Key message

  • The aphicidal activity of carlina oxide, the main component of C. acaulis root oil, was studied.

  • Lethal and sub-lethal effects of carlina oxide were investigated on M. dirhodum.

  • Carlina oxide LC50 was 1.06 mL L-1 and the LC90 was 2.58 mL L-1.

  • Being exposed to the aphid LC90 showed little toxicity on A. aphidimyza and C. carnea.

Introduction

The aphid Metopolophium dirhodum (Walker) (Hemiptera: Aphididae) is an important pest of cereals, especially wheat and barley (Honěk 1994). In addition to the injury to plant tissue caused by sucking plant sap, which reduces grain quality, this insect pest is also an important vector of viral diseases such as barley yellow dwarf virus (BYDW) (Holt et al. 1984). Protection against aphids is based on the application of synthetic insecticides, mainly pyrethroids, organophosphates, and neonicotinoids (Gong et al. 2021a). However, the frequent application of synthetic insecticides leads, similarly to other pests, to the emergence of resistant populations. For example, Gong et al. (2021b) reported resistant populations of M. dirhodum against the insecticides thiamethoxam, imidacloprid, abamectin, and omethoate. In addition, the use of non-selective pesticides has a negative effect on aphid predators (Aphidoletes spp., Chrysoperla spp., Syrphus spp. Episyrphus spp., and Epistrophe spp.) and parasitoids (Praon spp. Aphidius spp., Aphelinus spp., etc.) (Honěk and Kocourek 1988; Takada 2002; Wojciechowicz-Zytko 2009).

For these reasons, it is necessary to search for new active substances characterized by new mechanisms of action (MoA) and, at the same time, tolerable to non-target organisms. Botanical insecticides also belong to promising products replacing synthetic insecticides. These plant protection preparations use secondary metabolites as active substances, which plants synthesize as part of their natural defense against pathogens and pests (Pavela and Benelli 2016). These metabolites also include essential oils (EOs) which are partially responsible for the taste and aroma of plants. In addition to many health benefits, they also exhibit bactericidal, fungicidal, and insecticidal effects, which have been proven in a wide number of studies (Isman and Grieneisen 2014; Pavela 2018; Benelli et al. 2020b).

Carlina acaulis L., a plant that naturally grows in the calcareous soils of southern and central Europe, belongs to the Asteraceae (Compositae) family (Tutin et al. 1976). It is well-known for its traditional medicinal use and possesses various beneficial health effects (Herrmann et al. 2011; Stojanović-Radić et al. 2012; Strzemski et al. 2019; Belabbes et al. 2020). The EO extracted from the roots of C. acaulis is primarily composed of the polyacetylene 2-(3-phenylprop-1-ynyl) furan, commonly known as carlina oxide. Polyacetylenes are a class of plant secondary metabolites involved in defense against insults and attacks of fungal, viral, and insecticidal origin (Spinozzi et al. 2023b).

Researchers have conducted experiments using C. acaulis EO, carlina oxide, and formulations containing these substances to test their efficacy against arthropods and nematodes. These include vectors of pathogens such as Culex quinquefasciatus Say and Musca domestica L., agricultural pests such as Lobesia botrana (Denis & Schiffermüller), Bactrocera oleae (Rossi), Ceratitis capitata (Wiedemann), Meloidogyne incognita (Kofoid & White), and stored-products pests such as Acarus siro L., Alphitobius diaperinus (Panzer), Oryzaephilus surinamensis L., Prostephanus truncatus (Horn), Rhyzopertha dominica (F.), Sitophilus oryzae L., Tribolium confusum Jacquelin du Val, T. castaneum (Herbst), Tenebrio molitor L., and Trogoderma granarium Everts (Pavela et al. 2020; Rizzo et al. 2021; Kavallieratos et al. 2022; Spinozzi et al. 2023a). These studies have also demonstrated that carlina oxide shows limited toxicity to non-target species and holds promise for being safe based on LD50 and LC50 values determined on rats and human cells, respectively (Pavela et al. 2020, 2021; Benelli et al. 2022).

