Whole genome scanning of a Mediterranean basin hotspot collection provides new insights into olive tree biodiversity and biology.

Olive tree (Olea europaea L. subsp. europaea var. europaea) is one of the most important species of the Mediterranean region and one of the most ancient species domesticated. The availability of whole genome assemblies and annotations of olive tree cultivars and oleaster (O. europaea subsp. europaea var. sylvestris) has contributed to a better understanding of genetic and genomic differences between olive tree cultivars. However, compared to other plant species there is still a lack of genomic resources for olive tree populations that span the entire Mediterranean region. In the present study we developed the most complete genomic variation map and the most comprehensive catalog/resource of molecular variation to date for 89 olive tree genotypes originating from the entire Mediterranean basin, revealing the genetic diversity of this commercially significant crop tree and explaining the divergence/similarity among different variants. Additionally, the monumental ancient tree 'Throuba Naxos' was studied to characterize the potential origin or routes of olive tree domestication. Several candidate genes known to be associated with key agronomic traits, including olive oil quality and fruit yield, were uncovered by a selective sweep scan to be under selection pressure on all olive tree chromosomes. To further exploit the genomic and phenotypic resources obtained from the current work, genome-wide association analyses were performed for 23 morphological and two agronomic traits. Significant associations were detected for eight traits that provide valuable candidates for fruit tree breeding and for deeper understanding of olive tree biology.

Investigating the in vivo biocontrol and growth-promoting efficacy of Bacillus sp. and Pseudomonas fluorescens against olive knot disease.

Olive knot disease, caused by Pseudomonas savastanoi, poses a significant threat to olive cultivation, necessitating sustainable alternatives to conventional chemical control. This study investigates the biocontrol effectiveness of Bacillus sp. (Og2) and Pseudomonas fluorescens (Oq5), alone and combined, against olive knot disease. Olive plants were sprayed with 5 ml of the bacteria until uniformly wet, with additional application to the soil surface. Pathogen injection occurred 24 h later. The results revealed that treating plants with a combination of both bacteria provided the highest reduction in disease severity (89.58 %), followed by P. fluorescens alone (69.38 %). Significant improvements were observed in shoot height, particularly with the combination of Bacillus sp. and P. fluorescens. The root length of olive seedlings treated with P. fluorescens and Bacillus sp., either alone or in combination, was significantly longer compared to the control and pathogen-treated seedlings. In terms of root dry weight, the most effective treatments were treated with P. fluorescens was the highest (82.94 g) among all treatments followed by the combination of both isolates with seedlings inoculated with P. savastanoi. These findings underscore the potential of Bacillus sp. and Pseudomonas fluorescens as effective biocontrol agents against olive knot disease and promoting olive seedlings growth, providing a sustainable and environmentally friendly approach to disease management.

Interactions at the Oviposition Scar: Molecular and Metabolic Insights into Elaeagnus angustifolia's Resistance Response to Anoplophora glabripennis.

The Russian olive (Elaeagnus angustifolia), which functions as a "dead-end trap tree" for the Asian long-horned beetle (Anoplophora glabripennis) in mixed plantations, can successfully attract Asian long-horned beetles for oviposition and subsequently kill the eggs by gum. This study aimed to investigate gum secretion differences by comparing molecular and metabolic features across three conditions-an oviposition scar, a mechanical scar, and a healthy branch-using high-performance liquid chromatography and high-throughput RNA sequencing methods. Our findings indicated that the gum mass secreted by an oviposition scar was 1.65 times greater than that secreted by a mechanical scar. Significant differences in gene expression and metabolism were observed among the three comparison groups. A Kyoto Encyclopedia of Genes and Genomes annotation and enrichment analysis showed that an oviposition scar significantly affected starch and sucrose metabolism, leading to the discovery of 52 differentially expressed genes and 7 differentially accumulated metabolites. A network interaction analysis of differentially expressed metabolites and genes showed that EaSUS1, EaYfcE1, and EaPGM1 regulate sucrose, uridine diphosphate glucose, α-D-glucose-1P, and D-glucose-6P. Although the polysaccharide content in the OSs was 2.22 times higher than that in the MSs, the sucrose content was lower. The results indicated that the Asian long-horned beetle causes Russian olive sucrose degradation and D-glucose-6P formation. Therefore, we hypothesized that damage caused by the Asian long-horned beetle could enhance tree gum secretions through hydrolyzed sucrose and stimulate the Russian olive's specific immune response. Our study focused on the first pair of a dead-end trap tree and an invasive borer pest in forestry, potentially offering valuable insights into the ecological self-regulation of Asian long-horned beetle outbreaks.

