Haplotype diversity of Heterodera koreana (Tylenchida: Heteroderidae), affecting bamboo in Korea

In a survey of plant-parasitic nematodes in agricultural fields, cyst-forming nematodes were found in soil planted bamboo in Korea. The aim of this study was to identify the cyst nematodes based on morphological and molecular characteristics. As the results, the morphology and morphometrics of cysts and second-stage juveniles (J2s) were consistent with those of previous descriptions of Heterodera koreana. In phylogenetic analyses based on DNA sequences, these cyst nematodes were clustered together with clade of H. koreana in internal transcribed spacer (ITS) region, and large subunit D2-D3 segments (LSU D2-D3). These nematodes were clustered together with clade of H. koreana in cytochrome c oxidase subunit I (COI) gene, but a haplotype was different when compared with previous reported haplotypes (haplotype A-C) in Japan. This study showed these cyst nematodes were identified as H. koreana, and a new haplotype of H. koreana is distributed in Korea. We suggest that the new haplotype of H. koreana name as haplotype D.

H. koreana and H. sojae were rst discovered in Republic of Korea, and were reported as a novel cyst nematode species.Since then, the nematodes distributions were reported in other countries (Kang et al., 2016;Vovlas et al., 1992).H. koreana was described as Afenestrata koreana at the rst time by Vovlas et al. (1992), but the genus Afenestrata was subsequently changed to Heterodera (Mundo-Ocampo et al., 2008).Because the genus Afenestrata was not classi ed as a speci c clade and was clustered together with Heterodera spp. in phylogenetic study, synonymisation of Afenestrata with Heterodera was proposed (Mundo-Ocampo et al., 2008).Recently, the bamboo is cultivated as crop for tea, seasoned vegetables in Korea, and damage by the Korean cyst nematode is expected.Since the Korean cyst nematode (H.koreana) rstly reported, there were no further ndings or studies of the cyst nematode.The study of the cyst nematode based on molecular characteristics is required because it is di cult to identify the Heterodera species using morphological and morphometrical characteristics.
In this study, we identi ed the cyst nematodes extracted from bamboo using the morphological characteristics and the molecular characteristics based on DNA barcoding gene such as LSU D2-D3 expansion segments, ITS region, and mitochondrial DNA CO gene.Furthermore, we specialized the haplotype of the Korean cyst nematode based on the COI gene and named the new haplotype.

