Kimchi cabbage can be damaged by two cyst nematodes, the clover cyst nematode (CCN; H. trifolii) and the sugar beet cyst nematode (SBCN; H. schachtii), which are classified as quarantine nematodes in Korea (Animal and Plant Quarantine Agency, 2022; Mwamula et al., 2018). The economic loss caused by these two cyst nematodes is estimated at 16.4 million USD in Korea (unpublished data). Similarly, legume crops have been damaged by two cyst nematodes: H. glycines and H. sojae. They have caused pod declines and yield loss (31.2 million USD) (unpublished data) in soybean in Korea (Han and Cho, 1980; Kang et al., 2016, 2021). Although it is impossible to eradicate nematodes in fields, various management strategies are used to reduce the damage caused by cyst nematodes (Riggs and Schuster, 1998).

Currently, two species of cyst nematodes that parasitized Kimchi cabbage are managed as regulatory control pests and diseases (Kwon et al., 2018). Control methods include chemical and agricultural control methods, and chemical compounds are effective in suppressing the cyst nematodes on Kimchi cabbage (Lee et al., 2018). Agricultural control is an effective method of controlling the population of cyst nematodes because the host range of cyst nematodes is narrow (Riggs and Schuster, 1998). Crops that are not hosts for cyst nematodes, resistant varieties, and nematode-inhibiting cover crops can be used for intercropping (Cook and Noel, 2002; Ko et al., 2020; Miller et al., 2006). In Korea, the government is conducting a public control project that focuses on reducing the density in the soil and suppressing the spread of cyst nematodes by using green fertilizer crops such as white mustard and nematicidal agents, and paying loss compensation for fallow farmland where the number of nematodes exceeds 1,000 individuals in 500 cm³ soil (Ko et al., 2020). In order to apply control methods to stop the spread and mitigate damage, it is very important to accurately separate and identify CCN and SBCN in fields growing Kimchi cabbage, and to quickly identify new outbreaks.

The life cycle of cyst nematodes consists of several stages: egg, juvenile, and adult. The adult female lays eggs (Moens et al., 2018). Second-stage juveniles hatch from these eggs and invade the roots of host plants in the soil (Turner and Subbotin, 2013). After three molts, the juveniles mature into adult females and males (Subbotin et al., 2010). The female dies, and her body forms a protective cyst containing the eggs (Koenning and Sipes, 1998). Cysts containing many eggs should be extracted from soils while studying the taxonomy, physiology, ecology, and control of the PPNs to establish the integrated pest managements against the nematodes. Several methods, including sieving and centrifugal flotation, have been used to extract cysts from soil (Barker, 1985). The paper strip method (PSM) is very simple, inexpensive, and accurate, but can be time-consuming (European and Mediterranean Plant Protection Organization, 2013). To conduct bioassays on nematodes, large numbers of cysts should be rapidly extracted. The centrifugal flotation method (CFM) combined with a sieve is a faster extraction method. A sugar solution is typically used for nematode extraction in CFM (Rodriguez-Kábana and King, 1975). The sugar solution used in this case acts as a hypertonic solution and may influence the viability of nematodes. However, there is an absence of study on the efficiency of cyst extraction by specific gravity of sugar solution (SGSS) and the impact of sugar solutions on eggs hatching rate in cyst nematodes.

This study was conducted to find the optimal SGSS for extracting cysts. To keep the nematodes suspended and isolate them, the minimum specific gravity of the extract used is 1.084 or higher (Andrássy, 1956). Generally, extracts with a specific gravity of 1.15 or 1.18 are used for nematode isolation, and extracts with a specific gravity of 1.28 are used for isolation cysts of cyst nematodes (van Bezooijen, 2006). Therefore, when applying the CFM using extracts with four different specific gravity, the effect of SGSS on the cyst extraction efficiency of cyst nematodes (CCN and SBCN) was investigated. In addition, whether high-specific sugar solutions affect egg hatching was evaluated.

