Chunyan Li

Blue honeysuckle (Lonicera caerulea L.) plants produce small fruit that are used as food and medicine. In September 2024, 100 kg of blue honeysuckle 'Lanjingling' (China National Plant Variety Protection (CNPVP) 20200389) fruits were harvested in Harbin, China (126.48°E, 45.87°N), and 20% of the fruits showed postharvest fruit rot symptoms, leading to whole-fruit rotting with skin browning and necrotic lesions (Fig. 1. A). Small (1 to 2 mm) samples of infected tissue were obtained from five randomly selected fruits. Samples were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaClO) for 3 min, rinsed three times with sterile distilled water, dried with paper towel, and plated on 9 cm Petri dishes containing potato dextrose agar (PDA). Purified cultures were obtained using the single-spore technique. After 5 d at 28°C, the colonies displayed yellow brown aerial mycelium on the PDA plates (Fig. 1. B, C). Conidiophores were transparent and smooth, while conidial heads were brown and nearly spherical (Fig. 1. D). The entire surface was fertile, producing mostly spherical to subglobose conidia with diameters of 2.12 to 3.24 μm (n = 50) (Fig. 1. E). The internal transcribed spacer region (ITS, included ITS1+5.8S +ITS2, GenBank PQ606583), β-tubulin (TUB, GenBank PQ611757), and nuclear large subunit rRNA (LSU, GenBank PQ620103) genes were partially amplified with their respective primers (ITS1/ITS4 (White et al. 1990), Btub2Fd/Btub4Rd (Glass and Donaldson, 1995) and LROR/LR7 (Rehner and Samuels, 1994). BLAST analysis revealed that the sequences of the three genes showed 99.30 to 100% homology (526/526 nt, 141/142 nt, and 882/882 nt) with the MH859926, AY819971, and MH876373 sequences for isolates of Aspergillus ochraceus. In a phylogenetic tree constructed by Maximum likelihood method based on ITS and TUB sequences, the isolate LDGS-6 was located in the same clade with A. ochraceus (Fig. 2). For pathogenicity, twenty healthy blue honeysuckle 'Lanjingling' fruits were superficially sterilized with 75% ethanol and washed with distilled water. Ten fruits were inoculated with a 10 μL conidial suspension of isolate LDGS-6 (106 spores/mL) and ten with sterile distilled water (control). After fruits were incubated in 9 cm Petri dishes at 28°C and 75% relative humidity in the dark for 7 d, inoculated fruits displayed rot symptoms while control fruits did not (Fig. 1. F and G). The experiment was replicated three times. The causal agent was isolated from inoculated fruits, and it was identified as the original isolate based on morphological traits and ITS, TUB, and LSU sequencing, whereas no Aspergillus-like strains were isolated from control fruits, thus confirming Koch's postulates. In conclusion, based on molecular, morphological, and pathogenic analysis, A. ochraceus is the causal agent of the fruit rot disease on blue honeysuckle fruits (Ou et al. 2024). A. ochraceus was previously reported in China in Shengzhou nane (on Prunus salicina var. taoxingli) (Ou et al. 2024). To our knowledge, this is the first report of postharvest fruit rot caused by A. ochraceus on blue honeysuckle fruits in China. The annual fruit yield production of trees over 5 years old can reach about 40,000 tons during the high-yielding years in China (Sun et al. 2024), but its storage is challenged by pathogen infection. Future work should focus on monitoring its occurrence and spread and developing effective control strategies.

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.

