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.
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