This study reports an efficient inoculation protocol that allowed cytological analysis of the infection process of the rice false smut pathogen Ustilaginoidea virens. Examination of serial semithin and ultrathin sections of infected spikelets showed that the primary infection sites for the pathogen were the upper parts of the three stamen filaments located between the ovary and the lodicules. The stigma and lodicules were also occasionally infected to a limited extent. The pathogen infected the filaments intercellularly and extended intercellularly along the filament base. The host cells were degraded gradually. The pathogen did not penetrate host cell walls directly and did not form haustoria. In the balls the ovary remained alive and was never infected. This suggests that the pathogen is a biotrophic parasite that grows intercellularly in vivo.
Rice false smut is a unique fungal disease, and confusion remains about the disease cycle and infection process. Although there have been many hypotheses and much speculation, experimental evidence is still lacking (Wang et al., 2004). Pathogen growth in various media is very slow, and chlamydospores survive for just a few months. It has also been reported that the pathogen can infect many different hosts, including rice and some weeds, such as Panicum trypheron (Shetty & Shetty, 1987), but its life cycle in natural environments remains unclear.
The characteristic symptom of rice false smut is the formation of ball-like colonies in spikelets, which begin to appear 10–15 days after rice anthesis. At the start, the balls consist of white hyphae, which then form a thick, yellow, loose outer layer of chlamydospores in summer and early autumn, and an olive to black, hard outer layer in late autumn. Sclerotia often form on the colony surfaces, especially in later autumn, with lower temperatures and high temperature differences between day and night (Ikegami, 1963b; Miao, 1992).
Inoculation protocols using spray or injection inoculation with chlamydospores at the germination, seedling or booting stages proved to be successful (Ikegami, 1962, 1963a; Miao, 1992). More recently, chlamydospores were replaced as the inocula by conidia produced in liquid culture, and inoculation focused on the late booting stage using improved protocols, resulting in a much higher disease index (Fujita et al., 1989; Wang et al., 1996; Zhang et al., 2004; Ashizawa et al., 2011; Tanaka et al., 2011). The rice stigma, ovary, and even the young grain, were assumed to be the primary infection site (Padwick, 1950; Wang, 1992), but it was subsequently found by histological observation that the pathogen can attack the root at the seedling stage and lead to symptomless colonization of the entire plant, as detected by molecular techniques (Schroud & TeBeest, 2005; TeBeest, 2010). The pathogen was also found to infect rice coleoptiles intercellularly at the earlier germination stage in the laboratory of the present study (data not shown). To date, the fungal life cycle and the primary infection sites in nature have remained a puzzle.
Knowledge of the pathogen’s life history and infection process in nature is critical for disease control. Here, an efficient inoculation protocol was used to gain a higher infection rate, making possible the examination of the pathogen infection mechanism at the cytological level. The primary infection site and the mode of pathogen growth were examined using infected spikelets at different stages and mature smut balls collected in the field.
Materials and methods
Pathogen isolates, rice cultivars and inoculation protocol
Isolate ZJ0202 of U. virens, which was isolated from the indica–japonica hybrid rice cultivar Yongyou 6 in Xiangshan, Zhejiang, was used as the inoculum. After 5 days of culture in potato sucrose (PS) broth at 26°C on a shaker at 130 rpm, hyphae were filtered and conidia were collected from the filtrate by centrifugation. The conidia were then resuspended in fresh PS medium to a density of 2–5 × 105 conidia mL−1 and used as the inoculum.
Artificial inoculations were carried out in the greenhouse. Yongyou 12, an indica–japonica hybrid rice cultivar, was grown under normal conditions in a paddy field at the Experimental Farming Station of Zhejiang University until 2 weeks before the heading stage, when plants were transplanted into plastic pots and put under a 14-h-light/10-h-dark regime in the greenhouse at 30°C. The inoculation protocol described by Fujita et al. (1989) and Ashizawa et al. (2011) was employed with minor modification. One week before heading, 1–2 mL inoculum suspension was injected into 40 swollen sheaths of flag leaves on the main stems or main tillers with sterile injectors. The inoculated plants were placed in the plant growth chamber for 2 days at 20°C with 85–95% relative humidity (RH) and then for 5 days at 25°C with 3000 lux illuminance from white fluorescent tubes. The plants were then transferred into the greenhouse and kept at 28–30°C and about 80% RH. Eight of the injected panicles were sampled at each time point (3, 5, 10 and 15 days after inoculation) and others were used to count the infection percentage (infected spikelets/total spikelets) 25 days after inoculation.
