Background and aims: Acute oak decline (AOD), a decline syndrome affecting mature oaks, involves bacterial pathogens which likely act as opportunists under host stress. Trees displaying symptoms (bleeding cankers) appear in localized clusters, not whole stands. This study investigates the potential involvement of local scale factors, in interaction with large scale environmental drivers, in influencing onset and progression of AOD. Methods: AOD-symptomatic (n=30) and asymptomatic trees (n=30) across three UK oak woodlands were assessed for tree characteristics, their surrounding context, and soil properties. Results: Tree health status was linked to significant differences in soil and tree properties across sites. Symptomatic trees exhibited greater loss of crown density, lower local stand (0 to 20 m) basal area and shallower depth to gleying. Significant differences in soil properties included lower concentrations of Olsen P, total N, and exchangeable Mg in symptomatic trees, alongside higher exchangeable Fe, especially at 40 to 50 cm depth. Depth to gleying and exchangeable Fe were identified as the most influential predictors of AOD. Conclusions: AOD symptomatic trees may experience seasonal soil water saturation closer to the surface compared to asymptomatic trees, resulting in a higher proportion of their roots being exposed to an anoxic, iron reducing environment. This study is the first to report such an association between gleying depth, likely seasonal water saturation, and symptom status for AOD. It is unclear whether water balance and associated soil nutrient variations are predisposing factors or consequences of declining tree health, though the identified local scale factors likely contribute to AOD. A feedback loop is conceptualised where declining tree health worsens soil conditions, creating a negative cycle that accelerates tree decline. ### Competing Interest Statement The authors have declared no competing interest.

Acute oak decline (AOD), a decline syndrome affecting mature oaks, involves bacterial pathogens which likely act as opportunists under host stress. Trees displaying symptoms (bleeding cankers) appear in localized clusters, not whole stands. This study investigates the potential involvement of local-scale factors, in interaction with large-scale environmental drivers, in influencing onset and progression of AOD. AOD-symptomatic (n = 30) and asymptomatic trees (n = 30) across three UK oak woodlands were assessed for tree characteristics, their surrounding context, and soil properties. Tree health status was linked to significant differences in soil and tree properties across sites. Symptomatic trees exhibited greater loss of crown density, lower local stand (0–20 m) basal area and shallower depth to gleying. Significant differences in soil properties included lower concentrations of Olsen P, total N, and exchangeable Mg in symptomatic trees, alongside higher exchangeable Fe, especially at 40–50 cm depth. Depth to gleying and exchangeable Fe were identified as the most influential predictors of AOD. AOD symptomatic trees may experience seasonal soil water saturation closer to the surface compared to asymptomatic trees, resulting in a higher proportion of their roots being exposed to an anoxic, iron-reducing environment. This study is the first to report such an association between gleying depth, likely seasonal water saturation, and symptom status for AOD. It is unclear whether water balance and associated soil nutrient variations are predisposing factors or consequences of declining tree health, though the identified local-scale factors likely contribute to AOD. A feedback loop is conceptualised where declining tree health worsens soil conditions, creating a negative cycle that accelerates tree decline.

Stone fruits ( Prunus spp.) are economically and nutritionally valuable commodities, with production concentrated in the temperate regions. In the UK, limited to cherries and plums, production trends highlight a resurgence in cherry yields due to modern practices. However, bacterial cankers, caused by diverse strains of P . syringae , pose significant threats to Prunus spp., affecting both commercial orchards and ornamental plant nurseries. This disease, characterised by cankers, gummosis, and necrotic lesions in woody tissues, incurs severe economic losses, with young orchards particularly vulnerable. Surveys have revealed the emergence of novel pathogens in P . syringae populations, with predominant groups varying across continents. Advances in molecular techniques have improved pathogen classification and resistance screening in Prunus cultivars is increasing. Addressing bacterial cankers requires integrating knowledge of pathogen lifecycles, virulence factors, and host-pathogen interactions into management strategies. Current research focuses on pathogen evolution, breeding resistant tree cultivars, and implementing new biocontrol strategies to mitigate the economic and ecological impacts. © The Authors 2025

