1. Introduction
Maize (
Zea mays L.) is a major crop grown all over the world and is considered the most significant grain crop [
1]. Its high production is due to its several functions as a human and animal food source [
2]. Maize is a staple food source for approximately 200 million people globally, contributing an estimated 15% of protein and 20% of calories to the human diet [
2]. Murdia et al. [
3] reported that maize can be cultivated in a variety of agro-climatic zones, which makes it an adaptable crop, in contrast to many other crops. In South Africa, it is produced throughout the country in diverse environments [
4]. The top three provinces in South Africa for growing maize, which collectively contribute to nearly 83% of the country’s total output, are North West, Mpumalanga, and KwaZulu-Natal [
5]. South Africa [
6] is ranked among the top 10 maize-producing countries in the world [
6,
7]. Since 1995, maize has been grown on about three to four million hectares of agricultural land, accounting for about 60 to 70% of the arable area [
8]. However, the production of this crop is threatened by alien invasive weeds that release allelochemicals that inhibit seed germination and growth.
Mexican poppy [
Argemone ochroleuca (Sweet)] is an alien invasive weed with potential biological inhibition properties to other plants [
9]. Dar et al. [
10] reported that both the shoot and root parts of
A. ochroleuca released water-soluble phytotoxins that decreased the germination of
Farsetia aegyptia Turra,
Salvia aegyptiaca,
Hordeum vulgare, and
Medicago sativa. Essential oils of
A. ochroleuca showed significant phytotoxic activity against
Lactuca sativa and
Peganum harmala, thus inhibiting their germination and decreasing seedling growth [
11]. These findings are supported by the study by Mlombo et al. [
12] and Nxumalo et al. [
13], who reported the inhibition of the germination and seedling length of common bean and maize, and millet, respectively.
A study by Paul and Begum [
14] reported that the leaf and root extracts of
A. mexicana decreased the germination of
Brassica napus,
Triticum, and
Vigna mungo. Namkeleja et al. [
15] reported that
A. mexicana extract concentrations of 25, 50, 75, and 100% significantly decreased the mean germination of
Brachiaria dictyoneura by 65, 50, 12.5, and 7.5%, respectively, and the mean germination of
Clitoria ternatea by 77.5, 77.5, 37, and 25%, respectively. This was also observed by Miranda-Arámbula et al. [
16], who reported that aqueous extracts of
A. mexicana decreased the germination of tomato and lettuce.
Brahmachari et al. [
17] reported that terpenoids, alkaloids, phenolics, and flavonoids were among the metabolites discovered through the chemical characterization of
A. mexicana. Joshi et al. [
18] also reported various kinds of secondary metabolites that included alkaloids, flavonoids, glycosides, phenol, lignins, saponins, sterols, and tannins in the phytochemical screening of
A. mexicana leaves. The presence of allelochemicals such as phenolics and other secondary metabolites like terpenoids, and alkaloids could be responsible for inhibiting seed germination and seedling growth, and could potentially reduce the yield potential of maize. It was, however, reported that the presence and concentrations of these phytochemicals vary with the plants’ growing environment [
19]. Hence, the aim of this study was to characterize the phytochemistry of the shoots and roots of
A. ochroleuca and determine whether the same extracts could inhibit the germination of maize seeds. The expected outcomes include the identification of key phytochemicals in
Argemone ochroleuca extracts as well as evidence showing that these extracts inhibit maize seed germination.
3. Results
There were no significant differences among the independent trials, hence the data were pooled and reanalyzed. All data for the maize seed germination variables treated with the water, acetone, and hexane extracts were not normally distributed, except for the germination speed of the hexane extract, hence the data were transformed accordingly.
3.1. Effects of Argemone ochroleuca Water Extracts on Maize Seed Germination
The interaction between the water extract concentrations and plant part factors (roots and shoots) were highly significant (
p ≤ 0.01) for germination speed, mean germination time, germination index, plumule length, and radicle length, whereas the interactions were statistically significant (
p ≤ 0.05) for the germination percentage and mean daily germination. The concentration and plant parts as separate factors were highly significant (
p ≤ 0.01) for all germination variables and seedling length variables. Generally, increasing the water extract concentrations of both the root and shoot extracts significantly decreased the germination variables and seedling length variables, with significant root extract effects being observed at 70% and above for the germination variables and 50% and above for the plumule and radicle seedling lengths, whereas significant shoot extract effects were observed at 40% and above for the germination and seedling length variables (
Table 1 and
Table 2).
