Chemical compositions, Fumigant and Repellent Activities, of Essential oils from three Indigenous medicinal plants and their mixture, against stored grain pest, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

 

Naima Boukraa^1,2*, Segni Ladjel^1,3, Mohamed Bilal Goudjil^1,3, Amar Eddoud^4,5,
Kifah Waleed Mustafa Sanori⁶

¹Kasdi Merbah University, Faculty of Applied Sciences, Process Engineering Laboratory, Ouargla, Algeria.
²Kasdi Merbah University, Faculty of Natural Sciences and Life Sciences, Biological Sciences Department, Ouargla, Algeria.

³Kasdi Merbah University, Faculty of Applied Sciences, Department of Process Engineering,

Ouargla 30000, Algeria.

⁴Kasdi Merbah University, Faculty of Natural Sciences and Life Sciences, Department of Agronomic Sciences, Ouargla, Algeria.

⁵Kasdi Merbah University, Faculty of Natural Sciences and Life Sciences, Saharan bio-resources laboratory, Ouargla, Algeria.

⁶Universiti Sains Malysia, School of Languages Literacies and Translation, Penang, Malaysia.

 

ABSTRACT:

The chemical composition of the essential oils (EOs) of Artemisia herba-alba Asso, Rosmarinus officinalis L and Juniperus pheonicea L and its fumigant and repellent activities against Tribolium castaneum (Herbst) were investigated as well as their mixture. Dried leaves and flowers were subjected to hydrodistillation using a Clevenger-type apparatus and the chemical composition of the volatile oils was studied by gas chromatography-mass spectrometry (GC-MS). A total of 117 components were identified from the three EOs and their mixture. The main compounds identified for EOs of A. herba alba, R. officinalis, J. pheonicea and the mixture were Car-3-en-5-one (12.51%), Camphor (34.37%), α-Pinene (15.49%), and camphor (13.95%), respectively. it Interestingly we discovered that the EO extracted from A. herba alba from Khenchela had a new chemotype with the appearance of new compounds in the mixture of EOs. Results of fumigant toxicity showed that after 24 h exposure time, all EOs had pronounced toxicity against T. castaneum, with LC₅₀ value of 107.199, 155.724 and 156.589μl/l air for A. herba-alba, J. pheonicea and R. officinalis, respectively, while the LC50 of the mixture was 131.570µl/l air. However, at the concentration 2µl and after 30 min exposure time, the EOs of A. herba alba and J. phoenicea showed significantly more repellent effects up to 100.00±0.00 and 90.00±10.00%, respectively, followed by the mixture and the R. officinalis EO with the percentages of repulsion equal to 76.67±11.55 and 41.67±10.41% . Data analysis by CompuSyn showed that the synergy, antagonism or additive effect of the EOs in both tests depend on the concentration of the bioinsecticides and the exposure time. The results indicated that the tested EOs and their mixture had the potential to be developed as bioinsecticides and repellent for the control of T. castaneum adults.

 

 

 

INTRODUCTION:

Stored-grain insect pests damage agricultural stored products in granaries as well as storehouses and account worldwide loss reaches up to 10-40 % annually^1,2,3. The continuous increasing pressure of human population expansion has also created a critical problem of food scarcity¹. In addition, the red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is one of the major pests of stored product in tropical and semitropical areas^4,5,6. The secreta of T. castaneum can cause: flour agglomeration, change in the color and as a result it goes bad. added to that, the secreta contains the benzoquinone which is a kind of carcinogenic substance. It has the characteristics of rapid reproduction, strong adaptation, and worldwide distribution. This pest causes extensive  loss of wide range of commodities including grain, flour, peas, beans, cacao, nuts, dried fruits, and spices, but milled grain products like flour appear to be their preferred food^4,6,7,8,9. In such situation, protection  for stored grain and agricultural products against insect infestation is  a must¹⁰. Synthetic chemical insecticides and fumigants are commonly used to control pestiferous insects throughout the world⁶. However; the use of synthetic insecticides and fumigants raised concerns which include chronic poisoning of applicators, farmworkers, and even consumers; development of resistance and resurgence in pestiferous insect populations, adverse effects on the environment such as ozone depletion and pollution.^5,11,12. Therefore, it is an utmost priority to search for alternatives insecticides, which are deem environmentally safe, biodegradable and specific against these pests³. Amongst such plant-derived products, the use of essential oils (EOs) extracted from aromatic plants to control these pests has been investigated and is well documented, since last two decades^14,3. EOs are a complex mixture of volatile compounds produced as secondary metabolites¹⁵. Some studies have assessed the ability of the EOs and their constituents as fumigants and repellents against a number of insect pests^2,14, while some formulations of EOs are in use as bioinsecticides ¹, whereby  having the  advantages over conventional fumigants in terms of low mammalian toxicity, easy biodegradability and local availability^5,6,4.

