Full Length
Protective mild isolates of
cucumber mosaic virus
obtained from chili pepper in bali
Dewa Nyoman NYANA
1, Gede SUASTIKA
2, I Gede Rai Maya TEMAJA
3and *Dewa Ngurah
SUPRAPTA
31
Doctorate Program on Agricultural Science, School of Postgraduate Udayana University.
2
Department of Plant ProtectionFaculty of Agriculture, Bogor Agricultural University, Bogor Indonesia.
3
Laboratory of Biopesticide, Faculty of Agriculture, Udayana University, Denpasar Bali, Indonesia.
*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.
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-
NYANA et al. 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 with 700 l isopropylalcohol and incubation at –80oC 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 double-stranded satRNA was stained with ethidium bromide and visualized under UV. The mild isolates containing double-stranded 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
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
Number of CMV mild isolates containing satRNA
Number of protective isolate against severe
CMV
Badung 17 5 1 (N1) 0
Bangli 16 2 0 0
Buleleng 31 4 0 0
Denpasar 23 7 1 (N2) 1 (N2)
Gianyar 18 6 1 (N3) 0
Jembrana 27 5 0 0
Karangasem 26 8 0 0
Klungkung 22 6 1 (N4) 1 (N4)
Tabanan 14 0 0 0
Total 194 43 4 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
Percentage of symptomless plants after challenge
inoculation*
Results of ELISA test **
20 days 30 days 40 days
N1 --- 100 100 100 0.226
N2 --- 100 100 100 0.248
N3 --- 100 100 100 0.238
N4 --- 100 100 100 0.227
N1 V3 80 60 40 0.474
N2 V3 100 100 100 0.255
N3 V3 80 60 60 0.460
N4 V3 100 100 100 0.258
--- V3 0 0 0 0.426
--- --- 100 100 100 0.106
*The number of plant tested was 10 per treatment. **Absorbance values were measured at 405 nm.
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
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.
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satRNA
100 bp 500 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 N1), Denpasar (satRNA-N2), 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 CMV-V3 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
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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