Evidence of occurring alfalfa mosaic virus in potato plants in Assam, India

Abstract

Alfalfa mosaic virus (AMV, family Bromoviridae, genus Alfamovirus) has an extensive host range. The reports of AMV available in India were dated far back as 1979 and 1981 found in alfalfa and brinjal crops respectively. In January 2019, field surveys were conducted for viral diseases infecting potato in Sonitpur and Jorhat districts of Assam state of India. Severe yellow mosaic or calico pattern symptom, consistent with infection with AMV were observed with an incidence of approximately 25% of the plants found in farmer’s fields. Sixty different symptomatic leaf samples including those associated with AMV observed were collected at random and were analysed to detect the presence of AMV. Leaf samples were frozen in liquid nitrogen and total RNA extracted from them were analyzed by one step polymerase chain reaction to detect the presence of AMV reported in potato inducing similar symptoms using a specific pair of primers for coat protein gene. An expected amplicon size of 351 bp was observed in 70% of the symptomatic leaf samples when the PCR products were analyzed on a 1.2% agarose gel. The PCR product for one sample each from the surveyed districts was eluted, purified and sequenced. The sequence results obtained were compared with those deposited in GenBank database. Blastn analysis of the sequenced isolates submitted to GenBank revealed nucleotides similar to AMV Iran isolate sequences. To our knowledge, this is the first report of AMV infecting potato in India.

Full text

SHORT COMMUNICATION
Evidence of occurring alfalfa mosaic virus in potato plants
in Assam, India
Mohamad Hussam Halabi
1
•John Oladeji Oladokun
1
•Palash Deb Nath
1
Received: 18 September 2019 / Accepted: 26 November 2019
ÓIndian Virological Society 2019
Abstract Alfalfa mosaic virus (AMV, family Bromoviri-
dae, genus Alfamovirus) has an extensive host range. The
reports of AMV available in India were dated far back as
1979 and 1981 found in alfalfa and brinjal crops respec-
tively. In January 2019, field surveys were conducted for
viral diseases infecting potato in Sonitpur and Jorhat dis-
tricts of Assam state of India. Severe yellow mosaic or
calico pattern symptom, consistent with infection with
AMV were observed with an incidence of approximately
25% of the plants found in farmer’s fields. Sixty different
symptomatic leaf samples including those associated with
AMV observed were collected at random and were anal-
ysed to detect the presence of AMV. Leaf samples were
frozen in liquid nitrogen and total RNA extracted from
them were analyzed by one step polymerase chain reaction
to detect the presence of AMV reported in potato inducing
similar symptoms using a specific pair of primers for coat
protein gene. An expected amplicon size of 351 bp was
observed in 70% of the symptomatic leaf samples when the
PCR products were analyzed on a 1.2% agarose gel. The
PCR product for one sample each from the surveyed dis-
tricts was eluted, purified and sequenced. The sequence
results obtained were compared with those deposited in
GenBank database. Blastn analysis of the sequenced iso-
lates submitted to GenBank revealed nucleotides similar to
AMV Iran isolate sequences. To our knowledge, this is the
first report of AMV infecting potato in India.
Keywords AMV Potato Assam India
Potato (Solanum tuberosum L.) is seen as a basic compo-
nent part of almost every household daily meal in north-
eastern region (NER) states of India. Among states in NER,
Assam in the leading state contributing to the country total
production of potato with respect to total area and pro-
duction strength. In India, potato has emerged as fourth
most important food crop after rice, wheat and maize [2].
Coincidentally, potato is one of the most important and
largely cultivated horticultural crops of Assam [4]. At
present, India production stands at 43.77 million tonnes
while Assam state accounted for 2.39% of the total pro-
duction [5]. Despite the relevance of this crop to Assam
and India at large, like many other countries of the world,
potato production is greatly hampered by pathogen espe-
cially those caused by viruses. Viral diseases are prevalent
throughout the India but are most severe in North-eastern
plains and plateaux where population of aphid vectors is
high throughout the crop season [7]. Of all viral diseases
known, Tomato leaf curl New Delhi virus (ToLCNDV),
Potato leaf roll virus (PLRV) and Potato virus Y (PVY) are
the most important viruses in India.
Recently in Assam, potato tubers of different cultivars
were noticed in the market to be smaller than the usual
sizes we are familiar with; which we presumed to be
associated to running out or degeneration of seed stocks
and suspected to be caused by viral diseases as affirmed by
many research findings. In an attempt to confirm and find
answers to the cause of this sudden change and to deter-
mine the prevalence of potato viruses in the region which
are limited, we conducted field surveys for viruses infect-
ing potato in January 2019 in two different districts of
Assam state, namely, Sonitpur and Jorhat where potato was
largely grown. While on the field, we observed severe
yellow mosaic or calico pattern (Fig. 1) and other known
virus-like symptoms associated with potato viral diseases
&Mohamad Hussam Halabi
mohamad.halabi@aau.ac.in
1
Faculty of Agriculture, Department of Plant Pathology,
Assam Agricultural University, Jorhat, Assam 785013, India
123
VirusDis.
https://doi.org/10.1007/s13337-019-00560-3