 

The present study was carried for to identification of chemical constituents as well as  determining the  fumigant and repellent  activities of the EOs of Artemisia herba-alba Asso (Asteraceae), Rosmarinus officinalis L (Lamiaceae), and Juniperus phoenicea L (Cupressaceae) against the red flour beetle T. castaneum (Herbst), alone and together, for evaluating their synergistic, antagonistic or  additive effects and the possible interactions between chemical constituents of these oils. The selection of species was made on the basis of their abundance and availability in Algeria.

 

MATERIAL AND METHODS:

Plant material:

Leaves and flowers of R. officinalis L, J. phoenicea L and A. herba Alba Asso were harvested during September 2017 from the region of Khenchela in East of Algeria. Taxonomic authentication was performed by Plant taxonomists in the Department of Agronomy at Kasdi Merbah University, Ouargla, Algeria. Collected samples were dried in the shade, at room temperature (20-25°C) under good ventilation after that woody stems were separated.

 

Extraction of EOs:

EOs were obtained by steam distillation.100g of air-dried plant material of each individual species was put into water (1:10 w/v plant material/water volume ratio) for 3 hours, using a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia¹¹.  Anhydrous sodium sulphate was used to remove water after extraction. The resulting EOs were kept in separate glass flasks at 4°C in dark condition for further analysis. The EO contents (%) were expressed as volume of EO vs weight of fresh leaves (v/w).

 

Rearing of test insects:

Red flour beetles (T. castaneum Herbst) were collected from infested grains purchased from local market and brought to the laboratory; and its identity was confirmed by Pr. Sekour Makhlouf, Department of agronomy, faculty of Natural Sciences and Life Sciences, Kasdi Merbah University, Ouargla, Algeria.

 

Insect laboratory culture were established in glass containers containing a mixture of disinfected wheat flour and beer yeast at a ratio of 10:1(w: w), under controlled temperature 29±1°C and 70–8% relative humidity in darkness following procedures by Liang et al. (2017)⁶. Adult unsexed insects, 7-14 days old, were used for subsequent experiments.

 

Gas Chromatography mass spectrometry (GCMS):

This step was carried out at Process Engineering Laboratory, Kasdi Merbah University Ouargla, to qualify and quantify the chemical composition of the samples. The chromatographic analysis of the EOs and their mixture was performed with a typical gas chromatograph (Bruker SCION 436 GC) coupled to a mass spectrometer. Fragmentation is performed by electronic impact at 70 eV. The column used is a HP-5MS capillary column (15m x 0.25mm). The film thickness is 0.25μm. The stationary phase of the column consists of: 5% Phenyl and 95% dimethylpolysiloxane.

 

Operating conditions are indicated as the following:

Injector temperature (1:50 split mode): 250°C

Temperature programming: 70°C to 280°C at 10°C/min;

The vector gas used is Helium with a flow rate of 1.5 ml/min.

 

The temperatures of the quadripole source are set at 250°C and 220°C, respectively. The linear retention indices (RI) for all compounds were determined using nalkanes as standards. The identification of different constituents was achieved by comparing their mass spectra with those of the reference products contained in available computerized libraries (NIST and Wiley 2014).

