Olive Tissues Research Articles

Characterization of the Metabolic Profile of Olive Tissues (Roots, Stems and Leaves): Relationship with Cultivars' Resistance/Susceptibility to the Soil Fungus Verticillium dahliae.

Verticillium wilt of olive (VWO) is one of the most widespread and devastating olive diseases in the world. Harnessing host resistance to the causative agent is considered one of the most important measures within an integrated control strategy of the disease. Aiming to understand the mechanisms underlying olive resistance to VWO, the metabolic profiles of olive leaves, stems and roots from 10 different cultivars with varying levels of susceptibility to this disease were investigated by liquid chromatography coupled to mass spectrometry (LC-MS). The distribution of 56 metabolites among the three olive tissues was quantitatively assessed and the possible relationship between the tissues' metabolic profiles and resistance to VWO was evaluated by applying unsupervised and supervised multivariate analysis. Principal component analysis (PCA) was used to explore the data, and separate clustering of highly resistant and extremely susceptible cultivars was observed. Moreover, partial least squares discriminant analysis (PLS-DA) models were built to differentiate samples of highly resistant, intermediate susceptible/resistant, and extremely susceptible cultivars. Root models showed the lowest classification capability, but metabolites from leaf and stem were able to satisfactorily discriminate samples according to the level of susceptibility. Some typical compositional patterns of highly resistant and extremely susceptible cultivars were described, and some potential resistance/susceptibility metabolic markers were pointed out.

Confronting stresses affecting olive cultivation from the holobiont perspective.

The holobiont concept has revolutionized our understanding of plant-associated microbiomes and their significance for the development, fitness, growth and resilience of their host plants. The olive tree holds an iconic status within the Mediterranean Basin. Innovative changes introduced in olive cropping systems, driven by the increasing demand of its derived products, are not only modifying the traditional landscape of this relevant commodity but may also imply that either traditional or emerging stresses can affect it in ways yet to be thoroughly investigated. Incomplete information is currently available about the impact of abiotic and biotic pressures on the olive holobiont, what includes the specific features of its associated microbiome in relation to the host's structural, chemical, genetic and physiological traits. This comprehensive review consolidates the existing knowledge about stress factors affecting olive cultivation and compiles the information available of the microbiota associated with different olive tissues and organs. We aim to offer, based on the existing evidence, an insightful perspective of diverse stressing factors that may disturb the structure, composition and network interactions of the olive-associated microbial communities, underscoring the importance to adopt a more holistic methodology. The identification of knowledge gaps emphasizes the need for multilevel research approaches and to consider the holobiont conceptual framework in future investigations. By doing so, more powerful tools to promote olive's health, productivity and resilience can be envisaged. These tools may assist in the designing of more sustainable agronomic practices and novel breeding strategies to effectively face evolving environmental challenges and the growing demand of high quality food products.

Functional Characterization of Four Olive Squalene Synthases with Respect to the Squalene Content of the Virgin Olive Oil.

The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.

Foliar Application of Potassium Salts to Olive, with Focus on Accompanying Anions.

Potassium (K) is an essential element, which is often supplied to horticultural crops via foliar spraying. Some studies have investigated the effect of different foliar-applied K compounds; however, most studies have focussed on crop quality and yield parameters, or were performed with isolated leaf cuticles. The aim of this study was to evaluate the rates of the foliar ion penetration and leaf surface deposition of 130 mM K sprays of compounds with markedly different point of deliquescence (POD) and efflorescence (POE) values, the rates having been previously estimated in climate chamber trials. Shoots of field-grown, commercial olive trees were sprayed with K-nitrate (KNO3), K-sulphate (K2SO4), K-chloride (KCl), K-phosphate (K3PO4), K-carbonate (K2CO3) and K-bicarbonate (KHCO3), and leaf samples were collected after 3 and 24 h. Cation and anion concentrations were determined in the leaf tissues, and in a preliminary leaf water wash for estimating surface-deposited ion concentrations. No significant leaf tissue K increments were recorded between the K sprays. Olive tissue anion concentrations showed different patterns, and a chloride (Cl-) increase was detected 3 h after the foliar KCl supply. On the other hand, the foliar K applications led to leaf nitrate changes regardless of the K source supplied. High amounts of K and accompanying ions were recovered in the washing liquid of the foliar K-supplied leaves. Some foliar K treatments increased the leaf surface concentration of sulphate and chloride, suggesting a potential effect on leaf cell anion extrusion. Hence, despite no evidence of foliar K uptake, an effect of leaf anion concentrations was observed, indicating that foliar nutrient sprays may influence leaf and leaf surface anion balance.

