Pesticidal potential of some wild plant essential oils against grain pests Tribolium castaneum (Herbst, 1797) and Aspergillus flavus (Link, 1809)

2.1 Plant material

Fresh aerial parts of Chenopodium ambrosioides L., Conyza sumatrensis (Retz.) Erigeron canadensis L. and Tagetes minuta L. were collected from two different areas of Abbottabad, Pakistan (Table 1). The plant samples were identified by Dr. Abdul Nazir and voucher specimens were submitted to the herbarium of the Environmental Sciences Department, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan. Plant material was maintained at −20 °C until essential oil extraction.

2.2 Essential oil extraction

Steam distillation was used to extract essential oils from the plant material, as described earlier (Azeem et al., 2019). Briefly, fresh aerial parts were chopped with a knife and 1,500 g was subjected to steam distillation in a stainless-steel distillation apparatus. Distilled water was added to the distillation vessel that did not have direct contact with the plant material which was packed in a meshed container fitted in the vessel above water level. The vessel was heated on an electrical heating mantle and the distillate containing the plant’s volatile compounds, along with condensed steam, was collected in a separating funnel for 4 h. The layer of essential oil at the top of the water was separated through decantation. The remaining distillate was further used to collect essential oil through liquid–liquid extraction (LLE) by using HPLC grade n-hexane. A small amount of anhydrous magnesium sulphate was added to the n-hexane extract for the complete elimination of water. After filtration, the n-hexane was evaporated under vacuum in a rotary evaporator. The essential oil isolated through decantation and LLE were combined and weighed to determine the percent yield. Purified samples were stored at −20 °C until insecticidal and anti-fungal experiments and chemical analysis.

2.3 Chemical profiling of essential oils

The chemical composition of the essential oils was determined by a Hewlett Packard 6890 N gas-chromatograph (GC) coupled with a HP 5973 mass spectrometer (MS, Agilent Technologies Inc. USA). The GC was equipped with a DB-5 column (30 m length, 0.25 mm internal diameter and 0.25 µm stationary phase film thicknesses). The injector was operated at 235 °C and the oven temperature was programmed as follows: isothermally at 40 °C for 2 min, then increased by 4 °C per min to 240 °C, and afterwards held isothermally at 240 °C for 8 min. The carrier gas was high purity Helium (99.99%) with a constant flow of 1 mL/min. Injections of 1 µL essential oil solutions (1 mg/mL) were made in splitless mode for 30 sec. Mass spectral detection was performed by an electron ionization system operated at the ionization energy of 70 eV. The ion source temperature of MS was set at 180 °C and the solvent delay for an injection was set for 5 min. The mass spectra of separated compounds were scanned from 30 to 400 amu.

GC peak areas were used to determine the percentage composition of essential oils without using correction factors. The identification of the essential oils’ constituents were initially carried out by comparing their mass spectra and retention indices with those available in National Institute of Standards and Technology (NIST) 2008 MS library and from the literature (Adams, 2007). In order to calculate retention indices of essential oil constituents, a standard solution of straight chain alkanes (C₉-C₂₄) was injected in the GC–MS using the same parameters used for essential oil analysis. Finally, wherever possible, the identification of compounds was achieved by injecting solutions of authentic pure standards.

2.4 Insect rearing

Red flour beetles were maintained in the laboratory following a reported procedure (Bouda et al., 2001; Dal Bello et al., 2001). Briefly, T. castaneum adults were collected from infested rice purchased from a local market in Abbottabad. Adult beetles were provided insecticide-free wheat flour and maintained in plastic jars (18 × 10 × 10 cm) in an incubator at 25 ± 3 °C and 60 ± 10 % relative humidity (RH) with alternating light–dark cycles of 12 h. A cheese cloth was used to close the jar mouths. The adults obtained from the infested rice were transferred to another jar by sieving after two weeks. Once new adult beetles started emerging, they were transferred to separate jars. The jars with eggs and larvae were observed regularly to separate newly emerged T. castaneum adults in fresh jars containing flour. The process was continued until sufficient numbers of beetles were obtained for experiments.

