Optimization of a simple, low-cost one-step reverse transcription recombinase polymerase amplification method for real-time detection of potato virus A in potato leaves and tubers.

Vegetative propagation of potatoes makes it possible for potato viruses to be transmitted through tubers. Potato virus A (PVA) is one of these viruses, which belongs to the Potyvirus genus in the Potyviridae family. Potato tuber yield can be reduced by 30-40% by PVA alone. Losses can be further exacerbated by potato virus X and/or potato virus Y infection. PVA is transmitted primarily by several species of aphids in non-persistent manner. With the aim of resolving this problem, we developed one-step reverse transcription-recombinase polymerase amplification (RT-RPA), a highly sensitive and cost-effective method for detecting PVA in both potato tubers and leaves. Detection and amplification are performed using isothermal conditions in this method. There was good amplification of the coat protein gene in PVA with all three primers tested. To conduct this study, a primer set that can amplify specific 185 base pair (bp) product was selected. PVA detection was optimized by 30-min amplification reactions, which showed no cross-reactivity with other potato viruses. A simple heating block or water bath was used to amplify PVA product using RT-RPA at a temperature range of 38-42 °C. In comparison to conventional reverse transcription-polymerase chain reaction (RT-PCR), the newly developed RT-RPA protocol exhibited high sensitivity for both potato leaves and tuber tissues. Using cellular paper-based simple RNA extraction procedure, the virus was detected in leaf samples as efficiently as purified total RNA. We also found that combining LiCl-based RNA precipitation with cellular paper discs allowed us to successfully optimize RNA extraction for one-step RT-RPA for detecting PVA in tubers. Tests using this simplified one-step RT-RPA method were successfully applied to 300 samples of both leaves and tubers from various potato cultivars. In our knowledge, this is the first report of an RT-RPA assay utilizing simple RNA obtained from either cellular disc paper or LiCl coupled with cellular disc paper to detect PVA. As a result, this method was equally sensitive and specific for detecting PVA in potatoes. The developed RT-RPA assay is more versatile, durable, and do not require highly purified RNA templates, thus providing an effective alternative to RT-PCR assays for screening of germplasm, certifying planting materials, breeding for virus resistance, and real-time monitoring of PVA.

Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato.

Potatoes are developed vegetatively from tubers, and therefore potato virus transmission is always a possibility. The potato leafroll virus (PLRV) is a highly devastating virus of the genus Polerovirus and family Luteoviridae and is regarded as the second-most destructive virus after Potato virus Y. Multiple species of aphids are responsible for the persistent and non-propagating transmission of PLRV. Due to intrinsic tuber damage (net necrosis), the yield and quality are drastically diminished. PLRV is mostly found in phloem cells and in extremely low amounts. Therefore, we have attempted to detect PLRV in both potato tuber and leaves using a highly sensitive, reliable and cheap method of one-step reverse transcription-recombinase polymerase amplification (RT-RPA). In this study, an isothermal amplification and detection approach was used for efficient results. Out of the three tested primer sets, one efficiently amplified a 153-bp product based on the coat protein gene. In the present study, there was no cross-reactivity with other potato viruses and the optimal amplification reaction time was thirty minutes. The products of RT-RPA were amplified at a temperature between 38 and 42 °C using a simple heating block/water bath. The present developed protocol of one-step RT-RPA was reported to be highly sensitive for both leaves and tuber tissues equally in comparison to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. By using template RNA extracted employing a cellular disc paper-based extraction procedure, the method was not only simplified but it detected the virus as effectively as purified total RNA. The simplified one-step RT-RPA test was proven to be successful by detecting PLRV in 129 samples of various potato cultivars (each consisting of leaves and tubers). According to our knowledge, this is the first report of a one-step RT-RPA performed using simple RNA extracted from cellular disc paper that is equally sensitive and specific for detecting PLRV in potatoes. In terms of versatility, durability and the freedom of a highly purified RNA template, the one-step RT-RPA assay exceeds the RT-PCR assay, making it an effective alternative for the certification of planting materials, breeding for virus resistance and disease monitoring.

Establishment of a one-step reverse transcription recombinase polymerase amplification assay for the detection of potato virus S.

Potato virus S (PVS) is a noteworthy threat to the propagation of healthy seed potatoes. Accurate and speedy detection is critical for effective PVS management. In the present study, an isothermal-based one-step reverse transcription-recombinase polymerase amplification (RT-RPA) approach was developed to detect PVS infection in potato leaves and tubers. A primer set based on the coat protein gene successfully amplified a 158 bp product out of three primer sets examined. The amplification reaction took less than 30 min to complete with no account of cross-reactivity with major potato viruses. Additionally, amplification of RT-RPA products was performed on the heating system and/or water bath at 38-42 °C. The results of sensitivity analysis revealed that one-step RT-RPA has shown 100 times higher sensitivity than routine RT-PCR for the detection of PVS in infected leaves. Furthermore, ten times higher sensitivity of RT-RPA was observed in infected tubers. The methodology was simplified further by the use of template RNA extracted using a cellular disc paper-based extraction method that detected the PVS more effectively than purified total RNA. PVS was detected in 175 samples (leaves and tubers each) of several potato varieties using this innovative technique. To our acquaintance, this is the first report of one-step RT-RPA using a basic RNA extract derived through cellular disc paper that is significantly sensitive and precise for PVS detection in potatoes. The advantages of one-step RT-RPA in terms of proficiency, robustness, and the availability of a highly pure RNA template make it an attractive choice for seed accreditation, resistance breeding, and field inspections.

