Allelopathic Effects of Lantana camara L. Leaf Aqueous Extracts on Germination and Seedling Growth of Capsicum annuum L. and Daucus carota L.

Allelopathy is the chemical interactions between plants that might lead to either stimulation or inhibition of growth, community structure, and plant invasions. Lantana camara L. is a noxious invasive weed that negatively affects seed germination, seedling growth, and increases the mortality of the crop plant. The objective of this work was to assess allelopathic effect of L. camara leaf aqueous extract on germination and seedling growth of Capsicum annuum (pepper) and Daucus carota (carrot). The aqueous extract of Lantana leaf samples was used as a source of allelopathic effects. Data were collected for germination and seedling growth parameters. The result indicated that the highest concentration of the allelopathic extract (20 mg/L) has demonstrated significantly the highest germination inhibition rate GIR (60.00%), germination speed V (2.54 U/day) for D. carota as GIR (70.00%), mean germination time MGT (0.36 days), and GI (0.67%) for C. annuum seeds. The highest concentration of the allelopathic extract (20 mg/L) has recorded the highest plumule inhibition rate PIR (59.63%) and radical inhibition rate RIR (48.95%) for D. carota seeds, as well as PIR (27.47%) and RLR (79.49%) for C. annuum. The largest negative allelopathic index (−60.00% or allelopathic intensity of 60.00%) was recorded for D. carota seeds, whilst (−63.43% or allelopathic intensity of 63.43%) was recorded for C. annuum seed germination. For D. carota seed germination, the first principal component (PC1) has got high positive loads from GI (0.36), RLR (0.31), GR (0.34), allelopathic index AI (0.34), relative length of plumule RLP (0.24), and V (0.30). By contrast, PC1 for D. carota seed germination has got the highest negative component loads recorded by GIR (−0.34), PIR (−0.24), MGT (−0.35), and RIR (−0.31). In allelopathic effect on C. annum seed germination, the first principal component (PC1) has got high positive scores from relative length of radical RLR (0.31), RLP (0.33), germination rate GR (0.33), V (0.33), and AI (0.33). Likewise, the high negative component loads were recorded by GIR (−0.33), PIR (−0.33), RIR (−0.31), and MGT (−0.32). The result of the present study demonstrated that GIR, PIR, and RIR were directly related to negative allelopathic activity.


