Proceedings of the 5th International Conference on ...

Proceedings of the 5th International Conference on "Olive Culture, Biotechnology and Quality of Olive Tree Products"

OLIVEBIOTEQ 2014 3-6 November, 2014 Amman – Jordan

Edited by Dr. Salam Ayoub, NCARE Dr. Mohammad Ajlouni, AARINENA

Organizing Committee: Dr. Fawzi Al-Sheyab - Chair, NCARE, Jordan Dr. Mohamad Al Ajlouni - AARINENA Dr. Halim Ben Haj Salah - ICARDA Dr. Mostafa Qrunfleh - Faculty of Agriculture, University of Jordan Dr. Muien Qaryouti - NCARE, Jordan Dr. Salam Ayoub – NCARE, Olive Network, Jordan Dr. Mohammed AlQasem - NCARE, Jordan Dr. Nawaf Freihat - Faculty of Agriculture, Jordan University of Science and Technology Dr. Khalid Al-Absi, Faculty of Agriculture, Mu’tah University, Jordan. Dr. Taleb Abu-Zahra - Faculty of Agricultural Technology, Al-Balqa' Applied University, Jordan Mr. Mohmoud Al Ouran - Jordan Farmers Union, Jordan Eng. Jamal Al Batsh - Ministry of Agriculture, Jordan Eng. Islam Maghaireh - Agricultural Engineers Association, Jordan Eng. Nidal Samain - Jordan Olive Oil Producers Association Ms. Ruba Daghmish - Jordan Olive Products Exporters Association (JOPEA), Jordan International Advisory Committee: El Sheyab, F., Jordan Solh, M., ICARDA Chartzoulakis K., NAGREF, Greece Caruso T., University of Palermo, Italy Ismaili - Alaoui M., IAV, Morocco Roussos S., IRD, France Barjol J.L., IOC, Spain Sebastiani L., Scuola Super. Sant' Anna, Italy Boujnah, D., AARINENA Olive Network Coordinator, Tunisia Scientific Committee: Abdel-Wali M., Jordan Al-Absi K., Jordan Alaoui M., Morocco AlQasem M., Jordan Aparicio R., Spain Ayoub S., Jordan Belaj A., Spain Boubaker H., Morocco Caballero J., Spain Centritto M., Italy Chalak L., Lebanon Chartzoulakis K., Greece Ferguson L., USA Fernandez E., Spain Freihat N., Jordan

Fernandez-Escobar R., Spain Gargouri K., Tunisia Gucci R., Italy Kalaitzaki A., Greece Kamoun N., Tunisia Koubouris G., Greece Lanza B., Italy Lopez-Lopez A., Spain Ozkaya M., Turkey Ouazzani N., Morocco Parra-López C., Spain Pereira, José A., Portugal Perica S., Croatia Psarras G., Greece Qrunfleh M., Jordan

Proceedings of the 5th Int. Conf. Olivebioteq 2014

Rapoport H.F., Spain Roussos S., France Rugini E., Italy Rusan M., Jordan Sayadi S., Spain Sebastiani L., Italy Sergeeva V., Australia Servili M., Italy Shdiefat S., Jordan Stefanoudaki E., Greece Tous J., Spain Vita Serman F., Argentina Xiloyannis C., Italy

2

th

OLIVEBIOTEQ 2014

5 International Conference for Olive Tree and Oil Production

Organizers Association of Agriculture Research Institutions in the Near East & North Africa (AARINENA) National Center for Agricultural Research and Extension (NCARE) International Center for Agricultural Research in the Dry Areas (ICARDA)

In cooperation with Ministry of Agriculture, Jordan

Supporters International Olive Council (IOC) Scientific Research Support Fund, Jordan

Sponsors Jordan Olive Products Exporters Association (JOPEA) Greater Amman Municipality

Conference Secretariat Ms. Hazar Nazzal, AARINENA Mrs. Hala Hamati, ICARDA Mr. Hisham Athamneh, NCARE


3

Table of Contents

12

PREFACE INTRODUCTORY PRESENTATIONS What IOC consider people should really need to know about olive oil J. L. Barjol

15

Olive Sector in Jordan J. Albatsh

16

GENETIC RESOURCES / BREEDING / PROPAGATION Olive genetic resources: current status of characterization, evaluation and use in breeding programs A. Belaj

18

Centennial Olive Trees in Lebanon: a Substantial Patrimony L. Chalak, F. Malas, B. Hamadeh, L. Essalouh, B. Khadari

19

Towards the identification of SSR marker linked to vigor in olive tree (Olea europea L.) H. Zaher, B. Boulouha, M. Baaziz, F. Gaboun, S.M. Udupa

26

Search for new duel use olive clones in the Central East of Tunisia: Characterization for pomological and chemical traits I. Laaribi, M. Mezghani Aïachi, H. Gouta, M. Ayadi, F. Labidi, M. Mars

32

Preliminary Evaluation of Oil Content and Fatty Acids Profile for Olive Varieties Cultivated in Lebanon M. El Riachy, M.Breidi. G. Abou-Sleymane, L.Chalak

39

Evaluation of some olive cultivars under Kuwait environmental conditions H. Al-Menaie, O. Al-Ragam, H. Mahgoub, M. Al-Hadidi, A. Al-Shatti and M. Al-Zalzalah

45

Evaluation of nutrient uptake in different olive cultivars grafted on Gemlik rootstock M. Azimi, M. Ozkaya, H. Çolgecen, H. Buyukkartal

51

Histological evaluation of graft compatibility in Olea europea L. scion/rootstock combinations M. Azimi, H. Colgecen, M. Ozkaya, H. Buyukkartal

56


4

Somatic embryogenesis and plant regeneration from radicles of olive (Olea europea L) zygotic embryos, cv. Chemlal. K. Titouh, K. Hadj Moussa, M. Khelifi-Slaoui, L. Khelifi Use of morphological descriptors and principal component analysis for studying the variability of olive descendants issued from controlled pollination I. Laaribi, M. Mezghani Aïachi, F. Labidi, M. Mars

63 69

Performance of olive hybrids (Olea europaea L.) derived from hybridization program in Tunisia I. Guellaoui, F. Ben Amar, M. Boubaker, M. Ayadi, R. Ayadi, A. Yengui, N. Ben Belgacem

76

Selection of the best olive genotypes from several crosses among cultivated varieties B. Alfei, A. Paoletti, A. Rosati, A. Santinelli, G. Pannelli

81

Characterization of ancient olive genotypes in Emilia-Romagna region: Molecular genotyping, chemical and sensory properties of monovarietal olive oils L. Morrone, A. Rotondi, D. Beghè, T. Ganino, A. Fabbri 89 Preliminary results of olive improvement using gamma radiation A. Chaari, M. Maalej, A. Chelli-Chaabouni, S. Baccari

96

Assessment of variability for three olive varieties in Montenegro B. Lazović, M. Adakalić, D. Bandelj, T. Perović

101

Genotype effect on embryogenic capacity and plant regeneration from olive (Olea europaea L.) callus, cv. Picual K.Titouh, L. Khelifi, K. H. Moussa, S. Cerezo-Medina, J. Mercado, F. Pliego-Alfaro

107

Micropropagation of 'Chemlali' olive (Olea europaea L.) by in vitro germination of zygotic embryos K. H. Moussa, K. Titouh, M. Khelifi-Slaoui, L. Khelifi

114

MOLECULAR BIOLOGY / BIOTECHNOLOGY Hydroxytyrosol – An attractive olive phytochemical produced through metabolic engineering in E. coli F. Ververidis, E. Trantas, Th. Pavlidis, E. Navakoudi, A. Kontaratou, E. Mpalantinaki

122

Identification of the genes in olive (Olea europaea cv. “Koroneiki”) that may be involved in the biosynthesis of hydroxytyrosol N. Mougiou, E. Trantas, A. Argiriou, F. Ververidis, A.M. Makris, K.E. Vlachonasios

129


5

A de novo transcriptome approach to identify the gene expression in the development of the pollen tube in olive (Olea europaea L.) D. Iaria, A. Chiappetta, V. Vizzarri, I. Muzzalupo

135

BIOLOGY / PHYSIOLOGY Chemical and histological analysis of olive tree tissues to understand seasonal starch distribution E. Cauli, G. Bandino, P. Sedda, R. Zurru and M. Mulas

143

The adaptations and acclimation mechanisms to salt-stress in roots of salt-sensitive (Leccino) and salt-tolerant (Frantoio) olive cultivars L. Rossi, A. Francini, A. Minnocci, L. Sebastiani

150

Metabolic profiling and gene-expression analysis in salt stressed olive trees (Olea europea L.) cultivars Leccino and Frantoio L. Rossi, M. Borghi, A. Francini, D. Xie, L. Sebastiani

155

Effect of Sodium Chlorid on olive seedlings under controlled conditions S. Baccari, A. Chaari-Rkhis, A. Chelli-Chaabouni

161

Frost tolerance and recovery ability of eleven olive cultivars in central Italy E.M. Lodolini , B. Alfei, A. Santinelli, T. Cioccolanti, D. Neri

167

Chilling and heat requirements of different olive varieties (Olea europaea L.) grown in the south of Tunisia I. Zouari, M. Aïachi-Mezghani, A. El-Aroui, F. Labidi, M. Mars

172

PLANTING SYSTEMS / ORCHARD MANAGEMENT Olive planting systems and mechanization J. Tous

181

Characterizing olive tree geometry features using Unmanned Aerial Vehicle (UAV) images J. Torres-Sanchez,, F. Lopez-Granados, N. Serrano, O. Arquero, R. Fernandez-Escobar, J.M.Pena

194

Effect of Planting Density on the Behaviour of Sourani Olive Tree under Kuwaiti Environment O. Al-Ragam, H. Al-Menaie, H. Mahgoub, M. Al-Hadidi, A. Al-Shatti and M. Al-Zalzalah

199

A comparative study of hand-held harvesting machine with traditional methods used for olive harvesting in Jordan R. Ahmad, S. Ayoub

206


6

Adaptation of different varieties of olive tree to high-density planting systems O. Arquero, N. Serrano, M. Vinas, M. Lovera

212

IRRIGATION / STRESS PHYSILOGY / NUTRITION An environmental-friendly approach for a productive olive orchard management C. Xiloyannis, A. Palese

218

Physiological responses to deficit irrigation of young olive trees (Olea europaea, L.) grown in semi-arid area of Morocco A. Bouizgaren, L. Sikaoui, H. Boula, A. El Antari, M.Karrou, V. Nangia, T. Oweis

226

Effect of switching from surface to drip irrigation on the performance of mature olive trees in a dry area of Morocco A. El Antari, L. Sikaoui1, A. Bouizgaren, M. Karrou, H. Boulal, M. Idrissi, Y. Ouguas, V. Nangia, T. Oweis

233

Responses of olive varieties to restrictive water regimes M. Aïachi-Mezghani, C. Masmoudi –Charfi, I. Zouari, F. Labidi, L. Attia, M. Gouiaa

241

The effect of different irrigation strategies on olive oil (Memecik cv.) antioxidant content and activity D. Sevim, O. Koseoglu, U. Kaya, M. Parlak, N. Pouyafard, T. Çakır, E. Akkuzu

249

The effect of foliar fertilization on carbohydrates status of 'Chemlali' olive (Olea europaea L.) leaves cultivated under rain-fed conditions I. Zouari, M. Aïachi-Mezghani, B. Mechri, F. Labidi, F.Attia, D. Boujneh, H. Chehab, M. Hammami

256

Development and validation of an irrigation scheduling tool for olives to improve water use efficiency at farm level K. Chartzoulakis, G. Psarras, I. Kasapakis, M. Bertaki

262

Arbuscular mycorrhizal fungus (Glomus deserticola) enhance drought tolerance of olive tree (Olea europaea) W. Khabou, T. Gargouri, S. Kammoun

267

Variability of the floral biology of four olive cultivars grown under two watering regimes in an arid region of Tunisia M. Aïachi-Mezghani, A. Diab, A. Laroui, I. Zouari, I. Laaribi, F. Labidi, L. Attia, M. Mars

274

Efficacy of partial root zone drying application on olive tree in arid climate L.Trablesi, S.Maktouf, M.Ghrab, M.Khlifi, N.Soua, K.Gargouri

282


7

Physiological responses of young olive (Olea europaea L. cv Ayvalık) trees to water stress N. Pouyafard, E. Akkuzu, U. Kaya

291

Effect of deficit irrigation strategies on canopy temperature and leaf water potential in olive (Memecik Cv.) U. Kaya, T. Cakır, M. Parlak, N. Pouyafard, M. Gurbuz, G.P. Mengu, E. Akkuzu

298

Ecophysiological response of young 'Chemlali' olive plants under three irrigation regimes. M. Gouiaa, F. Zaouay , D. Boujnah

304

Rainfed olive production as influenced by low quantity of applied water during the first stage of fruit growth R. Razouk, A. Kajji, M. Karrou

311

PEST AND DISEASE CONTROL Integrated Pest Management of olive tree in Tunisia M. Ksantini

318

Effective, environmentally-sensitive pest management in olive production V. Sergeeva

319

Evaluation of susceptibility of the most cultivated olive trees cultivars in Tunisia to Verticillium wilt disease (Verticillium dahlia) M.A. Triki, H. Hassairi, F. Ben Amar, Y. Gharbi, I. Hammami, S. Krid, W. Khabou, A. Rhouma and R. Gdoura

326

PESTOLIVE: a Mediterranean research project for understanding and managing soil-borne parasites on olive using historical and ecological approaches T. Mateille, M. Achouri, M. Ater, A. Belaj, G. Besnard, P. Castillo, E. Chapuis, R. De La Rosa, F. De Luca, A.M. D’Onghia, H. El Maraghi, C. El Modafar, A. El Mousadik, A. El Oualkadi, Z. Ferji, N. Horrigue-Raouani, R.M. Jimenez-Diaz, M. Kadiri, S. Kallel, B. Khadari, B.B. Landa, L. Leon, M. Montes-Borrego, A. Moukhli, J.A. Navas-Cortes, A. Öcal, N. Sasanelli, J. Tavoillot, M.A. Triki, A. Troccoli, E. Tzortzakakis, M. Ulas, N. Vovlas, T. Yaseen

331

Combined effectiveness of tebuconazole-trifloxystrobin against infections of Fusicladium oleagineum in comparison to conventional products V. Vizzarri, N. Iannotta , I.Muzzalupo, T. Belfiore

337

Learning from olive evolution and cultivation to understand the diversity of associated plant- parasitic nematodes communities in Morocco N. Ali, E. Chapuis, J. Tavoillot, M. Aït Hamza, Z. Ferji, A. El Mousadik, A. El Oualkadi, G. Besnard, A. El Bakkali, A. Moukhli, B. Khadari, C. El Modafar, M. Ater, T. Mateille

344


8

Effect of olive varieties and rearing substrates on plant-parasitic nematode communities in southern Morocco olive nurseries M. Ait Hamza, Z. Ferji, N. Ali, H. Tazi, J. Tavoillot, A. Moukhli, H. Lakhtar, S. Roussos, H. Boubaker, A. El Mousadik and T. Mateille

351

Current status of Olive Weevil Rhynchites cribripennis Desbrochers (Coleoptera: Attelabidae) in Montenegro T. Perovic, S. Hrncic, M. Cizmovic

358

OLIVE OIL QUALITY/ HEALTH / TABLE OLIVE Olive Oil Quality and Health A. Kiritsakis, D. Gerasopoulos, E. L. Iorio and K. Kiritsakis

363

Hepatoprotective activity of oleocanthal extracted from olive oil amurca in the rat S. Janakat, A. Al Amour

364

Preliminary results of olive zoning study in the province of Sassari: the peculiarities of cv Bosana oils produced in different growing areas L. Morrone, A.Rotondi,N. Di Virgilio,B.Alfei, C.Cantini, E.Vagnoni, P.Duce

371

Nutritional value and health benefits of table olives F. Gungor, A. Yildırım

378

Comparison of single strain starter culture and a selected inoculum enrichment in the processing of natural table olives M. Campus, P. Sedda, E. Cauli, R. Comunian, A. Paba, E. Daga, S. Schirru, R. Zurru, G. Bandino

385

OLIVE MILL WASTE MANAGEMENT Alternative technologies for olive mill wastewater management with emphasis on soil application K. Chartzoulakis, G. Psarras, N. Kalogerakis, F. Santori

392

The different points of view of olive mill wastewater in Turkey: A result of New Ecological Paradigm R. Tunalioglu, R.Yildirim

400

Valorization of olive husk into valuable organic amendment: does initial C/N ratio affect the quality of the produced compost? K. Azim , S. Roussos , C. Périssol, I. Thami Alami , B. Soudi

404

Production of extracellular lignocellulolytic enzyme by Streptomyces Sp grown on olive pomace L. Medouni-Haroune, M. Kecha, F. Zaidi, S. Roussos, V. Desseau

412


9

Influence of olive mill wastewater applied at different quantities on an alkaline soil fertility R. Razouk, K. Oubella

420

Performance of Jet-Loop reactors with ultrafiltration membrane system (JACTO.MBR) for olive mill wastewaters biotreatment B. Ribeiro, I. Torrado, L. Baeta-Hall, A. Amer, M. Rusan, A. Eusébio

426

ECONOMICS / MARKETING Olive Oil Promotion: A must for the future. M. Rappou

434

Performance of the olive trees and economic profitability of olive production in the Haouz region in Morocco: effect of drip irrigation with full and deficit water regimes compared to flooding method A. Ait Hmida , L. Sikaoui , M. Karrou, V. Nangia

440

Estimating price and income elasticity of olive oil demand in Libya for the period 1980-2010 K. Elbeydi , A. Hamuda

445

The diversity of the Italian monovarietal extra virgin olive oils: from trained panel to consumer experience B. Alfei, M. Magli, A. Rotondi, L. Morrone

451

Analysis of efficiency in organic and conventional Italian olive farms using F.A.D.N. dataset N. Galluzzo

460

Olive growing in Morocco: Competitive agribusiness M. Ismaili Alaoui

467

LIST OF PARTICIPANTS

468


10

Acknowledgements We wish to thank all the institutions, organizations, sponsors and colleagues who supported the OLIVEBIOTEQ 2014 conference. We are also grateful to the owners of olive farms and mills who gratefully accepted to host the technical tour visits. We also thank the International Advisory Committee, the Scientific and Organizing Committees for the support given in the organization of the Conference. A special thank goes to our colleagues from the National Center for Agricultural Research and Extension (NCARE), International Center for Agricultural Research in the Dry Areas (ICARDA) and Association of Agriculture Research Institutions in the Near East & North Africa (AARINENA) who sustained our efforts with enthusiasm and encouragement.


11

Preface The olive tree is among the oldest known cultivated trees in the word. It is a symbol of peace and it was mentioned in the literature of many religious. The olive tree has played a vital part in the life of human kind and has been substantial for the development of culture and food. The olive tree is a functional plant in the agricultural system of many countries and has acquired a huge socio–economic importance over the centuries. Owing to its longevity and adaptability, it plays a major role as a feature of the landscape and as a crop that grows even in harsh soil and difficult climatic conditions. Emphasizing that olive cultivation governs the existence and standard of living of millions of families who are dependent on the measures taken to maintain and expand the consumption of olive products and to enhance the world economy for such products. Olive oil and table olives are essential basic commodities in the regions where olive growing is established, and they are basic constituents of the Mediterranean diet and recently also of other diets. OLIVEBIOTEQ 2014 brings together scientists, technicians, managers, exporters, policy makers, and students to exchange experience of new approaches to olive production and marketing, and to develop new thinking for approaches to a sustainable and more profitable global olive industry. This fifth gathering of international olive industry leaders build on a series of past OLIVEBIOTEQ conferences:  OLIVEBIOTEQ-2004 in Errachidia, Morocco ‘Biotechnology and olive product quality in the Mediterranean’;  OLIVEBIOTEQ-2006 in Palermo, Italy;  OLIVEBIOTEQ-2009 in Sfax, Tunisia;  OLIVEBIOTEQ-2011 in Crete, Greece. OLIVEBIOTEQ-2014 Conference was held in Amman, Jordan at the Holiday Inn Hotel during the period from 3-6 November 2014. OLIVEBIOTEQ-2014 conference as an international forum for olive sector aiming at promoting scientists, technicians, students as well as managers, exporters and policy makers to exchange experiences, ideas and knowledge in the olive sector and developing further cooperation for a new, sustainable and profitable olive industry around the world. This conference was a good opportunity to meet at Amman, Jordan, the beautiful Mediterranean country with old civilization, long olive history, and hospitality, to discuss the participants’ experiences and disseminate the results of these discussions to all global professionals to improve the olive industry in very sustainable, economical, and environmentally sound way. The health and nutritional related properties of olive products were highlighted as well as their role as functional foods attempting to promote and increase the olive products consumption. The conference was attended by more than 100 participants from 22 countries including: Algeria, Australia, Brazil, China, Egypt, France, Greece, Italy, Kuwait, Lebanon, Libya, Proceedings of the 5th Int. Conf. Olivebioteq 2014

12

Montenegro, Morocco, Palestine, Portugal, Spain, Syria, Tunisia, Turkey, United Kingdom, USA and Yemen, in addition to 33 participants from Jordan; the host country. The Jordan Olive Products Exporters Association organized olive products exhibition during the conference for 5 leading producers, this exhibition included extra-virgin olive oil and table olive products. The following topics were covered by the conference:  Genetic resources / breeding / propagation;  Biology / physiology / biotechnology;  Planting systems / pruning / harvesting;  Irrigation / stress physiology / nutrition;  Pest and disease control;  olive oil quality and health / table olives;  Olive mill waste management;  Economics and marketing. More than 80 papers were submitted to the scientific committee for the proceedings. The scientific committee had done tremendous work in selection and revision of the papers. We wish to thank all authors of oral and poster contributions, the invited speakers and all members of the scientific committee that made a special effort working hard all along the evaluation process to make possible this publication. We hope that all participants were enriched by the presentations of the conference and have enjoyed the stay and visits to olive orchards, mills and the historical places of Jordan. We are confident that this proceeding will give us the opportunity to update our knowledge on olive culture and its derivative products that is gaining growing importance in the scientific community and the private sector around the world.


