Root-lesion nematodes (Pratylenchus spp.) are one of the important groups of plant-parasitic nematodes. They have a wide distribution and a broad host rang, rank third worldwide among all plant-parasitic nematodes, and can cause significant yield suppression in soybean. Recent nematode surveys revealed that soil samples from a soybean field in North Dakota contained root-lesion nematodes which differ from any other known species of root-lesion nematodes. The ND isolate on soybean represents a new root-lesion nematode species that has never been reported in the literature and is named Pratylenchus dakotaensis, paying homage to the state in which it was discovered. Twenty soybean varieties were evaluated for resistance to the new species but none of the varieties tested were resistant. More soybean varieties need to be screened to identify varieties with high resistance to this new species. Distinction between root-lesion nematode species based on morphology is difficult and time consuming. Real-time and conventional PCR assays were developed to identify P. dakotaensis using nematode individuals and soil DNA. However, no real-time PCR assay is available for detection and quantification of this species directly from infected pant roots. The damage threshold and impact level of this species on soybean plant growth and yield are unknown.

• Evaluate ten additional soybean varieties to determine the levels of resistance to the new root-lesion nematode, P. dakotaensis detected in North Dakota.
• Establish a system to culture and increase pure population of P. dakotaensis for further investigating the nematode impact on soybean.
• Develop a real-time PCR assay to detect and quantify P. dakotaensis directly in DNA extracts from soybean plant roots.

• The resistance or susceptibility of ten soybean cultivars to this new root-lesion nematode species detected in ND will be disclosed.
• A system for producing and increasing pure population of P. dakotaensis will be established.
• A new molecular diagnostic assay will be developed to detect and quantify this new species directly in DNA extracts of soybean roots.

Update:
2024 Mid-Year report

Resistance of Soybean Varieties to Pratylenchus dakotaensis, a New Root-Lesion Nematode Species Infecting Soybean

Principle Investigator: Dr. Guiping Yan
Co-PI: Dr. Sam Markell


Research Overview and Objectives

Root-lesion nematodes (Pratylenchus spp.) are one of the important groups of plant-parasitic nematodes and can cause significant yield suppression. The new root-lesion nematode species P. dakotaensis was detected in soybean fields in North Dakota. This nematode was found to infect and reproduce well on soybean. The objectives of this research are to 1) evaluate ten soybean varieties to determine the levels of resistance to the new root-lesion nematode, P. dakotaensis detected in North Dakota, 2) establish a system to culture and increase pure population of P. dakotaensis for further investigating the nematode impact on soybean, and 3) develop a real-time PCR assay to detect and quantify P. dakotaensis directly in DNA extracts from soybean plant roots.

Completed Work

A greenhouse experiment was set up in small cone-tainers after confirming the species of P. dakotaensis with PCR. The greenhouse trial consisted of 12 treatments and 5 replications in a completely randomized design. Ten soybean varieties, a positive control (Barnes) and an unplanted naturally infested soil as negative control were included (Table 1). The soil was well mixed and then three sub-samples were taken from different spots to determine the initial nematode population. After that, the cone-tainers were filled with equal amount of infested soil. Pre-germinated seeds were sown in the cone-tainers. The initial population of root-lesion nematode in the soil was 625 nematodes per kg soil. The trial was harvested after nine weeks and the final nematode population was determined by extracting from both soil and roots using Whitehead tray method. The varieties were classified into different categories based on ratio of final population densities (density in a test variety/density in the susceptible check). The variety JF30-93N was used as the susceptible check as it had the highest final mean population density among all varieties. In this ranking, ratios were expressed in percentage; less than or equal to 25%: Resistant; 26% to 50 %: Moderately Resistant; 51 to 75%: Moderately susceptible; and greater than or equal to 76%: Susceptible (Chowdhury et al. 2022). Data were analyzed using SAS 9.4 version. The second trial was also set up and harvested. The final nematode population for each variety will be determined and the varieties will be ranked in the same rating scale.

