First report of Aphelenchoides bicaudatus (Imamura, 1931 ) Filipjev and Schuurmans Stekhoven, 1941 associated with grass in South Africa
Article Category: Research Note
Published Online: Dec 30, 2022
Page range: 414 - 423
Received: Apr 13, 2022
Accepted: Aug 17, 2022
DOI: https://doi.org/10.2478/helm-2022-0035
Keywords
© 2022 E. Shokoohi, N. A. G. Moyo, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Grasslands are one of the most critical biomes in South Africa (Le Roux et al., 2011; Richardson et al., 2020), which cover the northern parts of the Western Cape Province. Grasslands constitute a significant component of the natural vegetation. The interface between grasslands and other biomes contributes substantially to their floristic and faunal diversity and to their important role in the agricultural economy, including livestock. The grasslands of South Africa are also home to most of the human population across the country (Le Roux et al., 2011; Richardson et al., 2020).
Therefore, the aims of the present work were 1) to study the morphology of
Specimens were collected at the Kirstenbosch National Botanical Garden in Cape Town (S: 33° 59’ 13.19”; E 18° 25’ 29.39”) and Magoebaskloof (S: 23°52’40.368”; E: 29°56’14.459”) from the rhizosphere of grass plants (Family: Poaceae;
DNA extraction was done using the Chelex method (Straube & Juen, 2013). Five specimens of the analyzed species were hand-picked with a fine tip needle and transferred to a 1.5 ml Eppendorf tube containing 20 μl double distilled water. The nematodes in the tube were crushed with the tip of a fine needle and vortexed. Thirty microliters of 5 % Chelex® 50 and 2 μL of proteinase K were added to the microcentrifuge tube that contained the crushed nematodes and mixed. These separate microcentrifuge tubes with the nematode lysate were incubated at 56 °C for two hours and then incubated at 95 °C for 10 minutes to deactivate the proteinase K and finally spin for 2 min at 16000 rpm (Shokoohi, 2021). The supernatant was then extracted from the tube and stored at –20 °C. Following this step, the forward and reverse primers, 988F (5ʹ-CTCAAAGATTAAGCCATGC-3ʹ) and 1912R (5ʹ-TTTACGGTCAGAACTAGGG-3ʹ) for 18S rDNA (Holterman et al., 2006), and TW81 (5ʹ-GTTTCCGTAGGTGAACCTGC-3ʹ), and AB28 (5ʹ-ATATGCTTAAGTTCAGCGGGT-3ʹ) for ITS rDNA (Joyce et al., 1994) were used in the PCR reactions for partial amplification of the 18S and ITS rDNA region, respectively. PCR was conducted with eight μl of the DNA template, 12.5 μl of 2X PCR OneTaq® Quick-Load® 2X Master Mix with Standard Buffer (Inqaba Biotec, South Africa), one μl of each primer (10 pmol μl-1), and ddH2O for a final volume of 30 μl. The amplification was processed using an Eppendorf Mastercycler gradient (Eppendorf, Hamburg, Germany), with the following program: initial denaturation for 3 min at 94 °C, 37 cycles of denaturation for 45 s at 94°C; 54 °C and 53°C annealing temperatures for 18S rDNA and ITS rDNA for 30 s, respectively; extension for 45 s to 1 min at 72 °C, and finally an extension step of 6 min at 72 °C followed by a temperature on hold at 4 °C. After DNA amplification, four μl of product from each tube was loaded on a 1 % agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and one mM EDTA) for evaluation of the DNA bands. The bands were stained with the SafeView™ Classic stain (Applied Biological Materials Inc. (abm); Canada) and visualized and photographed on a UV transilluminator. The PCR products for 18S rDNA and ITS rDNA were stored at –20 °C. Finally, the PCR products were purified and sequenced by Inqaba Biotech (South Africa). The obtained ribosomal DNA sequences were analyzed and edited with BioEdit (Hall, 1999) and aligned using CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were generated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The HKY+Γ (gamma distribution of rate variation with a proportion of invariable sites) model was selected using jModeltest 2.1.10 (Guindon & Gascuel, 2003; Darriba et al., 2012). Analysis using the GTR+G+I model was initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 106 generations for 18S and ITS rDNA. The trees were visualized with the TreeView program (Page, 1996). Also, as outgroups,
To evaluate the morphological variations between the populations of A. bicaudatus, principal component analyses (PCA) were conducted using XLSTAT software (Addinsoft, 2007). Various morphometric features obtained from fixed nematodes, including body length, a (body length/greatest body diameter), b (body length/ neck length), c (body length/tail length), cˈ (tail length/anal body diameter), V (% anterior end to vulva/body length), stylet length, and tail length were included in the PCA analyses (Table 2). The morphometric measurements for the different populations were taken from their original descriptions. The measures were normalized using XLSTAT software before their analysis (Addinsoft, 2007). The scores values were determined for each species based on each of the principal components, and the scores for the first two components were used to form a two-dimensional plot (PC1 and PC2) of each isolate based on the eigenvalues given by the software XLSTAT.
