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Molecular and phenotypic characterization of Hemicriconemoides rosae (Rathour et al., 2003) from mustard rhizosphere in India

Molecular and phenotypic characterization of Hemicriconemoides rosae (Rathour et al., 2003) from... Background Nematodes belonging to Genus Hemicriconemoides, commonly known as sheathoid nematodes, dam- aged many fruits, vegetables and cash crops, worldwide. A survey has been conducted in the agricultural fields of Bulandshahr district to find out the plant-parasitic nematodes load. Results A total of 85 soil samples were collected from mustard fields of Bulandshahr district of Uttar Pradesh and pro - cessed for the presence of plant-parasitic nematodes and they were isolated by ‘Cobb sieving method’. Among all the collected soil samples, one soil sample found to be positive for the genus Hemicriconemoides and docketed as HCN. The earliest identification based on morphology revealed the species to be Hemicriconemoides rosae which was recov- ered from the mustard field for the first time. The morphology was found to be more consistent with rose population as compared to sugarcane population of the Hemicriconemoides rosae. Further, the Hemicriconemoides rosae was identified on the basis of molecular and phylogenetic analysis based on the concatenated matrix. In addition, correla- tion analysis of the Hemicriconemoides rosae based on morphometric parameters was done. The results revealed the importance of body length and its relation with other morphometric parameters, and they were found significant. Besides this, de Manian index a showed highest correlation with the body length in adult females. In all the studied ratios, a is very important for the evaluation of the females of a specific species of genus Hemicriconemoides. The data from the principal component analysis (PCA) revealed the high intraspecific and interspecific variations between the species of genus Hemicriconemoides. However, less intraspecific variations were present between the rose population and mustard population of H. rosae. Conclusions The study revealed the new host, i.e. mustard crops, for H. rosae showing dissimilarity in morphology with the sugarcane population. Keywords Hemicriconemoides rosae, Mustard, Morphology, Taxonomy, Correlation, PCA have been evolved in such a way that they have devel- Background oped an accessory structure known as stylet with the There are some nematodes which show association with help of which they are associated with the plants and the plants over millions of years, have resulted in the able to derive the nutrients from them. Genus Hemic- evolution of plant-parasitic nematodes (PPNs). They riconemoides described by Chitwood and Birchfield (1957) comprises of the species which does not belong to either the genus Hemicycliophora (De Man, 1921) or *Correspondence: Himani Sharma Criconemoides (Taylor, 1936). The demarcating charac - sharmahimani1995hariom@gmail.com teristics possessed by members of this genus are the pres- Nematology Laboratory, Department of Zoology, Chaudhary Charan ence of an extra cuticular sheath in the mature females, Singh University, Meerut 250004, India presence of large body annules and having lateral and © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 2 of 15 sublateral ridges throughout the length of males giving plants irrespective of fruit and vegetable (Edward & the appearance of slight caudal alae. These nematodes Misra, 1964; Edward et  al., 1965; Dasgupta et  al., 1969; are commonly known as ‘sheathoid’ nematodes due to Crozzoli et  al., 1998; Siddiqi, 2000; Jain, 2009; Van den the presence of an outer accessory sheath with smooth Berg et al., 2015). Among all the Hemicriconemoides spe- annuli. cies, H. rosae was first described from the rose fields of The species of this group have been reported world - Bareilly district, Uttar Pradesh, India (Rathour et  al., wide; however, some of them are native (Geraert, 2003), then reported from the sugarcane fields of Meerut 1966). The nematodes of this genus showed obligatory district, Uttar Pradesh, India, by Khan et  al. (2019). A ectoparasitism (Geraert, 1966). Though they are obligate survey was conducted in the agricultural fields of Buland - parasites, they could be migratory, semi-sedentary and shahr district (28°40′ North and 77°86′ East at an altitude sedentary depending upon the environmental conditions of 209  m ASL), Uttar Pradesh, India. One species of the (Geraert, 1966).The nematodes of this group were found genus Hemicriconemoides recovered from the rhizos- to be associated with higher plants, mainly woody peren- phere of mustard fields. The earliest identification based nials, vines, etc.(Christie, 1953; Jenkins & Taylor, 1967), on morphological and some morphometric characters and several other plants as a result of which rot, necrosis revealed that the isolated population could be H. rosae of leaves, suppressed root growth, root lesions and coarse (Rathour et al., 2003), as it showed resemblance with the roots, etc., are common symptoms observed in case of original description. The occurrence of H. rosae in the these nematodes. sugarcane and rose fields is very common. However, the The genus Hemicriconemoides (Chitwood & Birchfield, species was first time reported from the mustard fields. 1957) comprises of 54 species (Khan et  al., 2019; Maria For more precise identification, molecular and phyloge - et al., 2018). Besides this, there were some species which netic analysis was done. Furthermore, to look for the var- was synonymized by the Geraert (2010). It is interest- iations between the species of genus Hemicriconemoides ing to note that 13 species have been reported from the reported worldwide with the studied isolate, principal agricultural rich fields of India, i.e. Hemicriconemoides component analysis (PCA) was also done. asymmetricus recovered from rose (Rathour et  al., 2003; Uttar Pradesh), H. communis isolated from the orange, Methods mango, mulberry, litchi and peach plants (Edward & Collection and nematode isolation Misra, 1964; Uttar Pradesh), H. conicaudatus recovered The present investigation was carried out in the fields of from peach fruits rhizosphere (Phukan & Sanwal, 1983; Bulandshahr district, Uttar Pradesh. The soils were col - Assam), H. dipterocarpus recovered from Dipterocarpus lected from the meadows, pastures, gardens and agri- tuberculatus (Mohilal et  al., 2004; Manipur), H. doon- cultural fields of district Bulandshahr (28° 40′ North and ensis from litchi (Srivastava et  al., 2000; Uttar Pradesh), 77° 86′ East, elevation of 209 m ASL) of Meerut Division. H. longistylus from tree fern (Rahman, 1990; Assam), H. A total of 85 soil samples were collected at a minimum mangiferae from litchi, mango amla and ashoka tree (Sid- depth of 5–10  cm (Southey, 1974) from the fields. Dur - diqi, 1961; all India), H. mehdii from grasses (Suryawan- ing the collection of samples, it was made sure that the shi, 1971; Maharashtra), H. neobrachyurus from oak, rice samples were collected from the previous sample at a dis- pomegranate and rose (Dhanachand & Jairajpuri, 1980; tance of 5 km. Soil samples (250gm) were collected in the Manipur), H. rosae from rose and sugarcane (Khan et al., transparent polyzipper bags labelled with the date, host 2019; Rathour et al., 2003; Uttar Pradesh), H. rotundoides plant, soil type and locality. Further, the soil was brought from Hibiscus rosasinensis (Geraert, 2010;Uttar Pradesh), to Nematology Laboratory, Department of Zoology, H. sunderbanensis from mangrove (Ganguly & Khan, Chaudhary Charan Singh University, Meerut, India, for 1982; West Bengal) and H. variabilis from peach (Rah- further processing. man & Ahmad, 1995; Assam). It is noteworthy to men- tion that only three species out of thirteen have been Nematode extraction and identification reported with males, i.e. H. communis, H. mangiferae The nematodes were extracted from the soil, using modi - and H. variabilis. The morphological identification of fied Cobb’s (1918) sieving method and Baermann’s fun - these species is a tedious task and requires expertise. In nel techniques (Goodey, 1957; Southey, 1986). To isolate some Hemicriconemoides species, males are absent which the plant-parasitic nematodes, 250 g of the soil was taken increases the need for the proper identification of the and then all lumps were broken gently in order to avoid females and juveniles (if present) (Ashokkumar & Vadi- the damage to nematodes. After this, 250 gm of soil was velu, 1990). taken in a 5 L bucket which was filled up to 3–4 L water. There are various species of genus Hemicriconemoides After topping up with water, it was swirled thoroughly by which were encountered from the rhizosphere of various hand to suspend all particles. The resulting suspension Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 3 of 15 was washed through a 2 mm aperture sieve. The mixture 95 °C for 10 min. The lysates were chilled on ice and then was allowed to settle for about 25  s sedimentation time. centrifuged at 6000 × g for 2  min, and 3 μL of superna- After this, the supernatant was decanted through a bank tant was used for PCR (Bhat et al., 2021). The ITS rDNA of sieves differing in their aperture size mainly 60  µm, was amplified using primers 18S: 5′-TTG ATT ACG TCC 150  µm, 200  µm, 250  µm, 300  µm, 350  µm and 400  µm, CTG CCC TTT-3′ (forward) and 28S: 5′-TTT CAC TCG respectively. Then, the residues on the sieves were washed CCG TTA CTA AGG -3′ (reverse) (Vrain et  al., 1992) and thoroughly with a gentle jet of water and finally collected partial sequence of LSU (28S) gene, D2–D3 domains in a beaker. Water was added to the remaining heavy resi- were amplified using primers, and D2A (5’-ACA AGT dues and thoroughly mixed, and again the supernatant ACC GTG AGG GAA AGTTG-3’) and D3B (5’-TCG GAA was decanted through the same bank of sieves. The sieves GGA ACC AGC TAC TA-3’) (De Ley et al., 1999) primer were washed, and the residues on the sieves were added pairs were used. The 30  µl PCR product consisted of to the previously collected. The sievings were shaken gen - 10 µl nuclease free distilled water, 1 µl each forward and tly to suspend all particles before pouring through a 90p reverse primer, 3  µl DNA extract and 15  µl Dream Taq aperture nylon screen supported by polyethylene ring Green PCR master mix 2X (Thermo Scientific). The PCR (Flegg, 1967), which was immediately placed on a Baer- amplifications were carried out using Verti 96-well fast mann funnel containing enough water just to submerge thermocycler (AP Scientific) with heated lid pre-set at the screen surface and debris. After 20  h, about 25  ml 95ºC and subjected to the following cycling profile: For of water containing the extracted nematodes from the the ITS rDNA region, PCR conditions included 1 cycle funnel was taken for examination under a stereoscopic of initial denaturation at 94 °C for 5 min; followed by 35 microscope at × 25 or × 50 magnification. cycles of 94  °C (denaturation) for 1  min, 55  °C (anneal- ing) for 1 min 30 s or 52 °C (annealing) for 30 s and 72 °C Morphology and morphometry analysis (primer extension) for 1  min, followed by a final exten - The adult females were heat killed in Ringer’s solution sion at 72 °C for 15 min to confirm all of the PCR prod - and then fixed in 50% TAF for 24  h. They were then ucts are complete length. For the D2D3 rDNA region, fixed in 100% TAF for 5–7  days (Courtney et  al., 1955) PCR conditions included 1 cycle of initial denaturation at and dehydrated in Seinhorst I and II (Seinhorst, 1959). 