on the leaves in almost 25% of the plants found in farmer’s
field. Also, most fields visited are either having potato
grown solely or the combination of potato-tomato, potato-
cowpea or potato-pepper. In an intercrop field, similar
symptom expressed on potato was also expressed on
intercrop plants especially on cowpea plants (Fig. 2). The
potato cultivars grown were recorded as local landraces
(small table potato from Jorhat), Kufri Himalini, Kufri
Pukhraj and Kufri Jyoti (recommended potato cultivars
from Sonitpur).
Sixty symptomatic leaf samples (both calico and other
virus-like symptoms) were randomly collected in sample
bag and kept in ice pack while in transit to the virology
laboratory of Department of Plant Pathology, Assam
Agricultural University and kept at -80 °C prior to
detection. Total RNA was isolated by freezing each leaf
sample kept in a small sample bag in liquid nitrogen and
grinded to fine powder. Then, each powdered sample was
transferred into a 1.5 ml eppendorf tube. After this,
appropriate volume of TRIzol
TM
reagent (ThermoFisher
SCIENTIFIC) was added to each eppendorf tube according
to the procedure described by the manufacturer [3].
Extracted RNA was then subjected to One-step reverse
transcription-polymerase chain reaction (RT-PCR) assay
using PrimeScript
TM
kit (TAKARA BIO INC
Ò
Japan) with
a pair of AMV coat protein specific primers AMV-F (50-
CCATCATGAGTTCTTCACAAAAG-30) and AMV-R
(50-TCGTCACGTCATCAGTGAGAC-30;[8]. RT-PCR
composition contained 1 ll of target RNA was mixed with
12.5 ll29RT-PCR containing dNTPs mixture (final
concentration was 400 lM), One Step enhancer solution;
1ll PrimeScript one-step enzyme mix containing Prime-
Script RTase, DNA polymerase (TaKaRa Ex Taq HS),
RNase inhibitor; specific forward and reverse primers and
RNase free water bringing the total reaction volume to
25 ll. The temperature programme for amplification
reactions was as follows: synthesis of cDNA 50 °C for
30 min followed by initial denaturation for 3 min at 94 °C,
followed by 30 cycles of 95 °C for 30 s, annealing at 52 °C
for 30 s, and extension temperature at 72 °C for 30 s. The
final extension was at 72 °C for 5 min. A GeneAmp 9700
thermocycler was used for RT-PCR amplifications. PCR
products were separated on a 1.2% agarose gel, stained
with ethidium bromide, and visualized under UV light. An
amplicon, one sample from each surveyed district was
eluted and purified using a NucleoSpin
Ò
Gel and PCR
Clean-up Midi Kit (TAKARA BIO INC
Ò
Japan) for
sequencing. The obtained sequences were compared with
the isolates available at the National Centre for Biotech-
nological Information (NCBI) GenBank by BLASTn
analysis.
Multiple nucleotide sequence alignment was performed
by using Clustal W (MEGA 6.0) and a phylogeny tree was
constructed for comparative sequence analysis of these
isolates with other isolates available in the Genebank.
The gel electrophoresis results showed an expected
amplicon size of 351 bp. Of 60 samples tested, 42 were
positive to AMV (Fig. 3) while others (18) were negative
but positive to other potato viruses (data not presented in
this report). This confirms the symptom (calico) to be
caused by AMV. BLASTn analysis of the sequenced PCR
products submitted to GenBank revealed highest identity of
99.45% (MK931302) and 97.78% (MK931303)
Fig. 1 Symptom observed in AMV infected potato leaves in the
surveyed districts showing severe yellow mosaic or calico pattern
Fig. 2 Cowpea plants expressing virus-like symptom similar to
AMV infected potato plants from the surveyed field
Fig. 3 RT-PCR amplification with AMV specific primers: the
samples 2, 3, 5, 7, 8, 9, 10 and 11 were positive showing specific
amplification of an AMV band with expected size, M: 100 bp DNA
ladder
M. H. Halabi et al.
123

nucleotides which is similar to AMV Iran isolate sequences
available in the GenBank (KX535488) obtained from
alfalfa (Fig. 4).
The detection of AMV in potato leaf samples indexed is
an indication that the virus is present in the host in India.
Other viruses infecting potato reported elsewhere in India
from the surveyed areas such as Potato leaf roll virus
(PLRV), PVY, Potato virus M (PVM), Potato virus X
(PVX) and Potato virus S (PVS) were also tested and
positive but not included in this report due to further work
is still ongoing. AMV is known to have a very wide host
range, including pepper, tomato, cucurbits, beans, potato,
alfalfa, clover, tobacco, several other crops and weeds. It is
transmitted by seed, aphids as well as mechanically. AMV;
genus Alfamovirus, family Bromoviridae, whose genome is
composed of three single-stranded positive-sense RNA
molecules (RNA1, RNA2 and RNA3). RNA1 and RNA2
encode proteins are essential for replication (P1 and P2)
while RNA3 encodes for the movement (MP) and coat
(CP) proteins, the latter being translated from a subge-
nomic RNA4 (sgRNA4) produced by transcription of the
negative-sense strand of RNA3 [1]. However, regular
surveillance of viral diseases is a key target control effort in
managing viruses, because, it is significantly important to
accurately survey viral disease distribution. AMV is
reported to have limited economic impact in potato unless
a tuber necrosis inducing strain is present [6]. Therefore,
the existence of AMV on potato should be given more
attention since it a major crop of preference in the NER. To
our knowledge, this is the first report of AMV naturally
infecting potato in India. In addition, this is also the first
report of AMV in NER of India. Hence, further studies will
make it possible in determining the geographical distribu-
tion and incidence of AMV in potato fields and as well
determine the routes through which this virus may have
been introduced in India.
Acknowledgements This work was financially supported by Indian
Council for Cultural Relations (ICCR) and Assam Agricultural
University (AAU).
Compliance with ethical standards
Conflict of interest There is no conflict of interest to be declared.
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jurisdictional claims in published maps and institutional affiliations.
Fig. 4 Phylogenetic trees derived from 351 nucleotide sequence of
the coat protein of the AMV isolates with the other previously
reported AMV isolates. Phylogenetic analyses were performed
employing the maximum likehood method packaged in the MEGA
6.0 software
Evidence of occurring alfalfa mosaic virus in potato plants in Assam, India
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