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

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:

Figure 3:

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:

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:

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

The male was not found.

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.

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.

The species is named after the host of its isolation.

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.