected with GC-MS. The measured
concentration of 2,4-DTBP increased with pepper growth. The results
based on seed toxicity revealed that low-concentration (\textless{}50
mg\selectlanguage{ngerman}·L-1) 2,4-DTBP improved lettuce seed germination rates and that
high-concentration (\textgreater{}300 mg·L-1) 2,4-DTBP had significant
(p\textless{}0.05) inhibitory effects. The results obtained from
sensitive cucumber plants showed that the average plant height in the
treatments containing 2,4-DTBP was lower (p\textless{}0.05). Significant
increases in reactive oxygen species (ROS) levels and reductions in the
photosynthetic rate (Pnet) were also detected (p\textless{}0.05). Leaf
transpiration (Tr), stomatal conductance (Gs) and root activity also
decreased significantly compared with those of the control. The
comparison results between the 2,4-DTBP treatment and the pepper CC
treatment showed that the cucumber was more significantly damaged in the
latter. Similar trends also occurred in pepper. Our results indicate
that the in situ sampler in this study can reflect the actual
concentrations of possible allelochemicals in rhizosphere soil. 2,4-DTBP
is one of the most important allelochemicals in pepper, and the
accumulation of 2,4-DTBP may be an important factor inducing continuous
cropping obstacles in pepper.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Title: In situ sampling revealed that 2,4 DTBP contributes to
continuous cropping obstacles in pepper}

Ning Wang\textsuperscript{a,b}, Hongyan Yang\textsuperscript{a,b}, Chun
Lei Wang\textsuperscript{a,b}, Min
Zhao\textsuperscript{a,b}11Corresponding authors E-mail address:
82191513@163.com; Tel.: +86 451 82191513; Fax: +86 451 82191513.

\textsuperscript{a}\emph{Key Laboratory of Enzyme and Enzyme-like
Material Engineering of Heilongjiang Province, Harbin 150040, China}

\emph{\textsuperscript{b}College of Life Sciences, Northeast Forestry
University, Harbin 150040, China}

\textbf{Abstract}

Continuous cropping (CC) obstacles resulting from the increase in
planting frequency restrict the sustainable development of the vegetable
industry. To understand the mechanisms causing continuous cropping
obstacles in depth, we analyzed pepper (\emph{Capsicum annuum} L.)
rhizosphere soils. Samples were collected using a new type of in situ
sampler composed of microporous ceramic tubes. After elution, the
samples were qualitatively and quantitatively analyzed via gas
chromatography-mass spectrometry (GC-MS) and high-performance liquid
chromatography (HPLC). Then, the effects of the main possible
allelochemicals on sensitive plants were investigated. Fourteen
chemicals, including 2,4-di-tert-butyl-phenol (2,4-DTBP), diisobutyl
adipate (DIBA), dibutyl phthalate (DBP), and limonene, were detected
with GC-MS. The measured concentration of 2,4-DTBP increased with pepper
growth. The results based on seed toxicity revealed that
low-concentration (\textless{}50 mg\selectlanguage{ngerman}·L\textsuperscript{-1}) 2,4-DTBP
improved lettuce seed germination rates and that high-concentration
(\textgreater{}300 mg·L\textsuperscript{-1}) 2,4-DTBP had significant
(\emph{p} \textless{}0.05) inhibitory effects. The results obtained from
sensitive cucumber plants showed that the average plant height in the
treatments containing 2,4-DTBP was lower (\emph{p} \textless{}0.05).
Significant increases in reactive oxygen species (ROS) levels and
reductions in the photosynthetic rate (\emph{P\textsubscript{net}} )
were also detected (\emph{p} \textless{}0.05). Leaf transpiration
(\emph{T\textsubscript{r}} ), stomatal conductance
(\emph{G\textsubscript{s}} ) and root activity also decreased
significantly compared with those of the control. The comparison results
between the 2,4-DTBP treatment and the pepper CC treatment showed that
the cucumber was more significantly damaged in the latter. Similar
trends also occurred in pepper. Our results indicate that the in situ
sampler in this study can reflect the actual concentrations of possible
allelochemicals in rhizosphere soil. 2,4-DTBP is one of the most
important allelochemicals in pepper, and the accumulation of 2,4-DTBP
may be an important factor inducing continuous cropping obstacles in
pepper.

\textbf{Key words:} in situ sampling; allelopathy; allelochemical;
pepper, continuous cropping obstacle

\textbf{Introduction}

Pepper (\emph{Capsicum annuum} L.) is an economically important
vegetable crop species that is widely cultivated in temperate and
tropical regions of the world. Its global annual yield is approximately
3.7 million tons (FAO, 2019). Pepper production in China accounts for
50.8\% of global pepper production. Therefore, the stable development of
the pepper industry has an important effect on vegetable industry
development.

To ensure a sustainable supply, planting frequency in one year or the
number of successive planting years has increased, which has resulted in
continuous cropping (CC) obstacles that restrict the sustainable
development of the vegetable industry (Li et al., 2019). This problem
occurred in the pepper industry (Khan et al., 2015). CC obstacles slow
crop growth and development, decrease plant vigor, increase the
frequency of disease and directly decrease crop yield and quality (Li et
al., 2015a, 2015b; Li et al., 2018; Li and Liu, 2018). This means that
CC obstacles significantly influence the sustainable development of
agriculture and have become one of the crucial problems to be solved in
the field of agricultural science.

