First Report of Diplodia bulgarica Causing Black Rot Canker on Apple in China.

Abstract

Xinjiang, a major apple-producing region in, is renowned for its high-quality apples in China. In May 2024, symptomatic branches showing black rot canker were collected from commercial 'Golden Delicious' (Malus domestica) and Xinjiang Wild Apple (Malus. sieversii) trees in orchards located in Ili Kazakh Autonomous Prefecture (sites: 43.57°N, 81.92°E and 44.43°N, 80.78°E, respectively). The prevalence of symptomatic trees ranged from 30% to 40% (five-point sampling; n=50). External symptoms included charcoal-like cankers with bark peeling on primary and secondary branches, while internal examination revealed dark brown xylem with firm consistency. To isolate the causal pathogen, symptomatic branch samples (n = 14) were cut into 5 mm length pieces, surface-disinfected in 75% ethanol and rinsed with sterile water, and then transferred onto potato dextrose agar (PDA). Plates were incubated at 25°C for 3 days. Colonies consistent with Botryosphaeriaceae species (Phillips et al. 2013) (n = 20) were consistently obtained, and pure cultures were obtained by transferring single hyphal tips to fresh PDA. Colonies exhibited light gray coloration, irregular margins, and abundant aerial mycelium. To induce pycnidia formation, two isolates (ZM305-5 and ZM316-5) were cultured on pine needle agar (PNA) under near-ultraviolet light (340 nm) at 25°C for 20 days. Conidia (n = 50) were thick-walled, ovoid, initially hyaline, and turned dark brown at maturity, mostly aseptate, with the size of mature conidia ranging from 24.99 to 29.07 (27.14) × 13.76 to  15.78 (14.71) μm. Molecular identification was performed by sequencing the internal transcribed spacer (ITS) using primers ITS1/ITS4, results showing 100% identity with the Diplodia bulgarica holotype (CBS 124136). To further confirm the identity, the translation elongation factor 1-alpha (tef1) and the beta-tubulin (tub2) gene were sequenced using EF1-688F/EF1-1251R and Bt2a/Bt2b, respectively (Phillips et al. 2012). Sequences were deposited in GenBank (accession nos. PV362629-PV362631 for ITS, PV388909-PV388912 for tub2, and PV419584-PV419587 for tef1). A maximum likelihood (ML) multi-locus phylogenetic analysis clustered Chinese isolates with reference strains of D. bulgarica. Pathogenicity tests were conducted on 1-year-old shoots (n = 5) of 5-year-old saplings trees by wounding the bark of test shoots and inoculating mycelial plugs of these isolates. Control branches were mock-inoculated with sterile PDA plugs. After 30 days, dark-brown necrotic lesions developed in inoculated branches with length ranging from 31.5 to  45.5 mm, whereas the negative controls remained asymptomatic. The experiment was performed twice with similar results. Koch's postulates were fulfilled by re-isolating the pathogen from lesion margins and confirming its identify via morphology and ITS sequence. D. bulgarica was first described affecting M. sylvestris in Bulgaria (Phillips et al. 2012) and later associated with branch cankers on M. domestica in Iran (Abdollahzadeh 2015), India (Nabi et al. 2020), Germany (Hinrichs-Berger et al. 2021), Türkiye (Eken 2021), and California (Elfar et al. 2023). The pathogen has also been identified as the cause of postharvest fruit rot (Eken 2022). To our knowledge, this is the first report of D. bulgarica causing black rot canker on apple in China, which providing important information for developing detection and disease control strategies in apple industry.