Edamame, also known as vegetable soybean, is a high-fiber, low-sugar snack that has gained popularity in the USA in recent years. Not only does edamame have a high protein content but it also contains all the essential amino acids the human body requires for proper nutrition (Dixit et al., 2011). Compared to traditional soybeans grown for oil or grain production that are harvested at reproductive maturity, edamame is a shorter-season crop that is harvested before the seed is mature. Edamame is harvested when the pods at the upper nodes are filled with green seeds, before the pods start to turn yellow and senesce. Because edamame is most commonly marketed in the pod, the condition and quality of both the pods and seeds is of major importance. Although edamame has been grown in the USA since the late 1800s (Conlon, 2009), its popularity has recently increased because it is a high protein, vegetable-based, healthy food. From 2003 to 2007, US gross sales of edamame increased from $18m to $30m (Bernick, 2009), with the majority of the product purchased frozen, either in-pod or shelled.
Soybean protein is comparable to animal-sourced protein and contains all the amino acids necessary for a complete human diet with less cholesterol and saturated fats than animal protein (Dixit
Because edamame is harvested in the pods prior to complete seed maturation, pest management concerns and strategies are different from those routinely used in traditional soybean production systems. Plant-parasitic nematodes are a significant pest concern to the profitability of soybean production in the state of Arkansas (Kirkpatrick et al., 2014), and in the southern USA (Wrather and Koenning, 2003, 2006). Two nematodes of major concern for soybean production in Arkansas include the soybean cyst nematode (
Although there have been studies on quality components of edamame (Li et al., 2012; Zeipina et al., 2017), the effect of nematodes on these quality traits has not be examined. A direct and widely used method of observing effects of nematode infection on a host is by identifying the nematode reproduction index (rate of initial to final nematode population densities (RI = Pi/Pf)) and relate the RI to the impact of plant yield. A similar approach can be taken to observe effects of RI on the quality of a plant host. Model systems have been used to describe the impact of nematodes on plant growth and yield (Seinhorst, 1988; Van de Berg, 2012). One of the most useful aspects of a disease-pressure model system is the estimation of a point at which the yield is threatened, also known as the damage threshold. Understanding the damage threshold of a specific host and cultivar allows growers or crop advisors to assess properly the nematode-associated risks and aid in determining when nematode management is needed. Many factors, in addition to nematode population density, play a role in determining the threshold including soil type, temperature, time, and host (McSorely and Duncan, 1995).
The goals of this study were to document the nematodes that were present in Arkansas edamame fields and determine the effect of two major nematode species on edamame yield and quality. The objectives were to: survey edamame production areas in Arkansas for plant-parasitic nematodes, determine the effects of root-knot and soybean cyst nematode initial population densities on the yield and food quality of edamame in microplots and evaluate edamame breeding lines from the University of Arkansas soybean breeding program for host suitability to
Commercial edamame fields under contract with the American Vegetable Soybean and Edamame, Inc. (33 fields in 2013 and 31 fields in 2014) were surveyed for the presence and density of plant-parasitic nematodes. All sites were sampled within three weeks after edamame harvest. Samples were collected using a 2.5-cm-diameter soil probe (Soil Sampler, Step Made, USA) to a depth of 15 to 20 cm. Six cores were collected for each individual sample along the rows where the soybean stalks remained using a zig-zag pattern (Van Bezooijen, 2006) with one site representing 4 to 8 hectares, averaging a total of 500−600 cm3 of soil. Samples were placed in plastic bags, labeled with field name and location within field, transported to the laboratory in an insulated ice chest and stored in a cooler at 10−13°C (Barker et al., 1969). Soil samples were processed within two weeks of collection date. For each sample, a 100-cm3 aliquot from the mixed composite sample was assayed using a semi-automatic elutriator (Byrd et al., 1976) equipped with 40-mesh (0.4 mm) sieve over a 400-mesh (0.037 mm) final collection sieve. Soil collected on the 400-mesh sieve was processed using centrifugation flotation (Jenkins, 1964) and nematodes were identified to genus using a dissecting microscope at 60−80×.