In relation to the untreated control, the A. ochroleuca root extracts decreased the germination percentage (18–33%), germination speed (11–21%), mean germination time (13%), mean daily germination (15–29%), germination index (17%), plumule length (20–44%), and radicle length (23–52%), whereas the A. ochroleuca shoot extracts decreased the germination percentage (21–55%), germination speed (18–48%), mean germination time (11–32%), mean daily germination (17–62%), germination index (14–43%), plumule length (24–73%), and radicle length (32–82%), respectively. There were highly significant differences between the plant parts on all of the germination and seedling length variables, with extracts from the A. ochroleuca shoots significantly reducing all variables when compared to those from the roots.
3.2. Effects of Argemone ochroleuca Hexane Extracts on Maize Seed Germination
The interaction between the concentration and plant part of the A. ochroleuca hexane extract was statistically significant (p ≤ 0.05) for the germination percentage, germination speed, mean germination time, mean daily germination, plumule length, and radicle length, whereas the interaction was not statistically significant (p > 0.05) for the germination index. The concentration and plant parts of A. ochroleuca as separate factors were not statistically significant (p > 0.05) for all germination variables and seedling length variables. Increasing the concentration of the shoot extracts significantly decreased the germination percentage, germination speed, mean germination time, mean daily germination, plumule length, and radicle length. At a 2.5 g/L concentration and above, the germination variables and seedling variables were reduced by the shoot extracts. The hexane root extracts had no effect on the germination variables, but stimulated the radicle length at a concentration of 7.5 g/L.
In relation to the untreated control, the
A. ochroleuca shoot extracts decreased the germination percentage (27–38%), germination speed (31–43%), mean germination time (10–12%), mean daily germination (24–31%), plumule length (27%), and radicle length (26%), whereas the
A. ochroleuca root extracts stimulated the radicle length by 29% (
Table 3 and
Table 4).
3.3. Effects of Argemone ochroleuca Acetone Extracts on Maize Seed Germination
The interaction between the
A. ochroleuca acetone extract concentrations and plant parts was not significant (
p > 0.05) for all of the maize germination and seed length variables, except for the plumule length. The concentration as a separate factor was statistically significant (
p < 0.05) for all germination variables and was highly significant (
p ≤ 0.01) for seedling length variables, whereas plant parts as a separate factor were not statistically significant (
p > 0.05) for all germination and seedling length variables. Increasing the extract concentrations significantly decreased the germination percentage and seedling radicle length, whereas other measured variables were not different from the untreated seeds. There was a concentration-dependent response on all of the germination variables, with lower concentrations having a stimulative effect whereas higher concentrations had inhibitory effects. In relation to the untreated control, the
A. ochroleuca extracts decreased the germination percentage (12–16%) and stimulated the radicle length by 40% (
Figure 1 and
Figure 2). The acetone root extract decreased the plumule length by 25% whereas the shoot extracts stimulated the plumule length by 34% (
Figure 3).
3.4. Phytochemicals in Argemone ochroleuca
The GC-MS of the shoot and root extracts of
A. ochroleuca identified 19 different compounds (
Table 5 and
Table 6). The acetone extracts identified six chemical compounds from the shoots and ten from the roots, while the hexane extracts identified one from the shoots and four from the roots. Most constituents were less than 10%, and the ones that showed a high quantity were recorded. These were 9,12-octadecadienoic acid (Z,Z) followed by 9,12,15-octadecatrienoic acid, (Z,Z,Z)-, n-hexadecanoic acid, and á-sitosterol. n-Hexadecanoic acid in the shoot extracts was found to be 3%, while the same compound in the root extracts was 5%, both appearing at different retention times. 9,12-Octadecadienoic acid (Z,Z)- in the acetone shoot extracts was 2%, while in the root extracts, it was 8%, both appearing at different retention times. Hexane extracts identified nonane, 4-ethyl-5-methyl-chemical compound from the shoots and four chemical compounds in the roots. Most of the chemical constituents were less than 5%, and the ones that showed a high quantity were recorded as nonane, 4-ethyl-5-methyl-, and undecane, 2,2-dimethyl. There were no similar chemical compounds between the acetone and hexane extracts.
