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Journal of Plant Research

, Volume 132, Issue 3, pp 439–455 | Cite as

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

  • Silvia Novelli
  • Angelo Gismondi
  • Gabriele Di Marco
  • Lorena Canuti
  • Valentina Nanni
  • Antonella CaniniEmail author
Regular Paper

Abstract

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.

Keywords

Olive quick decline syndrome Olive tree Plant defense Reactive oxygen species Secondary metabolites Xylella fastidiosa 

Abbreviations

HC

Healthy Cellina di Nardò

HL

Healthy Leccino

IC

Infected Cellina di Nardò

IL

Infected Leccino

ROS

Reactive oxygen species

OQDS

Olive quick decline syndrome

Introduction

Olive (Olea europaea L.) is a plant species, belonging to the Oleaceae family, which is mainly distributed in tropical and warm temperate regions of the World. However, the 98% of olive cultivations are restricted to the Mediterranean area (Peralbo-Molina and De Castro 2013). About 1,200 different cultivars of this species are known and 538 are distributed in the Italian peninsula (Bartolini 2012). Indeed, Italy is among the largest producer of olive oil, producing about 2.733 tons of oil per year from olives cultivated on 1,448 hectares of land (ISTAT 2015). Apulia (Southern Italy) is traditionally the most productive region for this crop, providing 37% of the national olive oil (Alba et al. 2009).

Xylella fastidiosa (Wells) Raju is a Gram-negative bacterium which presents six subspecies: fastidiosa, multiplex, sandyi, morus, tashke and pauca (Almeida and Nunney 2015; Baldi and La Porta 2017; Giampetruzzi et al. 2015; Giampetruzzi et al. 2016; Marcelletti and Scortichini 2016; Schaad et al. 2004; Schuenzel et al. 2005; Simpson et al. 2000; Su et al. 2016; Yuan et al. 2010). X. fastidiosa was associated with different plant diseases, including Pierce’s disease in grapevine, Citrus variegated chlorosis in citrus plants and Leaf scorch disease in almond, coffee, oleander, elm, sycomore, pecan, pear, mulberry, maple and oak (Hopkins and Purcell 2002; Janse and Obradovic 2010; Purcell 2013). In some of these plant species (e.g. Vitis vinifera, Citrus reticulate), X. fastidiosa virulence mechanism was hypothesized as follows: the bacterium colonizes the xylem vessels of the host and, by forming compact biofilms, occludes its vascular system, leading to plant tissue desiccation and death. The disease starts with leaf scorch and withering of scattered branches in the upper part of the crown and then progress into the rest of canopy, conferring a burned-like phenotype (Choi et al. 2013; Hopkins 1989; Janse and Obradovic 2010; Martelli 2016; Purcell and Hopkins 1996; Rodrigues et al. 2013; Saponari et al. 2013; Simpson et al. 2000). For this reason, X. fastidiosa has been included in the list of quarantine pathogens of the European Plant Protection Organization (EPPO) since 1981.

X. fastidiosa has been identified as a plant biotrophic pathogen (Charkowski 2016), although Rodrigues et al. (2013) have also documented that it behaves like a necrotrophic organism in the early stages of infection. However, it is difficult to classify such type of phytopathogen bacteria, due to the complexity of their infection process (Kraepiel and Barny 2016).

Olive infection by X. fastidiosa subsp. pauca, the so-called olive quick decline syndrome (OQDS), was recorded for the first time in the Southern of Italy, in 2013. In this context, the death of thousands of centenarian olive trees was documented (Saponari et al. 2013). In addition, this plant disease has caused severe economic consequences; ISTAT data reveal a 10.4% loss in olive oil production from 2012 to 2015, linked to olive diseases (i.e. OQDS and Bactrocera oleae) (ISTAT 2015). As it is very difficult to control X. fastidiosa spread, after its first appearance in Apulia (Italy), several measures of containment were adopted by European Community (Directive 2000/29/EC) and Italian National Phytosanitary Service, to prevent and limit its diffusion (Martelli 2016; Piano degli Interventi art. 1 c. 4 dell’OCDPC 225/2015). Since olive represents a novel host for X. fastidiosa, the exact mechanism of infection in this species and the molecular response activated by plant to contrast pathogen colonization are still unclear. Therefore, the identification of the factors potentially involved in olive resistance to X. fastidiosa could be advantageous in the setup of eradication strategies against OQDS. In particular, it is worthy of note that not all olive cultivars are equally sensitive to this bacterium; for instance, cv. Leccino exhibits very mild symptoms when infected, suggesting the existence of an innate resistance to OQDS for this variety (Baù et al. 2017; Boscia et al. 2014, 2017; Giampetruzzi et al. 2016; Martelli et al. 2015). On the other hand, Ogliarola Salentina and Cellina di Nardò cultivars are deeply damaged, desiccated and prone to death in presence of X. fastidiosa (Martelli 2016).

According to all this evidence, in the current research the interaction between olive and X. fastidiosa was investigated, analysing the colonization mechanism of the pathogen in the host plant and relative consequences on its tissues.

Then, since it is well documented that plants evolved several defense strategies against environmental biotic stresses (Hammond-Kosack and Jones 1996), the second objective of this work aimed to individuate which were the endogenous molecular mediators able to determinate OQDS resistance in Leccino cultivar. In particular, we examined if reactive oxygen species (ROS) exerted a defensive role against X. fastidiosa, acting both directly as oxidative agents and indirectly like cell secondary messengers. Moreover, we also decided to gain information on synthesis and bioactivity of antimicrobial and antioxidant plant molecules in olive tissues exposed to the pathogen. Finally, we supposed the possibility that OQDS infection could modulate the concentration of olive mRNAs involved in pathogen plant response, redox state homeostasis and metabolism. To do it, both on healthy and infected Leccino and Cellina di Nardò plants, we monitored: (a) level of ROS; (b) total content of secondary metabolites; (c) quantitation of specific bioactive molecules; (d) tissue antioxidant activity; (e) expression of the most distinctive plant defense genes. For this reason, in the present work, we used a multi-methodological approach, which included both techniques already applied in literature to study X. fastidiosa infection (e.g. scanning electron microscopy, liquid chromatography, qPCR/transcriptomic analysis) (Cardinale et al. 2018; Giampetruzzi et al. 2016; Luvisi et al. 2017) and not (e.g. Western Blotting, ROS detection).

