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Microbial competition within plant tissues affects invading pathogens' fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce's disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography-mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

Xylella fastidiosa is one of the most dangerous plant‐pathogenic bacteria globally, affecting various host species. Plum leaf scald (PLS) is the primary disease affecting plum trees in Brazil, with plant eradication efforts significantly increasing in recent years. This study aims to elucidate the disease dynamics and its vector in three monitored orchards from 2010 to 2015 and associate the pattern of pathogen dispersal with mechanical pruning. The incidence was lower in Porto Amazonas, starting at 0% and reaching 14% after 5 years. In contrast, the orchards in Araucaria and Palmeira showed an initial incidence around 5% in the first year, exceeding 80% by the end of the study. Sharpshooter leafhoppers, the vectors, were collected at ground and plant canopy level during the last 3 years. In 2013, more leafhoppers were found near the ground, but their numbers equalized across heights in the following years. Kernel smoothing revealed spatial dependence, and an autologistic model indicated an aggregated infection pattern with increased PLS risk for neighbouring plants. The border effect at the epidemic's onset was linked to pathogen entry via external vectors. The disease's progression along rows was attributed to effective mechanical pathogen transmission through pruning. Therefore, in regions with several summer prunings, it is recommended to sanitize pruning shears to prevent pathogen transmission. Constant monitoring and control of the vector are essential for effective disease management. [ABSTRACT FROM AUTHOR]

Background: In the context of pathogen surveillance, it is crucial to ensure interoperability and harmonized data. Several surveillance systems are designed to compare bacteria and identify outbreak clusters based on core genome MultiLocus Sequence Typing (cgMLST). Among the different approaches available to generate bacterial cgMLST, our research used an assembly-based approach (chewBBACA tool). Methods: Simulations of short-read sequencing were conducted for 5 genomes of 27 pathogens of interest in animal, plant, and human health to evaluate the repeatability and reproducibility of cgMLST. Various quality parameters, such as read quality and depth of sequencing were applied, and several read simulations and genome assemblies were repeated using three tools: SPAdes, Unicycler and Shovill. In vitro sequencing were also used to evaluate assembly impact on cgMLST results, for six bacterial species: Bacillus thuringiensis, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, Vibrio parahaemolyticus and Xylella fastidiosa. Results: The results highlighted variability in cgMLST, which not only related to the assembly tools, but also induced by the intrinsic composition of the genomes themselves. This variability observed in simulated sequencing was further validated with real data for six of the bacterial pathogens studied. Conclusion: This highlights that the intrinsic genome composition affects assembly and resulting cgMLST profiles, and that variability in bioinformatics tools can induce a bias in cgMLST profiles. In conclusion, we propose that the completeness of cgMLST schemes should be considered when clustering strains. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a xylem‐limited bacterial plant pathogen transmitted by insect vectors. It infects a wide range of plant species and causes devastating diseases. Botanical gardens are global repositories of plant diversity exposed to local biotic and abiotic stresses. We used molecular diagnostic tools for the detection of X. fastidiosa in a collection of New Zealand indigenous plants grown in a X. fastidiosa‐infected Californian area to determine if any species were infected with this bacterial pathogen and to help inform biosecurity responses. To this end, 130 New Zealand indigenous plant species comprising 72 genera, growing in the University of California Botanical Garden at Berkeley, California, were screened for X. fastidiosa. Multiple PCR‐based methods were used to detect the pathogen at the subspecies and sequence‐type levels directly from plant material; nine plant species tested positive by at least two PCR‐based methods. All nine infections were identified as X. fastidiosa subsp. multiplex sequence types 6 or 7 by the amplification of seven housekeeping genes. Three strains were cultured in vitro and their whole genome sequences were obtained; these strains belonged to three distinct clades within subspecies multiplex, indicating that the infections were not transmitted among these New Zealand indigenous plant species. The information gathered in this study will help to assess the risk of X. fastidiosa to New Zealand indigenous flora and the potential spillover to crops of agricultural importance. The work also shows the applied value of botanical garden collections as sentinels for international plant pathogen biosecurity risk assessment. [ABSTRACT FROM AUTHOR]

Spittlebugs and froghoppers (Hemiptera: Cercopoidea) are insects feeding on xylem, which potentially can cause significant economic damage worldwide by transmitting plant pathogenic bacteria such as Xylella fastidiosa. Australia and New Zealand are currently free from X. fastidiosa, but they are home to at least 45 native spittlebug species. Among these, the Australian natives Bathyllus albicinctus (Erichson, 1842) and Philagra parva (Donovan, 1805) are particularly widespread and can be found across southern and eastern Australia, with B. albicinctus also in New Zealand. The potential that both species might be capable of vectoring Xylella fastidiosa poses a substantial biosecurity risk if the bacterium were to invade these regions. In this study, we examined 87 spittlebug nymphs collected across 12 different host plant species, in five locations in Victoria, Australia. Our objective was to explore the factors influencing bacterial communities within and between these widespread spittlebug species, considering geographic location, insect phylogenetics, and host plant associations. We employed COI barcoding to assess insect genetic variation and 16S high throughput sequencing (HTS) metabarcoding to analyse bacterial microbiome diversity across various host plants. Our findings revealed minimal genetic divergence among spittlebug individuals in the same species, highlighting conspecificity despite conspicuous morphological divergences. On the other hand, we recorded significant variation in bacterial communities harboured by Bathyllus albicinctus nymphs feeding on different plants, even when these were collected within close proximity to each other. Therefore, host plant association appeared to shape the bacterial communities of spittlebugs more than insect genetic divergence or geographical location. These diverse bacterial communities could potentially facilitate transmission of plant pathogenic bacteria, underscoring the risk of widespread transmission among numerous plant hosts through insect-plant interactions. This study emphasizes the critical need to understand these complex interactions, particularly in the context of biosecurity. [ABSTRACT FROM AUTHOR]

Global change is believed to be a major driver of the emergence of invasive pathogens. Yet, there are few documented examples that illustrate the processes that hinder or trigger their geographic spread. Here, we present phylogenetic, epidemiological and historical evidence to explain how European vineyards escaped Xylella fastidiosa (Xf), the vector-borne bacterium responsible for Pierce's disease (PD). Using Bayesian temporal reconstruction, we show that the export of American grapevines to France as rootstocks to combat phylloxera (~1872–1895) preceded the spread of the Xf grapevine lineage in the USA. We found that the time of the most recent common ancestor in California dates to around 1875, which agrees with the emergence of the first PD outbreak and the expansion into the southeastern US around 1895. We also show that between 1870 and 1990, climatic conditions in continental Europe were mostly below the threshold for the development of PD epidemics. However, our model indicates an inadvertent expansion of risk in southern Europe since the 1990s, which is accelerating with global warming. Our temporal approach identifies the biogeographical conditions that have so far prevented PD in southern European wine-producing areas and predicts that disease risk will increase substantially with increasing temperatures. [ABSTRACT FROM AUTHOR]

This study investigated the phenology and population dynamics of potential insect vectors of Xylella fastidiosa Wells et al. and other Auchenorrhyncha species in olive and citrus groves of Chania province, Crete, Greece. Although X. fastidiosa has not been reported in Greece, its introduction could cause serious diseases in many crops, including olives and citrus. Olive groves of Olea europaea L. 'Koroneiki' were sampled systematically using sweep net and Malaise traps over 24 months. One citrus grove was sampled for one year using a Malaise trap. Sweep net samples were taken from the herbaceous cover, tree canopy, and field borders of olive groves. Auchenorrhyncha were more abundant on the herbaceous cover compared to the canopy and field margins. Aphrophoridae species were mostly found on the herbaceous cover and in low numbers during fall (October–December) and spring (April–May). Cicadellidae species, such as Euscelis spp., were frequently found on the herbaceous cover of both olive and citrus groves. One Aphrophoridae and several Cicadellidae species were recorded in the citrus grove. Altitude was found to influence the population abundance of some Auchenorrhyncha species in olive groves. These results provide information for effective integrated management of insect vectors and their vector-borne pathogens. [ABSTRACT FROM AUTHOR]

Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), causes significant losses in citrus crops. An efficient pathogen detection system is critical for epidemiology studies, particularly when a large sample size is involved. In this study, we report the development of an immunomolecular assay, immunocapture real-time polymerase chain reaction (IC-qPCR), targeting the spiralin gene for direct detection of S. citri without DNA isolation. This method can use either plant sample extracts or media in which S. citri was cultivated. The IC-qPCR protocol demonstrated a limit of detection for pure S. citri culture at a Ct value of 36.523 with a 103-fold dilution factor, making it equally sensitive as qPCR, which exhibited signal disappearance at a 10–3 dilution (Ct value of 37.484). In contrast, the immunological double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test produced positive results up to a 10–2 dilution only. For S. citri-infected citrus samples, the established IC-qPCR protocol had a limit of detection at 36.46 Ct with a 1/64-fold dilution factor, matching the sensitivity of qPCR, where signal disappearance occurred at a 1/64 dilution (Ct value of 37.21). On the other hand, the immunological DAS-ELISA test yielded positive results only up to a 1/16 dilution, with optical density (OD) values of 0.364 and 0.113 for 1/16 and 1/32 dilutions, respectively. The IC-qPCR assay shows no cross-reaction for any other highly related spiroplasma species and bacteria affecting citrus trees including Candidatus liberibacter, Xylella fastidiosa, and Xanthomonas campestris pv. citri. Therefore, IC-qPCR assay provides an alternative quick and very sensitive method to screening S. citri, with the advantage of not requiring any concentration or DNA purification steps while still allowing an accurate diagnosis of CSD. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa subsp. pauca (Xfp) is a significant threat to Mediterranean agriculture, particularly impacting olive trees in southern Italy, causing Olive Quick Decline Syndrome. Resistant olive cultivars, such as 'Leccino' and 'Fs-17', have been identified as alternatives to restore the oliviculture within the infected areas. 'Frantoio' and 'Cipressino' are included in ongoing studies on genetic resistance to Xfp. The mechanization of olive harvesting is essential for reducing production costs in the olive oil sector. Two systems, trunk shakers and over-the-row machines, are used depending on the tree density and canopy structure, with super-high-density systems offering advantages in terms of cost and efficiency. This study investigates the feasibility of using simple and non-destructive indices to assess the optimal mechanical harvesting time. Different physical ripening indices, including detachment force, fresh weight, pigmentation, and firmness, were measured on four olive cultivars ('Fs-17', 'Leccino', 'Frantoio', 'Cipressino') in southern Italy over two years. The study found that the pigmentation index had a strong relationship with the detachment index, particularly for 'Fs-17', and 'Leccino', providing a reliable non-destructive measure for optimal harvesting time. The results indicate that the optimal harvesting times for mechanical harvesting are early September for 'Cipressino', early October for 'Fs-17', and mid-October for 'Frantoio' and 'Leccino'. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa (Xf), a gram-negative bacterium, is a notorious, world-wide plant pathogen with an extended latent period that presents a challenge for early disease detection and control interventions. We used thermal imaging of tissue-cultured, experimentally Xf-infected blueberry plants to identify visually pre-symptomatic leaves and compared the minimum force required to dislodge symptomatic leaves from infected plants to leaves on uninfected (control) blueberry plants. For two different blueberry cultivars and one pathogenic isolate of X. fastidiosa, we found no statistical difference between the mean downward force for leaf dislodgement, regardless of symptom category, on Xf-infected blueberry plants. That force was about 50% to 30% of the mean force to remove leaves from uninfected blueberry plants depending on the cultivar. These results indicate that visually pre-symptomatic leaves may be just as readily lost under field conditions as visually symptomatic leaves, both of which are important for early disease detection. Second, some thermally symptomatic and visually symptomatic leaves appeared to self-prune (abscise) and this may be an unrecognized early symptom of Xf-caused disease in blueberries. Last, it is possible that the self-pruning of visually asymptomatic leaves may occur in other agriculturally and culturally important plants infected by X. fastidiosa, but this remains an unrecognized early disease symptom. [ABSTRACT FROM AUTHOR]

Pistachio (Pistacia vera L.) is a dioecious, anemophilous, and drought-resistant fruit tree species. It is cultivated in new Mediterranean areas, including the regions of southern Italy (Apulia and Basilicata). It has been estimated that over 40,000 ha are suitable for pistachio cultivation in areas infected by Xylella fastidiosa subsp. pauca. As a newly introduced species, knowledge of its biological reproductive behaviors in its new areas of spreading is essential for appropriate agronomic planning and management. This two-year study (2022 and 2023), carried out in the countryside of Stigliano (MT, Italy), had the objective of evaluating the flowering phenograms, pollen production, and assessing protocols for the conservation and extension of pollen viability, of the most widespread cultivars. A slight delay was observed in the blooming phenograms, compared to other cultivation Mediterranean areas, such as Spain or Sicily. Furthermore, the overlap between female and male phenograms was partial. No significant differences were observed in the polliniferous aptitude of the two male cultivars. Among the different protocols tested, the pollen storage at 33% relative humidity and a temperature of −80 °C maintained the pollen germinability above 50% for up to three weeks. These findings highlight the importance of controlled environmental conditions in preserving pollen viability over extended periods, providing valuable insights for agricultural and botanical research that relies on maintaining pollen viability for breeding and genetic studies. [ABSTRACT FROM AUTHOR]

Site selection analysis is a fundamental methodology for the regeneration of Xylella fastidiosa subsp. pauca (Xfp) infected areas, with the introduction of Xfp immune/resistant tree crop species. The diffusion of these species could be assessed by combining ecological optima data, climate and soil attributes of the study area, and GIS tools. The study aimed to evaluate the potential suitability of eight Xfp immune tree crops, including Neglected and Underutilized Species (NUS) drought-resistant and new species, as follows: carob, hawthorn, prickly pear, mulberry, loquat, walnut, persimmon, and avocado. The use of GIS tools allowed the integration of different layers, such as climate and soil, to contribute to the identification of suitable areas for the cultivation of these tree crops helping the policy-makers to define plans for land use at a regional scale. Following the ecological optima, which represents the ideal environmental conditions for each species, this analysis provided valuable insights into the compatibility of the selected tree crops with the prevailing environmental factors in the affected area. Carob revealed its remarkable adaptability and drought resistance, presenting the broadest suitability. Hawthorn and Loquat also exhibited high adaptability, indicating their potential contribution to agricultural diversification and ecological balance. Conversely, crops like Avocado, Prickly pear, and Walnut, despite their economic value, demonstrated limited adaptability due to their specific soil and climate requirements. These findings can potentially contribute to the development of strategies for the policymakers, aimed at diversifying and enhancing the resilience of agricultural systems, facing the problem of emerging quarantine pathogens and the incoming climate change, and highlighting the possibility of opening new cultivation scenarios in the zones affected by Xfp. [ABSTRACT FROM AUTHOR]