13

INTRODUCTORY PRESENTATIONS


14

WHAT IOC CONSIDER PEOPLE SHOULD REALLY NEED TO KNOW ABOUT OLIVE OIL J. L. Barjol International Olive Council (IOC), Madrid, Spain. Abstract Since 1959, the IOC is the international, intergovernmental forum for debating olive and olive oil issues. Member governments enter into a commitment to apply IOC standards in their international trade. Nowadays, the IOC has 17 members that account for 97% of world olive oil production and 96% of world exports. World consumption has almost doubled in 25 years. At present, around 3 million tones of olive oil are produced in the world, of which 0.8 million tones go into international trade (intra‐European Union trade excluded). More than 80% of the world’s olive oil is produced by five countries: Spain (45% share of the total) followed by Italy and then Greece, Tunisia and Turkey. The top importers are the United States, the European Union (excluding intra‐ European Union trade), Brazil (newcomer), Japan, China (newcomer), Canada and Australia. These are followed by a long list of other countries since olive oil is now a globalised product. The IOC is mandated to fix the designations and definitions of olive oil to be used by member governments in their international trade. A clear distinction has to be made between the family of virgin olive oils (extra virgin, virgin and ordinary virgin plus lampante virgin which is not fit for direct human consumption) and olive oil (blend of refined lampante virgin olive oil with extra virgin or virgin olive oil) and olive pomace oil (blend of refined crude pomace oil extracted from olive pomace, the by‐product of virgin olive oil extraction, with extra virgin or virgin olive oil). IOC standards fix purity criteria to guarantee that the oil has been obtained solely from olives and quality criteria (including sensory evaluation performed by a recognized panel of trained tasters) to classify the different types of virgin olive oils. Methods and limits are also specified in the standards alongside the criteria. All are regularly updated on the basis of scientific research validated by scientific peers as well as in ring tests to make sure there are no problems due to the geographical or varietal origin of oils. Virgin olive oils, olive oil and olive pomade oil are predominantly made up of monounsaturated fatty acids. Their positive impact on health is well documented (especially in preventing cardiovascular disease and decreasing LDL‐cholesterol without decreasing HDL‐cholesterol) and justifies the issue of a qualified health claim. The minor components (antioxidants and vitamins) present in virgin olive oils (and to a certain extent in olive oil and olive pomade oil because they are blended with virgin olive oils) have a high biological value. They help to prevent various diseases (cancer, diabetes, high blood pressure …), combat ageing and protect from memory decline and neurodegenerative diseases (Alzheimer’s, Parkinson’s). Keywords: Olive oil, International Olive Council, quality, standards, international trade, health.


15

OLIVE SECTOR IN JORDAN J. Albatsh Ministry of Agriculture, Jordan Abstract Jordan is considered as one of the homelands and natural habitat of olive cultivation. The olive industry is one of the most important components of the Jordanian agricultural sector. The area planted with olive trees in 2013 reach to 125,986 hectare, representing 77% of the total area planted with fruit trees, and 44% of the actually cultivated area in Jordan. The sector also constitutes a pillar to socio-economic development in the country, since it is a major tool for poverty and unemployment alleviation. Thus, olive farming is an important income source for 80 thousand Jordanian families. Jordan investments in olives sector is more than one billion JOD including the value of cultivated lands and related industries. Olive tree is mainly cultivated within two areas in Jordan; first, the western highlands (rain fed) crossing Jordan from North to South and second, the Northern Western deserts area (irrigated). According to the Ministry of Agriculture 2013 statistics, around 77.3 % of the total area of olives is rain fed area, where 22.7% of the total area is under permanent irrigation. In addition, around 87% of the trees are at the production stage and some 80% of their production is converted into oil. Jordanian olive oil is characterized by a natural flavor and excellent chemical composition compared to other oils over the world. There is high potential for producing best quality of virgin olive oil in both rain-fed and irrigated areas. Olives are mainly utilized for olive oil and table olives. The most dominating olive cultivars in Jordan are: Nabali, Rassei and Souri. The latter is mainly grown in Jerash and Ajloun. These local cultivars give oil with distinguished chemical and sensory features as witnessed by world experts in tasting. According to MOA statistics, the estimated olive production during this season 2014/2015 is about 178 thousand tons; 36 thousand tons of this harvest consumed as green and black table olive. The rest quantities are used for oil extraction to produce 25 thousand tons of virgin olive oil. Domestic production of olives oil is usually fluctuated from year to year, due to alternate bearing phenomena. However, domestic supply of olive oil can meet consumption needs at average price. Jordan has achieved self-sufficiency of olive oil in 2000. Olive’s sector operates around 131 mills with total capacity of (369 tons) per hour. More than 90% of those mills are recent and supplied with modern lines which preserve the quality of oil. It's worth to be mentioned that Jordan has joined the International Olive Council (IOC) in December 2002 to meet the requirements of the coming era and to cope up with the international changes in olive industry and olive oil market around the world. The Ministry of Agriculture in cooperation with the European Programme for Industrial Development and IOC had trained four olive oil tasting panels and two table olive tasting panels. That is to up-grade the quality of Jordanian olive oil and table olive to meet the Jordanian standard which leads to improve the competitiveness of Jordanian olive oil and table olive and increase its ability to penetrate the international markets, as well as promoting its consumption at the local level. Keywords: Jordan, olive area, olive oil, olive cultivars, production, consumption


16

GENETIC RESOURCES BREEDING PROPAGATION


17

OLIVE GENETIC RESOURCES: CURRENT STATUS OF CHARACTERIZATION, EVALUATION AND USE IN BREEDING PROGRAMS A. Belaj IFAPA Centre “Alameda del Obispo”, Córdoba, Spain Corresponding author: [email protected]

Abstract Unlike most other fruit species, as a consequence of tree longevity and lack of turnover with new genotypes, the genetic patrimony of olive is very large. More than 1,200 cultivars maintained ex situ in 79 international and national repositories, are still actively cultivated. In addition, in situ conservation of ancient olive trees has recently been considered as an alternative approach to preserve this unexploited reservoir of genetic diversity. Wild relatives and related subspecies might also be very interesting source of genes for some agronomic traits as resistance to stresses. In spite of this rich genetic biodiversity, the knowledge on olive genetic is still limited. In this paper we report the efforts to characterize, identify and exploit the species genetic resources. Up to date, the application of molecular tools has been mainly related to the analysis of genetic variability in Olea europaea complex and for the olive oil origin traceability. While, the recent generation and application of genomic tools can be of great help for understanding the molecular basis of fruit and oil quality and that of traits of agronomical importance looking for cultivars with more desirable phenotypes for any of these traits. However, due to the long period of time needed and to the difficulties of field trials, there is a certain gap between the molecular and phenotypic evaluation of this diversity. And there is a general need of comparative cultivar field trials in Mediterranean countries. Finally, wild olives are also being characterized at molecular and phenotypic level and the possibility to introgress new and superior alleles into cultivated varieties is under exploration. Keywords: Olea europaea, genetic patrimony, olive cultivars, identification, evaluation, olasters


18

CENTENNIAL OLIVE TREES IN LEBANON: A SUBSTANTIAL PATRIMONY L. Chalak1,*, F. Malas1, B. Hamadeh2, L. Essalouh3, B. Khadari4,5 1

The Lebanese University, Faculty of Agricultural Sciences, Dekwaneh, Beirut, Lebanon. Lebanese Agricultural Research Institute, Fanar Station, Beirut, Lebanon. 3 Montpellier SupAgro, UMR 1334 AGAP, 34398 Montpellier, France. 4 INRA, UMR 1334 AGAP, F-34398 Montpellier, France. 5 Conservatoire Botanique National Méditerranéen (CBNMED), UMR 1334 AGAP, 34398 Montpellier, France. 2

*Corresponding author: [email protected]

Abstract This study aimed to assess the centennial olive trees growing across Lebanon, with the perspective of conservation of the ancient germplasm. The survey indicated the existence of numerous centennial olive trees distributed in different agro-climatic areas, from 80 to 1350 meters altitude across the country. Centennial olives were found in large size orchards and scattered as well as in young orchards, road hedges and gardens for ornamental purposes. Yet, no reliable information is available regarding the age of the centennials, but they can considered as 500 to 1000 years old. Among these, only six centennials located in Bcheale village in the north of the country at 1000 meters altitude are considered as “millenials” or “monumentals” by the Ministry of Tourism, while the remaining ones widespread across the countries are still ignored. As a preliminary morphological characterization of the trees conducted on 292 centennials spread in 48 orchards, a large variability was recorded for foot, trunk and central cavity sizes. Principal component analysis showed that foot and trunk circumferences as well as central cavity diameter were the most discriminating descriptors. Most of the 48 orchards were clustered together in one pool sharing similar traits. The most outstanding orchards were located in 10 locations distributed across Lebanon. At the tree level, six single trees located in North and Mount Lebanon were well differentiated by their large sized foot, trunk and central cavity diameter. These centennials should be further characterized using morphological and agronomical descriptors in order to understand their performance through time and to valorize them in selection programs. Keywords: Olea europaea L., centennials, Lebanon, distribution, trees characteristics.

Introduction Olive history in Lebanon is very old dating back to the old era (Zohary and Hopf, 2000). Centennial and millennial trees are still growing across the country (Mahfoud, 2007). Few of these old trees have an important historical and ornamental value and are already classified as monumental. Few of these ancient olive trees have been recently assessed and compared to traditional local cultivars by using SSR markers, they were found to clearly match the widespread traditional common variety called "Baladi" (Chalak et al., in press). This ancient germaplasm may be linked to the beginning stages of olive growing in the country playing an important role in the domestication process. Other findings in Italy and Spain supported the hypothesis that ancient olive trees might be unknown traditional cultivars that remained uncharacterized and suggested they might Proceedings of the 5th Int. Conf. Olivebioteq 2014

19

represent early stages in the domestication processes of the olive tree in the Mediterranean (Baldoni et al., 2006; Erre et al., 2010; Diez et al., 2011). Unfortunately, the ancient olive trees of Lebanon are threatened of disappearing due to their increasing ornamental value and to the progressive transformation of traditional olive groves into new commercial orchards. Hence, the conservation and characterization of the ancient Lebanese olive germplasm become a priority task. In the present study we undergone a survey of the old groves spread in different agroclimatic areas of the country with a general assessment of the groves status. Also we established a morphological characterization of the centennials by examining mainly the tree dimensions. Further, a set of old groves is recommended for evaluation, valorization and preservation purposes. Materials and Methods Survey and characterization of the groves. A survey was carried out throughout Lebanon during the summer 2013 to identify ancient olive groves or trees growing across Lebanon, particularly in the principal areas of olive production (Fig. 1). Forty eight groves were visited spread between 80 to 1350 m of altitude, 35⁰46'30'' to 35⁰50'34'' of longitude, and 33⁰44'28 to 34⁰12'22'' of latitude.

Fig. 1. Distribution of the 48 centennial olive groves surveyed in Lebanon. General information. A questionnaire following directive and semi-directive method was addressed to the owners, habitants and municipalities in order to assess the general status of the centennial olive trees. It covered in particular the trees age, origin, restoration, land estate, field maintenance, production and phytosanitary status. Personal observations were also noted. Moreover, municipalities and old people were asked for old texts regarding ancient olive trees.


20

Morphologic characterization. A total of 292 trees growing in the 48 groves, with a circumference > 2.3 m and assumed to have more than 300 years old according to information given initially by farmers, were examined for their morphological characteristics. Tree traits were examined including the canopy height , central cavity diameter, trunk circumference at 1.3 m height, trunk foot circumference, crown projection diameter and total height of tree. Results and Discussion Groves distribution and size. Old groves were found in various agro-climatic areas at altitudes ranging from 80 m (Bhannine in the North) to 1350 m (Bcheale village in the North) where minimal winter temperature may sink to -0.01°C. The annual rainfall ranked between 185 mm (Fakehe stand in the province of Baalbek-Hermel) and 600 mm (Deir Mimas in the South). It is worthy noted that groves located in Bcheale, Qabaait and Chaqra groves are covered with snow during winter. Large old groves were found in both South and North Lebanon, either in terraces or in straight lands (Fig. 2). One of the largest centennial groves was located in Deir Marchayna (Zgharta) where 1,400 centennials are still growing in around 2 ha, thus in mixture with other olive trees of all ages. Then comes Kfar Matta with 400 centennials extended in 1.2 ha followed by Marjaayoun (South) with around 225 centennials, the valley of Tair Filsay (South) where more than 100 centennials were scattered among young olive trees. On the other hand, large old groves in Kfar Hamam and Hasbayya (South) were remarkably homogenous with hundreds of centennials having apparently the same age. Centennials were also found as scattered trees in road hedges, family gardens and yards growing mostly as ornamentals. The six old trees located in Bcheale village in North Lebanon and classified as “millennials” or “monumentals” by the Ministry of Tourism are still standing. Other centennials are still surviving as single in many locations such as Haret Jandal (Mount Lebanon), Abou Samra (Tripoli) and Ezki (Minieh-Denneieh). Age, history and related information. Yet no accurate information has been published on the age of the Lebanese olive centennials. Only little oral information given by the habitants and few short texts in newspapers are available. In 1964 Al Nahar newspaper published a short article about the centennials of Kfar Matta, saying they are dating back to 3100 years ago. According to an unpublished study on Bcheale centennials, the oldest trees were estimated to be 1500 years old where others in the same village are thought to be 500 years old (Bou Yazbeck, Pers. Comm.). Many touristic websites mentioned the groves of Bcheale, Chaqra (South) and Kaoukaba (South) as housing centennial olive trees. In some areas, centennials are still designated by the historic era to which they belong. For instance most of the centennials in Mount Lebanon are called Maani, where the Maani’s introduced the olives around 1500 years ago. In the South, most of the centennials are called Romanian going back to the time of Romanians 2000 years ago.


21

Bcheale

Chaqra

Abou Samra

Kfar Matta

Fig. 2. Centennials believed to be the oldest in Lebanon.


22

Property estate. Most of the visited centennial groves are of familial property inherited from ancestors through time. Some other centennials located in the North such as Deir Marchayna, Ezki, and Bcheale are of endowment property. Only Bcheale centennials are maintained by the Ministry of tourism while all others are not yet considered by any governmental or non-governmental organizations. Agricultural practices. Most of the centennial groves as well as the scattered ones are apparently healthy and well maintained in terms of growth without any particular visual problem or nutrient deficiencies. Some centennials are restored by cutting back the trunk and grafting of new varieties (e.g. Qarkaf) while many of them, mostly in the North and Mount Lebanon, are just cut at 50 cm from the base to let the trunk grow its new branches for rejuvenation and production increase. It is worthy to note the neglected and abandoned status of the unique centennial available in Ezki (North) and the three centennials in Kafra (South); however these old trees are still surviving. Production. All the centennial groves visited are still productive and harvested. Olives and oil produced by centennials are usually consumed by the owners, except for the large sized exploitation while productions of the centennials and young groves are mixed together before being commercialized. Recently some information circulated regarding the exportation to USA of Lebanese oil said being harvested on “the world’s oldest living olive trees, Bcheale centennials” (www.dailystar.com.lb/.../193293-bechealehs-ancient-t...). Threats. Many old groves are progressively transformed into new commercial orchards. Others are eradicated to be replaced by buildings and roads. As mentioned above, single centennials are still standing in many areas in the country, alone, witnessing the existence in the past of old groves. In the laste few decades, old olive trees have been increasingly used as ornamentals in the yards of mansions and houses, hotels and touristic resorts, schools and universities, rings and edges of road edges thus after been hollowed out from their original place. Unfortunately, numerous centennials are frequently seen nowadays on the highway sides prepared in large containers to be sold for decoration. Morphological characterization. A total of 292 centennials growing in 48 groves were examined in this study for the morphological characteristics of the tree (data not shown). On the 48 groves studied, only 8% had polycormic trunk, while 92% were monocormic. Large foot circumferences were found in the visited groves varying from 3.25 m (e.g. Aayta Ech Chaab) to 18.7 m (e.g. Haret Jandal). Trunk circumferences (at 1.3 m height from the ground) were comprised between 2.3 m (e.g. Bhannine) to 15.2 m (e.g. Haret Jandal) while most groves have trunk circumferences comprised between 2.3 m and 4.94 m. Trunk cavity was absent in Hmaire, Al Qaouzah, Fakehe and Chhim groves while small to large trunk cavities were found in the other groves with diameter comprised between 0.07 m (e.g. Ouadi Jezzine) to 3.4 m (e.g. Haret Jandal, Kaoukaba, and Kfar Matta. Tree height ranged from 2.85 m (e.g. Al Bireh, Akkar) to 8.1 m (e.g. Hasbayya, South) while many trees had an average height of 5.82 m. The canopy height was comprised between 2.4 m to 7.06 m while most of the trees had an average of 4.49 m. Diameter of crown projection ranged between 5 m (e.g. Hmaire) to 13.7 m (e.g. Haret Jandal) while the majority of the trees had an average of 8.13 m (e.g. Kaftoun, Kfar Kila,and Balde). In some groves such as Al Bireh (Akkar), some centennials had trunk circumference (2.3 m) quietly three times smaller than foot circumference (6.03 m), thus due to the old practice of cutting back the trunk for rejuvenation purpose and leaving just one sucker to grow as a trunk. Most distinguished centennial groves. A two-dimensional principal component analysis was conducted to differentiate between the 48 centennial groves studied on the base of the tree characteristics (Fig. 3). Principal coordinate PC1 clustered the groves according to the foot circumference while the second coordinate PC2 clustered them on the base of central cavity diameter and trunk circumference. The majority of the groves were clustered Proceedings of the 5th Int. Conf. Olivebioteq 2014

23

together in one pool sharing similar traits while few others are found out in terms of large sized foot and trunk circumferences and central cavity diameter. The most outstanding groves were: Kafra (South) with a foot circumference of 12.9 m; Kaoukaba (South) with a central cavity diameter of 1.54 m; Balde (North) with a foot circumference of 12.74 m and a central cavity diameter of 1.33 m; Bcheale (North) with a foot circumference of 12.05 m, a trunk circumference of 6.32 m and a central cavity diameter of 1.88 m; Deir Marchayna (North); Baawarta (Mount Lebanon); Kfar Matta (Mount Lebanon); Tair Filsay (South); Kfar Chouba (South); Kfar Kila (South) with a foot circumference 8.67 m; trunk circumference 3.54 m; central cavity diameter 1.5 m.

