Effects of 13C isotope-labeled allelochemicals on the growth of the invasive plant Alternanthera philoxeroides

The secondary metabolites of indigenous plants have significant allelopathic inhibitory effects on the growth and development of invasive alien plants. Methyl palmitate (MP) and methyl linolenate (ML) were used as exogenous allelopathic substances. The research investigated the differences of inhibitory effects of MP and ML on the growth of seedlings of Alternanthera philoxeroides, and calculated their morphological characteristics, biomass, physiological indicators and the response index (RI). The synthetical allelopathic index (SE) of 1 mmol/L MP was the smallest (− 0.26) and the allelopathic inhibition was the strongest; therefore, it was selected as a 13C-labeled allelochemical. The distribution of 1 mmol/L MP in different parts of A. philoxeroides and the correlation between the biomass ratios of roots, stems and leaves and the 13C content were studied by 13C stable isotope tracing experiments. Atom percent excess (APE) between roots, stems and leaves of A. philoxeroides treated with 1 mmol/L MP were significantly different in terms of magnitude, with leaves (0.17%) > roots (0.12%) > stems (0.07%). The root, stem and leaf biomass ratios of invasive weeds had great significant positive correlation with 13C content (p < 0.01, R2 between 0.96 and 0.99). This current research provides a new idea and method for the control of A. philoxeroides, but large-scale popularization remains to be studied.


Material and methods
Source of experimental materials.The invasive plant, Alternanthera philoxeroides, was used for the experiment, and the plant specimen number is ANUB001553.We comply with relevant institutions, national and international standards and laws in the collection of plants.A. philoxeroides with good growth and maturity were dug up in Nongcui Garden of Anhui Agricultural University (31° 52′ N, 117° 16′ E) and washed in the plant physiology laboratory. A. philoxeroides of uniform size and diameter of about 3 mm were selected, cut into rhizomes of length 3 cm along the stem nodes with scissors, disinfected in 10% solution of sodium hypochlorite for 10 min, and rinsed in distilled water to prevent mildew during their cultivation.Various circular sponges with a diameter of about 15 cm were selected, and taken holes evenly and place them in the bottom 1/5 of a 500 mL beaker to fix the A. philoxeroides and conserve water.The reagents methyl palmitate (MP) with purity > 99% and methyl linolenate (ML) with purity > 99.5% were purchased from Shanghai Titan Scientific Co., Ltd and Hefei Shang Cheng experiment supplies Ltd.

Preparation of exogenous allelochemicals solutions.
The solid powders of MP and ML were weighed on a ten thousand scale and emulsified in a beaker with distilled water in a constant temperature water bath at 40 °C with stirring.After cooling, it was shaken in the ultrasonic cleaner for 5 min to form an emulsion 35 .The emulsion was fixed in a 1000 mL volumetric flask, transferred to a wide-mouth flask and configured as 0.01, 0.1, 0.5, 1 mmol/L of MP (abbreviated as MP0.01, MP0.1, MP0.5, MP1) and ML (abbreviated as ML0.01, ML0.1, ML0.5, ML1) of which 13 C-labeled MP1 solution (purchased from cambridge isotope laboratories inc, chemical purity ≥ 98%) solutions and stored in a refrigerator at 4 °C for backup.Distilled water was used as a control treatment.

