Documentation of Phytotoxic Compounds Existing in Parthenium hysterophorus L. Leaf and Their Phytotoxicity on Eleusine indica (L.) Gaertn. and Digitaria sanguinalis (L.) Scop

The utilization of the invasive weed, Parthenium hysterophorus L. for producing value-added products is novel research for sustaining our environment. Therefore, the current study aims to document the phytotoxic compounds contained in the leaf of parthenium and to examine the phytotoxic effects of all those phytochemicals on the seed sprouting and growth of Crabgrass Digitaria sanguinalis (L.) Scop. and Goosegrass Eleusine indica (L.) Gaertn. The phytotoxic substances of the methanol extract of the P. hysterophorus leaf were analyzed by LC-ESI-QTOF-MS=MS. From the LC-MS study, many compounds, such as terpenoids, flavonoids, amino acids, pseudo guaianolides, and carbohydrate and phenolic acids, were identified. Among them, seven potential phytotoxic compounds (i.e., caffeic acid, vanillic acid, ferulic acid, chlorogenic acid, quinic acid, anisic acid, and parthenin) were documented, those are responsible for plant growth inhibition. The concentration needed to reach 50% growth inhibition in respect to germination (ECg50), root length (ECr50), and shoot length (ECs50) was estimated and the severity of phytotoxicity of the biochemicals was determined by the pooled values (rank value) of three inhibition parameters. The highest growth inhibition was demarcated by caffeic acid, which was confirmed and indicated by cluster analysis and principal component analysis (PCA). In the case of D. sanguinalis, the germination was reduced by 60.02%, root length was reduced by 76.49%, and shoot length was reduced by 71.14% when the chemical was applied at 800 μM concentration, but in the case of E. indica, 100% reduction of seed germination, root length, and shoot length reduction occurred at the same concentration. The lowest rank value was observed from caffeic acids in both E. indica (rank value 684.7) and D. sanguinalis (909.5) caused by parthenin. It means that caffeic acid showed the highest phytotoxicity. As a result, there is a significant chance that the parthenium weed will be used to create bioherbicides in the future.

control weeds using secondary metabolites derived from plants or other natural sources, helping to safeguard both people and the environment. On the other hand, bioassays are typically created to examine a plant species' potential allelopathic effects. Because of the influence of several environmental circumstances, a plant that exhibits severe phytotoxicity toward the target plant species in laboratory conditions may not exhibit the same level of toxicity in the field context [21].
Some previous reports reveal the herbicidal potential of P. hysterophorus extracts in different plant species. As per our initial screening trials, parthenium leaf extracts have been examined to be a potential source of different allelochemicals with herbicidal and phytotoxic effects. However, inadequate evidence is available on the phytotoxicity of specific bioactive compounds which are identified in P. hysterophorus on the growth and development of crabgrass and goosegrass, which are the major weeds of rice and many of the field crops. Therefore, the main objective of this study is to evaluate the phytotoxicity of seven identified compounds from parthenium on the germination and growth of these weeds. The identification of its phytotoxic compounds was analyzed by using LC-ESI-QTOF-MS = MS (liquid chromatograph electrospray ionization quadrupole time of flight mass spectrometry).

Identified Compounds from P. hysterophorus Leaf Methanol Extract through LC-MS Analysis
The identified compounds from P. hysterophorus leaf methanol extract through LC-MS analysis and their relative proportions of P. hysterophorus leaf with methanolic extract from positive and negative polarity analyses are listed in Tables 1 and 2

Documentation of Phytotoxic Compounds from P. hysterophorus Leaf Methanol Extract through LC-MS Analysis
The LC-MS analyses of P. hysterophorus leaf methanol extract revealed the presence of many compounds, such as terpenoids, flavonoids, amino acids, pseudo guaianolides, carbohydrates, and phenolic acids. Among them, phenolic acids are responsible for plant growth inhibition. The list of proposed phytotoxic compounds (caffeic acid, ferulic acid, vanillic acid, quinic acid, parthenin, chlorogenic acid, and p anisic acid) with their retention time, molecular formula, polarity, and mass fragment (m/z) is presented in Table 3.

