Bacterial extracellular vesicles (BEVs) are released from the surface of bacterial cells and contain a diverse molecular cargo. Studies conducted primarily with bacterial pathogens of mammals have shown that BEVs are involved in multiple processes such as cell-cell communication, the delivery of RNA, DNA, and proteins to target cells, protection from stresses, manipulation of host immunity, and other functions. Until a decade ago, the roles of BEVs in plant-bacteria interactions were barely investigated. However, recent studies have shown that BEVs of plant pathogens possess similar functions as their mammalian pathogen counterparts, and more research is now devoted to study their roles and interactions with plants. In the following methods chapter, we provide five well-validated assays to examine the interaction of BEVs with the plant immune system. These assays rely on different markers or immune outputs, which indicate the activation of plant immunity (defense marker gene expression, reactive oxygen species burst, seedling inhibition). Furthermore, we offer assays that directly evaluate the priming of the immune system following BEV challenge and the effectiveness of its response to subsequent local or systemic infection. Altogether, these assays provide a thorough examination to the interactions of BEVs and the plant immune system.

Gram-negative bacteria form spherical blebs on their cell periphery, which later dissociate from the bacterial cell wall to form extracellular vesicles. These nano scale structures, known as outer membrane vesicles (OMVs), have been shown to promote infection and disease and can induce typical immune outputs in both mammal and plant hosts. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated plants at 2, 6 and 24 h post challenge. The most pronounced transcriptional shift occurred at the first two time points tested, as reflected by the number of differentially expressed genes and the average fold change. OMVs induce a major transcriptional shift towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors. Comparing the response of Arabidopsis to OMVs and to purified elicitors, revealed that OMVs induce a similar suite of genes and pathways as single elicitors, however, pathways activated by OMVs and not by other elicitors were detected. Pretreating Arabidopsis plants with OMVs and subsequently infecting with a bacterial pathogen led to a significant reduction in pathogen growth. Mutations in the plant elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) co-receptor, did not significantly affect the immune priming effect of OMVs. All together these results show that OMVs induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change may facilitate resistance to bacterial infection.

Potato common scab (CS) and peanut pod wart diseases cause substantial economic losses every year in Israel. In this comprehensive study on the Streptomyces spp. population, isolates were collected during 2004–2016 from potato tubers with symptoms grown in Israel, seed tubers imported from Europe and from peanut pods with symptoms grown in Israel. A total of 142 isolates were characterized by PCR using three primer sets ( txtA , tomA and nec1 genes) and by three pathogenicity tests. Seven species were identified among the isolates, S . bottropensis , S . europaeiscabiei , S . griseus , S . scabiei , S . sampsonii , S . turgidiscabies , and S . venezuelae . S . europaeiscabiei was the most dominant among isolates from imported seed tubers, while S . scabiei and S . turgidiscabies were isolated from tubers grown in Israel. Isolates originating from peanuts were significantly more virulent than those from imported seed tubers, as determined by a pathogenicity assay on radish plants. The presence of pathogenic Streptomyces was detected by quantitative PCR (qPCR) in 289 soil samples collected from 31 commercial potato fields with CS history. Eighty percent to 100% of samples collected from fields with a high disease incidence on tubers (50%) gave positive results in qPCR, whereas in samples collected from fields with low CS incidence (2%–30%) the qPCR results were very variable. Potato seed lots imported from Europe during 2008–2021 were found contaminated with CS symptoms at different levels on visual examination. The results of this study indicate that Streptomyces species, in particular S . europaeiscabiei , were introduced to Israel through seed lots imported from Europe.

