Preliminary study on bacteria isolated from bleeding cankers of Aesculus hippocastanum in Emilia Romagna Region

Since 2017, Aesculus hippocastanum is listed as vulnerable in the European Red List of Trees because its extinction is likely in the near future given current knowledge about its population trends, and recent threats. In particular, among biotic stresses, horse-chestnut declines is caused by the leaf-miner moth, Cameraria ohridella, and by Pseudomonas syringae pv. aesculi (Psae), the causal agent of bleeding cankers across Europe, at present never reported in Italy. In this study, from horse-chestnut tree showing typical bleeding canker symptoms of Psae, several bacterial colonies were isolated (both from bark trunk and fresh liquid oozing) on different substrates (KB, NSA and EMB) amended with cycloheximide (100 µg/ml) and subsequently purified by sub-culturing to obtain axenic isolates. On Psae and Brenneria spp. like colonies were carried out: 1) in planta assays to test their ability to induce hypersensitive reaction (HR) on leaves of tobacco plant; 2) carbon source utilization screening by using the Biolog GenIIITM system; 3) quantitative real-time PCR analyses to identify Psae with primers AM-Aes1F/AM-Aes1R and Psa2F/Psa2R; 4) PCR analyses to identify both Brenneria nigrifluens using the primers F1/C3 and B. rubrifaciens with primers BR1/BR3; 5) 16S rRNA gene sequence analysis. From these preliminary analyses, no isolates were identified as Psae and also attempts to isolate Phytophthora spp. on PAR selective substrate were unsuccessful. Pathobiotic mechanisms involved in bacterial diversity of symptomatic tissue that harboured opportunistic or animal pathogens were discussed. This work was partially supported by University of Bologna RFO funds, Department of Agricultural and Food Sciences.