sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ ORMAN FAKÜLTESİ DERGİSİ
time consuming, through the need to obtain pure bacterial cultures from diseased
horse chestnut tissues which may be colonised by a range of bacterial genera
(Green et al., 2009). Recently, real-time PCR has proven to be a very useful tool
for the detection of plant pathogenic fungi and bacteria, being highly sensitive,
specific and rapid, with the added capacity for quantification of the pathogen in
host tissues (Schaad and Frederick, 2002; Vandroemme et al., 2008). The first aim
of this study was to develop and test a robust, reliable, quantitative real-time PCR
assay which is specific to P. syringae pv. aesculi, and to demonstrate its use for
detecting the bacterium in inoculated and naturally infected horse chestnut trees
(Green et al., 2009). Also, briefly discussed are ongoing projects aimed at i)
characterising the key genetic and physiological factors determining virulence and
epiphytic fitness of P. syringae pv. aesculi on European horse chestnut and ii)
tracing the epidemiological origins of this devastating bacterium.
2. MATERIALS AND METHODS
For the development of the real-time PCR assay, a total of 65 bacterial isolates
were collected from various parts of diseased or healthy horse chestnut in Britain,
the DNA extracted and the partial gyrase B gene region amplified using universal,
degenerate primers. The amplified fragments were sequenced and aligned with
other bacterial gyrase B gene sequences available in GenBank to design real-time
PCR primers specific to P. syringae pv. aesculi. The specificity and sensitivity of
the real-time PCR primers was tested using nine strains of P. syringae pv. aesculi,
17 other strains of P. syringae, 11 other non-pathogenic Pseudomonas spp. and 14
other species of bacteria isolated from horse chestnut trees. The ability of the realtime
primers to amplify and quantify DNA of P. syringae pv. aesculi in diseased
horse chestnut tissues was also demonstrated.
3. RESULTS
The real-time primer pair and reaction conditions developed to detect P.
syringae pv. aesculi amplified all isolates of P. syringae pv. aesculi, but did not
amplify the DNA extracted from the included reference bacteria or horse chestnut
when 1 ng of DNA was used as the template. 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. The real-time PCR assay
detected and quantified DNA of P. syringae pv. aesculi in sixteen out of seventeen
tissue samples collected from naturally infected or artificially inoculated horse
chestnut, with generally good consistency between the two PCR runs in terms of Ct
values and quantity of pathogen DNA detected.
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