Design and Synthesis of Pyridine and Thiazole Derivatives as Eco-friendly Insecticidal to Control Olive Pests.

Treatment of p-tosyloxybenzaldehyde (1) with ethyl cyanoacetate afforded ethyl 2-cyano-3-(4-{[(4-methylphenyl)sulfonyl]oxy}phenyl)acrylate (2) which reacted with some active methylene derivatives under microwave irradiation in presence of ammonium acetate yielded pyridine derivatives 3-7. On the other hand, when treatment of compound 1 with thiosemicarbazide gave 4-tosyloxybenzylidenethiosemicarbazone (8), which allowed to react with some active methylene compounds, such as: ethyl bromoacetate, chloroacetonitrile or phenacyl bromide derivatives gave thiazole derivatives 9-13. The structure of all products were confirmed by elemental and spectroscopic analyses such as IR, 1 H-NMR, 13 CNMR and mass spectra. The advanced of this method are short reaction time (3-7 min), excellent yield, pure products, and low-cost processing. In the final category, the toxicological characteristics of all compounds were tested towards Saissetia oleae (Olivier, 1791) (Hemiptera: Coccidae). With respect to the LC50 values. It has been found that compound 3 possesses the highest insecticidal bioefficacy compared with other products, with values of 0.502 and 1.009 ppm, for nymphs and adults female, respectively. This study paves the way towards discovering new materials for potential use as insecticidal active agents.

The Contribution of Theoretical Prediction Studies to the Antioxidant Activity Assessment of the Bioactive Secoiridoids Encountered in Olive Tree Products and By-Products.

Assessment of the antioxidant activity of different types of natural compounds is a complex research area that encompasses various in vitro tests and in vivo studies. Sophisticated modern analytical tools permit the unambiguous characterization of the compounds present in a matrix. The contemporary researcher, knowing the chemical structure of the compounds present, can carry out quantum chemical calculations that provide important physicochemical information assisting the prediction of antioxidant potential and the mechanism behind the activity of target compounds before further experimentation. The efficiency of calculations is steadily improved due to the rapid evolution of both hardware and software. It is possible, consequently, to study compounds of medium or even larger size, incorporating also models that simulate the liquid phase (solution). This review contributes to the establishment of theoretical calculations as an inherent part of the antioxidant activity assessment process, having as a case study the complex mixtures of olive bioactive secoiridoids (oleuropein, ligstroside, and related compounds). The literature indicates great variability in theoretical approaches and models used so far for only a limited number of this group of phenolic compounds. Proposals are made for standardization of methodology (reference compounds, DFT functional, basis set size, and solvation model) to facilitate comparisons and communication of findings.

Combining Zeolites with Early-Maturing Annual Legume Cover Crops in Rainfed Orchards: Effects on Yield, Fatty Acid Composition and Polyphenolic Profile of Olives and Olive Oil.

Under climate change threats, there is a growing need to adapt the conventional agronomic practices used in rainfed olive orchards by sustainable practices, in order to ensure adequate crop yield and olive oil quality and to preserve soil health. Therefore, for two years, the effects of conventional tillage practice (T) and two sustainable soil management strategies, a leguminous cover crop (LC) and its combination with natural zeolites (ZL), on the yield, fatty acid composition, polyphenolic profile and quality indices of olive fruits and oil were evaluated. Crop yield was significantly increased by LC and ZL in the first year. Although in the second year no significant differences were verified, the cumulative yield increased significantly by 31.6% and 35.5% in LC and ZL trees, respectively. LC enhanced the moisture and size of olives, while ZL increased, in general, the concentrations of oleuropein, verbascoside, caffeic acid and epicatechin, as well the oleic/linoleic ratio in fruits and the levels of 3,4-dihydroxyphenylglycol, tyrosol, verbascoside and caffeic acid in olive oil. Despite the higher concentration of total phenols in the fruits and oil from T trees in the warmer and dryer year, the quality of the oil decreased, mainly when compared with ZL, as evidenced by the peroxide value and K232 and K270 coefficients. In short, both sustainable soil management strategies appear to be promising practices to implement in olive orchards under rainfed conditions, but the innovative strategy of combining zeolites with legume cover crops, first reported in the present study, confers advantages from a nutritional and technological point of view. Nevertheless, studies subjected to the long-term use of these practices should be conducted to ensure the sustainability of the crop yield and olive oil quality.