4. Discussion
This study showed that the
A. ochroleuca shoots and roots have allelopathic effects that inhibit maize seed germination. Similar results have been reported in other allelopathic studies due to the inhibition of allelochemicals found in the plants [
24,
25]. Wang et al. [
26] reported that the enzyme activity and chlorophyll content of leaves from
Amygdalus pedunculata varieties decreased with an increase in the aqueous leaf extract concentrations of
Amorpha fruticosa,
Hedysarum mongolicum,
Sabina vulgaris, and
Hippophae rhamnoides. Mlombo et al. [
12], working with common bean seeds, observed the allelopathic effects of
A. ochroleuca aqueous extracts on seed germination and early seedling growth. The allelopathic effects of other weed extracts such as
Argemone mexicana,
Amaranthus hybridus,
Parthenium hysterophorus, and
Datura stramonium on wheat were recorded by Gella et al. [
27]. Kawawa et al. [
28] explained the phenomenon as due to the allelochemicals inherent in these plants when explaining the suppressed germination and growth responses of
Cassia occidentalis seeds to
Psidium guajava extracts. In a study by Namkeleja et al. [
15], it was observed that the
A. mexicana leaf and seed extracts significantly decreased the seed germination, shoot length, root length, and seedling length of
Brachiaria dictyoneura and
Clitoria ternatea.In the current study, it was also observed that there was a difference in the allelopathic response of the maize seed to the water, hexane, and acetone extracts. These findings were similar to findings by Akter et al. [
29], who reported that aqueous extracts of
Heliotropium indicum leaves and roots were more inhibitory compared to the methanol extracts for seed germination
Lagenaria siceraria. Sayed et al. [
30] reported that the aqueous, ethanol, and acetone extracts of honey weed significantly decreased the germination percentage of brinjal, okra, amaranth, and cucumber. These findings are concurrent with the findings from M’barek et al. [
31], who reported that acetone extracts of
Tetraclinis articulate inhibited the germination of barley, lettuce, radish, and tomato. Wang et al. [
26] reported that the aqueous leaf extract concentrations of
Amorpha fruticosa,
Hedysarum mongolicum, and
Sabina vulgaris at lower concentrations significantly promoted the seed germination and seedling growth of two
Amygdalus pedunculata varieties. This is in agreement with the findings in the current study where lower extract concentrations stimulated the germination and germination variables, with the effect much more prominent in the hexane root extracts. This may be attributed to the stress effect of these extracts on plants, and may stimulate plants to defend themselves by increasing the concentration of some metabolites such as amino acids, proteins, phenolics, and carbohydrates, a phenomenon known as hormesis [
26]. The concentration-dependent responses of the plants to the water, hexane, and acetone fractions revealed that all three fractions contained allelochemicals, but that the greatest potential was in the water-soluble fraction [
32]. The method used in extraction has also been reported by other studies to influence the extract yield, hence the resultant allelochemical composition of the extract [
33].
There was a difference in the allelopathic effects of the different plant parts whereby the shoot extracts were inhibitory when compared to the root extracts. It was also reported in a study by Paul and Begum [
14] that the leaf and root extracts of
Argemone mexicana had different suppressive effects on the germination of wheat, blackgram, and rapeseed, whereas in some cases, the root extracts were more suppressive, and in others, the leaf extracts had more suppressive effects. This was also observed by Huang et al. [
34], who reported that lower concentrations of
Alternanthera philoxeroides root extracts stimulated the root length, shoot length, and fresh weight of
Zoysia matrella, while higher concentrations inhibited the variables. This was also reported by Sitthinoi et al. [
35], who observed that different extraction solvents and plant parts from where the extract was made showed differences in the inhibitory effect on the germination and seedling growth of rice. These findings conform to a study conducted by Netsere [
36], who reported that there were differences in the allelopathic response of different plant parts of
Parthenium hysterophorus on the germination and seedling growth of maize and sorghum. Similar findings were reported by Sitthinoi et al. [
35], who reported that the shoot extracts from jungle rice showed higher inhibitory effects on the seedling dry weight and root length of rice compared to the root extracts. Favaretto et al. [
37] explained that the consistence of leaves as a source of the allelochemicals in various studies could not only be explained by their role as storage organs, but also that they are the most metabolically active plant organs in the plant body, hence it is reasonable that they have a greater diversity of allelochemicals and thus a greater allelopathic effect. Based on the results from the current study, it is evident that