Pseudomonas syringae pathovar aesculi (E-Pae) causes bleeding canker disease in the woody tissue of European horse chestnut (HC). Comparative genomic analysis of E-Pae with a related leaf-infecting strain (I-Pae) and other P. syringae strains identified candidate virulence genes for colonisation of woody tissue, including a sucrose uptake and utilisation system (scrYABCDBR cluster) found in 162 of 206 P. syringae strains spanning the pangenome. Growth analysis using sucrose as sole carbon source showed that I-Pae (lacking the gene cluster) was unable to grow whereas E-Pae could grow. P. savastanoi pv. phaseolicola 1448A and P. syringae pv. morsprunorum R15244 were compromised in growth despite the presence of the gene cluster. Sucrose utilisation assays using scrB and scrY mutants and complemented strains confirmed the importance of the cluster for sucrose metabolism in vitro. Pathogenicity assays in HC revealed the sucrose gene cluster is important for symptom development in the woody tissue. While the scr genes contribute to disease causation, they were not essential for pathogen fitness when compared to hrpL and hopAB1 mutants. E-Pae caused disease symptoms in HC leaves, suggesting the strain has the potential to infect leaves as well. However, it was notable that the scrB mutant of E-Pae caused increased disease symptoms, possibly highlighting a niche adaptation strategy for I-Pae to cause leaf spots in HC as well as constraining E-Pae to predominantly infect the woody tissue.

The specific association of the potentially plant-pathogenic Pseudomonas syringae with Peltigera lichens raises questions about the factors driving this host specificity. To explore this, the metabolic profile of seven lichen species belonging to three genera (Cladonia, Peltigera and Stereocaulon) was analysed using LC-MSMS. In addition, we assessed the growth of P. syringae strains in media supplemented with extracts from each lichen species. This revealed that Peltigera exhibits lower metabolite richness compared to other genera, but shows a higher chemical investment in specific compounds. Growth kinetics showed comparable P. syringae growth across lichen-supplemented media, except for Cladonia arbuscula and Cladonia sp., where the former exhibited lower growth rates. Inhibition assays with lichen extracts showed no inhibition of P. syringae. The lichen metabolome is predominantly composed of lipids and organic acids. Furthermore, specific compounds, such as aminoglycosides, may facilitate P. syringae presence in Peltigera by inhibiting Bacillus subtilis and other antagonists. In addition, compounds absent in Peltigera, like anthracene, might serve as a carbon source inhibitor like Bacillus velezensis.

Bacteriophages (phages) are viruses that infect and lyse bacteria and have the potential for controlling bacterial diseases. Isolation of phages targeting the cherry pathogen Pseudomonas syringae pv. syringae (Pss) led to five distinct phage genotypes. Building on previous in vitro coevolution experiments, the coevolution of the five phages (individually and as a cocktail) with Pss on cherry leaves was conducted in glasshouse and field experiments. Phages effectively reduced Pss numbers on detached leaves, with no evidence of phage resistance emerging in the bacterial population. Field application of phages in a cherry orchard in Southeast England evaluated phage survival, viability and impact on bacterial populations and the microbial community. The bacterial population and phages persisted in the leaf and shoot environment as long as the bacterial host was present. In contrast to in vitro studies, the plant environment constrained the emergence of phage resistant Pss populations. Application of phage cocktail in the orchard did not affect the cherry leaf microbiome. These observations provide essential knowledge for using phage treatments to control bacterial diseases while minimising the impact on the plant microbiome, highlighting phages' potential to safely control bacterial diseases in trees.

