1. INTRODUCTION

The root-lesion nematode, Pratylenchus spp. is a migratory endoparasitic nematodes with over 350 hosts, including import crops such as soybeans, potatoes, corn, bananas, wheat, and cabbage. The genus Pratylenchus Filipjev, 1936 contains approximately 100 diverse species (Handoo et al. 2021), with 16 species recorded in the Republic of Korea (Park 1996;Park et al. 2002;Choi et al. 2006;Kim et al. 2006;Park et al. 2009;Kim and Chun 2014).

Research on nematodes in Korean agricultural environments is being conducted (Ko et al. 2021). In addition, nematodes are an important factor in investigating the microbial environment in the Korean plant ecosystem (Eo et al. 2018). The large number of hosts as well as its migratory endoparasitic habits make this genus a very important root-lesion nematode in crops. During the survey of soil nematodes, Pratylenchus hippeastri was isolated from Giant pussy willow (Salix chaenomeloides Kimura). Pratylenchus hippeastri, also known as the amaryllis lesion nematode, has been previously reported from the USA (De Luca et al. 2010;Handoo et al. 2020), China (Wang et al. 2016), and South Africa (Knoetze et al. 2019;Shokoohi 2019). The host range of this nematode includes amaryllis, bromeliads, grapevines, strawberry and apples (De Luca et al. 2010;Wang et al. 2016;Knoetze et al. 2019;Handoo et al. 2020;Brenes-Campos et al. 2022), as well as Acer (Chen et al. 2014) and cape willow trees, Salix mucronata (Shokoohi 2019). This study reports the first detection of this species in Korea from Giant pussy willow, or S. chaenomeloides.

2. MATERIALS AND METHODS

Collection methods of nematode. Soil and roots were collected from a Giant pussy willow tree in Andong (36°25ʹ48.0ʺN 128°38ʹ20.0ʺE), Gyeongsangbuk-do, Korea. Nematodes were separated from soil and roots using the modified Baermann funnel method (Kang 2016).

Morphological analysis. In the analysis, twelve nematodes with fully grown and confirmed genital organs were used. Individual nematodes were mounted in water on temporary glass slides, measured, and digitally photographed using an Olympus, BX53 light microscope with differential interference contrast optics and an Olympus DP73 digital camera. Additional measurements were taken from specimens processed by either the lactophenol method (Franklin and Goodey 1949) or the quick glycerol method (Seinhorst 1959).

Molecular analysis. Each nematode individuals was picked and thirty nematodes were transferred to a 2.0 mL microcentrifuge tube. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen Inc., CA, USA). Two rRNA fragments, namely LSU D2/D3 and ITS region, were amplified, respectively. The primers for D2/D3 expansion segments amplification were D2A (5ʹ-ACAAGTACCGTGGGGAAAGTTG-3ʹ) and D3B (5ʹ-TCGGAAGGAACCAGCTACTA-3ʹ) (Subbotin et al. 2006). The primers for ITS amplification were TW81 (5ʹ-GTTTCCGTAGGTGAACCTGC-3ʹ) and AB28 (5ʹ-ATATGCTTAAGTTCAGCGGGT-3ʹ) (Subbotin et al. 2008). The genomic DNA was used as a template for PCR as follows: after an initial 5-minute denaturation step at 94°C, a 40-cycle amplification (94°C for 1 minute, 56°C [D2/D3 expansion segments] to 58°C [ITS region] for 1 minute, and 72°C for 1 minute) was conducted. The final extension step was continued for 10 minute at 72°C. To confirm the successful amplification of DNA by PCR, electrophoresis was performed using 0.5× TAE buffer on a 1% agarose gel. The PCR product was subsequently purified using a PCR purification kit (Bioneer Co., Korea). The amplicons were cloned into pGEM-T Easy Vector system (Promega, USA), and the resultant plasmid DNA was isolated using a Plasmid Miniprep System (Bioneer Co., Korea). Both strands of the PCR amplicons were cycle-sequenced with an ABI PRISM BigDye Terminator version 1.1 Cycle Sequencing kit and ABI Prism ABI 377 Genetic analyzer (PE Applied Biosystems, CA, USA).

Phylogenetic analysis. The LSU D2/D3 expansion segments and ITS sequences of P. hippeastri were compared using the BLAST sequence alignment software of the NCBI database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/). Sequences were also aligned using the ClustalW implemented MEGA 11.0 software. Results of closest nucleotide sequences were selected to BLASTn analysis. The seqences of the ITS region exhibited 99% similarity to P. hippeastri (FN554886.1) and 28S rDNA D2/D3 expansion segment were found to be 99% identical to those of P. hippeastri (MK749422.1). Phylogenetic analyses were performed using MEGA version 11.0 (Tamura et al. 2021) with the neighbor-joining (NJ) statistical method and the Jukes-Cantor model.

