First Report of Colletotrichum graminicola Causing Maize Anthracnose in Austria

Colletotrichum graminicola causes maize anthracnose leaf blight and stalk rot. Stalk rot causes blackening of lower stalks, dark brown pith, and often leads to sudden plant death and lodging before grain maturity. As the infection progresses, the degradation of pith tissue weakens the plant's structural integrity, leading to lodging and significant economic losses (Wise et al., 2016). On October 14, 2024, maize stem samples displaying black, irregularly shaped lesions were collected from a cultivated field in Lipsch, southern Austria. Disease incidence in this field was estimated at approximately 1%, and severity ranged from 10% to 80%. Stem sections (~50 mm²) were excised, surface-disinfected for 90 s in 20% commercial bleach (v/v), and rinsed three times with sterile distilled water. The samples were then transferred to potato dextrose agar (PDA) Petri dishes supplemented with ampicillin (100 μg/ml) and lactic acid (1.5 ml/l) and incubated at 25°C for 2 days. Subsequently, fungal colonies were subcultured onto half-strength PDA medium and incubated for an additional 2 days before being transferred to PDA and grown for 10 to 15 days (Sukno et al., 2008). Individual spores were isolated and cultured on PDA for 6 days. Morphological characterization revealed dark gray aerial mycelium with orange-colored spore masses. One hundred conidia of each isolate were examined, revealing conidia that are falcate, slightly curved, and tapered at the tips, measuring 29.85 ± 2.62 × 6.13 ± 0.66 μm. These characteristics are consistent with the description of C. graminicola. Two isolates, AU-8423-4 and AU-8423-15, were selected for molecular analysis. They were grown in PDB at 25°C under light and agitation for three days. DNA extraction was performed using a modified CTAB method (Baek & Kenerley, 1998; Irfan et al., 2013). The internal transcribed spacer (ITS) region of rDNA and the manganese-type superoxide dismutase gene (Sod2) were amplified using the primers ITS4 and ITS5 (White et al., 1990) and SOD625/SOD507 (Fang et al., 2002) and consequently sequenced. BLAST analysis showed that the sequences were 100% identical to each other and to those of C. graminicola strains in GenBank, including that of strain M1.001 which is commonly used for molecular genetic studies. Phylogenetic analysis including closely related Colletotrichum spp. confirmed the clustering of AU-8423-4, AU-8423-15, and M1.001 into a unique clade. All sequences were deposited in GenBank (accession numbers: PV200080 - PV200083). The combination of genotypic, phylogenetic, and phenotypic analyses confirmed that the isolates belonged to C. graminicola. To fulfill Koch's postulates, maize plants (Mo940) at the V3 developmental stage were placed horizontally in a tray for inoculation. Twenty droplets (7.5 μl each) of a conidial suspension (3 × 10⁵ conidia/ml) were applied to the surface of the third leaf. The trays were sealed to maintain humidity and incubated overnight at 23°C. The following day, the plants were returned to a vertical position and maintained in a growth chamber at 25°C with 80% relative humidity under a 16 h light/8 h dark cycle (Vargas et al., 2012). After six days, inoculated leaves developed brown, elongated lesions with necrotic centers, consistent with C. graminicola infection. In contrast, control plants inoculated with water showed no symptoms. Fungal strains reisolated from infected leaves were morphologically identical to the original isolates. To our knowledge, this is the first report of C. graminicola causing maize anthracnose in Austria. Recently, C. graminicola has also been reported in several nearby European countries, including Spain, Portugal, Switzerland and Bosnia and Herzegovina (Rogério et al., 2023). The rapid spread of the pathogen across various regions of Europe serves as a warning of the potential for increased economic losses which could severely impact maize crops across the continent. This situation underscores the urgent need for effective monitoring and intervention strategies to mitigate the risks associated with maize anthracnose.