1. Introduction
Conyza bonariensis (L.) Cronquist or Erigeron bonariensis is an invasive plant of the Asteraceae family, native to South America. It is often found in tropical and subtropical regions and is widespread in most warm regions of Europe, Africa, Asia, the Caribbean and Central America [1] . C. bonariensis is a plant known for its medicinal properties and is therefore widely used in traditional medicine for the treatment of rheumatism, dental pain and headaches; it is also attributed with anti-ulcerogenic and anticoagulant activities. C. bonariensis is rich in essential oils (EOs), with an oil content of 0.1% - 0.5% for the whole plant. Studies conducted on the chemical composition showed that its EO is rich in terpenes (limonene and (E)-β-farnesene) and acetylenic compounds (cis-Lachnophyllum ester and Matricaria ester) [2] [3] . However, to the best of our knowledge, no chemical study was reported on the plant acclimatized in the African Sub-Saharan Region.
Recently, we reported electron-deficient synthetic alkynes bioactive on root-knot nematode Meloidogyne incognita [4] . We found that the conjugation of electron-withdrawing carbonyl groups to an alkyne triple bond was extremely proficient in inducing nematode paralysis and death. Naturally occurring acetylenics are of particular interest since many of them display important biological activities. They are of great interest for medicine, pharmacology, medicinal chemistry, and pharmaceutical industries and could be valorised in pest management [5] . This evidence prompted us to investigate herein the nematicidal and insecticidal activities of a plant from Asteraceae family, known to be rich in acetylenic compounds.
Callosobruchus maculatus F. (Coleoptera: Bruchidae) is an insect pest that attacks cowpea seed stock, resulting in rapid crop deterioration. Losses caused by this insect can be as high as 36.4% after two months of storage, and as high as 100% within few months [6] [7] . Phytoparasitic nematodes are microscopic worms that cause plant diseases whose typical symptoms are stunted growth, wilting, leaf discoloration, and deformation of plant organs. This results in reduced crop quantity and quality [8] . Root-knot nematodes of the genus Meloidogyne are for instance involved in the decline of tomato production in Togo [9] .
If synthetic pesticides are usually used to control insects and nematodes, concerns about their safety to environment and human health are more and more risen. Plant-based insecticide and nematicide would be affordable and less dangerous to the environment and food security. In the present study, chemical constituents of essential oils along with the crude oil of C. bonariensis were evaluated for their insecticidal and nematicidal activities on the adult stages of stored-bean pest C. maculatus and juvenile stages of M. incognita. We therefore chose in this work to determine the chemical composition of essential oils (EO) and selectively extracted acetylenic components from different parts of C. bonariensis by GC-MS and LC-MS. On the other hand, this study evaluated the biological activities of the different extracts on cowpea weevils and parasitic root-knot nematodes.
2. Materials and Methods
2.1. Materials
Chemicals and instruments
Methanol, diethyl ether, deuterated chloroform and hexane used were of high-performance liquid chromatography grade. Reactions were monitored by TLC on 0.25 mm E. Merck silica gel plates (60F-254) visualized under UV light and by applying a phosphomolybdic acid solution in EtOH followed by heat. High-quality reagents were purchased at the highest quality that was commercially available and was used without further purification.
Plant material
Plant materials of C. bonariensis were collected in the middle of May 2021 at Dagni Koudzragan in Togo with geographical coordinates Lat: 7.13396 N 7˚8'2.27178'' long: 0.67799 E 0˚40'40.75512''. The biomass was left under air conditioning for different extractions.
2.2. Methods
Essential oil and solvent extractions
Harvested aerial parts and flowers of C. bonariensis were dried at a temperature of about 15˚C under air conditioning for 5 days. Furthermore, the obtained biomass was submitted to 4 h water steam-distillation using a Clevenger-type apparatus. Resulting essential oils were dried over anhydrous sodium sulphate before being transferred to Teflon-sealed cap dark glass vials and stored at 4 C until use.
