First Description of <i>Diplodia mutila</i> Causing Botryosphaeria Stem Blight of Highbush Blueberry cv. Brigitta in the Maule Region, Central Chile

Highbush blueberry (Vaccinium corymbosum L.) is an important economic fruit crop in Chile, with an approximate total area of 17,000 ha, i.e., 5,626 ha in the Maule region (35°26'S, 71°40'W), Central Chile (www.odepa.cl, 2025). Botryosphaeria stem blight is a threat to blueberry production in Chile, with incidence between 15 and 45%, attributed to Botryosphaeriaceae spp., including Neofusicoccum arbuti, N. australe, N. nonquaesitum, and N. parvum (Espinosa et al. 2009; Perez et al. 2014; Millas et al. 2023). In January 2023, five bushes of 12-year-old blueberry cv. Brigitta, from a commercial plantation, with and incidence of 8% was assessed across the 5 ha, in Longaví, Maule region, were submitted to the Fruit Pathology Lab at the University of Talca. Symptoms included decline, basal canker, stem and cane blight (red flagging) (Polashok et al. 2017). In cross-section, lignified stems have brown U or V-shaped necrotic or discolored hard tissues. Diseased stems were disinfected according to Díaz and Latorre (2014), and internal tissues were cut into small pieces (3 x 3 mm) and placed on potato dextrose agar (PDA) and 0.1% Igepal CO-630. After 5 days incubation at 20°C in darkness, 10 pure cultures were transferred to PDA. Four isolates developed white to white-grey fast-growing colonies with abundant aerial mycelium, and abundant aggregated black pycnidia after 28 days at 20°C. Conidia were one-cell, hyaline, thick-walled, ellipsoidal to cylindrical with a size of (27.7-) 25.1 ±1.5 (-22.4) x (14.7-) 13.2 ± 1.3 (-11.4) µm with a ratio 1.9 length/width. To determine the species level, three isolates were amplified and sequenced using ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R pair-primers of the internal transcribed spacer (ITS1-5.8S-ITS2) region, a beta-tubulin (tub), and the translation elongation factor 1-α (TEF) DNA regions, respectively (Alves et al., 2004; Dissanayake et al. 2015). The sequences showed a 99% similarity with sequences of D. mutila CBS 112553 ex-type. The sequences of D. mutila isolates Bot-ara-1 to Bot-ara 3 were deposited in GenBank (ITS: PV032958 to PV032960; BT: PV037028 to PV037030; TEF: PV037031 to PV037033). A phylogenetic tree, constructed using the maximum parsimony method using the concatenated sequences of the three regions, grouped the three isolates in the same clade with CBS 112553 ex-type. Pathogenicity tests were conducted in the field (Longaví, Maule region) using D. mutila isolates (Bot-ara-1 to Bot-ara-3) on blueberry of five-year-old cv. Brigitta in July. Twenty dormant stems (five per bush) were pruned (tip) with shears, and 80 µL of conidial suspension (105 conidia/mL) was placed on each fresh pruning using a micropipette. Additionally, 20 dormant stems (40 cm long) were wounded with a sterile cork borer and inoculated with mycelial plugs of D. mutila (5 days old). An equal number of stems inoculated with sterile water or sterile PDA media were used as controls. Necrotic lesions of 61 to 90 mm in length and dieback symptoms were observed after five months in stems inoculated with conidial suspension. Necrotic lesions of 53 to 67 mm in length were measured after three months on stems inoculated with mycelial plugs. D. mutila was reisolated from all samples (100%) and reidentified using morphological characteristics and sequencing of ITS region. Control stems remained symptomless. To our knowledge, this is the first report of D. mutila causing Botryosphaeria stem blight in Chile and worldwide.