Aphids are globally important pests causing damage to a broad range of crops. Due to insecticide resistance, there is an urgent need to develop alternative control strategies. In our previous work, we found Pseudomonas fluorescens PpR24 can orally infect and kill the insecticide-resistant green-peach aphid (Myzus persicae). However, the genetic basis of the insecticidal capability of PpR24 remains unclear. Genome sequencing of PpR24 confirmed the presence of various insecticidal toxins such as Tc (toxin complexes), Rhs (rearrangement hotspot) elements, and other insect-killing proteases. Upon aphids infection with PpR24, RNA-Seq analysis revealed 193 aphid genes were differentially expressed with down-regulation of 16 detoxification genes. In addition, 1325 PpR24 genes (542 were upregulated and 783 downregulated) were subject to differential expression, including genes responsible for secondary metabolite biosynthesis, the iron-restriction response, oxidative stress resistance, and virulence factors. Single and double deletion of candidate virulence genes encoding a secreted protease (AprX) and four toxin components (two TcA-like; one TcB-like; one TcC-like insecticidal toxins) showed that all five genes contribute significantly to aphid killing, particularly AprX. This comprehensive host-pathogen transcriptomic analysis provides novel insight into the molecular basis of bacteria-mediated aphid mortality and the potential of PpR24 as an effective biocontrol agent.

Tree bacterial diseases are a threat in forestry due to their increasing incidence and severity. Understanding tree defence mechanisms requires evaluating metabolic changes arising during infection. Metabolite extraction affects the chemical diversity of the samples and, therefore, the biological relevance of the data. Metabolite extraction has been standardized for several biological models. However, little information is available regarding how it influences wood extract’s chemical diversity. This study aimed to develop a methodological approach to obtain extracts from different tree species with the highest reproducibility and chemical diversity possible, to ensure proper coverage of the trees’ metabolome. A full factorial design was used to evaluate the effect of solvent type, extraction temperature and number of extraction cycles on the metabolic profile, chemical diversity and antibacterial activity of four tree species. Solvent, temperature and their interaction significantly affected the extracts’ chemical diversity, while the number of extraction cycles positively correlated with yield and antibacterial activity. Although 60

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. syringae (Pss) strains within PG2 have a reduced repertoire of type III effectors (T3Es) but produce several phytotoxins. Effectors within the cherry pathogen Pss 9644 were grouped based on their frequency in strains from Prunus as the conserved effector locus (CEL) common to most P. syringae pathogens; a core of effectors common to PG2; a set of PRUNUS effectors common to cherry pathogens; and a FLEXIBLE set of T3Es. Pss 9644 also contains gene clusters for biosynthesis of toxins syringomycin, syringopeptin and syringolin A. After confirmation of virulence gene expression, mutants with a sequential series of T3E and toxin deletions were pathogenicity tested on wood, leaves and fruits of sweet cherry (Prunus avium) and leaves of ornamental cherry (Prunus incisa). The toxins had a key role in disease development in fruits but were less important in leaves and wood. An effectorless mutant retained some pathogenicity to fruit but not wood or leaves. Striking redundancy was observed amongst effector groups. The CEL effectors have important roles during the early stages of leaf infection and possibly acted synergistically with toxins in all tissues. Deletion of separate groups of T3Es had more effect in P. incisa than in P. avium. Mixed inocula were used to complement the toxin mutations in trans and indicated that strain mixtures may be important in the field. Our results highlight the niche-specific role of toxins in P. avium tissues and the complexity of effector redundancy in the pathogen Pss 9644.

Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges in vitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest.

Beyond their role in horizontal gene transfer, conjugative plasmids commonly encode homologues of bacterial regulators. Known plasmid regulator homologues have highly targeted effects upon the transcription of specific bacterial traits. Here, we characterise a plasmid translational regulator, RsmQ, capable of taking global regulatory control in Pseudomonas fluorescens and causing a behavioural switch from motile to sessile lifestyle. RsmQ acts as a global regulator, controlling the host proteome through direct interaction with host mRNAs and interference with the host's translational regulatory network. This mRNA interference leads to large-scale proteomic changes in metabolic genes, key regulators, and genes involved in chemotaxis, thus controlling bacterial metabolism and motility. Moreover, comparative analyses found RsmQ to be encoded on a large number of divergent plasmids isolated from multiple bacterial host taxa, suggesting the widespread importance of RsmQ for manipulating bacterial behaviour across clinical, environmental, and agricultural niches. RsmQ is a widespread plasmid global translational regulator primarily evolved for host chromosomal control to manipulate bacterial behaviour and lifestyle.

