First Report of Alternaria tenuissima Causing Leaf Blight on Sugarcane in China.

Sugarcane (Saccharum officinarum L.) is the main sugar crop in China. Yunnan is the second largest sugarcane production province in China. In December 2018, leaf blight was first observed on almost every leaf of sugarcane on 'Huanan 54-11', 'Baimei' and 'Chongan' in Kaiyuan (103°27' E, 23°72' N), Yunnan. In October 2019, during our survey in the field in Lingcang (100°08' E, 23°88' N), Yunnan, this disease was also observed on 'ROC 25'. Symptoms of the disease initially appeared as wilted, which seemed to be cause by water stress. As the disease progressed, irregular straw-yellow and blighted lesion ran throughout the leaf lamina from leaf tip to entire leaf sheath, many small black conidia formed in the dead leaf tissue under humid conditions. Symptomatic leaf tissues were surface-sterilized with 70% ethanol for 30 s, 0.1% HgCl2 for 1 min, and rinsed with sterilized water three times, air dried on sterile filter paper, and plated on potato dextrose agar (PDA). Six isolates were obtained from six symptomatic leaf samples and were transferred onto potato carrot agar (PCA). Colonies on PDA were white with loose aerial hyphae at first, then turned to dark olive or dark. Colonies on PCA were grayish with sparse hyphae, then turned to dark gray. Conidiophores were brown, simple or branched, and produced numerous conidia in short chains. Conidia (n = 50) were obclavate to obpyriform or ellipsoid, brown to dark brown, with a cylindrical short beak at the tip (2.3 to 17.3 µm in length), and 15.3 to 46.6 μm × 4.2 to 17.9 μm, 2 to 7 transverse septa and 0 to 3 longitudinal septa. Morphologically, the isolates were identified as Alternaria tenuissima (Simmons 2007). Two representative isolates C4 and C5 were selected for molecular identification. The internal transcribed spacers (ITS), Histone 3 genes and plasma membrane ATPase were amplified with primer pairs ITS1/ITS4, H3-1a/H3-1b and ATPDF1/ATPDR1, respectively (Glass et al. 1995; Lawrence et al. 2013). The sequences were deposited in GenBank (ITS, MT679707-MT679708; Histone 3, MT710929-MT710930; ATPase, MT833928-MT833929). BLAST searches showed ≥99% nucleotide identity to the sequence of A. tenuissima (ITS, 100% to MN822571; Histone 3, 100% to MN481955; ATPase, 99% to JQ671875, 100% to MH492703, respectively). Thus, the fungus was identified as A. tenuissima based on morphological and molecular characteristics. For pathogenicity tests, five healthy 2-month-old potted sugarcane leaves were wounded with one sterile needle and inoculated with 20 μl of suspension of 106 conidia/ mL, and five plants were inoculated with distilled water as the controls. Plants were placed in a greenhouse at 25 to 35°C. After two months, the leaf wound inoculated with the putative pathogen displayed blighted as those observed in the field whereas the controls remained symptomless. The fungus was reisolated from symptomatic leaves with the same morphological and molecular traits as the original isolates. The fungus was not isolated from the control plants. Pathogenicity tests were repeated two times. A. tenuissima causing leaf blight on barley in China was reported in 2008 (Luo et al. 2008). Leaf spot disease of sugarcane caused by A. tenuis has been recorded in Maharashtra (Patil et al. 1974). To our knowledge, this is the first report on A. tenuissima affecting leaf blight on sugarcane in Yunnan Province, China. Identification of the causes of the disease is important to develop effective disease management strategies. The author(s) declare no conflict of interest. Funding: This research was supported by Sugar Crop Research System (CARS-170303), the Yunling Industry and Technology Leading Talent Training Program "Prevention and Control of Sugarcane Pests" (2018LJRC56), and the Yunnan Province Agriculture Research System. References: Glass, N. L., et al. 1995. Appl. Environ. Microbiol. 61:1323. Lawrence, D. P., et al. 2013. Mycologia 105:530. Luo, Z., et al. 2008. Acta Phytophy. Sin. 35(5): 469-470. Patil, A.O., et al. 1974. Res. J. Mahatma Phule Agric. Univ. 5(2): 122-123. Simmons, E. G. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands. Caption for supplementary Figure 1 Supplementary Figure S1. Disease symptoms of sugarcane leaf blight disease and morphological characteristics of Alternaria tenuissima. (A) Typical straw-yellow and blighted lesions on naturally-infected leaves of sugarcane; (B) Infected symptoms on wounded leaves of sugarcane two months after artificial infection with A. tenuissima; (C) Colony of A. tenuissima on PDA; (D) Colony of A. tenuissima on PCA; and (E-F) Sporulation and conidia of A. tenuissima on PCA. (Scale bars = 100 μm; 20 μm).