Soil samples infected with CCN and SBCN were used in this study. Since 2017, two species of nematodes have been reproduced using Kimchi cabbage plants in soil. Infected soil was blended with non-infected soil in a ratio of approximately 1:5 to reduce the overall nematode density. Fifteen soil samples with CCN and nine soil samples with SBCN, each weighing 100 cm³, were prepared. We used sieving and CFM to extract cysts from the soil samples. The soil samples were mixed with 4 liter tap water, and the suspension was sequentially filtered through 850 μm and 250 μm mesh sieves (Eisenback and Zunke, 1998). The residue on the 250 μm mesh sieve was transferred into a 50 mL falcon tube using a wash bottle containing tap water, and kaolin powder (0.5 g) was added. The tube was placed in a centrifugal machine (MF-550, Hanil Science Industrial, Incheon, Korea) and rotated at 3,000 rpm for 4 min. Next, we removed the supernatant, and the sugar solution with one of four specific gravities (1.15, 1.20, 1.25, and 1.30) was added to each tube. When preparing the sugar solution, we used commercial white sugar, and measured the specific gravity using a hydrometer with a range of 1.1 to 1.2 and 1.2 to 1.3 (Densimètre, Alla France, Chemillé en Anjou, France). The tubes were then rotated in a centrifuge at 3,000 rpm for 2 min. The supernatant was filtered through a 250 μm mesh sieve, and the residue on the sieve was transferred to a square petri dish (100 mm × 100 mm). Cysts in the sediment of the tube were extracted using PSM. The number of cysts in the supernatant and sediment was observed under a stereomicroscope (MZ5, Leica, Wetzlar, Germany). PSM was used for cyst extraction from the control soil samples (European and Mediterranean Plant Protection Organization, 2013). The soil samples were mixed with 4 liter of tap water and the suspension was filtered through 850 μm and 250 μm mesh sieves. The residue on the 250 μm mesh sieves was filtered using filter paper. The debris on the paper was observed under a stereomicroscope to determine the number of cysts. The cyst extraction efficiency was calculated using the following formula:

An in vitro assay was performed to determine whether SGSS affected egg hatching, and the efficiency was only evaluated to CCN J2s. The cysts extracted using CFM were transferred to a Baermann funnel, and hatched second-stage juveniles (J2s) in the funnel were collected in a 15 ml centrifuge tube (120 mm length × 17 mm diameter) once a week for 4 weeks. The collected J2s were stored at 4°C using a refrigerator. After 4 weeks, the unhatched eggs inside the cysts were collected in a 15-ml centrifuge tube. The number of hatched J2s and unhatched eggs were counted under a stereomicroscope (MZ5, Leica). The egg-hatching rate was calculated using the following formula:

The cyst extraction efficacies of both methods are described in Table 1. Using CFM, a large amount of the cysts of CCN (maximum 93%) in soil was floated in the supernatant (Table 1, Fig. 1). The extraction efficiency was higher using PSM (100%) than CFM (87-93%). This may be because some cysts were deposited after centrifugation. The SGSS affected the cyst extraction rates when using CFM. The largest number of cysts was extracted with 1.30 SGSS (93%), followed by 1.25 (92%), 1.20 (89%), and 1.15 (84%) SGSS. The cyst extraction efficiency of 1.30 SGSS was improved by 9% and 4% compared to those of 1.15 and 1.20 SGSS, respectively (df = 4, P = 0.000592). However, the efficiency of 1.30 SGSS was not significantly different from that of 1.25 SGSS. In an experiment using cysts of SBCN, the cyst extraction efficiency of 1.30 SGSS (93%) was higher than that of 1.20 (87%), and the extraction efficiency in 1.30 SGSS was improved by 6% compared to that of 1.20 SGSS (df = 2, P = 5.98e-05). The result in the SBCN experiment was consistent with that of CCN (Table 2); the cyst extraction efficiency was improved at a higher specific gravity.

The egg-hatching rates by cyst extraction method and SGSS in CFM are shown in Table 3. The overall egg-hatching rate of CCN was observed to be approximately 20%. The hatching rate for 1.25 SGSS (27%) was the highest, followed by 1.20 (24%), 1.15 (23%), and 1.30 (20%) SGSS and PSM (18%). Using CFM with sugar solution, the egg-hatching rate was higher than that of PSM. However, the rates were not significantly different between treatments. This result indicate that the egg-hatching rate of cyst nematode was not affected by sugar solutions at the test concentrations. Eggs of cyst nematodes that are not adversely affected by the sugar solution can be used for bioassays.