95 °C for 3 min; followed by 35 cycles of 95 °C (denatura- Further processing was done by following the protocols tion) for 45 s, 55 °C (annealing) for 45 s and 72 °C (primer of Bharti et  al. (2020). To prevent nematodes from flat - extension) for 1  min, followed by a final extension at tening, they were mounted in a small drop of glycerine 72  °C for 10  min to confirm all of the PCR products are on permanent clean glass slides with an extra amount complete length. The amplified products were electro - of paraffin wax (Bhat et  al., 2019). The phase contrast phoreses in 1% agarose (w/v) gel using 1 × TAE buffer microscope’s inbuilt software was used to perform obser- buffered agarose gel stained with ethidium bromide vations and measurements are in micrometre (μm). De (45 min, 100 V) was used to assess amplification success Manian indices (de Man, 1881) and other ratios were (Bharti et  al., 2020). The amplified PCR products were calculated. A drawing tube connected to a Nikon micro- purified and sequenced by Bioserve Pvt. Ltd., Hyderabad scope with differential interference contrast optics was (India). BioEdit was used to edit the newly discovered used to create line drawings. Images were captured with sequences (Hall, 1999). Sequences were deposited under a Nikon Digital Sight DS-U1 camera and DIC optics on the accession numbers: ® ® a Nikon microscope. Adobe Photoshop CS was used to edit the micrographs. Phylogenetic analysis The ITS (Internal transcribed spacer) and D2D3 Molecular characterization sequences were BLAST (Basic Local Alignment Search DNA isolation Tool) for % similarity matches with the previously sub- The DNA was extracted from single virgin females (not mitted sequences in the NCBI (National Centre for having plug at the vulva). The single virgin female from Biotechnology Information) (Altschul et  al., 1990). The each species/strain was first washed with Ringer’s solu - conserved regions, i.e. D2D3 and ITS, have been exploited tion followed by washing in Phosphate buffer saline (pH for establishing the relationship among the Hemicri- 7.2). The female from each species/strain was trans - conemoides species. For each amplified rDNA region ferred into a sterile Eppendorf tube (500 µl) with extrac- (ITS and LSU), an alignment of our data with sequences tion buffer 20  µl (ddH20 17.7  µl, 10X Taq buffer with from other closely related species was generated using (NH ) SO 2  µl, 1% tween2 µl, and proteinase K 0.1  µl). default ClustalW parameters in MEGA 7.0 (Kumar et al., 4 2 4 This buffer was frozen at − 80  °C for several days before 2016) and manually optimized in BioEdit (Hall, 1999). being incubated at 65  °C for 1  h and subsequently at The best fit models of nucleotide substitution used for Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 4 of 15 Morphological characterization the phylogenetic analysis were selected using jModel- Present specimen (HCN) showed close resemblances to Test 2.1.10 (Darriba et al., 2012). Sequences were concat- the Hemicriconemoides rosae (Rathour et  al., 2003) in enated with the R package ‘concatipede’ v1.0.0 (Vecchi morphological observations with respect to the presence & Bruneaux, 2021).Bayesian inference (BI) was used to of a prominent and elevated labial disc, long stylet, phar- infer the phylogenetic trees from the datasets. Aglenchus ynx distinct and separated from intestine. Besides this, agricola were utilized as out-group taxa, with all charac- vulva was enclosed with well-developed membranous teristics being equally weighted and gaps being treated as sheath, presence of conoid tail which is dorsally convex missing data (Subbotin et  al., 2005; Van den Berg et  al., at the tail tip, and hence, it was considered as the same 2014; Khan et al., 2019). MrBayes 3.2.7 was used to pro- (Fig.  1). Line drawings were prepared for comprehensive duce Bayesian phylogenetic reconstructions (Ronquist analysis (Fig.  2). However, few morphometrical charac- et  al., 2012). The General Time Reversible substitution ters showed little variation from each other. model with gamma distributed rate variation across sites and a proportion of invariable sites (GTR + G + I) was used as the optimal nucleotide substitution model for the Female analyses. One tree was preserved per 1000 generations Small in size 0.37–0.47  mm in length. Body straight to after running Markov chain Monte Carlo generations slightly arcuate ventrally after heat killed. Sheath closely (MCMC) for 1 × 10 cycles. The tree was visualized and fit to the body except on tail and frequently protrud - saved with FigTree 1.4.4 (Rambaut, 2018). ing forward over the lip region. Sheath is not annulated at vulval region. Cuticle smooth and covered by outer accessory layer/sheath with smooth annules. Body Statistical analyses annuli distinct and rounded (Fig.  1). Lip region rounded Correlation analysis and linear regression analysis were with a prominent labial disc, not set off with two annuli, performed to resolve the relationship between vari- first annuli smaller than second (Fig.  1, Table  1). Sty- ous morphometrical parameters using GraphPad Prism let long (50-58  µm), well developed with stylet knobs software (version 6.0). Principal component analysis (2.4–4.0  µm) prominently anchor-shaped anteriorly and (PCA) was done to find out the morphological varia - rounded posteriorly, 4.8–6.9  µm wide (Fig.  1, Table  1). tions between the isolated nematodes in the present Dorsal pharyngeal gland opening situated at the base investigation and closely related species of the nema- of stylet knobs. Median bulb observed with prominent todes. The measurements of the Hemicriconemoides spe - valve. Procorpus and metacorpus found to be fused. cies were collected from their original descriptions. The Excretory pore situated from 5–11 annuli posterior 2D plot was formed by using the PC1 and PC2 values of to the base of pharynx. Hemizonid not found. Sheath each isolate based on eigenvalues given by the software annuli smooth, slightly indented over the whole length. MINITAB 20 (Nisa et al., 2021). Anastomoses found absent. Vulva posterior, located 8–13 annuli from terminus and 90–94% of body length (Fig.  1, Table  1). Flap like structure absent on vulva but Results with a prominent membranous sheath. Spermatheca well In the present investigation, HCN strain recovered from developed and oval shape filled with sperm cells (Fig.  1). one soil sample out of 85 soil samples collected from the Vagina observed distinct and straight. Body found to be rhizosphere of mustard from Bulandshahr district. The tapering posterior to anus. Tail shape conoid and found strain was found to be plant-parasitic nematodes due to dorsally convex with the sheath enclosing the tip, taper- presence of long stylet on the labial region. The pH of ing gradually to a more pointed tip. Anus position was the soil was 5.4, and the texture of the soil was found to uncertain still it was located just posterior to vulva 2–5 be alluvial. Most of the sampling was done in the winter annuli (Table 1). season mainly in January–March. The strains HCN bear double cuticle, which is a characteristic feature of Hemic- riconemoides, thus belong to same genus. Male Based on morphology and morphometry, these nema- Males are absent. todes were found close to Hemicriconemoides rosae, hence described as the same. In the present study, only Comparative study of H. rosae strain (HCN) and H. rosae females of the strain HCN were recovered as noted in the (Rathour et al., 2003) original descriptions. For the morphological characteri- As far as females was concerned, most of the morpho- zations, permanent slides were prepared and deposited metric parameters of isolate (HCN) and original spe- in the Nematology Laboratory, Department of Zoology, cies was found to be approximately similar. The other Ch. Charan Singh University, India. Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 5 of 15 Fig. 1 Photomicrographs of Hemicriconemoides rosae isolate HCN. A, B Anterior region of female; C Whole female; D Posterior region of female; E Female gonad; F Vulval region; and G Anal and tail region. The arrows indicate the stylet, stylet knob and nerve ring (B), female gonad (E), vulval region and spermatheca of female (F) and anal region and tail (G) measurements, i.e. a (13.6–16.9 vs 16.0–17.6), b (4.6–5.6 of the pharynx (72–94 vs 102 µm) and number of annuli vs 4.9–5.4), V (90–95 vs 92–94.5), stylet length (50–58 posterior to vulva (8–13 vs 6–9) which showed deviation vs 50–55  µm), metenchium length (40–49 vs 44.8  µm), from the original description. telenchium length (9–11.8 vs 7.7  µm), stylet knob width Hemicriconemoides rosae isolate (HCN) was compared (4.8–6.9 vs 6.9  µm), labial disc diameter (4.3–5.9 vs with H. rosae Khan et al.(2019) sugarcane population and 5.1 µm), R (102–115 vs 116), Rst (12–20 vs 12–14), Roes three closely related species of Hemicriconemoides, and it (20–22 vs 21–26) of present strain, were found close to differs in some morphological and morphometric charac - the original description H. rosae (Rathour et  al., 2003). ters with compared species. The comparison of morpho - All the body parameters were found to be close except metrics is presented in Tables 1 and 2 and is described as body length (376–472  µm vs 470–510  µm), the length follows: Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 6 of 15 Fig. 2 Line drawings of Hemicriconemoides rosae from rhizosphere of mustard. A, B Anterior region of female; C: Female gonad and tail; D: Entire adult female; E body annuli in mid-body region; and F Vulva region The present isolate HCN of Hemicriconemoides rosae terms of number of sheath annuli, anus position, ante- recovered from mustard were found to show some vari- rior secretory to excretory position and the presence of ations from the H. rosae (Khan et  al., 2019) sugarcane males. H. communis was found to have more annuli than population. The parameters which show deviations were: H. rosae (102–115 vs 110–130), smaller VL/VB (0.9–2.0 average body length (376–472 vs 323-486  µm), ratio a vs 1.3–2.0). The position of anus is uncertain in H. rosae; (13.6–16.9vs 12.2–18.7), ratio b (4.6–5.6 vs 3.88–5.93), however, anus is clearly visible in H. communis. Males are telenchium length (9–11.8 vs 5.1–10.8 µm), stylet (50–58 present in H. communis, whereas in H. rosae no males vs 52-61 µm) and annulus width (3.6–5.2 vs 2.9–3.8 µm) were observed (Table 2). (Table 1). Based on morphological characterization, H. rosae Hemicriconemoides rosae HCN females were differenti - HCN was separated from the H. wessoni Chitwood ated from the H. communis Edward and Misra (1964) in and Birchfield (1957) in terms of ratio a, c and other Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 7 of 15 Table 1 Morphometrics of Hemicriconemoides rosae isolate HCN from the mustard (present population), rose population (Rathour et al., 2003) and sugarcane population (Khan et al., 2019). All measurements are in µm (except ratio and percentage) and in the form of mean ± standard deviation (range) Character Mustard population Rose population (Rathour Sugarcane isolate HCN et al., 2003) population (Khan et al., 2019) n 15 6 32 L 443 ± 25 (376–472) 490 (470–510) 398 (323–486) a (Length/Diameter at mid-body) 15.3 ± 0.9 (13.6–16.9) 16 (16–17.6) 15.4 (12.2–18.7) b (Length/Pharynx length) 5.1 ± 0.3 (4.6–5.6) 5.2 (4.9–5.4) 4.8 (3.9–4.9) c (Length/Tail) 16.4 ± 1.7 (14.1–20) – – c’ ( Tail/diameter at anus) 22 ± 1.8 (19.4–25) – – V (anterior to vulval/total body length) × 100 92 ± 1.4 (90–95) 93.2 (92–94.5) 91.5 (89.9–92.7) M (ratio between the length of prorhabdion and stylet in %) 85.6 ± 2.5 (81.2–91.5) 85.3* 85.6 (82.2–90.4) EP (Excretory pore to anterior end) 116 ± 5.5 (102–123) – – WEP (Diameter at excretory pore) 28 ± 0.9 (25–29) – – NR (Nerve ring) 71 ± 2.4 (64–74) – – PL (Pharynx length) 87 ± 5.4 (72–94) 102* 84 (71–103) EBL (oesophageal bulb length) 15.8 ± 2.2 (11.5–18.7) – – EBW (oesophageal bulb width) 12 ± 1.8 (8.8–15) – – Tail 27 ± 2.7 (23–31) – – Diam. at anus 19.8 ± 1.3 (18–23) – – Diam. at mid body 29 ± 2.0 (26–33) 26.5* 25.9 (23.4–29.6) V’ (Distance from the anterior end to vulva) 409 ± 26 (340–444) – – Stylet 54 ± 2.4 (50–58) 52 (50–55) 55 (52–61) Stylet Knob height 2.8 ± 0.5 (2.4–4.0) 2.6* 2.7 (2.3–3.8) Stylet Knob width 5.7 ± 0.7 (4.8–6.9) 6.9* (4.8–6.9) 6.2 (4.8–7.4) Metenchium length 44 ± 2.3 (40–49) 44.8* 47 (44–51) Telenchium length 10.3 ± 0.8 (9–11.8) 7.7* 8.0 (5.1–10.8) R ( Total number of body annules) 108 ± 4.1 (102–115) 116* 116 (109–124) Rst (Annules from anterior extremity to base of stylet) 13.9 ± 2.0 (12–20) 13 (12–14) 16.6 (15–18) Roes (Annules from anterior extremity to base of oesophagus) 21 ± 0.8 (20–22) 23.5 (21–26) 