Phytochemical composition and insecticidal effects of native plant extracts against the mango mealybug, Rastrococcus invadens Williams (

The mango mealybug Rastrococcus invadens Williams causes enormous damage to fruit trees in Burkina Faso, with losses of up to 90% for mango trees and 53% for citrus fruits. The objective of this study was to evaluate the insecticidal effects of native plant extracts against this insect pest. The insecticidal activity and biological effectiveness of the extracts were evaluated on Petri dishes and mango leaves previously contaminated by spraying them with the native plant extracts. The experimental was a completely randomized Fischer block, and compared six doses of the hydroalcoholic extracts of A. conyzoides and H. suaveolens with abamectin 18 EC and water (control). These treatments were repeated up to ten times. The determination of the efficiency coefficients was achieved using Abbott's formula (1925). Both extracts of A. conyzoides and H. suaveolens were effective, but the one based on A. conyzoides at 50 gl -1 was the most effective with 100% mortality in 48 hours compared to 95.00% for H. suaveolens’ extract in 72 h. Their respective LC50 were 12.50 gl -1 and 17.49 gl -1 , showing that A. conyzoides’ extract is more toxic to R. invadens . The high content in phenols, flavonoids, alkaloids, etc. of leafy stems of A. conyzoides and of H. suaveolens could help to sustainably control R. invadens in an integrated approach and effectively protect mango orchards.


INTRODUCTION
The mealybug Rastrococcus invadens Williams (Homoptera: Pseudococcidae) is a major insect pest of fruit trees including mango and citrus.This mealybug causes enormous damage to the mango tree, up to 90% loss of mangoes and 53% loss of fruit yield in citrus fruits (FAO, 2022) .Currently, this insect pest is distributed in over 27 countries in Africa, Asia, and French Guiana in Southern America (CABI, 2022).Nymphs and females feed on fruit and leaves leading to severe damage.These produce honeydew which causes sooty mold, leading to a reduction in production.Rastrococcus invadens is a polyphagous insect pest attacking more than forty-five plant species in twenty-two families, including economically important crops such as mango and citrus, Citrus spp.(Nébié et al., 2018, Fall et al. 2019).The means of control undertaken throughout Africa, such as the destruction of the attacked organs or insecticide treatments, have gradually proven to be ineffective with toxicological risks and more growing invasion due to the great dispersion of host plants and to climate change (Germain et al., 2015, Azrag et al., 2023).Treatment with synthetic insecticides is not always effective and safe and leads the mango orchards owners to alternative practices, mainly the usage of botanicals.Synthetic insecticides, such as Thiametoxam (neonicotinoid) and Chlorpyrifos-ethyl (organophosphorus) evaluated in real environments, have proven effective against the insect pest, but remain incompatible with the action of parasitoids associated with R. invadens (Nébié, 2011).Furthermore, the resistance to insecticides has been demonstrated for many years by several authors and the use of these pesticides is proving to be dangerous for ecosystems and human health (Ahmad et al., 2010;Abd El-Rahman et al., 2021).These pesticides even annihilate the action of parasitoids used in biological control programs against this insect pest in West Africa (Mano et al., 2022, Mohamed et al.,2022).However, organic insecticides based on extracts and essential oils from indigenous plants could constitute a sustainable alternative.Numerous studies have previously revealed the insecticidal potential of plant extracts which, according to Mano and Nana (2022) also preserve natural enemies.For example, neem and Mahogoni extracts were reported to exhibit good control of the mango leafhopper Idioscopus clypealis, (Shawan et al., 2018).The same was shown with neem oil against the same insect pest on the mango tree (Ferdous and Jahan, 2020).The use of plant extracts in plant protection is non-polluting and less toxic compared to synthetic insecticides.This justifies the importance of the present study, which aimed at evaluating the insecticidal effects of native plant extracts against the mango mealybug, R. invadens.

