First report of <i>Lasiodiplodia pseudotheobromae</i> and <i>Diaporthe unshiuensis</i> causing stem rot on <i>Perilla frutescens</i> in China

Perilla frutescens (L.) Britt., an annual Lamiaceae herb, possess high medicinal, healthcare, and industrial value (Wang et al. 2020). Severe basal stem rot was recently observed on cultivated P. frutescens in Shaowu (27.34°N, 117.49°E), Fujian province, China, causing mortality ranging from 30% to 100%. Early symptoms included dark brown to black discoloration on the basal stem epidermis; mid-late stages showed brown to black discoloration of vascular bundles, followed by foliar wilting and extensive plant death. Eleven symptomatic stems were sampled, and 32 lesion marginal segments (5 × 5 mm) were obtained. Segments were surface-sterilized with 5% sodium hypochlorite for 1 min, rinsed thrice with sterile water, and air-dried. All tissues were cultured on PDA at 25°C in the dark for 7 days. Isolates were purified via single-spore separation and reincubated under identical conditions. A total of 36 isolates were obtained, including 25 Lasiodiplodia-like isolates produced compact colonies with dense aerial mycelia that changed from white/grayish-white to dark gray on PDA, and formed hyaline, ellipsoidal, unicellular conidia (22.3–27.4 μm × 12.2–15.6 μm, n = 30) on OMA (Guo et al. 2020; Wang et al. 2024). The 11 Diaporthe-like isolates developed white, fluffy aerial mycelia that grayed with age, with off-white to gray colony reverses and dark gray or pale brown centers, and yielded aseptate, smooth, ellipsoidal to clavate, biguttulate alpha conidia (5.3–7.2 μm × 1.3–3.1 μm, n = 30) on Oat Meal Agar (Zhou et al. 2025). The isolation frequency of Lasiodiplodia and Diaporthe were 69.4% and 30.6%, respectively. Genomic DNA of representative isolates (1401-10, Lasiodiplodia; 1411-11, Diaporthe) was extracted via CTAB (Stewart &amp; Via, 1993). Partial internal transcribed spacer (ITS) (White et al. 1990), translation elongation factor (TEF-1α) (Carbone &amp; Kohn, 1999), and beta-tubulin (TUB2) (Glass &amp; Donaldson, 1995) sequences were amplified, with GenBank accessions PV639174/PZ287758, PZ280354/PZ295460, and PZ280355/PZ295461, respectively. Isolate 1401-10 showed 100% similarity to Lasiodiplodia pseudotheobromae ITS (MH057185), TEF-1α (OR760715), and TUB2 (KX034522), isolate 1411-11 showed 100%, 99.64%, and 99.36% similarity to Diaporthe unshiuensis ITS (MT043829), TEF-1α (KJ623300), and TUB2 (MK691289), respectively. A maximum likelihood phylogenetic tree (combined ITS, TEF-1α, and TUB2; MEGA6) clustered 1401-10 with L. pseudotheobromae and 1411-11 with D. unshiuensis (bootstrap value = 98%, Chen et al. 2021; Guo et al. 2020). Perilla seedlings were inoculated with 1% (v/v) wheat grain inoculum according to Zhong et al. 2015. Seedlings treated had pathogen-free wheat grain were served as control. The assay consisted of three biological replicates with five plants each, and the experiment repeated twice. Seedlings were covered with plastic film for 24 h and incubated at 25°C under a 12 h light/dark cycle. Ten days later, inoculated stems showed typical field symptoms, while controls remained asymptomatic. The same fungi were re-isolated from diseased stems, confirming pathogenicity. To our knowledge, this is the first report of these two pathogens causing Perilla basal stem rot in China. As a major P. frutescens producer, China faces serious threats from the emerging disease, effective management strategies are needed given their broad host ranges.