25.9 (21–30) Ran (Annules from anus to tail terminus) 6.9 ± 0.6 (6–8) – – Rex (Annules from anterior extremity to excretory pore) 28 ± 2.0 (25–33) – 28 (22–30) Rv (Annules from vulva to tail terminus) 9.6 ± 1.5 (8–13) 7.5 (6–9) 7.5 (7–9) MBL (Median bulb length) 14.7 ± 1.6 (11.5–16.4) – – MBW (Median bulb width) 12.8 ± 1.3 (10.9–15.5) – – Lip Length 5.9 ± 0.2 (5.2–6.6) 5.7* 5.8 (5.0–6.5) Lip Width 5.1 ± 0.5 (4.3–5.9) 5.1* 5.4 (4.5–6.0) D % (excretory pore/pharynx length × 100) 134 ± 10.9 (115–163) – – E % (excretory pore/Tail) × 100 430 ± 49 (365–505) – – VL (distance from vulva to terminus) 35 ± 6.1 (23–27) 34.7* 33.7 (28.6–39.5) VB (body diameter at vulva) 24 ± 1.8 (21–28) 21.6* 18.8 (15.0–21.6) VL/VB (distance from vulva to terminus/body diameter at vulva) 1.5 ± 0.3 (0.9–2.0) 1.6* 1.8 (1.3–2.2) Sty%L ( Stylet length/body length × 100) 12.3 ± 1.0 (10.7–14.8) 10.7* 14.0 (12.0–16.2) First lip diam 8.6 ± 0.5 (8.2–9.9) 8.5* 8.8 (8.0–10.6) Second lip diam 12.1 ± 0.7 (10.1–14.2) 12.3* 11.7 (10.0–13.3) First body annulus diam 14.6 ± 0.4 (12.3–15.2) 14.8* 14.2 (12.1–15.8) Second body annulus diam 16.9 ± 0.9 (14.5–19.9) 17.2* 15.7 (13.5–18.8) Third body annulus diam 18.1 ± 1.2 (16.2–21.3) 18.9* 17.2 (15.2–21.0) Annulus width 4.4 ± 0.4 (3.6–5.2) 4.3* 3.4 (2.9–3.8) Values were determined in holotype specimens Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 8 of 15 Table 2 Comparative morphometrics of adult female isolate HCN with original Hemicriconemoides rosae population and other species of the Hemicriconemoides genus Characters Authors L a b V PL Stylet R Rst Roes Rv Ran Sty%L Annulus W H. rosae Present isolate 443 15.3 5.1 92 87 54 108 13.9 21 9.6 (8–13) 6.9 (6–8) 12.3 4.4 (3.6–5.2) HCN, Mustard (376–472) (13.6–16.9) (4.6–5.6) (90–95) (72–94) (50–58) (102–115) (12–20) (20–22) (10.7–14.8) H. communis Edward and 480 16 4.5 93 109* 54* 115 16* 26* 8* 7* 10.8* 3.5* Misra (1964) (340–500) (12–25) (4–5) (89–96) (51–58) (110–130) H. wessoni Chitwood 429.2 (382.5– 12.7 4.6 92 – 54 (76–83) – – 9* 5* 15.9* (4–5) and Birchfield 499.8) (10.7–15) (4.0–5.5) (89–93.5) (50–60) (1957) H. califor- Pinochet and 440 17 4.1 91 – 80 119 – – 11 (10–12) 5 (4–6) – – nianus Raski (1975) (410–460) (16–19) (3.4–4.6) (90–92) (77–83) (112–127) H. macrodorus Volvas et al. 691 17 4.4 95 152 101 137 23 – 13 (2–14) 8 (7–9) 15 (13–18) 5.8* (2000) (548–761) (15–21) (3.8–5.2) (94–96) (140–171) (90–110) (127–148) (18–27) H. chitwoodi Esser (1960) 540 15.5 3.8 90.1 – 91 124 – – (12–16) (8–11) – – (480–590) (13.4–17) (3.5–4.1) (88.1–91.0) (85–95) (116––133) H. paratai- Decraemer 499 15.2 4.2 90 120.2 79.4 138.1 25.4 – 9.1 (8–10) 5 (4–10) 15.9* 3.5* wanensis and Geraert (440–535) (13.8–16) (3.9–4.5) (88.7–91) (107–129) (77–82) (129–145) (23–29) (1992) H. paracamel- Maria et al. 563 16.9 4.4 88.9 127 83 132 23.1 34.5 (31–37) 14.8 (13–16) 8 (5.0–10.0) 14.7 – liae (2018) (519–604) (15.3–18.5) (4.1–5.0) (87.7–90.1) (119–135) (80–85) (124–140) (22–26) (13.7–16.3) H. kanayaensis Nakasono 571 21.5 4.8 88.9 – 74 150 – – 18 (16–21) 12 (11–15) 12.9* 4.6* and Ichinohe (500–631) (18.7–24.4) (3.3–5.6) (87.5–91.5) (66–79) (142–164) (1961) H. phoenicis Van den Berg 630 21.6 4.7 46 133 88 130 23 32 (28–36) 14 (13–19) 10 (9–15) 14 (12.6–16.3) 5.5 (4.5–6.5) et al. (2015) (549–699) (16.7–24.3) (4–5.7) (27–62) (117–149) (81–97) (123–137) (21–25) H. litchi Edward and 480 15 4.6 92 107* 63 130 12* 25* 12* 7* 13.1* 3.5* Misra (1964) (450–500) (13–18) (3–5) (91–93) (60–65) (128–133) H. strictathe- Esser (1960) 560 – – – – 79 138 18.8* 27.3* (11–13) – – – catus (510–590) (73–83) (127–152) H. cocophilus Dasgupta (460–500) (14–15) (4.0–5.0) (91–92) – (50–57) (108–143) – – (9–10) (8–9) – (3–5) et al. (1969) H. brachyurus Dasgupta (400–540) (13–17) (4.3–5.2) (93–95) 96* (56–64) (98–119) – – (7–9) (6–7) – – et al. (1969) All measurements are in µm (except ratio and percentage) and in the form of mean ± standard deviation (range) Values were determined in holotype specimens L = Body length, PL = Pharynx length, a(L/BD), b(L/PS), V% (anterior to vulval/total body length) × 100, Stylet = Stylet length, Stylet knob W = Stylet knob width, R = Total number of body annules, Rst = Annules from anterior extremity to base of stylet, Roes. = Annules from anterior extremity to base of oesophagus, Rv = Annules from vulva to tail terminus, Ran = Annules from anus to tail terminus, Annulus W = Annulus width, Sty%L = Stylet length/body length*100 Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 9 of 15 Principal component analysis parameters. The ratio a of H. wessoni (12.3–14.8) was Principal component analysis was done using morpho- found to be smaller when compared with H. rosae (13.6– metric data of the nematode isolates, i.e. H. commu- 16.9). The ratio c of H. wessoni (16.2–22.9) is larger when nis (Edward and Misra 1964), H. wessoni (Chitwood & compared with H. rosae (14.1–20). Besides this, other Birchfield, 1957), H. californianus (Pinochet & Raski, characteristics of the H. rosae isolate HCN showed dif- 1975), H. macrodorus (Volvas et al., 2000), H. chitwoodi ferences with the H. wessoni such as tail shape and labial (Esser, 1960), H. parataiwanensis (Decraemer & Ger- disc. The labial disc in case of H. rosae was found to be aert, 1992), H. paracamelliae (Maria et  al., 2018), H. elevated, whereas in case of H. wessoni, slight or no ele- kanayaensis (Nakasono & Ichinohe, 1961), H. phoenicis vation was observed. The pharyngeal bulb in H. rosae (Van den Berg et  al., 2015), H. litchi (Edward & Misra, showed no overlapping; however, in H. wessoni dorsal 1964), H. strictathecatus (Esser, 1960), H. cocophilus overlapping was present. The shape of the tail in H. rosae (Dasgupta et al., 1969), H. brachyurus (Dasgupta et al., was found to be conoid and the membranous sheath 1969) isolated from different geographical areas and the enclosed the tail tip dorsally, whereas in H. wessoni the studied isolate HCN. tail shape was found to be conoid but membranous The results of the PCA showed that there are vari - sheath may or may not enclose the tail tip (Table 2). ations in the morphometry between the Hemicri- The H. rosae isolate HCN showed differences from conemoides rosae isolate HCN recovered from mustard the H. minutus Esser (1960) in terms of de Manian indi- in the present investigation and the Hemicriconemoides ces, i.e. a and b, stylet and pharyngeal bulb. The ratios a rosae (Khan et  al., 2019) isolated from the sugarcane (13.6–16.9 vs 10.3–16.4) and b (4.6–5.6 vs 3.0–3.8) of H. and with the other species of Hemicriconemoides spe- rosae were found to be higher than H. minutus. The stylet cies (Dasgupta et  al., 1969). However, the analysed length of H. rosae was found to be smaller than H. minu- parameters of morphometry showed less variations tus (50–58 vs 70.5–80.5  µm). The pharyngeal bulb of H. between the present isolate HCN with the originally rosae showed no overlapping, while in case of H. minutus described Hemicriconemoides rosae (Rathour et  al., overlapping was present. Fig. 3 The principal component analysis (PCA) of different populations of Hemicriconemoides species based on female specimens with different body parameters as factors. The biplot was based on two major principal components; PC1 and PC2. L = Body length, PL = Pharynx length, a (L/BD), b(L/PS), V% (anterior to vulval/total body length) × 100, Stylet = Stylet length, Stylet knob W = Stylet knob width, R = Total number of body annules, Rst = Annules from anterior extremity to base of stylet, Roes. = Annules from anterior extremity to base of oesophagus, Rv = Annules from vulva to tail terminus, Ran = Annules from anus to tail terminus, Annules W = Annules width, Sty%L = Stylet length/body length*100. The ellipses represent the group of nematodes/isolates coming together showing fewer variations Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 10 of 15 2003) (Fig.  3). Besides this, the isolates showed dif- Table 3 Loading scores of the variables and factor score of the observations for females of Genus Hemicriconemoides spp ferences from the other Hemicriconemoides species (Fig. 3). Characters Loading Species Factor The PCA based on the morphometry of females scores of the score of the variables observations showed an accumulated variability of 66.2% (Fig.  3). The contribution of PC1 and PC2 was found out to be Females Females 43.3% and 22.9%, respectively (Fig.  3). Four parameters, Variable PCA1 PCA2 PCA1 PCA2 annulus width (r = -0.06), b (r = -0.16), stylet knob width L 0.23 0.39 HCN, Mustard − 1.96 0.65 (r = -0.15) and V% (r = -0.17) were found to be negatively a 0.25 0.37 H. rosae, rose − 1.82 1.20 correlated across nematode/species in PC1. Ten charac- b − 0.16 0.43 H. rosae, sugar- − 1.51 − 0.57 ters out of fourteen characters were found to be positively cane correlated across isolates, and the remaining characters V − 0.17 − 0.32 H. promissus − 3.59 0.80 were negatively correlated considering PC1 (Fig.  3). The PL 0.33 − 0.03 H. litchi − 0.51 0.14 highest coefficient of correlation was observed in annuli Stylet 0.33 − 0.26 H. silvaticus 2.54 − 1.20 from anus to tail terminus (r = 0.38) and annuli from R 0.32 0.14 H. strictathecatus 0.46 − 0.11 anterior extremity to base of stylet (r = 0.37) in PC1. Con- Rst 0.37 − 0.12 H. brachyurus − 2.59 − 0.34 sidering PC2, nine characters out of fourteen were found Roes 0.38 − 0.13 H. californianus 2.58 − 2.50 to be positively correlated and the remaining were nega- Rv 0.33 0.13 H. macrodorus 3.57 − 0.31 tively correlated (Fig.  3, Table  3). The highest coefficient Ran 0.15 0.32 H. chitwoodi 1.89 − 2.73 of correlation was observed in b (r = 0.43) PC2. Stylet Knob width − 0.15 0.03 H. wessoni − 3.33 0.62 Sty%L 0.27 − 0.35 H. minutus − 0.47 − 2.44 Molecular analysis Annulus width − 0.06 0.27 H. ortonwilliamsi − 2.49 1.68 The Hemicriconemoides species isolate HCN examined H. communis − 1.02 0.31 in present study has been characterized molecularly by H. cocophilus − 2.59 − 1.41 the sequences of two genes, i.e. ITS rDNA (695  bp) and H. parataiwan- 1.74 − 1.66 D2D3 fragments of 28S rDNA (708  bp). The ITS and ensis D2D3 sequences of Hemicriconemoides rosae isolate H. paracamelliae 2.65 0.46 HCN were deposited in NCBI GenBank with accession H. kanayaensis 3.22 2.99 number ON844213 and ON844212, respectively. H. phoenicis 3.24 4.41 Pairwise distances (Kimura 2-parameter) based on the ITS regions revealed the genetic distances between the Hemicriconemoides species. The present isolate HCN (ON844213) genetic distance with previously described phylogenetic relatedness of the Hemicriconemoides Hemicriconemoides rosae (MK371815, MK371814, and rosae with other species of Hemicriconemoides was MK371816) was found to be 0.0000. The present isolate established from the comparison of concatenated Hemicriconemoides rosae HCN (ON844213) showed sequences (Fig.  4, Additional file  3: S3). There are two greatest genetic distance (0.1915) from H. kanay- major clades in the rDNA phylogenetic tree of Hemic- aensis (MG029568). The average overall genetic dis - riconemoides. It is interesting to note that the phyloge- tance between in-group species was 0.1675 ranging netic analysis was in agreement with the morphology from 0.0000 to 0.3055 between H. rosae (MK371815, and molecular analysis. The first cluster includes the MK371814, MK371816) and H. kanayaensis (MG029568) Hemicriconemoides brachyurus, H. cocophilus, H. (Additional file 1: S1). ortonwilliamsi, H. minutus, H wessoni, H. macrodorus The average overall genetic distance (Kimura 2-param - and H. promissus. The second cluster includes the eter) based on D2D3 regions between in-group spe- Hemicriconemoides rosae HCN, Hemicriconemoides cies was 0.1336 ranging from 0.0000 between H. rosae rosae and Hemicriconemoides species. A thorough (MK371813) and H. rosae (MK371811) to 0.2326 analysis of sequences of the 28S and ITS rDNA gene between H. minutus (KF856516) and H. parataiwanensis of the HCN isolate described in this study reveals clear (MG029573) (Additional file 2: S2). relatedness with 18 other Hemicriconemoides species. Bayesian inference phylogenetic analyses based on the concatenated sequences (ITS and 28S rDNA) showed Phylogenetic