In situ control of root-bacteria interactions using optical trapping in transparent soil.

Bacterial attachment on root surfaces is an important step preceding the colonization or internalization and subsequent infection of plants by pathogens. Unfortunately, bacterial attachment is not well understood because the phenomenon is difficult to observe. Here we assessed whether this limitation could be overcome using optical trapping approaches. We have developed a system based on counter-propagating beams and studied its ability to guide Pectobacterium atrosepticum (Pba) cells to different root cell types within the interstices of transparent soils. Bacterial cells were successfully trapped and guided to root hair cells, epidermal cells, border cells, and tissues damaged by laser ablation. Finally, we used the system to quantify the bacterial cell detachment rate of Pba cells on root surfaces following reversible attachment. Optical trapping techniques could greatly enhance our ability to deterministically characterize mechanisms linked to attachment and formation of biofilms in the rhizosphere.

Landscape Epidemiology of Potato Blackleg.

Potato blackleg is a common bacterial disease that causes serious losses in potato (Solanum tuberosum) production worldwide. Despite this, relatively little is known of the landscape epidemiology of this disease. This study provides the first national-scale analysis of spatial and spatiotemporal patterns of blackleg incidence rates and associated risk factors for disease at the landscape scale. This was achieved through a combination of ArcGIS and interpretable machine learning applied to a longitudinal dataset of naturally infected seed potato crops from across Scotland. We found striking differences in long-term disease outcomes across the country and identified that features (variables) related to the health status and management of mother crops (seed stocks), matching features in daughter crops, and the characteristics of surrounding potato crop distributions were the most important predictors of disease, followed by field, bioclimatic, and soil features. Our approach provides a comprehensive overview of potato blackleg at a national scale, new epidemiological insights, and an accurate model that could serve as the basis of a decision support tool for improved blackleg management.

The role of l-arabinose metabolism for Escherichia coli O157:H7 in edible plants.

Arabinose is a major plant aldopentose in the form of arabinans complexed in cell wall polysaccharides or glycoproteins (AGP), but comparatively rare as a monosaccharide. l-arabinose is an important bacterial metabolite, accessed by pectolytic micro-organisms such as Pectobacterium atrosepticum via pectin and hemicellulose degrading enzymes. However, not all plant-associated microbes encode cell-wall-degrading enzymes, yet can metabolize l-arabinose, raising questions about their use of and access to the glycan in plants. Therefore, we examined l-arabinose metabolism in the food-borne pathogen Escherichia coli O157:H7 (isolate Sakai) during its colonization of plants. l-arabinose metabolism (araBA) and transport (araF) genes were activated at 18 °C in vitro by l-arabinose and expressed over prolonged periods in planta. Although deletion of araBAD did not impact the colonization ability of E. coli O157:H7 (Sakai) on spinach and lettuce plants (both associated with STEC outbreaks), araA was induced on exposure to spinach cell-wall polysaccharides. Furthermore, debranched and arabinan oligosaccharides induced ara metabolism gene expression in vitro, and stimulated modest proliferation, while immobilized pectin did not. Thus, E. coli O157:H7 (Sakai) can utilize pectin/AGP-derived l-arabinose as a metabolite. Furthermore, it differs fundamentally in ara gene organization, transport and regulation from the related pectinolytic species P. atrosepticum, reflective of distinct plant-associated lifestyles.

Total mRNA sequence dataset from Pectobacterium atrosepticum colonising potato or radish roots.

Pectobacterium atrosepticum (Pba) is a gram-negative bacterium that causes blackleg and tuber soft rot of potato but can also asymptomatically colonise other (non-host) plant species. The aim of this study was to investigate the molecular processes and responses involved in Pba-host (potato) and Pba-non-host (radish) interactions, under laboratory conditions. To achieve this, we used total mRNA-sequencing to measure the gene expression patterns from all three species: Pba, potato and radish. We employed an end-point dual transcriptome approach. We used hydroponically grown potato (Solanum tuberosum var. Estima) and oil radish (Raphanus sativa var. Bento) roots inoculated with Pba SCRI1039 for 14 days compared to un-inoculated control plants or cultured bacteria. Total RNA was extracted from replicates of the two plant species and the bacterium using a Macherey-Nagel Nucleospin Plant RNA kit. The RNA from the 17 samples was then subjected to total mRNA-sequencing (paired-end) on an Illumina NovaSeq 6000™ sequencing platform. This gave between 39.2-58.1M reads per sample. The high-quality reads obtained were mapped to the corresponding reference genomes using Bowtie2 and the percentages of bacterium and plant transcripts calculated. This dataset constitutes the raw read fastq files and can be used to inform on genes active in plant rhizosphere-microbe interactions.

The endophytic lifestyle of Escherichia coli O157:H7: quantification and internal localization in roots.

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.

A multi-repeat adhesin of the phytopathogen, Pectobacterium atrosepticum, is secreted by a Type I pathway and is subject to complex regulation involving a non-canonical diguanylate cyclase.

