First laboratory confirmation of Xylophilus ampelinus in Slovenia*

Abstract

Bacterial blight of grapevine is caused by a slow-growing bacterium Xylophilus ampelinus . It has been suspected to occur in Slovenia on the basis of visual observation of characteristic symptoms in the 1960s. In the present study, symptoms were recorded in an infected vineyard during three consecutive years (2002/2004). Samples from this vineyard were tested by nested-PCR and isolation of bacteria on media was attempted. In the first year, angular lesions on leaves were highly expressed and an isolate morphologically similar to X. ampelinus was obtained from one sample. It was purified and identified as X. ampelinus using biochemical and nutritional tests, fatty acid analysis, immuno-fluorescence, nested PCR and partial sequencing of the 16S rRNA gene. The 16S rDNA sequence showed 99-100% homology to known sequences of X. ampelinus strains, including the type strain. Pathogenicity of the isolate was confirmed in tissue-cultured and potted grapevine plants. In the following two years, symptoms of bacterial blight were only faintly expressed. Using isolation on media and nested-PCR, 23 and 17 extracts prepared from 10 and 8 grapevines, respectively, were analysed. In 2003, no positive sample was found, but X. ampelinus was again isolated and identified by colony morphology and nested-PCR in 2004.

Bacterial diseases of grapevine

“…Bacterial blight occurs mainly in the Mediterranean region of Europe (Italy, Greece, Spain), in South Africa and more recently in Slovenia (Dreo et al 2005, Grall & Manceau 2003, OEPP/EPPO 2009, Serfontein et al 1997. Isolates derived from different sources show very little physiological and biochemical differences (Dreo et al 2005, Willems & Gillis 2006. The physiological and molecular bases for virulence of X. ampelinus have not been determined.…”

“…fastidiosa may still be difficult, since endophytic bacteria that grow faster than the these pathogens may overgrow them in vitro on nutrient agar during isolation. Methods for isolation of these grapevine pathogens have been described, e. g., by Dreo et al (2005), Serfontein et al (1997) and Willems & Gillis (2006) for X. ampelinus, and by Aguilar et al (2008), Almeida et al (2004) and Fry et al (1990) for X. fastidiosa subsp. fastidiosa.…”

PM 7/96 (1): Xylophilus ampelinus

“…The fatty acid profiling of X. ampelinus is fairly simple. Major acids are 8:0 3 OH (2%), 14:0 (3%), 16:0 (24%), 16:1 w7c (41%) and 18:1 w7c (28%) (Dreo et al. , 2005).…”

Development of a real‐time PCR‐based method for detection of Xylophilus ampelinus

“…Plates were incubated at 24 ° C for 10 days and checked for the presence of small yellow colonies from the sixth day onwards. Characteristic yellow, slow-growing colonies were purified and identified as X. ampelinus using molecular methods and fatty acid profiling as described previously ( Janse, 1991;Dreo et al ., 2005).…”