Bioecological Traits of Spittlebugs and Their Implications for the Epidemiology and Control of the Xylella fastidiosa Epidemic in Apulia (Southern Italy).

Spatial-temporal dynamics of spittlebug populations, together with transmission biology, are of major importance to outline the disease epidemiology of Xylella fastidiosa subsp. pauca in Apulian olive groves. The spread rate of X. fastidiosa is mainly influenced by (i) the pathogen colonization of the host plant; (ii) the acquisition of the pathogen by the vector from an infected plant, and its inoculation to healthy plants; (iii) the vector population dynamics and abundance at different spatial scales; and (iv) the dispersal of the vector. In this contribution we summarize the recent advances in research on insect vectors' traits-points ii, iii, and iv-focusing on those most relevant to X. fastidiosa epidemic in Apulia. Among the vectors' bioecological traits influencing the X. fastidiosa epidemic in olive trees, we emphasize the following: natural infectivity and transmission efficiency, phenological timing of both nymphal and adult stage, the role of seminatural vegetation as a vector reservoir in the agroecosystem and landscape, and preferential and directional dispersal capabilities. Despite the research on X. fastidiosa vectors carried out in Europe in the last decade, key uncertainties on insect vectors remain, hampering a thorough understanding of pathogen epidemiology and the development of effective and targeted management strategies. Our goal is to provide a structured and contextualized review of knowledge on X. fastidiosa vectors' key traits in the Apulian epidemic, highlighting information gaps and stimulating novel research pathways on X. fastidiosa pathosystems in Europe. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Non-Targeted Spectranomics for the Early Detection of Xylella fastidiosa Infection in Asymptomatic Olive Trees, cv. Cellina di Nardò.

Olive quick decline syndrome (OQDS) is a disease that has been seriously affecting olive trees in southern Italy since around 2009. During the disease, caused by Xylella fastidiosa subsp. pauca sequence type ST53 (Xf), the flow of water and nutrients within the trees is significantly compromised. Initially, infected trees may not show any symptoms, making early detection challenging. In this study, young artificially infected plants of the susceptible cultivar Cellina di Nardò were grown in a controlled environment and co-inoculated with additional xylem-inhabiting fungi. Asymptomatic leaves of olive plants at an early stage of infection were collected and analyzed using nuclear magnetic resonance (NMR), hyperspectral reflectance (HSR), and chemometrics. The application of a spectranomic approach contributed to shedding light on the relationship between the presence of specific hydrosoluble metabolites and the optical properties of both asymptomatic Xf-infected and non-infected olive leaves. Significant correlations between wavebands located in the range of 530-560 nm and 1380-1470 nm, and the following metabolites were found to be indicative of Xf infection: malic acid, fructose, sucrose, oleuropein derivatives, and formic acid. This information is the key to the development of HSR-based sensors capable of early detection of Xf infections in olive trees.

Xylella fastidiosa's relationships: the bacterium, the host plants, and the plant microbiome.

Xylella fastidiosa is the causal agent of important crop diseases and is transmitted by xylem-sap-feeding insects. The bacterium colonizes xylem vessels and can persist with a commensal or pathogen lifestyle in more than 500 plant species. In the past decade, reports of X. fastidiosa across the globe have dramatically increased its known occurrence. This raises important questions: How does X. fastidiosa interact with the different host plants? How does the bacterium interact with the plant immune system? How does it influence the host's microbiome? We discuss recent strain genetic typing and plant transcriptome and microbiome analyses, which have advanced our understanding of factors that are important for X. fastidiosa plant infection.

HPLC-MS/MS method applied to an untargeted metabolomics approach for the diagnosis of "olive quick decline syndrome".