Materials and Methods
Nematode isolation.Soil samples were collected from bamboo rhizosphere of three elds a depth of approximately 15 cm soil in Sacheon city, Gyeongsangnam-do in Korea using soil sampler (diameter 2 cm).The samples were labeled as BC348, DR437, and DR1256, respectively (Table 1).Cysts were extracted by sieving method using 20 and 60 mesh sieves (Kang et al., 2016).After extraction, cysts and J2s were transferred into a watch glass containing tap water using forceps and pipette under a stereomicroscope (MZ205; Leica, Wetzlar, Germany) and were kept at 4℃ until further use.Morphological analysis.For light-microscopic observations, J2s were killed and xed by addition of 80 o C FG 4:1 xative (Southey, 1986).The nematodes were xed for at least 24 hr, then processed according to a Seinhorst method (Cid Del Prado Vera and Subbotin, 2012;Seinhorst, 1959).Specimens were mounted on Cobb slides and sealed with a para n ring and glycerin (Cobb, 1917).Vulval cones were cut under a stereomicroscope (M205; Leica, Wetzlar, Germany), and were transferred to glycerin on slide glasses.The nematodes were observed, measured, and photographed with the aid of a compound microscope (DM5000; Leica, Wetzlar, Germany) equipped with microscope digital camera (DFC450; Leica, Wetzlar, Germany).Also, overall shape of cysts, shape of annule, vulval cone, head and lateral eld were observed.The nematodes were identi ed morphologically based on Heterodera species identi cation key authored by Subbotin et al. (2010).Molecular analysis.To extract genomic DNA, each single cyst of three different populations was transferred to a slide-glass on a small drop of distilled water, opened and its contents crushed using a lter paper chip (2 mm x 2 mm) and forceps.Using forceps, the chip having crushed eggs and J2s was transferred into a PCR tube containing 30 µl lysis buffer (sterilized triple distilled water, 1 M Tris-HCl, 10% Triton-X 100, 100 µg/ml Proteinase K, 2 M KCl, 1 M MgCl 2 ) for extracting the nematode DNA (modi ed Iwahori et al. 2000).The tubes were incubated in a Thermal cycler (PTC-200, MJ Research, Alameda, CA, USA) at 60 o C for 1hr and 94 o C for 10 min.Two ribosomal RNA fragments, i.e. the LSU D2-D3 segments, ITS regions, and CO gene of mitochondrial genome were ampli ed.Primers for D2-D3 segments ampli cation were D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') (Subbotin et al., 2006).Primers for ITS ampli cation were TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') (Subbotin et al., 2000).A primer set of JB3 (5'-TTTTTTGGGCATCCTGAGGTTTAT-3') and JB5 (5'-AGCACCTAAACTTAAAACATAATGAAAATG-3') for CO gene was used in the PCR reaction (Derycke et al., 2005).
The PCR condition was as follows: pre-denaturation stage; 94°C for 5 min, cycling stage (n = 40); denaturation at 94°C for 1 min, annealing at 56°C (D2-D3 segments), 58°C (ITS region), and 57°C (CO gene) for 1 min, respectively, and extension at 72°C for 2 min.The nal extension was continued at 72°C for 10 min.In order to verify the PCR amplicon, electrophoresis was performed using 0.5x TAE buffer on 1% agarose gel.The amplicon was subsequently puri ed using commercial PCR Puri cation Kit (Qiagen, Valencia, CA).All strands of the PCR amplicons were cycle-sequenced with an ABI PRISM BigDye Terminator version 1.1 Cycle Sequencing Kit and electrophoresed in each direction on an ABI Prism ABI 377 Genetic Analyzer (PE Applied Biosystems, USA).The newly obtained sequences were submitted to the GenBank database (Table 1)., 2000).The newly obtained and published sequences for each gene were aligned using Clustal W with default parameters (Thompson et al., 1994).Sequence alignments were manually edited using BioEdit (Hall et al. 1999).The alignment quality was examined by naked-eyes, and optimized manually by adjusting the ambiguous nucleotide positions.Models of base substitution were evaluated using MODELTEST3.7 combined with PAUP4.0 (Huelsenbeck and Ronquist, 2001;Posada and Crandall, 1998;Swofford, 2003).The Akaike-supported model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in phylogenetic analyses.Bayesian analysis was performed to con rm the tree topology for each gene separately using MrBayes 3.1.2running the chain for 1 × 10 6 generations and setting the 'burn-in' at 2500 (Huelsenbeck and Ronquist, 2001).The MCMC (Markov Chain Monte Carlo) method was used within a Bayesian framework to estimate posterior probabilities of the phylogenetic trees (Larget and Simon, 1999), and generate a 50% majority-rule consensus tree.
The posterior probabilities are given on appropriate clades.Trees were visualized using TreeView (Page, 1996).
Data of COI gene analysis.The sequences of the COI gene were assembled and aligned using MEGA version X (