Materials and methods

Plant material

Olive petioles and leaves (taken from symptomatic and non-symptomatic plants) were collected from two farms, located in the province of Lecce (Apulia, Italy; see acknowledgement), which possessed both X. fastidiosa infected orchards and fields with healthy (not infected) organisms. A total of 16 individuals were sampled (4 infected Leccino, 4 infected Cellina di Nardò, 4 healthy Leccino, 4 healthy Cellina di Nardò). The two farms provided both cultivars, which characterized distinct orchards positioned in close areas. Sampling was performed after authorisation of “Ministero delle Politiche Agricole, Alimentari e Forestali, ex. DG Sviluppo Rurale, ex. DISR V-Produzioni vegetali”, according to their guidelines (DG DISR-DISR 05-Prot. Uscita N. 0023466 del 03/10/2016). Olive trees used in this work satisfied the following parameters: (i) same area of origin (ii) same environmental condition of growth; (iii) same management regime; (iv) common age (25 years). In particular, infected plants (monitored by Mipaaf since OQDS has appeared in 2014 in these areas) were selected by Apulia Region Institution—phytosanitary agronomists, after evaluation of the same stage of the disease (whose symptoms were leaf scorching, presence of some desiccated branches, level 1 of the infection severity scale; Luvisi et al. 2017). On the contrary, healthy samples did not show any of these phenotypic traits. The genetic identity of the trees (Leccino or Cellina di Nardò cultivars) was confirmed by studying their microsatellite (Short Tandem Repeat, STR) profiles, according to the molecular method widely described in Gismondi and Canini (2013), using primers (DCA3, DCA7, DCA9, DCA16 and DCA18) and experimental references (samples of Leccino and Cellina di Nardò) reported in OLEA scientific database (http://www.oleadb.it/). Absence of other pathogens (Colletotrichum acutatum, Colletotrichum gloeosporioides, Phaeoacremonium spp., Botryosphaeria dothidea, Phytophthora ssp. and Phaeomoniella chlamydospora) was confirmed in all samples, following the same method reported in Luvisi et al. (2017). All samples (20 mature leaves, comprehensive of petioles, collected from six random portions of branches per tree) were preserved at − 80° C until their use, except that for ROS detection assay where fresh tissues were immediately used.

Xylella fastidiosa detection by ELISA assay and PCR analysis

The presence of X. fastidiosa in plant material was specifically detected by the PathoScreen ELISA Kit (Agdia), according to the manufacturer’s instructions and using a SUNRISE microplate reader (TECAN). Five different samples per tree were subjected to this analysis.

On the other hand, for the genetic detection of the bacterium, total DNA, including X. fastidiosa genome, was purified from plant tissues by NucleoSpin Plant II Kit (Macherey–Nagel), according to manufacturer’s instructions. Extracted DNA was used, as template, to amplify four specific gene regions of X. fastidiosa (sigma factor of RNA polymerase, RST; unknown protein, HL5; two 16S rDNA portions, SSX and XF; Table S1). We decided to use these 4 different PCR assays to be obtain a further confirmation of the presence of X. fastidiosa in our samples. PCR reactions were carried out in 50 µL of final volume, using the primer pairs reported in Table S1. Amplifications were done in a IQ5 thermocycler (BIO-RAD) set as follows: 5 min at 94° C; 40 cycles including a phase at 94 °C for 1 min, another step at melting temperature (Table S1) for 30 s and the last one at 72° C for 1 min. Finally, an additional extension step was performed at 72 °C for 10 min. PCR products were separated by agarose gel electrophoresis and visualized under UV-light, after staining with ethidium bromide.

Immunohistochemistry, scanning electron and light microscopy

In order to perform X. fastidiosa immunohistochemical analysis on plant material, as reported in Fuoco et al. (2012), olive samples were subjected to snap frozen, cut in sections (10 μm thickness) by a rotative cryostat (Leica CM 1900, Heerbrugg, Switzerland), mounted on SuperFrost Plus slides and dried at room temperature (RT). Sections were then fixed with 4% paraformaldehyde for 30 min and washed 3 times in PBS. Subsequently, 20 μL of primary antibody against X. fastidiosa (code 07319/02, Loewe, Germany), diluted 1:1,000 in PBS, were applied on each slide and incubated, in a humid chamber, for 30 min at RT, as reported by Cariddi et al. (2014). Samples were carefully washed with 0.1% Tween20 in PBS for three times. Finally, 10 μL of FITC-conjugated goat anti rabbit secondary antibody (code 07201, Loewe, Germany), diluted 1:150 in PBS, were distributed on each section, incubated and rinsed as previously described. A drop of phosphate glyceric buffer (1.8 mM Na2HPO4; 5.8 mM NaH2PO4; 50% glycerol; pH 7.6) was pipetted on each section before the microscopy analysis. Images were captured using a FluoView 1000 Olympus IX81 inverted Confocal Laser Scanning Microscope (Shinjuku-ku, Tokyo, Japan). FITC and lignin were excited by an argon ion laser set at 488 nm and 543 nm, respectively. Their signals were obtained recovering the emissions between 500–530 nm and 570–600 nm, in that order. For scanning electron microscopy (SEM) study, olive petiole sections were firstly fixed with 3% glutaraldehyde, then washed three times, for 5 min each, in 0.2 M phosphate buffer (pH 7) and finally post fixated with 1% osmium tetroxide in 0.2 M phosphate buffer for 2 h. After washing with the same buffer, samples were dehydrated with increasing concentrations (25, 50, 75 and 95%) of ethyl alcohol for 10 min per treatment. Then, they were metalized 2 min at 25 mA, using a sputter coater (EMITECH K550X, Quorum Technologies Ltd, West Sussex, United Kingdom) with a gold target. Finally, samples were observed by FE-SEM, Field Emission Scanning Electron Microscope (SUPRA 35, Carl Zeiss SMT, Oberkochen, Germany) with a SE (Second Electron) detector. The instrument was set at 5 keV as gun Voltage and 8 mm as working distance. Images of olive vascular tissues were captured by Fujifilm Finepix S1000fd camera or by a light microscope (Nikon ECLIPSE E100) (40–60 × enlargement) connected to a camera (Progress capture pro 28.0.1). Per each sample, cell wall thickness of 15 random xylem vessels was measured using ImageJ program (Fuji software) and applying the following formula: (area of the internal lumen/area of the whole vessel) × 100. All microscopy analyses were performed in triplicate for each cultivar condition.

ROS detection

The 2′,7′-dichlorofluorescein diacetate (DCF-DA) molecule was used as marker for the detection of ROS in plant tissues, as suggested by Sandalio et al. (2008). Indeed, DCF-DA, converted in DCF when reacts with reactive species, releases fluorescence at 530 nm after excitation at 488 nm. In this specific protocol, fresh leaf sections were incubated in 10 mM TRIS–HCl (pH 7.5), containing 25 mM DCF-DA, in agitation at 37 °C, in the dark, for 30 min. Then, in the same conditions, samples were washed four times with the same buffer. Finally, labelled tissues were placed on a microscope slide and observed by FluoView 1000 Olympus IX81 inverted Confocal Laser Scanning Microscope (Shinjuku-ku, Tokyo, Japan). Chlorophyll was detected by exciting the tissues at 635 nm and recovering the emissions between 655 and 755 nm. Negative (AA) and positive (H2O2) controls were performed, incubating the plant sections with 1 mM of ascorbate and 5 mM of H2O2 in PBS, respectively, for 1 h, in agitation, at 37 °C, before treatment with DCF-DA.

