Primers designed for the detection of grapevine pathogens spreading with propagating material by quantitative real-time PCR

The use of healthy planting stock is a basic prerequisite to prevent spreading of several grapevine diseases including viroids, viruses, phytoplasmas, bacteria and fungi which systemically and frequently infect their host plant in latent form. To obtain pathogen-free plants several protocols have been developed which include various diagnostic methods that include biological, -serological and molecular assays to detect and identify, and curative treatments to eliminate the various pathogens (Bisztray et al. 2012, Szegedi et al. 2012). Due to its high sensitivity, cost efficiency and easy application the polymerase chain reaction (PCR) has become the most widely used diagnostic protocol during the last 25 years in plant pathology (Louws et al. 1999). To increase the sensitivity, reliability and specificity of conventional PCR methods several novel technologies have been introduced into the plant quarantine studies during the recent years. In addition to increased sensitivity, most of these methods allow also the simultaneous detection of multiple pathogens. Novel molecular diagnostic techniques


Introduction
The use of healthy planting stock is a basic prerequisite to prevent spreading of several grapevine diseases including viroids, viruses, phytoplasmas, bacteria and fungi which systemically and frequently infect their host plant in latent form.To obtain pathogen-free plants several protocols have been developed which include various diagnostic methods that include biological, -serological and molecular assays to detect and identify, and curative treatments to eliminate the various pathogens (Bisztray et al. 2012, Szegedi et al. 2012).Due to its high sensitivity, cost efficiency and easy application the polymerase chain reaction (PCR) has become the most widely used diagnostic protocol during the last 25 years in plant pathology (Louws et al. 1999).To increase the sensitivity, reliability and specificity of conventional PCR methods several novel technologies have been introduced into the plant quarantine studies during the recent years.In addition to increased sensitivity, most of these methods allow also the simultaneous detection of multiple pathogens.

Novel molecular diagnostic techniques
Loop-mediated isothermal amplification: In spite of the high convenience, sensitivity and cost-efficiency of conventional PCR several novel molecular techniques have been developed and introduced to increase the sensitivity, reliability and/or applicability of diagnostic and identification protocols.The loop-mediated isothermal amplification (LAMP) originally developed by Notomi and coworkers (2000) uses a set of inner and outer primers and Bst polymerase that amplifies DNA at 65 o C. The elongation is followed by a colourimetric reaction using, e. g., hydroxynapthol blue (Goto et al. 2009).Thus the use of thermal cycler, gel electrophoretic separation and ethidiumbromide staining, and even DNA extraction is not necessary for scoring the results.Among grapevine pathogens this protocol has already been used for the detection of Xylella fastidiosa (Harper et al. 2010) and phytoplasmas (Kogovšek et al. 2015, Tomlinson et al. 2010).Further improvement includes addition of reverse transcriptase into the reaction which allows detection of RNA targets, RNA viruses and viroids.Among grapevine viruses an RT-LAMP based method was developed for GLRaV-3 and proved to be as sensitive as nested PCR (Walsh and Pietersen, 2013).Besides its high specificity and sensitivity a great advantage of LAMP is that it can be easily used in mobile laboratories, for example in the field and state border quarantine stations.