Introduction
Te term allelopathy derived from Greek words, namely, allele and pathy (meaning "mutual harm" or "sufering," respectively) [1].Allelopathy is the interaction between plants in neighbourhood that might lead to either stimulation or inhibition of growth by allelochemicals that are released through volatilization, eluviations, and decomposition of the plant or root exudates during growth [2].Allelochemicals can also indirectly afect plants through the inhibition of microorganisms, including nitrogen fxing and nitrifying bacteria [3] and ectomycorrhizae [4].Allelopathy widely exists in nature and plays a vital role in crop cultivation systems, controlling weeds, and preventing crop disease and insect infection.Diferent groups of plants, like algae, lichens, crops, and annual and perennial weeds have widely known allelopathic interactions [4,5].
A signifcant portion of the agricultural land in developing countries particularly tropics is heavily infested by various native and alien (invasive) weeds [6], and controlling weeds is a big challenge to farmers [7].Te crop-weed interaction can be owing to competition alone, allelopathy, or both [8].Many allelochemicals are phytotoxic and have potential as herbicides or as templates for new herbicide classes.Allelopathic efects can be useful in some environmentally friendly techniques for controlling the weeds and help reduce the economic and environmental costs of using herbicides because herbicides create environmental pollution [9,10].Various reports of allelopathic interactions also reported including bioactive compounds [11], responses of plant proteomes to soil antibiotics [12], biofungicide [13,14], and biopesticide [15].
Allelochemicals can stimulate or inhibit the germination or/and growth of plants and increase the resistance of crops to biotic and abiotic stresses [2].Allelochemicals are mainly secondary metabolites present in diferent parts of plants.Chemicals that inhibit the growth of some species at certain concentrations can stimulate the growth of the same or diferent species at lower concentrations [16].Te allelopathic nature of the plants help them to be highly competitor for space, light, and nutrients with the nearby plants [9].Te allelochemicals are released into the neighboring environments and rhizosphere soil of the plants and neighbouring environments during rainfall leachates, decomposition of plant residues, root exudation, and volatilization from living plant parts [17][18][19].Te released allelochemicals may suppress the regeneration process of indigenous plant species by decreasing their germination and seedling growth and increasing their mortality.Te natural decomposition process of crop residues induced by microbes, dispels chemicals in soil which are potentially very toxic even though the primary substances are not toxic [20].
Allelopathic plants inhibit or suppress germination, growth, development, or metabolism of crops due to secretion of allelochemicals into the rhizosphere of neighboring crop plants [21].Various phenolic compounds inhibited cell division.It is also possible that cell elongation was afected by extracts of weed residues.Many phytotoxic allelochemicals have been isolated, identifed, and found to infuence a number of physiological reactions.Tese allelochemicals afected many cellular processes in target plant species, including disruption of membrane permeability [22], ion uptake [23], inhibition of electron transport in both photosynthesis and the respiratory chain [24], damage to DNA and protein, alterations of some enzymatic activities [25,26], and ultimately leading to programmed cell death [25].
Lantana camara is an invasive weed belonging to Verbenaceae family [27].It interrupts the regeneration process of indigenous plant species by decreasing their germination, reducing their seedling growth, and increasing their mortality [27,28].Te extracts, essential oil, leachates, residues, and rhizosphere soil of L. camara suppressed the germination and growth of other plant species [28,29].Terefore, the allelopathic property of L. camara may support its invasive potential and the formation of dense monospecies stands [29].
Te elevated temperature has been contributing to the allelopathy of L. camara [30], which indicates that global warming may increase the threat of the invasion of the species into additional nonnative areas.Direct negative allelopathic efects of Lantana spp. on some crops have been reported previously [5,8,31].However, these efects may vary depending on the species variety.Farmers in the Hararghe region of eastern Ethiopia cultivate diferent vegetables including carrot and pepper for which information regarding the allelopathic efect of Lantana spp. is insufcient.Terefore, this research was initiated to evaluate the allelopathic efect of Lantana leaf extract on germination and seedling growth of Capsicum annuum and Daucus carota.

Preparation of the Aqueous Extracts.
Te leaf sample was air dried at room temperature for 10 days and dried leaf sample was ground to fne powder by using mortar and pestle.Ten aqueous extraction was done with distilled water by dissolving 120 grams of the powder in 500 mL of distilled water in a 500 ml fask.Te extract was then fltrated through Whatman no. 1 flterpaper, and the fltrate was dried in rotary evaporator at 50 °C under reduced pressure to evaporate water and obtain the extracts in a somewhat dried form [32]. Aqueous extracts of diferent concentrations (10,15, and 20 mg/L) were then prepared by dissolving the dried crude extract in distilled water.Te control experiment, without the use of the aqueous extract was made with distilled water.Te crude extract was stored at 4 °C until use.All experiments were replicated three times.

Seed Germination and Seedling Growth Bioassays.
Te experiment was carried out in sterile 20 cm diameter Petri dishes, in which moistened Whatman No. 3 paper was used as a germination support.Before being used, the tested seeds were chosen as healthy and then disinfected with 70% ethanol.Afterwards, 10 seeds were placed in each Petri dish before being treated with a sufcient amount of various concentrations of the aqueous extract, while the control was only treated with distilled water.Te experiment was carried out under laboratory conditions for a period of 2 months in three replications for each treatment.A seed is considered germinated when the radicle appears.Germination was recorded daily, and the results were determined by measuring diferent germination and seedling growth parameters like root length (cm), number of leaves, and seedling dry weight.Germination parameters like germination kinetics, germination index, inhibition rate, and the allelopathic index (AI) were calculated as follows: 2 Scientifca

Germination Rate (GR).
Te germination rate was calculated according to the formula given by Come [33]: where Ng is the number of germinated seeds and Ns is the number of seeds sown.

Germination Speed (V).
Te germination speed is calculated by the following formula proposed by Come [33]: where V is the speed of germination and N1 is the number of seeds germinated at time T1.

Mean Germination Time (MGT)
. Te mean germination time is calculated as described by Ranal et al. [34]: where n i and t i are consecutively the numbers of newly germinated seeds in the last time and the time from the beginning of the experiment to the last observation and k is the last time of germination.

