Insecticidal Activity of an Indian Botanical Insecticide ULTRA ACT ® against the Olive Pest Bactrocera oleae (Diptera: Tephritidae) in Tunisia

Bactrocera oleae is the most economically damaging insect of olive in the Mediterranean. As a reliable alternative to synthetic chemical insecticides, botanical pesticides from plant extracts are also considered natural control methods safe for the environment and human health. In practical applications up to date, studies demonstrating toxically effects of biological control agents on the insect have been carried out in organic farming systems based on some industrial microbial formulations. But much less attention has been given to botanical insecticides against B. oleae. Thus, a certified botanical insecticide issue from various plants and approved per Indian and International organic standards was tested against larvae and pupae of the olive pest Bactrocera oleae in the east of Tunisia under laboratory conditions. The experiments were conducted with 3 normal treatments and 1 control. Two techniques were used for larvae and pupae: direct contact using concentration from 0.5; 0.750; 1 and 1.5 mg/l and fumigation treatment using 2 mg/l conducted in rectangular boxes (25˚C - 27˚C, 75% - 85% RH and 14:10 LD photo period). For larval stages, LC50 and LC90 values for Ultra-Act were 0.45 and 1.22 mg/L in direct contact and 1.5 - 2 mg/l with fumigation, respectively. For pupal stage, the ID25 and ID50 values were estimated to be 0.71 mg and 1.26 mg respectively corresponding to the inhibition of emergence of 25% and 50% of B. oleae adults from pupa. Our results indicate that sprayed Ultra-Act product has the potential to control the three olive fly instar larvae. Pupal emergency was decreased significantly with fumigation treatment. In conclusion, Ultra-Act is a promising botanical insecticide against B. oleae that could be used as a successful alternative for chemicals in integrated control methods of this pest in Tunisia.


Introduction
The intensive use of pesticides has been the major practice in crop protection for decades to control pests and plant pathogens to avoid yield losses. Bactrocera oleae Gmel. (Dipt., Tephritidae) is the major pest of commercial olives worldwide (Daane and Johnson, 2010) [1]. The insect accounts for approximately 30% -40% of loss of olive production and the affected olive fruit is unsuitable for processing a good quality of olive oil. It is estimated that in the Mediterranean region (Fimiani, 1989) [2], currently, the most common control practices against this pest are based on the use of synthetic insecticides (Katsoyannos, 1992 [3]; Nardi et al., 2006 [4]; Daane and Johnson, 2010 [1]). But this has created problems related to adverse environmental effects, resistance of the insect pest and negative impact on non-target organisms and natural enemies in addition to high operational costs. Besides, common insecticides such as dimethoate failed to protect olives for as long a period after the last spray. Research for biological control alternatives against this pest is based essentially on mass trapping methods and the use of natural product-based pesticides such as the Bacillus thuringiensis (Bt), spinosad that offer a more sustainable solution to pest control than synthetic alternatives (Lambert and Peferoen, 1992 [5]; Federici and Sakano, 2006 [6]; Sameh et al., 2009 [7]). Strategies involving semiochemical-based products have been pursued in the development of integrated pest management strategies for B. oleae (Mazomenos and Haniotakis, 1981 [8]; Gil-Ortiz, 2012 [9]; Canale et al., 2014 [10]). However, still heavy B. oleae infestations were recorded in case of enormous populations so that further applications are required for efficient control (Saour, 2004) [11].
Insecticides of botanical origin are considered eco-friendly products for control of crop pests and have been reported as easily biodegradable, effective safe control, not toxic to human and domestic animals and useful for control. In this regard, interest in plant botanical derivatives has been extensively used for insect control (Balandrin, 1985 [12]; Perich et al., 1994 [13]; Giatropoulos et al., 2018 [14]). More than 1200 plant species are already known to have insecticidal properties (Sukumar et al. 1991) [15] and the current state of knowledge on larvicidal plant species has been reviewed (Shallan et al., 2005) [16]. The Neem (Azadirachta indica A. Juss) belonging to the Meliaceae family is one of the most recognized plant biopesticide in progress replacing toxic insecticides (Chaudhary et al., 2017) [17] with highly larvicidal toxically effects on insects tephritidae (Vi-Advances in Chemical Engineering and Science jayalakshmi et al., 2016). Manufactured products from plant based biopesticide offer today an important perspective of being widely used according to their share in the global market. Numerous products are produced today by relatively small companies of local value and developed on the basis of active substances in plant species for the protection of harmful insects.
The product ULTRA ACT  is an organic certified botanical pesticide considered as an eco-friendly alternative to unsafe chemicals. Various plant species, seeds and animal wastes used in organic farming systems are applied in manufacturing formulation (ISO9001 certified company Vivekon, 2015) [18]. Regarding the importance of botanical plant insecticides for the insect pest control, ULTRA ACT  is an organic certified botanical pesticide developed by Vivekon Company, India that provide a new opportunities for plant protection with new Insecticides of botanical origin. In the ecological context adopting the organic farming system sustainable and environmentally, this product is a biologically active compound mentioned as an eco-friendly plant-derived insecticide. ULTRA-ACT  is a combination of various plants, seeds and animal extracts. The pesticidal efficacy, environmental safety and farmers acceptability of ULTRA-ACT for control of common crop insect pests such as whitefly, mealy bug, mites, jassid thirps, armyworms, round worm, thread worm caterpillar, borer larva, paddy larva and all other similar sucking insect pests., has led to its adoption into various crops for insect pest control programmes in many countries. The present paper aims to evaluate the effectiveness of ULTRA ACT based biopesticide as a sustainable means to control the olive fly B. oleae under laboratory conditions. The research study intends to examine the larvicidal activity of the product and to discuss its fumigant insecticidal potential on the pupal growth of the insect.

