Progress in the study of false smut disease in rice

Abstract

Rice false smut disease is an increasing concern for research and production, not only because of the increasing epidemic occurrence in rice production, but also the intriguing specific pathogenesis of the disease to be a unique pathological system to enrich the molecular mechanism of plant-microbe interaction. Progresses have been achieved in the pathogen phylogenetic placement, the alternative hosts, the pathogen morphology and diversity, the toxins generated by false smut balls, the artificial inoculation method, and the pathogen transformation as well as rice resistance to the disease. However, it is still controversy on the infection process. It is not clear how the life cycle of this pathogen is coupled with the disease cycle. This review summarized our current understanding on the pathogen, the pathogenesis, and the rice resistance to the disease. Future work should pay attention to developing a more rapid and effective system to evaluate rice resistance and susceptibility to the disease, screening of rice germplasm for disease-resistance breeding, studying the resistance inheritance, and investigating the molecular mechanism of rice-false smut fungus interaction.

Full text

Journal of Agricultural Science and Technology A 2 (2012) 1211-1217
Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Progress in the Study of False Smut Disease in Rice
Xiaoyi Guo1, Yan Li1, Jing Fan1, Liang Li1, Fu Huang2 and Wenming Wang1
1. Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China
2. Department of Plant Pathology, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Received: August 30, 2012 / Published: November 20, 2012.
Abstract: Rice false smut disease is an increasing concern for research and production, not only because of the increasing epidemic
occurrence in rice production, but also the intriguing specific pathogenesis of the disease to be a unique pathological system to enrich
the molecular mechanism of plant-microbe interaction. Progresses have been achieved in the pathogen phylogenetic placement, the
alternative hosts, the pathogen morphology and diversity, the toxins generated by false smut balls, the artificial inoculation method,
and the pathogen transformation as well as rice resistance to the disease. However, it is still controversy on the infection process. It is
not clear how the life cycle of this pathogen is coupled with the disease cycle. This review summarized our current understanding on
the pathogen, the pathogenesis, and the rice resistance to the disease. Future work should pay attention to developing a more rapid
and effective system to evaluate rice resistance and susceptibility to the disease, screening of rice germplasm for disease-resistance
breeding, studying the resistance inheritance, and investigating the molecular mechanism of rice-false smut fungus interaction.
Key words: Rice, false smut disease, false smut ball, disease cycle, resistance, review.
1. Introduction
Rice false smut, also known as pseudo-smut, or
green smut, has been recorded in all rice-growing
countries worldwide [1-3]. It is categorized as a minor
disease due to its sporadic occurrence. However, the
disease has emerged as an increasing concern for rice
production and pathologists since the widespread
cultivation of hybrid rice and heavy application of
nitrogenous fertilizer [4], and became the most
devastating rice grain disease in the past two decades
in the major rice-growing regions in China [5].
Ustilaginoidea virens (Uv), the agent causing false
smut disease in rice, belongs to Ascomycete. Infection
by the fungus transforms individual grains of the
panicle into a yellowish smut ball, which changes to
yellowish orange, green, olive green and finally to
greenish black. The smut balls are often covered by
sclerotia that eventually fall to the ground, leaving
abundance of powdery chlamydospores. Uv is found
Corresponding author: Wenming Wang, professor,
research field: plant pathology. E-mail:
j316wenmingwang@sicau.edu.cn.
to produce both sexual (ascospores) and asexual
(chlamydospores) stages in its life cycle with multiple
propagules [6, 7]. Recently, Villosiclava virens was
proposed as the new name for the teleomorph of the
false smut fungus [8]. The damages by false smut
disease include the reduction of yield, the
contamination of grains and straws with ustiloxins, the
mycotoxins produced by Uv on diseased tissues and
the antimitotic cyclic peptides from its
chlamydospores, which are poisonous to both human
and animals [9, 10]. This review summarized the
research progress on the disease so as to help us to
explore the best research point in rice-Uv interaction.