Centrifugal sugar-flotation methods are typically used when studying animal parasites, such as nematodes, coccidians, and cryptosporidiosis. In animal-parasitic nematodes, using an SGSS of 1.30 is the most effective method for extracting nematode eggs through centrifugal sugar flotation from pet feces (Medeiros et al., 2018). For PPNs, 1.18 to 1.23 SGSS is commonly used to extract cysts and juveniles in the soil using CFM (Barker, 1985; Eisenback and Zunke, 1998), but there is no study on the efficiency of cyst extraction by specific gravity. Our results showed that the cyst extraction of PPNs can be extracted more efficiently in 1.30 SGSS than in 1.20 SGSS, which was used previously by other research groups. Using a sugar solution with 1.30 specific gravity in CFM can improve cyst extraction efficiency by 4-6%. Heterodera sojae, the white soybean cyst nematode, has a thicker and denser cyst wall in terms of thickness, composition, and texture compared to other cyst nematode species, which may require a higher specific gravity for separation (Han et al., 2020; Kang et al., 2016). Additionally, CCN is significantly larger in all morphological characteristics compared to H. glycines and H. sojae (Han et al., 2020). Therefore, using high specific gravities of SGSS can sometimes be helpful in isolation methods for species identification of cyst nematodes.

To extract PPNs and animal parasites eggs, many kinds of solvent, such as sugar solution, zinc sulfate, and magnesium sulfate floatation fluid, are used in centrifugal floatation (Barker, 1985; European and Mediterranean Plant Protection Organization, 2013; Farr and Luttermoser, 1941; Hirano, 1963; Sawitz, 1942). The extraction efficiency of PPNs could be different by solvents, which are used in CFM (Farr and Luttermoser, 1941). Therefore, it is essential to investigate the efficiency of cyst separation based on the solvents used in centrifugal flotation in further research.

For tapeworms and zooplankton, which are animal parasites, the sugar solution has no direct effect on egg hatching after CFM (Lukic et al., 2016; Matsuo and Kamiya, 2005). For PPNs, to the best of our knowledge, the effect of sugar solution on egg hatching in CFM has not been assessed till now. In our experiment, the hatching of cyst nematode eggs was not affected by sugar solutions ranging from 1.15 SGSS to 1.30 SGSS. Cyst nematode eggs are enclosed in hard cuticle (Baldwin and Handoo, 2018), cyst has a gelatinous matrix inside cysts, and the matrix is attached near the eggs (Meyer and Wergin, 1998). For these features, egg hatching in cyst nematodes, such as CCN and SBCN, is estimated to be not affected by high gravity of sugar solution. Therefore, the use of 1.30 SGSS in CFM can be widely adopted as an efficient method for extracting cysts of various cyst-forming nematodes, including Heterodera spp. and Globodera spp., in soil samples.

The egg hatching of cyst nematode varies depending on the species and various factors, including host plants, inorganic ions, and temperature. The hatching rate of SBCN (H. schachtii) is higher than that of CCN (H. trifolii) under the same host plant conditions, and the eggs hatching rates were different by root diffusates of the host plant (Steel et al., 1982). The cumulative hatching rates of cereal cyst nematode (H. avenae) eggs were 49% under 30 mM ZnCl₂ and 13% under 10 mM FeCl₃, with the maximum hatching rate observed at pH 8.5 (Li et al., 2020). CCN, SBCN, and soybean cyst nematode (H. glycines) also exhibited similar results to H. avenae with ZnCl₂ (Steel et al., 1982; Tefft and Bone, 1985). Studies on rice cyst nematodes (H. sacchari and H. oryzicola) revealed that the cumulative egg-hatching rate in distilled water was below 10% during one month, but the hatching rate was increased when plant root diffusates were treated (Ibrahim et al., 1993). When investigating the cumulative egg hatching of SBCN within the temperature range of 5°C to 30°C in tap water condition, the highest rates (20.8%) were observed at 25°C (Vandenbossche et al., 2015). In this study, we used tap water to hatch cyst nematode eggs under in vitro. The hatching rates of CCN eggs were 18% in the control group (PSM), and ranged from 20% to 27% in the group using CFM. We confirmed that the egg-hatching rate of CCN collected from field soil at 15 cm depth was observed to be around 20% in another study (Han et al., 2020). Comparing with previous study, the hatching rate of CCN eggs was never low in tap water conditions. Therefore, our results suggest that can be used in CFM when required accurate detection of cyst nematodes in soil using as high specific gravities of SGSS. It can also help you prepare for experiments with eggs and second-stage juveniles because it doesn’t affect hatchability.