First report of leaf spot disease caused by Neopestalotiopsis chrysea on Ligustrum lucidum in China

Ligustrum lucidum Ait., native to temperate Asia, is one of the most popular ornamental plants in China, especially used as street or green space trees (Wang et al., 2018). It also has important medical values for the treatments of several human chronic diseases (Xia et al., 2012). In March 2024, serious leaf spot disease of L. lucidum occurred in the green space at Guiyang University, Guizhou Province, China (26°55'87"N, 106°78'10"E). The spot starts either from the main vein or the edge of leaf, forming light grey halos with dark to black margins. In severe cases, it can cause the whole leaf turn to wilt. In a 0.08-ha field, the disease incidence was 55-65%, seriously reduced the plant values. To identify the causal agent, ten L. lucidum leaves with typical symptoms were randomly collected from five plants. Diseased leaves were cut into 5 mm × 5 mm pieces and washed in 75% ethanol for 3 min, 4% sodium hypochlorite solution for 30 s, and then rinsed three times with sterile water. Subsequently, tissues were placed in the center of Petri plates containing PDA medium and cultured in the dark at 28°C for 5 days. Twelve pure cultures were obtained by monosporic isolation. Nine isolates (NZ-1 to NZ-9) showed similar morphology on PDA, colonies were fluffy, round shape, and white. The conidia were fusiform, composed of one transparent cell at the top, three brown cells and one basal cells. The average size of conidia was 16.28 to 25.19 μm × 3.88 to 6.62 μm (n = 50). Based on the morphological characteristics, the fungus was identified as Pestalotiopsis spp. (Maharachchikumbura et al., 2014). For molecular identification, DNA was extracted, and PCRs were performed with the primers ITS1/ITS4 (White et al., 1990), Bt2a-F/Bt2b-R (Glassanddon-Aldson, 1995), and TEF1-728F/TEF1-986R (Carbone and Kohn, 1999). Sequences of the ITS region, TUB2, and TEF1 genes (from isolates NZ-1, NZ-4, and NZ-7) were deposited in GenBank (NZ-1: PP758651, PP779766, and PP779767; NZ-4: PQ136409, PQ153040, and PQ153039; and NZ-7: PQ136410, PQ153042, and PQ153041). BLASTn showed that the sequences ITS, TUB2, and TEF1 of NZ-1, NZ-4, and NZ-7 had >98% nucleotide identities (ITS: 99.63%, 532/534 bp, KT783663; 99.64%, 556/558, KU534877; 100%, 521/521, MT459336; TUB2: 98.54%, 474/481, OQ410711; 99.14%, 460/464, KT783666; 99.12%, 450/454, KT783666, and TEF1: 99.18%, 243/245, KT783668; 99.14%, 231/233, KT783667; 99.56%, 224/225, JX399051) with Neopestalotiopsis chrysea, respectively. A multilocus (ITS, TUB2, and TEF1) phylogenetic analysis indicated that three isolates were N. chrysea. To test pathogenicity, forty-five healthy leaves of nine intact plants were inoculated with the same pathogens by spraying 200 μL of conidia suspension (1×106 conidia/mL) of N. chrysea NZ-1, NZ-4, and NZ-7. The other three plants were treated with sterile water as a negative control. All plants were incubated in a greenhouse at 25°C and 75% relative humidity. The test was performed three times. After 8 days, all leaves inoculated with N. chrysea NZ-1, NZ-4, and NZ-7 showed similar symptoms to those observed in the green space. In contrast, no symptoms appeared on water-treated leaves. Then, pathogens were reisolated from inoculated L. lucidum plants and confirmed to be N. chrysea based on the morphological characterization and molecular analyses. To our knowledge, this is the first report that N. chrysea causing leaf spots on L. lucidum in China. Further study should be directly toward developing effective control measures.