Hydroponic experiment.
A double layer of filter paper was laid in the petri dishes and a small amount of distilled water was added.10 rhizomes of A. philoxeroides were placed in each petri dish and incubated in a light incubator (SPX-250, Shanghai) (25 °C, 12 h D/12 h L, 80% relative humidity) for 7 days.The rhizomes of A. philoxeroides germinated and grew into seedlings.The round sponges were soaked in distilled water to absorb water and placed in 500 mL beakers.The experiment was conducted in a randomized complete block design (RCBD).5 seedlings of A. philoxeroides were transplanted in each beaker at the perforation of the sponge, and the seedlings were cultivated in a light incubator for 45 days.Every 2 days, 5 mL of different concentrations of Growth bioassay.At the end of the experiment, the morphological characteristics, biomass changes and physiological indicators of A. philoxeroides were measured.The roots, stems and leaves were cut and put into envelope bags respectively, and the root length and stem length were measured with a steel ruler, and the leaf area was measured with a leaf area meter (Yaxin-1241 leaf area meter, Beijing Yaxin LIYI Technology Co., LTD), and the number of nodes and leaves were counted.After the experiments were completed, the fresh weights of the shoot and root parts of the grasses were weighed and placed in an oven at 70 °C for 48 h to dry to a constant weight, and the dry weights of the shoot and root parts were weighed.Total chlorophyll, chlorophyll a, b and carotenoids contents were determined by colorimetric method using ethanol extraction 36 .The mid-vein of the leaves of A. philoxeroides was removed, 0.2 g of the fresh cut sample was weighed, a little CaCO 3 and quartz sand were added, then 10 mL of anhydrous ethanol was added, and the plant tissue was ground until it turned white.After resting, the solution was filtered and set the volume to be 50 mL with anhydrous ethanol, a small amount of pigment extract was rinsed on the cuvette.The solution was added to 4/5 of the cuvette, and anhydrous ethanol was used as a blank control.The absorbance was determined colorimetrically using a UV spectrophotometer (T6 New Century, Beijing General Analytical Instrument Co.) tuned to the corresponding wavelength.Finally, the relevant chlorophyll content was calculated by the formula.We weighed 0.3 g of fresh leaves, crushed and centrifuged, placed in an ice-water mixture, and the supernatant was taken.The malondialdehyde (MDA) content, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activity indexes of A. philoxeroides leaves were determined by the kit (Nanjing Jiancheng) 37 .Related indicator formula: Calculation formula for chlorophyll and carotenoids 36 : where Total Chl.Content is total chlorophyll content, Chl. a is chlorophyll a content, Chl.b is chlorophyll b content, Car is carotenoid content, A 663 , A 645 and A 470 are the absorbance of chlorophyll extracts at 663 nm, 646 nm and 470 nm, respectively.

The indices of allelopathic effects and synthetical allelopathic index. The response index (RI),
referring to the method of Bruce Williamson et al. 38 : where C is the control value and T is the value of different concentrations of MP and ML of A. philoxeroides; when RI < 0, it means inhibition, and the larger the absolute value, the higher the inhibition; when RI > 0, it means promotion, and the larger the absolute value, the better the promotion; the absolute value represents the strength of the allelopathy of various concentrations of MP and ML.
The synthetical allelopathic index (SE) 39 is the arithmetic mean of the response index under different concentrations of MP and ML treatments: when SE < 0, it means that different concentrations of MP and ML have an inhibitory effect on A. philoxeroides; when SE > 0, it means that it has a positive effect.
Plant stable isotope 13 C determination.The roots, stems and leaves of 13 C-labeled 1 mmol/L MP treated A. philoxeroides were dried, ground in a mortar and pestle and passed through a 50 mesh sieve.0.5 g of the sample was weighed and wrapped in dry tinfoil, and then the δ 13 C (‰) value of the sample was determined using an Isotope Ratio Mass Spectrometer (Thermo Fisher Scientific, model MAT-253, Germany).The following formula was used to calculate the correlation between the proportion of carbon storage, atomic percentage excess of 13 C and biomass ratio and 13 C content: (1) Root-shoot ratio = Root dry weight/ Shoot dry weight  where R PDB = 0.011237, PDB standard: PDB(Pee Dee Belemnite) is a Cretaceous marine fossil, Belemnitella americana, collected from the Pee Dee formation in South Carolina, which carbon and oxygen isotopic compositions of carbonate rocks are usually determined using PDB standards 41 : where R sample is the isotope ratio of the sample where 13 C atom % (labeled sample) represents the atomic percentage of 13 C measured in the labeled sample; 13 C atom % (control sample) represents the atomic percentage of 13 C measured in the unlabeled sample 42 : where C stock represents the C amount in different parts of plants, APE represents the atom percent excess of 13 C in different parts of plants exceeds 42 .

