First report of Diplodia mutila causing canker and branch dieback of walnut trees in Xinjiang, China.

Abstract

Walnut (Juglans regia L.) has become an important economic fruit tree in China. In April 2022, branch dieback was observed in 15-year-old walnut trees (cv Wen 185) in a commercial orchard in Aksu, Xinjiang, China (40°21'55''N, 80°1'48''E), with an incidence of 2% (4 out of 200 trees) of affected trees. The symptoms observed, included depressed and shrunken cankers, twigs and branches dieback. Cross-sections of diseased branches revealed dark-brown wedge-shaped lesions. To isolate the potential causal pathogen, four specimens were isolated from diseased branches, and small pieces taken from the edge of canker samples (0.5 × 0.5 cm), were disinfected by immersion in 75% ethanol for 30 s and 2% NaClO solution for 3 min, and rinsed three times with sterile water. The disinfected wood samples were then placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for 2-3 days. Then, we applied the mycelial tip purification method and repeated this purification process until a single colony was formed. Four pure isolates (3-1-51A, W-2-54, K-1-43A, K-1-43B) developed white to white-gray fast-growing colonies with abundant aerial mycelium after seven days at 25°C on PDA and gradually became dark olive green over subsequent growth stages. Conidia production was then induced on 2% w/v water agar containing sterilized pine needles under near-U/V light (Alves et al., 2004). The conidia were initially hyaline, thick-walled, oblong to ovoid with one septum and a size range of 19.47-24.16 × 9.78-13.51 µm (n = 40). Based on morphological characteristics these isolates were tentatively identified as Diplodia mutila (Fr.) Mont. (Alves et al., 2004).To confirm the pathogen identified, the representative isolate 3-1-51A was amplified and sequenced using specific primer pairs (ITS1/ITS4, EF1-986/EF1-728E, BT2a/BT2b) to the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB) (White et al. 1990; Carbone and Kohn 1999; O' Donnell and Cigelnik 1997), respectively. The sequences showed 100% similarity to two D. mutila strains CBS230.30 and CBS112553. Maximum likelihood analysis was performed based on a concatenated dataset (ITS + TEF1-α + TUB) gene using MEGA 11. 0 and isolate 3-1-51A formed a single clade with the reference ex-type of D. mutila. The isolate 3-1-51A was deposited into GenBank as OP006733, OP373140, and OP373139 for ITS, TEF1-α, and TUB, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate 3-1-51A on one-year-old healthy walnut branches cv. Wen 185 of walnut trees (n=5). Five twigs of healthy walnut branches were cleaned, submerged in 1% NaClO for 15 minutes and then dried. Then, a sterile hole punch (5mm in diameter) was used to create a wound in the middle of each walnut branch, and placing mycelial plugs(3 days old; 5 mm in diameter) and sealed with parafilm. An equal number of twigs inoculated with sterile agar plugs served as controls. On the 7th day after inoculation, dark brown coloration was developed on the branches with symptoms of shrinkage dryness, and dieback. D. mutila isolate was re-isolated only from the inoculated branches. In negative control twigs, lesions and re-isolated were absent, thus fulfilling Koch's postulates. D. mutila has been previously reported causing canker and branch dieback in walnut trees in Chile (Díaz et al. 2018) and California (Chen et al. 2014). Previously, D. seriata (Zhang et al. 2017), Botryosphaeria dothidea (Guo et al. 2016), Lasiodiplodia pseudotheobromae (Guo et al. 2016), Dothiorella gregaria (Liu et al. 1986) and Neofusicoccum parvum (Yu et al. 2015) have been identified on walnut trees in China. To our knowledge, this is the first report of canker and branch dieback caused by D. mutila in walnut trees in Xinjiang, China. Further studies are now required to better understand the etiology of canker and branch dieback on walnut trees from different areas in China.