Considering these findings, herein we evaluated the acute toxicity of carlina oxide on adults of the aphid M. dirhodum. Furthermore, sublethal effects caused by being exposed to selected concentrations of carlina oxide on aphid fertility and potential natality were investigated. At the same time, to better estimate the environmental safety of this compound, its effectiveness was tested on two important natural enemies of aphids, i.e., Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae) and Chrysoperla carnea Stephens (Neuroptera: Chrysopidae).

Materials and methods

Chemicals

Carlina oxide was obtained by hydrodistillation of C. acaulis roots (Minardi & Figli S.r.l., Bagnacavallo, Ravenna, Italy) (yield of 0.75%, w/w); it was a yellowish oil with a density of 1.063 g/mL. Specifically, 1 kg of dry roots and 10 L of distilled water were inserted in a 20 L round-bottom flask and carlina oxide was collected after 6 h of hydrodistillation with a Clevenger-type apparatus. Once obtained, the compound was stored at—20 °C until chemical analysis and biological assays. GC–MS analysis was performed to assess the purity of the compound (98.1%, Fig. 1), adopting the same method by Spinozzi et al. (2023a). The chemical structure was confirmed by MS and NMR analyses and comparing the results with those of a chemical standard obtained in the authors’ laboratory (Benelli et al. 2019).

Fig. 1
figure 1

Carlina oxide chromatogram obtained by GC–MS analysis displaying a purity of 98.1% based on total peak areas

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Insects

Metopolophium dirhodum

M. dirhodum adults (wingless females, 1–2 days old) were obtained from laboratory mass-rearing (Crop Research Institute, Czech Republic). Colonies of M. dirhodum aphids were maintained for > 20 generations on wheat plants (Triticum aestivum L.) at a temperature of 21 ± 3 °C, 65 ± 5% R.H., and a16:8 (L:D) photoperiod.

Aphidoletes aphidimyza

Third instar larvae were obtained from established laboratory breeding (Crop Research Institute, Czech Republic). Adults were placed in insect cages of dimensions 35 × 35 × 60 cm where they were allowed to oviposit on leaves near Myzus persicae (Sulzer) aphids that were on Brassica oleracea var. gongylodes L. Predatory larvae fed on aphids developing on kohlrabi plants ad libitum until reaching the 3rd instar, when they were used for experiments. Breeding was maintained at a temperature of 21 ± 3°C, 65 ± 5% R.H. and a 16:8 (L:D) photoperiod.

Chrysoperla carnea

Second instar larvae were purchased from a commercial biofactory (Koppert, Holland). Larvae were used in experiments immediately after delivery.

Bioassays

Acute toxicity against Metopolophium dirhodum

Aphid adults were transferred with a fine paintbrush to sown wheat plants (BBCH scale 11, 5 planted in a standard peat substrate, pots with a diameter of 9 cm) at the rate of 15 adults/pot. Between the transfer and application was a 3 h time gap, when aphids were allowed to freely settle on plant leaves and feed. Carlina oxide was emulsified using Tween 20 (Sigma Aldrich, Czech Republic), when stock emulsions were subsequently prepared using a Witeg HG15A homogenizer (5000 revolutions/min) in a concentration range of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL L−1. The concentrations used were estimated based on preliminary tests. To reduce the surface tension of the spray liquid, Tween 20 was used as a surfactant (3.0 mL L−1). The emulsions were applied to the plants using a laboratory Sprayer Sge1 (Biostep, Fisher, Czech Republic) at a dose of 5 mL/pot (corresponding to the equivalent of 500 L ha−1). Only water with Tween 20 (3.0 mL L−1) was used as a negative control. The commercially available product Neudosan (Neudorff W.GmbH. KG, Germany, also in potassium salts of fatty acids 515 g kg−1) was chosen as a positive control at the concentration recommended by the manufacturer (20 mL L−1).