This scientometric study reviews the scientific literature and CABI distribution records published in 2022 to find evidence of major disease outbreaks and first reports of pathogens in new locations or on new hosts. This is the second time we have done this, and this study builds on our work documenting and analyzing reports from 2021. Pathogens with three or more articles identified in 2022 literature were Xylella fastidiosa, Bursaphelenchus xylophilus, Meloidogyne species complexes, 'Candidatus Liberibacter asiaticus', Raffaelea lauricola, Fusarium oxysporum formae specialis, and Puccinia graminis f. sp. tritici. Our review of CABI distribution records found 29 pathogens with confirmed first reports in 2022. Pathogens with four or more first reports were Meloidogyne species complexes, Pantoea ananatis, grapevine red globe virus, and Thekopsora minima. Analysis of the proportion of new distribution records from 2022 indicated that grapevine red globe virus, sweet potato chlorotic stunt virus, and 'Ca. Phytoplasma vitis' may have been actively spreading. As we saw last year, there was little overlap between the pathogens identified by reviewing scientific literature versus distribution records. We hypothesize that this lack of concordance is because of the unavoidable lag between first reports of the type reported in the CABI database of a pathogen in a new location and any subsequent major disease outbreaks being reported in the scientific literature, particularly because the latter depends on the journal policy on types of papers to be considered, whether the affected crop is major or minor, and whether the pathogen is of current scientific interest. Strikingly, too, there was also no overlap between species assessed to be actively spreading in this year's study and those identified last year. We hypothesize that this is because of inconsistencies in sampling coverage and effort over time and delays between the first arrival of a pathogen in a new location and its first report, particularly for certain classes of pathogens causing only minor or non-economically damaging symptoms, which may have been endemic for some time before being reported. In general, introduction of new pathogens and outbreaks of extant pathogens threaten food security and ecosystem services. Continued monitoring of these threats is essential to support phytosanitary measures intended to prevent pathogen introductions and management of threats within a country. [ABSTRACT FROM AUTHOR]

Olive Quick Decline Syndrome (OQDS) associated with Xylella fastidiosa subsp. pauca is one of the most destructive diseases of olive trees in the Salento Peninsula (Southern Italy), particularly on the cultivars Cellina di Nardò and Ogliarola Salentina. This study proposes the NuovOlivo protocol as a management strategy to permit coexistence between X. fastidiosa subsp. pauca and olive drupes and extra-virgin oil production. Thirty-two private olive orchards affected by OQDS and cultivated following the standard agronomic techniques in use in the area were surveyed during the 2019–2023 olive-growing seasons. Tested cultivars included Cellina di Nardò, Ogliarola Salentina, Coratina, Ascolana Tenera, Nociara, Leccino, and Bella di Cerignola. At the beginning of the protocol application, the susceptible plants showed OQDS symptom severity of 40–80% and did not produce olives or oil, while the resistant(?)/tolerant cultivars exhibited a 2–8% leaf scorch and a drupe production less than 1–2 kg/plant. After the removal of dry branches in January–February, plants were sprayed two times per year (preferably in March and October) with NuovOlivo®, a mixture of aqueous botanical extracts esterified in the presence of sodium hydroxide with vegetable oils and activated at the time of use with sodium bicarbonate. In all the orchards, a slow-release fertilizer was distributed, and weeds were controlled by mowing or chopping. Upon eventual appearance, the dry twigs were removed. Treated olive trees produced new vegetation, rebuilt their foliage, reduced OQDS symptoms, and turned out cluster inflorescence and drupes. The drupes yield was 6.67–51.36 kg per plant, with an average of 13.19% in extra-virgin olive oil (free acidity 0.01–0.2%). Plants used as controls showed OQDS symptoms and were unproductive, and newly formed shoots were desiccated. The proposed protocol promotes, supports, and restores new vegetation, flowers, fruits, and oil production of the treated olive plants affected by OQDS without losing susceptible olive plants. The Apulian landscape and economy, based on olive presence and production, could be also safeguarded. [ABSTRACT FROM AUTHOR]

The initial outbreak of Xylella fastidiosa subsp. pauca (Xfp) on olive groves in Salento (Apulia, Italy) dates back to the years 2008 and 2009 when extensive twig and branch diebacks were observed in the area of Gallipoli area (province of Lecce). Subsequently, the bacterium also spread northwards to other areas of Apulia. In many cases, entire olive groves, also including the centennial ones, died. After the crown collapse, in many cases, it has been observed that the suckers are resprouting at the base of the trunk. After two to three years, such suckers usually died as well. However, during the last four to five years, in the first Xfp outbreak area, a complete restoration of the crown of the Xfp-susceptible cultivars Ogliarola salentina and Cellina di Nardò has been noticed. Such trees or olive groves also started to yield again. To monitor this tree resilience phenomenon, together with local non-profit organizations, a survey in the province of Lecce has been carried out to find olive groves for which any curative or agronomical practices have been applied since the bacterium outbreak. Resilient olive groves are scattered in many municipalities all over the province of Lecce. The phenomenon regards both young and adult olive groves and also includes some centennial trees. In many cases, the trees are yielding fruits, and farmers started to cultivate them again. Olive resilience in Salento is already being studied and can represent a significant opportunity to restore the local and valuable olive germplasm. [ABSTRACT FROM AUTHOR]

Simple Summary: In our research conducted in seven citrus orchards in Amazonas State, Brazil, we recorded the presence of eight sharpshooter species. One of the identified species is known to transmit the bacterium Xylella fastidiosa, which is responsible for causing citrus variegated chlorosis (CVC). Among the eight species found, five were newly recorded in citrus plants within the State, and three were entirely new to the northern region. This research emphasizes the importance of mapping the locations of these insects to gain insights into their behavior. Doing so will enable the development of strategies to effectively manage the threat of CVC in local citrus crops. This research study reports new records of eight species of Cicadellini (Hemiptera, Cicadellidae, Cicadellinae) across seven municipalities in Amazonas State, Brazil. Among these species, one is known as a vector of CVC (Provancherana corniculata). Additionally, five species are being reported for the first time in citrus for the State (Erythrogonia sexguttata, Hortensia similis, Provancherana corniculata, Scopogonalia amazonensis, and Scoposcartula oculata), and three species (Diedrocephala variegata, Macugonalia moesta and Xyphon reticulatum) are reported for the first time in the northern region. This research highlights the lack of information on sharpshooter occurrence in the Amazonas region, emphasizing the need for further investigations in this area. [ABSTRACT FROM AUTHOR]

The detection of Xylella fastidiosa in Portugal occurred through prospection in 2019 in Vila Nova de Gaia, Porto, in plants of Lavandula dentata L. Currently, in Portugal, there are 18 Xylella fastidiosa Demarcated Zones (DZs). The main objective of this study is to gain a comprehensive understanding of this problem within the defined Demarcated Zones in the North Region of Portugal from 2019 to June 2023. This work comprised two phases: the prospection phase (inspecting plants, sampling, and submission of samples to the laboratory) and the research phase (collecting and organizing data and statistical treatment). Our findings provide essential insights, suggesting that the Northern Region of Portugal is highly conducive to Xylella fastidiosa. Portugal has Xf-preferred hosts such as the olive tree, orange tree, Laurus nobilis, Rosa spp., Nerium oleander L., Pelargonium sp., Hedera helix L., and Lavandula dentata L. Portugal's favorable environmental factors such as temperature and humidity can have an important role in influencing the interaction between bacteria and hosts. Nevertheless, it is crucial to consider variations in the behavior of insect vectors, as these variations can limit the prevalence of the disease. Activities like the transport of infected planting materials from the first Demarcated Zone of the Area Metropolitana do Porto could be associated with the introduction and spread of Xylella fastidiosa, potentially triggering new disease outbreaks in the country. Our findings confirm the alarming spread of Xylella fastidiosa across Portugal. Factors such as the presence of insect vectors, abundance of host plants, and climate suitability play pivotal roles in Xylella fastidiosa dispersal. We recommend that countries identified with relatively high risk, like Portugal, undertake thorough individual risk analyses. The implementation of preventive measures and, if necessary, the enhancement of surveillance systems for early detection of Xylella fastidiosa in plants and insect vectors are crucial steps. [ABSTRACT FROM AUTHOR]

