Plant-parasitic nematodes (PPNs) are serious threat as potent soil pathogens that cause 15% to 50% yield loss, which leads to food scarcity and economic crisis worldwide (Fatemi et al., 2023; Jian et al., 2023). Approximately 4,100 PPNs species were identified with many being significant soil pathogens affecting agriculture and horticulture globally (Perry and Moens, 2013). Pratylenchus sp. commonly called root lesion nematodes (RLNs) are polyphagous migratory endoparasites (Khan and Quintanilla, 2023; Khan et al., 2023). Due to the wide host plant range (400 plant species), these Pratylenchus spp. occurrence are widespread globally and are listed as the third most common PPN in soil next to cyst (Heterodera spp., Globodera spp.) and root-knot nematodes (Meloidogyne spp.) (Jones et al., 2013). Also RLNs feed on host plant roots creating a internal channel that aids secondary infection by entry of pathogenic soil-borne fungi and microbes, resulting in early dying of the crop plants (Esteves et al., 2015).
Pratylenchus penetrans is a particularly economically damaging PPN widespread in temperate regions and affecting various crops (Baidoo et al., 2017). Due to the wide host range, P. penetrans infected field were limited for crop rotation raising the risk of increased population densities. Other hand understanding the prevalence of RLNs in the cultivation field becomes crucial for mitigating yield loss and implementing effective nematode management. Number of research reports from the past decade have indicated the potential use of DNA-based polymerase chain reaction (PCR) techniques for the identification of RLNs from genus to species level. The challenges in species-level identification of P. penetrans by conventional methods based on morphological characteristics highlight the necessity for alternative molecular approaches. Most PCR-based methods target regions such as internal transcribed spacer (ITS), mitochondrial cytochrome c oxidase subunit I (mtCOI), 18S, and 28S D2-D3 expansion region of ribosomal DNA as a molecular target to differentiate various species of PPN within the same genus (Fatemi et al., 2023).
The use of species-specific primers (SSPs) in PCR technologies represents a significant advancement in the sensitive and precise detection of P. penetrans. Studies on SSP for the detection of P. penetrans have been reported by conventional PCR method and other species of Pratylenchus genus (Al-Banna et al., 2004; Peetz and Zasada, 2016; Uehara et al., 1998; Waeyenberge et al., 2009). A couple of studies on quantitative PCR (qPCR) assay have adopted SSP combinations for P. penetrans, designed within 28S rDNA to diagnose soil samples (Sato et al., 2010). However, limited research detailing the techniques to identify and quantify P. penetrans, and the described methods are not easily accessible in field samples. Earlier research by (Baidoo et al., 2017) developed a significant tool for diagnosing P. penetrans in soil using the primer set which produced a PCR amplicon of 111 bp (Baidoo et al., 2017). Small size of PCR amplicon poses challenges for differentiation using conventional PCR, during the preliminary screening of soil samples in epidemiological studies.
Addressing this concern, the present study focused on SSPs that produce larger PCR amplicons, allowing for more precise identification and diagnosis of P. penetrans. Also, to check the efficiency of SSP in field scale, the study also aims to include the collection of field samples infested with Pratylenchus sp. and identify the species by its molecular traits, further the targeted P. penetrans and non-targeted Pratylenchus sp. from the cultivation filed were used to check the accuracy and its specificity. This emphasizes the necessity for a more efficient, accurate, reliable, and robust molecular approach to detect and quantify P. penetrans in infested plant and soil samples.
Materials and Methods
Sample collection and nematode isolation
A survey was conducted across various locations in the Republic of Korea. Rhizosphere soils were collected from cultivation field as described (Park et al., 2009). Soil samples were mixed in a poly bag (70 cm × 20 cm) and about 100 cm3 of soil were added to 4 L of tap water for nematode isolation. After 12 h, nematodes were isolated using sieve and funnel techniques (Baermann, 1917; Barker and Davis, 1996; Fatemi et al., 2023). The soil suspensions were sequentially filtered through sieves with pore sizes of 850 μm and 38 μm, and the material remaining on the 38 μm pore size of sieve was transferred to a 100 mL beaker. The extracted liquid was then poured into a Baermann funnel and left for 48 h at 25°C. Finally, the nematodes that settled at the bottom were collected in a clean 15 mL centrifuge tube and examined using a stereomicroscope (LEICA MZ 12 microscope, Leica Microsystems GmbH, Wetzlar, Germany) and the morphological traits were recorded using LEICA DM 5000 B + CTR 5000 microscope system.
