Soybean cyst nematode (SCN) is a very important soil-borne disease for soybean production in North Dakota (ND) and can cause 15-30% yield loss without obvious above-ground symptoms. Managing SCN becomes crucial to reduce economic losses for farmers. Enhancement of soil-based natural suppression could be an alternative means to manage SCN or as a part of an integrated pest management program. Nematode-suppressive soils are often recognized or suspected when population densities of the nematode decline greatly over time after initial establishment. Field observations of suspected nematode-suppressive soils must be verified by greenhouse tests and specific soil suppressiveness is transferable. Soils suppressive to SCN have been reported in a number of locations in the U.S. and other areas in the world. However, soil suppressiveness to SCN and biocontrol agents associated with it have not been investigated in ND. During our previous work for increasing SCN populations for HG type tests, we found 15 field soil samples with low SCN densities produced zero or very low SCN cysts whereas the other samples at the similar levels produced high numbers of cysts. We suspect some of the fields may have suppressiveness to SCN, which is transferrable to conducive soil. Thus, we propose to sample and assay soybean fields for SCN population densities, and select and evaluate ten soybean fields with no reproduction of SCN or declined SCN populations to identify suppressive soil to manage SCN in ND.

1. Sample and assay soybean fields with no reproduction of SCN or declined populations from previous work to select ten soybean fields still having low SCN densities.
2. Evaluate these soybean fields for SCN reproduction to identify suppressive soil to manage SCN in North Dakota.

1. The fields with no reproduction of SCN or declined SCN populations will be identified.
2. The levels of soil suppressiveness in these fields to SCN will be disclosed.
3. The information will be made available to soybean farmers.

Updated November 30, 2021:
Identification of Suppressive Soil for Managing Soybean Cyst Nematode in North Dakota

PI: Guiping Yan, Ph.D.
Collaborators: Drs. Sam Markell and Berlin Nelson

Objectives of the research

1. Sample and assay soybean fields with no reproduction of SCN or declined populations from previous work to select ten soybean fields still having low SCN densities.
2. Evaluate these soybean fields for SCN reproduction to identify suppressive soil to manage SCN in North Dakota.

Completed work

A total of 23 fields were sampled in May 2021 and soybean cyst nematodes (SCN) were extracted and quantified from the soil. From these soil samples, 10 fields with SCN egg numbers between 0 and 640 eggs per 100 cc of soil from Cass, Dickey, Lamoure, Nelson, Richland, and Trial counties in North Dakota were selected for subsequent experiments. These fields were previously reported to have high SCN egg numbers, but were found to have low numbers at the time of soil collection. Table 1 showed the reduction of SCN eggs in those selected field samples compared with the data collected from 2016.

After selecting 10 field soil samples, an experiment was set up in August, 2021, with four treatments; T1: field soil was autoclaved at 121°C for 2 hours, and 2,000 SCN eggs were inoculated, T2: 90% autoclaved-field soil mixed with 10 % non-autoclaved field soil and inoculated 2,000 SCN eggs, T3: non-autoclaved field soil inoculated with 2000 SCN eggs, and T4 (control): non-autoclaved field soil with no inoculation. The soil was mixed thoroughly for each treatment, and a SCN population from Richland county was used for the inoculation. Susceptible soybean cultivar Barnes was used for the host of SCN. All treatments were replicated four times and were planted in plastic cone-containers each containing an average of 100 cc of soil in a growth chamber maintained at 27 °C. Those entries were terminated 60 days after planting in October, 2021. After the termination of the experiment, plant parts above the soil surface were removed, and cysts were extracted from plant roots and soil in each container using the sieving and decanting method. White females were identified and counted under the dissecting microscope.

To confirm the results from the first experiment, the soil was collected again in October 2021 from the 10 fields selected. The second experiment was started in November 2021 by using the new soil sampled from the same location as before. A hatching experiment was conducted to hatch SCN eggs into 2nd stage juveniles (J2) in an incubator as inoculum, and an experiment with three field soils was set up in the growth chamber (Figure 1). Each of the soybean seedlings (cultivar Barnes) for treatments 1-3 was inoculated with 700 J2 after six days of planting.