For the solvent extraction; dried plant roots (200 g) were extracted with chloroform for 30 min by ultrasonication and left overnight at room temperature for maceration (3 × 600 mL). After 24 h, the extract was filtered and evaporated under reduced pressure to dryness at 40˚C to give a gummy product that was suspended in MeOH/H2O (3:1). The mixture is subsequently partitioned with hexane (3 × 600 mL) to eliminate fats. The methanol/water phase was evaporated at reduced pressure (40˚C) to eliminate methanol before being partitioned with Et2O (3 × 100 mL). The Et2O and hexane extracts rich in acetylenic compounds were concentrated to dryness under reduced pressure.
Essential oil analysis by gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC/MS) analysis was performed on an Agilent 5973 equipment with a 60 m × 0.25 mm × 0.25 µm HP1-MS apolar column at an initial temperature of 60˚C isothermal for 10 min, then at a final temperature of 300˚C for 20 min; the gradient was 2˚C/min. The carrier gas (helium) flow rate was 1 mL/min. The mass spectrometer detector was HED/EM (High Energy Dynode/Electron Multiplier 0 - 3000 V) with an energy of 70 eV; the other parameters remained the same. One microliter of essential oil diluted with hexane (10 thousand times) was injected. Unknown mass spectra of peaks obtained from chromatograms were compared to known spectra from literature in NIST Database. Available compounds were eluted for retention time confirmation. However, Unavailable compounds were confirmed by Kovats indices calculation using alkanes.
LC-MS analysis of Diethyl and hexane phases
The diethyl and hexane phases obtained from C. bonariensis plant were analyzed by reverse-phase liquid chromatography on an Agilent 1200 series LC system using a Kinetex EVO C18, 100 Å, 5 µm, 150 × 2.1 mm (Phenomenex, Castel Maggiore, Italy). The LC conditions were as follows: flow rate: 0.3 mL/min; solvent A: 0.1% formic acid in bi-distilled water; solvent B: methanol; and gradient was from 10% to 100% B over 10 min and kept 10 min. Eight microliters of filtered samples dissolved in methanol were then analyzed by Electrospray ionization in positive and negative modes using an Agilent 6520 Time of Flight (TOF) MS. Mass spectral data were acquired in the m/z range of 100 - 1500 with an acquisition rate of 1.35 spectra/s, averaging 10,000 transients. The source parameters were adjusted as follows: drying gas temperature 250˚C, drying gas flow rate 5 L/min, nebulizer pressure 45 psi. Based on the original acquisition files, we performed a pre-processing step with MetAlign software used for automated baseline correction and alignment of all extracted mass peaks across all samples. Results were stored as CSV file. ESI/QTOF MS data were then analyzed using the molecular feature extraction algorithm of the MassHunter Workstation software (version B 03.01 Qualitative Analysis, Agilent Technologies, Santa Clara, CA, USA). The molecular feature extraction algorithm took all ions into account exceeding 1000 counts with a charge state equal to one. Blank runs showed maximum 10 features with intensity threshold at 1000 counts. Isotope grouping was based on the common organic molecules model.
Nuclear magnetic resonance analysis of isolated compounds
Nuclear magnetic resonance (NMR) spectra of different isolated compounds were recorded on an Inova 500 spectrometer (Varian, Palo Alto, CA, USA). The chemical shifts (δ) are reported in part per million downfield from tetramethylsilane (TMS), which was used as internal standard, and the spectra were recorded in deuterated chloroform (CDCl3).
Isolation of Lachnophyllum ester and Lachnophyllum lactone: Nuclear magnetic resonance (NMR) analysis
Isolation of the Lachnophyllum ester by recrystallisation
(Z)-Lachnophyllum ester was obtained by dissolving C. bonariensis EO in hexane. One milliliter of EO was dissolved in 10 mL of hexane and kept at −20˚C. After 24 h, (Z)-Lachnophyllum ester crystallizes and the supernatant solvent is removed. (Z)-Lachnophyllum ester crystals are purified with 10 mL of hexane 6 times to obtain almost pure crystals as demonstrated by GC-MS analysis. The crystals obtained were used for chemical analysis and nematicide tests.
