Tithonia diversifoliais a plant from the Asteraceae family that, due to the large quantity of hydrogenated monoterpenes present in its essential oil, can be used to prepare a product capable of combating agricultural pests. Therefore, the aim of this study was to carry out a phytochemical screening of the components of the essential oil produced by hydrodistillation of the leaves of Tithonia diversifoliaand to verify its possible acaricidal applications in the control of the spider mite Tetranychus urticae (Koch) (Acari: Tetranychidae) and insecticidal applications in the control of the corn weevil Sitophilus zeamais (Motsch) (Coleoptera: Curculionidae), both via fumigation. Gas chromatography coupled with mass spectrometry revealed the presence of 32.67% β-pinene (6,6-dimethyl-2-methylenebicycle, heptane), 24.74% α-pinene (2,6,6-trimethylbicycle, hept-2-ene) and 22.69% limonene (1-methyl-4-(1-methyleneyl)-cyclohexene), among other components, in the essential oil of T. diversifolia. Fumigation tests revealed 52% mortality of spider mites at a concentration of 20 µL/L air and 96% at 80 µL/L air. The insecticidal activity tests on the corn weevil showed no mortality, but the repellency tests on this same insect showed promising results close to 100% at a concentration of 10 µL/L air, suggesting further studies to implement its use in greenhouses to control spider mites and in silos to repel corn weevil due to its volatility.
Research Article
Chemical Composition and Acaricidal and Insecticidal Activity of the Essential Oil of Tithonia Diversifolia (Asteracea) (Hemsl.) a. Gray
https://doi.org/10.21203/rs.3.rs-4638600/v1
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Essential oil
Tithonia diversifolia
Tetranychus urticae
Sitophilus zeamais
Agribusiness is one of the main economic activities worldwide, but it suffers enormous losses caused by approximately 50,000 species of phytopathogens, among which insects and mites are notable (Zikankuba et al. 2019). The spider mite, an agricultural pest with a cosmopolitan distribution, is responsible for enormous damage to more than one hundred agricultural crops, mainly fruit trees such as strawberries, pineapples, beans, corn, watermelons, passion fruit and others of enormous economic importance in tropical agriculture (Adesanya et al. 2021), and the maize weevil is the main pest of grains, seeds and cereals in storage facilities such as maize, rice, sorghum, wheat and cassava, which also causes quantitative and qualitative losses, jeopardizing their commercialization (Zhang et al. 2020).
Chemical pesticides are currently the most common method used to control agricultural pests, but they are routinely limited by the development of resistance in pests, human poisoning and the impact they can have on the entire ecosystem (Garcia et al. 2020). Thus, the search for safe and effective products from natural sources, especially plant-derived products, has been encouraged in recent years in agriculture as an alternative to traditional chemical pesticides (Alonso-Gato et al. 2021). In this sense, the essential oils of various plants have shown promising results in controlling agricultural pests (Silva and Farias 2020; Pedrotti et al. 2019).
The essential oil of Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae) has already been evaluated for its biopesticidal activity (Maina et al. 2018; Wanzala et al. 2018; Githinji et al. 2021; Akeumbiwo et al. 2023), making it, due to its volatilization, an alternative for the control of some agricultural pests, but more research is needed to expand its potential in the control of mites, such as spider mites, Tetranychus urticae Koch (Acari: Tetranychidae) in greenhouses and insects, corn weevils Sitophilus zeamais Motsch (Coleoptera: Curculionidae) in seed and grain storage silos. Therefore, the aim of this study was to analyse the chemical composition of the essential oil of T. diversifolia and its acaricidal effects on spider mites and insecticidal effects on corn weevils via fumigation.
Plant collection and ethnobotanical identification
Tithonia diversifolia leaves were collected on August 30, 2022, at Fazenda São Jorge (latitude: 19.503 S, longitude: 41.065 W and altitude: 193 m), which is located in the municipality of Itaguaçu, southeastern Espírito Santo state, Brazil. A total of 10 kg of leaves (fresh mass) was removed before flowering, allowing a higher level of secondary metabolites to be concentrated in the leaves (Gama 2014). The taxonomic classification of the plant was carried out at the Mello Leitão Museum of Biology Herbarium, with specimen registration number MBML 55380.
Essential oil extraction and analysis
Ten kilograms of leaves were collected, crushed and stored in a freezer in 1 kg blocks at -20 °C for 3 days. The essential oil was then obtained by steam distillation in a Clevenger-type apparatus at 60 °C for 3-4 hours (Oliveira et al. 2020) using a 5-litre flask. After extraction, the oil was stored in an amber glass bottle in a refrigerator at -5 °C until analysis. Equation 1 (Santos et al. 2004) was used to calculate the oil yield.
