First Report of Leaf Spot on Sorghum bicolor Caused by Alternaria tenuissima in China

Abstract

Sorghum bicolor (L.) is an important crop used in renewable fuels, which is widely planted in Africa, North America, and Asia for its resistance to stress (Dahlberg 2019). A foliar disease on sorghum was observed in 2019, affecting approximately 11,000 ha, in Daqing, Heilongjiang Province, China. Disease incidence averaged approximately 50% among all affected fields and caused 10 to 20% economic loss. At the initial stage of the infection, small brown spots with light yellow halos appeared on the leaves, which gradually enlarged and coalesced to form large areas of lesions, 2 to 20 mm long and 2 to 5 mm wide, on leaves in the advanced stage of the infection. To identify the pathogen, 10 symptomatic leaves were collected and surface disinfected with 1% sodium hypochlorite for 1 min followed by 70% ethanol for 30 s, rinsed in sterile distilled water, and dried on filter paper. Leaf spot fragments were then placed onto potato dextrose agar (PDA) and incubated at 28°C in darkness for 3 days. Four fungal isolates were obtained from the diseased leaves by a single-spore isolation method. The fungal colonies of the isolates had round margins with abundant, fluffy, and olive-green to brown aerial mycelia after 7 days on PDA. Conidiophores were pale brown with septa, straight or flexuous, single or in clusters, with branches forming occasionally. Numerous pale brown, obclavate to obpyriform shaped conidia were produced on conidiophores and occurred in long chains. Conidia ranged in size from 14.5 to 49.6 × 7.5 to 17.6 μm, with longitudinal and transverse septa varying from zero to three and from one to six, respectively (n = 50). Based on morphological characteristics, all the fungal isolates were preliminarily identified as Alternaria spp. (Simmons 2007). The internal transcribed spacer (ITS) region of rDNA and the partial coding sequences of allergen (Alt) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes of isolates were amplified and sequenced with primers ITS1/ITS4, Alt a/b, and G3PDH a/b, respectively (Woudenberg et al. 2013). DNA sequences from amplification of ITS from two isolates were deposited in GenBank (MN816298 and MN939684). BLAST analysis of the sequences from isolates AT1 and AT2 (MN816298 and MN939684) showed 99.64% (557/559 bp) and 100% (558/558 bp) similarity with the ITS sequence (MN907695) of Alternaria tenuissima (ZZ9-5-1). Sequences of Alt (MT109294) and G3PDH (MT109295) of isolate AT2 were 99.59% (486/488 bp) and 99.67% (596/598 bp) similar to the Alt and G3PDH sequences from A. tenuissima SY-4 (MK593137.1) and 15-238 (LC134315.1), respectively. Therefore, the pathogen was identified as A. tenuissima on the basis of the morphological characteristics and the sequences of ITS, Alt gene, and G3PDH gene. Pathogenicity of the isolates was tested on leaves of five sorghum seedlings by spraying with aqueous conidial suspensions (105 conidia/ml), whereas negative control plants were sprayed with sterile distilled water and then incubated in chambers at 25°C and 95% relative humidity. Plants inoculated with the fungus developed brown lesions on leaves within 8 days, whereas the negative controls were symptomless. To fulfill Koch’s postulates, reisolations from margins of necrotic leaf spot tissue of all tested strains were streaked on V8 agar (Blagojevic et al. 2015). Fungi isolated from lesions showed the same colony morphology as described above and were identified by morphological characters or DNA sequences as A. tenuissima. The control leaves remained asymptomatic, and the pathogen was not reisolated. The pathogenicity test was repeated with similar results. A. tenuissima could potentially cause leaf spots on many plants (Sun et al. 2019). However, this is the first report of A. tenuissima causing leaf spots on sorghum in China. Identification of this disease pathogen is important to prevent its spread to other areas.