Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae)

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(1)UNIVERSIDAD POLITÉCNICA DE MADRID Escuela Técnica Superior de Ingenieros Agrónomos. Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae). TESIS DOCTORAL. Paloma Bengochea Budia Ingeniera Agrónoma 2012.

(2) DEPARTAMENTO DE PRODUCCIÓN VEGETAL: BOTÁNICA Y PROTECCIÓN VEGETAL Escuela Técnica Superior de Ingenieros Agrónomos. Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae). TESIS DOCTORAL. Paloma Bengochea Budia Ingeniera Agrónoma. Directora: Mª del Pilar Medina Vélez Dra. Ingeniera Agrónoma. Madrid, 2012.

(3) Tribunal nombrado por e Magfco. Y Excmo. Sr rector de la Universidad Politécnica de Madrid, el día de de 2012.. Presidente D. Vocal D. Vocal D. Vocal D. Secretario D. Suplente D. Suplente D.. Realizada la lectura y defensa de la Tesis el día. de. de. en Madrid, en la Escuela Técnica Superior de Ingenieros Agrónomos.. Calificación:. El Presidente. Los Vocales. El Secretario. 2012.

(4) A mis padres, a mi hermano y a mis abuelas.

(5) Gracias….. A todos aquellos que me habéis apoyado y/o ayudado durante antes y durante la elaboración de esta Tesis…espero no olvidarme de ninguno… A la Universidad Politécnica de Madrid, porque sin la beca que me concedieron no hubiese realizado esta Tesis, y sobre todo… A todo el personal de la Unidad de Protección de Cultivos, sin cuyo apoyo y ayuda no hubiese podido realizar este trabajo ni sobrevivir a todos los “problemillas” surgidos durante estos años. Por todas esas comidas terapéuticas en las que arreglamos el mundo, nos reímos y nos desahogamos. Por vuestra amistad. Gracias… A mi tutora, Pilar Medina, por su apoyo incondicional y su ánimo en todos estos años. Por estar siempre dispuesta a ayudarme y aclarar todas mis dudas existenciales. Por animarme a escribir esta Tesis en inglés, que ha sido un reto. Por dejarme hacer millones de cursos y llevarme a tantos Congresos. Por escucharme cuando lo he necesitado. A Flor, Ángeles, Elisa y Pedro, por transmitirnos a todos vuestro entusiasmo por la entomología, vuestras sugerencias y aportaciones. A Luis, por tener siempre a punto a mis “pobres” Psyttalias para los ensayos. A mis compañeros de laboratorio: los siguieron otros caminos: Sara, Guille, Edu, Raquel, Cherre (tu consejo para las larvas de la Ceratitis no tiene precio) y con los que sigo codo con codo compartiendo horas de laboratorio y se han convertido en buenos amigos: al incondicional equipo desayuno con los que empezar el día es otra cosa, y a los que se unen de vez en cuando: Mar, Yara, Nacho, Andrea, Jader, Agus y Rosa. A Fermín, que además es un gran apoyo y un grandísimo amigo; porque siempre está dispuesto a echarme una mano y a escucharme; por todos los momentos en que en el laboratorio nos hemos reído a más no poder (la fuga de las chrysopas, el pobre piticli…) A Román Zurita, el “guardián de los campos”, por resolver todos los problemas tecnológicos que nos surgen y por habernos ayudado a transformar la enfermería del águila en el invernadero en que mis olivos se han refugiado durante estos años. A Ángela Alonso y Ezequiel Cabrera, que me ayudaron a identificar los hongos que aparecía en la dieta de las pobres Bactroceras. Al equipo INIA, especialmente a Manuel por resolver las dudas que me surgían sobre el olivar y a Ismael, por ayudarme con todas mis dudas estadísticas. A Jose Luis Porcuna y Mamen Alaurín, por permitirme visitar en insectario de Silla y darme las primeras calabazas para comenzar mi cría de Aspidiotus. A Manuel Ruiz-Torres y Bárbara Castellanos, que me mandaron aceitunas desde Jaén y Cáceres para poder hacer mis ensayos..

(6) A Andrew Jessup, de la sede de la FAO/IAEA de Seibersdorf, por dejarme visitar las instalaciones para aprender cómo criar la mosca y enviarnos material siempre que necesitábamos… y aun así la mosca se resistió… A Carmen Calleja por resolver las dudas moleculares. A Ian, Mª José, Olivier, Pieter y a todos aquellos que han contribuido a que esta Tesis tenga unas pocas menos de faltas de ortografía… A Josep Jacas y Alberto Urbaneja por aceptarme dos meses en el IVIA. A todas las personas que conocí allí y que me acogieron como una más: Laura, Francesc, Sara, Alejandro, Óscar, Óscar, Pablo, Joel, Elena, María, Paco, Miquel, Consuelo… (mil perdones si me olvido de alguien). A Tati, que me introdujo en el mundo de los ácaros y además fue un gran apoyo el tiempo que estuve allí. A Pili, Poli, César y Helga que me adoptaron para ir a San Sebastián. Moltes gracies a tots! A Guy Smagghe por acogerme por dos veces en la Universidad de Gante y permitirme introducirme en el “mundo molecular”. A todos aquellos que me ayudaron en el laboratorio el primer año, el segundo o los dos y que con paciencia me traducían las conversaciones en dutch: Marteen, Rick, Didier, Patrick, Luck, Peter, Dorin, Ivan, Hanneke, Jisheng, Ruben, Moises, Astrid, Sara… (y alguno más de los que espero me perdonen pero se me ha olvidado el nombre…). A Jochem, el “spanishsitter”, que además intentó ayudarme a distinguir los Chilocorus de las Chilocoras (aunque no tuviésemos mucho éxito), a Pieter, por los buenos momentos, y sobre todo a Olivier: sin tu ayuda (y vigilancia) quién sabe qué habría salido de las PCR, que al llegar a Gante me parecían máquinas dificilísimas de entender. Gracias por enseñarnos Flandes y compartir tantos ratos buenos con nosotros. Dankjewel! A Marta y de nuevo a Fermín, el resto del “spanish team”, porque sin vosotros las estancias en Gante hubiesen sido muy diferentes y probablemente menos divertidas. Por haberme hecho poner cada mañana al mal tiempo buena cara (en el sentido más literal de la frase). A todos los jolgorianos, con quienes paso tan buenos momentos sea donde sea A Eva, Marta, Juli, Jesús, Eva, Isa e Irene, por una amistad que viene de lejos. A mis frijos, Cris y Ángeles, porque son un apoyo incondicional pese a la distancia. A Ana, Bea y Marta, que me animaron tanto a seguir con esta beca. A Yuse, Sergio, Bea, Eva, Sergio y demás agrónomos. A Sara, Susi, Berta y Gema, mis compis agronómicas desde el primer día de carrera. A Miguel, Cris, Alberto, Elena, Inés, Irene, Yoli, Mariano, Javi y demás ruteros con los que siempre parece que he quedado ayer aunque pueda haber pasado un año. A Ceci, Freya, Vero y Vera, las amigas al otro lado del charco. A mis padres y mi hermano. Por haberme animado a realizar el doctorado cuando yo no tenía las cosas muy claras, pero sobre todo por su gran apoyo diario y su cariño. A mis abuelas, que son todo un ejemplo, y al resto de mi familia..

(7) La paciencia es la madre de la ciencia….

(8) Index. Index INDEX. i. RESUMEN. vii. SUMMARY. ix. 1. INTRODUCTION. 1. 1.1 The olive tree. 1. 1.1.2 The origin of the crop. 2. 1.1.3 Geographical distribution. 3. 1.1.4 Importance of the crop. 3. 1.1.4.1 Economic importance. 4. 1.1.4.2 Social importance. 4. 1.1.4.3 Environmental importance. 5. 1.1.5 Olive growing in Spain. 5. 1.1.6 Pests and diseases: characteristics of the most important pests and diseases of olive groves 1.1.6.1 The olive fruit fly (Bactrocera oleae). 6 10. 1.1.6.2 The olive moth (Prays oleae). 14. 1.1.6.3 The black scale (Saissetia oleae). 15. 1.1.6.4 The olive leaf spot (Spilocaea oleagina). 16. 1.2 Control of pests and diseases. 17. 1.2.1 Integrated Pest Management. 18. 1.2.2 Organic farming. 21. 1.2.3 Integrated Protection in olive groves. 23. 1.2.4 Organic olive farming. 28. 1.3 Side-effects of pesticides on non-target organisms. 28. 1.4 Natural enemies used in the experiments. 30. 1.4.1 Psyttalia concolor. 30. 1.4.2 Chilocorus nigritus. 35. i.

