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Bacterial diseases affecting wheat production in Uruguay are an issue of growing concern yet remain largely uninvestigated in the region. Surveys of 61 wheat fields carried out from 2017 to 2019 yielded a regional collection of 63 strains identified by 16S rRNA gene analysis as Xanthomonas spp. A real-time PCR protocol using species-specific primers previously reported allowed the identification of 44 strains as X. translucens, the causal agent of bacterial leaf streak (BLS) in wheat and other cereal crops. Multilocus sequence analysis (MLSA) of four housekeeping genes (dnaK, fyuA, gyrB, and rpoD) revealed that these strains were most closely related to X. translucens pv. undulosa, the pathovar that is most commonly associated with BLS of wheat. Multilocus sequence typing (MLST) was applied to examine the genetic diversity among X. translucens strains. Strains were assigned to four different sequence types, three of which have been previously reported globally. Additionally, 17 Xanthomonas strains not belonging to X. translucens were obtained from diseased wheat leaves. Phylogenetic analysis showed that these strains are closely related to Xanthomonas prunicola, and clustered together with previously uncharacterized Xanthomonas strains isolated from wheat in Minnesota, US. In planta pathogenicity assays carried out on a BLS susceptible wheat cultivar showed that X. translucens pv. undulosa strains caused brown necrosis symptoms typical of BLS, while non-translucens Xanthomonas sp. strains elicited an atypical symptom of dry necrosis. These findings suggest that local wheat fields are affected by X. translucens pv. undulosa, and by a new wheat pathogen within the Xanthomonas genus.

As with many phytopathogenic bacteria, the virulence of Xanthomonas campestris pv. campestris, the causal agent of black rot disease in cruciferous plants, relies on secretion of a suite of extracellular enzymes that includes cellulase (endoglucanase), pectinase, protease, and amylase. Although the role in virulence of a number of these enzymes has been assessed, the contribution of amylase to X. campestris pv. campestris virulence has yet to be established. In this work, we investigated both the role of extracellular amylase in X. campestris pv. campestris virulence and the control of its expression. Deletion of XC3487 (here renamed amyAXcc), a putative amylase-encoding gene from the genome of X. campestris pv. campestris strain 8004, resulted in a complete loss of extracellular amylase activity and significant reduction in virulence. The extracellular amylase activity and virulence of the amyAXcc mutant could be restored to the wild-type level by expressing amyAXcc in trans. These results demonstrated that amyAXcc is responsible for the extracellular amylase activity of X. campestris pv. campestris and indicated that extracellular amylase plays an important role in X. campestris pv. campestris virulence. We also found that the expression of amyAXcc is strongly induced by starch and requires activation by the global posttranscriptional regulator RsmA. RsmA binds specifically to the 5′-untranslated region of amyAXcc transcripts, suggesting that RsmA regulates amyAXcc directly at the posttranscriptional level. Unexpectedly, in addition to posttranscriptional regulation, the use of a transcriptional reporter demonstrated that RsmA also regulates amyAXcc expression at the transcriptional level, possibly by an indirect mechanism.

BACKGROUND Induced resistance against several plant pathogens was reported using different beneficial plant growth-promoting microorganisms. The potential of five fungal isolates, Trichoderma harzianum GT 3-2, Fusarium equiseti GF 18-3, F. equiseti GF 19-1, Phoma sp. GS 10-1 and Phoma sp. GS 14-1, to stimulate tomato growth and resistance against bacterial speck disease caused by Pseudomonas syringae pathovar (pv.) tomato DC3000 was evaluated. RESULTS Based on the results of disease severity and growth promotion experiments, GF 18-3 exhibited the best results among all fungal isolates. Treatment with barley grain inocula (BGI) and culture filtrate (CF) of the isolates promoted tomato growth and suppressed the pathogen in pot trials. Furthermore, expressions of the pathogenesis-related genes (PR-1, β-1,3-glucanase A, β-1,3-glucanase B and LOX) were relatively higher than the control in the leaves of tomato plants treated with both BGI and CF. The transcription levels remained consistently higher than the control plants for 6 days post-inoculation with pathogen. CONCLUSION Taken together, the results indicate that the tested fungal isolates have the potential to promote tomato growth and induce systemic resistance against the bacterial speck disease. Analysis of certain PR gene expression revealed significant activation in both BGI and CF treatments, leading to stimulated resistance against the pathogen. © 2021 Society of Chemical Industry.

