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

INFORMAZIONI SU QUESTO ARTICOLO

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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.
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).
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).

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).
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).

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).
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).

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.
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.

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.
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.

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).
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
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