2.1. Extraction Yield and Phytochemical Analysis
The yield of essential oil from
B. cinerea varies between 0.080 and 0.87% according to several studies [
7]. However, the study of Guaouguaou et al. [
17] showed that an extraction that lasted 6 h gave a yield of (0.92%). The yield obtained in our study was 0.39%.
The essential oil’s GC-MS analysis revealed the presence of 21 components. The oil was distinguished by a significant concentration of oxygenated monoterpenes. (89.03%) and low content of hydrocarbon monoterpenes (10.64%). These results are in agreement with other studies: Chlif et al. [
18] reported that the components identified in the dry aerial parts of
B. cinerea were oxygenated monoterpenes (56.67%), monoterpene hydrocarbons (12.08%); in a different study, the components identified were oxygenated monoterpenes (95.40%) followed by monoterpene hydrocarbons (2.17%) [
19]. In another research, the essential oil of this species was shown to contain oxygenated monoterpenes (82.3%) and monoterpene hydrocarbons (14.5%) [
20].
Twenty one constituents representing about 99.97% of the EO were identified (
Figure 1 and
Table 1), of which the major components were thujone (24.9%), lyratyl acetate (24.32%), camphor (13.55%), and 1,8-cineole (10.81%). Comparing these results with others reported in several studies (
Table 2), it can be seen that the sample studied is distinct by its lyratyl acetate content, which represents almost a quarter of the totality. However, in the majority of cases, thujone or one of its isomers remain the major compounds, with the presence of santolina triene, 1,8-cineol, and camphor. The essential oil components of the plant might vary according on environment conditions, soil type and utilized portion, and harvest season.
2.2. Antimicrobial Activity
The findings of the EOs’ antibacterial and antifungal activities are provided in
Table 3 and
Table 4, respectively.
B. cinerea EO exerted significant inhibitory activity against all microbial strains tested compared to synthetic antibiotics (streptomycin and fluconazole).
The yeast C. albicans was very sensitive to the EO with an inhibition diameter of (42.33 mm), followed by S. aureus (31.33 mm). In addition, the EOs showed lower zones of inhibition against E. coli and B. subtills, which were, respectively, 26.33 mm and 25 mm. However, P. aeruginosa showed a little resistance to the essential oil tested compared to the other bacterial species. The mycelia strain, which appears to be very sensitive to the oil used, is F. oxysporum (88.44%), compared to the two species of Aspergillus (A. flavus (48.44%); A. niger (36.55%)).
The results obtained in the microdilution method show that P. aeruginosa was very sensitive to B. cinerea EO, inhibited by a very low dose (0.0018 mg/mL). The concentration of 0.0037 mg/mL was sufficient to stop the growth of E. coli, while B. subtilis appeared to be the least sensitive with an inhibition starting from (0.037 mg/mL). Similarly, MICs between 0.0149 and 0.06 mg/mL were sufficient to arrest the growth of mycelia strains. In general, among all the strains studied, the two gram-negative bacteria (E. coli and P. aeruginosa) remained the most sensitive to essential oils extracted from B. cinerea. The present study reveals that essential oils extracted from the aerial parts of B. cinerea have the potential to be an antimicrobial agent against several pathogenic microorganisms.
Previous studies have been carried out on the antimicrobial effect of the essential oil of
B. cinerea, or one of its major compounds, against strains belonging to the same genera as those examined in this study. Ghouti et al. [
22] revealed that
B. cinerea oil limits the development of
B. subtilis by a minimal concentration of (MIC = 0.303 mg/mL). Similarly, the growth of
B. subtilis,
E. coli, and
S. aureus was stopped at 1/500
v/
v concentration [
23]. In addition, Chouikh [
24] found that the strains of
E. coli and
S. aureus showed great sensitivity to the concentrations (1/1, 1/2, 1/4, 1/8) of EO (flowering period) where the diameter of inhibition ranged between (21 mm to 50 mm), while
P. aeruginosa showed stiff resistance with every concentration of essential oil.
The effect of the essential oil of
B. cinerea also inhibits the growth of mycelial fungi. Reference [
25] reported that the oil of this plant inhibits the growth of
A. niger (50.80%) at a concentration of 1/250
v/
v, but complete inhibition of its growth requires a concentration of 1/100
v/
v. Similarly, Boussoula et al. [
23] showed that oil stops the growth of
A. niger at 1/250
v/
v. It was shown in another study that the activity of essential oils of
B. cinerea is high against a group of human pathogenic fungi, including
Candida albicans, with minimum inhibitory concentrations of 3.2 to 4.7 mg/mL [
26].
According to these observations, it can be speculated that the strong antimicrobial activity exerted by the EOs of
B. cinerea can be attributed only to the major compounds (thujone, lyratyl acetate, camphor, and 1,8-cineole), which account for about 73.58% of its total composition, and it could be the result of synergies between the different constituents of the EO [
27]. Thujone (major compound) or one of its isomers (A or B) have been reported as molecule inhibiting the growth of several microbial strains including (
S. aureus,
P. aeruginosa,
C. albicans, E. coli,
A. niger) [
28,
29,
30,
31].
The percentage of Lyratyl acetate in the studied essential oils is very significant (24.04%). Casiglia [
32] showed that the essential oils of
Anthemis secundiramea, containing (Z)-lyratyl acetate (14.6%) as a major compound, exhibits significant antibacterial action against a variety of pathogens, with very low MIC values (μg/ mL) (
B. subtilis 12.5;
S. aureus 25;
E. coli 50;
P. aerogenosa 100;
C. albicans 50;
F. oxysporum 12.5, and
A. niger 6.25). Other EOs, containing (Z)-lyratyl acetate as the majority compound, inhibited the growth of
A. flavus (74.4%) by a concentration of 20 μL/petri dish for 6 days) [
33].
The antimicrobial activity of camphor depends on its synergistic action with 1,8-cineole or other compounds existing in EOs [
34,
35]. Silver nanoparticles produced greenly from camphor extract, on the other hand, demonstrated their potential to favorably suppress isolated bacterial pathogens of both American and European foulbrood [
36].
1,8-cineole inhibited the growth of
A. niger (52.3 ± 43%) and
F. oxysporum (72.2 ± 2.6%) by a concentration of 0.918 mg/mL. In addition, its presence in the essential oil in large quantities generates a very remarkable antimicrobial power [
37]. Wagner et al. [
38], showed that the EOs of
Lavandula dentata, containing eucalyptol (or 1,8-cineole) (34.33%), fenchone (17.78%), and camphor (15.75%), had significant fungicidal action against
Cercospora kikuchii,
Cercospora sojina, and
Septoria glycines plant pathogenic fungi, with diameters of the inhibition zone of 34.00, 29.50, and 22.00 mm at a concentration of 5 μL/disc, respectively.
According to the results of later studies, it is assumed that the strong antimicrobial activity of the
B. cinerea EOs is strongly due to the presence of lyratyl acetate, 1,8-cineole, and monoterpene alcohols (
p-Mentha-1(7),8(10)-dien-9-ol(5.62%), 4-Terpineol (2.02%), (-) Borneol (1.52%); α-terpineol (1.67%)). Indeed, Dorman et al. [
39] tested a large number of pure constituents of EOs against 25 different genera of bacteria and showed that thymol is the compound with the broadest spectrum of antibacterial activity followed by carvacrol and α-terpineol.
The EO’s antibacterial action was thought to include simultaneous cytomembrane breakdown, which resulted in the release of intracellular constituents including protein and K
+ [
40]. As for their mode of action, EOs cause cell wall damage by establishing a membrane potential across the cell wall and disrupting ATP assembly [
41]. However, the inhibitory power of the molecules remains inferior to that exerted by EOs containing these molecules as majority compounds or in large quantities [
40]; this boosted effect can be explained by the synergic effect of the molecules contained in the EO.
2.3. Insecticidal Activity
Figure 2 and
Figure 3 present the results of the action of
B. cinerea oils tested by inhalation and by contact on the mortality of
C. maculatus. Indeed, at the lowest concentration (1 μL/L of air) tested by inhalation, and after 24 h of exposure, the essential oils of
B. cinerea cause a mortality of 63.33 ± 15.28% of the adults of
C. maculatus, while the toxicity by contact induces 80 ± 10% mortality. At the highest concentration (20 μL/100 g),
B. cinerea EOs showed significantly higher action compared to the control and caused 100% mortality in both tests, respectively. Statistical analysis (
Table 5) shows that the LC50 value in the inhalation test (0.72 μL/L of air) is higher than that observed in the contact test (0.61 μL/L of air).