Pectobacterium brasiliense (Pbr) infects a wide range of crops worldwide, causing potato blackleg and soft rot and vegetable soft rots. This study aimed to characterize the genetic diversity and virulence variability among 68 Pbr strains isolated from either symptomless potato progeny tubers, diseased potato plants, ware potatoes wash water, or vegetables grown in Israel, as well as strains isolated from symptomless seed tubers grown in Europe, or diseased potato plants grown in France. The collection was typed using PCR and TaqMan real-time PCR analyses, dnaX sequence analysis, pulsed-field gel electrophoresis (PFGE), and pectolytic activity. dnaX phylogeny grouped almost all strains in a common genetic clade related to Pbr, which was distinct from the other Pectobacterium species. PFGE analysis identified two main clusters, including one major group of 47 strains with 95%-100% similarity. Maceration assays on two potato cultivars showed significant differences between strains but with no correlations with the source of the strains nor the status of the host (with/without symptoms). Molecular (dnaX sequences and PFGE profiles) and phenotypic analyses (tuber maceration tests) showed that the tested Pbr strains are not a homogeneous group. Analysis of the tested Pbr strains isolated from potato and vegetables grown in fields with a history of potato cultivation suggests that seed tubers imported from Europe may be the main source for Pbr in Israel. To the best of our knowledge, this is the first study that describes biodiversity and population structure of P. brasiliense isolated from potato and vegetables under hot climate conditions.

Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast where they confront plant innate immunity. ABSTRACT Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S. Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H2O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S. Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H2O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S. Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and dl-β-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H2O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1, as did Salmonella apoplastic colonization in H2O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

Acidovorax citrulli is the causal agent of bacterial fruit blotch disease of cucurbits. Strains of this pathogen are distributed into two major groups: Group I strains have been mainly isolated from melon and other non-watermelon cucurbits, while Group II strains have been mainly recovered from watermelon. Here we report the characterization of strains T1 and EP isolated from diseased tomato and eggplant plants, respectively, and further confirmed to belong to A. citrulli species. Based on PCR, PFGE, and rep-PCR, these strains showed high similarity to the Group II strain 7a1. Sequencing and comparative analyses revealed that the genomes of T1 and EP aligned with that of the Group II model strain AAC00-1, over 97.88% and 99.22%, respectively. The virulence of T1, EP, and 7a1 determined on tomato, eggplant, and watermelon was similar and significantly higher than that of Group I strain M6. In contrast, M6 was more virulent on melon. Expression levels of seven virulence genes measured 24 hr after inoculation of tomato, eggplant, watermelon, and melon showed that the expression pattern was generally similar in strains 7a1, T1, and EP, whereas for M6 the expression was high only on melon. Overall, our results indicate that the solanaceous strains belong to Group II. To the best of our knowledge, this is the first study that reports characterization of A. citrulli strains isolated from solanaceous species. The fact that A. citrulli is able to naturally colonize and cause disease in non-cucurbit crops poses additional challenges for management of this important pathogen.

