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28Russian Journal of Nematology, 2021, 29 (2), 101 - 109
Morphological and molecular characterisation of Boleodorus volutus Lima & Siddiqi, 1963 from South Africa with the first SEM observations of
the species
Ebrahim Shokoohi
University of Limpopo, Green Biotechnologies Research Centre of Excellence, Private Bag X1106, 0727,
Sovenga, Limpopo province, South Africa e-mail: [email protected] a
Accepted for publication 12 June 2021
Summary. During a survey on plant-parasitic nematodes in South Africa, a population of Boleodorus was recovered in association with grasses. This species characterised by a conical lip region lacking incisures. Stylet length 9-11 ^m, lateral field with four incisures, post-vulval sac less than the corresponding body diameter. Tail conically elongated, often hooked at the end, 51-58 ^m long. The nblast analysis based on the D2-D3 segment of 28S rDNA of the South African population of B. volutus, revealed 98% similarity to the North American B. volutus (MT994501). The phylogenetic analysis put the South African population of B. volutus together with the other B. volutus and unidentified Boleodorus with a 100 posterior probability support. In addition, the SEM photographs are provided for the first time for B. volutus. This is the first complete report of this species in South Africa. Key words: morphology, Tylenchidae, 28S rDNA.
The genus Boleodorus Thorne, 1941 is a member of the family Tylenchidae (Geraert, 2008; Qing & Bert, 2019). These nematodes are not economically significant, but they are considered herbivores (Bongers & Bongers, 1998) or algal/moss feeders (Siddiqi, 2000). The genus Boleodorus comprises 30 nominal species (Geraert, 2008). Additionally, one species, B. abnormus Khan & Basir, 1964, has been considered as species inquirenda by Geraert (2008). Originally, B. volutus Lima & Siddiqi, 1963 has been described from England (Lima & Siddiqi, 1963). Afterwards, this species has been reported from Germany (Lelifeldt & Sturhan, 1994; Handelmann et al., 2001; Sturhan & Hohberg, 2016), The Netherlands (Bongers, 1988), Poland (Brzeski, 1998), Pakistan (Hussain & Iqbal, 2017), Iran (Alvani et al., 2013), Belgium (Geraert, 1971), Australia (Nobbs, 2003), Sweden (Boström & Sohlenius, 2005), Bulgaria (Peneva et al., 2011), Slovac Republic (Renco et al., 2020) and Morocco (Ali et al., 2017). However, no morphometrics were given for the records.
The genus Boleodorus has been reported previously from South Africa (Jansen van Rensburg, 2010; Durand et al., 2012). However, there is no
morphological relevant information available for B. volutus from South-Africa. Therefore, the aims of the study are: i) to study B. volutus using morphology; and ii) to study the phylogenetic position of B. volutus using 28S rDNA.
MATERIAL AND METHODS
Nematode extraction and processing.
Rhizosphere soil samples were collected from a wild grass in a natural area. Nematode extraction was achieved using the Baermann (1917) funnel technique. Extracted individuals were fixed with a hot 4% formaldehyde solution with the temperature about 80°C (except those specimens used for molecular analyses) and transferred to anhydrous glycerin utilising the method of De Grisse (1969) and mounted on permanent glass slides.
Scanning electron microscopy (SEM). Specimens preserved in glycerin were selected for observation under SEM, according to Shokoohi et al. (2007) and Shokoohi & Abolafia (2011). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed
© Russian Society of Nematologists, 2021; doi: 10.24412/0869-6918-2021-2-101-109 Published online 10 December, 2021
Fig. 1. Boleodorus volutus Lima & Siddiqi, 1963. A: Anterior end of the female. B: Lip region (superficial view). C: Lip region and stylet. D: Female reproductive system. E-H: Entire female. I-L: Female posterior end.
with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).