Automatic Detection of Olive Tree Canopies for Groves with Thick Plant Cover on the Ground.

Marking the tree canopies is an unavoidable step in any study working with high-resolution aerial images taken by a UAV in any fruit tree crop, such as olive trees, as the extraction of pixel features from these canopies is the first step to build the models whose predictions are compared with the ground truth obtained by measurements made with other types of sensors. Marking these canopies manually is an arduous and tedious process that is replaced by automatic methods that rarely work well for groves with a thick plant cover on the ground. This paper develops a standard method for the detection of olive tree canopies from high-resolution aerial images taken by a multispectral camera, regardless of the plant cover density between canopies. The method is based on the relative spatial information between canopies.The planting pattern used by the grower is computed and extrapolated using Delaunay triangulation in order to fuse this knowledge with that previously obtained from spectral information. It is shown that the minimisation of a certain function provides an optimal fit of the parameters that define the marking of the trees, yielding promising results of 77.5% recall and 70.9% precision.

Nutraceutical Role of Polyphenols and Triterpenes Present in the Extracts of Fruits and Leaves of Olea europaea as Antioxidants, Anti-Infectives and Anticancer Agents on Healthy Growth.

There is currently a worldwide consensus and recognition of the undoubted health benefits of the so-called Mediterranean diet, with its intake being associated with a lower risk of mortality. The most important characteristics of this type of diet are based on the consumption of significant amounts of fruit, vegetables, legumes, and nuts, which provide, in addition to some active ingredients, fiber and a proportion of vegetable protein, together with extra virgin olive oil (EVOO) as the main sources of vegetable fat. Fish and meat from poultry and other small farm animals are the main sources of protein. One of the main components, as already mentioned, is EVOO, which is rich in monounsaturated fatty acids and to a lesser extent in polyunsaturated fatty acids. The intake of this type of nutrient also provides an important set of phytochemicals whose health potential is widely spread and agreed upon. These phytochemicals include significant amounts of anthocyanins, stilbenes, flavonoids, phenolic acids, and terpenes of varying complexities. Therefore, the inclusion in the diet of this type of molecules, with a proven healthy effect, provides an unquestionable preventive and/or curative activity on an important group of pathologies related to cardiovascular, infectious, and cancerous diseases, as well as those related to the metabolic syndrome. The aim of this review is therefore to shed light on the nutraceutical role of two of the main phytochemicals present in Olea europaea fruit and leaf extracts, polyphenols, and triterpenes, on healthy animal growth. Their immunomodulatory, anti-infective, antioxidant, anti-aging, and anti-carcinogenic capabilities show them to be potential nutraceuticals, providing healthy growth.

Freezing effect on the oleuropein content of olive leaves extracts obtained from microwave-assisted extraction.

This work aimed to investigate the effect of freezing on the oleuropein content obtained from olive leaves extracts. The extracts were obtained by microwave-assisted extraction using different solvents, pH, temperatures and microwave irradiation time. Afterward, HPLC was used to identify and quantify the amount of oleuropein in the extracts. A part of the extracts was immediately analyzed, and another was frozen for a week. The experimental results highlighted that the storage condition has a significant (p < 0.05) effect on the oleuropein content. Regardless of the extraction condition, the frozen storage was responsible for a decrease in the oleuropein content, ranging from 5.38 to 70.09%. These results indicate that it is important to consider the degradation of oleuropein in frozen olive leaf extracts so that subsequent applications are suitable.

Characterization, source apportionment, air/plant partitioning and cancer risk assessment of atmospheric PAHs measured with tree components and passive air sampler.

