Ustilaginoidea virens is a flower-infecting fungus that forms false smut balls in rice panicle. Rice false smut has long been considered a minor disease, but recently it occurred frequently and emerged as a major disease in rice production. In vitro co-cultivation of U. virens strain with young rice panicles showed that U. virens enters inside of spikelets from the apex and then grows downward to infect floral organs. In response to U. virens infection, rice host exhibits elevated ROS accumulation and enhanced callose deposition. The secreted compounds of U. virens can suppress rice pollen germination. Examination of sectioning slides of freshly collected smut balls demonstrated that both pistil and stamens of rice flower are infected by U. virens, hyphae degraded the contents of the pollen cells, and also invaded the filaments. In addition, U. virens entered rice ovary through the thin-walled papillary cells of the stigma, then decomposed the integuments and infected the ovary. The invaded pathogen could not penetrate the epidermis and other layers of the ovary. Transverse section of the pedicel just below the smut balls showed that there were no fungal hyphae observed in the vascular bundles of the pedicel, implicating that U. virens is not a systemic flower-infecting fungus.
Rice false smut is a severe and widespread disease in major rice-growing areas of Asia, Africa and America [1-4]. It has been in the Unite States since at least 1906 [
A successful infection is indicated by the appearance of smut balls in rice panicle, which usually takes about 20 days after the infection to occur. Rice false smut disease causes yield loss and reduces grain quality. The yield losses have been attributed to not only the smut ball incidence but also the chaffiness and reduction in 1000- grain weight [
The natural infection process of U. virens remains unclear, which hampers studies on the disease. However, several research groups have reported that the artificial inoculation method can be used to evaluate disease index of fungal infection in greenhouse [9-11]. The artificial inoculation experiments demonstrated that the late booting stage is the critical period for U. virens infection. The artificial inoculation method uses the mixture of conidia and hyphae produced in liquid culture to inject the leaf sheath of flag leaf at the late booting stage, leading to a much higher disease index and even more reproducibility than the field trial.
In contact with plants, pathogens can trigger an array of reactions deployed in plants to prevent pathogens’ invasion, many of which appear to involve the production of reactive oxygen species (ROS) [12,13]. High levels of ROS are often accumulated rapidly after recognition of the pathogen [14,15]. ROS have been implicated in playing a vital role in plant defenses against pathogen invasion not only in direct antimicrobial roles but also in cellular signaling associated with the induction of defense gene expression [
The frequent occurrence of rice false smut makes it a serious problem in rice production recently, but the progress in understanding the routes of fungal infection was much slower than we expected. Knowing fungal infection process is critical for exploring the relationship between U. virens and rice host, also it is important to control the occurrence of the disease in rice production. Here, we monitored the early processes of fungal infection and detected the ROS levels and callose deposition in rice floral organs upon the recognition of U. virens infection. We also examined the invasion sites at the late stage of U. virens infection and provided evidence to show degradation of the integuments of ovary may be the critical strategy for U. virens to proliferate in the infected rice spikelets.
Rice variety Huangxiuzan was grown in the experimental station of Plant Protection Institute, Guangdong Academy of Agricultural Science, Guangdong Province, China. The experiment field was protected by a metal net, and there was an automatic water-spraying system underneath the net and above the field, this system sprayed mistlike water for 30 seconds per hour from 10 o’clock in the morning to 4 o’clock in the afternoon to increase humidity in the atmosphere during the infection period from artificial inoculation of rice plants with U. virens to emergence of the smut balls on rice panicles. PSB (potato and sucrose broth) medium was used to culture U. virens strain for inoculation. U. virens was cultured at 28˚C for 10 days, the whole culture was stirred three times in a stirrer, each for 15 sec, to break down hyphae to small pieces, the resultant mixture of hyphae and spores were used to inoculate rice panicles. We used a syringe with needle to inoculate rice plants at booting stage, each plant was injected with 2 - 4 ml of inoculum until the liquid flooded out from the top of leaf sheath. Smut balls appeared in rice panicles about 20 days after inoculation.