 

Fumigant effect:

The EOs and their mixture were tested for their fumigant activity on mortality of T. castaneum adults, following the method of Papachristos et Stamopoulos (2002)¹⁶ with slight modifications. Glass jars of 60 mL capacity with screwed plastic caps were used as exposure chambers.  Twenty 7-14-day-old T. castaneum adults were placed at the bottom of each container to expose them to the various concentrations of fumigants. A small piece of cotton was attached to the underside of the screw cap to serve as a diffuser, on which varying doses (2.5, 5, 10 and 20 µl) of fumigants were applied to achieve final concentrations of 44.66, 83.33, 166.66 and 333.33µl/l of air respectively, with respect to  the volume of the jars, while the control diffuser was left untreated. Thereafter, the jars were kept in the ambient conditions of temperature and humidity. Insect mortalities were determined and calculated after 24, 48, 72 and 96 hours of exposure time.  Test insects were considered dead when no leg or antennal movements were observed. Three replicates were performed for each dose and control.

 

Repellent effect:

Repellency effects of the EOs against T. castaneum were assessed by the area preference method described by Jilani and Saxena. (1990)¹⁷. Range-finding studies were run to determine the appropriate testing concentrations. The solutions of EOs were prepared by dissolving 0.25, 0.5, 01 and 2µl of plant EO in 0.5 ml acetone. Acetone alone was used as the negative control.

 

Whatman filter papers (diameter 9cm) were cut into two equal halves and 0.5ml of each concentration was applied separately on one half of the filter paper as uniformly as possible by using a micropipette. The other half of the filter paper was treated with an equal volume of acetone as a control. The treated and control paper disc were air-dried to remove the solvent. Both treated and untreated sides were attached to their opposites with cellophane tape and placed at the bottom in glass Petri dishes (9cm in diameter). Twenty unsexed adults of T. castaneum were released at the center of each filter paper disc and the lid was sealed with Parafilm®. Three replicates set for each concentration, and insects were used only once. Counts of the insects present on each strip were made 30 min from the beginning of experiment.  Percentage Repellency (PR) of each volatile oil and mixture was calculated using the following formula¹⁸;

 

PR = Nc – Nt / [(Nc + Nt)] × 100 Nc

 

Where, Nc is the number of insects present in the negative control half paper after the exposure interval and Nt is the number of insects present in the treated one after the exposure interval.

 

The averages were then assigned to different classes (0 to V) using the following scale (percentage repellency). Class, % repellency: 0, >0.01 to 0.1; I, 0.1–20.0; II, 20.1–40.0; III, 40.1–60.0; IV, 60.1–80.0; and V, 80.1–100¹⁹.

 

Data analysis:

The statistical analysis was performed using the SPSS software (version 24). The means were compared by the ANOVA test, followed by a multiple comparison using the Tukey test. The value of P > 0.05 were considered significant 20. The concentration-mortality data were analyzed by Probit analysis 21, to estimate lethal concentration values LC₅₀ using the same software. The effect of the combination of the three EOs was evaluated using CompuSyn® software version 1.0 22, which allows the calculation of the combination index (IC). The choice of this method is based on its sensitivity for the categorization of qualitative analysis of interactions between substances. The effects of the combination of EOs were classified according to IC values based on the categories proposed by 23: synergy (IC - < 0.70); moderate synergy (IC 0.70 to 0.90); additive (IC 0.90 to 1.10); moderate antagonism (IC 1,10–1,45) and antagonism (IC > 1,45).

