A Simple Precipitation Approach for

Isolation and Enrichment of Sugarcane

Streak Mosaic Virus

Viswanathan Chandran & Prabu

Gajjeraman

Sugar Tech
An International Journal of Sugar Crops
and Related Industries

ISSN 0972-1525

Sugar Tech
DOI 10.1007/s12355-013-0225-x

1 23
Your article is protected by copyright and
all rights are held exclusively by Society for
Sugar Research & Promotion. This e-offprint
is for personal use only and shall not be self-
archived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com”.

1 23
 Author's personal copy
Sugar Tech
DOI 10.1007/s12355-013-0225-x
SHORT COMMUNICATION
A Simple Precipitation Approach for Isolation and Enrichment
of Sugarcane Streak Mosaic Virus
Viswanathan Chandran • Prabu Gajjeraman
Received: 14 March 2013 / Accepted: 4 June 2013
Ó Society for Sugar Research & Promotion 2013
Abstract Sugarcane streak mosaic virus (SCSMV) is a
more prevalent sugarcane infecting virus reducing the total
sugar production of the world which necessitates rapid
screening and detailed studies on SCSMV pathogenicity. A
simple reliable, user friendly approach for purification and
enrichment of SCSMV from infected sugarcane leaf tissue
was developed as alternative to ultracentrifugation
approach. The quality and fold enrichment of viral particle
was confirmed by RT-PCR and sequence analysis of
SCSMV coat protein region. This approach offers 20.03-
fold increase in sensitivity for their detection and high
viral: host genome ratio of 102 and 103 in infected leaf
tissue and purified viral fraction, respectively.
Keywords Enrichment  Purification  RT-PCR 
Streak mosaic virus  Sugarcane
Sugarcane represents third largest crop in terms of value next
to rice and wheat in India. Among the sugarcane diseases,
mosaic is an important viral disease of sugarcane with sig-
nificant yield losses in India (Prabu et al. 2008; Parameswari
et al. 2013). Sugarcane mosaic virus (SCMV) and Sugarcane
streak mosaic virus (SCSMV) are the two major RNA viruses
causing mosaics. SCSMV is more prevalent than SCMV in
most of the Asian countries including India (Bagyalakshmi
et al. 2012; Parameswari et al. 2013). The analysis of the single
stranded positive sense RNA genome of SCSMV isolates
from different geographical regions and lack of serological
V. Chandran  P. Gajjeraman (&)
Plant Functional Genomics Unit, Department of Biotechnology,
Karpagam University, Eachanari Post, Coimbatore 641021,
Tamil Nadu, India
e-mail: prabugajjeraman@gmail.com
cross reactions between SCSMV and other potyviruses sup-
ports that SCSMV represents a new genus, namely Poacevi-
rus, as approved by International Committee on Taxonomy of
Viruses (ICTV) in the family Potyviridae (Li et al. 2011).
Recent reports showed that SCMV and SCSMV and/or sug-
arcane grassy shoot phytoplasma (SCGS) and SCSMV are
widespread all over India (Prabu et al. 2008; Viswanathan and
Rao 2011; Bagyalakshmi et al. 2012).
Molecular basis for understanding the mechanism of
SCSMV infection has been a lacuna in screening and devel-
opment of resistant plant types due to (1) lack of serological
cross reactions between SCSMV and other potyviruses (2)
lack of methodology to isolate significant amount of virus
particle (3) limited experimental information on SCSMV
pathogenicity. In addition, the infectivity of a SCSMV virus
can be determined comparatively based on their titer value,
where in many cases the virus titer and its ability to multiply
need not correspond with the symptomatic exposure of the
infection (Jarosova and Kundu 2010). Thus, estimating the
virus titer has become inevitable for studying any virus par-
