J Gen Plant Pathol (2012) 78:255259
DOI 10.1007/s10327-012-0389-3
FUNGAL DISEASES
Rice false smut pathogen, Ustilaginoidea virens, invades through
small gap at the apex of a rice spikelet before heading
Taketo Ashizawa Mami Takahashi
Michiyoshi Arai Tsutomu Arie
Received: 7 February 2012 / Accepted: 10 April 2012 / Published online: 13 June 2012
The Phytopathological Society of Japan and Springer 2012
Abstract Ustilaginoidea virens, the false smut pathogen
of rice, produces false smut balls on spikelets after heading.
To clarify how the fungus invades spikelets during the
booting stage, we developed a fungal strain that expresses a
green fluorescent protein gene and injected conidia from
this strain into rice sheaths. Observations at 48 h post-
inoculation showed many conidia were present on spikelet
surfaces, and the conidia had germinated and the hyphae
have gradually grown by 120 h post-inoculation. By 144 h,
hyphae had invaded spikelets through their apices, via the
small gap between the lemma and palea and had already
reached all floral organs.
Keywords Rice
Spikelet
Villosiclava
Green
fluorescent protein
Conidia
Rice false smut caused by Ustilaginoidea virens (Cooke)
Takahashi (teleomorph Villosiclava virens) (Kepler et al.
2012; Tanaka et al. 2008; White et al. 2000) is a serious
disease of agricultural rice. False smut balls, green with
numerous chlamydospores, appear on rice spikelets, and
contain ustiloxins that are poisonous to animals (Nakamura
et al. 1992; Suwa 1915). Chlamydospores in paddy soils
T. Ashizawa (&)
M. Takahashi
M. Arai
Hokuriku Paddy Cropping System Project Team,
Hokuriku Research Center, National Agricultural Research
Center, National Agriculture and Food Research Organization,
1-2-1 Inada, Joetsu, Niigata 943-0193, Japan
e-mail: toketa@affrc.go.jp
T. Arie
Laboratory of Plant Pathology, Tokyo University
of Agriculture and Technology, 3-5-8 Saiwaicho,
Fuchu, Tokyo 183-8509, Japan
are the primary source of infection (Ikegami 1963). The
fungus then colonizes tissues of the growing points of til-
lers during the vegetative stage of rice growth (Ikegami
1963) and is believed to be transferred to young panicles in
leaf sheaths. As already reported by Ashizawa and Kataoka
(2005), the fungus is present in panicles at the booting
stage because nested-PCRs targeting the species-specific
ITS region of ribosomal DNA have confirmed the presence
of the fungus in whole panicles before rice heading. In
addition, spray inoculation tests of rice plants in fields
indicated that the infection did not occur after heading of
plants (Fujita et al. 1989). After rice heading, we were able
to clearly discriminate infected from uninfected spikelets
on a panicle because infected spikelets contained the white
fungus and finally formed smut balls (Ikegami 1961). Our
hypothesis is that infection occurs in spikelets on panicles
in the leaf sheath before heading. However, how the fungus
invades spikelets during the booting (reproductive) stage
has been unknown.
A green fluorescent protein (GFP) from the jellyfish
Aequorea victoria (Prasher et al. 1992; Shimomura et al.
1962) provided a means of labeling the fungus to observe
its behavior in plant tissues under in vivo conditions under
fluorescence microscopy. Visualization of the GFP-labeled
fungus is effective and allows temporal analysis of inva-
sion. A GFP-labeled strain of Ustilaginoidea virens was
previously developed by Agrobacterium tumefaciens-
mediated transformation (Zhang et al. 2006) or electro-
poration (Tanaka et al. 2011). We have also developed a
GFP-labeled strain of U. virens by polyethylene-glycol-
mediated protoplast transformation and observed the initial
infection of rice spikelets before heading.
