Branch blight of Juglans sigillata caused by Juglanconis appendiculata in China.

Juglans sigillata Dode, an endemic walnut species native in southwest China, is mainly used as nuts in Sichuan Province (Jin et al., 2019). In May 2021, symptoms of branches blight were observed in an orchard measuring 10 hectares located in Mianyang City, Sichuan Province (31°5' 25″N, 105°27'36″E, 365 m above sea level). About 40% of plants were diseased in the quadrat consisting of twenty walnut trees, and 20% of branches were dead on each affected tree. Initially, light brown spots appeared; then, the spots expanded to surround the whole branches; finally, the branches changed from brown to reddish-brown and died. Four symptomatic branches were sampled randomly from different trees. Next, four fungal isolates were obtained from the acervuli of each branch using the single-conidium isolation (Chomnunti et al. 2014) and cultured on potato dextrose agar (PDA). The Petri dishes were placed in an incubator and cultured at 25 °C under a 12-h photoperiod. Colonies were initially white with thin aerial mycelia and gradually turned dark grey with irregular margins. Conidiomata were acervular, black and scattered, with a diameter of 0.3 - 0.7 mm. Conidiophores were narrowly cylindrical, simple or branched at the base, 30 - 43 × 3 - 8 µm (x = 36.5 × 5.5 µm, n = 40). Conidiogenous cells were annellidic with distinct annellations. Conidia were unicellular, brown when mature, narrowly ellipsoid with gelatinous sheaths and truncate scars at the base, 17 - 32 × 7 - 12 µm (x = 27 × 9 µm, n = 40). The genomic DNA of a representative isolate SICAUCC 22-0064 was extracted, and the internal transcribed spacer (ITS) region, guanine nucleotide-binding protein subunit beta gene (ms204), translation elongation factor 1-alpha (tef1-α), and partial sequences of β-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers V9G/LR5 (de Hoog & van den Ende 1998), MS-E1F1/MS-E5R1 (Walker et al. 2012), EF1-728F (Carbone & Kohn 1999)/TEF1LLErev (Jaklitsch et al. 2005), and T1/BtHV2r (Voglmayr et al. 2017), respectively. The sequences of ITS, ms204, tef1-α, and tub2 were deposited in NCBI with accession numbers ON000068, ON112376, ON112374, and ON112375, respectively. With the consideration of the sequence lack of ms204 and tub2 in the ex-type strain (D96) of Juglanconis appendiculata Voglmayr & Jaklitsch, the isolate D140 was used for nucleotide blast. The results showed 99.68%, 100%, 100%, and 100% identities of ITS, ms204, tef1-α, and tub2 with D140 (accession numbers KY427138, KY427157, KY427207, KY427226). Phylogenetic analysis based on a combined dataset showed 100% bootstrap with J. appendiculata, and the morphology was consistent with the asexual stage of J. appendiculata (Voglmayr et al., 2017). To verify Koch's postulates, five branches wounded by pin-prick were sprayed with conidial suspension (1 × 105 conidia/mL) in each plant, and three repetitions were performed on healthy 2-year-old potted plants. The same number of branches were sprayed with sterile distilled water as controls. The plants were placed in a greenhouse at 25 ℃ under 90% relative humidity and a 12-h fluorescent light/dark regime. After five weeks, all the inoculated branches showed brown necrosis similar to that observed in the field, and no symptoms occurred on the controls. The pathogens were re-isolated from the necrotic lesions and identified by morphology and phylogeny. J. appendiculata has been reported on Juglans nigra and J. regia in Austria, France, Spain and Greece (Farr & Rossman 2022). This paper is the first report of branch blight on Juglans sigillata caused by J. appendiculata in China. This result may develop the understanding of walnut diseases and lay a foundation for further management.