Isodon serra is a hepatoprotective herb widely used in traditional Chinese medicine. In July 2024, plants at a cultivation base in Maoming (21°41'11" N, 110°29'12" E), China, exhibited stem rot-like symptoms, with a field incidence rate of 50%. Initial disease presentation involved foliar chlorosis with brown stem lesions, progressing to leaf desiccation, internodal browning, and eventual plant collapse. Pathogen isolation was accomplished by first surface-sterilizing symptomatic stem segments with 75% ethanol for 30 seconds, followed by treatment with 1% NaClO for 2 minutes. The segments were rinsed thoroughly 3 times with sterile water finally and then cultivated on potato dextrose agar (PDA) at 28°C. The experiment was repeated 3 times, with isolations performed from multiple infected stem tissue segments, resulting in a total of 3 pure cultures with consistent morphology. Hyphal growth initiated within 12h, achieving complete medium colonization within 3-4 days while colony morphology transitioned from white to brownish-black after 7 days. The average diameter of pycnidium was 80.283 μm (n=20); spherical or oblate in shape; a hole on the top was for the release of condidium. The condidium were long oval or elliptical in shape with the average of 8.83 ± 1.39 μm long × 5.87 ± 1.17μm wide (n = 20); colorless transparent; warty protuberance on the surface. Chlamydospores are spherical, colorless and transparent. The morphological features consistent with Macrophomina sp. characteristics (Wu et al., 2022). The genomic DNA was extracted from a representative isolate by Fungi Genomic DNA Extraction Kit for molecular identification. The primer sets ITS4/ITS5, LROR/LR5, EF1-728F/EF1-986R and Bt2a/Bt2b (Wu et al. 2022) were used to amplify the internal transcribed spacer (ITS), large subunit ribosomal RNA gene (LSU), translation elongation factor-1α (tef-1α) and β-tubulin regions (β-TUB). The outcome of a blast of sequences showed 100% identity to M. euphorbiicola sequences (MN270855.1, MN270854.1, MH716048.1 and MF457657.1) in β-tubulin regions. Three groups of specific primers (MeTefF/MeTefR, MsTefF/MsTefR and MpTefF/MpTefR) (Santos et al., 2020) corresponding to the translation elongation factor-1 α were used for verification. The result showed that only the primer of M. euphorbiicola (MeTefF/MeTefR) was able to amplify the band. The result of multilocus sequence analysis (MLSA) (Altschul et al. 1990) constructed by ITS, LSU, tef1-α and β-tubulin also showed 100% homology to M. euphorbiicola MFLUCC 23-00057. Both morphological and molecular characteristics confirmed the fungi was M. euphorbiicola. The sequences were submitted in Genbank under accession numbers (PV544976(ITS), PV549410(LSU), PV595098(tef1-α), PV595099(TUB)). Three 25-cm-tall Isodon serra seedlings each were used for the treatment and control. A combined wound and spore suspension method was applied: treated plants were needle-wounded at the stem base, inoculated with M. euphorbiicola plugs, and drenched with 10 mL of spore suspension (1×10 6 spores/mL). Controls received PDA plugs and sterile water. All plants were kept at 30°C, 90% RH, and a 12-h photoperiod. Symptoms appeared around day 4, followed by wilting and death by day 7, while controls stayed healthy. Re-isolation and PCR with specific primers confirmed M. euphorbiicola as the causal agent. This is the first report of Macrophomina euphorbiicola as the causal agent of stem rot on Isodon serra.
Li et al. (Sun,) studied this question.