Introduction

False smut of rice, caused by Ustilaginoidea virens (Che.) Tak. (teleomorph: Villosiclava virens) (White et al. 2000; Ashizawa et al. 2010), is a serious disease of rice worldwide (Rush et al. 2000; Ahonsi and Adeoti 2002; Bischoff et al. 2004; Atia 2004; Tsuda et al. 2006; Zhou et al. 2008; Brooks et al. 2009; Ladhalakshmi et al. 2012). The pathogen produces both sexual ascospores and asexual chlamydospores in its life cycle. The teleomorph of U. virens had been named Claviceps virens (Sakurai ex Nakata) and Claviceps oryaze-sativae (Hashioka) because its teleomorphic characteristics are similar to those of Claviceps (Hashioka 1971). However, the molecular phylogenetic analysis, which based on both large subunit of the rRNA gene and acetaldehyde dehydrogenase gene sequences, revealed that members of Ustilaginoideae are distinct from teleomorphic genera of Clavicipitaceae and should be recognized as a monophyletic group within Hypocreales (Bischoff et al. 2004; Tanaka and Tanaka 2008). As a result, it was suggested Villosiclava virens as the new name for the teleomorph of U. virens (Tanaka et al. 2008), which was accepted and used in recent reports (Ashizawa et al. 2012; Fu et al. 2012; Tang et al. 2013). The typical symptoms of false smut appear in rice panicles at maturity, with diseased spikelets, developing into spore balls that have a velvety appearance. The colour of the spore balls is initially orange, becoming yellowish green or greenish black at maturity (Lee and Gunnell 1992). Aside from the loss of production, false smut is also a threat to food safety as the chlamydospores of U. virens produce mycotoxins (Koiso et al. 1992; Miyazaki et al. 2009; Shan et al. 2012). Given that rice is the staple food of half 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 depend on a better understanding of the developmental process of this pathogen, and the establishment of disease.

Although early research by Ikegami (1962) indicated that it was possible to infect plants by inoculating coleoptiles with spores at the earliest stages of seed germination, it was also found that the infection levels became very low after the coleoptiles reached 10 mm in length. Field observations and the results of more recent inoculation experiments, combined with histological studies of the smut ball, now suggest that the most likely route of U. virens infection is when rice plants are at the booting stage (Sonoda et al. 1997; Ashizawa and Kataoka 2005; Ashizawa et al. 2012). Furthermore, experiments using a mixture of conidia and hyphae from U. virens cultured in vitro demonstrated that serious cases of false smut could arise when the inoculation was made by injecting the panicles at the booting stage (Zhang et al. 2004), with up to 100 % of the panicles becoming infected. A similar method was utilized to assess the resistance of rice plants to false smut infection, where a conidial spore suspension was injected into the leaf sheaf of mature plants (Ashizawa et al. 2011).

The advent of genetic transformation has opened new possibilities for studying plant pathogen interactions and several techniques, including Agrobacterium mediated transformation (Zhang et al. 2006) and electroporation (Tanaka et al. 2011), have been developed for the transformation of U. virens. A recent study utilized a transgenic strain expressing green fluorescent protein gene (GFP) (Ashizawa et al. 2012) to observe the initial infection of rice panicles before heading. It was found that the hyphae of U. virens are able to invade the spikelet apices, via a small gap between the lemma and palea, and thereby colonize the other floral organs. Furthermore, a recent cytological study (Tang et al. 2013) indicated that the pathogen infected the filaments intercellularly and extended intercellularly along the filament base.

Despite these advances, our understanding of the infection strategy of U. virens remains fragmentary and the pattern of colonization and distribution of U. virens in artificially inoculated rice panicles requires further investigation. The relationship between inoculum and disease development is also poorly defined, and the disease cycle, which begins with the infection of plant tissues and culminates in the formation of false smut balls, has not been fully characterized. The current study was initiated to resolve these issues using a U. virens P3 isolate labelled with EGFP. The relationship between different inoculations, including inoculum load and inoculation site, and diseases occurrence were investigated, and the process of panicle invasion was observed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), which provided further insights into the complexity of the plant-pathogen interaction.

