First Report of Leaf Spot of Small-leaved Boxwood Caused by <i>Pseudonectria foliicola</i> in China

Small-leaved boxwood [Buxus sinica (Rehd. et Wils.) Cheng] is an ornamental shrub in China, accounts for 30% of the global planting area. Volutella leaf and stem blight, caused by ascomycete fungi Pseudonectria foliicola and P. buxi, is one of the major diseases affecting the health and ornamental qualities of boxwood. Its widespread occurrence and disfiguring symptoms have caused substantial economic losses to the ornamental industry (Riwera et al., 2018). A leaf spot disease was observed on small-leaved boxwood in Shouguang, Weifang City, Shandong Province, China (37°28′ N, 119°14′ E), in Dec 2024. The incidence in the nursery was approximately 90% (diseased plants/m2). Number of diseased leaves per plant was &gt;50%. Leaf spots were nearly round or irregular, greyish-white. The lesions sometimes coalesced, and were often surrounded by a yellow halo of irregular width. Diseased leaves were cut into small pieces (2x2 mm) and soaked in 75% ethanol for 1 min, rinsed with sterile water, cultured on PDA culture medium and incubated at 25°C for 7 days. Seven isolates with similar colony morphology were obtained from 20 diseased leaves. The colonies were initially white and later turned salmon-pink in color, producing fluffy white aerial mycelium on PDA medium. The conidiophores produced whorled branches with a wider base and a thinner tip. The conidia were colourless, unicellular, circular to elliptical in shape, measuring 1.33–8.67 × 1.23–5.33 μm (n = 30), with an average size of 4.10 × 2.52 μm. DNA was extracted from mycelium according to the method of (TRANS-EasyPure Microbiome DNA Isolation Kit) and β-tubulin (Tub2), the internal transcriptional spacer (ITS), and the large subunit ribosomal RNA (NL) of the 28S rRNA gene were amplified and sequenced using the primer pairs Tubulin-F/Tubulin-R, ITS1/ITS4 and NL1/NL4, respectively (Spetik et al., 2020, Baysal-Gurel et al., 2014). BLASTn analysis revealed that the obtained sequences of the Tub2 amplicon (PV207327), ITS amplicon (PV192930), and NL amplicon (PV192931) showed 100% (306/306 bp), 99.45% (539/542 bp), and 99.47% (563/566 bp) identity to sequences of P. foliicola (KM232035.1, MN091009.1 and JF937574.1) in the NCBI database. A multilocus phylogenetic tree constructed by the MEGA 11 analysis showed that the HY-001 of isolates were assigned to the group of P. foliicola. To determine pathogenicity of these strains, the surface of small-leaved boxwood leaf (plant height = 25-30 cm, 10 leaves/treatment/plant, inoculating 10 plants) was sprayed with 1 ml of spore suspension (105 spores per ml/plant). Another 10 plants treated with sterilized distilled water were used as negative control. The plants were grown at 25 ± 2°C with 14 hours of light per day. Only the plants inoculated with P. foliicola began to show symptoms at 7 day after inoculation. The symptoms were similar to those observed on the diseased plants in the field. P. foliicola was re-isolated from inoculated diseased leaves with 100% homology. However, no symptoms were observed on the negative control plants. The results confirm that P. foliicola is the pathogen responsible for boxwood leaf spot. This is the first report of leaf spot of small-leaved boxwood caused by P. foliicola in China (Shi and Hsiang, 2014). When boxwood leaf spot disease is severe, it leads to premature leaf drop. This not only impairs the ornamental value but even leads to death. Our study laid a theoretical foundation for the prevention and control of this disease.