Isolation of Lachnophyllum lactone by preparative HPLC
LC-MS analysis of diethyl-ether fraction revealed the two compounds as major components: Lachnophyllum ester and Lachnophyllum lactone. Preparative HPLC was used to isolate the latter. Compounds were eluted on a C18 column with an isocratic mobile phase MTBE: Hex (1:4). A diode array detector with absorption wavelengths of 254, 310 and 360 nm coupled with a refractive index detector was used. The HPLC pump was stabilized at a flow rate of 2 mL/min. Different obtained fractions were submitted to thin layer chromatography before nuclear magnetic resonance (NMR) analysis.
Insecticidal and nematicidal bioassays
Insecticidal activity
Insecticidal activity was evaluated on young generations of cowpea bruchids, C. maculatus. The rearing was carried out by introducing 50 adult couples on 200 g of healthy cowpea seeds sterilized at a temperature of −18˚C for 3 days in freezer. The insects were removed after 48 h and infected seeds were put in incubation from which first generation was emerged after 21 days. Newly emerged adults were used for insecticidal activities. To determine insecticidal activity of essential oil of C. bonariensis, 20 couples of newly emerged adults were selected and placed in one-liter jars with different concentration (1, 2, 3 and 5 µL) of essential oil which were placed on 5 cm diameter filter paper discs. For each dose, 4 trials were carried out in addition to 4 controls that had not received the product. After 24 hours, insects were removed and placed in Plexiglas petri dishes for another day to monitor their agony and mortality rate of adults in each treatment was determined by following the formula:
Mortality rate (%) = (Ntr − Nte)/NT × 100
where Ntr is number of dead insects in the treatment, Nte is number of dead insects in control and NT is total number of insects tested [10] .
Nematicidal activity
Population of Meloidogyne incognita originally obtained from tomato (Solanum lycopersicum L.) roots harvested in a greenhouse was used for rearing on tomato plants cv. Belladonna, a cultivar that is very susceptible to root-knot nematodes. All plants were maintained in a greenhouse at 25˚C - 28˚C, 60% relative humidity, and 16h photoperiod in plastic pots (18 cm diameter) containing a 10:1 (v/v) mixture of peat and perlite. After 40 days, the plants were uprooted, and the roots were washed free of soil and cut into 2 cm pieces. Eggs were extracted using the sodium hypochlorite procedure [11] and second instar larvae were obtained using the modified Baermann method at 28˚C. All J2 hatched within the first 3 days were discarded and subsequent generations were collected and used in the experiments.
Nematicidal activities of essential oils of different parts of C. bonariensis, diethyl ether extract and isolated Lachnophyllum ester were tested for loss of mobility of J2, and EC50 values were calculated. Stock solutions of tested samples were prepared in methanol, while the final solutions were obtained by dilution with water containing the surfactant polysorbate 20 (Tween-20). Final concentrations of methanol and Tween-20 in each well never exceeded 1.0% and 0.3% v/v, respectively, because preliminary tests showed that the mobility of nematodes exposed to these concentrations was similar to the mobility of nematodes maintained in pure water [12] . For the assays, 96-well microplates were used. The test well constituted of 200 µL solution containing 25 - 30 J2s. After 24, 48 and 72 h, mortality (motility) of nematodes was checked under a reversed microscope (Zeiss, Germany). Every sample was replicated 6 times and the experiment was repeated at least twice at different times. The percentages of dead J2 were corrected by eliminating the natural death in the water Tween 20 0.3%/MeOH (2:98 v/v) control (less than 5% of total number of J2) according to the Schneider Orellis formula: [13]
and they were analyzed (ANOVA) after being combined over time. Since ANOVA indicated no significant treatment by time interaction, means were averaged over experiments. Corrected percentages of death J2 treated with tested compounds were subjected to nonlinear regression analysis using the loglogistic equation proposed by Seefeldt et al. in 1995 [14] :
where C = the lower limit, D = the upper limit, b = the slope at the EC50, and EC50 = the test compounds concentration required for 50% death/immotility of nematodes after elimination of the control (natural death/immotility). In the regression equation, the test compounds concentration (% w/v) was the independent variable (x) and the immotile J2 (percentage increase over water control) was the dependent variable (y). The mean value of the six replicates per compound concentration and immersion period was used to calculate the EC50 value.
