Root-lesion nematodes (Pratylenchus spp.) are one of the impo1tant groups of plant-parasitic nematodes. They have a wide distribution and a broad host range, and can cause significant yield suppression in soybean. Recent nematode surveys in soybean fields of N011h Dakota (ND) led to the first detection of a new root-lesion nematode species in ND that had never been repo1ted in the literature. This species is now named as Pratylenchus dakotaensis. Twenty soybean cultivars were evaluated for their reactions to the new species and none of the cultivars tested were resistant, indicating that this nematode can infect and reproduce well on soybean. To manage the new species P. dakotaensis, it is important to have a rapid and sensitive method for detecting this nematode from infested fields. Real-time and conventional PCR assays were developed to detect and identify P. dakotaensis. However, PCR-based molecular diagnosis often requires advanced instruments and software making on-site field detection infeasible. Recombinase polymerase amplification (RP A) is a novel isothermal nucleic acid amplification technology and several studies using RP A have demonstrated high sensitivity and specificity for detecting root-knot nematode and pine wood nematode. However, there are no RP A assays available for detecting root-lesion nematodes. We propose to develop a new RPA assay for rapid detection of the new root-lesion nematode species P. dakotaensis from nematode individuals and also from field soil DNA extracts.

1. Develop a new recombinase polymerase amplification assay for detection of Pratylenchus dakotaensis from nematode individuals; and
2. Develop a new recombinase polymerase amplification assay for rapid and direct detection of P. dakotaensis in DNA extracts from field soils.

1. A new recombinase polymerase amplification assay will be developed for detection of P. dakotaensis from nematode individuals; and
2. A new recombinase polymerase amplification assay will be developed for rapid and direct detection of P. dakotaensis in DNA extracts from field soils.

Updated November 30, 2022:
Develop a New Recombinase Polymerase Amplification Assay for Rapid Detection of the Root-lesion Nematode Pratylenchus dakotaensis from Soil

Principle Investigator: Dr. Guiping Yan

Objectives of the Research

1. Develop a new recombinase polymerase amplification assay for detection of Pratylenchus dakotaensis from nematode individuals.
2. Develop a new recombinase polymerase amplification assay for rapid and direct detection of Pratylenchus dakotaensis in DNA extracts from field soils.

Completed Work

A new recombinase polymerase amplification (RPA) assay was developed to detect a recently identified and named root-lesion nematode species, Pratylenchus dakotaensis, on soybean in North Dakota (ND) from both nematode individuals and DNA extracts from field soils.

Pratylenchus dakotaensis nematodes were extracted from 200 g of soil which was obtained from the maintenance pots of P. dakotaensis in the greenhouse. The extracted nematodes were picked manually to extract DNA from them using proteinase K method. A total of 16 primers (eight forward and eight reverse) were designed from the internal transcribed spacer (ITS)-rDNA sequence of P. dakotaensis (accession number KX889989) acquired from GenBank. These primers were aligned with the sequence of P. dakotaensis along with 19 ITS sequences of other Pratylenchus spp. Two probes were designed to work with these primers according to the instruction of TwistDx company. The specificity of these primers and probes was evaluated in in-silico analysis with the sequences used to design the primers and ITS sequences of 16 other important Pratylenchus spp. collected from GenBank. Primer and probe specificity was predicted in silico by evaluating the primer-template duplex stability (?G) values calculated using the software OligoAnalyzer 3.1.

The primer set (ICF4/ICR3) with probe ICP1 was selected for further experiment since they worked well when combined. TwistAmp basic and exo kits were used and reactions with both kits were conducted at 39.50C for 20 minutes. Threshold level with the exo kit (real-time detection) was determined to distinguish the positive amplification from the negative sample. The basic kit (end-point detection) required a purification step to observe the bands properly after gel electrophoresis, so the purification of amplified products was done. Specificity of the assay was determined in the lab experiment with both basic and exo kits. Sensitivity was investigated to establish a detection limit with the serially diluted DNA of a single nematode which was picked manually, and also with the serially diluted DNA extracted from artificially inoculated sterile soil. For the soil DNA-based method, a single nematode was inoculated into sterile soil and then DNA was extracted from the soil using Qiagen DNeasy PowerSoil Pro Kit.

After the assay was developed and optimized to detect single nematodes, it was tested with field samples collected from different places of ND. A total of 19 soil samples were collected from fields in two counties (Sargent and Richland) of ND, infested with Pratylenchus spp. based on our previous research. The collected field samples were sub-sampled and DNA was extracted from each field soil using the soil DNA extraction kit as mentioned above.