Allelopathic Effects of the Phytochemicals on D. sanguinalis and E. indica
Significant killing effects of the chemicals on the test weed species were observed. The chemicals and their mixtures produced varying degrees of inhibitory effects on the germination, root growth, and hypocotyl elongation of D. sanguinalis and E. indica. The P. hysterophorus leaf extracts have a range of chemical compounds. Among them, phenolic compounds cause dermatitis, autotoxicity, and the suppression of other plants.
There was a correlation between the quantity and kind of compounds detected in each plant and herbicidal activity.

Allelopathic Effects of the Phytochemicals on D. sanguinalis and E. indica
Significant killing effects of the chemicals on the test weed species were observed. The chemicals and their mixtures produced varying degrees of inhibitory effects on the germination, root growth, and hypocotyl elongation of D. sanguinalis and E. indica. The doses required for a 50% growth inhibition (EC 50 ) of the weeds, as indicated by EC g50 (germination), EC r50 (root), and EC s50 (shoot) growth, were computed and found different from the control.

Effects on Germination and Early Growth of D. sanguinalis
In the concentration-response bioassay, the inhibitory magnitude was increased for all compounds by increasing the concentration of chemicals from 100 µM to 1600 µM (Table 4). At the lowest concentrations (100 and 200 µM) of all tested compounds, less significant effect was found on the germination of D. sanguinalis, relative to the control except caffeic acid, which significantly suppressed the growth when applied with 200 µM; whereas for other chemicals, the germination percentage was significantly suppressed at rates higher than 400 µM. The germination of D. sanguinalis was severely decreased from 800 µM of chlorogenic acid, ferulic acid, parthenin, and vanillic acid. For quinic acid, anisic acid and a combination of compounds (mixture) inhibited growth when treated with 1600 µM. No germination was observed when treated with1600 µM of caffeic acid. Tested compounds did not exceed the doses to obtain EC 50 employed in this study except caffeic acid, chlorogenic acid, and parthenin, which produced the highest growth inhibition at 100, 48, and 60%, respectively, and the lowest inhibition was caused by anisic acid (32%). Therefore, caffeic acid was the highest toxic in comparison to other chemicals in all the concentrations, followed by parthenin and chlorogenic acid.
Therefore, the phytochemicals have significant allelopathic effects on the root growth of the tested weeds. The root growth was significantly (p ≤ 0.05) reduced by caffeic acid, chlorogenic acid, quinic acid, parthenin, and a combination of their mixtures at all concentrations. Table 5 shows that caffeic acid, quinic acid, and parthenin were very toxic, reducing root development even at the lower doses (100 µM). An increase in the dose of these chemicals resulted in a higher degree of growth inhibition. The caffeic acid caused 76% inhibition at 800 µM, and from the 1600 µM concentration, no root was visible. Parthenin, quinic acid, chlorogenic acid, and a mixture of compounds, on the other hand, reduced the root growth by 60, 46, 47, and 47%, respectively, at a dose of 800 µM. The weakest inhibition (47%) was observed from ferulic acid even at the highest concentration.  A more or less similar pattern of effects on shoot length also occurred due to the treatments (Table 6). However, the shoot elongation of D. sanguinalis was not significantly decreased by a lower (400 µM) concentration of all compounds except caffeic acid, quinic acid, parthenin, and their mixtures. Vanillic acid, ferulic acid, and chlorogenic acid exhibited an adverse effect on the shoot elongation at 800 µM and beyond. On the other hand, only anisic acid exhibited a 43% inhibition at the highest concentration.  Table 7 shows some remarkable differences among the allelochemicals in terms of D. sanguinalis growth inhibition. The differences were apparent from the rank values of composites. Caffeic acid (R e = 909.5) and parthenin (R e = 2569.4) exposed higher inhibitory influences on the germination and development of D. sanguinalis; in other words, these compounds showed the most phytotoxic impact, which indicates that less concentration is needed to suppress this plant. While anisic acid (R e = 14845.8), ferulic acid (R e = 8878.8), and quinic acid (R e = 8647.4) showed the weakest phytotoxicity compared to other chemicals. Consequently, it was apparent that the growth inhibitory effect of these compounds was the lowest. It means that anisic acid, ferulic acid, and quinic acid inhibit 50% of D. sanguinalis by more concentration than other tested compounds. According to Re value, the ranking of phytochemicals was caffeic acid < parthenin < chlorogenic acid < mixture < vanillic acid < quinic acid < ferulic acid < anisic acid. It can be mentioned here that the phytochemicals inhibited the growth of root length more than the growth of the shoot length and percent germination. The sum of EC r50 value for all compounds was 7811.2, whereas that of germination and shoot length were 32,597.9 and 54,700.8, respectively.

