First report of a leaf spot disease caused by Stagonosporopsis cucurbitacearum on Lonicera caerulea L. in Heilongjiang Province, China

Blue honeysuckle (Lonicera caerulea L.) has been widely used in food, medicine, health products, cosmetics, materials, and other products. Between September 2022 and September 2023, a leaf spot disease was observed on approximately 20% of blue honeysuckle plants of the 'Lanjingling' cultivar grown in a 0.67 ha field in the city of Yichun (47.67° N, 129.32° E) in Heilongjiang Province, China. The leaves of the infected plants first displayed ~ 1 mm small tawny-colored dots. Then, they developed into ~ 5 mm irregular spots with conspicuous yellow borders that expanded to cover the entire leaf. To identify the causal agent, one symptomatic leaf was collected randomly from each of ten plants. Small (3 to 4 mm) segments of infected tissue were surface sterilized using 75% ethanol for 30 s and 5% sodium hypochlorite (NaClO) for 3 min, and then rinsed three times with sterile distilled water, dried in a sterile paper towel, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA). Nine isolates (LD-211 to LD-219) were isolated on the PDA plate with an isolation frequency of 90%, and all isolates displayed a morphology consistent with Stagonosporopsis spp. (Jiang et al. 2015). Conidia were hyaline, ellipsoidal, aseptate or one-septate, and 4.20 to 12.60 × 3.00 to 4.60 μm (n = 50) (Garampalli et al. 2016). Pycnidia were globose to subglobose, brown, ostiolate. The ascospores were fusoid to ellipsoidal, uniseptate, and hyaline, matching characteristics of Stagonosporopsis spp. (Jiang et al. 2015). PCR amplification of LD-211 and LD-212 genomic DNA was performed with ITS1/ITS4 (White et al. 1990) and TUB2Fd/TUB4Rd (Glass and Donaldson 1995) primers. Genes of the ITS (PP600304, PP600305) and TUB (PP624325, PP624326) sequences were identical among both isolates. The ITS and TUB sequences had 100% (497/498 nt, 497/498 nt) and 98% (297/304 nt, 297/304 nt) similarity to Stagonosporopsis cucurbitacearum sequences (KM216012, LC485291). In a neighbor-joining phylogenetic tree based on the concatenated sequences of ITS and TUB genes, two isolates LD-211 and LD-212 were located in the same clade with S. cucurbitacearum (Jiang et al. 2015). Therefore, based on morphological characteristics and molecular phylogeny, LD-211 and LD-212 were identified as S. cucurbitacearum. A pathogenicity test was performed using nine healthy two-year-old blue honeysuckle Lanjingling plants. The leaves were surface disinfected with 75% alcohol and then wiped with sterilized water three times. Three of the plants were inoculated with 10 mL of LD-211 or LD-212 conidial suspension (1 × 106 spores/mL), and three of the plants were sprayed with sterile water as an experimental control. All plants were placed in a greenhouse (28℃, 75% relative humidity, 12 h light and dark cycle), and the experiment was replicated three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 days, whereas no symptoms were detected on water-treated leaves. The pathogens, reisolated from infected leaves, displayed the same morphological and molecular traits and were again identified as S. cucurbitacearum, thereby confirming Koch's postulates. S. cucurbitacearum has been reported to cause leaf spot on luohanguo (Siraitia grosvenorii) (Jiang et al. 2015). To the best of our knowledge, this is the first report of blue honeysuckle leaf spot caused by S. cucurbitacearum in China. Heilongjiang Province has the largest blue honeysuckle germplasm resources in China (Sun et al. 2024). Critical information about the characteristics of blue honeysuckle leaf spot could provide a theoretical basis to support its control and prevention.