Preliminary results

After the harvest of the first experiment, for the total of 160 cone-containers, white females were extracted from plant roots and soil in each of the containers and then counted. Five field samples (HG 21-1A, HG 21-1B, HG 66, HG 119-2, HG 21-3) were found to have high numbers of white females ranging from 160 to 421 per 100 cc of soil in the non-inoculated treatment (T4) with 100% natural field soil, indicating that they supported SCN reproduction and might not possess suppressiveness against SCN. Egg and juvenile counting that is being conducted is necessary to confirm the results based on the numbers of white females.

Out of the other five field samples, one of the field samples from Richland County (HG 21-2A) showed a significant reduction in SCN white females in 100% field soil (T3 and T4) as compared to 10% field soil mixed with autoclaved soil (T2), indicating the potential to be suppressive. For the other four soil samples, the non-inoculated field soils had very little numbers of white females ranging from 0 to 2 per 100 cc of soil but no significant difference was found in other treatments of these soils. This might be due to the possibility of error in counting white females because some of the white females changed the color to brown during the counting period and it is difficult or almost impossible to distinguish the newly changed brown females from old brown females already present in the field soils. Counting of eggs and juveniles that is underway is essential for confirming the results based on the numbers of white females.

Work to be completed

SCN cysts from the first experiment are stored in the suspension vials and some of them were already crushed and SCN eggs and juveniles are being quantified under a microscope. After getting all the data from the first experiment (SCN white females, eggs, and juveniles), reproductive factors comparing the initial and final populations of SCN eggs and juveniles will be obtained, which is a major factor for determining suppressiveness in the experiment. For the second experiment, we carefully examined the possible errors from the first experiment and tried to minimize them. Those five field soils that showed the higher number of white females in the first experiment will be discarded from the second experiment if the egg and juvenile counts support the results from the white females.

The second experiment started in the middle of November. It is the repetition of the first experiment with some changes. To prevent white females from changing color and make counting more consistent after the harvest, the second experiment is being conducted on weekly basis (2-3 field soils per week). We will hatch SCN eggs into second-stage juveniles (J2) and inoculate 600-700 J2 per plant instead of SCN eggs. For the second experiment, we may add more field samples from the initial 23 field soils which had fewer SCN eggs to increase the chance to identify suppressive soils. The second experiment will be harvested after two months of planting and in a similar way as in the first experiment. White females, eggs, and juveniles will be identified and quantified, and compared among the treatments to find the suppressiveness. The data from the second experiment will be compared with the results of the first experiment to evaluate the efficacy of the experiments. Enhancement of soil-based natural suppression could be an alternative means to manage SCN or as a part of an integrated pest management program.

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Updated June 30, 2022:
Identification of Suppressive Soil for Managing Soybean Cyst Nematode in North Dakota

