Effects of Temperature and Relative Humidity on the Effectiveness of Peanut bud necrosis virus Inoculation on Peanut
The difference in PBND incidence in the rainy and dry seasons in Thailand has led to a hypothesis that temperature and relative humidity might be the causal factors. The objective of this study was to investigate the effects of temperature and relative humidity on the effectiveness of mechanical inoculation of PBNV on peanut. Two experiments were conducted in the rainy and dry seasons during 2004 to 2005, using the susceptible peanut genotype Tainan 9. The first experiment evaluated the effect of low temperature on the effectiveness of PBNV transmission by mechanical inoculation. The second experiment compared two temperature and relative humidity conditions in the rainy season and in the dry season. The results showed that exposing the PBNV-inoculated plants to low temperature (25°C, 90% RH) during a 12 h light period and 20°C, 90% RH during a 12 h dark period) for all the time during the experimental period did not increase the infected plants. Two climate conditions representing the dry season (daytime, 32°C, 44% RH, night-time, 22°C, 65% RH) and the rainy season (daytime, 36°C, 46% RH, night-time, 25°C, 70% RH) in Thailand also showed no difference in the infected plants. These results indicated that low temperature or relative humidity did not affect the transmissibility of PBNV. The difference in disease incidences in the rainy and the dry seasons in Thailand could not be explained by the difference in climatic conditions but could possibly be accounted for by the difference in vector infestation.
July 06, 2010; Accepted: August 16, 2010;
Published: November 12, 2010
Peanut bud necrosis disease (PBND) caused by Peanut bud necrosis virus (PBNV)
and transmitted by Thrips palmi Karny, is a serious disease of peanut
in many countries in Asia (Satyanarayana et al.,
1996; Reddy et al., 1995). The disease can
cause substantial yield losses in peanut (Dwivedi et al.,
1995). PBNV is now recognized as a distinct species in the genus Tospovirus
(Reddy et al., 1992; Van
Regenmortel et al., 2000). Currently, there is no practical control
measure for PBNV on peanut. However, the disease incidence can be reduced by
some cultural practices such as adjustment of planting date to the period with
low levels of vector activity, intercropping with fast growing cereals (Reddy
et al., 2000) and close spacing (Basu, 1995;
Buiel, 1996; Wongkaew, 1995). Irrigation
may effect the distribution of thrips and consequently can reduce PBND incidence
(Bhatnagar et al., 1995). Insecticide application
to control the vector failed to control the disease and the rapid acquisition
of resistance to insecticides by thrips had been observed (Boiteux
et al., 1993). Therefore, cultivar resistance appears to be the most
effective strategy for control disease.
Successful inoculation of PBNV is necessary to evaluate the levels of resistance
of genotypes. Although, field screening of peanut genotypes for resistance to
the disease has been reported, a low disease incidence of a genotype in field
screening could not be discerned whether it was due to the effect of resistance
to the virus or resistance to the vector or the collective effect of both (Dwivedi
et al., 1995). Therefore, an efficient method for mechanical transmission
of the virus is required in breeding for PBNV resistance. Sap inoculation is
a standard method for resistance evaluation. However, the method has been reported
to yield different results for the same genotype. Dwivedi
et al. (1995) reported PBND incidences on a susceptible check in
the range of 78-94% by mechanical inoculation. Buiel and
Parlevliet (1996) studied the occurrence of mature plant resistance and
found the incidences between 12 and 96% on a susceptible peanut genotype depending
on the age of the plants. In, screening peanut genotypes for resistance to PBNV,
Pensuk et al. (2002) obtained the transmission
efficiency between 60-100% on susceptible genotypes by the mechanical inoculation
under greenhouse conditions. The inconsistency of results from mechanical inoculation
has made the PBNV resistance evaluation less efficient and has caused difficulties
in the genetic studies of PBNV resistance. To improve the efficiency of mechanical
transmission of the virus, a better understanding of the factors causing the
variation of the results is needed.