Measurements of females of
| Province | Western Cape | Limpopo |
|---|---|---|
| Locality | Cape Town | Magoebaskloof |
| n | 8 females | 4 females |
| L | 455.3 ± 64.5 (409 – 529) | 451.7 ± 56.7 (415 – 517) |
| a | 29.1 ± 2.0 (27.1 – 31.1) | 28.9 ± 0.7 (28.2 – 29.6) |
| b | 4.6 ± 0.5 (4.1 – 5.1) | 4.4 ± 0.3 (4.1 – 4.7) |
| M | 47.9 ± 3.4 (44.1 – 50.6) | 49.4 ± 3.6 (45.5 – 52.6) |
| c | 16.3 ± 2.0 (14.1 – 17.8) | 15.2 ± 1.3 (14.3 – 16.7) |
| c’ | 3.2 ± 0.6 (2.6 – 3.6) | 3.3 ± 0.3 (3.1 – 3.6) |
| V (%) | 68.8 ± 1.6 (67 – 71) | 68.7 ± 1.7 (67 – 70) |
| Lip region height | 2.8 ± 0.9 (2 – 4) | 3.1 ± 0.5 (3 – 4) |
| Lip region width | 5.7 ± 0.1 (5.6 – 5.8) | 5.4 ± 0.4 (5.1 – 5.8) |
| Stylet length | 11.1 ± 1.7 (10 – 13) | 9.6 ± 0.6 (9.5 – 10) |
| Conus | 4.7 ± 0.3 (4.5 – 5.0) | 4.8 ± 0.3 (4.5 – 5.0) |
| Mid of median bulb to anterior end. | 47.3 ± 3.2 (45 – 51) | 48.3 ± 2.6 (46 – 51) |
| Median bulb diameter | 9.0 ± 0.8 (8.5 – 10.0) | 9.2 ± 0.8 (8.0 – 10.0) |
| Median bulb length | 11.2 ± 0.7 (11 – 12) | 10.9 ± 1.0 (10 – 12) |
| Pharynx length | 88.3 ± 11.2 (76 – 98) | 93.2 ± 7.3 (86 – 100) |
| Neck | 99.0 ± 8.7 (89 – 104) | 103.3 ± 7.5 (95 – 110) |
| Nerve ring from anterior end | 71.7 ± 0.6 (71 – 76) | 73.0 ± 2.0 (71 – 75) |
| Excretory pore from anterior end | 61.3 ± 3.2 (60 – 65) | 60.3 ± 1.5 (59 – 62) |
| Body diameter at median bulb | 12.5 ± 1.8 (11 – 15) | 12.3 ± 1.5 (11 – 14) |
| Body diameter at mid body | 15.6 ± 1.5 (14 – 17) | 15.7 ± 2.1 (14 – 18) |
| Body diameter at anus | 8.8 ± 0.7 (8 – 9) | 9.1 ± 0.1 (8 – 10) |
| Anterior branch of reproductive system | 179.3 ± 18.0 (167 – 200) | 199.7 ± 53.2 (167 – 261) |
| Post-vulval uterine sac | 47.0 ± 2.6 (45 – 50) | 46.0 ± 1.0 (45 – 47) |
| Vagina length | 6.3 ± (6 – 7) | 6.7 ± 0.6 (6 – 7) |
| Rectum | 14.3 ± (12 – 16) | 14.7 ± 1.5 (13 – 16) |
| Tail length | 28.0 ± (24 – 31) | 29.7 ± 1.2 (29 – 31) |
Morphological important characters of females of
| Present study | Imamura, 1931 (Filipjev and Schuurmans Stekhoven (1941)) | Siddiqui and Taylor (1967) | Jen |
Kim |
Israr |
|
|---|---|---|---|---|---|---|
| Population | South Africa | Japan | USA | Taiwan | South Korea | Pakistan |
| L(μm) | 455.3 ± 64.5 (409-529) | 430 (380 - 470) | 460 (410-550) | 499.12 ±67.95 (376-637) | 517.9 ±3.8 (513.6-522.6) | 360 |