DNA extraction, amplification, and phylogenetic analysis
DNA for the molecular identification of the Pratylenchus sp. extracted based on Maafi et al. (2003). Based on the distinct morphology female adult nematodes were put in 100 μL of double distilled water over glass slide and individual nematodes were crushed using the small Wattman filter paper in forceps under stereomicroscope. Transferred to PCR tube containing 10 μL DNA extraction buffer and digested at 65°C (30 min), followed by 95°C (10 min) in thermocycler was used for PCR amplification. D2-D3 expansion region of 28S rRNA gene was amplified using D2A forward/D3B reverse primer sets and PCR products were purified, later subjected to direct sequencing (Subbotin et al., 2006). PPNs (P. penetrans, P. neglectus, P. loosi, Tylenchulus semipenetrans, and Helicotylenchus sp.) sequences were aligned using ClustalW module of Mega 11 version 11.01.13 with other corresponding gene sequences of Pratylenchus sp. acquired from GenBank (https://www.ncbi.nlm.nih.gov) (Supplementary Table 1). Outgroup for the phylogenetic relation taxa were chosen based on our previous literature (Ko et al., 2021). Alignments for each gene fragment and combined alignment containing all genes were separately analyzed with Bayesian inference performed using MrBayes 3.2.7 (Ronquist et al., 2012).
Nematode preparation and genomic DNA extraction from nematodes and soil
PPNs species isolated from field samples and identified were used in this study (Table 1, Supplementary Table 2). The P. penetrans used in the study were from perilla (Perilla futescens Britten var. Japonica Hara) cultured in a greenhouse by Crop Protection division at NAAS, Rural Development Administration, South Korea (Ko et al., 2021). Further DNA from 100 hand-picked nematodes from greenhouse sample and single nematode from field soil were used for molecular identification with slight modifications in the method described (Al-Banna et al., 2004). Briefly, the nematodes in extraction buffer were frozen with liquid nitrogen, homogenized in a sterile mortar, and then extracted by chloroform:isoamyl alcohol (24:1) and phenol:chloroform:isoamyl alcohol (25:24:1) as described (Al-Banna et al., 2004). For PCR and qPCR protocols optimization, including limit of quantification and limit of detection using standard curve, the hand-picked nematodes as were inoculated in the sterile autoclaved soil (0.5 g) and the genomic DNA from the inoculated soil and the field soil samples were extracted using kit method as per the manufacturers protocol (Norgen Biotek Corp., Thorold, ON, Canada) (Gorny et al., 2019).
Design and development of species-specific primer
The DNA sequences of field isolates and the reference targeted P. penetrans and non-targeted Pratylenchus sp. sequence from National Center for Biotechnology Information (NCBI) GenBank were subjected BLAST algorithm (http://www.ncbi.nlm.nih.gov/) and aligned by ClustalW module of Mega 11 (version 11.01.13). The putative conserved DNA sequences of P. penetrans, which diverged from other Pratylenchus sp., were identified using DNASTAR Lasergene software (DNASTAR, Madison, WI, USA). SSPs are designed using the Integrated DNA Technologies online software (https://sg.idtdna/pages). From 12 primer sets designed and screened for specificity and efficiency to amplify P. penetrans in different samples using conventional PCR. Finally, sense and antisense primers used in this study were D-Pp7F (5′-CCTAGCTTGCAAGCAACAATGTT-3′) and D-Pp12R (5′-TTTACGCCGAGAGTGGGATTG-3′). Five different P. penetrans isolates, six other Pratylenchus sp., three Heterodera sp., and one Meloidogyne sp. were used to assess the specificity of the SSPs (Table 1). Non-target nematodes, such as Helicotylenchus sp., Heterodera sp., and Meloidogyne sp., commonly found in radish, cabbage, and potato fields, served as controls in the study. The detection efficiency of the SSP in soil was evaluated using fine-textured sandy-loam soil, and the DNA from different population densities P. penetrans inoculated in soil were used. Varying numbers of P. penetrans were inoculated into 0.5 g of sterile autoclaved soil, and DNA was isolated as previously described. All experiments were performed in triplicates to confirm the repeatability and robustness of the SSPs (Baidoo et al., 2017).
