African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 4962

Full Length Research Paper

Multiplex RT-PCR identification of five viruses associated with the watermelon crops in the Brazilian Cerrado

Raimundo Wagner de Souza Aguiar
  • Raimundo Wagner de Souza Aguiar
  • Plant Production Graduate Program, Federal University of Tocantins, Gurupi-TO, Brazil.
  • Google Scholar
Adelmo Rodrigues Martins
  • Adelmo Rodrigues Martins
  • Plant Production Graduate Program, Federal University of Tocantins, Gurupi-TO, Brazil.
  • Google Scholar
Vitor L. Nascimento
  • Vitor L. Nascimento
  • Plant Production Graduate Program, Federal University of Tocantins, Gurupi-TO, Brazil.
  • Google Scholar
Aristóteles Capone
  • Aristóteles Capone
  • Plant Production Graduate Program, Federal University of Tocantins, Gurupi-TO, Brazil.
  • Google Scholar
Lara Teixeira Melo Costa
  • Lara Teixeira Melo Costa
  • Departament of Biotechnology, Universidade Federal do Tocantins, Gurupi, TO, 77413-070, Brazil.
  • Google Scholar
Fabrício Souza Campos
  • Fabrício Souza Campos
  • Departament of Biotechnology, Universidade Federal do Tocantins, Gurupi, TO, 77413-070, Brazil.
  • Google Scholar
Rodrigo Ribeiro Fidelis
  • Rodrigo Ribeiro Fidelis
  • Plant Production Graduate Program, Federal University of Tocantins, Gurupi-TO, Brazil.
  • Google Scholar
Gil Rodrigues dos Santos
  • Gil Rodrigues dos Santos
  • Plant Production Graduate Program, Federal University of Tocantins, Gurupi-TO, Brazil.
  • Google Scholar
Renato de Oliveira Resende
  • Renato de Oliveira Resende
  • Department of Cell Biology, Universidade de Brasilia, Brasilia-DF, Brazil.
  • Google Scholar
Tatsuya Nagata
  • Tatsuya Nagata
  • Department of Cell Biology, Universidade de Brasilia, Brasilia-DF, Brazil.
  • Google Scholar


  •  Received: 03 September 2018
  •  Accepted: 13 December 2018
  •  Published: 21 January 2019

 ABSTRACT

Watermelon plants can be naturally infected by several viruses in single or mixed infections, of which the diagnosis is difficult and require specific techniques. This study aims to detect and to verify the presence of Cucumber mosaic virus (CMV), Papaya ringspot virus type W (PRSV-W), Watermelon mosaic virus (WMV), Zucchini lethal chlorosis virus (ZLCV), Zucchini yellow mosaic virus (ZYMV), and the main cucurbit viruses in Brazil using multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay. Oligonucleotides were designed towards the conserved regions of the virus genomes. In the duplex-PCR, it was possible to detect all the virus combinations, except ZLCV with ZYMV. The amplified product sizes were 644 bp (CMV), 535 bp (WMV-2), 398 bp (PRSV-W), 244 bp (ZLCV), and 214 bp (ZYMV). To test the efficacy of the methodology, we analyzed plants with virus symptoms from four municipalities in the state of Tocantins, located at Brazilian Cerrado. Mixed infections were detected in 80% of samples in all the municipalities. The multiplex RT-PCR assay can be used to detect and differentiate watermelon viruses that affect crops in the state of Tocantins.

 

Key words: Cucumovirus, multiplex RT-PCR, Orthotospovirus, potyvirus, watermelon.


