Real-time PCR, a great tool for fast identification

Eur J Plant Pathol https://doi.org/10.1007/s10658-018-1487-7

Real-time PCR, a great tool for fast identification, sensitive detection and quantification of important plant-parasitic nematodes Andrea Braun-Kiewnick & Sebastian Kiewnick

Accepted: 20 April 2018 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Abstract Plant-parasitic nematodes can cause significant damage to agricultural crops and forests worldwide, resulting in major economic losses. Some nematode species do not occur in all areas and are regulated as quarantine organisms. To avoid introduction and spread of these organisms, fast, simple and reliable detection and identification methods are needed, that help plant diagnostic services such as reference centres or national plant protection organizations (NPPOs) to rapidly identify suspicious nematodes. Real-time PCR is one of the fastest, most sensitive and reliable methods to fulfil this task. It is a DNA-based method that is easy to learn with the only requirement of having a specific thermocycler (Real-time Platform) and the appropriate chemistry. Real-time PCR provides very sensitive detection and species-specific identification with the potential to quantify target organisms if required. Following DNA extraction, results can be seen in 1–3 h and management decisions applied. Real-time PCR can be used for highthroughput analysis of many samples and in some cases for multiplexing, allowing for identification of more than one species in a single reaction. Over the past 15 years, real-time PCR methods have been developed

A. Braun-Kiewnick Agroscope, Research Division Plant Protection, CH-8820 Waedenswil, Switzerland S. Kiewnick (*) Julius Kuehn Institute, Federal Research Center for Cultivated Plants, Institute for Plant Protection in Field Crops and Grassland, Messeweg 11/12, 38104 Braunschweig, Germany e-mail: [email protected]

for the main plant-parasitic nematodes, in particular the regulated species. This paper reviews the achievements in plant nematology diagnostics using real-time PCR as the method of choice for fast and reliable detection, identification and even quantification of plant parasitic nematodes. Keywords Quantitative PCR . Survey or monitoring studies . Import controls

Introduction The phylum nematoda is the most populated after arthropods and consists of currently about 80,000 described species, of which some originated about one billion years ago (Postnikova et al. 2015; Wang et al. 1999). While most nematodes are multicellular, freeliving organisms that consume bacteria, fungi, other nematodes and protozoa, or are saprophytes that degrade dead plant material in soil and thus are important organisms in ecosystem stability, about 44% of the described species are parasites of animals and 15% are parasites of plants (Lambert and Bekal 2002). Plantparasitic nematodes are recognized as major agricultural pathogens and known to attack plants and cause great economic losses in agricultural crops worldwide. Rootknot nematodes (RKN) of the genus Meloidogyne are among the most damaging and economically important plant parasitic nematodes. They can cause worldwide crop losses of 12–14% or more than US $100 billion per year (Sasser 1990; Castagnone-Sereno et al. 2013). As a

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result of RKN feeding, large galls are formed on the roots of host plants (Anonymous 2016a, b). There are just above 100 Meloidogyne species described at the moment and four represent a major threat to agricultural crops: Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica and Meloidogyne hapla. Meloidogyne incognita is the most common and destructive nematode species found in all agricultural regions worldwide and is considered as Bthe single most damaging crop pathogen in the world^ (Trudgill and Blok 2001). A recent review on root-knot nematodes in Europe by Wesemael et al. (2011) showed that out of 90 species described then, 23 have been found in Europe. M. incognita, M. arenaria and M. javanica are the most prevalent in southern European countries and in protected cultivation systems in the northern parts of Europe. In addition, some previously neglected or newly described Meloidogyne spp. such as M. enterolobii, M. ethiopica and M. luci can develop into major problems and need to be considered as new risk for agriculture in tropical and temperate climates (Elling 2013). Further important genera belonging to the top ten plant parasitic nematodes in molecular plant pathology (Jones et al. 2013) are cyst nematodes comprising Globodera and Heterodera spp., followed by root lesion nematodes (Pratylenchus spp.), the burrowing nematode (Radopholus similis), the stem nematode Ditylenchus dipsaci and the pine wilt nematode (Bursaphelenchus xylophilus). Finally, the reniform nematode (Rotylenchulus reniformis), the virus vector

