Morphological and Molecular Characterization of Hoplolaimus tuberosus n. sp. (Nematoda: Hoplolaimidae) Associated with Potato in Uganda

A survey was conducted to determine the occurrence and distribution of plant-parasitic nematodes on potatoes in Uganda in key potato-growing agroecological zones. Soil and root samples were collected, and nematodes were extracted using the modified Baermann technique (Whitehead and Hemming, 1965). Morphological and morphometric assessment comprised of mounting and examining individual nematodes on temporary slides and then retrieving them for DNA extraction to correlate morphology with molecular data. Morphological vouchers linked with molecular sequences are deposited in BOLD (https://v4.boldsystems.org/index.php/MAS_Management_DataConsole?codes=RQHPL). Other specimens were heat-killed and fixed using a 4% formaldehyde solution and then gradually processed to anhydrous glycerin as per the method described by De Grisse (1969), but the step of nematodes staying overnight in ethanol was skipped. Micrographs of both temporary and permanently fixed specimens were captured, and all measurements were taken from permanently fixed specimens using an Olympus BX50 DIC microscope (Olympus Optical) equipped with an HD Ultra camera. Drawings were created using an Olympus BX51 DIC microscope (Olympus Optical) equipped with a drawing tube. For SEM analysis, 300 mL of freshly prepared fixative solution (4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3) was transferred into a glass staining block containing nematodes. The block was microwaved for 4 seconds, and the fixed specimens were stored at 4°C for three days. The specimens underwent eight 20-minute dehydration cycles in graded ethanol solutions (30%, 50%, 65%, 75%, 85%, 90%, 95%, and 98%), followed by three 10-minute washes in 100% ethanol. Subsequently, the nematodes were critical point-dried using liquid CO₂, mounted on stubs with carbon tabs, coated with a 10 nm gold layer (120 seconds at 15 mA), and imaged with a JSM-840 electron microscope at 12 kV. (Singh et al., 2018). The soil texture and organic carbon content was analyzed of the type locality. The organic carbon was determined according to the Walkley and Black method (Walkley and Black, 1934) through oxidation with 1N K2Cr7O7 (7%) in the presence of concentrated H2SO4 (96%), and back-titration using ferrous sulphate using ferroin as indicator. Soil texture was analyzed using the pipette method as outlined by Van Reeuwijk (1993) following removal of organic matter using H₂O₂ and carbonates using NaAc.

Nematode DNA was extracted from nematodes recovered from temporarily mounted specimens. The nematodes were washed thrice with distilled water and cut into three pieces using a sterile needle. These pieces were then placed in a PCR tube containing 20μl of worm lysis buffer (50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2, 0.45% NP-40 (Tergitol Sigma), 0.45% Tween-20), followed by freezing at −20°C for 10 minutes. After freezing, 1 μl of proteinase K (1.2 mg/ml) was added, and the mixture was incubated at 65°C for 1 hour and then at 95°C for 10 minutes, followed by centrifugation of the lysate at 15,000 g for 1 minute. The DNA samples were stored at −20°C until required for PCR analysis. DNA amplification was conducted using a Bio-Rad T100™ thermocycler with a master mix containing 10μl dH2O, 12.5 μl DreamTaq (Thermo Fisher Scientific), 0.5 μl of each forward and reverse primer (10 μl, and 2 μl of extracted DNA was used. Primers for D2–D3 of 28S amplification were forward primer D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG-3′) and the reverse primer D3B (5′-TCC TCG GAA GGA ACC AGC TAC TA-3′) (Nunn, 1992). PCR conditions were pre-denaturation at 94°C for 4 min followed by 40 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min and finished with one cycle at 72°C for 10 min. For the internal transcribed spacer (ITS) region, TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATTGCTTAAGTTCAGCGGGT-3′) primers were used (Joyce et al., 1994). The PCR conditions were pre-denaturation at 94°C for 4 min followed by 40 cycles of 94°C for 1 min, 57°C for 1 min, 72°C for 1 min and finished with one cycle at 72°C for 10 min. For COI, the primers used were JB3: 5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′ and JB4.5: 5′-TAA AGA AAG AAC ATA ATG AAA ATG-3′ (Bowles et al., 1992) and the PCR conditions were 1 min at 94°C, 1min at 45°C, and 1 min at 72°C for 38 cycles. The primers SSU18A: 5′-AAAGATTAAGCCATGCATG-3′ and SSU26R: 5′-CATTCTTGGCAAATGCTTTCG-3′ (Mayer et al., 2007) were used to amplify the 18S gene following thermal profiles described in Singh et al. (2018). PCR products were purified enzymatically, as detailed by Singh et al. (2020), and sent for sequencing in both forward and reverse directions to Macrogen (https://dna.macrogen.com). Contigs from the sequences were assembled using Geneious Prime software, version 2024.0.7 and deposited in GenBank.

