First Report of Husk Black Spot of Macadamia Caused by Fusarium proliferatum in China

Macadamia (Macadamia integrifolia), native to eastern Australia (Dahler et al. 1995), is an economically important nut crop widely valued for its high nutritional content and is often referred to as the “king of nuts” (Guo et al. 2025; Liu et al. 2019). Yunnan Province, China, is the world’s largest macadamia-producing region, accounting for 54.3% of global and 90% of national cultivation areas (Nie et al. 2023). Intensive cultivation has resulted in increasing outbreaks of pests and diseases, which significantly reduce yield and nut quality. Husk black spot is a prevalent disease in Dehong Prefecture, Yunnan, affecting over 50% of orchards and causing yield losses of 20-30% (Akinsanmi et al. 2016). Early symptoms appear as small brown lesions on the husk. As the disease progresses, lesions expand, coalesce, become sunken and necrotic, and often develop epidermal cracking. At the mid-stage, lesions cover more than half the husk surface, which turns black and rotten with visible white mycelia, and the kernel becomes soft and shriveled. In the late stage, fruits shrivel, crack, turn charred black, and internal tissues decay completely, leading to extensive fruit drop or mummification. For pathogen isolation, surface-sterilized diseased husk tissues were incubated under high humidity (Liang et al. 2023). Mycelia emerging from tissue margins were transferred to potato dextrose agar (PDA). A single-spore isolate, designated OCG-2, was obtained for subsequent studies. On PDA, colonies were initially white, fluffy with abundant aerial mycelium, and had an even margin. With incubation time, colonies developed pale purple, pink, or tan pigmentation. Hyphae were hyaline, smooth, septate, and 2-5 μm in diameter. Two types of conidia were observed: abundant microconidia, aseptate or 1-septate, clavate to reniform, measuring (9.1 ± 1.2) × (3.3 ± 0.5) μm (n = 30); and fewer macroconidia, 3-5-septate, slightly curved and falcate, with a distinct basal foot cell, measuring (34.5 ± 6.4) × (3.6 ± 0.5) μm (n = 30). These morphological characteristics are consistent with those of Fusarium proliferatum (Cai et al. 2022). Molecular identification was performed via sequencing of the internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α), and β-tubulin (tub2) genes. The ITS (528 bp), TEF1-α (624 bp), and tub2 (478 bp) sequences of isolate OCG-2 were deposited in GenBank under accession numbers PX840715 (ITS), PZ363029 (TEF1-α), and PZ369712 (tub2). All sequences were aligned using the MUSCLE algorithm implemented in MEGA12, and maximum likelihood (ML) phylogenetic trees were constructed in the same software with the GTR+G nucleotide substitution model; bootstrap support values were calculated from 1000 replicates. BLASTn analysis against the GenBank nucleotide database revealed that the sequence of isolate OCG-2 shared ≥99% identity with reference sequences of F. proliferatum. The concatenated ML phylogenetic tree, constructed using the combined ITS, TEF1-α, and tub2 sequences, further confirmed this identification: isolate OCG-2 formed a strongly supported monophyletic clade (bootstrap value 98%) with reference strains of F. proliferatum (including strains M14 and CBS 263.54), within a larger well-supported cluster containing additional F. proliferatum isolates and Gibberella moniliformis. This clade was clearly separated from closely related species including F. annulatum, F. concentricum, and F. fujikuroi, with Aspergillus carneus strain CBS 494.65 as the outgroup. Combined morphological and molecular data confirmed strain OCG-2 as Fusarium proliferatum. Pathogenicity tests were conducted on surface-sterilized healthy macadamia fruits. Each treatment included 5 fruits with three biological replicates. Inoculation was performed by placing a 5-mm mycelial plug from 7-d-old OCG-2 cultures onto three small wounds per fruit arranged in an inverted triangular pattern; control fruits received sterile PDA plugs. All fruits were incubated at 25 °C under high humidity. Small black spots appeared around inoculation sites at 3 days post-inoculation (dpi). These spots expanded and coalesced into large black lesions by 5-7 dpi. By 9-11 dpi, lesions covered over 50% of the fruit surface, with tissue rot and dense white mycelia, matching field symptoms. Control fruits remained asymptomatic. The pathogen was successfully re-isolated from 100% of symptomatic fruits, and the re-isolated fungus was morphologically and molecularly identical to OCG-2, fulfilling Koch’s postulates. Several pathogens have been reported to cause husk black spot or fruit rot on macadamia, including Calonectria pentaseptata (Jiang et al. 2020a), Phytophthora heveae (Jiang et al. 2020b), Lasiodiplodia theobromae (Li X et al. 2022), and Colletotrichum fructicola (Li J et al. 2023), indicating a complex etiology of this disease. In this study, we confirmed F. proliferatum as a new causal agent of the disease by fulfilling Koch’s postulates. Previous studies have documented the potential for interspecific interactions among Fusarium species during plant colonization (Reyes et al. 2020; Qian et al. 2017; Tai et al. 2021). This raises the possibility that F. proliferatum may interact with other pathogens in macadamia orchards, either as a co-infecting agent or as a secondary colonizer of pre-damaged tissues. Clarifying such interactions will require further field surveys and co-inoculation experiments. To our knowledge, this is the first report of Fusarium proliferatum as a causal agent of husk black spot on macadamia. This finding provides a scientific basis for future studies on the epidemiology and management of this disease.