First report of Erwinia amylovora in Tuscany, Italy

Copyright: © 2021 D. Migliorini, F. Pecori, A. Raio, N. Luchi, D. Rizzo, C. Campani, L. Neri, A. Santini. This is an open access, peer-reviewed article published by Firenze University Press (http:// www.fupress.com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Fire blight symptoms were observed on 2-year-old pear trees (Pyrus communis 'Williams' and 'Red Williams') in October 2020 during an orchard phytopathological survey in south-eastern Tuscany (43.37966 N, 11.81162 E), an important fruit production area.
Plants were obtained in early spring 2020 from a fruit tree nursery located in a "protected area" within the Emilia Romagna region, where the pathogen causing this disease has been present since 1994 (EPPO, 114/1995). Plant material was standard, i.e. without phytosanitary certification proving disease-free status. At the time of the survey, the young pear trees showed a variety of symptoms, including shoot blight, stem canker or complete dieback, dried terminal shoots and shepherd's crook (Figure 1). During autumn 2020, 21 infected plants were observed (19 Pyrus communis, two Malus domestica) out of more than 400 plants surveyed. Symptomatic shoot samples were collected, transferred to the laboratory on ice, and were processed for bacteria isolation and molecular analyses. Tissue samples were processed according to EPPO standard protocol (2013), as described below. Fragments of symptomatic tissues were surface sterilized in 1% NaClO solution, washed in sterilized distilled water and then ground in an antioxidant maceration buffer. Macerated tissues were enriched in liquid King's B medium (Sigma-Aldrich) and were incubated at 25°C for 48 h. To obtain single colonies, enriched suspensions were streaked onto Levan medium and nutrient glucose agar (NGA; 28 g L -1 nutrient agar, 5 g L -1 glucose: Oxoid), and incubated at 27°C for 48-72 h. Bacterial isolates were selected on the basis of colony morphology, and were purified and evaluated by KOH tests (Buch, 1982) to identify Gram negative bacteria. Isolates showing white mucoid colonies and levan production on Levan medium, were identified as putative Erwinia amylovora.
Sequencing of 16S rDNA (GenBank accession no. MW786972; 1392 bp;  (Table 1). Alignments were made using Geneious Prime (version 11.0.9) and phylogenetic analysis was performed with MEGA (version 10.2.2), using the Maximum Likelihood method and Tamura-Nei model. The trees obtained for both genes confirmed the species identification (Figures 2 and 3).
The presence of the pathogen was also confirmed by extraction of DNA with CTAB 2% (Li et al., 2008) and All processed samples were identified as E. amylovora. All symptomatic plants were removed and destroyed. In order to check for others possible outbreaks intensive monitoring started in the original orchard and the surrounding areas. This is the first report of E. amylovora in Tuscany. This pathogen may pose a serious threat to apple and pear production in this area. Presence of this pathogen in Tuscany is also a clear example of the spread of a quarantine pathogen by the plants-for-planting pathway. This record supports the need to use certified plant material, especially when nurseries are located in protected areas.  Tamura -Nei model (Tamura and Nei, 1993). The tree with the greatest log likelihood (-2704.47) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Number of boostrap replicates = 500. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 11 nucleotide sequences. There were a total of 1285 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The sequence obtain in the present study is marked with a black dot.

Figure 3.
Phylogenetic analysis based on recA gene sequences from diverse Erwinia spp. strains. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei, 1993). The tree with the greatest log likelihood (-1905.63) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Number of boostrap replicates = 500. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 16 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There was a total of 399 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The sequence obtain in the present study is marked with a black dot.