Kfar Kila 1,5

Kaoukaba 2 Bcheale 0,5

Bhannine

Kfar Chouba

Al Bireh Kaoukaba 1

Aayta Ech Chaab Kfar Hamam Arde Kaftoun Deir El Amar Bkefteen Kfar Yachit Btourram Al Qaouzah

Mghairiye Mghairiye

Balde

Marjaayoun Beit Hoaouch Al Jarmaq Hasbayya

Ouadi El Leimoun Chaqra Ouadi Jezzine

-0,5

Joun Bater

Qabaait Qana Deir Mimas

Kfar Matta Tair Filsay Deir Marchayna

Gharifa

Baawarta

Ain Yaaqoub Aanout Hmaire

Kafra

-1,5 -2,7

-1,7

-0,7

0,3

1,3

2,3

3,3

Fig. 3. Bi plot on the F1 and F2 axes of PCA for centennial olive trees based on tree traits. Groves of the south are shown in green color, those of the north in red, Mount Lebanon in blue. Most distinguished centennials. According to the individual data collected on each of the 292 centennials, many trees were differentiated for their highest sizes of foot and trunk circumferences and central cavity diameter. The most particular distinguished trees could be listed as following: Haret Jandal (Mount Lebanon) with foot circumference 18.7 m, trunk circumference 15.2 m, central cavity 3.4 m; Bcheaale NLC1 (North) with foot circumference 21 m, trunk circumference 13.5 m, central cavity diameter 4 m; Bater MLC265 (Mount Lebanon) with foot circumference 10 m, trunk circumference 6.45 m, central cavity diameter 2 m; Ezki tree (North) with foot circumference 10 m, trunk circumference 12.3 m, central cavity diameter 2.9 m; Joun MLC228 (Mount Lebanon) with foot circumference 10 m, trunk circumference 6.5 m, central cavity diameter 0.9 m; Abou Samra (North) with foot circumference 9.7 m, trunk circumference 5.1 m, central cavity diameter 1.4 m.


24

Conclusions This is the first report on the assessment of centennial olive groves growing across Lebanon, with the perspective of valorization, conservation and sustainable utilization of the ancient germplasm. Most of the 48 groves studied were sharing similar traits while other groves were outstanding in terms of large foot, trunk and cavity sizes. Moreover, several centennials were differentiated within the groves studied for their large dimensions. Centennials will be further submitted to molecular characterization by DNA markers in order to assess their genetic diversity (Sefc et al., 2000; de La Rosa et al., 2002; Khadari et al., 2008) and to understand the role of the ancient olive germplasm in the global domestication process (Besnard et al., 2013). Furthermore, centennials should be submitted to a detailed morphological and agronomical characterization in order to understand their performance despite their age and to valorize them in selection programs. For that end, the preservation of the centennial groves of Lebanon is crucial. References Baldoni, L., Tosti, N., Ricciolini, C., Belaj, A., Arcioni, S., Pannelli., G., Germana, M.A., Mulas, M. and Porceddu, A. 2006. Genetic structure of wild and cultivated olives in the central Mediterranean basin. Annals of Botany 98: 935–942. Besnard G., Khadari B., Navascués M., et al. (2013) The complex history of the olive tree: from Late Quaternary diversification of Mediterranean lineages to primary domestication in the northern Levant. Proc Roy Soc Lond Ser B 280: 2012-2833 Chalak L., Haouane H., and Khadari B (in press) Lebanese ancient olive trees and their relatedness with the local variety "Baladi". Proc. VII International Symposium on Olive Growing, San Juan, Argentina, 2012. Acta Hort. de La Rosa, R., James, C.M. and Tobutt, K.R. 2002. Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae. Mol. Ecol. Notes 2: 265-267. Dı´ez, C.M., Trujillo, I., Barrio, E., Belaj, A., Barranco, D. and Rallo, L. 2011. Centennial olive trees as a reservoir of genetic diversity. Annals of Botany 108(5): 797-807. Erre, P., Chessa, I., Mun˜oz-Diez, C., Belaj, A., Rallo, L., Trujillo, I. 2010. Genetic diversity and relationships between wild and cultivated olives (Olea europaea L.) in Sardinia as assessed by SSR markers. Genetic Resources and Crop Evolution 57: 41– 54. Khadari, B., Charafi, J., Moukhli, A. and Ater, M. 2008. Substantial genetic diversity in cultivated Moroccan olive despite a single major cultivar: a paradoxical situation evidenced by the use of SSR loci. Tree Genetics and Genomes 4: 213-221. Mahfoud, S., 2007. Green Gold, the Story of Lebanese Olive Oil. 164 pp. Sefc, K.M., Lopes, M.S., Mendonça, D., Rodrigues Dos Santos, M., Laimer Da Cámara Machado, M. and Da Cámara Machado, A. 2000. Identification of microsatellites loci in Olive (Olea europaea L.) and their characterization in Italian and Iberian trees. Mol Ecol 9:1171–1173. Zohary D. and Hopf M., 1994. Domestication of Plants in the Old World, second ed. Clarendon Press, Oxford. http://www.discoverlebanon.com/en/panoramic_views/history-olive-oil.phpref http://www.dailystar.com.lb/.../193293-bechealehs-ancient-t...


25

TOWARDS THE IDENTIFICATION OF SSR MARKERS LINKED TO VIGOR IN OLIVE TREE (Olea europea L.) H. Zaher1,2,*, B. Boulouha1, M. Baaziz2, F. GABOUN3, S.M. Udupa4 1

INRA Marrakech, Morocco Faculty of Sciences Semlalia, Marrakech, Morocco 3 INRA Rabat, Morocco 4 INRA/ICARDA Cooperative Research Project, ICARDA, Rabat, Morocco 2


Abstract In this study, a progeny resulting from a cross between "Menara" and "Arbequina" varieties was characterized by both morphological and SSR markers. The Bulk Segregant Analysis (BSA) was used to establish linkage between molecular markers and vigor parameters (tree height, perimeter of trunk diameter and canopy circumference). Thirty SSR primers were used to compare the parents and bulks of individuals of different vigor. Eleven SSR primers were selected for their ability to detect polymorphism between the two parents and bulks. Results of morphological analysis showed highly significant genotypic differences for all measured parameters. Principal component analysis revealed highly significant phenotypic variation where the first two axes represented 85.32 % of the total variation. For association analysis, results obtained by the General Linear Model showed that some of the used SSR markers are significantly associated with the phenotypic traits studied. Two SSR markers, DCA9 (p 65%), as ‘Soury’ (Iaal) and ‘Baladi’ (Ijd Ibrine) showed the minimum of 64.9%. Proceedings of the 5th Int. Conf. Olivebioteq 2014

41

Table 2: FA composition (%) of VOO samples from the studied olive varieties. Variety (origin) ‘Baladi’ (Kousba) ‘Soury’ (Iaal) ‘Bou Chawkeh’ (Fakehe) ‘Ayrouni’ (Amioun) ‘Dal’ (Bakkifa) ‘Baladi’ (Lebaa) ‘Edlbi’ (Lebaa) ‘Baladi’ (Ijd Ibrine) ‘Nabali’ (Kfarchakhna) ‘Soury’ (Ain Baal) ‘Roumani’ (Qana) ‘Baladi’ (Aitaroun) IOOC (IOOC, 2003) Minimum (%) Maximum (%) Mean (%) Standard Deviation (%)

C14:0 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 ≤ 0.05 0.01 0.02 0.01 0.00

C16:0 11.5 12.0 10.4 14.3 12.0 12.4 12.1 13.1 15.5 13.4 12.3 11.3 7.5-20 10.4 15.5 12.5 1.4

C16:1 0.4 0.5 0.7 0.7 0.6 0.4 0.7 0.8 0.9 0.5 0.4 0.4 0.3-3.5 0.4 0.9 0.6 0.2


C18:0 C18:1 4.0 69.0 4.5 64.9 1.9 73.7 3.4 68.0 2.3 67.1 3.5 70.8 3.3 67.5 3.1 64.9 2.7 65.3 3.6 67.5 3.8 71.9 3.4 73.5 0.5-5 55-83 1.9 64.9 4.5 73.7 3.3 68.7 0.7 3.2

C18:2 11.4 14.4 10.3 9.9 14.4 8.4 12.8 15.0 11.6 11.2 7.8 8.0 3.5-21 7.8 15.0 11.3 2.5

C18:3 C20:0 C20:1 C22:0 C24:0 0.6 0.6 0.4 0.1 0.1 0.6 0.6 0.4 0.1 0.1 0.8 0.3 0.4 0.1 0.1 0.6 0.5 0.3 0.1 0.1 0.8 0.4 0.3 0.1 0.1 0.6 0.6 0.4 0.2 0.1 0.5 0.5 0.3 0.1 0.1 0.5 0.4 0.2 0.1 0.1 0.7 0.4 0.3 0.1 0.1 0.5 0.5 0.3 0.1 0.1 0.5 0.5 0.3 0.1 0.1 0.5 0.5 0.3 0.1 0.1 ≤1 ≤ 0.6 ≤ 0.4 ≤ 0.2 ≤ 0.2 0.5 0.3 0.2 0.1 0.1 0.8 0.6 0.4 0.2 0.1 0.6 0.5 0.3 0.1 0.1 0.1 0.1 0.1 0.0 0.0

42

The MUFA in all studied varieties represents the most important group of FAs (69.6%) followed by saturated fatty acids (SFA) (16.5%) and PUFA (11.9%) (data not shown). A high percentage of MUFAs in all studied varieties explains the nutritional benefits of VOO (Mensink and Katan, 1992). D’Imperio et al. (2007) proved that the ratio between SFA and unsaturated fatty acids (UFA) can contribute to cultivar characterization, since it is known that a particular FA composition, together with other compounds, can be used to differentiate monovarietal VOO. In this study, a genetic variability was observed in this ratio with values between 4.2 and 6.7 with a mean value of 5.0. On the other hand, MUFA/PUFA ranged between 4.2 and 8.8 with a mean of 6.2 and C18:1/C18:2 (O/L) comprised between 4.3 and 9.3 with a mean of 6.5. High ratios of MUFA/PUFA (>7) and O/L (>8) such as ‘Baladi’ (Lebaa) (MUFA/PUFA= 7.9; O/L= 8.4), ‘Baladi’ (Aitaroun) (MUFA/PUFA= 8.7; O/L= 9.2) and ‘Roumani’ (Qana) (MUFA/PUFA=8.8; O/L= 9.3) are associated with high oxidative stability and low rancidity of olive oil (Tous and Romero, 1993), and affect, in combination with other minor compounds, the flavor and health properties of VOO (Maestro-Durán and BorjaPadilla, 1990). As to the linoleic/linolenic (L/Ln) ratio, it varied in this study between 12.3 and 28.1 with an average of 18.8. Actually, this diet is of particular interest for cardiovascular health and should constitute 1–2% of adults' caloric intake with a ratio of 10/1 for L/Ln (Kiritsakis, 1998). This study is still ongoing relying on duplications and covering different ripening stages in order to have an accurate and comprehensive oil profile for the local olive varieties in Lebanon. References Bouaziz, M., Jemai, H., Khaboub, W., and Sayadi, S. 2010. Oil content, phenolic profiling and antioxidant potential of Tunisian olive drupes. J. Sci. Food Agric. 90:1750–1758 Del Río, C., Caballero, J.M., and García-Fernández, M.D. 2005. Capitulo 13, Libro segundo. In: Rallo, L., et al. (Eds.), Variedades de olivo en España, Consejería de Agricultura y Pesca, Ministerio de Agricultura Pesca y Alimentación, Mundi-Prensa S.L., Madrid, pp 348–356. D’Imperio, M., Dugo, G., Alfa, M., Mannina, L., and Segre, A.L. 2007. Statistical analysis on Sicilian olive oils. Food Chem. 102:956–965. European Union Commission Regulation EEC/2568/91. 1991. On the Characteristics of Olive and Olive Pomace Oils and Their Analytical Methods. El Riachy, M., Priego–Capote, F., Rallo, L., Luque de Castro, M.D, and León, L. 2012. Phenolic composition of virgin olive oils from cross breeding segregating populations. Eur. J. Lipid Sci. Technol. 114:542–551. Garcia-González, D., Aparicio-Ruiz, R., and Aparicio, R. 2009. Olive Oil. In: Gourmet and Health Promoting Specialty Oils. Eds: Robert A. Moreau Afaf Kamal-Eldin. AOCS PRESS, Urbana, lllinois, pp. 33–72. Instruction Manual Soxtherm SE 416 (Gerhardt Gmbh). 2004. No. D 53639. IOOC. 2003. Trade Standards applying to olive oil and olive-pomace oil. COI/T15/NC No 3. IOOC, 2006. International Oil Olive Council trade standard applying to olive oil and olivepomace oil. International Olive Oil Council. Madrid. Spain. Kiritsakis, A.K. 1998. Flavor components of olive oils a review. JAOCS 75:673–681.


43

León, L., De la Rosa, R., Gracia, A., Barranco, D., and Rallo, L. 2008. Fatty acid composition of advanced olive selections obtained by crossbreeding. J. Sci. Food Agric. 88:1921–1926. Manai, H., Haddada, F.M., Trigui, A., Daoud, D., and Zarrouk, M. 2007. Compositional quality of virgin olive oil from two new Tunisian cultivars obtained through controlled crossings. J. Sci. Food Agric. 87:600–606. Maestro-Durán, R., and Borja-Padilla, R. 1990. La calidad del aceite de oliva en relación con la composición y maduración de la aceituna. Grasas Aceites 41:171–178. Mensink, R.P., Katan, M.B. 1992. Effect of dietary fatty acids on serum lipids and lipoproteins. A meta-analysis of 27 trials. Arterioscler. Thromb. Vasc. Biol. 12:911– 919. Padula, G., Rosati, A., Pandolfi, S., Giordani, E., Bellini, E., Mennone, C., et al. 2006. Fatty acid composition of oils from olive selections derived from a breeding program and cultivated in Metaponto and Spoleto. Proc 2nd Int Seminar Olivebioteq, MarsalaMazara del Vallo, Vol. I, pp. 187–190. Ranalli, A., de Mattia, G., Ferrante, M.L., Giansante, L. 1997. Incidence of olive cultivation area on the analytical characteristics of the oil. Note 1. Riv. Ital. Sostanze Grasse 74:501–508. Tous, J., and Romero, A. Variedades de Olivo. Fundación ‘La Caixa’, Barcelona (1993).


44

EVALUATION OF SOME OLIVE CULTIVARS UNDER KUWAIT ENVIRONMENTAL CONDITIONS H. Al-Menaie*, O. Al-Ragam, H. Mahgoub, M. Al-Hadidi, A. Al-Shatti, M. Al-Zalzalah Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, PO Box 24885, Sa fat 13109, Kuwait *Corresponding author: [email protected]

Abstract An adequate knowledge of the best olive cultivars in the arid area is mandatory to efficiently utilize them for greenery development, desertification control, table olive, and olive oil production. As a preliminary effort, twenty six olive cultivars were subjected to performance evaluation in the coastal site of (Salmiya), Kuwait. Various growth parameters such as plant height, stem girth, number of shoots, and length of shoots were examined. It was found that except the cultivar Shauki, all other cultivars under observation survived under Kuwait environmental conditions. Moreover, all the varieties studied including Picual, Picholine, Pendulino, Coratina, Arbequina, and Frantoio flowered and produced fruits, indicating their ability to reproduce under the harsh climatic conditions of Kuwait. Keywords: Olea europaea, cultivars, growth rate, fruit traits. Introduction Olea europaea forms an important cultural element and an indispensable culinary component of many civilizations since time immemorial. It is a native plant of Mediterranean region and it is associated with victory, sanctity, strength, fertility, glory, and peace. The health beneficial properties in alleviating the effects of several degenerative diseases and its economic value have driven the market for olives tremendously, and olive cultivation has become an area of wide interest among different nations. They are primarily cultivated for their valuable fruit and oil. Furthermore, they are planted with the end in view to enhance the greenery and control desertification. Previously, several research groups have studied the influence of varietal difference and particular crop management practices on various growth parameters in olive cultivars (Hammami et al., 2011; Levin and Lavee, 2005). Nevertheless, the cultivar and environment interaction was an area of scrutinized research considering the impact of environment and climatic conditions on the productivity of various olive cultivars (Fernandez-Escobar et al., 2008; Di Vaio et al., 2013). Olive, being a drought- resilient crop could be a major fruit crop in Kuwait. Olive trees were first introduced and evaluated by Kuwait Institute of Scientific Research (KISR) in 1985. Later in 1990, approximately 300 seedlings were planted in Green Island in the waterfront project. Nevertheless, an extensive and rigorous evaluation incorporating more number of olive cultivars has not been carried out in previous studies under Kuwait environmental conditions. Currently, the knowledge of the best suitable cultivars is vital to harness the maximum production of table olives and olive oil from olives. Therefore in this study, growth and productivity of different introduced olive cultivars were evaluated under Kuwait environmental conditions. Various growth and quality determining parameters such as plant height, stem girth, leaf, and fruit characteristics were studied.


45

Materials and Methods Twenty-six imported olive cultivars were planted in a randomized complete block design with five single-plant replications at the KISR waterfront site, Salmiya, Kuwait. The weather conditions during the investigations were harsh and fluctuating. The annual precipitations were below normal, and the relative humidity was very low during summer. The dust storms were also unusually severe and more frequent than normal. Plants were irrigated using brackish water at the Urban Demonstration Garden (UDG), Salmiya. A compound fertilizer 15:15:15 was applied twice a year (March and May) to all the plants under observation. Lateral branches arising from the main stem up to 50 cm from ground were pruned to remove dead branches and unwanted vegetative growth, thus, facilitating the development of strong canopy and desired structure. During the study period, no incidences of pests and disease infestation were noticed. Parameters studied covered the survival percentage, plant height, stem diameter, shoots number, nodes number and internodes length, tree vigor, canopy density, growth habit, foliage characteristics, fruit weight, shape and color at maturity. Results The planned observations on growth and quality parameters were carried out for 26 different olive cultivars at three-month intervals. Survival percentage, height, and stem diameter Out of the 26 cultivars planted, all five plants of 13 cultivars survived (100% survival) in spite of the extreme environmental conditions during the duration of the study; whereas, all the plants of the Shauki variety died. The other cultivars showed survival percentages ranging from 20% to 80% (Table 1). After 79 months of planting in the coastal area of the waterfront testing site, Salmiya, the average height growth rate of different cultivars ranged from 12.2% recorded in Dan variety to 39.0% in Picual; whereas, Pendulino and South Verdale recorded 358.6 cm and 192.5 cm of height, respectively (Table 1). Shoots number, nodes number and internodes length Picual cultivar produced the highest (94.4) number of shoots followed by Yunani (85.0), Tufahi (80.0), Frantoio (79.0), Arbequina (74.2), Nepali (74.0), South Australia Verdale (73.8), Jalat (73.2), Picholine (71.6), Barnea (65.0), Del Morocco (63.6), Toumaee (61.8), Dan (58.4), and UC 13A6, which produced the lowest number of shoots (10.8) . The number of new shoots varied from 7.8 per plant in Istambuli to 50.6 per plant in Picual. The average rate of growth of new shoots ranged between 13.11% in Shauki and 148.21% in Pendulino. The average number of nodes and internodes length of these new shoots ranged between 8.6 (Koroneiki) and 16.4 (Picholine) and of 1.2 and 3.2 cm, respectively (Table 1).


