Characterization of the olive endophytic community in genotypes displaying a contrasting response to Xylella fastidiosa.

BACKGROUND: Endophytes mediate the interactions between plants and other microorganisms, and the functional aspects of interactions between endophytes and their host that support plant-growth promotion and tolerance to stresses signify the ecological relevance of the endosphere microbiome. In this work, we studied the bacterial and fungal endophytic communities of olive tree (Olea europaea L.) asymptomatic or low symptomatic genotypes sampled in groves heavily compromised by Xylella fastidiosa subsp. pauca, aiming to characterize microbiota in genotypes displaying differential response to the pathogen. RESULTS: The relationships between bacterial and fungal genera were analyzed both separately and together, in order to investigate the intricate correlations between the identified Operational Taxonomic Units (OTUs). Results suggested a dominant role of the fungal endophytic community compared to the bacterial one, and highlighted specific microbial taxa only associated with asymptomatic or low symptomatic genotypes. In addition, they indicated the occurrence of well-adapted genetic resources surviving after years of pathogen pressure in association with microorganisms such as Burkholderia, Quambalaria, Phaffia and Rhodotorula. CONCLUSIONS: This is the first study to overview endophytic communities associated with several putatively resistant olive genotypes in areas under high X. fastidiosa inoculum pressure. Identifying these negatively correlated genera can offer valuable insights into the potential antagonistic microbial resources and their possible development as biocontrol agents.

Advances in Plant Disease Detection and Monitoring: From Traditional Assays to In-Field Diagnostics.

Human activities significantly contribute to worldwide spread of phytopathological adversities. Pathogen-related food losses are today responsible for a reduction in quantity and quality of yield and decrease value and financial returns. As a result, "early detection" in combination with "fast, accurate, and cheap" diagnostics have also become the new mantra in plant pathology, especially for emerging diseases or challenging pathogens that spread thanks to asymptomatic individuals with subtle initial symptoms but are then difficult to face. Furthermore, in a globalized market sensitive to epidemics, innovative tools suitable for field-use represent the new frontier with respect to diagnostic laboratories, ensuring that the instruments and techniques used are suitable for the operational contexts. In this framework, portable systems and interconnection with Internet of Things (IoT) play a pivotal role. Here we review innovative diagnostic methods based on nanotechnologies and new perspectives concerning information and communication technology (ICT) in agriculture, resulting in an improvement in agricultural and rural development and in the ability to revolutionize the concept of "preventive actions", making the difference in fighting against phytopathogens, all over the world.

Molecular Responses to Cadmium Exposure in Two Contrasting Durum Wheat Genotypes.

Cadmium is a heavy metal that can be easily accumulated in durum wheat kernels and enter the human food chain. Two near-isogenic lines (NILs) with contrasting cadmium accumulation in grains, High-Cd or Low-Cd (H-Cd NIL and L-Cd NIL, respectively), were used to understand the Cd accumulation and transport mechanisms in durum wheat roots. Plants were cultivated in hydroponic solution, and cadmium concentrations in roots, shoots and grains were quantified. To evaluate the molecular mechanism activated in the two NILs, the transcriptomes of roots were analyzed. The observed response is complex and involves many genes and molecular mechanisms. We found that the gene sequences of two basic helix-loop-helix (bHLH) transcription factors (bHLH29 and bHLH38) differ between the two genotypes. In addition, the transporter Heavy Metal Tolerance 1 (HMT-1) is expressed only in the low-Cd genotype and many peroxidase genes are up-regulated only in the L-Cd NIL, suggesting ROS scavenging and root lignification as active responses to cadmium presence. Finally, we hypothesize that some aquaporins could enhance the Cd translocation from roots to shoots. The response to cadmium in durum wheat is therefore extremely complex and involves transcription factors, chelators, heavy metal transporters, peroxidases and aquaporins. All these new findings could help to elucidate the cadmium tolerance in wheat and address future breeding programs.

Accumulation of Azelaic Acid in Xylella fastidiosa-Infected Olive Trees: A Mobile Metabolite for Health Screening.

