First Report of Yeast-Spot Disease of Soybean Seeds Caused by Eremothecium coryli in Serbia

Over the last 15 years, the area planted with soybeans (Glycine max) in Serbia has increased drastically, from 131,000 hectares in 2005 to 230,000 in 2019, and the average yield reached 3.2 t/ha in 2020. The Province of Vojvodina is the most important soybean production region with 95% of the total soybean area in Serbia (www.stat.gov.rs). During the 2021 growing season, soybean seeds with various kinds of symptoms including colour changes, light and dark brown spots, blotching, necrosis, and shriveling were collected from soybean field before harvest of soybean cv. Dukat in the Tamiš locality (South Banat District, Vojvodina Province: GPS: 44°56'12.936"N 20°43'24.216"E) in Serbia. The incidence of symptomatic seeds was estimated at 6.4%. Symptomatic soybean seeds were surface disinfected with 2% NaOCl for 2 min, rinsed in sterile water, dried on sterile filter paper, placed on potato dextrose agar (PDA) and were incubated at 25°C in the dark for 10 to 14 days. The identification of fungi at the genus level based on morphological characteristics revealed the presence of species of Macrophomina, Botrytis, Cercospora and Alternaria, which were previously reported as pathogens of soybean seed in Serbia (Krsmanović et al. 2020). Also, seven white to slightly creamy colonies with yeast-like morphology were observed around seeds expressing discoloration and necrotic and sunken spots. Ten days later, microscopic observations of yeast-like colonies revealed the presence of globose budding cells (diameter of 20 to 28 μm) mostly single or rarely in short chains. Also, two to eight needle-shaped ascospores (52 to 80 μm in length) were arranged lengthwise in many cylindrical to naviculate asci (60 to 96 x 8 to 12, avg. 72.4 x 9.2 µm). Ascospores were with a unilateral, slender, flexuous, whip-like appendage. The morphology of the different fungal structures indicated that the pathogen was Eremothecium coryli (Pelgion) Kurtzman and it was further supported by molecular identification. Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). Sequence analysis of ITS region revealed that the Serbian isolate ND2/21 (GenBank Accession No. OL958602) shared the highest nucleotide identity of 100% with E. coryli isolate (Accession No. KY103387). For pathogenicity test, fresh soybean seeds (cv. Sava) were surface-disinfected with 2% NaOCl and rinsed in sterile water before inoculation. The seeds were pierced 3-4 times with a sterile insect pin through a drop of yeast suspension (concentration 106 ascospores/ml) of one selected single-spore isolate (ND2-21). Similarly, control seeds were pierced with sterile insect pins through a drop of sterile distilled water. Five inoculated seeds and control (five replicates per treatment) were arranged uniformly in a Petri dish (9 cm diameter) and incubated at 22 to 25°C in the dark and kept under >95% relative humidity during the first 48 h. Twenty days after inoculation, small brown necrotic lesions were visible on the soybean seeds. Re-isolation from symptomatic seeds on PDA dishes yielded yeast-like colonies with the same morphological characteristics as those used for inoculation, thus confirming Koch's postulates. The control seeds had no symptoms. This fungus is widely known as a pathogen of yeast spot disease on soybean seeds (Heinrichs et al. 1976; Kimura et al. 2008), but to our knowledge, it has never been reported in Serbia. Considering that invasive species Nezara viridula L. and Halyomorpha halys (STÅL, 1855), the vectors of this fungus, were reported in our country (Kereši et al. 2012; Šeat 2015) and that their mass appearance has been documented in recent years (Konjević et al. 2020), the presence of this pathogen has the potential to cause considerable damage and severe yield losses, resulting in significant economic impact on soybean production in Serbia