African Journal of
Bacteriology Research

  • Abbreviation: J. Bacteriol. Res.
  • Language: English
  • ISSN: 2006-9871
  • DOI: 10.5897/JBR
  • Start Year: 2009
  • Published Articles: 121

Review

Next generation sequencing platforms for potato virus hunting, surveillance and discovery

Alinda Alfred K.
  • Alinda Alfred K.
  • Department of Biological Sciences, School of Natural Sciences (SONAS), Masinde Muliro University of Science and Technology (MMUST), Kakamega, Kenya.
  • Search for this author on:
  • Google Scholar
Okoth Patrick K.
  • Okoth Patrick K.
  • Department of Biological Sciences, School of Natural Sciences (SONAS), Masinde Muliro University of Science and Technology (MMUST), Kakamega, Kenya.
  • Search for this author on:
  • Google Scholar
Onamu Rose
  • Onamu Rose
  • Department of Biological Sciences, School of Natural Sciences (SONAS), Masinde Muliro University of Science and Technology (MMUST), Kakamega, Kenya.
  • Search for this author on:
  • Google Scholar
David Read
  • David Read
  • Agricultural Research Council, Biotechnology Platform (ARC/BTP). Onderstepoort, Pretoria, South Africa.
  • Search for this author on:
  • Google Scholar
Genevieve Thompsons
  • Genevieve Thompsons
  • Agricultural Research Council, Biotechnology Platform (ARC/BTP). Onderstepoort, Pretoria, South Africa.
  • Search for this author on:
  • Google Scholar
Muoma John O.
  • Muoma John O.
  • Department of Biological Sciences, School of Natural Sciences (SONAS), Masinde Muliro University of Science and Technology (MMUST), Kakamega, Kenya.
  • Search for this author on:
  • Google Scholar


  •  Received: 31 January 2020
  •  Accepted: 10 June 2020
  •  Published: 30 June 2020

 ABSTRACT

Potato (Solanum tuberosum L.) is a key alternative to maize crop in Kenya. However, pests and diseases affect the yields. Information on Irish potato virology is continually patchy. Viral disease dynamics require constant updating to track new and novel agents. Efforts to mitigate viruses and crop breeding for tolerance can be determined this way. In Kenya, key potato viruses include: Potato Leaf Roll Virus (PLRV), Potato Virus X (PVX), Potato Virus S (PVS) and Potato Virus Y (PVY). Detection of these viruses has been through symptomatology, serology and nucleic-acid approaches. Molecular biology has revolutionary developments in sequencing technologies influencing diagnosis of plant viruses. Massive parallel sequencing has promoted detection, identification and discovery of novel viruses in plants without use of antibodies or prior virus knowledge. Complete viral genomes can be sequenced from asymptomatic and symptomatic samples. Viral metagenomics, diversity and genome variability can be deduced this way. Next generation sequencing platforms bring robustness, timeliness and affordability to virus detection. However, few studies have attempted to utilize it in unravelling potato virology beyond the routine detectable agents in the country. The current study reviews diagnosis of Irish potato viruses in Kenya against the techniques used, comparing them to next generation sequencing.

 

Key words: Deep sequencing, next generation sequencing, reverse transcriptase polymerase chain reaction (RT-PCR), serology.


 INTRODUCTION

Potato is ranked the fourth most important food crop globally, with a production of 388 million tons in 2017, after rice (770 million tons), wheat (771 million tons) and maize (1.1 billion tons). It is the third most important food crop since maize as a food crop is at 14% (FAOSTAT, 2019). The IPBO  (2019)  documents  that;  Africa  potato production has increased. Kenya is among the top 6 leading producers alongside Algeria, Egypt, South Africa and Morocco. Pests and disease production constraints have been documented (CIP, 2019; FAOSTATS, 2019).
 
Viruses contribute to over 47% of the total plant emerging infectious  diseases  (Anderson  et  al.,  2004).
 
These viruses are widespread and threaten as emerging crop virus infections (Craig et al., 2004). Potato viruses contribute to reductions in quality and quantity of potato in Sub Saharan Africa (SSA). Losses due to the viruses have been experienced in many SSA countries. For instance; Potato Virus Y has devastated production in Kenya, Uganda, Ethiopia and South Africa; Potato Leaf Roll Virus and Potato Virus X (havocked Kenya and Uganda); while Potato Virus A damaged production in Kenya (Gildemacher et al. 2009; Ibaba and Gubba, 2011; Wangai and Lelgut, 2013). The Kenyan Irish potato tonnage has had an unsteady precedence between the years 2000 and 2010 (FAOSTATS, 2011). Potato viral infections manifest symptoms in some cases while other viruses have total asymptomatic characteristics. Visual diagnosis seldom differentiates one viral infection from the other. Among potato viral diseases, stunting, necrosis, mosaic, and leaf roll are most important and caused by viruses such as Potato Virus X, Potato Virus M (PVM), Potato Virus S (PVS), PVY, Potato Virus A (PVA), Potato aucuba virus, potato leaf roll virus (PLRV), mop-top virus and Potato apical leaf roll virus (APLRV) (Awasthi and Verma, 2017).
 
Experimental virus indexing has documented high prevalence of potato viruses (Gildemacher, 2012; Machangi et al., 2004; Olubayo et al., 2010). The viruses responsible for majority of yield reductions are Potato leaf roll virus (PLRV), Potato virus Y (PVY), and Potato virus X (PVX) that occur in combination with mild viruses like Potato virus A (PVA), Potato virus M (PVM) and Potato virus S (PVS) (Kabira et al., 2006; Schulte-Geldermann et al., 2012). PVY is the most important virus globally (Lacomme et al., 2017), despite Potato Leaf Roll Virus being considered the most economically potent virus. However, studies have shown that Potato virus Y and Potato Leaf Roll Virus are the most significant viruses infecting South African potatoes (Denner et al., 2012).  
 
Characterization of these viruses has been based on biological assay, microarray, electron microscopy, nucleic acid based techniques like PCR and serological techniques such as enzyme linked immunosorbent assay (ELISA) (Boonham et al., 2008; Ng et al., 2011). Any investigation of viral dynamics in wild plant species needs clear background of plant biochemical and structural features. ELISA and Reverse Transcriptase-Polymerase Chain Reaction for a phloem-restricted nature study model system (plant virus BYDV-PAV) makes the findings relevant to detection sensitivity in plant-microorganism systems. The methodological approaches tested show importance of optimizing and assessing virus detection techniques for application in wild plant hosts. Such information is critically needed (Lacroix et al., 2016). This is consistent with similar studies (Kunta et al., 2014; Pereira and Lister 1989; Rashed et al., 2014; Sanchez-Navarro et al., 2007) in other plants that have not been well exploited.
 