Germination Index.
Te germination index (GI) is a quantitative expression of germination which relates to the daily germination rate at the maximum value of germination [35], it is calculated by the following equation: where N n is the percentage of germination on n th day.

Relative Length of Radicles (RLR).
Te relative length of radicles is calculated according to the formula given by Rho and Kil [36]: where Rr is the relative length of radicle; Lr is the average length of radicles of treated plants and Lc is the average length of radicle of control plants.

Relative Length of Plumule (RLP). According to Rho
and Kil [36] this parameter is calculated by the following formula: where Rs is the relative length of plumule; Lp is the average length of plumule of treated plants; and Lc is the average length of plumule of control plant.
2.3.8.Allelopathic Index (AI) to Refect the Intensity of the Allelopathic Efect.AI was calculated according to the work of Luo et al. [38] as follows: where GRt is the germination rate in treatment or allelopathic extract concentration t and GRc is the germination rate of the control (given sterile distilled water).An AI < 0 refects a negative allelopathic efect of L. camara leaf extract on seed germination, and an AI > 0 value indicates a promoting efect on the germination, while the absolute value of AI indicated the allelopathic intensity.

Statistical Analysis.
All the experiments were performed in a completely randomized design.Data were subjected to one-way analysis of variance and were presented as mean separated at p < 0.05 applying least signifcant diference (LSD) test and statistical analysis was conducted using SAS version 9.2 software package.

Allelopathic Index for D. carota and C. annum Seed
Germination.Te allelopathic index (AI) for D. carota seed germination (Table 2 and Figure 1) and C. annuum seed germination (Table 2 and Figure 2) demonstrated negative allelopathic activity as the concentration of the allelopathic extract from L. camara leaf increases 10 to 20 mg/L.Te allelopathic intensity was found to be the highest (60%) for D. carota and (63.43%) for C. annuum seed germination (Table 2).Te large negative allelopathic activity of L. camara leaf extract might contribute to the retardation of growth and yield loss of crop plants near L. camara invasive shrub.

Correlation of Germination Parameters and Allelopathic
Index of L. camara Leaf Extract.Te Pearson's correlation coefcient was used to assess the association of D. carota and C. annuum seed germination parameters and the allelopathic index of L. camara leaf aqueous extract (Tables 3 and  4).Te negative allelopathic index (AI) for D. carota seed germination (Table 3) was signifcant and negatively correlated with RIR (−0.762),GIR (−1.00), and germination speed (−0.807), indicating that the negative allelopathic activity increases with increasing RIR, GIR, and germination speed.However, the allelopathic index was found to be signifcant and positively correlated with RLR (0.762), germination rate (1.00), MGT (0.909) and germination index (0.944), indicating that the negative allelopathic activity decreases with increasing rate of germination, germination index, MGT, and RLR during D. carota seed germination.Tus, germination inhibition rate (GIR), radicle inhibition rate (PIR), and germination speed were found to be the best parameters or traits that can be used as indicators of allelopathic activity during D. carota seed germination.
Te negative allelopathic index (AI) for C. annuum seed germination (Table 4) was signifcant and negatively correlated with plumule inhibition rate (−0.903), radicle inhibition rate (−0.835), germination inhibition rate (−0.987), and the mean germination time (−0.922),indicating that the negative allelopathic activity increases with increasing PIR, RIR, GIR, and MGT in C. annuum seed germination.Contrastingly, the allelopathic index was found to be signifcant and positively correlated with RLP (0.904), RLR (0.835), germination rate (0.987), and germination speed (0.987), indicating that the negative allelopathic activity decreases with increasing rate of relative length of plumule, relative length of radicle, germination rate, and germination speed.
Since the correlation coefcient indicated the existence of association among traits.It does not indicate the direct and indirect relationships among them.Terefore, the principal component analysis (PCA) was used to identify the most discriminating traits showing allelopathic activity of the plant extract (Table 5).Te eigen values corresponding to sum of squares of variances greater than one being accounting for the majority of the variances were considered for interpretation of the result [39].Te PCA of allelopathic activity of L. camara on seed germination of D. carota and C. annum (Table 5) demonstrated that only PC1 (with eigenvalue >1) account for 95% of the variances for D. carota, while for C. annum seed germination, PC1 (with eigen value > 1) accounted for about (99%) of the variations; hence, the frst PC was sufcient for interpretation of the relationship allelopathic index and germination parameters.

