Molecular Characterization of Onion Yellow Dwarf Virus (Garlic Isolate) with Production of Virus-free Plantlets
Ahmed M. Soliman,
Sabry Y.M. Mahmoud
Rehab A. Dawood
Garlic samples showing symptoms of Onion Yellow Dwarf Virus (OYDV) were obtained from previous study and tested by indirect-enzyme linked immunosorbent assay (I-ELISA), transmitted to Chenopodium amaranticolor and then confirmed by immunosorbent electron microscopy (ISEM) for the presence of OYDV. PCR primers were used to amplify about 1.1 kb fragment from the viral genome using RT-PCR from infected garlic plants, such fragment were not obtained from healthy- looking plants and/or virus-free seedlings of shoot-tips. The amplified products of OYDV was cloned into pGEM®-T Easy vector and transformed into Escherichia coli strain DH5a. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank. The sequence analysis showed that; nucleotide sequence of OYDV-EG [Egyptian isolate (HM473189)] had 82-96% similarity compared with ten reported OYDV isolates. Thus, a method of identification and detection by RT-PCR of OYDV was established. This study also aimed to obtain OYDV-free plants from infected garlic plants using cloves subjected to electrotherapy, thermotherapy, chemotherapy or meristematic dissection followed by in vitro culture. A combination treatment with electro- and chemotherapy (15 mA/10 min + 20 mg L-1 virazol) was found to more effective on viral elimination and survival of explants. ELISA tests showed that 85% of the plantlets that survived were OYDV-negative.
June 20, 2011; Accepted: July 27, 2011;
Published: October 25, 2011
Many viruses are known to infect garlic (Allium sativum) and a few of
them can seriously reduce crop yields and quality. Losses of around 25-50% due
to natural infections have been reported (Lot et al.,
1998; Dovas et al., 2001a). Whatever their
primary means of transmission are based on vegetative propagation of the crop
favors their dissemination and accumulation in bulbs (AVRDC,
1997). Onion Yellow Dwarf Virus (OYDV), Leek Yellow Stripe Virus (LYSV)
(genus, Potyvirus , family Potyviridae), Garlic common latent virus (GarCLV),
Garlic latent virus (GarLV) and Shallot Latent Virus (SLV) (genus, Carlavirus)
have been reported to infect garlic, often as mixed infections (Dovas
et al., 2001b; Chen et al., 2004;
Meenakshi et al., 2006; Shahraeen
et al., 2008). OYDV, an aphid-borne potyvirus, is one of the major
viral pathogens of onion and garlic. In garlic, OYDV produces symptoms of mild
chlorotic stripes to bright yellow stripes depending on virus isolate and cultivars.
Reduction in growth and bulb size also occurs (FAO/IPGRI,
1997). OYDV is recognized as a major element of the virus disease complex
in garlic (Takaichi et al., 2001).
In previous study, OYDV was isolated and detected in garlic leaves by ELISA
and immunocapture/reverse. Transcription-polymerase chain reaction (IC- PCR)
(Mahmoud et al., 2007). Since ELISA can not be
routinely used for detection of OYDV, a RT- PCR based method was standardized
for detection of OYDV in leaves and bulbs of garlic. Group-specific primers
used for detection of potyviruses for the amplification of 3' terminal region
and part of NIB gene (Pappu et al., 1993; Gibbs
and Mackenzie, 1997) did not work, therefore, specific primers from conserved
region of RNA-dependent RNA polymerase gene and 3' UTR region of viral RNA of
OYDV isolates were designed and synthesized (Meenakshi et
al., 2006). The method was validated to detect OYDV in garlic plants
. In order to regenerate virus-free garlic, meristem tip culture is a well established
method for eliminating viruses from garlic (Sang-II
et al., 2002; Hwang and Lee, 2008) and thermotherapy
has been found in many cases to aid in garlic virus elimination (Peiwen
et al., 1994). Electrotherapy was then recommended in cleaning of
Cucumber mosaic virus (CMV), Arabia mosaic virus (ArMV), Grapevine fern leaf
virus (GFLV), Chicory yellow mottle (ChYMV) and Tobacco mosaic virus (TMV).
