Abiotic Transmission of Rice yellow mottle virus Through Soil and Contact Between Plants
The roles of guttation fluid, irrigation water, contact
between plants and transplantation into contaminated soil in the transmission
of Rice yellow mottle virus (RYMV) were assessed. RYMV presence
and infectivity were tested by Enzyme-Linked Immunosorbent Assay (ELISA)
and by inoculation to susceptible rice cultivar BG90-2. The virus was
readily detected in guttation fluid collected from infected rice plants.
Transmission tests from this fluid led to high disease incidence (86.6%).
Irrigation water collected at the base of infected plants growing in pots
was less infectious, as inoculations led to disease incidences below 40%.
No virus was detected and could be transmitted from field-irrigation water.
Up to 44% healthy rice plants whose leaves were in contact with those
of infected plants became infected but, no transmission occurred through
intertwined roots. Transplantation of rice seedling into virus-contaminated
soil also led to plant infection. However, virus survival in the soil
decrease rapidly and infectivity was completely lost 14 days after soil
contamination. Altogether, these results indicated that high planting
densities of rice are likely to favour secondary spread of rice yellow
mottle disease. Transplantation of rice seedlings not earlier than 2 weeks
after soil preparation should prevent soil transmission of the virus.
Although guttation fluid is highly infectious its contribution to virus
infectivity in irrigation water is negligible as field-irrigation water
was not found to be an infectious source for RYMV.
Rice (Oryza sativa L. and O. glaberrima Steud.)
is a staple crop in most countries in Africa. Since the early 1990s, rice
production is severely affected by rice yellow mottle which is one the
most damaging disease of rice in the continent. The disease is caused
by Rice yellow mottle virus (RYMV) first reported in Kenya 40 years
ago (Bakker, 1970). RYMV is now widespread all over Africa south of the
Sahara (Kouassi et al., 2005). Common symptoms induced by RYMV
are yellow discoloration and mottling of the leaves of infected rice plants.
Additionally, symptoms may also include stunting, reduced tillering and
poor panicle exertion (Hull and Fargette, 2005; Kouassi et al.,
2005). Therefore, yield may be dramatically reduced by 25 to 100% (Konate
et al., 1997; Calvert et al., 2003).
RYMV belongs to the genus Sobemovirus of plant viruses
(Hull and Fargette, 2005). It is a stable and highly infectious virus
which is easily transmitted by mechanical inoculation. The longevity in
vitro is 56 days and the dilution end point fluctuates between 10-6
and 10-9 depending on the inoculum source (Hull and Fargette,
2005). RYMV induces systemic infections in rice and wild host species
and can invade the seeds produced by infected plants. However, it has
been demonstrated that seed transmission does not occur (Konate et
al., 2001; Allarangaye et al., 2006).
Current control measures against RYMV are mainly directed
to breeding for resistance. Sources of resistances to the virus have been
identified in a limited number of cultivars from the two cultivated rice
species O. sativa and O. glaberrima (Ndjiondjop et al.,
1999; Ioannidou et al., 2000). Unfortunately, RYMV isolates capable
to overcome the identified resistances have been reported at relatively
high frequencies (Traore et al., 2006a), which is a threat to the
success of genetic control of rice yellow mottle disease. Consequently,
in addition to using resistant rice cultivars, RYMV control should also
include other means such as phytosanitary measures in an integrated management
strategy. However, phytosanitary measures themselves are not fully known
due to limited knowledge concerning the epidemiology of the disease.
RYMV transmission in the field involve a few insect species,
mainly chrysomelid beetles (Bakker, 1974), mammals such as cows, donkeys
and rats (Sarra and Peters, 2003) and man himself through some cropping
practices (Traore et al., 2006b). Wind-mediated transmission of
RYMV has been also reported by Sarra et al. (2004). Several other
means of transmission and sources of infection are suspected. These, include
irrigation water flowing from infected fields to healthy ones, contact
between plants, using dung from cows that fed upon infected stubble and
planting rice in contaminated soil (Abo et al., 2000; Calvert et
In this study, we studied the involvement of irrigation
water, contact between plants and contaminated soil in the abiotic transmission
of RYMV. Implications of the findings in the epidemiology and management
of rice yellow mottle disease are discussed.
