Protective mild isolates of cucumber mosaic virus obtained from chili pepper in bali.
Agricultural Science Research Journal Vol. 2(6) pp. 280 – 284 June 2012
Available online http://www.resjournals.com/ARJ
ISSN-L: 2026-6073 ©2012 International Research Journals
Full Length
Protective mild isolates of cucumber mosaic virus
obtained from chili pepper in bali
Dewa Nyoman NYANA1, Gede SUASTIKA2, I Gede Rai Maya TEMAJA3 and *Dewa Ngurah
SUPRAPTA3
1
Doctorate Program on Agricultural Science, School of Postgraduate Udayana University.
Department of Plant Protection Faculty of Agriculture, Bogor Agricultural University, Bogor Indonesia.
3
Laboratory of Biopesticide, Faculty of Agriculture, Udayana University, Denpasar Bali, Indonesia.
2
*Corresponding author email: biop@dps.centrin.net.id
ABSTRACT
Chili pepper (Capsicum annum L.) is usually affected by two viral diseases showing typical yellowing
and mosaic symptoms, respectively. The yellowing symptom had been known to be caused by infection
of Pepper yellow leaf curl virus (PepYLCV), while the mosaic symptom is associated with the infection
of three viruses, namely Tobacco mosaic virus (TMV), Chili vein mottle virus (ChiVMV), and Cucumber
mosaic virus (CMV). However, CMV was found to be the main virus responsibly for mosaic symptom in
chili pepper grown in Bali. Among chili plants showing mosaic symptoms, there were some plants
expressing very mild symptoms that predicted to be infected by mild isolates of CMV. Double-stranded
(ds) RNA analysis of 43 samples of chili pepper plants showing very mild symptoms indicated that four
samples contained satellite RNA (satRNA) of about 400 bp. Two of them (designated as isolates N2 and
N4) had significant protective effect against other CMV isolate naturally induced severe mosaic disease.
These mild CMV isolates may be useful as biological control agent for mosaic disease management of
chili pepper in Bali or other area of Indonesia.
Keywords: Satellite RNA, double stranded RNA analysis, biological control agent.
INTRODUCTION
Chili pepper being cultivated in Bali is mainly in the
species Capsicum annum L. but a few belong to C.
frutescent L. Both plant species are cultivated in lowland
and in the upland. The fruit can be used as vegetables
and spices. Spicy chili pepper is very popular in Bali as
well as in Southeast Asia as a food flavor enhancer. In
Indonesia, chili pepper can be grown throughout the year,
either in mono culture or in mix cropping systems with
other crops. The low quality of seed, high production
costs, fluctuating in market price, and plant diseases are
the major constraints for chili pepper production. Viral
disease is one of the most important diseases on chili
pepper that may cause significant yield losses.
There were two different viral diseases found in chili
pepper in Bali, namely yellowing disease that has been
known to be associated with infection of Pepper yellow
leaf curl virus (PepYLCV), family Geminiviridae, genus
Begomovirus (De Barrow et al., 2008) and mosaic
disease that has been observed in the last five years.
Retno (2011) showed that 29.18% of the chili plants with
mosaic symptoms were infected by Cucumber mosaic
virus (CMV), family Bromoviridae, genus Cucumovirus.
Cucumber mosaic virus has a worldwide distribution
and a very wide host range. It has been known that the
virus has the widest host range of any known plant virus
(191 hosts in 40 families) (Gallitelli, 2000). It can be
transmitted from plant to plant both mechanically by sap
and by aphids in a stylet-borne manner (Palukaitis and
Garcia-Arenal, 2003). Some strains of CMV have been
reported to harbor satellite RNAs (satRNAs) which are
small, linear molecules ranging from 332 to 405
nucleotides long. The satRNAs are dependent on CMV
NYANA et al.
for their replication, encapsidation, and dispersion, but
they are not necessary for the life cycle of the virus
(Garcia-Arenal and Palukaitis, 1999). The presence of a
satRNA results in a decreased accumulation of CMV in
the tissues of infected plants and modifies the symptoms
induced by CMV, according to a complex interaction
among the strain of CMV, the variant of satRNA and the
species of host plant (Garcia-Arenal and Palukaitis 1999).
