LAUSR

Idesia polycarpa is a deciduous tree, widely grown in Guizhou Province for the high oil yield and potential economic value of its fruits (Zhang et al. 2023). In September 2023, a severe black spot disease was observed on I. polycarpa in the planting base in the state-owned forest farm of Dushan County, Guizhou Province, with an incidence of more than 60% (n=300). In the early stage of the disease, dark spots of irregular shape and size appeared on the leaves. As the disease progressed, the color of the central area of the spot gradually became lighter, and the edge remained dark black, with black conidia appearing on the surface. The black spots gradually merged until the leaves withered and fell off. Symptomatic leaves (n=10) were selected and surface-disinfected with 2% NaClO and 75% ethanol for 30 s, rinsed with sterile water, dried, and placed on potato dextrose agar (PDA) medium and incubated at 25°C for 7 d. Fungal colonies of 6 obtained isolates were white on PDA, and the reverse side appeared yellowish. The conidia were unicellular, ellipsoid, hyaline and measured 5.3 μm (4.1 to 7.6) × 2.6 μm (1.2 to 3.3) (n=50). The morphological characteristics of the isolates were consistent with descriptions of Didymella spp. (Chen et al. 2015). For molecular identification, DNA was extracted from three representative isolates (DS011, DS012, DS013) and the internal transcribed spacer (ITS) region and partial sequences of beta-tubulin (TUB2), RNA polymerase II subunit B (RPB2), and 28S large subunit rDNA (LSU) genes were amplified with primers ITS1/ITS4 (White et al. 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), RPB2-5F2/fRPB2-7cR (Liu et al. 1999) and LR7/LROR (Rehner and Samuels 1994), respectively. Sequences of ITS, TUB2, RPB2 and LSU were deposited in GenBank, with accession numbers (DS011: PV491363, PV567359, PV567360 and PV544964; DS012: PV771606, PV809725, PV809723 and PV779100; DS013: PV771630, PV809726, PV809724 and PV779353). BLAST results showed the sequences of DS011, DS012 and DS013 all exhibited > 99% identity, and DS011 had the highest identity with D. segeticola (NR172383 [ITS], 486/486; OR861528 [TUB2], 352/354; OR878476 [RPB2], 883/885; KP330452 [LSU], 1310/1310). A phylogenetic tree constructed with MEGA-X through Maximum Likelihood method indicated isolates DS011, DS012 and DS013 clustered with D. segeticola NC 20-852. Based on these results, the three isolates were identified as D. segeticola. Isolate DS011 was deposited in the Forest Protection Laboratory, Guizhou University. To confirm pathogenicity, spore suspension (1×106 spores/mL) of DS011 was sprayed on healthy leaves of 14-month-old potted I. polycarpa seedlings (n=6), while an equal volume of sterile water was sprayed as control (n=3). Then they were placed at 20°C and 70-80% RH. After 7 d, black spot symptoms appeared on leaves inoculated with the isolate DS011 while control plants remained asymptomatic. The isolates recovered from symptomatic leaves were identical to D. segeticola based on morphological and molecular analysis, fulfilling Koch’s postulates. Leaf spot disease caused by D. segeticola has been previously observed in tobacco (Guo et al. 2020) and Rosa roxburghii (An et al. 2024) in China, but to our knowledge, this is the first report of D. segeticola causing black spot of I. polycarpa in China, which would support a theoretical basis for manage the disease which might lead to growth restriction and oil production decline in I. polycarpa.