Evaluating the Phytotoxicity of Methanolic Extracts of Parthenium hysterophorus L. on Selected Crops and Weeds

Herbicides made from natural molecules may be a good environmentally friendly alternative to synthetic chemical herbicides for weed control. As a result, this investigation was carried out to ascertain the phytotoxicity of Parthenium hysterophorus L. as well as to identify its phenolic components. Germination of seeds and development of seedlings of Vigna subterranea (L.) Verdc, Raphanus sativus (L.) Domin, Cucurbita maxima Duchesne., Cucumis sativus L., Solanum lycopersicum L., Capsicum frutescens L., Zea mays L., Abelmoschus esculentus (L.) Moench, Daucus carota L., Digitaria sanguinalis (L.) Scop and Eleusine indica (L.) Gaertn were investigated using P. hysterophorus leaf, stem, and flower methanol extracts. Six concentrations (25, 50, 75, 100, and 150 g L−1) were comparison to the control (distilled water). The concentration of extracts increased, the rate of the seed sprouting and seedling growth decreased. EC50 values showed that the extraction of leaf of P. hysterophorus (811) was phytotoxic in comparison to the stem (1554) and flower (1109) extract. According to PCA analysis, Raphanus sativus, Solanum lycopersicum, Capsicum frutescens, Abelmoschus esculentus, Daucus carota, Digitaria sanguinalis, and Eleusine indica were all very susceptible to allelochemicals. A LC-MS analysis revealed that the P. hysterophorus leaf extract contained 7 phenolic compounds that were responsible for inhibition. These studies also revealed that the leaf of P. hysterophorus is a major source of phytotoxicity, which could be valuable in the future for developing a natural herbicide.


36
Parthenium (Parthenium hysterophorus L.) is a noxious herb that has now invaded 46 countries and 37 extended its spread from a few islands to around the world, there have been eleven minor and eight 38 major introductions [1]. Its high invasiveness is associated with several factors, including a higher num-39 ber of seeds production, highly competitive and rapidly expanding, biological plasticity of the life cycle, 40 allelopathic ability and high survival ability against biotic and abiotic stresses [2][3][4][5]. 41 Allelopathy is described as chemical's positive or negative impacts substances formed primarily 42 by plant, microbe, and fungal secondary metabolism on the growth and establishment of neighbouring 43 plants or microorganisms, as well as the dynamical processes of agricultural and natural eco-systems 44 [6]. It's a complicated phenomenon that's influenced by a variety of internal and external circumstances. 45 Due to its intricacy, the explanation is a difficult endeavour that necessitates knowledge from a variety 46 of professions [7]. Allelochemicals are plants that release secondary metabolites into the environment. 47 They are anti-inflammatory substances that belong to a variety of chemical classes, primarily phenolic 48 compounds and terpenoids [8]. Bhadoria [9] provided a more comprehensive summary of allelochem-49 icals' that affects plant growth and development. All plant organs (stems, leaves, rhizomes, roots, flow-50 ers, pollen, fruits, seeds) contain allelochemicals, which are released through volatilization, leaf leach-51 ing, plant material breakdown, and root exudation. In some way, membrane stability, cell division, 52 elongation, shape and permeability, enzyme activity, and respiration of plants are all influenced. Pho- 53 tosynthesis, protein synthesis, nucleic acid metabolism, and other direct and indirect ways of action 54 cause seed sprouting suppression and limited seedling development [8]. In addition, the microbial 55 breakdown of soil allelochemicals has an impact on the effective dose of allelochemicals that can inhibit 56 plants [10,11]. 