Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.

Bacterial canker is a major disease of Prunus avium (cherry), Prunus domestica (plum) and other stone fruits. It is caused by pathovars within the Pseudomonas syringae species complex including P. syringae pv. morsprunorum (Psm) race 1 (R1), Psm race 2 (R2) and P. syringae pv. syringae (Pss). Psm R1 and Psm R2 were originally designated as the same pathovar; however, phylogenetic analysis revealed them to be distantly related, falling into phylogroups 3 and 1, respectively. This study characterized the pathogenicity of 18 newly genome‐sequenced P. syringae strains on cherry and plum, in the field and laboratory. The field experiment confirmed that the cherry cultivar Merton Glory exhibited a broad resistance to all clades. Psm R1 contained strains with differential specificity on cherry and plum. The ability of tractable laboratory‐based assays to reproduce assessments on whole trees was examined. Good correlations were achieved with assays using cut shoots or leaves, although only the cut shoot assay was able to reliably discriminate cultivar differences seen in the field. Measuring bacterial multiplication in detached leaves differentiated pathogens from nonpathogens and was therefore suitable for routine testing. In cherry leaves, symptom appearance discriminated Psm races from nonpathogens, which triggered a hypersensitive reaction. Pathogenic strains of Pss rapidly induced disease lesions in all tissues and exhibited a more necrotrophic lifestyle than hemibiotrophic Psm. This in‐depth study of pathogenic interactions, identification of host resistance and optimization of laboratory assays provides a framework for future genetic dissection of host–pathogen interactions in the canker disease.

Pseudomonas syringae is a bacterial species complex associated with plants and aquatic environments that has been reported to cause disease on over 180 plant species (Berge et al., 2014). The pathogen is important globally as it infects most major crops. Host-adapted strains are responsible for damaging disease epidemics when invading new territories, for example the outbreak of horse chestnut bleeding canker in northern Europe (Green et al., 2010; Steele et al., 2010) and kiwifruit canker in New Zealand (McCann et al., 2017). P. syringae strains were traditionally classified based on host of isolation into groups of pathogenic varieties (pathovars) that generally infect one or a few related plant species (Sarkar et al., 2006). Classification is now supported by genotypic data, leading to the proposal for distinct genomospecies, phylogroups or phylogenomic species based on DNA-DNA hybridization, multilocus sequence analysis (MLSA) or more recently, whole genome sequence data comparisons (Gomila et al., 2017). Nineteen phylogenomic species

19Bacterial canker is a major disease of cherry and other stone fruits caused by several pathovars of 20Pseudomonas syringae. These are P.s pv. morsprunorum race 1 (Psm R1), P.s pv. morsprunorum 21 race 2 (Psm R2) and P.s pv. syringae (Pss). Psm R1 and R2 were originally designated as races of 22 the same pathovar, however phylogenetic analysis has revealed them to be distantly related. This 23 study characterised the pathogenicity of P. syringae on cherry and plum, in the field and the 24 laboratory. The field experiment identified variation in host cultivar susceptibility to the different 25 pathogen clades. The cherry cultivar Merton Glory exhibited a broad resistance to all clades, whilst 26 cultivar Van showed race-specific resistance. Psm R1 may be divided into a race structure with 27 some strains pathogenic to both cherry and plum and others only pathogenic to plum. The results of 28 laboratory-based pathogenicity tests were compared to results obtained on whole-trees. Only cut 29 shoot inoculations were found to be sensitive enough to detect cultivar variation in susceptibility. 30

Fusarium oxysporum is a globally distributed soilborne fungal pathogen causing root rots, bulb rots, crown rots and vascular wilts on a range of horticultural plants. Pathogenic F. oxysporum isolates are highly host specific and are classified as formae speciales. Narcissus is an important ornamental crop and both the quality and yield of flowers and bulbs can be severely affected by a basal rot caused by F. oxysporum f. sp. narcissi (FON); 154 Fusarium isolates were obtained from different locations and Narcissus cultivars in the United Kingdom, representing a valuable resource. A subset of 30 F. oxysporum isolates were all found to be pathogenic and were therefore identified as FON. Molecular characterisation of isolates through sequencing of three housekeeping genes, suggested a monophyletic origin with little divergence. PCR detection of 14 Secreted in Xylem (SIX) genes, previously shown to be associated with pathogenicity in other F. oxysporum f. spp., revealed different complements of SIX7, SIX9, SIX10, SIX12 and SIX13 within FON isolates which may suggest a race structure. SIX gene sequences were unique to FON and SIX10 was present in all isolates, allowing for molecular identification of FON for the first time. The genome of a highly pathogenic isolate was sequenced and lineage specific (LS) regions identified which harboured putative effectors including the SIX genes. Real-time RT-PCR, showed that SIX genes and selected putative effectors were expressed in planta with many significantly upregulated during infection. This is the first study to characterise molecular variation in FON and provide an analysis of the FON genome. Identification of expressed genes potentially associated with virulence provides the basis for future functional studies and new targets for molecular diagnostics.

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Plant diseases caused by bacteria remain problematic for the global horticultural industry due to a lack of effective control measures (Sundin et al., 2016). The genus Prunus contains over 400 species, a selection of which are grown for top fruit, ornamental use and timber production (Bortiri et al., 2001). Bacterial canker, caused by members of the Pseudomonas syringae species complex, can be a major limiting factor in the cultivation of Prunus spp. (Omrani et al., 2019;Vicente et al., 2004). The disease is primarily characterized by necrosis, gummosis and/ or dieback of woody plant tissues. In addition, the pathogens colonize other plant tissues where they exist epiphytically or invade to cause leaf and fruit spots, and blossom blight. These tissues can act as reservoirs

Nicotinamide adenine dinucleotide (NAD + ) has emerged as a key component in prokaryotic and eukaryotic immune systems. The recent discovery that Toll/interleukin-1 receptor (TIR) proteins function as NAD + hydrolases (NADase) links NAD + -derived small molecules with immune signaling. We investigated pathogen manipulation of host NAD + metabolism as a virulence strategy. Using the pangenome of the model bacterial pathogen Pseudomonas syringae , we conducted a structure-based similarity search from 35,000 orthogroups for type III effectors (T3Es) with potential NADase activity. Thirteen T3Es, including five newly identified candidates, were identified that possess domain(s) characteristic of seven NAD + -hydrolyzing enzyme families. Most Pseudomonas syringae strains that depend on the type III secretion system to cause disease, encode at least one NAD + -manipulating T3E, and many have several. We experimentally confirmed the type III-dependent secretion of a novel T3E, named HopBY, which shows structural similarity to both TIR and adenosine diphosphate ribose (ADPR) cyclase. Homologs of HopBY were predicted to be type VI effectors in diverse bacterial species, indicating potential recruitment of this activity by microbial proteins secreted during various interspecies interactions. HopBY efficiently hydrolyzes NAD + and specifically produces 2′cADPR, which can also be produced by TIR immune receptors of plants and by other bacteria. Intriguingly, this effector promoted bacterial virulence, indicating that 2′cADPR may not be the signaling molecule that directly initiates immunity. This study highlights a host-pathogen battleground centered around NAD + metabolism and provides insight into the NAD + -derived molecules involved in plant immunity.