Carbonylated protein content

Protein extraction from sampled leaves was performed exactly as reported in Di Marco et al. (2014). Total protein content was estimated by spectrophotometric assay according to Bradford method (Bradford 1976). Carbonylated proteins were labelled by OxyBlot Protein Oxidation Detection Kit (Millipore, Vimodrone, Italy), according to manufacturer’s guidelines and revealed by Western Blotting analysis, as widely described in Gismondi et al. (2014). For carbonyl quantitation, β-Actin (Rabbit polyclonal anti-β-actin, Sigma-Aldrich, Milan, Italy) was used as normalizing internal loading control. Results were expressed in arbitrary unit, as amount of carbonylated proteins normalized for β-Actin signal level.

Quantitation of simple phenols, flavonoids and tannins

Simple phenol and flavonoid concentrations were identified in plant samples (powder of leaves and petioles), respectively, by Folin-Ciocalteu (Singleton and Rossi 1965) and Chang et al. (2002) methods, opportunely modified as reported in Di Marco et al. (2014). On the other hand, tannin level was detected in the powder of leaves and petioles, according to Impei et al. (2015) modified protocol. All measurements were revealed using a spectrophotometer (Uvikon 860 UV–visible; Kontron) and calibration curves adequately obtained with increasing amounts (0–250 mg/L) of gallic acid, quercetin and catechin, employed as standards. For simple phenol and flavonoid quantitation, results were expressed as mg of standard equivalents (mg SE; gallic acid and quercetin, respectively) per g of sample fresh weight (g SFW). Tannin data were reported as catechin equivalents (mg CE) per g of sample fresh weight (g SFW).

High pressure liquid chromatography (HPLC) analysis

Secondary metabolites were extracted from olive leaves and petioles according to Silva et al. (2006) method, after adequate modifications. In particular, 2.5 g of powdered sample were resuspended, for 30 min in agitation, in 50 mL of N-hexane and, then, centrifuged at 12.000 rpm for 5 min. The supernatant was discarded, while the pellet was diluted in 10 mL of 2% sodium bisulphite and left under agitation for 30 min. After centrifugation at 12.000 rpm for 5 min, the new pellet was resuspended with 50 mL of MetOH:dH2O (80:20, v/v), for 30 min in agitation. The sample was finally centrifuged at 12,000 rpm for 5 min and the supernatant was collected and passed through a 0.45 μm filter (Sartorius, Minisart Syringe Filter). The purified solution was completely dried under steam of nitrogen and resuspended with 2 mL of pure MetOH. 20 μL of extract were injected and analysed in an HPLC system (Shimadzu, Japan) interfaced with a diode array detector (SPD-M20A model) and equipped with Phenomenex Luna C18(R) column (3 μm × 150 mm × 4.60 mm). Samples were eluted by an aqueous gradient whose flow rate was 0.95 mL/min. In particular, 0.1% phosphoric acid in pure water and 100% methanol were used as A and B solvent, respectively. The run lasted 70 min and presented the following settings: t0 = 15% Phase B; t20 = 35% Phase B; t55 = 90% Phase B; t68 = 15% Phase B; and t70 = 15% Phase B. Only for oleanoic acid detection, the analysis was carried out using an isocratic method (phase A: 5% water; phase B: 95% MetOH) at 0.40 mL/min flow rate and run of 35 min. Diode array detection was performed by scanning the whole visible spectrum (190–800 nm) and subtracting the interference signal of the methanol (background signal) to the absorbance profile of each sample. In total, 20 different plant secondary metabolites were identified and quantified, according to their retention time and principal peak of light absorbance (as reported in Table S2), compared to pure standards (Sigma-Aldrich, Milan, Italy) and their relative calibration curves (0.1–12.5 mg mL−1), as μg of standard equivalents per g of sample fresh weight (μg SE g SFW−1).

FRAP assay

The antioxidant activity of each extract was estimated against Fe3+ 2,4,6-tripyridyl-S-triazine (TPTZ) molecule, according to the spectrophotometric methods widely described in Impei et al. (2015) and based on Benzie and Strain (1999) protocol. In particular, 2 g of olive powdered leaves were suspended in 20 mL of 0.1% EtOH, left in agitation for 2 h and, then, centrifuged for 5 min at 8.000 rpm. The supernatant was filtrated with 0.22 μm sieve (Sartorius, Minisart Syringe Filter) and used for the antiradical assay. Data were expressed as μmol iron sulfate equivalents per mg of sample fresh weight (μmol ISE mg SFW−1).

qPCR analysis

For qPCR investigations, total RNA was extracted from fresh leaf material, using Pure Link RNA Mini Kit (Ambion; Thermo Fisher Scientific, Waltham, MA, USA) as suggested by manufacturer. Then, RNA was retro transcribed in cDNA as follows: 2.5 μg of RNA, previously treated with DNase I according to manufacturer’s guidelines (Promega, Italy) and denatured at 65 °C for 2 min, were added to 10 pmol dNPTs, 200 U M-MLV reverse transcriptase (Promega, Milan, Italy), 1X reverse transcriptase buffer, 10 mM DTT, 0.5 μg random examers and 20 U RNase inhibitor and incubated, at 37 °C, for 90 min. Each qPCR reaction was carried out mixing 25 ng of cDNA, 1X SYBR Green (Kapa SYBR Fast qPCR kit; Kapa Biosystems, Woburn, MA, USA) and 5 pmol of forward and reverse primer (Table S3). Amplifications were performed in a thermal cycler (IQ5, BIO-RAD iCycler) set as reported: (i) initial denaturation at 95 °C for 2 min; (ii) 45 cycles of denaturation at 95 °C for 15 s, primer annealing at 60 °C for 15 s and extension at 72 °C for 30 s; (iii) production of dissociation curve, from 50 to 90 °C (rate: 0.5 °C every 5 s). For each analysed gene, the relative amount of transcript was measured using the 2−ΔΔCt formula (Livak and Schmittgen 2001), where the threshold cycle (Ct) of the target gene was firstly normalized with the internal reference gene (Tubulin, ΔCt) and then for the respective value obtained in the control sample (ΔΔCt), i.e. the healthy Cellina di Nardò sample which was considered as unit (100%). In our study, the validation of 2−ΔΔCt method was carried out by ΔCt variation analysis at different template concentrations, as widely described in Livak and Schmittgen (2001); relative ΔCt equations were reported in Table S3.