Pseudomonas syringae pv. actinidiae is a phytopathogen that causes devastating bacterial canker in kiwifruit. Among five biovars defined by genetic, biochemical, and virulence traits, P. syringae pv. actinidiae biovar 3 (Psa3) is the most aggressive and is responsible for the most recent reported outbreaks; however, the molecular basis of its heightened virulence is unclear. Therefore, we designed the first P. syringae multistrain whole-genome microarray, encompassing biovars Psa1, Psa2, and Psa3 and the well-established model P. syringae pv. tomato, and analyzed early bacterial responses to an apoplast-like minimal medium. Transcriptomic profiling revealed i) the strong activation in Psa3 of all hypersensitive reaction and pathogenicity (hrp) and hrp conserved (hrc) cluster genes, encoding components of the type III secretion system required for bacterial pathogenicity and involved in responses to environmental signals; ii) potential repression of the hrp/hrc cluster in Psa2; and iii) activation of flagellum-dependent cell motility and chemotaxis genes in Psa1. The detailed investigation of three gene families encoding upstream regulatory proteins (histidine kinases, their cognate response regulators, and proteins with diguanylate cyclase or phosphodiesterase domains) indicated that cyclic di-GMP may be a key regulator of virulence in P. syringae pv. actinidiae biovars. The gene expression data were supported by the quantification of biofilm formation. Our findings suggest that diverse early responses to the host apoplast, even among bacteria belonging to the same pathovar, can lead to different virulence strategies and may explain the differing outcomes of infections. Based on our detailed structural analysis of hrp operons, we also propose a revision of hrp cluster organization and operon regulation in P. syringae. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

In Montenegro, stone fruit species are grown on intensive and semi-intensive commercial plantations. However, almond production is mainly organized on family gardens and for household consumption. During two seasons (2017–2018), we surveyed apricot, peach, nectarine, sweet cherry, Japanese plum, and almond orchards for the presence of bacterial diseases at different geographical locations in Montenegro. From leaf, petiole and fruit lesions, branch or twig cankers, and necrotizing buds, a total of 29 isolates were obtained and subjected to identification based on their morphological, pathogenic, biochemical, and molecular characteristics. Pathogenicity of the isolates was confirmed by reproducing the symptoms on leaves, fruits, and twigs of the corresponding host plants. The biochemical tests indicated that the isolates belong to Pseudomonas syringae. However, isolates’ characterization showed variation in their phenotypic and molecular features. The presence of the syrB gene and ice nucleation activity grouped most of the isolates within pathovar syringae. The results of rep-PCR using the BOX primer revealed high genetic diversity of isolates. Multilocus sequence analysis (MLSA), using four housekeeping genes, showed that 27 isolates belong to the genomic species 1, P. syringae sensu stricto, corresponding to P. syringae phylogroup 2. However, isolates from the same phylogroup 2 did not form a monophyletic group. One strain isolated from apricot was most distinct and similar to members of genomic species 2, phylogroup 3. All tested isolates showed significant levels of resistance to copper sulfate and high level of sensitivity to streptomycin sulfate in vitro.

Background and Purpose: Diarrhoeagenic E. coli (DEC) is one of the germs responsible for childhood diarrhea in developing countries. This study aims at determining the prevalence of the five main pathotypes of DEC isolated from faeces of children under five years old with diarrhea or not, living in the city of Koula-Moutou. Methodology: Isolates of E. coli were phenotypically screened on chromIDTM agar and molecularly by multiplex PCR to detect the presence of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorragic E. coli (EHEC) and enteroinvasive E. coli (EIEC). The evaluation of their sensitivity to 12 β-lactam antibiotic molecules was carried out by Kirby Bauer method. This method has also made it possible to characterize phenotypically the different β-lactamases produced. Results and Conclusion: Overall, at least one DEC pathovar was detected in the 63 E. coli strains with phenotypic and molecular frequencies of 63.5% and 68.5% respectively. Thus, ETEC (28.3%) and EHEC (28.3%) were the most frequent DEC in diarrheal isolates. ETEC/EHEC hybrid was recorded in both groups with rates of 7.5% in diarrheal cases and 10.0% for controls. The results showed produced carbapenemase type β-lactamases (31.7%), followed by ESBL (24.4%) and few produced high level penicillinases (4.9%). The DEC, in particular ETEC and EHEC are most likely the epidemiological agents responsible for childhood diarrhea in this study.

