First report of Neopestalotiopsis chrysea causing leaf black spot on Macadamia integrifolia in China

Macadamia nut (Macadamia integrifolia) is a crop with significant nutritional and economic value. In November 2024, one of leaf spot symptoms was observed in Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China (23°50′~25°20′ N, 97°31′~98°43′ E), with a disease incidence of 35% in field. The disease initially manifests along the main veins or leaf margins, forming irregular lesions. These lesions have grayish centers surrounded by dark brown to black margins. As the lesions expand and coalesce, they cover large areas of the leaf surface. In severe cases, the leaves become wilted and defoliated, with black acervuli visible on the affected spots. Single-spore isolation was used to isolate the pathogen and four strains were obtained. The fungal colonies on PDA exhibited a circular, white, cottony appearance with an undulated surface. After 18 days, black, globular acervuli emerged on the colony's surface. Conidia 18.59 to 25.96 × 4.50 to 6.54 µm (x̄ = 22.61 × 5.55 μm) (n = 50), fusiform, straight to slightly curved, 4-septate; basal cell obconic to conic, hyaline, thin and smooth-walled, 2.72 to 5.61 μm long (x̄ = 4.13 μm); second cell from base pale brown, 4.06 to 5.95 μm (x̄ = 4.81 μm); third cell darker brown, 3.57 to 5.76 μm (x̄ = 4.61 μm); fourth cell darker, 3.74 to 5.64 μm (x̄ = 4.70 μm); apical cell 2.23 to 4.37 μm long (x̄ = 3.34 μm), hyaline, conic to obconic; apical appendages 16.34 to 23.17 μm long (x̄ = 18.97 μm), 2–3, tubular, arising from the apex; basal appendage, 3.30 to 7.08 μm long (x̄ = 4.82 μm), filiform. Morphological characteristics were consistent with published descriptions of Neopestalotiopsis chrysea. The internal transcribed spacer (ITS) regions, large subunit (LSU), and translation elongation factor 1-alpha (TEF1-α) were amplified with the primers ITS1/ITS4, LR0R/LR5, and EF1-728F/EF-2, respectively. The obtained sequences from swfu06 showed similarity with those from Neopestalotiopsis chrysea, ITS (KT783664, 553/551, 99.82%), TEF1-α (OQ410712, 500/513, 99.80%), LSU (KM116261, 874/896, 100.00%) in GenBank，respectively. All the sequences were deposited in GenBank: ITS (PQ803208, PQ803209); LSU (PQ804966, PQ804967); TEF1-α (PQ867804, PQ867805). A neighbor-joining phylogenetic tree was constructed using MEGAX, incorporating Neopestalotiopsis sp. sequences from GenBank. The pathogen was found to cluster within the N. chrysea clade with 99% bootstrap support. To test pathogenicity, Five healthy 2-year-old seedlings were selected, and four leaves per seedling were inoculated with 200 μl of conidial suspension per leaf (106 conidia/ml) with syringe. Five control plants were inoculated with ddH2O. All inoculated seedlings were covered with plastic bags for 48 h then being placed in the nature. All the inoculated leaves showed symptoms similar to those observed in the field, whereas the control leaves were asymptomatic for 7 days. N. chrysea (swfu07) was reisolated from the lesions, whereas no fungus was isolated from the control leaves. This disease will lead to early leaf drop, affecting photosynthesis and fruit yield, and posing a threat to the macadamia nut industry. This is the first report that N. chrysea causing leaf spots on M. integrifolia in China. This is of great significance for ensuring the sustainable development of macadamia nut industry. To reduce economic losses, early monitoring and disease management should be strengthened in the field.