Results
Morphological analysis.Morphological characters and morphometric features of cysts, vulval cone of cysts and J2s were examined and measured for species identi cation.Lemon-shaped cysts, which are variable in size (377-903 µm) with distinct neck and vulval cone, were observed (Fig. 1A).Cuticle appeared light to dark brown in colour.Stylet and other pharyngeal structures were indistinct.Gelatinous egg sac was not observed and the cyst cuticle has irregular zigzag pattern on mid-body.Vulval cone with lacking-fenestration was observed and covered with tuberculate pattern (Fig. 1B and 1C).J2s had a cylindrical body, tapering posteriorly, straight of slightly ventrally curved after xation (Fig. 1D).Stylet with a length of 20-21 µm was well developed, stylet-knobs were oviform (Fig. 1E and 1G), and the body length ranged from 454 to 545 µm (Table 2).The J2s had three incisures in the lateral eld (Fig. 1F).Anus and hyaline part of tail were distinct, and hyaline terminal section was average 50.9 (45.5-55.0)µm long (Fig. 1H).(1.0-2.9) 1.1 ± 0.1   Phylogenetic tree based on COI gene of mtDNA was described in Fig. 4. The average nucleotide compositions were as follows: 24.50% A, 8.68% C, 14.18 G and 52.64% T. Using Rotylenchus eximius and R. urmiaensis as the outgroup taxa, the molecular phylogenetic relationship of the dataset, which contains three Korean populations and previously registered data in NCBI, was closed to H. koreana.However, three Korean populations were not clustered together with Japanese haplotype of H. koreana, haplotype A-C, and were classi ed to a new clade of H. koreana.
Data of COI gene analysis.We identi ed four haplotypes (Haplotype A-C, and Korean haplotype) in the 46 COI sequences (357 bp), and the dataset included 21 polymorphisms (Table 3).The network analysis showed a radial-shaped haplotype network with the most common Haplotype A (48%) and B (43%) occupying a central position with the rest of the haplotypes radiating differing from it by up to 13 substitutions (Fig. 5).Haplotype A and C were found in Japan.Haplotype B was found in both Japan and USA.The Korean haplotype was newly found in this study in Korea.We analyzed haplotype diversity and nucleotide diversity by regional populations.In Japan populations, the haplotype diversity was 0.5317 ± 0.0319 and the nucleotide diversity was 0.0090 ± 0.0053.The Tajima's D statistic was negative for Japan populations (-0.68579).The Korean and USA populations were no diversities in the haplotype and the nucleotide, because there was no variation in regional populations.
Pairwise F ST , which is a xation index between regional populations, was calculated to estimate in genetic differentiation that can be caused by the genetic structure.The Korean population had a high xation index value 1.000 with USA populations and 0.680 with Japanese populations, indicating a differentiated genetic structure to the Korean population.On the other hand, the Japanese and USA populations had a low differentiation with a xation index of 0.177.The AMOVA showed that variation by region was responsible for 60.46% of the total variation.The remaining 39.54% of the variance was explained by the variation among populations within region (7.76%) and variation within populations (31.78%) (Table 4).

Discussion
During a PPNs survey in 2020, three populations of Heterodera species were isolated from rhizosphere of bamboo in Republic of Korea.Three cyst-forming nematodes were identi ed as H. koreana using morphological identi cation key for Afenestrata sensu stricto group and phylogenetic analysis ( Morphology and morphometrics of the cysts and the J2s of three Korean populations were consistent with the described H. koreana in China (Wang et al., 2012).However, these populations differed from original descriptions in Republic of Korea and Japanese population by the shorter cyst body length, the longer J2s body length, tail length, and length of hyaline region (Sekimoto et al., 2017;Vovlas et al., 1992).The 'c' value and 'tail length' in Korean populations were inconsistent with the original description of Iranian population (Maa and Tehari, 2015).These differences in morphology could be explained as a result of intraspeci c variation (Wang et al., 2012).
Recently, molecular and phylogenetic analysis based on the DNA barcoding genes such as LSU D2-D3 segments, ITS region, and mtDNA COI gene are very important to speed up and simplify the identi cation of animals including plant-parasitic nematodes.However, our phylogenetic study based on the COI showed the haplotype of the Korean populations was not clustered together with Japanese populations (Fig. 4).
In previous study, three CO haplotypes of H. koreana were found in Japan, and named as haplotypes A, B and C (Table 3).The haplotype A is dominant in Japan (Sekimoto et al., 2017).However, our study showed the new haplotype of H. koreana was present in Korea because the haplotype of the Korean populations was distinguished from Japanese haplotype, and the AMOVA analysis also showed that the regional differences (60.46%) between Korean populations and Japanese population was greater than that between total populations (31.78%) (Table 4).Thus, we suggest that the new haplotype of H. koreana is present in Republic of Korea and name as haplotype D (Table 3 and Fig. 5).
To investigate associations between genetic haplotype and phenotype in the nematodes, we performed morphological comparisons between the Korean haplotype and the haplotypes reported in China and Japan.Our study showed that the whole length and hyaline portion in J2s in the Korean populations were similar to that of the Chinese population (Wang et al., 2012) (Table 2).However, our results showed that the Korean population had a longer J2 body length (499 ± 15.5 vs. 458 ± 17.3 µm) and a longer hyaline length of the J2 tail (51 ± 3.0 vs. 41 ± 3.2 µm) than those of the Japanese population (Sekimoto et al., 2017) (Table 2).Thus, we suggest that the differences in the haplotype of the CO gene sequence may be affect to the morphological differences within the species, H. koreana.The studies on the association between genotype of LSU D2-D3, ITS regions, CO , and morphological characteristics were conducted in marine nematodes.The results showed that highly divergent genotype clusters were accompanied by morphological differences (Derycke et al., 2008), and in the CO gene and ITS region particularly (Fonseca et al., 2008).Because the sequence of the Chinese haplotype is absent in NCBI Genbank, the study on the association between genetic haplotype and phenotype is required.In addition, the various haplotypes could be distributed in Republic of Korea due to the similar morphology between original description of H. koreana in Republic of Korea and those of the Japanese population (Sekimoto et al., 2017;Vovlas et al., 1992).The haplotype may be associated with phenotypes of the nematodes like ecotype, and pathotype.Therefore, we require the study on correlations between haplotype and phenotype of the nematodes in further study.
The   Phylogenetic relationships between Heterodera species.Bayesian 50% majority rule consensus tree as inferred from the analysis of the CO gene sequence alignment under the TVM+I+G model.Posterior probabilities over 50% are given for appropriate clades.Newly obtained sequences are indicated by bold font.