Statistical analysis.
The raw data were organized and statistically analyzed using Excel 2016, and one-way ANOVA was performed on the experimental treatment data using SPSS26.The effects of different concentrations of MP and ML on the response variables (Morphological indicators, biomass, physiology and 13 C-related indicators of different parts of the plant) of A. philoxeroides were assessed using LSD and Duncan's method.The relationship between 13 C content and biomass ratio of root, stem, leaf and total biomass was analyzed using onedimensional linear regression.Origin2022 was used for plots and analyses to determine the extent of the effects of MP and ML on different variable indicators of A. philoxeroides.

Results
Effect of allelochemicals on the morphological characteristics.Different concentrations of MP had significant effects on stem length (F = 10.63,P < 0.001) and leaf area (F = 12.88, P < 0.001) of A. philoxeroides, and the most significant inhibition of leaf area was observed with MP1 (77.72%) (Table 1).with a higher concentration of MP solution (0.1-1 mmol/L), root length was shorter with high concentration inhibition, and MP1 had a significant inhibition of root length (35.58%) compared to the control.Except for MP0.1 concentration, the node number and leaf number decreased with increasing concentration.Different concentrations of ML had significant effects on stem length and leaf area, and ML1 treatment had the greatest inhibition of leaf area (73.87%).Root length, node number and leaf number were inhibited by ML (0.1-1 mmol/L) compared to the control, and the inhibition tended to diminish and then increase.ML1 had the strongest inhibitory effect on root length and leaf number (48.33% and 38.68%), and ML0.1 had the most significant inhibition on node number (21.67%).
(  2).The inhibition increased with increasing concentration, and MP1 and ML1 were extremely inhibitory (50.91% and 50.91%).Different concentrations of MP and ML had an inhibitory effect on root dry weight, and the most inhibitory effect of MP0.5 and ML1 was significant (40.74% and 33.33%).Compared to control, root-shoot ratio, plant water content and degree of succulence were promoted, and ML significantly increased plant water content and degree of succulence.
Effect on photosynthetic pigment content of leaves.Apart from ML0.5 concentration, there was no significant effect of MP and ML on leaf of total chlorophyll, chlorophyll a, b and carotenoid contents of A. philoxeroides compared with the control (Fig. 1).The total chlorophyll content showed a trend of increasing, then decreasing and then increasing with the increase of MP concentration (Fig. 1a).As the concentration of ML increased the total chlorophyll content decreased and then increased, and the ML0.5 treatment had the lowest content (41.56%).All were smaller than the control content.Chlorophyll a content increased with rising MP concentration (Fig. 1b), and chlorophyll b and carotenoid contents of leaves treated with different MP concentrations did not differ significantly (Fig. 1c,d).Leaf chlorophyll a and b content declined with higher concentration of ML.ML0.

