Abstract: Viruses are major diseases affecting potato plantations in Egypt. Different molecular and serological methods for virus detection were used to detect the Potato virus X (PVX), Potato virus Y (PVY) and Potato leaf roll virus (PLRV). ELISA, RT-PCR, immunocapture RT-PCR (IC-RT-PCR) and RT-PCR-ELISA were the detection methods used. Naturally virus-like infected potato samples were collected from different locations at El-Munofia Governorate in Egypt. PVX and PVY were isolated and identified biologically through host range and indicator plants. All detection methods mentioned above were sensitive enough to detect the three potato viruses in potato tissues. Using ELISA method, PVX, PVY and PLRV were detected in 4, 7 and 10 out of 26 samples (15.4, 26.9 and 38.5%), respectively. Mixed infection was found between PVX/PVY; PVY/PLRV and also among PVX/PVY/PLRV. According to the results, it is the use of molecular methods to confirm certain serological results i.e., the virus especially when low concentration in the infected samples. Using the advantage of IC with RT-PCR-ELISA will reduce the costs and avoid RNA degradation. Additionally, RT-PCR-ELISA could be the method of choice among the molecular methods.
INTRODUCTION
Potato (Solanum tuberosum L.) is used worldwide for human and animal consumption and as raw material for starch and alcohol production. Nowadays, one of the most important aspects of potato production is tuber quality that includes biological traits (Carputo et al., 2005).
In Egypt, potato is one of the most important and economic vegetable crop. The total potato harvested area of Egypt reached about 100,000 ha; this area produced 2,500 tons ha-1 with an average of 25 tons ha-1 (FAO, 2006). Potato suffers from many diseases, which decrease the production of this crop. A wide range of diseases, including fungal, bacterial, viral and nematode diseases, impacts potato crop. More than 25 different viruses or virus diseases have been reported and all are pathogenic to potato (Salazar, 1977). Among several factors responsible for the low potato production, potato diseases like early and late blights, potato scab, black scurf and viral diseases i.e., PVX, PVY and PLRV are most important (Qamar and Khan, 2003).
PLRV (genus Polerovirus, family Luteoviridae), like all other luteoviruses has a monopartite, single stranded RNA genome. PLRV is transmitted by aphids in a circulative non-propagative manner and is mainly restricted to phloem tissues of infected plants (Mayo and Ziegler-Graff, 1996). PLRV has genomic RNA of ~5.9 kb that contains six large Open Reading Frames (ORFs). A 5’-located gene cluster contains three ORFs that code for a polypeptide of 28 kDa with unknown functions (ORF0) and two proteins of 70 kDa (ORF1) and 108 kDa protein (ORF1/2) that appear to be replication-associated proteins (Mayo and Ziegler-Graff, 1996). Within the 3’-located gene cluster, ORF3 encodes the 23 kDa Coat Protein (CP). ORF4, which encodes a 17 kDa product (P4), is contained within the CP gene, but in a different reading frame. P4 is thought to be a movement protein (Tacke et al., 1993). PLRV ORF5 is separated from the upstream CP gene by a single amber termination codon.
PVY is an important pathogen in solanaceous crops. It belongs to the genus Potyvirus, of which it is the type species, in the plant virus family Potyviridae (Shukla et al., 1994). PVY has a single positive-sense genomic RNA ~10 kb long and forms flexuous virions. The genomic RNA contains a unique ORF encoding a polyprotein which is processed into functional viral proteins by virus-encoded proteases (P1, HC-Pro and NIa) (Riechmann et al., 1992). It is naturally transmitted by aphids in a non-persistent manner with great efficiency, causing epidemics in potato, tomato, pepper, tobacco and other solanaceous plants (De Bokx and Huttinga, 1981). The CP gene is the gene most frequently used for studies of genetic diversity in potyviruses (Shukla et al., 1994).
