Infection And Developmental Processes Of Ustilaginoidea Virens In Rice

Rice false smut, which caused by Ustilaginoidea virens, is a ubiquitous disease in rice planting areas worldwide. Given that rice is the stable food for half of the world’s population, false smut presents a constant threat to global food supplies. The development of durable and environmentally friendly strategies for the control of rice false smut disease depends on a better understanding of the developmental process of the disease. The current study focused on resolving these issues and the main results were as followes.The effects of U. virens and the filtrate on rice seed germination and seedling growth were evaluated. Results showed that the chlamydospore was a powerful dose-dependent inhibitor of rice seed germination and seedling growth. However, no significant difference observed on seed germination and seedling growth when the seeds treated by conidia or hyphae, respectively. There were no significant effect on seed germination and seedling growth for both WX98and GY129cultivars, when seed treated by the culture filtrate of U. virens. Two pairs of specific primers, UV-F3/UV-R3and UV279-F/UV693-R, were designed and screened for U. virens detection. The primers of UV279-F/UV693-R showed high sensitivity for detection of U. virens by polymerase chain reaction (PCR), and the limit was25pg/μL of U. virens DNA sample. Based on the primers, a stable and rapid PCR detection method was developed for detection of U. virens from rice seeds. In artificially infected/contaminated seeds, the detection sensitivity was3.25×103chlamydospores per seed (the quantity of chlamydospores adhere to the surface of seed) or20μg of hyphae per seed (the weight of hyphae mixed with seed), respectively. Seventy-four seed samples from different rice planting regions in China were detected by PCR, and the results showed that39.2%and13.5%of seed sapmles were U. virens positive by seed-washing method (detection of U. virens on the seed surface) and seed-grinding method (detection of U. virens in the seed) by PCR, respectively.An inoculation method was designed in greenhouse. The method combined the cultivation of the main culm of rice until the plants reached the booting stage (about7days before heading), with a quicker method to produce a quantity of hyphae-conidia that were then injected into the leaf sheath. The optimum inoculation condition was1-2mL inoculum adjusted to a concentration of2×106conidia per milliliter with PS injected into the mid-point of rice panicles. After inoculation, all the rice plants were kept at26/32℃(night/day), covered with a plastic film and manually sprayed (15min) every eight hours to maintain the environment at90-95%relative humidity (RH). After5days the plastic film was removed and the plants were grown under normal greenhouse conditions at25-36℃and80-100%RH.The developmental processes of U. virens in rice panicles were characterized using a strain labeled by enhanced green fluorescent protein (EGFP). The collected spikelet samples were used to probe the infection progress by confocal laser scanning microscopy and scanning electron microscopy. The images showed that the primary colonization site of U. virens was at the base of the filaments in the inner spikelets infected by hyphae at24hours post inoculation (hpi). The accumulation of hyphae reached its highest level at168hpi, 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.In response to U. virens infection, the accumulation of reactive oxygen species (ROS) was significantly increased in rice. The infection of U. virens suppressed rice pollen germination and pistil development, and led to producing a large number of false smut balls and bleached grains. To detect the ROS in healthy, infected and bleached spikelets, H2O2was stained and visualized by exposuring to DAB. The absorbance of oxidized DAB at465nm was measured, and a significant increase in infected spikelets solution was detected compared to the healthy spikelets. After DAB treatment, brown oxidized DAB precipitates could be detected readily on the location of U. virens in infected spikelets but not on the mock-infected spikelets. DAB oxidation seemed to be associated with locations colonized by the fungus at spikelets.Presumably, both seed and soil polluted by pathogen could be the major primary infection sources of false smut in fields. The study was initiated to prove this issue. Results from CLSM observation, visual assessments and detection by polymerase chain reaction indicated that primary infection sources were still not fully understood.