Apple canker has decreased yields of the economically important apple (Malus domestica Borch) crop in China in recent years. Pathogen identity is highly challenging and the disease is poorly understood. Specimens of 339 fungi were isolated from apple trees in the primary apple-producing region in the Tarim Basin during the current study. A total of 9 species of Cytospora and 5 species of Botryosphaeriaceae were identified by morphological observation and multi-locus phylogenetic analyses (ITS, ACT, TEF, and TUB for Cytospora; ITS, TEF, and TUB for Botryosphaeriaceae). Cytospora pyri from the Cytospora genus was the dominant species causing apple canker in the Tarim Basin. Cytospora melnikii, Cytospora tritici, Cytospora euonymina, Diplodia seriata, and Botryosphaeria dothidea have been described as the cause of apple canker in China. Apple (Red Fuji) branches were utilized to assess the pathogenicity of 24 representative fungal isolates from the 14 species and branches from seven distinct woody plants, Korla pear, walnut, Chinese date, Xinjiang poplar, sand jujube, Populus euphratica, and willow, were utilized to analyze the host range. The main pathogenic fungal species of apple canker around the Tarim Basin were identified and biological characteristics explored. Pathogen diversity and regional source diversity were assessed with host range and pathogenicity. The aim was to provide a theoretical foundation for the prevention and treatment of apple canker.

Apple (Malus pumila Mill.) is an important fruit crop in Xinjiang, China. In September 2021, apple tree canker was observed in a 21-year-old commercial apple orchard cv. Fuji in Xinjiang (38°17'51.43"N, 77°9'50.81"E) , northwest of China. Of the 200 plants surveyed, 25% were symptomatic. The diseased trees showed branch dieback and cankers. The cankers observed on the wood were sunken, shriveled, and discolored. After the bark was peeled off, the diseased wood was dark brown, and the necrosis was obvious on the cross-section of the diseased branch. To identify the causal agent, five symptomatic trees were collected and analyzed in the laboratory. Apple wood samples (0.5×0.5 cm) were surface-disinfected with 1% v/v sodium hypochlorite and 75% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA), and incubated in the dark at 25 °C for 5 days. Conidia were induced on sterilized pine needles covered with 2% w/v water agar under near-UV light. The colonies of five isolates were white to gray with sparse aerial mycelium that gradually became dark olive green in the later stage. Conidia were initially hyaline but becoming brown at maturity, 1-septate, oval, rounded at both ends, and with dimensions of 24.9-32.1 × 15.1-21.5 µm (n =50) and the aspect ratio of 1.6. Based on the cultural and morphological features of Phillips (2002), the isolates were identified initially as Diplodia mutila (Fr. : Fr.) Mont. To confirm species identification, genomic DNA was extracted from the representative isolate SC-8A. The primer ITS1/ITS4, EF1-728F/EF1-986R and BT2a/BT2b were used to amplify the rDNA sequences of, respectively, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1-α) gene, and a portion of beta-tubulin (tub2) gene. The nucleotide sequences indicated ≥99% identity to D. mutila (CBS 112553) for three DNA regions. Consensus sequences were deposited in GenBank. as accession numbers OM618108, OM676657 and OM676658 for ITS, EF1-α and tub2, respectively. To fulfill Koch’s postulates, pathogenicity tests were performed using isolate SC-8A on one year old branches of cv. Fuji (n=5). Wounds were created in the middle of the branches using a sterilized hole punch (5mm diameter) and were immediately inoculated with mycelial plugs of the same diameter. For the control treatment, sterile agar plugs were used (n=5) in the branches. The inoculated and control branches were wrapped with sterile parafilm. On the 10th day after inoculation, canker lesions appeared on the inoculated branches, but no lesions were observed in the negative control. D. mutila was re-isolated from 100% of the inoculated shoots and was not re-isolated from any of the negative controls, the Koch's postulates were met. Previously, D. mutila has been reported in Canada (Úrbez-Torres et al., 2016), Argentina (Lódolo et al., 2022) and Chile (Díaz et al., 2022) causing Botryosphaeria canker and dieback in apples. To our knowledge, this is the first report of D. mutila causing Botryosphaeria canker and dieback in apple trees in China.

