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

Abstract

'Lanjingling' [China National Plant Variety Protection (CNPVP) 20200389] is the first new nationally registered cultivar of blue honeysuckle (Lonicera caerulea L.) developed by the Northeast Agricultural University for the fresh-fruit market (Zhu et al. 2022). Between September 2022 and September 2023, a leaf spot disease was observed on approximately 30% of blue honeysuckle Lanjingling plants grown in a 0.02 ha field in the city of Harbin (45.07° N, 128.14° E) in Heilongjiang Province, China. The leaves of the affected plants initially displayed gray-colored spots that gradually expanded into irregular white blotches with dark brown borders affecting the whole leaf. To identify the causal agent, total 10 symptomatic leaves were randomly collected from ten individual plants in representing entire filed. Small (3 to 4 mm) segments of infected tissue from 10 leaves from different plants 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 paper towel, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA). Seven isolates (LD-366 to LD-372) from each piece were isolated on the PDA plate, and all colonies displayed a conidial morphology consistent with Pestaloid taxa. They have undulated margins, white to pale in color, with moderate aerial mycelium on the surface. The conidia were ellipsoid, straight to slightly curved, four septate, and measuring 12.30 to 33.40 × 2.10 to 7.60 μm (n = 50). The apical cell was cylindrical and hyaline, with two to three tubular apical appendages, unbranched, and filiform. To confirm this identification, PCR amplification of two representative strains LD-366 and LD-367 genomic DNA were performed with ITS1/ITS4 (White et al. 1990), TUB2Fd/TUB4Rd (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone and Kohn 1999) primers. Sequences of LD-366 and LD-367 ITS (PP697761, PP697762), TUB (PP700299, PP700300), and TEF (PQ567378, PQ567379) revealed 99.09, 98.78, and 98.78% (547/552 nt, 547/552 nt; 438/440 nt, 438/440 nt; 243/246 nt, 243/246 nt) consistency with Pestalotiopsis disseminata sequences (MT374688, MT374713, and MT374700), respectively. Phylogenetic analysis of the combined sequences were performed using maximum-likelihood (ML) and Bayesian inference (BI) methods (Jiang et al. 2022), isolates LD-366 and LD-367 were located in the same clade with P. disseminata. A pathogenicity test was performed using six healthy, two-year-old blue honeysuckle Lanjingling plants. Three plants were inoculated with either the LD-366 or the LD-367 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control. All plants were placed in a greenhouse (28℃, 75% relative humidity, 12 h light and dark cycle), and each experiment was replicated three times. Typical leaf spot symptoms were first observed on inoculated leaves after 10 days. Two pathogens, reisolated from infected leaves, displayed the same morphological and molecular traits based on the ITS, TUB and TEF sequences as the initial isolates and were again identified as P. disseminata, thereby confirming Koch's postulates. P. disseminata previously caused gray blight disease on Euonymus japonicus plants in China (Wang et al. 2023). This is the first report of blue honeysuckle leaf spot caused by P. disseminata in China, and it is crucial to continue developing efficient control strategies for blue honeysuckle growing.