Abundance on and access time to the host plant are the pivotal factors in Xylella fastidiosa transmission to olive by the meadow spittlebug Philaenus spumarius. Therefore, olive varieties suitable for the vectors, i.e., plants providing all the necessary cues to the insect for their location, settling and acceptance, and devoid of antixenotic defenses, could be more susceptible to infection than varieties non- or less suitable for the vector. Here we evaluated whether a bacterium-susceptible olive variety, Ogliarola Salentina, could be a more suitable host for P. spumarius than the two resistant varieties Leccino and FS-17. We carried out: (i) an evaluation of between-hosts and within-host preference; (ii) an insect survival analysis; (iii) an Electrical Penetration Graph (EPG)-assisted analysis of the probing behavior; (iv) light microscopy of the tissues the spittlebugs had access to; (v) an analysis of the xylem sap primary metabolites. In choice tests, the insect exhibited a significant preference for Ogliarola Salentina. In addition, spittlebugs displayed longer xylem sap ingestion bouts on the bacterium-susceptible variety compared to resistant genotypes, possibly because of differences in the xylem sap chemical profile rather than xylem anatomy. Spittlebugs preference for Ogliarola over both Leccino and FS-17 could be a relevant and so far overlooked component of the low disease prevalence in these two olive varieties reported in Southern Italian olive orchards. Overall, our data point toward the importance of incorporating studies on vector-plant interaction and host traits of resistance to the vector in research on genotypes resistant to X. fastidiosa. [ABSTRACT FROM AUTHOR]

BACKGROUND: Xylella fastidiosa is an important causative agent of Olive Quick Decline Syndrome in the Apulia region of Italy. The current study evaluated the bioefficacy of three entomopathogenic fungal strains: Beauveria bassiana SGB7004, Metarhizium robertsii SGB1K, and Akanthomyces lecanii SGB4711 against Philaenus spumarius the main vector of this pathogen, under laboratory conditions. Pathogenicity bioassays were performed by dipping nymphs and adults of P. spumarius in an aqueous suspension of powdered fungal culture (PFC) or conidial suspension (CS) of the three fungal strains. RESULTS: Both B. bassiana SGB7004 and M. robertsii SGB1K affected the viability of nymphs, resulting in more than 80% mortality at 48 h post treatment, while the effect of A. lecanii SGB4711 was not statistically significant. On adults, all three biocontrol strains were effective in a time‐ and concentration‐dependent manner. The PFCs of B. bassiana SGB7004, M. robertsii SGB1K, and A. lecanii SGB4711 at the highest concentration tested (120 mg mL−1) resulted in 97%, 83% and 27% mortality at the trial endpoint (120 h), respectively. Mycelial growth was observed on 38.5%, 37.0% and 61.5% of dead insects treated with B. bassiana SGB7004 (2.3 × 108 CFU mL−1), M. robertsii SGB1K (3.8 × 106 CFU mL−1) and A. lecanii SGB4711 (5.4 × 108 CFU mL−1), respectively. None of the PFCs of the tested strains was pathogenic when injected into nymph spittle. CONCLUSIONS: Beauveria bassiana SGB7004 and M. robertsii SGB1K significantly affected the survival of P. spumarius nymphs and adults, while A. lecanii SGB4711 was not effective on nymphs and only slightly effective against adults. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Xylella fastidiosa is a multi‐host bacterium that can be detected in hundreds of plant species including several crops. Diseases caused by X. fastidiosa are considered a threat to global food production. The primary method for managing diseases caused by X. fastidiosa involves using insecticides to control the vector. Hence, it is necessary to adopt new and sustainable disease management technologies to control not only the insect but also the bacteria and plant health. We demonstrated that N‐acetylcysteine (NAC), a low‐cost cysteine analogue, is a sustainable molecule that can be used in agriculture to decrease the damage caused by X. fastidiosa and improve plant health. RESULTS: Using 15N‐NAC we proved that this analogue was absorbed by the roots and transported to different parts of the plant. Inside the plant, NAC reduced the bacterial population by 60‐fold and the number of xylem vessels blocked by bacterial biofilms. This reflected in a recovery of 0.28‐fold of the daily sap flow compared to health plants. In addition, NAC‐treated citrus variegated chlorosis (CVC) plants decreased the oxidative stress by improving the activity of detoxifying enzymes. Moreover, the use of NAC in field conditions positively contributed to the increase in fruit yield of CVC‐diseased plants. CONCLUSION: Our research not only advances the understanding of NAC absorption in plants, but also indicates its dual effect as an antimicrobial and antioxidant molecule. This, in turn, negatively affects bacterial survival while improving plant health by decreasing oxidative stress. Overall, the positive field‐based evidence supports the viability of NAC as a sustainable agricultural application. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a destructive bacterial plant pathogen affecting hundreds of plant families, including important crops such as olive and citrus trees and vines. The pathogenicity of this bacterium rests in a group of genes that encode toxins, enzymes, fimbrial/afimbrial adhesins, cell–cell aggregation proteins and other virulence factors. We investigated the contribution of mobile genetic elements (MGEs) to the pathogenicity of X. fastidiosa. Using computational tools, we analysed 94 X. fastidiosa genomes corresponding to the three most important subspecies to identify insertion sequences (ISs) and prophages. Results indicate that the distribution and diversity of ISs are modest, with a few families widespread throughout the subspecies of X. fastidiosa. The prophages are abundant, and play a more significant role in the pathogenicity of X. fastidiosa. Prophages are common in the subspecies of X. fastidiosa and contribute to numerous genes related to pathogenicity. The phylogenetic analyses suggest that prophages are recently acquired and that the rearranging of pathogenicity genes triggered by them could generate strains with new pathogenic capacities. Finally, the contribution of MGEs to the pathogenesis of X. fastidiosa was evidenced by determining their association with virulence genes using a Bayesian framework for phylogenetic analysis of character evolution. [ABSTRACT FROM AUTHOR]

Since recently, a decline of holm oak (Quercus ilex L.) is observed in Salento (South Italy), this caused concern regarding the protection of native biodiversity and local forests. Symptomatic trees show browning of leaves, thinning of canopy, and loss of branches. The aim of this study was to understand the biotic factors involved in the observed decline. During the monitoring activity, the use of phenotypic descriptors highlighted the presence of two types of decline with distinct symptoms among oaks in rural and urban areas, which could be classified as Acute Oak Decline (AOD) and Chronic Oak Decline (COD), respectively. Following this, diagnostic analyses were carried out to assess the presence of various holm oak pathogens on twigs, trunk, and soil. These analyses revealed the presence of several Botryosphaeriaceae oak pathogens, present ubiquitously between the two environmental contexts, with Diplodia seriata being overall the most prevalent. Contrarily, bacteria associated with AOD were only present in the rural areas and included: Brenneria goodwinii , Gibbsiella quercinecans , and Rahnella victoriana. As far as known to the authors, this is the first time these pathogens have been found in the Mediterranean coastal regions. Lastly, soil analyses performed to detect the presence of Phytophthora cinnamomi were negative. Our results highlight the presence of several pathogens, which indicates that oak decline is a multifactorial phenomenon for which the predisposing, initial, and contributing factors are not yet known particularly for the recently described AOD and COD. Finally, the identification of AOD-related bacteria in Salento is a cause of concern for the need of urgent management actions aimed to contain their spread and to preserve the forest heritage of the region. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa ssp. fastidiosa (Xff) is the causal agent of Pierce's disease of grapevine, a management-intensive and potentially deadly disease. However, different stains and other subspecies, such as Xylella fastidiosa ssp. multiplex (Xfm), exist in the same regions and vary in capacity to cause disease. All strains differ in the fatty acids that comprise cell membranes, as these allow adaptations to specific host microenvironments. Therefore, studies were initiated to observe the fatty acid profiles of different Californian Xf isolates via fatty acid methyl ester (FAME) analysis. Observations revealed that the four Xff strains had similar FAME profiles that were distinct from those of the three Xfm strains, even in isolates that originated from the same host plant species. These data show consistent differences between Xff and Xfm strains and demonstrate the potential that FAME profiling has for Xylella subspecies identification of novel isolates. [Figure: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