57 Herbicides have been the least expensive and principal method of weed control in developing 58 countries for about 50 years [12]. Herbicides, on the other hand, pose significant risks to agriculture, 59 human health, and the environment. However, increasing crop production without using chemical 60 herbicide is an urgent challenge in crop production. Manual weed management is the most effective 61 and long-term solution for weed management. So, accurate weed control is necessary for food security 62 throughout the world. Therefore, researchers are motivated to seek alternatives because of the labour 63 movement from agriculture to others, and weed biotypes resistant to traditional synthetic pesticides 64 [13]. This strategy will aid in reducing reliance on chemical herbicides, reducing the likelihood of weed 65 resistance to herbicides, reducing health risks and environmental damage, and strengthening the na-66 tional economy. In the meantime, there are a variety of possible allelochemicals in aerial sections (e.g. 67 leaves) of Parthenium weed have been confirmed by several earlier studies; among them p-anisic acid 68 (C8H8O3), p-coumaric acid (C9H8O3), caffeic acid (C9H8O4), ferulic acid (C4H4O4), fumaric acid (C4H4O4), 69 p-hydroxybenzoic acid (C7H6O3), neochlorogenic acid (C16H18O9), protocatechuic acid (C7H6O4), aerulic 70 acid, chlorogenic acid (C16H18O9) and vanillic acid (C4H4O4) are the most important [14,15] sprouting 71 and development of a plant species in abundance, natural plants are included and different crops and 72 pasture species can be inhibited by these chemicals [16]. Wheat, maize, and horse gram [5], lentil [17,18] 73 and other field crops. Hassan et al., [19] showed an inhibitory impact when exposed to parthenium 74 extract. Dhawan and Gupta [15] reported that the extraction of diverse active phytochemicals with fla-75 vonoid concentrations works best using methanol as an extraction solvent. 76 However, there is insufficient evidence on the influence of Parthenium methanolic extracts on the 77 sprouting and seedlings development of several crops especially Bambara groundnut weeds. The Bam-78 bara groundnut is a new crop for Malaysia, but there is information lacking on the suppression of alle-79 lopathy on Bambara groundnut weeds by different parts of P. hysterophorus. The current study aimed 80 to find out the allelopathic capacity of Parthenium in a laboratory experiment to evaluate the allelopa-81 thic suppression of weeds by P. hysterophorus in Bambara groundnut weeds. The research was directed 82 with the following objectives (1) to evaluate the phytotoxicity of methanol extracts made from the aerial 83 portions of P. hysterophorus on target species in order to develop bioherbicides based on natural prod-84 ucts (2) LC-MS was used to identify its phenolic derivatives. 85  Leaf, stem, and floral parts of parthenium was applied at different concentration viz., 0, 25, 50, 75, 92 100, and 150 g L -1 [42]. All treatments were arranged in a completely randomized design (CRD) and 93 repeated four times. 94

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For extraction of the leaf of P. hysterophorus plants, plant materials were taken from Ladang In-96 foternak farm in Sungai Siput, Perak, Malaysia, and also grown in the net house of field 15 at University 97 of Putra Malaysia, Selangor, Malaysia. The above-ground part of the plants was collected just before 98 maturity, rinsed several times using tap water to eliminate dust elements, then air-dried at ambient 99 temperature (24-26 0 C) for three weeks. The leaves, stems, and flowers were divided and bulked up into 100 three main parts. In a laboratory blender, both bulked plant components were ground into fine dust 101 and sieved through a 40-mesh sieve. 102 The inhibitory action of P. hysterophorus was investigated on nine plant species. Bambara ground-103 nut (Vigna subterranea L. Verdc), radish (Raphanus sativus L. Domin), sweet gourd (Cucurbita maxima 104 Duchesne), tomato (Solanum lycopersicum L.), cucumber (Cucumis sativus L.), chili (Capsicum frutescens 105 L.), corn (Zea mays L.), carrot (Daucus carota L.) and okra (Abelmoschus esculentus L. Moench) and two 106 weed species goosegrass (Eleusine indica L. Gaertn)] and [crab grass (Digitaria sanguinalis L. Scop). Crop 107 seeds were attained from Sin Seng Huat Seeds Sdn Bhd company in Malaysia, while seeds of grasses 108 were personally picked from the Universiti Putra Malaysia's agricultural field. The seeds were cleaned, 109 air-dried, and stored in airtight containers maintain at -18° C. The vegetable crops are chosen for the 110 determination of ecological effects of allelopathic substances as they represented commonly used spe-111 cies in the field that's are recommended by US EPA [54]. They belong to different plant families and 112 can provide great genetic diversity. The seeds germinated 86-95% of the time, according to a random 113 test. 114