 INTRODUCTION

Watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) is a crop of significant importance to Brazil due to its economic and social aspects, mainly in the northern and northeastern regions. However, the crop in these regions is affected by viruses (Aguiar et al., 2013,  2015). The most important viruses that affect the watermelon plants are: (i) cucumovirus as Cucumber mosaic virus (CMV) (Roossinck, 2001); (ii) potyviruses as Papaya ringspot virus type W (PRSV-W) (Barnett, 1991; Bateson et al., 2002),  Watermelon  mosaic  virus (WMV) (Desbiez and Lecoq, 2004; Purcifull and Hiebert, 1979), and Zucchini yellow mosaic virus (ZYMV) (Desbiez and Lecoq, 1997; Harth et al., 2017); and (iii) orthotospovirus as Zucchini lethal chlorosis viruses (ZLCV) (Camelo-García et al., 2015; Lima et al., 2016). These viruses are widely distributed in the cucurbit cultivation areas in Brazil, limiting the yield and fruit quality (Aguiar et al., 2013, 2015; Soares et al., 2016). Moreover, they have been reported to cause not only single but mixed infections in the cucurbits (Aguiar et al., 2015; Ali et al., 2012; Soares et al., 2016), that contribute not only to the maintenance of the population diversity but also associated with the predominance of these viruses in commercial crops (Gómez et al., 2009; Kumar et al., 2010). Therefore, the development of methods that allow the simultaneous detection and identification of different viruses are desirable for the routine diagnosis of cucurbit virus infections, particularly, one that would require less time, work, and costs.
 
Several methods are used to detect viruses such as enzyme-linked immunosorbent assay (ELISA), double-antibody sandwich ELISA (DAS-ELISA), reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), nucleic acid spot hybridization (NASH), macroarrays and microarray technologies (Ge et al., 2013). However, all these methods are slow and expensive to carry out in a standard research laboratory. To overcome these shortcomings and  increase the diagnostic capacity of PCR, a variant method termed multiplex PCR (mPCR) has been developed wherein one target sequence can be amplified by including more than one single pair of primers in the reaction (Elnifro et al., 2000). Reverse Transcription reaction followed by mPCR (mRT-PCR) can be used for the simultaneous detection of multiple viruses, and it is more efficient and economical than the other conventional methods (Kwon et al., 2014; Zhao et al., 2016).
 
Multiplex systems have been developed for the detection of multiple viral infections. Several genera have already been detected, for instance, astrovirus, rotavirus and reovirus (Jindal et al., 2012), carlavirus (Nam et al., 2015), crinivirus (Wintermantel and Hladky, 2010), nodavirus (Senapin et al., 2010), potyvirus, and allexivirus (Kumar et al., 2010), in animals (Jindal et al., 2012; Senapin et al., 2010) and plants (Kumar et al., 2010; Nam et al., 2015; Wintermantel and Hladky, 2010). According to the literature, the identification of viruses using mRT-PCR with specific primers has been widely used to detect viruses associated with different important crops around the world such as grapevine (Vitis vinifera) (Hajizadeh et al., 2012), pepino (Solanum muricatum) (Ge et al., 2013), potato (Solanum tuberosum L.) (Agindotan et al., 2007; Rigotti and Gugerli, 2007), Prunus spp (Jarosova and Kundu, 2010), sugarcane (Saccharum spp) (Xie et al., 2009) and tomato (Solanum lycopersicum L.) (Chen et al., 2011).
 
In the present study, we developed the mRT-PCR method for the simultaneous detection of five watermelon viruses (PRSV-W, WMV, ZYMV, CMV, and ZLCV), in the leaf tissue samples from infected watermelon from four different municipalities of Tocantins state, located at the Brazilian Cerrado.


 MATERIALS AND METHODS

Plant and virus samples
 
Leaf samples from the infected watermelon plants in the reproductive stage, at the main watermelon producing regions of the municipalities of Gurupi, Porto Nacional, Lagoa da Confusão, and Formoso do Araguaia of Tocantins state (Figure 1) were collected as described by Aguiar et al. (2015).
 