analysis a clear monophyly of the group formed by the isolate ITS and D2D3 regions are well-conserved regions in HCN and already described Hemicriconemoides rosae the nematodes and are used to ascertain the relation- Khan et al. (2019) (Fig. 4, Additional file 3 : S3). ship between closely and distantly related species. The Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 11 of 15 Fig. 4 Phylogenetic relationships within populations and species of Hemicriconemoides as inferred from Bayesian analysis based on the concatenated sequences of ITS and 28S rRNA. Numbers at the nodes indicate Bayesian posterior probability. Newly obtained sequences of H. rosae underlined. The scale bar represents 0.01 substitutions per nucleotide position Relationship between various morphometric parameters analysed parameters, no correlation has been observed The results obtained from the two tailed Pearson’s cor - with the de Man indices except a ratio of female which relation analysis revealed that some parameters of showed positive correlation (r = 0.6978, p = 0.0038) Hemicriconemoides rosae isolate HCN showed signifi - (Fig.  5, Table  4). The index a also showed positive cor - cant correlation with other morphometric parameters relation with other parameters, i.e. excretory pore (Table  4). The morphometric parameters which were (r = 0.7389, p = 0.0017, Table  4), oesophageal bulb focused in the study to understand the relation between (r = 0.6953, p = 0.0040, Table  4), vulva-anterior end various parameters were body length, excretory pore, (r = 0.7538, p = 0.0012, Table  4). However, index a tail, pharynx stylet, de Manian indices a, b, c and c’, showed no correlation with other indices (Table  4). The nerve ring, vulva width and other parameters. In this, 15 index b showed positive correlation with the index c females of the isolate HCN were analysed to figure out (r = 0.5689, p = 0.0269, Table  4) and negative correlation the correlation between the morphometric data. Each with pharynx (r = − 0.5443, p = 0.0359, Table  4). Among morphometric parameter was replicated 3 times to get all the parameters, the c’ showed positive correlation the final results. with the tail (r = 0.5966, p = 0.0189, Table  4) and nega- The results based on the two-tailed Pearson’s corre - tive correlation with index c (r = − 0.6779, p = 0.0055, lation suggested that body length has highest correla- Table  4). These observations suggested that body length, tion in comparison with others with the morphometric vulva position, de Manian index ‘a’ are important for the data. Though body length showed correlation with the evaluation of the females of Hemicriconemoides rosae. Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 12 of 15 Table 4 Correlation of morphometric data of Hemicriconemoides rosae isolate HCN was determined by two tailed Pearson’s correlation L a b c c’ V L 1 0.6978** 0.3446 0.3492 0.1044 0.3261 a 0.6978** 1 0.2484 0.3704 − 0.01959 0.3505 b 0.3446 0.2484 1 0.5689* − 0.4858 0.1374 c 0.3492 0.3704 0.5689* 1 − 0.6779** 0.5414* c’ 0.1044 − 0.01959 − 0.4858 − 0.6779** 1 − 0.08350 V 0.3261 0.3505 0.1374 0.5414* − 0.08350 1 EP 0.9651*** 0.7389** 0.3727 0.3506 0.1033 0.3744 NR 0.5820* 0.4458 − 0.3041 − 0.05316 0.4115 0.5198* PL 0.5990* 0.4690 − 0.5443* − 0.1700 0.4952 0.1685 EBL 0.6816** 0.6953** 0.1143 0.4009 − 0.2365 0.1240 T 0.5458* 0.2028 0.05948 − 0.3330 0.5966* − 0.04380 Body diameter: anus 0.5670* 0.4774 − 0.07373 − 0.1201 0.09106 0.1515 Body diameter: mid-body 0.5379* 0.4317 0.2305 − 0.01556 0.009445 − 0.1235 VDesh 0.9715*** 0.7538** 0.3453 0.4479 0.07187 0.5407* Stylet 0.5498* 0.1846 − 0.1253 − 0.1770 0.1626 − 0.1398 MBL 0.5448* 0.4334 − 0.02157 0.09194 0.1999 0.5293* WV 0.5872* 0.4855 0.1949 0.1943 0.06458 0.1955 Bold and asterisk (*) values represent the statistical difference. p < 0.05 was considered a statistically significant difference Shows statistical significant; Pearson’s correlation Shows p < 0.05, ** shows p < 0.01, *** shows p < 0.001 2010; Khan et  al., 2019) and is associated with many crops. The H. rosae (Rathour et  al., 2003) possessed very less morphological and morphometric characters which were used for the comparison with the studied isolate HCN. The morphological data of the HCN isolate showed much resemblance with the original description of the H. rosae (Rathour et al., 2003); however, it showed more deviation from the sugarcane population of H. rosae (Khan et  al., 2019) (Table 1). The various morphometric characters are not suffi cient for the delineation of the species. Molecular charac- Fig. 5 Relationship between the body length with ratio ‘a’ as terization is essential in order to validate the taxonomic determined by regression analysis of females of Hemicriconemoides position as well as the authenticity and phylogenetic isolate HCN. The symbol represents the values of ratio ‘a’ at specific body length. The solid line denotes linear regressions between the relationships among the species of a particular genus body length and ratio ‘a’. and between different nematode orders. The molecu - lar analysis based on the conserved regions ITS and D2D3 revealed the present isolate HCN to be H. rosae Discussion as it showed very less genetic distance from the already Hemicriconemoides was first described by Chitwood & described H. rosae (ITS:0.0000, D2D3: 0.0014, 0.0129) Birchfield, 1957 and further redescribed by Dasgupta (Additional file 1: S1 and Additional file 2: S2). et  al., 1969. These nematodes are commonly referred to The phylogenetic analysis was found to be consistent as sheathoid nematodes due to the body cuticle of female with the molecular data which revealed that the present covered by an outer accessory layer/sheath with smooth isolate to be H. rosae (Fig.  4). The phylogenetic analysis annules which is missing in juveniles. They are mostly based on the concatenated sequences (ITS and D2D3) distributed in temperate areas of the world mainly in region revealed that mustard population of H. rosae HCN Africa, America, Australia and South Asia and South formed a monophyletic clade with the sugarcane popula- Europe. This genus contains 54 valid species (Geraert, tion of H. rosae with a Bayesian posterior probability 1. Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 13 of 15 The phylogenetic tree revealed that H. rosae formed a sugarcane and rose fields is very high in the western separate branch without including any other Hemicri- Uttar Pradesh, India (Khan et  al., 2019; Rathour et  al., conemoides species, however, the position of H. rosae in 2003). The present investigation was carried out in the the phylogenetic tree was close to H. promissus, H. mac- fields of mustard which is an alarming sign as mus - rodorus, H. wessoni and H. minutus which found to be tard is one of the cash crops of India having economic consistent with the previous observations (Khan et  al., importance. From the studies, it is clear that species 2019). of genus Hemicriconemoides possess certain charac- The PCA showed large intraspecific and interspecific teristics which cause potent damage to the crops, ulti- variations. These variations were found to be independ - mately affecting the yield (Inserra et  al., 2014; Maria ent of the collection localities of soil samples. It was et al., 2018). The information revealed from the present evident from the previous studies that there were large investigation can be exploited by taxonomists for the intraspecific variations present among the nematodes comparison, identification and correlation between the which was also observed among the present isolate HCN morphometric parameters of the species. (mustard population) and Hemicriconemoides rosae (sug- arcane population) isolates of the present investigation Supplementary Information (Khan et al., 2019). However, less intraspecific variations The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s41936- 023- 00338-6. were observed between present isolate HCN and origi- nally described Hemicriconemoides rosae (Rathour et al., Additional file 1: S1. Pairwise distances (Kimura 2-parameter) between 2003) (Fig. 3, Table 3). species of Hemicriconemoides based on ITS (Internal transcribed spacer) The results obtained from the analysis of morpho - regions. Out-groups were not included. metric data based on two tailed Pearson’s correlation Additional file 2: S2. Pairwise distances (Kimura 2-parameter) of the between Hemicriconemoides species based on D2D3 (Large ribosomal revealed that in case of females of H. rosae, de Manian subunit, 28S) regions. Out-groups were not included. index a is very important for the evaluation as it showed Additional file 3: S3. List of GenBank accession numbers of the Hemicri- positive correlation with body length (Fig.  5, Table  4). conemoides species used in the phylogenetic tree. Similar results were observed in the genus Eucepha- lobus and Acrobeles (Amirzadi et  al., 2011, 2013). The Acknowledgements results of Fortuner (1990) also exhibited the highest cor- The authors are thankful to the Department of Zoology, Chaudhary Charan relation between the de Manian indices especially a and Singh University, Meerut, for providing necessary laboratory facilities for conducting the experiments. The authors are grateful to local farmers for their b and the length. Furthermore, the other ratios, i.e. V assistance in sample collection. The authors are also thankful to Dr. Abhilash also important along with the de Manian indices, i.e. a, Prabhat for his assistance in statistical analysis. The authors extended their c and c’ which is also consistent with the previous find - sincere thanks to Lovely Bharti for helping in morphological identification. ings (Fortuner, 1984). It was found from the previous Author contributions studies that a, c and c’ de Manian indices are essential The study was designed by HS and AKC. Interpretation of the data was done for the genus Helicotylenchus Steiner, 1945. Similar find - by HS. Manuscript writing was done by all authors. Tables and reference set- tings were done by HS. Errors and grammatical mistakes in manuscript were ings were obtained by Amirzadi et  al. (2013) for genus removed and edited by AKC. Then, final manuscript was read and approved by Acrobeles von Linstow (1877) where correlation between both authors. de Manian indices (b and c’) and body length was found Funding positive. The present study on the females of genus The authors declare that no funds, grants or other support were received dur- Hemicriconemoides also revealed the positive correla- ing the preparation of this manuscript. tion between the body length and ratio a (Fig. 5, Table 4). Availability of data and materials The reason behind that some morphological characters The data and material of this manuscript are available from corresponding showed highest degree of correlation; however, some of author on reasonable request. them showed least might be due to control of those char- acters by a specific gene cluster (Amirzadi et  al., 2013). Declarations It can be concluded from the results that there are some Ethics approval and consent to participate characters which showed high correlation with body This article does not contain any studies with human participants or animals. length which results in the increase or decrease in the characters with the body length (Fig. 5, Table 4). Consent for publication Not applicable. Conclusions Competing interests The authors declare that they have no competing interests. The present isolate HCN were identified as H. rosae based on the morphological, molecular and phy- logenetic analysis. The infection of H. rosae in the Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 14 of 15 Received: 23 August 2022 Accepted: 4 April 2023 with a revision of the key to species of Hemicriconemoides. Nematologica, 11, 157–161. Esser, R. P. (1960). Three additional species in the genus Hemicriconemoides Chitwood & Birchfield, 1957 (Nemata: Tylenchida). Nematologica, 5, 64–71. Flegg, J. J. M. (1967). Extraction of Xiphinema and Longidorus species from soil References by a modification of Cobb’s decanting and sieving technique. Annals of Bhat, A. H., Chaubey, A. K., Shokoohi, E., & Mashela, P. W. (2019). Study of Stein- Applied Biology, 60, 429–437. ernema hermaphroditum(Nematoda, Rhabditida) from the West Uttar Fortuner, R. (1984). Morphometrical variability in Helicotylenchus Steiner, 1945. Pradesh, India. Acta Parasitologica, 64, 720–737. https:// doi. org/ 10. 2478/ 5: On the validity of ratios. 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Molecular and phenotypic characterization of Hemicriconemoides rosae (Rathour et al., 2003) from mustard rhizosphere in India