Field microplot experiments were conducted at two locations in 2014 to examine the effect of
Location 1 was at the University of Arkansas Southwest Research and Extension Center near Hope, AR. Microplots were made from square clay flue tiles (37.5 cm×37.5 cm) buried 122 cm deep in a grid pattern of 5×10 and spaced 91 cm apart. Each microplot was filled with a mixture of silt loam topsoil (48% sand, 50% silt, 2% clay) mixed 50:50 (v/v) with coarse sand to a volume of 21,093.75 cm3. Prior to filling the plots, the soil mix was steam pasteurized for 30 min at 70°C. The entire block was enclosed in metal fencing to deter pests. During the first month after seeding, the block was covered with nylon netting to deter birds. Location 2 of this study was a previously established plot on the Arkansas Agricultural Research and Extension Center in Fayetteville, AR. At this site, the microplots were of the same size and material as in Location 1. Existing soil from each plot was removed with a shovel and the inside surface of each tile was drenched with a 10% chlorine bleach (NaOCl) solution two months prior to refilling and planting. The microplots were filled with soil (86% sand, 11% silt, 3% clay, bulk density of 1.2 g/cm3) obtained from the Arkansas River Valley near Van Buren, AR, that had been pasteurized as described above. The site was fenced to deter pests.
Inoculum of
Inoculum for
Immediately after inoculation, six seeds of the edamame cultivar “8080”, which was the most common cultivar grown by Arkansas growers, were planted in each plot about 7 cm apart. At 18 days after seedling emergence, the plant population was adjusted to four plants per plot. Plots were treated with a mild insecticidal soap to discourage an aphid infestation (“Bug B Gon”, Ortho®) two weeks after seedling emergence. A complete fertilizer (Scott’s Osmocote® 14-14-14) was applied as directed in label one week after seedling emergence as suggested by soil test results. All plots were hand-watered as necessary throughout the study.
The nematode population density from all plots at both locations was assayed four weeks after planting to monitor populations halfway through the season. Soil samples from each microplot were obtained by collecting six individual cores using a soil probe (2.5-cm-diameter) 15 cm deep. To avoid contamination between plots, the soil probe was washed and sterilized with 10% chlorine bleach after each plot was sampled. The risk of significant contamination was further minimized by first taking the samples from the nematode-free control plots and then sampling each treatment separately from low to high population density. The cores were bulked for each plot and a 100-cm3 subsample was assayed using a semi-automatic elutriator followed by centrifugal flotation. Juvenile and egg counts were recorded for each plot. Soil was sampled from each plot again on the same day as harvest before the plants were removed from the plots to quantify nematode densities at time of harvest. Cross contamination was prevented in the same manner as described above. The roots from
Soybeans in the microplots were harvested by hand-picking all pods from each plant at soybean growth stage R6 (Fehr, 1971) that occurred 73 days after planting (Location 1) and 75 days after planting (Location 2). The number of pods per plant was recorded, and all pods from each plot were bulked, placed into a paper bag and labeled. The number of pods containing one, two and three seed and total weight of all pods were recorded per plot. All pods were hand-shelled, and the beans were bulked for each plot. The total fresh seed weight was recorded.
The fresh seeds from all pods were arranged in foil boats by plot and put in a blast freezer (Air-O-Chill, Elextrolux Professional SpA, Pordenone, Italy) at −25°C for 20 min. The seeds were then placed in a freeze dryer (Virtis Genesis, SP Scientific, PA, USA) at −45°C for a total of 120 hr (Rayaprolu et al., 2015). The moisture content was calculated as the weight difference before and after freeze drying. The freeze-dried seeds were ground using an electric coffee bean grinder for approximately 30 sec and sieved through a 30-mesh (0.595 mm) sieve. The sieved flour was used for analysis of protein (AOAC 990.03), lipid (AACC 30-26) and starch (Megazyme kit, AACC 76-13.01, 1995) as follows: the total amount of protein in each sample was measured using the nitrogen combustion methodology (CLG-PRO4.03) (AOAC method 992.23, AOAC, 1990). The lipid extraction protocol was modified from the AACC method, “Crude Fat in Soy Flours” (AACC method 30-26, AACC, 1995). Filter paper (WhatmanTM 4, 150 mm, Cat No 1004 150) was folded, weighed, and a 2-gram sample of the flour was wrapped in the filter paper. The wrapped sampled was inserted into a paper tube and placed in a soxhlet apparatus placed under an enclosed vent hood. The system included condensers, filter tubes and 500-mL glass collection beakers on a series of hot plates (AACC method 30-26). Approximately 150 mL of petroleum ether was added to each sample via the condensers, and the samples were run through a 5-hour cycle to ensure removal of all soluble lipids. The samples wrapped in filter paper were then aired dried for a minimum of 8 hr and weighed. The amount of lipid in each sample was calculated by the change in sample flour weight to four decimal places. The starch content was analyzed using a Megazyme©, 2011 Total Starch kit (amyloglucosidase/