This negative effect of CC obstacles on crop growth is mainly caused by
the accumulation of allelochemicals, causing self-allelopathy and
allelopathy in the soil (Inderjit, 2006; Wang et al., 2019). Most
allelochemicals in the soil are plant secondary metabolites. These
natural compounds, such as sugar, amino acids, organic acids, phenolic
compounds, flavonoids, and lipids, are mainly secreted by plant roots
(Dong et al., 2018; Oburger et al., 2018).

Of these compounds, the effects of allelopathy from phenolic compounds
on crop growth and development are particularly obvious (Batish et al.,
2004; Khan et al., 2015; Verbon \& Liberman, 2016). Chen et al. (2020)
found that the accumulation of phenols in soil significantly inhibited
strawberry growth and reduced the abundance of various beneficial
bacterial groups; as a result, the quality and yield of strawberry fruit
decreased. Wu et al. (2009) reported that the soil microbial ecological
environment changed due to the accumulation of ferulic acid in cucumber
soil, further inhibiting cucumber growth. Batish et al. (2007) found
that when \emph{Chenopodium murale} and wheat were intercropped, the
phenolic substances secreted by \emph{C. murale} roots hindered the
growth and development of wheat. Thus, the study of allelochemicals and
root system allelopathy contributes to understanding the mechanisms of
and solving the problems caused by CC obstacles.

Determining how to sample allelochemicals in the soil is crucial for
further analysis. Currently, root exudate sampling methods include the
hydroponic method, tube method, agar culture method, air culture method,
filter paper adsorption method, plant root box method, field
investigation method, pot culture method, microsuction cup method, and
others, the most popular of which is hydroponics (Wang et al., 2019).
Many important advances in root exudate studies have been achieved with
this method. However, because this method does not consider the
decomposition and transformation of root exudates by soil
microorganisms, it is difficult to simulate the true concentrations of
root exudates in the soil with hydroponics. Therefore, it is still
challenging to compare the results obtained under hydroponic conditions
with those from the real soil environment (Oburger \& Jones, 2018).
Instead, in situ soil collection has become the most promising technical
means of solving the above problems because the exudates are undisturbed
and remain closer to the actual secretion conditions. Proctor \& He et
al. (2017) transplanted \emph{Rhododendron groenlandicum} from an
ombrotrophic bog to a special root trap for cultivation and sampled root
exudates without soil disturbance using a set of negative-pressure
vacuum devices. Qualitative and quantitative analysis of the root
exudates of different species revealed the differences in root exudates
among different species and the variability in root exudates from roots
with different morphologies. Oburger et al. (2013) invented an in situ
culture box in which plant roots grew naturally into a hydroponic
compartment containing an exchange membrane and root exudates were
extracted under negative pressure. Then, the low-molecular-weight
organic matter in the root exudates was quantitatively analyzed. In
addition, an in situ root box was also used to culture individual roots
to analyze the exudates with the aid of filter paper or resin (Haase et
al., 2007; Shi et al., 2011).

In this study, to understand the allelopathy mechanism that results in
pepper CC obstacles, we invented a new, noninvasive in situ sampler
based on the principle of capillarity that extracts substances from the
soil and identified the main potential pepper allelochemicals by
qualitative and quantitative analyses. The mechanism of the main
potential allelochemical was explored.

\textbf{MATERIALS AND METHODS}

\textbf{Noninvasive in situ sampler}

The in situ sampler, composed of microporous ceramic, was made by
Yizhong Ceramic Technology Co., Ltd. (Yixing, China). The specifications
of the in situ sampler were as follows: inner diameter, 10 mm; external
diameter, 20 mm; length, 14 cm; average adsorption volume of tube wall,
33 cm\textsuperscript{3}; and intratubal hollow volume, 11
cm\textsuperscript{3}. The intratubal volume of the in situ sampler was
filled with nonionic XAD-4 resin (Solarbio, Bei Jing, China) for
sampling and extraction.

\textbf{Collection of potential allelochemicals}

Two wooden incubators with volumes of 1 m × 1 m× 0.3 m were placed in a
greenhouse and then filled to a 20-cm depth with soil that had not been
previously planted with pepper. Pepper seedlings of uniform height and
good growth status were planted in one of the wooden incubators in a 5×5
pattern at a planting distance of 10-15 cm. The other wooden incubator
was not planted with pepper seedlings to provide a control treatment.
Nine in situ samplers were inserted into the soil in each incubator in a
3×3 arrangement. During each growth period, three of the in situ
samplers were removed to analyze the components extracted from the
rhizosphere soil. The XAD-4 resins removed from the sampler were eluted
with 300 mL of methanol solution. The eluates were merged and
concentrated to 5 mL by nitrogen purging. Then, the concentrates were
filtered with a 0.45-\selectlanguage{greek}µ\selectlanguage{english}m filter membrane. The filtrates were stored at
-20\selectlanguage{ngerman}°C for gas chromatography--mass spectrometry (GC-MS) and
high-performance liquid chromatography (HPLC) analysis.

