First Report of <i>Fusarium incarnatum</i> Causing Walnut Brown Leaf Spot in Xinjiang, China

Walnut (Juglans regia L.), an economically important woody oil crop, is widely cultivated in southern Xinjiang. In August 2024, field surveys conducted across five walnut orchards (covering approximately 20 hectares) in Kalatal Town, Aksu Prefecture, Xinjiang (80.4933°E, 40.7665°N) revealed that 30-70% of trees exhibited leaf necrosis symptoms. The initial symptoms appeared as small, irregular brown necrotic lesions along leaf margins, which progressively expanded across the entire leaf surface as the disease developed. A total of 12 symptomatic leaves were collected from three walnut orchards. Leaf segments (4×4 mm) and twig sections (10 mm in length) were excised from lesion margins, surface-sterilized sequentially in 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min, rinsed 7-8 times with sterile distilled water, dried with sterile filter paper towels, and placed on potato dextrose agar (PDA) for 5-7 d at 28°C. Eight single-spore strains were obtained using the single-spore isolation method. After 4 days of incubation, the colonies initially appeared white to light pink, gradually developing a distinct pink pigmentation upon maturation. The macroconidia produced by this strain were falcate or elliptical, measuring 4.6-16.6 µm× 1.6-4.0 µm (n = 50). Genomic DNA was extracted from the representative isolate KY11 using the cetyltrimethylammonium bromide (CTAB) protocol. Four genetic loci were amplified and sequenced: the elongation factor 1-α (EF-1α) gene, internal transcribed spacer (ITS) region, 28S large ribosomal subunit (LSU) rRNA gene, and calmodulin (CAL) gene, along with the second largest subunit of RNA polymerase II (RPB2) gene. Amplification was performed using the following primer pairs: EF1-α: EF1-728F/EF1-986R, ITS: ITS1/ITS4, LSU: LR0R/LR5, CAL: CL1/CL2A, RPB2: fRPB2-5F/fRPB2-7Cr (White et al. 1990; Schoch et al. 2012; Xia et al. 2019). The sequences of isolate KY11 were deposited in GenBank with the following accession numbers: ITS(PV441516), EF-1α(PV524932), LSU(PV441478), CAL (PV536123), RPB2 (PV524934). BLASTn analysis revealed high sequence similarity with F. incarnatum references: 99.8% (ITS: MN871572.1), 99.61% (EF-1α: ON292430.1), 99.22% (LSU: MG274307.1), 100% (CAL: GQ505503.1), and 98.61% (RPB2: MT889350.1). A maximum likelihood phylogenetic tree was constructed based on concatenated sequences of EF-1α, CAL, and RPB2 using RAxML 8.0.0 software. Phylogenetic analysis confirmed that isolate KY11 clusters within the same clade as F. incarnatum, thereby supporting its taxonomic classification. (Garcia-Estrada et al.,2021). Isolate KY11 was identified as F. incarnatum based on morphological and sequence analyses. Pathogenicity tests were conducted on 3-month-old walnut seedlings to fulfill Koch's postulates. The inoculation procedure was as follows: three fully expanded healthy leaves per seedling were surface-sterilized with 75% ethanol, wounded with a sterile needle, and inoculated with a KY11 spore suspension at a concentration of 106 spores/mL. Control plants were mock-inoculated with sterile water. All plants were maintained at 28°C with 70% relative humidity under a 12-h light/dark cycle. After 12 days, inoculated leaves developed symptoms identical to field observations, while controls remained asymptomatic. The experiment consisted of two independent trials with five seedlings each. F. incarnatum was successfully re-isolated from symptomatic leaves and confirmed through both morphological and molecular methods, thereby fulfilling Koch's postulates and verifying its pathogenicity. This pathogen has been reported to cause cucumber fruit rot in Mexico (Garcia-Estrada et al., 2021), maize leaf blight (Xu et al., 2022), and luffa leaf spot (Chen et al., 2025) in China. To our knowledge, this represents the first report of F. incarnatum causing walnut brown spot in China, providing fundamental information for disease diagnosis and the development of control strategies.