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First Report of Neopestalotiopsis clavispora Causing Root and Crown Rot on Strawberry Plants in Argentina

Obregón, V. G., Meneguzzi, N. G., Ibañez, J. M., Lattar, T. E., Kirschbaum, D. S.
Plant disease 2018 v.102 no.9 pp. 1856
DNA primers, Fragaria ananassa, Neopestalotiopsis clavispora, appendages, color, conidia, crown rot, culture media, death, discoloration, ethanol, field crops, fungi, greenhouses, humidity, internal transcribed spacers, leaf spot, leaves, mycelium, pathogenicity, plastic bags, roots, sodium hypochlorite, strawberries, Argentina, China, Spain
Argentina cultivates ≈1,300 ha of strawberries (Fragaria × ananassa Duch.) and produces ≈45,500 t year-round, especially in the provinces of Corrientes and Tucuman, two of the major strawberry production areas of the country (Kirschbaum et al. 2017). Since August 2013, severe damage has been observed on strawberry plants from commercial production fields, in the northern provinces of Corrientes (Bella Vista) and Tucuman (Famailla), Argentina. The main symptom was observed in leaves and consisted in reddish necrotic interveinal zones, with rust-colored areas in older leaves, starting from the margins and covering the whole leaf as the disease developed. This symptom was associated with necrotic areas and discoloration of roots and crown, resulting in plant death. To isolate the causal agent, symptomatic roots and crown were surface disinfested in 2% sodium hypochlorite (NaClO) for 1 min, 70% ethanol for another 1 min, rinsed three times with sterile distilled water, and finally dried in laminar a flow cabinet. Small pieces of crown and root tissue were then plated on potato dextrose agar (PDA) and incubated at 24 to 25°C until mycelium and reproductive structures were formed. After 7 to 10 days, white cottony circular fungal colonies were observed at the upper side of the medium and light-yellow coloration at the reverse side. The cultures were covered with dark acervuli (100 µm average width) after 7 days of incubation. Conidia were fusiform to ellipsoid, 8.6 × 24.6 μm (width × length) n = 50, with 5 versicolor cells (consisting of hyaline apical and basal cells, and three brown median cells, the second and third being darker than the fourth one). One basal and 2 to 4 apical appendages were observed on conidia. According to colony and conidia morphology, the isolates were identified as Neopestalotiopsis clavispora (Maharachchikumbura et al. 2012). The genus and species were confirmed by sequencing the internal transcribed spacer (ITS) region with primers ITS1 and ITS4. BLAST analysis of four isolates (two from Bella Vista and two from Famailla) showed 100% similarity with N. clavispora (GenBank, accession nos. KR052094 and KM264343). Pathogenicity tests were performed on young healthy plants cultivated in pots with sterile substrate (four plants per isolate and four control plants). A 5-mm-diameter mycelial plug grown on PDA was deposited on a small wound performed on the crown, and sterile PDA plugs were used as a control. These plants were covered with plastic bags for 5 days to maintain humidity (80%) and were cultivated in a greenhouse at 25 ± 2°C. The first symptoms appeared on leaves 10 days after inoculation and were similar to those found in field crops. Control plants did not show symptoms. This assay was repeated three times. The fungus was reisolated from crown symptomatic tissue, fulfilling Koch’s postulates. N. clavispora has been recently reported as causal agent of leaf spot on strawberry in China (Zhao et al. 2016) and of root and crown rot on strawberry in Spain (Chamorro et al. 2016). This is the first report of N. clavispora on strawberry in Argentina.