Kimchi-cabbage is one of the important vegetables in Korea, serving as a key ingredient in the traditional Korean fermented dish known as Kimchi. This crop is cultivated throughout Korea, with a total cultivation area of 30,537 ha (Ministry of Agriculture, Food and Rural Affairs, 2023). Gangwon-do accounts for 23% (6,940 ha) of the total Kimchi-cabbage cultivation area in Korea and is a main producing area for highland Kimchi-cabbage, with 4,069 ha of cultivation area during the summer season. Therefore, Kimchi-cabbage is considered a significant agricultural industry in Gangwon. However, severe damage has recently occurred to the crop due to plant pathogens such as verticillium wilt, bacterial soft rot, Turnip mosaic virus, and cyst nematodes (Dumin et al., 2021; Kwon et al., 2018; Mwamula et al., 2018; Park et al., 1998, 2023).
Cyst nematodes are among the most important plant-parasitic nematodes in agriculture, with major genera including Heterodera, Globodera, Punctodera, and Cactodera (Choi, 2001). Specifically, the genus Heterodera can cause significant economic losses in various crops (Subbotion et al., 2010). In Korea, seven Heterodera species have been reported, including soybean cyst nematode (H. glycines, HG), white soybean cyst nematode (H. sojae, HSo), sugar beet cyst nematode (H. schachtii, HS), clover cyst nematode (H. trifolii, HT), rice cyst nematode (H. elachista and H. oryzae), and Korean cyst nematode (H. koreana) (Choi, 2001; Kang et al., 2016; Mwamula et al., 2018; Vovlas et al., 1992). Recently, cyst nematodes have been causing damage to Fabaceae and Brassicaceae crops in Korea.
In the USA, HG is responsible for causing annual losses of more than US $1.5 billion, and cereal cyst nematode (H. avenae) causes annual losses of US $3.4 million in wheat production (Moens et al., 2018). Heterodera schachtii has caused yield losses of €90 million in European countries (Müller, 1999). In Korea, HG is widely distributed in soybean fields and is causing an estimated economic loss of US $30.77 million (Kang et al., 2021). Since 2011, Kimchi-cabbage has been adversely affected by cyst nematodes in Korea, resulting in estimated yield losses of US $15 million in 2023 (unpublished data). Two cyst nematodes, HS and HT, have been inflicting damage on Kimchi-cabbage in Korea, leading to symptoms such as stunted growth, foliar wilting, and suppression of head-formation (Mwamula et al., 2018). While the damage caused by HS and HT to Kimchi-cabbage is known, the extent of their distribution has not yet been investigated.
The objective of this study was to assess the incidence and distribution of HS and HT in the main producing area of Kimchi-cabbage in Korea. Furthermore, this research aimed to identify the dominant cyst nematode species responsible for the damage observed in Kimchi-cabbage.
Materials and Methods
Soil sampling and cyst nematodes isolation
From 2018 to 2021, soil samples were collected from 469 Kimchi-cabbage fields in eight provinces of Korea (Gyeonggi-do, Gangwon-do, Chungcheongnam-do, Chungcheongbuk-do, Jeollabuk-do, Jeollanam-do, Gyeongbuk-do, and Gyeongnam-do). For each field, 30 subsamples were taken and mixed at a depth of 15 cm using a hand trowel, and approximately 1 liter of soil was collected. The soil samples were stored in a 10°C cold room until nematode extraction. Cyst nematodes were extracted using a sieving method. 100 ml of each soil sample was measured using a plastic beaker, then mixed with 4 l of tap water in a 5 l plastic container to create a soil suspension. The suspension was filtered through 20 mesh (850 μm) and 60 mesh (250 μm) USA Standard Test Sieve (Gilson Co., Lewis Center, OH, USA) in sequence, and the residue on 60 mesh sieve was transferred to a square Petri dish. The number of cysts present in the dish was counted under a stereo-microscope (MZ12, Leica, Wetzlar, Germany). All detected cysts were placed into a 5 ml tube containing 1 ml of tap water, and the cyst walls were disrupted using a homogenizer to examine the total number of eggs.