In order to establish the pure culture of P. dakotaensis, Gamborg’s B5 medium along with the corn explant has been used. The corn variety found to be the host of this root-lesion nematode was used in the culture. The corn seeds were kept in agar medium for germination. After the roots were 1.5-inch long, they were cut and allowed to grow in Gamborg’s B5 medium. Once the root explants covered the petri-dish, they were inoculated with the target nematodes (i.e. 10, 50, and 100 individuals) (Fig. 1). Two other root-lesion nematode species, namely P. scribneri and P. penetrans, have been kept for pure culture following the same procedure and are used as controls. The plates will be kept for five months and their final numbers will be calculated and compared.

The set of primers designed previously in our lab were tested for their specificity and sensitivity with the DNA extracted from the infected soybean roots. The infected plant roots used were obtained from the first greenhouse trial in small cone-tainers. After the primers were found to bind with the targeted DNA, the DNA extracted from the roots artificially inoculated with nematodes was analyzed for presence of potential PCR inhibitors. The roots used for inoculation were uninfected roots grown in autoclaved soil. The extracted DNA were subjected to two-fold dilutions followed by real-time PCR amplification with the original DNA and diluted DNA samples. Bovine Serum Albumin (BSA) was added to the reaction mixture to nullify the effect of the PCR inhibitors.

After optimizing the conditions, the primers (IC-ITSF/ITS1R) were tested for the specificity with P. dakotaensis. The specificity test was also conducted with other root-lesion nematode species as well as common plant-parasitic nematodes reported in ND. Sensitivity test was performed to determine the detection limit of the real-time PCR assay with serially diluted DNA extracted from the roots artificially inoculated with single nematodes. DNA was extracted in triplicates and real-time PCR was performed in two technical replicates for each dilution. The extracted DNA from roots was serially two-fold diluted up to 1/512th dilution level.

Progress of Work and Results to Date

In the first greenhouse trial with small cone-tainers, none of the tested varieties were found to be resistant. Five varieties were susceptible, three moderately susceptible and three moderately resistant to P. dakotaensis (Table 1). JF30-93N had the highest mean population density among all varieties tested. NS2031NR2 had the lowest mean final population density and statistically had no significant difference than NS0773NLL and P11T36E.

All the infected roots of 11 soybean varieties were found to have average Cq values between 24.02 to 26.09 from the real-time PCR assay developed. The melting curve had only one peak signifying that the amplification was of single PCR product only (Fig. 2). So, the primers were found to be specific to the DNA extracted from P. dakotaensis-infected soybean roots. Additionally, the presence of inhibitors in the DNA samples extracted from roots was identified by serial two-fold dilutions and the use of Bovine Serum Albumin (BSA) reduced the effect of the PCR inhibitor.

The primers were also found to be specific to only the P. dakotaensis-inoculated soybean roots with average Cq value 26.53. Other three root-lesion nematode species (P. scribneri, P. penetrans, P. neglectus), common plant-parasitic nematodes found in ND (Paratylenchus spp., Paratrichodorous spp., Helicotylenchus spp., Xiphinema spp., Heterodera glycines) and negative controls were not amplified by the set of primers as their average Cq values ranged from 34.44 to 35.18 or no amplification was observed (Table 2). For sensitivity test, the real-time PCR could detect up to 1/128h dilution of DNA of one P. dakotaensis individual inoculated in uninfected root with average Cq value ˜ 33 (Table 3).

Work to be Completed

The second greenhouse trial was harvested and the nematodes will be extracted. So, the nematodes will be identified, counted and the data will be analyzed. The final data taken from both the experiments will be analyzed and final conclusions will be drawn. The rankings of the varieties for resistance to this new species will be summarized and the results will be made available to growers.

For the pure culture development, the petri-dishes will be harvested after incubation of five months after inoculation. The nematodes will be counted and reproductive factor of the root-lesion nematodes through this procedure will be calculated. This will help to increase the pure population of this nematode and produce enough inoculum for future research.