Validation of qPCR assay
D-Pp7F and D-Pp12R primer sets in qPCR assay for identifying P. penetrans were performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific Korea Co., Ltd., Seoul, Korea). The qPCR SYBR premix of AccuPower 2× GreenStar qPCR Master Mix (Bioneer, Daejeon, Korea) along with primer concentration of 5 pM and 1 μL of DNA template for each reaction (20 μL PCR reactions) as per the manufacturer’s protocol. Briefly, PCR conditions included an initial denaturation at 95°C for 15 min, and 40 cycles comprising 95°C denaturation for 15 s, 60°C annealing for 30 s, and extension at 72°C for 30 s, followed by default melt curve analysis. The qPCR results were analyzed and interpreted using AccuSEQ Real-time PCR Software (Sato et al., 2011).
Sensitivity and standard curve validation based on DNA concentration
DNA from 2,000 nematodes were serially diluted (log5) with ddH2O representing the concentration range from 200 ng to 0.0005 ng respectively. Further qPCR assay used serially diluted DNA as template and double distilled H2O as no template control (NTC). Similar pattern of DNA dilution (log10) was made from DNA (150 ng) of 2,000 P. penetrans inoculated in 0.5 g of autoclaved soil (concentration range from 150 ng to 0.0015 ng) were performed in triplicates. A standard correlation plot was generated by plotting the obtained Cq values from each dilution level against the respective logarithmic starting quantity. The serially diluted DNA used for qPCR also served as the DNA template to check the limit of detection in conventional PCR (Chowdhury and Yan, 2021).
Validation of standard curve based on nematode density
The relative Cq values from the different population densities of P. penetrans in soil were determined in order to validate and correlate SSP qPCR assay. This assay included varying numbers of P. penetrans nematodes, ranging from 1 to 500 (1, 5, 10, 50, 125, 250, and 500 nematodes), introduced into 0.5 g of autoclaved sterile soil and the DNA was extracted as mentioned earlier. The resulting Cq values were graphed against the logarithm of the number of P. penetrans to determine amplification efficiency.
Sensitivity of SSP to detected P. penetrans in field soil samples
The efficiency of the D-Pp7F/D-Pp12R primer sets in detecting and quantifying P. penetrans in field soil samples was determined using qPCR assays. A total of 15 soil samples from different cultivation fields (potato, radish, cabbage, etc.) that were naturally infested with P. penetrans and other PPN were collected (Table 1). Approximately 100 cm3 of soil was air-dried to remove moisture, and large particles such as stones, roots, and debris were removed before homogenization with a porcelain mortar and pestle to achieve a smooth texture. Further, homogenized soil was taken for genomic DNA extraction using commercial soil DNA isolation kit (Norgen Biotek Corp.), followed by the manufacturer’s protocol as mentioned. The reliability of the experiments was checked by three biological and technical replicates. The isolated DNA from the field soil samples was used for conventional PCR and qPCR amplification in three technical replicates. DNA isolated from sterile soil without P. penetrans inoculation served as a control in the PCR reactions, while DNA of P. penetrans isolated from perilla plants and soil was used as a positive control in this experiment (Baidoo et al., 2017; Yan et al., 2012).
Results
Molecular characterization
The infestation of Pratylenchus sp. in the cultivation field was confirmed by the molecular identification by the results of phylogenetic analysis (Fig. 1). Briefly, the soils from the cultivation fields were found to infest with Pratylenchus sp. and the respective isolates after the species identification were submitted to the NCBI database as nematode isolates (Supplementary Table 2). The isolate included five P. penetrans, two P. neglectus, one P. loosi, one P. vulnus, and one Tylenchulus semipenetrans respectively. All the DNA sequences had clustered along with respective species retrieved from GenBank. The obtained results were highly correlated with the morphometric characteristic identification and BLASTN analysis (Supplementary Result 1).