46

Table 1. Survival %, plant height, stem diameter, growth rate, nodes number, internode length of the olive cultivars under evaluation. -, no growth was recorded. Cultivar

Survival %

Plant height (cm)

Stem diameter (cm)

Shoots number

Growth rate (%/month)

Nodes number

Internodal length (cm)

Dan

40

310.99

43.0

26.4

60.29

12.2

1.8

Istambuli

60

208.53

37.2

7.8

45.83

11.0

2.3

Tufahi

20

256.28

47.0

20.2

62.32

9.2

2.9

Totumaee

100

242.89

45.5

26.4

53.52

16.0

3.2

Nepali

40

293.01

39.9

26.8

29.61

15.6

2.9

Sourani

100

201.75

43.6

29.0

63.24

10.2

2.3

Yunani

80

373.89

91.1

33.0

31.68

12.6

2.2

Jalat

80

243.23

32.0

19.6

19.63

10.8

2.2

Dead

Dead

Dead

Dead

Dead

Dead

Dead

Arbequina

40

279.31

47.0

37.0

44.87

13.2

1.6

Arecuzzo

80

207.45

40.8

24.4

64.06

9.6

2.1

Azapa

100

239.3

29.1

18.2

50.00

10.6

2.1

Barnea Black Italian Coratina

100

328.98

51.6

30.8

61.96

12.2

2.7

100

181.15

19.3

19.6

33.03

14.4

2.1

100

323.2

60.9

36.4

56.79

10.0

2.7

Correggiola Del Morocco Frantoio

100

279.64

50.3

20.6

73.02

9.6

2.4

40

272.31

49.8

20.8

53.33

8.8

1.8

100

299.47

57.7

34.6

87.10

13.0

2.8

Koroneiki

100

255.23

42.2

30.6

32.81

8.6

1.7

Leccino

80

196.7

22.1

15.6

59.02

10.4

1.8

Manzanillo

100

273.45

54.8

35.0

86.15

12.6

2.1

Pendulino

100

358.6

66.7

30.0

148.21

13.6

2.2

Picual

100

296.24

66.2

50.6

60.00

39.0

2.7

Picholine

100

282.89

61.4

38.6

44.44

16.4

2.7

Verdale

20

192.5

14.4

-

-

-

-

UC 13A6

40

232.2

30.4

20.6

72.50

9.6

1.4

264

45.8

Shauki

Mean

L. S.D 0.05 for cultivars (Plant height

55.98

L.S.D 0.05 for cultivars (Stem diameter)

24.8

Tree vigor, canopy density, growth habit and foliage characteristics The cultivars exhibited different foliage characteristics with the shape of the leaves as elliptic, lanceolate, or elliptic-lanceolate. The upper face of the leaves was dark green in all the cultivars; whereas, the lower face was grey green or green grey. Cultivars did not differ in leaf texture. The studied cultivars presented considerable differences in their vigor, growth habit, and canopy density. The tree vigor was weak in three cultivars (Jalat, Proceedings of the 5th Int. Conf. Olivebioteq 2014

47

Arbequina, and Coratina), medium in 22 cultivars (e.g., Dan and Istambuli), and strong in one cultivar (Yunani) (Table 2). The growth habit was found to be spreading in seven cultivars (e.g., Nepali and Sourani) and erect in 19 cultivars (e.g. Azapa and Barnea). The canopy density was medium in 22 cultivars (e.g., Tufahi and Picual), sparse in three cultivars (Jalat, Tourani, and Arbequina), and dense in one cultivar (Black Italian). The yield production was low in 12 cultivars (e.g., Dan and Tufahi), medium in eight cultivars (e.g., Picholine and Frantoio), and heavy in two cultivars (Picual and Leccino). Moreover the cultivars Istambuli, Nepali, Azapa, and Del Morocco were found to be suitable for greenery purposes (Table 2). Fruit characteristics Fruit weight, shape, and color at maturity were recorded for all the cultivars under study (data not shown). According to the International Olive Oil Council (IOOC) methodology for primary characterization of olive varieties, the fruit shape was characterized as elongated (L/W > 1.45) in 18 cultivars (e.g. Tufahi and Dan), ovoid (L/W= 1.25-1.45) in five cultivars (e.g. Istambuli and Azapa), and globes (L/W < 1.25) in three cultivars (Nepali, Pendulino, and South Aus. Verdal). The fruit weight was high (4-6 gm) in five cultivars (e.g., Dan and Tufahi), medium (2-4gm) in 19 cultivars (e.g. Barnea and Coratina), or small (< 2 gm) in two cultivars (Totumaee and Pendulino). The fruit color was violet in eleven cultivars (e.g., Dan, Istambuli), black in eleven cultivars (e.g., Tufahi and Totumaee), and red in four cultivars (Correggiola and Azapa).


48

Table 2. Tree characteristics of olive cultivars. Cultivar Dan Istambuli Tufahi Totumaee Nepali Sourani Yunani Jalat Shauki Arbequina Arecuzzo Azapa Barnea Black Italian Coratina Correggiola Del Morocco Frantoio Koroneiki Leccino Manzanillo Pendulino Picual Picholine South Australian Verdale UC13A6

Vigor Medium Medium Medium Medium Medium Medium Strong Weak Medium Weak Medium Medium Medium Medium Weak Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium

Habit Spreading Erect Erect Spreading Spreading Spreading Spreading Erect Erect Spreading Erect Erect Erect Erect Erect Erect Erect Erect Erect Spreading Erect Erect Erect Erect Erect

Canopy Density Medium Medium Medium Medium Medium Sparse Medium Sparse Medium Sparse Medium Medium Medium Dense Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium

Yield production Light Greenery Light Medium Greenery Medium Medium Light Light Light Light Greenery Light Light medium Medium Greenery Medium Medium Heavy Light Light Heavy Medium Light

Medium

Erect

Medium

Light

Discussion All the varieties tested under the Kuwait environmental conditions survived, except the cultivar Shauki. Survived trees showed significant growth demonstrated by growth parameters such as plant height, stem girth, number of shoots, and length of shoots. The intensity and extent of their growth varied among cultivars similarly to previous works (Barranco Navero et al., 2000). All varieties flowered and produced fruits, proving their capacity to carry out their reproductive cycle under Kuwait environmental conditions. Thus, these cultivars could be used in the production of table olives and oil. It would be necessary to monitor their growth until they reach the adult stage, and evaluate the first significant productions and olive characteristics, in order to complete the characterization of the varieties.


49

The results obtained in this study confirmed the high adaptability of olive tree to harsh environments of Kuwait which is characterized by high temperatures, drought conditions and strong winds (Bhat et al., 2012). It is also indicated the high possibility for growing olives under Kuwait environment to fill the gap in table olive and olive oil increased consumption and to fulfill the public interest in this crop. References Barranco D., Touzani C., Cimato A., Castaneda C., Fiorino P., Serafini F., Rallo L., and Trujillo I. 2000. World Catalogue of Olive Varieties. International Olive Council, Madrid, pp 360. Bhat N.R., Al-Manaie H., Suleiman M.K., Al-Mulla L., Famiani L., and D'Cru T. 2012. Performance and water requirement of young olives (Olea europaea L.) in the harsh environment of Kuwait. Arch. Agron. Soil Sci. 1: 39-50. Di Vaio C., Nocerino S., Paduano A., and Sacchi R. 2013. Influence of some environmental factors on drupe maturation and olive oil composition. J. Sci. Food. Agri. 93: 1134-9. Fernandez-Escobar R., Prado A.M., and Rapoport.H.F. 2008. Nitrogen status influence on olive tree flower quality and ovule longevity. Environ. Exp. Bot. 64: 113-119. Hammami S.B.M., Manrique T., and Rapoport, H.F. 2011. Cultivar-based fruit size in olive depends on different tissue and cellular processes throughout growth. Sci. Hortic. 130: 445-451. Levin A.G., and Lavee S. 2005. The influence of girdling on flower type, number, inflorescence density, fruit set, and yields in three different olive cultivars (Barnea, Picual, and Souri). Aust. J. Agri. Res. 56: 827-831. Bati C.B., Godino G., Monardo D., and Nuzzo D. 2006. Influence of propagation techniques on growth and yield of olive tree cultivars ‘Carolea’ and ‘Nocellara Etnea’. Sci. Hortic. 109: 173-182.


50

EVALUATION OF NUTRIENT UPTAKE IN DIFFERENT OLIVE CULTIVARS GRAFTED ON 'GEMLIK' ROOTSTOCK M. Azimi1, M.T. Özkaya1*, H. Çölgeçen2, H.N. Büyükkartal3 1

Ankara University, Faculty of Agriculture, Department of Horticulture, 06110 Diskapi Ankara, Turkey 2 Bülent Ecevit University, Faculty of Arts and Science, Department of Biology, 67100 Incivez, Zonguldak, Turkey 3 Ankara University, Faculty of Science, Department of Biology, 06100 Tandoğan, Ankara, Turkey *Corresponding author: [email protected]

Abstract The olive (Olea europaea L.) varieties are cultivated by grafted or self-rooted plants. ‘Gemlik’ cvs is an easy-to-root variety so can be used as clonal rootstocks. However there could be grafting incompatibility problem between cvs. due to some problems in vascular system. Self-rooted ‘Ayvalik’ (A) and ‘Gemlik’ (G) cvs and six grafted (T-budding) combinations; ‘Ayvalik’/‘Gemlik’ (A/G), ‘Domat’/‘Gemlik’ (D/G), ‘Gemlik’/‘Gemlik’ (G/G), ‘Memecik’/‘Gemlik’ (M/G), ‘Nizip Yaglik’/‘Gemlik’ (N/G) and ‘Sari Ulak’/‘Gemlik’ (S/G) were used as plant materials. These plants had been grown in the greenhouse for two years, in pots containing a mixture of soil: sand: manure (1:1:1). The leaf samples for nutrient analysis were collected at the end of the second year after grafting. According to the plant nutrient analysis, results showed that there were significant differences (p zero). Preliminary observations were carried out on the genotypes' susceptibility to peacock eye disease at Carassai (April 2010) and at Jesi (June 2011), using a scale from zero (no infection) to 5. In May 2012 cold damage following an exceptionally cold February was also evaluated using a scale from zero (no damage) to four (damage affecting even trunk). Results Productive and vegetative characteristics of selected genotypes Genotypes MOT14 and MOE, grown at Carassai and Jesi respectively, were excluded from the selection, despite having achieved high yields, because they were insufficiently replicated. At Carassai, genotype MO13 had the highest cumulative yield, more than three times the average of that field (Table 1). Genotype MOT12 had a cumulated yield of almost three times the average, followed by genotypes MT6, MT14 and MO54, with yield more than double the field average. At Jesi, genotypes MO28, MT6, MOD, MO59 and MTO18 yielded more than twice the field average. Proceedings of the 5th Int. Conf. Olivebioteq 2014

82

Table 1. Trunk growth, cumulated yield and yield efficiency of all studied genotypes in both fields. Genotype MO1 MO4 MO5 MO6 MO7 MO8 MO9 MO10 MO12 MO12A MO13 MO14 MO15 MO17 MO18 MO19 MO20

(1)

CARASSAI Annual diamater Cumulated increments yield mm ± e.s. g ± e.s. 7.9 0.5 2433 353 7.5 7860 8.2 0.7 7443 1107 9.7 0.4 5007 547 7.4 0.6 850 153 9.0 0.9 2250 427 7.4 2700 6.2 0.3 2762 305 11.4 0.7 7308 941 10.2 0.4 6072 36 7.9 0.4 12822 929 5.9 0.5 6332 518 10.8 0.3 3570 130 8.6 0.1 8683 1373 7.6 0.3 1566 200 9.1 0.9 5289 862 6.3 0.3 833 167

Yield efficiency g/cm² ± e.s. 26 10 78 22 64 27 49 14 10 6 22 13 30 15 27 16 43 22 35 18 103 50 91 35 21 12 83 17 12 5 53 10 13 9

MO22 MO24 MO25 MO26 MO27 MO28 MO32 MO33 MO35

7.3 12.3 15.5 4.3 7.9 9.8 11.0 7.7 5.3

0.7 0.8 0.8 0.5 0.5 0.4 0.5 0.8 0.6

4184 2105 4647 2170 3228 4038 1358 3429 733

694 551 1179 551 544 541 366 637 188

44 13 22 34 36 24 9 26 8

23 5 7 14 10 9 4 11 6

MO39 MO40 MO42 MO45 MO46 MO47 MO48 MO52 MO53 MO54 MO55 MO57 MO59

7.6 9.5 5.1 9.6 8.9 8.6 12.2 7.4 8.5 7.6 9.6 10.1 13.2

0.5 0.2 0.2 0.6 0.8 0.4 0.6 1.2 0.5 0.6 1.4 1.0 0.4

1752 750 226 1800 1020 265 8143 510 3858 8796 1103 1400 7015

319 250 37 416 215 60 541 40 1046 1193 260 306 586

15 14 10 15 16 12 39 16 36 138 7 12 35

9 5 6 7 5 9 14 10 10 45 6 2 18

7.9 7.1 6.9 8.7

0.5 0.5 0.5

2600 3402 1830 8675

721 243 876

36 56 32 91

17 23 9 15

7.5 11.4 8.8 10.5

0.5 0.3 0.1 0.5

2550 11598 10510 1567

492 1273 230 780

40 69 96 12

9 16 45 7

6.4 8.8 9.9 10.3 9.2

0.3 0.5 0.3 1.0 0.4

150 10238 437 6978 9047

0 993 71 1776 421

4 77 4 38 64

2 34 1 23 22

4.9 8.7

0.6 0.4

202 193

77 58

13 5

8 4

(5)

MOA MOB MOC MOD MOT10 MOT12 MOT14 MT1 MT5 MT6 MT7 MT10 MT14 MT17 MTO8

(2) (*)

(3)

(4)


JESI Annual diamater Cumulated increments yield mm ± e.s. g ± e.s.

Genotype MO4 MO5 MO6

10.9 11.4 7.5

MO8 MO9 MO10 MO12 MO12A MO13

9.4 15.5 10.9 11.8 11.7 10.7

MO15 MO17 MO18 MO19 MO20 MO21 MO22

6.6 10.3 11.4 11.6 7.0 8.9 7.5

MO25 MO26 MO27 MO28 MO32 MO33 MO35 MO36

11.5 12.0 6.6 12.6 10.8 9.4 10.7 16.6

MO42 MO45 MO46 MO47 MO48 MO52 MO53 MO54 MO55 MO57 MO59 MO64 MOB MOC MOD MOE

MT3 MT5 MT6 MT7 MT10 MT14 MT15

(1)

(4)

(3) (*)

(2)

0.7 0.7 0.7

1573 967 1368

1.0 1.7 1.4 0.5

115 3020 1903 2842 2729 3620

0.4 0.5 0.3 1.2 0.5

1.5 0.9 0.9 1.0 0.6

9.7 13.9 11.9 11.8 9.3 8.0 6.5 6.2 9.1 8.3 14.0 12.5

0.9 0.5 0.8 0.7 0.7

8.1 4.3 7.9 16.8

0.5

12.8 12.8 10.3 9.7 10.9 8.3 14.4

0.9 0.9 1.0

0.9

0.6 0.7 0.2 0.7

600 3326 3314 5475 577 910 2045 4605 3700 3430 7294 3731 465 575 4250

2395 2204 1462 1499 2123 0 570 2155 238 893 5985 2750 2402 600 6173 12050

100 840 6228 1475 3553 1344 2705

Yield efficiency g/cm² ± e.s.

371 216 506

12 9 13

4 3 4

626 1110 716 983

3 13 15 17 20 29

2 4 7 4 7 6

9 26 16 30 6 8 28

5 9 8 11 5 4 13

27 27 43 35 16 4 5 17

17 10 11 12 9 2 1 4

23 15 12 12 17 0 6 49 2 12 27 17

11 5 4 6 4 0 5 18 1 4 8 6

24 35 52 43

9 27 29 14

1 4 56 9 29 18 11

0 2 25 3 7 6 7

513 434 3975 285 388

1316 899 1172 345 246

463 362 299 363 492

140 355 1300

699 2611

1240 384 1801 292

83

MTO9 MTO13 MTO15 MTO18 Field average

9.2 7.5 10.8 11.8 8.7

0.5 0.3 0.8 1.0

1291 1433 2372 3042 3869

257 176 644 367

12 16 13 18 35

5 8 7 5

MTO9

8.6

0.8

698

157

10

4

MTO15 MTO18 (5) Field average

12.6 13.5 10.5

1.0 0.8

1159 5480 2590

275 925

4 19 19

2 2

In brackets the position of the top five genotype in the scoring. Standard error is reported only for those genotypes for which we had 3 or more replications. * Productive genotypes not considered due to insufficient repetitions (less than three). Assuming cumulated yield as the most important parameter and considering the yield of both fields (i.e. Carassai and Jesi), MT6 was the most productive genotype. At Carassai this genotype was the third most productive, while in Jesi it was the second most productive. The next four best genotypes, in decreasing order, were: MOD, MO13, MO59 and MO17. To these selected genotypes, we added MOT12, MT14 and MO54 which were within the top five most productive (cumulated yield) genotypes at Carassai, and MO28 and MTO18 at Jesi. The total number of selected genotypes was therefore 10. At Carassai MO54 had high yield efficiency, almost four times the field average, distantly followed by MO13 and MOD. At Jesi, the most efficient was MT6, with efficiency almost three times greater than the field average, followed by MOD and MO54. The latter, therefore, was among the top genotype for efficiency in both fields. High efficiency might suggest good suitability to super high density olive orchards. Among genotypes selected for high cumulated yield, only MO54 had lower diameter increments than field average in both fields, with high yield efficiency. These results agree almost perfectly with previous findings showing MO13, MO17, MT6, MO54, MOT12, MOD, MT14 as the most productive genotypes at Carassai (Alfei et al., 2009); data from Jesi further confirm the results since the most productive genotypes in Jesi were the same as in Carassai, except for MO4, MO5, MO48, MO54 and MT14. Some genotypes were very productive at Jesi (i.e. above field average) but not at Carassai (MO28 and MTO18). Genotypes MO17, MO28 and MOD had higher cumulated yield also in Spoleto (Alfei et al., 2008). Fruit characteristics of selected genotypes None of the selected genotypes had fruits as large as those of Ascolana Tenera (6.16 g) (Alfei et al., 2008) (Table 2). MTO18 had the largest fruits (i.e. 5.0 g), larger than in most of the parent cultivars (Carboncella 1.94 g, Gentile di Chieti 2.26 g, Intosso 3.75 g, Leccino 1.88 g, Dritta 2.03 g) (Alfei et al., 2008). MO59 had fruits of less than 1g. MOT12 had a pulp/pit ratio (7.0) similar to that of the best parents (Ascolana tenera 8.20, Intosso 7.68). MO13 had the highest oil content on dry matter, slightly lower than the average of the six parents (44.5%), while MO59 had the lowest oil content. Fruits from MO13, MO28, MO54, MT14 and MTO18 had pigmentation index lower than 1, and are therefore late-maturing genotypes. Among them, MT14 had the highest pulp firmness.


