Heterodera fengi, Wang, Honghong, Zhuo, Kan, Ye, Weimin, Zhang, Hongling, Peng, Deliang & Liao, Jinling, 2013

Wang, Honghong, Zhuo, Kan, Ye, Weimin, Zhang, Hongling, Peng, Deliang & Liao, Jinling, 2013, Heterodera fengi n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China a new cyst nematode in the Cyperi group, Zootaxa 3652 (1), pp. 179-192 : 180-190

Heterodera fengi n. sp.

Figs. 1–4

Measurements. See Table 1.

Material examined. Type-material: Holotype vulval cone, three paratype vulval cones, one paratype male and ten paratype J2s are deposited in the USDA Nematode Collection, Beltsville, Maryland; two paratype vulval cones, one paratype male and ten paratype J2s in the Canadian National Nematode Collection, Ottawa, Canada. Other voucher specimens are available at the Plant Nematode Research Laboratory, College of Natural Resources and Environment, South China Agricultural University, Guangzhou, China.

Stage Character Holotype Paratype

Cyst

n 25

L (including neck) 524.7 481.5±90.5 (370.4–587.5) L (excluding neck) 417.2 371.5±63.7 (280.0–465.0) W 307.3 288.0±31.4 (225.8–335.0) Neck 107.5 95.9±20.2 (66.1–122.5) L (including neck)/Diam. ratio 1.7 1.7±0.2 (1.4–1.9)

Vulval plate

n 10

Fenestrate length 42.5 48.4±9.0 (40.0–65.0) Semifenestrate length 17.5 21.9±4.6 (16.3–30.0) Fenestrate width 53.75 50.9±5.0 (45.0–62.5) Vulval slit length 47.5 47.0±5.3 (40.0–60.0) Vulval-anal distance 33.75 33.7±4.4 (27.5–42.5) Underbridge 95 91.7.0±6.9 (85.0–105.0)

Female

n 10

L (including neck) 467.1±38.2 (429.0–547.5) L (excluding neck) 378.6±39.2 (340.0–450.0) W 270.8 ±41.9 (204.8–343.0) Neck 98.3±11.0 (85.0–116.4) L (including neck)/Diam. ratio 1.8 ±0.2 (1.3–2.1) Stylet (n=3) 23.4±0.5 (23.0–24.0) DGO (n=2) 6.1±0.2 (1.3, 2.1) Anterior end to median bulb (n=3) 59.8±10.9 (48.8–70.5) Length of median bulb (n=3) 25.9±1.7 (24.2–27.6) Width of median bulb (n=3) 23.4±2.1 (21.4–25.6)

J2

n 20

L 481.5±23.3 (440.0–520.0) a 23.4±1.3 (20.7–26.3) b 4.6±0.5 (3.9–5.2) b' 3.3±0.2 (2.8–3.5) c 6.9±0.4 (6.2–7.6) c' 5.0±0.2 (4.7–5.4) Stylet length 23.2±0.6 (22.0–24.0) Lip height 4.8±0.3 (4.5–5.0) Lip diam. 9.6±0.5 (9.0–10.0) DGO 4.9±0.3 (4.5–5.3) Anterior end to excretory pore 103.3±5.6 (95.0–113.8) Anterior end to median bulb 69.4±3.1 (60.0–73.0) ......continued on the next page Description. Cyst. Lemon-shaped with distinct vulval cone, variable in size. Cuticle light brown to dark brown. Neck prominent, stylet and other pharyngeal structures indistinct. Remnant subcrystalline layer present. Cuticle with irregular zig-zag pattern. Ambifenestrate. Underbridge present, thicker in central part and bifurcate at ends. Bullae absent. Eggs packed in the body. Anus small, but distinct.

White female. Body lemon-shaped, white, covered with subcrystalline layer. Head set off, with two annuli. Stylet strong, with rounded and backwardly sloping knobs. Median bulb massive. Excretory pore indistinct. Egg sac large, 1.5–2 times size of female.