\textbf{GC-MS analysis of potential allelochemicals in pepper root
exudates}

GC-MS analysis of the methanol extract was conducted with an Agilent
7890A GC system coupled to an Agilent 5975C mass selective detector
(MSD) (Agilent, Technologies, Santa Clara, CA, USA). The GC was equipped
with a two-way purged splitter installed after the analytical column (30
m × 0.25 mm, 0.25 \selectlanguage{greek}μ\selectlanguage{english}m, HP-5MS, J \& W Scientific, USA). The inlet
temperature was 250\selectlanguage{ngerman}°C. The column temperature was initially held at 50°C
and was gradually increased to 280°C at a rate of
10°C·min\textsuperscript{-1}. High-purity helium was used as the carrier
gas at a flow rate of 1 mL·min\textsuperscript{-1}. The injection volume
was 1 \selectlanguage{greek}μ\selectlanguage{english}L in splitless mode.

The mass spectrometry conditions were the following: electron
bombardment source, electrospray ionization; bombardment voltage, 70 eV;
scanning range m/z, 30-600 amu; scanning speed, 0.2 s whole-process
sweep; ion source temperature, 200\selectlanguage{ngerman}°C; and quadrupole temperature, 150°C.

\textbf{Reconfirmation of in situ sampling effects}

To evaluate in situ sampling, a given mass of 2,4-DTBP (400 mg·kg) was
added to the soil. The traditional root soil extraction method (Yao et
al., 2020) was used to quantitatively determine the content of 2,4-DTBP
in pepper soil. Briefly, forty-five grams of soil was extracted with 100
ml of ethyl acetate for 24 h and centrifuged at 10 000 \emph{g} (15
min). The supernatant was concentrated to 5 ml by rotary evaporation and
filtered through a 0.45-\selectlanguage{greek}μ\selectlanguage{english}m filter. The filtrate was used for further
comparison with in situ sampling. Five in situ samplers were put into
one pot (3.571 L), and 5 pots were prepared. Water was added to the pot
three times every day (total: 2 L). After 48 h, samplers were removed
for quantitative analysis of 2,4-DTBP. The pepper CC soil was also
treated with the same method.

\textbf{Quantification analysis of 2,4-DTBP}

The quantitative analysis of 2,4-DTBP in the concentrated solution was
performed by an external standard method with an Agilent 1200s HPLC
instrument (Agilent Co., Palo Alto, CA, USA) equipped with a C18
reversed-phase column. The mobile phase was methanol, and the flow rate
was 1.0 mL min\textsuperscript{-1}. The column temperature was 38\selectlanguage{ngerman}°C, and
the injection volume was 20 \selectlanguage{greek}μ\selectlanguage{english}L.

\textbf{Detection of 2,4-DTBP allelopathy}

Lettuce seeds were placed on filter paper soaked with 2 mL of methanol
solution containing 2,4-DTBP at concentrations of 200, 300, 500, 800,
and 1,000 mg\selectlanguage{ngerman}·L\textsuperscript{-1}. Fifty lettuce seeds were sown in
each of three Petri dishes using filter paper as the substrate. Methanol
solution without 2,4-DTBP was used as a blank control. All Petri dishes
were placed in a dark room at 25°C for 6 days. The germination time was
defined as the time until the radicle had protruded beyond the seed coat
by at least 1 mm. The lengths of the radicle and hypocotyl of the
lettuce seedlings were measured on the 17th day after germination.

Six treatments with 4 potential allelochemicals were established in the
lettuce seed germination tests. In the treatments, the concentrations of
2,4-DTBP were 0, 10, 20, 30, 40, and 50 mg·L\textsuperscript{-1}. The
concentrations of DIBA, DBP and limonene were 15
mg·L\textsuperscript{-1}. The lettuce seed germination rate was
determined every 8 h from 24 to 48 h.

\textbf{Greenhouse experiments}

The phenolic compound 2,4-DTBP was obtained from Sigma Chemical Company
(St. Louis, MO, USA). The 2,4-DTBP solution (1.18
g·L\textsuperscript{-1}) was prepared with methanol, and distilled water
and methanol were used as controls. The experimental soil and pepper CC
soil were obtained from the Horticultural Subacademy of the Heilongjiang
Academy of Agricultural Sciences. The four treatments were designated CK
(water), Me-CK (methanol), 2,4-DTBP, and pepper CC soil. Solutions of
2,4-DTBP, water and methanol were sprayed onto the soil. Cucumber
(\emph{Cucumis sativus} L. cv. holland No. 83-16) seeds were germinated
in perlite. Ten-day seedlings with uniform growth vigor were selected
for all treatments, and there were three replicates per treatment. The
content of 2,4-DTBP in the soil was 23.6 mg·kg\textsuperscript{-1}.
During the experiment, the air temperature was maintained at 25±2°C, and
the relative humidity was between 50\% and 70\%. The plant height was
measured at 3-day intervals. The experiment ended 25 days after
inoculation with the 2,4-DTBP solution.

Four-week-old pepper seedlings were selected to test the effects of
2,4-DTBP on pepper. The same four treatments used for cucumber were
established. After 3 weeks, the physiological indexes of pepper were
measured.