2. The Agent Causing False Smut Disease in
Rice
2.1 The Pathogen
Rice false smut disease is caused by the fungal
pathogen Ustilaginoidea virens, which produces both
sexual ascospores and asexual chlamydospores in its
life cycle [11, 12]. The anamorph form,
Ustilaginoidea virens (Cooke) Takahashi, is widely
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Progress in the Study of False Smut Disease in Rice
1212
used for description of the causing agent of false smut
in rice and maize [3, 13]. The teleomorph had been
named Claviceps virens Sakurai ex Nakata and
Claviceps oryzae-sativae Hashioka because the
teleomorphic characteristics of U. virens are similar to
those of Claviceps [14]. However, phylogenetic
analyses using sequences of the large subunit of the
ribosomal RNA gene suggested that members of
Ustilaginoideae are distinct from teleomorphic genera
of Clavicipitaceae and should be recognized as a
monophyletic group within Hypocreales [15].
Molecular phylogenetic studies have also revealed that
Ustilaginoidea species formed a paraphyletic group,
and not congeneric with Claviceps based on the
ALDH1-1 gene, which encodes a member of the
aldehyde dehydrogenase family [16]. Based on
morphological and biological characteristics of the
teleomorph, Tanaka et al. [8] suggested Villosiclava
virens as the new name for the teleomorph of
Ustilaginoidea virens [8], which is accepted and the
name was used by recent reports [2, 6, 17, 18].
2.2 Diversity of the Pathogen
Strains isolated from different regions or different
varieties are distinguishable in genetic diversity and in
virulence to rice. Zhou et al. [4] analyzed 110 U. virens
isolates sampled from Liaoning and Beijing of North
China using amplified fragment length polymorphism
(AFLP) markers and revealed that these isolates can be
divided into three groups according to the genetic
distance and the isolates from the same region can be
placed into one group. The populations among different
locations where sexual stage of the pathogen is rare to
be found within ecological region are similar, and the
variation of this pathogen has mainly arisen via asexual
mechanisms [4]. 59 isolates from Sichuan of China on
three rice hybrids could be classified into six groups
based on their virulence to the tested rice hybrids [19].
2.3 False Smut Ball and Chlamydospore
False smut balls on rice panicles are the typical
symptom of the disease. The interiors of the false smut
balls are intertwined with hyphae at early stage and
then chlamydospores are formed. The chlamydospores
are almost smooth when young, and become warty
when mature [20]. Under bright-field light microscopy,
the conidia are found to be round to eliptical and warty
on the surface with diameters approximately ranging
from 3 to 5 mm. Under scanning electron microscopy,
the globose to irregularly rounded conidia are
ornamented with prominent spines. The spines are
pointed at the apex or irregularly curved, and
approximately 200-550 nm long [6, 20]. Under
transmission electron microscopy, both the spined
conidia and hyphae displayed lipid globules and
vacuoles in the cytoplasm enclosed by an
electron-transparent cell wall. Whereas, on the surface
of conidia, the electron-dense spines displayed
obclavate or irregularly protruding shapes with varying
heights along the conidial cell wall. Hyphae had
concentric bodies that showed an electron-transparent
core surrounded by an electron-dense layer [20]. In
addition, the chlamydospores produced in culture
medium are found prone to germinate in distilled water
and produces secondary spores. The conidia are
holoblastically and sympodially produced at the apex of
each conidiophore cell [6].
2.4 Sclerotium
The fungus has several kinds of propagules. In
addition to the chlamydospores in smut balls, sclerotia
are often formed on the colony surfaces, especially in
later autumn, with relatively lower temperatures and
high temperature differences between day and night
[21, 22]. The sclerotia existing on or under the soil
surface can germinate and form fruit-body, then
produce ascospores that contribute to the primary
infection sources [7]. Recently, Fu et al. [6] described
their observation on sclerotia in detail. The sclerotia
produced on the mature false smut balls are black
horseshoe-shaped and irregular oblong or flat, ranged
from 2 to 20 mm. The outer sclerotium wall appears

Progress in the Study of False Smut Disease in Rice
1213
rough at a low resolution under scanning electron
microscop, whereas, the surface is full of projections at
a higher magnification. The interior of each sclerotium
is intertwined with compact hyphae [6].
2.5 Ustiloxin
False smut balls formed on rice panicles produce a
number of metabolites that are toxic to both plants and
animals. The toxicity of the water extract from the
false smut balls was first reported to rabbits [23].