Disclosing the molecular basis of salinity priming in olive trees using proteogenomic model discovery.

Plant responses to salinity are becoming increasingly understood, however, salt priming mechanisms remain unclear, especially in perennial fruit trees. Herein, we showed that low-salt pre-exposure primes olive (Olea europaea) plants against high salinity stress. We then performed a proteogenomic study to characterize priming responses in olive roots and leaves. Integration of transcriptomic and proteomic data along with metabolic data revealed robust salinity changes that exhibit distinct or overlapping patterns in olive tissues, among which we focused on sugar regulation. Using the multi-crossed -omics data set, we showed that major differences between primed and nonprimed tissues are mainly associated with hormone signaling and defense-related interactions. We identified multiple genes and proteins, including known and putative regulators, that reported significant proteomic and transcriptomic changes between primed and nonprimed plants. Evidence also supported the notion that protein post-translational modifications, notably phosphorylations, carbonylations and S-nitrosylations, promote salt priming. The proteome and transcriptome abundance atlas uncovered alterations between mRNA and protein quantities within tissues and salinity conditions. Proteogenomic-driven causal model discovery also unveiled key interaction networks involved in salt priming. Data generated in this study are important resources for understanding salt priming in olive tree and facilitating proteogenomic research in plant physiology.

Cell Localization of DPI-Dependent Production of Superoxide in Reproductive Tissues of the Olive Tree (Olea europaea L.)

Reactive Oxygen Species (ROS) are compounds derived from oxygen with important implications in biological processes in plants, some of them related to reproduction. Among ROS, superoxide is the primary oxidant, since an array of other ROS are eventually derived from this anion. Therefore, analysis of the molecular systems able to generate this molecule and the cellular compartmentalization of these events is of paramount importance. We have used the fluorochrome DCFH2-DA and the chromogenic substrate NBT in association with DPI (a specific inhibitor of Rboh enzymes generating superoxide in plants) in combination with confocal microscopy and stereomicroscopy, respectively to identify cell localization of ROS in general, and superoxide accumulation in olive reproductive tissues. A significant production of both ROS and superoxide has been described, showing a fairly precise spatial and temporal location throughout olive flower development. The reduction of the NBT signal after the addition of DPI suggests that the generation of superoxide is largely due to Rboh or other flavin oxidase activity. At the subcellular level, accumulation of O2●− has been located in the plasma membrane of mature pollen and germinated pollen, as well as in the rough endoplasmic reticulum and in mitochondria.

Phenolic Compounds and Triterpenes in Different Olive Tissues and Olive Oil By-Products, and Cytotoxicity on Human Colorectal Cancer Cells: The Case of Frantoio, Moraiolo and Leccino Cultivars (Olea europaea L.).

Phenolic and triterpenoid compounds of the olive tree are recognized as having a key role in health promotion, thanks to their multiple protective actions in humans. To expand the source of these bioactive compounds, the phenolic and triterpenoid profiles of leaf, branch, destoned fruit, destoned pomace, shell, seed, and extra virgin olive oil from the Frantoio, Leccino, and Moraiolo olive cultivars were simultaneously characterized by HPLC-DAD-MS. Overall, 43 molecules were quantitated and expressed on the obtained dry extracts. Oleuropein was mainly concentrated in branches (82.72 g/kg), fruits (55.79 g/kg), leaves (36.71 g/kg), and shells (1.26 g/kg), verbascoside (4.88 g/kg) in pomace, and nüzhenide 11-methyl oleoside (90.91 g/kg) in seeds. Among triterpenoids, which were absent in shells, the highest amount of oleanolic acid was found in olive leaves (11.88 g/kg). HCT-116 colorectal cells were chosen to assess the cytotoxicity of the dry extract, using the phytocomplex from Frantoio, which was the richest in phenols and triterpenoids. The IC50 was also determined for 13 pure molecules (phenols and terpenoids) detected in the extracts. The greatest inhibition on the cell’s proliferation was induced by the branch dry extract (IC50 88.25 μg/mL) and by ursolic acid (IC50 24 μM). A dose-dependent relationship was observed for the tested extracts.