3. Results and Discussion
3.1. Essential Oil Extractions
Extraction of fresh aerial part of C. bonariensis plant gave a yellow EO with a yield of 0.12% of fresh material. The flower gave 0.3% while a percentage yield of 0.01% was found for the root. When studying the same plant, Barbosa et al. [3] reported yields varying from 0.04% to 0.32% for the extraction of different parts of C. bonariensis harvested in Brazil. Maia et al. [2] reported percentage yields between 0.1% - 0.5% for 5 samples harvested in different geographic areas. The extraction yield found herein is within the general found range and still high for a medicinal endemic plant. This work, report for the first time, EO extraction from C. bonariensis acclimatized in West Africa.
3.2. Chemical Composition of the Extracted Essential Oils
GC/MS analysis of the aerial part and root of the EO identified 35 compounds (Table 1) representing more than 95% of the crude essential oils. Major components were methyl lachnophylum ester and lactone, limonene, trans β-farnesene, trans β-ocimene, β-caryophylene and germacrene D. As expected, acetylenic compounds were the major components of the EO. Monoterpene hydrocarbons made up to 21.69% and sesquiterpene hydrocarbons 18.75% for the aerial part while the root showed less.
Chemical composition of the aerial part of the EO of C. bonariensis acclimatized in Tunisia and Italy was studied by Hammami et al. [15] . For Tunisian chemotype, nearly 90% terpenes were found and major compounds were caryophyllene oxide (18.7%), spathulenol (18.6%) and α-curcumene. The major compounds of the plant harvested in Sardinia, Italy, were cis-Lachnophyllum ester (14.2%) and (E)-β-farnesene (12.0%). We found in this work the same components even though the previous work did not identify Lachnophyllum lactone. This may be explained by the less sensitivity of this compound to electronic ionization and absence of the lactone in previous NIST mass spectra libraries. Slight difference in chemical composition of the EO of the plant acclimatized in Togo was probably caused by harvesting location combined with the extraction method. In fact, Hammami et al. [15] demonstrated that neophytadiene (53.2%) was the major component when supercritical fluid extraction is used while caryophyllene oxide (18.7%) was the major compound when hydrodistillation extraction was used on a sample harvested in Tunisia.
The results obtained from a chemotype of different parts of C. bonariensis plant from Brazil [3] showed the presence of methyl Matricaria ester (76.4%), manool (25.3%), limonene (29.6%) and carvone (21.1%) as major compounds of the root, stem, flower and leaf, respectively. cis-Lachnophyllum ester which is the major compound of our work was not identified but its trans isomer was identified at 21.3% in the root. These findings highlight the need to evaluate chemical composition of EOs from different regions before use. Likewise, Maia et al. [2] identified in different chemotypes harvested in Amazon region of Brazil, limonene (58.4%), (E)-β-farnesene (30.9%), manool (25.3%) and carvone (21.1%) in different parts of C. bonariensis .
3.3. LC-MS/Q-TOF Analysis of Different Extracts of C. bonariensis
To check the overall acetylenic components of the West African acclimatized C. bonariensis, different selective extracts were made. With an initial chloroform extract, diethyl ether and hexane fractions were obtained by liquid-liquid extraction from aerial and root parts of the plant. After evaporation, extracts were submitted to liquid chromatography coupled with a quadrupole time of flight detector (LC-MS/QTOF). Four fractions were analyzed: aerial part diethyl fraction (CBAPET2O) and hexane fraction (CBAPHex); and root diethyl fraction (CBRET2O) and hexane fraction (CBRHex). Chromatograms and exact mass
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Table 1. Volatile compounds identified in the essential oil of C. Bonariensis.
extractions of potential acetylenic components are shown in Figures S1-S4. Lachnophyllum methyl ester, Lachnophyllum lactone, Lachnophyllum ethyl ester, Matricaria lactone and Matricaria ethyl ester were identified in the different extracts (Table 2).