Injection of the water extract of false smut balls into
mice can cause damages to liver and kidney quite
similar to those observed in lupinosis caused by
phomopsin A, a mycotoxin produced by Phomopsis
leptostromiformis [9]. The toxic substance, named
ustiloxin, was isolated, and its structure was
determined by a combination of X-ray
crystallographic and amino acid analyses. Its structure
and biological activity are closely related to those of
phomopsin A, causing the lupinosis [24].
Subsequently, five ustiloxins, including ustiloxin A, B,
C, D and E, were isolated [25]. Ustiloxins exhibit
phytotoxic and mycotoxic effects through their potent
inhibition of microtubule assembly [26]. Ustiloxin A,
structurally resembles phomopsin A, inhibits the
formation of a particular intra-chain cross-link in
beta-tubulin and the alkylation of tubulin by iodo
acetamide. In addition, ustiloxin A inhibits exposure
of hydrophobic areas on the surface of the tubulin
molecule via strongly stabilizing the binding of
colchicine to tubulin [27]. Ustiloxin D was
synthesized via an unprecedented ethynyl aziridine
ring-opening of phenol derivatives including 15 steps
from commercially available compounds [28]. A
sensitive method to detect the presence of ustiloxin A
was developed for routine monitoring of the
contamination of ustiloxin A in forage rice silage [29].
3. Artificial Inoculation and Pathogenesis
3.1 Artificial Inoculation
It is necessary to develop an efficient inoculation
method for the investigation of the rice-Uv interaction.
Up to date, quite a few reports consistently conclude
that inoculation by injection of spores into the sheath
during booting stage is sufficient and efficient to
induce disease symptoms [30-32]. Inoculums can be
obtained by inoculation of Uv on XBZ solid medium
and transferring into potato sucrose (PS) broth at
26 C [31, 33]. After filtered the hyphae, conidia can
be collected from the filtrate by centrifugation and
re-suspending the conidia in fresh PS medium to a
density of 2-5 × 105 conidia mL-1 and used as the
inoculums [17]. Nevertheless, the conidia and
suspension of hyphae fragments obtained by breaking
the mycelia with a high-speed blender were also used
as inoculums in some labs [19]. The concentration of
the conidia is not as important as temperature and
humidity for the disease development. After injection
of the conidia, the inoculated plants should be put at
16 C for 2 days and then at 26 C with 100%
humidity for 5 days for the best development of the
false smut balls [34].
Wang et al. [35] obtained a white smut strain that is
virulent to rice plants. With this strain, they found that
disease severity is higher by injection than by
spraying inoculation as assessed by percent of infected
panicles. Low temperature exposure after inoculation
has a strong stimulatory effect on disease
development [35].
3.2 The Procedure of Pathogenesis
In spite of the increasing importance of false smut
disease, detail on the procedure of pathogenesis by Uv
has not been described until recently. The
chlamydospores survived through the winter in paddy
soils are believed to be the primary source of infection.
The chlamydospores germinate on coleoptile
epidermal cells of rice seedlings, and the infection
hyphae invade intercellular spaces and reach the
meristematic tissues of tillers during the vegetative
stage, and transfer to young panicles in leaf sheaths to
infect the developing florets [21]. It is hypothesized

Progress in the Study of False Smut Disease in Rice
1214
that the infection occurs in spikelets of panicles in the
leaf sheath before heading [11]. The fungus does exist
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 [36]. Conversely,
infection does not occur after heading by spraying
inoculation in fields [5, 32, 37]. After heading, it is
easy to clearly identify infected spikelets from
uninfected one on a panicle because the infected
spikelets contain the white fungus and finally form
smut balls [5, 21]. The pathogen can attack the root at
the seedling stage and lead to asymptomatic
colonization in the entire plant, as detected by
molecular techniques and histological observation [38,
39]. The pathogen is also found to infect rice
coleoptiles intercellularly at the earlier germination
stage [17].