Distinct Physiological Roles of Three Phospholipid:Diacylglycerol Acyltransferase Genes in Olive Fruit with Respect to Oil Accumulation and the Response to Abiotic Stress.

Three different cDNA sequences, designated OepPDAT1-1, OepPDAT1-2, and OepPDAT2, encoding three phospholipid:diacylglycerol acyltransferases (PDAT) have been isolated from olive (Olea europaea cv. Picual). Sequence analysis showed the distinctive features typical of the PDAT family and together with phylogenetic analysis indicated that they encode PDAT. Gene expression analysis in different olive tissues showed that transcript levels of these three PDAT genes are spatially and temporally regulated and suggested that, in addition to acyl-CoA:diacylglycerol acyltransferase, OePDAT1-1 may contribute to the biosynthesis of triacylglycerols in the seed, whereas OePDAT1-2 could be involved in the triacylglycerols content in the mesocarp and, therefore, in the olive oil. The relative contribution of PDAT and acyl-CoA:diacylglycerol acyltransferase enzymes to the triacylglycerols content in olive appears to be tissue-dependent. Furthermore, water regime, temperature, light, and wounding regulate PDAT genes at transcriptional level in the olive fruit mesocarp, indicating that PDAT could be involved in the response to abiotic stresses. Altogether, this study represents an advance in our knowledge on the regulation of oil accumulation in oil fruit.

Unraveling salt-responsive tissue-specific metabolic pathways in olive tree.

Salinity is a serious constraint that reduces olive crop productivity. Here, we defined metabolite and gene expression changes in various tissues of olive trees (cv. "Chondrolia Chalkidikis") exposed to 75 mM NaCl for 45 days. Results showed that salinity induced foliar symptoms and impaired growth and photosynthetic parameters. The content of Na+ and Cl- in roots, xylem, phloem and leaves increased, although the Na+ levels in old leaves and Cl- in young leaves remained unaffected. Mannitol was accumulated in roots and old leaves challenged by salinity. NaCl-treated trees have a decreased TCA-associated metabolites, such as citric and malic acid, as well as changes in phenylpropanoid-associated metabolites (i.e., pinoresinol and vanillic acid) and genes (OePLRTp2 and OeCA4H). Salt treatment resulted in hydroxyl-decarboxylmethyl eleuropein aglycone accumulation and OeGTF up-regulation in new leaves, possibly suggesting that oleuropein metabolism was modified by NaCl. Tyrosine metabolism, particularly verbascoside levels and OePPO and OehisC expressions, was modulated by salinity. Both genes (e.g., OeAtF3H and OeFNSII) and metabolites (e.g., apigenin and luteolin) involved in flavonoid biosynthesis were induced in old leaves exposed to NaCl. Based on these data, we constructed an interaction scheme of changes in metabolites and transcripts across olive tissues upon salinity. Particularly, several metabolites involved in carbohydrate metabolism were reduced in roots, while many sugars, carbohydrates and flavonoids were increased in leaves. This study provided a framework for better understanding the possible mechanisms that govern the tissue-specific response of olive tree to salinity stress, with insights into molecules that can be used for olive crop improvement projects.

Biophenolic Profile Modulations in Olive Tissues as Affected by Manganese Nutrition.

Manganese (Mn) is an essential element that intervenes in several plant metabolic processes. The olive tree, and its fruits and leaves, are known as a source of nutraceuticals since they are rich in biophenols. However, there is still a serious lack of data about biophenolic distribution in olive stems and roots under Mn fertilisation. In this context, our study aimed to examine the effects of Mn fertilisation on the biophenolic profile in the leaves, stems, and roots of the ‘Istarska bjelica’ olive cultivar. The experiment was set up in a greenhouse, during a period of five months, as a random block design consisting of three treatments with varying Mn concentrations in full-strength Hoagland’s nutrient solution (0.2 µM Mn, 12 µM Mn, and 24 µM Mn). The obtained results indicate that the amount of Mn in the examined olive plant tissues was significantly higher under 12 µM Mn and 24 µM Mn treatments compared to that of the 0.2 µM Mn treatment. While the concentration of biophenols varied in roots depending on the compound in question, a strong positive impact of the increased Mn concentration in nutrient solution (12 µM Mn and 24 µM Mn) on the concentrations of the main biophenolic compounds was observed in stems. The concentration of oleuropein in leaves almost doubled at 24 µM Mn, with the highest Mn concentration, as compared to the 0.2 µM Mn treatment. The obtained results led to the conclusion that the supply of Mn could enhance the concentration of some biologically active compounds in olives grown hydroponically, implying a critical need for further investigation of Mn fertilisation practices in the conventional olive farming system.