HomePlant DiseaseVol. 104, No. 8First Report of Pectobacterium parmentieri, One of the Causal Agents of Potato Blackleg and Tuber Soft Rot Diseases, in Israel PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Pectobacterium parmentieri, One of the Causal Agents of Potato Blackleg and Tuber Soft Rot Diseases, in IsraelL. Tsror (Lahkim), O. Erlich, S. Lebiush, I. Galilov, M. Hazanovsky, L. Chalupowicz, M. Reuven, O. Dror, and S. Manulis-SassonL. Tsror (Lahkim)†Corresponding author: L. Tsror (Lahkim); E-mail Address: [email protected]http://orcid.org/0000-0001-9759-3575Agricultural Research Organization (ARO), Department of Plant Pathology and Weed Research, Gilat Research Center, MP Negev 85280, IsraelSearch for more papers by this author, O. ErlichAgricultural Research Organization (ARO), Department of Plant Pathology and Weed Research, Gilat Research Center, MP Negev 85280, IsraelSearch for more papers by this author, S. LebiushAgricultural Research Organization (ARO), Department of Plant Pathology and Weed Research, Gilat Research Center, MP Negev 85280, IsraelSearch for more papers by this author, I. GalilovAgricultural Research Organization (ARO), Department of Plant Pathology and Weed Research, Gilat Research Center, MP Negev 85280, IsraelSearch for more papers by this author, M. HazanovskyAgricultural Research Organization (ARO), Department of Plant Pathology and Weed Research, Gilat Research Center, MP Negev 85280, IsraelSearch for more papers by this author, L. ChalupowiczARO, Department of Plant Pathology and Weed Research, The Volcani Center, Rishon LeZion 7528809, IsraelSearch for more papers by this author, M. ReuvenARO, Department of Plant Pathology and Weed Research, The Volcani Center, Rishon LeZion 7528809, IsraelSearch for more papers by this author, O. DrorARO, Department of Plant Pathology and Weed Research, The Volcani Center, Rishon LeZion 7528809, IsraelSearch for more papers by this author, and S. Manulis-SassonARO, Department of Plant Pathology and Weed Research, The Volcani Center, Rishon LeZion 7528809, IsraelSearch for more papers by this authorAffiliationsAuthors and Affiliations L. Tsror (Lahkim)1 † O. Erlich1 S. Lebiush1 I. Galilov1 M. Hazanovsky1 L. Chalupowicz2 M. Reuven2 O. Dror2 S. Manulis-Sasson2 1Agricultural Research Organization (ARO), Department of Plant Pathology and Weed Research, Gilat Research Center, MP Negev 85280, Israel 2ARO, Department of Plant Pathology and Weed Research, The Volcani Center, Rishon LeZion 7528809, Israel Published Online:16 Jun 2020https://doi.org/10.1094/PDIS-02-20-0226-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Potato (Solanum tuberosum) blackleg and tuber soft rot diseases in Israel, caused by Pectobacterium and Dickeya spp., originate from seed tubers imported from Europe. These diseases are of great concern due to the warm climatic conditions during the growing season that favor disease expression and may result in establishment of the pathogens in potato fields and their spread to weeds and other crops (Tsror (Lahkim) et al. 2009, 2012). P. parmentieri was recently reported in Europe as an emerging threat to potato production, among other Pectobacterium and Dickeya spp. (Suárez et al. 2017; van der Wolf et al. 2017; Zoledowska et al. 2018). Surveys for pectinolytic bacteria were carried out during spring 2019 in an experimental plot located at Gilat Research Center Israel (Agricultural Research Organization), where different imported seed lots were planted, and during 2017 in a commercial packhouse. P. parmentieri was detected in diseased potato plants (cv. Sifra) and in wash water sampled during the process of packing ware potato, respectively. Plant material was surface sterilized with 0.3% (v/v) hypochlorite for 3 min, and 10 segments from the stem base of each plant were homogenized with 10 ml of sterile distilled water. Sample homogenates were plated on crystal violet pectate medium (CVP). The wash water surveys were done during May, July, and August 2017, with processed tubers (cvs. Sifra, Winston, Allians, Evolution, and Rosanna) obtained from commercial spring plots, located in Western Negev, in which imported seeds from Europe were planted in January. Wash water samples (200 ml) were filtered through rough filter paper followed by fine filtering using Millipore filter paper (45 µm). The Millipore filter paper was resuspended in 2 ml of sterile distilled water, and suspensions were plated on CVP. Single colonies obtained on CVP medium were purified by repeated subculturing on nutrient agar (Difco), and selected strains were further characterized. Most of the isolated colonies were identified as Pectobacterium carotovorum subsp. carotovorum, P. carotovorum subsp. brasiliense, or Dickeya solani; however, one isolate from the diseased plants and three isolates from the wash water were characterized as Pectobacterium parmentieri. DNA extracted from these colonies reacted positively in a qPCR assay based on the YD repeat protein gene PW7011F/R specific primers (Kim et al. 2012) and also in a TaqMan assay based on the mdh sequence (van der Wolf et al. 2017). Sequencing the gapA gene (Cigna et al. 2017) identified the isolate as P. parmentieri (GenBank accession no. MN887102), with 100% similarity to P. parmentieri strain SCC3193 (CP003415.1). All four isolates caused maceration of potato tubers at 30°C (Tsror (Lahkim) et al. 2013). In a pathogenicity assay with the isolate from the diseased plant and one of the isolates from the wash water, conducted at 30°C on potato plants (cv. Allians), wilting symptoms appeared 48 h after stem inoculation (10 μl of bacterial suspension at 108 cells/ml). Reisolated bacteria from the inoculated plants were confirmed as P. parmentieri by PCR. This is the first report on the presence of P. parmentieri in Israel. These findings are of phytosanitary significance due to the recent reports from Europe indicating that P. parmentieri has an important role in the pectinolytic bacterial complex causing potato blackleg and thus has the potential to cause disease under warm climate conditions as well.The author(s) declare no conflict of interest.References:Cigna, J., et al. 2017. Plant Dis. 101:1278. https://doi.org/10.1094/PDIS-12-16-1810-RE Link, ISI, Google ScholarKim, M. H., et al. 2012. Plant Dis. 96:253. https://doi.org/10.1094/PDIS-06-11-0511 Link, ISI, Google ScholarSuárez, M. B., et al. 2017. Plant Dis. 101:1029. https://doi.org/10.1094/PDIS-01-17-0013-PDN Link, ISI, Google ScholarTsror (Lahkim), L., et al. 2009. Eur. J. Plant Pathol. 123:311. https://doi.org/10.1007/s10658-008-9368-0 Crossref, ISI, Google ScholarTsror (Lahkim), L., et al. 2012. Plant Pathol. 61:161. Crossref, ISI, Google ScholarTsror (Lahkim), L., et al. 2013. Plant Pathol. 62:1097. https://doi.org/10.1111/ppa.12030 Crossref, ISI, Google Scholarvan der Wolf, J. M., et al. 2017. Plant Pathol. 66:571. https://doi.org/10.1111/ppa.12600 Crossref, ISI, Google ScholarZoledowska, S., et al. 2018. Plant Dis. 102:154. https://doi.org/10.1094/PDIS-05-17-0761-RE Link, ISI, Google ScholarThe author(s) declare no conflict of interest.Funding: This study was supported by the Israeli Ministry of Agricultural and Rural Development grant no. 20‐02‐0051.DetailsFiguresLiterature CitedRelated Vol. 104, No. 8 August 2020SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionApple scab lesions on h(11) Malus baccata ‘Jackii’ found in 2011 at Skierniewice, Poland (A. Patocchi et al.). Photo credit: A. Patocchi. Healthy `ōhi`a seedling growing from a crack in a lava field (M. A. Hughes et al.). Photo credit: K. Hughes. Metrics Article History Issue Date: 28 Jul 2020Published: 16 Jun 2020First Look: 25 Mar 2020Accepted: 24 Mar 2020 Pages: 2288-2288 Information© 2020 The American Phytopathological SocietyFundingIsraeli Ministry of Agricultural and Rural DevelopmentGrant/Award Number: 20‐02‐0051KeywordsvegetablesSolanum tuberosumThe author(s) declare no conflict of interest.Cited bySurvey of Soft Rot Pectobacteriaceae Infecting Potatoes in South Africa5 November 2022 | Potato Research, Vol. 215Characterization of Pectobacterium brasiliense strains from potato and vegetables in Israel30 August 2021 | Plant Pathology, Vol. 70, No. 9Wild Potatoes: A Genetic Reservoir for Potato BreedingDiseases Caused by Pectobacterium and Dickeya Species Around the World5 January 2021