Mandal et al. (2001) reported that the common
factors causing the variability in mechanical transmission of Tomato spotted
wilt virus (TSWV) were the inoculum source, the buffer composition, the additives
used in the preparation of inoculum, the growth stage of assay plants, the methods
of inoculum application to the assay plants and the environment after inoculation.
These factors were assumed to cause the variability in mechanical transmission
of PBNV as well. However, in the studies of Pensuk et
al. (2002), the same source of inoculum, the same method of inoculum
preparation, the same growth stage of the assay plants and the same method of
inoculation were used. Yet, the results were still variable and a near 100%
disease incidence could not be achieved at will. It was hypothesized that environmental
conditions would have been the cause of the variability in PBNV transmission.
In Thailand, it was also found that the incidence of the disease was much lower
in the rainy season than in the dry season (Wongkaew, 1995).
Because crop grown in the dry season experiences low temperature and low relative
humidity at the early growth stage, these factors were suspected to favor disease
development resulted in a high disease incidence in the dry season. This study
aimed to investigate the effects of temperature and relative humidity on the
effectiveness of PBNV inoculation on peanut in order to develop a protocol that
enhances the efficiency and reliability of mechanical transmission of the virus.
MATERIALS AND METHODS
This study consisted of two experiments in the rainy and dry seasons during 2004 to 2005. The first experiment (Experiment 1) evaluated the effect of low temperature on the effectiveness of PBNV transmission by mechanical inoculation. The second experiment (Experiment 2) compared two temperature and relative humidity conditions that represented the conditions in the rainy season during 8 September 2004 and the dry season during 25 February 2005 in Thailand on the success of mechanical transmission of PBNV to peanut.
In both studies, the susceptible peanut genotype Tainan 9 was used. The test plants were grown in plastic pots and filled with a soil mixture (soil, sand and compost at 2:1:1 v/v/v). Each pot contained four plants. Each treatment consisted of 3 or 4 replicates depending on the trial, with 8 plants (two pots) per replicate. The test plants were inoculated at 7 days after planting. Two new unexpanded quadrifoliate leaves were inoculated per plant.
In preparing the inoculum, leaves of infected peanut plants showing typical
symptom of PBND were initially collected from a field. A virus clone was then
isolated from a single lesion that appeared as primary symptom on an infected
peanut plant. The isolated virus was propagated in Tainan 9 peanut plants that
were kept in a screened house. Identity of the virus was confirmed by ELISA
test using PBNV antiserum as a reference (Do Nascimento
et al., 2006). The inoculum was prepared by grinding systemically infected
peanut leaves in 0.05 M phosphate buffer, pH 7.0, containing 0.2% 2-mercaptoethanol
(1:10 w/v) using a chilled pestle and mortar. Debris was removed by squeezing
the ground extract through a pad of nonabsorbent cotton. Celite was added to
the plant extract to a final concentration of 1%. The extract was kept chilled
during inoculation. Inoculation was done by dripping 200 μL of the inoculum
onto the unfolding leaves of each plant and rubbed thoroughly. Prior to inoculation,
the plants were maintained under dark condition for 3 h. The plants were sprayed
with distilled water after inoculation.
For experiment 1, the treatments included maintaining the inoculated plants
in an environmental growth chamber (Contherm Model CAT 610) that was set at
25°C, 90% RH during a 12 h light period (a light intensity of 1,000 lk)
and 20°C, 90% RH during a 12 h dark period and outside the growth chamber
at room temperature. The test plants maintained in the growth chamber were divided
into 4 groups by the length of time they were kept in the growth chamber after
inoculation, i.e., 24, 48, 72 h and all the period after inoculation until the
test was terminated. The plants were visually scored for PBNV symptoms at 14
and 28 days after inoculation. Those showing systemic symptoms were considered
as infected plants. At 28 days after inoculation, a newly formed leaflet from
each plant was assayed by direct antigen coating enzyme-linked immunosorbent
assay (DAC-ELISA) as described by Wongkaew (1993) to
confirm PBNV infection. The primary antiserum specific to PBNV was obtained
from S. Wongkaew (Khon Kaen University, Thailand) and was cross absorbed with
healthy plant extract at a 1:1000 (v/v) dilution. Goat-anti-rabbit IgG conjugated
to alkaline phosphatase (Sigma 99H9235) at a dilution of 1:2000 was used as
the secondary antibody. Extracts from healthy and infected Tainan 9 peanut leaves
were used as the negative and positive controls, respectively. Absorbance (405
nm) at least 2 folds higher than those of the healthy control was considered
positive for the virus. The data were obtained using an ELISA plate reader Model
Microplate Manager® 4.0 Bio-Rad Laboratories, Inc. The experiment
was repeated 5 times. Trail II was conducted in 3 replicates, while other trials
were conducted in 4 replicates. The disease incidence was determined as the
infected plants. Data were analyzed statistically after being transformed by
an arc sine transformation.
