Georgios C. Koubouris

Microsatellite high-resolution melting (SSR-HRM) analysis for genotyping and molecular characterization of an Olea europaea germplasm collection

Olea europaea L. has been cultivated in the Mediterranean region for thousands of years and is of major economic importance. The origin of olive cultivars remains as complex to trace as their identification. Thus, their molecular characterization and discrimination will enable olive germplasm management. In addition, it would be a useful tool for authentication of olive products. High-resolution melting (HRM) analysis, coupled with five microsatellite markers, was integrated to facilitate molecular identification and characterization of main O. europaea cultivars collected from the National Olive Tree Germplasm Collection established in Chania, Greece. The five microsatellite loci used were highly informative and generated a unique melting curve profile for each of the 47 cultivars and for each microsatellite tested. In particular, three microsatellite markers (DCA03, DCA09 and DCA17), which generated 29 HRM profiles, were sufficient to genotype all the olive cultivars studied, highlighting their potential use for cultivar identification. Furthermore, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method well suited for molecular characterization of olive cultivars.

Ultraviolet-B radiation or heat cause changes in photosynthesis, antioxidant enzyme activities and pollen performance in olive tree

The present study attempts to determine how some physiological and reproductive functions of olive tree (Olea europaea L., cv. Koroneiki) respond to enhanced UV-B radiation or heat. Enhanced UV-B radiation was applied to (1) three-year-old potted plants in an open nursery (corresponded to ca. 16% ozone depletion), and (2) in vitro cultured pollen samples (220 μmol m−2 s−1, PAR = 400−700 nm + UV-B at 7.5, 15.0, or 22.5 kJ m−2 d−1). Potted olive plants were also subjected to high temperature (38 ± 4°C) for 28 h to mimic heat levels regularly measured in olive growing areas. A significant effect of UV-B on photosynthetic rate was observed. However, enhanced UV-B radiation did affect neither chlorophyll nor carotenoid content, supporting previous reports on hardiness of the photosynthetic apparatus in olive. Increased superoxide dismutase activity was observed in UV-B-treated olive plants (+ 225%), whereas no effect was found in the plants under heat stress. Neither UV-B and nor heat did affect H2O2 accumulation in the plant tissues. However, the same treatments resulted in enhanced lipid peroxidation (+ 18% for UV-B and + 15% for heat), which is likely linked to other reactive oxygen species. The increased guaiacol peroxidase activity observed in both treatments (+ 32% for UV-B and + 49% for heat) is related to the defense against oxidative membrane damage. The observed reduction in pollen germination (20–39%) and tube length (11–44%) could have serious implications on olive yields, especially for low fruit-setting cultivars or in years and environments with additional unfavorable conditions. UV-B and heat effects described here support the hypothesis that plant response to a given stressor is affected by the overall context and that a holistic approach is necessary to determine plant strategies for climate change adaptation.

Influence of cross-pollination on the development of parthenocarpic olive (Olea Europaea) fruits (shotberries)

The impact of three different pollination treatments (self-, cross-, free-) on the degree of shotberry (seedless fruit) formation of the olive tree cultivars (cvs) Koroneiki, Kalamata, Mastoidis and Amigdalolia was studied for three consecutive years. Controlled crosses were made for the cross-pollination treatments, while for the free pollination treatment flowers were allowed to receive pollen from more than 40 cultivars present in the field. Significant differences were recorded between treatments, cultivars and years. The lowest degree of shotberry formation was observed in free-pollinated trees while the highest was in self-pollinated trees of all cultivars. Low air temperature incidents during the flowering period increased shotberry formation. Cultivars Koroneiki and Mastoidis were reciprocally the most effective pollinator varieties in reducing shotberries. Cultivar Koroneiki could be considered as the most suitable pollinator to reduce shotberries in Kalamata. When cv. Amigdalolia was cross-pollinated by cvs Koroneiki and Mastoidis the degree of shotberry formation was lower when compared to cross-pollination by cv. Kalamata.