Three labs reported that a green fluorescence
protein (GFP)-labeled strain of Uv was developed by
Agrobacterium-mediated transformation,
electroporation or polyethylene glycol
(PGE)-mediated protoplast transformation,
respectively [18, 40, 41]. By using the GFP-tagged
strain, Ashizawa et al. [18] observed that the infection
of Uv initiates with conidia landing and germinating
on the outer spikelet surface, then hyphae grow on the
outer spikelet surface and extend from spikelet apex
onto the inner spikelet surface, finally, hyphae grow
onto the floral organs and eventually form smut ball
[18]. At 72 hours post inoculation (hpi), many
germinated conidia are observed to present on spikelet
surfaces. By 120 hpi, the hyphae have gradually
grown to form mycelia on the spikelets. At 144 hpi,
hyphae have grown extensively to the apices of
spikelets, and then colonize onto the inner surfaces of
the lemma and palea through the small gap between
the lemma and palea. After heading, the floral organs
are covered with hyphae by 9 dpi, and finally all the
organs are completely covered with hyphae by 11 dpi
[18]. In addition, by examination of serial semi-thin
and ultra-thin sections of infected spikelets, Tang et al.
[17] identified that the primary infection sites for the
pathogen are the upper parts of the three stamen
filaments located between the ovary and the lodicules,
where the pathogen intercellularly grows inside the
filaments and extends along the filament base. After
the fungal infection, the host cells are degraded
gradually. However, the pathogen does not penetrate
host cell walls directly and does not form haustoria.
The stigma and lodicules are also occasionally
infected. In the smut balls, the ovary remains alive and
is never infected [17].
4. Resistance in Rice
Rice resistance to false smut disease is relatively
less reported and the inheritance of resistance is rarely
recorded. A few reports claimed that resistance level
is significantly different among different varieties
[42-44]. To study the inheritance of the resistance to
rice false smut, Li et al. [45] developed a population
of 157 recombinant inbred lines (RILs) derived from
the cross between resistant cultivar IR28 (Oryza sativa
supsp. indica) and susceptible landrace Daguandao (O.
sativa supsp. japonica) [45]. By using the RIL
population, a model of mixed two major genes and
polygenes (model E-1-3) is proposed to explain the
inheritance of the resistance against rice false smut
disease. The two major genes have equivalent additive
effect of 11.41, and their heritability is about 76.67%,
while the heritability of polygenes is about
22.86% [45].
5. Alternative Hosts
In addition to rice, maize and several weeds
common to rice fields are found to be the alternative
hosts of Uv. It was reported that Uv also attacks
Digitaria marginata, a common rice weed, which
occurs in 85% of the rice fields [46]. False smut
disease was also found on Panicum trypheron, a
common grass around paddy fields. Cross inoculation
studies revealed that chlamydospores from P.

Progress in the Study of False Smut Disease in Rice
1215
trypheron infected rice and vice versa. Thus P.
trypheron is an important source of inoculums
between seasons [47]. False smut disease was also
found on the tassels of Corn (Zea mays L.), but was of
little economic importance [13]. In addition, the
fungus was also reported to attack Echinochloa
crusgalli and Imperata cylindrica, two common
weeds on irrigation canals in Egypt [1].
6. Future Prospective and Conclusions
Generally, the study on certain disease includes the
pathogen, the host and the host-pathogen interaction.
Plants respond to pathogen infection with a
two-branched innate immune system [48]. The first
branch recognizes and responds to microbe-associated
molecular patterns (MAMPs), the molecules common
to many classes of microbes including non-pathogens,
resulting in MAMP-triggered immunity (MTI). The
second responds to pathogen virulence factors, either
directly or through their effects on host targets,
resulting in effector-triggered immunity (ETI) [48].
As for rice smut disease, the resistance mechanism is
still unknown. Studies on the pathogen side have
made great progresses in pathogen purification,
culture conditions, and the life cycle, as well as the
morphology of the chlamydospore, sclerotium and
ascospore [6, 20, 49]. Methods for transformation of
Uv for transgenic analysis and artificial inoculation
have been developed for better investigation of the
disease [34, 40, 41]. Comparing with that of the
pathogen, less progress is made on the host side,
although it does exist different sensitivities to Uv
attack among different rice varieties [43-45]. There
still misses a link for the disease cycle with the life
cycle of the pathogen, although it seems that Uv firstly
colonizes on the spikelet surface and extends into the
floret through the small gap between palea and lemma
and then infects filaments of the stamen and
eventually occupies the whole inside space of the
floret to form the smut balls [17, 18]. It is imaginable
that ascospores or chlamydospores enter the booting
sheath along with water flowing on the top leaves.
