First Report of <i>Aspergillus tubingensis</i> Causing Maize Ear Rot in Serbia

Maize is the most significant cereal which in Serbia in last decade was harvested on approximately 1.15 million hectares in Serbia with average yield 5300 kg ha-1 and average total amount of production 5.7 million tons (Đokovic et al. 2019). Aspergillus section Nigri poses significant challenges in species classification due to the similarity in morphological and chemical characteristics among them (Ismail, 2017). The first symptoms were observed on maize in September 2017, 2 to 3 weeks after silking in the form of powdery or velvety black mycelium on the surface of kernels at the top of the ear. With favorable weather conditions, the mycelium colonizes along rows of kernels. Disease incidence ranged from 5 to 10% in Zemun Polje, Serbia (44.8713° N, 20.3230° E). After surface disinfection with bleach/distilled water 1:3, isolation of the fungus was performed by direct plating on Potato Dextrose Agar (PDA). Pure colonies were used to obtain monosporial cultures. For identification, fragments of colonies were transferred to Malt Extract Agar (MEA) and Czapek Yeast Autolysate Agar (CYA) and incubated at 25°C in the dark for 7 days. Colonies on CYA were black, cottony with a whitish border, averaging about 56 mm in diameter. Conidia on MEA were wrinkled in texture, globular in shape, and had a mean diameter of 4.2 µm (n=50) which is consistent with description of A. tubingensis (Samson et al. 2007). For molecular identification internal transcribed spacer region (ITS), polymerase II second largest subunit (RPB2) and calmodulin gene (CAM) were amplified (Samson et al. 2014) using the primer pairs ITS1/ITS4 (White et al. 1990), 5F/7CR (Liu et al., 1999), and CMD5/CMD6 (Hong et al., 2005), respectively. BLAST analyses sequence of the ITS region showed 100% similarity as well as the same max score with different A. tubingensis (MH858714) and A. niger (MH892847). BLAST analyses of CAM and RPB2 gene showed that our isolate shared 100% similarity with isolates of A. tubingensis (NRRL 365-EF661149 and NRRL 4851-EF661054, respectively). Concatenated Maximum Parsimony tree (with 1000 bootstrap replicates) including sequences from reference isolates from A. section Nigri and isolate 4473 confirmed its identity as A. tubingensis. Sequences of ITS, RPB2 and CAM were submitted to GenBank under accession numbers OQ456474, OQ737939, OQ426520, respectively. The pathogenicity of isolate 4473 was carried out in the field in Zemun Polje. For inoculations, conidial concentrations (1×10^6 spores/ml) were injected into the silk channel three days after 50% of them had reached silking. Isolate 4473 was used to inoculate twenty ears, with four replicates (Reid et al. 1996). The inoculum was prepared from 7-day-old PDA colonies, and 2 ml of a conidial suspension was used. Control plants were inoculated with sterile water. After three weeks, all the inoculated ears developed symptoms similar to those in the field, while the control ears showed no signs of infection. We successfully reisolated the fungus, using morphological and molecular techniques we confirmed it was identical to the original isolate, fulfilling Koch's postulates. To our knowledge, this is the first report of A. tubingensis on maize kernels in Serbia. Considering that the countries of Southeast Europe are experiencing climate change, which contribute to an increase in the frequency of Aspergillus species and the infections they cause, an improved understanding of their presence, symptoms, morphology and genetic profiles is necessary (Levic et al. 2013).