Monitoring Xylella fastidiosa is critical for eradicating or at least containing this harmful pathogen. New low-cost and rapid methods for early detection capability are very much needed. Metabolomics may play a key role in diagnosis; in fact, mobile metabolites could avoid errors in sampling due to erratically distributed pathogens. Of the various different mobile signals, we studied dicarboxylic azelaic acid (AzA) which is a key molecule for biotic stress plant response but has not yet been associated with pathogens in olive trees. We found that infected Olea europaea L. plants of cultivars Cellina di Nardò (susceptible to X. fastidiosa) and Leccino (resistant to the pathogen) showed an increase in AzA accumulation in leaf petioles and in sprigs by approximately seven- and sixfold, respectively, compared with plants negative to X. fastidiosa or affected by other pathogens. No statistically significant variation was found between the X. fastidiosa population level and the amount of AzA in either of the plant tissues, suggesting that AzA accumulation was almost independent of the amount of pathogen in the sample. Furthermore, the association of AzA with X. fastidiosa seemed to be reliable for samples judged as potentially false-negative by quantitative polymerase chain reaction (cycle threshold [Ct] > 33), considering both the absolute value of AzA concentration and the values normalized on negative samples, which diverged significantly from control plants. The accumulation of AzA in infected plants was partially supported by the differential expression of two genes (named OeLTP1 and OeLTP2) encoding lipid transport proteins (LTPs), which shared a specific domain with the LTPs involved in AzA activity in systemic acquired resistance in other plant species. The expression level of OeLTP1 and OeLTP2 in petiole samples showed significant upregulation in samples positive to X. fastidiosa of both cultivars, with higher expression levels in positive samples of Cellina di Nardò compared with Leccino, whereas the two transcripts had a low expression level (Ct > 40) in negative samples of the susceptible cultivar. Although the results derived from the quantification of AzA cannot confirm the presence of the erratically distributed X. fastidiosa, which can be definitively assessed by traditional methods, we believe they represent a fast and cheap screening method for large-scale monitoring.

Combined Effect of Cadmium and Lead on Durum Wheat.

Cadmium (Cd) and lead (Pb) are two toxic heavy metals (HMs) whose presence in soil is generally low. However, industrial and agricultural activities in recent years have significantly raised their levels, causing progressive accumulations in plant edible tissues, and stimulating research in this field. Studies on toxic metals are commonly focused on a single metal, but toxic metals occur simultaneously. The understanding of the mechanisms of interaction between HMs during uptake is important to design agronomic or genetic strategies to limit contamination of crops. To study the single and combined effect of Cd and Pb on durum wheat, a hydroponic experiment was established to examine the accumulation of the two HMs. Moreover, the molecular mechanisms activated in the roots were investigated paying attention to transcription factors (bHLH family), heavy metal transporters and genes involved in the biosynthesis of metal chelators (nicotianamine and mugineic acid). Cd and Pb are accumulated following different molecular strategies by durum wheat plants, even if the two metals interact with each other influencing their respective uptake and translocation. Finally, we demonstrated that some genes (bHLH 29, YSL2, ZIF1, ZIFL1, ZIFL2, NAS2 and NAAT) were induced in the durum wheat roots only in response to Cd.

Evaluation of Phytochemical and Antioxidant Properties of 15 Italian Olea europaea L. Cultivar Leaves.

Olive leaf extracts are of special interest due to their proven therapeutic effects. However, they are still considered a by-product of the table olive and the oil industries. In order to learn possible ways of exploiting this waste for health purposes, we investigated the phytochemical profiles and antioxidant activities in the leaves of 15 Italian Olea europaea L. cultivars grown in the same pedoclimatic conditions. The phenolic profiles and amounts of their seven representative compounds were analyzed using HPLC ESI/MS-TOF. The antioxidant activities were determined using three different antioxidant assays (DPPH, ORAC, and superoxide anion scavenging assay). Wide ranges of total phenolic content (11.39-48.62 g GAE kg-1 dry weight) and antioxidant activities (DPPH values: 8.67-29.89 µmol TE mg-1 dry weight, ORAC values: 0.81-4.25 µmol TE mg-1 dry weight, superoxide anion scavenging activity values: 27.66-48.92 µmol TE mg-1 dry weight) were found in the cultivars. In particular, the cultivars Itrana, Apollo, and Maurino, showed a high amount of total phenols and antioxidant activity, and therefore represent a suitable natural source of biological compounds for use in terms of health benefits.