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Abstract

Background Nematodes belonging to Genus Hemicriconemoides, commonly known as sheathoid nematodes, dam- aged many fruits, vegetables and cash crops, worldwide. A survey has been conducted in the agricultural fields of Bulandshahr district to find out the plant-parasitic nematodes load. Results A total of 85 soil samples were collected from mustard fields of Bulandshahr district of Uttar Pradesh and pro - cessed for the presence of plant-parasitic nematodes and they were isolated by ‘Cobb sieving method’. Among all the collected soil samples, one soil sample found to be positive for the genus Hemicriconemoides and docketed as HCN. The earliest identification based on morphology revealed the species to be Hemicriconemoides rosae which was recov- ered from the mustard field for the first time. The morphology was found to be more consistent with rose population as compared to sugarcane population of the Hemicriconemoides rosae. Further, the Hemicriconemoides rosae was identified on the basis of molecular and phylogenetic analysis based on the concatenated matrix. In addition, correla- tion analysis of the Hemicriconemoides rosae based on morphometric parameters was done. The results revealed the importance of body length and its relation with other morphometric parameters, and they were found significant. Besides this, de Manian index a showed highest correlation with the body length in adult females. In all the studied ratios, a is very important for the evaluation of the females of a specific species of genus Hemicriconemoides. The data from the principal component analysis (PCA) revealed the high intraspecific and interspecific variations between the species of genus Hemicriconemoides. However, less intraspecific variations were present between the rose population and mustard population of H. rosae. Conclusions The study revealed the new host, i.e. mustard crops, for H. rosae showing dissimilarity in morphology with the sugarcane population. Keywords Hemicriconemoides rosae, Mustard, Morphology, Taxonomy, Correlation, PCA have been evolved in such a way that they have devel- Background oped an accessory structure known as stylet with the There are some nematodes which show association with help of which they are associated with the plants and the plants over millions of years, have resulted in the able to derive the nutrients from them. Genus Hemic- evolution of plant-parasitic nematodes (PPNs). They riconemoides described by Chitwood and Birchfield (1957) comprises of the species which does not belong to either the genus Hemicycliophora (De Man, 1921) or *Correspondence: Himani Sharma Criconemoides (Taylor, 1936). The demarcating charac - sharmahimani1995hariom@gmail.com teristics possessed by members of this genus are the pres- Nematology Laboratory, Department of Zoology, Chaudhary Charan ence of an extra cuticular sheath in the mature females, Singh University, Meerut 250004, India presence of large body annules and having lateral and © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 2 of 15 sublateral ridges throughout the length of males giving plants irrespective of fruit and vegetable (Edward & the appearance of slight caudal alae. These nematodes Misra, 1964; Edward et  al., 1965; Dasgupta et  al., 1969; are commonly known as ‘sheathoid’ nematodes due to Crozzoli et  al., 1998; Siddiqi, 2000; Jain, 2009; Van den the presence of an outer accessory sheath with smooth Berg et al., 2015). Among all the Hemicriconemoides spe- annuli. cies, H. rosae was first described from the rose fields of The species of this group have been reported world - Bareilly district, Uttar Pradesh, India (Rathour et  al., wide; however, some of them are native (Geraert, 2003), then reported from the sugarcane fields of Meerut 1966). The nematodes of this genus showed obligatory district, Uttar Pradesh, India, by Khan et  al. (2019). A ectoparasitism (Geraert, 1966). Though they are obligate survey was conducted in the agricultural fields of Buland - parasites, they could be migratory, semi-sedentary and shahr district (28°40′ North and 77°86′ East at an altitude sedentary depending upon the environmental conditions of 209  m ASL), Uttar Pradesh, India. One species of the (Geraert, 1966).The nematodes of this group were found genus Hemicriconemoides recovered from the rhizos- to be associated with higher plants, mainly woody peren- phere of mustard fields. The earliest identification based nials, vines, etc.(Christie, 1953; Jenkins & Taylor, 1967), on morphological and some morphometric characters and several other plants as a result of which rot, necrosis revealed that the isolated population could be H. rosae of leaves, suppressed root growth, root lesions and coarse (Rathour et al., 2003), as it showed resemblance with the roots, etc., are common symptoms observed in case of original description. The occurrence of H. rosae in the these nematodes. sugarcane and rose fields is very common. However, the The genus Hemicriconemoides (Chitwood & Birchfield, species was first time reported from the mustard fields. 1957) comprises of 54 species (Khan et  al., 2019; Maria For more precise identification, molecular and phyloge - et al., 2018). Besides this, there were some species which netic analysis was done. Furthermore, to look for the var- was synonymized by the Geraert (2010). It is interest- iations between the species of genus Hemicriconemoides ing to note that 13 species have been reported from the reported worldwide with the studied isolate, principal agricultural rich fields of India, i.e. Hemicriconemoides component analysis (PCA) was also done. asymmetricus recovered from rose (Rathour et  al., 2003; Uttar Pradesh), H. communis isolated from the orange, Methods mango, mulberry, litchi and peach plants (Edward & Collection and nematode isolation Misra, 1964; Uttar Pradesh), H. conicaudatus recovered The present investigation was carried out in the fields of from peach fruits rhizosphere (Phukan & Sanwal, 1983; Bulandshahr district, Uttar Pradesh. The soils were col - Assam), H. dipterocarpus recovered from Dipterocarpus lected from the meadows, pastures, gardens and agri- tuberculatus (Mohilal et  al., 2004; Manipur), H. doon- cultural fields of district Bulandshahr (28° 40′ North and ensis from litchi (Srivastava et  al., 2000; Uttar Pradesh), 77° 86′ East, elevation of 209 m ASL) of Meerut Division. H. longistylus from tree fern (Rahman, 1990; Assam), H. A total of 85 soil samples were collected at a minimum mangiferae from litchi, mango amla and ashoka tree (Sid- depth of 5–10  cm (Southey, 1974) from the fields. Dur - diqi, 1961; all India), H. mehdii from grasses (Suryawan- ing the collection of samples, it was made sure that the shi, 1971; Maharashtra), H. neobrachyurus from oak, rice samples were collected from the previous sample at a dis- pomegranate and rose (Dhanachand & Jairajpuri, 1980; tance of 5 km. Soil samples (250gm) were collected in the Manipur), H. rosae from rose and sugarcane (Khan et al., transparent polyzipper bags labelled with the date, host 2019; Rathour et al., 2003; Uttar Pradesh), H. rotundoides plant, soil type and locality. Further, the soil was brought from Hibiscus rosasinensis (Geraert, 2010;Uttar Pradesh), to Nematology Laboratory, Department of Zoology, H. sunderbanensis from mangrove (Ganguly & Khan, Chaudhary Charan Singh University, Meerut, India, for 1982; West Bengal) and H. variabilis from peach (Rah- further processing. man & Ahmad, 1995; Assam). It is noteworthy to men- tion that only three species out of thirteen have been Nematode extraction and identification reported with males, i.e. H. communis, H. mangiferae The nematodes were extracted from the soil, using modi - and H. variabilis. The morphological identification of fied Cobb’s (1918) sieving method and Baermann’s fun - these species is a tedious task and requires expertise. In nel techniques (Goodey, 1957; Southey, 1986). To isolate some Hemicriconemoides species, males are absent which the plant-parasitic nematodes, 250 g of the soil was taken increases the need for the proper identification of the and then all lumps were broken gently in order to avoid females and juveniles (if present) (Ashokkumar & Vadi- the damage to nematodes. After this, 250 gm of soil was velu, 1990). taken in a 5 L bucket which was filled up to 3–4 L water. There are various species of genus Hemicriconemoides After topping up with water, it was swirled thoroughly by which were encountered from the rhizosphere of various hand to suspend all particles. The resulting suspension Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 3 of 15 was washed through a 2 mm aperture sieve. The mixture 95 °C for 10 min. The lysates were chilled on ice and then was allowed to settle for about 25  s sedimentation time. centrifuged at 6000 × g for 2  min, and 3 μL of superna- After this, the supernatant was decanted through a bank tant was used for PCR (Bhat et al., 2021). The ITS rDNA of sieves differing in their aperture size mainly 60  µm, was amplified using primers 18S: 5′-TTG ATT ACG TCC 150  µm, 200  µm, 250  µm, 300  µm, 350  µm and 400  µm, CTG CCC TTT-3′ (forward) and 28S: 5′-TTT CAC TCG respectively. Then, the residues on the sieves were washed CCG TTA CTA AGG -3′ (reverse) (Vrain et  al., 1992) and thoroughly with a gentle jet of water and finally collected partial sequence of LSU (28S) gene, D2–D3 domains in a beaker. Water was added to the remaining heavy resi- were amplified using primers, and D2A (5’-ACA AGT dues and thoroughly mixed, and again the supernatant ACC GTG AGG GAA AGTTG-3’) and D3B (5’-TCG GAA was decanted through the same bank of sieves. The sieves GGA ACC AGC TAC TA-3’) (De Ley et al., 1999) primer were washed, and the residues on the sieves were added pairs were used. The 30  µl PCR product consisted of to the previously collected. The sievings were shaken gen - 10 µl nuclease free distilled water, 1 µl each forward and tly to suspend all particles before pouring through a 90p reverse primer, 3  µl DNA extract and 15  µl Dream Taq aperture nylon screen supported by polyethylene ring Green PCR master mix 2X (Thermo Scientific). The PCR (Flegg, 1967), which was immediately placed on a Baer- amplifications were carried out using Verti 96-well fast mann funnel containing enough water just to submerge thermocycler (AP Scientific) with heated lid pre-set at the screen surface and debris. After 20  h, about 25  ml 95ºC and subjected to the following cycling profile: For of water containing the extracted nematodes from the the ITS rDNA region, PCR conditions included 1 cycle funnel was taken for examination under a stereoscopic of initial denaturation at 94 °C for 5 min; followed by 35 microscope at × 25 or × 50 magnification. cycles of 94  °C (denaturation) for 1  min, 55  °C (anneal- ing) for 1 min 30 s or 52 °C (annealing) for 30 s and 72 °C Morphology and morphometry analysis (primer extension) for 1  min, followed by a final exten - The adult females were heat killed in Ringer’s solution sion at 72 °C for 15 min to confirm all of the PCR prod - and then fixed in 50% TAF for 24  h. They were then ucts are complete length. For the D2D3 rDNA region, fixed in 100% TAF for 5–7  days (Courtney et  al., 1955) PCR conditions included 1 cycle of initial denaturation at and dehydrated in Seinhorst I and II (Seinhorst, 1959). 