Insect Rearing
Larvae of second and third instar of B. oleae were obtained from laboratory-established colony from infested olive fruits collected in a Tunisian olive farming grove in Sousse Government (Bouficha). Fruits were put in aerated plastic boxes and supplemented with distilled water and examined each day separately under binocular to isolate larvae with available stage and vivacity.

Preparation of ULTRA ACT  Tests
The commercial formulation of ULTRA ACT  product was dissolved in distilled water and used in all experiments. Two techniques were used: direct contact for larvae and fumigation treatment for pupae. Four concentration were used for each technique 0.5; 0.750; 1 and 1.5 mg/l. Each concentration preparation was kept in a small glass bottle (50 ml) and stored at room temperature 25˚C until experimental use.

Bioassays
Effects of ULTRA ACT  on larval mortality Advances in Chemical Engineering and Science For direct contact experiment, ten fruits with larvae were put in a separate set of petri dishes. A total of 5 dishes were used for each concentration treatment as well as 5 dished take up as control without insecticide. As method of treatment, the liquid insecticide product is applied directly to larvae using a small jet sprayer. The bioassay was conducted with 3 replicates. Dishes were covered with a tissue permeable to provide aeration and to avoid the escape of the larvae. The data of the larval mortality was assembled after 24, 48 and 72 hours for 2nd and 3th instar of B. oleae. The dead larvae were removed after every adding up. The percent corrected mortality was calculated using Abbott's formula (Abott, 1925) [19] and Log probit analysis was used to determine the median lethal concentration LC50 and 90% lethal concentration LC90 of the product.
Effects of ULTRA ACT  on pupal emergency Fumigation treatment of a box rearing habitat aims to test effect of insecticide residues on pupa before they can mature into adult and emerge in nearby area with fruits. For fumigation treatment test, a total of twenty infested fruits with their nearby pupa (olive fruit and pupae) were put into an aerated box that was previously pulverized with the insecticide. Five replicates of each concentration were run under the same conditions along with untreated control.
Pupal emergency was numbered by counting emerged adults from pupa after one week of pupation. Emerged adults were removed after each count. Pupa missing emergency were considered as inhibited. The inhibition percentages of adult emergence were also corrected in accordance with Abbott (Abott, 1925) [19]. The inhibition doses (ID) ID25 and ID50 (doses causing 25% and 50% inhibition of adult emergency) were determined with corresponding 95% fiducially limits (95% FL) by means of a non linear regression.
Effects of ULTRA ACT  on pupal weight ULTRA ACT  was administrated by fumigant application at its ID25 and ID50 on 1-day newly ecdysed pupae (<24 h). Three replicates with 20 pupa were used in each ID treatment and 20 pupa used as control. The pupal weight was registered each 2days after treatment until the emergence of adults.