Collection of young false smut balls
Young false smut balls were collected from the artificially inoculated plants as well as from plants of three susceptible cultivars grown in the field: Yongyou 12, Xiushui 134 (an indica rice in Xiangshan, Zhejiang) and Fengyouxiangzhan (an indica hybrid rice, in Mianxian, Shaanxi). All the spikelets in the inoculated panicles were stripped off and the infected ones were selected. In the field, the panicles with young rice false balls, i.e. white balls just protruding from the lemma and palea, were collected from early September to October. They were stripped out in the laboratory to find spikelets in the early stages of infection. False smut balls at different developmental stages were collected, such as small ones with an intact, white, enveloping membrane, and mature ones yellow, brown or dark green in colour. False smut balls were immersed in 2·5% glutaraldehyde in 100 mm phosphate buffered saline (PBS, pH 7·2) for 48 h at 4°C. Samples for transmission electron microscopy were cut into small pieces and transferred into fresh 2·5% glutaraldehyde solution for 24 h.
Anatomy and observation of rice false smut balls
The pre-fixed samples of inoculated panicles were washed thoroughly and lemmas and paleas removed carefully with sharp tweezers. They were then immersed in lactophenol (phenol:lactic acid:glycerol:water, 1:1:2:1, v/v) containing 0·1% trypan blue, boiled for 10 min in a water bath, and then destained in lactophenol for 5 min in the boiling water bath. The samples were mounted on glass slides and observed under a stereoscopic microscope or light microscope to select the infected spikelets for further study. The big false smut balls were cut into small pieces with different organs, such as ovary, anther, etc., by sharp blades in various directions for further sample preparation. Others were dissected to check the ovaries and anthers.
Sample preparation for light microscopy (LM) and transmission electron microscopy (TEM)
The pre-fixed samples were cut into small pieces of 2 × 2 × 3 mm, washed for 1 h in PBS buffer (pH 7·2, 100 mm) with several buffer changes, and then post-fixed in 1% osmium tetroxide (OsO4) for 2 h at room temperature. After washing several times in double-distilled water, the samples were dehydrated stepwise in 30, 50, 70, 80, 90, 100 and 100% ethanol for 20 min at each step. The samples were immersed gradually and finally embedded in Spurr resin. Polymerization was performed at 70°C for 16 h.
For LM, the embedded intact samples were cut into serial 2-μm sections. The sections were mounted on glass slides and stained with 0·05% methylene blue (w/v), and then observed and recorded with a Nikon E200 microscope.
For TEM, the samples were located by LM and then cut into ultrathin sections of 6–7 nm. After contrasting using a standard protocol, they were examined with a Hitachi H-7650 transmission electron microscope.
The pedicels beneath the false smut balls and the stems of naturally severely infected panicles were also cut into species of 1 × 2 mm. These were treated as described above to examine if there were pathogen hyphae in them.
Sample preparation for scanning electron microscopy (SEM)
The pre-fixed samples were cut into small pieces and washed several times in PBS buffer (pH 7·2, 100 mm) for a total of 1 h and post-fixed in 1% OsO4 for 2 h at room temperature. After washing several times in double-distilled water, the samples were dehydrated stepwise in 30, 50, 70, 80, 90, 100 and 100% acetone for 30 min at each step. The samples were transferred to isoamyl acetate for 2 h and then dried in a Hitachi HCP-2 critical point drier. The samples were mounted on sample plates and coated with Eiko IB-5 ion coater and observed with a Philips XL30 ESEM.
Fifteen days after inoculation, symptoms appeared in all inoculated panicles. On average, 26·6% of spikelets were infected and formed false smut balls (511 of 1921 spikelets in seven panicles), indicating that the inoculation was successful and making it easy to find infected spikelets. After removing lemmas and paleas, all fixed samples were stained and checked by light microscopy to select the infected flowers (Fig. 1). After observation and recording, the infected spikelets were treated further for LM and TEM analysis.
LM observation revealed that the pathogen hyphae appeared initially on the surfaces of anthers and were particularly dense along the anther grooves (Fig. S1). Serial semithin transverse sections were cut throughout the intact infected spikelets. After examination of all sections it was found that all infection sites were on the upper parts of the filaments 5 days after inoculation. Both LM and TEM observation showed that the pathogen attached to the surfaces of the filaments and then ‘pressed’ into the cell gaps (Figs 2 and 3). The pathogen hyphae then extended mainly into the central vascular tissues along the cell walls. The individual filament cells appeared to be separated into many ‘isolated islands’ by a large number of radial hyphae (Figs 2 and 3). Some epidermal cells appeared to be digested completely and the spaces were occupied by pathogen hyphae. No haustoria were detected and the pathogen hyphae did not penetrate directly into the host cell wall.
With the exception of the filaments, no other spikelet parts were infected until 10 days after inoculation. Dense hyphae tightly contacted anthers, stigmas, styles, ovaries and lodicules, but no primary infection site was detected on these parts in the 19 young balls examined by serial sections (Fig. S2). This suggested that the primary infection sites were rice stamen filaments at the heading stage.
