First report of anthracnose on Fagus sylvatica caused by Colletotrichum fioriniae in Italy

Abstract

European beech (Fagus sylvatica) is one of the most common deciduous forest species in Italy and across Europe, important for its economic, cultural and environmental value (Siddique et al., 2021). In June 2023, leaves showing anthracnose symptoms (Figure 1) were collected from the UNESCO site “Faggeta Vetusta di Monte Raschio” located in Oriolo Romano, Viterbo in central Italy. To identify the causal agent, leaves were surface sterilised by dipping successively in 70% ethanol for two minutes, 1% sodium hypochlorite for three minutes and 70% ethanol for one minute, and finally rinsed three times with sterile water (SDW). Slices obtained with a sterile scalpel were plated on potato dextrose agar plates and incubated at 25°C for 8 to 15 days. Developing fungal colonies had white to grey cottony mycelium and were pale orange with dark flecking from the reverse side (Figure 2). Conidia were hyaline, smooth-walled, aseptate, narrowly elliptical pointed at both ends, measuring 10.5-21 μm (mean 16.4 μm) × 3.5-7 μm (mean 4.3 μm) (Figure 3). The morphological characteristics fit published descriptions of Colletotrichum fioriniae (Shivas & Yu, 2009; Damm et al., 2012). Genomic DNA of three representative isolates (FAG14, FAG15 and FAG16) were extracted from 100 mg of fresh mycelium using the NucleoSpin®PlantII kit manufacturer's protocol for fungi (Macherey-Nagel,Germany). The ribosomal internal transcribed spacer (ITS), β-tubulin (TUB2), actin (ACT), partial chitin synthase (CHS-1), histone 3 (HIS3) and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions were amplified and sequenced. A BLASTn search of the sequences (GenBank Accession Nos. OR587916-18 for ITS, OR596663-65 for TUB2, OR596666-68 for ACT, OR596669-71 for CHS-1, OR596672-74 for HIS3, OR596675:77 for GAPDH) revealed 100% identity with C. fioriniae. Pathogenicity tests were done using pure cultures of the FAG14 isolate. Healthy beech leaves were surface sterilised and inoculated with 10 μL of a spore suspension (106 conidia/mL) on the surface. Leaves inoculated with SDW were used as a control. The inoculated samples were placed in closed sterile boxes and kept at 25°C and 100% relative humidity. Seven to ten days after inoculation, the leaves had necrotic spots, whereas the controls remained healthy (Figure 4). To fulfil Koch's postulates, C. fioriniae was reisolated from the symptomatic leaves and its identity was confirmed both morphologically and molecularly. To the best of our knowledge, this pathogen has only previously been reported on Fagus sylvatica in seedlings in a nursery in Poland (Pszczółkowska et al., 2017). Thus, this is the first report of C. fioriniae causing anthracnose on adult trees of Fagus sylvatica in a natural forest in Italy. This study is part of the Agritech National Research Center and funded by the European Union Next-Generation EU.