Preliminary Results

After the primers and probes were designed using the sequence alignment with ITS sequences of Pratylenchus spp. and P. dakotaensis, they were evaluated in in-silico analysis for specificity. All the primers and probes evaluated were predicted to have strong and specific binding with the ITS sequence of P. dakotaensis based on primer-template duplex stability (?G) values. Since ?G values less than -31 kcal/mol predict stable primer-template hybrid formation, the primer set (ICF4/ICR3) and probe ICP1, having ?G value of -64.68, -57.54 and -86.23 respectively, were predicted to strongly hybridize with the ITS sequence of P. dakotaensis, but did not hybridize with ITS sequences of other Pratylenchus spp. Values of ?G greater than -31 kcal/mol are indicative of unstable primer-template binding resulting in unidirectional or non-logarithmic amplification, which are not competitive with specific amplification.

The primers ICF4 and ICR3 could amplify single nematode DNA of P. dakotaensis using the basic kit at 39.5 0C in 20 minutes (Figure 1A). This primer set also worked well with probe ICP1 and amplified single nematode DNA of P. dakotaensis using the exo kit at 39.5 0C in 20 minutes (Figure 1B). Hence, the primer set (ICF4 and ICR3) and probe (ICP1) were used for further experiment. Threshold level was determined two times during the experiment using eight negative controls in the first time and six negative controls in the second time. In both times, the threshold level was found very similar. Any amplification value greater than this threshold level is considered as positive or amplified and any amplification lower than this threshold is considered negative or not-amplified in the assay with exo kit.

Specificity test using exo kit with P. dakotaensis, seven other Pratylenchus spp., and plant-parasitic nematodes in six other genera revealed that RPA assay only amplified P. dakotaensis above threshold level but not other nematodes. The test using basic kit with P. dakotaensis and four other Pratylenchus spp. showed the similar result, which indicate that the designed primers and probe are specific to P. dakotaensis. Since the basic kit requires the purification step which is time consuming and needs more chemical reagents, the further experiment including sensitivity and field sample testing was done using the exo kit only.

From the sensitivity test, the RPA assay could detect up to 1/4 nematode DNA when DNA was extracted from a single nematode using proteinase K method and serially diluted. For the DNA extracted from artificially inoculated sterile soil using the soil DNA extraction kit, it could amplify up to 1/32 nematode DNA when a single nematode was inoculated into soil and DNA was extracted from soil and serially diluted. The difference in the sensitivity may be attributed to the different methods of DNA extraction, resulting in different DNA extraction efficiency.

The developed RPA assay was tested with 19 different field soil samples including 8 samples from field HG 50 previously recorded to be infested with P. dakotaensis (Figure 2A) and 11 samples from three fields (Cogswell, HG 73 and HG 105) in other locations previously recorded with other Pratylenchus spp. (Figures 2B and 2C). The results showed that the RPA assay amplified HG 50 field soil samples above threshold level but not other field samples. Higher amplification was found for the samples with higher nematode counts observed microscopically from HG 50 field samples and vice versa.

Work to be Completed

Some of the experiments have not been repeated but need to be repeated at least once such as sensitivity, specificity, and field sample testing. Using the same methods as described above, the repetitions of those experiments will be conducted. Similarly, sensitivity tests using basic kit will be carried out to justify why the exo kit is more practical than the basic kit. The soil sample HG 50 used in the experiment seemed to be very clayey and will be sent to Agvise Laboratories for soil texture analysis. Information generated from this research will improve detection efficiency for the root-lesion nematode species P. dakotaensis in soybean fields in North Dakota. Rapid detection of nematode species is critical for nematode risk assessment and management.

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Updated June 30, 2023:
a. Background information

Root-lesion nematodes (Pratylenchus spp.) are destructive plant pathogenic nematodes found worldwide. They pose a significant threat to various crops, including soybeans. In soil surveys conducted in 2015 and 2016 in North Dakota, soil samples from Richland County revealed the presence of root-lesion nematodes, ranging from 125 to 2,000 per kg of soil. Further analysis from combining morphological and molecular tests identified a new species of root-lesion nematode different from any known species in the literature. This newly identified nematode species was named Pratylenchus dakotaensis in 2021, paying homage to the state of its discovery. Subsequent evaluations done in the NDSU Nematology lab demonstrated that 13 out of 20 tested soybean cultivars were susceptible to this new nematode species, indicating its ability to infect and reproduce on soybean plants.
Effective management of the root-lesion nematode is crucial, necessitating a rapid and sensitive method for detecting the nematode in infested fields. Traditional morphological identification of root-lesion nematodes is time-consuming and challenging due to similarities among closely related species. To address this, real-time and conventional PCR assays were developed for detecting and identifying P. dakotaensis in 2021. However, PCR-based molecular diagnosis typically requires advanced instruments and more time, making on-site field detection impractical. A novel isothermal nucleic acid amplification technology called recombinase polymerase amplification (RPA) offers potential for on-site field detection of this new root-lesion nematode species in field samples.