Though convincing experimental evidence is lack for
this hypothesis, there does exist air-borne spores of
the fungus and exhibits the daily maximum recurring
at 22:00 pm [50]. The hypothesis that water flowing
brings the primary infection source into the sheath is
consistent with the observation that the disease is
much more severe when rice heading stage is located
in rainy and high humidity days. In addition to rice,
maize and at least four gramineae weeds, there quite
possibly exist more weeds common in the rice fields
being the alternative hosts of Uv to produce plenty of
chlamydospores at rainy days as the primary infection
source. Thus, study foci in the future should be on: (1)
the development of a more rapid and effective system
to evaluate rice resistance and susceptibility to the
disease. Current sheath injection method is limited to
booting stage only and is time consuming; (2) the
screening of resistance rice germplasms that can
efficiently control false smut disease and be used for
breeding of resistance varieties; (3) resistant
mechanisms of the resistance germplasms; (4) the host
ranges and possible colonizing organs other than
florets and (5) the mechanisms of pathogenesis by Uv.
References
[1] Atia MMM, Rice false smut (Ustilaginoidea virens) in
Egypt, Journal of Plant Diseases and Protection 111
(2004) 71-82.
[2] D. Ladhalakshmi, G.S. Laha, R. Singh, A. Karthikeyan,
S.K. Mangrauthia, Isolation and characterization of
Ustilaginoidea virens and survey of false smut disease of
rice in India, Phytoparasitica 40 (2012) 171-176.
[3] M.C. Rush, A.K.M. Shahjahan, J.P. Jones, Outbreak of
false smut of rice in Louisiana, Plant Dis. 84 (2000) 100.
[4] Y.L. Zhou, Y.J. Pan, X.W. Xie, L.H. Zhu, J.L. Xu,
Genetic diversity of rice false smut fungus, ustilaginoidea
virens and its pronounced differentiation of populations
in North China, J. Phytopathology 156 (2008) 559-564.
[5] G. Deng, Present status of research on false smut in
China, Plant Prot 15 (1989) 39-40. (in Chinese)
[6] R. Fu, L. Ding, J. Zhu, P. Li, A.P. Zheng, Morphological
structure of propagules and electrophoretic karyotype
analysis of false smut Villosiclava virens in rice, J.
Microbiol. 50 (2012) 263-269.

Progress in the Study of False Smut Disease in Rice
1216
[7] R.A. Singh, K.S. Dubey, Sclerotial germination and
ascospore formation of Claviceps oryzae-sativae in India,
Indian Phytopathology 37 (1984) 168-170.
[8] E. Tanaka, T. Ashizawa, R. Sonoda, C. Tanaka,
Villosiclava virens gen. nov., comb. nov., the teleomorph
of Ustilaginoidea virens, the causal agent of rice false
smut, Mycotaxon 106 (2008) 491-501.
[9] K. Nakamura, N. Izumiyama, K. Ohtsubo, Y. Koiso, S.
Iwasaki, "Lupinosis"-like lesions in mice caused by
ustiloxin, produced by Ustilaginoieda virens: A
morphological study, Nat Toxins 2 (1994) 22-28.
[10] Y. Koiso, Y. Li, S. Iwasaki Ustiloxina, Antimitotic cyclic
peptides from false smut balls on rice panicles caused by
Ustilaginoidea virens, The Journal of Antibiotics 47
(1994) 765-773.
[11] G.L. Wang, Studies on the infection period and the
infection gate of the chlamydospores of Ustilaginoidea
virens (Cooke) Tak. on rice, Acta Phytophylacica Sinica
19 (1992) 97-100. (in Chinese)
[12] A. Biswas, False smut disease of rice: A review,
Environment and Ecology 19 (2001) 67-83.
[13] H.K. Abbas, G. Sciumbato, B. Keeking, First report of
false smut of corn (Zea mays) in the Mississippi Delta,
Plant Dis. 86 (2002) 1179.
