Isolation, identification and artificial inoculation of Ustilago esculenta on Zizania latifolia

Abstract

The smut Ustilago esculenta is an endophytic fungus that invades Zizania latifolia (Jiaobai) and plays an important role in inducing the host stem to form a swollen gall. The aims of this study were to investigate isolation, identification and inoculation methods of U. esculenta from Jiaobai. The spore suspension culturing method was the best method of isolating U. esculenta, and yeast-like fungal strains and septate hyphal strains were obtained. The universal primers ITS1 and ITS4 amplified a ribosomal DNA spacer element from all tested strains, while specific primers for Lam16A gene generated a 442 bp product only with septate hyphae strains. De novo transcriptome assembly results showed that the yeast-like fungal strains did not match Ustilago, and genomic analysis suggested the septate hyphae strains were highly similar to known smut fungi. Therefore, the septate hyphae strains were identified as U. esculenta, which indicated that the ITS primers were not species-specific but that Lam16A-specific primers can distinguish U. esculenta accurately and easily. In vitro, U. esculenta grew quickly on PDA medium at 28 °C in darkness. Furthermore, an inoculation method was established to produce swollen stems by injecting a spore suspension of U. esculenta at OD600 = 2.5 into the basal culm internode of male Jiaobai.