Identification of an unknown pathogen in a commercial strawberry cultivation

Abstract

Strawberry (Fragaria × ananassa Duch.) is an economically important fruit crop around the globe. However, strawberries are susceptible to various diseases caused by bacteria, viruses, and fungi, leading to substantial yield losses. Recently a severe outbreak of a disease was observed in a strawberry field in Nuwara Eliya district. The objective of the present study was to identify the causative agent of the said disease and to characterize the pathogen using morpho-molecular data analysis. Early symptoms of this disease include different size reddish to dark brown lesions irregularly distributed on the abaxial surfaces of the leaves. In some cases, the leaves become reddish in color. As the disease progress, leaves become completely necrotic and reddish color lesions were visible on the crown region, ultimately resulting the plant death. Pathogen was isolated using standard procedure into pure cultures and white, cottony colonies layered with concentric circles were observed on PDA media. The colonies turned dark with time. Ten days after inoculation, black color spores formed and concentrated forming black color acervuli scattered over the PDA. Conidia had two to three apical appendages, and one basal appendage. Conidia harbored versicolor median cells and were 17 – 25 μm × 6.5 – 8.5 μm in dimensions. The pathogenicity test of the fungal isolates conducted on 50 young strawberry cultivars showed that the fungal isolates caused typical symptoms. A fungus with significant similarity to the original strain was re-isolated from the inoculated strawberry crowns. Total genomic DNA was isolated and sequences of the PCR products of the internal transcribed spacer region (ITS1-5.8S-ITS4) were obtained. Sequences were similar to Neopestalotiopsis sp. with above 97% query coverage and above 97% percent identity. The best matches with the highest query coverage (99%) were multiple Neopestalotiopsis clavispora accessions (OR913022.1, OR913021.1, OQ594895.1 etc), and the reference sequence matched with many Neopestalotiopsis sp. with 97% query coverage. Therefore, Neopestalotiopsis sp. can be tentatively identified as the causative agent of strawberry plant infection based on morpho- molecular data.