First Report of Leaf Blight Caused by Alternaria tenuissima on Senna nomame in China.

Abstract

Senna nomame (Makino) T. C. Chen is an annual plant in the Fabaceae. This plant can be used in a tonic, as a diuretic, and for the prevention of obesity due to the presence of anthraquinones, flavonoids, and lipase inhibitors isolated from the aerial parts and seeds (Hatano et al. 1997). In June to September 2019, a severe foliar blight was observed on the leaves of 1-year-old S. nomame landrace plants in Qinglong (40.41°N, 118.95°E) in Qinhuangdao City, Hebei Province, China. The incidence of leaf blight was as high as 67% in the fields (n≥3). Symptoms began with small, brown spots at the margins and tips of leaves, with gray or yellowish-brown spots in the center of leaves. The spots gradually expanded to irregular large yellowish-brown lesions, and the leaves gradually withered. The pathogen was isolated from 20 leaves with typical symptoms from 10 individual plants. Leaf pieces (2 to 4 mm2) were excised from the junction of diseased and healthy tissues, disinfected in 75% ethanol for 15 s, rinsed in sterile distilled water, and placed on potato dextrose agar (PDA) plates. Colonies of 69% of the isolated fungi had round margins, and the olive-green fluffy aerial mycelia began to sporulate after 3 days at 28°C. On potato carrot agar (PCA), pure cultures formed yellowish brown mycelium with a light-colored, taupe-white center. Conidiophores were brown, simple or branched, and produced numerous conidia in short chains of three to six conidia. The conidia (n=50) were inverted pear-shaped or orbicular-ovate, light brown to brown, with a cylindrical short beak at the tip, and 19.9 to 30.4 μm (mean 25.4±3.6 μm) × 10.4 to 17.1 μm (mean 13.4±1.9 μm), with two to four transverse septa and zero to three longitudinal septa. The fungal isolates U-2, U-2-1, and U-2-2 were further characterized by sequencing of the rDNA ITS (MN712241, MZ781312, MZ781313), actin (ACT) (MN752246, MZ593671, MZ593672), calmodulin (CAL) (ON811636 to ON811638), ATPase (ON872785 to ON872787), and Alt a 1 (ON792172 to ON792174) genes using ITS1/ITS4, ACT-512F/ACT-738R, CALDF1/CALDR1, ATPDF1/ATPDR1, and Alt-for/Alt-rev primers for PCR amplification, respectively (Yang et al. 2009; Elfar et al. 2018). The sequences of the amplicons showed 99% to 100% identity with Alternaria tenuissima isolates: ITS (569/570 bp; MK560480 ), ACT (243/243 bp; MK593135), Alt a 1 (509/512 bp; MK593137), CAL (717/721 bp; MG925128), ATPase (1196/1197 bp; MG740623). Therefore, based on morphological characteristics and DNA sequence data, the isolates were identified as A. tenuissima. For pathogenicity tests, leaves on 10 healthy 1-year-old potted S. nomame plants were inoculated by wounding with a sterile needle and sprayed with a conidial suspension (2×105 spores/mL). Sterile water was used as the control. Inoculated plants were incubated in a greenhouse at 28°C with a 12 h photoperiod (75% to 80% relative humidity). The pathogenicity test was repeated three times. Lesions were observed on inoculated plants seven to nine days after inoculation, but no lesions were observed on control plants. A. tenuissima was successfully re-isolated from the symptomatic leaves and identified by morphology and sequencing of PCR amplicons. A. tenuissima has caused brown leaf spot on kiwifruit (Li et al. 2019) in China and pigeonpea (Sharma et al. 2012) in India. To our knowledge, this is the first report of A. tenuissima causing leaf blight on S. nomame in China. This new finding is essential in the diagnosis and management in field production.