DPp7F12R SSP: specificity assay
The alignment of D2-D3 expansion segment of 28S rRNA from P. penetrans mined a unique putative conserved DNA sequence of polymorphism, was used in SSP for the PCR-based detection and quantification of P. penetrans in soil samples. The SSP consisted of a sense primer DPp7F (GC: 38%) and antisense primer DPp12R (GC: 52%). This primer set amplified a PCR product of 300 bp specifically within the D2-D3 expansion region. Homology and specificity check using BLAST algorithm result showed no matches with non-targeted nematode species of genus Pratylenchus sp. and other plant-parasitic or free-living nematodes. In addition, SSP doesn’t match with the any of other soil microbes, whereas the homology hits exhibited a 100% similarity only with P. penetrans (E value = 0.001). Initial 12 primer sets were designed and all the primer sets were evaluated for its specificity towards P. penetrans detection using conventional PCR. The obtained result has screened only four primer sets that possess the specificity to detect the targeted P. penetrans (Supplementary Table 3). All four primer sets and their combinations were examined for their efficiency in amplifying the DNA template of P. penetrans along with the non-targeted P. neglectus. The conventional PCR significantly amplified DNA of P. penetrans. The potential SSP sets DPp7F12R were selected based on the preliminary PCR test (Supplementary Fig. 1), the optimized annealing temperature and the primer concentration were set as 60°C and 5 pM, respectively (Supplementary Figs. 2 and 3).
Detection sensitivity of SSP by PCR methods
The novel primer set designed for qPCR, based on the D2-D3 expansion region of 28S rDNA of P. penetrans, demonstrated high specificity. It produced a PCR amplicon size of 300 bp, amplifying only the target DNA of P. penetrans and non-targeted other 10 nematode species included in this study. The gel picture of conventional PCR product evidently shows the specificity of SSPs for P. penetrans (Supplementary Fig. 4). The threshold Cq values for the positively amplified P. penetrans isolates obtained from infested agricultural soil ranged from 21.61 to 22.71. This amplification was characterized by a single melt curve peak at 86°C, confirming that a single amplicon was generated using the DPp7F12R primer set (Fig. 2A, Supplementary Fig. 5A). Also, respective Cq values and the amplification details were illustrated (Table 1). These results highlight the effectiveness and precision of the designed primer set in specifically identifying P. penetrans in agricultural samples.
Identification of P. penetrans from nematode-infected soil sample
Furthermore, the detection sensitivity and specificity of the target nematode were assessed through real-time amplification of total genomic DNA isolated from 15 field soil samples infested with P. penetrans and other nematode species. The results demonstrated specific amplification of P. penetrans in the infested soil, with Cq values ranging from 25.84 to 27.82 (Table 1). This amplification corresponded to a single amplicon, indicated by a melt peak at 86°C (Fig. 2B). The qPCR results suggest that the designed SSPs are effective for detecting and quantifying P. penetrans specifically in total DNA extracted from field soils. Furthermore, the sequence information from the amplified PCR products obtained using the DPp7F12R SSPs was analyzed using the BLASTN algorithm. This analysis significantly discriminated and identified the presence of Pratylenchus sp. in the field soils (Fig. 2B, Supplementary Fig. 5B). Obtained qPCR results have highly coincided with the conventional PCR results showing the positive amplifications specifically for P. penetrans (Supplementary Fig. 4). These findings confirm the assay’s capability for accurate identification and quantification of P. penetrans, reinforcing its potential as a reliable tool for monitoring nematode infestations in agricultural settings.