Rapid and sensitive detection of potato virus X by one-step reverse transcription-recombinase polymerase amplification method in potato leaves and dormant tubers.

Potato virus X (PVX), is a serious threat to global potato production. A simple and rapid detection method is imperative for PVX diagnosis and early management. In this study, an isothermal one-step reverse transcription-recombinase polymerase amplification (RT-RPA) method was optimized for the quick and convenient detection of PVX in potato leaves and tubers. Our results revealed that this one-step RT-RPA method was highly efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR). The amplification reaction was free from cross-reactivity with other common potato viruses and completed within 30 min. Moreover, this RT-RPA assay did not require a thermocycler based specific temperature phase amplification and can be easily performed using a simple heating block or water bath at a temperature range of 39-42 °C. The sensitivity assay demonstrated that the developed one-step RT-RPA method was 100 times more sensitive than a routine one-step RT-PCR. Initially, the purified total RNA as the template isolated from infected leaves of potato was used for the detection of PVX. One-step RT-RPA was later performed using cellular disc paper-based simple RNA extract as a template that could detect the virus more efficiently than purified total RNA. The performance of the one-step RT-RPA assay was further evaluated using 500 field samples of leaves and tubers representing different cultivars and geographical regions. To our knowledge, this is the first report of rapid, sensitive, and reliable detection of PVX infection by one-step RT-RPA using cellular disc paper-based simple RNA extract from leaves and dormant tubers of potato. It is superior to the common RT-PCR assay in terms of its versatility, quickness, and independence of highly purified RNA template and can be adopted as a substitute to RT-PCR as an effective technique for seed potato certification, quarantine, breeding, and field surveys.

Molecular characterization and infectivity analysis of tomato leaf curl New Delhi virus isolates infecting potato.

Nucleotide sequence of complete genome of a new isolate (KAN-6) of tomato leaf curl New Delhi virus (ToLCNDV) from Kanpur, Uttar Pradesh, India was determined. Sequence analysis indicated that it shared maximum identity to ToLCNDV isolates from pumpkin and ashgourd. Infectious clones of isolate KAN-6 along with two other ToLCNDV isolates (MOD-21 & FAI-19) obtained from potato fields of Modipuram and Faizabad, India were produced and used in symptom expression studies in N. benthamiana and potato plants through agro-inoculation. These isolates produced different symptoms both in N. benthamiana and potato. Severe symptoms of yellow mottling, downward curling and stunted growth were observed in N. benthamiana plants inoculated with KAN-6. MOD-21-inoculated plants also showed downward curling, stunted growth, but yellow mottling was observed only in older leaves whereas FAI-19-inoculated plants produced only downward curling symptoms. In case of potato, typical symptoms of apical leaf curl disease were observed in cultivar Kufri Pukhraj inoculated with MOD-21 and KAN-6 that are similar to those produced by virus-infected plants in the field. However, MOD-21 produced more prominent yellow mosaic symptoms as compared to KAN-6. FAI-19 produced only restricted yellow spots in Kufri Pukhraj. Only mild symptoms appeared in KAN-6 and no symptoms were observed in MOD-21- and FAI-19-inoculated Kufri Bahar plants which is known to show lowest seed degeneration under field conditions. Analysis of genomic components indicated that these isolates had 94.8-94.9% and 87.9-97.3% identity among them in DNA A and DNA B, respectively. The results of the study indicate the association of ToLCNDV isolates of different symptomatology with apical leaf curl disease of potato. This is also a first experimental demonstration of Koch's postulate for a begomovirus associated with apical leaf curl disease of potato.Author names: Please confirm if the author names (Swarup Kumar Chakrabarti) are presented accurately and in the correct sequence (given name, middle name/initial, family name).Yes. It is correct. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02752-5.

Development of a visual detection method for Potato virus S by reverse transcription loop-mediated isothermal amplification.