Based on these results, Hernandez et al. (1999)
built a device to apply electric current in the cleaning of a viral complex
in garlic. Other applications of the same technology have been reported in potato
(Mahmoud et al., 2007; Dhital
et al., 2008), grapevine (Guta et al.,
2010) and banana plants (Hernandez et al., 2002)
for Potato leaf-roll virus (PLRV) and Potato virus Y (PVY), Grapevine leaf roll
associated virus (GLRaV) and Banana Streak Virus (BSV), respectively.
This study aimed to identify the OYDV infecting garlic in Egypt by using viral
cDNA cloning, sequencing and phylogenetic analysis of OYDV and also to find
appropriate methods of virus elimination using tissue culture techniques together
with other treatments.
MATERIALS AND METHODS
Virus isolate and plant materials: OYDV-garlic (Allium sativum L.
cv. Balady) cloves used in this study was obtained from Virology laboratory,
Faculty of Agricultutre, Ain Shams University. (Mahmoud
et al., 2007). Garlic cloves were cultivated under greenhouse (Fig.
1a) to obtain materials for further studies.
The virus isolate was re-identified depending on symptomatology and differential
hosts by inoculation using 0.05 M borate buffer, pH 8.1 containing activated
charcoal (1:100, w/v) and sodium diethyldithiocarbamate (1:1000, w/v). Also,
serologically by I- ELISA using OYDV-specific polyclonal antibodies prepared
by Mahmoud et al. (2007). Leaves and bulbs infected
materials resulted in greenhouse were used in molecular detection and virus
Extraction of total RNA from plant tissues: Total RNA was isolated
from the infected garlic plants using Simply P total RNA Extraction Kit obtained
from BioFlux according to manufacture s instructions.
PCR primers: The PCR primers obtained from Meenakshi
et al. (2006) and the designed for two conserved regions among the
OYDV genome were used. One primer present at the 3`-end of RNA-dependent RNA
polymerase gene (forward primer) and the other one present in the 3`-UTR region
(reverse primer). The sequence of the reverse primer designed from the 3`-UTR
region was OYDVVKBR-5`-GTCTCYGTAATTCACGC-3` with degeneracy at one point. The
sequence of forward primer designed from RNA-dependent RNA polymerase gene was
OYDVVKBF-5`- ATAGCAGAAACAGCTCTTA-3` (Meenakshi et al.,
Reverse transcription-polymerase chain reaction (RT-PCR): Total RNA extracted from infected garlic plants was used as template for RT-PCR amplification reaction using QIAGEN One Step RT-PCR Kit. Reverse transcription reaction started with incubation at 5°C for 30 min, followed by denaturation at 95°C for 15 min. PCR amplification was performed by 30 cycles in a thermal cycler starting with denaturation at 95°C for 1 min, primer annealing at 48°C for 45 sec and extension at 72°C for 1 min with final extension at 72°C for 10 min. Five microliters aliquots of RT-PCR products were analyzed on 1% agarose gels in 0.5X TBE buffer. One killobite DNA ladder (Fermentas) was used to determine the size of RT-PCR products. Gels were stained with ethidium bromide and visualized by UV illumination using Gel Documentation System (Gel Doc 2000, Bio-Rad, USA). The expected size of the PCR product was 1.1 kb.
Cloning and sequencing of RT-PCR product: Amplified fragment covering
the RNA dependent RNA polymerase gene, CP gene and 3`UTR from Egyptian isolate
were extracted using Gel Extraction kit (QIAGEN). The PCR product was ligated
into pGEM®-T Easy vector
(Promega, USA) and the recombinant plasmids were introduced into E. coli
strain DH5a as described by manufactures instructions. The nucleotide
sequence of clones having ~ 1.1 kb insert were selected for dideoxy sequencing.
