First Report of <i>Phyllosticta capitalensis</i> Causing Leaf Spot Disease on <i>Fagopyrum dibotrys</i> in Sichuan Province, China

Fagopyrum dibotrys is an annual or perennial dicotyledonous plant in the family Polygonaceae. Due to its rich content of secondary metabolites and functional components, F. dibotrys is a traditional multipurpose plant, particularly in China (Zhang et al. 2021). In July 2023, at Sichuan Agricultural University's F. dibotrys Nursery, a 720 m2 farmland, in Ya’an, Sichuan Province, China (29.98°N, 102.99°E), 87.7% of cultivated F. dibotrys plants exhibiting disease susceptibility were observed. The symptoms appeared as ​chlorotic lesions​ on the adaxial leaf surfaces and ​uredinial pustules​ on the abaxial surfaces. In the early stage, the leaves develop irregular, yellow-brown spots that gradually expand, causing complete leaf yellowing with green margins, and leading to wilting. To identify the pathogen via tissue isolation and serial dilution plating, a 2 mm × 2 mm sample from three leaves was excised and cultured on PDA with streptomycin-penicillin (100 μg/mL); equivalent samples serially diluted (10⁻¹ to 10⁻³) were plated on PDA. Five isolates (SAU-1 to SAU-5) were obtained from each leaf on PDA plates. All colonies displayed consistent morphology: flat margins, olivaceous-black pigmentation, and a farinose to granular surface with slate-grey aerial hyphae. Lactophenol cotton blue staining revealed light brown vegetative hyphae and irregular blue aerial hyphae, measuring approximately 70-130 µm, and bearing translucent, elliptical, or spherical swollen structures. Conidia were blue, transparent, smooth, ellipsoidal, or spherical, 10.1-12.8 × 5.0-5.7 µm or 5.6-7.0 × 6.1-8.2 µm. Those results of the pathogen are consistent with those of Phyllosticta species described by Yan et al. (Yan et al. 2021). The primers ITS1/ITS4, T1/T22, NL1/NL4, and EF1-728F/EF1-986R were used to amplify the internal transcribed spacer (ITS; White et al. 1990), β-tubulin (Kerry et al. 1997), 28S rDNA, and translation elongation factor 1-α (EF1-α; Slippers et al. 2004), respectively, of a representative strain SAU-1. Analysis using BLASTn and GenBank showed that the ITS, EF1-α, β-tubulin, and 28S rDNA sequences exhibited high homology (over 99%) with Phyllosticta species. Using neighbor-joining analysis using MEGA11, phylogenetic trees showed that the obtained strain clustered with P. capitalensis strain CPC 18848, P. capitalensis strain GZ, P. capitalensis strain ARC, and P. capitalensis strain GZSN202005-1, respectively, suggesting the pathogen is P. capitalensis. To satisfy Koch’s postulates, P. capitalensis cultures were diluted to an OD600 of 1.0 and sprayed onto the adaxial surface of wild F. dibotrys leaves, and distilled water was applied as a control. Five days after spraying, inoculated, wild F. dibotrys leaves developed symptoms similar to natural field infections, while the control plants were healthy. The disease portion was isolated and cultured again. The re-isolated strain showed identical morphological characteristics and molecular profiles to the original isolate, thus fulfilling Koch's postulates. Phyllosticta, a key perithecial fungal genus, causes leaf spots and diseases on a wide range of hosts (Wikee et al. 2011). Although pathogens have been documented to infect F. tataricum (Li et al. 2021; Tang et al. 2022), this study is the first to report Phyllosticta capitalensis in F. dibotrys in Sichuan Province, China. This identification enhances the knowledge required for potential germplasm conservation and Phyllosticta control in cultivated F. dibotrys.