U. virens was cultured in PSB medium at 28˚C for 8 days, the culture was filtered with three-layer of lens papers to exclude hyphae, U. virens conidia were collected and adjusted to the concentration of 1 × 107 conidia per milliliter. Panicles at booting stage were peeled out and rinsed in the filter-treated conidia solution for 10 minutes. After the treatment, the panicles were cultured in 1/2 MS medium for 5 days, spikelets were used to observe conidium germination and examine the early infection process of U. virens.
U. virens-infected spikelets were opened carefully under a dissection microscope, the processes of hyphae infection of the anthers and stigma were observed under stereomicroscope (Zeiss Stemi SV11, Carl Zeiss,Germany) and corresponding images were taken. Anthers and stigma from in vitro U. virens-infected spikelets were pretreated and mounted to a metal stub (10 mm in diameter). The specimens were sputter-coated with gold particles (approximately 30 nm in thickness) and examined with a scanning electron microscope (JSM-6360 LV, JEOL Ltd, Japan).
Smut balls collected from the field were first fixed in FAA (Formaldehyde acetic acid ethanol) solution for 24 hours. After several times of dehydration and rehydration, the specimens were embedded in epoxy resin and cut with a glass knife into semi-thin sections. The semi-thin sections were carried out aniline blue staining according to the procedure described by Hood and Shew [
To detect ROS production on the rice flower organs during U. virens infection, spikelets from infected panicles and uninfected control panicles were stained with 3, 3’- Diaminobenzidine tetrahydrochloride solution (1 mg of DAB-HCl per 1mL of water) for 3 h at room temperature. The samples were destained in ethanol:chloroform (4:1) and then kept in the dark in 60% glycerol until examination by light microscopy.
Callose deposition was monitored by potassium hydroxide-aniline blue staining. Briefly, the materials were vacuum infiltrated in 1 M potassium hydroxide for 2 h at room temperature, followed by staining with 0.05% (w/v) aniline blue in water. The stained materials were placed in glass slides and examined by means of epifluorescence microscopy (Leica DMRBE, Switzerland).
We used freshly cultured U. virens to inoculate rice panicles at the late booting stage, the concentration of inoculum was 107 conidia per milliliter (
Rice panicles were collected from U. virens infected plants at day 3 after infection, the panicles were immersed in a solution of 3, 3’-diaminobenzidine tetrahydrochloride (DAB-HCl) for 3 hours at room temperature, after destained in ethanol:chloroform solution, rice flower organs were observed under microscope to exam-
ine ROS accumulation. DAB forms a brown polymerization product in the presence of ROS. Flower organs from U. virens-infected panicles showed enhanced brown oxidized DAB precipitates (Figures 2(a)-(c)), in contrast, the flower organs from the control plants did not exhibit brown color (Figures 2(d)-(f)). We cut the ovaries from both the U. virens-infected and the control plants and examined the DAB oxidation under microscope, the ovaries from U. virens-infected plants displayed more DAB oxidation products than those from uninfected plants (Figures 2(a), (d)), especially, the base of the infected ovary showed the highest level of oxidation, indicating more ROS produced at this site. Probably, the base of the ovary is the place where the fungus obtains water and nutrients from host plant.
The stigma from U. virens-infected plants showed enhanced accumulation of ROS as evidenced by the brown DAB oxidation, whereas the stigma of the control plants did not show brown color (Figures 2(b), (e)). In addi-
tion, the anthers from U. virens-infected plants had higher levels of DAB oxidation than those of the uninfected plants (Figures 2(c), (f)), indicating that U. virens infection induced high levels of ROS accumulation at the places it invaded.