Olive quick decline syndrome (OQDS) is a disorder associated with bacterial infections caused by Xylella fastidiosa subsp. pauca ST53 in olive trees. Metabolic profile changes occurring in infected olive trees are still poorly investigated, but have the potential to unravel reliable biomarkers to be exploited for early diagnosis of infections. In this study, an untargeted metabolomic method using high-performance liquid chromatography coupled to quadrupole-time-of-flight high-resolution mass spectrometry (HPLC-ESI-Q-TOF-MS) was used to detect differences in samples (leaves) from healthy (Ctrl) and infected (Xf) olive trees. Both unsupervised and supervised data analysis clearly differentiated the groups. Different metabolites have been identified as potential specific biomarkers, and their characterization strongly suggests that metabolism of flavonoids and long-chain fatty acids is perturbed in Xf samples. In particular, a decrease in the defence capabilities of the host after Xf infection is proposed because of a significant dysregulation of some metabolites belonging to flavonoid family. Moreover, oleic acid is confirmed as a putative diffusible signal factor (DSF). This study provides new insights into the host-pathogen interactions and confirms LC-HRMS-based metabolomics as a powerful approach for disease-associated biomarkers discovery in plants.

Emergence of a Plant Pathogen in Europe Associated with Multiple Intercontinental Introductions.

Pathogen introductions have led to numerous disease outbreaks in naive regions of the globe. The plant pathogen Xylella fastidiosa has been associated with various recent epidemics in Europe affecting agricultural crops, such as almond, grapevine, and olive, but also endemic species occurring in natural forest landscapes and ornamental plants. We compared whole-genome sequences of X. fastidiosa subspecies multiplex from America and strains associated with recent outbreaks in southern Europe to infer their likely origins and paths of introduction within and between the two continents. Phylogenetic analyses indicated multiple introductions of X. fastidiosa subspecies multiplex into Italy, Spain, and France, most of which emerged from a clade with limited genetic diversity with a likely origin in California, USA. The limited genetic diversity observed in X. fastidiosa subspecies multiplex strains originating from California is likely due to the clade itself being an introduction from X. fastidiosa subspecies multiplex populations in the southeastern United States, where this subspecies is most likely endemic. Despite the genetic diversity found in some areas in Europe, there was no clear evidence of recombination occurring among introduced X. fastidiosa strains in Europe. Sequence type taxonomy, based on multilocus sequence typing (MLST), was shown, at least in one case, to not lead to monophyletic clades of this pathogen; whole-genome sequence data were more informative in resolving the history of introductions than MLST data. Although additional data are necessary to carefully tease out the paths of these recent dispersal events, our results indicate that whole-genome sequence data should be considered when developing management strategies for X. fastidiosa outbreaks.IMPORTANCEXylella fastidiosa is an economically important plant-pathogenic bacterium that has emerged as a pathogen of global importance associated with a devastating epidemic in olive trees in Italy associated with X. fastidiosa subspecies pauca and other outbreaks in Europe, such as X. fastidiosa subspecies fastidiosa and X. fastidiosa subspecies multiplex in Spain and X. fastidiosa subspecies multiplex in France. We present evidence of multiple introductions of X. fastidiosa subspecies multiplex, likely from the United States, into Spain, Italy, and France. These introductions illustrate the risks associated with the commercial trade of plant material at global scales and the need to develop effective policy to limit the likelihood of pathogen pollution into naive regions. Our study demonstrates the need to utilize whole-genome sequence data to study X. fastidiosa introductions at outbreak stages, since a limited number of genetic markers does not provide sufficient phylogenetic resolution to determine dispersal paths or relationships among strains that are of biological and quarantine relevance.

Draft Genome Sequence Resources of Three Strains (TOS4, TOS5, and TOS14) of Xylella fastidiosa Infecting Different Host Plants in the Newly Discovered Outbreak in Tuscany, Italy.