TECHNICAL REPORT
NORTH DAKOTA SOYBEAN COUNCIL
JUNE, 2022

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

Soybean cyst nematode (SCN) is one of the destructive diseases for soybean production in North Dakota (ND) and can cause 15-30% yield loss without obvious above-ground symptoms. It is very important to manage SCN to reduce economic losses for farmers. Host resistance and crop rotation are the common ways of managing SCN but still, they have their own limitations. The use of suppressive soil can be an alternative to manage this pathogen as a part of an integrated pest management program. Nematode suppressive soils are often found when population densities of the nematode decline greatly over time after initial establishment. These suppressive soils do not support the reproduction of the nematode and have very few or no nematode at all. Soils suppressive to SCN have been reported in a number of locations. However, soil suppressiveness to SCN has not been investigated in ND.
Our previous work for increasing SCN populations for HG type tests revealed that 15 field soil samples with low SCN densities produced zero or low SCN cysts whereas the other samples at similar levels produced a high number of cysts. We suspect some of these fields may be suppressive to SCN, which is transferrable to conducive soil. Hence, the objectives of this research were to evaluate these soybean fields with no reproduction of SCN or declined populations to select ten soybean fields still having low SCN densities and also to evaluate these 10 selected fields for SCN reproduction to identify suppressive soil to manage SCN in ND.
A total of 23 soybean fields, including the 15 fields mentioned above, were sampled from the upper 10 cm of soil in 2021. Soil samples were thoroughly mixed together into a composite sample for each field to ensure the even distribution of nematodes. SCN cysts were extracted from 100 cc of soil and the cysts were crushed to obtain SCN eggs which were quantified using a microscope. Ten of the field soil samples obtained from six counties (Cass, Dickey, Lamoure, Nelson, Richland, Traill) in ND were found to have SCN egg numbers between 0-640 eggs per 100 cc of soil. These fields were previously reported to have high SCN egg numbers but were found to have low numbers at the time of soil collection. Table 1 showed the reduction of SCN eggs in those field samples compared to the data collected in 2016. These ten fields were selected for subsequent greenhouse experiments to identify suppressive soil to SCN.
For the greenhouse experiments, each field soil sample was mixed thoroughly by hand. The experiments were conducted with four treatments; T1: inoculated 100 % autoclaved field soil, T2: inoculated (90% autoclaved field soil + 10 % natural field soil), T3: inoculated 100 % natural field soil, and T4 (control): non-inoculated 100% natural field soil. The soil was then mixed thoroughly for each treatment, and an SCN population from Richland county was used for inoculation. Susceptible soybean cultivar Barnes was used as the host of SCN and the seed was pre-germinated for 4 to 5 days before planting. All treatments were replicated four times in plastic cone containers each containing an average of 100 cc of soil in a growth chamber maintained at 27 °C.
A total of three trials were conducted. Trial 1 was carried out in August 2021 with inoculation of 2,000 SCN eggs per plant per pot, and harvested 60 days after planting and inoculation. In November 2021, trial 2 was conducted after collecting soil again from those ten fields. Trial 3 was performed in February 2022. Trials 1 and 3 were conducted in the same way but there were some changes in trial 2. The difference in trial 2 was that the experiment was set up on a weekly basis (2-3 field samples per week) and 600-650 second-stage juveniles (J2s) of SCN were used as inoculum instead of eggs. The purpose was to avoid any error in counting white females which might turn brown in a short time after harvest. A hatching experiment was conducted to hatch SCN eggs into J2s for trial 2 in an incubator at 270 C for 48 hrs.
After harvesting the experiments, plant parts above the soil surface were removed, and cysts were extracted from plant roots and soil in each container. White females (before becoming cysts) were identified and counted under a microscope. All white females and brown cysts were crushed, and eggs along with J2s were extracted and counted. The reproductive factor was calculated by dividing the final population density by the initial population density. Analysis of Variance (ANOVA) was conducted for the statistical analysis and grouping of means was done by Fisher’s least significant difference (LSD) at a=0.05 using R software. The counting data were transformed using log-transformation log(x+1) before doing ANOVA.
From trial 1, five field samples (HG 21-1A, HG 21-1B, HG 66, HG 119-2, HG 21-3) were found to have high numbers of white females ranging from 240 to 631 per 100 cc of soil in the non-inoculated 100% natural field soil treatment (Table 2). Similarly, the numbers of eggs in these field soils except in HG 66 were found to be high ranging from 363 to 1,564 per g of soil (Table 3). It indicates the four field samples (HG 21-1A, HG 21-1B, HG 119-2, HG 21-3) supported the SCN reproduction and might not possess suppressiveness against SCN. Two of these four field samples were tested in trial 2. Results of the count of the white females from the two field soils still showed high numbers of SCN cysts as in trial 1. Thus, these four field samples were not chosen for trial 3.
All the three trials consistently showed that five field samples (HG 64, HG 150, HG 193, HG 21-2A, and HG 66,) had low numbers of SCN white females (0-9.5 per 100 cc of soil) except HG 66 in trial 1, and low numbers of SCN eggs (0-33.75 per g of soil) in non-inoculated 100% natural field soil treatment (Tables 2 and 3).
Out of the five field samples, one sample from Richland County (HG 21-2A) showed a significant reduction in SCN white females and eggs at inoculated 100% natural field soil as compared to inoculated 100% autoclaved field soil in trials 1 and 3, indicating the potential to be suppressive (Table 2 and Table 3). This similar difference was not observed in trial 2, which might be due to the changes in trial 2 during the experimental setup. Similarly, the reproductive factor was also found significantly lowered in inoculated 100% natural field soil as compared to inoculated 100% autoclaved field soil in trial 1 and trial 3 (Fig 1). But the mixing of 10% field soil with 90% autoclaved field soil was not able to suppress SCN population significantly in HG 21-2A, which might be due to the lower amount of natural field soil mixed with autoclaved soil. Increasing the percentage of field soil to be mixed with autoclaved soil is necessary to verify whether the suppressiveness of this soil is transferrable to conducive soil.
This research found that HG 21-2A may be potentially suppressive to SCN among the selected 10 fields. Other fields considered for both trials 2 and 3 having very low SCN populations in non-inoculated natural field soils might be due to other factors. Further research can be done to investigate this field through microbiome analysis to identify and quantify the specific microbes such as fungi and bacteria that may be antagonistic to SCN for developing biocontrol agents.