2.5 Insecticidal bioassay through treated grains

The insecticidal activity of essential oils was tested through food poisoning method by adopting methods described by Bouda et al. (2001) and Hematpoor et al. (2017) with some modification. Essential oil (40 mg) was dissolved in 1 mL of ethanol and the resulting solution was applied on 10 g of rice and shaken well for 15 min by using a rotary shaker (Hashem et al. 2018). Thus, the concentration of essential oil on rice was 0.4% (w/w). For a control treatment, the same amount of rice were treated with 1 mL of ethanol. Both control and essential oil treated rice samples were dried at room temperature for 20 min prior to use in bioassays. The control and essential oil treated rice samples were placed at the center of two separate 90 mm glass Petri plates and fifteen unsexed adults T. castaneum (4–7 day old and 24 h starved) were released in each Petri plate. The Petri plates were covered with lids and incubated at 25 ± 3 °C and 60 ± 10 % RH with alternating light–dark cycles of 12 h. The numbers of live beetles in each Petri plate were counted after 24 h, 48 h, and 72 h exposure, followed by 24 h recovery time. A beetle was considered alive if it moved its legs or antenna upon prodding with a soft forceps. Five replicates of each essential oil or control were run in similar way and the experiment was carried out in duplicate. Chlorpyrifos solution was used as positive control. To find the lethal dose, the experiments were run using decreasing doses, such as 20 mg, 10 mg, 5 mg and 2.5 mg, and same number of replicates and experiments were employed for all doses tested.

2.6 Insecticidal bioassay through fumigation

The fumigant bioassay was carried out by using a published method (Khani & Rahdari, 2012; Negahban et al., 2007; Sriti Eljazi et al., 2017) with some modifications. Various concentrations of essential oils were prepared in ethanol, thus the fumigant concentration in gas tight jars were 2.5 mg/L to 40 mg/L (2.5 ppm − 40 ppm) by using a two-fold dilution in each step. One side of a filter paper strip (2 cm width and 6 cm length) was impregnated with 10 µL of essential oil solution. After drying for 5 min at room temperature, the untreated side of the filter paper strip was attached to the internal side of the jar lid and the gas-tight lid was placed on each of the glass jars each of 100 mL volume. The sample-bearing side of the filter paper strip was suspended inside the glass jar at the height of 2 cm from the bottom of the jar. This fumigation setup was made to prevent direct contact of the insects with the test substance or control. In each glass jar, fifteen unsexed adult beetles were released. They were 7–12 days old and starved for 24 h. The beetles were exposed to the vapors of the test essential oil or negative control (ethanol) or chlorpyrifos and the dead beetles were counted after 72 h of exposure time. In order to check the mortality of the beetles, they were removed from the testing jar and the insects showing no movement of legs upon piercing with a tweezer were considered dead. The fumigation bioassays were repeated twice using five replicates in each experiment. Total numbers of surviving insects in the control and test substance jar were counted and percent insect mortality was calculated by using the following formula:

% Mortality = [Numbers of live beetles in control treatment - Numbers of live beetles in test treatment] × 100/ [Numbers of live beetles in control treatment].

2.7 Anti-fungal bioassay

A strain of Aspergillus flavus was isolated from the contaminated food commodity by inoculating it on potato dextrose agar (PDA) at 28 ± 1 °C for 72 h. The isolated fungal colony was inoculated on fresh PDA Petri plates until a purified single colony was obtained. The fungal strain was identified based on morphological characteristics and maintained on PDA and stored at 4 °C until being used for anti-fungal bioassays. To determine anti-fungal activity of extracted essential oils, an earlier reported agar well diffusion method was used (Mekonnen et al., 2016). Briefly, sterilized molten PDA medium was poured into 90 mm sterilized Petri plates and left to solidify overnight on a clean bench. Fungal inoculum was prepared by making the fungal spore suspension in sterilized distilled water from the pure culture plate and then used for the experiment. An aliquot of 100 μL fungal inoculum containing 10⁵ conidia/mL was spread evenly on the PDA Petri plate with the help of glass spreader and the plate was incubated for 30 min so that the inoculum would be completely absorbed on the medium surface. One well of 6 mm diameter was dug in the center of the PDA Petri plate with the help of a sterilized cork-borer. Essential oil (10 μL) was added into the well whereas on the control Petri plate 10 µL dimethyl sulfoxide (DMSO) was added. Dithane M45 solution was used as positive control. All the plates were incubated in a laminar flow hood for 30 min, so that the essential oil and control samples could diffuse into the agar. The Petri plates were incubated at 28 ± 2 °C for 72 h. After the incubation period, the inhibition zone around the well was measured in mm with a scale and magnifying glass. At least five replicates of each essential oil and control were run and the experiment was conducted in a duplicate.

