Metarhizium brunneum (Ascomycota; Hypocreales) Treatments Targeting Olive Fly in the Soil for Sustainable Crop Production

Olive fly population was monitored weekly on two olive cultivars (Buža and Istarska bjelica), from June until mid October, in Rovinj and Livade (Istria, Croatia). The number of eggs, larvae, and pupae was established and the total and active infestation was calculated. The fruit infestations for early and for late harvesting were obtained based on the calculated regression equation. For the prediction of the changes in the oil quality parameters linear regression slopes, obtained by Koprivnjak et al., were used. We established a strong positive correlation between DD accumulation and cumulative capture of flies, as well as with the total and active fruit infestations. According to obtained results it can be stated that I. bjelica is less sensitive to decrease in total phenols amount, to increase in free fatty acids mass ratio and to increase in peroxide values comparing to Buža. Moreover, the differences in investigated parameters between earlier and late harvesting dates in I. bjelica are lower due to lower infestation predicted for both harvesting dates and due to lower sensitivity to the changes in quality parameters. Therefore, early harvesting date as a model for preventing fruit damage and as a model for preventing negative change in oil quality parameters is a valid tool. However, the effectiveness of this model could also depend on the characteristics of olive cultivar.Practical applications: Understanding the factors that affect the olive fly attack is the basis of scientific and practical interest in the production of olives and olive oil. Research of monitoring methods allows reliable forecasting and determining protection measures. Knowledge about this topic could contribute to the reduction of insecticides use and to the improvement of quality and food safety concept in olive oil production.Damaged olive fruits are proved to directly affect the quantitative and qualitative properties of olive oils. The most important fruit damage is caused by the olive fly (Bactrocera oleae Gmelin).

The infestation due to the olive fruit fly, Bactrocera oleae (Rossi), the key pest in most of world olive groves, has been monitored in six years, from 2004 to 2008 and in 2014, in eight coastal Sicilian olive groves consisting of ‘Cerasuola’ and ‘Nocellara del Belice’ cultivars. Infestation was recorded following the classical sampling method based on olive collection and dissection, in order to count live olive fruit fly instars (eggs, larvae and pupae) and exit holes. Four different infestation indexes, all of them calculated using data recorded at harvest, have been used to assess their relationship with the main three quality parameters of the olive oil obtained from the same olive samples processed within 24 hours by quality oil mills: free acidity (% oleic acid), peroxides (mEq O2 /kg of oil) and total phenols (mg·kg−1 oil). Each year and in each olive grove up to five plots were differently treated with different products allowed in organic agriculture, stopping sprays at least one month before harvest, obtaining different infestation levels. In this study a total of 43 theses were tested, 10 of them involving ‘Cerasuola’ and 33 ‘Nocellara del Belice’. The two indexes based on the exit holes produced by mature larvae in olives resulted strictly and positively correlated to the lowering of oil quality. Moreover, among the two infestation indexes based on the occurrence of exit holes at harvest, percentage of olives with exit holes and No. of exit holes per 100 olives, the latter is more sensitive in presence of high infestation levels. No significant relation between infestation indexes and total phenols resulted in our analyses. Nevertheless, when olives are harvested since the end of October to mid-November, and processed by quality oil mills, olives bearing up to 45 % of exit holes produced high quality extra virgin olive oil. Furthermore, all olives bearing up to 62 % of exit holes still produced extra virgin olive oil. Our results, in spite of the widespread prejudices involving an overestimation of damages due to low olive fly infestation, are very close to recent studies on correlation between olive fly attacks and olive oil quality. Late harvesting can lead to worse results at the same olive fly exit holes levels, confirming that timing and quality procedures of harvesting and oil extraction are important almost as much as olive fly control.