Statistics

Each result represents means ± standard deviation (sd) of four independent measurements (biological replicates), each one repeated three times by different experiments (technical replicates). The significance of the data was estimated by the one-way ANOVA analysis with Tukey’s honest significant difference Post-hoc test, using PAST software (p values: *< 0.05; **< 0.01; ***< 0.001).

Results and discussion

Plant innate immunity, responsible for the defence response against viruses, bacteria, fungi, nematodes, insects and even other plants, is based on the intersection of complex signaling pathways. In particular, this mechanism includes two main systems: the pathogen associated molecular patterns (PAMP)-triggered immunity (PTI), which is activated by extracellular pattern recognition receptors sensible to external stimuli (i.e. conserved microbial elicitors), and the effector-triggered immunity (ETI), induced by molecules (known as danger-associated molecular patterns; DAMPs) released from the pathogen into the host cells during the invasion and recognized through specific intracellular receptors of pathogen virulence. Generally, PTI and ETI stimulate similar responses, although ETI is qualitatively stronger and faster, including a cell death process named the hypersensitive response (HR) (Dangl et al. 2013; Katagiri and Tsuda 2010). During infection and parasitism events, we can register in plant cells several early and late phenomena which, respectively, are involved in activation and carrying out of the innate immunity, such as Reactive Oxygen Species (ROS) burst, production of nitric oxide, salicylic acid, jasmonic acid and ethylene, synthesis of antibiotic secondary metabolites, systemic acquired resistance induction, change in membrane permeability and ion fluxes, HR (Dixon et al. 1994; McDowell and Dangl 2000; Onaga and Wydra 2016; Wojtaszek 1997). However, as these mechanisms are deeply interconnected one another and partially overlapping, it is important to elucidate the specific defence system activated by each plant species to the different pathogens. For this reason, the present work aimed at investigating the molecular response evolved by olive trees to contrast X. fastidiosa infection.

Detection of X. fastidiosa in analysed samples

Both X. fastidiosa infected and healthy plant material belonging to Leccino and Cellina di Nardò olive cultivars was collected in Apulia Region. The genetic certification of the two cultivars was guaranteed by microsatellite analysis. In particular, the STR profiles of Leccino (DCA3: 243, 253; DCA7: 143, 149; DCA9: 162, 206; DCA16: 150, 174; DCA18: 177, 177) and Cellina di Nardò (DCA3: 232, 239; DCA7: 133, 151; DCA9: 186, 204; DCA16: 146, 156; DCA18: 171, 171) samples were compared with those of the experimental collection registered in OLEA scientific database, confirming their identity. At first, the presence of the bacterium in symptomatic olive tissues was checked by ELISA kit, using a specific antibody against X. fastidiosa. All symptomatic samples (100%) resulted to be infected, while no healthy specimen was positive to ELISA test. To corroborate this datum, as suggested by Loconsole et al. (2014), the pathogen was also detected in the samples by genetic analysis, using 4 different PCR assays (Fig. S1). Genetic results were perfectly coherent with those obtained by ELISA test. No template controls (NTC or negative CTR), as well as not infected materials (H), were used as negative samples both in ELISA and PCR analyses.

Distribution of X. fastidiosa in olive tissues

The presence of the pathogen into infected plant tissues was also confirmed by immunohistochemical analysis, monitoring FITC-fluorescent probes conjugated with anti-X. fastidiosa antibodies by confocal microscopy (Fig. 1). Healthy samples did not reveal, neither in transversal (Fig. 1a) nor in longitudinal sections (Fig. 1b), the presence of the microorganism, although it showed lignified elements (red signal). On the contrary, infected sections (Fig. 1c-h) clearly revealed the existence of X. fastidiosa into their tissues. Indeed, in these samples, the green signal (relative to X. fastidiosa) was specifically distributed inside the vascular vessels. Here, aggregates of X. fastidiosa cells, recognizable by their typical elongated shape, could be evidenced (Fig. 1e, g, white arrows). Moreover, as already suggested by Cardinale et al. (2018), our analysis evidenced that colonization events of adjacent vessels seem to be due to horizontal movements of the bacteria (Fig. 1e).
Fig. 1

Confocal microscopy analysis. Images of olive petioles (from healthy, H, or infected, I, Cellina di Nardò, C, or Leccino, L, samples) captured by confocal laser scanning microscope (CLSM) at different magnifications (a white dimensional bar was indicated in each panel), after immunohistochemistry treatments. In red is shown the lignin fluorescence and in green the signal corresponding to FITC-antibody directed against Xylella fastidiosa. a, b are representative images of HC (transversal) and HL (longitudinal) sections, respectively. c and d panels represent transversal sections of IC and IL samples, in that order. All the others panels are longitudinal sections of IC (e, g) and IL (f, h). White arrows indicate X. fastidiosa aggregates

The colonization of host plant tissues by X. fastidiosa was also confirmed by SEM analysis (Fig. 2). This approach evidenced the rod-like morphology of the bacteria; they were about 2 μm in length and 0.4 μm in width with a non-smooth surface, probably due to high concentrations of lipopolysaccharides (Fig. 2a, b, c). The strong adhesion among microorganisms, clearly visible in Fig. 2d and f, demonstrated that bacteria formed a compact biofilm into vascular tissues, maybe obstructing xylem flow (Fig. 2e, f) as suggested in literature (Baccari and Lindow 2011; Cardinale et al. 2018; Niza et al. 2015).
Fig. 2

SEM analysis. Representative SEM images of transversal (a, b, c, d) and longitudinal (e, f) sections of petiole vascular vessels present in olive samples infected with Xylella fastidiosa. In detail, a, b represent Leccino samples, while c, d, e and f are sections of Cellina di Nardò. Images were captured at different magnifications (a white dimensional bar was indicated in each panel). f panel is the enlargement of the central portion of e panel

In grapevines, the diffusion of X. fastidiosa into xylem network seems to occur thanks to the capability of the bacterium to digest the membranes of adjacent xylem cells, allowing them the passage from a vessel to another one (Newman et al. 2003). Indeed, Scarpari et al. (2003) suggest the existence, in X. fastidiosa, of putative pathogenicity-related genes which are involved in host tissue infection and codify for membrane degrading enzymes. On the basis of our results, it is not totally clear which is the exact diffusion mechanism of X. fastidiosa in olive; however, the theory proposed by Newman et al. (2003) could be highly probable also in olive, as supported by Fig. 1e which shows the transversal transition of microorganisms from a vessel to an adjacent one.