Xiphinema index, the false root-knot nematode (Nacobbus aberrans) and the white tip nematode Aphelenchoides besseyi are also of major interest and great economic importance. Fortunately, some of the nematodes do not occur in all agricultural significant areas. To avoid the introduction and spread of organisms harmful to plants or plant products (quarantine pests) to non-contaminated areas, the International Plant Protection Convention (IPPC) as well as the European Plant Protection Organization (EPPO) developed lists and regulations to prevent the occurrence of important plant parasitic nematode species (Table 1) by quarantine measures (www.eppo.org). However, recent reports on the spread of plant parasitic nematodes, mostly due to worldwide trading of plants and commodities (De Weerdt et al. 2011; CastagnoneSereno 2012; Braun-Kiewnick et al. 2016), demonstrate that pathways exist for the introduction of harmful organisms that have to be identified to avoid establishment and possible economic damage. In order to comply with these regulations and before phytosanitary measures and management strategies can help to prevent the spread of quarantine nematodes, fast and reliable detection and identification methods are required at borders and plant diagnostic services for import controls. Detection of nematodes Due to the fact that nematodes are present in soil and plants in a variety of life stages, various extraction

Table 1 Nematode species recommended for regulation or for conducting a pest risk assessment according to the EPPO A1, A2 and alert list (as approved by EPPO Council in March 2018) A1 list

A2 list

Alert list

Nacobbus aberrans

Aphelenchoides besseyi

Heterodera elachista

Radopholus similis (attacking citrus, formerly R. citrophilus)

Bursaphelenchus xylophilus

Meloidogyne ethiopica

Xiphinema americanum sensu stricto

Ditylenchus dipsaci

Meloidogyne luci

Xiphinema bricolense

Globodera pallida

Meloidogyne graminicola

Xiphinema californicum

Globodera rostochiensis Heterodera glycines Meloidogyne chitwoodi Meloidogyne enterolobii Meloidogyne fallax Meloidogyne mali Radopholus similis, not attacking citrus Xiphinema rivesi

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techniques to isolate these nematodes from their matrices are required. The matrix, i.e. soil, roots, and plant tissue (leaves, tubers, seeds) as well as an appropriate choice of method for extraction is of major importance for successful detection of a target species (Den Nijs and Van den Berg 2012). The currently available methods for nematode extraction with information on the suitability for specific nematode species are presented in the EPPO horizontal standard on PM 7/119(1) Nematode extraction (Anonymous 2013). However, depending on the purpose of the study, one should be aware that the efficiency of the chosen extraction method is critical for detection of the target nematode in question. This is especially important for government regulatory agencies where detection of quarantined organisms is essential (Sayler et al. 2012). DNA extraction methods After recovery of nematodes from plant parts or soil of suspicious samples and before identification by molecular methods, nucleic acid (DNA) extraction is required. Depending on the circumstance of use, the type of laboratory or other factors, DNA extraction can be done from single specimen (J2, adult stages) or from the whole nematodes suspension extracted from soil, roots or other plant parts (for examples see Table 2). Extraction can also be done from the content of cysts in floats after extraction from soil in the case of cyst nematodes (Reid et al. 2010, 2015) or directly from soil or plant