A number of variants of satRNA attenuate symptoms
induced by CMV in different plant species, whereas
others intensify them.
Dodds et al. (1985) found that some mild strains of
CMV were found to show cross-protection property to the
severe strains and also prevented the accumulation of
virions and double stranded-RNAs of the challenge
strain. The protection was likely to be systemic because
the protection effect was detected in leaves inoculated
with the challenge strain and also in later formed leaves.
Cross-protection is a phenomenon where pre-infection
with one virus can prevent symptom expression on plants
after subsequent infection with closely related strains of
the same virus (Kobori et al., 2005). There were some
successful evidences on the development and selection
of mild strains of plant viruses used for cross protection to
control viral diseases (Wu et al., 1989; Rezende and
Pacheco, 1998; Lecoq et al., 1991; Wang et al., 1991).
This study was conducted in order to find protective mild
isolates of CMV which could be used to protect the chili
pepper plant from the infection of severe strain of CMV.
MATERIAL AND METHODS
Collection of CMV mild isolates
Samplings to obtain the mild isolates of CMV were done
in nine regencies in Bali Island. The number of samples
taken at each regency varied from 14 to 31 samples. The
chili pepper plants showing no or mild symptoms found
nearby the plants with severe mosaic symptom under
natural condition were collected, grown in the plastic
bags and maintained in a green house. The presence of
CMV was confirmed based on direct enzyme linked
immunosorbent assay according to the manufacturer’s
protocols (Agdia Inc., USA) test and value of absorbance
was measured at 405 nm with ELISA Reader. Inoculation
on the leaves of Chenopodium amaranticolor Coste &
Reyn was also performed to confirm virus infection. The
samples associated with CMV were then confirmed for
their association with satellite RNA (satRNA).
Detection of satRNA
Detection of satRNA in mild isolate of CMV was done
through extraction of the double stranded-RNAs (White
and Kaper, 1989) and visualizing in the polyacrylamide-
281
gel electrophoresis (Peres et al., 1992). About 0.1 g of
chili pepper leaf sample was homogenized in 1 ml
extraction buffer (0.5 M glycine, 0.5 M NaCl, 5 mM EDTA,
pH 9.0) containing 1% sodium N-lauryl salcosinate and
2% sodium dodecylsulfate. The sap was clarified with
200 l phenol and 200 l chloroform and centrifugation at
14,000 rpm for 2 min. The total RNAs were precipitated
o
with 700 l isopropylalcohol and incubation at –80 C for
20 min. The RNAs were pelleted by centrifugation at
14,000 rpm for 10 min. The pellet was resuspended in 25
l dye buffer (containing 1 l of 0.2 mg/ml RNase) and
run in 5% acrylamide gel electrophoresis. The doublestranded satRNA was stained with ethidium bromide and
visualized under UV. The mild isolates containing doublestranded satRNA were then examined for their ability to
protect the chili plants from the infection of virulent strain
of CMV.
Cross-protection test
To assess the ability of CMV mild isolates to prevent
detrimental effects on chili pepper plants caused by
severe CMV isolate, the experiments were conducted
using local chili pepper seedlings (var. Cakra Putih)
grown in 1.2-liter pots in an insect proof net-house. The
method for cross-protection test was developed based on
the method of Kobori et al. (2005) with slight modification.
The selected mild CMV isolates (designated as CMV-N1,
-N2, -N3 and -N4) was sap inoculated on 12-day-old chili
pepper seedlings. Eighty to 100 seedlings with 10 in one
group were subject to each of the treatments. Twelve
days after inoculation, the first true leaves were
mechanically inoculated with sap from plants infected
with CMV severe isolate (designated as CMV-V3) as
“challenge inoculation”. Prior to inoculation, the leaves
were dusted with cellite powder. The sap was prepared
by macerating 0.5g leaf samples in 5 ml of 0.05 M
sodium phosphate buffer, pH 7.2. The sap was then
rubbed on to the leaves by sterile cotton bud, and then,
the inoculated leaves were washed with distilled water.
All the plants were observed for symptoms development
up to 40 days after challenge inoculation with 10 days
interval. Ten chili pepper plants were used for each CMV
mild isolate. The sap was also inoculated on the leaves of
C. amaranticolor, which was used as a local lesion host
to check the CMV infection.