Role of reactive oxygen species in olive defense

In plants, ROS mediate the resistance against both biotic and abiotic stresses but also participate to several developmental processes. In olive, it has been documented that Verticillium dahliae infection induces the up-regulation of ROS stress response genes, indicating that radical molecules strongly increase in plant districts in presence of this pathogen (Jiménez-Ruiz et al. 2017; Leyva-Pérez et al. 2018). For this reason, in the present work, total ROS levels (both apoplastic and intracellular) were monitored in our plant samples, following the green DCF signal by confocal fluorescence microscopy (Fig. 3). Healthy Leccino and Cellina di Nardò leaves showed comparable amounts of ROS; for both samples, the green fluorescence was homogeneously distributed in the whole cell tissue, as also happened for the chlorophyll (red signal). On the contrary, infected leaves of both cultivars showed an irregular distribution of ROS. In detail, only a few cells of infected Cellina di Nardò presented a strong increase of ROS production (or rather an accumulation of these reactive molecules), while the same phenomenon occurred in almost all infected Leccino cells. These results evidenced a different capacity of the two olive cultivars to synthesize oxygen radicals after exposure to X. fastidiosa. To assess the correctness of this assay, negative and positive controls were carried out on fresh olive tissues using ascorbic acid and hydrogen peroxide, respectively (Fig. S2).
Fig. 3

ROS detection. Representative images of healthy (H) and infected (I) leaf tissues (upper blade) of Cellina di Nardò (C) and Leccino (L) cultivars captured by CLSM. In red was reported the chlorophyll distribution and in green was identified the signal relative to DCF molecule which is a direct indicator of ROS. Merging of both signals is also reported. The white bar indicates the dimensional unit

In the same samples, the concentration of carbonylated proteins was measured, with the aim to understand if the change in apoplastic and intracellular ROS levels, induced in plant tissues by microbial infection, could determine damages on host cell components, besides oxidizing and degrading the pathogen. As indicated in Fig. 4a, the amount of carbonylated proteins increased in infected samples, with respect to the relative healthy tissues, probably as consequence of their upper levels of ROS. For the same reason, infected Leccino proteins showed more carbonylations than infected Cellina di Nardò ones.
Fig. 4

Carbonylated protein, flavonoid, simple phenols and tannin quantitation. a Protein carbonylation was quantified using OxyBlot kit, as described in Material and Method section. The amount of carbonylated proteins, normalized for β-actin levels, was reported as arbitrary unit for healthy (H) or infected (I) Cellina di Nardò (C) and Leccino (L) samples. b The graph reported the concentration of total flavonoids (dark grey bars) and simple phenols (light grey bars) detected in all samples (powder of leaves and petioles). Data were expressed as mg of standard equivalents per g of sample fresh weight (mg SE g SFW−1). c The total level of tannins (powder of leaves and petioles), expressed as mg of catechin equivalents per g of sample fresh weight (mg CE g SFW−1), were analysed and reported. All data were reported as mean ± SD of four independent measurements (biological replicates), each one repeated three times by different experiments (technical replicates). Asterisks indicate the significance of the results, as described in Statistics paragraph

Different models, already proposed in other plant systems, could be hypothesized to justify this event: (i) infected Leccino balanced ROS-induced oxidative stress by increasing the production of antioxidant compounds (i.e. secondary metabolites) (Shao et al. 2008); (ii) infected Leccino used ROS to directly oxidize X. fastidiosa cells and destroy them (De Gara et al. 2003); (iii) ROS were exploited as secondary messengers for the activation of signal transduction pathways, which specifically induced the transcription of plant defense related genes, and like agents able to modify the plant extracellular compartment (Orozco-Cárdenas et al. 2001). In our opinion, the latter could represent the principal ROS-dependent mechanism adopted by Leccino plants to contrast X. fastidiosa infection, since the second hypothesis, indicating ROS as potential plant antimicrobial agents, has not been totally acknowledged by literature (Pitzschke et al. 2006).

Variation of total secondary metabolite content in infected plants

Secondary metabolites are molecules produced by plants to protect themselves from biotic and abiotic stresses (Gismondi et al. 2017). Indeed, a lot of these compounds possess antimicrobial properties and are essential for plant disease resistance and tolerance: bacterial growth, aggregation and biofilm formation in plant tissues may be affected by xylem sap components. For instance, as reported by Luvisi et al. (2017), phenolic compounds, such as gallic and caffeic acids, may inhibit the in vitro growth of X. fastidiosa subsp. fastidiosa. In addition, catechin synhtesis seems to be promoted in grapevine xylem tissue after exposure to X. fastidiosa infection.

For these reasons, in order to evaluate the potential defense role of the secondary metabolites in olive plants exposed to X. fastidiosa, the total content of simple phenols, flavonoids and tannins was measured (Fig. 4b, c). Cellina di Nardò plants did not modify their flavonoid level after infection. On the contrary, infected Leccino samples increased this class of molecules of 18 mg SE g SFW−1, with respect to their healthy counterpart. Regarding the phenol content, no significant difference was observed between all samples, presenting a concentration range which varied from 55 to 64 mg SE g SFW−1 (Fig. 4b). Tannin quantitation showed the most interesting results (Fig. 4c). The basal levels of tannins observed in healthy Cellina di Nardò and healthy Leccino were unexpectedly very different, suggesting the existence of a cultivar-related variability. Moreover, after infection, cv. Cellina di Nardò did not modify the amounts of tannins, while infected Leccino plants significantly accumulated them (0.33 mg CE g SFW−1 in healthy Leccino vs 0.05 mg CE g SFW−1 in infected Leccino). The increase of flavonoids and tannins only in infected Leccino samples would suggest that these compounds have a crucial function in plant defense of this cultivar. Indeed, Cellina di Nardò variety, more sensitive to OQDS, did not show any significant modification in the synthesis of secondary metabolites when infected with the pathogen. Moreover, as plant metabolites are widely documented to be good antioxidants (Di Marco et al. 2014), it is also fascinating to hypothesize that their over-production in infected Leccino samples can be due to the necessity to reduce the strong oxidative environment generated by apoplastic and intracellular ROS only in infected tissues of this ecotype (Fig. 3). Finally, since tannins are produced by plants as deterrent for the herbivory, thanks to their capacity to interact, precipitate and inhibit predator’s proteins, the elevated synthesis of these molecules in Leccino plants affected by X. fastidiosa might be also required for blocking the enzymatic activity of the pathogen.

