Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Pseudomonas syringae Pathovars pisi and syringae

Pseudomonas syringae causes bacterial blight (BB) disease worldwide on economically important fruit and vegetable crops including field pea (Pisum sativum L.). The two pathovars responsible for BB in field pea are Pseudomonas syringae pathovar pisi (Psp) and syringae (Pss). In the field, both pathovars cause indistinguishable symptoms on field pea and require laboratory diagnosis to determine the causal pathovar. To aid in-field and laboratory diagnosis, accurate, and robust loop-mediated isothermal amplification (LAMP) assays for Psp and Pss were developed. The assays were able to detect Psp or Pss on live or heat-killed bacterial cells, plant exudates, seeds, and DNA extracts with no inhibitory effects. The two specific LAMP assays developed detected Psp and Pss accurately in less than 20 min and no cross-reaction was observed with 18 strains of closely related species of Pseudomonas syringae. Compared to the conventional PCR assays, the two LAMP assays were equally specific but have advantages of producing quicker and visual live results, enabling early detection and differentiation of Psp and Pss. Our results suggested a potential use of LAMP assays for laboratory testing and can be applied for in-field surveys.

Transposon Mutagenesis of Pseudomonas syringae Pathovars syringae and morsprunorum to Identify Genes Involved in Bacterial Canker Disease of Cherry

Bacterial canker of Prunus, affecting economically important stone fruit crops including cherry, peach, apricot and plum, is caused by the plant pathogen Pseudomonas syringae (P.s.). Strains from two pathovars—P.s. pv. syringae (Pss) and P.s. pv. morsprunorum race 1 (PsmR1) and 2 (PsmR2)—in three phylogenetically distant clades have convergently evolved to infect Prunus. The bacteria enter woody tissues through wounds and leaf scars, causing black necrotic cankers. Symptoms are also produced on blossom, fruit and leaves. Little is known about the mechanisms P.s. uses to colonise tree hosts such as Prunus. Here, we created transposon (Tn) mutant libraries in one strain of P.s. from each of the three clades and screened the mutants on immature cherry fruit to look for changes in virulence. Mutants (242) with either reduced or enhanced virulence were detected and further characterised by in vitro screens for biofilm formation, swarming ability, and pathogenicity on leaves and cut shoots. In total, 18 genes affecting virulence were selected, and these were involved in diverse functions including motility, type III secretion, membrane transport, amino acid synthesis, DNA repair and primary metabolism. Interestingly, mutation of the effector gene, hopAU1, led to an increase in virulence of Psm R2.

Isolation, Characterization, and Pathogenicity of Two Pseudomonas syringae Pathovars from Populus trichocarpa Seeds

Pseudomonas syringae is a ubiquitous plant pathogen, infecting both woody and herbaceous plants and resulting in devastating agricultural crop losses. Characterized by a remarkable specificity for plant hosts, P. syringae pathovars utilize a number of virulence factors including the type III secretion system and effector proteins to elicit disease in a particular host species. Here, two Pseudomonas syringae strains were isolated from diseased Populustrichocarpa seeds. The pathovars were capable of inhibiting poplar seed germination and were selective for the Populus genus. Sequencing of the newly described organisms revealed similarity to phylogroup II pathogens and genomic regions associated with woody host-associated plant pathogens, as well as genes for specific virulence factors. The host response to infection, as revealed through metabolomics, is the induction of the stress response through the accumulation of higher-order salicylates. Combined with necrosis on leaf surfaces, the plant appears to quickly respond by isolating infected tissues and mounting an anti-inflammatory defense. This study improves our understanding of the initial host response to epiphytic pathogens in Populus and provides a new model system for studying the effects of a bacterial pathogen on a woody host plant in which both organisms are fully genetically sequenced.

Characterization of Pseudomonas syringae pathovars from different sweet cherry cultivars by RAPD analysis

Pseudomonas syringae pvs., isolated from sweet cherry grown on different localities in Serbia, were genetically characterized using RAPD analysis. Four out of eleven tested primers (SPH1, DJP 17, DJ 15, and DJ 16) were selected on the basis of the differences between isolates within two pathovars - syringae and morsprunorum race 1. Cumulative RAPD analysis indicated heterogeneity within the population of both groups of tested isolates, revealing four different patterns in each group. RAPD analysis showed up to 24% differences among pv. syringae isolates, as well as 41% in comparison with the reference strain KFB0103 (pv. syringae), while differences of 15% among isolates pv. morsprunorum 1 race and 36% compared to the reference strain CFBP2119 (pv. morsprunorum 1) were observed. Isolates from locality Selenca exhibited three different genotypic patterns of pv. morsprunorum race 1 and one pattern of pv. syringae. Isolates of pv. morsprunorum collected in the same year from two plant organs (branches and leaves) of the cv. Vanda yielded two different patterns. The pv. morsprunorum on cv. Kordia and pv. syringae on cv. Regina were detected at Mikicevo locality. The same patterns were observed for isolates of pv. syringae from Kanjiza and Selenca, as well as from Gornji Tavankut in two years of isolation. Differences were noted between isolates from the same pathovar originating from Ljutovo and Mikicevo, as well as with respect to all other isolates of same pathovar.