Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.     

Biological control of Bactocera oleae (Diptera: Tephritidae) using a Greek Bacillus thuringiensis isolate

Bacillus thuringiensis, isolate 114A, was used in toxicity experiments against the wild population ofthe olive pest Bactroceria oleae (Gmelin). In laboratory experiments, spores and crystals of the B.t. were delivered to the insects with the food. Longevity, oviposition period, number of eggs produced, and percent hatch were recorded. Olive fruits from the oviposition test were dipped into a suspension containing spores and crystals of B. thuringiensis 114A after the eggs were deposited. In field experiments, four to six sprayings per year of B.t. 114A isolate were applied for three successive years. It was found that, in addition to the longevity of B. oleae, the oviposition period, number of eggs and percent egg hatch decreased. Also, the percentage of pupation and emergence was reduced when olive fruits with eggs in their mesocarp were dipped in the solution of spores and crystals. Field applications with the toxins of 114A isolate of B. thuringiensis have resulted in significant protection of the olive production.     

Insecticidal activity of strains of Bacillus thuringiensis on larvae and adults of Bactrocera oleae Gmelin (Dipt. Tephritidae).

The olive fly, Bactrocera oleae, is the key pest on olives in the Mediterranean area. The pest can destroy, in some cases, up to 70% of the olive production. Its control relies mainly on chemical treatments, sometimes applied by aircraft over vast areas, with their subsequent ecological and toxicological side effects. Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects, including the orders of Lepidoptera, Diptera, and Coleoptera and other invertebrates. The aim of this study was to search for isolates toxic to B. oleae. Several hundred B. thuringiensis isolates were obtained from olive groves and olive presses in different areas of Greece, Sardinia (Italy), and Spain and from cooperating scientists throughout the world. Some isolates were found toxic only to adults or larvae and some to both stages of the olive fly. In addition, the most toxic isolates were assayed on Opius concolor Szepl. (Hym. Braconidae), the most important parasitoid of the olive fruit fly. Only 3 isolates out of 14 gave significant mortality against this parasitoid. Several of the most toxic crystalliferous isolates may contain novel toxins since they gave no PCR products when probed with primers specified for 39 known toxin genes.     

Mating disruption for the control of the olive moth, Prays oleae (Bern) (Lep., Yponomeutidae) with the ajor sex pheromone component

Abstract: Mating disruption to control the olive moth Prays oleae (Bern) was tested from 1992 to 1996 in olive groves in Greece. The major sex pheromone component (Z)-7-tetradecenal was formulated in β-cyclodextrin (β-CD) and polyvinyl chloride (PVC) polymers. Pheromone trap catches were reduced by up to 96–100% in the mating-disruption plots. During the first year of mating disruption a treatment with Bacillus thuringiensis (var. kurstaki ) (Bt) was applied to reduce the first generation or larvae. Fruit damage in the mating disruption plots was lower compared with the fruit damage in Bt, insecticide and untreated plots. In high fruiting years, the proportion of fruit damage was lower than in low fruiting years. Mating disruption applied in the same olive grove over several years progressively reduced the olive moth population from year to year.     

Partial purification and insecticidal activity of toxic metabolites secreted by a Mucor hiemalis strain (SMU-21) against adults of Bactrocera oleae and Ceratitis capitata (Diptera: Tephritidae)

The secondary metabolites present in the methanol extract of a Mucor hiemalis strain (SMU-21) mycelia, cultured in liquid medium, were evaluated for toxicity to Bactrocera oleae (Gmelin) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) adults. Feeding and contact bioassays revealed that the methanol fraction of the crude supernatant was strongly toxic to both species. Symptoms of toxicity developed quickly; lethargy occurred 1-2 h posttreatment, and mortality reached 82-97% after 24 h. Both feeding and contact bioassays showed that B. oleae was more susceptible than C. capitata. Concentrations producing 50% mortality (LC50) to B. oleae and C. capitata after 24 h in feeding bioassays were 0.52 and 1.28 mg/ 0.1 ml diet, respectively, but 34.8 and 64.0 microg/ cm2, respectively, after 4 h in contact bioassays. Solvent partition, precipitation, and chromatographic procedures were used to isolate the active principles from the crude supernatant. These resulted in the isolation of one high-pressure liquid chromatography fraction with insecticidal activity on B. oleae flies equal to the initial crude supernatant.     

Pheromone trap catches of the olive moth, Prays oleae (Bern.) (Lep., Plutellidae) in relation to olive phenology and degree-day models

Abstract:  The population dynamics of the olive moth, Prays oleae (Bern) (Lep., Plutellidae) was monitored using pheromone traps containing (Z)-7-tetradecenal, from 2000 to 2002 in abandoned and commercial olive groves of Bursa in north-western Turkey. The carpophagous generation lasted significantly longer than the phyllophagous generation. The degree-days required for the anthophagous generation were significantly shorter than that for the phyllophagous and carpophagous generations. The relationship between temperature accumulations and pheromone trap captures of male P. oleae in Bursa olive groves showed a significant sigmoid relationship for the phyllophagous and anthophagous flights. Although there was generally a linear relationship between cumulative male catch and olive tree phenology, the use of the prediction model for insecticide treatments was insufficient. Cumulative male catch–degree days together with the olive tree phenology model can be used as a forecasting method for proper timing of insecticide applications against anthophagous and carpophagous larvae in olive groves.     

Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 10(6) cells ml(-1). Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding delta-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The presence of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2-, 6.6-, and 14.6-fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), and K122(pBCcry1Aa + pBScry1Ia), respectively. Furthermore, determination of the Synergistic Factor between Cry1Aa and Cry1Ia showed that they act synergistically. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control.     

Density and structure of Saissetia oleae (Hemiptera: Coccidae) populations on citrus and olives: relative importance of the two annual generations

Saissetia oleae (Olivier) (Hemiptera: Coccidae) populations were studied and compared in citrus (Citrus spp.) and olive (Olea europaea L.) groves to determine the number of generations, crawler emergence periods and changes in population density during the year. Ten citrus and four olive groves were sampled regularly between March 2003 and December 2005 in eastern Spain, covering an area of 10,000 km2. Each sample consisted of 16 branches and 64 leaves. Saissetia oleae populations presented a similar trend in both crops during the three years of study. Populations peaked in July, when crawlers emerged after the egg-laying period, and decreased during several months due to mortality of first instars in summer. A second crawler emergence period, with lower numbers and more variability from year to year, occurred between October and March. Populations did not increase during this period, probably because most eggs and crawlers perished during the winter and also because females that gave rise to this fall-winter generation were half as big and fecund as spring females. No differences were found between the size of mature females that had developed on citrus and on olives during the spring. Considering this population pattern, the best seasonal period to apply pesticides to control S. oleae would be at the end of July, when populations are synchronous, all crawlers have already emerged, and first instars predominate.     

Tracing the history of an enzyme polymorphism: the case of alcohol dehydrogenase-2 (Adh-2) of the olive fruit fly Bactrocera oleae

In the olive fruit fly Bactrocera oleae, previous studies have described a one-locus three-allele electrophoretic polymorphism of the enzyme alcohol dehydrogenase and provided evidence that the polymorphism is under the influence of selection. A recent study has shown that this species carries a two-locus duplication for alcohol dehydrogenase. Here, we show that the polymorphism maps at one of the duplicated loci, Adh2, and identify the nucleotide and, therefore, the inferred amino acid differences among the three allozymes. At the amino acid level, the polymorphism is of the simplest possible form: there is no intra-allozyme variation, and interallozyme differences are restricted to one amino acid for two pairs of alleles and to two amino acids for the third pair. Consideration of the amino acid residues at the sites that segregate in B. oleae in four congeneric species and the phylogenetic trees produced from the nucleotide sequences of the Adh2 gene of these species point to the same allozyme as the ancestral form of the polymorphism. Interestingly, this allozyme comprises less than 1% of the gene pool of present-day natural populations of B. oleae, where the other two allozymes appear to form a stable polymorphism. Previous studies have shown that the frequency of the rare allozyme rises rapidly in laboratory colonies maintained on artificial diet and declines again when the artificial diet is replaced with olive fruit, the natural substrate of B. oleae. The geographical distribution of several congeneric species suggests that B. oleae originated in the Indian subcontinent, where the olive tree is practically absent. The poor performance of the ancestral allele on the olive fruit suggests the possibility that the decline of this allele and the concomitant rise of the presently common alleles might be associated with the expansion of the insect's geographical distribution to areas where the olive tree has become its main and perhaps sole host. The estimated age of the polymorphism is compatible with this hypothesis, but firmer support could be difficult to obtain.     

Fungal Diversity Associated to the Olive Moth, Prays Oleae Bernard: A Survey for Potential Entomopathogenic Fungi

Olive production is one of the main agricultural activities in Portugal. In the region of Trás-os-Montes, this crop has been considerably affected by Prays oleae. In order to evaluate the diversity of fungi on Prays oleae population of Trás-os-Montes olive orchards, larvae and pupae of the three annual generations (phyllophagous, antophagous and carpophagous) were collected and evaluated for fungal growth on their surface. From the 3,828 larvae and pupae, a high percentage of individuals exhibited growth of a fungal agent (40.6%), particularly those from the phyllophagous generation. From all the moth generations, a total of 43 species from 24 genera were identified, but the diversity and abundance of fungal species differed between the three generations. Higher diversity was found in the carpophagous generation, followed by the antophagous and phyllophagous generations. The presence of fungi displaying entomopathogenic features was highest in the phyllophagous larvae and pupae, with Beauveria bassiana as the most abundant taxa. The first report of Beauveria bassiana presence on Prays oleae could open new strategies for the biocontrol of this major pest in olive groves since the use of an already adapted species increases the guarantee of success of a biocontrol approach. The identification of antagonistic fungi able to control agents that cause major olive diseases, such as Verticillium dahliae, will benefit future biological control approaches for limiting this increasingly spreading pathogen.     

Mitotic and polytene chromosome analysis in Dacus oleae (Diptera: Tephritidae).

The present study constitutes the first attempt to construct a photographic map of the polytene chromosomes of Dacus oleae, a pest of the olive tree that causes serious financial damage in all olive oil producing countries. The map was constructed by using the larval fat body cells, the chromosomes of which are representative of the polytene chromosomes of other polytene tissues. In addition, the mitotic chromosomes of brain ganglia were examined, permitting tentative correlations between mitotic and polytene elements. This investigation shows that D. oleae is suitable for cytogenetic analysis in both mitotic and polytene chromosomes, a fact that may prove very useful for obtaining more detailed genetic information on the pest's natural populations.     

The effect of gamma rays on the gonads of the olive fruit fly, Dacus oleae (Gmelin)

Pupae of the olive fruit fly, Dacus oleae (Gmelin) 1 to 2 days before adult emergence were irradiated with the suitable sterilizing dose of 80 Gy gamma rays. At intervals of 5, 10, 15, 20, 25 and 30 days after adult emergence, anatomical and biometrical studies were performed to determine the extent of recovery of D. oleae gonads during one month of adult life. There were some indications of gonad recovery after two weeks. This recovery was observed as a decrease in the percentage deviation from the corresponding controls of 20-day-old adult gonad (especially those of males). Generally, female gonads are more sensitive to gamma-rays than those of males.     

Evaluation of Fopius arisanus as a biological control agent for the olive fruit fly in California

1 The egg‐prepupal parasitoid Fopius arisanus (Hymenoptera: Braconidae) was evaluated in quarantine facilities as a potential biological control agent for the olive fruit fly Bactrocera oleae (Diptera: Tephritidae) in California, U.S.A. 2 Nontarget testing of two weed biological control agents confirmed that F. arisanus will not attack Tephritidae that feed in inflorescences or galls. It may, however, pose risks to native Tephritidae that feed in fruit. 3 Females preferentially oviposited in eggs, although first‐instar B. oleae were also attacked. Low lifetime reproductive potential and high rates of direct mortality inflicted on host eggs indicate that rearing on B. oleae may prove difficult. 4 In multiparasitized B. oleae, F. arisanus prevailed in competition against two species of larval–pupal parasitoids, Diachasmimorpha kraussii and Psyttalia concolor (both Hymenoptera: Braconidae). 5 The broad host‐range of F. arisanus with respect to fruit‐feeding Tephritidae may preclude its introduction to California, as may its low fecundity and its intrinsic competitive superiority over larva l–pupal parasitoids, which include specialists on B. oleae that are currently being introduced to California. High rates of direct mortality, however, point to potential uses in augmentative biological control. Whether or not F. arisanus is released in California, its biology as a parasitoid of B. oleae has been little studied to date and the results herein may be applied in other regions worldwide where B. oleae is a problem.     

Olive fruit fly (Diptera: Tephritidae) ovipositional preference and larval performance in several commercially important olive varieties in California

The olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae),is an invasive pest of olives (Olea spp.) in the United States. The objectives of this study were to determine whether B. oleae exhibits ovipositional preference under California field conditions similar to that demonstrated in European populations and whether the resulting larvae fare better in preferred varieties. Female B. oleae exhibited strong ovipositional preference for certain varieties of the domesticated olive, Olea europaea L, and the resulting larvae performed better by some measures in preferred varieties than in lesser preferred varieties. Ovipositional preference was observed in the field from 2003 to 2005, and laboratory assays were conducted to evaluate larval performance in 2005 and 2006. Among the olive varieties tested, Sevillano, Manzanillo, and Mission olives were the most heavily infested during three consecutive years. The larval performance measurements used were pupal yield, pupal weight, larval developmental time, and pupal emergence time. Ovipositional preference and pupal yield do not seem associated. There were significant differences in pupal emergence time, but these also measures did not reflect ovipositional preference. Two measures on performance did seem related to ovipositional preference; there were significant effects of variety on pupal weight and larval developmental time. Pupae developing in Manzanillo and Sevillano olives were heavier than those developing in less preferred varieties, and larval developmental time was significantly shorter in Sevillano olives relative to the other varieties. Oviposition preference and enhanced larval performance has implications for the pest status of this invasive insect in California.     

Factors affecting male Prays oleae (Lepidoptera: Yponomeutidae) captures in pheromone-baited traps in olive orchards

Effects of trap design, height and side of trap placement on olive trees, pheromone doses in dispensers, aging of dispensers in the field, and secondary pheromone components were evaluated for the development of an effective pheromone monitoring system for Prays oleae (Bernard) Lesne (Lepidoptera: Yponomeutidae) in olive orchards. Field trials showed that trap design, pheromone dose, and trapping side affected male captures, whereas dispenser age, trap height, and secondary components had no influence. Pherocon 1C and Delta traps baited with 1 mg of (Z)-7-tetradecenal captured more male moths than Pherocon II or Funnel traps. Placement of traps at different cardinal directions significantly affected captures, but this trend was not consistent and varied with flight period and trap position internal or external to the tree canopy. Moth phenology as determined by pheromone traps from early April to mid-October was consistent with published field data. Results indicate that Pherocon 1C or Delta traps baited with 1 mg of (Z)-7-tetradecenal provide an effective tool for monitoring the flight activity of P. oleae and the time of application of control measures.     

Insecticidal activity of Citrus aurantium fruit, leaf, and shoot extracts against adult olive fruit flies (Diptera: Tephritidae)

Solvent extracts of differing polarity from Citrus aurantium (L.) (Rutaceae) fruit, leaves, and shoots were evaluated for biological activity against adults of the olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae). Using a petri dish residual exposure bioassay, we found that the petroleum ether extract from fruit alone showed insecticidal activity against the flies. The extract of the three fruit tissues (flavedo [peel], albedo, and flesh) indicated that bioactivity was limited to the flavedo, and this activity was significantly higher than that of the whole fruit extract. The most effective extract was obtained when fresh flavedo was used, whereas extracts of oven-dried flavedo were inactive. Fruit maturity also affected bioactivity; extracts of ripe fruit were more effective than those of unripe fruit. Our results suggest that C. aurantium flavedo contains secondary metabolites with insecticidal activity against B. oleae adults.     

An inapparent infection with a probable picornavirus in several stocks of laboratory reared and naturally occurring populations of Dacus oleae Gmel. pupae in Greece

Pupae from several stocks of wild and laboratory reared olive fruit fly, Dacus oleae, were fractionated by a series of steps designed to identify occluded and nonoccluded viruses. Two different size of particles were isolated, the smaller of which contained a single-stranded RNA molecule of about 2.8 x 10(6). This small RNA virus was found to inapparently infect a Ceratitis capitata continuous cell line.     

Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.