In this study, ambient air and olive tree components (leaf and branch) were simultaneously collected and analyzed for polycyclic aromatic hydrocarbons (PAHs) to investigate their levels and accumulations, temporal variations, possible sources, air-plant partitioning and cancer risks for 12 months. During the sampling period, total of 14 PAH (∑14PAH) concentrations measured in the olive leaves (dissolved and particle phase) and braches (1- and 2-year-old) were 593 ± 472, 81 ± 67, 558 ± 273 and 316 ± 213 ng/g dry weight (DW), respectively. Similarly, the average ∑14PAH concentrations measured in the ambient air was found to be 15 ± 16 ng/m3. Generally, 4-, 5- and 6- ring PAHs were the dominant groups for all tree components, while 2- and 3-ring PAHs were predominant in the air samples. Ring distributions and molecular diagnosis ratios were employed to determine PAH sources in the sampling site. Petroleum and combustion-related sources were found to be important. The Pearson correlation coefficient was allowed to figure out the affinity between PAH levels in the sampling materials and meteorological factors. Temperature and mixing layer height were found to be effective factors on the concentrations. Atmospheric PAH levels were also predicted to employ a bark-air exchange model for determining the PAH movement direction. The predicted/measured ratios were above 1.0. This was probably due to utilizing the branch values rather than bark values in the model. Finally, the risk of cancer has been evaluated. The calculated cancer risks via inhalation were at low levels for adults and children.

Use of activated carbons for detoxification of a lignocellulosic hydrolysate: Statistical optimisation.

In order to improve bioethanol production from hydrolysates from olive tree pruning residues the present study assessed a detoxification method using the same waste material. The objective was to develop optimal removal without diminishing fermentable sugars. A statistical optimisation study was performed for the production of activated carbons using the same olive tree pruning residue as raw material. The effect of the impregnation ratio, activation temperature and the time needed for maximum removal of the phenolic compounds was assayed. The conditions for maximum adsorption were as follows: 799.18 °C, IR 4.88 and 101.38 min of activation, removing 55%, 64% and 41% of total phenolic compounds, HMF and furfural, respectively. The activated carbon obtained under optimum conditions (Aop) was submitted to adsorption kinetics and equilibrium assays. Experimental kinetic data were well represented by a pseudo-first order kinetic and intraparticle diffusion model, which helped explain the adsorption mechanism. Experimental isotherms were analysed with Langmuir, Freundlich and Temkin models, and Langmuir best fitted the experimental data. Hydrolysate from olive tree pruning residues, detoxified with the optimum activated carbon, was fermented with a commercial Saccharomyces cerevisiae yeast, obtaining a 100% theoretical maximum ethanol yield and a 56% bioconversion of fermentable sugars.

Combined Antimicrobial Effect of Bio-Waste Olive Leaf Extract and Remote Cold Atmospheric Plasma Effluent.

A novel strategy involving Olive Leaf Extract (OLE) and Cold Atmospheric Plasma (CAP) was developed as a green antimicrobial treatment. Specifically, we reported a preliminary investigation on the combined use of OLE + CAP against three pathogens, chosen to represent medical and food industries (i.e., E. coli, S. aureus and L. innocua). The results indicated that a concentration of 100 mg/mL (total polyphenols) in OLE can exert an antimicrobial activity, but still insufficient for a total bacterial inactivation. By using plain OLE, we significantly reduced the growth of Gram positive S. aureus and L. innocua, but not Gram-negative E. coli. Instead, we demonstrated a remarkable decontamination effect of OLE + CAP in E. coli, S. aureus and L. innocua samples after 6 h. This effect was optimally maintained up to 24 h in S. aureus strain. E. coli and L. innocua grew again in 24 h. In the latter strain, OLE alone was most effective to significantly reduce bacterial growth. By further adjusting the parameters of OLE + CAP technology, e.g., OLE amount and CAP exposure, it could be possible to prolong the initial powerful decontamination over a longer time. Since OLE derives from a bio-waste and CAP is a non-thermal technology based on ionized air, we propose OLE + CAP as a potential green platform for bacterial decontamination. As a combination, OLE and CAP can lead to better antimicrobial activity than individually and may replace or complement conventional thermal procedures in food and biomedical industries.

Chemical, Thermal and Antioxidant Properties of Lignins Solubilized during Soda/AQ Pulping of Orange and Olive Tree Pruning Residues.

