Istrska Belica Research Articles

Changes in the anthocyanin pathway related to phenolic compounds and gene expression in skin and pulp of cv. 'Istrska belica' (Olea europaea L.) during ripening

The purpose of research was to study in detail the dynamics of the anthocyanin pathway during the ripening of olives, comprising the relative gene expression of nine enzymes and the contents of twelve phenolic compounds. The analyses were conducted on cv. 'Istrska belica' at seven maturity stages, separately in the pulp and the skin. Most phenolic compounds showed a higher content in the skin than in the pulp. Results showed that the accumulation of dihidroquercetin and dihydromyricetin started at the latest maturity stages. The most abundant phenolics evaluated in the current study present in both tissues were cyanidin-3-O-rutinoside and delphinidin-3-O-glucoside, both presented at all maturity stages, even when colour was not yet visible in the skin or pulp. Gene expression of enzymes revealed tissue-specific regulation during ripening. Genes expressions for phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavonoid 3-hydroxylase and flavonoid 3′-hydroxylase showed higher levels in the skin than in the pulp, and an upregulation during ripening in both tissues. Anthocyanidin synthase was the only gene with the highest expression at the beginning of ripening, with extreme decrease between second and third maturity stage, which suggests that the enzyme is mainly synthesized at the beginning of ripening and that enzyme activation starts at latest maturity stages. Our research contributes to a better understanding of the dynamics of phenolic accumulation and the relative gene expression of enzymes involved in the anthocyanin pathway in reveals tissue-specific changes during olive fruit ripening. The previous results are also supported by physical changes, which are reflected in a statistical increase in fruit weight, a decrease in fruit firmness and also by changes in appearance observed during ripening. Understanding the accumulation of anthocyanins could, through further study, help to improve the quality of the fruit and therefore the quality of olive products.

Phytochemical screening of the olive variety ‘Istrska Belica’ infected by fungus Venturia oleaginea

Phytochemical screening of the olive variety ‘Istrska Belica’ infected by fungus Venturia oleaginea

An Insight into an Olive Scab on the "Istrska Belica" Variety: Host-Pathogen Interactions and Phyllosphere Mycobiome.

The olive tree is one of the most important agricultural plants, affected by several pests and diseases that cause a severe decline in health status leading to crop losses. Olive leaf spot disease caused by the fungus Venturia oleaginea can result in complete tree defoliation and consequently lower yield. The aim of the study was to obtain new knowledge related to plant-pathogen interaction, reveal mechanisms of plant defense against the pathogen, and characterize fungal phyllosphere communities on infected and symptomless leaves that could contribute to the development of new plant breeding strategies and identification of novel biocontrol agents. The highly susceptible olive variety "Istrska Belica"' was selected for a detailed evaluation. Microscopy analyses led to the observation of raphides in the mesophyll and parenchyma cells of infected leaves and gave new insight into the complex V. oleaginea pathogenesis. Culturable and total phyllosphere mycobiota, obtained via metabarcoding approach, highlighted Didymella, Aureobasidium, Cladosporium, and Alternaria species as overlapping between infected and symptomless leaves. Only Venturia and Erythrobasidium in infected and Cladosporium in symptomless samples with higher abundance showed statistically significant differences. Based on the ecological role of identified taxa, it can be suggested that Cladosporium species might have potential antagonistic effects on V. oleaginea.

Effect of Brown Marmorated Stink Bug (Halyomorpha halys Stål.) Infestation on the Phenolic Response and Quality of Olive Fruits (Olea europaea L.)

