Insecticidal and fungicidal activity of Cymbopogon citratus (Poaceae) essential oil on Atta sexdens colonies

effect on a wide range of organisms, including leafcutter ants. The combination of insecticidal activity of a product and possible fungicidal activity constitutes an effective strategy against these insect pests, since both agents of this mutualistic interaction (both organisms are benefited) are affected, ensuring quicker collapse of the colony. The aim of this study was to evaluate the insecticidal and fungicidal activity of Cymbopogon citratus essential oil applied on Atta sexdens colonies under laboratory conditions. A nebulized spray system with three concentrations (15, 30 and 60%) and a control treatment were used to evaluate the effect of C. citratus on A. sexdens colonies. The C. citratus essential oils showed both insecticidal activity against the leafcutter ants ( A. sexdens ) and fungicidal activity against their symbiotic fungi. Colonies exposed to this essential oil showed considerable reduction in the weight of fungal mass, and mortality of both the queen and worker ants, which proves to be promising for the development of new insecticides for management of this insect pest.


INTRODUCTION
Leafcutter ants in the genera Atta and Acromyrmex (Hymenoptera: Formicidae) are known in both forest and agricultural production systems as one of the most destructive herbivores, due to their abundance, wide distribution, and potential for damage [1][2][3].In Brazil, Atta and Acromyrmex species are the main pests that affect Eucalyptus spp.and Pinus spp.crops [4].
Species from these genera occur in the whole Neotropical realm; however, the largest number of colonies and diversity are found in Brazil [5].These ants infest regions from the southern United States (USA) to northern Argentina [6].The symbiotic relation of these ants with the basidiomycete Leucoagaricus gongylophorus (Möller) Singer involves intense foraging and the cutting of fresh parts of plants, which serve as a substrate for the fungi, kept as the main nutritional source and habitat for the ant descendants [7].
Chemical methods are still most used for control of leafcutter ants, due to their higher efficiency.Some formicides are registered for managing leafcutter ants in Brazil, and they are applied in four distinct ways: dried powder formulations, formulations in thermal nebulization, formicides of liquid formulation, and the application of toxic baits [8].In planted forests, toxic baits are most used for control as this method is considered safe, practical, and economical [1].This method overcomes the challenges of ant colony architecture since the bait is transported in and uniformly distributed in the chambers by the ants themselves [2].
Botanical insecticides have been studied as an alternative to synthetic formicides, as these botanical agents are considered environmentally safer [6,[9][10][11], therefore, there is a constant search for alternatives that are efficient and do not cause negative impacts [12].Essential oils represent an alternative for pest control because they have certain desirable properties, such as biodegradability, selectivity to pest targets [13][14], and toxic effects on a wide diversity of organisms [10,[15][16], including leafcutter ants [17][18][19][20].
The Cymbopogon genus belongs to the monocotyledon gramineous plant group, which produces the most important essential oils of the Poaceae (Graminae) family.This family contains around 180 species, subspecies, varieties, and subvarieties widely distributed across temperate and tropical regions of the world [21].Cymbopogon citratus is native to Sri Lanka and the south of India.It is widely grown in tropical areas of America and Asia and contains several phytoconstituents, such as flavonoids, phenolic compounds, and terpenoids, responsible for different biological activities [22].Among these activities, C. citratus essential oil has been used for pathogen and insect control [23][24][25].
For that reason, combining this insecticide activity with a possible fungicide activity of a product for leafcutter ant control constitutes a great strategy against these insects, since both mutualistic agents are affected, ensuring quicker collapse of the colony [35].Therefore, the purpose of this study was to evaluate the insecticide and fungicide potential of C. citratus (Poaceae) essential oil applied on Atta sexdens (Hymenoptera: Formicidae) colonies under laboratory conditions.

Essential Oil
The C. citratus essential oil was extracted through hydrodistillation on industrial scale and obtained from the Destilaria Bauru Ltda.company (Catanduva, São Paulo, Brazil).