RESULTS AND DISCUSSION
Protective mild isolates of CMV
During the survey, we found chili pepper plants
expressing no or very mild symptoms among the plants
developed mosaic symptom caused by CMV (Table 1).
The symptom expressions may be influenced by plant
282
Agric. Sci. Res. J
Table 1. Number of mild isolate of Cucumber mosaic virus (CMV) obtained from naturally infected chili pepper
(Capsicum annum) plants in Bali and mild CMV isolates that protected against severe strain.
Sampling
location
Number of plants
with mild symptom
Number of plant
infected by CMV
17
16
31
23
18
27
26
22
14
194
5
2
4
7
6
5
8
6
0
43
Badung
Bangli
Buleleng
Denpasar
Gianyar
Jembrana
Karangasem
Klungkung
Tabanan
Total
Number of CMV
mild isolates
containing satRNA
1 (N1)
0
0
1 (N2)
1 (N3)
0
0
1 (N4)
0
4
Number of protective
isolate against severe
CMV
0
0
0
1 (N2)
0
0
0
1 (N4)
0
2
Table 2. Protective properties of four Cucumber mosaic virus (CMV) mild isolates against a
severe CMV-V3 isolate in chili pepper (Capsicum annum).
Mild isolates
Challenged
CMV
isolate
N1
--N2
--N3
--N4
--N1
V3
N2
V3
N3
V3
N4
V3
--V3
----*The number of plant tested was 10 per treatment.
**Absorbance values were measured at 405 nm.
Percentage of symptomless
plants after challenge
inoculation*
20 days
30 days
40 days
100
100
100
100
100
100
100
100
100
100
100
100
80
60
40
100
100
100
80
60
60
100
100
100
0
0
0
100
100
100
host (genotype and growth condition), growth
environment (temperature, nutrition, light, etc.), and virus
strains (Hull, 2002). The fact that the plants with different
symptoms were usually found in the same field (the same
chili pepper cultivar and age, as well as growth
environment), suggested that several virus strains are
likely present in the natural population of the CMV
infected chili peppers in Bali.
In this study, 194 chili pepper plants that showed mild
or no symptom grown adjacent to the plants showing
severe mosaic symptom were collected from nine
regencies in Bali (Table 1). The direct ELISA test results
revealed that 22.16 % of the samples (43 plants) were
infected with CMV (Table 1) These virus isolates were
considered to be CMV mild isolates because they were
obtained from naturally infected, asymptomatic chili
Results of
ELISA test **
0.226
0.248
0.238
0.227
0.474
0.255
0.460
0.258
0.426
0.106
pepper plants.
Double-stranded RNA analyses revealed that only
9.3% (4 of 43 plants) of the mild isolates contain satRNA
(Table 1; Figure 1). By using 100 base DNA ladder
(BioRad, USA), the size of satRNA was about 400 bp.
According to Collmer and Howell (1992) this size is in the
range size of CMV satRNAs.
As a result of this screening trial, we obtained four
promising mild isolates designated as CMV-N1, CMV-N2,
CMV-N3, and CMV-N4. These four mild isolates of CMV
were tested for their detrimental effect on the chili plants
under an aphid-proof net house condition. Results
showed that all four mild isolates tested, did not produce
prominent mosaic symptoms on chili pepper plants. This
result confirmed that all four CMV mild isolates containing
satRNAs have no detrimental effects on chili pepper
NYANA et al.
500 bp
283
satRNA
100 bp
Figure 1. Profile of satRNA in mild strains of Cucumber mosaic virus (CMV) obtained from
chili pepper (Capsicum annum) in Bali. From left to right: 100 b DNA Ladder (BioRad),
samples from Bangli, Buleleng, Karangasem, Badung (satRNA-N1), Denpasar (satRNAN2), Gianyar (satRNA-N3), Klungkung (satRNA-N4), Jembrana, and positive control for
satRNA-contained CMV isolate (CMV-T2) (Nyana et al., 2005).
Figure 2: A. Symptom of CMV occurred on chili pepper inoculated with virulent CMV-V3.