For real-time PCR assay, the tests for specificity and sensitivity will be repeated one more time for validation. Then, the stand curve will be generated using artificially inoculated nematodes and will also be validated. The plant root samples from Barnes included in objective 1 will be collected, DNA will be extracted directly from the plant roots and assayed by the real-time PCR and standard curves. Nematodes will be extracted, identified and counted using microscopy from the remaining roots of the same Barnes’ plants used in DNA extraction. Correlation analysis will be conducted to determine the relationship between the real-time PCR detection and traditional microscopic counting methods. Hence, all the objectives will be achieved by June 30, 2024.

Other Relevant Information

Extraction of DNA from plant roots resulted in co-extraction of substances that inhibited PCR amplification. Bovine serum albumin was added to the real-time PCR reactions to suppress the activity of the PCR inhibitors. The project is going well based on the plan proposed.

Summary
The first half of the research project showed significant progress and promising findings. The results obtained so far are encouraging and suggest that we are on track to meet the overall objectives within the given time frame. The second half without any doubt looks challenging and exciting. We will definitely build that upon the present findings and address the remaining research questions. In addition, we have shared some of our findings in a conference and continue to do so to broaden the reach of our work. This proposed research will enable us to improve nematode detection efficiency, establish a system for producing pure P. dakotaensis population for future research, and help farmers select the resistant varieties in infested fields to control the nematode disease.

View uploaded report

Updated June 29, 2024:
Resistance of Soybean Varieties to Pratylenchus dakotaensis, a New Root-Lesion Nematode Species Infecting Soybean

TECHNICAL REPORT
NORTH DAKOTA SOYBEAN COUNCIL
JUNE 2024

Principle Investigator: Dr. Guiping Yan. Dept. of Plant Pathology, NDSU

a. Background Information
Root-lesion nematodes (Pratylenchus spp.) pose a significant threat to soybean production because they can cause yield losses by infecting and damaging plant roots. These are endo-migratory nematodes that infect soybean roots and above ground symptoms are only visible after severe damage, making them difficult to manage. During a soil survey in 2017, a new root-lesion nematode species was reported in North Dakota with population densities 125-2000 per kg soil. It was different from other reported Pratylenchus species based on both morphology and sequencing of the genomic regions.
When the infested soil was planted with a cultivar of soybean (Barnes) in a greenhouse bioassay, a reproductive factor as high as 5.04 was observed. Hence, soybean was a good host to this nematode and it can be a potential threat to ND soybean production. Effective management strategies must be deployed in time before it causes significant yield losses. For this, timely identification and quantification of the nematode species P. dakotaensis is necessary. However, the traditional methods of identification and quantification require a lot of time and expertise in nematology. Therefore, there is a necessity to develop an efficient detection assay that can rapidly identify P. dakotaensis from the infected soybean roots as more than 50% of the nematode population was found to reside in the root versus soil.

b. Research Objectives

• Evaluate ten additional soybean varieties to determine the levels of resistance to the new root-lesion nematode, P. dakotaensis detected in North Dakota.
• Establish a system to culture and increase pure population of P. dakotaensis for further investigating the nematode impact on soybean.
• Develop a real-time PCR assay to detect and quantify P. dakotaensis directly in DNA extracts from soybean plant roots.

