Diptera Tephritidae) Male and Female

IOBC / WPRS

Working group “Integrated Protection of Olive Crops”

OILB / SROP Groupe de travail “Protection Intégrée des Olivaies”

Proceedings of the meeting

Comptes rendus de la réunion

at / à

Florence (Italy)

26-28 October 2005

Edited by: Argyro Kalaitzaki

IOBC wprs Bulletin Bulletin OILB srop Vol. 30 (9), 2007

The content of the contributions is in the responsibility of the authors

The IOBC/WPRS Bulletin is published by the International Organization for Biological and Integrated Control of Noxious Animals and Plants, West Palearctic Regional Section (IOBC/WPRS)

Le Bulletin OILB/SROP est publié par l‘Organisation Internationale de Lutte Biologique et Intégrée contre les Animaux et les Plantes Nuisibles, section Regionale Ouest Paléarctique (OILB/SROP)

The Publication Commission of the IOBC/WPRS:

Horst Bathon Luc Tirry Federal Biological Research Center University of Gent for Agriculture and Forestry (BBA) Laboratory of Agrozoology Institute for Biological Control Department of Crop Protection Heinrichstr. 243 Coupure Links 653 D-64287 Darmstadt (Germany) B-9000 Gent (Belgium) Tel +49 6151 407-225, Fax +49 6151 407-290 Tel +32-9-2646152, Fax +32-9-2646239 e-mail: [email protected] e-mail: [email protected]

Address General Secretariat:

Dr. Philippe C. Nicot INRA – Unité de Pathologie Végétale Domaine St Maurice - B.P. 94 F-84143 Monfavet cedex France

ISBN 92-9067-205-4 http://www.iobc-wprs.org i

Preface

This bulletin contains the proceedings of the European meeting of the IOBC/WPRS Working Group “Integrated Protection of Olive Crops” that was held in Florence, Italy, October 26-28 2005 in the Polo Scientifico of Sesto Fiorentino. This is the second meeting of the new period of activity of the group which was started in 2003 in Chania.

Approximately, 86 scientists from 12 different olive growing countries (Italy, Spain, Portugal, Greece, UK, Austria, USA, Montenrgro, Slovakia, Egypt, Tunisia, Iran) attended the meeting. During the meeting, three plenary lectures, 30 oral contributions and 40 posters were presented. Topics in this Bulletin include: Bactrocera oleae (behaviour, chemical ecology, monitoring, population dynamics, chemical, biological and biotechnical control methods, side effects), other pests of olive groves and olive agroecosystem aspects, diseases and advanced IPM strategies in olive groves.

On behalf of the working group and all attendances I would like to express my gratitude to the Local Organizing Committee: Prof. Antonio Belcari (Chairperson), Dr. Marzia Cristiana Rosi, Dr. Patrizia Sacchetti and Prof. Giuseppe Surico from University of Florence, Dr. Bruno Bagnoli from Istituto Sperimentale per la Zoologia agraria of Florence, Dr. Ruggero Petacchi from Biolabs, Sant’ Anna School of Advanced Studies of Pisa and Prof. Alfio Raspi from University of Pisa, for organizing this meeting. I would like also to thank the public and private sponsors who supported financially this working group meeting and contributed substantially to its success.

I believe that this Bulletin will be a useful tool for scientists and technicians who have as a target the development of integrated control strategies for pests in olive groves, the preservation of the complex of natural enemies and the reduction of pesticides inputs.

Argyro Kalaitzaki

Convener of the Working Group Integrated Protection of Olive Crops ii iii

Contents

Preface ...... i Contents ...... iii List of participants ...... ix

Opening Session

The olive growing and olive oil sector in Tuscany L. Zoppi ...... 1 Arsia activities in the field of olive crop protection M. Ricciolini & M. Toma ...... 5

Bactrocera oleae: Behaviour, Chemical Ecology, Monitoring, Population Dynamics

Chemical ecology of bacterial relationships with fruit flies David C. Robacker ...... 9 Effect of age and mating status on the antennal sensitivity of Bactrocera oleae (Rossi)

(Diptera Tephritidae) male and female. A. De Cristofaro, G. Rotundo, A. Belcari, G.S. Germinara ...... 23 The relationship between olive fly adults and epiphytic bacteria of the olive tree Granchietti A., A. Camèra, S. Landini, M.C. Rosi, M. Librandi, P. Sacchetti, G. Marchi, G. Surico, A. Belcari...... 25 Field assessment of different combinations of ammonia-based attractants and a synthetic female sex pheromone for the monitoring and control of the olive fruit fly, Bactrocera oleae Gmel. (Diptera: Tephritidae) in Apulia, southern Italy A. De Cristofaro, M. Cristofaro, F. Tenaglia, A. Fenio, C. Tronci ...... 31 Inhibitory effect of water assumption on attraction to ammonia, protein baits and bacteria in Bactrocera oleae (Gmelin) Vincenzo Girolami, Alessia Piscedda, Damiano Emer, Andrea Di Bernardo, Luca Mazzon & Rita Signorini ...... 33 Attractiveness to the olive fly of Pseudomonas putida isolated from the foregut of Bactrocera oleae Patrizia Sacchetti, Serena Landini, Aurelio Granchietti, Alessandra Camèra, Marzia Cristiana Rosi, Antonio Belcari ...... 37 Preliminary notes on the gall midges (Diptera: Cecidomyiidae) associated with the olive fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae) Raffaele Sasso Gennaro Viggiani ...... 43 Augmentative releases of Eupelmus urozonus Dalm. against the olive fruit fly and observations on its facultative hyperparasitism G. Delrio, A. Lentini, A. Satta ...... 47 On the use of the exotic oo-pupal parasitoid Fopius arisanus for the biological control of Bactrocera oleae in Italy Riccardo Moretti, Elena Lampazzi, Placido Reina and Maurizio Calvitti ...... 49 iv

Presence of a symbiotic bacterium in the olive fly Bactrocera oleae (Gmelin) Vincenzo Girolami, Andrea Squartini, Luca Mazzon, Alessia Piscedda, Caterina Capuzzo ...... 61 Histopathological observations in the midgut and behaviour of olive fruit fly (Bactro- cera oleae Gmelin) adults treated with a strain of Bacillus thuringiensis Berliner Luca Ruiu, Gavino Delrio, Ignazio Floris, Alberto Satta, Mario Solinas ...... 67 Some biological aspects of the Bactrocera oleae (Rossi) rearing Angelo Canale, Roberto Canovai, Augusto Loni, Alfio Raspi ...... 73 Bait stations field test for Bactrocera oleae (Gmelin) in the Balearic islands (Spain) M.A. Miranda, E. Martinez, M. Monerris, A. Alemany ...... 77 Molecular markers as useful tools for population genetics of the olive fly, Bactrocera oleae D. Segura, C. Callejas, M. D. Ochando ...... 79 Susceptibility to Bactrocera oleae (Gmelin) of some Sicilian olive cultivars Roberto Rizzo, Virgilio Caleca ...... 89 Behavioural responses of the olive fly, Bactrocera oleae, to chemicals produced by Pseudomonas putida in laboratory bioassays Serena Landini, Aurelio Granchietti, Michele Librandi, Alessandra Camèra, Marzia Cristiana Rosi, Patrizia Sacchetti, Antonio Belcari ...... 101

Bactrocera oleae: Chemical, Biological and Biotechnical Control Methods, Side Effects

Sterile insect technique (SIT) – an environmentally friendly approach to controlling major fruit-fly pests M. Kozαnek, C. Caceres ...... 109 Tests on the effectiveness of kaolin and copper hydroxide in the control of Bactrocera oleae (Gmelin) Virgilio Caleca, Roberto Rizzo ...... 111 Resistance to organophosphates in Bactrocera oleae in Grecee and Cyprus John Tsitsipis, John T. Margaritopoulos, Panagiotis Skouras, Konstantinos Mathiopoulos and Nikos Serafides ...... 119 A Beauveria bassiana-based bioinsecticide for the microbial control of the olive fly (Bactrocera oleae) Massimo Benuzzi, Enrico Albonetti, Fabio Fiorentini, Edith Ladurner ...... 125 tm Bait applications effect of Spinosad Success 0.24CB (GF-120)" formulation, on Bactrocera oleae Gmel. (Dacuol), and impact on other non target organisms in olive trees P.V. Vergoulas, D. Prophetou-Athanassiadou, E. Alimi, H. Ben Salah, C. Mavrotas, C. Jousseaume, M. Miles ...... 131 Effect of several insecticides for control of Bactrocera oleae (Gmelin) (Diptera: Tephritidae) to arthropods fauna of olive grove V. Alexandrakis, K. Varikou, A. Kalaitzaki, D. Lykouressis ...... 133 Mass trapping experiments with two different “Attract and Kill” devices for Bactrocera oleae (Gmelin) Nino Iannotta, Massimiliano Pellegrino, Enzo Perri, Luigi Perri, Fausto De Rose .. 135 Tests on the effectiveness of mass trapping by Eco-trap (Vyoril) in the control of Bactrocera oleae (Gmelin) in organic farming Virgilio Caleca, Roberto Rizzo, Isabella Battaglia, Manuela Palumbo Piccionello .. 139 v

Control trials of Bactrocera oleae (Gmel.) (Diptera Tephritidae) in the district of Bar in Montenegro Tatjana Perović, Snježana Hrnčić, Antonio Franco Spanedda, Alessandra Terrosi, Claudio Pucci, Biljana Lazović, Mirjana Adakalić ...... 147 Kaolin protects olive fruits from Bactrocera oleae Gmelin infestations unaffecting olive oil quality E. Perri, N. Iannotta, I. Muzzalupo, B. Rizzuti, A. Russo, M.A. Caravita, M. Pellegrino, A. Parise, P. Tucci ...... 153 Spinosad treatment for Bactrocera oleae (Gmel.) control and olive oil quality in the Montenegrin cv Žutica Maurizio Servili, Sonia Esposto, Stefania Urbani, Biljana Lazovic, Mirjana Adakalic, Tatjana Perovic, Snježana Hrncic, Claudio Pucci, Antonio Franco Spanedda, AlessandraTerrosi, Enzo Perri ...... 155 Effect of the olive fruit fly and the olive antrachnose on oil quality of some Portuguese cultivars A. Sousa, J.A. Pereira, S. Casal, B. Oliveira, A. Bento ...... 159 Biological control of olive fruit fly in California by Psyttalia cf. concolor (Szepligeti) from Moscamed, Guatemala V.Y. Yokoyama, G.T. Miller, P. Rendon, J. Sivinski ...... 161 Psyttalia concolor (Szépligeti) mass-rearing: new acquisitions Angelo Canale, Augusto Loni, Alfio Raspi ...... 163 The effects of treatments against Bactrocera oleae (Gmelin) on the entomo-fauna of the olive ecosystem Nino Iannotta, Tiziana Belfiore, Pietro Brandmayr, Stefano Scalercio ...... 169

Other Pests of Olive Groves and Olive Agroecosystem Aspects

Inventory and role of the third generation parasitoids of Prays oleae Bern. (Lepido- ptera, Yponomeutidae) in Sfax region (South of Tunisia) Imen Blibech, Mohiedine Ksantini and Taieb Jardak ...... 175 Mating disruption of the olive pyralid moth, Euzophera pinguis Ortiz A., A. Perabá, A. Quesada, A. Sánchez ...... 179 Effect of chemical control on over-wintered population of olive psyllid Euphyllura olivina Costa (Homoptera, Aphalaridae) in Iran (Tarom-Sofla region, Qazvin province) H. Nouri ...... 181 Factors affecting male Prays oleae (Lepidoptera: Yponomeutidae) captures in

pheromone-baited traps in olive orchards N.G. Kavallieratos, C.G. Athanassiou, G.N. Balotis, G.Th. Tatsi, B.E. Mazomenos .... 187 Mating disruption trials for the olive moth, Prays oleae (Bern.), (Lep.:Yponomeutidae) in Trás-os-Montes olive groves (northeast of Portugal) A. Bento, J.A. Pereira, J.E. Cabanas, M. Konstantopoulou, L.M. Torres, B.E. Mazomenos ...... 189 Resistance of olive cultivars to carpophagous generation of Prays oleae Lentini, G. Delrio, S. Deliperi ...... 191 Optimization of the field performance of released Trichogramma spp. in olive groves, in Egypt

E.M. Hegazi, A. Herz, S.A. Hassan, E. Agamyy, W.E. Khafagi, S. Mostafa, N. Khamis ...... 193 vi

Distribution and spatial pattern of Saissetia oleae (Olivier) on the olive tree in the northeast of Portugal J.A. Pereira, A. Bento, L.M. Torres ...... 195 Twig dieback in olive trees associated with Resseliella oleisuga (Targioni Tozzetti) (Diptera Cecidomyiidae) and Libertella sp. Gabriella Frigimelica, Alessio Rainato, Luca Mazzon, Vincenzo Girolami ...... 197 Bionomics of Resseliella oleisuga (Targ.-Tozz.) in Tuscany (Diptera Cecidomyiidae) B. Bagnoli, D. Benassai, E. Mosconi ...... 203 Effect of eriophyides mites on the sensitivity of some olive tree varieties Chatti, M. Ksantini, T. Jardak ...... 205

Effect of cereal cover crops on Araneae population in olive orchard M. Cárdenas, J.A. Barrientos, P. Garcνa, F. Pascual, M. Campos ...... 207 Coccinellidae communities: diversity and dynamics in organic and integrated olive groves from Tràs-os-Montes (northeast of Portugal) S.A.P. Santos, J.A. Pereira, A. Raimundo, A.J.A. Nogueira, L.M. Torres ...... 209 Coccinellids associated with olive groves in north-eastern Portugal M.F. Gonçalves, S.A.P. Santos, A. Raimundo, J.A. Pereira, L.M. Torres ...... 211

Diseases

Current problems related to olive diseases in the Mediterranean basin E.C. Tjamos, P. Antoniou, S.E. Tjamos, E.J. Paplomatas ...... 215 Olive viruses and strategies for producing virus-free plants M. Saponari, G. Bottalico, G. Loconsole, G. Mondelli, A. Campanale, V. Savino, G.P. Martelli ...... 217 Fungal agents responsible for olive dieback in Iran M. Salati, H. Afshari Azad, A. Javadi Estahbanati ...... 219 Comparison between real-time PCR and semi-selective medium in monitoring Verticillium dahliae microsclerotia in the olive rhizosphere and suppression of the pathogen by compost Giuseppe Lima, Filippo De Curtis, Anna Maria D’Onghia, Franco Nigro ...... 221 Foliar application of phosetyl-al for controlling olive verticilliosis: Realistic goal or false hope? F. Nigro, P. Gallone, F. Palmisano, P. Sumerano, A. Ippolito ...... 225 Host-derived resistance for biological control of verticillium wilt of olive Colella, C. Miacola, M. Amenduni, M. D’Amico, G. Bubici, M. Cirulli ...... 227 Characterization of Colletotrichum species causing olive anthracnose in Italy. S.O. Cacciola, G.E. Agosteo, R. Faedda, S. Frisull, G. Magnano Di San Lio ...... 229 A symbiotic relation found between Pseudomonas savastanoi and Pantoea aglomerans in the knots formed on olive G. Marchi, G. Casati, G. Surico, A. Sisto, A. Evidente ...... 231 Epidemiological study of olive scab in Calabria Giovanni Enrico Agosteo, Rocco Zappia ...... 233 Non–conventional chemical control of olive anthracnose Giovanni Enrico Agosteo, Luigi Scolaro, Giovanni Previtera ...... 245 Control olive powdery mildew (Leveillula taurica) with the use of soft fungicides V.A. Bourbos, E.A. Barbopoulou ...... 249 vii

Phytophthora species associated with root rot of olive in Sicily S.O. Cacciola, G. Scarito, A. Salamone, A.S. Fodale, R. Mulé, G. Pirajno, G. Sammarco ...... 251 Susceptibility of olive genotypes to Pseudomonas savastanoi (Smith) Nino Iannotta, Donatella Monardo, Maria Elena Noce, Luigi Perri ...... 253 Detection of Verticillium dahliae in irrigation water E. Rodrìguez, M. Campos, M.L. Fernández, J.A. Ocampo, J.M. Garcìa-Garrido ..... 259

Advanced IPM Strategies in Olive Groves

Olive fruit fly biology and cultural control practices in California Victoria Y. Yokoyama, Gina T. Miller ...... 263 Studies towards an enhanced food attractant for fruit flies, especially for the olive fruit fly Bactrocera oleae (Gmelin). A. Gally, M. Vamvakias, N. Ragoussis ...... 271 New technology for auto-dissemination of pheromones and pesticides: potential for control of olive fly and olive moth Philip Howse ...... 273 Effectiveness of different copper products against the olive fly in organic olive groves Marzia Cristiana Rosi, Patrizia Sacchetti, Michele Librandi, Antonio Belcari ...... 277 Establishment of TEAM (Tephritidae of Europe, Africa and Middle East), a new international working group on fruit flies of economic importance N. Papadopulos, A. Bakri, S. Quilici, M. Bonizzoni, B. Barnes, Y. Gazit, S.A. Lux, D. Nestel, M. Cristofaro, R. Pereira, M.A. Miranda, N. Kouloussis ...... 283 Application of forecasting models of olive fly (Bactrocera oleae Gmel.) (Diptera, Tephritidae) infestation in Montenegro Snježana Hrnčić, Claudio Pucci, Antonio Franco Spanedda, Alessandra Terrosi, Tatjana Perović, Biljana Lazović, Mirjana Adakalić ...... 285 Application of internet and mobile technologies in pest management: a case study of Bactrocera oleae control in Tuscany Guidotti, S. Marchi, A. Bo, M.Ricciolini, R. Petacchi ...... 289 Integrated protection system against Bactrocera oleae (Gmelin) in organic production Nino Iannotta, Tiziana Belfiore, Enzo Perri, Luigi Perri, Vincenzo Ripa ...... 291 Differences in insects within the olive orchard agroecosystem under integrated management regime in south Spain Cotes, F. Ruano, P. Garcνa, F. Pascual, A. Tinaut, A. Peρa, M. Campos ...... 297 New biodegradable controlled-released pheromone dispenser for Bactrocera oleae (Gmelin) I. Navarro-Fuertes, R. Gil Ortiz, P. Moya Sanz, V. Navarro-Llópis ...... 299 Increased olive oil yield and quality in Montenegrin cv Žutica by Bactrocera oleae Gmel. (Diptera Tephritidae) control and improved harvest techniques Biljana Lazović, Mirjana Adakalić, Tatjana Perović , Snježana Hrnčić, Claudio Pucci, Alessandra Terrosi, Antonio Franco Spanedda ...... 301 Integrated olive pest management in Iran H. Nouri ...... 307 Can spring-preventive adulticide treatments be assumed to improve Bactrocera oleae (Rossi) management? Giorgio Ragaglini, Diego Tomassone, Ruggero Petacchi ...... 309

Participants

Name Address ADAKALIC Mirjana Biotechnical Institut, Center for Plant Protection, Bjelisi bb, 85000 Bar, Montenegro, e-mail: [email protected] AGOSTEO Giovanni Enrico Dip. di Agrochimica e Agrobiologia - Università Mediterranea di Reggio Calabria, piazza San Francesco di Sales 4, Gallina-Reggio Calabria, 89061, Italy, e-mail: [email protected] ALEXANDRAKIS NAGREF - Institute of Olive Tree and Subtropical Plants, Venizelos Agrokipio, Chania, Greece, 73100, Greece, e-mail: [email protected] ATHANASSIOU Agricultural University of Athens, 75 Iera Odos Christos G. (Votanikos), Athens, 11855, Greece, e-mail: [email protected] BAGNOLI Bruno ISZA, via di Lanciola 12 A, Cascine del Riccio, Firenze, 50125, Italy, e-mail: [email protected] BENTO Albino Antonio Instituto Politecnico de Braganca Escola Superior Agraria, Campus de Sta Apolonia 172, Braganca, 5301-855, Portugal, e-mail: [email protected] BENUZZI Massimo Intrachem Bio Italy S.p.a.- R& D Department, via Calcinaro 2085/7, Cesena (FC), 47023, Italy, e-mail: [email protected] BLIBECH HADRI Imen Unite de protection des plantes et de l environnement, Institut del Olivier, Route de l’Aeroport 3000 Sfax, Tunisia, e-mail: [email protected] BRACCINI Piero Arsia, via Pietrapiana, 30 - 50121 Firenze, FI, Italy, e-mail: [email protected] CACERES Carlos IAEA, Seibersdorf Laboratory, A-4222 Seibersdorf, Vienna, Austria, e-mail: [email protected] CALECA Virgilio Università Palermo - Dip. Entomologia, viale delle scienze, Palermo, 90128, Italy, e-mail: [email protected] CALVITTI Maurizio ENEA C.R. Casaccia-UTS Biotec Agro, S.M. di Galeria, via Anguillarese, Italy, e-mail: [email protected] x

CAMERA Alessandra DIBA- Università di Firenze, via Maragliano 77, Firenze, 50144, Italy, e-mail: [email protected] CAMPOS Mercedes Dept. of Agroecology and Plant Protection, Consejo Superior de Investigaciones Cientificas, Estacion Experimental del Zaidin (C.S.I.C.), Granada, 18008, Spain, e-mail: [email protected] CANALE Angelo Dip. di Coltivazione e Difesa delle Specie legnose "G. Scaramuzzi", Universita degli Studi di Pisa, Via San Michele degli Scalzi 2, Pisa, 56124, Italy e-mail: [email protected] CHATTI Amel Institute de l'Olivier, Rue l'Aéroport Km 1.5, Sfax, 3000, Tunisia, e-mail: [email protected] CIRULLI Matteo Dip. di Biologia e Patologia Vegetale, Università degli Studi di Bari, Via G. Amendola, 165/A, Bari,70126, Italy, e-mail: [email protected] COTES RAMAL Belèn Dep. of Agroecology and Plant protection, Consejo Superior de Investigaciones Cientìficas, Estación Experimental del Zaidìn, Profesor Albareda 1, Granada, 18008, Spain, e-mail: [email protected] CRISTOFARO Massimo ENEA C.R. Casaccia-UTS Biotec Agro, S.M. di Galeria, Via Anguillarese 301, Roma,00060, Italy, e-mail: [email protected] DE CRISTOFARO Antonio Dip. Scienze Animali Vegetali e dell’ Ambiente, Università del Molise – Entomologia, Via Francesco de Sanctis, Campobasso, 86100, Italy, e-mail: [email protected] DELIPERI Salvatore Dip. di Protezione delle Piante, Università degli Studi di Sassari, Via De Nicola, Sassari, 07100, Italy, e-mail: [email protected] DELRIO Gavino Dip. di Protezione delle Piante, Università degli Studi di Sassari, Via De Nicola, Sassari, O7100, Italy, e-mail: [email protected] ESPOSTO Sonia Dip. Scienze Economico-Estimative e degli Alimenti, Università degli Studi di Perugia, Via S.Costanzo, Perugia, 06126, Italy, e-mail: [email protected] FRIGIMELICA Gabriella Dip. di Agronomia Ambientale e Produzioni Vegetali, Università di Padova, Agripolis, Viale dell'Università 16, Legnaro, Padova, 35020, Italy, e-mail: [email protected]

GILORTIZ Ricardo Centro de Ecologìa Quìmica Agrìcola, Universita di Politècnica de Valencia, campus de Vera, Edificio 9/B, Valencia, 46022, Spain, e-mail: [email protected] GIROLAMI Vincenzo Dip. di Agronomia Ambientale e Produzioni Vegetali, Università di Padova, Viale dell'Università 16, Legnaro, Padova, 35020, Italy, e-mail: [email protected] GRANCHIETTI Aurelio Dip. Biotechnologie Agrarie, Università di Firenze, Via Maragliano 77, Firenze, 50144, Italy, e-mail: [email protected] GUIDOTTI Diego Aedit S.r.l., Viale Rinaldo Piaggio, Pontedera (PI), 56025, Italy, e-mail: [email protected] HAFEZ Hisham Dep. of Economic Entomology, Faculty Agriculture Biological Control, Alexandria University, El Shatby, Alexandria, 21545, Egypt, e-mail: [email protected] HEGAZI Esmat Dep. of Economic Entomology, Faculty Agriculture Alexandria University, El Shatby, Alexandria,21545, Egypt, e-mail: [email protected] HERZ Annette Institute for Biological Control, Federal Biological Research Centre for Agriculture and Forestry, Heinrichstr. 243, 64287 Darmstadt, Germany, e-mail: [email protected] HOSSEININEJAD Seyed Plant Pest and Diseases Research Institute, P.O Box 1454- Abbas 19395, Tehran, 19395, Iran, e-mail: [email protected] HOWSE Philip Exosect Ltd., 2 Venture Road, Southampton, S016 7NP, United Kingdom, e-mail: [email protected] HRNCIC Snjezana Biotechnical Institut-Center for Plant protection, Kralja Nikole bb, 81000 Podgorica, Montenegro, e-mail: [email protected] IANNOTTA Nino IRSA, Vermicelli, 87036, Rende (CS), Italy, e-mail: [email protected] JARDAK Taieb Unite de Protection des Plantes et de l’Environnement, Institut de l Olivier, Route de l’Aeroport, 3000 Sfax, Tunisia, e-mail: [email protected] KALAITZAKI Argyro Institute of Olive Tree and Subtropical Plants, Agrokipio, Chania, 73100, Greece, e-mail: [email protected] xii

KOZANEK Milan Institute of Zoology - Slovak Academy of Sciences, Dubravská cesta 9, Bratislava, 84206, Slovakia, e-mail: [email protected] LANDINI Serena Dip. Biotechnologie Agrarie - Università di Firenze, Via Maragliano 77, Firenze, 50144, Italy, e-mail: [email protected] LAZOVIC Biljana Biotechnical Institut-Center for plant protection, Bjelisi, CS, 85000 Bar, Montenegro, e-mail: [email protected] LIBRANDI Michele Dip. Biotechnologie Agrarie- Università di Firenze, Via Maragliano 77, Firenze, 50144, Italy, e-mail: [email protected] LIMA Giuseppe Dip. di Scienze Animali, Vegetali e dell'Ambiente - Università del Molise, Via Francesco De Sanctis, Campobasso, 86100, Italy, e-mail: [email protected] LONI Augusto Dip. di Coltivazione e Difesa delle Specie legnose "G. Scaramuzzi", Università di Pisa, Via San Michele degli Scalzi 2, Pisa, 56124, Italy, e-mail: [email protected] MAGNANO Di San Lio Dip. di Agrochimica e Agrobiologia - Università Mediterranea di Reggio Calabria, piazza San Francesco di Sales 4, Gallina-Reggio Calabria, 89061, Italy, e-mail: [email protected] MARCHI Guido Dip. Biotechnologie Agrarie - Università di Firenze, Piazzale delle Cascine 28, Firenze, 50144, Italy, e-mail: [email protected] MAVROTAS Costas Dow Agro Sciences, Vouliagmenis Avenue 85, City Plaza Center,Glyfada, Athens, 73100, Greece, e-mail: [email protected] MAZZON Luca Dip. Di Agronomia ambientale e Produzioni Vegetali - Università di Padova, Viale dell'Università 16, Legnaro (Padova), 35020, Italy, e-mail: [email protected] MIRANDA-CHUECA Lab.Zoology, University of the Balearic Islands, Miguel Angel Cra.Valldemossa Km 7.5, Palma, 7122, Spain, e-mail: [email protected] MORETTI Riccardo ENEA C.R. Casaccia, Via Catullo 7, Ariccia, 00040, Italy, e-mail: [email protected] MOYA SANZ Pilar Centro de Ecologìa Quìmica Agrìcola – Universidad Politècnica de Valencia, Campus de Vera, Edificio 9/B, Valencia, 46022, Spain, e-mail: [email protected]

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NAVARRO-FUERTES Centro de Ecologìa Quìmica Agrìcola - Universidad Ismael Politècnica de Valencia, Campus de Vera, Edificio 9/B, Valencia, 46022, Spain, e-mail: [email protected] NAVARRO-LLOPIS Centro de Ecologìa Quìmica Agrìcola - Universidad Vicento Politècnica de Valencia, Campus de Vera, Edificio 9/B, Valencia, 46022, Spain, e-mail: [email protected] NIGRO Franco Dip. Di Biologia e Patologia Vegetale- Università d Bari, Via Amendola, 165/A, Bari, 70126, Italy, e-mail: [email protected] NOURI Hossain Agricultural and Natural Resources Research Center, Qazvin, Iran, e-mail: [email protected] OCHANDO M. Dolores Departemento de Génetica, Universidad Complutense, Facultad de Ciencias Biòlogicas, Madrid, 28040, Spain, e-mail: [email protected] ORTIZ Antonio EPS de Linares, Universidad de Jaen EUP, C/ Alfonso X el Sabio 28, Linares (Jaen), Spain, e-mail: [email protected] PEDUTO Francesca Dip. Biotechnologie Agrarie - Università di Firenze, P.le delle Cascine 28, Firenze, 50144, Italy, e-mail: [email protected] PENNINO Giuseppe Regione Sicilia, Via S.Giuseppe La Rena 30/B, Catania, 95121, Italy, e-mail: [email protected] PEREIRA JOSE Alberto Instituto Politecnico de Braganca Escola Superior Agraria, Campus de Sta Apolonia 172, Braganca, 5301-855, Portugal, e-mail: [email protected] PEROVIC Tatjana Biotechnical Institut-Center for plant protection, Bjelisi bb, 85000 Bar, Montenegro, e-mail: [email protected] PERRI Enzo Istituto Sperimentale per l' Olivicoltura, C.da Li Rocchi - Vermicelli - 87036 Rende, Cosenza, 87036, Italy, e-mail: [email protected] PETACCHI Ruggero Scuola Superiore di Studi Universitari e di Perfezionamento "S. Anna", c/o Polo S. Anna Valdera- Viale Rinaldo Piaggio Pontedera, Pontedera ( PI), 56025, Italy, e-mail: [email protected] PISCEDDA Alessia Dip. Di Agronomia Ambientale e Produzioni Vegetali - Università di Padova, Viale dell'Università 16, Legnaro (Padova), 35020, Italy, e-mail: [email protected] xiv

PUCCI Claudio Dipartimento di Protezione delle Piante Università degli Studi della Tuscia, Viterbo, 01100, Italy, e-mail: [email protected] RAGAGLINI Giorgio Scuola S. Anna – Biolabs, Viale Rinaldo Piaggio, Pontedera (PI), 56025, Italy, e-mail: [email protected] RAGOUSSIS Nikitas VIORYL S.A., 36 Viltaniotis St., Athens, 145 64, Greece, e-mail: [email protected] RAINATO Alessio Dip. Di Agronomia ambientale e Produzioni Vegetali - Università di Padova, Viale dell'Università 16, Legnaro (Padova), 35020, Italy, e-mail: [email protected] RAITI Giovanni Regione Sicilia, Via S.Giuseppe La Rena 30/B, Catania, 95121, Italy, e-mail: [email protected] RASPI Alfio Dip. di Coltivazione e Difesa delle Specie legnose "G. Scaramuzzi" - Università di Pisa, Via San Michele degli Scalzi 2, Pisa, 56124, Italy, e-mail: [email protected] RICCIOLINI Massimo Arsia, Via Pietrapiana 30, Firenze, 50121, Italy, e-mail: [email protected] RIZZO Roberto Università Palermo, Viale delle Scienze, Palermo, 90128, Italy, e-mail: [email protected] ROBACKER David USDA, Kika de la Garza SARC- 2413 E – Higway, Weslaco, 78596, USA, e-mail: [email protected] RODRIGUEZ Navarro Estacion Experimental del Zaidin (C.S.I.C.), Profesor Albereda 1, Spain, e-mail: [email protected] RUANO Francisca University of Granada, Depatment of Animal Biology and Ecology, Univ. of Granada, Granada, 18071, Spain, e-mail: [email protected] SANTOS Sónia A.P. Escola Superior Agraria de Braganca, Campus de Sta Apolonia, Apt 1172, Braganca, 5301-854, Portugal, e-mail: [email protected] SAPONARI Maria Dip. di Biologia e Patologia Vegetale - Università d Bari, Via Amendola, 165/A, Bari, 70126, Italy, e-mail: [email protected] SERVILI Maurizio Dip. Scienze Economico-Estimative e degli Alimenti - Università degli Studi di Perugia, Via S.Costanzo, Perugia, 06126, Italy, e-mail: [email protected]

STIMILLI Giuliano ASSAM-Regione Marche, Via Alpi, 21, Ancona, 60100, Italy, e-mail: [email protected] SURICO Giuseppe Dip. Biotechnologie Agrarie, Piazzale delle Cascine, 28 - 50144 Firenze, Firenze, 50144, Italy, e-mail: [email protected] TETRADIS Giorgio VIORYL S.A., 28 Km Athens, Lamia Nat. Road, Athens, Greece, e-mail: [email protected] TJAMOS Elefterios Department of Plant Pathology, Agricultural University of Athens, 75 Iera Odos str.-Votanikos, Athens, 11855, Greece, e-mail: [email protected] TSITSIPIS Ioannis University of Thessaly, Fytokou Street, Nea Ionia Magnissias, 3844, Greece, e-mail: [email protected] VIGGIANI Gennaro Dip. di Entomologia e Zoologia Agraria ‘’Filippo Silvestri’’, Università Napoli- via Universitá 100, Portici, 80055, Italy, e-mail: [email protected] YOKOYAMA Victoria USDA-ARS-SJVASC, 9611 S. Riverbend Avenue, Perlier- California, 93648, USA, e-mail: [email protected] ZOPPI Luciano Regione Toscana, Assessorato all'Agricoltura, Via di Novoli 26, Firenze, Italy, e-mail: [email protected]

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 1-3

The olive growing and olive oil sector in Tuscany

L. Zoppi Regione Toscana, Assessorato all’Agricoltura, Via di Novoli, 26, Firenze, Italy

In this paper I will try to briefly outline the main characteristics of olive growing and olive oil in Tuscany, and its importance for the region, and to describe the initiatives that are being taken to safeguard and promote a product that is so closely bound up with the region and its image. The saleable output of the olive and olive oil sector makes up just 5% of the region’s total agricultural and lifestock production. However, the importance of the olive growing and olive oil sector extends far beyond purely economic considerations. It also encompasses essential environmental, landscape, social and cultural functions. I will now try to briefly analyze each of these aspects. In Tuscany about 100,000 hectares of land are planted with olives, over 90% of which are in hilly or low mountain areas. From a climatic point of view, Tuscany lies on the northern boundary of the olive growing area. The olives are not fully ripe when they are harvested, which, when taken together with the olive varieties grown and various other natural and human factors, contributes to determining the properties of Tuscan olive oil, in particular its typical fruity flavour. Thanks to the beneficial influence of the sea and the protection afforded by the Apennines, the climate in Tuscany’s hill areas is fairly mild, somewhere between that of Liguria (more temperate) and Umbria (more continental). Having said that, olive crops are sometimes damaged by very low minimum temperatures. The icy spell in January 1985, for instance, destroyed the majority of the region’s olive groves. The work that was done to repair the damage, with the replacement of almost all the olive groves, is a concrete demonstration of the interest and affection that Tuscans have for olive trees. There are about 15 million olive trees in Tuscany, 90% of which are one of a limited number of varieties: Frantoio (48%) cultivated for the quality of the product; Moraiolo (22%) cultivated for the yield; Leccino (16%) cultivated for their adaptability to the climate; Maurino Pendolino cultivated for pollination purposes. Tuscan olive oil is traditionally produced from a blend of these olive varieties, which are normally grown alongside each other, together with a host of other minor varieties (over 80). A census and various in-depth studies have been conducted in order to build up a composite picture of the immense genetic patrimony of the region’s olive groves: selected and reproduced locally for centuries, they are at one with the environment and, together with human efforts, help give Tuscan olive oil its distinctive character. There are almost 80,000 olive producers in the region; the average size of the plot of land planted with olives is less than 1.5 hectares. The quality and properties of Tuscan extra virgin olive oil are heavily influenced by geographic factors (terrain and climate), the varieties grown, but also by human factors. The cultivation, processing and conservation methods are part of traditions that date back hundreds of years.

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Olive growing normally involves extensive cultivation techniques and a very limited use of fertilizers and plant protection products. There has recently been a further drop in the 3 amount of chemical products used and an increase in organic production methods. This is partly due to the widespread application of the agro-environmental measures introduced by EU regulation 2078 (1992), which were then worked into the rural development programme according to the provisions of EU regulation 1257 (1999). The olives are harvested early, commencing at the end of October or the beginning of November and normally finishing within a month or month and a half. Early harvesting is a traditional practice in Tuscany, and is partly done to satisfy the taste of regional consumers, who generally have a marked liking for “new” oil and regard it as a fundamental ingredient in some of Tuscany’s typical dishes. Harvesting is normally by hand (known as brucatura). The olives are picked directly from the tree and not from the ground. Before being pressed, the harvested olives are kept in appropriate conditions (in shallow crates or on mats) for a very short period of time (just a few days). There are a large number of olive mills in Tuscany (over 450), and their even distribution throughout the region is such that even small batches of olives can be pressed quickly. This is a big help to growers, who can do several pressings in the course of a single harvest, which benefits the overall quality of the product. A period of restructuring is currently underway in the sector, which has led to some closures and to the modernization of many mills, with the introduction of the more productive continuous milling process. The majority of olive oil produced in Tuscany is obtained with this method. The production of olive oil of Tuscan origin is quantitatively limited, and amounts on average (though it varies considerably from year to year) to about 20,000 tons per year, which is approximately 3% of national production. The fine quality of Tuscan olive oil, combined with a deep-rooted olive tree and olive oil “culture”, has undoubtedly contributed to the success of the product at home and abroad, and the word “Tuscan” has been long and widely used as a marketing ploy. In March 1998 the word “Tuscan”, when used in connection with extra virgin olive oil, was officially registered with the EU as Indicazione Geografica Protetta (I.G.P.) in accordance with regulation no. 2081 (1992). Besides providing a general degree of protection for all Tuscan producers, the I.G.P. Toscano has also enabled further valorization of the region’s olive oil through official recognition of a number of Denominazioni di Origine Protette (D.O.P.), which are more restricted denominations within the I.G.P. itself. In accordance with EU regulation 2081 (1992), the region has acquired three important D.O.P.s for extra virgin olive oil: Chianti Classico, Terre di Siena and Terre di Lucca. The proposal to establish two further D.O.P.s, Colline di Firenze and Seggiano, are at the preliminary national stage. The I.G.P. Toscano and the D.O.P.s are important opportunities for promoting the region’s top-quality olive oil, and may be accompanied by other forms of certification, especially regarding certain environmentally friendly production processes, for instance the use of the term “organic” (in compliance with EU regulation 2092 (1991)) or “integrated”, which the Region of Tuscany has disciplined with Regional Law 25 (1999). One of the strong points of the Tuscan olive growing and olive oil sector is undoubtedly the well- established olive and olive oil culture in the region, which manifests itself in a host of interesting initiatives at a local, national and international level. One tangible indication of the importance given to olive oil by people in Tuscany is the number of olive oil tasters in the region, which is much higher than in other regions – over 500 tasters registered in a special regional list – and as many as 14 tasting groups that apply panel test methodology as per EU regulation 2568 (1991). To round off this analysis of the Tuscan olive growing and olive oil sector it is also necessary to mention the important work 3 being done by various bodies offering services in the regional system of agricultural development, in particular the public-sector Regional Agency for Development and Innovation in Agriculture and Forestry, known by its Italian acronym ARSIA. 4 Thanks also to the assistance of various scientific institutions, a large number of studies and research projects regarding the main technical and economic aspects of the production process have been carried out. Of particular importance as regards technical assistance and research activities in recent years are regional projects aimed at improving the quality of olive oil; these have been financed by the European Community, with a deduction from production grants, and by the central government. These projects, run directly by the Region of Tuscany from 1997 to 2004, involved various bodies, including ARSIA, various associations of olive growers, ARPAT and the Unioncamere Toscana. One of the most significant actions was a project to limit the olive fly by using environmentally friendly techniques such as mass capture. Others included the opening of special points around the region to provide technical assistance; the organization of courses, meetings and study trips; research into the characterization of Tuscan olive varieties; and chemical and organoleptic analysis of the region’s oils.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 5

Arsia activities in the field of olive crop protection

M. Ricciolini & M. Toma ARSIA - Agenzia Regionale per lo Sviluppo e l’innovazione nel settore Agricolo-forestale, Via Pietrapiana, 30- 50121 Firenze, Italy.

The ARSIA is the Regional Agency for Development and Innovation in Agriculture and Forestry. It is a technical unit set up by the Region of Tuscany to mediate between the fields of agricultural production, research and specialised technology. The agency encourages innovation and helps provide technological support for the growing, processing and sale of agricultural products. It operates through a service network for technicians, manufacturers and rural farming areas. The ARSIA is engaged in numerous activities in a wide range of sectors. The agency promotes research and experimentation into issues regarding the development of olive growing in Tuscany, by inviting the submission of proposals. Two studies have been financed: the first one concerned the natural enemy of Saissetia oleae in Tuscany and the increase of the Metaphycus bartletti (ISZA) population, the second studied the effect of Azadiracta indica extracts on the female fertility of Bactrocera oleae (Siena University). At the preliminary meeting of the consultation groups for the next call for proposals, new studies were announced regarding Bactrocera oleae, Saissetia oleae, Pseudomonas syringae pv savastanoi and Cicloconium oleaginum. In the field of training, the ARSIA has funded three scholarships for the olive and olive oil Master’s course at Pisa University. Numerous courses have also been organized for teachers and technicians working in the olive and olive oil chains. As far as technical innovation is concerned, the agency conducts trials of organic and chemical protection techniques in its testing centres, testing new insecticides or technology developed for use against Bactrocera oleae infestation. Since 1993, with the collaboration of the region’s olive growers’ associations (AIPROL and OTA) and with scientific assistance from the S.Anna School of Advanced Studies in Pisa, the ARSIA has carried out several trials in different areas of the region, using the mass trapping method to provide protection against B. oleae attack. The agency has built up a modern and friendly service called Agroambiente.info, which provides assistance to technicians and olive farmers. The service is based on a network of over 260 control points monitored each week by 20 technicians. A weekly report for each area is published on the internet, in the press, via teletext and, in the eventuality of an alert, directly on the mobile phone of growers with text messages.

Bactrocera oleae: Behaviour, Chemical Ecology, Monitoring, Population Dynamics

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 9-22

Chemical Ecology of Bacterial Relationships with Fruit Flies

David C. Robacker ARS, USDA, Crop Quality and Fruit Insects Research, Kika de la Garza Subtropical Agricultural Research Center, 2413 E. Hwy 83, Weslaco, Texas, 78596, USA

Abstract: The nature of relationships between fruit flies (Diptera: Tephritidae) and bacteria has been controversial. Theories of obligate symbioses have given over to facultative mutualism, accidental, and predator-prey depending on circumstances. Fruit flies are attracted to bacteria to quench drive states including protein hunger and others that are poorly understood. Following chemical functional group characterizations indicating attractive principals were mostly chemicals containing ionizable nitrogen, a novel technique was devised to identify ammonia, 1-pyrroline, acetic acid, and several amines, pyrazines, and alcohols from bacterial odors. Mixtures of these chemicals in the same concentrations as in bacterial odors were about 80-90% as attractive as the odors to Mexican fruit flies. Volatiles produced by bacteria attractive to fruit flies were found to vary with bacteria taxon at all levels of classification and with culturing medium. Interactions of attractiveness of the chemicals are consistent with the need for fruit flies to forage for various bacteria species on various substrates. The information obtained in these studies is useful for development of fruit fly lures, improvement of fly cultures, and understanding of our natural world.

Key words: symbiosis, fruit fly, Tephritidae, bacteria, semiochemical, attractant

Introduction

Relationships between fruit flies (Diptera: Tephritidae) and bacteria are poorly understood. Petri (1910) reported symbiosis of the bacterium (Pseudomonas savastanoi) that causes olive knot disease with the olive fruit fly (Bactrocera oleae) and described how the bacteria are transferred through all life stages of the fly. Later, Yamvrias et al. (1970) were unable to find P. savastanoi associated with the olive fly, casting doubt on the validity of Petri’s work. However, the presence of bacteria in the fly that were transferred through life stages has been confirmed by later workers (Girolami, 1973, 1983; Mazzini and Vita, 1981). Allen et al. (1934) described a similar symbiosis between the bacterium (Pseudomonas melophthora) that can cause apple rot and the apple maggot fly (Rhagoletis pomonella). Again, later workers were unable to identify this bacterium from the apple maggot fly (Huston, 1972; Dean and Chapman, 1973). Despite the uncertainty regarding the identities of the bacteria studied by Petri (1910) and Allen et al. (1934), these studies became the basis for the prevailing view during most of the 20th century that fruit fly associations with bacteria were generally obligate symbioses (Buchner, 1965). As research into bacterial associations accelerated during the last quarter of the 20th century, it became more apparent that bacteria in fruit fly digestive tracts reflected bacteria found in the flies’ environment (Howard, 1989). Studies in which numerous species of bacteria were found associated with a single fly and these same bacteria could be isolated from fruit fly hosts became common (Yamvrias et al., 1970; Tsiropoulos, 1983; Fitt and O’Brien, 1985; Drew and Lloyd, 1989; Martinez et al., 1994) and the theory that bacteria maintained obligate symbioses with fruit flies began to erode. By the end of the century, few believed that any fruit fly associations with bacteria represented obligate symbioses (Howard, 1989; Drew and Lloyd, 1989).

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New theories arose to replace obligate symbioses. Huston (1972) considered bacteria accidental symbiotes because the species associated with the apple maggot fly were determined by the species in its habitat. Drew et al. (1983) concluded that bacteria serve as a protein source for fruit flies after a series of studies in which Drew and his colleagues found that flies seeded their environment with bacteria then later foraged on the colonies, that bacteria were more numerous in the crop and midgut than in the feces indicating digestion, and that flies fed a diet of bacteria as their protein source produced as many eggs as those fed standard breeding diet. Robacker and Moreno (1995) hypothesized that metabolites emitted during bacterial breakdown of protein elicits attraction due to an innate neural association of bacterial odor with the presence of protein. Rather than completely abandoning close symbioses, others consider that bacteria and fruit flies engage in facultative mutualistic symbioses in some cases (Lauzon et al. 2000). Compelling evidence for these types of symbioses are formation of biofilms in fruit fly intestines, specialized organs in fly gastrointestinal tracts where bacteria are harbored, and vertical transmission of bacteria through all life stages of the flies (Stammer, 1929; Mazzini and Vita, 1981; Lauzon, 2003), as had been described originally by Petri (1910). The major difference now is that most associations are thought to be flexible. As examples, perhaps more than one bacteria species could fulfill the role of symbiont in a particular fly (Lauzon, 2003), and maybe no bacteria are necessary if diet already contains digestible nutrients (Howard, 1989). However, the theory that an obligate ‘coevolved’ bacterium symbiosis exists in the olive fly has not been disproved and continues to garner evidence (Capuzzo et al., 2005). As a corollary to symbiosis is the question of what roles bacteria play in mutualistic symbioses with fruit flies. Among the proposed functions of bacteria are that they may biosynthesize essential amino acids (Miyazaki et al., 1968), serve as food (Drew and Lloyd, 1989) or indicators of food (Robacker and Moreno, 1995), convert unusable biochemicals into nutrients that flies can digest (Lauzon et al., 2000), detoxify allelochemicals in food and otherwise protect the gut from ingested toxins (Lauzon et al., 2003), and fix atmospheric nitrogen in the gut of flies (Behar et al. 2005). Quite possibly, different bacteria may take on different roles and maintain different levels of association within the life history of a single species of fruit fly. Putting the question of symbiosis aside, two undeniable facts make the study of fruit fly relationships with bacteria of great relevance: bacteria are ubiquitous in fruit flies and they are attractive to fruit flies. The ubiquity of associations between fruit flies and bacteria was documented above (Yamvrias et al., 1970; Tsiropoulos, 1983; Fitt and O’Brien, 1985; Drew and Lloyd, 1989; Martinez et al., 1994). Demonstrations that fruit flies are attracted to bacteria have been published for numerous species of both flies and bacteria (Drew and Lloyd, 1989; Jang and Nishijima, 1990; Robacker et al., 1991; MacCollom et al., 1992; Epsky et al., 1998). Despite nearly universal recognition that bacteria are attractive to fruit flies, relatively little has been accomplished regarding development of fruit fly lures from bacteria-produced attractants. One reason is that identification of chemicals produced by bacteria that are attractive to fruit flies has been nearly as problematic as determination of the nature of bacteria/fruit fly relationships. Production of ammonia by bacteria and its attractiveness to fruit flies has been common knowledge for most of the last century (Jarvis, 1931). Interestingly, several early studies suggested that ammonia was not really that attractive to fruit flies. Gow (1954) and Drew and Fay (1988) each believed chemicals produced by bacteria other than ammonia were primarily responsible. Gow (1954) even went so far as to hypothesize that the primary attractants of bacterial cultures were water soluble, nitrogen- containing chemicals other than ammonia. Unfortunately, his idea was largely ignored for 40 years as no serious studies to determine the identities of those chemicals were undertaken. 11

Studies done later to identify the attractive principals of bacterial fermentations showed a lack of understanding of why bacteria were attractive and therefore what type of chemicals would be involved (Robacker, 1998a). Because of the lack of understanding, bioassays were not designed properly so flies were not usually primed to respond to bacteria. Also, the chemistry methods were not appropriate for the type of chemicals that were involved. To make matters worse, often chemists performed the studies without cooperation with biologists or vice versa so chemicals were identified but not tested as attractants, or bioassays were done on bacterial fermentations but no identifications of the attractive principals were conducted. All of this led to identification of the wrong chemicals including numerous alcohols, aldehydes, ketones, pyrazines, sulfides, carboxylic acids, and aromatics. In this paper I review work done in my lab over the past 15 years to identify chemicals produced by numerous species and strains of bacteria that are attractive to the Mexican fruit fly. Based on the results I propose a theory tying together the chemistry of bacterial odors with the ecology of fruit fly associations with bacteria.

Material and methods

Bacteria culturing Staphylococcus aureus was isolated from laboratory colony Mexican fruit flies (Robacker et al., 1991). Enterobacter agglomerans was isolated from wild R. pomonella and Anastrepha ludens (Robacker et al., 2004). Most other bacteria were obtained from the American Type Culture Collection (Rockville, Maryland, U.S.A.). Some Bacillus thuringiensis strains were obtained from the Institut Pasteur (Paris, France). Most bacteria were cultured in tryptic soy broth (TSB) (Robacker et al., 1991). E. agglomerans was cultured in Petri plates using media with glucose and uric acid (uric acid medium) (Robacker and Lauzon, 2002), casein peptone (protein medium), glucose and casein peptone (TSB), or sucrose (carbohydrate medium (Robacker et al., unpublished) as the carbon sources.

Attractiveness bioassays Two types of bioassays were used. One was a cage-top bioassay in which fruit flies in a screen cage were exposed to odor sources loaded onto filter papers and placed on the top of the cages, elevated to prevent contact chemoreception (Robacker et al., 1991). The numbers of flies beneath the filter papers were counted every minute for 10 minutes and compared with the numbers that were beneath papers loaded with water, solvent, or uninoculated culturing medium as appropriate. The second was a wind-tunnel bioassay in which odor sources were placed in the upwind end of a plexiglas tunnel screened on each end to allow airflow through the chamber and flies were released in the downwind end of the tunnel. The numbers of flies that moved upwind and contacted the odor source within 5 min of sample introduction were recorded (Robacker and Lauzon, 2002).

Chemistry Bacterial cultures were aqueous samples for most work. Collection of volatiles from bacteria samples was done using solid phase microextraction with a PDMS fiber (Supelco, Inc., Bellefonte, Pennsylvania, U.S.A.). The fiber was exposed in the headspace above the bacteria sample then chemicals were thermally desorbed from the fiber in a heated injection port of a gas chromatograph (GC). Various GC models were used over the course of this work. For most analyses, on-column injection was conducted into a fused silica retention gap connected to the analytical GC column. The analytical column was usually a DB-1 with a 5 micrometer 12

film (J & W Scientific, Folsom, California, U.S.A.). Detection and identification of eluted chemicals was by flame ionization, flame thermionic, or mass spectrometry (MS). Details of methods can be found in various publications (Robacker and Flath, 1995; Robacker and Bartelt, 1997; Robacker et al., 2004).

Results and discussion

Establishment of a physiological link to protein hunger Tryptic soy broth cultures of S. aureus were more attractive to sugar-fed Mexican fruit flies than to flies deprived of sugar (Figure 1) (Robacker and Garcia, 1993). The effect increased as the period of deprivation increased. Protein deprivation had the opposite effect. Attraction of flies to S. aureus increased as the amount of protein in their adult diet diminished (Figure 2) (Robacker and Moreno, 1995). These experiments indicated that there is a physiological link between attraction of Mexican fruit flies to tryptic soy broth cultures of S. aureus and hunger for sugar and protein. This finding led to a hypothesis that bacteria odor elicits innate foraging for proteinaceous food. Sugar hunger inhibits this response because carbohydrate hunger is critical to immediate survival and the need for energy overrides the need for protein. Only after a sugar meal can flies resume protein foraging behavior. Note, however, that this relationship may not hold in cases in which attraction to bacteria may be occurring for another reason such as to replenish gut microflora (Lauzon, 2003). Evidence that finding protein is not the only reason fruit flies seek bacteria is that protein-fed Mexican fruit flies, even flies satieted on a complete breeding diet, continue attraction to S. aureus cultures at higher rates than to water controls, although at lower rates than protein-deprived flies (Robacker and Garcia, 1993; Robacker and Moreno, 1995).

3 2

0 sugar fed 1 2 3 days of sugar deprivation

Figure 1. Effect of sugar deprivation on attraction of Mexican fruit flies to tryptic soy broth (TSB) cultures of S. aureus. Attractiveness = number of flies attracted to the bacteria culture relative to the number attracted to uninoculated TSB.

Isolation and characterization of attractive principals Experiments were conducted to determine if the attractive principals from S. aureus cultures became dissolved in the aqueous medium as cells were cultured. Pellets containing cells were 13

obtained from bacterial cultures by centrifugation. Supernatant was filtered to remove the remaining cells. Supernatants were >3X more attractive than resuspended pellets in cage-top bioassays indicating that attractive principals produced by the bacteria were concentrated in the aqueous medium (Robacker et al., 1993).

0 sugar 12481632 only % protein in diet

Figure 2. Effect of protein in adult diet on attraction of Mexican fruit flies to tryptic soy broth (TSB) cultures of S. aurues. Attractiveness = number of flies attracted to the bacteria culture relative to the number attracted to uninoculated TSB.

8 7 6 5 4 3 2 1 0 2 3 4 5 6 7 8 9 10 11 12 pH Figure 3. Effect of pH of bacterial supernatant on attraction of Mexican fruit flies to tryptic soy broth cultures of S. aureus. Attractiveness = number of flies attracted to a pH treatment relative to the number attracted to water.

The pH of filtered supernatants of S. aureus cultures was manipulated to determine the effect on attractiveness (Figure 3) (Robacker et al., 1993). Preparations with pH between 7-10 14

were the most attractive to sugar-fed Mexican fruit flies. This indicated that the attraction was due to chemicals that contained protonizable nitrogen because these chemicals would be ionized and thus nonvolatile at the lower pH’s. Similar results were obtained with numerous other species of bacteria (Robacker and Bartelt, 1997; Robacker et al., 1998). Additional chemical functional group tests also implicated nitrogenous chemicals as the attractive principals of bacterial odor. These included formation of nonvolatile salts of the attractive chemicals at low pH that could be completely dried then reactivated by dissolution in water at high pH, retention on strong cation exchange media at low pH then elution at high pH, and lack of retention of attractive principals on reversed-phase HPLC unless paired with a negative counter ion (Robacker et al., 1993).

Identification of attractive principals Over 50 chemicals were identified by SPME and GC-MS from cultures of S. aureus, K. pneumoniae, C. freundii, E. agglomerans, and bird feces, a known fruit fly food, containing unidentified bacteria (Robacker and Flath, 1995; Robacker and Bartelt, 1997; Robacker et al., 2000; Robacker and Lauzon, 2002; Robacker et al., 2004). Chemicals that fit the profile of attractive principals were ammonia, several water-soluble aliphatic amines including methylamine, dimethylamine trimethylamine, 2-methylpropanamine, 2-methylbutanamine, and 3-methylbutanamine, the aromatic amine indole, imines including 1-pyrroline and 2,3,4,5-tetrahydropyridine, and pyrazines including pyrazine, 2,5-dimethylpyrazine and trimethylpyrazine. Among numerous other chemicals were acetic acid and other short-chain carboxylic acids, alcohols including 3-methylbutanol, 2-ethylhexanol, and 2-phenylethanol, phenol, 3-hydroxybutanone, and dimethylsulfide. Cage-top bioassays were conducted to establish attractiveness of these chemicals to Mexican fruit flies (Robacker and Flath, 1995; Robacker and Bartelt, 1997; Robacker and Lauzon, 2002; Robacker et al., 1996, 2000, 2004). Ammonia, most of the amines, 1-pyrroline, and acetic acid were the most attractive chemicals. All chemicals containing nitrogen were attractive to sugar-fed flies and dimethylamine, 1-pyrroline, and indole were also attractive to sugar-starved flies. Most chemicals without nitrogen were slightly attractive to sugar-starved flies and acetic acid and 3-hydroxybutanone were also attractive to sugar-fed flies. These results indicate that, for bacterial odors attractive primarily to sugar-fed, protein-hungry fruit flies, the attractive principals were mostly chemicals that contained protonizable nitrogen including ammonia, amines, 1-pyrroline, and pyrazines. Exceptions to this rule are known. In the case of E. agglomerans that is attractive to sugar-starved as well as sugar-fed Mexican fruit flies, other chemicals such as 3- hydroxybutanone probably also play important roles in attraction (Robacker et al., 2004). Bird feces containing bacteria were attractive to both sugar-fed and sugar-starved Mexican fruit flies and pH had little effect on attractiveness (Robacker et al., 2000). Phenol and 2- ethylhexanol may have significant attractive effects in this case. Phenol has also been implicated in attractiveness of K. pneumoniae (Robacker and Bartelt, 1997) and acetic acid may contribute to attactiveness of S. aureus and Bacillus thuringiensis coreanensis (Robacker et al., 1998). These exceptions indicate that attraction of fruit flies to bacteria undoubtedly has roots that spread far beyond need for protein.

Evaluation of the analytical method Standard methods used to identify semiochemicals involved in insect behavior have not been successful in identification of the attractive chemicals in bacterial odors. Collection of volatiles above bacterial cultures with activated charcoal and extraction with organic solvent resulted in identification of numerous organic chemicals but no amines (Lee et al., 1995; 15

DeMilo et al., 1996). My initial attempts to identify chemicals from bacterial odor also involved standard methods including collection of volatiles with Porapak Q, elution of chemicals from the adsorbent with organic solvent, and analysis using splitless injection onto commonly used thin-film GC columns (Robacker et al., 1993; Robacker and Flath, 1995). Although extracts prepared this way contained numerous chemicals and were very similar to the bacteria cultures as sensed by human olfaction, the extracts were not attractive to Mexican fruit flies and no amines were found by GC-MS. Standard methods did not work because of several problems. First, the most important chemicals were dissolved in the aqueous culturing media and could not be extracted out by solvent-solvent partitioning (Robacker et al., 1993). Direct analysis of the bacteria cultures by GC was not a viable alternative because aqueous samples are notoriously difficult to analyze by GC (Robacker, 1998a). SPME with a PDMS fiber overcame these problems because organic chemicals, especially amines, have a high affinity for the fiber while water does not appreciably adsorb (Robacker, 1998a). The adsorbed chemicals can then be thermally desorbed in the GC injector without introduction of water onto the column. However, another problem was that highly polar chemicals such as short-chain amines adsorb irreversibly onto active sites in injector liners and on GC columns. This problem was reduced by using on- column injection onto a thick-film GC column (Robacker and Flath, 1995). Thick-film columns effectively cover most of the active sites so that even trace amounts of ammonia and amines pass through the column (Robacker, 1998a). In addition, thick-film columns greatly increase retention of low-molecular weight chemicals allowing baseline resolution of ammonia and short-chain amines (Robacker and Flath, 1995). Also, the absence of a solvent peak allows easy quantitation of these small molecules. To verify that our method using SPME and a thick-film GC column successfully identified the attractive principals, synthetic mixtures of the chemicals found in the bacterial odors were constructed. Mixtures were prepared such that headspace concentrations of key nitrogenous chemicals, acetic acid, and phenol, were equal to concentrations in headspace above bacterial cultures of S. aureus, K. pneumoniae, and C. freundii. These synthetic mixtures were 81-89% as attractive as their respective bacterial cultures (Robacker and Flath, 1995, Robacker and Bartelt, 1997). A synthetic mixture of bird feces odor that contained phenol and 2-ethylhexanol in addition to nitrogenous chemicals was 96% and 80%, respectively, as attractive as the fecal extract to sugar-fed and sugar starved Mexican fruit flies (Robacker et al., 2000). Thus, our analytical method successfully determined the attractive chemicals in bacterial odors.

Variation in volatiles production with bacteria taxon The analytical method was used to survey attractive chemicals produced by bacteria over both broad and narrow levels of classification. Volatiles were identified from seven genera representing four major taxonomic categories of bacteria: Enterbacter, Klebsiella, and Citrobacter, facultatively anaerobic, gram-negative rods; Alcaligenes, aerobic, gram-negative rods and cocci; Staphylococcus and Micrococcus, gram-positive cocci; and Bacillus, endospore-forming, gram-positive rods (Robacker and Flath, 1995; Robacker and Bartelt, 1997; Robacker et al.; 1998). Within the genus Bacillus, volatiles were identified from 5 species and within B. thuringiensis they were identified from 4 subspecies (Robacker et al., 1998). Results showed that production of volatiles was not strongly tied to bacteria relatedness. Some chemicals were produced relatively uniformly by all taxa whereas others varied at almost every level of classification. Ammonia and 2,5-dimethylpyrazine were produced in similar amounts by all of the bacteria (Table 1). Other chemicals including trimethylamine, 3- 16

methylbutanamine, 1-pyrroline and acetic acid varied by as much as 1000 fold from genus to genus. Within Bacillus, similar trends were observed among the species B. sphaericus, B. subtilis, B. megaterium, B. popilliae, and B. thuringiensis. Trimethylamine and 2,5- dimethylpyrazine were relatively uniform among the species while 3-methylbutanamine, cyclohexylamine, and 1-pyrroline varied widely. Even within the single species B. thuringiensis, chemicals such as trimethylamine and 1-pyrroline varied by as much as 20 fold while other chemicals such as 2,5-dimethylpyrazine and acetic acid varied only a little (Table 2). These results testify to the great diversity of metabolic capabilities among bacteria. They also demonstrate that some chemicals such as ammonia and 2,5-dimethylpyrazine are to be expected in volatiles of almost any bacterium that is studied. Finally results suggest that numerous other attractive chemicals are yet to be discovered.

Table 1. Concentrations (µg/ml) of chemicals in supernatant filtrates of various genera of bacteria.

Staph Micro Bac Alcal Enter Kleb Citro ammonia 650 1000 1400 650 900 600 600 TMA 2.5 0.003 0.02 0.002 0.003 0.04 0.02 3-MBA 1.6 0.04 1.3 0.03 0.01 0.5 0.08 1-pyrroline - 0.02 0.003 0.003 0.003 0.2 0.2 2,5-DMP 5 1.9 2.5 2.3 3.4 2.8 4.8 acetic acid 60 13 490 210 71 0 0 Abbreviations: Staph, Staphylococcus; Micro, Micrococcus; Bac, Bacillus; Alcal, Alcaligenes; Enter, Enterbacter; Kleb, Klebsiella; Citro, Citrobacter; TMA, trimethylamine; 3-MBA, 3-methylbutanamine; 2,5-DMP, 2,5-dimethylpyrazine.

Table 2. Concentrations (µg/ml) of chemicals in supernatant filtrates of subspecies of Bacillus thuringiensis.

shan kon darm cor TMA 0.08 0.16 0.18 0.01 1-pyrroline 0.02 0.001 0.002 0.002 2,5-DMP 1.0 0.78 0.92 1.4 acetic acid 660 380 320 1000 Abbreviations: shan, B. t. shandongiensis; kon, B. t. konkukian; darm, B. t. darmstadtiensis; cor, B. t. coreanensis; TMA, trimethylamine; 2,5-DMP, 2,5-dimethylpyrazine.

A case involving E. agglomerans provides a final example of how volatiles vary with bacteria relatedness. Two strains of this bacterium that differed in their ability to metabolize uric acid, the primary nitrogen source in bird feces, were isolated from apple maggot flies 17

(Lauzon et al., 2000). When the two strains were cultured on a medium that contained uric acid as its principal nitrogen source, the uricase (+) strain produced more ammonia, trimethylpyrazine and 3-hydroxybutanone (not detected in the uricase (-) strain) (Robacker and Lauzon, 2002). A uricase (+) strain isolated from the Mexican fruit fly produced more indole than the uricase (+) apple maggot strain but only the apple maggot strain produced 3- hydroxybutanone (Robacker et al., 2004). Further investigation showed that the apple maggot strain contained indole-producing and no-indole cell types that alternated predominance in plated subculturings. Thus, batches of plates of this strain contained either mostly indole- producing cell types, or the other, so that volatiles produced by the strains varied widely from batch to batch of culturings. The indole-producing culturings also emitted amounts of ammonia, 3-methylbutanol, 2,5-dimethylpyrazine and 2-phenylethanol that were very similar to those emitted by the indole-producing Mexican fruit fly strain. However, they consistenly produced 3-hydroxybutanone whereas the Mexican fruit fly strain never emitted this chemical. This E. agglomerans case further illustrates that production of volatiles that constitute bacterial odors cannot be predicted by bacteria taxonomy, except that certain chemicals such as ammonia are produced almost ubiquitously.

Variation in volatiles production with culturing medium Volatiles produced by bacteria also vary with the culturing medium. The apple maggot uricase (+) strain of E. agglomerans was cultured on media that differed in their primary source of nutrients (Robacker et al., unpublished). Bacteria cultured on the uric acid medium produced much more ammonia and 2-nonenol than those cultured on the other three media. Culturings on either uric acid or carbohydrate media resulted in much greater production of trimethylamine, and culturings on tryptic soy broth contained much greater amounts of 2,5- dimethylpyrazine, compared with the other media. These results indicate that volatiles produced by the same bacterium in nature are likely to differ depending on the fermentation substrate.

Chemical interactions in attraction of fruit flies to bacteria Considering differences in volatiles produced by different bacteria species and strains of the same species and differences caused by bacteria growing on different substrates, an interesting chemical ecology question is ‘how do fruit flies manage to find the bacteria they need?’. Work in my laboratory over the past 10 years has addressed this question in the Mexican fruit fly. Part of the answer appears to be the way chemicals in the bacterial odors are perceived and the signals integrated in the central nervous system of the flies. The approach was to quantitate attraction of sugar-fed, protein-hungry flies to one of more of the attractive chemicals in various combinations (Figure 4). As discussed above, ammonia and most of the amines were attractive when tested individually. Combinations of ammonia with either methylamine or putrescine were much more attractive than any of the three chemicals alone indicating synergistic effects (Robacker and Warfield, 1993). Putrescine was chosen for testing because it is a possible decomposition product of arginine (Wakabayashi and Cunningham, 1991) and is commonly found as a volatile emitted by fermentations of fruit and protein baits for fruit flies (Heath et al., 1995). Combinations of methylamine with putrescine were only slightly more attractive than either alone indicating additive effects (Robacker and Warfield, 1993). To some extent, various amines could substitute for methylamine with little change in attractiveness, but they could not substitute for ammonia or putrescine (not shown). Finally, 1-pyrroline was highly attractive by itself (Robacker et al., 2000) and greatly increased attractiveness of mixtures of ammonia, methylamine and putrescine (Robacker et al., 1997). 1-Pyrroline also could substitute for 18

putrescine in combinations with ammonia and methylamine (Robacker, 2001) (not shown). Wakabayashi and Cunningham (1991) had similar results with melon fly (Bactrocera cucurbitae). They presented evidence that mixtures of ammonia with either putrescine or pyrrolidine were much more attractive than any chemicals alone.

0.8

0.6

0.4 attractiveness 0.2

0 NH3 MA put 1-pyr NH3 + NH3 + MA + NH3 + all 4 MA put put MA + put

Figure 4. Synergistic and additive interactions of chemicals in attraction of Mexican fruit flies. Attractiveness = attraction to each chemical or combination relative to attraction to all four chemicals.

These results indicate that ammonia interacts synergistically with at least some amines that can be found as products of bacterial fermentations. Other nitrogen-containing chemicals had either additive effects with each other, no effects but could substitute for each other, or in some cases inhibitory effects with each other (Robacker, 1998b). Very little work has been directed toward interactions of chemicals that do not contain nitrogen. However, phenol added attractiveness to a mixture of the nitrogenous components of the odor of C. freundii (Robacker and Bartelt, 1997), as discussed above. Acetic acid enhanced the attractiveness of ammonia, methylamine and putrescine to sugar-starved, protein-starved Mexican fruit flies, but not to other hunger-state groups of flies (Robacker et al., 1996). Other non-nitrogenous chemicals such as 3-hydroxybutanone have not been tested in combinations but may play roles in attraction of flies in undetermined drive states. Although the neural physiology of these interactions has not been investigated, it is probable that the observed behavioral effects are the result of both peripheral reception and central nervous system integration of antennal signals (Robacker, 1998b).

Chemical ecology of attraction of fruit flies to bacteria These findings lead me to hypothesize that fruit fly appetitive search for bacterial fermentations is a function of: 1) the specific need for bacteria; 2) the chemical signature of the fermentation; and 3) the workings of the fly’s nervous system. The specific need is the drive that triggers the fly to search for bacteria. It could include search for bacteria as food (Drew and Lloyd, 1989), as indicators of food (Robacker and 19

Moreno, 1995), to replenish gut microflora (Lauzon, 2003), to detoxify allelochemicals in food (Lauzon et al., 2003), etc., as discussed in the introduction. The chemical signature of a bacterial fermentation provides information to the fly as to what type of nutrients are present. It can vary according to the species and strain of bacterium and the substrate on which the bacteria are growing. Flies searching for bacteria as protein or as indicators of protein could find the fermentations they need by responding to odors containing ammonia, amines or 1-pyrroline, i.e. common metabolic breakdown products of proteinaceous foodstuffs. Flies searching for a specific strain of E. agglomerans to build up their gut biofilm may respond to fermentations emitting 3-hydroxybutanone, a chemical that is emitted only by this bacterium. As a final example, flies needing a specific essential amino acid may search for chemicals that are metabolic byproducts from its biosynthesis and therefore would be attracted to bacteria actively synthesizing that amino acid. The way the fruit fly’s nervous system interprets the blends of chemicals in bacterial odors greatly enhances the ability of the flies to find the fermentations they need. This is best demonstrated by the synergism of ammonia with amines and 1-pyrroline (Figure 4). Because ammonia is ubiquitous in bacterial fermentations (every bacterium we studied has produced this chemical), it is a focal point in the attraction process, i.e. it is attractive by itself and it synergizes attractiveness with at least some amines and 1-pyrroline. To illustrate using an example from the previous paragraph, flies would be able to locate fermentations containing protein by responding additively to ammonia, each of several amines, and 1-pyrroline. However, the synergistic neural response to these chemicals makes the searching process much more efficient. Also, the capability of at least some amines to substitute for each other in their synergism with ammonia means that flies are empowered to find a protein fermentation if it emits any of several amines. I indicated above that specific amines may give flies information about specific amino acids. At present, there is no evidence if that occurs or if it might involve specific synergisms with ammonia or other chemicals. Also, it is not known if attractive bacteria-produced chemicals including 3-hydroxybutanone, acetic acid, phenol, and 2-ethylhexanol (and several other alcohols) act independently or synergistically with other chemicals in bacterial odors. The effects of drive states on responses to bacterial odors should not be overlooked. These states seem to turn on or turn off the flies’ motor systems that control responses to the odors (Robacker, 1998b). In at least one study, sugar-hunger greatly depressed attraction while protein-hunger stimulated attraction of Mexican fruit flies to bacterial odors (Robacker and Garcia, 1993) indicating that need for protein was the driving force in that case. Other types of chemicals, such as 3-hydroxybutanone, may stimulate specific receptors and brain centers that are activated when flies are primed by drive states for particular bacteria that emit that chemical. This discussion is a synthesis attempting to tie together findings from numerous studies cited earlier in this paper, and is by no means conclusive. I only hope to stimulate others to examine this hypothesis and develop research to confirm or refute my ideas.

Benefits of these studies Studies of bacteria associated with fruit flies have already benefitted entomological pursuits. Information from these studies has left its mark on development of new fruit fly lures including both the BioLure MFF lure (Suterra LLC, Inc., Bend, Oregon, U.S.A.) (Heath et al., 1995) and the AFF lure (Advanced Pheromone Technologies, Inc., Marylhurst, Oregon) (Robacker and Warfield, 1993). The potential for improvement of lures by addition of novel bacteria-produced chemicals remains great. Improvement of fruit flies that are mass-reared for release of sterile males is another potential use of information from studies of fruit fly and 20

bacteria relationships. New studies have shown that addition of probiotic bacteria to diets fed to Mediterranean fruit flies aids reparation of the gut damaged by irradiation (Niyazi et al., 2004; Lauzon and Potter, 2005). Finally, these studies enhance understanding of our natural world. Knowledge of these complex relationships may lead to advancements in entomology, microbiology, and even medicine in ways that no one has yet imagined.

Acknowledgements

I have had numerous collaborators and technical assistants over the years to whom I am greatly indebted: Jose Garcia, Maura Rodriguez, William Warfield, Daniel Moreno, Aleena Tarshis Moreno, Roger Albach, Bianca Chapa, Xiaodun He, and Joe Patt (ARS, Weslaco, Texas, U.S.A.); A. J. Martinez (APHIS, Mission, Texas); Robert Flath (ARS, Albany, California, U.S.A.); Robert Heath (ARS, Miami, Florida, U.S.A.); Robert Bartelt (ARS, Peoria, Illinois, U.S.A.); Michael Kaufman (Michigan State University, East Lansing, Michigan, U.S.A.); and Carol Lauzon (California State University, Hayward, California). I also thank Professor Antonio Belcari (Universita degli Studi di Firenze, Firenze, Italy) for giving me the opportunity to present this work at the 2nd European Meeting of the IOBC/WPRS Study Group “Integrated Protection of Olive Crops”, Firenze, Italy, 2005.

