First Report of Leaf Spot on Lonicera japonica Caused by Aspergillus niger in China.

Honeysuckle (Lonicera japonica Thunb, LJ) is a common medicinal and edible plant (He et al. 2022). It has been utilized in various industries such as biomedicine, animal husbandry, and food production (Li et al. 2014; Su et al. 2020). In June 2023, a significant leaf lesion was observed on approximately 20% of honeysuckle "Juhua No.1" leaves in a 3.33-ha field at the base of Julu County, Hebei province, China. Almost all leaves were infected. Leaf spot disease occurred in the field honeysuckle throughout the flowering period, especially after picking. The disease mainly infected the leaves of honeysuckle, forming irregular spots on the edge of the leaf surface with black-brown edges, the midrib and lateral veins were affected (Figure S1A). In advanced stages, the entire leaf would become necrotic. For pathogen isolation, small pieces (4×4 mm) of the infected tissue from diseased leaves were surface sterilized with 75% ethanol and 5% sodium hypochlorite, rinsed with the sterile water, incubated on PDA. Finally, six isolated pathogens were obtained. Hyphae were white. The mycelium was multicellular, had diaphragm. Conidiophores protruded from the stroma, started as spherical structures and gradually developed into radial, black-brown formations. Spore acrosome was subglobose, bilayered pedicels covering acrosome, 40-60 µm in diameter, yellowish brown (Figures S1B, S1C). Based on morphological and cultural characteristics, the leaf spot disease fungus was tentatively identified as Aspergillus spp. (Wei 1979). To test the pathogenicity of pathogen, leaves of three healthy potted honeysuckle "Juhua No.1" plants were inoculated by sprayed with conidial suspensions (10⁶ spores/ml) (Figure S1D). Negative controls were established by inoculating leaf with sterile distilled water. All plants were incubated in a greenhouse at 28 ± 2℃. The experiment was replicated three times. After 10 days, typical leaf spot symptoms were observed on inoculated leaves, whereas no symptoms were found on the control groups. The re-isolated fungus from the inoculated leaves displayed the same morphological traits (Figures S1E-S1H), again identified as Aspergillus spp., confirming Koch's postulates, designated as H2. To confirm the pathogen's identity, genomic DNA was extracted from the pathogenicity isolate H2. The 18S rDNA and the ITS genes were amplified and sequenced using primer pairs S1/S2 (Zhang et al. 2018) and ITS1/ITS4 (Zhang et al. 2023), respectively. Results of BLAST searches showed that the 18s rDNA and ITS sequences of H2 were highly homologous (>99%) with Aspergillus niger. The close genetic relationship indicated that H2 belonged to the genus Aspergillus (Figure S2a). We further sequenced the whole genome of H2. The sequence data were available in the NCBI GenBank (Accession number: PRJNA1117256). We also analyzed the ANI (Yoon et al. 2017) and digital DNA-DNA blotting (dDDH) (Figures S2b, S2c). The ANI values of H2 compared to Aspergillus niger CBS 554.65 and Aspergillus niger KJC3 were higher than 95%. The dDDH values of H2 compared to Aspergillus niger CBS 554.65 and Aspergillus niger KJC3 were higher than 70%. Above all results, honeysuckle leaf spot disease was identified as Aspergillus niger. This is the first report of leaf spot on Lonicera japonica caused by Aspergillus niger in China. Our findings expand the geographical range of A.niger-infected plants, also provide reference for scientific prevention and control of honeysuckle leaf spot disease.