First report of Fusarium concentricum causing wilt on Podocarpus macrophyllus in China.

Abstract

Podocarpus macrophyllus (Thunb.) D. Don is used in many fields, including landscape, medicine, and forest interplanting. In July 2019, shoot blight was observed on P. macrophyllus at three nurseries in Harbin, China. Approximately 15% of plants had symptoms of the disease, which included rapid, synchronized death of leaves on individual branches. Eventually the whole plant wilted. Leaves and stems turned dark blue to brown. Ten infected vascular tissue samples from 10 individual plants were surface-disinfested in 0.5% NaOCl for 5 min, rinsed 3 times in sterile distilled water, and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 26°C. Six similar fungal isolates from ten samples were isolated and subcultured. Single-conidium isolates were generated with methods reported previously (Leslie and Summerell 2006). Colonies on PDA consisted of densely floccose aerial hyphae with light yellow and pinkish pigments. Microconidia were oval to obovoid or allantoid, 3.8 to 11.8 μm in length and 2.8 to 4.6 μm in width, mostly non-septate on carnationleafagar (CLA). Macroconidia were naviculate-to-fusiform slender, 24.9 to 57.2 μm in length and 2.8 to 4.5 μm in width with 3- to 5- septate, with a beaked apical cell and a foot-shaped basal cell. According to these morphological characteristics, all isolates were identified as Fusarium spp. (Aoki et al. 2001 ). Genomic DNA was extracted from a representative isolate LHS1. Theinternal transcribed spacer regions (ITS), translation elongation factor 1-alpha gene (TEF-1ɑ)andβ-tubulin (TUB2) gene were amplified using the primers ITS1 and ITS4 (Yin et al. 2012)，EF1-728F/EF1-986R (Carbone and Kohn 1999)and T1/Bt2b (Glass and Donaldson 1995), respectively. DNA sequences of LHS1 were deposited in GenBank (accession nos. MT914496 for ITS, MT920920 for TEF-1ɑ and MT920921 for TUB2, respectively). MegaBLAST analysis of the ITS, TEF-1a, and TUB2 sequences indicated 100%, 97.7% and 100% similarity withFusarium concentricum isolate CBS 450.97 (accession no. MH862659.1 for ITS, MT010992.1 for TEF-1a, and MT011040.1 for TUB2, respectively). To determine pathogenicity, P. macrophyllus plants were grown in 10-cm pots containing a commercial potting mix (five plants/pot). At the 10 to 12 leaf stage, 10 healthy plants (2 pots) were inoculated by spraying 5 ml of a conidial suspension (4×106spores/ml) onto every plant. Ten plants treated with sterile distilled water served as a control. Thetestwasrepeatedtwice. All plants were placed in a humidity chamber (>95% RH, 26℃) for 48 h after inoculation and then transferred to a greenhouse at 22/28°C (night/day). All inoculated wilted with leaves and stems turning dark blue to brown 15 days after inoculation. No symptoms were observed on the control plants. The fungus was re-isolated and confirmed to be F. concentricum according to morphological characteristics and molecular identification. To our knowledge, this is the first report of F. concentricum on P. macrophyllus in world. The disease caused a large number of plants to wilt and die, seriously impacting the ability of the horticulture industry to produce P. macrophyllus. Although this pathogen causes leaf and shoot blight symptoms, it is not clear if the pathogen is also a vascular wilt disease. The occurrence of the new disease caused by F. concentricum highlights the importance of developing management strategiestoprotect P. macrophyllus.