tissue samples (e.g. galls). Single nematodes can either be crushed in molecular grade water on a glass slide with a pipette tip, or by adding glass beads and an equal volume of worm lysis buffer to release the nucleic acid (Gamel et al. 2017). Lysis buffers containing Proteinase K and β-mercaptoethanol can easily be prepared and are highly recommended, because they effectively degrade proteins and other cell wall material and usually release DNA in 2 to 3 h. Lysis buffer protocols have been used in a number of studies (Holterman et al. 2006, 2011; Kumari and Subbotin 2012; Kiewnick et al. 2014), including studies on quantitative DNA-based monitoring of nematode assemblages (Vervoort et al. 2012) and nematodes in complex DNA backgrounds (RybarczykMydłowska et al. 2012; Kiewnick et al. 2015). The advantage of using lysis buffer is that it provides sufficient and clean DNA for downstream quantitative PCR assays without any further purification or PCR inhibitor removal step. Other DNA extraction protocols include buffers based on sodium hydroxid and Tween 20 to degrade cell walls and Tris-HCl and EDTA to neutralize DNA and bind proteins (Xin et al. 2003; Sayler et al. 2012). DNA extraction can also be done by commercial kits or automated systems. An overview of different DNA extraction methods is given in Table 2. DNA extraction is an important step before downstream molecular identification and is as critical as nematode extraction from soil or plants. The extraction method used depends on the purpose of the study and the nematode species targeted. Each method can also have an

Table 2 Available protocols for rapid DNA extraction and detection of target nematodes obtained from soil in different matrices Type of DNA extraction

Material

Purification needed

Source

Reference

Lysis buffer I

Single specimen

No

n. a.

Holterman et al. 2006

NaOH

Single specimen

No

n. a


GenElute Kit

Single specimen

Yes

Sigma Aldrich


Purelink Kit

Single specimen

Yes

Thermo Fisher Scientific


Lysis buffer

Nematode suspension

No

n. a.

Rybarczyk-Mydłowska et al. 2012; Kiewnick et al. 2015; Braun-Kiewnick et al. 2016

Lysis buffer

Nematode suspension

No

n. a.

Sayler et al. 2012; Xin et al. 2003

API Buffer

Floats with PCN

Yes

Qiagen

Reid et al. 2010, 2015

QIAamp DNA Blood Maxi kit or lysis buffer Purelink DNA extraction kit

Single specimen or floats with PCN Freeze dried nematode suspension

Yes

Qiagen

Gamel et al. 2017

Yes

Invitrogen

Oliveira et al. 2017

n. a. = not applicable; PCN = potato cyst nematodes

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influence on yield and/or purity of DNA and PCR conditions, etc. Some of the problems encountered with DNA extraction are e.g. laborious steps in the extraction protocols, load of sample (e.g. amount of soil/extraction), and efficiency of kits (% loss), detection limit, and PCR inhibitors. Solutions to these problems are the use of clean suspensions, cysts, and lysis buffers, kits with purification step (e.g. magnetic beads that catch and release pure DNA), self-made, modified or commercially available kits (e.g. www.cleardetections. com). In addition, the use of appropriate negative and positive amplification controls as described in EPPO diagnostic standards (e.g Anonymous 2016b) help to identify possible PCR inhibitors and consequently to improve and adjust DNA extraction protocols. Standard nematode diagnostics for identification After the initial detection of suspicious nematodes based on their morphology and efficient recovery from soil or plant parts, successful identification of the nematode in question is needed. Classical taxonomy relies usually on the different life stages of nematodes, highly trained diagnosticians or experienced taxonomists. For morphological identification, features of adult stages and morphometric data (body length, stylet length, etc.) and appropriate identification keys (Castillo and Vovlas 2007; Hunt and Handoo 2009; Anonymous 2016a, b) are required. Differential host plant tests can also be utilized to identify nematode species, but they require space and are time consuming but can provide material for molecular identification (Blok and Powers 2009). Biochemical tests mainly involve protein-based techniques such as isozyme analysis of young female stages for Meloidogyne spp. or antibody-based enzyme-linked immunosorbent assays (ELISA) for distinguishing between species of nematodes. Ahmed et al. (2016) recently reviewed principles of different assays and their advantages, disadvantages, current applications and their importance for nematode identification. Phenotyping by isozyme analysis at the protein level (e.g. carboxyl esterase, malate dehydrogenase, superoxide dismutase, glutamate oxaloacetate transaminase, etc.) is widely used to separate species of cyst and root-knot nematodes (Esbenshade and Triantaphyllou 1990; Karssen et al. 1995; Blok and Powers 2009; Hunt and Handoo 2009). However, Agudelo et al. (2011) who