(9) Index. 2. OBJECTIVES. 41. 3. GENERAL MATERIALS AND METHODS. 43. 3.1 Environmental conditions of insect rearing and laboratory experiments. 43. 3.2 Insect rearing. 44. 3.2.1 Psyttalia concolor. 44. 3.2.1.1 Mass-rearing of Ceratitis capitata. 45. 3.2.1.1.1 Adults’ cage. 45. 3.2.1.1.2 Eggs handling. 45. 3.2.1.1.3 Larvae rearing. 46. 3.2.1.2 Mass-rearing of Psyttalia concolor 3.2.1.2.1 Parasitization. 47 47. 3.2.2 Chilocorus nigritus. 48. 3.2.2.1 Mass-rearing of scales 3.2.3 Bactrocera oleae. 49 51. 3.3 Common characteristics of the experiments. 52. 3.4 Parameters evaluated. 54. 3.4.1 Mortality. 54. 3.4.2 Life span. 54. 3.4.3 Effects on reproductive parameters. 54. 3.5 Statistical analysis. 56. ii.

(10) Index. 4. LETHAL AND SUBLETHAL EFFECTS OF KAOLIN PARTICLE FILMS AND COPPER-BASED COMPOUNDS ON THE NATURAL ENEMIES PSYTTALIA CONCOLOR AND CHILOCORUS NIGRITUS. 59. 4.1 Introduction and objectives. 59. 4.2 Material and methods. 60. 4.2.1 Chemicals. 60. 4.2.1.1 Kaolin. 62. 4.2.1.2 Copper. 64. 4.2.2 Laboratory tests. 65. 4.2.2.1 Residual contact on glass surfaces. 65. 4.2.2.2 Oral toxicity. 67. 4.2.2.3 Treatment of parasitized pupae. 68. 4.2.2.4 Treatment of the parasitization surface. 69. 4.2.3 Extended-laboratory experiments 4.2.3.1 Treatment of olive tree leaves. 71 71. 4.2.3.2 Treatment of the parasitization surface and olive tree leaves 4.2.4 Semi-field experiment. 72 73. 4.2.5 Dual choice and no-choice experiments. 75. 4.2.5.1 Psyttalia concolor. 75. 4.2.5.2 Chilocorus nigritus. 76. 4.3 Results. 79. 4.3.1 Direct mortality. 79. 4.3.2 Life span. 80. 4.3.3 Emergence. 84. 4.3.4 Beneficial capacity. 85. 4.3.5 Dual choice and no choice experiments. 88. 4.3.5.1 Psyttalia concolor. 88. 4.3.5.2 Chilocorus nigritus. 92. iii.

(11) Index 4.4 Discussion. 95. 4.4.1 Lethal and sublethal effects of kaolin, Bordeaux mixture and copper oxychloride 4.4.2 Effects of kaolin treated surfaces in dual choice and nochoice experiments 4.5 Appendix (tables of results). 5. ECDYSONE AGONISTS: EFFICACY AND ECOTOXICOLOGY ON BACTROCERA OLEAE AND PSYTTALIA CONCOLOR. INSECT TOXICITY BIOASSAYS AND MOLECULAR DOCKING APPROACHES 5.1 Introduction. 96 102 104. 111 111. 5.1.1 The ecdysone receptor. 112. 5.2 Objectives and procedures. 113. 5.3 Materials and methods. 114. 5.3.1 Insect bioassays. 114. 5.3.2 EcR-LBD sequence. 116. 5.3.3 Confirmation of expression of EcR in the ovaries. 122. 5.3.4 Modeling of PcEcR-LBD and docking studies. 123. 5.4 Results. 124. 5.4.1 Efficacy and toxicological effects of methoxyfenozide, tebufenozide and RH-5849 5.4.2 BoEcR-LBD sequence. 124 127. 5.4.3 PcEcR-LBD sequence, phylogenetic tree and expression in the ovaries 5.4.4 Modeling of BoEcR-LBD and PCEcR-LBD and docking studies. 130 135. 5.5 Discussion. 142. 5.5.1 Effciacy and toxicology of insect gorwth regulators on Bactrocera oleae and Psyttalia concolor, respectively 5.5.2 Molecular docking studies 5.6 Appendix (tables of results). 143 148 151. 6. CONCLUSIONS. 155 iv.

(12) Index. 7. REFERENCES. 157. APPENDIX. 183. Index of figures. 183. Index of tables. 189. Acronyms. 191. v.

(13) Index. vi.

(14) Resumen /Summary. RESUMEN. Los tratamientos fitosanitarios en el olivar siguen siendo hoy en día uno de los métodos de control más empleados en la lucha contra las principales plagas y enfermedades que afectan a este cultivo: la mosca del olivo, Bactrocera oleae (Rossi), el prays, Prays oleae (Bernard), la cochinilla del olivo, Saissetia oleae (Olivier), y el repilo, provocado por el hongo Spilocaea oleagina Fries. Sin embargo, y como la nueva legislación en materia fitosanitaria se dirige hacia una gestión integrada de las plagas y enfermedades, continúa siendo importante evaluar y conocer los efectos que los pesticidas tienen sobre los enemigos naturales presentes en los diferentes agrosistemas.. Una parte de este trabajo ha consistido en el estudio de los efectos directos e indirectos del caolín y dos formulados a base de cobre (caldo bordelés y oxicloruro de cobre), mediante diferentes ensayos de laboratorio, laboratorio extendido y semicampo en los enemigos naturales Psyttalia concolor (Szèpligeti)., parasitoide de la mosca del olivo, y Chilocorus nigritus (F.), depredador de diaspídidos. Este depredador se ha utilizado en lugar de C. bipustulatus (L.), que es la especie que se encuentra en los olivares. El caolín actúa fundamentalmente como repelente de los insectos y/o disuade la oviposición. En el olivar se emplea para el control de la mosca y el prays. El cobre se emplea en el control de enfermedades fúngicas y bacterianas, como el repilo y otras enfermedades del olivar. En ninguno de los ensayos realizados se encontraron diferencias estadísticamente significativas con respecto a los controles, excepto cuando se evaluó la toxicidad oral de los productos en las hembras de P. concolor. En este caso, el caolín y el oxicloruro de cobre causaron una mortalidad mayor de las hembras a las 72 horas del tratamiento, y tanto el caolín como las dos formulaciones de cobre redujeron la supervivencia. Los parámetros reproductivos sólo se vieron vii.

(15) Resumen /Summary. afectados negativamente por la ingesta de caolín. Además de los ensayos anteriores, en el caso del caolín, por su particular modo de acción, se plantearon un ensayo de elección y otro de no elección. Tanto las hembras de P. concolor como los adultos de C. nigritus mostraron una clara preferencia por las superficies no tratadas con el producto cuando se les ofrecía la posibilidad de elegir entre una superficie tratada y otra sin tratar. Cuando esa posibilidad no existía, no se detectaron diferencias estadísticamente significativas entre los tratamientos y los controles.. Además se ha comprobado también la eficacia y la selectividad de tres insecticidas reguladores del crecimiento (metoxifenocida, tebufenocida y RH-5849) sobre B. oleae y P. concolor, respectivamente. Además de estudios para evaluar la toxicidad en laboratorio de los insecticidas, se extrajo RNA de los insectos y con el cDNA obtenido se secuenció y clonó el dominio de unión a ligando (LBD) del receptor de ecdisona de ambos insectos. Posteriormente, se obtuvo la configuración en tres dimensiones del LBD de ambas proteínas y se estudió el acoplamiento de las moléculas de los tres insecticidas en la cavidad que forman las 12 α-hélices que constituyen el LBD de cada una de las proteínas. Tanto los ensayos de toxicidad como las técnicas moleculares han demostrado que metoxifenocida y tebufenocida no tienen ningún efecto nocivo ni en B. oleae ni en P. concolor. RH-5849, sin embargo, resultó inocuo para el parasitoide pero redujo notablemente la supervivencia de los adultos de la mosca, especialmente cuando entraron en contacto con el residuo fresco. El estudio del acoplamiento de la molécula de este insecticida ha mostrado que podría más o menos encajar en la cavidad que forman las hélices del LBD de la proteína de B. oleae, por lo que la búsqueda de nuevos insecticidas para el control de la mosca del olivo podría realizarse tomando como modelo la molécula de RH-5849.. viii.

