Distribution and molecular characterization of potato virus Y (PVY) strains infecting potato (Solanum tuberosum) crop in Kashmir (India)

Abstract

For breeding resistant cultivars, understanding the nature and distribution of PVY strains is indispensable. In present study, during the course of survey two hundred potato samples showing symptoms of vein clearing, mosaic, stunting, mottling, curling and vein banding were collected from 4 major potato growing districts of Kashmir valley. The disease incidence ranged from 16 to 27.33% with maximum in district Srinagar (27.33%). All the samples were serologically tested for PVY infection using DAS-ELISA and 74 tested positive for PVY infection. Out of 74 positive samples forty samples were re-confirmed by RT-PCR by amplifying 900 bp using coat protein (CP) gene specific primers. The PCR-positive samples were further characterized into different strains using strain specific primers. The strains NTN, N and O were reported and among them NTN strain was found to be most prevalent throughout the valley. The phylogenetic analysis of selected isolates carried out with known PVY strains also confirmed that the isolates belong to the N, NTN and O strains of PVY. The study will help in developing point of care strain specific diagnostics and also in devising the strategy for developing PVY resistant varieties, because when we have the complete information about the virus and its strains it will help us in screening the germplasm against each strain and, therefore, eventually development of a multi-strain resistant variety.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13337-021-00722-2.

Potato, (Solanum tuberosum L.), an auto tetraploid member of family Solanaceae, is a starchy, tuberous crop ranks fourth in the world in terms of production [1]. Potato is susceptible to wide range of biotic stresses and among them bacterial, fungal and viral diseases cause significant economic losses. Viruses are one of the most detrimental pathogens infecting potato crop and more than 40 viruses have been reported to be infecting the crop. The most important viruses are potato virus Y (PVY), potato leaf roll virus (PLRV), potato virus A(PVA), potato virus X (PVX), potato virus S(PVS) and potato virus M (PVM) [2]. Among viruses, PVY causes huge economic yield losses up to 80% under severe conditions [3]. It belongs to family Potyviridae and genus Potyvirus. The host range of PVY is wide and generally infects the members of families, Asteraceae, Chenopodiaceae, Amaranthaceae, and Fabaceae and particularly the members of Solanaceae [4]. The PVY virion is about 730 nm long and 11 nm wide [5]. The genome is monopartite single-stranded positive sense RNA, nearly 10 kb in length with single large open reading frame (ORF) flanked by 5` and 3` untranslated regions (UTRs) [6]. It is transmitted through aphids and vegetative propagation [7].

The PVY had three major strains viz., PVYN, PVYO and PVYC and the most common strain among them is PVYO. Presently, potato crop is infected with most common recombinant strains viz., PVYN: O, PVYNTN and PVYN-Wi. The infection with recombinant strains is economically detrimental for growth of potato industry, as recombinant strains of the virus were reported to cause tuber necrotic ringspot disease (PTNRD) in many potato-growing regions of the world [8]. In India, the PVY has been also a threat to the cultivation of potato, according to the cultivar, virus strain and environment and causes noteworthy yield losses, up to the extent of 100% [9]. However only few reports are available in context to strain specific characterization of PVY infection in India, particularly in Jammu and Kashmir. Keeping in view the present study was devised to know the status of PVY infection and distribution of various PVY strains in major potato growing districts of Kashmir.

During 2018–2019, the survey was conducted in four major potato growing districts namely Anantnag, Baramulla, Srinagar and Budgam, to know the distribution of PVY infection in potato fields of Kashmir Valley, India. The area was divided into four major potato growing districts representing entire Kashmir. Each district was divided into two blocks and each block was further divided into two locations. The field disease incidence was calculated using below mentioned formula [10]. 50 samples were randomly collected from each district, making a total 200 samples showing various symptoms along with healthy were collected and analysed in laboratory.

$$\text{Percent}\text{disease}\text{incidence}=\frac{\text{Number}\text{of}\text{diseased}\text{potato}\text{plants}}{\text{Total}\text{number}\text{of}\text{potato}\text{plants}\text{observed}}\times 100$$

The samples were tested serologically for the presence of PVY virus by using specific DAS-ELISA detection kit (Agdia USA, Catalog no. SRA 20,001). The results were first assessed by naked eye and then by measuring the absorbance at 405 nm wavelength using ELISA reader (Thermo Scientific).

