PEST MANAGEMENT: DISEASES
Germination and Infection of Rice
Roots by Spores of Ustilaginoidea virens
P. Schroud and D.O. TeBeest
ABSTRACT
False smut, caused by the fungus, Ustilaginoidea virens, is found in most of the
counties in Arkansas in which rice is grown. The velvety spore balls, indicative of the
disease, are found on susceptible cultivars near maturity and most of the cultivars grown
in Arkansas have been rated as susceptible or moderately susceptible although plants
are symptomless until heading. The disease cycle is not well described for this plant
disease. The purpose of this research project is to examine the mechanism(s) by which
rice plants are infected in the fields with the long-term goal of clarifying the disease
cycle in Arkansas. Black spores were isolated from false smut-infected panicles collected
in 2005 and used to measure germination and behavior on rice roots. The results of
the germination tests indicate that some spores germinate in liquid suspensions within
four hours while others only begin to germinate 40 hours later. Some spores germinated
rapidly on rice roots and produced infection structures that penetrated roots within 48
hours, others germinated more slowly. These results, though preliminary in nature,
suggest that spores coming into contact with rice roots may be a mechanism through
which rice is infected in the field.
INTRODUCTION
False smut has now been found in most of the counties in which rice is grown in
Arkansas (Cartwright, 2002; Wilson, 2005). False smut appears to be spreading within
the state. Diagnostic symptoms of the disease are the velvety spore balls that appear
on the panicles at harvest time. The spore balls can be up to 1.5-cm in size at maturity.
There are no known symptoms of infection prior to the appearance of the spore balls
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near harvest. Recent review of the literature and the presence of false smut within
Arkansas indicate that a clearer understanding of false smut disease is needed to help
control its spread and importance.
False smut is caused by the fungus, Ustilaginoidea virens. The disease cycle, which
begins with the infection of plant tissues and culminates in new sporulation, has not been
fully characterized (Lee and Gunnell, 1992). In false smut, spore balls replace one or
more kernels on mature heads as a result of infection of the plant (Fig. 1). From one to
more than 20 spore balls can be found on a single infected rice panicle. The spore balls
can be pale yellow, orange, or greenish-black in color depending on their age. Mature
spore balls usually are olive-black in color. The spore balls are composed of three sporeproducing layers surrounding a hard core called a sclerotium (or pseudomorph) at the
center of some of the spore balls (Lee and Gunnell, 1992). Spores are readily released
from the spore balls and often are deposited on other seeds of an infected panicle to
such an extent that these seeds are often blackened (Fig. 1). Spore balls can survive
up to four months in soil and it is presumed that the sclerotia can survive much longer
(Lee and Gunnell, 1992). In addition, spores (sometimes called chlamydospores) found
in the spore balls can produce one or more secondary spores after germination. Spores,
secondary spores, and ascospores are all reportedly capable of infection (Anonymous,
1992). This fungus is believed to invade the ovary at early flowering, however, Ikegami
(1962) reported that plants could be infected by inoculating coleoptiles at the earliest
stages of seed germination, but that the levels of infection were very low after coleoptiles
reached 10 mm in length. Later inoculations of the panicles with spores during booting
or at flowering were less successful in establishing the disease.
Control measures are not generally warranted for this disease although fungicides applied at heading have been reported to be successful in controlling this disease
(Cartwright, 2002). Recent reports (Cartwright, 2002; Wilson, 2005 ) have shown that
many of the rice cultivars grown in Arkansas are susceptible or moderately susceptible
to false smut.
The objectives of this research were 1) to determine if viable spores of U. virens
could infect root tissues of the rice cultivar, ‘Francis’, because we were unable to obtain
infection of rice panicles and seeds by inoculating flowers as previously reported, and
2) to describe, after histological examination, the initial steps in the infection process
of root tissues.
PROCEDURES
Viability of Spores
The viability of the black spores from spore balls collected in 2005 from panicles
found in Arkansas rice fields was assessed by placing spores in water, 1% sucrose, and
0.8% nutrient broth and measuring the number of spores that produced visible germ
tubes. Samples were collected from the solutions 0, 4, 8, 12, and 24 hours after suspension and observed under a microscope at 200X magnification.
