Rapid diagnosis tools for detection of root-knot nematodes play an important role in the disease control and eradication programme. Recombinase polymerase amplification (RPA) assays were developed targeting the IGS rRNA gene of the pacara earpod tree root-knot nematode, Meloidogyne enterolobii. The RPA assays using TwistAmp® Basic and TwistAmp® exo kits allowed detection of M. enterolobii from gall tissues and crude nematode extracts of all stages of target species without a DNA extraction step. The results of real-time RPA assays using a real-time fluorescent detection of a series of crude nematode extracts showed reliable detection with sensitivity of 1/10 of a second-stage juvenile in a RPA reaction tube after 15-20 min. The RPA assay provides affordable, simple, fast and sensitive detection of M. enterolobii.
Nearly 100 valid species of root-knot nematodes have been described to date (Hunt & Handoo, 2009). They include agriculturally important species Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949, M. javanica (Treub, 1885) Chitwood, 1949, M. arenaria (Neal, 1889) Chitwood, 1949, and M. hapla Chitwood, 1949, which are distributed worldwide in agricultural areas. The pacara earpod tree root-knot nematode, M. enterolobii Yang & Eisenback, 1983, is another agricultural pest but with more limited distribution. Meloidogyne enterolobii is a tropical or subtropical nematode and has a broad host range, including cultivated plants and weeds. It is now considered to be one of the most important root-knot nematode species as it displays wide virulence and can develop on crop genotypes carrying several root-knot nematode resistance genes in economically important crops, including tomato, soybean, cowpea and peppers, and has a higher pathogenicity and reproductive potential than either M. incognita or M. arenaria (Anonymous, 2014, 2016). Consequently, this species was added to the European and Mediterranean Plant Protection Organization A2 list of pests recommended for regulation as quarantine pests. The nematode is recorded from several countries of Africa, Asia, Central America and the Caribbean, North and South Americas. In Europe it has been reported in a few glasshouse crops in France and Switzerland (Yang & Eisenback, 1983; Kiewnick et al., 2009; Anonymous, 2016). In North America, M. enterolobii has been found in the USA in Florida (Brito et al., 2004), North Carolina (Ye et al., 2013), and in Mexico (Ramírez-Suárez et al., 2014).
Presently, M. enterolobii is considered under the highest A rating in California as a pest that has not been known to occur or is under official control in the State of California. This nematode has never been detected in regulatory samples collected during the California Department of Food and Agriculture’s (CDFA) nematode control and phytosanitary certification programmes or through state-wide nematode surveys of host plants grown in agricultural production sites and nurseries in California. California has a suitable climate and hosts for M. enterolobii and, if introduced, the species is likely to become widespread (Chitambar, 2016).
The use of DNA analysis is now essential for accurate identification of root-knot nematodes at the species level. Several molecular techniques have been developed for identification of M. enterolobii, including sequencing of the IGS rRNA gene (Blok et al., 1997), the intergeneric mitochondrial region between the COII and lRNA genes (Blok et al., 2002; Brito et al., 2004), ITS rRNA (Brito et al., 2004), COI, COII, partial 28S rRNA and 18S rRNA genes (Kiewnick et al., 2014), conventional PCR with specific primers (Long et al., 2006; Tigano et al., 2010), loop-mediated isothermal amplification (LAMP) (Niu et al., 2012; He et al., 2013) and real-time PCR with specific primers (Kiewnick et al., 2015; Braun-Kiewnick et al., 2016).
