Main content area

First Report of Leaf Blotch Caused by Phaeosphaeria avenaria on Leymus chinensis (Chinese Rye Grass) in China

Zhang, Y. W., Duan, T. Y., Nan, Z. B.
Plant disease 2018 v.102 no.7 pp. 1447
Bromus, DNA primers, Hordeum jubatum, Leymus chinensis, Lolium, Phaeosphaeria avenaria, agar, air drying, asci, ascospores, coatings, conidia, culture media, ethanol, forage grasses, fungi, genes, greenhouses, humidity, internal transcribed spacers, leaf blotch, leaves, meadows, mycelium, nutritive value, oats, palatability, pathogen identification, pathogenicity, pathogens, perennials, phylogeny, polymerase chain reaction, polymethylmethacrylate, ribosomal DNA, seeds, sequence analysis, sodium hypochlorite, solar radiation, spraying, steppes, sterilizing, temperature, triticale, Canada, China, Northern European region, United States
Leymus chinensis (Poaceae) is a perennial grass species that grows widely in northeast China. It is widely studied as a forage grass due to its nutritional richness, high yield, and palatability (Hu et al. 2012). Leaf blotch is a widespread devastating disease in the northern United States, Canada, and northern Europe that often infects cereals and common grasses such as triticale, oat, Hordeum jubatum, and so on (Ueng et al. 2003). In August of 2014 to 2016, a leaf blotch on L. chinensis was found in the Hulunbeir meadow steppe of Inner Mongolia (49°19′20″N, 119°56′31″E), China. About 80% of plants had lesions scattered on the leaves. Ten plants with severe lesions were examined. Lesions were oval to spindly, black brown to tawny on the leaf surface, and often resulted in gray withered leaves, 2.1 to 8.5 (±1.6) × 6.5 to 13.3 (±1.8) mm. The pathogen was morphologically identified using an Olympus (Japan) microscope. Pseudothecium were in longitudinal rows, slightly raised on sheaths near nodes and the middle of the leaves, with a 130 to 165 (±8.5) μm height and a 135 to 170 (±9.4) μm globose to subglobose shape. The wall of the pseudothecium surface was textura angularis with a slime coating. Pseudothecium were dark brown and formed black dots on the leaf surface. Asci (n = 30) were broadly cylindrical or cylindric-clavate in a broad hymenium, bitunicate, and subsessile, and the size was 55.5 to 90 (±8.1) × 9.5 to 15.5 (±1.8) μm, with eight overlapping linearly biseriate ascospores. Ascospores (n = 30) were straight or slightly curved with three septa, columniform with obtuse ends, and 20.5 to 25 (±1.3) × 4.0 to 6.5 (±0.62) μm. The ascospores were yellowish-brown to dark brown. However, the asexual stage was not observed. Based on morphological characteristics, the fungal pathogen was identified as closely related to Phaeosphaeria avenaria (Phookamsak et al. 2014; Shoemaker and Babcock 1989). Fungal cultures were isolated from leaf lesions, and leaves with lesions were surface sterilized with a short wash in 75% ethanol and 1% NaClO for 1 min, rinsed three times in sterile water, air dried on sterile filter paper, cut into 0.5 to 1 cm² leaf pieces, cultured on water agar for 3 to 5 days at 23°C, and then subcultured onto potato dextrose agar (PDA) for 14 days at the same temperature. White-pinkish colonies were formed on PDA after 2 weeks. To confirm morphological identification, the fungal genomic DNA was extracted directly from 4-week-old fungal mycelium with a UNIQ-10 fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) region was amplified using primer pair ITS4 and ITS1 (Phookamsak et al. 2014). The partial small subunit nuclear rDNA (SSU gene) was amplified with primers NS1 and NS4 (Phookamsak et al. 2014). The obtained sequences were deposited in GenBank (MF991941 and MF784260 for ITS and SSU, respectively). Sequence analysis revealed that the ITS sequence was 97% identical to Phaeosphaeriaceae sp. (KT268961.1), and the sequence of SSU was a 99% match to P. avenaria f. sp. avenaria (EU189208.1). The phylogenetic trees were built, and our sequences clustered with P. avenaria or Phaeosphaeriaceae sp. Thus, the isolates were identified as P. avenaria based on the morphological characteristics and sequence data. To fulfill Koch’s postulates and confirm pathogenicity, L. chinensis seeds were planted in pots and grown in a greenhouse (26 ± 2°C, normal daylight). After 4 weeks, healthy plants were inoculated by spraying an approximately 5 × 10⁶ conidia/ml spore suspension onto the foliage, and control plants received a similar application of sterilize distilled water. Six pots (three plants per pot) were used for each treatment. Inoculated plants were covered with a Plexiglas cylinder for 24 h to enhance humidity (90 to 100%) and maintained in a greenhouse at 20 to 25°C. Symptoms started appearing on leaves 10 days after inoculation, whereas control plants remained asymptomatic. The symptoms and morphology were similar to P. avenaria on naturally infected L. chinensis plants. The same fungus was reisolated from symptomatic leaves but not from the controls. Thus, leaf blotch of L. chinensis was caused by P. avenaria. To our knowledge, this is the first report of P. avenaria occurring on L. chinensis in China.