Introduction
Pseudostellaria heterophylla from the family of Caryophyllaceae,
a famous genuine herb in China has been historically used as herbal medicine (Surhone et al.
2010; Wu et al. 2019a). However, P. heterophylla were severely affected
by replant problems, which also known as the consecutive monoculture problems
in production. Consecutive monoculture practice usually resulted in P. heterophylla plants with lower root
weight and fewer numbers of roots, leading
to sharp reducing of yield and medicinal value (Feng et al. 2010). Therefore, methods of alleviating and reducing
replant problems in P. heterophylla
have become an important research topic.
Previous studies have shown that
replant problems affecting the production of traditional Chinese medicine on
herbs involve obstruction of nutrient absorption, changes in soil
physiochemical properties, the structural imbalance in rhizosphere microbial
community and allelopathy in plants (Einhellig 1996;
Lin et al. 2011; Li et al. 2012; Chen et al. 2021). Recent advances have found that the allelotoxic substances in rhizosphere soil are one of the
critical factors triggered the generation of replant problems (Marschner and Timonen 2005; Mccully
2007). Identifying and screening of specific allelotoxic
substances in plants have thus become the necessary works for deeply revealing
the formation mechanism of replant problems.
Secondary metabolites from plants
are important sources of plant allelochemicals (Lovett and Hoult 1998).
Currently, the main method for identification of allelochemicals is gas
chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass
spectrometry (HPLC-MS) (Fu et al.
2017). In Tagetes patula
L, there were 25 and 27 potential allelochemicals were confirmed in roots
and rhizosphere soil extracts by GC-MS, respectively (Kumar et al. 2017). Although GC-MS technology was widely used in plant
metabolomics, it could only analyze about 20% of organic matter that was
thermally stable or gasified, making its application in the separation and
analysis of allelochemicals greatly limited (Kong and Xu 2003). Recently,
multi-stage combined techniques could widely identify complex metabolic
processes in plants and intermediate products, thereby providing an important
analytical platform for global metabolomics studies (Sangwan et al. 2015). Using high-performance
liquid chromatography-mass spectrometry (HPLC-MS/MS) and other high-throughput
metabolomics methods, a large number of plant-specific allelochemicals were
identified from plants. The allelopathic potential of plant residue and root
exudates of S. oleraceus on flavonoid
composition and nodulation on two legumes were studied by HPLC-MS/MS (Gomaa et al. 2015). Tandem
quadrupole-time-of-flight mass spectrometry (QTOF-MS) and ultrahigh-performance
liquid chromatography (UHPLC) is of great advantage and is an important tool
for the analysis and identification of the chemical composition of complex
samples (Nguyen et al. 2006; Eugster et al.
2011).
In previous studies, the imbalance
in rhizosphere microorganisms induced by allelochemicals has found relate to
replant disease formation in P.
heterophylla. However, until now potential allelochemicals (group) in P. heterophylla have still not been
clearly identified. To further understand the induction and formation
mechanisms for replant problems in P.
heterophylla, in this study, UHPLC-QTOF-MS were used for a detailed
analysis of differences in the unplanted
control soil and the rhizosphere soil of P.
heterophylla. Simultaneously, a detailed detection of the secondary
metabolite spectrum of plant tissue cultures and tissue culture rhizosphere in P. heterophylla without interference
from environmental factors was carried out. The combination of the two sets of
experiments was used to provide a detailed understanding of the release and
accumulation process of secondary metabolites in the rhizosphere by P. heterophylla. Further, the metabolite
groups that may be intimately associated with replant problems in P. heterophylla were identified. This
study provides a foundational dataset for screening and examination of
allelochemicals that probably cause replant problems of P. heterophylla.
Materials
and Methods
Materials
The rhizosphere soil of the species "Zherong No. 2" of P.
heterophylla (RS) was collected in Zherong
County, Ningde City, Fujian Province (119.9288E,
27.1418N) in July 2016. The P.
heterophylla was planted from November 2015 to July 2016 (The material was identified
as Pseudostellaria
of Caryophyllaceae by Linkun Wu, College of Life Sciences, Fujian Agriculture and
Forestry University). Sampling method: there was removed 0~5 cm deep surface
soil and then removed root soil by shaking the root method. The rhizosphere
soil close to the root of P. heterophylla
was collected and the obtained rhizosphere soil was mixed thoroughly. In
addition, the adjacent unplanted control soil sample (UCS) was collected as a
control. The samples were randomly collected from 6 points as independent
replicates.