In the Mediterranean basin, organophosphate (OP) insecticides have been used intensively to control olive fly populations. Acetylcholinesterase (Ace) is the molecular target of OP insecticides, and three resistance-associated mutations that confer different levels of OP insensitivity have been identified. In this study, genotypes of olive fly Ace were determined in field-collected populations from broad geographical areas in Turkey. In addition, the levels of asymmetry of wing and leg characters were compared in these populations. Our study revealed the existence of a genetically smooth stratification pattern in OP resistance allele distribution in the olive fly populations of Turkey. In contrast to earlier findings, the frequency of Δ3Q was found to be lower in the Aegean region, where the populations have been subjected to high selection pressure. Results based on the morphological differences among the samples revealed a similar pattern for both sides and did not demonstrate a clear separation. The frequencies and geographic range of resistance alleles indicate that they were selected in the Aegean coast of Turkey and then spread westward towards Europe. One possible explanation for the absence of morphological asymmetry in olive fly samples might be the presence of modifier allele(s) that compensate for the increase in asymmetry.

Simple SummaryOlive cultivation is extensive throughout the Mediterranean region and essential for the rural economy, local heritage and the environment. To control the olive fruit fly, a major threat for this cultivation, pesticides, in the form of bait or cover sprays, are applied. These pesticide applications can potentially impact pollinators that forage in the nearby areas or live inside the olive orchards. Based on current practice, olive trees supply shadow, water, and still support some flowering plants at the period of the year with the highest temperature and minimum nectar and pollen flow, which can be beneficial for bees. In this study pesticide residues were monitored in honeybees, bumblebees, honey and olive oil, after placement of bee colonies in Greek olive orchards where applications to control the olive fruit fly took place. Variations of concentrations were evidenced, for the three active ingredients that were applied. In limited cases, concentrations in bees higher than the median lethal dose can possibly be attributed to bait dose rates or broad foraging of bees in nearby orchards with similar applications. Determined olive oil residues corroborated that those pesticides were applied in the olive orchards.In 2017 and 2018, a field survey was initiated on Greek olive orchards to investigate the attractiveness of bait spray applications and the impact of cover and bait sprays applied against the olive fruit fly Bactrocera oleae (Diptera: Tephritidae), on the honeybee, Apis mellifera L. and bumblebees Bombus terrestris, by investigating the pesticides’ residual prevalence. Bee colonies were evenly distributed in three sites located on coastal areas of Western Crete and visited almost weekly between July and October. Samples collected, were analyzed using existing or developed-optimized liquid and gas chromatographic methods. In bee samples, concentrations varied from 0.0013 to 2.3 mg/kg for dimethoate, from 0.0013–0.059 mg/kg for its metabolite omethoate, and from 0.0035 to 0.63 mg/kg regarding the pyrethroids, β-cyfluthrin and λ-cyhalothrin. In one bee sample dimethoate concentration exceeded both acute oral and contact median lethal dose (LD50). Residue findings in bees, along with verified olive oil residues corroborated that those insecticides had been applied in the olive orchards and transferred to bees. The possibility of non-target effects of the bait sprays to the bees, as well as the impact of the contaminated olive to the bees are discussed.

A Bactrocera oleae (Rossi) damage estimation model to anticipate pest control strategies in olive production