Recently, a number of nucleic acid amplification techniques were developed that do not require the use of thermal cycling equipment (Craw & Balachandran, 2012). Several novel diagnostic applications have been published using the recombinase polymerase amplification (RPA) technique (Piepenburg et al., 2006; James & Macdonald, 2015; Daher et al., 2016). RPA represents a hugely versatile alternative to polymerase chain reaction (PCR). RPA uses a highly efficient displacement polymerase that amplifies a few copies of target nucleic acid in 20 min at a constant temperature (37-42°C). The advantages of RPA include highly efficient and rapid amplification and a low constant operating temperature. RPA products can be carried out by using fluorescent probes in real time or detected by agarose gel electrophoresis or lateral flow assay. Several tests using RPA have demonstrated high sensitivity and specificity for detecting small amounts of various plant viral, bacterial, and fungal DNA (Miles et al., 2015; Lau et al., 2016; Londoño et al., 2016; Babu et al., 2018). To my knowledge, there are no publications describing any RPA test for the detection of plant-parasitic nematodes. This article describes the development and validation of RPA assays for rapid and specific detection of M. enterolobii. The study included three stages: i) testing and selection of RPA specific primer combinations; ii) validation of sensitivity and specificity for real-time RPA assay; and iii) practical evaluation of real-time RPA assay with field samples.
Materials and methods
Two isolates of M. enterolobii were obtained from Drs J. Brito (Division of Plant Industry, Florida Department of Agriculture and Consumer Services (FDACS), Gainesville, FL, USA) and P. Roberts (University of California Riverside (UCR), Riverside, CA, USA) for RPA assay development. Second-stage juveniles (J2) and males from root and soil samples were extracted using the centrifugal-flotation method (Coolen, 1979). The ITS and IGS rRNA genes were sequenced from each isolate to confirm its identity. DNA of several root-knot nematodes: M. arabicida López & Salazar, 1989 (Costa Rica), M. ethiopica Whitehead, 1968 (Brazil), M. hispanica Hirschmann, 1986 (Brazil), M. izalcoensis Carneiro, Almeida, Gomes & Hernández, 2005 (El Salvador), M. morocciensis Rammah & Hirschmann, 1990 (Brazil), and M. paranaensis Carneiro, Carneiro, Abrantes, Santos & Almeida, 1996 (Brazil), provided by Dr Valerie Williamson (University of California Davis (UCD), Davis, CA, USA); M. arenaria, M. floridensis Handoo, Nyczepir, Esmenjaud, van der Beek, Castagnone-Sereno, Carta, Skantar & Higgins, 2004 and M. javanica (all from Florida, USA) provided by Dr J. Brito (FDACS); and M. incognita and M. hapla (all from California, USA) from the CDFA collection were also used in this study-for-specificity experiment. These nematode samples were previously identified by morphological and molecular methods (Pagan et al., 2015).
Eight DNA samples named here as field samples and identified as positive for the presence of M. enterolobii were provided by Dr W. Ye (Agronomic Division of the North Carolina Department of Agriculture and Consumer Services (NCDA), Raleigh, NC, USA) from North Carolina and Dr J. Brito (FDACS) from Florida, USA. The nematode species from field samples were identified by sequencing of the NAD5 barcode gene (Janssen et al., 2016), partial 28S rRNA gene (Tenente et al., 2004) or isozymes (Brito et al., 2008). These samples were used to evaluate and validate the practical application of RPA assay.
Free-living and plant-parasitic nematodes were extracted using the centrifugal-flotation method from several field samples collected in California, and their extracts were used as a background non-target DNA in an experiment of real-time RPA assays.
Two methods were used for destruction and homogenisation of nematodes. The first method included cutting J2 or males using a dental stainless-steel needle into 15 μl (samples with proteinase K) or 20 μl (samples with crude nematode extract) distilled water on a glass slide under a binocular microscope. Cut nematodes in water were transferred into a 0.2 ml Eppendorf tube. In the experiment with extraction using proteinase K, 3 μl of this enzyme (600 μg ml−1) (Promega) and 2 μl of 10× PCR buffer (Taq PCR Core Kit, Qiagen) were added to each tube. The tubes were incubated at 55°C (30 min) and 95°C (10 min) consecutively. The second method included a homogenisation of different stages of nematodes in a Screw Cap microtube (Sarstedt) with 250 μl of distilled water and a glass bead (5 mm) using a Mini BeadBeater (Biospec Product) (speed: 42; time: 1 min) (Fig. 1). Several different extracts in a total volume of 250 μl of water were prepared: i) five J2; ii) 50 J2; iii) single female; and iv) gall tissue with one or more females and egg masses. Different dilutions were prepared from nematode extracts for sensitivity assays. Crude extract of non-target and non-root-knot nematodes were obtained from homogenisation of several hundred soil free-living and plant parasitic nematodes in 250 μl of water.