c. Materials and Methods

To meet the objectives, soil samples were collected from a soybean field in Richland County during the summer 2023 where P. dakotaensis was first reported. These samples were properly homogenized into a composite sample to assure uniform distribution of nematodes. The initial nematode population density was then determined from three subsamples and the species was confirmed through PCR. For the resistance screening, ten varieties grown in the region, namely NS 2031NR2, NS 60743NXR2, NS 0571NLL, NS 0773NLL, P04A98E, P06A38E, P11T36E, P17A87E, Ashtabula, and JF30-93N were selected. Furthermore, Barnes was used as a positive control and an unplanted control was used as a negative control. In addition to this, the cultivar having highest mean population density was used as a susceptible check in each trial.
The greenhouse trial consisted of twelve treatments and five replications in a completely randomized design. The large cone-type containers were filled with equal amount of the well mixed infested soil which was 500 g in Trial 1. Pre-germinated seeds were sown in the containers. The plants were subsequently grown in a controlled greenhouse environment for 15 weeks at 22 °C with a photoperiod of 16 hours. The experiment was repeated with 700 g of soil in each cone-type containers and same greenhouse conditions. Trial 2 was harvested 12 weeks after planting as the plants started turning yellow.
The aerial portion of each plant was removed at harvest, and soil with roots were taken for nematode extraction. The plant roots were separated from soil and nematodes were extracted from soil by Sugar Floatation method. The roots were washed under continuous stream of water to make them free from soil particles and blotted dry. Then the roots were weighed and cut into 1 to 2-cm pieces, mixed thoroughly and nematodes were extracted from roots using the Whitehead tray nematode extraction method. The extracted nematodes were collected in a nematode suspension vial through a 20 µm-aperture sieve. The root-lesion nematodes were identified to genus level and quantified under an inverted light microscope. The final population densities of root-lesion nematodes from both roots and soil were then averaged across the five replicates of each variety. The reproductive factor was determined by dividing the final mean population density by the initial population density (Table 1). In the second trial, 0.6 to 0.8 g of roots from each plant in one of the replications were separated for DNA extraction.
The varieties were classified into four different categories based on ratios of final mean population densities: Resistant (R), Moderately Resistant (MR), Moderately Susceptible (MS) and Susceptible (S) using the rating scale published (Chowdhury et al. 2022). The ratio of final population densities was cucullated by dividing the final mean population density in a test variety by the final density in the susceptible check. The variety having highest final mean population density among all varieties was used as the susceptible check. In this ranking, ratios were expressed in percentage, with less than or equal to 25%: R; 26% to 50 %: MR; 51 to 75%: MS; and greater than or equal to 76%: S.
In order to develop the pure culture of P. dakotaensis in lab, Gamborg’s B5 medium along with the corn explant was used. The corn variety used was found to be the host of this nematode. Corn seeds were kept in agar medium for germination. After the roots were 1.5-inch, they were cut and allowed to grow in Gamborg’s B5 medium. Once the root explant covered the whole petri-dish, they were inoculated with different numbers (10, 40, and 80) of nematodes (Table 3). Additionally, two other root-lesion nematode species, namely P. penetrans and P. scribneri were kept for pure culture as controls following the same procedures. The plates were incubated at 25°C for 5 months, and their final number and reproductive factor were calculated. Moreover, a susceptible cultivar (Barnes) was grown in the greenhouse with P. dakotaensis-infested soil to increase the population which was used for obtaining pure population from plant roots.
To develop the real-time PCR (qPCR) assay for detecting and quantifying this new species directly in DNA extracts from infected roots, the primer set (IC-ITS1F/IC-ITS1R) was tested for its specificity with P. dakotaensis. The specificity test was conducted with other root-lesion nematode species (P. scribneri, P. neglectus and P. penetrans) reported in the region and other common plant-parasitic nematodes. A two-fold serial dilution using DNA extracted from the roots inoculated with a single nematode was prepared to determine the detection sensitivity. 0.5 µl of PCR enhancer (Bovine Serum Albumin: BSA) was added to the reaction mixture and the DNA was extracted in triplicate and qPCR was performed in duplicate for each dilution factor. A standard curve was generated through sequential three-fold dilution of DNA extracted by inoculating 32 P. dakotaensis individuals in 0.2g of non-infected soybean roots. DNA extraction was done using the FastDNA Spin Kit and qPCR reaction was performed for each level of dilution. Melting curve analysis was done to monitor the specificity of the assay.
To validate the assay, varying number of P. dakotaensis (1, 5, 10, 15, and 20) were picked and added to 0.2 g of non-infected roots. DNA was extracted and qPCR was run for each level of inoculation. The numbers assayed by qPCR was plotted with the inoculated numbers of P. dakotaensis. In addition, nematodes from 11 soybean varieties’ infected roots from the greenhouse bioassay were manually extracted and counted thrice under a microscope. DNA was extracted from each root sample in triplicate and assayed by the qPCR and standard curve. Correlation analysis was conducted to determine relationship between the nematode numbers determined by the qPCR assay and traditional microscopic method.