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Plants
Seeds of the rice cultivar, Francis, were obtained from the Foundation Seed Program of the University of Arkansas, Division of Agriculture. Seeds were germinated
on filter paper disks in 9-cm petri dishes placed beneath fluorescent lamps (15-hour
photoperiod) at 24°C until roots were approximately 1-cm long.
Inoculum
Spores of the fungus used in the germination and root inoculation experiments
were obtained from spore balls collected in fields in 2005 in Lonoke County, Ark. Spore
balls were collected in the field, air-dried for several weeks, and then placed into storage at -20°C until needed. Spore suspensions (approximately 2 million spores per ml)
for the experiments were obtained by placing 1 spore ball in 10 to 15 ml of deionized
water, 1% sucrose, or 0.8% nutrient broth as needed. Spores were suspended by vortexing for 30 to 45 seconds. The experiment was repeated three times, one spore ball was
used for all treatments in each repetition. Inoculum concentrations were determined
and standardized with a hemacytometer for each of the experiments.
Germination
Germination of spores from the spore balls was assessed at 25°C in water and 1%
solutions of sucrose and 0.8% nutrient broth. Spore suspensions were made by vortexing
spore balls briefly to fully suspend spores in the solutions. Tubes containing the spore
suspensions were then incubated beneath a 20W GE fluorescent lamp (F20T12-PL/AQ)
with a 15-hour photoperiod. At the requisite times, the tubes were again briefly vortexed
and samples of spores were taken from the solutions and placed on glass slides for
microscopic examination. Spores were considered to have germinated if germ tubes
were visible external to the spore wall.
Inoculation of Roots
Roots were collected from germinated seedlings and surface sterilized in 0.6%
sodium hypochlorite solution for five minutes, rinsed briefly in distilled water, and then
placed in spore suspensions for 5 minutes. After 5 minutes, roots were removed from
the spore suspensions, rinsed briefly in distilled water, and incubated in distilled water
until needed. At selected time intervals, ten roots were collected from distilled water
in which they were incubated and placed directly into 95% ethanol.
Statistical Analysis
Statistical analyses were accomplished using the mixed procedure of the SAS
system.
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Images
Root samples were examined after additional clearing with 95% alcohol, rehydrated in distilled water, and then stained in aqueous aniline blue for 3 minutes.
Samples were briefly de-stained in water to eliminate excess stain from root surfaces.
Roots were examined microscopically and color images of spores, germinated spores,
and infections were obtained with a Zeiss Axioskop 2+ microscope using automontage
functions with planachromat objectives at 10X magnification. Images were recorded
on a JVC digital camera, model KYF75U.
RESULTS AND DISCUSSION
Viability of Spores
The data from replicated experiments show that the black spores collected in 2005
and used in this study germinated very rapidly in water, sucrose, and nutrient broth solutions (Table 1). Within four hours, germination of spores in a sucrose solution, nutrient
broth, and water was approximately 30%. In water, 30.6%, 34.4%, 38.8%, and 33%
germinated after 4, 8, 12, and 24 hours, respectively. In 1% sucrose, 29.6%, 36.8%,
39.6%, and 43.0% of the spores germinated after 4, 8, 12, and 24 hours, respectively.
In nutrient solution, 30.8%, 37.9%, 40%, and 42.7% of the spores germinated after 4,
8, 12, and 24 hours, respectively. Statistical analysis revealed no statistical differences
in germination over time in any of the germination media and a high degree of variation in the percentage of the spores that had germinated between replications within
treatments and between treatments. We attributed these differences to variation within
the spores collected from different spore balls used in the experiments. We consider it
important to note that spore germination was asynchronous in all treatments and that
the average germination rate of spores was less than 50% in all treatments.