Insects and insecticides
The mealybugs were collected from untreated mango tree plants using bags in the city of Bobo Dioulasso in April 2023.They were bred in the laboratory (T°: 38 ± 2 °C; RH: 70%; PP : 12/12h) until the adults of the F1 generation were obtained.Identification was made by possible using the Williams (1989) and Williams and Granara De Willink (1992)

Preparation of extracts
Powders from the leafy stems of A. conyzoides and H. suaveolens were used as solid-liquid extraction substrate.For this purpose, 50 g of each leafy stem powder were extracted by maceration with 0.5 L of 80% ethanol in 1 L Erlenmeyer flasks under constant mechanical stirring using a laboratory shaker for 24 h.An additional volume of 125 ml of the extracting solvent was added under a percolator and filtered through cotton.The total extracts obtained were dried in an oven (40 ± 1 °C) to 100 gl -1 and used as insecticide products.

Evaluation of insecticidal activity
The host mango tree leaves were used as a trophic support during the various tests in the laboratory from April to June 2023.The insecticidal activity of the extracts was evaluated under laboratory conditions in a Fischer block with four treatments [Abamectin (manufactured by the company SAVANA based in Burkina Faso), water control, extracts from A. conyzoides and H. suaveolens] repeated ten times.Mango leaves were previously cut into leaf discs and placed in Petri dishes.Each box received 10 adult mealybugs.and foliar contamination of 0.6 ml of extract.The number of dead or weakened individuals was counted under a binocular microscope after 24; 48 and 72 hours of exposure.As for dose effects, concentrations of 12.5; 25 and 50 gl -1 of the most active extracts were tested on Petri dishes as previously.

Evaluation of the biological effectiveness of insecticides
Four treatments including the extracts of A. conyzoides 50 SL and H. suaveolens 50 SL were compared with abamectin 18 EC and water as a control in the same device above.The infestation was recorded from the average number of R. invadens per mango leaf.The mortalities' means of the mealybug after 72 hours of treatment were calculated and submitted to the Abbott formula (1925) for the determination of the efficiency coefficient.

Phytochemical screening
The chemical screening of the extracts was carried out by the qualitative method described by Ciulei (1982).These are general and/or specific chemical reaction tests for coloring or complexation in a liquid medium.The aim is to highlight the bioactive phytochemical groups involved in the insecticidal activities observed.Quantitative analysis was performed following the standard procedure for phenols (Ainsworth and Gillespie, 2007), flavonoids (Chang et al., 2002) and alkaloids (Puro et al., 2018).

Statistical analysis of data
R software version 4.0.3 was used for data analysis.The Chapiro test verifying the normality of data and that of Fligner-Killeen attesting the homogeneity of variances allowed the analysis of variance of mortalities.The means were compared using the Tukey test at 5% threshold.Lethal concentrations LC50 and LC90 were determined using logistic regression by probit analysis.

Extraction yield
The yields of hydroalcoholic extracts were higher in A. conyzoides.A mass of extract of 16.50 g or 8.25% was obtained with A. conyzoides compared to 12.86 g or 6.43% for H. suaveolens.

Insecticidal activity of the extracts on R. invadens
As demonstrated by the analysis of variance (Table I), the organic extracts studied induced mortality on adults of R. invadens with very highly significant differences (P<.001;F ≥ 244.10).The extracts increased mortality proportionally to their doses.The mortalities induced by the extract of A. conyzoides varied from 50.00% with 12.5 g/L to 92.50% with 50 g/L in 24 hours and 50.11% with 12.5 g/L to 100% with 50 g/L in 72 hours.As for those of H. suaveolens extracts, the transition from 12.5 g/L to 50 g/L resulted in mortality rates varying from 37.50 to 90.00% in 24 hours, from 47.50% to 92.50% in 48 hours and 50.00% to 95.00% in 72 hours.

Biological effectiveness of plant extracts on R. invadens
The data presented in Figure 1 showed variable biological effectiveness coefficients following the different treatments.The highest biological effectiveness (100.00%) was recorded with the extract of A. conyzoides 50 SL, followed by that of H. suaveolens 50 SL (94.75%) which is in close proximity to Abamectin 18 EC (79.00%).

Phytochemical composition of extracts
The chemical characterization test of the two active extracts showed the presence of polyphenols, flavonoids, leucoanthocyanins, tannins, alkaloids, saponins, quinones and anthraquinones and essential oils (Table III).Leucoanthocyanins, quinones and anthraquinones were absent in A. conyzoides while sterols and terpenes were not detected in H. suaveolens.Quantitative analysis indicated phenolic, flavonoid and alkaloid contents respectively of 0.50; 143.29; 125.48 mg/g of dry matter of H. suaveolens versus 7.54; 29.25 and 2.33 mg/g of dry matter of A. conyzoides.

