First report of Stemphylium eturmiunum causing leaf spot on Eucalyptus tricarpa in Australian native vegetation

Eucalyptus spp. dominate Australia’s most environmentally and economically significant ecosystems due to their ecological resilience (through post-fire epicormic regeneration), and widespread occurrence in remnant vegetation adjacent to agricultural crops, where they can influence disease dynamics and biosecurity risk (Keane et al. 2000). In March 2025, leaves from epicormic growth of two mature Eucalyptus tricarpa trees exhibiting concolorous 35 mm 2 (15% leaf area) asymmetrical brown, sunken necrotic lesions with dark margins were collected from native vegetation near Anglesea, Victoria (38°22’27” S, 144°14’43” E). Approximately 10% of epicormic leaves of adjacent E. tricarpa within a 100 m radius of the site displayed similar lesions. The leaves were surface sterilized (80% ethanol) and sections from lesion margins were cultured (25°C, one week). Hyphal tips were subcultured onto Potato Dextrose Agar (PDA). One lesion yielded three morphologically distinct isolates, including Alternaria sp., Allelochaeta sp., and Stemphylium sp., the second lesion yielded a further two distinct isolates, including Readeriella sp., and Neofusiccocum eucalypticola . All species isolated are known Eucalyptus pathogens (Crous et al. 2018; Keane et al. 2000), except Stemphylium sp. (UOM26001), which was examined further. This genus comprises important agricultural pathogens but are unknown from Australian native vegetation (Vaghefi et al. 2020). Two week-old PDA cultures had pale salmon mycelium and brown sporulation rings. Microscopic examination revealed hyaline few-septate hyphae, and mid-brown vertically and transversely 2-4-septate globose conidia 23.6 ± 3.38 x 17.6 ± 2.59 µm (21.0 - 30.9 x 12.3 - 22.8 µm), n=16, consistent with Stemphylium spp. (Simmons 2001). DNA was extracted via a modified Qiagen Plant MiniKit protocol (Vaghefi et al. 2024), and PCR and Sanger sequencing was performed for the internal transcribed spacer region and intervening 5.8S (ITS), calmodulin and gapdh with primers ITS1/ITS4 (White et al. 1990), caldf1/caldr2 (Carbone and Kohn 1999), and Gpd1/Gpd2 (Berbee et al. 1999). Sequences were deposited in GenBank as PZ054204 (ITS), PZ055902 (cal), PZ055903 (gapdh). BLAST showed all sequences were identical to Stemphylium eturmiunum reference sequences, including ex-holotype (CBS 109845). Multi-locus phylogenetic analysis was performed using RaxML v8.2.11 and MrBayes v3.2.6 (GTR model and 10 000 bootstrap replicates). UOM26001 clustered with S. eturmiunum isolates, with bootstrap and posterior probability values of 96 and 1.00, respectively. To assess pathogenicity, detached leaf assays were conducted due to constraints in sourcing whole trees for representative in planta assays, and biosecurity restrictions for trials external to a laboratory space. Therefore, following a standard method for Eucalyptus spp. bioassays (Alcova et al. 2026) sprigs of E. tricarpa epicormic growth each had 2 leaves surface sterilized (1% NaClO) and inoculated with 1 mm2 plugs of one week old UOM26001 via wounding (abrasions with sterile scalpel) (n = 6) and non-wounding (n = 6) methods. Negative controls (n = 6) were sterilized, wounded, and inoculated with clean PDA. Sprigs were placed in water and incubated for two weeks (12 - 20°C, 90% humidity, 12hr photoperiod), pathogenicity assays were repeated 3 times, for a total of 6 replicates of each inoculation method, including the control. After 14 days, 3/6 wounded, and 2/6 non-wounded inoculated leaves displayed pale brown necrotic lesions, similar but not identical to the original lesion due to the field symptoms being caused by a disease complex rather than UOM26001 alone. Negative controls were symptom free. Sections from lesion margins were plated onto PDA and incubated for one week at 25°C. Colonies re-isolated from all inoculated symptomatic leaf lesions were identified as S. eturmiunum based on culture and conidia morphology, confirming pathogenicity. Stemphylium eturmiunum is known in Australia and globally as a pathogen of agricultural crops (Vaghefi et al. 2020), but this is the first report on native Eastern Australian vegetation, and on E. tricarpa globally. This suggests S. eturmiunum may threaten post-fire regeneration of native trees by destroying new photosynthetic surfaces. And while this report cannot fully confirm significance for E. tricarpa due to only a single isolate being recovered, the detection in native vegetation expands the host range of S. eturmiunum , indicating E. tricarpa remnant vegetation could act as a pathogen reservoir, facilitating crop infection in Australian agricultural settings.