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

Abstract

Blue honeysuckle (Lonicera caerulea L.) is a perennial plant of the Caprifoliaceae family and Lonicera genus, the largest genus in the plant kingdom. Between September 2021 and September 2022, a leaf spot disease was observed on ~20% of blue honeysuckles of the 'Lanjingling' cultivar grown in a 3.33 ha field at the Xiangyang base (126.96°E, 45.77°N) of the Northeast Agricultural University, Harbin (Heilongjiang Province, China). Leaf spots first presented black mildew centers, gradually covering large areas of the leaf until it eventually fell off. Small 3-4 mm segments of infected tissue from 50 randomly selected leaves were surface sterilized with 75% ethanol and 5% sodium hypochlorite, rinsed in sterile distilled water, and transferred to 9 cm Petri dishes containing potato dextrose agar (PDA) after drying. Finally, two isolated pathogens were obtained through single spore culture on PDA; they appeared as gray-black colonies and were named LD-12 and LD-121. The observed LD-12 and LD-121 conidia displayed a morphology consistent with Alternaria spp. They were obpyriform and dark brown, with 0-6 transverse and 0-3 longitudinal septa, measuring 6.00-17.70 μm × 9.30-42.30 μm and 5.70-20.70 μm × 8.40-47.70 μm for LD-12 and LD-121, respectively (n = 50). Genomic DNA was extracted from the two isolates for molecular verification, and PCR amplification was performed with ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Woudenberg et al. 2015), EFl-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-7CR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005) primers. Sequences of LD-12 ITS (OQ607743), GPD (OQ623200), TEF (OQ623201), RPB2 (OQ658509), and ALT (OQ623199) revealed 99-100% of identity with Alternaria tenuissima sequences (KC584567, MK451973, LT707524, MK391051, and ON357632). Sequences of LD-121 ITS (OQ629881), GPD (OQ850078), TEF (OQ850075), RPB2 (OQ850076), and ALT (OQ850077) revealed 99-100% identity with A. alternata sequences (MN826219, ON055384, KY094927, MK637444, and OM849255). Nine two-year-old healthy plants from the 'Lanjingling' cultivar were selected for a pathogenicity test. Three plants were inoculated with either the LD-12 or LD-121 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control condition (Mirzwa-Mróz et al., 2018; Liu et al., 2021). All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle, and each experiment was performed three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 d. The same pathogens reisolated from infected leaves displayed the same morphological and molecular traits. They were again identified as A. tenuissima and A. alternata, confirming Koch's postulate. A. tenuissima and A. alternata were previously reported on Orychophragmus violaceus (Liu et al., 2021) and L. caerulea (Yan et al., 2022) in China. This study is the first report of a blue honeysuckle leaf spot caused by A. tenuissima in China. In the future, effective biological and chemical control should be used to prevent blue honeysuckle leaf spots in China.