In plant screening, biological assay and microscopy are ancient (Matthews, 1991). This technique is dependent on high quality indicator and propagative host plants. Serology and PCR rely on known agents only (Coetzee et al., 2010; Yanagisawa et al., 2016; Zheng et al., 2017). Other than over-reliance on high quality indicators, test results are subjective and not confirmative in Electron microscopy. Introduction of serological assay (ELISA) used antibodies for detection of viruses (Clark and Adams, 1977), followed by PCR clonal amplification of DNA (Candresse et al., 1998). ELISA is cost effective, robust and amenable (Boonham et al., 2014); however, it has restricted use to universally known agents and is unable to detect novelty especially in viroids (Grothaus et al., 2006). Molecular diagnostics advantages include high turnover rate, ability to identify individual strains and scalability to high throughput (Martin, 2012). Viral RNA is reverse transcribed from RNA+/- strands by the RT enzyme to synthesize cDNA (Ali et al., 2014).
 
Whereas molecular techniques are highly sensitive and specific than serology, symptomatology is error prone due to an overlap in the manifest symptoms (Notomi et al., 2000). Potato Leaf Roll Virus is asymptomatic especially late infection of potato by the SymlessLS10 Potato Leaf Roll Virus isolate (Hühnlein et al., 2016). Unlike culture methods, real time PCR (qPCR) has demonstrated high reproducibility and less variability (Dong et al., 2016). This is consistent with similar studies by Hockman et al. (2017) that merit reverse transcriptase PCR (RT-PCR). However, these techniques are culpable of significant drawbacks in detecting unknown viral agents either in a new host or novel agents due to sole reliance on routine agents by sequence or antibodies. Advent of novel technologies such as Next generation sequencing has been used in diagnosis and characterization of new viruses affecting various plants (Prabha et al., 2013).


 CHARACTERIZATION OF POTATO VIRUSES IN AFRICA

Potato viruses have colonized numerous potato growing nations across Africa. Detection of the virus and viroids has utilized varied diagnostics in documenting incidence, prevalence and occurrence as shown in Table 1. In North Western Cameroon, studies exploring prevalence of six potato viruses applied Double Antibody Sandwich-ELISA (DAS-ELISA) to confirm six viruses (Potato Virus A, Potato Leaf Roll Virus, Potato Virus M, Potato Virus S, Potato Virus X and Potato Virus Y) as prevalent in the country (Njukeng et al., 2013). Less sensitive versions of ELISA, {Multi-array test strips (MALTS)}, have viral antibodies that were used to detect eight viruses in potato, but are less sensitive (Safenkova et al., 2016).
 
 
Similar studies investigating the incidence of Potato Virus X, Potato Virus S, Potato Mop Top Virus, Potato Virus M, Potato Virus A, Tomato Spindle Wilt Virus and Potato  Spindle  Tube   Viroid   in  South   Africa   applied reverse transcriptase polymerase chain reaction (RT-PCR) CP-gene amplification and whole genome amplification for PSTVd and detected only two viruses as present in the samples (Potato Virus S and Potato Virus X); while Potato Leaf Roll Virus was reported to have attained a reduced pathogenicity (Wiets, 2013). The study suggests that the other viruses failed to be detected by RT-PCR either due to failure of amplification or absence of the agents (Wiets, 2013). Potato Spindle Tuber Viroid (PSTVd) is distributed widely around the globe (CABI/EPPO, 2014). Primers have been designed specifically for pospiviroid amplicon generation in Real-time PCR or conventional PCR that is used for successful detection of PSTVd (Boonham et al., 2004; Bostan et al., 2004; Botermans et al., 2013; Shamloul et al., 1997; Verhoeven et al., 2004). Studies by Lezan, (2017) and Wiets (2013) are consistent in the use of ELISA as the routine testing tool by the South African Seed Potato Certification Scheme, for viruses such as PLRV. Though RT-PCR assay is amenable for epidemiological studies and certification schemes to detect Potato Leaf Roll Virus early in potato crops (Hossain et al., 2013), post agarose gel electrophoresis analysis is time consuming and less accurate.
 
RT-PCR and Next Generation Sequencing have been used in validating better tools for potato certification of Potato Leaf Roll Virus and identification of coding regions in potato viruses in the Sandveld region South Africa. Through the study, noncoding 5’ and 3’ regions of the genome were compared using Next Generation Sequencing other than identification of novel potato viruses, leading to endorsement of RT-PCR and Next Generation Sequencing as better tools in characterizing potato viruses (Lezan, 2017). Furthermore, the same study used Next Generation Sequencing (Ion Torrent Sequencing) to gain more information on Potato Leaf Roll Virus prior to tracing its ancestry in relevance to global strains. Similarly, RT-qPCR has been developed in South Africa for the detection of Potato Leaf Roll Virus in potato leaves and tubers (Espach, 2015), though it is comparatively expensive than ordinary RT-PCR (Coudray-Meunier et al., 2016). In studies to detect Potato Spindle-Tube Viroid, Potato Virus A, Potato Virus S, Potato Virus X, Potato Virus Y, Potato Leaf Roll Virus and Potato Virus M, Multiplex PCR has been successfully adopted (Kumar et al., 2017; Zhang et al., 2017).
 
In Central Tunisia, the presence of the six most economically important viruses: Potato Leaf Roll Virus, Potato Virus S, Potato Virus M, Potato Virus X, Potato Virus A and Potato Virus Y were determined across various incidence levels. Serological tests ranged from 0.5% (Potato Virus M) to 71% (Potato Virus Y). The variability of Potato Virus Y was analyzed by a combination of serotyping, indexing on tobacco and RT-PCR tests of the 2 genomic regions (5′NTR/P1 and CP/3′NTR). Serological samples revealed dominance of the PVYN strain (88.2% of total PVY positives). Furthermore, strains subjected to molecular typing revealed that 73.3% of the PVYN strains were PVYNTN variants having a recombination junction at the CP/3′NTR region across 94.4% of their totals, though no recombination junction was found in the genome of the isolates of PVYN group (Larbi et al., 2012).
 
In Eastern Africa, focus has been on the use of DAS-ELISA, NCM-ELISA and RT-PCR in the diagnosis of potato viruses. DAS-ELISA was adopted in Mbeya region of Tanzania to determine whether Potato leaf roll virus (PLRV), Potato virus S (PVS), Potato virus A (PVA), Potato virus Y (PVY), Potato virus X (PVX) and Potato virus M (PVM) are present in potato. Though DAS-ELISA confirmed the presence of all the six viruses, further analysis using RT-PCR dismissed occurrence of PLRV PVS and PVY (Evangelista, 2013.) Here, occurrence of PLRV, PVA, PVM, PVS, PVY and PVX was determined from 219 potato accessions in Mbeya regions of Kawetele,   Umalia,    Uyole,    Kikondo,    and    Rungwe (Mwakaleli) in Tanzania. Virus-like symptomatology was observed in most fields, including: leaf rolling, yellowish-green mosaic, and vein necrosis. The ten symptomatic and three asymptomatic leaves sampled from each field and tested by double antibody sandwich (DAS)-ELISA tested positive for PVS and PLRV in 55 and 39% of total samples, respectively. PVM and PVX were positive in 14 and 5% of most fields respectively. Co-infections of PLRV and PVS were detected in 14% of the samples. PVY and PVA were present in two localities. Mixed infections (3 or 4 viruses) were present in 5% of the crops. Twenty samples, from Mwakeleli and Uyole ELISA-positive more than one virus, were analysed using RT-PCR with virus-specific primers for amplifying the coat protein (CP) encoding gene. ELISA-positive leaf samples were subjected and tested positive for RT-PCR. ELISA-negative for the viruses PVA or PVX, were positive when tested by RT-PCR, indicating suitability in actual incidence of the viruses as opposed to DAS-ELISA. The PCR products 5 samples for each virus sequenced, reconfirmed presence of PVA, PVS, PLRV, PVX and PVM.
 