\textbf{Assay of antioxidant enzyme activity and malondialdehyde (MDA)
conten} \emph{t}

Following the method of Li et al. (2011), 0.2 g leaves were selected and
ground with 2 mL of cold PBS buffer (pH 7.8). After centrifugation at
4°C and 10,000 \emph{g} for 20 min, the supernatant was extracted to
determine the activity of superoxide dismutase (SOD). The reaction
mixture was composed of 2.7 mL of 14.5 mM methionine, 90 \selectlanguage{greek}μ\selectlanguage{english}L of 50 mM
phosphate buffer (pH 7.8), 10 \selectlanguage{greek}μ\selectlanguage{english}L of 20 \selectlanguage{greek}μ\selectlanguage{english}M riboflavin, 10 \selectlanguage{greek}μ\selectlanguage{english}L of 2.25 mM
nitro blue tetrazolium chloride and 30 \selectlanguage{greek}μ\selectlanguage{english}L of enzyme supernatant, and the
SOD activity was determined at 560 nm.

For the measurement of MDA, 0.2 g of leaf tissue was homogenized in 1.5
mL of 5\% trichloroacetic acid (TCA) after being ground with liquid
nitrogen. The homogenate was centrifuged at 10,000 \emph{g} for 10 min
at 4\selectlanguage{ngerman}°C. 1-mL aliquot of the supernatant was mixed with 2 mL of 0.6\%
thiobarbituric acid and heated at 95°C for 30 min. After being quickly
cooled in an ice bath, the absorbance of the supernatant was recorded at
450, 532 and 600 nm (Dhindsa et al., 1981).

\selectlanguage{english}\textbf{Assay of O\textsubscript{2}\textsuperscript{-} and
H\textsubscript{2}O\textsubscript{2} formation rates}

Following the method of Elstner and Heupel (1976), 0.2 g of leaves
ground with liquid nitrogen were homogenized in 65 mM phosphate buffer
(pH 7.8, 3 mL) and centrifuged at 4degC and 5,000 \emph{g} for 10 min. The
resultant supernatant (1 mL) was mixed with 0.1 mL of 10 mM
hydroxylamine chlorhydrate and 65 mM phosphate buffer (0.9 mL) for 20
min at 25degC. Sulfanilamide (17 mM, 0.5 mL) and alpha-naphthylamine (7
mM, 0.5 mL) were added to the mixture (0.5 mL). After 20 min at 25degC, 3
mL of ethyl ether was added to the mixture for centrifugation at 4degC and
2,000 \emph{g} for 15 min, and the absorbance was measured at 530 nm.

For the H\textsubscript{2}O\textsubscript{2} content assay, 2 g of
leaves was ground in liquid nitrogen together with 3 mL of acetone and
centrifuged at 4degC for 20 min (15,000 x g). One milliliter of
supernatant was mixed with 5\% titanium sulfate (2.5 mL) and ammonium
hydroxide (0.2 mL). After precipitation formation, the mixture was
centrifuged at 5,000\emph{g} for 20 min, and the supernatant was
discarded. Five milliliters of 1 M sulfuric acid was added to stop the
reaction. The absorbance was measured at 415 nm, and the formation rate
of H\textsubscript{2}O\textsubscript{2} was calculated from a standard
curve of H\textsubscript{2}O\textsubscript{2}reagent.

\textbf{Gas exchange measurements}

The net photosynthetic rate (\emph{P\textsubscript{net}} ),
intercellular CO\textsubscript{2} concentration
(\emph{C\textsubscript{i}} ), leaf transpiration
(\emph{T\textsubscript{r}} ) and stomatal conductance
(\emph{G\textsubscript{s}} ) were measured after 15 days of treatment
using a portable photosynthesis measurement system (CIRAS-1, PP System
Company, UK). The CIRAS-1 homemade light source and a
CO\textsubscript{2} cylinder were used to determine the gas exchange
parameters. The light intensity was 800
\selectlanguage{greek}μ\selectlanguage{english}mol\selectlanguage{ngerman}·m\textsuperscript{-2}·s\textsuperscript{-1}, and the concentration
of CO\textsubscript{2} in the fixed system was \selectlanguage{greek}μ\selectlanguage{english}L\selectlanguage{ngerman}·L\textsuperscript{-1}.

\textbf{Root activity}

For the root activity measurements, 0.2 g of root tip tissue was
homogenized in a 10-mL mixture of 0.4\% 2,3,5-triphenyltetrazolium
chloride (TTC) and phosphate buffer (pH 7.8). The mixture was placed at
37°C in the dark for 3 h. Then, 2 mL of sulfuric acid was added to the
mixture to terminate the reaction.

The grinding fluid was poured into a test tube. The residue was washed
three times with a small amount of ethyl acetate. Finally, the residue
was combined with 10 mL of ethyl acetate. The absorbance was measured
three times at 485 nm. The TTC reduction amounts were calculated from a
standard curve according to the formula:\(\text{root\ activity}=\selectlanguage{greek}\frac{TTC\ reduction\ quantity(\text{μg})}\selectlanguage{english}{root\ weigt\left(g\right)\times time(H)}\)

The roots of the plants involved in the chlorophyll fluorescence assay
were washed with distilled water to remove any impurities, and their
morphology was characterized under a microscope (Nikon, TE2000).