Cyclic diguanylate (c-di-GMP) is a second messenger controlling many important bacterial processes. The phytopathogen Pectobacterium atrosepticum SCRI1043 (Pba1043) possesses a Type I secretion system (T1SS) essential for the secretion of a proteinaceous multi-repeat adhesin (MRP) required for binding to the host plant. The genes encoding the MRP and the T1SS are tightly linked to genes encoding several putative c-di-GMP regulatory components. We show that c-di-GMP regulates secreted MRP levels in Pba1043 through the action of two genes encoding predicted diguanylate cyclase (DGC) and phosphodiesterase proteins (ECA3270 and ECA3271). Phenotypic analyses and quantification of c-di-GMP levels demonstrated that ECA3270 and ECA3271 regulate secreted MRP levels by increasing and decreasing, respectively, the intracellular levels of c-di-GMP. Moreover, ECA3270 represents the first active DGC reported to have an alternative active-site motif from the 'canonical' GG[D/E]EF. ECA3270 has an A-site motif of SGDEF and analysis of single amino acid replacements demonstrated that the first position of this motif can tolerate functional substitution. Serine in position one of the A-site is also observed in many other DGCs. Finally, another T1SS-linked regulator (ECA3265) also plays an important role in regulating secreted MRP, with an altered localization of MRP observed in an ECA3265 mutant background. Mutants defective in these three T1SS-linked regulators exhibit a reduction in root binding and virulence, confirming that this complex, finely tuned regulation system is crucial in the interaction with host plants.

Pectobacterium atrosepticum KDPG aldolase, Eda, participates in the Entner-Doudoroff pathway and independently inhibits expression of virulence determinants.

Pectobacterium carotovorum has an incomplete Entner-Doudoroff (ED) pathway, including enzyme 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) but lacking phosphogluconate dehydratase (Edd), while P. atrosepticum (Pba) has a complete pathway. To understand the role of the ED pathway in Pectobacterium infection, mutants of these two key enzymes, Δeda and Δedd, were constructed in Pba SCRI1039. Δeda exhibited significant decreased virulence on potato tubers and colonization in planta and was greatly attenuated in pectinase activity and the ability to use pectin breakdown products, including polygalacturonic acid (PGA) and galacturonic acid. These reduced phenotypes were restored following complementation with an external vector expressing eda. Quantitative reverse transcription PCR analysis revealed that expression of the pectinase genes pelA, pelC, pehN, pelW, and pmeB in Δeda cultured in pyruvate, with or without PGA, was significantly reduced compared to the wild type, while genes for virulence regulators (kdgR, hexR, hexA, and rsmA) remained unchanged. However, Δedd showed similar phenotypes to the wild type. To our knowledge, this is the first demonstration that disruption of eda has a feedback effect on inhibiting pectin degradation and that Eda is involved in building the arsenal of pectinases needed during infection by Pectobacterium.

Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen Pectobacterium atrosepticum.

Quorum sensing (QS) in vitro controls production of plant cell wall degrading enzymes (PCWDEs) and other virulence factors in the soft rotting enterobacterial plant pathogen Pectobacterium atrosepticum (Pba). Here, we demonstrate the genome-wide regulatory role of QS in vivo during the Pba-potato interaction, using a Pba-specific microarray. We show that 26% of the Pba genome exhibited differential transcription in a QS (expI-) mutant, compared to the wild-type, suggesting that QS may make a greater contribution to pathogenesis than previously thought. We identify novel components of the QS regulon, including the Type I and II secretion systems, which are involved in the secretion of PCWDEs; a novel Type VI secretion system (T6SS) and its predicted substrates Hcp and VgrG; more than 70 known or putative regulators, some of which have been demonstrated to control pathogenesis and, remarkably, the Type III secretion system and associated effector proteins, and coronafacoyl-amide conjugates, both of which play roles in the manipulation of plant defences. We show that the T6SS and a novel potential regulator, VirS, are required for full virulence in Pba, and propose a model placing QS at the apex of a regulatory hierarchy controlling the later stages of disease progression in Pba. Our findings indicate that QS is a master regulator of phytopathogenesis, controlling multiple other regulators that, in turn, co-ordinately regulate genes associated with manipulation of host defences in concert with the destructive arsenal of PCWDEs that manifest the soft rot disease phenotype.

Expansin-like Exl1 from Pectobacterium is a virulence factor required for host infection, and induces a defence plant response involving ROS, and jasmonate, ethylene and salicylic acid signalling pathways in Arabidopsis thaliana.

Expansins are encoded by some phytopathogenic bacteria and evidence indicates that they act as virulence factors for host infection. Here we analysed the expression of exl1 by Pectobacterium brasiliense and Pectobacterium atrosepticum. In both, exl1 gene appears to be under quorum sensing control, and protein Exl1 can be observed in culture medium and during plant infection. Expression of exl1 correlates with pathogen virulence, where symptoms are reduced in a Δexl1 mutant strain of P. atrosepticum. As well as Δexl1 exhibiting less maceration of potato plants, fewer bacteria are observed at distance from the inoculation site. However, bacteria infiltrated into the plant tissue are as virulent as the wild type, suggesting that this is due to alterations in the initial invasion of the tissue. Additionally, swarming from colonies grown on MacConkey soft agar was delayed in the mutant in comparison to the wild type. We found that Exl1 acts on the plant tissue, probably by remodelling of a cell wall component or altering the barrier properties of the cell wall inducing a plant defence response, which results in the production of ROS and the induction of marker genes of the JA, ET and SA signalling pathways in Arabidopsis thaliana. Exl1 inactive mutants fail to trigger such responses. This defence response is protective against Pectobacterium brasiliense and Botrytis cinerea in more than one plant species.