Activation of a gene network in durum wheat roots exposed to cadmium.

BACKGROUND: Among cereals, durum wheat (Triticum turgidum L. subsp. durum) accumulates cadmium (Cd) at higher concentration if grown in Cd-polluted soils. Since cadmium accumulation is a risk for human health, the international trade organizations have limited the acceptable concentration of Cd in edible crops. Therefore, durum wheat cultivars accumulating low cadmium in grains should be preferred by farmers and consumers. To identify the response of durum wheat to the presence of Cd, the transcriptomes of roots and shoots of Creso and Svevo cultivars were sequenced after a 50-day exposure to 0.5 µM Cd in hydroponic solution. RESULTS: No phytotoxic effects or biomass reduction was observed in Creso and Svevo plants at this Cd concentration. Despite this null effect, cadmium was accumulated in root tissues, in shoots and in grains suggesting a good cadmium translocation rate among tissues. The mRNA sequencing revealed a general transcriptome rearrangement after Cd treatment and more than 7000 genes were found differentially expressed in root and shoot tissues. Among these, the up-regulated genes in roots showed a clear correlation with cadmium uptake and detoxification. In particular, about three hundred genes were commonly up-regulated in Creso and Svevo roots suggesting a well defined molecular strategy characterized by the transcriptomic activation of several transcription factors mainly belonging to bHLH and WRKY families. bHLHs are probably the activators of the strong up-regulation of three NAS genes, responsible for the synthesis of the phytosiderophore nicotianamine (NA). Moreover, we found the overall up-regulation of the methionine salvage pathway that is tightly connected with NA synthesis and supply the S-adenosyl methionine necessary for NA biosynthesis. Finally, several vacuolar NA chelating heavy metal transporters were vigorously activated. CONCLUSIONS: In conclusion, the exposure of durum wheat to cadmium activates in roots a complex gene network involved in cadmium translocation and detoxification from heavy metals. These findings are confident with a role of nicotianamine and methionine salvage pathway in the accumulation of cadmium in durum wheat.

Glutathione S-transferase related detoxification processes are correlated with receptor-mediated vacuolar sorting mechanisms.

KEY MESSAGE: Triticum durum Glutathione S-transferase Z1 is specifically responsive to glyphosate. Its expression influences the receptor-mediated vacuolar sorting mechanisms involved in tolerance mechanisms. A zeta subfamily glutathione S-transferase gene from Triticum durum (cv Cappelli) (TdGSTZ1) was characterized as part of a complex detoxification mechanism. The effect of different abiotic stresses on TdGSTZ1 revealed that the gene is unexpectedly responsive to glyphosate (GLY) herbicide despite it should not be part of tolerance mechanisms. Its role in the non-target-site mechanism of GLY resistance was then investigated. To analyze the GLY and the TdGSTZ1 overexpression effects on vacuolar sorting mechanisms, we performed transient transformation experiments in Nicotiana tabacum protoplasts using two vacuolar markers, AleuGFPgl133 and GFPgl133Chi, labeling the Sar1 dependent or independent sorting, respectively. We observed that the adaptive reaction of tobacco protoplasts vacuolar system to the treatment with GLY could be partially mimicked by the overexpression of TdGSTZ1 gene. To confirm the influence of GLY on the two vacuolar markers accumulation and the potential involvement of the secretion pathway activity in detoxification events, Arabidopsis thaliana transgenic plants overexpressing the non-glycosylated versions of the two markers were analyzed. The results suggested that GLY treatment specifically altered different vacuolar sorting characteristics, suggesting an involvement of the receptor-mediated AleuGFP sorting mechanism in GLY resistance. Finally, the expression analysis of selected genes confirmed that the non-target-site GLY resistance mechanisms are related to vacuolar sorting.

