First report of leaf spot and shoot dieback of <i>Ligustrum japonicum</i> caused by <i>Botryosphaeria dothidea</i> in China

Ligustrum japonicum (Japanese privet) is an evergreen shrub native to East Asia. It is widely cultivated as an ornamental plant. Since September 2023, a necrotic leaf spot outbreak on L. japonicum was observed in Baihe Wetland Park (32°5'42"N, 112°28'13"E) in Nanyang, China. The disease caused sporadic and irregularly shaped brown lesions, with chlorotic halo and dark margin on leaves, and disease incidence was up to 29% among 120 plants surveyed. As the disease progressed, leaf lesions enlarged, became slightly sunken and dark brown, and sometimes coalesced. In severe cases, necrosis extended from leaves into adjacent shoots. To isolate the pathogen, small tissue pieces excised from the margin of necrotic lesions on symptomatic leaves were surface sterilized by immersing in 75% ethanol for 30 s followed by 1% NaClO for 1 min, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 3 days. A total of 22 fungal isolates were obtained. Three representative isolates (Bd1, Bd6, and Bd17) sampled at separate sites were selected for further investigation. On PDA, the colonies were white at first and gradually turned dark gray with irregular margins within 7 days at 25°C. Conidia were hyaline, aseptate, fusiform, measuring 19.6-24.4 × 4.4-8.6 μm (n = 100). These morphological characteristics were connected to Botryosphaeriaceae fungi (Slippers et al. 2014). For molecular identification, the rDNA internal transcribed spacer (ITS), 28S large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha gene (tef1-α), and β-tubulin genes (tub) were amplified using primers ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Rehner and Samuels 1994), EF1-688F/EF1-1251R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were deposited in GenBank under accession numbers OR922737, OR976075, OR976076 (ITS), OR977584, OR977713, OR978107 (LSU), OR993993 to OR993995 (tef1-α), OR947471, OR993991, OR993992 (tub). A phylogenetic tree was constructed using neighbor-joining method based on concatenated ITS, LSU, tef1-α, and tub sequences, together with sequences from type or reference strains. The three isolates clustered closely with the reference strains CBS 115476 and CBS 110302 of Botryosphaeria dothidea in phylogenetic analysis and were clearly separated from other Botryosphaeria species. The molecular analyses confirmed that the isolates are B. dothidea (Slippers et al. 2014). Pathogenicity experiments were performed on healthy L. japonicum seedlings (three-month-old, 60 to 80 cm tall) using conidial suspensions (106 conidia ml-1) as inoculum. For each isolate (Bd1, Bd6, and Bd17), the conidial suspension was sprayed onto the aerial parts (leaves and young shoots) of 10 seedlings. Control plants were sprayed with sterile distilled water. All plants were maintained in controlled-environment chambers at 25°C and 90% relative humidity under a 12 h light/12 h dark photoperiod. Initial symptoms on leaves were first observed at 5 days post inoculation. At 10 dpi, inoculated seedlings developed necrotic brown lesions mainly at the petiole bases and leaf margins. By 21 dpi most affected leaves had abscised, and some lesions occurred on young shoots leading to partial shoot dieback. All inoculated seedlings developed symptoms (30/30 total), whereas control plants remained symptomless. The three isolates caused similar symptoms with comparable disease incidence. B. dothidea was consistently reisolated from symptomatic leaves and shoots, thereby fulfilling Koch’s postulates. B. dothidea is a well-known pathogen that causes diseases in various plants, including Camellia oleifera (Hao et al. 2023), Quercus dentata (Liu et al. 2023), and Cornus officinalis (Zhang et al. 2023). This is the first report of B. dothidea as the foliar disease causal agent on L. japonicum in China. Identifying this pathogen contributes to a better understanding of the disease affecting L. japonicum in this region and provides a basis for future disease management strategies.