Occurrence of <i>Alternaria alternata</i> Causing Leaf Spot on <i>Buxus harlandii</i> in China

Boxwood (Buxus spp.) is extensively cultivated in China for ornamental landscaping, commonly utilized in the decoration of gardens and parks. Between October 2022 and 2023, a leaf spot was observed on Buxus harlandii in Nanyang City, Henan province (33°3'36″ N, 112°35'22″ E), with an incidence rate of 62% based on assessment of 50 randomly selected plants. The initial symptoms appeared as brown spots and expanding grayish-white, desiccated lesions on the leaves. As the disease progressed, these lesions spread throughout the plant, resulting in wilting and defoliation. Diseased leaves were collected, cut into 3 × 3 mm2 pieces. The tissues were disinfected with 75% ethanol solution for 30 s, followed by 1% NaClO solution for 60 s, placed on potato dextrose agar (PDA) plates. A total of 28 purified fungal isolates were obtained from 30 samples. These fungal isolates showed similar morphological phenotypes. The three strains (QSHY8, QSHY10, and QSHY16) from different areas were selected for identification. The colonies were gray to dark green with abundant aerial mycelium on the surface. Conidia were obclavate or spheroidal, dark brown, with 2 to 3 transverse septa and 1 to 3 longitudinal septa, measuring 12.3 to 27.1 × 2.7 to 5.9 µm (n=100), with conical beak lengths ranging from 0 to 2.9 μm. Conidial chains exhibited a unbranched linear morphology, comprising 4 to 12 conidia (70.2 to 192.5 µm).These morphological characteristics were consistent with the genus Alternaria (Woudenberg et al. 2015). The rDNA internal transcribed spacer (ITS), translation elongation factor (tef1), glyceraldehyde 3-phosphate dehydrogenase (gapdh), and RNA polymerase II second largest subunit (rpb2) were amplified and submitted to GenBank with accession No. OR352571, OR352576, OR352577 (ITS), OR360378 to OR360380 (tef1), OR360381 to OR360383 (gapdh), PP054843 to PP054845 (rpb2). The isolates were identical to each other at each locus. BLASTn analysis of strain QSHY8 showed that ITS, gapdh, and rpb2 regions revealed 100% identity (503/503 bp, 557/557 bp, 951/951 bp) with Alternaria alternata strain (PV444769, MK903028, MK605900), and 99.57% identity with PV054872 (231/232 bp) for tef1. A phylogenetic tree of concatenated markers sequences (ITS, tef1, gapdh, and rpb2) and ex-type strains sequences was constructed using MEGA with the neighbor-joining method. The three strains formed a clade with the strains CBS121456 of Alternaria alternata, distinct from other Alternaria spp. Healthy potted plants were used for the pathogenicity test. Five leaves per plant were wounded at a single spot each with sterile needles to breach the cuticle. A 20 µl drop of 106 conidia ml-1 suspension was placed onto each wound, and sterile water served as blank controls. All plants were grown in pots in climate incubators maintained at 28°C and 90% relative humidity. 10 days post-inoculation, lesions consistent with those observed in the field were present on the inoculated plants, while no symptoms appeared on the mock-inoculated controls. Isolates recovered from the symptomatic plants were identified as A. alternata. based on morphological and ITS sequence analyses, fulfilling Koch's postulates. Leaf spot of boxwoods (Buxus spp.) caused by A. alternata has been reported in Korea (Shin et al. 2022). This is the first report of A. alternata causing leaf spot on B. harlandii in China. Leaf spot significantly reduces ornamental value, and this finding provides valuable insights into the control of the disease.