Root-lesion nematodes (Pratylenchus spp.) are one of the important groups of plant-parasitic nematodes. They have a wide distribution and a broad host range, rank third worldwide among all plant-parasitic nematodes, and can cause significant yield suppression in soybean. Recent nematode surveys revealed that soil samples collected from a soybean field in Richland County, North Dakota contained root-lesion nematodes which differ from any other known species of root-lesion nematodes in both morphology and DNA sequences. The ND isolate on soybean represents a new root-lesion nematode species that has never been reported in the literature. Twenty soybean cultivars were evaluated for resistance to the new species. Our results indicated that one cultivar was resistant, seven were moderately resistant, nine were moderately susceptible, and three were susceptible to this species. However, the resistance levels of other soybean cultivars to this species are unknown. More soybean cultivars need to be screened to identify cultivars with high resistance to this new species. Additionally, at least two other rootlesion nematode species, P. neglectus and P. scribneri were present in soybean fields in ND. Distinction between different root-lesion nematode species is difficult and time consuming based on morphology. A real-time PCR assay is being developed for identifying this species using nematode individuals. However, this assay is not able to quantify this new species from soil DNA. A sensitive, rapid and reliable molecular diagnostic method is needed to quantify this new species directly in DNA extracts of infested field soil.

1. Evaluate ten additional soybean cultivars to determine the levels of resistance to the new root-lesion nematode species detected in North Dakota.
2. Develop a real-time PCR assay to detect and quantify this new root-lesion nematode species directly in DNA extracts from field soil.

The resistance or susceptibility of ten soybean cultivars to this new root-lesion nematode species detected in ND will be disclosed. A new molecular diagnostic system will be developed and validated to detect and quantify this new species directly in DNA extracts of field soil.

Update:
Resistance of Soybean Cultivars to a New Root-lesion Nematode Species in North Dakota

PI: Guiping Yan, Ph.D.


Objectives of the research

1. Evaluate ten additional soybean cultivars to determine the levels of resistance to the new root-lesion nematode species detected in North Dakota.
2. Develop a real-time PCR assay to detect and quantify this new root-lesion nematode species directly in DNA extracts from field soil.

Completed work

To evaluate the resistance levels of ten soybean cultivars to the new root-lesion nematode species, soil samples were collected from the field where the new species was first detected. Soil samples were then thoroughly mixed together into a composite sample to ensure even distribution of nematode population. Subsequently, three subsamples were collected from the composite sample. Nematodes were then extracted from the subsamples using sieving, decanting and sucrose centrifugation methods to determine the initial population density. To confirm the species identity, DNA were extracted from the root-lesion nematodes and species-specific polymerase chain reaction (PCR) was conducted.

Among the ten cultivars selected for resistance screening, two were from NorthStar Genetics (cultivars: NS 1492NR2 and NS 61624NXR2), two from NDSU soybean breeding program (Benson and Stutsman), and one from each of Channel (0916R2X), Integra Seed (50948N), Thunder Seed (SB-8807N), Proseed (50-90), Hefty Seed (H06X7), and Legacy Seed (LS-1138NRR2X). In addition to the ten cultivars, two positive controls were selected for comparison purposes, including the local soybean cultivar Barnes and the NorthStar Genetics cultivar NS 1911NR2. Moreover, a non-planted control was used as the negative control. After pre-germinating each of these seeds in a petri dish with water, the seeds were planted in large cone-type containers containing soil, naturally infested with the new species. Five replicates of each cultivar and the positive and negative controls were included. The plants are being maintained for growth in a greenhouse room at 22 °C.

To develop a real-time PCR (qPCR) assay to detect and quantify this new species from soil DNA, the primer set, IC-ITS1F/IC-ITS1R developed in our previous study was tested for its specificity using both nematodes and soil DNA extracts. DNA extracts from non-target root-lesion nematode species (Pratylenchus scribneri, P. neglectus and P. penetrans) were prepared and qPCR reactions were set up along with positive and negative controls. To construct the standard curve, pots with infested soil and soybean plants (Barnes) were maintained in the greenhouse for population increase. Nematodes were extracted from soil and varying number of the target nematodes (1, 4, 16, 64, and 256) including both adults and juveniles, were picked and added to 0.5 g of autoclaved soil. DNA extraction was done in triplicates for each level of inoculation using the Qiagen DNeasy PowerSoil Kit. The DNA extracts were used to set up a SYBR green based qPCR with 9 observations (3 biological replicates with 3 technical replicates) per inoculation level. DNA from uninoculated soil and ddH2O were used as negative controls and a DNA extract of the new species was used as a positive control. A dilution series using the DNA extract from a single nematode down to an equivalent of 1/128 of the nematode were prepared and qPCR reactions were run to determine the detection sensitivity.

Six field soil samples were collected from different counties of ND, and nematodes were manually extracted and counted to identify and quantify different types of nematodes in each sample. DNA extractions have been done in triplicate for these soil samples. A total of 15 soil samples naturally infested with the new root-lesion nematode species were collected from a soybean field in Richland County and nematodes were manually extracted from soil and counted twice for each sample under a microscope. From each soil sample, DNA extractions in triplicate haven been performed (45 extractions in total) and the samples are being stored at -20oC for further use in qPCR.