Pseudomonas syringae is the most common phytopathogenic bacterium with a wide range of target plants, which include important cereals such as wheat. One of the main pathogens of bacterial diseases of wheat is Pseudomonas syringae pv. atrofaciens. In some countries, wheat yield losses caused by this phytopathogenic bacterium reach 50%. Currently, the taxonomy of P. syringae includes more than 50 pathovars with varying degrees of adaptation to wheat lesions. One of them is Pseudomonas syringae pv. сoronafaciens. P. syringae pv. Coronafaciens is non-host pathogen for wheat. However, the infectionsof a wide range of crops, including wheat, with this pathogen attracts the attention of both researchers and specialiss of the agro-industrial complex. The study of the mechanisms of wheat resistance to host and non-host pathovars of P. syringae is of great interest, both in terms of in-depth study of the pathogen and in the perspective of selection of bacterial disease-resistant varieties of this strategically important grain crop for Ukraine. The aim of the study was to compare the antioxidant reactions of wheat seedlings of different winter wheat varieties under the grain exposition to P. syringae of different pathovars and their lipopolysaccharides (LPS). It was found that reactive oxygen species generation, as a mechanism of plant immune protection against phytopathogenic pseudomonads, is equally activated in the case of exposure to both host and nonhost pathovars and to a lesser extent in the case of the exposure with LPS of both pathovars. In grains of Favoritka variety (most sensitive to phytopathogenic pseudomonads) exposed to host pathovar, significant activation of antioxidant enzymes was observed. Exposure to the non-host pathovar causes sharp proline accumulation. Thus, the sensitivity of wheat seedlings to phytopathogenic host and non-host pathovars of phytopathogenic pseudomonads largely depends on the balanced functioning of the antioxidant defense system. Taken together, these data indicate the wheat cell oxidative metabolism as a target for selection of varieties resistant to phytopathogenic bacteria.

Summary The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l‐ and d‐rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o‐methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was reclassified as X. campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.

Thirty isolates of Pseudomonas syringae other than pathovar (pv.) phaseolicola, recovered from beans (18) and weeds (12) in a Northern Spanish region, were studied by a polyphasic approach, including phenotypic tests, detection of levansucrase and toxin (syringomycin and syringopeptin) genes, typing by RAPD (Random Amplified Polymorphic DNA) and PFGE (Pulsed-Field Gel Electrophoresis). The isolates were phenotypically heterogeneous, and both RAPD and PFGE, probed to be highly discriminative, as they were almost able to differentiate at the level of isolate. Phylogenetic analysis based on conserved genes of the core genome (rpoD, gltA and gyrB) distributed the isolates in three different clades (2A, 2B and 2C) belonging to phylogroup 2 of the P. syringae complex. According to toxin production, 18 of them, within clade 2B, were assigned to P. syringae pv. syringae and five of these caused symptoms after experimental inoculation on bean pods. Four of the five pathogenic isolates were obtained from weeds, supporting the role of weeds associated with bean crops, particularly Cyperus rotundus, Malva sylvestris, and Polygonum lapathifolium, as a reservoir of the pathogen. In conclusion, results of this study suggest that apart from P. syringae pv. phaseolicola, P. syringae isolates circulating in our region are mostly epiphytes, and support the role of weeds associated with bean crops as reservoir of pathogenic strains of P. syringae pv. syringae.

Populations of Phytophthora infestans, the oomycete causal agent of potato late blight in the United States, are predominantly asexual, and isolates are characterized by clonal lineage or asexual descendants of a single genotype. Current tools for clonal lineage identification are time consuming and require laboratory equipment. We previously found that foliar spectroscopy can be used for high-accuracy pre- and postsymptomatic detection of P. infestans infections caused by clonal lineages US-08 and US-23. In this work, we found subtle but distinct differences in spectral responses of potato foliage infected by these clonal lineages in both growth-chamber time-course experiments (12- to 24-h intervals over 5 days) and naturally infected samples from commercial production fields. In both settings, we measured continuous visible to shortwave infrared reflectance (400 to 2,500 nm) on leaves using a portable spectrometer with contact probe. We consistently discriminated between infections caused by the two clonal lineages across all stages of disease progression using partial least squares (PLS) discriminant analysis, with total accuracies ranging from 88 to 98%. Three-class random forest differentiation between control, US-08, and US-23 yielded total discrimination accuracy ranging from 68 to 76%. Differences were greatest during presymptomatic infection stages and progressed toward uniformity as symptoms advanced. Using PLS-regression trait models, we found that total phenolics, sugar, and leaf mass per area were different between lineages. Shortwave infrared wavelengths (>1,100 nm) were important for clonal lineage differentiation. This work provides a foundation for future use of hyperspectral sensing as a nondestructive tool for pathovar differentiation.