If Lachnophyllum and Matricaria methyl esters are frequently reported in Conyza species [3] [16] , Lachnophyllum and Matricaria ethyl esters identified herein are less reported. They were identified only in diethyl ether extracts. Ethyl esters were identified before in C. albida by Pacciaroni et al. [17] . Nevertheless, this is the first time the ethyl esters of Lachnophyllum and Matricaria are detected in C. bonariensis. More investigations are needed to isolate those compounds from C. bonariensis for structure elucidation. Furthermore, Matricaria and Lachnophyllum lactones were more abundant in solvent extract than essential oils. Frequently, lactones derivatives show more bioactivity than ester derivatives [18] [19] . Chloroform extraction with subsequent fractionations demonstrated in this work could raise C. bonariensis bioactivity.
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Table 2. Distribution of acetylenic compounds in different extracts of C. bonariensis.
3.4. Isolation of Two Known Acetylenic Compounds
cis-Lachnophyllum methyl ester isolation
The major component of the aerial part EO was isolated by recrystallisation in hexane. It was a white needle-like crystals that melt very rapidly at room temperature (around 28˚C). GC-MS, 1H, 13C, HMBC and HMQC NMR analyses confirmed the structure of the compound (Figure 1). At the best of our knowledge, this work is the first report of isolation of cis-Lachnophyllum methyl ester by recrystallisation.
Proton NMR spectrum showed the presence of a triplet at δ 2.36 representative of 2 H-8 protons coupled with the H-9 protons (J = 7 Hz) that appeared at δ 1.59. Coupling between H-8 and H-10 protons gave a multiplet at δ 1. More, a long-range coupling between H-8 protons and H-2 proton of the double bond is shown at δ 6.15 (J = 1 Hz). The other H-3 proton of the double bond at δ 6.19 gave a doublet with its neighbor. Their coupling constant J = 11 Hz confirmed the cis geometry of the C-2,3 double bond. Finally, the methyl of the ester appears as a singlet at δ 3.77.
On the decoupled 13C NMR spectrum (Figure S5 and Figure S6), we can easily observe C-10 methyl at δ 13.461, the two carbons C-9 and C-8 at 21.569 and 21.753 respectively. Acetylenic carbons C-7, 6, 5 and 4, were shown at δ 90.70; 86.570; 70.83; and 65.185, respectively. Furthermore, peaks of C-3 and 2 double bonds carbons were observed at δ 130.674 and 122.501, respectively. The peak δ 164.775 should be C-1 carbon. The last carbon of the acetylenic compound that is the methyl group of the ester is resolved at δ 51.593.
If from two previous spectra, the presence of multiple bonds is almost confirmed, there are still doubts regarding the long-range couplings. To elucidate these neighboring relationships, further studies were carried out in HMBC and HMQC. In HMBC spectrum, a spot at C-2 and H-8 intermediary was observed (Figure S2). This confirms the majority of multiplets obtained in 1H and 13C interpreted as arising from long distance couplings beyond seven bonds. HMQC spectra did not change this decision.
These magnetic resonance analyses confirmed the structure of the cis-Lachnophyllum methyl ester proposed in previous line. The trans isomer of this
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Figure 1. NMR spectra (a) and structure (b) of cis-Lachnophyllum methyl ester.
compound has already been isolated by Barbosa et al. [3] and analyzed by 1H NMR, they obtained almost the same peaks as in this work notably the carbons of double bond H-2 and 3 respectively at δ 6.83 and 6.32. The difference is in coupling constants of the two protons, i.e. 15.7 Hz against 11 Hz for our analyses. This difference confirms the identified double bond configurations. In the same paper, Matricaria methyl ester was isolated and the C-2 and C-3 carbons that were of cis geometry gave a coupling constant of J = 10.8, similar to our findings. Our experiment was isolated with a relatively easy method, Lachnophyllum methyl ester by recrystallization. The compound could serve as intermediary for chemical synthesis of bioactive compounds.
Lachnophyllum lactone isolation
LC-MS analysis showed that Lachnophyllum lactone was readily present in the roots of the plant. To isolate more acetylenic compounds, we performed a preparative HPLC on the diethyl ether extract that gave different fractions. Fortunately, a fraction showed to be a pure Lachnophyllum lactone as confirmed by mass spectrometry analysis and 1H, 13C analyses (Figure 2).
1H proton NMR analyses showed (400 MHz, CDCl3) δ 1.03 (t, 3H, J 5.2 Hz, CH3), 1.62 (sex, 2H, J 7.2 Hz, CH2), 2.43 (td, 2H, J 7.2, 2.4 Hz, CH2), 5.31 (t, 1H, J 2.4 Hz, CH), 6.22 (d, 1H, J 5.2 Hz, CH), 7.37 (d, 1H, J 5.2 Hz, CH); 13C NMR
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Figure 2. NMR spectra (a) and structure (b) of 4-Z Lactone Lachnophyllum.
(125 MHz, CDCl3) δ 168.9, 156.0, 142.6, 120.1, 104.6, 95.1, 74.8, 22.1, 21.8, 13.5; MS/MS (EI, 70 eV) m/z (%) 162.0 [M+] (precursor ion), 147.0 (23) [M − CH3]+, 133.0 (25) [M − C2H5]+, 119.1 (31) [M − C3H7]+, 105.1 (14) [M − C2H5 − CO]+, 91.1 (47) [M − C3H7 − CO]+, 82.0 (100) [C4H2O2]+. This compound is generally found in plants of the Asteraceae family. It was isolated in extracts of the aerial part Conyza canadensis [20] [21] ; also in the essential oil of root of Erigeron acris [22] .
3.5. Insecticidal Activity of C. bonariensis Essential Oil
After the determination of chemical composition of different extracts of C. bonariensis, we chose to test the aerial part EO with the higher yield on cowpea weevils C. maculatus. When we applied a concentration of 1 µL/L, a mortality of 56% of adults was observed after 24 hours (Figure 3). When concentrations were raised, we observed 73% mortality at 2 µL/L, 83% at 3 µL/L and 100% at 4 µL/L. To the best of our knowledge, no work reported before the insecticidal activity of C. bonariensis plant. Otherwise, some biological activities of the plant have been reported. The insecticidal activity of the plant may be due to its acetylenic compounds and limonene present in in the essential oil. [17] [23] [24] . Recently, (4Z)-Lachnophyllum lactone was demonstrated to have phytoxicity activity on Cuscuta species, obligate parasitic plants by Fernández-Aparicio. [24]
Compared to other studies on insecticidal activity of EOS against cowpea bruchid, results reported by Ilboudo et al. [25] showed that the EOs of Ocimum americanum, Hyptis suaveolens, Hyptis spicigera and Lippia multiflora with thymol were toxic to C. maculatus. Reported LD50 values were 0.23 mL/L, 1.30 mL/L, 5.53 mL/L and 6.44 mL/L, respectively. It could be concluded that C. bonariensis EO had a similar toxicity to the weevils as O. americanum and H. suaveolens, but more toxic than H. spicigera and L. multiflora. According to the work of Nyamador et al. [26] , EOs of Cymbopogon nardus and Cymbopogon giganteus acclimatized in Togo induced at the concentrations of 40 mL/L, 50%
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Figure 3. Nematicidal activity of essential oils of different parts of C. bonariensis.
and 90% mortality, respectively of C. maculatus adults after 24 hours. C. bonariensis could be classified among the best EOs against cowpea weevils. Noteworthy, C. bonariensis is an endemic not edible plant and could give cheap biopesticide.
3.6. Nematicidal Activity
EOs of different parts of Conyza bonariensis were tested on second instar (J2) juveniles of root-knot nematode of genus M. incognita. EC50 values are reported in Table 3.
EO of the whole plant was more effective than the EO of leaves and flowers. This difference would be due to variation in chemical composition of EOs of different parts. From the previous lines, we demonstrated that bioactive components were more abundant in root parts of the plant. The lack of nematicidal activity of the flowers and leave of the plant are explained by absence of those compounds. The effectiveness of the whole plant EO was probably due to the presence of acetylenic components that are the major compounds in the EO. Kimura et al. [27] reported the nematicidal activity of Matricaria and Lachnophyllum methyl esters. Those compounds present herein in the EO, could be responsible of bioactivity.
Nematicidal activity of diethyl ether extract of C. bonariensis root
To confirm our hypothesis of nematicidal activity of the detected acetylenic compounds by mass spectrometry, we tested the diethyl ether root extract (rich of acetylenic compounds) on M. incognita. The concentrations 250, 500 and 1000 mg/L were assessed. Results are reported in Figure 4.
Results showed that all tested concentrations induced nematode mortality. The LC50/72h value was estimated by probit analysis to 470 mg/mL. Chemical composition showed earlier in this work strong presence of two major acetylenic compounds including Lachnophyllum and Matricaria lactones. These two compounds that are biologically active and have several properties including nematicidal properties [27] explained the strong nematicidal activity of the plant.
Nematicidal activity of cis methyl ester Lachnophyllum
We also demonstrated the nematicidal activity of an isolated acetylenic compound. Lachnophyllum methyl ester isolated from the EO of the whole plant by recrystallisation was tested alone on J2 of M. incognita at concentrations of 75, 150, and 300 mg/L. Results are reported in Figure 5.
Results of Figure 5 revealed a high mortality rate of nematodes in contact with the different concentrations of the compound with an EC50/72h estimated to
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Table 3. Nematicidal activity of essential oils of different parts of C. bonariensis.
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Figure 4. Nematicidal activity of diethyl ether extract of C. bonariensis root.
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Figure 5. Nematicidal activity of cis-Lachnophyllum methyl ester.
78.61 mg/L. The efficacy of cis-Lachnophyllum methyl ester on nematodes was probably due to its chemical structure with conjugated triple and double bonds rising the compound bioactivity [28] . Previous studies reported several bioactivities of cis-Lachnophyllum methyl ester such as nematicidal [27] , antifungal [29] [30] and antibacterial [30] . Our work is the first report of nematicidal activity of this molecule to root-knot nematodes of Meloidogyne spp. This compound could be vaporized to control plant parasitic nematode after further studies.
4. Conclusion
The work in this study was conducted on the essential oil of the whole plant and the chloroform extract of the root of Conyza bonariensis. The chemical composition of the essential oil of C. bonariensis was determined to be more than 98%, with the major compounds Lachnophyllum cis ester (57.97%) and limonene (11.69%). The chloroform extract was partitioned and fractionated to give a 4-Z Lactone Lachnophyllum compound and by recrystallisation the cis ester Lachnophyllum was isolated from the essential oil. The structure of both compounds was determined by proton, carbon, HMQC and HMBC NMR. The neighbouring relationships confirmed the structures identified by other authors. On other hand, the essential oil of C. bonariensis has a very lightning insecticidal activity on insects as it induces 100% mortality at a dose of less than 4 µL/L during a 24 h application on Callosobruchus maculatus bruchids. The essential oil, partitioned extract and Lachnophyllum cis ester were very effective against J2 nematodes of the genus M. incognita with very high mortality rates of the different concentrations. Furthermore, Lachnophyllum cis ester and limonene also showed a synergistic effect against these nematodes. Thus, the use of plant extracts and biological molecules as biopesticides is interesting because they are natural and have little negative impact on health and the environment.
Supplements
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Figure S1. LC-MS Chromatograms of C. bonariensis aerial part hexane fraction (a) Total ion chromatogram; (b) Lachnophyllum methyl ester ion extract chromatogram; (c) Lachnophyllum lactone ion extract chromatogram.
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Figure S2. LC-MS Chromatograms of C. bonariensis aerial part diethyl fraction (a) Total ion chromatogram; (b) Lachnophyllum lactone ion extract chromatogram.
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Figure S3. LC-MS Chromatograms of C. bonariensis root hexane fraction (a) Total ion chromatogram; (b) Lachnophyllum methyl ester ion extract chromatogram; (c) Lachnophyllum lactone ion extract chromatogram.
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Figure S5. HMQC and HMBC of Lachnophyllum methyl ester.
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Figure S6. 13C NMR of Lachnophyllum methyl ester.