Materials and methods

Fungal material and culture conditions

The P3 strain of U. virens, which had been isolated from an infected rice spikelet by single spore isolation, and labeled with EGFP using the Agrobacterium-mediated transformation method (Fig. 1a), was obtained from Professor Liu (Institute of Plant Protection, Jiangsu Academy of Agricultural Science, People’s Republic of China). The EGFP labelled strain was equal to the wild type based on both its morphological characteristics and the results of pathogenicity tests (data not shown). The mixture of hyphae-fragment and conidia of EGFP-P3 was used as the inoculum. Although conidia alone can also cause infection, it was found that at equivalent conidia concentration the mixture of hyphae-fragment and conidia could cause more stable and severe symptoms which represented a reproducible system for inducing pathogenicity. Six mycelial plugs (5 mm) were taken from the edge of 15-day-old cultures grown on potato sucrose agar (PSA, which made from boiled extract of 200 g fresh potatoes, 20 g sucrose, and 20 g of agar per litre of distilled water) and placed into 100 ml flasks containing 50 ml of potato sucrose (PS, which made from boiled extract of 200 g fresh potatoes, 20 g of sucrose per litre of distilled water) liquid medium. The cultures were incubated in a rotary shaker (120 rpm) at 28 °C for 7 days. The hyphae and conidia were collected by filtration and centrifugation, respectively. The suspension of conidia was filtered and the conidia collected by centrifugation (3,000 × g, 5 min). The hyphae were then resuspended in the filtrate and pounded for 3 min with a JYL-C022 pounding machine, before the conidia were added back to the hyphae-fragment suspension. The resulting mixture was adjusted to a concentration of 2 × 106 conidia per millilitre with PS, and used for inoculation.

Fig. 1
figure 1

Spikelets of a susceptible host (YY9) infected with a strain of U. virens expressing EGFP: a Fluorescent hyphae and conidia viewed with CLSM, Bars = 50 μm; b–e, bright field illuminated stereomicroscopic images: b Uninoculated spikelet; c Inoculated spikelet showing conidia and hyphae on the glume surface at the point of inoculation (red arrow); d Inner structure of uninoculated spikelet; e Interior of spikelet containing fungal hyphae at 9 dpi; f–g SEM images of the surface of spikelets: f Uninoculated spikelet; g Hyphae and conidia (red arrow) on inoculated spikelet

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Plant materials and growth conditions

A hybrid rice cultivar, Yongyou 9 (YY9), known to be susceptible to false smut, was obtained from Professor Hu (State Key Lab. for Rice Biology, Zhejiang University, People’s Republic of China). A total of 480 rice seeds were surface sterilized in 75 % ethanol for 1 min and 1 % sodium hypochlorite (NaClO) for 5 min, and thoroughly rinsed in distilled water three times. The seeds were then planted in 150 × 200 × 50 cm pools filled with sterilized paddy soil. The experimental plants were grown in an air-conditioned greenhouse with temperatures ranging from 22 °C at night to a maximum of 36 °C during the day.

Inoculation and sampling

The panicles of the YY9 plants were inoculated at the booting stage (about 7 days before heading), by injecting the leaf sheath with the hyphae-conidia suspension. A range of inoculum volumes (0.2, 0.5, 1, 2 ml) were injected into three different sites on separate panicles, namely at the apex (Ap), the mid-point (M) and the base (Ba), as illustrated in Fig. 2 and Table 1. The number of 1, 2, 3, and 4 represent different inoculum volumes of 0.2, 0.5, 1, and 2 ml, respectively. Another panicle was selected for the microscopic investigation of the U. virens infection process, and injected with 2 ml inoculum at its mid-point. Sterile PS broth was used as a negative control for all the experiments. After inoculation, all the rice plants were kept at 26/32 °C (night/day), covered with a plastic film cover and manually sprayed (15 min) every 8 h to maintain the environment at 90–95 % relative humidity (RH). After 5 days the plastic film was removed and the plants grown under normal greenhouse conditions at 26–35 °C and 80–100 % RH.