The treated plants were placed in a greenhouse where the temperature was maintained at 21 ± 3°C, 65 ± 5% R.H., and a 16:8 (L:D) photoperiod. Each treatment was replicated 5 times. Mortality was assessed 48 h after application.

Inhibition of fertility and potential natality of Metopolophium dirhodum

The experiment was performed using the same method as for acute toxicity, except that adult females were treated with concentrations corresponding to the estimated lethal concentration that kills 30% (LC30) and 50% (LC50) of the population (i.e., 0.7 and 1.1 mL L−1, respectively). After 48 h, the surviving individuals were placed on new T. aestivum plants and the number of newly born nymphs was recorded for 7 days. Every each day the newborn nymphs were removed from the plants with a fine paintbrush to avoid issues on the following days. The experiment was located at a temperature of 21 ± 3°C, 65 ± 5% R.H. and a 16:8 (L:D) photoperiod. The experiment was repeated 5 times.

Fertility was expressed as the number of newly hatched nymphs per surviving treated female per day. Fertility inhibition then expresses the percentage by which the number of laid nymphs was reduced compared to the control.

Potential birth rates are then expressed by the number of hatched nymphs that a population of 100 treated females will produce in one day, assuming that their 30% or 50% mortality occurs within 24 h (for females treated with LC30 or LC50, respectively).

Potential natality was calculated according to the following formula: Nat = average number of nymphs laid by 100 treated females * predicted mortality coefficient, and mortality coefficient = 0.7 for aphids treated with concentrations corresponding to the estimated LC30 or by the coefficient 0.5 for aphids treated with concentrations corresponding to the estimated LC50.

Acute toxicity against Aphidoletes aphidimyza and Chrysoperla carnea

Third instar larvae of A. aphidimyza and 2nd instar larvae of C. carnea were treated with carlina oxide at concentrations corresponding to the LC50 and LC90 estimated for M. dirhodum (i.e., 1.1 and 2.6 mL L−1, respectively). Larvae (15 insects per replicate) were immersed in prepared stock solutions for 3 s and then placed in plastic cups (10 cm diameter) containing filter paper and covered with perforated lids. Larvae of C. carnea were kept individually in cups because of their cannibalistic tendencies. Different species of aphids were added to the cup as food for the test larvae in an ad libitum amount. The experiment was maintained at a temperature of 21 ± 3°C, 65 ± 5% R.H., and a 16:8 (L:D) photoperiod. The experiment was repeated 5 times. Mortality was assessed 48 h after application.

Data analysis

Metopolophium dirhodum mortality rates observed in acute toxicity experiments were adjusted according to Abbott (1925); then, LC50 and LC90 with 95% confidence interval (Cl95) were estimated through probit analysis (Finney 1971). Percentage data on the inhibition of aphid fertility and potential natality as well as mortality data of A. aphidimyza and C. carnea were transformed through arcsine square root transformation before being analyzed by ANOVA followed by Tukey’s HSD test (P ≤ 0.05). For all statistical analyses, software Biostat 5.9.8 was used.

Results

From an extraction and purification perspective, carlina oxide can be easily prepared by simple hydrodistillation, leading to a 98% purity (Fig. 1).

The effectiveness of carlina oxide from C. acaulis on the acute toxicity of M. dirhodum aphids is shown in Table 1. At the highest tested concentration of 3.0 mL L−1, a mortality of 96.7% was found, and it is evident that this concentration of carlina oxide had a similar effectiveness as the positive control we used. Probit analysis allowed the estimation of an LC50 of 1.06 mL L−1 and LC90 of 2.58 mL L−1.

Table 1 Acute toxicity of carlina oxide isolated from Carlina acaulis roots against Metopololphium dirhodum adults
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The effects of being exposed to different lethal concentrations of carlina oxide on M. dirhodum fertility and potential natality are shown in Table 2. Wingless adults were exposed to LC30 and LC50 estimated in acute toxicity tests. Tween-formulated emulsion applied at lethal concentrations reduced potential natality compared to the untreated control by more than 50%, with no significant difference observed between LC30 and LC50 application; on the other hand, the use of LC30 and LC50 of this compound allowed the fertility inhibition of the target pest by 35.68 ± 6.21% and 23.66 ± 10.58%.