References

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Robacker, D.C. 2001: Roles of putrescine and 1-pyrroline in attractiveness of technical-grade putrescine to the Mexican fruit fly (Diptera: Tephritidae). – Fla. Entomol. 84: 679-685. Robacker, D.C. & Bartelt, R.J. 1997: Chemicals attractive to Mexican fruit fly from Klebsiella pneumoniae and Citrobacter freundii cultures sampled by solid-phase microextraction. – J. Chem. Ecol. 23: 2897-2915. Robacker, D.C. & Flath, R.A. 1995: Attractants from Staphylococcus aureus cultures for Mexican fruit fly, Anastrepha ludens. – J. Chem. Ecol. 21: 1861-1874. Robacker, D.C. & Garcia, J.A. 1993: Effects of age, time of day, feeding history, and gamma irradiation on attraction of Mexican fruit flies (Diptera: Tephritidae), to bacterial odor in laboratory experiments. – Environ. Entomol. 22: 1367-1374. Robacker, D.C. & Lauzon, C.R. 2002: Purine metabolizing capability of Enterobacter agglomerans affects volatiles production and attractiveness to Mexican fruit fly. – J. Chem. Ecol. 28: 1549-1563. Robacker, D.C. & Moreno, D.S. 1995: Protein feeding attenuates attraction of Mexican fruit flies (Diptera: Tephritidae) to volatile bacterial metabolites. – Fla. Entomol. 78: 62-69. Robacker, D.C. & Warfield, W.C. 1993: Attraction of both sexes of Mexican fruit fly, Anastrepha ludens, to a mixture of ammonia, methylamine, and putrescine. – J. Chem. Ecol. 19: 2999-3016. Robacker, D.C., DeMilo, A.B. & Voaden, D.J. 1997: Mexican fruit fly attractants: Effects of 1- pyrroline and other amines on attractiveness of a mixture of ammonia, methylamine, and putrescine. – J. Chem. Ecol. 23: 1263-1280. Robacker, D.C., Garcia, J.A. & Bartelt, R.J. 2000: Volatiles from duck feces attractive to Mexican fruit fly. – J. Chem. Ecol. 26: 1849-1867. Robacker, D.C., Garcia, J.A., Martinez, A.J. & Kaufman, M.G. 1991: Strain of Staphylococcus attractive to laboratory strain Anastrepha ludens (Diptera: Tephritidae). – Ann. Entomol. Soc. Amer. 84: 555-559. Robacker, D.C., Lauzon, C.R. & He, X. 2004: Volatiles production and attractiveness to the Mexican fruit fly of Enterobacter agglomerans isolated from apple maggot and Mexican fruit flies. – J. Chem. Ecol. 30: 1329-1347. Robacker, D.C., Lauzon, C.R. & Patt, J. unpublished: Dependence on culturing medium of volatiles produced by Enterobacter agglomerans and effect on attractiveness to the Mexican fruit fly. – J. Chem. Ecol. In preparation. Robacker, D.C., Martinez, A.J., Garcia, J.A. & Bartelt, R.J. 1998: Volatiles attractive to the Mexican fruit fly (Diptera: Tephritidae) from eleven bacteria taxa. – Fla. Entomol. 81: 497- 508. Robacker, D.C., Moreno, D.S. & DeMilo, A.B. 1996: Attractiveness to Mexican fruit flies of combinations of acetic acid with ammonium/amino attractants with emphasis on effects of hunger. – J. Chem. Ecol. 22: 499-511. Robacker, D.C., Warfield, W.C. & Albach, R.F. 1993: Partial characterization and HPLC isolation of bacteria-produced attractants for the Mexican fruit fly, Anastrepha ludens. – J. Chem. Ecol. 19: 543-557. Stammer, H.J. 1929: Bakteriensymbiose der Trypetiden (Diptera). – Zeit. Morphol. Ökol. Tiere. 15: 481-523. Tsiropoulos, G.J. 1983: Microflora associated with wild and laboratory reared adult olive fruit flies, Dacus oleae (Gmel.). – Zeit. Angew. Entomol. 96: 337-340. Wakabayashi, N. & Cunningham, R.T. 1991: Four-component synthetic food bait for attracting both sexes of the melon fly (Diptera: Tephritidae). – J. Econ. Entomol. 84: 1672-1676. Yamvrias, C., Panagopoulos, C.G. & Psallidas, P.G. 1970: Preliminary study of the internal bacterial flora of the olive fly (Dacus oleae Gmelin). – Ann. Inst. Phytopath. Benaki. 9: 201-206. Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 23

Effect of age and mating status on the antennal sensitivity of Bactrocera oleae (Rossi) (Diptera Tephritidae) male and female*

A. De Cristofaro1, G. Rotundo1, A. Belcari2, G.S. Germinara1 1 Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Universitá degli Studi del Molise, Via De Sanctis, I-86100, Campobasso, Italy. 2 Dipartimento di Biotecnologie Agrarie, Universitá degli Studi di Firenze, Via Maragliano 77, I-50144 Firenze, Italy.

The olive fruit fly [Bactrocera oleae (Rossi) (Diptera Tephritidae)] control is really difficult; as a consequence, studies to set up alternative control methods (i.e. use of semiochemicals) were undertaken. While several researches were carried out on the sex pheromone, only few investigations have been focused on the compounds emitted by the host plant or associated bacteria and their possible use in modifying olive fruit fly behaviour. In order to correctly address future bioassays, in the present paper, preliminary to the study of the role and perception of plant and bacterial volatile compounds, EAG technique was used to determine the olfactory sensitivity of virgin and mated males and females of different age (1-3; 10-15; 27-32; 57-62; 87-92 days old) to 28 synthetic substances identified in olive leaves and fruits. EAG responses were submitted to ANOVA and cluster analysis. Responses of the different insect categories were compared using the t-test (P=0.01; P=0.05). Both sexes, independently from age and mating status, were able to perceive a wide variety of odours emitted by the olive plant. Considering the mean EAG response to all compounds, the olfactory sensitivity decreases with age advancement in virgin males and females while it is quite constant in mated ones. Virgin insects showed a higher number of EAG response groups than mated ones, with a tendence to decrease with the age. Contrary to mated females, a clear reduction of the EAG response groups was observed in mated males. The persistent olfactory sensitivity and selectivity of mated females might be related to the necessity of the oviposition site location. In addition, some electrophysiologically-active terpenes [(+)-α-pinene, (-)-β-pinene, R- (+)-limonene, L-(-)-limonene) were able to attract both sexes of B. oleae in preliminary wind- tunnel experiments. In an open field study, using sticky tablet traps baited with rubber septa dispensers containing different doses (0.1, 1.0, 10 mg diluted in mineral oil) of a synthetic compound, R-(+)-limonene showed the higher catch potency, trapping Olive fruit fly males and females in a 1:1 sex ratio. Researches focused to a practical utilization of the identified kairomone, alone or mixed with other compounds, like bacterial volatile compounds, in B. oleae monitoring and control (mass trapping, lure and kill) techniques, are still in progress.

* Research supported by MIUR (PRIN 2003).

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 25-30

Relationship between olive fly adults and epiphytic bacteria of the olive tree

Aurelio Granchietti,1 Alessandra Camèra,1 Serena Landini,1 Marzia Cristiana Rosi,1 Michele Librandi,1 Patrizia Sacchetti,1 Guido Marchi,2 Giuseppe Surico,2 Antonio Belcari1 1 Department of Agricultural Biotechnology, Section of General and Applied Entomology, University of Florence, Italy 2 Department of Agricultural Biotechnology, Section of Plant Pathology, University of Florence, Italy

Abstract: Culturable epiphytic bacteria were isolated in 2004 and 2005 from the twigs, leaves and fruits of olive trees, and their number compared with captures of Bactrocera oleae. The results obtained show a certain degree of correlation between the presence of bacteria on the phylloplane of the olive tree and the size of the B. oleae population in the olive grove. This suggests that olive epiphytic bacteria may play an essential role for fly fitness on the olive.

Keywords: Culturable bacteria, Bactrocera oleae, Olea europea, host plant/insect relationship.

Introduction

Fruit flies are serious pests throughout the world, and due to their economic importance they have been extensively studied from various points of view. Management of these pests is problematic because of their biology and reproductive potential; moreover, limited results have been obtained with the IPM approach. This is also due to incomplete understanding of some aspects of the fruit fly’s life history, in particular the mutual association the flies are capable of establishing with some micro-organisms, in particular bacteria. Fruit flies, both adults and larval stages, require amino acid sources for their normal development and for egg production. The availability of proteins thus affects the spread of these species directly. Some authors have suggested that honeydew meets this need, but in the 1980s other researchers began to show that this requirement is met by plant surface micro- organisms (Courtice & Drew, 1983). Ever since the beginning of the last century, bacteria have been observed in the alimentary canal of some species in the Tephritidae family. More recently several bacteria belonging to the Enterobacteriaceae family were found in the gut of certain tropical Dacinae species. Most of these bacteria were also isolated from the faeces of field-collected flies, host fruit, oviposition sites and larval tunnels of infested fruits (Lloyd et al., 1983). The most important studies of Bactrocera oleae have been carried out in Greece and Italy; in 1983 Tsiropoulos isolated bacteria from the gut of both wild and reared flies, while bacteria from the olive fly foregut have recently been investigated in Italy and epiphytic bacteria from olive trees have also been isolated (Belcari et al., 2003). Recently Capuzzo et al. (2005) found a new bacterial species, Erwinia dacicola, acting as an unculturable obligate symbiont of the olive fly. However the ecological significance of this mutual relationship is still little investigated, and it is not yet understood how bacterial association can affect the behaviour and physiology of the olive fly.

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In this paper the results of preliminary investigations in two olive groves in central Italy are reported. The aim is to improve knowledge of the ecological relationship between the epiphytic bacteria of olive trees and the olive fly, by monitoring both the B. oleae population and the fluctuations in epiphytic bacteria.

Materials and methods

Research was conducted in 2004 and 2005 in two different olive groves near Florence (central Italy). Only one olive grove was considered per year, and both consisted of local olive tree varieties. In 2005 climatic data were recorded by means of a weather station near the olive grove.

Monitoring the olive fly population The B. oleae population dynamic was monitored by trapping adults using chemiotropic traps. Traps were positioned in olive groves to evaluate the attractiveness of a volatile compound produced by Pseudomonas putida, a bacterium isolated in previous research from the olive fly and from the olive phylloplane. The traps were baited with bacterial filtrates, commercial hydrolysed proteins (Buminal) and tryptic soy broth (the bacterium growth medium), all of them in water solution. The weekly overall number of adults caught by the traps was considered as a measure of the population density of the flies. Baits were replaced once a week, and the number of flies captured was counted. In 2004 the monitoring lasted three months, while in 2005 traps were set from March to November.

Isolation of epiphytic bacteria from olive trees Bacteria were isolated separately from the surfaces of twigs, leaves and olives. Taking care to prevent any external contamination, disposable gloves, sterile bags and disinfected scissors were used. Plant samples were washed for 2 hours in phosphate buffer solution 0.05M at 170 rpm; decimal dilutions were plated in Petri dishes containing 15 ml of Nutrient Sucrose Agar (NSA) mixed with cycloheximide (70 mg/l). The dishes were incubated at 28°C and after 5 days the number of colonies was counted. Pure cultures of the main representative bacteria were isolated for subsequent identification. The dry weight of the samples was then obtained to compare the results of different samples. The bacterial load was expressed as a logarithmic of the Colony Forming Unit (CFU)/gram of dry weight. Isolation of bacteria lasted three months in 2004 and from March through to November in 2005 (the same period as the olive fly monitoring).

Results

Observations in 2004 The greatest proportion of the bacterial load was observed on twigs, followed by leaves and olives (Figure 1). However, in October the bacterial population on the surface of the olive fruit increased approximately tenfold, while it decreased on the twig and leaf surfaces. During this period, captures were very scarce because of the low population density of the olive fly in the majority of Tuscany’s olive groves. However, when the number of B. oleae adults captured is related to the epiphytic bacteria population in the different aerial plant parts, a similarity can be seen between the trend of fly captures and the bacterial presence on twigs (Figure 2). This relationship is more evident in the last ten days of September and throughout October, when the captures increased or drastically decreased, as shown on 19 October.

5 -1

Log CFU g 3

0 4-Oct 1-Sep 7-Sep 12-Oct 19-Oct 25-Oct 13-Sep 20-Sep 27-Sep 13-Aug 19-Aug 26-Aug CFU on olive fruit CFU on leaf CFU on twig

Figure 1. Fluctuations in the epiphytic bacteria load on leaves, twigs and olives in 2004.

8 1

0.9 7.5 0.8 7 0.7 6.5 0.6

6 0.5

0.4 log CFU g log-1 CFU 5.5 trap flies per 0.3 5 0.2 4.5 0.1

4 0 4-Oct 1-Sep 7-Sep 12-Oct 19-Oct 25-Oct 13-Sep 20-Sep 27-Sep 13-Aug 19-Aug 26-Aug CFU on twig olive flies/trap

Figure 2. Adult olive fly population and bacterial presence on twig surfaces in 2004.

Observations in 2005 As in the previous year, a higher bacteria load was found on twigs than on leaves and fruits. In spring the fly population was very low and it was impossible to compare the fly and

8.00 30

7.00 25

6.00 20 -1

5.00 15 log CFU g 4.00 10 flies per trap

3.00 5

2.00 0 3-Oct 6-Sep 4-Apr 11-Jul 25-Jul 8-Aug 2-May 13-Jun 28-Jun 18-Oct 31-Oct 19-Sep 18-Apr 13-Dec 21-Mar 21-Nov 22-Aug 16-May 30-May CFU on twig CFU on leaf CFU on olive fruit olive flies/trap

Figure 3. Fluctuations in the epiphytic bacteria population and captures of Bactrocera oleae adults in 2005.

8.00 30.00

7.00 25.00

6.00 20.00

5.00 15.00 log CFU g-1 log CFU flies per trap 4.00 10.00

3.00 5.00

2.00 0.00 4-Jul 5-Dec 18-Jul 1-Aug 8-Nov 20-Jun 10-Oct 24-Oct 12-Sep 26-Sep 19-Dec 16-Aug 29-Aug CFU on leaf CFU on olive fruit olive flies/trap

Figure 4. B. oleae adult population and bacteria load on leaves and fruit surfaces in 2005.

7.00 80

6.00 70

60 5.00 50 -1 4.00 40 3.00 log CFU g log CFU 30 2.00

20 or mm per °C trap flies

1.00 10

0.00 0 4-Jul 3-Oct 6-Sep 11-Jul 18-Jul 25-Jul 8-Nov 1-Aug 8-Aug 10-Oct 18-Oct 24-Oct 31-Oct 12-Sep 19-Sep 26-Sep 16-Aug 22-Aug 29-Aug rainfall average air temperature olive flies/trap CFU on olive fruit

Figure 5. B. oleae adult population, bacteria load on olive fruit surfaces and climatic data in 2005.

bacteria populations (Figure 3). By contrast, from the beginning of July the number of flies captured increased through to the end of September. After that period the adult population suddenly decreased following organophosphate spraying against olive fly larvae. This measure was adopted because the proportion of infested olives had exceeded the action threshold. A close relationship can be observed between the trends of the two populations, which is particularly evident for the leaves and fruits. Indeed, as shown in Figure 4, the first peak of adult captures (at the beginning of July) coincided with a remarkable increase in epiphytic bacteria, on leaves but in particular on fruits (about 100 times greater in a week). This trend continued until the adult peak of September (on the 19th). After this date, for the above-mentioned reason, the fly population decreased drastically, while there was no such trend for the bacteria. On the basis of the average temperature and rainfall recorded during the period, it can be noted that bacterial populations are influenced by climatic factors, in particular by rainfall. However, the bacterial load on olives seems to be more affected by the olive fly population caught in the orchard than by temperature or rainfall (Figure 5).

Discussion

As far as we know, the only research to date into the relationship between fruit flies and bacteria has been carried out in Australia in tropical Dacinae by Drew and colleagues (Drew et al., 1983; Lloyd et al., 1986). No investigation of the relationship between the fruit fly population and the load of epiphytic bacteria resident on the host plant has previously been conducted. Our results show for the first time a relationship between epiphytic micro- organisms and the olive fly population. It is known that B. oleae creates a close biological relationship with bacteria (Girolami, 1973). On the basis of our study this relationship appears to be more wide-ranging and also to involve other ecological factors. It can thus be assumed 30

that the olive fly is closely linked to these micro-organisms, which are one of the most important proteinaceous sources. At the same time, the olive fly plays an important role as a carrier for epiphytic bacteria, enhancing their spread and growth in the olive ecosystem. If we consider that the olive fly, insofar as it is a monophagous species, is strictly dependent on the olive plant and that no important proteinaceous source is available in the olive grove, especially for females during the preoviposition period, it seems that epiphytic bacteria play an essential role for fly fitness. Further investigations are needed to confirm our preliminary results. However, field trials carried out in central and southern Italy, in which copper products (acting as symbionticides) were used against the olive fly, confirm the importance of these associations.

References

Belcari, A., Sacchetti, P., Marchi, G. & Surico, G. 2003: La mosca delle olive e la simbiosi batterica. – Informatore Fitopatologico 9: 55-59. Capuzzo, C., Firrao, G., Mazzon, L., Squartini, A. & Girolami V. 2005: “Candidatus Erwinia dacicola”, a coevolved symbiotic bacterium of the olive fly Bactrocera oleae (Gmelin). – International Journal of Systematic and Evolutionary Microbiology 55: 1641-1647. Drew, R.A. I., Courtice, A.C. & Teakle, D.S. 1983: Bacteria as a natural source of food for adult of Fruit Flies (Diptera: Tephritidae). – Oecologia 60: 279-284. Girolami, V. 1973: Reperti morfo-istologici sulle batteriosimbiosi del Dacus oleae Gmelin e di altri Ditteri Tripetidi, in natura e negli allevamenti su substrati artificiali. – Redia 54: 269-273. Lloyd, A.C., Drew, R.A.I., Teakle, D.S., & Hayward, A.C. 1986: Bacteria associated with some Dacus species (Diptera: Tephritidae) and their host fruit in Queensland. – Aust. J. Biol. Sciences 39(4): 361-368. Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 31

Field assessment of different combinations of ammonia-based attractants and a synthetic female sex pheromone for the monitoring and control of the olive fruit fly, Bactrocera oleae Gmel. (Diptera: Tephritidae) in Apulia, southern Italy

A. de Cristofaro1, M. Cristofaro2, F. Tenaglia1, A. Fenio3, C. Tronci3 1Dipartimento SAVA, Università degli studi del Molise, Campobasso, Italy. 2ENEA C.R. Casaccia UTS BIOTEC, S.M. di Galeria (RM), Italy. 3Biotechnology and Biologica Control Agency, Sacrofano (RM), Italy.

Olive fruit fly, Bactrocera oleae (Gmelin), is a very serious pest of olives in the Mediterranean basin, where the majority of the world's olives are produced. This is particularly true for the Italian region of Apulia which alone produces about 12% of the total world olives and olive oil. Aim of the work was to evaluate the efficacy of six different combinations of ammonia- based fruit-fly attractants, ammonium acetate (AA), putrescine (PT), and trimethylamine (TMA) on the wild B. oleae population as a part of a FAO/IAEA Coordinated Research Program. Proposed treatments were compared with the widely used protein-based attractant Nu- Lure and with a synthetic female sex pheromone. The experiment took place in an olive plantation at Serracapriola, Apulia in autumn 2003; the tests were repeated in the same location during fall 2004 due to the scarce B. oleae population in 2003. Olive fruit fly males and females clearly showed to prefer NuLure, when compared to the different combinations of AA, PT and TMA. Between them, treatments E and F (respectively 4AB+PT and 2AB+PT) showed significantly higher scores. Female sex pheromone showed an extremely high performance even when compared with Nu-Lure, allowing its use for earlier detection of olive fruit fly male population.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 33-36

Inhibitory effect of water assumption on attraction to ammonia, protein baits and bacteria in Bactrocera oleae (Gmelin)

Vincenzo Girolami, Alessia Piscedda, Damiano Emer, Andrea Di Bernardo, Luca Mazzon, Rita Signorini University of Padua, Department of Environmental Agronomy and Plant Productions, Viale dell’Università, 16 - 35020 Legnaro, Padua, Italy. E-mail: [email protected]

Abstract: The influence of water and food availability on the attraction to ammonia and protein baits in Bactrocera oleae (Gmelin) has been investigated. Flies deprived of water for at least 48 hours frenetically respond to ammonia sources; no such response is observed in adults that have quenched their thirst with water or sugar solution. Crystalline sugar has no influence. It is probable that ammonia odors are utilized to search for puddles where bacterial fermentation occurs.

Key words: Bactrocera oleae, ammonium carbonate, behavior, attraction.

Introduction

The olive fly Bactrocera oleae (Gmelin) is the most important pest for olive trees in the Mediterranean basin. At the beginning of the past century, in the first experiences of olive fly control, molasses poisoned baits (Berlese, 1905) were used. These have now been replaced by more satisfactory protein or ammonia baits (Delrio et al., 1982). Since the beginning of the last century it was known that in high humidity conditions fruit flies are poorly attracted to alimentary baits, probably because they find food and water in the environment (Dal Guercio, 1930). The aim of this work is to understand whether attraction to protein and ammonia baits is negatively influenced by water availability, food abundance or both; the answer to bacterial colonies’ odors will also be investigated.

Material and methods

Flies were maintained at 23-25 °C with relative humidity lower than 70% in cubic tulle cages, generally containing 20 or 30 adults (sex ratio 1:1) fed with a saturated sucrose solution and water placed on the top of the cage. Before the test, the flies were divided into groups either deprived of water and sugar for 24 or 48 hours, or those normally reared. Only water, or only crystalline sugar, or only saturated sucrose solution were also offered to other groups of cages for 48 hours before the test. For the investigation, the cubic cage is inserted in a vertical plexiglass parallelepiped (15 cm x 15 cm x 50 cm) olfactometer in which air moves from the bottom to the top with a speed of about 1 m/s (Figure 1). The temperature and relative humidity of air entering from the outside, are controlled and an extractor fan eliminates the outgoing air, to avoid the contamination of the laboratory. To evaluate the attraction of the insects, a gauze (4x4 cm), is inserted at the base of the olfactometer after being dampened with different solutions or impregnated with salts.

33 34

The adults present on the bottom of the cage are counted: first, before the insertion of the gauze and then again at 30 and 60 seconds after its insertion. Since light is coming from the window with a frontal direction, flies tend to linger on the top of the cage and to go to the bottom only if they are responding to odors coming from the base of the olfactometer. Solutions of ammonium carbonate (0.1%), were routinely used. Ammonium hydrate, protein baits, bacterial colonies and filtered liquid media, in which Pantoea agglomerans has grown, were also preliminarily tested.

entering air fan air-conditioning

fan

outgoing air

tulle cage

odor

Figure 1. The vertical olfactometer

Results and discussion

Effects of food and water on attraction to ammonia Adults deprived of water and food for 48 hours have an immediate response to all the tested baits. No such response is observed if saturated sucrose solution is given to the same adults. At least half an hour must pass after sugar solution assumption for the inhibition of the response. Similar results are obtained if water is offered to adults deprived for 48 hours of food and water. For instance, (Figure 2) employing 30 adults per cages before the test, 2-4 flies linger on the bottom; after exposure to the odor, more than about 70% of the flies go to the bottom in few seconds. The same hungry adults, that previously have shown such a strong attraction to ammonia, give no response if water is offered. Only one day of water deprivation at room temperature is not sufficient to improve attraction. Similarly, newly emerged flies need 2 days of water deprivation before showing attraction.

no water and sugar after water n.° of flies on the bottom for 48 hours assumption at the start 3 2 after 30 sec 25 3 after 60 sec 27 2

30 B

25 B m

20 at the start 15 after 30 sec after 60 sec 10 n.° of flies on the botto flies on the of n.° 5 A A A A 0 no water and sugar for 48 hours after water assumption

Figure 2. Number of adults (of a total of 30) observed on the bottom of the cage, attracted by ammonium carbonate (0.1 %) after 30 and 60 sec. of exposure to the odor. If water is offered, adults no longer respond to ammonia solution. Different letters indicate significant differences among columns (according to G test, P< 0.001).

Nitrogen compounds attraction Olive flies deprived of water are attracted, in the above described olfactometer, in a similar way to the solution of ammonium carbonate and ammonium hydrate (minimum 50 ppm), protein baits (more than 1%) and other nitrogen compounds such as putrescine and urea. Water alone is not attractive even if flies (after 48 hours without water) are nearly dying of thirst. nevertheless crystalline ammonium carbonate exhibits strong attraction. No difference was found between females and males or mature and immature adults. Temperature and relative humidity variations, observed in the laboratory at different seasons, had no evident influence on the response to ammonia.

Bacterial colonies attraction A strong attraction of thirsty flies to bacterial colonies can be observed by opening a Petri dish with microorganisms at the bottom of the olfactometer, or using a gauze dampened with bacterial colonies or filtered grow liquid media; Pantoea agglomerans strain was used. Similar results are obtained using an embedded gauze with water collected in field puddles or with backwater inside forgotten bottles. Tap water had no attraction. As observed using ammonia, attraction to bacteria colonies, grow media, or backwater is completely inhibited if water is offered to the flies. All considerations reported refer to the olive fly Bactrocera oleae, but preliminary observation indicates a similar behavior in medfly Ceratitis capitata. In conclusion, only thirsty flies show a frenetic attraction to ammonium carbonate or hydrate, protein baits, bacterial products or backwater. As a first hypothesis, it may be that thirsty flies, searching for water, can easily perceive water puddles due to the emission of 36

nitrogen chemical volatiles that are produced during fermentation of organic matter in waters where algae easily grows.

References

Berlese, A., 1905: Probabile metodo di lotta efficace contro la Ceratitis capitata Wied., Rhagoletis cerasi L. e altri Tripetidi. – Redia 3: 386-388. Del Guercio, G., 1930: Le ricerche e le esperienze di Puglia dal 1910 al 1914 contro la mosca delle olive. – Redia 18: 171-399. Delrio, G., Prota, R., Economopoulos, P.V., Economopoulos, A.P., Haniotakis, G.E., 1982: Comparative study on food, sex and visual attractants for the olive fruit fly. – In: R. Cavalloro (ed.): Fruit flies of economic importance. Proceedings of the joint CEC/IOBC- wprs International Symposium, Athens (Greece), 16-19 November 1982. A.A. Balkema, Rotterdam: 465-472.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 37-42

Attractiveness to the olive fly of Pseudomonas putida isolated from the foregut of Bactrocera oleae*

Patrizia Sacchetti, Serena Landini, Aurelio Granchietti, Alessandra Camèra, Marzia Cristiana Rosi, Antonio Belcari Department of Agricultural Biotechnology, Section of General and Applied Entomology, University of Florence, Italy

Abstract: On the basis of laboratory bioassays, in 2003 and 2004 field trials aimed at evaluating the attractiveness of a bacterial filtrate to olive fly adults were performed in olive orchards in the Tuscan countryside. Pseudomonas putida isolated from the foregut of the olive fly was cultured in a liquid medium (Tryptic Soy Broth, TSB) in order to prepare a bait for testing. Elkofon-type traps were baited with different protein compounds diluted in water. The nitrogen baits consisted of commercial hydrolyzed protein (Buminal®), filtrates of the bacterial cultures and the TSB medium. Despite the low population density of the olive fly recorded in 2003 and 2004, the results show that bacterial cues attracted B. oleae adults to a comparable degree with respect to the commercial bait. Males and females were both attracted by bacterial filtrates.

Keywords: olive fly, attractants, monitoring, field trials, bacteria.

Introduction

The role played by epiphytic bacteria as a source of nitrogen compounds for some fruit flies is well known, as is the presence of bacteria in the gut of some species belonging to the family Tephritidae. Moreover, investigations on tropical species of fruit flies have shown that these insects are attracted by odours produced by phylloplane bacteria (Drew and Lloyd, 1989). The ecological role of the symbiotic relationships in the behaviour of the flies can therefore be usefully investigated with a view to improving control strategies against these pests. Few studies have focused on the relationship between Bactrocera oleae (Rossi) and the epiphytic bacteria of olive trees. Some authors have suggested that the attraction of olive fly to the host plant could be explained by the production of volatiles by microbial epiphytic flora (Scarpati et al., 1996). Furthermore, bacteria from the olive tree phylloplane and the olive fly foregut have been isolated in Greece and more recently in Italy (Tsiropoulos, 1983; Belcari et al., 2003). A number of species have been pinpointed, including Pseudomonas putida, which has been isolated both from the gut of wild and laboratory-reared flies and from the olive tree phylloplane. For this reason, P. putida was chosen in order to evaluate its possible attractiveness towards B. oleae adults. On the basis of laboratory bioassays, we assumed that volatiles produced by bacteria may provide a key to improving the current monitoring and control techniques of the olive fly. In this paper results obtained in two years of monitoring carried out in Tuscan olive groves are shown and discussed.

* Research supported by MIUR (PRIN 2003)

37 38

Materials and methods

In order to compare the attractiveness of liquid baits, Elkofon traps (by Phytophyl, N.G. Stavrakis, Greece) were chosen. These traps consist of two parts: a mushroom-shaped plastic top (epithem) with an entrance area (35 mm diameter) riddled with small holes; and a glass jar, containing the bait, screwed to the top. Different protein compounds diluted in water were tested. The nitrogen baits consisted of commercial hydrolyzed protein (Buminal®), filtrates of the P. putida cultures and tryptic soy broth (TSB), the medium used for culturing P. putida.

Preparation of the bacterial filtrate P. putida isolated from the olive fly foregut was grown in Tryptic Soy Broth (TSB) liquid medium for 6 days in an orbital shaker (at the temperature of 30±5°C). Bacterial cultures were centrifuged (10,000 rpm for 15 minutes) and the resulting supernatans were filtered through 0.45µm filters (FP 30/0,45 CA-S) mounted on syringes. The final filtration was carried out in a laminar flow hood to completely remove bacterial cells. About 1.5 l of bacterial filtrate was prepared each time. The bacterial filtrate was divided into samples containing the dosage needed for the field bait and these were stored at -20°C up to the preparation of the bait.

Trap set In 2003 traps were positioned in two olive groves, both located in San Casciano, a hilly area about 20 km from Florence. In the first one (Castellina olive orchard), water, 1% Buminal® solution (the concentration recommended by the manufacturer), 1% bacterial filtrate solution and 1% TSB (by Difco) solution were compared. Six traps for each treatment were positioned in the plot (in a random position chosen by computer). In the second olive grove (Marchi), 5 treatments were compared in order to highlight possible differences in the attractiveness of the TSB brand and the concentration: 1% Buminal® solution; 1% and 4% bacterial filtrate solutions (growth in TSB Difco); 1% and 4% bacterial filtrate solutions (growth in TSB Oxoid). Three traps for each treatment were randomly positioned in the plot (15 traps in total). In 2004 only TSB produced by Oxoid was used as a culture medium. The traps were baited with the following 8 treatments: 5%, 10% and 20% bacterial filtrate solutions, 5%, 10% and 20% TSB solutions, 1% Buminal® solution and water. The traps were positioned in Girone, a hill facing east about 2 km from Florence. Forty traps were randomly positioned in the experimental plot (8 treatments per 5 replications). In both years the liquid baits were removed and substituted with new ones every week. The caught insects were identified and counted. B. oleae adults were examined in order to distinguish males, gravid females and females without eggs. Non-target insects were identified up to order level.

Statistical analysis In 2004 the average number of captured flies was analyzed using the non parametric test analysis of the Kruskall-Wallis variance test. Where necessary, multiple comparisons were performed using the Mann-Whitney U test (Zar, 1999).

Results and discussion

Due to the low population density recorded in 2003, a total of just 67 adults were caught in 14 weeks’ sampling in the Castellina olive orchard; most of them (46) were captured by the traps 39

baited with Buminal® solution. The TSB baited traps caught 15 adults and the bacterial filtrate baited traps just 6 olive flies. No flies were captured by water. In the Marchi olive orchard, 4% bacterial filtrate solution, cultured in TSB Oxoid, caught a higher number of olive fly adults (161) than the 4% filtrate cultured in TSB Difco (89) or the 1% bacterial solution cultured in TSB Oxoid solution (71). Commercial hydrolyzed protein was the best attraction, catching 306 adults. The 1% bacterial filtrate cultured in TSB Difco trapped just 33 adults. In Figure 1 the weekly number of B. oleae adults caught during the sampling period by the five treatments is shown. Olive fly populations were very scarce up to the end of September and there were no relevant differences between treatments. In October the population density of the olive fly suddenly increased, peaking on the 15th. At this date Buminal® showed the highest captures (140 adults overall) followed by 4% Oxoid bacterial filtrate (94 adults) and 1% Oxoid bacterial filtrate (49 adults). The Difco bacterial filtrate yielded the lowest captures.

140

120

100

80 n. 60

0 1-Oct 8-Oct 3-Sep 15-Oct 22-Oct 29-Oct 10-Sep 17-Sep 24-Sep 13-Aug 20-Aug 27-Aug

1% Buminal® 1% BF Oxoid 4% BF (Oxoid) 1% BF (Difco) 4% BF (Difco)

Figure 1. Total number of B. oleae adults caught in the Marchi olive grove (2003)

A similar capture pattern was observed when examining the number of gravid females only. As with the overall adults, the highest number of gravid females as of 15 October was caught by Buminal® (85 females), followed by 4% Oxoid bacterial filtrate (64 females). In consideration of the good performance obtained in 2003 by the 4% bacterial filtrate solution cultured in Oxoid TSB medium, in 2004 the research focused on evaluating the possible effects of concentration on attractiveness. Once again, the very low population density – even lower than 2003 – caused very few captures. Only data concerning overall adults and females are shown since no statistical differences were evidenced in the number of males captured by different treatments. As in the previous year traps baited with water did not catch any flies. In Figure 2 the results of statistical analysis comparing the average number of adults captured in different treatments is shown. Traps baited with 5% and 10% bacterial filtrate 40

solutions captured the highest number of olive fly adults. No statistical differences emerge with respect to Buminal® and 10% TSB traps. A difference is evident between flies caught by 10% and 20% bacterial filtrate. As regards the attractiveness for the females, traps baited with Buminal® caught a lower number of adults compared to bacterial filtrates at 5% and 10% concentration, without there being any statistical difference (Figure 3). It is worth pointing out that 20% bacterial filtrate solution did not catch females but there are no statistical differences with the TSB medium at the same concentration.

0.60 b

0.50 ab

0.40

0.30 abc abc

0.20 ac ac

mean number of adults per trap c 0.10

0.00 1% Buminal 5% TSB 10% TSB 20% TSB 5% BF 10% BF 20% BF

Figure 2. Mean number of adults of B. oleae caught in Girone olive grove (2004); Kruskal-Wallis test: H (6, N = 312)=13.51053 p = 0.0356).

0.35 b

0.30 b

0.25

0.20 ab

0.15 ab a 0.10 ac mean number of females per trap per females mean number of 0.05 c

0.00 1% Buminal 5% TSB 10% TSB 20% TSB 5% BF 10% BF 20% BF

Figure 3. Mean number of females of B. oleae caught in Girone olive grove (2004); Kruskal-Wallis test: H (6, N = 312) = 16.51476, p = 0.0112 41

As regards non-target insects, all the treatments caught a very low number of parasitoid wasps and lacewings, usually present in large numbers in olive orchards. In Figure 4 the average number of Diptera Brachycera, except B. oleae, trapped by different treatments is drawn. The capture values increase as the concentration rises, evidently due to the attractiveness played by nitrogen odour sources. It is worth underlining that, unlike the response observed for the other Brachycera, B. oleae captures are not affected by protein concentration. This fact could be related to the different food preferences of B. oleae which feeds on protein sources usually available on the olive tree surface, in contrast to the majority of Brachycera.

60.00 d

dg 50.00

40.00 c

30.00

ab 20.00 b bf e

10.00 mean number of Diptera Brachycera per trap

0.00 1% Buminal 5% TSB 10% TSB 20% TSB 5% BF 10% BF 20% BF

Figure 4. Mean number of Diptera Brachycera (except B. oleae) caught in Girone olive grove (2004); Kruskal-Wallis test: H (6, N = 312) 153.2545, p<0.0001

Despite the reduced presence of B. oleae recorded in 2003 and 2004, the field trials demonstrated that P. putida bacterial filtrate acts as an attractant toward olive fly adults. The 10% bacterial filtrate yielded the best capture, even though there was no statistical difference with respect to the common protein bait. By contrast, the 20% bacterial filtrate solution seems to be unattractive to B. oleae females. On the basis of these initial field results, it can be affirmed that B. oleae showed a good response to the volatiles produced by P. putida, as has also been proven in laboratory bioassays (Landini et al., 2007). Finally, even though no strong differences appear, the results indicate that the odours produced by the bacterium may have a better specificity than the more generic odours emitted by other protein sources. This seems to open up new possibilities for improving monitoring techniques and control methods.

References

Belcari, A., Sacchetti, P., Marchi, G. & Surico, G. 2003: La mosca delle olive e la simbiosi batterica. – Informatore Fitopatologico 9: 55-59. 42

Drew, R.A.I. & Lloyd, A.C. 1989: Bacteria associated with fruit flies and their host plants. – In: Robinson, A.S. and Hooper, G. (eds.): Fruit flies, their biology, natural enemies and control. World Crop Pest, Elsevier, Amsterdam, 3A: 131-140. Landini, S., Granchietti, A., Librandi, M., Camèra, A., Rosi, M.C., Sacchetti, P., Belcari, A. 2007: Behavioural responses of olive fly, Bactrocera oleae, to chemicals produced by Pseudomonas putida in laboratory bioassays. – IOBC/wprs Bull. 30(9): 101-105. Scarpati, M.L., Lo Scalzo, R., Vita, G. & Gambacorta, A. 1996: Chemiotropic behavior of female olive fly (Bactrocera oleae Gmel.) on Olea europaea. L. – Journal of Chemical Ecology 22(5): 1027-1036. Tsiropoulos, G.J. 1983: Microflora associated with wild and laboratory reared adult olive fruit flies, Dacus oleae (Gmel.). – Zeit. Angew. Entomol. 96 (4): 337-340. Zar, J.H. 1999: Biostatistical analysis. Fourth edition. – Prentice Hall International Inc., New Jersey, pp. 663. Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 43-46

Preliminary notes on the gall midges (Diptera: Cecidomyiidae) associated with the olive fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae)

Raffaele Sasso, Gennaro Viggiani Dipartimento di Entomologia e Zoologia agraria “Filippo Silvestri”, Università degli Studi di Napoli “Federico II”, Via Università, 100 – 80055 Portici (Italia)

Abstract: A morphological and biological study of the gall midges associated with olive fruits infested by Bactrocera oleae (Gmelin) in Central and South Italy started in 1999. Beside Lasioptera berlesiana Paoli, widespread in the Mediterranean countries, other species were detected. The most common gall midge has been tentatively identified Clinodiplosis sp. The female deposits more eggs, commonly 6-8, mostly near, externally or internally, the emerging hole of the mature larva of B. oleae. The larvae develop in general gregariously in the olive fly tunnels as saprophagous species. An interesting lasiopterid, near the genus Lasioptera and apparently undescribed, is rather common. This species, which is also gregarious, appears strictly mycophagous. Another species, belonging to the Oligotrophidi, is under study. Finally, Asynapta furcifer Barnes has been obtained only from olives collected in Sicily. Several behavioral and phenological aspects of L. berlesiana were investigated. The observations carried out showed that the gall midge reproduces commonly on lentisk (Pistacia lentiscus L.) associated with leaf galls produced by Aceria stefanii (Nalepa), feeding on undetermined fungi, from end May to September, with a maximum of reproduction in July. Starting from the latter month, L. berlesiana is the only gall midge which reproduces on olive fruits of early cultivars, where the olive fly makes the first ovipositing wounds. In this narrow microhabitat, mostly occupied by the egg or by the very young larva of B. oleae, L. berlesiana oviposits, probably attracted by fungus or host plant tissue semiochemicals. In this peculiar situation the young gall midge larva can not avoid to prey the egg or the young larva inhabiting the same niche, and then continues her development, feeding on the invading fungi (commonly Camarosporium dalmaticum) and probably also on the decaying plant tissues. Subsequently during summer and fall the gall midge can oviposit in any other opening on the olive fruits. In conclusion, L. berlesiana, as the other gall midges associated with olive fruits infested by B. oleae, appears normally a mycophagous and/or a saprophagous species. The activity of the gall midges associated with the olive fruits may interfere with the oil quality.

Key words: Lasioptera, Clinodiplosis, Lasiopteridi, Oligotrophidi, Camarosporium

Introduction

The fly, Bactrocera oleae (Gmelin), causes scars for oviposition, tunnels for the larval development and exit holes in olive fruits. These openings allow other insects, as the gall midges, to profit to start they reproductions. Several insects of this group have been recorded from olive fruits in association with the olive fly (Barnes, 1932; Coutin & Katlabi, 1986), but almost all still remain poorly known. Some biological data are only known for Lasioptera berlesiana Paoli, widespread in the Mediterranean countries. This species is in fact associated both with the olive fly and with the fungus Camarosporium dalmaticum (Thüm.) Zachos at Tzavella-Klonari. The latter association is not well known; some aspect concerning the fungus transmission are controversial, as the feeding behaviour of the larvae.

43 44

In this preliminary paper information is given on the complex of gall midges associated with the olive fly fruits in Central and South Italy, in particular on their morphological and biological characterization.

Material and methods

Since 1999, olive fruits apparently infested by the olive fly were picked in some areas, mainly in Campania, but also in Tuscany, Sardinia, Calabria and Sicily. They were examined in the laboratory, dissecting the infested parts and recording the type and the stages of the gall midges found. Notes were taken also on the associated fungi. The larvae were partially preserved in alcohol 70% and others were not removed from the fruits, kept in vials or in small bottle, in order to obtain adults. For their study, larvae and adults were mounted on slides adopting specific methods. The associated with the the olive fly and the gall midges were collected and preserved for further identification.

Results and discussion

The gall midge complex The gall midge complex has been represented by Lasioptera berlesiana Paoli, Lasiopterid indet., Clinodiplosis sp., Oligotrophid indet. and Asynapta furcifer Barnes. The presence of L. berlesiana has been the first to be recorded starting from July-early August when on the fruits mostly oviposition scars are present. Later on, particularly in September, the dominant species has been Clinodiplosis sp. inhabiting only the olive fly larval galleries. The same behaviour seems to have A. furcifer. On the other side, L. berlesiana, Lasiopterid indet. and Oligotrophid indet. may be associated to both oviposition scars and larval tunnels.

Lasioptera berlesiana This species belongs to the Lasiopteridae, which are characterized by joint Vein R5 and R1, reaching the costal vein before the distal apex of the wing. Several species (L. kiefferiana Del Guercio, 1910; L. carpophila Del Guercio, 1918; L. brevicornis Melis, 1925) have been synony- mized under L. berlesiana, based on some intraspecific variations, concerning body colour and antennal segments. This gall midge shows antennae with antennomeres variable in number from 11 to 21. The annual reproductive activity of L. berlesiana on olive fruits starts in July on early cultivars as soon as they are suitable for the olive fly oviposition. In this narrow microhabitat, mostly occupied by the egg or by the very young larva of B. oleae, L. berlesiana oviposits, probably attracted by fungus or host plant tissue semiochemicals. In this peculiar situation the young gall midge larva can not avoid, at least in same cases, to prey the egg or the young larva inhabiting the same niche, and then continues her development, feeding on the invading fungi (commonly Camarosporium dalmaticum) and probably also on the decaying plant tissues. Subsequently during summer and fall the gall midge can oviposit in any other scar, hole or tunnel, present on the olive fruits, where fungi and/or decaying material is present. The adult gall midges very probably reach the olive tree from lentisk (Pistacia lentiscus L.), where it is associated with leaf galls caused by Aceria stefanii (Nalepa) and reproduces from end May to September, with a maximum in July feeding on undetermined fungi (Sasso & Viggiani, 2002). Biological notes on L. berlesiana have been provided mainly by Silvestri (1945, 1947, 1949), Solinas (1967), Coutin & Katlabi (1986) and Hepdurgun & Onder (1999).

Laspiopterid indet At first glance this rather small Lasiopterid appears rather similar to the species included in the genus Lasioptera, but it can be easily distinguishable mainly by the abdominal features lacking of the typical spoon structures and some male genitalia characters. At present the species can not placed in any of the known lasiopterid genera. The white, gregarious larvae, can be found associated with a white fungus in course of identification, both in the oviposition scars and in the larval galleries of B. oleae. This species, as L. berlesiana seems mostly active in July-early August.

Clinodiplosis sp. According to a preliminary identification of Dr. M. Skuhrava (pers. com.) this species belongs to the genus Dichodiplosis Rübsaamen and is very similar to D. langeni Rübsaamen, mycophagus on mummified fruits of Prunus spinosa. As other members of the Cecidomyiidi Clinodiplosini the wings show the vein R5 meeting with the costal vein nearly or beyond the apex of the wing. The orange full larva of this species has been by some authors confused with that of L. berlesiana (Coutin & Katlabi, 1986; Longo et al., 2004), which does not show 2 robust spines at the abdomen end. The female deposits more eggs, commonly 6-8, mostly near, externally or internally, the out hole of the mature larva of B. oleae. The larvae develop gregariously in the olive fly tunnels as saprophagous species.

Oligotrophid indet The wing of this species is similar to that of Clinodiplosis sp., but the male flagellomeres are not binodose. Other important characters for discrimination are found in the male genitalia. This gall midge reproduces as a solitary species. The whitish larvae are associated with fungi.

Asynapta furcifer Kieffer This species, recorded from Cyprus, Sicily and Palestine (Coutin & Katlabi, 1986), in our survey has been obtained only from infested olive fruits collected in Sicily, near Palermo. The orange larvae of this species are rather longer than those of L. berlesiana and Clinodiplosis sp., easily distinguishable for several morphological characters. They appear to be mycophagous and /or saprophagous. The fungi collected in the scars and tunnel produced by the olive fly are at present, as the associated gall midges, only partially identified. They belong to the genera Alternaria, Camarosporium and Cladosporium. According to this preliminary observations L. berlesiana does not seem associated only with Camarosporium dalmaticum, but also with other fungi of which their role is under study. Among the gall midges associated with the olive fruit infested by B. oleae the role of L. berlesiana still remains controversial. Originally this species was considered a pest (Paoli, 1907). Subsequently Koronéos (1939) observed and more precisely Silvestri (1945, 1949) observed that the species mostly during end June-July, beginning of August, when on the green olive fruits are present only oviposition scars of B. oleae, the gall midge may become in this very narrow microhabitat, occasionally predator of eggs and very young larvae of the olive fly. For this behaviour, Silvestri (1949) considered P. berlesiana a “friend” of the olive grower, even if the association of the gall midge with the fungus C. dalmatica causes severe fruit dropping. The same author supposed the transmission of the mentioned fungus by the female (infected eggs) or the larvae of the gall midge. This assumptions were subsequently supported by Solinas (1967), but in fact the obligate association L. berlesiana - C. dalmaticum was never demonstrated. Our observations confirm the view that C. dalmaticum and other fungi which cause “Marciumi” may invade the olive fruits through scars and galleries produced by B. oleae 46

(Gigante, 1934; Harpaz and Gerson, 1966), but not as the result of a kind of symbiosis between L. berlesiana- C. dalmatica. More probably, the gall midge, as other species, is attracted by fungus or plant tissue semiochemicals. The supposed zoophagy of L. berlesiana appears only accidental and without practical importance in the natural control of the olive fly. The complex of the gall midges associated with the olive fruits infested by B. oleae can increase deleterious biochemical reactions for the oil quality.

Acknowledgements

We thank Prof. Giovanni Mineo, University of Palermo and Prof. Alfio Raspi, University of Pisa, for their help in collecting material.

References

Barnes, H. F. 1933: Gall midges (Cecidomyidae) as enemies of mites. – Bull. Ent. Res. 24: 215-228. Coutin, R. & Katlabi, H. 1986: Cecidomyiidae. – In: Entomologie agricole, Y. Arambourg (Dir.): 95-113. Gigante, R.1934: Ricerche sulla morfologia, la biologia e la posizione sistematica del fungo che è stato descritto come “Macrophoma dalmatica”. – Boll. Staz. Pat. Veg. Roma 14: 125-171. Harpaz, I & Gerson, U. 1966: “Biocomplex” of the olive fruit fly (Dacus oleae Gmel.), the olive fruit midge (Prolasioptera berlesiana Paoli), and the fungus Macrophoma dalmatica Berl. & Vogl. – In: Olive fruits in the mediterranean Basin. Scripta Hierosolymitana 18: 81-26. Hepdurgun, B. & Onder, F. 1999: Investigations on the biology of the olive gall midges (Lasioptera berlesiana Paoli) (Diptera: Cecidomyiidae). – Türkiye Entomol. Dergisi. 23 (3): 191-202. Longo, O., Cavallo, C., D'Agnano, G., Schiamone, D. & Porcelli, F. 2004: Inusuale cascola di olive per azione combinata di tre parassiti. – L'informatore Agrario 60 (22): 57 Sasso, R. & Viggiani, G. 2002: Cecidomidi associati a foglie di lentisco (Pistacia lentiscus L.) infestate da Aceria stefanii (Nalepa) (Acarina: Eriophyoidea). – Boll. Lab. Ent. Agr. Filippo Silvestri. 57: 55-65. Silvestri, F. 1945: Contribution à la biologie de la petite Cécidomyie des olives (Prolasioptera berlesiana Paoli) en Italie. – Moniteur International de la Protection des Plantes 19: 73- 76. Silvestri, F. 1947: Nuove notizie sulla Cecidomia delle olive (Prolasioptera berlesiana Paoli). – Rendiconti Acc. Naz. Lincei 8 (2): 750-752. Silvestri, F. 1949: Un piccolo insetto amico degli olivicoltori. – Olearia 1: 3-10. Solinas, M. 1967: Osservazioni biologiche condote in Puglia sulla Prolasioptera berlesiana Paoli, con particolare riferimento ai rapporti simbiotici col Dacus oleae Gmel. e con la Spheropsis dalamatica (Thüm.) Gigante. – Entomologica 3: 129-176.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 47

Augmentative releases of Eupelmus urozonus Dalm. against the olive fruit fly and observations on its facultative hyperparasitism

G. Delrio, A. Lentini, A. Satta Dipartimento di Protezione delle Piante, Sezione di Entomologia agraria, Universitá degli Studi di Sassari, Via E. De Nicola, 07100 Sassari, Italy.

Experiments on biological control of the olive fruit fly (Bactrocera oleae) with augmentative releases of Eupelmus urozonus (lab-reared on pupae of the factitious host Ceratitis capitata) were carried out in two olive groves in Sardinia. In a grove of one hundred olive oil trees, manifesting low production and high infestation of olive fruit fly, 100 mated females of E. urozonus were released in August 1993. The olive fly parasitism, estimated by dissecting samples of infested fruits, showed a high activity of Pnigalio agraules in the first weeks of August (max 20% of parasitism), whereas E. urozonus prevailed later reaching 60% of parasitism in September. Despite the high rate of parasitism, at harvest all the olives were infested. In 1994, in another olive grove of 200 trees, showing high production and low initial infestation, a total of 3480 E. urozonus mated females were released weekly during September and October. The rate of P. agraules parasitism was very low in September and increased to a maximum of 10.5% in November, whereas E. urozonus was found sporadically. In these experiments E. urozonus behaved mainly as a hyperparasitoid of P. agraules, as an autoparasitoid of its own species, and only occasionally as a primary parasitoid of B. oleae pupae. Given the negative results on the biological control, observations were carried out in various olive groves between 1995-2004, in order to verify the parasitism behaviour of E. urozonus. In this case, only the data on the parasitoid ovipositions found in olives was taken into account. The eggs were found deposited in galleries of B. oleae containing preimaginal stages of P. agraules, E. urozonus and Eurytoma martellii (59%), recently dead (4%) and rotten (13%) larvae of B. oleae, live larvae of B. oleae (4%), and in B. oleae pupae (19%). These observations suggest that E. urozonus, in the olive agroecosystem, acts essentially as a hyperparasitoid and partly as a primary parasitoid of olive fly pupae. However, further research must be conducted to verify the primary parasitism on B. oleae larvae.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 49-60

On the use of the exotic oo-pupal parasitoid Fopius arisanus for the biological control of Bactrocera oleae in Italy

Riccardo Moretti, Elena Lampazzi, Placido Reina, Maurizio Calvitti C.R. ENEA Casaccia, UTS Biotec Agro, Via Anguillarese 301, 00060 Roma, Italy. E-mail: [email protected]

Abstract: With the aim to broaden the natural enemies complex available for the biological control of key tephritid fruit flies (Diptera) of the Mediterranean Basin, the South-East Asiatic oo-pupal parasitoid Fopius arisanus (Sonan) (Hymenoptera: Braconidae) was imported in Italy in 1999. Laboratory studies allowed to develop effective low cost rearing techniques of this parasitoid on the secondary host Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) using artificial devices. Moreover, we ascertained the suitability of a new host to the parasitization by F. arisanus: the olive fruit fly Bactrocera oleae (Gmelin) (Diptera: Tephritidae), key pest of olive groves in all the Mediterranean region. Preliminary field cage tests were carried out in 2000 to evaluate the survival and the parasitization ability in a typical olive area of central Italy. Results evidenced better performances of the parasitoid during autumn, rather than in summer. The parasitoid showed the shortest longevity in early summer (in average, 2d in July August and 8-10d in the period September- November). In 2001, and 2004 investigations were carried out in Central Italy to verify the capability of F. arisanus to mate and increase its population in open field. In 2001, about 10000 F. arisanus specimens were released in a 1ha olive grove in July, while 2000 specimens were released in a 0.5ha olive area in September 2004. In 2004, quality control tests have been also performed to ascertain the preservation of the performances in parasitizing B. oleae in field of the laboratory reared F. arisanus population. Finally, results from intrinsic competition with the autochthonous parasitoid E. urozonus were also evaluated to test the effects from the release of the exotic parasitoid on local B. oleae antagonists. The periodical recovery of olives during field tests allowed us to verify that the parasitoid was able to reproduce and gradually increase its population in field: in particular, we estimated parasitoid population to increase in field from 51.7 to 96.3 emerged specimens per 1000 olives in average from late September to late October 2004. Percent parasitism increased as well: the average level was in fact about 20% in September and 25% in October, with highest daily levels of 68 and 71% respectively. F. arisanus also maintained a sex ratio not significantly different from that obtained in laboratory (F/M=0,85). Quality control tests evidenced that the rearing conditions did not seem to affect F. arisanus ability in searching and parasitizing B. oleae in field. Furthermore, E. urozonus was consistently superior than F. arisanus in intrinsic competition (only 4% of the multiparasitized hosts emerged as the latter). Both field cage and open field tests have pointed out that hot-dry climatic conditions limit the activity of F. arisanus, while under higher humidity levels, normally recorded from early September, the parasitoid show a remarkable efficiency in parasitizing B. oleae being also able to establish n field until the end of the olive cropping season.

Key words: Bactrocera oleae, Fopius arisanus, biological control

Introduction

A research program targeting B. oleae aimed at evaluating the possibility that the exotic oo- pupal parasitoid F. arisanus (Hymenoptera: Braconidae) could represent an added value in biological control of this pest in the Mediterranean basin, both under economical and eco-

49 50

logical point of views. In particular, our studies on F. arisanus necessarily passed through a series of fundamental steps: 1) the study of its capability to recognize olives as a suitable substrate for host searching and parasitizing; 2) the assessment of host suitability for its development up to the adult stage; 3) the set up of an efficient and low cost mass rearing method; 4) the evaluation of the capability by unexperienced females to find and parasitize hosts in an olive grove and to survive under Mediterranean climate conditions; 5) quality control tests to ascertain its ability to parasitize hosts and mating in field after pluriennal rearing cycles under laboratory conditions; 6) the study of competitive interactions with autochthonous parasitoids of B. oleae; 7) the assessment of the ability to mate and reproduce in open field allowing for a seasonal establishment; 8) the experimentation of field employment strategies in order to enhance its potential in controlling B. oleae by inundative or seasonal inoculative releases (Van Lenteren & Bueno, 2003) and choice of the best environmental conditions for performing releases. Preliminary studies demonstrated the ability by F. arisanus to find and parasitize B. oleae eggs on olives and also showed the full suitability of this host for its postembrional development (Calvitti et al., 2002). However, 10-20% of the parasitized host eggs are usually killed by the parasitoid first instar larva, with no developing of any insects (Moretti et al., 2004). Contemporarily, a low cost rearing method on artificial devices was developed on the host Ceratitis capitata (Wiedemann) (Calvitti et al., 2002; Moretti and Calvitti, 2003). Field cage experiments carried out in 2000 and 2001 allowed to ascertain parasitoid survival and its capability to find and parasitize B. oleae in a confined experimental system under field climatic conditions (Antonelli et al., 2004). Tests evidenced that hot and dry conditions, quite common in early Mediterranean summer, could limit parasitoid survival while, F. arisanus exerted excellent parasitization activity on B. oleae from late summer to autumn reaching 80- 85% parasitism among insects emerging from the recovered olives. In the present work we report results from steps 5, 6 and 7 of our research program. These steps concern quality control assessment of the reared F. arisanus population, evaluation of competitive interactions with other beneficial organisms, in specific E. urozonus, and open field experiments testing its efficiency in parasitizing B. oleae and reproducing in a Mediterranean olive grove. Several laboratory investigations proved that F. arisanus win intrinsic competitions against most of the known opiinae species (Hymenoptera: Braconidae) (Bautista & Harris, 1997b), including even P. concolor (Wang & Messing, 2002; Wang et al., 2003), while information was wanting on competition displacement with larval- pupal parasitoids belonging to other Hymenoptera families. Consequently, we studied intrinsic competition between F. arisanus and E. urozonus (as a representative of B. oleae ectoparasitoids)

Materials and methods

Parasitoids The initial F. arisanus stock was provided from the U.S. Pacific Basin Agricultural Research Center, USDA-ARS (Hawaii, USA) in 1999 and from then on reared at our laboratories on the host C. capitata by using an artificial device resembling a fruit for host eggs parasitization (Calvitti et al., 2001; Moretti & Calvitti, 2003). Development up to the adult stage was then allowed by using a carrot based rearing medium. E. urozonus was obtained from olives recovered in central Italy in 2001. The identification of the collected specimens was kindly confirmed by G.A.P. Gibson (Canadian National Collection of Insects (CNC), Agriculture and Agri-Food Canada). Rearing of E. urozonus was even carried out at our laboratories on C. capitata by exposing host puparia 51

(Arambourg, 1964). In particular, hosts fixed to a cardboard slide using a non toxic paper glue were exposed to mated E. urozonus females 10-20d old, at a 5:1 ratio between hosts and parasitoids. Expositions lasted 24h and after that, the cardboard slide was recovered by removing parasitoids from its surface using an aspirator. Puparia were then maintained as previously described for F. arisanus until E. urozonus adults emergence, taking place about 30 d after the hosts escaping parasitization.

Quality control assessment of the reared F. arisanus population In order to evaluate eventual loss in quality of the reared F. arisanus population, a field cage test was carried out in 2004 under the same experimental conditions of tests performed in 2000 (unpubl. data). With this aim, before olive fruits ripening, three small size olive trees were individually isolated, by cages constituted by a cylindrical metallic structure, 3m high, with a circular 3m in diameter basis, covered with a fine mesh (0.5mm) net. In late September, once verified the absence of B. oleae infestation, 25 mated olive fly females were released inside each cage. Immediately after, 25 mated F. arisanus females were also released in each cage. A further analogous release of hosts and parasitoids was performed after a week. Two weeks after the first release, 1000 olives per cage were collected and then maintained in laboratory at 24±0.5°C and 50±4% R.H. until insect adults emergence. Host density was evaluated by the count of emerging olive flies and parasitoids from the recovered olives. Results in terms of percent parasitism by F. arisanus were compared with the ones obtained in 2000.

Intrinsic competition between F. arisanus and E. urozonus Intrinsic competitive interaction between F. arisanus and E. urozonus was investigated by exposing to the latter parasitoid C. capitata pupae developed from host eggs parasitized by the first one. Observations on the parasitization status were allowed by the dechorionation of C. capitata eggs after the exposure to F. arisanus (Moretti & Calvitti 2003). Parasitized hosts were collected and reared until pupation. Pupae were then presented to E. urozonus in a vertical position, through the insertion of the puparium into holes made on a plastiline basis, so that only a posterior one half of each puparium was accessible to parasitization. Sets of 10 puparia were each one exposed to a 10d old mated E. urozonus female and the parasitizations were directly observed. Immediately after the parasitization, each puparium was recovered and maintained until adult’s emergence. As control, puparia parasitized only by F. arisanus or E. urozonus were kept in similar conditions to evaluate mean percent conversion of parasitized hosts in parasitoid adults.

Open field experiments (2001-2004) Open field experiments had the purpose to evaluate the ability of F. arisanus to parasitize B. oleae, mate and seasonally establish, under typical climatic conditions of Mediterranean olive groves. Tests were performed in two sites all in central Italy. Site A was a 1ha orchard located next to Cerveteri (province of Rome) in an area where several olive groves are present and it was constituted by 120 olive trees about 30yr old arranged in 12 rows of 8 trees each. Olive grove of site B, 0.5ha large and constituted by 50 olive trees about 20yr old arranged in 10 rows of 5 trees each, was located at C.R. ENEA Casaccia (Rome) and was quite isolated from other olive orchards. Finally, site C was a 0.5ha olive grove quite similar to and about 1km far from the site B.

With the purpose to evaluate the field climatic conditions during the tests, a climatic data recorder (Campbell – mod. CR 10) was located in the experimental plots. Data on maximal, minimal and average temperature, relative humidity and rainfalls were recorded. 2001. Tests carried out in 2001 aimed at evaluating the capability of F. arisanus to parasitize B. oleae in open field under hot and dry climatic conditions, which, as already proved by field cage experiments, are detrimental to its survival. Experiments took place in site A. Infestation by B.oleae was monitored by recovering olive samples and searching for olive fly oviposition punctures. Once recorded the first evidence of pest ovipositions, 5 releases of F. arisanus were thus weekly performed. The first release took place on 6th July and the last on 2nd August. Each release was constituted by 2000 F. arisanus adults distributed in 20 test tubes (50ml Falcon), randomly placed in the orchard, each one containing about 50 males and 50 females 5±1d old. Totally, about 10000 specimens were released. Starting from 10th July, samples of 1000 olives were weekly recovered picking up 50 fruits at random from 20 trees. Percent olives presenting one or more oviposition puncture by B. oleae were counted to evaluate host density during the experiment. Olives were maintained inside cages at 24±1°C, 60±10% RH, until insect adults emergence. Every three days, B. oleae, F. arisanus and others native parasitoids were recorded and removed from the cages. The fifth and last olive fruit sample was recovered on 8th August. Consequently, results took into consideration emerging insects from a total amount of 5000 fruits. 2004. Performances of F. arisanus against B. oleae were tested in 2004, under favourable open field climatic conditions (September-October), as already found by our previous studies. Experiments were carried out in Site B and Site C. Infestation by B. oleae was monitored by collecting olive samples and counting percent oviposited olives. F. arisanus was then introduced in site B, as parasitized C. capitata puparia, to specifically test its capability to mate in open field and reproduce on B. oleae. With this purpose, Release Units (R.U.) were prepared in laboratory as follows. A group of medfly puparia (9±2d old) developed from eggs exposed to F. arisanus was sieved to obtain a small dimension size class (2mm maximum width) corresponding, in major part, to parasitized puparia (Ramadan et al., 1994). Small size puparia were then portioned into lots, each constituted by 2g of pupae which approximately correspond to 250 F. arisanus specimens (unpubl. data). Each lot was thus held in Release Unit (R.U.) constituted by a plastic cup (about 40c.c.) with a mesh-screen cover. The mesh size (1mm2) allowed for the passing through by F. arisanus adults but prevented the escape of medflies. R.U. were also protected by a plastic roof (10x10cm) against rain and other agents. As control, two R.U. were maintained in laboratory for assessing percent emergence and sex ratio of F. arisanus. Four R.U. were placed on one of the main branches of olive trees located randomly in Site B olive grove. Consequently, about 1000 parasitoids were used in a single release. Two releases were carried out, on 3rd and 10th September. At each parasitoid release, host density in the experimental olive grove was evaluated by dissecting about 200 olives and counting percent fruits infested by B. oleae eggs or first instar larvae. Starting from 16th September, samples of about 5000 olives were periodically collected from the site (50 fruits per 10 randomly chosen trees). Seven samples were collected every 5d, so that about 35.000 olives were totally retrieved. Each sample was placed inside a cage and kept in laboratory at 22±1°C and 60±10% RH. Emerged B. oleae and F. arisanus were daily recorded and removed from the cages. As control, olive samples were also recovered, following the same methodology, from site C, in order to have comparable data on B. oleae emergence without any parasitoids release. 53

Study of the insect population dynamics Data from the tests carried out in the two years were furnished by the periodical identification and registration of the insects emerged from the recovered olives. Data obtained from each cage allowed to have information on insect population dynamics within a limited time range. In fact, B. oleae and F. arisanus activity was stopped on the day of olive fruits recovery. Usually, after about 35d from the day of olive samples recovery, adult insect emergences had already ended. However, we could get information on a broader period of the cropping season by periodically sampling olives. Results were expressed as mean number of emerged insect adults per 1000 olives and used to draw curves describing host-parasitoid population dynamics. The graphics could not represent actual field population dynamics because olive samples have been carried in laboratory to facilitate insect recovery. Consequently, the time needed by insects to reach adult stage could be artificially shortened, especially from October on, when mean temperatures kept in laboratory were higher than in field. However, the obtained data can certainly represent the parasitization activity by the parasitoids on B. oleae and allow for an estimation of the pest individuals subtracted to the field population. Percent parasitism and sex ratio by F. arisanus were also calculated. At last, an estimation of the total amount of olive fruits in the sites was also carried out with the purpose to allow for the approximate calculation of the actual parasitoid number produced in the plot.

Statistical Analysis Statistical analysis were performed using SPSS software (SPSS; 2000). Descriptive statistics, mean and standard deviation (S.D.) were generated. Where applicable, one-Way Analysis of Variance was performed. Percentage data were transformed using arcsine-square root method before statistical analysis, but results presented are non transformed means.

100

73.38b 69.65b 80

40.40a 32.41a 40

0 2000 2004

% olives infested by B. oleae % parasitism by F. arisanus

Figure 1. Quality control assessment of the laboratory reared F. arisanus population: Comparison between percent parasitism of B. oleae obtained in 2000 and 2004 under the same field cage experimental conditions.

Results Quality control assessment Data on infestation of B. oleae inside field cages was not significantly different between 2000 and 2004 (Figure 1). Percent parasitism on B. oleae by F. arisanus did not significantly decrease from 2000 to 2004 tests (F = 0.126; df = 4; p = 0.74). Mean temperatures and 54

relative humidity of September 2000 (T: 28.21±2.97 max, 16.17±1.36 min; RH: 90.02±9.47 max, 39.53±15.03 min) and 2004 (T: 27.49±3.48 max, 17.55±1.12 min; RH: 88.44±10.02 max, 41.52±12.56 min) were just slightly different. Results clearly indicated that the parasitoid maintained in both the years the same performances in parasitizing B. oleae under field conditions, despite of pluriannual rearing cycles in laboratory by means of artificial devices for host exposure.

Intrinsic competition tests The comparison of multiparasitization and alone treatments allowed us to ascertain that E. urozonus clearly prevailed in intrinsic competition tests. In fact, hosts parasitized by F. arisanus and exposed to the autochthonous ectoparasitoid E. urozonus just rarely gave F. arisanus adults (about 4 %) while more than 60% of the exposed hosts developed as E. urozonus adults (Table 1). The remaining individuals did not survive beyond pupal stage.

Table 1. Development of E. urozonus on C. capitata pupae parasitized by F. arisanus: percent emerging F. arisanus and E. urozonus from multiparasitization and alone treatments.

% E. urozonus % F. arisanus N adults adults

Hosts parasitized by F. arisanus 50 72.80b

Host parasitized by E. urozonus 50 59.68a

Hosts parasitized by F. arisanus and 50 61.24a 4.00c then exposed to E. urozonus

Open field experiments

Climatic data relative to the field experiments of 2001 and 2004 are reported in Table 2.

Table 2. Data on temperature (mean, mean of maximal and minimal daily °C values), relative humidity (mean, mean of the maximal and minimal daily % values) and rainfall (mm) per month, during field tests in 2001 (site A) and 2004 (site B).

Year Month T Max T Min T RH Max RH Min RH Rainfall

July 25.0±1.9 31.2±2.8 19.4±1.9 44.7±18.6 58.0±8.7 30.0±3.8 0 2001 August 26.1±2.0 32.6±3.0 19.2±2.2 77.4±10.7 88.2±7.0 39.9±9.8 6.14

September 22.6±2.7 27.5±3.5 17.6±1.1 69.2±9.1 88.4±10.0 41.5±12.6 24.32 2004 October 18.9±4.2 23.8±3.1 16.2±1.4 76.8±10.4 91.4±9.1 39.4±14.8 100.75

2001. The count of the punctured olives allowed us to measure a percent infestation by B. oleae increasing from about 15 to 54% during July 2001. Results from insect emergences evidenced that F. arisanus successfully parasitized B. oleae (Figure 2). The emergence of the first parasitoid individual was registered on 11th August. Every three days, in average about 5.4 parasitoids and 49.4 olive flies emerged per 1000 olives from 13th to 25th August. In total, 27 F. arisanus and 245 B. oleae adults per 1000 olives were registered within the same period. Data on emergence of B. oleae adults confirmed a high level of olive fruits infestation by the pest. By estimating the total amount of olives present in the orchard as about 300-350 thousands we calculated that approximatively 8100-9450 parasitoids could have emerged in field starting from the 10000 released. Percent parasitism averaged about 10% and peaked at 14.29% of the emerged insects on 22nd August.

80 100%

90% 70 80% 60 s 70% 50 60% F. arisanu 40 50%

40% 30 30% 20 20% parasitism by Mean individuals / olives1000 10 10%

0 0% 10/8 13/8 16/8 19/8 22/8 25/8 date

F. arisanus B. oleae % parasitism by F. arisanus

Figure 2. Reproduction of F. arisanus on B. oleae in early summer: F. arisanus and B. oleae adults emerged every three days and percent parasitism by the parasitoid obtained from olive samples collected during July-August 2001 open field tests.

2004. Host density ranged 7 to 12% when F. arisanus releases had been carried out. Parasitoid emergence started in laboratory from 25th September, that is about 22d after the first parasitoid release (Figure 3). We ascertained the occurrence of two parasitoid emergence peaks at the end of September and at the beginning of November respectively. Between these peaks F. arisanus population decreased, reaching very low levels (next to zero individuals). This observation could therefore prove the subsistence of two field F. arisanus generations after the first release. About 5 parasitoids per 1000 olives daily emerged during the first generation peak, while second generation peaked at about 9 individuals per 1000 fruits. By estimating the total amount of olives present in the orchard as about 100.000 we calculated that approximatively 5172 and 9628 parasitoids could have emerged in field within F1 and F2 generations respectively. Moreover, considering that 2000 parasitoid had been initially released, results show the occurrence of a remarkable population increase in field. 56

Site B 2004: Releases of F. arisanus

100%

90% 30 s 80%

70%

60% 20 F. arisanus F. 50%

40%

10 30% 20% Mean individuals / 1000 olive / 1000 individuals Mean parasitism by parasitism 10%

0 0% 22/9 27/9 2/10 7/10 12/10 17/10 22/10 27/10 1/11 6/11 11/11 date F. arisanus B. oleae Percent Parasitism

Site C 2004: No parasitoid releases

10 Mean individuals / 1000 olives

0 22/9 27/9 2/10 7/10 12/10 17/10 22/10 27/10 1/11 6/11 11/11 16/11 date

B. oleae

Figure 3. Reproduction of F. arisanus on B. oleae in late summer-autumn. Above: daily emerged F. arisanus and B. oleae adults and percent parasitism by the parasitoid resulting from olive samples recovered during 2004 open field tests. Below: control treatment (no parasitoid releases).

In Figure 3, daily percent parasitism by F. arisanus among emerging insect adults is also shown. The maximal levels of parasitization were reached just a few days after the peaks in parasitoid adults emergence. In fact, contemporarily, the number of emerging B. oleae adults drastically decreased. Parasitism by F. arisanus on B. oleae averaged about 20% within the interval between first parasitoid adult and minimum in parasitoid individuals emergence, taking place about 25d later. Within this period percent parasitism reached 68% on 2nd October. Percent parasitism averaged about 34% during the emergence of the second field generation reaching the maximum on 4th November at about 71% of the emerged insects. Data shown in Figure 3 referring to the last 4-5d of insect emergences are merely indicative. In fact, due to the sampling procedure, ended on 13th October, the fate of host or parasitoid eggs oviposited after that date could not be shown. Consequently, the last emerging individuals just represent an artificial tail in insects population dynamics. 57

Results from site B are comparable with results from site C where parasitoid releases had not been performed. As shown in Figure 3, B. oleae population dynamic is quite different. Olive fly population increased during September-October reaching the maximum average level at the end of October (about 30-35 daily emerged adults). In average, a total amount of about 900 and 620 B. oleae adults emerged per 1000 recovered olives from site C and B respectively. Data on sex ratio among emerging F. arisanus adults clearly showed that the parasitoid was able to mate in field maintaining a sex ratio not significantly different from that usually obtained in laboratory and also confirmed by parasitoids emerging from R.U. kept in laboratory as control (Figure 4). In fact, 45.06% of the released F. arisanus were females, while females among field F1 and F2 adults were 43.18 and 45.95% respectively.

100%

45.06±2.2a 43.18±1.9a 45.95±2.0a

50%

54.94±2.2b 56.82±1.9b 54,05±2.0b

0% Released parasitoids Field F1 Field F2 % F. arisanus males % F. arisanus females

Figure 4. Sex ratio of F. arisanus after field release of pupae: percent males and females F. arisanus emerged from the olives recovered during September-October 2004 field test, compared with adults emerged in laboratory from a portion of the pupae used for the releases.

