Brenneria salicis Associated with Watermark Disease Symptoms on Salix alba in Italy

From 1999 to 2010, withering of white willow was observed on trees growing along roads or irrigation canals in Torino, Alessandria, and Vercelli provinces of Italy, with incidence varying from 15, 25, and 30%, respectively. In spring and autumn 2008, six samples from withering branches with bark cankers were collected. On the bark surface near the cankers, iridescent traces of dried ooze were found. Tissues immediately below the cankers were dark with water-soaked, olive-colored edges. In some cases, the xylem appeared affected. Small fragments taken from the affected tissue on both edges of bark alterations and darkened vessels were crushed into mortars with sterile saline. Ten-fold serial dilutions (10^-1, 10^-2) were also performed. Aliquots of 0.1 ml were plated on nutrient agar and incubated at 25°C for 4 days. Bacterial colonies were ivory to white, circular, and bright, with a diameter of ~2 mm. Isolates were negative for Gram staining, presence of arginine dehydrolase, oxidase, phenylalanine deaminase, urease, hydrolysis of gelatin and starch, nitrate reduction, acidity from d-arabinose, cellobiose, lactose, maltose, trehalose, xylose, and pectinolytic activity on potato slices; positive for the presence of catalase and levan, fermentative metabolism of glucose, acid production from aesculin, l-arabinose, dextrose, d-galactose, inositol, d-mannitol, α-methylglucoside, raffinose, salicin and sucrose, H₂S production from cysteine, and bright yellow pigment production on autoclaved potato tissue. They were not fluorescent on King's medium B and did not induce hypersensitivity reaction on tobacco leaf. Similar results were obtained with Brenneria salicis control strain, LMG 6089, except for acid production from α-methylglucoside (negative) and l-arabinose (negative). Acid production from α-methylglucoside has been reported for the Japanese strains of B. salicis, which do not produce acidity from inositol (4). Genomic DNA was extracted (1) from three isolates, and PCR reactions were performed with Es1A and Es4B primers (2) that amplify a 553-bp fragment from the 16S rDNA of B. salicis. The isolates showed a PCR product of expected size, like the positive control LMB 6089. On the basis of colony features, biochemical tests, and the PCR assay, we conclude that the isolates belong to B. salicis, a pathogen reported in Belgium, Germany, Great Britain, the Netherlands, Hungary, Japan, and New Zealand (2,3) but, as well as watermark disease symptoms, never previously reported in Italy. In summer 2009, pathogenicity tests were performed by inoculating young, white willow plants with B. salicis suspensions of ~1 to 2 × 10⁹ CFU/ml placed with a syringe at the intersection of 1-year-old branches on the trunk. However, a year later, no symptoms of disease have been noted on the inoculated plants. According to the literature, pathogenicity tests rarely lead to the expected results because the bacterium can survive for many years in latent form, breaking out only when proper environmental conditions occur (3). Also the tests with B. salicis LMG 6089 gave negative results. Further investigation is necessary to clarify the relationships between this bacterium and the environment in causing withering of white willows in Piedmont. References: (1) W. P. Chen and T. T. Kuo. Nucleic Acids Res. 21:2260, 1993. (2) L. Hauben et al. Appl. Environ. Microbiol. 64:3966, 1998. (3) M. Maes et al. Environ. Microbiol. 11:1453, 2009. (4) Y. Sakamoto et al. Plant Pathol. 48:613, 1999.