First Report of Willow Anthracnose Caused by Colletotrichum salicis in Poland.

Abstract

Growing interest in willow species of the genus Salix as a bioenergy crop has contributed to an increase in willow plantations in Poland (Sulima et al. 2017). In June 2016, terminal bud necrosis was observed in a willow plantation (Salix purpurea, S. vinimalis × S. schwerini) near Olsztyn (Poland). Brown to black discoloration of willow bark at a height of 150 to 170 cm and progressive necrosis of shoots located above the affected area were noted. The symptoms of disease were observed on two to three shoots per plant, and total infection rates in the plantation reached approximately 50 to 70% in S. vinimalis × S. schwerini and 20 to 30% in S. purpurea. Willow shoots (three to five per plant) were sampled in five locations in the plantation to identify the fungus responsible for the disease symptoms. The shoots with healthy, and diseased tissues were cut into 5-mm sections, surface disinfected with 70% ethanol for 1 min and 1% NaOCl, and placed on Petri plates with potato dextrose agar growth medium. The obtained fungal colonies were characterized by gray aerial mycelia, and the cultures contained cylindrical, hyaline, and smooth-walled conidia measuring 11 to 17 × 3 to 5 μm (n = 50). The cultured species was identified as C. acutatum based on its morphological characteristics (Damm et al. 2012). The taxonomic identity of the pathogen was confirmed by isolating DNA from representative strain SP17/2016 with the use of the Maxwell16LEV Plant DNA Kit (Promega, Madison, WI). The identity of the species was confirmed by next-generation sequencing with the use of the MiSeq platform (Illumina, San Diego, CA). The genomic DNA libraries were prepared using the Nextera XT kit (Illumina). The sequences were assembled using Velvet 1.2.10 and were annotated using Augustus 0.1.1, both implemented in Geneious 8.1.9, and were deposited in GenBank. The identification of the isolate to the species level of C. salicis was validated by performing a BLASTn search with six sequences in accordance with polyphasic analysis (Sharma and Shenoy 2016). The following matches were obtained: JFFI01000260.1 (region, 7506 to 7929; protein ID, KXH68653.1) for heat shock factor binding protein 1 (KY745736) with I = 99% and Q = 100%; JFFI01001175.1 (region, 118784 to 119087; protein ID, KXH62418.1) for cytochrome-c oxidase chain VIIc (KY745760) with I = 100% and Q = 100%; JFFI01001268.1 (region, 10848 to 11483; protein ID, KXH61539.1) for ubiquitin-conjugating enzyme E2 35 (KY745744) with I = 99% and Q = 100%; JFFI01001474.1 (region, 60131 to 61160; protein ID, KXH59678.1) for p450 monooxygenase (KY745746) with I = 100% and Q = 100%; JFFI01001277.1 (region, 73500 to 75238; protein ID, KXH59678.1) for glutathione synthetase (KY745725) with I = 99% and Q = 100%; and JFFI01002559.1 (region, 48713 to 49176; protein ID, KXH30318.1) for NADH:ubiquinone oxidoreductase 11.5kD subunit (KY745730) with I = 100% and Q = 100%, where I is “identity” and Q is “query coverage” (queries are our sequences). To confirm pathogenicity, the conidia (1 × 10⁷/ml) of the SP17/2016 isolate, obtained from 14-day cultures exposed to ultraviolet (UV) radiation (in the 12-h dark/12-h UV light cycle), were transferred to the shoots of 1-year-old S. purpurea and S. vinimalis seedlings in a greenhouse experiment (16-h light/8-h dark photoperiod, temperature of 24°C, plants generally watered every other day). The first disease symptoms (necrosis) on shoots were observed around 7 days after the experimental infection. The presence of C. salicis was confirmed by culturing the isolated pathogen, and its species identity was determined based on microscopic observations of morphological features and by qPCR with the use of primers specific for C. acutatum (Debode et al. 2009). To date, willow anthracnose caused by C. acutatum has been reported from California (U.S.A.) (Swain et al. 2012), and C. salicis has been isolated from willows in Germany, New Zealand, and the Netherlands (Damm et al. 2012). This is the first report documenting the presence of C. salicis, the causative agent of willow anthracnose, in Poland.