The incidence of Potato leafroll virus (PLRV), Alfalfa mosaic virus (AMV) and Potato virus Y (PVY) in potato crops was assessed visually and confirmed through direct tissue blot immunoassay at three locations, Elnaiya, Shambat and Elshehinab in Khartoum State, the main potato growing region in Sudan (Baldo et al., 2010).
CABI/EPPO 2019 reports (https://www.cabi.org/ISC/datasheet/43762) are keen in reporting viral densities from across the world. Using researcher inferences by varied characterization methods, the reports in Africa, for instance, shows Potato virus Y (PVY) densities cladding the continent but limited in Kenya. The report distribution map (https_www.cabi.org_isc_distribution_map.png) also documents historical research into the PVY potato mottling virus across the globe with similar elucidations on incidence, prevalence and distribution.
 
SEROLOGICAL DETECTION OF POTATO VIRUSES IN KENYA
 
Studies have always emphasized on the use of immunological or serological diagnosis of crop viruses in Kenya. Initial studies on Potato virus Y (PVY) to cross match disease incidence to host range and to compare results applied both ELISA and Electron Microscopy have been done (Bondole, 1992). The study documented prevalence of PVY as high, setting a standard for the use of the two tools in potato pathology. Studies by Were et al. (2013) in Kenyan highlands used ELISA to detect Potato virus S (PVS) (dominant virus), Potato Virus Y and Potato Virus X in potato. Furthermore, the study reported the same viruses in Solanum nigrum using DAS-ELISA while PLRV, PVM, PVS and PVY were detected in Solanum incunum weed species. The virus strains  PVYO, PVYN, recombinant strains PVYN-Wi and PVYNTN were also distinguished using ELISA. Additionally, Muthomi et al. (2009) document using serology to detect PLRV, PVS and PVY strains in potato. Okeyo, (2017) used CIP DAS-ELISA to detect 4/6 viruses (PVS, PVY, PLRV and PVM) in Irish potato in Kenya during his study of resistance in potato genotypes. No single study has gone further to use Next Generation Sequencing in confirming the incidence, prevalence and distribution of the viruses alongside Serology.
 
Monoclonal antibody DAS-ELISA has been used to confirm presence of Potato Virus Y strains in potato, in Eastern, Western, Central and the Rift valley regions of Kenya (Nyamwamu et al., 2014). Similar studies by Muthomi et al. (2011) and Were et al. (2014) used Serology to successfully document strains of PVY in Kenya. Also, Nyaboga et al. (2008) used NCM-ELISA to document key potato viruses including Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV), and Sweet potato chlorotic fleck virus (SPCFV). Four PVY strains: (PVYO {common}, PVYC {Stipple streak strain}, PVYN {Tobacco venial necrosis strain} and PVY NTN have also been documented using serology and molecular techniques (John et al., 2013).
 
Symptomatology has been used as the primary diagnostic tool for viral infections in potatoes. However, the symptoms are highly erratic due to an overlap in manifestation. A number of viruses are equally asymptomatic on infection, implying a great bias in using the tool. In plants, detecting disease agents by symptomatological methods, is less selective and requires intensive expertise to cross-match specific symptoms to an agent (Prabha et al., 2013). Studies using molecular diagnostic tools in potato viruses are scanty. A few publications have attempted to use the tool to portray viral ecogenomics and diversity in the Kenyan case.
 
The directorate of diagnostics and research laboratory, foundation of Plant services (UCDAVIS-FPS) determines that serology and nucleic acid based technologies are considered expensive per sample (Al Rwahnih et al., 2015), unlike modern diagnostics of plant viruses. This is consistent with individual sample analysis studies that are characteristic in immunological and some molecular based diagnostic tools such as qPCR, RT-PCR, RFLP, Multiplex PCR, Nested PCR and other variants (Marina et al., 2014).


 SUCCESS OF NGS IN VIRUS HUNTING AND DISCOVERY

Sequencing uses Capillary Electrophoresis (CE) principles to unravel the genetic code. Throughput, scalability, robustness and speed dictate the various generations of sequencing, with NGS as the most powerful (Capobianchi et al., 2013).  Sangers sequencing is one of the two first generation sequencing platforms that have been used for long to sequence of between 600 and 1000 base pairs (bp) per run, though it is comparatively costly and time consuming (Wu et al., 2015). Sequencing through short read approaches is divided into sequencing by ligation (SBL) and sequencing by synthesis (SBS) (Goodwin et al., 2016; Myllykangas et al., 2012).
 
The first High Throughput Sequencing platforms (under Second generation Sequencing) was Roche 454 FLX Pyrosequencing platform by 454 life sciences (http://www.454.com). Roche/454 sequencing had an initial market appearance in 2005. The method uses Sequencing by Synthesis (SBS) approach, known as pyrosequencing technique, that relies on the detection of a pyrophosphate released after each nucleotide is incorporated into the growing DNA strand by way of a luciferase enzyme (http://www.454.com). The Roche/454 has the ability to generate longer reads, easier to map onto any reference genomes; however, the technique is prone to Insertion and Deletion errors on the sequences as a result of homopolymeric regions (Margulies et al., 2005; Huse et al., 2007).
 
In 2007, Solexa GA released the genome analyzer of illumine (http://www.illumina.com). Illumina (Solexa Genome Analyzer (GA)) similarly applies sequencing by synthesis approach and is the most commonly used approach. It is based on a two-step adapter addition and bridge PCR amplification, resulting in clusters that are excited by laser technology (that emits a light signal specific to every added nucleotide), that is detected using CCD camera (coupled-charge device camera) translatable into the nucleotide sequence by computer programs (Shendure and Ji, 2008; Balasubramanian, 2015).
 
Life Technologies commercialized the Ion Torrent semiconductor sequencer in 2010 (https://www.thermofisher.com/us/en/home/brands/ion-torrent.html). Unlike Roche 454 pyrosequencing that relies on identifying a pyrophosphate, Ion Torrent is similar but relies on detecting hydrogen ions for sequencing (Rotheberg et al., 2011). Moreover, Supported Oligonucleotide Ligation and Detection (SOLiD) by Life Technologies (http://www.lifetechnologies.com) is another Next Generation Sequencing technique. In 2007, Applied Biosystems (ABI) bought SOLiD and developed ABI/SOLID technology that follows Sequencing by ligation (SBL) approach (Shendure and Ji, 2008). The ABI/SOLiD cascade consists of multiple sequencing rounds, starting by adapter addition to the DNA fragments, amplification by PCR emulsion, 8-mer florolabelling and ligation to the DNA fragments. The fluorescence color emitted is recorded and decoded into representative base sequences (Mardis, 2008). Wash and clean is used for the second generation platforms such as SOLiD, Illumina and Roche 454 (Schadt et al., 2010).
 
