Branch blight of Juglans regia caused by Palmiascoma qujingense in China

Juglans regia L. is one of the major cultivated walnut species in China for nuts and wood (Pollegioni et al. 2012). In June 2020, branches with blight symptoms were observed in an orchard at Chongzhou City (30°33'34″N, 103°38'35″E). In an orchard of 30 hectares, disease incidence was around 50%. A total of 15 plants were sampled and 40% of their branches were affected by this disease. Firstly, brown and irregular spots appeared, then the spots gradually expanded and encircled the branch, which eventually killed the branch. Five samples of diseased branches from different trees were collected and a single fungal isolate was obtained from each of the five samples using the single ascospore isolation (Chomnunti et al. 2014). Colonies of the five isolates on potato dextrose agar (PDA) were identical that initially appeared white on the top, becoming light to dark brown with age. On the host, ascostroma were black, globose to subglobose, short-papillate, ostiolate, 260 - 410 × 210 - 320 µm (x = 335 × 265 µm, n = 20). Asci were 8-spored, bitunicate, cylindrical, short pedicellate, 55 - 78 × 8 - 12 µm (x = 67.5 × 10 µm, n = 40). Ascospores were 1-septate, fusiform to ellipsoidal, slightly curved, guttulate, 12 - 17 × 3 - 5 µm (x = 14.5 × 4 µm, n = 40). These sexual morphological characteristics are consistent with the Palmiascoma qujingense Phook. & K.D. Hyde (Monkai et al. 2021). Asexual morphs were formed on PDA in incubator after 17 days (25℃, 90% relative humidity, 12-h photoperiod). Conidiomata were black, globose to subglobose, 220 - 300 × 240 - 380 µm (x = 270 × 310 µm, n = 20). Conidia were oblong to ellipsoidal, aseptate and smooth-walled, 3 - 7 × 2 - 4 μm (x = 4.9 × 3 μm, n = 50). The genomic DNA of a representative isolate SICAUCC 21-0013 was extracted, and the internal transcribed spacers (ITS) region, large subunit rDNA (LSU) region, small subunit rDNA (SSU) region, and the largest subunit of RNA polymerase II (rpb2) gene were amplified and sequenced with primers ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Rehner et al. 1994), NS1/NS4 (White et al. 1990), and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. The sequences were deposited in NCBI with accession numbers MZ983549, MZ959419, MZ951112, and MZ818772, respectively, which showed 100%, 100%, 99.14%, and 99.59% identities with P. qujingense KUMCC 19-0201 (holotype) (accession numbers MT477185, MT477186, MT477183, MT495782respectively). Phylogenetic analysis (maximum likelihood) based on a concatenated dataset showed 93% bootstrap support values with P. qujingense. To verify Koch's postulates, 9 healthy branches from three 1-year-old seedlings were inoculated with conidial suspension (106 conidia/ml) from 4-week-old cultures via pin-prick inoculation (Desai et al. 2019), and the same number of seedlings and branches were inoculated with sterile water as controls. Plants were placed in a greenhouse at 25℃ and 90% RH on a 12-h fluorescent light/dark regime. After 28 days, brown spots were formed on P. qujingense-inoculated branches and similar to those observed in the field, while the controls remained asymptomatic. The pathogen was re-isolated from the lesions and identified by morphology and phylogeny. To our knowledge, this is the first report of P. qujingense causing branch blight on J. regia in the world. This disease potentially impacts the growth and yield of J. regia, and control measures should be made.