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.

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.

Bacterial canker disease is a major limiting factor in the growing of cherry and other Prunus species worldwide. At least five distinct clades within the bacterial species complex Pseudomonas syringae are known to be causal agents of the disease. The different pathogens commonly coexist in the field. Reducing canker is a challenging prospect as the efficacy of chemical controls and host resistance may vary against each of the diverse clades involved. Genomic analysis has revealed that the pathogens use a variable repertoire of virulence factors to cause the disease. Significantly, strains of P . syringae pv. syringae possess more genes for toxin biosynthesis and fewer encoding type III effector proteins. There is also a shared pool of key effector genes present on mobile elements such as plasmids and prophages that may have roles in virulence. By contrast, there is evidence that absence or truncation of certain effector genes, such as hopAB , is characteristic of cherry pathogens. Here we highlight how recent research, underpinned by the earlier epidemiological studies, is allowing significant progress in our understanding of the canker pathogens. This fundamental knowledge, combined with emerging insights into host genetics, provides the groundwork for development of precise control measures and informed approaches to breed for disease resistance.

Pseudomonas syringae can cause stem necrosis and canker in a wide range of woody species including cherry, plum, peach, horse chestnut and ash. The detection and quantification of lesion progression over time in woody tissues is a key trait for breeders to select upon for resistance. In this study a general, rapid and reliable approach to lesion quantification using image recognition and an artificial neural network model was developed. This was applied to screen both the virulence of a range of P. syringae pathovars and the resistance of a set of cherry and plum accessions to bacterial canker. The method developed was more objective than scoring by eye and allowed the detection of putatively resistant plant material for further study. Automated image analysis will facilitate rapid screening of material for resistance to bacterial and other phytopathogens, allowing more efficient selection and quantification of resistance responses.

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry ( Prunus avium ) production worldwide. One important strategy for disease control is the development of resistant varieties. Partial varietal resistance in sweet cherry is discernible using shoot or whole tree inoculations; however, these quantitative differences in resistance are not evident in detached leaf assays. To identify novel sources of resistance to canker, we used a rapid leaf pathogenicity test to screen a range of wild cherry, ornamental Prunus species and sweet cherry × ornamental cherry hybrids with the canker pathogens, Pseudomonas syringae pvs syringae , morsprunorum races 1 and 2, and avii . Several Prunus accessions exhibited limited symptom development following inoculation with each of the pathogens, and this resistance extended to 16 P . syringae strains pathogenic on sweet cherry and plum. Resistance was associated with reduced bacterial multiplication after inoculation, a phenotype similar to that of commercial sweet cherry towards nonhost strains of P . syringae . Progeny resulting from a cross of a resistant ornamental species Prunus incisa with susceptible sweet cherry ( P . avium ) exhibited resistance indicating it is an inherited trait. Identification of accessions with resistance to the major bacterial canker pathogens is the first step towards characterizing the underlying genetic mechanisms of resistance and introducing these traits into commercial germplasm.

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. s yringae (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.