B. Chili pepper treated with mild isolate (CMV-N4) followed by challenge inoculation with
virulent CMV-V3.
plants.
The property of CMV mild isolates to protect the plants
from the infection by the severe isolate of CMV-V3 was
also tested. Chili pepper plants inoculated with CMV-V3
exhibited severe mosaic symptoms accompanied with
leaf malformation. The disease severity induced by CMVV3 in this experiment was similar to the symptoms of
naturally infected chili pepper plants in the field.
Chili pepper plants inoculated with either CMV-N2 or
CMV-N4 prior to inoculation with the severe isolate of
CMV-V3 apparently suppressed the expression of mosaic
symptoms and/or leaf deformations. However, 60% and
40% of protected plants with CMV-N1 and CMV-N3
respectively, induced severe mosaic symptoms on newly
developed leaves 40 days after challenge inoculation
(Table 2), demonstrating that CMV-N1 and CMV-N3 did
not protect chili pepper plants against the severe isolate
of CMV-V3 when compared with CMV-N2 and CMV-N4.
Further work to know the dsRNA sequence of the
respective mild isolate is necessary in order to
understand the characteristic of the dsRNA molecule.
Previous study done in Bali by Nyana et al. (2005)
showed that an isolate of CMV-MP1 that contains
satellite dsRNA effectively protected tobacco (Nicotiana
tabacum L.) plants from infection of a severe strain of
CMV-BV2. Kosaka et al. (2006) demonstrated the use of
attenuated isolate of Zucchini yellow mosaic virus
(ZYMV) designated as ZYMV-2002 to successfully
suppressed the infection with severe ZYMV on cucumber
(Cucumis sativus L.). Other work done by You et al.
(2005) showed that engineered mild strain of Papaya
ringspot virus derived from isolate PRSV-HA5-1
284
Agric. Sci. Res. J
effectively suppressed the infection by severe strains of
W-CI and P-HA in horn melon (Cucumis metuliferus
E.Mey) and squash (Cucurbita moschata L.) plants. The
use of mild isolates of virus can be a promising strategy
in plant virus control, and the mild isolates of CMV-N2
and CMV-N4 obtained from chili pepper grown in Bali can
be further developed and characterized to be used as
mild isolates to control severe strains of CMV in chili
pepper.
CONCLUSION
Two CMV mild isolates obtained from chili pepper in Bali
containing satRNA named CMV-N2 and CMV-N4 were
found to posses effective cross protection property
against severe CMV isolate, demonstrating that they
could be developed as biological control agents to control
CMV disease on chili pepper.
ACKNOWLEDGEMENTS
The authors would like to thanks the Directorate General
of Higher Education, Ministry of Education and Culture of
the Republic of Indonesia for providing research grant to
support this study in the fiscal year 2010.
REFERENCES
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expression of symptoms caused by plant viruses. Annu. Rev.
Phytopathol. 30: 419-422.
Dodds JA, Lee SQ, Tiffany M (1985). Cross protection between
strains of cucumber mosaic virus: Effect of host and type of
inoculums on accumulation of virions and double-stranded
RNA of the challenge strain. Virology 144 (2): 301-309.
Gallitelli D (2000). The ecology of Cucumber mosaic virus and
sustainable agriculture. Virus Res. 71:9-21.
De Barrow PJ, Hidayat SH, Frohlich D, Subandiyah
S, Shigenori U (2008). A virus and its vector, pepper yellow
leaf curl virus and Bemisia tabaci, two new invaders of
Indonesia. Biological Invasions 10 (4): 411-433.
Hull R (2002). Matthew’s Plant Virology. Academic Press, San
Diego, CA.
García-Arenal F, Palukaitis P (1999). Structure and functional
relationships of satellite RNAs of cucumber mosaic virus. In
Vogt PK, Jackson AO (eds.) Satellites and Defective Viral
RNAs. Berlin, Springer-Verlag. 37–63.pp.
Kobori T, Ryang BS, Natsuaki T, Kosaka Y (2005). A new
technique to select mild strains of Cucumber mosaic virus.
Plant Dis. 89: 879-882.
