A PESTICIDE EFFECT STUDY ON BACTROCERA OLEAE IN CENTRAL GREECE

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1 A PESTICIDE EFFECT STUDY ON BACTROCERA OLEAE IN CENTRAL GREECE D.G. Stavridis 1, P. Ioannidis 1, P.N. Deligeorgidis 2, N.P. Deligeorgidis 2 and C.G. Ipsilandis 3 1: Region of Central Greece, Dpt. of Rural Economy and Veterinary of Larissa, Larissa, Greece. 2: Technological Education Institute of Western Macedonia/Branch of Florina, Dpt of Crop Production. Terma Kontopoulou, Florina, Greece. 3: Regional Administration of Central Macedonia, Dpt. of Rural Economy and Veterinary of Regional Unity of Thessaloniki, Al. Papanastasiou 61,54453 Thessaloniki, Greece. ABSTRACT The purpose of this study was to estimate olive fruit fly s response in pesticides that are used for decades in control programs and to explore possible resistance adopted by olive fruit fly, resulting in ineffective pesticide application decreasing olive production in Meliviaarea (Larissa, Thessaly, Central Greece).Two different pesticides were used:dimethoate and Cypermethrin. After pesticide application, insect mortality was estimatedand LD 50 was calculated byprobit analysis. 3 different recordings of bioassays showed a progressive decrease in LD 50.Females showed greater LD 50 values, indicating a kind of resistance,since in nature are exposed for a longer period to pesticides.adult susceptibility to the particular tested pesticides was found to be different. Generally, flies were susceptible to Cypermethrin and showed resistance to Dimethoate. Key-words: monitoring, olive fly, probit analysis,susceptibility Introduction The olive fly Bactrocera oleaerossi, is a species of fruit fly which belongs to the Dacinae subfamily (Diptera, Tephritidae). It is a kind of phytophagous species, whose larvae feed on the fruit of olive trees, hence the common name. It is considered the most serious pest in the cultivation of olives. Now detected also in the United States, and its range coincided with the range of the olive tree in the Eastern Hemisphere: Africa, Southern Europe, Canary Islands, India, Western Asia. In the Western 1

2 Hemisphere, it is currently restricted to California.The olive fruit fly was first detected in North America infesting olive fruits in Los Angeles County. This species is associated with plants of the Olea europea. It is considered the most serious pest towards olives in regions where it presides, significantly affecting both the amount and quality of production in most olive growing areas. The impact of its attacks tend to worsen in the more humid and cooler growing areas, with significant variations depending on the variety grown, where it affects olive trees and areas which have hot summers and less drought. In Central Greece, olive tree arboriculture is now widespread and this resulted in the presence of olive flies in significant populations (Koufali, 2009). Adults are 4-5 mm long, being easily recognized in conjunction with other Tefritidae for the small angled dark spot at the apex of the wing and the length of the narrow, elongated anal cell. The adult male has a hardened wing at the top of the anal cell, which is longer than the female's. The third urite shows the pectorals. The adult female has a yellowish head with two strong circular spots under the antennas close to the compound eye, whilst the eyes are bluish-green. The chest can show various specks instead of the typical bands and lines. The mesonotum is bluish-gray with three blackish longitudinal lines. The humeral callus and areas mesopleurali metapleurali and mesoscutello are ivory. The wings are hyaline, with part of the pterostigma with brown specks at the apex. The abdomen is light brown with variable colourings: typically there are pairs of blackish spots on the first to fourth urotergit, which often come together in bands. The ovipositor is clearly visible, partly invaginated in the seventh urite which is always black. The chemical battle against the olive fruit fly can be implemented against larvae by using preventive treatments against adults. The treatments are carried out by spraying the olive trees with insecticides dimethoate and deltamethrin. The insecticide dimethoate is commonly used for its effectiveness and relatively low cost. It is also considered that this may be preferable because it would leave low residues in olive oil since it is water soluble and would pass through the amurca. In the near future, legislative authorization for the withdrawal of use of dimethoate is to be implemented. Among the products with low impact should be cited azadirachtin, a natural repellent extracted from the fruits of the Neem-tree. However, its effectiveness against the olive fruit fly has not yet been sufficiently tested. Among the organic insecticide literature rotenone is also mentioned, however the use of this active ingredient, not readily available, must be authorized by established competent bodies after demonstrating the need for it. The larvicidal treatment is carried out according to the criteria of the scheduled pest management, pest management and integrated pest management. Other alternative methods of insect control are described by Bueno and Jones (2002) and Tsolakis et al. (2011). Generally, in Greece, large numbers of pesticides are used in order to keep pests in low populations in a variety of cultivations and for many years (Deligeorgidiset al., 2007). Thesefacts highlight the priority of exploring possible resistance to pesticides (Kakaniet al., 2010).Vontaset al. (2002) and Hawkes et al. (2005) reported possible resistance adopted by olive fruit fly due to mutations in specific alleles for populations collected (among other) in many places in Greece. The purpose of this study was to estimate olive fruit fly s response in pesticides that are used for decades in control programs and to explore possible resistance adopted 2

