RESUMO
Hazelnut is a minor but rapidly increasing commercially grown species in Montenegro. In June 2021, severe infection, affecting more than 80% of the trees, was observed on 6-year-old hazelnut plants (Corylus avellana) cultivar Hall's Giant, in a 0.3ha plantation near Cetinje, central Montenegro. Numerous, small, 2-3mm in diameter, irregular, brown, necrotic spots, sometimes surrounded by a weak chlorotic halo, were observed on leaves. As the disease progressed, the lesions coalesced and formed large necrotic areas. Necrotic leaves remained attached to the twigs. Longitudinal brown lesions developed on twigs and branches, causing their dieback. Necrotic, unopened buds were noticed as well. No fruits were observed in the orchard. From the diseased leaf, bud and twig bark tissue, yellow, convex, and mucoid bacterial colonies were isolated on yeast extract dextrose CaCO3 medium and 14 isolates were subcultured. The isolates induced hypersensitive reaction in pelargonium leaves (Pelargonium zonale), were Gram-negative, catalase positive, oxidase negative, obligate aerobic, hydrolyzed starch, gelatin and esculin, did not reduce nitrate and did not grow at 37°C and in the presence of 5% NaCl, showing so the same biochemical profile of the reference strain Xanthomonas arboricola pv. corylina (Xac) NCPPB 3037. Using primer pair XarbQ-F/XarbQ-R (Pothier et al., 2011), a 402 bp product was amplified in all 14 isolates and the reference strain, confirming their affiliation to X. arboricola species. Additionally, the isolates were further identified by PCR analysis, using primer pair XapY17-F/XapY17-R (Pagani 2004; Pothier et al., 2011), resulting in a single band of 943 bp characteristic for Xac. The amplification and sequencing of the partial rpoD gene sequence of two selected isolates RKFB 1375 and RKFB 1370, were performed using a set of primers described by Hajri et al., 2012. The obtained DNA sequences showed that the isolates (GenBank Nos. OQ271224 and OQ271225) share 99.47% to 99.92% rpoD sequence identity with Xac strains CP076619.1 and HG992342.1 isolated from hazelnut in France and HG992341.1 in USA. Pathogenicity of all isolates was confirmed by spraying young shoots (20 to 30 cm long, with 5 to 7 leaves) on 2-year-old potted hazelnut plants (cv. Hall's Giant) using a handheld sprayer with the bacterial suspension (108 CFU/mL of sterile tap water), in three replicates. Sterile distilled water (SDW) and NCPPB 3037 Xac strain were used as negative and positive control, respectively. The inoculated shoots were incubated under plastic bags, providing high humidity conditions, in an acclimatized greenhouse at 22-26°C, for 72 h. Lesions surrounded by a halo appeared on leaves of all inoculated shoots within 5 to 6 weeks after inoculation, while leaves sprayed with SDW remained symptomless. Koch's postulates were confirmed by the re-isolation of the pathogen from the necrotic test plant tissue and identity checked by PCR using the primer set of Pothier et al., 2011. Based on pathogenic, biochemical, and molecular characteristics, the isolates from hazelnut plants in Montenegro were identified as X. arboricola pv. corylina. This is the first report of Xac affecting hazelnut in this country. Considering favorable environmental conditions, the pathogen can cause significant economic losses in hazelnut production in Montenegro. Therefore, phytosanitary measures have to be implemented to prevent introduction and spread of the pathogen in other areas.