This study had been carried out between 1986-1993 in some olive groves in Antalya province. The population dynamics of olive pest and their natural enemies were determined by beating, knock-down and visual observation methods. The population densities of olive kernel borer, olive bark beetle, olive fruit fly and black scale were determined by survey ant their parasitoids and parasitism rates were found out by culturing method. The presence periods of the natural enemies in olive orchards were tried to be determined. In olive groves 34 species of olive pests and 65 species of natural enemies as parasitoids, predators and entomopathogen were determined. The population level and effectiveness of some beneficial insects were found relatively high. Ageniaspis fuscicollis Dalm., Elasmus albipennis Thoms , E.fabellatus, Bracon variegator Spinole, Chelonella depressa Thoms, Phanerotoma sp., Chelonus oculator Panz, C.cingulipes, Phanerotomella kerteszii Szepl. and Pediobius sp. as parasitoids of olive kernel borer; Psyllaephagus euphyllurae Silv. as parasitoid and Anthocoris nemoralis Fabr., A.minki, Deraeocoris delagrangei Put., Heterotoma dalmatinum Wgn., Campyloneura virgula H.-S., Myrmecoris gracilis (T.Sahlb.), Mimocoris coarctatus (Ms.et Rey) and Orius niger (Wolff.) as predators of olive psylla; Eupelmus urozonus Dalm., Crytoptyx dacicida Masi. and Opius concolor Szepl. as parasitoids of olive fruit fly; Scutellista cyanea Motsch. and Metaphycus sp. as parasitoids and Chilocorus bipustulatus L. and Scymnus spp. as predators of black scale; Telenomus spp. as parasitoids of Agalmatium bilobum Fieb.; Cheiropachus quadrum F., Metacolus unifasciatus Forst., Rhaphitelus maculatus Walk., Eurytoma morio Boh., Dendrosotinus ferrigineus Marsh. and Ecphylus sp. as parasitoids and Nemosoma elongatum L., Denops albofasciatus (Charp.,), Opilo taeniatus Kol., C.virgula and M.gracilis as predators of olive bark beetle; Mesopolobus mediterraneus (Mayr.) and Platygaster sp. as parasitoids of olive leaf midge; Microterys masii Silv. as parasitoids of olive cottony scale and F.follicularis, Beauveria bassiana (Balsamo) Vuill as entomopathogen of F.follicularis ; Elachertus sp. as parasitoid of leopard moth; Exochus sp. as parasitoid and D.albofasciatus and O.taeniotus as predators of Apate monachus F.; Aphytis sp. as parasitoid of olive white fly; A.nemoralis as predator of olive thrips; Asilus sp. as predator of Calocoris trivialis Costa and C.annulus; Cybocephalus fodori E.-Y., Pharoscymnus pharoides Mars., C.bipustulatus and Scymnus spp. as predators of olive tree scale and olive scale; Chrysoperla carnea (Steph.), Anisochrysa genei Ramb., A.zelleri, A.prasina, Suarius nanus Mclachlan, Conwentzia hageni Banks, Nagusta goedeli (Klt.), Raphidia ressli Asp. et Asp. and Mantis religiosa L. as general predators of olive pest had been determined by this study. In olive orchards the natural enemies reached their maximum level between the second half of April and the first half of June. For this reason, it will be suitable to make the chemical control applications before or after this period for protecting and continuing the natural balance. The natural enemies of olive kernel borer, olive psylla and black scale were most active in April, May and June. For this reason if the population level of the natural enemies of target pest are rather important it will be advantageous to preserve them in these periods in olive orchards. If the parasitoids of olive fruit fly are active in olive orchards, the chemical applications should be delayed for protecting them between late June and the first half of September, because they are most active in these periods.

In this work, the influence of olive fruit fly (Bactrocera oleae Rossi) attack on the quality of 'Rosinjola' virgin olive oil (free fatty acids, peroxide value, specific extinction at 232 and 270 nm, and sensory evaluation) as well as on the fatty acid and volatile composition was investigated. Olive fruits were collected and divided into two groups according to the presence (100% infested) or absence (healthy, 0% infested) of infestation by olive fruit fly, and a third group was made by mixing the two previously described groups in equal proportions (50% infested). Each group of olive fruits was separately processed under the same conditions to produce corresponding virgin olive oils. The results of oil acidity, a sign of hydrolytic degradation of olive oils, showed that olive fruit fly infestation at the 50 and 100% levels significantly reduced the quality of oil. Sensory score and intensity of positive sensory characteristics of the investigated oils decreased with increasing fruit fly infestation, but defects in oils as a consequence of fruit fly attack were not detected. Considering the fatty acid composition, olive fly attack caused only a slight reduction of palmitic acid. The volatile profile of investigated olive oils showed that olive fly attack led to a decrease in total C6 volatiles (a decrease in C6 aldehydes, but an increase in C6 alcohols) and an increase in total C5 volatiles. The results confirmed the importance of healthy fruit in obtaining high-quality virgin olive oil, but, despite the olive fruit fly attack, 'Rosinjola' oil still maintained extra virgin olive oil category according to market quality parameters.