Real time sequencers from Pacific biosciences (PacBio) (http://www.pacificbiosciences.com) together with a miniature portable device of Oxford Nanopore MinION (http://www.oxfordnanopore.com) belong to Third generation Sequencing (TGS). This is characterized by two main approaches (Goodwin et al., 2016): Single molecule real time sequencing approach (SMRT) (Bentley et al., 2008) and synthetic approach relying on existing short reads technologies utilized by Illumina (Moleculo) (Harris et al., 2008) in construction of long reads. SMRT approach is the most common, being used in sequencers like Pacific Biosciences and Oxford Nanopore sequencing (MinION-sequencer). Pacific Biosciences uses fluorescent labelling, but instead of PCR amplification, it detects the floro-signals in real time (McCoy et al., 2014; Rhoads and Au, 2015). In Oxford Nanopore sequencing, the initial strand of a DNA molecule is attached to a hairpin on the complementary strand. The fragment goes through a protein nanopore, generating a current disturbance relative to a specific nucleotide base. This is translated into a sequence using a computer software (Mikheyev and Tin, 2014; Laehnemann et al., 2015; Laver et al., 2015). A comparison of the technologies is as shown in Table 2. Despite this availability, studies in Kenya are scanty in application of these techniques for viral pathogenesis in crops.
 
NGS extraction protocols include: total mRNA (Al Rwahnih et al., 2009; Wylie and Jones, 2011), sRNAs such as siRNAs (Kreuze et al., 2009) and dsRNAs in RNA virus infested material (Coetzee et al., 2010; Dodds et al., 1984). The most commonly utilized approach is total sRNA sequencing (Seguin et al., 2014; Wu et al., 2015). De Novo assembly of siRNAs can be applied to identify both RNA and DNA viruses, though, dsRNAs are only used in the identification of RNA viruses (Seguin et al., 2014; Wu et al., 2015). Virus hunting is faster through metagenomic analysis and deep sequencing, where a lot of known and unknown viruses have been identified from both short and long reads (Capobianchi et al., 2013; Espach et al., 2012).
 
The direct genetic genome analysis of the environmental sample is called metagenomics (Thomas et al., 2012). This analysis is also termed as ecogenomics as it attempts to sequence total nucleic acids including whole genomes of diseased samples with cheaper purification, cloning and screening steps for identification and diagnosis of viruses (Kreuze et al., 2009; Mokili et al., 2012). The use of metagenomics studies by Studholme et al. (2011) documents that discovery of viruses is possible through Next Generation Sequencing in impacting phylogenesis, pathogenesis and microbial evolution. Next Generation Sequencing approach is cost effective for generating high-throughput data (Marz et al., 2014) and requires no prior knowledge of symptomatic or asymptomatic samples to detect both known and novel viruses. Multiple viruses can be sequenced     using      Next     Generation     Sequencing  (Cox-Foster et al., 2007; Quan et al., 2008; Wu et al., 2015). Next Generation Sequencing includes the following steps: sample collection, fractionation, RNA/DNA extraction, DNA/cDNA sequencing, sequence assembly, binning, genome annotation, bioinformatics/statistical analysis, data storage and metadata sharing (Thomas et al., 2012).
 
Next Generation Sequencing studies have been used to evaluate viruses present in grapevine (Al Rwahnih et al., 2009), to identify unknown viruses (Adams et al., 2009; Coetzee et al., 2010) and in providing deep sequencing viral data of infected plants (Kreuze et al., 2009; Lotos et al., 2017) as shown in Table 3. Similar studies by Coetzee et al. (2010) and Ng et al. (2011) for virus diversity, document the use of Next Generation Sequencing in vector-enabled metagenomics of vector born viruses. This technique has been used for analysis of small interference RNA (siRNA) to identify viruses in infected plants (Kreuze et al., 2009). Also, Adams et al. (2009) used NGS for discovery of novel Cucumovirus from long reads of cDNA from a sample of Gomphrena globosa infected through mechanical inoculation with an unknown pathogen. Pallett et al. (2010) used  Roche  454 pyrosequencing of small RNA (sRNA) from leaves of wild Dactylis glomerata (cocksfoot grass), to document novel Cereal yellow dwarf virus, in wild cocksfoot grass. Likewise, Giampetruzzia et al. (2011) used Illumina sequencing for discovery of Grapevine berry inner necrosis virus (GINV) that was novel and classified as Grapevine Pinot gris virus.
 
 
NGS of dsRNAs on pooled samples detected numerous grapevine-infecting viruses including putative fungal viruses (Coetzee et al., 2010). Deductions by Giampetruzzia et al. (2011) analyzed small RNA of grapevines in the Trentino region (Italy) using Illumina HTS to discover Grapevine rupestris stem pitting-associated virus (GRSPaV), Hop stunt viroid (HSVd), Grapevine yellow speckle viroid 1 (GYSVd1), the Marafiviruses Grapevine rupestris vein feathering virus (GRVFV) and GSyV-1.  Biodiversity studies of viruses by Roossinck et al. (2010) using NGS for Tall Grass Prairie in Northeastern Oklahoma and Northwestern Costa Rica, documented  Potyviridae, Totiviridae, Bromoviridae, Endornaviridae, Luteoviridae Caulimoviridae, Chryso-viridae, Closteroviridae, Narnaviridae, Partitiviridae, Tymoviridae and some novel viruses.  However, no such studies have used this technique for discovery and documentation of viruses infecting Irish potato in Kenya.
 
Genome scanning, genome assembly and De novo genetic mapping can be explored as approaches (Capobianchi, et al., 2013; Gould and Stinchcombe, 2017; Li et al., 2017; Standage et al., 2016). Similarly, Prabha et al. (2013) and Studholme et al. (2011) document applications of HTS to differentiate viral diseases that are unknown and to show virus-host interactions in other plants. Similar studies by Barzon et al. (2011) document the use of Next Generation Sequencing as unbiased, as it needs no antibodies or any prior knowledge of sequence to diagnose. When parallel sequencing is done, the variations that can be determined   include:   viral   genome   variations,  in-host evolution and virus defense mechanism. An example of viruses detected by Illumina Sequencing is shown on the krona chart Figure 1.
 


 LIMITATIONS OF NEXT GENERATION SEQUENCING

Though sequencing siRNA is sensitive in identifying viruses of varied genome features and different nucleic acid types in low titers not readily detected by other methods, (Kreuze, 2014; Wu et al., 2015), assembly of full genomes or sequence coverage of the viral genome may be difficult (Kreuze, 2014) as small endogenous plant RNAs may interfere with short 21-24 base pairs (Boonham  et  al.,  2014).  Next  Generation   Sequencing platforms with short read lengths products may equally limit the ability to characterize large repeat regions accurately (Snyder et al., 2010). Lack of known reference genomes for a majority of sequences at times renders classification of reads impossible (Edwards and Rohwer, 2005). However, deep sequenced samples can undergo de novo assembly or mapped to reference genomes for viral discovery (Coetzee et al., 2010; Hwang et al., 2013; Kreuze et al., 2009; Maree et al., 2015). The methods assemble genomes of the majority species in the sample by ignoring technical errors and low-frequency variants. Poor sequence similarity leads to low sequencing depth or coverage to reference sequences as fewer reads cover the same fragment of DNA (Thomas et al., 2012).