Discussion

For successful biological control programs, laboratory-reared parasitoids should maintain natural behavior traits, such as the efficient host searching and parasitization behavior, once released in field (Gandolfi et al., 2003; Bautista and Harris, 1997a). The predisposition shown by F. arisanus to be easily mass reared on C. capitata, as known very easily reared as well, and also the absence of loss of any biological performances after more then 5 years of laboratory rearing are fundamental conditions to be considered in order to use this parasitoid as an effective biological control agent. The use of an artificial device quite similar to a fruit for host exposure to parasitization helped possibly in preserving useful behavioral traits under laboratory rearing conditions. Moreover, our findings on the intrinsic competition against E. urozonus are of fundamental importance for the purposes of our research project. In fact, competitive risk and non target impacts by introduced species need to be carefully evaluated before field releases of an exotic parasitoid, to prevent undesirable side effects on local entomophauna. Results 58

from our tests definitely evidenced that host parasitization by F. arisanus does not influence developmental success of E. urozonus eventually attacking the same host. These results may be probably extended to other autochthonous B. oleae parasitoids, as they are mainly general ectoparasitoids (as E. urozonus) and therefore usually not susceptible to the mechanisms involved in intrinsic competition strategy shown by F. arisanus, such as egg hatching inhibition and/or direct killing of others immature parasitoids (Bautista and Harris, 1997b). Furthermore F. arisanus is known to not recognize flower head infesting tephritids as potential hosts (Wang et al., 2004), as indeed many other larval parasitoids belonging to Opiinae usually do (Duan and Messing, 2000). Consequently, effects to non target organisms by F. arisanus release are probably minimal or null, thanks to its high specialization in exclusively parasitizing tephritid eggs on fruits. Results from 2001 test put in evidence the difficulty in reproducing on B. oleae by F. arisanus under hot and dry climatic conditions typical of Mediterranean basin early summer (Antonelli et al., 2004). Actually, survival of parasitoids native of tropical and subtropical areas is usually negatively affected by a correlated increase in temperature and decrease in humidity. Regarding to B. oleae parasitoids, even P. concolor, is strongly limited by hot and dry climatic conditions (Yokoyama et al., 2005). However, despite of a low survival, F. arisanus was able to establish in the olive grove, maintaining, in the next generation, a number of individuals just a little lower than the released population. Tests carried out in 2004 evidenced that F. arisanus was instead able to gradually and strongly increase its population on B. oleae during late summer-autumn, when higher humidity levels are normally recorded in the Mediterranean area. These conditions enabled in fact F. arisanus to efficiently mate in field. A balanced sex ratio is expected to benefit biological control because a high level of mating would increase rates of population growth following augmentative or inoculative releases of parasitoids (Purcell et al., 1993). The parasitoid was consequently capable to seasonally establish. Moreover, the analysis of the host density in correspondence to the parasitoid releases allowed us to ascertain an efficient capability by F. arisanus in finding and parasitizing B. oleae in field even if host density is low. The parasitization activity of F. arisanus led to a reduction in the B. oleae populations development. This result was perhaps reached also because not all the host eggs survive to the parasitization, allowing also an immediate preservation of olive fruits by the B. oleae larval trophic activity (Moretti et al., 2004). A decrease in pest emerging adults was evidenced in correspondence of the peaks in F. arisanus emergence from the olives recovered during the test. The comparison with results from control treatment (site C) allows to evidence a significant impact by the parasitoid on olive fly population dynamics, although other repetitions are needed to give to these findings statistical significance. Our results encourage us to carry further investigations particularly to test a seasonal inoculative release strategy using F. arisanus for controlling B. oleae combined maybe with other ecocompatible control strategies (Knipling, 1992). An anticipation of F. arisanus releases may be also attempted to allow for an earlier establishment of the parasitoid in the olive groves as it was found possible since July-August. Further research will focus on competitive interactions in field with other B. oleae antagonists such as E. urozonus and P. concolor to directly compare ability in controlling B. oleae. Moreover, we are investigating whether F. arisanus is able to overwinter under climatic conditions of Central Italy, taking into consideration that for projects where inundative releases are done every year, it is of crucial importance to know whether the biological control agent can survive to the winter in the country of introduction, and thus possibly establish outside the side of release (Babendreier et al., 2003). 59

Acknowledgements

We wish to thank the mass rearing unit (USDA-ARS, Honolulu) for providing the initial stock of F. arisanus and particularly R.C. Bautista for his valuable suggestions. We also thank M. Cristofaro (C.R. ENEA, Casaccia) and G.A.P. Gibson (Canadian National Collection of Insects (CNC), Agriculture and Agri-Food Canada). Finally, we are grateful to M. Antonelli for participating at the first phases of our research program on F. arisanus.

References

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 61-66

Presence of a symbiotic bacterium in the olive fly Bactrocera oleae (Gmelin)

Vincenzo Girolami1, Andrea Squartini2, Luca Mazzon1, Alessia Piscedda1, Caterina Capuzzo1 1 University of Padua, 1Department of Environmental Agronomy and Plant Productions, and 2 Department of Agrarian Biotechnology, Viale dell’Università, 16 - 35020 Legnaro, Padua, Italy

Abstract: The taxonomic identification of “Candidatus Erwinia dacicola”, the hereditary prokaryotic symbiont of the olive fly Bactrocera oleae, suggests the reported consideration. To avoid microbial contaminants, flies were surface-sterilized at larval stage and reared under aseptic conditions until adult emergence. B. oleae flies originating from different geographical areas and collected at different times of the year were tested. Bacteria were isolated from the cephalic oesophageal bulb, which is known to be a specific site of symbiont multiplication in the adults. Attempts at cultivation of the isolated bacteria ex situ were not productive at any stage. PCR amplification and sequencing of the entire 16S rRNA gene yielded a single sequence similar (97%) to Erwinia persicina and Erwinia rhapontici yet different from Pseudomonas savastanoi and, to a lesser extent, from “fruit flies associated bacteria”. Morphological differences exist among the pharyngeal bulbs of the olive fly and other fruit flies (belonging to Dacinae and Trypetinae) in which the presence of hereditary symbionts has not yet been demonstrated.

Key words: Bactrocera oleae, Candidatus Erwinia dacicola, bacteria, symbionts, rRNA gene, sequencing.

Introduction

The olive fly Bactrocera (= Dacus) oleae (Gmelin) (Diptera: Tephritidae) is widespread across the entire range of olive tree growth (the Mediterranean, South Africa, Asia and recently North America) (Commonwealth Institute of Entomology, 1996). The adult feeds on vegetable exudates, substances containing sugar such as honeydew, mature fruits and micro- organisms. B. oleae is considered the most important pest of olive trees; the use of chemical treatments is currently the main control strategy. At the beginning of the past century (1909) an association between the olive fly B. oleae and bacteria was first described by Petri. Adults harbour micro-organisms inside a cephalic organ (known as pharyngeal bulb or oesophageal bulb), connected to the pharynx, in which bacteria rapidly multiply forming masses that reach the midgut. The mother can transmit the symbionts to larvae during egg laying due to the presence of glands filled with bacteria in the ovipositor. The bacteria multiply inside intestinal coeca at all larval stages, but their location inside pupae is still unknown. Petri suggested that the symbiont might be ‘Bacterium’ (Pseudomonas) savastanoi, the causal agent of the olive knot disease. The basis of the symbiotic advantage for the flies was postulated to be a nutritional effect, both in terms of enhanced dietary protein hydrolysis and the synthesis of required amino acids lacking in the olive pulp (Tsiropoulos, 1980). Moreover, the adults reared on artificial media without bacteria have shown a lower vitality and fertility, accompanied by a shrinking of the oesophageal bulb (Girolami & Cavalloro, 1972).

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The recent use of DNA-based methodologies, combined with the recent development of DNA sequence databases, has clarified the systematic placement of the B. oleae symbiont. Due to its phenotype and 16S rRNA gene sequencing, the B. oleae symbiont represents a novel taxon within the family Enterobacteriaceae and the name "Candidatus Erwinia dacicola" was chosen according to the guidelines for in vitro unculturable bacteria (Capuzzo et al., 2005). Its 16S rRNA sequence is 97% similar to those of Erwinia rhapontici and Erwinia persicina, two species in the subgroup of Erwinia amylovora. This paper reports considerations mostly concerning entomological aspects of the experimentation that, waiting for the congress, have already appeared in a review of systematic microbiology.

Material and methods

Insect origin The flies used for the analyses were collected in mature larval stage from unripe olives in Apulia and the Garda lake area in 2001, and from Sicily and Sardinia in 2003. In Liguria pupae ware collected from overripe olives in April 2002 and from unripe olives in August of the same year.

Insect surface sterilization and rearing under microbiologically controlled conditions To avoid the contamination of micro-organisms different from the symbiont originated from food or from the environment, the samples analysed were surface-sterilized at the larval or pupal stage by immersion in a 1% sodium hypochlorite solution for 2-3 min, and pupae were kept inside sterile small vials (Eppendorf) until their emergence (Figure 1A). Each adult was then transferred in a larger sterile vial containing LBA or PCA agar media on the bottom and closed with a permeable membrane (Dialysis Cellulose Membrane, Sigma-Aldrich). A drop of sterile 50% glucose solution was applied in the back part of the cap to feed the fly (Figure 1B). All operations were carried out under a laminar flow hood.

Insect dissection The adults were dissected with sterile forceps under a stereoscope 2 or 3 days after emergence to extract the cephalic organ, the midgut or the ovipositor. For bacterial analysis the organs were transferred in sterile Eppendorf tubes, while for inoculation in agar media they were directly streaked onto the medium.

Attempts at cultivation of bacteria on different media The contents of the oesophageal bulbs and midguts were transferred directly onto agar media or suspended in liquid media (Capuzzo et al., 2005). Surface-sterilized insects were employed and, as control, two plates for each medium were streaked with the content of oesophageal bulbs and guts of some non-surface-sterilized flies.

DNA extraction, amplification, sequencing and phylogenetic analysis Bacterial DNA of the adult was extracted from the content of the oesophageal bulbs, midguts and ovipositor, as described by Palmano et al. (2000). Only flies in vials without the development of bacteria colonies on the agar medium in the bottom were employed. DNA was also extracted from whole surface-sterilized pupae and from the bacteria content of intestinal coeca of larvae collected inside unripe healthy olives. The universal bacterial 16S rRNA primers fD1 and rP1 (Weisburg et al., 1991) were used, yielding an amplicon of about 1500 bp as reported in Capuzzo et al. (2005). 63

The amplified products were sequenced and the additional primers fR2 (5’- CGTGTCTCAGTTCCAGTGTG-3’), fL2 (5’-GGAACTGCATTCGAAACTG-3’), rR2 (5’- CTCGTGTTGTGAAATGTTGG-3’) and rL2 (5’-AAGGCACTAAGGCATCTCTG-3’) were devised in order to walk through the entire 16S rRNA gene sequence (Capuzzo et al., 2005). A BLAST GenBank analysis of the sequences obtained was run through the NCBI website. The sequences were aligned with their closest relatives using the BioEdit sequence alignment editor (Hall, 1999). Phylogenetic trees were constructed using the neighbour- joining method (Saitou and Nei, 1987).

Sterile glucose solution

Teflon

Permeable membrane

Sterile paper

Surface sterilized pupa

Agar media

Figure 1. Microbiologically controlled conditions for single fly rearing: (A) Sterile vial for adult emerging; (B) Sterile vial for adults rearing.

Results and discussion

Bacterial cultivation attempts In the majority of cases, no colonies developed on the plates streaked with the content of the oesophageal bulbs, the midguts, or the ovipositors of adults reared in sterile conditions. Only occasionally, sporadic colonies appeared on the plates. However, their amplified 16S rRNA gene sequence displayed an ARDRA (Amplification Ribosomal DNA Restriction Analysis) profile completely different from the one of the unculturable bacteria present in the oesophageal bulb (Capuzzo et al., 2005). Despite the fact that the sterilized flies were maintained alive for a few days inside the vials before dissection, routinely no colonies developed, neither from faeces contact nor from contact with mouth or tarsi, confirming the validity of the sterilization method based on adults obtained from surface-sterilised pupae. Conversely, in all plates inoculated with non-surface-sterilized insects, numerous bacterial and fungal colonies developed. Of the adults that were held inside the vials that did not contain development of bacteria on the agar medium, their oesophageal bulbs and the midguts were found to contain profuse amounts of bacteria. These results confirm that the main 64

symbiont core is represented by a unculturable entity, and that if bacteria colonies do develop, they take origin from bacteria present by chance among the masses of symbionts.

DNA amplification and nucleotide sequencing A PCR product of the expected size of about 1500 bp was obtained in all cases with primers fD1/rP1. In total, 13 flies developed from mature larvae grown inside unripe or overripe olives from different Italian regions and collected at different periods of the year were analysed. The sequences were found to be all identical (GenBank accession number AJ586620) (Capuzzo et al., 2005). This confirms that a single bacterial species represents the entire, or at least the largest fraction of the symbiotic microflora of B. oleae. As the same gene sequence was also found inside the ovipositor, larvae coeca and pupae, the bacteria can be considered to be maternally transmitted. In addition, no evidence emerged to support the possibility that unculturable symbiotic bacteria could be ingested with food or from the environment since the sequences are identical independently of the ripeness of olives and of geographical placement.

Phylogenetic analysis The taxonomic position of the symbiont of B. oleae is shown, with respect to its closest relatives, in the neighbour-joining tree based on the 16S rRNA gene sequence (Figure 2). The B. oleae symbiont represents a distinct branch of the tree, well supported by a high bootstrap value. The symbiont is different from Pseudomonas savastanoi, the causal agent of the olive knot disease, as theoretically suggested by Petri in 1909 and later confirmed by Hellmuth (1956), Buchner (1965), and Hagen (1966), but questioned by Girolami and Cavalloro (1972).

Figure 2. Neighbour-joining tree showing the phylogenetic position of the B. oleae symbiont based on 16S rRNA gene sequence analysis. Bootstrap values > 50% are reported on nodes (Capuzzo et al., 2005). 65

B. oleae symbiont is not related to Buchnera aphidicola, the primary symbiont of aphids. Its lineage is instead closer to Pantoea agglomerans, Enterobacter cloacae and Klebsiella oxytoca (Figure 2) that can be considered ‘fruit-fly-associated bacteria’ (Lloyd et al., 1986) commonly present in the intestines of insects and vertebrates.

Conclusions

The sequencing of the sample shows that the symbiont “Candidatus Erwinia dacicola” is not P. savastanoi. Therefore, this work closes a secular debate on the identity of olive fly symbiont. It is interesting to note that Petri (1909) has only hypothesized that symbionts inside pharyngeal bulb could be P. savastanoi, causal agent of olive knot disease, because he obtained small hyperplasiae on olive twigs inoculated with the content of bulb. The symbiotic bacteria of the olive fly that fills the lumen of the midgut in masses, results unculturable in vitro. Therefore, all the bacterial species reported in literature that are obtained from the pharyngeal bulb or the gut of B. oleae can be considered minor components of the intestinal microflora isolated by chance and different from the symbiotic species “Candidatus Erwinia dacicola”. In the recent past, great importance was given to the “associated bacteria” commonly found in the intestine of fruit flies and diffused on the leaf surface or inside damaged fruits of the host plants (Courtice & Drew, 1984), and the existence of hereditary symbiosis in the fruit flies was questioned (Drew & Lloyd, 1989). It is probable that numerous species of fruit flies do not have hereditary symbiotic bacteria, but that they are present in the olive fly has been well known for one century (Petri, 1909). The pharyngeal bulb of the olive fly where the symbiotic bacteria multiplies is morphologically different from that of other known fruit flies. The pharyngeal bulbs of the genus Ceratitis, Rhagoletis, Anastrepha, Dacus and Bactrocera (oleae species excluded) are smaller than those of olive flies and were described as “Ceratitis type” bulbs in the work that demonstrated the presence of such an organ in all the Tephritidae (Girolami, 1973) and not only in the olive fly as previously considered (Buchner, 1965). Strong morphological differences of the pharyngeal bulb may be linked to the coevolution of the olive fly and symbiotic bacteria that may not have been present in other fruit flies belonging to the subfamily Dacinae and Trypetinae. In the subfamily Tephritinae an hereditary bacterial symbiosis has been described by Stammer (1929). Inside this subfamily, DNA analysis of symbiotic bacteria of species belonging to the genus Tephritis and Campiglossa demonstrate the presence of identical sequences inside each insect species in a way similar to that reported for the olive fly (Capuzzo, 2004).

References

Buchner, P. 1965: Endosymbiosis of Animals with Plant Microorganisms. – Interscience Publ., NY. Capuzzo, C. 2004: Presenza di batteri coevoluti in Bactrocera oleae (Gmelin) ed in altri Ditteri Tefritidi. Phd. – Thesis University of Padova. Capuzzo, C., Firrao G., Mazzon, L., Squartini, A., Girolami, V. 2005: ‘Candidatus Erwinia dacicola’, a coevolved symbiotic bacterium of the olive fly Bactrocera oleae (Gmelin). – Int. J. Syst. Evol. Microbiol. 55: 1641-1647. Commonwealth Institute of Entomology. 1996: – Distribution Maps of Pests. Series A: Map n. 74 (1st revision) Bactrocera oleae (Gmelin). 66

Courtice, A.C. & Drew, R.A.I. 1984: Bacterial regulation of abundance in tropical fruit flies (Diptera: Tephritidae). – Austr. Zool. 21: 251-266. Drew, R.A.I., Lloyd, A.C. 1989: Bacteria associated with fruit flies and their host plants. – In: Robinson, A.S. and Hooper, G. (eds.): "Fruit Flies their Biology, Natural Enemies and Control". World Crop Pests, Elsevier, Amsterdam, 3A: 131-140. Girolami, V., Cavalloro, R. 1972: Aspetti della simbiosi batterica di Dacus oleae (Gmelin) in natura e negli allevamenti di laboratorio. – Ann. Soc. Entomol. France 8 (3): 561-571. Girolami, V. 1973: Reperti morfo-istologici sulle batteriosimbiosi del Dacus oleae Gmelin e di altri Ditteri Tripetidi, in natura e negli allevamenti su substrati artificiali. – Redia 54: 269-294. Hagen, K.S. 1966: Dependence of the olive fly, Dacus oleae, larvae on symbiosis with Pseudomonas savastanoi for the utilization of olive. – Nature (Lond.) 209: 423-424. Hall, T.A. 1999: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. – Nucl. Acids. Symp. Ser. 41: 95-98. Hellmuth, H. 1956: Untersuchungen zur Bakteriensymbiose der Trypetiden (Diptera). – Z. Morphol. Oekol. Tiere 44: 438-517. Jukes, T.H., Cantor, C.R. 1969: Evolution of protein molecules. – In: Mammalian Protein Metabolism, H.N. Munro (ed.). Academic Press, NY.: 21-132. Lloyd, A.C., Drew, R.A.I., Teakle, D.S., Hayward, A.C. 1986: Bacteria associated with some Dacus species (Diptera: Tephritidae) and their host fruit in Queensland. – Aust. J. Biol. Sci. 39: 361-368. Mazzini, M., Vita, G. 1981: Identificazione submicroscopica del meccanismo di trasmissione del batterio simbionte in Dacus oleae (Gmelin) (Diptera, Trypetidae). – Redia 64: 277- 318. Palmano, S., Firrao, G., Locci, R. 2000: Sequence analysis of domains III and IV of the 23S rRNA gene of verticillate streptomycetes. – Int. J. Syst. Evol. Microbiol. 50: 1187-1191. Petri, L. 1909: Ricerche sopra i batteri intestinali della Mosca olearia. – Mem. Staz. Pat. veg. Roma. Saitou, N. & Nei, M. 1987: The neighbour-joining method: a new method for reconstructing phylogenetic trees. – Mol. Biol. Evol. 4: 406-425. Stammer, H. 1929: Die Bakteriensymbiose der Trypetiden (Diptera). – Z. Morphol. Ökol. Tiere 15. Tsiropoulos, G.J. 1980: Major nutritional requirements of adult Dacus oleae. – Ann. Entomol. Soc. Am. 73: 251-253. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J. 1991: 16S ribosomal DNA amplification for phylogenetic study. – J. Bacteriol. 173: 697-703.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 67-71

Histopathological observations in the midgut and behaviour of olive fruit fly (Bactrocera oleae Gmelin.) adults treated with a strain of Bacillus thuringiensis Berliner

Luca Ruiu1, Gavino Delrio1, Ignazio Floris1, Alberto Satta1, Mario Solinas2 1 Dipartimento di Protezione delle Piante – Entomologia Agraria, University of Sassari (Italy) 2 Dipartimento di Scienze Agrarie ed Ambientali-Entomologia, University of Perugia (Italy)

Abstract: Ultrastructural changes are usually observed in the midgut of various insect species after feeding on diets containing specific toxins from different Bacillus thuringiensis strains. A spore-crystal suspension of a Bacillus thuringiensis strain, previously known to be toxic against Olive Fruit Fly adults, was used to study the post-ingestion effects on the midgut ultrastructure of treated flies compared to untreated ones. Observations were carried out at different time intervals, until 72 h after feeding the bacterial suspension. Transmission electron micrographs showed a typical symptomathology involving a general disruption or disorganization of the midgut epithelial cells often ending in the cell lysis. Similar pathological changes in the intestine are known for other insect species belonging to the orders Lepidoptera, Coleoptera and Diptera. Behavioural observations were also carried out comparing treated to untreated flies. In the post feeding period, treated adults went through a progressive symptomatology which involved a general reduction in the activity, sluggish and shaky behaviour until general paralysis and death. The behavioural symptoms of intoxication paralleled the histopathology observed in the midgut of treated flies. Untreated adults looked healthy and did not show any pathological symptoms.

Key words: biological control, bioassays, behavioural observations, ultrastructural changes, electron microscopy.

Introduction

The Olive Fruit Fly, Bactrocera oleae Gmelin (Diptera Tephritidae), is an important pest of olives in the Mediterranean area which causes economically significant lost of production. Management of this species normally involves the use of chemical insecticides against larvae and adults. Ecological risks for the environment and human health, involved in the use of pesticides, promote the search for alternative strategies of olive fly control including the use of microbial agents. Bacillus thuringiensis is a sporeforming bacterium which produces parasporal bodies (crystals) containing specific insecticidal endotoxins. B. thuringiensis δ-endotoxins, once ingested by the insect, are solubilised and activated in the gut, and after binding specific plasma membrane receptors on the midgut epithelial cells, generate leaky pores in the membrane and a consequent inflow of ions and water, which eventually degenerate in the cell lysis (Knowles & Ellar, 1987). Ultrastructural changes in the midgut (such as microvillar disruption, vacuolization and disorganization of the cytoplasm, deterioration of muscular sheath, alteration of mitochondria and a general cell deterioration) have been studied in various insect species exposed to B. thuringiensis, especially Lepidopteran larvae (Lane et al., 1989; Rausell et al., 2000). Among Diptera, investigations have been carried out on Simulidae (Lacey & Federici, 1979) and

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Culicidae (Weiser & Zizka, 1994). Little work has been developed on flies even if toxicity of B. thuringiensis has been demonstrated on various fly species, such as the Housefly Musca domestica L. (Hodgman et al., 1993), the Medfly Ceratitis capitata Wied (Satta et al., 1998) the Mexican Fruit Fly Anastrepha ludens Loew (Robacker et al., 1996) and the Olive Fruit Fly B. oleae (Alberola et al., 1999). Histopathological effects of toxins from B. thuringiensis var. israelenisis on housefly larvae midgut were proved in vitro (Singh et al., 1986), as well as histopathological effects of other entomopathogenic bacteria were shown on adults (Trona et al., 2004). Ultrastructural changes in the gut were also studied on C. capitata adults fed on a spore-crystal mixture of B. thuringiensis var. morrisoni (Younes et al., 1996). Concerning the olive fruit fly, to our knowledge there is a unique report concerning the pathological effects in the larval midgut caused by B. thuringiensis strain ormylia (Dimitriadis & Domouhtsidou, 1996), whereas there are no investigations on adults. The aim of our work was to study the ultrastructural changes caused in the midgut of Olive Fruit Fly adults by a spore-crystal mixture of a Bacillus thuringiensis strain previously known to be toxic against this insect stage.

Materials and methods

Bacterial preparation Bacillus thuringiensis strain TE 37.18 (Alberola et al., 1999) was grown in Nutrient broth at 30°C and 180 rpm until complete sporulation and sporangia lysis. Spore-crystal mixtures were collected and washed in sterile milliQ water by three repeated cycles of centrifugation at 10,000 g and 4°C. Concentration, expressed as spore-crystal/ml, was quantified by using a Thoma chamber (E. Hartnack, Berlin, Germany).

Bioassays Single newly emerged B. oleae adults were kept in transparent plastic pot (3 cm Ø and 8 cm high) and fed daily on a liquid diet containing 30% sucrose and 1x109 B. thuringiensis strain TE 37.18 spore-crystal /ml, administered ad libitum by 20 µl capillary tubes. A control was run feeding flies with just a 30% sucrose solution. A total of 50 flies (25 treated and 25 as control) were involved in this study. Flies were randomly sacrificed for midgut observations at various time intervals until 72 h after the first food administration. Behavioural observations were also conducted during the bioassay, comparing treated and untreated flies through direct watching.

Electron microscopy Midgut portions, close to the pyloric valve, were excised from cold anaesthetized flies and immediately immersed in Karnosvsky’s (1965) fixative solution for 3 h at 4°C before being rinsed overnight in cacodylate buffer with 5% sucrose, fixed in 1% Osmium tetroxide for 1 h, rinsed again in cacodylate buffer, dehydrated in a progressive ethanol gradient until 90%, block stained with 1% uranyl acetate in 95% ethanol for 1 h, treated by two 15 min passages in absolute alcohol and embedded through propylene oxide in Epon-Araldite. L.K.B. “Nova” ultramicrotome was used to cut cross sections about 70 nm thick which were then stained with uranyl acetate and lead citrate before being observed and micrographed through the transmission electron microscope Zeiss EM 109.

Results and discussion

Exposition of B. oleae adults to a diet containing a spore-crystal mixture of B. thuringiensis strain TE 37.18 caused dramatic effects on midgut cell ultrastructure and on fly feeding and behaviour, which brought progressively flies to death. First evident behavioural symptoms of intoxication were observed 48 h after the first administration of the B. thuringiensis suspension. Then symptomathology became progressively more dramatic until fly died. At the beginning flies showed sluggish and shaky behaviour and decreased responsiveness to external stimuli, then they stopped feeding and sometimes lay on the back until general paralysis and death occurred. Electron microscopy observations of control flies revealed that midgut epithelial cells had a dense cytoplasm with well developed mitochondria, golgi bodies, rough endoplasmic reticulum, moderate numbers of lysosome-like structures and abundant microvilli projecting into the midgut lumen (Figure a). Epithelial cells were also tight adhering to the basal lamina, to which muscular sheath are also well attached, and the binding connective tissue appeared normally developed and compact. In treated flies first histopathological effects were observed 24 h after the first administration of the bacterial treated diet, and they became more and more extensive and dramatic with longer time of incubation. After 72 h numerous cells of susceptible flies were in advanced state of disruption. Thus corroborating the above reported behavioural symptomathology. In detail, the main hindgut alterations consisted of a considerable epithelial cell disorganization involving: vacuolization of the cytoplasm; deformation of endoplasmic reticulum; mitochondria irregularly enlarged, condensed and showing electron dense matrix; increased numbers of lysosome-like structures; disruption of microvilli (Figures b and c). Thereafter, the pathological symptoms further progressed until the infected cells released their content into the gut lumen (Figure d). Alteration of the muscular sheath and its associated connective tissue was also noted. The general alterations reported are reminiscent of those described for both flies and other insects exposed to different B. thuringiensis strains (Lane et al., 1989; Dimitriadis & Domouhtsidou, 1996; Younes et al., 1996; Trona et al., 2004). It is remarkable that the cell swelling, vacuolization and lysis observed suggest the influx of water in the cell. In treated flies affected and unaffected cells were often observed even adjacent to one another. This different cell susceptibility supports the hypothesis that specific membrane receptors recognise the bacterial toxins. However the sympomathology we noticed on adults, similarly to what previously reported for larvae (Dimitriadis & Domouhtsidou, 1996), was due to a spore-crystal mixture, hence further investigation is needed to discriminate which factors are responsible of the various cell alterations observed. Our investigations suggest that the mechanism of action involved with alteration of the cell membrane permeability is similar to those widely studied on Lepidopteran larvae (Lane et al., 1989).

Acknowledgements

This study was supported by Italian Ministero dell’Università e della Ricerca Scientifica (Research Program: “Biotecnologie innovative per il controllo di insetti nocivi mediante l’impiego di agenti microbiologici”. Coordinator: Prof. Ignazio Floris, University of Sassari – Italy).

Figures a, b, c, d. T.E.M. micrographs showing midgut cells from Bactrocera oleae adults sacrificed 72 h after being fed on a healthy diet (Fig. a) or on a diet containing a spore-crystal mixture of Bacillus thuringiensis, which display obvious pathological features, such as cytoplasm vacuolization and microvilli deterioration (Fig. b), mitochondrial alteration (Fig. c), and cell lysis (Fig. d). L, midgut lumen; LY, lysosome-like structures; M, mitochondria; MV, microvilli; P, peritrophic matrix; V, vacuoles. 71

References

Alberola, T.M., Aptosoglou, S., Arsenakis, M., Bel, Y., Delrio, G., Ellar, D.J., Ferrè, J., Granero, F., Guttmann, D.M., Kolias, S., Martinez-Sebastiàn, M.J., Prota, R., Rubino, S., Satta, A., Scarpellini, G., Sivropoulou, A. & Vasara, E. 1999: Insecticidal activity of strains of Bacillus thuringiensis on larvae and adults of Bactrocera oleae Gmelin (Dipt. Tephritidae). – Journal of Invertebrate Pathology 74: 127-136. Hodgman, T.C., Ziniu, Y., Ming, S., Sawyer, T., Nicholls, C.M. & Ellar, D.J. 1993: Characterization of a Bacillus thuringiensis strain which is toxic to the housefly Musca domestica. – FEMS Microbiology Letters 114: 17-22. Dimitriadis, V.K. & Domouhtsidou, G.P. 1996: Effects of Bacillus thuringiensis strain ormylia spore-crystal complex on midgut cells of Dacus (Bactrocera) oleae larvae. – Cytobios 87: 19-30. Knowles, BH & Ellar, DJ. 1987: Colloid-osmotic is a general feature of the mechanism of action of Bacillus thuringiensis delta-endotoxins with different insect specificity. – Biochim. Biophys. Acta 924: 509-518. Lane, N.J., Harrison, J.B., Lee, W.M. 1989: Changes in microvilli and golgi-associated membranes of lepidopteran cells induced by an insecticidally active bacterial delta- endotoxin. – Journal of Cell Science 93: 337-347. Lacey, L.A., Federici, B.A. 1979: Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simulidae). – Journal of Invertebrate Pathology 33: 171-182. Rausell C., De Decker N., Garcia-Robles I., Escriche B., Van Kerkhove E., Real M.D., Martinez-Ramirez A.C. 2000: Effect of Bacillus thuringiensis toxins on the midgut of the nun moth Lymantria monacha. – Journal of Invertebrate Pathology 75: 288-291. Robacker, D.C., Martinez, A.J., Garcia, J.A., Diaz, M. & Romero, C. 1996: Toxicity of Bacillus thuringiensis to Mexican Fruit Fly (Diptera: Tephritidae). – Journal of Economic Entomology 89: 104-110. Satta, A., Bazzoni, E., Delrio, G., Prota, R. & Rubino, S. 1998: Recenti acquisizioni sulla tossicità di Bacillus thuringiensis nei confronti di Ceratitis capitata Wied. in laboratorio. – Atti XVIII Congresso Nazionale Italiano di Entomologia (Maratea, 21-26 June 1998). Singh, GJP, Schouest, LP Jr & Gill, SS (1986) Action of Bacillus thuringiensis subsp. israelensis delta-endotoxin on the ultrastructure of the House fly larva neuromuscular system in vitro.– Journal of invertebrate Pathology 47: 155-166. Trona, F., Ruiu, L. Floris, I. & Solinas, M. 2004: Comparative behavioural and anatomo- pathological investigations on Musca domestica L. adults treated with a new strain of Bacillus sp. close related to B. thuringiensis (Berliner). – Entomologica, Bari, 38: 49-73. Weiser, J. & Zizka, Z. 1994: Effect of Bacillus thuringiensis beta exotoxin on ultrastructure of midgut cells of Culex siriens. – Cytobios 77: 19-27. Younes, M.W.F., Hashem, A.G., EL-Abassi, T.S., Abo-Houla, A.I.A. 1996: Effects of Bacillus thuringiensis var. morrisoni on the adult stage of Mediterranean Fruit Fly Ceratitis capitata (Wied.). – J. Union Arab Biol., Cairo, 5(A): 189-203.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 73-75

Some biological aspects of the Bactrocera oleae (Rossi) rearing

Angelo Canale, Roberto Canovai, Augusto Loni, Alfio Raspi Department of Tree Science, Entomology and Plant Pathology “G. Scaramuzzi”, University of Pisa, via S. Michele degli Scalzi, 2 – 56124 Pisa, Italy

Abstract: An olive fruit fly adults caging and egging system was proposed. This system of adult rearing facilitates eggs collection and reduces the labour requirements. In order to search for the optimal population density/cage, a comparison among three different densities (100, 250 and 400 specimens/cage) was performed. The density of 250 specimens/cage allowed the collection of about 150 eggs/day/cage, a number relatively higher than that obtained at the remaining two densities tested.

Key words: olive fruit fly, mass-rearing, population density.

Introduction

The possibility to produce an insect’s colony in the laboratory results very useful in providing specimens for physiological, ethological, genetics studies and for bio-control purposes. As regarding Bactrocera oleae (Rossi) (Diptera, Tephritidae), the key phytophage of the olive grove agro-ecosystem throughout the Mediterranean area, the critical phase in establishment of a laboratory population results the initial stage of colonization (Tsitsipis, 1977; 1982). In particular, the collection of a massive amount of eggs represents the key point to start. In order to deal with olive fruit fly rearing on artificial substrate, our attention was focused on the possibility to realize a simplified small-scale caging and egging system for adults of B. oleae.

Material and methods

The insects used in the study were collected in field during January 2005 by sampling infested olive drupes. The infested olives were stored and maintained in standard laboratory conditions, (21 ± 2 °C, 40-50 R.H., 16:8 photoperiod) in cylindrically shaped plexiglas cages (diameter 30 cm and length 40 cm) until the flies emergence, then water and a solid diet, composed by a mixture of 10 parts of sugar and 1 of yeast extract, has been supplied. Two generations were performed rearing the flies on ripe olives and some biological parameters were recorded (survival period of male and females, oviposition period, mean number of laied eggs). Moreover, a comparison among three population density of adults, 100, 250 and 400 specimens/cage (males and females, sex ratio 1:1), was performed. In details, the flies were subdivided in three cylindrically shaped plexiglas cages (diameter 30 cm, length 25 cm, volume 17 dm3) at the three different density above mentioned; adults were supplied by water and the solid diet above reported, and maintained in standard laboratory conditions. The cages (Figure 1) present the top surface and lower base covered by a perforated nylon net with hole sides measuring 0.2 mm and 2.0 mm, respectively. The lateral surfaces were mainly covered with the same nylon net as the upper surface, to consent a good aeration of the inner volume of the cage. The lower surface was placed on a removable oviposition dish, constituted by a plastic ring (35 cm diameter) covered with a paraffin-coated

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nylon net (0.2 mm hole side). Females of olive fruit fly can lay their eggs directly on the oviposition dish, through the holes of the lower nylon net surface, but they can’t escape from the same holes when the cage was removed. Eggs were so easily flushed twice a day by means of a spray-gun rinse, because eggs don’t remain glued on the paraffin-coated surface. After ten days from the flies introduction into the cage, the number of eggs daily produced was recorded for the successive ten days. The comparison among the number of eggs obtained from each population density was performed by the Student’s t test (Sokal & Rohlf, 1981).

30 cm

25 cm 1 1 1

35 cm Figure 1. Schematic representation of the caging and egging system described with more details in the text. 1: nylon net with hole sides measuring 0,2 mm; 2: nylon net with hole sides measuring 2,0 mm; 3: cylindrically shaped plexiglas cages; 4: removable oviposition dish.

Results and discussion

In standard laboratory conditions, it was observed that wild adults, obtained from field- collected pupae, live for periods over 3 months, with a mean survival period longer in the male (about 59 days) than the female (about 48 days). This data well fitted to the results of previous paper (Tzanakakis, 1989). Investigation of the oviposition period revealed that wild females start ovipositing on fresh olives on the 8th-10th day after emergence, with an oviposition period ranging 20–50 days after emergence. The proposed caging and egging system represented a small-scale rearing facilitating eggs collection and reducing the labour requirements. The population density of 250 specimens/cage allowed the collection of about 150 eggs/day/cage, through a period of ten days, a number relatively higher than that obtained at the remaining two densities tested (Table 1). In laboratory conditions, when B. oleae females oviposit directly inside the olive

drupes a major daily number of eggs/female can be obtained (Tzanakakis, 1989). However, notwithstanding the difficulty of females to oviposit directly on a rigid surface, from a point of view of a small-scale rearing our result could appear satisfactory, because the easy eggs collection and the marked labour reduction counterbalancing the low oviposition rate produced.

Table 1. Mean oviposition daily rate, produced by the three different population densities of B. oleae, through a period of ten days. Values followed by different letters are statistically different by the Student’s t test (P<0,05%).

B. oleae specimens/cage Mean of eggs/day Standard deviation 150 89.7 a 24.6 250 150.2 b 42.3 400 129.2 b 30.6

Acknowledgments

This research was financially supported by Italian M.I.U.R. (Ministry of Education, Universi- ty and Research) – P.R.I.N. - 2003.

References

Sokal, R.R., Rohlf, F.J. 1981: Biometry. – Freeman & Company Ed., 2nd edition, New York: 179-270. Tsitsipis, J. A. 1977: Development of a caging and egging system for mass rearing the olive fruit fly, Dacus oleae (Gmel.) (Dipera, Tephritidae). – Z. angew. Ent. 83: 96-105. Tsitsipis, J.A. 1982: Change of wild ecotype of olive fruit fly adaptation to lab rearing. – CEC/IOBC Symposium /Athens/Nov. 1982: 416-428. Tzanakakis, M. E. 1989: Dacus oleae. – In: Fruit flies, their biology, natural enemies and control, vol. 3B, Robinson A.S. & Hooper G. Eds., Elsevier: 105-118.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 77

Bait stations field test for Bactrocera oleae (Gmelin) in the Balearic islands (Spain)

M.A. Miranda, E. Martinez, M. Monerris, A. Alemany Department of Biology, Cra. Valldemossa, km 7.5, Universitat Illes Balears, Palma - Illes Balears, Spain

During the year 2004 it was concluded the last phase of the FAO/IAEA Co-ordinated Research Programme on Development of Improved Attractants and their Integration into Fruit Fly SIT Management Programmes. This project targeted on several economic species of fruit flies including Bactrocera oleae (Gmelin.). An important part of the research was focused in developing bait stations for the olive fly based on chemical and visual stimuli. A one-month field test was conducted in 2004 in an olive groove located in Palma of Majorca (Balearic Islands). The bait station was based in a plastic red sphere baited inside with an Ammonium Bicarbonate tablet (AB). Glue and two insecticides, Imidachloprid and Methomyl, were used separately as a killing agent. Two control treatments based on Multilure traps (MLT) baited with Nu Lure and Ammonium Bicarbonate were also included in the experiment. The results obtained showed that the best treatment for the males of B. oleae was Red sphere plus glue, followed by the MLT- NuLure; Red Sphere- AB- Methomyl; MLT- AB and Red Sphere- Imidachloprid. In the case of females, the treatments ranked as follows: Red Sphere with glue; Red Sphere Methomyl and MLT- NuLure, Red Sphere Imidachloprid and finally MLT- AB. In general, it seems that the red sphere coated with glue outperformed even the MLT baited either with NuLure or AB. These results suggest that the round shape combined with red colour and ammonia acts as an important synergistic stimuli, and is as effective as the ammonia released by hydrolysed proteins placed on yellow traps. 20

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 79-87

Molecular markers as useful tools for population genetics of the olive fly, Bactrocera oleae

D. Segura, C. Callejas, M. D. Ochando Departamento de Genética, Facultad de Ciencias Biológicas, Universidad Complutense, 28040- Madrid, Spain

Abstract: In pest populations, the distribution of genetic variability can reveal not only their history but also the direction and patterns of their evolution. An understanding of the within- and between- population genetic variability is crucial in the study of crop pests. Recently, molecular genetics is providing us with new and much more sensitive tools to face different questions related to the appropriate strategies for eradication or control. The tephritid Bactrocera oleae (Gmelin) is a harmful pest of olive crops; its larvae are monophagous and feed exclusively on olive fruits. Despite the economic importance of this species, very little is known about the genetic structure of its populations. In the present work, the genetic variability within and among different geographic populations was assessed using RAPD-PCR. A considerable level of intraspecific diversity was detected but the genetic differentiation among the populations was low. These results might be explained by the length time that has elapsed since B. oleae became established in the Mediterranean region, the large effective sizes expected of its populations, and gene flow among populations. The results suggest the existence of a single, large Mediterranean olive fly population and show the need for integrated control programs coordinated between different geographical areas.

Key words: molecular markers, genetic variability, Bactrocera oleae

Introduction

In pest populations, the distribution of genetic variability can reveal not only their history but also the direction and patterns of their evolution. An understanding of the within- and between- population genetic variability, i.e. knowledge of the genetic structure of its populations, is crucial in the study of crop pests. Recently, the role of genetics and molecular methods in studies related to insect pests has increased rapidly, and there is indication that this trend will continue. Molecular genetics is providing us with new and much more sensitive tools to face different questions related to the appropriate strategies for eradication or control. The revolution in molecular biology over the last thirty years has provided a number of relatively simple techniques that allow new and more sensitive ways of looking at genetic variation. DNA- and PCR-based methodologies offer great possibilities. In particular, the random amplification of polymorphic DNA by the polymerase chain reaction (RAPD-PCR, Williams et al., 1990, Welch and McClelland, 1990) using single primers of arbitrary nucleotide sequence has considerable appeal: it is generally faster and less expensive than other methods of detecting DNA sequence variation, no previous knowledge of the genome is required, the whole genome can be screened, minute quantities of DNA are sufficient, it is relatively cheap, and no radioactive material is used. Further, very high levels of polymorphism are generally detected. Problems concerning reliability can be eliminated by optimising experimental conditions and by following experimental protocols to the letter. Thus, it is not surprising that in recent years RAPD has been used to investigate different biological problems in all manner of organisms including insect pests.

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The tephritid Bactrocera oleae (Gmelin) (Diptera: Tephritidae), the olive fly is a harmful pest of olive crops; its larvae are monophagous and feed exclusively on olive fruits. The consequence is fruit drop, which seriously reduces the fruits’ oil content and quality. Production losses due to the olive fly have been estimated over 15 % of all production and for Mediterranean basin countries, where 98% of the world’s cultivated olive trees are found, represent a very important economic problem. However, despite its agro-economic importance, Bactrocera oleae is insufficiently understood genetically. Furthermore, articles related to population structure of this pest are scarce and mostly "descriptive" (Zouros and Krimbas, 1969; Tsakas and Krimbas, 1975; Bush and Kitto, 1979; Tsakas and Zouros, 1980; Loukas et al., 1985; reviews of Zouros and Loukas, 1989, and Loukas, 1989; Ochando et al., 1994, 2003; Nardi et al., 2005). But for an efficient control of this pest information on its population structure is crucial. Taking into account these considerations, the objective of this study has been to investigate the genetic variability present in Mediterranean populations of Bactrocera oleae. Through RAPD-PCR, we propose to obtain information about the inherited inter- and intra-population variation. This will in turn allow us to gain insights into the structure of the populations of this special insect, as well as into the possible evolutionary processes involved in the maintenance of its genetic variability.

Material and methods

A total of seven Mediterranean populations were assessed. Flies of five of the populations were collected by harvesting infested fruit from different areas of Spain and allowing the adults to emerge in the laboratory. Two other samples were obtained from Italy and Israel, and received in the laboratory as adult flies. Table 1 and Figure 1 show the population names, locations and their origins.

Table 1. Code populations, location, collection sites, year of capture and geographic coordinates.

Code Collection sites SL Year Latitude, longitude TAR Cambrils, Tarragona, Spain 1 1997 42º08’ N 01º23’E ALI Alicante, Spain 2 1998 38º20’ N 30º46’ O JAE Huelma, Jaén, Spain 3 1997 37º39’ N 04º32’ O SEV Sevilla, Spain 4 1998 37º22’ N 07º59’ O MAD Algete, Madrid, Spain 5 1998 40º35’ N 04º30’ O ITA Viterbo, Lazio, Italy 6 1998 42º24’ N 12º06’E ISR Iksal, Israel 7 2000 32º41’ N 35º19’ E

Twenty flies per population and primer were score. Genomic DNA was extracted from single flies according to Reyes et al. (1997). DNA amplifications were performed under the conditions reported by Williams et al. (1990), with slight modifications. For amplifications, seven primers were used: A-02, A-07, A-17, C-05, C-06, C-11 and C-18 (Operon Technologies Inc.). Amplification reactions were performed in 12.5 µl solutions containing 81

12.5 ng DNA, 3.5 pM of primer, 0.2 mM of each dNTP, 4 mM MgCl2, 1.25 µl buffer 10x and 0.6 units of Stoffel Fragment DNA polymerase (Applied Biosystems). A Peltier PTC-100 programmable thermocycler (M.J. Research, Watertown, MA) was used for PCR reactions. The thermal profile for RAPD-PCR was 94°C for 5 min for initial denaturation followed by 45 cycles of 94°C for 1 min, 36°C for 1 min and 72°C for 2 min, and finally 72°C for 6 min.

Figure 1. Map with location of the populations sampled

The amplification products were resolved according to their molecular size by electrophoresis in 2% agarose gels with TAE buffer (40 mM Tris-Acetate, 1mM EDTA pH 8.0) containing ethidium bromide (0.5 µg/ml). A 100 bp DNA Ladder Plus marker (MBI Fermentas) were used as molecular size standard. According to some authors, the main problem with RAPD is reproducibility. Therefore, all amplifications were repeated, the protocol carefully followed, and the same reagents used for all assays. All amplifications were consistently reproducible. Figures 2 is an example of the RAPD patterns generated. RAPD-PCR products were scored as either present or absent for each fly (intensity variations were not taken into account). For each population, the total number of bands and their frequencies were calculated, as well as the proportion of monomorphic and polymorphic markers. Similarity intra and interpopulation indices were also calculated (Nei and Li, 1979), using the RAPDplot program (RAPD-PCR software package (Black IV, 1997). 82

Figure 2. RAPD patterns of fly samples analyzed with primer OPC-06. Size marker 100bp Ladder Plus, columns 1, 15 and 29.

Nei’s (1972) genetic distances were obtained from marker frequencies using the RAPDdist program (RAPD-PCR software package), and these values were used to construct a dendrogram through the unweighted pair-group (UPGMA, Sneath and Sokal, 1973) method (NTSYSpc software v. 2.01b; Rohlf, 1997). The reliability of the tree was evaluated using 1000 bootstrap replicates (RAPDdist program).

Results and discussion

Table 2 shows the variability found in the Mediterranean populations of Bactrocera oleae analyzed. Polymorphism, with high values, range from 60 % for SEV population to 70 % for JAE population. In general, the polymorphism detected for this species is comparatively higher than that reported in other insect RAPD studies, including other tephritid flies species (see de Sousa et al., 1999; Lin et al., 1999; Zitoudi et al., 2001; Ochando et al., 2003). From the few population studies of B. oleae found in the literature (reviewed by Zouros and Loukas, 1989, Ochando and Reyes, 2000, Ochando et al., 2003), a high rate of genetic variability seems to be characteristic of the species.

Table 2. Polymorphism indices found with the different primers for the seven populations analyzed.

Populations/ ALI ISR ITA JAE MAD SEV TAR Primers

OPA02 0,40 0,73 0,53 0,73 0,53 0,60 0,60 OPA07 0,63 0,74 0,84 0,74 0,68 0,67 0,74 OPA17 0,87 0,75 0,81 0,81 0,87 0,81 0,87 OPC05 0,50 0,57 0,50 0,57 0,50 0,50 0,50 OPC06 0,91 0,74 0,74 0,83 0,78 0,83 0,87 OPC11 0,63 0,56 0,56 0,63 0,50 0,44 0,56 OPC18 0,42 0,50 0,33 0,58 0,50 0,33 0,67

Mean 0,63 0,66 0,62 0,70 0,62 0,60 0,69

The potential for ecological heterogeneity to promote genetic diversity, and perhaps divergence, has been proposed (Abrahamson and Weis, 1997, Downie et al., 2001). However, B. oleae does not meet such expectations: the olive fly is a strictly monophagous species. Moreover, as said, comparison with the information available on other Tephritidae species highly polyphagous, such as Ceratitis capitata, underscores the idea of the great variability of olive fly populations (Gasperi et al., 2002, Kourti, 2004a,b, Ochando et al., 2003, Callejas and Ochando, 2004). The high genetic variability of B. oleae populations is probably due to the length of time that has elapsed since they became established in the Mediterranean. It is thought that the olive fruit fly arrived in Europe more than two thousand years ago with the introduction of the olive tree from Western Asia and Africa by the Phoenicians (Ruiz, 1948). Another factor that may contribute to this genetic variability is the elevated effective size expected for these populations since olive groves cover wide expanses of territory. Olive flies can therefore maintain high population densities. With respect to the distribution patterns of the underscore variability the most evident characteristic of note is the low level of genetic differentiation among the seven population tested. Table 3 shows the intra (diagonal) and interpopulational similarity indices. The intrapopulation indices ranged from 0.62 (JAE population) to 0.67 (ISR, ITA and SEV populations) and are similar (however slightly higher) to interpopulation values (0.60 between JAE and ALI populations, and 0.65, between SEV and ITA and MAD populations). In brief, a considerable level of intraspecific diversity was detected but the genetic differentiation among populations was low. These results agree with previous data recorded for this species (Zouros and Loukas, 1989, Ochando et al., 1994, Callejas et al., 1998).

Table 3. Intra and interpopulation similarity indices.

ALI 0,63

ISR 0,61 0,67

ITA 0,62 0,64 0,67

JAE 0,60 0,62 0,63 0,62

MAD 0,61 0,64 0,63 0,62 0,66

SEV 0,64 0,64 0,65 0,63 0,65 0,67

TAR 0,61 0,64 0,64 0,62 0,64 0,64 0,66

ALI ISR ITA JAE MAD SEV TAR

According to historic data, the olive fly probably colonized the Mediterranean more than two thousand years ago: the time elapsed would be enough for diversifying selection to produce genetically different populations. Thus, the uniformity among the Mediterranean populations of olive fly might be due to gene flow. The existence of small and isolated 84

populations would lead to divergence between populations and homogeneity within them. Conversely, the presence of large, interconnected populations would result in less interpopulational differentiation yet greater diversity within these populations (Lin et al., 1999). Different authors have reported adult olive fly movements ranging from 200 m in the presence of olive hosts, to as much as 4000 m if hosts need to be searched out. Dispersals of up to 10 Km have been reported over open water in the Mediterranean (Rice et al., 2000). The gene flow among the Mediterranean populations could be an important factor influencing the genetic structure of B. oleae in this region, where large populations have long been established. Other aspect of interest in the population structure of the olive fly is the genetic relationships between populations of different geographic areas. Table 4 shows the genetic distances between each two of the studied populations.

Table 4. Nei´s genetic distances between populations.

Populations ALI ISR ITA JAE MAD SEV ISR 0.0649 ITA 0.0421 0.0441 JAE 0.0409 0.0527 0.0357 MAD 0.0413 0.0388 0.0377 0.0354 SEV 0.0284 0.0500 0.0329 0.0254 0.0298 TAR 0.0434 0.0454 0.0352 0.0422 0.0340 0.0415

Figure 3. UPGMA dendrogram based on Nei´s genetic distances. Bootstrap values, based on 1000 replications, are shown near the corresponding branches.

Based on these distances, an UPGMA dendrogram were constructed (Figure 3). Bootstrap values, based on 1000 replications, are shown near the corresponding branches. The bootstrap values were generally low, supporting the idea that these populations are genetically so similar that they are difficult to separate (Haymer et al., 1997; Clements et al., 2000). 85

Notwithstanding, some interesting points were revealed in relation to the phylogeographic structure of this species. The most easterly of the Mediterranean populations studied, i.e. that from Israel (ISR) differ somewhat to the remaining populations (in agreement with published data: Nardi et al., 2005, and unpublished data: Segura et al.), may be due to its higher proximity to the origin of this pest. Whereas, the Italian population cluster together with the Spanish ones. The genetic distances indicate that all populations of the Northern Mediterranean are very similar (genetic distance 0.0254- 0.0649), which can be explained by gene flow. In the Iberian Peninsula, as well as in Italy, olive groves cover huge expanses of territory. This, along with the data obtained in this study, strongly suggests the existence of a large olive fly population in Northern Mediterranean. Summarizing, the methodology used (RAPD-PCR) prove to be useful for characterizing the genetic structure of the studied populations. A substantial level of polymorphism was detected, predominantly diversity within populations and low genetic diversity among populations probably due to important gene flow among them. Thus, in our opinion strategies for integrated control programs need to be coordinated between different geographical areas.

Acknowledgements

This work was funded by the EC Project FAIR3 CT96-1972 "Development of standardized molecular techniques for the identification of insect quarantine pests" The authors are grateful to F. Alonso, J.A. Cortés, A. García-Ortiz, E. Hernández-Ortiz, J.M. Llorens, S. Olmedo, N. Papadopoulos and S. Sibbett for help in collecting part of the samples.

References

Abrahamson, W.G. & Weis, A.E. 1997: Evolutionary Ecology Across Three Trophic Levels: Goldenrods, Gallmakers, and Natural Enemies. – Monographs in Population Biology 29. Princeton University Press. pp 456. Black IV, W.C. 1997: FORTRAN programs fort the analysis of RAPD-PCR markers in populations. – Colorado State University, Ft. Collins. Bush, G.L. &. Kitto, G.B. 1979: Research on the genetic structure of wild and laboratory strains of the olive fly. – In: Development of Pest Management Systems for Olive Culture Program. FAO Report, Rome: FAO. GRE69/525. Callejas, C., Roda, P., Reyes, A. &. Ochando, M.D. 1998: Identificación genética de Dacus – Bactrocera- oleae Gmelin (Diptera: Tephritidae) mediante marcadores RAPD-PCR. – Bol. San. Veg. Plagas 24: 873-882. Callejas, C. & Ochando, M.D. 2004: Allozymic variability in Spanish populations of Ceratitis capitata. – Fruits 59, 181-190. Clements, K.M., Sorenson, C.E., Wiegmann, B.M., Neese, P.A & Roe, R.M. 2000: Genetic, biochemical, and behavioral uniformity among populations of Myzus nicotianae and Myzus persicae. – Entomologia Experimentalis et Applicata. 95: 269-281. De Sousa, G.B., De Dutari, G.P. & Gardenal, C.N. 1999: Genetic structure of Aedes albi- fasciatus (Diptera: Culicidae) populations in Central Argentina determined by random amplified polymorphic DNA-polymerase chain reaction markers. – J. Med. Entomol. 36: 400-404. Downie, D.A., Fisher, J.R. & Granett, J. 2001: Grapes, galls, and geography: the distribution of nuclear and mtDNA variation across host plant species and regions in a specialist herbivore. – Evolution 55: 1345-1362. 86

Gasperi, G., Bonizzoni, M., Gomulski, L.M., Murelli, V., Torti, C., Malacrida A.R. & Guglielmino, C.R. 2002: Genetic differentiation, gene flow and the origin of infestations of the medfly, Ceratitis capitata. – Genetica 116: 125-135. Haymer, D.S., He, M. & McInnis, D.O. 1997: Genetic marker analysis of spatial and temporal relationships among existing populations and new infestations of the Mediterranean fruit fly (Ceratitis capitata). – Heredity 79: 302-309. Kourti, A. 2004a: Estimates of gene flow from rare alleles in natural populations of medfly Ceratitis capitata (Diptera: Tephritidae). – Bulletin of Entomological Research 94: 449- 456. Kourti, A. 2004b: Patterns of variation within and between Greek populations of Ceratitis capitata suggest extensive gene flow and latitudinal clines. – J Econ Entomol. 93: 1186- 1190. Lin, H., Downie, D.A., Walker, M.A., Granett, J. & English-Loeb, G. 1999: Genetic structure in native populations of grape phylloxera (Homoptera: Phylloxeridae). – Ann. Entomol. Soc. Am. 92: 376-381. Loukas, M. 1989: Population genetics studies of fruit flies of economic importance, especially medfly and olive fruit fly, using electrophoretic methods. – In: Electrophoretic studies on agricultural pests. Loxdale, H.D. & J.D. Hollander (eds.). Clarendon Press, Oxford. Loukas, M., Economopulos, A.P., Zouros, E.& Vergini, Y. 1985: Genetic changes in artificially reared colonies of the olive fruit fly (Diptera: Tephritidae). – Ann. Entomol. Soc. Am. 78: 159-165. Nardi, F., Carapelli, A., Dallai, R., Roderick, G.K. & Frati, F. 2005: Population structure and colonization history of the olive fly, Bactrocera oleae (Diptera, Tephritidae). – Mol. Ecol. 14: 2729-2738. Nei, M. 1972: Genetic distance between populations. – Am. Nat. 106: 283-292. Nei, M. &. Li, W.H. 1979: Mathematical model for studying genetic variation in terms of restriction endonucleases. – Proc. Natl. Acad. Sci. U.S.A. 76: 5269-5273. Ochando, M.D., Callejas, C., Fernández, O.H. & Reyes, A. 1994: Variabilidad genética alo- enzimática en Dacus oleae (Gmelin) (Diptera: Tephritidae). I. Análisis de dos poblacio- nes naturales del sureste español. – Bol. San. Veg. Plagas 20: 35-44. Ochando, M.D. & Reyes, A. 2000: Genetic population structure in olive fly Bactrocera oleae (Gmelin): gene flow and patterns of geographic differentiation. – J. Appl. Ent. 124: 177- 183. Ochando, M.D., Reyes, A., Callejas, C., Segura, D. & Fernández, P. 2003: Molecular genetic methodologies applied to the study of fly pests. – Trends in Entomology 3: 73-85. Reyes, A., Linacero, R.& Ochando, M.D. 1997: Molecular genetic and integrated control: A universal genomic DNA microextraction method for PCR, RAPD, restriction and Southern analysis. – IOBC/wprs Bulletin 20(4): 274-284. Rice, R.E. 2000: Bionomics of the olive fruit fly Bactrocera (Dacus) oleae. – UC Plant Protection Quarterly 10: 1-5. Rohlf, J. 1997: Numerical Taxonomy and Multivariate Analysis System. NTSYS-pc v2.01b. – Department of Ecology and Evolution, New York. Ruiz, A. 1948: Fauna entomológica del olivo en España. Estudio sistemático y biológico de las especies de mayor importancia económica. – Trabajos del Instituto Español de Entomología. Madrid. Segura, D., Callejas, C. & Ochando, M.D. Bactrocera oleae: a single large population in the Mediterranean basin?. (submitted to Bull. Entomol. Res.). Sneath, P.H.A. & Sokal, R.R. 1973: Numerical Taxonomy. – Freeman, San Francisco. 87

Tsakas, S. & Krimbas, C.B 1975: How many genes are selected in populations of Dacus oleae. – Genetics 79: 675-679. Tsakas, S. & Zouros, E. 1980: Genetic differences among natural and laboratory reared populations of the olive fruit fly Dacus oleae (Diptera: Tephritidae). – Entomol. Exp. Appl. 28: 268-276. Welsh, J. & McClelland, M. 1990: Fingerprinting genomes using PCR with arbitrary primers. – Nucleic Acids Res.18: 7213-7218. Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A. & Tingey, S.V. 1990: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. – Nucleic Acids Res. 18: 6531-6535. Zitoudi, K., Margaritopoulos, J.T., Mamuris, Z. & Tsitsipis, J.A. 2001: Genetic variation in Myzus persicae populations associated with host-plant and life cycle category. – Entomol. Exp. Appl. 99: 303-311. Zouros, E. & Krimbas, C.B. 1969: The genetics of Dacus oleae. III. Amount of variation at two esterase loci in a Greek population. – Gen. Res. 14: 249-258. Zouros, E. & Loukas, M. 1989: Biochemical and colonization genetics of Dacus oleae (Gmelin). – In: Fruit Flies: their Biology, Natural Enemies and Control. Robinson, A.S. and G. Hooper (eds.). Elsevier, Amsterdam.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 89-99

Susceptibility to Bactrocera oleae (Gmelin) of some Sicilian olive cultivars

Roberto Rizzo, Virgilio Caleca Dipartimento S.EN.FI.MI.ZO., Sez. Entomologia, Acarologia e Zoologia, Università di Palermo, viale delle Scienze, 90128 Palermo, Italy. [email protected]

Abstract: Genetic resistance of olive germplasm could be an important tool in the control of Bactrocera oleae (Gmelin), the key pest in Mediterranean Basin olive groves. Up to now, no study carried out on olive varieties stressed a complete resistance to the attack of B. oleae, although differences among olive cultivars in the susceptibility to olive fruit fly could be usefully considered both in organic and conventional olive cultivation, to obtain quality productions and to reduce insecticides use. The present study was carried out at Castelvetrano (Trapani province, Sicily), in the olive germplasm collection of Ente di Sviluppo Agricolo of the Sicilian Region. From 2002 to 2005, the assessment of susceptibility was made recording infestation levels on 18 cv, representing the most widely cultivated in Sicily. Samplings were carried out every 11-20 days, starting from the second half of August to the end of October. Moreover, from 2003 to 2005 infestation levels were correlated with hardness and size of the olives, while in 2004-2005 further data on olive colouration were collected at different ripening stages. A positive correlation between infestation and olive sizes was found, resulting in higher infestation levels recorded on the cultivars producing larger olives. A negative correlation between hardness and infestation was found in the early olive growing, until they reached almost definitive sizes. B. oleae showed to have a clear preference for green drupes, instead of reddish or blackish ones. Among the cultivars producing larger olives, Nocellara del Belice resulted the susceptible to the olive fly attacks, while Nocellara messinese was the less infested. Among cultivars with medium and small-sized fruits Moresca, Vaddarica, Nasitana frutto grosso, Minuta and Bottone di gallo were the less suceptible.

Key words: olive fruit fly, fruit size, fruit hardness, organic farming.

Introduction

The olive fly, Bactrocera oleae (Gmelin), is considered the olive key pest in Mediterranean Basin. As a consequence of ECC incentives, during the last five years many olive growers changed cultivation strategy from conventional to organic method, although the olive fly control is very difficult, because of the limited availability of effective products as permitted by Council Regulation (EEC) No 2092/91. Several studies were carried out on the effectiveness of allowed products or of new natural substances (Belcari & Bobbio, 1999; Tsolakis & Ragusa, 2002; Petacchi & Minnocci, 2003; Saour & Makee, 2004) and on the susceptibility of different cultivars to the olive fly attacks (Donia, 1971; Neuenschwander et al., 1985; Iannotta, 1999). In Sicily many local cultivars are well characterized and regularly cultivated; nevertheless their resistance to the olive fly infestation is not yet deeply investigated. The aim of the present research is to assess the susceptibility of the most widespread Sicilian cultivars, to better control the olive fly and to give useful information for new olive groves planting.

89 90

Materials and methods

From 2002 to 2005, the research was carried out on 18 Sicilian cultivars, in the experimental olive grove “Campo Carboj” of ESA (Ente Sviluppo Agricolo, Regione Siciliana), located at Castelvetrano (Trapani Province). A list of the 18 cultivars is reported in Table 1; La Mantia et al. (2005) now consider Pizzo di corvo as junior synonym of Giarraffa. In the different years, the cultivars with not enough drupes for the minimum sampling were not included in the research. Each thesis consisted of three untreated plants for each cultivar, subjected to the same cultural practices. Male olive fly population was monitored by wing traps baited with the specific pheromone (1.7-dioxaspiro [5.5] undecane). During each year, two-three traps were placed in the field from the end of July-beginning of August to the end of October. Traps were checked every week, and pheromone dispensers were replaced every 30 days.

Table 1. Sicilian olive cultivars tested in the research; cultivars are listed in descending order of size

No CULTIVAR 2002 2003 2004 2005 SIZE*(cm3) 1 Pizzo di corvo (= Giarraffa) X X X X 11.1 l 2 Giarraffa X X X X 9.6 l 3 Tonda Iblea X 8.2 l 4 Nocellara messinese X X X X 7.7 l 5 Nocellara del Belice X X X X 6.6 l 6 Carbucia X 6.6 l 7 Moresca X X X X 5.3 m 8 Vaddarica X X X X 4.5 m 9 Nasitana frutto grosso X X X X 4.3 m 10 Cerasuola di Sciacca X X X X 4.2 m 11 Biancolilla Caltabellota frutto grosso X X 4.1 m 12 Calatina X X 3.0 m 13 Piricuddara X X X X 2.5 s 14 Biancolilla Caltabellotta frutto piccolo X X 2.2 s 15 Bottone di gallo X X X X 2.1 s 16 Castricianella rapparina X X X X 1.9 s 17 Minuta X X X X 1.9 s 18 Olivo di Mandanici X X X X 1.5 s TOTAL No PER YEAR 15 16 14 15 *average size ([(π/6) x max.D.] x min.D.2) at the end of October calculated from 2003 to 2005; l = large, m = medium, s = small.

From August to October, samples of 60 drupes (20 per tree) in 2002-2004 and 90 drupes (30 per tree) in 2005 were randomly collected every 11-20 days, at a 1.70 m height and around all the tree. In the laboratory olives were examined under a steromicroscopy, to check

the presence of oviposition punctures. Olives were also sectioned to record the presence of the different preimaginal stages. The number of eggs, larvae of the different stages, pupae and exit holes was recorded to calculate the total infestation. Furthermore, sterile stings (punctures not followed by oviposition) and empty galleries were counted. Moreover, during 2003-2005, on 30 sampled drupes of each cultivar, the following biometric data were recorded: hardness, maximum diameter (max.D.), minimum diameter (min.D.). The latter two measurements were used to calculate the olive volume ([(π/6) x max.D.] x min.D.2). A visual analysis of olive colour was carried out on 30 drupes of the last sample collected on 2003 and of all the 2004 samples, and on all the 90 olives collected during 2005. Three different colouration classes were adopted to classify olives: green, reddish (mostly during viraison), and blackish (completely mature olives). Climatic data from the agrometeorological station located at Castelvetrano (30 m a.s.l., Trapani Province) were kindly provided by S.I.A.S. (Servizio Informativo Agrometeorologico Siciliano of Government of the Sicily Region). Data on total infestation recorded at each sampling date were statistically evaluated by ANOVA followed by Tukey post-hoc test at confidence level p<0.05. Pearson linear correlation (p<0.05) total infestation/olive volume and total infestation/hardness was calculated at each sampling data from 2003 to 2005.

Results

2002 In all samplings the four cultivars with a large fruit were among the most infested cultivars with the exception of Nocellara messinese whose infestation did not statistically differ from the four smaller cultivars (Table 2).

Table 2. Total infestation in 15 Sicilian olive cultivars (listed in descending order of size) and statistical analysis (ANOVA 1-way followed by Tukey post-hoc test; p<0.05). Year 2002.

CULTIVAR 03/09/02 18/09/02 01/10/02 15/10/02 29/10/02 Pizzo di corvo 0.17 b c 0.73 a b 2.09 a 1.63 a b 1.59 b Giarraffa 0.43 a 0.94 a 1.47 b 1.29 b c d 1.41 b c d Nocellara messinese 0.04 c 0.26 c d 0.56 f 0.54 h 0.78 e f Nocellara del Belice 0.29 a b 0.46 b c 1.18 b c 1.64 a b 1.50 b c Moresca 0.13 b c 0.15 c d 0.59 d e f 1.13 c d e 0.22 f Vaddarica 0.11 b c 0.71 a b 0.60 e f 1.09 c d e f 0.79 e f Nasitana f.g. 0.19 b c 0.70 a b 1.01 c d e 1.04 d e f g 0.97 e Cerasuola di Sciacca 0.17 b c 0.70 a b 1.19 b c 0.59 g h 1.14 c d e Biancolilla Caltab. f.g. 0.03 c 0.10 d 1.06 b c d 1.81 a 1.14 c d e Piricuddara 0.03 c 0.24 c d 0.51 f 0.89 d e f g h 2.16 a Biancolilla Caltab. f.p. 0.07 b c 0.31 c d 1.13 b c 1.54 a b c 1.03 c d e Bottone di gallo 0.03 c 0.30 c d 0.49 f 0.70 e f g h 1.02 d e Castricianella rapp. 0.00 c 0.10 d 0.43 f 0.66 e f g h 1.17 c d e Minuta 0.01 c 0.26 c d 0.44 f 0.61 f g h 0.91 e Olivo di Mandanici 0.04 c 0.14 d 0.30 f 1.03 d e f g 0.86 e

In the last two sampling dates Moresca, Vaddarica and Nasitana f.g. probably because of their early dark colouration resulted among the less infested cultivars in the last two sampling dates. In the last sampling date Piricuddara, with olives still green in that moment, reached the hightest infestation although its small size.

0.40 a 0.35 ab abc ab 0.30 abc 0.25 abc abc abc 0.20 abc

per drupe per 0.15 abc abc 0.10 bc bc bc c c Average total infestation 0.05

0.00 Minuta Calatina Giarraffa Moresca Carbucia Vaddarica Piricuddara Tonda Iblea Nasitana f.g. Nasitana Pizzo di corvo Pizzo di Bottone di gallo Olivo di Mandanici Castricianella rapp. Nocellara del Belice del Nocellara Nocellara messinese Nocellara Mean Cerasuola di Sciacca Cultivars Mean±0.95*ES

Figure 1. Total infestation level in 15 Sicilian olive cultivars (listed in descending order of size) on October 31st 2003 (Different letters denote statistically significant differences; ANOVA 1- way followed by Tukey test; p<0.05)

2003 During this year, B. oleae infestation was very low, mostly due to the high temperatures. Significant differences among the cultivars were recorded only at the last sampling date October 31st (Figure 1). Eight out of sixteen cultivar did not show any statistical difference from other cultivars also in this date. The highest infestation level was recorded on Olivo di Mandanici, Piricuddara and Calatina, which differed from only 5 cultivars, and also on differing from Nocellara messinese and Nasitana f. g. least infested cultivars. Sterile stings were more abundant than the total infestation, in the first five sampling dates, while in the last date they were at the same level of total infestation, due to the need of adult females to get liquid food (Girolami, 1978). Biometric analysis on hardness and size of drupes from the different cultivars showed significant differences among them. The two cultivars with highest hardness values resulted Nocellara messinese and Piricuddara, characterized by large and small size of olives, respectively.

Correlations infestation/hardness and infestation/size were not significant, until the last sampling date. At this date (30/10/2003), a significant positive correlation was found between infestation and hardness, while a significant negative correlation was found between infestation and size (Table 3).

Table 3. Pearson linear correlation infestation/size and infestation/hardness of variously coloured drupes of 16 sicilian olives cultivars in 2003. (Values in bold indicate statistically significant correlation; p<0.5)

SIZE HARDNESS INFESTATION -0.05 0.03 4 September INFESTATION 0.06 -0.03 23 September INFESTATION 0.00 -0.03 15 October INFESTATION -0.17 0.10 30 October

Table 4. Total infestation in 14 Sicilian olive cultivars (listed in descending order of size) and statistical analysis (ANOVA 1-way followed by Tukey test; p<0.05) – Year 2004

CULTIVAR 03/09/04 20/09/04 06/10/04 26/10/04 Pizzo di corvo 0.00 b 0.28 b c 0.73 a b c 2.25 a Giarraffa 0.06 b 0.55 a 0.70 a b c d 1.38 b Nocellara messinese 0.00 b 0.02 d 0.88 a b 0.75 b c Nocellara del Belice 0.07 b 0.32 a b 0.95 a 2.80 a Moresca 0.18 a 0.08 b c d 0.28 d 0.73 b c Vaddarica 0.03 b 0.25 b c d 0.60 a b c d 0.80 b c Nasitana f.g. 0.00 b 0.08 b c d 0.53 a b c d 0.82 b c Cerasuola di Sciacca 0.00 b 0.13 b c d 0.50 b c d 1.18 b c Piricuddara 0.00 b 0.05 c d 0.37 c d 0.95 b c Biancolilla Caltab. f.p. 0.00 b 0.22 b c d 0.87 a b 0.83 b c Bottone di gallo 0.00 b 0.12 b c d 0.48 b c d 1.05 b c Castricianella rapp. 0.00 b 0.15 b c d 0.35 c d 0.93 b c Minuta 0.00 b 0.18 b c d 0.48 b c d 0.53 c Olivo di Mandanici 0.02 b 0.02 d 0.45 b c d 1.13 b c

2004 In all samplings cultivars with large drupes were the most attacked, excepting Nocellara messinese on September 20th. In the last date Nocellara del Belice and Pizzo di corvo recorded the highest infestation significantly differing from all cultivar, while Minuta was the least infested without significant differences from ten other cultivars (Table 4). In this year, in all the sampling dates, a significant positive correlation was found between the infestation levels and the olive size, indicating that olive flies prefer to oviposit

on larger drupes. The correlation between infestation and hardness resulted statistically negative on the 20th of September, and positive in the last date (Table 5). Moreover, limiting the analysis to the green olives, a significant positive correlation was confirmed between infestation and olive size, while a significant negative correlation was found only on September 20th (Table 6).

Table 5. Pearson linear correlation infestation/ Table 6. Pearson linear correlation between size and infestation/hardness of infestation/size and infestation/hard- variously coloured drupes of 14 ness of green drupes of 14 Sicilian Sicilian olives cultivars in 2004 olives cultivars in 2004 INFESTATION SIZE HARDNESS INFESTATION SIZE HARDNESS

0.12 -0.07 0.12 -0.07 3 September 3 September

0.21 -0.13 0.23 -0.16 20 September 20 September

0.14 0.10 0.16 -0.03 6 October 6 October

0.17 0.27 0.26 0.11 26 October 26 October Values in bold indicate statistically significant correlation (p<0,05)

2.2 a 2.0 1.8 1.6 b 1.4 b 26/10/04 1.2 1.0 0.8 Average infestation total 0.6 Green Reddish Blackish Coloration

Figure 2. Total infestation of differently coloured drupes of 14 Sicilian olives cultivar (Different letters denote statistically significant differences; ANOVA 1-way followed by Tukey post- hoc test; p<0.05)

The different results obtained in the last sampling date indicates that olive fly females prefer larger and green olives for oviposition; on October 26th, green olives resulted statistically more infested than the reddish and blackish ones (Fig. 2).

2005 During the first three sampling dates, infestation levels were low on all cultivars. Only on Nocellara del Belice the infestation resulted significantly higher at the first sampling date.

Excluding September 9th in all samples the infestation level of Nocellara del Belice was the highest of large-sized of cultivars, with the following statistically significant differences from: Nocellara messinese in all dates, Giarraffa in all dates except September 23rd, Pizzo di corvo in all dates except September 23rd and October 7th.