Blue honeysuckle (Lonicera caerulea L.) cultivation has gradually expanded in China but continues to be limited by challenges such as leaf spot disease. Between September 2022 and September 2023, a leaf spot disease was observed on approximately 30% of 'Lanjingling' blue honeysuckles grown in a 2.66 ha field (a total of about 11,000 plants) in Jiamusi city (130.47°E, 46.16°N), Heilongjiang Province, China. Affected plants displayed brown necrotic lesions on their leaves that gradually expanded in area until the leaves fell off the plant entirely. Small, 3 to 4 mm segments of infected tissue from 50 randomly selected leaves were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried on paper towels, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA) (Yan et al. 2022). Five pathogens (LD-232, LD-233, LD-234, LD-235, and LD-236) were isolated on PDA and displayed a conidia morphology consistent with Pseudopithomyces spp. (Perelló et al. 2017). The fungal colonies on PDA were villiform, white, and whorled and had sparse aerial mycelium on the surface with black conidiomata. The conidia were obpyriform and dark brown, had 0 to 3 transverse and 0 to 1 longitudinal septa, and measured 9.00 to 15.30 μm × 5.70 to 9.30 μm in size (n = 50). Genomic DNA was extracted from a representative isolate, LD-232, for molecular verification and PCR amplification was performed with ITS1/ITS4 (White et al. 1990), LROR/LR7 (Carbone and Kohn 1999), and RPB2-5F2/RPB2-7CR (Liu et al. 1999) primers. Sequences of LD-232 ITS (OR835654), LSU (OR835652), and RPB2 (OR859769) revealed 99.8% (530/531 nt), 98.8% (639/647 nt), and 99.8% (1015/1017 nt) shared identity with Pseudopithomyces chartarum sequences (OP269600, OP237014, and MK434892), respectively (Wu et al. 2023). Bayesian inference (BI) was used to construct the phylogenies using Mr. Bayes v. 3.2.7 to confirm the identity of the isolates (Ariyawansa et al. 2015). Phylogenetic trees cannot be constructed based on the genes' concatenated sequences because selective strains do not have complete rDNA-ITS, LSU, and RPB2 sequences. Therefore, based on the morphological characteristics and molecular phylogeny, LD-232 was identified as P. chartarum (Perelló et al. 2017; Wu et al. 2023). A pathogenicity test was performed with six healthy, two-year-old 'Lanjingling' blue honeysuckle plants. Three plants were inoculated by spraying the LD-232 conidial suspension (1 × 106 spores/ml) or clean water as an experimental control condition (Wu et al. 2023; Yan et al. 2023). All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle, and each experiment was replicated three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 days. The same pathogens were reisolated from infected leaves, displayed the same morphological and molecular traits, and were again identified as P. chartarum, confirming Koch's postulate. P. chartarum previously caused leaf spot disease on Tetrapanax papyrifer in China (Wu et al. 2023). To our knowledge, this is the first report of blue honeysuckle leaf spot caused by P. chartarum in China. Identification of P. chartarum as a disease agent on blue honeysuckle will help guide future management of leaf diseases for this economically important small fruit tree.

Blue honeysuckle (Lonicera caerulea L.) fruit is growing in popularity as a natural, functional 'super fruit', but its storage is challenged by pathogen infection. In June 2022, approximately 30% of 100 kg of blue honeysuckle fruits (cv. Lanjingling) obtained in Harbin, China (128.70°E, 44.87°N) showed postharvest fruit rot symptoms after 15 d of storage at 4°C, leading to whole fruit rotting with gray fungal growth (Fig.1 A). Small (1-2 mm) segments of infected tissue were obtained from 20 randomly selected fruits which were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in paper towel, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA). Five purified cultures were obtained and their front colonies were dark brown (Fig.1 C) on the PDA plates after 5 d at 25°C (Alam et al. 2019; Riquelme-Toledo et al. 2020). The conidia (n = 50) were single-celled, hyaline, either ellipsoid or ovoid, and measured 7.5-15.0 μm (11.7 μm average) × 6.0-11.4 μm (8.3 μm average). The conidiophores (Fig.1 E) were branched at the apex bearing bunches of conidia resembling grape clusters (Ellis 1971). For molecular confirmation, genomic DNA was extracted from a representative isolate LDGS-3 using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer region (ITS, GenBank ON952502), heat shock protein (HSP60, GenBank OP039103), the second-largest subunit of RNA polymerase II (RPB2, GenBank OP186114) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH, GenBank OQ658508) genes were partially amplified with the respective primers ITS1/ITS4, HSP60f/HSP60r, RPB2f/RPB2r, and G3PDH-F/G3PDH-R (Staats et al. 2005; White et al. 1990). BLAST analysis revealed that the sequences of the four genes showed 100% homology with the MH782039, MH796663, MN448501 and MH796662 sequences for isolates of Botrytis cinerea. Based on morphology and molecular characteristics, the isolate LDGS-3 was identified as B. cinerea. For pathogenicity, twenty healthy blue honeysuckle fruits (cv. Lanjingling) were superficially sterilized with 75% ethanol and washed with distilled water. Ten inoculated blue honeysuckle fruits, which were injected with 10 μL conidial suspension of isolate LDGS-3 (106 spores/mL) displayed fruit rot symptoms (Fig.1 B) inside 9 cm Petri dishes after 10 d at 4°C, while no symptoms were detected on ten fruits inoculated with sterile distilled water (Alam et al. 2019). The same isolate that was reisolated from infected fruits with the same morphological and molecular traits was also identified as B. cinerea, confirming Koch's postulates. B. cinerea was previously reported in Henan Province, China in hawthorn (Zhang et al. 2018). To our knowledge, this is the first report of postharvest fruit rot caused by B. cinerea on blue honeysuckle fruit in China, which will aid future management of this emerging postharvest disease.