Preliminary results

The average initial population density of the new species of root-lesion nematodes determined from the three subsamples was 1,200 nematodes per kg of soil and then the soil is being used for the greenhouse experiment. The identity of the root-lesion nematodes was confirmed to be the new root-lesion nematode species using the species-specific PCR diagnostic method.

The qPCR detected DNA specific to the new species with quantification cycle (Cq) values ranging from 19.87 to 29.28. No fluorescent signals were detected from the non-template water control and from the non-target nematode species. Figure 1 shows the single melting peak obtained at 81.5o C and the standard curve with the equation y = -3.5528x + 28.352 (R² = 0.9908 and E = 91%). The qPCR assay was sensitive and detected an equivalent of 1/32 of the DNA of a single nematode with a detection sensitivity curve depicting y = -3.4647x + 27.944 (R² = 0.9827 and E = 94%). For the soil samples naturally infested with the new root-lesion nematode species, nematode counts from the collected field soil samples ranged from 20 - 1,240 per kg of soil.

Work to be completed

The soybean plants in the greenhouse room will be harvested 15 weeks after planting. Nematodes will be extracted from the harvested soil and roots to determine the final population density. The experiment will be repeated with the same seed lots and the data for two repetitions of the experiment will be grouped for final analysis. With the aid of previous literature, numerical ratings and qualitative classification of resistant, moderately resistant, moderately susceptible, and susceptible will be determined for each of the cultivars tested.

For the qPCR assay development, field soil DNA extracts along with positive and negative controls will be used to set up qPCR reactions in triplicate and the data will be compared with manual counting. A qPCR estimate of the target nematode from the Cq value for each sample will be obtained using the standard curve equation. The equivalent quantity of the target nematode in each DNA template will be determined from a subsample of the same soil sample using manual nematode extraction and microscopic counting. A correlation curve will then be plotted between the nematode numbers determined by the traditional microscopic method and the numbers quantified by qPCR.

As proposed, the resistance or susceptibility of ten soybean cultivars to this new root-lesion nematode species detected in ND will be disclosed and made available to soybean farmers. A new molecular diagnostic system will be developed to detect and quantify this root-lesion nematode species directly in DNA extracts of field soil to improve detection efficiency.

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Update:

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RESISTANCE OF SOYBEAN CULTIVARS TO A NEW ROOT-LESION NEMATODE SPECIES IN NORTH DAKOTA

EXECUTIVE SUMMARY

NORTH DAKOTA SOYBEAN COUNCIL
JUNE, 2021

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

Research Conducted
Ten soybean cultivars used in ND were evaluated for resistance to a new root-lesion nematode (RLN) species recently discovered in a ND soybean field. The experiment was conducted under greenhouse conditions (Fig. 1A) and repeated to confirm research findings. A quantitative real-time PCR (qPCR) assay was developed to detect and quantify this new species in DNA extracts of field soil. Primers were tested for specificity and detection sensitivity was determined. A standard curve was generated. The qPCR assay was validated by comparing the numbers of nematodes in 15 field soil samples obtained by traditional microscopic counting and qPCR through correlation analysis.

Why the research is important to ND soybean farmers
Root-lesion nematodes are one of the most important groups of plant-parasitic nematodes. During our previous soil surveys in ND, a new RLN species was identified. In 2021, this new species was named as Pratylenchus dakotaensis, paying homage to the state in which it was discovered. RLNs have a wide host range including soybean. One of the most economical and effective ways to control RLNs is the use of cultivar resistance. This research aims to provide valuable information about resistance levels of soybean cultivars in ND to manage the new species, and an efficient DNA-based, qPCR assay for improving nematode detection to facilitate management strategies.

Final findings of the research
Resistance rating results were consistent between two trials. Although none of the cultivars tested were resistant or moderately resistant, three were found to be moderately susceptible and the remaining seven were susceptible (Fig. 1B). A new qPCR assay was developed for detection and quantification of this new species in DNA extracts of field soils. The assay was highly specific and sensitive, and had a high correlation between the numbers of nematodes in field soil samples determined through manual counting and qPCR (Fig. 2). This is the first report for detection and quantification of this species directly from field soil DNA.

Benefits/Recommendations to North Dakota soybean farmers and industry
Such research findings provide an insight into the virulence of the new RLN species on commercial soybean cultivars. Moderately susceptible cultivars identified may perform better than the susceptible cultivars. However, further research is necessary to find better performing resistant cultivars. The qPCR assay developed provides a rapid and efficient method for P. dakotaensis quantification in soil DNA and can serve as a valuable diagnostic tool for providing informed decisions to growers.

Fig. 1. A: soybean plants grown in a growth chamber maintained at 22 °C for resistance evaluation to the new root-lesion nematode species (Pratylenchus dakotaensis) identified in ND. B: classification of the resistance responses of ten soybean cultivars to this new species based on the two trials.

Fig. 2. Relationship between the numbers of Pratylenchus dakotaensis determined by qPCR and traditional microscopic counting method from 15 naturally infested field soil samples.

The proposed research on real-time PCR assay development will enable us to avoid time consuming steps for manual nematode extractions, microscopic identification and counting of the nematodes from field soil samples to improve detection and quantification efficiency. 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 cultivar resistance evaluation will help understand resistance or susceptibility of soybean cultivars used in ND and identify more cultivars that have resistance to this nematode species. This information is important to help farmers select the resistant cultivars in infested fields to control the nematode disease to increase soybean yield.