\textbf{Statistical analysis}

Three different sets of lettuce seeds exposed to different
concentrations of potential allelochemicals were used in the germination
experiments. Statistical analyses were performed using the SPSS 20.0
statistical software program. Data were assayed in triplicate and
expressed as the means \selectlanguage{ngerman}± standard errors. Differences were analyzed by
one-way analysis of variance (ANOVA) and Tukey's honestly significant
difference (HSD) test. Differences were considered very significant
at\emph{p} \textless{}0.05.

\textbf{Results}

\textbf{Analysis of potential allelochemicals in pepper rhizosphere
soil}

In this study, we designed an in situ sampler, which is shown in Fig.
S1. By the use of this in situ sampler, which relies on capillary
action, rhizosphere chemicals were continuously collected without
disturbing the plant roots. Twenty-eight organic compounds were detected
in the pepper rhizosphere soils by GC-MS analysis, including 16 esters,
4 olefins, 2 phenols, 2 hydrocarbons, 1 aromatic, 1 alcohol, 1 ketone
and 1 hydrazine (Table 1). Among them, their matching values to 14
standard chemical substances, including \selectlanguage{greek}γ-\selectlanguage{english}sitosterol, DIBA, and
tridecane, were higher than 90\%. Most of these compounds were
identified as the main allelochemicals in root exudates in previous
reports (Shui et al., 2010; Sun et al., 2013). The results showed that
it is feasible to use the in situ sampler developed in this study for
the qualitative analysis of root exudates.

An in situ sampler in combination with GC-MS was used to quantitatively
analyze the main chemical substances in pepper rhizosphere soil at
different growth stages in one growing season. 2,4-DTBP, DIBA, DBP and
limonene were the four most predominant substances throughout the whole
growing season (Fig. 1). The accumulation of 2,4-DTBP with pepper growth
was the most significant of all the compounds identified.

To test the effects of in situ sampling, we carried out reconfirmation
experiments using a direct extraction method from rhizosphere soil. The
results showed that the content of 2,4-DTBP in the soil of Y1 and the
measured values (23.6 mg\selectlanguage{ngerman}·kg\textsuperscript{-1}; 22.4
mg·kg\textsuperscript{-1}) from in situ sampling and direct extraction
were not significantly different (\emph{p} \textgreater{}0.05). For the
same pepper CC soil, the values (in situ sampling: 153.0
mg·kg\textsuperscript{-1}; soil: 161.19 mg·kg\textsuperscript{-1}) were
also not significantly different (\emph{p} \textgreater{}0.05).

\textbf{Effects of 2,4-DTBP on lettuce seed germination and growth}

According to the GC-MS results, 2,4-DTBP, DIBA, DBP and limonene were
the four main compounds in the rhizosphere. To test the allelopathy of
2,4-DTBP, we mixed these four substances and performed experiments with
0-50 mg·L\textsuperscript{-1} concentrations of 2,4-DTBP and fixed
concentrations of 15 mg·L\textsuperscript{-1} DIBA, DBP and limonene.
The germination of lettuce seeds was significantly promoted (\emph{p}
\textless{}0.05) at low concentrations (\textless{}50
mg·L\textsuperscript{-1}) (Fig. S3). To further clarify the allelopathic
threshold, six concentrations of 0, 200, 300, 500, 800 and 1,000
mg·L\textsuperscript{-1} 2,4-DTBP were established. When the
concentration of 2,4-DTBP was more than 300 mg·L\textsuperscript{-1},
the germination time of lettuce seeds was significantly prolonged, and
the growth of the radicle and hypocotyl was also significantly inhibited
(Table 2) (\emph{p} \textless{}0.05). The germination rate of lettuce
seeds decreased by 56.7\%, 68.2\% and 63.6\% at 2,4-DTBP concentrations
of 500, 800 and 1,000 mg·L\textsuperscript{-1}, respectively, compared
with that under no 2,4-DTBP exposure. The results showed that 2,4-DTBP
at concentrations above 300 mg·L\textsuperscript{-1} had a significant
inhibitory effect on seed germination.

\textbf{Effects of pepper CC soil and 2,4-DTBP on the physiological and
biochemical properties of cucumber and pepper leaves}