Olives ripen in the late autumn and represent a good source of nutrients that Halyomorpha halys uses to prepare for diapause. This is the first study to investigate the impact of H. halys infestation on the phenolic response and olive fruit quality in the pierced tissue of damaged fruits and in the non-pierced part of damaged fruits of ‘Istrska belica’ and ‘Pendolino’ cultivars. Both total phenolic content and antioxidant capacity contents significantly increased in the infested fruits of the cultivar ‘Istrska belica’. Total phenolic content in the pierced tissue of damaged fruits increased by 10.7%, while the content of AC in the non-pierced tissue of damaged fruits increased by 7.11% and in the pierced tissue of damaged fruits by 6.1% compared to control. A total of 44 individual phenolic compounds were identified, 21 of them increased in at least one cultivar after infestation. Huge increases in phenolic content were observed in both cultivars, particularly for flavones, secoiridoids, anthocyanins, and flavonols in the pierced tissue of damaged fruits. The most responsive individual phenolic compound in both cultivars was oleuropein. Its content in the pierced tissue of damaged fruits increased by 44.7% in the cultivar ‘Pendolino’ and for 82.6% in the cultivar ‘Istrska belica’.

Developing an Olive Biorefinery in Slovenia: Analysis of Phenolic Compounds Found in Olive Mill Pomace and Wastewater.

The valorization of olive pomace through the extraction of phenolic compounds at an industrial scale is influenced by several factors that can have a significant impact on the feasibility of this approach. These include the types and levels of phenolic compounds that are present, the impact that seasonal variation and cultivar type have on the phenolic compound content in both olive pomace and mill effluents and the technological approach used to process the olive crop. Chemical analysis of phenolic compounds was performed using an HPLC-diode-array detector (DAD)-qTOF system, resulting in the identification of 45 compounds in olive mill wastewater and pomace, where secoiridoids comprised 50–60% of the total phenolic content. This study examined three different factors that could impact the phenolic compound content of these processing streams, including cultivar types typically grown on local farms in Slovenia, the type of downstream processing used and seasonality effects. Olive crop varieties sourced from local farms showed high variability, and the highest phenolic content was associated with the local variety “Istrska Belica”. During processing, the phenolic content was on average approximately 50% higher during two-phase decanting compared to three-phase decanting and the type of compound present significantly different. An investigation into the seasonal effects revealed that the phenolic content was 20% higher during the 2019 growing season compared to 2018. A larger sample size over additional growing seasons is required to fully understand the annual variation in phenolic compound content. The methods and results used in this study provide a basis for further analysis of phenolic compounds present in the European Union’s olive crop processing residues and will inform techno-economic modelling for the development of olive biorefineries in Slovenia.

Influence of irrigation treatments on the yield and quality of ‘Istrska belica’ olive oil

In recent years, Slovenian olive growers and producers have faced problems in achieving constant yields and quality of olive oil due to extreme weather conditions, especially because of the more frequent occurrence of drought. A technically accomplished pilot automatic irrigation system for olive trees was established. In 2015, the impact of different irrigation treatments (0, 15, 33, 40 and 100% ETc (crop evapotranspiration)) on olive cultivar 'Istrska belica' were studied to determine the minimum amount of added water that has a positive effect on the yield and quality of the extra virgin olive oil produced. The study results showed that the quantity of applied water equal to 100% ETc had a significant effect on yield of olive fruits. The average fruit mass of the trees grown under full irrigation (100% ETc) was significantly higher than that of those given rain-fed and deficit irrigation treatments (15, 33 and 40% ETc), with no significant differences found between these treatments. The fatty acid contents showed the highest amount of oleic acid (75.61%) at 100% ETc and the lowest amount (74.69%) at 15% ETc, while the highest amount of linoleic acid (6.52%) was determined at 15% ETc and the lowest (5.98%) at 100% ETc. Total biophenol content and biophenol composition was determined. The 40% ETc irrigation system produced the highest amounts of total biophenols, total oleuropein and total ligstroside derivatives (1076, 562 and 388 mg kg-1, respectively), while the lowest results (883, 477 and 299 mg kg-1, respectively) were determined in the control system (0% ETc). The organoleptic assessment of the olive samples showed that the positive sensory attributes of fruity, bitter and pungent were more intensive at 33% ETc and were graded highest. Olive oil from non-irrigated trees (0% ETc) received the lowest sensory score.

