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.

Potato (Solanum tuberosum L.) is seen as a basic component 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 production 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 production [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 especially 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 determine the prevalence of potato viruses in the region which are limited, we conducted field surveys for viruses infecting 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 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).

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

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™ 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™ kit (TAKARA BIO INC^® Japan) with a pair of AMV coat protein specific primers AMV-F (5′-CCATCATGAGTTCTTCACAAAAG-3′) and AMV-R (5′-TCGTCACGTCATCAGTGAGAC-3′; [8]. RT-PCR composition contained 1 µl of target RNA was mixed with 12.5 µl 2 × RT-PCR containing dNTPs mixture (final concentration was 400 μM), One Step enhancer solution; 1 µl PrimeScript one-step enzyme mix containing PrimeScript 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 µl. 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^® Gel and PCR Clean-up Midi Kit (TAKARA BIO INC^® Japan) for sequencing. The obtained sequences were compared with the isolates available at the National Centre for Biotechnological 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) nucleotides which is similar to AMV Iran isolate sequences available in the GenBank (KX535488) obtained from alfalfa (Fig. 4).

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

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

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 subgenomic 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 distribution and incidence of AMV in potato fields and as well determine the routes through which this virus may have been introduced in India.

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Conflict of interest

There is no conflict of interest to be declared.