The wild-type U. virens UV-PD11 strain and the green fluorescent protein-labeled U. virens gfp-z1, gfp-z2, and gfp-z3 strains were stored at the Institute of Crop Protection, Guizhou University.

Potato sucrose agar medium (PSA) and potato sucrose medium (PSB) were prepared. A total of 200 g of fresh potatoes were cut into thin slices, boiled for 30 min, filtered with three layers of gauze, and supplemented with 20 g of sucrose and 20 g of agar powder in a beaker with 1,000 ml of purified water. The medium were sterilized using a high-pressure steam sterilization pot (Shanghai ShenAn Medical Equipment Factory, Shanghai, China) at 121°C for 30 min. The procedure for preparing PSB medium was the same as that for preparing PSA medium, except that agar powder was not added.

The UV-PD11, gfp-z1, gfp-z2, and gfp-z3 strains were each inoculated into PSA medium. After 10 days, the colony diameter was recorded and the average growth rate was calculated using the following Equation 1.

The seeds of the indica rice variety ‘Jinyou 785’ (Guizhou Jinnong Technology Co., Ltd., Guizhou, China) were disinfected with 75% ethanol (Shandong Chituma Medical Treatment Technology Co., Ltd., Shandong, China) and rinsed thoroughly with sterile water. Field management was carried out according to conventional rice cultivation measures, and fungicides were not used in the experimental area.

The panicle sheath instillation was performed as follows: 5 cm PSA medium of U. virens was inoculated into 100 ml of PSB medium and cultured under shaking at 28°C and 130 rpm for 6 days. The mycelium of U. virens was ground and filtered with three layers of gauze to obtain hypha spore suspension. The filtrate was diluted with PSB medium to a final concentration of 1 × 10⁶ spores/ml hypha spore suspension as the inoculum. Inoculation was performed at stages 6–8 of young panicle differentiation of rice, with each inoculum having a volume of 3 ml. The filtrate was dripped into the panicle sheath of rice at a rate of 40 drops per minute. The rice panicle sheath was wrapped with a plastic film for heat and moisture preservation, which was removed after 3 days.

The rice variety ‘Jinyou 785’ was inoculated with the hypha spore suspension of the strains gfp-z1, gfp-z2, gfp-z3, and UV-PD11 using panicle sheath instillation. The samples were taken at 3, 6, 8, 12, 15, and 18 days, and the spikelets on the rice panicles were cut. A single spikelet was cut with a double-edged blade and observed under a fluorescence microscope. The fluorescence light source of the microscope was turned on, and the excitation block was turned to the blue light source (wavelength 450–480 nm) to observe the colonization and expansion of U. virens in the rice floral organs (filament, stigma, style, and ovary). Each treatment included 7 rice plants and 3 replicates, with a total of 21 plants. The average rate of diseased panicles was calculated using Equation 2, and the significance was analyzed.

P_d, diseased panicle rate; N_d, number of diseased panicles; N_t, total number of panicles.

The bright green fluorescence of the rice false smut balls could be detected under a fluorescence microscope (Ningbo Yongxin Optical Co., Ltd., Ningbo, China), which was selected as the isolation material. The rice false smut balls were clamped with sterile tweezers, and the chlamydospore powder was knocked down to the surface of the rifampicin-containing PSA medium by tapping the tweezers. The culture medium was cultured in the dark at a constant temperature (28°C) for 3–4 days, and then white-spot colonies were observed on the surface of the PSA culture medium. These colonies were picked out and placed in fresh PSA medium after 12 days. The morphology of the colonies was observed, and the fluorescence intensity was detected under a fluorescence microscope. The labeled hyphae of U. virens were picked out with a sterile inoculation needle and gently applied to the center of the groove of the glass slide. After the cover glass was covered, water around the cover glass was removed with absorbent paper and observed under a fluorescence microscope. A high-pressure mercury lamp was turned on and preheated for 15 min before performing the observations. U. virens was observed under a low-power bright field and with the bright-field light source turned off. The fluorescent light source of the fluorescence microscope was then turned on and the blue light source (wavelength 450–480 nm) was selected using the excitation block dial.

DPS software was used to analyze the variance of the experimental results. Here, p < 0.05 was considered to reflect a significant difference, whereas p < 0.01 was considered to reflect a very significant difference.

