First Report of <i>Diaporthe eres</i> Causing Branch Rot of Sea Buckthorn ( <i>Hippophae rhamnoides</i> ) in China

In 2024, a plant disease survey in Linkou County, Mudanjiang City, Heilongjiang, China, found Hippophae rhamnoides plants with branch rot symptoms: branch ulcers and black eye-like lesions (Udayanga et al., 2011). Later, branches dried up, with lesions spreading, killing fruits and leaves. Diseased branches were washed, and 10 mm² bark pieces from the diseased-healthy junction were disinfected in 3% sodium hypochlorite for 2 min, then 75% ethanol for 60 sec, and inoculated onto PDA with 50 mg/L streptomycin. After incubation at 28°C for 2 days, several Diaporthe isolates with uniform morphology were purified via hyphal tip isolation. Two representative isolates, LK2024-SB001 and SW2024-SB002, were selected for detailed study due to their characteristic morphological and molecular features. Ten 2 × 2 mm² agar blocks per isolate were placed in 90 mm Petri dishes with 10 ml PDA and incubated for 3 days to form mycelial pads. After 15 days at room temperature, black fruiting bodies (2,000–5,000 μm high, 2,000–3,000 μm wide) formed. At 40x magnification on an Olympus BX43 microscope, these were identified as acervuli with transparent, smooth conidiophores (5–20 μm long) (Gomes et al., 2013). After 10–20 more days, orange conidial masses oozed from fruiting body tips. Microscopy revealed three conidia types: α-type (most abundant), spindle-shaped, aseptate, with oil-like droplets at each end, sized [means ± SD =(6.47 ± 0.65)×(2.53 ± 0.53) μm]; β-type, hooked or sickle-shaped, sized [means ± SD=(17.90 ± 4.39)×(1.50 ± 0.23) μm]; γ-type (least abundant), cylindrical, rod-shaped, with a middle constriction and oil-like droplets at both ends, sized [means ± SD=(9.15 ± 1.26)×(2.32 ± 0.36) μm] (Gomes et al., 2013; Udayanga et al., 2011). During our 2024 field surveys, similar branch rot symptoms were observed on Hippophae rhamnoides in other regions of Heilongjiang, where additional isolates were obtained and analyzed (unpublished data). Pathogenicity tests were conducted in a laboratory. Due to the difficulty of re-inoculation using needle piercing, a cross-scratch method was used. Fresh one- to two-year-old sea buckthorn branches were selected, and their smooth sections were evaluated. Each branch was scratched with 3–4 wounds. Experimental branches were inoculated with a spore suspension (108 conidia/mL), while control branches were treated with sterile water. Branches were kept humid and sprayed daily with sterile water. After 14 days, inoculated branches showed black spot ulcers matching field symptoms, while controls remained healthy. The pathogen was re-isolated from inoculated branches, matching the initial isolates, confirming pathogenicity. Molecular identification used ITS, TEF1-α, and TUB2 markers (Gomes et al., 2013). ITS was amplified with ITS1/ITS4 primers(White et al., 1990), TEF1-α with EF1-728F/EF1-986R(Carbone & Kohn, 1999), and TUB2 with Bt2a/Bt2b (Glass & Donaldson, 1995). Sequences were uploaded to GenBank and compared to Diaporthe eres reference sequences using the BLASTn tool on the NCBI database to determine sequence identity. For isolate LK2024-SB001, sequences (ITS: PQ578294; TUB2: PV274531; TEF1-α: PV267361) were 100% identical to ON520786, PV052969, and OQ910288, respectively (Chaisiri et al., 2021). For isolate SW2024-SB002, sequences (ITS: PQ620237; TUB2: PV344633; TEF1-α: PV344634) showed 98.9%, 100%, and 100% identity to GenBank sequences OM536179, PV052969, and OQ910288, respectively, based on BLAST comparisons. Sequence identity based on ITS, TEF1-α, and TUB2 sequences confirmed that isolates LK2024-SB001 and SW2024-SB002 belong to Diaporthe eres. The identification was further supported by characteristic morphological features consistent with previous descriptions of D. eres. The pathogen was identified as Diaporthe eres based on morphology and molecular data from these isolates. This study is the first to report the branch rot disease caused by Diaporthe eres on sea buckthorn plants, providing a basis for disease monitoring and management in sea buckthorn planting areas (Dai et al., 2024).