CMV, PRSV-W, WMV, ZYMV, and ZLCV were obtained from Embrapa CNPH and Universidade de Brasília (UnB); they were provided by Dr. Mirtes Lima and Dr. Tatsuya Nagata. All the viruses were kept in a greenhouse at Universidade Federal do Tocantins-Gurupi in Cucurbita pepo, Nicotine benthamiana, and Datura stramonium plants by mechanical inoculation (Aguiar et al., 2015; Lima et al., 2009).
 
Primer design
 
The primers used in this study (Table 1) were designed from the specific nucleotide sequences data obtained from National Center for Biotechnology Information (NCBI) GenBank and checked by BLAST to confirm virus specificity. 11 accessions to CMV, 18 accessions to PMV-W, 18 accessions to WMV, 13 accessions to ZYMV, and 2 accessions to ZLCV (Supplemental Table 1) were used. The sequences were aligned by CLUSTAL W multiple sequence alignment program (Higgins and Sharp, 1989, 1988; Thompson et al., 1994) to find the genome regions specific for each virus (Table 1). Self-annealing and primer-dimer formation were tested in silico by using OligoCalc software (Kibbe, 2007). The primers were used here in several duplex combinations (Figures 4 and 5).
 
RNA extraction and complementary DNA (cDNA) synthesis
 
Total RNA extraction was performed from the leaves of watermelon plants with symptoms of infection with CMV, PRSV-W, WMV, ZYMV, and ZLCV viruses (Figure 2) using the Plant RNA Purification Reagent Kit (Invitrogen Life Technologies, USA), according to the manufacturer's protocol. cDNA was synthesized from the total RNA extracted from the leaves with symptoms of infection of the viruses as previously described. First-strand cDNAs were synthesized using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions, with DEPC-treated water and random primers (NNNNNNN) (Invitrogen Life Technologies, USA) according to the supplied protocol.
 
Monoplex and multiplex PCR
 
PCR reactions were performed using the cDNA product with the addition of 0.4 μM of each virus-specific oligonucleotide, 10 μM of each dNTP, 2.5 μL of Taq DNA Polymerase Reaction Buffer, 2.0 mM of MgCl2, and 1U of Taq DNA Polymerase (Invitrogen Life Technologies, USA), to a total volume of 25 μL. 
 
 
 
Amplification of the fragments for virus detection was performed with an initial denaturation step at 94°C for 5 min followed by 35 cycles at 95 °C for 30 s, 52 °C for 1 min and 30 s, and 72 °C for 4 min. A final elongation step was performed at 72°C for 8 min and 4ºC at the end until the samples were analyzed.
 
For the mRT-PCR assay, some conditions were modified in order to optimize the Taq DNA Polymerase amplification of cDNA template on two or more simultaneous viruses. The amount of each oligonucleotide specific for each virus was changed to 0.5 mM and 2.5μM of each dNTP and 2.0 mM of MgCl2, to a total volume of 25 μL.
 
Analysis of mRT-PCR
 
The products of PCR (5 μL) were analyzed on 1% agarose gel in TBE buffer (0.1 M boric acid, 0.02 mM EDTA, pH 8.3) by electrophoresis at 80V. The molecular size of the amplified fragments was determined by comparison with 1kb Ladder Marker (Invitrogen Life Technologies, USA). The gel was stained with ethidium bromide solution (0.1 μL/mL) and visualized in a UV light transilluminator (Molecular Image LPIX-Locus Biotechnology). Moreover, the PCR products were sequenced to confirm the virus specificity and the nucleotides sequences of each target virus were compared with NCBI GenBank Database by using BLAST tool.
 

 


 RESULTS

A range of symptoms caused by watermelon viruses were observed in the collected plant samples from the different municipalities of Tocantins state (Figure 1). The most  frequently  here  observed  symptoms  were  ruffled edges, blistering, spur, leaf narrowing, shoestring, leaf distortion, leaf rolling, necrosis, mosaic, and stunting, corresponding to viral infections symptoms in watermelon (Ali et al., 2012).
 