In experiment 2, the inoculated plants were kept in two growth chambers that were set to the conditions representing the average climatic conditions in the rainy and the dry seasons in Thailand, respectively. For the condition representing the dry season, the growth chamber was set to 32°C, 44% RH, during an 11 h 30 min light period and 22°C, 65% RH, during a 12 h 30 min dark period. For the condition representing the rainy season, the environmental growth chamber was set to 36°C, 46% RH, during a 12 h 30 min light period and 25°C, 70% RH, during an 11 h 30 min dark period. The inoculated plants were kept in the growth chambers for four weeks then were visually checked for PBNV symptoms. Only plants showing clear systemic symptoms were considered to be infected. The experiment was repeated 3 times, all with 4 replicates. The disease incidence was determined as the percentage of infected plants and the differences between means of the two treatments were test statistically by t-test.
The experiment 1 showed no difference among treatments in the number of infected plants (Table 1), this indicated that keeping PBNV inoculated plants in low temperature for a certain period did not increase the percentage of infected plants over the conditions of room temperatures at different times of the year. Variations in the percentages of infected plants were still obtained in different trials. High percentages of infected plants were obtained in Trials II and III, i.e., 100% for all treatments in Trial II and 88 to 100% in Trial III, but lower percentages were showed in other trials. Trials I, II and V were conducted during the late-rainy season while Trials III and IV were conducted in the rainy seasons. The temperatures and relative humidity under room conditions in these trials also differed, so as the percentages of infected plants under room conditions. However, no association was observed between the temperature and relative humidity under room condition with the percentage of PBNV infected plants. The same was found for the treatments where the inoculated plants were kept under low temperature in the growth chamber for a certain period. In the treatment in which the inoculated plants were kept under the same control condition all the time after inoculation, the PBNV infected plants still varied in the different trials. These results indicated that, temperature and relative humidity were not the factor causing the variability of PBNV transmission by mechanical inoculation.
Experiment 2 was performed to compensate for the fact that in experiment 1
the environmental conditions outside the growth chamber could not be controlled
and these conditions might affect the symptom expression of the disease.
|| Incidences of PBNV infected plants obtained from mechanical
inoculation under different environmental conditions
|aGrowth chamber condition: 25°C, 90% RH during
a 12 h light period and 20°C, 90% RH during a 12 h dark period. bData
in trial I, IV and V were transformed using the arcsine transformation before
analysis; NS = Not significant.cData were recorded during the
experimental period, i.e., 28 days after inoculation
||Incidences of PBNV infected plants obtained from mechanical
inoculation under the environmental conditions representing the conditions
in rainy and the dry seasons in Thailand
|NS: Not significant
In this experiment, the environmental conditions representing the average temperature
and relative humidity conditions in the rainy season and the dry season were
set up using growth chambers (Table 2). The results from three
repeated trials showed that the two temperature and relative humidity conditions
did not cause a difference in the number of symptomatic plants. Again, variations
in the infected plants were observed among trials. The warm and humid condition
representing the rainy season tended to produce less symptomatic plants than
the cool and less humid condition representing the dry season in Trial I, but
the opposite was observed in Trial III. In Trial II, however, 97% infection
was obtained in both conditions. These results also confirmed the results of
experiment I in that temperature and relative humidity were not the causal factor
for the variability in PBNV transmission by mechanical transmission.
