(4Z)-Lachnophyllum Lactone, a Metabolite with Phytotoxic and Antifungal Activity against Pests Affecting Mediterranean Agriculture: A New Versatile and Easy Scalable Parallel Synthesis.

A methodology for the total and modulable synthesis of (4Z)-lachnophyllum lactone (1), on a gram scale, is reported for the first time. The present work started with the design of a retrosynthetic pathway for the target compound, with the key step identified in Pd-Cu bimetallic cascade cross-coupling cyclization. (4Z)-Lachnophyllum lactone (1) is an acetylenic furanone previously isolated, in a low amount, from the organic extract of the autotrophic weedConyza bonariensis. Tested against the stem parasitic weed Cuscuta campestris in a seedling growth bioassay, (4Z)-lachnophyllum lactone (1) showed almost 85% of inhibitory activity up to 0.3 mM in comparison with the control. At the same concentration, the compound displayed radicle growth inhibitory activity of the root parasitic weeds Orobanche minor and Phelipanche ramosa higher than 70 and 40%, respectively. Surprisingly, the compound showed a high percentage of inhibition, up to 0.1 mM, on C. bonariensis seed germination too. This versatile synthetic strategy was also used to obtain two further natural analogues, namely, (4E)-lachnophyllum lactone (8) and (4Z,8Z)-matricaria lactone (9), that showed, in most cases, the same inhibitory trend with slight differences, highlighting the importance of the stereochemistry and unsaturation of the side chain. Furthermore, all of the compounds showed antifungal activity at 1 mM reducing the mycelial growth of the olive pathogen Verticillium dahliae. The design and implementation of scalable and modulable total synthesis on a gram scale of acetylenic furanones allow the production of a large amount of these natural products, overcoming the limit imposed by isolation from natural sources. The results of the present study pave the way for the development of ecofriendly bioinspired pesticides with potential application in agrochemical practices as alternative to synthetic pesticides.

Correction: Velasco-Amo et al. Use of traC Gene to Type the Incidence and Distribution of pXFAS_5235 Plasmid-Bearing Strains of Xylella fastidiosa subsp. fastidiosa ST1 in Spain. Plants 2022, 11, 1562.

In the original publication [...].

Insights into the Methodological, Biotic and Abiotic Factors Influencing the Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees.

Vascular pathogens are the causal agents of some of the most devastating plant diseases in the world, which can cause, under specific conditions, the destruction of entire crops. These plant pathogens activate a range of physiological and immune reactions in the host plant following infection, which may trigger the proliferation of a specific microbiome to combat them by, among others, inhibiting their growth and/or competing for space. Nowadays, it has been demonstrated that the plant microbiome can be modified by transplanting specific members of the microbiome, with exciting results for the control of plant diseases. However, its practical application in agriculture for the control of vascular plant pathogens is hampered by the limited knowledge of the plant endosphere, and, in particular, of the xylem niche. In this review, we present a comprehensive overview of how research on the plant microbiome has evolved during the last decades to unravel the factors and complex interactions that affect the associated microbial communities and their surrounding environment, focusing on the microbial communities inhabiting the xylem vessels of olive trees (Olea europaea subsp. europaea), the most ancient and important woody crop in the Mediterranean Basin. For that purpose, we have highlighted the role of xylem composition and its associated microorganisms in plants by describing the methodological approaches explored to study xylem microbiota, starting from the methods used to extract xylem microbial communities to their assessment by culture-dependent and next-generation sequencing approaches. Additionally, we have categorized some of the key biotic and abiotic factors, such as the host plant niche and genotype, the environment and the infection with vascular pathogens, that can be potential determinants to critically affect olive physiology and health status in a holobiont context (host and its associated organisms). Finally, we have outlined future directions and challenges for xylem microbiome studies based on the recent advances in molecular biology, focusing on metagenomics and culturomics, and bioinformatics network analysis. A better understanding of the xylem olive microbiome will contribute to facilitate the exploration and selection of specific keystone microorganisms that can live in close association with olives under a range of environmental/agronomic conditions. These microorganisms could be ideal targets for the design of microbial consortia that can be applied by endotherapy treatments to prevent or control diseases caused by vascular pathogens or modify the physiology and growth of olive trees.

Molecular Characterization of Peronospora variabilis Isolates Infecting Chenopodium quinoa and Chenopodium album in Spain.