Lignification is a structural modification of xylem cell wall which is highly favoured during plant infection with pathogens causing vascular damages. Indeed, lignin apposition gives thickness, resistance and rigidity to plant vessels, representing a real barrier for the diffusion of microorganisms into the vascular system (Giampetruzzi et al. 2016; Niza et al. 2015; Pomar et al. 2004; Rodrigues et al. 2013). In order to verify if this phenomenon also occurred in olive trees infected by X. fastidiosa, light microscopy images of petiole xylem tissues were captured (Fig. 5a). Surprisingly only infected Leccino samples showed a strong thickening of vascular cell walls with lignin, while the pathology did not modify xylem lignification in Cellina di Nardò plants, as shown in Fig. 5b (where cell wall thickness was measured by ImageJ software). In addition, since lignin synthesis occurs thanks to the cross linking of phenolic monomers in presence of oxidants, such as hydrogen peroxide (Weir et al. 2005), the increased amount of ROS in infected Leccino samples could be also easily associated to the necessity of these plants in production of new lignin molecules. Furthermore, the high levels of tannins in infected Leccino specimens would confirm this theory, being them specific metabolites associated with woody tissues.
Fig. 5

Analysis of vascular tissues. a Representative transversal sections of healthy (H) or infected (I) Cellina di Nardò (C) and Leccino (L) petioles were reported. The light microscopy images showed structure, dimension and thickness of the xylem vessels present in each sample. The analysis evidenced that only IL vascular tissue presented cell walls strongly thickened by lignin (see white arrows). White boxes represent enlargement of the areas included in black boxes. The black bar indicates the dimensional unit (20 µm). b Cell wall thickness of xylem vessels was measured, by ImageJ software, in arbitrary units (A.U.), as the ratio between area of the internal lumen and area of the respective whole vessel x 100. The reduction of this ratio indicates a potential increase of lignin apposition (*p < 0.05 vs all samples)

HPLC–DAD analysis of plant compounds and antioxidant capacity

All plant samples were subjected to HPLC–DAD analysis for the quantitation of characteristic secondary metabolites, known for their involvement in plant defense (Min-Sun et al. 2016). Metabolic profiles of healthy and infected leaf tissues, belonging to cv. Cellina di Nardò and cv. Leccino, were reported in Table 1. Coherently with previous spectrophotometric data (Fig. 4b), flavonoids, such as quercetin, kaempferol and genistein, augmented in infected Leccino samples but not in infected Cellina di Nardò, with respect to their healthy counterparts, indicating a possible involvement of these compounds in plant defense. On the other hand, it was peculiar the presence of apigenin in infected Cellina di Nardò tissues, while it was totally missing in healthy Cellina di Nardò.
Table 1

HPLC–DAD profiles

Molecules

HC (μg SE g SFW−1)

IC (μg SE g SFW−1)

HL (μg SE g SFW−1)

IL (μg SE g SFW−1)

Luteolin

719.8 ± 35.9

706.2 ± 35.3

446.2 ± 22.3**

587.2 ± 29.3*

Oleuropein

1,417.4 ± 70.8

1,975 ± 98.7*

1,815.7 ± 90.7*

2,682.2 ± 134.1**

Oleanolic acid

25,458.6 ± 1,272.9

19,110.1 ± 955.5

22,098 ± 1,104.9

31,540.3 ± 1,577.01**

3 Hydroxytyrosol

200.5 ± 10

210.9 ± 10.5

265.2 ± 13.2*

283.7 ± 14.2

Salicylic acid

438.2 ± 21.9

287.9 ± 14.4**

355.6 ± 17.7*

441.8 ± 22.1

Syringic acid

108.4 ± 5.4

107.2 ± 5.4

87.6 ± 4.3

122.2 ± 6.1*

4 Hydroxy benzoic acid

66.6 ± 3.3

65.5 ± 3.3

75 ± 3.7

80.3 ± 4.01

ellagic acid

119.7 ± 5.9

43.6 ± 2.2**

64.46 ± 3.2**

139.3 ± 6.9**

Kynurenic acid

21.8 ± 1.1

15.7 ± 0.8

10.83 ± 0.5*

51.7 ± 2.6***

Vanillic acid

8.6 ± 0.4

3.7 ± 0.2*

3.62 ± 0.2*

12.4 ± 0.6**

Gallic acid

93.8 ± 4.7

137.9 ± 6.9*

114.9 ± 5.7

137.9 ± 6.9*

Chlorogenic acid

14.1 ± 0.7

14.2 ± 0.7

14.6 ± 0.7

14.2 ± 0.7

Caffeic acid

7.3 ± 0.4

6.2 ± 0.3

6.8 ± 0.3

6.2 ± 0.3

p-Coumaric acid

8 ± 0.4

n.d.

7.5 ± 0.4

n.d.

Kaempferol

28.4 ± 1.4

6 ± 0.3**

19.1 ± 0.9*

31.3 ± 1.5**

Quercetin

20.8 ± 1

13 ± 0.6*

12.6 ± 0.6*

41.2 ± 2**

Genistein

31.4 ± 1.6

36.7 ± 1.8

29.4 ± 1.5

92.9 ± 4.6**

Apigenin

n.d.

24.3 ± 1.2

6.4 ± 0.3

10.8 ± 0.5

Myricetin

13.7 ± 0.7

5.9 ± 0.3*

7.2 ± 0.4*

9.3 ± 0.5

Qualitative and quantitative analysis of specific secondary metabolites in leaf extracts of healthy (H) or infected (I) Cellina di Nardò (C) and Leccino (L) samples. The concentration of each plant molecule was detected by HPLC-DAD and expressed as μg of standard equivalents per g of sample fresh weight (μg SE g SFW−1). All results were reported as mean ± SD of four independent measurements (biological replicates), each one repeated three times by different experiments (technical replicates) (n.d.: not detected compound). Statistics was calculated for IC vs HC; for HL vs HC; for IL vs HL (*p < 0.05, **p < 0.01, ***p < 0.001)

Regarding the simple phenols, as expected by previous spectrophotometric assay, no significant difference between cultivars was found. The only significant variation was observed for p-coumaric acid which disappeared in infected tissues of both cultivars, with respect to the relative healthy counterpart, as observed in other cases of plant infection. Indeed, as reported by Min-Sun et al. (2016), p-coumaric acid, synthesized in phenyl propanoid pathway, is the precursor of different flavonoids involved in plant defense mechanisms. For this reason, after infection, it was expected that the level of p-coumaric acid was reduced, as a possible consequence of an increased production of flavonoids. Moreover, p-coumaric acid is also involved in chlorogenic acid generation, which has a strong antibacterial activity (Lou et al. 2011), suggesting another explaination to the observed phenomenon.

In all samples, the highest concentrations were reached by oleuropein and oleanolic acid, two typical molecular markers of olive extracts. In particular, oleanolic acid, known for its antimycotic, allelopathic and antimicrobial effects (Shanmugam et al. 2014), increased in infected Leccino, compared to healthy Leccino, while it decreased in infected Cellina di Nardò, with respect to healthy Cellina di Nardò, underlining the potential protective activity of this compound against X. fastidiosa in olive.