[14] Y. Hashioka, Rice diseases in the world, VIII. Diseases
due to hypocreales, ascomycetes (fungal diseases, no. 5),
Riso. 20 (1971) 235-258.
[15] J.F. Bischoff, R.F. Sullivan, K.M. Kjer, J.F. White, Jr.
Phylogenetic placement of the anamorphic tribe
Ustilaginoideae (Hypocreales, Ascomycota), Mycologia
96 (2004) 1088-1094.
[16] E. Tanaka, C. Tanaka, Phylogenetic study of
Clavicipitaceous fungi using acetaldehyde dehydrogenase
gene sequences, Mycoscience 49 (2008) 115-125.
[17] Y.X. Tang, J. Jin, D.W. Hu, M.L. Yong, Y. Xu,
Elucidation of the infection process of Ustilaginoidea
virens (teleomorph: Villosiclava virens) in rice spikelets.
Plant Pathology, 2012, DOI:
10.1111/j.1365-3059.2012.02629.x.
[18] T. Ashizawa, M. Takahashi, M. Arai, T. Arie, Rice false
smut pathogen, Ustilaginoidea virens, invades through
small gap at the apex of a rice spikelet before heading, J.
Gen. Plant Pathol. 78 (2012) 255-259.
[19] D.H. Lu, X.Q. Yang, J.H. Mao, H.L. Ye, P. Wang,
Characterising the pathogenicity diversity of
Ustilaginoidea virens in hybrid rice in China, Journal of
Plant Pathology 91 (2009) 443-451.
[20] K.W. Kim, E.W. Park, Ultrastructure of spined conidia
and hyphae of the rice false smut fungus Ustilaginoidea
virens, Micron 38 (2007) 626-631.
[21] H. Ikegami, Studies on the false smut of rice X, invasion
of chlamydospores and hyphae of the false smut fungus
into rice plants, Res. Bull Fac. Agric. Gifu. Univ. 18
(1963) 54-60.
[22] Q.M. Miao, Studies on infection route of rice false smut,
Journal of Yunnan Agricultural University 7 (1992)
40-42. (in Chinese)
[23] M. Suwa, Experimental ustilaginoidea toxicosis, Igaku
Chuo Zasshi 13 (1915) 661-686. (in Japanese)
[24] Y. Koiso, M. Natori, S. Iwasaki, Ustiloxin: A phytotoxin
and a mycotoxin from false smut balls on rice panicles,
Tetrahedron Letters 33 (1992) 4157-4160.
[25] Y. Koiso, Y. Li, S. Iwasaki, K. Hanaoka, T. Kobayashi,
Ustiloxins, antimitotic cyclic peptides from false smut
balls on rice panicles caused by Ustilaginoidea virens, J.
Antibiot (Tokyo) 47 (1994) 765-773.
[26] Y. Li, Y. Koiso, H. Kobayashi, Y. Hashimoto, S. Iwasaki
Ustiloxins, new antimitotic cyclic peptides: Interaction
with porcine brain tublin, Biochem. Pharmacol. 49 (1995)
1367-1372.
[27] R.F. Luduena, M.C. Roach, V. Prasad, M. Banerjee, Y.
Koiso, Interaction of ustiloxin A with bovine brain
tubulin, Biochem Pharmacol 47 (1994)1593-1599.
[28] P. Li, C.D. Evans, M.M. Joullie, A convergent total
synthesis of ustiloxin D via an unprecedented
copper-catalyzed ethynyl aziridine ring-opening by
phenol derivatives, Org. Lett. 7 (2005) 5325-5327.
[29] S. Miyazaki, Y. Matsumoto, T. Uchihara, K. Morimoto,
High-performance liquid chromatographic determination
of ustiloxin a in forage rice silage, J. Vet. Med. Sci. 71
(2009) 239-241.
