Within the plant-parasitic nematode (PPN) genus
In the current paper, we characterize a newly discovered
A soil sample mixed with some roots was collected using a shovel from 15 to 25 cm soil depth in a zig-zag pattern from a finger millet field [Eleusine coracana (L.) Gaertn.] in Kipkaren Estate, Eldoret, Kenya in February, 2019. The GPS coordinates of the field location are 00°30.395’ N; 035°14.825’E. Nematodes were extracted from 100 ml of soil by using modified Baermann’s method (Whitehead and Hemming, 1965). The extracted nematodes were stored at 4°C during the course of analysis.
Host roots were stained using acid fuchsin (Byrd et al., 1983). For this, clean roots were bleached in 2.5% NaOCl for 5 min, followed by rinsing the roots in running tap water, and boiling them in 30 ml distilled water with 1 ml of stock staining solution (0.35 g acid fuchsin, 25 ml acetic acid, and 75 ml distilled water) in a microwave for 30 sec. After cooling, excess fuchsin was drained and roots were washed with running tap water and de-stained by immersing them in 70% acidified glycerol, and finally observed under a stereo microscope for presence of stained PPN.
For morphological studies, live nematodes were heat-relaxed by quickly passing over a flame in a drop of water on a glass slide until nematode movement stopped and examined, photographed, and measured using an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an Olympus C5060Wz camera as described in Singh et al. (2018). After recording morphological information, each specimen was recovered from the slide and its genomic DNA was extracted. For fixing, the nematode suspension obtained after extraction was concentrated in a drop of water in a glass embryo dish, followed by addition of a few drops of Trump’s fixative [2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Sorenson buffer (Sodium phosphate buffer at pH = 7.5)] into it. The nematodes were then immediately heated in a microwave (700 watts) for about 4 sec and left for 1 hr at room temperature and finally at 4°C for 24 hr. This was followed by gradually transferring the nematodes to anhydrous glycerin, ready to be mounted on glass slides as described in Singh et al. (2018). For SEM, specimens fixed in Trump’s fixative were washed in 0.1 M phosphate buffer (pH = 7.5) and dehydrated in a graded series of ethanol solutions, critical-point-dried with liquid CO2, mounted on stubs with carbon tabs (double conductive tapes), coated with 25 nm gold, and photographed with a JSM-840 EM (JEOL) at 12 kV (Singh et al., 2018).
After morphological analysis, heat-relaxed nematodes were recovered from temporary slides and each individual nematode was cut into pieces in distilled water using a blade and the pieces were transferred to a PCR tube containing 20 µl of worm lysis buffer [50 mM KCl, 10 mM Tris at pH = 8.3, 2.5 mM MgCl2, 0.45% NP 40 (Tergitol Sigma), 0.45% Tween 20]. The PCR tubes were incubated at −20°C (10 min) followed by addition of 1 µl proteinase K (1.2 mg/ml), incubation at 65°C (1 hr) and 95°C (10 min), and finally centrifuging the mixture at 14,000 rpm for 1 min (Singh et al., 2018). PCR amplifications of the partial ITS, 18S, and D2-D3 expansion segment of 28S of rDNA were done using the primer pairs, Vrain2F: 5´-CTTTGTACACACCGCCCGTCGCT-3´/Vrain2R: 5´-TTTCACTCGCCGTTACTAAGGGAATC-3´ (Vrain et al., 1992), SSU18A: 5´-AAAGATTAAGCCATGCATG-3´/SSU26R: 5´-CATTCTTGGCAAATGCTTTCG-3´ (Mayer et al., 2007) and D2A: 5´-ACAAGTACCGTGAGGGAAAGTTG-3´/D3B: 5´-TCCTCGGAAGGAACCAGCTACTA-3´ (Nunn, 1992), respectively, using thermal profiles described in Singh et al. (2018, 2019). For amplification of the
The phylogenetic relationships of the new species with other related species were analyzed based on the partial sequences of ITS, 18S, 28S, and
Figures 1 and 2, Tables 1 and 2.