The Significance of Xylem Structure and Its Chemical Components in Certain Olive Tree Genotypes with Tolerance to Xylella fastidiosa Infection.

Olive quick decline syndrome (OQDS) is a devastating plant disease caused by the bacterium Xylella fastidiosa (Xf). Exploratory missions in the Salento area led to the identification of putatively Xf-resistant olive trees (putatively resistant plants, PRPs) which were pauci-symptomatic or asymptomatic infected plants belonging to different genetic clusters in orchards severely affected by OQDS. To investigate the defense strategies employed by these PRPs to contrast Xf infection, the PRPs were analyzed for the anatomy and histology of xylem vessels, patterns of Xf distribution in host tissues (by the fluorescent in situ hybridization technique-FISH) and the presence of secondary metabolites in stems. The xylem vessels of the PRPs have an average diameter significantly lower than that of susceptible plants for each annual tree ring studied. The histochemical staining of xylem vessels highlighted an increase in the lignin in the parenchyma cells of the medullary rays of the wood. The 3D images obtained from FISH-LSM (laser scanning microscope) revealed that, in the PRPs, Xf cells mostly appeared as individual cells or as small aggregates; in addition, these bacterial cells looked to be incorporated in the autofluorescence signal of gels and phenolic compounds regardless of hosts' genotypes. In fact, the metabolomic data from asymptomatic PRP stems showed a significant increase in compounds like salicylic acid, known as a signal molecule which mediates host responses upon pathogen infection, and luteolin, a naturally derived flavonoid compound with antibacterial properties and with well-known anti-biofilm effects. Findings indicate that the xylem vessel geometry together with structural and chemical defenses are among the mechanisms operating to control Xf infection and may represent a common resistance trait among different olive genotypes.

Xylem Embolism and Pathogens: Can the Vessel Anatomy of Woody Plants Contribute to X. fastidiosa Resistance?

The maintenance of an intact water column in the xylem lumen several meters above the ground is essential for woody plant viability. In fact, abiotic and biotic factors can lead to the formation of emboli in the xylem, interrupting sap flow and causing consequences on the health status of the plant. Anyway, the tendency of plants to develop emboli depends on the intrinsic features of the xylem, while the cyto-histological structure of the xylem plays a role in resistance to vascular pathogens, as in the case of the pathogenic bacterium Xylella fastidiosa. Analysis of the scientific literature suggests that on grapevine and olive, some xylem features can determine plant tolerance to vascular pathogens. However, the same trend was not reported in citrus, indicating that X. fastidiosa interactions with host plants differ by species. Unfortunately, studies in this area are still limited, with few explaining inter-cultivar insights. Thus, in a global context seriously threatened by X. fastidiosa, a deeper understanding of the relationship between the physical and mechanical characteristics of the xylem and resistance to stresses can be useful for selecting cultivars that may be more resistant to environmental changes, such as drought and vascular pathogens, as a way to preserve agricultural productions and ecosystems.

Optimization of SAW Sensors for Nanoplastics and Grapevine Virus Detection.

In this work, we report the parametric optimization of surface acoustic wave (SAW) delay lines on Lithium niobate for environmental monitoring applications. First, we show that the device performance can be improved by acting opportunely on geometrical design parameters of the interdigital transducers such as the number of finger pairs, the finger overlap length and the distance between the emitter and the receiver. Then, the best-performing configuration is employed to realize SAW sensors. As aerosol particulate matter (PM) is a major threat, we first demonstrate a capability for the detection of polystyrene particles simulating nanoparticulates/nanoplastics, and achieve a limit of detection (LOD) of 0.3 ng, beyond the present state-of-the-art. Next, the SAW sensors were used for the first time to implement diagnostic tools able to detect Grapevine leafroll-associated virus 3 (GLRaV-3), one of the most widespread viruses in wine-growing areas, outperforming electrochemical impedance sensors thanks to a five-times better LOD. These two proofs of concept demonstrate the ability of miniaturized SAW sensors for carrying out on-field monitoring campaigns and their potential to replace the presently used heavy and expensive laboratory instrumentation.