compared real-time PCR and esterase phenotype analysis of mature females doubt the feasibility of esterase phenotype analysis alone for identification of Meloidogyne spp., because the technique relies on the expression of a gene product occurring in mature females and this stage is not often found in soil samples, where second stage juveniles are more prevalent. Twodimensional polyacrylamide gel electrophoresis (2DPAGE) has been used to compare Heterodera avenae isolates (Ferris et al. 1994) and antibody-based serological techniques such as the use of polyclonal or monoclonal antibodies to test major Meloidogyne species (Ibrahim et al. 1996; Davies et al. 1996). Tastet et al. (2001) used 2D-PAGE to identify a major protein of Meloidogyne chitwoodi and M. fallax that was not found in several other Meloidogyne species, and following internal amino-acid sequencing, a peptide was synthesized and used to raise antisera in rabbits. They were able to distinguish M. chitwoodi and M. fallax from eight other Meloidogyne species in a dot-blot hybridization assay with soluble proteins extracted from a single female. However, this technique cannot be used to analyse soil samples as they contain mainly juvenile stages of nematodes and rarely females. Furthermore, matrixassisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) has been used to distinguish oat and lucerne races of the stem nematode, Ditylenchus dipsaci, in Australia (Perera et al. 2009). In addition, some species like Xiphinema spp., important virus vectors, are often present in great soil depths and low densities making it difficult to identify by classical diagnostics (Van Ghelder et al. 2015). Consequently, rearing on host plants is required for correct identification, resulting in a time-consuming process until final results are seen. Furthermore, some species are difficult to distinguish from each other as they are lookalikes. A good example being Meloidogyne enterolobii (Castagnone-Sereno 2012), as it is morphologically difficult to distinguish from M. incognita. Therefore, morphological identification relies mainly on expert knowledge, but expertise in nematode taxonomy has been declining over the past years (Boonham et al. 2008; Ahmed et al. 2016). Taxonomic experts are retiring, nematode species are renamed, and new species are being described resulting in revision of nematode identification keys. This requires repeated training for staff involved in nematode diagnostics and as a

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consequence, the education of biology technicians today focusses more on modern molecular-based techniques in support of classical nematology using a microscope and identification keys. Furthermore, manual counting is a laborious and highly time-consuming and thus expensive task. Molecular methods used for identification An objective and reliable identification and resolution to species level is necessary to derive appropriate control and management strategies. Therefore, molecular methods have become the preferred method of identification with some of them suitable for high throughput analyses. In addition, molecular methods are often independent of taxonomic expertise, more rapid, very specific and sensitive and thus less laborious and expensive than traditional skills and techniques (Table 4). Conventional PCR with emphasis on sequence characterised amplified regions (SCAR) based primers (Zijlstra 2000; Randig et al. 2002; Meng et al. 2004; Adam et al. 2007; Tigano et al. 2010) has been used successfully to identify nematodes on the species level. However, most assays are lacking sensitivity in detecting low amounts of target DNA in complex DNA backgrounds and the target species cannot be quantified. They are also often not suited for multiplexing to identify several nematode species within one sample or high throughput analyses (Kiewnick et al. 2013), and thus many single PCR assays have to be run in parallel to identify the correct nematode in a suspicious sample. Random amplification of polymorphic DNA (RAPD), where a short primer set anneals to several sites on the DNA has been used to distinguish between species and populations of Meloidogyne from different origins (Castagnone-Sereno et al. 1994). If two of the annealed short primers happen to be close and opposite to each other, they will produce an amplicon. Difference in the gel fingerprints of amplicons separates species or populations, but may lack reproducibility. Amplified fragment length polymorphism (AFLP) was used to identify the genetic variability within the tobacco cyst nematode complex (Marché et al. 2001). The technique involves a series of PCR steps in which separate sets of primers are used to selectively amplify some subsets of products of each preceding PCR step. All selected fragments are run on a gel to produce unique fingerprints. However, the