(16) Resumen /Summary. SUMMARY. 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. ix.

(17) Resumen /Summary. 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.. x.

(18) Introduction. Chapter 1 INTRODUCTION 1.1 The olive tree The olive tree, Olea europaea L., is a treelike species of the Oleaceae family, within cultivated olive trees, belonging to the sativa subspecies, and wild olive trees, subspecies sylvestris, are included. Plants of this family are mainly trees and bushes Figure 1: An olive grove in Castile‐La Mancha. and some of them produce essential oils in their flowers or fruits; only olive tree fruits are edible (Lombardo, 2003; Rapoport, 2008).. Its taxonomical classification is: class Angiosperms, subclass Ranunculidae, superorder Lamianae, order Lamiales, familiy Oleaceae, subfamily Oleoideae, genus Olea, species O. europaea L. (Devesa, 2005).. It is a long‐cycle crop, as it takes a long time to begin to produce olives, to reach its peak yield and to start its decline (Civantos, 2008). Its irregular production depends on climatic conditions, pests, diseases and the alternate bearing (named “vecería” in Spanish) (Orenga and Giner, 1998). According to Cirio (1997), olive growing is described as a considerable environmental‐variability depending crop, which is highly influenced by climatic, soil, biologic, agronomic, socioeconomic and cultural conditions.. 1.

(19) Introduction The genetic homogeneity of every cultivar is high because of the vegetative propagation techniques that have traditionally been used. On one hand, first olive farmers from every region used to select, among the wild olive trees, those which were the most productive, had the biggest fruits and the highest oil‐content, allowing the conservation of the characteristics of those original cultivars. On the other hand, the spreading of these first local cultivars allowed its hybridization with other from different regions, achieving the stability of the selected individuals through vegetative reproduction techniques (Barranco, 2008).. 1.1.2 The origin of the crop The olive crop was one of the first fruit trees cultivated by man. It has been claimed that its cultivation dated back to 4,000‐3,000 years BC in the area of Palestine. After that, it spread out to all the countries of the Mediterranean region. As a consequence of Columbus’s, Magellan’s and Elcano’s voyages, it started to be cultivated in the New World. Nowadays it is also grown in North America, South Africa, China, Japan and Australia (Lombardo, 2003; Civantos, 2008), although it is considered that around 98% of olive oil world heritage is located in the Mediterranean area (Civantos, 2008).. It is not clear when olive cultivation started in Spain, but the most accepted hypothesis pointed to Phoenicians and Greeks as its introducers. During the Roman period, Hispania olive oil trade spread out all over the western Roman Empire (Pajarón, 2007), which resulted in the expanse of the crop in the Betis Valley (nowadays known as Guadalquivir area), getting up to Sierra Morena. The railway‐ building during the 19th Century favoured olive cultivation in the interior areas of the country and filled out the Spanish olive crop map (AAO, 2011).. At the beginning of the second half of the last century, the olive growing system changed from a traditional into a modern one, due to the increase of labour salaries. This fact caused the replacement of the labor by machinery and the introduction of monoculture crops. However, concerning olive groves, these changes were not as big. 2.

(20) Introduction as in other crops because of the longevity of the trees, mostly affecting cultural work, while the structure of the plantation and the variety of the trees were maintained (Pajarón, 2007).. 1.1.3 Geographical distribution The olive tree habitat is located between 30º and 45º, both in the north and in the south, in regions with a Mediterranean climate (characterized by hot and dry summers), and up to 1,000 metres above sea level. In the southern hemisphere it is also founded in more tropical latitudes whose climate is modified by altitude (Cirio, 1997; Rotundo and De Cristofaro, 2003; Civantos, 2008).. Optimal climatic conditions are those whose minimum temperatures are not lower than ‐5ºC, the average precipitation is no more than 500‐550 mm and the soil has a balanced composition, is rich in organic matter and its pH is neutral or slightly basic. Thanks to its huge adaptation capacity, it is able to grow also in very poor soils and dry locations (<250mm yearly) (Cirio, 1997).. 1.1.4 Importance of the crop The olive tree is the iconic tree of the Mediterranean area where, along with vines and cereals, it helps define the most striking features of the agricultural landscape (Duarte et al., 2008). Apart from its economic, social and cultural importance, its environmental value must also be taken into account, because of the high level of biodiversity and low rates of soil erosion found in this agrosystem (Pajarón, 2007).. 3.

(21) Introduction. 1.1. 4. 1 Economic importance World olive growing is estimated at around 1,000 million of olive trees, occupying a surface of 10 million hectares. 98% is located in the Mediterranean basin, 1.2% in America, 0.4% in Asia and the other 0.4% in Oceania. Average world olive fruit production is estimated in 16 million tons, of which 90% goes to olive oil production and the other 10% to table olives (Civantos, 2008).. The main olive oil producing countries in the world are Spain (39%), Italy (22%), Greece (16%), Tunisia (6%), Turkey (5%), Syria (4%), Morocco (2%), Portugal (2%), Algeria (1%) and Jordanian (1%) (Civantos, 2008).. According to the data of the “International Olive Council” (IOC), the world olive oil production during the 2009/2010 campaign was 3,024,000 t, and the provisional figure for 2011, 2,948,000 t (IOOC, 2011).. 1.1.4.2 Social importance Traditional olive growing has a significant socio‐economical role, as it provides an important source of income and employment, particularly in marginal regions, strongly dependent upon agricultural activities (Duarte et al., 2008). As olive‐growing areas, many villages are trying to offer different activities for rural tourism, in order to earn money not only by selling their olives. In Spain, different initiatives are being encouraged, such as the opening of the “Museo de la Cultura del Olivo” (Olive Tree Culture Museum) in Baeza (Jaén) or the recognition of labels granted to some “guarantee of origin and quality” of some varieties. The promotion of the oleo‐tourism allows people to know more about the crop, the villages and their inhabitants, the properties of olive oil and its culinary uses (Anonymous, 2009a,b).. Some studies have been carried out about the olive oil benefits on human health. This product has been not only the basis of the Mediterranean diet for ages, but of 4.

(22) Introduction plenty more besides: it is used to make cosmetics, in religious rituals, in medicine and it also has an important role in mythology. Furthermore, during the last few decades it has been shown that diet is the most important environmental factor affecting the quality of life, and olive oil is necessary in order to reach a healthy old age and to prevent the most important causes of mortality all over the world (CIAS, 2008).. 1.1.4.3 Environmental importance Olive trees are essential in the Mediterranean ecosystem because their fruits are important foodstuffs for the fauna related to it. Moreover, because trees do not loose their leaves, and thanks to the shelter that foliage provides, a special microclimate is created within the olive canopy during the winter. This makes it warmer and more attractive than the outside (exposed to the wind and to low temperatures). Indigenous and wild flora, which includes around one thousand of herbaceous and woody species (Cirio, 1997), benefit from these special conditions as well (Saavedra, 1998).. 1.1.5 Olive growing in Spain Spain is the first olive oil and table olive producer and exporting country in the world (Civantos, 2008), having the longest olive grove surface (2,580,577 ha) and the largest number of olive trees (282,696,000) (AAO, 2011; MARM, 2011a). Because of its wider territorial spreading and its economical, social, environmental and health importance, the olive growing is one of the main sectors of the Spanish agricultural system. Olive trees can be found all over Spain, except in Galicia, Asturias and Cantabria (Civantos, 2008; AAO, 2011).. According to the data published by the Ministry of the Environmental and Rural and Marine Environs (Ministerio de Medio Ambiente y Medio Rural y Marino); from December 2011, Ministerio de Agricultura, Alimentación y Medio Ambiente), Spanish olive oil production in 2011 was estimated at 1,357,400 t and 485,300 t of table olives (MARM, 2011b). 5.