3 by olive fruit fly, resulting in ineffective pesticide application decreasing olive production in Larissa area (Thessaly, Central Greece). Materials and Methods Aiming to study olive fruit fly Bactrocera oleae Rossi (Diptera: Tephritidae) response in pesticide applications, in year 2005 infected olive fruits were collected from Melivia area (Larissa, Thessaly, Central Greece, Fig. 1) and transferred in laboratory, in controlled conditions 16L:8D photoperiod, 24±1 C air temperature and 70% relative humidity (Koufali, 2009). Olive fruits were placed in to plastic containers filled with sand and larvae were moving in the sand to develop pupa stage. Every day, sand was sifted through sieves and collected pupae were put into petri plates. Petri plates were inspected in a daily basis and newly developed adults were moved into cups of ten samples (five females and five males), covered with napkin. For feeding adult insects, a piece of cotton infused with sucrose solution 5% was put into cups. Adults 24h old were transported into a proper cage where narcosis was achieved by carbon dioxide application for 15 seconds. Carbon dioxide was not found to have any effect on the insecticide susceptibility (Hsu et al., 2004). Narcosis lasted for only 45 seconds and thus pesticides were applied rapidly by suitable pipettes into the scutellum of the insects. Two different pesticides were used:dimethoate and Cypermethrin. After pesticide application, insect mortality was monitored according to pesticide dosage. Pesticide was applied in two replications (with ten days interval) and total estimation of LD 50 was performed.threeseparate recordingsfor estimating LD 50 were made: at 48h, 6 days and 14 days. Statistics were based on probit analysis (Bliss, 1935) and performed in SPSS (2010) computer software. Probit analysis is a type of regression used with binomial response variables. It is very similar to logit, but is preferred when data are normally distributed. Most common outcome of a doseresponse experiment, in which probit analysis is used, is the LC 50 /LD 50 (Vincent, 2010). Pesticide dosage was measured in ng per insect. Results Table 1 presentsld 50 estimations through 3 recording periods. In case of Dimethoate, in 3 different recordings of bioassays, LD 50 values are decreasing from to 8.076ng per insect. In case of Cypermethrin, some X 2 values were high indicating a kind of heterogeneity in fly population. Table 2 presents pesticide applications and B. oleae response according to probit models for Dimethoate and Cypermethrin, for male and female adults. Females showed 2 to 3-fold greater LD 50 values (68.288±1.02 and50.331±1.03ng per insect for 1 st and 2 nd application respectively) than male flies (23.14±1.64and34.743±1.017ng per insect for 1 st and 2 nd application respectively) for Dimethoate. Differences for Cypermethrin were even greater (0.431±0.02and0.127±0.01ng per insect for 1 st and 2 nd application respectively for females and 0.159±0.01and0.022±0.01 for males respectively). Line equations describing probit model are also demonstrated in Table 2. Slope values were not so different for males and females except for 1 st application of Dimethoate. In general, X 2 values were found low, up to 4.72 for Cypermethrin 1 st application. 3

4 Discussion From the obtained results it is clear that LC 50 values for the treated adult females increased more than the treated adult males. That means that the adult males were more susceptible to the tested insecticides than the adult females. These findings are consistent with the results of Stark et al. (2004) that the males of Ceratitiscapitata were significantly more susceptible than the adult females. Other researchers reported that for other Bactrocera species always males were significantly more susceptible than the adult females, in agreement to our findings(hsu et al., 2004; Ahmad et al., 2010). This may be due to increased female resistance, since females generally live more than males in nature and are exposed for a longer period to pesticides. In such conditions resistant (mutant) alleles may be overexpressedas those described byvontaset al.(2002) and Hawkes et al.(2005). In successive recordings LD 50 tended to be lower and in accordance to the findings of Ahmad et al.(2010) and Moslehet al. (2011). High X 2 values and slope calculations indicated some heterogeneity (Ahmad et al., 2010) in part of the population tested. These findings lead us to the exclusion of some results. Obtained data demonstrated that adult susceptibility to the particular tested pesticides was found to be different based on the calculated LC 50 values. Generally, flies were susceptible to Cypermethrin and showed resistance to Dimethoate, since the last have been used widely in Greece for many years (Hawkes et al., 2005). In many cases gradient pesticide resistance was reported by researchers of various Bactroceraspecies (Hawkes et al., 2005; Ahmad et al., 2010; Kakaniet al., 2010 and Moslehet al., 2011). According to our findings resistance topyrethrin-type pesticides is not yet realized. As a conclusion, resistance to pesticides may result in totally ineffective pesticide applications. Thus, for countries like Greece, Italy and Spain new techniques must be practiced as various biological methods or semiochemicals like pheromones (Haniotakiset al., 1977)for controlling olive fly populations (Bueno and Jones, 2002; Delrioet al., 2003). References 1. Ahmad, S.F., R.R.Khan and M.K. Nadeem(2010) Evaluation of insecticide resistance in two strains of fruit fly, Bactrocerazonata (Saunders) (Tephritidae: Diptera), with fruit dip method. Pakistan Entomologist, Vol. 32(2), p.p Bliss, C.I. (1935)The calculation of the dosage-mortality curve. Annals of Applied Biology, Vol.22, p.p doi: /j tb07713.x 3. Bueno, A.M. and O. Jones(2002) Alternative methods for controlling the olive fly, Bactroceraoleae, involving semiochemicals.iobc WPRS Bulletin,Vol. 25(9),p.p Deligeorgidis,P.N., C.G. Ipsilandis, G. Kaltsoudas, G. Sidiropoulos, N.P. Deligeorgidis, M. Vaiopoulou and A. Vardiabasis(2007) Chemical control of Thripstabaci, Epitrixhirtipennis and Myzuspersicae in Tobacco fields in Northern Greece. Journal of Entomology, Vol. 4(6), p.p Delrio, G. A. lentini and A. Satta(2005)Biological control of olive fruit fly through inoculative releases of OpiusconcolorSzépl.IOBC WPRS Bulletin,Vol. 28(9),p.p