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The extracts were made according to the procedure published by [55] and [42]. Accurately 100 g 116 powder from leaves, stems, and flowers of parthenium were placed in a conical flask and allowed to 117 soak in 1L of 80% (v/v) methanol separately. After that, the conical flask was wrapped in paraffin and 118 shaken for 48 hours at 24-26°C room temperatures in an Orbital shaker at 150 rpm agitation speed. To 119 remove debris, cheesecloth in four layers were used to filter the mixtures and centrifuged for one hour 120 at 3000 rpm in a centrifuge (5804/5804 R, Eppendorf, Germany). A single layer of Whatman No. 42 filter 121 paper was used to filter the supernatant. A 0.2-mm Nalgene filter was used to filter the solutions once 122 more to avoid microbial development (Lincoln Park, NJ-based Becton Dickinson percent Labware). Us-123 ing a rotary evaporator (R 124, Buchi Rotary Evaporator, Germany), the solvents were evaporated from 124 the extract to dryness (a thick mass of coagulated liquid) under vacuum at 40° C and the sample was 125 then collected. From a 100 g sample of P. hysterophorus powder, the average extracted sample was 17.56 126 g. For the bioassay, each stock extract from P. hysterophorus leaves, stems, and flowers were diluted 130 in sterile distilled water to provide extract concentrations of 25, 50, 75, 100, and 150 g L -1 , while purified 131 water was served as control. All extracts were stored at 4° C in the dark until use. 132 For LC-MS analysis, 100% HPLC GRADE methanol (20 mL) was diluted with the crude sample 133 (20 mg) and filtered through 15-mm, 0.2-µm syringe filters (Phenex, Non-sterile, Luer/Slip, LT 134 Resources Malaysia). 135 136 Healthy, uniform seeds were gathered and treated with 0.2% potassium nitrate for 24 hours 137 (KNO3) before being rinsed with distilled water. Twenty Bambara groundnut and sweet gourd seeds 138 and thirty seeds of radish, cucumber, tomato, chili, corn, okra, carrot, crabgrass, and goosegrass were 139 set up in a sterilized Petri dish with Whatman No. 1 filter paper (90 × 15 mm). 10 mL of extract of each 140 concentration (25, 50, 75, 100, and 150 g L -1 ) was delivered in Petri dishes, distilled water serving as a 141 control. In a growth chamber, all Petri dishes were inserted. and incubated at 30 0 C/20°C (day/night) 142 temperature under fluorescent light (8500 lux) on photoperiod 12 h day/12 h night maintained 30-50% 143 relative humidity. To facilitate gas exchange, the petri dish lids were not sealed. The LC-MS was used to identify the chemical contents of the extracts. The phytochemical com-146 pounds of the methanol extracts were performed using LC-MS followed by [56]. LC-MS analysis was 147 performed using Agilent spectrometry equipped with a binary pump. The LC-MS was interfaced Ag-148 ilent 1290 Infinity LC system coupled to Agilent 6520 accurate-mass Q-TOF mass spectrometer with a 149 dual ESI source. Full-scan mode from m/z 50 to 500 was performed with a source temperature of 125°C. 150 The column of Agilent zorbax eclipse XDB-C18, narrow-bore 2.1x150 mm, 3.5 microns (P/N: 930990-151 902) was used with the temperature 30 o C for the analysis. A-0.1% formic acid in water and B -0.1% 152 formic acid in methanol were used as solvents. Isocratic elution was used to supply solvents at a total 153 flow rate of 0.1 mL minutes -1 . MS spectra were collected in both positive and negative ion modes. The 154 drying gas was 300 0 C, with a 10L min-1 gas flow rate and a 45-psi nebulizing pressure. Before analysis, 155 1 ml of concentration. sample extracts were diluted with methanol and filtered through a 0.22 m nylon 156 filter. The extracts were injected into the analytical column in 1 µl volume for analysis. The mass frag-157 mentations were discovered using an Agilent mass hunter qualitative analysis B.07.00 (Metabolom-ics-158 2019.m) tool and a spectrum database for organic chemicals. 159 2.7. Data collection 160 The germination percentage, radicle, and hypocotyl length were measured with a ruler at seven 161 days after seeding. The radicle and hypocotyl length was assessed by software Image J [57] while the 162 inhibition (%) of P. hysterophorus extracts on a radicle, and hypocotyl length was computed following 163 the formula used by Kordali [58]:

Germination and growth bioassays
Here, "I" is the percentage of inhibition, "C" control's mean growth and development and "A" is 166 the aqueous extracts' mean growth and development. 167

168
On pooled (two seasons) data, a one-way analysis of variance (ANOVA) was used to regulate any 169 significant variances among concentrations and control. To calculate the difference between the 170 concentration means, the Tukey test (SAS 9.4) with a 0.05 probability level was utilised. ECr50, ECg50, 171 and ECh50 were used to compute real dosages accomplished of suppressing 50% of germination, radicle 172 development, and hypocotyl growth. Based on the suppression of germination (percentage), radicle, 173 and hypocotyl development, Probit analysis was used to compute the ECg50, ECr50, and ECh50 values. 174 From each tested plant, a rank was determined by using the following equation to calculate an index 175 (Re) for each of the most active extracts and plants that are the most susceptible: Where Re is the plant's rank n, ECr50n, ECh50n and ECg50n are the amounts of plant extract n that 178 inhibit 50% germination, radicle, and hypocotyl length, respectively. The lowest Re value had the max-179 imum active tissue extracts and the utmost sensitive plants, while the highest Re value had the least 180 allelopathic effect of the extract. 181 The most common application of NTSYSpc 2.02e (Numerical Taxonomy and Multivariate Analysis 182 System) is to do various types of agglomerative cluster analysis of some type of similarity or dissimi-183 larity matrix and the quantity of extract sensitivity among the plants under investigation [59,60]. The 184 principal component analysis (PCA) was used to re-validate Johnson's cluster analysis [20]. When the P. hysterophorus stem and flower extract were applied at lower doses (25,50, and 75 g L -207 1 ), there was no significant reduction in germination (%). When the concentration was raised from 100 208 to 150 g L -1 , the sprouting was substantially decreased between 1-100 percent in the stem and 48-100 209 percent in the flower extract among the indicator plants, while it was 61-100 percent in the leaf extract 210 ( Figure 1& 2). Among them, leaf extract was affected in many crops than stem and flower extract. On 211 the other hand, germination (%), radicle, and hypocotyl length were significantly decreased at 50 to 100 212 g L -1 leaf extracts (Table 1). Increasing the concentration level eventually reduced the germination per-213 centage over time. Both extracts of Parthenium inhibit the germination percentage of examined indica-214 tor both weed species (Figure 2). 215 Methanol extracts had a significant phytotoxic influence on all the crops on radicle and hypocotyl 216 length studied at varied doses except sweet gourd, cucumber, and corn. Extraction of the leaf at doses 217 more than or equal to 50 g L -1 substantially decreased the radicle length of target plants (p≤0.05) (Table 218 1). With 100 to 150 g L -1 stem and flower extract, root development of certain plants was decreased by 219 more than half, whereas the uppermost concentration of the leaf extract (100 to 150 g L -1 ) resulted in no 220 root development such as Bambara groundnut, radish, chili, okra, etc (Table 1). From the concentration 221 level of 100 to 150 g L -1 Parthenium extract radicle length showed the inhibition level 53-100%, 36-100%, 222 and 10-100% were from leaf, stem, and flower, respectively ( Table 1, 2 & 3). As a result, the leaf extract 223 had a higher concentration than the others (Table 1). 224 Furthermore, we observed that the weed crabgrass and goosegrass were severely affected by leaf 225 and flower methanol extract in the doses of 50 to 150 g L -1 , but stem extract was affected by doses of 100 226 to 150 g L -1 . So, it was observed that severely affected weed by leaf than flower and stem plant parts. 