First Report of Leaf Spot on Chenopodium album Caused by Nigrospora pyriformis in China

Chenopodium album Linn. (Chenopodiaceae) is an herbaceous vegetable plant that originates from Western Asia. It is usually grown in gardens and considered as nutritious in the human diet (Poonia and Upadhayay 2015). Moreover, the leaves of C. album harbor several bioactive compounds, which are used in ethnomedical practices (Sikarwar et al. 2017). In July 2018, leaf spots were observed on leaves of C. album cultivated at a production base located in the Shuitang National Forest Park, Hezhang, Guizhou, China (27°04′56″ N, 104°38′36″ E). The emerging occurrence of the disease significantly reduced the yield of C. album at different small-scale farms in the region. At the selected sampling site, the disease incidence was 50 to 60% on a 0.067-ha field. A total of 12 leaf samples with symptomatic spots were collected. They were surface sterilized with 10% sodium hypochlorite solution for 30 s followed by 75% ethanol for 30 s, rinsed three times with sterilized distilled water, air dried, placed on potato dextrose agar, and incubated at 25°C in the dark for 10 days. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after 10 days. Conidia were produced from clusters of conidiophores. The single-cell conidia were initially pale brown with black pigments that accumulated with time, dimorphic, globose to subglobose, 8.2 to 13.3 μm in diameter or pyriform, 14.4 to 21.5 × 8.3 to 14.5 μm. The surface of the conidium was shiny, smooth, and aseptate. Morphological characteristics of the isolates matched the description of the Nigrospora genus (Barnett and Hunter 1998). Genomic DNA of four representative isolates was extracted according to the manufacturer’s instructions (Biomiga Fungal DNA Extraction Kit, U.S.A.). Subsequently, PCR amplifications were performed with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) for the internal transcribed spacer (ITS) region, which also includes flanking rRNA regions and the complete 5.8S rRNA gene (White et al. 1990). The obtained sequences were compared with other ITS sequences following alignment with the GenBank database. BLAST analysis of the ITS region showed more than 97% identity (alignment length: 524 bp) to Nigrospora pyriformis type specimen isolate CGMCC 3.18122 (accession no. NR_153469.1). A representative sequence (isolate CXYL) was deposited in GenBank (accession no. MN044951). In addition, homology was confirmed with a phylogenetic tree constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch’s postulates, conidial suspensions of the representative strain were prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 106 conidia/ml. Leaves of five 4-month-old healthy C. album plants were spray inoculated with the suspension (200 μl per leaf), and an additional five plants were sprayed with equal amount of 0.05% Tween 20 buffer as a control. After 15 days, symptomatic spots appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control. Pure cultures of N. pyriformis were isolated from infected leaves and identified with the methods mentioned above. Recently, Nigrospora leaf spot was reported on watermelon and Photinia serrulata in Guizhou Province, China (Chen et al. 2019). To our knowledge, this study is the first report of N. pyriformis as a pathogen on C. album causing leaf spots. The identification of the pathogen could provide relevant background for its future management.