Male. Body vermiform, straight or slightly curved ventrally after relaxation. Labial region hemispherical with a distinct labial disc and four postlabial annuli. Lateral field with four incisures, outer bands areolated. Well developed stylet with rounded basal knobs, sloping posteriorly. Median bulb with strong valve plates. Nerve ring broad, located slightly posterior to median bulb. Hemizonid ca 2 annuli long, situated 7–10 annuli anterior to excretory pore. Tail very short and bluntly rounded with protruding cloacal tubus. Phasmids indistinct. Spicules arcuate, each terminating with a bifurcate tip. Gubernaculum straight and simple.

J2. Body vermiform, straight or slightly ventrally curved after relaxation. Lip region offset, hemispherical, with a labial disc and three or four postlabial annuli. Body annuli 1.8–2.0 μm at mid-body. Lateral field with three incisures. Stylet robust with knobs slightly projecting or flat anteriorly. Median bulb ovoid and prominent. Hemizonid situated slightly anterior to excretory pore. Anus distinct. Tail elongate conical with rounded terminus, distinct hyaline terminal section 41.2 (35.0–45.0) μm long, occupying 58.9 (50.0–62.5)% of tail length. Genital primordium oval, 10.0 (9.5–10.2) μm long, 7.5 (7.2–8.2) μm wide, 200 (195.0–211.3) μm anterior to tail terminus. Phasmids pore-like, 4–7 annuli posterior to anus.

Diagnosis and relationships. Heterodera fengi n. sp. belongs to the Cyperi group. Cysts are characterized by prominent vulval cone with ambifenestrate, bifurcate underbridge that is thicker in middle and a 47.0 (40.0–60.0) μm long vulval slit, but without bullae; Second-stage juveniles are characterized by a 23.2 (22.0–24.0) μm long stylet with slightly projecting or anteriorly flattened knobs, three incisures in lateral field, a 70.2 (62.5–77.0) μm long tail with bluntly rounded terminus and hyaline portion forming 58.9 (50.0–62.5)% of the tail length; Males are characterized by a 25.1 (24.5–26.3) μm long stylet with rounded knobs sloping posteriorly, four incisures in lateral field, a 29.8 (27.5–31.3) μm long spicule with bifurcate tip.

The new species appears morphologically most similar to three species of the Cyperi group, i.e., H. graminophila Golden & Birchfield, 1972, H. oryzae Luc & Brizuela, 1961 and H. graminis Stynes, 1971 . Heterodera fengi n. sp. differs from H. graminophila by more incisures in the male lateral field (4 vs 3), the J2 stylet knob shape (slightly projecting or flat anteriorly vs rounded), the slightly longer J2 tail (62.5–77.0 μm vs 52.0–67.0 μm) and the longer J2 hyaline tail portion (35.0–45.0 μm vs 23.0–38.0 μm); from H. oryzae by the absence of bullae (vs presence), the underbridge ends shape (bifurcate vs dendroid-like), the slightly shorter vulvalanal distance (27.5–42.5 μm vs 33.5–64.0 μm), the slightly longer J2 tail length (62.5–77.0 μm vs 46.2–68.9 μm) and the J2 tail terminus shape (rounded vs pointed); from H. graminis by the longer vulval slit length (40.0–60.0 μm vs 27.0–46.0 μm), the presence of subcrystalline in cyst stage (vs absence), the presence of a large egg sac containing eggs (vs a small egg sac but no eggs), the J2 stylet knob shape (slightly projected or flat anteriorly vs concave), the longer J2 body (440.0–520.0 μm vs 343.0–444.0 μm), the longer J2 tail (62.5–77.0 μm vs 44.0–65.0 μm), the longer J2 hyaline region of tail (35.0–45.0 μm vs 23.0–36.0 μm) and shorter spicule (27.5–31.3 μm vs 31.0–35.0 μm).