Bacterial leaf streak caused by different pathovars of Xanthomonas translucens is the most important seedborne bacterial disease of small grain cereals. However, variations in the virulence-associated genomic areas of the pathogen remain uninvestigated. In this study, the diversity of transcription activator-like effectors (TALE) was investigated using the Southern blotting of BamHI-digested genomic DNAs in the Iranian strains of X. translucens. All 65 X. translucens strains were assigned into 13 genotypes, where 57 X. translucens pv. undulosa strains were placed in genotypes 1 to 8, and seven X. translucens pv. translucens strains were placed in genotypes 9 to 12. Interestingly, we did not find any TALE genes in the strain XtKm7 (genotype 13), which showed to be pathogenic only on barley. Virulence and aggressiveness of these strains in greenhouse conditions were in agreement with the TALE-based clustering of the strains in the pathovar level, though variations were observed in the aggressiveness of X. translucens pv. undulosa strains. In general, strains containing higher numbers of putative TALE genes were more virulent on wheat and barley than strains containing fewer. This is the first TALE-based genetic diversity analysis on X. translucens strains and provides novel insights into the virulence repertories and genomic characteristics of the pathogen. Further investigations using TALE mutagenesis and complementation analysis are warranted to precisely elucidate the role of each detected X. translucens TALE in bacterial virulence and aggressiveness either on wheat or barley.

Bacterial leaf streak (BLS) of wheat and barley has been a disease of increasing concern in the Upper Midwest over the past decade. In this study, intra- and interfield genetic and pathogenic diversity of bacteria causing BLS in Minnesota was evaluated. In 2015, 89 strains were isolated from 100 leaf samples collected from two wheat and two barley fields naturally infected with BLS. Virulence assays and multilocus sequence alignments of four housekeeping genes supported pathovar identifications. All wheat strains were pathogenic on wheat and barley and belonged to the same lineage as the Xanthomonas translucens pv. undulosa-type strain. All barley strains were pathogenic on barley but not on wheat. Three lineages of barley strains were detected. The frequency and number of sequence types of each pathovar varied within and between fields. A significant population variance was detected between populations of X. translucens pv. undulosa collected from different wheat fields. Population stratification of X. translucens pv. translucens was not detected. Significant differences in virulence were detected among three dominant sequence types of X. translucens pv. undulosa but not those of X. translucens pv. translucens. Field trials with wheat and barley plants inoculated with strains of known sequence type and virulence did not detect significant race structures within either pathovar. Knowledge of virulence, sequence types, and population structures of X. translucens on wheat and barley can support studies on plant–bacterial interactions and breeding for BLS disease resistance.

Pseudomonas syringae-secreted HopA1 effectors are important determinants in host range expansion and increased pathogenicity. Their recent acquisitions via horizontal gene transfer in several non-pathogenic Pseudomonas strains worldwide have caused alarming increase in their virulence capabilities. In Arabidopsis thaliana, RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) gene confers effector-triggered immunity (ETI) against HopA1pss derived from P. syringae pv. syringae strain 61. Surprisingly, a closely related HopA1pst from the tomato pathovar evades immune detection. These responsive differences in planta between the two HopA1s represents a unique system to study pathogen adaptation skills and host-jumps. However, molecular understanding of HopA1′s contribution to overall virulence remain undeciphered. Here, we show that immune-suppressive functions of HopA1pst are more potent than HopA1pss. In the resistance-compromised ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) null-mutant, transcriptomic changes associated with HopA1pss-elicited ETI are still induced and carry resemblance to PAMP-triggered immunity (PTI) signatures. Enrichment of HopA1pss interactome identifies proteins with regulatory roles in post-transcriptional and translational processes. With our demonstration here that both HopA1 suppress reporter-gene translations in vitro imply that the above effector-associations with plant target carry inhibitory consequences. Overall, with our results here we unravel possible virulence role(s) of HopA1 in suppressing PTI and provide newer insights into its detection in resistant plants.

