Fecha de publicación: 05/12/2023 Fuente: PubMed "stone fruits" Plant Dis. 2023 Dec 4. doi: 10.1094/PDIS-08-23-1584-PDN. Online ahead of print.ABSTRACTChile leads cherry exports in the southern hemisphere with a total of 415.315 t exported in the 2022 to 2023 season (IQonsulting, 2023). Cytospora canker, produced by Cytospora spp., causes destructive infections and limit the productivity of sweet cherry orchards (Luo et al. 2019). This study was focused on isolating Cytospora strains to identify and characterize the species present in sweet cherry. During the period 2019-2022, ten samples of stem or branch presenting canker, dieback, gummosis or dead buds, were collected from sweet cherry cultivars 'Skeena', 'Lapins', 'Santina', 'Sweetheart', and 'Regina', in the regions Ñuble and O'Higgins, Chile. Five mm pieces from the necrotic wood margins of the samples were rinsed with sterile deionized water, placed on potato dextrose agar (PDA, Difco) and incubated at 20±2 ºC for 5 days. One isolate was recovered from each sample, resulting in ten Cytospora-like strains. Single hyphal tips were transferred onto PDA plates and all isolates were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM). Colonies grown on PDA reached 89 mm in diameter in 10 d at 25 °C, showing irregular margin, lacking aerial mycelium, initially off-white to cream that turned greenish gray in the center, which darkens with age. After 20 days of culturing on pine needle agar (Chen et al. 2015), isolates produced conidiomata pycnidial, semi-immersed, black, and subglobose (362)445-555(681)×(357)528-700(1053) µm (n=10), generating amber slimy conidia masses; Conidiophores were phialidic, cylindrical, aseptate, hyaline (6.77)9-10.04(12.88)×(0.82)1.1-1.28(1.99) µm (n = 30); conidia were abundant, allantoid, hyaline to light brown, aseptate (3.39)4.28-4.57(5.36)×(0.69)0.96-1.09(1.47) µm (n = 30) (Supplementary Figure 1). No sexual morph was observed. With the exception of the strain RGM 3390, all the isolates shared morphological characters to the descriptions of Cytospora sorbicola Norphanph., Bulgakov, T. C. Wen & K. D. Hyde (Norphanphoun et al. 2017). Isolates were identified at species level, by sequencing DNA regions described by Pan et al. (2020): ITS1-5.8S-ITS2, LSU; act, tef-1α, and tub2 with the exception of the RBP2, because this region could not be amplified in seven out of ten isolates. The consensus tree included the concatenated sequences of the ten isolates and those of reference Cytospora species reported by Ilyukhin et al. (2023) using a maximum likelihood analysis with the tool IQ-TREE webserver. MLSA confirmed the taxonomic affiliation of nine of the isolates with C. sorbicola and one isolate with Cytospora sp. (RGM 3390), that might represent a novel species (Supplementary Figure 2). The isolates RGM 3399 and RGM 3400, were selected randomly for pathogenicity tests. Inoculations were performed on 2-year-old sweet cherry cv. 'Lapins' grow in pots in a greenhouse at 26±6°C. Seven plants per isolate were cut to about 6-cm length from the main stem, and inoculated onto fresh cuts with 5-mm mycelium PDA plugs of 5-d-old culture and wrapped in moist sterile cotton and parafilm to keep moisture. Six plants were inoculated with non-colonized PDA agar plugs as control. The average canker length 3 months after inoculation was 3.1 and 0.8 cm, for RGM 3389 and RGM 3400, respectively (Supplementary Figure 1). Symptomatic twigs were incubated in moist chambers at 20±2 ºC for 10 d, resulting in the re-isolation of Cytospora strains that produced pycnidia and conidia structures in agreement with C. sorbicola. Both strains were reidentified to fulfill Koch's postulates, control twigs remained asymptomatic and no fungus was isolated from these twigs. This is the first report of C. sorbicola causing canker on sweet cherry in Chile. Our findings suggest that this species could be the most recurrent in cherry in central Chile, coinciding with it found in California where C. sorbicola has been described as the main causal agent of Cytospora canker of stone fruits in California (Lawrence et al. 2018).PMID:38050404 | DOI:10.1094/PDIS-08-23-1584-PDN