Fig. 2
figure 2

Typical symptoms of false smut 15 dpi with U. virens at different sites on the panicle: a Inoculation sites on the panicle at booting stage: apex (Ap), mid-point (M) and base (Ba); b–d Top, ellipses indicate sites of injection; b–d Bottom, typical symptoms of U. virens observed on the corresponding rice panicles; e Top, black false smut balls exhibiting few chlamydospores formed at < 26 °C; e Bottom, yellow false smut balls with many chlamydospores formed at > 26 °C

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Table 1 Effect of different inoculation treatments on the severity of false smut infection (Cultivar:YY9)
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Histological observation and statistical analysis

Visual disease symptoms were assessed macroscopically at 21 dpi. The effect of the different inoculation sites and inoculum load was assessed by counting the number of diseased or bleached grains. Each sample consisted of three biological replicates taken in parallel, and each replicate was a pool of 36 panicles. To evaluate the reliability of the different inoculation treatments, the correlation coefficients of the YY9 data sets were calculated. All the data were subjected to analysis of variance and Duncan’s multiple range test (DMRT) at P = 0.05, using SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA).

Microscopic observation by CLSM and SEM

The spikelets from inoculated rice panicles were collected at 12, 24, 36, 84, 108, 134, 168, 192 and 216 hpi. Each time point sample consisted of three biological replicates taken in parallel, and each replicate was a pool of 20 spikelets. The spikelets were washed in sterile water, and the glume shells stripped with sharp tweezers. Spikelets exhibiting green fluorescence under the condition of CLSM were considered to be infected.

The CLSM was performed with a Nikon TE2000-E plus LSM5 PASCAL LASER scanning microscope equipped with an Argon LASER (Nikon D-eclipse C1, Japan). Image overlays of the two channels were captured as single optical sections (2-D) and a z-series of optical sections (3-D) using an excitation of 488 nm and an emission at 543 nm for EGFP. Red chlorophyll autofluorescence was visualized using an excitation of 543 nm emission filter. The resulting images were processed using the LSM 5 Pascal program (Carl Zeiss).

The SEM samples were first fixed with 2.5 % (v/v) glutaraldehyde in 50 mM phosphate buffer (pH 7.2) for 6–8 h at 4 °C, before being rinsed with the same buffer for 2 h, and then fixed in 1 % (w/v) osmium tetroxide in 50 mM phosphate buffer for 1 h. After dehydration in a graded acetone series, the samples were critical-point dried, mounted on stubs, sputter coated with gold-palladium, and viewed using a HITACHI S-3400N scanning electron microscope operating at 15 kV.

Results

Effect of inoculation site and inoculum load on disease severity

The experimental plants were assessed for U. virens infection at 21 dpi. All of the inoculated plants exhibited typical symptoms of false smut disease (Fig. 2), with the location of heaviest infection corresponding to the site of inoculation. Closer comparison of the infected panicles (Fig. 3) revealed that the disease severity increased with increasing inoculant load, with the higher levels of disease being observed when the inoculation volume was 1.0 ml or greater.

Fig. 3
figure 3

Panicles of a susceptible host (YY9) at 21 dpi with U. virens. Different samples were inoculated at various locations on the panicle, and with different volumes of inoculant. CK Non-inoculated, Ap1-4 apex inoculated, M1-4 mid-point inoculated, Ba1-4 base inoculated. The values 1, 2, 3, 4 indicate the different inoculum volumes (0.2, 0.5, 1, and 2 ml of a 2 × 106 conidia/ml suspension) respectively

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The diseased and bleached grains from each panicle were counted and the data subjected to statistical analysis (Table 1). The results revealed that an inoculation volume of 0.5 ml or greater significantly affected the number of heading panicles and also resulted in an increased number of diseased grains, the highest level being observed with the M3 treatment. The artificial inoculation also resulted in bleached grains, particularly with higher inoculant loads, the greatest number being 21.06 % for the Ba4 treatment. The ANOVA showed that there was also variation between the different inoculation sites, with the basal inoculation resulting in a greater number of diseased panicles at the lower volumes of inoculant (0.2 & 0.5 ml), the maximum being 83 % for Ba2, while at the higher volumes (1 ml & 2 ml) the mid-point inoculation produced the most severe symptoms with 30.69 % of the grains in treatment M3 being diseased compared with only 12.99 % for the Ap3 treatment. Although the number of bleached grains generally increased with the volume of inoculant, it is interesting to note that this was not true for the mid-point inoculation, where an increase of inoculant volume beyond 0.5 ml did not seem to result in the development of a greater number of bleached grains.