The acquired sequences were analyzed along with sequence data sets of other Hoplolaimus species available in GenBank. Multiple sequence alignments were conducted using MUSCLE with default settings, followed by manual trimming of the poorly aligned ends. Phylogenetic analysis using Bayesian inference (BI) was performed with MrBayes 3.2.6 (Huelsenbeck et al., 2001) under the GTR + I + G nucleotide substitution model. The analysis was executed for 1 × 10⁶ generations across four separate runs, with a burn-in of 20% and a subsampling frequency of 200 generations (Huelsenbeck et al., 2001). The resulting phylogenetic trees were visualized using Tree View, including the posterior probability (PP) values for the relevant clades. All phylogenetic programs were performed within Geneious Prime, version 2024.0.7.

Soil samples collected from two distinct potato fields; BUD1 (1°03′48.636″N, 34°15′19.404″E) and BUD5 (1°03′36.5394″N, 34°14′55.0674″E) in Budwale Sub-county, Mbale District, Eastern Uganda, revealed the presence of several nematode species, including an undescribed species belonging to the genus Hoplolaimus Daday, 1905. The BUD5 soil sample (the type locality) was characterized by a fine-textured clay soil with high clay content, low sand content, and moderate silt content (72% clay; 24% silt; 8% sand) and an organic content of 2.6%. Up to 50 – 75 specimens per 100 ml of soil were retrieved including juveniles, males, and females. The two fields were previously utilized for potato and bean cultivation in the two preceding growing seasons. This region is characterized by a humid subtropical climate, 15°C and 28°C temperature ranges, and a bi-modal precipitation pattern, with an average annual rainfall of 1500 mm (Jiang et al., 2014).

Hoplolaimus tuberosus n. sp. (Figs. 1–3; Table 1).

Figure 1:

Light- and scanning electron microscopy images of females of Hoplolaimus tuberosus n. sp. A, B, E: Anterior body in lateral view showing the position of the SE pore opening and the hemizonid, stylet and stylet knobs, and median bulb, (arrows indicate tulip-shaped stylet knobs); C, D, F: Head region showing lip annuli, oral disc and longitudinal striations on the basal lip annule, (arrows indicate the four lip annuli, horizontal arrow the basal lip annule); G, H, I, N: Vulva region in ventral and lateral views and the out stretched anterior and posterior gonads; J: Body habitus; K, L, M: Scutella (appointed with arrow), lateral view; O: Mid body lateral field, arrow showing zigzag longitudinal line formed by anastomoses; P, S: Lateral field in the posterior region; Q, R: Tail region showing anal opening and tail annuli.

Figure 2:

Light- and scanning electron microscopy images of Hoplolaimus tuberous n. sp. males. A, B, C, D and F: Anterior body in lateral view showing head region, lip annuli (arrows; horizontal arrow indicates the basal lip annule), stylet, medium bulb and the lateral field (arrow indicate zigzag longitudinal line formed by anastomoses); E: body habitus; H: Mid body lateral field; G, I, J, K: Lateral and ventral view of the tail region showing spicules and bursa.

Figure 3:

Illustrations of Hoplolaimus tuberosus n. sp.: A: Female whole body; B: Female anterior region; C: Male anterior region; D: Male whole body; E, F: Female tail region; G: Female reproductive system; H: Male reproductive system; I: Male spicule, tail, and bursa.

Hoplolaimus tuberosus n. sp. is characterized by moderately large sized females (1.2–1.6 mm); a robust stylet averaging 48 μm long; an offset head with 4–5 annuli, a tessellated basal lip annule forming 10–12 irregular blocks; hemizonid located twelve annuli posterior to excretory pore; a variable lateral field represented by 1 incisure (longitudinal zigzag line formed by anastomoses) at the anterior and posterior end and 2–3 irregular and incomplete striae at mid-body; a pharyngeal gland with six gland nuclei; and a short tail measuring 11–16 μm with 8–10 annuli. Males are present and slightly smaller than females; their basal lip annule divided into 2–4 longitudinal striae; and long spicules measuring 46–58 μm.

Using the Hoplolaimus key and compendium provided by Handoo and Golden (1992) along with recent descriptions, Hoplolaimus tuberosus n. sp. is found to be morphologically close to H. indicus Sher, 1963, H. dubius Chaturvedi and Khera, 1979, H. aegypti Shafiee and Koura, 1969, H. seinhorsti Luc, 1958, H. columbus Sher, 1963 and H. pararobustus Schuurmans Stekhoven and Teunissen, 1938, by having excretory pore anterior to hemizonid, lateral field formed by one incisure, one scutellum anterior and one posterior to the vulva and six gland nuclei (except H. pararobustus) (Table 2). It differs from all by having fewer tail annuli; 8–10 vs 17–27 for H. aegypti, 10–15 for H. seinhorsti, 8–22 for H. indicus, 16–22 for H. columbus, 7–15 for H. pararobustus and 10–15 for H. dubius. In addition, Hoplolaimus tuberosus n. sp. differs from H. indicus by the relatively longer stylet (45–51 μm vs 33–47 μm), different number of longitudinal striae on the basal lip annule (10–12 vs 6–20) and the longer spicules (46–58 μm vs 34–42 μm); from H. seinhorsti by the relatively longer stylet (45–51 μm vs 40–49 μm), and males (present vs unknown); from H. columbus by the relatively longer stylet (45–51 μm vs 40–48 μm) and males (present vs. rarely present); from H. aegypti by having shorter spicules (46–58 μm vs 54–65 μm); from H. dubius by the relatively longer body length (1.2–1.6 mm vs 1.05–1.27 mm in females), longer stylet (45–51 μm vs 31–42μm), and longer spicules (46–58 μm vs 37–44 um); and from H. pararobustus by more esophageal gland nuclei (6 vs 3), less longitudinal striae on the basal lip annule (10–12 vs 18–25), and a relatively longer stylet (45–51 μm vs 38–49 μm). It also clearly differs molecularly from all these species based on all the genes analyzed.