DISCUSSION
An insecticidal activity causing mortalities of 50.00% to 95.00% of R. invadens in 72 hours was obtained with the hydroalcoholic extracts of H. suaveolens.Toxicological studies also reported an LC 50 of 17.49 gl -1 .These results confirmed those of several authors.According to Thiam et al ., (2017) the aqueous extract of H. suaveolens at 187.5 gl -1 is effective against H. armigera.Extracts from this plant help to reduce losses (Johnson et al., 2018) .Some authors exhibited larvicidal activity against the larvae of the yellow fever mosquito Aedes aegypti (L) and Aedes albopictus (Conti et al., 2012 andTennyson et al., 2018).But what explains these biological activities detected in H. suaveolens?In this study, a wide range of chemical compounds were isolated from this plant.These were polyphenols, flavonoids, tannins,alkaloids,saponins, leucoanthocyanins, quinones and anthraquinones and essential oil.Previous studies indicated that the biological activities are due to compounds such as alphapinene, beta-pinene, sabinene, terpinolene, betacaryophyllene, and 4-terpineol (Conti et al., 2012).Phenolic compounds for example, which are the most abundant secondary metabolites synthesized by plants subjected to biotic and abiotic stresses to adapt and survive, are potentially toxic for many pests (Dai and Mumper, 2010).Furthermore, this study reports that extracts of A. conyzoides caused mortalities of 50.11% and 100% in 72 hours at 50 gl -1 with a CL 50 of 17.49 gl -1 .These data confirmed the results of several studies which have shown that the variety of Ageratum species has excellent biological properties.Arya et al. (2011) and Vasantharani et al. (2022) reported various biological properties, such as antifungal, cytotoxic, nematicidal, antifeedant and larvicidal activities.These occurring activities could be related to the presence of various phytochemicals, such as bromophenols, polysaccharides, tannins, flavonoids, phenols, phenolic acids and carotenoids (Chandraker et al., 2019;Puro et al., 2018).
Phytochemical screening of leafy stem powder of A. conyzoides indicated the presence of secondary metabolites such as sterols and terpenes, polyphenols, flavonoids, catechic and gallic tannins, alkaloids and saponins.These results are not very similar to those recently reported by Chabi-Sika et al. (2023) but very similar to those of Okereke et al. (2017) and Ndacnou et al. (2020).These authors demonstrated in A. conyzoides the presence of various phytochemical compounds such as terpenoids, alkaloids, flavonoids, chromenes, coumarin, saponins, tannins, glycosides, phenols and resins.Variations in phytochemical profile would be due to the screening methodology, the nature, polarity and concentration of solvents, the collection area, the nature of the soil and the stage and organs of the plant used (Nawaz et al., 2020, Lefebvre et al., 2021).Phenolic compounds produced by plants contribute to their antioxidant capacity compared to other natural resources.Polyphenolic compounds such as flavonoids highlighted in this study, possess various biological activities including the formation of complexes with the extracellular wall (Ichino et al., 2014).

CONCLUSION
This study aimed at evaluating the insecticidal effects of native plant extracts against the the mango mealybug, R. invadens.Hydroalcoholic extracts based on leafy stems of A. conyzoides and H. suaveolens at 50 gl -1 have effective control against R. invadens.The one from A. conyzoides is more toxic to the mealybug.These plant extracts are rich in phytochemical compounds such as phenols, flavonoids, alkaloids, etc. which are natural resources with insecticidal properties.These active ingredients could contribute in effectively controlling mango mealybug in an integrated approach.Their application in mango orchards could increase fruit yields while preserving the ecosystems and environment of tropical areas.Not having been able to handle the young stages of the mealybug and deepen the chemical analysis, further studies should focus on the mechanism of action of each extract.Knowledge of the mechanism of action will help in the development of complexes of irresistible insecticide principles against R. invadens and other related homopteran insect pests.

Table I :
Analysis of mortality of R. invadens caused by treatments NB: Means in the same column, assigned with the same letter, are not significantly different at the 5% threshold (Tukey test); ***: Highly significant

Table II :
Analysis of extracts toxicity on R. invadens Figure 1: Biological effectiveness of treatments using the Abbott formula