Nematode DNA extraction
To identify the cyst nematode species detected in 161 of the 469 Kimchi-cabbage fields, cysts were extracted from each field, and genomic DNA was extracted using a modified method from Iwahori et al. (2000). A single cyst was placed on a slide glass, and the eggs and juveniles inside were removed using a small piece of filter paper chip (2 × 2 mm2) (n = 3). A fresh filter paper chip was used to crush the eggs and juveniles by rubbing them to break the cell. The filter paper fragment was then placed into a 200 μl polymerase chain reaction (PCR) tube containing 10 μl of DNA extraction solution (1 ml triple distilled water, 10 μl 1 M Tris-HCl, 10 μl 10% Triton X-100, 9 μl 100 mg/ml Proteinase K, 2 μl 1 M KCl, and 2 μl 1 M MgCl2). The tube was placed in a thermal cycler (PTC-200, MJ Research, Alameda, CA, USA) and incubated at 65°C for 30 min followed by 94°C for 10 min to extract the genomic DNA.
Polymerase chain reaction
To identify the cyst nematode species, PCR was performed on the mitochondrial DNA cytochrome c oxidase subunit I (mtCOI) gene, commonly used for DNA barcoding. The mtCOI gene region of the cyst nematode was amplified using forward primer JB3 (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′) and reverse primer JB5 (5′-TAA AGA AAG AAC ATA ATG AAA ATG-3′) (Derycke et al. 2005). A total volume of 50 μl PCR reaction mixture was prepared in a 200 μl PCR tube, containing 1 μl template DNA, 0.5 μl of 10 pmol forward primer (JB3), 0.5 μl of 10 pmol reverse primer (JB5), 15 μl of PCR premix (Ready-2X-Go with Taqplus, Nanohelix, Daejeon, Korea), and 33 μl of triple distilled water. The PCR reaction was conducted in a thermal cycler (PTC-200, MJ Research) with a pre-denaturation step at 94°C for 10 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s. The PCR products were analyzed by electrophoresis on a 1% agarose gel in 1× TAE buffer (0.045 M Tris-borate, 0.001 M EDTA) at 100 V and 25 mA for 25 min, and the amplification was confirmed using a UV transilluminator (MLB-21, Maestrogen, Hsinchu, Taiwan).
DNA sequence analysis and phylogenetic tree construction
DNA sequencing of the PCR products was conducted using the sequencing service of Macrogen Inc., utilizing the ABI 3730xl System (Thermo Fisher Scientific Inc., Waltham, MA, USA). Based on the sequencing results obtained, the mtCOI gene sequence of the cyst nematode was determined using Chromas Lite (version 2.0), EditSeq 5.05 (DNAstar, Madison, WI, USA), and SeqMan 5.05 (DNAstar). The completed mtCOI gene sequence data of the cyst nematode was compiled with sequences registered in the National Center for Biotechnology Information (NCBI) to create a dataset, which was aligned using Clustal X (ver. 1.83) with default options. This dataset was executed in the Bayesian phylogenetic tree analysis program MrBayes (version 3.2.6) to construct a molecular phylogenetic tree, which was subsequently edited using Dendroscope (version 3.5.7). The outgroup for the cyst nematode was referenced from the study by Vovlas et al. (2015).
Precise distribution mapping of nematodes
The addresses of 161 fields, where cyst nematodes were detected among 469 Kimchi-cabbage fields, were organized sequentially in Microsoft Office Excel and converted into GPS coordinates using Google Sheets (Supplementary Table 1). The converted GPS coordinates were then inserted as circular markers into the map layer using the QGIS software (Quantum Geographic Information System, https://www.qgis.org). A Google Road Map layer (https://mt1.google.com/vt/lyrs=m&x={x}&y={y}&z={z}) was added to QGIS to generate a precise distribution map of cyst nematode infestations based on the field addresses.