227 The amount of inhibition rose when the concentration level was raised. Different components of Par-228 thenium reduced the shoot length of all examined plants by 27-100%, 61-100%, and 38-100%, respec-229 tively, at the doses of 100 to 150 g L -1 .    Table 4 showed the half inhibitory (EC50) impact of Parthenium plant parts with methanol extracts, 274 as well as the sensitivity of the evaluated starting growth parameters and plants. The efficacy of stem 275 extract (1554) was lower than that of leaf extract (811), and it was followed by flower extract (1109) in 276 all tested crops. The EC50 value showed some differences in sensitivity between the tested plant's re-277 sponses to the inhibitory influence of P. hysterophorus (Table 4). In case of leaf methanol extract corn, 278 Cucumber, and sweet gourd were only impacted at higher concentrations. The rank value of these 279 crops is 463, 144, and 108 respectively, it means that these crops are more tolerant, which shows that 280 more doses need to destroy these plants. On the other hand, Bambara groundnut, radish, tomato, car-281 rot, crabgrass, and goosegrass are more sensitive to leaf methanol extract next to chili (53) and okra 282 (41).   298 Cluster analysis was also used to categorize distinct groups of plants with comparable responses 299 to the inhibition of leaf, stem, and flower extracts by combining all three characteristics examined. Clus-300 ter analysis produced a dendrogram that revealed variation in sensitivity among the plants (Figure 3). 301  Plants may be divided into four classes based on how they react to leaf, stem, and flower extracts 310 (Table 5). According to Table 5, group IV comprises tolerant monocot plants, whereas the dicot plants 311 examined referred to the sensitive groups. Corn was recorded tolerant, whereas the moderately sweet 312 gourd, cucumber, and carrot had an intermediate reaction to the phytotoxicity. The most vulnerable 313 plants, on the other side, were Bambara groundnut, radish, tomato, crabgrass, goose grass, okra, and 314 chili. Overall, the dicot plants were shown to be more active against the Parthenium extract than the 315 monocots. 316 The principal component analysis (PCA), on the other hand, is a re-validation tool for cluster anal-317 ysis. Johnson uses PCA to estimate the total variation that exists in a set of characters [20]. As shown 318 by the eigenvector in the two-dimensional ( Figure 4) and three-dimensional ( Figure 5) graphical eluci-319 dations, the majority of the indicator plants were spread at short distances, while just two were dis-320 persed at long distances. Bambara groundnut and Corn were the accessions that were farthest from the 321 centroid, whilst other accessions were close to it.  330 The identified phenolic derivatives of P. hysterophorus plant parts with methanolic extracts through 331 LC-MS analysis are listed in Table 6. The leaf, stem, and flower extracts of P. hysterophorus have diverse 332 chemical compositions. A total of 7 Phenolic derivatives were detected from methanol extract of P. 333 hysterophorus different parts through LC-MS analysis (Table 6) ( Figure 6). These phenolic derivatives 334 are responsible for inhibition to other plants, autotoxic, and dermatitis. Parthenin and other phenolic 335 acids found in the leaf and flower extracts include vanillic acid, caffeic acid, quinic acid, anisic acid, 336 chlorogenic acid, and ferulic acid, contrary Parthenin, vanillic acid found in the stem extract. The 337 amount and kind of chemicals discovered in each plant were found to be proportional to herbicidal 338 action. As a consequence, the compound of the various plant parts inhibited indicator plant germina-339 tion and seedling growth, with the extraction of leaf having a greater inhibitory influence than the other 340 plant parts. 