First report of Alternaria alternata causing leaf spot of Cynanchum atratum Bunge in China.

Abstract

Cynanchum atratum Bunge belongs to Asclepiadaceae, and is distributed in North Korea, Japan and China. Its roots and rhizomes have antibacterial, antiviral, anti-inflammatory and anti-tumor effects. In July 2021, a leaf spot was observed in a 1.3 ha plantation of C. atratum in Harbin, Heilongjiang Province in China. The incidence was more than 85%. Initial symptoms were yellowing leaves with circular, or ellipsoid brown spots forming on leaf apexes or leaf margins. Small spots expanded and coalesced to form large circular or irregular, pale to light brown lesions, and leaves finally withered. Thirty, 5 × 5 mm, leaf pieces excised from the junction of symptomatic and healthy tissues were collected from different leaves with typical symptoms on ten plants, sterilized in 75% ethanol for 30s, then in 2% NaClO for 30s, rinsed in sterile water three times, placed on potato dextrose agar (PDA) plates, incubated for 5 days at 28°C in the dark, further purified by single spore method and transferred to new PDA and potato carrot agar (PCA) plates. Finally, 12 fungal isolates, most with similar morphology, were selected. After a 7-day incubation in the dark, colonies on PDA were 53 to 70 mm in diameter, circular and grayish brown. A total of 150 conidia were evaluated for morphology. Conidia were single or in chains, ovoid to inverted pear-shaped, with 2 to 6 transverse septa, 0 to 4 longitudinal or oblique septa, and measured 16.5 to 56.5μm × 9.0 to 16.5 μm. Beaks and supposititious beaks were mostly columnar, rarely conical, 0 to 22.5 μm × 2.5 to 4.0 μm. Conidiophores were solitary or clustered, pale brown, erect or bent, branched or unbranched, separated, 53.5 to 120.5 μm × 2.5 to 6.0 μm (Fig 1). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Two representative isolates (BW and BW2) were used for molecular identification. Internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit (RPB2), Alternaria major allergen (Alt a 1), translation elongation factor 1-alpha (TEF-1 α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced with the primers ITS1/ITS4 (White, et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou. 2013), Alt-F /Alt-R (Hong et al. 2005), TEF-F/TEF-R (Carbone and Kohn. 1999) and GDF/GDR (Templeton et al. 1992). The sequences obtained were deposited in GenBank (ITS: OM317915, ON534349; RPB2: OM296253, ON550475; Alt a 1: OM171248, O550474; TEF: OM238096, O550473; GAPDH: OM296217, ON550472). The phylogenetic analysis of maximum likelihood tree by MEGA 7 showed that the two isolates had 98% similarity with A. alternata CBS 916.96 (Fig 2). To test pathogenicity, 40-day-old plants were sprayed with spore suspensions (1×106 spores /mL) from 7-day-old cultures of BW and BW2. Each isolate was inoculated onto 3 leaves on 3 separate plants. Three other plants were sprayed with sterile distilled water as a control. The plants were incubated in the greenhouse (natural light, T: 25℃, H: 50%). After 15 days, the leaves turned yellow and irregular grayish spots appeared. The fungi reisolated from the inoculated leaves shared the same morphological and molecular features as A. alternata, fulfilling Koch's postulates. No fungi were isolated from the control group. This is the first time to report A. alternata causing leaf spot on C. atratum. Leaf spot can reduce the yields of C. atratum and this study provides a basis for the prevention and control of the disease.