84

Table 2. Fruit characteristics of the 10 selected genotypes. Genotype MO13 MO17 MO28 MO54 MO59 MOD MOT12 MT6 MT14 MTO18

Fruit weight Pulp/Pit ratio Oil content (FM) Oil content (DM) Pigmentation index g n % % n 2.7 3.1 3.4 3.3 0.8 3.2 3.1 3.0 3.0 5.0

5.2 4.4 5.4 6.1 2.6 4.7 7.0 5.3 5.5 5.0

17.4 14.2 17.3 15.4 10.9 15.7 15.1 16.7 17.3 11.1

42.9 37.7 38.8 40.3 24.6 40.9 41.6 40.7 38.4 31.3

0.4 1.9 0.5 0.1 1.1 1.7 2.0 1.9 0.6 0.9

Pulp firmness g 432 393 419 616 184 380 463 583 557 258

Oil chemical and taste characteristics of the selected genotypes Free acidity, peroxide number and spectrophotometric constants (i.e. K 232, K 270 e ∆K) were all within the legal limits for olive oil (Table 3). MOT12 had high phenols content, while MT6 had the lowest content, but yet higher than 300 mg/kg, the threshold value to attribute beneficial health properties to the oil. This genotype also had the lowest quality index (i.e. Oleic/(Palmitic+Linoleic)), due to a very low oleic acid content (i.e. 60%). MO13, MOD, MOT12 and MO17 had the highest quality index. Table 3. Oil characteristics of the 10 selected genotypes Genotype

Acidity Perosside n. (% Oleic) (meq oss/Kg)

∆K

K232

K270

Total phenols Oleic/(Palmitic+Linoleic) (mg/Kg) (n)

MO13

0.27

6.9

-0.002

1.583

0.124

401

4.0

MO17

0.40

5.8

-0.001

1.658

0.133

504

3.7

MO28

0.17

7.2

-0.002

1.822

0.146

596

2.9

MO54

0.23

6.7

-0.002

1.718

0.170

648

3.5

MO59

0.24

8.1

-0.009

1.618

0.186

648

3.1

MOD

0.30

6.8

0.000

1.728

0.134

407

3.8

MOT12

0.32

5.4

-0.001

1.796

0.174

779

3.8

MT6

0.17

8.6

0.000

1.763

0.137

356

1.6

MT14

0.26

8.0

-0.003

1.699

0.171

605

2.7

MTO18

0.22

2.4

-0.003

1.669

0.115

542

2.5

MO28 had the highest olive fruitiness and had grass, artichoke, almond and tomato flavours (Table 4). MO13, MO54, MT6 and MTO18 had mostly grass and tomato sensations and light artichoke and almond flavours, with medium-intensity olive fruitiness. All other oils had medium olive fruitiness and grass flavour, with predominant almond sensation in genotypes MO17, MOD, MOT12; artichoke sensation was dominant in MT14, and aromatic herbs was dominant in MO59; occasional apple sensations were detected in MOT12, MOD were reminiscent of berries. MOT12 oil was the most bitter and pungent, in line with its high phenol content. MT6 oil was more sweet than bitter. The final scoring was > 7 for all oils and up to 8 in MO28 and MT14. Proceedings of the 5th Int. Conf. Olivebioteq 2014

85

Table 4. Sensory evaluation of the oils from the 10 selected genotypes. Olive Genotyp Grass Artichok Tomat fruitines e Leaf e o s MO13 2.7 1.9 0.5 1.1 MO17 2.8 1.8 1.2 0.2 MO28 3.2 2.5 1.4 1.2 MO54 2.7 2.0 1.0 1.1 MO59 2.2 1.3 0.7 0.0 MOD 2.4 1.6 0.3 0.1 MOT12 2.6 1.9 1.1 0.3 MT6 2.7 2.1 0.7 1.6 MT14 2.9 2.6 1.6 1.1 MTO18 2.5 2.0 0.0 2.0

Almon d 0.7 1.8 1.3 0.4 0.9 1.7 1.8 0.6 1.4 0.0

Apple Berries 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0

Aromati c herbs

Bitter

0.5 0.0 0.0 0.3 1.0 0.0 0.0 0.0 0.0 1.0

1.9 2.1 2.0 2.5 2.7 2.1 3.3 1.9 2.7 2.0

Pungen Sweet t 2.1 2.4 2.5 2.2 2.5 2.2 3.1 2.1 2.5 2.0

1.9 1.6 1.7 1.8 1.4 1.7 1.0 2.2 1.7 2.0

Score 7.4 7.8 8.0 7.7 7.2 7.4 7.5 7.5 8.0 7.3

Susceptibility to the main diseases and to cold of selected genotypes The preliminary observations indicated that peacock eye was more severe at Carassai, compare to Jesi (Table 5). We report data only for the selected genotypes. MO59 was the most tolerant at Carassai. At Jesi all selected genotypes had medium sensitivity. Cold damage was limited at Carassai. Among the selected genotypes, MTO18 and MO54 were injured the least. At Jesi cold injuries were minor; MT6, MT14 and MTO18 had no damages. MO54 had the least overall damage and was below field average in both sites. Table 5. Susceptibility to peacock eye and cold of the 10 selected genotypes. Genotype MO13

Carassai Peacock eye Cold damage n n 4 2

Peacock eye n 2

Jesi Cold damage n 1

MO17

3

3

2

2

MO28

5

3

2

1

MO54

4

1

2

1

MO59

1

2

2

2

MOD

3

3

2

2

MOT12

5

3

MT6

4

2

2

0

MT14

4

2

2

0

MTO18

3

1

2

0

Field average

4

2

2

1

Discussion In the present study, the agronomic performance of the 64 genotypes was compared among the Carassai and the Jesi fields, as well as to previous results from another field in Spoleto (Alfei et al., 2008). MT6 had the highest yields when considering both fields. Only three genotypes (i.e. MO17, MO28 and MOD), among the 10 selected at Carassai and Jesi (i.e. Marche Region) were also selected at Spoleto (Umbria Region) for a higher cumulated yield. However the selection criteria in Spoleto were different and trees were grafted, so a full Proceedings of the 5th Int. Conf. Olivebioteq 2014

86

comparison is difficult. Among the selected genotypes, only MO54 had low vigor in both fields: this genotype may be therefore suitable for high density orchards. The agronomic and qualitative (i.e. of the oil) characteristics of the 10 genotypes selected in this study are summarized in table 6. Table 6. Summary table of the selected genotype characteristics. Tree Genotype

MO13

M

U

Age of first bearing E

MO17

M

U

E

H

MO28

MH

O

E

MO54

ML

U

E

MO59

H

W

MOD

M

MOT12

H

MT6 MT14 MTO18

Vigor Habitus

Sensitivity to pests and environmental stress

Fruit Oil Fruit content weight (FM) M H

Oil Pigmentation Pulp content time firmness (DM) H L M

Cold

Peacock eye

Suitability to harvesting systems

ML

MH

C

M

M

E

M

M

M

B

H

H

M

L

M

M

H

T

H

M

H

L

H

L

MH

C-O

E

L

L

L

M

L

ML

L

B

O

E

H

M

H

E

M

M

M

B

O

E

H

M

H

E

M

M

H

T

M

O

E

M

M

H

E

H

ML

MH

C

M

O

E

M

H

M

M

H

ML

MH

C

H

SW

L

H

L

M

M

L

L

M

T

Vigor: Low = L; Medium-Low = ML; Medium = M; Medium-High = MH; High = H Habitus: Upright = U; Open =O; Weeping = W; Semi-weeping = SW Age of first bearing: Early (E); Late (L). Fruit weight: ≤1,5 = Low (L); 1,6÷3,0 = Medium (M); ≥3,1 = High (H) Oil content (FM): ≤12 = Low (L); 12,1÷17,0 = Medium (M); ≥17,1 = High (H) Oil content (DM): ≤30 = Low (L); 30,1÷40,0 = Medium (M); ≥40,1 = High H) Pigmentation time: ≤0,5 = Late (L); 0,6÷1,5 = Medium (M); ≥1,6 = Early (E) Pulp firmness: ≤300 = Low (L); 301÷500 = Medium (M); ≥501 = High (H) Sensitivity to cold: 0÷1 = Low (L); 2 = Medium-Low (ML); 3 = Medium (M) Sensitivity to peacock eye: 0÷1 = Low (L); 2 = Medium-Low (ML); 3 = Medium (M); 4 = Medium-High (MH); 5 = High (H) Suitability to harvesting systems: C = Combs; B = Beating with sticks; T = Trunk shaker; O = Over the row The results confirm that it is extremely difficult to combine all desired superior traits in one genotype (Padula et al., 2008; Ripa et al., 2008; Alfei et al., 2008; Pannelli et al., 2009a; Paoletti et al., 2009; Pannelli et al., 2009b; Alfei et al., 2009), due to environmental and genotypic effects and their interactions. Further testing of the selected material is necessary, particularly the comparison with all parental cultivars, and other traditional cultivars to be replaced. This comparison should be carried out using self-rooted plant material grown in the areas where the parental cultivars are traditionally grown. New cultivars are also needed for super high density orchards, that are increasingly planted, but for which only few cultivars appear suitable. Commonly, it is considered that these cultivars have low vigor and small size, but it is not clear what characteristics lead to the low vigor. Recently it has been suggested that high branching frequency is a key trait, leading to reduced growth and higher production (i.e. resulting in higher yield efficiency), and therefore greater suitability to super high density Proceedings of the 5th Int. Conf. Olivebioteq 2014

87

orchards (Rosati et al., 2013). Some of the genotypes here selected had higher yield efficiency than the others and it might be interesting to evaluate their suitability to such orchards. Further studies on these materials, therefore, should consider their branching frequency and its role in making them suitable to super high density orchards. References Alfei B., Paoletti A., Rosati A., Santinelli A., Pannelli G. 2008. Agronomic and qualitative evaluation of new olive genotypes selected in central Italy. Advances in Horticultural Science, 2: 136-141. Alfei B., Paoletti A., Rosati A., Santinelli A., Pannelli G. 2009. Produttività e qualità dell’olio in nuovi genotipi ottenuti da incrocio e selezionati nelle Marche. Italus Hortus, 3: 95-100. Bongi G. 2004. Modelli produttivi in olivicoltura. Olivo e Olio, 9: 8-15. Padula G., Giordani E., Bellini E., Rosati A., Pandolfi S., Paoletti A., Pannelli G., Ripa V., De Rose F., Perri E., Buccoliero A., Mennone C. 2008. Field evaluation of new olive (Olea europaea L.) selections and effects of genotype and environment on productivity and fruit characteristics. Advances in Horticultural Science, 2: 87-94. Pannelli G., Rosati A., Paoletti A., Pandolfi S., Pellegrini A., Ripa V., De Rose F., Perri E., Buccoliero A., Mennone C., Padula G., Giordani E., Bellini E. 2009a. Valutazione agronomica di nuovi genotipi di olivo da incroci programmati coltivati in Metaponto, Rossano Calabro e Spoleto. Italus Hortus, 3: 11-34. Pannelli G., Paoletti A., Rosati A., Pandolfi S., Pellegrini A., Ripa V., De Rose F., Perri E., Buccoliero A., Mennone C., Padula G., Giordani E., Bellini E. 2009b. Caratteristiche degli alberi, dei frutti e dell’olio di progenie di olivo da incroci programmati selezionate nell’ambito del progetto Se.In.Ol.Ta.. Italus Hortus, 3: 45-54. Paoletti A., Rosati A., Pandolfi S., Pellegrini A., Pannelli G., Ripa V., De Rose F., Caravita M.A., Parise M.R., Perri E., Buccoliero A., Mennone C., Padula G., Giordani E., Bellini E. 2009. Valutazione qualitativa degli oli di nuovi genotipi di olivo da incroci programmati coltivati in Metaponto, Rossano Calabro e Spoleto. Italus Hortus, 3: 35-44. Ripa V., De Rose F., Caravita M.A., Parise M.R., Perri E., Rosati A., Pandolfi S., Paoletti A., Pannelli G., Padula G., Giordani E., Bellini E., Buccoliero A., Mennone C. 2008. Qualitative evaluation of olive oils from new olive selections and effects of genotype and environment on oil quality. Advances in Horticultural Science, 2: 95-103. Rosati A., Paoletti A., Caporali S., Perri E. 2013. The role of tree architecture in super high density olive orchards. Scientia Horticulturae 161: 24-29.


88

CHARACTERIZATION OF ANCIENT OLIVE GENOTYPES IN EMILIAROMAGNA REGION: MOLECULAR GENOTYPING, CHEMICAL AND SENSORY PROPERTIES OF MONOVARIETAL OLIVE OILS L. Morrone1*, A. Rotondi1, D. Beghè2, T. Ganino2, A. Fabbri2 1 2

IBIMET-CNR Via Gobetti 101, 40129 Bologna, Italy Dipartimento di Scienze degli Alimenti, Università degli Studi di Parma, Viale delle Scienze, 59/A 43100 Parma, Italy


Abstract Ancient olive trees of Olea europaea L. present in the piedmont area of the Emilian Apennins (Italy) were assessed in the course of several years. In this research, seven old accessions from Modena, Parma, Piacenza and Reggio Emilia Provinces were evaluated by SSR markers followed by chemical and sensory characterization of monovarietal olive oils. As to the genetic analysis, 14 SSR markers were used; they produced a total number of 72 alleles that permitted the discrimination of the seven studied genotypes and their comparison with genetic profiles of national and international cultivars. Genetic relationships were estimated by the unweighted pair-group method with arithmeric averaging (UPGMA). By microsatellite analysis five genotypes were identified, one of which corresponds to cv. “Frantoio”, while cv. “Carolea” appeared genetically distant from the others. The comparison reveals that only the known cultivar Frantoio can be found within the studied accessions. Monovarietal olive oils produced by a low-scale mill allowed to analyse their chemical and sensory properties according to EC Reg. 796/2002. Some of these oils were distinguished for their elevated contents in total phenols, other samples were also interesting by their high contents in oleic acid, above 79%. Sensory analysis emphasized the aromatic peculiarities of some monovarietal oils, characterized by a relevant intensity of fruitiness, bitterness and pungency. Moreover oils were singled out for their particular flavours (almond, apple, artichoke and green notes), ascribable to the different genetic matrices of the mother plants. Keywords: monovarietal olive oil, genetic matrix, SSR, phenols, sensory analysis, Olea europaea Introduction The Western provinces of the Emilia-Romagna region are characterised by various microclimates in which olive growing is possible. The presence of the species, albeit sporadic, in these territories for several centuries as a fruit crop is well documented, not merely by archaeological and written testimony, but also by a large number of very old trees, located in particular sites, favourable for growth and development of this species (Ganino et al., 2007; Ganino and Fabbri, 2008). The olive germplasm we retrieved comes from old stands and trees, all more than a century old, which survived thanks to favorable environmental conditions. Chemical and sensory properties of monovarietal olive oils, produced by olive genotypes identified during a clonal selection programme, represent very important selective parameters, in order to obtain oils of a high nutritional standard. Also the organoleptic profiles play a key role in the identification of genotypes able to produce oils provided of peculiar aromatic Proceedings of the 5th Int. Conf. Olivebioteq 2014

89

substances. Oil fatty acid composition and phenolic substances have a great importance from a health point of view, as diets rich in monounsatured fatty acid (oleic acid) may lower lowdensity lipoprotein (LDL) cholesterol (Beltrand et al., 2004). The nutritional importance of extra virgin olive oil is also attributed to its richness in phenolic substances which act as natural antioxidants and may contribute to the prevention of several human diseases (Bendini et al., 2007). In this research, genetic diversity of old olive trees of Emilia territory was studied utilising the SSR technique followed by chemical and sensory characterization of monovarietal olive oils produced using a low-scale mill. Materials and methods Numerous ancient accessions of Olea europaea L., present in the piedmont area of the Emilian Apennins (Italy), were described in the course of several years (Ganino et al., 2007; Ganino et al., 2008; Beghè et al., 2011). In this research a set of seven olive trees located in the territory of the Modena, Parma, Piacenza and Reggio Emilia Provinces, was selected. The accessions were named according to the location of retrieval, or to local denominations (Table 1). Genomic DNA was extracted from fresh leaves as described by De la Rosa et al. (2004). The samples were subjected to characterization with SSR markers according to the PCR conditions described by Beghè et al. (2011). For DNA amplification 14 couples of SSR primers were used which had shown a good discriminating capacity: DCA3, DCA4, DCA5, DCA9, DCA15, DCA16, DCA17, DCA18 (Sefc et al. 2000), UDO24, UDO43 (Cipriani et al. 2002), GAPU59, GAPU101, GAPU103 (Carriero et al. 2002) and EMO90 (De la Rosa et al. 2002). The amplification products were separated with a CEQ 2000 Genetic Analysis System (Beckman Coulter, Inc.) sequencer on acrylamide gel CEQ Separation Gel LPA-1 (Beckman Coulter, Inc.). To identify the genotypes the data obtained by SSR analysis were compared with SSR profiles of olive cultivars available in the SSR database of Parma University (Italy) (Beghè et al., 2009; Beghè et al., 2011 and unpublished data). The level of similarity/dissimilarity among the examined olive trees was obtained through the genetic similarity matrix utilizing Euclidean distance. Cluster analysis and construction of the dendrogram relative to genetic distances were obtained by using the unweighted pairgroup method with arithmetic mean (UPGMA) algorithm and XLSTAT 2009 software (AddinsoftTM1995–2009). Olives were hand harvested in the mid October-early November period. To reduce the effect of the degree of maturation on oils properties, all productions were transformed to a similar ripening index (RI) corresponding to about 50% pigmentation of the olive skin (Uceda and Hermoso, 1998). A low-scale mill (TEM, Firenze, Italy) was employed in order to obtain monovarietal olive oil under controlled conditions of temperature and time of the different technological phases (crushing, malaxation and extraction). Olive oils were stored in dark glass bottles at 15-18°C, no headspace was left in the bottles. After one month the samples were subjected to the following analyses: fatty acid composition, total phenols contents and panel test. Chromatographic analysis was performed in a Chrompack CP 9000 gas chromatograph using a capillary column (Stabilwax, Restek Corporation, USA). The phenols extract was obtained according to Pirisi et al., (2000), the total phenols content of the extracts was determined by the Folin–Ciocalteau spectrophotometric method at 750 nm (Jasco Proceedings of the 5th Int. Conf. Olivebioteq 2014

90

Spectrophometer V-500, Tokyo, Japan). All chemical analyses were repeated three times for each sample. Sensory analysis was performed by a fully-trained panel recognized by IOOC. A panel test was established using a standard profile sheet (IOOC/T20) modified by IBIMET-CNR in order to obtain a complete description of the organoleptic properties of the oils sampled (Cerretani et al., 2008). Table 1. List of accessions identified in Emilia-Romagna region, accession name and province of origin (Area Id.). Accession name Area id. Arcello Piacenza (IT) Vernasca Piacenza (IT) Cevola Parma (IT) Castellaro Parma (IT) Montanari Modena (IT) Nirano Modena (IT) Casola Reggio-Emilia (IT)

Results and discussion Molecular analysis The 14 oligonucleotides of the series DCA, UDO, GAPU and EMO have produced polymorphic and reproducible amplification fragments (data not shown). The allelic polymorphism has permitted the discrimination of the seven studied genotypes, and produced a total number of 72 alleles. The number of alleles at each locus ranged between 3, for marker DCA15 which resulted to be the least polymorphic, and 8, for marker DCA9, for an average of 5.14 for the investigated loci. The studied accessions were then compared with the SSR genetic profiles of olive cultivars present the SSR Database of Parma University (Italy). The comparison reveals that only the known cultivar Frantoio can be found within the studied accessions (data not shown). The genetic relationships among individuals is showed in the dendrogram produced by cluster UPGMA analysis at Euclidean distance (Fig. 1).

Fig. 1. UPGMA dendrogram of Olea europaea L. genotypes based on 14 SSR loci.


91

Out of the nine examined accessions five different genotypes, one of which corresponds to cv. “Frantoio” (also used as internal standard) (Fig. 1) while cv. “Carolea” (the other internal standard) appeared, as expected, genetically distant from the studied accessions. A genetically homogeneous cluster (indicated as “Frantoio”) can be singled out below the 10% maximum potential Euclidean distance, that is made of individuals which show genetic identity or whose allelic profiles differ by one or two alleles. The cluster “Frantoio”, grouped in addition to the cv. “Frantoio”, the accessions designated as Nirano, Montanari and Arcello. By comparing the allelic profiles of the studied trees, as shown in Table 2, it can be noticed that accession Arcello is distinguished by two alleles, at loci DCA9 and UDO43, and the differences are only for two base pairs from cv. “Frantoio” in the same loci (Table 2). Accessions Nirano and Montanari differed from “Frantoio” for one or two alleles of locus GAPU103 and of loci DCA9 and UDO43, respectively. These accessions are characterized by unique alleles, “176” of locus GAPU103 and “197” of locus DCA9; these alleles resulted as decisive to distinguish intra-cultivar variations (Fig. 1). Accessions Casola, Vernasca, and Castellaro, finally, are located in the dendrogram at a high Euclidean distance if compared to other regional accessions and to the internal standard cultivars, and can therefore be considered different genotypes. Chemical and sensory characteristics of monovarietal olive oils Oil analysis revealed the presence of an interesting genetic diversity in the studied genotypes. Table 2 reported some compounds involved in the nutritional value of extra virgin olive oil. Oils codified as Arcello and Casola were characterized by highest phenolic contents. Three monovarietal oils produced by Castellaro, Nirano and Vernasca exhibited medium contents superior to 200 mg of gallic acid/kg of oil, while Montanari and Cevola oils showed lower total phenol contents with 188.33 and 157.37 mg of gallic acid/kg of oil, respectively. All samples, except the oil produced by Castellaro genotype, show levels of oleic acid superior to 75% and genotypes codified as Montanari and Arcello were particularly interesting for their elevated contents in oleic acid, superior to 79%. It is important to underline that hydrophilic phenols are strongly connected with the organoleptic quality of extra virgin olive oil, and are responsible for pungent and bitter notes (Gutièrrez et al., 1992). The oil sensory profile is deeply influenced by the genetic matrix; the combined effect of the taste, odor and chemical response gives rise to the sensation perceived as “flavor” (Kilcast, 2003). Table 2. Total phenol and major fatty acids content of monovarietal oils studied. Total phenols Palmitic Linoleic Linolenic Oleic acid (mg gallic acid acid acid (C18:1) acid/Kg oil) (C16:0) (C18:2) (C18:3) Arcello 332.74±101.90 79.27±1.77 11.42±0.91 4.84±0.59 0.59±0.05 Montanari 188.33±30.72 79.30±0.71 11.50±0.07 5.23±0.68 0.63±0.09 Casola 328.47±28.98 78.80±0.58 11.57±0.34 5.85±0.36 0.66±0.04 Castellaro 247.15±57.92 68.45±1.86 14.94±0.73 10.86±2.66 0.90±0.08 Cevola 157.37±41.50 77.13±0.17 12.08±0.28 5.36±0.04 0.83±0.05 Nirano 266.54±125.38 75.72±4.67 14.13±4.42 5.54±0.19 0.75±0.11 Vernasca 220.65±34.54 75.12±2.56 13.58±1.49 7.02±0.70 0.68±0.15


92

From the sensorial point of view, oil codified as Vernasca is very interesting for its highest intensity of olive fruity, bitterness and pungency, moreover this oil exhibited green notes and artichoke flavour (Table 3). Table 3. Sensory intensities of the attributes used to characterize monovarietal oils studied. Other Pleasant notes Olive Green Bitterness Pungency pleasant (mainly fruity notes flavors perceived) Artichoke4.2 3.7 4.7 2.4 3.0 Arcello almond Montanari 3.8 2.5 3.8 2.7 3.4 Almond-apple Casola 4.1 4.1 4.7 3.3 3.6 Almond Castellaro 4.6 4.6 4.2 3.5 3.3 Artichoke Cevola 3.8 2.2 3.8 2.6 3.4 Almondl Nirano 4.3 3.7 5.0 2.4 2.8 Almond-apple Vernasca 5.2 6.0 6.5 3.4 2.7 Artichoke Other oils, codified as Montanari and Cevola were characterized by lower intensity of olive fruity, bitterness and pungency accompanied by almond-apple and almond notes, respectively. The lower intensities of bitterness and pungency of these samples were also confirmed by the lower contents of total phenols (188.33 in Montanari oil and 157.54 in Cevola oil) (Table 2). Comparing genetic results with chemical and sensory properties of oil produced, it is interesting to underline that accessions Casola, Vernasca and Castellaro which were located in the dendrogram at a high Euclidean distance and can therefore be considered different genotypes, were also characterized by different contents of oleic acid as well as of total phenols. Considering monovarietal oils produced by the genotypes grouped with “Frantoio” genotype: Nirano, Montanari and Arcello and the cv. “Frantoio”, it is important to note that Arcello and Montanari oils exhibited the highest levels of oleic acid (over 79%), while Nirano oil showed a lower oleic acid content (75.72%). In detail Arcello is distinguished by two alleles and the differences are only for two base pairs from cv. “Frantoio” in the same loci. The sensory profile of Arcello oil is very similar to classic “Frantoio” sensory profile characterized by intermediate intensities of olive fruity, bitterness and pungency, moreover “Frantoio” oil is characterized by almond and artichoke flavors. Conclusion These preliminary results revealed the important genetic diversity of the ancient olive trees of Emilia territory. The chemical analysis of the monovarietal oils produced from these ancient trees showed the good nutritional values in terms of oleic acid and total phenol contents. Moreover the sensory analysis underlined the richness in flavor notes of the oils studied. These results should be further validated by studying the productions of further crop seasons. Moreover, during the second step of the clonal selection process, these results will be compared with those of oils produced from the same selected genotypes cultivated in an experimental field where our germplasm has been collected and is under further evaluation, i.e., growing under the same environmental conditions.


