Bacterial Leaf Spot Caused by the Quarantine Pathogen Xanthomonas arboricola pv. pruni on Cherry Laurel in Central Italy

Abstract

In June 2010, 1-year-old potted plants of cherry laurel (Prunus laurocerasus L.) cv. Novita showing leaf spot symptoms were collected in a commercial nursery in the district of Pistoia (Tuscany, central Italy). Red-purple necrotic lesions (measuring a few millimeters up to 1 cm) surrounded by a brilliant light green halo were observed on the abaxial surface of symptomatic leaves. With age, the necrotic areas drop out, leaving a “shot-hole” appearance. Microscopic observation revealed the absence of fungal structures, whereas bacteria were isolated from symptomatic tissue on nutrient sucrose agar medium. Purified single colonies appeared mucoid, convex, and yellow on yeast extract-dextrose-CaCO3 agar (YDCA) medium, were positive to the KOH test, and induced hypersensitive responses on tobacco (cv. Virginia Bright). Three isolates were selected arbitrarily for further analysis. A fragment of approximately 500 bp of the 16S rRNA gene was amplified via PCR with the universal primer pair 27f/519r and sequenced. Subsequent database searches in the INSD (GenBank, EMBL, and DDBJ) indicated that the resulting sequences had 100% identity over 490 bp with the corresponding gene of a Xanthomonas sp. The isolates were further identified as Xanthomonas arboricola pv. pruni on the basis of quinate metabolism and starch hydrolysis tests and by sequencing the PCR products obtained with the gyrB (4) and X. arboricola pv. pruni-specific (3) primer sets. Pathogenicity tests were conducted on cvs. Novita and Caucasica following the detached leaf bioassay procedure (1) and by injecting with a hypodermic needle a bacterial suspension (1 × 107 CFU/ml) in the leaf mesophyll of 1-year-old potted plants (three plants per cultivar and three leaves per isolate on each plant). Incubation was carried out at 25°C under fluorescent lights with a 16-h photoperiod. After seven (detached leaves) and four (potted plants) days, all leaves inoculated with X. arboricola pv. pruni isolates showed brown necrotic spots delimited by a chlorotic margin. Reisolated bacteria on YDCA showed the same colony morphology as described above and tested positive to the X. arboricola pv. pruni-specific primer set, confirming the causal agent of the disease. Leaf tissue inoculated with sterile distilled water remained symptomless. Bacterial leaf spot on cherry laurel was reported in Lombardy (northern Italy) by the local plant protection service in 2005 but without a confirmatory diagnosis of the causal agent (2). To our knowledge, this is the first confirmed report on the occurrence of X. arboricola pv. pruni on cherry laurel in Italy. The pathogen could have a significant impact on the commercial cherry laurel production in the district of Pistoia, which is the most important area for ornamental plants nurseries (4,536 ha of cultivated surface in 2005) in Italy. X. arboricola pv. pruni is included in the EPPO A2 list of pests recommended for regulation to the member countries. References: (1) Anonymous. EPPO Bull. 36:129, 2006. (2) EPPO Reporting Service. Online publication. Retrieved from archives.eppo.org/EPPOReporting/2006/Rse-0606.pdf , 2006. (3) M. C. Pagani. Ph.D. diss. North Carolina State University. Online publication. http://repository.lib.ncsu.edu/ir/bitstream/1840.16/4540/1/etd.pdf , 2004. (4) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009.