341  In a dose-dependent way, all 351 portions of Parthenium extracts affected germination, radicle length, and hypocotyl length in the tested 352 species. Because of its exceptional strength, efficacy, and consistency in preventing germination and 353 seedling development, extracts of Parthenium leaf were the most promising. Plant extracts are hypoth-354 esized to decrease germination through having osmotic potential on the rate of absorption, which in 355 turn affects germination and, in particular, cell elongation [25]. 356 Wheat, maize, and horse gram seedling growth is inhibited by extracts of P. hysterophorus metha-357 nol extract. Its demonstrated greater inhibitory power, in comparison to the aqueous extract [26]. Dha-358 wan & Gupta [15] reported that the extraction of different active phytochemicals with flavonoid con-359 centration works best using methanol as an extraction solvent. The germination of V. radiata seeds were 360 tested for up to 120 hours using methanol crude Parthenium extracts and it was discovered that there 361 is a considerable difference in germination kinetics between the treatments of methanol crudes. [27]. 362 Tef germination was significantly reduced at intermediate to higher concentrations when Parthe-363 nium flower and leaf extracts were used. This suggests that inhibitory compounds are present in larger 364 concentrations in flower and leaf than in stem and root sections [28,29]. The fact that roots came into 365 direct touch with the extract and then with inhibitory compounds, as reported in previous research 366 with a variety of crops and weeds [30,31]. 367 The aerial parts extract of P. hysterophorus had a substantial influence on germination of seed, rad-368 icle and hypocotyl length reduction in this investigation. These effects grew stronger as the concentra-369 tion level increased. These discoveries are consistent with those of Mulatu et al. [32] and Mersie and 370 Singh [33] who discovered a robust link between greater P. hysterophorus aqueous extract concentra-371 tions and increased poisonousness to agronomic crops and weeds. The effects of secondary metabolites 372 generated by P. hysterophorus aerial parts on growth and development in Bambara groundnut weeds 373 and chosen species. Phytochemicals isolated from P. hysterophorus stems, leaves, and flowers methanol 374 extracts were competent to alter crops and weed seedling sprouting and development. Similarly, Mot-375 mainna et al.

Identified phenolic derivatives from LC-MS analysis
[34] discovered that P. hysterophorus extract had a considerable impact on the germination 376 and development of the weed species. The degree of inhibition raised when the concentration of the 377 extract was increased. Radicle growth is more vulnerable to allelopathic plant extracts than other or-378 gans due to radicles is the first tissue to be shown to phytotoxic chemicals and have a more absorbent 379 tissue than other parts [35,36], and/or the root apical meristem has a low mitotic division rate [37]. 380 Furthermore, allelopathic elements can suppress the production of radicle and epidermis by altering 381 genes involved in cellular characterization [38]. Parthenium extract was more effective than the B. alata 382 and C. rutidosperma extract [34]. This is in consistent with [39], who discovered that extracts of allelopa-383 thic plant have a stronger effect on radicle length than hypocotyl development. This could be due to 384 the roots are the initial to attract allelochemicals substances, from the atmosphere. 385 The survivability rate of the target plants was inhibited by varying doses of P. hysterophorus leaf, 386 stem, and flower methanol extracts. Maximum doses of methanol extracts included more inhibitory 387 chemicals, resulting in more inhibition. In the same way, Han et al. [40] had reported that the phytotox-388 icity of P. hysterophorus extracts was concentration-dependent, and phytotoxicity rose as extract con-389 centration was raised. It was also claimed that the leaf extract had a more inhibitory allelopathic activity 390 than other vegetative portions, and phytochemical research had already revealed a larger accumulation 391 of growth inhibitors in P. hysterophorus leaves [41]. At all doses examined, the extracts inhibited P. minor 392 germination, and when extract concentrations increased then inhibition increased [25]. However, ex-393 tracts from the leaves had a higher level of toxicity than extracts from the stem [42]. 