Description of Heterodera microulae sp. n. (Nematoda: Heteroderinae) from China – a new cyst nematode in the Goettingiana group

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Description of Heterodera microulae sp. n. (Nematoda: Heteroderinae) from China – a new cyst nematode in the Goettingiana group

Wenhao Li / Huixia Li * / Chunhui Ni / Deliang Peng / Yonggang Liu / Ning Luo / Xuefen Xu

Keywords : Goettingiana group, Heterodera , Morphology, New species, Phylogeny, Taxonomy

Citation Information : Journal of Nematology. Volume 52, Pages 1-16, DOI: https://doi.org/10.21307/jofnem-2020-097

License : (CC-BY-4.0)

Received Date : 05-July-2020 / Published Online: 24-November-2020

ARTICLE

ABSTRACT

A new cyst-forming nematode, Heterodera microulae sp. n., was isolated from the roots and rhizosphere soil of Microula sikkimensis in China. Morphologically, the new species is characterized by lemon-shaped body with an extruded neck and obtuse vulval cone. The vulval cone of the new species appeared to be ambifenestrate without bullae and a weak underbridge. The second-stage juveniles have a longer body length with four lateral lines, strong stylets with rounded and flat stylet knobs, tail with a comparatively longer hyaline area, and a sharp terminus. The phylogenetic analyses based on ITS-rDNA, D2-D3 of 28S rDNA, and COI sequences revealed that the new species formed a separate clade from other Heterodera species in Goettingiana group, which further support the unique status of H. microulae sp. n. Therefore, it is described herein as a new species of genus Heterodera; additionally, the present study provided the first record of Goettingiana group in Gansu Province, China.

Graphical ABSTRACT

Cyst-forming nematodes are the economical pests of cultivated crops and known to be reported from all the continents (Jones et al., 2013). The genus Heterodera was erected by Schmidt (1871) and currently contains about 80 species (Subbotin et al., 2010). Literature studies have indicated the presence of 14 Heterodera species from China mainland, including H. avenae (Chen et al., 1991), H. glycines (Liu et al., 1994), H. sinensis (Chen and Zheng, 1994), H. filipjevi (Li et al., 2010), H. koreana (Wang et al., 2012; Wang et al., 2012b), H. elachista (Ding et al., 2012), H. ripae (Wang et al., 2012a; Wang et al., 2012b), H. hainanensis (Zhuo et al., 2013), H. fengi (Wang et al., 2013), H. guangdongensis (Zhuo et al., 2014), H. zeae (Wu et al., 2017), H. sojae (Zhen et al., 2018), H. schachtii, and H. vallicola (Peng et al., 2020).

Due to overlapping morphological characters and phenotypic plasticity, it is difficult to distinguish closely related Heterodera species; therefore, sequence-based diagnosis is gaining more reliability for precise and accurate identification of cyst-forming nematodes (Peng et al., 2003). The internal transcribed spacer region of the ribosomal DNA (ITS-rDNA), the D2 and D3 expansion fragments of the 28S ribosomal DNA genes (D2-D3 of 28S-rDNA), and mitochondrial DNA (COI gene) units are good candidate genes for molecular taxonomic and phylogenetic studies (Subbotin et al., 2001; Subbotin et al., 2006; Madani et al., 2004; Vovlas et al., 2017). Based on morphomolecular characterizations, Handoo and Subbotin (2018) divided Heterodera into nine distinct groups such as Afenestrata, Avenae, Bifenestra, Cardiolata, Cyperi, Goettingiana, Humuli, Sacchari, and Schachtii group. Sequence analysis of these groups is significant to study the phylogenetic relationship and identifying the Heterodera species.

During 2018 and 2019, a population of cyst nematode was collected from the rhizosphere of Microula sikkimensis in Tianzhu county of Gansu Province, China. Considering the economic value of the cyst nematode, morphomolecular studies were performed; the preliminary studies indicated that the population belongs to Goettingiana group of Heterodera. The species characters were then compared with all the related species and concluded that this population possess unique characters and it is described herein as Heterodera microulae sp. n.

Materials and methods

Isolation and morphological observation of nematodes

The nematodes were extracted from root and soil samples of Microula sikkimensis in Tianzhu county, Gansu Province, China. Cysts and white females were collected using sieving-decanting method, while second-stage juveniles (J2s) were recovered from hatched eggs and kept in water suspension until further use (Hooper, 1970; Golden, 1990). Males were not found. For morphometric studies, second-stage juveniles were killed by gentle heating, fixed in TAF solution (formalin: triethanolamine: water = 7:2:91), and processed to ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969) and mounted on permanent slides. Vulval cones were mostly mounted in glycerin jelly. Measurements were made on mounted specimens using a Nikon Eclipse E100 Microscope (Nikon, Tokyo, Japan). Light micrographs and illustrations were produced using a Zeiss Axio Scope A1 microscope (Zeiss, Jena, Germany) equipped with an AxioCam 105 color camera and Nikon YS 100 with a drawing tube (Nikon, Tokyo, Japan), respectively.

Molecular analyses

DNA samples were prepared according to Maria et al. (2018). Three sets of primers (synthesized by Tsingke Biotech Co. Ltd., Xi’an, China) were used in the PCR analyses to amplify sequences of the ITS, D2-D3 expansion segments of 28S, and COI gene. The ITS region was amplified with TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (Maafi et al., 2003). The 28S D2-D3 region was amplified with the D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al., 2005; Ye et al., 2007). Finally, the partial COI gene was amplified using primers Het-coxiF (5′-TAGTTGATCGTAATTTTAATGG-3′) and Het-coxiR (5′-CCTAAAACATAATGAAAATGWGC-3′) (Subbotin, 2015). PCR conditions were as described by Ye et al. (2007), De Ley et al. (2005), and Subbotin (2015). PCR products were separated on 1% agarose gels and visualized by staining with ethidium bromide. PCR products of sufficiently high quality were purified for cloning and sequencing by Tsingke Biotech Co. Ltd., Xi’an, China. The PCR products were purified by the Tiangen Gel Extraction Kit (Tiangen Biotech Co. Ltd., Beijing, China), cloned into pMD18-T vectors and transformed into DH5α-competent cells, and then sequenced by Tsingke Biotech Co. Ltd (Xi’an, China).