Hoplolaimus tuberosus n. sp. was isolated from soil collected from the rhizosphere of potatoes (Solanum tuberosum) and the species epithet tuberosus is derived from the species epithet of the crop's name.

Type material was obtained from Budwale subcounty (field BUD5) (GPS location: 1° 3′ 36.5394″N; 34° 14′ 55.0674″E, elevation 1956 m a.s.l.), Mbale district, Eastern Uganda, associated with potatoes. The new species was also found in another field (BUD1), about 2 km away from the type locality, in the same sub-county. The conspecificity of both populations was supported by 99.5–100% identical sequences.

Female holotype (UGMD_104479), 2 females (UGMD_104480), 1 male and 2 females (UGMD_104481), 1 male (UGMD_104482), and 3 male paratypes (UGMD_104483) are deposited at Ghent University Museum, Zoology Collections, Belgium. Six additional slides containing four female paratypes and 3 male paratypes are deposited at UGent Nematode Collection of Nematology Research Unit of Ghent University, Belgium (UGnem349-354). Two slides, one containing two female paratypes and one with two male paratypes are deposited at Makerere University, Nematology Research Unit, Uganda. The video (virtual microscopy) of the holotype is available on https://nematodes.ugent.be/vce.html. Registered in ZooBank (http://zoobank.org/) with identifier: urn:lsid:zoobank.org:pub:2C7F5543-EDEA-4B6A-944B-7CE02C2B5ACA.

The amplification of the D2–D3 expansion segment of the 28S-rRNA, 18S-rRNA, ITS-rRNA, and COI of mtDNA genes produced fragments approximately 720, 890, 986, and 417 bp in length, respectively. The seven newly obtained sequences of the D2–D3 region from male and female specimens showed intraspecific variations of 0–4 bp, with a similarity of 99.5% to 100%. A phylogenetic tree including eight different species and seven unidentified Hoplolaimus sequences (Fig. 4A) revealed a maximally supported clade of H. tuberosus n. sp. and sequences of H. pararobustus, H. columbus, H. indicus, H. seinhorsti, and H. dubius (Clade 1). H. tuberosus n. sp. is in an unresolved position from these species, but the new sequences differ by 22–26 bp (3.6%), 25–35 bp (5%), 33 bp (5.4%), 31–55 bp (6%), and 33–42 bp (5.4–7.8%). Thus, the highest observed similarity was only 96.7%, specifically with H. pararobustus sequences (OP459422, OP459421, and OP459423) reported from Nigeria (Olajide et al., 2023).

Figure 4:

BI phylogenetic tree generated from the analysis of D2–D3 of 28S rRNA (A) and COI of mt DNA (B) sequences using GTR + G + I nucleotide substitution model. Bayesian posterior probabilities are given next to each node and sequences of Hoplolaimus tuberosus n. sp. are in bold. Clade numbers are taken from Olajide et al. (2023).

The obtained COI mtDNA sequences displayed an intraspecific variation of 13 nucleotides (95.7% similarity) and formed a well-supported sister relationship (PP: 0.98) with H. columbus, H. indicus, and H. seinhorsti (Fig. 4B). These sequences differed significantly from the aforementioned species by 56–57 bp (16–18%), 61 bp (18%), and 64–87 bp (21–25%), respectively. The closest match was H. columbus (KP864609) with a similarity of only 83.8% (Holguin et al., 2015).

The two obtained 18S rDNA sequences of H. tuberosus n. sp. (Fig. 5A) showed 99.9% similarity, differing by only 1 bp. In the resulting phylogeny, the sequences formed a well-supported clade (PP = 0.97) together with H. indicus, H. seinhorsti, H. columbus, and H. pararobustus, showing differences of 2–6 bp (0.2–0.7%), 4–16 bp (1.4–5%), 9–19 bp (1–2%), and 17–24 bp (2–2.4%), respectively.

Figure 5:

BI phylogenetic tree generated from the analysis of 18S-rRNA (A) and ITS-rRNA (B) sequences using GTR + G + I nucleotide substitution model. Bayesian posterior probabilities are given next to each node and sequences of Hoplolaimus tuberosus n. sp. are in bold. Clade numbers are taken from Olajide et al. (2023).

Finally, ITS sequence analysis revealed a maximally supported sister relationship with H. columbus and H. seinhorsti (Fig. 5B; clade 1), showing differences of 55–182 bp (12.6–19.2%) and 174–185 bp (19%), respectively. The most similar sequence was identified as H. columbus (AB933482), with only 87.4% similarity.