Potato blackleg and tuber soft rots in Israel, caused by Pectobacterium and Dickeya spp., originating from seed tubers imported from Europe, are of a great concern due to the warm climatic conditions during the growing season that favour…

SummaryPantoea agglomerans (Pa), a widespread commensal bacterium, has evolved into a host‐specific gall‐forming pathogen on gypsophila and beet by acquiring a plasmid harbouring a type III secretion system (T3SS) and effectors (T3Es). Pantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) elicits galls on beet and gypsophila. HsvG and HsvB are two paralogous T3Es present in both pathovars and act as host‐specific transcription activators on gypsophila and beet, respectively. PthG and PseB are major T3Es that contribute to gall development of Pag and Pab, respectively. To establish the minimal combinations of T3Es that are sufficient to elicit gall symptoms, strains of the nonpathogenic bacteria Pseudomonas fluorescens 55, Pa 3‐1, Pa 98 and Escherichia coli, transformed with pHIR11 harbouring a T3SS, and the phytopathogenic bacteria Erwinia amylovora, Dickeya solani and Xanthomonas campestris pv. campestris were transformed with the T3Es hsvG, hsvB, pthG and pseB, either individually or in pairs, and used to infect gypsophila and beet. Strikingly, all the tested nonpathogenic and phytopathogenic bacterial strains harbouring hsvG and pthG incited galls on gypsophila, whereas strains harbouring hsvB and pseB, with the exception of E. coli, incited galls on beet.

