Alternaria tenuissima Causing Leaf Necrosis on Polygonatum cyrtonema in Jinzhai, China

Abstract

Polygonatum cyrtonema Hua is one of the most important traditional Chinese medicine herbs used for treating stomach weakness, palpitation, diabetes, and dry cough and is commonly planted in the mountain area of southeast and southwest China. During June of 2018 and 2019, necrotic lesions on the leaf apex and leaf margin of P. cyrtonema were observed in seven greenhouses, affecting approximately 2 ha, in Jinzhai County, Anhui Province, China. Disease incidence averaged approximately 30% among all affected greenhouses. The symptoms initially appeared on the leaf apex and leaf margin as small dark brown spots surrounded by light yellow halos. Over time, the lesions enlarged and coalesced to form large areas of necrotic lesions on leaves. To identify the pathogen, 15 symptomatic leaves were collected and surface disinfested with 2% sodium hypochlorite for 2 min followed by 70% ethanol for 30 s, rinsed in sterile water, and dried on filter paper. Plant tissues were then transferred onto potato dextrose agar (PDA) and incubated at 28°C for 3 days in the dark. Five fungal isolates were obtained from the diseased P. cyrtonema leaves by a single-spore isolation method. Colonies of the isolates had round margins and thick fluffy aerial mycelia with olive-green to brown coloration after 5 days on PDA. Conidiophores were pale brown, straight or flexuous, septate, single or in clusters. Conidia were obclavate or oval, multicellular, pale brown, and ranged from 4.91 to 15.83 × 17.29 to 47.32 μm (n = 50), with longitudinal and transverse septa varying from zero to three and from one to six, respectively. Conidia beaks were cylindrical, pale brown, and measured 2.00 to 7.46 × 2.00 to 5.28 μm. On the basis of morphological characteristics, the fungal isolates were preliminary identified as Alternaria spp. (Simmons 2007). To further identify the fungal isolates, the internal transcribed spacer (ITS) region of rDNA of each isolate was amplified and sequenced using the primers ITS4/ITS5 (Guo et al. 2012). The ITS sequence (accession no. MN249629) was 99.45% similar to the reference sequences of Alternaria alternata (KU527784) and A. tenuissima (KX664408) in GenBank, respectively. Partial regions of β-tubulin 2 (TUB2) and histone 3 (H3) genes were sequenced using primers T1/T2 (O’Donnell and Cigelnik 1997) and H3-1a/H3-1b (Glass et al. 1995), respectively. Sequences of TUB2 and H3 (MN256456 and MN256455) of the isolate were 100 and 99.80% similar to the fungus A. tenuissima (JQ811941 and MH647724), respectively. To complete Koch’s postulates, a pathogenicity test of the A. tenuissima isolates was performed on sterile leaves of P. cyrtonema. Ten milliliters of conidial suspension (5 × 10⁵ spores/ml) from each of the five isolates was sprayed on five 6-month-old plants. As a control, five plants were sprayed with sterile water. All treated plants were placed in a moist chamber at 26°C, with 90% relative humidity and an 8-h photoperiod of fluorescent light. Ten days later, 90% of the inoculated plant leaves developed brown spots and necrotic lesions similar to those observed in the field, whereas the control plants were asymptomatic. The same pathogens were again isolated from the necrotic leaves and confirmed as A. tenuissima by DNA sequencing. A. tenuissima is known to cause leaf spot and necrosis on many economically important crops, including lotus (Nelumbo nucifera) and kiwifruit (Actinidia spp.), in China (Li et al. 2019; Zhang et al. 2019). To our knowledge, this is the first report of A. tenuissima causing leaf necrosis on P. cyrtonema in Jinzhai, China.