Infection of Rice Roots by Black Spores
Experiments were conducted to determine the viability of spores and behavior of
germinated spores on rice root surfaces. The data show that approximately 15% of the
spores found on the root surfaces had germinated on the root surface and in the matrix
of the root surface within 24 hours after inoculation (Table 2). Between 2 and 7 days
after inoculation, only 16% to 25% of the spores found on the root surface had germinated. These results are in contrast to the data describing germination of spores in water,
sucrose, or nutrient broth solutions in which germination was more rapid and reached
higher levels (Table 1). Germination resulted in the formation of thick germ tubes, many
of which branched several times similar to the germination showed in Fig 2D.
Spores germinated on the root surface by the formation of a germ tube (Fig. 2A).
This was followed by the formation of hyphae that rapidly branched (Figs. 2B,C, and
D). The first visible evidence of infection of rice roots occurred between 24 and 48 hours
after inoculation. By 48 hours after inoculation, approximately 12 % of the spores that
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had germinated on roots had produced an infection hyphae within root cells (Table 2).
The results also show that the fungus grows through the root cells in all directions from
the site of entry (Fig. 2). The data and microscopic examination of roots also showed
that after 48 hours, some spores had already shown evidence of infecting roots while
other spores were just beginning to produce germ tubes. This shows that germination
of the black spores on root surfaces was not synchronous as had been found in water
and in sucrose or nutrient broth solutions.
Ikegami (1962) has previously shown that the highest level of infection, as measured by the incidence of spore balls on panicles, occurred when coleoptiles between
3- and 4-mm in length were inoculated while plants grown from coleoptiles inoculated
when they were longer than 10-mm were not infected. Zhou et al (2003) were able to
detect U. virens in stems, sheaths, and florets near injection sites using PCR techniques
after they artificially inoculated plants by injecting leaf sheaths and boots at the booting stage.
Covered smut of barley, caused by Ustilago hordei, can cause considerable losses
in barley when it infects heads and displaces seeds with fungal fruiting masses similar
to false-smut spore balls. Paradoxically, artificial inoculation of clean seeds with spores
often resulted in very low levels of infection (Tapke, 1942). This difficulty was solved
by development of a specialized inoculation technique that used vacuum to help place
spores beneath the hulls of seeds and resulted in higher levels of infection and more
effective study of that disease. In addition, Willits and Sherwood (1999) have recently
detected U. hordei in leaves, necks, and flowers of plants grown from seeds inoculated
as described by Tapke and Bever (1942). It is therefore possible that spores of U. virens
found on the surface of seeds can be a possible source of infection of rice plants similar
to the mechanism for infection of barley by U. hordei and we are currently investigating that hypothesis.
SIGNIFICANCE OF FINDINGS
Free moisture is a common requirement for germination of spores of many plant
pathogens. The requirement for free moisture for spores of U. virens appears to be
relatively short, from 4 to 24 hours. However, it is also clear that spore germination is
not synchronous and in this study never reached 100%.
Careful observation of rice panicles naturally infected by false smut revealed that
healthy seeds adjacent to spore balls were often heavily infested on the surface by spores
of false smut in the field. The fungus could be considered seed-borne as a result. It is
also possible that the disease may be dispersed from field to field by planting infected
and/or infested seed.
This work is the first known report of infection of roots by spores of U. virens.
Histological examinations show that roots of rice seedlings were infected by germinating spores of U. virens within a few hours after inoculation. This work does not clarify
whether root infection can lead to infection of panicles and production of spore balls
on plants grown from infested seeds. Those experiments are in progress.
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Based on previous reports (Lee and Gunnell, 1992; Ikegami, 1962) and our histological data, it now appears possible that the fungus can infect rice plants in at least
two separate ways: through roots and through flowers.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the support of the Arkansas Rice Research
and Promotion Board, the Arkansas Agricultural Experiment Station, Dr. R. Cartwright,
Dr. Y. Jia, Dr. F. Spiegel, and Mr. C. Parsons for their contributions to this work.