A. ochroleuca contains allelochemicals that affect the germination, plumule, and radicle length of maize.
Profiled chemicals in the roots and shoots indicated various chemicals including fatty acids, sterols, and terpenoides. Previous studies have reported that the
Argemone species contain phytochemicals such as benzoic acid, coptisine, cinnamic, protopine, berberine [
38,
39], and phenolic compounds such as salicylic acid, vanillic acid, and p-hydroxy benzoic acid [
40]. These chemicals have been reported to potentially inhibit the germination and seedling vigor of crops [
9]. Tahir et al. [
25] reported that moringa extracts were rich in octadec-9-enoic acid, nonacosane, methyl 12,15-octadecadienoate and 9-octadecenoic acid, and methyl ester, (E)-; in the same study, it was reported that these extracts inhibited and stimulated the germination and seedling growth of wheat and
Sinapus arvensis.
The presence of 9,12,15-octadecatrienoic acid, (Z,Z,Z)-, n-hexadecanoic acid, octadecadienoic acid (Z,Z)-, and á-sitosterol in the extracts of
A. ochroleuca with their allelopathic effects on seed germination in the present study is supported by several studies. Wang et al. [
41] reported similar compounds as the current study, which were extracted and identified from
Humulus scandens root extracts and had potential allelopathic effects on
Alternanthera philoxeroides. Tahir et al. [
25] also reported that 9,12-octadecadienoic acid (Z,Z), identified from
Pistacia atlantica Kurdica hexane leaf and seed extracts, was a possible allelochemical that caused inhibition in the germination of
Triticum aestivum and
Sinapis arvensis seeds. 9,12-Octadecadienoic acid (Z,Z)- was identified as an effective allelochemical that had a significant allelopathic effect on weed suppression [
42]. Tahir et al. [
25] further reported that 9,12-octadecadienoic acid was significantly correlated with the inhibition in the germination of
Triticum aestivum and
Sinapis arvensis seeds. 9,12,15-Octadecatrienoic acid (Z,Z,Z) was extracted from
Carduus nutans and n-hexadecanoic acid was extracted from
Cirsium creticum and
Carduus nutans and identified as some of the chemicals that had an allelopathic effect on the germination of
Raphanus sativus,
Lactuca sativa, and
Lepidium sativum [
43]. According to studies conducted by Zhang et al. [
44] and Yuan et al. [
45], the identified n-hexadecanoic acid in the
Solidago canadensis and
Spartina alterniflora aqueous extracts had significant allelopathic effects on the growth of wheat and algae. Zhang et al. [
44] also noted that the higher the n-hexadecanoic acid concentration, the greater the inhibitory effect on the shoot and root lengths of wheat. Verma et al. [
42] identified 9,12,15-octadecatrienoic acid, methyl ester, and phytol from sunnhemp, 9,12-octadecadienoic acid (Z,Z)- from sesame and neophytadiene, and 9,12-octadecadienoic acid (Z,Z) from pearl millet, which were effective as allelochemicals when used in weed control. The presence of different allelochemicals may be responsible for the inhibitory effects of various extracts on the germination and seedling growth of the
Microcystis aeruginosa blooms [
41]. Several phenolic compounds present in
A. ochroleuca are known to be biologically active, and they could also be potential allelochemicals [
25,
42]. Since more than one compound with allelopathic potential was discovered in this plant in the present study, it could be speculated that synergistic effects of these potential allelochemicals could exist for this plant to exert its potential allelopathic effects on other plants [
46]. As allelopathic substances, these chemical compounds should be further investigated as well as their effects on the germination of seeds. Some of these chemical compounds have been identified and used in pharmaceutical studies and in the development of bioherbicides [
47,
48].