The content variation of salycilic and kynurenic acids showed, in all samples, the same trend observed for oleanolic acid. The high dose of both these plant compounds could justify part of the resistance evidenced in cv. Leccino to OQDS. Indeed, while kynurenic acid was demonstrated to be involved in plant defense mechanisms, salicylic acid represents, in plants, an important signaling molecule for the induction of apoptosis, hyper sensitive response and ROS synthesis (Min-Sun et al. 2016). In particular, this last bioactivity of salicylic acid could be associated to ROS over-production observed in infected Leccino samples.

As previously explained, endogenous antioxidants have to modulate ROS accumulation and removal in plant tissues, especially in presence of biotic agents which induce oxidative stress (Torres et al. 2006). As in X. fastidiosa infected samples a variation of both ROS and antiradical compound levels were observed, a free radical scavenging assay, the FRAP test, was carried out. Results showed that healthy Cellina di Nardò, infected Cellina di Nardò, healthy Leccino and infected Leccino presented reducing properties of 311.0 ± 15, 243.9 ± 12.2, 495.8 ± 24.7 and 287.9 ± 14.3 μmol ISE per mg of SFW, respectively. These data agreed with previous experiments (Figs. 3, 4a), suggesting that, in both cultivars, X. fastidiosa infection induced an increase of ROS which proportionally reduced the whole tissue antiradical level. Indeed, coherently with ROS concentration, cell antioxidant status lessened in infected Leccino, with respect to healthy Leccino (Δ = − 207.9), more than in infected Cellina di Nardò, compared to healthy Cellina di Nardò (Δ = − 67.1). Indeed, although infected Leccino samples presented higher concentrations of total and specific secondary metabolites (i.e. total tannins, total flavonoids, genistein, oleuropein) than Cellina di Nardò, the general antioxidant level of both samples could be considered quite comparable, suggesting that in the resistant cultivar the plant compounds were probably more prone to act as direct antimicrobials rather than antioxidants.

Gene expression related to plant defense

In plants, as in animals, innate immunity represents the first strategy of inducible defense towards microbial infection. In this context, receptor-mediated recognition of pathogen associated molecular patterns triggers the activation of signaling pathways that manage plant systemic defense against pathogens (Nürnberger et al. 2004). According to these notions, to ascertain the existence in olive of defense mechanisms activated by X. fastidiosa, the expression levels of different genes involved in plant response to infections were monitored both in healthy and diseased samples of Leccino and Cellina di Nardò. Since olive strategies against X. fastidiosa have not been clarified yet, a screening of the expression of genes commonly involved in plant response to biotic stress was performed. In detail, among all, we selected 17 genes specifically associated to plant defense, redox homeostasis and secondary metabolism (Table S3), in order to confirm or reject the main hypotheses previously assumed in this work and understand which cell pathway was mainly involved in such type of infection.

All qPCR results, expressed in percentage with respect to healthy Cellina di Nardò sample (considered as unit, onefold change), were reported in Table 2. α-Tubulin was used as internal control gene since its expression did not change between healthy and infected samples. According to their expression trend, studied genes were classified in four clusters.
Table 2

qPCR analysis

Genes

HC

IC

HL

IL

CPK11

1

0.57 ± 0.04

0.474 ± 0.028*

0.79 ± 0.034

ICS-1

1

0.125 ± 0.004*

0.111 ± 0.008*

0.29 ± 0.02

LEU-PT

1

54,350.285 ± 1,606.04***

42.371 ± 0.31**

312,825.80 ± 17,542.79***

FRK-1

1

3.386 ± 0.19*

0.085 ± 0.009*

0.66 ± 0.19*

NH01

1

5.115 ± 0.04*

0.84 ± 0.002

97.68 ± 1.71**

PR1

1

161.456 ± 0.051***

0.929 ± 0.009

16.50 ± 1.35**

NHL10

1

546.849 ± 0.28***

0.188 ± 0.023*

582.05 ± 22.60***

CPK6

1

6.475 ± 0.15*

2.93 ± 0.083*

40.36 ± 0.79**

RbohD-1

1

13.784 ± 0.52**

0.063 ± 0.00005*

1,937.52 ± 63.97***

C1-LIKE

1

70.9 ± 0.0,035***

0.635 ± 0.009

0.38 ± 0.015

PR5

1

0.005 ± 0.0,003***

0.006 ± 0.0,003***

0.0004 ± 0.000009**

CRK18

1

1.366 ± 0.021

2.75 ± 0.087*

0.29 ± 0.009*

CRK7

1

2.514 ± 0.038*

2.14 ± 0.079*

0.81 ± 0.008*

PHI1

1

16.223 ± 0.16**

28.94 ± 3.09**

20.46 ± 0.347

CRK37

1

101.828 ± 0.33***

0.19 ± 0.008*

0.061 ± 0.0005

PR2

1

2.858 ± 0.093*

5.50 ± 0.016*

1.65 ± 0.037*

CPK4

1

1.596 ± 0.022

2.63 ± 0.047*

1.36 ± 0.106

mRNA expression of seventeen plant defense genes (see SM3 for gene name abbreviations) was monitored, by qPCR, in healthy (H) or infected (I) Cellina di Nardò (C) and Leccino (L) plants. After normalization with Tubulin (used as internal loading control), the amount of each transcript was expressed in all samples as percentage, compared to the respective mRNA levels detected in HC and used as unit (onefold change). Results were reported as mean ± SD of four independent measurements (biological replicates), each one repeated three times by different experiments (technical replicates). Statistics was calculated for IC vs HC; for HL vs HC; for IL vs HL (*p < 0.05, **p < 0.01, ***p < 0.001)

The first group included genes whose expression, after infection, decreased in Cellina di Nardò and increased in Leccino: CPK11 and ICS-1. Respectively, they represent a kinase protein, usually induced by environmental stressors and probably able to activate transcription factors, and an isochorismate synthase, which would explain the increased amount of secondary metabolites in infected Leccino samples. These genes might represent the key elements determining the resistance of cv. Leccino to X. fastidiosa, with respect to Cellina di Nardò variety.

The second cluster was represented by genes which extraordinarily increased their expression in infected tissues of both cultivars: LEU-PT, FRK1, NH01, PR-1, NHL-10, CPK-6 and RbohD-1. This evidence suggested that these genes, mostly coding for protein kinases, were surely involved in olive defense mechanism to microbial attack, independently from the ecotype. Particular attention should also be paid to RbohD-1 gene, which was documented to be responsible for apoplastic ROS burst in response to microbe associated plant molecular patterns (Pogany et al. 2009; Torres et al. 2002; Zhang et al. 2007). The expression levels of this plant NADPH oxidase, increasing in infected Cellina di Nardòand infected Leccino samples of about 13.8% and 30,754.4%, respectively, compared to their healthy counterparts, were perfectly coherent with the gain of total ROS synthesis previously observed in infected samples, especially in cv. Leccino.

