First report of Botryosphaeria dothidea causing root rot of sugar beet in Serbia.

Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not. is predominantly recognized as a pathogen of various woody plants, inducing symptoms of stem canker, dieback, and fruit rot worldwide. However, sporadic reports suggest its impact on field crops, including B. dothidea associated with stem canker in soybean and tobacco (Bian et al. 2015; Chen et al. 2021), as well as B. quercuum on sugar beet (Alfieri et al. 1984). In September 2023, during a survey of root rot pathogens of sugar beet (Beta vulgaris L.) in Rimski Šančevi, Serbia (N 45°19´57″; E 19°49'58″), 3% of collected samples exhibited root rot symptoms. Externally, the lesions exhibited a dark brown coloration. On cross-section, the tissue displayed a gradient of discoloration ranging from light to dark brown throughout the roots. The roots were completely rotted. From these samples, two fungal isolates (SR28/II and SR4/III) were obtained from rotted internal root fragments, after washing, surface disinfection (70% ethanol), and plating on potato dextrose agar (PDA). Colonies on PDA were fluffy with abundant aerial mycelium, surface light to dark grey-brown, reverse black in the centre and grey-brown towards the irregular margin, after 7 days at 25°C in the dark. To induce sporulation, isolates were cultivated on 2% water agar with pine needles and incubated under continuous near ultraviolet (NUV) light at room temperature for 30 days. Conidia were hyaline, aseptate, fusiform, subtruncate at the base, subobtuse at the apex, and measured (19.56-) 24.12 - 26.31 (-28.99) x (5.13-) 5.94 - 6.54 (-7.44) µm (mean 25.06 x 6.26 µm, n=100), consistent with description of B. dothidea (Phillips et al. 2013). For molecular identification, DNA was extracted from mycelium of 7-day-old cultures, and internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha gene (TEF) and partial β-tubulin gene (TUB) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Multiple sequence alignment and BLAST analyses showed that our isolates had 100% sequence similarity with reference isolates of B. dothidea, including ex-type isolate CBS 115476 (= CMW 8000) in ITS (AY236949), TEF (AY236898), and TUB (AY236927). Maximum likelihood phylogeny of concatenated sequences confirmed the identity of the isolates as B. dothidea. Sequences of ITS, TEF and TUB of isolate SR4/III were submitted to GenBank under accession numbers PP908658, PP911334, PP911333, respectively. The pathogenicity of obtained isolates was assessed on 3-month-old sugar beet plants grown in sterile substrate in the greenhouse. For inoculation, the upper parts of the roots were wounded (10x3 mm) 1 cm above the substrate, using a sterilized nail, and 2x2 mm mycelial plugs of 7-day-old culture grown on PDA were inserted and sealed with parafilm. Control plants were inoculated with sterile PDA plugs. Six plants were used per isolate and for control. After 3 weeks of incubation, the inoculated plants were removed from the substrate and brown rot symptoms were observed upon cross-sectional examination, whereas control roots remained asymptomatic. The pathogen was successfully reisolated and morphologically identified as B. dothidea, fulfilling Koch's postulates. The production of sugar beet, an important industrial crop in Serbia valued for its sugar content, is imperiled by various pathogens. To our knowledge, this is the first report of B. dothidea causing root rot of sugar beet in Serbia and the world. Given its widespread distribution on other hosts and potential aggressiveness, the presence of B. dothidea in sugar beet should not be neglected.