The new species is also closely related to other members of the Cyperi group, including H. oryzicola Rao & Jayaprakash, 1978 , H. elachista Ohshima, 1974 , H. pakistansis Maqbool & Shahina, 1986 and H. raski Basnet & Jayaprakash, 1984 . It can be distinguished from H. oryzicola by the absence of bullae (vs presence), the J2 stylet knob shape (slightly projecting or flat anteriorly vs rounded), the longer J2 hyaline region of tail (35.0–45.0 μm vs 22.0–29.0 μm) and the longer spicule (27.5–31.3 μm vs 19.0–25.0 μm); from H. elachista by the absence of bullae (vs presence), the longer J2 body length (440.0–520.0 μm vs 330.0–450.0 μm), the longer J2 stylet (22.0–24.0 μm vs 16.0–21.0 μm) and the longer male stylet (24.5–26.3 μm vs 20.0–21.0 μm); from H. pakistansis by the larger fenestrae length and width (40.0–65.0 × 45.0–62.5 μm vs 25.0–38.0 × 15.0–20.0 μm), the longer J2 stylet (22.0– 24.0 μm vs 16.8–18.0 μm), the smaller J2 DGO (4.5–5.3 μm vs 5.6–6.8 μm), the male stylet basal knob shape (rounded sloping posteriorly vs anteriorly directed) and more postlabial annuli of male (4 vs 3); from H. raski by the shorter cyst (370.4–587.5 μm vs 710.0–835.0 μm), the lower L/W ration of cyst (1.4–1.9 vs 2.7–3.5), the presence of large egg sac in female (vs absent), the longer J2 stylet (22.0–24.0 μm vs 17.5–20.0 μm), the J2 stylet knob shape (slightly projected or flat anteriorly vs rounded), the tail terminus shape of J2 (bluntly rounded vs acutely pointed), the shorter spicule (27.5–31.3 μm vs 33.5–36.0 μm) and longer male stylet (24.5–26.3 μm vs 20.5–22.0 μm).

Compared with the other three species reported from bamboo, namely H. bamboosi (Kaushal & Swarup, 1988) Wouts & Baldwin, 1998 , H. koreana (Vovlas, Lamberti & Choo, 1992) Mundo-Ocampo, Troccoli, Subbotin, Cid, Baldwin & Inserra, 2008 , and H. hainanensis Zhuo, Wang, Ye, Peng & Liao, 2013 , the new species can be easily distinguished from them by the presence of fenestrae (vs afenestrate). In addition, Heterodera fengi n. sp. further differs from H. bamboosi by the presence of a prominent vulval cone (vs absence of vulval cone) and more incisures in the male lateral field (4 vs 3); from H. koreana and H. hainanensis by the longer J2 stylet (22.0–24.0 μm vs 16.0–20.0 μm in H. koreana and 15.8–17.5 μm in H. hainanensis ) and more lip annuli of J2 (3–4 vs 2).

Type-locality and habitat: Heterodera fengi n. sp. was collected from roots and rhizosphere of bamboo ( Phyllostachys pubescens Mazel ), which were collected from Guangzhou City (latitude N23°8’, longitude E113°17’), Guangdong Province, China from 2009 to 2012. The soil type is sandy loam and the local climate is subtropical.

Etymology: The species name is given to show respect to Professor Feng Zhixin for his outstanding contributions to our knowledge of plant nematodes.

Molecular profiles and phylogenetic status. The sequenced LSU D2D3 and ITS region are 787 bp and 1039 bp respectively. A Blastn search of H. fengi n. sp. on the LSU D2D3 revealed high-scoring matches with some Heterodera species, the most similar being with Heterodera sp. GX665 (GenBank accession number JX 081322 ), which is an unidentified species isolated from the rhizosphere of an unknown weed in Guangxi Province in China. The identities of these two sequences are 96.9% (756/787) with five insertions/deletions (0.6%). Compared with H. elachista ( HM560842 ), a member of the Cyperi group, H. fengi n. sp. has 28 variable sites with 96.8% identity (756/784) and five insertions/deletions (0.6%). A Blastn search of H. fengi n. sp. on the ITS region also revealed similarities with some Heterodera species, but the identities were less than 80%. The highest match was H. elachista ( JN202917 ) with only 79.2% identity (920/1161) and 109 insertions/deletions (9.4%). Compared with Heterodera sp. GX665 (GenBank accession number JX 081325 ), H. fengi n. sp. has 126 variable sites with 74.5% identity (863/1158) and 169 insertions/deletions (14.6%).