3. RESULTS

Pratylenchus hippeastriInserra, Troccoli, Gozel, Bernard, Dunn, and Duncan, 2007

왕버들뿌리썩이선충 (신칭) (Figs. 1-4; Table 1)

Description. Female (n=12) : Body slender, slightly curved ventrally after relaxation with length=424.1± 16.0 μm and maximum body diameter=19.0±3.8 μm (Figs. 1A, 2A). Lip region flatten with two annuli. The stylet is strong with length=15.3±0.4 μm, basal knobs ellipsoidal or slightly concave with a height=1.8± 0.1 μm and width=2.9±0.1 μm (Fig. 1B). Pharyngeal glands overlapping intestine ventrally (Fig. 2B). Spermatheca rectangular without sperm. Vulval lips usually slightly raised in some specimens. Vulval body diameter= 16.9±0.7 μm and distance between vulva to anus=73.0±16.6 μm, anal body width=11.5±1.0 μm. Tail conoid with bluntly pointed or subhemispherical, smooth terminus with length=24.7±2.7 μm (Figs. 1C, 2C). De Man ratios are a=22.8±2.0 μm, b=5.7±0.2, b′=4.1±0.1, c=16.7±1.5, c′=2.2±0.1, V=79.3±0.4 (Table 1).

Male : Not found.

Molecular characters and phylogenetic analyses. The sequenced LSU D2/D3 expansion segments of P. hippeastri were 748 bp in length. A Blastn search of the LSU D2/D3 expansion segments sequence revealed high matches with the originally reported sequences of P. hippeastri (KP161608.1 and MK749422.1). The identities of the LSD D2/D3 expansion segments and the originally reported sequence were 99.5% without insertions or deletions. The phylogenetic tree based on the LSU D2/D3 expansion segments sequences showed that P. hippeastri was nested within the same clade with all the reported P. hippeastri (Fig. 3). The sequenced ITS segments of P. hippeastri were 932 bp in length. A Blastn search of the ITS sequence revealed high matches with the originally reported sequences of P. hippeastri (FJ713000.1). The identities of ITS segments and the originally reported sequence were 99.6% without insertions/deletions. The phylogenetic tree based on the ITS sequence showed that the sequence of P. hippeastri obtained in this study and all the reported P. hippeastri sequences formed a high degree of similarity (Fig. 4). The result was indicated as P. hippeastri with a high probability, but the number of matching data was small. Compared with the results of the LSUD2/D3 expansion segments sequence, it is assumed that it is simply the result of a small number of data.

4. DISCUSSION

In this study, the morphometric and morphological characteristics of P. hippeastri from Korea were found to be consistent with the original report, except for a shorter body and tail length (424.14±16.0 μm and 24.7±2.7 μm vs. 614±22.4 μm and 33.3±3.0 μm, respectively) (De Luca et al. 2010). The body and tail length of P. hippeastri from Korea were identified to be similar to those from China (Wang et al. 2016) and Japan (Gu et al. 2014). The body and tail length of P. hippeastri from China was 447.8±28.2 μm and 25±1.9 μm, respectively. While those from Japan were 479.9± 31.9 μm and 29.5±3.1 μm, respectively.

The variability in body length within a species can be affected by nutritional or environmental factors (Castillo and Vovlas 2007). The body lengths of P. hippeastri from different region varied, while the ratio of V (%, distance of vulva from anterior) is very similar: among specimens form Korea (79.3±0.4 μm), China (78.2±1.3 μm) (Wang et al. 2016), Japan (77.2±1.0 μm) (Gu et al. 2014), California (77±0.8 μm) (Inserra et al. 2007), and Florida (77.7±1.2 μm) (De Luca et al. 2012), respectively. Pratylenchus hippeastri may be better adapted to warm tropical regions (Wang et al. 2016). Consequently, P. hippeastri found in tropical ornamentals such as amaryllis and bromeliads (Inserra et al. 2007;De Luca et al. 2012) were larger than those found on apple trees from China (Wang et al. 2016). Significant variation in length has previously been observed in Pratylenchus species. Bogal et al. (2021) reported that the body length of seven populations of P. penetrans collected in Europe ranged from 381 to 625 μm. P. hippeastri was previously identified as an unrecorded species of Pratylenchus in Korea (NIBR 2010). The identification relied on 28S rDNA analysis along with four photos. However, the specimens lacked information on morphological measurements, locality, or host plant details. Therefore, this is the first comprehensive report of P. hippeastri in Korea. P. hippeastri, a root-lesion nematodes, can damage apples, strawberries, and grapevines as well as willows native to Korea, also a possibility of infection with other unknown species of plants, requiring further research and quarantine.

Key to Pratylenchus Species in Korea