2.8 Data analysis

For all bioassays at least five replicates were used and average results presented. In case of>20% insect mortality in control replicates the experiment was repeated, however, in case of 1–20% insect mortality in control replicates Abbott’s formula was used to calculate observed corrected mortality (Abbott, 1925). The lethal doses (LD₅₀ and LD₉₀) of essential oils were calculated by using probit analysis (Finney, 1971). In order to find the statistical difference between bioactivity of different essential oils, the data were analyzed by one way ANOVA (analysis of variance) with a Bonferroni post hoc test. The data of two separate experiments were analyzed using a t test. The statistical tests, probit analysis and relative median potency analysis were performed by using computer software SPSS 20 (IBM, USA). The lethal dose estimates for tested essential oils were considered significantly different (P < 0.05) from the baseline essential oil if confidence limits for relative median potency ratios did not overlap with the value 1 (Gaire et al. 2019).

3.1 Yield of essential oils

The highest yield of essential oil was obtained from T. minuta (0.61%) whereas aerial parts of C. ambrosioides had the least amount (0.11%; Table 1).

3.2 Chemical composition of essential oils

A number of major compounds were found in the essential oil of C. sumatrensis, including 37.7% cis-lachnophyllum ester, 21.6% limonene, 13.4% germacrene D, 6.6% trans-β-farnesene and 5.7% trans-β-ocimene, representing 85.0% of the essential oil (Table 2). In the E. canadensis essential oil sample, limonene (43.4%), germacrene D (12.9%), matricaria ester (9.6%) trans-β-ocimene (7.7%), and cosmene (7.1%) were the major constituents, representing 80.7% of the essential oil (Table 2). cis-β-Ocimene (26.3%), dihydrotagetone (9.9%), cis-tagetenone (15.8%), and trans-tagetenone (19%) were identified as the major constituents in the essential oil of T. minuta (Table 2). The major compounds in the C. ambrosioides essential oil were α-terpinene (41.4%), p-cymene (14.7%), and germacrene D (16.2%).

3.3 Insecticidal activity through treated grains

The food poisoning insecticidal bioassay revealed that the bioactivity of essential oils was both dose- and time-dependent. At a dose of 40 mg, C. sumatrensis essential oil showed 56 ± 1.6% and 100.0% mortality of T. castaneum adults after 24 h and 48 h that was significantly greater (P < 0.05) than other three essential oils (Fig. 1). However, when the exposure time increased to 72 h, all tested essential oils exhibited 100% mortality against T. castaneum, similar to the positive control (P > 0.05). On testing the 20 mg dose and 72 h exposure time, C. sumatrensis and E. canadensis essential oils exhibited mortality comparable to the positive control (P > 0.05), whereas C. ambrosioides and T. minuta showed 84.0 ± 1.6% and 88.0 ± 2.5% mortality, respectively, which were significantly lower than (P < 0.05) positive control (Fig. 1).

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Fig. 1

Mortality percentages of T. castaneum exposed to 10 g of rice grains treated with different concentrations of C. ambrosioides, C. sumatrensis, E. canadensis, T. minuta essential oils and chlorpyrifos for different periods of time. Columns bearing different lower case letters are significantly different (P < 0.05) from each other when the mortality percent of different substances were compared with each other at the same tested dose independent of exposure time by ANOVA post-hoc Bonferroni test. The error bars denote standard error of the mean (n = 5).

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C. ambrosioides showed 44.0 ± 1.6%, 69.3 ± 2.6%, and 100.0% mortality at the 40 mg dose after 24 h, 48 h, and 72 h exposure time, respectively, whereas at the 10 mg dose the mortality was only 4.0 ± 1.6%, 11.0 ± 0.6%, and 37.0 ± 2.6%, respectively (Fig. 1). C. sumatrensis essential oil showed the highest insecticidal activity against T. castaneum, exhibiting 100% insect mortality after 48 h and 72 h exposure time when 40 mg and 20 mg doses were administrated, respectively. At a dose of 10 mg this essential oil showed 16.0 ± 1.6%, 57.0 ± 1.6%, and 95.3 ± 2.4% mortality after 24 h, 48 h, and 72 h, respectively (Fig. 1).