Germination and Early Growth of E. indica Treated with Detected Allelochemicals
In the concentration-response bioassay, the inhibitory magnitude was increased for all compounds with increasing concentration from 100 µM to 1600 µM (Table 8). At the lowest concentrations (100 µM) of all tested compounds, less significant effect was found on the germination of E. indica, except by caffeic acid, chlorogenic acid, and the compound mixture. It significantly suppressed inhibition when applied at 200-1600 µM except for quinic acid and anisic acid. The germination of E. indica severely decreased from 800 µM of caffeic acid, chlorogenic acid, and parthenin.  On the other hand, vanillic acid, ferulic acid, quinic acid, anisic acid, and a combination of compounds inhibited the weed growth when treated with 1600 µM. However, no germination of weed seeds was observed when treated with 800 µM of caffeic acid. Tested compounds did not produce a significantly lower value of EC 50 with the investigational doses used in this study except by caffeic acid, chlorogenic acid, and parthenin. The maximum growth inhibition of these compounds was observed at the rate of 100, 64, and 77%, respectively, and the lowest inhibition was found from anisic acid (15%). Therefore, it is obvious from the analysis that caffeic acid inhibited the most in all the concentrations, followed by parthenin and chlorogenic acid.
Identified allelochemicals have significant allelopathic effects on the root growth of the tested plant at varying doses (Table 9). All chemicals except vanillic acid significantly inhibited root elongation (p ≤ 0.05) at doses from 100 to 400 µM. However, at doses of more than 400 µM concentration, it suppressed the weed growth heavily. Table 9 shows that caffeic acid, quinic acid, and parthenin were strongly active, reducing root development even at the lowest concentration (100 µM). The caffeic acid produced 100% inhibition at an 800 µM concentration and above, while no root was visible, but 75 and 79% inhibition were observed from quinic acid and parthenin, respectively. The weakest phytotoxic effect (48%) on root development was noticed from chlorogenic acid at the highest concentration, while the rest of the compounds caused slightly more than 50% inhibition at the highest concentration. A similar pattern of effect on shoot length was noticed as was on germination and root length ( Table 10). The hypocotyl elongation of E. indica was not significantly decreased by a lower concentration (400 µM) of all compounds except caffeic acid, and vanillic acid. These compounds exhibited an adverse effect on the shoot elongation at the 800 µM and beyond. On the other hand, only anisic acid exhibited a 45% inhibition at the highest concentration. Table 11 shows some remarkable differences among the identified allelochemicals in terms of the growth inhibition of E. indica. The differences were apparent from the rank values of composites. Caffeic acid (R e = 684.7) and parthenin (R e = 1637.66) showed the highest phytotoxicity on the germination and development of E. indica; in other words, these compounds showed the most phytotoxic impact, as indicated by the lower concentrations needed to suppress this plant. While anisic acid (R e = 19553.25), ferulic acid (R e = 7970.02), and a mixture (R e = 5613.8) showed the weakest phytotoxicity compared to the others. The anisic acid, ferulic acid, and mixture of these compounds inhibit 50% of E. indica at a higher concentration than other tested compounds. The overall ranking, according to Re value, is caffeic acid < parthenin < vanillic acid < quinic acid < chlorogenic acid < mixture < ferulic acid < anisic acid. It is clear from the findings that the growth of root length is more affected by the chemicals than the growth of shoot length and percent germination. The sum of EC r50 values for all compounds was 9557.51, whereas the values for germination and shoot length were 27,149.61 and 48,847.3, respectively.

