First Report of Neopestalotiopsis vacciniicola Causing Leaf Spot on Rhododendron delavayi in China

In March 2025, leaf spot was observed on nursery-grown Rhododendron delavayi in Dali, Yunnan, China (25°40′52″N, 100°18′01″E); of 300 plants randomly surveyed from the nursery population, 108 were affected. Adaxial lesions began as dark brown to purplish flecks, enlarged into tan-centered necrotic spots, coalesced, and caused premature leaf abscission; abaxial discoloration was less conspicuous. Nine symptomatic leaves were randomly collected from six plants showing typical symptoms. Lesion-margin pieces (ca. 5 × 5 mm) were disinfected in 70% ethanol for 30 s and 1% NaOCl for 1 min, rinsed, dried, and plated on potato dextrose agar (PDA). Twenty-one Neopestalotiopsis-like colonies were recovered from 27 pieces (77.8%); seven isolates were obtained. S1-1, S1-2, and S1-3 from different leaves had similar morphology. On PDA at 25°C, representative S1-1 formed white to creamy, cottony colonies after 7 d. Conidia of S1-1 were fusoid to slightly curved, mostly 4-septate, with three pigmented median cells and hyaline end cells, 13.5–26.0 × 5.9–8.4 μm (mean ± SD = 20.0 ± 3.2 × 7.0 ± 0.6 μm; n = 30), excluding appendages. They bore two to three tubular, unbranched, filiform apical appendages, 5.2–24.8 μm long, and one basal appendage. ITS, tef1-α, and tub2 were amplified from all three isolates using ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Within the retained intervals, all three isolates had identical ITS, tef1-α, and tub2 sequences. GenBank accessions were PX894796, PX905804, and PX905805 for S1-1; PZ583457, PZ683319, and PZ683320 for S1-2; and PZ583458, PZ683321, and PZ683322 for S1-3. Compared with ex-type CAA1055/MUM 21.35 (MW969751, MW959103, and MW934614), exact matches were 511/512 bp (99.8%) for ITS, 253/254 bp (99.6%) for tef1-α, and 419/419 bp (100%) for tub2. A partitioned maximum-likelihood analysis of concatenated ITS/tef1-α/tub2 sequences (40 taxa; 1,268 characters) placed S1-1, S1-2, and S1-3 in an exclusive clade with CAA1053, CAA1054, and ex-type CAA1055/MUM 21.35 (SH-aLRT/UFBoot = 94.1/99) (Santos et al. 2022). Two independent S1-1 experiments used seed-grown potted R. delavayi plants (3 years old; no named cultivar; n = 2 and 3, respectively), sprayed to runoff with 1 × 10⁶ conidia/mL in 0.05% Tween 80 (3 unwounded leaves per plant); 1 and 2 control plants received carrier only. Each treated leaf was sealed in a 0.012-mm-thick PVC bag for 48 h; plants were kept at 25°C, 85% RH, and a 12-h photoperiod. By day 7, inoculated leaves in both experiments had expanding necrotic spots (6/6 and 9/9); controls were asymptomatic (0/3 and 0/6). Experiment 2 ended at day 7; lesions enlarged through day 14 in experiment 1. Symptoms on inoculated leaves were generally similar to those observed in the nursery, although purplish margins were less conspicuous on inoculated plants. The fungus was reisolated; SR1-1 matched S1-1 morphologically, and its ITS sequence was identical to that of S1-1. S1-1 is maintained in the authors’ laboratory collection in Kunming, China, and is available from the corresponding author. The disease may reduce the marketability of R. delavayi nursery stock under humid production conditions and warrants monitoring. To our knowledge, this is the first experimentally confirmed report of N. vacciniicola causing leaf spot on R. delavayi.