Figure 2
Figure 2 Phylogenetic relationships within population and species of Heterodera.Bayesian 50% majority rule consensus tree from two runs as inferred from the analysis of the D2-D3 of 28S rDNA gene sequences under the GTR+I+G model.Posterior probability values more than 50% are given in appropriate clades.Newly sequenced samples are indicated by bold font.

Figure 3 Phylogenetic
Figure 3Phylogenetic relationships within population and species of Heterodera.Bayesian 50% majority rule consensus tree from two runs as inferred from the analysis of the ITS rRNA gene sequences under the TVM+I+G model.Posterior probability values more than 50% are given in appropriate clades.Newly sequenced samples are indicated by bold font.

Table 1
For phylogenetic study, the sequences of three populations compared with GenBank nematode sequences using the BLAST homology search program.The closest sequences were selected for phylogenetic analyses.Outgroup taxa for each dataset was chosen according to previous phylogenetic study for cyst-forming nematodes (Mwesige et al., 2020; Kang et al., 2016), Cryphodera brinkmani Karssen & van Aelst, 1999 and Meloidogyne alni Turkina & Chizhov, 1986 (De Luca et al., 2013; Subbotin et al.

Table 2
Morphometric comparison of Heterodera koreana populations from Republic of Korea, Japan, China and Iran.All measurements are in µm and in the form: mean ± s.d.(range).
Molecular and phylogenetic analysis.The LSU D2-D3 segments, ITS region, and COI gene of the mtDNA were ampli ed as indicated in methodology section.The sequenced LSU D2-D3 segments, ITS region, and COI are 751-755, 902-908, and 424-425 bp, respectively.A BLASTn search on the LSU D2-D3 segments and ITS region revealed similarities with the Afenestrata group of Heterodera species such as H. Koreana and H. hainanensis.The highest match of LSU D2-D3 segments sequences was H. koreana (LC202092), with 100% identities and no gaps.The ITS region results also revealed the most similar species with H. koreana (KX640828), with 99.89% identities (901/902) and no insertions/deletions.In addition, a BLASTn search of H. koreana on the COI revealed high-scoring matches with H. koreana (LC202153), which is the species isolated from Phyllostachys nigra var.henonis in Iwate in Japan.The identities between the Korean population (this study) and H. koreana (LC202153) were 98.21% (385/392), with no insertions/deletions.The molecular phylogenetic relationships of Korean populations of H. koreana were shown in Figs.2, 3, and 4. The phylogenetic tree of the LSU D2-D3 segments region dataset of Heterodera species is shown in Fig.2.The average nucleotide compositions were as follows: 19.33% A, 21.47% C, 33.92 G and 25.28% T. Using Cryphodera brinkmani and Meloidodera alni as the outgroup taxa, the molecular phylogeny strongly supported monophyly of Heterodera species.Phylogenetic tree inferred from ITS region dataset was in Fig.3.The average nucleotide compositions were as follows: 19.17% A, 22.44% C, 29.04% G and 29.35% T. Three Korean populations of Heterodera species were close to H. koreana when C. brinkmani and M. alni as the outgroup taxa were used.The results showed that BC348, DR437, and DR1256 population belong to 'Afenestrata' group clade.

Table 3
Variable position in the four CO haplotypes of Heterodera koreana populations.

Table 4
Analysis of molecular variance of three populations of Heterodera koreana.Group refers to the pooling of Japan and USA together and Korea alone.