The nucleotide sequence was compared and analyzed using DNAMAN Sequence Analysis
Software (Lynnon BioSoft. Quebec, Canada) with those of OYDV isolates available
Virus-free garlic production
Plant materials and tissue culture: Garlic plants cultivated under greenhouse
conditions were harvested. Bulbs were air-dried and stored for two months before
treatments, except on electrotherapy treatment, whereas the harvested bulbs
were treated directly then air-dried and stored. The infection of harvested
bulbs with OYDV was 100% as estimated by I-ELISA. Bulbs were split into individual
cloves and the outer dry papery scales removed. Cloves were surface sterilized
for 2 min in 70% ethanol and 15 min in 15% sodium hypochlorite with 2-3 drops
of Tween 80 and then washed three times (2 min for each one) in sterile water.
Isolated basal plates were used as explants and cultured on MS basal medium
containing 0.2 mg L-1 BA. After 4 weeks of growth on initiation media,
explants were transferred to shoot multiplication media containing 0.5 mg L-1
BA for 6 weeks (2 subcultures).
Meristem tip culture: Meristem culture was conducted from basal plates
explants cultured on MS basal medium containing 0.2 mg L-1 BA as
previously reported. The shoots were excised from basal plates and were cut
to about 2 mm. Meristem tips were excised in sterilized conditions under a binocular,
measured along their base (meristem size) and plated on MS basal medium without
plant growth regulators. After 4 weeks of growth on initiation media, explants
were transferred to multiplication media containing 0.4 mg L-1 BA.
Thermotherapy: Two temperature regimes 37±1 and 38±1°C
were used to analyze the effect of therapeutic treatments on viral elimination.
The micropropagated shoots were cultivated in the shooting media then subjected
to the previous temperature and incubated for 3 weeks at 16 h light and 8 h
Electrotherapy: The whole bulb was electric shocked by connected it
to the electrodes for electric current intensity-time combinations: 5, 10 or
15 milliamper (mA) for 5 or 10 min according to Mahmoud
et al. (2007). Electricity was supplied by electrophoresis power
supply (LABCONCO power supply 433- 3240). Shoot apices were excised from cloves
and cultured as previously reported.
Chemotherapy: Virazol was used to analyze the effect of antiviral compound on viral elimination; the micro propagated shoots were cultivated in the shooting media supplemented with 10, 20, 30 and 40 mg L-1 of virazol for 3 weeks.
Combined therapy: For study the combination effects between chemo and electrotherapy the 15 mA/10 min, electric-treated cloves were removed from bulbs and basal plates were isolated as mentioned above. Basal plates were cultured in shooting media supplemented with 20 mg L-1 of virazol and the others were cultured on basal MS media and then subjected to thermotherapy as mentioned above (Fig. 4). After the end of each treatment, samples were tested by I-ELISA then shoot clumps were transferred to control media and then dissected into single shoots and placed on MS medium supplemented with 0.2 mg L-1 NAA for root induction (Fig. 5).
Virus detection: After 3 weeks survive plantlets were indexed for OYDV
by I-ELISA. Polyclonal antibody was prepared on previous work (Mahmoud
et al., 2007). Then the percentages of survival and virus elimination
were calculated. Therapy efficiency % was calculated by survival plantlets percentage
multiply by virus-free plantlets percentage according to Mahmoud
et al. (2007).
Isolate verification: OYDV-G isolated from garlic infected plants showing
irregular yellow striping to complete yellowing and downward curling, crinkling
and stunting (Fig. 1a). Samples gave positive reactions with
indirect-ELISA (I-ELISA) were mechanically inoculated on Ch. amaranticolor
leaves and gave chlorotic local lesions (Fig. 1b). Lesions
were extracted and inoculated on Narcissus sp. for propagation (Fig.
1c). By ISEM in the same sample extracts, it was observed filamentous virus
particles (Fig. 1d).
||Symptoms induced by OYDV. (a) Garlic plant showing yellow
strips, curling and stunting; (b) Ch. amaranticolor showed
chlorotic local lesions; (c) Narcissus sp. showed yellow stripes
and (d) OYDV occurs as filamentous particles in sap
||(a) Schematic diagram showing the PCR primes positions.