DNA extraction, PCR, and phylogenetic analysis. DNA extraction was done using the Chelex method (Straube & Juen, 2013). Five specimens of each species were hand-picked with a fine tip needle and transferred to a 1.5 ml Eppendorf tube containing 20 ^l 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 ^l of proteinase K were added to each of the microcentrifuge tubes that contained the crushed nematodes and mixed. These separate microcentrifuge tubes with the nematode lysate were incubated at 56°C for 2 h and then incubated at 95°C for 10 min to deactivate the proteinase K and finally spin for 2 min at 28,672 g (Shokoohi, 2021). The supernatant was then extracted from each of the tubes and stored at -20°C. Following this step, the forward and reverse primers, D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3'), D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') (De Ley et al., 1999), were used in the PCR reactions for partial amplification of the 28S rDNA region. PCR was conducted with 8 ^l of the DNA template, 12.5 ^l of 2X PCR Master Mix Red (Promega, USA) for the South African specimens, 1 ^l of each primer (10 pmol ^l-1), and ddH2O for a final volume of 30 ^l. The amplification was processed using an Eppendorf master cycler 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; 56°C annealing temperatures for 28S rDNA; 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, 4 ^l of product from each tube was loaded on a 1% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for evaluation of the DNA bands. The bands were stained with RedGel and visualised and photographed on a UV transilluminator. The amplicons of each gene were stored at -20°C. Finally, the PCR products were purified for sequencing by Inqaba Biotech (South Africa). Also, as an outgroup, Ditylenchus dipsaci (MG676657) was selected based on Qing & Bert (2019). The ribosomal DNA sequences were analysed and edited with BioEdit (Hall, 1999) and aligned using CLUSTAL W (Thompson et al., 1994). The length of the alignments was 1194 bp for 28S rDNA. Phylogenetic trees were generated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The GTR + I + G model was selected using
jModeltest 2.1.10 (Guindon & Gascuel, 2003; Darriba et al., 2012). The selected model was then initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 106 generations. The pairwise genetic distance and Tajima's test were calculated using Mega-X software (Kumar et al., 2018). The partial 28S rDNA of B. volutus was deposited in GenBank under their accession numbers: MW704025 and MW704026.
DESCRIPTION
Boleodorus volutus Lima & Siddiqi, 1963 (Figs 1-3)
Measurements. See Table 1.
Female. Body curved ventrally after heat relaxation. Lip region conical, continuous with body contour, lacking annuli, anteriorly flattened. A basal annuli of the lip region slightly wider than the adjacent annulus. Labial plate undivided. Lips fused, oral opening circular. Amphids small, oblique shaped, curved dorsally, appear behind the lateral lips, between the labial papillae, in Figure 2C amphid aperture covered with dust. Four cephalic papillae present on the lip region. Six small labial papillae present on the labial plate (Fig. 3). Lateral fields start from 11-13 annuli (7-8 ^m) posterior to the lip region with two incisures, with four incisures at mid-body, the two inners lines are faint, the three bands are 2.4 ± 0.4 (2.1-2.8) ^m wide at mid-body, i.e. , approximately 14-16% of the corresponding body diameter. Regular areolation of lateral fields (outer bands) observed in the pharyngeal region. Cuticle 0.7 ± 0.1 (0.5-1) ^m thick, clearly annulated, one annulus 0.5 ± 0.1 (0.4-0.5) ^m wide at mid-body. Conus 41-44% of the stylet length, shaft 55-59% of the stylet length, and knobs 6% of the stylet length. Stylet delicated, 2.3-3.1 times longer than labial region diameter. Basal knobs weakly developed, flanged and slightly sloping backwards, knob width 0.6 ^m length. Pharynx tylenchoid with procorpus cylindrical. Median pharyngeal bulb weakly developed, broadly oval, 14 x 6 ^m, located at 55.9 ± 6.9 (48-66) ^m from anterior end. Isthmus 18 ± 1.5 (17-20) ^m long, encircled by nerve ring at anterior to mid-point. Basal bulb pyriform, pharyngeal glands developed, only one nucleus visible close to the base of the basal bulb. Nerve ring enveloping isthmus at the middle, at 57-73% of the neck from anterior end. Excretory pore located at the anterior end of the basal bulb level, at 75-88% of the neck length from anterior end. Excretory duct clearly sclerotized. Hemizonid distinct, one annulus long, located one
Fig. 2. Boleodorus volutus Lima & Siddiqi, 1963, scanning electron microscopy. A-C: Anterior end of the female (lateral and sub-ventral view, respectively). D: Vulval region. E: Vulva-anus distance. F: Female posterior end. G: Anus (arrow; lines indicate lateral field).