Korla pear (Pyrus sinkiangensis Yü) is an important commercial fruit tree that originated in China (Zhou et al. 2020). In April 2020, a survey was conducted in Aksu region, Xinjiang (40°55'37"N, 80°28'42"E), China. Some Korla pear trees (>15 years old) exhibited symptoms of branch dieback and branch cankers. Cankers observed on the trunk and branches of the tree were sunken, dark ulcerative lesions sometimes exhibiting signs of stromata erumpent through the bark and exuding yellow to reddish-orange spore tendrils. Of the 180 plants surveyed, 80% were symptomatic. Thirty samples of symptomatic tissues of infected branches were taken to the laboratory. Bark and cortical wood samples containing necrotic and healthy tissue were excised with flame-sterilized scalpels, surface disinfected with 75% ethanol and 1% NaClO, placed on PDA plates, and incubated at 25°C. A total of 30 fungal isolates were obtained. Among them, 28 isolates were identified as Valsa mali var. pyri (Lu. 1992) based on morphological and molecular identification, and two isolates (ALE6T-GP21 and ALE7T-GP23) were identified as Valsa nivea (Hoffm.) Fr. Valsa nivea isolates had a fine villi form mycelium that was initially white, turned grayish-green over time and grew close to the medium surface. Cultures also contained black ostiolate pycnidia in a stroma that consisted of multiple irregular locules. Conidiophores were hyaline, occasionally branched at the bases and (15.50-)16.48-17.94(-18.50)×(1.00-)1.13-1.37(-1.50) μm (n=20). Conidiogenous cells were phialidic and subcylindrical that taper towards the apex. Conidia were hyaline, banana-like and (5.47-)6.13-6.97(-7.64)×(1.02-)1.06-1.20(-1.23) μm (n=10). The molecular characteristics are consistent with the previous description of V. nivea (Adams et al. 2006). The internal transcribed spacer (ITS), transcription elongation factor (tef-1α) and β-tubulin (Tub2) gene were sequenced using ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b primers, respectively (Zhang et al. 2014). BLAST (Basic Local Alignment Search Tool) searches against the NCBI database revealed that the ITS sequence had 99.83% homology (ON843984.1 and ON843987.1), tef-1α gene had 99.22% homology (MH015266.1 and MH015267.1), and the Tub2 sequence had 99.57% and 100% homologies (KT934364.1 and KT934364.1) with V. nivea sequences. The amplified sequences of ITS region (OK442665 and OK442666), tef-1α (OK510871 and OK510872) and Tub2 (OK510869 and OK510870) were deposited in the GenBank. A phylogenetic analysis was performed using MEGA7 that shows 100% bootstrap support that ALE6T-GP21 and ALE7T-GP23 were V. nivea. A pathogenicity trial was conducted with isolate ALE6T-GP21 inoculated onto 1-year-old shoots of 15-year-old Korla pear trees in Alar city, Xinjiang, China. Five shoots were inoculated by making 5-mm deep wounds using a sterile scalpel then inoculating with a 50 μL conidia suspension (1×106 mL−1). Additionally, five shoots served as the negative control and were inoculated in the same way using 50 μL ddH2O. The trees were kept under ambient conditions. Inoculated branches developed symptoms 18 days post inoculation, whereas the control branches showed no symptoms. V. nivea was re-isolated from the symptomatic areas and the isolate confirmed as ALE6T-GP21 by sequence analysis. Currently, the proven hosts of V. nivea are Populus, Elaeagnus, Juglans, Malus and Salix (Adams et al. 2006; Wang et al. 2020). To our knowledge, this is the first report of pathogenic V. nivea occurring on P. sinkiangensis in the world. It will provide a basis for research into the occurrence, distribution of V. nivea on Korla Pear.