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First report of Neopestalotiopsis rosae causing leaf spot and crown rot on strawberry ( Fragaria × ananassa ) in Paraguay

2023; Wiley; Volume: 48; Issue: 2 Linguagem: Inglês

10.1002/ndr2.12239

2044-0588

Y. A. Fernández‐Ozuna, A. R. Gini Álvarez, Horacio D. Lopez-Nicora, Andrea Alejandra Arrúa Alvarenga, Adans A. Colmán,

Yeasts and Rust Fungi Studies

In Paraguay, strawberry is produced from an area of c. 400 ha in Areguá in the Central Department. In 2021, five strawberry plants (cv. Sweet Charlie) with Neopestalotiopsis-like symptoms were collected from two commercial fields. Disease incidence in these fields was c. 30% (estimated from 500 plants) with c. 30% of the leaf area of infected plants being affected by disease symptoms. Necrotic leaf spots, in which fruiting bodies eventually developed, were initially observed. Crown and root necrosis were also observed and plants eventually died. Light tan and sunken spots with acervuli developed on fruits (Fig. 1). Diseased leaf tissue was surface disinfected in 2.5% NaOCl for three minutes, rinsed with sterile water, and air-dried. Samples were plated onto potato dextrose agar medium and incubated at 25°C. Five monosporic isolates were obtained through hyphal tip transfer and grown at 25°C for 12 days. Fungal colonies were white, circular with wavy, cottony edges on the upper surface and white to pale yellow on the reverse (Fig. 2). Black acervuli appeared ten days after plating. Conidia were ellipsoid to fusiform, with five versicolor cells, basal and apical cells were hyaline, whereas the three middle cells were brown. Spore measurements were conducted by randomly selecting 30 spores per isolate. Conidia length ranged from 24.5 to 30.2 μm (mean = 27.58 ±1.76 μm), width ranged from 7.3 to 10.2 μm (8.35 ±0.76 μm). The conidia featured a basal appendage and two to four apical appendages (primarily three), with mean lengths of 3.9 to 6.9 μm (5.25 ±1.05 μm) and 22.9 to 30.6 μm (27.37 ±2.19 μm), respectively. The fungus was identified as Neopestalotiopsis sp. following the description by Maharachchikumbura et al. (2014). DNA was extracted from each isolate and the ITS (White et al., 1990) and β-tub (Glass & Donaldson, 1995) regions amplified. Consensus sequences and alignments were generated using DNA Sequence Contig Assembler Software (Hepperle, 2011) and deposited in GenBank with Accession Nos. OP586603-OP586607 and OP588447 OP588451 for ITS and β-tub, respectively. BLASTn analysis revealed 98.6% and 99.6% identity with ITS (MK895143) and β-tub sequences (MK903339) of N. rosae isolated in Florida, respectively. A phylogenetic tree of Bayesian inference using concatenated sequences placed all the isolates with others from strawberry with a high posterior probability confirming its identity (Fig. 3) Three-month-old plants (cv. Sweet Charlie) were used to test Koch's postulates. Conidia from each isolate (n = 5) were suspended in sterile water and adjusted to a concentration of 1×105 conidia/mL. In a growth chamber, plants (three replicates) were either sprayed with 10 mL of the inoculum or drench inoculated around the base of plants previously wounded in the crown with a sterile needle. Sterile water was used for the control. Plants were maintained at 25°C and 85% relative humidity (Sigillo et al., 2019). Foliar symptoms and crown and root rot were observed 14 and 30 days after inoculation, respectively (Fig. 4; Wu et al., 2020). No symptoms were observed on control plants. The pathogen was reisolated from inoculated plants and identified morphologically. The pathogenicity test was performed three times with the same results. To our knowledge, this is the first report of N. rosae causing leaf spot and crown and root rot on strawberry in Paraguay. Accurate diagnosis of this disease will help technicians and producers implement adequate techniques to control this pathogen and mitigate severe damage to strawberry production in Paraguay.