Nitric oxide (NO) is a signaling molecule that regulates various processes, including plant growth and development, immunity, and environmental interactions. Using high throughput RNA-seq data, we explored the role of the NO-induced ATILL6 gene in plant growth and defense using functional genomics. The atill6 mutant and wild-types were challenged with either oxidative (H2O2, MV) or nitro-oxidative (CySNO, GSNO) stress conditions, and the phenotypic results showed that ATILL6 gene differentially regulates cotyledon development frequency (CDF) as well as the root and shoot lengths of the plants. To investigate whether ATILL6 plays a role in plant basal or resistance (R)-gene-mediated defense, the plants were challenged with either virulent or avirulent strains of Pseudomonas syringae pathovar tomato (Pst) DC3000. The atill6 line showed a susceptible phenotype, higher pathogen growth, and highly reduced transcript accumulation of PR1 and PR2 genes. These results suggested that ATILL6 positively regulates plant basal defense. Furthermore, after the inoculation of atill6 with avirulent Pst (DC3000), the expressions of the PR1 and PR2 genes decreased, suggesting a positive role in R-gene-mediated resistance in protecting the plant from further spread of disease. We also investigated the role of ATILL6 in systemic acquired resistance (SAR), and the results showed that ATILL6 positively regulates SAR, as the mutant line atill6 has significantly (p ≤ 0.05) lower transcript accumulation of PR, G3DPH, and AZI genes. Overall, these results indicate that the NO-induced ATILL6 gene differentially regulates plant growth and positively regulates plant basal defense, R-gene-mediated resistance, and SAR.

A sensitive and specific assay was developed to detect bacterial leaf and flower spot disease on zinnia caused by Xanthomonas campestris pv. zinniae (Xcz). A draft genome sequence was determined for Xcz strain E4, isolated from zinnia in Jilin province, China. Comparative genomics analyses were utilized to identify a sequence specific to Xcz in the unique gene 0834. This sequence region was amplified as 1044 bp DNA fragment using designed primers 0834F and 0834R. Using this primer set, specific PCR products were only amplified from DNA from Xcz strains and not from other pathovars of X. campestris, other species of Xanthomonas, or from other genera such as Pseudomonas and Bacillus. The limit of PCR detection in pure culture suspension was approximately 1.7 × 103 CFU/ml per reaction. Specific amplification of the fragments for Xcz was sensitive relatively to the technique used, detecting as low as 0.1 pg template DNA. The PCR technique was also applied to detect the Xcz pathogen in naturally infected leaves and seeds of zinnia.

Plant cytokinins (CKs) are essential for many central cellular processes and play important roles in the interaction between bacteria and plants. Perception of CK is executed by the CHASE domain in the histidine kinase sensors of a class of two-component regulatory systems. Despite advances in understanding the structural basis for CK perception by the sensor AHK4 in Arabidopsis, the molecular mechanism of CK binding by other sensors is unclear. Here, we report the crystal structure of the CHASE domain in the histidine kinase PcrK of the bacterial plant pathogen Xanthomonas campestris pathovar campestris, which senses plant CK, determined at 2.55 A resolution. The structure reveals that the PcrK has an AHK4-like overall topology and assembles into a homodimer. Strikingly, detailed structural analysis unveils two unique features of the PcrK ligand binding pocket: the size of the pocket is restricted for CK binding, and the PcrK applies a positively charged arginine but not a negatively charged aspartate to recognize the ligand. We propose a model to explain how the PcrK accommodates CK-sized compounds through conformational changes, providing a potential mechanistic framework for understanding ligand recognition by the PcrK.

Various pathovars (pv.) of Xanthomonas campestris (Xc) are prevalent in South Korea, of which pv. campestris causing black rot threatens the production of cabbage in this major cabbage prod...