(b) Agarose gel electrophoresis analysis of RT-PCR amplified products. M:
1 kb DNA ladder (Fermentas); L1 to L4: Garlic samples infected with Egyptian
isolate of OYDV, L5: Healthy garlic sample (negative control)
RT-PCR: The used PCR primers amplified one fragment covering three different
regions among the viral genom. The amplified PCR fragment included the viral
RNA dependent RNA polymerase gene, CP gene and 3`UTR as shown in Fig.
2a. The PCR amplification was carried out using the total RNA isolated from
infected garlic plants. Electrophoresis analysis of RT-PCR product showed a
single amplified fragment of ~ 1.1 kb and no fragments were amplified from the
RNA extracted from symptomless or healthy plants (Fig. 2b).
Cloning of RT-PCR fragment into pGEM®-T
Easy vector: The pGEM®-T
Easy Vector System is convenient system for the cloning of PCR products. This
vector is characterized by adding a 3` terminal thymidine to both ends. These
single 3`-T overhangs at the insertion site greatly improve the efficiency of
ligation of a PCR product into the plasmids by preventing recircularization
of the vector and providing a compatible overhang for PCR products generated
by certain thermostable polymerases. This allows PCR inserts to ligate efficiently
with the vector (Mezei and Storts, 1994; Robles
and Doers, 1994).
Isolation of recombinant plasmids: Several white colonies resistant to ampicillin were selected to test for recombinant plasmids containing the OYDV-PCR product. Restriction enzyme digestion with Eco RI released the cloned gene at the expected size.
Sequence analysis: Nucleotide sequencing of the RT-PCR amplified fragment
in the recombinant plasmid was completed to determine if this RT- PCR fragment
was OYDV or not and to compare the sequence from this isolate with those of
other OYDV isolates available in GenBank.
||A phylogenetic tree showing relationships among reported isolates
of OYDV and the Egyptian isolate based on the nucleotide sequences. Horizontal
distances indicate degree of relatedness
The nucleotide sequence of the Egyptian isolate of OYDV was submitted in the
GenBank under Accession No. HM473189. Multiple sequence alignment of the nucleotide
sequence of OYDV Egyptian isolate (HM473189)] with the corresponding sequence
of ten different OYDV isolates available in GenBank [Japan (AB000840 and AB000841);
China (AJ292224 and FJ765739); UK (AJ293278 and AJ409310); India (EU045556 and
EU045558); Australia (DQ925454) and Argentina (X89402)] were analyzed using
DNAMAN software (Fig. 3). Sequence comparisons showed the
percentage of similarity ranged from 82-96% of the ten reported isolates of
OYDV with the Egyptian isolate of OYDV. The similarity of the nucleotide sequences
suggested that the architecture of the potyviruses is highly conserved.
Plant regeneration and virus eradication: To investigate the effect of meristem size, thermal, chemical and electrical shock treatments on the generation rate of plantlets during the tissue culture, the regeneration and growing pattern of each regenerated plantlets were observed. As indicated in Table 1, the rate of regeneration was retarded by using small sizes of meristems and thermal treatments. The rate of regeneration and growth of shoots were not greatly retarded by treatment of virazol supplemented in the culture media. Electrical shock treatment enhancement the regeneration rate until using 10 mA/10 min, then decreased after treatment with 15/5 and 15/10 (mA/min). On the other hand, electrical shocked plant materials and then cultured on virazol (20 mg L-1) resulted 80% as are generation rate and good survival especially when 15 mA current was used (Table 1; Fig. 4). Where as, the combination of electrotherapy (15 mA/10 min) with thermotherapy (37°C for 3 weeks), the survival plants percentage reached to 60 % only.
||Final assessment of efficiency of virus elimination treatments
for OYDV eradication from garlic plants
||Effect of Electrotherapy and virazole antivirals on in
vitro propagated garlic plantlets
Multiplication and rooting of the treated plantlets: The treated shoot
clumps were transferred to control media (Fig. 5a) and then
dissected into single shoots and placed on MS medium supplemented with 0.2 mg/LNAA
for root induction (Fig. 5b).