An outbreak of Xylella fastidiosa was discovered in late 2018 in northern Italy affecting several plant species. Multilocus sequence typing analyses detected the presence of strains clustering in X. fastidiosa subsp. multiplex and harboring a hitherto uncharacterized sequence type, ST87. Three cultured strains (TOS4, TOS5, and TOS14) were subjected to high-throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated that they belong to the subspecies multiplex. The genetic information generated for these newly discovered strains further supports the evidence that sequence types are associated with the emergence of X. fastidiosa in Europe, posing major challenges for predicting the main threatened European and Mediterranean crops and plant species.

Draft Genome Resources of Two Strains ("ESVL" and "IVIA5901") of Xylella fastidiosa Associated with Almond Leaf Scorch Disease in Alicante, Spain.

An outbreak of Xylella fastidiosa subsp. multiplex sequence type ST6 was discovered in 2017 in mainland Spain affecting almond trees. Two cultured almond strains, "ESVL" and "IVIA5901," were subjected to high throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated they belong to the subspecies multiplex, and pairwise comparisons of the chromosomal genomes showed an average nucleotide identity higher than 99%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The availability of these draft genomes contribute to extend the European genomic sequence dataset, a first step toward setting new research to elucidate the pathway of introduction and spread of the numerous strains of this subspecies so far detected in Europe.

Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California.

Strain differentiating marker profiles of citrus tristeza virus (CTV) isolates from California have shown the presence of multiple genotypes. To better define the genetic diversity involved, full-length genome sequences from four California CTV isolates were determined by small-interfering RNA sequencing. Phylogenetic analysis and nucleotide sequence comparisons differentiated these isolates into the genotypes VT (CA-VT-AT39), T30 (CA-T30-AT4), and a new strain called S1 (CA-S1-L and CA-S1-L65). S1 isolates had three common recombination events within portions of genes from VT, T36 and RB strains and were transmissible by Aphis gossypii. Virus indexing showed that CA-VT-AT39 could be classified as a severe strain, whereas CA-T30-AT4, CA-S1-L and CA-S1-L65 were mild. CA-VT-AT39, CA-S1-L, and CA-S1-L65 reacted with monoclonal antibody MCA13, whereas CA-T30-AT4 did not. RT-PCR and RT-qPCR detection assays for the S1 strain were developed and used to screen MCA13-reactive isolates in a CTV collection from central California collected from 1968 to 2011. Forty-two isolates were found to contain the S1 strain, alone or in combinations with other genotypes. BLAST and phylogenetic analysis of the S1 p25 gene region with other extant CTV sequences from the NCBI database suggested that putative S1-like isolates might occur elsewhere (e.g., China, South Korea, Turkey, Bosnia and Croatia). This information is important for CTV evolution, detection of specific strains, and cross-protection.

Identification and Characterization of Citrus tristeza virus Isolates Breaking Resistance in Trifoliate Orange in California.

Most Citrus tristeza virus (CTV) isolates in California are biologically mild and symptomless in commercial cultivars on CTV tolerant rootstocks. However, to better define California CTV isolates showing divergent serological and genetic profiles, selected isolates were subjected to deep sequencing of small RNAs. Full-length sequences were assembled, annotated and trifoliate orange resistance-breaking (RB) isolates of CTV were identified. Phylogenetic relationships based on their full genomes placed three isolates in the RB clade: CA-RB-115, CA-RB-AT25, and CA-RB-AT35. The latter two isolates were obtained by aphid transmission from Murcott and Dekopon trees, respectively, containing CTV mixtures. The California RB isolates were further distinguished into two subclades. Group I included CA-RB-115 and CA-RB-AT25 with 99% nucleotide sequence identity with RB type strain NZRB-G90; and group II included CA-RB-AT35 with 99 and 96% sequence identity with Taiwan Pumelo/SP/T1 and HA18-9, respectively. The RB phenotype was confirmed by detecting CTV replication in graft-inoculated Poncirus trifoliata and transmission from P. trifoliata to sweet orange. The California RB isolates induced mild symptoms compared with severe isolates in greenhouse indexing tests. Further examination of 570 CTV accessions, acquired from approximately 1960 and maintained in planta at the Central California Tristeza Eradication Agency, revealed 16 RB positive isolates based on partial p65 sequences. Six isolates collected from 1992 to 2011 from Tulare and Kern counties were CA-RB-115-like; and 10 isolates collected from 1968 to 2010 from Riverside, Fresno, and Kern counties were CA-RB-AT35-like. The presence of the RB genotype is relevant because P. trifoliata and its hybrids are the most popular rootstocks in California.