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IDENTIFICATION OF SUPPRESSIVE SOIL FOR MANAGING SOYBEAN CYST NEMATODE IN NORTH DAKOTA

EXECUTIVE SUMMARY
NORTH DAKOTA SOYBEAN COUNCIL
JUNE, 2022

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

Research conducted
A total of 23 soybean fields from eight counties in ND were sampled for SCN population densities that were previously reported to have decreasing populations over past years. Ten fields with lower SCN population were selected for greenhouse experiments (Fig. 1). Three experiments were conducted each in four replications. Susceptible soybean cultivar Barnes was used as the host of SCN. After 60 days of growth and inoculation, cysts were extracted from plant roots and soil, and white females were identified and counted under a microscope. Cysts were then crushed and SCN eggs and juveniles were extracted and counted. The reproductive factor was calculated by dividing the final nematode population density by the initial population density.

Why the research is important to ND soybean farmers?
Soybean cyst nematode is the most damaging pathogen of soybean. Management of this disease is crucial to reduce the economic losses to the farmers. Enhancement of soil-based natural suppression could be an alternative means to manage SCN or as a part of an integrated pest management program. This project targets the integrated pest management approach to managing SCN through identification of suppressive soil. This is the first study in ND to investigate suppressive soil for SCN management.

Final findings of the research
Two of the greenhouse experiments (Trial 1 and Trial 3) consistently showed that one of the fields from Richland county has the potential to be suppressive against SCN as they significantly reduced the SCN white females (per 100 cc of soil) and SCN eggs (per g of soil) in inoculated 100% field soil (suppressive treatment) as compared to inoculated 100 % autoclaved field soil (conducive treatment). The reproductive factor values were also found to be significantly reduced in HG 21-2A in those two trials (Fig. 2) in the suppressive treatment. Four fields out of the 10 selected fields showed lower SCN populations in non-inoculated 100% natural field soil in all the trials.

Benefits/Recommendations to North Dakota soybean farmers and industry
Such research findings can be beneficial because suppressive soil (HG 21-2A) can reduce nematode numbers in infested fields for SCN management. Further research on its transferability should be performed. Soil microbiome analysis will help identify specific microbes playing a key role in the suppressiveness against SCN for developing biocontrol agents. Biocontrol agents and bionematicides have become increasingly attractive as they are highly specific to the target nematodes and friendly to the environment.

SCN is a destructive pest on soybean. Managing SCN is crucial to reduce economic losses for farmers. Crop rotation, cultivar resistance and chemical nematicides are common management strategies. However, crop rotation does not allow soybean farmers to produce sufficient soybean every year. Resistant cultivars could be short-lived as SCN is adapting to the cultivars and new virulent fortns can develop and overcome the existing resistance. Most effective chemical nematicides have been restricted because of their harmfulness to the environment or cost. This proposed project for identifying suppressive soil will allow for biological control of SCN in infested soybean fields. Developing biocontrol agents and bionematicides has become increasingly attractive as they are highly specific to the target nematodes and friendly to the environment. Enhancement of soil-based natural suppression could be an alternative means to manage SCN or as a part of an integrated pest management program.