Micro and macroarray techniques:
The microarray and macroarray technologies involve the hybridization of PCR amplified and labelled samples to immobilized oligonucleotide probes specific for the various pathogens.Engel and coworkers (2010) printed 70-mer oligonucleotides of 570 probes specific for 44 grapevine viruses onto the surface of a microscope slide.These probes included both family-, and species-specific sequences.Thus each virus was covered by several specific sequences.Viral sequences were enriched by random primed PCR prior to hybridization.Using this step the detection becomes more sensitive than labelling total plant RNA.Since microarrays allow the detection of ten-thousands of specific sequences in a single hybridization step, this technique allows the detection of mixed infections or even the complete virus population present in the tested plant.Thompson and coworkers (2014) used the macroarray technology to detect and identify grapevine viruses.They used similarly 60-70mer oligonucleotide virus-specific probes.Approximately 1600 probes specific for 38 viruses and plant internal controls were immobilized onto a nylon membrane and used for the subsequent hybridization experiment with PCR enriched and labelled plant cDNA.Although macroarrays allow the application of approximately only one tenth of probes than that of microarrays, it is still far sufficient to detect viruses infecting grapevines and the method does not require expensive equipments and the membrane blots can be reused up to 20-30 times.Results of the micro-, and macroarray detections were consistent with ELISA and/or reverse transcription PCR assays.The great advantages of array technologies over the conventional and real-time PCR that they it cover much longer nucleotide sequences (several 70-mer oligonucleotides for a given virus) thus the reaction is highly specific for the given pathogen.In simple nucleic-acid amplification based detections the reactions are determined by short (usually 21-25 nucleotide long) primers thus a few mutations may cause false negative results.A further hybridization-based technique applied a "polyprobe" established from four tandemly cloned viroid sequences.This probe was then labelled with digoxygenin (DIG) and hybridized to grapevine RNA extract blotted onto nylon membrane.This method allowed the simultaneous detection of four viroids (Zhang et al. 2012).To detect fungal diseases Martos and coworkers (2011) dotblotted the PCR products onto a nylon membrane followed by hybridization with a Phaeomoniella-specific probe.The low density-array uses microwell plates (with 384 sample wells) precoated with a set of virus specific primers and TaqMan probes thus allowing the simultaneous detection of several viruses from a large number of samples by quantitative realtime PCR (Osman et al. 2008).
Barcodes: A recent technique called barcoding combines PCR amplification and sequencing.A basic prerequisite of a barcode is that it should contain highly conserved regions for a given taxonomic group but sequences bordered by these regions should be variable enough to discriminate species or strains.In the first step of barcoding a relatively short DNA fragment is amplified using primers designed for the conserved borders.Subsequently, this fragment is sequenced that allows the precise identification of the given organism.DNA barcodes for phytoplasmas based on tuf gene sequences (Makarova et al. 2012) and for fungi based on the ITS region (Schoch et al. 2012) have already been published, and barcode database for plant pathogens has also been established (Bonants et al. 2010, www.qbol.org).This database contains data for some grapevine pathogenic bacteria such as Xylella fastidiosa subsp.fastidiosa and Xanthomonas viticola as well.
Deep (Next generation) sequencing: Fast evolution in sequencing techniques established the possibility to get metagenomic information of the investigated plants.Next generation sequencing serves as a base of finding all of the expressed RNA (including the pathogens also) in the host and get a virome of the plant.Different platforms were used for the description of new grapevine viruses: Roche 454 for Grapevine Syrah-1 virus (Al Rwahnih et al. 2009), while Illumina for Grapevine virus F (Al Rwahnih et al. 2012, Al Rwahnih et al. 2013).This later platform was used also to create the viromes of a vineyard (Coetzee et al. 2010).RNA interference, the RNA based defense reaction of the plant, opens a new possibility in virus diagnosis using deep sequencing of small RNA libraries of infected plants (Donaire et al. 2009, Kreuze et al. 2009).During virus infection small interfering RNAs having sequence similarity to the infecting viruses are formed and sequencing them make it possible to identify viroids (Navarro et al. 2009) or viruses (Pantaleo et al. 2010) even if they are alien on the plant or never described (Giampetruzzi et al. 2012, Wu et al. 2012, Zhang et al. 2011).Deep sequencing offers a unique opportunity to reveal any viruses or viroids present in the sample, either expected or not.It needs a major investment at the beginning, running costs are relatively high, but barcoding the libraries for pooled sequencing, improvement of data analysing pipelines (Ho and Tzanetakis, 2014) and serious drop in the sequencing costs can make this method suitable for large scale sample testing in the near future.
Changes in the expression profile of grapevine host genes: Pathogen infections may cause specific changes in the expression profiles of infected grapevine plants (Albertazzi et al. 2009, Choi et al. 2010b, Espinosa et al. 2007, Fung et al. 2008, Hren et al. 2009, Santi et al. 2013, Zhao et al. 2011).Therefore analysis of host mRNAs may provide valuable information for pathogen diagnosis.Choi and coworkers (2010a) found four grapevine genes which were specifically up-regulated following Xylella fastidiosa infections, thus monitoring these genes indicates latent infections prior to symptom development.In symptomless Eutypa latainfected grapevine plants 10 genes were found which were differentially up-, or down-regulated (Camps et al. 2014).
GC-MS analysis of volatile compounds in healthy and galled (Agrobacterium vitis-infected) grapevine cuttings provided further indirect evidence for the pathogen-induced changes in plant gene expression.Diseased plants accumulated styrene, a phenylalanine derivative that was not present in healthy samples (Blasioli et al. 2010).In phytoplasma infected grapevine leaves genes involved in plant defence mechanisms such as callose synthase, sugar transporters and cell wall invertase showed higher expression (Santi et al. 2013).
The quantitative real-time PCR: Soon after the introduction of conventional PCR a more developed technique, the quantitative real time-PCR (q-PCR) has also been introduced for the detection of plant pathogens (Boonham et al. 2014, Christensen et al. 2013, Hren et al. 2010, Schena et al. 2004).The q-PCR is more sensitive than the conventional PCR method and the amplification and detection is combined in one step, thus subsequent gel-electrophoretic separation of the amplified product is not necessary.The amplification is continuously detected by various fluorescent reporters bound to the primers (Molecular bacon and Scorpion probes) or to internal complementer oligonucleotide sequences (TaqMan® probes) or bound specifically only to the amplified double stranded DNA (SYBR ® Green).Until annealing or insertion of primer or probe sequences to the target DNA the light emission is blocked by specific quenchers.The quenchers are removed during amplification from the reporters allowing the fluorescent light emission.Due to its high sensitivity and specificity the application of q-PCR has been widely used in plant quarantine laboratories despite of its relatively high costs.
At present the various versions (TaqMan ® , SYBR Green ® ) of q-PCR protocols combined with traditional PCR are most extensively applied in pathogen detection and identification.In a previous survey we have collected a primer "databank" for the detection of the most important grapevine pathogens by conventional PCR methods (Manduláné Farkas et al. 2014).As a subsequent part of this work here we summarize primer data available for q-PCR detection of grapevine pathogens spreading with propagating material (Tables 1-5).Be aware that these published reverse oligos are in sense orientation, for PCR reaction reverse complementer of them must be used i Be aware that these published forward and reverse oligos are mixed up j The forward oligos are the same as in the previous paper