Threat of establishment of non-indigenous potato blackleg and tuber soft rot pathogens in Great Britain under climate change.

Potato blackleg and soft rot caused by Pectobacterium and Dickeya species are among the most significant bacterial diseases affecting potato production globally. In this study we estimate the impact of future temperatures on establishment of non-indigenous but confirmed Pectobacterium and Dickeya species in Great Britain (GB). The calculations are based on probabilistic climate change data and a model fitted to disease severity data from a controlled environment tuber assay with the dominant potato blackleg and soft rot-causing species in GB (P. atrosepticum), and three of the main causative agents in Europe (P. carotovorum subsp. brasiliense, P. parmentieri, Dickeya solani). Our aim was to investigate if the European strains could become stronger competitors in the GB potato ecosystem as the climate warms, on the basis of their aggressiveness in tubers at different temperatures. Principally, we found that the tissue macerating capacity of all four pathogens will increase in GB under all emissions scenarios. The predominant Pectobacterium and Dickeya species in Europe are able to cause disease in tubers under field conditions currently seen in GB but are not expected to become widely established in the future, at least on the basis of their aggressiveness in tubers relative to P. atrosepticum under GB conditions. Our key take-home messages are that the GB potato industry is well positioned to continue to thrive via current best management practices and continued reinforcement of existing legislation.

Root cap influences root colonisation by Pseudomonas fluorescens SBW25 on maize.

We investigated the influence of root border cells on the colonisation of seedling Zea mays roots by Pseudomonas fluorescens SBW25 in sandy loam soil packed at two dry bulk densities. Numbers of colony forming units (CFU) were counted on sequential sections of root for intact and decapped inoculated roots grown in loose (1.0 mg m(-3)) and compacted (1.3 mg m(-3)) soil. After two days of root growth, the numbers of P. fluorescens (CFU cm(-1)) were highest on the section of root just below the seed with progressively fewer bacteria near the tip, irrespective of density. The decapped roots had significantly more colonies of P. fluorescens at the tip compared with the intact roots: approximately 100-fold more in the loose and 30-fold more in the compact soil. In addition, confocal images of the root tips grown in agar showed that P. fluorescens could only be detected on the tips of the decapped roots. These results indicated that border cells, and their associated mucilage, prevented complete colonization of the root tip by the biocontrol agent P. fluorescens, possibly by acting as a disposable surface or sheath around the cap.

Detection of the Bacterial Potato Pathogens Pectobacterium and Dickeya spp. Using Conventional and Real-Time PCR.

Blackleg and soft rot of potato, caused by Pectobacterium and Dickeya spp., are major production constraints in many potato-growing regions of the world. Despite advances in our understanding of the causative organisms, disease epidemiology, and control, blackleg remains the principal cause of down-grading and rejection of potato seed in classification schemes across Northern Europe and many other parts of the world. Although symptom recognition is relatively straightforward and is applied universally in seed classification schemes, attributing disease to a specific organism is problematic and can only be achieved through the use of diagnostics. Similarly as disease spread is largely through the movement of asymptomatically infected seed tubers and, possibly in the case of Dickeya spp., irrigation waters, accurate and sensitive diagnostics are a prerequisite for detection. This chapter describes the diagnostic pathway that can be applied to identify the principal potato pathogens within the genera Pectobacterium and Dickeya.

The effects of volatile microbial secondary metabolites on protein synthesis in Serpula lacrymans.

The effects of volatile secondary metabolites produced by Trichoderma pseudokoningii, Trichoderma viride and Trichoderma aureoviride on growth rate and protein synthesis in two Serpula lacrymans isolates were investigated. Mycelial growth was affected to differing degrees, depending on the specific interactive microbial couplet involved. Protein synthesis by both S. lacrymans (Forfar) and S. lacrymans (H28) was affected by the volatile secondary metabolites of T. aureoviride and T. viride, but not by those of T. pseudokoningii. Mycelial growth and the original pattern of protein synthesis resumed when the antagonists were removed. It is probable that volatile secondary metabolites have played an important role during the evolution of microorganisms in the context of community, population and functional dynamics.

Draft genome sequences of 17 isolates of the plant pathogenic bacterium dickeya.

Dickeya (formerly Erwinia chrysanthemi) species cause diseases on a wide range of crops and ornamental plants worldwide. Here we present the draft sequences of 17 Dickeya isolates spanning four Dickeya species, including five isolates that are currently unassigned to a species.

Draft Genome Sequences of Four Dickeya dianthicola and Four Dickeya solani Strains.

Dickeya dianthicola and "Dickeya solani" are currently the dominant bacterial pathogens of potatoes in Europe. Here, we present the draft genome sequences of four strains of each pathogen.