Signaling Cross-Talk between Salicylic and Gentisic Acid in the 'Candidatus Phytoplasma Solani' Interaction with Sangiovese Vines.

"Bois noir" disease associated with 'Candidatus Phytoplasma solani' seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma-grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of 'Ca. P. solani'-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in 'Ca. P. solani-positive plants compared to -negative ones during the observed period.

Effects of Cadmium on Root Morpho-Physiology of Durum Wheat.

Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.] can accumulate a high level of Cd in grains with a significant variability depending on cultivars. Understanding how this toxic element is distributed in cereal tissues and grains is essential to improve the nutritional quality of cereal-based products. The main objective of this work was to investigate roots of durum wheat plants (cv. Iride) exposed to different Cd concentrations (0.5 and 5.0 µM) to identify the mechanisms involved in Cd management. Results showed that the root morphology was altered by Cd treatment both at macroscopic (increased number of tips and primary root length) and ultrastructural levels (cell membrane system damaged, cell walls thickened and enriched in suberin). On the other side, Cd was localized in vesicles and in cell walls, and the metal colocalized with the phytosiderophore nicotianamine (NA). Overall, data suggest that Cd is chelated by NA and then compartmentalized, through vesicular trafficking, in the root thickened walls reducing Cd translocation to the aerial organs of the plant.

Xylella fastidiosa and Drought Stress in Olive Trees: A Complex Relationship Mediated by Soluble Sugars.

Xylella fastidiosa (Xf) subsp. pauca "De Donno" is the etiological agent of "Olive Quick Decline Syndrome" (OQDS) on olive trees (Olea europaea L.); the presence of the bacterium causes xylem vessel occlusions inducing a drought stress and the development of leaf scorch symptoms, which may be worsened by water shortage in summer. In order to evaluate how the two stress factors overlap each other, the carbohydrate content and the expression patterns of genes related to carbohydrate metabolism have been evaluated in two olive cvs trees (Cellina di Nardò, susceptible to Xf, and Leccino, resistant to Xf) reporting transcriptional dynamics elicited by Xf infection, drought, or combined stress (drought/Xf). In the Xf-susceptible Cellina di Nardò plants, Xf and its combination with drought significantly decrease total sugars compared to control (-27.0% and -25.7%, respectively). In contrast, the Xf-resistant Leccino plants show a more limited reduction in sugar content in Xf-positive conditions (-20.1%) and combined stresses (-11.1%). Furthermore, while the amount of glucose decreases significantly in stressed Cellina di Nardò plants (≈18%), an increase was observed in Leccino plants under drought/Xf combined stresses (+11.2%). An opposite behavior among cvs was also observed for sucrose, as an accumulation of the disaccharide was recorded in stressed Leccino plants (≈37%). The different response to combined stress by Xf-resistant plants was confirmed considering genes coding for the sucrose or monosaccharide transporter (OeSUT1, OeMST2), the cell wall or vacuolar invertase (OeINV-CW, OeINV-V), the granule-bound starch synthase I (OeGBSSI) and sucrose synthase (OeSUSY), with a higher expression than at least one single stress (e.g., ≈1-fold higher or more than Xf for OeMST2, OeINV-CW, OeINV-V, OeGBSSI). It is probable that the pathways involved in drought stress response induce positive effects useful for pathogen resistance in cv Leccino, confirming the importance of investigating the mechanisms of cross-talk of biotic and abiotic responses.

Detection of Ampelovirus and Nepovirus by Lab-on-a-Chip: A Promising Alternative to ELISA Test for Large Scale Health Screening of Grapevine.