Xanthomonas campestris pv. campestris (Xcc) secretes a suite of effectors into host plants via the type III secretion system (T3SS), modulating plant immunity defenses. Strain Xcc8004 causes black rot in brassica plants, including Arabidopsis thaliana, making it a classical model for the study of Xanthomonas pathogenesis. XopLXcc8004 was defined as a T3SS effector (T3SE) since its homologues XopLXcv85-10 from Xanthomonas campestris pv. vesicatoria (Xcv85-10) contribute to virulence in host plants. Except for its virulence on Chinese radish plants, little was previously known about the regulation and function of XopLXcc8004. Here, we tested the role of XopLXcc8004 in the pathogenicity of Xcc8004 on different host plants including Arabidopsis. We found that it was required for full virulence of Xcc8004 in Chinese cabbage. XopLXcc8004 promoted bacterial infection in Arabidopsis and suppressed bacterial flagellin (flg22)-induced FRK1 transcription, reactive oxygen species (ROS) burst, callose deposition, and pathogenesis-related marker gene expression, but it did not affect mitogen-activated protein kinases (MAPKs) cascade. Early and prolonged expression of XopLXcc8004 affected Arabidopsis growth and development. We demonstrated that XopLXcc8004 is a virulence factor and interferes with innate immunity of Arabidopsis by suppressing pathogen-associated molecular pattern-triggered immunity (PTI) signaling, independent of MAPKs.

International audience; In a prospective, nationwide study in France of Escherichia coli responsible for pneumonia in patients receiving mechanical ventilation, we determined E. coli antimicrobial susceptibility, phylotype, O-type, and virulence factor gene content. We compared 260 isolates with those of 2 published collections containing commensal and bacteremia isolates. The preponderant phylogenetic group was B2 (59.6%), and the predominant sequence type complex (STc) was STc73. STc127 and STc141 were overrepresented and STc95 underrepresented in pneumonia isolates compared with bacteremia isolates. Pneumonia isolates carried higher proportions of virulence genes sfa/foc, papGIII, hlyC, cnf1, and iroN compared with bacteremia isolates. Virulence factor gene content and antimicrobial drug resistance were higher in pneumonia than in commensal isolates. Genomic and phylogenetic characteristics of E. coli pneumonia isolates from critically ill patients indicate that they belong to the extraintestinal pathogenic E. coli pathovar but have distinguishable lung-specific traits.

Summary Both Solanum tuberosum and Ralstonia solanacearum phylotype IIB originated in South America and share a long‐term co‐evolutionary history. However, our knowledge of potato bacterial wilt pathogenesis is scarce as a result of the technical difficulties of potato plant manipulation. Thus, we established a multiple screening system (virulence screen of effector mutants in potato, growth inhibition of yeast and transient expression in Nicotiana benthamiana) of core type III effectors (T3Es) of a major potato pathovar of phylotype IIB, to provide more research perspectives and biological tools. Using this system, we identified four effectors contributing to virulence during potato infection, with two exhibiting multiple phenotypes in two other systems, including RipAB. Further study showed that RipAB is an unknown protein with a nuclear localization signal (NLS). Furthermore, we generated a ripAB complementation strain and transgenic ripAB‐expressing potato plants, and subsequent virulence assays confirmed that R. solanacearum requires RipAB for full virulence. Compared with wild‐type potato, transcriptomic analysis of transgenic ripAB‐expressing potato plants showed a significant down‐regulation of Ca2+ signalling‐related genes in the enriched Plant–Pathogen Interaction (PPI) gene ontology (GO) term. We further verified that, during infection, RipAB is required for the down‐regulation of four Ca2+ sensors, Stcml5, Stcml23, Stcml‐cast and Stcdpk2, and a Ca2+ transporter, Stcngc1. Further evidence showed that the immune‐associated reactive oxygen species (ROS) burst is attenuated in ripAB transgenic potato plants. In conclusion, a systematic screen of conserved R. solanacearum effectors revealed an important role for RipAB, which interferes with Ca2+‐dependent gene expression to promote disease development in potato.

Made available in DSpace on 2019-10-06T16:09:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like + strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production. Departamento de Microbiologia Imunologia e Parasitologia Escola Paulista de Medicina Universidade Federal de São Paulo Instituto de Microbiologia Paulo de Góes Universidade Federal do Rio de Janeiro Laboratório Regional de Santo André VIII Instituto Adolfo Lutz Centro de Bacteriologia Núcleo de Doenças Entéricas Instituto Adolfo Lutz Laboratório de Bacteriologia Instituto Butantan Departamento de Microbiologia e Imunologia Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Microbiologia e Imunologia Universidade Estadual Paulista (UNESP) Instituto de Biociências CNPq: #150392/2017-6 FAPESP: #2015/05971-0

