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Identification of Alternaria alternata as the Leaf Spot-Causing Agent of Chicory in China

Chicory (Cichorium intybus L.) is a perennial Asteraceae herb that has been widely cultivated as a forage for livestock in many provinces of China. Since 2009, leaf spots have been observed on chicory in Chongqing, China. In April 2018, severe leaf spotting was observed at an incidence of ∼75% in a chicory (cv. Jiangjun) field in Rongchang district, Chongqing. Infected leaves initially exhibited small brown spots and then enlarged into larger, circular to elliptic, brown necrotic lesions of 5 to 15 mm diameter that coalesced to form large necrotic areas. Forty symptomatic leaves were collected and surface sterilized with 0.1% HgCl₂ solution for 3 min, rinsed three times with sterile distilled water, and air dried. Small (3 to 5 mm) sections of symptomatic leaf tissue that included the leading edge of the lesion were placed on potato dextrose agar. A fungus was consistently isolated from symptomatic leaf samples (87.5% isolation rate). Colonies were initially white and then turned gray to grayish black. Conidia were pale to brown, obpyriform to long ellipsoid, slightly constricted near some septa, with a body length of 10 to 26 µm and a body width of 6 to 14 µm. Conidia had one to four transepta and zero to three longisepta. Conidiophores were straight or flexuous, light brown to brown, and measured 10 to 28 × 2 to 5 µm. Morphological characteristics matched the descriptions of Alternaria alternata (Woudenberg et al. 2013). To confirm the identity of the fungus, the internal transcribed spacer region (ITS), the 28S large subunit ribosomal RNA (LSU), actin (ACT), and major allergen Alt a 1 (Alt a 1) genes were amplified with primers ITS1/4, LSU1Fd/LR5, ACT-512F/783R, and Alt-for/rev, respectively, of eight representative isolates were sequenced (Woudenberg et al. 2015). BLAST results showed 100% identity of the ITS (526/526 nucleotides), LSU (900/900 nucleotides), and ACT (219/219 nucleotides) sequences with those of A. alternata (KX064978, MH877754, and MH892480) and 99.78% identity of the Alt a 1 (463/464 nucleotides) sequence with A. alternata (MG598797). Based on morphology and DNA sequence analysis, the associated fungus was A. alternata. Representative sequences of one isolate (ACH2) were uploaded to GenBank (MK893994, MK896854, MK896856, and MK896855). Pathogenicity tests were performed on 10 heathy, 1-month-old potted chicory plants by spraying leaves with 5 ml of a conidial suspension (approximately 10⁵ conidia/ml) of isolate ACH2. Control plants were inoculated with sterile distilled water. After 7 days, similar leaf spots to those in the field were observed on inoculated leaves. No leaf spots developed on control plants. A. alternata was reisolated from inoculated leaves, thus completing Koch’s postulates. A. alternata was previously reported on chicory in Greece, Italy, and Turkmenistan (Farr and Rossman 2019). However, to our knowledge, this is the first report of leaf spot on chicory caused by A. alternata in China. Chicory is an important forage in south China. This disease has affected forage quality, reduced yield, and resulted in economic losses to planting enterprises and farmers. Therefore, effective prevention and control measures are urgently needed.