Sequence alignment and phylogenetic analysis

The newly obtained sequences for each gene (ITS-rDNA, D2-D3 region of 28S-rDNA, and COI gene) were compared with known sequences of Heterodera using BLASTn homology search program. Outgroup taxa for phylogenetic analyses were selected based on the previously published studies (Subbotin et al., 2001; Maafi et al., 2003; Mundo-Ocampo et al., 2008; Kang et al., 2016; Madani et al., 2018; Vovlas et al., 2017). The selected sequences were aligned by MAFFT (Kazutaka and Standley, 2013) with default parameters and edited using Gblock (Castresana, 2000). Phylogenetic analyses were based on Bayesian inference (BI) using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). The GTR + I + G model was selected as the best-fit model of DNA evolution for both 28S D2-D3, ITS, and COI regions using MrModeltest version 2.3 (Nylander, 2004), according to the Akaike information criterion (AIC). BI analysis for each gene was initiated with a random starting tree and run with four Markov chains for 1,000,000 generations. The Markov chains were sampled at intervals of 100 generations and the burn-in value was 25%. Two runs were performed for each analysis. After discarding burn-in samples, the remaining samples were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) were given on appropriate clades. The phylogenetic consensus trees were visualized using FigTree v.1.4.3 software (http://tree.bio.ed.ac.uk/software/figtree/) (Rambaut, 2016). The species in Goettingiana group and their localities, hosts, and GenBank accession numbers used in this study were presented in Table S1.

Table S1.

Goettingiana group species, locality, host plants, and GenBank accession number used in this study.

10.21307_jofnem-2020-097-t0S1.jpg

Results

Systematics

Heterodera microulae sp. n. (Figures 1–4; Measurement Table 1)

Description

Cyst

It is lemon-shaped with an obtuse vulval cone, neck extruding, and cuticle thick with an irregular zig-zag pattern. The color was white to pale to medium brown; remnants of the subcrystalline layer were rarely present. The egg sac was usually absent (Figs. 1G, 3B, C). The vulval cone was ambifenestrate-like waning crescent moon and separated by a well-developed vulval bridge. The anus area was distinct, bullae were absent (Figs. 1F, 3D, E). The vulval slit was longer than fenestral width (39.00 vs 37.75 µm); the underbridge was weak and often lost during cone preparation.

Figure 1:

Line drawing of H. microulae sp. n. A: Anterior region of second-stage juvenile; B: Head of second-stage juvenile; C: Stylet of second-stage juvenile; D: Tail of second-stage juvenile; E: Cyst; F: Fenestration in vulval cone.

10.21307_jofnem-2020-097-f001.jpg
Figure 3:

Light micrographs of H. microulae sp. n. A: immature female on the root; B: Cyst; C: Cysts; D-E: Fenestration in vulval cone; F: Egg (scale bar: A, D = 50 µm; B = 100 µm; C = 200 µm; E, F = 20 µm).

10.21307_jofnem-2020-097-f003.jpg

Female

The female was lemon-shaped, pearl white, or pale yellow in color. It was rarely rounded with a protruding neck and vulva, the subcrystalline layer was present, and the egg sac absent (Figs. 2A, B, 3A). There was a labial region with two annuli. Labial sclerotization was weak, the stylet was strong, and basal knobs were rounded and anteriorly flattened. The excretory pore was indistinct, median bulb was rounded and massive, and other parts of the pharynx were not clearly discernable. There was vulval slit in a cleft on the cone terminus (Fig. 2C, D).

Figure 2:

Light micrographs of H. microulae sp. n. A: females attached on M. sikkimensis; B: yellow and white females; C: Anterior region of female; D: Vulval region of female (scale bar: A = 2 mm; B = 1 mm; C, D = 20 µm).

10.21307_jofnem-2020-097-f002.jpg

Second-stage juvenile

The body was straight or slightly curved ventrally after heat treatment (Fig. 4A). The lip region was offset and rounded, measuring 3.90 to 5.50 (4.63) µm in height and 9.65 to 12.75 (11.01)-µm wide. The cephalic framework was strongly sclerotized (Figs. 1B, 4C). The stylet was strong; knobs were well developed, rounded and flat, or slightly concave anteriorly (Figs. 1C, 4C). The dorsal esophageal gland orifice measured from 5.32 to 6.32 (5.61) µm posterior to the stylet knob. Median bulb was rounded with a strong valvular apparatus. The pharyngeal glands were well developed, overlapping the intestine dorsoventrally (Figs. 1A, 4B). The hemizoind was distinct from one to three annuli long (Fig. 4H), the excretory pore was situated 102.46 to 130.79 (114.40) µm from the anterior end, and one to two annules were posterior to the hemizonid (Fig. 4G). There was a lateral field with four incisures (Figs. 1D, 4G). The dorsal gland nucleus and subventral gland nuclei were distinct (Fig. 4E, F). Genital primordium situated at 59 to 62% of body length behind the anterior end, with two distinct nucleate cells (Fig. 4I). The tail was conoid, gradually tapering to a finely rounded terminus. The hyaline portion was irregularly annulated occupying 50% of tail length. Phasmid was absent (Figs. 1E, 4D).

Figure 4:

Light micrographs of second-stage juvenile of H. microulae sp. n. A: Entire body; B: Anterior region of; C: Head region; D: Tail region; E: Posterior pharyngeal region arrow showing the position of dorsal gland nucleus; F: Posterior pharyngeal region arrow showing the position of subventral gland nuclei; G: Lateral field; H: Hemizonid; I: Genital primordium; J: Excretory pore (scale bar: A = 100 μm, B, H, G = 50 μm, C, D, E, F, I, J = 20 μm).

10.21307_jofnem-2020-097-f004.jpg

Eggs

Body hyaline without any markings was presented; juveniles folded six times (Fig. 3F).

Male

The male was not found.

Type material

Holotype and paratype material (20 cysts, 20 females, and 20 second-stage juveniles) were deposited in the nematode collection of the Department of Plant Protection, Biocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou, China.

Type host and locality

Heterodera microulae sp. n. was collected from the roots and rhizosphere soil of Microula sikkimensis Hemsl. (Boraginaceae, Tubiflorae, Metachlamydeae) in Tianzhu county of Gansu Province, China. The geographical position is N 37°11′46″; E 102°47′6″. This site was located in continental highland with the vegetation type of meadow grassland and the soil is composed of chernozems. The climatic parameters of the site include 450 mm of average rainfall and an approximate −2 air temperature.