Out of 74 PVY positive samples 40 samples (10 from each district) were used for reconfirmation by RT- PCR using coat protein (CP) specific primers. Total RNA was extracted from infected leaves of potato by crushing the samples in liquid nitrogen and then using TRIzol RNA extraction kit (Thermo Fisher Scientific). The First strand cDNA was synthesized by superscript reverse transcriptase (Invitrogen, USA) using oligo(dt) primer as mentioned in the manufacturers protocol. The CP gene specific primers were designed using BioEdit and Primer3 plus software’s from previously reported strains of PVY which are available at National Centre for Biotechnological Information (NCBI) (Supplementary Table 1). Two-five microliters of cDNA was used for PCR amplification using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). The PCR was also used to identify different strains of PVY using strain specific primers taken from previous study of Lorenzen et al. [11] (Supplementary Table 1). The reaction was carried out with 20 µL of total volume containing 2 µL of cDNA, 0.5µL of 4 primers (Forward and reverse) of 4 strains (NTN, N, O, N:O), 4 μL of HF buffer, 0.4 μL of 10 mM dNTPs, 0.3µL of Phusion DNA polymerase and the final volume was made by adding 11.3 µL of autoclaved millipore water then the mixture was subjected to multiplex PCR amplification with thermal conditions: initial denaturation at 95 °C for 3 min, followed by 40 main cycles (95 °C for 10 s, 55 °C for 10 s, 72 °C for 30 s) and finally 1 cycle of single extension (72 °C for 1 min), the samples were analysed. The CP as well as strain specific amplicons were visualized on agarose 1% gel using 1 kb DNA ladder (Thermo Scientific) and amplified products were eluted by using QIAquick® Gel extraction kit (Qiagen company).

The purified PCR amplicons of CP as well as strain specific products (nine isolates) representing each district, were sequenced in sense and antisense direction (sequencing (geneilabs.com). The final sequences were assembled into contigs to generate the complete gene sequence of CP using DNA Baser. Nucleotide sequences were confirmed through basic local alignment search tool (BLAST) analysis (http://www.ncbi.nlm.nih.gov/). The dendrograms were constructed using the MEGA7 (Molecular Evolutionary Genomics Analysis Version 7) software with Maximum Likelihood method with bootstrap of 1000 replicates.

PVY associated symptoms were recorded in all the four surveyed districts of Kashmir valley. The various symptoms observed were mosaic, stunting, mottling, vein clearing, curling and vein-banding (supplementary Fig. 1). The disease incidence based on the symptoms ranged from 16 to 27.33 per cent. Maximum being in district Srinagar (27.33%) and minimum in district Budgam (16.0%).

Out of total 200 potatoes leaf samples showing various symptoms only 74 were tested positive for PVY infection through DAS-ELISA. Based on DAS-ELISA results, the maximum PVY incidence was observed in Srinagar (52.00%) and minimum in Budgam (24%).

Based on DAS-ELISA results ten positive samples were further selected from each district for reconfirmation through RT-PCR using PVY coat protein (CP) specific primers. The amplicon of ~ 900 bp of CP was amplified and no non-specific amplification was found in any sample (Fig. 1A). The sequence of 900 bp of coat protein gene obtained and BLAST analysis revealed the nucleotide sequence similarity varied between 88 to 100 percent with earlier reported PVY isolates. The highest identity at nucleotide level was observed with the isolates of Hungry (X54661) and Vietnam (DQ925437).

Fig. 1.

A Amplicon of 900 bp of Coat Protein gene from samples infected with PVY from four potato growing districts of Kashmir valley, M-1 kb ladder, L1-SL1, L3-SL2, L4-SL3, L5-SL4, L6-F2, L7-F4, L8-F8, L9-Ant4, L10-Ant6, L11-Ant7, L12-SS1, L13-FGH3, L14-FGH8, L15-SS6. B Multiplex PCR Product separated on 1% Agarose gel, NTN & O strain: M-1 Kb ladder, L1-SS1, L2-FGH2, L3-FGH3, L4-SL1, L5-SL3, L6-F2, L7-F3, L8-Ant4, L9-Ant5, L10-Ant9 (452 bp & 267 bp respectively). C NTN & N strain: M-1 Kb ladder, L1-SL1, L3-SL2, L4-SL3, L5-SS1, L6-F2, L7-F3, L8-F5, L9-Ant4, L10-Ant6, L11-Ant7, L12-FGH8, L13-SS6 (452 bp & 398 bp respectively)

After amplification with the CP specific primers, the ten representative isolates from each district were also further analyzed using strain-specific primers. The PCR-based studies revealed that the PVY positive samples were of PVY-N, PVY-O and PVY-NTN strains (Table 1). The amplicon of 452 bp, 328 bp and 267 bp were amplified from PVYNTN, PVYN, PVYO strains respectively (Fig. 1B). Mixed infection of strains was also present in some of the samples, which was observed and inferred when multiple bands were got in the PCR experiment (Fig. 1C) (Supplementary Table 2). Purified PCR product got after amplification with strain specific primers of NTN, N and O strains, were sent for custom sequencing. The sequences received were compared with sequences of PVY obtained from NCBI gene bank. The BLASTn result of all the three strains of PVY (NTN, N, O) showed 89–99% sequence similarity with their respective strains of PVY previously published (AB711143, KJ946936, MF405303, AB711145, HQ912896, KC61470 and D00441). The strain specific sequences of all nine isolates were deposited in GenBank and received under the following accession numbers (MN913419, MN913420, MN913421, MN913422, MN913423, MN913424, MN913425, MN913426 and MN913427).