Results
Incidence of cyst nematode in main Kimchi-cabbage production areas
The results of the cyst nematode incidence investigated in the main Kimchi-cabbage production areas (28 cities across 8 provinces) are shown in Table 1. Among the 469 surveyed fields, 188 fields (40%) were found to be infested with cyst nematodes. The average infestation density was 393 cysts per 500 cm3 of soil, with an average of 37,759 eggs. By province, Gangwon-do had the highest cyst nematode infestation rate at 78%, with an average of 419 cysts and 39,988 eggs per 500 cm3 of soil. This was followed by Gyeongsangbuk-do (17%), Gyeonggi-do and Gyeongsangnam-do (7%), and Jeollanam-do (3%). No cyst nematodes were detected in the cabbage fields of Chungcheongnam-do, Chungcheongbuk-do, or Jeollabuk-do. Among the cities, Taebaek city had the highest infestation rate at 89%, with an average density of 522 cysts and 49,734 eggs. This was followed by Samcheok (77%), Jeongseon (75%), Yeongwol (40%), Muan (40%), Andong (29%), Gumi, Haman, and Hwaseong (20%), Mungyeong (17%), and Jindo (3%). No cyst nematodes were detected in cabbage fields from other regions.
Identification and distribution of cyst nematode species detected in main Kimchi-cabbage production areas
The results of molecular identification using PCR for cyst nematodes detected in 161 Kimchi-cabbage fields in Korea are presented in Table 2 and Fig. 1. Four cyst nematode species were identified as follows: HS, HT, HG, and HSo. Six infestation types were observed, including single-species infestations by each of the four cyst nematode species, as well as mixed infections of HT + HG and HG + HSo. The most prevalent infestation type was single-species infestation by HT, which accounted for 78% of all cases, followed by HG (11%), HS (4%), HT + HG (4%), HSo (2%), and HG + HSo (1%). In Gangwon-do, which had the highest cyst nematode infestation rate, three cyst nematode species were identified. The most common was HT (85%), followed by HG (7%) and HS (4%). Additionally, 6 fields (4%) showed mixed infestations of HT and HG. In Taebaek city, where the highest detection rate of cyst nematodes was recorded, HT dominated with a detection rate of 95%, while HG appeared either as a single-species infestation (4%) or in mixed infestation with HT (1%). The cyst nematodes HT and HS, known to cause significant damage to cabbage, were detected only in Gangwon-do. HG was found in Gyeonggi-do, Gangwon-do, Chungcheongbuk-do, Gyeongbuk-do, and Gyeongnam-do, while HSo was detected exclusively in Jeollanam-do. These findings indicate that, by 2021, Kimchi-cabbage damage caused by cyst nematodes was limited to the Gangwon-do.
Discussion
Cyst nematodes that can damage Brassicaceae crops, such as HS and HT, have been reported (Inserra et al., 1993; Okada et al., 2021). HS and HT are among the cyst nematodes causing significant damage to crops, and are distributed globally, including in the United States, European countries, and countries in Asia (Subbotin et al., 2010). In Korea, HS was first reported in 2011 in highland Kimchi-cabbage fields in Taebaek, Gangwon-do, and began to cause damage (Kim et al., 2016). The nematode spread to highland Kimchi-cabbage fields in Samcheok and Jeongseon (Kwon et al., 2018). In 2017, a cyst nematode morphologically similar to HS but with yellow-colored females (in contrast to the white-colored females of HS) was first detected in highland Kimchi-cabbage fields in Jeongseon (Maas et al., 1982; Mwamula et al., 2018; Wang and Riggs, 1999). The cyst nematode detected in Jeongseon was identified as HT based on morphological characteristics, COI gene sequence analysis, and phylogenetic tree analysis (Ko et al., 2018; Mwamula et al., 2018). Since 2017, both HS and HT have caused damage to Kimchi-cabbage in the highland areas of Gangwon-do. However, nationwide surveys targeting Kimchi-cabbage fields, including those in highland Gangwon-do, only focused on the distribution of HS between 2011 and 2013.