Callose plays important roles in response to multiple biotic and abiotic stresses, and can be induced upon the recognition of pathogen infection. We collected rice panicles at day 5 after U. virens inoculation to clarify callose deposition. Flower organs from U. virens infected plants were first recorded under bright light of microscope (Figures 2(g), (j) and (m)), and then stained with aniline blue to observe callose deposition. The infected rice flower including anthers, stigma and ovary, showed bright aniline blue staining (
Booting stage is the critical time for U. virens infection. In transverse sections of anthers from U. virens infected plants, the pollen mother cells (PMC), both the cell wall and the contents, were slightly stained by aniline blue (
U. virens secretes compounds including ustiloxins into media, and ustiloxins are toxic to humans and livestock. To verify the effects of secreted compounds on rice pollen development, we used the supernatant of old-cultured U. virens to treat rice pollen grains to investigate the germination rate. Rice pollen grains were first treated with germination solution (15% sucrose, 20 mg/L borate and 40 mg/L CaCl2), after treatment for 10 min, about 75% of pollen grains germinated (
grains were selected and the pollen tube length was recorded (
To investigate the routes of U. virens infection and analyze the infection effects on rice flower development, we performed semi-thin sectioning of smut balls. The crosssection showed the stigma with two branches were enclosed by hyphae (
In transverse section of the anthers embedded in the mycelia of smut balls, four locules of an anther were still in shape and visible, but the contents were decomposed and replaced with hyphae (
Rice ovary contains an ovule consisting of the nucellus and an embryo sac containing an egg cell and a central cell. The nucellus is covered by inner and outer integuments, each typically with two layers of cells (
We did serial cross-sectioning of the smut balls at different positions, in transverse section of mid-region of the ovary persisted in the smut ball and wrapped by mycelia, hyphae were observed in the ovary. The intruded hyphae digested the integuments and grew around the nucellus at the position of integuments without infecting the ovular vascular cells (
Rice spikelet is supported by the pedicel that connects to the main stem of the panicle. Transverse section of the pedicel from the control rice plant showed normal vascular bundle (phloem and xylem) and parenchyma (
infected by U. virens and formed smut ball, in this case, some changes to the cells of the pedicel should occur, in other words, hyphae would cause the xylem or phloem tissues of the infected rachilla to become at least partially dysfunctional, and the traces of hyphae should be observed. We carried out cross-sectioning of the pedicel of the spikelet with smut ball at the position just right below the rudimentary glumes. In transverse section, the phloem, xylem and the parenchyma cells were clearly observed and well organized, we did not observe any trace of hyphae in either the vascular tissues or the thin-walled parenchyma cells (
Global agricultural change has brought about many improvements to rice production, including irrigation changes, high-yield variety utilization and heavy application of fertilizers [3,25]. Although these changes have led to a significant increase in rice yield, they also provide opportunities to pathogens that may cause new diseases or enhance the severity of the existed diseases. Recently, rice false smut has emerged as one of the major diseases worldwide; its impact on rice production has increased in importance over time and attracted much concern of researchers. However, details of the infection process and the relationship between U. virens and rice host are scarce.
The routes of U. virens infection have been debated for many years and the infection process of this fungus is still poorly understood. Recently, Ashizawa et al. [
Colonized inside of the spikelets, U. virens hyphae infect pistil and stamens including filaments. Tang et al. [
It is well known that U. virens secretes mycotoxins such as ustilaginoidins and ustiloxins [29-31], which are toxic to humans and live stocks. Pathogen infection caused the developmental arrestment and content decomposition of the pollen mother cells. In consequence, no mature pollen grains were produced. Furthermore, the supernatant of pathogen liquid culture inhibited pollen grain germination and suppressed pollen tube elongation. These inhibitory effects are probably due to the functions of the secreted compounds. Previous research reported that ustiloxins are able to not only inhibit tubulin polymerization in a concentration-dependent manner but also induce the depolymerization of preformed microtubules [
Flower-infecting fungi can initiate systemic invasion through the apical meristems. This group of fungi includes many smut fungi that are either seedborne such as Ustilago nuda f.sp. bordei or capable of entering through vegetative tissues such as the anther smut pathogen Microbotryum violaceum [
This research was supported by grants from the Important Direction Research of Chinese Academy of Sciences Knowledge Innovation Project, no. KSCX2-EW-N-06, and the Hundred Talent Program of Chinese Academy of Sciences to J.X.L.