(23) Introduction During the last years, the Spanish olive grove system has undergone an updating of techniques which are increasing its productivity. In the 60’s and the 70’s, old trees or trees planted in marginal areas were pulled out and replaced with other crops which were more suitable or more profitable. Simultaneously, the Spanish administration established the “Plan of the restructuring of productivity and conversion of olive groves” (Plan de Reestructuración Productiva y de Reconversión del Olivar) in which proceedings to improve or increase the olive grove productivity figured. As a consequence, areas well adapted to this crop increased the surface dedicated to them and their production, while the less adapted ones cut down on it (Civantos, 2008). There are also other changes concerning this crop, such as the increase of irrigated olive groves, the use of higher denseness plantation (2,000 – 3,000 trees per hectare), the choosing of the trees whose trunks are better adapted for mechanical harvesting and the development of a nursery industry dedicated to obtain plants with just one trunk and an early fruiting. Furthermore, farmers are nowadays aware of the importance of using the suitable growing and oil‐making techniques that guarantee a better quality of the olive oil produced (Rallo, 1998).. 1.1.6 Pests and diseases: characteristics of the most important pests and diseases of olive groves From a phytosanitary approach, olive groves can be considered as simple agricultural systems. This is due to their environmental stability, the orientation of its production, the small number of really harmful parasitic, the tolerance to produced damages, and the abundance of natural enemies (Cirio, 1997). Because of these facts, the number of chemical treatments remains still low compared to other crops (Alvarado et al., 2008). However, the olive tree grows closely related to several biotic and abiotic factors which not only establish the specificity of the present organisms, but also determine their population changes. A single change in one of them affects the whole agrosystem balance (Iannotta, 2003; Alvarado et al., 2008). For example, although more than 225 potential damage organisms have been cited since olive tree cultivation started (Haniotakis, 2005), the most important pests affecting this crop are the olive fruit fly and the olive moth. However, from the 1960’s up to nowadays, as a. 6.

(24) Introduction consequence of the abuse in the use of pesticides to fight against these two pests, another one, the black scale, has increased its population, causing different damage. Troubles brought on by other pests, like other scales, mites, etc., have also risen up due to this traditional pest management system (Alvarado et al., 2008).. Losses caused by pests, diseases and weeds are estimated to reach 30% of the crop, of which 15% are due to the action of insects. Amongst them, 10% are attributed to the main olive grove pests (Haniotakis, 2005).. According to this author, four pest categories have been established in the Mediterranean area: ‐. Key or major pests: they are the most damage‐causing ones. An annual monitoring of them is required. The olive fruit fly, Bactrocera oleae (Rossi) (Diptera, Tephritidae), is the only one considered in this category.. ‐. Secondary important pests: their losses have an occasional or local importance. The olive moth Prays oleae (Bernard) (Lepidoptera, Yponomeutidae) and the black scale Saissetia oleae (Olivier) (Homoptera, Coccidae) are included in this category.. ‐. Pests of a limited economic or localized importance: pests that change over time and cause locally and/or occasionally economic losses.. ‐. Pests without much economic importance: they rarely cause damage or losses.. The main olive grove phytophagous and pathogens are included in Tables 1 and 2.. 7.

(25) Introduction Table 1: Main olive grove phytophagous and their eating habits1,2 Key pests and secondary important pests Olive fruit fly (mosca del olivo): Bactrocera oleae (Rossi) Olive moth (polilla, prays): Prays oleae (Bernard) Black scale (cochinilla de la tizne, caparreta): Saisseta oleae (Olivier). Monophagous Olygophagous Polyphagous. Pests of economically moderate importance Olive bark beetle (barrenillo, palomita): Phloeotribus scarabaeoides (Bernard) Olive bark borer (barrenillo negro): Hylesinus oleiperda (F.) Olive pyralid, jazmines moth, olive leaf moth (polilla del jazmín, glifodes): Palpita unionalis (Hübner) Olive pyralid moth (abichado): Euzophera pingüis (Haworth) Olive gall mite (sarna, erinosis o acariosis del olivo): Aceria oleae (Nalepa). Olygophagous Olygophagous Olygophagous Olygophagous Monophagous. Secondary pests with local or temporary importance Apple mussel scale, oystershell scale (serpeta o coma del manzano): Lepidosaphes ulmi (L.) Olive parlatoria scale, (parlatoria, piojo violeta): Parlatoria oleae (Colvee) Olive psyllid (algodón, tramilla): Euphyllura olivina (Costa) Olive weevil, oziorrinco (otiorrinco): Otiorhynchus cribricollis (Gyllenhal) White grubs, European cockchafer (gusanos blancos): Melolontha papposa Illiger, Ceramida cobosi (Bagena) Olive thrips (arañuelo): Liothrips oleae (Costa) Olive midge (mosquito de la corteza): Resseliella oleisuga (Targioni‐ Tozzetti) Cicada (cigarra): Cicada barbara (Stal) Leopard moth (zeuzera o taladro amarillo): Zeuzera pyrina (L.) Birds, rodents, rabbits and hares (Oryctolagus cuniculus (L.) and Lepus europaeus Pallas) 1. Polyphagous Polyphagous Monophagous Polyphagous Polyphagous Monophagous Olygophagous Polyphagous Polyphagous Polyphagous. Phytophagous have been arranged in groups, according to their economic importance and their diet habits (monophagous, olygophagous or polyphagous). 2 The Spanish name of the pests is indicated in brackets. Sources: (Iannotta, 2003; Alvarado et al., 2008).. 8.

(26) Introduction Table 2: Olive grove pathogenic agents and abiotic diseases. Significance of the damage caused by them1,2 Bacteria Olive knot disease, tuberculosis of olive tree (tuberculosis del olivo): Pseudomonas savastanoi pv. savastanoi. Moderate‐ High. Foliar diseases‐Fungus Olive leaf spot (repilo): Spilocaea oleagina Fries (= Cyclonium oleaginum Cast.) Anthracnose (antracnosis, aceituna jabonosa): Colletotrichum gloeosporioides Penz. (= Gloeosporium olivarum Alm.) Dalmatian disease (escudete de la aceituna): Camarosporium dalmaticum (= Sphaeropsis dalmatita Thüm.) Cercosporosis or leaf spot disease on olive (emplomado de la aceituna): Pseudocercospora cladosporioides (= Cercospora cladosporioides Sacc.) Coin canker (lepra): Phlyctema vagabunda Desm.(= Gloeosporium olivae Petri) Sooty mould, fumagina (fumagina, negrilla): Capnodium elaeophilum Prill. Other fruit rots (otras podredumbres del fruto): Fusarium, Alternaria, Cladiosporium… Other foliar fungal (otras micosis foliares): Stictis, Leveillula, Phylactina Trunk decay (caries del tronco): Fonus, Phellinus, Polyporus, Stereum Chancre (chancro). High Moderate Low Moderate Low Low Low Not important Low Low. Root fungus Verticillium wilt, soil borne fungus (verticilosis): Verticillum dahliae Thick root rot fungus (podredumbre de raíces gruesas): Armillaria, Rosellinia, Omphalotus Thin root rot fungus (podredumbre de raicillas): Phytophtora, Cylindrocarpon, Fusarium. High Low Moderate‐ Low. Virus Malformation, yellowish (malformaciones, amarilleo): unidentified virus Latent infections, yellowish (infecciones latentes, amarilleo): Nepovirus, Cucumovirus, Oleavirus. Not important Not important. Nematode Root lesions: nodes, galls (nódulos, agallas): Meloidogyne, Pratylenchus…. Not important. Phanerogam Mistletoe, Jopo, Cuscuta. Not important Abiotic diseases. Lack of essential nutrients : boron, iron, potassium Other damages: frost, drought, flooding… 1. Moderate‐ Low High ‐ Low. The table has been organised according to the different pathogen agents or the causes of abiotic diseases. Their economic importance, according to the damage caused, has also been pointed out as high, moderate, low or unimportant. 2 The Spanish name of the pests is indicated in brackets. Sources: Iannotta, 2003; Trapero and Blanco, 2008; Trapero et al., 2009. 9.