ticles in detail. Hence, it is essential step to isolate and enrich
the SCSMV for many applications, like to isolate specific
antibody production, in vitro characterization by mechanical
inoculation and systematic studies on SCSMV.
Techniques based on selective amplification using poly-
merase chain reaction (PCR), DNA hybridization, immuno-
capture RT-PCR, direct antigen coating-enzyme linked
immunosorbent assay (DAC-ELISA) and dot-blot immuno-
binding assays, (Hema et al. 2003; Prabu et al. 2008; Para-
meswari et al. 2013) have been used for identification and
classification of virus in host sugarcane plants. However
majority of these methods depend on the viral titre in the
infected tissue and the quantity of amplifiable viral genome in
the background of host genome. Although several protocols
like sucrose density ultracentrifugation and differential
123
 Author's personal copy
centrifugation (Hema et al. 1999; Yang et al. 2012), and
chromatographic techniques (ion exchange, liquid and
monolithic columns; Rupar et al. 2010) are exist for the
enrichment of virus fractions, but are complex, time con-
suming and of high cost or not applicable to most of labs
working in this area due to technical difficulties. This
prompted us to look for alternative, user friendly enrichment
methods that result in high viral: host genome ratio.
In our study, we tried to take advantage of the precipi-
tation ability of virus and developed PEG/NaCl mediated
precipitation method without the need of ultracentrifuga-
tion, to achieve the enriched purification of SCSMV virus
as described for tobacco mosaic virus titer analysis
(Kathiria et al. 2010). In the present study, we describe an
efficient and simple reliable approach for enrichment of
SCSMV from infected sugarcane tissues. The quality and
fold enrichment of viral RNA was confirmed by PCR and
sequence analysis of SCSMV coat protein region.
As shown in Fig. 1a, streak mosaic disease symptomatic
sugarcane cultivar, Co86032 (10 month old) were collected
from sugarcane fields of Tamil Nadu Agricultural Uni-
versity, Coimbatore and were diagnosed for SCSMV
infection by RT-PCR using SCSMV coat protein specific
primers as described in our earlier studies (Prabu et al.
2008). The collected plant samples were maintained in
Karpagam University, Coimbatore, India under insect-
proof condition maintained at 25 °C during the day and
20 °C at night, with a photoperiod of 16 h of daylight.
For SCSMV viral particle purification, fresh leaf tissues
(1.0 g) were homogenized in homogenization buffer
(1 ml g-1 of fresh tissue) containing 0.5 M phosphate buffer
(pH 7.0) and 143 mM b-mercaptoethanol. Two volumes of
water-saturated chloroform/butanol (1:1, v/v) were added
with the homogenate and centrifuged at 3,0009g for 15 min.
To the aqueous phase, 4 % PEG-8000 (w/v, final concentra-
tion) was added and kept on ice for 10 min for precipitating
the viral particles. Virus was collected by centrifugation in a
microfuge, resuspended in 50 ll of 10 mM phosphate buffer
Fig. 1 a Streak mosaic disease symptomatic sugarcane leaf showing
chlorotic streaks. b PCR amplification of SCSMV coat protein gene
partial fragment (1,197 bp) using SCSMV-CPf and SCSMV-CPr
primers. Lane M, 100 bp DNA ladder Mix (MBI Fermentas); Lane 1,
Water control; Lane 2, RNA from enriched viral fraction (50 ng ll-1)
123
Sugar Tech
(pH 7.0), and centrifuged again to remove insoluble materials.
Finally, virus was precipitated again with 50 ll each of 40 %
PEG-8000 and 10 % NaCl (w/v), collected by centrifugation
at 3,0009g for 15 min and suspended in 20 ll of 10 mM
