Isolation and identification of Pseudomonas syringae pv. aesculi causing bleeding canker of horse chestnut in the UK

Abstract

Since 2003 the incidence of stem bleeding on horse chestnut ( Aesculus hippocastanum ) has risen markedly in the UK. Symptoms include rusty-brown, or black, gummy liquid seeping from bark, and necrotic phloem with a mottled orange-brown colour. Some bleeding cankers have been so extensive that infected tissue encircled branches or trunks, causing dieback or tree mortality in 3–4 years. Previous episodes of horse chestnut bleeding canker were caused by Phytophthora spp., but considered uncommon and only recorded in southern England (Strouts & Winter, 2000). However, reports made to Forest Research Disease Diagnostic Advisory Service indicate the disorder is now present in England, Scotland and Wales, with a similar upsurge in bleeding canker also recorded in the Netherlands, Belgium and France. Culturing from the margins of the necrotic phloem on to various selective media rarely yielded Phytophthora spp. Instead a Gram-negative bacterium was isolated consistently. Ten isolates were characterized and were fluorescent on King’s medium B, produced levan on sucrose nutrient agar, were negative for oxidase and arginine dihydrolase, non-pectolytic and induced a hypersensitive response in tobacco (LOPAT+ — +). Fatty acid analysis and sequencing of the gyrase B ( gyrB ) gene (Sarkar & Guttman, 2004) indicated a single strain of P. syringae . The gyrB sequence was identical to that of P. syringae pv. aesculi (NCPPB3681) isolated from foliage of A. indica in India (Durgapal & Singh, 1980) and to a P. syringae strain isolated from leaf-spot symptoms on A. indica in Surrey in 2005. REP PCR analysis (Schaad et al ., 2001) showed all UK isolates shared identical profiles. Of the 25 amplicons identified using BOX-, ERICand REP PCR, 20 were common to NCPPB3681, indicating that all the strains were closely related. Suspensions of four UK isolates (1 × 10 7 CFU mL − 1 ) were inoculated through stem cuts into 5-year-old A. hippocastanum saplings. After 4 months, necrotic bark lesions (3–7 cm 2 ) formed around the inoculation points with some bleeding; no necrosis occurred with the water controls. Re-isolation and identification confirmed Koch’s postulates. This is the first report of P. syringae pv. aesculi outside India, and the impact of the bacterium on A. hippocastanum in the UK appears to be widespread and damaging.