b. Research objectives

• Develop a new recombinase polymerase amplification assay for detection of Pratylenchus dakotaensis from nematode individuals.
• Develop a new recombinase polymerase amplification assay for rapid and direct detection of P. dakotaensis in DNA extracts from field soils.

c. Research findings

To achieve the objectives, P. dakotaensis nematodes were extracted from soil samples (200g) using standard techniques such as hand sieving, decanting, and sugar centrifugal floatation. DNA was extracted manually from the nematodes and sixteen primers with eight forward and eight reverse were designed from the internal transcribed spacer (ITS)-rDNA sequence of P. dakotaensis. Four forward primers and three reverse primers were selected based on high alignment scores when aligned with the ITS sequences of 20 Pratylenchus spp. Two probes were designed to work in conjunction with these primers, following the instructions from TwistDx. After the primers and probes for the assay were designed, they were evaluated in silico analysis for specificity. All seven primers and two probe sets were predicted to have strong and specific annealing with the ITS-rDNA sequence of P. dakotaensis based on primer-template duplex stability (?G) values of the in-silico analysis.

The forward primer ICF4 and reverse primer ICR3 could amplify single nematode DNA of P. dakotaensis using RPA-Basic kit at 39.5 0C in 20 minutes (Fig. 1A). These primers set worked well with the probe (ICP1) and amplified single nematode DNA of P. dakotaensis using RPA-Exo kit at 39.5 0C in 20 minutes (Fig. 1B). So, the primer set (ICF4 and ICR3) and probe (ICP1) were used for further experiments. The threshold level was established for RPA-Exo to distinguish positive and negative samples and any amplification value higher than the threshold level was regarded as positive or amplified, and lower than the threshold level was considered as negative or not-amplified.

Specificity tests using P. dakotaensis, six isolates of other Pratylenchus spp., and other plant-parasitic nematodes in six genera revealed that the amplification value for only P. dakotaensis but no other nematodes was higher than the threshold level suggesting that it exclusively amplified only P. dakotaensis in RPA-Exo (Fig. 2). RPA-Basic with P. dakotaensis along with four other Pratylenchus spp. also showed the similar result, with a specific band (approx. 200 bp) in only P. dakotaensis but not in other nematodes. This meant the designed primers for RPA-Basic and RPA-Exo were specific to P. dakotaensis.

The sensitivity of the assays was investigated to determine the detection limit using DNA obtained from two methods; DNA extracted from nematode individuals and DNA extracted from inoculated sterile soil directly. when DNA extracted from the single nematode with Proteinase K method was diluted to 1/2, 1/4, and 1/10, RPA-Basic can detect up to 1/2 nematode DNA whereas RPA-Exo can detect up to 1/4 nematode DNA (Fig. 3A and 3B). It showed higher sensitivity from RPA-Exo than RPA-Basic. From DNA extracted directly from the soil inoculated with the nematode with a commercial soil DNA extraction kit, DNA from single nematode was diluted to 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/100, and 1/1000, it amplified up to 1/32 nematode DNA in RPA-Exo (Fig. 3C). The difference in the sensitivities could be attributed to the different methods of DNA extraction. Since RPA-Exo was more sensitive than RPA-Basic and didn’t require extra purification steps as in RPA-Basic, RPA-Exo was selected for further experiments to test different field samples.

After the RPA assay was developed, it was tested with 19 different field samples from ND including eight samples from the soybean field where P. dakotaensis was previously reported and 11 samples from other locations where other Pratylenchus spp. were previously detected. All the experiments were conducted at least in two repetitions each with at least two biological replicates. RPA-Exo amplified only the field samples with P. dakotaensis but not the other field samples with other Pratylenchus spp. (Fig. 4). Higher amplification values (fluorescence signals) were found for higher nematode counts observed microscopically from the P. dakotaensis-infested field samples and vice versa.

d. Benefits to ND soybean farmers and Industry

This developed RPA assay can help to improve the detection efficiency of this new species in soybean fields in North Dakota. Rapid and accurate detection of root-lesion nematodes is critical for nematode risk assessment and effective management. This assay will become increasingly important as soybean growers become more aware of this nematode disease to increase soybean yield.




Fig. 1. RPA amplified single nematode DNA of P. dakotaensis at 39.5 0C in 20 minutes. 1A: RPA detection using the Basic kit with the primers ICF4/ICR3, showing a specific band (approx. 200 bp). L: 100 bp ladder, 1: single nematode DNA, P: Positive control DNA (2 nematode DNA of P. dakotaensis), and N: nuclease free water. 1B: RPA detection using the Exo kit with the primers and probe (ICP1), showing the fluorescence signals from all nematode samples amplified above the water control.





Fig. 2. RPA assay exclusively amplified only P. dakotaensis but no other nematodes. Specificity test using RPA assay where 1: Positive control (5-nematode P. dakotaensis), 2: dd H20 (negative control), 3-7,14: other genera nematodes, and 8-13: other Pratylenchus spp. tested.




Fig. 3. Sensitivity tests with the dilutions of single DNA of P. dakotaensis. 3A: DNA from the nematodes where the assay with Basic kit detected up to 1/2 DNA of single nematode. 3B: DNA from the nematodes where the assay with Exo kit detected up to 1/4 DNA of single nematode. 1: Positive control (5-nematode DNA), 2: single nematode DNA, 3-6: different dilutions of the single nematode DNA, 7: ddH2O (negative control). 3C: DNA directly from the inoculated sterile soil where the Exo kit detected up to 1/32 DNA of single nematode. 1: single nematode DNA, 2-6 and 9-11: different dilutions of the single nematode DNA, 7: positive control (6 nematodes inoculated into the soil), and 8: ddH2O (negative control).




Fig. 4. Detection of P. dakotaensis from different field soils using RPA-Exo kit and DNA extracts from soil. 4A: The assay detected P. dakotaensis from the field soil samples (HG 50) with the target nematode but not from the field (Cogswell) without the target nematode. Eight HG 50 field samples and positive control (DNA from 5 nematodes of P. dakotaensis) detected above the threshold level and water but Cogswell field samples were amplified below threshold and water. 4B: The assay did not detect P. dakotaensis from field samples (HG 105-1 to HG 105-3, HG 73-1 to HG 73-3) without P. dakotaensis. All field samples without P. dakotaensis were detected below the threshold level and water whereas the positive control (DNA from a single nematode of P. dakotaensis) was detected above the threshold level.

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View uploaded report 2

a. Research Project Description
Root-lesion nematodes are destructive plant-parasitic nematodes, posing a significant threat to various crops including soybean. Effective management of the nematodes is crucial, necessitating a rapid and sensitive method for detecting them in infested fields. Traditional morphological identification is time-consuming and even PCR-based molecular diagnosis makes on-site field detection impractical. Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technique, representing a competent alternative to PCR for developing fast, portable nucleic acid detection assays.

b. Research Conducted
This research aimed to develop a RPA assay for detecting Pratylenchus dakotaensis, a newly discovered and named root-lesion nematode species from ND soybean fields (Fig. 1). Species-specific primers and probes targeting the internal transcribed spacer region were designed and evaluated for specificity through DNA sequence analysis and lab experiments. Sensitivity of the assay was determined using serially diluted DNA from individual nematodes and DNA from artificially infested sterile soil. The RPA assay was tested with 19 field soil samples infested with Pratylenchus spp. The RPA assay's validity was confirmed by comparing with the nematode numbers in the field samples using traditional microscopy.

c. Findings of the research
RPA assays were developed for detecting P. dakotaensis from nematode individuals and DNA extracts from field soils. RPA with Basic and Exo kits were able to detect P. dakotaensis from DNA extracted from single nematode (Fig. 2). The RPA-Exo kit detected the nematode at 39.50C in 20 minutes and was more sensitive than the Basic kit. The assay detected up to an equivalent of 1/32 of single nematode DNA extracted from artificially infested soil. Specificity tests showed that only P. dakotaensis DNA and none of other control nematode species tested, were amplified above the threshold level. When the assay was tested with 19 field soil samples collected in ND, it showed a similar trend between amplification values from RPA-exo and nematode counts from traditional microscopic method. RPA exclusively amplified soil DNA extracts from P. dakotaensis-infested field samples but not DNA from field soils infested with other Pratylenchus spp.

d. Benefits
A new RPA assay was developed for rapid detection of P. dakotaensis from infested field soils, and it has great potential for on-site field application. Such research findings will improve the nematode species detection efficiency in soybean fields and increase the nematode detection capacity. Rapid and accurate detection of the root-lesion nematodes is critical for nematode risk assessment and effective management.


Fig. 1. A soybean field from which Pratylenchus dakotaensis samples were collected.

Fig. 2. RPA amplified single nematode DNA of P. dakotaensis. A: RPA detection using the Basic kit with the primers ICF4/ICR3, showing a specific band (approx. 200 bp). B: RPA detection using the Exo kit with the primers and probe (ICP1), showing the fluorescence signals from all nematode samples amplified above the water (negative) control.

Results of the proposed research will improve detection efficiency for this new root-lesion nematode species P. dakotaensis in soybean fields in North Dakota. Rapid and accurate detection of root-lesion nematodes is critical for nematode risk assessment and effective management, and will become increasingly important as soybean growers become more aware of this new nematode disease to further increase soybean yield.