The olive fruit fly, Bactrocera oleae, is the most important pest of olives in olive growing regions worldwide, especially in the Mediterranean basin and North America. Despite the economic importance of the olive fly, the colonization route of this species is unclear. We used nuclear microsatellite markers and mitochondrial DNA to provide information about the population structure and invasion route of olive fly populations in Turkey, as representative of the Eastern Mediterranean region. Adult fly samples were collected from 38 sublocations covering all olive growing regions in Turkey. The simple sequence variability data revealed a significant genetic variability in olive fly populations and a certain degree of differentiation between Mediterranean and Aegean populations. Mediterranean populations harbor higher levels of microsatellite variation than Aegean populations, which points to the eastern part of the Mediterranean as the putative source of invasion. mtDNA results suggest olive flies from the western part of Turkey are closely related to Italo-Aegean flies of the Mediterranean basin and the olive fly populations have invaded the northern part of the Mediterranean basin through western Turkey. In addition, finding specific American haplotypes in high frequencies might indicate that Turkey is the possible source of American olive fly populations. In order to more precisely characterize the population structure and invasion routes of this organism, more DNA-based sequence analysis should be carried out worldwide.

Escudete, which is caused by Botryosphaeria dothidea, is a disease that is widely distributed in the Mediterranean basin, but is of little general importance. Nevertheless, serious attacks have been observed on occasion, which have caused a considerable reduction in the quality of table olives. The incidence of the pathogen has been associated with damage caused by the olive fly (Bactrocera oleae) and the presence of a possible vector agent, i.e., the midge Prolasioptera berlesiana, whose larvae can feed on fly eggs (although the role the midge may play in the spread of this disease is not well known). Therefore, it is necessary to clarify these interactions to adopt appropriate disease control measures. Studies were conducted in olive orchards planted with the Gordal Sevillana, Picudo, and Hojiblanca olive cultivars. Field surveys were carried out in order to sample their fruits for laboratory analysis, and several bioassays were also performed. Moreover, the population of B. oleae adults was monitored using traps that were baited with food attractants. The results indicated that the three agents developed and evolved in parallel under field conditions. Thus, the midges were attracted by the oviposition punctures caused in fruits by olive fruit flies, regardless of whether the punctures contained eggs. All the investigated olive fruits in which midges were present inside punctures created by olive fruit flies exhibited typical symptoms of escudete, which is necessary for the development of this disease. Forty-eight hours after fly punctures were artificially simulated in the olive fruits, 48.0% of them contained a midge, whereas no midges appeared in the artificially created shapeless wounds in the fruits. This indicates that an olive fly egg is not required for the development of midges; however, they do prefer punctures made by B. oleae. Moreover, when the olive fruits were incubated in a humid chamber, the B. dothidea fungus only appeared in those fruits that contained midges, thus indicating a close relationship between these two agents. Additionally, the midges were able to complete their entire development from egg to adult under controlled conditions, and they fed on the pure cultures of the B. dothidea fungus. Furthermore, although no pathogens were present in the immature midges, some of the pathogens could have been isolated from the inner tissues of the adult female midges. The fact that mycangia is present in the abdomen of P. berlesiana supports the hypothesis that their relationship with B. dothidea may be mutualistic and that they may act as a vector for the fungus.

Olive anthracnose, caused by Colletotrichum fungi, and the olive fruit fly Bactrocera olea are, respectively, the most important fungal disease and pest affecting olive fruits worldwide, leading to detrimental effects on the yield and quality of fruits and olive oil. This study focuses on the content of hydroxytyrosol (HYT) and its derivatives (the "olive oil polyphenols" health claim) in olive oils extracted from fruits of 'Galega Vulgar' and 'Cobrançosa' cultivars, naturally affected by olive anthracnose and olive fly. The olives, with different damage levels, were harvested from organic rainfed orchards, located in the center of Portugal, at four harvest times over three years. Galega oils extracted from olives with a higher anthracnose and olive fly incidence showed no conformity for the extra virgin olive oil (EVOO) and virgin olive oil (VOO) categories, presenting high acidity and negative sensory notes accompanied by the disappearance of oleacein. Conversely, no sensory defects were observed in Cobrançosa oils, regardless of disease and pest incidence levels, and quality criteria were still in accordance with the EVOO category. The total HYT and tyrosol (TYR) content (>5 mg/20 g) allows for the use of the "olive oil polyphenols" health claim on the label of all the analyzed Cobrançosa olive oils.