technique is complex and labour-intensive and thus expensive. Recently, Ahmed et al. (2016) eminently summarized all of the advantages and disadvantages of these single methods and their importance for nematode identification. The invention of loop-mediated isothermal amplification (LAMP) about 15 years ago (Notomi et al. 2000) provided a new approach to molecular diagnosis, since it does not need a thermal cycler or other expensive tools for data generation or evaluation and thus is very valuable for field-testing. LAMP uses a set of four to six primers that form loops to generate new priming sites and a highly processive DNA polymerase with strand displacement activity (BstDNA polymerase) to separate the DNA strands and amplify DNA with high specificity under isothermal conditions (63–65 °C) within 0.5– 1.5 h. DNA products can be visualized either by gel electrophoresis or by addition of a fluorescent dye to a positive LAMP reaction which produces a color change and allows detection with the naked eye, UV light or an optical color scanning device. Although LAMP is fast and easy to perform with a detection sensitivity comparable to or better than conventional PCR or even approaching that of real-time PCR (Tomlinson et al. 2007) it has not been widely accepted in Europe and has only been used in a limited number of studies for nematode identification, such as for rapid detection of Bursaphelenchus xylophilus from individual nematodes or wood samples (Leal et al. 2015; Kikuchi et al. 2009), Meloidogyne enterolobii in soil and roots (Niu et al. 2012), Radopholus similis from plant tissue (Peng et al. 2012), Tylenchulus semipenetrans in soil (Lin et al. 2016), and Pratylenchus zeae from individual nematodes (Liu et al. 2017). Next to high costs/sample, the main reason why LAMP has not become a prevalent method probably is that it is not well suited for highthroughput detection (Boonham et al. 2008). DNA barcoding offers accurate identification and focuses on strengthening the link between traditional and molecular taxonomy (Bonants et al. 2013; Kiewnick et al. 2014). Its two aims are assigning unknown individuals to species, and secondly enhancing the discovery of new species. Creation of the BBarcode^ involves PCR amplification and sequencing of a conserved gene sequence, such as the mitochondrial Cytochrome oxidase I, II gene and SSU or LSU sequences of the ribosomal DNA. These barcode sequences are then

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aligned to assign individuals to distinct taxa (Holterman et al. 2006, 2009; Vervoort et al. 2012; RybarczykMydłowska et al. 2012; Kiewnick et al. 2014). DNA-Barcoding and direct nucleic acid sequencing (de-novo sequencing) using Next-generation sequencing technologies are becoming more evident in nematode identification (Boonham et al. 2008; Ahmed et al. 2016). However, at the moment, both technologies are state of the art, but very expensive. Their purpose also goes beyond identification of the target nematode in question. They are well suited for phylogeny or evolutionary studies, microbiome research and ecological community studies. Real-time PCR assays for nematode identification and choosing appropriate target regions for assay designs Real-time PCR assays are well suited for detection, identification and even quantification of plant-parasitic nematodes. Real-time PCR assays have been developed for the root-knot nematodes Meloidogyne javanica (Berry et al. 2008), M. chitwoodi and M. fallax (Zijlstra and Van Hoof 2006), M. minor (De Weerdt et al. 2011, Table 3). They have been developed for the lesion nematodes Pratylenchus zeae (Berry et al. 2008), P. thornei (Yan et al. 2012; Mokrini et al. 2014), P. scribneri (Huang and Yan 2017), and for P. penetrans (Mokrini et al. 2013) for species specific identification and differentiation of Ditylenchus dipsaci, D. destructor, and D. gigas (Jeszke et al. 2015), for the identification of the dagger nematode Xiphinema elongatus (Berry et al. 2008), as well as X. index, X. diversicaudatum, X. vuittenezi, X. italiae (Van Ghelder et al. 2015). Real-time PCR assays have also been developed for species level identification of cyst nematodes of the genus Globodera and Heterodera (Gamel et al. 2017; Madani et al. 2005, 2008, 2011; Nakhla et al. 2010; Nowaczyk et al. 2008; Papayiannis et al. 2013; Reid et al. 2010, 2015; Toumi et al. 2015), for the reniforme nematode Rotylenchus reniformis (Sayler et al. 2012) and the pinewood nematode Bursaphelenchus xylophilus (Ye and Giblin-Davis 2013; François et al. 2007; Leal et al. 2007; Cao et al. 2005). While most papers published using real-time PCR and nematodes are based on the noncoding internal transcribed spacer regions ITS-1 or ITS-2 or on the intergenic spacer regions IGS1 or IGS2 located between