 

RESULTS AND DISCUSSIONS:

Chemical composition:

The yields of yellow EOs from R. officinalis, A. herba alba and J. phoenicea were 0.4, 0.88 and 0.99 % (v/w) respectively. The composition of the EOs and their mixture have been summarized in (Table 1). Total of 117 constituents were identified for the three EOs and their mixture (accounting for 88,29-94.09% of the samples), It was found that EO from A. herba alba from Khenchela had a new chemotype with Car-3-en-5-one (12.51%) as  major constituent, followed by Camphor (12.18%) and 1-(2-Ethyl-3-cyclohexenyl) ethanol (8.08%). These results are compared with others obtained in Algeria, which indicate that EO of A herba alba harvested from Tamanrasset had Davanon chemotype²⁴. while, EO from Saida and Djelfa had camphor chemotype^25,26. Furthermore, Badreddine and Baouindi (2016)²⁷; Qnais et al. (2016)²⁸; Sharifian et al. (2012)¹⁴  reported that EO of A. herba alba from Tunisia, Jordan and Iran respectively, had β-thujone chemotype; while the EO from Morocco had α-terpineol chemotype²⁹. Moreover, EO of R. officinalis was composed of Camphor (34.37%) as major constituent, along with eucalyptol (14.46%) and α-pinene (12.94%). All these compounds were previously detected as major constituent in EOs of             R. officinalis from Tunisia 30, Spain 31 and even from China 32, with different proportions.

 

Table 01: Retention time (RT), chemical constituents of EOs extracted from A. herba alba, J. phoenicea, R. officinalis and their mixture.

RT: Retention Time; A: A. herba alba; B: J. pheonicea; C: R. officinalis; D: Mixture of Eos

 

Table 2:  Result of Probit analysis to calculate LC₅₀ values of EOs extracted from A. herba alba, J. phoenicea, R. officinalis and their mixture against T. castaneum after 24, 48, 72 and 96 h exposure time respectively.

 

Major compounds of J. phoenicea L EOs were α-Pinene (15.49%), α-Terpinyl acetate (9.54%) and 3-Carene (9.63%) , these findings conflicted with those obtained by Ramdani et al. (2013) ³³,  where terpinolene (0-13%), Δ3-Karen (0-12.4%) and the β-phellandrene (0-7.3%) were identified as prominent components of  J. phoenicea EOs belonging to different regions in Algeria. Moreover, α–pinene was detected as the main constituent in EOs from different origins worldwide ^34,35,36,37. These differences in the EOs compositions might arise from differences in environmental (climatic, seasonal, geographical) factors ¹¹. The chemical composition of the mixture of the three EOs was different compared to the chemical composition of the EOs, with the appearance of new compounds such as 4-Thujanol as a second main constituent (9.02%), while camphor (13.95%), and α-Pinene (8.36%) are the first and the third main compounds, respectively. In the same sample, it was observed that there was an increase in the total constituents’ number, which may result from the interactions between the constituents of the three EOs.

 

Fumigant and Repellent activities of EOs:

It was found that the EOs of all selected plant species and their mixture had pronounced fumigant activities against T. castaneum adults (Table 2). Toxicity data indicate a remarkable difference in susceptibility of this stored pest to treatment by EOs and their mixture in function of the concentrations of the samples, plant species and exposure time. No mortality was noted in the control.  Probit analysis showed that at a 24 h exposure time, A. herba-alba was highly toxic toward T. castaneum adults with LC₅₀ value of 107.199 µl/L air. Whereas, the mixture of EOs possessed fumigant activity with LC₅₀ value of 131.570 µl/L air.  On the other side, EOs of J. phoenicea and R. officinalis were less efficacious, with LC₅₀ values of 155.724 and 156.589µl/L, respectively.

 

Generally, mortality rates increased and LC₅₀ values, decreased when the EOs concentration and exposure time increased. After 96h of treatment the corresponding LC₅₀ values were 66.455, 74.389, 97.833 and 109.127 μl/l air for R. officinalis, A. herba alba, J. phoenicea and the mixture of EOs respectively.

 

The results obtained from the CompuSyn analysis (Table 03), showed that there was a synergistic effect between the three EOs at the concentration of 41.67 µl/l after 24 hours. while, moderate synergism was observed at the concentration of 83.33 µl/l after 24 hours. However; a slight synergistic effect was recorded at the highest and lowest concentrations after 24 and 48 hours, respectively.