Table 7. Total infestation in 15 Sicilian olive cultivars (listed in descending order of size) and statistical analysis (ANOVA 1-way followed by Tukey test; p<0.05). Year 2005

CULTIVAR 26/08/05 8/09/0523/09/05 7/10/05 20/10/05 31/10/05 Pizzo di corvo 0.00 b 0.00 a 0.15 a b 0.49 a b c 0.63 e f g h 2.85 b c Giarraffa 0.00 b 0.03 a 0.12 a b c 0.46 b c d 0.88 d e f g 2.36 c Nocellara messinese 0.00 b 0.00 a 0.00 c 0.16 e f 0.92 d e f 1.51 d Nocellara del Belice 0.05 a 0.00 a 0.17 a 0.71 a 2.43 a 5.24 a Moresca 0.00 b 0.00 a 0.07 a b c 0.14 f 0.30 h i 1.15 d e Vaddarica 0.00 b 0.00 a 0.00 c 0.13 f 0.50 g h i 0.64 e Nasitana f.g. 0.00 b 0.02 a 0.10 a b c 0.09 f 0.21 h i 0.88 e Cerasuola di Sciacca 0.00 b 0.02 a 0.03 a b c 0.48 a b c d 1.68 b 3.52 b Biancolilla Caltab. f.g. 0.02 a b 0.02 a 0.13 a b c 0.52 a b 1.62 b 2.12 c Calatina 0.00 b 0.00 a 0.02 b c 0.39 b c d e 0.54 f g h i 1.31 d e Piricuddara 0.00 b 0.00 a 0.02 b c 0.27 c d e f 1.57 b c 1.36 d e Bottone di gallo 0.00 b 0.05 a 0.02 b c 0.24 d e f 0.24 h i 1.13 d e Castricianella rapp. 0.00 b 0.00 a 0.00 c 0.20 e f 0.98 d e 2.56 c Minuta 0.00 b 0.00 a 0.00 c 0.07 f 0.20 i 1.01 d e Olivo di Mandanici 0.00 b 0.02 a 0.00 c 0.08 f 1.17 c d 2.19 c

In the last three dates Nocellara del Belice showed an infestation higher than that one in all other cultivars excepting Biancolilla Caltabellotta f. g. and Cerasuola di Sciacca on October 7th. Among medium and small size cultivars these last two cultivars plus Piricuddara, Castricianella rapparina and Olivo di Mandanici were the most attacked. In variously coloured olives the positive correlation between infestation and olive sizes resulted statistically significant from September 23rd to the end of samplings; the correlation between infestation and drupe hardness was significantly negative on the August 26th, September 8th, October 20th and 31st, while it was significantly positive on the October 7th (Table 8). In green olives positive correlation between infestation and size is confirmed as statistically significant from September 23rd to the last sampling date. Differently from analysis in variously coloured fruits, infestation and hardness in green olives resulted positively correlated on October 20th, and not significant on October 7th and 31st (Table 9). These data to indicate that, when the olives are all unripe and green, B. oleae preferably oviposits on softer olives. When olives are completely grown in size, oviposition occurs mostly on larger and still green drupes. Infestation on olives characterized by different colouration (Figures 3-4) resulted statistically higher on green olives than on reddish and blackish ones, on October 20th and 31st.

Table 8. Pearson linear correlation infestation Table 9. Pearson linear correlation infestation /size and infestation/hardness of /size and infestation/hardness of variously coloured drupes of 15 green drupes of 15 Sicilian olives Sicilian olive cultivars in 2005 cultivar in 2005 INFESTATION SIZE HARDNESS INFESTATION SIZE HARDNESS

26 August 0.09 -0.09 26 August 0.09 -0.09

8 September -0.11 -0.11 8 September -0.01 -0.11

23 September 0.10 -0.04 23 September 0.11 -0.03 7 October 0.25 0.10 7 October 0.29 -0.05

20 October 0.14 -0.30 20 October 0.23 0.15

31 October 0.26 -0.13 0.57 -0.11 31 October Values in bold indicate statistically significat correlation (p<0,05)

2.8 a 2.6 3 b 2.4

2.2 2.0

31/10/05 1.8 b 1.6

1.4 Average total infestation Average total 1.2 Green Reddish Blackish Coloration

1.4 a 1.2 4 1.0

0.8 b

0.6

20/10/05 c 0.4

0.2 Average total Infestation Average total 0.0 Green Reddish Blackish Coloration

Figures 3-4. Total infestation of differently coloured drupes of 15 Sicilian olives cultivar (Different letters denote statistically significant differences; ANOVA 1-way followed by Tukey post- hoc test; p<0.05)

Discussion

The results of our research confirm that none of tested Sicilian olive cultivars is resistant to olive fly attack. Nevertheless, a range of susceptibility among the different cultivars was found. The sizes of drupes is considered by several authors one of the most important factors in the choice of olives by B. oleae female (Pucci & Ambrosi, 1981; Jimenez, 1988). The positive significant correlation between infestation levels and olive sizes seems to confirm this relationship. Moreover, the infestation level on cultivars characterized by large drupe size resulted usually higher than that one recorded on cultivars bearing small olives (Figure 5). Olive hardness was proved to be another important factor in determining the choice of drupes for oviposition by B. oleae females (Martin, 1948; Orphanidis et al., 1958). The occurrence of a negative significant correlation between infestation and hardness was confirmed mostly during the early developing and ripening period of the olives, when all drupes are completely green, showing that hardness play an important role until the end of August- half of September. Afterwards, when olives reach their nearly final sizes and become softer, they turn dark- coloured. Also the olive colouration seems to play a role in females choice (Katsoyannos, 1989). Indeed, green olives resulted more infested than brown ones, as found also by Orphanidis et al. (1959) and Cirio (1971). As a result, cultivars such as Giarraffa and Pizzo di corvo, charachterized by large olives and early ripening period, were the most infested in September. Afterwards, the complete viraison of olives from these two cultivars, lead the olive fly females to prefer other cultivars for oviposition.

2002 2003 2004 2005 Average 2002-05

Olivo di Mandanici fg a cd c cd

Minuta g a d e e

Castricianella rapparina fg a cd c cd Bottone di gallo efg a cd de de

Piricuddara d a d c c

Cerasuola di Sciacca de a cd b b Nasitana frutto grosso cd a cd e de

Vaddarica def a cd e de

Moresca g a d de e

Nocellara del a a a a Belice bc

Nocellara a cd cd de Messinese fg

Giarraffa ab a bc c b

Pizzo di a ab c b corvo a

0,00,51,01,50,0 0,5 1,0 0,0 0,5 1,0 1,5 0,0 0,5 1,0 1,5 2,0 0,0 0,5 1,0 1,5

Figure 5. Mean values of total infestation in September and October in 13 Sicilian olive cultivars (listed in ascending order of size) from 2002 to 2005, and statistical analysis (repeated measurements ANOVA followed by Tukey test, p<0.05)

In the cultivars Moresca, Nasitana f. g. and Vaddarica, characterized by medium size olives and early maturation, almost 50% of olives on the trees is already blackish, in the first week of October; these three cultivars thus avoided the most harmful olive fly attack, commonly occuring at the end of October. Among the tested cultivars, Nocellara del Belice resulted the most susceptible to the olive fly attack, both for the large olive size and the still green colouration at the end of October. On the other hand, Nocellara messinese, in spite of the large olive size, resulted one of the less attacked cultivar, with infestation levels similar to the small cultivars (Figure 5). As Nocellara del Belice and Nocellara messinese have also in common a high fruit hardness and a green colouration until the end of October, but their susceptibility is highly different, other (physical, chemical) factors are surely involved in determing this difference. Also in the susceptibility to B. oleae attacks Giarraffa and Pizzo di corvo did not show statistically significant differences in mean values of the all the years, according to La Mantia et al., (2005) considering them synonyms. Among the cultivars producing small olives, Minuta showed the lowest susceptibility, probably due to both the small olive size and to the brown colouration that more than 50% of olives had in the first half of October. The wide range of susceptibility level shown by tested cultivars could be useful in organic olive growing. In the most susceptible cultivars, to limit damages due to B. oleae early harvesting and effective interventions are necessary; less susceptible cultivars (Nocellara messinese, Moresca, Vaddarica, Nasitana f. g., Minuta, Bottone di gallo) could be suggested for new organic olive plantings for oil or table olives production.

Acknowledgements

We thank E.S.A. (Regione Siciliana), Dipartimento di Colture Arboree (University of Palermo), Dr. Dario Parrivecchio, Fabio Castronovo and Vito Mazzara for their help in samplings, Dr. Gabriella Lo Verde for the critical reading of the text. Research funded by University of Palermo ex quota 60% (“Il controllo degl’insetti fitofagi nell’agricoltura biologica e convenzionale”).

References

Belcari, A. & Bobbio, E. 1999: L’impiego del rame nel controllo della mosca delle olive Bactrocera oleae. – Informatore fitopatologico 49 (12): 52-55. Cirio, U. 1971: Reperti sul meccanismo stimolo-risposta nell'ovideposizione del Dacus oleae Gmelin (Diptera Trypetidae). – Redia 52: 577-600. Donia, A. R., El Sawaf, S. K., Abou Ghadir, M. F., Sawaf, S. K. E. L., Ghadir, M. F. Abou 1971: Number of generations and seasonal abundance of the olive fruit fly, Dacus oleae (Gmel.) and the susceptibility of different olive varieties to infestation (Diptera: Trypetidae). – Bulletin de la Societe Entomologique d'Egypte 55: 201-209. Girolami, V. 1978: Note demo-ecologiche su Dacus oleae Gmelin. – Notiziario sulle Malattie delle Piante 98-99 (III Serie 25-26): 11-25. Iannotta, N. 1999: Suscettibilità di diverse cv di olivo alla mosca e all'occhio di pavone. – L'Informatore Agrario 48: 69-73. Jimenez, A. 1988: Influencia de la variedad de olivo en el comportamiento ovipositor de Dacus oleae Gmel.. – Boletin de Sanidad Vegetal. Plagas 14: 95-98. Katsoyannos, B. I. 1989: Response to Shape, Size and Color. – In: Robinson A.S., G. Hooper (Eds.) “Fruit Flies their Biology, Natural Enemies and Control”: 307-324.

La Mantia, M., Lain, O., Caruso, T., Testolin, R. 2005: SSR-based DNA fingerprints reveal the genetic diversità on Sicilian olive (Olea europeae L.) germplasm. – Journal of Horticultural Science and Biotechnology 80 (5) 628-632. Martin, H. 1948: Observations biologiques et essais de taitements contre la mouche de l’olive (Dacus oleae Rossi) dans la province de Terragone (Espagne) de 1946 à 1948. – Bull. Soc. ent. Suisse 21: 361-402. Neuenschwander, P., Michelakis, S., Holloway, P., Berchtold, W. 1985: Factors affecting the susceptibility of fruits of different olive varieties to attack by Dacus oleae (Gmel.) (Diptera Tephritidae). – Zeitschrift für Angewandte Entomologie 100: 174-188. Orphanidis, P. S., Alexopoulou, P.S., Plytas, F.M., Tsakmakis, A.A. 1958: La duretée de la surface du fruit de l’olive en corrélation avec l’intensité de l’attaque du Dacus. Expérience s préliminaires d’application de résines synthétiques pour l’augmentation de la duretée. – Ann. Inst. Phytopathol. Benaki (N.S.) 1: 223-228. Orphanidis, P. S., Phytizas, E. A., Tsakmakis, A.A. 1959: Quelques observations sur l’intensité de l’attaque du Dacus, en fonction du degré de maturation de l’olive. – Ann. Inst. Phytopathol. Benaki (N.S.) 2: 144-148. Petacchi, R. & Minnocci, A. 2002: Olive fruit fly control methods in sustainable agriculture. – Acta Horticulturae 2002: (586): 841-844 Pucci, C. & Ambrosi, G. 1981: Ovideposizione del Dacus oleae (Gmel.) e dimensioni delle drupe. – Frustala Entomologica (N.S.) 4: 181-194. Saour, G. & Makee, H. 2004: A kaolin-based particle film for suppression of the olive fruit fly Bactrocera oleae Gmelin (Dip., Tephritidae) in olive groves. – Journal of Applied Entomology 128: 28-31. Tsolakis, H. & Ragusa, E. 2002: Prove di controllo di Bactrocera oleae (Gmelin) (Diptera, Tephritidae) con prodotti a basso impatto ambientale. – Phytophaga 12: 141-147.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 101-105

Behavioural responses of the olive fly, Bactrocera oleae, to chemicals produced by Pseudomonas putida in laboratory bioassays

Serena Landini, Aurelio Granchietti, Michele Librandi, Alessandra Camèra, Marzia Cristiana Rosi, Patrizia Sacchetti, Antonio Belcari Department of Agricultural Biotechnologies, Section of General and Applied Entomology, University of Florence, Italy

Abstract: The behavioural responses of the female olive fly, Bactrocera oleae (Rossi), to bacterial odours were studied in laboratory bioassays with a Y-tube olfactometer and a wind tunnel. In both experiments females showed a significant response to odours emitted by bacterial filtrates compared to a commercial bait usually employed in the field as an attractant for adult fruit flies.

Key words: Bactrocera oleae, volatiles, bacteria, Pseudomonas putida, Y-tube olfactometer, wind tunnel.

Introduction

Fruit flies belonging to the Bactrocera genus establish close interactions with micro- organisms, especially with bacteria, that can evolve into symbiotic relationships (Drew & Lloyd, 1989). Due to their co-evolution, fruit flies involved in these associations have developed recognition mechanisms to enhance their fitness. It has been demonstrated that B. tryoni uses chemicals produced by epiphytic bacteria for orientation towards host plants (Drew, 1987a; Drew, 1987b). Recently a relationship was found between the olive fly, Bactrocera oleae (Rossi), and bacteria living on the olive phylloplane (Granchietti et al., 2005). It is also known that micro-organisms are essential for the normal development of the fly (Manousis & Ellar, 1988). On this basis we hypothesized that adult olive flies may be attracted by volatiles produced by bacteria. Pseudomonas putida is a ubiquitous bacterium of the olive phylloplane, and it has also been isolated on the oesophageal bulb of both field and lab-reared olive flies (Belcari et al., 2003). The effect of odours produced by P. putida on the behaviour of the olive fly were studied in olfactometer and wind tunnel laboratory bioassays.

Materials and methods

P. putida was cultured in liquid Tryptic Soy Broth medium (TSB) for 6 days in an orbital shaker. The culture was centrifuged, filtrated and tested at concentrations of 1% in Y-tube olfactometer tests and 5, 10 and 20% in wind tunnel experiments. The odours produced by the bacterial filtrate diluted at different concentrations were compared with a commercial proteinaceous bait (Buminal®) and the Tryptic Soy Broth medium (TSB). In all the experiments females were tested separately. In the Y-tube olfactometer an air flow enters the paired arms and the two streams converge longitudinally in the common arm, generating two distinct flows that remain in contact along the middle line of the arm. The insect, in the common arm, can thus perceive the two separate odours. Several treatments were tested: water (W), the commercial bait Buminal® (PB), P. putida bacterial filtrate (BF) and the Tryptic Soy Broth medium (TSB).

101 102

All the odour sources were tested at 1% concentration. Different pairs of treatments were tested: Buminal® and bacterial filtrate were compared with water (PB-W, BF-W) and with each other (BF-PB); bacterial filtrate was also compared with the medium (BF-TSB). Bioassays were analyzed using a video-tracking software (X-bug, University of Palermo, Italy) to register the walking behavioural parameters of the flies in the common and paired arms of the Y-tube olfactometer. In the common arm the pre-choice time (s) (time spent before entering the paired arms), linear speed (mm/s) and angular speed (°/s) were measured. All the parameters were analyzed using the Kruskall-Wallis test and comparisons made between the experiments. In the paired arms only data concerning the first visit to one of the arms were measured, that is the duration and the walking parameters. Data were analyzed using the Kruskall-Wallis test, and the different treatments were compared. The same treatments tested in the Y-tube olfactometer were used in wind tunnel experiments, though the TSB medium and the bacterial filtrate were used at higher concentrations of 5, 10 and 20% (BF5, BF10, BF20, TSB5, TSB10, TSB20). In the wind tunnel bioassays treatments were tested singly. Insects were observed for 5 minutes, and the number of flying and non-flying females was recorded. The results were analyzed using the chi-square goodness-of-fit test; the expected frequencies were defined at the proportion of 1:1. All the indicated statistical analyses were done using exact tests based on the Monte- Carlo simulation (Sokal & Rohlf, 1995).

Results

In the common arm, there were significant differences between the treatments with regard to pre-choice time, linear speed and angular speed, as evidenced by the Kruskall-Wallis test (pre-choice time H=10.080, p= 0.021; linear speed H= 13.703, p= 0.006; angular speed H= 13.703, p= 0.006).

Table 1. Behavioural responses of B. oleae females in the Y-tube olfactometer common arm exposed to bacterial filtrate and other nitrogen sources.

Response variable Treatments Average Pre-choice time (s) PB-W 8.11 a BF-W 9.23ab BF-PB 23.26bc BF-TSB 23.53 c Linear speed (mm/s) PB-W 16.26 a BF-W 26.64 a BF-PB 11.71b BF-TSB 11.05 b Angular speed (°/s) PB-W 205.41 a BF-W 205.04a BF-PB 252.84ab BF-TSB 303.74 b Kruskal-Wallis Test; p-value estimated by Monte Carlo Simulation; generated random samples: 1000. (PB-W n=26; BF-W n=23; PB-BF n= 29; BF-TSB n= 27). 103

Comparisons between pre-choice times show that females enter a paired arm faster when the protein bait Buminal® or P. putida bacterial filtrate was contrasted with water, without any other odour source (PB-W, BF-W); there were no significantly different times between these treatments. Flies walk for a longer time in the common arm when the bacterial filtrate is present with another nitrogen source, as in the case of the BF-PB and BF-TSB combinations. In these experiments the duration is similar, though the time employed to enter one of the arms in the BF-PB experiment is about twice as long as the BF-W experiment; in addition, it is not significantly different (Table 1). In the paired arms the analysis of parameters considered during the first path indicates that the duration of the visit in the arm with the bacterial filtrate (BF) is significantly longer compared to the duration in the arm with the nitrogen bait Buminal® (PB). Similarly, in the experiments where the bacterial filtrate (BF) is compared to the medium (TSB), the linear speed is higher in the arm treated with the bacterial filtrate (Table 2).

Table 2. Behavioural responses of B. oleae females in the Y-olfactometer paired arms exposed to bacterial filtrate and other nitrogen sources.

Arm 1 Arm 2 Response variable Comparisons p Trt avg Trt avg Duration (s) PB-W PB 54.2164 W 77.4492 0.693 BF-W BF 71.3891 W 35.8850 0.753 BF-PB BF 73.9094 PB 9.1569 0.037 BF-TSB BF 58.6243 TSB 12.9138 0.185 Linear speed (mm/s) PB-W PB 12.1083 W 7.6514 0.867 BF-W BF 21.3591 W 15.8475 0.674 BF-PB BF 7.8306 PB 10.0500 0.228 BF-TSB BF 8.1371 TSB 11.4262 0.031 Angular speed (°/s) PB-W PB 278.0367W 349.1343 0.153 BF-W BF 330.2091W 321.2308 0.826 BF-PB BF 331.9719PB 288.3577 0.236 BF-TSB BF 311.5114TSB 268.9192 0.095 Kruskal-Wallis Test; p-value estimated by Monte Carlo Simulation; generated random samples: 1000.

In the wind tunnel experiments (Table 3) there was a significant difference in the proportions of flying and non-flying females between bioassays with nitrogen bait (PB) and those with the bacterial filtrate at concentrations of 10 and 20% (BF10, BF20).

Discussion

The results of these preliminary bioassays show that olive fly females respond to odours present in the bacterial filtrate. In the olfactometer experiments all the parameters measured in the common arm (pre-choice time, liner speed and angular speed) evidence that the behavioural responses of the olive flies to the bacterial filtrate are no different than they are to the proteinaceous bait Buminal®. This bait is employed in IPM programmes and the response 104 of B. oleae to hydrolysed proteins contained in this product is well documented (Monaco, 1981; Delrio et al., 1983).

Table 3. Behavioural responses of B. oleae females in wind tunnel experiments with bacterial filtrate and other proteinaceous sources at different concentrations.

Flying Non flying Treatments N χ2 p females % females % Water 25 56.0 44.0 0.360 0.630 PB 18 88.9 11.1 10.889<0.001 TSB5 10 80.0 20.0 3.600 0.106 TSB10 10 60.0 40.0 0.400 0.747 TSB20 8 62.5 37.5 0.500 0.729 BF5 14 57.1 42.9 0.286 0.808 BF10 12 83.3 16.7 5.333 0.036 BF20 11 90.9 9.1 7.364 0.016 Goodness-of-fit chi square test; p-value estimated by Monte Carlo simulation; generated random samples: 1000.

When the bacterial filtrate is combined with the proteinaceous bait (BF-PB) the average pre-choice time increases as well as the angular speed, while the linear speed decreases showing a localised walking behaviour. This is probably due to the presence of an additional proteinaceous source that may reduce or confuse the olfactory response of the flies. However most of the parameters are not significantly different to those of the BF-W experiment. A similar behaviour is evident in the BF-TSB experiment, where the presence of the tryptic soy broth again seems to induce a localised walking behaviour. Here, however, the results are different to both experiments PB-W and BF-W. Comparison between the behavioural responses of females in the paired arms shows differences for some parameters in the experiments where the bacterial filtrate was used with the proteinaceous bait or the tryptic soy broth (bioassays BF-PB and BF-TSB). B. oleae females stay longer in the arm with the bacterial filtrate than in the one with the bait (experiment BF-PB) and show higher linear speed when the bacterial filtrate is compared with the tryptic soy broth (experiment BF-TSB). However, it should also be borne in mind that the incomplete response of B. oleae to bacterial odours for some parameters could depend on the filtrate concentration; the Y-tube olfactometer experiments were performed with a bacterial filtrate concentration of 1%. Indeed, the bacterial filtrate concentration could have a role in inducing a response from the olive flies, as evidenced in wind tunnel experiments. In these latter experiments the response towards bacterial odours varies with the filtrate concentration. The wind tunnel results show that the percentage of flying females is higher in bioassays performed with bacterial filtrates at 10 and 20 percent as well as with Buminal®, while females do not display interest in the tryptic soy broth treatment at different concentration. The preliminary results deriving from the olfactometer and wind tunnel tests evidence a behavioural response of the olive fly females to chemicals fermented by P. putida and they encourage further studies in the laboratory and in the field aimed at developing new monitoring and control techniques.

105

Acknowledgements

This research was supported by PRIN 2003.

References

Belcari, A., Sacchetti, P., Marchi, G. & Surico, G. 2003: La mosca delle olive e la simbiosi batterica. – Informatore Fitopatologico 53 (9): 55-59. Delrio, G., Prota, R., Economopoulos, P.V., Economopoulos, A.P. & Haniotakis, G.E. 1983: Comparative study on food, sex and visual attractants for the olive fruit fly. – In: Fruit flies of economic importance, Proceedings of the CEC/IOBC International Symposium, Athens, Greece, 16-19 November 1982: 465-472. Drew, R.A.I. 1987: Relationship of fruit flies (Diptera: Tephritidae) and their bacteria to host plants. – Annals of the Entomological Society of America 80 (5): 629-636. Drew, R.A.I. 1987: Behavioural strategies of fruit flies of the genus Dacus (Diptera: Tephritidae) significant in mating and host-plant relationships. – Bulletin of Entomo- logical Research 77(1): 73-81. Drew, R.A.I. & Lloyd, A.C. 1989: Bacteria associated with fruit flies and their host plants. – In: Robinson A.S. & Hooper G. (eds.), Fruit flies, their biology, natural enemies and control, Vol. 3A, World Crop Pest, Elsevier, Amsterdam: 131-140. Granchietti, A., Camèra, A., Landini, S., Rosi, M.C., Librandi, M., Sacchetti, P., Marchi, G., Surico, G., & Belcari, A. 2005: Relationship between olive fly adults and epiphytic bacteria of the olive tree. – IOBC/WPRS Bull. 30(9): 25-30. Manousis, T. & Ellar, D.J. 1988: Dacus oleae microbial symbionts. – Microbiological Sciences 5 (5): 149-152. Monaco, R. 1981: Prove sulla attrattività delle esche proteiche. – Informatore Fitopatologico 31 (1-2): 67-71. Sokal, R.R. & Rohlf, F.J. 1995: Biometry. The principles and practice of statistics in biological research. – W.H. Freeman & Co., N.Y. 887 pp.

Bactrocera oleae: Chemical, Biological and Biotechnical Control Methods, Side Effects

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 109

Sterile insect technique (SIT) – an environmentally friendly approach to controlling major fruit-fly pests

M. Kozanek1, C. Caceres2 1 Institute of Zoology, Slovac Academy of Sciences, Bratislava, Slovakia. 2 International Atomic Energy Agency, Wien, Austria.

The sterile insect technique (SIT) developed in the 1950s has since been sucessfully used for many insect key pests worldwide. The area wide approach is one of the most important prerequisite for successful application of SIT programme. SIT control strategies include release of male insects mass produced in specialized facilities that have been sterilized by irradiation. Mass production of males can be accomplished most effectively through genetic sexing strains of the pest. Genetic sexing strains GSS are based upon selectable characters linked to the male sex by using Y-autosome translocation. In the case of medfly, temperature sensitive lethal strain (tsl) is used which include temperature sensitive lethal (tsl) mutation in addition of recessive pupal colour mutation (wp). To avoid of the occurrence of recombinants during the mass rearing, the filter rearing system (FRS) has been developed. The FRS consists of small colony, physically cleaned of recombinants, which is “bridged” to a large operational colony via a short series of colony amplifications, called an amplification bridge. The operational colony provides rearing material to much larger colony – release stream – which is dedicated to providing all the flies for sterilization and field release. The first SIT program to control fruit flies (medfly) in Europe was running by Italian National Agency for New Technology, Energy and Environment in Italy, with experimental campains at Capri and Procida islands. Since1993, European Union is supporting Madeira- Med program using SIT as the control strategy for medfly control to level below the economic threshold in Madeira and Porto Santo Islands. Madeira-Med has operational units for field activities, Medfly mass production, quality control, fly handling and releases, public relations and administration. In last four decades, several successful SIT programmes to eradicate or control fruit flies were running all over the world. The melon fly, Bactrocera cucurbitae, has been completely eradicated from Okinawa, Japan in 1993. Following the expansion of target areas during the eradication campaign, the number of flies produced was increased from 5 million to 280 million per week. The Moscamed programme in Guatemala began in 1975. Since than, the programme has evolved into the largest medfly control and eradication effort in the Americas, if not even the world. The Mendoza medfly programme started in 1990 with the aim to eradicate Medfly in Mendoza province and subsequently from the whole territory of Argentina. The production of sterile males grew continually from 70 to 200 million per week. Shortly after initiation of the Programme, substance degree of Medfly suppression was achieved. Beside of numerous SIT programmes dedicated to control medfly in different parts of the world, this insect pest area-wide control technique is used also against several other fruit fly pests. One of the perspective candidates for SIT control is also olive fruit fly. In last decade, substantial progress was achieved in the development of rearing technology to produce sterile flies.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 111-117

Tests on the effectiveness of kaolin and copper hydroxide in the control of Bactrocera oleae (Gmelin)

Virgilio Caleca, Roberto Rizzo Dipartimento SENFIMIZO, Sezione di Entomologia, Acarologia e Zoologia, Università di Palermo, Viale delle scienze, 90128 Palermo ITALY. E-mail: [email protected]

Abstract: Repellent and antiovipositional products in the control of Bactrocera oleae (Gmelin) finds a great interest in organic farming, because of the lack of effective products able to kill the olive fly immature stages. In 2003 in Castelvetrano (Trapani province, Sicily), tests on the effectiveness of Surround WP, a product containing 95% of kaolin, were carried out on three table olive cultivars, Nocellara del Belice, Moresca and Tonda Iblea. In 2004, in the same field and on the same cultivars, BPLK kaolin was tested too. In the second year the two products containing kaolin were also tested on Cerasuola cultivar in an organic olive grove located in Trapani, comparing them with copper hydroxide. At Castelvetrano both in 2003 and in 2004 B. oleae infestation levels of the plots treated with the two products containing kaolin were statistically lower than those of the control plots. In this site, in 2004 Surround WP protected olives significantly better than BPLK kaolin, limiting olive fly harmful infestation up to 17-23% vs. 68-87% of BPLK plots. At Trapani in 2004, the two products containing kaolin and copper hydroxide showed statistically significant differences from the untreated control, but not among themselves, limiting the harmful infestation up to 3-37% vs. 87% of the control. The different results of 2004 recorded by Surround WP and BPLK kaolin in the two olive groves seems linked to the different rainfall of the period after the last treatment, 64 mm in three rainy days at Castelvetrano and 41 mm in eight rainy days at Trapani; BPLK kaolin was probably washed away more than Surround WP. The tested products containing kaolin and copper hydroxide are effectively able to limit B. oleae infestation to a very good level for olive oil production, moreover, considering the earlier harvesting of table olives, these products give a new opportunity for controlling the olive fly also in the organic olive groves for table olives production.

Key words: olive fruit fly, repellent, antiovipositional, organic farming, Sicily

Introduction

The use of repellent and antiovipositional products in the control of Bactrocera oleae (Gmelin) finds a great interest in organic farming, because of the lack of effective products able to kill the olive fly preimmaginal stages. From 1937 to 1953 Russo and some other entomologist (Russo, 1937; Russo and Fenili, 1949; Russo, 1954) tested the effectiveness of clay and Bordeaux mixture against the olive fly, obtaining a similar protection, suggesting their use for early ripening olives to harvest before autumnal rainfall. Visual and chemical stimuli lead the female olive fly to oviposit into fruits (Katsoyannos and Kouloussis, 2001; Rotundo et al., 2001; Solinas et al., 2001); so the clay, especially white clays as kaolin, disrupts ovipositing females, while copper salts through their antibacterial action make fruits less attractive to ovipositing females because of the lack of some bacterial compounds on the surface of fruits (Tsanakakis, 1985; Belcari et al. 2003), furthermore the

111 112

presence of the particles of these products on fruit surface could be another obstacle for the fruit recognition of the female olive fly. More recently some authors tested copper products (Prophetou-Athanasiadou et al, 1991; Belcari and Bobbio, 1999; Petacchi and Minnocci, 2002; Tsolakis & Ragusa, 2002) and kaolin (Saour & Makee, 2004) against B. oleae obtaining interesting results. The aim of this research is to test the effectiveness of kaolin against the olive fly, comparing it with copper hydroxide, in different conditions of infestation level.

Material and methods

In 2003 in the table olive germplasm collection (0.6 ha) of “Ente di Sviluppo Agricolo della Regione Siciliana” and “Dipartimento di Colture Arboree” (University of Palermo) located in Castelvetrano (Trapani province, Sicily), tests on the effectiveness of Surround WP (Engelhard Co. U.S.A.), a product containing 95% of kaolin, were carried out on three table olive cultivars, Nocellara del Belice, Moresca and Tonda Iblea. In 2004, in the same Castelvetrano field and on the same cultivars, BPLK kaolin (Goonvean Ltd. U.K.) was tested too. This is a 100% kaolin product utilised for ceramic and other purposes, but never used in agriculture. In the second year the two products containing kaolin were also tested on Cerasuola cultivar in a 1 ha organic olive grove located in Trapani, comparing them with copper hydroxide (Cuprantol Ultramicron) containing 35% of copper. Olive trees were sprayed twice both in Trapani in 2004 and Castelvetrano in 2003, whereas in the last site in 2004 three treatments were done. First treatment was realised after reaching the threshold of 5% of total infestation, but in any case never later than the first week of September. The second treatment was done when the fruit were no more covered by the kaolin. The doses were 5 kg of kaolin products and 0.3 kg of copper hydroxide per hl of water. Sampled trees in each thesis were seven at Castelvetrano and eight at Trapani; samples consisted of ten olives from each of the trees of the plot. Collected fruits were analysed under the stereomicroscope to detect eggs, larvae, pupae, exit holes, empty galleries and punctures without oviposition. The infestation level was expressed as “harmful” infestation (3rd instar larvae, pupae, exit holes in absence of larvae and pupae) and “total” infestation (harmful infestation plus eggs and other larvae). In each olive grove two traps with 1,7-dioxaspiro[5.5]undecane were placed to monitor the presence of male olive flies. Thermopluviometric data concerning Castelvetrano Seggio and Trapani Fontanasalsa weather stations were kindly provided by SIAS, Servizio Informativo Agrometeorologico Siciliano (Government of the Sicilian Region). Data concerning fruit infestation were statistically analysed by T-test (p<0.05), repeated measurents ANOVA, 1-way ANOVA and Tukey post-hoc test (p< 0.05).

Results

In 2003 at Castelvetrano the temperature of July and August was high, in the last month exceeding 35°C in 22 days; after the conspicuous rainfall of 16th-19th of September (66 mm) the kaolin was still abundant on the fruits; the rainfall of 18th October (71 mm) washed away most of the clay, but the close harvest-time led us not to treat again. The captures of B. oleae males were very low until 16th of September, raising up to 38 males per week from 14th to 28th of October.

113

Vertical bars show confidence intervals of 95% 0,8

Moresca 0,6 Tonda Iblea Nocellara a del Belice

attacks per olive 0,4

B. oleae b a' 0,2

0,0 b' Average number of

-0,2 Surround W P Surround WP Surround WP Surround WP Control Control Control Control

Sep-4-03 Sep-23-03 Oc t-15-03 Oct-30-03

Figure 1. Trend of Bactrocera oleae total infestation in olives of three different table cultivars at Castelvetrano in 2003 (Different letters denote statistically significant differences; T-test, p<0.05)

In this year, as shown in Figure 1 B. oleae infestation level was low; nevertheless, in the last sampling date the total infestation was significantly higher in the control trees than in those treated with Surround WP, both in Moresca and Nocellara del Belice plots. In 2004 at Castelvetrano, the temperature in August was cooler than in 2003; the rainfall from July 20th to October 26th was 146 mm, 64 of them from 13th to 16th of October, after the last treatment performed in October 6th. The olive fly males caught by pheromone traps ranged between 2 and 5 in July and August, increasing up to 21 at the end of September. In this site, B. oleae infestation levels of the plots treated with the two products containing kaolin were lower than those of the control plots, with statistically significant differences both in total and in harmful infestation (Figures 2 & 4), except the harmful infestation of BPLK kaolin in Moresca cultivar which was not statistically different from untreated trees. As shown in Figures 2 & 4, at Castelvetrano Surround WP protected olives significantly better than BPLK kaolin, limiting olive fly total infestation in the three cultivars up to 0.3-0.5 attacks per olive (27-40% infested fruits) vs. 1-1.8 attacks per olives (70-93% of infested fruits) in BPLK plots, and 1.4-4.4 attacks per olives (70-100% of infested fruits) in untreated trees; in this site harmful infestation reached 0.22-0.25 attacks per olive (15-23% infested fruits) in Surround WP plots, 0.9-1.5 attacks per olives (68-87% of infested fruits) in BPLK plots and 1.2-4 attacks per olives (61-100% of infested fruits) in untreated trees. 114

V ert i cal ba rs sh ow con fide nce in terv al s of 9 5% 6 Cv. NOCELLARA DEL BELICE 5 Mean values of the whole period Untreated 1.91 a 4 BPLK Kaolin 0.86 b Surround WP 0.17 c attacks per olive attacks 3 B. oleae 2

0 Average number of

-1 Sep-20-04 Oct-6-04 Oct-26-04

Figure 2. Trend of Bactrocera oleae total infestation in Nocellara del Belice cultivar at Castelvetrano in 2004 (Different letters denote statistically significant differences; repeated measurements ANOVA, p<0.05)

V ert i cal ba rs sh ow con fide nce in terv al s of 9 5% 2.0

1.8 Cv. MORESCA Mean values of 1.6 the whole period Untreated 0.87 a 1.4 BPLK Kaolin 0.58 b Surround WP 0.23 c 1.2 attacks per olive 1.0

0.8 B. oleae 0.6

0.4

0.2

0.0

-0.2 Average number of of number Average

-0.4 Sep-20-04 O ct-6-04 Oct -26 -04

Figure 3. Trend of Bactrocera oleae total infestation in Moresca cultivar at Castelvetrano in 2004 (Different letters denote statistically significant differences; repeated measurements ANOVA, p<0.05) 115

V ert i cal ba rs sh ow con fide nce in terv al s of 9 5% 3.0 Cv. TONDA IBLEA Mean values of 2.5 the whole period Untreated 1.10 a BPLK Kaolin 0.58 b Surround WP 0.23 c 2.0 attacks per olive per attacks 1.5 B. oleae 1.0

0.5

0.0 Average number of

-0.5 Sep-20-04 O ct-6-04 Oct -26 -04

Figure 4. Trend of Bactrocera oleae total infestation in Tonda Iblea cultivar at Castelvetrano in 2004 (Different letters denote statistically significant differences; repeated measurements ANOVA, p<0.05)

Vertical bars show confidence intervals of 95% 2,2

2,0 Cv. CERA SUOLA Mean values of 1,8 the whole period 1,6 Untreated 0.74 a Copper Hydroxide 0.33 b 1,4 BPLK Kaolin 0.28 b

attacks per olive 1,2 Surround WP 0.19 b

1,0 Treatments

B. oleae 0,8

0,6

0,4

0,2

0,0

-0,2 Average number of number Average -0,4 Sep-7 Sep-21 Oct-2 Oct-9 Oct-16 Oct-23 Oct-30

Figure 5. Trend of Bactrocera oleae total infestation at Trapani in 2004 (Different letters denote statistically significant differences; repeated measurements ANOVA, p<0.05) 116

In 2004 at Trapani, the trend of the mean temperature was similar to Castelvetrano one; the rainfall from July 12th to October 30th was 118 mm, 41 of them after the last treatment (October 3rd) distributed in eight rainy days. Mean captures of males by pheromone traps remained below 5 individuals per week until mid-October, reaching afterwards 13 males per trap per week. The total infestation in the untreated plot was clearly higher than in the other three plots since October 2nd; in BPLK kaolin and copper hydroxide plots the total infestation sharply increase only in the last date (Figure 5). Nevertheless both the total and the harmful infestation of the plots with the two products containing kaolin and copper hydroxide recorded statistically significant differences from the untreated control, but not among themselves. The two sprays of the three products limited the total infestation at the harvesting up to 0.48-1.22 olive fly attacks per olive (45-87% of infested fruits) vs. 1.6 attacks per olive (98% of infested fruits) in the control, while the harmful infestation in the three sprayed plots was much more different from that one in the control reaching 0.03-0.48 olive fly attacks per olive (3-37% of infested fruits) vs. 1.43 fly attacks per olive (87% of infested fruits) in the control.

Discussion

The tested products containing kaolin and copper hydroxide are effectively able to limit B. oleae infestation to a very good level for olive oil production, moreover, considering the earlier harvesting of table olives (before mid October), these products give a new opportunity for controlling the olive fly also in the organic olive groves for table olives production. The different results of 2004 recorded by Surround WP and BPLK kaolin in the two olive groves seems linked to the different rainfall of the period after the last treatment, 64 mm in three rainy days at Castelvetrano and 41 mm in eight rainy days at Trapani; BPLK kaolin was probably washed away more than Surround WP. The kaolin clay has some advantages: unlike copper hydroxide it has no environmental toxicity; thanks to the white coating due to kaolin sprays the need of further treatments is easily detectable by the growers watching the fruits in the field.

Acknowledgements

We thank Ente di Sviluppo Agricolo della Regione Siciliana, Dipartimento di Colture Arboree (University of Palermo) and Giovanni Curatolo for providing the research fields, labour and machinery for treatments. Thanks to Marco Corcella, Isabella Battaglia and Dario Parrivecchio for their help in samplings. Research funded by University of Palermo Fondi di Ateneo ex quota 60% (“Il controllo degl’insetti fitofagi nell’agricoltura biologica e convenzionale”) and Assessorato Agricoltura e Foreste della Regione Siciliana (“Innovazioni della filiera olivicola da mensa”)

References

Belcari, A. & Bobbio, E. 1999: L’impiego del rame nel controllo della mosca delle olive Bactrocera oleae. – Informatore fitopatologico 49 (12): 52-55. Belcari, A., Sacchetti, P., Marchi, G., Surico, G. 2003: La mosca delle olive e la simbiosi batterica. – Informatore Fitopatologico 53(9): 55-59. Katsoyannos, B. I. & Kouloussis, N. A. 2001: Captures of the olive fruit fly Bactrocera oleae on spheres of different colors. – Entomologia Experimentalis et Applicata 100: 165–172. 117

Petacchi, R. & Minnocci, A. 2002: Olive fruit fly control methods in sustainable agriculture. – Acta Horticulturae 2002 (586): 841-844 Prophetou-Athanasiadou, D. A., Tsanakakis, M. E., Myroyannis D., Sakas G. 1991: Deterrence of oviposition in Dacus oleae by copper hydroxide. – Entomologia Experimentalis et Applicata 61: 1-5. Rotundo, G., Germinara, G. S., De Cristofaro, A., Rama, F. 2001: Identificazione di composti volatili in estratti da diverse cultivar di Olea europaea L. biologicamente attivi su Bactrocera oleae (Gmelin) (Diptera: Tephritidae). – Bollettino del Laboratorio di Entomologia Agraria “Filippo Silvestri” 57: 25-34. Russo, G. & Fenili, G. 1950: Esperimenti antidachici eseguiti in Marina di Ascea (Salerno) nel 1949. – Olearia (5-6): 1-12. Russo, G. 1937: Primi esperimenti di un nuovo metodo di lotta contro la Mosca delle Olive. – L’Olivicoltore, Roma 14 (11): pp. 3 Russo, G. 1954: Reperti biologici, sistemi e metodi di lotta sui principali insetti dannosi all’olivo. – Bollettino del Laboratorio di Entomologia Agraria “Filippo Silvestri” 13: 64-95. Saour, G. & Makee, H. 2004: A kaolin-based particle film for suppression of the olive fruit fly Bactrocera oleae Gmelin (Dip., Tephritidae) in olive groves. – Journal of Applied Entomology 128: 28-31. Solinas, M., Rebora, M., De Cristofaro, A., Rotundo, G., Girolami, V., Mori , N., Di Bernardo, A. 2001: Functional morphology of Bactrocera oleae (Gmel.) (Diptera: Tephritidae) tarsal chemosensilla involved in interactions with the host-plant. – Entomologica 35: 103-123. Tsanakakis, M. E. 1985: Considerations on the possible usefulness of olive fruit fly symbionticides in integrated control in olive groves. – In: Cavalloro R. & Crovetti A. “Proceedings of Integrated control in olive groves” CEC7FAO/IOBC Int. Joint Meeting, Pisa 3-6 April, 1984: 386-393. Tsolakis, H. & Ragusa, E. 2002: Prove di controllo di Bactrocera oleae (Gmelin) (Diptera Tephritidae) con prodotti a basso impatto ambientale. – Phytophaga 12: 141-148.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 119-123

Resistance to organophosphates in Bactrocera oleae in Greece and Cyprus

John Tsitsipis1, John T. Margaritopoulos1, Panagiotis Skouras1, Konstantinos Mathiopoulos2, Nikos Serafides3 1 Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Volos, Greece 2 Deparment of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece 3 Agricultural Research Institute, Nicosia, Cyprus

Abstract: The control of the olive fruit fly Bactrocera oleae (Gmelin) (Diptera: Tephritidae) in Greece has been based mostly on bait sprays with organophosphate insecticides for more than 40 years. In the present study, a two-year survey to monitor the development of resistance to dimethoate in B. oleae field populations collected from Greece and Cyprus was performed. A laboratory susceptible strain was used as the reference population. Considerable variation in the resistance ratios to dimethoate was recorded ranging from 6.3 to 61.9. The highest levels of resistance were observed in populations from Crete, while the lowest in those from Cyprus. In mainland Greece moderate to high resistance was recorded. This variation could be attributed to different insecticide pressure but also to migration.

Keywords: Bactrocera oleae, dimethoate, insecticide resistance

Introduction

The most noxious insect pest of the fruit of the olive tree, Olea europaea L. (Oleaceae), in many regions of the world, is the olive fruit fly Bactrocera oleae (Gmelin) (Diptera: Tephritidae). The insect lays its eggs in the olive fruit and the larvae feed and grow in the mesocarp. The infested fruits drop before they become mature being unsuitable for eating (table olives) or the quality of olive deteriorates (oil producing olives) (Michelakis & Neuenschwander, 1983). Although there is a contemporary interest for environmentally compatible control strategies (Gilmore, 1989; Navrozidis et al., 2000; Broumas et al., 2002; Petacchi et al., 2003; Saouer & Makee, 2004) the control of B. oleae is mostly based on insecticides, particularly organophosphates (OPs) (for review on chemical control of tephritid flies see Roessler 1989). Among them dimethoate has been used more often due to its low residual persistence in olive oil. Recently, pyrethroids and the macrolytic lactone Spinosad are also used against the fly in some countries (Collier & Van Steenwyk, 2003; Haniotakis, 2005). The extensive and long-term use of insecticides for the control of B. oleae, apart from side effects on beneficial organisms (Roessler, 1989), could give rise to insecticide resistance problems. This phenomenon has been studied in various tephritid pests around the world. In his review Keiser (1989) reported that in Hawaii natural populations or laboratory strains of the oriental fruit fly Bactrocera dorsalis Hendel and the melon fly Bactocera cucurbitae Coquillett developed resistance to DDT and methoxychlor but not to malathion. By contrast, the Mediterranean fruit fly Ceratitis capitata (Wiedemann) was found susceptible to these insecticides. Hsu et al. (2004) conducting laboratory selection experiments with a B. dorsalis populations from Taiwan produced resistant lines not only to malathion but also to other OPs

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and to insecticides of other chemical groups (carbamates and pyrethroids). In Israel, resistance to OPs and to carbamate methomyl has been found in Dacus ciliatus Loew (Maklakov et al., 2001). Koren et al., (1984) reported a low level tolerance (2-3X) to malathion in females of C. capitata and none in males after selection for 18 generations in the laboratory. Orphanidis et al., (1980) selected C. capitata with DDT and malathion for 80 generations and found also a low level of tolerance. Recently, the ability of B. oleae to develop resistance to OPs has been well documented. Vontas et al. (2001) reported a 9-fold resistance to dimethoate in a natural population of B. oleae from south-central Greece compared to a susceptible laboratory strain. An altered achetylocholinesterase (AChE) has been reported as the major resistance mechanism of B. oleae to OPs (Vontas et al., 2001; 2002). Two point mutations in the AChE gene were found, which together produce a 16-fold resistance in insecticide sensitivity. These mutations have been reported in homozygous state in almost all samples from Greece and Albania (OPs are used extensively in these countries). Resistance-associated alleles where found at lower frequencies in western Mediterranean where OPs are not used extensively. The mutations were not found in samples from South Africa (Hawkes et al., 2004). The present study, therefore, aimed at examining the resistance level of B. oleae populations from Greece (mainland and islands) and Cyprus to dimethoate.

Material and methods

Bactrocera oleae During 2003-2004 14 populations of B. oleae were collected from various regions of mainland Greece and islands and three from Cyprus and assayed with dimethoate. Almost all populations from Greece and Cyprus were collected from orchards in which bait sprays with dimethoate or fenthion were regularly performed. Two Greek populations were collected from organic olive orchards. In Cyprus, however, fewer (1-2) bait sprays are usually performed than in Greece (3-6). In addition, a laboratory population which has not received insecticides for 30 years (Democritus laboratory strain) was used as the susceptible control strain (LS). In each region and sampling date olives infested by B. oleae were collected from an orchard. The fruits were put in paper bugs and transferred to the laboratory. The olives were put on plastic trays containing a thin layer of dry heat sterilized (100°C for 1 h) sawdust and kept at 23°C and L16: D8. The sawdust was used as a pupation substrate for the larvae. The trays were inspected daily and the pupae were transferred to cages (8 cm X 8 cm Χ 8 cm). The two opposite sides of the cages were covered with toule to allow aeration while the other with plexiglas. On the day of adult emergence, solid diet was provided (sugar: yeast hydrolysate, 3 : 1) and water via a plastic vial the opening of which was covered with a cotton wool. One to three day old adults were assayed with the insecticides as described below.

Bioassays Dimethoate (40EC) was delivered in 1 µl of acetone to the mesonotum of each adult fly by a 10-µl Hamilton microsyringe. The insects before the application of the insecticide were anaesthetised by carbon dioxide. About 20-30 adults per dose were tested and 6-8 doses were used (including the control treated with acetone only). In each dose an equal number of males and females were tested. The treated adults were kept in the aforementioned cages at 23°C and L16:D8 and mortality was scored 24 h post-treatment. ED50 values and 95% confidence intervals were calculated by probit analysis using the program SPSS ver. 10 (SPSS Inc. 1999). Differences in ED50 values between populations were considered significant if the 95% confidence intervals did not overlap.

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Results and discussion

Significant variation was observed in ED50 values between populations ranging from 13 to 123 ng / insect, although the differences were not found significant in all pair wise comparisons (Table 1). Significant heterogeneity (i.e. within population variation) was observed in two populations as revealed by the X2 values. According to Resistance Ratio (RR) values (ED50 of a given population / ED50 of the laboratory susceptible strain) the populations could be categorised in four groups: low (RR <10), moderate (RR between 11 and 20), high (RR between 20 and 30) and very high (RR >30) resistance ratios. In Greece, 50 % of the examined populations showed RR values between 11 and 20, 29 % between 20 and 30 and 21% more than 30. The highest RR values were observed in two populations from the Crete island (Kentri: 64 and Sitia: 46). Most of the populations from Crete were ranked among the most resistant ones. Contrary to that observed in Crete, one population from another Greek island (i.e. Samos,) showed lower RR value (17.1). A different situation was observed in Cyprus, since the four populations examined showed low resistance level (RR ranged from 7 to 8). Such low RR values were not recorded elsewhere even in the populations collected from the two organic orchards in Greece. Apart from the clear separation of the populations from Crete and Cyprus, no other grouping according to resistance level and geographical region appeared. Also, high within region variation was observed. For example Anifi in southern Greece, is only 5 km away from Prosimni and the populations collected from these two sites are ranked as 5th and 9th in regard to their RR values. Also, differences between collection years within the same sampling site were observed (see in Table 1 the two populations from Kavala). The present study showed clearly that populations of B. oleae have developed resistance to the commonly used dimethoate, although in many cases the insecticide still works adequately (ED50 lower than the recommended 300 ng/insect for cover sprays). The different resistance status of resistance in Greece and Cyprus could be attributed to the control management strategy that is adopted. In more than 95% of olive orchards in Greece the control of the fly is under the responsibility of the government and area-wide bait sprays with dimethoate and/or fenthion have been performed for more than 40 years. During 2003-04 a mean number of 2-6 bait sprays were performed in the regions sampled in the present study (Data from the Greek Ministry of Agriculture) with the highest number of sprays occurring in eastern Crete. It is known, hoever, that farmers also perform additional cover sprays in certain areas in Crete (e.g. Sitia). By contrast, the management in Cyprus is more flexible and it is under farmer responsibility. Usually 1-2 bait sprays are performed each year according to the pressure of the B. oleae populations (Data form the Ministry of Agriculture of Cyprus). The fact, therefore, that populations from Cyprus are characterised by a low exposure history to OPs explain to a certain extent the low resistance level found. However, the high selection pressure by OPs in Greece does not explain the within region variation observed, i.e. two fold difference in resistance ratio between populations sampled from localities 5-10 km apart from each other. In addition, populations from two orchards in mainland Greece which are not sprayed with OPs for many years showed significant higher ED50 than the LS strain, with RR values ranging from 13.6 to 22.8. The former case may be the result of isolation between sympatric populations. Whether ecological factors or physical barriers are involved needs further investigation. In the latter case, presumably ecological factors such as migration and gene flow have been involved. A possible scenario which could explain the resistance level observed in non sprayed orchards is an influx (either via migration or commerce) of resistant individuals from areas of high incidence of resistant alleles. The same situation has been also observed in another fly, Drosophila simulans (Sturtevant) (Diptera: Drosophilidae) (Windelspecht et al., 1995).

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In conclusion, an efficient control management of B. oleae requires information on the OP resistance status, the possible resistance development in newly introduced insecticides as well as information on migration/dispersal and gene flow. Towards that direction the use of classical bioassays combined with molecular diagnostic tools (Hawkes et al., 2005) is of primary importance. Such information will prove indispensable to plan and follow a resistance management scheme.

Table 1. Toxicity of dimethoate to field populations of Bactrocera oleae from Greece and Cyprus compared to a laboratory susceptible strain (LS).

Regiona N Collection date LD50 in ng / insect Slope X2 P value RRc (95% C.I.) b Kentri, Crete 156 9.x.2004 122.60 (85.34 – 199.54) A 1.52 7.53 0.11 61.9 Sitia, Crete 123 28.xi.2004 92.15 (67.42 - 212.88) A 3.08 1.57 0.67 46.1 Kyparissia, SG 205 6.xii.2003 66.08 (47.56 – 118.95) AB 1.80 4.94 0.18 33.1 Mirtos, Crete 140 9.xi.2004 59.59 (45.21 - 82.86) AB 2.50 6.42 0.17 29.8 Anifi, SG 160 21.iv.2004 51.62 (39.51 - 76.57) ABC 2.44 4.48 0.21 25.8 Kavala, NG 178 3.xi.2004 46.86 (38.29 - 59.89) ABC 3.16 0.91 0.82 23.5 Fitoko, CG 142 24.xi. 2004 44.91 (37.14 - 55.34) BC 5.54 3.04 0.39 22.5 Heraklion, Crete 199 27.x.2003 38.49 (33.02 - 44.29) CD 4.88 5.80 0.22 19.3 Prosimni, SG 189 11.xi.2003 38.49 (33.86 - 44.39) CD 5.72 0.31 0.96 19.3 Kavala, NG 109 21.xi.2003 37.00 (29.50 - 45.86) CDE 5.00 0.08 0.99 18.5 d Vrachneika, SG 125 8.xii.2004 36.56 (29.96 - 45.29) CDE 4.45 1.61 0.66 18.3 Samos, A 112 12.xii.2004 34.07 (25.61-47.11) CDE 2.65 2.34 0.51 17.1

Kala Nera, CG 150 27.x.2003 32.66 (26.39 - 43.15) CDE 3.30 4.85 0.30 16.4 d Argalasti, CG 112 15.i.2004 27.65 (22.07 -34.28) DE 4.90 1.66 0.65 13.9 Pafos, Cyprus 128 13.xii.2004 15.05 (14.00 - 15.92) F 18.12 0.13 0.94 7.6 Deftera, Cyprus 143 22.xii.2004 12.87 (11.58 – 14.29) F 6.45 1.94 0.59 6.5 Mazotos, Cyprus 186 22.xi.2004 12.45 (8.28 - 19.53) F 5.12 15.91 0.01 6.3 LS 194 5.x.2004 1.98 (1.60 - 2.35) G 4.11 2.71 0.61 - aNG = North Greece, CG = Central Greece, SG = South Greece, A = eastern Aegean island, LS = b laboratory susceptible strain. ED50 values followed by different letter differ significantly (P<0.05). dc Resistance ratio = ED50 of the population / ED50 of the LS.

Acknowledgements

We thank Nikolaos Pirianian and Matthaios Papayiannis for assistance in the sample collection and the bioassays.

References

Broumas, T., Haniotakis, G., Liaropoulos, C., Tomazou, T. & Ragoussis, N. 2002: The efficacy of an improved form of the mass-trapping method, for the control of the olive fruit fly, Bactrocera oleae (Gmelin) (Dipt., Tephritidae): pilot-scale feasibility studies. – J. Appl. Entomol. 126: 217-223. Collier, T.R. & Van Steenwyk, R. 2003: Olive fruit fly in California: prospects for integrated control. – California Agriculture 57: 28-32.

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Gilmore, J.E. 1989: Sterile Insect Technique (SIT). – In: World Crop Pests. Fruit flies; Their Biology, Natural Enemies and Control Vol. IIIB, eds. Robinson and Hooper: 353-363. Haniotakis, G.E. 2005: Olive pest control: present status and prospects. – IOBC/WPRS 28(9): 1-9. Hawkes, J.N., Hemingway, W.R. & Vontas, J. 2005: Detection of resistance- associated point mutations of organophosphate-insensitive acetylcholinesterase in the olive fruit fly, Bactrocera oleae (Gmelin) Pestic. – Biochem. Physiol. 81: 154-163. Hsu, J.C., Feng, H.T. & Wu, W.J. 2004: Resistance and synergistic effects of insecticides in Bactrocera dorsalis (Diptera: Tephritidae) in Taiwan. – J. Econ. Entomol. 97: 1682- 1688. Keiser, I. 1989: Insectiside resistance status. – In: World Crop Pests. Fruit flies; Their Biology, Natural Enemies and Control Vol. IIIB, eds. Robinson and Hooper: 337-344. Koren, B., Yawetz, A. & Perry A.S. 1984: Biochemical properties characterizing the development of tolerance to malathion in Ceratitis capitata Wiedemann (Diptera: Tephritidae). – J. Econ. Entomol. 77: 864 – 867. Maklakov, A., Ishaaya, I., Freidberg, A., Yawetz, A., Horowitz, A.R. & Yarom, I.. 2001: Toxicological studies of organophosphate and pyrethroid insecticides for controlling the fruit fly Dacus ciliatus (Diptera : Tephritidae). – J. Econ. Entomol. 94: 1053-1058. Michelakis, S.E. & Neuenschwander, P. 1983: Estimates of the crop losses caused by Dacus oleae (Gmel.)(Diptera, Tephritidae) in Crete, Greece. – In: Fruit Flies of Economic Importance, ed. Cavalloro: 603-611. Navrozidis, E.I, Vasara, E., Karamanlidou, G., Salpiggidis, G.K. & Koliais, S.I.. 2000: Biological control of Bactocera oleae (Diptera: Tephritidae) using a Greek Bacillus thuringiensis isolate. – J. Econ. Entomol. 93: 1657-1661. Orphanidis, P. S., Kalmoukos, B. & Kapetanakis, E. 1980: Development of resistance in Ceratitis capitata Wied. in laboratory under intermittent pressure of organophosphorus and chlorinated insecticides. – Ann. Instit. Phytopathol. Benaki (N.S.) 12: 198-207. Petacchi, R., Rizzi, I., & Guidotti, D. 2003: The 'lure and kill' technique in Bactrocera oleae (Gmel.) control: effectiveness indices and suitability of the technique in area-wide experimental trials. – Int. J. Pest Manag. 49: 305-311 Roessler, Y. 1989: Control; insecticides; insecticidal bait and cover sprays. – In: World Crop Pests. Fruit flies; Their Biology, Natural Enemies and Control Vol. IIIB, eds. Robinson and Hooper: 329-336. Saour, G. & Makee, H. 2004: A kaolin-based particle film for suppression of the olive fruit fly Bactrocera oleae Gmelin (Dip., Tephritidae) in olive groves. – J. Appl. Entomol. 128: 28-31. SPSS Inc. (1999) SPSS Base 10.0 for Windows User’s Guide. – SPSS Inc. Chicago, IL. Vontas, J., Cosmidis, N., Loukas, M., Tsakas, S., Hejazi, M.J., Ayoutanti, A. & Hemingway, J. 2001: Altered acetylcholinesterase confers organophosphate resistance in the olive fruit fly Bactrocera oleae. – Pestic. Biochem. Physiol. 71: 124-132. Vontas, J., Hejazi, M.J., Hawkes, N.J., Cosmidis N., Loukas M. & Hemingway J. 2002: Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase in the olive fruit fly Bactrocera oleae. – Insect Mol. Biol. 11: 329-336. Windelspecht, M., Richmond, R.C. & Cochrane, B.J. 1995: Malathion resistance levels in sympatric populations of Drosophila simulans (Diptera: Drosophilidae) and Drosophila melanogaster differ by two orders of magnitude. – J. Econ. Entomol. 88: 1138-1143

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 125-130

A Beauveria bassiana-based bioinsecticide for the microbial control of the olive fly (Bactrocera oleae)

Massimo Benuzzi, Enrico Albonetti, Fabio Fiorentini, Edith Ladurner Intrachem Bio Italia S.p.A., R&D Department, Via Calcinaro 2085/7, I-47023 Cesena, Italy

Abstract: In 2003-2004, different certified Italian testing facilities conducted several GEP trials on the efficacy of the B. bassiana-based bioinsecticide Naturalis against Tephritid flies (Ceratitis capitata, Rhagoletis cerasi, and Bactrocera. oleae). In this paper we report the results of the 6 efficacy trials conducted against B. oleae. The efficacy of weekly applications of the product at 125-130 ml/hl in reducing the percentage of fruits damaged by B. oleae at harvest was comparable to or higher than that of the chemical reference treatment in 5 out of 6 trials. The efficacy of 14-day-interval applications at 125 and 250 ml/hl was comparable to that of the chemical standard in 4 out of 6 trials. Furthermore, promising results were obtained when Naturalis was used in an integrated strategy. The bioinsecticide can therefore be considered a new and reliable tool for the control of olive flies.

Key words: entomopathogen, tephritid flies, organic agriculture, integrated pest management

Introduction

Beauveria bassiana (Balsamo) Vuillemin (Deuteromycetes, Moniliales) is an entomopathogenic fungus, recognized in 1835 by Agostino Bassi as the causal agent of the white muscardine disease of the silk worm, Bombyx mori (L.). B. bassiana affects a wide range of arthropod species, such as coleopterans, mites, homopteran and heteropteran hemipterans (Knauf, 1992; Lacey et al., 1999; Benuzzi et al., 2001; Wright & Kennedy, 1996). The microrganism acts primarily by contact. When the fungal conidia become attached to the insect’s cuticle, they germinate producing penetration hyphae, which penetrate the cuticle and proliferate in the insect’s body. High humidity or free water, and thus thorough wetting of the plants, is essential for conidial germination (Hallsworth & Magan, 1999; Benuzzi & Santopolo, 2001). Infection can take between 24 and 48 hours, depending on the temperature (BCPC, 2004). With the proliferation of the fungus inside the insect’s body usually also the production of toxins starts, leading to the insect’s death within 3-5 days. The B. bassiana mycelium multiplies by feeding on the host and consuming its nutrients. After the insect’s death, conidiospores are produced on the outside of its body and new conidia are released on the outside of the insect cadaver (BCPC, 2004). B. bassiana can affect all developmental stages of its host, eggs, immature stages, and adults, provided that they are present on the outside of the plants. The formulated bioinsecticide Naturalis is a concentrated suspension of spores of the B. bassiana strain ATCC 74040. The original strain has been isolated from Anthonomus grandis (Boheman), a curculionid beetle attacking cotton, in the Lower Rio Grande valley, Texas, USA (BCPC, 2004). The distinctive characteristic of this strain consists in not producing any toxic substance. The penetration hyphae perforate the cuticle mechanically and physically by means of special enzymes, causing the dehydration and finally the death of the insect. Several years of experimental applications of this entomopathogenic B. bassiana strain against the boll weevil showed that also other insect pests, such as whiteflies, thrips and leafhoppers, are susceptible to infection (Knauf, 1992; Wright & Kennedy, 1996). Given

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these results, in the USA, the registration of Naturalis, obtained in 1995, was extended to other phytophagous insects. In Italy, Naturalis was registered in April, 2000. The formulated product (henceforth f.p.) contains 7.16 g active substance per 100 g f.p., i.e. at least 2.3 x 107 viable spores / ml f.p. This bioinsecticide, a suspension of conidiospores in natural oil, can be used also in organic farming. The olive fly, Bactrocera oleae (Gmelin) is the key-pest on olive trees in the Mediterranean environment. The reduced availability of pesticides, residue issues, the growing demand of reliable alternatives to chemical control, and finally the increasing area of organic olive groves in Italy, promoted the research of new biological tools to control the olive fly (Delrio, 1992; Konstantopoulou & Mazomenos, 2005). In order to identify new control methods for organic farming (that may be used also in IPM strategies), in 2003-2004, under the supervision of the R&D Department of Intrachem Bio Italia S.p.A., different certified Italian testing facilities conducted several trials on the efficacy of the B. bassiana- based bioinsecticide Naturalis against Tephritid flies (mediterranean fruit fly, Ceratitis capitata Wiedemann; cherry fly, Rhagoletis cerasi L.; olive fly, B. oleae). All trials were carried out in compliance with the EPPO guidelines and with the Principles of Good Experimental Practices (GEP). In this paper we report the results on the efficacy of Naturalis, used alone and in integrated strategies, at different doses and application intervals, in reducing the percentage of fruits damaged by B. oleae at harvest, recorded in 6 trials carried out in Italy in 2003-2004.

Material and methods

Location of study sites The location of the different study sites, the testing facilities that conducted the trials, and the main characteristics of the study olive groves (cultivars, row x plant spacing) are reported in Table 1.

Table 1. Location of the different study sites, testing facilities, and main characteristics of the study olive groves (cultivars, row x plant spacing).

Trial Testing Location of study Row x plant Year Cultivars no. facility site spacing (m) 1 2003 G.Z. Srl Bisceglie (Bari) Coratina 6 x 6 2 2003 Agri 2000 Scrl Cerignola (Foggia) Coratina 6 x 6 Nocellare etnea, 3 2003 CO.R.AGRO Mineo (Catania) 8 x 6 Biancolilla 4 2004 G.Z. Srl Trani (Bari) Coratina 10 x 10 Nocellare etnea, 5 2004 CO.R.AGRO Mineo (Catania) 8 x 6 Biancolilla 6 2004 Agri 2000 Scrl Tursi (Matera) Coratina 5 x 5

Treatments and data assessment The treatments tested in the different trials are reported in Table 2. To compare the different treatments, in each trial, a fully randomised block design was used with 4 replicates per treatment, and with 3 (trial no. 4), 4 (trial no. 1, 2, 3, and 6) or 5 plants (trial no. 6) per plot. Treatments were applied starting from the beginning of the olive fly susceptibility period of the crop (BBCH 75: fruit size about 50% final size, stone starts to

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Table 2. Description of treatments, number of applications, and time intervall between successive applications in the different efficacy trials conducted in 2003-2004.

Time interval Treatment Form., Dose No. No. between appl. (a.i.) conc. a.i. (ml/hl) appl. (days) Trial no. 1 – Bisceglie (Bari), 2003 1 Naturalis (B. bassiana) SC 7.2 125 9 6 2 Naturalis (B. bassiana) SC 7.2 125 5 10 3 Naturalis (B. bassiana) SC 7.2 125 3 15 4 Rogor L40 (dimethoate) EC 405 100 1 - 5 Untreated control – – – – Trial no. 2 – Cerignola (Foggia), 2003 1 Naturalis (B. bassiana) SC 7.2 125 9 7 2 Naturalis (B. bassiana) SC 7.2 125 7 14 3 Naturalis (B. bassiana) SC 7.2 125 5 21 4 Rogatox (dimethoate) EC 403 g/l 150 2 30 5 Untreated control – – – – Trial no. 3 – Mineo (Catania), 2003 1 Naturalis (B. bassiana) SC 7.2 125 7 7 2 Naturalis (B. bassiana) SC 7.2 125 4 14 3 Naturalis (B. bassiana) SC 7.2 125 3 21 4 Lebaycid (fenthion) EC 48.5% 100 3 21 5 Untreated control – – – – Trial no. 4 – Trani (Bari), 2004 1 Naturalis (B. bassiana) SC 7.2 130 9 7 2 Naturalis (B. bassiana) SC 7.2 250 5 14 Rogor L40 (dimethoate)+ EC 405 130 2 30 3 Naturalis (B. bassiana) SC 7.2 100 4 7 4 Rogor L40 (dimethoate) EC 405 100 2 30 5 Untreated control – – – – Trial no. 5 – Mineo (Catania), 2004 1 Naturalis (B. bassiana) SC 7.2 125 8 7 2 Naturalis (B. bassiana) SC 7.2 250 4 14 Danadim (dimethoate)+ EC 40% 140 2 14 3 Naturalis (B. bassiana) SC 7.2 130 2 14 4 Danadim (dimethoate) EC 40% 140 2 14 5 Untreated control – – – Trial no. 6 – Tursi (Matera), 2004 1 Naturalis (B. bassiana) SC 7.2 125 5 7 2 Naturalis (B. bassiana) SC 7.2 250 3 14 Lebaycid (fenthion)+ EC 509.2 g/l 100 1 – 3 Naturalis (B. bassiana) SC 7.2 130 2 7 4 Lebaycid (fenthion) EC 509.2 g/l 100 1 – 5 Untreated control – –

lignificate) through fruit ripening. At harvest, in each plot, the number of fruits damaged by B. oleae was counted on a sample of 100 (trial no. 3 and 5), 200 (trial no. 2), 300 (trial no. 1 and 4) or 1,000 (trail no. 6) randomly selected fruits, and the percentage of damaged fruits was

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Table 3. Mean percentage of fruits damaged by B. oleae and mean efficacy (%) recorded in the different treatments and trials.

Dose (ml/hl), % damaged % efficacy No. Treatment (a.i.) no. applications fruits (mean) (mean) Trial no. 1 – Bisceglie (Bari), 2003¹ 1 Naturalis (B. bassiana) 125, 9 18.3 c 61.7 2 Naturalis (B. bassiana) 125, 5 23.3 c 52.5 3 Naturalis (B. bassiana) 125, 3 30.8 b 37.1 4 Rogor L40 (dimethoate) 100, 1 18.3 c 62.3 5 Untreated control - 49.2 a - Trial no. 2 – Cerignola (Foggia), 2003¹ 1 Naturalis (B. bassiana) 125, 9 6.5 d 78.4 2 Naturalis (B. bassiana) 125, 7 15.5 c 50.5 3 Naturalis (B. bassiana) 125, 5 23.0 b 27.0 4 Rogatox (dimethoate) 150, 2 13.3 cd 59.5 5 Untreated control - 32.5 a - Trial no. 3 – Mineo (Catania), 2003² 1 Naturalis (B. bassiana) 125, 7 5.8 a 79.0 2 Naturalis (B. bassiana) 125, 4 12.0 b 58.2 3 Naturalis (B. bassiana) 125, 3 15.0 b 47.0 4 Lebaycid (fenthion) 100, 3 7.5 a 73.0 5 Untreated control - 29.3 d - Trial no. 4 – Trani (Bari), 2004¹ 1 Naturalis (B. bassiana) 130, 9 25.7 c 68.1 2 Naturalis (B. bassiana) 250, 5 36.7 b 54.2 Rogor L40 (dimethoate)+ 100, 4 3 23.1 c 71.2 Naturalis (B. bassiana) 130, 2 4 Rogor L40 (dimethoate) 100, 2 22.3 c 72.0 5 Untreated control - 81.0 a - Trial no. 5 – Mineo (Catania), 2004³ 1 Naturalis (B. bassiana) 125, 8 50 b 39.5 2 Naturalis (B. bassiana) 250, 4 37 c 55.4 Danadim (dimethoate)+ 140, 2 3 30 d 64.4 Naturalis (B. bassiana) 130, 2 4 Danadim (dimethoate) 140, 2 32 cd 61.3 5 Untreated control - 82 a - Trial no. 6 – Tursi (Matera), 2004¹ 1 Naturalis (B. bassiana) 125, 5 7.8 c 92 2 Naturalis (B. bassiana) 250, 3 35.3 b 62 Lebaycid (fenthion)+ 100, 1 3 7.8 c 92 Naturalis (B. bassiana) 130, 2 4 Lebaycid (fenthion) 100, 1 36.8 b 61 5 Untreated control - 94.0 a - ¹ Different letters indicate statistically significant differences (Student-Newman-Keuls test: P<0.05). ² Different letters indicate statistically significant differences (Tukey HSD test: P<0.05). ³ Different letters indicate statistically significant differences (Duncan’s multiple range test: P<0.05). determined. Furthermore, in each trial, the efficacy according to Abbott (Abbott, 1925) of the different treatments in reducing percent fruit damage was calculated. 129

Statistical analysis In each trial, the percentage of fruits damaged by B. oleae was compared across treatments using one-way ANOVA, followed by the Student-Newman-Keuls test (trial no. 1, 2, 4, and 6), the Tukey HSD test (trial no. 3), or Duncan’s multiple range test (trial no. 5) for posthoc comparison of means.

Results and discussion

When applied alone, the efficacy of the B. bassiana-based insecticide in reducing the percentage of fruits damaged by the olive fly at harvest (total no. trials = 6), was usually comparable to and sometimes even higher than that of the chemical standard (Table 3). The efficacy of weekly applications of the formulated B. bassiana strain at 125-130 ml/hl was comparable to that of the chemical reference treatment in 4 out of 6 trials, and significantly higher than that of the chemical standard in trial no. 6. For an efficient control of B. oleae on olive, we therefore recommend applications of the B. bassiana-based bioinsecticide at 5-7-day intervals. The number of required applications may vary with climatic conditions and infestation level. Even when the time interval between successive applications of Naturalis was increased to 14 days, the efficacy of the B. bassiana-based treatments was still comparable to that of the chemical reference treatment in 4 out of 6 trials. In this case, a slight dose-response effect seems to exist: at the dose of 125 ml/hl, the efficacy of Naturalis ranged from 37.1 to 58.2% (trial no. 1-3), while at 250 ml/hl it varied from 54.2 to 62.0%. The results on the use of the B. bassiana formulation in an integrated strategy are not yet conclusive, because a statistically significant increase in the efficacy of the integrated strategy compared to the chemical reference treatment was recorded only in 1 trial out of 3. In order to establish recommendations for the use of the product in integrated pest management strategies, further studies were conducted in 2005 (data processing in progress). Given these results and the promising results obtained in other trials with the same bioinsecticide against C. capitata and R. cerasi (unpublished), the product can be considered a new and reliable tool for the control of tephritid flies, especially olive flies. In fact, in 2005, the registration of Naturalis in Italy was extended also to these pests. Further studies are needed to establish whether mechanisms other than simple contact action are involved in the activity of B. bassiana against the olive fly (Konstantopoulou & Mazomenos, 2005).

