First report of leaf spot disease caused by Cladosporium pseudocladosporioides on Lonicera caerulea L. in Heilongjiang Province, China

Blue honeysuckle (Lonicera caerulea L.) has contributed to maintaining the forest's ecological balance and remarkable frost-resistant abilities, helping it withstand extremely cold conditions (-46 °C) and a wide pH range (5 to 8) (Sharma and Lee 2021). Between September 2022 and September 2023, leaf spots were observed on approximately 30% of blue honeysuckle plants of the 'Lanjingling' cultivar grown in a 1.13 ha field in Da Hinggan Ling Prefecture (50.32° N, 124.13° E) in Heilongjiang Province, China. The leaves of the affected plants displayed black-colored spots. To identify the causal agents, 10 healthy and symptomatic leaves were randomly collected from ten healthy and infected individual plants, respectively. Small (3 to 4 mm) segments of the symptomatic tissues were immersed in 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in a paper towel, and plated on 9-cm Petri dishes containing potato dextrose agar (PDA). Ten fungal colonies developed on the PDA plates with an isolation frequency of 100% from 10 symptomatic leaves, and all colonies displayed a morphology consistent with Cladosporium spp. (Bensch et al. 2018). Cladosporium-like fungi were not isolated from healthy leaves. Dark olive-colored mycelia were observed, with straight unbranched conidiophores bearing terminal light brown-colored limoniform conidia (1.80 to 4.50 × 2.10 to 12.60 μm) and surrounded by a thin line of white mycelium (Delisle-Houde et al. 2024). To confirm this identification, PCR amplification of two representative strains LD-299 and LD-300 genomic DNA was performed with ITS1/ITS4 (White et al. 1990) and ACT512F/ACT783R (Carbone and Kohn 1999) primers. Basic local alignment search tool (BLAST) analyses of the National Center for Biotechnology Information database showed that sequences of the ITS (PP600316, PP600317) and ACT (PP624334, PP624335) all revealed 100% (493/493 nt, 493/493 nt; 181/181 nt, 181/181 nt) shared identity with Cladosporium pseudocladosporioides strain ex-type MF473195 and HM148674 (Bensch et al. 2010), respectively. Using a neighbor-joining phylogenetic analysis based on the ITS and ACT sequences, isolates LD-299 and LD-300 clustered in the same clade of C. pseudocladosporioides. Therefore, based on its morphological characteristics and molecular phylogeny, the two isolates were identified as C. pseudocladosporioides (Cosseboom and Hu 2023). A pathogenicity test was performed using nine healthy two-year-old blue honeysuckle Lanjingling plants. Three plants were inoculated with either the LD-299 or the LD-300 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control (Aydoğdu et al. 2023). All plants were cultured in a greenhouse (28℃, 75% relative humidity, 12 h light and dark cycle), and each experiment was replicated three times. Typical leaf spot symptoms were first observed on the inoculated leaves after 10 days. Morphological and molecular characterization of re-isolated pathogens from the artificially infected leaves indicated that the two isolates were identical, thereby confirming Koch's postulates. Cladosporium pseudocladosporioides previously caused leaf spot disease on artichoke (Cynara scolymus) in Türkiye (Aydoğdu et al. 2023). To the best of our knowledge, this is the first report of C. pseudocladosporioides causing leaf spots on blue honeysuckle in China. Blue honeysuckle production losses due to the leaf spots are critical for growers. Therefore, further focus should be given to investigate the host range and geographic distribution of C. pseudocladosporioides.