The fungi Botryosphaeriaceae are involved in olive declines in both the world hemispheres and in all continents where this species is cultivated. In Salento (Apulia, Italy), the Botryosphaeriaceae Neofusicoccum mediterraneum and N. stellenboschiana have been reported as the agents of a branch and twig dieback that overlaps with olive quick decline syndrome caused by Xylella fastidiosa subsp. pauca. In this study, we report the finding of Diplodia seriata, another Botryosphaeriaceae species, in Salento in Xylella fastidiosa-infected olive trees affected by symptoms of branch and twig dieback. Given that its presence was also reported in olive in the Americas and in Europe (Croatia) with different degrees of virulence, we were prompted to assess its role in the Apulian decline. We identified representative isolates based on morphological features and a multilocus phylogeny. In vitro tests showed that the optimum growth temperature of the isolates is around 25–30 °C, and that they are highly thermo-tolerant. In pathogenicity trials conducted over eleven months, D. seriata expressed a very low virulence. Nonetheless, when we imposed severe water stress before the inoculation, D. seriata significatively necrotized bark and wood in a time frame of 35 days. Moreover, the symptoms which resulted were much more severe in the trial performed in summer compared with that in autumn. In osmolyte-supplemented media with a water potential from −1 to −3 Mpa, the isolates increased or maintained their growth rate compared with non-supplemented media, and they also grew, albeit to a lesser extent, on media with a water potential as low as −7 Mpa. This suggests that olives with a low water potential, namely those subjected to drought, may offer a suitable environment for the fungus' development. The analysis of the meteorological parameters, temperatures and rainfall, in Salento in the timeframe 1989–2023, showed that this area is subjected to a progressive increase of temperature and drought during the summer. Thus, overall, D. seriata has to be considered a contributor to the manifestation of branch and twig dieback of olive in Salento. Coherently with the spiral decline concept of trees, our results suggest that heat and drought act as predisposing/inciting factors facilitating D. seriata as a contributor. The fact that several adverse factors, biotic and abiotic, are simultaneously burdening olive trees in Salento offers a cue to discuss the possible complex nature of the olive decline in Salento. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa subsp. pauca ST53 (XFP), the causal agent of olive quick decline syndrome (OQDS), was thoroughly investigated after a 2013 outbreak in the Salento region of Southern Italy. Some trees from Ogliarola Salentina and Cellina di Nardò, susceptible cultivars in the Gallipoli area, the first XFP infection hotspot in Italy, have resprouted crowns and are starting to flower and yield fruits. Satellite imagery and Normalized Difference Vegetation Index analyses revealed a significant improvement in vegetation health and productivity from 2018 to 2022 of these trees. Lipid molecules have long been recognized as plant defense modulators, and recently, we investigated their role in XFP-positive hosts and in XFP-resistant as well as in XFP-susceptible cultivars of olive trees. Here, we present a case study regarding 36 olive trees (12 XFP-positive resprouting, 12 XFP-positive OQDS-symptomatic, and 12 XFP-negative trees) harvested in 2022 within the area where XFP struck first, killing millions of trees in a decade. These trees were analyzed for some free fatty acid, oxylipin, and plant hormones, in particular jasmonic and salicylic acid, by targeted LC-MS/MS. Multivariate analysis revealed that lipid markers of resistance (e.g., 13-HpOTrE), along with jasmonic and salicylic acid, were accumulated differently in the XFP-positive resprouting trees from both cultivars with respect to XFP-positive OQDS symptomatic and XFP-negative trees, suggesting a correlation of lipid metabolism with the resprouting, which can be an indication of the resiliency of these trees to OQDS. This is the first report concerning the resprouting of OQDS-infected olive trees in the Salento area. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa has been identified as the causal agent of several horticultural plant diseases that have resulted in major economic and cultural heritage losses. In the last decade, X. fastidiosa emerged as a destructive phytopathogen on olive trees in the Apulia region, Italy, prompting widespread surveillance throughout the Mediterranean basin. The present paper reports monitoring efforts for X. fastidiosa in Palestine on olive for 5 years (2017–2022) and 1 year (2022) on stone fruit trees, as a result of international collaboration projects. No signs of olive quick decline syndrome were observed on olive trees in all the Palestinian olive‐growing lands. This observation was confirmed by molecular tests using LAMP technology and PCR. In addition, 500 leaf samples from stone fruit trees (almond, apricot, peach, nectarine and plum) were tested using LAMP and PCR. All of these samples were negative for X. fastidiosa, even though few of the samples from almond trees in Idna (Hebron governorate) and apricot trees in Bal'a (Tulkarm governorate) showed leaf scorch‐like symptoms. This study provides confirmation that these important horticultural crops in Palestine (olive and stone fruits) are still free of X. fastidiosa. Preventive measures and surveillance of these and other horticultural crops such as grapevine and citrus trees are strongly recommended. [ABSTRACT FROM AUTHOR]

Key message: New defense elicitor peptides have been identified which control Xylella fastidiosa infections in almond. Xylella fastidiosa is a plant pathogenic bacterium that has been introduced in the European Union (EU), threatening the agricultural economy of relevant Mediterranean crops such as almond (Prunus dulcis). Plant defense elicitor peptides would be promising to manage diseases such as almond leaf scorch, but their effect on the host has not been fully studied. In this work, the response of almond plants to the defense elicitor peptide flg22-NH2 was studied in depth using RNA-seq, confirming the activation of the salicylic acid and abscisic acid pathways. Marker genes related to the response triggered by flg22-NH2 were used to study the effect of the application strategy of the peptide on almond plants and to depict its time course. The application of flg22-NH2 by endotherapy triggered the highest number of upregulated genes, especially at 6 h after the treatment. A library of peptides that includes BP100-flg15, HpaG23, FV7, RIJK2, PIP-1, Pep13, BP16-Pep13, flg15-BP100 and BP16 triggered a stronger defense response in almond plants than flg22-NH2. The best candidate, FV7, when applied by endotherapy on almond plants inoculated with X. fastidiosa, significantly reduced levels of the pathogen and decreased disease symptoms. Therefore, these novel plant defense elicitors are suitable candidates to manage diseases caused by X. fastidiosa, in particular almond leaf scorch. [ABSTRACT FROM AUTHOR]

Simple Summary: Zelus renardii Kolenati (Hemiptera, Reduviidae, Harpactorinae), the Leafhopper Assassin Bug (LAB), first appeared in Europe in 2011 and is now well-acclimatized. The LAB is a promising mass-rearing candidate for the inundative biocontrol strategy of Xylella fastidiosa pauca ST53 vectors. We reared LABs for two subsequent years, using live adult Drosophila melanogaster Meigen (Diptera, Drosophilidae) (Dm) as living prey versus several artificial diet formulations (D0, D1, D2, D3, and D4), to identify the best formulation. An artificial medium can minimize rearing costs and make the process sustainable and safer. The rearing of LAB is feasible with live prey, oligidic, meridic, and holidic artificial formulations. All diets, except for D2, showed a favorable trend according to the species' reproductive potential. Scoring accumulated degree days in LAB's rearing made it possible to predict the time required to complete post-embryonic development as a function of temperature. Mass production is a prerequisite for using natural enemies in integrated pest management and organic farming. Natural enemies in agroecosystems include predators that prey on insects, which they can subdue while maintaining adequate pest population densities. The Leafhopper Assassin Bug (LAB), Zelus renardii, can be a natural enemy in agroecosystems, selecting its prey for size and mobility. Some of LAB's prey include Philaenus spumarius (L.), Bactrocera oleae (Rossi), Drosophila suzukii (Matsumura), and Macrohomotoma gladiata Kuwayama, suggesting this reduviid for biocontrol agent in various contexts. We reared LABs for two subsequent broods offering living prey and artificial diets. Our data show that the rearing of Z. renardii is feasible with oligidic, meridic, and holidic artificial formulations. Four artificial diets allowed the complete post-embryonic development of LABs in captivity for two successive generations. The accumulated degree-days (ADDs) accurately predict the growth of LABs based on heat accumulation, estimating that up to three generations could grow per year in captivity at the experimented T°C. [ABSTRACT FROM AUTHOR]