(27) Introduction In intensive farming systems, the high‐density plantations and the continuous presence of fresh shoots during the whole vegetative cycle allow phytophagous species to have a constant availability of food. Apart from traditional damaging organisms, others which had never caused significant damage on the crop could be now categorized as potential pests or disease‐causing agents (Torrell and Celada, 1998).. 11.6. 1 The olive fruit fly (Bactrocera oleae ) Bactrocera oleae distribution is primarily limited to the regions where cultivated and wild olive trees are found. Today, the olive fruit. fly. is. reported. throughout. the. Mediterranean basin, Africa and from Middle East to India (Guerrero, 2003; Alvarado et al., 2008). It is also found in all the countries where olive crop has been introduced during. Figure 2: Female of B. oleae (Anonymous, 2009c). recent years, as USA, China, but it has not been observed in South America and Australia (Civantos, 1999; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Daane and Johnson, 2010).. It was recorded attacking olives in biblical times and has long been a major pest in the Mediterranean basin. Larvae are monophagous on olive fruits in the genus Olea, including O. europaea (cultivated and wild), O. verrucosa and O. chrysophylla (Daane and Johnson, 2010). However, it has also been reared in laboratory on Ligustrum and Jasminum berries (Civantos, 1999; Alvarado et al., 2008) and on tomatoes (Navrozidis and Tzanakakis, 2005).. As soon as adults emerge, they look for the sweetened and nitrogenous substances they need as nutritional requirements. They feed on a variety of organic sources including insect honeydews (for example, black scale honeydew), plant nectar, plant. 10.

(28) Introduction pollens and fruits exudates (De Andrés, 1991), and also bird dung, bacteria and yeasts. Females lay their eggs in ripening but also in green fruits, in which the newly hatched larvae feed upon the pulp. They pupate inside the olive or exit to do it on the ground (Daane and Johnson, 2010). Larval development is largely temperature dependent. It has been reported that the lower temperature threshold is 6ºC and the upper one is 35ºC, while the optimal temperature ranges from 20º to 25ºC. Relative humidity is only important if its value is low and temperatures are high during a long period (De Andrés, 1991; Civantos, 1999; Rotundo and De Cristofaro, 2003; Alvarado et al., 2008).. The number of annual generations depends not only on the temperature, but also on the relative humidity, on the microclimate within the olive canopy and on the availability and quality of olive fruits. This results in variation in the reported number of generations per season within the fly’s endemic range, which encompasses a variety of climatic regions. Two to three generations have been reported in continental climate areas, while three generations are always common in coastal regions (Civantos, 1999), or even four (De Andrés, 1999; Alvarado et al., 2008). Some authors have also reported up to six or seven generations (De Andrés, 1991).. Several studies have shown that olive cultivars vary in their susceptibility to the olive fruit fly. Some of the factors that possibly play a role include fruit size and weight, colour, fruit epicarp hardness, surface covering (mainly aliphatic waxes), phenological stage of the crop, and chemical factors (Daane and Johnson, 2010). Oil destined cultivars are less susceptible to olive fly attack than table olive cultivars (Civantos, 1999; Rotundo and De Cristofaro, 2003; Alvarado et al., 2008). It has also been demonstrated that the most susceptible fruits in a tree are the biggest and those which are in the outer part of the tree’s crown (Alvarado et al., 2008).. The relative importance of the economic damage provoked depends either on the olive fly population density or on the period of the year considered (De Andrés, 1991; Civantos, 1999; Alvarado et al., 2008; Daane and Johnson, 2010). In areas of the world where the olive fruit fly is established, it has been reported as responsible for losses of up to 80% of oil value and 100% of some table cultivars. It has been estimated to 11.

(29) Introduction damage 5% of total olive production, resulting in economic losses of approximately USD 800 million per year (Haniotakis, 2005; Daane and Johnson, 2010). In table olives damage. is. more. important. because. oviposition stings on fruits totally reduce their value (Alvarado et al., 2008). Therefore, the tolerable infestation level is near zero larvae per. fruit. (Daane. and. Johnson,. 2010).. Economic damage can be classified as direct Figure 3: Detail of a B. oleae larva in an olive fruit. Microorganism growth can be observed in the feeding gallery. and indirect:. Direct damage: premature fruit dropping or loosing of fruit weight resulting from larvae feeding the pulp. The production rates can decrease between 5 and 10 % Indirect damage: they are referred to the lowered quality and value of pressed oil due to increased acidity as a result of microorganism growth inside olive tree fruits (bacteria, yeasts and mould).. In the Mediterranean area, it seems that none of the olive fruit fly’s predators or parasitoids is able to totally control the pest (González‐Núñez, 2008). According to the earlier surveys, it appeared that sub‐Saharan Africa might provide a rich source of natural enemies of B. oleae. The most recent surveys suggest that a smaller group best represents the primary parasitoids attacking olive fruit fly in its native range. Most of these wasps are synovigenic, koinobiont, larval‐pupal or larval‐prepupal parasitoids in the Opiinae subfamily. The exception is the idiobiont larval ectoparasitoid Bracon celer Szépligeti. (Hymenoptera, Braconidae). Some chalcid parasitoids have also been reared from olive fruit fly, although many of the species are polyphagous parasitoids that may opportunistically attack the olive fruit fly (Daane and Johnson, 2010). In the Mediterranean basin the most common parasitoids found are the Hymenoptera Eupelmus urozonus Dalman (Eupelmidae), Pnigalio mediterraneus (Ferriere and Delucchi) (Eulophidae), Eurytoma martellii Domenichini (Eurytomidae), Psyttalia concolor (Szèpligeti) (Braconidae) and Cyrtoptyx latipes (Rondani) (Pteromalidae) 12.

(30) Introduction (Iannotta, 2003; Rotundo and De Cristofaro, 2003). The most commonly found are E. urozonus and P. mediterraneus, but they are not effective enough to control B. oleae populations (Civantos, 1999). Neonate larvae of the olive fruit midge, Lasioptera berlesiana Paoli (Diptera, Cecydomiidae), feed on the eggs of B. oleae. The problem is that females, at the time of egg‐laying, also introduce the fungus S. dalmatica (C. dalmaticum), which causes Dalmatian disease in olive fruits. There seems to be a large number of B. oleae larvae and pupae predators too. Predators attack B. oleae when third instar larvae drop to the ground to pupate beneath the trees. This group of predators includes ants, Carabidae, Staphyllinidae, spiders and earwigs (González‐ Núñez, 2008; Daane and Johnson, 2010). Some authors also take into account the role of insectivorous birds and birds which feed on olive fruits because they can decrease B. oleae populations of wild, overgrown and ornamental olive trees (González‐Núñez, 2008).. Over the last four decades B. oleae management has been based on the use of different insecticides (such as organophosphates, pyrethroids and spinosad). However, the fly has already evolved resistance to dimethoate (Daane and Johnson, 2010) and spinosad (Kakani et al. 2010). Furthermore, the continued use of such products has been questioned in recent years, especially by environmentalists. For example, in the case of dimethoate, which is used to control both B. oleae and the anthophagus generation of the olive moth, a strong and dramatic effect on the abundance of different trophic groups has been reported (Santos et al., 2010). In addition, residues of pesticides have been detected both in olive oil and in the environment where olives are grown. These facts have caused concern in most olive growing countries and have lead to a concerted effort to reduce the amount of pesticides used in this crop (Montiel‐Bueno and Jones, 2002).. 13.

(31) Introduction. 1.1.6.2 The olive moth (Prays oleae ) Prays. oleae. (Bernard). (Lepidoptera:. Yponomeutidae) is considered the second important pest in olive groves. It is found throughout the Mediterranean basin. Although its main host plant is the olive tree, it can also Figure 4: Adult of P. oleae (Anonymous, 2009d). feed on other Oleaceae species (Rotundo and De Cristofaro, 2003; Alvarado et al., 2008).. It has three generations per year, synchronized with the olive tree phenology. The first one infests the leaves (phyllophagous), the second one the flowers (antophagous) and the third one the fruits (carpophagous). The most harmful is the third one (Iannotta, 2003) because feeding damage causes premature fruit dropping. Chemical treatments against the first or the second generation are only justified when trees are young or when moth population is high and the number of flowers is low (Rotundo and De Cristofaro, 2003; Alvarado et al., 2008).. Climatic conditions are very important to the olive moth development and determine its presence in the different geographical regions (Civantos, 1998a). Cold weather during the winter (<10ºC) or hot weather (>30ºC) and a high relative humidity percentage (> 70%) during the summer control the populations. A relative humidity percentage below 50% makes survival difficult for the eggs (Alvarado et al., 2008; Civantos, 1998a; Rotundo and De Cristofaro, 2003).. Parasitism rate is high and varies among generations, years and geographical areas. It is responsible for between 10 and 50% of moth population mortalities. Amongst their parasitoids, the Hymenoptera Ageniaspis fuscicollis var. praynsicola (Silvestri) (Encyrtidae), Chelonus eleaphilus Silvestri (Braconidae) (both of them specific to prays), Diadegma armillata (Gravenhorst) (Ichneumonidae), Apanteles xanthostigmus (Haliday) (Braconidae), the Eulophidae Pnigalio mediterraneus (Ferriere and Delucchi). 14.