Therapy efficiency (TE): The greatest TE, 68%, was obtained from garlic
cloves which exposed to 15 mA for 10 min, then shoot apices were excised and
cultured on media supplemented with 20 mg L-1 of virazol. This is
a result of 80% regenerated plantlets of which, 85% were (virus-free Table
||Micropropagation stages of treated garlic plantlets. (a) Multiplication
stage and (b) Rooting stage
Garlic is an economically important crop for several Egyptian agricultural
regions. Egyptian growers traditionally produce their own garlic propagative
material. This fact accounts for the observed heavy viral infection and implies
a potentially high reduction in yield and quality of this crop. If the farmers
use their own material as it happens in Egypt, 100% infection is most likely
to occur. To face this problem, a strategy for production of virus-free garlic
propagative material was needed. The occurrence of OYDV infecting garlic plants
in Egypt has been reported previously (El-Kewey et al.,
2004; Mahmoud et al., 2007). Our study extends
the information provided in earlier reports to viral occurrence in onion and
garlic plants in Egypt. In contrast to other virus genera, serology is not a
very good parameter for virus differentiation among viruses of the genus Potyvirus,
as serological cross reactions often cause misinterpretation of results (Conci
et al., 1999). Although, serology can be used for Potyvirus detection,
it is not suitable for potyvirus taxonomy (Shukla and Ward,
1988). These observations support the application of molecular techniques
for characterization of the garlic viruses, as demonstrated by others (Lot
et al., 1998; Tsuneyoshi et al., 1998).
The sequence of the coat protein gene has been used as an efficient tool in
defining the Potyvirus species (Shukla and Ward, 1988).
The specific primers from RNA-dependent RNA polymerase region and 3'-UTR successfully
detected OYDV in infected garlic plants. This result confirmed our previous
findings in 2007 (Mahmoud et al., 2007) when
used specific primer for amplification of common central region of OYDV cp gene
which produced an amplicon of 601 bp in all the samples indicating the presence
of OYDV in the tested samples. Peiwen et al. (1994)
reported that yields of virus-free garlic increased 25 to 80 and 35 to 89%,
respectively, compared with infected garlic. However, re-infection in plants
in the field is the major factor that discourages use of virus-free clones.
Traditional techniques applied for cleaning of viral diseases in plants, i.e.,
meristems culture, thermotherapy and chemotherapy fail to produce enough quantities
of clean material in most species. Alternative procedures using electric current
treatments have become in an efficient tool to overcome this problem. Black
et al. (1971) demonstrated a relation between growths stimulation
in tomato plants that were treated with low current densities (3-15 μA/plants)
during 4, 5, 12 and 24 h and the ion concentrations detected. Quacquerelli
et al. (1980) applied electric current to Cactanucia tree stakes
showing intense mosaic symptoms caused by virus, proving that treatments of
500 V/5-10 min lead up to 90% of cleaned plants. Those results settled the basis
for the electrotherapy concept. It's known that molecular structures, protein
or nucleoprotein, could be redefined as molecular machines. The molecular machines
inside the cells can be treated, or they behave, as harmonic oscillators in
thermal bathroom. If the viral particles behave as oscillators, then we should
hope the population's of viral particles half energy is proportional to the
magnitude RT, where R is the gases constant and T is the absolute temperature,
because RT is the media energy, for particle mole, for a oscillator in balance
in a thermal bath (Schneider, 1991). In particular,
we assume that the viral particles behave as quantum oscillators, that is to
say, they can take alone discreet securities of energy. Starting from this reasoning
a theoretical model is developed to find the dependence between the absorbance
and the electrical power in each explant. In conclusion, the study suggests
that RTPCR-based detection using specific primers from conserved regions
of OYDV is a more sensitive technique than ELISA, based on our previous findings.
Also meristem culture, thermotherapy and chemotherapy are not efficient techniques for OYDV eradication from garlic, whereas electrotherapy seems to be more attractive and alternative method. It avoids the time- consuming meristem excision especially when combined with chemotherapy. Results might lead to wider application for the eradication of other viruses from other hosts.
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