RPA primer design and testing
A total of 15 RPA primers specific to M. enterolobii and universal to root-knot nematodes were manually designed based on species sequence polymorphisms in the IGS rRNA gene. Primers were synthesised by Integrated DNA Technologies. The primers were firstly screened in different combinations using the TwistAmp® Basic kit (TwistDx). Reactions were assembled according to the manufacturer’s instructions. The lyophilised reaction pellets were suspended in 29.5 μl of rehydration buffer, 2.4 μl of each forward and reverse primers (10 μM), 1 μl of DNA template obtained using the first method of nematode extraction and 12.2 μl of distilled water. For each sample, 2.5 μl of 280 mM magnesium acetate were added and mixed. Tubes were incubated at 37°C (4 min) in a MyBlock Mini Dry Bath (Benchmark Scientific) then inverted 8-10 times to mix, briefly centrifuged and returned to the incubator block (37°C, 56 min). Amplification products were purified with QIAquick PCR Purification Kit (Qiagen). Six μl of product were run in a 1% TAE-buffered agarose gel (100 V, 40 min) and visualised with ethidium bromide. Amplification products were directly sequenced by Quintara Biosciences using amplification primers.
Real time RPA assay
Three TwistAmp® exo probes were initially designed according to the manufacturer’s instructions. Probes were synthesised by Biosearch Technologies. Probes were tested in the same conditions as described below and only one probe was selected for the assay (Fig. 2) based on best amplification results. The real time detection of RPA assay products was accomplished using TwistAmp® exo kit (TwistDx). The reaction mixture for each RPA assay contained 2.1 μl of each forward and reverse primers (10 μM), 0.6 μl (10 μM) of exo probe, 1 or 5 μl template and water bringing the volume to 47.5 μl per sample. DNA templates were obtained using the first and second method of nematode extraction. Magnesium acetate solution provided with the kit in a volume of 2.5 μl was added to the lid of the tube and the lids were closed carefully. The tubes were briefly centrifuged to spin the magnesium acetate into the reaction mixture and initiate reactions simultaneously. The reaction tubes were incubated at 37°C for 4 min in a MyBlock Mini Dry Bath, then inverted vigorously 8-10 times to mix, and briefly centrifuged. The tubes were immediately placed in Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System to incubate at 39°C for 25-30 min (Fig. 1). The fluorescence signal was monitored in real-time, and measured every 20 s (cycle) using the FAM channel. Positive control with M. enterolobii and negative control without any nematode DNA were included in runs. Three replicates of each variant in at least two runs were performed for sensitivity and specificity experiments.
Real time RPA assay of field samples
To evaluate and validate the practical application of the RPA assay, samples containing DNA of root-knot nematodes obtained from nematological field surveys in California, Florida and North Carolina, commercial samples shipped to the CDFA from Florida and other states, and control samples containing M. enterolobii DNA were tested. Two replicates were performed for this experiment.
Among several combinations of 15 primers, the forward species-specific primer for M. enterolobii paired with the reverse universal tropical root-knot nematode group RPA primer was found to be optimal (Table 1; Fig. 2) and used for the assay. These primers are modified versions of the primers proposed by Long et al. (2006) for conventional PCR. The primer set reliably and specifically amplified the target gene fragment of the IGS region. Using the TwistAmp® Basic kit, the RPA generated a strong band with ca 240 bp in length and a weak band with ca 500 bp in length on a gel (Fig. 3). The amplicon of ca 240 bp had a unique sequence for M. enterolobii that was confirmed by direct sequencing of this product. Sequence identity of a weak band was not identified. Non-specific weak bands were also observed for some samples with other root-knot nematode species (data not shown). Analytic detection of all nematode stages of M. enterolobii was confirmed with several samples from Florida provided by the FDACS and a sample of unknown origin provided from the UCR using the TwistAmp® Basic kit (data not shown).