This study was performed to investigate the distribution of antimicrobial resistance genes and extra-intestinal virulence determinants in a collection of 98 Escherichia coli strains isolated from rectal swabs of healthy children. Forty-six isolated strains were resistant to at least one of the tested antibiotics (usually active against enterobacteria). They were mainly resistant to ampicillin and ticarcillin (42.97%), tetracyclin (26.5%), and trimethoprim/sulfamethoxazole (18.4%). No resistance to the third generation of cephalosporins, carbapenems, aminoglycosides and colistin was found. Resistance to penicillins was encoded by blaTEM-1 (n=34) and blaSHV-1 genes (n=4). Tetracyclin resistance was encoded by tetB (n=12), tetA (n= 5), and tetC (n=1) genes. Amongst resistant quinolones isolated (n=5), chromosomal mutations in gyrA and parC genes were detected in four isolates and qnrS1 gene in two strains. Nine plasmid replicon types were detected; IncFIB (n=36) and IncI1 (n=7) were the most frequent ones. Isolates frequently belonged to phylogenetic groups A (51.1%) and D (27.5%). Extra-intestinal pathovar (n=38) occurred mainly in B2 phylogroup (P=0.0002). Amongst them, two isolates (non-extended-spectrum-β-lactamase (ESBL)-producers) belonged to the pandemic clone ST131. A significant distribution of virulence determinants and pathogenicity island marker was observed within strains belonging to B2 and D phylogroups. Interestingly, our results showed that ExPEC strains, including ST131 pandemic clone, are present within fecal isolates in healthy children. These findings highlight the importance of intestinal microbiota as a reservoir for virulent and resistant strains. Thus, reinforcing hand hygiene and antibiotic rational use is imperative to avoid the diffusion of these pathogens in the community.

The Gram-negative bacterium Xanthomonas translucens infects a wide range of gramineous plants with a notable impact on small grain cereals. However, genomics-informed intra-species population structure and virulence repertories of the pathogen have rarely been investigated. In this study, the complete genome sequences of seven X. translucens strains representing an entire set of genetic diversity of two pathovars X. translucens pv. undulosa and X. translucens pv. translucens is provided and compared with those of seven publicly available complete genomes of the pathogen. Organization of the 25 type III secretion system genes in all the 14 X. translucens strains was exactly the same, while TAL effector genes localized singly or in clusters across four loci in X. translucens pv. translucens and five to six loci in X. translucens pv. undulosa. Beside two previously unreported endogenous plasmids in X. translucens pv. undulosa, and variations in repeat variable diresidue (RVD) of the 14 strains, tal1a of X. translucens pv. translucens strain XtKm8 encode the new RVDs HE and YI which have not previously been reported in xanthomonads. Further, a number of truncated tal genes were predicted among the 14 genomes lacking conserved BamHI site at N-terminus and SphI site at C-terminus. Our data have doubled the number of complete genomes of X. translucens clarifying the population structure and genomics of the pathogen to pave the way in the small grain cereals industry for disease resistance breeding in the 21st century’s agriculture.

The environmental bacterium Burkholderia gladioli pv. cocovenenans (B. cocovenenans) has been linked to fatal food poisoning cases in Asia and Africa. Bongkrekic acid (BA), a mitochondrial toxin produced by B. cocovenenans, is thought to be responsible for these outbreaks. While there are over 80 species in the Burkholderia genus, B. cocovenenans is the only pathovar capable of producing BA and causing human death. However, the genomic features of B. gladioli and the evolution of the BA biosynthesis gene cluster, bon, in B. cocovenenans remain elusive. In this study, 239 whole genome sequences (WGSs) of B. gladioli, isolated from 12 countries collected over 100 years, were used to analyze the intra-species genomic diversity and phylogenetic relationships of B. gladioli and to explore the origin and evolution of the bon gene cluster. Our results showed that the genome-wide average nucleotide identity (ANI) values were above 97.29% for pairs of B. gladioli genomes. Thirty-six of the 239 (15.06%) B. gladioli genomes, isolated from corn, rice, fruits, soil, and patients from Asia, Europe, North America, and South America, contained the bon gene cluster and formed three clades within the phylogenetic tree. Pan- and core-genome analysis suggested that the BA biosynthesis genes were recently acquired. Comparative genome analysis of the bon gene cluster showed that complex recombination events contributed to this toxin biosynthesis gene cluster’s evolution and formation. This study suggests that a better understanding of the genomic diversity and evolution of this lethal foodborne pathovar will potentially contribute to B. cocovenenans food poisoning outbreak prevention.