Little is known about the interactions between the environments and the symptomatic
expression of the recently described PBNV that is now recognized as a member
of the genus Tospovirus. PBND in Thailand is prevalent mainly during
the dry season (Wongkaew, 1995), thus, we hypothesized
that the environmental factors prevailing in dry season might favor the disease
incidence. To test this hypothesis, the inoculated plants were exposed to a
warm temperature, i.e., under room condition and a cooler temperature, i.e.,
under growth chamber condition, after inoculation. Different times of incubating
the inoculated plants in the growth chamber were also examined, as, if it works,
the shortest incubating time would be preferred to speed up the process of peanut
breeding lines evaluation for PBNV resistance. However, the disease incidences
obtained from all treatments were not significantly different in all the 5 repeated
trials. These results indicated that low temperature did not favor the PBNV
transmission. These results differed somewhat from the study of Mandal
et al. (2001) on the transmission of TSWV. They reported that the
maximum rate of transmission of TSWV from tobacco to peanut achieved at 15 days
postinoculation (DPI) under greenhouse condition was 66.6%, whereas a 100% transmission
was achieved by 10 DPI under growth chamber condition. Llamas-Llamas
et al. (1998) studied the effect of temperature on symptom expression
and accumulation of TSWV in Nicotiana tabacum and reported that, in
general, virus accumulation in the inoculated leaves of all plants was higher
at a lower temperature. Llamas-Llamas et al. (1998)
also reported that TSWV replication was higher at 20°C than at 36°C,
but the disease symptoms were more severe at 36°C. These finding has led
to their conclusion that there is not necessarily a direct relationship between
virus accumulation and symptom expression.
Since, low temperature may not be the only factor that favors bud necrosis
incidence, a second experiment was conducted in which both temperature and relative
humidity that characterize the average conditions of the rainy and the dry seasons
in Thailand were controlled. The inoculated plants were kept in the growth chamber
throughout the experiment to avoid exposing the plants to varying environmental
conditions. The results in three repeated trials indicated that the disease
incidence was not affected by the environmental conditions under which the inoculated
plants were kept. Variation in the percentages of infected plants among the
trials also could not be accounted for by temperature and relative humidity
as the same conditions of the two treatments were maintained in all the 3 repeated
trials. The difference in the incidences of PBND in the rainy and the dry seasons
in peanut production areas in Thailand, therefore, could not be accounted for
by the difference in temperature and relative humidity in the two seasons. However,
Tsai et al. (1995) reported that T. palmi
was able to tolerate a low temperature (56% mortality when held at 0°C for
15 h) much better than a high temperature (100% mortality at 45°C for 15
h). Tsai et al. (1995) also showed that T.
palmi could not complete its development at 35°C or higher temperatures.
This might partially explain why T. palmi populations are higher in the
winter and spring and lower in the summer in Florida (Tsai
et al., 1995). As temperatures during the rainy season in Thailand
are generally above 35°C, T. palmi populations in peanut production
areas of the country are expected to be low and this could account for the low
PBND incidence in these areas.
The results of the present study indicated that low temperature or low relative
humidity did not affect the transmissibility of PBNV by mechanical inoculation.
A 100% PBND incidence from mechanical inoculation was obtained in some trials
but not the others. The less than perfect infection by mechanical inoculation
might be attributed to the disease escape and/or the subliminal infection as
suggested by Do Nascimento et al. (2006). The
low PBND incidence occurring in the rainy season in Thailand could not be accounted
for by the high temperature and high relative humidity conditions prevailing
in the season, but might be explained by the low vector population and hence,
low vector infestation in the rainy season.
This study was funded by the Senior Research Scholar Project of Prof. Dr. A. Patanothai under the Thailand Research Fund and the work was carried out with the assistance of the Peanut Project, Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand.
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