To test the effect of 2,4-DTBP on plant growth, cucumber was first
selected as the test object. Four treatments, namely, a no-addition
control (CK), a methanol solvent-addition control (Me-Ck), a 2,4-DTBP
treatment (2,4-DTBP) and a pepper CC soil treatment (pepper soil), were
established. The 2,4-DTBP (23.6 mg·kg\textsuperscript{-1}) and pepper CC
soil (2,4-DTBP:153.0 mg·kg\textsuperscript{-1}) treatments both
significantly inhibited the growth of cucumber plants (\emph{p}
\textless{}0.05). Compared with that in the CK treatment, the plant
heights of cucumber in the 2,4-DTBP and pepper soil treatments were 34
cm and 45 cm lower, respectively. The growth rate and leaf area of
cucumber treated with 2,4-DTBP and pepper soil were significantly lower
than those of the corresponding control groups (Fig. 2 and Fig. 3)
(\emph{p} \textless{}0.05). In particular, the growth rate of plants in
the pepper soil was only 1.57 cm·d\textsuperscript{-1}, which was 41.9\%
of that in the control group, and the leaf area of plants in the pepper
soil was only 17.6\% (17.5 cm\textsuperscript{2}) of the leaf area of
the CK plants. The root activity in the pepper soil was significantly
different from that in the other treatments. Compared with CK, the root
activity of the cucumber in the pepper soil treatment was 55.2\% lower.
2,4-DTBP decreased root activity by 19\% compared with CK, but the
difference was not significant. Based on the results for root activity,
we further used fluorescence imaging to assess the root damage. The
results showed that the fluorescence intensities for the CK, Me-CK,
2,4-DTBP and pepper soil treatments were 19.16, 21.61, 30.32, and 33.93
a.u., respectively. The fluorescence intensities of 2,4-DTBP and pepper
soil were significantly higher than those of the two control groups
(Fig. 4) (\emph{p} \textless{}0.05). The root activity of pepper
decreased with continuous cropping years and showed significant
differences between the treatments. Compared with CK, the root activity
of Y1, Y2 and Y3 was 1.49, 0.93 and 0.28 mg
TTF·g\textsuperscript{-1}·h\textsuperscript{-1}, respectively. The root
fluorescence intensities resulting from the Y1, Y2 and Y3 treatments
were 14.53, 19.94 and 24.00 a.u., respectively, which were significantly
higher than those in CK (6.94 a.u.) (Fig. 5). In this study, there were
no significant differences in root activity and root fluorescence
intensity between CK and Me+CK.

Compared with those in CK and Me-CK, the SOD activity and MDA content in
cucumber leaves grown in pepper soil significantly increased after 15
days of treatment, but the SOD activity and MDA content in cucumber
leaves in the 2,4-DTBP treatment did not increase significantly
(\emph{p} \textless{}0.05). These results indicate that the damage to
the cucumber leaf membrane was more serious in the pepper CC soil than
in the 2,4-DTBP treatment. The 2,4-DTBP and pepper soil treatments
significantly increased the level of reactive oxygen species (ROS) in
the soil, i.e., the contents of H\textsubscript{2}O\textsubscript{2} and
O\textsubscript{2}\selectlanguage{english}\textsuperscript{-}(\emph{p} \textless{}0.05). The
content of O\textsubscript{2}\selectlanguage{english}\textsuperscript{-} was significantly
different between the two treatments (Table 3). Compared with that in
CK, the 2,4-DTBP and pepper soil had significant effects on
leaf\emph{P\textsubscript{net}} , decreasing it by 18.6\% and 41\%,
respectively (\emph{p} \textless{}0.05). 2,4-DTBP and pepper soil
significantly reduced the C\emph{\textsubscript{i}}(\emph{p}
\textless{}0.05). The \emph{T\textsubscript{r}}
and\emph{G\textsubscript{s}} in the 2,4-DTBP treatment were reduced by
36.4\% and 33.3\%, respectively. The \emph{T\textsubscript{r}}
and\emph{G\textsubscript{s}} in the 2,4-DTBP treatment decreased by
50.8\% and 53.3\%, respectively (Table 4).

The results from pepper showed that the activity of SOD and the content
of MDA and O\textsubscript{2}\selectlanguage{english}\textsuperscript{*-} in pepper
significantly increased when grown in CC soil for different years. The
ROS level of pepper leaves was significantly increased (Table 3).
The\emph{P\textsubscript{net}} and WUE of pepper leaves were
significantly affected by pepper CC years. The
\emph{P\textsubscript{net}} values of Y1, Y2 and Y3 were 4.9, 0.91 and
0.82 \selectlanguage{greek}μ\selectlanguage{english}mol\selectlanguage{ngerman}·m\textsuperscript{-2}·s\textsuperscript{-1}, and the WUE
values of Y1, Y2 and Y3 were 3.12, 1.74 and 1.53, respectively.
The\emph{G\textsubscript{s}} of Y2 and Y3 were 22.39 and 22.10
mmol·m\textsuperscript{-2}·s\textsuperscript{-1}, and
the\emph{T\textsubscript{r} of} Y2 and Y3 were 0.52 and 0.53
mmol·m\textsuperscript{-2}·s\textsuperscript{-1}, respectively. The CC
years also had significant effects on the intracellular
CO\textsubscript{2} concentration (C\textsubscript{i}), which increased
with the increase in CC years (Table 4). In general, with the increase
in CC years, the photosynthesis of pepper was affected more
significantly. The CC of pepper has a strong physiological toxic effect
on itself. Compared with the allelopathic effect of CC of pepper on
cucumber, the allelopathic effect of pepper was more significant.

\textbf{Discussion}

\textbf{In situ sampling for the detection of possible allelochemicals
in pepper rhizosphere soil}

Considering the biological specificity of plant root structures and the
spatial diversity of soil structures, the continuous, noninvasive
extraction of plant root exudates under natural conditions is an urgent
technical problem and a challenge for allelopathy research. Currently,
most studies on root exudates are carried out by extracting the
hydroponic solution from around the root or by indirectly converting the
crude extract into an aqueous solution for analysis. Because these
techniques separate plants from the soil environment, root growth, root
cell activity and other physiological characteristics may change. When
root metabolism changes, the natural compounds secreted by plant roots
also change (Lucas Garcia et al., 2001; Canarini et al., 2016).