PVX, the prototype member of the potexvirus genus, is a rod-shaped virus containing single stranded RNA which is capped, polyadenylated and contains five ORFs (Huisman et al., 1988). Open Reading Frame (ORF) 1 encodes a 166 kDa protein which functions as a replicase. ORFs 2, 3 and 4 encode proteins of 25, 12 and 8 kDa, respectively and are known as the triple gene block. These gene products are thought to be involved in cell-to-cell movement of the virus. The fifth ORF encodes the CP, which is 25 kDa in size (Hefferon et al., 1997). PVX is readily transmitted by contact of plant parts in the field and by the cutting knife before planting (Bostan and Haliloglu, 2004).
Attempts to produce virus-free, high-yielding planting material are being made around the world by many laboratories, a prerequisite of which is the availability of accurate and rapid methods for detection of very low levels of virions and viral RNA molecules, for this reason, extensive research has been undertaken into potato viruses.
In Egypt, PVX, PVY and PLRV are common virus diseases of potato. Infections with these viruses may result in the loss of certification for potato seed and affect quality and yield in commercial production. In this study, we present biological, serological and molecular diagnostic tools to detect and differentiate potato viruses (PVX, PVY and PLRV).
MATERIALS AND METHODS
Source of Viruses
Field collected potato samples showed positive results against PVX, PVY
and PLRV using serological method were used as source of viruses. Datura
stramonium and Datura metel were mechanically inoculated with PVX
and PVY crude sap, respectively.
Host Range and Symptomatology
Sixteen plant species belonging to four families (Amaranthace, Chenopodiacea,
Fabaceae and Solanaceae) were mechanically inoculated with crude sap of PVX
or PVY. Four seedlings of each host plant were inoculated and observed daily
for symptoms development.
Serological Diagnosis of Potato Viruses
PVX, PVY and PLRV were tested in the field-collected samples using ELISA.
PVX and PLRV were tested using direct ELISA methods (DAS-ELISA) (ELISA kit,
Sanofi, Sante Animal, Paris France). PVY was tested using indirect ELISA method
(DAC-ELISA) according to Mahmoud et al. (1996).
Molecular Diagnosis
RNA Extraction
Total RNA was extracted from naturally infected and non-infected potato
tissues using two methods: CTAB method described by Gibbs
and Mackenzie (1997) with some modifications and RNA Reagent kit (Biobasic,
cat No. BS410) according to manufacturer’s instructions.
Complementary DNA (cDNA) Synthesis
Complementary DNA was prepared by mixing 2 μg of total RNA, 1.5 μL
of 10 μM of the complementary primers (pPVXc2, PVYCPcEcoRI and pPLRVc2,
Table 1) and nuclease-free water to a final volume of 15 μL.
The reaction tubes were heated at 70°C for 5 min and then the tubes were
cooled on ice. The following components were added to the annealed primer/template:
5 μL of 5X M-MLV reaction buffer [250 mM of Tris-HCl (pH 8.3), 375 mM of
KCl, 15 mM of MgCl2 and 50 mM of DTT (dithiothretol)], 2 μL
of 10 mM dNTPs, 25 units of RNasin®
ribonuclease inhibitor and 200 units of Moloney Murine Leukemia Virus-Reverse
Transcriptase (MMLV-RT) enzyme (Promega) and nuclease-free water to final volume
of 25 μL. The tubes were mixed gently by flicking the tubes and incubated
for 60 min at 37°C.
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) amplification was performed in a final volume
of 25 μL of 2.5 μL of cDNA, 2.5 μL of 2.5 mM of dNTPs, 2.5 μL
of 10X buffer, 2.5 μL of 25 mM MgCl2, 1 μL of each forward
and reverse primer at 10 μM, 0.2 μL Taq DNA polymerase and water.