Effects of extreme drought on the vegetative and productive behavior of olive ‘Istrska belica’

Effects of extreme drought on the vegetative and productive behavior of olive ‘Istrska belica’

Intra-varietal variability and genetic relationships among the homonymic East Adriatic olive ( Olea europaea L.) varieties

Abstract The intensive exchange and diffusion of plant material over the whole Mediterranean Basin including the East Adriatic region, has contributed to the confusion in denomination of olive varieties, and formation of a large number of synonyms and homonyms. In the present study, we report on clonal diversity and genetic relationships among a total of 190 olive trees sampled along the East Adriatic coast in Slovenia (SVN), Croatia (HRV) and Montenegro (MNE). Samples belonged to the six East Adriatic olive varieties with homonymic denomination: ‘Istrska belica’ (SVN), ‘Bjelica’ (HRV), ‘Žutica’ (MNE), ‘Crnica’ (SVN), ‘Crnica’ (HRV), and ‘Crnica’ (MNE). Results showed polymorphism in all twelve microsatellite markers (DCA3, DCA9, DCA11, DCA14, DCA16, DCA18, EMO03, EMO90, GAPU101, GAPU103A, GAPU71B and UDO99-019), revealing a total of 83 alleles. A total of 36 different genotypes in the panel of 190 samples were found. Genetic profiling of six homonymic East Adriatic olive varieties revealed four different varieties: 1) ‘Istrska belica’ (SVN), 2) ‘Bjelica’ (HRV)/‘Žutica’ (MNE), 3) ‘Crnica’ (HRV)/‘Crnica’ (MNE) and 4) ‘Crnica’ (SVN). Detailed clonal and genetic relationship analyses demonstrated genetic uniformity of ‘Istrska belica’ (SVN), negligible low diversity of ‘Crnica’ (SVN), and moderate diversity of ‘Crnica’ (HRV)/‘Crnica’ (MNE). Polyclonal property was assigned to only ‘Bjelica’ (HRV)/‘Žutica’ (MNE) variety. High genetic similarity between ‘Bjelica’ (HRV)/‘Žutica’ (MNE), and ‘Crnica’ (HRV)/‘Crnica’ (MNE), with different distribution of clones within them, reflects the past selection and propagation processes in Croatia and Montenegro. For the first time, international synonymy of ‘Žutica’ (MNE) and ‘Bjelica’ (HRV), and homonymy of ‘Istrska belica’ (SVN) and ‘Bjelica’ (HRV)/‘Žutica’ (MNE), as well as ‘Crnica’ (SVN) and ‘Crnica’ (MNE, HRV) were reliably confirmed with DNA markers.

Variations of phenolic compounds and sensory properties of virgin olive oils from the variety 'Istrska belica'

The olive variety 'Istrska belica' is well known for its numerous positive properties, such as resistance to low temperature and high oil content. The aim was to determine the variations in the levels of phenolic compounds and sensory properties during storage of 'Istrska belica' virgin olive oil. The profile of the phenolic compounds and sensory properties of 'Istrska belica' olive oil were further compared with those for other varieties, including 'Leccino' and 'Maurino'. The content of phenolic compounds of the olive oils decreased after 1 year and 2 years of storage. After 2 years of storage, the levels of oleuropein and the ligstroside derivates significantly decreased, while the end-stage compounds tyrosol and hydroxytyrosol increased. These data show that after 1 year of storage, the 'Istrska belica' olive oil preserves similar intensities for bitterness and pungency, and similar oleuropein and ligstroside derivates levels. In contrast to the other oils analysed, the intensities of bitterness and pungency of 'Istrska belica' olive oil decreased greatly only after 2 years of storage. Moreover, the phenolic compounds content, and oleuropein and ligstroside derivates levels, and the intensities of bitterness and pungency were the highest in fresh 'Istrska belica' olive oil, compared to the other olive oils analysed. Overall, 'Istrska belica' olive oil has important advantages over olive oil from other varieties that are grown in the Istria region.