We used the false smut isolate U. virens U2003-1
(Ashizawa et al. 2010) to obtain protoplasts. Hyphal tips of
U2003-1 were transferred into potato dextrose broth (PDB)
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a d

b e

c f

Fig. 1 Deposition, colonization, and invasion processes of a GFP-
tagged Ustilaginoidea virens isolate, U2003-1, on surface of spikelets
of rice variety Yumeaoba, viewed with fluorescence microscopy.
Inoculated rice panicles were examined at 24144 h post-inoculation
(hpi) before heading and 9 and 11 days post-inoculation (dpi) after
heading. a Numerous conidia on surface of spikelet, 48 hpi. Arrows
indicate conidia that have not germinated. Upper right inset is a detail
of spores on spikelet surface. White bar 20 lm, yellow bar 10 lm.
b Hyphae (arrow) on surface of spikelet, 120 hpi. Bar 50 lm. cg
Hyphae at 144 hpi. c Hyphae at small gap at spikelet apex. Black
arrows indicate mycelium on outer surface of spikelet. Blue arrow
indicates hyphae growing through the small gap (between lemma and
palea) into the interior of spikelet. Bar 200 lm. White arrows indicate
hyphae on inner surface of spikelet. d Hyphae on inner surface of
spikelet. Bar 200 lm. e Hyphae (arrows) on anther. Bar 50 lm.
f Hyphae on stigma (upper arrows) and anther (lower arrow). Bar
50 lm. g Hyphae (arrow) on lodicules. Bar 200 lm. h Hyphae
covering anther, 9 dpi. Bar 50 lm. i Hyphae covering all floral
organs, 11 dpi. Bar 100 lm. j Hyphal locations on rice floral organs
based on observations in eh: 1 upper anther (corresponding to e, h),
2 stigma (corresponding to f), 3 lower anther (corresponding to f),
4 lodicule (corresponding to g). Bar 1 mm

(Difco, Franklin Lakes, NJ, USA) and incubated at 28 C
for 7 days with shaking at 120 rpm. Hyphae were then
harvested from the liquid culture. The tubes were incubated
on a horizontal shaker at 120 rpm for 4 days to obtain a
conidial suspension (Ashizawa et al. 2011).
Plasmid pMK412 (Watanabe et al. 2007) carrying the
engineered GFP (EGFP) and the hygromycin B phospho-
transferase gene was used for fungal transformation. The
plasmid was propagated in Escherichia coli DH5a and
purified using a Plasmid Mini Kit (Qiagen, Tokyo, Japan).
One microgram per milliliter of the purified plasmid was
linearized with 750 U XbaI in 50 lL TrisEDTA (TE) at
37 C for 4 h and used to transform the fungus without
inactivation of the enzyme.
Hyphae of U2003-1, grown in PDB as described, were
filtered and washed with OM buffer (1.2 M MgSO4,
10 mM sodium phosphate, pH 6.8). Washed hyphae were
resuspended in 10 mL of filter-sterilized (pore diameter

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g i
h j
Fig. 1 continued
0.2 lm) OM buffer containing 30 mg/mL of lysing
enzyme (Sigma, St. Louis, MO, USA) in a 50 mL conical
tube. The tube was incubated at 28 C for 3.5 h by gentle
horizontal shaking (60 rpm). The protoplast solution fil-
tered through two-layered tissue paper was overlaid with
7.5 mL of ST buffer (1.2 M sorbitol, 10 mM TrisHCl, pH
7.5) in a 50 mL conical tube. The tube was centrifuged at
40009g for 10 min. The protoplast layer (middle layer)
was transferred to a new 50 mL conical tube and mixed
with 15 mL of STC buffer (1.2 M sorbitol, 10 mM Tris
HCl, 20 mM CaCl2, pH 7.5). The protoplast solution was
centrifuged at 20009g for 10 min, and suspended in
800 lL of STC buffer adjusted to a concentration of
5 9 107 protoplasts/mL.