Mixtures were incubated for 2 min at 94°C, followed by 35 cycles of 1 min
at 94°C, 1 min at 55°C and 2 min at 72°C, with a final incubation
of 10 min at 72°C and then the reaction was held at 4°C. Five microliter
aliquots were separated by electrophoresis in 1 and 1.5% agarose gel in 0.5X
TBE buffer using 100 bp DNA ladder (Gibco BRL, cat No. 15628-019) and 100 bp+1.5
Kb DNA ladder (SibEnzyme, cat No. M23). The gel was visualized with UV illumination
using Gel Documentation System (Gel Doc 2000, BioRad).
Immunocapture RT-PCR (IC/RT-PCR)
A volume of 100 μL of PVX, PVY and PLRV antisera was diluted to 1:
500 in coating buffer (0.035 M sodium, bicarbonate and 0.015 M sodium carbonate,
pH. 9.6). Then was added to each PCR tubes and incubated at 4°C overnight.
Polymerase Chain Reaction (PCR) tubes were washed with Phosphate-Buffered Saline-Tween
(PBS-T) buffer, pH 7.4. Infected leaves were homogenized with grinding buffer
(PBS-T, 2 % PVP).
The homogenate (100 μL) was added to the antiserum-precoated PCR tube
and incubated at 37°C for 3 h. PCR tubes were washed 3 times then, dried
and 10 μL of distilled water were added and boiled at 100°C for 5 min.
Five microliter of immunocapture reaction was used directly into RT-PCR. Finally,
5 μL of the products were analyzed by electrophoresis as described before.
| Table 1: | Designed primers for RT-PCR and capture cDNA probes |
| P: Primer; v1: Viral sense primer; c2: Complementary sense primer; p3 Biotin: Biotin labeled viral cDNA capture probe; p4 Biotin: Biotin labeled viral cDNA capture probe; S: Sense; AS: Antisense. Nucleotide at degenerate positions are represented by a single letter of the IUPAC ambiguity code, D = A, G, T; H = A, C, T; K = G, T; M = A, C; N = A, C, G, T; R = A, G; W = A, T; Y = C, T | |
RT-PCR-ELISA
Dig-Labeling
DIG-labeled DNA probe was prepared by PCR, using the primers pPVXv1 and
pPVXc2. PCR was carried out in a 50 μL reaction mixture containing Tris-HCl
(pH 8.4), 20 mM, KCl 50 mM, MgCl2 1.5 mM, dGTP, dATP, dCTP each 0.1
mM, dTTP 0.065 mM, DIG-dUTP 0.035 mM (Boehringer Mannheim Corp., Indianapolis,
IN, USA, cat. No. 1636120), primers 1 μL each, Taq DNA polymerase 2 units
(Gibco-BRL) and 2.5 μL template DNA.
Preparation of Biotin-Labeled cDNA Capture Probe
DNA oligonucleotides (21-23 nt in length; capture probe primers, Table
1) were synthesized and biotinylated at Life Technologies, Inc. The DNA
sequences of capture probes were complementary to the internal nucleotide sequence
of amplified DNA.
PCR-DIG Detection
Two hundred microliters of each mixture were pipetted into an ELISA microtiter
plate well precoated with streptavidin and was covered with self adhesive tape
(ScotchTM, St. Paul, MN, USA). Microtiter plate was kept in a shaking
water bath at 55°C for 3 h. The biotin-labeled capture probe was
added to the hybridization buffer to a final concentration of capture probe
of 7.5 nM. The hybridization solution was removed and the wells were washed
six times with washing solution. Two hundred microliter of polyclonal anti-DIG
Fab fragments conjugated to peroxidase diluted 1:100 in Tris-HCl (pH 7.5) were
added to each well and the microtiter plates were shaken gently at 37°C
for 30 min. Wells were washed six times with washing solution.Two hundred microliter
of substrate solution were added to each well and microtiter plates were incubated
for 30 min at 37°C in the dark. The absorbances of hybridized products were
measured at 405 nm in an ELISA-reader (ELX 800, Biotek Instruments, Inc.).