References

Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. – J. Econ. Entomol. 18: 265-267. BCPC 2004: Beauveria bassiana biological insecticide (fungus). – In: The Manual of Biocontrol Agents, 3rd edition, ed. L.G. Copping: 43-46. Benuzzi, M. & Santopolo, F. 2001: Naturalis: bioinsetticida a base di Beauveria bassiana. – Informatore Fitopatologico 4: 61-64. Benuzzi, M., Albonetti, E. & Baldoni P.G. 2001. Prova di lotta biologica su fragola contro il ragnetto rosso (Tetranichus urticae) con un formulato a base di Beauveria bassiana. – Notiziario sulla protezione delle piante 13: 39-44. Delrio, G. 1992: Integrated control in olive groves. – In: Biological Control and Integrated Crop Protection: Towards Environmentally Safer Agriculture, Proc. Int. Conf. IOBC/WPRS, Veidhoven, Netherlands: 67-76. 130

Hallsworth, J.E. & Magan, N. 1999: Water and temperature relations of growth of the entomogenous fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus. – J. Invert. Pathol. 74: 261-266. Knauf, T.A. 1992: Naturalis-L: a biorational insecticide for boll weevil and whitefly control. – Proc. Beltwide Cotton Conference 1: 21-32. Konstantopoulou, M.A. & Mazomenos, B.E. 2005: Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata. – BioControl 50: 293-305. Lacey, L.A., Horton, D.R., Chauvin, R.L. & Stocker, J.M. 1999: Comparative efficacy of Beauveria bassiana, Bacillus thuringiensis, and aldicarb for control of Colorado potato beetle in an irrigated desert agroecosystem and their effects on biodiversity. – Entomologia Experimentalis et Applicata 93: 189-200. Wright, J.E. & Kennedy, F.G. 1996: A new biological product for control of major greenhouse pests. – Proc. Brighton Crop Protection Conference. Pests & Diseases: 886-892.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 131

tm Bait applications effect of Spinosad Success 0.24CB (GF-120)" formulation, on Bactrocera oleae Gmel. (Dacuol), and impact on other non target organisms in olive trees

P.V. Vergoulas1, D. Prophetou-Athanassiadou2, E. Alimi3, H. Ben Salah4, C. Mavrotas5, C. Jousseaume1, M. Miles6 1 DowAgrosciences, Sophia Antipolis, France. 2 Professor of Entomology, Thessaloniki University, Greece. 3 Conseiller Agricole, Tunis, Tunisia. 4 Entomologist in State Tunisian Research Institute, Tunis, Tunisia. 5 DowAgroSciences, Athens, Greece. 6 DowAgroSciences, Abingdon, UK

In 2002, two field studies were organized to evaluate the effect of spinosad bait treatments on beneficial arthropods and to compare them with the standard organophosphate bait treatments of fenthion and dimethoate. SuccessTM (GF-120, a prepackaged bait which contains 0.24g spinosad/L), was tested in North Greece by Thessaloniki University on olive trees to evaluate the effect on the whole range of non target insects present in the field. It was also tested on caper plants by the State Tunisian Research Institute to evaluate the effect on the parasitoid Opius concolor. In the North Greece in olive trial, GF-120 at 1250 mL/ha was statistically equal to the untreated to Chilocorus sp., Chrysopa sp., Coccinella sp., Coccinellidae predators and Hymenopteran parasitoids. All GF-120 treatments were safer and statistically different than fenthion to Chilocorus sp., Coccinella sp., Chrysopa sp., Coccinellidae predators and Hymenoptera parasitoids, while statistically equal to fenthion on Syrphidae predators. In Tunisia in caper plants, GF-120 was safe to Opius concolor and equal to the untreated. The standard, dimethoate bait treatment, was toxic to Opius concolor. It was conclued that GF-120 ready made bait, at its proposed recommended use rates, was safe to a wide range of non target beneficial arthropods.

TMTrademark of DowAgroSciences

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 133

Effect of several insecticides for control of Bactrocera oleae (Gmelin) (Diptera: Tephritidae) to arthropods fauna of olive grove

V. Alexandrakis1, K. Varikou1, A. Kalaitzaki1, D. Lykouressis2 1 Institute of Olive Tree and Subtropical Plants, National Agricultural Research Foundation, Agrokipio, 731 00 Chania, Crete, Greece. 2 Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, Iera Odos 75, 188 55 Athens, Greece.

In 2002-2004 field studies have been organized to evaluate the effect of bait sprays with several insecticides, for control of Bactrocera oleae (Gmelin), on arthropods fauna of the olive grove. The experiments have been carried out in Chania (Crete, Greece), by the Institute of Olive Tree and Subtropical Plants on 96.000 olive trees in Glossa region. The insecticides that were used were the pyrethroids: deltamethrin 2.5% (Decis flow 2.5), b cyfluthrin 2.5% (Bulldock 025 SC), b cypermethrin 10% (ATO 10 EC), l cyhalothrin (Karate), a cypermethrin 10% (Fastac 10 SC), z cypermethrin 10% (Fury 10 EW), the organophosphate insecticides: dimethoate 40% (Dimethoate 40 EC) and fenthion 50% (Fenthion 50 EC) and the selective insect control product produced by the fermentation of the naturally occurring soil bacterium Saccharopolyspora spinosa (Spinosad) (NAF 85 and GF-120). The results showed that more than 170 different arthropod species were recorded, which belongs to 50 insects families and 12 orders of arthropods. Heteroptera species were found to be the most abundant. From the recorded species 23 were beneficial of the families Braconidae (Praon sp., Opius concolor Szepl., Apanteles sp., Dacnusa sp., Chelonus eleaphilus Silv.), Eupelmidae (Eupelmus urozonus Dalm.), Scelionidae (Telenomus sp.), Eulophidae (Pnigalio sp.), Syrphidae (Syrphus sp.), Coccinelidae (Lindorus lophanthae (Blaisdell), Chilocorus bipustulatus L., Adalia bipunctata L., Coccinella septempunctata L., Scymnus sp.), Chrysopidae (Chrysoperla carnea Stephens) and Anthocoridae (Orius leavigatus Fieber). Among the tested chemicals, spinosad had the lowest toxicity on the Hymenoptera species than the other chemicals. Pyrethroids group had the highest toxicity on Hymenoptera followed by the organophosphate insecticides. All the tested chemicals had the same effect on the Diptera species. On the Lepidoptera were not found differences among the tested insecticides and their toxicity on them was very low. On Coleoptera only pyrethroids group showed high toxicity.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 135-138

Mass trapping experiments with two different “Attract and Kill” devices for Bactrocera oleae (Gmelin)

Nino Iannotta, Massimiliano Pellegrino, Enzo Perri, Luigi Perri, Fausto De Rose C.R.A. Institute for Olive Growing - 87036 Rende, Cosenza, Italy

Abstract: Different experiments using mass-trapping performed over the last years have shown good efficacy for the control of olive-flies, thus providing a potential solution to active infestation and a consequent reduction in olive damage. However, the substantial cost of the attract and kill system, and especially their installation and employment in large areas where they show the maximum efficacy, has rendered their commonplace use impractical. In the present report, we compared the efficacy of two different types of traps (type 1, already commercially available, and type 2, which is in an experimental phase), both produced by Agrisense that were used in smaller numbers than usually employed per hectacre (150 and 100, respectively, instead of 400). The traps were primed by sexual attraction (Spiroketal) and by olfactory attraction (ammonium salts). Lambda-cyhalothrin, which is allowed under organic farming legislation, was used as an insecticide. The two types of traps were compared to an untreated plot. There were no significant differences in the adult population present in the field in the different treatment groups. However, examination of the active and total infestation showed that the type 1 trap was more efficacious. This improved efficacy was evident until October, when the harvesting time is generally considered optimal.

Key words: mass trapping, olive fly, attract and kill

Introduction

In olive-growing, the increasing pressures demanded by organic production requires the cessation of chemical treatments against phytophagous insects and pathogens. In southern Italy, Bactrocera oleae is the principal phytophagous insect involving the olive ecosystem and causes considerable damage to the quality and quantity of oil production. Accordingly, control systems must be implemented in order to obtain high quality oil and to render it competitive from an economic standpoint. Among the different control systems utilized against Bactrocera oleae, mass-trapping has appeared only relatively recently and appears very interesting. This method has been experimented in Italy for more than 10 years and provides satisfactory results when applied to large areas and accompanied by appropriate agronomical practices such as early harvesting, which increase its effectiveness. Moreover, a system based on the use of traps primed by olfactory and sexual attractants assures a high selectivity due to the use of specific attractants and, consequently, it is considered highly environmentally friendly. Traps also completely lack any toxicological risk for humans, since there is no contact with the active agent. Additionally, traps are perfectly compatible with the present legislation on organic cultivation in Italy, which has recently allowed the use of selected insecticides (deltametrine and lambda cyhalothrin) in mass capture devices. The aim of the present study was to test the effectiveness of mass capture devices against Bactrocera oleae and to evaluate these devices, considered more effective and less expensive, as either purchase cost or as overheads. The new devices have been constructed keeping product cost low and last for the entire application period (from June to harvesting time). This

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eliminates the need to repeatedly change the traps and results in savings in the costs for new traps as well as the labor for their replacement. In these tests, we utilized the new “attract and kill” mass-trapping devices, which are less expensive and are more simple to manage compared to older models. The new devices were assessed in the Calabria region in Italy, which is highly infested with the above-mentioned Diptera.

Materials and methods

The test were performed in the olive collection of the C. R. A.- Experimental Institute for Olive Growing in Rende, Cosenza on 20-year-old plants from June to December 2004. This plant collection field was divided into two areas, where 100 “attract and kill” traps (AgriSense) per hectare were installed in June. In the first region (area A), already available commercial devices were utilized (type 1), while the new experimental devices (type 2) were used in the second area (area B). Both devices were loaded with an olfactory attractant (ammonium carbonate) and a sexual attractant (Spiroketal), but differ in their inert support. The former is made up of funnel-shaped cardboard, while the latter is constructed using double plastic sheets. Another distant field in the same environmental and cultivation conditions was used as a control, untreated area (area C). Three chromotropic traps per placed per hectare in order to monitor the trend of the adult population during the entire time where tests were carried out. From the time of stone hardening (mid-July) and until November, 200 olive samples were picked in duplicate every 10 days to monitor active and total infestation. Meteorological data were collected by an observation post equipped with electronic sensors. The data were compared by analysis of variance tests.

Results

Figure 1 shows the results on adult population present in the three treatment areas. It is evident that a lower number of captures were found in area A compared to the other two areas that demonstrated significant differences during the period from September to November. Zones B and C showed a highly similar trends in terms of the adult population.

Active infestation A 70 B 60 C 50 40 30 20 Active inf. % inf. Active 10 0 12/8 13/9 5/10 26/10 16/11

Figure 1. Adult of olive fly population

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Captures A 60 B 50 C 40 30 20 N° adults/trap N° 10 0 2/7 2/8 2/9 28/9 19/10 9/11 30/11

Figure 2. Percentage of active olive fly infestation.

Figure 2 shows the results in active infestation. A lower percentage of infestation was recorded in area A, which persisted until harvesting time (end of October-beginning of November); the amount of active infestation was within the 20% limit. In contrast, in areas B and C the level of infestation appeared to be higher during the entire observation period and statistically significant differences were observed with respect to area A during the optimum harvesting period. During the first days of November, the active infestation increased considerably in all areas.

Total infestation A 90 B 80 C 70 60 50 40 30 Total inf. % inf. Total 20 10 0 12/8 13/9 5/10 26/10 16/11

Figure 3. Percentage of total olive fly infestation.

In Figure 3 the total infestation is shown in which a lower incidence in area A was observed compared to zones B and C.

Discussion

Our results demonstrated a greater effectiveness of the traditional device (type 1) with respect to the newer model. This improved efficacy is reflected in the data on the adult population, in 138

addition to that regarding active infestation, which is responsible for qualitative and quantitative worsening of olive production. In conclusion, our data support the effectiveness of mass-trapping by using devices that decrease the level of active infestation by about 50% compared to untreated groves. In fact, active infestation was restrained within 20% until harvesting time, a percentage that is still compatible with the production of high quality oil.

Acknowledgements

This research has been carried out with funding from the MIUR as part of the OLIVIBIO project.

References Broumas, T., Haniotakis, G. 1986: Further studies on the control of the olive fruit fly by mass trapping. – II Intern. Symp. Fruit Flies, Crete Sept. 1986: 561-565. Broumas, T., Haniotakis, G., Yamvrias, C., Stavrakis, G. 1990: Comparative studies of a mass trapping method and various bait sprays for the control of the olive fruit fly. First year results. – In: Pesticides and alternatives, Casida J.E. (ed.): 205-215. Broumas, T., Liaropoulos, C., Yamvrias, C., Haniotakis, G. 1984: Experiments on the control of the olive fruit fly by mass trapping. In: Integrated Pest Control in Olive-groves. R. Cavalloro & A. Crovetti (eds.), Apr. 84, A.A. Balkema,: 84-93. Delrio, G., Lentini, A., Bandino, G., Sedda, P.G. 1990: Experiments on the control of the olive fruit fly by mass trapping in Sardinia. – Atti del Convegno “IOBC Fruit Fly Open Meeting, Working Group on Fruit Flies of Economic Importance”, Sassari, 26-27 November 1990. Delrio, G., Lentini, A. 1993: Applicazione della tecnica delle catture massali contro il Dacus oleae in due comprensori olivicoli dalla Sardegna. – Atti del Convegno “Olivicoltura”, Firenze 1991: 41-45. Haniotakis, G. 1984: Control of the olive fruit fly, Dacus oleae Gmel. (Dipt., Tephritidae) by mass trapping: present status – Prospect. – Proc. VII Circum-Mediterranean Plant Protection Organisation, Chanea, Crete, Greece, Sept.: 24-28. Haniotakis, G., Kozyrakis, M., Bonatsos, M. 1986: Area-wide management of the olive fruit fly by feeding attractants and sex pheromones on toxic traps. – II Inter. Symp. Fruit Flies/Crete, Sept. 1986: 549-560. Haniotakis, G., Kozyrakis, M., Fitsakis, T., Antonidaki, A. 1991: An effective mass trapping method for the control of Dacus oleae (Dipt.: Tephritidae. – J. Econ. Entom. 84: 564-569. Iannotta, N. 2003: La difesa fitosanitaria. – In: “Olea Trattato di olivicoltura”, ed. Ed agricole- Il sole 24ore: 393-407. Iannotta, N., Monardo, D., Perri, L., Alessandrino, M. 2002: Trappolaggio massale di Bactrocera oleae (Gmel.) con un nuovo dispositivo bersaglio (attract and kill). – Atti Conv. Int. Olivicoltura, Spoleto: 444-448. Iannotta, N., Perri, L. 1993: Prova di mass trapping nel controllo di Dacus oleae (Gmel.) in Calabria. – Convegno “Olivicoltura”, Firenze 1991: 41-45. Iannotta, N., Perri, L., Rinaldi, R. 1994: Control of the olive-fly by mass-trapping in Calabria. – Acta Horticulturae 356: 411-413. Petacchi, R., Rizzi, I., Guidotti, D., Toma, M. 2000: Informatizzazione della raccolta e gestione dei dati nei programmi finalizzati al controllo della mosca dell’olivo: l’esperienza della Regione Toscana nella tecnica delle “catture massali”, in corso di stampa su l’Informatore Agrario. Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 139-145

Tests on the effectiveness of mass trapping by Eco-trap (Vyoril) in the control of Bactrocera oleae (Gmelin) in organic farming

Virgilio Caleca, Roberto Rizzo, Isabella Battaglia, Manuela Palumbo Piccionello Dipartimento SENFIMIZO, Sezione di Entomologia, Acarologia e Zoologia, Università di Palermo, Viale delle scienze, 90128 Palermo ITALY. E-mail: [email protected]

Abstract: Tests on the effectiveness of mass trapping by Eco-trap (Vyoril) in the control of Bactrocera oleae (Gmelin) in organic farming were carried out in 2003 and 2004. The tests took place into two organic olive groves located in Agrigento and Trapani (Sicily); in both years the olive cultivar was Cerasuola. In Agrigento, it was considered the effectiveness of Eco-trap vs. bottle traps baited with diammonium phosphate; while in Trapani the effectiveness of Eco-trap added to other products admitted in organic farming (two products containing kaolin and one containing copper) was evaluated. In 2003, year with a low B. oleae population density, no statistically significant difference resulted among Eco- trap, bottle traps with diammonium phosphate and control. In 2004 B. oleae infestations were high; although some statistically significant differences among plots with Eco-traps and plots without them emerged, the additional power of Eco-trap in controlling B. oleae resulted very limited in plots sprayed with kaolin products and more consistent in the plot with copper hydroxide. The economic advantage of the use of Eco-trap, also in comparison with repellent and antiovipositional products, still remains doubtful.

Key words: olive fruit fly, attractive, repellents, antioviposition.

Introduction

The control of the olive fruit fly, Bactrocera oleae (Gmelin), in organic farming is mostly realized by mass trapping. In the organic farming, the Council Regulation (EEC) No. 2092/91 permits the use of two pyrethroids (deltamethrin and lambda-cyalothrin) only in traps to control B. oleae and Ceratitis capitata (Wiedemann). In organic olive groves, mass trapping can be made by using poisoned bait, or various traps, such as: chromotropic traps, bottle traps with food attractants and traps with ammoniacal salts, sexual pheromone plus pyrethroids. One of the most utilized trap is Eco-trap (Vyoril), a polyethylene small bag (15 x 20 cm) covered with special paper treated with deltamethrin, containing 70gr of ammonium bicarbonate and provided with a sexual pheromone (1,7-dioxaspiro-5,5-undecano) dispenser. The aim of the research was to test the effectiveness of Eco-trap in the control of B. oleae infestation in Sicilian organic olive groves, in comparison with bottle-traps baited with diammonium phosphate or in addition to others repellent products permitted in organic olive groves.

Materials and methods

Eco-trap vs. bottle traps baited with diammonium phosphate (2003) In 2003 the test on the effectiveness of Eco-trap, was carried out in an olive grove (cultivar Cerasuola) of 6.6 ha in Licata, Agrigento province (Sicily); the field was divided into 3 plots (Figure 1):

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1. 2 ha with Eco-trap (one trap for each tree along the perimeter and one every 2 trees inside the plot); 2. 2.6 ha with bottle traps containing a 4% diammonium phosphate water solution (the same trap density of the preceding plot); 3. 2 ha untreated plot; initially, sprays of copper hydroxide should be done in a half of this plot, but the total infestation did not exceed the limit of 5% until the 17th October; so, there was no need to treat, and all the plot was untreated. To survey the infestation level, olives were sampled (every two weeks) collecting 100 drupes from the trees of the central part of each plot (Figure 1).

OOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOOOO OOOOOOOOOOOOOO OOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOEco-trap OOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOPhosphate OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOBottle-traps OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOControl OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOO OOOOOOOOOOOOOOOOOOOOO OOOO OOOO O

Figure 1. Licata olive grove (2003); grey area = sampled trees.

Assessment of the additional power of Eco-trap to others products permitted in organic olive groves (2004) In 2004 tests on the effectiveness of Eco-trap, were carried out in an olive grove (cultivar Cerasuola) of about 2 ha in Trapani (Sicily); the field was divided into 2 parcels, one with Eco-trap and one without, each of them divided into four smaller plots (Figure 2): 1. the first one was unsprayed; 2. the second one was sprayed twice (7th September and 3rd October) with Surround WP (containing 95% of kaolin);

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3. the third one was sprayed twice with BPLK kaolin; 4. the last one was sprayed twice with copper hydroxide. To survey the infestation level, olive trees were weekly sampled collecting 60 drupes in each of the 8 plots (10 drupes from each of the six sampled trees). Collected drupes were examined at the stereoscopic microscope to pick out the preimmaginal stages of the fly, the exit holes and the empty tunnel. The infestation pointed out on the drupes was subdivided in: 1. active infestation (eggs, alive first and second instar larvae); 2. harmful infestation (third instar larvae, pupae, exit holes and the empty tunnel); 3. total infestation (active infestation added to harmful infestation).

Plots without Eco-trap Plots with Eco-trap ↓↓ OOO OOO OXXO OOOO OOOOOOOO O Unsprayed → OOXOXXXO OXXXXXXOOO OOOOOOOO OOOOOOOOOOO OOOOOOOO OOOOOOOOOOO OXOX OXO OOOXXXOXOOO Surround WP → OOOXXOXO OOOOOXXOOOO OOOOOOO OOOOOOOOOO OOOOOOO OOOOOOOOOOOO OOXOOXX OOOOXOOOOXOO Other BPLK Kaolin → OXOOXOX OOOOXX XXOOO olive OOOOOO OOOOOOOOOOOO trees OOOOO OO OOOOOOOOO with OOOOXX OOO OOXOXOXOO Eco-trap Copper Hydroxide→ OOXXXXO OXOXOXOOO OOO OO ↑↑ Plots without Eco-trap Plots with Eco-trap

Other olive trees with Eco-trap

X Sampled tree O Not sampled tree

Figure 2. Trapani olive grove (2004).

Results and discussions

In 2003, year with low olive fly infestation, the three plots of Licata olive grove did not result statistically different in the whole period (Figure 3). In 2004 B. oleae infestation reached high levels in Trapani olive grove. Comparing the total infestation of the whole period in the 8 theses, 5 theses (4 sprayed with kaolin products and one with Eco-trap and copper hydroxide) recorded the lowest infestation without statistically significant difference among them; the thesis sprayed with copper hydroxide reached a higher infestation level without statistically difference with the thesis sprayed with BPLK kaolin, while the two unsprayed plots (with or without Eco-trap) reached the highest infestation level without statistically difference between them (Table 1).

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The two couples of plots unsprayed or treated with Surround WP did not show any statistically difference both in the total and harmful infestation analysing the whole period through repeated measurements ANOVA, while in the last sampling date plots with Eco-trap showed a significantly lower infestation (1-way ANOVA) (Figures 4 & 5).

Vertical bars show confidence intervals of 95% 0,25

Rep ea ted me as uremen ts A NOV A 1-w ay ANOVA 0,20 Average of the w hole period 10-Nov-03 Untreated 0.02 a 0.11 a Eco-traps 0.01 a 0.06 a Phosphate Bottle-traps 0.03 a 0.16 a 0,15

attacks per olives 0,10

0,05 Bactrocera oleae oleae Bactrocera 0,00

Number of -0,05

-0,10 8-Aug-03 20-Aug-03 18-Sep-03 1-Oct-03 17-Oct-03 10-Nov-03

Figure 3. Trend of B. oleae total infestation in Licata olive grove in 2003.

Table 1. Total infestation (no. attacks per olive), in Trapani olive grove in 2004 (1-way ANOVA within each column; different letters denote statistically significant differences)

ANOVA repeated Theses 25thAug 7thSept 21stSept 2ndOct 9thOct 16thOct 23rdOct 30thOct 6th Nov measurement (for the whole period)

Untreated 0.00 0.00 a 0.10 a 0.47 a 0,45 ab 0.98 a 1.57 a 1.60 ab 2.78 a 0.88 a

Eco-trap 0.00 0.00 a 0.07 ab 0.33 ab 0,58 a 0.88 a 1.40 a 1.95 a 2.00 b unsprayed 0.80 a

Surround WP 0.00 0.02 a 0.00 b 0.13 bc 0,23 bc 0.10 c 0.38 bc 0.48 e 1.07 c 0.27 c

Eco-trap + 0.00 0.00 a 0.02 ab 0.12 c 0,22 c 0.10 c 0.18 c 0.28 e 0.7 c Surround WP 0.18 c

BPLK kaolin 0.00 0.00 a 0.03 ab 0.06 c 0,13 c 0.37 b 0.38 bc 0.97 cd 0.97 c 0.32 bc

Eco-trap + 0.00 0.02 a 0.00 b 0.12 c 0,12 c 0.23 bc 0.17 c 0.63 de 1.08 c BPLK kaolin 0.26 c Copper 0.00 0.02 a 0.02 ab 0.10 c 0,13 c 0.28 bc 0.52 b 1.22 bc 2.02 b hydroxide 0.48 b Eco-trap + copper 0.00 0.02 a 0.00 b 0.06 c 0,02 c 0.07 c 0.17 a 0.55 e 0.7 c 0.17 c hydroxide

143

Vertic al bars show c onf idence intervals of 95% 4,0

3,5 Repeated measurements ANOVA 1-way ANOVA 3,0 Av erage of the whole period 6-Nov Untreated 0.88 a 2.78 a Eco-trap 0.80 a 2.00 b 2,5 attacksolives per 2,0

1,5

Bactrocera oleae 1,0

0,5 Number of of Number 0,0

-0,5 25-Aug 7-Sep 21-Sep 2-Oct 9-Oct 16-Oct 23-Oct 30-Oct 6-Nov

Figure 4. Trend of B. oleae total infestation in untreated and Eco-trap plots (Trapani, 2004).

Vertical bars show conf idence interv als of 95% 1,6

1,4

1,2 Repeated measurements ANOVA1- way A N OV A Av e rag e of the wh ole p eri od 6- No v 1,0 Surround WP 0.27 a 1.07 a Eco-trap + Surround WP attacks per olives per attacks 0.18 a 0.67 b 0,8

0,6

0,4 Bactrocera oleae

0,2

Number of 0,0

-0,2 25-Aug 7-Sep 21-Sep 2-Oct 9-Oct 16-Oct 23-Oct 30-Oct 6-Nov

Figure 5. Trend of B. oleae total infestation in Surround WP and Eco-trap + Surround WP plots (Trapani, 2004).

144

Vertical bars show conf idence interv als of 95% 1,6

1,4 R epeat e d m ea su rem e nt s AN OVA1-way ANOVA 1,2 Av erage of the whole period 6-Nov BPLK Kaolin 0.33 a 0.97 a Eco-trap + BPLK Kaolin 0.26 b 1.08 a 1,0 attacks per olives per attacks 0,8

0,6

0,4 Bactrocera oleae

0,2

Number of 0,0

-0,2 25-Aug 7-Sep 21-Sep 2-Oct 9-Oct 16-Oct 23-Oct 30-Oct 6-Nov

Figure 6. Trend of B. oleae total infestation in BPLK kaolin and Eco-trap + BPLK kaolin plots (Trapani, 2004).

Vertical bars show conf idence interv als of 95% 2,5

Repeated measurements ANOVA1-way ANOVA 2,0 Av erage of the whole period 6-Nov Copper Hy droxide 0.48 a 2.01 a Ecotrap + Copper Hy droxide0.17 b 0.68 b 1,5 attacks per olives

1,0

0,5 Bactrocera oleae

0,0 Number of of Number

-0,5 25-Aug 7-Sep 21-Sep 2-Oct 9-Oct 16-Oct 23-Oct 30-Oct 6-Nov

Figure 7. Trend of B. oleae total infestation in copper hydroxide and Eco-trap + copper hydroxide plots (Trapani, 2004).

145

In the two couples of plots treated with BPLK kaolin and copper hydroxide, the total infestation of the whole period was significantly lower in plots with Eco-trap (Figures 6 & 7). The statistical analysis of the total infestation on November 6th showed a significant lower level in the plot with copper hydroxide with Eco-trap vs. the plot copper hydroxide without Eco-trap (Figure 7); on the contrary, in the couple of plots sprayed with BPLK kaolin no difference resulted (Figure 6). The additional power of Eco-trap in controlling B. oleae resulted very limited in plots sprayed with kaolin products and more consistent in the plot with copper hydroxide. Results of this research confirm that the use of Eco-trap has not a clear effectiveness in reducing B. oleae infestation (Viggiani, 2001); in case of low pressure of the olive fly its use does not provide a significant reduction, and we confirm that the same happens, in any case, in the more external trees of the field or in small fields (Tsolakis & Ragusa, 2005); in case of high pressure of the tephritid the inner parts of the olive groves with Eco-traps, although some statistically significant differences, did not show so good results balancing their high cost. Using Eco-traps their cost is every year the same, but in years with low level of B. oleae infestation their use will result unnecessary, while in years with high infestation levels, their use, also in organic olive groves, could be effectively replaced by a more flexible strategy consisting of 1-3 sprays with repellent products such as kaolin or copper products.

Acknowledgements

We thank Massimo Carlino and Giovanni Curatolo landholders of two olive groves, and Marco Corcella for his help in collecting field data. Research funded by University of Palermo Fondi di Ateneo ex quota 60% (“Il controllo degl’insetti fitofagi nell’agricoltura biologica e convenzionale”) and Assessorato Agricoltura e Foreste della Regione Siciliana (“Innovazioni della filiera olivicola da mensa”).

References

Tsolakis, H. & Ragusa, E. 2005: Osservazioni preliminari sulla tecnica attratticida e sugli effetti della sua combinazione con altre tecniche di controllo di Bactrocera oleae Gmelin. – Atti XX Congresso Nazionale Italiano di Entomologia, Perugia-Assisi, giugno 2005: 272. Viggiani, G. 2001: Controllo della mosca delle olive con ecotrappole. – Informatore agrario 57(25): 69-73.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 147-151

Control trials of Bactrocera oleae (Gmel.) (Diptera Tephritidae) in the district of Bar in Montenegro

Tatjana Perović1, Snježana Hrnčić2, Antonio Franco Spanedda3, Alessandra Terrosi3, Claudio Pucci3, Biljana Lazović1, Mirjana Adakalić1 1 Centre of Subtropical Cultures, Bjelisi bb, 85000 Bar, Montenegro; 2 Biotechnical Institute Centre of Plant Protection, Kralja Nikole bb 81000 Podgorica, Montenegro; 3 Università degli Studi della Tuscia – Dipartimento di Protezione delle Piante, Via S. Camillo de Lellis, 01100 Viterbo – Italy.

Abstract: The aim of this paper was an evaluation of the efficacy of bioinsecticides based on Spinosad for olive fly control. The investigations were carried out in olive-grove (Žutica variety), under agroecological condition of Bar (Montenegro), during 2004. The following insecticides were applied as bait spray: Success™, GF 120 fly bait™ and Decis™. From the beginning of September five treatments were administered on average intervals of 7-10 days. First treatment was applied when the gravity index Z exceeded the threshold level (Z>0.10). Efficacy of applied insecticides was evaluated weekly and expressed as to their effect on level of infestation. Obtained results show that insecticides based on Spinosad aren’t sufficient for control of olive fly.

Key words: Bactrocera oleae, treatment, proteinic bait, Spinosad, Deltamethrin

Introduction

In Montenegro, as in the other Mediterranean countries, B. oleae is the most important pest of olive crop, causing substantial damage every year, often responsible for up to 50% of yield losses (Mijušković, 1955). The control of the pest since the 60s was based on ground spraying of the entire tree canopy using parathion insecticides. Following decades characterised the use of bait treatments with organofosfate insecticides of broad spectrum activity – dimethoate and fenthion. In the last 10-15 years protein hydrolysate bait sprays containing delthametrin insecticide were used as aerial strip treatment. The modern tendency in protecting olive-trees from infestation of B. oleae takes into account the development of new techniques and methods able to enhance all those biotical factors of natural control that are more selective and that influence as little as possible the ecosystem’s balances. In this context there was considerable experimental activity with the purpose to evaluate the effect of use of the natural substances on reducing the olive-fly infestation. In this contribution the effect of compounds of microbial origin on fly infestation in 2004, particularly favourable for Tephritidae, was investigated.

Material and methods

The olive-grove where the research was carried out is situated in the district of Bar (Monte- negro) and the main present cultivar is Žutica.

147 148

The experimental programme involved the identification of four plots: three of them were treated with proteinic bait while the remaining one acted as untreated control. The first two sections were treated with bio insecticides based on Spinosad, whose active principle derives from an, Saccharopolyspora spinosa, present in the ground and active for ingestion and contact on different species of insects including apple tree leaf miners. The third section received a treatment based on Deltamethrin. In all of theses the plants were sprinkled with a 1 litre of solution at half canopy, on the southern part. The active principles compared were: 1. Spinosina A+D at the concentration of 0.02% of a.i. (Success™) at the dose of 750 cc/hl and added to a proteinic bait (Buminal); 2. Spinosina A+D at the concentration of 11.6% of a.i. (GF 120 Fly bait™) at the dose of 90 ml/hl; 3. Deltamethrin at the concentration of 2.39% of a.i. (Decis™) with dose of 100 g/hl added to a proteinic bait (Buminal). Population was monitored in the period from the beginning of July to the end of October. At the end of June 3 plants per plot were chosen. On each tree a yellow-sticky trap was placed at the medium height of the canopy, on the southern side. Olive fly adults were counted weekly. Average temperature was recorded in the same capture week. Olive fly infestation was assessed at the same period. The olive sampling was performed by randomly picking 40 drupes per plant, from trees with yellow-sticky trap (total of 120 fruits for each portion). Olive fruits were observed by stereomicroscope with the aim of quantifying infestation on the basis of a number of eggs, 1st + 2nd + 3rd instar larvae, pupae, empty cocoons and abandoned galleries.

Table 1. Synoptic table of treatments.

PLOT A B C D

Active ingredients deltamethrin spinosyn A + D spinosyn A + D Control

A.i. concentration 2.8 % 0.02 % 11.6 % dosis: dosis: dosis: 100 ml/hl 750 cc/hl 120 cc/hl (DECIS) (GF 120 FLY BAIT) (SUCCESS) + 1000 g/hl of + 1000 cc/hl of MODE OF proteinaceous bait proteinaceous bait Not treated EMPLOY (BUMINAL) (BUMINAL)

quantity: quantity: quantity: 0,5-1 litres/tree on 0,5-1 litres/tree on 0,5-1 litres/tree on southern side of southern side of southern side of canopy canopy canopy 03 September 2004 DATE OF 11 September 2004 TREATMENT 23 September 2004 28 September 2004 07 October 2004

The treatment threshold was defined using the “female” forecast model and treatments were applied when the gravity index Z exceeded the threshold level equal to 0.10 value. 149

The first intervention was carried out at the beginning of September and the following four continued on average intervals of 7-10 days (Table 1.). The effect of applied insecticides was evaluated weekly.

Results and discussion

Level of infestation in all treatments, during investigation period, is shown in Figure 1. In all four sections, in summer time, the quantity of captures and infestations remained at a low level because of the elevated temperatures. An increase in the infestation level was recorded at the end of August. Infestation levels in all sections was the same– under 20% up to September 21st. However the infestation, as verified in October, had such a sudden increase that at harvest it was on values of 80-90% in the those treated with Spinosad, and even 100% in the control. Instead, in the section treated with Deltamethrin, the infestation at harvest was much more restrained, on values of 30%. Considering the poor effects of Spinosad added to a proteinic bait treatments, it would be better to carry out more research using the same active principles with covering treatments realised on all foliage. Spinosad is the only approved compound for control of olive fly in California and its weekly application is recommended. It is also approved for organic certification in the EU. Studies of Vergoulas et al., (2004, 2004a) show that Spinosad has an excellent profile on non- target beneficial arthropods. Different tests carried out in California, Greece, Cyprus, Tunisia and Turkey showed heterogeneous effects. The Olive Oil Source (2002) reported that spinosad has demonstrated mixed success in controlling the fly. They notify that table olive growers in Tulare, sprayed spinosad in two week intervals from June continuing in some cases up to the day before picking, had obtained excellent control. In the same paper they referred to the experience of Nick Sciabica who delayed the first application until July 11th and the following reapplications of Spinosad in 7-14 days intervals with poor effect. Some Californian table olive growers reported that control by weekly application of GF-120 was not satisfactory and their olives were rejected by processors (Johnson et al., 2005a). In another paper the same authors (Johnson et al., 2005b) noted high efficacy of spinosad. In their field trials mean mortalities of flies 4 to 21 day after treatment with mixture of 1.5:1 of water to GF-120 ranged from 90.6 to 99.2% in August and 83.4 to 97.5% in September. Their studies indicate that the mixture killed a greater percentage of the test population for 21 day than 4:1 mixture. Few more authors reported excellent effects of spinosad on olive fly. Mavrotas et al., (2003) in the trials carried out in Greece, Cyprus, Tunisia and Turkey noted high efficacy of GF 120 bait spraying for the control of olive fruit fly. Varikou et al., (2004) reported that the combinations of the mass trapping systems (mts) with chemicals were equal to combinations of mts with Spinosad bait applications. Studies of Alexandrakis et al., (2005) show that Spinosad applied as complementary sprays in Mass trapping field provided significant control for olive fly in comparison to the classical control. Poullot & Warlop, (2002) noted high efficacy of spinosad in laboratory trial. Olive growing in Montenegro has an increasing impact due to the interest shown by farmers as well as by governmental institutions. That is the reason for searching for more rational tools for cultivation and in particular for phytosanitary measures, in order to reach good quality standards of the product. It is clear that in the sector of plant protection priority must be given to those means that might guarantee a low toxicological and ecological impact. Spinosad is a new product of natural origin, which can be adopted for controlling olive fly. Although first results were not so satisfactory, we need to continue experimental trials by means of more effective techniques for employing this useful product. 150

control (untreated) 100%

80%

infestation 60%

40%

20%

l l l l t t t t u u u u g g g g g p p p p c c c c J J J J u u u e e e e - - - - A A A Au Au S S S S O O O O 5 3 0 7 ------1 2 2 1 9 7 4 1 7 4 1 9 5 2 9 6 1 2 3 1 2 2 1 1 2

Spinosad (Success) 100% = treatment 80% infestation 60%

40%

20%

l l l l g g p p p p t u u u u u e c -Ju -Se -Se -Se 5 1-Aug9-Aug7-A 4-A 7-S 5-O 13-J 20-J 27-J 1 2 31-Aug 14 21 29 12-Oct19-Oct26-Oct

Spinosad (GF 120 Fly Bait) 100% = treatment 80%

infestation 60%

40%

20%

g g p p t ul u u ug e c ct -Jul 3-Jul0-J -A -Sep -O 5 1-A 9-A 7 9-Se 5-Oct2-O 1 2 27-Jul 17 24-Aug31-Aug 14-Sep21-S 2 1 19-Oct26

Deltamethrin (Decis) 100% = treatment 80%

infestation 60%

40%

20%

l l l g p p t t t ul u ug ug e e ep c c -J -Ju -A -A -Sep -S -Oc 5 0-Ju 1-Aug9-Aug 7 1-S 5-Oct2-O 6 13-J 2 27 17 24 31-Au 14-S 2 29 1 19-O 2

Figure 1. Trend of infestation level in the different plots of the experimental field. 151

References

Alexandrakis, V., Varikou, K., Kalaitzaki, A. 2005: Study of trapping systems for control of Bactrocera oleae (Gmelin) (Diptera Tephritidae) in Crete olive groves. – Paper presented at Researching Sustainable Systems – International Scientific Conference on Organic Agriculture, September 21-23, 2005, Adelaide, Australia. Organic eprints. http://orgprints.org/4226/. Johnson, W.M., Daane, M.K., Nadel, H. 2005: Current Status of Olive Fly Management in California Table Olives. – 79th Annual Western Orchard Pest & Disease Management Conference, 5 - 7 January 2005, Portland, OR. Abstracts. Johnson, W.M., Nadel, H., Daane, M., K. 2005a: Impacts of weathered GF-120 residues on olive fly mortality in San Joaquin Valley, CA. – Ten-Minute Papers, Section F. Crop Protection Entomology, Annual Meeting, http://esa.confex.com/esa/2005/techprogram/paper_21898.htm. Mavrotas, C., Alexandrakis, V., Prophetou, D., Kovaios, D., Varikou, K., Vergoulas, P. 2003: GF-120* Naturalyte insect control product field performance for control of olive fruit fly (Bactrocera oleae Gmel.) on olive trees by bait application in the Mediterranean countries. – 1st European meeting of the IOBC/WPRS study group “Integrated Control in Olives”, 29-31 may 2003, Chania, Crete. Abstracts: 15. Mijušković, M. 1955: Ogledi suzbijanja maslinine mušice parationskim sredstvima na Crnogorskom Primorju. – Zaštita bilja. 31: 45-49. Olive Oil Source 2002: The Olive Fly- Bactrocera (Dacus) oleae; current news. http://www.oliveoilsource.com/olive_fly.htm. 1-7. Poullot, D. & Warlop, F. 2002: Strategies de lutte contre les adultes de la mouche de l’olive; Essais d’insecticides biologiques en laboratoire. – Phytoma 555: 38-40. Varikou, K., Alexandrakis, V., Mavrotas, C., Kouletakis, A. 2004: New records on the control of fruit fly Bactrocera oleae (Gmel.). – 5th International Symposium on Olive Growing, 27 September - 2 October 2004, Izmir, Turkiye. Abstract. 59. Vergoulas, P.V., Prophetou-Athanassiadou, D., Alimi, E., Salah, B., Mavrotas, C. 2004: Effect of spinosad bait applications on non target organisms in olive trees and caper plants in Greece and Tunisia, 2002. – 5th International Symposium on Olive Growing, 27 September - 2 October 2004, Izmir, Turkiye. Abstract. 60. Vergoulas, P.V., Prophetou-Athanassiadou, D., Koveos, D., Vasiliou, G., Mavrotas, C., Miles, M., Athanassiadou, E. 2004a: Effect of spinosad, alpha-cypermethrin and fenthion bait applications on non target organisms in olive trees in Greece, 2003. – 5th International Symposium on Olive Growing, 27 September-2 October 2004, Izmir, Turkiye. Abstract. 88.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 153

Kaolin protects olive fruits from Bactrocera oleae Gmelin infestations unaffecting olive oil quality

E. Perri, N. Iannotta, I. Muzzalupo, B. Rizzuti, A. Russo1, M.A. Caravita, M. Pellegrino, A. Parise, P. Tucci C.R.A. Istituto Sperimentale per l’Olivicoltura, Rende (CS), Italy. 1 Dipartimento di Chimica, Universitΰ della Calabria, Arcavacata di Rende (CS), Italy.

The efficacy of the processed kaolin “Surround WP” to control olive fruit fly, Bactrocera oleae Gmelin, field infestations was investigated in east Calabria. The preliminary results showed that fruit infestation levels were significantly reduced on kaolin-treated trees compared with untreated trees. The promising results of these experiments points to the feasibility of using particle film technology composed of a non-toxic material, to avoid olive fly damage as an alternative to the applications of rotenone in organic orchards. Finally, kaolin treatment unaffected the nutritional and sensory quality parameters of the corresponding virgin olive oils obtained by a laboratory scale olive mill, thus satisfying the present quality requirements.

153

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 155-158

Spinosad treatment for Bactrocera oleae (Gmel.) control and olive oil quality in the Montenegrin cv Žutica

Maurizio Servili1, Sonia Esposto1, Stefania Urbani1, Biljana Lazovic2, Mirjana Adakalic2, Tatjana Perovic2, Snježana Hrncic3, Claudio Pucci4, Antonio Franco Spanedda4, AlessandraTerrosi4, Enzo Perri5 1 Universita degli Studi di Perugia, Dipartimento di Scienze Economiche e degli Alimenti – Sezione di tecnologie e biotecnologie. Borgo XX Giugno, 00125 Perugia, Italy 2 Biotechnical Institute Centre of Subtropical Cultures, Bjelisi bb, 85000 Bar, Montenegro 3 Biotechnical Institute Center of Plant Protection, Kralja Nikole bb 81000 Podgorica, Montenegro 4 Università degli Studi della Tuscia – Dipartimento di Protezione delle Piante, Via S. Camillo de Lellis, 01100 Viterbo – Italy 5 C.R.A. Instituto Sperimentale per l’ olivicoltura - Cosenza – Italy

Abstract: Nowadays the experimentation of natural origin products assumes the first role in the researches aimed to focus on B. oleae’s new check strategies, more selective in their action and with less risk of secondary harmful effects. This issue tested the effect of treatments made with Spinosad and Deltamethrin added to proteinic fly bait in a high infestation year (2004). The olive grove, made of cv Žutica trees, was divided in 5 sections, so organized: 1. one tested with GF 120 Flybait; 2. one with Success + Buminal; 3. one with Deltamethrin + Buminal; 4. one with Dimethoate; 5. witness. The treatments were made to exceeding of Z>0.1, with Z calculated by the expression Z = 0.039 (Fm – 9.7) – 0.186 (Tm – 22.1) where Fm is the average number of females/week captured by means of a yellow chromo tropic trap and Tm represents the mean temperature of the capture week. Excluding the one treated with Dimethoate, which was submitted to a single intervention in the middle of September, for the other four 5 interventions each have been made. In each of the 5 plots, 3 olive – trees have been chosen at random. On the 26/10/2004, from the canopy were randomly withdrawn samples of 1.5 kg of olives, which were submitted to oil extraction made by pressure after 24-48 hours from the harvest. The obtained oil samples have been analyzed pointing out following parameters: free acidity, peroxides number, phenols and ortho – diphenols. From the Analysiss of the obtained results it appears that the oil coming from the olives treated with GF 120 Flybait is an extra-virgin with qualitative levels like the one made of Deltametrina and Dimethoate treated olives. This has to be related to the experimentation year, a loss of production occurred, due to more than 30% dropped olives, except the Dimethoate treated part, where the dropped olived results neared 5% even if the oil qualitative properties in all treated thesis, seem to allow the classification as extra-virgin oil.

Key words: integrated control, Žutica, fatty acid composition, phenols

Introduction

One of the main problems in olive growing is protection of olive fruit against olive fly, B. oleae. In olive growing in Montenegro olive fly in some years affects production because of the fruit drop. The most important Montenegrin olive variety is Zutica which is very sensitive to the attack of olive fly. Olive fly infestation has a negative effect on the oil quality and production. Olive oil quality is of great concern for its biological and nutritional value. The infestation produces an increase in the acidity and peroxide value and decrease in the total poliphenols, while the influence on the acidic composition of the oil appears to be less

155 156

evident (Perri et al., 1996; Montemurro et al., 2002). The consumer requires detailed quality characteristics, safety and absence of pesticide. Many studies were conducted for estimation of the level of the pesticides at harvest in olives and in the oil (Leandri et al., 1993; Molinari et al., 1994), but there is much work to be done on the influence of the treating substances on the olive oil quality parameters and composition of fatty acids. Those parameters have wide variability that can be determined by the environmental conditions, cultural techniques, genetical characteristics and extraction modalities (Montedoro and Servili, 1993). At present experimentation with natural origin products assumes check strategies, more selective in their action and with less risk of secondary harmful effects. This issue tested the effect, in a high infestation year (2004), of treatments made with Spinosad (new natural product of microbic origin not toxic to mammals) compared to the most used insecticides for controlling olive fly, that is to say Deltamethrin added to proteinaceous fly bait and Dimethoate.

Material and methods

The experimental field is situated in the city of Bar on 8 m above sea level and 500 m distant from the sea. The olive grove, made of cv Zutica trees, has been divided in 5 sections, so organized: Spinosad (GF 120 Flybait); Spinosad (Success) + proteinacoeus bait (Buminal); Deltamethrin (Decis) + proteinacoeus bait (Buminal); Dimethoate (Rogor); Untreated control. The treatments have been made to exceeding of Z>0.1, with Z calculated by the expression Z = 0.039 (Fm – 9.7) –0.186 (Tm – 22.1) where Fm is the average number of females/week captured by means of a yellow chromotropic trap and Tm represents the mean temperature of the capture week. Excluding the plot treated with Dimethoate, which was submitted to a single intervention in the middle of September, for the other four have been made 5 interventions each. In each of the 5 plots, 3 olive-trees have been chosen at random. On the 26/10/2004, from the canopy were randomly collected samples of 1,5 kg of olives, which have been submitted to oil extraction made by pressure after 24 – 48 hours from the harvest. The obtained oil samples have been analyzed pointing out the following parameters: free acidity, peroxides number, phenols and ortho-diphenols, as well as acid composition.

Results and discussion

To obtain quality olive oil it is of great importance to achieve reliable information on the olive agronomic management from which olive oil samples is obtained. In general infestation provokes important biochemical changes in the fruit, that can make questionable production of good quality olive oil. Hydrolitic and oxidative processes, acidity and number of peroxides, show progressive increment depending on the level of infestation (Pucci and Terrosi, 2002). Obtained results confirm the negative influence of infestation of B. oleae (Gmel.) on the oil quality. All the important components of chemical characteristisc of the oil (acidity, peroxide value, total phenols) were in relation to the level of infestation in control, and to the effectiveness of the treatments. 157

Table 1. Main olive oil chemical parameters in relation to the different treatments for olive fly control.

Treatments Peroxide Total Orto Free acidity value phenoles diphenoles Dimethoate 0.28 ± 0.02 4.9 ± 0.2 272.7 ± 1.4 101.1 ± 2.5 Spinosad (GF120 Flybait) 0.31 ± 0.03 5.8 ± 0.4 291.8 ± 1.5 135.9 ± 3.3 Spinosad + proteinaceous 0.33 ± 0.02 5.7 ± 0.6 124.0 ± 1.6 51.5 ± 3.1 bait (Success + Buminal) Deltamethrin + proteinacoeus 0.32 ± 0.02 6.1 ± 0.2 109.0 ± 1.6 45.5 ± 2.6 bait (Decis + Buminal) Control (untreated) 1.20 ± 0.10 9.5 ± 0.3 80.3 ± 2.0 38.4 ± 3.1

Table 2. Olive oil fatty acids composition in relation to the different treatments for olive fly control.

Spinosad Spinosad (GF Deltametrin + Fatty acids Dimethoate (SUCCESS)+ 120 proteinacoeus Control composition proteinacoeus (Rogor) FLYBAIT) bait bait Myristic 0.005 + 0.001 0.005± 0.001 0.012 ±0.004 0.008 ± 0.04 0.005±0.000 Acid Palmitic 11.800 + 0.296 11.42 ±0.341 13.180 ±1.339 11.600 ±0.550 11.241±0.173 Acid Palmitoleic 0.200 + 0.016 0.917± 0.071 1.199 ±0.183 1.028 ±0.024 1.073 ±0.045 Acid Margaric 0.030 + 0.001 0.023 ±0.002 0.016 ±0.006 0.023 ±0.003 0.028 ±0.002 Acid Eptadecenoic 0.050 +0.008 0.0460 ±0.04 0.037 ±0.009 0.050 ±0.009 0.059 ±0.001 Acid Stearic 1.320+ 0.840 1.684 ±0.940 3.186 ±0.856 2.965 ±0.326 1.448 ±0.325 Acid Oleic 77.880 + 0.196 77.743±0.278 69.004 ±1.427 75.057 ±0.815 77.579±0.566 Acid Linoleic 7.660 + 0.325 6.543 ±0.422 11.952 ±1.663 7.623 ±0.100 7.081 ±0.651 Acid Linolenic 0.371 +0.020 0.486 ±0.010 0.579 ±0.058 0.470 ±0.026 0.482 ±0.016 Acid Arachic 0.325 + 0.021 0.411 ±0.047 0.348 ±0.171 0.336 ±0.027 0.407 ±0.020 Acid Eicosenoic 0.160 + 0.012 0.237 ±0.021 0.206 ±0.068 0.193 ±0.055 0.238 ±0.010 Acid Behenic 0.020 + 0.012 0.044 ±0.017 0.024 ±0.004 0.104 ±0.027 0.018 ±0.003 Acid Lignoceric 0.150 + 0.010 0.090 ±0.033 0.060 ±0.017 0.081 ±0.021 0.099 ±0.006 Acid

From the analysis of the obtained results it appears that the oil coming from the olives treated with Spinosad (both GF120 Flybait and Success) is an extra-virgin with qualitative levels like the one made of Deltamethrin and Dimethoate treated olives. This has to be related to the percentage of olives with the exit holes of the B. oleae. 158

The benefical effects of olive oil beside high unsaturate/saturated acid ratio is also due to vitamins, carotenoides and phenolic compounds (Gimeno et al., 2002). From our results the phenoles and orto-diphenoles values differ among treatments, with the highest content in Spinosad (GF120 Flybait) and Dimethoate. Fatty acid composition was not much affected by the various treatments (Table 2.). In the obtained results that refer to acidic composition of the oil the only one differing from the others comes from Deltamethrin treated olives. In particular, the ratio between oleic and linoleic acid was altered. Content of oleic acid was for about 8 to 11 % lower in the fruits treated with Deltametrin comparing to the other treatments, while the content of linoleic acid was about 36 to 45% higher respectively. This effect might have occurred due to the low production of sampled trees, which allowed a more rapid ripening favourable to linoleic than oleic acid. Moreover, we should not forget that in the experimentation year, a loss of production occurred, due to more than 30% dropped olives, except the Dimethoate treated part, where the dropped olives result near 5%, even if the oil qualitative properties in all the treated thesis, seem to allow the classification as extra-virgin oil. The experimental work aimed to contribute to the employment of effective tools in order to not only preserve plants from pest infestation but also guarantee high quality of product with no risk for human health. From this point of view, we can state that results emerging from trials with Spinosad based products are quite satisfactory.

References

Gimeno, E., Castellote, A.L., Lamuela-Raventos, R.M., De la Torre, M.C., Lopez-Sabater, M.C. 2002: The effects of harvest and extraction methods on the antioxidant content (phenolics, α-tocopherol, and β-carotene) in virgin olive oil. – Food Chemistry 78(2): 207-211. Leandri, A., Pompi, V., Parlati, M.V., Iannotta, N. 1993: Residui di fitofarmaci presenti sulle olive e nell'olio dopo trattamenti larvacidi al Dacus oleae. – Atti del Convegno su Tecniche, norme e qualita in olivicoltura. Potenza 15-17 Dicembre: 815-822. Molinari, G.P., Cavanna, S., Fontana, G. 1994: Trattamenti all'olivo con fenthion e rischio di residui in olive ed olio. – Atti Giornate Fitopatologiche. 1:39. Montemurro, N., Benedetto, P., Lacertosa, G., Castoro, V., Martelli, S. 2002: Quality olive oils production in Basilicata Region: Chemical Characteristics Investigations. – Acta Hort., ISHA. 586: 533-536. Montedoro, G. F. & Servili, M. 1993: Innovazioni technologiche nella estrazione dell' olio di oliva. – Atti del Convegno su Techniche, norme e qualità in olivicoltura. Potenza 15-17 Dicembre: 149-161. Perri, E., Iannotta, N., Parlati, M.V., Zaffina, F. 1996: Influenza dell' infestazione dacica sulla composizione dei composti fenolici di oli di cultivar calabresi. – Atti del Convegno: L'olivicoltura mediterranea: Stato e prospettive della coltura e della ricerca, Cosenza, Italy 1996: 555-560. Pucci, C., Terrosi, A. 2002: Il controllo della mosca dell' olivo (Bactrocera oleae (Gmel.) Diptera Tephritidae): stato dell’ arte e prospettive. – Medjunarodna manifestacija maslini i maslinovom ulju: tekuće zeleno zlato Istre, Tar, Hrvatska: 25-37.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 159

Effect of the olive fruit fly and the olive antrachnose on oil quality of some Portuguese cultivars

A. Sousa1, J.A. Pereira1, S. Casal2, B. Oliveira2, A. Bento1 1 CIMO/Escola Superior Agraria de Bragança, P.O. box 1172, 5301-855 Bragança, Portugal. 2 Serviço de Bromatologia, Faculdade de Farmácia do Porto, Rua Anνbal Cunha, 164, 4050-047 Porto, Portugal.

The olive fruit fly, Bactrocera oleae (Gmelin), and the olive anthracnose, Colletotrichum sp., cause damage on fruits with repercussion on olive oil quality. The aim of this work was to examine the effect of olive fruit fly and olive anthracnose on oil quality of five Portuguese olive cultivars (Galega vulgar, Cordovil de Castelo Branco, Cobranosa, Madural and Verdeal Transmontana). In Galega vulgar and Cordovil de Castelo Branco three groups of olives were constituted, one with olives infested by olive fly (FO), another with olives attacked by anthracnose (AO) and other with health olives (HO). In the other cultivars HO and OF are compared. Fat content (in dry matter), acidity, specific extinction coefficients (232 and 270 nm) and fatty acid composition were determined. Our results showed that HO had the highest fat content. AO oil presented the worst quality, presenting acidity values twice as much as HO. FO oils showed an increase in acidity 50% higher than HO. No differences were observed concerning fatty acid composition of HO and FO oils. However, the oil produced with AO showed the lowest percentage of monounsaturated fatty acids and the greatest value of saturated fatty acids. Oleic acid was higher on oil produced with HO. Work partially financed by demonstrative project AGRO 482 “Protecção contra pragas do olival numa σptica de defesa do ambiente e do consumidor”.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 161

Biological control of olive fruit fly in California by Psyttalia cf. concolor (Szepligeti) from Moscamed, Guatemala

V.Y. Yokoyama1, G.T. Miller1, P. Rendon², J. Sivinski3 1 U.S. Department of Agriculture, Agric. Research Service, San Joaquin Valley, Agric. Res. Center, 9611 S. Riverbend Ave., Parlier, CA 93648, USA. ² USDA, Animal and Plant Health Inspection Serv., Plant Protection and Quarantine, Programa MOSCAMED, 4A Avenida 12-62, Zona 10,Guatemala City, Guatemala. 3 USDA, ARS, Center for Medical, Agric., and Veterinary Entomol., P.O. Box 14565, Gainesville, FL 32604, USA.

The larval parasitoid, Psyttalia cf. concolor (Szepligeti), was imported into California, USA, from Moscamed, Guatemala, and shown to have potential for biological control of olive fruit fly, Bactrocera oleae (Gmelin). Calculated percentage parasitism of olive fruit fly 3rd instars in field cage tests ranged from 4% in a dry and warm inland valley area, to 29% in a humid and cool coastal area. Small field releases of the parasitoid resulted in 5% parasitism based on the number of parasitoid adults reared from olive fruit fly infested olives collected 1 wk after releases in a coastal area. In laboratory tests at constant temperature, parasitoid adult survival decreased with an increase in temperature and correlated decrease in humidity when provided with water (48 d at 15°C and 12 d at 35°C) or with no water (4 d at 15°C and 0 d at 35°C). In greenhouse tests, at fluctuating diurnal and nocturnal temperatures, parasitoid adult survival with food and water was 21 d at ≈26°C and 4 d at ≈36°C, and was 4 d at ≈26°C and ≤1 d ≈36°C without food and water.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 163-167

Psyttalia concolor (Szépligeti) mass-rearing: new acquisitions

Angelo Canale, Augusto Loni, Alfio Raspi Department of Tree Science, Entomology and Plant Pathology “G. Scaramuzzi”, University of Pisa, via S. Michele degli Scalzi, 2 – 56124 Pisa, Italy.

Abstract: Techniques of Psyttalia concolor mass-rearing utilised in Entomology laboratories at University of Pisa are described. A synthesis of a pluriannual study on parameters that play an essential role in optimising the rearing technique, as well as ensuring more correct utilization of the parasitoid in field conditions, is also reported.

Key words: parasitoid, Olive Fruit Fly, biological control.

Introduction

Psyttalia concolor (Szépligeti) (Hymenoptera Braconidae) is a koinobiont, endophagous solitary parasitoid of larvae of Tephritidae (Diptera). The first reported finding of the species dates back to 1910, when Marchal obtained the Braconidae from olives infested by Bactrocera oleae (Rossi) in Tunisia (Marchal, 1910). The first rearing of Ceratitis capitata (Wiedemann) on an artificial substrate and the subsequent acquisitions concerning the Braconidae’s ability to successfully parasitize the larvae of this phytophage in laboratory conditions, made it possible to set up the rearing of P. concolor, using the Mediterranean Fruit Fly as host. The parasitoid was subsequently utilized in various Mediterranean areas for biological control of B. oleae, with the inundative and/or propagative method (for a synthesis see Neuenschawander et al., (1986) and Raspi, (1995)). In Italy, P. concolor can be found spontaneously, in late autumn, on B. oleae in Sicily, southern Sardinia and in various localities of coastal Tuscany (Raspi, 1995; Raspi et al., 1996; Loni et al., 2005). Since 1990 a rearing of P. concolor on C. capitata has been stably present at the Agricultural Entomology Section of the “G. Scaramuzzi” Tree Science, Entomology and Plant Pathology Department - University of Pisa (Raspi & Loni, 1994). It has therefore been possible both to undertake studies on the relations between the parasitoid and the hosts and also to utilize the parasitoid in experiments aiming at control of the Olive Fruit Fly (Canale, 1998, 1999, 2001, 2003; Loni, 1997; Loni & Canale, 2005-2006; Raspi & Canale, 2000; Raspi & Loni, 1994). Useful acquisitions have been thereby obtained, which should lead to more detailed knowledge of the parameters that play an essential role in optimising the rearing technique as well as ensuring more correct utilization of the parasitoid in field conditions. In this paper, a synthesis of our acquisitions, based on bibliographical data, is reported.

Material and methods

Host and parasitoid rearing technique The host production unit is composed of a variable number of cylindrical 30 cm diameter and 30 cm long PVC cages, each containing about 2000 C. capitata adults (sex-ratio 1:1). One end of each cylinder is closed with a 0.2 mm nylon mesh wall, and the open end of a black

163 164

polythene bag is fixed with adhesive tape over the other end of each cylinder. Each bag is slit open at its base, and closed with a tie. Adults are fed on a dry diet composed of 10 parts of icing sugar and 1 part of yeast extract. Water is provided in a screw-top plastic jar, modified so that when the jar is upturned, a film of water is always present on the edge of the screw cap. Cages are placed above one another to form a pyramid, with each of the nylon walls jutting out over a water-filled plastic gutter designed to collect eggs that fall into it when females lay eggs by inserting the ovipositor into the mesh. Oviposition is favoured by the fact that the nylon wall, which is the only wall allowing light to enter the cage, is illuminated 12 hours a day by a neon light source. Eggs are collected every 2 days and distributed into 10 plastic rectangular bowls (50 x 15 x 2 cm) each containing 0.5 kg artificial culture medium. The medium is composed of: Alfalfa meal g 1000, yeast g 300, sugar g 390, citric acid (crystalline) g 32, sodium benzoate g 22 and water 0.5-0.6 l. The bowls are placed on wooden racks inside cabinets with wooden frames and nylon sides. Mature larvae “jump” out of the medium and fall into the removable net forming the bottom of the cabinet, then they are divided into equal amounts for parasitization by P. concolor and to renew the Ceratitis breeding supply. Cages for P. concolor breeding are made of 40 cm diameter and 50 cm long transparent Plexiglas tubes, into which 1500 adults are introduced (male/female ratio 0.3-0.5). Closures of the cylindrical cages are the same as for Ceratitis cages. Adults are fed on a semi- solid diet of a mixture of honey, sugar and pollen grains. Water is supplied as for Ceratitis. Two 6 cm diameter holes are bored in the upper part of every cage, and plugged with corks each having a foam rubber cylinder glued to its tip that protrudes into the cage. Nylon mesh bags, each containing up to 600-800 Ceratitis fully-grown larvae destined to be parasitized, are fixed to these cylinders by means of simple elastic bands. Each sleeve thus obtained is exposed to parasitization for 15-45 minutes as a function of the population density present in the cage, which varies over time.

Relations between the parasitoid and the host For a detailed description of the methodology adopted in each of the below mentioned studies, we remand to the cited publication.

Results and discussion

Parasitoid/host ratio, exposure time, host instar, host species In laboratory conditions, on C. capitata as host, superparasitism (laying in an already parasitized host) of both second instar and fully-grown larvae is very common. The superparasitism degree of the host is determined above all by the parasitoid/host ratio and by exposure times of the parasitoid to the host: the proportion of superparasitized hosts increases at lower host availability (Canale, 1998) (Table 1) and/or longer exposure time (Canale, 2001) (Table 2). Superparasitism does not necessarily have adverse consequences on the parasitoid progeny. On fully-grown larvae of C. capitata as hosts a number of 2 parasitoid eggs/host appears to represent the optimal conditions to obtain the maximum quantity of offspring (30- 40%) (Table 1), while the laying of a single egg on second instar larvae is sufficient to secure an offspring (Raspi & Canale, 2000) (Table 3). From a mass-rearing standpoint, to provide a greater number of C. capitata fully-grown larvae (or to shorten the exposure time) in order to reduce the superparasitism degree to a minimum is wasteful. In laboratory conditions, P. concolor can successfully parasitize second and third instar larvae of both C. capitata and B. oleae (Canale, 1999; Raspi & Canale, 2000). In general, this

Table 1. The effect of different parasitoid/host ratios of P. concolor (24 hours of host exposure time) on the parasitization (≥1 parasitoid egg) and superparasitization (≥ 2 parasitoid eggs) of 165

fully-grown C. capitata larvae. (One-way ANOVA; Duncan test - P < 0.05; n = 150). (Modified from CANALE, 1998).

Ratio Parasitized hosts Superparasitized hosts Mean of eggs/host P. concolor p/h (%) (%) (n ± sd) progeny (%) 1/1 99.06 a 96.2 a 5.91 (±0.94) a 2.6 a 1/5 89.8 ab 87.5 a 3.28 (±1.38) b 14.8 b 1/10 81.2 b 52.5 b 2.04 (±0.88) bc 37.3 c 1/20 44 c 6 c 1.1 (±0.34) c 4.01 a

Table 2. The effect of two exposure times (1/1 p/h ratio) of P. concolor on the eggs distribution pattern among C. capitata fully-grown larvae. (One-way ANOVA; Duncan test - P < 0.05; n = 150). (Modified from CANALE, 2001).

Exposure Parasitized hosts Superparasitized hosts Mean of eggs/host P. concolor time (%) (%) (n ± sd) progeny (%) 30 min 77.3 a 51.3 a 1.86 (±0.13) a 20 a 60 min 94.7 b 92 b 3.72 (±0.63) b 10 b

Table 3. The effect of different exposure times (1/10 p/h ratio) of P. concolor on the eggs distribution pattern among second instar C. capitata larvae. (One-way ANOVA; LSD test - P < 0.05; n = 150). (Modified from RASPI & CANALE, 2000). Exposure Parasitized hosts Superparasitized hosts Mean of eggs/host P. concolor time (%) (%) (n) progeny (%) 1-hour 36.7 a 4 a 1.1 a 11 a 6-hours 40.6 a 16 b 1.49 b 12 a 12-hours 52.7 b 31.3 c 1.61 b 7.5 b

suggests the convenience to release the parasitoid in the field when the host population consists mostly of young larvae. Moreover, on fully-grown larvae of B. oleae and C. capitata the mean duration of parasitoid development at 18 °C is similar, with a mean value of 40.6 days utilizing B. oleae and 40.9 days using C. capitata (Loni & Canale, 2005-2006), suggesting that that both species represent an equally suitable resource for development of the Braconidae. Factors eliciting the ovipositor-probing behaviour In laboratory conditions, a combination of both mobile host larvae and host substrate induced the highest level of probing responses in P. concolor, suggesting that more than one cue is necessary to elicit the optimal response (Canale, 2003) (Figure 1). Moreover, the finding that mobile larvae induced a higher level of ovipositor-probing behaviour than immobilized larvae suggests that host movement likely played a major role in eliciting ovipositor-probing behaviour in P. concolor. Developmental rate at different temperatures The P. concolor developmental temperature range lies between 15 and 30°C, with a mean development time lasting from 77.25 to 14.62 days (Table 4). The optimal temperature lies between 23 and 25°C, where development time ranges from 21.52 to 17.87. The P. concolor low temperature threshold (cumulated for both sexes) is 11.7°C or 11.85°C with line 166

regression and thermal summation, respectively. Therefore, this parasitoid appears to be well adaptable to mild climates.

a 100 90 80 70 60 b 50 40 30 c 20 c Wasps probing (%) 10 c 0 Substrate + Mobile host Substrate only Immobile host Control mobile host Treatments

Figure 1. Ovipositor-probing responses of P. concolor to artificial dishes containing both C. capitata larvae and substrate, C. capitata larvae alone, substrate alone, immobilized C. capitata larvae alone or neither (control). Thirty wasps were tested for each treatment. (Pairwise χ2 test, P < 0.01). (Modified from CANALE, 2003). Table 4. Development time of P. concolor at various constant temperatures. (Modified from LONI, 1997).

Temperature P. concolor Mean total developmental Emergence (°C) (n) time (dd ± sd) (%) 13 0 0 0 15 10 77.25 (±4.5) 0.4 18 846 42.68 (±1.96) 33.84 20 796 33.87 (±1.89) 31.84 23 867 21.52 (±1.42) 34.68 25 905 17.87 (±1.36) 36.16 28 233 15.38 (±1.43) 9.32 30 18 14.62 (±0.78) 0.72 33 0 0 0

References

Canale, A. 1998: Effect of parasitoid/host ratio on superparasitism of Ceratitis capitata (Wiedemann) larvae (Diptera, Tephritidae) by Opius concolor Szépligeti (Hymenoptera, Braconidae). – Frustula Entomologica, n. s. XXI (XXXIV): 137-148. Canale, A. 1999: Studi bio-etologici su parassitoidi di Ditteri Tefritidi di interesse economico: il superparassitismo in Opius concolor Szépligeti (Hymenoptera Braconidae). – Ph. D. Thesis, Università di Pisa: pp. 94. Canale, A. 2001: Effect of exposure time on superparasitism of Ceratitis capitata (Wiede- mann) (Diptera Tephritidae) fully-grown larvae by Psyttalia concolor (Szépligeti) (Hymenoptera Braconidae). – Frustula Entomologica, n. s. XXIV (XXXVII): 167-172. 167

Canale, A. 2003: Psyttalia concolor (Szépligeti): role of host movements and host substrate on ovipositor-probing behaviour. – Bulletin of Insectology 56 (2): 211-213. Loni, A. 1997: Developmental rate of Opius concolor (Hym.: Braconidae) at various constant temperatures. – Entomophaga 42 (3): 359-366. Loni, A. & Canale, A. 2005-2006: Duration of development and reproductive success of Psyttalia concolor (Szépligeti) (Hymenoptera Braconidae) on different hosts: preliminary note. – Frustula Entomologica, n. s. XXVIII-XXIX (XLI-XLII), in press. Loni, A., Canovai, R. Canale, A., Raspi, A., 2005: Presenza di Psyttalia concolor (Szépligeti) (Hymenoptera Braconidae) in Toscana. – Atti del XX Congresso Nazionale Italiano di Entomologia, Assisi (PG), 13-18 Giugno 2005: 387. Marchal, P. 1910: Sur un Braconide nouveau, parasite de Dacus oleae. – Bulletin de la Societe Entomologique de France 13: 243-244. Neuenschwander, P., Michelakis, S., Kapatos, E. 1986: Dacus oleae (Gmel.). – In: Entomologie oleicole. Arambourg, Y. (ed.). Conseil Oleicole International, Madrid: 115- 159. Raspi, A. & Loni, A. 1994: Alcune note sull’allevamento di Opius concolor (Szèpl.) (Hymenoptera: Braconidae) e su recenti tentativi d’introduzione della specie in Toscana ed in Liguria. – Frustula entomologica, n. s. XVII (XXX): 135-145. Raspi, A. 1995: Lotta biologica in olivicoltura. – In: Atti Convegno Tecniche, norme e qualità in olivicoltura, Potenza (Italia), 15 -17 Dicembre 1993: 483-495. Raspi, A., Canovai, R., Antonelli, R., 1996: Andamento dell’infestazione di Bactrocera oleae (Gmelin) in oliveti del Parco regionale della Maremma. – Frustula entomologica, n. s. XIX (XXXII): 189-198. Raspi, A. & Canale, A. 2000: Effect of superparasitism on Ceratitis capitata (Wiedemann) (Diptera, Tephritidae) second instar larvae by Psyttalia concolor (Szépligeti) (Hymenoptera, Braconidae). – Redia 83: 123-131.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 169-172

The effects of treatments against Bactrocera oleae (Gmelin) on the entomo-fauna of the olive ecosystem

Nino Iannotta1, Tiziana Belfiore2, Pietro Brandmayr2, Stefano Scalercio1 1 C.R.A. Institute for Olive Growing - 87036 Rende, Cosenza, ITALY 2 Department of Ecology, University of Calabria - 87036 Rende, Cosenza, ITALY

Abstract: Only a limited number of studies have examined the efficacy and environmental effects of treatments against Bactrocera oleae in organic production of olives, especially in terms of the insect community present in these ecosystems. Herein, we compared the effects of a conventional insecticide (dimethoate) with a plant protection system allowed by organic legislation (mass trapping plus rotenone), and an untreated, control field against B. oleae. The experiments were performed in the Calabria region in Italy (on the Ionic side of Cosenza), in a grove (Mirto-Crosia) with extensive active insect infestation. In two conventionally treated fields with a surface area of 2 hectares each, two treatments were performed (in August and September) utilizing dimethoate (150 gr in 100 lt water). In another 2 hectare area, mass-trapping devices (attract and kill) were installed and treatment with rotenone (Rotena 300 gr in 100 lt water) was performed in September. The entomo-fauna present in the different areas was evaluated by insect capture utilizing cromotropic traps. The integrated protection system (mass-trapping plus rotenone treatment) led to a reduction in the total number of insects in comparison to the traditional method (dimethoate treatment). It proves the negative effect of organic system on the olive ecosystem entomo-fauna.

Key words: Bactrocera oleae, organic farming, entomo-fauna, olive agroecosystem, diversity.

Introduction

During recent years, more ecologically friendly olive growing techniques have become relatively widespread and are used in both integrated and organic farming methods. These cultivation methods are guided by regulations that list the active ingredients and describe how they should be applied. The regulations for organic farming techniques are instituted by experimental evaluation of efficacy, although only a few studies have examined their environmental impact, with particular reference to the entomo-fauna in olive groves. It is well-known that, especially in southern regions, olive fruit fly (Bactrocera oleae) is a key phytophagous insect that massively infest olive groves and provoke heavy damage. Thus, appropriate control measures are essential. In the present report, we evaluated the impact of treatment against B. oleae on several groups of arthropod fauna present in olive groves carried out with conventional pesticides (dimethoate) for integrated cultivation compared to mass- trapping and rotenone, which is allowed in organic farming.

Materials and methods

The analyses were carried in an olive field in the Calabria in southern Italy where infestation by olive fruit flies is particularly extensive (Mirto-Crosia in Cosenza) due to climatic conditions that are particularly favorable for its development. Two fields treated by conventional methods, each with a surface area of 2 hectares, differed in their varietal composition (A, several cultivars; B, single cultivar); two treatments using dimethoate (150

169 170

gm in 100 liters water) were made in August and September. In another field (C) with the same surface area, biological traps for mass-trapping were installed (150 per hectare; Agrisense attract and kill model). In this case, rotenone treatment (300 gr Rotena in 100 lt water) was performed in September. Evaluation of entomo-fauna in the different areas was carried out by capture using cromotropic traps. The taxonomic groups examined were among those most frequently encountered in these groves and included Neuroptera, Diptera Syrphidae, Coleoptera Coccinellidae, Lepidoptera, and Mecoptera Panorpidae. The results are presented as the mean of the number of individuals captured in traps per unit of time exposed (activity density; Brandmayr and Brunello Zanitti, 1982). The meteorological conditions of the fields were also monitored using electronic equipment.

Results

The data are summarized in Figure 1. From these results, it is evident that the mass-trapping device is less efficient compared to the other two conventional treatment groups. Nonetheless, no differences were observed regarding the number of macrotaxa present. Table 1 detailed the data relevant to the five macrotaxa found, from which it can be deduced that mass-trapping led to a smaller number of Lepidoptera, Neuroptera, Diptera, and Coleoptera; only Mecoptera showed a greater number of individuals. Almost all the species of Lepidotteri were present at a lower frequency in area C, and the Shannon index, a measure of diversity, was also lower in the same area.

350

300

250

200

150

100

0 Thesis A Thesis B Thesis C Individuals 274 343 215 Macrotaxa 555

Figure 1. Overall biomass observed in the three treatment areas.