95 °C for 3 min; followed by 35 cycles of 95 °C (denatura- Further processing was done by following the protocols tion) for 45 s, 55 °C (annealing) for 45 s and 72 °C (primer of Bharti et  al. (2020). To prevent nematodes from flat - extension) for 1  min, followed by a final extension at tening, they were mounted in a small drop of glycerine 72  °C for 10  min to confirm all of the PCR products are on permanent clean glass slides with an extra amount complete length. The amplified products were electro - of paraffin wax (Bhat et  al., 2019). The phase contrast phoreses in 1% agarose (w/v) gel using 1 × TAE buffer microscope’s inbuilt software was used to perform obser- buffered agarose gel stained with ethidium bromide vations and measurements are in micrometre (μm). De (45 min, 100 V) was used to assess amplification success Manian indices (de Man, 1881) and other ratios were (Bharti et  al., 2020). The amplified PCR products were calculated. A drawing tube connected to a Nikon micro- purified and sequenced by Bioserve Pvt. Ltd., Hyderabad scope with differential interference contrast optics was (India). BioEdit was used to edit the newly discovered used to create line drawings. Images were captured with sequences (Hall, 1999). Sequences were deposited under a Nikon Digital Sight DS-U1 camera and DIC optics on the accession numbers: ® ® a Nikon microscope. Adobe Photoshop CS was used to edit the micrographs. Phylogenetic analysis The ITS (Internal transcribed spacer) and D2D3 Molecular characterization sequences were BLAST (Basic Local Alignment Search DNA isolation Tool) for % similarity matches with the previously sub- The DNA was extracted from single virgin females (not mitted sequences in the NCBI (National Centre for having plug at the vulva). The single virgin female from Biotechnology Information) (Altschul et  al., 1990). The each species/strain was first washed with Ringer’s solu - conserved regions, i.e. D2D3 and ITS, have been exploited tion followed by washing in Phosphate buffer saline (pH for establishing the relationship among the Hemicri- 7.2). The female from each species/strain was trans - conemoides species. For each amplified rDNA region ferred into a sterile Eppendorf tube (500 µl) with extrac- (ITS and LSU), an alignment of our data with sequences tion buffer 20  µl (ddH20 17.7  µl, 10X Taq buffer with from other closely related species was generated using (NH ) SO 2  µl, 1% tween2 µl, and proteinase K 0.1  µl). default ClustalW parameters in MEGA 7.0 (Kumar et al., 4 2 4 This buffer was frozen at − 80  °C for several days before 2016) and manually optimized in BioEdit (Hall, 1999). being incubated at 65  °C for 1  h and subsequently at The best fit models of nucleotide substitution used for Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 4 of 15 Morphological characterization the phylogenetic analysis were selected using jModel- Present specimen (HCN) showed close resemblances to Test 2.1.10 (Darriba et al., 2012). Sequences were concat- the Hemicriconemoides rosae (Rathour et  al., 2003) in enated with the R package ‘concatipede’ v1.0.0 (Vecchi morphological observations with respect to the presence & Bruneaux, 2021).Bayesian inference (BI) was used to of a prominent and elevated labial disc, long stylet, phar- infer the phylogenetic trees from the datasets. Aglenchus ynx distinct and separated from intestine. Besides this, agricola were utilized as out-group taxa, with all charac- vulva was enclosed with well-developed membranous teristics being equally weighted and gaps being treated as sheath, presence of conoid tail which is dorsally convex missing data (Subbotin et  al., 2005; Van den Berg et  al., at the tail tip, and hence, it was considered as the same 2014; Khan et al., 2019). MrBayes 3.2.7 was used to pro- (Fig.  1). Line drawings were prepared for comprehensive duce Bayesian phylogenetic reconstructions (Ronquist analysis (Fig.  2). However, few morphometrical charac- et  al., 2012). The General Time Reversible substitution ters showed little variation from each other. model with gamma distributed rate variation across sites and a proportion of invariable sites (GTR + G + I) was used as the optimal nucleotide substitution model for the Female analyses. One tree was preserved per 1000 generations Small in size 0.37–0.47  mm in length. Body straight to after running Markov chain Monte Carlo generations slightly arcuate ventrally after heat killed. Sheath closely (MCMC) for 1 × 10 cycles. The tree was visualized and fit to the body except on tail and frequently protrud - saved with FigTree 1.4.4 (Rambaut, 2018). ing forward over the lip region. Sheath is not annulated at vulval region. Cuticle smooth and covered by outer accessory layer/sheath with smooth annules. Body Statistical analyses annuli distinct and rounded (Fig.  1). Lip region rounded Correlation analysis and linear regression analysis were with a prominent labial disc, not set off with two annuli, performed to resolve the relationship between vari- first annuli smaller than second (Fig.  1, Table  1). Sty- ous morphometrical parameters using GraphPad Prism let long (50-58  µm), well developed with stylet knobs software (version 6.0). Principal component analysis (2.4–4.0  µm) prominently anchor-shaped anteriorly and (PCA) was done to find out the morphological varia - rounded posteriorly, 4.8–6.9  µm wide (Fig.  1, Table  1). tions between the isolated nematodes in the present Dorsal pharyngeal gland opening situated at the base investigation and closely related species of the nema- of stylet knobs. Median bulb observed with prominent todes. The measurements of the Hemicriconemoides spe - valve. Procorpus and metacorpus found to be fused. cies were collected from their original descriptions. The Excretory pore situated from 5–11 annuli posterior 2D plot was formed by using the PC1 and PC2 values of to the base of pharynx. Hemizonid not found. Sheath each isolate based on eigenvalues given by the software annuli smooth, slightly indented over the whole length. MINITAB 20 (Nisa et al., 2021). Anastomoses found absent. Vulva posterior, located 8–13 annuli from terminus and 90–94% of body length (Fig.  1, Table  1). Flap like structure absent on vulva but Results with a prominent membranous sheath. Spermatheca well In the present investigation, HCN strain recovered from developed and oval shape filled with sperm cells (Fig.  1). one soil sample out of 85 soil samples collected from the Vagina observed distinct and straight. Body found to be rhizosphere of mustard from Bulandshahr district. The tapering posterior to anus. Tail shape conoid and found strain was found to be plant-parasitic nematodes due to dorsally convex with the sheath enclosing the tip, taper- presence of long stylet on the labial region. The pH of ing gradually to a more pointed tip. Anus position was the soil was 5.4, and the texture of the soil was found to uncertain still it was located just posterior to vulva 2–5 be alluvial. Most of the sampling was done in the winter annuli (Table 1). season mainly in January–March. The strains HCN bear double cuticle, which is a characteristic feature of Hemic- riconemoides, thus belong to same genus. Male Based on morphology and morphometry, these nema- Males are absent. todes were found close to Hemicriconemoides rosae, hence described as the same. In the present study, only Comparative study of H. rosae strain (HCN) and H. rosae females of the strain HCN were recovered as noted in the (Rathour et al., 2003) original descriptions. For the morphological characteri- As far as females was concerned, most of the morpho- zations, permanent slides were prepared and deposited metric parameters of isolate (HCN) and original spe- in the Nematology Laboratory, Department of Zoology, cies was found to be approximately similar. The other Ch. Charan Singh University, India. Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 5 of 15 Fig. 1 Photomicrographs of Hemicriconemoides rosae isolate HCN. A, B Anterior region of female; C Whole female; D Posterior region of female; E Female gonad; F Vulval region; and G Anal and tail region. The arrows indicate the stylet, stylet knob and nerve ring (B), female gonad (E), vulval region and spermatheca of female (F) and anal region and tail (G) measurements, i.e. a (13.6–16.9 vs 16.0–17.6), b (4.6–5.6 of the pharynx (72–94 vs 102 µm) and number of annuli vs 4.9–5.4), V (90–95 vs 92–94.5), stylet length (50–58 posterior to vulva (8–13 vs 6–9) which showed deviation vs 50–55  µm), metenchium length (40–49 vs 44.8  µm), from the original description. telenchium length (9–11.8 vs 7.7  µm), stylet knob width Hemicriconemoides rosae isolate (HCN) was compared (4.8–6.9 vs 6.9  µm), labial disc diameter (4.3–5.9 vs with H. rosae Khan et al.(2019) sugarcane population and 5.1 µm), R (102–115 vs 116), Rst (12–20 vs 12–14), Roes three closely related species of Hemicriconemoides, and it (20–22 vs 21–26) of present strain, were found close to differs in some morphological and morphometric charac - the original description H. rosae (Rathour et  al., 2003). ters with compared species. The comparison of morpho - All the body parameters were found to be close except metrics is presented in Tables 1 and 2 and is described as body length (376–472  µm vs 470–510  µm), the length follows: Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 6 of 15 Fig. 2 Line drawings of Hemicriconemoides rosae from rhizosphere of mustard. A, B Anterior region of female; C: Female gonad and tail; D: Entire adult female; E body annuli in mid-body region; and F Vulva region The present isolate HCN of Hemicriconemoides rosae terms of number of sheath annuli, anus position, ante- recovered from mustard were found to show some vari- rior secretory to excretory position and the presence of ations from the H. rosae (Khan et  al., 2019) sugarcane males. H. communis was found to have more annuli than population. The parameters which show deviations were: H. rosae (102–115 vs 110–130), smaller VL/VB (0.9–2.0 average body length (376–472 vs 323-486  µm), ratio a vs 1.3–2.0). The position of anus is uncertain in H. rosae; (13.6–16.9vs 12.2–18.7), ratio b (4.6–5.6 vs 3.88–5.93), however, anus is clearly visible in H. communis. Males are telenchium length (9–11.8 vs 5.1–10.8 µm), stylet (50–58 present in H. communis, whereas in H. rosae no males vs 52-61 µm) and annulus width (3.6–5.2 vs 2.9–3.8 µm) were observed (Table 2). (Table 1). Based on morphological characterization, H. rosae Hemicriconemoides rosae HCN females were differenti - HCN was separated from the H. wessoni Chitwood ated from the H. communis Edward and Misra (1964) in and Birchfield (1957) in terms of ratio a, c and other Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 7 of 15 Table 1 Morphometrics of Hemicriconemoides rosae isolate HCN from the mustard (present population), rose population (Rathour et al., 2003) and sugarcane population (Khan et al., 2019). All measurements are in µm (except ratio and percentage) and in the form of mean ± standard deviation (range) Character Mustard population Rose population (Rathour Sugarcane isolate HCN et al., 2003) population (Khan et al., 2019) n 15 6 32 L 443 ± 25 (376–472) 490 (470–510) 398 (323–486) a (Length/Diameter at mid-body) 15.3 ± 0.9 (13.6–16.9) 16 (16–17.6) 15.4 (12.2–18.7) b (Length/Pharynx length) 5.1 ± 0.3 (4.6–5.6) 5.2 (4.9–5.4) 4.8 (3.9–4.9) c (Length/Tail) 16.4 ± 1.7 (14.1–20) – – c’ ( Tail/diameter at anus) 22 ± 1.8 (19.4–25) – – V (anterior to vulval/total body length) × 100 92 ± 1.4 (90–95) 93.2 (92–94.5) 91.5 (89.9–92.7) M (ratio between the length of prorhabdion and stylet in %) 85.6 ± 2.5 (81.2–91.5) 85.3* 85.6 (82.2–90.4) EP (Excretory pore to anterior end) 116 ± 5.5 (102–123) – – WEP (Diameter at excretory pore) 28 ± 0.9 (25–29) – – NR (Nerve ring) 71 ± 2.4 (64–74) – – PL (Pharynx length) 87 ± 5.4 (72–94) 102* 84 (71–103) EBL (oesophageal bulb length) 15.8 ± 2.2 (11.5–18.7) – – EBW (oesophageal bulb width) 12 ± 1.8 (8.8–15) – – Tail 27 ± 2.7 (23–31) – – Diam. at anus 19.8 ± 1.3 (18–23) – – Diam. at mid body 29 ± 2.0 (26–33) 26.5* 25.9 (23.4–29.6) V’ (Distance from the anterior end to vulva) 409 ± 26 (340–444) – – Stylet 54 ± 2.4 (50–58) 52 (50–55) 55 (52–61) Stylet Knob height 2.8 ± 0.5 (2.4–4.0) 2.6* 2.7 (2.3–3.8) Stylet Knob width 5.7 ± 0.7 (4.8–6.9) 6.9* (4.8–6.9) 6.2 (4.8–7.4) Metenchium length 44 ± 2.3 (40–49) 44.8* 47 (44–51) Telenchium length 10.3 ± 0.8 (9–11.8) 