Eight hundred microliters of the protoplast suspension
was mixed with 200 lL of XbaI-digested pMK412 in a
15 mL conical tube. One milliliter of polyethylene glycol
(PEG) solution [60 % (w/v) PEG 4000, 10 mM TrisHCl,
50 mM CaCl2] was added twice to the mixture by droplet,
kept for 18 min at 25 C and transferred to ice for 2 min.
Forty milliliters of STC-50 buffer (1.2 M sorbitol, 10 mM
TrisHCl, 50 mM CaCl2, pH 8.0) was gently added and
mixed, and centrifuged at 30009g for 10 min. The proto-
plast solution was suspended in 10 mL of YG1/2SC
257
1
2
3
medium [0.5 % (w/v) yeast extract, 2.0 % (w/v) glucose,
0.6 M sorbitol, 25 mM CaCl2], and incubated at 28 C for
3 h. The protoplast suspension was mixed with 300 mL of
YG20S agar [0.5 % (w/v) yeast extract, 2.0 % (w/v) glu-
cose, 20 % (w/v) sucrose, 1.5 % (w/v) agar] containing
100 lg/mL of hygromycin B. After incubation at 28 C for
710 days, emergent colonies were transferred to new
YG20S agar containing 200 lg/mL of hygromycin B.
Three GFP-transformants of U2003-1 were obtained. GFP-
tagged transformants were selected using fluorescence
microscopy (Leica, DN4000B, Tokyo, Japan) with UV
filter set GFP Plant MZ FLIII and used for further study.
Pathogenicity of the GFP-tagged U2003-1 to rice plants
was previously tested and confirmed, and the fungus
formed normal false smut balls (data not shown).
We cultivated the main culms of plants of susceptible
rice variety Yumeaoba for use in a rapid method to obtain
conidial suspensions (Ashizawa et al. 2011). Briefly, five
rice seeds were sown in 400 g of artificial soil (Honensu-
baido1, Honen Agri, Niigata, Japan) in a pot (diameter
10.5 cm). Rice plants were allowed to grow only along
their main culms, and emergent tillers were cut (Satake
1972). For inoculation of rice plants, eight dried barley
seeds on which the isolate was growing were added to a
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a emerged), 2 mL of conidial suspension (5 9 105 conidia/
b excised at 9 and 11 days post-inoculation (dpi).
c onto the inner surfaces of the lemma and palea (Fig. 1d).
Fig. 2 Diagram of infection route from conidial deposition to floral
organ invasion by the false smut fungus. a (1) Conidia (black spot) are
deposited on outer spikelet surface at 48 h post-inoculation (hpi). Bar
1 mm. b (2) Conidia germinate, and hyphae (black lines) develop on
outer spikelet surface until 120 hpi. Hyphal development near the
spikelet apex (red circle) is crucial for further invasion. Bar 1 mm. c (3)
Hyphae elongate from the small gap (yellow arrow) at the spikelet apex
at 144 hpi onto the inner spikelet surface (black arrows). Bar 1 mm.
Upper right inset is a detail of the small gap (yellow arrow). (4) Hyphae
attach to the floral organs (red circle). Blue bar 200 lm
50 mL conical tube containing 25 mL of PDB and 2 %
sucrose (w/v). The cultured broth was filtered through
two layers of tissue paper to obtain a conidial suspension.
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At the booting stage of rice growth (when flag leaves have
mL) were then injected onto the leaf sheaths of rice plants.
Plants were transferred to an incubator at 16 C for 2 days,
then moved to a moist chamber with 100 % relative
humidity at 26 C for 5 days. After inoculation, plants
were moved to a greenhouse controlled at 25/20 C (day/
night). Nonheading, inoculated panicles within leaf sheaths
were excised at 24, 48, 72, 96, 120, and 144 h post-inoc-
ulation (hpi) to examine the infection process of U. virens.