Where:
TO = oil content (mL of essential oil in 100 g of dry biomass) or extraction yield (%)
Vo = volume of oil extracted (ml)
Bm = plant biomass (leaves) (grams)
U = biomass moisture, wet basis (%).
Analysis of the volatile components of the essential oil of Tithonia diversifolia was carried out at the Central Analysis Laboratory 1 (UFES/Alegre) using gas chromatography (Shimadzu GCMS-QP2010 SE; Japan) with a capillary column (Support Rx-5Sil MS; 30 m x 0.25 mm, film thickness 0.25 µm). The chromatographic analyses were carried out with a drag gas (helium) flow rate of 1 ml/min and a split ratio of 1:10. After 1 min at 60 °C, the temperature was increased to 250 °C (4 °C/min) and maintained at 250 °C for 15 min. Mass spectra were obtained at 70 eV. For analysis, 30 µL of pure essential oil was diluted in 970 µL of hexane, and 1 µL of this solution was injected into the device. The volatile constituents were identified on the basis of their retention indices (Kovats, 1965) and their mass spectra, which were compared with reference data (Adams, 2001).
Obtaining and rearing T. urticae
The population of Tetranychus urticae used in the bioassays was established from collections in strawberry plantations in 2011 in the municipality of Guaçuí, ES (20° 46' 36.48” S and 41° 40' 37.92” O) and reared at 25 ± 1 °C, R.U. 70 ± 10% and 12 h photophase on pigeon pea plants, Canavalia ensiformes L. DC (Fabaceae), which were replaced every five days with healthy plants.
Evaluation of the toxicity of T. diversifolia essential oil to T. urticae via fumigation
To evaluate the acaricidal effect of T. diversifolia oil, tests to analyse the toxicity of T. diversifolia essential oil via fumigation on adult females of T. urticae were carried out at a temperature of 27.4 ± 1.9 °C, a relative humidity of 61.5 ± 7.0% and a 12 h photophase at the Center for Scientific and Technological Development in Phytosanitary Pest and Disease Management (NUDEMAFI) located on the Alegre campus of the Federal University of Espírito Santo. Leaf discs of pigeon pea 3 cm in diameter were placed in 50 ml plastic cups lined internally with a thin layer of damp absorbent cotton so that the leaf discs remained turgid during the test and to prevent the mites from escaping from the leaf area. Afterwards, 10 adult females of T. urticae between 24 and 48 hours of age were carefully transferred from the brood to leaf discs located at the bottom of the plastic cups. Then, small plastic cups with leaf discs containing adult females of T. urticae were placed at the bottom of 120 ml glass containers and used as fumigation chambers. The essential oil of T. diversifolia was applied with an automatic pipettor to 18 cm2 filter paper attached to the underside of the lid of the glass containers (fumigation chamber), leaving the mites exposed to the vapour of the essential oil for 48 hours. For sealing, glass jars (purge chambers) were wrapped in PVC film. After 48 hours of exposure to the oil, the numbers of live and dead mites were counted. The tests were carried out using a completely randomized design with five doses of T. diversifolia essential oil (5 µL/L, 10 µL/L, 20 µL/L, 40 µL/L and 80 µL/L air) and a negative control (no essential oil), with each treatment having five replicates. To assess mite mortality, a binocular stereomicroscope (ECZ-BLACK) was used at 80x magnification, and mites that showed no movement after light contact with tweezers on their cephalothorax were considered dead.
Obtaining and rearing Sitophilus zeamais
The insects were collected from a cornfield in the municipality of Alegre in 2012. The insects were reared and maintained from pure populations obtained from the laboratory of the Center for Scientific and Technological Development in Pest and Disease Management (NUDEMAFI) at the Center for Agricultural Sciences and Engineering of the Federal University of Espírito Santo (CCAE-UFES) in Alegre, Espírito Santo, Brazil, located at 20° 45' 49“ S latitude and 41° 31' 58” W longitude. To synchronize the age of the weevils, 6 glass containers with a capacity of 2 litres were used, containing 100 g of white maize and 50 unsexed adult insects to lay the eggs. To prevent the insects from escaping, the rearing containers were closed with organza-type fabric, thus allowing them to breathe. The containers containing the insects were kept at 26 °C (± 2 °C) with 70% relative humidity and a 12-hour photophase. After 10 days of confinement, the adult weevils were removed from the glass containers and discarded, after which the containers containing the seeds were stored until the next generation emerged (Rossetto, 1972).