Potato blackleg and tuber soft rots in Israel, caused by Pectobacterium and Dickeya spp., originating from seed tubers imported from Europe, are of a great concern due to the warm climatic conditions during the growing season that favour disease expression and may result in the establishment of the pathogens in potato fields and their spread to weeds and other crops (Tsror et al., 3). In a previous survey in Israel, Dickeya solani was isolated only from Cyperus rotundus, out of symptomless plants of 12 weed species (Tsror et al., 4). Recently, Pectobacterium carotovorum subsp. brasiliense (Pcb) was reported as an emerging threat in Western Europe and the primary blackleg-causing pathogen for some countries in the region (van der Wolf et al., 6). The pathogen is also responsible for considerable disease in Israel (personal information). To study the dissemination of Pcb to weeds, a survey was conducted in potato fields where Pcb-infected plants were detected during the spring of 2018. Symptomless weed plants from 13 genera and 10 families, namely Solanum nigrum (Solanaceae), Polygonum equisetiforme (Polygonaceae), Centaurea procurrens, Sonchus oleraceus (Asteraceae), Lolium rigidum, Phalaris brachystachys, Avena sterilis (Poaceae), Malva nicaeensis (Malvaceae), Amaranthus blitoides (Amaranthaceae), Chenopodium murale (Chenopodiaceae), Chrozophora tinctoria (Euphorbiaceae), Orobanche aegyptiaca (Orobanchaceae) and Erucaria rostrate (Brassicaceae), were randomly collected from areas where potato plants infected by Pcb or D. solani had been identified. Roots or stems (in the case of O. aegyptiaca) of 6-15 plants of each weed were washed, surface sterilised, macerated in sterile distilled water and the suspensions were plated on crystal violet pectate medium. Cavity forming bacteria were transferred to nutrient agar for further characterisation. Pcb was isolated only from latently infected Malva nicaeensis plants and with an incidence of 16.7%. DNA extracted from these colonies reacted positively in a PCR assay using BR1f/L1r specific primers (Duarte et al., 2) and also in a TaqMan assay based on araC sequence (van der Wolf et al., 6). Sequencing the gapA gene (Cigna et al., 1) identified the isolate as Pcb (GenBank Accession No. MK086015). A maceration assay on potato tubers at 30°C (Tsror et al., 5) was positive. This is the first report of latent infection of Pectobacterium carotovorum subsp. brasiliense in one of the most prevalent weeds in potato fields in Israel. Malva nicaeensis may serve as an alternative host for Pcb allowing the pathogen to survive in the absence of the host crop. This study was supported by the Israeli Ministry of Agricultural and Rural Development (grant no. 20-02-0051).

Cell-to-cell communication mediated by the diffusible signal factor (DSF) is a common form of gene regulation and plays an important role in virulence of many plant pathogenic bacteria including Xanthomonas spp. Here we describe several approaches to study the involvement of DSF-dependent QS system of the plant pathogenic bacteria Xanthomonas campestris pv. pelargonii (Xhp) as an example of the Xanthomonas spp. The methods described include detection and measurement of DSF, movement in planta, colonization, and aggregate formation.

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is an important disease of tomatoes. Seedlings supplied by nurseries may serve as a source of primary inoculum in commercial production units. In the nursery, infections originate from contaminated seeds, and the pathogen may also spread and develop during the nursery stage. In the nursery, seedlings are irrigated through overhead irrigation systems and fertilizers are incorporated in the irrigation water (fertigation). It was hypothesized that addition of fertilizer to the irrigation water influences the host-pathogen interactions and alters the development of the disease in the nursery. The specific goals of the research were to 1) study the effect of fertigation on disease severity and 2) examine whether rinsing the foliage with fresh water soon after fertigation affects seedling infection. Results showed that even a single fertigation cycle prior to Cmm inoculation significantly increased disease severity as compared with seedlings that were not fertigated. The pH of the fertigation solution had no significant effect on disease severity. Rinsing with fresh water soon after fertigation decreased disease severity by similar to 75% compared with unrinsed plants.

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single‐molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR , ELISA , Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis.