Kosaka Y, Ryang BS, Kobori T, Shiomi H, Yasuhara H,
Kataoka M (2006). Effectiveness of an attenuated Zucchini
yellow mosaic virus isolate for cross- Protecting cucumber.
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Zucchini yellow mosaic virus in squash by cross protection.
Plant Dis. 75: 208-211.
Nyana DN, Suastika G, Natsuaki KT, Sayama H (2005). Control
of cucumber mosaic virus on tobacco by attenuated-CMV. J.
ISSAAS 11(3): 97-102
Palukaitis P, García-Arenal F (2003). Cucumoviruses.
Advances in Virus Res. 62: 241-323.
Peres RD, Gilling MR, Gunn LV (1992). Differentiation of
biologically distinct Cucumber mosaic virus isolates by PAGE
of double-stranded RNA. Intervirology 34: 23-29.
Retno B (2011). Incidence of mosaic virus and collection of mild
isolates of CMV from chili pepper grown in Bali. Master
Thesis. Study Program on Agricultural Biotechnology, School
of Postgraduate Udayana University, Denpasar (in
Indonesian language).
Rezende JAM, Pacheco DA (1998). Control of Papaya ringspot
virus- type W in zucchini squash by protection in Brazil. Plant
Dis. 82: 171-175.
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Effectiveness of cross protection by a mild strain of Zucchini
yelow mosaic virus in cucumber, melon and squash. Plant
Dis. 75: 203-207.
White JM, Kaper JM (1989). A simple method for detection of
viral satellite RNAs. J. Virol. Methods 23: 83-94.
Wu G, Kang L, Tien P (1989). The effect of satellite RNA on
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Available online http://www.resjournals.com/ARJ
ISSN-L: 2026-6073 ©2012 International Research Journals
Full Length
Protective mild isolates of cucumber mosaic virus
obtained from chili pepper in bali
Dewa Nyoman NYANA1, Gede SUASTIKA2, I Gede Rai Maya TEMAJA3 and *Dewa Ngurah
SUPRAPTA3
1
Doctorate Program on Agricultural Science, School of Postgraduate Udayana University.
Department of Plant Protection Faculty of Agriculture, Bogor Agricultural University, Bogor Indonesia.
3
Laboratory of Biopesticide, Faculty of Agriculture, Udayana University, Denpasar Bali, Indonesia.
2
*Corresponding author email: biop@dps.centrin.net.id
ABSTRACT
Chili pepper (Capsicum annum L.) is usually affected by two viral diseases showing typical yellowing
and mosaic symptoms, respectively. The yellowing symptom had been known to be caused by infection
of Pepper yellow leaf curl virus (PepYLCV), while the mosaic symptom is associated with the infection
of three viruses, namely Tobacco mosaic virus (TMV), Chili vein mottle virus (ChiVMV), and Cucumber
mosaic virus (CMV). However, CMV was found to be the main virus responsibly for mosaic symptom in
chili pepper grown in Bali. Among chili plants showing mosaic symptoms, there were some plants
expressing very mild symptoms that predicted to be infected by mild isolates of CMV. Double-stranded
(ds) RNA analysis of 43 samples of chili pepper plants showing very mild symptoms indicated that four
samples contained satellite RNA (satRNA) of about 400 bp. Two of them (designated as isolates N2 and
N4) had significant protective effect against other CMV isolate naturally induced severe mosaic disease.
These mild CMV isolates may be useful as biological control agent for mosaic disease management of
chili pepper in Bali or other area of Indonesia.
Keywords: Satellite RNA, double stranded RNA analysis, biological control agent.
INTRODUCTION
Chili pepper being cultivated in Bali is mainly in the
species Capsicum annum L. but a few belong to C.
frutescent L. Both plant species are cultivated in lowland
and in the upland. The fruit can be used as vegetables
and spices. Spicy chili pepper is very popular in Bali as
well as in Southeast Asia as a food flavor enhancer. In
Indonesia, chili pepper can be grown throughout the year,
either in mono culture or in mix cropping systems with
other crops. The low quality of seed, high production
costs, fluctuating in market price, and plant diseases are
the major constraints for chili pepper production. Viral
disease is one of the most important diseases on chili
pepper that may cause significant yield losses.
