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A. Belaj, Z. Satovic, I. Trujillo and L. Rallo

Eighty-two Spanish olive cultivars from the World Germplasm Bank of the Centro de Investigación y Formación Agraria (CIFA) Alameda del Obispo in Cordoba (Spain) were analysed by RAPD markers to assess their genetic relatedness and to study patterns of genetic variation. The dendrogram based on unweighted pair group cluster analysis using Jaccard's index included two major groups that consisted mostly of cultivars from the southern and central part of Spain. Clustering together of cultivars from the Levante zone was also observed. The pattern of genetic variation among olive cultivars from three different Spanish zones (Levante, central and Andalusia) was analysed by means of the analysis of molecular variance (AMOVA). Although most of the genetic variability was attributable to differences of cultivars within each zone (95.88%), significant φ-values among zones (φst = 0.041; p < 0.001) suggested the existence of phenotypic differentiation. These results are consistent with the predominantly allogamous nature of Olea europaea L. species. Significant values of φst for the pair Andalusia/Levante indicate the presence of differentiation. The negative value of φst observed in the case of the Andalusia/central pair suggests that some varieties from central Spain are more similar to the Andalusian ones than to the varieties of their own geographic area, and vice versa.

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George D. Nanos, Elizabeth Agtsidou and Evangelos M. Sfakiotakis

Ripening of detached mature-green and black-ripe olives (Olea europaea L., cv. Conservolea) was studied during storage at 0, 5, 10, or 20 °C in air or air plus 100-200 μL·L-1 propylene. Green olive skin h° remained unchanged after 24 days at 0 or 5 °C in air or air + propylene, while olives partially lost their green color at 10 °C and developed purple color at 20 °C together with a substantial flesh softening. Propylene partially delayed flesh softening only at 10 °C. Respiration of green and black olives increased with storage temperature. Black olives had higher respiration rate than green olives. Propylene had no substantial effect on green or black olive respiration rate, except for an increase in respiration and ripening rates of green olives kept at 20 °C. Ethylene production rate of air- or air + propylene-treated green olives was almost undetectable. Black olives had higher ethylene production rate than green olives and this rate significantly increased with storage temperature. Addition of propylene had only minor effect on ethylene production of black olives. No climacteric respiratory rise or autocatalytic ethylene production was observed in green and black olives.

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N. Nikoloudakis, G. Banilas, F. Gazis, P. Hatzopoulos and J. Metzidakis

Random amplified polymorphic DNA (RAPD) markers were used to study the genetic diversity and to discriminate among 33 Greek olive (Olea europaea L.) cultivars. Three feral forms from Crete and five foreign cultivars recently introduced into Greece were also included. Nineteen primers were selected which produced 64 reproducible polymorphic bands in the 41 olive genotypes studied, with an average of 3.4 informative markers per primer. The RAPD markers resulted in 135 distinct electrophoretic patterns, with an average of 7.1 patterns per primer. Based on either unique or combined patterns, all genotypes could be identified. Genetic similarities between genotypes were estimated using the Dice similarity index and these indicated that a high degree of diversity exists within the Greek olive germplasm. Using the unweighted pair-group method (UPGMA) most cultivars were clustered into two main groups according to their fruit size or commercial use (table or olive oil). However, poor correlation was detected between clustering of cultivars and their principal area of cultivation. RAPD marker data were subjected to nonmetric multidimentional scaling (NMDS) which produced results similar to those of the UPGMA analysis. The results presented here contribute to a comprehensive understanding of cultivated Greek olive germplasm and provide information that could be important for cultural purposes and breeding programs.