Quinoa is an expanding crop in southern Spain. Downy mildew, caused by Peronospora variabilis, is the most important quinoa disease in Spain and worldwide. In Spain, this disease has also been observed on the weed Chenopodium album. The objectives of this study were to unravel the origin of the P. variabilis isolates currently infecting quinoa in southern Spain and to study their genetic diversity. We hypothesized that P. variabilis isolates infecting quinoa in Spain could have been introduced through the seeds of the quinoa varieties currently grown in the country or, alternatively, that these isolates are endemic isolates, originally infecting C. album, that jumped to quinoa. In order to test these hypotheses, we sequenced the internal transcribed spacer (ITS), cytochrome c oxidase subunit 1 (cox1), and cox2 regions of 33 P. variabilis isolates infecting C. quinoa and C. album in southern Spain and analyzed their phylogenetic relationship with isolates present in other countries infecting Chenopodium spp. cox1 gene sequences from all of the Spanish P. variabilis isolates were identical and exhibited nine single-nucleotide polymorphisms (SNPs) compared with a single P. variabilis cox1 sequence found at GenBank. Phylogenetic analyses based on the ITS ribosomal DNA region were not suitable to differentiate isolates according to their geographical origin or host. The cox2 sequences from P. variabilis Spanish isolates collected from C. quinoa and C. album were all identical and had a distinctive SNP in the last of four polymorphic sites that distinguished Spanish isolates from isolates from other countries. These results suggest that P. variabilis infecting quinoa in southern Spain could be native isolates that originally infected C. album.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Complete Circularized Genome Resources of Seven Strains of Xylella fastidiosa subsp. fastidiosa Using Hybrid Assembly Reveals Unknown Plasmids.

Xylella fastidiosa is a vascular plant pathogenic bacterium native to the Americas that is causing significant epidemics and economic losses in olive and almonds in Europe, where it is a quarantine pathogen. Since its first detection in 2013 in Italy, mandatory surveys across Europe revealed the presence of the bacterium also in France, Spain, and Portugal. Combining Oxford Nanopore Technologies and Illumina sequencing data, we assembled high-quality complete genomes of seven X. fastidiosa subsp. fastidiosa strains isolated from different plants in Spain, the United States, and Mexico. Comparative genomic analyses discovered differences in plasmid content among strains, including plasmids that had been overlooked previously when using the Illumina sequencing platform alone. Interestingly, in strain CFBP8073, intercepted in France from plants imported from Mexico, three plasmids were identified, including two (plasmids pXF-P1.CFBP8073 and pXF-P2.CFBP8073) not previously described in X. fastidiosa and one (pXF5823.CFBP8073) almost identical to a plasmid described in a X. fastidiosa strain from citrus. Plasmids found in the Spanish strains here were similar to those described previously in other strains from the same subspecies and ST1 isolated in the Balearic Islands and the United States. The genome resources from this work will assist in further studies on the role of plasmids in the epidemiology, ecology, and evolution of this plant pathogen.

Xylella fastidiosa Infection Reshapes Microbial Composition and Network Associations in the Xylem of Almond Trees.

Xylella fastidiosa represents a major threat to important crops worldwide including almond, citrus, grapevine, and olives. Nowadays, there are no efficient control measures for X. fastidiosa, and the use of preventive measures and host resistance represent the most practical disease management strategies. Research on vessel-associated microorganisms is gaining special interest as an innate natural defense of plants to cope against infection by xylem-inhabiting pathogens. The objective of this research has been to characterize, by next-generation sequencing (NGS) analysis, the microbial communities residing in the xylem sap of almond trees affected by almond leaf scorch disease (ALSD) in a recent X. fastidiosa outbreak occurring in Alicante province, Spain. We also determined community composition changes and network associations occurring between xylem-inhabiting microbial communities and X. fastidiosa. For that, a total of 91 trees with or without ALSD symptoms were selected from a total of eight representative orchards located in five municipalities within the X. fastidiosa-demarcated area. X. fastidiosa infection in each tree was verified by quantitative polymerase chain reaction (qPCR) analysis, with 54% of the trees being tested X. fastidiosa-positive. Globally, Xylella (27.4%), Sphingomonas (13.9%), and Hymenobacter (12.7%) were the most abundant bacterial genera, whereas Diplodia (30.18%), a member of the family Didymellaceae (10.7%), and Aureobasidium (9.9%) were the most predominant fungal taxa. Furthermore, principal coordinate analysis (PCoA) of Bray-Curtis and weighted UniFrac distances differentiated almond xylem bacterial communities mainly according to X. fastidiosa infection, in contrast to fungal community structure that was not closely related to the presence of the pathogen. Similar results were obtained when X. fastidiosa reads were removed from the bacterial data set although the effect was less pronounced. Co-occurrence network analysis revealed negative associations among four amplicon sequence variants (ASVs) assigned to X. fastidiosa with different bacterial ASVs belonging to 1174-901-12, Abditibacterium, Sphingomonas, Methylobacterium-Methylorubrum, Modestobacter, Xylophilus, and a non-identified member of the family Solirubrobacteraceae. Determination of the close-fitting associations between xylem-inhabiting microorganisms and X. fastidiosa may help to reveal specific microbial players associated with the suppression of ALSD under high X. fastidiosa inoculum pressure. These identified microorganisms would be good candidates to be tested in planta, to produce almond plants more resilient to X. fastidiosa infection when inoculated by endotherapy, contributing to suppress ALSD.

