First Report of Fusarium kyushuense Causing Leaf Spot on Sweet Persimmon in China

Sweet persimmon (Diospyros kaki L.) is a fruit of significant nutritional and commercial value in Asia. In summer 2023, leaf spots were observed affecting 20 to 30% of sweet persimmon trees in a commercial orchard located in Gongcheng City, Guangxi, China. Initially, the infected leaves exhibited sparse light brown spots on their upper surface, which subsequently evolved into brown circular to irregular lesions encircled by a yellow halo. Eventually, these lesions became densely distributed across the leaves leading to insufficient nutrient accumulation in the fruit. To isolate the pathogen, diseased leaves were cut into small pieces (5×5 mm), disinfected with 75% ethanol for 15 seconds, followed by 1% NaClO for 1minute, rinsed three times with sterile water, and then transferred onto potato dextrose agar (PDA) plates. The plates were then incubated in darkness for 3 days at 25°C. Pure cultures were obtained using the hyphal-tip method and single-spore isolation. On PDA, the colonies initially appeared fluffy and white after 24 hours, turning yellowish or red after 3 days. Macroconidia (average length of 26.1 μm in length × 4.3 μm in width, n = 50) exhibited dorsiventral curvature and were hyaline, with 3 to 5 septa. Microconidia (average length of 9.45 μm in length × 3.4 μm in width, n = 50) were hyaline, aseptate, and oval. Two representative isolates, Gxfky1 and Gxfky2, were selected for further molecular analyses. Their internal transcribed spacer (ITS) region rDNA gene were amplified via PCR and sanger sequenced (GenBank Accession Nos. PP506475, PP506593) using the primer pair ITS1/ITS4 (White et al. 1990), showing more than 99% sequence identity with Fusarium kyushuense type-material strain NRRL3509 (NR_152943) according to BLASTn analysis in NCBI. To further confirm the identity of the isolates, four gene sequences were amplified: RPB1 (PP532864, PP532865), RPB2 (PP532866, PP532867), TEF1 (PP580505, PP580506), and TUB2 (PP532862, PP532863), using the F5/G2R, 5f2/11ar, EF1/EF2, and T1/T2 primer sets, respectively (O'Donnell et al., 1997; O'Donnell et al., 2010). A multi-locus maximum likelihood phylogenetic analysis revealed that Gxfky1 and Gxfky2 clustered with strains F. kyushuense with 100% bootstrap support. Pathogenicity tests using Gxfky1 and Gxfky2 were conducted on leaves of two-year-old sweet persimmon plants using non-wound inoculation. Specifically, 5-mm mycelial plugs and sterile agar plugs were placed on six leaves and secured with cling film, with six plugs each for the inoculation treatment and negative control, respectively. They were then incubated in a greenhouse at room temperature (25 ± 2°C) with a relative humidity of 70 to 80%. After 5 days, the same symptoms on naturally infected plants were observed on leaves inoculated with mycelium, while no symptoms were observed on the controls. The same fungus were reisolated from the inoculated leaves and identified based on morphology and the TEF1 gene sequence, thus fulfilling Koch's postulates. Fusarium kyushuense has previously been reported to cause diseases in various plant species, including maize (Cao et al., 2021), rice (Wang et al., 2024), and tobacco (Wang et al., 2013). To our knowledge, this is the first report of F. kyushuense causing leaf spot on sweet persimmon in China, which expands the known host range of this pathogen.