 

Table 03: Effect of combination of EOs of A. herba alba, J. phoenicea, R. officinalis against T. castaneum at different concentrations after 24, 48, 72 and 96 h exposure time.

*Within each sample (EOs or mixture), means in the same column followed by the same letters do not differ significantly (P > 0.05) in the Tukey test.

**Combination Index (CI) - < 0.70 = synergism; 0.70–0.90 = moderate synergism; 0.90–1.10 = additive; 1.10–1.45 = moderate antagonism; > 1.45 = antagonism. a Percent control obtained according to²³.

In contrast, an additive effect was observed at the concentration of 166.67µl/l after 24 hours of treatment. With increasing time, a slight antagonism was noted at the concentrations 83.33 and 333.33µl/l after 48 hours and 333.33 after 72 hours. Then, moderate antagonism was registered at the concentration 41.67µl/l and 83.33 µl/l after 96 hours. Finally, antagonist effect was observed at the concentrations 166.67µl/l after 72 hours and 41.67, 83.33 and 166.67µl/l after 96 hours.

 

Our results indicate that the nature of the effect was influenced by the concentration of the mixture used in the treatment, and the time of exposure to that treatment. Comparing the effects recorded for all concentrations between the first and the last day of treatment confirms that the potency of the EOs alone increased over time; in contrast to the potency of the mixture of EOs, which decreased after 96 h of treatment.

 

The results revealed that the EOs of A. herba alba, J. phoenicea and R. officinalis and their mixture were repulsive against adults of T. castaneum (Table 4). The EO of A. herba alba showed the most repulsive activity with a repulsion percentage of 80.00±10.00% (class V), at the lowest test concentration of 0.25µl/l against T. castaneum after 2 h of exposure, followed by the mixture of EOs which had a repulsion equal to 61.67 ± 31.75 % (class IV), whereas for the same concentration, the results indicated that the EO of J. phoenicea had an approximate repulsive activity with the EO of R. officinalis against the same insect with a repulsion percentage equal to 23.33±2.89 and 25.00±8.66% respectively, (Class II). At the highest concentration (2µl) the EOs of A. herba alba and J. phoenicea showed significantly more repellent effects up to 100.00 ± 0.00 and 90.00±10.00%, respectively (class V). Less significant repellent effects were noted for the mixture and the R. officinalis EO, thus recorded percentages of repulsion equal to 76.67±11.55 (Class IV) and 41.67± 10.41 (Class III) respectively. Based on the data of the repellent action against T. castaneum, adults were highly dependent on the sample concentration, an increase in repulsion was noted with the increase of concentrations.

 

Analysis of the Compusyn repulsive effect data has awakened a synergy between the three EOs at the concentration of 0.25µl, while a low synergy was observed at the concentration of 0.5µl. Moreover, moderate antagonism was stated at concentrations 1 and 2µl.

 

Under this study, Sharifian et al. (2012)¹⁴ reported that A. herba-alba oil might have potential as a control agent against T. castaneum, C. maculatus, and R. dominica. Similar to this study, Badreddine and Baouindi. (2016)²⁷ assessed the fumigant and repellent effects of EO from the same plant against Trogoderma granarium and T. castaneum adults. Several investigations showed the effectiveness of EO from Artemisia species which include A. maritima Linn, A. anethoides, and A. dubia as an insecticidal and repellent agent against T. castaneum adults^2,6,38. However, studies by Hashemi  and Rostaefar (2014)³⁹, showed that EO from J. communis possesses a potential for the management of T. castaneum and Rhyzopertha dominica. On the other hand, Sener et al. (2009)⁴ motioned that rosemary EO could be recommended as a potential source of environment-friendly botanical insecticide in the control of the confused flour beetle. Besides, EO of R. officinalis exhibited fumigant toxicity on T. castaneum, Sitophilus granarius, Callosobruchus maculatus, and Plodia interpunctella adults⁸.

