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

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.

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.

Species	Locality	Host-plant	Marker	Accession number
H. goettingiana	Germany	Pisum sp.	ITS	AF274414
H. goettingiana	Lorestan, Doroud, Akbar Abad, Iran	Trifolium repens	ITS	AF498374
H. goettingiana	Monopoly, Bari province, Italy	Pisum sativum	ITS	KY129827
H. sp	Yuekou village, Tianmen county, Hubei province, China	–	ITS	HM560794
H. sp	Morroco	–	ITS	AY347918
H. carotae	Creances, France	Daucus sp.	ITS	AF274413
H. carotae	South Africa	Daucus carota	ITS	MG976790
H. cruciferae	Brielle, The Netherlands	Brassica sp.	ITS	AF274411
H. urticae	Diksmuide, Belgium	Urtica sp.	ITS	AF274412
H. cruciferae	Moscow, Russia	Brassica oleracea	ITS	GU126668
H. sp	Xinzhuang village, Huangzhong county, Qinghai province, China	–	ITS	EU623623
H. microulae sp. n.	Tianzhu county, Gansu province, China	Microula sikkimensis	ITS	MT573437
H. microulae sp. n.	Haiyan county, Qinghai province, China	–	ITS	HM560791
H. microulae sp. n.	Haibei city, Qinghai province, China	–	ITS	HM370425
H. microulae sp. n.	Xining city, Qinghai province, China	–	ITS	HM370423
H. persica	Tehran, Dizin, Iran	Heracleum persicum	ITS	AF498377
H. scutellariae	Bremen, Germany	Circaea lutetiana	ITS	AY368994
H. circeae	Muenster, Germany	Scutellaria galericulata	ITS	AY368995
H. carotae	Ontario province, Canada	Daucus carota	28S	KX463292
H. carotae	Ontario province, Canada	Daucus carota	28S	KX463293
H. sp	Yuekou village, Tianmen county, Hubei province, China	–	28S	HM560857
H. sp	Iran	–	28S	GU456692
H. cruciferae	Iran	–	28S	KP114546
H. urticae	Belgium	–	28S	DQ328696
H. goettingiana	Iran	–	28S	DQ328697
H. microulae sp. n.	Haiyan county, Qinghai province, China	–	28S	HM560855
H. microulae sp. n.	Haomen village, Menyuan county, Qinghai province, China	–	28S	HM560856
H. microulae sp. n.	Tianzhu county, Gansu province, China	Microula sikkimensis	28S	MT573436
H. carotae	South Africa	Daucus carota	COI	MN820659
H. carotae	Mesola, Forli-Cesena province, Italy	Daucus carota	COI	KX463299
H. carotae	Mesola, Forli-Cesena province, Italy	Daucus carota	COI	KX463300
H. carotae	Margherita, di Savoia, Italy	Daucus carota	COI	KX463301
H. carotae	Margherita, di Savoia, Italy	Daucus carota	COI	KX463302
H. carotae	Ontario province, Canada	Daucus carota	COI	KX463303
H. carotae	Ontario province, Canada	Daucus carota	COI	KX463304
H. carotae	Ontario province, Canada	Daucus carota	COI	KX463305
H. carotae	Ontario province, Canada	Daucus carota	COI	KX463306
H. carotae	Mexico	Daucus carota	COI	MG563227
H. carotae	Mexico	Daucus carota	COI	MG563229
H. carotae	Switzerland	–	COI	MG563231
H. carotae	Switzerland	–	COI	MG563232
H. carotae	France	–	COI	MG563233
H. carotae	Belgium	–	COI	KC172916
H. urticae	Faulkner county, Arkansas, USA	Stellaria media	COI	MK093155
H. urticae	Faulkner county, Arkansas, USA	Stellaria media	COI	MK093156
H. cruciferae	California, USA	–	COI	MG563230
H. cruciferae	Moscow region, Russia	–	COI	MG563234
H. goettingiana	Monopoly, Bari province, Italy	Vicia faba	COI	KY129829
H. goettingiana	Monopoly, Bari province, Italy	Pisum sativum	COI	KY129830
H. goettingiana	Monopoly, Bari province, Italy	Medicago lupulina	COI	KY129831
H. microulae sp. n.	Tianzhu county, Gansu province, China	Microula sikkimensis	COI	MT576084

Note: Newly added sequences are indicated by bold font.

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.

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).

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).

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).

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 2.