MATERIALS AND METHODS
Plant inoculation: This study was conducted during two consecutive
years 2003-2004 at Kamboinse agricultural research station (Burkina Faso).
In all experiments, RYMV isolate Bf5 from our virus collection and rice
cultivar BG90-2 highly susceptible to RYMV, were used. The virus isolate
first was propagated by mechanical inoculation. Inoculum was prepared
by grinding 1 g of frozen infected rice leaves in 10 mL of 100 mM phosphate
buffer pH 7. Carborundum (600 mesh) was added to the inoculum and the
mixture was rubbed onto leaves of 14 day-old plants. Inoculations were
done in an insect-proof screenhouse with temperature between 25 and 30°C
and 80-90% relative humidity. Symptoms developed fully at 21 days post-inoculation
(dpi) and infected plants were used as source of inoculum in the experiments.
Testing the presence of RYMV in guttation fluid and irrigation water:
Guttation fluid was collected from infected rice plants. To ease fluid
collection, the plants were previously covered with a plastic bag overnight.
Samples of standing water were collected from pots in which infected plants
were grown. Five days before collecting the samples, the plants were irrigated
either by sprinkling the water from top (top-irrigation) or by pouring
it directly into the pots (pot-delivered water). Field samples of irrigation
water were also collected in plots where rice yellow mottle incidence
was 80 to 100%. Altogether, irrigation water was sampled in 20 mL vials
from 10 fields.
To test the presence of RYMV, samples of guttation fluid
and irrigation water were analyzed by double antibody sandwich enzyme
Linked-immunosorbent assay (DAS-ELISA) (Clark and Adams, 1977). A high
titre polyclonal antibody prepared in rabbit (Traore, 2006) was used for
coating microtitration plates. The same antibody was coupled to alkaline
phosphatase and used as conjugate. All samples were tested in triplicates
and were considered positive if absorbance readings (A405 nm) from each
replicate were more than the negative/positive threshold (mean A405 nm
readings from healthy controls plus three times standard deviations).
Biological tests were also conducted to diagnose RYMV in samples of guttation
fluid and irrigation water. Each sample was mixed with carborundum (600
mesh) and rubbed onto leaves of 10 two-week-old rice seedlings. Appearance
of symptoms was monitored for 45 dpi and leaves were taken from inoculated
plant for serological confirmation tests.
Testing RYMV transmissibility through contact between leaves and between
roots: RYMV-infected rice plants grown in plastic pots in the greenhouse
were pruned to get rid of all symptomless leaves. The effect of contact
between leaves on transmission of the virus was tested by displaying 20
groups of five pots containing healthy rice seedlings in circles around
an infected plant. The close positions of the pots allowed the leaves
of all healthy seedlings to be in contact with those of diseased plants.
To test RYMV transmissibility by contract between roots,
three to five rice seeds were sown in plastic pots. At 14 days post-germination,
the plants were thinned to two per pot. A transparent plastic tube was
placed over one of the plants to isolate its above-ground part from that
of the second plant. In this way, the two plants were in contact through
the roots only. Therefore, RYMV was inoculated to the non-isolated plant.
The experiment was done in 100 replicates and isolated plants were observed
during 45 days for symptom development. RYMV detection performed by ELISA
and bioassays was positive in leaves, stems and roots of inoculated plants
when tested at 21 days post-inoculation.
Tests of RYMV transmission through contaminated soil: Infected
leaves were pooled and cut into small pieces from which inoculums were
prepared. In each case, four extracts were prepared by grinding leaf pieces
(100, 10, 1 and 0.1 g, respectively) in one litre of tap water. Extracts
obtained were referred to as E1, E2, E3 and E4, respectively. Corresponding
healthy leaf extracts were also prepared in the same way to be used as
negative controls. Each extract including plant debris was mixed with
5 kg of moistened sterile soil in a bucket and all soil preparations were
done the same day. Then, the two following experiments were performed:
In experiment 1, the transmissibility of RYMV by sowing
rice seeds in contaminated soil was tested. Seeds were sown in the buckets
containing contaminated and healthy soil (dilution E1), respectively.
Twenty seeds were put per bucket and treatments were run in triplicates
for each leaf extract.