Infection process and colonization pathway

With the exception of the sparse white mycelium near the site of inoculation, stereomicroscopy (Fig. 1b–e) revealed little evidence of external symptoms of U. virens infection in intact spikelets at 9 dpi (Fig. 1c). However, when the spikelet was dissected the true extent of the infection was exposed with an extensive white mycelial mass enclosing most of the flower organs (Fig. 1e).

Although the EGFP labelled U. virens strain was only weakly fluorescent under UV light, it could readily be detected by CLSM (Fig. 1a). The flourescence of the EGFP labelled strain was used to monitor the progress of the infection (Fig. 4), and CLSM showed that the hyphae initially began to invade the inner spikelets at 24 hpi, and that the extent of the infection peaked at 168 hpi, with 86.7 % of the spikelets being infected. After this point the infection ceased to spread, possibly suggesting a transition from vegetative phase to a reproductive one.

Fig. 4
figure 4

Infection of U. virens in a susceptible host (YY9) at various times post inoculation. The samples for each time point consisted of three biological replicates taken in parallel, with each replicate being a pool of 20 spikelets. The 2 ml inoculum (EGFP-P3) was injected at the mid-point of the panicles and the degree of infection was assessed by fluorescence microscopy. Bars represent standard errors from three independent replicates

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The CLSM images in Fig. 5 show the progress of the infection in greater detail. At 12 hpi, no green flourescence was detected within inoculated spikelets (Fig. 5a), indicating that hyphal growth was limited to the outer surface of the glume, lemma and palea. Furthermore, the hyphae seemed to be randomly distributed lacking any directional growth. At 36 hpi, the hyphae near the edges of the glume and lemma had reached the adaxial surfaces and become attached to the surface of the filaments (Fig. 5b), confirming that infection had occurred. At 84 hpi, a dense mycelial network formed on the inner surfaces of the filaments, which extended to the anthers and pistil tissue (Fig. 5c). As the hyphae cluster increased further, the fungus extended to the anthers along the filament at 108 hpi (Fig. 5d). The mycelium then expanded slowly, mainly within the anther groove (Fig. 5e), and to a lesser degree on the stigma, while the uninoculated ones showed no green fluorescence (Fig. 5h). When the pathogen reached the top of the anthers (Fig. 5f), the hyphae cluster filled the inter-anther space, although few hyphae were present under the anther surface. The mycelium then expanded quickly until it completely enclosed the inner spikelets. At this stage, the tissue autofluorescence could be observed in the pistil and anthers (Fig. 5g), and the pathogen did not penetrate the host cell walls. It may suggest that the pathogen is a biotrophic parasite and could not penetrate the tissues of the flowering organs.

Fig. 5
figure 5

CLSM images of an EGFP labelled U. virens strain in rice spikelets at selected stages of development: a No green fluorescence in the inner spikelets at 12 hpi; b Primary infection in the basal part of the filaments at 36 hpi; c Hyphae extend along the filament at 84 hpi; d Hyphae extend to the anther along the filament at 108 hpi; e Hyphae colonize the inter-anther space the surface of pistil at 134 hpi; f Hyphae extend along the inter-anther space to the anther apex (white arrow) at 168 hpi; g Cross-section of spikelet (z-stacked images, 13.5 μm) at 9 dpi, the whole inner spikelet is enclosed by hyphae. The pistil (white arrow) and anthers (blue arrow) which showed faint autofluorescence remained alive; h Uninoculated inner spikelet. Bars = 100 μm

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To further clarify the infection process, CLSM was used to investigate the location of U. virens throughout the entire panicle, including the spikelets, stems and false smut balls (Fig. 6). Fluorescence was only observed in samples from the false smut balls (Fig. 6c), which suggests that the U. virens could not spread from the spikelets to the stem.