All collected data were analyzed using general linear models (SAS Institute, Inc., 2014). Differences among treatments were compared using least significant difference (LSD) at
In total, 24 advanced breeding lines of edamame from the University of Arkansas, Division of Agriculture soybean breeding program were obtained from Dr Pengyin Chen, Department of Crops, Soils, and Environmental Sciences, University of Arkansas, Fayetteville, AR. Three seeds of each line were planted in 10-cm-diameter clay pots filled with pasteurized sandy loam soil (86% sand, 11% silt, 3% clay, bulk density of 1.2 g/cm3). One week after planting, plant populations were adjusted to one plant per pot. In all trials, the pots were arranged in a randomized complete block design to account for possible variability in environmental conditions at different positions on the greenhouse bench. Five replications of each line were infested with nematodes as described below. Plants were watered and fertilized with Scott’s Osmocote® 14-14-14 (Scott’s MiracleGroTM) as needed for active growth. Greenhouse temperatures ranged from 26–29 °C for the trials conducted in June–July, 2013, and 24–27°C for the trial repeated in October–December, 2013. All lines were evaluated in a third trial May–June, 2014.
A population of
After 45 days, the plants were cut at the soil line and the stem and root tissue was weighed for fresh weight. The roots were then rated for root galling severity on a 0–10 scale where 0=no galls, and 10=100% of the roots covered with galls (Barker, 1982). Following gall ratings, roots were placed whole into a 500-mL wide-mouth Erlenmeyer beaker and stirred with an electric mixer in 0.5% NaOCl for 4 min. The solution and roots were poured through a 100-mesh (0.149 mm) over 500-mesh (0.025 mm) stacked sieves and rinsed with tap water. The eggs collected on the 500-mesh sieve were quantified at 60× magnification. The roots were then dried for 48 hr at 60°C in a plant drier and weighed. Root weights were used to calculate the nematode reproduction per gram of dry root tissue.
The same stock culture of
After 30 days, plants were cut at the soil line and each pot was extracted separately by placing the roots and soil into a 4.7-liter bucket. Water was added at high pressure, and the soil and roots were mixed vigorously and poured through 30 over 60-mesh nested sieves with each sieve rinsed thoroughly (Hussey et al., 1991). Cysts were collected from the 60-mesh sieve in 100-mL beakers and stored at 4°C until quantified. The contents of the beaker were poured into a counting dish, and cysts were counted. After extraction, the roots of each plant were dried for 48hr in a large plant drier at 60°C and weighed.
The five replications within each trial were analyzed in an ANOVA using JMP ® 14.1.0 (SAS Institute Inc., 2018) for both root-knot and soybean cyst nematode trials, independently.
Data for the three root-not trials were recorded as gall rating and average egg count per gram of root tissue across all five replicates. For the cyst trials, the average number of cysts collected from the five replicates was calculated for each trial and an ANOVA was used for each trial independently. Lines that performed significantly different within each trial were identified using LSD analysis (
A total of 203 soil samples were collected and processed from edamame fields in two years. The nematode genera found in both years of the survey were
Average population density of nematode genera (when present) identified in Arkansas edamame fields.
County | No. of fields |
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Faulkner | 1 | 0 a | 0 | 94 | 42 | 83 | 42 | 0 |
Johnson | 1 | 42 | 83 | 83 | 42 | 0 | 0 | 0 |
Logan | 10 | 0 | 42 | 50 | 42 | 80 | 42 | 0 |
Lonoke | 9 | 278 | 54 | 77 | 88 | 236 | 125 | 125 |
Pulaski | 1 | 0 | 125 | 83 | 250 | 167 | 125 | 0 |
White | 7 | 42 | 52 | 0 | 0 | 0 | 42 | 0 |
Yell | 4 | 339 | 0 | 369 | 42 | 83 | 42 | 0 |
Percent prevalent b | 36.36 | 39.39 | 57.58 | 39.39 | 30.3 | 21.21 | 3.03 | |
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Johnson | 7 | 2731 | 38 | 51 | 142 | 50 | 38 | 0 |
Logan | 3 | 0 | 0 | 38 | 102 | 128 | 0 | 0 |
Lonoke | 7 | 637 | 346 | 66 | 109 | 99 | 38 | 0 |
White | 8 | 0 | 61.2 | 38 | 67 | 66 | 154 | 77 |
Yell | 6 | 38 | 58 | 53 | 84 | 38 | 0 | 0 |
Percent prevalent | 22.58 | 38.71 | 48.39 | 80.65 | 58.06 | 19.35 | 3.23 |
Notes: aMean number of vermiform nematodes per 100 cm3 soil in each collected sample in the respective county; bthe percent of fields that contained the nematode genera across all sampled locations.