7.7* 8.0 (5.1–10.8) R ( Total number of body annules) 108 ± 4.1 (102–115) 116* 116 (109–124) Rst (Annules from anterior extremity to base of stylet) 13.9 ± 2.0 (12–20) 13 (12–14) 16.6 (15–18) Roes (Annules from anterior extremity to base of oesophagus) 21 ± 0.8 (20–22) 23.5 (21–26) 25.9 (21–30) Ran (Annules from anus to tail terminus) 6.9 ± 0.6 (6–8) – – Rex (Annules from anterior extremity to excretory pore) 28 ± 2.0 (25–33) – 28 (22–30) Rv (Annules from vulva to tail terminus) 9.6 ± 1.5 (8–13) 7.5 (6–9) 7.5 (7–9) MBL (Median bulb length) 14.7 ± 1.6 (11.5–16.4) – – MBW (Median bulb width) 12.8 ± 1.3 (10.9–15.5) – – Lip Length 5.9 ± 0.2 (5.2–6.6) 5.7* 5.8 (5.0–6.5) Lip Width 5.1 ± 0.5 (4.3–5.9) 5.1* 5.4 (4.5–6.0) D % (excretory pore/pharynx length × 100) 134 ± 10.9 (115–163) – – E % (excretory pore/Tail) × 100 430 ± 49 (365–505) – – VL (distance from vulva to terminus) 35 ± 6.1 (23–27) 34.7* 33.7 (28.6–39.5) VB (body diameter at vulva) 24 ± 1.8 (21–28) 21.6* 18.8 (15.0–21.6) VL/VB (distance from vulva to terminus/body diameter at vulva) 1.5 ± 0.3 (0.9–2.0) 1.6* 1.8 (1.3–2.2) Sty%L ( Stylet length/body length × 100) 12.3 ± 1.0 (10.7–14.8) 10.7* 14.0 (12.0–16.2) First lip diam 8.6 ± 0.5 (8.2–9.9) 8.5* 8.8 (8.0–10.6) Second lip diam 12.1 ± 0.7 (10.1–14.2) 12.3* 11.7 (10.0–13.3) First body annulus diam 14.6 ± 0.4 (12.3–15.2) 14.8* 14.2 (12.1–15.8) Second body annulus diam 16.9 ± 0.9 (14.5–19.9) 17.2* 15.7 (13.5–18.8) Third body annulus diam 18.1 ± 1.2 (16.2–21.3) 18.9* 17.2 (15.2–21.0) Annulus width 4.4 ± 0.4 (3.6–5.2) 4.3* 3.4 (2.9–3.8) Values were determined in holotype specimens Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 8 of 15 Table 2 Comparative morphometrics of adult female isolate HCN with original Hemicriconemoides rosae population and other species of the Hemicriconemoides genus Characters Authors L a b V PL Stylet R Rst Roes Rv Ran Sty%L Annulus W H. rosae Present isolate 443 15.3 5.1 92 87 54 108 13.9 21 9.6 (8–13) 6.9 (6–8) 12.3 4.4 (3.6–5.2) HCN, Mustard (376–472) (13.6–16.9) (4.6–5.6) (90–95) (72–94) (50–58) (102–115) (12–20) (20–22) (10.7–14.8) H. communis Edward and 480 16 4.5 93 109* 54* 115 16* 26* 8* 7* 10.8* 3.5* Misra (1964) (340–500) (12–25) (4–5) (89–96) (51–58) (110–130) H. wessoni Chitwood 429.2 (382.5– 12.7 4.6 92 – 54 (76–83) – – 9* 5* 15.9* (4–5) and Birchfield 499.8) (10.7–15) (4.0–5.5) (89–93.5) (50–60) (1957) H. califor- Pinochet and 440 17 4.1 91 – 80 119 – – 11 (10–12) 5 (4–6) – – nianus Raski (1975) (410–460) (16–19) (3.4–4.6) (90–92) (77–83) (112–127) H. macrodorus Volvas et al. 691 17 4.4 95 152 101 137 23 – 13 (2–14) 8 (7–9) 15 (13–18) 5.8* (2000) (548–761) (15–21) (3.8–5.2) (94–96) (140–171) (90–110) (127–148) (18–27) H. chitwoodi Esser (1960) 540 15.5 3.8 90.1 – 91 124 – – (12–16) (8–11) – – (480–590) (13.4–17) (3.5–4.1) (88.1–91.0) (85–95) (116––133) H. paratai- Decraemer 499 15.2 4.2 90 120.2 79.4 138.1 25.4 – 9.1 (8–10) 5 (4–10) 15.9* 3.5* wanensis and Geraert (440–535) (13.8–16) (3.9–4.5) (88.7–91) (107–129) (77–82) (129–145) (23–29) (1992) H. paracamel- Maria et al. 563 16.9 4.4 88.9 127 83 132 23.1 34.5 (31–37) 14.8 (13–16) 8 (5.0–10.0) 14.7 – liae (2018) (519–604) (15.3–18.5) (4.1–5.0) (87.7–90.1) (119–135) (80–85) (124–140) (22–26) (13.7–16.3) H. kanayaensis Nakasono 571 21.5 4.8 88.9 – 74 150 – – 18 (16–21) 12 (11–15) 12.9* 4.6* and Ichinohe (500–631) (18.7–24.4) (3.3–5.6) (87.5–91.5) (66–79) (142–164) (1961) H. phoenicis Van den Berg 630 21.6 4.7 46 133 88 130 23 32 (28–36) 14 (13–19) 10 (9–15) 14 (12.6–16.3) 5.5 (4.5–6.5) et al. (2015) (549–699) (16.7–24.3) (4–5.7) (27–62) (117–149) (81–97) (123–137) (21–25) H. litchi Edward and 480 15 4.6 92 107* 63 130 12* 25* 12* 7* 13.1* 3.5* Misra (1964) (450–500) (13–18) (3–5) (91–93) (60–65) (128–133) H. strictathe- Esser (1960) 560 – – – – 79 138 18.8* 27.3* (11–13) – – – catus (510–590) (73–83) (127–152) H. cocophilus Dasgupta (460–500) (14–15) (4.0–5.0) (91–92) – (50–57) (108–143) – – (9–10) (8–9) – (3–5) et al. (1969) H. brachyurus Dasgupta (400–540) (13–17) (4.3–5.2) (93–95) 96* (56–64) (98–119) – – (7–9) (6–7) – – et al. (1969) All measurements are in µm (except ratio and percentage) and in the form of mean ± standard deviation (range) Values were determined in holotype specimens L = Body length, PL = Pharynx length, a(L/BD), b(L/PS), V% (anterior to vulval/total body length) × 100, Stylet = Stylet length, Stylet knob W = Stylet knob width, R = Total number of body annules, Rst = Annules from anterior extremity to base of stylet, Roes. = Annules from anterior extremity to base of oesophagus, Rv = Annules from vulva to tail terminus, Ran = Annules from anus to tail terminus, Annulus W = Annulus width, Sty%L = Stylet length/body length*100 Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 9 of 15 Principal component analysis parameters. The ratio a of H. wessoni (12.3–14.8) was Principal component analysis was done using morpho- found to be smaller when compared with H. rosae (13.6– metric data of the nematode isolates, i.e. H. commu- 16.9). The ratio c of H. wessoni (16.2–22.9) is larger when nis (Edward and Misra 1964), H. wessoni (Chitwood & compared with H. rosae (14.1–20). Besides this, other Birchfield, 1957), H. californianus (Pinochet & Raski, characteristics of the H. rosae isolate HCN showed dif- 1975), H. macrodorus (Volvas et al., 2000), H. chitwoodi ferences with the H. wessoni such as tail shape and labial (Esser, 1960), H. parataiwanensis (Decraemer & Ger- disc. The labial disc in case of H. rosae was found to be aert, 1992), H. paracamelliae (Maria et  al., 2018), H. elevated, whereas in case of H. wessoni, slight or no ele- kanayaensis (Nakasono & Ichinohe, 1961), H. phoenicis vation was observed. The pharyngeal bulb in H. rosae (Van den Berg et  al., 2015), H. litchi (Edward & Misra, showed no overlapping; however, in H. wessoni dorsal 1964), H. strictathecatus (Esser, 1960), H. cocophilus overlapping was present. The shape of the tail in H. rosae (Dasgupta et al., 1969), H. brachyurus (Dasgupta et al., was found to be conoid and the membranous sheath 1969) isolated from different geographical areas and the enclosed the tail tip dorsally, whereas in H. wessoni the studied isolate HCN. tail shape was found to be conoid but membranous The results of the PCA showed that there are vari - sheath may or may not enclose the tail tip (Table 2). ations in the morphometry between the Hemicri- The H. rosae isolate HCN showed differences from conemoides rosae isolate HCN recovered from mustard the H. minutus Esser (1960) in terms of de Manian indi- in the present investigation and the Hemicriconemoides ces, i.e. a and b, stylet and pharyngeal bulb. The ratios a rosae (Khan et  al., 2019) isolated from the sugarcane (13.6–16.9 vs 10.3–16.4) and b (4.6–5.6 vs 3.0–3.8) of H. and with the other species of Hemicriconemoides spe- rosae were found to be higher than H. minutus. The stylet cies (Dasgupta et  al., 1969). However, the analysed length of H. rosae was found to be smaller than H. minu- parameters of morphometry showed less variations tus (50–58 vs 70.5–80.5  µm). The pharyngeal bulb of H. between the present isolate HCN with the originally rosae showed no overlapping, while in case of H. minutus described Hemicriconemoides rosae (Rathour et  al., overlapping was present. Fig. 3 The principal component analysis (PCA) of different populations of Hemicriconemoides species based on female specimens with different body parameters as factors. The biplot was based on two major principal components; PC1 and PC2. L = Body length, PL = Pharynx length, a (L/BD), b(L/PS), V% (anterior to vulval/total body length) × 100, Stylet = Stylet length, Stylet knob W = Stylet knob width, R = Total number of body annules, Rst = Annules from anterior extremity to base of stylet, Roes. = Annules from anterior extremity to base of oesophagus, Rv = Annules from vulva to tail terminus, Ran = Annules from anus to tail terminus, Annules W = Annules width, Sty%L = Stylet length/body length*100. The ellipses represent the group of nematodes/isolates coming together showing fewer variations Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 10 of 15 2003) (Fig.  3). Besides this, the isolates showed dif- Table 3 Loading scores of the variables and factor score of the observations for females of Genus Hemicriconemoides spp ferences from the other Hemicriconemoides species (Fig. 3). Characters Loading Species Factor The PCA based on the morphometry of females scores of the score of the variables observations showed an accumulated variability of 66.2% (Fig.  3). The contribution of PC1 and PC2 was found out to be Females Females 43.3% and 22.9%, respectively (Fig.  3). Four parameters, Variable PCA1 PCA2 PCA1 PCA2 annulus width (r = -0.06), b (r = -0.16), stylet knob width L 0.23 0.39 HCN, Mustard − 1.96 0.65 (r = -0.15) and V% (r = -0.17) were found to be negatively a 0.25 0.37 H. rosae, rose − 1.82 1.20 correlated across nematode/species in PC1. Ten charac- b − 0.16 0.43 H. rosae, sugar- − 1.51 − 0.57 ters out of fourteen characters were found to be positively cane correlated across isolates, and the remaining characters V − 0.17 − 0.32 H. promissus − 3.59 0.80 were negatively correlated considering PC1 (Fig.  3). The PL 0.33 − 0.03 H. litchi − 0.51 0.14 highest coefficient of correlation was observed in annuli Stylet 0.33 − 0.26 H. silvaticus 2.54 − 1.20 from anus to tail terminus (r = 0.38) and annuli from R 0.32 0.14 H. strictathecatus 0.46 − 0.11 anterior extremity to base of stylet (r = 0.37) in PC1. Con- Rst 0.37 − 0.12 H. brachyurus − 2.59 − 0.34 sidering PC2, nine characters out of fourteen were found Roes 0.38 − 0.13 H. californianus 2.58 − 2.50 to be positively correlated and the remaining were nega- Rv 0.33 0.13 H. macrodorus 3.57 − 0.31 tively correlated (Fig.  3, Table  3). The highest coefficient Ran 0.15 0.32 H. chitwoodi 1.89 − 2.73 of correlation was observed in b (r = 0.43) PC2. Stylet Knob width − 0.15 0.03 H. wessoni − 3.33 0.62 Sty%L 0.27 − 0.35 H. minutus − 0.47 − 2.44 Molecular analysis Annulus width − 0.06 0.27 H. ortonwilliamsi − 2.49 1.68 The Hemicriconemoides species isolate HCN examined H. communis − 1.02 0.31 in present study has been characterized molecularly by H. cocophilus − 2.59 − 1.41 the sequences of two genes, i.e. ITS rDNA (695  bp) and H. parataiwan- 1.74 − 1.66 D2D3 fragments of 28S rDNA (708  bp). The ITS and ensis D2D3 sequences of Hemicriconemoides rosae isolate H. paracamelliae 2.65 0.46 HCN were deposited in NCBI GenBank with accession H. kanayaensis 3.22 2.99 number ON844213 and ON844212, respectively. H. phoenicis 3.24 4.41 Pairwise distances (Kimura 2-parameter) based on the ITS regions revealed the genetic distances between the Hemicriconemoides species. The present isolate HCN (ON844213) genetic distance with previously described phylogenetic relatedness of the Hemicriconemoides Hemicriconemoides rosae (MK371815, MK371814, and rosae with other species of Hemicriconemoides was MK371816) was found to be 0.0000. The present isolate established from the comparison of concatenated Hemicriconemoides rosae HCN (ON844213) showed sequences (Fig.  4, Additional file  3: S3). There are two greatest genetic distance (0.1915) from H. kanay- major clades in the rDNA phylogenetic tree of Hemic- aensis (MG029568). The average overall genetic dis - riconemoides. It is interesting to note that the phyloge- tance between in-group species was 0.1675 ranging netic analysis was in agreement with the morphology from 0.0000 to 0.3055 between H. rosae (MK371815, and molecular analysis. The first cluster includes the MK371814, MK371816) and H. kanayaensis (MG029568) Hemicriconemoides brachyurus, H. cocophilus, H. (Additional file 1: S1). ortonwilliamsi, H. minutus, H wessoni, H. macrodorus The average overall genetic distance (Kimura 2-param - and H. promissus. The second cluster includes the eter) based on D2D3 regions between in-group spe- Hemicriconemoides rosae HCN, Hemicriconemoides cies was 0.1336 ranging from 0.0000 between H. rosae rosae and Hemicriconemoides species. A thorough (MK371813) and H. rosae (MK371811) to 0.2326 analysis of sequences of the 28S and ITS rDNA gene between H. minutus (KF856516) and