Bacterial angular leaf spot disease (ALS) caused by Pseudomonas syringae pv. lachrymans (Psl) is one of the biological factors limiting cucumber open-field production. The goal of this study was to characterize cytological and transcriptomic response of cucumber to this pathogen. Plants of two inbred lines, B10 (susceptible) and Gy14 (resistant), were grown, and leaves were inoculated with highly virulent Psl strain 814/98 under growth chamber conditions. Microscopic and transcriptional evaluations were performed at three time points: before, 1 and 3 days post inoculation (dpi). Investigated lines showed distinct response to Psl. At 1 dpi bacterial colonies were surrounded by necrotized mesophyll cells. At 3 dpi, in the susceptible B10 line bacteria were in contact with degraded cells, whereas cells next to bacteria in the resistant Gy14 line were plasmolyzed, but apparently still alive and functional. Additionally, the level of H2O2 production was higher in resistant Gy14 plants than in B10 at both examined time points. In RNA sequencing more than 18,800 transcripts were detected in each sample. As many as 1648 and 2755 differentially expressed genes (DEGs) at 1 dpi as well as 2992 and 3141 DEGs at 3 dpi were identified in B10 and Gy14, respectively. DEGs were characterized in terms of functional categories. Resistant line Gy14 showed massive transcriptomic response to Psl at 1 dpi compared to susceptible line B10, while a similar number of DEGs was detected for both lines at 3 dpi. This suggests that dynamic transcriptomic response to the invading pathogen may be related with host resistance. This manuscript provides the first transcriptomic data on cucumber infected with the pathovar lachrymans and helps to elucidate resistance mechanism against ALS disease.

We report the genome sequence of Xanthomonas arboricola pv. juglandis strain CPBF 427, which was isolated from early-season buds of a diseased walnut tree, suggesting overwinter potential. This study provides a consistent genomic reference for this pathovar and may contribute to addressing the overwinter survival of these walnut pathogens.

Temperature impacts plant immunity and growth but how temperature intersects with endogenous pathways to shape natural variation remains unclear. Here we uncover variation between Arabidopsis thaliana natural accessions in response to two non-stress temperatures (22°C and 16°C) affecting accumulation of the thermoresponsive stress hormone salicylic acid (SA) and plant growth. Analysis of differentially responding A. thaliana accessions shows that pre-existing SA provides a benefit in limiting infection by Pseudomonas syringae pathovar tomato DC3000 bacteria at both temperatures. Several A. thaliana genotypes display a capacity to mitigate negative effects of high SA on growth, indicating within-species plasticity in SA—growth tradeoffs. An association study of temperature x SA variation, followed by physiological and immunity phenotyping of mutant and over-expression lines, identifies the transcription factor bHLH059 as a temperature-responsive SA immunity regulator. Here we reveal previously untapped diversity in plant responses to temperature and a way forward in understanding the genetic architecture of plant adaptation to changing environments., Author summary Temperature has a profound effect on plant innate immune responses but little is known about the mechanisms underlying natural variation in transmission of temperature signals to defence pathways. Much of our understanding of temperature effects on plant immunity and tradeoffs between activated defences and growth has come from analysis of the common Arabidopsis thaliana genetic accession, Col-0. Here we examine A. thaliana genetic variation in response to temperature (within the non-stress range—22 oC and 16 oC) at the level of accumulation of the thermoresponsive biotic stress hormone salicylic acid (SA), bacterial pathogen resistance, and plant biomass. From analysis of 105 genetically diverse A. thaliana accessions we uncover plasticity in temperature-modulated SA homeostasis and in the relationship between SA levels and plant growth. We find that high SA amounts prior to infection provide a robust benefit of enhancing bacterial resistance. In some accessions this benefit comes without compromised plant growth, suggestive of altered defence–growth tradeoffs. Based on a temperature x SA association study we identify the transcription factor gene, bHLH059, and show that it has features of a temperature-sensitive immunity regulator that are unrelated to PIF4, a known thermosensitive coordinator of immunity and growth.

Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn’s disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn’s disease and non-Crohn’s tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.

Journal of plant diseases and protection 128(1), 249–259 (2020). doi:10.1007/s41348-020-00378-y, Published by Springer, Heidelberg