Cluster Analysis and Assessment of Principal Component Analysis
The allelopathic activities of examined compounds and their combination in bioassay were clustered into four interpretable groups, according to the dendrogram (group I-V) as indicated. In the dendrogram, there was a coefficient cut-off at 0.65 for ease of interpretation ( Figure 3). Group I consisted of caffeic acid, which was characterized by the most inhibitory effects and with low-rank values. Parthenin and quinic acid are in group II with stronger inhibitory effects; Group III is comprised of vanillic acid, anisic acid, and mixture; group IV consists of ferulic acid; and chlorogenic acid is in group V, which had moderate inhibitory effects. The compounds under groups IV and V demonstrated a relatively weak phytotoxic effect in comparison with other groups.

Cluster Analysis and Assessment of Principal Component Analysis
The allelopathic activities of examined compounds and their combination in bioassay were clustered into four interpretable groups, according to the dendrogram (group I-V) as indicated. In the dendrogram, there was a coefficient cut-off at 0.65 for ease of interpretation ( Figure 3). Group I consisted of caffeic acid, which was characterized by the most inhibitory effects and with low-rank values. Parthenin and quinic acid are in group II with stronger inhibitory effects; Group III is comprised of vanillic acid, anisic acid, and mixture; group IV consists of ferulic acid; and chlorogenic acid is in group V, which had moderate inhibitory effects. The compounds under groups IV and V demonstrated a relatively weak phytotoxic effect in comparison with other groups. The effects of D. sanguinalis and E. indica were responsible for the majority of the differences observed in the cluster. The two-dimensional and three-dimensional ( Figure  4) graphical elucidations confirmed that the maximum of the phytochemicals was discrete at low distances, the only two were discrete at long distances as represented by the eigenvector. The furthest accessions from the centroid were 3 and 4, whereas others were close to the centroid.  The effects of D. sanguinalis and E. indica were responsible for the majority of the differences observed in the cluster. The two-dimensional and three-dimensional ( Figure 4) graphical elucidations confirmed that the maximum of the phytochemicals was discrete at low distances, the only two were discrete at long distances as represented by the eigenvector. The furthest accessions from the centroid were 3 and 4, whereas others were close to the centroid.

Cluster Analysis and Assessment of Principal Component Analysis
The allelopathic activities of examined compounds and their combination in bioassay were clustered into four interpretable groups, according to the dendrogram (group I-V) as indicated. In the dendrogram, there was a coefficient cut-off at 0.65 for ease of interpretation ( Figure 3). Group I consisted of caffeic acid, which was characterized by the most inhibitory effects and with low-rank values. Parthenin and quinic acid are in group II with stronger inhibitory effects; Group III is comprised of vanillic acid, anisic acid, and mixture; group IV consists of ferulic acid; and chlorogenic acid is in group V, which had moderate inhibitory effects. The compounds under groups IV and V demonstrated a relatively weak phytotoxic effect in comparison with other groups. The effects of D. sanguinalis and E. indica were responsible for the majority of the differences observed in the cluster. The two-dimensional and three-dimensional ( Figure  4) graphical elucidations confirmed that the maximum of the phytochemicals was discrete at low distances, the only two were discrete at long distances as represented by the eigenvector. The furthest accessions from the centroid were 3 and 4, whereas others were close to the centroid.