Ten days after inoculation, most infected flowers were filled with pathogen hyphae and some colonies began to grow out of the spikelets (Fig. 1). At the initial infection sites, most of the filament cells had been digested and recognition of the original filament appearance became very difficult (Figs 2 and 3). Serial semithin section analysis showed that the primary infection sites were expanded along the filaments to the filament bases. Surprisingly, the pathogen seemed just to infect the filaments of the three special stamens located between the ovary and the lodicules near the lemma (Fig. 4). The other three filaments remained intact, but surrounded and enveloped, while some were extruded and shrunken (Fig. 4). This was validated by further anatomical examination and semithin sections of more infected spikelets.
Fifteen days after inoculation, most of the false smut balls had grown to their final size. It was found by examination of serial semithin sections that ovaries, styles and lodicules in all the balls remained alive. Secondary hyphae were found infecting styles in just one case and lodicules in just two cases out of a total of 36 balls examined, and hyphal extension was just limited to a few layers of lodicule cells (Fig. 5) and to a few stigma cells (Fig. 6).
The mature false smut balls collected from the rice fields were also examined with serial sections by LM, TEM and SEM. It was found that the infection process was similar to that with artificial inoculation. The pathogen hyphae penetrated and extended into the base of the stamen filaments. The ovaries in all the fresh balls remained green and alive (Fig. S3).
At later stages of ball development it was observed that hyphae entered or filled the anther loculi and most of the pollen appeared well developed. Examination of serial sections revealed that the hyphae entered the cavities through the cracks formed by breakage of the cleft cells. No hyphae were found to penetrate into the outer and inner surfaces of the anthers (Fig. S4). In most false smut balls collected from the field, anther loculi were shrunken, and pollen grains were underdeveloped. Few or no hyphae were found in the loculi.
It was surprising that the pathogen hyphae always extended along the filament from top to base, but never in the reverse direction into the anther connective cells, although the filament and the connective tissue are continuous and similar in tissue organization (Fig. S4).
The rigid rice lemmas were also examined under SEM. Infection sites were never detected, but occasionally some hyphae and chlamydospores were found to be attached to the surfaces (Fig. S5). This suggests that the pathogen cannot infect and enter the hard coats.
The rice ovary and young grains were reported to be infected by the pathogen (Padwick, 1950). In the past few years at the laboratory of the present study, more than 1000 fresh false smut balls at different developmental stages have been cut transversely by sharp blades. In all the balls without exception there were six anthers present at the centre. This indicates that the pathogen infection occurred long before anthesis and the anthers were enveloped and enclosed in the spikelets. Rice grains were never found to be infected or forming the ball-like colonies.
The pedicels beneath the false smut balls and stems of naturally severely infected panicles were also checked by both LM and TEM. No pathogen hyphae were visible in them (Fig. S6). This suggests that all or most of the false smut balls may arise from infections at the rice booting stage.
The debate on the pathogen infection process has been going on for a long time because of the lack of experimental evidence from cytological examinations. One of the important reasons for this has been the lack of an efficient and reproducible inoculation method. In recent years the inoculation protocol has improved gradually (Fujita et al., 1989; Wang et al., 1996; Ashizawa et al., 2011). The present study employed the protocol with a minor modification of a low-temperature treatment (2 days at 20°C), and a high percentage of infected spikelets were obtained. This enabled enough infected spikelets to be obtained for cytological research and greatly decreased the amount of work required. Serial sections with various false smut balls at different stages allowed whole balls to be observed during their development in different orientations. It was confirmed that the upper parts of the three filaments located between the lodicules and the ovary are the primary infection sites. This limitation of sites suitable for infection may explain why inoculation and infection in nature are difficult. Previous research showed that inoculation must be carried out at the booting stage, and that infection was very difficult to establish after the panicles headed (Wang et al., 2004). This may be related to the structure and physiological state of the filament.
After fertilization, rice pistils will take up large amounts of nutrients and photosynthesis products to form grains. The filaments will elongate out of the spikelets and die after flowering. Therefore, the pistil or its stigmas and ovary have been considered as the infection sites for the false smut pathogen when pathogen hyphae were visible on the surfaces of anthers, stigmas and ovaries (Wang, 1992). It is an unexpected finding that the primary infection occurs on the filaments. The development of the pathogen requires a lot of nutrients and the filaments appear too thin and short-lived to support it. Further work will be focused on the metabolic changes induced in the filaments by infection and on the question of why this target tissue is so susceptible to the pathogen attack.
During anthesis of cereal plants, the filaments normally elongate rapidly and synchronously with the enlarging lodicules to push the anthers out of the lemmas. In filament elongation, extension is limited to the epidermis and one or two subepidermal cell layers (Keijzer et al., 1996). Thus, its cell organization and adhesion must be unique and fragile, which may render it susceptible to pathogen attack. It was observed that the fungus could enter the lodicule or the bases of stigmata with a low probability, but extension of the hyphae was limited, which also implies that the filament tissue or its surface is much more fragile than stigmas and lodicules.