The morphological characteristics and growth rates of the wild-type strain UV-PD11 and green fluorescent protein-labeled strains gfp-z1, gfp-z2, and gfp-z3 were measured to determine whether there were differences in the biological characteristics. The colony of UV-PD11 was generally round in shape with dense white hyphae, a raised center resembling a hat, and bright yellow color at the back, which turned dark green at the late stage of culture. The colonies of the strains gfp-z1, gfp-z2, and gfp-z3 were round overall and the hyphae were dense. The colony back of the green fluorescent protein-labeled strains was bright yellow, which were similar to those of the U. virens strain UV-PD11. There were no significant difference in the growth rate among the strains gfp-z1, gfp-z2, gfp-z3, and UV-PD11, indicating that there was no significant differences in morphology and growth rate between strains labeled with green fluorescent protein and wild-type strains (Supplementary Figure 1).

The growth of the apical hyphae of the strains gfp-z1, gfp-z2, gfp-z3, and UV-PD11 was compared to determine the morphological differences between the obtained transformants and the wild-type strain UV-PD11. The results showed that the hyphae of the strains gfp-z1, gfp-z2, gfp-z3, and UV-PD11 were pale yellow. There was no significant difference in hyphal thickness and diaphragm number of U. virens, and many branches were bifurcated (Supplementary Figure 2). Therefore, there was no significant difference in hyphal morphology between the strains gfp-z1, gfp-z2, and gfp-z3 and the wild-type strain UV-PD11.

To explore the changes in the pathogenicity of U. virens, the rice variety ‘Jinyou 785’ was inoculated with the strains gfp-z1, gfp-z2, gfp-z3, and UV-PD11 using panicle sheath instillation. The average rate of diseased panicles was determined, and the significance of its differences among the strains was analyzed. The average rates of infected panicles of the strains gfp-z1, gfp-z2, gfp-z3, and UV-PD11 were 90.48, 80.95, 85.71, and 90.48%, respectively. There was no significant difference in pathogenicity among the strains gfp-z1, gfp-z2, gfp-z3, and UV-PD11 (Supplementary Figure 3).

In the horizontal and vertical sections of the rice false smut balls, the anthers and stigma of rice were clearly visible. The anthers were butterfly-shaped and the stigma was radial, all of which were tightly embedded by the white hyphae of U. virens. Under the fluorescence microscope, strong fluorescence could be detected in the infected area. The stigma and anther were tightly wound by U. virens, and the stigma was brown when the rice false smut ball was transected (Figure 6).

U. virens inoculated rice spikelets and uninoculated rice spikelets were sampled, observed under a stereoscope, and photographed to record the formation process of rice false smut balls. The entire floral organ of rice was wrapped by U. virens, which led to an inability of the rice to bloom normally. A massive structure (floral organ – U. virens complex) was observed under the stereoscope, which gradually expanded and finally opened the glumes, forming rice false smut balls that were two to three times larger than those observed in normal uninfected rice. The rice false smut ball was bright yellow at the early stage, which then turned into yellow-green and dark green at the later stage. Meanwhile, the chlamydospore on the surface of the rice false smut balls changed from a nondormant to a dormant state. The floral organs of normal rice grains were scattered, the anthers, filaments, and stigma were clearly visible under a stereomicroscope. At the later stage, the filaments stretched, the glume expanded and then extended outward, the anthers burst, and the pollen fell off and scattered randomly on floral organs, such as rice stigma and filaments. After pollination, the glume closed and the successfully pollinated ovary gradually expanded and filled (Figure 7).

The rice false smut balls with green fluorescence were isolated to obtain a strain of U. virens. As shown in Figure 8, the colonies of the obtained U. virens were white at the early stage and pale yellow at the late stage. The back of the colony was orange and the center of the colony was dark green, which were the typical characteristics of U. virens at the late stage. Observation of the strain under a fluorescence microscope revealed that the colony, conidia, and hyphae of the strain gfp-R1 emitted bright fluorescence. The obtained gene encoding green fluorescent protein was stably inherited and expressed in U. virens. This indicated that rice false smut produced via inoculation with the strain labeled by green fluorescent protein could be preserved, so that the U. virens with strong pathogenicity and expressing gfp could still be obtained at the later stage.

Rice false smut could be produced through inoculation of rice via panicle sheath instillation at stages 6–8 of rice young panicle differentiation (as shown in Figure 9), which could potentially explain the natural infection pathway of U. virens in the field. Based on findings in this study, a schematic of the process of U. virens infection was constructed (Figure 9). The conidia of U. virens were produced from sclerotia or chlamydospore, which became an important infection source. In the field, during the sixth to eighth stages of rice young panicle differentiation, the conidia of U. virens could enter the panicle bracts through the panicle sheath gap via rain or dew. The conidia of U. virens germinated on the surface of rice hulls to produce hyphae, which entered the glumes through the gap between the palea and lemma. U. virens randomly attached to the surface of the floral organs, causing pollen abortion, and absorbed nutrients from the rice floral organs to form rice false smut balls.