The virus-specific oligonucleotides were designed towards the conserved regions of CMV, WMV, PRSV-W, ZYMV, and ZLCV obtained from the sequences deposited in NCBI GenBank. The most conserved genome regions used for the development of oligonucleotides of the potyviruses (PRSV-W, ZYMV, and WMV) correspond to the regions of the protein coat genes. The oligonucleotides were developed for the rapid differentiation and detection of the watermelon viruses with each analyzed virus having a specific amplicon size.
 
The oligonucleotides designed to identify the watermelon virus were confirmed by both monoplex (single PCR) and mRT-PCR, in agarose gel electrophoresis (Figure 3). Using duplex combinations of oligonucleotides, the mRT-PCR assay was used for virus identification in infected plants samples from four different locales of watermelon production in the state of Tocantins (Figures 4 and 5).
 
In Gurupi, 80% of the leaf samples were positive for mixed infections (occurrence of two or more virus species) (Figure 4A). In all plants, the fragments of the expected size were amplified, especially PRSV- W, which was detected by mixed or single PCR in 80% of samples, WMV virus in 30%, ZLCV in 20%, and CMV in 10% samples (Figure  4A  and  Table  2).  It was observed that ZYMV was not present in the Gurupi samples.
 
 
 
 
In the city of Porto Nacional, 80% of the leaf samples had mixed infections as detected by mRT-PCR (duplex type) (Figure 4B). The PRSV-W was present in 70% of the samples with 30% of them in the mixed form (Figure 3B and Table 2). Interestingly, all the five viruses were found in  the  samples  from  the  city of  Porto  Nacional.
 
In the city of Lagoa da Confusão, 100% of the leaf samples were found to have mixed infections by mRT-PCR (duplex type) (Figure 5A). WMV was present in 80% of the samples, followed by PRSV-W in 70% and ZLCV in 50%. Besides, the absence of CMV and ZYMV was observed at this locale (Figure 5A and Table 2).
 
Finally,  80%  of  the  leaf  samples   from   the   City  of  Formoso do Araguaia were identified with mixed infections by mRT-PCR (duplex type) (Figure 5B). PRSV-W was present in 80% of the samples, followed by ZCLV in 40%, WMV in 30%, CMV in 20%, and ZYMV in 10% of the samples. So, all the viruses were detected in this locale.
At the state level, 85% of the total infections were of mixed type while the rest were of single type (Table 2). The CMV occurred in 20% and ZLCV in 32.5% of the total samples analyzed (Table 2).


 DISCUSSION

Watermelon culture is affected by several viruses that directly influence its yield and fruit quality. In the present study, CMV, PRSV-W, WMV, ZYMV, and ZLCV were detected that affect watermelon yield in several places, such as Asia (Kwon et al., 2014; Zhao et al., 2016), Africa (Levi et al., 2016), and North America (Ali et al., 2012). Here we focused on the occurrence of these viruses in the Brazilian Cerrado in the  state  of  Tocantins,  a  large producer of this crop. It is important to consider that the identification of a specific virus affecting watermelon crops is very difficult, or impossible, because of the possibility of asymptomatic or mixed virus infections, different virus strains, and confusion with abiotic disorders, such as nutrient deficiencies (Ali et al., 2012).
 
The design of oligonucleotides for effective detection of CMV, WMV, PRSV-W, ZYMV, and ZLCV becomes necessary because of the natural occurrence of these viruses in mixed infections, an interesting phenomenon of occurrence of synergism between two or more virus species (Ali et al., 2012). Thus, specific target oligonucleotides must be designed for the conserved regions of each virus genome. The oligonucleotides used here could detect and differentiate mixed viral infections by amplifying the products during mRT-PCR (duplex type) without interacting between them, the ideal for this technique (Wei et al., 2008). Even for ZLCV and ZYMV whose fragment sizes (244 and 214 bp, respectively) are very close, it is possible to differentiate the amplicons on agarose gel. It is important to note that alterations in the concentrations  of  oligonucleotides,   cDNA,  and/or Mg2+ source, for instance, may improve the efficiency of PCR (Arezi et al., 2003), as the results here show.
 
