First report of leaf brown spot caused by on in China.

The leaves of Lithocarpus polystachyus (Wall. ex A. DC.), an economically significant tree species in China, are commonly referred to as 'sweat tea' due to their high dihydrochalcone content, which holds biomedical importance, particularly in the treatment of diabetes (Hou et al. 2011). In January 2024, brown spots on L. polystachyus leaves were widely observed in Ningxiang (28°23'N, 112°59'E), Hunan Province, China. According to the investigation, the incidence rate of this disease was about 74% (222/300 plants surveyed). On each infected plant, nearly 60% leaves had symptoms. The disease initially presented as small yellow lesions that eventually developed into large brown patches with dark brown edges. More than 80% of the area was covered by leaf lesions, which eventually turned into leaf necrosis. To ascertain the pathogenic species responsible for this disease, pathogen isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected by immersing in 75% ethanol followed by 0.1% HgCl2. Small pieces (0.5 × 0.5 cm) were then excised and placed onto PDA medium, and incubated at 28°C for 6-9 days. Sterilized dissecting needles were used to pick mycelia from the edge of the colonies and placed onto PDA for strains purification. On the PDA, the colony color of upper side initially appeared white (Rayner 1A1), and then turned grey (Rayner 11C1), while the reverse side turnd faint yellow (Rayner 4A3). Black pycnidia were induced on PDA at 28°C under a 12 h/12 h light/dark cycle for 12 days. Alpha conidia were 5.37 _8.84 × 1.53 _3.19 μm (average: 6.77 × 2.37 μm, n = 50), hyaline, fusiform or ellipsoidal. Beta conidia were 13.61 _23.45 μm × 0.94 _1.47 μm (average: 18.78×1.18 μm, n = 50), hyaline, aseptate, filiform, straight or hamate. Morphologically, the fungi were identified as Diaporthe sp. (Guarnaccia and Crous 2017). To further affirm the identification of the pathogen, the genomic DNA was extracted from representative isolate, referred to as Dip, for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), β-tubulin (TUB2) and histone H3 (HIS) genes were amplified from genomic DNA using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and CYLH3F/H3-1b, respectively, to sequence for BLAST (Huang et al. 2015). The results showed that the ITS (GenBank: PP502145), EF1-α (GenBank: PP505773), TUB2 (GenBank: PP505774) and HIS (GenBank: PP505772) sequences of Dip isolate, respectively, showed 100% (469/469 bp), 98.47% (257/261 bp), 97.63% (617/632 bp), and 100% (379/379 bp) identity to their counterparts (GenBank: MW504747, ON049530, MW514138 and ON113058) in Diaporthe phoenicicola. The Maximum Likelihood tree was built based on the ITS, EF1-α, HIS and TUB2 sequences using MEGA11.0. Isolate Dip clustered with D. phoenicicola. The fungus was finally identified as D. phoenicicola by combining morphological and molecular characteristics. To confirm the pathogenicity of the isolated D. phoenicicola to induce brown spot, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspension (1×105 conidia per mL) was inoculated on 12 unwounded leaves collected from six 3-years-old plants, and sterile water was as control. The inoculated leaves were then incubated in chambers at 28℃ and 90% humidity with a 12 h photoperiod. The experiment was repeated three times. The results showed that inoculated leaves other than control developed brown spot symptoms within six days after inoculation. The test proved D. phoeicicola as the causal agent of this brown spot disease on L. polystachyus. The pathogen was exclusively re-isolated from the infected leaves and showed identical morphological characteristics to those of the original pathogens. To our knowledge, this is the first report of leaf brown spot of L. polystachyus caused by D. phoenicicola in China. This disease severely delays the plant development of L. polystachyus and significantly reduces the yield and quality of sweat tea. Our findings will contribute to the control of brown spot of L. polystachyus.