Fig. 6
figure 6

CLSM images of panicles at 21 dpi with an EGFP labelled U. virens strain: a Infected panicle, showing that even though many spikelets are bleached not all of the spikelets have been affected; b, d, e & f Cross and longitudinal sections of panicle stems showing no green fluorescence in either inoculated (b, d, & e) or uninoculated (f) samples; c Infected spikelets exhibiting strong fluorescence. Bars = 100 μm

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The infection process was also monitored using SEM. Shortly after inoculation, the hyphae-conidia mixture could be detected on the surface of spikelets (Fig. 1g), while uninoculated spikelets just displayed a warty (Fig. 1f). At 36 hpi, the pathogen invaded the interior of spikelets and formed a hyphal cluster attached to the filaments (Fig. 7a). It is interesting to note that the hyphae and filaments adhered together tightly to form a mycelial stroma. At this stage, a small number of infection hyphae formed, most of which colonized the surface of basal filaments before extending towards the pistil and anthers. At 84 hpi (Fig. 7b), many hyphae could also be detected on the surface of the ovary, although no infection hyphae were observed. At 134 hpi (Fig. 7c and d), the pathogen accumulated to form a hyphae cluster, which extended upward along the anther groove to its apex. A large number of hyphae were observed on the surface of ovary and stigma, while few were detected under the anther surface. At 168 hpi (Fig. 7e), the hyphae had extended to the anther apex and began to spread over the surface of anthers. By 192 hpi the fungal growth was so extensive that it enclosed almost the whole of the inner spikelet (Fig. 7f). A comparison of the infected spikelet and the non-infected control indicated that the U. virens infection colonized the surfaces of the flower organs and the space between them (Fig. 7g and h).

Fig. 7
figure 7

SEM images of the infection process of U. virens in rice spikelets: a, b Hyphae (Hy), mycelial stroma (white arrow) and infection hyphae (pink arrow) on the surfaces of filaments at 36 hpi and ovaries (Ov) 84 hpi; c & d Hyphae (white arrow) extend along the anther (An) groove and on the surface of the pistils at 134 hpi; e, f Hyphae extend to the anther apex (pink arrow) at 168 hpi (e) and begin to cover the inner surface of spikelet at 192 hpi (f); g Infected spikelet at 192 hpi; h, Uninoculated spikelet

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Discussion

There have been several recent reports regarding the infection processes of U. virens in different rice cultivars (Schroud and TeBeest 2006; Ashizawa et al. 2012; Tang et al. 2013). However, these studies have been limited in their scope, so it has been difficult to establish an overview of the infection processes that results in damaged panicles, or to what extent different rice cultivars are affected. The present study provided a more comprehensive insight into the colonization of the rice panicle by U. virens, not only assessing the physiological state of different tissues during the growth of the pathogen, but also investigating the infection processes. This approach revealed several specific infection events that have not been described previously.

A range of hyphae-conidia suspensions were used as inocula, and similar to previous reports (Zhang et al. 2004; Yang et al. 2011), the highest concentration (2 × 106 conidia/ml) was found to produce the heaviest infections, causing severe symptoms of false smut disease. It was noted that the germination of conidiospores and the appearance of infection hyphae in the inner spikelets at 24 hpi occurred much more rapidly than the 72 hpi previously reported (Ashizawa et al. 2012). This discrepancy might be explained by differences in the experimental technique, with the direct injection of a high inoculant load in the current study resulting in more rapid establishment of infection during the initial stages, since the fungal propagules were immediately in direct contact with the spikelets surface. The other reason might be explained by the use of a mixture of hyphal-fragment and conidia in this study, which provide a more favourable microenvironment for U. virens infection.

The improved method of artificial inoculation developed in this study also showed that temperature was an important factor affecting the morphological characteristics of false smut balls (Fig. 2e). When the temperature after inoculation was < 26 °C, black false smut balls with few chlamydospores often developed. However, when the temperature was > 26 °C yellow false smut balls with large number of chlamydospores emerged. This study demonstrated that the temperature is also an important parameter in false smut balls formation.