The lodicules are two diminutive bodies that can expand several times in size at anthesis, but their vascular tissue did not elongate. Pathogen hyphae were found not to reach the vascular tissue, and as a consequence may obtain insufficient nutrients for ball development. Although large amounts of pathogen hyphae were in tight contact with the ovaries, the pathogen never penetrated into the ovaries. This suggests that the filaments are the exclusive primary infection sites.
In necrotrophic pathogens, mycotoxins are always found to kill the host cells and to penetrate the host cell walls with infection hyphae during their extension (Koiso et al., 1994, 1998; Horbach et al., 2011). The pathogen of rice false smut was reported to contain large amounts of mycotoxins (Koiso et al., 1994, 1998). However, the observations in the present study showed that in fresh mature balls, the ovaries and filament cells remained alive. Most typical biotrophic and hemibiotrophic pathogens of plants, such as powdery mildew and rust, invade host cells and form special organs for nutrient uptake, the haustoria (Horbach et al., 2011). During the infection and development of rice false smut, no typical infective hyphae or haustoria, which could penetrate host cell walls and enter the host cell through a hole, were detected. This implies that U. virens is a biotrophic parasite with a distinct nutrient-uptake mechanism. Ustilaginoidea virens appears to be a biotrophic pathogen which also produces mycotoxins, which makes the infection mechanism unusual.
In previous studies two kinds of infection mode were reported, including infection of the spikelets and grains (Padwick, 1950), but in observations at the present laboratory over the past decade, infected grains, i.e. false smut balls containing a young embryo and endosperm, have never been found. In the rainy season the balls often decayed and their fine structure was difficult to recognize, which may have led to misinterpretation. The pathogen could not penetrate very young ovaries, and so it is also unlikely to be able to penetrate the grains with their harder seed coats.
In the present study the inoculations were carried out 5–7 days before heading, which was also suggested as the suitable time for fungicide spraying to control the disease (Wang et al., 2004). In the present study it was found that pollen development in the artificially inoculated spikelets was much better than that in the naturally infected balls from the field. This means that fungicides should be applied much earlier to control the disease.
There is evidence in the literature that the pathogen could infect rice seedlings (Ikegami, 1962, 1963a; Miao, 1992; TeBeest, 2010) and that the pathogen could be detected by molecular methods in adult plants (Ashizawa & Kataoka, 2005; TeBeest, 2010). Ikegami (1963a) found that the pathogen could penetrate into the young coleoptiles and grew intercellularly along sieve tubes, up to the mid-tillering stage. The hyphae did not develop at the ear primordium. The present laboratory has obtained similar results in the past few years. In the current study, there were also no hyphae detected in the pedicel of the infected spikelets and stems of naturally diseased panicles. Thus, the direct cytological evidence supports the deduction that U. virens specifically infects rice filaments at the booting stage. In recent years pathogen transformants with a GFP tag and a naturally albinic mutant have been obtained (Wang et al., 2008; Tanaka et al., 2011). Combination of those isolates and the improved inoculation protocol (Ashizawa et al., 2011) will provide more evidence for the infection mechanism.
The infection routes of Claviceps spp., species closely related to U. virens, are clear (Pazoutova, 2001; Tanaka et al., 2008). The characteristic feature of all Claviceps spp. during infection is their strict organ specificity (Scheffer & Tudzynski, 2006). They usually infect host stigma and then extend intercellularly along the transmitting tissues of the style to the tip of the ovarian axis (rachilla) (Tudzynski & Scheffer, 2004). Ustilaginoidea virens also infects a specific organ, the rice filament, at a specific stage of development. The two plant targets, pistil and filament, have different structures and functions (Keijzer et al., 1996; Tudzynski & Scheffer, 2004). This suggests that their infections have different biochemical mechanisms. Both the pathogens infect and grow in intercellular mode, and do not form haustoria. Claviceps species also produce mycotoxins – the ergot alkaloids, tri- or tetracyclic derivatives of prenylated tryptophan – but they are biotrophic parasites and do not kill the infected organ during development (Lorenz et al., 2009). Ustilaginoidea virens also did not kill the filament in the present study. These results suggest that the two kinds of pathogen share many common mechanisms in pathogenesis, and the previous research on cereal ergot may aid the study of rice false smut.
This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (200903039-5) and the opening fund of the State Key Laboratory for Rice Biology, Zhejiang University. Dr Luo Chaoxi, Huazhong Agricultural University, gave useful advice on the inoculation protocol in details. Dr Dudler of University of Zurich and Dr Schaffrath of RWTH Aachen University read and revised the manuscript carefully. Dr Tanaka of Ishikawa Prefectural University provided help with references.