 CONCLUSION

Potato viruses rely on both mechanical or vector borne transmission modalities. A great number have multiple host ranges across the plant taxa. Studies have shown that, viruses like PSTVd have wild hosts in the solanaceous family and other plants. Vectors have the capability of availing the viruses to and from infected or healthy crop irrespectively. Known virus agents can be detected using serology and Polymerase Chain Reaction (PCR). However, the tools are not potent for discovery or novelty studies aiming to detect undocumented virome. Next generation sequencing is an emerging tool to plant molecular biologists in determining whole virus genomes, and undertaking viral metagenomic studies for novel viruses. Serology and PCR diagnostics have had challenges in detecting unknown virus agents. These techniques are extremely costly per sample other than being time consuming to run. With the constant change in genetics of viruses, novelty and un-targeted viruses are missed out. Next Generation Sequencing, through sample pooling by RNAtaq-Seq protocols, is able to analyse numerous samples in a single barcoded run. This cuts down costs as samples are run simultaneously, saves time as the various platforms have a higher throughput rate and increases robustness by being able to detect both known and unknown virome, leading to discovery. Adopting Next Generation Sequencing to boost serology and PCR diagnostics will allow for total documentation of viral entities infecting Irish potato in Kenya. Virus disease etiology will be opened and an understanding of virus-host interactions enabled. Furthermore, antagonistic and synergistic or mutualistic virus relationships will be opened up upon determination of the total virome.


 RECOMMENDATIONS

Serology relies on familiarity for it to be effective, hence would be robust with up to date viral genomes. This is also important for Polymerase Chain Reaction (PCR) and PCR variants used in the Molecular diagnostics of Irish potato viruses. Next Generation Sequencing has justified ability in cataloguing familiar and new novelties in the world of virome discovery. This can be adopted and used to avail information on the potato viruses in the country. Disease dynamics have to be conducted to ascertain causality of disease Vis a Vis the viral agent tied to it. Since Next Generation Sequencing avails copious data and metadata on genome discovery within a sample, it is prudent for further studies to be conducted on disease symptomatology, virus-host interactions and virus-vector mediation to determine the pathology. Furthermore, stacked viral influence and effect on symptomatology and infection modalities in potatoes has to be conducted as an extrapolation of disease dynamics. This is to rule out misdiagnosis and enhance documentation of viral interactions in potato hosts. It is mandatory to conduct a countrywide potato viral discovery studies using deep massively parallel Next Generation Sequencing techniques such as: Illumina, SOLiD, Roche 454 pyrosequencing, Ion Torrent, Oxford Nanopore and Pacific Biosciences sequencing technologies.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



 REFERENCES

Adams IP, Glover RH, Monger WA, Mumford RA, Jackeviciene E, Navalinskiene M, Samuitiene M, Boonham N (2009). Next-generation sequencing and metagenomic analysis. A universal diagnostic tool in plant virology. Molecular Plant Pathology 10:537-545.
Crossref
 

Al Rwahnih Maher, Daubert S, Golino D, Islas C, Rowhani A (2015). Comparison of next generation sequsencing versus biological indexing for the optimal detection of viral pathogens in grapevine. Phytopathology 105:758-763.
Crossref

  
 

Al Rwahnih Maher, Daubert S, Golino D, Rowhani A (2009). Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus. Virology Elsevier Inc 387:395-401.
Crossref

  
 

Ali M, Hameed S, Tahir M (2014). Luteovirus: Insights into pathogenicity. Archives of Virology 159:2853-2860.
Crossref

  
 

Anderson PK, Cunningham AA, Patel NG, Morales FJ, Epstein PR, Daszak P (2004). Emerging infectious diseases of plants: Pathogen pollution, climate change and agrotechnology drivers. Trends in Ecology and Evolution 10s:535-44.
Crossref

  
 

Awasthi LP, Verma HN (2017). Current status of viral diseases of potato and their ecofriendly management - A critical review. Virology Research and Reviews 1(4):1-16.
Crossref

  
 

Balasubramanian S (2015) .Solexa sequencing: Decoding genomes on a population scale. Clinical Chemistry 61:21-24.
Crossref

  
 

Baldo NH, Elhassan SM, Elballa MMA (2010). Occurrence of viruses affecting potato crops in Khartoum State-Sudan. Potato Research 53(1):61-67.
Crossref

  
 

Barzon L, Lavezzo E, Militello V, Toppo S, Palu' G (2011). Applications of next-generation sequencing technologies to diagnostic virology. International Journal of Molecular Sciences12:7861-84.
Crossref

  
 

Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, et al (2008). Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53-59.

  
 

Bondole BM (1992). Potato virus y (PVY) in Irish potatoes (Solanum tuberosum) and tree tomato (Cyphomandra betaceae) and the influence of potato susceptibility to the virus and the aphid virus-vector on the spread of PVY Mosaic in Kenya. Uonbi.erepository found at 

View

  
 

Boonham N, Glover R, Tomlinson J, Mumford R (2008). Exploiting generic platform technologies for the detection and identification of plant pathogens. European Journal of Plant Pathology 121:355-363.
Crossref

  
 

Boonham N, Kreuze JF, Winter S, van der Vlugt R, Bergervoet J, Tomlinson J, Mumford RA (2014). Methods in virus diagnostics: From ELISA to next generation sequencing. Virus Research 186: 20-31.
Crossref

  
 

Boonham N, Pe'rez LG, Me'ndez MS, Peralta EL, Blockley A, Walsh K, Barker I, Mumford RA (2004). Development of a real-time RT-PCR assay for the detection of potato spindle tuber viroid. Journal of Virological Methods 116:139146.
Crossref

  
 

Bostan H, Nie X, Singh RP (2004). An RT-PCR primer pair for the detection of pospiviroid and its application in surveying ornamental plants for viroids. Journal of Virological Methods 116:189193.
Crossref

  
 

Botermans M, van de Vossenberg BT, Verhoeven JThJ, Roenhorst JW, Hooftman M, Dekter R, Meekes ETM (2013). Development and validation of a real-time RT-PCR assay for generic detections of pospiviroids. Journal of Virological Methods 187:4350.
Crossref

  
 

CABI/EPPO (Centre for Agriculture and Bioscience International/ European and Mediterranean Plant Protection Organization) (2019). Potato Virus Y (Potato mottle). Distribution Maps of Plant Diseases. 

  
 

CABI/EPPO (Centre for Agriculture and Bioscience International/European and Mediterranean Plant Protection Organisation) (2014). Potato Spindle Tuber Viroid. Distribution Maps of Plant Diseases No. 729. CABI Head Office, Wallingford, UK.