Salmonella enterica serovar Typhimurium, a human enteric pathogen, has the ability to multiply and survive endophytically in plants. Genes encoding the type III secretion system (T3SS) or its effectors (T3Es) may contribute to its colonization. Two reporter plasmids for T3E translocation into plant cells that are based on hypersensitive response domains of avirulence proteins from the Pantoea agglomerans-beet and Xanthomonas euvesicatoria-pepper pathosystems were employed in this study to investigate the role of T3Es in the interaction of Salmonella ser. Typhimurium 14028 with plants. The T3Es of Salmonella ser. Typhimurium, SipB and SifA, which are translocated into animal cells, could not be delivered by Salmonella ser. Typhimurium into cells of beet roots or pepper leaves. In contrast, these effectors were translocated into plant cells by the phytopathogenic bacteria P. agglomerans pv. betae, Erwinia amylovora, and X. euvesicatoria. Similarly, HsvG, a T3E of P. agglomerans pv. gypsophilae, and XopAU of X. euvesicatoria could be translocated into beet roots and pepper leaves, respectively, by the plant pathogens but not by Salmonella ser. Typhimurium. Mutations in Salmonella ser. Typhimurium T3SS genes invA, ssaV, sipB, or sifA, did not affect its endophytic colonization of lettuce leaves, supporting the notion that S. enterica cannot translocate T3Es into plant cells.

Pantoea agglomerans, a widespread epiphytic bacterium, has evolved into a hypersensitive response and pathogenicity (hrp)-dependent and host-specific gall-forming pathogen by the acquisition of a pathogenicity plasmid containing a type III secretion system (T3SS) and its effectors (T3Es). Pantoea agglomerans pv. betae (Pab) elicits galls on beet (Beta vulgaris) and gypsophila (Gypsophila paniculata), whereas P. agglomerans pv. gypsophilae (Pag) incites galls on gypsophila and a hypersensitive response (HR) on beet. Draft genome sequences were generated and employed in combination with a machine-learning approach and a translocation assay into beet roots to identify the pools of T3Es in the two pathovars. The genomes of the sequenced Pab4188 and Pag824-1 strains have a similar size (∼5 MB) and GC content (∼55%). Mutational analysis revealed that, in Pab4188, eight T3Es (HsvB, HsvG, PseB, DspA/E, HopAY1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on beet and gypsophila. In Pag824-1, nine T3Es (HsvG, HsvB, PthG, DspA/E, HopAY1, HopD1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on gypsophila, whereas the PthG effector triggers HR on beet. HsvB, HsvG, PthG and PseB appear to endow pathovar specificities to Pab and Pag, and no homologous T3Es were identified for these proteins in other phytopathogenic bacteria. Conversely, the remaining T3Es contribute to the virulence of both pathovars, and homologous T3Es were found in other phytopathogenic bacteria. Remarkably, HsvG and HsvB, which act as host-specific transcription factors, displayed the largest contribution to disease development.

Clavibacter michiganensis ssp. michiganensis (Cmm) causes substantial economic losses in tomato production worldwide. The disease symptoms observed in plants infected systemically by Cmm are wilting and canker on the stem, whereas blister-like spots develop in locally infected leaves. A wide repertoire of serine proteases and cell wall-degrading enzymes has been implicated in the development of wilt and canker symptoms. However, virulence factors involved in the formation of blister-like spots, which play an important role in Cmm secondary spread in tomato nurseries, are largely unknown. Here, we demonstrate that Cmm virulence factors play different roles during blister formation relative to wilting. Inoculation with a green fluorescent protein (GFP)-labelled Cmm382 indicates that penetration occurs mainly through trichomes. When spray inoculated on tomato leaves, the wild-type Cmm382 and Cmm100 (lacking plasmids pCM1 and pCM2) strains form blister-like spots on leaves, whereas Cmm27 (lacking the chp/tomA pathogenicity island) is non-pathogenic, indicating that plasmid-borne genes, which have a crucial role in wilting, are not required for blister formation. Conversely, mutations in chromosomal genes encoding serine proteases (chpC and sbtA), cell wall-degrading enzymes (pgaA and endX/Y), a transcriptional regulator (vatr2), a putative perforin (perF) and a putative sortase (srtA) significantly affect disease incidence and the severity of blister formation. The transcript levels of these genes, as measured by quantitative reverse transcription-polymerase chain reaction, showed that, during blister formation, they are expressed early at 8-16 h after inoculation, whereas, during wilting, they are expressed after 24-72 h or expressed at low levels. Plant gene expression studies suggest that chpC is involved in the suppression of host defence.