Phylogenetic Diversity and Phenotypic Characterization of Phlyctema vagabunda (syn. Neofabraea alba) and Neofabraea kienholzii Causing Postharvest Bull's Eye Rot of Apple in Northern Italy.

Bull's eye rot, caused by Phlyctema vagabunda and Neofabraea species, is one of the most important postharvest diseases of apple. South Tyrol (northern Italy) is the largest continuous apple-producing area in Europe, with approximately 1 million tons being produced yearly and conserved in technologically advanced storage facilities for several months. Still, studies on the pathogen species causing postharvest bull's eye rot of apple, as well as their diversity and biology, are lacking for this region. Therefore, the main purpose of the present work was to identify and characterize fungal isolates obtained from decayed apple fruit with symptoms of bull's eye rot that were collected in 2018 and 2019 in different packinghouses in South Tyrol. Among more than 1,000 fungal isolates that were obtained, 419 could be assigned to the genera Phlyctema and/or Neofabraea based on rot symptoms on apple fruit and colony morphology on potato dextrose agar. A smaller subset of 101 representative isolates was further analyzed by DNA sequencing of the internal transcribed spacer region. Furthermore, partial segments of the ß-tubulin gene, the translation elongation factor 1α gene, and the 16S mitochondrial ribosomal RNA gene were studied. The phylogenetic analyses, including sequences of reference species, showed that P. vagabunda is the dominant species associated with bull's eye rot of apple in the study area, whereas Neofabraea kienholzii was found only on a small number of apple fruit samples. The combination of multilocus sequence data revealed 11 unique genotypes that belonged to P. vagabunda and four to N. kienholzii. To the best of our knowledge, this study is the first to report N. kienholzii as a postharvest pathogen of apple in Italy. Finally, a pathogenicity test demonstrated different degrees of virulence among selected isolates of P. vagabunda and N. kienholzii on the cultivar Golden Delicious. The present study emphasizes the importance of accurate species identification, because different species may vary in their biological and pathogenic characteristics, and consequently require distinct disease management strategies, both in the field and during the postharvest stages.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Colletotrichum salicis Causing Bitter Rot of Apple in Italy.

Apple (Malus domestica Borkh.) is the most important fruit crop in South Tyrol (northern Italy), with a total production of 905.089 tons in 2019 (Chamber of Commerce, Industry, Crafts and Agriculture of Bozen-Bolzano 2020). Symptoms of bitter rot were observed on organic apples of the cultivar 'Roho 3615'/Evelina® collected in a packinghouse in South Tyrol at the end of March 2018 after approximately six months of storage in controlled atmosphere. Lesions were circular and light brown with orange conidial masses. Tissue samples were removed under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to Petri dishes with potato dextrose agar (PDA), and incubated in the dark at room temperature for two weeks. Single spore cultures were obtained by adapting the procedure of Choi et al. (1999). Pure cultures were grown in quadruplicate on PDA at 20°C in the dark for two weeks. The colony appearance on the upper side was mostly flat with a distinct margin, the surface was covered with short, floccose aerial mycelium, and the color ranged from light gray to dark gray, while the reverse side appeared yellow. From each replicate culture, conidia were harvested, and the length and width of 50 randomly selected conidia were measured using a compound light microscope coupled to a digital camera (Leica DMLS, Leica Microsystems, Wetzlar, Germany). Conidia were cylindrical to fusiform, pointed at one end, and measured 10.0 to 19.5 × 2.5 to 5.0 µm (14.5 ± 1.9 × 3.9 ± 0.7 µm [mean ± SD]) in consistency with Damm et al. (2012). In order to determine the species of isolate 18-DSS-BS-EL-1-012, a multi-locus DNA sequence analysis was performed. Genomic DNA was extracted by following the protocol described by Cassago et al. (2002). Four loci, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3) and the internal transcribed spacer (ITS) region of the rRNA operon were amplified by PCR and Sanger sequenced (Damm et al. 2012; Lévesque and de Cock 2004). The obtained DNA sequences of ACT, GAPDH, HIS3 and ITS were 186, 150, 317 and 505 bp long and were submitted to GenBank under the accession numbers MT347599, MT347600, MT347598 and MT337388, respectively. A MegaBLAST analysis resulted in 100% sequence identity at all four loci with a type culture of Colletotrichum salicis (CBS 607.94; GenBank accession numbers: JQ949781, JQ948791, JQ949451 and JQ948460), which belongs to the C. acutatum species complex (Damm et al. 2012). A pathogenicity test was performed with twelve 'Golden Delicious' apples by wounding the fruit with a sterile piercing tool and inoculating 20 µl of spore suspension (104 conidia per ml) from a 21-day-old PDA culture. Inoculated fruits were incubated for 21 days in a moist chamber at 20°C in the dark. The symptoms were recorded at 3, 5, 7, 10, 14 and 21 days post-inoculation (dpi). Symptoms appeared after 7 days on all inoculated fruits and resembled those observed on the original fruit, while mock-inoculated controls with sterile water remained symptomless. Fungal colonies resembling the original culture were re-isolated from lesions on the apple and plated on PDA. Their identity was confirmed by DNA sequence analysis of the ITS region, thereby proving Koch's postulates. Bitter rot occurs globally and is considered one of the most important diseases of apple that has the potential to cause significant crop losses (Sutton et al. 2014). Other Colletotrichum species have been commonly reported from apple in Europe, such as C. godetiae and C. fioriniae, whereas C. salicis has been reported solely in New Zealand and Belgium (Damm et al. 2012; Grammen et al. 2019). To the best of our knowledge, this is the first report of C. salicis causing bitter rot of apple in Italy.