First Report of Diaporthe eres causing Dieback Disease of Nerium oleander in China and the World

Nerium oleander L. (Apocynaceae) is an evergreen shrub widely cultivated as an ornamental plant throughout China. In July 2024, dieback symptoms were observed on N. oleander in the roadside green belt of Wangcun Town, Nanyang City, Henan Province (32.98°N, 112.52°E). Disease incidence across the surveyed area ranged from 23% to 37%. Symptomatic plants exhibited progressive chlorosis, leaf abscission, wilting, and acropetal stem necrosis. Affected tissues showed dark brown to black discoloration. Ten symptomatic tissues were collected. Tissue segments (5 × 5 mm) excised from the advancing lesion margins were surface-sterilized sequentially in 75% ethanol for 30s, followed by 0.1% mercuric chloride for 3 min, and rinsed three times with sterile distilled water. Segments were plated on PDA and incubated at 28 °C for 14 days. Three single-spore isolates (JZT21, JZT22, JZT23) were obtained, all displaying identical morphological characteristics. On PDA, colonies of isolate JZT21 showed slightly raised centers with dense white mycelium after 3–5 days, turning light gray with radial growth by day 7. Colony margins were villous with abundant aerial mycelium and white granular protrusions. After 14 days, spherical to subspherical black conidiomata developed. Alpha conidia were unicellular, translucent, fusiform, measuring 4.2-6.5 × 1.3-2.7 µm (mean = 5.3 × 2.0 μm, n = 30). Beta conidia were hyaline, aseptate, filiform and hamate, measuring 20.6-46.6 × 1.8-3.3 µm.（mean = 27.5 × 2.5 μm, n = 30). Gamma conidia and the sexual morph were not observed. For molecular identification, ITS, TEF1-α, TUB2, HIS3, and CAL gene regions were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), Bt2a/Bt2b (Glass and Donald-son 1995), CYLH3F/H3-1b (Glass and Donald-son 1995, Crous et al. 2004), and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. Sequences were deposited in GenBank under accession nos. PX204941 (ITS), PX229922 (TEF1-α), PZ276404 (TUB2), PZ126008 (HIS3), and PX229919 (CAL). BLAST searches revealed 100% identity with Diaporthe eres strain AR5193. Phylogenetic analysis using maximum likelihood methods confirmed that the isolate clustered within a well-supported clade with authentic D. eres specimens. Pathogenicity tests were conducted using isolate JZT21 on three 3-month-old N. oleander plants grown under greenhouse conditions. Stems were wounded and inoculated with 10 μL of spore suspension (1 × 106 conidia/mL). Three control plants received sterile water. Plants were maintained at 25 ℃ with a 12-h photoperiod and 75% relative humidity. The experiment was repeated three times. Over 14 days inoculation, all plants developed dieback symptoms identical to those observed in the field, while control plants remained asymptomatic. The fungus was successfully re-isolated from symptomatic tissues and confirmed as D. eres by morphological and molecular methods thereby fulfilling Koch's postulates. Diaporthe eres has been previously reported causing diseases on various hosts worldwide. To the best of our knowledge, this is the first report of D. eres causing dieback disease on N. oleander in China and the world. This finding expands the known host spectrum of D. eres and provides critical epidemiological baseline data for developing integrated disease management strategies in urban and peri-urban landscapes.