This scientific report provides an update of the Xylella spp. host plant database, aiming to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp. Upon a mandate of the European Commission, EFSA created and regularly updates a database of host plant species of Xylella spp. The current mandate covers the period 2021–2026. This report is related to the 10th version of the database published in Zenodo in the EFSA Knowledge Junction community, covering literature published from 1 July 2023 up to 31 December 2023, and recent Europhyt outbreak notifications. Informative data have been extracted from 39 selected publications. Sixteen new host plants, five genera and one family were identified and added to the database. They were naturally infected by X. fastidiosa subsp. fastidiosa or unknown either in Portugal or the United States. No additional data were retrieved for X. taiwanensis, and no additional multilocus sequence types (STs) were identified worldwide. New information on the tolerant/resistant response of plant species to X. fastidiosa infection were added to the database. The Xylella spp. host plant species were listed in different categories based on the number and type of detection methods applied for each finding. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method either by sequencing or pure culture isolation (category A), reaches now 451 plant species, 204 genera and 70 families. Such numbers rise to 712 plant species, 312 genera and 89 families if considered regardless of the detection methods applied (category E). [ABSTRACT FROM AUTHOR]

The European Commission requested the EFSA Panel on Plant Health to deliver a risk assessment on the likelihood of pest freedom from Union quarantine pests and pests subject to measures adopted pursuant to Article 30 of Regulation (EU) No 2016/2031 for the maple veneer sheets manufactured according to the process set out by Canada, with emphasis on the freedom from Davidsoniella virescens and Phytophthora ramorum (non‐EU isolates). The assessment was conducted for veneer sheets of up to 0.7 mm and up to 6 mm thickness, taking into account the different phases in the veneer production in a systems approach. Some of those phases, taken alone, including the heat treatment of logs in a water bath, the cutting into thin veneer sheets and the final high heat drying of veneer sheets are expected to be effective against some of the pests, without uncertainties, making the system approach fully effective. The panel considers that no insects would survive cutting of logs into thin veneer sheets of 0.7 mm and that Xylella fastidiosa will not survive the temperatures in the water bath and final drying of veneers. The degree of pest freedom for the different groups of organisms is generally very high with slightly lower degree of pest freedom for veneer sheets of 6 mm thickness because of lower temperatures reached in the final drying of veneer sheets compared to thinner sheets. P. ramorum is not expected to survive the high heat drying of thin veneer sheets, but it may survive the lower temperatures inside thicker veneer sheets. The Expert Knowledge Elicitation (EKE) indicated, with 95% certainty, that between 9989 and 10,000 veneer sheets (thickness 6 mm) per 10,000 will be free from living P. ramorum. For D. virescens, the EKE indicated, with 95% certainty, that between 9984 and 10,000 veneer sheets (0.7 mm) per 10,000 and that between 9954 and 10,000 veneer sheets (6 mm) per 10,000 will be free from living inoculum. For other relevant groups of pests, the greatest likelihood of pest presence was observed for wood decay fungi. The EKE indicated, with 95% certainty, that between 9967 and 10,000 veneer sheets (0.7 mm) per 10,000 and that between 9911 and 10,000 veneer sheets (6 mm) per 10,000 will be free from living wood decay fungi. [ABSTRACT FROM AUTHOR]

Outbreak response to quarantine pathogens and pests in the European Union (EU) is regulated by the EU Plant Health Law, but the performance of outbreak management plans in terms of their effectiveness and efficiency has been quantified only to a limited extent. As a case study, the disease dynamics of almond leal' scorch, caused by Xylella fastidiosa, in the affected area of Alicante, Spain, were approximated using an individual-based spatial epidemiological model. The emergence of this outbreak was dated based on phylogenetic studies, and official surveys were used to delimit the current extent of thé disease. Different survey strategies and disease control measures were compared to determine their effectiveness and efficiency for outbreak management in relation to a baseline scenario without interventions. One-step and two-step survey approaches were compared with different confidence levels, buffer zone sizes, and eradication radii, including those set by the EU legislation for X. fastidiosa. The effect of disease control interventions was also considered by decreasing the transmission rate in the buffer zone. All outbreak management plans reduced the number of infected trees (effectiveness), but large differences were observed in the number of susceptible trees not eradicated (efficiency). The two-step survey approach, high confidence level, and the reduction in lhe transmission rate increased the efficiency. Only the outbreak management plans with the two-step survey approach removed infected trees completely, but they required greater survey efforts. Although control measures reduced disease spread, surveillance was the key factor in the effectiveness and efficiency of the outbreak management plans. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa (Xf) is a quarantine plant pathogen capable of colonizing the xylem of a wide range of hosts. Currently, there is no cure able to eliminate the pathogen from a diseased plant, but several integrated su-alegies have been implemented for containing the spread of Xf. Nanotechnology represents an innovative strategy based on the possibility of maximizing the potential antibacterial activity by increasing the surface-to-volume ratio of nanoscale formulations. Nanoparticles based on chitosan and/or fosetyl-Al have shown different in vitro antibacterial efficacy against Xf subsp. fastidiosa (Xff) and pauca (Xfp). This work-demonstrated the uptake of chitosan-coated fosetyl-Al nanocrystals (CH-nanoFos) by roots and their localization in the stems and leaves of Olea europaea plants. Additionally, the antibacterial activity of fosetyl-Al, nano-fosetyl, nano-chitosan, and CH-nanoFos was tested on Nicotiana tabacum cultivar SRI (Petite Havana) inoculated with X/7 Xfp, or Xf subsp. multiplex (Xfin). The bacterial load was evaluated with qPCR. and the results showed that CH-nanoFos was the only treatment able to reduce the colonization of Xff, Xfin, and Xfp in tobacco plants. Additionally, the area under the disease progress curve, used to assess symptom development in tobacco plants inoculated with Xff, Xfin, and Xfp and treated with CH-nanoFos, showed a reduction in symptom development. Furthermore, the twitching assay and bacterial growth under microfluidic conditions confirmed the antibacterial activity of CH-nanoFos. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa (Xf) is a major phytosanitary threat to global agricultural production. The complexity and difficulty of controlling Xf underscore the pressing need for novel antibacterial agents, i.e., bacteriophages, which are natural predators of bacteria. In this study, a novel lytic bacteriophage of Xf subsp. pauca, namely Xylella phage MATE 2 (MATE 2), was isolated from sewage water in southern Italy. Biological characterization showed that MATE 2 possessed a broad-spectrum of antibacterial activity against various phytobacteria within the family Xanthomonadaceae, a rapid adsorption time (10 min), and high resistance to a broad range of pH (4-10) and temperatures (4-60°C). Most importantly, MATE 2 was able to suppress the growth of Xf subsp. pauca cells in liquid culture for 7 days, demonstrating its potential as an effective antibacterial agent against Xf. The genomic and electron microscopy analyses revealed that MATE 2 is a new species tentatively belonging to the genus Carpasinavirus within the class Caudoviricetes, with an isometric capsid head of 60 ± 5 nm along with a contractile tail of 120 ± 7.5 nm. Furthermore, the high-throughput sequencing and de novo assembly generated a single contig of 63,695 nucleotides in length; representing a complete genome composed of 95 Open Reading Frames. Bioinformatics analysis performed on MATE 2 genome revealed the absence of lysogenic mediated genes, and genes encoding virulence factors, antibiotic resistance, and toxins. This study adds a new phage to the very short list of Xfinfecting lytic phages, whose in-vitro antibacterial activity has been ascertained, while its efficacy on Xf-infected olive trees in the field has yet to be determined. [ABSTRACT FROM AUTHOR]

