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Abstract Background Understanding the interaction between environmental conditions, crop yields, and soil health is crucial for sustainable agriculture in a changing climate. Management practices to limit disease are a balancing act. For example, in potato production, dry conditions favour common scab (Streptomyces spp.) and wet conditions favour blackleg disease (Pectobacterium spp.). The exact mechanisms involved and how these link to changes in the soil microbiome are unclear. Our objectives were to test how irrigation management and bacterial pathogen load in potato seed stocks impact: (i) crop yields; (ii) disease development (blackleg or common scab); and (iii) soil microbial community dynamics. Methods We used stocks of seed potatoes with varying natural levels of Pectobacterium (Jelly [high load], Jelly [low load] and Estima [Zero – no Pectobacterium]). Stocks were grown under four irrigation regimes that differed in the timing and level of watering. The soil microbial communities were profiled using amplicon sequencing at 50% plant emergence and at harvest. Generalised linear latent variable models and an annotation-free mathematical framework approach (ensemble quotient analysis) were then used to show the interacting microbes with irrigation regime and Pectobacterium pathogen levels. Results Irrigation increased blackleg symptoms in the plots planted with stocks with low and high levels of Pectobacterium (22–34%) but not in the zero stock (2–6%). However, withholding irrigation increased common scab symptoms (2–5%) and reduced crop yields. Irrigation did not impact the composition of the soil microbiome, but planting stock with a high Pectobacterium burden resulted in an increased abundance of Planctomycetota, Anaerolinea and Acidobacteria species within the microbiome. Ensemble quotient analysis highlighted the Anaerolinea taxa were highly associated with high levels of Pectobacterium in the seed stock and blackleg symptoms in the field. Conclusions We conclude that planting seed stocks with a high Pectobacterium burden alters the abundance of specific microbial species within the soil microbiome and suggest that managing pathogen load in seed stocks could substantially affect soil communities, affecting crop health and productivity. Video Abstract

Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonisation. As such, we compared the ability of the VTEC strain, E. coli O157:H7 ‘Sakai’, to colonise the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum) and prickly lettuce (L. serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analysed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonisation. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 hour) transcriptional response to extracts as well as longer-term (10 days) plant colonisation or persistence. We show that propagation temperature (37 versus 18 oC) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilisation. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress or translational stalling. A large proportion of differentially regulated genes are uncharacterised (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

Antimicrobial peptides constitute an important factor in the defense of plants against pathogens, and bacterial resistance to these peptides have previously been shown to be an important virulence factor in Dickeya dadantii, the causal agent of soft-rot disease of vegetables. In order to understand the bacterial response to antimicrobial peptides, a transcriptional microarray analysis was performed upon treatment with sub-lethal concentration of thionins, a widespread plant peptide. In all, 36 genes were found to be overexpressed, and were classified according to their deduced function as i) transcriptional regulators, ii) transport, and iii) modification of the bacterial membrane. One gene encoding a uricase was found to be repressed. The majority of these genes are known to be under the control of the PhoP/PhoQ system. Five genes representing the different functions induced were selected for further analysis. The results obtained indicate that the presence of antimicrobial peptides induces a complex response which includes peptide-specific elements and general stress-response elements contributing differentially to the virulence in different hosts.

The exopolysaccharide amylovoran is one of the major pathogenicity factors in Erwinia amylovora, the causal agent of fire blight of apples and pears. We have previously demonstrated that the RcsBCD phosphorelay system is essential for virulence by controlling amylovoran biosynthesis. We have also found that the hybrid sensor kinase RcsC differentially regulates amylovoran production in vitro and in vivo. To further understand how the Rcs system regulates E. amylovora virulence gene expression, we conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and on immature pear fruit. Array analyses identified a total of 648 genes differentially regulated by RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls expression of amylovoran biosynthetic genes in vivo but negatively controls expression in vitro. Besides amylovoran biosynthesis and regulatory genes, cell-wall and cell-envelope (membrane) as well as regulatory genes were identified as the major components of the RcsBC regulon, including many novel genes. We have also demonstrated that transcripts of rcsA, rcsC, and rcsD genes but not the rcsB gene were up-regulated when bacterial cells were grown in minimal medium or following infection of pear fruits compared with those grown in Luria Bertani medium. Furthermore, using the genome of E. amylovora ATCC 49946, a hidden Markov model predicted 60 genes with a candidate RcsB binding site in the intergenic region, 28 of which were identified in the microarray assay. Based on these findings as well as previous reported data, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora.

Two-dimensional polyacrylamide gel electrophoresis of the secreted proteins of Erwinia carotovora subsp. atroseptica revealed alow-abundance protein that was identified by mass spectrometry as a homologue of a Xanthomonas campestris avirulence protein with unknown function. The predicted Svx protein has an N-terminal signal sequence and zinc binding-region signature, and the mature protein is post-translationally modified. A 2D difference gel electrophoresis (DIGE) showed that the protein is secreted by the type II (out) secretion apparatus, which is also responsible for the secretion of the major known virulence factors, PelC and CelV. Transcription of the svx gene is under Nacyl- homoserine lactone-mediated quorum-sensing control. The svx gene was inactivated by transposon insertion. The mutant showed a decrease in virulence in potato plant assays, demonstrating a role for Svx in the pathogenicity of E. carotovora subsp. atroseptica. These results show that Svx is a previously unidentified virulence determinant which is secreted by the out machinery and is regulated by quorum sensing, two systems employed by several other virulence factors. Thus, the type II secretory machine is a conduit for virulence factors other than the main pectinnases and cellulase in E. carotovora subsp. atroseptica.

Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T4SS). This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably, this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria. [ABSTRACT FROM AUTHOR]