Several researchers around the world developed methods with mRT-PCR to simultaneously identify and differentiate plant viruses for various crops, for instance  the identification of pospiviroids in grapevine, with detection rate superior to 60% in four of five viroid samples analyzed (Hajizadeh et al., 2012), potexvirus, carlavirus, and tobamovirus in pepino, with 5% to 40% virus detection (Ge et al., 2013), potyvirus and polerovirus in sugarcane (Xie et al., 2009), cucumivirus and tobamovirus in tomato, with detection superior to 75% (Chen et al., 2011); potyvirus and allexivirus in onion (Kumar et al., 2010) and garlic (Nam et al., 2015), and crinivirus in tomato, lettuce (Lactuca sativa L), and melon (Cucumis melo L.) (Wintermantel and Hladky, 2010). Here, we detected all the five viruses tested (CMV, PRSV-W, WMV, ZLCV, and ZYMV) in almost all locales, except CMV in Lagoa da Confusão and ZYMV in Lagoa da Confusão and Gurupi.
 
ZYMV virus was found only in two samples, in the mixed infection form, at Porto Nacional and Formoso do Araguaia, in combination with PRSV-W and CMV. It is important to mention that the distance between the locations of these two cities is large enough, and infection is not possible by the same host insects from one place to another. Moreover, PRSV-W had the highest number of infected leaves samples, was present in all cities studied, with infection of 75% of the samples tested here (Table 2), and these results indicate the predominance of this virus in the commercial plantations of cucurbits in Tocantins state. Besides, the quality of our multiplex results is similar to that demonstrated by other studies in cucurbits (Kwon et al., 2014; Wang et al., 2010). Moreover, the capacity of PRSV-W infection and association with other viruses makes it difficult to identify the infection by symptoms. Another key factor is the mixed infections caused by this virus (Figures 3 and 4), which was obtained in all combinations (PRSV-W + WMV; PRSV-W + CMV; PRSV-W + ZLCV; and PRSV-W + ZYMV).
 
Cucurbit crops are cultivated throughout the world with watermelon being mainly produced in South America; however, the yield and fruit quality are affected by viral diseases (Romay et al., 2014). CMV, PRSV-W, WMV, ZYMV, and ZLCV are the main viruses that infect watermelon through aphids (CMV, PRSV-W, WMV) and trips (ZLCV) as vectors (Romay et al., 2014). In Brazil, ELISA method is often used to detect watermelon virus. Previous studies report PRSV-W and WMV as the main viruses infecting watermelon and other cucurbits in different regions such as the northeast (Moura et al., 2001; Silveira et al., 2009; Soares et al., 2016), southeast (Lima and Alves, 2011), midwest (Lima and Alves, 2011) and north (Aguiar et al., 2013, 2015; Lima and Alves, 2011). On the other hand, it was reported in Paraiba that ZYMV  was   the   main  virus  infecting  watermelon  crop (Soares et al., 2016) contrary to other reports from the same region (Moura et al., 2001; Silveira et al., 2009). Here, we mainly described PRSV-W and, especially in Tocantins, it has been reported that CMV causes more damage to watermelon plants (Aguiar et al., 2013), and the pattern of infection frequency is the same: PRSV-W with the highest rate followed by WMV, ZLCV, CMV, and ZYMV (Aguiar et al., 2015). For instance, PRSV-W was detected in 22% of symptomatic plants by ELISA method, while mRT-PCR found it in 75% (Table 2). It is worth mentioning that weeds can act as natural reservoirs of these watermelon viruses, particularly ZYMV and PRSV-W (Aguiar et al., 2018). The distribution of virus in the state can be attributed to the mode of transmission, non-persistent by several species of aphids, of these viruses (CMV, PRSV-W, WMV, and ZYMV). In this way, the potyviruses (PRSV-W, WMV, and ZYMV) were detected in all the samples analyzed, in single or mixed infections, and these results indicate the prevalence of potyvirus infecting cucurbits in Tocantins.
 