Table 2 Sublethal effect of carlina oxide on fertility and potential natality of Metopolophium dirhodum aphids
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From a non-target point of view, the effect of the application of carlina oxide concentrations corresponding to LC50 and LC90 on aphid predators A. aphidimyza and C. carnea is reported in Table 3. For carlina oxide, only low mortality (less than 10%) was observed for both non-target species, and this mortality was not higher than the negative control.

Table 3 Acute toxicity of carlina oxide LC estimated on Metopolophium dirhodum against two natural enemies of aphids
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Discussion

The control of aphids represents a major challenge due to their fast reproduction capacity and the significant crop losses they cause (Ikbal and Pavela 2019). In fact, they can damage many crops worldwide and carry dangerous pathogenic viruses (Luo et al. 2022). Pyrethroids, neonicotinoids and carbamates are currently used to protect crops from these pests. However, aphids are becoming resistant to traditional products due to different mechanisms which avoid the toxic effect of insecticides (Bass and Nauen 2023). Specifically, M. dirhodum resistance phenomena mainly rely on detoxification enzymes genes expression (Gao et al. 2021). In this regard, we evaluated the insecticidal potential of carlina oxide, a natural product with well-demonstrated efficacy against other target insects, to find an eco-friendly alternative for the treatment of this dangerous pest.

Herein, carlina oxide was prepared by simple hydrodistillation, leading to a > 98% purity. This is certainly a factor favoring its future industrial application as ingredient of botanical insecticides (see also Spinozzi et al. 2023c). When carlina oxide was tested for on M. dirhodum at 3.0 mL L−1, it was able to cause > 96% mortality, with a similar effectiveness comparable to the positive control; the LC50 and LC90 were 1.06 and 2.58 mL L−1. Despite the negative impact of M. dirhodum adults on many crops worldwide, only few EOs and related botanical constituents have been evaluated for their insecticidal potential against this important pest (Ikbal and Pavela 2019). As a general trend, the tested botanicals showed lower toxicity with respect to carlina oxide. For example, phytol, (E)-nerolidol and spathulenol isolated from Stevia rebaudiana Bertoni showed LC50 values of 1.4, 3.5, and 4.3 mL L−1, respectively, which were higher with respect to those estimated for carlina oxide (Benelli et al. 2020a). Chopa and Descamps (2012) determined even higher LC50 values of 76.2 mL L−1 and 15.2 mL L−1 for Tagetes terniflora Kunth and Salvia officinalis L. EOs, respectively.

Carlina oxide has been proven to be highly effective against a relatively wide range of insect species (Spinozzi et al. 2023a). However, this is the first study exploring its insecticidal activity against aphids. The low LC50 values obtained depend probably on the contact toxicity exerted by carlina oxide. The exact mode of action related to the insecticidal action has not been determined yet, but it is probably linked to the formation of reactive oxygen species and free radicals following its reaction with sunlight. The propynyl chain of the molecule is embedded with a triple bond moiety which could be the main responsible for radicals’ production (Spinozzi et al. 2023a). The latter are highly reactive and can cause oxidative damages and insect death (McLachlan et al. 1984). The photosensitization is typical of polyacetylenes, and the class of compound carlina oxide belongs to (Gommers and Geerligs 1973; Konovalov 2014). Moreover, it has been demonstrated that this compound can inhibit the acetylcholinesterase (AChE) of insects (Benelli et al. 2019).