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Table 1. Abundance of selected macrotaxa within surveyed sites reported as density of activity (DA), DA = (individuals / n° of pitfall traps) x (10 / days of traps exposure).

MACROTAXA Thesis A Thesis B Thesis C Lepidoptera 32.61 35.23 21.18 Neuroptera Chrysopidae 14.13 13.62 6.78 Diptera Syrphidae 23.83 37.78 30.74 Coleoptera Coccinellidae 3.12 3.87 0.95 Mecoptera Panorpidae 3.43 9.65 282.01

Discussion

Our results demonstrate that mass-trapping integrated with rotenone leads to a more efficient reduction in the number of insects present compared to treatment with dimethoate. In particular, from the taxonomic groups the number of Lepidoptera captured was lower in the mass-trapping field (Table 2). A similar trend was observed regarding the number of Coccinellidae, Syrphidae, and Neuroptera. Only the number of Mecoptera Panorpidae was higher in field C. In conclusion, mass-trapping combined with rotenone for control of olive fruit flies in organic farming provokes negative effects on the entomo-fauna in olive groves. The negative effects of mass-trapping were even greater with respect to conventional pesticide treatments.

Table 2. Species of Lepidoptera present at the three treatment areas.

Thesis Thesis Thesis Spodoptera exigua 1 1 0 SPECIES A B C Ochlodes venatus 0 1 0 Lasiommata megera 21 29 27 Argynnis pandora 0 1 0 Pieris brassicae 21 16 25 Aricia agestis 0 1 0 Lampides boeticus 5 12 7 Carcharodus alceae 0 1 0 Colias crocea 5 15 3 Paradrina clavipalpis 0 1 0 Pieris rapae 1 9 2 Leptotes pirithous 0 1 0 Palpita unionalis 7 6 2 Spialia sertorius 0 1 0 Pararge aegeria 1 0 2 Menophra japygiaria 6 0 0 Agrochola lychnidis 0 0 2 Autographa gamma 1 0 0 Pyrgus malvoides 0 0 2 Idaea obsoletaria 1 0 0 Polyommatus icarus 2 6 0 Lycaena phlaeas 1 0 0 Scopula minorata 14 5 0 Macdunnoughia Coenonympha confusa 1 0 0 pamphilus 1 5 0 Nomophila noctuella 1 0 0 Nodaria nodosalis 14 4 0 Rhodometra sacraria 1 0 0 Prays oleae 3 2 0 Individuals 108 121 70 Sloperia proto 0 2 0 Species richness 20 21 9 Gegenes pumilio 0 2 0 Shannon index 2.39 2.48 1.55

References

Bagnoli, B. 2000: Indagini sull’impatto di dispositivi per la cattura massale di adulti di Bactrocera oleae sull’entomofauna utile dell’oliveto. – Prog. Reg. A.R.S.I.A. Firenze. 172

Brandmayr, P. & Brunello Zanitti, C. 1982: Le comunità a Coleotteri Carabidi di alcuni Querco-Carpineti della bassa pianura del Friuli. Quaderni sulla “Struttura delle Zoocenosi Terrestri”. 4. – I boschi della Pianura Padano-Veneta: 69-124. Castro Rodas, N. 2004: Individuazione di bioindicatori entomologici nell’agroecosistema oliveto. – Tesi di Perfezionamento. Scuola Superiore S. Anna, Classe di Scienze sperimentali settore di Agraria. Castro Rodas, N. & Petacchi, R. 2002: Risultati preliminari sull’individuazione di bioindicatori entomologici nell’agroecosistema oliveto. – Atti XIX Congresso Italiano di Entomologia, Catania, 10-15 giugno 2002: 267-272. Iannotta, N. 2003: La difesa fitosanitaria ed i parassiti. – In: AA.VV., OLEA, Trattato di olivicoltura: 391-407. Petacchi, R., & Minocci, A. 1993: Analisi sulla composizione dell’entomofauna dell’oliveto e sull’impatto provocato da diverse strategie di lotta antidacica. – Atti Convegno su “Tecniche,norme e qualità in olivicoltura”, Potenza, 15-17 Dicembre 1993: 509-525. Pimentel, D.A., Stachoww, U., Takacs, D.A., Brubaker, H.W., Dumas, A.R., Meaney, J.J., O’Neil, J.A.S., Onsi, D.E. & Corsilius, D.B. 1992: Conserving biological diversity in agricultural and forestry systems. – Bioscience 42: 354-364. Ricketts, T.H., Daily, G.C. & Ehrlich, P.R. 2002: Does butterfly diversity predict moth diversity? Testing a popular indicator taxon at local scales. – Biological Conservation 103: 361-370.

Other Pests of Olive Groves and Olive Agroecosystem Aspects 182

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 175-178

Inventory and role of the third generation parasitoids of Prays oleae Bern. (Lepidoptera, Yponomeutidae) in Sfax region (South of Tunisia)

Imen Blibech, Mohiedine Ksantini and Taieb Jardak Olive Institute, Department of Plant Protection and Environment, B.P. 1087 3000, Sfax, Tunisia

Abstract: The olive moth Prays oleae Bern. (Lepidoptera, Hyponomeutidae) is considered as the most important olive pest in the costal regions of the center and the south of Tunisia. However, abiotic factors such as droughtness and biotic factors such as parasitoids and predators play a great role in the reduction of P. oleae population. The follow-up of the pest adults and its parasitoids emergence is achieved by the installation of trap bands in undulating cardboard (10 X 20 cm) infested branches at the rate of 25 cardboards per tree. Fifty trees were examined for the collection of P. oleae larvae and chrysalids that are isolated individually in hemolyses tubes closed with an absorbent cotton to facilitate the aeration to follow the emergence of P. oleae adults and its parasitoids. The emergence of P. oleae adults is spreading on about thirty days in the laboratory (month of March) and forty days (second week of March until the end of April) on the field. However, the emergence of parasitoids spreading on all the flight period of the insect with temporary cadences of emergencies especially during the maximum of the flight, coinciding with the presence of P. oleae larvae and as consequence a relatively high rate of parasitism in the nature (61.33%). The daily follow-up of emergencies in laboratory has permitted to establish the curves of P. oleae and its parsitoids flight and to compare them to those in the field. The inventory of the third generation parasitical fauna of P. oleae has been composed of 4 known species: Chelonus eleaphilus (67.39%), Angitia armillata (17.45%), Apanteles xanthostigmus (0.25%), Ageniaspis (14.12%) and of 6 new species: 1 species of the genus Apanteles, 2 species of the genus Itoplectis and 2 species of the genus Dibrachys. The predators captured are essentially spiders, ants and larvae of Chrysoperla carnea.

Key words: Prays oleae, parasitoids, parasitism

Introduction

Most agricultural crops are attacked by numerous pests and consequently they are increasingly protected using agrochemicals, which cause environmental pollution and reduction of natural enemies in olive production ecosystem. The olive is with no exception, being host to a diverse range of specific insect pests such the olive moth (Prays oleae), the pyralid moths (Palpita unionalis and Euzophera pinguis), the olive beetle (Phloeotribus scarabaeoides), the olive scale (Saissetia oleae) and the olive fly (Bactrocera oleae). To control the key pest of olive crops, some alternatives were recommended to provide minor risk to the environments and to the biodiversity; in such situations, when the olive infestation is below the economic threshold levels, curative treatments were not necessary, much more the use of organic production system increase the densities and activities of native natural enemies. A rich diversity of olive pest parasitoids is known to occur in several parts of Africa. Although a considerable amount of biological information has been accumulated regarding native parasitoids and predators in the Mediterranean region.

175 176

The knowledge of these natural parasitoids is of a great importance in order to know and classify responsible species in relation with different biotopes and to determine their effect in the control of olive pests for a possible management strategies integration by studying the possibilities of importing effective parasitoids attacking the pest in its native home range and the monitoring of these natural enemies in olive production systems and the evaluation of their effectiveness under field conditions. In the research aspect, we evaluated the importance and the efficacy of parasitoids belonging to third generation of Prays oleae Bern. in Sfax region in the south east of Tunisia which is known by the olive monoculture.

Materials and methods

Biotopes of experiments The experiments were carried out in two olive growing sites of Siris - Zaghden in the North of Sfax at 20km and Chaffar - Kallel in the South of Sfax at 30 km. In these groves, the cultivated variety is the Chemlali with a plantation density of 24 m x 24 m in rain fed conditions and well conducted agricultural practices such the tillage and the pruning. Monitoring of populations of the olive moth and its parasitoids in the field For the studies on the third generation population inventory of P. oleae and its parasitoids, we used 25 trap bands per tree with dimensions of 10 x 20 cm for one trap. Theses traps on undulating cardboard were fixed on 50 trees in both biotopes of Siris in 2002 and Chaffâr in 2003. To start on the following method in field, the appropriate phenological stage of the olive tree is that of buds appearance. Laboratory rearing Parasitoids communities of the olive moth were studied in the two olive growing regions by sampling larvae and pupae from the phylophagous generation and subsequent rearing in the laboratory into plastic tubes closed with cotton until emergence of adult parasitoids. Inventory of the third generation parasitical fauna of Prays oleae The emergence of adults and parasitoids has been controlled every day and the identification of species was achieved. Key natural enemies were identified by comprehensive literature reviews on natural enemy complexes occurring in the olive grove habitat in the involved regions as well as by field collections.

Results and discussion

Identification of key natural enemies in olive grove habitats In the biotope of Siris, 131 parasitoids were identified in 2002; they were composed of two known species: Angitia armillata (Hymenoptera, Ichneumonidae), Chelonus eleaphilus (Hymenoptera, Braconidae) and Apanteles xanthostigmus (Hymenoptera, Braconidae). Whereas, in the biotope of Chaffâr in 2003, 1270 insects were examined under binocular; the same known species such in Siris and six new parasitoids were found for the first time in Tunisia that represent 0.77% of the total parasitoids; 2 species of the genus Apanteles (Hymenoptera, Braconidae), 2 species of the genus Itoplectis (Hymenoptera, Ichneumonidae, subfam. Pimplinae) and 2 species of the genus Dibrachys (Hymenoptera, Chalcidoidea, Pteromalidae). Parasitism rates The parasitism rates observed in larvae and pupae of the phylophagous generation of Prays oleae, in each biotope, are shown in the Tables 1 and 2. 177

Table 1. Parasitism rate of the phylophagous generation of Prays oleae in Siris biotope, (2002)

Sex Prays oleae Parasitoids ratio Total Apanteles Chelonus parasitism Angitia armillata F/ F+M xanthostigmus eleaphilus (%) Male Female % Nb % Nb % Nb %

39 34 46.5 11 4.76 144 62.33 3 1.29 68.39

Table 2. Parasitism rate of the phylophagous generation of Prays oleae in Chaffâr biotope, (2003)

Sex Prays oleae Parasitoids ratio Total Apanteles Angitia Chelonus Ageniaspis New parasitism F/ xanthostigmus armillata eleaphilus sp. parasitoids (%) Male Female F+M Nb % Nb % Nb % Nb % Nb % %

28 20 41.6 2 0.26 13 17.46 525 67.39 110 14.12 6 0.77 61.33

It appears from these results that the parasitism rate of the phylophagous generation of Prays oleae is very important in infested olive grove habitats in Sfax region (exceed 50%). A rich diversity of olive moth parasitoids is occurring in the two biotopes and known in several parts of Tunisia; Angitia armillata, Chelonus eleaphilus and Apanteles xanthostigmus. The most important parasitoid obtained in Siris is Angitia armillata (62.33%) however in Chaffâr the most important parasitoid is Chelonus eleaphilus (67.39%). We conclude from these results that the parasitoids showing the best biological characteristics, especially parasitism rate which obviously influence biological control, were Angitia armilata and Chelonus eleaphilus in Sfax region. Nevertheless, the new parasitoids identified led to the conclusion that the fauna in the olive growing regions in the south of Tunisia are rich and diversified and should be preserved from chemical pollution. Biological control was indispensable in this case and many experiments are necessary in each geographical by involving, for example habitat management strategies on natural enemies, preferably in several olive growing regions such as the creation of vegetation islands by sawing seeds of wild flowering plants (Foeniculum vulgare, Daucus carota, Polygonum aviculare, Borago officinalis, Medicago sp.) and to study the effect of the presence of these plants on species composition and abundance of natural enemies on infestation of the olive trees by target pests.

References

Abou-Elkhair, S.S., Stefanos, S.S., Nasr, F.N., Youssef, A.A., Shehata, W.A. 2002: Survey of biological control agents of the olive leaf moth, Palpita unionalis Hübn. (Lep.: Pyralidae), and olive moth, Prays oleae Bern. (Lep.: Yponomeutidae) in olive orchards in Egypt. – 2nd Inter. Conf., PPRI.,Cairo, Egypt, 21-24 December, 2002. Agamy, E., Bento, A., Hafez, B., Hassan, S.A., Hegazi, E., Herz, A., Jardak, T., Ksantini, M., C. Lozano, T. Morris, M. Campos, J.A. Pereira, A. Bento 2002: Detection by ELISA of 178

predators of Prays oleae (Lepidoptera: Yponomeutidae) in a Portuguese olive orchard. – IV International Symposium on Olive Growing. ISHS Acta Horticulturae 586. Jorge, S., Bento, A., Torres, L. 2003: Preliminary results on the effect of the creation of vegetation islands with flowering plants on beneficial insects associated with the olive agroecosystem. – 1st European Meeting of the IOBC/WPRS Study Group: “Integrated Protection of Olive Crops”, 29. - 31. May 2003, Chania (Crete). Konstantopoulou, M., Mazomenos, B., Nasr, F., Pereira, J. A., Torres, L., Youssef, A. 2003: TRIPHELIO - an international research project for sustainable control of Lepidopterous pests in olive groves. – 1st European Meeting of the IOBC/WPRS Study group “Integrated Protection of Olive Crops”, 29. - 31. May 2003, Chania (Crete). Nasr, F.N., Abou-Elkhair, S.S., Stefanos, S.S., Youssef, A.A., Shehata, W.A. 2002: New record of some biological control agents of Palpita unionalis Hübn. (Lepidoptera: Pyralidae) and Prays oleae Bern. (Lepidoptera: Yponomeutidae) in olive groves in Egypt. – J. Biol. Pest Control 12 (2): 129.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 179

Mating disruption of the olive pyralid moth, Euzophera pinguis

A. Ortiz1, A. Perabá1, A. Quesada1, A. Sánchez2 Department of Inorganic and Organic Chemistry. University of Jaén, Spain. 1 EUP Linares. Alfonso X El Sabio 28. 23700 Linares (Jaén), Spain. 2 Paraje las Lagunillas s/n 23071 Jaén, Spain.

A two years field experiment were conducted to determine efficacy in disrupting sexual communication of the olive pyralid moth (OPM) Euzophera pinguis Haworth (Lepidoptera: pyralidae) in infested olive groves. Shin-Etsu pheromone Rope-type dispensers were placed at the beginning of 2004 and 2005 first flight periods at a rate of 500 dispensers per hectare, about 2-3 ropes per tree, in two 3 ha olive plots. A total of 50 g pheromone of a blend of (Z)- tetradecen-1-ol (Z9-14:OH) and (Z,E)-9,12-tetradecadien-l-ol acetate (ZETA) was applied/ha. Under field conditions, pheromone release-rates from dispensers were measured in laboratory weekly over 7 months. Pheromone was trapped on Tenax TA from an air stream, thermally desorbed and quantified by gas chromatography. The success of mating disruption was evaluated using two parameters: inhibition of males capture in pheromone traps and reduction of infestation in susceptible infestation sites (bark crevices and wounds). In pheromone-treated blocks, captures of E. pinguis were reduced from 95% to complete shut-down of pheromone trap catch in all of the plots for at least 180 days. In addition, a reduction of 40-70% was recorder in the infestation levels on wounds during pheromone treatment.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 181-186

Effect of chemical control on over-wintered population of olive psyllid Euphyllura olivina Costa (Homoptera, Aphalaridae) in Iran (Tarom-Sofla region, Qazvin province)

H. Nouri Iran, Qazvin Agricultural and Natural Resources Research Center.P.O.Box- 34185-618

Abstract: Chemical control is one of the most important strategies in Integrated Pest Management (IPM). Studies on the chemical control with emphasis on effectiveness of emulsifiable oil on dormant olive psylla, Euphyllura olivina Costa, were carried out in Tarom-Sofla region (Qazvin province) during 1996-1997. Emulsifiable oil (1 and 2%), Azinphos methyl 0.002 with oil 1% and without, Etrimfos 0.001/5 with oil 1% and without compared to control treatment were tested in randomized complete block design in 3 replications. Results showed that emulsifiable oil 2% is preferred to control olive psylla especially on preoviposition period, because of economic aspects and its stability and less because of its side effect on environment and natural enemies.

Keywords: Olea europea (L.), Olive psylla (Euphylura olivina Costa), chemical control.

Introduction

Olive psylla is one of the major pests of olive crops in Roodbar and other olive plantation regions in Iran, which causes plant weakness by sucking the plant/ flower nectar. In the other hand it causes yield reduction by honey dew secretion on blossoms that prevents flowers fecundation/fertilization(Chermiti, 1992). It has one generation per year in Tarom-Sofla region (Qazvin province) and causes damage on olive trees particularly in orchards which proper pruning principles and plant nutrition are not considered. Mating may start, in consideration with temperature fluctuation, in February and March. Olive psylla causes increase of smut/soot infestation by its honey dew. Chemical control must be start when pest population is higher than economic injury level in regions with special possibility of pest damage, and for this purpose a contact pesticide with fumigation effect is recommended at the beginning of flowering stage (Taherzadeh, 1994). When inflorescence infestation is about 10%, and the average number of pupa per infested inflorescence is 5, they litter 32% more than a common situation (Nouri, 1996). 1-5 pupa per bunch causes a little damage, 6-8 pupa per bunch causes about 30% littering and when population reach to 30 pupa per bunch, damage will reach to 40% (Saeb & Farzaneh, 1993). Farahbakhsh and Moieni (1975) reported that when lately wax threads are seen on foliage, Thiodan and Malathion can be applied for spraying against olive psylla because they are less harmful, less durable and probably cheaper than Soopracid, Diazinon and Phentoate. Taebi et al. (1991) in Pesticides Types Determination Committee of Plant Protection Organization suggested Diazinon 60% EC and Malathion 57% EC in one turn for olive psylla in the proportion of 1⁄1000 and 2.5⁄1000 in the beginning of oviposition.

181 182

Materials and methods

Spraying operations The experimental olive orchard has been recognized in Tarom- Sofla (Callag valley) during 1996 and 1997, and each treatment was applied on three adjoining trees in Randomized Complete Block Design. One tree was regarded among adjoining treatments as a blank. The emolsifiable oil (volk) with sulfunation degree 92 and Azinphos methyl (Guzathion M) 20% EC that produced by Melli Keshavarz company and Etrimfos (Okamet) 50% EC produced by Ni Kaiako Japanese industry, were used. The capacity of the used spraying machine was 100 lt.

Sampling method Before spraying operations, 27 trees were randomly selected, and hitting-shoot was carried out on an external and an internal branch. For applying this method a cardboard funnel, which its upper opening diameter was 50cm and its lower opening diameter was 6cm, was used. In each sampling tow hit-shoot was carried out on test tree in each treatment. After spraying operations, hitting-shoot were repeated to count the number of living adult population of olive psylla. Considering olive psylla population control symptom is cotton threads unappearance, so coincident with seeing cotton threads, 5 branches with 15-20 cm long were separated from each experimental tree, that totally 15 branches were cut in each treatment and then were labeled and transferred to laboratory, and in the next stage separation and counting of infested and uninfected branches was done.

Statistical analysis For this purpose, variance analysis and average competition of sampling data was carried out by Mstatc software. Effectiveness percentage (infestation reduction) of various tested chemical compounds on overwintered adult population control was calculated by Abbot (1952) formula. Effectiveness percentage (% Mortality) = Number of living insects in control plots - the number of living insects in sprayed plots / number of living insects in control plots.

Results and discussion

Olive psylla population density reduced strongly in 1996 in Tarom-Sofla, due to the sever changes in temperature in summer of 1995. In Table 1, 2, 3 & 4 have been presented the variance analysis and the average competition results of various chemical compounds effectiveness on adult insects by branch sampling method. Variance analysis and average competition by Donken method in 1996 shows that there is an important difference between experimental treatments in probability level 5% and the best result is related to 2% volk application. Azinphos methyl had most branch infestation and with control treatment placed in class a, while other treatments had the same effectiveness rate and located in class b. The results indicate that oil application causes infestation reduction in the rate of 60-65%, due to its longer establishment and strength on olive trees, especially when olive psylla is at the beginning of mating stage. In Tables 5, 6, 7 & 8 evaluation, variance analysis and average competition results of various chemical compounds effectiveness on adult insects have been presented by branch sampling method in year of 1997. In second test year, there isn’t an important difference between experimental treatments. The best result same as 1996 is related to 2% oil. 183

Table 1. Evaluation of various chemical compounds effectiveness on adult olive psylla by shoot counting in Tarom-Sofla region (1996).

The number of uninfested The number of infested shoots Treatments shoots I II III Total I II III Total Volk oil 2% 9 10 8 27 6 5 7 18 Volk oil 1% 7 9 6 22 8 6 9 23 Etrimfos 0.001/5 5 5 7 17 10 10 8 28 Etrimfos+oil 1% 8 8 5 21 7 7 10 24 Azinphos methyl 0.002 3 5 1 9 12 10 14 36 Azinphos methyl +oil 1% 8 9 6 23 7 6 9 22 Check 1 3 2 3 8 12 13 12 37 Total 43 48 36 127 62 57 69 188 Median 6.1 6.9 5.1 18.1 8.9 8.1 9.9 62.9

Table 2. Effectiveness percentage (infestation reduction) of various chemical compounds on adult olive psylla by shoot sampling in Tarom-Sofla region (1996).

Effectiveness percentage Median of Treatments (infestation reduction)1 effectiveness I II III percentage Volk oil 2% 50 76.9 66.7 64.5 Volk oil 1% 58.3 69.2 50 59.2 Etrimfos 0.001/5 41.6 38.4 58.3 46.1 Etrimfos+oil 1% 66.7 46.1 41.7 51.5 Azinphos methyl 0.002 0 38.4 8.3 15.6 Azinphos methyl +oil 1% 66.7 69.2 50 61.9 1- It was done by Abbot formula.

Table 3. Variance analysis of various chemical compounds effectiveness on adult olive psylla (effectiveness percentage) by shoot sampling in Tarom-Sofla region (1996).

S.O.V df SS MS F Replication 2 231.33 115.66 Treatment 5 2583.85 516.77 5.30* Error 10 975.02 97.5 Total 17 3790.2 * Significant in 5% level.

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Table 4. Evaluation of various chemical compounds effectiveness on adult olive psylla by shoot counting in Tarom-Sofla region (1996).

Treatment Class Medians Differences Volk oil 2% 6 46.5 b 13.03 ns Volk oil 1% 4 59.2 b 7.67 ns Etrimfos 0.001/5 2 46.1 b -5.40 ns Etrimfos+oil 1% 3 51.5 b – Azinphos methyl 0.002 1 15.6 a 35.93** Azinphos methyl +oil 1% 5 61.9 b 10.47 ns ** Significant in 1% levels, ns isn’t significant.

Table 5. Evaluation of various chemical compounds effectiveness on adult olive psylla by shoot counting in Tarom-Sofla region (1997).

The number of uninfested The number of infested shoots Treatments shoots I II III Total I II III Total Volk oil 2% 8 11 8 27 7 4 7 18 Volk oil 1% 2 6 3 11 13 9 12 34 Etrimfos 0.001/5 8 9 8 25 7 6 7 20 Etrimfos+oil 1% 6 8 8 22 9 7 7 23 Azinphos methyl 0.002 3 5 13 19 12 12 2 36 Azinphos methyl +oil 1% 5 3 10 18 10 12 5 27 Check 1 1 0 0 1 14 15 15 44 Total 33 40 50 123 72 65 55 192 Median 4.7 5.7 7.1 17.6 10.3 9.3 7.9 27.4

Table 6. Effectiveness percentage (infestation reduction) of various chemical compounds on adult olive psylla by shoot sampling in Tarom-Sofla region (1997).

Effectiveness percentage Median of Treatments (infestation reduction)1 effectiveness I II III percentage Volk oil 2% 50 73.3 53.3 58.9 Volk oil 1% 7.2 40 20 22.4 Etrimfos 0.001/5 50 60 53.3 54.4 Etrimfos+oil 1% 35.7 53.3 53.3 47.4 Azinphos methyl 0.002 14.3 20 86.7 4.3 Azinphos methyl +oil 1% 28.6 20 66.7 38.4 1-It was done by Abbot formula.

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Table 7. Variance analysis of various chemical compounds effectiveness on adult olive psylla (effectiveness percentage) by shoot sampling in Tarom-Sofla region (1997).

S.O.V df SS MS F Replicatin 2 736.27 358.13 Treatment 5 1022.08 204.42 1.36 ns Error 10 1500.98 150.09 Total 17 3259.33 * ns isn’t significant

Table 8. Effectiveness of various chemical compounds on adult olive psylla (effectiveness percentage) by shoot sampling in Tarom-Sofla region (1997).

Treatment Class Medians Differences Volk oil 2% 6 58.9 a 11.43 ns Volk oil 1% 1 22.4 a -25.03 ns Etrimfos 0.001/5 5 54.4 a 7.00 ns Etrimfos+oil 1% 4 47.4 a Azinphos methyl 0.002 3 40.3 a -7.10 ns Azinphos methyl +oil 1% 2 38.4 a -9.00 ns * ns isn’t significant.

In consideration with obtained results in tests during year 1996-1997, envioromental protection, chemicals use reduction, more economical profit of emolsifiable oils in relation to pesticides, more durability of emolsifiable oils, negation of the insect oviposition sites and finally direct control of the olive psylla overwintered adult population, 2% volk application are suggested at the beginning of the mating stage and exactly before oviposition. Volk application 2%, regarding its apply conditions that are refered in this test, can be used as a safe method with other suggested methods in Integrated Pest Management program for olive psylla that has been presented by Katsoyannos (1992) to obtain the best results and includes: crop unplanting and weed control in tree crown area, modification of nitrogen fertilizers use, irrigation in a tree required amount, fumgus control and olive trees prunning.

Acknowledgment

Special thanks to financial credit of this test by National Research Projects budget of Agricultural Research, Education and Promotion Organization.

References

Abbot, W.S. 1925: A method of computing the effectiveness of an insecticicde. – J. Econ. Entomol. 18: 265-267. Chermiti, B. 1992: An approach to the assessment of the harmfulness of the olive psyllid, Euphyllura olivina costa (Hom., Aphalaridae). – Olive. 43: 34-42. Farahbakhsh, G. & Moieni, M. 1975: The major pests of olive in Iran, olive psylla. – The Plant Pests and Diseases Institute publication. 30-40. 186

Katsoyannos, P. 1992: Olive pests and their control in the Near East. – Division of Food and Agricultural Organization of the United Nations. 177 pp. Mustafa, T.M. 1989: Bionomics of the olive psylla, Euphyllura olivina Costa (Hom., Psyllidae) in Jordan. – J. Biol. Sci. Res. 20(1): 159-165. Nouri, H. 1996: Evaluation of olive psylla biology in Tarom-Sofla region (Qazvin province). – Final Report by Plant Pests and Diseases Research Section of Qazvin Agricultural Research Center. 17pp. Saeb, H. & Farzaneh, A. 1993: Evaluation of olive psylla population changes and its natural enemies recognition. – Annual Research Report by Plant Pests and Diseases Research Section of Gilan Agricultural Research Center.79-96. Taebi, M., Nickhoo, F., Sepehr, K. & Mirzaloo, M.R. 1991: The index of important plant pests and diseases and weeds of country major crops and recomened pesticides types and their application methods determination committee devices. – Plant Protection Organization. 340pp. Taherzadeh, M.R. 1994: The educational and promotional method role in olive orchards development and improvement. – The First Olive Confrance of Agricultural Ministry. 25-26.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 187

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

N.G. Kavallieratos,1 C.G. Athanassiou2, G.N. Balotis3, G.Th. Tatsi4, B.E. Mazomenos5 1 Laboratory of Agricultural Entomology, Department of Entomology and Agricultural Zoology, Benaki Phytopathological Institute, 8 Stefanou Delta, 14561, Kifissia, Attica, Greece. 2 Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, 75 Iera Odos, 11855, Athens, Attica, Greece. 3 Institute of Agricultural Sciences, 182 Kifissias Avenue, 15124, Amaroussion, Attica, Greece. 4 Technological Educational Institute of Larissa, Department of Plant Production, 41110, Larissa, Greece. 5 Chemical Ecology and Natural Products Laboratory, Institute of Biology, N. C. S. R. “Demokritos”, P.O. Box 60228, 15310, Aghia Paraskevi, Attica, Greece.

The effects of trap design, height and site of trap placement on the olive tree, pheromone doses in the dispensers, aging of the dispensers in the field and secondary pheromone components were evaluated for the development of an effective pheromone monitoring system for the olive moth Prays oleae (Bernard) Lesne in olive orchards. Field trials showed that trap design, pheromone dose and trapping side, affected male captures, while 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.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 189

Mating disruption trials for the olive moth, Prays oleae (Bern.), (Lep.:Yponomeutidae) in Trás-os-Montes olive groves (northeast of Portugal)

A. Bento1, J.A. Pereira1, J.E. Cabanas1, M. Konstantopoulou2, L.M. Torres3, B.E. Mazomenos2 1 CIMO / Escola Superior Agrária de Bragança, Quinta Santa Apolónia, Apartado 1 172, 5301-855 Bragança, Portugal. 2 Chemical ecology & Natural Products Laboratory, Institute of Biology NCSR “Demokritos” Aghia Paraskevis, Attikis Greece. 3 Universidade de Trás-os-Montes e Alto Douro, Quinta de Prados, 5000-911 Vila Real, Portugal.

The olive moth, Prays oleae (Bern), is one of the most serious olive pests in the Medi- terranean basin. The objective of the present study was to integrate environmentally safe methods for the control of the pest. Trials were carried out for three consecutive years (2002- 2004) in an olive grove about 20 ha, in the ecological production region at Romeu (North of Mirandela). The trees were of medium size, about 60 years old and mainly of the Cobranosa and Verdeal Transmontana cultivars. In the flower generation when 10% of the flowers were open, the entire grove was sprayed with Bacillus thuringiensis, (var. kurstaki) to reduce the larvae population. Within the grove, two 7 ha plots, one treated with pheromone during the fruit generation (MD-plot) and the other used as control, untreated (CO-plot), were selected. The distance between the two plots was approx. 300 m. Pheromone dispensers were installed at the onset of the fruit generation (3 June 2002, 5 June 2003 and 8 June 2004) in the MD- plot, and the dose of pheromone applied was 40 g/ha. Results were evaluated by fruit injury and by capture of male P. oleae in Delta traps baited with polyethylene vials, loaded with 1 mg of synthetic pheromone. The P. oleae pheromone is a single component the Z-7 tetradecenal; in mating disruption treatments the pheromone was formulated in ί-cyclodextrin and dispensed from polyethylene vials. During the flower generation, either male captures in pheromone traps or flower infestation were similar in both plots. However, during the fruit generation, male catches were higher at the CO-plot, with a maximum of 497.0±97.20 and 259.2±81.16 individuals per trap and per week, respectively in 2003 and 2004, than on the MD-plot (18.8±4.60 and 4.4±3.17). The rate of male disorientation was between 73.77 - 97.04% in 2003 and 96.21 - 97.42%, in 2004. Fruit infestation was significantly different between plots, with a maximum of 82.9% - 54.7% and 20.7±12.7 - 16.7±8.81 of infested fruits in the CO and MD plots, respectively for 2003 and 2004. The overall crop yield was similar in both plots. The results obtained suggest that the mating disruption method applied against the fruit generation of P. oleae has the potential to reduce the moth population and to minimize losses due to the pest.

This study was conducted with financial support from the EU, contract ICA4-CT-2001-1004 “Sustainable control of Lepidopterous pests in olive groves – Integration of egg parasitoids and pheromones”.

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Resistance of olive cultivars to carpophagous generation of Prays oleae

A. Lentini, G. Delrio, S. Deliperi Dipartimento di Protezione delle Piante, Sezione di Entomologia agraria, Universitá degli Studi di Sassari, Via E. De Nicola, 07100 Sassari, Italy.

During 1999-2005, the resistance of 3 olive cultivars to the carpophagous generation of olive kernel borer (Prays oleae Bern.) was studied in a grove in Sardinia. At the end of June the infestation level, determined as percentage of olives with penetrating larvae, varied between 4.1% and 75.2%. In the large drupe variety “Manna” the infestation level was constantly higher than in the smaller drupe varieties “Bosana” and “Semidana”. The autumn olive fall caused by the mature larvae varied from 0.5% to 25.5%, depending on variety and year. The reduction of June infestation was due to both the high fall in the post-setting stage, that occurred with greater frequency in infested olives, and to intrinsic factors not yet identified which caused larval mortality. The percentage of larvae eliminated by physiological fruit drop in post setting was higher in the cv Bosana (average value 77.97%) compared to the cv Semidana (61.59%) and the cv Manna (54.79%). On the contrary, the summer reduction of infestation due to larval mortality inside of the drupe was higher in the cv Manna (average value 30.01%) and the cv Semidana (25.10%) compared to the cv Bosana (11.86%). However, the overall reduction of infestation was higher in the cv Bosana, whereas in the other two varieties no differences were found. The observations made, highlight the fact that the damage caused by the olive kernel borer to the cv Bosana and the cv Semidana is very limited whereas, in certain cases, in the Manna losses of over 10% in autumn production were recorded. Heavier damage to the cv Manna is attributed to two factors: higher attacks, due to the olive kernel borer’s preference for the large drupe variety, and secondarily its lower varietal resistance.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 193

Optimization of the field performance of released Trichogramma spp. in olive groves, in Egypt

E.M. Hegazi1, A. Herz2, S.A. Hassan2, E. Agamyy3, W.E. Khafagi4, S. Mostafa5, N. Khamis1 1 Faculty of Agriculture, Alexandria University, Egypt. 2 Institute for Biological Control, Darmstadt, Germany. 3 Faculty of Agriculture, Cairo University, Egypt. 4 Plant Protection Research Institute, Alexandria, Egypt. 5 Central Lab. of Pesticide, Cairo, Egypt.

The dose and daily emergence patterns of three endemic species, Trichogramma cordubensis (TC), T. euproctidis (TEU) and T. bourarachae and the commercial available species, T. evanescens (TE) were monitored under lab and field conditions. Peaks of adult emergence varied according to the species and date of testing. The results suggested that TB and TC were the most adapted species for warm field weather than the other test species. The pattern of adult emergence and duration of adults availability of TB and TC seem to be more suitable for utilization in inundative releases in olive farms of arid area. The results suggest the importance of such studies to select and management of available endemic wasp species to achieve successful control of the target pests.

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Distribution and spatial pattern of Saissetia oleae (Olivier) on the olive tree in the northeast of Portugal

J.A. Pereira1, A. Bento1, L.M. Torres2 1 CIMO/Escola Superior Agrαria de Bragança. P.O. box 1172. 5301-855 Bragança, Portugal. 2 Universidade de Trás-os-Montes e Alto Douro. Quinta de Prados, 5000-911 Vila Real, Portugal.

The black scale, Saissetia oleae (Olivier), is a major olive tree pests throughout Portugal. In order to improve the knowledge on the pest population dynamics, as a basis for its optimal control, a study was conducted in the northeast region of the country on the within-plant distribution of the various insect stages and the spatial pattern of these stages on the host tree. The experimental work was carried out from April 1997 to December 1999, on two olive groves located near Mirandela, unsprayed for several years and non-irrigated. In each grove, ten trees were random selected and eight twigs about 30 cm in length were collected from each tree, on a biweekly basis from April to November and monthly from November to April. Twigs were taken from the four cardinal points and inside and outside of the tree canopy. A sub-sample of 20 leaves and 20 cm of branch was obtained from each of such samples and the scales present were counted, distinguishing the various stages of development and their position on the leaf (lower and upper side) Taylor’s power law and Iwao`s patchiness regression technique were user to analyse the spatial pattern of the insect. The results showed that the immatures were located mainly on the lower side surface of the leaves, whilst the adults were preferentially located on the branches. In general, the number of scales was higher inside the tree canopy, but no preference was shown in respect to the cardinal points. The spatial pattern of S. oleae, which could be adequately described by Taylor’s power law and Iwao`s regression methods, was generally aggregated. Also it was shown that the degree of aggregation decreased with the development of the insect and, in general, was higher in the inside of the tree canopy and in the lower side surface of the leaves.

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Twig dieback in olive trees associated with Resseliella oleisuga (Targioni Tozzetti) (Diptera Cecidomyiidae) and Libertella sp.

Gabriella Frigimelica, Alessio Rainato, Luca Mazzon, Vincenzo Girolami University of Padua, Department of Environmental Agronomy and Plant Productions, Viale dell’Università, 16 , 35020 Legnaro, Padua, Italy

Abstract: Twig dieback has been observed in olive trees growing in the Euganei and Berici hills. Dieback was related to the presence of the gall midge Resseliella oleisuga and fungi causing canker. The biological cycle of the cecidomyiid and the presence of associated fungi has been observed for two years. The occurrence of the fungus Libertella sp. and larvae of R. oleisuga on wounds resulted correlated, suggesting a close insect-fungus association.

Key words: olive tree diseases, Resseliella oleisuga, associated fungi, Libertella sp.

Introduction

The gall midge Resseliella oleisuga (Targioni Tozzetti, 1886) (Diptera Cecidomyiidae) is a pest occurring on olive tree (Olea europaea) in the whole Mediterranean basin (Skuhrava, 1994), but found also on Fraxinus and Phillyrea (Tremblay, 1994; Pollini, 1998). R. oleisuga have been reported only in Southern Italy and Sicily (Skuhrava, 1995), where sporadically causes strong infestations (Liotta, 1981; Brogi and Galligani, 1987). The cecidomyiid lay eggs in wounds of the bark on the twigs. The flat larvae develop under the bark, feeding on the cambium. A purple discoloration of the bark and twig dieback is associated. Mature larvae leave the host after about 18 days, to pupate into the soil (Shazli and Mustafa, 1980). Emergence of the adults takes place after about 8 days (Shazli and Mustafa, 1980). Recent studies carried out in Tuscany (Central Italy) report R. oleisuga as completing at least 3-4 generations per year (Bagnoli, 1982; 2005). The aim of the work is to verify the presence of R. oleisuga in Northern Italy (Veneto Region, the Euganei and Berici hills) and to investigate the role of the insect-fungus association in the olive twig dieback.

Material and methods

Experimental areas and sampling The study was carried out in the Euganei and Berici hills, two olive-growing areas of the Veneto Region. Eleven olive groves in the Euganei and two in the Berici hills were examined. The total extension of the two sampled areas was approximately the same (10 ha each). At least 40 symptomatic twigs were collected monthly from infested olive trees and brought to the laboratory for further investigations. The same number was collected as a control group in the second year. Sampling has been carried out for two consecutive years in the periods February-September 2004 and January-August 2005.

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Laboratory study The occurrence of eggs or larvae of the insect, and the state of cankers (expanding or scarred) was recorded for each wound on the sampled twigs. Fungal isolation was carried out on healthy twigs (control, n=232) and infested lesions (n=1194), with a distinction between actively expanding lesions (n=429) and lesions with callus formation (n=765). From each sample, six small pieces of inner bark tissues, including both healthy and necrotic tissues, were aseptically collected at the edges of cankers and moved to a PDA medium (Difco® Potato Dextrose Agar), then incubated at 25 ± 1°C and then exposed to the natural photoperiod for one week. Emerging colonies were transferred on fresh PDA medium in order to isolate the developing fungi. The same protocol was applied to the healthy twigs kept as control (Garberlotto et al., 1992). Fungal colonies were identified by in vitro morphological features and reproductive structures. The fungal isolation frequency (IF) was calculated for each month (January- September) as the number of colonies of each fungal type per the number of samples (n° total colonies / n° bark fragments) (Frisullo et al., 2002). A pathogenicity test was performed as described by Kuhlman and Bhattacharyya (1984). A total of ten branches of three healthy olive trees were inoculated, with each branch being inoculated twice, respectively with PDA colonised by the mycelium of Libertella sp. and with sterile PDA (control). The longitudinal length of the induced cankers was measured after eight months.

Statistical analyses Differences between the IF of each fungus isolated from both control and symptomatic twigs were tested by a χ2-test (P=0.05). In the pathogenicity test, the differences between the lesion lengths due to Libertella sp. and control inoculations were tested by a t-test (P=0.05).

Results and discussion

Occurrence of R. oleisuga The gall midge was found in all observed olive growing areas in the Euganei and Berici hills. The specimens found are the most northern so far collected, enlarging the known distribution area of the species, which until now was reported only for Southern Italy (Skuhrava, 1994; 1995). Further studies on the distribution of this insect will be carried out in Northern Italy.

Life cycle of R. oleisuga During the summer the wounds seem to be infested only by eggs. Larvae occur in the infested branches only from autumn to spring (Figure 1). It seems that eggs enter the diapause during early summer as no egg hatch has been observed in laboratory conditions during this season. The number of branches infested by larvae shows one first peak in spring (40%), and the highest peak in August (56%) (Figure 1). Mature larvae of R. oleisuga overwinter in the soil, according to the literature (Tremblay, 1994; Pollini, 1998). As in Tuscany (Bagnoli, 1982; 2005), three or four generations (or more overlapping during the year) are completed per year according to the present research in the Euganei and Berici areas.

Associated fungi The total isolation frequency (IF) was calculated as total number of colonies of each fungal type per number of samples and ranges between 13% in healthy branches to 102% in infested twigs. Thirteen fungal taxa having IF>1% were isolated from the lesions.

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(%) 40

30 Resseliella oleisugaResseliella

20 Frequency of Frequency

0 JFMAMJJAS

larvae eggs

Figure 1. Occurrence of R. oleisuga (larvae and eggs).

60 18

50 )

(% 40 12 sp. (%)

30 Libertella Resseliella oleisugaResseliella

20 6 Frequency of Frequency of of Frequency

0 0 JFMAMJJAS

larvae Libertella sp.

Figure 2. Presence of larvae and the fungus Libertella sp. on symptomatic twigs during the season (mean of two years for each month).

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The fungus Libertella sp. was never found in the controls and the 3,8% IF value in scarred wounds increased to 11% in expanding lesions, suggesting that it could have a role in the canker formation and the subsequent twig dieback. In this respect, the inoculation of the Libertella mycelium caused cankers in all the inoculated twigs similar to those observed in the field on infested trees (data not shown). The length of the necrosis occurring on twigs inoculated with Libertella sp. was significantly higher than the controls and Libertella sp. was isolated from all the inoculated branches. The identification of the Libertella genus has been confirmed by the Centraalbureau voor Schimmelcultures (Utrecht, Holland). Libertella sp. shows a higher IF value in wounds hosting feeding larvae (IF=13,5%) than in wounds abandoned by the insect (IF=7%). The correlation between the occurrence of the fungus and the larvae (Figure 2) further confirms that the successful colonisation of Libertella sp. is related to the larval activity. This close association, not yet reported in literature, is similar to the insect-fungal organisms relationships reported for the gall midge species Thomasiniana theobaldi Barnes on Rubus sp. (Wilson & Green, 1944; Pitcher, 1952; Stoyanov; 1963; Tremblay, 1994; Anfora et al., 2005).

References

Anfora, G., Carlin, S., Guerriero, A., De Cristofaro, A., Versini, G., Ioriatti, C. 2005: Resseliella theobaldi (Barnes) (Diptera Cecidomyiidae): analisi chimiche ed elettro- fisiologiche sui semiochimici coinvolti nel rapporto fitofago-pianta ospite. – Proceedings XX National Italian Entomological Congress, Perugia-Assisi, June 13th-18th: 218. Bagnoli, B. 1982: Alcune precisazioni sulla biologia di Thomasiniana oleisuga (Targ.) (Dipt. Cecidomyiidae) in Toscana. – Ann. Ist. Sperim. Zool. Agr. 7: 77-84. Bagnoli, B., Bennassai, D., Mosconi, E. 2007: Bionomics of Resseliella oleisuga (Targ.- Tozz.) in Tuscany (Diptera Cecidomyiidae). – IOBC/WPRS Bull. 30(9): 203. Brogi, P. & Galligeni, L. 1987: Il “moscerino suggiscorza” dell’olivo. – Inf. Fitopat. 12: 19- 22. Dahl, C., Krivosheina, N.P., Krzeminska, E., Lucchi, A., Nicolai, P., Salamanna, G., Santini, L., Skuhrava, M., Zwick, P. 1995: Diptera Blepharicenomorpha, Bibionomorpha, Psychodomorpha, Ptychopteromorpha. – In: Minelli, A., Ruffo, S., La Posta, S. (eds.), Checklist delle specie della fauna italiana, 65. Calderini, Bologna. Frisullo, S., Lops, F., Carlucci, A. 2002: Indagini sui funghi endofiti nei rametti di olivo apparentemente sani con foglie e malati defogliati. – Proceedings of the National Congress: “L’Endofitismo di funghi e batteri patogeni in piante arboree e arbustive”. Sassari, May 19th-21th: 113-125. Garbelotto, M., Frigimelica, G., Mutto Accordi, S. 1992: Vegetative compatibility and conversion to hypovirulence among isolates of Cryphonectria parasitica from northern Italy. – Eur. J. For. Path. 22: 337-348. Kuhlman, E., Bhattacharyya, H. 1984: Vegetative incompatibility and hypovirulence conversion among naturally occurring isolates of Cryphonectria parasitica. – Phytopathol. 74: 659-664. Lotta, G. 1981: Problemi entomologici dell’olivo. – Inf. Fitopat. 1-2: 11-17. Pitcher, R.S. 1952: Observations on the raspberry cane midge (Thomasiniana theobaldi Barnes) and its association with cane blight. – Rev. Appl. Entomol. 40: 141-143. Pollini, A. 1998: Manuale di entomologia applicata. – Edagricole, Bologna. 1462 pp. (cfr. 817). Shazli, A. & Mustafa, T.M. 1980: Frequency of Thomasiniana oleisuga Targ. (Dipt. Cecido- myiidae) and its parasites and predators in Amman, Jordan. – Z. Ang. Ent. 89: 269-277. 201

Skuhrava, M. & Skuhravý, V. 1994: Gall midges (Diptera Cecidomyiidae) of Italy. – Entomol. 28: 45-76. Stoyanov, D. 1963: Studies on raspberry midge Thomasiniana theobaldi Barnes in Bulgaria. – Izv. Inst. Zasht. Rast. 4: 41-66. Targioni-Tozzetti, A. 1886: Notizie sommarie di due specie di Cecidomidei, una consociata a un Phytoptus, ad altri acari ed ad una Thrips in alcune galle del nocciolo (Corylus avellana L.), una gregaria sotto la scorza dei rami di olivo, nello stato larvale. – Boll. Soc. ent. ital. 18: 419-431. Tremblay, E. 1994: Entomologia applicata. – Liguori Editore, Napoli. III/1: 152 pp. Wilson, G.F. & Green, D.E. 1944: Observation on two Raspberry troubles. – J. R. Hort. Soc. 69 (3): 79-86.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 203

Bionomics of Resseliella oleisuga (Targ.-Tozz.) in Tuscany (Diptera Cecidomyiidae)

B. Bagnoli, D. Benassai, E. Mosconi C.R.A. - Istituto Sperimentale per la Zoologia Agraria,Via Lanciola, 12/A, 50125 Firenze, Italy.

Research carried out during different years in various olive-growing areas allowed deeper knowledge on ethology and ecology of Resseliella oleisuga (Targ.-Tozz.) (Diptera Cecido- myiidae) in Tuscany. From weekly examination of olive branches artificially wounded emerged that adult presence and oviposition are practically continuous from April to October. Under suitable laboratory conditions preimaginal development period averages 25-30 days whereas in the field the duration of the life-cycle, even for population from eggs laid in the same period, varies considerably. This is predominantly due to the length of the larval instar and to the time the mature larva spends before inside the branch and then in the soil. The life-cycle and specially larvae behaviour are very affected by weather conditions. During the spring-summer period a part of the population completes its life-cycle (egg to adult) in 35-50 days. Thus between April and October there can be until three-four generations overlapping. Two main peaks of adult presence are usually observed: the first in July and the second in September. From laboratory rearing data sex ratio appears greatly unsettled in favour of females (as far as 10:1) that show a potential fecundity of over 100 eggs per individual. Every solution of continuity of the bark in twigs with a diameter of 3-15 mm can be used by females for laying eggs. At the same time R. oleisuga females can exploit pruning cuts and oviposition hurts of Auchenorrhyncha. One wound can be used by different females for successive ovipositions. In Tuscany the frequency and the seriousness of the species infestations seem tight associated with hail storms. Larval population of R. oleisuga are usually subjected to attack of the predator Pyemotes ventricosus (Newport) (Acari Pyemotidae), the ectoparasite Eupelmus sp. (Chalcidoidea Eupelmidae) and the endoparasites Platygaster sp. and Leptacis sp. (Proctotrupoidea Platygasteridae).

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Effect of eriophyides mites on the sensitivity of some olive tree varieties

A. Chatti, M. Ksantini, T. Jardak Olive Tree Institute, BP 1087- 3000 Sfax, Tunisia.

In the framework of the integrated protection concept or more recently the integrated production, a big attention is allowed to the preventive measures to control pests and diseases, among them, the choice of resistant or tolerant varieties to the destructives and diseases attacks. In this context we have studied the resistance to the eriophydes mites attacks of 15 olive trees varieties in the collection of the experimental station of Taous (Sfax- Tunisia). These varieties were composed of 7 table olive tree cultivars (Meski, Picholine, Manzanille, Zarrazi, Touffahi, Lucque and Blanquetta) and 8 oils olive tree varieties: Chemlali (clones C 236 and C 340), Chemlali, Zalmati, Chemlali Zarzis, Chemlali Ontha, Ouslati, Koroneiki and Azeitira. The trees are 4 years old and planted 6 x 6 meters with drip irrigation. The resistance to the mites was apprehended by the eriophydes density by mm2 of leaves, which is estimated with an uniform sampling on threes at the rate of five branches/ tree each 15 days during the year 2004. According to the maximum density of eriophydes mites /mm2 of leaves and depending on their degree of resistance, we were able to classify the mites varieties in three varieties can be classified on tree categories: – Category n° 1: Resistant varieties when the density is inferior to 3 mites/ mm2 of leaf. The varieties were: Oueslati (oil variety) and Touffahi, Lucque and Blanqetta (table varieties). – Category n°2: moderately resistant varieties when the density is between 3 and 12 individus/mm2 of leaf. The varieties included in this category were: Zalmati, Chemlali (clone 340) and the table varieties Manzanille, Picholine and to fine double Zarrazi and Azeitira. – Category n°3: sensitive varieties when the density is superior to 12 mites/ mm2 of leaf. This category contains the majority of oils varieties such as: Chemlali, Chemlali Zarzis, Chemlali Ontha Chemlali (clone c 236), Koroneiki and the table olive variety Meski. This work shows the less sensitivity to mites of the local variety Oueslati and a high sensitivity of certain local varieties particularly the Chemchali of Gafsa, the Chemlali Ontha of Tataouine, the Chemlali of Zarzis, the Meski and the foreign variety Koroneiki. It shows equally a different sensitivity between clones of the same variety (Chemlali).

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 207

Effect of cereal cover crops on Araneae population in olive orchard

M. Cárdenas1, J.A. Barrientos2, P. Garcνa3, F. Pascual4, M. Campos1 1 Department of Agroecology and Plant Protection. Estaciσn Experimental del Zaidνn. CSIC. Granada, Spain. 2 Department of Animal Biology and Ecology. Autonomic University of Barcelona. Barcelona, Spain. 3 Department of Statistic and I.O., Spain. 4 Animal Biology and Ecology. University of Granada. Granada, Spain.

Spiders, Araneae, are one of the most common predator orders in olive orchards. The potential bioindication of spiders as indicators of soil management was tested in field trials on olive orchards of south Spain. Field trials were carried out in five olive zones: two with cereal cover and three without cover and ploughed. In all zones 4 plots of 5 trees per plot were sampled. Each tree was sampled by beating method and by pitfall traps. The study was carried out during two years, 1999 and 2000, from April to October. The total captures were greater in zones with cereal cover and the results showed that there was a signification tendency in Kruskal-Wallis test for total number of spiders between covered and ploughed olives zones. Nevertheless, only in 2000 these differences were significant. The analysis of the samples per plots showed no significant differences for plot 1, significant differences at plot 2 for the family Linyphiidae, with more captures in zones with cover, and it was the same for Loxocelles rufescens (Sicariidae) at plot 4. There was a signification tendency for Thyene imperialis (Salticidae) at plot 3. The spider taxonomic composition was very similar, including 16 families and 63 species represented at the three ploughed zones and 17 families and 61 species represented at the two cereal covered olives zones. These results indicated the important role of cereal covers in the abundance and diversity of spiders in olives orchards. These cover crops play an important role as a shelter of spiders and for their alternative preys and this management makes possible a faster potential answer opposite to pests increase.

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Coccinellidae communities: diversity and dynamics in organic and integrated olive groves from Trás-os-Montes (northeast of Portugal)

S.A.P. Santos1, J.A. Pereira1, A. Raimundo2, A.J.A. Nogueira3, L.M. Torres4 1 Escola Superior Agrária de Bragança, P.O. box 1172. 5301 Bragança, Portugal. 2 Universidade de Évora, P.O. box 94. 7001 Évora, Portugal. 3 Universidade de Aveiro, 3810-193 Aveiro, Portugal. 4 Universidade de Trás-os-Montes e Alto Douro. Quinta de Prados, 5000-911 Vila Real, Portugal.

Coccinellidae are well known predators in agroecosystems. In olive groves they may exert control against scales, such as the black-scale, Saissetia oleae (Olivier) and other minor pests. The aims of this work were i) to study the diversity of Coccinellidae species in two olive groves with different plant protection systems (integrated plant protection – Paradela grove, and organic growing guidelines – Valbom-dos-Figos grove); ii) to analyse the dynamics of these predators, and iii) to compare the differences between groves. The experimental work was carried out from April 2002 to November 2003. Weekly, in each grove, five plots of ten olive trees per plot were randomly selected and one branch was sampled per tree using the beating technique. The captured Coccinellidae were identified to species level. Experimental results showed the existence of differences between olive groves and years. A total of 17 species belonging to nine genera were identified. In Paradela, Rhyzobius chrysomeloides (Herbst.) was the most abundant species representing 40%, followed by Scymnus (Pullus) mediterraneus Khnz., with 17%, Scymnus (Pullus) subvillosus Gze. and Stethorus punctillum (Ws.), both with 10% of total captured individuals. In Valbom-dos-Figos, the community of Coccinellidae was more diversified and Scymnus (Scymnus) interruptus Gze. was the dominant species with 56% of total captures, followed by Rhyzobius chrysomeloides (Herbst.), with 19% and Chilocorus bipustulatus L., with 10%.

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Coccinellids associated with olive groves in north-eastern Portugal

M.F. Gonçalves1, S.A.P. Santos2, A. Raimundo3, J.A. Pereira2, L.M. Torres1 1 Universidade de Tràs-os-Montes e Alto Douro. Quinta de Prados, 5000-911 Vila Real, Portugal. 2 CIMO/Escola Superior Agrària de Bragança. P.O. box 1172. 5301 Bragança. Portugal. 3 Universidade de Évora. P.O. box 94. 7001 Évora. Portugal.

In order to point out quantitatively and qualitatively the main species of Coccinellidae present in olive groves in north-eastern Portugal, six groves were sampled from August to October, in 2002, and three of these groves were sampled again, from March to July, in 2003. Samples were collected on a weekly or fortnightly basis, by beating two branches per tree, from each of 25 trees, randomly selected per grove and date. A total of 710 individuals belonging to 12 species were captured: Chilocorus bipustulatus L., Exochomus nigromaculatus (Gze.), Exo- chomus quadripustulatus L., Scymnus (Sc.) interruptus Gze., Sc.(Pullus) subvillosus (Gze.), Hyperaspis reppensis Herb., Oenopia lyncea (Oliv.), O. conglobata (L.), Coccinella septempunctata (L.), Sospita oblongoguttata (L.), Rhyzobius chrysomeloides Herb. and R. lophantae (Blaisdell). Sc.(Pullus) subvillosus predominated (50,6%), followed by R. chrysomeloides (23,9%), Sc. interruptus (13,4%), E. quadripustulatus (6,8%) and C. bipustulatus (1,8%). The other species were scarcely represented (less than 1%). Captures were obtained over the whole period of the trial, although in greatest numbers during August and September (89,7%), in 2002, and from the middle of March to the middle of April and from the end of June to the end of July (88,9%), in 2003. Sc.(Pullus) subvillosus was present mainly (94,7%) from the beginning of August to the end of October and during July, while S. interruptus occurred in greatest numbers (91,6%) from the beginning of August to the end of September and during July. R. chrysomeloides predominated (83,4%), from the beginning of August to the end of October and from the middle of March to the middle of April. E. quadripustulatus occurred mainly (97,9%) from the beginning of March to the end of July, while C. bipustulatus was captured principally (92,3%), from the beginning of August to the middle of October.

Work partially financed by demonstrative project AGRO 296 “Protecção integrada da oliveira nas regiões de Tràs-os-Montes e Beira Interior”.

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Diseases

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 215

Current problems related to olive diseases in the Mediterranean basin

E.C. Tjamos, P. Antoniou, S.E. Tjamos, E.J. Paplomatas Agricultural University of Athens, Votanikos 11855, Athens, Greece.

Verticillium wilt constitutes the main olive disease problem in the Mediterranean basin with severe symptoms usually in irrigated groves. The situation has been made more complex by the appearance of a defoliating strain initially in the USA and currently in the Mediterranean regin with emphasis in Spain. Recent screening of olive germplasm for selecting resistant cultivars or rootstocks provided promising data. Various cultural measures, soil solarization or chamber solarization have been suggested. Studies on chemicals show that currently available fungicides are unable to control the disease regardless of references that post fruit- setting foliar treatments of phosetyl-Al may be beneficial. As for olive scab disease, chemical control includes copper fungicide during the main infection seasons of spring and autumn. Strobilurin-based fungicides, less effective as pretective are more efficient as curative compared to organocupric fungicides. As for Mycocentrospora cladosporioides it has been widely spread among several Mediterranean countries. The fungus frequently appears on the lower surface of the older leaves, while green or mature olives are also occasionally attacked. Preliminary trials with different copper oxychlorides in Italy indicated that four treatments (February, April, end of August and late September) in cv Leccino are required to effectively reduce severity of the disease. We believed that Clitocybe olearia and to a lesser extend Armillaria mellea were the main root rot and wood decay agents but recently Phomitiporia punctata (P. mediterranea) is spreading in old olive orchards causing symptoms similar to esca of grapevines. As for olive knot the disease is widespread all over the Mediterranean basin with severity directly related to the susceptibility of the varieties, to the degree of wounding from frost, hailstorm and harvesting injuries accompanied by rainy or wet weather. Concerning phytoplasmas several authors in Italy have characterized phytoplasmas in olive trees showing symptoms of yellowing, shortening of internodes, witches’ broom, bud abortion, little leaf, hypertrophied inflorescences, decline and fasciation. DNAs extracted from leaf veins were amplified in PCR reactions using universal or group-specific primers constructed on 16S rRNA phytoplasma sequences and restricted with five different enzymes. It appears that phytoplasmas are ubiquitous in the areas surveyed, but a clear correlation between a given syndrome and the presence of one or more phytoplasmas did not emerge. As for olive viral diseases olive is hosting up to 13 different viruses while other viruses, which are either non mechanically transmissible or occur in low concentration in plant tissues, may be also present. This is supported by the widespread occurrence of double-stranded RNAs (dsRNAs) in plants negative to biological tests. Molecular hybridization tests on dsRNA- positive samples collected in Italy (Apulia), revealed the presence of the three nepoviruses (Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV) and Strawberry latent ring spot virus (SLRSV), plus Olive leaf yellowing associated virus (OLYaV) and Olive latent virus-1 (OLV-1). The most common virus in southern Italy is OLYaV, the main virus in central Italy is SLRSV whereas CLRV was detected in five samples from Latium, Umbria and Sicily. Problems related to the dispersal of pathogens by exporting olive plant material in southern hemisphere countries will be also discussed.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 217

Olive viruses and strategies for producing virus-free plants

M. Saponari1, G. Bottalico2, G. Loconsole2, G. Mondelli2, A. Campanale1, V. Savino1,2, G.P. Martelli1,2 1 Istituto di Virologia Vegetale CNR- Sezione di Bari, Italy. 2 Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi di Bari, Italy.

Surveys carried out in a number of Mediterranean olive-growing countries have disclosed a high incidence of viral infections, mostly in symptomless trees (Felix et al., 2002; Saponari et al., 2002; Faggioli et al., 2005; Fadel et al., 2005). Although the impact of these infections on the crop is largely unknown, they affect marketing of propagating material (rooted plants, budsticks, seedlings, seeds), because, according to the Conformitas Agraria Communitatis (CAC) enforced in the European Union, nursery productions must be free from a number of detrimental “pests”, including viruses. It means that only virus-tested or virus-free mother plants can be used by nurserymen for propagation. Implementation of preventive measures in the framework of certification schemes, such as sanitary selection and sanitation, represents the only strategy currently available to restrain spreading of olive viruses. In Italy, sanitary improvement programmes are underway for the production virus-tested and virus-free mother plants. Selected plants fitting the requirements of a legislative decree issued in June 1993 by the Ministry of Agriculture represent “primary sources” (nuclear stocks) which, following registration by a Technical Committee of the Ministry of Agriculture, enter the certification system. Sanitary selection, development of diagnostic tools and sanitation treatments are the main objectives of our studies. As to laboratory testing our aim was to set up a simple and sensitive protocol for the simultaneous detection of olive-infecting viruses from field plants and in vitro-grown explants. Multiplex hybridisation of crude sap proved useful for the detection of Cherry leaf roll virus (CLRV), Strawberry latent ringspot virus (SLRV), Arabis mosaic virus (ArMV), Olive latent ringspot virus (OLRV) and Olive latent virus 1 (OLV-1). As to sanitation, trials are underway to determine the behaviour of different cultivars towards in vitro culture and different temperatures, i.e. (i) exposure of infected in vivo plants to low temperature (5-6 °C) followed by heat therapy (35-38 °C for 2-3 months), excision of shoot tips and their transfer to a growth medium; (ii) in vitro heat therapy (35-38 °C for 15-20-25 days) followed by meristem tip culture; (iii) meristem tip culture directly from in vitro-grown plantlets. The combined use of these techniques can efficiently eliminate virus infections, especially those by Olive leaf yellowing-associated virus (OLYaV), one of the most widespread viruses found so far.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 219

Fungal agents responsible for olive dieback in Iran

M. Salati1, H. Afshari Azad2, A. Javadi Estahbanati2 1 Agricultural Research Center of Golestan, Iran. 2 Plant Pests & Diseases Research Institute, Tehran, Iran.

Olive dieback is prevalent in most olive orchards of Iran. Symptoms produced is different from those which has been reported sofar from the country. In this disorder, both young and old grove twigs are affected and there is a distinguished border between healthy and affected areas. In all, 416 samples collected from Golestan province showing disease symptoms were collected and examined. Three species of fungus belonging to Sphaeropsis, Fusicoccum, and Phoma were isolated from the infected tissue and their pathogenicity were approved by stem wounding method. The morphometric characteristics of first isolate is similar to Sphaeropsis malorum which was isolated from grapevines. However, the measured characteristics for identification of the other two genus up to species level of the fungus is not enough and needs futher studies. S. malorum was the most frequent isolate which produced dark colony on PDA,with brown mycelium. Pycnidia with one cavity, round or flask shaped to elongate, with neck and ostiole, light brown which turne darker, 140-200 x 150-250 µm. Picnidial wall consists of 3-4 layers of deeply pigmented angular cells, with thick-walled cells on the outside and thin-walled, rounded cells on the inside. Conidiogenous cells holoblastic, hyaline, elongated and smooth. Conidia clavate, usually one end thiner than the other, unicellular, hyaline, 4-7 x 15-24 µm, which produced 1-2 septa at germination.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 221-224

Comparison between real-time PCR and semi-selective medium in monitoring Verticillium dahliae microsclerotia in the olive rhizosphere and suppression of the pathogen by compost

Giuseppe Lima1, Filippo De Curtis1, Anna Maria D’Onghia2, Franco Nigro2 1 Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Università degli Studi del Molise, Via F. De Sanctis, 86100 - Campobasso, Italy, E-mail: [email protected] 2 Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi di Bari, Italy, E-mail: [email protected]

Abstract: Trials were conducted on young olive plants grown in soil artificially contaminated by different inoculum density (0, 5, 30, 60 and 100 microsclerotia/g of soil) of Verticillium dahliae. In blind trial experiments, the pathogen was monitored in the contaminated soil up to 100 days by both semi-selective medium and real-time nested Scorpion PCR. Linear regression analysis revealed significantly high correlations (R2 >0.8; P <0.01) between the two diagnostic methods. The molecular technique was very reliable and accurate and drastically reduced the time of the diagnosis. In the rhizosphere of plants contaminated with 30 microsclerotia/g of soil, the incorporation of 15% (w/w) of a compost obtained from cured olive oil by-products significantly reduced the inoculum density of V. dahliae. The suppressive activity was improved adding a Trichoderma viride based biofungicide to the compost. The real-time Scorpion PCR as well as composts from olive by-products seem interesting tools for potential application in eco-compatible agriculture systems.

Key words: Verticillium dahliae, Olea europaea, diagnosis, compost

Introduction

Verticillium wilt caused by Verticillium dahliae Kleb. is one of the most important diseases of olive (Olea europaea L.). In the last years, favoured by inadequate cultural practices and crop production intensification, a progressive disease increment was observed in most olive growing areas (Jiménez-Díaz et al., 1998). Since fungicides are often ineffective, the use of pathogen free planting material and cultivation soils are considered key factors for an efficient disease prevention (Nigro et al., 2005). Soil health testing before new plantations and phytosanitary certification are among the most effective measures. However, laborious and time consuming current diagnostic methods represent severe constraints to this achievement. The rapid identification and quantification of the pathogen in situ is a goal for both nursery growers and farmers. For this purpose, several methods, mainly based on pathogen DNA analyses, were developed. Among the new methods, the real-time Scorpion PCR seems very promising for V. dahliae diagnosis in both soil and plant (Nigro et al., 2004). In addition, the increasing demand for eco-compatible control methods has stimulated researches on biological and integrated control of V. dahliae, too (Tjamos, 2000). One of the most promising alternatives to reduce pathogen inoculum in the soil is represented by composts and amendments (Niaounakis & Halvadakis, 2004). The aims of this research were to evaluate the reliability of the real-time Scorpion PCR technique (Nigro et al., 2004) for detection and monitoring of V. dahliae microsclerotia in the soils of olive plants and to assess the suppressive activity against the pathogen of a selected

221 222

compost obtained from cured olive oil by-products (Ranalli et al., 2002; Lima et al., 2004). Material and methods

Preparation of experiments on olive plants Different composts were obtained in a composting pilot plant in Molise (Central Italy) using olive humid husks produced by a two-phase oil mill plant. A compost, obtained by composting the following materials (w/w), was tested: OL (olive leaves) 8%, OHH (olive humid husks) 67%, and CHH (composted, one year-old, humid husks) 25%. Composting conditions, quality and antagonistic activity in vitro against different fungal pathogens were previously reported (Ranalli et al., 2002; Lima et al., 2004).According to the schedule of each experiment, the selected compost was mixed (0, 15, 30, 60 or 100% w/w) with an orchard sterilised soil (SOS: pH 7.42, sand 36%, silt 15%, clay 47.9% and organic matter 1.1%). In the first year, the mixtures were artificially contaminated with 30 V. dahliae microsclerotia (MS)/g, while in the second year, using only a mixture of 15% of compost and 85% of soil, the following V. dahliae inoculum densities were applied: 0, 5, 30, 60 and 100 MS per gram. Compost applied at 15% was also tested in combination with a biofungicide (Trichoderma viridae TV1- Agribiotec, Italy). The substrate was dispensed in 2-liters plastic pots (3 replicates of 6 pots for each treatment) and olive plants (cv. Leccino from 2-years rooted cuttings), previously wounded by removing few mm of the apical roots, were planted into them. The plants were kept under a laterally opened screen-house and climatic conditions were costantly monitored.

V. dahliae microsclerotia (MS) V. dahliae MS for atificial contamination of substrates were prepared as reported by Hawke & Lazarovits (1994). A mixture of MS in equal proportion of 4 V. dahliae isolates from infected discolored olive wood was used.

Monitoring of V. dahliae MS In order to quantify the density of V. dahlie MS in the rhizosphere, at 20-days interval, soil samples were collected from each pot and air dried for 30 days. Each sample was then splitted in two subsamples and according to a blind-trial scheme each treatment was examinated to verify the presence of V. dahliae MS by both the semi-selective medium (Shetty et al., 2000) and real-time nested Scorpion PCR (Nigro et al., 2004).

Statistical analyses Data were submitted to variance analysis and means compared using Duncan’s multiple range test. Data on MS inoculum density, obtained from classical dilution plating method, and threshold cycle values of real-time nested Scorpion PCR, were submitted to simple linear regression analysis. The averaged cycle threshold values were plotted against the log- transformed number of MS/g of soil and the correlation coefficient (R2) of the curve was calculated by using the least square fit method (Snedecor & Cochran, 1980).

Results and discussion

Comparison between real time PCR and semi-selective medium According to previous results (Nigro et al., 2004), the molecular method confirmed its reliability for a rapid and accurate detection of V. dahliae in the soil of olive plants. The cycle threshold values of analysed soil samples were related to the dynamic of V. dahliae MS assessed by semi-selective media. Linear regression analysis revealed a significantly high correlation between the conventional and molecular methods (Figure 1). The R2 values indicated the possibility of estimating the number of MS/g of soil by using the nested 223

Scorpion PCR technique and hence avoiding the laboriousness of the plating method. The ability to quickly ascertain the presence or absence of the pathogen in the soil would have significant benefits on both olive tree farmers and nursery growers.

ID [Log10 (Ms/g+1)]= 1.8247 – 0.0444 x Cycle Threshold ID [Log 10 (Ms/g+1)] = 1.8436 – 0.0452 x Cycle Threshold 1.6 1.6 2 R = 0.808 R2= 0.887 1.4 p < 0.0000 1.4 p < 0.0000

1.2 1.2

1.0 1.0

0.8 0.8 (Ms/g +1)] 0 days 40 days 10 0.6 0.6 0.4 0.4

ID [Log 0.2 0.2 0.0 0.0 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 Cycle Threshold Cycle Threshold

Figure 1. Relationships between inoculum density (ID) of V. dahliae MS found by semi-selective medium in the rhizosphere of olive plant and the corresponding threshold cycles of real-time PCR on soil samples few hours (0 days) or 40 days after contamination with different ID of MS. Soil samples were air-dried for 30 gg before analyses.

Suppressive activity of compost in the rhizosphere of olive plants A single application of 15% (w/w) of cured compost or biofungicide TV1 (T. viridae), separately or in combination, significantly reduced the density of V. dahliae MS in the rizosphere during the first 20-60 days with respect to the untreated control (Figure 2a). The inoculum density of V. dahliae MS found on semi-selective media (Figure 2a) was also related to threshold values of real-time Scorpion PCR (Figure 2b).

Uncontaminated SOS SOS a Uncontaminated SOS SOS b SOS+15% CP SOS+TV1 SOS+15% CP SOS+TV1 SOS+TV1+15%CP SOS+TV1+15%CP 30 40 25 20 30 15 20 10 10 5 0 Threshold cycle 0 Recovered MS/g soil MS/g Recovered 04060 04060 Days from application of treatments Days from application of treatments

Figure 2. V. dahliae Microsclerotia (MS) recovered on semi-selective medium (a) and corresponding threshold cycles of real-time Scorpion PCR (b) on soil samples from the rhizosphere of olive plants. SOS= Sterilised Orchard Soil; CP= Compost 15% w/w; TV1= (T. viridae). The substrates were contaminated by 30 V. dahliae MS/g. Bars represent the standard deviation from the mean. Soil samples were air-dried for 30 gg before analyses.

As previously reported (Lima et al., 2004), the suppressive activity of the composts seems mainly caused by the beneficial residual microbial population selected during the 224

composting process. However, additional mechanisms, as antifungal activity of phenolic compounds (El-Masry et al., 2002) or induction of resistance in the plant (Paplomatas et al., 2005) could be involved. The results of our investigations indicate that composted olive by- products, besides enhancing the physico-chemical and biological characteristics of soil (Niaounakis & Halvadakis, 2004), have got a high potential in order to control V. dahliae and, perhaps, other soil-borne pathogens in eco-compatible agriculture systems.

Acknowledgements

This research was partially funded by the Molise Region Government (Italy) “Virgin Olive Oil Quality Improvement" (EC Reg. 528/99 – G Action). We thanks Prof. G. Ranalli and Agribiotec srl (Cavezzo, Modena, Italy) for supplying the composts and the Biofungicide TV1, respectively.