Location affected many of the tested variables, therefore data were analyzed and reported by site independently. The mean second-stage juvenile (J2) population density at harvest ranged from 79 to 110/100 cm3 soil at Hope and 2 to 1,018 J2s/100 cm3 of soil at Fayetteville in all infested treatments (Table 2). Root galling severity was significantly greater at the two higher initial infestation rates at Hope, and at the highest infestation rate at Fayetteville, compared to the lower infestation levels. No galling was seen in the uninfested control plots. Host suitability, as indicated by egg production on roots, was high in all infested treatments at both locations with some variability in the Hope location. Plant top dry weight declined with increasing initial nematode infestation levels at the Hope location, while only the highest initial density resulted in lower plant weights at Fayetteville (Fig. 1A). Dry root weights were not significantly different between treatments (data not shown). Fresh pod weight and fresh weight of shelled seed were suppressed by the highest initial infestation level at the Hope location with no significant effect in the Fayetteville location (Fig. 1B,C). Other data including the number of pods per plant, the number of seeds per 30 grams of pods and the ratio of 1-bean, 2-bean and 3-bean pods were not affected by
Initial infestation density (eggs per microplot) |
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Gall ratinga | Average number of eggs from one root ball |
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0 | 0 a,b | 0 a | 0 a |
1,000 | 92 b | 6.2 b | 1,681,720 b |
10,000 | 79 b | 8.3 c | 1,510,267 b |
100,000 | 110 b | 9.1 c | 682,973 a,b |
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0 | 0 a | 0 a | 0 a |
1,000 | 2 a | 5.2 b | 200,720 b |
10,000 | 297 b | 7.0 b | 859,840 b |
100,000 | 1,018 c | 9.7 c | 1,559,313 c |
Notes: aRoot gall rating scale from 1 to 10 where 0 = no galling and 10=100% root system with severe galling; bMeans within columns for each location with the same letter do not differ significantly (p ≤ 0.05) by LSD.
More J2s and cysts were recovered from both soil and roots in plots with the highest initial infestation levels at both locations (Table 3). The highest initial infestation level resulted in lower edamame plant top dry weights and lower average number of pods than for the all other treatments at the Hope location. There were no treatment effects on plant dry weight or pods at Fayetteville (Fig. 2A,B). The number of seeds per 30 grams of pods was greater at the highest initial infestation level at Hope, and higher than the intermediate infestation rate at Fayetteville (Fig. 2C). This measurement indicates that average seed size of the edamame was significantly smaller in higher nematode density treatments. Pod weight and fresh seed weight were suppressed at the highest initial infestation level compared to the control and lowest initial infestation level at both locations (Fig. 2D,E).
Initial infection level (eggs per microplot) |
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Cysts per 100 cm3 soil | Cysts from roots in microplots at harvest |
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0 | 0 a | 0 a | 0 a |
1,000 | 2 a | 8 a | 97 a |
10,000 | 26 a | 67 a | 745 a |
100,000 | 199 b | 850 b | 7,674 b |
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0 | 176 a | 14 a | 240 a |
1,000 | 102 a | 12 a | 220 a |
10,000 | 531 a | 36 a | 774 a |
100,000 | 2,068 b | 254 b | 3,923 b |
Note: aMeans within columns for each location with the same letter do not differ significantly (p ≤ 0.05) by LSD.
