First Report of Neopestalotiopsis clavispora Causing Calyx and Receptacle Blight on Strawberry in China.

Abstract

Strawberry (Fragaria × ananassa) is widely cultivated in Liaoning Province, the most important strawberry production area in China. In November 2020, extensive brown spotting on the calyces and receptacles and dark brown necrotic lesions on the leaves of 'YanLi' strawberry plants were observed in the greenhouse, College of Horticulture, in Shenyang Agriculture University of Liaoning Province, China. The incidence ranged from 15% to 20% in the greenhouse. As the disease progressed, the spots extended to cover the entire receptacles and entire leaves. Fresh samples (5 mm2) were cut from the margin of healthy and infected tissues. The isolation and purification of methods were as described previously (Obregón et al. 2018). Fungal isolations are suborbicular with irregular edges. The colonies showed white aerial mycelium at the early stage and formed concentric circles after 5 days culture at 28°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 7~10 days culture. Acervulus was 162.4 µm in average diameter, n= 30. Conidia were spindle shape, five cells, four septa, 7.0 µm (average width) and 24.0 µm (average length), n=30. The apical and basal cells are lighter in color, most of them are hyaline, the middle three cells are darker in color, mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 30.8 µm in average length, and the basal cell has a small appendage, 9.0 µm in average length, n=30 (Fig. 1c-g). For fungal identification to species level, genomic DNA was extracted by the 2% CTAB method. Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. Purified PCR products were sequenced in single direction. BLASTn searches of GenBank, using the partial ITS (MW861730), β-tubulin (MW856302) and TEF-1α (MW861729) sequences of representative isolate NP102 as the queries, revealed 97%, 100% and 100% sequence identity, respectively, to Neopestalotiopsis clavispora strain WJ31 accessions MG729690, MG740736 and MG740759. The phylogenetic tree was constructed by MEGA X (Fig. 2) (Kumar et al. 2018). Pathogenicity assay was carried out by spraying a spore suspension (1 × 106 spores/ml) of 8-day-old culture of fungus on six healthy 'YanLi' strawberry plants. An additional six control plants were only sprayed with sterile distilled water, and the inoculated plants were maintained in a light incubator at 25 ± 1°C, 90% relative humidity, with a photoperiod of 12 h. Calyx and receptacle blight and leaf spot symptoms resembled those observed in the greenhouse on all inoculated tissues after 10 days (Fig. 1h and i), whereas calyces, receptacles and leaves on control plants remained asymptomatic. The experiment was repeated twice. Neopestalotiopsis clavispora was successfully reisolated from the infected tissues, fulfilling Koch's postulates. Researchers in Spain (Chamorro et al. 2016) reported N. clavispora for the first time on strawberry. After two years, N. clavispora was found in Argentina (Obregón et al. 2018). The reports characterized it mainly as a root rot pathogen. In January 2021, Neopestalotiopsis spp. were reported that it is a pathogen of strawberry leaves and fruits in Florida strawberry fields (Juliana et al. 2021). Our studies indicated that isolate NP102 was more aggressive. In the warm and humid greenhouse, the disease struck suddenly at the early stages of strawberry fruiting. Unlike previous reports, symptoms were a calyx and receptacle blight as well as a leaf spot. This pathogen can cause serious economic losses to strawberry growers. To the best of our knowledge, this is the first report of calyx and receptacle blight by N. clavispora on strawberry in China.