d. Research Results and Discussion

The first and second trials yielded similar resistance rating results with some differences in mean final population densities and reproductive factors. In Trial 1, the mean final population density and reproductive factor values of P. dakotaensis for the varieties ranged from 1,520 to 3,163 nematodes per container from both soil and roots and from 1.07 to 2.23, respectively. In Trial 2, these two parameters varied from 2,881 to 6,239 nematodes per container and from 1.52 to 3.28, respectively. The variability could be linked to the variation in initial soil quantity. The second trial used more amount of soil and expectedly had a greater number of initial inoculum than the first trial. Therefore, all of the varieties in Trial 2 had higher final population densities and reproductive factors compared to Trial 1.
In Trial 1, NS 2031NR2, NS 60743NXR2, NS 0571NLL, NS 0773NLL, P06A38E, P11T36E, and Ashtabula were moderately susceptible, P04A98E was moderately resistant, P17A87E and JF30-93N were susceptible. In Trial 2, NS 2031NR2, NS 60743NXR2, NS 0773NLL, P06A38E, P11T36E, P17A87E, and JF30-93N were moderately susceptible, NS 0571NLL was moderately resistant, P06A38E and Ashtabula were susceptible. The varieties (P04A98E and NS 0571NLL) that showed moderate resistance in one of the trials were only 2% or 4% less than 50% being classified as moderately susceptible. The variation in ratings could be due to minor difference in environment conditions (e.g. soil water content) and nematode populations (e.g. penetration ability). Combined results of the two trials revealed that one variety Ashtabula (76%) was susceptible and the remaining varieties were moderately susceptible to P. dakotaensis (Figure 1). The positive control Barnes (90%) was rated as a susceptible cultivar, supporting our previous results. None of the screened varieties were found to be resistant or moderately resistant.
A new system to culture and increase the population of P. dakotaensis was adopted using Gamborg’s (GB5) media and corn explant in our lab. It was successful to increase the population of P. dakotaensis up to 10.42 times (Table 3). In the greenhouse bioassays, soybean (Barnes) had the average reproductive factor of 2.44.
The developed qPCR assay was specific to the target nematode P. dakotaensis. Inoculated soybean roots produced an average Cq (quantification cycle) value of 25.97 and a single peak at 81.5° C in melting curve analysis (Figure 2). Other three root-lesion species (P. scribneri, P. penetrans and P. neglectus), common plant-parasitic nematodes (Paratylenchus sp., Paratrichodorous sp., Helicotylenchus sp., Xiphinema sp., Heterodera glycines), and uninfected soybean roots were not amplified by the primers. The qPCR assay could detect up to 1/128th of a single P. dakotaensis individual per 0.2 g soybean roots. The generated standard curve (y = -3.3674x + 26.946) showed a high amplification efficiency (E = 98.1%) and had a strong correlation between the Cq values and log number of nematodes added to uninfected roots (R² = 0.9738) (Figure 3). The standard curve was validated by a strong, positive correlation between the numbers of P. dakotaensis determined by qPCR and numbers of P. dakotaensis (1, 5, 10, 15, and 20) added to 0.2 g of uninfected soybean roots (y = 0.6439x + 1.14, R² = 0.8407) (Figure 4). Moreover, a positive correlation was found between qPCR estimates and manual counts of the root samples from infected roots of 11 soybean varieties including Barnes from the greenhouse bioassay.

e. Benefits to ND Soybean Farmers and Industry
The research successfully evaluated ten soybean varieties for resistance reactions to this newly identified root-lesion nematode species in ND. In addition to this, a new method for culturing and increasing the pure population of P. dakotaensis was developed, which will facilitate future investigations in its impacts on soybean growth and yield. Moreover, a specific and sensitive qPCR assay was developed, enabling the direct identification and quantification of P. dakotaensis in DNA extracts from soybean plant roots. These findings will help growers to choose soybean varieties rationally and also provide a crucial tool for early detection as well as quantification of this species which will play an important role in effective management of the nematode disease. Further research should focus on identifying resistance levels of more soybean varieties and identifying new sources of resistance enhancing overall crop performance.