the ribosomal genes including the intragenic spacers, only a few papers focus on coding genes or gene regions (Table 3). Coding genes used to design real-time PCR assays for nematode identification are for instance the heat shock protein (Hsp90) (Madani et al. 2011), the β1,4 endoglucanase gene (Mokrini et al. 2013), the MspI satellite DNA (François et al. 2007), sequence characterized amplified (SCAR) based genes (Agudelo et al. 2011), or the cytochrome oxidase subunit I (COI) gene (Kiewnick et al. 2015). Furthermore, Gamel et al. (2017) established new molecular markers based on microsatellite loci for simultaneous detection of three nematode species across two genera. The variable non-coding ITS region or the IGS regions may be appropriate for genus level identification of root-knot nematodes, but due to its intraindividual variation they do not seem suitable for species level identification by barcoding or realtime PCR (Kiewnick et al. 2014, 2015). Although Madani et al. (2008) successfully developed a multiplex real-time PCR assay to detect and differentiate the potato cyst nematodes Globodera rostochiensis and G. pallida and to identify the tobacco cyst nematode G. tabacum in a single assay, they question the sole use of ITS sequences for developing new realtime PCR assays due to its intrinsic heterogeneity (multiple copies of diverged rRNA) and the existence of different haplotypes (Madani et al. 2011). According to the authors molecular identification of Globodera species in unknown samples particularly when extracted from soil and not specifically associated with host plant roots will be tenuous if based solely on the relatively homogeneous ITS base sequence. Recently, it was demonstrated that barcoding regions such as the small (SSU) and large subunit (LSU) of the ribosomal DNA genes are better suited for the development of species-specific real-time PCR assays (Rybarczyk-Mydłowska et al. 2012). The large subunit ribosomal DNA (LSU rDNA, D2D3 region) and the small subunit ribosomal DNA (SSU rDNA, D1-D2 region) are more conserved and thus suitable DNA regions for this purpose due t o l e s s i n t e r-s p e c i es v a r i a t i o n (Ryb a r c z y k Mydłowska et al. 2012). Also suitable and more frequently used for species level identification/ differentiation and barcoding are two short DNA loci of the COI and COII regions. Particularly the cytochrome oxidase subunit I gene is a less variable coding region located in the mitochondrial DNA of

M. chitwoodi M. fallax X. index X. diversicaudatum X. vuittenezi X. italiae D. dipsaci D. destructor D. gigas Bursaphelenchus xylophilus

ITS

P. penetrans P. pseudocoffeae P. kumamotoensis

LSU rRNA (variable regions; D2-D3 expansion segments of nuclear 28S rDNA)

ITS

SYBR Green I

TaqMan LNA probe

M. enterolobii

IGS-2

TaqMan

SYBR Green I

Rotylenchus reniformis TaqMan, SYBR Green TaqMan (Hydrolysis P. crenatus Probe) P. penetrans P. neglectus P. scribneri SYBR Green

ITS-1

Single nematodes

Single nematodes

Single nematodes

Single nematodes, Cysts

Single

Single

Single

Single

Single

Single/Duplex

Single

Jeszke et al. 2015

Koyama et al. 2016

Kiewnick et al. 2015

Huang and Yan 2017

Oliveira et al. 2017

Sayler et al. 2012

1 individual, regardless of Ye and Giblin-Davis 2013 life stage

>5 individuals (about 100 fg DNA) Nematodes suspensions Plasmid or > 3 from 200 g soil specimen/200 g soil sample Single nematodes DNA of 1/128 of a single nematode Suspensions from 100 ml 1 J2/rxn in 1000 other soil nematodes Suspensions from 10 g >2 individuals/10 g soil soil

