First report of Phaeocytostroma ambiguum causing root and stem base rot on maize in Bulgaria

Maize (Zea mays) is the second most grown cereal crop in Bulgaria, with an average area of about 500 000 ha. Damage was observed in October 2014 on a plot in the region of Silistra (northeastern Bulgaria). Symptoms consisted of scattered plants with suppressed growth and small cobs due to a root and stem base rot. On some of the damaged parts (except cobs), black conidiomata were visible. The isolations on potato dextrose agar (PDA) revealed a relatively fast-growing fungus. Single-conidia colonies grown in the dark at 25°C on PDA were fluffy and covered the plates (d = 90 mm) in about one week. The conidiomata both on plant parts and medium released two types of conidia morphologically identical to those of Phaeocytostroma ambiguum (Mont.) Petr. (Lamprecht et al. 2011). The predominant type (alpha-conidia) were pale to medium brown, ellipsoid to pyriform, with an ovoid to truncate base, averaged 13.8 × 6.8 μm. The second type (beta-conidia) were sparse, predominantly formed in conidiomata on plant tissue, and were pale brown, subcylindrical, straight to curved and measured 15 to 28 × 1.25 to 2 μm. Pathogenicity of two isolates was proven by artificial inoculation of maize seedlings (4-week old, 3 plants per pot, d = 10 cm) by pouring the substrate with a mix of sterile water (100 ml) and a blended 3-week-old colony grown on PDA, followed by incubation in a moist chamber (72 h). Additionally, detached maize cobs (milk and dough stages) were also inoculated by placing colonized PDA plugs (5 mm) under the corn husks and covering the inoculation site with plastic tape before incubation under moist conditions (48 h). All inoculated objects and the negative controls (inoculated with sterile water or sterile PDA plugs) were incubated at room temperature (22 to 25°C). A positive reaction consisting of necrosis around the inoculation point was first found on the cobs after one week. After another 2-week period, the symptomatic zone enlarged and was covered by numerous emerging, small, black stromata. The single-necked conidiomata were fully mature after about 40 days. Symptoms of wilting and root necrosis were registered in the seedlings test about a month after the inoculation. The subsequent reisolations revealed the same fungus from all inoculated samples but not from negative controls. The genomic DNA of one isolate was extracted using a fast DNA extraction method (Vercauteren et al. 2010). The rDNA-ITS region was amplified using primers ITS1 and ITS4 (White et al. 1990) and the PCR product was sequenced. This sequence was BLAST searched at GenBank, resulting in a 100% homology to all P. ambiguum ITS sequences present in the database, and was subsequently submitted to GenBank (Accession No. KT966745). To our knowledge, this is the first report of P. ambiguum (Mont.) Petr. as the causal agent of root and stem base rot on maize in Bulgaria. The pathogen’s capacity to inoculate the cobs should also be noted.