EFFICIENCY OF Citrus limon L. RIPE PEEL EXTRACT IN THE CONTROL OF Raoiella indica (HIRST, 1924) (PROSTIGMATA: TENUIPALPIDAE)

Purpose: This study aimed to evaluate the acaricidal potential of the aqueous extract obtained from the ripe peels of Citrus limon on adults of Raoiella indica. Theoretical Reference : R. indica is a phytophagous mite that primarily attacks plants of the Arecaceae family. As it is considered a relatively new pest in Brazil, there are few control methods available, with synthetic chemical control being the primary one. Method: The ripe peels of C. limon were dried and ground into a fine powder, which was used to formulate extract concentrations of 0.00, 1.00, 1.64, 2.70, 4.44, 7.30, and 12.00%. The extracts were sprayed on 12 adult females of R. indica per experimental unit, with 7 repetitions for each concentration. Mortality was assessed at 24, 48, and 72 hours after spraying. Results and Conclusion: The mortality of R. indica increased as the concentrations of the ripe peel extract of C. limon increased, reaching 88% for the highest concentration after 72 hours of application. Research

Actually, the mite can be found in basically all the Brazilian states, with high dispersal potential, and affecting tillages of coconuts, bananas, açai palms, native and exotic palm species and ornamental plants (NUVOLONI et al., 2021;CRUZ et al., 2023).This species promotes severe attacks with significant damage at high population levels (ALCÍVAR et al., 2020), principally due to its polyphagous habits, feeding on the leaf's lower surface, extracting sap through the host's stomata (DI PALMA et al., 2022;CARRIÇO et al., 2024).This specialized feeding habit interferes in the host's photosynthesis, cell breathing, stomatal opening and closing and water balancing processes (DI PALMA et al., 2022;JOSHI et al., 2023), at first, giving the leaf a tanned aspect, and later, causing the vegetable tissues necrosis (FIDELIS et al., 2022;CUZZUOL et al., 2023;JOSHI et al., 2023).
In other countries, the red palm mite management is made basically with synthetic chemical control.However, in Brazil there are still few registered control methods, being only the two commercial products VERTIMEC ® 18 EC and ABAMECTIN 72 EC NORTOX as chemical control, and the predator mite, Neoseiulus barkeri (Hughes, 1948) (Mesostigmata: Phytoseiidae), as biological control (CUZZUOL et al., 2023;AGROFIT, 2024).Due to growing environmental concerns regarding the improper use of insecticides and acaricides, particularly concerning the elimination of non-target organisms, the development of resistant populations, and environmental contamination, advances have been made in the study of alternative methods for pest control (SILVA et al., 2023).In this context, the use of specific plants that possess insecticidal/acaricidal properties, including those derived from agro- 4 industrial residues such as peels and seeds, has offered a potential and promising alternative for pest population management and the utilization of plant residues (CUZZUOL et al., 2023;PIFFER et al., 2023;CARRIÇO et al., 2024).
Due to the economic importance of the red palm mite, as well as the increasing demand for the use of alternative and sustainable products in pest management in agriculture, the present study aimed to evaluate the efficacy of the aqueous extract of Sicilian lemon Citrus limon ((L.) Burm.fil., 1768) (Rutaceae) ripe peels, on adult of Raoiella indica.

THEORETICAL REFERENCE
Raoiella indica was described for the first time in 1924, in coconut tree leaves in India.Was reported in the Americas for the first time in 2004, and spread out fast across the continent (SANTOS et al., 2020).R. indica is a coconut and palm tree species primary plague, however, it has been frequently associated with injuries in banana trees and many species from different families of ornamental plants in several countries (CRUZ et al., 2023).
Usually, it's management is made using synthetic chemical products (CUZZUOL et al., 2023), however, the indiscriminate use can promote the emergence of resistant populations, eliminate natural enemies, intoxicate humans and the surrounding fauna, and pollute the environment (MARCHIORI et al., 2023).Thus, it's necessary to search for new effective methods that are less harmful to the environment (CARRIÇO et al., 2024).
Species of the Rutaceae family, primarily in the genus Citrus, are rich in flavonoids, volatile oils, d-limonene and other secondary substances that have high repellent power and acaricidal/insecticidal potential, therefore, being an excellent alternative in the red palm mite management (LIAO et al., 2023;ALIMI et al., 2024).6 The dishes were kept in BOD-type climate chambers under conditions of 25 ± 1 °C, relative humidity of 70 ± 10 %, and a 12-hour photoperiod.