Table 1. Measurements of Boleodorus volutus isolated from Free State province, South Africa. All measurements are in |im and in the form: mean + s.d. (range).
annulus anterior to excretory pore. The reproductive system with monodelphic-prodelphic, well developed; anterior branch 132.8 ± 25.1 (104-150) ^m long, post-vulval uterine sac occupying 56-73% of the vulval body diameter. Vulva posterior, 104.5 ± 16.3 (76-122) ^m to anus. Vulva without a distinct epiptygma. Vagina with internal walls slightly sclerotized, 5.9 ± 1.0 (5-7) ^m long. Spermatheca 12-14 * 8-10 ^m long, oval to round, offset, filled with small ameboid sperm, sperm size 2.0-3.8 ^m long, ovaries with multiple rows of oocytes, 68 ^m long (n = 1), oviduct clear in one specimen (11.6 ^m long). Crustaformeria with four cells in each row and well developed (21 ^m long), uterus tubular, 38-50 ^m long. Phasmids not visible. Tail conoid-elongated, terminus hooked, the lateral field ended 11-13 ^m posterior to the anus.
Male. Not found.
2 f.im
Fig. 3. Schematic view of lip region for Boleodorus volutus.
Diagnosis and relationships. Boleodorus
contains several species, of which B. thylactus Thorne, 1941 being the most distributed worldwide (Geraert, 2008). Two species, namely B. thylactus and B. volutus have already been studied by Geraert (1971). He indicated that B. thylactus and B. volutus are very similar to each other. However, the lip region and tail length are distinguishing characters for the mentioned species. The lip region in B. volutus is flattened, whereas in B. thylactus bears a depression at the middle of the lip region, which is visible under light microscopy photographs. This character has been studied by SEM, as indicated by Geraert (1971). Another distinctive character that helps to separate the mentioned species is the tail length. Tail length in B. thylactus is 62-88 ^m, whereas tail length in B. volutus is 35-60 ^m (see Geraert, 2008). However, Geraert (2008) provided a body habitus after heat relation as a separation character in the key to species (body ventrally arcuate to open or closed C-shape for B. thylactus and body spirally coiled for B. volutus), which is not reliable as in the present study both habitus were observed for B. volutus.
Locality and habitat. Specimens of B. volutus were collected in Parys, Free State province of South Africa (S 26°54'37.336", E 27°27'24.582"), associated with wild grass.
Collection materials. Three voucher slides (BV-SA1-3) of B. volutus were deposited in the Nematology laboratory collection of the University of Limpopo, South Africa.