The proliferation of U. virens in inoculated rice spikelets has been studied previously using microscopy (Ashizawa et al. 2012; Tang et al. 2013), histological staining and PCR analysis (Cai and Zhou 2009). Although the results showed that the speed of hyphal grow within spikelets could vary, little is known about the effect of inoculation volume and location on the incidence of disease. This may be due to the difficulties with the determination of panicles variables, with providing well-defined infection conditions during the flowering time, and with the measurement of the infection level in a single panicle. In this study, statistical analysis of rice cultivar YY9 infected with U. virens showed a positive relationship between these variables and the incidence of disease, which has not been reported previously. The hyphae-conidia suspension of the pathogen elongated around the inoculation site. At lower volumes of inoculum (0.2 and 0.5 ml), inoculation at the apex or the middle resulted in a number of infected spikelets surrounding the inoculation sites. Basal areas were not affected (Fig. 3), because panicles were enclosed by flag leaves at the booting stage. At higher volumes of inoculum, a positive correlation between inoculum volume and the incidence of disease could be observed (Table 1). This showed that the treatments of different inoculation volumes and inoculation sites were feasible. Furthermore, many bleached grains developed after the artificial inoculation, which was again in contrast to previous reports that detected none. Filtrates from U. virens cultures were injected into non-inoculated panicles to test if substances secreted by the fungus produced the bleached grains. However, statistical analysis (data not shown) indicated that the filtrate had no influence and that therefore the presence of the pathogen was required to produce the bleached spikelets.

The route by which U. virens penetrates rice panicles has long been an issue of debate. A recent study (Ashizawa et al. 2012) has indicated that U. virens follows a specific route, with the hyphae colonizing the outer surface of the spikelet, and then entering the inner spikelet surface from the apex. However, the present study indicates that the infection process follows a different pattern involving specific zones at different stages of development (Figs. 5 and 7). Consistent with a previous report (Tang et al. 2013), both CLSM and SEM observations showed that the pathogen initially attaches itself to the surface of the filaments (Figs. 5b, c, and 7a). Several discrete structures were formed by U. virens during the infection of spikelets, including mycelial stroma and infection hyphae (Fig. 7a). These specialized infection structures, which are commonly found in other pathogens such as Claviceps purpurea (White et al. 2000; Pazoutova 2001; Scheffer and Tudzynski 2006), have not been reported for U. virens previously. Furthermore, the growth of the hyphae appeared to be limited to the intercellular space and the cell surface without actually entering the intracellular space of the inner spikelets (Figs. 5 and 7). The inner spikelets were colonized from the base of the filaments and moved upwards to the apex of the anthers quickly enclosing all the floral organs. A possible explanation for this behaviour is that it allows pathogen to prevent the spikelet from heading in the shortest time possible. Like the interaction between Fusarium graminearum and wheat spikelets (Brown et al. 2010; Sella et al. 2013), the growing point of rice panicles provides a favourable environment for U. virens colonization and infection because it is very rich in nutrients. Both the CLSM and SEM images suggest that U. virens penetrates and colonizes the spikelet displaying strict organ specificity. From a practical perspective, these observations could indicate that all or most false smut balls arise from infections during the booting stage. The capacity of confocal microscopy to image optical sections at different depths also allowed the investigation of fungal growth deep within the plant tissue during the later stages of infection (Fig. 6). In accordance with a previous study (Tang et al. 2013), which used both LM and TEM, it was found that both the pedicels beneath the false smut balls and the stems of severely infected panicles exhibited no evidence of the green flourescence fungi, although it is possible that the bright autofluorescence (Fig. 6b) of the rice stems could have prevented the visualization of hyphae in the samples observed. It is also possible that the timing of certain infection events could coincide with the translocated of proteins into the plant cells, as has been observed in rust fungi and oomycete pathogens (Kemen et al. 2005; Dodds et al. 2004; Birch et al. 2006). The intracellular growth of the infecting hyphae, and the extended interaction zone established could be the site at which such translocation of proteins into the host cell occurs. Further work, including increased numbers of samples and molecular analysis at different stage of rice panicle development is required to characterize the infection process more completely.

In summary, this study provides both an overview and novel insights into the U. virens-rice interaction in panicles, integrating the progress of visual symptoms with histopathological aspects of the U. virens infection and colonization process. It also serves as a possible platform for future research to identify the specific periods essential for U. virens pathogenicity and also how different host cultivars might affect these stages of infection. The threat posed by changes in the behaviour of this pathogen as a consequence of altered cultural practices associated with intensified rice production is also an issue of major concern. Investigations of the evolutionary events that have led to the ability of U. virens to infect spikelets could therefore also be a fruitful avenue for further research. Although the current study has improved our knowledge of panicle colonization and the primary infection of U. virens, these results, obtained in a sterile artificial system, require further investigation under natural conditions. It is possible that a more detailed understanding of the primary infection site of U. virens could result in the development of control strategies that can efficiently combat false smut disease in rice crops.