Etymology

The species is named after the host of its isolation.

Diagnosis and relationships

Heterodera microulae sp. n. is characterized by having lemon-shaped cysts that have protruding necks and obtuse vulval cones. The cysts are 414 to 543-µm long and 305 to 456-µm wide having ambifenestrate vulval cone and bullae are absent. Females are white in color with a subcrystalline layer. Second-stage juveniles are straight or slightly curved ventrally with four incisures in the lateral field. The juveniles are 506 to 628-µm long having strong stylets with well-developed rounded stylet knobs, genital primordium situated at 59 to 62% of body length, and tail 49 to 61-µm long with a hyaline portion of 24 to 33 µm. Eggs are hyaline without any markings; juveniles inside the eggs form sixfold.

The new species belongs to the Goettingiana group of Heterodera; currently, the group contains seven valid species, viz, Heterodera goettingiana (Liebscher, 1892), H. carotae (Jones, 1950), H. cruciferae (Franklin, 1945), H. circeae (Subbotin and Turhan, 2004), H. scutellariae (Subbotin and Turhan, 2004), H. urticae (Cooper, 1955), and H. persica (Maafi et al., 2006).

The new species differs from H. goettingiana by having a shorter fenestral length (31 µm vs 35 µm), absence of bullae (vs few), weak underbridge (vs 117 µm), longer J2s body length (568 µm vs 486 µm), stylet knobs rounded and flat or slightly concave anteriorly vs smoothly rounded to slightly hook-shaped with a recurved anterior surface, longer distance of median bulb from the anterior end (MB) (86 µm vs 70 µm), shorter excretory pore distance from the anterior end (114 µm vs 158 µm), and shorter length of hyaline tail portion (29 µm vs 37 µm).

The new species is differentiated from H. carotae by having a bigger size of cysts (495 × 384 µm vs 408 × 309 µm), shorter vulval slit length (39 µm vs 47 µm), longer J2s body length (568 µm vs 422 µm), stylet knobs rounded and flat or slightly concave anteriorly vs concave anterior face, higher MB value (86 µm vs 66 µm), longer excretory pore distance from the anterior end (114 µm vs 99 µm), and longer tail length (57 µm vs 52 µm).

The new species differs from H. cruciferae by having a bigger size of cysts (495 × 384 µm vs 429 × 333 µm), slightly shorter fenestral length (31 µm vs 34 µm), shorter vulval length (39 µm vs 45 µm), longer J2s body length (568 µm vs 431 µm), higher MB value (86 µm vs 68 µm), longer excretory pore distance from the anterior end (114 µm vs 101 µm), longer tail length (57 µm vs 50 µm), and longer length of hyaline tail portion (29 µm vs 25 µm).

The new species differs from H. persica by a shorter fenestral length (31 µm vs 47 µm), absence of bullae (vs present), shorter vulval slit length (39 µm vs 49 µm), longer J2s body length (568 µm vs 440 µm), stylet knobs (flat or concave anteriorly vs projecting slightly anteriorly, convex posteriorly), longer stylet (26 µm vs 23 µm), higher MB value (86 µm vs 70 µm), longer excretory pore distance from the anterior end (114 µm vs 103 µm), longer tail length (57 µm vs 47 µm), and longer length of hyaline tail portion (29 µm vs 24 µm).

Compared with H. urticae, the new species has a smaller size of cysts (495 × 384 µm vs 492 × 435 µm), vulval cone obtrusive (vs unobtrusive) and absence of egg sac (vs presence), shorter fenestral length (31 µm vs 38 µm), shorter vulval slit length (39 µm vs 46 µm), longer J2s body length (568 µm vs 541 µm), shorter DGO (8 µm vs 5 µm), and shorter excretory pore distance from the anterior end (114 µm vs 130 µm).

The new species differs from H. circeae having a smaller size of cysts (495 × 384 µm vs 555 × 397 µm), a shorter fenestral length (31 µm vs 43 µm), vulval slit length (39 µm vs 48 µm), longer J2s body length (568 µm vs 434 µm), stylet knobs (rounded and slightly sloping posteriorly vs rounded and flat or slightly concave anteriorly), higher MB value (86 µm vs 70 µm), longer excretory pore distance from the anterior end (114 µm vs 101 µm), longer tail length (57 µm vs 52 µm), and longer length of hyaline tail portion (29 µm vs 26 µm).

The new species differs from H. scutellariae, having smaller cysts (495 × 384 µm vs 560 × 424 µm), by a shorter fenestral length (31 µm vs 35 µm), vulval slit length (39 µm vs 43 µm), longer J2s body length (568 µm vs 408 µm), higher MB value (86 µm vs 62 µm), longer excretory pore distance from the anterior end (114 µm vs 89 µm), longer tail length (57 µm vs 47 µm), and longer length of hyaline tail portion (29 µm vs 25 µm).

Additionally, comparative morphological and morphometric characters of H. microulae sp. n. with other valid species of Goettingiana group are listed in Table 2.

Table 1.

Morphometrics of H. microulae sp. n.

10.21307_jofnem-2020-097-t001.jpg
Table 2.

Main morphological character of represent species from the Goettingiana group (all measurements are in µm).

10.21307_jofnem-2020-097-t002.jpg

Molecular characterization and phylogenetic relationships

The H. microulae sp. n. sequences of D2-D3 region of 28 S (734 bp), ITS (993 bp), and COI (415 bp) gene were obtained and submitted to the GenBank.

The D2-D3 of 28S-rRNA sequence (accession no. MT573436) of H. microulae sp. n. showed 97.09% (19-bp difference), 97.66 to 98.49% (11-17-bp difference), 98.38% (9-bp difference), 98.62% (9-bp difference), 98.45% (11-bp difference), and 99.86 to 100% (0-1-bp difference) sequence identities with H. goettingiana (DQ328697), H. carotae (KX463292 and KX463293), H. cruciferae (KP114546), H. urticae (DQ328696), Heterodera sp. RH-2010 (GU456692) from Iran, and Heterodera sp. DP-2010 (HM560856 and HM560855) from Qinghai, China, respectively. The Bayesian phylogenetic tree of the D2-D3 of 28S gene (Fig. 5) represented a highly supported (posterior probability PP = 100) clade of Heterodera species, where Goettingiana group species occupied a basal position. It is noted that H. microulae sp. n. clustered together with Heterodera sp. DP-2010 (HM560855, HM560856) from Qinghai, China and forms a 100% supported clade.

