Hazelnut (Corylus avellana L.) is one of the most economically important tree crops in Italy. Xanthomonas arboricola pv. corylina (Xac) causes bacterial blight of hazelnut (4). During early summer 2010, a survey of three orchards (5 ha total) containing 4-year-old hazelnut trees (cv. Tonda di Giffoni) in Viterbo Province, Latium region, Italy, showed an 80 to 100% incidence of bacterial blight. Initially, water-soaked, necrotic spots were visible on leaves, fruit involucres, and shells, followed by lateral shoot dieback and development of cankers as longitudinal bark cracks on twigs, branches, and main trunks. Brown necrosis of the cambium was observed when bark tissue was removed. By late summer, necrosis had extended down main branches to the trunk, causing complete girdling of branches. Symptomatic tissues were collected from leaves, branches, and trunks, sections were surface-sterilized in 1% NaOCl for 1 min followed by two rinses in sterile distilled water (SDW, each for 1 min), and each section was then crushed in SDW. A loopful of the suspension was streaked onto yeast extract-dextrose-calcium carbonate agar medium (YDCA). Thirty six (12 from each type of tissue) yellow-mucoid, shiny, round bacterial colonies, each approximately 2 mm in diameter, were subcultured on YDCA. All strains were gram-negative and aerobic; negative for indole, lecithinase, urease, tyrosinase, and nitrate reduction; and positive for catalase, growth in 2% NaCl in nutrient broth, and growth at 35°C. All strains produced dark green pigment on succinate-quinate (SQ) medium. Inoculum of each of 15 isolates was prepared in nutrient broth, and washed cells from late log-phase cultures used to prepare a bacterial suspension of each isolate for inoculation of 2-year-old potted hazelnut plants cv. Tonda di Giffoni. A suspension of 106 CFU/ml for each isolate was sprayed onto leaves (10 ml/plant), and drops of inoculum were placed on wounds made on twigs with a sterile scalpel (0.10 μl/wound). For each isolate, three plants were inoculated per inoculation method. Inoculations with two reference strains of Xac (Xaco 1 from central Italy (3) and NCPPB 2896 from England) and SDW were performed on the same number of plants for positive and negative control treatments, respectively. Inoculated plants were maintained at 26 ± 1°C in a greenhouse. After 21 days, all inoculated plants had developed symptoms on leaves, while cankers developed on twigs after 40 days. Positive control plants developed the same symptoms, while negative control plants remained asymptomatic. Bacteria recovered from lesions on plants inoculated with the test strains or positive control strains had the same morphological and physiological characteristics as the original strains. No bacteria were recovered from negative control plants. Total DNA was extracted from bacterial suspensions and 16S rDNA amplified using universal primers (2). Sequences (GenBank Accession Nos. JQ861273, JQ861274, and JQ861275 for strains Xaco VT3 to VT5) had 99 to 100% identity with 16S rDNA sequences of Xac strains in GenBank. In Italy, Xac was reported by Petri in 1932 in Latium, and later in other regions on several hazelnut cultivars (1). However, to our knowledge, this is the first report of the disease causing severe damage in Italy. References: (1) M. Fiori et al. Petria 16:71, 2006. (2) J. R. Lamichhane et al. Plant Dis. 95:221, 2011. (3) J. R. Lamichhane et al. Acta Horticol.:In press. 2012. (4) OEPP/EPPO Bull. 179:179, 2004.
Isolation and characterization of Erwinia piriflorinigrans causal agent flower necrosis of red poppy