Real time RPA detection and sensitivity
In the results of analysis of experiment series, which included 25 runs containing positive controls and negative controls with water and non-target DNA, the threshold level for the reliable M. enterolobii detection was established as equal to 30 cycles (= 10 min) and baseline to 500 000 (ΔRn) fluorescence level with the TwistAmp® exo kit using Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System. Samples that produced an exponential amplification curve above the threshold were considered as positive for M. enterolobii and below the threshold were considered as negative. Detection of all nematode stages of M. enterolobii was confirmed with samples provided by FDACS and UCR using the TwistAmp® exo kit (Fig. 4A).
The sensitivity of the assay was tested in variants with serial dilutions (1, 1:2, 1:10 and 1:100) of crude nematode extractions obtained from extracts with five J2 and 50 J2 per 250 μl of water. At least three replicates of each variant were included in runs. The reliable detection level of M. enterolobii for the samples was estimated as 1/10 of a J2 in a RPA reaction tube (Fig. 4B). The detection was confirmed in the presence of non-target background crude nematode extract (Fig. 4C). The assay allowed detecting a crude extract of one female per 250 μl of water in a sample or 1/50 of female in a RPA reaction tube (Fig. 4D). This method also detected nematodes in an extract from plant gall tissues containing up to single females with an egg-mass per 250 μl of water (Fig. 4D).
Real-time RPA specificity
The RPA assay was tested for specificity using DNA extracted from other related root-knot nematodes. These nematodes included: M. arabicida, M. arenaria, M. ethiopica, M. floridensis, M. hapla, M. hispanica, M. incognita, M. izalcoensis, M. javanica, M. morocciensis and M. paranaensis. The experiment was repeated in two runs. The RPA showed high specificity to M. enterolobii only and no cross-reactions were observed against other root-knot nematode species (Fig. 4E).
Real time RPA assay of field samples
The assay was validated using eight DNA extracts from field samples from Florida and North Carolina containing M. enterolobii alone or in a mixture with other nematode species and 10 DNA extracts from field samples containing J2 of other non-target root-knot nematode species. The RPA assay with several runs showed strong signals for the presence of M. enterolobii in eight field samples with this nematode and signals below the threshold level in all samples in which DNA of this nematode was absent (Fig. 4F).
In this study, an RPA method for the affordable, simple, fast and sensitive detection of M. enterolobii is developed and described. RPA assay was developed using Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR. This method allows simultaneous analysis of samples in a 96-well plate. Currently, the cost of a RPA reaction is estimated to be in the range US$4.3-5.5 (Daher et al., 2016; Londoño et al., 2016), and this cost is higher than for PCR. However, the total price of detection for single samples by RPA may be comparable with PCR if lower labour costs due to the shorter time of RPA assay and an exclusion of time-consuming DNA extraction procedure are considered.
The real-time RPA assay requires establishing the baseline value and threshold level for M. enterolobii detection. Because of the presence of a fluorescent background, a baseline for reliable detection of a target nematode had to be experimentally established. The threshold level in this study was estimated based on analysis of the fluorescent background of negative samples in 25 independent runs and fluorescent signals of positive samples. Two parameters correlating to the amount of template were considered: the onset time of detectable amplification and fluorescence intensity. In the Flex Real-Time PCR System, threshold cycle was estimated as 30 cycles (=10 min) and baseline value was equal to 500 000 (ΔRn) fluorescence level. These values may vary across other real-time PCR instruments.
RPA has good flexibility to be adapted to various detection systems. For example, instead of using a cost-effective heat block, portable fluorescence readers (ESEQuant, Qiagen; Genie, OptiGene; T8-ISO, TwistDX; and others) can be used, which are simpler and less expensive than a real-time PCR machine and can be run on battery power for use in the field (James & Macdonald, 2015; Daher et al., 2016).
