First Report of Twig Dieback Caused by Neopestalotiopsis rosae on Highbush Blueberry in the Continental United States.

Abstract

Highbush blueberry (Vaccinium corymbosum) is an important fruit crop for pick-your-own agritourism farms in the Midwest. Declining or diseased plants are a major concern for pick-your-own farms, as consumers prioritize healthy plants and organic practices (Norby and Retallick 2012). In August 2023, leaf spot and dieback symptoms were observed sporadically on the current year's growth throughout an organic berry agritourism farm in Eastern Iowa. Small red spots on the twigs and leaves progressed into larger lesions with gray centers, and brown borders with no observable acervuli or pycnidia. Symptomatic samples from 20 individual 'Duke' blueberry plants were collected for disease identification. Tissue was cut from lesion edges, surface sterilized with 10% bleach for 1 min, rinsed thrice in sterilized water, cultured on potato dextrose agar amended with 800 mg/L yeast extract (PDAY) and 100 µg/ml streptomycin, then kept at 20°C. Twenty isolates were identified morphologically, and three were submitted for molecular analysis. Isolated colonies had white aerial mycelia, irregular margins, and were light yellow underneath. Black, globular acervuli produced five-celled fusoid conidia that ranged from straight to slightly curved. The center three cells were light to dark brown. The basal cell was generally conical, with a small basal appendage. The apical cell ranged from conical to cylindrical, with 2-3 appendages. The spores averaged 23.7 µm long (range 19.7-30.1 µm) and 5.9 µm wide (range 5.0-6.5 µm), n=50. BLAST analysis demonstrated that sequences of ITS (ITS1-F/ITS4; PP764834, PQ344945-46), TUB2 (Bt2a/Bt2b; PP786462, PQ356370-71), and TEF1-α (EF1-526-F/; PP789161, PQ356368-69) were ~98 to 100% similar to KM199360, KM199430, and KM199524 published for Neopestalotiopsis rosae. Using MEGA 11 program (Kumar et al. 2018), maximum likelihood analysis based on the concatenated ITS, TUB2, and TEF1-α sequences placed all three isolates in a high-confidence cluster with N. rosae (Baggio et al. 2021; Jayawardena et al. 2016; Lee et al. 2019), confirming their identification. Pathogenicity of one isolate, NPB-1, was confirmed by inoculating three twigs on three 1-year-old V. corymbosum 'Blue Gold' plants (n=9). Twigs were pierced using a sterile 20-gauge needle with a mycelium plug to mimic pruning wounds, or a PDAY plug as a control (Borrero et al. 2018). Inoculated and control plants were kept at 28°C, 50% relative humidity, with an 18-hour photoperiod. Ten days post inoculation (dpi), inoculated plants showed symptoms of reddish-brown spots along wounded stems which were not present on control plants. At 35 dpi, inoculated plants displayed blight symptoms like the field-observed ones. Inoculated twigs had internal discoloration beyond the inoculation point, unlike the control twigs. N. rosae was successfully reisolated 2 cm away from the inoculation site and confirmed by sequencing in all inoculated twigs (n=9), but not in control plants (n=9). Neopestalotiopsis spp. have been previously reported to cause twig blight on blueberries in Korea, Chile, Uruguay, China, and Spain (Lee et al. 2019; Borrero et al. 2018; Chen et al. 2016; González et al. 2012; Espinoza et al. 2008). To our knowledge, this is the first report of N. rosae causing twig dieback on V. corymbosum in the US, and this knowledge will aid in proper diagnosis and treatment development.