Discussion
Te use of Daucus carota as a representative of storage root crops and Capsicum annuum as storage fruit vegetable so as to demonstrate diferential response to the allelopathic efect of L. camara.It was found that for D. carota the inhibition on root is lower than that of shoot.However, for C. annuum inhibition of root elongation is higher than that of shoot.Te fact that D. carota is a storage root crop might have made the growth of root more vigorous than shoot.However, it must be confrmed with further study.Te maximum concentration of the allelopathic extract (20 mg/L) has demonstrated signifcantly the highest GIR and MGT indicating that GIR and MGT of the extracts increased with increasing extract concentration.Tis fnding was in agreement with a number of previous studies like Maiti et al. [40] who reported the inhibitory efect of aqueous leaf extracts and leaf leachates of L. camara on seed germination metabolism of mungbean seeds.Similar result was also reported by Nandi and Dalal [41] the allelopathic efect of L. camara on germination and seedling growth of radish (Raphanus sativus L.) and spinach (Spinacia oleracea L.).Aqueous extracts of all parts of Lantana camara have strong allelopathic efect on the germination of Pennisetum americanum, Lactuca sativa and Setaria italica [42].

Scientifca
In the present study the allelopathic inhibitory efect was increasing with the concentrations of the extracts and higher concentration had the stronger inhibitory efect whereas the lower concentration showed stimulatory efect in some cases.Te inhibitory efect was much in root than in shoot during seed germination.Similar fnding was alsao reported by Ahmed et al. [4] who investigated the allelopathic efects of crop plants including Brassica juncea (L.) Czern; Cucumis sativus L.; Phaseolus mungo L.; Raphanus sativus L.; Vigna unguiculata (L.) Walp., and Cicer arietinum L. Te   6 Scientifca allelochemicals released from L. camara may provide the species with a competitive advantage against the native plants and may also suppress the regeneration process of native plant species and contribute to establishing their habitats as invasive plant species and formation of monospecies stands [29].
According to Gniazowska and Bogtek [43] seed germination may also be inhibited due to hampered resource mobilization by allelochemicals during early stages of seed germination.It is also possible that allelochemicals such as some phenolic compounds impair the synthesis and/or activity of gibberellic acid [44], which regulates the production of amylase [45] so that seed germination is negatively afected.Based on the results of germination that tells us seed viability, C. annuum was more susceptible to the extracts than D. carota.Tis implies that diferent plant species may have varying sensitivity to allelochemicals of a given plant species.
Te allelopathic efects of L. camara leaf extract on plumule and radicle growth is shown in Table 2. Compared to the lowest extract concentration, the lengths of plumule and radicle of the tested plants were signifcantly (p < 0.05) higher than the higher extract concentration treated seeds (Table 2).Tis fnding agrees with that of Corsato et al. [46] and Rigon et al. [47] who, respectively, studied the allelopathic efects of leaf extracts of sunfower and castor on Bidens pilosa and, canola and raddish.Barreto et al. [48] also previously reported that radicle length of canola seedlings decreases with increasing concentration of soybean leaves extracts.Tey also explained that the length of the radicle is aindicator of the allelopathic efect as this plant part is very sensitive and in direct contact with the extracts.Tis observation of reduced initial seedling growth suggests that L. camara leaf extract possesses allelopathic compounds.