Food composition percentage on a dry weight basis from edamame seed in
Treatment (eggs per plot) | Moisture | Protein | Lipid | Starch |
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0 | 71.37 a | 38.24 a | 11.16 a | 10.29 a |
1,000 | 70.37 a | 38.23 a | 12.83 a | 10.56 a |
10,000 | 68.04 b | 36.89 b | 14.58 a | 10.08 a |
100,000 | 68.83 b | 36.46 b | 14.66 a | 10.06 a |
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0 | 76.04 a | 36.53 a | 10.02 a | 9.27 a |
1,000 | 75.41 a | 36.44 a | 10.27 a | 9.56 a |
10,000 | 76.19 a | 36.92 a | 12.10 a | 9.43 a |
100,000 | 75.75 a | 36.47 a | 10.40 a | 9.58 a |
Note: aMeans within columns for a site differ significantly at p ≤ 0.05 by LSD.
Food composition percentage on a dry weight basis from edamame seed in
Treatment (eggs per plot) | Moisture | Protein | Lipid | Starch |
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0 | 70.15 a,b | 37.57 a | 10.14 a | 10.73 a |
1,000 | 70.61 a | 37.57 a | 10.85 a | 10.46 a |
10,000 | 69.59 a,b | 36.86 a,b | 10.38 a | 10.91 a |
100,000 | 66.24 b | 36.18 b | 11.66 a | 10.94 a |
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0 | 74.87 a | 35.86 a | 8.03 a | 12.62 a |
1,000 | 75.33 a | 36.37 a | 7.82 a | 12.39 a |
10,000 | 74.82 a,b | 35.71 a | 8.87 a,b | 13.01 a |
100,000 | 74.46 b | 34.66 b | 9.62 b | 13.25 a |
Note: aMeans within columns for a site differ significantly at p ≤ 0.05 by LSD.
The gall ratings from the three trials were not significantly different, therefore gall rating data are presented as the average. The maximum root gall rating over the three trials was 8.6 (“R08-4014”, in Trial 3), and the minimum gall rate was 1 (“R07-589”, in Trial 1). When comparing ratings across the three trials, the mean root galling rates were consistently lower in edamame lines “R07-589”, “V96-7198”, “R07-7645” and “R07-7722” compared to the other evaluated lines (Fig. 3), including the widely used commercial cultivar, “8080”. There was significant variation in the egg count per gram of root between the three root-knot trials (
There were no significant differences between the two repetitions, so the data were combined for analysis. The highest number of cysts per plant, 332, was collected from “Randolph” and the lowest cyst count of 195 was collected from “R07-7645”. All the evaluated soybean lines were suitable hosts for
In the field survey, many nematode genera were found associated with edamame production.
In the microplot study,
It was observed at edamame harvest that there was nematode contamination in the cyst nematode study at the Fayetteville location. Cyst nematodes were detected in the uninfested control treatment at levels not significantly different than the two lowest cyst-treated plots. The contamination of cyst is likely due to residual cysts from previous trials that were not eradicated in the decontamination prior to conducting the trial. This theory was confirmed by performing a host differential test using cysts collected from the uninoculated control plots and identifying the cysts to be of race 14 (H.G. type 1.3), as opposed to the Race 5 used in this experiment (data not shown). The data collected at Fayetteville did not have a successful control, and results may not be reliable. However, the repetition study in Hope does indicate there are possible effects of both root-knot and cyst nematode on edamame quality parameters.
In both soybean cyst and root-knot resistant assays, all lines were overall good hosts for
“R07-589” produces a dark brown seed coat, a unique characteristic in vegetable soybean cultivars. The seed of edamame varieties that are currently on the market commercially in the USA are a vivid green, but there is no current market demand for dark-colored edamame. It is common for disease-resistant soybean lines to have the trait for a dark-colored bean (Shannon et al., 2004). Dark brown soybeans may contain a lower amount of protein but more oil per gram of seed tissue than green soybeans. In comparison, black soybeans contain the most protein (Shurtleff and Aoyagi, 2009). Black and dark brown soybeans are commonly used in Asian culture for both human and animal consumption. These components of edamame breeding line “R07-589” could provide a unique breeding and marketing opportunity not only for its moderate resistance to
Edamame continues to grow in production and popularity in the state of Arkansas. Our studies document the presence of parasitic nematodes and their potential threat to the relatively new industry and that these nematodes are a pest of concern on edamame. Our study also indicates that if the nematode pressure is high enough, nematodes could have a significant impact not only on yield but also seed composition such as protein and moisture levels of edamame, which, in turn, could reduce the marketability of the crop. Where