Nematodes extracted from pinewood logs, wood chips, pine trees, wood-packing materials Single nematodes

Yan et al. 2012

Berry et al. 2008

De Weerdt et al. 2011

Madani et al. 2005

Madani et al. 2008; Nakhla et al. 2010

Nowaczyk et al. 2008

Source

>1 J2 Zijlstra and Van Hoof 2006 >100 fg DNA 1 J2 in 10–1000 other Van Ghelder et al. 2015 nematodes (in a buffer volume ≤ 2 ml)

1 individual/g sterile soil

>1/40 J2

>1 J2

>5 J2

1 J2 Content of 1 Cyst

Content of 1 Cyst

Sensitivity

Larval and adult stages of 1 individual/rxn single nematodes (0.016 ng DNA)

0.5 g soil (Power Soil DNA isolation kit) TaqMan, MGB Probes Single/Multiplex Potato tuber; Flower roots TaqMan Single Larval and adult stages

Single

Single

SYBR Green I

Single

SYBR Green I

Single

SYBR Green I2 TaqMan, MGB Probe

Multiplex

TaqMan

ITS-1

ITS-1

ITS-1

ITS-1

ITS

Meloidogyne spp. Pratylenchus zeae Xiphinema elongatum P. thornei

ITS

ITS

ITS

ITS

Cysts

Single

TaqMan1

ITS

G. rostochiensis G. artemisiae G. rostochiensis G. pallida G. tabacum G. pallida H. schachtii M. minor

Single/Multiplex Matrix

Assay type

Target gene or gene region Nematode species

Table 3 Real-time PCR assays for plant parasitic nematodes and characteristics as described in original publications

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A. besseyi A. fragariae A. ritzemabosi A. subtenuis M. enterolobii

H. avenae H. latipons Bursaphelenchus xylophilus

β-1,4 endoglucanase

SSU

COI

H. glycines (SCN)

SCAR

TaqMan

Single

Single

Multiplex

TaqMan

SYBR Green I

Single

Single

Cysts /single nematodes J2/eggs/females

Single nematodes

Single nematodes, nematode suspensions or wood samples Single nematodes, cysts or floats with cysts

Nematode suspensions from 100 ml soil Single nematodes

LNA hydrolysis probe3 Single SensiFAST Probe Hi-ROX TaqMan

Nematodes suspensions (soil/flower bulbs)

Single

Single nematodes Nematode suspensions

Single

Sensitivity

1 J2 in mix

1 J2

Ye 2011

Agudelo et al. 2011

Gamel et al. 2017

François et al. 2007

1 pg target DNA or 1 J2

1 J2

Toumi et al. 2015

Kiewnick et al. 2015

Rybarczyk-Mydłowska et al. 2012

Mokrini et al. 2014

Mokrini et al. 2013

Madani et al. 2011

Source

1 J2 in 1000 other nematodes 1 individual nematode

>5 individuals in 1000 other nematodes

1 individual nematode

1 individual nematode

Single nematodes or cysts 1 J2/ Content of 1 Cyst

Single

SensiFAST Probe Hi-ROX4 SYBR Green I

TaqMan

Multiplex

Single/Multiplex Matrix

1) Taqman: TaqMan® probe with an Applied Biosystems™ label on the 5’ end and minor groove binder (MGB) and non-fluorescent quencher (NFQ) on the 3’ end. 2) SYBR Green I: SYBR Green I is a DNA double-strand-specific dye. During each phase of DNA synthesis, the SYBR Green I dye, which is included in the reaction mix, binds to the amplified PCR products; the amplicon can be detected by its fluorescence. 3) LNA Hydrolysis Probe: Short (8- to 9-mer) fluorescently-labelled hydrolysis probes (synthesized from locked nucleic acid (LNA)). 4) Applied Biosystems, California, USA