Genome-Wide Analysis Provides Evidence on the Genetic Relatedness of the Emergent Xylella fastidiosa Genotype in Italy to Isolates from Central America.

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp. pauca.

Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca.

BACKGROUND: The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. RESULTS: A global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. CONCLUSIONS: Collectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

Isolation and Partial Characterization of a Novel Cytorhabdovirus from Citrus Trees Showing Foliar Symptoms in Iran.

Citrus ringspot is a graft-transmissible disease, and at least two taxonomically distinct viral species are associated with this syndrome: Citrus psorosis virus (CPsV) and Indian citrus ringspot virus (ICRSV). Neither of these two viruses was detected, however, by serological or molecular assays in symptomatic tissues from citrus trees in southern Iran, where the ringspot syndrome is widespread. By contrast, electron microscopy and molecular assays revealed the presence of a rhabdovirus-like virus, which was graft transmitted to several citrus species and mechanically to herbaceous hosts. Virus particles were bacilliform and resembled rhabdovirus nucleocapsids deprived of the lipoprotein envelope. Partial sequences of the viral nucleoprotein and RNA polymerase genes showed a distant genetic relatedness with cytorhabdoviruses. This virus appears to be a novel species, for which the name Iranian citrus ringspot-associated virus (IrCRSaV) is suggested.

Infectivity and transmission of Xylellua fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae) in Apulia, Italy.

Discovery of Xylella fastidiosa from olive trees with "Olive quick decline syndrome" in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brullé were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy.

A survey in natural olive resources exposed to high inoculum pressure indicates the presence of traits of resistance to Xylella fastidiosa in Leccino offspring.

Introduction: The epidemic spread of the harmful bacterium Xylella fastidiosa causing the "olive quick decline syndrome", decimating olive trees in southern Italy, in the region of Apulia, prompted investigations to search for olive genotypes harbouring traits of resistance. Methods: A prospecting survey was carried out to identify, in the heavily infected area of Apulia, olive genotypes bearing resistance. Given the limited genetic diversity in the commercial olive groves with few cultivars widely cultivated, surveys targeted predominantly spontaneous olive genotypes in natural and uncultivated areas. Trees, selected for the absence of symptoms, were subjected to diagnostic tests and parentage analysis to disclose their genetic background. Transcriptomic analyses were also employed to decipher the molecular pathways in resistant genotypes. Artificial inoculations were carried out to confirm the resistant phenotypes of four open-pollinated seedlings of the cultivar Leccino. Results: Among the 171 olive collected genotypes, 139 had unique simple sequence repeat (SSR) profiles, with the cultivars Leccino, Cellina di Nardò, and Ogliarola salentina being the most frequent candidate parents. Among the Leccino progeny (n. 61), 67% showed a highly resistant (HR), resistant (R), or tolerant (T) phenotype to infection by X. fastidiosa. The occurrence of such phenotypes among those deriving from Cellina di Nardò and Ogliarola salentina was 32% and 49%, respectively. Analyses of the transcriptomic profiles of three Leccino-bearing genotypes, naturally infected and not showing symptoms, unravelled that a total of 17,227, 13,031, and 4,513 genes were found altered in the expression, including genes involved in photosynthesis, cell wall, or primary and secondary metabolism. Discussion: Indeed, transcriptomic analyses showed that one of these genotypes (S105) was more resilient to changes induced by the natural bacterial infection than the remaining two (S215 and S234). This study consolidates the evidence on the presence and heritage of resistance traits associated with the cv. Leccino. Moreover, valuable insights were gathered when analysing their transcriptomic profiles, i.e., genes involved in mechanisms of response to the bacterium, which can be used in functional genetic approaches to introduce resistance in susceptible cultivars and initiate strategies in olive-breeding programs through marker-assisted selection.

