First Report of <i>Fusarium proliferatum</i> Causing Leaf Spot on <i>Juglans regia</i> in Xinjiang, China

In southern Xinjiang, walnut (<i>Juglans regia</i> L.) is the primary source of income for farmers and a key crop in the region's specialty fruit and forestry industries. In September 2024, leaf spotting was observed in several walnut orchards in Aral, Xinjiang. In the early stages of the disease, small brown spots appeared on the leaf tips and margins, which later expanded irregularly across the entire leaf, eventually causing the leaves to wither and necrotize completely. Diseased leaves were collected from four walnut (Xinwen185) orchards in Aral (81.2997°E, 40.5583°N) for pathogen isolation. Leaf sections (4×4 mm) were excised from lesion margins and subjected to surface sterilization through sequential treatment with 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min. They were then rinsed eight times by immersion in 50 mL of sterile distilled water with gentle agitation for 30 s each time. After drying with sterile filter paper, the segments were transferred onto potato dextrose agar (PDA) plates and incubated at 28 ℃for 5-7 days. Twelve single-spore strains were obtained through single-spore isolation technique. After 6 days of incubation, the isolates produced abundant aerial white mycelia and acquired a purple pigmentation. The hyphae were hyaline with septation. The isolates produced numerous oval unicellular microconidia without septa, 4.8 to 18.6 × 1.5 to 4.2 µm (n = 50) and very few macroconidia with three to four septa (21.6 to 46 × 3.1 to 4.3 µm [n = 30]), narrowed at both ends (Fig.S1). These morphological characteristics are consistent with those described for <i>F. proliferatum</i> (Leslie and Summerell, 2006). Genomic DNA was extracted from a representative isolate KY7 using the cetyltrimethylammonium bromide (CTAB) protocol. Four genetic loci were amplified and sequenced: the elongation factor 1-α (TEF1) 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: TEF1: EF1-728F/EF1-986R, ITS: ITS1/ITS4, LSU: LR0R/LR5, CAL: CL1/CL2A, RPB2: fRPB2-5F/fRPB2-7Cr. The sequences of the isolate KY7, were deposited in GenBank under the following accession numbers: ITS (PV441515), TEF1 (PV524931), LSU (PV441477), CAL (PV524935), RPB2(PV524933). BLASTn analysis revealed that isolate KY7 showed 99.62% (ITS: EF453150.1), 98.84% (TEF1:KX656210.1), 99.89% (LSU: PP336543.1),99.73% (CAL: AF291057.1) and 99.66% (RPB2: MN193893.1) with <i>F. proliferatum</i>. A maximum likelihood phylogenetic tree was constructed based on concatenated sequences of EF-1α, CAL, and RPB2 using Fast tree 2.2 software. The isolate KY7 clustered within the same evolutionary clade as <i>F. proliferatum</i>. Based on morphological and molecular analyses, isolate KY7 was identified as <i>F. proliferatum</i> (Fig.S2) (Leslie and Summerell, 2006). Three-month-old walnut seedlings were selected for pathogenicity testing of isolate KY7. The leaves were surface-sterilized with 75% ethanol, rinsed three times with sterile water, and then wounded with a sterile needle. For the inoculation group, each plant was sprayed with 5 mL of KY7 spore suspension (1×106 spores/mL), while the control group was sprayed with an equal volume of sterile water. All plants were maintained at 28℃ with 80% relative humidity under a 12 h photoperiod. The experiment consisted of two independent trials, with each trial including five inoculated seedlings and five control seedlings (resulting in a total of ten inoculated and ten control plants). Ten days after inoculation, irregular brown spots consistent with field observations appeared on the inoculated leaves, whereas the control plants remained asymptomatic (Fig.S1). The same pathogen was re-isolated from the diseased leaves, fulfilling Koch's postulates. F. proliferatum has been reported to cause leaf blight in Phoenix dactylifera in Tunisia (Namsi et al., 2021) and leaf spot in Juglans regia in Hebei Province, China (Wang et al., 2022). In Xinjiang, characterized by arid conditions, low rainfall, and abundant sunlight (Chang et al., 2022), the pathogen's ability to infect walnuts has raised concerns among local farmers. In contrast, disease management practices in Hebei Province are likely more targeted. The identification of <i>F. proliferatum</i> in this study enables the development of tailored management strategies, providing a critical foundation for precise disease control. This will assist growers in selecting effective fungicides and formulating optimized application protocols. To our knowledge, this is the first report of <i>F. proliferatum</i> causing walnut leaf spot in Xinjiang, China.