Soil contains immense stocks of carbon, which may accelerate climate change if released. Soil microbes affect these carbon stocks by producing decomposition-catalyzing enzymes, a capacity varying across different microbial groups. Consequently, establishing links between global variation in microbial communities and functions should substantially enhance future projections of soil carbon. To this end, we here reveal global patterns in soil microbial community function using nearly 13,000 observations of microbial biomass, community structure, and enzyme activities (>100,000 measurements). We find total biomass and fungal and Gram-negative bacterial dominance increase with latitude, whereas Gram-positive bacteria predominate near the equator. Enzyme stoichiometry correspondingly suggests greater nitrogen and carbon limitation at higher latitudes. Comparing microbial and enzyme patterns, fungal biomass indicates nitrogen limitation, whereas Gram-negative bacterial biomass indicates carbon limitation. Together, microbial community structure explains significant variation in enzyme profile uncaptured by climate, soil properties, or landcover. Soil microbial communities dominated by fungi and Gram-negative bacteria exhibit less enzyme activity per unit biomass, with two- to four-fold variation in temperature- and biomass-normalized activity rate observed across the Earth. Significant functional differences thus arise with global turnover in microbial communities, indicating that community structure merits a central position in process-based soil models.

Summary Many strains of Pseudomonas colonise plant surfaces, including the cherry canker pathogens, Pseudomonas syringae pathovars syringae and morsprunorum. We have examined the genomic diversity of P. syringae in the cherry phyllosphere and focused on the role of prophages in transfer of genes encoding Type 3 secreted effector (T3SE) proteins contributing to the evolution of virulence. Phylogenomic analysis was carried out on epiphytic pseudomonads in the UK orchards. Significant differences in epiphytic populations occurred between regions. Nonpathogenic strains were found to contain reservoirs of T3SE genes. Members of P. syringae phylogroups 4 and 10 were identified for the first time from Prunus. Using bioinformatics, we explored the presence of the gene encoding T3SE HopAR1 within related prophage sequences in diverse P. syringae strains including cherry epiphytes and pathogens. Results indicated that horizontal gene transfer (HGT) of this effector between phylogroups may have involved phage. Prophages containing hopAR1 were demonstrated to excise, circularise and transfer the gene on the leaf surface. The phyllosphere provides a dynamic environment for prophage‐mediated gene exchange and the potential for the emergence of new more virulent pathotypes. Our results suggest that genome‐based epidemiological surveillance of environmental populations will allow the timely application of control measures to prevent damaging diseases.

Wheat, the world’s most widely cultivated crop, is highly susceptible to Take-all disease caused by the soil ascomycete fungus Gaeumannomyces tritici (Gt). Crop rotations using different wheat cultivars can influence Take-all build up (TAB) in the following year’s wheat crop. The exact mechanism for this is unknown, but there may be a bacterial biocontrol component associated with TAB control. We cultured and characterised Pseudomonas isolates from the rhizosphere and endosphere of second-year field-grown wheat, in plots which, in the previous season, had been used to culture High-TAB cultivar Hereward or Low-TAB cultivar Cadenza. Analysis of two bacterial loci, wsm and fecB, showed that these genes were significantly and differentially associated with first-year wheat-planting regime: wsm was more abundant within the High-TAB Hereward-derived isolates, while fecB was more prevalent within the Low-TAB Cadenza-derived isolates. These findings were supported by a gyrB phylogenetic analysis and an in vitro Gt antagonism assay. These data show that the wheat cultivar grown in the first year plays an especially important role in the selection of the associated Pseudomonas spp. found in the second-season wheat-root environment, emphasising the importance of understanding agronomic practice in improving plant health and food security.