Table 6 shows that the EO of
B. cinerea (EOBC) caused a significant reduction in fecundity. Thus, the application of the low concentration (1 μL/100 g) resulted in an oviposition reduction rate of 95.98% when compared to the control group. When applying a concentration of (20 μL/100 g), the rate of egg laying reduction is 100%. The number of eggs laid per female
C. maculatus in the control jar is 207.33 ± 12.5. For emergence, a significant reduction rate of 91.34% was observed using (20 μL/100 g).
Figure 2.
The effects of inhaling essential oil of B. cinerea (EOBC) on the mortality of adults of C. maculatus.
Figure 2.
The effects of inhaling essential oil of B. cinerea (EOBC) on the mortality of adults of C. maculatus.
EOBC: Essential oil of B. cinerea
Figure 3.
The effects of B. cinerea essential oil on the mortality of adults of C. maculatus by contact.
Figure 3.
The effects of B. cinerea essential oil on the mortality of adults of C. maculatus by contact.
EOBC: Essential oil of B. cinerea
Table 6.
Effects of B. cinerea EOs on fecundity and adult emergence of C. maculatus.
Table 6.
Effects of B. cinerea EOs on fecundity and adult emergence of C. maculatus.
Dosage (μm/L) | Egg Laying Reduction Rate (%) | Adult Inhibition Rate (% IR) |
---|
Control | 0 a | 0 a |
1 | 95.9b a | 100 b |
5 | 98.87 c | 100 b |
10 | 100 c | 100 b |
20 | 100 c | 100 b |
2.4. Cowpea Weevil Insecticidal Activity
The results presented in
Table 7 show that the
B. cinerea EOs present a medium repulsive effect on the adults of
C. maculatus with an average repulsion of 38.33 ± 11.38%, classifying them, respectively, in class III according to the classification of McDonald et al. [
42].
Using the contact and inhalation test, the total mortality was very important; the LC50 was 0.62 and 0.73 µL/L, respectively. In the bibliography, several works have been conducted on the insecticidal activity of EOs or certain volatile compounds against C. maculatus, all using several protocols, inhalation, fumigation, contact, and repulsion.
Some EOs contain majority compounds resembling those obtained in the
B. cinerea EOs used in the present study. Using the fumigation technique, the EOs of
Rosmarinus officinalis L., composed mainly of α-pinene (22.64%), camphor (21.84%), 1,8-cineole (21.53%), and camphene (9.18%), show an insecticidal effect against
C. maculatus with an LC
50 = 15.69 µL/L air [
43]. In a recent study [
44], it was reported that the EOs of
Salvia officinalis, containing α-thuyone (24.27%), camphor (18.10%), 1–8 cineol (14.38%), and β thujone (7.38%) as major components, were toxic against
C. maculatus, while batches treated with 16 μL/L of EOs air resulted in 33.5% of eggs hatched, while 24% evolved into larvae and 19.5% was the number of emergences. It was also shown previously that EOs isolated from
Artemisia herba-alba had an LD
50 of 11.670% against the insect tested using the contact test, the major compounds of these oils were chrysanthenone (31.40%), camphor (15.97%), alpha-thuyone (14.90%), the 1,8-cineole (4.57%), and camphene (3.95%) [
45].
EOs containing high amounts of certain volatile compounds have been used to control the insect pest
C. maculatus. The oil extracted from
E. caryophyllus (74.31% of eugenol), has an LC
50 value of 1.27 μL/20 g obtained by contact test, and an LC
50 of 20.27 μL/L air obtained by fumigation test [
46]. Similarly, the EOs of
Syzygium aromaticum L. (87.4% of eugenol) and
Cinnamomum zeylanicum L. (73.1% of eugenol), respectively, have an LD
50 of 78.2 and 131 μL/Kg [
47]. In contrast, the use of EOs of
I. verum (88.85% of E-anethole) against
C. maculatus showed an LC
50 = of 9.62 μL/20 g by contact test and an LC
50 of 22.36 μL/L of air by fumigation test [
46].
Previous studies have also reported the insecticidal effect of a few molecules against
C. maculatus. The fumigant effect of α-bisabolol on
C. maculatus oviposition was five eggs at a concentration of LC
50 = 2.47 μL L
−1 compared to 79.25 eggs at hexane application [
48], while the use of citral (geranial +Neral) at a concentration of 90 μg/mL leads to a mortality of (30.00%), oviposition (55.71%), and emergence(1.51%) [
49].