A model for predicting the phenology of Philaenus spumarius.

The design and implementation of Philaenus spumarius control strategies can take advantage of properly calibrated models describing and predicting the phenology of vector populations in agroecosystems. We developed a temperature-driven physiological-based model based on the system of Kolmogorov partial differential equations to predict the phenological dynamics of P. spumarius. The model considers the initial physiological age distribution of eggs, the diapause termination process, and the development rate functions of post-diapausing eggs and nymphal stages, estimated from data collected in laboratory experiments and field surveys in Italy. The temperature threshold and cumulative degree days for egg diapause termination were estimated as 6.5 °C and 120 DD, respectively. Preimaginal development rate functions exhibited lower thresholds ranging between 2.1 and 5.0 °C, optimal temperatures between 26.6 and 28.3 °C, and upper threshold between 33.0 and 35 °C. The model correctly simulates the emergence of the 3rd, 4th, and 5th nymphal instars, key stages to target monitoring actions and control measures against P. spumarius. Precision in simulating the phenology of the 1st and 2nd nymphal stages was less satisfactory. The model is a useful rational decision tool to support scheduling monitoring and control actions against the late and most important nymphal stages of P. spumarius.

Rapid differentiation of citrus Hop stunt viroid variants by real-time RT-PCR and high resolution melting analysis.

The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ by five to six nucleotides located within the variable (V) domain referred to as the "cachexia expression motif". Sensitive hosts such as mandarin and its hybrids are seriously affected by cachexia disease. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson's Special mandarin and/or direct nucleotide sequence analysis of amplicons from RT-PCR of HSVd-infected plants. Two independent high throughput assays to segregate HSVd variants by real-time RT-PCR and High-Resolution Melting Temperature (HRM) analysis were developed: one based on EVAGreen dye; the other based on TaqMan probes. Primers for both assays targeted three differentiating nucleotides in the V domain which separated HSVd variants into three clusters by distinct melting temperatures with a confidence level higher than 98%. The accuracy of the HRM assays were validated by nucleotide sequencing of representative samples within each HRM cluster and by testing 45 HSVd-infected field trees from California, Italy, Spain, Syria and Turkey. To our knowledge, this is the first report of a rapid and sensitive approach to detect and differentiate HSVd variants associated with different biological behaviors. Although, HSVd is found in several crops including citrus, cachexia variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid diagnosis for cachexia and non-cachexia variants is, thus, important for the management of HSVd in citrus and reduces the need for bioindexing and sequencing analysis.

Exploring the xylem-sap to unravel biological features of Xylella fastidiosa subspecies pauca ST53 in immune, resistant and susceptible crop species through metabolomics and in vitro studies.