H. parataiwanensis of the HCN isolate described in this study reveals clear (MG029573) (Additional file 2: S2). relatedness with 18 other Hemicriconemoides species. Bayesian inference phylogenetic analyses based on the concatenated sequences (ITS and 28S rDNA) showed Phylogenetic analysis a clear monophyly of the group formed by the isolate ITS and D2D3 regions are well-conserved regions in HCN and already described Hemicriconemoides rosae the nematodes and are used to ascertain the relation- Khan et al. (2019) (Fig. 4, Additional file 3 : S3). ship between closely and distantly related species. The Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 11 of 15 Fig. 4 Phylogenetic relationships within populations and species of Hemicriconemoides as inferred from Bayesian analysis based on the concatenated sequences of ITS and 28S rRNA. Numbers at the nodes indicate Bayesian posterior probability. Newly obtained sequences of H. rosae underlined. The scale bar represents 0.01 substitutions per nucleotide position Relationship between various morphometric parameters analysed parameters, no correlation has been observed The results obtained from the two tailed Pearson’s cor - with the de Man indices except a ratio of female which relation analysis revealed that some parameters of showed positive correlation (r = 0.6978, p = 0.0038) Hemicriconemoides rosae isolate HCN showed signifi - (Fig.  5, Table  4). The index a also showed positive cor - cant correlation with other morphometric parameters relation with other parameters, i.e. excretory pore (Table  4). The morphometric parameters which were (r = 0.7389, p = 0.0017, Table  4), oesophageal bulb focused in the study to understand the relation between (r = 0.6953, p = 0.0040, Table  4), vulva-anterior end various parameters were body length, excretory pore, (r = 0.7538, p = 0.0012, Table  4). However, index a tail, pharynx stylet, de Manian indices a, b, c and c’, showed no correlation with other indices (Table  4). The nerve ring, vulva width and other parameters. In this, 15 index b showed positive correlation with the index c females of the isolate HCN were analysed to figure out (r = 0.5689, p = 0.0269, Table  4) and negative correlation the correlation between the morphometric data. Each with pharynx (r = − 0.5443, p = 0.0359, Table  4). Among morphometric parameter was replicated 3 times to get all the parameters, the c’ showed positive correlation the final results. with the tail (r = 0.5966, p = 0.0189, Table  4) and nega- The results based on the two-tailed Pearson’s corre - tive correlation with index c (r = − 0.6779, p = 0.0055, lation suggested that body length has highest correla- Table  4). These observations suggested that body length, tion in comparison with others with the morphometric vulva position, de Manian index ‘a’ are important for the data. Though body length showed correlation with the evaluation of the females of Hemicriconemoides rosae. Sharma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 12 of 15 Table 4 Correlation of morphometric data of Hemicriconemoides rosae isolate HCN was determined by two tailed Pearson’s correlation L a b c c’ V L 1 0.6978** 0.3446 0.3492 0.1044 0.3261 a 0.6978** 1 0.2484 0.3704 − 0.01959 0.3505 b 0.3446 0.2484 1 0.5689* − 0.4858 0.1374 c 0.3492 0.3704 0.5689* 1 − 0.6779** 0.5414* c’ 0.1044 − 0.01959 − 0.4858 − 0.6779** 1 − 0.08350 V 0.3261 0.3505 0.1374 0.5414* − 0.08350 1 EP 0.9651*** 0.7389** 0.3727 0.3506 0.1033 0.3744 NR 0.5820* 0.4458 − 0.3041 − 0.05316 0.4115 0.5198* PL 0.5990* 0.4690 − 0.5443* − 0.1700 0.4952 0.1685 EBL 0.6816** 0.6953** 0.1143 0.4009 − 0.2365 0.1240 T 0.5458* 0.2028 0.05948 − 0.3330 0.5966* − 0.04380 Body diameter: anus 0.5670* 0.4774 − 0.07373 − 0.1201 0.09106 0.1515 Body diameter: mid-body 0.5379* 0.4317 0.2305 − 0.01556 0.009445 − 0.1235 VDesh 0.9715*** 0.7538** 0.3453 0.4479 0.07187 0.5407* Stylet 0.5498* 0.1846 − 0.1253 − 0.1770 0.1626 − 0.1398 MBL 0.5448* 0.4334 − 0.02157 0.09194 0.1999 0.5293* WV 0.5872* 0.4855 0.1949 0.1943 0.06458 0.1955 Bold and asterisk (*) values represent the statistical difference. p < 0.05 was considered a statistically significant difference Shows statistical significant; Pearson’s correlation Shows p < 0.05, ** shows p < 0.01, *** shows p < 0.001 2010; Khan et  al., 2019) and is associated with many crops. The H. rosae (Rathour et  al., 2003) possessed very less morphological and morphometric characters which were used for the comparison with the studied isolate HCN. The morphological data of the HCN isolate showed much resemblance with the original description of the H. rosae (Rathour et al., 2003); however, it showed more deviation from the sugarcane population of H. rosae (Khan et  al., 2019) (Table 1). The various morphometric characters are not suffi cient for the delineation of the species. Molecular charac- Fig. 5 Relationship between the body length with ratio ‘a’ as terization is essential in order to validate the taxonomic determined by regression analysis of females of Hemicriconemoides position as well as the authenticity and phylogenetic isolate HCN. The symbol represents the values of ratio ‘a’ at specific body length. The solid line denotes linear regressions between the relationships among the species of a particular genus body length and ratio ‘a’. and between different nematode orders. The molecu - lar analysis based on the conserved regions ITS and D2D3 revealed the present isolate HCN to be H. rosae Discussion as it showed very less genetic distance from the already Hemicriconemoides was first described by Chitwood & described H. rosae (ITS:0.0000, D2D3: 0.0014, 0.0129) Birchfield, 1957 and further redescribed by Dasgupta (Additional file 1: S1 and Additional file 2: S2). et  al., 1969. These nematodes are commonly referred to The phylogenetic analysis was found to be consistent as sheathoid nematodes due to the body cuticle of female with the molecular data which revealed that the present covered by an outer accessory layer/sheath with smooth isolate to be H. rosae (Fig.  4). The phylogenetic analysis annules which is missing in juveniles. They are mostly based on the concatenated sequences (ITS and D2D3) distributed in temperate areas of the world mainly in region revealed that mustard population of H. rosae HCN Africa, America, Australia and South Asia and South formed a monophyletic clade with the sugarcane popula- Europe. This genus contains 54 valid species (Geraert, tion of H. rosae with a Bayesian posterior probability 1. Shar ma and Chaubey The Journal of Basic and Applied Zoology (2023) 84:16 Page 13 of 15 The phylogenetic tree revealed that H. rosae formed a sugarcane and rose fields is very high in the western separate branch without including any other Hemicri- Uttar Pradesh, India (Khan et  al., 2019; Rathour et  al., conemoides species, however, the position of H. rosae in 2003). The present investigation was carried out in the the phylogenetic tree was close to H. promissus, H. mac- fields of mustard which is an alarming sign as mus - rodorus, H. wessoni and H. minutus which found to be tard is one of the cash crops of India having economic consistent with the previous observations (Khan et  al., importance. From the studies, it is clear that species 2019). of genus Hemicriconemoides possess certain charac- The PCA showed large intraspecific and interspecific teristics which cause potent damage to the crops, ulti- variations. These variations were found to be independ - mately affecting the yield (Inserra et  al., 2014; Maria ent of the collection localities of soil samples. It was et al., 2018). The information revealed from the present evident from the previous studies that there were large investigation can be exploited by taxonomists for the intraspecific variations present among the nematodes comparison, identification and correlation between the which was also observed among the present isolate HCN morphometric parameters of the species. (mustard population) and Hemicriconemoides rosae (sug- arcane population) isolates of the present investigation Supplementary Information (Khan et al., 2019). However, less intraspecific variations The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s41936- 023- 00338-6. were observed between present isolate HCN and origi- nally described Hemicriconemoides rosae (Rathour et al., Additional file 1: S1. Pairwise distances (Kimura 2-parameter) between 2003) (Fig. 3, Table 3). species of Hemicriconemoides based on ITS (Internal transcribed spacer) The results obtained from the analysis of morpho - regions. Out-groups were not included. metric data based on two tailed Pearson’s correlation Additional file 2: S2. Pairwise distances (Kimura 2-parameter) of the between Hemicriconemoides species based on D2D3 (Large ribosomal revealed that in case of females of H. rosae, de Manian subunit, 28S) regions. Out-groups were not included. index a is very important for the evaluation as it showed Additional file 3: S3. List of GenBank accession numbers of the Hemicri- positive correlation with body length (Fig.  5, Table  4). conemoides species used in the phylogenetic tree. Similar results were observed in the genus Eucepha- lobus and Acrobeles (Amirzadi et  al., 2011, 2013). The Acknowledgements results of Fortuner (1990) also exhibited the highest cor- The authors are thankful to the Department of Zoology, Chaudhary Charan relation between the de Manian indices especially a and Singh University, Meerut, for providing necessary laboratory facilities for conducting the experiments. The authors are grateful to local farmers for their b and the length. Furthermore, the other ratios, i.e. V assistance in sample collection. The authors are also thankful to Dr. Abhilash also important along with the de Manian indices, i.e. a, Prabhat for his assistance in statistical analysis. The authors extended their c and c’ which is also consistent with the previous find - sincere thanks to Lovely Bharti for helping in morphological identification. ings (Fortuner, 1984). It was found from the previous Author contributions studies that a, c and c’ de Manian indices are essential The study was designed by HS and AKC. Interpretation of the data was done for the genus Helicotylenchus Steiner, 1945. Similar find - by HS. Manuscript writing was done by all authors. Tables and reference set- tings were done by HS. Errors and grammatical mistakes in manuscript were ings were obtained by Amirzadi et  al. (2013) for genus removed and edited by AKC. Then, final manuscript was read and approved by Acrobeles von Linstow (1877) where correlation between both authors. de Manian indices (b and c’) and body length was found Funding positive. The present study on the females of genus The authors declare that no funds, grants or other support were received dur- Hemicriconemoides also revealed the positive correla- ing the preparation of this manuscript. tion between the body length and ratio a (Fig. 5, Table 4). Availability of data and materials The reason behind that some morphological characters The data and material of this manuscript are available from corresponding showed highest degree of correlation; however, some of author on reasonable request. them showed least might be due to control of those char- acters by a specific gene cluster (Amirzadi et  al., 2013). Declarations It can be concluded from the results that there are some Ethics approval and consent to participate characters which showed high correlation with body This article does not contain any studies with human participants or animals. length which results in the increase or decrease in the characters with the body length (Fig. 5, Table 4). Consent for publication Not applicable. Conclusions Competing interests The authors declare that they have no competing interests. The present isolate HCN were identified as H. rosae based on the morphological, molecular and phy- logenetic analysis. 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The Journal of Basic and Applied ZoologySpringer Journals

Published: Apr 26, 2023

Keywords: Hemicriconemoides rosae; Mustard; Morphology; Taxonomy; Correlation; PCA

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