OBTAINING THE SOLUTIONS
To prepare the extracts, ripe fruits of Sicilian lemon were collected from the fruiticulture sector of IFES -Itapina Campus.The peels were removed and sanitized in a 5 % (v/v) sodium hypochlorite solution.They were then dried in a forced air circulation oven at 60 °C until reaching a constant mass.Subsequently, these materials were ground in a Willey-type knife mill, obtaining a fine powder, which was stored in Becker glassware, sealed with plastic wrap, and covered with aluminum foil to prevent contact with the external area and photodegradation.

CHEMICAL CHARACTERIZATION
The methodology described by Swain & Hillis (1959) was used, with adaptations, to calculate the total phenolic compounds content.The crude extracts were diluted in distilled water (1 mg/mL).In a test tube, 0.5 mL of the diluted extract, 8 mL of distilled water, and 0.5 mL of the Folin-Ciocalteau reagent were added.The solution was vortexed, and after 3 minutes, 1 mL of 7.5 % sodium carbonate solution was added, and the mixture was vortexed again.After 1 hour of incubation in the dark, absorbance readings were performed in triplicate using a spectrophotometer at 750 nm.Gallic acid was used as a standard to construct a calibration curve.
From the obtained straight-line equation, the total phenolic compound content was calculated, expressed in µg GAE/mg extract (micrograms of gallic acid equivalent per milligram of extract).
The total flavonoids content was determined by the colorimetric method with aluminum chloride (AlCl3), performed according to the methodology adapted from Perdigão ( 2012).An aliquot of 2 mL of the extract was diluted in distilled water (1 mg/mL) and transferred to a 25 mL volumetric flask.Then, 0.6 mL of glacial acetic acid, 10 mL of pyridine and water solution (1:4, v/v), and 2.5 mL of 7.5 % (w/v) aluminum chloride solution were added, completing the volume up to 25 mL with distilled water.After 30 minutes, the samples were read at 420 nm in a spectrophotometer.A blank was also prepared using all the above reagents except the aluminum chloride sample.Quercetin concentrations used to establish the standard curve were The total tannins were quantified according to the methodology of Pansera et al. (2003).
The crude extracts were diluted in distilled water (1 mg/mL).In a test tube, 1 mL of the diluted extract and 1 mL of the Folin-Denis reagent were added.The solution was homogenized, and after 3 minutes, 1 mL of 7.5 % sodium carbonate solution was added, and the mixture was vortexed.After 1 hour of incubation in the dark, the reaction tubes were centrifuged at 2,000 RPM for 5 minutes.The supernatant was subjected to absorbance readings in a spectrophotometer at 750 nm.Gallic acid was used as a standard to construct a calibration curve.
From the obtained straight-line equation, the total tannin content was calculated, expressed in µg GAE/mg extract (micrograms of gallic acid equivalent per milligram of extract).

DIRECT APPLICATION TEST
The extracts were prepared according to previously stipulated concentrations on a logarithmic scale, adjusted based on a value previously empirically tested in a pre-test, aiming to observe 95 % mortality of R. indica individuals.Upon observing such mortality, a logarithmic scale interval was schematically outlined, obtaining concentrations through arithmetic progression, following the model suggested by Carvalho et al. (2017).The following concentrations were determined: 0.00 % (control), 1.00 %, 1.64 %, 2.70 %, 4.44 %, 7.30 %, 12.00 %.For the dilution of each concentration and formulation of different extract treatments, the predetermined quantity in grams of the fine powder was transferred to an Erlenmeyer flask containing 1 mL of alcohol (solubilizer), 1 mL of Tween ® 80 adhesive spreader (0.05 % v/v), and the volume was completed with distilled water to reach 100 mL.Subsequently, the extract was kept under homogenization on a magnetic stirrer (240 rpm) for a period of 24 hours, taking care to seal and protect from light.Each concentration was applied on adult female R. indica

STATISTICAL ANALYSIS
The design used was completely randomized (CRD).For eachsolution, mortality data were corrected using the formula of Abbott (1925), and subsequently subjected to Probit analysis (p ≤ 0.05), using the statiscial program R. From the obtained equations, the LC50 and LC90 were calculated for each solution applied on R. indica.

