Infection processes of Ustilaginoidea virens during artificial inoculation of rice panicles

Ustilaginoidea virens is a ubiquitous plant pathogen that causes rice false smut disease, one of the most destructive diseases of rice (Oryza sativa L.) production. However, data concerning the effect of inoculation on disease development and the infection process of this pathogen are not comprehensive. In this study, the developmental processes of U. virens in rice panicles were characterized using an enhanced green fluorescent protein (EGFP) labelled strain. A mixture of hyphae and conidia of U. virens was used to inoculate rice panicles by leaf sheath injection during the booting stage of rice plants grown in a greenhouse. The panicles were assessed to determine the relationship between artificial inoculation and disease occurrence. Increasing volumes of inocula (0.2, 0.5, 1, and 2 ml of a mixture of hyphae-fragment and 2 x 10(6) conidia/ml suspension) caused more severe infections, and small differences were also observed for the different inoculation sites at the base, apex and mid-point of rice panicles. The optimum inoculation condition was 1-2 ml inoculum injected into the mid-point of rice panicles. Spikelet samples were collected as the disease progressed and observed by confocal laser scanning microscopy and scanning electron microscopy. The images collected showed that the primary site of U. virens colonization was at the base of the filaments with the inner spikelets becoming infected by hyphae at 24 h post inoculation (hpi). The accumulation of hyphae reached its highest level at 168 hpi, before the rice heading stage, as the infection extended upward from basal filaments to the anther apex, and then enclosed all the floral organs to produce a velvety smut ball.