Plasmids drive bacterial evolutionary innovation by transferring ecologically important functions between lineages, but acquiring a plasmid often comes at a fitness cost to the host cell. Compensatory mutations, which ameliorate the cost of plasmid carriage, promote plasmid maintenance in simplified laboratory media across diverse plasmid-host associations. Whether such compensatory evolution can occur in more complex communities inhabiting natural environmental niches where evolutionary paths may be more constrained is, however, unclear. Here we show a substantial fitness cost of carrying the large conjugative plasmid pQBR103 in Pseudomonas fluorescens in the plant rhizosphere. This plasmid fitness cost could be ameliorated by compensatory mutations affecting the chromosomal global regulatory system gacA/gacS , which arose rapidly in plant rhizosphere communities and were exclusive to plasmid carriers. These findings expand our understanding of the importance of compensatory evolution in plasmid dynamics beyond simplified lab media. Compensatory mutations contribute to plasmid survival in bacterial populations living within complex microbial communities in their natural environmental niche.

Pseudomonas syringae is a bacterial complex that is widespread through a range of environments, typically associated with plants where it can be pathogenic, but also found in non-plant environments such as clouds, precipitation, and surface waters. Understanding its distribution within the environment, and the habitats it occupies, is important for examining its evolution and understanding behaviours. After a recent study found P. syringae living among a range of vascular plant species in Iceland, we questioned whether lichens could harbour P. syringae. Sixteen different species of lichens were sampled all over Iceland, but only one lichen genus, Peltigera, was found to consistently harbour P. syringae. Phylogenetic analyses of P. syringae from 10 sampling points where lichen, tracheophyte, and/or moss were simultaneously collected showed significant differences between sampling points, but not between different plants and lichens from the same point. Furthermore, while there were similarities in the P. syringae population in tracheophytes and Peltigera, the densities in Peltigera thalli were lower than in moss and tracheophyte samples. This discovery suggests P. syringae strains can localize and survive in organisms beyond higher plants, and thus reveals opportunities for studying their influence on P. syringae evolution.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

At the level of the gene, mutation is the raw material for natural selection. However, at the level of the gene regulatory network (GRN), variation is revealed to selection via promiscuous regulator activity ('crosstalk'), which creates opportunities for genetic innovation that can facilitate adaptation. Many genetic and environmental features can contribute to increasing potential for crosstalk by facilitating non-cognate interactions between regulatory elements. If a novel interaction provides a fitness benefit, rewired GRNs with strengthened affinity for newly forged connections can be selected. Here, we identify factors that facilitate opportunities for crosstalk and rewiring between GRNs, consider whether features of some GRNs make them more 'rewireable' than others and if these features might constrain evolution towards convergent outcomes. We explore patterns from laboratory and natural microbial populations that show changes within GRNs during adaptation. Finally, we discuss the prospects and open questions in the field.

Upon infection, DNA viruses can be sensed by pattern recognition receptors (PRRs), leading to the activation of type I and III interferons to block infection. Therefore, viruses must inhibit these signaling pathways, avoid being detected, or both. Papillomavirus virions are trafficked from early endosomes to the Golgi apparatus and wait for the onset of mitosis to complete nuclear entry. This unique subcellular trafficking strategy avoids detection by cytoplasmic PRRs, a property that may contribute to the establishment of infection. However, as the capsid uncoats within acidic endosomal compartments, the viral DNA may be exposed to detection by Toll-like receptor 9 (TLR9). In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we showed that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts. Finally, we demonstrated that the cancer-associated human papillomaviruses show a reduction in CpG dinucleotides within a TLR9 recognition complex. IMPORTANCE Viruses must avoid detection by the innate immune system. In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we demonstrated that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts.