Morphological and molecular characterizations of Heterodera oryzae in Korea
Online veröffentlicht: 06. Nov. 2020
Seitenbereich: 1 - 12
DOI: https://doi.org/10.21307/jofnem-2020-113
Schlüsselwörter
© 2020 Rose Mwesige et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Rice [
Four species of cyst nematodes infect rice viz
In a survey to identify plant-parasitic nematodes on rice in South Korea, cyst nematodes were found in two rice fields in Dang-jin city and this required a detailed study of the samples in order to get accurate information on the nematode species. Nematode species identification is a requirement before designing and undertaking any management strategy in crop production. Species identification using morphological characters alone is very difficult and does not give correct results. A combination of both morphological and molecular approaches is very crucial for proper and accurate identification (Subbotin et al., 2015). The use of mitochondrial DNA cytochrome c oxidase subunit I (mt
Soil samples were taken from two rice fields in Dang-jin city – South Korea using soil auger from about 15 cm in depth and placed in plastic bags. Samples were labeled as SG936 and SG153 (Table 1). Cysts were extracted from 100 cm3 of a soil sample using the sieving method (20 and 60 mesh). To establish pure cultures, second-stage juveniles (J2s) obtained from a single cyst were used to infect rice plants growing in pots in a plant growth room kept at 25°C. The plants were left to grow for five months before extracting cysts from pot soil. After extraction, cysts were collected with forceps into a watch glass containing tap water using a stereomicroscope (MZ12; Leica, Wetzlar, Germany) and were kept at 4°C until further use.
In total, 13 cysts for SG936 and 25 cysts for SG153 were used for full body measurement, vulval cone making, and J2s collection. Full shapes for cysts were captured to measure their length and width.
Vulval cones were cut under a stereomicroscope, transferred to glycerin on slide glasses for microscopic examination. Important characters for distinguishing cyst nematode species such as the underbridge length, presence of bullae, fenestral length, semifenestral width, and vulval slit length were observed under a light microscope, imaged and measured (Sekimoto et al., 2017).
Important taxonomic diagnostic characters for J2s were obtained by temporarily mounting juveniles on slide glasses for examination under a light microscope (DM5000; Leica, Wetzlar, Germany). An automatic camera attached to a LEICA DM5000 compound microscope was used to capture images for measurements. J2s measurements were made for body length, middle body width, stylet length, tail length, hyaline tail length, and excretory pore distance from the anterior part of the body. Measurements were recorded in µm, arithmetic mean, standard deviation, and measurement ranges obtained. Nematodes were identified morphologically based on
To extract DNA, a single cyst was transferred to a glass slide on a small drop of distilled water, opened and its contents crushed using a filter paper chip (2 mm × 2 mm) and forceps. Using forceps, the chip having crushed eggs and J2s was transferred into a PCR tube containing 30 µl lysis buffer (autoclaved triple distilled water, 1 M Tris-HCl, 10% Triton-X 100, 100 µg/ml Proteinase K, 2 M KCl, 1 M MgCl2) for extracting nematode DNA (Iwahori et al., 2000). The tubes were then incubated in a PCR machine (PTC-200; MJ Research, Alameda, CA, USA) at 60°C for 1 hr and 94°C for 10 min. The DNA was stored at −20°C for later use in PCR reactions.
The internal transcribed spacer (ITS) region was amplified using 0.5 µl each of primers TW81 [5′-GTTTCCGTAGGTGAACCTGC-3′] and AB28 [5′-ATTGCTTAAGTTCAGCGGGT-3′] (Joyce et al., 1994). In a PCR tube containing 15 µl of PCR mixture ready to use (Ready-2x-Go with Taqplus; NanohelixTM, Daejeon, Korea) was added 2 µl of nematode DNA extract, 32 µl of triple distilled water, and 0.5 µl of each primer making a total volume of 50 µl. Cycling included pre-denaturation step at 94°C for 5 min followed by 40 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min, and finished with one cycle at 72°C for 10 min.
Primers D2A [5′-ACAAGTACCGTAGGGAAAGTT-3′] and D3B [5′-TCGGAAGGAACCAGCTACTA-3′] (De Ley et al., 1999) were used to amplify the region. PCR conditions were pre-denaturation at 94°C for 6 min followed by 40 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min and finished with one cycle at 72°C for 6 min.
A primer set of JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB5 (5′-AGCACCTAAACTTAAAACATAATGAAAATG-3′) (Derycke et al., 2005) was used in the PCR reaction. PCR conditions were pre-denaturation step at 94°C for 4 min followed by 40 cycles of 94°C for 30 sec, 57°C for 30 sec, and 72°C for 45 sec and finished with one cycle at 72°C for 5 min.
PCR products were analyzed by electrophoresis on 1% agarose gel with 1X TAE buffer for 25 min. The gels were visualized using UV transilluminator (UVCI-1100; Major science, New Taipei City, Taiwan).
After amplification, PCR products were purified using PCR purification kit following the manufacturer’s manual (NucleoSpin Gel and PCR clean up; Macherey-Nagel, Duren, Germany). Purified DNA was sent for sequence analysis at GenoTech Corporation (Daejeon, Korea). Obtained sequences were edited using EditSeq application to remove low quality bases and assembled in SeqMan computer program. Assembled sequences were posted in National Center for Biotechnology Information (NCBI) to obtain similarity match with other
Morphological characters and morphometric features of J2s (stylet length, body length, tail length, and number of lateral lines) together with cysts and their vulval cone features were examined and measured for species identification. The color of cysts varied from light to dark brown having either oval or lemon shape. Vulval cones were ambifenestrate with bullae and a strong underbridge (Figs. 1, 2). Second-stage juveniles had a strong stylet with an average length of 21 µm. The body length ranged from 414 to 478 µm and 433 to 495 µm for SG936 and SG153, respectively (Table 2). The juveniles for the two samples had three incisors in the lateral field (Figs. 1F, 2F).
Nematode samples used in this study.
| Population code | Collection date | Host plant | Coordinates |
|---|---|---|---|
| SG936 | July 27, 2016 | Rice | 36.902188, 126.661624 |
| SG153 | August 16, 2017 | Rice | 36.901005, 126.671008 |
Morphometrics of second-stage juveniles and cysts for the
| Population | SG936, South Korea, This study | SG153, South Korea, This study |
|
|
|
|---|---|---|---|---|---|
| Cysts ( |
13 | 25 | 50 | 50 | 39 |
| Length (excl. neck) | 498.9 ± 96.4 (307.0-617.2) | 523.3 ± 54.5 (438.9-643.3) | 714.8 ± 71.5 (442-999) | 446 ± 30.3 (328-557) | 431 ± 48 (340-586) |
| Width | 429.3 ± 97.3 (251.2-550.1) | 360.2 ± 47.2 (288.3-447.8) | 426.6 ± 53.8 (229-655) | 322.6 ± 26.0 (229-449) | 311 ± 48 (225-540) |
| Length/width | 1.2 ± 0.1 (1.0-1.4) | 1.5 ± 0.2 (1.3-1.8) | 1.71 ± 0.12 (1.1-2.3) | 1.4 ± 0.1 (1.1-1.9) | 1.4 ± 0.2 (1.1-1.9) |
| Vulval areas ( |
11 | 16 | 10 | 10 | 10 |
| Fenestral length | 36.5 ± 4.7 (28.2-40.9) | 34.2 ± 4.9 (24.8-43.6) | 38.8 ± 1.85 (32-46) | 29.3 ± 2.0 (23.36) | 40 ± 6.5 (30-50) |
| Semifenestral width | 34.8 ± 4.4 (29.3-44.2) | 35.7 ± 4.4 (25.9-41.0) | 51.6 ± 5.20 (36-69) | 32.0 ± 2.5 (26-39) | 31.2 ± 4.7 (26-42) |
| Vulval slit length | 33.1 ± 5.0 (27.6-45.6) | 34.3 ± 3.8 (27.8-41.3) | 50.7 ± 4.0 (41-65) | 37.5 ± 2.8 (26-46) | 40 ± 3.8 (32-45) |
| Vulval bridge width | 9.0 ± 4.5 (4.4-17.9) | 7.2 ± 3.3 (4.0-15.0) | 12.14 ± 1.9 (6-16) | 5.8 ± 2.0 (2-16) | – |
| Underbridge length | 80.1 ± 7.2 (70.8-96.4) | 83.0 ± 7.9 (63.4-95.3) | 112.5 ± 9.0 (87-153) | 72.0 ± 2.9 (65-78) | 85 |
| Vulva to anus distance | 34.9 ± 3.0 (30.5-41.6) | 36.6 ± 5.9 (30.1-50.3) | – | – | – |
| J2 ( |
11 | 11 | 20 | 20 | 10 |
|
|
450.6 ± 16.6 (414.7-478.6) | 472.4 ± 19.3 (433.1-495.1) | 554 ± 9.4 (523-589) | 402 ± 10.0 (377-450) | 391 ± 11 (372-410) |
|
|
23.4 ± 0.7 (22.4-24.6) | 23.3 ± 1.6 (20.1-25.9) | 34.0 ± 1.03 (30.1-39.3) | 24.6 ± 0.9 (21.3-28.1) | 22.5 ± 1.0 (20.8-24) |
|
|
– | – | |||
|
|
6.8 ± 0.3 (6.3-7.4) | 6.8 ± 0.3 (6.3-7.2) | 9.4 ± 0.3 (8.6-10.7) | 7.0 ± 0.4 (5.4-8.4) | 6.7 ± 0.3 (5.9-7.2) |
| Stylet (S) | 21.1 ± 0.5 (20.4-22.4) | 21.1 ± 0.8 (19.5-22.6) | 20.5 ± 1.15 (16-25) | 19.2 ± 0.8 (16-21) | 20 ± 0.7 (18-21) |
| Labial region height | 4.7 ± 0.5 (3.8-5.4) | 4.7 ± 0.3 (4.3-5.3) | – | – | 3 |
| Labial region diam. | 9.0 ± 0.7 (7.8-10.1) | 9.1 ± 0.3 (8.7-9.6) | – | – | 7.8 ± 0.4 (7-8) |
| DGO | 7.3 ± 0.7 (6.5-8.4) | 6.0 ± 0.8 (4.9-7.7) | – | – | 5.2 ± 0.8 (4-7) |
| Anterior end to | |||||
| Excretory pore | 97.5 ± 4.8 (87.5-103.0) | 99.3 ± 6.6 (84.8-110.8) | – | – | 84 ± 2.8 (79-88) |
| Median bulb valve (MB) | 65.4 ± 2.7 (62.1-72.6) | 64.4 ± 2.2 (60.6-68.0) | – | – | 59 ± 3.6 (52-67) |
| Body diam. at | |||||
| Mid-body | 19.2 ± 0.8 (18.1-20.7) | 20.4 ± 1.9 (18.2-24.3) | 16.3 ± 0.43 (15-18) | – | 17.4 ± 0.8 (16-19) |
| Anus (BDA) | 12.6 ± 0.9 (11.7-14.5) | 13.3 ± 0.8 (12.0-14.6) | 12.3 ± 0.72 (10-15) | 11.4 ± 0.8 (10-13) | 10.2 ± 0.6 (9-11) |
| Hyaline region (H) | 36.9 ± 3.2 (29.3-41.0) | 35.8 ± 3.4 (31.6-42.4) | 32.8 ± 1.3 (26-44) | – | 32 ± 3.0 (25-39) |
| Tail length (Tl) | 66.9 ± 3.8 (60.5-73.4) | 69.6 ± 4.7 (60.6-78.9) | 58.8 ± 2.25 (49-66) | 57.8 ± 2.9 (47-70) | – |
| H/S | 1.7 ± 0.1 (1.4-1.9) | 1.7 ± 0.2 (1.4-2.0) | 1.6 ± 0.11 (1.4-2.2) | – | 1.6 ± 0.1 (1.3-1.9) |
| L/MB | 6.9 ± 0.2 (6.6-7.2) | 7.3 ± 0.3 (6.9-8.1) | – | – | 6.7 ± 0.4 (5.6-7.4) |
| Tl/H | 1.8 ± 0.2 (1.6-2.5) | 2.0 ± 0.1 (1.7-2.2) | – | – | – |
Figure 1:
Morphological characteristics of cyst, vulval cone, and second-stage juvenile of
Figure 2:
Morphological characteristics of cyst, vulval cone, and second-stage juvenile of
ITS region, LSU D2-D3 (28S) segments, and
Figure 3:
PCR results for ITS region, 28S D2-D3, and
Similarity search of the ITS sequences obtained from our study population revealed higher similarities with
Similarity search of the 28S sequences obtained in our study in NCBI GeneBank database ranged from 99.73 to 100% identity with
A BLASTn search with the studied nematode sequences on the mt
Phylogenetic relationships of the studied species are indicated in Figures 3-5. Figure 3 shows a phylogenetic tree based on the ITS region data set of
Figure 4:
Phylogenetic relationship between two
Figure 5:
Phylogenetic relationship between two
The phylogenetic tree of the 28S region dataset of
The
Using identification key by Subbotin et al. (2010) for
DNA barcoding has increasingly become common for efficiency and accuracy in nematode species identification (Amiri et al., 2003). The 28S, ITS,
Molecular analysis of mtDNA is very important to speed up and simplify the identification of animals such nematodes (Herbert et al., 2003a, 2003b; Lambshead, 2004). This is because the mtDNA is less exposed to recombination, lacks introns, and has a relatively high mutation rate compared to nuclear genome (Saccone et al., 1999). The features of mtDNA bring about a significant variation in mtDNA sequences between species than within species (Elsasser et al., 2009), facilitating phylogeny construction to address confusion in species boundaries and population variations (Blok and Powers, 2009).
The morphology of second stage juveniles and cysts and molecular analysis of