Figure 5:

Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from 28S D2/D3 extension region under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *Identified as Heterodera sp. by Ye et al. (unpublished) and Peng et al. (unpublished) in the GenBank.

10.21307_jofnem-2020-097-f005.jpg

The ITS-rDNA sequence (accession no. MT573437) divergence of H. microulae sp. n. with other Goettingiana group species is as follows: 0.20% (2-bp difference), 0.4 to 0.5% (4-bp difference), 3.02% (29-bp difference), 5.01% (48-bp difference), 5.11% (49-bp difference), 7.45% (72-bp difference), 6.77 to 6.95% (67-68-bp difference), 6.29 to 7.25% (66-70-bp difference), and 7.41 to 8% (74-77-bp difference) for Heterodera sp. DP-2010 (HM560791), H. goettingiana (HM370423, HM370425), H. persica (AF498377), H. scutellariae (AY368995), H. circeae (AY368994), H. urticae (AF274412), H. carotae (AF274413; MG976790), H. cruciferae (AF274411; GU126668), and H. goettingiana (KY129827; AF274411; AF498374), respectively. The Bayesian phylogenetic tree of the ITS gene (Fig. 6) represented a highly supported (posterior probability PP = 100) clade of Heterodera species. As in the 28S tree, the ITS tree also positioned the Goettingiana group species. H. microulae sp. n. (MT573437) clustered with H. persica (AF498377), H. scutellariae (AY368994), H. circeae (AY368995), Heterodera sp. DP-2010 (HM560791), and H. goettingiana (HM370423, HM370425) from Qinghai, China with high-probability support (pp = 91%). It is also noted that sequences of H. goettingiana (HM370423, HM370425) from Qinghai, China, clustered outside with other H. goettingiana (KY129827, AF274411, and AF498374) subclades and should be considered a misidentification. However, H. microulae sp. n. (MT573437) is clustered with H. sp. DP-2010 (HM560791) and H. goettingiana (HM370423, HM370425) from Qinghai, China, with 100% support. It is also noted that H. microulae sp. n. (MT573437) clustered with two Chinese populations of Heterodera species (HM560791; HM370425) with 100% support.

Figure 6:

Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from ITS region under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *Identified as Heterodera goettingiana by Peng et al. (unpublished); **Identified as Heterodera sp. by Peng et al. (unpublished); ***Identified as Heterodera goettingiana by Huang et al. (unpublished) in the GenBank.

10.21307_jofnem-2020-097-f006.jpg

The COI gene sequence of H. microulae sp. n. showed 87.21 to 89.53% (differing from 36 to 44 bp), 88.19% (differing from 43 bp), 88.67 to 88.92% (differing from 46 to 47 bp), and 88.67 to 89.40% (differing from 44 to 47 bp), sequence identities with H. goettingiana (KY129829-KY129831), H. urticae (MK093155 and MK093156), H. cruciferae (MG563230 and MG563234), and H. carotae (KX463299-KX463306, MG563227, MG563229, MG563231-MG563233, and MN820659), respectively. The Bayesian phylogenetic tree of the COI gene (Fig. 7) represented a highly supported (posterior probability PP = 100) clade of Heterodera species. In this tree, H. microulae sp. n. clustered with H. goettingiana, H. urticae, H. cruciferae, and H. caratae with 98% support; however, H. microulae sp. n. formed a separate clade from those sequences.

Figure 7:

Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from COI gene under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *KC172916 identified as H. pratensis by Toumi et al. (2013) and later corrected to Heterodera carotae by Madani et al. (2018).

10.21307_jofnem-2020-097-f007.jpg

Discussion

Taxonomy of Heterodera species has been revised extensively in the past; Baldwin and Mundo-Ocampo (1991) placed 23 Heterodera species into Goettingiana group. However, Sturhan (1998) and Subbotin et al. (2001) used J2’s lateral field characters and host preferences to separate Heterodera species into different groups (such as Bifenestra, Cyperi, and Humuli groups). The key morphological characters of the Goettingiana group include lemon-shaped cysts having a protruding neck, ambifenestration, and absence of bullae (small bullae occasionally present); some species may have an egg sac, vulval slit length > 35 µm, a thin vulval bridge, fenestral length (30-45 µm), and a weak underbridge. There were second-stage juveniles with body length > 400 µm, stylet length > 20 µm, tail length > 45 µm, hyaline tail portion > 20 µm, and lateral field with four lines (Subbotin and Turhan, 2004). The new species also belong to the Goettingiana group and morphologically very close to H. urticae; however, morphometrics of J2s body lengths, DGO and excretory pore position, fenestral length, vulval slit length, and cyst width can be used to differentiate both species.

Phylogenetically, it is evident that H. microulae sp. n. is a member of Goettingiana group. In our analyses, it is also noted that Heterodera sp. DP-2010 (HM560791, HM560855, and HM560856) and H. goettingiana (HM370423 and HM370425) from Qinghai, China, formed a well-supported molecular clade with the H. microulae sp. n. Moreover, the nucleotide differences of these sequences with our new species sequences are also very low (2-4-bp difference for ITS and 0-1 bp for 28S). Previously, Escobar-Avila et al. (2018) indicated that the sequences of H. goettingiana (HM370423 and HM370425) from Qinghai, China, might be a case of misidentification. Based on our phylogenetic and sequence analysis results, we regard Heterodera sp. DP-2010 (HM560791, HM560855, and HM560856) and H. goettingiana (HM370423 and HM370425) as H. microulae sp. n.

Heterodera microulae sp. n. is isolated from Microula sikkimensis, it is a biennial herbaceous plant that grows in forests, meadows, and forest edges at altitudes of 2,200 to 4,700 m, and it is widely distributed in South and East Asian countries (Pi et al., 2014). H. microulae sp. n. was found in Gansu and Qinghai Provinces, but we speculate that it is likely to be found in some localities that are characterized by low temperature, high rainfall, and high altitude.

The present study described a new species found in the rhizosphere of M. sikkimensis; further research is needed to understand the distribution and biology of the new species. In addition, plenty of leguminous crops (pea, kidney bean, pole bean, etc.) are growing in the same locality. Therefore, host-suitability tests of H. microulae sp. n. are an open research field to investigate the damage potential of this species.