The Ampelovirus Grapevine leafroll-associated virus 3 (GLRaV-3) and the Nepovirus Grapevine fanleaf virus (GFLV) are pathogens reported in many grapevine-growing areas all over the world, main causal agents of grapevine leafroll disease and grapevine fanleaf disease, respectively. Prevention of virus spread thanks to rapid diagnosis of infected plants is a key factor for control of both diseases. Although serological (e.g., enzyme-linked immunosorbent assay-ELISA test) and molecular methods are available to reveal the presence of the viruses, they turn out to be quite expensive, time-consuming and laborious, especially for large-scale health screening. Here we report the optimization of a lab-on-a-chip (LOC) for GLRaV-3 and GFLV detection, based on an electrochemical transduction and a microfluidic multichamber design for measurements in quadruplicate and simultaneous detection of both targets. The LOC detect GLRaV-3 and GFLV at dilution factors more than 15 times higher than ELISA, providing a higher sensitivity in the detection of both viruses. Furthermore, the platform offers several advantages as easy-to-use, rapid-test, portability and low costs, favoring its potential application for large-scale monitoring programs. Compared to other grapevine virus biosensors, our sensing platform is the first one to provide a dose-dependent calibration curve combined with a microfluidic module for sample analysis and a portable electronics providing an operator-independent read-out scheme.

Screening of Olive Biodiversity Defines Genotypes Potentially Resistant to Xylella fastidiosa.

The recent outbreak of the Olive Quick Decline Syndrome (OQDS), caused by Xylella fastidiosa subsp. pauca (Xf), is dramatically altering ecosystem services in the peninsula of Salento (Apulia Region, southeastern Italy). Here we report the accomplishment of several exploratory missions in the Salento area, resulting in the identification of thirty paucisymptomatic or asymptomatic plants in olive orchards severely affected by the OQDS. The genetic profiles of such putatively resistant plants (PRPs), assessed by a selection of ten simple sequence repeat (SSR) markers, were compared with those of 141 Mediterranean cultivars. Most (23) PRPs formed a genetic cluster (K1) with 22 Italian cultivars, including 'Leccino' and 'FS17', previously reported as resistant to Xf. The remaining PRPs displayed relatedness with genetically differentiated germplasm, including a cluster of Tunisian cultivars. Markedly lower colonization levels were observed in PRPs of the cluster K1 with respect to control plants. Field evaluation of four cultivars related to PRPs allowed the definition of partial resistance in the genotypes 'Frantoio' and 'Nocellara Messinese'. Some of the PRPs identified in this study might be exploited in cultivation, or as parental clones of breeding programs. In addition, our results indicate the possibility to characterize resistance to Xf in cultivars genetically related to PRPs.

Secondary Metabolites in Xylella fastidiosa-Plant Interaction.

During their evolutionary history, plants have evolved the ability to synthesize and accumulate small molecules known as secondary metabolites. These compounds are not essential in the primary cell functions but play a significant role in the plants' adaptation to environmental changes and in overcoming stress. Their high concentrations may contribute to the resistance of the plants to the bacterium Xylella fastidiosa, which has recently re-emerged as a plant pathogen of global importance. Although it is established in several areas globally and is considered one of the most dangerous plant pathogens, no cure has been developed due to the lack of effective bactericides and the difficulties in accessing the xylem vessels where the pathogen grows and produces cell aggregates and biofilm. This review highlights the role of secondary metabolites in the defense of the main economic hosts of X. fastidiosa and identifies how knowledge about biosynthetic pathways could improve our understanding of disease resistance. In addition, current developments in non-invasive techniques and strategies of combining molecular and physiological techniques are examined, in an attempt to identify new metabolic engineering options for plant defense.

Increase in ring width, vessel number and δ18O in olive trees infected with Xylella fastidiosa.