Virus detection by rapid and reliable techniques can be important tools in plant virology for disease control in production fields to phytosanitary barriers. Current panorama of watermelon production in Brazil and the world indicates that an effective simultaneous detection and differentiation of CMV, PRSV-W, WMV, ZLCV, and  ZYMV is required due to the natural occurrence of these viruses in mixed infections. To our knowledge, this is the first report on the use of mRT-PCR for simultaneous detection of these five viruses that affect watermelon crop in Tocantins, an important agricultural region in Brazil. The mRT-PCR assays developed in this study will provide a simple method for the detection of multiple viruses in watermelon culture in Brazil. Moreover, the specificity of the oligonucleotides allows the development of epidemiological, spatial, and temporal distributions of the virus under study.


 CONFLICT OF INTERESTS

The authors did not declare any conflict of interest.

 


 ACKNOWLEDGEMENT

This study was funded by the Universidade Federal do Tocantins and Universidade de Brasília.


 ABBREVIATIONS

CMV, Cucumber mosaic virus; DAS-ELISA, double-antibody sandwich ELISA; ELISA, enzyme-linked immunosorbent assay; M-MLV, Moloney Murine Leukemia Virus; mPCR, multiplex PCR; mRT-PCR, reverse transcription  reaction followed by mPCR; NASH,  nucleic acid spot hybridization; PRSV-W, Papaya ringspot virus type W; qRT-PCR, quantitative real-time PCR; RT-PCR, reverse transcription polymerase chain reaction; WMV, Watermelon mosaic virus; ZLCV, Zucchini lethal chlorosis virus; ZYMV, Zucchini yellow mosaic virus.



 REFERENCES

Agindotan BO, Shiel PJ, Berger PH (2007). Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan® real-time RT-PCR. Journal of Virological Methods 142(1-2):1-9.
Crossref

 

Aguiar RWS, Evangelista MP, Ramos ACC, Pascoal PV, Barros HB, Santos MM (2013). Damage and Symptoms Associated with Watermelon Virus in The State of Tocantins, Brazil. Bioscience Journal 29(5):1632-1639.

 
 

Aguiar RWS, Rodrigues A, Portella ACF, Lopes MM, Lima MF, Resende RO, Nagata T (2015). Serological Identification of Virus Production Fields in the Tocantins State in Watermelon. Brazilian Archives of Biology and Technology 58(2):192-197.
Crossref

 
 

Aguiar RWS, Alves GB, Queiroz AP, Nascimento IR, Lima MF (2018). Evaluation of weeds as virus reservoirs in watermelon crops. Planta Daninha 36:1-10.
Crossref

 
 

Ali A, Abdalla O, Bruton B, Fish W, Sikora E, Zhang S, Taylor M (2012). Occurrence of viruses infecting watermelon, other cucurbits, and weeds in the parts of southern United States. Plant Health Progress doi:10.1094/PHP-2012-0824-01-RS
Crossref

 
 

Arezi B, Xing W, Sorge JA, Hogrefe HH (2003). Amplification efficiency of thermostable DNA polymerases. Analytical Biochemistry 321(2):226-235.
Crossref

 
 

Barnett OW (1991). Potyviridae, a proposed family of plant viruses. Archives of Virology 118(1-2):139–141.
Crossref

 
 

Bateson MF, Lines RE, Revill P, Chaleeprom W, Ha CV, Gibbs AJ, Dale JL (2002). On the evolution and molecular epidemiology of the potyvirus Papaya ringspot virus. Journal of General Virology 83(10):2575–2585.
Crossref