Identification of Soil Properties Associated with the Incidence of Banana Wilt Using Supervised Methods.

Over the last few decades, a growing incidence of Banana Wilt (BW) has been detected in the banana-producing areas of the central zone of Venezuela. This disease is thought to be caused by a fungal−bacterial complex, coupled with the influence of specific soil properties. However, until now, there was no consensus on the soil characteristics associated with a high incidence of BW. The objective of this study was to identify the soil properties potentially associated with BW incidence, using supervised methods. The soil samples associated with banana plant lots in Venezuela, showing low (n = 29) and high (n = 49) incidence of BW, were collected during two consecutive years (2016 and 2017). On those soils, sixteen soil variables, including the percentage of sand, silt and clay, pH, electrical conductivity, organic matter, available contents of K, Na, Mg, Ca, Mn, Fe, Zn, Cu, S and P, were determined. The Wilcoxon test identified the occurrence of significant differences in the soil variables between the two groups of BW incidence. In addition, Orthogonal Least Squares Discriminant Analysis (OPLS-DA) and the Random Forest (RF) algorithm was applied to find soil variables capable of distinguishing banana lots showing high or low BW incidence. The OPLS-DA model showed a proper fitting of the data (R2Y: 0.61, p value < 0.01), and exhibited good predictive power (Q2: 0.50, p value < 0.01). The analysis of the Receiver Operating Characteristics (ROC) curves by RF revealed that the combination of Zn, Fe, Ca, K, Mn and Clay was able to accurately differentiate 84.1% of the banana lots with a sensitivity of 89.80% and a specificity of 72.40%. So far, this is the first study that identifies these six soil variables as possible new indicators associated with BW incidence in soils of lacustrine origin in Venezuela.

Use of traC Gene to Type the Incidence and Distribution of pXFAS_5235 Plasmid-Bearing Strains of Xylella fastidiosa subsp. fastidiosa ST1 in Spain.

Xylella fastidiosa (Xf) is a phytopathogenic bacterium with a repertoire of self-replicating genetic elements, including plasmids, pathogenicity islands, and prophages. These elements provide potential avenues for horizontal gene transfer both within and between species and have the ability to confer new virulence traits, including the ability to colonize new host plants. However, they can also serve as a 'footprint' to type plasmid-bearing strains. Genome sequencing of several strains of Xf subsp. fastidiosa sequence type (ST) 1 from Mallorca Island, Spain, revealed the presence of a 38 kb plasmid (pXFAS_5235). In this study, we developed a PCR-based typing approach using primers targeting the traC gene to determine the presence of pXFAS_5235 plasmid or other plasmids carrying this gene in a world-wide collection of 65 strains X. fastidiosa from different subspecies and STs or in 226 plant samples naturally infected by the bacterium obtained from the different outbreaks of Xf in Spain. The traC gene was amplified only in the plant samples obtained from Mallorca Island infected by Xf subsp. fastidiosa ST1 and from all Spanish strains belonging to this ST. Maximum-likelihood phylogenetic tree of traC revealed a close relatedness among Spanish and Californian strains carrying similar plasmids. Our results confirm previous studies, which suggested that a single introduction event of Xf subsp. fastidiosa ST1 occurred in the Balearic Islands. Further studies on the presence and role of plasmids in Xf strains belonging to the same or different subspecies and STs can provide important information in studies of epidemiology, ecology, and evolution of this plant pathogen.