Robustness and quantification threshold in detection of P. penetrans
The standard curve was generated from the serial dilution (1:5 dilution) of genomic DNA extracted from 2,000 P. penetrans, with DNA concentrations (200 ng to < 0.01 ng). The assay produced a clear and distinct qPCR amplification curve with a melt peak at 86°C, indicating a single and specific PCR amplicon using the DPp7F12R, and demonstrated the robustness of the SSPs. Further, clear amplification was observed across all dilutions, with a profound efficiency of 97% for qPCR. Linear curve indicated a strong negative linear regression between Cq values and the corresponding dilutions (y = 1.533x + 27.9276, R2 = 0.97263, P < 0.0001), as shown (Fig. 3A, Supplementary Table 4, Supplementary Fig. 6A). Limit of quantification for P. penetrans using DPp7F12R was determined to be around 0.002 ng, indicating the qPCR assay is suitable for quantifying very low numbers of P. penetrans in the soil. Further, the results obtained using the soil DNA, serial dilution (1:10) reflected a significant linearity (y = 1.91546x + 29.76379, R2 = 0.955575, P < 0.0001) demonstrated the robustness of SPPs as shown (Fig. 3B, Supplementary Fig. 6B). Also, the respective gel pictures showed the amplified PCR products from the hand-picked P. penetrans DNA. And the field soil DNA containing P. penetrans mentioned respective template DNA concentrations used in qPCR were shown (Fig. 3). When compared with the pure culture DNA and soil DNA the limit of quantification (LOQ) of P. penetrans were almost same around 0.002 ng respectively.
Validation of linearity of qPCR assay based on population density standard plot
The population standard curve of Cq values against varying populations of P. penetrans inoculated in 0.5 g of sterilized soil (1, 5, 10, 50, 125, 250, and 500 P. penetrans). A strong linearity in the relationship, represented by y = 4.48607x + 39.54108, with an R2 = 0.9924 (P < 0.001). Therefore, increase in population of P. penetrans would reflect in respective decrease in the Cq values, ranging from 26.47 to 39.09. Also, there was no amplification in the non-inoculated sample controls, confirming the specificity of the assay for P. penetrans (Fig. 4, Supplementary Table 5, Supplementary Fig. 7) and ensuring that the results obtained from inoculated samples are accurate and indicative of true P. penetrans populations.
Limit of detection by single nematode DNA
The threshold sensitivity of SSPs to detect P. penetrans in soil estimated by plot generated with the Cq values against the (1:10) serial dilution of DNA from 0.5 g of soil inoculated with single P. penetrans (Fig. 5, Supplementary Table 6, Supplementary Fig. 8). The Cq values from 13 to 22 respectively were recorded based on the concentrations of DNA template and reflected a strong correlation mentioning the significant reliability and sensitivity of DPp7F12R down up to 0.0001 ng of genomic DNA extracted from the soil (y = 2.08396x + 15.05914, R2 = 0.97876, P < 0.0001).
Discussion
RLNs are distributed worldwide, and their infestation has been reported with severe impact on economic loss (Jian et al., 2023). Numerous research studies reported the wide host range of RLNs more than 400 plants and listed as the third most critical soil pathogen, following root-knot and cyst nematodes (Jones et al., 2013). Reports indicate an economic threshold of 1-2 RLNs per gram of infested soil for radish and potato (Castillo and Vovlas, 2007). A recent survey by our research team (Nematology Laboratory, Crop Protection Division, Rural Development Administration, Republic of Korea) for epidemiological study accounted for 147 soil samples across the country and found that a few samples were infested with RLNs (data not shown). The present investigation included some of the infested soil samples for the molecular identification of Pratylenchus sp. using the D2-D3 region of the 28S rDNA gene, as detailed in the meta-data (Table 1). The molecular characteristics by phylogenetic analysis provided evidence of P. penetrans in the radish and potato fields (Fig. 1, Supplementary Fig. 9, Supplementary Results 1 and 2). In the results, the major clades in the phylogenetic tree significantly correlate with the findings of Subbotin et al. (2008). Along with the targeted P. penetrans, a few other Pratylenchus sp. were identified and used as non-target controls in the SSPs study (Table 1). Considering the adverse impact of P. penetrans on cultivated crops, the present study developed a robust conventional and qPCR assay to detect and discriminate different RLNs and non-targeted PPNs directly from soil samples. The SSPs mined from D2-D3 region were used to quantify P. penetrans in the soil, thereby overcoming the time consumed by conventional identification. qPCR assay is considered as a molecular tool in plant pathology and nematode pest management diagnostics, bypassing potential challenges linked to precise