DNA characterisation. The nblast result showed South African population of B. volutus has 98% similarity with the American (Maryland) population of B. volutus (MT994501). Additionally, South African B. volutus showed 99% similarity with unidentified American (JQ005001) population of
Characters Females
n 7
L 461.7 ± 59.8 (379-554)
a 27.2 ± 4.0 (23.7-31.5)
b 4.7 ± 0.8 (2.9-5.3)
c 9.5 ± 2.0 (7.8-14.7)
c' 5.1 ± 0.8 (4.2-6.5)
V 68 ± 5.3 (58-76)
Lip region L 2.8 ± 0.5 (2-3)
Lip region W 4.4 ± 0.7 (3.5-5.4)
Stylet length 9.6 ± 1.1 (8.5-11)
m 42.7 ± 2.5 (41-44)
DGO 1.6 ± 0.2 (1.4-1.9)
Isthmus length 18.3 ± 1.5 (17-20)
Median bulb 55.9 ± 6.9 (48-66)
Bulb length 16.5 ± 3.5 (14-19)
Bulb width 9.5 ± 0.7 (9-10)
MB 58.4 ± 6.3 (54-63)
Pharynx length 101.4 ± 17.3 (91-127)
Neck length 104.5 ± 16.1 (83-140)
Neck base diameter 16.1 ± 1.8 (14-18)
Nerve ring to the anterior end 75.4 ± 5.8 (69-80)
Excretory pore to the anterior end 82.8 ± 8.5 (71-90)
Midbody diameter 16.9 ± 3.2 (14-21)
Anal body diameter 10.2 ± 2.1 (7-13)
Vagina length 5.9 ± 1.0 (5-7)
Anterior genital branch 132.8 ± 25.1 (104-150)
Post-vulval uterine sac 10.8 ± 2.3 (8-13)
Head-anus length 465.6 ± 52.3 (406-504)
Vulva-anus length 100.2 ± 19.9 (66-122)
Tail length 48.3 ± 9.7 (30-63)
iggj MK639376 Basiria gracilis 93if]
100
100
79
90
78
100
100
L
87
71
97
MK639375 Basiria gracilis DQ328717 Basiria gracilis KX156326 Basiria graminophila - KP730046 Neothada cancellata
KX156323 Neopsilenchus magnidens
100_I MK639379 Neopsilenchus magnidens
MK639380 Neopsilenchus magnidens KM502982 Discopersicus iranicus 80j MK639378 Boleodorus sp. MK639377 Boleodorus sp.
MW056183 Boleodorus thylactus
97
JQ005021 Boleodorus sp.
100
68
99
100 100 64
DQ328718 Boleodorus sp.
r JQ005002 Boleodorus sp. JQ005003 Boleodorus sp.
KP313830 Boleodorus thylactus
JQ005001 Boleodorus sp.
MW704025 Boleodorus volutus MW704026 Boleodorus volutus
MT994501 Boleodorus volutus
100
100
100
£
99
100
100
iool
KP730043 Tylenchus naranensis JQ005014 Filenchus sp. JQ005017 Filenchus annulatus KM817178 Coslenchus oligogyrus |r DQ328719 Coslenchus costatus 100 KM817175 Coslenchus franklinae ~ AY780979 Aglenchus agricola ~ KX789727 Atetylenchus sp. MK639384 Psilenchus hilarulus MK639383 Psilenchus hilarulus _KX156310 Malenchus nanellus
100
100
100 [
100
1001— KX156308 Malenchus ovalis 100_r KX156324 Lelenchus leptosoma KX156322 Lelenchus leptosoma KY776629 Labrys chinensis 1 KY776628 Labrys chinensis EU915491 Cephalenchus hexalineatus EU915492 Cephalenchus hexalineatus
100 |
100
c
EU915490 Eutylenchus excretorius AY633444 Deladenus siricidicola KX094978 Deladenus posteroporus
MG676657 Ditylenchus dipsaci
03
Fig. 4. The Bayesian tree inferred from known Boleodorus and newly sequenced B. volutus from South Africa based on the 28S rDNA region.
Table 2. Genetic pairwise distance estimating for different populations of Boleodorus including B. volutus. Down is pairwise genetic distance; the upside is a standard error.