Peroxidase Enzyme Fractions as Markers of Somatic Embryogenesis Capacities in Olive (Olea europaea L.).

As part of the search for biochemical markers of somatic embryogenesis in tissue cultures of olive (Olea europaea L.), peroxidases (POXs) in both the soluble and ionically wall-bound fractions were studied in two reputed olive cultivars (cvs.): “Picholine Marocaine” and “Dahbia”. In order to carry out embryogenesis induction, proximal cotyledons were cultured in modified olive medium (OMc) supplemented with 25 μM indole-3-butylic acid (IBA) and 2.5 μM 2-isopentenyladenine (2iP), while distal leaf fragments (somatic explants) were cultured in OMc supplemented with 4.56 µM zeatin riboside (ZR) and 10.25 µM 1-naphthaleneacetic acid (NAA). Regarding embryogenic potentials, the zygotic explants (cv. Picholine Marocaine: 43.39%; cv. Dahbia: 53.41%) were more regenerative than the somatic explants (cv. Picholine Marocaine: 13.05%; cv. Dahbia: 19.51%). The enzyme assay showed a higher POX activity in embryogenic calluses (ECs) than in nonembryogenic calluses (NECs) for the zygotic explants in both studied cultivars. When expressed as units per milligram of proteins (U mg−1 proteins), the highest total POXs activities (soluble POXs + ionically wall-bound POXs) were found in the ECs derived from the zygotic explants; for cv. Dahbia, 65% of the enzyme activities came from the ionically wall-bound fractions. Polyacrylamide gel electrophoresis showed that the ECs of the highly active cv. Dahbia were characterized by highly active isoperoxidases that were revealed in four migration zones, particularly a doublet in the A4 zone (Rf 0.70–0.73) present in the ionically wall-bound POXs. The fast-moving anodic POXs of the ionically wall-bound fractions could be adopted as an early electrophoretic test to determine the embryogenesis capacities in olive tissue culture materials. As biochemical markers, the POX enzyme and its profile in fractions, i.e., as soluble POXs and ionically wall-bound POXs, can offer a valuable tool for improving the tissue culture of olive via somatic embryogenesis.

Olive (Olea europaea L.) Genetic Transformation: Current Status and Future Prospects.

Olive (Olea europaea L.) is the most characteristic and important oil crop of the Mediterranean region. Traditional olive cultivation is based on few tens cultivars of ancient origin. To improve this crop, novel selections with higher tolerance to biotic and abiotic stress, adaptable to high-density planting systems and resilient to climate change are needed; however, breeding programs are hindered by the long juvenile period of this species and few improved genotypes have been released so far. Genetic transformation could be of great value, in the near future, to develop new varieties or rootstocks in a shorter time; in addition, it has currently become an essential tool for functional genomic studies. The recalcitrance of olive tissues to their in vitro manipulation has been the main bottleneck in the development of genetic transformation procedures in this species; however, some important traits such as fungal resistance, flowering or lipid composition have successfully been manipulated through the genetic transformation of somatic embryos of juvenile or adult origin, providing a proof of the potential role that this technology could have in olive improvement. However, the optimization of these protocols for explants of adult origin is a prerequisite to obtain useful materials for the olive industry. In this review, initially, factors affecting plant regeneration via somatic embryogenesis are discussed. Subsequently, the different transformation approaches explored in olive are reviewed. Finally, transgenic experiments with genes of interest undertaken to manipulate selected traits are discussed.