[30] J.C. Zhang, Z.Y. Chen, B.X. Zhang, Y.F. Liu, F. Lu,
Inoculation techniques used for inducing rice false smut
efficiently, Acta Phytopathologica Sinica 34 (2004)
463-467. (in Chinese)
[31] S. Wang, Y.J. Bai, X.Z. Liu, M.S. Zhu, X.B. Guo, The
comparative study on inoculums and inoculation
protocols of rice false smut, Liaoning Agricultural
Sciences (1996) 20-29. (in Chinese)
[32] Y. Fujita, R. Sonoda, H. Yaegashi, Inoculation with
conidiospores of false smut fungus to rice panicles at the
booting stage, Ann. Phytopath Soc. Japan 55 (1989)
629-634. (in Japanese)
[33] Y.L. Zhou, Q. Zhang, Preliminary report on the isolation
technique of Ustilagonoidea virens (Cooke) Tak, Chinese
Journal of Rice Science 13 (1999) 186-188. (in Chinese)
[34] T. Ashizawa, M. Takahashi, J. Moriwaki, K. Hirayae, A
refined inoculation method to evaluate false smut
resistance in rice, J. Gen. Plant Pathol. 77 (2011) 1-5.
[35] S. Wang, M. Li, H. Dong, X.Z. Liu, Y.J. Bai, Sporulation,
inoculation methods and pathogenicity of Ustilaginoidea
albicans, the cause of white rice false smut in China, J.
Phytopathology 156 (2008) 755-757.
[36] T. Ashizawa, Y. Kataoka, Detection of Ustilaginoidea

Progress in the Study of False Smut Disease in Rice
1217
virens in rice panicles before and after heading in the
field using nested-PCR technique with species-specific
primers, Jpn. J. Phytopathol 71 (2005) 16-19. (in
Japanese)
[37] D.W. Wang, S. Wang, J.F. Fu, Research advance on false
smut of rice, Liaoning Agricultural Sciences (2004)
21-24. (in Chinese)
[38] D. TeBeest, Infection of rice by Ustilaginoidea virens,
Phytopathology 100 (2010) 125.
[39] P. Schroud, D.O. TeBeest, Germination and infection of
rice roots by spores of Ustilaginoidea virens, AAES
Research Series 540 (2005) 143-151.
[40] Z. Zhang, X. Du, R. Chai, X. Mao, H. Qiu,
Agrobacterium tumefaciens-mediated transformation of
the pathogen of Ustilaginoidea virens, Chin. J. Rice Sci.
20 (2006) 440-442. (in Chinese)
[41] E. Tanaka, T. Kumagawa, C. Tanaka, H. Koga, Simple
transformation of the rice false smut fungus Villosiclava
virens by electroporation of intact conidia, Mycoscience
52 (2011) 344-348.
[42] D. Lu, J. Mao, P. Wang, Z. He, F. Chen, Resistant
difference of indica rice hybrid varieties to rice false smut,
Acta Phytophylacica Sinica 35 (2008) 289-294. (in
Chinese)
[43] R. Sonoda, Y. Fujita, H. Yaegashi, Varietal difference in
resistance of rice to false smut, Ann. Phytopathol Soc.
Jpn. 58 (1992) 561-562.
[44] K. Kurauchi, Y. Kudo, T. Kimura, T. Uemura, Difference
in resistance to false smut disease between rice cultivars
in Aomori prefecture, Ann. Rept. Plant Prot. North Jpn.
57 (2006) 17-21. (in Japanese)
[45] Y.S. Li, Z. Zhu, Y.D. Zhang, L. Zhao, C.L. Wang,
Genetic analysis of rice false smut resistance using mixed
major genes and polygenes inheritance model, Acta
Agron. Sin. 34 (2008) 1728-1733.
[46] S.A. Shetty, H.S. Shetty, An alternative host for
Ustilaginodea virens (Cooke) Takahashi, IRRI
Newsletter 10 (1985) 11.
[47] S.A. Shetty, H.S. Shetty, Role of Panicum trypheron in
annual recurrence of false smut of rice trans., Br. mycol.
Soc. 88 (1987) 409-411.
[48] J.D. Jones, J.L. Dangl, The plant immune system, Nature
444 (2006) 323-329.
[49] L. Chen, D.W. Hu, M.J. Chen, Electron microscope
observation of rice false smut balls and colony
microstructure of Ustilaginoidea virens, Mycosystema 26
(2007) 89-96.
[50] T. Sreeramulu, B.P.R. Vittal, Periodicity in the air-borne
spores of the rice false smut fungus, Ustilaginoidea
virens, Trans. Br. Mycol. Soc. 49 (1966) 443-449.