Species Accession number 1 2 3 4 5 6 7 8 9 10 11 12
1 B. volutus MW704025 0.00 0.00 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01
2 B. volutus MW704026 0.00 0.00 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01
3 B. volutus MT994501 0.00 0.00 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01
4 Boleodorus sp. JQ005021 0.03 0.03 0.03 0.01 0.01 0.01 0.00 0.00 0.00 0.01 0.00
5 Boleodorus sp. JQ005001 0.00 0.00 0.00 0.03 0.01 0.01 0.01 0.01 0.01 0.01 0.01
6 Boleodorus sp. JQ005002 0.03 0.03 0.03 0.03 0.03 0.01 0.01 0.01 0.01 0.01 0.01
7 Boleodorus sp. JQ005003 0.03 0.03 0.03 0.04 0.02 0.03 0.01 0.01 0.01 0.00 0.01
8 B. thylactus MW056183 0.03 0.03 0.03 0.00 0.03 0.02 0.03 0.00 0.00 0.01 0.00
9 Boleodorus sp. MK639378 0.04 0.04 0.04 0.01 0.03 0.03 0.03 0.01 0.00 0.01 0.00
10 Boleodorus sp. MK639377 0.04 0.04 0.04 0.01 0.03 0.03 0.03 0.01 0.00 0.01 0.00
11 B. thylactus KP313830 0.02 0.02 0.03 0.03 0.02 0.03 0.00 0.03 0.03 0.03 0.01
12 Boleodorus sp. DQ328718 0.03 0.02 0.03 0.01 0.03 0.02 0.03 0.00 0.00 0.00 0.03
Boleodorus. Besides, South African B. volutus showed 94% and 97% similarity with a Chinese (MW056183) and Iranian (KP313830) population of B. thylactus. Genetic pairwise distance estimation showed ranges from 0.00 to 0.04 for Boleodorus species. However, the genetic distance among B. volutus from South Africa and the USA was estimated at 0.00. The phylogenetic analysis using 28S rDNA (Fig. 4) placed South African B. volutus close to B. volutus (MT994501) from the USA with a 0.77 posterior probability value. However, all Boleodorus species form a group with the support of a 1.00 posterior probability value. The Tajima's Test for three sequences, including B. volutus (MW704025; MW704026) from South Africa and B. volutus (MT994501) from the USA, showed 715 identical sites, no divergence, and one unique difference in each of the tested sequences (P < 0.05). The result indicates a high similarity of the South African and American B. volutus, which confirms its identification.
DISCUSSION
The South African population resembles the original description of B. volutus provided by Lima & Siddiqi (1963). Compared to the Pakistanian population of B. volutus (Hussain & Iqbal, 2017), they differ in the lower range for the tail length (3063 vs 40-50 ^m). Compared to the Iranian B. volutus (Alvani et al., 2013), they differ in lower range for the tail length (30-63 vs 51-64 ^m).
Our phylogenetic analysis using 28S rDNA (Fig. 4) placed the South African B. volutus together with the other descriptions of B. volutus. Molecularly
characterised species of Boleodorus grouped together. However, more studies are needed with different genes, such as mtDNA, to determine the real position of the Boleodorus members. This result agrees with previous results (Qing & Bert, 2019). According to the literature, this is the first complete record of B. volutus from South Africa. In conclusion, the lip region (flattened) and short tail are the distinguishing characters for B. volutus compared to B. thylactus.
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E. Shokoohi. Морфологическая и молекулярная характеристика Boleodorus volutus Lima & Siddiqi, 1963 из Южной Африки с помощью первых СЭМ наблюдений этого вида.
Резюме. Во время исследования нематод, паразитирующих на растениях в Южной Африке, была обнаружена популяция рода Boleodorus, ассоциированного с травами. Данный вид характеризуется конической областью губ без инцизур. Длина стилета составляет 9-11 мкм, латеральное поле с четырьмя насечками, поствульварный мешок меньше соответствующего диаметра тела. Хвост конической формы, часто крючковатый на конце, длиной 51-58 мкм. Анализ nblast, основанный на сегменте D2-D3 28S рДНК южноафриканской популяции B. volutus, выявил 98% сходства с североамериканским B. volutus (MT994501). Филогенетический анализ распределил южноафриканскую популяцию B. volutus с другими B. volutus и неопределеннными до вида представителями Boleodorus с апостериорной вероятностью 100. Кроме того, впервые представлены фотографии B. volutus, выполненные на сканирующем электронном микроскопе. Это первое полное сообщение о данном виде в Южной Африке.