A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed to detect the Potato virus S (PVS) in potato. Two sets of six novel primers that recognize the coat protein gene sequence of the PVS were designed and RT-LAMP assay was optimized for the parameters such as different concentrations of primers, MgSO4, betaine, dNTPs, Bst DNA polymerase, temperature and duration. The RT-LAMP was carried out under isothermal conditions without the thermal cycler using PVS infected leaf and tuber samples, LAMP specific primers with amplification at 65 °C for 60 min, and 80 °C for 5 min. The results were assessed by gel electrophoresis and visual observation of colour change using SYBR Green I dye. The detection limit of the developed RT-LAMP assay was determined and compared with a conventional reverse transcription-polymerase chain reaction (RT-PCR). RT-LAMP was found 100 times more sensitive than RT-PCR. The optimized RT-LAMP assay is robust, reliable, sensitive and convenient for the detection of the PVS in infected potato tubers including asymptomatic plants. No cross-reactions were observed with healthy plants and other potato viruses. The assay is economical and can be employed in large scale testing of potato plants against PVS under healthy seed potato production programme.

An insight into differentially regulated genes in resistant and susceptible genotypes of potato in response to tomato leaf curl New Delhi virus-[potato] infection.

Apical leaf curl disease, caused by tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) is one of the most important viral diseases of potato in India. Genetic resistance source for ToLCNDV in potato is not identified so far. However, the cultivar Kufri Bahar is known to show lowest seed degeneration even under high vector levels. Hence, microarray analysis was performed to identify differentially regulated genes during ToLCNDV-[potato] infection in a resistant (Kufri Bahar) and a susceptible cultivar (Kufri Pukhraj). Under artificial inoculation conditions, in Kufri Pukhraj, symptom expressions started at 15days after inoculation (DAI) and then progressed to severe symptoms, whereas no or only very mild symptoms were observed in Kufri Bahar up to 35 DAI. Correspondingly, qPCR assay indicated a high viral load in Kufri Pukhraj and a very low viral load in Kufri Bahar. Microarray analysis showed that a total of 1111 genes and 2588 genes were differentially regulated (|log2 (Fold Change)|>2) in Kufri Bahar and Kufri Pukhraj, respectively, following ToLCNDV-[potato] infection. Gene ontology and mapman analyses revealed that these altered transcripts were involved in various biological & metabolic processes. Several genes with unknown functions were 5 to 100 fold expressed after virus infection and further experiments are necessary to ascertain their role in disease resistance or susceptibility. This study gives an insight into differentially regulated genes in response to ToLCNDV-[potato] infection in resistant and susceptible cultivars and could serve as the basis for the development of new strategies for disease management.

Uniplex and duplex PCR detection of geminivirus associated with potato apical leaf curl disease in India.

Apical leaf curl disease has emerged as a new disease in potato during the last decade in India due to a change in planting date and an increased whitefly population. Its incidence is on the rise threatening the cultivation of potato across the country. Hence, a PCR assay was developed for the detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV-Potato) which is the causal agent of apical leaf curl disease in potato. Primers specific to the coat protein (AV1) and replicase (AC1) gene regions were designed and used for standardization of the PCR. Some of the primers (LCVCPF1/LCVCPR1, LCVREPF2/LCVREPR2, LCrep1F/LCrep2R) could detect the virus in 2.4-0.24pg of total DNA of infected plant. A duplex PCR assay was optimized with the selected coat protein gene specific primers and primers specific to potato urease gene, a housekeeping gene served as an internal check. The suitability of these primers was examined for detection of the virus in 80 potato apical leaf curl disease samples from 11 different potato growing states of India and also from micro-plants grown in tissue culture. The selected coat protein primer pair (LCVCPF1/LCVCPR1) was found to be conserved in all 80 isolates except for a few isolates, which had a single nucleotide substitution in the forward primer sequence. These substitutions did not interfere with amplification of the coat protein gene. The primers could detect the virus using a print-capture PCR assay both in the presence and absence of an internal control. These results indicate the robustness of the PCR assay for virus indexing of mother stocks in the seed production system.

Complete Genome Sequence of Potato leafroll virus Isolates Infecting Potato in the Different Geographical Areas of India Shows Low Level Genetic Diversity.

Five Potato leafroll virus (PLRV) isolates were collected from five states representing different potato growing parts of India. The ssRNA genome sequences of these isolates were determined. The genome comprised of 5,883 nucleotides and deduced genome organization resembled other PLRV isolates. About 97.6-98.7 % similarities was observed within the Indian isolates and were more close to European, Canadian, African, American and Czech isolates (95.8-98.6 %) than to an Australian isolate (92.9-93.4 %). These isolates were 43.7-53.1 % similar to other poleroviruses and 29.1-29.3 % to Barley yellow dwarf virus, a luteovirus. Out of five isolates, the isolate PBI-6 was recombinant one as detected by RDP3 software. Multiple sequence alignment of nucleotide and amino acid sequences of different ORFs indicated that the ORF 3 and ORF 4, corresponding to coat protein and movement proteins are more conserved than other ORFs. Amino acid changes specific to Indian isolates were observed and it was more in ORF 2 than in ORF 0, ORF 3 and ORF 4. This is the first report of complete genome sequence of PLRV isolates from India, which reveals low level genetic diversity.