Discussion
The P. hysterophorus extracts contained a large number of chemicals that were discovered using phytochemical screening, some of which had previously been recognized as toxins in other studies [28][29][30][31][32][33]. Furthermore, a variable number of chemicals were also present in different plant parts of P. hysterophorus. The leaf has a stronger inhibitory impact since it contains more harmful chemicals than the other plant parts. The suppressive influence of extracts, according to Verdeguer et al. [34] is determined by the extract's chemical makeup as well as the plant sections to which it is applied. These findings are consistent with those of Javaid and Anjum [35] and Verma et al. [36] who discovered that the main causes of the inhibition of plant growth are parthenin and other phenolic acids including caffeic acid, vanillic acid, anisic acid, chlorogenic acid, and para-hydroxybenzoic acid.
In this investigation, tested the phytotoxicity of all previously identified allelopathic compounds. The pure compound bioassay (chemicals purchased from the market) demonstrated that all of the examined compounds and their mixtures were physiologically dynamic and toxic, reducing seed germination and development in crabgrass and goosegrass. These results confirmed that the compounds found in P. hysterophorus are potential allelochemicals and that they are most likely responsible for P. hysterophorus' herbicidal behavior. Caffeic acid, chlorogenic acid, and parthenin were the most active of the compounds tested (Tables 2 and 6). In fact, the plant's allelochemicals have yet to be discovered.
According to Bajwa et al. [42] and Guo et al. [43] the extracts from allelopathic plant species produce much higher total phenolics than extracts from non-allelopathic plant species. The most vital and prevalent plant allelochemicals in the environment are phenolic derivatives [44]. Numerous papers have focused on the allelopathic and phytotoxic characteristics of phenolic and flavonoid chemicals [42,45]. Phenolic derivatives are an important class of allelopathic chemicals with a wide range of allelopathic actions. Regardless of dose, these components exhibited the most negative impact on seed germination and the development of barnyard grass [46]. Plant growth and development are inhibited by phenolic acids, which are one of the principal groups of metabolites implicated in allelopathic interactions in the soil atmosphere [47]. Amarowicz et al. [48] discovered that phenolics from the Jerusalem artichoke (Helianthus tuberosus L.) influenced lettuce development. According to Braga et al. [49], flavonoids inhibited the growth of standard target species (STS), such as Lactuca sativa (lettuce), Lycopersicon esculentum (tomato), and Allium cepa (Onion). Parthenin, chlorogenic acid, and ambrosian were also found to be favorably connected with germination inhibition and radicle elongation inhibition [42]. Caffeic acid, chlorogenic acid, ferulic acid, gallic acid, p-coumaric acid, 4-hydroxy-3-methoxybenzoic acid, m-coumaric acid, syringic acid, and vanillic acid were found as phytotoxins in parthenium, which cause allelopathic effects on crops [50]. Caffeic acid was shown to be the most effective inhibitor, as measured by thin-layer chromatography, melting point, infrared spectrum studies, and seedling emergence reduction [38,51].
P. hysterophorus extracts were found to have a higher inhibitory effect than individual compounds and even a mixture of all identified components [52]. The extracts' stronger inhibitory effects could be owing to unique chemical combinations that work in an additive or synergistic manner. This shows that undiscovered extract components may have a synergistic effect on phytotoxic action, if not direct activity [53]. It can be speculated that in addition to the established phenolic and flavonoid components, unknown chemicals are responsible for the overall allelopathic impact of extracts. Mixtures of phenolic compounds were less suppressive as compared to the allelopathic activity of individual phenolic compounds (Tables 2 and 6), which might be due to the fact that the allelopathic effect is regulated by concentration interactions, chemical combinations, and test species sensitivity because growth inhibition in mixes is lower than in individual component chemicals [54].

Conclusions
From the LC-MS analysis, many compounds, such as terpenoids, flavonoids, amino acids, pseudo guaianolides, and carbohydrate and phenolic acids, were identified from positive and negative polarity analysis. Among them, seven known phenolic derivatives were documented from the P. hysterophorus leaf methanol extract, which was responsible for plant growth inhibition. Seed germination and the development of crabgrass and goosegrass was reduced by all of the compounds, indicating that all combinations of all compounds were physiologically active. Caffeic acid and parthenin had the maximum phytotoxicity on crabgrass and goosegrass during germination and seedling development; indicating that a lower dosage is required to inhibit this plant. In comparison to the others, anisic acid, ferulic acid, and combination demonstrated the least phytotoxicity. This means that these chemicals need to inhibit to a greater extent than other chemicals on crabgrass and goosegrass germination and seedling growth to achieve the same effect. Overall, the ranking values were caffeic acid < parthenin < vanillic acid < quinic acid < chlorogenic acid < mixture < ferulic acid < anisic acid. Among these tested compounds, caffeic acid, chlorogenic acid, and parthenin were found to be the most active, and thus might be appropriate candidates for developing bioherbicides.

Site Description
The experimentation was conducted in the weed science laboratory of the Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia. Liquid Chromatography-Mass Spectrophotometry (LC-MS) analysis was carried out at Monash Universiti, Malaysia.

