Leaf Spots on bodhi tree (Ficus religiosa) caused by Diaporthe tulliensis

Ficus religiosa L., known as bodhi tree, is an ornamental tree and widely planted as an avenue and roadside tree due to ovate-rounded leaves with narrow, elongated tips. During 2018-2021, circular to oval-shaped leaf spots with pale white centers and brown-black edges surrounded by a chlorotic halo were observed on the leaves of more than 200 bodhi trees all year round in a park in Zhanjiang, Guangdong (N 21°15'22.29''; E110°23'1.03''). The leaf disease incidences were usually 15-80%, in severe cases, up to 100% in autumn and winter every year, and some trees shed all leaves(Fig S1). Repeated annual defoliation may weaken the tree and decrease the aesthetic value in the landscape. Diseased tissues (5 × 5 mm) of five symptomatic infected leaves were surface sterilized in 3% hydrogen peroxide solution for 3 min, rinsed thrice with sterile water, plated on potato dextrose agar (PDA) amended with ampicillin (50mg/L), and incubated at 25-28 ℃ in the dark for 3-7 days. Five strains with similar morphology were obtained by transferring hyphal tips of the colonies to fresh PDA and further isolating by single spore method. Fungal colonies were flat and spreading, with sparse, white aerial mycelium, and black pycnidial conidiomata semi-immersed in PDA after 30-days incubation at 25-28 ℃ in dark. Conidiophores were hyaline and α-conidia were single-celled, oval to fusiform, guttulate, 5.3 × 2.5 μm (n>50), similar to Diaporthe sp. (Crous et al. 2015), but no β and γ -conidia were observed. The internal transcribed spacer(ITS), large subunit ribosomal RNA gene(LSU), calmodulin (CAL) and β-tubulin(TUB) gene regions of a representative strain were amplified using specific primers reported by White et al. (1990), Gao et al (2017) and Gomes et al (2013), and submitted to GenBank (ITS: OM200852, LSU: OM228732, CAL: OM244761, TUB: OM244760). NCBI Blastn showed above 99% identity to D. tulliensis (anamorph: Phomopsis heveicola ) isolates of ITS (MT974186, MN393590 and KX457967), LSU (KR936131), CAL (MW759801), and TUB (KR936132 and MN399886), respectively (Crous et al. 2015; Huang et al. 2021; Gong et al. 2020; Bai et al. 2017). Based on the concatenated ITS, CAL, and TUB, a Maximum Likelihood phylogenetic analyses using MEGA 10.1.8 clustered the fungus with D. tulliensis in a clade with a 93% bootstrap support(Fig S2). Therefore, the fungus was identified as D. tulliensis based on morpho- molecular characteristics. Healthy detached leaves were sanitized thrice with 70% alcohol, and rinsed with sterile water. PDA plugs with actively growing 10-days-old mycelium were placed on predetermined sites, put into a sealed box with above 80% relative humidity and incubated at room temperature (25-28℃). Each isolate was inoculated at 25 needle-wounded and unwounded sites, PDA plugs without mycelium served as controls. Symptomatic spots appeared on all wounded leaves by 7 days post-inoculation (dpi) and on all unwounded leaves by 12 dpi. No symptoms appeared on controlled leaves. Pure cultures were recovered from inoculated leaves and showed identical morpho-molecular criteria to the original isolates. More than 70 pathogenic fungi are known to cause diseases on F. religiosa while there is no record of D. tulliensis infecting bodhi according to the U.S. National Fungus Collection (Farr and Rossman 2022). This report could provide basic understanding and alerting role for horticulturist in daily management.