First Report of Colletotrichum nymphaeae Causing Anthracnose on Juglans regia Fruits in Southern Brazil

Juglans regia L. (Juglandaceae) is the most cultivated walnut species in the world. The species is economically important to temperate climatic regions because of its edible nuts and high quality of wood (McGranahan and Leslie 1991). In South America the nut production is important, mainly in Chile, where 37 thousand hectares (ODEPA 2016) are cultivated. In southern Brazil it is cultivated in small farms for subsistence and small-scale sales. Since 2016, fruits with anthracnose symptoms were observed, showing dark (∼1.0 in diameter) irregular and depressed spots, in a walnut orchard (28°58′16″ S, 52°00′38″ W) in Anta Gorda, Rio Grande do Sul, Brazil. The disease affected approximately 50% of the fruits, and the incidence was higher in rainy and hot summers. In January 2019, 20 symptomatic fruits were collected, disinfested for 1 min in 70% ethanol solution and 1% sodium hypochlorite, and subsequently washed in sterile water. The fruits were placed in a humidity chamber and incubated at 25 ± 2°C with a 12-h photoperiod. A single spore was used to obtain a pure culture, and the fungus was grown on Mathur culture medium (Mathur et al. 1950) for 7 days, with a 12-h photoperiod at 25 ± 2°C. The colonies showed pale salmon coloration on the underside of the plate, white aerial mycelium, orange conidial mass, and 8.4 mm/day mycelial growth (n = 6). The conidia were fusiform, hyaline, and unicellular, and the size 10.9 to 14.9 µm (mean = 13.3 µm, n = 30) × 3.7 to 5.4 µm (mean = 4.6 µm, n = 30). These morphological characteristics are consistent with the Colletotrichum acutatum species complex. After 15 days, acervuli were observed on Mathur culture medium, without the presence of setae. The appressoria were ovate and the size 9.0 to 15.0 µm (mean = 11.5 µm, n = 30) × 6.5 to 11.7 µm (mean = 9.5 µm, n = 30). To verify Koch’s postulates, the pathogenicity test was carried out with 18 healthy fruit in the middle stage of development arranged in previously sterilized boxes containing two sheets of filter paper. Each fruit was inoculated with 100 μl of a spore suspension (10⁶ conidia/ml), divided into six uniform drops, without wounding. The control was inoculated with sterile water. The boxes were kept at 25 ± 2°C, with a 12-h photoperiod. Symptoms identical to the collected fruits were observed 3 days after inoculation and were not observed in the control. The pathogen was recovered from inoculated fruits, fulfilling the Koch’s postulates. The β-tubulin (βTUB) and actin (ACT) genes were amplified and sequenced with TUB2F/TUB4R and ACT512F/ACT783R primers. The sequences of βTUB (MK953924) and ACT (MK953925) genes were deposited in GenBank and showed a close genetic identity to the ex-epitype culture of C. nymphaeae (CBS 515.78), a 99% match to JQ949518 (ACT), and a 100% match to JQ949848 (βTUB). Morphological characteristics and molecular features identified the isolate as C. nymphaeae, based on the description in Damm et al. (2012). The fungal culture was deposited at the herbal SMDB of the Universidade Federal de Santa Maria (SMDB18317). In Brazil, C. nymphaeae has been reported causing anthracnose in Malus domestica B. (Velho et al. 2014). According to our knowledge, this is the first report of C. nymphaeae causing anthracnose on J. regia fruits in Brazil.