Continuation of
Arabis mosaic virus, GFLV: Grapevine fanleaf virus, GFkV: Grapevine fleck virus, GLRaV-1, -2, -3, -4, -5, and -9: Grapevine leafroll-associated virus-1, -2, -3, -4, -5, and -9, respectively, GRSPaV: Grapevine rupestris stem pitting-associated virus, GVA: Grapevine virus A, GVB: Grapevine virus B, GVD: Grapevine virus D, TBRV: Tomato black ring virus, ToRSV: Tomato ringspot virus ** For TaqMan oligonucleotide probes (show in bold) we only refer for the sequence and not show either the fluorophore or the quencher.a ARMV in this case was tested in lily leaf not in grapevine b two different probes mixed together were used for succesful detection of highly variable isolates c In order to increase sensitivity different probes were used to detect all or at least the majority of diverse isolates d Two different probes were used separately e Two different forward primers mixed together were used for succesful detection of variable isolates f The mixture of the multiple primers were used to increase the efficiency of the detection g Oligos were used in different combinations h

Table 2 .
Virus specific primers

Table 2 Table 3 .
Phytoplasma specific primers *n. g.: not given, **: non fluorescent MGB quencher, ***: In BN detection, due to the low sensitivity of the 210F/280R primers, prior to qPCR an RT-PCR has to be performed using 190F/660R primers.

Table 5 .
Fungus specific primers *forward and reverse primers are fromShena et al. 2002