Essential oil analysis
The analysis of the chemical constituents of the essential oil was carried out in the Organic Chemistry Research Laboratory of the Department of Chemistry at the Universidade Federal de Sergipe, SE, Brazil.
Chemical constituents were analyzed using a GC-FID-MS (GCMSQP2010 Ultra, Shimadzu Corporation, Kyoto, Japan) equipped with an AOC-20i (Shimadzu) automatic injection sampler.Separations were made through Rtx®-5MS Restek a fused-silica capillary column (5% diphenyl / 95% dimethyl polysiloxane) (30 m × 0.25 mm) with 0.25 µm of film thickness in a helium (5.0) constant flow rate of 1.2 mL.min -1 .The samples (1.0 µL of the ethyl acetate solution) were injected with a split ratio of 1:10.Oven temperature began at 60°C (for 4 min) and increased at a rate of 3 °C.min - up to 220°C, and then increased to 280°C at a rate of 20 °C.min -1 .The injector and interface temperatures were both at 280°C.
The mass spectrometry (MS) data (total ion chromatogram, TIC) were obtained in the full scan mode (m/z 40-550) with a scan rate of 0.3 scan/s by electron ionization (EI) at 70 eV.The temperature of the ion source was 200°C.The percentage composition of each constituent was estimated by the area of each component divided by the total area of all components in the sample.The percentage of compounds was calculated through the peak areas of GC-MS and arranged in the GC elution order.
Retention rates were obtained by injecting a mix of linear hydrocarbons (C7-C30), and compounds were identified by comparing the retention rates [36] through computerized comparison of the mass spectra obtained with those registered in the mass spectra database of three libraries of the equipment: WILEY8, NIST107, and NIST21, along with the mass spectra from literature [37].

Insects
The Atta sexdens colonies were obtained from mass rearing at the Forest Entomology Laboratory of the Department of Forestry Sciences at the Universidade Federal de Sergipe, São Cristóvão, SE, where they were kept under a controlled environment (25 ± 10 ºC and 70 ± 10% RH).Artificial colonies consisted of three chambers: fungus garden (1000 mL), waste chamber (500 mL), and feeding chamber (500 mL).Food was provided periodically: corn flakes and Hibiscus spp.flowers.The colonies were kept in the laboratory until the fungus garden achieved a volume of 1000 mL, ideal for performing bioassays.

Nebulization bioassay
The experiment was carried out at the Forest Entomology Laboratory of the Department of Forestry Sciences at the Universidade Federal de Sergipe.
A nebulization system containing three concentrations (15, 30, and 60 %) and a control treatment, with three repetitions, was used to evaluate the effect of C. citratus essential oil on Atta sexdens colonies [38].
For each colony, 5 mL of the mixed product was used, the commercial standard.The control treatment consisted of 5 mL of mineral oil alone.The concentrations were prepared from an essential oil solution in proportions of 15, 30 and 60% and an oil adjuvant (mineral oil) that were manually homogenized to form a mixture.They were then placed in the nebulization unit I-205®.
All colonies were weighed before nebulization and after each evaluation to verify change in fungus weight after the application.Evaluations occurred at 1, 2, 3, 7, 14, 28, 35, 42, 50, and 60 days after application of the treatments through the following parameters: change in fungus weight; ant intoxication; ant mortality; ant activities, including food transport; chamber shifts of the fungus; fungus in the waste chamber; presence of filamentous fungi in the symbiotic fungus; queen mortality; and general aspects of the colony [39].

Statistical analysis
All of the parameters were submitted to an analysis of variance, then the means on the colony weight were compared by the Tukey test at 5% probability and mortality data to regression analysis, being the models selected, according to the significance of the regression coefficients (t, P < 0.05) and the coefficient of determination (R 2 ).The statistical analyses were performed by the usage of SISVAR 5.6 software [40].

Essential oil Characterization
Twenty-three components were identified in description of the profile of the volatile organic compounds in C. citratus essential oil, corresponding to 99.4% of the total composition (Table 1).The major constituents were monoterpenes: geranial (51.9%) and neral (41.1%), the structures are presented in Figure 1.(Adams, 2007).a NI = not identified.a tr = trace, < 0.1%; % of the peak area calculated by GC-MS.

Nebulization Bioassay
The colony weight data did not differ between treatments and the control (F3,11 = 0.549; df = 3; P > 0.663) before nebulization (Table 2).After the nebulization process, the treatments differed from the control, affecting the colonies' weight (F3,11 = 10.788;df = 3; P < 0.0035) (Table 2).The treatments (mean ± standard error of the mean) followed by the same letter did not differ significantly from each other by the Tukey test (P < 0.05).
The C. citratus essential oil concentration evaluated on the Atta sexdens colonies was effective, since it caused colony and queen mortality (Figure 2).The queens and colonies subjected to the treatment died in the course of 19 days, in contrast with the colonies exposed only to the control treatment, which showed no change in the observed parameters after nebulization, with no intoxication or mortality of worker ants.
After nebulization, all treatments with C. citratus essential oil concentrations showed intoxication and ant mortality, absence of flower-cutting and incorporation in the symbiotic fungi, reduction in the symbiotic fungal volume, and growth of filamentous fungi.
Major signs of intoxication included fungal oxidation, reduction in walking activity of the ants, transfer of immature pupae to the upper part of the fungus garden, transfer of dead ants to the waste chamber or leaving dead ants in the fungus chamber, absence of flower-cutting behavior, absence of handling the corn flakes and their further incorporation in the fungi, and increase in humidity in the fungus chamber.In addition, filamentous fungi grew in the fungus chamber.All the colonies were weighed at the end of evaluations to confirm reduction in the symbiotic fungi.There was a significant difference between the control and the other treatments (F3,11 = 10.788;df = 3; P > 0.003) (Table 2 and Figure 3).