(32) Introduction and P. pectenicornis (L.), and other Trichogrammatidae species stand out. They parasitize larvae, pupae or eggs (Civantos, 1998a; González‐Núñez, 2008). Among their predators, the chrysopids Chrysoperla carnea (Stephens) and Dichochrysa flavifrons (Brauer) (Neuroptera, Chrysopidae) are very important (González‐Núñez, 2008). They feed on the eggs (BOJA, 2002; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Alvarado et al, 2008), although they can also feed on larvae of the phyllophagous and the anthophagous generations (González‐Núñez, 2008). Also different spider species, especially mites which feed on eggs and larvae (Civantos, 1998a), ants, Heteroptera and Coleoptera are important predators of the olive moth (González‐Núñez, 2008). Bacteria, fungi, protozoa and virus can also affect the pest, but they are not usually efficient enough (Civantos, 1998a).. 1.1.6.3 The black scale (Saissetia oleae ). The. black. scale,. Saisettia. oleae. (Olivier). (Homoptera, Coccidae), is spread all over the world, but mainly in the Mediterranean basin. It is found in olive groves and citrus, but also in some other trees and bushes. It prefers shady areas and humid environments (Rotundo and De Cristofaro, 2003; Figure 5: S. oleae adult females. Alvarado et al., 2008).. They feed on the trees by piercing of host tissues and sucking the sap. Although direct damage is not really significant, the black fungi developing on the honeydew they deposit on trees (sooty mould) are responsible for reducing photosynthesis and can be referred to as contamination, often rendering plants or fruits unmarketable (Iannotta, 2003; Alvarado et al., 2008). Furthermore, honeydew is one the of B. oleae adults’ favourite sweet foodstuff, so it can also attract them (Guerrero, 2003).. Saissetia oleae holds a high number of natural enemies which parasitize different nymphal stages and even preovipositional females. They are mainly Hymenoptera 15.

(33) Introduction parasitoids of the genus Metaphycus (M. helvolus (Compere) and M. lounsburyi (Howard); Encyrtidae) and other native Aphelinidae, such as Coccophagus lycimnia (Walker), C. semicircularis (Föster) and C. scutellaris (Dalman), which also parasitize other Coccidae species (González‐Núñez, 2008). Among predators, the most important are the Hymenoptera Scutellista cyanea (=S. caerulea) (Mostchulsky) (Pteromalidae), whose larvae feed on the eggs under the female scale, and some Coleoptera Coccinellidae, such as Chilocorus bipustulatus (L.) (Iannotta, 2003; Rotundo and De Cristofaro, 2003; Alvarado et al., 2008), Brumus quadripustulatus (L.), Rhyzobius spp., Scymnus spp. The Lepidoptera Eublemma scitula Rambur (Noctuidae) and the Neuroptera Coniopteryx spp. (Coniopterygidae) are important as well (González‐ Núñez, 2008).. 1.1.6.4 The olive leaf spot (Spilocaea oleagina ) The olive leaf spot, caused by the fungus Spilocaea oleagina Cast., is the most common disease in Spanish olive orchards (Trapero et al., 2009). Its importance is due both because of the extensive areas where it can be found. Figure 6: Olive leaf spot. and because of the damage caused when development conditions are in its favour (rainy years, high density and poorly aerated plantations and olive groves irrigated or close to wet areas) (Guerrero, 2003; Pajarón, 2007; Trapero and Blanco, 2008). Despite the fact that the fungus is only pathogenic on olive trees, morphologically similar fungus have been described as disease‐causing on Ligustrum, Phillyrea and Quercus species (Trapero et al., 2009).. Typical symptoms of the disease are the black circular spots on the adaxial surface of the leaves, often surrounded by a yellowish hallow. Leaves fall prematurely and death of twigs may ensue. Conidia are spread out mainly by the rain. Once they are on the susceptible tissues of the plant, they germinate only if there is water available or. 16.

(34) Introduction the relative humidity is higher than 98% and temperature swung by around 0º and 27ºC, being 15ºC the optimum. Consequently, cultural practices helping aeration of the trees are very important (Trapero et al., 2009). Premature defoliation has serious consequences on the plant vegetative activity and yield. It could reduce either the differentiation rate of auxiliary buds into flower‐bearing shoots or the productivity of the trees. Sometimes, it can also infect fruit peduncles and provoke their premature fall, which decreases their quality and fatty yield. Olive oil quality from these fruits, however, remains unaffected (Guerrero, 2003; Pajarón, 2007; Trapero and Blanco, 2008).. 1.2 Control of pests and diseases. The use of different substances with insecticide properties dates from Roman and Greek times. During the 19th Century, artificial fertilizers were developed. They were cheap, powerful and easy to transport in bulk. Similarly, it also occurred in the 1940’s with chemical pesticides, leading to the decade being referred to as the “Pesticide era” (MARM, 2006). Indeed, the synthesis of the DDT in 1939 seemed to have solved all pest problems (Dent, 1991; Casida and Quistad, 1998).. Traditional agricultural systems are based on the use of different chemical products, without taking into account the possible negative impacts caused by their widely and uncontrolled use and abuse. For example, environmental contamination, intoxications or the appearance of resistances to insecticides among the pests have already been reported in different crops, including olive groves (Cirio, 1997; De Ricke, 1998; Chamorro and Sánchez, 2003; Iannotta, 2003; Alvarado et al., 2008).. In contrast to these chemical‐based traditional pest management practices, once farmers and researchers realized it was necessary to rationalise the use of pesticides, the concept of “Integrated Pest Management” (IPM) appeared. Furthermore, as a reaction to agriculture growing reliance on synthetic fertilizers, the organic movement had already started between 1930 and 1940 (MARM, 2006). 17.

(35) Introduction. 1.2.1 Integrated Pest Management. In 1967, the FAO (Food and Agriculture Organization of the United Nations) defined IPM as “the careful consideration of all available pest control techniques and subsequent integration of appropriate measures that discourage the development of pest populations and keep pesticides and other interventions to levels that are economically justified and reduce or minimize risks to human health and the environment. IPM emphasizes the growth of a healthy crop with the least possible disruption to agro‐ecosystems and encourages natural pest control mechanisms” (FAO, 2011a).. Later on, in 1976, the International Organisation for Biological and Integrated Control of Noxious Animals and Plants (IOBC) defined Integrated Production: “Integrated Production is a concept of sustainable agriculture based on the use of natural resources and regulating mechanisms to replace potentially polluting inputs. The agronomic preventive measures and biological/physical/chemical methods are carefully selected and balanced taking into account the protection of health of both farmers and consumers and of the environment”. The aim of the IOBC is the promotion of biological and integrated methods to fight against pest, diseases and weeds (IOBC, 2011).. Integrated Protection (IP) fits among the different Integrated Production measures. The aims of IP are both to protect the environment and to be profitable for farmers, although both situations are not always compatible. IP is based on three practices (Cirio, 1997):. ‐. Crop monitoring: that is, a routinely checking process against defined objectives and targets. It takes place periodically and evaluates and verifies standards across a range of plantation activities. It is a rigorously documented process that will normally result in a programme of improvements.. 18.