In Europe, the recently reported plant pathogen Xylella fastidiosa subsp. multiplex affects several wild, ornamental, and cultivated trees causing scorch diseases. In 2018, the sequence type 87 was reported in Tuscany on Mediterranean shrubs and trees. Although spittlebugs (Hemiptera: Aphrophoridae) were already identified as main vectors of this bacterium in Europe, their role in the transmission of this subspecies has not been ascertained yet. In this study the ability of Philaenus spumarius and Neophilaenus campestris to acquire and transmit Xylella fastidiosa subsp. multiplex sequence type 87 from and to Rhamnus alaternus was evaluated in two-year semi-field experiments. To acquire the bacterium, insects were confined on wild, naturally infected R. alaternus shrubs for 120 h. Then, they were transferred to healthy plants and maintained in cages for 96 h. To follow the infection, plant samples were collected every two months for three times. Tested plants were destroyed at the end of experiments and roots, twigs and leaves were analysed. Philaenus spumarius showed a significantly higher survival rate than N. campestris. The infection status of both insects and plants was assessed through molecular analysis. P. spumarius and N. campestris were able to infect healthy plants although the acquisition rate and the estimated probability of transmission appeared to be low. These findings provide new accounts on the role of two polyphagous insect vectors in spreading a quarantine organism, which is lethal to a huge number of plant species. However, further studies are needed to disclose more specific interactions within this complex pathosystem. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a plant‐pathogenic bacterium that poses a serious threat to the production of economically important plant species including grapes, almonds, olives and a broad range of amenity plants, causing significant economic losses worldwide. While multiple molecular detection assays have been developed for X. fastidiosa, there is a lack of molecular tools available for detection and differentiation of the closely related pear pathogen, Xylella taiwanensis. In this study, we present a novel conventional PCR assay with primers that can amplify both Xylella species. The amplified product could be sequenced and used for discrimination between the two species and the subspecies within the fastidiosa species. This PCR assay was designed using a genome‐informed approach to target the ComEC/Rec2 gene of both Xylella species, ensuring a higher specificity than other previously developed PCR assays. A test performance study across five national plant diagnostic laboratories in Australia and New Zealand demonstrated this assay's high sensitivity and specificity to all known species and subspecies within the Xylella genus. This PCR assay can be used for Xylella identification at the species and subspecies level and is compatible with Sanger sequencing and nanopore sequencing for rapid turnaround time. The newly developed conventional PCR assay presented here offers rapid detection and accurate identification of both Xylella species from plant, insect vector or bacterial samples, enabling timely implementation of biosecurity measures or disease management responses. [ABSTRACT FROM AUTHOR]

This research focused on studying the dynamics of the bacterial pathogen Xylella fastidiosa in almond trees across different developmental stages. The objective was to understand the seasonal distribution and concentration of X. fastidiosa within almond trees. Different tree organs, including leaves, shoots, branches, fruits, flowers, and roots, from 10 X. fastidiosa-infected almond trees were sampled over 2 years. The incidence and concentration of X. fastidiosa were determined using qPCR and isolation. Throughout the study, X. fastidiosa was consistently absent from fruits, flowers, and roots, whereas it was detected in leaves as well as in shoots and branches. We demonstrate that the absence of X. fastidiosa in the roots is likely linked to the inability of this isolate to infect the peach-almond hybrid rootstock GF677. X. fastidiosa incidence in shoots and branches remained consistent throughout the year, whereas in leaf petioles, it varied across developmental stages, with lower detection during the early and late stages of the season. Similarly, viable X. fastidiosa cells were isolated from shoots and branches at all developmental stages, but no successful isolations were achieved from leaf petioles during the vegetative and nut growth stage. Studying the progression of almond leaf scorch symptoms in trees with initial infections showed that once symptoms emerged on one branch, symptomless branches were likely already infected by the bacterium. Therefore, selectively pruning symptomatic branches is unlikely to cure the tree. This study enhances our understanding of X. fastidiosa dynamics in almond trees and may have practical applications for its detection and control. [ABSTRACT FROM AUTHOR]

In 2013, an outbreak of Xylella fastidiosa (Xf) was identified for the first time in Europe, in the extreme south of Italy (Apulia, Salento territory). The locally identified subspecies pauca turned out to be lethal for olive trees, starting an unprecedented phytosanitary emergency for one of the most iconic cultivations of the Mediterranean area. Xf pauca (Xfp) is responsible for a severe disease, the olive quick decline syndrome (OQDS), spreading epidemically and with dramatic impact on the agriculture, the landscape, the tourism and the cultural heritage of this region. The bacterium, transmitted by insects that feed on xylem sap, causes rapid wilting in olive trees due to biofilm formation, which obstructs the plant xylematic vessels. The aim of this review is to perform a thorough analysis that offers a general overview of the published work, from 2013 to December 2023, related to the Xfp outbreak in Apulia. This latter hereto has killed millions of olive trees and left a ghostly landscape with more than 8000 square kilometers of infected territory, that is 40% of the region. The majority of the research efforts made to date to combat Xfp in olive plants are listed in the present review, starting with the early attempts to identify the bacterium, the investigations to pinpoint and possibly control the vector, the assessment of specific diagnostic techniques and the pioneered therapeutic approaches. Interestingly, according to the general set criteria for the preliminary examination of the accessible scientific literature related to the Xfp outbreak on Apulian olive trees, fewer than 300 papers can be found over the last decade. Most of them essentially emphasize the importance of developing diagnostic tools that can identify the disease early, even when infected plants are still asymptomatic, in order to reduce the risk of infection for the surrounding plants. On the other hand, in the published work, the diagnostic focus (57%) overwhelmingly encompasses all other possible investigation goals such as vectors, impacts and possible treatments. Notably, between 2013 and 2023, only 6.3% of the literature reports addressing the topic of Xfp in Apulia were concerned with the application of specific treatments against the bacterium. Among them, those reporting field trials on infected plants, including simple pruning indications, were further limited (6%). [ABSTRACT FROM AUTHOR]

The pathogen Xylella fastidiosa subsp. fastidiosa has circulated through California's vineyards since its introduction from Central America in the 1800s. This pathogen is responsible for a bacterial disease called Pierce's disease (PD) of grapevine. With no known cure, PD has had devastating effects on some vineyards. Important factors that impact disease severity and persistence include: the presence of insect vectors, grapevine cultivar, management, ecology, and winter temperatures. Removal of infected vines is critical for reducing pathogen spread but relies on accurate and rapid pathogen detection. In this study, we foster a greater understanding of disease symptom emergence by way of a 3-year field inoculation project in Napa Valley. Although PD emergence and symptom progression have been studied in greenhouse and experimental plots, there is a large knowledge gap in quantifying disease progression under commercial conditions. After inoculating 80 mature Vitis vinifera vines in April 2017, we measured bacterial populations and six symptom types at four locations within each plant throughout the subsequent three growing seasons. The main foci of the project were understanding X. fastidiosa movement through the plants, infection, overwinter curing, and symptom development. We observed greater winter recovery than expected, and shriveled grape clusters proved to be a more reliable early indication of infection than other more commonly used symptoms. Although there were differences among wine grape cultivars, this work suggests that disease progression in the field may not fit the paradigm of predominant leaf scorch and low recovery rates as neatly as has been previously believed.

While there are documented host shifts in many bacterial plant pathogens, the genetic foundation of host shifts is largely unknown. Xylella fastidiosa is a bacterial pathogen found in over 600 host plant species. Two parallel host shifts occurred-in Brazil and Italy-in which X. fastidiosa adapted to infect olive trees, whereas related strains infected coffee. Using 10 novel whole-genome sequences from an olive-infecting population in Brazil, we investigated whether these olive-infecting strains diverged from closely related coffee-infecting strains. Several single-nucleotide polymorphisms, many derived from recombination events, and gene gain and loss events separated olive-infecting strains from coffee-infecting strains in this clade. The olive-specific variation suggests that this event was a host jump with genetic isolation between coffee- and olive-infecting X. fastidiosa populations. Next, we investigated the hypothesis of genetic convergence in the host shift from coffee to olive in both populations (Brazil and Italy). Each clade had multiple mutations and gene gain and loss events unique to olive, yet no overlap between clades. Using a genome-wide association study technique, we did not find any plausible candidates for convergence. Overall, this work suggests that the two populations adapted to infect olive trees through independent genetic solutions.