Interaction between Bactrocera oleae (Diptera: Tephritidae) infestation and fruit mineral element content in Olea europaea (Lamiales: Oleaceae) cultivars of global interest

Olive fruit fly Bactrocera oleae (Gmelin) (Diptera: Tephritidae) infestation levels were quantified in rainfed adult olive (Olea europaea L.) (Lamiales: Oleaceae) trees of seven cultivars originating from Spain, Italy, Greece, Portugal and France to determine their relationships with fruit length, width, weight, oil and mineral element content and to investigate the effects of infestation on fruit properties. Fruit from the cultivars Koroneiki, Mastoidis, Picholine, Manzanilla, Arbequina, Branquita and Leccino was collected in November 2013 in Greece. Marked genotypic variation was observed for both total and alive fruit infestation with Manzanilla being the most susceptible and Arbequina the most resistant among the cultivars studied. Marked differences were recorded in the fruit mineral element content between the cultivars studied. B. oleae infestation was positively correlated with the fruit length, width, fresh weight, and K and Fe content. Also, B. oleae infestation caused significant changes in the P, K, Fe and Mg concentration in fruits, while an overall decreasing trend was observed for N. B. oleae infestation caused no significant changes in the fruit oil content of the studied cultivars. Results presented in this study supplemented with targeted integrated research on breeding resistant genotypes and developing improved pest control tools could contribute to important savings of resources as well as improvement of yields and food quality and safety.

A model based on S-allele dominance relationships to explain pseudo self-fertility of varieties in the olive tree

Self-fertility is largely decreased and even prevented by various mechanisms because, broadly, it causes inbreeding depression, although some species have retained self-reproduction regimes. Species of plants that display the self-incompatible sporophytic type of self-incompatibility may rarely self-pollinate. It is only possible in the absence of foreign compatible pollen. In the olive tree with a sporophytic mechanism, we will show that three co-dominant S-alleles R1, R3 and R5 do not lead to the same level of self-fertility. All varieties that carry R1 are less self-fertile than those that carry R5, whatever the other S-alleles, while those carrying R3 are intermediate. S-allele pair-wise combinations that differ by two or three levels of dominance, and not the other combinations allow self-fertility, and moreover each S-allele R1, R3 and R5 decreases, maintains and enhances the self-fertility rate, respectively.

Effects of Carbon Inputs on Chemical and Microbial Properties of Soil in Irrigated and Rainfed Olive Groves

Abstract Olive tree pruning residue and olive waste represent a great amount of organic materials that are produced during a short period. The application of organic materials to land is a common practice in sustainable agriculture in recent years. However, its implementation in olive groves under different irrigation regimes has not been systematically tested. The aim of this work was to study the effect of alternative carbon input techniques (e.g., wood shredded, pruning residues, returning of olive mill wastes the field with compost) on soil chemicals (e.g., pH, EC, total organic carbon, total nitrogen, inorganic nitrogen, humic and fulvic acids, available P, and exchangeable K), and microbial properties (e.g., soil basal microbial respiration and microbial biomass carbon) in relation to irrigation conditions (e.g., irrigated and rainfed olive orchards). The results showed that changes in soil quality in olive orchards due to carbon inputs depend on irrigation conditions. Soil carbon content remarkably reduced by the addition of organic material in irrigated plots compared to control whereas they were substantially increased in rainfed plots. Microbial parameters appeared to be reliable indicators of changes in soil management over the short period of this study. Nutrient concentrations and microbial properties of soil show a decreasing trend with increasing distance from tree trunk regardless the irrigation conditions and carbon input practices. This fact indicates the influence of a tree canopy area and root density on the soil surface properties. Soil depth significantly influenced soil attributes; major decreases were recorded for SOC, inorganic nitrogen, and microbial properties, indicating the high potential of surface soil in olive groves to sequester carbon. Conversely, the addition of organic matter and irrigation conditions did not contribute to subsoil C content. Proper management of alternative carbon inputs in soil can positively affect soil productivity in Mediterranean olive groves.

Reply to Saumitou-Laprade et al. (2017) “Controlling for genetic identity of varieties, pollen contamination and stigma receptivity is essential to characterize the self-incompatibility system of Olea europaea L.”. Eva:https://doi.org/10.1111/eva.12498

This study was carried out to examine the validity of previous studies on the intercompatibility of olive and to compare the approach and techniques used for proposing the diallelic self‐incompatibility system and the sporophytic self‐incompatibility system. Analysis of the literature indicates that the mating system of the olive tree is a controversial issue and requires further studies to clearly and fully comprehend it. All possible approaches should be used to maximize reliability of the final conclusions on the olive mating system.