The species "Zherong No. 2" of P.
heterophylla was cultivated using plant tissue cultures method in MS medium
for 65 days at 22°C under 3000 Lux light intensity for the collection of plant
tissue cultures (PTC) and rhizosphere tissue culture medium (RTCM) (Fig. 1).
Extraction
of compounds in the UCS and RS
The UCS and RS were naturally air-dried and
filtered through 100 mesh sieve. A 30 g soil samples in sextuplicate were
weighed and extracted in 150 mL of methanol, ethyl acetate and n-hexane
using a shaker at 120 r/min for 24 h, respectively and filtered. The filtered
liquid supernatant was evaporated to 10 mL using a rotary evaporator under
reduced pressure at 40°C. Subsequently, the extractions in methanol, ethyl
acetate and n-hexane were mixed and dried under nitrogen gas and then
redissolved in 10 mL solution of acetonitrile: water (1: 1 ratio). The
supernatant was filtered through 0.22 μm nylon
filters and stored under 20°C. When injecting, taking 2 μL from each sample and
pooling as QC samples, and then taking 2 μL supernatant for the UHPLC-QTOF-MS
analysis. The extraction method of two soil samples was the same as described
above.
Extraction
of RTCM and PTC
The RTCM and PTC were completely separated. The
PTC were then ground with plant tissue grinder (Shanghai Jingxin,
Tissuelyser-24). Five g of PTC and 20 g of RTCM were weighted and extracted in
150 mL methanol, ethyl acetate and n-hexane using the same method for
soil extraction.
UHPLC-QTOF-MS
analysis
The compounds in extractions were
separated using a Waters UHPLC BEH Amide column (1.7 μm,
2.1 × 100 mm) under Agilent 1290 Ultra High-Performance Liquid System. Water
(containing 25 mM ammonium acetate
and 25 mM ammonia) (A) and
acetonitrile (B) were used as mobile phase following the optimal elution
procedure: 0 ~ 1 min, 15% A; 12 ~ 12.1 min,
35 ~ 60% A；15 ~ 15.1 min,
60 ~ 15% A；20 min, 15% A. Mass spectral data were recorded on
AB Sciex Triple TOF
6600 high-resolution Mass Spectrometer equipped with an ESI source operating in
negative ion mode. Bombardment energy: 35 eV, 15 secondary spectra every 50ms.
The ESI ion source parameter was set to as following: atomization pressure (GS1): 60 Pa, auxiliary pressure: 60 Pa, air curtain
pressure: 30 Pa, temperature: 550°C, spray voltage: -4500 V. 2 μL
extractions were injected for UHPLC-QTOF-MS analysis.
Data processing
The statistical analyses were performed using
SIMCA software (V14.0, MKS Data Analytics Solutions, Umea, Sweden).
Unsupervised principal component analysis (PCA), partial least squares
discriminant analysis (PLS-DA) and supervised
(orthogonal) partial least squares (OPLS-DA) were used to observe the overall
distribution of samples and the stability of the whole analysis process, to
distinguish the overall differences of metabolic profiles among groups and to
identify the different metabolites among groups. In OPLS-DA analysis, variables
with VIP greater than 1 were considered as differential variables. The
identification of compounds of interest was conducted by searching against the
Traditional Chinese Medicine database (TCM, AB SCIEX).
%20289-294_files/image002.jpg)
Fig. 1: Flow chart of experimental design. UCS (unplanted control soil); RS
(rhizosphere soil of P. heterophylla).
TC (tissue culture). RTCM (rhizosphere tissue culture medium). PTC (plant
tissue cultures)
Results
Identification
of critical metabolites in rhizosphere of P.
heterophylla in field conditions
To develop a preliminary understanding of the set
of compounds in the rhizosphere of P.
heterophylla, the molecules in the RS and UCS were profiled and totally 878
entities were obtained. By searching against TCM databases, 636 compounds were
identified of which 176 compounds which were significantly accumulate in the RS
(set A) while some compounds that were decreased. Through classification
analysis of the set A, we found that these compounds are mainly long-chain
fatty acids, organic acids, terpenoids and steroids while decreased compounds
are mainly amino acids and their derivatives, alkaloids and sugars (Fig. 2a, b
and c). The set A probably secreted by the root system or products of microbial
action on root exudates, while the decrease of the compounds probably resulted
by the utilization of plant.