Conclusion
Te present study has used Daucus carota as a representative of storage root crops and, Capsicum annuum as storage fruit vegetable so as to demonstrate diferential response to the allelopathic efect of L. camara.It was found that for D. carota the inhibition on root is lower than that of shoot.However, for C. annuum inhibition of root elongation is higher than that of shoot.Te fact that D. carota is a storage root crop might have made the growth of root more vigorous than shoot.However, it must be confrmed with further study.Te allelopathic efect was found to be increasing as the concentration of the allelopathic extract increases from 10 to 20 mg/L for both D. carota and C. annuum seed germinations.Te germination inhibition rate (GIR), radicle inhibition rate (PIR) and germination speed were found to be the best parameters or traits that can be used as indicators of allelopathic activity during D. carota seed germination.Te the negative allelopathic activity increases with increasing PIR, RIR, GIR, and MGT in C. annuum seed germination.Te highest negative scores in PCs indicate direct relationship between the negative allelopathic index and negative score factors while the highest positive scores in PCs indicate inverse relationship between negative allelopathic activity and positive score factors in C. annum seed germination.Te large negative allelopathic activity of L. camara leaf extract might contribute for retardation of growth.
Growth of D. carota and C. annuum Seeds.Te impacts of L. camara leaf extracts on seed germination of D. carota and C. annum were assessed using germination parameters including germination rate (GR), germination inhibition rate (GIR), germination speed (V), mean germination time (MGT), and germination index (GI) (Table1).For both bioassayed plants the control group has recorded signifcantly the highest GR (100% for D. carota, and 83.33% for C. annum); germination speed (2.40 U/day for D carota, and 8.33 U/day C. annuum); and GI (16.89% for D. carota, and and 0.61% for C. annuum).On the other hand, the maximum concentration of the extract has demonstrated signifcantly the highest GIR (60% for D. carota, and 70% for C. annuum); MGT (3.83 for D. carota, and 0.36 for C. annuum) indicating that GIR and MGTof the extracts increased with increasing extract concentration.Tus, MGT and GIR can be recommended as the best indicators of allelopathic efect of seed germination if corroborated with further works.3.2.Efect of L. camara Aqueous Leaf Extract on Seedling Growth of D. carota and C. annuum.Te allelopathic efect of L. camara leaf extract on seedling growth of both Scientifca bioassayed plants including D. carota and C. annuum are shown in Table 2. Te least RLP (40.37%) and RLR (51.05%) were recorded for D. carota; RLP (72.53%) and RLR (29.51%) for C. annuum were recorded with maximum concentration (20 mg/L) of L. camara leaf extract.Te reduction in the relative growth of both plumule and radicle with increase in concentration of the extract might indicate the growth inhibitory efect of allelochemicals in the crude leaf extract of L. camara.Te highest PIR (59.63%),RIR (48.95%), negative allelopathic index or allelopathic intensity (|AI|) (60.00) for D. carota; PIR (27.47%),RIR (70.49%), and allelopathic intensity (|AI|) (63.43) for C. annuum were recorded with the maximum concentration of the allelopathic extract (20 mg/L) indicating that PIR, RIR, and |AI| are the best parameters to measure the negative allelopathic efect on seed germination and seedling growth of crop plants.

Table 1 :
Allelopathic efect of L. camara leaf extract on germination of D. carota and C. annuum seeds.Likewise, the high negative component loads were recorded by GIR (−0.33),PIR (−0.33),RIR (−0.31), and MGT (−0.32).Terefore, the highest negative scores in PCs indicate direct relationship between the negative allelopathic index and negative score factors.However, the highest positive scores in PCs indicate inverse relationship between negative allelopathic activity and positive score factors in C. annum seed germination.

Table 2 :
Allelopathic efect of L. camara leaf extract on seedling growth of D. carota and C. annuum.

Table 3 :
Correlation coefcient of germination and allelopathic index of D. carota., the germination rate; GIR, the germination inhibition rate; MGT, the mean germination time; GI, the germination index; RLP, the relative length of plumule; RLR, the relative length of radicle; PIR, the plumule inhibition rate; RIR, the radicle inhibition rate; AI, the allelopathic index.* Signifcant at P < 0.05; * * signifcant at P < 0.01. GR

Table 4 :
Correlation coefcient of germination and allelopathic index of C. annuum.
GR, the germination rate; GIR, the germination inhibition rate; MGT, the mean germination time; GI, the germination index; RLP, the relative length of plumule; RLR, the relative length of radicle; PIR, the plumule inhibition rate; RIR, the radicle inhibition rate; AI, the allelopathic index.* Signifcant at P < 0.05; * * signifcant at P < 0.01.

Table 5 :
Te principal component analysis (PCA) for the allelopathic activity of L. camara leaf aqueous extract on germination of D. carota and C. annum seeds.