LAUSR

Litsea cubeba, an economical important tree species originally from China, produces fruit from which essential oils are extracted and extensively used in the chemical industry (Zhang et al. 2020). In August 2021, a large-scale outbreak of black patch disease was first observed on the leaves of Litsea cubeba in Huaihua (27°33'N; 109°57'E), Hunan province, China (disease incidence 78%). A second outbreak in 2022, in the same area, lasted from June to August. Symptoms consisted of irregular lesions that initially appeared as small black patches near the lateral veins. These lesions grew along the lateral veins and formed feathery patches until almost the entire lateral veins of the leaves were infected by the pathogen. The infected plants grew poorly and eventually the leaves desiccated and the tree defoliated. To identify the causal agent, the pathogen was isolated from nine symptomatic leaves from three trees. Symptomatic leaves were washed with distilled water three times. Leaves were cut into small pieces (11 cm), surface sterilized with 75% ethanol for 10s and 0.1% HgCl2 for 3 min, and then washed 3 times in sterile distilled water. Surface disinfected leaf pieces were placed onto potato dextrose agar (PDA) medium with cephalothin (0.2 mg/ml) and incubated at 28°C for 4-8 days (about 16h light, 8h dark). Seven morphologically identical isolates were obtained, from which five were selected for further morphological examination and three for molecular identification and pathogenicity test. Strains from grayish white colonies with a granular surface and grayish black wavy edges; bottom of the colonies turned black over time. Conidia were hyaline and nearly elliptical, unicellular. The sizes of conidia ranged from 8.59 to 15.06 μm (n=50) in length and 3.57 to 6.36 μm (n=50) in width. These morphological characteristics are consistent with the description of Phyllosticta capitalensis (Guarnaccia et al. 2017, Wikee et al. 2013). To further confirm the identity of this pathogen, genomic DNA of three isolates (phy1, phy2 and phy3) were extracted to amplify the internal transcribed spacer (ITS) region, the 18S rDNA region, the transcription elongation factor (TEF), and actin (ACT) gene with ITS1/ITS4 (Cheng et al. 2019), NS1/NS8 (Zhan et al. 2014), EF1-728F/EF1-986R (Druzhinina et al. 2005) and ACT-512F/ACT-783R (Wikee et al. 2013) primers, respectively. Sequence similarity indicated that these isolates were highly homologous to Phyllosticta capitalensis. The ITS (Genbank No. OP863032, ON714650 and OP863033), 18S rDNA (Genbank No. OP863038, ON778575 and OP863039), TEF (Genbank No. OP905580, OP905581 and OP905582) and ACT (Genbank No. OP897308, OP897309 and OP897310) sequences of isolates Phy1, Phy2 and Phy3 shared up to 99%, 99%, 100% and 100% similarities with their counterparts (Genbank No. OP163688, MH051003, ON246258 and KY855652) in Phyllosticta capitalensis, respectively. To further confirm their identity, a neighbor-joining phylogenetic tree was generated using MEGA7. Based on morphological characteristics and sequence analysis, the three strains were identified as P. capitalensis. To fulfill Koch's postulates, conidial suspension (1×105 conidia per mL) collected from three isolates were independently inoculated on artificially wounded detached leaves and leaves on trees of Litsea cubeba. Leaves were inoculated with sterile distilled water as negative controls. The experiment was repeated three times. All pathogen-inoculated wounds exhibited necrotic lesions within 5 days on detached leaves and 10 days on the leaves growing on trees after inoculation, whereas no symptoms were observed on the controls. The pathogen was exclusively re-isolated from the infected leaves and showed identical morphological characteristics to those of the original pathogens. P. capitalensis is a destructive plant pathogen that has been shown to cause leaf spots or black patch symptoms on variety of host plants around the world (Wikee et al. 2013), including oil palm (Elaeis guineensis Jacq.), tea plant (Camellia sinensis), Rubus chingii and castor (Ricinus communis L.). To our knowledge, this is the first report of black patch disease of Litsea cubeba caused by P. capitalensis in China. This disease causes severe leaf abscission in fruit development stage of Litsea cubeba and leads to a large amount of fruit drop.