The indices of allelopathic effects at different levels on A. philoxeroides. The response index (RI)
was used to evaluate the overall allelopathic effect of MP and ML on A. philoxeroides by the indicators of stem length, root length, node number, leaf number, leaf area, shoot dry weight, root dry weight, root-shoot ratio, plant water content and degree of succulence (Table 4).As shown by the RI means, stem length, root length, node number, leaf number, leaf area, shoot dry weight, root dry weight, had negative values (− 0.13 to − 0.59) and root-shoot ratio, plant water content and degree of succulence had positive values (0.07-0.18).The synthetical allelopathic index (SE) of different concentrations of MP and ML were negative (− 0.04 to − 0.26), indicating that different concentrations of MP and ML had inhibitory effects on the growth of A. philoxeroides.MP1 and ML1 had the smallest SE (− 0.26 and − 0.24, respectively) and the strongest inhibition.The SE (0.1-1 mmol/L) reduced with the increase of MP concentration.It tended to decline and then increase and then decrease with the increase of ML concentration.
Carbon storage in different parts of A. philoxeroides.Carbon stocks of CK treatments of A. philoxeroides stems were significant (p < 0.05) compared to roots, leaves and total plant, and carbon stocks of CK and MP roots, stems, leaves and total plant showed the same trend (Fig. 2).The CK group and the MP group treated stems had the largest carbon stocks, the carbon stocks of roots and leaves were equal, the carbon stocks of stems were about twice as large as those of roots and leaves, and the carbon stocks of the total plant were larger in the MP treatment than in the CK.The carbon stocks of different parts of MP treated plants were insignificant, but the carbon stocks of stems in CK treatment were significantly greater than those of roots and leaves, and increased by 65.22% and 57.52%, respectively.www.nature.com/scientificreports/Atom percent excess of different parts of A. philoxeroides.With MP treatment, there was a highly significant effect (p < 0.05) on the atom percent excess (APE) between roots, stems and leaves of A. philoxeroides (Fig. 3).Compared to the total plant, both stems and leaves were significantly affected, excluding the APE of roots which was not significant.The average APE size of the roots, stems and leaves was in the order of leaves (0.17%) > roots (0.12%) > stems (0.07%).APE was significantly lower by 27.64% and 56.01%for roots and stems, respectively, compared to leaves, and by 39.21% for stems compared to roots.Compared to total plant APE, leaves significantly increased by 38.65% and stems significantly decreased by 39.01%.

Correlation analysis of root, stem, leaf biomass ratio and total biomass with 13 C content.
A linear regression of the root, stem and leaf biomass ratios of A. philoxeroides on the associated 13 C content showed a highly significant positive correlation (p < 0.01) (Fig. 4).The R 2 of root, stem, leaf and total biomass fitted by the model were all above 0.95, where the linear coefficients were ranked in order of magnitude as R 2 stem (0.99) > R 2 leaf (0.98) = R 2 total (0.98) > R 2 root (0.96).The average increase was ranked in order of total plant (0.4538) > leaf (0.1067) > root (0.0565) > stem (0.0316).Overall, the one-dimensional linear regression of root, stem and leaf biomass ratios, total biomass and 13 C content were fitted with high accuracy, and the 13 C content tended to increase with increasing biomass, which could predict the 13 C content of A. philoxeroides.