identification and quantification of PPN populations (Gorny et al., 2019). We report the development of a highly sensitive conventional and qPCR assay involving SSPs (DPp7F12R), which is highly conserved for P. penetrans. Other studies have reported SSP for P. penetrans with a lower PCR amplicon size (111 bp), poses challenges in conventional PCR and phylogenetic construction for molecular identification (Baidoo et al., 2017; Sato et al., 2011). In contrast, the DPp7F12R, thereby circumventing (300 bp PCR amplicon) the complications associated with the previously reported SSPs. As the result, this method reduces processing time and is cost-effective, making it useful for accurately detecting and measuring P. penetrans in studies with large sample sizes. From the obtained results, this qPCR assay efficiently quantified P. penetrans directly from field soil (Fig. 2, Supplementary Fig. 5). The SSPs can potentially detect down to a single nematode in the soil samples, demonstrating its sensitivity. According to earlier reports, the D2-D3 region of the Pratylenchus genus is highly stable, and the SSPs developed can detect and quantify P. penetrans populations from different geographical locations (Al-Banna et al., 2004). Similar studies have suggested that SSPs mined from the ITS region of the Pratylenchus genus exhibit variations in gene length due to high polymorphism across different populations (Orui, 1996; Waeyenberge et al., 2009). Thus, ITS has limitations in developing SSPs for P. penetrans and may not be suitable for quantification across different geographical locations (Baidoo et al., 2017). Regression analysis of the P. penetrans population showed a significant correlation (P < 0.001), suggesting the SSPs capability to detect and quantify down to a single target P. penetrans in both 0.5 g of artificially inoculated and naturally infested soil samples. Similar strong regression correlations have been recorded for the accurate quantification of P. neglectus in soil samples (Yan et al., 2008, 2013). Other primer sets, such as PP1/PP2, reported earlier, were not capable of detecting and quantifying P. penetrans in soil samples collected from different locations (Huang and Yan, 2017). The limit of detection in the qPCR test using species-specific primers was the DNA from a single nematode diluted to 1 in 1,000 (10−3). The sensitivity of DPp7F12R was found to be comparable to previous findings for other SSPs used for nematode species identification by conventional and real-time PCR (Huang and Yan, 2017). Therefore, the investigated primer set can detect and identify less than a single nematode using both PCR methods described, with several other research studies, such as those by Berry et al. (2007), demonstrating the detection of less than a single P. zeae and P. scribneri by Huang and Yan, respectively. Thus validated DPp7F12R primers can accurately discriminate P. penetrans from other genetically closely related Pratylenchus species in agricultural soil samples in South Korea. Further, SSPs do not amplify other species of PPNs in soil (Table 1). Earlier report mentioned the possibility of more than one species of Pratylenchus genus can infest the same cultivation area; in such cases, the present primer set can efficiently be used for molecular identification and quantification purposes. Cq values obtained from the pure culture was 23.4, indicating a reduction in the hindrance caused by polymerase inhibitors. This finding was similar to previous reports, on SPPs to quantify M. hapla from soil using qPCR assay (Gorny et al., 2019). High cycle threshold (Ct) values were recorded as a result of delayed amplification, thought to be significantly influenced by PCR inhibitors in the DNA extracted from soil and further, the present issue can be minimized by using polyvinylpolypyrrolidone in DNA extraction protocol. In continuation, limit of detection revealed varying population densities in the qPCR assay (1 to 500 nematodes), with DNA from a single nematode amplified at a Cq of 39.21 and a single melt peak at 86°C, indicating the presence of polymerase inhibitors along with a low quantity of target DNA template in the PCR samples. Additionally, these results highlight the quantification of P. penetrans in inoculated soil, suggesting a significant detection limit. Based on the evaluated data, the DPp7F12R SSPs-based qPCR can be considered reliable for quantifying P. penetrans populations, showing varying fold changes. The SSPs developed in this study would be an invaluable tool for the molecular detection, identification, and quantification of P. penetrans, even at very low densities in field soil samples. Therefore, this qPCR assay can serve as a significant diagnostic method with high accuracy and reliability for monitoring P. penetrans infestations in cultivation areas, making it a valuable asset in soil pest management systems.
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