93

Acknowledgment We thank Matteo Mari and Mafalda Govoni for their technical support References Beghè D., Amendola A.P., Ganino T., Rotondi A., and Fabbri A. 2009. Una banca dati genetica (SSR) per il germoplasma olivicolo dell'Emilia Romagna. In: Acta Italus Hortus. I° Convegno nazionale dell’olivo e dell’olio. Portici, 1-2/10/2009, 1:105-108 Beghè D., Ferrarini A., Ganino T., and Fabbri A. 2011. Molecular characterization and identification of a group of local Olea europaea L. varieties. Tree Genet. Genomes 7, 1185–1198 Beltràn G., Del Rio C., Sànchez S., and Martìnez L. 2004. Influence of harvest date and crop yield on the fatty acid composition of virgin olive oils from cv. Picual. J. Agric. Food Chem. 52, 3434-3440. Bendini A., Cerretani L., Carrasco-Pancorbo A., Gomez-Caravaca, A.M., Segura-Carettero A., Fernàndez-Gutierrez A. and Lercker G. 2007. Phenolic molecules in virgin olive oil: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules, 12, 1679-1719. Carriero F.; Fontanazza G.; Cellini F.; and Giorio G. 2002. Identification of simple sequence repeats (SSRs) in olive (Olea europaea L.). Theor. Appl. Genet., 104, 301-307. Cerretani L., Salvador M.D., Bendini A., and Fregapane G. 2008. Relationship between sensory evaluation performed by Italian and Spanish official panels and volatile and phenolic profiles of virgin olive oils. Chemosensory Perception, 1(4), 258-267. Cipriani G., Marrazzo M.T., Marconi R., Cimato A., Testolin R., 2002. Microsatellite markers isolated in olive (Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars. Theor Appl Genet 104:223-228 De la Rosa R., James C.M., and Tobutt K.R. 2004. Using microsatellites for paternity testing in olive progenies. HortScience 39:351-354. De La Rosa R.; James C.M.; and Tobutt K.R. 2002. Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae. Mol. Ecol. Notes, 2 (3), 265-267. Ganino T., Beghe' D., Valenti S., Nisi R., and Fabbri, A. 2007. RAPD and SSR markers for characterization and identification of ancient cultivars of Olea europaea L. in the Emilia region. Genetic Resources and Crop Evolution, 54: 1531-1540 Ganino T., and Fabbri, A. 2008. Genetic characterization of Olea europaea L. germplasm in Northern Italy. Proceedings of the 5th International Symposium on Olive Growing, Acta Horticulturae, 1:95-102. Gutièrrez-Rosales F., Perdiguero S., Gutièrrez P., and Olìas J.M. 1992. Evaluation of the bitter taste in virgin olive oil. J. Am. Oil Chem. Soc. 69: 394-395. Kilcast D. 2003. Sensory Science, Chemistry in Britain, 39, 62-64. Rotondi A., Bendini A., Cerretani L., Mari M., Lercker G., and Gallina Toschi T. 2004. Effect of olive ripening degree on the oxidative stability and organoleptic properties of Nostrana di Brisighella extravirgin olive oil. J Agric Food Chem 52:3649-3654. Uceda M. and Hermoso M. 1998. La calidad del aceite de oliva. In El cultivo del olivo. Barranco, D., Fernàndez-Escobar, R, and Rallo, R. Eds. Junta de Andalucìa Editiones Mundi-Prensa: Madrid, Spain, 547-572.


94

Pirisi F.M., Cabras P., Cao C.F., Migliorini M., and Muggelli M. 2000. Phenolic compounds in virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and quantification procedures. Journal of agricultural and food chemistry, 48(4), 1191-1196. Sefc K.M., Lopes M.S., Mendonça D., Rodrigues Dos Santos M., Laimer Da Câmara Machado M., Da Câmara Machado A. 2000. Identification of microsatellite loci in olive (Olea europaea) and their characterization in Italian and Iberian olive trees. Mol Ecol 9:11711173.


95

PLANT BREEDING OF PRELIMINARY RESULTS

OLIVE

USING

GAMMA

RADIATION:

A. Chaari-Rkhis1,*, M. Maalej2, A. Chaabouni3, S. Baccari2 1

Olive Institute (University of Sfax) BP 1087. 3000 Sfax. Tunisie. Faculty of Sciences (University of Sfax)/Route de la Soukra.3000 Sfax Tunisia. 3 National Institute of Agronomic Research . Tunis. (University of Carthage)/H. Karray Street.3049 Ariana .Tunisia. 2


Abstract Both climate change and intensification of olive cultivation encourage olive genetic improvement to make available ecotypes that respond to current and future needs of the Tunisian farmers. Application of Gamma rays on olive may be at the origin of obtaining new interesting cultivars. Thus, in vitro vegetative explants (axillaries buds) of Chemlali Sfax variety has been subject to a mutagenic treatment by gamma rays at increasing doses (0.5, 10, 15, 20, 25, 30, 40 and 50 Gray). After two months of in vitro culture, it was found that doses of 5 to15 Gray regenerate the highest explants viability rates. After three subcultures, in vitroshoots with an average of 2 to 3 cm of length corresponding to an average of 3 to 4 nodes were regenerated. We proceeded then to their ex vitro rooting which allowed us to generate about thirty olive trees which were planted and followed up in the field. After 2 years, seven olive trees are in bloom. Keywords: olive tree, in-vitro culture, Gamma Rays, Lethal Dose Introduction The genetic improvement of Tunisian olive currently stands as a priority to develop cultivars suitable for both new techniques production and stress conditions. Olives slightly benefited from research programs on genetic improvement by conventional ways ( Fiorino 1992; Rallo 1995; Trigui 1996 ; Msallem and Helali 2000) and even less by biotechnological techniques (Rugini et al 2011). The principal cause of the limitation of using conventional breeding techniques for olive tree is the small size of its flowers and its long period of juvenility. Thus, application of mutagenic treatments to induce somatic mutations of olive may answer the best current and future needs of olive grove. Mutations can be induced by physical or chemical agents and some plant agronomic traits have been already improved by those techniques such as for some fruits trees (Sanada and Amano 1998; Harten 1998; Trigiano and Gray 2005 ; Tulmann Neto et al. 2011; Akshatha and Chandrashekar 2014 ... ). Advances in plant tissue culture and molecular biology can be integrated with those techniques to make it more efficient. Indeed, using tissue culture is particularly suited because a large number of explants can be used for the induction of mutations in a small space, and several subcultures can be carried out in a short time to dissociate chimeras and increase mutants’ plant population. Therefore, mutation technique combined with in-vitro culture could be successfully applied for genetic improvement of several crops (FAO/IAEA 1997). Proceedings of the 5th Int. Conf. Olivebioteq 2014

96

It is in this option that application of Gamma radiation has been considered for mutagenesis induction in the Tunisian olive oil cultivars. We present in this paper the preliminary data related to the determination of radio sensitivity of olive to gamma rays and the behavior of the treated vegetative material. Material and methods Plant material and establishment of in-vitro cultures Chemlali Sfax, the most cultivated Tunisian olive variety was chosen to test the sensibility of olive to Gamma-rays. Thus, stem fragments with a single node, taken in the fields in 2009, were used to establish in-vitro culture on MM culture media ( Chaari- Rkhis et al 2011) for culture initiation. Cultures were maintained in a growth chamber at 25 ± 1 °C and under cool white fluorescent lamps (45 μmol m−2 s−1) with a 16 h photoperiod. After 10 weeks, the developed shoots from each explant (3 to 5 cm in length) were divided into nodal explants with 1 pair of buds and subcultured twice under the same culture media and environmental conditions. Gamma Rays application Gamma rays produced by a 60Cobalt source were used. The regenerated vitro-shoots were cutting in several leafy single nodes portion (about 1-cm-long) and irradiated with eight doses: 0, 5, 10, 15, 20, 25, 30, 40 and 50 Gray (Gy). The explants were put on Petri dishes, containing MM nutrient media, at 5 per box. 20 boxes for each dose were used. After 2 months, we estimated the rate of explants survivors to determine the suitable lethal dose LD50 (dose at which 50% of explants remained alive). Rooting of in vitro-shoots Explants that survived after applying gamma rays were grown on MM nutrient medium (Chaari 2013; Chaari et al. 2011). After three successive subcultures in order to eliminate chimera, the in vitro-shoots with 2 to 3 cm of length and between 3 and 4 nodes were put in plates (with small holes) containing sterile substrate (Peat) for their rooting. The bases of these in vitro-shoots were first soaked for a few seconds in an Indol Butyric Acid solution (4000 ppm) to stimulate rooting. The plates were maintained in closed glass case (1 m3) of which the bottoms were heated to 25 ° C ± 1 ° C. They were kept in an atmosphere of 24 ° C ± 1 ° C and a relative humidity of about 80%. After 2 months, young rooted olives were removed, planted in plastic bags (8 l) containing sand and peat (1:1) and held in an adequate atmosphere for a few months (at 25 ° C ± 1 ° C and 80% to 50% RH) to be well acclimatized. A year after their livestock (in 2012), olive plantlets, about 50 cm high, was planted on the field with a spacing of 4 m/4 m. Data analysis Statistical analyses were performed using analysis of variance (SPPS Version 20 Windows). Comparisons of the means were determined by Duncan test at p≤0.05. Result and discussion LD determination Up to 30 Gy, percentages of surviving explants, two months after applying gamma radiations, are higher than 50%. Thus, for such type of explants, 30 Gy present the LD for olive tree (Fig.1). Higher doses cause necrosis or loss of regenerative capacity (Table1). Likewise, doses of gamma radiation applied to other species are comparable and generally low Proceedings of the 5th Int. Conf. Olivebioteq 2014

97

(between 20 and 50 Gy) sometimes very low (less than 10 Gy) (Predieri 2001; Sangsiri et al. 2005; Noor et al. 2009 ...). 120

Survival rate %

100 80

b 60

c

40

d

20 0 0

5

10

15

20

25

30

40

50

Gamma rays dose (Gy)

Fig. 1: Explants survival rate after 2 months of treatment. In our case and owing to the registered growth rates, only doses less or equal to 15 Gy gamma rays permit to regenerate a measurable elongation (more than 1 cm). As for the nodes number, their average is superior to 2 for all explants treated with doses of Gamma rays less than 15 Gy (Table 1).

Table 1: Growth of explants (± SE) after 2 months of Gamma rays application. Length average (cm) Nodes number average. Control (0 Gy) 5Gy 10 Gy 15Gy 20Gy 25Gy 30Gy 40Gy 50Gy

2,96± 1,12a 1,84± 1,56 ab 1,96± 1,36 ab 1,24± 1,02b 0,42± 0,29c 0,36± 0,57c 0,3± 0,51c 0,24± 0,48d 0,03±0,08e

3,88± 1,15a 2,31± 1,30 a 2,38± 1,31a 2± 0,11b 1,2± 0,62bc 0,78± 0,88c 0,69± 0,79c 0,38± 0,72c 0,13± 0,34d

Growth of irradiated materiel Irradiated in vitro-shoots were subcultured to new nutrient media after their fragmentation (Chaari-Rkhis et al. 2011; Chaari-Rkhis 2013). This last manipulation was done three times to be sure of the elimination of eventual chimera (Sen and Alikamanaglu 2012). Thus, in Table 2 are reported length and nodes number averages according to the Gamma ray doses applied after Proceedings of the 5th Int. Conf. Olivebioteq 2014

98

the third subculture. It is noted that only vegetative materiel which have undergone a lower dose than 15 Gy grew well with an average of more than 2 cm elongation. Weak or no growth was observed for the plant material treated with higher doses (Table 2). The same result was reported by Sen and Alikamanaglu (2012) on sugar beet which showed a decrease of regeneration rate and fresh weights with an increase in radiation dose and concluded that optimal doses for mutation induction were between 15 and 20 Gy for this species. Table 2: Average growth of in vitro-shoots (± SE) after the 3th subculture. Length ( cm) Nodes Number Control (0 Gy) 3.96±1.12a 4.88±1.15a 5 Gy 2.84±1.56ab 3.31±1.30b 10 Gy 2.96±1.36ab 3.38±1.31b 15 Gy 2.24±1.02b 2.69±1.11b 20 Gy 1.42±0.29c 1.2±0.62c 25 Gy 0.36±0.32d 0.78±0.88c 30 Gy 0.32±0.51d 0.69±0.79c 40 Gy 0.24±0.23d 0.38±0.72c 50 Gy 0±0e 0±0d

Rooting Only in vitro-shoots whose initial explants underwent Gamma rays of 5, 10 and 15 Gy were used for rooting seen their satisfactory vegetative growth. The roots began to appear the first month and after about two months, 90% of in vitro-shoots have been well rooted. After about six months, when the plants, well acclimated, reached an average height of about 50 cm, they were planted in the field. It is noteworthy that during both phases rooting and livestock no visual sign of difference between the olive trees was detected. Planting in the field 40 trees were planted in the field. It should be noted that for the control no Gamma rays treatment was used for those trees, for Z7, Z8 and Z9 trees the initial explants were treated respectively with 10 Gy, 5 Gy and 15 Gy ( Table 3). After about two years, only 35 olive trees survived (12.5% from Z9 are missing). Currently, twigs and leaves of trees do not exhibit visible morphological differences. The heights and diameters of trees are not significantly different. But, in the end of the second year of plantation, 7 plants were in bloom. However, the use of molecular biology techniques are needed to help check first the existence of the mutation and then try to discover its nature (Harten 1998; Ki-Hong et al. 2008). Table 3: Average growth of olive trees after planting Olive Dose of Number of Height Name Gamma trees Average (cm) Control 0 3 131.83±77.1ab Z8 5Gy 1 180±0a Z7 10Gy 29 145.93±24.5a Z9 15Gy 2 128.5±25.6b


Trunk diameter average (cm) 12.26±4.1a 13.62±0a 13.27±3.4a 14.52±2.1a

99

References Akshatha K.,and Chandrashekar R. 2014. Gamma Sensitivity of forest plants of western Ghats. J environ radioactivity 132: 100-107. Chaari-Rkhis A. 2013. Production de plants d’olivier par la technique de la culture in vitro. Invention Patent 22361 INNORPI/Tunisia 10p. Chaari Rkhis A., Maalej M., Drira N., and Standardi A. 2011. Micropropagation of olive tree Olea europaea L. cv ‘Oueslati’. Turk. J. Agric. For. 35: 403-412. FAO/IAEA. 1997. In vitro techniques for selection of radiation-induced mutants adapted to adverse environmental conditions. IAEA 312. D2.RC.545- Working Material Reproduced by the IAEA.Vienne. Austria. Fiorino A. 1992. L’amélioration génétique de l’olivier. Oliveae 41: 55-62 Harten A.V. 1998. Mutation Breeding, Theory and Practical Application. Cambridge University Press. London. p.111-134. Ki-Hong J., Gynheung A., and Pamela C.R. 2008. Nature Reviews. Genetics 22: 91-101. Msallem M., and Hellali R. 2000. Amélioration génétique par croisement de la variété d’olive de table "Meski" : Synthèse de trois campagnes. Revue Ezzaitouna 6 (1-2): 59-73. Noor N.M., Clyde M.M., Rao V.R., and Jeevamoney J. 2009. Radiosensitivity and in vitro studies of Citrus suhuiensis. In: Induced Mutation in Tropical Fruit Trees/ IAEA-TEC DOC-1615: 17-32. Predieri S. 2001. Mutation induction and tissue culture in improving fruits. Plant Cell, Tiss Organ Cult. 64: 185–210. Rallo L. 1995. Seleccion y mejora genetica del olivo en Espana. Olivae 59: 56-63. Rugini E., De Pace C., Gutierrez-Pesce P., and Muleo R. 2011. Olea. In: Wild crop relatives: Genomic and breeding resources, Temperate fruits. C. Kole Ed. Springer-Verlag Berlin Herdelberg, Chap.5: 79-117 Sanada T., and Amano E. 1998. “Induced mutation in fruit trees”, Somaclonal variation and induced mutations in crop improvement (Jain et al Eds) Kluwer Academic Publishers, pp401-419. Sangsiri C., Sorajjapinum W., and Srivivesc P. 2005. Gamma radiation induced mutations in mungbean. Sci Asia. 31: 251-255. Sen A., and Alikamanoglu S. 2012.Analysis of drought-tolerant sugar beet (Beta vulgaris L.) mutants induced withgamma radiation using SDS-PAGE and ISSR markers. Mutation Research 738–739: 38– 44 Trigiano, R.N., and Gray D.J. 2005. Plant Development and Biotechnology. CRC. Press. New York. pp. 73-83. Trigui A. 1996. L'amélioration génétique de l'olivier: Méthodologie et résultats préliminaires obtenus en Tunisie. Revue Ezzaitouna 2 (1 et 2) : 10-34. Tulmann Neto A., Ando A., Figueira A., Latado R.R., Dos Santos P.C., Correa L.S., Peres L.E.P., Hauagge R., Pulcinelli C.E., Ishiy T., Ferreira Filho A.W.P., and Camargo C.E.O. (in memoriam). 2011. Genetic Improvement of Crops by Mutation Techniques in Brazil. Plant Mutation Reports 2 (3): 24-37.