References

EL-Masry, M.H., Khalil, A.I., Hassouna, M.S. & Ibrahim, H.A.H. 2002: In situ and in vitro suppressive effect of agricultural composts and their water extracts on some phytopathogenic fungi. – World J. Microbiol. Biotechnol. 18: 551-558. Hawke, M.A. & Lazarovits, G. 1994: Production and manipulation of individual microsclerotia of Verticillium dahliae for use in studies of survival. – Phytopathology 84: 883-890. Jiménez-Díaz, R.M., Tjamos, E.C. & Cirulli, M. 1998: Verticillium wilt of major tree hosts. Olive. – In: A compendium of verticillium wilts in tree species. Eds. Hiemstra and Harris, Ponsen & Looijen, Wageningen, The Netherlands: 13-16. Lima, G., Piedimonte, D., De Curtis, F., Abobaker-Elgelane, A., Nigro, F., D’Onghia, A.M., Alfano, G. & Ranalli, G. 2004: Suppressive effect of cured compost from olive oil by- products towards Verticillium dahliae and other fungal pathogens. – 5th International Sym- posium on Olive Growing, Izmir, Turkiye, Sett. 27– Oct. 2. (in press on Acta Hortic). Niaounakis, M. & Halvadakis, C.P. 2004: Olive Mill Waste Management – Literature Review and Patent Survey. – G. Dardanos Publ., Athens (GR): 430 pp. Nigro, F., Gallone, P., Barham, H., Schena, L., Ippolito, A. & Salerno, M. 2004. Diagnosis of olive verticillium wilt by Real-Time Scorpion PCR. – 5th International Symposium on Olive Growing, Izmir, Turkiye, sett. 27-Oct. 2. (in press on Acta Hortic). Nigro, F., Gallone, P., Romanazzi, G., Schena, L., Ippolito, A. & Salerno, M.G. 2005: Incidence of verticillium wilt on olive in Apulia and genetic diversity of Verticillium dahliae isolates from infected trees. – J. Pl. Pathol 87: 13-23. Paplomatas, E.J., Tjamos, S.E., Malandrakis, A.A., Kafka, A.L. & Zouvelou, S.V. 2005: Evaluation of compost amendments for suppressiveness against verticillium wilt of eggplant and study of mode of action using a novel Arabidopsis pathosystem. – European J. Plant Pathol. 112: 183-189. Ranalli, G., Principi, P., Zucchi, M., da Borso, F., Catalano, L. & Sorlini, C. 2002: Pile composting of two phase centrifuged olive husks: bioindicators of the process. – In: Micro- biology of composting. Eds. Insam, Riddech and Klammer, Springer-Verlag, Berlin, Heidelberg. Shetty, K.G., Subbarao, K.V., Huisman, O.C. & Hubbard, J.C. 2000: Mechanism of broccoli- mediated verticillium wilt reduction in cauliflower disease. – Phytopathology 90: 305-310. Snedecor, G.W. & Cochran, W.G. 1980: Statistical Methods. – The Iowa State University Press, USA. Tjamos, E.C. 2000: Strategies in developing methods and applying techniques for the biological control of Verticillium dahliae. – In: Advances in Verticillium Research and Disease Management. Eds. Tjamos, Rowe, Heale and Fravel,. A.P.S., St. Paul, MN, U.S.A: 249-252. Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 225

Foliar application of phosetyl-al for controlling olive verticilliosis: Realistic goal or false hope?

F. Nigro1, P. Gallone1, F. Palmisano2, P. Sumerano3, A. Ippolito1 1 Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi di Bari, via Amendola 165/A - 70126 Bari, Italy. 2 Centro Ricerca e Sperimentazione in Agricoltura “Basile Caramia”, Via Cisternino, 281 - 70010 Locorotondo (BA), Italy. 3 Consorzio di Difesa e Valorizzazione delle Produzioni Agricole della provincia di Brindisi, via Tor Pisana 96-98 - 72100 Brindisi, Italy.

The results of field treatments to assess the efficacy of foliar application of Phosetyl-Al for controlling verticillium wilt of olive are reported. Field experiments were established on three different olive groves, naturally infected by Verticillium dahliae (Kleb.), in the three years 2001–03. In the first two fields, located at Torre S. Susanna and Taranto (Apulia, Southern Italy) there were 5-year old plants, cv Picholine, grown under an intensive farming system; in the third field, located at Ostuni (Apulia, Southern Italy), there were mature plants (20-year old) of the cv Leccino. On the whole, treatments included two (February and August), three (February, June, and October) and four (February, June, August, and October) applications of Phosetyl-Al (3000 ppm) which were repeated for each year of the trial by using a normal motorized sprayer. In each field there were five replications arranged in a randomized complete block design; in the field located at Torre S. Susanna and Taranto each plot consisted of 80 plants, whereas in the third field (Ostuni) there were six plants per plot. Untreated plants were used as controls. Plants were chosen among those showing wilting symptoms on the 50% of the canopy. Infection was ascertained by traditional isolation procedure and by molecular methods, using a nested Scorpion-PCR protocol. Visual scoring of verticillium wilt severity was done by means of an empirical scale at four months intervals for each plot. To summarize the progress of disease severity, the area under the disease progress curve (AUDPC) was calculated and used to compare the effect of different treatments. Analysis of variance was computed over four years to determine the main effect of each treatment, as well as interactions among them for AUDPC. At two of the three locations, Torre S. Susanna and Taranto, where young plants were grown, four Phosetyl-Al sprays decreased AUDPC by 16.7%, and 22.8%, respectively, as compared with untreated control plants. At the third location (Ostuni), on mature plants, disease severity was significantly more restricted as compared to the untreated control. The application of fungicide (4 sprays) reduced the AUDPC by 44.5%. However, the combined effect treatments/reading date calculated over the four years trial, resulted significant, indicating a general lowering of disease severity also in the untreated control. Therefore, it seems that Phosetyl-Al sprays can speed up the symptoms remission, a natural phenomenon already described in literature. In addition, no differences in the presence of the pathogen in the xylem, as determined by nested Scorpion-PCR and traditional techniques, were observed. Based on our data, Phosety-Al foliar sprays resulted scarcely effective on young plants and insufficient to meet a good control of verticilliosis on mature olive plants.

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Host-derived resistance for biological control of verticillium wilt of olive

C. Colella, C. Miacola, M. Amenduni, M. D’Amico, G. Bubici, M. Cirulli Dipartimento di Biologia e Patologia vegetale, Università degli studi di Bari, Via G. Amendola 165/A, 70126 Bari, Italy.

Fifty seven wild olive accessions collected from the Mediterranean basin were screened under greenhouse conditions for resistance to Verticillium wilt caused by Verticillium dahliae Kleb. Plants were inoculated at thirteen months after their emergence by dipping the root system in a conidial suspension of the fungus (4 x 10 6 conidia/ml) after shaking off the soil from roots and washing them under running water. One defoliating and one non-defoliating V. dahliae isolates, both obtained from diseased plants in southern Italy, were used. Plants of the highly susceptible cv Cima di Mola, frequently used as rootstock in Apulia, were included as control in this experiment. Disease reaction of tested accessions was evaluated on the basis of external symptoms, vascular browning and by calculating the area under disease progress curve (AUDPC). On the basis of AUDPC values and severity of external symptoms, the tested accessions were grouped into four phenotypic groups: highly resistant, moderately resistant, susceptible and highly susceptible. Most accessions showed different levels of resistance/susceptibility to both V. dahliae pathotypes. A minor part was resistant/susceptible to one of the two pathotypes only. Three accessions showed high type resistance to both V. dahliae pathotypes. Forty resistant plants were selected from accessions that had shown the highest levels of resistance. From each of these plants, clones were obtained by in vitro micro-propagation. The M-1 clones were inoculated with the defoliating pathotype using the same procedures adopted to test the original accessions. Ten M-1 clones, showed the high type resistance characteristics of their original mother plants, while the other ones showed different levels of disease severity. This research provided the identification of new olive rootstocks highly resistant to Verticillium wilt which could be included in breeding programmes for resistance of olive to V. dahliae.

This work was supported by the European Commission (Framework Programme 5; Project reference number QLRT 1999-1523). Project “Verticillium wilt in tree species; developing essential elements for integrated and innovative management strategies“.

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Characterization of Colletotrichum species causing olive anthracnose in Italy.

S.O. Cacciola1, G.E. Agosteo2, R. Faedda3, S. Frisullo4, G. Magnano Di San Lio2 1 Dipartimento S.En.Fi.Mi.Zo.,Viale delle Scienze 2, Università di Palermo, 90128 Palermo, Italy. 2 Dipartimento di Agrochimica e Agrobiologia, Università mediterranea di Reggio Calabria, Piazza S. Francesco di Sales 2, 89061 Reggio Calabria, Italy. 3 Dipartimento di Scienze e tecnologie fitosanitarie, Universita di Catania, Via S.Sofia 100, 95125 Catania, Italy. 4 Dipartimento di Scienze Agro-Ambientali, Chimica e Difesa vegetale, Università di Foggia, Via Napoli 25, 71100 Foggia, Italy.

Over 300 Colletotrichum isolates from a wide range of hosts and geographical origins, including 220 isolates from drupes and leaves of olive with symptoms of anthracnose collected in various regions of southern and central Italy, were examined for morphological, cultural and physiological characters as well as for electrophoretic banding patterns of eight mycelial isozymes and RAPD profiles obtained with 16 decamer primers . Most of the isolates had been previously identified as either C. gloeosporioides (Penz.) Penz. & Sacc. or C. acutatum Simmonds. Isolates of other species of Colletotrichum, such as C. musae (Berk. & M. A. Curtis) von Arx, C. coccodes (Wallr.) Hughes and C. circinans (Berk.) Vogl., were included in this study as out-group isolates. Cluster analysis of RAPD and isozyme profiles was perfomed with the UPGMA algorithm and was supported by bootstrap analysis. RAPD and electrophoretic profiles identified the same discrete groups. All the isolates from strawberry produced fusiform conidia, grew slowly on agar-media, showed an optimum growth temperature of about 24 °C, were benomyl-resistant (MIC ≥ 102 µg ml-1) and formed a distinct molecular group (C. acutatum sensu stricto). This group comprised also olive isolates from Portugal and Spain. A group, identified as C. gloeosporioides sensu stricto, comprised isolates from diverse hosts, including olive isolates from various italian regions. Most olive isolates from Sicily were in this group. Other olive isolates from various regions of southern and central Italy were in three separate groups, genetically distinct from C. gloeosporioides sensu stricto, but conformed to a broad concept of this species (i. e. cylindrical conidia, fast growth, optimum growth temperature ≥ 27°C, benomyl MIC ≤ 1 µg ml-1). Olive isolates from regions of southern Italy where olive anthracnose is endemic (i.e. Calabria and Apulia) clustered together and probably represent a species genetically and biologically distinct from both C. acutatum and C. gloeosporioides. Surprisingly, this molecular group included also rhododendron isolates from Italy and northern Europe as well as sweet cherry isolates from Norway, previously identified as C. acutatum by other Authors.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 231

A symbiotic relation found between Pseudomonas savastanoi and Pantoea aglomerans in the knots formed on olive

G. Marchi1, G. Casati1, G. Surico1, A. Sisto2, A. Evidente3 1 Dipartimento di Biotecnologie Agrarie-Patologia Vegetale, Università di Firenze, Piazzale delle Cascine 28, 50144 Firenze, Italy. 2 Istituto di Scienze delle Produzioni Alimentari-CNR, Via Amendola 122/O, 70126 Bari Italy. 3 Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli Federico II, Via Università 100, 8055 Portici, Italy.

We report a case of symbiosis that can be interpreted in different ways (mutualism, com- mensalism, or inquinilism), of Pantoea agglomerans (ex Erwinia herbicola) with Pseudo- monas savastanoi in the knots (or tubercles) formed by the latter on olive. P. agglomerans is known as one of the most common components of the saprophytic prokaryote microflora on both the phylloplane and the rhizoplane of many plant species. Nevertheless, some sets of strains of this bacterium were also described as primary pathogens on some agricultural crops, e.g. the pv. gypsophilae on Gypsophila paniculata, the pv. milletiae on Wisteria sinensis, and the pv. betae on Beta vulgaris, all of which cause galls on their respective hosts, while for other strains of this bacterium it has been supposed that they operate as secondary pathogens in some pathogenic processes, either because they enhance the predisposition of a host to infection, or because they change the virulence of certain plant-pathogenic bacteria. In the present case it was ascertained first of all that P. agglomerans occurs in intact tubercles of olive knot sometimes in even greater numbers that the primary pathogen itself, and that it occurs in a high proportion of tubercles. When some isolates of this bacterium were inoculated on healthy olive, they multiplied in olive tissues and remained vital for a long time but did not lead to the formation of tubercles. By contrast, when P. agglomerans was co- inoculated with P. savastanoi in ratios of 1:1 or 1:100, its multiplication was abundant, and equal or indeed even superior to that of P. savastanoi. Moreover, the tubercles that formed at these co-inoculation sites were larger than those formed by P. savastanoi when inoculated alone. It thus appears that the presence of P. agglomerans at plant sites where tubercles of olive knot are forming and developing hinders, at least in the initial phases of bacterial reproduction, the multiplication of P. savastanoi (possibly as a result of competition for space or for nutrients) but at the same time makes the tubercles larger, so that more space and more nutrients are available for both bacteria. This last could be explained by the fact, ascertained in our laboratory, that the olive isolates of P. agglomerans produce indolacetic acid in culture.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 233-244

Epidemiological study of olive scab in Calabria

Giovanni Enrico Agosteo, Rocco Zappia Department of Agrochemistry and Agrobiology, Mediterranean University of Reggio Calabria, 89061 Gallina – Reggio Calabria, Italy. E-mail: [email protected]

Abstract: Olive scab caused by Spilocaea oleagina (Cast.) Hughes, is a widespread disease in all olive-growing areas of the Mediterranean region. A study was carried out in the years 2000-2001 and 2001-2002 in Calabria (southern Italy) with the aim of clarifying some epidemiological aspects of this disease. Disease incidence and severity were assessed in different olive-growing areas and on different cultivars. Early infections were detected in laboratory according to the method proposed by Loprieno and Tenerini (1959). A different degree of susceptibility to olive scab among cultivars was found. Both intensity and period of maximum incidence of the disease varied in each year and among locations indicating a strong effect of climatic and environmental conditions. In both years results showed that disease incidence increases from late autumn to a maximum in late winter and early spring. Independently on the period of infection, intensive defoliation occurs during spring, with warm weather condition. Results suggest that the treatments to prevent olive scab should be scheduled according to the periods of infection that may vary on the basis of the annual climatic course, with particular regard to rainfall. In southern Italy, with dry autumns and mild winters, seem useful to move the usual autumnal treatments on winter.

Key words: Spilocaea oleagina, Olive leaf spot, Peacock eye disease.

Introduction

Olive scab, also named peacock eye disease, caused by Spilocaea oleagina (Cast.) Hughes, is a major disease of olive in the Mediterranean region. The pathogen develops colonies beneath the cuticle and over the outer wall of the epidermal cells. On the upper leaf surface it produces round spots arranged in concentric circles that vary in size and colour, from brown to yellow and green. The infected leaves fall prematurely and the branches totally bare. Climatic factors have a considerable influence on the development of olive scab. The conidia require sufficient moisture to germinate. A film of water, from rainwater, dew or fog, is necessary to the fungus to begin an infectious process on the leaf surface. High temperatures in the summer restrict the pathogen development inside the leaf. The period of incubation may vary greatly from approximately two weeks, under the most favourable conditions, to months, as for spring infections that are followed by a hot and dry season. The amount of rain and, more importantly, the distribution of rainy days, have a great influence on disease incidence that varies greatly in consequence of local environmental conditions. Sporulation of the scab lesions occurs prevalently during autumn and spring. There are one or two main infection periods, either during autumn and winter (in areas with dry summers and mild winters) or in spring and early summer (in areas with colder winters) or in both seasons (Graniti, 1990). The availability of epidemiological data is considered of great importance to guide growers in chemical control and very useful to define time and frequency of applications. In Calabria, where high susceptible varieties are diffusely grown, olive scab is widespread and numerous applications of copper fungicides are used to control the disease. However the disease has been poorly investigated and no data are available on its epidemiology.

233 234

A two years research program was conducted in Calabria, in the years 2000-01 and 2001- 02, in different locations and on different cultivars, with the aim to define some epidemiological aspects, like disease incidence and severity, time of infections, quantity and time of leaf shedding.

Materials and methods

The disease was monitored on 10 olive cultivars in 13 different locations of 6 important olive growing areas in Calabria (Table 1). Data were collected from four non-treated plants per single location. Disease incidence and severity were detected at intervals of 15-30 days, starting from the new annual vegetation until the next spring. In many places the research interested the same plants for two consecutive years. In the second year of the research (2001- 02), field observations and samplings were stopped earlier than the first one. In some places the evolution of the infections was differentiated between autumnal and spring vegetation. Leaves of comparable age (100 for each plant) were detached at random from the medium- lower parts of differently oriented sides of the trees. Latent infections were detected in laboratory, by dipping the leaves for two minutes in 5% NaOH or KOH hot solution (50- 60°C) according to the method proposed by Loprieno and Tenerini (1959). For young leaves the solution was maintained at room temperature. Disease severity was assessed on the basis of a five levels infection scale, where 0 = no symptoms, 1= 1-2, 2= 3-4, 4= 5-7, 6= >7 spots per single leaf.

Table 1. Areas of investigation and selected cultivars g Areas Locations Cultivars Year 2000-2001 Upper Ionian area Sibari (CS) Cassanese Francavilla (CS) Carolea Crotone Strongoli (KR) Tonda di Strongoli Cirò Marina (KR) Dolce di Rossano Andali (KR) Carolea Rocca Bernarda (KR) Nocellara messinese Gioia Tauro plain Molochio (RC) Ottobratica Molochio (RC) Sinopolese Gioia Tauro (RC) Nocellara messinese Rizziconi (RC) Cassanese Lower Ionian area Ferruzzano (RC) Nocellara del Belice Camini (RC) Grossa di Gerace Year 2001-2002 Upper Ionian area Sibari (CS) Cassanese Francavilla (CS) Carolea Crotone Strongoli (KR) Tonda di Strongoli Cirò Marina (KR) Dolce di Rossano Andali (KR) Carolea Lamezia Terme Maida (CZ) Carolea Maida (CZ) San Benedetto Vibo Valentia Francica (VV) Cassanese Francica (VV) Ottobratica Gioia Tauro plain Molochio (RC) Sinopolese 235

In some locations, to assess the amount and time of defoliation caused by the disease, the state of infections was followed directly in the field on two plants per place. Four differently oriented shoots per plant were selected, marked and periodically assessed for new vegetation, leaves fall and leaf spots of S. oleagina, visible with naked eye. To differentiate any possible cause of defoliation, all the events that may get involved in leaves fall were analogously recorded.

Results

The time course of the early detected percentage of infection on spring leaves is represented in the Figures 1-6 and 7-12, for the years 2000-01 and 2001-02 respectively. The disease progress on the autumnal vegetation, for two selected localities, is reported in the Figures 13 and 14.

100 90 80 70 60

50 40 30 20 10 0

0 0 0 00 /00 0 00 /00 01 01 5/ 6/ 1/ 3/ 4/ /0 /0 /07 /08/00 /09/00 /10/ /1 /12 /01/01 /02/01 /0 /0 8 8 8 8 8 08 0 0 08 08 08 0 0 08 08 08 0 Figure 1. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2000. Sibari (CS) cv Cassanese, (2000-2001). Each value was the mean of four data (4 plants, 100 leaves per plant).

100 90 80 70 60

20 10 0 0 0 /00 1/01 08/05/00 08/06 08/07/00 08/08/0 08/09/00 08/10/0 08/11/00 08/12/00 08/0 08/02/01 08/03/01 08/04/01 08/05/01 Figure 2. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2000. Francavilla (CS) cv Carolea, (2000-2001). Each value was the mean of four data (4 plants, 100 leaves per plant). 236

100 90 80

70 60 Tonda di Strongoli Dolce di Rossano 50 Carolea 40 Nocellara messinese 30

10 0

0 0 0 1 0 0 0 /00 /00 /00 /00 /0 /01 /01 9 0 1 2 2 3 4 /06/ /07/ /08/ /0 8 8 8 8 2 2 2 28/0 28/1 28/1 28/1 28/01/01 2 28/0 28/0 Figure 3. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2000. Area of Crotone: Strongoli (cv Tonda di Strangoli), Cirò Marina (cv Dolce di Rossano), Andali (cv Carolea), Rocca Bernarda (cv Nocellara messinese), (2000-2001). Each value was the mean of four data (4 plants, 100 leaves per plant).

100

60 Nocellara Messinese

50 Cassanese 40

0 0 0 0 0 0 0 0 0 1 1 0 00 0 0 0 00 0 00 0 0 /20 /2 /20 /20 /20 /2 /20 /2 /20 /20 4 6 7 9 1 12 2 3 3/03/20003/0 3/05 3/0 3/0 3/0 3/10 3/1 3/ 3/0 3/0 1 1 1 1 1 13/08/20001 1 1 1 13/01/20011 1

Figure 4. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2000. Gioia Tauro (RC), cv Nocellara messinese; Rizziconi (RC), cv Cassanese, (2000-2001). Each value was the mean of four data (4 plants, 100 leaves per plant).

In general, the disease incidence on spring leaves increased progressively from autumn to the maximum levels in late winter or early spring. In humid areas with mild temperatures in summer, such as the areas of Gioia Tauro and Vibo Valentia, characterized by the presence of very long plants and high foliages covering (“olive wood”), the infections, on the new vegetation of susceptible cultivars, were detected starting in spring and developing during summer (Figures 4, 5, 10, 11). 237

In humid areas (Sibari, Lamezia Terme, Gioia Tauro plain and Vibo Valentia) on susceptible cultivars including Carolea, Cassanese, Sinopolese, Nocellara messinese the disease incidence exceeded values of 50% up to 70-100% (Figures 1, 4, 5, 7, 10, 11, 12). In less humid, dry or windy areas disease incidence values were lower. In this locations maximum values were under 30% on Carolea, the most important cultivar in Calabria (Figures 2, 3, 8, 9). The cvs Ottobratica, San Benedetto and Grossa di Gerace showed, on the contrary, a high resistance to the disease with the maximum values of incidence ranging from 1 to 16% in humid areas (Figures 5, 6, 10).

100 90 80 70 60 Sinopolese 50 Ottobratica 40 30 20 10 0

00 00 01 01 1 /00 7/00 /00 / 0/00 / 2/00 / / /0 /01 06 08 /03 /05 07/ 07/0 07/ 07/09 07/1 07/11 07/1 07/01 07/02 07 07/04/0107 Figure 5. Time course of the percentage of leaf infections by Spilocaea oleagina on the new spring vegetation of the year 2000. Molochio (RC) cvs Ottobratica and Sinopolese, (2000-2001). Each value was the mean of four data (4 plants, 100 leaves per plant).

50 45 40 35 30 Nocellara del Belice 25 Grossa di Gerace 20 15 10

5 0

0 0

/08/0 /09/0 9 9 0 0 09/10/0009/11/0009/12/0009/01/0109/02/0109/03/0109/04/0109/05/0109/06/01 Figure 6. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2000. Ferruzzano (RC), cv Nocellara del Belice; Camini (RC), cv Grossa di Gerace, (2000-2001). Each value was the mean of four data (4 plants, 100 leaves per plant).

238

100 90 80 70 60 50 40 30 20 10 0

1 1 1 1 1 1 1 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 /2 /2 /2 /2 /2 /2 /2 /2 /2 4 5 6 7 8 0 1 2 1 /0 /0 /0 /0 /0 /1 /1 /1 /0 0 0 0 0 0 0 0 0 0 3 3 3 3 3 30/09/20013 3 3 3 Figure 7. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2001. Sibari (CS) cv Cassanese, (2001-2002). Each value was the mean of four data (4 plants, 100 leaves per plant).

1 01 001 001 001 001 00 0 001 001 002 2 2 2 2 /2 2 2 2 /07/ 08 /01/ 30/04/ 30/05/ 30/06/ 30 30/ 30/09/2 30/10/2001 30/11/ 30/12/ 30 Figure 8. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2001. Francavilla (CS) cv Carolea, (2001-2002). Each value was the mean of four data (4 plants, 100 leaves per plant).

Disease incidence was in general lower in 2001-02 than 2000-01, a less humid and rainy year. The distance, between the incidence values recorded in the two consecutive years, varied in relation to cultivars and location. In Francavilla, on “Carolea” (Figures 2 and 8), the percentage of leaf infection was 27% in January 2001 and 4% in the same period of the next year. In Sibari, on “Cassanese”, the percentage of leaf infection was of about 60 and 50 % respectively (Figures 1 and 7). Disease severity values were strictly correlated with the values of disease incidence. On susceptible cultivars the higher values of incidence were accompanied by the maximum 239

values of the severity scale (4-6). On resistant cultivars low values of severity index, ranging from 1 to 2, corresponded to low incidence values.

20 Tonda di Strongoli 15 Dolce di Rossano Carolea 10

1 1 2 01 01 0 0 00 001 001 00 00 002 /2 /2 /2 7/2 2/2 1/2 /06/2 /0 /08 /09 /1 /0 /02 21/05/2 21 21 21 21 21/10/200121/11/200121 21 21 Figure 9. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2001. Strongoli (KR), cv Tonda di Strangoli; Cirò Marina (KR), cv Dolce di Rossano; Andali (Kr), cv Carolea, (2001-2002). Each value was the mean of four data (4 plants, 100 leaves per plant).

100 90 80 70 60 San Benedetto 50 Carolea 40 30 20 10 0

1 2 01 02 001 002 002 /20 2 /20 2 2 9/ 2/ /08/20009 0 /01/200/02 0 4/ 8/ 5/ 20/07/200103/08/200117/08/200131 1 2 12/10/200126/10/200109/11/200123/11/200107/12/200121/12/200104/01/200218 01 1 01/03/ Figure 10. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2001. Maida (CZ) cvs Carolea and San Benedetto (2001-2002). Each value was the mean of four data (4 plants, 100 leaves per plant).

At Sibari, Francavilla and Rizziconi the appearance of new spots was followed directly in the field, on spring and autumnal leaves (Figure 15). At Sibari and Francavilla, on “Cassanese” and “Carolea” respectively, spots appeared prevalently from December to May while in the Gioa Tauro plain, on “Nocellara messinese”, they appeared prevalently in autumn. This may be related to the mild summers favouring the evolution of spring infection. On the new annual vegetation, independently from the periods of leaf infection, defoliation occurs prevalently in spring, with warm weather conditions. In the area of Sibari 240

on “Cassanese”, disease incidence revealed with naked eye began to increase in December and reached a maximum in March (81% of infected leaves). Defoliation began to increase in March. In July, 88% of infected leaves of the annual vegetation was fallen (Figure 16). At Sibari and Francavilla about 80% of defoliation, on “Cassanese” and “Carolea” respectively, occurred in 3-4 months, from March to June (Figure 17).

100 90 80 70 60 Cassanese 50 Ottobratica 40 30 20 10 0

01 01 01 01 02 02 6/01 2/01 04/ 0 08/ 09/ 11/ 1 01/ 02/ 7/ 17/ 17/05/01 17/ 17/07/01 17/ 17/ 17/10/01 17/ 17/ 17/ 1 Figure 11. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2001. Francica (VV) cvs Cassanese and Ottobratica, (2001-2002). Each value was the mean of four data (4 plants, 100 leaves per plant).

100 90 80 70 60 50 40 30 20 10 0

30/04/01 30/05/01 30/06/01 30/07/01 30/08/01 30/09/01 30/10/01 30/11/01 30/12/01 Figyre 12. Time course of the percentage of leaf infection by Spilocaea oleagina on the new spring vegetation of the year 2001. Molochio (RC), cv Sinopolese, (2001-2002). Each value was the mean of four data (4 plants, 100 leaves per plant).

Discussion

Olive scab was confirmed to be a major disease in all olive-growing areas in Calabria, causing a heavy leaf drop on susceptible cultivars. Climatic factors have a great influence on both the 241

incidence and the severity of the disease. The great variability of the Mediterranean climate, with particular reference to rain and temperature, determine an annual variability in disease appearance. The clonal heterogeneity of some cultivars may also contribute to the variability of disease incidence. In both years of investigation, 2000-01 and 2001-02, winter was mild and in 2001 the weather was very dry. Results showed that in dry autumns and mild winters maximum infection occurs in winter or late winter. In humid areas, with mild summer, like the Gioia Tauro and Vibo Valentia areas, infections may progress in summer and the disease first appears in autumn.

100 90 80 70 60 50 40 30 20 10 0 0 0 1 2000 /2000 /2001 10/ 10/200 /11/2000 12 12/200 01/2001 01 02/200 03/2001 0/ 1/ 02/ 22/ 11 01/ 21/ 1 30/ 19/ 1

Figure 13. Time course of the percentage of leaf infection by Spilocaea oleagina on the autumnal vegetation of the year 2000. Sibari (CS) cv Cassanese. Each value was the mean of four data (4 plants, 100 leaves per plant).

100 90 80 70 60 50 40 30 20 10 0

0 0 1 1 1 0 0 0 20 200 1/2000 2/20 2/ 1/ 1/200 3/2001 3/20 /1 /1 /1 /0 /0 /0 02/10/2000 22/10/2000 11 01 21 10 30/0 19/02/2001 11 31 Figure 14. Time course of the percentage of leaf infection by Spilocaea oleagina on the autumnal vegetation of the year 2000. Francavilla (CS) cv Carolea. Each value was the mean of four data (4 plants, 100 leaves per plant).

242

Sibari (CS), cv Cassanese 100 90 80 70 60 Spring leaves 50 Autumnal leaves 40 30 20 10 0 Sept-Nov Dec-Feb Mar-May June-Aug

Francavilla (CS), cv Carolea 100 90 80 70 Spring leaves 60 Autumnal leaves 50 40 30 20 10 0 Sept-Nov Dec-Feb Mar-May June-Aug

Rizziconi (RC), Nocellara messinese 100 90 80 70 60 Spring leaves 50 Autumnal leaves 40 30 20 10

0 Sept-Nov Dec-Feb Mar-May June-Aug

Figure15. Time of appearance of olive scab spots. Percentage of spots detected on spring and autumnal vegetation of the year 2000, according to the period of appearance. Sibari (CS), cv Cassanese; Francavilla (CS), cv Carolea; Rizziconi (RC), cv Nocellara messinese. For each location values are the means of the data obtained from two plants (four shoots per plant).

Independently of the period of infection, intensive defoliation of the annual leaves occurs during spring. On susceptible cultivars, a total defoliation may occur in the first year. Results suggest that the treatments to prevent olive scab should be scheduled according to the periods of infection that in turn depend on annual climatic course, expecially as far as rainfall is concerned. The early detection of infection is useful to guide chemical control. According 243

with our results, in southern Italy, with dries autumns and mild winters, chemical treatments traditionally performed in autumn, should be better make in winter.

incidence defoliation 100 90 80 70 60 50 40 30 20 10 0 0 0 0 0 1 1 1 1 0 0 /0 /0 /0 0 0 0 9 0 3 /0 /1 /11/00 /12/00 /01/01 /0 0 1 0 1 1 1 31/07/ 31/08/ 3 3 3 3 3 28/02/013 30/04/0131/05/0130/06/ 31/07/ 31/08/

Figure 16. Area of Sibari, cv Cassanese. Spring vegetation of the year 2000. Time course of disease incidence (%) and defoliation (%) caused by Olive scab. Values are the means of the data obtained from two plants (four shoots per plant).

Area of Francavilla, cv Carolea 50 45 40 35 30 25 20 15 10 5 0

01 01 01 01 0 2001 0 0 0 2/2 / 4/2 6/2 0 /03/2001 0 /05/2001 /0 /07/2 26/ 18 07/04 27/ 17 06/06/2001 26 16

Area of Sibari (CS), cv Cassanese 50 45 40 35 30 25 20 15 10 5 0

2000 2001 2001 2001 12/ 01/ /02/ 04/ 27/ 27/ 27 27/03/2001 27/ 27/05/2001 27/06/2001 Figure 17. Percentage distribution of defoliation caused by S. oleagina on the annual vegetation of the year 2000. Area of Sibari (CS), cv Cassanese; Area of Francavilla Values are the means of the data obtained from two plants (four shoots per plant). 244

Acknowledgements This work was funded by the Regional Consortium of Olive-growing Associations (Co.R.Ass.Ol.) and by the Consorzio per la Ricerca e le Applicazioni di Tecnologie innovative (CRATI s.c.r.l.). We thank the following agronomists of Co.R.Ass.Ol. involved in field observations and samplings: C. Andiloro, A. Balzetti, D. Barone, L. Cirolla, S. Colosimo, A. Mendicino, B. Paglia, C. Preiato, A. Sergi, G.F. Sirianni.

References

Graniti, A. 1990: Plant diseases in the Mediterranean region. – Phytoparasitica 18(1): 57-65. Loprieno, N. & Tenerini, I. 1959: Metodo per la diagnosi dell'"Occhio di pavone" dell'olivo (Cycloconium oleaginum Cast.). – Phytopath. Z. 34: 385-392.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 245-248

Non-conventional chemical control of olive anthracnose

Giovanni Enrico Agosteo, Luigi Scolaro, Giovanni Previtera Department of Agrochemistry and Agrobiology, Mediterranean University of Reggio Calabria, 89061 Gallina – Reggio Calabria, Italy. E-mail: [email protected]

Abstract: Chemical control of olive anthracnose in the Gioia Tauro plain in Calabria (southern Italy) traditionally requires a high numbers of sprays in autumn. In 2002 and 2003, field trials were conducted on the cvs Cassanese and Ottobratica respectively, with the aim of comparing the traditional copper fungicides with alternative compounds. In the first year, we tested azoxystrobin, heliocuivre (a terpenic formulate of copper hydroxide), sodium bicarbonate and the electrolytic chlorine oxidant amuchina. Sodium bicarbonate and amuchina were also tested in addition with pinolene, a water-emulsifiable organic concentrate obtained from pine resin, to increase both adherence and persistence. In the second year we tested azoxystrobin, trifloxystrobin, heliocuivre and a copper peptidate formulation (Peptiram 5). In both trials a tetra-cupric copper oxychloride formulate was used as reference product. Azoxystrobin proved to be the most effective product in both years. All the compounds tested, with the exception of trifloxystrobin, reduced significantly the incidence of fruit rot. The addition of pinolene enhanced the effectiveness of copper formulates and sodium bicarbonate. The low dosage copper formulates were comparable to copper oxycloride in reducing the incidence of olive anthracnose.

Key words: chemical control, azoxystrobin, sodium bicarbonate, copper fungicides.

Introduction

Anthracnose is the most important olive disease in the Gioia Tauro plain in Calabria (southern Italy), a humid area where olive is grown intensively and environmental conditions favour the epidemic development of the disease (Martelli, 1959; Cacciola et al., 1996). Fruit attacks occur in autumn, with the first rain, when drupes begin to change in colour. Chemical control is traditionally based on copper sprays in autumn, repeated until harvesting. The persistence of fungicides is limited by the high humidity conditions and the effectiveness of the treatments has proved generally to be unsatisfactory. The stop to copper sprays before harvest, coinciding with the period of maximum infection intensity (major susceptibility to the disease of the drupes and most favourable climatic conditions for the rapid development of the disease), cause a disease explosion during drupes ripening. Moreover, recently, was imposed a limitation to the annual amount of copper ions per hectare in organic farming. The aim of the present work was to test the efficacy of more adhesive formulates, new fungicides and natural compounds (to be used also in pre-harvesting) as an alternative to the traditional copper formulates.

Materials and methods

Field trials were carried out in 2002 and 2003 in the Gioia Tauro area, on 10 and 8-years-old trees of the cvs Cassanese and Ottobratica, respectively. A random-blocks experimental design with four replicates per each treatment and three plants per plot was adopted. Treatments of the first and of the second year respectively, dosages of fungicides, and number and date of applications are reported in table 1. A tetra-cupric copper oxychloride formulate

245 246

was used as reference product. Azoxystrobin and trifloxystrobin are Qo1 STAR synthetic fungicides, derivate from strobilurin A, a fungal metabolite with antimicrobial propriety; heliocuivre is a more adhesive liquid formulate of copper hydroxide mixed with terpenic derivatives (pine resin); copper peptidates are new low dosage copper formulates, containing copper ions chelated to aminoacids and peptides. With the last two formulates is possible to reduce the dosage rate of copper ions per hectare. Sodium bicarbonate is a natural mineral compound included in the list of fungicides authorized in organic farming. The electrolytic chlorine oxidant amuchina is a source of active chlorine (analogously to sodium hypochlorite), highly utilized in numerous sanitation systems, including the disinfection of vegetables before eating. Both sodium bicarbonate and the electrolytic chlorine oxidant may be used until harvesting without residue problems. In the first year, pinolene (di-1-p- menthene), a water-emulsifiable organic concentrate obtained from pine resin and used as adhesive to increase treatments persistence was added to some fungicides solutions. Pinolene was tested also alone, as control. High volume sprayings (1000 L ha-1) were made using high- pressure, hand-held spray guns. The effectiveness of the treatments was evaluated as percentage of infected fruits, by random collecting one lot of 100 drupes per plant. Drupes were collected on the 9th December 2002 and on the 15th November 2003, respectively. Disease incidence was determined after two days of incubation of the drupes in humid conditions at room temperature.

Results and discussion

Results are reported in table 2 and 3, for 2002 and 2003, respectively. In 2002 all the fungicides reduced significantly the incidence of the disease. Azoxy- strobin (2 sprays) proved to be the most effective product, also compared to the reference product (3 sprays). The terpenic formulate of copper hydroxide (3 sprays), was as effective as the copper oxycloride. The effectiveness of sodium bicarbonate (3 sprays) was greatly enhanced by the addition of pinolene and comparable to the electrolytic chlorine oxidant and the terpenic formulate of copper hydroxide (2 sprays). The addition of pinolene enhanced also the performance of the copper oxychloride treatment. Conversely it reduced the effectiveness of the electrolytic chlorine oxidant. In 2003 azoxystrobin proved again to be the most effective product in reducing disease incidence. On the contrary the disease incidence on trees treated with trifloxystrobin did not differentiate from the control. All the copper fungicides differentiated from the control and no significant difference was observed among the low dosage copper fungicides and copper oxychloride. The effectiveness of the chemical treatments varied with the susceptibility of the olive cultivars and the climatic conditions favouring the disease spread. In 2002, on “Cassanese”, a medium susceptible cultivar, control of anthracnose was satisfactory or very satisfactory. In 2003, on “Ottobratica”, a highly susceptible cultivar, control was not fully satisfactory, showing that two autumnal sprays were not sufficient. In the present, there are not authorized fungicides alternatives to copper formulates for the control of olive anthracnose in Italy. In previous tests, copper compounds, compared to numerous fungicides, proved to be the most effective products (Pennisi et al., 1993). In the present trials azoxystrobin showed to be more effective than copper compounds. The new low dosage copper formulates were in general comparable to traditionally copper salts in reducing the incidence of fruit rot. The addition of pinolene to copper fungicides and to sodium bicarbonate enhanced the effectiveness of the treatments. Unlike copper salts, sodium bicarbonate and electrolytic chlorine oxidant may be sprayed on ripening drupes within twenty days before harvesting. 247

Table 1. Treatments, fungicides dosages, number and dates of sprays

F.p. concentration F.p. dosages number Active ingredients (%) Formulated products dates of sprays (g hl-1) (Kg ha-1) of sprays

2002 Tetra-cupric copper oxychloride (40) Neoram, Dow Agrosciences 500 5 3 2002-10-15; 2002-11-8, 22 Tetra-cupric copper oxychloride (40) Neoram, Dow Agrosciences 500 5 2 2002-10-15; 2002-11-22 Pinolene (96) Vapor gard, Intrachem 250 2,5 3 2002-10-15; 2002-11-8, 22 Tetra-cupric copper oxychloride (40) + pinolene (96) Neoram, Vapor Gard 500+250 5+2,5 2 2002-10-15; 2002-11-22 Azoxystrobin (22,9) Quadris, Solplant 100 1 2 2002-10-15; 2002-11-22 Sodium bicarbonate Solvay 1000 10 3 2002-10-15; 2002-11-8, 22 Sodium bicarbonate + pinolene Solvay, Vapor gard 1000+250 10+2,5 3 2002-10-15; 2002-11-8, 22 Electrolytic chlorine oxidant (1,1% active chlorine) Amuchina 2000 20 3 2002-10-15; 2002-11-8, 22 Electrolytic chlorine oxidant + pinolene (96) Amuchina, Vapor gard 2000+250 20+2,5 3 2002-10-15; 2002-11-8, 22 Copper hydroxide (40) + terpenic derivatives Heliocuivre, Intrachem 150 1,5 3 2002-10-15; 2002-11-8, 22 Copper hydroxide (40) + terpenic derivatives Heliocuivre, Intrachem 150 1,5 2 2002-10-15; 2002-11-22 Water control / / / / / 2003 Tetra-cupric copper oxychloride (40) Neoram Dow Agrosciences 500 5 2 2003-09-26; 2003-10-24 Azoxystrobin (22,9) Quadris, Solplant 100 1 2 2003-09-26; 2003-10-24 Trifloxystrobin (50) Flint, Bayer 25 0,25 2 2003-09-26; 2003-10-24 Copper hydroxide (40) + terpenic derivatives Heliocuivre, Intrachem 150 1,5 2 2003-09-26; 2003-10-24 Copper peptidates (5) Naturam 5, Bayer 650 6,5 2 2003-09-26; 2003-10-24 Control / / / / / 247 248

Table 2. Effect of treatments on the incidence of olive anthracnose on drupes. Year 2002, cv Cassanese

Number of Incidence Active ingredients sprays (%)* Azoxystrobin 2 2,2 f Copper hydroxide + terpenic derivatives 3 6,9 ef Tetra-cupric copper oxychloride + pinolene 2 7,1 ef Tetra-cupric copper oxychloride 3 7,3 ef Tetra-cupric copper oxychloride 2 8,2 df Sodium bicarbonate + pinolene 3 13,3 de Electrolytic chlorine oxidant (1,1% active chlorine) 3 15,1 de Copper hydroxide + terpenic derivatives 2 15,1 de Electrolytic chlorine oxidant + pinolene 3 20,0 bd Sodium bicarbonate 3 28,2 bc Pinolene 3 30,6 ab Control / 41,5 a *Values followed by the same letters don’t differ significantly (P=0,05)

Table 3. Effect of treatments on incidence of olive anthracnose on drupes. Year 2003, cv Ottobratica

Number of Incidence Active ingredients sprays (%)* Azoxystrobin 2 44,7 c Copper peptidate 2 53,7 bc Copper hydroxide + terpenic derivatives 2 55,2 bc Tetra-cupric copper oxychloride 2 57,1 bc Trifloxystrobin 2 60,1 ab Control / 71,6 a *Values followed by the same letters don’t differ significantly (P=0,05)

Acknowledgements

This work was funded by the Regional Consortium of Olive-growing Associations (Co.R.Ass.Ol.).

References

Cacciola, S.O., Agosteo, G.E. Pane, A. & Magnano di San Lio, G. 1996: Osservazioni sull'epidemiologia dell'antracnosi dell'olivo in Calabria. – Informatore Fitopatologico 6: 27-32. Martelli, G.P. 1959: La lebbra delle olive. Presenza e diffusione in Calabria. – Italia agric. 96: 905-914. Pennisi, A.M., Agosteo, G.E. & Grasso, S. 1993: Chemical control of the olive rot caused by Glomerella cingulata. – Bulletin OEPP/EPPO Bulletin 23: 467-472. Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 249

Control olive powdery mildew (Leveillula taurica) with the use of soft fungicides

V.A. Bourbos, E.A. Barbopoulou NAGREF, Institute of Olive Tree and Subtropical Plants of Chania, Lab. of Plant Pathology and Ecotoxicology of Plant Protection Products, Agrokipio, 73100 Chania, Crete, Greece.

Olive powdery mildew caused by the fungus Leveillula taurica (Lev.) Arnaud. could cause, under favourable conditions, serious damages in young vegetation. Infection is more severe at the leaves of new vegetation of rejuvenated olive trees, young nursery trees and olive leaf- cuttings. This work studies the possibility to control the pathogen with the use of soft fungicides. Wetting sulphur in the dose of 400 g/hl of the commercial product Thiovit 80 WP and sodium bicarbonate in the dose of 300 g/hl with the wetting agent Agral 90 in the dose of 25 ml/hl were used. These products could be also used in organic oliveculture after authorisation from the organization for control and certification of organic products. The fungicide pyrifenox in the dose of 20 ml/hl of the commercial product Dorado 20 EC was used as reference product. The trial took place at rejuvenated olive trees of Koroneiki cultivar. Estimation of the effectiveness was based on the measurement of infected leaves as well as on the percentage of leaf fall. Taking as criterion leaf infection, sulphur as well as sodium bicarbonate controlled, under the conditions of the experiment, the pathogen with an effectiveness ranged from 99.4 to 99.7% that did not differ statistically significant from reference product (99,7%) while infection at the control plots increased at 89,4-91,5%. Regarding the criterion of leaf fall, effectiveness ranged at 97.1% for sulphur and from 98 to 98.1% for sodium bicarbonate.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 251

Phytophthora species associated with root rot of olive in Sicily

S.O. Cacciola1, G. Scarito1, A. Salamone1, A.S. Fodale 2, R. Mulé2, G. Pirajno1, G. Sammarco1 1 Dipartimento S.En.Fi.Mi.Zo., Universitá di Palermo, Viale delle Scienze 2, 90128 Palermo, Italy. 2 C.R.A. – Istituto Sperimentale per l’Olivicoltura, Palermo, Italy.

Phytophthora root and crown rot of olive trees has been recognized as an emerging phytopathological problem in many olive-growing areas in the Mediterranean region, probably as a consequence of the increasing use of irrigation. A survey aimed at determining both the diffusion of root and crown rot in commercial orchards and the Phytophthora species associated to these diseases is being carried on in Sicily. The species of Phytophthora were identified by using traditional morphological as well as biochemical (polyacrylamide gel electrophoresis of total mycelial proteins and isozymes) and molecular (ITS sequences of rDNA) criteria. The following species of Phytophthora were recovered from both young and mature (10- to 12-year-old) olive trees with symptoms of chlorosis, defoliation and wilting: P. inundata, P. megasperma, P. nicotianae, and P. palmivora. The last two species were found associated with root rot of fine roots on both nursery plants and mature trees originated from rooted cuttings. P. palmivora was more common than P. nicotianae. There are other recent reports of this tropical species on olive in southern Italy and Spain. P. megasperma, which has been previously reported in other olive-growing countries including Greece and Spain, has been recovered from roots and basal stem cankers of young plants. All Sicilian isolates of this species were referred to the BHR (Broad Host Range) group on the basis of DNA sequencing. P. inundata, a species formally described only recently, has been found associated with root rot on trees subjected to flooding. This is the first report of P. inundata on olive in Italy, where this species has already been recovered from roots of ornamental palms and peach.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 253-258

Susceptibility of olive genotypes to Pseudomonas savastanoi (Smith)

Nino Iannotta, Donatella Monardo, Maria Elena Noce, Luigi Perri C.R.A. Institute for Olive Growing- 87036 Rende, Cosenza, ITALY

Abstract: Previous studies have found that different olive genotypes have different susceptibility to P. savastanoi. In the present report, we have examined a large number of other olive varieties for their susceptibility to Pseudomonas. The study was performed using a collection of varieties from Mirto in Cosenza (Italy), where all of the genotypes tested were subjected to the same environmental and agronomic conditions. The stage of infection was estimated on the basis of the quantity of tubercles present on branches. The results showed a different behavior among the various cultivars and displayed varying severities of disease. Of the 262 cultivars tested, 15 Italian cultivars were highly damaged; of the 43 non- Italian cultivars, 9 showed extensive damage. Eighty- six Italian varieties showed no signs of infection, while the remaining genotypes were partially damaged.

Key words: genotype, susceptibility, olive, Pseudomonas savastanoi

Introduction

For several years the C.R.A.- Experimental Institute for Olive Growing of Rende in Cosenza has been carrying out a large-scale investigation on the different genotypic responses to the major and widespread parasites of olives. The present study was facilitated by the possibility to observe different cultivars under the same environmental and agronomic conditions using an experimental field in which more than 260 Italian varieties, over 50 foreign varieties, and about 150 several other accessions of olives are cultivated. Within the framework of olive diseases, we have previously observed that many cultivars have different susceptibility to challenge with Pseudomonas savastanoi. In 2002, we published a preliminary report in which the different susceptibility of 125 Italian and 20 foreign cultivars was described. After three years, all the cultivars present in the field were again submitted to screening, thus studying, older and larger plants. During this time, several adverse meteorological took place, including record minimum temperatures, which are well- known to influence the onset of the disease. In the present study, we detail our results on the susceptibility of various genotypes to Pseudomonas savastanoi by evaluation of the tubercles presence on vegetative organs.

Materials and methods

The investigation was carried out in the spring of 2005 in the germplasm conservation field, located at Mirto- Crosia in Cosenza, where plants are cultivated under the same environmental and growing conditions. Five plants are present for each Italian cultivar, while four are present for each non- Italian cultivar. The response to pathogen was evaluated by examining the symptomatology according to the following scale:

1 – tubercles present on less than 20% of branches; 2 – tubercles present on 20% to 50% of branches;

253 254

3 – tubercles present on 50% to 70% of branches; 4 – tubercles present on 70% to 100% of branches. Whether conditions were monitored using a meteorological observation post equipped with electronic sensors.

Results and discussion

In Table 1, the data on the Italian cultivars are detailed.

Table 1. Italian cultivars (P = affected plants/ observed plants; I = total score; % = percentage of infection).

Infection Cultivar Infection Cultivar P I % P I % 1 Abunara 0/4 0 0.00 40 Cellina di Nardo 4/5 38 63.33 2 Aitana 0/4 0 0.00 41 Cellina di Rotello 0/4 0 0.00 3 Americano 1/5 1 1.67 42 Cerasella 3/5 13 21.67 4 Annarea 1/4 1 2.08 43 Cerasuola 5/5 34 56.67 5 Arnasca 4/5 28 46.67 44 Ciciarello 3/5 12 20.00 6 Ascolana dyra 0/5 0 0.00 45 Cima di Melfi 4/5 21 35.00 7 Ascolana semitenera 0/4 0 0.00 46 Cima di Mola 4/5 23 38.33 8 Ascolana tenera 5/4 20 33.33 47 Colombina 0/4 0 0.00 9 Augellina 4/5 15 25.00 48 Coratina 3/5 6 10.00 10 Aurina 0/4 0 0.00 49 Corneglia 0/4 0 0.00 11 Bianchella 1/5 2 3.33 50 Cornia 0/4 0 0.00 12 Bianchera 0/5 0 0.00 51 Corniola 0/4 0 0.00 13 Borgese 4/5 26 43.33 52 Corniolo 0/5 0 0.00 14 Borgiona 2/4 17 35.42 53 Coroncina 1/5 5 8.33 15 Bosana 4/5 12 20.00 54 Correggiolo 0/5 0 0.00 16 Bottoni di Gallo 1/5 1 1.67 55 Corsicana da olio 5/5 42 70.00 17 Brandofino 5/5 39 65.00 56 Crognalegna 4/5 23 38.33 18 Buscionetto 4/5 22 36.67 57 Cucca 3/5 7 11.67 19 Cacaredda 0/4 0 0.00 58 Cucco 4/5 22 36.67 20 Cacaridduni 4/4 34 70.83 59 Dolce Agogia 4/5 11 18.33 21 Caiazzana 1/4 1 2.08 60 Dolce di Andria 0/4 0 0.00 22 Calatina 1/5 1 1.67 61 Dolce di Cassano 3/5 12 20.00 23 Canino 4/5 34 56.67 62 Dritta 3/5 9 15.00 24 Capena 5/5 33 55.00 63 Dritta di Loreto 2/4 3 6.25 25 Capolga 0/4 0 0.00 64 Faresana 1/4 2 4.17 26 Caprina Casalanguida 0/4 0 0.00 65 Favarol 4/5 12 20.00 27 Caprina vastese 0/4 0 0.00 66 Fecciaro 2/5 12 20.00 28 Carboncella 4/5 26 43.33 67 Feligna 5/5 40 66.67 29 Carbonchia 0/4 0 0.00 68 Femminella di Torraca 3/4 7 14.58 30 Cariasina 5/5 14 23.33 69 Fosco 0/4 0 0.00 31 Carmelitana 3/5 9 15.00 70 Frangivento 4/5 21 35.00 32 Carolea 4/5 25 41.67 71 Frantoio 4/5 18 30.00 33 Carpinetana 0/4 0 0.00 72 F.S.17 4/5 15 25.00 34 Casaliva 5/5 34 56.67 73 Gaggiolo 5/5 38 63.33 35 Cassanese 5/5 19 31.67 74 Gentile dell’ Aquila 4/4 14 29.17 36 Castiglionese 3/5 8 13.33 75 Gentile di Chieti 3/5 12 20.00 37 Castricianella rapparina 1/4 1 2.08 76 Gentile di Larino 0/5 0 0.00 38 Cavalieri 0/4 0 0.00 77 Gentile nera Colletorto 0/4 0 0.00 39 Cazzinicchio 4/5 10 16.67 78 Geracese 3/5 9 15.00 255252

Infection Cultivar Infection Cultivar P I % P I % 79 Ghiannara 1/4 1 2.08 131 Nera di Villacidro 2/5 15 25.00 80 Giardino 2/5 4 6.67 132 Nerba 4/4 14 29.17 81 Giarfara 3/4 7 14.58 133 Nocellara del Belice 5/5 24 40.00 82 Giarraffa 4/5 31 51.67 134 Nocellara etnea 5/5 13 21.67 83 Giusta 0/4 0 0.00 135 Nocellara etnea ovale 0/4 0 0.00 84 Gnagnaro 0/4 0 0.00 136 Nocellara messinese 4/5 11 18.33 85 Gragnan 1/5 1 0.00 137 Nociara 4/5 11 18.33 86 Grappolo 0/4 0 0.00 138 Nolca 4/5 17 28.33 87 Grossa di Spagna 2/5 6 10.00 139 Nostrana 3/5 15 25.00 88 Grossa di Venafro 0/4 0 0.00 140 Nostrana campana 1/4 3 6.25 89 Grossale 0/4 0 0.00 141 Nostrana di Brisighella 5/5 30 50.00 90 I/77 0/4 0 0.00 142 Nostale di Fiano r. 4/5 31 51.67 91 Imperiale 3/5 9 15.00 143 Nostrale di Rigali 3/5 24 40.00 92 Intosso 3/5 13 21.67 144 Ogliara 0/4 0 0.00 93 Itrana 4/5 34 56.67 145 Ogliarola barese 4/5 29 48.33 94 Laurina 0/5 0 0.00 146 Ogliarola del Bradano 2/5 15 25.00 95 Lavagnina 0/5 0 0.00 147 Ogliarola del Vulture 5/5 35 58.33 96 Lea 4/5 19 31.67 148 Ogliarola garganica 3/4 11 22.92 97 Leccino 5/5 20 30.00 149 Ogliarola messinese 5/5 20 33.33 98 Leccio del Corno 1/5 3 5.00 150 Ogliarola Montalbano 4/5 36 60.00 99 Lezze 1/5 3 5.00 151 Ogliarola Salentina 2/5 18 30.00 100 Lumiaru 3/4 12 25.00 152 Ogliastro grande 0/4 0 0.00 101 Mafra 4/5 17 30.00 153 Oliastro 4/5 23 38.33 102 Maiatica di Ferrandina 2/5 6 10.00 154 Olivago 3/5 20 33.33 103 Mandanici 1/5 2 3.33 155 Oliva grossa 0/4 0 0.00 104 Mantonica 0/4 0 0.00 156 Olivastra seggionese 0/5 0 0.00 105 Marina 2/5 15 25.00 157 Olivastro Bucchinico 0/4 0 0.00 106 Marina pugliese 0/5 0 0.00 158 Olivastro frentano 2/4 5 10.42 107 Marzio 0/5 0 0.00 159 Olivo da olio 0/4 0 0.00 108 Maurino 2/5 4 6.67 160 Olivo da salare 3/4 11 22.92 109 Mele 4/5 12 20.00 161 Olivo del Mulino 4/5 30 50.00 110 Mignola 5/5 26 43.33 162 Olivo della Madonna 0/5 0 0.00 111 Mignolo 5/5 25 41.67 163 Orbetanna 0/5 0 0.00 112 Minna di vacca 0/4 0 0.00 164 Ortice 0/4 0 0.00 113 Minuta 5/5 37 61.67 165 Ortolana 4/5 23 38.33 114 Minutella 4/5 20 33.33 166 Ottobratica 3/5 7 11.67 115 Monaca 1/4 1 2.08 167 Ottobrina 3/5 25 41.67 116 Mora 0/5 0 0.00 168 Paesana bianca 1/4 4 8.33 117 Moraiolo 5/5 18 30.00 169 Paesana nera 1/4 1 2.08 118 Moraiolo T Corsini 2/5 4 6.67 170 Pampagliosa 0/5 0 0.00 119 Morchiaio 0/4 0 0.00 171 Pasola 5/5 19 31.67 120 Morellona di Grecia 0/4 0 0.00 172 Passulunara 1/5 1 1.67 121 Moresca 5/5 39 65.00 173 Pendolino 5/5 18 30.00 122 Morinello 0/5 0 0.00 174 Pennulara 0/4 0 0.00 123 Nasitana (frutto grosso) 0/5 0 0.00 175 Peppino Leo 1/5 3 5.00 124 Nebba 2/4 18 37.50 176 Peranzana 4/5 26 43.33 125 Nebbia 5/5 25 41.67 177 Perciasacchi 4/4 5 10.42 126 Nebbio di Chieti 0/5 0 0.00 178 Pescarese 5/5 14 23.33 127 Nebbio di Pescara 2/4 5 8.33 179 Piangente 0/5 0 0.00 128 Negrera 1/5 4 6.67 180 Piantone di Falerone 1/5 1 1.67 129 Nera di Cantinelle 4/5 21 35.00 181 Piantone di Moiano 0/5 0 0.00 130 Nera di Gonnos 3/5 15 25.00 182 Pidicuddara 2/5 2 3.33 253256

Infection Cultivar Infection Cultivar P I % P I % 183 Pignola 0/5 0 0.00 223 San Benedetto 0/4 0 0.00 184 Pisciottana 5/5 42 70.00 224 San Felice Acquasparta 4/5 26 43.33 185 Pizz’e carroga 5/5 27 45.00 225 San Francesco 0/5 0 0.00 186 Pizzutella 1/4 5 10.40 226 Santa Caterina 5/5 33 55.00 187 Posola 0/4 0 0.00 227 Sant’ Agatese 4/5 17 28.33 188 Posolella 0/4 0 0.00 228 Sant’ Agostino 4/5 23 38.33 189 Precoce 0/5 0 0.00 229 Santa Maria 0/4 0 0.00 190 Procanica 5/5 33 55.00 230 Santomauro 3/5 22 36.67 191 Provenzale 1/4 1 2.08 231 Sargano 5/5 19 31.67 192 Puntella 0/4 0 0.00 232 Semidana 3/5 13 21.67 193 Racioppa 1/4 1 2.08 233 Sessana 2/4 14 29.17 194 Racioppella 0/5 0 0.00 234 Simona 5/5 50 83.33 195 Raggiala 3/5 12 20.00 235 Sinopolese 4/5 14 23.33 196 Raja 4/5 27 45.00 236 Sirole 4/5 26 43.33 197 Raja sabina 2/5 10 16.67 237 Sperone di gallo 0/5 0 0.00 198 Rajo 2/5 6 10.00 238 Taggiasca 5/5 44 73.33 199 Rastellina 0/5 0 0.00 239 Tendellone 1/5 5 8.33 200 Raza 0.5 0 0.00 240 Termite di Bitetto 4/5 22 36.67 201 Razzo 0/5 0 0.00 241 Terza grande 1/5 3 5.00 202 Razzola 5/5 37 61.67 242 Toccolana 2/5 10 16.67 203 Reale 5/5 30 50.00 243 Tombarello 0/4 0 0.00 204 Remugnana 0/4 0 0.00 244 Tonda di Alife 0/4 0 0.00 205 Resciola di Venafro 0/4 0 0.00 245 Tonda di Cagliari 3/5 9 15.00 206 Riminino 3/5 13 21.67 246 Tonda di Filadelfia 5/5 32 53.33 207 Ritonnella 1/4 1 2.08 247 Tonda di Filogaso 2/5 5 8.33 208 Rizza 4/5 41 68.33 248 Tonda di Strongoli 4/5 19 31.67 209 Rizzitella 1/4 2 4.17 249 Tonda dolce 0/4 0 0.00 210 Romanella molisana 0/5 0 0.00 250 Tonda dolce di 0/4 0 0.00 211 Romanella molisana 1/5 2 3.33 Partanna 212 Rosciola 1/5 2 3.33 251 Tonda iblea 4/5 14 23.33 213 Rosciola coltodino 0/4 0 0.00 252 Tondina 4/5 39 65.00 214 Rosciola di Rotello 0/4 0 0.00 253 Tortiglione 4/5 10 16.67 215 Rosciola laziale 3/5 10 16.67 254 Toscanina 4/5 8 13.33 216 Rossanese 3/5 10 16.67 255 Tunnulidda 0/4 0 0.00 217 Rustica 0/4 0 0.00 256 Uccellara 5/5 23 38.33 218 Saligna 0/4 0 0.00 257 Vallanella 4/5 20 33.33 219 Salvia 4/5 22 36.67 258 Verdello 4/5 11 18.33 220 Salviana 4/5 32 53.33 259 Vicio 0/4 0 0.00 221 Sammartinara 0/4 0 0.00 260 Zaituna 3/5 21 35.00 222 Sammartinenga 0/4 0 0.00 261 Zimbimbo 5/5 28 46.67 262 Zinzifarica 0/5 0 0.00

Among the 262 Italian cultivars observed, 15 showed a high percentage of infection (more than 60%) and 32 showed a percentage of infection ranging from 40% to 60%. Lower values of infection (between 10% and 40%) were observed in 93 varieties, while 86 genotypes had no symptoms of disease. In Table 2 the data obtained for the non- Italian cultivars are shown. As can be easily inferred, 4 of these cultivars showed no sign of attack by the pathogen, 3 were completely affected, 9 displayed a high percentage of infection (more than 60%) and 3 showed intermediate (between 40 and 60%) amount of infection. 257252

Table 2. Non Italian cultivars (P= affected plants/ observed plants; I= total score; %= percentage of infection).

Infection Cultivar Infection Cultivar P I % P I % 1 Arbequina 3/4 7 14.58 23 Manzanilla 4/4 39 81.25 2 Azeteira 1/4 2 4.17 24 Massabi 2/4 7 14.58 3 Bardhi 0/2 0 0.00 25 Megaritki 4/4 12 25.00 4 Barnea 4/4 45 93.75 26 Mixan 2/4 2 4.17 5 Bidh el Hammam 3/4 28 58.33 27 Negrinha (de Freixo) 4/4 44 91.67 6 Bouteillan 4/4 11 22.92 28 Oblica 4/4 12 25.00 7 Chalchidikidis 4/4 18 37.50 29 Picholine 3/4 34 70.83 8 Chetani 0/4 0 0.00 30 Picholine marocaine 3/4 9 18.75 9 Cornicabra 1/4 12 25.00 31 Picual 4/4 6 12.50 10 Drobnica 4/4 48 100.00 32 Plominca 2/4 20 41.67 11 Galegagrada de Serpa 4/4 39 81.25 33 Preveza 2/4 14 29.17 12 Gordal Sevillana 4/4 46 95.83 34 Rosulja 2/4 8 16.67 13 Hoji blanca 0/4 0 0.00 35 Salonenque 0/4 0 0.00 14 Kalamata 2/2 16 33.33 36 Sigoise 4/4 30 62.50 15 Karidolia 4/4 44 91.67 37 Souri 3/4 18 37.50 16 Kokermadh i Berat 2/4 3 6.25 38 Souruni 2/4 8 16.67 17 Konservolia 2/4 16 33.33 39 Tanche 2/4 5 10.42 18 Koroneiki 4/4 48 100.00 40 Vasilikada 4/4 48 100.00 19 Lechin 1/4 1 3.13 41 Verdale 4/4 39 81.25 20 Leucocarpa 4/4 20 41.67 42 Verdelho 4/4 11 22.92 21 Luques 0/2 0 0.00 43 Yacauti 3/4 9 18.75 22 Manaki 1/4 2 4.17

The present study shows that there is a large diversity in the susceptibility of olive cultivars to P. savastanoi. This different susceptibility is evident under the same agro environmental conditions and confirms a different response to the pathogen in relation to the ratio plant/ parasite and to low temperatures during the observation period (2003-2005).

References

Iannotta, N. 2003: La difesa fitosanitaria. – In: Olea Trattato di olivicoltura. ed. Ed agricole- II Sole 24 ore: 393-407. Iannotta, N., Lombardo, N., Monardo, D. & Perri, L. 2002: Prime osservazioni sulla suscettibilita di cultuvar di olivo alla “rogna” (Pseudomonas savastanoi). – Atti Conv. Int. Olivicoltura, Spoleto: 483-488. Lavermicocca, P., Surico, G., Varvaro, L. & Babelegoto, N.M. 1987: Attivita fitormonica, criogena e antimicrobica dei batteri epifiti dell’oleandro. – Phitop. medit. 26: 65-72. Prota, U. 1996: Le malattie dell’ olivo e relativa difesa. – Atti Conv. „L’olivicoltura Medi- terranea“, Cosenza, 451-471. Surico, G. 1998: Malattie crittogamiche dell’olivo: Epidemiologia e prospettive di lotta. – Int. Course on Olive Growing. Scandicci (FI): 231-237. Varvaro, L. & Surico, G. 1978: Comportamento di diverse cultivar d’ olivo (Olea europaea L.) alla inoculazione artificiale con Pseudomonas savastanoi (Smith). – Phytop. Medit. 17: 174-177. 252258

Varvaro, L. & Balestra, G.M. 1993: Alcune considerazioni sulla presenza di popolazioni batteriche patogene e saprofite sul filloplano dell’olivo. – Atti Conv. “Tecniche, norme e qualita in olivicoltura”, Potenza. Varvaro, L. & Martelia, L. 1993: Virulent and avirulent isolates of Pseudomonas syringae sub. savastanoi as colonizer of olive leaves: evaluation of possible biological control of the olive knot pathogen. – Bull. OEPP/EPPO 23: 423-427. Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 259

Detection of Verticillium dahliae in irrigation water

E. Rodrìguez, M. Campos, M.L. Fernández, J.A. Ocampo, J.M. Garcìa-Garrido Estación Experimental del Zaidìn. Consejo Superior de Investigaciones Cientìficas. C/ Profesor Albareda, 1. 18008. Granada, Spain.

The surface of irrigation olive orchards have extended considerably in the last years. The spread and increase of Verticillium wilt (Verticillium dahliae Kleb.) in olive crop is associated, among other causes, to the transformation of great surfaces of unirrigated land into irrigated one. The relationship between watering and the increase of Verticillium wilt in soil is well documented; nevertheless the dispersion of the pathogen by water is not well known. Nowadays our studies about the incidence of Verticillium wilt in olive crop in southern Spain point to water as an effective source of dissemination of the disease. Therefore the objective of this work is to detect and quantify the propagules of the pathogen in irrigation water in olive grove. For this purpose water samples were collected from rivers and drippers to be assayed for V. dahliae content using a nested PCR assay. This PCR assay uses sets of primer pairs that produce specific markers for the cotton-defoliating (D) and nondefoliating (ND) pathotypes of the pathogen. The results have shown that nested PCR assay is an accurate procedure for detecting the fungus in irrigation water.

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Advanced IPM Strategies in Olive Groves

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 263-269

Olive fruit fly biology and cultural control practices in California

Victoria Y. Yokoyama, Gina T. Miller U.S. Department of Agriculture, Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, 9611 S. Riverbend Ave., Parlier, CA 93648

Abstract: Olive fruit fly, Bactrocera oleae (Gmelin), first found in California, USA in 1998 was investigated in laboratory and field studies. Mortality of 1-5, 6-8, 9-11, and 12-14 d-old immature insects in olives was 19-75, 13-58, 5-27, and 0-7% when exposed to 15oC and 65% relative humidity (RH), and was 14-31, 8-32, 16-38, 4-22% when exposed to 25oC and 35% RH, respectively. Mortality decreased with an increase in age except for 9-11 d-old larvae exposed to 25oC and 35% RH and 6-8 d-old larvae exposed to 15°C and 65% RH. Mortality was 100% in all immature stages in fruit exposed to 5°C and 85% RH and 35°C and 25% RH. The pre-ovipositional period for adult females was 13.0 ± 4.0 d; peak oviposition occurred at 19.7 ± 1.8 d; and, egg laying ended after 63.7 ± 3.8 d (mean ± SEM) at 23°C. Olive fruit fly completed development from the egg to the adult stage from fruit with a mean volume of 0.17 ± 0.01 cm³ (mean ± SEM). The number of adults trapped in baited yellow panel traps with male attractant was higher in olive trees with irrigation water at the base (39.9 ± 8.7 adults per trap per week) than in olive trees without irrigation water (27.7 ± 6.4 adults per trap per week) (mean ± SEM) in the absence of fruit in the canopy. The highest numbers of adults were collected between 2 and 9 October. The daily mean temperature (≈15°C) and relative humidity (≈ 74%) was similar in trees with and without water at the base. Percentage mortality of olive fruit fly 3rd instars was greater than young (0-4 d-old) and old (9-12 d-old) pupae after immersion in water and sand for 1-5 d, and young pupae were in general more susceptible than old pupae.

Key words: biology, survival, sanitation, irrigation

Introduction

Olive fruit fly, Bactrocera oleae (Gmelin), was discovered in Los Angeles, California, U.S.A. in 1998 (Rice, 2000). High populations of olive fruit fly occur in the coastal areas of the state but only low numbers have been detected in the San Joaquin Valley of California, the center of canned olive, Olea europaea, production in the U.S. Quarantine strategies to mitigate pest populations in harvested fruit transported to processing plants were developed by Yokoyama and Miller (2004), and other methods of control have been investigated by Yokoyama et al., (2004) including attract and kill traps and biological control. The effect of climatic factors such as temperature and relative humidity on olive fruit fly mortality needs further investigation in relation to the geographical location of olive production in the state. Furthermore, olive fruit fly ovipositional behavior and cultural control practices such as the effect of orchard irrigation on survival of immatures needs additional study.

Materials and methods

Mortality of immatures by temperature and humidity Olives were placed in cages with olive fruit fly adults of varying ages that were reared from olives in the laboratory. The adults oviposited in the fruit for 1-3 d. The infested fruit was placed on top of rigid co-polymer plastic mesh (7.0 mm openings) suspended inside a 739 ml

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plastic container (about 15 cm wide by 15 cm long by 5 cm deep). The center of the lid of the container was removed, and nylon organdy cloth was held in place over the top with the outer lid band. Thirty infested olives were placed into each container, and held at about 21ºC for periods of 1 to 14 d. After the appropriate time period, the infested fruit was placed in environmental chambers (Model E32560, Lab-Line, Melrose Park, IL) for the following test temperatures, relative humidities (RH), and durations: 5ºC and 85% RH, 7-8 wks; 15ºC and 65% RH, 5-6 wks; 25ºC and 35% RH, 5-6 wks; and, 35ºC and 25% RH, 3 wks. Each container of infested fruit from 1 through 14 days-old was considered a replicate and three replicates were used for each temperature and relative humidity tested. A control container with the same infested fruit as the test container was prepared at the same time and placed in an environmental chamber at 21ºC and 60% RH. The instar present in the fruit at the time of exposure to each temperature and humidity was determined by comparing the age of the immature stage to those reared in fruit exposed to oviposition and held at 21ºC and 60% RH. Sixty infested fruit were dissected each day from 5 to 15 d and the larval stages measured. Larvae ≤1.5, 2-2.5, and ≥3.0 mm were considered as 1st, 2nd, and 3rd instars, respectively. At the end of the 3-8 wk exposure period, the containers were held at about 23ºC for three weeks. The total number of adults that emerged in each container was recorded and reported as the mean ± SEM of 4-9 replicates. The mortality of each life stage in each container was based on adult emergence in the control container and only the data from replicates with a control that resulted in ≥10 adults were used. Percentage mortality was calculated by dividing the number of adults in the test container by the number of adults in the control container at the end of the test multiplying by 100, and subtracting the product from 100. Percentage mortality for each life stage was compared between each temperature and humidity tested with a t-test and the response to each temperature and relative humidity tested was compared among the life stages using a one way ANOVA and Tukey’s multiple comparison test (GraphPad Software, 2004).

Female ovipositional period Newly emerged olive fruit fly adults that were reared from olives in the laboratory were placed in a screened cage (30.5 cm wide by 30.5 cm long by 30.5 cm high) (about 8-12 females and 12-19 males per cage) with five mature green olives. Three cages were prepared and each cage was considered a replicate. The fruit was removed from the cage and replaced with new olives every 1-4 d. The fruit exposed to oviposition was inspected for ovipositional sites. Each ovipositional site was dissected and inspected for eggs. The total number of eggs per 1-4 d per cage were recorded and reported in a figure as the mean per five olives for 1-6 d periods after adult emergence.

Development in immature fruit Immature fruit was harvested from ornamental olive trees located east of the airport in Los Angeles, California. The trees were infested with an established olive fruit fly population. Three replicates of 25 fruit were measured between the stem and blossom end (height) and across the greatest width of the cross section (width). The estimated volume of each fruit was calculated using the formula for the volume of an elliptical spheroid, where volume = 1/6 times height times diameter2 (Mutschler et al., 1986). Three replicates of olives (99-100) including the measured olives were placed on top of rigid plastic mesh inside a 739 ml plastic container (about 15 cm wide by 15 cm long by 5 cm deep), and the top covered with nylon organdy cloth secured with the lid band. The olives were held at about 23°C for 50 d. Calculated fruit volume (cm3) and total number of pupae and adults that emerged from the immature fruit were reported as the mean ± SEM. 265

Field irrigation and trapped adults An orchard with Mission type olives used for oil in Arroyo Grande, California was used to study the effect of standing water on presence of olive fruit fly adults. A circular basin with a bank on the outer edge about 1.2 m from the trunk was trenched around the base of the tree and lined with polyethylene film (0.15 mm). The basin was filled with water for 5 trees with fruit and 4 trees without fruit randomly located in the orchard. For control trees without water at the base, 3 trees were randomly selected with fruit and 4 trees were selected without fruit. Two temperature loggers (Model XTI08-5+37, Intermountain Environmental, Logan, Utah) with external thermistors (Model TMC6-1T, Intermountain Environmental, Logan, Utah) on extension cables (1.8 m long) and two humidity loggers (Model SRHA08, Inter- mountain Environmental, Logan, Utah) were placed at random in two separate trees in each test with water and either fruit or no fruit, and each of the controls with no water and either fruit or no fruit. The weekly means for temperature and humidity recorded every two minutes by each logger were reported as the mean ± SEM and compared between the trees with and without water or fruit with a t-test. A clear plastic packet (10.5 cm wide by 10.5 cm high) of ammonium bicarbonate bait (15-20 g) (Vioryl, Athens-Lamia, Greece) was pierced three times with a straight needle at the top and stapled lengthwise to the vertical edge of the single-fold Pherocon ® AM trap (Trécé, Adair, Oklahoma). The trap was folded in half with the bait packet on the inside and loosely fastened by the bottom corner tab and a wire hanger at the top center, which allowed complete ventilation through the top, side, and bottom of the trap. Pheromone (1,7-dioxaspiro[5,5] undecane, 80 mg) (Vioryl, Athens-Lamia, Greece) in a plastic dispenser (1.7 cm wide by 4.8 cm long) was pierced on the end with a straight needle (1.2 mm diameter) to open the dispenser, and was attached to the plastic coated wire used to hang the trap. The trap was suspended in a shaded area of the canopy of test trees with and without water at the base or fruit. The traps were collected weekly from 4 September to 30 October 2002. The total number of olive fruit fly adults in each trap were counted and reported as the mean ± SEM of 9 collection periods of 3-5 traps. Mean number of adults trapped in trees with or without water at the base, or with and without fruit, throughout the trapping period were compared using a one way ANOVA and Tukey’s multiple comparison test (GraphPad Software, 2004).

