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First Report of Diaporthe ambigua associated with dead arm disease on grapevine in Chile.

Grapevine (Vitis vinifera L.) is one of the most important fruit crops in Chile based on economic value. Phaeomoniella chlamydospora and Botryosphaeriaceae species have been reported as the major causal agents associated with dieback symptoms in Chile commercial vineyards (Díaz and Latorre 2014; Besoain, 2018; Larach et al. 2020). Recently Eutypa lata has been reported attacking Chilean vineyards with dieback symptoms (Lolas et al. 2020). In this study, two commercial cv. Cabernet Sauvignon vineyards, located in O'Higgins Region of Chile, showing dead cordons, dead spur with a grayish color, canker, and vascular necrosis were sampled in fall 2018, with a high incidence of symptoms was observed. Four symptomatic wood samples were analyzed from these vineyards. Pieces of wood (<1 cm2) were taken from the advance zone of the canker lesions, disinfected with 70% ethanol, rinsed in sterile distilled water, dried, and transferred to two media in Petri plates, potato dextrose agar acidified with 0.5 ml of 96% lactic acid (APDA) and malt extract agar, and incubated for at least seven days at 24°C in darkness. From mycelium obtained from monosporic culture, two isolates were selected and morphologically identified as Diaporthe sp. To induce sporulation, these two isolates were grown in APDA under near-ultraviolet light (λ = 320 nm) at room temperature. After 30 days, the development of pycnidia was observed. Both Diaporthe sp. isolate presented alpha-conidia ellipsoidal with an obtuse apex, biguttulate (n=30) of 6.7 µm ± 0.33 µm x 3.3 µm ± 0.32 µm. No Beta-conidia or perhitecia were observed. DNA was extracted from the monosporic mycelium. The ribosomal internal transcribed spacer (ITS), β-tubulin (BT) gene, and elongation factor (EF) gene were amplified using ITS4/ITS5, Bt2a/Bt2b, and EF1-728F/EF1-986R primer pairs, respectively. PCR products were sequenced and identified as Diaporthe ambigua Nitschke (PUCV2140 and PUCV2141), showing 100% sequence identity with ITS MH864620.1, 99.8% with BT MG281142.1, and 100% with EF KC343738.1 sequences from D. ambigua. Sequences were deposited in GenBank (ITS: accession numbers MW301136, MW301137; BT: MW323445, MW323446 and EF: MW308305, MW308306). Two pathogenicity tests were performed with strains PUCV2140 and PUCV2141 using 2-year-old V.vinifera cv. Cabernet Sauvignon. In each test, three plants were used per isolate, considering one plant as an experimental unit. In the first test, a 5 mm mycelial plug from a 6-day-old APDA culture was inoculated using an oblique cut made in the bark with a sterile scalpel and done at the middle of the trunk. In the second test, the trial was done under the same described conditions previously, but in this case, one-year-old semi-lignified shoots were inoculated between two internodes, using mycelial plugs, one shoot for each plant. Injured plants but treated with sterile APDA plugs were used as controls. Plants were placed in natural conditions, and after three months from inoculations, plants showed a cortical canker and brown vascular lesions. Non-inoculated plants remained asymptomatic. The lengths of the cankers were 22.0 ± 1.8 mm and 10.5 ± 0.6 mm, after inoculations of the trunk and cane, respectively. The vascular lesions were 37.0 ± 3.3 and 18.0 ± 2.0 mm, in trunk and cane inoculations, respectively. D. ambigua was re-isolated and reidentified morphologically from the inoculated symptomatic plants, confirming Koch's postulate. Also, the plants inoculated on the trunk showed premature leaf drop. To our knowledge, this is the first report of D. ambigua associated with dieback affecting grapevines in Chile. Previous D. ambigua was reported causing fruit rots (Auger et al. 2013; Díaz et al. 2017) and cordon dieback in kiwifruit (Díaz and Latorre, 2018), and stem canker and dieback in blueberry (Elfar et al. 2013) in Chile. This study reports a new species of fungi for Chile associated with the dead arm in vineyards. D. ambigua is a pathogen in essential crops in our country. Therefore, it is important to study its prevalence in the future.