  
 

Candresse T, Cambra M, Dallot S, Lanneau M, Asensio M, Gorris MT, Revers F, Macquaire G, Olmos A, Boscia D, Quiot JB, Dunez J (1998). Comparison of monoclonal antibodies and PCR assays for the typing of isolates belonging to the D and M serotypes of plum pox virus. Phytopathology 88:198-204.
Crossref

  
 

Capobianchi MR, Giombini E, Rozera G (2013). Next-generation sequencing technology in clinical virology. Clinical Microbiology and Infection 19:15-22.
Crossref

  
 

Coetzee B, Freeborough MJ, Maree HJ, Celton JM, Rees DJ, Burger JT (2010). Deep sequencing analysis of viruses infecting grapevines: virome of a vineyard. Virology 400(2):157-63.
Crossref

  
 

Coudray-Meunier C, Fraisse A, Martin-Latil S, Delannoy S, Fach P, Perelle S (2016). A novel high-throughput method for molecular detection of human pathogenic viruses using a nanofluidic real-time PCR system. PLoS ONE 11: 1-17.
Crossref

  
 

Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, Moran NA, Quan P, Briese T, Hornig M, Geiser DM, Martinson V, van Engelsdorp D, Kalkstein AL, Drysdale A, Hui J, Zhai J, Cui L, Hutchison SK, Simons JF, Egholm M, Pettis JS, Lipkin WI (2007). A metagenomic survey of microbes in honey bee colony collapse disorder'. Science 318: 283-287.
Crossref

  
 

Craig G, Webster, Stephen J, Wylie, Michael GK, Jones (2004). Diagnosis of plant viral pathogens. 86(12):10604-1607 Available at 

View

  
 

Denner FDN, Venter SL, Niederwieser JG (2012). Guide to potato production in South Africa. Pretoria, SA: ARC-Roodeplaat Vegetable and Ornamental Plant Institute. 

  
 

Dodds JA, Morris TJ, Jordan RL (1984). Plant viral double-stranded RNA'. Annual Review of Phytopathology 22:151-168.
Crossref

  
 

Dong M, Fisher C, Anez G, Rios M, Nakhasi HL, Hobson JP, Beanan M, Hockman D, Grigorenko E, Duncan R (2016). Standardized methods to generate mock (spiked) clinical specimens by spiking blood or plasma with cultured pathogens. Journal of Applied Microbiology 120:1119-1129.
Crossref

  
 

Edwards RA, Rohwer F (2005). Opinion: viral metagenomics. Nature reviews. Microbiology 3: 504.
Crossref

  
 

Espach A (2015). Final report. The validation of virus-specific real-time RT-PCR assays for commercial use in the potato industry.

  
 

Espach Y, Maree HJ, Burger JT (2012). Complete Genome of a Novel Endornavirus Assembled from Next Generation Sequence Data. Journal of Virology 86:13142.
Crossref

  
 

Evangelista C (2013). Viruses Occurring in Potatoes (Solanum tuberosum) in Mbeya Region, Tanzania. University of Helsinki.

  
 

Department of Agricultural Sciences. Plant Production Science/ Plant Pathology January, 2013.

  
 

FAOSTAT (2011). Available at View

  
 

FAOSTAT (2019). Available at View

  
 

Giampetruzzia A, Roumia V, Robertoa R, Malossinib U, Yoshikawac N, Nottea PL, Terlizzi F, Credid R, Saldarelli P (2011). A new grapevine virus discovered by deep sequencing of virus-and viroid-derived small RNAs in Cv Pinot gris. Virus Research 163:262-8.
Crossref

  
 

Gildemacher PR (2012). Innovation in seed potato systems in Eastern Africa.PhD Thesis Wageningen University, Netherlands. pp. 12-13. Available at View

  
 

Gildemacher PR, Paul D, Baker I, Kaguongo W, Woldegiorgis G, Wagoire WW, Wakahiu M, Leeuwis C, Struik PC (2009). A description of seed potato systems in Kenya, Uganda and Ethiopia. Potato Research 86:373-382.
Crossref

  
 

Goodwin S, McPherson JD, Richard McCombie W (2016). Coming of age: Ten years of next-generation sequencing technologies. Nature Reviews Genetics 17:333-351.
Crossref

  
 

Gould BA, Stinchcombe JR (2017). Population genomic scans suggest novel genes underlie convergent flowering time evolution in the introduced range of Arabidopsis thaliana. Molecular Ecology 26:92-106.
Crossref

  
 

Grothaus DG, Bandla M, Currier T, Giroux R, Jenkins RG, Lipp M, Shan G, Stave JW, Pantella V (2006). Immunoassay as an analytical tool in agricultural biotechnology. Journal of AOAC International 89:913-928.
Crossref

  
 

Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Braslavsky I, Causey M, Colonell J, DiMeo J, Efcavitch JW, Giladi E (2008). Single molecule DNA sequencing of a viral genome. Science 320:106-9.
Crossref

  
 

Hockman D, Dong M, Zheng H, Kumar S, Huff MD, Grigorenko E, Beanan M, Duncan R (2017). Comparison of multiplex PCR hybridization-based and singleplex real-time PCR-based assays for detection of low prevalence pathogens in spiked samples. Journal of Microbiological Methods. Elsevier B.V 132:76-82.
Crossref

  
 

Hossain B, Nasir IA, Tabassum B, Husnain T (2013). Molecular characterization, cloning and sequencing of coat protein gene of a Pakistani Potato leaf roll virus isolate and its phylogenetic analysis. African Journal of Biotechnology 12:1196-1202.

  
 

Hühnlein A, Schubert J, Zahn V, Thieme T (2016). Examination of an isolate of Potato leafroll virus that does not induce visible symptoms in the greenhouse. European Journal of Plant Pathology 145: 829-845.
Crossref

  
 

Huse SM, Huber JA, Morrison HG, Sogin ML, Welch DM (2007). Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biology 8:R143.
Crossref

  
 

Hwang YT, Kalischuk M, Fusaro AF, Waterhouse PM, Kawchuk L (2013). Small RNA sequencing of Potato leafroll virus-infected plants reveals an additional subgenomic RNA encoding a sequence-specific RNA-binding protein. Virology 438:61-69.
Crossref

  
 

Ibaba JD, Gubba A (2011). Diversity of potato virus Y isolates infecting solanaceous vegetables in the province of KwaZulu. Natal in the Republic of South Africa. Crop Protection 30:1404-1408.
Crossref

  
 

IPBO (International Plant Biotechnology Outreach) (2019). Potato in Africa. RE, Silvia Travella, (IPBO/VIB) D/2019/12.267/3. Technologie park 19 9052 Ghent Belgium Tel. + 32 9 292 80 44 

View

  
 

John O, Kiarie N, Solomon S, Muthoni J, Otieno SP (2013). Potato virus Y (PVY) and potato virus X (PVX) resistance breeding in Kenya: Applicability of conventional approaches. Agriculture and Biology Journal of North America 4:398-405.
Crossref

  
 

Kabira JN, Wakahihu M, Wagoire W, Gildemacher P, Lemaga B (2006). Guidelines for production of healthy seed potatoes in East and Central Africa. Edited by Lusike Wasilwa, Kenya Agricultural Research Institute, Nairobi, Kenya. pp. 1-28. Available at 

View

  
 

Kreuze Jan F, Perez A, Untiveros M, Quispe D, Fuentes S (2009). Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1-7.
Crossref

  
 

Kreuze JF (2014). siRNA deep sequencing and assembly: Piecing Together Viral Infections in Detection and diagnostics of plant pathogens. Springer Netherlands, pp. 21-38.
Crossref

  
 

Kumar R, Jeevalatha A, Baswaraj R, Kumar R, Sharma S, Nagesh M (2017). A multiplex RT-PCR assay for simultaneous detection of five viruses in potato. Journal of Plant Pathology 99:37-45.