In this study, our in situ sampler was placed at the same depth as the
roots of the plants. The tube wall came into close contact with the root
system without causing physical damage to the root system in the
collection process. Meanwhile, the capillary action of the tube wall
naturally brought the root exudates from the soil into the tube without
any external force. The in situ sampler used in this study collected the
allelochemicals secreted by the soil roots without disturbance. After
sampling with the in situ samplers, the samples were analyzed
qualitatively and quantitatively. We also compared the concentrations of
the exudates collected by the in situ sampler with the concentrations of
exudates obtained through direct soil extraction analysis. The results
showed that the differences in the concentrations of 2,4-DTBP between
the samples from the in situ sampler and direct soil extraction were not
significant. The results of the reconfirmation experiment with direct
extraction and in situ sampling show that the in situ sampler could be
suitable for sample collection. This result illustrated that the in situ
sampling technique could reliably and accurately reflect the actual
concentration of allelochemicals from the plant in the soil (Holz et
al., 2018; Oburger \& Jones, 2018). The in situ sampler developed in our
study is potentially suitable for noninvasive dynamic detection and
analysis of root exudates.

\textbf{Potential allelochemical analyses in pepper rhizosphere soil}

By in situ sampling and GC-MS analysis of the pepper root exudates at
different growth stages, twenty-eight potential allelochemicals were
identified in the pepper root exudates. Most of them have been detected
in a variety of plants. For example, dibutyl phthalate, isooctyl
phthalate, 2,4-DTBP, benzyl benzoate, DBP, adipic acid, and diisobutyl
ester were identified in the root soil of eggplant, garlic/eggplant CC
or \emph{Bidens pilosa} (Zhou et al., 2011, Wang et al., 2014, Shen et
al. 2018). In this study, through the combination of in situ sampling
and GC-MS analysis, 2,4-DTBP was identified in pepper soil (Fig. 1). The
accumulation of 2,4-DTBP gradually increased as the pepper plants grew.
The accumulation of allelochemicals is closely related to CC obstacles
(Agegnehu et al., 2016; Chen et al., 2020). Through seed germination
tests and plant growth tests with sensitive plants, we deduced that
2,4-DTBP is the main allelochemical substance in pepper rhizosphere
soil. To the best of our knowledge, this is the first report that
2,4-DTBP is one of the main allelochemicals in pepper. According to
previous reports, 2,4-DTBP has a wide range of biological functions. For
example, 2,4-DTBP promotes senescence in human gastric adenocarcinoma
cells by inhibiting the activity of the HDAC6 enzyme, induces mitotic
mutations, produces polynuclear cells, increases the proportion of
polymerized tubulin and has anticancer activity (Song et al., 2018).
2,4-DTBP is also an excellent antibacterial agent that is used as a
biological fumigant to prevent and control diseases caused
by\emph{Botrytis cinerea} and \emph{Rhizopus stolonifer} (Zhang et al.,
2020). 2,4-DTBP can change the cell membrane properties
of\emph{Streptococcus pyogenes} and increase the sensitivity of \emph{S.
pyogenes} to antibiotics. In addition, 2,4-DTBP can be used in
combination with antibiotics to achieve antibacterial efficacy and is
considered a nonbactericidal and nontoxic drug candidate (Viszwapriya et
al., 2016). In this study, we found that 2,4-DTBP is one of the main
allelochemicals in pepper. The results of this study could aid in
advancing functional research on 2,4-DTBP and solving the CC problem.

When the 2,4-DTBP concentration exceeded 300 mg·L\textsuperscript{-1},
2,4-DTBP significantly affected the rate and time of germination and the
length of the radicle and hypocotyl of lettuce seeds. However, when the
2,4-DTBP concentration was below 50 mg·L\textsuperscript{-1}, 2,4-DTBP
significantly promoted the germination of lettuce seeds. This typical
allelopathic phenomenon in 2,4-DTBP, i.e., high-concentration inhibition
and low-concentration promotion, is similar to the results of previous
studies on various allelochemicals. For example, Wang et al. (2007)
found that low concentrations of cinnamic acid promoted the growth of
cucumber seedlings under saline-alkali conditions, while high
concentrations of cinnamic acid inhibited the growth of cucumber
seedlings.

\textbf{The effect of pepper CC soil and 2,4-DTBP on the physiological
growth of cucumber and pepper}

Phenolic compounds are very important and common allelochemicals in the
ecosystem and generally exhibit obvious allelopathy. Ye et al. (2006)
found that the oxidative stress caused by cinnamic acid had a
significant inhibitory effect on cucumber growth and leaf area and
increased the incidence of cucumber wilt. Huang et al. (2020) showed
that the main phenolic substance (p-hydroxybenzoic acid (pHBA)) in the
soil after three consecutive years of cucumber cropping reduced the
activity of root meristems, thereby inhibiting the growth of cucumber
roots. Most studies have suggested that allelochemicals have inhibitory
effects on plant growth (Maberly, 2014; Asaduzzaman et al., 2016;
Hussain and Reigosa, 2015). CC obstacles are an important ecological
factor restricting vegetable production. The measures to solve or reduce
the negative effects caused by CC are rotation or intercropping of
different varieties of vegetables. As the main vegetable product,
cucumber is usually intercropped with pepper (Dong et al, 1994; Zhao et
al, 2016). The pepper CC soil and 2,4-DTBP had significant inhibitory
effects on cucumber growth (Fig. 3). In particular, the cucumber plant
height, leaf area and root vitality in the continuous pepper cropping
soil treatment were significantly reduced compared with those in the CK
treatment. The inhibitory effect of pepper CC soil was the most
significant in this study, indicating that this inhibitory effect was
not caused only by the individual effect of 2,4-DTBP.