There were two different viral diseases found in chili
pepper in Bali, namely yellowing disease that has been
known to be associated with infection of Pepper yellow
leaf curl virus (PepYLCV), family Geminiviridae, genus
Begomovirus (De Barrow et al., 2008) and mosaic
disease that has been observed in the last five years.
Retno (2011) showed that 29.18% of the chili plants with
mosaic symptoms were infected by Cucumber mosaic
virus (CMV), family Bromoviridae, genus Cucumovirus.
Cucumber mosaic virus has a worldwide distribution
and a very wide host range. It has been known that the
virus has the widest host range of any known plant virus
(191 hosts in 40 families) (Gallitelli, 2000). It can be
transmitted from plant to plant both mechanically by sap
and by aphids in a stylet-borne manner (Palukaitis and
Garcia-Arenal, 2003). Some strains of CMV have been
reported to harbor satellite RNAs (satRNAs) which are
small, linear molecules ranging from 332 to 405
nucleotides long. The satRNAs are dependent on CMV
NYANA et al.
for their replication, encapsidation, and dispersion, but
they are not necessary for the life cycle of the virus
(Garcia-Arenal and Palukaitis, 1999). The presence of a
satRNA results in a decreased accumulation of CMV in
the tissues of infected plants and modifies the symptoms
induced by CMV, according to a complex interaction
among the strain of CMV, the variant of satRNA and the
species of host plant (Garcia-Arenal and Palukaitis 1999).
A number of variants of satRNA attenuate symptoms
induced by CMV in different plant species, whereas
others intensify them.
Dodds et al. (1985) found that some mild strains of
CMV were found to show cross-protection property to the
severe strains and also prevented the accumulation of
virions and double stranded-RNAs of the challenge
strain. The protection was likely to be systemic because
the protection effect was detected in leaves inoculated
with the challenge strain and also in later formed leaves.
Cross-protection is a phenomenon where pre-infection
with one virus can prevent symptom expression on plants
after subsequent infection with closely related strains of
the same virus (Kobori et al., 2005). There were some
successful evidences on the development and selection
of mild strains of plant viruses used for cross protection to
control viral diseases (Wu et al., 1989; Rezende and
Pacheco, 1998; Lecoq et al., 1991; Wang et al., 1991).
This study was conducted in order to find protective mild
isolates of CMV which could be used to protect the chili
pepper plant from the infection of severe strain of CMV.
MATERIAL AND METHODS
Collection of CMV mild isolates
Samplings to obtain the mild isolates of CMV were done
in nine regencies in Bali Island. The number of samples
taken at each regency varied from 14 to 31 samples. The
chili pepper plants showing no or mild symptoms found
nearby the plants with severe mosaic symptom under
natural condition were collected, grown in the plastic
bags and maintained in a green house. The presence of
CMV was confirmed based on direct enzyme linked
immunosorbent assay according to the manufacturer’s
protocols (Agdia Inc., USA) test and value of absorbance
was measured at 405 nm with ELISA Reader. Inoculation
on the leaves of Chenopodium amaranticolor Coste &
Reyn was also performed to confirm virus infection. The
samples associated with CMV were then confirmed for
their association with satellite RNA (satRNA).
Detection of satRNA
Detection of satRNA in mild isolate of CMV was done
through extraction of the double stranded-RNAs (White
and Kaper, 1989) and visualizing in the polyacrylamide-
281
gel electrophoresis (Peres et al., 1992). About 0.1 g of
chili pepper leaf sample was homogenized in 1 ml
extraction buffer (0.5 M glycine, 0.5 M NaCl, 5 mM EDTA,
pH 9.0) containing 1% sodium N-lauryl salcosinate and
2% sodium dodecylsulfate. The sap was clarified with
200 l phenol and 200 l chloroform and centrifugation at
14,000 rpm for 2 min. The total RNAs were precipitated
o
with 700 l isopropylalcohol and incubation at –80 C for
20 min. The RNAs were pelleted by centrifugation at
14,000 rpm for 10 min. The pellet was resuspended in 25
l dye buffer (containing 1 l of 0.2 mg/ml RNase) and
run in 5% acrylamide gel electrophoresis. The doublestranded satRNA was stained with ethidium bromide and
visualized under UV. The mild isolates containing doublestranded satRNA were then examined for their ability to
protect the chili plants from the infection of virulent strain
of CMV.