 

In addition, some compounds identified in the tested EOs also demonstrated bioactivities against many insects. Chaubey (2012)³ reported that α-pinene and β-caryophyllene, had high fumigant toxicity against T. castaneum adults and larvae. 1,8-cineole screened from rosemary and common sage was the most potent fumigant toward confused flour beetle, followed by β-thujone from common sage and p-cymene from thyme⁴. The compounds α-pinene and D-limonene reached the same level (Class V) of repellency as N, N-diethyl-3-methylbenzamide (DEET) against L. bostrychophila at 63.17nL/cm² after 2 h treatment. α-pinene and D-limonene were also found to have contact and repellent activities against T. castaneum and L. bostrychophila¹³.

 

Table 04: Repellent effects of essentials oils from A. herba alba, J. phoenicea, R. officinalis and their Mixed formulation against T. castaneum adults.

* Within each sample (EOs or mixture), means in the same column followed by the same letters do not differ significantly (P > 0.05) in the ANOVA test.

 

Previously Guo et al. (2015)⁴⁰ reported that Estragole, 1,8-cineole, and limonene showed weaker repellent activity than the EO of Etlingera yunnanensis rhizomes against L. bostrychophila adults. However, it has been suggested that this occurs since plants usually present defenses as a suite of compounds, not as individual ones. Accordingly, the insecticidal effects of the EOs and their mixture cannot be explained by the action of their major components only, even minor compounds may be involved in the insecticidal activities of the corresponding oils and may have a synergistic effect enhancing the effectiveness of the major constituents through a variety of mechanisms^41,18,1,13. To the authors’ knowledge, this is the first experiment evaluating the contribution of the combined effect of EOs from R. officinalis, A. herba alba and J. phoenicea in the insect fumigation and repellency against T. castaneum. However; several previous experiments on insect toxicity are consistent with our results showing that the combined effect of bioactive substances on insects is synergistic, additive or antagonistic depending on the substances and the insect species. Bedini et al. (2016) and Zibaee and Khorram (2015)^42,43 stated that Eucalyptus globulus and R. officinalis EOs were toxic to Periplaneta americana (L.), Blattella germanica (L.), Supella longipalpa, Culex pipiens, Anopheles stephensi and Musca domestica adults, their mixed formulation showed enhancement in the adulticidal activity against the same insects. Such activity has been reported in EOs from sesame, Amyris, sandalwood, Helichrysum, cedar wood, and black pepper that exhibited synergistic effects in Aedes aegypti (Diptera: Culicidae)⁴⁴. Similarly, Chaubey (2012)³ Found that α-Pinene and β-Caryophylene in binary combination shows synergism, reduces the egg-laying capacity, inhibits pupation and adult emergence in T. castaneum., The same was observed in a study made by Araújo et al. (2016)⁴⁵, that reported synergistic interactions in all combinations (1:1) between carvacrol, thymol and eugenol against Rhipicephalus microplus larvae, while synergistic interactions and moderate synergistic effects were noted for R. sanguineus s.l. larvae²³ observed a synergistic or additive effect for combinations of thymol and carvacrol, depending on the concentration, against D. nitens larvae, while for A. sculptum larvae they observed a moderate synergistic or additive effect. However, the combinations of thymol with (E)-cinnamaldehyde and carvacrol with (E)-cinnamaldehyde presented an antagonistic effect at all concentrations of each substance, against both tick species and only D. nitens, respectively. Recently, Novato et al. (2019)²⁰ demonstrated that the combinations of thymol + carvacrol (3.125mg/mL), as well as carvacrol + eugenol and eugenol + thymol (6.25 mg/mL), had a synergistic effect on engorged females, while the other associations had an additive effect.

 

These findings indicate that different species can respond distinctly to the association of EOs or their components, so that an effect found against one organism cannot be extrapolated to others²³. Besides this, factors such as concentrations, the testing methods, route of penetration and source of the EOs can also interfere. Therefore, various aspects must be considered when investigating the interactions of EOs compounds^46,20.