(36) Introduction ‐. Application of the Economic Injury Level (EIL), which is the point when economic damage that occurs from insect injury equals the cost of managing insect populations; it is the breakeven point (Alvarado et al., 2008). Damage that occurs below that point is not worth the cost of preventing it. Because these insect or injury levels are not wanted to be reached, a point that is set well below the EIL is used, usually meaning when an insecticide can be applied. This “take action” level is known as the Economic Threshold (ET).. ‐. Evaluation of the proper control methods both for effectiveness and risk. Good practices related to crop protection include those that use resistant cultivars and varieties, crop sequences, associations, and cultural practices that maximize biological prevention of pests and diseases; maintaining regular and quantitative assessment of the balance status between pests and diseases and beneficial organisms of all crops; adopt organic control practices where and when applicable; applying pest and disease forecasting techniques where available; determining interventions following consideration of all possible methods and their short‐ and long‐term effects on farm productivity and environmental implications in order to minimize the use of agrochemicals (FAO, 2011b).. Biological control will always try to exploit agrosystem trophic food chains (Urbaneja and Jacas, 2008). Therefore, all the measures which protect auxiliars should be favoured. Special attention should be paid to cultural practices and pesticide applications, which can control pests but also have a negative impact on natural enemies (Jiménez et al., 2002). For that reason, it is very important to know the biological and phenological cycles of auxiliary fauna, their role in pest control and the side effects of pesticides on them (Civantos, 1998b). Furthermore, regional regulations of integrated production have established at least two natural enemies whose protection and increase are important (González‐Núñez, 2008).. Pesticides should only be applied as a last resort when there are no adequate non‐ chemical alternatives and their use is economically justified. They should be as specific as possible for the target and have the least side effects on human health, non‐target 19.

(37) Introduction organisms and the environment. Their use should be kept at minimum levels, e.g., by partial applications (Civantos, 1998b; Hassan, 1998; BOJA, 2002; Malavolta et al., 2002; FAO, 2011a).. In 2009, the European Parliament approved the new legislation about the sustainable use of pesticides and their trade (Regulation EC 1107/2009 of the European Parliament and the Council of 21 October 2009 concerning the placing of plant protection products on the market. It repeals the Council Directives 97/117/EEC and 91/414/EEC and the Directive 2009/128/EC of the European Parliament. It establishes a framework for Community action to achieve the sustainable use of pesticides). The compromise deal on the proposed regulation will put in place a system where a positive list of approved active substances in pesticides will be drawn up. Pesticides will then be licensed at the national level based on this list. The deal allows exemptions for banned active ingredients to be used in pesticides for up to five years, if they are proven essential for crop survival. Certain types of banned active ingredients (candidates for substitution) have to be replaced within three years, if safer alternatives exist. The compromise deal on the framework Directive requires Member States to adopt National Action Plans with quantitative targets, measures and timetables. The deal prohibits pesticide use, or at least requires it to be kept to a minimum, in specific areas used by the general public or by vulnerable groups (IEEP, 2009; OJEU, 2009a,b; Palomar, 2009; Ruiz‐Torres, 2009).. In Spain, IP practices are carried out under the supervision of public regulated organisms, the ATRIAS (“Agricultural Integrated Treatment Groups”: “Agrupaciones de Tratamientos Integrados en Agricultura”, in Spanish), since 1979. They control the crops and decide the proper treatments according to the methodology tuned up by Plant Health Services (Chamorro and Sánchez, 2003).. 20.

(38) Introduction There is a national logo which identifies products produced under IP techniques, as well as some other different Autonomous Communities’ logos (BOE, 2004; MARM, 2004a,b).. Figure 7: National and Autonomous Integrated Protection logos (BOE, 2004; MARM, 2004b). 1.2.2 Organic farming Despite having started at the beginning of the XXth century, organic farming was not recognized as a feasible agricultural method until the beginning of the seventies (MARM, 2006).. There is a worldwide umbrella organization for the organic movement which unites more than 750 member organizations in 116 countries, the International Federation of Organic Agriculture Movement (IFOAM). It actively participates in international agricultural and environmental negotiations with the United Nations and multilateral institutions to further the interests of the organic agricultural movement worldwide. According to IFOAM, organic agriculture can be defined as “A production system that sustains the health of soils, ecosystems and people. It relies on ecological processes, biodiversity and cycles adapted to local conditions, rather than the use of inputs with adverse effects. Organic agriculture combines tradition, innovation and science to 21.

(39) Introduction benefit shared environment and promote fair relationships and good quality of life for all involved” (IFOAM, 2011).. Just as in IPM, plant health should be achieved through the maintenance of preventive measures. For example, the choice of appropriate species and varieties resistant to pest and diseases, suitable crop rotations, mechanical and physical methods and the protection of natural enemies. In case of needing a treatment to a crop, plant protection products may only be used if they have been authorised for its use in organic production (there is a restricted list of products and substances that can be used). All products and substances should have plant, animal, microbial or mineral origin, except if products are not available in sufficient quantities or if alternatives are not possible. If they are not identical to their natural form, they may only be authorised whether their conditions for use preclude any direct contact with the edible part of the crop. Each European Union Member State regulates the use of these products within their territory (OJEU, 2007).. The council Regulation (CE) 834/2007 regulates on organic production and labelling of organic products. It repeals the previous Regulation (EEC) 2092/91. In order to ensure that organic products are produced in accordance with requirements laid down under the Community legal framework of organic production, activities performed by operators at all stages of production, preparation and distribution of organic products should be submitted to a control system set up and managed on official controls (OJEU, 2007). In Spain, the MARM has a specific program whose main aims are to promote organic farming, to improve the trade, the consumption and the knowledge about organic products, as well as to get better collaboration among institutions and farmers. Autonomous Communities are directly responsible for regulations (MARM, 2007; OJEU, 2007).. 22.

(40) Introduction There is a European Union logo, a national one and some others from some Autonomous Communities. There is also a logo which certifies Spanish organic products for export (OJEU, 2007; Anonymous, 2008; MARM, 2011c).. Figure 8: Spanish, Autonomous Communities and European Union logos. Certification for European organic products (ECO CERT, SHC) (Anonymous 2008, MARM, 2011c). 1.2.3 Integrated Protection in olive groves Integrated Olive Production in Spain has specific regulations in six Autonomous Communities (Andalusia, Balearic Islands, Catalonia, Extremadura, Murcia and Valencian Community). The number of hectares growing under this production system is 290,505 ha, which accounts for11% of the total olive crop hectares in our country (MARM, 2011e,f).. Within the IOBC, there is a specific Working Group (WG) focus on olive crops, the “Integrated Protection of Olive Crops”, which was initiated in 1991. The main goal of the group is to promote collaboration in multidisciplinary research on the development, evaluation and implementation of integrated control strategies for olive pests and diseases. An important priority is the exchange of knowledge and the main ultimate targets are to minimize the impacts of olive crop protection on the environment, to increase sustainability and also to support the production of higher quality products (IOBC, 2011). IOBC has also published specific guidelines for 23.

(41) Introduction integration production of olives. The purpose of these guidelines is to define the basic requirements of integrated production in olives in such a generalised way that these rules can be applied in all geographical regions (Malavolta et al., 2002).. Table 3 summarizes some of the specific integrated production practices of olive groves.. Table 3: Integrated pest management in olive crops Crop monitoring As olive grove areas tend to be big, in order to simplify pest and diseases monitoring they are divided into homogeneous smaller areas. Control plots, as more representative of the whole area as possible, are then the monitoring units. Different traps are placed in the control plot and vegetative parts of the trees or fruits, depending on the phenological stage and the pest or the disease evaluated, are periodically sampled Economic threshold levels Tolerable thresholds are somewhat debatable because several factors influence them (the region, the variety, the destination of the harvest, etc.). That is the reason why the specific pest or disease, their secondary effects and the particular conditions of each farm should be taken into consideration when evaluating. Data from similar areas can be extrapolated Sources: (Cirio, 1997; Civantos, 1998b; Civantos, 1999; BOJA, 2002; Chamorro and Sánchez, 2003; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Romero et al., 2006; Moretti et al., 2007; Pajarón, 2007; Alvarado et al., 2008; González‐Núñez, 2008; Trapero and Blanco, 2008; IAEA, 2009; Trapero et al., 2009; Bento et al., 2010; Delrio, 2010).. 24.