RPA has some important advantages over PCR methods, the first being that it uses crude nematode extract for the analysis instead of DNA extracts, which are required for PCR assays. The second advantage is that results are available in 15-20 min for RPA vs 1.5-3.0 h for PCR assays. The RPA assay has also some important advantages over other isothermal technique such as LAMP, because it uses shorter primers and a shorter run time than LAMP technology. Real-time RPA detection is performed with fluorescent probes in a closed-tube assay format, avoiding downstream cross-contamination, which is often problematic with LAMP (Daher et al., 2016). When comparing the practical applicability of RPA, PCR and LAMP techniques, RPA is the easiest to perform and the reagents are very stable.
Although the reliable detection limit of this RPA assay is comparable with those for conventional and real-time PCR (Braun-Kiewnick et al., 2016), there remains the capacity to improve this parameter by adjusting 250 μl of water to a smaller sample volume before destruction and homogenisation of nematodes, as well as by increasing the template amount in a RPA reaction mixture. Nevertheless, other improvements could be made to the technique. In the described approach, we tested crude nematode and plant gall extracts for RPA. Testing of nematode extracts directly from soil samples with quantification could be proposed as a future development of this method. However, detailed analysis of some possible factors and inhibitors influencing the efficiency of RPA should be carefully tested and considered. Thus, the RPA assays have great potential for being applied and implemented in testing programmes on root-knot nematodes in diagnostics laboratories, contributing a timely and reliable identification of these pests and thereby minimising the risk of their spread.
The author thanks Drs J. Brito (FDACS), W. Ye (NCD), P. Roberts (UCR) and V. Williamson (UCD) for providing root-knot nematode samples, P. Woods (CDFA) for assistance with Flex Real-Time PCR System, and Drs C. Blomquist (CDFA), J.J. Chitambar (CDFA) and anonymous reviewers for critical reading and correction of the manuscript. This work was supported by a FarmBill grant (16-8506-2005-CA).
Braun-KiewnickA.ViaeneN.FolcherL.OllivierF.AnthoineG.NiereB.SappM.van de VossenbergB.ToktayH. & KiewnickS. (2016). Assessment of a new qPCR tool for the detection and identification of the root-knot nematode Meloidogyne enterolobii by an international test performance study. European Journal of Plant Pathology 14497-108. DOI: 10.1007/s10658-015-0754-0
BritoJ.A.KaurR.CetintasR.StanleyJ.D.MendesM.L.McAvoyE.J.PowersT.O. & DicksonD.W. (2008). Identification and isozyme characterization of Meloidogyne spp. infecting horticultural and agronomic crops, and weed plants in Florida. Nematology 10757-766. DOI: 10.1163/156854108785787253
ChitambarJ. (2016). Meloidogyne enterolobii Yang and Eisenback 1983. http://blogs.cdfa.ca.gov/Section3162/?p=2376.
JanssenT.KarssenG.VerhaevenM.CoyneD. & Bert W. (2016). Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution. Scientific Reports 622591. DOI: 10.1038/srep22591
KiewnickS.HoltermanM.van den ElsenS.van MegenH.FreyJ.E. & HelderJ. (2014). Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives. European Journal of Plant Pathology 14097-110. DOI: 10.1007/s10658-014-0446-1
KiewnickS.JürgE.FreyJ.E. & Braun-KiewnickA. (2015). Development and validation of LNA-based quantitative real-time PCR assays for detection and identification of the root-knot nematode Meloidogyne enterolobii in complex DNA backgrounds. Phytopathology 1051245-1249. DOI: 10.1094/PHYTO-12-14-0364-R
TiganoM.de SiqueiraK.Castagnone-SerenoP.MuletK.QueirozP.dos SantosM.TeixeiraC.AlmeidaM.SilvaJ. & CarneiroR. (2010). Genetic diversity of the root-knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava-damaging species. Plant Pathology 591054-1061. DOI: 10.1111/j.1365-3059.2010.02350.x