DISCUSSION
The chemical composition of C. citratus essential oil and its insecticidal and fungicidal potential on A. sexdens colonies under laboratory conditions were investigated in this study.The main compounds found in the essential oil were geranial and neral, compounds that have been reported in studies conducted by several researchers as the most abundant, even in plants from different regions [31,33,[41][42].Cymbopogon is an important essential oil genus of aromatic grasses of the Poaceae family [43].Cymbopogon citratus (D.C.) Stapf is one of most widely distributed species, with proven larvicidal [33] and insecticidal [26] activity and antibacterial, antioxidant, antifungal, and anticarcinogenic properties [44].These characteristics are attributed to the constituents of the essential oil extracted from the leaves, especially citral, an isometric mixture of neral and geranial [45].Most of the insecticidal properties of the C. citratus essential oil are due to citral, as already shown in other studies [46][47][48].
The C. citratus concentrations applied on the A. sexdens colonies caused queen and colony deaths.This may be attributed to the mechanisms of essential oil activity, which may cause neurotoxic effects in insects due to terpenoids, involving several mechanisms, notably through gamma-aminobutyric acid (GABA), octopamine synapses, and acetylcholinesterase (AChE) inibition [49].Several constituents of essential oils have been identified as inhibitors of AChE, including monoterpenes like neral, geranial, and linalool [50][51][52] present in the C. citratus essential oil used.AChE inhibition triggers an imbalance because hydrolysis of the ACh in the synapses does not occur.This causes an abnormally large accumulation of ACh, resulting in large nerve stimulation, which leads to behavioral shifts, asphyxiation, hyperactivity, and finally death [53].
Atta sexdens colonies exposed to Lippia sidoides (Verbenaceae) essential oil through nebulization showed several behavioral changes, such as: fungal oxidation, reduction in ant movement, transfer of dead ants to the waste chamber, absence of flower-cutting behavior, increase in humidity in the fungus chamber, growth of filamentous fungi, just like the inactivity of the colonies and survival of the queens, through nearly the entire period of evaluation [38].These changes were observed in this study as well.The transfer of immature pupae to the upper part of the fungus garden to avoid a contaminated environment from the essential oil and from filamentous fungi showed a disturb in equilibrium of the treated colonies from essential oils [55].
The volume of symbiotic fungi was reduced after exposure to C. citratus, indicating its fungicidal activity.The citral applied by fumigation on the symbiotic fungus Leucoagaricus gongylophorus had higher fungistatic activity.The concentration of this compound necessary to inhibit 50% of mycelial growth was around 3.3 times less when compared to the concentration of the essential oil of chemotype citral of Myrcia lundiana; the citral compound also showed a fungicidal effect in the 123.21 µL -1 concentration by fumigation [19].In vitro experiments with the L. gongylophorus fungus in a growth medium containing several concentrations of azadirachtin showed less growth in fungus weight, even under low concentrations of this compound [35].
The reduction in the final weight of the fungi observed in this study and others occurs through low production of hyphae, with direct effects on the colony, since hyphae make nutrients available for queens and worker ants [35,56].The leafcutter ants feed on liquid nutrients found on the gongylidia, which are rich in carbohydrates and necessary to feed the queen [57].
In general, the antifungal activity of citral may be due to its capacity to penetrate fungal cells, changing mitochondria morphologically and modifying respiratory rates [58][59].The bioactivity of the essential oil has been reported on phytopathogenic fungi [34].
Mortality of the colonies and their queens occurred in the period of 19 days in this study.In A. sexdens colonies treated with different indoxacarb concentrations, also under laboratory conditions, mortality rates of around 60% occurred; however, sulfluramid caused 100% mortality in the period of 21 days after application [60].In the field, Atta sexdens rubropilosa treated with commercial sulfluramid products showed a reduction in nest activities three days after application of the bait.The average total paralysis of activity in the treated nests occurred in 20.55 days, while untreated nests showed normal activity [61], similar to the results found in this study.

CONCLUSION
This study highlights the potential of Cymbopogon citratus essential oil as an insecticide for control of leafcutter ants and as a fungicide for control of symbiotic fungi.This essential oil caused significant losses in colony weight and mortality of the queen and worker ants, showing promise for development of new insecticides for leafcutter ant management.

Figure 2 :
Figure 2: Insecticidal and fungicidal activity of Cymbopogon citratus essential oil at different concentrations in Atta sexdens colonies maintained in the laboratory.

Table 2 :
Weights of Atta sexdens (Hymenoptera: Formicidae) colonies maintained in the laboratory before and after application of Cymbopogon citratus essential oil at different concentrations.