Extract Preparation
Parthenium leaves were collected from the Ladang Infoternak farm in Sungai Siput, Perak, Malaysia. Plants leaf was collected randomly during the vegetative stage (15-20 days old plants), rinsed with tap water numerous times to remove dust particles, and air-dried for three weeks at room temperature (24-26 • C). In a laboratory blender, plant leaves were mashed into a fine powder and sieved through a 40-mesh sieve.
The extracts were made according to the procedure described by Ahn and Chung [55] and Aslani et al. [56]. An amount of 100 g leaf powder of parthenium was placed in a conical flask and allowed to soak in 1 L of 80% (v/v) methanol. The conical flask was wrapped in paraffin and shaken for 48 h at 24-26 • C room temperature in an orbital shaker at a 150 rpm agitation speed. To remove debris, cheesecloth in four layers were used to filter the mixtures. The supernatant was centrifuged for one hour at 3000 rpm in a centrifuge (5804/5804 R, Eppendorf, Germany). A single layer of Whatman No. 42 filter paper was used to filter the supernatant. A 0.2-mm Nalgene filter (Lincoln Park, NJ-based Becton Dickinson percent Labware) was used to filter the solutions one more time to exclude microbial development. Using a rotary evaporator (R 124, Buchi Rotary Evaporator, Germany), the solvents were evaporated from the extract to dryness (a thick mass of coagulated liquid) under vacuum at 40 • C and the sample was then collected. From a 100 g sample of P. hysterophorus powder, the average extracted sample was 17.56 g, which was estimated as per the following formula [57]: [Extract weight (g)/powder weight (g)] × 100 = Extraction percentage (1) All extracts were stored at 4 • C in the dark until use. For LC-MS analysis, 100% HPLC GRADE methanol (20 mL) was diluted with the crude sample (20 mg) and filtered through 15-mm, 0.2-µm syringe filters (Phenex, Non-sterile, Luer/Slip, LT Resources Malaysia).

Identification of Phytotoxic Compounds from P. hysterophorus Leaf Methanol Extract
The analysis of the phytochemical compounds of the methanol extracts was performed using LC-MS followed by Schimanski et al. [58]. LC-MS analysis was carried out using Agilent spectrometry equipped with a binary pump. The LC-MS was interfaced with the Agilent 1290 Infinity LC system coupled to Agilent 6520 accurate-mass Q-TOF mass spectrometer with a dual ESI source. Full-scan mode from m/z 50 to 500 was set with a source temperature of 125 • C. The column of Agilent zorbax eclipse XDB-C18, narrow-bore 2.1 × 150 mm, 3.5 microns (P/N: 930990-902) was used at the temperature of 30 • C for the analysis. A-0.1% formic acid in water-and B-0.1% formic acid in methanol-were used as solvents. Isocratic elution was used to supply solvents at a total flow rate of 0.1 mL minutes −1 . MS spectra were collected in both positive and negative ion modes. The drying gas was 300 • C, with a 10 mL min-1 gas flow rate and a 45-psi nebulizing pressure. Before analysis, sample extraction was diluted with methanol and filtered through a 0.22 m nylon filter. The extracts were injected into the analytical column in 1 µL volume for analysis. The mass fragmentations were discovered using an Agilent mass hunter qualitative analysis B.07.00 (Metabolom-ics-2019.m) tool and a spectrum database for organic chemicals.

Plant Materials and Compounds
These detected seven phytotoxic compounds were purchased from Bio-solutions Sdn Bhd, Kuala Lumpur, Malaysia. The source of all chemicals is Sigma-Aldrich (St. Louis, MO, USA). The seeds of two weed species, crabgrass and goosegrass, were collected from UPM agricultural field and then kept in a refrigerator for 15 days at 4 • C for further use.

