RESUMEN
Maize (Zea mays L.) is one of the major crops in China. In July 2020, leaf blight was observed on approximately 18% of maize plants at the 852 Farm, Heilongjiang province. Symptoms appeared as yellow necrotic lesions on leaf tips and margins, which later expanded to the entire. The disease was first observed on the lower leaves of the plants and then progressed up the plant. Thirty symptomatic leaves were collected in 2020, and tissue samples between healthy and necrotic area (4 × 2 mm) were surface disinfected with 1% NaOCl for 3 min, 70% ethanol for 10 s, and washed three times with sterile water. Disinfected tissues were placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter) and incubated at 25°C for 1 week. Cultures were purified using the hyphal-tip technique for morphological and molecular analyses. Morphological characteristics were observed on 1-week-old PDA cultures grown at 25°C. Mycelium changed from cream colored to pale pink or reddish, and the back of plate turned pink with time. Hyphae were hyaline, 2 to 7 µm wide, with clamps at primary septa. Hyaline basidiospores were sphere to ellipsoid, and ranged from 8 to 10 × 6 to 9 µm. Based on the presence of clamp connections and morphological features, the fungus was preliminarily identified as Limonomyces roseipellis (Stalpers et al. 1982). To confirm the identity of L. roseipellis, primers ITS1/ITS4 (White et al. 1990), MS1/MS2 (White et al. 1990), and LR0R/LR5 (Vilgalys and Hester 1990) were used to amplify the internal transcribed spacer (ITS) region, the partial mitochondrial small subunit rDNA (mtSSU) and nuclear large subunit rDNA (nuLSU), respectively. These sequences were deposited in GenBank (GenBank accession no.s MW067756, MW322806 and MW386178). The ITS sequence had 99.55% nucleotide identity (660 bp/668 bp) with L. roseipellis isolate EF82 (GenBank accession no. MK918632). The mtSSU sequence was 99.69% identical (634 bp/636 bp) to that of L. roseipellis strain SY-LQG101 (Genbank accession no. KF824718). The nuLSU sequence was 99.14% identical (924 bp/932 bp) to that of L. roseipellis (GenBank accession no. EU622844). A single basidiospore was isolated and cultured on PDA for pathogenicity testing. To fulfill Koch's postulates, ten healthy, surface-disinfected maize plants grown in pots (four to five leaves stage) were sprayed with basidiospore suspension (1×106 spores/ml); another ten healthy surface-disinfected maize plants sprayed with distilled water to serve as controls. Plants were sealed in plastic bags immediately after inoculation and maintained at 90% relative humidity in a mist chamber for 24 h at 25°C with a 12-h light cycle (Nicoli et al. 2016). Plants were moved and maintained in the greenhouse and observed for disease development. The experiment was conducted twice. Leaf blight symptoms appeared on all inoculated plants 3 to 5 days postinoculation and were consistent with symptoms observed in the field. No disease symptoms were observed on control plants. The pathogen was reisolated from diseased plants, and species identification was confirmed by the morphological and molecular method described. L. roseipellis has been reported to infect Cynodon dactylon, Lolium perenne and Festuca arundinacea, respectively. To our knowledge, this is the first report of the identification of L. roseipellis as a pathogen of maize in China, and this report will assist with monitoring distribution of the disease to assist with developing management recommendations.