Mortality of immatures by immersion About 100 ml sand (30 grit) was placed in a 400 ml glass beaker and saturated with de- ionized water. Three olive fruit fly life stages, either late 3rd instars, 0-4 d-old pupae, or 9-12 d-old pupae that had emerged from infested olives were placed on top of the sand. The immature stages were covered with 50 ml of sand. Filter paper (5.5 cm diameter) was placed on top of the sand and water was added to a total volume of 175 ml and the filter paper removed. The beakers were held at 23°C for daily intervals from 1-5 days. One control beaker without water, and three replicate beakers with water were prepared for each of the three immature stages (14-150 insects) tested at each duration of exposure. At the end of each immersion period the immature stages were collected by passing the contents of the beaker through a sieve with 1.0 mm openings. The immature stages were placed in plastic petri dishes (100 mm diameter by 15 mm high) with a water moistened filter paper (90 mm diameter) placed at the bottom. The petri dishes were held at about 23°C until adult emergence. The total number of adults was counted and the percentage mortality was calculated by dividing the mean number of adults in each treatment by the number of adults in the control and subtracting from 1 and multiplying by 100. The percentage mortality among each duration of immersion of each immature stage were compared using a one way ANOVA and Tukey’s multiple comparison test (GraphPad Software, 2004). 266

Results and discussion

Mortality of immatures by temperature and humidity The duration of olive fruit fly egg, 1st, 2nd, and 3rd instars was 1-5, 6-8, 9-11, and 12-14 d based on larval size when reared at 21ºC. The number of adults that emerged from each replicate 1-14 d after oviposition for which the corresponding control had at least 10 adults emerge was combined with replicates of similar durations to calculate percentage mortality for each life stage. Exposure to 5ºC and 85% RH, or 35ºC and 25% RH caused 100% mortality of the egg and three larval instars with 839 and 831 adults emerging from the controls, respectively. These observations are consistent with Kapatos & Fletcher (1984) who showed that high temperatures were related to high olive fruit fly mortality. Tsitsipis (1980) found that constant temperatures in the range of 12.5 to 30ºC allowed larval and pupal development, but many adults did not emerge completely from the puparia. Mortality of eggs, 1st, 2nd, and 3rd instars in olives was 19-75, 13-58, 5-27, and 0-7% when exposed to 15 oC and 65% RH, and was 14-31, 8-32, 16-38, 4-22% when exposed to 25oC and 35% RH, respectively (Table 1). No significant differences were found in the response of each life stage between 15ºC and 25ºC. However, the 3rd instar was significantly (P < 0.05) more tolerant of exposure to 15ºC and 65% RH than the 1st instar. Mean percentage mortality decreased with an increase in age except for 2nd instars exposed to 25oC and 35% RH, and 1st instars at 15ºC and 65% RH. Tsitsipis (1977) reported that optimum temperature for egg development was 27.5ºC. In our investigation, egg mortality was highly variable and ranged from 0 to 61% in four replicates at 25ºC. High egg mortality was probably caused by exposure to low humidity (35% RH) in the tests at 25ºC. Tsitsipis & Abatzis (1980) showed that no eggs hatched below 90% RH at constant humidities and the lower the humidity and the longer the exposure, the lower the egg hatch. Mortality of the immature stages observed in our study may have been caused in part by transferring each life stage from rearing conditions at 21ºC into the temperature and humidity conditions of each test. An abrupt change in environmental conditions may have been disruptive to normal development.

Table 1. Percentage mortality (mean ± SEM) of olive fruit fly eggs, and 1st -3rd instars (4-9 replicates of 10-85 insects) reared to each life stage at 21ºC and exposed to 15ºC and 65% relative humidity (RH) or 25ºC and 35% RH.

Temp. % RH Life Stage Age (d) % Mortality (ºC) 15 65 Egg 1-5 32.9 ± 16.2 1st Instar 6-8 41.6 ± 9.5 2nd Instar 9-11 16.5 ± 7.5 3rd Instar 12-14 3.5 ± 2.8 25 35 Egg 1-5 30.6 ± 12.4 1st Instar 6-8 22.9 ± 8.2 2nd Instar 9-11 29.7 ± 5.7 3rd Instar 12-14 14.6 ± 4.3

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Female ovipositional period Olive fruit fly females laid the first eggs 13.0 ± 4.0 d, the highest number of eggs 19.7 ± 1.8 d, and the last eggs 63.7 ± 3.8 d (mean ± SEM) after emergence from the pupal stage. The mean number of eggs laid per five olives per 1-6 d periods for 8-12 females are shown in Figure 1. The interval of the pre-ovipositional period is about two weeks from the time of female adult emergence from the pupae to first egg laying. The rapid increase in numbers of eggs laid shortly after the pre-ovipositional period suggests that control measures should be initiated shortly after detection of the first adults to prevent establishment of a new pest population. The long period of ovipositional activity allows the pest to infest olives for an extended duration and supports the development of multiple generations during the growing season. Under these conditions the pest population can rapidly develop into damaging numbers. However, other physical and biological factors such as weather and natural enemies may help suppress population growth.

Mean 20 five olives no. eggsper 10

0 0 10 20 30 40 50 60 70 80 Female age (days)

Figure 1. Ovipositional period for newly emerged olive fruit fly adults at 23ºC.

Development in immature fruit The mean ± SEM diameter, length, and volume of fruit collected from trees infested with olive fruit fly was 1.10 ± 0.02 cm, 0.82 ± 0.01 cm, and 0.17 ± 0.01 cm³, respectively. The mean ± SEM number of adults and pupae that emerged from the infested fruit was 50.7 ± 1.4, and 1.3 ± 0.3, respectively. Dominici et al. (1986) showed that fruit weight was the most important attribute statistically related to olive fruit fly oviposition and the probability of more than one ovipositional site per fruit was dependent on weight and markedly so during fruit ripening. Sharaf (1980) reported that olive fruit fly oviposition began when olives attained the size of peas in June. In our investigation, the eggs and larval stages that developed from fruit with a mean volume of 0.17 cm³ successfully completed development to adults. The data indicates that immature fruit is susceptible to olive fruit fly infestation early in the growing season and perhaps shortly after bloom. Emergence of adults during the bloom period will produce gravid adults within two weeks (Figure 1) and the small fruit can be utilized for the F1 populations. Protection of young fruit should be considered in areas of high pest populations.

Field irrigation and trapped adults The number of olive fruit fly adults trapped in baited yellow panel traps with male attractant were higher in olive trees with water (39.9 ± 8.7 adults per trap per week) than in olive trees 268

without water at the base (27.7 ± 6.4 adults per trap per week) (mean ± SEM) without fruit in the canopy (Table 2). No significant differences were found in total adult captures throughout the trapping period among trees with and without fruit or water at the base. The mean number of adults captured on 4 September was less than all other trap dates. The highest populations were collected between 2 and 9 October. The daily mean temperature (≈15°C) and relative humidity (≈74%) was not significantly different between trees with and without fruit and between trees with and without water at the base. Irrigation in olive orchards in California varies among growers and regions. The data suggests that the availability of water may affect the number of adults found in the orchard. In our study the total number of olive fruit fly adults captured throughout the season was lower in trees without fruit and water than other test conditions. Such observations suggest that irrigation methods that minimize the availability of water and good orchard sanitation such as removing non-harvested fruit are good cultural practices to help control the pest.

Table 2. Mean (± SEM) temperature and percentage relative humidity (7-14 replicates, 12 September to 30 October 2002), and number of olive fruit fly adults trapped in yellow sticky traps (9 collection periods of 3-5 traps, 4 September to 30 October 2002) in olive trees with and without fruit in the canopy or water in a basin at the base of the tree

Treatment Total no. Temp. (ºC) % RH Fruit Water adults Yes Yes 15.4 ± 0.6 73.3 ± 2.9 35.0 ± 7.4 No Yes 15.0 ± 0.6 74.4 ± 3.2 39.9 ± 8.7 Yes No 15.0 ± 0.6 71.6 ± 4.5 39.4 ± 6.4 No No 15.0 ± 0.6 75.0 ± 3.5 27.7 ± 6.4

Mortality of immatures by immersion Percentage mortality of olive fruit fly 3rd instars was greater than young (0-4 d-old) and old (9-12 d-old) pupae after immersion in water and sand for 1-5 d (Table 3). After immersion for 2 d, the percentage mortality among all life stages was significantly different. In general, the larvae were more susceptible to immersion than the pupae. Young pupae were in general more susceptible than old pupae to immersion at all durations except after 5 d. Significantly higher mortality occurred after 3 d and 4 d of immersion for young and old pupae, respectively.

Table 3. Percentage mortality of olive fruit fly late 3rd instars, and young and old pupae immersed in water saturated sand for 1-5 d based on 3 replicates of 9-142 adults in controls.

% Mortality Lifestage Age (d) Immersion (d) 1 2 3 4 5 3rd Instar 13-15 84.0 66.7 98.8 100.0 100.0 Pupae 0-4 50.0 77.2 98.0 97.1 66.7 Pupae 9-12 1.6 7.7 1.0 74.8 83.0 269

The water immersion tests were developed to simulate potential control using flood irrigation in orchards. However, our findings show that the presence of water near olive trees without fruit in the canopy, may increase adult olive fruit fly populations (Table 2) and may offset any beneficial effect of the flood irrigation control method. Periods of high rainfall in which the soil in the orchard may become saturated with water, especially during winter when adults are not present, may have deleterious effects on any immature stage on or in the ground. Under these conditions water saturated soil will help reduce overwintering populations in the orchard and the resultant emergence of adults in the spring.

Acknowledgements

We are grateful to Gail E. Sergent and Donal Patrick Dwyer, USDA, ARS, SJVASC, Parlier, California for assistance with this project; and, to Gloria Medley, Arroyo Grande, California for use of her orchard to accomplish the field irrigation study.

References

Dominici, M., Pucci, C. & Montanari, G.E. 1986: Dacus oleae (Gmel.) ovipositing in olive drupes (Diptera, Tephrytidae). – J. Appl. Ent. 101: 111-120. GraphPad Software. 2004: GraphPad Prism, Version 4.02. – GraphPad Software, San Diego, California. Kapatos, E.T. & Fletcher, B.S. 1984: The phenology of the olive fly, Dacus oleae (Gmel.) (Diptera, Tephritidae), in Corfu. – Z. angew. Ent. 97: 360-370. Rice, R.E. 2000: Bionomics of the olive fruit fly Bactrocera (Dacus) oleae. – Univ. California Coop. Ext., UC Plant Protection Quarterly 10: 1-5. Sharaf, N.S. 1980: Life history of the olive fruit fly, Dacus oleae Gmel. (Diptera: Tephritidae), and its damage to olive fruits in Tripolitania. – Z. angew. Ent. 89: 390-400. Tsitsipis, J.A. 1977: Effect of constant temperatures on the eggs of the olive fruit fly, Dacus oleae (Diptera, Tephritidae). – Ann. Zool. Ecol. Anim. 9: 133-139. Tsitsipis, J.A. 1980: Effect of constant temperatures on larval and pupal development of olive fruit flies reared on artificial diet. – Environ. Entomol. 9: 764-768. Tsitsipis, J.A. & Abatzis, C. 1980: Relative humidity effects, at 20º, on eggs of the olive fruit fly, Dacus oleae (Diptera: Tephritidae), reared on artificial diet. – Entomol. Exp. Appl. 28(1): 92-99. Yokoyama, V.Y. & Miller, G.T. 2004: Quarantine strategies for olive fruit fly (Diptera: Tephritidae): Low temperature storage, brine, and host relations. – J. Econ. Entomol. 97: 1249-1253. Yokoyama, V.Y., Miller, G.T. & Sivinski, J. 2004: Quarantine control strategies for olive fruit fly in California. – Proceedings of the 6th International Fruit Fly Symposium 6-10 May 2002, Stellenbosch, South Africa, pp. 241-244.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 271

Studies towards an enhanced food attractant for fruit flies, especially for the olive fruit fly Bactrocera oleae (Gmelin)

A. Gally, M. Vamvakias, N. Ragoussis VIORYL Chemical & Agricultural Industry, Research S.A., 28th Km Athens-Lamia Nat. Road, 190 14 Afidnes, Greece.

Hydrolyzed vegetable proteins are the most common lures used in large scale programs for the management of various fruit flies, especially for the olive fruit fly Bactrocera oleae (Gmelin) and Mediterranean fruit fly Ceratitis capitata (Weidemann). Most of the commercial lures are produced by chemical or enzymatic hydrolysis of corn or soy gluten, (NuLure, Buminal, Dacus Bait etc) but also various other vegetables proteins are used. Among the volatiles present in proteinaceous lures ammonia appears to play an important role in attracting the fruit flies. It has been reported that raising the pH of the standard lure preparation, the attractiveness of the product increases significantly. The increase of the attractiveness of the lures at higher pH, is not solely attributed to the corresponding increase in the ammonia release. Some other volatiles released on basification are also involved in the increase of the attractiveness of the lures. In the search for more effective lures several papers reporting studies for the characterization of the volatile components of the proteinaceous lures have been published. The aim of all these studies was the identification of the volatiles that might be responsible for the attractiveness of the hydrolyzed proteins and potentially could be added to the commercial products to boost their efficiency or to be the base for the development of new more efficient lures. In the present work, a) very mild techniques for the isolation of the volatiles are used in order to avoid the formation of artefacts and the decomposition of the biologically active compounds and b) the evaluation of the attractiveness of the identified compounds is made in combination with solid ammonium bicarbonate as Fa source of ammonia. The commercial lures analysed during the present study are a) Dacus bait and Alma Dacus from the Greek market, b) Buminal from Italian and Spanish market and c) Mazoferm (corn step liquor) in liquid and solid form mainly used in USA and South America for the attraction of other fruit flies. Several new compounds have been identified for the first time in the above products. The main future of all the analyses was the presence in high percentage of various pyrazines and sulphur compounds. New solid dispensers were then prepared using compressed solid ammonium bicarbonate in which the new compounds were incorporated by a special technique. The effectiveness of these dispensers to attract olive fruit flies (Bactrocera oleae) was evaluated in an olive orchard 40 Km in the North of Athens using a) Yellow sticky panels (20 X 30 cm) loaded with the above dispensers and b) Dry yellow bottom MacPhail traps, loaded with one DDVP dispenser and one new dispenser. As control the same type of traps loaded with only ammonium bicarbonate dispenser, were used. Results on the use of the new dispensers as attractants for the olive fruit fly (Bactrocera oleae) will be presented in details.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 273-275

New technology for auto-dissemination of pheromones and pesticides: potential for control of olive fly and olive moth

Philip Howse Exosect Ltd., 2 Venture Road, Chilworth Science Park, Southampton SO16 7NP, UK, E-mail:[email protected];

Abstract: ExoSex technology utilises inert particles of materials that have the ability to adhere to the arthropod cuticle. The ExoSex AutoconfusionTM system has been developed as an insect control method that differs from all other mating disruption systems in contaminating the target pest with electrostatically chargeable powder formulated with pheromone. This technique can be used for control of most moth pests. The ExoLureTM system utilises adhesive particles as carriers for synthetic or biological pesticides and can be used as a lure & kill technique for insect pest control. Lure & kill systems using slow-acting insecticides or mycopathogens have proved efficent in control of tephritid fruit flies including Ceratitis capitata and Bactrocera species.

Introduction

The use of charged particle technology for insect pest control was first conceived in 1992, and details published in a Patent application (UK Patent application No. EP0650322, P.E.Howse). Preliminary trials were carried out using a lure and kill technique against Cydia pomonella and tephritid fruit flies using a pheromone lure to attract insects into dispensers and electrostatically charged powder formulated with slow-acting insecticide as a killing agent (Howse & Underwood, 2000). The latter was spread by contact to non-contaminated insects. Extensive field trials have been carried out on control of various Lepidoptera using female sex pheromone as the sole active ingredient. Having proven the efficacy of the technique, regulatory approval has been granted for use against certain species in the USA and Britain, and further applications are in progress. One of the first applications to be explored was the use of electrostatically chargeable particles (EntostatTM)as carriers for slow-acting insecticides. Recent research and development has led to the use of magnetic particles (EntomagTM) which also adhere strongly to the insect cuticle. The success of the technology suggests other applications that are now under investigation. These include methods for the dissemination of biopathogens from bait stations and, coupled with this, the use of sterile insects as carriers for semiochemicals or biopathogen (Chandler & Howse, 2005).

Autoconfusion for mating disruption The Exosex AutoconfusionTM technique utilises particles of inert materials that have the ability to adhere to the arthropod cuticle. The technique has now been successfully evaluated in the field against a range of lepidopteran species, including Cydia pomonella, Lobesia botrana, Lymantria dispar, Euproctis chrysorrhea and Chilo suppressalis. Auto-Confusion TM acts by attracting the target pest to dispensers in which they contaminate themselves with electrostatically charged powder formulated with semiochemicals. The amount of pheromone used per hectare is of the order of milligrams, similar to quantities used in conventional monitoring traps and the number of Exosex dispensers used is only 15 - 30 per hectare

273 274

Male moths are attracted to the Exosex dispenser, which contains Entostat powder and synthetic female pheromone. As a result of powder adhering to the antennae and other parts of the body, pheromone receptors become habituated to pheromone. Males then leave the dispenser having picked up Entostat powder and female pheromone. In addition to habituation raising the threshold for perception of the female pheromone, there are other phenomena that contribute to mating disruption. These include false pheromone trails produced by the males, which are now acting as mobile dispensers, inhibition of courtship behaviour at close range, and delay in mating, which is known to reduce fecundity and egg viability in insects such as codling moth (Cydia pomonella) (Knight, 1997)

The theoretical basis for autoconfusion Theoretical calculations show that there are approximately 1.5 x 1010 particles per gram of powder. The particle size is in the range of 5-20 microns diameter. Taking into account the known threshold responsiveness of Cydia pomonella it can be calculated that there is theoretically sufficient pheromone in one particle resting on the surface of the antenna to initiate habituation. An insect carrying approximately 1,800 particles would then be liberating sufficient pheromone in the short term to constitute an attractive source for another male moth. This means that the contents of one ExoSex dispenser (c. 3 g) of formulated powder are theoretically capable of contaminating about 21 million male moths with enough pheromone to make them attractive sources to other males. In any mating disruption technique the main constraints are the cost of the materials and the labour-intensive process of placing dispensers in the crop. In these respects, the Exosex system is considerably more cost-effective than other techniques. In the case of codling moth, for example, the total amount of pheromone dispensed per hectare is between 80 and 200 milligrams in 25 dispensers. By comparison, most conventional techniques dispense between 45 and 192 grams per hectare, i.e. around 1000 times as much, in 400-1000 dispensers.

Lure and kill techniques The ExolureTM system utilises adhesive particles as carriers for synthetic or biological pesticides. Insecticides with topical action (and entomopathogens) are held onto the cuticle for long periods increasing their lethality and making it feasible to use very low amounts of a.i. Preliminary trials were carried out against Cydia pomonella (Howse & Underwood 2000) and tephritid fruit flies using a lure (pheromone, trimedlure or food lure) to attract insects into dispensers containing electrostatically charged powder formulated with slow-acting insecticide as a killing agent. The latter was readily picked up and spread by contact to non- contaminated insects. Magnetic powders were subsequently found to have the property of adhering to the insect cuticle. Lure & kill techniques using electrostatic and magnetic powders in discrete dispensers are now being studied with the aim of developing targeted control systems for mediterranean and olive fruit flies. Field trials have been carried out by on Mediterranean fruit fly, oriental, melon and olive fruit flies in Spain and Mauritius (Chandler, 2004; Underwood & Howse, unpublished report). Trials with medfly (Ceratitis capitata) using dispensers with the male attractant Trimedlure, showed that 88-100% of males in field cages carried particles, and 52-100% of females. Similar trials in open orchard sites showed that just less than 50% of both males and females were contaminated. The number contaminated would have been diluted by individuals from outside the trial area, suggesting a high level of uptake and male to female transfer in the trial plot. Good results have been achieved in other pioneering field trials on medfly, Melon fly, Natal fruit fly and Peach fruit fly, using either the slow-acting insecticide Sulfluramid or 275

spores of the entomopathogen Metarhizium as active ingredients. In one trial in citrus orchards in Mallorca, using protein-baited traps to catch medfly females, catches declined in the treatment zone from 243 to 19 per week, while numbers in a control site actually increased over the same period.

Conclusions The Exosect technique of coating insects with certain types of powder that readily adhere to the cuticle has proved effective against a range of insect pests. Studies on the dynamics of powder pick-up and transfer to other individuals show that particles formulated with semiochemicals or insecticidal materials can be distributed throughout a pest population by the insects themselves. The autoconfusion technique for mating disruption and the lure & kill technique using adhesive powders offer promising and economically viable means of controlling populations of olive moth and olive fly respectively.

References

Chandler, J.C. 2004: Fruit flies – problems, economic importance and current and proposed control methods. – International Pest control 46 (3): 162-165. Chandler, J.C. & Howse, P.E. 2005: Cost reduction in SIT programmes using Exosect auto dissemination as part of area-wide integrated pest control. – International Pest control 47 (5): 257-260. Howse, P.E. & Underwood, K.L. 2000: Environmentally-safe pest control using novel bioelectrostatic techniques: initial results and prospects for area-wide usage. – In: Area- Wide Control of Fruit Flies and other Insect Pests. Ed. Tan, K.H,. Penerbit University Sains Malaysia, Penang: 295-299. Knight, A. 1997: Delay of mating of codling moth in pheromone disrupted orchards. – IOBC/ wprs Bulletin 20 (1): 203-206.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 277-281

Effectiveness of different copper products against the olive fly in organic olive groves

Marzia Cristiana Rosi, Patrizia Sacchetti, Michele Librandi, Antonio Belcari University of Florence, Department of Agricultural Biotechnology, Section of General and Applied Entomology, via Maragliano, 77, 50144, Italy.

Abstract: Different copper products were tested against the olive fly in organic olive groves during the 2004 and 2005 growing season. The active infestation level was reduced in all treated experimental plots with comparable effectiveness for all products. Furthermore, the results indicate a higher level of young larvae mortality as an effect of the copper treatment. This supports the hypothesis that copper acts as a symbioticide as well deterring oviposition.

Key words: Bactrocera oleae, sprays, symbionticide activity

Introduction

The possibility of using copper products as a symbionticide for bacteria associated with the olive fly was first proposed by Tzanakakis (1985). Later a certain activity of copper as an adult oviposition deterrent was also demonstrated (Prophetou-Athanasiadou et al., 1991). From the 1990s onwards, the use of copper products against the olive fly became widespread in organic olive crops (Belcari & Bobbio, 1999; Sacchetti et al., 2004; Iannotta, 2004). In this paper we report some results regarding the evaluation of the effectiveness of different copper products against Bactrocera oleae in an organic farm located in Calabria, southern Italy, where the insect usually causes severe damage to production.

Materials and methods

In 2003 and 2004 trials were conducted in two experimental one-hectare olive groves forming part of a wider olive orchard planted with the local Carolea cultivar. In each field an experimental design of 4 randomized blocks was established; each block consisted of 4 plots (thesis), making a total of 16 plots (4 theses repeated 4 times). Three different copper products were compared: Bordeaux mixture (Disperss® by Cerexagri), copper peptidate (Naturam 5® by Sicit 2000) and copper hydroxide (Ridox DF® by Siapa). The fourth plot was the untreated control. Each plot consisted of 36 olive trees, but only 16 trees in the central part were sampled in order to avoid the falsification of results due to the potential drift of sprays. To assess the level of infestation, 2 fruits/tree were collected for a total of 32 olives/plot. Every week a sample of 512 fruits was collected and dissected in the lab. Infested olives were then grouped into: active infestation (alive eggs, alive 1st and 2nd instar larvae), damaging infestation (alive 3rd instar larvae, olives with exit hole produced by full grown larvae) and total infestation (active infestation and damaging infestation). Sprays were applied above the action threshold, established at 5% of active infestation. In 2003, the threshold was reached on 17 September, and the treatments were carried out accordingly on 20 September; in 2004 the threshold was reached on 11 August, and the treatments were applied the following day.

277 278

Results and discussion

Trials performed in 2003 (S. Basile olive orchard) Figure 1 illustrates the active infestation in treated and untreated plots. Until 10 September the infestation level in all the plots was low (not exceeding 4% infestation). From this date on the infestation increased beyond the threshold for action and treatment was applied on 17 September. The effectiveness of treatments is amply demonstrated by the evident differences in the percentage of infested olives in the treated plots compared to the control plot. In the weeks through to harvesting (22 October), the infestation in the treated plots was very low, whereas in the control plot the infestation reached values of nearly 10%.

spray 10

4 infested olives(%)

0 9-Jul 1-Oct 8-Oct 3-Sep 16-Jul 23-Jul 30-Jul 6-Aug 15-Oct 22-Oct 10-Sep 17-Sep 24-Sep 13-Aug 20-Aug 27-Aug

Control Naturam Disperss Ridox

Figure 1. Active infestation in treated and untreated plots (S. Basile olive orchard, 2003).

Figure 2 shows the ratio between the number of young dead larvae and the total number of young larvae sampled in each thesis after spraying. Naturam and Ridox produced a high percentage of mortality in young larvae, whereas the use of Disperss made no difference. Statistical analysis established the significance of the mean values of Naturam and Ridox compared with control values (Control vs Naturam: Mantel-Haenszel test = 22.058, p = 0.000003; Control vs Ridox: Mantel-Haenszel test = 6.212, p= 0.013). No differences were found between Disperss and the control (Control vs Disperss: Mantel-Haenszel test = 0, p = 1).

Trials performed in 2004 (S. Domenico olive orchard) In Figure 3 it can be observed how the active infestation varies in different plots. As in the previous year, after spraying the level of infestation in the treated plots did not exceed 4% infestation and the effectiveness of the copper products lasted for roughly 60 days after the experimentation period was concluded. In the control plot the active infestation reached values of 13% soon after the date of spraying, showing a difference between the treated and 279

untreated plots (sampling of 18 August). After this date the level of infested olives in the control plots decreased slowly through to 8 September, probably due to high temperatures and a low RH level.

b 0.9

0.8

0.7 b, c 0.6

0.5

0.4

0.3 a a, c 0.2

0.1

0 Control Naturam Disperss Ridox

Figure 2. Ratio of dead young larvae out of total number of larvae in each thesis (S. Basile olive orchard, 2003).

18 spray 16

infested olives(%) 6

0 6-Oct 1-Sep 8-Sep 21-Jul 28-Jul 4-Aug 15-Sep 22-Sep 29-Sep 11-Aug 18-Aug 25-Aug

Control Naturam Disperss Ridox

Figure 3. Active infestation in treated and untreated plots (S. Domenico olive orchard, 2004). 280

Figure 4 shows the ratio between the number of young dead larvae and the total number of young larvae sampled in each thesis after spraying. In 2004, in contrast to the previous year, all the products showed a good degree of effectiveness on larval mortality, as confirmed by statistical analysis (Control vs Naturam: Mantel-Haenszel test = 18,541, p = 0,00002: Control vs Disperss: Mantel-Haenszel test = 25,065, p=0.000001; Control vs Ridox: Mantel- Haenszel test = 19,379, p=0,00001). There were no substantial differences in the relative effectiveness of the three products with regard to larval mortality.

0.9 b b 0.8 b

0.7

0.6

0.5

0.4

0.3 a 0.2

0.1

0 Control Naturam Disperss Ridox

Figure 4. Ratio of dead young larvae out of the total number of larvae in each thesis (S. Domenico olive orchard, 2004).

The results obtained in both years show the remarkable effectiveness of copper treatments in reducing infestation caused by the olive fly in experimental plots. Our previous hypothesis that copper acts as a symbionticide is also confirmed. In fact, statistical analysis revealed a high mortality rate in young larvae in treated plots, demonstrating that the bacterial symbiosis was interrupted; it is well known that young larvae cannot survive without the presence of bacteria in the gut. In consideration of the fact that the density of the fly was low in both years, the experiment needs to be repeated in years when there is a higher level of infestation in order to ascertain the real effectiveness of this kind of treatment. Further investigation is also required to evaluate the side effects of copper products on the soil fauna and on the useful fauna in this agroecosystem.

References

Belcari, A. & Bobbio, E. 1999: L’impiego del rame nel controllo della mosca delle olive, Bactrocera oleae. – Informatore Fitopatologico 12: 52-55. 281

Iannotta, N. 2004: La difesa dell’olivo dai principali fitofagi con metodi conformi al Reg. CEE 2092/91. – In: La difesa dai fitofagi in condizioni di olivicoltura biologica. Atti Accademia nazionale dell’Olivo e dell’Olio, Spoleto, 29-30 October 2002: 49-62. Prophetou-Athanasiadou, D.A., Tzanakakis, M.E., Myroyannis, D. & Sakas, G. 1991: De- terrence of oviposition in Dacus oleae by copper hydroxide. – Ent. Exp. Appl. 61: 1-5. Sacchetti, P., Belcari, A. & Del Pianta, R. 2004: Utilizzo di prodotti ad azione antibatterica per il controllo della mosca delle olive. – In: La difesa dai fitofagi in condizioni di olivicoltura biologica, Atti Accademia nazionale dell’Olivo e dell’Olio, Spoleto, 29-30 October 2002: 23-33. Tzanakakis, M.E. 1985: Considerations on the possible usefulness of olive fruit fly symbionticides in integrated control in olive groves. – In: Cavalloro R. and Crovetti A. (eds.): Integrated Pest Control in Olive-Groves. Proc. CEC/FAO/IOBC. International Joint Meeting, Pisa, 3-6 April 1984: 386-393.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 283

Establishment of TEAM (Tephritidae of Europe, Africa and Middle East), a new international working group on fruit flies of economic importance

N. Papadopulos, A. Bakri, S. Quilici, M. Bonizzoni, B. Barnes, Y. Gazit, S.A. Lux, D. Nestel, M. Cristofaro1, R. Pereira, M.A. Miranda, N. Kouloussis 1 ENEA C.R. Casaccia UTS BIOTEC, S.M. di Galeria (RM), Italy.

The Family Tephritidae includes 194 taxa: among them, numerous fruit flies species of tremendous economic importance can be found. Olive fruit fruit fly (Bactrocera oleae) is considered the most damaging pest in olives. Mediterranean fruit fly (Ceratitis capitata) is oligophagous damaging the fruits from more than 300 plant species and varieties. In addition, several other species mainly restricted to the genera Ceratitis and Bactrocera are considered target pests in the majority of tropical and subtropical regions worldwide. Most of those species are very well studied, and some of them (like C. capitata and B. oleae), due to their relevant economic importance, allow a wide range of multidisciplinary research approaches. For this reason, during the Meeting of the Working Group on Fruit Flies of the Western Hemisphere, held in Florida in May 2004, a group of scientists from 10 countries (8 Mediterranean, Kenya and Reunion Island for France) developed the idea of establishing the Fruit Fly Scientific Group. The first meeting of the group TEAM (Tephritidae of Europe, Africa and the Middle East) was held on 11th of May 2005 in the facilities of the IAEA in Vienna, Austria. The main goals of TEAM are: (a) to set up an independent scientific group dealing with fruit flies research and management in Europe, Africa and Middle East; (b) to provide a platform for interaction promoting collaboration and communication among scientists, growers and companies from different Countries with common interests on fruit flies; (c) to increase funding possibilities through cooperative research inputs; (d) facilitate the divulgation of TEAM activities setting up a web site and periodical newsletters; and (e) organize scientific meetings every 2-3 years.

283

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 285-288

Application of forecasting models of olive fly (Bactrocera oleae Gmel.) (Diptera, Tephritidae) infestation in Montenegro

Snježana Hrnčić1, Claudio Pucci2, Antonio Franco Spanedda2, Alessandra Terrosi2, Tatjana Perović3, Biljana Lazović3, Mirjana Adakalić3 1 Biotechnical Institute Centre of Plant Protection, Kralja Nikole bb 81000 Podgorica, Montenegro; 2 Università degli Studi della Tuscia – Dipartimento di Protezione delle Piante, Via S. Camillo de Lellis, 01100 Viterbo – Italy; 3 Centre of subtropical cultures Bjelisi bb, 85000 Bar, Montenegro

Abstract: In the paper has been investigated the possibility of application of two forecasting models for olive fly infestation. The first model was based on one hand on the number of caught females by means of yellow sticky traps and average weekly temperature and on the other hand it was based on infestation levels. The second model has considered male captured on pheromone traps. Obtained results indicate that both methods are applicable.

Key words: forecasting model, Bactrocera oleae, yellow-sticky trap, sex-pheromone trap

Introduction

In defining the control strategies for B. oleae we can usefully make use of instruments able to describe its population’s dynamics considering the course of environmental parameters. There have been many the attempts to work in this direction: from the most simple phenological models to the latest applications aimed to forecast the dynamics of the population of preimmaginal stages and the damage entity when the number of individuals captured and some climatic parameters are known (Ricci et al., 1979; Ballatori et al., 1980; Bagnoli et al., 1982; Pucci and Terrosi, 2002; Lo Duca et al., 2003). This contribution’s aim is to verify the applicability of two statistical models for forecasting the seriousness of infestation based, the first one, on the average number of females captured with chromotropical yellow sticky traps while the second one is based on the average number of males captured by means of pheromone traps.

Material and methods

Researches were carried out in the year 2004 in Montenegro, in an olive-grove of cv Žutica situated at a distance of 400m from the sea, in a flat land. From the end of June 6 plants were chosen. Sex-pheromon trap (Dacotrap) was placed on 3 plants at medium height of the canopy. The pheromone dispensers were replaced every four weeks. At the other 3 plants were placed yellow sticky traps at medium height and south side of the canopy. The number of individual olive flies captured were counted weekly. At the same time the olive sampling was performed by randomly picking 40 drupes per plant from the canopy of same tree chosen for flies trapping. Then olive fruits have been observed by stereomicroscope with the aim of quantifying infestation by counting the number of eggs, 1st 2nd and 3rd instar larvae, pupae, empty cocoons and abandoned galleries.

285 286

All over the investigation period some climatic parameters (temperature, humidity and rainfall) were recorded, too. Both models, based on the technique of canonical analysis, describe with sufficient precision the relationship between the number of individuals weekly captured and average temperature of the week on one hand, and infestation of the canopy’s drupe and olives dropped on the other. The first model – “female forecasting model” – based on the capture of females with yellow sticky trap was completed in the olive area of Northern Lazio and then successfully applied in different olive areas in Italy and abroad (Croatia) (Pucci, 1993; Castoro et al., 1996). This model define an index of the gravity of infestation Z which was calculated by formula: Z = 0.039(Fm-9,7) – 0,187(Tm-22,1) where: Fm is the average number of females weekly captured by means of yellow sticky chromotropic traps Tm is the average of the seven daily mean temperatures recorded in the same capture week. The second model – “male forecasting model” – based on the capture of males with pheromonical traps, also completed in Central Italy, has been applied for the first time in the olive areas of Montenegro (Lo Duca et al, 2003). This model define an index of the gravity of infestation Z which was calculated by formula: Z =0.027Mm – 0,399Tm + 8,71 where: Mm is the average number of males weekly captured by means of sex-pheromone traps Tm is the average of the seven daily mean temperatures recorded in the same capture week.

2 100% infestation of untreated plants

1,5 Z index 90% 1 treatment threshold 80% 0,5 3-terms moving average of Z index 70% values 0 60% -0,5 50% -1 40% infestation

Z index values -1,5 30% -2 20% -2,5 10% -3 0%

l l l l t t t t u u u u g g g g g p p p p c c c c J J J J u u u u u e e e e O O O O - - - - -A A A A A -S -S -S -S - - - - 6 3 0 7 3 0- 7- 4- 1- 7 4 1 8 5 2 9 6 1 2 2 1 1 2 3 1 2 2 1 1 2

Figure 1. Trend of index value (based on females captures) plotted against infestation level.

287

Results and discussion

Both models have allowed us to define an index of the gravity of infestation (Z), whose trend is correlated with the development of infestation. The results point out how both models are able to forecast with sufficient precision the infestation’s seriousness: in particular the model based on the weekly average female captures necessitates, if Z exceeds the value of 0.1, of immediate treatment execution (Figure 1). The model based on the captures of males with sex pheromone traps, if Z>-1, points out three weeks in advance the development of a dangerous infestation allowing the operator to easily organize even interventions in advance (Figure 2). Both models have the advantage that farmers are released from onerous operations of sampling and dissection of the drupes. It’s desirable that these models should be applied on different cv and in different olive areas.

2 100% infestation of untreated plants 1,5 90% Z index

1 treatment threshold 80%

0,5 3-terms moving average of Z index 70% values 0 60% -0,5 50% -1 40% infestation

Z index values Z index -1,5 30% -2 20% -2,5 10% -3 0%

l g g p p ug u u ep e e ct ct ct -Jul -Aug -S 6 7-Ju3-A 0-A 7-A 7 5-Oct2-O 9-O 6-O 13-Jul20-Jul2 1 1 24-Aug31 14-S 21-S 28-Sep 1 1 2

Figure 2. Trend of index value (based on males captures) plotted against infestation level.

References

Bagnoli, B., Belcari, A., Ghilardi, G., Piccoli, A., Pucci, C., Quaglia, F., Ricci, C. 1982: Osservazioni sulle catture di femmine di Dacus oleae (Gmel.) a mezzo di cartelle cromotropiche e sull’andamento dell’infestazione. Ann. Ist. Sper. Zool. Agr. 6: 93-103. Ballatori, E., Pucci, C., Ricci, C. 1980: Relation entre l’infestation des olive et les captures d’adultes de Dacus oleae (Gmel.) par piège chromotropiques. Redia. 63: 417-439. Castoro, V., Pucci, C. 1996: Applicazione di un modello statistico di previsione della gravità dell’infestazione di B. oleae (Diptera Tephritidae) nell’ambiente olivicolo materano: esperienze condotte nel biennio 1994-1995. Atti Giornate Fitopatologiche, Numana 22- 24 aprile 1996. I: 505-512. Lo Duca, P., Spanedda, F., Terrosi, A. 2003: A forecasting model of the olive-fruit fly infestation based on monitoring of males. Working Group “Integrated Protection of olive Crops” Proceedings of the meeting at Chania (Greece), 29-31May, 2003. Edited by: 288

Argyro Kalaitzaki, Venizelos Alexandrakis, Kyriaki Varikou. ISBN 92-9067-181-3 [XV+178 pp.] – IOBC Bulletin 2005. 28(9): 59-66. Pucci, C. 1993: Applicazione della tecnica dell’analisi canonica nella previsione dell’infestazione dacica. M.A.F., Atti del Convegno “Olivicoltura”, Firenze 1991 (Coord. M. Crovetti), ed. Ist. Sper. Pat. Veg., Roma. 49-61. Pucci, C., Terrosi, A. 2002: Il controllo della mosca dell' olivo (Bactrocera oleae (Gmel.) Diptera Tephritidae): stato dell’arte e prospettive. Medjunarodna manifestacija maslini i maslinovom ulju: tekuće zeleno zlato Istre, Tar, Hrvatska. 25-37. Ricci, C., Pucci, C., Ballatori, E., Forcina, A. 1979: Alcuni aspetti della dinamica delle popolazioni di adulti di Dacus oleae (Gmel.) e analisi della relazione tra infestazione e catture con cartelle cromotropiche. Notiz. Malattie Piante. 100 (3° serie, n.26): 261-282.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 289

Application of internet and mobile technologies in pest management: a case study of Bactrocera oleae control in Tuscany

D. Guidotti1, S. Marchi1, A. Bo1, M.Ricciolini2, R. Petacchi3 1 Aedit s.r.l., Pontedera (PI), Italy. 2 ARSIA, Via Pietrapiana, 30 50121, Firenze, Italy. 3 Scuola Superiore Sant'Anna, Pontedera (PI), Italy.

Sustainable agriculture integrates three main goals of environmental health, economic profit- ability, and socio-economic equity. In particular, consulting companies and extension services at Regional level play an important role in pest prevention, implementing new research findings into ordinary agricultural practices. This work can greatly be facilitated by software systems that can process large amounts of data and perform numerous combinations of a variety of factors, which affect agriculture management planning. Decision Support Systems (DSS) is a special type of such systems and can support sustainable agriculture, particularly pest control, when farmers are assisted by specialized consults. The application of technical assistance services, based on Short Message Service (SMS) and aimed to assist farmers in pest management, may help in reducing chemical treatments against olive fruit fly (Bactrocera oleae Gmelin). Different communication strategies have been tested in different Italian regions. From 2003 to present, a partnership among Aedit s.r.l., Scuola Superiore Sant’Anna and ARSIA (the Regional Agency for Development and Innovation in Agriculture and Forestry of Tuscany), has tested an SMS service on a large number of farmers. Dedicated software was developed for assisting technical advisors in editing personalized messages, with the final objective to guide pest management strategies at farm level. Here we discuss how integrate spatial information, biological and meteorological data from monitoring networks, and DSS with a human supervised SMS generation. Continued development will focus on improving olive responses to biotic and abiotic stresses and testing the model's functionality as a decision support tool for strategic and tactical farm management.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 291-295

Integrated protection system against Bactrocera oleae (Gmelin) in organic production

Nino Iannotta, Tiziana Belfiore, Enzo Perri, Luigi Perri, Vincenzo Ripa C.R.A. Institute for Olive Growing - 87036 Rende, Cosenza, ITALY

Abstract: Organic olive farming has gained a large success in Italy and immense surface areas have converted traditional production into organic techniques. In such practices however, the regions of southern Italy must cope with numerous olive fruit fly attacks without the use of chemical pesticides. In this study we estimated the efficacy of a organic control system consisting in the use of a mass- trapping supplement and rotenone treatment in areas highly infested by dipterous. The system was compared to traditional methods of control that utilize dimethoate and to untreated groves. Experiments were conducted in the Ionic region of Cosenza in 2004 in a field with several olive cultivars. The integrated organic system showed a good efficacy in control of olive fruit flies. The efficacy was more apparent in untreated groves compared to those treated with dimethoate in terms of both active and total infestation.

Key words: protection system, organic production, olive fly

Introduction

In past years, the success enjoyed in Italy by organic olive growing has led to a considerable increase in the amount of cultivated areas that adhere to such practices, especially in southern regions. However, in these temperate climate areas strong parasite attacks often occur, among which the olive fly (Bactrocera oleae) represents the most dangerous olive pest. Control of this phytophagous insect, already difficult in conventional olive growing using chemical pesticides, becomes even more problematic in organic farming conditions, where only the use of products listed in Table IIB of European Council Regulation EEC 2092/91 is permitted. Based on previous experience, it is well-known that the use of these products, when utilized alone, have little efficacy. For this reason, in high infestation environments an integrated system that relies on different methodologies for minimizing the damage due to the olive fruit fly within limits compatible with the achievement of an high quality product (olive oil) is desirable. In the present report, we assessed the efficacy of an integrated pesticide system by using mass-trapping together with rotenone treatment and early harvesting. This system was compared to traditional methods of pesticide control using chemical agents in a experimental field located in the Ionic areas of Cosenza.

Materials and methods

The experiments were performed in 2004 in the experimental olive collection field of Mirto- Crosia (CS) in a young olive orchard having a surface area of 12 hectares. The field was divided into three treatment areas: A, sprayed with dimethoate at a concentration of 150 g/hl (traditional system); B, mass-trapping + rotenone (Rotena, 300 g/hl); C, sprayed with water. Section B was further divided into two parts with the aim of testing two different mass- trapping capture devices based on an “attract and kill” mechanism (Agrisense). Both types of

291 292

devices were primed with olfactory (ammonium bicarbonate) and sexual (Spiroketal) attractants and poisoned with lambdacyhalothrin. The first type is already available on the market and is composed of funnel-shaped devices (B1), while the second type is simpler and made up of two plastic sheets united by attractants (B2). The insecticide treatments (dimethoate and rotenone) were performed on September 3 2004. The adult population present in the field was monitored with chromotropic traps that were read every 10 days. Drupe samples (n=200) were also collected every 10 days to determine the amount of active infestation. Meteorological data were collected by an observation post equipped with electronic sensors. The data were compared by analysis of variance tests.

Results

In Figure 1, data on the adult population present in the different groups is shown. Group A, or those treated with dimethoate, tended to have the lowest number of adults per trap, particularly in the period following treatment (September 3). Slight differences were also present between the two different types of mass-trapping: the commercial devices (B1) were somewhat more efficacious compared to the experimental device (B2), although these differences were not statistically significant.

Captures A 120 B1 100 B2 80 C 60 40

N° adults/trap N° 20 0 jul aug aug se p se p oct oct nov nov 15 05 26 16 30 14 28 11 25

Figure 1. Adult of olive fly population. A: sprayed with dimethoate, B1: mass-trapping (funnel- shaped devices) + rotenone, B1: mass-trapping (plastic sheets) + rotenone, C: sprayed with water.

Figure 2 shows the active infestation at various time periods. From this data, it was evident that the best results were obtained using the integrated systems. This more favorable trend of active infestation was observed during in the entire period of observation and showed statistically significant differences until the optimum harvesting time (mid October). During this time, the pre-imago population in the drupes did not exceed the limit of 20%. Figure 3 shows the data concerning the total infestation in which all treatment groups show a lower level of infestation compared to the untreated control group. Figure 4 shows humidity and temperature trend.

293

Active infestation A 70 B1 60 B2 50 C 40 30 20 Active inf. % inf. Active 10 0 jul aug aug se p se p oct oct nov nov 15 05 26 16 30 14 28 11 25

Figure 2. Percentage of active infestation. A: sprayed with dimethoate, B1: mass-trapping (funnel- shaped devices) + rotenone, B1: mass-trapping (plastic sheets) + rotenone, C: sprayed with water.

Total infestation A 100,0 B1 80,0 B2

60,0 C

40,0

Total inf. % inf. Total 20,0

0,0 jul aug aug se p se p oct oct nov nov 15 05 26 16 30 14 28 11 25

Figure 3. Percentage of total infestation. A: sprayed with dimethoate, B1: mass-trapping (funnel- shaped devices) + rotenone, B1: mass-trapping (plastic sheets) + rotenone, C: sprayed with water.

Discussion

Based on these observations, the effectiveness of integrated system on controlling olive fruit fly attacks was evident. Its efficacy was apparent not only compared to untreated controls, but also when areas treated with traditional pesticides (treatment with dimethoate) were considered. To date, such traditional treatments have been considered as the most reliable and are approved for treatment of olive groves. The control of active infestation within the 20% limit can be considered satisfactory, since it is compatible with the production of high quality olive oil. Achieving such limits without the use of traditional pesticides in an area characterized by very severe olive fruit fly attacks is certainly noteworthy.

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35,0 100

90 30,0

25,0 70

60 20,0 UR % Media T. °C Media 50 °C

UR % Poli. (UR % Media) 15,0 Poli. (T. °C Media) 40

30 10,0

5,0 10

0,0 0

t t t tt tt c ic lug lug go se se se o o ov ov d - - -a -set 7-ott 8- n n 1-lug 8 5-ago 2-set 9 6- 3- 0- 14-ott21- 2 4-nov1- 5- 2-dic 9-di 6- 3-dic 15 22-lug29-lug 12-ago19-ago26 1 2 3 1 18-nov2 1 2 30-dic

Figure 4. Humidity and temperature

Acknowledgements

This research has been carried out with contribution of MIPAF in OLIBIO project.

References

Bagnoli, B., Petacchi, R. 2001: Problematiche relative all’utilizzo di prodotti di origine vegetale per il controllo della mosca delle olive. – Relazione al Convegno “L’olivicoltura biologica e la lotta contro la mosca delle olive”, Scuola Superiore Sant’Anna di Studi Universitari e di Perfezionamento, Pisa, 24/04/2001. Baldacchino, F., Simeone V., 2001: Controllo della mosca delle olive in olivicoltura biologica in Puglia: esperienze preliminari. – Relazione al Convegno “L’olivicoltura biologica e la lotta contro la mosca delle olive”, Scuola Superiore Sant’Anna di Studi Universitari e di Perfezionamento, Pisa, 24/04/2001. Cristofaro, M., Di Ilio, V., Marchini, D., Nobili, P., Dallai, R., Cirio, U. 1996: Effects of an azadirachtin based compound on the fecundity of mediterranean fruit fly Ceratitis capitata: structural and ultrastructural analysis. – XX Iint. Congr. of Entomology, Firenze, August: 474. Iannotta, N. 2001: La lotta naturale ai parassiti dell’olivo. – Olivo e olio n° 5: 16-26. Iannotta, N. 2001: Esperienze di lotta contro Bactrocera oleae (Gmel) con metodi conformi al Reg. CEE 2092/91. Relazione al convegno “L’olivicoltura biologica e la lotta contro la mosca delle olive”, Scuola Superiore Sant’Anna di Studi Universitari e di Perfezio- namento, Pisa, 24/04/2001. Iannotta, N. 2002: Il controllo della mosca delle olive (Bactrocera eleae Gmel.) con metodi consentiti in coltivazione biologica. – GRIFA Int. Cong. “Biological products: which guarantees for the consumers”, Milano: 57-60. 295

Iannotta, N. 2003: La difesa fitosanitaria. – In “Olea Trattato di olivicoltura”, ed. Ed agricole- Il sole 24ore: 393-407. Iannotta, N. 2004: La difesa dell’olivo dai principali fitofagi con metodi conformi al Reg. 2092/91. – In: “La difesa dai fitofagi in condizioni di olivicoltura biologica”. Ed. Accademia Nazionale dell’olivo e dell’Olio, Spoleto: 49-62. Iannotta, N., Lombardo, N., Maiolo, B., Parlati, M.V., Scazziota, B. 2000: Controllo di Bactrocera oleae (Gmel.) con un prodotto fitosanitario naturale compatibile con la produzione biologica. – Atti Conv. “Produzioni alimentari e qualità della vita”, Sassari (in press) Iannotta, N., Madeo, A., Monardo, D., Perri, L., Scazziota, B. 2002: Lotta contro Bactrocera oleae (Gmel.) con alcuni principi attivi ammessi in coltivazione biologica. – Atti Conv. Int. Olivicoltura, Spoleto: 433-438. Iannotta, N., Monardo, D., Perri, L., Tocci, C., Zaffina, F. 2000: Esperienze di lotta alla Bactrocera oleae (Gmel.) con sistemi conformi al Reg. CEE 2092/91. – Atti Sem. “Metodi e sistemi innovativi dell’olivicoltura biologica e sostenibile”, Rende (Cs):123- 126. Iannotta, N., Monardo, D. 2004: Suscettibilità di cultivar di olivo a Spilocaea oleagina (Cast.) Hugh. e correlazione con il contenuto di oleuropeina nelle foglie. – Conv. “Germoplasma Olivicoli e tipicità dell’olio”, Perugia 5 Dicembre 2003: 216-220 Tsolakis, H., Ragusa, E., Ragusa Di Chiara, S. 1999: Effetti dell’olio di Neem (Azadirachta indica A. Juss) su Bactrocera oleae (Gmelin) (Diptera, Tephritidae) in prove di laboratorio e di campo. – Phytophaga 9 Suppl.: 65-75.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 297

Differences in insects within the olive orchard agroecosystem under integrated management regime in south Spain

B. Cotes1, F. Ruano1, P. Garcνa2, F. Pascual3, A. Tinaut3, A. Peρa1, M. Campos1 1 Estación Experimental del Zaidνn, CSIC, Granada, Spain. 2 Department of Statistics, University of Granada, Granada, Spain. 3 Department of Animal Biology and Ecology. University of Granada. Granada, Spain.

Interactions between insects (Class Insecta) and plants involve three quarter of the global biodiversity and they are crucial elements for the agroeconomy. Andalusian Legislation has established practices for the integrated management regime in the olive orchard. The obligatory practices are carried out by farmers; nevertheless there are suggested practices, which can be applied voluntarily to help reducing the impact on the agroecosystem. These significant differences among irrigation treatments, ploughed intensity, insecticide use and presence of a vegetal cover could be responsible for substantial variation in the structure of the insect community between the sampled olive orchards. The principal aim of our study is to compare the presence and abundance of insects in the canopy in the olive orchards under integrated management regime. The sampling was conducted in May and July 2003 in six commercial olive orchards with different cultural methods under integrated management regime in south Spain. In each olive orchard, 20 trees were distributed aleatory on 4 blocks. Each tree was sampled in the canopy by beating branches. Our results showed that the different management practices affect the relative abundance of the insect orders. In the canopy, Homoptera, Hymenoptera and Diptera were the most abundant, although showed significant differences in abundance among orchards during the two samples. The most important observation is the abundance of Homoptera, and principally the psyllid Euphyllura olivina (Costa), which was majority during the two sampling in the orchards with soil tillage, without vegetal cover and in some case insecticide use. However, other orchards under less intensive farming showed better balanced abundance of insects.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 299

New biodegradable controlled-released pheromone dispenser for Bactrocera oleae (Gmelin)

I. Navarro-Fuertes, R. Gil Ortiz, P. Moya Sanz, V. Navarro-Llópis Centro de Ecologìa Quìmica Agrìcola, Universidad Politécnica de Valencia. Campus de Vera, s/n. Edificio 9B. Lab. 111. 46022, Valencia, Spain.

A new type of eco-friendly, hand-applied pheromone (1,7-dioxaspiro[5.5]undecane) dispenser for monitoring and controlling Bactrocera oleae has been developed. Several types of dispensers, based on microporous and mesoporous materials and with different additives, in changing proportions, were designed and evaluated in order to obtain that possessing the optimal pheromone emission. Dispensers were subjected to a procedure of accelerated aging in a temperature and wind speed controlled chamber. Residual pheromone remaining in the dispensers was periodically evaluated by gas-liquid chromatography and release rates were determined. In addition, a comparative study between our dispensers and the commercial B. oleae Long Life Lure from Agrisense (Pontypridd, UK) was also carried out in laboratory.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 301-305

Increased olive oil yield and quality in Montenegrin cv Žutica by Bactrocera oleae Gmel. (Diptera Tephritidae) control and improved harvest techniques

Biljana Lazović 1, Mirjana Adakalić 1, Tatjana Perović 1, Snježana Hrnčić 2, Claudio Pucci3, Alessandra Terrosi 3, Antonio Franco Spanedda3 1 Biotechnical Institute, Center for Subtropical Cultures, Bjeliši bb, 85000 Bar, Montenegro, SCG 2 Biotechnical Institute, Center for Plant Protection, Kralja Nikole bb, 81000 Podgorica, Montenegro, SCG 3 Università degli Studi della Tuscia- Dipartimento di Protezione delle Piante, Via S. Camillo de Lellis. 01100 Viterbo - Italy

Abstract: The present inquiry represents the basis for wider research into the qualitative and quantitative characterization of the typical olive oil of Montenegro. In the considered biotope the 65% of plants is composed of cv Žutica. The key insect is B. oleae, which is, in some years, able to nullify quantitatively the entire production because of the olives dropping off the trees. However the entire production is usually harvested and the product is the clear oil, both because of the infestation and elevated temperature and the long storage periods of olive that cause a remarkable increase in degrees of acidity. The work, carried out in 2004, consisted of studying the inolation on weekly samples of healthy olives harvested from the canopy and processed for olive oil extraction after 24-48 hours. This has allowed us to identify that the right moment for harvest is the third week of October. Finally the qualitative Analysiss of the oil drawn from the plant are presented in comparison to the qualitative characteristics of the output usually obtained by Montenegrin olive-growers after different stock periods. The results obtained represent the first and basic stage for the realization of research projects aimed to improve the techniques of production and protection of oil yield.

Key words: inolation of fruits, fruit growth, storage, fatty acid, Žutica

Introduction

Žutica is the most important olive cultivar in Montenegro. It predominates in olive assortment with 65%, while in the areas of Bar and Ulcinj it is present with 98%. It is an old variety with some example 2000 years old, 'Velja maslina' in Ivanovići near Budva. Žutica is a good cultivar for oil production, which is of about 21% in the fruit. It is also used for conservation in traditional ways as green and black. The name of the cultivar comes from its colour, which before maturing is yellow. Sloppy terrains and poor soils characterize the conditions for olive growing in Montenegro. Traditional ways of growing, with high trees rarely pruned and tree structure that does not allow regular application of agricultural treatments, cause big problem for harvesting. B. oleae is another problem and in some years affects production because of the fruit dropping. Picking the fruits from the ground forms part of the harvest. Nets are rarely used and fruits fall, because of damage or maturity, on the ground, from where they are occasionally collected. Those conditions with elevated temperature and the olives’ long storage periods cause a remarkable increase in degrees of acidity.

301 302

However, Montenegro is directed to EU, with efforts to accept and to follow EU rules, so the good quality olive oil production would also be very important for olive development aspects and from an economical point of view as well. Investigations of this kind have not be conducted in Montenegro before. Olive oil was determined only with aim to characterize some varieties of interest (Miranović, 1971, 1979, 1994; Lazović, 2001). This work represents the basis for wider research to take place on quantitative and qualitative characterization of the typical olive oil of Montenegro.

Material and methods

The experimental field is situated in the city of Bar 8 m above sea level and 500 m distant from the sea. An old orchard, of area 10 x 8m, was regenerated in the 60s by decapitation on the level of trunk neck and 1 m of height respectively. In the 80s this orchard was split into 2 parts by street construction. One part of the orchard, with 48 trees, is in use by the Center for Subtropical Cultures, while the other part with 51 trees is in possession of the School of Agriculture. In the first part, used by the Center, trees were 7-8 m in height and 18 trees were severely pruned in 2004, to the height of about 4 m. Fertilization was done with 1 kg/tree of NPK (8:16:24) by the end of January, and twice with 0,5 kg/tree of nitrogen in March and May. In another part of orchard (School of Agric.) trees were 8-10 m in height and 44 trees were severely pruned in 2003, with about 5 m of height in experimental year. No irrigation was applied and orchard soil cover maintenance was made by regular grass cutting. For determining oil increment dynamics, samples of healthy fruit, about 200 g, were taken from 8 trees from the middle portion and all sides of the crown. After measuring fresh weight, samples were dried on 105 °C to the constant weight, grounded and oil was extracted by Foss let (Soxlet method). At harvesting time (26.10.) one sample of about 6 kg of fruits was taken, divided into 4 portions and from each portion oil was extracted at laboratory olive mill with one week of distance. In extracted oil free acid, peroxides and humidity were determined. All Analysiss were conducted at laboratories of the Center for Subtropical Cultures in Bar Montenegro and at the University of Tuscia in Viterbo, Italy.

Results and discussion

Fruits of cv Žutica were characterized as small (Miranovic, 1971, 1979) to the middle size (Lazovic, 2001) and this what was confirmed with those investigations. Fruit growth dynamics presented on Figure 1, shows permanent increment tendency. The most intensive growth was in more than one-month period, from the middle of September to last week of October. Average fruit volume rose for 1.5 g, corresponding to about 40.98 % of their weight in the observed period. Average fruit ripening of cv Žutica is in the beginning of November (Lazovic, 2000), and from the Figure 1. could be seen that fruit volume was reaching its maximum at the same period when harvesting was done. Proof for the right time of harvesting we also achieved by following the increment of oil dynamics in the fruit of cv Žutica, presented on Figure 2. Cv Žutica is characterized with high content of oil in the fruit of about 21% of fresh olives (Miranović, 1971, 1979, 1994; Lazović, 2001). Oil content presented on dry mass showed permanent increment during two months of observation. Very intensive oil increment was found in the period from the end of August, later than in findings of Tombesi (1994), to the middle of October. The most intensive oil accumulation was during 20 days of September 303

and in first half of October, when oil quantity increased by about 12% per period. From the middle of October there was very low increment or almost stagnation in oil accumulation. Cv Žutica reached its maximum of fruit inoliation by the end of October which is more than one month earlier in comparison to the most important Italian olive oil varieties (Cimato et al., 1996), but in accordance with the results of the maximal oil content regarding to the proper harvesting time in order to anticipate olive fly attack during autumn (Iannotta et al., 1996). However, this experiment should be followed during a longer period of time and in different areas where cv Žutica is grown to conclude that the early maximal inolation is the property of the cultivar.

4 3,5 3 2,5 2 1,5

fruit weight in g fruit weight 1 0,5 0 24-Aug 7-Sep 21-Sep 5-Oct 19-Oct 2-Nov measuring dates

Figure 1. Average value of fruit growth dynamic in cv Žutica in 2004.

60 50 40 30 20 10 0 oil content in dry mass (%) mass in dry content oil 5-Oct 7-Sep 12-Oct 19-Oct 26-Oct 14-Sep 21-Sep 28-Sep 24-Aug 31-Aug measuring dates

Figure 2. Oil content increment dynamic in fruits of cv Žutica in 2004.

Analysis of oil extracted from collected fruits. Quality parameters in olive oil extracted in one week periods, from fruit collected from the ground are presented in Figures 3 and 4. Acidity is alteration caused by olive oil hydrolysis due to microbic, enzymatic or microbic-hydric action. Microbes growing in the fruit after insect attacks, enzymatic activities such of lipase, or microbes developing in presence of water are the main factors for increasing acidity (Michelakis, 1992). Infestation by the olive fly (B. oleae) is a major cause of the high free fatty acids (FFA) content in olives. Falling from the trees causes bruises of the olives, which together with long storage times results in lipolysis increasing FFA content in the produced oil (Mulk et al. 2003). 304

14,00 12,00 10,00 8,00 6,00 4,00 2,00

free fatty acids content (%) 0,00 27-Oct 3-Nov 10-Nov 17-Nov measuring dates

Figure 3. Free fatty acid content in oil depends on the length of time the fruits are stored before processing.

Our results confirm previous opinion that low oil quality is also connected with the technique of collecting fruits by picking from the ground. Figure 3. shows increase of acidity (expressed as oleic acid gr/100 g of oil), with time of storage. Oil obtained from fruits one day after collecting had acidity less than 2%, while after one week it was about 6%, results obtained by analysing oil from the mills in Montenegro. Our results differ from those that Conti (1996) obtained from the fruits after 2 weeks of storage, where the higher content of FFA was only in fruits with 80% of infestation, of 3,62, while in our samples after two weeks FFA content was about 8. Humidity in oils (Figure 4) obtained from those samples had also increasing tendency depending on the fruit storage period.

0,40 0,35 0,30 0,25 0,20 0,15 humidity (%) 0,10 0,05 0,00 27-Oct 3-Nov 10-Nov 17-Nov measuring dates

Figure 4. Humidity in the oil of cv Žutica related to the fruit storage period.

In conclusion it can be said that for improving yield and olive oil quality in Montenegro there is the necessity for permanent investigation of the olive oil accumulation in the fruit regarding proper harvesting times, as well as it should be monitored more closely qualitative characteristics of olive oil in the relation olive fly control, harvesting technique and stocking period.

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Aknowledgement

Those investigations and Analysiss were conducted thank to Prof. Pucci from University of Study of Tuscia who helped from the initiation of the trial. Thanks also to his collaborators for their kind hospitality in the laboratory for oil Analysiss.

References

Cimato, A., Modi, G., Mattei, A., Alessandri, S. 1996: L'olio Toscano: Elementi di peculiarita'. – Atti del Convegno: L'olivicoltura mediterranea: Stato e prospettive della coltura e della ricerca, Cosenza, Italy: 629-638. Conti, B. 1996: Studi bio-etologici su Bactrocera oleae (Gmel.) (Diptera Tephritidae) e considerazioni sulla qualità dell' olio finale prodotto in un oliveto condotto in assenza di trattamenti antidacici. – Atti del Convegno: L'olivicoltura mediterranea: Stato e prospettive della coltura e della ricerca, Cosenza, Italy. 525-536. Iannotta, N., Parlati, M.V., Pandolfi, S., Perri, L., Zaffina, F. 1996: Individuazione dell’ epoca ottimale di raccolta della ‘Carolea’ e della ‘Sassanese’, in diversi areali calabresi, nella attuazione di lotta agronomica contro Bactrocera oleae (Gmel.). – Atti del Convegno: L'olivicoltura mediterranea: Stato e prospettive della coltura e della ricerca, Cosenza, Italy. 487-505. Lazović, B. 2000: Rodnost ispitivanih sorti masline (Olea europaea L.), Jugoslov. – Vocarstvo. 34 br. 131-132 (2000/3-4): 167-175. Lazović, B. 2001: Osobine ploda nekih sorti masline (Olea europaea L.). – Poljoprivreda i šumarstvo, Podgorica. 47(3-4): 15-25. Michelakis, N. 1992: Olive oil quality improvement in Greece. Past, present and future. – Olivae. 42: 22-30. Miranović, K. 1971: Važnije karakteristike nekih sorti maslina na Crnogorskom primorju. – Poljoprivreda i šumarstvo, Titograd. 17(3): 85-94. Miranović, K. 1979: Elajografska proučavanja autohtonih maslina u Bokokotorskom podrejonu. – Poljoprivreda i šumarstvo, Titograd. 24 (3-4): 97-106. Miranović, K. 1994: Studies of elayographic properties of olive cv. Žutica. – Acta Horti- culturae 356: 74-77. Muik, B., Lendl, B., Molina-Diaz, A., Ayora-Cañada, M.J. 2003: Direct, reagent-free determination of free fatty acid content in olive oil and olives by Fourier transform Raman spectrometry. – Analytica Chimica Acta 487: 211-220. Tombesi, A. 1994: Olive growing and metabolism. – Acta Horticulturae 356: 225-231.

Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 p. 307

Integrated olive pest management in Iran

H. Nouri Agricultural and natural resources research center, Qazvin, IRAN.

Olive black scale and olive psyllid were key pests on olive groves, the olive fruit fly was first recorded in October 2004 in Iran. Chemical treatments still prevail for S. oleae and E. olivina are also applied. In the most regions native parasites and predators are capable of maintaining S. oleae and E. olivina populations at low levels if they are not destroyed by hazardous chemical treatments. Based on researches results in Iran and Mediterranean countries, we provided a time table including major treatments such as biological control and accessory treatments such as pruning, moderate use of nitrogen fertilizers, using moderate irrigation and cover spray at the end of winter with emphasis using emulsifiable oils.

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Integrated Protection of Olive Crops IOBC/wprs Bull. 30(9), 2007 pp. 309-314

Can spring-preventive adulticide treatments be assumed to improve Bactrocera oleae (Rossi) management?

Giorgio Ragaglini, Diego Tomassone, Ruggero Petacchi Scuola S. Anna, Landscape Entomology Lab, Viale Rinaldo Piaggio 33, Pontedera (PI – Italy), e-mail: [email protected], [email protected], [email protected].

Abstract: We used pheromone traps network for monitoring adult’s behaviour from January to July until the beginning of the infestation. In this work our aims was: 1) to identify those areas where B.oleae infestation can be dangerous since early summer and define a regional map of infestation risk level during the early summer; 2) to put in evidence the relationship between spring adult’s captures with captures occurred in the previous autumn (backward) and with captures occurred at the infestation beginning during the following July (forward). On the base of our results we can hypothesize to carry out spring-preventive adulticide treatments in order to decrease the population density before the infestation starts.

Key words: monitoring network, olive fruit fly, overwintering, spatial analysis, degree day model.

Introduction

Currently researchers focus their studies on B. oleae management during the infestation season, while the ecology and biology of overwintering populations are not very well-known. Under field condition, the relationship between B. oleae spring adult’s population, developed from the pupae in the soil, and early summer adult’s population, that infested the new olives production, has not been shown until now. Some authors observed flights of male and female adults during the spring season from March to May (Lupo, 1943; Melis, 1953; La Face, 1959; Neuenschwander et al., 1986) and in previous studies we also observed peaks of captured adults during spring (April) at landscape scale (Ragaglini et al, 2004). Moreover Melis (1953) observed that all the pupae survived, from those collected in autumn, developed in adults before May. Other authors (Raspi et al., 1996) reports that during April about 60% of caught females had mature eggs. At present some hypothesis could explain the ecological significance of male and female adults in the olive grove during spring season: 1) adults may migrate on large scale from the interior hill zones to the coastal plain ones (Lupo, 1943) to find favourable climatic conditions or available host fruits; 2) adults moves on a small scale from the olive groves to feed on other host plants (Del Rio et al., 1977); 3) females, after mating, enter reproductive diapause till the olive fruits are suitable (Raspi et al, 1997; Raspi et al, 2002). In our previous studies at regional scale we identified areas where high infestation levels occurred since early summer (Petacchi et al., 2004), and in one of these areas we carried out a landscape scale investigation by use of a pheromone traps network. We observed large peaks of captured adults during the spring, than the captures decries during May and rise up in late June (Ragaglini et al., 2004). On the base of these data we suppose that captures occurred during April were caused by adults developed from overwintering pupae in the soil and that these adults complete another generation before July.

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Materials and methods

Overwintering populations were studied at landscape scale (Ragaglini & Petacchi, 2004) monitoring Bactrocera oleae using a pheromone traps network from winter till the early summer. For the identification of the areas with high infestation risk since July we used infestation data from the Regional monitoring network, focusing on the first generation infestation beginning and active infestation thresholds.

Landscape scale The area (400ha) was located in S. Vincenzo (Livorno – Italy) and landscape refers to a heterogeneous area which differs in land form, land use and management. On the whole experimental area olive harvesting usually finishes before December, so until July no host fruits are available. 45 pheromone traps (type DACOTRAP, produced by ISAGRO Italia) network was used to sample adults from 2003 to 2005. Samples were carried out weekly all over the year (including winter) except during 2003 when we sampled from late winter (March) to early summer (July). From capture’s dynamic we observed that peaks of captured adults occurred during the spring (April) of each year. The day in which traps catches amounts up to 50% of the total captures occurred during spring is used to identify the mean data of adults’ emergence (Figure 1). To verify if the insect can complete one generation from the harvesting time to the spring captures time and another generation from the spring captures time to the beginning of the infestation, we used a degree day model based on daily temperatures. The model was run backward and forward from observed data of adults’ emergence. The parameters of the model are 8.99°C such as minimum development temperature and 379.01°D such as accumulation threshold (Crovetti et al., 1982).

Regional scale Spatial data analysis and geostatistical techniques permitted us to put in evidence those zones were the insect can cause high infestation levels even since July (Guidotti et al., 2005). In this work we characterized the infestation risk level during early summer using three years of Bactrocera oleae infestation data (2002, 2003 an 2004) from the Tuscan monitoring network agroambiente.info (Petacchi et al., 2004). For each year we considered the week when the infestation starts and the weeks when active infestation exceeds 5% and 10% thresholds (Petacchi et al., 2004). Data analysis was carried out using geostatistic and spatial analysis techniques: semi- variography was used to study autocorrelation and spatial variability; kriging interpolation model was used to predict continuous spatial value from discrete points network (Petacchi et al., 2004); zonal-statistic was used to refer predicted values to the polygonal shape of Communes.

Results

Landscape scale The mean data of adult’s emergence occurred always in April (Figure 1). In particular during 2003 50% of captures occurred at the beginning of April (Figure 1), while in 2004 it occurred at the end of the same month and in 2005 during the second week of April.

311

1,00 100%

0,90 90%

0,80 80% 0,70 70%

0,60 60%

0,50 50% 0,40 40%

0,30 30% 0,20 20% normalized catches rate catches normalized

0,10 10% (%) catches trap cumulative

0,00 0%

3 4 4 /4 5 /3 /4 4 5 6 7/3 5/ 2/4 8/5 9/6 3/ 7/ 4/5 7/6 7 4 4/ 1/ 21/2 22/ 2 27/5 20 2 21/2 21/3 19/ 16/5 2003 2004 2005

= normalized catches = cumulated catches

Figure 1: In graphics are represented normalized trap catches (continuous line) and cumulated trap catches (dashed line) of B.oleae during the spring of 2003, 2004 and 2005; the circle show the day when catches amount up to 50% of total.

The degree day model running backward from these date showed that a generation could be completed starting from the half of previous October (Figure 2) when peak of captures and peak of eggs’ deposition occurred, as showed by the collected data (2004) and by the monitored farms of the area (www.agroambiente.info, 2002, 2003 and 2004). The degree day model running forward from the spring emergence date showed that a generation could be completed at the end of May or beginning of July, when captures rose again after a period of minimum of captures (Figure 2).

120

110

100

catches (n°) catches 50

0 27/9 27/1 27/3 27/5 27/7 27/9 27/1 27/3 27/5 27/7 27/9 27/1 27/3 27/5 27/7 27/9 27/1 27/3 27/11 27/11 27/11 27/11 Date

. = observed date % = estimated date Í = accumulation of 379,01°D Figure 2: Graphic refers to the degree day model simulation and shows date predicted (%) from the observed ones (.); the arrows point to the amount of 379,01 °D; catches amount during the three years is represented by the filled area. 312

Regional scale Spatial analysis permitted us study infestation data from the regional monitoring network. We used geostatistic kriging interpolation to describe B.oleae infestation spatial pattern for 2002, 2003 and 2004 (Petacchi et al, 2004). From those results we estimated the map of the infestation risk during early summer (Figure 3) showing that the risk since July is higher in the coastal areas (dark grey levels). In particular in the darkest grey zones (Val di Cornia and Maremma) Active Infestation exceeded the 10% threshold every year since July. Our experimental area is included in the very high class of risk (Figure 3).

San Vincenzo

Infestation risk level during early summer.

Very high High Possible Low Very low (Never)

Figure 3: Map of infestation risk during early summer; classes of risk are represented by grey scale.

Discussion

The degree day model simulation shows that spring adults can derive from adults observed during the harvesting season. So we suppose that spring captured adults derive from previous autumn population. In fact these results confirm and better explain previous experiences of Michelakis (1981) and especially of Melis (1953) that observed, in the same experimental area, every adults emerged in the spring period from pupae collected during the autumn. The degree day model simulation also shows that spring adults can complete a generation before July. We suppose that spring captured adults can complete another generation, probably in not collected olive fruits. According to this hypothesis Melis observed that no one of the emerged adults survived until July (Melis, 1953) and Raspi observed that in this period about 60% of caught females had mature eggs (Raspi et al., 1996). Spatial data analysis put in evidence those coastal zones of Tuscany where high infestation levels can occur since early summer (July). Therefore we suppose that, in coastal areas, the high density of B. oleae 313

population in early summer are due to better climatic condition for overwintering pupae in the soil and for spring emerged adults, thus the risk of infestation since July is very high. In conclusion we think that it is possible to carry out spring preventive adulticide treatments to improve IPM or organic management in areas where high infestation is expected. In the future we will provide to verify our hypothesis by investigations aimed at clarifying biological aspect of spring populations and testing the effectiveness of preventive treatments.

Acknowledgements

The works was supported by the Tuscany Region Project “Improvement of Olive Quality” (EC Reg. 528/99). The authors wish to thank Massimo Ricciolini, Massimo Toma, Angelo Bo and Sabina Palchetti from ARSIA (Tuscany Region Agency on Agricultural Development) for the collaboration in managing the monitoring network and geo-referencing of farms. We are grateful to all the farm advisors who participated in the project.

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