Acknowledgements

This research was supported by the National Natural Science Foundation of China No. 31760507 and the Special R & D Fund for Public Benefit Agricultural, No. 201503114. The authors thank the assistance of the Institute of Plant Protection of China for the light micrographs and Dr. Maria Munawar from the Department of Biological Sciences, University of Lethbridge, Canada, for English polishing in this paper.

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  7. De Ley, P. , Tandingan De Ley, I. , Morris, K. , Abebe, E. , Mundo-Ocampo, M. , Yoder, M. , Heras, J. , Waumann, D. , Rocha-Olivares, A. , Burr, A. H. J. , Baldwin, J. G. and Thomas, W. K. 2005. An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 360:1945–1958.
  8. Ding, Z. , Namphueng, J. , He, X. F. , Peng, D. L. and Huang, W. K. 2012. First report of the cyst nematode (Heterodera elachista) on Rice in Hunan Province, China. Plant Disease 96:151 151.
  9. Escobar-Avila, I. M. , Lopez-Villegas, E. O. , Subbotin, S. A. and Tovar-Soto, A. 2018. First report of carrot cyst nematode Heterodera carotae in Mexico: morphological, molecular characterization, and host range study. Journal of Nematology 50:229–242.
  10. Franklin, M. T. 1945. On Heterodera cruciferae n. sp. of Brassicas, and on a Heterodera strain infecting Clover and Dock. Journal of Helminthology 21:71–84.
  11. Golden, A. M. 1990. “Preparation and mounting nematodes for microscopic observations”, In Zuckerman, B. M. , Mai, W. F. and Krusberg, L. R. (Eds), Plant Nematology Laboratory Manual University of Massachusetts Agricultural Experiment Station, Amherst, MA, 197–205.
  12. Handoo, Z. A. and Subbotin, S. A. 2018. “Taxonomy, identification and principal species”, In Perry, R. N. , Oens, M. and Jones, J. T. (Eds), Cyst Nematodes CAB International, pp. 365–397.
  13. Hooper, D. J. 1970. “Handling, fixing, staining, and mounting nematodes”, In Southey, J. F. (Ed.), Laboratory Methods for Work with Plant and Soil Nematodes 5th ed., London: Her Majesty’s Stationery Office, pp. 39–54.
  14. Huelsenbeck, J. P. and Ronquist, F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:1754–1755.
  15. Jones, F. G. W. 1950. A new species of root eelworm attacking carrots. Nature 4185:81–81.
  16. Jones, J. T. , Haegeman, A. , Danchin, E. G. J. , Gaur, H. S. , Helder, J. , Jones, M. G. K. , Kikuchi, T. , Manzanilla-López, R. , Palomares-Rius, J. E. , Wesemael, W. M. L. and Perry, R. N. 2013. Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology 14:946–961.
  17. Kang, H. , Eun, G. , Ha, J. , Kim, Y. , Park, N. , Kim, D. and Choi, I. 2016. New cyst nematode, Heterodera sojae n. sp. (Nematoda: Heteroderidae) from Soybean in Korea. Journal of Nematology 48:280–289.
  18. Kazutaka, K. and Standley, D. M. 2013. MAFFT Multiple Sequence Alignment Software Version 7: improvements in performance and usability. Molecular Biology & Evolution 30:772–780.
  19. Li, H. L. , Yuan, H. X. , Sun, J. W. , Fu, B. and Sun, B. J. 2010. First Record of the cereal cyst nematode Heterodera filipjevi in China. Plant Disease 94:1505.
  20. Liebscher, G. 1892. Beobachtungen über das Aufreten eines Nematoden an Erbsen. Journal für Landwirtschaft 40:357–368.
  21. Liu, W. Z. , Liu, Y. and Duan, Y. X. 1994. Morphological observation of soybean cyst nematodes in China. Journal of Shenyang Agricultural University 2:164–167.
  22. Maafi, Z. T. , Subbotin, S. A. and Moens, M. 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5:111–111.
  23. Maafi, Z. T. , Sturhan, D. , Subbotin, S. A. and Moens, M. 2006. Heterodera persica sp. n. (Tylenchida: Heteroderidae) parasitizing Persian Hogweed Heracleum persicum (Desf. ex fisch.) in Iran. Russian Journal of Nematology 14:171–178.
  24. Madani, M. , Palomares-Rius, J. E. , Vovlas, N. , Castillo, P. and Tenuta, M. 2018. Integrative diagnosis of carrot cyst nematode (Heterodera carotae) using morphology and several molecular markers for an accurate identification. European Journal of Plant Pathology 150:1023–1039.
  25. Madani, M. , Vovlas, N. , Castillo, P. , Subbotin, S. A. and Moens, M. 2004. Molecular Characterization of cyst nematode species (Heterodera spp.) from the mediterranean basin using RFLPs and Sequences of ITS-rDNA. Journal of Phytopathology 152:229–234.
  26. Mathews, H. J. P. 1970. Morphology of the nettle cyst nematode Heterodera Urticae Cooper, 1955. Nematologica 16:503–510.
  27. Mathews, H. J. P. 1975. Heterodera carotae. CIH descriptions of plant parasitic nematodes. Set 5, 61:4.
  28. Maria, M. , Cai, R. H. , Ye, W. M. , Powers, T. O. and Zheng, J. W. 2018. Description of Gracilacus paralatescens n. sp. (Nematoda: Paratylenchinae) found from the rhizosphere of Bamboo in Zhejiang, China. Journal of Nematology 50:611–622.
  29. Mundo-Ocampo, M. , Troccoli, A. , Subbotin, S. A. , Cid, J. , Baldwin, J. G. and Inserra, R. N. 2008. Synonymy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterisation of H. koreana comb. n. and H. orientalis comb. n. (Tylenchida: Heteroderidae). Nematology 10:611–632.
  30. Nylander, J. A. A. 2004. MrModeltest v.2.3 Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden.
  31. Peng, D. L. , Subbotin, S. A. and Moens, M. 2003. rDNA restriction fragment length polymorphism of Heterodera avenae in China. Acta Phytopathologica Sinica 4:323–329.
  32. Peng, D. L. , Ricardo, H. , Zheng, J. W. , Chen, S. L. and Li, H. M. 2020. Current occurrences and integrated management of cyst forming nematodes in China. Monitoring and management of transboundary crop nematodes: proceedings of the first belt and road international nematology symposium, pp. 7–8.
  33. Pi, L. , Xing, Y. , Hu, F. , Chi, X. , Li, Y. , Han, T. , Zhao, X. and Han, F. 2014. The study on mineral elements in Microula sikkimensis from the Qinghai-Tibet Plateau. Spectroscopy Letters 48:375–380.
  34. Rambaut, A. 2016. FigTree v.1.4.3, available at: http://tree.bio.ed.ac.uk/ software/figtree/.
  35. Schmidt, A. 1871. Über den Rübennematoden. Zeitschrift der Vereinte Rübenzuckerindustrie Zollverein 21:1–19.
  36. Seinhorst, J. W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67–69.
  37. Stone, A. R. and Course, J. A. 1974. Heterodera goettingiana. CIH descriptions of plant parasitic nematodes. Set 4, 47:4.
  38. Stone, A. R. and Rowe, J. A. 1976. Heterodera cruciferae. CIH description of plant-parasitic nematodes. Set 6, 90:4.
  39. Sturhan, D. 1998. Notes on the taxonomy and phylogeny of Heteroderidae parasitising Gramineae. Nematologica 44:585–152..
  40. Subbotin, S. A. 2015. Heterodera sturhani sp. n. from China, a new species of the Heterodera avenae species complex (Tylenchida: Heteroderidae). Russian Journal of Nematology 23:145–152..
  41. Subbotin, S. A. and Turhan, D. S. 2004. Heterodera circeae sp. n. and H. scutellariae sp. n. (Tylenchida: Heteroderidae) from Germany, with notes on the goettingiana group. Nematology 6:343–355.
  42. Subbotin, S. A. , Sturhan, D. , Chizhov, V. N. , Vovlas, N. and Baldwin, J. G. 2006. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology 8:455–474.
  43. Subbotin, S. A. , Mundo-Ocampo, M. , Baldwin, J. G. , Hunt, D. J. and Perry, R. N. 2010. Systematics of cyst nematodes (Nematoda: Heteroderinae). Nematology Monographs and Perspectives 8:1–351.
  44. Subbotin, S. A. , Vierstraete, A. , De Ley, P. , Rowe, J. , Waeyenberge, L. , Moens, M. and Vanfleteren, J. R. 2001. Phylogenetic Relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA. Molecular Phylogenetics & Evolution 21:1–16.
  45. Toumi, F. , Waeyenberge, L. , Viaene, N. , Dababat, A. , Nicol, J. M. , Ogbonnaya, F. and Moens, M. 2013. Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi . European Journal of Plant Pathology 136:613–624.
  46. Vovlas, A. , Santoro, S. , Radicci, V. , Leonetti, P. , Castillo, P. and Palomares-Rius, J. E. 2017. Host-suitability of black medick (Medicago lupulina L.) and additional molecular markers for identification of the pea cyst nematode Heterodera goettingiana . European Journal of Plant Pathology 149:193–199.
  47. Wang, D. , Chen, L. J. and Duan, Y. X. 2012a. Description of a new record species of Heterodera from China (Tylenchida, Heteroderidae). Zoological Research 33:57–59.
  48. Wang, H. H. , Zhuo, K. , Zhang, H. L. and Liao, J. L. 2012b. Heterodera koreana, a new record species from China. Acta Phytopathologica Sinica 42:551–555.
  49. Wang, H. H. , Zhuo, K. , Ye, W. M. , Zhang, H. L. , Peng, D. L. and Liao, J. L. 2013. Heterodera fengi n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China-a new cyst nematode in the Cyperi group. Zootaxa 3652:179–192.
  50. Wu, H. Y. , Qiu, Z. Q. , Mo, A. S. , Li, J. Q. and Peng, D. 2017. First Report of Heterodera zeae on Maize in China. Plant Disease 101:1330 1330.
  51. Ye, W. M. , Giblin-Davis, R. M. , Davies, K. A. , Purcell, M. F. , Scheffer, S. J. , Taylor, G. S. , Center, T. D. , Morris, K. and Thomas, W. K. 2007. Molecular phylogenetics and the evolution of host plant associations in the nematode genus Fergusobia (Tylenchida: Fergusobiinae). Molecular Phylogenetics and Evolution 45:123–141.
  52. Zhen, H. Y. , Peng, H. , Kong, L. A. , Hong, B. Y. , Zhu, G. L. , Wang, R. H. , Peng, D. L. and Weng, Y. H. 2018. Heterodera sojae, a new cyst nematode record in China and its parasitism to legume crops. Scientia Agricultura Sinica 51:93–104.
  53. Zhuo, K. , Wang, H. H. , Zhang, H. L. and Liao, J. L. 2014. Heterodera guangdongensis n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China-a new cyst nematode in the Cyperi group. Zootaxa 3881:488–500.
  54. Zhuo, K. , Wang, H. H. , Ye, W. M. , Peng, D. L. and Liao, J. L. 2013. Heterodera hainanensis n. sp. (Nematoda: Heteroderinae) from bamboo in Hainan Province, China-a new cyst nematode in the Afenestrata group. Nematology 15:303–314.
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FIGURES & TABLES

