INTRODUCTION
The genus potyvirus (Family Potyviridae) includes a large group of plant
pathogen viruses that encompasses 111 recognized and 86 tentative species infecting
more than 30 plant families (Fauquet et al., 2005).
Potyvirus particles are flexuous rods of ~700-900 nm in length, contain
single-stranded RNA genome (~10 kb). Genomic RNA has a 5’ untranslated
region (5’ UTR), a single Open Reading Frame (ORF) and a 3’ UTR which
has a polyadenylated (poly A) tail. The whole genome encodes a single large
polyprotein that is subsequently processed into ten functional proteins (Adams
et al., 2005). Potyviruses are mainly transmitted by aphids in a
non-persistent manner and infect a wide range of plants in which they causes
significant losses, making the potyvirus genus interesting in a worldwide
agricultural concern. Although, they are most prevalent in tropical and subtropical
countries (Shukla et al., 1988).
Potato (Solanum tuberosum, Family solanaceae) a most important tuber
crop cultivated in India is very rich in starch contents. In Mediterranean country,
one of the most important aphid transmitted viruses is Potato virus Y
(PVY), the type member of the potyvirus group. Potato virus Y causes
a severe green mosaic on the leaves and stunting in infected plants. This virus
also causes serious diseases worldwide on pepper, tomato, tobacco and other
alternate crops. The predicted crop losses due to PVY infections range from
10-80% (De Bokx and Huttingah, 1981). Conserve amino
acid motifs of Nib and coat protein gene have recently becomes a useful tool
for taxonomic classification of distinct potyviruses and related isolates (Zheng
et al., 2010).
This current study focused on the characterization of geographically contiguous
Potato virus Y Rajasthan (Northern India) isolate PS with morphological,
serological methods. Concurrently molecular characterization done by cloning
and sequencing of core Nib and CP genes to compare with other potato virus isolates
described from other areas of the world.
MATERIALS AND METHODS
Survey and collection of leaves samples: The youngest leaves from 25
potato plant fields exhibiting typical potyvirus infection symptoms
i.e., leaf curling, mosaic and stunted growth (Fig. 1a) were
collected during Oct-2012 to Dec-2012 from Rajasthan, India. The samples were
cleaned, cut, rolled in a piece of tissue study and then stored at -20°C
in a deep-freezer till further use.
Mechanical inoculation: Virus inoculum was prepared by grinding 1 g
of young diseased leaves of potato in 5 mL of 0.04 M NaHPO4 containing
0.2% sodium diethyldithiocarbamate. Prior to inoculation, 75 mg mL-1
of carborundum and of activated charcoal were added to the sap extract (Morel
et al., 2000). For an experiment near about 40 N. benthamiana
plant grown on insect-free green house was taken and first three leaves of approximately
four week old plants were mechanically inoculated.
Virus purification and electron microscopy: Leaves harvested from naturally
infected potato and mechanically inoculated N. benthamiana at four week
post-inoculation were used for virus particle purification as described by (Chen
et al., 2003). Purified PVY samples applied on Formvar-coated grids
were stained with 1% uranyl acetate (Christie et al.,
1987) and examined under a JEOL 100S electron microscope operating at 80-100
KV (50,000xmagnification).
|
| Fig. 1(a-b): |
(a) Potato plant showing characteristics mosaic and stunting
growth and (b) Healthy plant |
Serological tests: Serological relationships of this virus isolate using
antisera against Potato virus Y specific antiserum were investigated
by employing plate trapped antigen enzyme-linked immunosorbant assay (PTA-ELISA)
(Mowat and Dawson, 1987). In PTA-ELISA, leaf antigens
were extracted in coating buffer (1:10 w/v; 200 mL well-1) and purified
virus (200 ng well-1) was used. The primary antibodies were used
at a dilution of 1:500 and Protein A conjugated to alkaline phosphatase at 1:1000
and horseradish peroxidase (HRP) conjugate at 1:1000 dilution were used as the
secondary antibody. The reaction was developed using P-Nitrophenyl Phosphate
(PNP) and 3, 3`, 5, 5`-tetramethyl benzidine (TMB) as a substrate. The absorbance
was measured at 405 and 450 nm in a Labtech LT-4000 ELISA Reader, respectively.
