First Report of Brown Leaf Spot Caused by Neofusicoccum parvum on Photinia × fraseri Dress (Christmas berry) in China.

Abstract

Christmas berry (Photinia × fraseri Dress), known as red tip photinia, is an important landscape color plant in South China (Zhu et al., 2021). In summer 2021, brown leaf spots of Christmas berry were observed with an incidence of 45-70% on the campus of Nanchang Institute of Technology, Jiangxi Province (28°41'32.61"N, 116°1'53.75"E), China. These spots have a negative influence on the photosynthetic activity and severely reduce the ornamental value of these plants. Lesions originally occurred on the margins or tips of the leaves as red patches (Figure 1 A) and then developed into irregular reddish-brown necrotic spots with yellow halos (Figure 1 B) that finally were grey lesions with dark brown ring circles surround with yellow halos (Figure 1 C). Ten leaf samples with typical symptoms from five plants were collected. The tissue between the healthy and necrotic area (ca. 5 mm × 5 mm) was cut with a sterile scalpel and surface sterilized in 70% alcohol for 45 s, followed by 2% NaClO for 2 min and washed in sterile deionized water three times, The sterilized leaf parts were placed on potato dextrose agar (PDA) and incubated at 25℃ for 3 to 5 days. A total of 28 fungal isolates were obtained from ten symptomatic leaves, which were classified into five species by morphological characteristics. Neofusicoccum was the genus with the highest isolation frequency (50%). For pathogenicity tests, healthy leaves of Christmas berry were surface-sterilized, wounded using a sterile needle and inoculated with mycelial plugs (6 mm in diameter) on the left site of the leaves. Five representative isolates were chosen and ten leaves were used for every isolate. The controls on the same leaves on the right side of the leaves were treated with sterile PDA plugs. All samples were placed in an artificial climate box (RH 90±3%, 27±1℃, 12 h light) for 3 days. Only the leaves inoculated with isolate NH7 showed dark brown leaf spots (Figure 1 D). The control leaves and those inoculated with the other four isolates remained symptomless. To confirm pathogenicity, mycelial plugs of isolate NH7 were applied on punctured leaves of Christmas berry in the field as the artificial climate box (Figure 1 E-H). Inoculation with a sterile PDA plug served as control. All the leaves were covered with plastic bags for 48 h to maintain high relative humidity. Seven days later, symptoms similar to those observed in the field developed on all leaves inoculated with isolated NH7, while the controls remained symptomless. To fulfill Koch's postulates, fungal isolates were reisolated from symptomatic leaves and identified by morphological and molecular characteristics. The colony of isolate NH7 developed aerial hyphae, which had a grey center surrounded by grey-white hyphae (Figure 1 I). Conidia were aseptate, primarily fusiform and measured 11.1-19.8 × 3.2-7.1 µm (n = 50) (Figure 1 J). For further confirmation of the identity, five genes, including ITS (White et al., 1990), RPB2 (Pavlic et al., 2009), tub2 (Glass & Donaldson, 1995), and tef1 (Carbone & Kohn, 1999) were sequenced. The sequences were deposited in GenBank (OL584411 for ITS, OL606622 for ACT, OL694623 for tub2, OM141481 for RPB2 and OM141482 for tef1), Based on the phylogenetic tree analysis using IQ-TREE, isolate NH7 clustered with Neofusicoccum parvum (Figure 2). N. parvum has been reported to cause leaf spot disease on different plants including Myristica fragrans (Jayakumar et al., 2011), Ginkgo biloba (Mirhosseini et al., 2014), Vitis heyneana (Wu et al., 2015), rubber tree (Liu et al., 2017) and Geodorum eulophioides (Du et al., 2021). To our knowledge, this is the first report of N. parvum causing leaf spot disease on Christmas berry in China. As one of the most widely planted ornamental shrubs in China, the detailed knowledge of the pathogens targeting Christmas berry is critical.