LITERATURE CITED
Cartwright, R.D, C.E. Parsons, B.J. Dodgen, F.N. Lee, and E.A. Sutton. 2002. Rice
disease monitoring and on-farm variety evaluation in Arkansas. In: R.J. Norman
and J.-F. Meullenet (eds.). B.R. Wells Rice Research Series 2001. University of
Arkansas Agricultural Experiment Station Research Series 495:138-143. Fayetteville, Ark.
Ikegami, H, 1962. Studies on the false smut of rice. V. Seedling inoculation with the
chlamydospores of the false smut fungus. Annals of the Phytopathological Society
of Japan. 27:16-23.
Lee, F.N. and P.S. Gunnell. 1992. False Smut. p. 28 In: R.K. Webster and P.S. Gunnell (eds.). Compendium of Rice Diseases. American Phytopathological Society,
St. Paul, Minn.
Tapke, V.F. and W.M Bever. 1942. Effective method of inoculating seed barley with
covered smut (Ustilago hordei). Phytopathology 32:1015-1021.
Willets, D.A. and J.E. Sherwood. 1999. Polymerase chain reaction detection of Ustilago hordei in leaves of susceptible and resistant barley varieties. Phytopathology
89:212-217.
Wilson, C.E., Jr., R.D.Cartwright, J.W. Gibbons, A.L. Richards, D.L. Frizzell, J.W.
Branson, S. Runsick, and C.E. Parsons. 2005. Evaluation of rice varieties for
performance and disease reaction on farms. In: R.J. Norman, J.-F. Meullenet, and
K.A.K. Moldenhauer (eds.). B.R. Wells Rice Research Studies 2004. University
of Arkansas Agricultural Experiment Station Research Series 529:149-158. Fayetteville, Ark.
Zhou, Y.L., K. Izumitsu, R. Sonada, T. Nakazaki, E. Tanaka, M. Tsuda, and C.
Tanaka. 2003. PCR-based specific of Ustilaginoidea virens and Ephelis japonica.
J. Phytopathology 151:513-518.
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Table 1. Germination of black spores of Ustilaginoidea
virens in water, 1 % sucrose, and 0.8% nutrient broth.
Germination medium
Time
Water
1% Sucrose
0.8% Nutrient broth
0.0z
30.6
34.4
38.8
33.0
0.0
29.6
36.8
39.6
43.0
0.0
30.8
37.8
40.0
42.7
(hrs)
0
4
8
12
24
z All
values are not significantly different according to the Mixed Procedure, SAS statistical
systems, P = 0.05, (0 hours removed before analysis).
Table 2. Germination and infection of rice root surfaces by
black spores of Ustilaginoidea virens of the rice cultivar, Francis.
Days after inoculation
Spore germination
---------------------------------------- (%)------------------------------------------
1
2
3
4
7
15.7z
16.3
23.7
19.3
25.0
Germinated spores with infection hyphae
0
11.8
12.3
11.5
8.1
z All
values are not significantly different according to the Mixed Procedure, SAS statistical
systems, P = 0.05, (0 hours removed before analysis).
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Fig. 1. An image of a rice panicle infected by Ustilaginoidea virens
in the field showing the black spore balls found near harvest. Seeds located
on the same panicle, spike, or spikelets near the spore ball are often
visibly blackened by spores from the nearby spore ball.
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A
B
C
D
Fig. 2. Images of Ustilaginoidea virens spores (black-stage) on rice roots at
various stages of development 43 hours after inoculation. All samples were
stained with aniline blue (bars = 10µm). A. germ-tube initials (arrowheads)
forming on black-stage spores of U. virens; B. a germ-tube (≈40µm in length)
formed by a green-stage spore of U. virens. The double arrows indicate a septation
in the germ tube. C. Penetration of the root epidermis by hyphae of U. virens (double
arrowheads) and a germ-tube initial (single arrowhead) produced by black-stage spores
of U. virens. D. A black-stage U. virens spore (double arrowhead) that has germinated
and produced septate, branching hyphae that have penetrated the root tissue adjacent to
another spore (single arrowhead) of U. virens which has produced a germ-tube initial.
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