Walnut (Juglans regia) is a deciduous tree of the Juglandaceae family, widely cultivated in China, and provides value in a variety of ways, including the usage of the wood and nuts, and offers substantial economic, social, and environmental advantages (Wang et al, 2017). Nevertheless, a fungal disease of causing walnut trunk rot was observed in approximately 30% of 50 counted ten-year-old J. regia in Chongzhou City (30°33'34″N, 103°38'35″E, 513 m), Sichuan Province, China, and this disease has greatly delete healthy growth of walnut. The infected bark exhibited purple necrotic lesions, and the sick parts were surrounded by water-soaked plaques. From 10 trunks of the 10 diseased trees, 20 isolated fungal colonies were the same. The ascospores placed in 60 mm plates were almost entirely covered with mycelium within 8 days, colonies on the PDA changed from initial pale to white, ad then turned yellowish to light orange or rosy to yellow-brown (25℃, 90% relative humidity, 12-h photoperiod). On the host, Ectostromata were immersed to erumpent, globose to subglobose, purple and brown, and measured 0.6 - 4.5 × 0.3 - 2.8 mm (x̄ = 2.6 × 1.6 mm, n = 40); Ascomata were flask-shaped to subglobose, dark brown, and measured 0.1 - 0.6 × 0.1 - 0.4 mm (x̄ = 0.35 × 0.25 mm, n = 40); Asci were numerous, cylindrical to subclavate, contained 8 uniseriate ascospores, and measured 80 - 150 × 10 - 20 μm (x̄ = 115 × 15 μm, n = 40), and Ascospores were ellipsoid, 2-celled, dark brown to black, plump or attenuated towards, apices with 1 large drop per cell, and measured 14 - 20 × 6.5 - 9 μm (x̄ = 17 × 7.8 μm, n = 40). These morphological characteristics are consistent with the species Myrmaecium fulvopruinatum (Berk.) Jaklitsch & Voglmayr (Jaklitsch et al. 2015). The genomic DNA of a representative isolate SICAUCC 22-0148 was extracted. The ITS, LSU region, tef1-α, rpb2 genes region were amplified using the primer pairs ITS1/ITS4 primers (White et al. 1990), LR0R/LR5 (Moncalvo et al. 1995), EF1-688F/986R (Alves et al. 2008), fRPB2-5f/fRPB2-7cr (Liu et al. 1999), respectively. The sequences were deposited in NCBI with accession numbers ON287043 (ITS), ON287044 (LSU), ON315870 (tef1-α), and ON315871 (rpb2), rspectively, which showed 99.8, 99.8, 98.1, and 98.5% identities with M. fulvopruinatum CBS 139057 holotype (accession numbers KP687858, KP687858, KP688027, and KP687933 respectively). Based on the analyses of phylogenies and morphologies, the isolates were identified as M. fulvopruinatum. The pathogenicity of SICAUCC 22-0148 was tested by inoculating surface-sterilized trunk wounds of four-year-old trees of J. regia with a mycelial plug (Desai et al. 2019). Sterile PDA plugs were used as controls. Wounds were covered with a film, to ensure humidity and prevent contamination. Each inoculation was repeated twice and included two plants, control and inoculated. A month later, the symptoms observed on inoculated trunks were similar to those in the wild, and M. fulvopruinatum was re-isolated from the inoculated trunk, confirming Koch's postulates. Previous research has reported M. fulvopruinatum as an important fungal species that cause canker delete symptoms on Chinese sweet chestnut in China (Jiang et al. 2018). We carried the taxonomy work of the fungi that caused trunk rot on walnut, and this is the first time that M. fulvopruinatum has been linked to walnut trunk rot on J. regia. Trunk rot of walnut will not only cause weakening of trees, but also affect the yield and quality of walnuts, bringing huge economic losses. This study was supported by the Sichuan Science and Technology Program under Grant 2022NSFSC1011. References: Alves, A., et al. 2008. Fungal Diversity 28:1-13. Desai, D.D., et al. 2019. International Journal of Economic Plants 6:147-149. Jaklitsch., W.M., et al. 2015. Fungal Diversity 73(1):159-202. Jiang, N., et al. 2018. Mycosphere 9(6):1268-1289. Liu, Y.L., et al. 1999. Mol Biol Evol 16:1799-1808. Moncalvo, J.M., et al. 1995. Mycologia 87:223-238. Wang, Q.H., et al. 2017. Australasian Plant Pathology 46:585-595. White, T.J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.
Keywords: Causal Agent; Fungi; Juglans regia; Pathogen detection; Subject Areas.