100

ASSESSMENT OF VARIABILITY IN THREE OLIVE VARIETIES FROM MONTENEGRO B. Lazović1,*, M. Adakalić1, D. Bandelj2, 3, T. Perović1 1

University of Montenegro, Biotechnical Faculty, Center for Subtropical Cultures, Topolica bb, 85000 Bar, Montenegro 2 University of Primorska, Science and Research Centre, Institute of Olive culture, Garibaldijeva 1, SI6000 Koper, Slovenia 3 University of Primorska, Faculty of Mathematics, Natural Sciences and Information Technologies, Glagoljaška 8, SI-6000 Koper, Slovenia *Corresponding author: [email protected]

Abstract In this study, we compared the capacity of morphological and molecular markers for assessing the varietal identity and connection of 16 selected clones/accessions of three local varieties: Crnica, Lumbardeška and Barkinja, from Montenegro. A study of morphological parameters showed differences between the tested clones, especially for degree of imperfect flowers (30.31 to 77.10%), fruit weight (2.89 to 5.36 g), stone weight (0.58 to 0.80 g) and oil content in dry matter (19.08 to 39.47%). The UPGMA analysis of 16 morphological traits and oil content showed variability and inter connection among the accessions of the three varieties. Analysis with nine SSR markers showed higher efficiency in the characterization of close individuals and revealed variability within Crnica variety and presence of synonymy. Keywords: olive, morphological characteristics, SSR markers, Montenegro Introduction Olive is a very important crop for Montenegro. It occupies about a quarter of the area under fruit plantation, with about 450,000 fruit bearing trees (Monstat, 2012). Intensive production of olives favors the use of few varieties with stable and regular yield. This could lead to genetic erosion and abandonment of locally adapted olive varieties (Bronzini de Caraffa et al., 2002; Ozkaya et al., 2006). The study of less common varieties is important, because they may have traits necessary to meet the challenges of modern growing (Cantini et al., 1999). For study of these varieties it is important to distinguish them morphologically and genetically to assure propagation of true-to-type material. The morphological traits have been used for identification, characterization and evaluation of varieties in different collections (Lazovic, 2003; Miranović, 1986; Trentacoste and Puertas, 2011). They are still considered essential for the germplasm evaluation, although are strongly influenced by the environment (Cantini et al., 2008). For precise genetic characterization, molecular markers are used. Moreover, microsatellite markers are able to differentiate closely related individuals developed through clone selection (Zaher et al., 2011). The combination of microsatellite marker profiles and morphological characters serve as reliable tool for detailed description of varieties. The olive varieties present in the Boka Kotorska sub-region represent the wealth of genetic resources of Montenegro, also a basis for further selection of clones. For a long history of olive growing on the Montenegrin coast, varieties are mostly vegetative propagated and Proceedings of the 5th Int. Conf. Olivebioteq 2014

101

disseminated with probable mixing. The emergence of varietal homonymous and synonymous and clonal selection complicates the identification and characterization of varieties in olive (Bandelj et al., 2002). The aim of this work was to evaluate the level of variability of accessions of three local olive varieties with morphological, agronomic, and molecular markers. Materials and Methods Plant material. The materials for analyses were three local olive varieties from Boka Kotorska. In the period from 2008 to 2011, 16 individuals of olive varieties: Crnica (CRN 19), Lumbardeška (LUM 1-4) and Barkinja (BAR 1-3), were studied. Morphological and oil content analyses. In the sample of 40 leaves, inflorescences, fruits and endocarps, and 10 twigs, the parameters of: leaf (length-LL, LW-width and shape indexLI), internode (length-INT), inflorescence (length INFL, number of flowers - FL and percentage of aborted flowers - AF), fruit (length-FL, width-FW, shape index-FI, weight-FWe, flesh ratio - FR) and endocarp (length - EL, width - EW, shape index - EI and weight - EWe) were measured according to the olive descriptor (Barranco et al., 2000). The oil content expressed as a percentage of oil on dry pulp (ODM) and fresh weight (OFM) was also measured in a Soxtec apparatus with use of diethyl ether. DNA analyses. Genomic DNA from fresh leaves of 14 examined accessions of olive varieties Crnica (9), Lumbardeška (2) and Barkinja (3) was extracted using the CTAB method (Kump et al., 1992). DNA was analyzed with nine microsatellite loci from four sets (Sefc et al., 2000; De la Rosa et al., 2002; Carriero et al., 2002; Cipriani et al., 2002). Amplification of microsatellites with Polymerase Chain Reaction (PCR) was performed according to the protocol of Bandelj et al. (2004). Detection of PCR products was carried out with an automated sequencer ABI Prism 3130 DNA Genetic Analyser (Applied Biosystems) using the internal standard GeneScan™-LIZ® 500 (Applied Biosystems). Data analyses. Morphological data. For 18 traits the analysis of variance (ANOVA) was performed using the Statistix 7.0. To determine the significance of differences LSD test at a 5% significance level was applied. The hierarchical cluster analysis was performed and a dendrogram was constructed using the unweighted pair-group average method (UPGMA) with the squared Euclidean distance in Statistica 5.0. Molecular data. The values of the frequencies of alleles (n), effective number of alleles (ne), observed (Ho) and expected (He) heterozygosity and fixation index (F)were calculated using the computer program Ganalex 6.4 (Peakall and Smouse, 2006). The probability of identity (PI) and null alleles (r) were calculated by a computer program IDENTITY 1.0 (Wagner and Sefc, 1999) and polymorphic information content (PIC) with program CERVUS 3.0.3 (Kalinowski et al., 2007). For estimation of the similarity between accessions Jaccard's coefficient was used. The dendrogram was constructed using UPGMA method by NTSYS 2.0 program (Rohlf, 1998).

Results and Discussion Morphological characteristics. The study was carried out to assess the similarities of 16 olive accessions supposed to belong to three local olive varieties Crnica, Lumbardeška and Barkinja. Studied accessions exhibited morphological differences and similarities in characteristics analyzed (Table 1). In the all studied accessions the internodes length and leaf length was medium, while the leaf width was medium (1-1.15) in all with exception of LUM 4 sample, which had a broad leaf (1.55). The leaf shape was elliptic-lanceolate in 14 accessions, Proceedings of the 5th Int. Conf. Olivebioteq 2014

102

and elliptic (25) and it is able to sequester into the soil high amount of carbon. In fact, a correct residues management is suggested as a realistic strategy to mitigate the greenhouse effect (Lal, 1997) (Table 3). In order to reach a nutritional aim in the short period, “high quality” material should be chosen. These residues have a low content of products that do not decompose easily, high nitrogen content and a low C/N ratio. These features determine relevant CO2 emissions and high release of mineral nitrogen in the soil due to the quick residue decomposition. Therefore, the shredding time is very important and has to be aimed to synchronize the needs of the olive trees with the nutrients release by the residues avoiding possible contamination of the groundwater avoiding water competition between olive trees and cover crops (Pastor et al., 2000). Often it should be recommended to use legumes in advanced stages of development (>C/N, >lignification) or a mixture of both low and high quality organic materials such as Leguminosae with Graminaceae in order to achieve simultaneously both nutritional and soil carbon enrichment aims. Particularly, this association combines the ability of cereals to limit erosion, thanks to their quick establishment on the ground, with legumes capacity to fix gaseous nitrogen. The suggested mixture allows a huge nitrogen accumulation in both epigeous and root biomasses as well as a significant CO2 sequestration (Table 3). Pruning residues Pruned material can represent an important source of organic dry matter (Table 3) according to the age of the trees, plant density, training system and intensity of the pruning. It Proceedings of the 5th Int. Conf. Olivebioteq 2014

219

represents a “low quality” material. If it is cut and buried in the soil, it can provide a material able to build up in the years SOM and release nutrients useful for the plants. This is true especially when pruning material is combined with cover crops. Such mixture produces an “intermediate” quality organic material, functional for SOM conservation, for improvement of activity and complexity of microbial biomass (Table 4), for nutritional aims, and as mulching against soil erosion. Sustainable soil management can also significantly modify the composition of bacterial communities from the phyllosphere and carposphere of olive trees increasing bacterial biodiversity and to trigger in the plant mechanisms of resistance against biotic and abiotic stresses. Particularly, 31 bacterial species were classified from the pulp (mesocarp) of fruits picked from olive trees grown under sustainable conditions against the 2 species found in olives of the conventional orchard (Table 5) (Pascazio et al, in press). In olive orchards subjected to phytosanitary problems, pruning residues can be used as structural material in farm composting. During the composting with other kind of agricultural wastes (olive mill wastewater, olive leaves and twigs, pomace, livestock manure, herbaceous residues) an effective sanitation process of the pruning material occurs. The use of this material in the composting may also overcome the reluctance of its direct use in the open field induced by eventual phytotoxicity problems due to the production of phenols during its degradation process. External carbon sources Among the different typologies of organic materials, stabilized manure is the best thanks to its several benefits as fertilizer and amendment. In the past the use of stabilized manure was very common in the olive groves. Often, olive growers owned animals and produced stabilized manure with no added cost. In areas where livestock presence is low, compost can be used with positive effects on olive tree productive performances and soil quality and fertility. The compost production by mixing pomace, olive mill wastewater, olive leaves and twigs coming from fruit cleaning, straw and poultry manure is recently increased. This is an effective example of carbon sources recycling within the olive-oil chain. On the other hand, the use of compost is not widely diffused because of its high price due to transport expenses from composting plants to farms. For this reason, it should be recommended the diffusion of simple on-farm composting technologies producing low-cost compost. Irrigation strategies for water saving and containment of soil respiration Irrigation significantly increases the vegetative growth of the olive tree and its yield response. In particular, water availability allows plants to go into production early, and consistently produce satisfactory yields and high quality (pulp/stone ratio, fruit size) (Table 6). Since productive performance of the olive trees is not affected by moderate levels of water stress, it is recommended, especially in arid and semi-arid conditions, to contain the irrigation volumes and distribute them by micro-irrigation in order to save water. Sustained deficit irrigation (SDI) distributes a reduced water volume, as percentage of ETc, throughout the whole irrigation season. Many authors found that a restitution ranging from 66 to 75% of ETc is enough to obtain good yields similar to those harvested from fully irrigated trees. However, phenolic compounds in oils significantly decreased passing from the lowest to the highest irrigation levels. Regulated deficit irrigation (RDI), firstly proposed by Chalmers et al. (1981), reduces water supplies during specific periods characterized by a less plant sensibility to water stress with minimal effects on yield. While water deficit can reduce fruit and oil yields due to Proceedings of the 5th Int. Conf. Olivebioteq 2014

220

the effect on flowering, fruit set, and oil accumulation phases, many researchers agree in identifying pit hardening, generally occurring in midsummer, as the less sensitive phenological stage of olive tree. On the other hand, in environments characterized by good spring rainfall and deep soil profiles, irrigation applied from the beginning of pit hardening to early fruit version? could control tree vigour while maintaining crop yield and oil quality. Therefore, the optimal irrigation volume should be established every year taking into account the expected yield and the climatic pattern. Drip irrigation combined with water stress strategies allow to limit the wet area and reduce the irrigation period during the annual cycle. As a consequence, natural CO2 emissions from soil due to root and microorganism respiration can be contained. Nutrient management in olive orchards A well balanced and appropriate fertilization should take into account some steady points: a) real nutrient needs of olive trees along the different stages of plant life cycle; b) soil nutrient availability and tree nutritional status; c) synchronization between nutrient requirements by the plants and their availability in soil volume where roots are present; d) fertilization techniques and their efficiency (application to the soil, fertigation, canopy spray); e) soil management techniques (spontaneous or seeded cover crops, recycling of pruning material within the orchard, use of manure or compost) and water availability linked to natural conditions (rainfall) or irrigation practice. A nutrient balance approach should be used to define the right amount of mineral elements to apply by means of fertilization (Table 7). The balance has to take into account nutrient input and output in the orchard system. Pruning material, when cut and left on the ground of the orchard, has to be considered as an input together with senescent leaves. Instead, cover crops show nutritional element intake during the first years of the orchard life. About 60% of nutrient requirements by the cover crops occur at the beginning of annual vegetative cycle of olive trees and at fruit set. During these phenological phases trees are sensitive to mineral feed competition. Among inputs, irrigation water can be a significant source of mineral elements. Therefore, water analysis has to be carried out and a provisional irrigation schedule should be defined before fertilization plan draft. Also soil mineralization can assure remarkable amount of nitrates which are the nitrogen form easily used by the plants. So, a sustainable fertilization should be taken into account the actual soil nitrate availability (about 20 ppm in the first 30 cm soil layer) and assure its steady concentration during the annual plant cycle thus avoiding excessive consumption by the plants and losses by leaching. Soil nitrate monitoring (0-50 cm layer) should be performed with field rapid tests according to a time schedule based on the rhythm of nutrient uptake by the plants during the annual cycle. Pruning material, if moved away from the orchard system, has to be considered an output together with yield. Particularly, in young olive groves it is necessary to take into account also requirements for growth of above and below-ground parts (immobilization in permanent structures) which can be considerable. Soil and leaf analyses are useful tools to guide appropriate choices in fertilization scheduling. The former should be performed every 4-5 years to monitor soil parameters which vary slowly over time. Acknowledgements This research was part of the LIFE11 ENV/GR/942 OLIVE-CLIMA Project Proceedings of the 5th Int. Conf. Olivebioteq 2014

221

Table 1. Atmospheric CO2eq fluxes: comparison between two management systems, Sustainable (cover crops + pruned material recycling) and Conventional (tillage) (mean 20012008). The symbol + indicates CO2 losses while – indicates CO2 sequestration (Palese et al., 2013).

Sustainable System Conventional System

Total annual Annual Net Primary Productivity Total emissions Difference * the pruning material was burned

CO2 (t ha-1 year-1) -37.84 -15.91 +22.39 +26.11* -15.45 +10.20

Table 2. Vertical infiltration of water (measured at 12 cm soil depth in correspondence of the ploughpan), soil macroporosity (measured in 0-30 cm soil layer and expressed as percentage of total porosity) and rain water stored during the rainy season in cover cropped and tilled soils for 10 years (Palese et al, 2014). Soil management Cover crops Tillage

Vertical infiltration rate of water (mm d-1) 160 13

Soil macroporosity (%) 9.5 5.4

Rain water stored into the soil up to 2 m depth (m3 ha-1) 4250 2936

Table 3. Dry matter and CO2 fixed in a mature olive orchard (156 plants ha-1) by spontaneous cover crops and pruning material (Palese et al., 2013). Spontaneous plant soil cover (aboveground part + belowground part) Pruning material

Dry matter (t ha-1) 7.5

CO2 (t ha-1) 13.2

3.3

5.9


222

Table 4. Total fungi and bacteria counts (Colony Forming Units - CFU) in soil of olive orchards grown under different management systems: Sustainable (cover crops + pruned material recycling) and Conventional (tillage) (Sofo et al., 2014). Orchard management Sustainable Conventional

Fungi Bacteria CFU g-1 dry soil 214000 35600000 29000 10000000

Table 5. Classification of the bacterial species from olive fruit pulp (mesocarp) identified on the basis of their genomic sequences (NCBI BLAST® hits) (Pascazio et al., in press) Orchard management

Number of species

Phylum

8

Proteobacteria

5

Firmicutes

5

Proteobacteria

4

Actinobacteria

2

Proteobacteria

1

Actinobacteria

1

Proteobacteria

1

Proteobacteria

1

Proteobacteria

1

Proteobacteria

1

Proteobacteria

1

Uncultured bacteria

2

Proteobacteria

Sustainable

Conventiona l

Class

Order

Family

Enterobacteriales Enterobacteriaceae Proteobacteria Bacilli Proteobacteria Actinobacteri dae Proteobacteria Actinobacteri dae Proteobacteria αProteobacteria Proteobacteria Proteobacteria Proteobacteria

Lactobacillales

Enterococcaceae

Enterobacteriales Enterobacteriaceae Actinomycetales

Species

Rahnella

aquatilis

Enterococcus

unknown

Kluyvera

intermedia

Microbacteriaceae Curtobacterium unknown

Enterobacteriales Enterobacteriaceae Actinomycetales

Genus

Microbacteriaceae

Averyellaa

dalhousiens

Frondihabitans

suicicola

Hafnia/Rahnell a Methylobacteriacea Methylobacteri e um

Enterobacteriales Enterobacteriaceae Rhizobiales

Enterobacteriales Enterobacteriaceae Enterobacteriales Enterobacteriaceae

alvei unknown unknown

Pantoea

Serratia/Rahnel unknown la

Enterobacteriales Enterobacteriaceae

Serratia

unknown

Enterobacteriales Enterobacteriaceae Proteobacteria

Pantoea

agglomeran s


223

Table 6. Olive and oil yields, and fruit characteristics (mean 2001-2008) of an olive orchard grown under the Sustainable management. Seasonal irrigation volume: 342.5 mm. Orchard plant density: 156 trees per hectare (Palese et al., 2013). Parameter Yield (kg tree-1) Mean fruit fresh weight (g) Flesh/Stone ratio Flesh incidence on the total fruit fresh weight (%) Olives felt within the equatorial classes ranging from 1 to 20 mm (%) Oil yield (%)

Sustainable management 62.4 3.8 6.1 85 93 20

Table 7. An example of nitrogen balance in a mature olive orchard (156 plant ha-1; average yield = 54 kg plant-1 fresh weight; pruning material = 36 kg plant-1 fresh weight; senescent leaves = 11.7 kg plant-1 fresh weight; N concentration in senescent leaves = 0.76% dry weight; seasonal irrigation volume = 3000 m3 ha-1 year-1; N–NO3 concentration in irrigation water = 8 ppm). Balance item Irrigation water (around 19 m3 plant-1 year-1)

Requirements Supplies g N plant-1 137*

Permanent tree structures Yield 413 Pruning material 182 Senescent leaves 88 Total 683 Difference 411 Integrative fertilization by fertigation (efficiency coeff.: 1.2) 493 * Use efficiency equal to that of mineral fertilizers (N used/N applied=0.90) ** N recycling equal to 50%

91** 44** 272

References Beaufoy, G., 2002. The environmental impact of olive oil production in the European Union: Practical options for improving the environmental impact. EU Report. Celano G., Dumontet S., Xiloyannis C., Nuzzo V., Dichio B., Arcieri M., 1998. Green manure plant biomass evaluation and total mineral nitrogen in the soil of a peach-orchard system. Acta Hort. 465, 579-586.


224

Chalmers, D.J., Mitchell, P.D., and van Heek, L. 1981. Control of peach tree growth and productivity by regulated water supply, tree density and summer pruning. J. Am. Soc. Hortic. Sci. 106:307-312. Duarte, F., Jones, N., Fleskens, L., 2008 Traditional olive orchards on sloping land: sustainability or abandonment? J. Environ. Manage. 89 (2), 86-98. Lal R., 1997. Residue management, conservation tillage and soil restoration for mitigating greenhouse effect by CO2-enrichment. Soil & Tillage Research 43, 81-107. Palese A.M., Pergola M., Favia M., Xiloyannis C., Celano G., 2013. A sustainable model for the management of olive orchards located in semi-arid marginal areas: some remarks and indications for policy makers. Environmental Science and Policy, 27: 81-90. Palese A.M., Vignozzi N., Celano G., Agnelli A.E., Pagliai M., Xiloyannis C., 2014. Influence of soil management on soil physical characteristics and water storage in a mature rainfed olive orchard. Soil & Tillage, 144: 96-109. Palese, A.M., Celano, G., Xiloyannis C., 2011. Esigenze minerali e tecniche di concimazione. Collana divulgativa dell’Accademia Nazionale dell’Olivo e dell’Olio, Spoleto (PG), p. 27. Available on: http://www.accademiaolivoeolio.com. Pascazio S., Crecchio C., Ricciuti P., Palese A.M, Xiloyannis C., Sofo A., 2015. Phyllosphere and carposphere bacterial communities in olive plants subjected to different cultural practices. International Journal of Plant Biology, in press. Pastor, M., Castro, J., Humanes, M.D., Muñoz, J. 2000. Gestione del suolo nell’olivicoltura dell’Andalusia. L’Informatore Agrario 3, 83-92. Sofo A., Ciarfaglia A., Scopa A., Camele I., Curci M., Crecchio C., Xiloyannis C., Palese A.M., 2014. Soil microbial diversity and activity in a Mediterranean olive orchard managed by a set of sustainable agricultural practices. Soil Use and Management, 30: 160-167. Xiloyannis, C., Martinez Raya, A., Kosmas, C., Favia, M.F., 2008. Semi-intensive olive orchards on sloping land: requiring good land husbandry for future development. Journal of Environment Management 89 (2), 110–119.


