First Report of Brown Spot on Lonicera japonica Caused by Enterobacter ludwigii in China

Honeysuckle (Lonicera japonica Thunb.) is an important traditional medicinal plant widely cultivated in China, with major production areas distributed across Shandong, Henan, Hebei, Hunan, and other provinces. It possesses significant economic value due to its use in pharmaceuticals, cosmetics, and functional beverages. Beginning in 2025, young honeysuckle plants at Hebei Agricultural University (38°05′N; 115°48′E) were affected by a highly infectious brown spot disease. Irregular brown spots appeared on leaves, leading to desiccation and defoliation; by June 2025, all 32 plants showed symptoms (100% incidence). To isolate the pathogen, tissue segments from lesion margins of five plants were surface-sterilized (75% ethanol, 30 s; 2% NaOCl, 30 s), rinsed, and plated on LB agar at 28°C for 48 h. A bacterial isolate (A-5) was purified, forming milky white, circular, opaque colonies (0.2–0.3 cm diameter). To further confirm the identification, total DNA was extracted, and the isolate A-5 was amplified by PCR for sequencing of the gyrase B (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3'/5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3') (Weisburg et al. 1991) and 16S rRNA (5'-AGAGTTTGATCCTGGCTCAG-3'/5'-GGTTACCTTGTTACGACTT-3') (Weisburg et al. 1991) regions. The gyrase B sequence (CP094841.1) and the 16S rRNA sequence (JF766425.1) showed 98% (844/860 bp) and 99% (968/971 bp) identity, respectively, to the ex-type strain Enterobacter ludwigii (MF623858). Based on the morphological and molecular data, the pathogen was identified as E. ludwigii. Pathogenicity of the A-5 bacterial isolate was tested on honeysuckle plants by leaf infiltration of a bacterial suspension (1×10⁸ CFU/mL) into the adaxial leaf surface using a sterile syringe, with 1 mL per leaf. Each treatment included three biological replicates, with three plants per replicate. Sterilized water was used as a negative control. The control plants remained healthy throughout the experiment. Inoculated plants (A-5) developed brown patches at 3 days post-inoculation (dpi). The strain was successfully reisolated from symptomatic tissues using the methods described above, and it was confirmed to be E. ludwigii, fulfilling Koch's postulates. To our knowledge, this is the first report of brown spot on honeysuckle caused by E. ludwigii in China, expanding its known host range beyond the previously reported hosts, including chrysanthemum (Günnan et al. 2007), watermelon (Wang et al. 2018), and maize (Liu et al. 2019). These findings not only extend the geographic distribution of E. ludwigii as a plant pathogen but also provide crucial references for developing scientific control strategies against honeysuckle brown spot.