Leaf Blast, a New Disease on Pennisetum sinese Caused by Pyricularia pennisetigena in China

Abstract

Pennisetum sinese Roxb, named king grass or ‘sugarcane grass’, belonging to the Poaceae family, is widely cultivated in China because of its use for livestock feed, bioenergy, water-soil conservation and phytoremediation. In September 2018, leaf blast on P. sinese was observed in a pasture in Zhanjiang, Guangdong province (21.15°N, 110.30°E ). The lesions initially were round, water-soaked, oval or spindle-shaped, then later turned to typical eye-shaped, whitish in the center with brown-black necrotic borders surrounded by a yellow halo (Fig. S1). Almost all of plants were infected and most of the spots appeared on the blades in the lower part of the plants. The leaves gradually turned yellow and withered, which affected the growth and quality of the herbage. Diseased leaves were surface-sterilized with 75% ethanol for 30s, followed by 3% hydrogen peroxide solution for 3 mins, and rinsed three times with sterile water, then placed in a hermetic container with moistened filter paper, and kept at 21-24℃ to stimulate conidiogenesis. After conidiation, conidial suspensions were prepared with sterile water and evenly spread onto PDA, and incubated at 25 ℃ in the dark to collect single-spore isolates. Colonies on PDA were fuscous black with grey centers, broad white and flat. Conidiophores were solitary, erect, straight or curved, unbranched, medium brown, smooth, 2 septate, and sometimes up to 5 septate. Conidia with a protruding and truncating hilum on basal-cells were pyriform to obclavate, 2 septate, 22.2-28.8 × 7.1-9.6 μm ( 24.9±1.7 × 8.5±0.6 μm) (Fig S2). The internal transcribed spacer region (ITS), nrRNA gene large subunit (LSU), actin gene (ACT) and RNA polymerase II largest subunit gene (RPB2) were PCR amplified, and the amplicons were sequenced by Sangon Biotech (Shanghai) Co., Ltd. (ITS: OM294657, LSU: OM294656, ACT: OM304642, RPB2: OM304643). BLASTn result showed above 99% nucleotide identity with the ITS (KM484935 465/465 bp), LSU (MH412641 862/863 bp), ACT (XM_029891619 418/421 bp) and RPB2 (XM_029892603 894/898 bp) of Pyricularia pennisetigena strains. Maximum likelihood (ML) analysis based on ITS using MEGA revealed that the fungal isolate clustered in P. pennisetigena clade with 99% bootstrap support (Fig S3). Based on morpho-molecular criteria, the fungus was identified as P. pennisetigena (Klaubauf et al., 2014; Gómez Luciano et al, 2019; Wang et al., 2019). Four healthy leaves of a 2-month-old plant were surface disinfected, and 2 leaves were wounded with a sterile needle (3 wound sites each leaf), then one leaf was inoculated with 10 μl 105 conidia/ml suspension on each site, and on the other leaf, sterile water was used as the control inoculation. The similar inoculations were conducted on the other 2 unwounded leaves. Pathogenicity tests were repeated three times. Seven days later, all inoculated leaves (wounded and unwounded) showed typical symptoms, while no symptoms were observed with the controls on both the wounded and unwounded leaves. The same fungus was recovered and confirmed by morphology and the ITS and LSU sequences. P. pennisetigena is pathogenic on a wide range of Poaceae plants in Brazil, Japan, Mali, Philippines, United States, China (Klaubauf et al., 2014; Reges et al., 2016, Wang et al., 2019). Han et al (2020) reported bacterial leaf blast on king grass while this is first report of P. pennisetigena causing leaf blast. This finding is a warning to prevent and control of this disease and its infectivity to Poaceae plants.