225

PHYSIOLOGICAL RESPONSES TO DEFICIT IRRIGATION OF YOUNG OLIVE TREES (Olea europaea, L.) GROWN IN SEMI ARID AREA OF MOROCCO. A. Bouizgaren1*, L. Sikaoui1, H. Boulal2, A. El Antari1, M.Karrou2, V. Nangia3 and T. Oweis3 1

Unit of Plant Breeding, National Institute for Agronomic Research (INRA), PO. Box 533, Gueliz 40000, Marrakesh, Morocco. 2 International Center of Agricultural Research in Dry Area (ICARDA) 3 International Center of Agricultural Research in Dry Area (ICARDA), Amman, Jordan *

Corresponding author: [email protected]

Abstract The scarcity of irrigation is the major constraint facing the olive production in Morocco. Under such conditions, deficit irrigation is one of the techniques leading to maximizing olive yields per unit of irrigation water. This work was conducted to investigate physiological responses, to deficit irrigation, of young olive trees (Olea europaea L.) cultivated under semiarid conditions of Morocco. The experiment was carried out at the Experiment Station of INRA Marrakech. Three watering regimes were studied: FDI= Full drip irrigation (100% of ETc), DDI= Deficit drip irrigation (70% of ETc) and FU= Flood irrigation (farmer’s conventional technique). The physiological parameters, leaf relative water content (LRWC), Leaf chlorophyll content (LCC), total leaf mineral content (TLMC), leaf protein content (LProtC), leaf soluble sugar content (LSSC), leaf proline content (LProlC) and stomatal conductance were measured at three phenological stages: stage 1 (first flowers open), stage 2 (fruit (fruit about 10% of final size) and satge 3 (fruit about 90% of final size, and suitable for pinking green). The results showed that at the three phenological stages, the low values of LRWC, LCC, TLMC and stomatal conductance were recorded under FU regime. The High values of LProlC, LSSC, LProtC were recorded by FDI regime, suggesting that the young trees are more exposed to water stress under this irrigation technique despite the high amount of applied water. For most of those physiological parameters no significant differences were noted between FDI and DDI regimes. These latter regimes allowed, respectively, 30% and 80% of water saving compared to FU regime. Keywords: young olive trees, deficit irrigation, physiological parameters Introduction The scarcity of water resources in Morocco is of growing concern for the agricultural sector, particularly in semi-arid areas, because of the increasing population, high drought frequency and severity due to the effects of climate change. Moreover, these areas are characterized by high evaporative demand (about 1500 mm/ year), and low and irregular rainfall (200-300 mm/year). In this semi arid area, the new olive orchards are planted under irrigation to ensure the supply of water required for growth and development of the crop. However, a large newly planted area is irrigated by a traditional inefficient surface irrigation system which leads to high water losses. Under conditions of water scarcity and drought, deficit irrigation at selected phenological phases can lead to saving water and maximizing yields per unit of water for a given crop Proceedings of the 5th Int. Conf. Olivebioteq 2014

226

(Tognetti et al. 2006) without significant yield losses. Therefore, to understand how young olive trees respond to water restrictions and drought, a study was conducted on the physiological responses of this crop to deficit irrigation under semi arid conditions of Morocco. Material and methods The experiment was conducted in a three years old olive orchard (Olea europaea L. cv Menara) installed in 2010 at Sâada Experiment station of INRA, Morocco, located 9 km from Marrakech city (32° N, 8°08 W, 411 m altitude). The climate is a typical Mediterranean. Spring and summer seasons at the experimental site are normally characterized by severe drought stress associated with high temperatures (Table 1). The soil has a loamy clay texture and it is over 2 m deep. Table 1: Mean climatic variables in the experimental year. Climatic variable Rainfall September–March (mm) Rainfall April– August June (mm) Mean daily min temp January–February (°C) Mean daily max temp July–August (°C) Total rainfall 12 months (mm) Total 12 months ET0 (mm)

2013 164.2 26.8 11.5 45.5 191.0 1508

Three watering regimes were studied: FDI= Full drip irrigation (100% x ETc), DDI= Deficit drip irrigation (70% x ETc), and FU=Flood irrigation (farmer’s practice corresponding to ‘460% x ETc’ and irrigation frequency ‘2 to 3 weeks’). For drip irrigation treatments (FDI and DDI), the frequency of irrigation was two days; while it was around one month in the case of flood irrigation during summer. The full drip irrigation regime received the crop water requirement (ETc). The ETc was calculated using the equation: ETc = ETo x Kc x Kr /Ne Where ETo is the reference evapotranspiration, Kc is the crop coefficient for olive tree, Kr is the coefficient to correct for incomplete soil cover and Ne is the efficiency of irrigation network. ET0 was estimated using Penman-Monteith equation and daily meteorological data collected from an automatic weather station located 50 m away from the experiment plot. The Kc values used are the ones reported by Orgaz and Fereres (1997); while the value of Kr was estimated at 0.8 (Fereres and Golhamer, 1990). The amounts of irrigation applied for the three watering regimes FDI, DDI and FU were 116 mm, 81.2 mm and 538 mm, respectively. A randomized complete-block design was used with three replications of 21 trees each. The spacing between trees was 8 m x 8 m. The physiological parameters were measured at three phenological stages: stage 1 (first flowers open), stage 2 (fruit about 10% of final size) and stage 3 (fruit about 90% of final size, and suitable for pinking green): Leaf relative water content (LRWC): LRWC was defined as follows (Makela et al., 1998): LRWC(%)=((FW-DW)/(TW-DW)) x100. Where FW (fresh weight), TW (turgid weight) and DW (dry weight). Proceedings of the 5th Int. Conf. Olivebioteq 2014

227

Leaf proline content (LProlC): is determined using the method described by Bates et al. (1973). Leaf total Chlorophyll contents (LTCC): Chlorophyll concentration was measured by the method described by Arnon (1949). Leaf protein content (LprotC): LProtC was determined using the method described by Bradford (1976). Leaf soluble sugar content (LSSC): The content of soluble sugars was determined according to the method of Dubois et al. (1956). Stomatal conductance: Stomatal conductance to H2O (g mm s-1) were measured in leaves, using leaf porometer (model sc-1). Statistical analysis Data were statistically analyzed using the analysis of variance and mean comparisons were carried out using the Student-Newman-Keuls test when significant treatment effects occurred. Results Leaf relative water content (LRWC): A significant variation was shown among the three studied irrigation regimes at the three phonological stages studied (Figure 1). The high values of this parameter were obtained under FDI; but the difference with DDI was not statistically significant. The lowest values of LRWC were obtained under FU regime.

Fig. 1: LRWC of young olive trees under three irrigation regimes for the three phenological stages (Values with the same letter do not differ significantly (P> 0.05)) Leaf proline content (LProlC): At the three phenological stages, the accumulation of proline in leaves varied significantly between the three irrigation regimes (Table 2). The FU regime induced high values of LPC which reached 21.1, 23.6 and 31.1 mg. g FW-1 at stages 1 , 2 and 3 respectively. The FDI and DDI regimes did not differ significantly. Leaf chlorophyll content (LTCC): The variation of the irrigation regimes induced a significant effect on LTCC of young olive trees (Table 2). The high values of this parameter Proceedings of the 5th Int. Conf. Olivebioteq 2014

228

were obtained under FDI regime with 22.7, 15.1 and 23.3 mg. ml-1 at stages 1, 2 and 3, respectively that did not differ significantly with those obtained under DDI regime. Leaf Proteins content (LProtC): Water regime affected significantly the LProtC of young olive trees at stages 2 and 3 (Table 2). The high accumulation of proteins in young olive leaves was noted under FU regime with 14.9, 23.3, and 54.3 mg. mg FW-1 at stages 1, 2 and 3, respectively. Soluble sugar (LSSC): A significant variation was shown among the irrigation regimes for the LSSC at the three phenological stages (table 2). Within the same phenological stage, the low and high values were achieved under FDI and FU regimes, respectively. At stage 3, the LSSC varied between 289.2 and 350.1 mg. g FW-1 for FDI and FU regimes, respectively, and we did not observe any significant difference between DDI and FDI regimes. Table 2: Physiological parameters of young olive trees measured at three phonological stages and under three different water regimes. LProlC LTCC LProtC Phenological Irrigation LSSC (mg. g FW (mg. ml (mg. mg FWstages regimes (mg. g FW-1) 1 1 1 ) ) ) FDI 15.4 a 22.7 a 14.2 a 160.6 a DDI 17.2 a 22.4 a 14.8 a 174.4 ab Stage1 FU 21.1 b 16.4 b 14.9 a 185.3 b P 0.018 0.05) Stomatal conductance: The average values of stomatal conductance measured at the three studied phonological stages are shown in figure 2. Significant variations were recorded among the three irrigation regimes during the three phenological stages. The high and low values were recorded under FDI and FU regimes, respectively.


229

Fig. 2: Stomatal conductance of young olive trees in relation with irrigation regimes Total mineral leaves content (TMLC): The total mineral content in leaves of the young olive trees measured at stage 3 varied significantly with the variation of the irrigation regimes (Figure 3). The highest mean values of TMLC were observed under full drip irrigation, while the lowest were obtained under flood irrigation.

Fig. 3: Total mineral leaves content of young olive trees in relation with irrigation regimes Discussion In the semi arid area where the experiment was carried out proper management of irrigation water, such as the application of deficit irrigation, increases water use efficiency and decreases


230

water consumption. However, water stress causes changes in a number of physiological and biochemical processes governing plant growth and productivity (Alexieva et al., 2001). Our results showed that at the three phenological stages, the LRWC depended on the irrigation regime. The lowest values of this parameter were recorded under FU regime, because the low frequency of irrigation with high amounts of water in summer under high temperatures certainly increased the loss of water by percolation and evaporation and created the conditions of water deficit in the root zone. The stomatal conductance was negatively affected by FU regime. Fernández (1997) showed that the olive tree is able to restrict water loss by closing its stomata. DDI induced a stomatal close in comparison with FDI. This is in relation with the soil water potential which controlled significantly stomatal conductance (Lu and Zhang 1999). We noted that FU regime induced a significant increase in accumulation of proline and sugar in leaves. The proline accumulation is frequently observed during drought stress and is thought to play a multifunctional role in defence mechanisms ( Boussadia et al., 2013). Sugars are also among the important osmolytes contributing to the osmotic adjustment of plants under water or saline stress (Ashraf and Harris, 2004). A significant decrease in LCC was shown under FU regime in comparison with drip irrigation regimes. In general, this decrease is related to water deficit which occurred under flood irrigation and caused a decrease in the concentration of chlorophyll pigments in the leaves of young olive trees. This phenomenon can be explained either by the degradation of the pigments by the hydrolytic enzymes, or by the inactivation of the biosynthesis of these pigments (Farooq et al., 2009). We noted an increase in proteins accumulation under FU regime at stages 2 and 3. Galau and closed (1992) reported that water deficit induced an increase in proteins content in olive trees by an increase of nitrogen (Garg, 2003). This induction of proteins by water deficit depends on the stage and the genotype of the tree (Riccardi et al., 1998). The total mineral content in leaves of the young olive trees varied significantly with irrigation regimes and the highest values were noted under FDI, while the lowest were obtained under FU regime which induce a limitation in nutrient uptake and decreasing their concentrations in leaves tissue. Similar results were reported by Mehanna et al., (2012) when the applied water stress on olive orchard. We conclude that deficit drip irrigation did not induce a significant effect on all physiological parameters measured in comparison with full drip irrigation. It allowed 30% of water saving compared to full drip irrigation and 80% of water saving compared to flood irrigation techniques. References Alexieva V., Sergiev I., Mapelli S, Karanov E. 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ 24: 1337– 1344. Arnon D.I. 1949. Copper enzymes in isolated chloroplasts: ployphenol-oxydase in Beta vulgaris L. Plant Physiol. 24:1-15. Ashraf M ., Harris P.J.C. 2004. Potential biochemical indicators of salinity tolerance in plants. Plant. Sci. 166: 3-16. Bates L., Waldren R.P., Teare J.D. 1973. Rapid determination of free proline for water stress studies. Plant Soil. 39:205-207. Proceedings of the 5th Int. Conf. Olivebioteq 2014

231

Boussadia O., Bchir A., Steppe K., Van Labeke M.C , Lemeur R., BrahamM. 2013. Active and passive osmotic adjustment in olive tree leaves during drought stress. European Scientific Journal. Vol.9, No.24, 423- 439. Bradford M.M. 1976. A rapid and sensitive method for the quantification of microgram qauntities of protein utilizing. The principale of protein dye binding. Anal. Bioch. 72: 248-257. Dubois F., Gilles K.A, Hamilton J.K., Rebers P.A. Smith F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356. Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., Basra, S.M.A., (2009). Plant drought stress: effects, mechanisms and management. Agron. Sustain. Dev., 29: 185–212. Fereres E. and Goldhamer D. 1990. Decious ruit and nut tress, p. 987-1017. In: B.A. Stewardt and D.R. Nielsen (eds.). Irrigation of Agricultural Crops-Agronomy Monograph N°. 30 Amer.Soc.Agron., Madison. Fernández J.E., Moreno F., Girón I.F., Blázquez O.M. 1997. Stomatal control of water use in olive tree leaves. Plant and Soil 190: 179-192. Fisher G, Shah M, van Velthuisen H., 2002. Climate change and agricultural vulnerability. Vienna (Austria): IIASA. Galau, G., Close, T.J., 1992. Sequence of the cotton group 2 LEA/ RAB/ dehydrin proteins en coded by lea3 cDNCs. Plant physiology. 98: 1523-1525. Garg B.K., 2003. Nutrient uptake and management under drought: nutrient-moisture interaction. Curr. Agric. 27: 1–8. Lu C. Zhang, J. 1999. Effect of water stress on photosystem II photochemistry and its thermostability in wheat plants. Journal of Experimental Botany, Vol. 50, N° 336: 11991206. Makela P., Munns R., Colmer T.D., Condon A.G., Peltonen-Sainio P. 1998. Effect of foliar applictions of glycinebetaine on stoamatal conducatnce, abscissic acid and solute concentrations in leaves of salt or drought-stressed tomato. Aust. J. Plant physiol. 25: 655663. Mehanna H.T., Stino, R.G., Ikram, S.E., Gad El-Hak, A.H. 2012. The Influence of Deficit Irrigation on Growth and Productivity of Manzanillo Olive Cultivar in Desert Land. Journal of Horticultural Science & Ornamental Plants 4 (2): 115-124, 2012 Orgaz F. and Fereres E. 1997. El cultivo del olivo. In: D. Barranco, R., Fernández-Escobar and L. Rallo (eds).. Mundi Prensa, Madrid. p. 251-272 Riccardi F., Gazeau, P., De Vienne D., Zivi, M. 1998. Protein changes in response to progressive water deficit in maize: quantitative variation and polupeptide identification. Plant physiology. 117: 125-126. Tognetti R., d'Andria R., Lavini A., Morelli G., 2006. The effect of deficit irrigation on crop yield and vegetative development of Olea europaea L. (cvs. Frantoio and Leccino). European Journal of Agronomy, 25: 356-364.


232

EFFECT OF SWITCHING FROM SURFACE TO DRIP IRRIGATION ON THE PERFORMANCE OF MATURE OLIVE TREES IN A DRY AREA OF MOROCCO A. El Antari1*, L. Sikaoui1, A. Bouizgaren1, M. Karrou2, H. Boulal2, M. Idrissi1, Y. Ouguas1, V. Nangia3, T. Oweis3 1

INRA, CRRA Marrakech, BP 533 Morocco ICARDA, Rabat, Morocco 3 ICARDA, Amman, Jordan 2


Abstract In Morocco, the area of irrigated olive trees increased in the last decades to reach 35% of the total (around one million hectares). Most of this irrigated area is cultivated under traditional inefficient surface irrigation system. To overcome this inefficiency of water use, new water saving irrigation methods, such as drip irrigation, have been promoted by the government. The objective of this research is to study the response of mature olive trees to switching from conventional to drip irrigation. For this purpose, a trial was conducted in 2012 in a 36-years old irrigated olive orchard (cv. Picholine Marocaine) at the Tessaout experimental station of INRA Marrakech characterized by a hot and dry summer Mediterranean climate. The cumulative rainfall of the site during 2012 was 293 mm. Five treatments were applied: Deep tillage under full irrigation or 100 % of crop water requirements (FWR), Deep tillage under 70% of FWR, Minimum tillage under 100% of FWR and 70% of FWR and flood irrigation (Farmer technique). The amounts of irrigation applied were 936 mm for flooding treatment, 524 mm for 100% FWR and 367 mm for 70% FWR. The results showed that the average olive fruit weight was higher under drip irrigation compared to flood irrigation and it was 4,5 g and 5,2 g, respectively. However, the effects of irrigation treatments on the composition of the total fatty acids of olive oils were significant on stearic and Mono/Poly insaturated ratio. The highest values of bitterness were obtained with flood irrigation treatment. The sensorial quality test showed that olive oil was more intense under deficit irrigation regimes than under the other treatments. The same trend was observed with bitter and pungent attributes. Specific aromas were also improved under deficit irrigation and it’s more appreciated by consumer. From the preliminary results, it seems that drip irrigation saved water and improved olive oil quality and composition. Keywords: olive, irrigation, yield, olive oil, olive oil quality Introduction The olive tree exhibit a fairly high resistant to water stress. Generally, the olive is grown in marginal areas with low water availability. The management of water supplies that are currently declining would be an obligation to ensure better production quality and quantitaty. Indeed, the great challenge for the coming decades (Centritto et al., 2000; Fereres and Soriano, 2007) is to increase crop production in the countries that suffering from low rainfall during the most critical phonological phases for yield production, with less water available for irrigation. Proceedings of the 5th Int. Conf. Olivebioteq 2014

233

Some studies suggest that the intakes of water through irrigation strategies help and improve the productivity of trees (Patumi et al., 1999, Tognetti et al. 2006). But they differ in terms of the effect of irrigation on olive oil quality. Moreover, it is widely known that the quality characteristics of extra-virgin olive oils derive from concomitant action of olive variety, climatic conditions, degree of maturation and agronomic practices related to irrigation (Servilli, et al., 2007; Gómez-Rico et al., 2009). The aim of the present work was to evaluate the effect of different irrigation practices on the quality and composition of olive oil from mature tree in south of Morocco. Material and methods Experimental site: The experiment was conducted in a 36-years old irrigated olive orchard (var. Picholine Marocaine) at the experimental Station of INRA in Tessaout (Marrakech region). The soil is classified as silty clay. The climate is Mediterranean with hot and dry summer. The average annual rainfall is 266 mm with a drought period from May to August. Treatments: Two soil tillage treatment are tested: deep tillage and minimum tillage. The deep tillage is conducted at 60 cm depth and 1 meter from the trunk. The minimum tillage is performed by one pass of chisel. Two irrigation methods were studied: drip irrigation with two water regimes (100% and 70% of crop evapotranspiration ‘ETc’), compared to flood irrigation (traditional system used by most of the farmers). In total, five treatments were tested: - T1= Flood irrigation (farmer’s use) - T2= Minimum tillage with drip irrigation (100% x ETc) - T3= Minimum tillage with drip irrigation (70% x ETc) - T4= Deep tillage with drip irrigation (1000% x ETc) - T5= Deep tillage with drip irrigation (70% x ETc) The ETc was calculated following the equation: ETc = ETo x Kc x Ke / Ne. The amount of water applied were 913 mm, 515 mm and 360 mm flood irrigation, full irrigation (Drip x100%) and (Drip x70%) respectively. Experimental design: Drip irrigation and flood irrigation systems are used in two adjacent plots. For drip irrigation, the trial was installed in randomized complete block design with three replicates and including 12 subplots (two tillage treatments x two irrigation regimes x three replicates). Each subplot included 10 trees. The planting density was 208 trees ha-1 in the case of flooding treatment with a row spacing of 8 m x 6 m and 156 trees ha-1 for drip irrigation treatment with 8 m x 8 m row spacing. In both systems, olive trees had the same age and a severe pruning was performed. Parameters measured: Olive yields were estimated on the entire trees per block. To study the fruit and oil parameters, samples of 4 kg of olives per tree (three trees per plot) were harvested at different stages of maturity. Fruits biometry were measured, and then triturated Proceedings of the 5th Int. Conf. Olivebioteq 2014

234

for oil analysis. The oil was extracted by the “Abencor” system. The extracted oil was analyzed for chemical and sensory parameters. The chemical parameters were: water and oil contents, viscosity, fatty acids and total polyphenols. Results and discussion Olive Yield: Estimated yields are reported in Figure 1. Significant variation was noted among treatments (P