Experiment 2 was conducted to assess the effect of
time of transplantation of rice seedlings into contaminated soil on the
transmission of RYMV. All seedlings were transplanted at 14 days after
sowing. The experimental design was composed 100 buckets arranged into
four blocks of 25 buckets each (one block per leaf extract). In every
block, buckets were further arranged into sub-blocks of five, three of
which contained soil mixed with the adequate dilutions of RYMV-infected
leaf extracts whereas the two remaining contained soil mixed with healthy
leaf extracts. At day zero (when leaf extracts were incorporated into
the soil), rice seedlings were transplanted in a first group of four sub-blocks
(corresponding to extracts E1, E2, E3 and E4, respectively). A total of
10 seedlings were transplanted per bucket. Four other transplantations
were done similarly at days 2, 4, 8 and 14, respectively. During the whole
experiment, rice plants were watered once a day, all buckets receiving
similar amounts of water.
Statistical analyses: Data on disease incidence (proportions of
infected plants) were analysed by using the X2 test for difference
between proportions (Fleiss, 1981). Differences in mean disease incidence
were compared using analysis of variance after angular-transformation
of the data to take into account the correction for normality (Zar, 1999).
Detection and infectivity of RYMV in guttation fluid and irrigation
water: ELISA detection of RYMV in guttation fluid and irrigation water
samples was successful in guttation fluid only. The virus was readily
detected in guttation fluid samples with absorbance readings (A405 nm)
higher than 1.2 after 2 h of substrate incubation (Table
1). By contrast, all irrigation water samples gave
Detection and infectivity
of RYMV in guttation fluid and irrigation water
a: Irrigation water
was collected from the field and from pots in the greenhouse
one day after irrigating rice plants by sprinkling or delivering
the water directly into pots, b: Lowest and highest
absorbance readings (A405 nm) after 2 h of substrate incubation.
The positive/negative threshold for virus detection is indicated
in parenthesis, c: No. of infected plants/total number
of susceptible rice plants inoculated in each case; NT: Not
A405 nm readings that were below the positive/negative threshold,
indicating the lack of virus detection. In mechanical inoculation tests,
guttation fluid and all water samples, except irrigation water collected
from the fields, were found to be infectious. Inoculated rice seedlings
showed mottle symptoms between 8 and 12 dpi. As indicated by the X 2test,
proportions of infected plants depended on virus inoculum sources (X2
= 44.98, p<0.01, df = 4). Guttation fluid was the most infectious as disease
incidence reached 86% and was more than twice that of any other case.
None of the inoculated plants became infected when irrigation water collected
from the field was used.
Transmission of RYMV by contact between plants: Up to 44% of the
healthy rice plants which were in contact with RYMV-infected plants through
the leaves became infected and showed clear yellow mottle symptoms. First
symptoms appeared 2 weeks after the plants were brought into contact.
Serological tests conducted on the remaining symptomless plants (56%)
were all negative. When diseased and healthy plants were put into contact
through their roots only, no virus transmission occurred. Despite the
fact that the roots were strongly intertwined, none of the 100 plants
tested became infected, as indicated by the absence of symptoms and the
lack of virus detection by ELISA.
Transmission of RYMV through contaminated soil: Sowing rice seeds
in soil contaminated with RYMV-infected leaf extracts did not result in
any infection of the emerged seedlings. All seedlings remained symptomless
throughout the time of the experiment. Therefore, one leaf was taken from
each seedling and the batch was used to prepare an extract. This extract
appeared to be serologically negative, confirming the absence of RYMV
in the plantlets.
On the contrary, rice yellow mottle infections occurred
clearly following transplantation of rice seedlings in soil contaminated
with infected leaf extracts
Rice yellow mottle
incidence in plots where rice seedlings were transplanted into
a: Leaf extracts
(E1, E2, E3 and E4) were prepared by grinding infected rice
leaves (100, 10, 1 and 0.1 g, respectively) in 1 litre of water.
Control (E1) was prepared as E1 but with uninfected leaves and
all extracts were mixed with soil in pots, b: Rice
seedlings were transplanted into contaminated soil at different
times: same day (Day-0) and two, four, eight and fourteen days
later (Day-2, Day-4, Day-8 and Day-14, respectively)
(Table 2). Typical yellow mottle symptoms
appeared on transplanted plantlets one week after transplantation. As
revealed by analysis of variance, there were significant effects of leaf
extract (F = 71.92, df = 4, p< 0.0001) and time of transplantation (F
= 28.20, df = 4, p< 0.0001). Especially, on the one hand, the disease
was induced only if rice seedlings were transplanted in soil contaminated
with infected extracts E1 and E2. On the other hand, disease incidence
dropped sharply from Day-0 to Day-14 at which time no disease could be
Guttation fluid from RYMV-infected plants was first reported to be a
source inoculum by Bakker (1974). But its role in the spread of rice yellow
mottle disease in the field was not known. Recently, Sarra et al.
