First Report of Alternaria Black Spot Caused by Alternaria alternata on Blue Honeysuckle in Poland

Abstract

Blue honeysuckle (Lonicera caerulea L. var. kamtschatica Sevast.) is becoming increasingly popular in Poland. Each year, the growing area of this plant increases significantly because it produces one of the earliest crops, and its berries contain a high concentration of antioxidants (i.e., vitamins A and C, potassium, calcium, phenolic compounds, and anthocyanins). Symptoms of Alternaria leaf spot were observed on blue honeysuckle plants growing in allotment gardens in Kanie (52°08′08″N, 20°47′14″E, Mazovia Province) and Kopalino (54°47′34″N, 17°50′02″E, Pomerania Province) in 2015 to 2016. The initial infections were noted on leaves at the end of June. Under favorable conditions, complete defoliation was observed at the beginning of August. Symptoms on leaves consisted of oval to irregularly shaped spots, 1 to 4 mm in diameter. They were initially black and later turned brown with a dark brown to black border. Over time, the center of the spots developed a gray shade. The spots were surrounded by a yellowish halo. The lesions gradually enlarged and sometimes coalesced. In 2015 and 2016, infected leaves were collected and sections (3 × 4 mm) were excised from the margin of diseased leaf tissue, disinfested with 1% sodium hypochlorite for 2 min, transferred to potato dextrose agar (PDA), and incubated at 25 ± 2°C. Twenty single-spored isolates were obtained from the developed colonies of the fungus. On potato carrot agar (PCA), cultures were olive brown with sparse, white-gray aerial mycelium. The conidiospores were obpyriform to obclavate, light brown to dark brown, with 1 to 6 transepta and 0 to 2 longisepta. After 9 days on PCA, 100 conidia were measured to be 12 to 46 × 7 to 17 µm. Morphological characteristics were similar to those described for Alternaria alternata (Fr.) Keissl (Simmons 2007). For the pathogenicity test, four isolates were grown on salt nutrient agar (SNA) for 10 days. Inoculations were performed on 2-year-old plants of blue honeysuckle cultivars Wojtek and Duet growing in pots. Plant leaves (three plants per isolate) were rinsed with sterile water. Next, they were inoculated with a spore suspension (∼10⁶ spores/ml) of each isolate using a pipette. Control plants were treated with sterile water. All plants were incubated in a moist chamber (Sanyo) at 27°C during the day and 20°C during the night, 90% relative humidity, and 16-h photoperiod. After 10 days, first leaf spots developed on the inoculated plants. The control plants remained symptomless. The pathogen was successfully reisolated from the spots and confirmed to be identical with the original fungal isolate, fulfilling Koch’s postulate. To confirm the morphological identification, genomic DNA was extracted from four single-spore isolates (JK8, JK2.3, JK 4.2, and Alt4). The internal transcribed spacer (ITS1, 5.8S, and ITS2) region of rDNA and the translation elongation factor (TEF1-α) gene were amplified with primers ITS1 and ITS4 (White et. al 1990) and EF1-728F (Carbone and Kohn 1999) and EF2 (O’Donnell et al. 1998), respectively. One representative sequence for the ITS and one for the TEF1-α gene were deposited in GenBank (accession nos. MF564200 and MF564197), respectively. BLAST analysis showed 99% identity with A. alternata sequences published in GenBank at the ITS (AF347031) and TEF1-α (KC584634) regions. Based on the morphology and molecular studies, the causal agent of Alternaria black spot on the blue honeysuckle was identified as A. alternata. To our knowledge, this is the first report of this pathogen on blue honeysuckle worldwide.