First Report of <i>Curvularia dactylocteniicola</i> Causing Curvularia Leaf Spot of Maize in China

Curvularia leaf spot (CLS) caused by Curvularia lunata is one of the most destructive foliar diseases of maize in China. Typical symptoms of this disease are small, round or oval lesions with a pale center and a brown edge surrounded by a yellow halo. In 2021, a large number of diseased maize leaves with typical symptoms were collected from three regions (Yanan City, Weinan City, and Ankang City) in Shaanxi Province, China, in order to study the genetic diversity of C. lunata. Small pieces (2 × 2 mm) were excised from the lesion margins, surface-sterilized with 2% sodium hypochlorite for 2 min, and then rinsed with sterile water three times. The sterilized pieces were transferred to potato dextrose agar (PDA) medium amended with rifampicin (50 μg/ml) and incubated at 25°C in the dark for 3 to 5 days. Eventually, a total of 89 purified isolates were obtained by using the hyphal-tip technique. To confirm all isolates as C. lunata, genomic DNA was extracted using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) and internal transcribed spacer (ITS) (White et al. 1990), translation elongation factor 1-alpha (TEF) (Manamgoda et al. 2012), and glyceraldehyde-3-phosphate dehydrogenase (GPD) (Berbee et al. 1999) loci were amplified and sequenced using the Sanger sequencing method. However, the phylogenetic analysis indicated that seven isolates ( SNC21-7, SNC21-68, SNC21-71, SNC21-76, SNC21-85, SNC21-86, and SNC21-90) were clustered with C. dactylocteniicola into one branch, rather than with C. lunata, based on the three loci. The isolate SNC21-7 was from Ankang City and the other six isolates were from Yanan City. The gene sequences of the seven isolates were deposited in GenBank (ITS:PV942344 to PV942350; TEF:PV976810 to PV976816; GPD:PV983684 to PV983690). These isolates were further transferred to PDA and incubated in the dark at 25°C for 7 days for morphological identification. The front surface of the colony was smoke grey with cottony mycelia, and the back was fuscous black. Conidiophores were unbranched, septate, straight or flexuous, and geniculate in the upper part. Conidia were ellipsoidal, pale brown, straight to curved, three-septate, and measured 16.36 to 33.57 × 7.01 to 9.87 μm (n = 30) in size. These cultural and morphological features were consistent with the description of C. dactylocteniicola (Marin-Felix et al. 2017). To complete Koch’s postulates, the pathogenicity test of the isolate SNC21-7 was carried out by spraying a conidial suspension (1 × 105 conidia/ml) onto the leaves of 28-day-old maize cultivar Zhengdan 958 (n = 5). Five non-inoculated plants were sprayed with sterile distilled water as a control. Each plant was covered with a plastic bag for 2 days to maintain high humidity, and then left in a greenhouse at 25 to 28 °C. After 2–3 days of pathogen inoculation, small, water-soaked spots appeared on all inoculated plants and gradually enlarged into 2–3 mm circular lesions. The lesions were pale in the center and brown in the margin with a yellow halo around them. The control plants remained asymptomatic. The pathogen was reisolated from the inoculated plants and identified as C. dactylocteniicola based on morphological and molecular analyses. This fungus has ever been reported as a pathogen of maize in Sri Lanka (Ferdinandez et al. 2024). To the best of our knowledge, this is the first report of Curvularia leaf spot caused by C. dactylocteniicola on maize in China.