Dothiorella sarmentorum Causing Branch Dieback of English Walnut in Maule Region, Chile.

Abstract

English walnut (Juglans regia), cv. Chandler is the most cultivated tree nut in Chile, with 43,734 ha. In Maule Region, central Chile, English walnut plantings have expanded over an additional 7,000 ha in the last five years. During a routine orchard survey in 2019, branch and twig dieback symptoms were observed in two commercial orchards located in San Rafael (10 years old) and Longaví (12 years old) in the Maule Region, with an incidence of 45% to 65% of affected trees, respectively. Symptomatic branch samples (n = 15) were collected from the two commercial orchards and transported to the laboratory in a cooler and then surface sterilized in 96% ethanol for 3 s and briefly flamed. Cross-section of symptomatic branches revealed brown to dark-brown wedge-shaped wood cankers. Small (5 mm) pieces of wood from the edge of cankered tissues were placed on Potato Dextrose Agar (PDA, 2%) amended with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (PDAm) (Díaz and Latorre 2014) and incubated at 25°C for five days in the dark. Pure cultures were obtained by transferring a hyphal tip from growing colonies to fresh PDA media. Each fungal isolate was recovered from a single diseased branch (47%). Seven isolates (Dsar-1 to Dsar-7) developed dark to olive-brown fast-growing colonies with scarce aerial mycelium after seven days at 25°C on PDA. These isolates showed a dark-olive color on the reverse side of Petri dishes and developed abundant, aggregated, and dark-brown pycnidia after 15 days at 25°C. Conidia were hyaline and aseptate, dark brown, 1-septate, with a brown wall, ovoid with a broadly rounded apex and truncated base, (17.5-) 19.5 ±1.2 (-22.0) x (7.6-) 8.9 ± 0.6 (-10.1) µm (n = 30). These isolates were tentatively identified morphologically as Dothiorella sp. (Phillips et al. 2005). Molecular identification was performed using ITS1/ITS4 and EF1-728F/EF1-986R primers (White et al. 1990; Dissanayake et al. 2015) of the internal transcribed spacer (ITS1-5.8S-ITS2) region and part of the translation elongation factor (EF1-) genes, respectively. A MegaBlast search in GenBank showed a 100% similarity to isolate CBS 115038, the ex-type of Dothiorella sarmentorum. The sequences were added to GenBank (OM161950 to OM161956 for ITS; OM177188 to OM177194 for EF1-). Pathogenicity of two isolates (Dsar-2 and Dsar-7) was tested in the orchard on freshly made pruning wounds on attached branches of 2-year-old-pruned English walnut trees cv. Chandler. A second pathogenicity test was done on freshly made pruning wounds in 1-year-old rooted cuttings (n=15) (40 cm of long) of English walnut cv. Chandler. Each pruning wound was inoculated with 40 µL conidial suspension (105 conidia/mL). Sterile distilled water was used as a control treatment. Both pathogenicity tests were repeated once. After seven months for attached branches and four months for rooted plants, necrotic streaks with a mean length of 81.3 and 44.5 mm were observed below the inoculated pruning wounds, respectively. No necrotic streaks were observed in any of the control wounds. Dothiorella sarmentorum was 100% reisolated from symptomatic tissues of inoculated branches and molecularly identified (EF1-), thus fulfilling Koch's postulates. Recently, D. sarmentorum has been reported causing English walnut dieback in Spain (López-Moral et al. 2020). To our knowledge, this is the first report of D. sarmentorum causing branch dieback of English walnut in Chile. Further studies are needed to know the impact and extent of canker and branch dieback of walnut in commercial orchards in the Maule Region, central Chile.