Whole, longitudinal, and cross sections of the panicles,
and/or 0.51 mm thick sections of the panicle tissues were
cut with a stainless steel razor blade, then observed with
fluorescence microscopy. After heading, panicles were
At 24 and 48 hpi, the inoculated panicles in the leaf
sheaths were well wetted by the conidial suspension of
GFP-tagged Ustilaginoidea virens isolate U2003-1, and
GFP-tagged conidia were observed on the surfaces of
spikelets (Fig. 1a). No germinated conidia were observed
on any panicle surfaces or in the remaining conidial sus-
pensions in leaf sheaths. At 72, 96, and 120 hpi, the conidia
of GFP-tagged U. virens deposited on the spikelet surfaces
were observed to have germinated and formed hyphae
(Fig. 1b). At 24120 hpi, no GFP-tagged U. virens were
observed in the floral organs of the inner spikelets. At 144
hpi, hyphae had grown from the apices of spikelets through
the small gap between the lemma and palea (Fig. 1c) and
Furthermore, GFP-tagged U. virens had reached floral
organs such as the anthers (Fig. 1e, f, j-1, j-3), stigmata
(Fig. 1f, j-2), filaments and lodicules (Fig. 1g, j-4). At
24144 hpi, the fungus was not observed in any inner tis-
sues/cells of the spikelet organs, the panicle axis, primary
and secondary branch pedicels, or the rudimentary glumes.
After heading of rice plants, the floral organs were covered
with hyphae by 9 dpi (Fig. 1h, j-1), and finally all the
organs were completely covered with hyphae by 11 dpi
(Fig. 1i). The hyphae in the spikelets appeared the same as
the typically observed symptoms; the initial infected
spikelets containing white hyphae.
The route of spikelet invasion of U. virens is outlined in
Fig. 2; (1) conidia land on the outer spikelet surface
(Fig. 2a), (2) hyphae develop and grow on the outer
spikelet surface (Fig. 2b), (3) then grow from spikelet apex
onto the inner spikelet surface (Fig. 2c). (4) Finally,
hyphae had grown onto the floral organs.
We developed GFP-tagged U. virens to monitor fungal
invasion of rice panicles. Consistently green-fluorescing U.
virens was clearly distinguishable from the red or hyaline
to yellow plant tissues using fluorescence microscopy.
After inoculation and the 2-day, 16 C incubation, the
conidia deposited on the spikelets and other panicle tissues
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had not germinated. Hyphae grew later, forming mycelia
on the surfaces of spikelets to enable hyphal invasion into
the floral organs in the spikelets. In fact, conidia in the
inoculum suspension were still found within a leaf sheath
with an unheaded panicle at 144 hpi, but germination was
poor, and the hyphae were too short to reach floral organs
(photo not shown). Interestingly, when cultured hyphae
were injected into leaf sheaths, no infection occurred, nor
did any false smut balls form. Thus, conidial deposition
and subsequent hyphal development may be a crucial step
for infection. The growth through the small gap also
indicates that U. virens infects rice plants without pene-
trating the cells and/or growing endophytically. The small
gap invasion process may contribute to the random
occurrence of false smut balls on panicles (Sonoda et al.
1988), because each random infection event results in a
spikelet with well-developed hyphae attached near the
small gap. During infection of the inner floral organs,
lodicule invasion, in particular, may be important for fur-
ther colonization by the hyphae. It is important for the
fungus to stop the lodicules from swelling, which pushes
apart the lemma, opening the spikelet (anthesis). Other air-
borne microorganisms could then invade the spikelets.
Interfering with lodicule swelling thus allows U. virens to
prevent contamination from competing microorganisms, as
well excessive drying and UV exposure. In fact, by
911 dpi, invading hyphae of U. virens had covered all the
floral organs including the lodicules. Our findings should
contribute to breeding rice varieties that are resistant to
false smut, through selection of lines with much smaller or
closed gaps and to the development of new fungicides to
suppress invasion of spikelets at the booting stage.
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