| a | 29.1 ±2.0 (27.1-31.1) | 31.5 (31.3-31.7) | 28.0 (25-31) | 33.03 ± 2.42 (27.00-38.64) | 28.3 ± 0.5 (27.7-28.8) | 30.1 -32.7 |
| b | 4.6 ±0.5 (4.1-5.1) | 7.4 (6.8-8.4) | 8.2 (7.3-9.6) | 9.0 ±0.7 (7.5-10.0) | 7.3 ±0.0 (7.3-7.4) | 7.2-8.8 |
| c | 16.3 ±2.0 (14.1-17.8) | 10.6 (9.4-12.6) | 11.4(9.8-13.7) | 11.94 ± 0.93 (10.16-14.80) | 11.3 ±0.5 (10.7-11.9) | 11.3-12.0 |
| c' | 3.2 ±0.6 (2.6-3.6) | 4.4* | 4.7* | 5.41 ±0.56 (4.13-7.14) | 4.6 ±0.1 (4.4-4.8) | 2.9-3.7 |
| Tail (μm) | 28.0 ±3.6 (24-31) | 44* | 41 | 37-43 | 45.9 ±2.5 (43.1 -48.8) | 30-31 |
| V(%) | 68.8 ± 1.6 (67-71) | 70.4 (61.7-90.2) | 67.5 (65-70) | 68.53 ± 1.20 (64.90-71.83) | 66.0 ± 0.2 (65.7-66.4) | 66.8-67.2 |
| 11.1 ±1.7(10-13) | 10* | 11.2(10-12) | 10.38 ±0.63 (9-12) | 11.2 ± 0.5 (10.4 - 11.7) | 10-11 |
Morphological characterization (Eight females in a good state of preservation)
Fig. 1
(A) anterior end; (B) entire female; (C) reproductive system; (D) female posterior end; (E) Post-vulval uterine sac.
The nBlast test of 18S rDNA showed 98 % similarity of the test population with the South Korean population of
The phylogenetic analysis using 18S and ITS rDNA, placed the South African
Fig. 2
Phylogenetic position of
Fig. 3
Phylogenetic position of
An accumulated variability of 74.86 % was observed in female based PCA, specifically, 42.66 % in the PC1 and 32.20 % in the PC2 (Fig. 4). The variables b (
Fig. 4
PCA plot of different populations of
Loading factor of the variables of the different populations of
| PC1 | PC2 | |
|---|---|---|
| L | 0.293 | 0.714 |
| a | 0.435 | -0.784 |
| b | 0.865 | -0.037 |
| c | -0.862 | -0.008 |
| c′ | 0.866 | 0.323 |
| Tail | 0.811 | 0.417 |
| V | 0.073 | -0.668 |
| Stylet | -0.483 | 0.852 |
Factor score of the variables of the different populations of
| Observation | PC1 | PC2 |
|---|---|---|
| South Africa | -3.296 | -0.120 |
| Japan | 1.298 | -1.668 |
| USA | 0.332 | 1.408 |
| Taiwan | 1.912 | -0.479 |
| South Korea | 0.476 | 2.367 |
| Pakistan | -0.723 | -1.507 |
The genus
Additionally, principal component analysis using morphometric features of species of
The results indicated that there is intraspecific morphological variation across
Three permanent microscope slides, containing the females of