Some agroforestry residues such as orange and olive tree pruning have been extensively evaluated for their valorization due to its high carbohydrates content. However, lignin-enriched residues generated during carbohydrates valorization are normally incinerated to produce energy. In order to find alternative high added-value applications for these lignins, a depth characterization of them is required. In this study, lignins isolated from the black liquors produced during soda/anthraquinone (soda/AQ) pulping of orange and olive tree pruning residues were analyzed by analytical standard methods and Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (solid state 13C NMR and 2D NMR) and size exclusion chromatography (SEC). Thermal analysis (thermogravimetric analysis (TGA), differential scanning calorimetry (DSC)) and antioxidant capacity (Trolox equivalent antioxidant capacity) were also evaluated. Both lignins showed a high OH phenolic content as consequence of a wide breakdown of ß-aryl ether linkages. This extensive degradation yielded lignins with low molecular weights and polydispersity values. Moreover, both lignins exhibited an enrichment of syringyl units together with different native as well as soda/AQ lignin derived units. Based on these chemical properties, orange and olive lignins showed relatively high thermal stability and good antioxidant activities. These results make them potential additives to enhance the thermo-oxidation stability of synthetic polymers.

Evolutionary transcriptomics reveals the origins of olives and the genomic changes associated with their domestication.

The olive (Olea europaea L. subsp. europaea) is one of the oldest and most socio-economically important cultivated perennial crop in the Mediterranean region. Yet, its origins are still under debate and the genetic bases of the phenotypic changes associated with its domestication are unknown. We generated RNA-sequencing data for 68 wild and cultivated olive trees to study the genetic diversity and structure both at the transcription and sequence levels. To localize putative genes or expression pathways targeted by artificial selection during domestication, we employed a two-step approach in which we identified differentially expressed genes and screened the transcriptome for signatures of selection. Our analyses support a major domestication event in the eastern part of the Mediterranean basin followed by dispersion towards the West and subsequent admixture with western wild olives. While we found large changes in gene expression when comparing cultivated and wild olives, we found no major signature of selection on coding variants and weak signals primarily affected transcription factors. Our results indicated that the domestication of olives resulted in only moderate genomic consequences and that the domestication syndrome is mainly related to changes in gene expression, consistent with its evolutionary history and life history traits.

Arbuscular mycorrhizal fungus inocula from coastal sand dunes arrest olive cutting growth under salinity stress.

Cultivation of olive trees covers large coastal areas of land in Mediterranean regions, many of them characterized by low soil fertility and exposed to salinity and seasonal drought. In this frame, we developed mixed community inocula of arbuscular mycorrhizal fungi (AMF) derived from the extreme, seasonally arid environments of six Mediterranean sand dunes and evaluated their effects, in the form of community inocula, on rooted semi-woody olive tree cuttings (Olea europaea cv. Koroneiki). The plantlets were grown in the greenhouse for 10 months under 50 mM and 100 mM concentrations of NaCl, successively applied to induce osmotic stress. Inoculation had a positive effect on plant growth and nutrient uptake. However, the three best-performing inocula in early colonization and in plant growth enhancement also resulted in high plant sensitivity to high salinity, which was not observed for the other three inocula. This was expressed by decreased nutrient uptake and drastically lower plant growth, plant photosynthesis, and stomatal conductance (generally an over 50% reduction compared to no salinity application). Amplicon sequencing analysis of the olive plants under salinity stress showed that the AMF communities in the roots were clearly differentiated by inoculation treatment. We could not, however, consistently associate the plant responses observed under high salinity with specific shared AMF community membership or assembly attributes. The observed physiological overreaction to osmotic stress may be an adaptation trait, potentially brought about by host selection coupled to abiotic environmental filtering, in the harsh conditions from which the AMF inocula were derived. The overreaction may, however, be undesirable if conveyed to allochthonous plants at an agronomic level.

Olive tree represents a new host of a subgroup 16SrVII-B phytoplasma associated with witches' broom disease in Brazil.