In the third group were included only two genes, C1-LIKE and PR-5, which appeared less expressed in infected tissues of both cultivars; probably, they were not involved in plant defense or shut down by the pathogen.

Finally, CRK-18, CRK-7, PHI-1, CRK-37, PR-2 and CPK-4 genes were considered as another independent group. After infection, they all increased their expression in Cellina di Nardò, while their mRNA levels appeared reduced in Leccino, meaning that these genes were maybe not responsible for the resistance of Leccino plants to X. fastidiosa. This result was quite unexpected because Cysteine-rich Receptor-like Kinases (CRKs) are widely documented to be regulated, in a positive manner at transcriptional level, by biotic and abiotic stresses. Indeed, CRKs are about 40 cell surface plant receptor-like kinases which, presenting an extracellular domain characterized by a cysteine-rich motif whose function is still unknown, perceive the environmental stimuli and convert them into transduction signals triggering adequate cell responses (Lehti-Shiu et al., 2009; Wrzaczek et al., 2010). In particular, several works reported that these kinases are specifically involved in plant defense to pathogen (Acharya et al. 2007; Bourdais et al., 2015; Chen et al. 2004; Yadeta et al., 2017; Yeh et al., 2015). However, in our study, they seem to be not implicated in Leccino resistance to X. fastidiosa.

Recently, Giampetruzzi et al. (2016) performed a global quantitative transcriptome analysis on Ogliarola salentina and Leccino olive samples infected by X. fastidiosa. Obviously, the huge amount of their data cannot be compared with those obtained in the present study, which focused its attention only on specific target genes. However, Giampetruzzi et al. (2016) observed that several kinases were up-regulated in infected Leccino, with respect to infected Ogliarola salentina, and that both cultivars activated a strong re-modelling of the cell wall after infection, as we also detected in our samples by qPCR (e.g. CPK11), spectrophotometric (e.g. tannin level) and microscopy (e.g. lignin thickness) analyses.

Simultaneously to the present study, Sabella et al. (2018) produced a paper where they suggested that the resistance of Leccino cultivar to X. fastidiosa could be due to its high lignin content, compared to Cellina di Nardò. Our results strongly support this evidence but they also propose, in this context, the potential role of ROS as inducer of lignin polymerization, direct agents in degrading and killing the pathogen, and secondary messengers activating the expression of genes (e.g. protein kinases and metabolic enzymes) involved in plant defense. Moreover, our work hypothesizes the involvement of other plant elements (e.g. tannins, specific phenolic metabolites, RbohD-1) in the defense mechanism activated by Leccino plant against X. fastidiosa.

Conclusions

The present research aimed to understand the interaction between olive and X. fastidiosa. In particular, colonization mechanism and distribution of the bacterium in host plant tissues were studied. In order to individuate alternative strategies for the preservation of Apulia millennial olive trees, which represent the most ancient existent arboreal patrimony, olive plants belonging to cultivars showing signs of resistance to OQDS (i.e. cv. Leccino) were analysed and compared to more sensitive varieties (i.e. cv. Cellina di Nardò). Our results suggested the molecular mechanisms adopted by Leccino plants to adapt themselves to X. fastidiosa infection. As summarized in Fig. 6, under biotic stress, Leccino plants, increasing ROS production, modulate their own gene expression and cause specific damages on pathogen cells. This last pathway can lead to the activation of both plant defense genes, codifying for proteins involved in direct (i.e. PR1) and indirect (i.e. RbohD-1) microbial resistance, and metabolism genes. Among them, many are implicated in the biosynthesis of secondary metabolites (i.e. ICS-1) able to: i) defeat X. fastidiosa thanks to their antibiotic activity (i.e. oleanolic acid); ii) maintain stable ROS levels, sufficient to induce plant defense but not dangerous for cell integrity, by their antioxidant potential (i.e. genistein); iii) give hardness to vascular vessels, preventing microbial spread (i.e. tannin and lignin). However, all previous conjectures, for instance the supposed antibiotic effects of specific plant metabolites and tannins against X. fastidiosa might be validated, in future studies, by isolating the pathogen from infected olive material and treating it with these pure compounds. Thus, the present work can be considered as a starting point for further investigations on this topic, in order to limit the dangerous effects of X. fastidiosa infection, in terms of ecologic, economic and biological value.
Fig. 6

Proposed model. Hypothetical defense molecular mechanism activated by olive plants, belonging to cv. Leccino, after infection with Xylella fastidiosa. The pathogen, located in xylem vessels, induces ROS over-production in the cytoplasm (C) and in the apoplast of the surrounding parenchymal tissues. ROS may both cause direct damages on bacteria and act as secondary messengers, modulating host gene expression (N: nucleus). This phenomenon leads to the transcription of plant defense genes which can both stimulate ROS synthesis (i.e. RbohD-1) and reduce pathogen’s offensive (i.e. PR-1). On the other hand, even metabolism genes are overexpressed (i.e. ICS-1), increasing the concentration of secondary metabolites. Among them, some plant compounds (i.e. oleanolic acid) show antibiotic activity against X. fastidiosa, others (i.e. genistein) balance ROS levels by their antioxidant potential and, finally, few molecules (i.e. lignin) are involved in the reduction of bacterial spread in adjacent vascular vessels

Notes

Acknowledgments

The authors thank Dr. Daniele Giaffreda for his work as intermediary with Agricultural Farms, the Agricultural Farm of Ing. Niccolò Coppola-srl-S.S. 101, Km 34.5-Tenuta di Torre Sabea 73014 Gallipoli (LE) and the Agricultural Farm of Cosimo Tornesello-Contrada Monaci Gallipoli-73048 (LE) which kindly provided olive samples, the Advanced Microscopies Center (AMC) of the University of Rome “Tor Vergata”, Biology Department, Dr. Elena Romano for her technical expertise in confocal microscopy analysis and Dr. Francesco Basoli for his contribution in SEM analysis A deep gratitude to “Ministero delle Politiche Agricole, Alimentari e Forestali, ex. DG Sviluppo Rurale, ex. DISR V-Produzioni vegetali”, “Regione Puglia-Area Politiche per lo Sviluppo Rurale, Servizio Agricoltura, Ufficio Osservatorio Fitosanitario” and “Regione Lazio-Direzione Regionale Agricoltura, Servizio Fitosanitario Regionale, Innovazione in Agricoltura” for authorisation to collect samples and work on this topic (DG DISR-DISR 05-Prot. Uscita N. 0023466 del 03/10/2016).

Supplementary material

10265_2019_1108_MOESM1_ESM.pdf (826 kb)
Supplementary material 1 (pdf 825 kb)

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© The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiologyUniversity of Rome “Tor Vergata”RomeItaly

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