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

Species	H. goettingianaa	H. carotaeb	H. cruciferaec	H. persicad	H. urticaee	H. circeaef	H. scutellariaeg	H. microulae sp. n.
Host	Pisum sativum L.	Daucus carota var.sativa	Brassica oleracea L.V.capitata	Heracleum persicum Desf.ex	Urtica dioica L.	Circaea lutetiana	Scutellaria galericulata	Microula sikkimensis
Locality	Germany	England	England	Iran	Northern Ireland	Germany	Germany	China
Cyst
Size	521 × 372	408 × 309	429 × 333	533 × 380	492 × 435	555 × 397	560 × 424	495×384
Fenestral length	35	31	34	47	38	43	35	31
Underbridge length	117	90	85	104	Weak	83	86	Weak
Bullae	Few	Absent	Absent	Present	Absent	Absent	Absent	Absent
Vulval slit length	39	47	45	49	46	48	43	39
Second-stage juvenile
Body length	486	422	431	440	541	434	408	568
a	25	21	–	23	23	22	21	25
Stylet knobs	Smoothly rounded to slightly hooked shaped with recurved anterior surface	Concave anterior face	Anterior face flat to concave	Rounded or projecting slightly anterior, convex posteriorly	Slightly concave anteriorly	Rounded and slightly sloping posteriorly	Slightly convave anteriorly	Rounded and flat or slightly concave anteriorly
Stylet length	25	24	24	23	27	25	24	26
Lateral line	4	4	4	4	4	4	4	4
DGO	5	5-6	–	6	8	6	5	5
MB	70	66	68	70	–	70	62	86
Excretory pore from anterior end	158	99	101	103	130	101	89	114
Tail	60	52	50	47	58	52	47	57
Hyaline portion of tail length	37	28	25	24	29	26	25	29

Notes: Data from: a(Stone and Course, 1974); b(Mathews, 1975); c(Stone and Rowe, 1976); d(Mathews, 1970); e,f(Subbotin and Turhan, 2004); g(Maafi et al., 2006).

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.

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 (HM560761), 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.

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).

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.

Table 1.

Morphometrics of H. microulae sp. n.

Stage	Character	Holotype	Paratype
Cyst
	n		20
	L (excluding length)	521.79	495.50 ± 41.01 (413.93-543.23)
	Diam.	419.33	384.29 ± 43.30 (304.96-455.51)
	L/Diam	1.27	1.30 ± 0.09 (1.12-1.45)
	Fenestral length	30.72	31.14 ± 1.36 (28.33-32.78)
	Fenestral width	36.16	37.75 ± 1.61 (35.46-40.07)
	Vulval slit length	35.53	39.00 ± 2.78 (35.16-44.38)
Female
	n		20
	Length		454.21 ± 28.32 (381.62-496.48)
	Width		326.47 ± 31.42 (256.32-421.63)
	Length/width		1.40 ± 0.12 (1.28-1.56)
Second-stage juveniles
	n		20
	Body length		567.73 ± 43.24 (505.62-627.92)
	Body width at mid-body		23.19 ± 1.31 (20.39-25.43)
	a		24.54 ± 1.34 (21.97-27.67)
	b		4.37 ± 0.33 (3.91-5.00)
	c		10.07 ± 1.18 (8.56-12.52)
	c′		4.26 ± 0.33 (3.63-4.90)
	Lip-region height		4.63 ± 0.44 (3.90-5.50)
	Lip-region diam.		11.07 ± 0.82 (9.65-12.75)
	Stylet length		25.73 ± 1.21 (24.07-28.92)
	Stylet base height		2.53 ± 0.33 (2.04-3.19)
	Stylet base width		5.30 ± 0.54 (4.39-6.09)
	Median bulb from the anterior end (MB)		85.57 ± 5.02 (76.37-95.26)
	Opening of dorsal pharyngeal gland from the stylet base (DGO)		5.13 ± 0.72 (4.03-6.41)
	Excretory pore from the anterior end (EP)		114.40 ± 6.89 (102.46-130.79)
	Median bulb width (MBW)		12.33 ± 1.49 (10.51-15.82)
	Diam. at the anus		13.23 ± 1.00 (11.24-15.26)
	Tail length		56.67 ± 3.75 (48.90-60.80)
	Hyaline portion tail		28.63 ± 1.91 (24.29-30.60)
	L/MB		6.65 ± 0.37 (5.76-7.53)
	TL/H		1.98 ± 0.10 (1.64-2.08)
Egg
	n		20
	Length		111.61 ± 8.02 (100.21-124.65)
	Width		50.34 ± 6.71 (35.64-71.51)
	Length/width		2.26 ± 0.38 (1.43-3.26)

Note: All measurements are in μm, and in the form: mean ± standard (range).