Olive trees exhibiting slow development, yellowing, and high intensity of shoot proliferation with small leaves were observed in commercial plantings, in the municipality of Extrema, Minas Gerais (MG) state in 2015. The incidence of symptomatic plants was about 70% and diseased trees presented yield reduction. Here we report the association of symptomatic olive trees with a phytoplasma and describe its molecular identification. Symptomatic plants (38 trees) were sampled in three growing areas located in the same municipality. The samples consisted of bunch of leaves and young shoots. The total DNA was extracted using DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). Phytoplasma detection was conducted by nested PCR with primers P1/16S-SR (Lee et al. 2004) followed by R16F2n/R16R2 (Gundersen and Lee 1996). PCR assays generated amplicons (~1.2 kb) from 28 trees out of 38 symptomatic plants, confirming the association of phytoplasma with diseased plants. The disease was named olive witches' broom. The genomic fragments amplified by nested PCR were cloned into Escherichia coli DH5α and sequenced. The sequence representative of the olive phytoplasma was designated OWB-Br01 (Olive Wiches' Broom-Brazil 01) and deposited in GenBank under accession number MH141985. This sequence shared 99% sequence identity with phytoplasmas affiliated with 16SrVII group. According to the iPhyClassifier online tool (Zhao et al. 2009) the olive witches'-broom phytoplasma was classified as a variant of subgroup 16SrVII-B with a pattern similarity coefficient of 0.99. The phylogenetic tree showed that OWB-Br01 phytoplasma emerges from the same branch of the reference phytoplasma of the 16SrVII-B subgroup (Erigeron witchesá¾½-broom phytoplasma - GenBank AY034608), indicating that the olive tree phytoplasma is a member of the 16SrVII-B subgroup. The pathogenicity test was performed with 28 healthy plants (cultivar Arbequina) grown in pots, which were grafted by simple english forklift with scions obtained from olive plants (Arbequina) six years old, naturally infected by the phytoplasma. The initial symptoms were observed four months after grafting and at eight months 22 grafted plants exhibited slow growth, yellowing, and small leaves as those naturally observed in the fields. Molecular characterization allowed identify the phytoplasma as a member of the 16SrVII-B subgroup. In Brazil, representatives of the 16SrVII group were previously reported in association with diverse botanical species. Thus, a strain of 16SrVII-C subgroup was identified in sunn hemp (Flôres et al. 2013); the reference phytoplasma of 16SrVII-D subgroup was found in erigeron plants (Flôres et al. 2015); and the representative of 16SrVII-F was detected in the wild species Vernonia brasiliana. (Fugita et al. 2017). Specifically regarding subgroup 16SrVII-B, the reference phytoplasma of this subgroup was described from erigeron and periwinkle (Barros et al. 2002), while other members of this subgroup were reported in cauliflower (Pereira et al. 2016a) and ming aralia (Pereira et al. 2016b). The disease here studied is a threat since olive planting is in large expansion in Brazil. A potential control option could be use of propagative material from sources free of the pathogen. Based on our findings, olive tree represents a new host for subgroup 16SrVII-B phytoplasma, which is different from 16Sr groups previously reported as associated with olive witches' broom in other countries.

Antifungal Potency and Modes of Action of a Novel Olive Tree Defensin Against Closely Related Ascomycete Fungal Pathogens.

Antimicrobial peptides play a pivotal role in the innate immunity of plants. Defensins are cysteine-rich antifungal peptides with multiple modes of action. A novel Oleaceae-specific defensin gene family has been discovered in the genome sequences of wild and cultivated species of a perennial olive tree, Olea europaea. OefDef1.1, a member of this defensin family, potently inhibits the in-vitro growth of ascomycete fungal pathogens Botrytis cinerea and three Fusarium spp. OefDef1.1 rapidly permeabilizes the plasma membrane of the conidial and germling cells of B. cinerea. Interestingly, it induces reactive oxygen species and translocates to the cytoplasm only in the germlings but not in the conidia. In medium containing a high concentration of Na1+, antifungal activity of OefDef1.1 is significantly reduced. Surprisingly, a chimeric OefDef1.1 peptide containing the γ-core motif of a Medicago truncatula defensin, MtDef4, displays Na1+-tolerant antifungal activity. In a phospholipid-protein overlay assay, the chimeric peptide exhibits stronger binding to its phosphoinositide partners than OefDef1.1 and is also more potent in inhibiting gray mold disease on the surface of Nicotiana benthamiana and lettuce leaves than OefDef1.1. Significant differences are observed among the four ascomycete pathogens in their responses to OefDef1.1 in growth medium with or without the elevated concentration of Na1+. The varied responses of closely related ascomycete pathogens to this defensin have implications for engineering disease resistance in plants.

Plant defense factors involved in Olea europaea resistance against Xylella fastidiosa infection.