phosphate buffer (pH 7.0). The virus particles were quantified
by measuring the optical densities of 0.234 and 0.191 at 260
and 280 nm, respectively using a BioPhotometer plus (Ep-
pendorf, Germany). The purified SCSMV viral particles had
an A260/280 ratio of around 1.22 in accordance with earlier
studies using ultracentrifugation (Hema et al. 1999).
In order to determine the identity of purified SCSMV
virus, primers SCSMV-CPf (50 -GGAGCAAACACACAGG
GAAGGAAGGATTT-30 ) and SCSMV-CPr (50 -CCTCCTC
ACTGGGCAGGTTGATTGT-30 ) were used to amplify a
partial gene encoding for coat protein of SCSMV by RT-
PCR based on the sequences obtained in our earlier studies
(Prabu et al. 2008). In brief, total RNA was isolated from the
purified fraction of virus and infected plant leaf tissue using
QIAamp Viral RNA Mini Kit and RNeasy plant mini kit
(QIAGEN, Germany), respectively. Total RNA (1 ll) and
1 ll primer SCSMV-CPf (15 lM) were boiled for 5 min,
snap chilled on ice and reverse-transcribed in a 10 ll assay
reaction using 19 PCR buffer, 6.5 mM MgCl2, 1 mM
dNTPs, 10 U RNase inhibitor, and 25 U MuLV reverse
transcriptase at 42 °C for 30 min followed by incubating at
95 °C for 5 min. The cDNA was amplified by PCR in a 50 ll
reaction consisting of 10 ll reverse transcribed ssDNA, 4 ll
PCR buffer, 4 ll MgCl2 (25 mM),1 ll primer SCSMV-CPr
(15 lM), 2.5 U Taq DNA polymerase and 30.5 ll RNase
free water. PCR conditions used were initial denaturation
at 95 °C for 2 min followed by 40 cycles of denaturation at
94 °C for 1 min, annealing at 54 °C for 1 min, extension at
72 °C for 2 min and final extension at 72 °C for 10 min and
analyzed on 1 % agarose gel.
Amplification using SCSMV coat protein specific primers
resulted in the amplicon of expected size 1,197 bp (Fig. 1b).
The PCR amplicon was purified using QIAEX II gel
extraction kit (QIAGEN, Germany), sequenced at the DNA
sequencing service of Eurofins, Bengaluru, India and sub-
mitted in NCBI GenBank database with accession numbers
of KF051273 and KF051274. The sequences (GenBank
accession numbers. KF051273, KF051274) were compared
and identified with known SCSMV coat protein gene
sequences in GenBank using the Basic Local Alignment
Search Tool (BLAST) software (http://www.ncbi.nlm.nih.
gov/blast).
To evaluate the enrichment level of purified SCSMV virus,
serial dilutions of total RNA from enriched viral fraction and
infected sugarcane leaf tissue were used as template for end-
point RT-PCR detection. In brief, 1 ll (50 ng ll-1) RNA
samples of enriched fraction and of infected leaf tissue were
serially diluted with sterile RNase free water up to 107 and
subjected to PCR analysis as described above, and visualized
 Author's personal copy
Sugar Tech
Fig. 2 Comparison of purification and enrichment level of SCSMV
from a enriched viral fraction b plant tissue in end point RT-PCR
detection using SCSMV-CPf and SCSMV-CPr primer pair. Lane M,
100 bp DNA ladder Mix (MBI Fermentas); Lane 1, water control;
under UV after 1 % (w/v) agarose/EtBr gel electrophoresis.
All reactions were performed in triplicates with negative
(water) reaction control.
By this precipitation approach, we could obtain addi-
tionally 386 lg ml-1 of RNA yield from enriched viral
fraction compared to same amount of infected plant tissue
(1.0 g). The primers, SCSMV-CPf and SCSMV-CPr detec-
ted the presence of SCSMV in template concentration as low
as 50 pg ll-1 (Fig. 2a) and 500 pg ll-1 (Fig. 2b) for enri-
ched viral fraction and infected plant tissues, respectively.
Hence, this detection limits could be used to estimate the
ratio of SCSMV RNA in total tissue RNA which is about