RESULTS AND DISCUSSION
The slope of the concentration/mortality curve was 1.44, estimating an LC90 of 23.67 %, with a confidence interval between 14.03 % and 62.05 % (Table 1).The concentration of 12 %, the highest used in the experiment, showed mortality of 88 % of R. indica (Figure 2).The toxicity of the aqueous extract of Sicilian lemon peel on adult red palm mites may be associated with the chemical components present in the solution, identified through chemical characterization (Table 1).In a study, Ribeiro et al. (2019) evaluated the use of essential oil obtained from C. limon peels on the two-spotted spider mite, Tetranychus urticae, (Koch, 1836) (Trombidiformes: Tetranychidae), observing high mortality of the individuals.In addition to its toxicity, Sicilian lemon oil also acted to reduce the fertility of the mite.
The acaricidal and sublethal effects found by the authors were mainly attributed to the compounds α-terpineol and linalool, which are part of the terpenes group, being the latter considered a neurotoxic compound.The mode of action is not fully understood; however, it is inferred that it increases the activity of sensory nerves, causing hyperexcitability of motor nerves leading to seizures and paralysis (DUROFIL et al., 2021;BAVA et al., 2022).
Additionally, in literature, the presence of terpenes in extracts is often directly associated with the mortality of insects and mites (LIU et al., 2021;STRAVOPOULOU et al., 2021;ALAM et al., 2022;KOTSINIS et al., 2023).
The tannins, the compounds with more expressive concentration in the C. limon extract, are constituted by complex structure molecules (BULE et al., 2020;SAHAKYAN et al., 2020), which permits the easier interaction with proteins, enzymes and alkaloids (EMAM & IBRAHIM, 2020), so that their different forms can have different biochemical effects in herbivore arthropods, causing mortality and deterrence (CHEN et al., 2022).Therefore, tannins can act as endogenous growth inhibitors (EL-ASWAD et al., 2023), anti-nutritional substances and can also act in the defense against biotic and abiotic stresses (PRATYUSHA, 2022).In situations of herbivore animals and pathogens attacking, the plants can produce phenolic compounds as a method of repelling harmful species or attracting natural enemies species (CHOWDHARY et al., 2021).Furthermore, it can intoxicate herbivores organism, causing interferences in biochemical processes, damaging their survival, growing, feeding, reproduction and physiological functions (HÝBL et al., 2021;YANAR et al., 2022;PUNIA et al., 2023).
The flavonoids have important roles in the plant metabolism, and can also act as defense substances against pests and pathogens attacks (STEC et al., 2021).Upon entering the arthropods' organism, it can cause biochemical interferences, affect their behavior, reduce the resistance, damage metabolic and physiological processes, such as growing, development, reproduction and oviposition, and lead to death by acute toxicity, feeding deterrence, reducing the nutrient absorption or corrupting metabolic pathways (GAUTAM et al., 2023;YANG et al., 2023;NGATSI et al., 2024;VOLF et al., 2024).
In addition to the mortality obtained in the tests, through visual observation, it was noted that some individuals of R. indica showed the behavior of climbing the edge of the arenas and cotton, suggesting a repellent effect of the extract.The absence of egg-laying on the plates was also observed, suggesting the ovicidal effect of the extract.However, further studies are still needed regarding the effect of compounds present in the Sicilian lemon ripe peels, as well as field tests to ascertain the efficacy of the extract in the management of the red palm mite.

CONCLUSION
The Citrus limon ripe peels aqueous extract was efficient in the management of Raoiella indica under laboratory controlled conditions, showing 88 % of mite mortality in the concentration of 12 %.In the chemical characterization was found a high concentration of tannins, inferred as the responsible for the extract's acaricidal effect.

Figure 1
Figure 1Location of the experimental site.
photoperiod.The acaricidal effect was evaluated at 24, 48, and 72 hours after spraying, and the cumulative total number of dead R. indica individuals was counted at the end of this period.

Figure 2
Figure 2 Mortality of R. indica under different concentrations of C. limon ripe peels aqueous extract.

Table 1
Chemical composition of C. limon ripe peels aqueous extract.