First report of a new species, Chrysofolia galloides sp.nov., causing black spots on gallnuts of Rhus punjabensis var. sinica in China

Abstract

In June 2021 and 2022, 30-40% ensiform galls of Rhus punjabensis var. sinica (Diels) Rehd. et Wils. showing similar black spots were observed at a same woodland in Zhuyu town (E 107"42', N 32"10', altitude 560 m), Wanyuan, Southwest China. The lesions were usually dotted, rounded to irregular black or blackish brown spots, and scattered to gather into patches on the gall surfaces; the central areas of the lesions were often depressed. The infected galls often grow slowly or even fall off early. Fresh symptomatic galls were randomly collected in June 2021 (9 galls) and 2022 (13 galls). Tissues from the infected galls (ca. 3×5 mm) were surface-disinfected by 75% ethyl alcohol and 10% sodium hypochlorite (NaOCl), then plated on potato dextrose agar (PDA) at 25°C in the dark for six days. Nine isolates from different galls showed similar colony morphology. Similar dominant fungal colonies were isolated from 44.0% and 38.5% of the infected galls in 2021 and 2022, respectively. After sub-culturing of hyphal tips on PDA and oat meal agar (OA)，the colonies showed cottony-felty, annular, whitish to grey-brown after 10 days. Four isolates, IFRDCC1022 and IFRDCC1023 from 2021, and IFRDCC1024 and IFRDCC1025 from 2022 were randomly selected for molecular and morphological evaluations. Selected isolates were cultured on OA and incubated at 21°C at a 12 h/12 h day/night regime to induce sporulation (Jiang et al. 2020). Pycnidia (up to 500 µm diam.) developed within six days or longer. Conidiogenous cells lined the inner cavity of base, which were hyaline, occasionally brownish, ampulliform, 6-12 × 2-4.5 µm. Conidia were solitary, hyaline, guttulate, elliptic, sometimes subcylindric or ovoid, straight to curved, aseptate, (3.5-) 4-6.5 (-8) × 2-3.5 (-4) µm (n=60). Respective DNA fragments were amplified and sequenced with primers ITS1/ITS4 for ITS, LR0R/LR5 for LSU, EF1-728F (Carbone and Kohn 1999) /EF-2 (O’Donnell et al. 1998) for tef1-α, and Bt2a/ Bt2b for tub2 (Glass and Donaldson 1995). The obtained sequences were deposited in GenBank (accession Nos. ITS: OR363211 - OR363214, LSU: OR363215 - OR363218, tef1-α: OR344509 - OR344512, and tub2: OR344513 - OR344516). The morphological characteristics of the new isolates were consistent with the genus Chrysofolia (Crous et al. 2015). The conidiogenous cells (6-12 × 2-4.5 µm) are larger than those of C. colombiana Crous, Rodas & M.J. Wingf. 2015 (5–8 × 2–3 μm) and C. barringtoniae Suwannar., Kumla & Lumyong 2016 (3–7.5 × 2–3 μm), while shorter and wider than those of C. coriariae S.Y. Wang, Yong Wang bis, and Y. Li 2022 (5–20 × 1–3.5 μm). Maximum likelihood (ML), Maximum parsimony (MP) and Bayesian phylogenetic trees generated from the datasets all showed similar topologies, four strains formed a well-supported clade (BS = 99-100%, PP=0.99-1) in Chrysofolia genus, and clearly separated from the other three Chrysofolia spp. and clustered as sister to C. coriariae with high supported values (BS = 97-100%, PP=0.97-1). Based on morphological characteristics and DNA phylogeny, the new isolates were described as a new species, Chrysofolia galloides T. Ma & Z. X. Yang, sp. nov. (Mycobank: MB850312). The two isolates from 2021 (IFRDCC1022 and IFRDCC1023) were randomly selected for pathogenicity tests. Mycelial plugs (ca. 6×6 mm) of a two-week-old colony were placed onto the surface of wounded and unwounded healthy ensiform galls with six or seven replicates. Sterile PDA agar plugs were used as controls. The inoculated galls were incubated at 25°C in sterile plastic containers (21.4×14.0×5.0 cm) lined with wet filter paper for high humidity. The typical symptom appeared within 3 days on 83.3-100% wounded galls, while the unwounded galls and the controls remained asymptomatic. All wounded galls and 20.0-85.7% of unwounded galls showed symptoms within 5 days, while the controls remained asymptomatic. The fungus was reisolated from the lesions and showed identical morphology and 100% sequence similarity of ITS, LSU, tef1-α and tub2 with the initial isolates. No fungus was isolated from the controls. Neofusicoccum parvum is the first fungal pathogen demonstrated to induce brown spots on Chinese gallnuts (Ma and Yang 2022). Therefore, C. galloides is the second fungus confirmed to cause black spots lesions on gallnuts. These findings provide foundations for the comprehensive management of gall production, which have significant economical implication for Chinese gallnut production.