1:7.69 9 102, 1:15.4 9 103 in enriched fraction (20.03 fold
increase) and infected plant tissues respectively.
Collectively, the present study reports an effective and
simple precipitation technique for purification and enrich-
ment of SCSMV particles with as low as 1 g of tissue
sample as alternative to time consuming ultracentrifugation
based techniques. This method increases the quantity of
amplifiable viral genome in the background of host genome
and other host interfering components of PCR detection.
The present method uses inexpensive materials which can
be easily accessible to any laboratory and enables a very
quick method for estimating viral titer value.
Acknowledgments Authors are thankful to Dr. R. Vasanthakumar,
Chairman, Shri. K. Murigaiah, Advisor and Dr. P. Lakshmanaperu-
malsamy, Registrar, Karpagam Univerisity, Coimbatore, Tamil Nadu,
India for their encouragement and support during the course of study.
References
Bagyalakshmi, K., B. Parameswari, C. Chinnaraja, R. Karuppaiah, V.
Ganesh Kumar, and R. Viswanathan. 2012. Genetic variability
Lane 2, undiluted RNA (50 ng); Lane 3, 10-1 dilution (5 ng); Lane 4,
10-2 dilution (0.5 ng); Lane 5, 10-3 dilution (50 pg); Lane 6, 10-4
dilution (5 pg); Lane 7, 10-5 dilution (0.5 pg); Lane 8, 10-6 dilution
(0.05 pg); Lane 9, 10-7 dilution (0.005 pg)
and potential recombination events in the HC-Pro gene of
Sugarcane streak mosaic virus. Archives of Virology 157:
1371–1375.
Hema, M., J. Joseph, K. Gopinath, P. Sreenivasulu, and H.S. Savithri.
1999. Molecular characterization and interviral relationships of a
flexuous filamentous virus causing mosaic disease of sugarcane
(Saccharum officinarum L.) in India. Archives of Virology 144:
479–490.
Hema, M., N. Kirthi, P. Sreenivasulu, and H.S. Savithri. 2003.
Development of recombinant coat protein antibody based IC-
RT-PCR for detection and discrimination of Sugarcane streak
mosaic virus isolates from Southern India. Archives of Virology
148: 1185–1193.
Jarosova, J., and J.K. Kundu. 2010. Validation of reference genes as
internal control for studying viral infections in cereals by
quantitative real-time RT-PCR. BMC Plant Biology 10: 146.
Kathiria, P., C. Sidler, A. Golubov, M. Kalischuk, L.M. Kawchuk,
and I. Kovalchuk. 2010. Tobacco mosaic virus infection results
in an increase in recombination frequency and resistance to viral,
bacterial, and fungal pathogens in the progeny of infected
tobacco plants. Plant Physiology 153: 1859–1870.
Li, W., Z. He, S. Li, Y. Huang, Z. Zhang, D. Jiang, X. Wang, and Z.
Luo. 2011. Molecular characterization of a new strain of
Sugarcane streak mosaic virus (SCSMV). Archives of Virology
156: 2101–2104.
Parameswari, B., K. Bagyalakshmi, R. Viswanathan, and C. Chinna-
raja. 2013. Molecular characterization of Indian Sugarcane
streak mosaic virus isolate. Virus Genes 46: 186–189.
Prabu, G.R., P.G. Kawar, G.B. Dixit, and D. Theerthaprasad. 2008.
First report of a phytoplasma and virus pathogens associated
with sugarcane grassy shoot disease in India. Sugarcane
International 26: 14–16.
Rupar, M., M. Ravnikar, M.T. Znidaric, P. Kramberger, L. Glais, and
I.G. Aguirre. 2010. Fast purification of the filamentous Potato
virus Y using monolithic chromato-graphic supports. Journal of
Chromatography A 1272: 33–40.
Viswanathan, R., and G.P. Rao. 2011. Disease scenario and management
of major sugarcane diseases in India. Sugar Tech 13: 336–353.
Yang, S., T. Wang, J. Bohon, M.È.L. Gagné, M. Bolduc, D. Leclerc,
and H. Li. 2012. Crystal structure of the coat protein of the
flexible filamentous Papaya mosaic virus. Journal of Molecular
Biology 422: 263–273.
123