 
 

Camelo-García VM, Lima ÉFB, Rezende JAM (2015). Identification of natural hosts of Zucchini lethal chlorosis virus. Tropical Plant Pathology 40(5):345-349.
Crossref

 
 

Chen S, Gu H, Wang X, Chen J, Zhu W (2011). Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting tomato using 18S rRNA as an internal control. Acta Biochimica et Biophysica Sinica 43(6):465-471.
Crossref

 
 

Desbiez C, Lecoq H (2004). The nucleotide sequence of Watermelon mosaic virus (WMV, Potyvirus) reveals interspecific recombination between two related potyviruses in the 5' part of the genome. Archives of Virology 149(8):1619-1632.
Crossref

 
 

Desbiez C, Lecoq H (1997). Zucchini yellow mosaic virus. Plant Pathology 46(6):809-829.
Crossref

 
 

Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE (2000). Multiplex PCR: Optimization and Application in Diagnostic Virology. Clinical Microbiology Review 13(4):559-570.
Crossref

 
 

Ge B, Li Q, Liu G, Lu M, Li S, Wang H (2013). Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR. Archives of Virology 158(6):1181-1187.
Crossref

 
 

Gómez P, Sempere R, Elena S, Aranda M (2009). Mixed Infections of Pepino Mosaic Virus Strains Modulate the Evolutionary Dynamics of this Emergent Virus. Journal of Virology 82(23):12378-12387.
Crossref

 
 

Hajizadeh M, Navarro B, Bashir NS, Torchetti EM, Di Serio F (2012). Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids. Journal of Virological Methods 179(1):62-69.
Crossref

 
 

Harth JE, Simmons HE, Stephenson AG (2017). Vertical infection of Zucchini yellow mosaic virus via pollen transmission occurs at a lower frequency than ovule transmission. European Journal of Plant Pathology 147(3):717-720.
Crossref

 
 

Higgins DG, Sharp PM (1989). Fast and sensitive multiple sequence alignments on a microcomputer. Computer Applications in the Biosciences 5(2):151-153.
Crossref

 
 

Higgins DG, Sharp PM (1988). CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73(1):237-244.
Crossref

 
 

Jarosova J, Kundu J (2010). Simultaneous detection of stone fruit tree viruses by one-step multiplex RT-PCR. Scientia Horticulturae 125(1):68-72.
Crossref

 
 

Jindal N, Chander Y, Patnayak DP, Mor SK, Ziegler AF, Goyal SM (2012). A Multiplex RT-PCR for the detection of Astrovirus, Rotavirus, and Reovirus in Turkeys. Avian Disease 56(3):592-596.
Crossref

 
 

Kibbe WA (2007). OligoCalc: An online oligonucleotide properties calculator. Nucleic Acids Research 35:43-46.
Crossref

 
 

Kumar S, Baranwal V, Joshi S, Arya M, Majumder S (2010). Simultaneous Detection of Mixed Infection of Onion yellow dwarf virus and an Allexivirus in RT-PCR for Ensuring Virus Free Onion Bulbs. Indian Journal of Virology 21(1):64-68.
Crossref

 
 

Kwon JY, Hong JS, Kim MJ, Choi SH, Min BE, Song EG, Kim HH, Ryu KH (2015). Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses. Journal of Virological Methods 206:133-139.
Crossref

 
 

Levi A, Coffey J, Massey L, Guner N, Oren E, Tadmor Y, Ling KS (2016). Resistance to Papaya ringspot virus-watermelon strain (PRSV-W) in the desert watermelon Citrullus colocynthis. HortScience 51(1):4-7.

 
 

Lima MF, Alves RC (2011). Levantamento de vírus em cucurbitáceas no Brasil, no período 2008–2010. EMBRAPA.