Consumption of Tritordeum Bread Reduces Immunogenic Gluten Intake without Altering the Gut Microbiota.

Gluten proteins are responsible for the wheat breadmaking quality. However, gluten is also related to human pathologies for which the only treatment is a gluten-free diet (GFD). GFD has gained popularity among individuals who want to reduce their gluten intake. Tritordeum is a cereal species that originated after crossing durum wheat with wild barley and differs from bread wheat in its gluten composition. In this work, we have characterized the immunogenic epitopes of tritordeum bread and results from a four-phase study with healthy adults for preferences of bread and alterations in the gut microbiota after consuming wheat bread, gluten-free bread, and tritordeum bread are reported. Tritordeum presented fewer peptides related to gluten proteins, CD-epitopes, and IgE binding sites than bread wheat. Participants rated tritordeum bread higher than gluten-free bread. Gut microbiota analysis revealed that the adherence to a strict GFD involves some minor changes, especially altering the species producing short-chain fatty acids. However, the short-term consumption of tritordeum bread does not induce significant changes in the diversity or community composition of the intestinal microbiota in healthy individuals. Therefore, tritordeum bread could be an alternative for healthy individuals without wheat-related pathologies who want to reduce their gluten consumption without harming their gut health.

Primer Choice and Xylem-Microbiome-Extraction Method Are Important Determinants in Assessing Xylem Bacterial Community in Olive Trees.

Understanding the unique and unexplored microbial environment of xylem sap is starting to be of relevant importance for plant health, as it could include microbes that may protect plants against xylem-limited pathogens, such as Verticillium dahliae and Xylella fastidiosa. In this study, we evaluated the effects that the method for extracting the xylem bacterial communities, the plant age and the PCR primers may have on characterizing the xylem-bacterial-community composition by using an NGS approach. Xylem sap was extracted from xylem vessels by using a Scholander pressure chamber, or by macerating wood shavings that were obtained from xylem tissues by using branches from 10-year-old olive trees, or the entire canopy of 1-year-old olive plantlets. Additionally, we compared four different PCR-primer pairs that target 16S rRNA for their efficacy to avoid the coamplification of mitochondria and chloroplast 16S rRNA, as this represents an important drawback in metabarcoding studies. The highest amplifications in the mitochondria and chloroplast reads were obtained when using xylem woody chips with the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. To the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (<27.48%), no chloroplast sequences and the highest numbers of bacterial OTUs identified (i.e., 254 and 266, respectively). Interestingly, only 73 out of 172 and 46 out of 181 genera were shared between the xylem sap and woody chips after amplification with PCR2 or PCR4 primers, respectively, which indicates a strong bias of the bacterial-community description, depending on the primers used. Globally, the most abundant bacterial genera (>60% of reads) included Anoxybacillus, Cutibacterium, Pseudomonas, Spirosoma, Methylobacterium-Methylorubrum and Sphingomonas; however, their relative importance varied, depending on the matrix that was used for the DNA extraction and the primer pairs that were used, with the lowest effect due to plant age. These results will help to optimize the analysis of xylem-inhabiting bacteria, depending on whether whole xylematic tissue or xylem sap is used for the DNA extraction. More importantly, it will help to better understand the driving and modifying factors that shape the olive-xylem-bacterial-community composition.

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.

Legacy effects of temporary grassland in annual crop rotation on soil ecosystem services.

The introduction of temporary grassland into an annual crop rotation is recognized to improve soil ecosystem services, and resulting legacies can be beneficial for the following crops. In this context, the aim of the present study was to evaluate legacy effects of introducing temporary grassland into an annual crop rotation on five ecosystem services (i) soil structure maintenance (aggregate stability), (ii) water regulation (saturated hydraulic conductivity), (iii) biodiversity conservation (microbial biomass and microbial metabolic activity, as well as microorganism, enchytraeid, springtail and earthworm communities), (iv) pathogen regulation (soil suppressiveness to Verticillium dahliae), and (v) forage production and quality. Three crop rotation schemes, maintained for twelve years, were compared in four random blocks, one being an annual crop rotation without grassland (0%), another with a medium percentage of grassland (50%, corresponding to 3 years of continuous grassland in the crop rotation), and a third one with a high percentage of grassland in the crop rotation (75%, corresponding to 6 years of continuous grassland in the crop rotation). The results showed that the grassland introduction into an annual crop rotation improved, whatever the duration of the grassland, soil structure maintenance and biodiversity conservation, while it decreased pathogen regulation and did not modify water regulation. Comparing the two crop rotations that included grassland, indicated a stronger beneficial grassland legacy effect for the higher proportion of grassland concerning soil structure maintenance and biodiversity conservation. By contrast, water regulation, pathogen regulation and forage production were not affected by the legacy of the 75% grassland during the rotation. Overall, our findings demonstrated the extent to which grassland legacies are affecting the current state of soil properties and possible ecosystem services provided. To improve ecosystem services, soil management should take legacy effects into account and consider longer timeframes to apply beneficial practices.