Bioassay
Individual chemicals and their mixtures were tested for their inhibitory effects on the germination and early growth of the weed species. Six different concentrations of the chemicals were achieved by dissolving the appropriate amount of chemicals in distilled water, i.e., 1600, 800, 400, 200, 100, and 0 µM (control), which were then sonicated at 60 kHz for one hour at 30 • C in an ultrasonic bath. The precise process for making various chemical concentrations includes dissolving the right amount of powder based on their molecular weight, such as the molecular weight of caffeic acid, i.e., 180.16 g. Thus, 1 mol equals 180.16 g. Therefore, a 1 molar solution will result from diluting 1 liter of distilled water by 180.16 g caffeic acid. Consequently, 1600 moles = (1600 × 180.16) = 540,480 g. In this manner, 540.48 mg of powder is required to create a 1000 mL solution in distilled water [59].
Healthy and uniform weed seeds were gathered and soaked for 24 h in 0.2% potassium nitrate (KNO3), then rinsed with distilled water and incubated at room temperature (24-26 • C) until the radicle emerged by about 1 mm. Thirty uniform pre-germinated seeds were inserted in disposable plastic 9.0-cm-diameter Petri dishes with two sheets of Whatman No. 1 filter paper. After that, the filter paper on the Petri dishes was wetted and soaked with 5 mL of six different chemical solutions. In the same way, 5 mL of pure water was treated as a control. The Petri dishes were then incubated under fluorescent light (8500 lux) in a growth chamber at 30/20 • C (day/night) with a 12 h/12 h (day/night cycle). The relative humidity ranged from 30% to 50%. To facilitate gas exchange and avoid anaerobic conditions, the lids of the Petri dishes were not sealed.

Data Measurement
Seed germination was counted, and root and shoot lengths of the weed species were measured after 1 week of seed placement with a ruler. The radicle and hypocotyl length was measured using Image J software (https://imagej.nih.gov/ij/docs/guide/user-guide.pdf; accessed on 10 July 2022) [60]. The inhibitory effect of P. hysterophorus extracts on germination, radicle length, and hypocotyl length was computed following the equation [25]: where "I" is the percentage of inhibition, "C" is the control's mean, and "A" is the treatment (extract) mean of germination, radicle length, and hypocotyl length.
To find discrete groupings of allelochemicals with similar phytotoxicity, the most common application of NTSYSpc 2.02e (Numerical Taxonomy and Multivariate Analysis System) was used to perform various types of agglomerative cluster analyses and to estimate some type of similarity or dissimilarity matrix to further define the level of sensitivity to chemical compounds among the plants under investigation [59,60].
Effective dosages capable of suppressing 50% of germination, root length, and shoot length were calculated using EC g50 , EC r50 , and EC h50 , respectively. The EC g50 , EC r50 , and EC h50 values were calculated using Probit analysis based on the percent of root and shoot length inhibition, respectively. The following equation was used to create an index (Re) for each of the most active extracts and the most sensitive plants for each plant tested: EC g50n (germination) + EC r50n (root) + EC h50n (shoot) = Rank (R e ) where Re is the plant's rank n and EC g50n , EC r50n , and EC h50n are the amounts of plant extract n that inhibit 50% of germination, root length, and shoot length, respectively. The lowest Re value had the most active chemical and the most sensitive plants, while the highest Re value had the least inhibition effect on the chemicals.

Identified Compounds from P. hysterophorus Leaf Extract
The identified compounds and their relative proportions of the P. hysterophorus leaf with methanolic extract from positive and negative polarity analyses are listed in Tables 1 and 2.

Details of the Phytotoxic Compounds
Details of the phytotoxic compounds (i.e., retention time, m/z, mass, polarity, synonyms, chemical formula and structure and biological activity with proper citations) of P. hysterophorus leaf with methanolic extracts through LC-MS analysis are available in Table 3.

Statistical Analysis
The data (germination percentage, root length, and shoot length) is transformed by the log transformation {log 10 (x + 1)} system. The variance homogeneity was evaluated using Levene's test. The data normality was analyzed using Shapiro-Wilk tests and after transformation, the data is assumed to be normally distributed. Two-way analysis of variance (ANOVA) was performed (two factors: concentrations and chemical compounds; fixed factor: weed species) using R-studio software to evaluate whether there was a significant difference between each treatment and the control, after that, the LSD test was used to separate the treatment and control means at 0.05 probability levels

Data Availability Statement:
The data presented in this study are available in this article.