Figure 1:

Line drawing of H. microulae sp. n. A: Anterior region of second-stage juvenile; B: Head of second-stage juvenile; C: Stylet of second-stage juvenile; D: Tail of second-stage juvenile; E: Cyst; F: Fenestration in vulval cone.

Full Size   |   Slide (.pptx)

Figure 2:

Light micrographs of H. microulae sp. n. A: females attached on M. sikkimensis; B: yellow and white females; C: Anterior region of female; D: Vulval region of female (scale bar: A = 2 mm; B = 1 mm; C, D = 20 µm).

Full Size   |   Slide (.pptx)

Figure 3:

Light micrographs of H. microulae sp. n. A: immature female on the root; B: Cyst; C: Cysts; D-E: Fenestration in vulval cone; F: Egg (scale bar: A, D = 50 µm; B = 100 µm; C = 200 µm; E, F = 20 µm).

Full Size   |   Slide (.pptx)

Figure 4:

Light micrographs of second-stage juvenile of H. microulae sp. n. A: Entire body; B: Anterior region of; C: Head region; D: Tail region; E: Posterior pharyngeal region arrow showing the position of dorsal gland nucleus; F: Posterior pharyngeal region arrow showing the position of subventral gland nuclei; G: Lateral field; H: Hemizonid; I: Genital primordium; J: Excretory pore (scale bar: A = 100 μm, B, H, G = 50 μm, C, D, E, F, I, J = 20 μm).