RNA extraction and RT-PCR: Total RNA (Host and Viral) was extracted
from 100 mg of symptomatic and healthy leaves using the TRIZOL method and resuspended
in 30 μL nuclease free water. The RNA was subjected to 25 μL reaction
mixture of cDNA containing 5 μL of RNA, 1 μL of oligo d(T)16,
5 μL of 5X reaction buffer, 2 μL of 2.5 mM dNTPs (2.5 mM), 0.5 μL
of AMV Reverse (20 U μL-1) Transcriptase and 1 μL of MgCl2
(25 mM). cDNA synthesis was carried out at 42°C for 1 h followed by
72°C for 10 min in a thermal cycler. Simultaneously PCR performed in a 25
μL reaction mixture containing 3 μL of cDNA, 2.5 μL 10X reaction
buffer, 2.5 μL of dNTPs (2.5 mM) and 1 μL MgCl2 (25 mM),
1 μL of 20 pmol each of potyvirus group specific published primers
against coat protein and Nib protein gene. These primers are MJ1(F)-5’-TGGTHTGGTGYATHGARA
AYGG-3’ and MJ2(R)-5’ TGCTGCKGCYTTCATYTG-3’ (Chen
et al., 2001) for CP gene and Nib(F)-5’-GTITGYGTIGAYGAY TTYAAYAA-3’
and Nib(R)-5’-TCIACIACIGTIGAIGG YTGNCC-3’ (Babu
et al., 2012) for Nib gene. PCR was performed in thermal cycler with
the programme 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec,
55°C for 1 min (with CP specific degenerate primer) and 57°C for 1 min
(with Nib specific degenerate primer), 72°C for 1 min and a final extension
of 72°C for 10 min. The amplified products were analyzed on 1% agarose gel,
stained with ethidium bromide.
Cloning and sequencing: The amplified PCR products were purified using
the Gel Extraction Kit (QIAGEN) and the product was cloned into the pGEM-T Easy
vector (Promega, USA) following the manufacture‘s protocols. The ligated
mix were used to transformed Escherichia coli DH5α. Resulting recombinants
clones were selected on Luria agar medium containing Ampicillin (100 μg
mL-1) and X-gal/IPTG (50 μg mL-1-40 mM) (Sambrook
and Russell, 2001). The clones were then subjected to sequenced by automated
ABI sequencer.
Phylogenetic analysis: The nucleotide sequence was compared with other
sequences from NCBI databases using BLASTn; (http://www.ncbi.nlm.nih.gov/blast).
Highest and lowest match scores and closest matching sequence from different
isolates of Potato virus Y was considered. From the blastn sequences
phylogenetic trees were constructed with the neighbor-joining method with 1,000
bootstrap value to determine the reliability of tree using MEGA 4.0 software.
RESULTS
Particle morphology and biological properties: Results of the electron
microscopy (Fig. 2) showed that particles were flexuous rods
with an average dimension of 750x15 nm, similar to the members of Family potyviridae.
Out of 40 N. benthamiana plants 29 were shown typical curling and mosaic
symptoms caused by virus inoculums. This resulted in the development of same
kind of symptoms as observed in naturally infected potato plants in the fields.
|
| Fig. 2: |
Electron micrograph of purified virus particles causing mosaic
disease on potato were stained with 1% uranyl acetate |
| Table 1: |
Absorbance values obtained by PTA-ELISA with 2 protein A enzyme
conjugates and the reaction of their substrates with adsorbed components
from sample extracts |
 |
| aI: Sap from infected leaf extracted in 5 mL of
carbonate buffer (pH 9.8) g-1 of tissue; 1/10 = I diluted 1.10
with extraction buffer; H: Sap from uninfected leaf extracted as I; B: Extraction
buffer control, bA405 values were recorded after overnight
incubation of substrate at 5°C. A450 values were recorded
after 20 min incubation of substrate at ambient temperature. Absorbance
values are the means of 3 wells. cPA-ALP: Protein A-alkaline
phosphatase conjugate; PA-HRP: Protein A-horseradish peroxidase conjugate.
Wells received sample followed by detecting antiserum, protein A enzyme
conjugate and substrate, dNPP: p-nitrophenyl phosphate, TMB:
3,3`, 5, 5`-tetramethyl benzidine; wells received successively sample and
substrate |
Detection and identification of plant viruses by PTA-ELISA: Potato
virus Y a species of potyvirus group were detected by PTA-ELISA using
the procedure described above (Table 1). Absorbance values
were accepted as positive when the reading was greater than twice the mean absorbance
of the virus-free control sample. The absorbance values of the wells receiving
sample extract and substrate were only similar to those of wells containing
extracts from virus-free tissue and extraction buffer controls. Serological
results shown absorbance values at least twice the mean for virus-free samples
were obtained with both dilutions of each sample therefore method was particularly
valuable in identifying isolates of PVY.
