First report of <i>Neopestalotiopsis rosae</i> causing crown rot of strawberry ( <i>Fragaria × ananassa</i> Duch.) in three production areas in Costa Rica

In September 2022, symptoms were observed in three-month-old strawberry plantations (Fragaria × ananassa Duch. cv. San Andreas) in Alajuela (10°9'23.832″N, 84°12'58.571″W and 10°9'15.084″N, 84°13'11.928″W), Cartago (9°56'27.564″N, 83°54'13.283″W and 9°55'30″N, 83°54'32.724″W), and San José (9°58'43.68″N, 83°59'16.98″W), Costa Rica with high severity. Affected plants showed foliar edge chlorosis, circular lesions, generalized necrosis, crown browning, and progressive wilting, with an incidence of 25% (n=20). Leaf lesions coalesced, leading to plant death. For isolation, samples were taken from symptomatic plants. Crowns were cut into small pieces (5 × 5 mm), disinfected with 75% ethanol for 30 s, 0.5% NaClO for 1 min, and rinsed twice with sterile distilled water. Pieces were plated on Petri dishes with PDA, incubated at 26 ºC (± 2 ºC) in the dark. After four days, colonies with grayish-white mycelium and black globular acervuli were subcultured and purified via single-spore isolation (Chakravarthi et al. 2020). Nineteen isolates were obtained from 25 plants. Conidia were fusiform to clavate, 5-celled with two dark median cells and hyaline end cells, consistent with the genus Neopestalotiopsis (Prasannath et al. 2020). The average size of conidia was 38.28 to 21.40 × 10.95 to 5.78 μm (n = 20). Colonies reached 90 mm after 8 days. DNA extraction was performed from seven pure cultures coded as NEO-A4, NEO-B6, NEO-C3, NEO-C5, NEO-D1, NEO-D5 and NEO-E5 from plants from different plantations. Molecular identification was performed using primers EF1-526/EF1-1567 for partial amplification of translation elongation factor 1-alpha (tef1) and Bt2a/Bt2b for beta-tubulin gene (tub2) (Maharachchikumbura et al. 2012). Obtained sequences were deposited in GenBank with accession numbers PP957619 to PP957625 (979 to 985 bp) and PV296038 to PV296044 (432 to 454 bp). BLASTn showed that tef1 and tub2 sequences of all isolates had 99.90% identity with the Neopestalotiopsis clavispora sequences [KU096881 (tef1), KU096880 (tub2)] of Chamorro et al. (2016). However, these were re-identified as Neopestalotiopsis rosae (Baggio et al. 2021). Phylogenetic analysis indicated that all isolates are N. rosae. Pathogenicity tests were performed on three-month strawberry plants San Andreas variety for each identified isolate. Four plants were inoculated with a 5 ml conidial suspension (1 × 106 conidia ml-1) prepared from 8-day old cultures. This was done by spraying a wound made with a sterile scalpel over the crown and then wrapping it with Parafilm. Four plants were inoculated with distilled sterile water, as negative controls. Each test was performed twice for a total of eight plants inoculated with each selected isolate. Plants were maintained under greenhouse conditions (&gt;80 % RH, 27 ± 2°C). After a month, older leaves showed chlorosis and necrosis. All isolates caused varying levels of disease. Control plants remained asymptomatic. Cross-sections in 100% of inoculated plants showed crown lesions like those observed in the field. The pathogen was reisolated with consistent morphology and symptoms matching field observations, fulfilling Koch’s postulates. This is the first report of N. rosae causing crown rot of strawberry in Costa Rica. This pathogen represents a serious and emerging threat to strawberry production and could potentially spread to other crops such as blueberries, mangoes, and ornamentals in the region.