Verticillium dahliae Inoculation and in vitro Propagation Modify the Xylem Microbiome and Disease Reaction to Verticillium Wilt in a Wild Olive Genotype.

Host resistance is the most practical, long-term, and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plant's microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remains unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless of whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless of infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are the first to show a breakdown of resistance to Vd in a wild olive that potentially may be related to a modification of its xylem microbiome and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance, which can be of use to fight against main vascular diseases of olive.

Evaluation of Established Methods for DNA Extraction and Primer Pairs Targeting 16S rRNA Gene for Bacterial Microbiota Profiling of Olive Xylem Sap.

Next-generation sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted, or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a non-biased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from "Picual" and "Arbequina" olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UniFrac distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignment, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction kit, the combination of 799F/1193R primers amplifying the hypervariable V5-V7 region, and the Silva 132 database for taxonomic assignment. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella, and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting the xylem sap of olives or other woody crop species.

Tritordeum breads are well tolerated with preference over gluten-free breads in non-celiac wheat-sensitive patients and its consumption induce changes in gut bacteria.

BACKGROUND: The ingestion of wheat and other cereals are related to several gut disorders. The specific components responsible for non-celiac wheat-sensitivity (NCWS) may include gluten and other compounds. Tritordeum is a new cereal derived from crossing durum wheat with a wild barley species, which differs from bread wheat in its gluten composition. In the present work, we examined the response of NCWS patients to tritordeum bread Gastrointestinal symptoms as well as tritordeum acceptability, gluten immunogenic peptides excretion, and the composition and structure of the intestinal microbiota were evaluated. RESULTS: Gastrointestinal symptoms of the subjects showed no significant change between the gluten-free bread and the tritordeum bread. Participating subjects rated tritordeum bread higher than the gluten-free bread. Analysis of the bacterial gut microbiota indicated that tritordeum consumption does not alter the global structure and composition of the intestinal microbiota, and only a few changes in some butyrate-producing bacteria were observed. CONCLUSIONS: All the results derived from acceptability, biochemical and microbiological tests suggest that tritordeum may be tolerated by a sub-set of NCWS sufferers who do not require strict exclusion of gluten from their diet. © 2020 Society of Chemical Industry.

First report of Verticillium wilt caused by Verticillium dahliae on Russian olive (Elaeagnus angustifolia) in Iran.