Fumigation of S. zeamais with T. diversifolia essential oil for toxicity tests
The methodology described by Aslan et al. (2004) with adaptations was used to evaluate the fumigant effect of T. diversifolia essential oil on S. zeamais adults. Fifteen grams of white maize, Zea mays L. (Poaceae), was added to 500 mL glass containers, and they were infested with 20 unsexed S. zeamais adults aged between 0 and 15 days. Five different concentrations of T. diversifolia essential oil (5 µL/L, 10 µL/L, 20 µL/L, 40 µL/L and 80 µL/L air) and a negative control (no essential oil) were pipetted onto the underside of the lid of the containers. After 48 hours, live and dead insects were counted, and the percentages of insect mortality were determined, with individuals who did not move when touched with a brush being considered dead.
Repellency tests of S. zeamais with T. diversifolia essential oil by fumigation
Two free-choice tests were carried out using a methodology adapted from Nunes and Rizental (2015). In the first test, six 120 ml plastic arenas were used, which were symmetrically connected to a central container of the same volume by plastic tubes. In the central arena, 20 adult S. zeamais insects aged between 5 and 10 days were released, and 10 g of corn was added to the other six connected arenas. The undersides of the lids of the six peripheral arenas containing corn received 2 cm2 sheets of filter paper on which five different concentrations of T. diversifolia essential oil (5, 10, 20, 40 and 80 µL/L air) and the control (no essential oil) were pipetted (automatic pipette). After 24 hours of exposure, the number of insects attracted to the negative control and to the different concentrations of essential oil was counted. Three replicates were used in this first test to calculate the repellency index (RI%). The RI% was calculated for each of the essential oil doses using the equation model adopted by Guerra et al. in 2019 using the following equation:
Where:
IR (%) = the repellency index.
T = number of insects on the treated surface.
C = number of insects on the control surface.
For the second repellency test, five plastic containers connected to a central container were used. In this second test, five replicates were carried out with the same concentrations of T. diversifolia essential oil as in the previous test but without the negative control. This second test was carried out to determine the minimum concentration of essential oil that the insects could tolerate. After 24 hours, the number of insects attracted to the different concentrations of essential oil was observed.
Statistical analysis
The results of all the bioassays were subjected to regression tests, the data obtained were subjected to analysis of variance (ANOVA), and the means were compared using the Tukey test at the 5% probability level using GraphPad Prism 6.0 statistical software.
Chemical analysis of the essential oil
Extraction yield of T. diversifolia essential oil was 0.02%. Figure 1 shows the chromatogram of the essential oil of T. diversifolia, and Table 1 shows the results of the chromatographic analysis. The main components were β-pinene, α-pinene and limonene, which together made up 80.1% of the oil components.
Table 1. Results of chromatographic analysis of essential oils from T. diversifolia leaves.
|
Compound |
Retention time (RT) |
Calculated Kovats |
Note (%) |
|
β-pinene |
10,69 |
977 |
32,67 |
|
α-pinene |
8,85 |
935 |
24,74 |
|
Limonene |
13.16 |
1030 |
22,69 |
|
(E)-β-cymene |
13,75 |
1043 |
5,64 |
|
Sabinene |
10.61 |
975 |
5.03 |
|
Borneol |
19.98 |
1170 |
2,38 |
|
Camphene |
9h45 |
950 |
2.21 |
|
Spatulenol |
38,23 |
1582 |
1,58 |
|
β-caryophyllene |
31,52 |
1420 |
1.14 |
|
Total |
- |
- |
98.08 |
Acaricidal activity of T. diversifolia essential oil on T. urticae via fumigation
The results obtained for acaricidal activity after fumigation of different concentrations of T. diversifolia essential oil can be seen in Figure 2 on a base 10 logarithmic scale.
The results showed that the percentage mortality rate of T. urticae was directly proportional to the increase in the concentration of T. diversifolia essential oil in the fumigation test after 48 hours of exposure.
Repellent activity of T. diversifolia essential oil on S. zeamais via fumigation
After the insecticidal evaluation by fumigation of T. diversifolia essential oil showed no mortality in insect pests (S. zeamais) of stored grains (data not shown), the repellency tests showed promising results. The repellency indices of S. zeamais to different concentrations of T. diversifolia essential oil and the percentages of insects present in each of the arenas containing different concentrations of essential oil are shown in Figure 3.