Pesticide applications are still one of the most common control methods against the main olive grove pests and diseases: the olive fruit fly, Bactrocera oleae (Rossi), the olive moth, Prays oleae (Bernard), the black scale, Saissetia oleae (Olivier), and the olive leaf spot, caused by the fungus Spilocaea oleagina Fries. However, and because the new pesticide legislation is aimed at an integrated pest and disease management, it is still important to evaluate and to know the ecotoxicology of pesticides on the natural enemies of the different agrosystems. A part of this work has been focusses on evaluating the direct and indirect effects of kaolin particle films and two copper-based products (Bordeaux mixture and copper oxychloride) through different laboratory, extended laboratory and semi-field experiments. Two natural enemies have been chosen: Psyttalia concolor (Szèpligeti), a parasitoid of the olive fruit fly, and Chilocorus nigritus (F.), predator of Diaspididae. This predator has been used instead of C. bipustulatus (L.), which is the species found in olive orchards. Kaolin mainly acts as a repellent of insects and/or as an oviposition deterrent. It is used in olive groves to control the olive fruit fly and the olive moth. Copper is applied against fungal and bacterial diseases. In olive groves it is used against the olive leaf spot and other diseases. No statistical differences were found in any of the experiments performed, compared to the controls, except when the oral toxicity of the products was evaluated on P. concolor females. In this case, kaolin and copper oxychloride caused a higher mortality 72 hours after the treatments, and both kaolin and the two copper formulations decreased females’ life span. Reproductive parameters were only negatively affected when kaolin was ingested. Apart from these experiments, due to the uncommon mode of action of kaolin, two extra experiments were carried out: a dual choice and a no-choice experiment. In this case, both P. concolor females and C. nigritus adults showed a clear preference for the untreated surfaces when they had the possibility of choosing between a treated surface and an untreated one. When there was no choice, no statistical differences were found between the treatments and the controls. Furthermore, the efficacy and the selectivity of three insect growth regulators (methoxyfenozide, tebufenozide and RH-5849) on B. oleae and P. concolor, respectively, have also been evaluated. In addition to laboratory experiments to evaluate the toxicity of the insecticides, also molecular approaches were used. RNA of both insects was isolated. cDNA was subsequently synthesized and the complete sequences of the ligand biding domain (LBD) of the ecdysone receptor of each insect were then determined. Afterwards the three dimensional structures of both LBDs were constructed. Finally, the docking of the insecticide molecules in the cavity delineated by the 12 α-helix that composed the LBD was performed. Both toxicity assays and molecular docking approaches showed that either methoxyfenozide or tebufenozide had no negative effects nor on B. oleae nor on P. concolor. In contrast, RH-5849 had no deleterious effect to the parasitoid but decreased olive fruit fly adults’ life span, especially when they were in contact with the fresh residue of the insecticide applied on a glass surface. The docking study of RH-5849 molecule has shown a very light hindrance with the wall of the LBD pocket. This means that this molecule could more or less adjust in the cavity. Thus, searching of new insecticides for controlling the olive fruit fly could be based on the basic lead structure of RH-5849 molecule.

The susceptibility of preimaginal and adult olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae), to a strain of the mitosporic ascomycete Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) and the insecticidal activity of its crude extract to olive fruit fly adults were investigated. Strain EAMb 09/01-Su caused 60% mortality to B. oleae adults, with average survival time (AST) of 8.8 d. In soil treatments against pupariating third-instar larvae, preimaginal B. oleae mortality reached 82.3%, whereas preimaginal mortality targeting puparia was 33.3%. The crude extract of EAMb 09/01-Su strain caused 80.0% adult mortality when administered per os, with AST of 27.7 h. The crude extract was demonstrated to be quite thermostable and photoresistant. These results indicate that M. brunneum EAMb 09/01-Su strain and its crude extract show potential to be used in an integrated pest management olive fruit fly management strategy targeting both adults and preimaginals.

Incidents of using naphthalene in olive orchards as a repellent of olive fly (Bactrocera oleae) have recently been recorded. Naphthalene, the primary ingredient of mothballs, is described to be diluted in plastic bottles and hanged on the trees releasing its characteristic odor which is supposed to repel olive fly. Since naphthalene has been characterized as possibly carcinogenic to humans and animals, it was considered essential to monitor olive oils regarding their potential contamination with this specific pollutant. To this goal, a simple, sensitive and reliable analytical method for the determination of naphthalene in olive oil has been developed. The method involves extraction with ethyl acetate: acetonitrile (1:1) and determination of naphthalene using gas chromatography triple quadrupole mass spectrometry. The method was validated in four fortification levels, 0.005, 0.01, 0.05, and 0.1 mg/kg in accordance with the EU requirements. The obtained results were acceptable as far as validation criteria are concerned, given recoveries between 71% and 87% and RSDs between 5% and 24%. The limit of determination for naphthalene was set at 0.005 mg/kg based on the lowest concentration level being validated with acceptable accuracy. The analytical technique was successfully applied to 75 olive oil samples collected from oil mills in Greece and no positive results of naphthalene were detected.

The olive fly (Bactrocera oleae) is the most important olive tree (Olea europaea) pest. In the Mediterranean basin, where 98% of its main hosts are concentrated, it causes major agricultural losses, due to its negative effect on production and quality of both olive and olive oil. Previous phylogeographic analyses have established that Mediterranean olive fly populations are distinct from other Old World populations, but did not agree on the specific population substructure within this region. In order to achieve a higher resolution of the diversity of olive fly populations, particularly in Central and Western Mediterranean (home to 70% of the world production), we comparatively analyzed a set of samples from Portugal in the context of published mitochondrial sequences across the species' worldwide range. Strong evidence of population substructure was found in the Central and Western Mediterranean area, with two clearly separate phylogenetic branches. Together with previously published data, our results strongly support the existence of at least three distinct Mediterranean populations of the olive fly, raise the possibility of additional regional substructure and suggest specific avenues for future research. This knowledge can be instrumental in the development of better management and control strategies for a major pest of Mediterranean agriculture.

The olive fruit fly Bactrocera oleae is one of the most serious and economically damaging insects worldwide, affecting the quality and quantity of both olive oil and table olives. Third instar larvae and pupae of several Tephritidae flies were reported to be susceptible to entomopathogenic nematodes (EPNs), but few studies have been carried out on the olive fruit fly. Laboratory assays were conducted to evaluate the susceptibility of B. oleae larvae and pupae to two commercial EPN species, Steinernema feltiae and Heterorhabditis bacteriophora and two indigenous Italian strains of H. bacteriophora and Steinernema carpocapsae. Moreover, an olive assay in the soil was performed to evaluate the capability of EPN strains to enter inside the olive fruits and interact with B. oleae during the pupal stage or the emergence of adults in the winter diapause. The susceptibility assays with B. oleae were performed in well plates, filled with sterile soil (n = 30 for each EPN strain and insect stage). Adult emergence and mortality were recorded daily for 15 days. Dead pupae and adults were dissected to assess nematode infection. The most noteworthy result was obtained with S. feltiae which was able to infect more than 80% of larvae and it killed the pupae inside olive fruits and the adults during their emergence with the same efficacy. Since this Tephritidae fly spends several months in the soil, the use of EPNs, in particular, S. feltiae may represent a promising method to control this pest.