From 2011 to 2013, the National Institute of Agricultural Sciences, Rural Development Administration, conducted a survey on the incidence of cyst nematodes in Kimchi-cabbage fields across six provinces (Gangwon-do, Gyeongsangbuk-do, Jeollanam-do, Jeollabuk-do, Chungcheongnam, and Chungcheongbuk). The results revealed the presence of cyst nematodes in Kimchi-cabbage fields in Gangwon-do, Jeollabuk-do, Chungcheongnam-do, and Chungcheongbuk-do, with an average density of 33 cysts per 500 cm3 of soil (Lee et al., 2014). In this study, from 2018 to 2021, we investigated the nationwide distribution of cyst nematodes in Kimchi-cabbage fields across eight provinces (excluding Jeju) (Table 1, Supplementary Table 1). As a result, cyst nematodes (Heterodera spp.) were detected in five provinces: Gyeonggi-do, Gangwon-do, Jeollanam-do, Gyeongbuk-do, and Gyeongnam-do, with an average infestation density of 393 cysts and 37,759 eggs per 500 cm3 of soil. Compared to the survey conducted from 2011 to 2013, the average infestation density of cyst nematodes in Kimchi-cabbage fields from 2018 to 2021 was 12 times higher. This increase is likely due to the continued cultivation of the host crop in infested fields, which may have contributed to the growth of the nematode population (Jun and Park, 2001; Kang et al., 2015). However, the survey from 2011 to 2013 did not provide detailed descriptions of cyst nematode incidence by province and city/county, making direct comparisons impossible.
Between 2014 and 2017, surveys on cyst nematode occurrence focused only on highland Kimchi-cabbage fields in Gangwon-do. The detection rates varied by region, with the highest observed in Samcheok (65.7%), followed by Yeongwol (50%), Taebaek (25.8%), Jeongseon (16.7%), and Gangneung (10%) (Kwon et al., 2018). In this study, the detection rates in the Gangwon-do were 95% in Taebaek, 76% in Samcheok, and 73% in Jeongseon, with HT newly detected in one field in Gangneung and HS in one field in Yeongwol. Notably, Taebaek, Samcheok, and Jeongseon showed a significant increase in cyst nematode infestations, likely due to the long-term continuous cropping of Kimchi-cabbage, which has led to higher detection rates. However, no previous studies have examined the relationship between continuous cabbage cultivation and the increase in cyst nematode densities, highlighting the need for additional research.
The cyst nematodes detected in Kimchi-cabbage fields of Korea were identified using PCR analysis. Lee et al. (2014) previously identified cyst nematodes detected in Kimchi-cabbage fields from 2011 to 2013 using internal transcribed spacer 1 region sequences. All cyst nematodes found in Taebaek, Jeongseon, and Samcheok were identified as HS, making it the dominant species. The detection rates for HS were highest in Jeongseon (46.4%), followed by Taebaek (29.6%) and Samcheok (26.0%). In contrast, this study used phylogenetic tree analysis based on the COI gene sequences, a recommended DNA barcode marker for animals, to identify species (Blok and Powers, 2009; Vovlas et al., 2015). The results revealed four cyst nematode species: HS, HT, HG, and HSo, with HT being the dominant species (Table 2, Fig. 1). The detection rates in Kimchi-cabbage fields of Korea were 78% for HT, 11% for HG, 4% for HS, and 2% for HSo, with 4% and 1% of the fields showing mixed infections of HT + HG and HG + HSo, respectively. Heterodera trifolii is known to prefer warmer temperatures than HS and has a capacity for rapid reproduction through parthenogenesis (Karakas, 2011; Maas and Heijbroek, 1982). Over the past 40 years, temperatures in Korea have continued to rise due to global warming, which may explain the shift from HS being the dominant species in the previous study in 2011-2013 to HT in this study (Khanal and Land, 2023; Seo et al., 2020). However, further research is needed to understand the precise effects of temperature on the proliferation and life cycle of HS and HT in Korea.
Surveys on the occurrence of cyst nematodes in highland Kimchi-cabbage fields in Taebaek, Samcheok, and Jeongseon from 2014 to 2017 revealed that HS and HT were predominantly detected in the fields near major roads (Kwon et al., 2018). In contrast, this study confirmed that HS and HT were distributed across numerous fields beyond the major road areas (Fig. 2). This suggests that soil from fields near the main roads may have been transferred to other fields during cabbage harvesting, as soil adhered to vehicle tires likely spread to various areas (Kwon et al., 2016). Therefore, to effectively manage HS and HT, which pose a significant threat to highland Kimchi-cabbage, it is crucial to consider proactive sanitary approaches to limit the spread by harvest vehicles as well.
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