Morphometric data for fixed female
Female character | |
---|---|
|
19 |
Body length ( |
690 ± 44 (620-760) |
|
27.3 ± 3.0 (23.0-33.0) |
|
5.8 ± 0.3 (5.0-6.0) |
|
5.1 ± 0.4 (4.5-6.1) |
|
66.0 ± 12.0 (49.0-91.0) |
|
0.6 ± 0.1 (0.3-0.8) |
|
58 ± 1 (55-60) |
Lip region height | 4.6 ± 0.3 (4.2-5.3) |
Lip region width | 8.2 ± 0.3 (7.7-8.7) |
Stylet length | 25.3 ± 0.9 (22.5-26.6) |
Cone length | 12.5 ± 0.7 (10.8-13.7) |
Shaft length | 10.3 ± 0.5 (9.4-11.2) |
Stylet knob height | 2.6 ± 0.4 (1.9-3.4) |
Cone % Stylet | 49.3 ± 1.8 (44.8-52.6) |
Dorsal gland opening from stylet base | 5.4 ± 0.6 (4.1-6.3) |
Anterior end to secretory-excretory pore | 107 ± 6.2 (93.2-118) |
Anterior end to pharynx-intestine junction | 118 ± 5.3 (105-128) |
Anterior end to pharyngeal gland end | 135 ± 6.0 (121-144) |
Anterior end to vulva opening | 400 ± 24.8 (341-408) |
Maximum body diameter (MBD) | 25.5 ± 2.9 (21.5-31.7) |
Anal body diameter (ABD) | 16.9 ± 1.4 (15.2-19.9) |
Tail length | 10.8 ± 2.1 (6.9-13.4) |
Phasmid to tail tip length | 31.6 ± 3.1 (25.1-36.3) |
Number of annuli between phasmid and tail tip | 20 ± 3 (12-26) |
Number of tail annuli | 7 ± 1 (5-9) |
Comparison of some important female characters and information on availability of males of 23
Species | Body length (mm) | Lip region | Lip annuli number | Stylet length (µm) | Cone% of stylet | Spermatheca in matured females | V% | Male | Tail annuli number | Phasmid position |
---|---|---|---|---|---|---|---|---|---|---|
|
0.6-0.7 | Broadly rounded, setoff to continuous | 4-5 | 24-25 | 42-45 | Not seen | 56-59 | Not found | 8-11 | 8-14 annuli anterior to anus |
|
0.8-1.0 | Broadly rounded, slightly setoff | 4-5 | 26-28 | 43-51 | Rounded, filled with sperms | 54-57 | Found | 13-16 | 7-15 annuli anterior to anus |
|
0.5-0.9 | Truncate, blunt anterior, not setoff | 4-5 | 20-31 | 42-51 | Rounded, filled with sperms | 54-66 | Found | 8-15 | 3-4 annuli posterior to anus |
|
0.6-0.8 | Rounded, not setoff | 4 | 26-29 | 46-50 | Rounded, filled with sperms | 54-59 | Found | 6-8 | 9-12 annuli anterior to anus |
|
0.5-0.8 | Broad, rounded, not setoff | 4-5 | 18-26 | – | Present | 52-62 | Found | 5-11 | 8-14 annuli anterior to anus |
|
0.7-1.2 | Hemispherical, slightly setoff | 5-7 | 25-35 | 39-45 | Rounded, filled with sperms | 54-59 | Found | 9-14 | 1-3 annuli anterior to anus |
|
0.8-1.1 | Hemispherical, slightly setoff | 7-8 | 30-33 | 45-48 | Rounded, filled with sperms | 55-56 | Found | 13-14 | 1-4 annuli anterior to anus |
|
0.6-0.9 | Hemispherical, not setoff | 5 | 24-32 | – | Rounded, filled with sperms | 55-63 | Found | 12 | 5 annuli posterior to anus |
|
0.5-0.8 | Hemispherical, well setoff | 3-4 | 21-28 | – | Small and empty | 57-67 | Not found | 6-7 | 7-14 annuli anterior to anus |
|
0.7-0.9 | Conoid, blunt, not setoff | 4 | 25-34 | 43-46 | Rounded, filled with sperms | 53-64 | Found | 7-9 | 8-14 annuli anterior to anus |
|
|
|
|
|
|
|
|
|
|
|
|
0.9-1.2 | Hemispherical, slightly setoff | 4 | 29-35 | – | Rounded, filled with sperms | 53-59 | Found | 8-12 | 15-20 annuli anterior to anus |
|
0.6-1.0 | Hemispherical, not setoff | 4-5 | 22-32 | – | Developed, filled with sperms | 53-61 | Found | ca 8 | 13-14 annuli anterior to anus |
|
0.7-1.0 | Rounded, slightly setoff | 4-5 | 21-27 | 43-47 | Rounded, filled with sperms | 51-60 | Found | 7-15 | 1-6 annuli anterior to anus |
|
0.5-0.7 | Rounded, not setoff | 4-5 | 21-25 | 44-52 | Rounded, filled with sperms | 60-66 | Found | 9-17 | 3-4 annuli anterior to anus |
|
