First report of leaf spot disease caused by Alternaria alternata on Lonicera caerulea L. in China.

Abstract

Relatively few studies investigating plant diseases in blue honeysuckle (Lonicera caerulea L.) have been conducted in China. In September 2021, a leaf spot disease was observed on approximately 10% of blue honeysuckle 'Lanjingling' leaves in a 0.02 ha field plantation in Tiekuang Street (124.36°E, 40.12°N) in Dandong City, Liaoning Province, China. The main symptom consisted of leaf spots with black mildew centers typically surrounded by yellow halos. Small pieces (3-4 mm) of the infected leaves were plated onto potato dextrose agar (PDA) medium as described by Wang et al. (2020) and six purified cultures were obtained through single spore culture on PDA. The observed conidia, consistent with the morphology of Alternaria alternata, were obpyriform and dark brown, measuring 5.8 to 15.3 μm × 7.9 to 42.5 μm, with 1-6 transverse septa and 0-3 longitudinal septa (n = 50) (Simmons 2007). For molecular verification, genomic DNA was extracted from a representative isolate LD-8. The ITS (GenBank OL454815), GPD (GenBank OL601993), TEF (GenBank OL538256), RPB2 (GenBank OL601966), and Alt (GenBank OL538257) genes were partially amplified with the respective primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Woudenberg et al. 2015), EFl-728F/EFI-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-7CR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005). BLAST analysis revealed that these genes shared 99%-100% identity with OK345332, MK451977, MN756011, KU933459, and MN655781, respectively. A greenhouse experiment was conducted using six, healthy two-year-old blue honeysuckle 'Lanjingling' plants to observe disease development (Mirzwa-Mróz et al. 2018). After 10 d, we noted typical leaf spot symptoms on inoculated leaves sprayed with a conidial suspension (106 spores/mL) while no symptoms were detected on uninoculated leaves. The same isolate, reisolated from infected leaves with the same morphological and molecular traits, was also identified as A. alternata, confirming Koch's postulates. The fungus was previously reported in cockscomb plants in Heilongjiang Province, China (Wang et al. 2020). To our knowledge, this is the first report of leaf spot disease caused by A. alternata in blue honeysuckle grown in China. This study will provide a basis for future development of effective protection strategies against blue honeysuckle leaf spot in China.