Russian olive, also known as, Persian olive or oleaster (Elaeagnus angustifolia L.) is a species in the Elaeagnaceae family native to western and central Asia. In some orchards in Iran, intercropping Russian olive or Prunus trees with vegetables is a common practice. In 2018, about 130 ha of E. angustifolia orchards in Shahrood, Semnan Province, Iran showed branch wilting and dieback. Symptoms on affected trees started with yellowing of the lower leaves, followed by wilting and finally death of affected branches. Sections of stems indicated brown or black streaks in the vascular tissues under the bark. Isolations were made from discolored vascular tissues by surface-disinfesting small pieces of tissue with 0.5% sodium hypochlorite for 2 min, plating them onto potato dextrose agar amended with 25 mg/l streptomycin sulfate and incubated in the dark for 14 days at 25°C. Fungi consistently isolated from symptomatic tissues. Fungal isolates were identified as Verticillium dahliae Kleb. based on characteristics of verticillate conidiophores, hyaline, elliptical, single celled conidia measuring 4.7-6.0 × 2.3-3.4 µm (n = 100) and irregular, dark microsclerotia measuring 27-34 × 22-26 µm (n = 50) that developed after 14 days of growth at 25°C in the dark. The identification of two isolates was further confirmed by performing real-time PCR assay using a pair of specific primers for internal transcribed spacer (ITS) region of V. dahliae as previously described (Hiemstra et al. 2013). In addition, the molecular subdivision of isolates was further determined to discriminate D and ND molecular types (Keykhasaber 2017). According to molecular assays, isolates were identified as V. dahliae and grouped with ND types. The pathogenicity of isolates was evaluated by root-dipping one-year E. angustifolia seedlings (10 seedlings) into conidial suspensions of 1×107 cfu/ml. Inoculated plants were transplanted in pots containing autoclaved soil and maintained in a greenhouse at 25°C until symptoms appearance. Two seedlings were treated with sterile distilled water as controls. All inoculated seedlings started to show wilting symptoms similar to those present in naturally affected trees within 30 days after inoculation and died thereafter. Furthermore, V. dahliae was consistently isolated from symptomatic tissues. No symptoms were observed on the control plants. The pathogenicity test was repeated twice with similar results. To the best of our knowledge, this is the first report of Verticillium wilt on Russian olive trees in Iran. In Iran, Verticillium wilt is the cause of serious losses in many woody and herbaceous plants with economic importance including many trees belonging to the genus Prunus that are highly susceptible to the disease. In Shahrood (Semnan Province), most agricultural fields have a potato- or tomato- growing history. Verticillium wilt may become an important economic problem in many Russian olive and Prunus orchards in the future since their cultivation is expanding rapidly in many agricultural areas previously dedicated to tomato and potato crops, the majority of which are infested with V. dahliae. References Hiemstra, J. A., Korthals, G. W., Visser, J. H. M., Dalfsen, P. v., Sluis, B. J. v. d., and Smits, A. P. 2013. Control of Verticillium in tree nurseries through biological soil disinfestation. Pages 62-62 in: 11th International Verticillium Symposium, Georg-August-Universität,Göttingen, Germany, 5-8 May 2013, B. Koopmann and A. von Tiedemann, eds. DPG Spectrum Phytomedizin, Göttingen. Keykhasaber, M., Faino L., van den Berg, G.C.M., Hiemstra, J. A., Thomma, B. P. H. J. 2017. A robust method for discriminating defoliating and the non-defoliating pathotypes of V. dahliae. . In; Keykhasaber M. thesis 62-84. Sun, M., and Lin, Q. 2010. A revision of Elaeagnus L. (Elaeagnaceae) in mainland China. J. Systematics and Evolution 48:356-390.

Phylogenetic inference enables reconstruction of a long-overlooked outbreak of almond leaf scorch disease (Xylella fastidiosa) in Europe.

The recent introductions of the bacterium Xylella fastidiosa (Xf) into Europe are linked to the international plant trade. However, both how and when these entries occurred remains poorly understood. Here, we show how almond scorch leaf disease, which affects ~79% of almond trees in Majorca (Spain) and was previously attributed to fungal pathogens, was in fact triggered by the introduction of Xf around 1993 and subsequently spread to grapevines (Pierce's disease). We reconstructed the progression of almond leaf scorch disease by using broad phylogenetic evidence supported by epidemiological data. Bayesian phylogenetic inference predicted that both Xf subspecies found in Majorca, fastidiosa ST1 (95% highest posterior density, HPD: 1990-1997) and multiplex ST81 (95% HPD: 1991-1998), shared their most recent common ancestors with Californian Xf populations associated with almonds and grapevines. Consistent with this chronology, Xf-DNA infections were identified in tree rings dating to 1998. Our findings uncover a previously unknown scenario in Europe and reveal how Pierce's disease reached the continent.

Complete Circularized Genome Data of Two Spanish strains of Xylella fastidiosa (IVIA5235 and IVIA5901) Using Hybrid Assembly Approaches.

Xylella fastidiosa is an economically important plant pathogenic bacterium of global importance associated, since 2013, with a devastating epidemic in olive trees in Italy. Since then, several outbreaks of this pathogen have been reported in other European member countries including Spain, France, and Portugal. In Spain, the three major subspecies (subsp. fastidiosa, multiplex, and pauca) of the bacterium have been detected in the Balearic Islands, but only subspecies multiplex in the mainland (Alicante). We present the first complete genome sequences of two Spanish strains: X. fastidiosa subsp. fastidiosa IVIA5235 from Mallorca and X. fastidiosa subsp. multiplex IVIA5901 from Alicante, using Oxford Nanopore and Illumina sequence reads, and two hybrid approaches for genome assembly. These completed genomes will provide a resource to better understand the biology of these X. fastidiosa strains.

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.