 

Several studies about the modes of toxic action of EOs indicated that the insecticidal activity of EOs and their components can affect different and specific targets causing inhibition of many biological mechanisms⁴⁸. Zarrad et al. (2015)⁴¹ found that EOs of Citrus aurantium and limonene showed a high Acetylcholinesterase (AChE) inhibition rate of Bemisia tabaci. Similarly, Olmedo et al. (2015)⁴⁸ revealed that the toxicity of Tagetes flifolia EO and its major compounds (E)-anethole and estragole against T. castaneum, might be associated with inhibition of AChE activity. The authors also revealed that EO from T. flifolia and (E)-anethole exhibited oxidative stress, thus altering the level of malondialdehyde (MDA). Other studies suggested that the phenylpropanoids could act also by binding to the octopamine receptors. Enan (2001)⁴⁶ demonstrated that Eugenol, α-terpineol and cinnamic alcohol accelerate heartbeat and increase cAMP production in the nervous system of Periplaneta americana, also, similar changes were observed following treatment with octopamine. Earlier, several reports described the impact of EO and their compounds on biological parameters such as fecundity, fertility. Fatiha et al. (2014)⁴⁷ as well as Douiri et al. (2014)⁴⁹ showed the effectiveness of the various oil treatments, including A. herba-alba Asso and R. officinalis EOs, on Callosobruchus chinensis and C. maculates, respectively. They reported that the effect of the oils reduced egg laying and egg hatching. Regarding EO mixture, Faraone et al. (2015)⁵⁰ suggested that synergism and antagonism between EOs, is contingent on the chemical compositions, properties or mode of action of these bioinsecticides. In addition to the chemical composition, it was concluded that the interaction between different compounds may lead to changes in the structural conformation, and it may result in the reduction of the biological activity giving hence an antagonistic effect⁵¹. An alternative explanation may be that the antagonistic relationship between EOs may result from competition for a possible primary target⁵¹. Conversely, the observed synergism may be the result of the insect being overwhelmed by the mixture of EOs that attack different target sites⁵⁰. This phenomenon of multi-target synergistic effect may occur, involving enzymes, substrates, metabolites and proteins, receptors, ion channels, transport proteins, ribosomes, DNA/RNA and physicochemical mechanisms⁵¹. This highlights the need for a better understanding of synergistic and antagonistic effects in a holistic approach.

 

EOs and their mixture could be efficient alternatives to conventional insecticides because of their fast degradability properties, regional availability and act at low doses^8,49, but their natural origin does not imply that they are safe and non-phytotoxic in nature. Thus, to develop a practical application for the EOs and the combined formulation as novel insecticides, further researches on the safety and effectiveness of the EO for humans and treated seeds (germination and seedling growth) are needed^1,2,41,47. However, considering their high volatility, they are not recommended for long-term protection of stored products, hence the need to develop methods of stabilization⁴⁹.

 

CONCLUSION:

GCMS analysis of this study revealed that EO of A. herba alba from Khenchela is of a new chemotype; Car-3-en-5-one. In addition, the mixture of three EOs revealed the appearance of new constituents as major and minor compounds, which necessitates further investigations about the modes of interactions between constituents of EOs. Our results demonstrated that the EOs of A. herba alba, J. phoenicea, R. officinalis and their mixed formulation had pronounced fumigant and repellent effect against T. castaneum adults. However, A. herba alba EOs was more toxic and repellent to the red flour beetle than the other bioinsecticides. About bioactivity of the mixed formulation, it was concluded that the synergy, antagonism or additive effect of the EOs in the fumigant and repellent activities depend on their concentration and exposure time. The present findings may be utilized for the development of botanical insecticides as supplementary and replacement to synthetic ones.

 

ACKNOWLEDGEMENT:

We thank Professor Sekour Makhlouf member of the department of agronomy, faculty of Natural Sciences and Life Sciences, Kasdi Merbah University, Ouargla, for his assistance.  

 

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Received on 29.07.2020                    Modified on 19.08.2020

Accepted on 12.09.2020                   ©AJRC All right reserved