394 Different plant species' susceptibility to inhibitory chemicals has been documented for a variety of 395 causes. Msafiri et al. [43] observed that both tested species showed substantial allelopathic effects of P. 396 hysterophorus seed and leaf aqueous extract on seed sprouting, root and hypocotyl length, fresh and dry 397 mass. According to Kobayasi [44] because of each species' have biological characteristics. The seed 398 structure and seed coat penetrability can also play a role in different reactions to similar allelopathic 399 extract [45]. Higher concentration reduced the seedling length of all the test crops but, sweet gourd, 400 corn, and cucumber were less sensitive than other crops. This may be due to genotypic variation in 401 response to the higher concentration of extracts. Similar results of inhibitory effect were observed by 402 Aslani et al. [42]. These findings agreed with Aslani et al. [46], phytotoxic compounds are more vulner-403 able to smaller plants, he said. These findings matched those of numerous prior research that found 404 that phytotoxin reactions differed by species. 405 The phytochemical screening revealed a huge number of compounds in the P. hysterophorus ex-406 tracts, some of which have previously been identified as poisons in several investigations [23,47,48]. 407 Furthermore, various plant sections of P. hysterophorus contained a different number of compounds. 408 The quantity of toxic compounds was more in the leaf than the other plant parts; as a result, the leaves 409 have a stronger inhibitory effect. P. hysterophorus leaves released allelochemicals into the soil by leach-410 ing or decomposition, and have the potential to impair the development of other plants by altering the 411 physicochemical properties of soil, according to Dogra & Sood,[49]. Arowosegbe & Afolayan [50] also 412 found that beetroot (Beta vulgaris L.), Turnip (Brassica rapa L.), and carrot (D. carota L.) were all inhibited 413 more by Aloe ferox Mill. leaf than by the root extract. The suppressive influence of extracts, according 414 to Verdeguer et al. [51] is determined by the extract's chemical makeup as well as the plant sections to 415 which it is applied. These findings are consistent with those of Javaid and Anjum [52] and Verma et al. 416 [53] who discovered that parthenin and other phenolic acids such as caffeic acid, vanillic acid, anisic 417 acid, chlorogenic acid, and para hydroxy benzoic acid are the most responsible for plant growth inhi-418 bition. 419

420
The study demonstrated that methanol extracts of P. hysterophorus had phytotoxicity on the ger-421 mination, growth, and development of tested plants. When the concentration of extracts was raised, 422 the rate of germination and seedling growth reduced in comparison to the control, indicating that P. 423 hysterophorus was phytotoxic. Moreover, the extracts' phytotoxic effects were reliant on the target spe-424 cies, concentration of extracts, and plant types. Fifty percent inhibitory concentrations (EC50) value of 425 P. hysterophorus leaf extract showed more phytotoxic than the stem and flower extract. According to the 426 findings, it was clear that the highly susceptible plants were Raphanus sativus, Solanum lycopersicum, 427 Capsicum frutescens, Abelmoschus esculentus, Daucus carota, Digitaria sanguinalis, and Eleusine indica. On 428 the other hand, 7 known phenolic derivatives were identified from the P. hysterophorus extract which 429 was responsible for inhibition. Given the hopeful results of P. hysterophorus extract, this plant could 430 possibly be studied next in the hopes of developing a herbicide based on natural products for green 431 agriculture that is sustainable. However, in order to offer farmers with useful suggestions, P. hysteroph-432 orus impacts in actual crop field settings must be validated after bioassay trials. To see if it may be used 433 to develop future alleloherbicides as structural leads, more research on isolation, characterization, and 434 determination of the herbicidal activity of the chemical components in P. hysterophorus extract, particu-435 larly from the leaf extract, is needed.