Olive quick decline syndrome (OQDS) is a dangerous plant disease, caused by the bacterium Xylella fastidiosa, which targets olive (Olea europaea). Since field observations suggested that some olive cultivars (i.e. Leccino) were more resistant to OQDS than others (i.e. Cellina di Nardò), the plant defense strategies adopted by olive to contrast X. fastidiosa infection were investigated. In the present study, ELISA and genetic approaches were used to confirm plant infection, while microbial colonization mechanism and distribution in host plant tissues and reactive oxygen species (ROS) levels were examined by light, scanning electron and confocal microscopy analyses. Spectrophotometric and chromatographic techniques were performed to measure secondary metabolites content and qPCR assay was carried out for monitoring plant gene expression variation. Our analysis showed that X. fastidiosa caused accumulation of ROS in Leccino samples compared to Cellina di Nardò. Moreover, the infection induced the up-regulation of defense-related genes, such as NADPH oxidase, some protein kinases, pathogen plant response factors and metabolic enzymes. We also found that Leccino plants enhanced the production of specific antioxidant and antimicrobial molecules, to fight the pathogen and avoid its spreading into xylem vessels. We provided new information on OQDS resistance mechanism applied by Leccino cultivar. In particular, we evidenced that high concentrations of ROS, switching on plant defence signalling pathways, may represent a key factor in fighting X. fastidiosa infection.

Volatolomics approach by HS-SPME-GC-MS and multivariate analysis to discriminate olive tree varieties infected by Xylella fastidiosa.

INTRODUCTION: Xylella fastidiosa (Xf) is a pathogenic bacterium that causes diseases in olive trees. Therefore, analytical methods for both the characterisation of the host/pathogen interaction and infection monitoring are needed. Volatile organic compounds (VOCs) are emitted by plants relate to their physiological state, therefore VOCs monitoring can assist in detecting stress or infection states before visible signs are present. OBJECTIVE: In this work, the headspace-solid phase microextraction-gaschromatography-mass spectrometry (HS-SPME-GC-MS) technique was used for the first time to highlight VOCs differences between healthy and Xf-infected olive trees. METHODOLOGY: VOCs from olive tree twig samples were extracted and analysed by HS-SPME-GC-MS, and hence identified by comparing the experimental linear retention indexes with the reference values and by MS data obtained from NIST library. Data were processed by principal component analysis (PCA) and analysis of variance (ANOVA). RESULTS: The HS-SPME step was optimised in terms of adsorbent phase and extraction time. HS-SPME-GC-MS technique was applied to the extraction and analysis of VOCs of healthy and Xf-infected olive trees. More than 100 compounds were identified and the differences between samples were evidenced by the multivariate analysis approach. The results showed the marked presence of methyl esters in Xf-infected samples, suggesting their probable involvement in the mechanism of diffusible signal factor. CONCLUSION: The proposed approach represents an easy and solvent-free method to evaluate the presence of Xf in olive trees, and to evidence volatiles produced by host/pathogen interactions that could be involved in the defensive mechanism of the olive tree and/or in the infective action of Xf.

Copper tolerance and antibiotic resistance in soil bacteria from olive tree agricultural fields routinely treated with copper compounds.

BACKGROUND: Heavy metal pollution may act as persistent selective pressure that favors the spread of antimicrobial resistance in natural environments. The aim of this study was to isolate and identify metal-tolerant bacteria from soils in olive tree fields routinely treated with copper-derived compounds and to evaluate the tolerance of bacterial strains to other metals and their resistance to clinically relevant antibiotics. RESULTS: Five hundred and ninety-five bacterial isolates from 45 olive tree agricultural fields were studied. Minimum inhibitory concentrations (MICs) ≥ 16 mmol L-1 were detected for copper (57% of isolates), zinc (37%) and lead (62%), while only 3% had MICs ≥ 12 mmol L-1 for nickel. Ninety-six metal-tolerant strains were selected for identification and antibiotic resistance determination. Most isolates belonged to the genera Pseudomonas (37%), Bacillus (23%) and Chryseobacterium (20%), while 6% were identified as Variovorax, 4% as Stenotrophomonas and 2% as Serratia or Burkholderia. Highest copper tolerance was detected among Pseudomonas. Over 75% of the strains with high copper tolerance were also resistant to vancomycin, 50% to ampicillin and 40% to erythromycin or trimethoprim/sulfamethoxazole. CONCLUSION: Bacteria from olive soils are tolerant to metals, mainly copper, but also zinc and lead, as well as resistant to clinically important antibiotics, which could be a troublesome issue in clinical settings. © 2019 Society of Chemical Industry.