RESULTS
Samples collected from naturally infected potato plants showed variable symptoms including stunting, vein clearing, severe and mild mosaic, yellowing, deeping veins, necrosis, leaf cup shape and crinkling (Fig. 1). Obtained samples were primary investigated using ELISA techniques to direct this research on the three major potato viruses (PVX, PVY and PLRV) some of positive plants were used in further studies (Table 2).
Mechanical Transmission
PVX and PVY symptoms appeared after 28 days of incubation period. PVX-inoculated
plants (N. tabaccum and D. stramonium), showed sever mosaic,
vein banding, mild mosaic, while PVY-inoculated plants (N. tabaccum and
D. metel) showed veinal necrosis and severe mosaic.
Host Range and Symptomatology
Sixteen plant species belonging to four families were mechanically inoculated
with PVX and PVY (Table 3). The tested plants showed different
symptoms and serologically reacted against PVX and PVY antisera. Mild and severe
mosaic, vein banding, leaf malformation and mottling symptoms appeared on different
hosts after 28 days incubation period. External local lesions appeared on Gompherena
globosa L. and Chenopodium amaranticolor for PVX and PVY, respectively.
Severe mosaic was the most severe symptoms appeared on Nicotiana tabaccum
cv. White burley for PVX and PVY. Variable symptoms (vein banding and mild mosaic)
appeared on D. stramonium for PVX and D. metel (veins clearing,
severe mosaic, malformation and leaf cup shape) for PVY. The following hosts
were inoculated with PVX, they did not show any symptoms and no serological
reaction was detected against PVX antiserum: Leguminaceae: Vacia faba
cv. Giza 67, Phaseolus vulgaris cv. Potima; Solanaceae: Capsicum annuum
L. cv. California wonder, D. metel L., Lycopersicon esculentum cv.
Castle rock, Physalis floridana L., Ch. amaranticolor, Ch.
Quinoa and ch. album (Table 3).
| Fig. 1: | Naturally infected potato plants showing different potato viral symptoms. (a) Severe mosaic, (b) Yellowing, © Deeping veins, crinkle and necrosis, (d) Mild mosaic, (e) Leaf roll and (f) Healthy potato |
| Table 2: | Detection of PVX, PVY and PLRV from naturally infected potato samples using DAS-ELISA and DAC-ELISA (A 405 nm) |
| 1: The mean A 405 nm value corresponds to 2 replicates, 2: Positive threshold = 2 times the mean A405 nm value of negative controls, 3: Potato leaves infected with PVX, PVY and PLRV reacted positively with PVX, PVY and PLRV antisera, respectively, 4: Healthy potato leaves | |
| Table 3: | Host range, symptoms and ELISA reaction of PVX and PVY after mechanical inoculation |
| 1 and 2: Correspond to PVX-infected and healthy samples, respectively | |
Also, some hosts inoculated with PVY, did not show any symptoms or serological reaction against PVY antiserum. These hosts were as follow: Vicia faba L. cv. Giza 2, N. benthamiana L., Ch. quinoa Wild, Ch. album L., Gompherna globosa and D. staramonium L.
Serological Diagnosis of Potato Viruses
Twenty six samples of naturally infected potato were serologically tested
against PVX and PLRV antisera using direct ELISA method (DAS-ELISA) while, PVY
antiserum using indirect ELISA method (DAC-ELISA). PVX and PLRV were detected
in 4 and 10 out of 26 samples (15.4 and 38.5%), respectively. However, PVY was
detected in 7 out of 26 samples 26.9%, (Table 2).
Immunocapture RT-PCR (IC/RT-PCR)
Immunocapture RT-PCR was used for the detection of PVX, PVY and PLRV in
potato plants. IC-RT-PCR amplified products were ~300, 801 and 420 bp for PVX,
PVY and PLRV, respectively (Fig. 2).
Molecular Diagnosis
Some of the serologically screened samples were tested at the molecular
level. Total RNA was successfully extracted from healthy and infected plants
with PVX, PVY and PLRV.