Full Size   |   Slide (.pptx)

Figure 5:

Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from 28S D2/D3 extension region under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *Identified as Heterodera sp. by Ye et al. (unpublished) and Peng et al. (unpublished) in the GenBank.

Full Size   |   Slide (.pptx)

Figure 6:

Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from ITS region under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *Identified as Heterodera goettingiana by Peng et al. (unpublished); **Identified as Heterodera sp. by Peng et al. (unpublished); ***Identified as Heterodera goettingiana by Huang et al. (unpublished) in the GenBank.

Full Size   |   Slide (.pptx)

Figure 7:

Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from COI gene under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *KC172916 identified as H. pratensis by Toumi et al. (2013) and later corrected to Heterodera carotae by Madani et al. (2018).

Full Size   |   Slide (.pptx)

REFERENCES

  1. Baldwin, J. G. and Mundo-Ocampo, M. 1991. “Heteroderinae, cyst- and non-cyst-forming nematodes”, In Nickle, W. R. (Ed.), Manual of Agricultural Nematology. New York, Basel, and Hong Kong: Marcel Dekker, pp. 275–362.
  2. Castresana, J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17:540–522.
  3. Chen, P. S. and Zheng, J. W. 1994. Primary report on a new species-Heterodera sinensis sp. nov. from China. Scientia Agricultura Sinica 27:88–89.
  4. Chen, P. S. , Wang, Z. M. and Peng, D. L. 1991. Preliminary report of identification on the cereal cyst nematodes of wheat in China. Scientia Agricultura Sinica 24:89–89.
  5. Cooper, B. A. 1955. “A preliminary key to British species of Heterodera for use in soil examination”, In Kevan, D. K. M. E (Ed.), Soil Zoology. London: Butterworths, pp. 269–280.
  6. De Grisse, A. T. 1969. Redescription on modifications de quelques techniques utilisees dans letude des nematodes phytoparasitaires. Mededlingen Rijksfaculteit der Landbouwwetenschappen Gent, 34:351–369.
  7. De Ley, P. , Tandingan De Ley, I. , Morris, K. , Abebe, E. , Mundo-Ocampo, M. , Yoder, M. , Heras, J. , Waumann, D. , Rocha-Olivares, A. , Burr, A. H. J. , Baldwin, J. G. and Thomas, W. K. 2005. An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 360:1945–1958.
  8. Ding, Z. , Namphueng, J. , He, X. F. , Peng, D. L. and Huang, W. K. 2012. First report of the cyst nematode (Heterodera elachista) on Rice in Hunan Province, China. Plant Disease 96:151 151.
  9. Escobar-Avila, I. M. , Lopez-Villegas, E. O. , Subbotin, S. A. and Tovar-Soto, A. 2018. First report of carrot cyst nematode Heterodera carotae in Mexico: morphological, molecular characterization, and host range study. Journal of Nematology 50:229–242.
  10. Franklin, M. T. 1945. On Heterodera cruciferae n. sp. of Brassicas, and on a Heterodera strain infecting Clover and Dock. Journal of Helminthology 21:71–84.
  11. Golden, A. M. 1990. “Preparation and mounting nematodes for microscopic observations”, In Zuckerman, B. M. , Mai, W. F. and Krusberg, L. R. (Eds), Plant Nematology Laboratory Manual University of Massachusetts Agricultural Experiment Station, Amherst, MA, 197–205.
  12. Handoo, Z. A. and Subbotin, S. A. 2018. “Taxonomy, identification and principal species”, In Perry, R. N. , Oens, M. and Jones, J. T. (Eds), Cyst Nematodes CAB International, pp. 365–397.
  13. Hooper, D. J. 1970. “Handling, fixing, staining, and mounting nematodes”, In Southey, J. F. (Ed.), Laboratory Methods for Work with Plant and Soil Nematodes 5th ed., London: Her Majesty’s Stationery Office, pp. 39–54.
  14. Huelsenbeck, J. P. and Ronquist, F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:1754–1755.
  15. Jones, F. G. W. 1950. A new species of root eelworm attacking carrots. Nature 4185:81–81.
  16. Jones, J. T. , Haegeman, A. , Danchin, E. G. J. , Gaur, H. S. , Helder, J. , Jones, M. G. K. , Kikuchi, T. , Manzanilla-López, R. , Palomares-Rius, J. E. , Wesemael, W. M. L. and Perry, R. N. 2013. Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology 14:946–961.
  17. Kang, H. , Eun, G. , Ha, J. , Kim, Y. , Park, N. , Kim, D. and Choi, I. 2016. New cyst nematode, Heterodera sojae n. sp. (Nematoda: Heteroderidae) from Soybean in Korea. Journal of Nematology 48:280–289.
  18. Kazutaka, K. and Standley, D. M. 2013. MAFFT Multiple Sequence Alignment Software Version 7: improvements in performance and usability. Molecular Biology & Evolution 30:772–780.
  19. Li, H. L. , Yuan, H. X. , Sun, J. W. , Fu, B. and Sun, B. J. 2010. First Record of the cereal cyst nematode Heterodera filipjevi in China. Plant Disease 94:1505.
  20. Liebscher, G. 1892. Beobachtungen über das Aufreten eines Nematoden an Erbsen. Journal für Landwirtschaft 40:357–368.
  21. Liu, W. Z. , Liu, Y. and Duan, Y. X. 1994. Morphological observation of soybean cyst nematodes in China. Journal of Shenyang Agricultural University 2:164–167.
  22. Maafi, Z. T. , Subbotin, S. A. and Moens, M. 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5:111–111.
  23. Maafi, Z. T. , Sturhan, D. , Subbotin, S. A. and Moens, M. 2006. Heterodera persica sp. n. (Tylenchida: Heteroderidae) parasitizing Persian Hogweed Heracleum persicum (Desf. ex fisch.) in Iran. Russian Journal of Nematology 14:171–178.
  24. Madani, M. , Palomares-Rius, J. E. , Vovlas, N. , Castillo, P. and Tenuta, M. 2018. Integrative diagnosis of carrot cyst nematode (Heterodera carotae) using morphology and several molecular markers for an accurate identification. European Journal of Plant Pathology 150:1023–1039.