Occurrence of endophytic fungi causing recalcitrance of olive cultivar ‘Istrska belica’ during shoot culture establishment

The primary aim of this study was to establish a micropropagation procedure for the Slovenian frost-tolerant olive cultivar ?Istrska belica?. Establishing an in vitro culture was very difficult due to constant contaminations, tissue browning and stunted shoot growth. A sterile shoot culture was finally achieved by washing with running tap water, immersing in a mixture of ascorbic and citric acid and sterilizing with 70% ethanol and dichloroisocyanuric acid. Shoot growth was optimal on DKW medium supplemented with 4 mg/L of 2iP. Even in optimized conditions, sporadic fungal outbursts occurred. Fungi were isolated and their taxonomic origin was determined by morphological observation and molecular identification. Based on BLAST queries in the NCBI database, five genera of fungi were identified: Cladosporium, Chaetomium, Preussia, Biscogniauxia and Sistotrema, the last three genera being isolated from olives for the first time. A detailed literature search was performed to provide data on previous reports of these genera in relation to their putative endophytic presence and their possible pathogenic status. This is the first study reporting the presence of endophytic fungi in olive tissue culture. The information provided in this work can be very useful for the optimization of micropropagation protocols of recalcitrant olive cultivars and can potentially improve field performance of nursery plants.

Migration of nutrients from soil to plant in olive orchards

Olive growing is gaining much interest in Slovenia. In thgis work we report the results of the 5 year experiments, on 9 locations, with the cultivar “istrska belica”. The results of soil and plant analyzes of the olive orchard on carstic soil indicated some unexpected facts about macro-nutrient mobility: although the phosphorus in soil is very low, its values in plants are almost optimal. A similar pattern also occurs with magnesium. On the other hand, we would expect low foliar potassium values, as reported in literature for rainfed orchards on calcareous soils, yet they are not. Also calcium levels are not as expected for orchards on carstic soil (higher values), and thus indicate calcium deficiency. It would definitely be interesting to study it in more details. We found that the results show a negative correlation between phosphorus and potassium plant mobility and soil organic matter.

GENOMIC AND EST MICROSATELLITE LOCI DEVELOPMENT AND USE IN OLIVE: MOLECULAR TOOLS FOR GENETIC MAPPING AND ASSOCIATION STUDIES

Identification of QTLs (quantitative trait loci) related to agronomic traits based on segregating population and/or on a collection of unrelated genotypes needs the use of molecular markers. Here, we aimed to develop and characterize genomic and expressed sequence tag (EST)-derived microsatellite markers in olive ( Olea europaea L.) and assess their Mendelian inheritance on the pseudo-F 1 population derived from the cross 'Oliviere' * 'Arbequina'. Based on an enriched genomic DNA library constructed on the French cultivar 'Oliviere', we designed 211 primer pairs among the total 584 identified SSR loci. Following 454 sequencing of cDNA fragments obtained on the Slovenian cultivar 'Istrska belica', we designed 113 primer pairs among 976 identified as EST-SSR loci. We screened these SSR markers for their informativeness for the saturation of the previously published linkage map based on the same cross, designed for bearing regularity study (in Montpellier, France). A set of 59 genomic and 40 expressed SSR loci were selected to genotype the 147 offspring. Twenty four (25.53%) SSRs show a Mendelian distortion segregation at P<0.05. From genotyped SSRs, 85 (90%), 64 (68%) and 94 (100%) SSR loci are now available for completing respectively 'Oliviere' and 'Arbequina', and the integrated maps published earlier.

Paternity analysis of the olive variety "Istrska belica" and identification of pollen donors by microsatellite markers.

