First report of colletotrichum graminicola causing maize anthracnose in Bosnia and Herzegovina

Colletotrichum graminicola is the causal agent of maize (Zea mays) anthracnose. The most common symptoms are leaf blight, top dieback, and stalk rot (Munkvold and White 2016). Owing to its negative impact on maize production, it is important to study the distribution and propagation of this disease. In September 2018, maize stem samples exhibiting symptoms of anthracnose stalk rot (glossy, black, and irregularly shaped lesions) were collected from a field in the Odžak (Posavska Mahala) region of northeastern Bosnia and Herzegovina. Stem samples, approximately 50 mm2, were dissected and surface disinfected for 90 s in 20% sodium hypochlorite (v/v) and rinsed three times in sterile distilled water. The samples were transferred to one-half-strength acidified potato dextrose agar (PDA) Petri dishes supplemented with ampicillin (100 µg/ml) and incubated for 4 to 6 days at 25°C (Sukno et al. 2008). Single-spore isolates from three distinct stem pieces were cultured on PDA. The aerial mycelium was dark gray with orange-colored spore masses, and conidia were falcate, slightly curved, tapered toward the tips, and produced in acervuli with setae. The length and width of 50 conidia were measured and ranged from 25 to 35 µm (average, 29.8 µm; standard deviation, ± 2.0 µm) and from 7 to 11 µm (average, 9.0 µm; standard deviation, ± 0.7 µm), respectively. These morphological characteristics are consistent with C. graminicola (Bergstrom and Nicholson 1999). Two isolates were grown in potato dextrose broth for 3 days at 25°C, and total genomic DNA was extracted following the protocol of Baek and Kenerley (1998). The internal transcriber spacer (ITS) region of rDNA was amplified using universal fungal primers ITS4 and ITS5 and consequently sequenced. GenBank BLAST analysis revealed that the sequences were 100% identical to ITS rDNA sequences of C. graminicola isolates. One sequence was deposited in GenBank under accession number MK955539. These results were consistent with the initial identification of C. graminicola. To confirm Koch’s postulates, maize plants (vegetative stage V3) were placed horizontally in a tray for inoculation and 20 droplets (7.5 µl) of a suspension of 3 × 105 conidia/ml were placed on the surface of the third leaf. The trays were closed to retain moisture and incubated overnight at 23°C. The next day, the plants were returned to a vertical position and incubated in a growth chamber at 25°C with 80% humidity and a light cycle of 16 h of light and 8 h of dark (Vargas et al. 2012). After 6 days, the leaves of the inoculated plants presented brown elongated lesions with chlorotic margins and necrotic centers consistent with C. graminicola infection. Controls inoculated with water did not exhibit any symptoms. Microscopic analysis of the surface of infected leaves revealed acervuli, setae, and conidia that were identical to the original isolates. To our knowledge, this is the first report of C. graminicola causing maize anthracnose in Bosnia and Herzegovina (Arsenijević et al. 1996). Previous reports have shown that maize anthracnose is an important disease in Croatia (Palaversic et al. 2009), and it has been reported in Slovenia. Because it may also be a threat for neighboring countries such as Serbia, Romania, and Hungary, where the cultivation of maize is an important component of the economy, continued surveillance is needed.