FIRST REPORT OF THE INVASIVE PEST DROSOPHILA SUZUKII(MATSUMURA, 1931)(DIPTERA DROSOPHILIDAE)ON OLEA EUROPAEAL. IN ITALY

The fruit crop pest Drosophila suzukii(Matsumura, 1931)(Diptera: Drosophilidae) is a highly polypha-gous invasive pestlisted in the EPPO A2 list. This specieshas been reported as host of a variety of cultivatedplantsandwild fruits.Herein,we report the record of someD.suzukiiviable specimensthatwere found in olives directly collected from the tree branches in four differentsites of Latium and Umbria (Italy). Adults emerging from these pupae were collected and morphological and molecular analysiswere carried out to confirm the speciesidentity. Olive tissues alterations wereobserved in associationwiththe presence of the species.The analysis of the available literature showed us that this is the first record of D. suzukiias a phytophagous species on the olive tree in Italy and probably worldwide

Analysis of OeMVK Gene Expression in Different Olive Tissues Using Real Time PCR

Olive tree ( Olea europaea L.) that is a member of the Oleaceae family, is an evergreen, small tree that have been cultivated since prehistoric times in the eastern Mediterranean region. Olive is one of the vital components of Mediterranean diet. The pharmacological properties of olive oil, the olive fruit, and its leaves have been recognized as important components of a healthy diet as well as medicine because of their active role in diseases management. Mevalonate Kinase (MVK; EC 2.7.1.36; ATP:(R)- mevalonate 5-phosphotransferase) is the first enzyme in the plant isoprenoid biosynthesis in MVA (Mevalonate) pathway. In this study the MVK (Mevalonate Kinase) gene was cloned successfully from Olea europea , and named OeMVK (accession number: MH427085). The ORF (Open Reading Frame) of OeMVK was 1164 bp. OeMVK protein was consisted of 387 amino acids. Homologous sequence analysis showed that amino acid sequence of OeMVK had the highest identity of 97% with Catharanthus roseus mevalonate kinase 2a. Real-time PCR assay demonstrated that OeMVK was constitutively expressed in all tissues of olive with a similar transcription level with a slightly increase in unripe fruit. The molecular characterization of olive MVK gene and its expression level were speculated by taking the all olive tissues into account.

Dual pathway for metabolic engineering of Escherichia coli to produce the highly valuable hydroxytyrosol.

One of the most abundant phenolic compounds traced in olive tissues is hydroxytyrosol (HT), a molecule that has been attributed with a pile of beneficial effects, well documented by many epidemiological studies and thus adding value to products containing it. Strong antioxidant capacity and protection from cancer are only some of its exceptional features making it ideal as a potential supplement or preservative to be employed in the nutraceutical, agrochemical, cosmeceutical, and food industry. The HT biosynthetic pathway in plants (e.g. olive fruit tissues) is not well apprehended yet. In this contribution we employed a metabolic engineering strategy by constructing a dual pathway introduced in Escherichia coli and proofing its significant functionality leading it to produce HT. Our primary target was to investigate whether such a metabolic engineering approach could benefit the metabolic flow of tyrosine introduced to the conceived dual pathway, leading to the maximalization of the HT productivity. Various gene combinations derived from plants or bacteria were used to form a newly inspired, artificial biosynthetic dual pathway managing to redirect the carbon flow towards the production of HT directly from glucose. Various biosynthetic bottlenecks faced due to feaB gene function, resolved through the overexpression of a functional aldehyde reductase. Currently, we have achieved equimolar concentration of HT to tyrosine as precursor when overproduced straight from glucose, reaching the level of 1.76 mM (270.8 mg/L) analyzed by LC-HRMS. This work realizes the existing bottlenecks of the metabolic engineering process that was dependent on the utilized host strain, growth medium as well as to other factors studied in this work.

EFFECT OF CYTOKININS ON IN VITRO SHOOT PROLIFERATION OF OLIVE CULTIVARS “EARLIK” AND “BARI ZAITOON-2”

In olive, micro-propagation is an effective technique for mass multiplication of disease free, true to type plants, but shoot proliferation in mature tissues of olive is a major difficulty encountered during culture establishment. Thus an experiment was designed with an objective to study the effect of different cytokinins (BAP and Zeatin) on shoot proliferation of two olive cultivars (Earlik and BARI Zaitoon-2). In olive Rugini medium, BAP and Zeatin were added alone and in combination at a concentration of 1 mg L-1 and 2 mg L-1. Experiments were arranged according to completely randomized design (CRD) with three replications per treatment. Results showed that both the olive cultivars performed well when BAP + Zeatin were supplemented to the medium at 2+2 mg L-1 concentration. However, the cultivar “BARI Zaitoon-2” had the highest shooting percentage (90.67 %) and number of nodes per shoot (8.33) in treatment containing BAP (2 mg L-1) + Zeatin (2 mg L-1), whereas, “Earlik” at same concentration had more shoot length (9.10 cm) and number of shoots per explant (3.67). Thus, it was concluded that the findings of these results will help in the future for developing shoot proliferation protocols for other olive cultivars. Moreover, these protocols can also be further be used in other woody plants micro propagation

Measurement of kinaesthetic properties of in-brine table olives by microstructure of fracture surface, sensory evaluation and texture profile analysis (TPA).