The leading olive variety in Slovenia is “Istrska belica” (Olea europaea L.), which currently represents 70% of all olive trees in productive orchards. Paternity analysis based on microsatellite markers was used for genotyping and identification of the potential pollen donors of “Istrska belica” and for assessing the proportion of self-fertilization in monovarietal olive orchards in the Slovene Istria. Seven microsatellite loci were used for genotyping thirty-one olive embryos from “Istrska belica” trees and for all potential pollen donor varieties, which are grown in the region and could participate as pollinators. Genotyping results and allele identification were performed using the FaMoz software. The most probable pollen donor was assigned to 39% of all analyzed embryos. Among all analyzed embryos no single case of self-fertilization was confirmed. According to the present results, the variety “Istrska belica” was in all cases fertilized by foreign pollen. The results will contribute to defining the new guidelines for farmers regarding the proper management and growing practice in monovarietal olive groves.

Assessment of Slovenian table olives produced by traditional technology

Table olives are prepared from the sound fruits of varieties of the cultivated olive tree ( Olea europaea L.) that are chosen for their production of olives whose volume, shape, flesh-to-stone ratio, fine flesh, taste, firmness and ease of detachment from the stone make them particularly suitable for processing. The world production in the crop year 2006/2007 is estimated at 1.8 million tons of table olives and in the last fifteen years it has constantly increased. Most of the Slovenian Istria table olive production is based on a modification of the Spanish style. Results of qualitative and quantitative sensory assessments of two olive cultivars, ‘Storta’ and ‘Istrska belica’, are presented. Sensory characteristics were determined after four and six months of fermentation. Texture differences between the two production systems were revealed. Significant decrease in hardness was determined after six months of fermentation with initial alkaline treatment. The effect of cultivar type was also evident. After ...

Chemical changes in extra virgin olive oils from Slovenian Istra after thermal treatment

Changes in the physico-chemical parameters of extra virgin olive oils after heating for 142h at 100°C with an air flow 10L/h were investigated. The experimental study was carried out on the two predominant olive cultivars in Slovenian Istra – cv. Istrska belica and cv. Leccino. The data obtained showed that oils from Istrska belica were more stable than those from Leccino. Peroxide values and spectrophotometric data showed higher amounts of oxidation products in oils from Leccino than in those from Istrska belica. After thermal treatment fatty acid composition was changed more in Leccino oils; particularly the amounts of polyunsaturated fatty acids dropped significantly, while α-tocopherol was completely depleted in all samples. The content of total biophenols decreased from 598mg/kg to 241mg/kg in Istrska belica oils and from 391mg/kg to 176mg/kg in Leccino oils. HPLC data showed that transformation of secoiridoid biophenols to the simple biophenols, tyrosol and hydroxytyrosol took place.

Assessment of genetic variability of olive varieties by microsatellite and AFLP markers

Fourteen developed microsatellite markers were characterized for their use in genotyping and diversity studies of olive varieties. After optimisation of microsatellite assay and allele sizing, ninety-six alleles were found in nineteen varieties, with an average of 6.8 alleles per locus. The characteristics of the microsatellite markers were used to identify markers that can be reliably applied for variety genotyping. Such features were the generation of complex banding patterns supported by underlying allele sequences, `short allele dominance', an unstable repeat structure and a low number of alleles. AFLP analysis was performed on the same set of olive varieties using eight primer pair combinations. The genetic relationships among nineteen olive varieties were compared on the basis of microsatellite and AFLP polymorphisms. Genetic distances between all pairwise combinations of the varieties were calculated using Jaccard's coefficient of similarity and dendrograms were constructed by the UPGMA method. The results of clustering analysis with both molecular systems showed the common genetic background of Tuscan varieties, and genetic divergence within Slovene olive germplasm. Slovenian varieties ‘Buga’, ‘Storta’ and ‘Samo’ might represent regionally selected olives, while ‘Zelenjak’ and ‘Crnica’ are probably derived from the Central Italian region. The predominant local ‘Istrska belica’ was introduced to Slovenia independently from the other regional varieties and showed the lowest genetic similarity with the other regional varieties.