As reported in earlier research, low doses or concentrations of EOs can significantly reduce insect fertility (Benelli et al. 2018), among other sublethal effects (Giunti et al. 2022). This phenomenon is of practical importance. Indeed, applying a smaller amount of the active ingredients (AIs) makes the application of botanical insecticides cheaper in practice, and also thanks to the ability of EOs to inhibit fertility, the number of pests can be reduced below the threshold of economic harm. We previously found that carlina oxide from C. acaulis inhibits the fecundity of Musca domestica L. (Diptera: Muscidae) (Pavela et al. 2020) and Tetranychus urticae Koch (Acari: Tetranychidae) adults (Rizzo et al. 2024). However, information on the effectiveness of this EO on aphid fertility is still lacking. Therefore, we studied this phenomenon in M. dirhodum. In fact, parthenogenetic reproduction of this aphid, which typically develops from unfertilized eggs, combined with viviparity causes a rapid grow of their population.

When wingless M. dirhodum adults were exposed to the LC30 and LC50 estimated in acute toxicity tests, sublethal effects on aphid fertility and potential natality were noted. Tween-formulated emulsion applied at both LC30 and LC50 reduced potential natality compared to the untreated control by more than 50%. Therefore, we showed that being exposed to the above-mentioned concentrations can reduce the abundance of aphid colonies on plants. That means that weakened colonies can then be further reduced by aphid natural enemies. This approach fully matches the Integrated Pest Management (IPM) concept, given that green insecticides and biological control can be used simultaneously to better manage the pest (Ehler 2006). Therefore, it is important that carlina oxide, as the active ingredient (AI) of potential botanical insecticides, is also friendly to non-target organisms (Giunti et al. 2022). Studying the effect of insecticides AIs on non-target organisms is important for estimating their environmental safety. Indeed, the preference for a selective insecticide or selective application are key decisions for the preservation of natural enemies. Where, for some reason, the insecticide cannot show enough effect, preserved natural enemies can significantly reduce the outbreak and resurgence of a given pest (Torres and Bueno 2018).

Based on these considerations, we evaluated the non-target effect of carlina oxide against the aphid natural enemies A. aphidimyza and C. carnea. When exposing non-target predators to carlina oxide concentrations corresponding to aphid LC50 and LC90, only low mortality (i.e., < 10%) was observed on both species of non-target biocontrol agents, and this mortality was not higher than the negative control. The product we used, which was applied as part of the positive control, contained a salt of fatty acids as active substance. This product is commonly used in organic farming and is therefore generally considered friendly to non-target organisms. However, as was evident from our experiments, this product was friendly to A. aphidimyza, but it showed toxicity > 80% on C. carnea larvae. Overall, it can be concluded that carlina oxide was friendly for the aphid predators A. aphidimyza and C. carnea. This is also outlined by our previous findings, when non-target impact was evaluated through experiments on Daphnia magna Straus (Cladocera: Crustacea) adults. Carlina EO and carlina oxide showed lower toxicity if compared to cypermethrin (Benelli et al. 2019).

However, we are fully aware that further tests on non-target organisms and on the behavior of carlina oxide in the environment (e.g., adhesion/absorption to soil and organic matter, and bioaccumulation capacity) should be carried out to clarify the possible environmental impacts of the applications of botanical insecticides based on carlina oxide and their synthetic analogs (Spinozzi et al. 2023a), which are currently the subject of our further research.

Conclusions

This work represents the first evidence for the aphicidal activity of carlina oxide. This compound showed excellent efficacy against M. dirhodum, in a comparable manner to that of a commercial insecticide. Furthermore, minimal toxicity for natural enemies of aphids A. aphidimyza and C. carnea was showed. Therefore, it is possible to conclude that carlina oxide is safe for these insects in concentrations effective against aphids. The agrochemical exploitation of this compound will be assured by in field cultivation and/or synthetic procedures that have been recently developed by our research group.

Author contributions

RPa, RPe, FM, and GB conceived and designed the research. MN, RP, ES, MF, RP, FM, and RR conducted the experiments and/or analyzed the data. MN, RP, ES, MF, FM, and GB wrote the original draft. MN, RP, and RR contributed to writing, review, and editing. All authors approved the final version of the manuscript.