  
 

Kunta M, da Graca JV, Malik NSA, Louzada ES, Setamou M (2014). Quantitative distribution of "Candidatus Liberibacter asiaticus" in the aerial parts of the Huanglongbing-infected citrus trees in Texas. HortScience 49:65-68.
Crossref

  
 

Lacomme C, Glais L, Bellstedt DU, Dupuis B, Karasev A, Jacquot E (2017). Potato virus Y: biodiversity, pathogenicity, epidemiology and management. Springer International Publishing. 
Crossref

  
 

Lacroix C, Renner K, Cole E, Seabloom EW, Borer ET, Malmstrom CM (2016). Methodological guidelines for accurate detection of viruses in wild plant species. Applied and Environmental Microbiology 82:1966-1975.
Crossref

  
 

Laehnemann D, Borkhardt A, McHardy AC (2015). Denoising DNA deep sequencing data-high-throughput sequencing errors and their correction. Brief Bioinformatics 17:154-79.
Crossref

  
 

Larbi I, Djilani-Khouadja F, Khamassy N, Fakhfakh H (2012). Potato virus surveys and wide spread of recombinant PVYNTN variant in Central Tunisia. African Journal of Microbiology Research 6(9):2109-2115.
Crossref

  
 

Laver T, Harrisona J, O'Neill PA, Moorea K, Farbosa A, Paszkiewicz K, Studholme DJ (2015). Studholme. Assessing the performance of the oxford nanopore technologies MinION. Biomolecular Detection and Quantification 3:1-8.
Crossref

  
 

Lezan W (2017). An investigation into the Potato leafroll virus problem in the Sandveld region, South Africa. 

  
 

Li Z, Guo B, Yang J, Herczeg G, Gonda A, Balazs G, Meril€a J (2017). Deciphering the genomic architecture of the stickleback brain with a novel multilocus gene-mapping approach. Molecular Ecology 26:1557-1575.
Crossref

  
 

Lotos L, Olmos A, Orfanidou C, Efthimiou K, Avgelis A, Katis N, Maliogka VI (2017). Insights into the etiology of Polerovirus-induced pepper yellows disease. Phytopathology 107. 
Crossref

  
 

Machangi JM, Olubayo FM, Njeru RW, Nderitu JH, El-Bedewy R, Yobera DM, Aura JA (2004). Occurrence of four major potato viruses in three main potato growing areas in Kenya. 6th Trienieal Conference, April 2004. African Potato Association (APA), pp. 273-281.

  
 

Mardis ER (2008). Next-generation DNA sequencing methods. Annual Review of Genomics and Human Genetics 9:387-402.
Crossref

  
 

Maree HJ, Pirie MD, Oosthuizen K, Bester R, Jasper D, Rees G, Burger JT (2015). Phylogenomic analysis reveals deep divergence and recombination in an economically important grapevine virus. PLoS ONE 10: 1-19.
Crossref

  
 

Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB (2005). Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376-380.

  
 

Marina B, Henryk C, Ahmed H (2014). Historical Perspective, Development and Applications of Next-Generation Sequencing in Plant Virology. Viruses 6:106-136.
Crossref

  
 

Matthews REF (1991). Plant Virology (Third Edition), Academic Press, 1991. Page xix, ISBN 9780124805538. 
Crossref

  
 

Martin IC (2012). Molecular Diagnostics in Plant Disease Diagnostic Clinics. What's the Status? Fungal Genomics and Biology 2:1.

  
 

Marz M, Beerenwinkel N, Drosten C, Fricke M, Frishman D, Hofacker IL, Hoffmann D, Middendorf M, Rattei T, Stadler PF, Töpfer A (2014). Challenges in RNA virus bioinformatics. Bioinformatics 30:1793-1799.
Crossref

  
 

McCoy RC, Taylor RW, Blauwkamp TA, Kelley JL, Kertesz M, Dmitry Pushkarev, Dmitri AP, Anna-Sophie F-L (2014). Illumina TruSeq synthetic long-reads empower de novo assembly and resolve complex, highly-repetitive transposable elements. PLoS ONE 9:e106689.
Crossref

  
 

Mikheyev AS, Tin MMY (2014). A first look at the oxford nanopore MinION sequencer. Molecular Ecology Resources 14:1097-1102.
Crossref

  
 

Mokili JL, Rohwer F, Dutilh BE (2012). Metagenomics and future perspectives in virus discovery. Current Opinion in Virology 2:63-77.
Crossref

  
 

Muthomi JW, Kinyungu TN, Nderitu JH, Olubayo FM (2011). Incidence of aphid- Transmitted viruses in farmer-produced seed potato tubers in Kenya. African Journal of Horticultural Science 5:18-25.

  
 

Muthomi JW, Nyaga JN, Olubayo FN, Nderitu JH, Kabira JN, Kiretai SM, Aura JA, Wakahiu M (2009). Incidence of aphid transmitted viruses in farmer based seed potato production in Kenya. Asian Journal of Plant Sciences 8:166-171.
Crossref

  
 

Myllykangas S, Buenrostro J, Ji HP (2012). Overview of sequencing technology platforms, bioinformatics for high throughput sequencing. 
Crossref

  
 

Ng TFF, Duffy S, Polston JE, Bixby E, Vallad GE (2011). Exploring the diversity of plant DNA viruses and their satellites using vector enabled metagenomics on whiteflies. PLoS ONE 6(4):e19050.
Crossref

  
 

Njukeng PA, Chewachong GM, Sakwe P, Chofong G, Nkeabeng LW, Demo P, Njualem KD (2013). Prevalence of Six Viruses in Potato Seed Tubers Produced in Informal Seed System in the North West Region of Cameroon. Cameroon Journal of Experimental Biology 09(01):44-49.
Crossref

  
 

Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Nobuyuki A, Tetsu H (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63.
Crossref

  
 

Nyamwamu PA, Mukoy B, Osogo AK, Omuse CN, Ajanga S, Were HK (2014). Distribution and Biological Characterization of Potato Virus Y in Kenya. Journal of Agri-Food and Applied Sciences 2(9):258-264.

  
 

Nyaboga EN, Ateka EM, Bulimo WD (2008). Serological detection of virus diseases of sweet potato in Kenya. Journal of Applied Biosciences 7:222-229.

  
 

Okeyo OG (2017). Response of Potato Genotypes to Virus infections and effectiveness of Positive selection in management of seed borne potato viruses. UoNbi.erepository. 
Crossref

  
 

Olubayo F, Kibaru A, Nderitu J, Njeru R, Kasina M (2010). Management of aphids and their vectored diseases on seed potatoes in Kenya using synthetic insecticides, mineral oil and plant extract. Journal of Innovation and Development Strategy 4(2):1-5.

