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

Li, Wenhao; Li, Huixia; Ni, Chunhui; Peng, Deliang; Liu, Yonggang; Luo, Ning; Xu, Xuefen

doi:10.21307/jofnem-2020-097

Figures & Tables

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.

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).

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).

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).

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 (unpublished) and Peng et al. (unpublished) in the GenBank.

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 (unpublished); **Identified as Heterodera sp.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 (unpublished); ***Identified as Heterodera goettingiana by Huang et al. (unpublished) in the GenBank.

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).

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)

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

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

Species	H. goettingiana ^a	H. carotae ^b	H. cruciferae ^c	H. persica ^d	H. urticae ^e	H. circeae ^f	H. scutellariae ^g	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

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

Figures & Tables

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Morphometrics of H_ microulae sp_ n_

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

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

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