Characterization of viral genome: PCR amplification products (323 and
353 bp) were observed from all the infected samples tested using Nib specific
primers and CP specific primers (Fig. 3b).
|
| Fig. 3(a-b): |
(a) RT-PCR results of infected S. tuberosum plants
with potyvirus group specific primers of Nib protein gene and (b)
RT-PCR results of infected S. tuberosum plants with potyvirus
group specific primers of coat protein gene |
| Table 2: |
Percentage identities of nucleotide sequences of the partial
Nib gene of Potato virus Y infecting with other Potato virus Y
isolates |
 |
The recombinant plasmids harboring cDNA inserts were sequenced as described
in the methods section. The authenticity of these clones was confirmed by sequenced
using T7 and SP6 universal primers and the sequences were deposited in the GenBank
(KC753451 and KC753448). After sequencing, the consensus sequence was compared
to the sequences registered in the NCBI database using blastn (http://www.ncbi.nlm.nih.gov/blast)
which confirmed the presence of Potato virus Y.
Table 2 and 3 summarize the relative nucleotide
sequences similarities between Nib and CP gene of viral isolate under study
with other viral isolates available in GenBank. It showed that the sequences
of our isolate (PVY-PS) have 93-97% identity with different isolates of Potato
virus Y worldwide. The phylogenetic trees were constructed from the sequences
of CP gene and Nib gene (Fig. 4b) reveals our isolate (PS)
positioned in a separate monophyletic cluster.
The result suggested that potato samples act as a natural host for Potato
virus Y. This is the first time that Potato virus Y has been isolated
and characterized from Rajasthan (India).
|
| Fig. 4(a-b): |
(a) Phylogenetic tree representing our Potato virus Y
isolate (PS, Nib gene) positioned in a separate monophyletic cluster with
different viral isolates using neighbor-joining method with 1000 bootstrap
replicates of Mega 4.0 Software and (b) Phylogenetic tree representing our
Potato virus Y isolate (PS, CP gene) positioned in a separate monophyletic
cluster with different viral isolates using neighbor-joining method with
1000 bootstrap replicates of Mega 4.0 Software |
| Table 3: |
Percentage identities of nucleotide sequences of the partial
coat protein gene of Potato virus Y infecting with other Potato
virus Y isolates |
 |
DISCUSSION
A virus naturally infecting potato was characterized at biological, serological
and molecular levels. Structure analysis reveals the purified virus appeared
as flexuous rods with an average dimension of 750x15 nm which is consistent
with the morphology of potyviruses. Subsequently, glasshouse-grown Nicotiana
spp., was inoculated mechanically with purified isolate from infected leaves,
approximates 80% of plants show mosaic symptoms showed occurrence of potyviruses.
The virus showed a serological relationship with PVY specific antiserum while
when used a ranges of other potyvirus specific antiserum were not give
any absorbance with same substrate as above (data not shown). The existence
of many conserved domains in the viral Nib and CP region facilitates the designing
of numerous degenerate primers for RT-PCR based detection of the virus genome
(Chen et al., 2001). The amplified products were
cloned in pGEMT Easy vector, sequenced using T7 and SP6 universal primers. The
sequences were deposited in the GeneBank (KC753451 and KC753450). BLASTn showed
a similarity of the PVY-PS with other Potato virus Y with a highest similarity
of 97% (Table 2-3). Viral sequences also
showed similarity with all other Potato virus Y isolates, with a similarity
ranging from 93-97% to confirm the presence of Potato virus Y isolate.
The phylogenetic tree was constructed by using the neighbor-joining method of
the MEGA 4.0 Software. The constructed trees from the sequences of Nib and CP
gene reveals our isolate (PVY-PS) positioned in a separate monophyletic cluster
which forms a clade, meaning that it consists of an ancestral species and all
its descendants.
The group specific PCR and subsequent molecular analysis of amplified regions
has been used for rapid detection and identification of potyviruses. Thus RT-PCR
with degenerate primers designed to amplify a short conserved region of the
potyviruses seem to be useful for the detection and identification of the
potyvirus infecting S. tuberosum Rajasthan, India. This is the first
time biological, sequence characterization and identification of the viral isolate
infecting S. tuberosum in Rajasthan, India which is essential for the
timely management of the disease and for the development of viral resistant
strategies. This survey of the incidence of mosaic diseases in potato plants
in the extensively cultivated areas of Rajasthan province revealed that PVY
was the predominant mosaic virus.
CONCLUSION
It can conclude that the diseased plants showed typical symptoms of severe
mosaic of the leaves, stunted and retardation of the plant growth. Positive
results were also obtained by biological, serological and RT-PCR methods to
detect and identify the potyvirus. The sequences of RT-PCR fragments
confirmed that a potyvirus associated with mosaic disease on potato plants
in Rajasthan (India). Potyvirus infecting potato plants in Rajasthan
are closely related to Potato virus Y (PVY). This survey represents the
usual disease incidence that is prevalent in this region.
ACKNOWLEDGMENT
The authors are thankful to University Grant Commission, Government of India
for financial support. The authors are also grateful to Supervisor Dr. R.K.
Gaur for helping in manuscript preparation.
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