(2004) indicated that guttation fluid had no significant effect on wind-mediated
transmission of RYMV. Present results are consistent with those of Bakker
(1974). Guttation fluid reacted strongly in serological tests and also
a high disease incidence was obtained when it was used as source of inoculum.
Infectiousness of top-irrigation and pot-delivered irrigation waters is
probably due to the fact that they contained some guttation fluid. It
is likely that top-irrigation water contained more guttation fluid swept
from the leaves by irrigation water coming from above the plants. Pot-delivered
irrigation water was expected to contain less guttation fluid which self
fell from the leaves. As a result, a higher disease incidence was obtained
upon inoculation of top-irrigation water (Table 1). Although
the disease was induced by inoculating both types of irrigation water,
serological tests conducted on these waters were negative. This indicated
that the biological test of RYMV is more sensitive that serological detection
of the virus. Like top-irrigation and pot-delivered irrigation water,
field-irrigation water probably contained some infectious guttation fluid
fallen from infected plants. However, although collected from highly infested
plots, field-irrigation water was ELISA negative and no infection was
induced from it (Table 1). This result is consistent
with the view that the virus from infectious guttation fluid was likely
too much diluted beyond its dilution end point. Consequently, irrigation
water flowing from infested plots is not to be considered as inoculum
source for the infection of plants in downstream plots.
RYMV was transmitted at a relative high rate (44%) though
contact between leaves of infected and healthy rice plants. This is consistent
with earlier reports by Abo et al. (1998, 2000) and Sarra et
al. (2004). The latter authors found virus transmission through contact
between leaves can increase by five to eightfold when rice planting densities
were doubled from 16 to 33 plants m-2. In contrast to contact
between leaves, no transmission was obtained through intertwined roots
of diseased and healthy rice plants. This result does not support the
findings of Abo et al. (2000). In order to induce any infection,
the roots needed to be wounded. In present experiments, such wounds were
not produced by the intertwining of the roots and they could not be produced
by any other factor as rice plants were grown in sterile soil. In field
conditions, root-feeding organisms such as nematodes can produce wounds,
thus leading to possible infection, even if nematodes themselves are not
reported to be vectors of RYMV (Bakker, 1974).
Sowing rice seeds into contaminated soil did not result
in any infection of the emerged seedlings. Therefore, direct seeding rice
fields instead of using the seedbed system is a way to reduce primary
infections by RYMV. It was recently reported that the process of setting
rice seedbeds up and transplantation of seedlings into the fields is a
major cause of RYMV dissemination (Traore et al., 2006b). Unfortunately,
direct seeding is not advisable in irrigated rice system because it causes
lower yields compared to transplantation of rice seedlings and also because
of more difficulties in controlling the weeds.
Transplantation of rice seedlings into contaminated soil
can lead to the establishment of rice yellow mottle disease. Notably,
this occurs with soil contaminated with highly concentrated inoculum such
as E1 and E2. This may happen in the field if highly infected rice stubble
is incorporated to the soil during ploughing (Reckhaus and Andriamasintseheno,
2001). Sarra (2005) also reported that contamination of the soil can result
from the use of dung taken from cattle which fed upon infected plant material.
Fortunately, our results indicated that the survival of the virus in the
soil decreased rapidly (Table 2). At two weeks after
the soil was contaminated, transplantation of rice seedlings did not result
in any infection. Consequently, as a prophylactic control measure, transplantation
of rice seedlings should be done at least two weeks after the field has
The authors are grateful to Mr. Moussa Sio Samake and
Mr. Tieman Traore at the Regional Centre of Agronomic Research of Sikasso
(Mali) for their skilful technical assistance. Fruitful discussions with
Dr. Abdoulaye Hamadoun, head of the Sikasso regional centre for agronomic
research and Dr. Yacouba Doumbia, head of lowland rice programme are also
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