Discussion
Alternanthera philoxeroides is a malignant weed with strong invasive properties, and the indigenous plants are able to secrete secondary metabolites into the surrounding area, producing allelopathic inhibition, whose effects are mainly reflected in seed germination and seedling growth 14 .Therefore, we selected methyl palmitate (MP) and
The inhibition effect of shoot dry weight and root dry weight of A. philoxeroides were enhanced with increasing MP and ML concentrations, which were positively correlated with the concentration of allelochemicals.Plant roots are affected by allelochemicals, which inhibit the division and elongation of root tip cells, thus reducing the ability to absorb surrounding nutrients and affecting aboveground biomass accumulation.Another cause  is that allelopathic substances are able to reduce the net primary productivity of A. philoxeroides, resulting in a reduction in belowground and aboveground biomass accumulation 51 .Chlorophyll a and chlorophyll b absorb red light and blue-violet light respectively, and carotenoids have the function of absorbing light energy and dissipating the remaining photosynthetic energy, which are closely related to plant organic matter accumulation and growing development 52 .It was concluded that MP and ML inhibited the total chlorophyll content, chlorophyll a, b and carotenoids of A. philoxeroides, and ML0.5 inhibited the total chlorophyll most significantly.Presumably due to the occurrence of allelopathic stress persecution in plants, the ultrastructure of chloroplasts is harmed, leading to chlorophyll degradation, chlorophyll content reductions, light and dark reactions are affected, resulting in metabolic dysregulation and lowered photosynthetic rate 53,54 .The reduction in chlorophyll accumulation was reported to be associated with the deletion of Phosphatidylglycerol (PG), which could be a result of allelochemicals inhibiting the up-regulation of chlorophyll biosynthesis pathway genes and accelerating the induction of down-regulation of degradation pathway genes expression, resulting in less chlorophyll accumulation 55 .
Reactive oxygen species (ROS) are generated in plants under unfavorable conditions, which cause membrane lipid peroxidation to produce malondialdehyde (MDA) and damage cell membranes and macromolecules such as proteins, hence affecting normal growth and development of plants 56 .At the same time, in plants under stressful environments, there is an enzymatic protection system, namely the antioxidant defense system.Antioxidant enzymes in plants majorly include superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), which can remove the excess free radicals and maintain the balance of reactive oxygen species production and removal in plant cells 57 .Therefore, SOD, CAT, POD activity and MDA content are often used as important indicators to determine whether or not a plant is damaged by adversity and the extent of damage 58 .The research results revealed that the leaf MDA content was greater than the control under MP and ML allelopathic stress, implying that the plant cells were attacked by free radicals and the cells were harmed so that the balance of reactive oxygen species was disrupted.The activities of CAT and POD in leaves of A. philoxeroides treated with different concentrations of MP were larger than those of the control group due to the differences in exogenous allelopathic substances and solution concentrations.SOD enzyme activities were more than the control group, except for MP0.01 and MP0.1 treatments, which were smaller than the control group.The CAT and POD enzyme activities of A. philoxeroides leaves under ML concentration treatment were greater than the control, but the SOD enzyme activity was less than the control, showing that the antioxidant defense systems activity was affected by the stress concentration in addition to the exogenous allelochemicals.Different concentrations of different exogenous allelopathic substances enhanced the resistance of A. philoxeroides to a certain extent, and the increase of enzyme activity strengthened the scavenging ability against free radicals 59 .This is explained by the fact that the different concentrations of MP and ML will stimulate the leaves of A. philoxeroides to strengthen the metabolism and the activity of the antioxidant enzyme protection system in the organism under the condition of adversity.The SOD, CAT, and POD activities will be promoted to scavenge the excessive oxygen radicals in the body in order to withstand the damage of the external environment.That was the adaptive mechanism of A. philoxeroides under allelopathic stress, demonstrating that allelochemicals have a stressful effect to some extent 60 .The allelopathic substances of linoleic acid, palmitic acid and ethyl palmitate in sweet potato aqueous extracts increased CAT and POD enzyme activities and decreased SOD enzyme activities in A. philoxeroides, which is consistent with the above findings 61 .
The response index (RI) and synthetical allelopathic index (SE) represent the intensity of allelopathic effects of various plants 38,62 .In terms of the mean values of different morphological characteristics and biomass indicators, RI of all indicators were negative, apart from the positive values of plant water content, root-shoot ratio and degree of succulence RI.Leaf area > Stem length > Shoot dry weight > Root dry weight > Root length = Leaf number > Node number, where the leaf area had the slightest RI and the strongest allelopathic inhibition.As shown by the synthetical allelopathic index, the SE of MP and ML treated with different concentrations were negative, indicating that both allelopathic substances had inhibitory effects on the growth of A. philoxeroides.The magnitude of inhibition was, MP1 > ML1 > ML0.1 > MP0.5 > ML0.5 > MP0.01 > ML0.01 > MP0.1, and the MP1 (SE = − 0.26) concentration had the strongest inhibitory effect on A. philoxeroides.
The 13 C labeling was based on the selection of 1 mmol/L MP with obvious inhibitory effect, and the distribution of 13 C was detected in the roots, stems and leaves of A. philoxeroides, which could indicate that the exogenous allelopathic substances could be directly absorbed.In the natural state, some indigenous plants produce secondary metabolites that interact with surrounding soil microorganisms and alter the community structure of plant inter-rooted microorganisms, leading to soil colony dysbiosis and soil acidification, thus indirectly affecting the growth of invasive weeds 63,64 .But in most cases, the allelopathic substances secreted by indigenous plants are mainly absorbed and utilized by the roots of invasive weeds and participate in cellular metabolic processes to inhibit their growth and development 65 .The distribution of the atom percent excess (APE) in different parts of A. philoxeroides was different, leaf > root > stem, and plant roots and stems produced more lignin and cellulose, which reduced the synthesis and utilization of allelopathic substances; it also related to the transport of the phloem of the stem, and A. philoxeroides enhanced photosynthesis in order to resist the stress damage of allelopathic substances, which led to the accumulation of more 13 C photosynthetic products in the leaf part 66 .The distribution of 13 C in the different parts of A. philoxeroides showed that the distribution of allelopathic substances absorbed by the plant in roots, stems and leaves was linked to the allelopathic inhibition of morphological characteristics (stem length, root length, node number, leaf number and leaf area).The highest 13 C content in leaves had the strongest inhibitory effect on invasive weeds.The maximum accumulation of 13 C in leaves also indicated some inhibitory effect on pigment content and enzyme activity of leaves.Allelopathic stress affected the enzyme activity of the leaf cells of invasive weeds, causing an increase in intracellular reactive oxygen radicals (Table 3), reducing chlorophyll content on cystoid membranes (Fig. 1), and lowering light uptake and conversion.This led to impaired electron transport and oxidative phosphorylation, which reduced photosynthesis and