E. canadensis essential oil at the 40 mg dose exhibited 46.6 ± 2.1, 84 ± 1.6, and 100.0% mortality of T. castaneum after 24 h, 48 h, and 72 h exposure time, respectively. This oil killed 97.3 ± 1.6% and 100.0% of the insects at 20 mg and 40 mg doses, respectively, but only at the longer exposure time (Fig. 1). Although T. minuta essential oil showed relatively good insecticidal activity at the highest tested dose and the longest exposure time, it failed to control T. castaneum at lower doses and shorter exposure times (Fig. 1).

Overall all the tested essential oils exhibited low activity after 24 h exposure time, thus the LD₅₀ of all oils was in the range of 33.9 – 44.8 mg/10 g rice (Table 3). After 48 h exposure, the LD₅₀ and LD₉₀ of C. sumatrensis essential oil against T. castaneum were 7.5 mg and 27.4 mg, respectively, and these values differed significantly (P < 0.05) from the lethal dose determined for other plant essential oils (Table 3 & Table S-1b). The essential oils of C. ambrosioides and T. minuta exhibited similar LD₅₀ and LD₉₀ values (P > 0.05), whereas E. canadensis lethal doses were significantly different (P < 0.05) from the other three oils (Table 3, Table S-1b).

When the exposure time increased to 72 h, the essential oil from C. sumatrensis exhibited 3.7 mg and 8.9 mg LD₅₀ and LD₉₀ values, respectively, against T. castaneum which were similar (P > 0.05) to E. canadensis and significantly different from C. ambrosioides and T. minuta (Table 3, Table S-1c). The LD₅₀ and LD₉₀ values of T. minuta and C. ambrosioides were similar (P > 0.05) and the highest among all plants tested (Table 3). Based on relative median potency analysis the most toxic essential oil was C. sumatrensis, whereas C. ambrosioides exhibited the least activity against T. castaneum adults (Table S-1).

3.4 Insecticidal activity through fumigation

The fumigation bioassay revealed that the essential oils from all plants showed good to excellent bioactivity against adult T. castaneum but only at the higher tested concentration. E. canadensis and C. sumatrensis exhibited 100% mortality at 40 mg/L, which was significantly higher (P < 0.05) than other two essential oils but similar to positive control (P < 0.05). C. sumatrensis vapors exhibited excellent bioactivity among all the tested essential oil to 10 mg/L and thus showed significantly higher activity (P < 0.05) compared to other oils (Fig. 2). In the fumigation bioassay the LD₅₀ and LD₉₀ values exhibited by C. sumatrensis were 6.6 mg/L and 15.8 mg/L, respectively, which were significantly lower (P < 0.05) than all tested essential oils (Table 4, Table S-2). The LD₅₀ and LD₉₀ values of E. canadensis were 10.6 mg/L and 29.6 mg/L, respectively, which were significantly higher than C. canadensis and significantly lower than C. ambrosioides and T. minuta (Table 4, Table S-2).

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Fig. 2

Mortality percentages of T. castaneum exposed to the vapors of different concentrations of C. ambrosioides, C. sumatrensis, E. canadensis, and T. minuta essential oils and chlorpyrifos for 72 h. Columns bearing different lower case letters are significantly different (P < 0.05) from each other when the mortality percent of different substances were compared with each other at the same tested dose by ANOVA post-hoc Bonferroni test. The error bars denote standard error of the mean (n = 5).

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3.5 Anti-fungal activity

The essential oils extracted from C. ambrosioides and E. canadensis exhibited the highest activity against A. flavus, showing 15.7 mm and 16.5 mm zones of inhibition, respectively. C. sumatrensis essential oil was found to be the least active against A. flavus with a significantly smaller (P < 0.05) zone of inhibition against A. flavus (Fig. 3).

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Fig. 3

Antifungal activity of different test substances against A. flavus. Different alphabets (a-e) indicate significant differences (P < 0.05) when comparison was made between anti-fungal activity of different treatments by ANOVA post-hoc Bonferroni test. The error bars denote standard error of the mean (n = 5).

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Insecticidal and anti-fungal bioassays were repeated twice by using the same numbers of replicates, however, no significant difference (P > 0.05, t-test) was found between the results of the two independent experiments.