First Report of an Alternaria alternata Isolate That Causes Leaf Spot Disease on Ilex crenata var. convexa in China

Abstract

Ilex crenata var. convexa is an evergreen, obtuse-toothed, and cultivated shrub belonging to the Ilex family and is often used as a ground cover or hedge in landscape greening and as a potted plant. In three surveys conducted in 2019 in Qingdao city, Shandong Province, China, many I. crenata var. convexa plants with obvious brown spots on the leaves were seen in a large area of the city, and the incidence was about 60%. The lesions usually first emerged at the leaf apex, gradually enlarged, and finally led to withering and necrosis of the leaves. To isolate the causative pathogenic fungus of this disease, leaf tissues (5 × 5 mm) were cut from the margins of lesions, surface disinfected with 70% ethanol, placed on potato dextrose agar, and incubated at 28°C with a 12-h photoperiod for 9 days. Colonies had round margins with some grayish/blackish aerial mycelia. Based on its dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa (Simmons 2007; Zhang 2003), the fungus was identified as Alternaria sp. Single spores were isolated to obtain the pure culture of the fungus. Fifty spores and 50 conidiophores were measured. Conidiophores arose singly and were 22.3 to 56.5 μm long and 3.1 to 5.7 μm wide. Conidia ranged from 17.4 to 37.9 × 7.2 to 12.9 μm and had three to six transverse and zero to three longitudinal septa, with a beak ranging from 4.6 to 13.2 μm long. To further identify the fungal species, the internal transcribed spacer (ITS) region of rDNA was PCR amplified using primers ITS1/ITS4 and sequenced. A BLAST search showed 100% similarity with those of Alternaria alternata (accession no. MN044802.1) and A. tenuissima (accession no. MK967997.1). The ITS sequence of rDNA amplified by primers ITS1/ITS4 could not be used to differentiate between A. tenuissima and A. alternata (Zheng et al. 2015). Hence, the partial coding sequence of the histone 3 gene was amplified using primers H3-1a/H3-1b (Zheng et al. 2015) and sequenced. The resulting 409-bp sequence was identical to that of A. alternata histone 3 gene (accession no. MG012317.1) and was deposited in GenBank (accession no. MN161248). Therefore, the Alternaria isolate was identified as A. alternata based on its morphological characteristics as well as ITS and histone 3 gene sequences. To further verify Koch’s postulates, three healthy I. crenata plants were inoculated with an aqueous spore suspension (6 × 10³ spores/ml) by spraying the whole plants until run-off. Three other healthy I. crenata plants were sprayed with sterile water and served as controls. All plants were kept in a humid chamber at 25°C for 6 days. Starting 6 days after inoculation, symptoms identical of those observed in the field were observed on the leaves inoculated with the spores, whereas the control plants remained symptomless. Moreover, a fungus with the same colony and conidial morphology was reisolated from the diseased leaves. To our knowledge, this is the first report of an A. alternata isolate that causes leaf spot disease on I. crenata var. convexa in China. Leaf spot disease largely damages the aesthetic aspect of I. crenata plants. This report provides a foundation for further studies on A. alternata infecting I. crenata.