The main finding of this study was that the essential oil of T. diversifolia has acaricidal activity on T. urticae according to fumigation and repellent activity on the coleopteran S. zeamais by increasing its concentration in a controlled environment. In 2022, Devi and colleagues also demonstrated the fumigant activity of the essential oil of this plant against adults of the rice weevil Sitophilus oryzae L. (Coleoptera: Curculionidae) at concentrations of 95.77 and 74.68 µL/L and against the brown stag beetle Tribolium castaneum Herbst. (Coleoptera: Tenebrionidae) at concentrations of 102, 98 and 81 µL/L air after 24, 48 and 72 h, respectively, demonstrating that the essential oil of T. diversifolia could be used in the management of these insects, which are considered to cause great damage to stored grain. Njuguna et al. (2022) demonstrated the action of monoterpenes in T. diversifolia essential oil through toxicity tests on three plant parasites with satisfactory results: the cotton louse Aphis gossypii Glover (Homoptera: Aphididae), the tobacco thrips Thrips tabaci Lind (Insecta: Thripidae) and the whitefly Bemisia tabaci Gennad (Hemiptera: Aleyrodidae). This biopesticidal activity of the essential oil was also detected in bacteria (Xanthomonas oryzae pv. oryzae and Pseudomonas fuscovaginae) at a concentration of 125 µg/mL and fungi (Bipolaris oryzae and Fusarium moniliforme) at 5000 µg/mL, which are agricultural pests that affect rice seeds in storage, demonstrating that the essential oil of T. diversifolia can be used as an alternative to synthetic fumigants (Dongmo et al. 2021). Arantes et al. 2024 also showed the pesticidal activity of the main components of T. diversifolia essential oil (α-pinene and β-pinene) in controlling the cattle tick Rhipicephalus (Boophilus) microplus.
The repellent activity of T. diversifolia essential oil was also demonstrated in tests carried out on S. zeamais in this study. In the first test (with a control), a repellency index close to 100% was found at a concentration of 10 µL/L air. The arenas without T. diversifolia essential oil were preferred by 65% of the insects, followed by the lowest concentration of 5 µL/L air (approximately 33%), as shown in graph 2. Based on the results of the first test and the clear evidence of the insects' preference for arenas without essential oil, the minimum concentration of essential oil that the insects could tolerate was determined in a second test without the control, and it can be seen that the concentration of 20 µL of essential oil per litre of air is already unbearable for approximately 100% of the insects. Graph 3 shows the repellent activity of T. diversifolia essential oil on S. zeamais in stored grain. The data obtained corroborate those of the study by Oyewole et al. (2008), in which the essential oil of T. diversifolia leaves showed repellent activity at different concentrations against the bites of Anopheles gambiae Giles (Diptera: Culicidae), Aedes aegypti L. (Diptera: Culicidae) and Culex quinquefasciatus L. (Diptera: Culicidae), and the author attributed this repellency to the presence of monoterpenes such as α-pinene, β-pinene, limonene, (E)-β-ocimene, sabinene, camphene, spatulenol and β-caryophyllene found in the essential oil of T. diversifolia. β-Pinene, the main compound found in the essential oil of the plant under study, showed repellency against the German cockroach Blattella germanica (Blattodea: Blattellidae) in studies carried out by Han et al. in 2012. In 2023, Liao and colleagues reported that limonene also had potent repellent effects on the flour beetle Tribolium confusum DuVal (Coleoptera: Tenebrionidae), a pest of stored grain. The identified chemical composition of the essential oil of T. diversifolia may explain the effects on the pests observed in this study. Secondary metabolites such as terpenes, which are important inhibitors of agricultural pests and perform defence functions in the plant kingdom, were found to be present in large quantities (Yousaf, 2018). Most of the compounds found in the essential oil of T. diversifolia, such as β-pinene, α-pinene and limonene, are terpenes related to physiological disturbances in cellular respiration, synapses and digestion, leading to the death of the pest and causing repellency by interfering with the insects' sensory systems, making the environment less attractive to them, but more data must be added to more specifically determine these actions (Pavela R. and Benelli G. 2016).
The essential oil yield of T. diversifolia (0.02%) was the same as that found by Akeumbiwo et al. (2023) but differed from that found by Lamaty et al. (1991) for plants collected in Cameroon, for which a yield of 0.01% was reported. This finding indicates a difference in yields depending on location, soil quality, climatic conditions at the time of harvest, leaf condition, time and extraction technique (Oyewole et al. 2008).
In conclusion, the acaricidal activity of the essential oil by fumigation on the spider mite (T. urticae) was verified, as was the repellent activity of the oil on the corn weevil (S. zeamais), making it possible to carry out further studies into the use of the chemical components present in the essential oil of T. diversifolia in the control of arthropods in closed environments, such as greenhouse plantations and environments used for grain storage. This study contributes to improving the knowledge and use of biomolecules found in plants that are potentially effective in controlling agricultural pests and thus minimizing the use of some chemical pesticides.
The authors declare that are no financial or non-financial conflict of interests that are directly or indirectly related to this study.
Acknowledgments
This work received funding from the Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES, grant number 289/2021 and 1041/2022 P: 2022-LDB7K), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: grant number 314722/2021-1).
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