Identification
of critical metabolites released from P.
heterophylla under tissue culture conditions
The compounds in the RTCM (set B) and PTC (set C) were
analyzed. 1,011 and 1,447 compounds were identified in the set B and C by
searching against TCM databases, respectively. Of which, 177 compounds were
simultaneously identified in the set B and C. Classification analysis showed
that these common substances were mainly organic acids, long-chain fatty acids,
amino acids and their derivatives, which are probably released into the tissue
culture medium during plant growth (Fig. 3a, b and c).
Integrated analysis of rhizosphere exudates in field and tissue condition
reveals candidate allelochemicals set of P.
heterophylla
Venn diagram was used to analyze the relationship
between the release and accumulation of secretions (Fig. 4a, b and c) and 21
compounds were detected both in the set A, B and C. These substances are mainly
organic acids, long-chain fatty acids and alkaloids. Moreover, there were 31
compounds were detected both in the set A and set B. These compounds are mainly
organic acids, long-chain fatty acids and flavonoids. Furthermore, 153
compounds were detected both in the set B and set C. Most of these substances
are organic acids, long-chain fatty acids, amino acids and their derivatives.
These results showed that the compounds that were secreted from P. heterophylla plants into the
rhizosphere are main esters, long-chain fatty acids, organic acids, terpenoids
and alkaloids.
Previous reports and classifies of
allelochemicals were compared with these results to determine the potential
allelochemicals secreted by P.
heterophylla into the rhizosphere. Twenty one compounds indicated the
potential allelopathic characteristics based on structural analysis, while 13
compounds such as pyruvaldehyde, succinic acid and
benzoic acid were simultaneously present in the set A, set B and set C. A total
of 8 compounds such as indole, vanillic acid and salicylic acid, were presented
in both the set B and set C. Majority of these potential allelochemicals belong
to benzoic acid and its derivatives, water-soluble organic acids, alkaloids,
flavonoids and so on (Fig. 5).
Discussion
Currently, researchers believe that
allelopathy is a main causes of replant problems and mediated rhizosphere
microbial ecology imbalance by allelopathy (Wu et al. 2016; Zhang et al.
2021). Therefore, the isolation and identification of allelochemicals is the
prerequisite for the accurate and comprehensive understanding of allelopathy
and is the key to understanding the cause of replant problems. In order to
exclude complex conditions in the field and accurately analyze allelochemicals
from P. heterophylla, UHPLC-QTOF-MS
were employed to combined analyze the %20289-294_files/image004.png)
Fig. 4: Distribution analysis of compounds of interest in P. heterophylla tissue and rhizosphere. (A) A set of compounds that
are significant accumulated in the rhizosphere soil after the planting of P. heterophylla (set A). (B) A set of
compounds in the rhizosphere tissue culture medium of P. heterophylla (set B). (C) A set of compounds in the plant tissue
cultures of P. heterophylla (set C)
%20289-294_files/image006.png)
Fig. 2: Comparison and classification of compounds in the
RS and UCS. (A) The types of compounds that are decreased in the rhizosphere
soil after the planting of P.
heterophylla. (B) Black: the non-significantly different compounds; Blue:
the decreased compounds. Red: the significant accumulated compounds. (C) The
types of compounds that are significant accumulated in the rhizosphere soil
after the planting of P. heterophylla
%20289-294_files/image008.png)
Fig. 3: Metabolic profiling of compounds in P.
heterophylla plant tissue cultures and rhizosphere tissue. (A) A set of
compounds in the rhizosphere tissue culture medium of P. heterophylla (set B). (B) A set of compounds in the plant tissue
cultures of P. heterophylla (set C).