 
 

Lima MF, Nagata T, Neves FM, Inoue-Nagata AK, Moita AW, Sousa C, Vecchia MD, Rangel MG, Dias RCS, Dutra LS, Ávila AC (2009). Serology detection of Melon yellowing-associated virus (MYaV) in melon producing areas of the Brazilian Northeast. Horticultura Brasileira 27(4):478-483.
Crossref

 
 

Lima RN, Oliveira AS, Leastro MO, Blawid R, Nagata T, Resende RO, Melo FL (2016). The complete genome of the tospovirus Zucchini lethal chlorosis virus. Virology Journal 13:3-7.
Crossref

 
 

Moura MCCL, Albersio J, Lima A, Oliveira VB, Fátima M, Gonçalves B (2001). Serological identification of virus species infecting cucurbits in producing areas of the State of Maranhão, Brazil. Fitopatologia Brasileira 26(1):90-92.
Crossref

 
 

Nam M, Lee YH, Park CY, Lee MA, Bae Y, Lim S, Lee JH, Moon JS, Lee S (2015). Development of Multiplex RT-PCR for simultaneous detection of garlic viruses and the incidence of garlic viral disease in garlic genetic Resources. Plant Pathology Journal 31(1):90-96.
Crossref

 
 

Purcifull DE, Hiebert E (1979). Serological distinction of watermelon mosaic virus isolates. Phytopathology 69(2):112-116.
Crossref

 
 

Rigotti S, Gugerli P (2007). Rapid identification of potato virus Y strains by one-step triplex RT-PCR. Journal of Virological Methods 140(1-2):90-94.
Crossref

 
 

Romay G, Lecoq H, Desbiez C (2014). Cucurbit crops and their viral diseases in Latin America and the Caribbean islands: A review. Journal of Plant Pathology 96(2):227-242.

 
 

Roossinck MJ (2001). Cucumber mosaic virus, a model for RNA virus evolution. Molecular Plant Pathology 2(2):59–63.
Crossref

 
 

Senapin S, Molthathong S, Phiwsaiya K, Jaengsanong C, Chuchird N (2010). Application of high resolution melt (HRM) analysis for duplex detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in shrimp. Molecular and Cellular Probes 24(5):291-297.
Crossref

 
 

Silveira LM, Queiroz MA, Lima JA, Nascimento AK, Neto ISL (2009). Serological survey of virus in cucurbit species in the Lower Middle São Francisco River Basin, Brazil. Tropical Plant Pathology 34(2):123-126.
Crossref

 
 

Soares MGO, Soares JA, Cezar MA, Cardoso TAL, Lima JAA (2016). Occurrence of pathogens in watermelon and pumpkin crops in the State of Paraiba. Revista Verde de Agroecologia e Desenvolvimento Sustentável 11(1):7-13.
Crossref

 
 

Thompson JD, Higgins DG, Gibson TJ (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22(22):4673-4680.
Crossref

 
 

Wang W, Zhang H, Yu X, Wu Y, Zhang W, Zhang C (2010). Establishment and application of multiplex RT-PCR for simultaneous detection of five watermelon viruses ZYMV, WMV, TMV, SqMV and CMV. Acta Phytopathologica Sinica 40(1):27-32.

 
 

Wei T, Lu G, Clover G (2008). Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses Journal of Virological Methods 151(1):132-139.
Crossref

 
 

Wintermantel WM, Hladky LL (2010). Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR. Journal of Virological Methods 170(1-2):106-114.
Crossref

 
 

Xie Y, Wang M, Xu D, Li R, Zhou G (2009). Simultaneous detection and identification of four sugarcane viruses by one-step RT-PCR. Journal of Virological Methods 162(1-2):64-68.
Crossref

 
 

Zhao L, Liu Y, Wu Y, Hao X (2016). Rapid Detection of watermelon viruses by reverse transcription loop-mediated isothermal amplification. Journal of Phytopathology 164(5):330-336.
Crossref

 

 




          */?>