  
 

Pallett DW, Ho T, Cooper I, Wang H (2010). Detection of cereal yellow dwarf virus using small interfering RNAs and enhanced infection rate with cocksfoot streak virus in wild cocksfoot grass (Dactylis glomerata). Journal of Virological Methods 168:223-7.
Crossref

  
 

Pereira AM, Lister RM (1989). Variations in virus content among individual leaves of cereal plants infected with Barley yellow dwarf virus. Phytopathology 79:1348-1353.
Crossref

  
 

Quan P, Briese T, Palacios G, Lipkin WI (2008). Rapid sequence-based diagnosis of viral infection'. Antiviral Research 79:1-5.
Crossref

  
 

Prabha K, Baranwal VK, RK Jain (2013). Applications of Next Generation High Throughput Sequencing Technologies in Characterization, Discovery and Molecular Interaction of Plant Vi-ruses. Indian Journal Virology. 
Crossref

  
 

Rashed A, Workneh F, Paetzold L, Gray J, Rush CM (2014). Zebra chip disease development in relation to plant age and time of "Candidatus Liberibacter solanacearum" infection. Plant Disease 98:24-31.
Crossref

  
 

Rhoads A, Au KF (2015). PacBio sequencing and its applications. Genomics, Proteomics and Bioinformatics 13:178-289.
Crossref

  
 

Roossinck MJ, Saha P, Wiley GB, Quan J, White JD, Lai H, Chavarrıoa F, Shen G, Roe BA (2010). Ecogenomics: Using massively parallel pyrosequencing to understand virus ecology. Molecular Ecology 19(Suppl. 1):81-88.
Crossref

  
 

Rotheberg JM, Hinz W, Rearrick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M, Hoon J (2011). An integrated semiconductor device enabling non-optical genome sequencing. Nature 475: 348-52.
Crossref

  
 

Safenkova IV, Pankratova GK, Zaitsev IA, Varitsev YA, Vengerov YY, Zherdev AV, Dzantiev BB (2016). Multiarray on a test strip (MATS): rapid multiplex immunodetection of priority potato pathogens. Analytical and Bioanalytical Chemistry 408:6009-6017.
Crossref

  
 

Sanchez-Navarro JA, Cañizares MC, Cano EA, Pallás V (2007). Plant tissue distribution and chemical inactivation of six carnation viruses. Crop Protection 26:1049-1054.
Crossref

  
 

Schadt EE, Turner S, Kasarskis A (2010). A window into third-generation sequencing. Human Molecular Genetics19:R227-240.
Crossref

  
 

Schulte-Geldermann E, Gildemacher PR, Struik PC (2012). Improving seed health and seed performance by positive selection in three Kenyan potato varieties. American Journal of Potato Research 89(6):429-437.
Crossref

  
 

Seguin J, Rajeswaran R, Malpica-López N, Martin RR, Kasschau K, Dolja VV, Otten P, Farinelli L, Pooggin MM (2014). De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs. PLoS ONE 9:1-8.
Crossref

  
 

Shamloul AM, Hadidi AF, Zhu SF, Singh RP, Sagredo B (1997). Sensitive detection of potato spindle tuber viroid using RT-PCR and identification of a viroid variant naturally infecting pepino plants. Canadian Journal of Plant Pathology 19:8996.
Crossref

  
 

Shendure J, Ji H (2008). Next-generation DNA sequencing. Nature Biotechnology 26:135-1145.
Crossref

  
 

Snyder M, Du J, Gerstein M (2010). Personal genome sequencing: Current approaches and challenges. Genes and Development 24:423-431.
Crossref

  
 

Standage DS, Berens AJ, Glastad KM, Severin AJ, Brendel VP, Toth AL (2016). Genome, transcriptome and methylome sequencing of a primitively eusocial wasp reveal a greatly reduced DNA methylation system in a social insect. Molecular Ecology 25:1769-1784.
Crossref

  
 

Studholme DJ, Glover RH, Boonham N (2011). Application of high throughput DNA sequencing in phytopathology. Annual Review of Phytopathology 49:87-105.
Crossref

  
 

Thomas T, Gilbert J, Meyer F (2012). Metagenomics - A guide from sampling to data analysis'. Microbial Informatics and Experimentation 2:1-12.
Crossref

  
 

Verhoeven JThJ, Jansen CCC, Willemen TM, Kox LFF, Owens RA, Roenhorst JW 2004). Natural infections of tomato by citrus exocortis viroid, columnea latent viroid, potato spindle tuber viroid and tomato chlorotic dwarf viroid. European Journal of Plant Pathology 110:823831.
Crossref

  
 

Wangai A, Lelgut D (2013). Status of potato viruses in Africa. Kenya Agricultural Research Institute, NPBRC, P. O. Box Njoro, Kenya (KALRO).

  
 

Were HK, Kabira JN, Kinyua ZM, Olubayo FM, Karinga JK, Aura J, Lees AK, Cowan GH, Torrance L (2014). Occurrence and Distribution of Potato Pests and Diseases in Kenya. Potato Research.
Crossref

  
 

Were HK, Kabira JN, Kinyua ZM, Olubayo FM, Karinga JK, Aura J, Torrance L (2013). Occurrence and Distribution of Potato Pests and Diseases in Kenya. Potato Research 56(4):325-342.
Crossref

  
 

Wiets GR (2013). An investigation of prevalence and the detection and race identification of South African potato viruses. Stellenbosch University View

  
 

Wu Q, Ding S, Zhang Y, Zhu S (2015). Identification of viruses and viroids by next generation sequencing and homology-dependent and homology-independent algorithms. Annual Review of Phytopathology 53:425-444.
Crossref

  
 

Wylie SJ, Jones MGK (2011). The complete genome sequence of a Passion fruit woodiness virus isolate from Australia determined using deep sequencing, and its relationship to other potyviruses. Archives of Virology 156:479-482.
Crossref

  
 

Yanagisawa H, Tomita R, Katsu K, Uehara T, Atsumi G, Tateda C, Kobayashi K, Sekine K (2016). Combined DECS analysis and next-generation sequencing enable efficient detection of novel plant RNA viruses. Viruses 70:1-11.
Crossref

  
 

Zhang W, Zhang Z, Fan G, Gao Y, Wen J, Bai Y, Qiu C, Zhang S, Shen Y, Meng X (2017). Development and application of a universal and simplified multiplex RT-PCR assay to detect five potato viruses. Journal of General Plant Pathology 83:1-13.
Crossref

  
 

Zheng Y, Gao S, Padmanabhan C, Li R, Galvez M, Gutierrez D, Fuentes S, Ling K, Kreuze JF, Fei Z (2017). VirusDetect. An automated pipeline for efficient virus discovery using deep sequencing of small RNAs. Virology. Elsevier 500:130-138.
Crossref

  

 




Copyright © 2024 Author(s) retain the copyright of this article.

This article is published under the terms of the Creative Commons Attribution License 4.0

Back to Vol. 12 No. 1
Back to articles
SUBSCRIBE TO RSS
SUBMIT MANUSCRIPT
CONFERENCES
          */?>