(C) The types of common compounds
compounds of the plant tissue culture
(PTC), rhizosphere tissue culture medium (RTCM) and the significant accumulated
compounds in the rhizosphere soil after the planting of P. heterophylla (set A). Compared to previous methods (Qin et al. 2009), our method precluded the
effects of microorganisms and determined which compounds were significantly
accumulated in rhizosphere soil and those directly originated from plant
secretions and which ones had cumulative effects. Our study identified 21
potential allelochemicals that caused replant problems in P. heterophylla. Among these compounds, 13 compounds were simultaneously
present in the PTC, RTCM and set A. These compounds may be secreted by the
plants into the rhizosphere and are potential allelochemicals with cumulative
effects on the soil. In addition, the eight compounds were present in the PTC
and RTCM but not significantly accumulated in rhizosphere soil, which may be
original compounds exuded by the plants. These compounds (groups) did not
undergo metabolic conversion by the soil or microorganisms.
The potential allelochemicals found in this study can be divided into
eight categories, which include water-soluble organic acids; long-chain fatty
acids; simple phenols, benzoic acid and its derivatives; cinnamic acid and its
derivatives; flavonoids; alkaloids and other compounds (Rice 1984). Currently,
research on allelochemicals in replant problems in P. heterophylla have mainly focused on simple phenols and
water-soluble organic acids (Zhao et al.
2015; Wu et al. 2017). No studies
involving alkaloids and flavonoids in replant problems in P. heterophylla have been conducted to date, despite extensive
investigations on other plants. Paszkowski and Kremer
(1988) isolated six types of flavonoids from velvetleaf and found that these
compounds have significant inhibitory effects of the germination and radicle
growth in all tested species using 1 mM
concentrations. Ambika (2002) studied the inhibitory effects of Chromolaena odorata weed on crop growth and found
phenols, alkaloids and amino acids were mainly responsible allelochemicals in
this regard. Therefore, more attention should be paid to these compounds in
future.
%20289-294_files/image010.png)
The potential allelochemicals that were found in this study such as
succinic acid and vanillic acid have been proven to be allelochemicals in
relevant studies on P. heterophylla
replant problems (Zhao et al. 2015;
Wu et al. 2017; Wu et al. 2019b). Although berberine,
oleanolic acid and other compounds were not reported in related studies on
replant problems in P. heterophylla,
these compounds have been proven by other studies involving other plants to be
allelochemicals. Dai et al. (2013)
found that berberine shows concentration-dependent inhibitory effects on the
growth of the Microcystis aeruginosa
905, and inhibition rates increase with higher berberine concentrations. Wang et al. (2016) isolated oleanolic acid
from the leaf extraction of Alstonia scholaris and found that it could inhibited the
activity of gram-negative bacteria. Rasmussen and Einhellig
(1977) found that p-coumaric and
ferulic acids synergistically inhibited the germination and growth of sorghum
seeds, resulting in greater toxicity. Therefore, the compounds identified in
this study may be classical as broad-spectrum allelochemicals.
The present study has identified 21
potential allelochemicals that may be utilized as a dataset for preliminary
screening and examination of allelochemicals that cause replant problems
involving P. heterophylla. However,
whether this set of allelochemicals can cause replant problems in P. heterophylla remains unclear. Thus,
further investigation is needed to determine if a single substance or
coordination by multiple substances are needed to elicit their effects.
Conclusion
Our study primarily found that 21
potential allelochemicals might be directly secreted from P. heterophylla plants, which had not been transformed or degraded.
Although the function of these allelochemicals needs to be verified by further
experiments, our study provided data sets information for identifying
allelochemicals that resulted in the replant problems of P. heterophylla. At the same time, this study also provokes for
further elucidating the formation mechanism of replant problems of P. heterophylla and its rhizosphere
ecological process.
Acknowledgments
This study was supported by the China Agriculture
Research System of MOF and MARA, the engineering technology research center of
characteristic medicinal plants of Fujian (Grant No. PP201804).
Author
Contributions
ZYZ designed the experiments; JF, BZ, HYL and MJL
executed the experiments and wrote the manuscript; JF, BZ, HYL and MJL analyzed
the data; MJL, LG, FJF and JMW discussed the results.
Conflict of Interest
All authors
declare no conflict of interest.
Data Availability
Data presented
in this study will be available on a fair request to the corresponding author.
Ethics Approval
Not applicable
to this paper.
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