RT-PCR
PCR products showed that the amplified fragments of ~300 and 420 bp were
obtained for the partial coat protein genes of PVX and PLRV, respectively. While,
801 bp was obtained for the coat protein gene of PVY and no fragments were amplified
from the RNA extracted from healthy plants (Fig. 3).
| Fig. 2: | Agarose gel electrophoresis analysis of IC-RT-PCR of PVX, PVY and PLRV. M: 100 bp DNA ladder; L1: IC-PCR of PVX infected sample with partial CP primer; L2: IC-PCR of PVY infected sample with CP primer and L3: IC-PCR of PLRV infected sample with CP primer |
| Fig. 3: | Agarose gel electrophoresis analysis of RT-PCR amplified products of (a and b) PVX and PLRV, respectively. M: 100 bp DNA ladder; L1: Positive control; L2: Negative control; L 3 to L 7: Potato samples collected from field and © PVY. M: DNA ladder; L1: PVY-positive control; L2, L3 and L4: Three samples positive ELISA and PCR; L5: Negative control; L6 and L7: Two samples negative ELISA but positive PCR; from L8 to L14: Potato samples collected from field |
RT-PCR-ELISA
DIG-labeled RT-PCR amplified cDNAs of PVX and PLRV from infected potato
leaves were analyzed by agarose gel electrophoresis (Fig. 4).
The DIG-labeled PVX-cDNA and PLRV-cDNA product was detected up to 10-5
dilution (Fig. 5). Color development was absent with products
from healthy tissues, or buffer control samples.
| Fig. 4: | Agarose gel electrophoresis analysis of (a) PVX and (b) PLRV DIG-labelled RT-PCR. M: 100 bp DNA ladder; L1 to L4: DIG-labelled RT-PCR amplified product from PVX or PLRV infected plants and L5 (b) Negative control |
| Fig. 5: | Colorimetric detection of (a) DIG-labelled PLRV and (b) PVX-cDNA (b) products as shown by absorbance values of each assay. A: Positive control, B to G: Potato samples and H: Negative control |
DISCUSSION
Mechanical Transmission
Mechanical transmission by sap inoculation to herbaceous indicator plants
can be done with minimal facilities and characteristic symptoms produced by
these plants allow both the detection and identification of many viruses (Horvath,
1993).
Host Range and Symptomatology
Visual inspection is relatively easy when symptoms clearly are characteristic
of a specific disease. However, many factors such as virus strain, host plant
cultivar/variety, time of infection and the environment can influence the symptoms
exhibited (Matthews 1980). Obtained data are similar to
those obtained by Salazar (1977) and Soliman etal.
(2002). Systemic symptoms were appeared on N. tabacum L. cv. White Burley;
N. glutinosa L.; N. rustica L. and (Solanum tuberosum L.
cvs. Nicola and Spunta). On the other hand, Phaseolus vulgaris L.,
Chenopodium quinoa wild, Chenopodium amaranticolor L. and
Chenopodium album L. are not reacted positively against PVX infection
under greenhouse conditions. Similar results were also recorded by Yousef
(1992) and Salazar (1977).
Serological Diagnosis
Many investigators have used ELISA test for serological diagnosis of PVX,
PVY and PLRV, (Varveri, 2006; Li
et al., 2006; Boukhris-Bouhachem et al.,
2007).
Molecular Diagnosis
The most common method for the detection of plant viruses is ELISA because
it is rapid and inexpensive. However, the serological methods have the drawbacks
of limited availability of antisera and questions regarding the specificity
of antisera produced from preparations containing virus mixtures. Raising antisera
is also time-consuming. By contrast, a diagnostic technique using RT-PCR can
be rapidly implemented in independent laboratories after the basic protocol
and primer sequences are made available. However, RT-PCR requiring separate
amplification of each virus of interest are potentially expensive and resource
intensive (Peiman and Xie, 2006).