Xylella fastidiosa (Xf) Wells, Raju et al., 1986 is a bacterium that causes plant diseases in the Americas. In Europe, it was first detected on the Salento Peninsula (Italy), where it was found to be associated with the olive quick decline syndrome. Here, we present the results of the first tree-ring study of infected and uninfected olive trees (Olea europaea L.) of two different cultivars, one resistant and one susceptible, to establish the effects induced by the spread of the pathogen inside the tree. Changes in wood anatomical characteristics, such as an increase in the number of vessels and in ring width, were observed in the infected plants of both the cultivars Cellina di Nardò (susceptible to Xf infection) and Leccino (resistant to Xf infection). Thus, whether infection affects the mortality of the tree or not, the tree shows a reaction to it. The presence of occlusions was detected in the wood of both 4-year-old branches and the tree stem core. As expected, the percentage of occluded vessels in the Xf-susceptible cultivar Cellina di Nardò was significantly higher than in the Xf-resistant cultivar Leccino. The δ 18O of the 4-year-old branches was significantly higher in infected trees of both cultivars than in noninfected trees, while no variations in δ 13C were observed. This suggests a reduction in leaf transpiration rates during infection and seems to be related to the occlusions observed in rings of the 4-year-old branches. Such occlusions can determine effects at leaf level that could influence stomatal activity. On the other hand, the significant increase in the number of vessels in infected trees could be related to the tree's attempt to enhance water conductivity in response to the pathogen-induced vessel occlusions.

Biochemical Changes in Leaves of Vitis vinifera cv. Sangiovese Infected by Bois Noir Phytoplasma.

Bois noir is a disease associated with the presence of phytoplasma 'Candidatus Phytoplasma solani' belonging to the Stolbur group (subgroup 16SrXII-A), which has a heavy economic impact on grapevines. This study focused on the changes induced by phytoplasma in terms of the profile and amount of secondary metabolites synthesized in the phenylpropanoid pathway in leaves of Vitis vinifera L. red-berried cultivar Sangiovese. Metabolic alterations were assessed according to the disease progression through measurements of soluble sugars, chlorophyll, and phenolic compounds produced by plant hosts, in response to disease on symptomatic and asymptomatic Bois noir-positive plants. Significant differences were revealed in the amount of soluble sugars, chlorophyll, and accumulation/reduction of some compounds synthesized in the phenylpropanoid pathway of Bois noir-positive and negative grapevine leaves. Our results showed a marked increase in phenolic and flavonoid production and a parallel decrease in lignin content in Bois noir-positive compared to negative leaves. Interestingly, some parameters (chlorophyll a, soluble sugars, total phenolic or flavonoids content, proanthocyanidins, quercetin) differed between Bois noir-positive and negative leaves regardless of symptoms, indicating measurable biochemical changes in asymptomatic leaves. Our grapevine cultivar Sangiovese results highlighted an extensive modulation of the phenylpropanoid biosynthetic pathway as a defense mechanism activated by the host plant in response to Bois noir disease.

The Xylella fastidiosa-Resistant Olive Cultivar "Leccino" Has Stable Endophytic Microbiota during the Olive Quick Decline Syndrome (OQDS).

Xylella fastidiosa is a highly virulent pathogen that causes Olive Quick Decline Syndrome (OQDS), which is currently devastating olive plantations in the Salento region (Apulia, Southern Italy). We explored the microbiome associated with X. fastidiosa-infected (Xf-infected) and -uninfected (Xf-uninfected) olive trees in Salento, to assess the level of dysbiosis and to get first insights into the potential role of microbial endophytes in protecting the host from the disease. The resistant cultivar "Leccino" was compared to the susceptible cultivar "Cellina di Nardò", in order to identify microbial taxa and parameters potentially involved in resistance mechanisms. Metabarcoding of 16S rRNA genes and fungal ITS2 was used to characterize both total and endophytic microbiota in olive branches and leaves. "Cellina di Nardò" showed a drastic dysbiosis after X. fastidiosa infection, while "Leccino" (both infected and uninfected) maintained a similar microbiota. The genus Pseudomonas dominated all "Leccino" and Xf-uninfected "Cellina di Nardò" trees, whereas Ammoniphilus prevailed in Xf-infected "Cellina di Nardò". Diversity of microbiota in Xf-uninfected "Leccino" was higher than in Xf-uninfected "Cellina di Nardò". Several bacterial taxa specifically associated with "Leccino" showed potential interactions with X. fastidiosa. The maintenance of a healthy microbiota with higher diversity and the presence of cultivar-specific microbes might support the resistance of "Leccino" to X. fastidiosa. Such beneficial bacteria might be isolated in the future for biological treatment of the OQDS.