Infection of horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi and its detection by quantitative real‐time PCR

Abstract

Pseudomonas syringae pv. aesculi is a pathogenic bacterium causing bleeding canker disease of horse chestnut ( Aesculus hippocastanum ). This is a serious disease which has been affecting horse chestnut in several European countries over the last five years; however, very little is known about the biology of the causal agent. One of the obstacles to studying this pathogen is the lengthy procedure associated with confirming its presence on the host. In this study, P. syringae pv. aesculi was isolated from lesions on different parts of horse chestnut and its pathogenicity confirmed on horse chestnut saplings using two inoculation techniques. Real-time PCR primers were developed based on gyrase B gene sequence data for the specific detection of P. syringae pv. aesculi . Primer specificity was tested on isolates of the target pathogen as well as on a broad range of related non-target bacteria and other bacterial spp. which inhabit horse chestnut. The real-time primers reliably amplified P. syringae pv. aesculi down to 1 pg of extracted DNA, with and without the presence of host DNA, and also amplified unextracted DNA in whole cells of the bacterium down to at least 160 colony forming units. Detection and quantification of the target pathogen in phloem and xylem of both naturally infected and inoculated horse chestnut tissues was also demonstrated. This quantitative real-time PCR assay provides the facility to study several important aspects of the biology of P. syringae pv. aesculi on horse chestnut including its potential for dissemination in different substrates.

Understanding Bacteriophage Specificity in Natural Microbial Communities

“…Finally, we found two phages collected from the horse chestnut leaf that are capable of infecting a previously characterized strain of P. syringae pathovar aesculi (Pae), the causal agent of bleeding canker disease in horse chestnut trees. These phages were not, however, capable of infecting the other three strains of Pae we tested, suggesting that there is variation in susceptibility of Pae strains to phages despite the relatively low genetic diversity typically found among isolates of this rapidly emerging pathogen [71]. …”

Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages

“…Because of both the observed positive correlation between motility and resistance to phages and the epidemiological significance of the species (Hirano and Upper, 2000;Webber et al, 2008;Green et al, 2009), we chose to focus exclusively on 10 bacterial isolates from the leaf interior that had 499% sequence similarity to known isolates of P. syringae. Importantly, these isolates were all sampled from separate leaves to decrease the probability of pseudoreplication.…”

A TaqMan real-time PCR assay targeting the cytochrome o ubiquinol oxidase subunit II gene for detection of several pathovars ofPseudomonas syringae

“…Cho et al (2010) developed a TaqMan realtime PCR method for detection of P. syringae pv. phaseolicola while Green et al (2009) reported a SYBR Greenbased real-time PCR assay for quantitative detection of P. syringae pv. aesculi.…”

“…Because post-PCR processing is required, the assays can not be easily automated for high throughput (Weller et al, 2000). Real-time PCR has proven to be a very useful tool for accurate detection of plant pathogenic bacteria (Weller et al, 2000;Fanelli et al, 2007;Tambong et al, 2008;Gervasi & Scortichini, 2009;Green et al, 2009;Cho et al, 2010;Gottsberger, 2010). Although real-time PCR systems have been reported for several plant pathogenic bacteria, the works of Cho et al (2010) and Green et al (2009) are among the very few, to the best of our knowledge, to target pathovars of P. syringae.…”

“…Although real-time PCR systems have been reported for several plant pathogenic bacteria, the works of Cho et al (2010) and Green et al (2009) are among the very few, to the best of our knowledge, to target pathovars of P. syringae. Green et al (2009) reported a SYBR Green real-time PCR assay for identification/detection of P. syringae pv. aesculi based on the gyrase B gene and Cho et al (2010) targeted the site-specific recombinase gene of P. syringae pv.…”