(42) Introduction Table 3: Integrated pest management in olive crops (continuation) Control methods Agricultural practice. Pest or disease controlled New plantations. Tuberculosis, verticillium wilt, olive leaf spot, Selecting best varieties for local growing anthracnose, black scale, olive fruit fly and conditions olive moth Verifying that both the soil and the new Verticillium wilt plants are pathogen‐free Planting trees in a density that provides Olive leaf spot, anthracnose a good aeration Cultural techniques Avoiding nitrogen over‐fertilisation. Verticillium wilt, black scale, olive leaf spot Fruit fly and olive moth pupae could be Limiting heavy tillage practices destroyed by tillage; however, limiting tillage practices increases populations of beneficials. Olive fly populations increase while beneficial Herbicide applications populations decrease It avoids nitrogen run off and increases Maintaining permanent soil covering biodiversity Limiting water inputs in irrigated Verticillium wilt, root fungus, white grubs, plantations black scale, olive leaf spot Olive pruning Black scale, bark beetles, Euzophera pingüis, tuberculosis, fungal diseases. Achieving a good aeration Disinfecting pruning tools and ensuring that there are not damage Removing the pruned branches and destroyed them Pruning during the dormant season. Olive knot disease Zeuzera pyrina and Cossus cossus larvae and Phloeotribus scarabaeoides adults Different pests. Harvesting Early harvest B. oleae Do not mixing fruits collected from olive B. oleae trees with those lying on the ground Sources: (Cirio, 1997; Civantos, 1998b; Civantos, 1999; BOJA, 2002; Chamorro and Sánchez, 2003; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Romero et al., 2006; Moretti et al., 2007; Pajarón, 2007; Alvarado et al., 2008; González‐Núñez, 2008; Trapero and Blanco, 2008; IAEA, 2009; Trapero et al., 2009; Bento et al., 2010; Delrio, 2010).. 25.

(43) Introduction Table 3: Integrated pest management in olive crops (continuation) Control methods Agricultural practice. Pest or disease controlled Biological control. Releases of the Hymenoptera Psyttalia concolor, Fopius arisanus, P. lounsburyi, Eupelmus urozonus Releases of the parasitoids Methaphycus swirskii, Diversinervus elegans, M. barletti, M. helvolus, M. lounsburyi and the predators Rhyzobius forestieri, Brumus quadripustulatus Releases of the fungus Bauveria bassiana Balsamo and Metarhizium anisopliae Metchnikoff Releases of different nematode species Applications of the fungus Talaromyces flavus Klöcker Inoculative releases of the fungus Fomes spp. and Agrobacterium tumefaciens Releases of the parasitoid Trichogramma spp., the predator Chrysoperla carnea and the parasporal crystals of the bacterium Bacillus thuringiensis var. kurstaki Releases of the parasporal crystals of the bacterium Bacillus thuringiensis var. kurstaki Management of the agroecosystem to maximize the effect of native or introduced biological control agents (conservative or natural control): sowing plants that attract natural enemies, offering honey or other sugar sources…. B. oleae. S. oleae. B. oleae Z. pyrina Verticillium wilt Vegetable parasites. P. oleae. Palpita unionalis. Different olive pests. Biotechnical methods Mass trapping (traps baited with pheromones) Z. pyrina, C. cossus Mass trapping (traps baited with food lures); “Attract and kill” (integrating the sexual pheromone and ammonium bicarbonate as lures), sexual confusion B. oleae and releases of sterile males (Sterile Insect Technique) Sources: (Cirio, 1997; Civantos, 1998b; Civantos, 1999; BOJA, 2002; Chamorro and Sánchez, 2003; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Romero et al., 2006; Moretti et al., 2007; Pajarón, 2007; Alvarado et al., 2008; González‐Núñez, 2008; Trapero and Blanco, 2008; IAEA, 2009; Trapero et al., 2009; Bento et al., 2010; Delrio, 2010).. 26.

(44) Introduction Fruit flies, such as B. oleae, are not attractive targets for classical biological control. This is partly because of several features in their life histories which make conditions very difficult for parasitoids. Adults of many fruit fly species disperse widely when they emerge and leave their parasitoids behind. This also happens when fruits disappear from crops and fruit flies disperse widely to other areas. Some examples of failures and successes in efforts to establish parasitoids in countries have been demonstrated by many years of extensive biological control programs conducted in the Pacific region and other countries outside it (Peters, 1996).. Conservative biological control programs are effective against some olive grove pests. For example, a study carried out by Boccaccio and Petacchi (2009) showed that landscape structure and natural or semi‐natural woodland play a role in enhancing B. oleae parasitoid activity. Some other experiments have studied the effect of different attractive sources (sugars, yeasts, etc.) on the abundance of olive pests predatory arthropods and the possible enhancement of their activity (Bento et al., 2004), or the effect of the establishment of vegetation patches which produce flowers (Jorge et al., 2005).. Pesticide applications in olive crops are sometimes necessary against B. oleae, P. oleae, olive leaf spot and anthracnose, less frequently with S. oleae and rarely with the rest of the pests (Iannotta, 2003). The most used products in these agrosystems are syntheticc insecticides, like organophosphates and pyrethroids against pests, and copper‐based compounds against diseases (MARM, 2011c). However, it should be pointed out that their use is lower compared to other crop systems (Cirio, 1997).. Spanish regulation has established the predator C. carnea as one of the two natural enemies whose protection and increase is important in olive groves. The other natural enemy should be chosen among the most important natural enemies in each region (González‐Núñez, 2008).. 27.

(45) Introduction. 1.2.4 Organic olive farming. Organic olive groves occupy a surface of 126,328.26 ha (4.9% of the total), chiefly in Cordoba (Andalusia), where a 53.30% of organic crop is located (according to the MARM, in 2010 there were 1,650,866 ha of organic farming in Spain) (MARM, 2011d).. Agricultural practices to fight against olive pests and diseases are similar to those previously described for IP systems. However, in organic olive systems there is a lack of a wide range of effective products to control some of them, as it occurs with B. oleae. In this case, the interest on using repellent and antiovipositional products, as well as products able to kill both their larvae and eggs, has increased in the last years (Caleca and Rizzo, 2006; Caleca et al., 2008).. 1.3 Side-effects of pesticides on non-target organisms. The effects of chemical pesticides on predators and parasites are much less known than on herbivorous. However, literature on natural enemy/pesticide research has increased at an exponential rate since the late 1950s.. It is necessary to test the possible negative effects of pesticides on non‐target arthropods, not only for regulatory requirements before a product is able to be registered, but also for knowing whether a plant protection product is suitable for using in IPM programs. In Europe, according to the Council Regulation (EC 1107/2009), the objective of protecting human and animal health and the environment should take priority over the objective of improving plant production. Therefore, it should be demonstrated, before plant protection products are placed on the market, that they present a clear benefit for plant production and do not have any harmful effect on human or animal health, including that of vulnerable groups, or any unacceptable effects on the environment. Regarding the effects on the environment, the following requirements have to be considered: the fate and distribution of the products in the. 28.

(46) Introduction environment, their impact on non‐target species, including the ongoing behaviour of those species, and their impact on biodiversity and the ecosystem (OJEU, 2009a). With the aim of developing and validating test methods to assess the side‐effects of plant protection products to non‐target arthropods, the IOBC, the BART (Beneficial Arthropod Testing Group) and the EPPO (European and Mediterranean Plant Protection Organisation) in collaboration with the Council of Europe), decided in 1994 to set up a Joint Initiative (JI) (Barret et al., 1994). JI activities started in 1995 and different reports and conferences resulted in the publication of a guidance document for regulatory testing and interpretation of semi‐field and field studies with non‐target arthropods. They describe test systems, treatments, validity criteria of the studies, information on test organisms, test procedures, test conditions, biological observations, data analyses and reporting for selected terrestrial non‐target arthropods (Candolfi et al., 2000).. In 1974, the working group of the OILB/SROP, “Pesticides and Beneficial Organisms” was founded. Their main objective was to coordinate the developing of standard methodologies to evaluate side‐effects on the most important natural enemies and to choose selective pesticides to be used in IPM programs (Hassan, 1998). Pesticides are selected according to a sequential process which assumes that harmless pesticides in laboratory tests will be also harmless in semi‐field and field, and they do not need additional studies. Since 1980, standard guidelines to test side‐effects of pesticides on natural enemies; rearing methods for beneficial arthropods; comparison of results of laboratory, semi‐field, field experiments, and results of the joint programs to test the side‐effects of pesticides on beneficial organisms have been published (IOBC, 2011).. The measurement of the acute toxicity of pesticides to beneficial arthropods has traditionally relied on the determination of an acute median lethal dose or concentration. However, these tests can only be a partial measure of their deleterious effects. In addition to direct mortality induced by pesticides, their sublethal effects on arthropod physiology and behaviour must be considered for a complete analysis of their impact (Desneux et al., 2007). In extended laboratory and semi‐field experiments, treated vegetal material is used. In the case of extended laboratory assays, plants are 29.