Xylella fastidiosa subsp. pauca ST53 (Xfp) is a pathogenic bacterium causing one of the most severe plant diseases currently threatening the olive-growing areas of the Mediterranean, the Olive Quick Decline Syndrome (OQDS). The majority of the olive cultivars upon infections more or less rapidly develop severe desiccation phenomena, while few are resistant (e.g. Leccino and FS17), being less impacted by the infections. The present study contributes to elucidating the basis of the resistance phenomenon by investigating the influence of the composition of the xylem sap of plant species on the rate of bacterial multiplication. Xylem saps from Xfp host and non-host species were used for growing the bacterium in vitro, monitoring bacterial growth, biofilm formation, and the expression of specific genes. Moreover, species-specific metabolites, such as mannitol, quinic acid, tartaric acid, and choline were identified by non-targeted NMR-based metabolomic analysis in olive, grapevine, and citrus. In general, the xylem saps of immune species, including grapevine and citrus, were richer in amino acids, organic acids, and glucose. The results showed greater bacterial growth in the olive cultivar notoriously susceptible to Xfp (Cellina di Nardò), compared to that recorded in the resistant cultivar Leccino. Conversely, higher biofilm formation occurred in Leccino compared to Cellina di Nardò. Using the xylem saps of two Xfp-immune species (citrus and grapevine), a divergent bacterial behavior was recorded: low planktonic growth and biofilm production were detected in citrus compared to the grapevine. A parallel evaluation of the expression of 15 genes showed that Xfp directs its molecular functions mainly to virulence. Overall, the results gained through this multidisciplinary study contribute to extending the knowledge on the host-pathogen interaction, while confirming that the host response and resistance mechanism have a multifactorial basis, most likely with a cumulative effect on the phenotype.

Nicotiana benthamiana as a model plant host for Xylella fastidiosa: Control of infections by transient expression and endotherapy with a bifunctional peptide.

Transient expression of genes encoding peptides BP134 and BP178 by means of a Potato virus X (PVX) based-vector system, and treatment with synthetic peptides by endotherapy, were evaluated in the control of Xylella fastidiosa infections, in the model plant Nicotiana benthamiana. Transient production of BP178 significantly decreased disease severity compared to PVX and non-treated control (NTC) plants, without adverse effects. Plants treated with synthetic BP134 and BP178 showed consistently lower levels of disease than NTC plants. However, the coinfection with PVX-BP134 and X. fastidiosa caused detrimental effects resulting in plant death. The levels of X. fastidiosa in three zones sampled, upwards and downwards of the inoculation/treatment point, significantly decreased compared to the NTC plants, after the treatment with BP178, but not when BP178 was produced transiently. The effect of treatment and transient production of BP178 in the induction of defense-related genes was also studied. Synthetic BP178 applied by endotherapy induced the expression of ERF1, PR1a, PAL, PALII and WRKY25, while the transient expression of BP178 overexpressed the Cath, Cyc, PR4a, 9-LOX and Endochitinase B genes. Both treatments upregulated the expression of PR1, PR3, PR4 and CycT9299 genes compared to the NTC or PVX plants. It was concluded that the effect of BP178, either by endotherapy or by transient expression, on the control of the X. fastidiosa infections in N. benthamiana, was due in part to the induction of the plant defense system in addition to its bactericidal activity reported in previous studies. However, the protection observed when BP178 was transiently produced seems mainly mediated by the induction of plant defense, because the levels of X. fastidiosa were not significantly affected.

Susceptible and resistant olive cultivars show differential physiological response to Xylella fastidiosa infections.

Olive quick decline syndrome (OQDS) is a severe disease, first described in Italy in late 2013, caused by strains of Xylella fastidiosa subsp. pauca (Xfp) in susceptible olive cultivars. Conversely, resistant olive cultivars do not develop OQDS but present scattered branch dieback, which generally does not evolve to severe canopy decline. In the present study, we assessed the physiological responses of Xfp-infected olive trees of susceptible and resistant cultivars. Periodic measurements of stomatal conductance (gs) and stem water potential (Ψstem) were performed using a set of healthy and Xfp-infected plants of the susceptible "Cellina di Nardò" and resistant "Leccino" and "FS17" cultivars. Strong differences in Δgs and ΔΨstem among Xfp-infected trees of these cultivars were found, with higher values in Cellina di Nardò than in Leccino and FS17, while no differences were found among healthy plants of the different cultivars. Both resistant olive cultivars showed lower water stress upon Xfp infections, compared to the susceptible one, suggesting that measurements of gs and Ψstem may represent discriminating parameters to be exploited in screening programs of olive genotypes for resistance to X. fastidiosa.