5 6. Haniotakis, G.E., B.E.Mazomenos andj.m. Tumlinson (1977)A sex attractant of the olive fruit flydacusoleae and its biological activity under laboratory and field conditions. EntomologiaExperimentalisetApplicata, Vol. 21, p.p Hawkes, N.J., R.W. Janes,J. Hemingway and J.Vontas(2005)Detection of resistance-associated point mutations of organophosphate-insensitive acetylcholinesterase in the olive fruit fly, Bactroceraoleae (Gmelin).Pesticide Biochemistry and Physiology, Vol. 81(3), p.p Hsu, J.C., H.T.Feng and W.J. Wu(2004)Resistance and synergistic effect to insecticides in Bactroceradorsalis (Diptera: Tephritidae) in Taiwan. Journal of Economic Entomology, Vol. 97(5), p.p Kakani, E.G., N.E. Zygouridis, K.T. Tsoumani, N. Seraphides, F.G Zalom and K.D. Mathiopoulos,(2010)Spinosad resistance development in wild olive fruit fly Bactroceraoleae(Diptera: Tephritidae) populations in California. SOCI, Pest Management Science, Vol. 66, p.p Koufali, N. (2009)Heattolerance of the olive fruit fly, Bactrocera (Dacus) oleae (Rossi), underlaboratory conditions. MScThesis. AristotleUniversity of Thessaloniki. 11. Mosleh Y.Y., S.F.M. Moussa and L.H.Y. Mohamed(2011)Comparative toxicity of certain pesticides to peach fruit fly, BactrocerazonataSaunders (Diptera: Tephritidae) under laboratory conditions. Plant Protection Science, Vol. 47(3),p.p SPSS, (2007). SPSS Manual, SPSS Inc, Chicago IL. 13. Stark,J.D., R.I. Vargas and N.W. Miller (2004)Toxicity against three economically important tephritid fruit fly species (Diptera: Tephritidae) and their parasitoids (Hymenoptera: Braconidae). Journal of Economic Entomology, Vol. 97, p.p Tsolakis, H., E. Ragusa. and P. Tarantino(2011)Control of Bactroceraoleaeby low environmental impact methods: NPC methodology to evaluate the efficacy of lure-and-kill method and copper hydroxide treatments. Bulletin of Insectology, Vol. 64 (1), p.p Vincent, K. (2010)Probit analysis. The San Francisco State University.Dpt of Biology Vontas, J.G.,M.J. Hejazi, N.J. Hawkes, N. Cosmidis, M. Loukas and J. Hemingway(2002) Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactroceraoleae. Insect Molecular Biology, Vol. 11(4), p.p

6 Tables and figures Table 1.LD 50 estimations through 3 recording periods (in ng per insect). Pesticide/recording Probit data Dimethoate 1 st rec. LD 50 =35.288±1.44 x= y X 2 =6.027 Dimethoate 2 nd rec. LD 50 =21.788±0.83 x= y X 2 =0.271 Dimethoate 3 rd rec. LD 50 =8.076±0.53 x= y X 2 =0.899 Cypermethrin 1 st rec. LD 50 =0.574±0.05 b= 1.02 X 2 =3.91 Cypermethrin 2 nd rec. X 2 >>10 Cypermethrin 3 rd rec. X 2 >>10 Table 2.Pesticide applications (1 st and 2 nd ap.) and B. oleae response (LD 50 total estimation in ngper insect) according to probit models for the pesticidesdimethoate and Cypermethrin, for male and female adults. Pesticide/application Probit data Females Males Dimethoate 1 st ap. LD ± ±1.64 Xequation x= y x= y X Dimethoate 2lmm n n nd ap. LD ± ±1.017 Xequation x= y x= y X Cypermethrin 1 st ap. LD ± ±0.01 Xequation x= y x= y X Cypermethrin 2 nd ap. LD ± ±0.01 Xequation x= y x= y X Figure 1.Melivia area (Larissa, Thessaly, Central Greece) 6