SCAR

G. rostochiensis G. pallida H. schachtii M. arenaria

Microsatellite loci

MspI satellite DNA

COI

P. thornei

β-1,4 endoglucanase

TaqMan

Hsp90

G. rostochiensis G. pallida G. tabacum P. penetrans

Assay type

Target gene or gene region Nematode species

Table 3 (continued)

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Eur J Plant Pathol

organisms. It provides very good discriminatory power between species of closely related organisms (Kiewnick et al. 2014). Real-time PCR assays based on those gene regions (SSU and COI) allowed for detection and correct identification of target nematode DNA in complex DNA backgrounds obtained from nematode suspensions containing thousands of nematode specimens, including closely related species (Rybarczyk-Mydłowska et al. 2012; Kiewnick et al. 2015). The rRNA genes (SSU and LSU) have been preferred in many nematological studies due to the availability of sequences from more conserved regions for universal primer design (Ahmed et al. 2016) and the availability of sequences of these two genes from described taxa in public databases. This also explains the, up to now, low number of assays based on mitochondrial genes. In terms of resolution, however, COI is capable of discriminating between species more than either of the rRNA genes. A combination of the SSU and LSU genes however may significantly improve the resolution, thereby achieving better detection levels (RybarczykMydłowska et al. 2012; Bonants et al. 2013; Kiewnick et al. 2016). All of the above mentioned DNA regions seem suitable for the development of real-time PCR assays for nematode identification. According to Braun-Kiewnick et al. (2016) and Ahmed et al. (2016) the purpose of the study or investigation is important before designing a new assay or using an existing assay. If e.g. the study requires species-level or even population level resolution or quantification, mitochondrial DNA based markers are useful for real-time PCR assays (COI, Nad5, 16S, COI and Nad2). This is necessary for instance for quarantine organisms or import controls. If however, the study requires family or genus level identification (community-level analysis) any marker within the rRNA gene can be suitable (SSU [18S] or LSU [28S]). Gamel et al. (2017) demonstrated how important a thorough evaluation of the sensitivity and specificity with a large number of closely related species and populations of the same species obtained from different geographical regions can be. While most previously developed assays aimed at differentiating between one or two target species with a limited number set of populations tested,

newly developed assays should cover a sufficient number of species and populations to guarantee a high specificity and no potential cross reaction (Rybarczyk-Mydłowska et al. 2012; Kiewnick et al. 2015; Gamel et al. 2017). A prerequisite must be a collection of populations representing possible sources for new introductions and recently described species that are closely related to the target species. Consequently, sufficient validation data should be made available to ensure efficient prevention of new introductions of quarantine species or populations into pest free areas. Conclusion: Why real-time PCR above other methods? For detection, identification and quantification of target nematodes, especially in the case of regulated nematodes and for national reference centres, plant inspection services or plant diagnostic labs, real-time PCR is a very useful diagnostic tool due to its many advantages compared with other techniques (Table 4). Detection is very specific on the species and even population level when appropriate target regions are chosen (Rybarczyk-Mydłowska et al. 2012; Kiewnick et al. 2015; Gamel et al. 2017). Detection in a given matrix such as a nematode suspension extracted from a 100 mL soil sample is also very sensitive (≤ 1–5 individual nematodes) independent of nematode life stage (Tables 3 and 4). Detection is also possible with cysts in floats obtained from soil samples when host plants are not available. Quantification is possible, thus threshold based control strategies can be derived or zero tolerance levels in the case of quarantine organisms. Quantification for environmental monitoring is also possible with high sensitivities when individual numbers range from practically absent to thousands of specimen per kg of soil (Kiewnick et al. 2015). Real-time PCR is easy to perform and results are readily available (≤ 3 h) following DNA extraction when compared to conventional methods. Results can be seen in real-time, during a single PCR run, without the need for post PCR analysis, which saves time and costs (gel documentation). The system can easily be adapted for high-throughput analyses of many samples at a time (96er or 384er format). In the case of high-throughput analysis, no taxonomical experts for nematode identification are needed also saving time until results are obtained.

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