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Genet Teshome Mekuria, Margaret Sedgley, Graham Collins and Shimon Lavee

A sequence-tagged site (STS) was developed to identify a genetic marker linked to resistance to olive leaf spot caused by the pathogen, Spilocea oleaginea (Cast) (syn. Cycloconium oleaginum Cast.). The STS was based on a randomly amplified polymorphic DNA (RAPD) marker of about 780 base pairs (bp) linked to olive leaf spot resistance. Several primer pairs were developed to flank the sequence, and one pair produced the expected polymorphism between resistant and susceptible individuals tested, and was used as an STS marker. This primer pair was tested against parents and 34 individuals from a population segregating for resistance to olive leaf spot, and 12 commercial olive (Olea europaea L.) cultivars showing various levels of resistance to the disease. The STS marker was present in 71.4% of the parents and progeny that were designated as resistant, and was absent in 87% of the parents and progeny showing susceptibility. These primers were also able to distinguish cultivars such as `Koroneiki' and `Leccino', that are reported to show resistance to olive leaf spot, from `Barouni' and `Mission', that are reported to be susceptible. This is the first report of a STS marker for olive, and its use will assist greatly in screening olive progeny for resistance to leaf spot in breeding programs.

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Jenny R. Guerin, Susan M. Sweeney, Graham G. Collins and Margaret Sedgley

The National Olive Variety Assessment (NOVA) collection, established at the Roseworthy Campus of the University of Adelaide, contains six replicate trees of 100 olive (Olea europaea L.) accessions grown in the same environment. The DNA fingerprints of these accessions were compared, using randomly amplified polymorphic DNA (RAPD), to those of a number of cultivars obtained from international collections. A total of 86 uniquely named accessions in the NOVA collection resulted in 58 different genotypes. Different names were synonyms for the same genotype, and homonyms were also found where accessions with the same name had different DNA fingerprints. A rapid and efficient protocol was developed to identify unknown olive genotypes using a two-stage process. Data from DNA fingerprints were added to a matrix already containing binary data from recognized standard cultivars. The estimated probability of any given RAPD profile randomly occurring at this stage ranged between 6 × 10-4 and 2 × 10-8. In the second stage, the approximate identity of the unknown genotype, revealed by the resulting dendrogram, was confirmed by comparing it with appropriate standards under identical conditions of DNA amplification and band separation. The data collected in this report form the basis of a genetic database that can be used for the identification of olive samples.

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A. Belaj, I. Trujillo, R. de la Rosa, L. Rallo and M.J. Giménez

Random amplified polymorphic DNA (RAPD) analysis was performed on the main Mediterranean cultivars of olive (Olea europaea L.) from the Germplasm Bank of the Centro de Investigación y Formación Agraria “Alameda del Obispo” in Cordoba, Spain. One hundred and ninety reproducible amplification fragments were identified using 46 random primers followed by agarose gel electrophoresis. Some 63.2% of the amplification products were polymorphic, with an average of 2.6 RAPD markers obtained for each primer. The combination of polymorphic markers resulted in 244 banding patterns. The high degree of polymorphism detected made identification of all the cultivars (51) possible by combining the RAPD banding patterns of just only four primers: OPA-01, OPK-08, OPX-01, and OPX-03. Cultivar-specific RAPD markers and banding patterns were also found. A dendrogram based on unweighted pair-group method cluster analysis was constructed using a similarity matrix derived from the RAPD amplification products generated by the 46 primers. Three major groups of cultivars could be distinguished by RAPD analysis: 1) cultivars from east and northeast Spain, 2) Turkish, Syrian, and Tunisian cultivars, and 3) the majority of common olive cultivars in Spain. The dendrogram thus showed a good correlation between the banding patterns of olive cultivars and their geographic origin. A higher level of polymorphism was observed when polyacrylamide gel electrophoresis was used to separate the amplification products. Thus, adequate use of RAPD technology offers a valuable tool to distinguish between olive cultivars.