A series of transformations occur in olive fruit both during ripening and processing. In particular, significant changes in the microstructural composition affect the flavour, texture, nutrients and overall quality of the end product. Texture is one of the sensory quality attributes of greatest importance to consumer acceptance. In the present work, kinaesthetic properties of in-brine table olives of three cultivars of Olea europaea L. (Bella di Cerignola, Peranzana and Taggiasca cvs) were provided by several measurements of olive tissue texture by sensory, rheological and microstructural approaches. Olives at the same stage of ripening and processed with the same technology, but belonging to different cultivars, showed significant differences at microstructural, sensorial and rheological levels. To describe the relationship between the three variables, multiple regression analysis and principal component analysis were chosen. Differences in microstructure were closely related both in terms of hardness measured by texture profile analysis and hardness measured by sensory analysis. The information provided could be an aid for screening and training of a sensory panel. © 2018 Society of Chemical Industry.

Identification of reference genes for real-time quantitative polymerase chain reaction based gene expression studies on various Olive (Olea europaea L.) tissues

ABSTRACTReference genes are essential for the normalisation of the expression data of quantitative real-time PCR for the purposes of validation. Although several reference genes have been validated for the olive, comprehensive analyses including an excessive number of candidate reference genes still require study in various olive tissue samples. In this work, a total of 40 candidate reference genes were tested for their stability in 8 different olive tissues (root, apical bud, lateral bud, pedicel, young leaf, mature leaf, fruit mesocarp, and seed) with the utilisation of the most popular software programs including GeNorm, NormFinder, BestKeeper, and ΔCt. The analyses of expression stability of candidate reference genes using quantitative real-time PCR demonstrated ubiquitin-conjugating enzyme (UBC1) as the most stable reference gene for the studied tissues of the olive. The GeNorm software also calculated the optimum reference gene combinations as two which consist of UBC1 and the Clathrin adaptor complex medium subunit (CLATHRIN) genes. This study provides the most stable reference gene combination for normalisation of target genes for quantitative real-time PCR gene expression studies on the olive.

Advances in olive fruit cell and tissue development

Trabajo presentado en el International Symposium on Physiological principles & their application to fruit production (Pome and Stone Fruit Section Environmental Physiology of Fruit Crops Working Group), celebrado en Genece (NY, US) del 26 al 28 de marzo de 2014.

Evaluation of a Pomegranate Peel Extract as an Alternative Means to Control Olive Anthracnose.

Olive anthracnose is caused by different species of Colletotrichum spp. and may be regarded as the most damaging disease of olive fruit worldwide, greatly affecting quality and quantity of the productions. A pomegranate peel extract (PGE) proved very effective in controlling the disease. The extract had a strong in vitro fungicidal activity against Colletotrichum acutatum sensu stricto, was very effective in both preventive and curative trials with artificially inoculated fruit, and induced resistance in treated olive tissues. In field trials, PGE was significantly more effective than copper, which is traditionally used to control the disease. The highest level of protection was achieved by applying the extract in the early ascending phase of the disease outbreaks because natural rots were completely inhibited with PGE at 12 g/liter and were reduced by 98.6 and by 93.0% on plants treated with PGE at 6 and 3 g/liter, respectively. Two treatments carried out 30 and 15 days before the expected epidemic outbreak reduced the incidence of the disease by 77.6, 57.0, and 51.8%, depending on the PGE concentration. The analysis of epiphytic populations showed a strong antimicrobial activity of PGE, which sharply reduced both fungal and bacterial populations. Because PGE was obtained from a natural matrix using safe chemicals and did not have any apparent phytotoxic effect on treated olive fruit, it may be regarded as a safe and effective natural antifungal preparation to control olive anthracnose and improve olive productions.