ROS, mainly H\textsubscript{2}O\textsubscript{2} and
O\textsubscript{2}\selectlanguage{english}\textsuperscript{-}, are byproducts of many metabolic
processes and play important signaling roles in all organisms (Asada,
2006). ROS are involved in many biological processes, such as plant
system resistance, stomatal closure, programmed cell death, germination
and root development. Allelochemicals can induce excessive ROS
production (Xu et al., 2008). To reduce the damaging effects of ROS,
plants have evolved enzymatic antioxidants, such as SOD. In many crops,
such as tomato, kidney bean and green algae, environmental stress has
been found to stimulate SOD activity (Zlatev et al., 2006; Hayat et al.,
2018; Singh et al., 2018). The accumulation of membrane lipid
peroxidation products (i.e., MDA) and ROS in plant tissues indicates
that the permeability and integrity of plant cell membranes have been
mostly destroyed (Sidhu et al., 2016). In plant cells, the oxidative
effect of allelochemicals can increase the production of ROS, leading to
the inhibition of photosynthesis (Garcia-Sanchez et al. 2012; Das \&
Roychoudhury, 2014). In this study, the pepper CC soil and 2,4-DTBP
treatments both increased the ROS levels in the cucumber leaves.
Similarly, these two treatments significantly reduced the net
photosynthetic rate, transpiration rate, stomatal conductance, and
intracellular CO\textsubscript{2} concentration in the plants and
inhibited the photoconductive effect (Table 4). In particular, the
decrease in stomatal conductance was more pronounced than the other
effects, indicating that the closure of stomata may be an important
factor leading to the decrease in photosynthesis and transpiration
(Moles et al., 2016). The accumulation of ROS in cells under
allelochemical stress can lead to pigment loss, reduced photosynthetic
CO\textsubscript{2} assimilation, and decreases in protein and RNA
levels (Rao et al., 2006). Therefore, the significant inhibitory effect
of pepper CC soil and 2,4-DTBP on cucumber growth may be the result of
diminished photosynthesis (Zhu et al., 2017). The SOD activity and MDA
and ROS contents of the pepper CC soil treatment were significantly
higher than those of the other treatments, indicating that the cell
membrane structure of the cucumber leaves was more severely damaged,
which in turn affected the photosynthesis and physiological growth of
the cucumbers more significantly.

The mechanisms of CC obstacles are very complicated, but the
accumulation of allelochemicals in the soil is certainly the main
causative factor (Ahammeda et al., 2012; Huang et al., 2020). Many
studies have found that CC leads to the continuous accumulation of
phenols, which change the properties of the soil and have significant
inhibitory effects on plant growth (Asao et al., 2008; Halvorson et al.,
2009). In this study, the 2,4-DTBP content of the pepper CC soil was
much higher than that of the unplanted soil and higher than that of
other substances in the pepper CC soil. Experiments with pepper CC soil
showed that the allelopathy of pepper CC was very obvious (Fig. S2). The
self-toxicity of pepper CC soil is an inevitable phenomenon; therefore,
we used cucumber, a common crop-rotation crop, for allelopathy tests
involving CC obstacles. Then, we used pepper to test this effect. The
results showed that the CC of pepper had a significant effect on the
growth, photosynthesis and other physiological indicators of both
cucumber and pepper. Thus, we deduce that the accumulation of 2,4-DTBP
is an important obstacle to the CC of pepper.

\textbf{Conclusion}

Plant root exudates are closely related to CC obstacles, and there are
many challenges in analyzing the components of root exudates accurately
and in real time. In this study, we developed a new nondisturbing in
situ sampler based on the capillary principle for the extraction and
analysis of pepper root exudates. The sampler effectively collected
possible allelochemicals without damaging the roots in the soil. The
quantitative analysis of 2,4-DTBP showed that the content of 2,4-DTBP in
the in situ sampler was close to that in the soil, indicating that the
new sampling method could truly reflect the contents of the chemical
substances secreted by roots into the soil. 2,4-DTBP had a typical
allelopathic effect on seed germination. The growth, photosynthesis and
root activity of cucumber were significantly affected by the 2,4-DTBP
and pepper CC soil treatments. The 2,4-DTBP and pepper CC soil
treatments significantly increased the activities of antioxidant enzymes
and the contents of MDA and ROS in plant leaves. The results revealed
that 2,4-DTBP is an important allelochemical in pepper CC soil. The
accumulation of 2,4-DTBP may be a significant factor in CC obstacles.

\textbf{ACKNOWLEDGEMENTS}

This work was supported by the National Natural Science Foundation of
China (No. 51678120). The author would also like to acknowledge Dr. Cui
Daizong for providing comments that greatly improved the manuscript.

Data Accessibility:

The sampling method, the data of manuscript, supplemental information:
Dryad doi:10.5061/dryad.zcrjdfnd6.

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