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Slavko Perica, Nacer Bellaloui, Carl Greve, Hening Hu and Patrick H. Brown

Boron (B) remobilization, mannitol and glucose concentrations, and the effect of B application on changes in soluble carbohydrates were investigated in various organs of bearing `Manzanillo' olive trees (Olea europaea L. `Manzanillo'). Following foliar 10B application to leaves of various ages, there was significant 10B export out of the treated leaves, and significant 10B enrichment in nontreated adjacent organs, including inflorescences and fruit. Results demonstrated that B can be remobilized from leaves of various ages, and that foliar-applied B is phloem mobile in olive. Soluble carbohydrate analysis determined that mannitol and glucose are the predominant sugars in all organs analyzed and that the mannitol concentration in the leaves is adequate to account for all B transport. This is consistent with observations in other species, where the presence of mannitol is known to facilitate phloem B transport through formation of a mannitol-B complex. Previous reports have indicated that B application can alter carbohydrate metabolism. In the present study, foliar B application significantly suppressed glucose concentration in the leaf petioles of all ages and increased mannitol in petioles of the current-year-developed leaves.

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Genet Teshome Mekuria, Graham Collins, Margaret Sedgley and Shimon Lavee

Olive leaf spot is a disease of olive (Olea europaea L.) caused by the fungal pathogen, Spilocea oleaginea Cast. Progeny derived from crosses among susceptible, resistant, and semiresistant parental lines were assessed in the field for 8 years and classified as either resistant or susceptible. DNA from some of the progeny of this segregating population was used to identify molecular markers linked to olive leaf spot disease using the randomly amplified polymorphic DNA (RAPD) technique and bulked segregant analysis (BSA). Two DNA bulks were constructed, each containing 13 progeny showing either resistance or susceptibility for the disease, and screened for polymorphisms using 100 primers. One primer produced two polymorphic bands, one of ≈700 base pairs (bp) from the susceptible bulk and the other of ≈780 bp from the resistant bulk. The 780 bp marker appeared in 70.6% of the segregating progeny and 100% of parents showing resistance to leaf spot disease, while the 700 bp marker appeared in 47.1% of the segregating progeny and 100% of the parents showing susceptibility. These markers can be used as screening tools in olive improvement programs.

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Claudio Cantini, Riccardo Gucci and Balilla Sillari

An alternative method to managing olive (Olea europaea L.) orchards for oil production is described. Using the coppiced system, the orchard is divided into 10 plots and all trees in one plot are coppiced year 1, all trees in a second plot are coppiced year 2, etc. In this way, the olive orchard consists of 10 different-aged plots after 10 years. Then a new cycle is started by coppicing trees in plot 1 in year 11, those in plot 2 in year 12, and so on. Since hardly any pruning is done after coppicing, the main advantages of this innovative management method are to reduce labor costs and the need for skillful labor, without negative effects on fruit yield, oil yield, or alternate bearing. Pesticide application, weed control, and fertilization were performed according to standard commercial practice. As a result, this system is more convenient than other training systems used for olive trees, it is suitable for renewing old trees, and can be adopted under many cultural conditions. The coppiced management system is compatible with soils of low fertility and is sustainable for long-term olive oil production.

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Kiyoshi Banno, George C. Martin and Robert M. Carlson

Citrate-phosphate buffer induced olive (Olea europaea L.) leaf abscission at pH 3, 5, or 7. Of several sources of P-induced leaf abscission, NaH2PO4 was the most effective. Sensitivity to P was shown by leaf abscission, leaf desiccation, or both among 32 species representing 22 genera. Applied P accumulates in petioles, but its effect on abscission does not seem to depend on ethylene production. The low P content in the ethephon molecule may have an additive effect in ethephon-induced abscission. Stem-fed NaH2PO4 or ethephon resulted in total leaf and fruit abscission. Of the chemicals applied as a foliar treatment, only NaH2PO4 caused fruit abscission with minimal leaf loss. Adding Al2O3 to adsorb P in treatment solutions delayed the abscission effect of ethephon and NaH2PO4. Adding glycerol to NaH2PO4 increased fruit abscission from 50% to ≈80% and leaf abscission from ≈9% to 18%. The presumed effect of glycerol is from slowing the drying rate and thereby increasing P penetration into the fruit abscission zone. The pedicel-fruit cavity is a collection basin for spray accumulation that is not present in the leaf petiole attachment to the stem. This morphological difference probably leads to greater absorption of abscission-inducing materials by fruit. Chemical name used: (2-chloroethyl)phosphonic acid (ethephon).