Table 1.

Distribution of PVY strains in different Districts of Kashmir valley

Location	Positive samples (on the basis of CP primers)	Strain type (on the basis of strain specific primers by using Multiplex PCR)
		NTN	O	N	N:O
Baramulla	7	5	5	4	0
Srinagar	11	9	7	5	0
Budgam	5	3	1	2	0
Anantnag	8	8	8	5	0

Phylogenetic analysis was carried out to compare the nucleotide sequences of our isolates with those of different PVY strains for further confirmation. On the basis of CP phylogenetic tree, two major clusters were formed, which was further sub divided into three sub clusters. Our isolate was clustered in sub cluster I along with other isolates from USA (EJ204166), Hungary (X54611), Brazil (JQ924287) and Vietnam (DQ925437) (supplementary Fig. 2).

Similarly for strain, all the nine isolates were phylogenetically analysed and the phylogenetic analysis grouped the NTN isolates into two major cluster and all the four NTN isolates were grouped in cluster-I along with isolates from Japan (AB711143), USA (AY884982) and UK (KC614702). Likewise, the N isolate of PVY formed one major cluster with SL3.N forming sub cluster with Egyptian isolate (D00441). O isolate also formed one major cluster and three sub clusters, with all our PVY O isolates grouping in third cluster with isolates from China (GQ200836), Poland (KX356070) and UK (KC614702) (Fig. 2).

Fig. 2.

Phylogenetic analysis of identified PVY strains (PCR product) by Maximum Likelihood method. The tree depicts the relationship of PVY strains from Kashmir highlighted with color symbols and in bold letters with other strains from different regions of world

Potato crop is prone to systematic infections by viruses, mycoplasmas, fungi, bacteria and nematodes. The degenerative effect of viral disease is more devastating in potato (as it is vegetatively propagated) and there is no control measure of these diseases at field level [12]. The PVY shows global presence in potato growing regions and being economically important virus causing yield loses up to 100%. The degree of yield loss depends on various factors viz., virus strain, time of infection, temperature and the potato cultivar [13]. The PVY is present globally and cause severe crop losses in countries like India, Pakistan, Europe, Canada, America etc [14]. The most important factor related to yield loss is the strain of PVY as they differ in pathogenicity. Thus, the awareness and understanding of genetic variability in PVY is of paramount importance for identification and exploitation of resistance sources and deployment of R gene over space and time. Survey conducted revealed the occurrence of the disease caused by PVY in almost all the potato growing areas of Kashmir, with field disease incidence ranging from 16 to 27.33%. Incidence of PVY in district Anantnag, Baramulla, Budgam and Srinagar was recorded in variable proportions. The presence of variable incidence of PVY in different areas has also been observed by Ghorai et al. [15]. The symptomatology is a basic step towards disease diagnosis but is not a reliable method to confirm viral diseases as similar kind of symptoms are produced due to various biotic and abiotic factors, as reported by Batool et al. The association of PVY with each disease sample was further established through RT-PCR. The results of RT-PCR revealed the presence of the PVY in almost all the potato growing areas of Kashmir with varying incidence. Limited information is available regarding the prevalence of PVY strains in various potato growing states of India especially in J&K. The most common strain of PVY (PVY-O) has been identified in India to be infecting potato crop [16] and a recombinant strain of PVY-N:O has also been characterized to cause infection in potato grown in India [17]. In Jammu & Kashmir, A northern most part of India, occurrence and complete genome characterization of tuber necrosis strain group (PVY-NTN) in Kashmir has been reported by Hamid et al. [18]. However, no information regarding status of PVY strains was available in Kashmir. In present study the phylogenetic analysis of CP and strain specific sequences of PVY from Kashmir valley were most closely related to PVY isolates from Brazil, USA, China and other countries. In host plants, establishing the relation between symptoms expression and specific viral strain infection can be further complicated by presence of mixed infections which offers scope for recombination and evolution [19]. The NTN, N and O strains were also found to be most prevalent in the Kashmir valley with most of the isolates showing mixed infection. In present study the presence of mixed infection indicates a continued possibility of the emergence of new recombinant strains as Potyviruses have relatively high recombination and evolution rate [20]. To the best of our knowledge, this is the first report of O & N strains of PVY in Kashmiri potato with nucleotide evidence, as the NTN strain has been already reported by Hamid et al. [18].

The present study gave first-hand information regarding the distribution and prevalence of PVY in general and its strains in particular in potato growing regions of Kashmir valley. Among the various strains (NTN, N and O) reported NTN was found to be most prevalent throughout the valley. The study will help in designing the resistance breeding programme for of potato cultivars grown in India and will also help in developing the Point-of –care diagnostics for easy certification of seed lots.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

We would like to acknowledge ICAR-IISS, Mau, India for funding this project.

Declarations

Conflict of interest

The authors declare no conflict of interests.