First Report of Leaf Spot Caused by Phyllosticta styracicola on Nageia nagi in China.

Abstract

Nageia nagi (Thunb.) Kuntze belongs to the family Podocarpaceae with shiny green branches and leaves, which is widely distributed in East Asia and the Southern Hemisphere. The leaves, roots and fruits of N. nagi have been used as herbal medicine to treat rheumatism, arthritis and venereal diseases (Abdillahi et al. 2011). In September 2022, leaf spot symptoms were found on approximately 30% of the leaves of N. nagi trees in a community located at the Economic and Technological Development Zone, Nanchang City, Jiangxi Province, China. Following the initial infection, the leaf lesions extended outwards from the top in a circular pattern, appearing as a dark brick color, and later changed to yellow and became dry, with a darker brown margin surrounding them. Ten symptomatic leaves were randomly selected, and a small piece of leaf tissue (5mm ×5mm) located between the health and infected tissues was cut and surface-desinfected with 70% ethanol for 30 s and 1% sodium hypochlorite (NaClO) for 30 s sequentially. After rinsing three times in sterile distilled water, all the small pieces of leaves were placed on potato dextrose agar (PDA) plates, followed by incubation at 28℃ for 3 days. Ten isolates, cultured on each PDA plate, appeared olive green with a granular surface, and an uneven white edge, and finally turned greenish black. The conidia were hyaline, with ellipsoidal to subglobose shapes and spore sizes of 5.5-8.3 × 7.2-12.0 μm (width × length) (=7.2±0.71 × 9.9±1.3 μm, n=40). These morphological characteristics are consistent with those of Phyllosticta species. To confirm the species, three representative isolates, JFRL 03-768, JFRL 03-769 and JFRL 03-770 were selected for further identification. The internal transcribed spacer (ITS) region, actin (ACT), translation elongation factor 1-alpha (TEF1-a), and glyceradehyde-3-phosphate dehydrogenase (GPD) genes of the three isolates were amplified and sequenced with the primers V9G/ITS4 (Carbone and Kohn 1999), ACT-512F/ACT-783R (Carbone and Kohn 1999), EF-728F/EF-2 (O´Donnell et al. 1998) and Gpd1-LM/Gpd2-LM (Myllys et al. 2002; Guerber et al. 2003), respectively. All sequences had been deposited into GenBank (ITS: OQ195332, OQ195333 and OQ195334; ACT: OQ207621, OQ207622 and OQ207623; TEF1-a: OQ207624, OQ207625 and OQ207626; GPD: OQ207627, OQ207628 and OQ207629). A maximum likelihood phylogenetic tree was constructed using the IQtree V1.5.6 (Ngugen et al. 2015) based on the concatenation of multiple sequences (ITS, ACT, TEF1-a and GPD). In the cluster analysis, the representative isolates (JFRL 03-768, JFRL 03-769 and JFRL 03-770) were positioned within a clade comprising of Phyllosticta styracicola. Subsequently, the pathogenicity of P. styracicola was determined by wound inoculation of healthy 2 year-old N. nagi plants, and this experiment was repeated for three times. Briefly, for each isolates, six disinfected leaves were wounded with a sterile scalpel, and then inoculated with 10-μl drop of the conidial suspension (1 × 106 conidia/ml). Another six disinfected leaves were inoculated with 10-μl drop of sterile water as a control group, and all plants were incubated at 28°C with 80% humidity. After 15 days, a similar spot lesion appeared on the leaves of the experimental group. P. styracicola was successfully re-isolated, and then subjected to morphological identification and molecular sequencing (ITS, ACT, TEF1-a and GPD genes). Whilst, the control leaves showed no symptoms. Previous studies have reported that P. styracicola could result in the development of lesions on Styrax grandiflorus leaves in China (Zhang et al. 2013). To our knowledge, this is the first report that P. styracicola can cause leaf spot on N. nagi in China.