Homologous recombination plays a key function in the evolution of bacterial genomes. Within Xylella fastidiosa, an emerging plant pathogen with increasing host and geographic ranges, it has been suggested that homologous recombination facilitates host switching, speciation, and the development of virulence. We used 340 whole-genome sequences to study the relationship between inter- and intrasubspecific homologous recombination, random mutation, and natural selection across individual X. fastidiosa genes. Individual gene orthologs were identified and aligned, and a maximum likelihood (ML) gene tree was generated. Each gene alignment and tree pair were then used to calculate gene-wide and branch-specific r/m values (relative effect of recombination to mutation), gene-wide and branch-site nonsynonymous over synonymous substitution rates (dN/dS values; episodic selection), and branch length (as a proxy for mutation rate). The relationships between these variables were evaluated at the global level (i.e., for all genes among and within a subspecies), among specific functional classes (i.e., COGs), and between pangenome components (i.e., accessory versus core genes). Our analysis showed that r/m varied widely among genes as well as across X. fastidiosa subspecies. While r/m and dN/dS values were positively correlated in some instances (e.g., core genes in X. fastidiosa subsp. fastidiosa and both core and accessory genes in X. fastidiosa subsp. multiplex), low correlation coefficients suggested no clear biological significance. Overall, our results indicate that, in addition to its adaptive role in certain genes, homologous recombination acts as a homogenizing and a neutral force across phylogenetic clades, gene functional groups, and pangenome components. IMPORTANCE There is ample evidence that homologous recombination occurs frequently in the economically important plant pathogen Xylella fastidiosa. Homologous recombination has been known to occur among sympatric subspecies and is associated with host-switching events and virulence-linked genes. As a consequence, is it generally assumed that recombinant events in X. fastidiosa are adaptive. This mindset influences expectations of how homologous recombination acts as an evolutionary force as well as how management strategies for X. fastidiosa diseases are determined. Yet, homologous recombination plays roles beyond that of a source for diversification and adaptation. Homologous recombination can act as a DNA repair mechanism, as a means to facilitate nucleotide compositional change, as a homogenization mechanism within populations, or even as a neutral force. Here, we provide a first assessment of long-held beliefs regarding the general role of recombination in adaptation for X. fastidiosa. We evaluate gene-specific variations in homologous recombination rate across three X. fastidiosa subspecies and its relationship to other evolutionary forces (e.g., natural selection, mutation, etc.). These data were used to assess the role of homologous recombination in X. fastidiosa evolution.

The lack of sustainable strategies for combating Xylella fastidiosa (Xf) highlights the pressing need for novel practical antibacterial tools. In this study, Lactococcus lactis subsp. lactis strain ATCC 11454 (L. lactis), known for its production of nisin A, was in vitro tested against Xf subsp. pauca. Preliminary investigations showed that nisin A was involved in a strong antagonistic activity exhibited by L. lactis against Xf. Thus, the efficacy of nisin A was comprehensively assessed through a combination of in vitro and in planta experiments. In vitro investigations employing viable-quantitative PCR, spot assay, turbidity reduction assay, fluorescence microscopy, and transmission electron microscopy demonstrated nisin's robust bactericidal effect on Xf at a minimal lethal concentration of 0.6 mg/mL. Moreover, results from fluorescence and transmission electron microscopies indicated that nisin directly and rapidly interacts with the membranes of Xf cells, leading to the destruction of bacterial cells in few minutes. In in planta tests, nisin also demonstrated the ability to tackle Xf infections within Nicotiana benthamiana plants that remained asymptomatic 74 days post inoculation. Furthermore, RPLC-ESI-MS/MS analyses showed that nisin translocated to all parts of the plants and remains intact for up to 9 days. For the first time, this study underscores the nisin-based strategy as a realistic and eco-friendly approach to be further investigated against Xf infections in the field. [ABSTRACT FROM AUTHOR]

Current practices for structural analysis of extremely large‐molecular‐weight polysaccharides via solution‐state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non‐specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year. [ABSTRACT FROM AUTHOR]

An unprecedented plant health emergency in olives has been registered over the last decade in Italy, arguably more severe than what occurred repeatedly in grapes in the United States in the last 140 years. These emergencies are epidemics caused by a stealthy pathogen, the xylem-limited, insect-transmitted bacterium Xylella fastidiosa. Although these epidemics spurred research that answered many questions about the biology and management of this pathogen, many gaps in knowledge remain. For this review, we set out to represent both the U.S. and European perspectives on the most pressing challenges that need to be addressed. These are presented in 10 sections that we hope will stimulate discussion and interdisciplinary research. We reviewed intrinsic problems that arise from the fastidious growth of X. fastidiosa, the lack of specificity for insect transmission, and the economic and social importance of perennial mature woody plant hosts. Epidemiological models and predictions of pathogen establishment and disease expansion, vital for preparedness, are based on very limited data. Most of the current knowledge has been gathered from a few pathosystems, whereas several hundred remain to be studied, probably including those that will become the center of the next epidemic. Unfortunately, aspects of a particular pathosystem are not always transferable to others. We recommend diversification of research topics of both fundamental and applied nature addressing multiple pathosystems. Increasing preparedness through knowledge acquisition is the best strategy to anticipate and manage diseases caused by this pathogen, described as "the most dangerous plant bacterium known worldwide.". [ABSTRACT FROM AUTHOR]

Plant diseases significantly impact food security and food safety. It was estimated that food production needs to increase by 50% to feed the projected 9.3 billion people by 2050. Yet, plant pathogens and pests are documented to cause up to 40% yield losses in major crops, including maize, rice, and wheat, resulting in annual worldwide economic losses of approximately US$220 billion. Yield losses due to plant diseases and pests are estimated to be 21.5% (10.1 to 28.1%) in wheat, 30.3% (24.6 to 40.9%) in rice, and 22.6% (19.5 to 41.4%) in maize. In March 2023, The American Phytopathological Society (APS) conducted a survey to identify and rank key challenges in plant pathology in the next decade. Phytopathology subsequently invited papers that address those key challenges in plant pathology, and these were published as a special issue. The key challenges identified include climate change effect on the disease triangle and outbreaks, plant disease resistance mechanisms and its applications, and specific diseases including those caused by Candidatus Liberibacter spp. and Xylella fastidiosa. Additionally, disease detection, natural and man-made disasters, and plant disease control strategies were explored in issue articles. Finally, aspects of open access and how to publish articles to maximize the Findability, Accessibility, Interoperability, and Reuse of digital assets in plant pathology were described. Only by identifying the challenges and tracking progress in developing solutions for them will we be able to resolve the issues in plant pathology and ultimately ensure plant health, food security, and food safety. [ABSTRACT FROM AUTHOR]

The meadow spittlebug Philaenus spumarius L. (Hemiptera: Aphrophoridae) is a xylem‐sap feeder and the most abundant and widespread vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) throughout Europe. Control strategies for the disease are based on containment and eradication measures regulated by the European Union. Within the context of biological control, the use of natural enemies aims at limiting the insect vector population in agroecosystems. Spiders are generalist predators with potential as biological control agents occupying all the habitats within crops. Crab spiders (Thomisidae) could be potential natural enemies against P. spumarius. The functional response (FR) of a predator represents the intake rate as a function of food availability and allows estimating feeding behaviour parameters such as the prey handling time and attack rate. However, spiders often kill more prey than they consume, unveiling their capacity as natural enemies. We assessed and compared the FR of Xysticus acerbus (Thomisidae) fed on P. spumarius in the laboratory considering (1) the total number of dead individuals and (2) only the consumed ones. We found that although both FRs were of type‐II, they significantly differed. The values of attack rate and handling time for consumed were 0.1 and 3.6, respectively, while for dead, the attack rate was 0.3 and 1.1, being both significantly different. Moreover, the number of overkilled individuals reached a plateau at the highest prey densities. Xysticus acerbus could be a potential natural enemy of P. spumarius. Nevertheless, we suggest that the effect of including the killed but not consumed prey individuals in FR studies using wasteful killing predators should be considered and further studied. [ABSTRACT FROM AUTHOR]