  25. Madani, M. , Vovlas, N. , Castillo, P. , Subbotin, S. A. and Moens, M. 2004. Molecular Characterization of cyst nematode species (Heterodera spp.) from the mediterranean basin using RFLPs and Sequences of ITS-rDNA. Journal of Phytopathology 152:229–234.
  26. Mathews, H. J. P. 1970. Morphology of the nettle cyst nematode Heterodera Urticae Cooper, 1955. Nematologica 16:503–510.
  27. Mathews, H. J. P. 1975. Heterodera carotae. CIH descriptions of plant parasitic nematodes. Set 5, 61:4.
  28. Maria, M. , Cai, R. H. , Ye, W. M. , Powers, T. O. and Zheng, J. W. 2018. Description of Gracilacus paralatescens n. sp. (Nematoda: Paratylenchinae) found from the rhizosphere of Bamboo in Zhejiang, China. Journal of Nematology 50:611–622.
  29. Mundo-Ocampo, M. , Troccoli, A. , Subbotin, S. A. , Cid, J. , Baldwin, J. G. and Inserra, R. N. 2008. Synonymy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterisation of H. koreana comb. n. and H. orientalis comb. n. (Tylenchida: Heteroderidae). Nematology 10:611–632.
  30. Nylander, J. A. A. 2004. MrModeltest v.2.3 Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden.
  31. Peng, D. L. , Subbotin, S. A. and Moens, M. 2003. rDNA restriction fragment length polymorphism of Heterodera avenae in China. Acta Phytopathologica Sinica 4:323–329.
  32. Peng, D. L. , Ricardo, H. , Zheng, J. W. , Chen, S. L. and Li, H. M. 2020. Current occurrences and integrated management of cyst forming nematodes in China. Monitoring and management of transboundary crop nematodes: proceedings of the first belt and road international nematology symposium, pp. 7–8.
  33. Pi, L. , Xing, Y. , Hu, F. , Chi, X. , Li, Y. , Han, T. , Zhao, X. and Han, F. 2014. The study on mineral elements in Microula sikkimensis from the Qinghai-Tibet Plateau. Spectroscopy Letters 48:375–380.
  34. Rambaut, A. 2016. FigTree v.1.4.3, available at: http://tree.bio.ed.ac.uk/ software/figtree/.
  35. Schmidt, A. 1871. Über den Rübennematoden. Zeitschrift der Vereinte Rübenzuckerindustrie Zollverein 21:1–19.
  36. Seinhorst, J. W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67–69.
  37. Stone, A. R. and Course, J. A. 1974. Heterodera goettingiana. CIH descriptions of plant parasitic nematodes. Set 4, 47:4.
  38. Stone, A. R. and Rowe, J. A. 1976. Heterodera cruciferae. CIH description of plant-parasitic nematodes. Set 6, 90:4.
  39. Sturhan, D. 1998. Notes on the taxonomy and phylogeny of Heteroderidae parasitising Gramineae. Nematologica 44:585–152..
  40. Subbotin, S. A. 2015. Heterodera sturhani sp. n. from China, a new species of the Heterodera avenae species complex (Tylenchida: Heteroderidae). Russian Journal of Nematology 23:145–152..
  41. Subbotin, S. A. and Turhan, D. S. 2004. Heterodera circeae sp. n. and H. scutellariae sp. n. (Tylenchida: Heteroderidae) from Germany, with notes on the goettingiana group. Nematology 6:343–355.
  42. Subbotin, S. A. , Sturhan, D. , Chizhov, V. N. , Vovlas, N. and Baldwin, J. G. 2006. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology 8:455–474.
  43. Subbotin, S. A. , Mundo-Ocampo, M. , Baldwin, J. G. , Hunt, D. J. and Perry, R. N. 2010. Systematics of cyst nematodes (Nematoda: Heteroderinae). Nematology Monographs and Perspectives 8:1–351.
  44. Subbotin, S. A. , Vierstraete, A. , De Ley, P. , Rowe, J. , Waeyenberge, L. , Moens, M. and Vanfleteren, J. R. 2001. Phylogenetic Relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA. Molecular Phylogenetics & Evolution 21:1–16.
  45. Toumi, F. , Waeyenberge, L. , Viaene, N. , Dababat, A. , Nicol, J. M. , Ogbonnaya, F. and Moens, M. 2013. Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi . European Journal of Plant Pathology 136:613–624.
  46. Vovlas, A. , Santoro, S. , Radicci, V. , Leonetti, P. , Castillo, P. and Palomares-Rius, J. E. 2017. Host-suitability of black medick (Medicago lupulina L.) and additional molecular markers for identification of the pea cyst nematode Heterodera goettingiana . European Journal of Plant Pathology 149:193–199.
  47. Wang, D. , Chen, L. J. and Duan, Y. X. 2012a. Description of a new record species of Heterodera from China (Tylenchida, Heteroderidae). Zoological Research 33:57–59.
  48. Wang, H. H. , Zhuo, K. , Zhang, H. L. and Liao, J. L. 2012b. Heterodera koreana, a new record species from China. Acta Phytopathologica Sinica 42:551–555.
  49. Wang, H. H. , Zhuo, K. , Ye, W. M. , Zhang, H. L. , Peng, D. L. and Liao, J. L. 2013. Heterodera fengi n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China-a new cyst nematode in the Cyperi group. Zootaxa 3652:179–192.
  50. Wu, H. Y. , Qiu, Z. Q. , Mo, A. S. , Li, J. Q. and Peng, D. 2017. First Report of Heterodera zeae on Maize in China. Plant Disease 101:1330 1330.
  51. Ye, W. M. , Giblin-Davis, R. M. , Davies, K. A. , Purcell, M. F. , Scheffer, S. J. , Taylor, G. S. , Center, T. D. , Morris, K. and Thomas, W. K. 2007. Molecular phylogenetics and the evolution of host plant associations in the nematode genus Fergusobia (Tylenchida: Fergusobiinae). Molecular Phylogenetics and Evolution 45:123–141.
  52. Zhen, H. Y. , Peng, H. , Kong, L. A. , Hong, B. Y. , Zhu, G. L. , Wang, R. H. , Peng, D. L. and Weng, Y. H. 2018. Heterodera sojae, a new cyst nematode record in China and its parasitism to legume crops. Scientia Agricultura Sinica 51:93–104.
  53. Zhuo, K. , Wang, H. H. , Zhang, H. L. and Liao, J. L. 2014. Heterodera guangdongensis n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China-a new cyst nematode in the Cyperi group. Zootaxa 3881:488–500.
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