Characterization of Fungi Associated With Wood Decay of Tree Species and Grapevine in Greece

A two-year survey was conducted to identify fungi associated with wood decay in a range of tree species and grapevine. Fifty-eight fungal strains isolated from plants of 18 species showing typical wood decay symptoms were characterized by morphological, physiological, and molecular analyses. By 5.8S rRNA gene-ITS sequencing analysis, these isolates were classified into 25 distinct operational taxonomic units, including important phytopathogenic species of the phyla Pezizomycotina and Agaricomycotina, such as Fomitiporia, Inonotus, Phellinus, Inocutis, Fuscoporia, Trametes, Fusarium, Eutypa, Phaeomoniella, Phaeoacremonium, and Pleurostomophora spp. The white rot basidiomycetes Fomitiporia mediterranea (20 isolates, 34.5%) and Inonotus hispidus (6 isolates, 10.3%) were the most prevalent. Pathogenicity tests revealed for the first time that certain fungal species of the genera Fomitiporia, Inonotus, Phellinus, Pleurostomophora, and Fusarium caused wood infection of various tree species in Greece and worldwide. To the best of our knowledge, this is the first report of F. mediterranea as the causal agent of wood decay in pear, pomegranate, kumquat, and silk tree. This is also the first record of Inonotus hispidus, Phellinus pomaceus, Pleurostomophora richardsiae, and Fusarium solani in apple, almond, avocado, and mulberry tree, respectively, whereas P. richardsiae was associated with wood infection of olive tree for the first time in Greece. Cross pathogenicity tests with F. mediterranea strains originated from grapevine applied on other woody hosts and from olive on grapevine demonstrated partial host specificity of the fungus. The potential of F. mediterranea to transinfect hosts other than those originated, along with the host range extension of the fungus, is discussed.

Evaluation of Greek grapevine cultivars for resistance to Phaeomoniella chlamydospora

In the present study, four Greek (Agiorgitiko, Asyrtiko, Roditis and Xinomavro) and one international (Soultanina) grapevine cultivars (Vitis vinifera L.) were screened for their resistance to Phaeomoniella chlamydospora. Artificial inoculation was carried out by drilling a hole into the trunk and injecting a concentrated conidial suspension into the vessels. Disease reactions were evaluated in an 87-day assessment period, on the basis of external symptoms (disease incidence, disease severity and mortality) and by calculating the relative areas under disease progress curves (relative AUDPC). The extension of vascular browning as well as the isolation ratio along the inoculated vine trunks were also taken into account as additional parameters for evaluating resistance. The results indicated that the resistance of grapevine cultivars to P. chlamydospora varied significantly. ‘Agiorgitiko’ and ‘Soultanina’ were susceptible, whereas ‘Asyrtiko’ and ‘Xinomavro’ were resistant; ‘Roditis’ showed an intermediate level of resistance. Cultivars’ resistance was mostly distinguished in terms of the extension of vascular browning and pathogen isolation ratio. On the contrary, the disease incidence, final disease severity, mortality and relative AUDPC provided less distinctive efficiency in resistance evaluation. The robust methodology presented here could be useful in rapid evaluation experiments for future screening programs to search and recognize natural resistant sources within grapevine genotypes against P. chlamydospora.

FLOWER PERFORMANCE AND FRUIT PERSISTENCE IN ORGANIC 'KALAMATA' OLIVE

Organic olive farming is characterized by products with high biological value and increased market prices but in the same time is doubted for tree nutrition inefficiency and lower yield consistency. For two consecutive years, we studied flower biology in an irrigated ‘Kalamata’ olive orchard. Additionally, fruit persistence, from fruit set to harvest was monitored in monthly intervals. Flowering was realized between mid-April and mid-May with duration affected by air temperature. The average number of flowers/inflorescence was 22 and flower fertility was 78%. 52% of the staminate flowers were located in the basal section of inflorescence, 33% in the middle part and only 15% in the apical fraction. In vitro pollen germination was 58.9% and mean pollen tube length was 196 μm. 64% of the fruit set was abscised between late May and late June while during the next 6 months to December 21% of the initial fruit load was dropped. The average fruit yield was 35.7 kg/tree. Tree productivity of organic olive compared to conventional farms is also discussed.