Cross-protection test
To assess the ability of CMV mild isolates to prevent
detrimental effects on chili pepper plants caused by
severe CMV isolate, the experiments were conducted
using local chili pepper seedlings (var. Cakra Putih)
grown in 1.2-liter pots in an insect proof net-house. The
method for cross-protection test was developed based on
the method of Kobori et al. (2005) with slight modification.
The selected mild CMV isolates (designated as CMV-N1,
-N2, -N3 and -N4) was sap inoculated on 12-day-old chili
pepper seedlings. Eighty to 100 seedlings with 10 in one
group were subject to each of the treatments. Twelve
days after inoculation, the first true leaves were
mechanically inoculated with sap from plants infected
with CMV severe isolate (designated as CMV-V3) as
“challenge inoculation”. Prior to inoculation, the leaves
were dusted with cellite powder. The sap was prepared
by macerating 0.5g leaf samples in 5 ml of 0.05 M
sodium phosphate buffer, pH 7.2. The sap was then
rubbed on to the leaves by sterile cotton bud, and then,
the inoculated leaves were washed with distilled water.
All the plants were observed for symptoms development
up to 40 days after challenge inoculation with 10 days
interval. Ten chili pepper plants were used for each CMV
mild isolate. The sap was also inoculated on the leaves of
C. amaranticolor, which was used as a local lesion host
to check the CMV infection.
RESULTS AND DISCUSSION
Protective mild isolates of CMV
During the survey, we found chili pepper plants
expressing no or very mild symptoms among the plants
developed mosaic symptom caused by CMV (Table 1).
The symptom expressions may be influenced by plant
282
Agric. Sci. Res. J
Table 1. Number of mild isolate of Cucumber mosaic virus (CMV) obtained from naturally infected chili pepper
(Capsicum annum) plants in Bali and mild CMV isolates that protected against severe strain.
Sampling
location
Number of plants
with mild symptom
Number of plant
infected by CMV
17
16
31
23
18
27
26
22
14
194
5
2
4
7
6
5
8
6
0
43
Badung
Bangli
Buleleng
Denpasar
Gianyar
Jembrana
Karangasem
Klungkung
Tabanan
Total
Number of CMV
mild isolates
containing satRNA
1 (N1)
0
0
1 (N2)
1 (N3)
0
0
1 (N4)
0
4
Number of protective
isolate against severe
CMV
0
0
0
1 (N2)
0
0
0
1 (N4)
0
2
Table 2. Protective properties of four Cucumber mosaic virus (CMV) mild isolates against a
severe CMV-V3 isolate in chili pepper (Capsicum annum).
Mild isolates
Challenged
CMV
isolate
N1
--N2
--N3
--N4
--N1
V3
N2
V3
N3
V3
N4
V3
--V3
----*The number of plant tested was 10 per treatment.
**Absorbance values were measured at 405 nm.
Percentage of symptomless
plants after challenge
inoculation*
20 days
30 days
40 days
100
100
100
100
100
100
100
100
100
100
100
100
80
60
40
100
100
100
80
60
60
100
100
100
0
0
0
100
100
100
host (genotype and growth condition), growth
environment (temperature, nutrition, light, etc.), and virus
strains (Hull, 2002). The fact that the plants with different
symptoms were usually found in the same field (the same
chili pepper cultivar and age, as well as growth
environment), suggested that several virus strains are
likely present in the natural population of the CMV
infected chili peppers in Bali.
In this study, 194 chili pepper plants that showed mild
or no symptom grown adjacent to the plants showing
severe mosaic symptom were collected from nine
regencies in Bali (Table 1). The direct ELISA test results
revealed that 22.16 % of the samples (43 plants) were
infected with CMV (Table 1) These virus isolates were
considered to be CMV mild isolates because they were
obtained from naturally infected, asymptomatic chili
Results of
ELISA test **
0.226
0.248
0.238
0.227
0.474
0.255
0.460
0.258
0.426
0.106
pepper plants.