View uploaded report

View uploaded report 2

Resistance of Soybean Varieties to Pratylenchus dakotaensis, a New Root-Lesion Nematode Species Infecting Soybean

EXECUTIVE SUMMARY

NORTH DAKOTA SOYBEAN COUNCIL
JUNE 2024

Dr. Guiping Yan, Principal Investigator, Dept. Plant Pathology, NDSU

Importance of the Research
Root-lesion nematodes are among important nematode pests affecting soybean production. A new root-lesion nematode, Pratylenchus dakotaensis that was reported in North Dakota soybean fields showed significant reproduction in greenhouse bio-assays and resided more in roots than soil. Using resistant varieties and detecting nematodes early are key management strategies. Similarly, it is important to increase the nematode population in controlled environment to know its impact on yields. Therefore, this research focuses on identifying resistance levels of soybean varieties, developing a molecular assay to detect it directly from roots, and establish a system to increase its population in lab and greenhouse.
Research Conducted
Resistance levels of ten soybean varieties used in the region were assessed for P. dakotaensis through repeated greenhouse experiments using naturally infested soil (Fig. 1). A rapid qPCR assay was developed to detect and quantify this new species directly from infected soybean roots. The specificity and detection limit of the assay were also determined. A standard curve was generated. The assay was validated through correlation analysis between numbers of nematodes artificially inoculated and estimates made by using the qPCR assay. A system for culture and increase of this nematode was developed using corn explants in Gamborg’s GB-5 medium.
Research Findings
Among those ten varieties screened, nine were found to be moderately susceptible, one was found to be susceptible, and none of them were resistant or moderately resistant in combined analysis (Fig. 2). The resistance rating results were mostly similar in both experiments. A new DNA-based molecular assay was developed for detection and quantification of this new species from infected soybean roots directly and it is sensitive and specific (Fig. 3). There was a strong, positive correlation between the numbers of nematodes inoculated into roots and detected by the developed qPCR assay. Furthermore, the population of this new nematode species can be increased in lab using corn explants and GB5 medium whereas Barnes can be used in greenhouse to increase its pure population.
Benefits
These findings can help growers to choose soybean varieties rationally to avoid susceptible or moderately susceptible varieties having higher nematode reproduction. This research also provides a crucial tool for early and rapid detection and quantification of this new nematode species from soybean roots in ND. Further research should focus on identifying resistant soybeans and new sources of resistance. Efficient nematode detection is essential for effective management of the nematode disease.


Figure 1. Soybean plants in the large cone-type container greenhouse trial for resistance responses to Pratylenchus dakotaensis.

Figure 2. Classification of the resistance responses of ten soybean varieties to the new root-lesion nematode species (Pratylenchus dakotaensis) based on combined data of the two trials.

Figure 3. Pratylenchus dakotaensis melting curve profile. A single melting peak was observed at 81.5°C and no amplification was observed for negative controls, indicating that the qPCR assay is specific.

Root-lesion nematodes are one of the important nematode pests on soybean. The proposed research will enable us to improve detection and quantification efficiency and capability, and establish a system for producing and increasing pure P. dakotaensis population for future research to determine the damage threshold and impact of this new species on soybean. The molecular diagnostic services will become increasingly important as growers become more aware of the damage caused by these root parasites. Results of the proposed research on variety resistance evaluation will help identify varieties that are resistant to this new species. This information is important to help farmers select the resistant varieties in infested fields to control the nematode disease to increase soybean yield.