0.9-1.1 | Hemispherical, slightly setoff | 4-5 | 27-30 | 43-50 | Empty | 48-57 | Not found | 14-18 | 3 annuli anterior to 5 annuli posterior of anus |
|
0.5-0.8 | Angularly hemispherical, slightly setoff | 3-4 | 20-27 | 56-58 | Rounded, filled with sperms | 53-66 | Found | 9-14 | 1 annule anterior to 4 annuli posterior of anus |
|
0.6-0.7 | Hemispherical, setoff | 4-5 | 20-23 | 43-50 | Rounded, filled with sperms | 55-58 | Found | 6-10 | 5-12 annuli anterior to anus |
|
0.9-1.6 | Hemispherical, well setoff | 6-7 | 33-50 | 50-56 | Rounded, filled with sperms | 52-58 | Found | 8-17 | 7 annuli anterior to 3 annuli posterior of anus |
|
1.0-1.3 | Rounded, slightly setoff | 4 | 35-42 | – | Developed, filled with sperms | 51-56 | Found | 12 | 8-10 annuli anterior to anus |
|
0.6-0.8 | Rounded, well setoff | 3 | 24-25 | 43-48 | Rounded, filled with sperms | 56-60 | Found | 10-11 | Anal level to 8 annuli anterior |
|
0.5-0.9 | Hemispherical, not setoff | 4 | 20-29 | – | Not seen | 47-59 | Not found | 9-13 | 6-7 annuli anterior to anus |
|
1.1-1.2 | Flattened anteriorly, not setoff | 4-5 | 29-29 | 43-47 | Rounded, filled with sperms | 45-55 | Found | 10-16 | 8-14 annuli anterior to anus |
Body moderately large (0.6-0.8 mm), habitus spiral to 6-shaped when heat relaxed. Lateral field differentiation starting as single areolated band, gradually forming two areolated bands up to level of metacorpus valve and further continuing as three areolated bands up to isthmus level, after which bands becomes smooth till tail terminus. Longitudinal cuticular striations in anterior region absent. Labial region hemispherical with 4 annuli, not offset from body but appears to have a slight depression under LM.
Not found.
This new species is morphologically closest to Rotylenchus abnormecaudatus Van den Berg and Heyns, 1974 and
The new species is also comparable to Rotylenchus cypriensis Antoniou, 1981,
The species epithet refers to its host. Wimbi originates from Swahili and is used as a common name for finger millet in Eastern Africa.
The new species was found parasitizing the host plant
Female holotype (Slide: WT 3796) and seven female paratypes in two slides (Slides: WT 3797 and WT 3798) are deposited at the National Plant Protection Organization, Wageningen Nematode Collection (WaNeCo), the Netherlands. One slide containing 10 female paratypes is deposited at Ghent University Museum, Zoology Collections, Belgium. An additional slide containing eight female paratypes is also deposited at UGent Nematode Collection (Slide: UGnem-300) of Nematology Research Unit of Ghent University, Belgium.
Several stained spiral-shaped nematodes were observed in the host roots which are
Five sequences of up to 770 bp without any sequence variation were produced (MW074362-MW074366). They were found to be most similar to a sequence of
Six sequences of up to 903 bp without sequence variation were produced (MW074378-MW074383). The closest
Five sequences of up to 973 bp without sequence variation were produced (MW074373-MW074377) which were found closest to a
Five sequences of up to 414 bp, without any sequence variation, were produced (MW074357-MW074361) and the closest
A total of 15
Morphologically, the new species is easily separated from all the African species, except for
The quantitative and qualitative impact of this newly discovered parasite of Kenyan finger millet remains to be studied, so as to assess the level of crop losses caused by the pest. This is especially important in the context of finger millet as it represents an emerging cereal crop of potentially great importance in the tropical and the sub-tropical regions.