Figure 2 .
Figure 2. Effects of different treatments on carbon storage in roots, stems, leaves and total plant of A. philoxeroides.The CK-R, CK-S, CK-L, and CK-T represent the root, stem, leaf, and total plant of the control group; The MP-R, MP-S, MP-L, and MP-T represent the root, stem, leaf, and total plant of 1 mmol/L MP treatment.

Figure 3 .
Figure 3.Effect of MP treatment on root, stem, leaf and total plant APE of A. philoxeroides.The MP-R, MP-S, MP-L, and MP-T represent the root, stem, leaf, and total plant of 1 mmol/L MP treatment groups; The APE is the atom percent excess (%).

Figure 4 .
Figure 4. Correlation fit of root, stem and leaf biomass ratio and total biomass with 13 C content in A. philoxeroides.Biomass ratios: root biomass (solid squares; solid lines); stem biomass (solid circles; double dotted lines); leaf biomass (solid triangles: dotted lines); R 2 correlation factor.Biomass: total plant biomass (solid squares; solid lines); R 2 correlation factor. )

Table 1 .
Effect of different concentrations of MP and ML on morphological indexes of A. philoxeroides.

of allelochemicals on MDA levels and the enzymatic activity of leaves.
5 significantly inhibited total chlorophyll content, chlorophyll a, b and carotenoids of A. philoxeroides (41.55%, 40.92%, 43.11% and 26.85%).Different concentrations of MP and ML had no significant effect on MDA content of A. philoxeroides leaves, and different concentrations of ML had non-significant effect on CAT and POD enzyme activities of leaves (Table3).Treatment with different concentrations of MP significantly promoted leaf CAT enzyme activity, with MP0.01 increasing 254.12% compared to the control.Compared to CK, there was a meaningful increase in leaf POD activity by MP0.01 and MP0.1 with 102.00% and 129.59%, and the promotion effect diminished with increasing MP concentration.SOD activity of A. philoxeroides leaves was significantly inhibited by MP0.01, MP0.1 and ML0. 1 concentrations with 69.44%, 88.68% and 74.32% inhibition, respectively.

Table 2 .
Effects of different concentrations of MP and ML on the biomass indexes of A. philoxeroides.