A technique that combines the technical advantages of PCR with the practical advantages of ELISA, called immunocapture (IC)-PCR, was developed for the detection of several different plant viruses (Nolasco et al., 1993). The benefits of using IC in with RT-PCR to remove inhibitory plant compounds and to make nucleic acid extractions unnecessary have been well recognized (Harper et al., 1999).
Colorimetric detection of hybridized PCR products combine the specificity of
DNA probe hybridization, the sensitivity of colorimetric or luminescent detection
and the ease of sample handling in microplates (Marinho
et al., 2003). Comparison of sensitivity among ELISA, PCR, IC-PCR
and ELISA-PCR for PVX, PVY and PLRV is showing in Table 4.
Using ELISA method, PVX, PVY and PLRV were detected in 4, 7 and 10 out of 26
samples (15.4, 26.9 and 38.5%), respectively. Mixed infection was detected between
PVX, PVY and PLRV (e.g., samples 3-6 for double infection and 2, 9 and 12 for
mixed infection with the three viruses. The serologically positive samples were
confirmed as positive by the molecular methods. Except for samples no 1 and
22, PVY was detected in these 2 samples using ELISA and PCR-ELISA but not using
RT-PCR. The negative results obtained by RT-PCR could be due to RNA degradation
during extraction. Also, certain serologically negative samples (no virus was
detected) were shown to be positive using the molecular methods. The absorbance
at 405 nm for these samples was higher than the absorbance of the healthy control
but less than the threshold (more than twice the healthy control). In a comparison
between ELISA and the other molecular methods, ELISA have the advantage of low
coasts and less precautions, however, the molecular methods are more sensitive.
| Table 4: | Comparison among ELISA, PCR, IC-PCR and RT-PCR-ELISA |
| 1: The mean A405 nm value corresponds to 2 replicates, 2: Positive threshold = 2 times the mean A405 nm value of negative controls, *: Potato leaves infected with PVX, PVY and PLRV reacted positively with PVX, PVY and PLRV antisera, respectively, (Positive control), **: Healthy potato leaves (Negative control) | |
According to present data the method works well when the virus is in a high enough concentration in the infected samples. Accordingly, we advice the use of molecular methods to confirm certain serological results i.e., the virus is in low concentration in the tested samples. According to the results in Table 4, RT-PCR-ELISA could be the method of choice among the molecular methods. Using the advantage of IC with RT-PCR-ELISA, this will reduce the coasts and avoid RNA degradation.
The results obtained in this study demonstrated that the successful use of RT-PCR-ELISA to detect, directly, plant viruses in sap extract from infected leaf tissue and indicated its feasibility as a rapid laboratory assay for detecting PVX and PLRV. No more than 10 h are required for positive identification of the virus from infected tissue. RT-PCR-ELISA using a microtiter plate has reported, recently, for the detection of the plant viruses and viroids: potato virus Y (Hataya et al., 1994), tomato spotted wilt (Weekes et al., 1996), plum pox (Olmos et al., 1997) and potato spindle tuber (Shamloul and Hadidi, 1999). Moreover, PCR-ELISA has been reported to be sensitive and specific for the detection of the targeted DNA (Kawai et al., 1994; Shamloul et al., 2001). Besides, the safety of non-radioisotopic detection, the method of microtiter well hybridization has advantages of speed, suitability for a large number of samples, visual examination and adaptation to automation (Shamloul and Hadidi, 1999). The detection of DIG-labelled RT-PCR products by microwell capture hybridization assay is more sensitive than the sensitivity of analysis of the amplified product by agarose gel electrophoresis. Consequently, the use of ethidium bromide is no longer needed.
Colorimetric PCR uses microtiter plates and ELISA plate reading equipment, thus conferring two additional advantages. First, as the PCR and hybridization steps become more standardized, it will be relatively simple to perform quantitative analysis of viral infection rates within plants. Second, many virus detection facilities already use microtiter plates and plate reading equipment, as a result, it should be fairly easy for old facilities to convert to this new assay. Existing equipment can be re-used and technicians already know some of the techniques used in colorimetric PCR (Rowhani et al., 1996).