Statistical Analysis
Results were presented as means and standard deviation (S.D). The homogeneity of variances was checked by a Levent's test. The significance between different series was tested using Student's t-test at 5% level. Data were subjected to one-way or two-way analysis of variance (ANOVA) followed by a posthoc Tukey test HSD. All analyses were carried out using the SAS package (SAS Institute, 2013).

Toxicity Effect on Larvae
Trials with ULTRA ACT  on B. oleae larvae showed highly significant results Advances in Chemical Engineering and Science (Tukey HSD) at 1 mg/l and 1.5 mg/l, showing 69.3 ± 1.8 (%) and 88.2 ± 3.36 (%) of the 2nd larval mortality respectively (p < 0.001), and 58.2 ± 3.2 (%) and 72.4 ± 1.4 (%) of the 3th larval mortality respectively (p < 0.001) ( Table 1). The second and third instars of B. oleae are both affected by the insecticidal product, in comparison with controls where there is no difference between arbitrary mortality due to the experiment whether; 3.25 ± 2.2 (%) and 2.66 ± 2.3 (%) of the 2nd and the 3th larval stage respectively (p > 0.05). Therefore, results revealed that earlier larval instars (L2) of B. oleae are more susceptible to ULTRA ACT  than older instars (L3) (p < 0.001) ( Table 1).
The concentrations resulting in 50% and 90% mortality of the larvae were determined with 95% confidence limits (95% FL, R 2 = 0.99) using an exponential regression equation (Figure 1

Fumigant Toxicity on Pupa
ULTRA ACT  product applied on newly ecdysed pupae causes an inhibition of adult emergence with a dose-response effect ( Figure 2). Indeed, our data revealed that the inhibitory effect of adult's emergency from treated pupae, vary between 20.19% ± 2.35% for the lowest concentration (0.5 mg/l) to 68.56% ± 3.15% for the higher concentration (1.5 mg/l). The Tukey HSD test indicated that there was a significant relationship between the different tested doses (p <0.001). In addition, the inhibition recorded in controls was 4.86% ± 1.19% ( Figure 2

Weight of Pupae
The weight of pupae was significantly affected by ULTRA ACT  fumigation.
Data showed that respective ID25 and ID50 caused a significant reduction in the weight of pupa compared to controls during the pupal stage at 6 (p < 0. 0001) and 2 days (p < 0.0001) respectively. Pupae obtained from fumigation treatment were significantly heavier than those in control box (F 2,19 = 3.42; P < 0.0025).
Furthermore, there was no significant difference between the weight of pupa for the two tested doses ID25 and ID50 after 6 days of pupal stage (p > 0.05) ( Table   2).

Discussion
The olive pulp was fed to the larva of Bactrocera oleae by incorporation into ULTRA ACT  at various concentrations under controlled conditions. 1 mg/L ULTRA ACT  caused a significant dose-dependent increase in mortality of   (Schmutterer, 1990;

Conclusion
This study demonstrated significant improvement in the biological control program of Bactrocera oleae, the major pest of olives worldwide, using a new based biopesticide from botanical origin; ULTRA ACT  . Important steps for conducting experimental method have been provided. Insect sampling, rearing material and conditions are provided as well as the experimental design for laboratory bioassay focused on lethal and sublethal effects of the product where two techniques are used: direct contact for larvae and fumigation treatment for pupae.
Interesting results show larvicidal activity from inhibiting doses I25 and I50. Indeed, statistical data revealed a significant fumigant inhibitory effect of adult's emergency from newly treated pupae. Further discussions on ULTRA ACT  potential on the pupal growth of the insect are provided. Manufacturing process of the botanical product ULTRA-ACT  may consequently standardize ratios of selective bioactive toxic substances that can significantly increase the biological insecticidal efficacy and enhance the potential fumigant insecticidal effect. Prospects for biotechnological production of insecticides from botanical plants in India grant a new opportunity for biological control against the olive fly insect pest. In this study, it appears that the pesticidal efficacy of the manufactured product ULTRA-ACT  proposes multiple undoubted benefits not only due to its environmental safety and manageable use against common Indian crop insect pests but particularly due to its importance likely related to the presence of bioactive metabolites with insecticidal potential against larvae and pupa of B. oleae insect pest in organic olive crops.