Double-stranded RNA analyses revealed that only
9.3% (4 of 43 plants) of the mild isolates contain satRNA
(Table 1; Figure 1). By using 100 base DNA ladder
(BioRad, USA), the size of satRNA was about 400 bp.
According to Collmer and Howell (1992) this size is in the
range size of CMV satRNAs.
As a result of this screening trial, we obtained four
promising mild isolates designated as CMV-N1, CMV-N2,
CMV-N3, and CMV-N4. These four mild isolates of CMV
were tested for their detrimental effect on the chili plants
under an aphid-proof net house condition. Results
showed that all four mild isolates tested, did not produce
prominent mosaic symptoms on chili pepper plants. This
result confirmed that all four CMV mild isolates containing
satRNAs have no detrimental effects on chili pepper
NYANA et al.
500 bp
283
satRNA
100 bp
Figure 1. Profile of satRNA in mild strains of Cucumber mosaic virus (CMV) obtained from
chili pepper (Capsicum annum) in Bali. From left to right: 100 b DNA Ladder (BioRad),
samples from Bangli, Buleleng, Karangasem, Badung (satRNA-N1), Denpasar (satRNAN2), Gianyar (satRNA-N3), Klungkung (satRNA-N4), Jembrana, and positive control for
satRNA-contained CMV isolate (CMV-T2) (Nyana et al., 2005).
Figure 2: A. Symptom of CMV occurred on chili pepper inoculated with virulent CMV-V3.
B. Chili pepper treated with mild isolate (CMV-N4) followed by challenge inoculation with
virulent CMV-V3.
plants.
The property of CMV mild isolates to protect the plants
from the infection by the severe isolate of CMV-V3 was
also tested. Chili pepper plants inoculated with CMV-V3
exhibited severe mosaic symptoms accompanied with
leaf malformation. The disease severity induced by CMVV3 in this experiment was similar to the symptoms of
naturally infected chili pepper plants in the field.
Chili pepper plants inoculated with either CMV-N2 or
CMV-N4 prior to inoculation with the severe isolate of
CMV-V3 apparently suppressed the expression of mosaic
symptoms and/or leaf deformations. However, 60% and
40% of protected plants with CMV-N1 and CMV-N3
respectively, induced severe mosaic symptoms on newly
developed leaves 40 days after challenge inoculation
(Table 2), demonstrating that CMV-N1 and CMV-N3 did
not protect chili pepper plants against the severe isolate
of CMV-V3 when compared with CMV-N2 and CMV-N4.
Further work to know the dsRNA sequence of the
respective mild isolate is necessary in order to
understand the characteristic of the dsRNA molecule.
Previous study done in Bali by Nyana et al. (2005)
showed that an isolate of CMV-MP1 that contains
satellite dsRNA effectively protected tobacco (Nicotiana
tabacum L.) plants from infection of a severe strain of
CMV-BV2. Kosaka et al. (2006) demonstrated the use of
attenuated isolate of Zucchini yellow mosaic virus
(ZYMV) designated as ZYMV-2002 to successfully
suppressed the infection with severe ZYMV on cucumber
(Cucumis sativus L.). Other work done by You et al.
(2005) showed that engineered mild strain of Papaya
ringspot virus derived from isolate PRSV-HA5-1
284
Agric. Sci. Res. J
effectively suppressed the infection by severe strains of
W-CI and P-HA in horn melon (Cucumis metuliferus
E.Mey) and squash (Cucurbita moschata L.) plants. The
use of mild isolates of virus can be a promising strategy
in plant virus control, and the mild isolates of CMV-N2
and CMV-N4 obtained from chili pepper grown in Bali can
be further developed and characterized to be used as
mild isolates to control severe strains of CMV in chili
pepper.
CONCLUSION
Two CMV mild isolates obtained from chili pepper in Bali
containing satRNA named CMV-N2 and CMV-N4 were
found to posses effective cross protection property
against severe CMV isolate, demonstrating that they
could be developed as biological control agents to control
CMV disease on chili pepper.
ACKNOWLEDGEMENTS
The authors would like to thanks the Directorate General
of Higher Education, Ministry of Education and Culture of
the Republic of Indonesia for providing research grant to
support this study in the fiscal year 2010.
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