The molecular phylogenetic relationships of the new species are shown in Figures 4 and 5. Figure 4 represents a phylogenetic tree based on LSU D2D3 from a multiple alignment of 673 total characters with 417 constant characters (62.0%). The average nucleotide composition is as follows: 17.31% A, 22.31% C, 34.53% G and 25.86% T. When using Cryphodera brinkmani Karssen & van Aelst, 1999 as the outgroup taxon, the molecular phylogeny strongly supports monophyly of Heterodera (100% posterior probability). H. fengi n. sp. is placed in a 95%-supported monophyletic clade with H. elachista in the Cyperi group. These species form a 100%-supported monophyletic clade with H. oryzicola , another member in the Cyperi group, and Heterodera sp. GX665.

Figure 5 represents a phylogenetic tree based on ITS-rRNA from a multiple alignment of 1120 total characters with 411 constant characters (36.7%). The average nucleotide composition is as follows: 16.99% A, 23.31% C, 30.52% G and 29.18% T. Using H. circeae Subbotin & Sturhan, 2004 as the outgroup taxon, this tree inferred two main clades. H. fengi n. sp. is also close to H. elachista and other species including H. oryzicola , H. cyperi and H. mothi belonging to the Cyperi group with 100% support.

The RFLP-ITS-rRNA profile obtained for H. fengi n. sp. is presented in Figure 7 and Table 2. This RFLP-ITS pattern distinguished H. fengi n. sp. from RFLP profiles of other known Heterodera species, as shown in Subbotin et al. (2010b), especially from other species of the Cyperi group, including H.cadiolata , H. cyperi , H.elachista , H. mothi , and H. oryzicola ( Table 2). A size comparison of fragments indicates that the enzyme Ava I is the most useful to distinguish the new species from these five Heterodera species.

FIGURE 5. The 10001st Bayesian tree inferred from D2D3 of 28S rDNA under GTR+I+G model (lnL=4226.0581; AIC=8472.1162; freqA=0.1731; freqC=0.2231; freqG=0.3453; freqT=0.2586; R(a)=0.4768; R(b)=3.1814; R(c)=1.3864; R(d)=0.236; R(e)=6.0188; R(f)=1; Pinva=0.3884; Shape=0.6589). Posterior probability values exceeding 50% are given on appropriate clades.

FIGURE 6. The 10001st Bayesian tree inferred from ITS-rRNA under GTR+I+G model (lnL=10678.832; AIC=21377.6641; freqA=0.1699; freqC=0.2331; freqG=0.3052; freqT=0.2918; R(a)=1.0035; R(b)=4.0744; R(c)=1.9286; R(d)=0.5549; R(e)=3.4884; R(f)=1; Pinva=0.2339; Shape=1.8049). Posterior probability values exceeding 50% are given on appropriate clades.

AB28 for Heterodera fengi n. sp. and five other Heterodera species within the Cyperi group.

Species Unrestricted Alu I Ava I Bsh 1236I Bsu RI

(Bst uI) (Hae III)

H. fengi n. sp. 1139 592,351,192 647,492 443,250,218, 474,252,

101,26 213,200

H.cadiolata 1025 437,387,201 1025 600,275,150 318,224,182,141, 80,32,24,13,11

H.cyperi 1103 398,326,193, 1103 753,219,131 455,253,193,97, 148,38 52,29,24

H.elachista 1074 338,325,187, 1074 319,220,205, 552,236,183, 179,28,17 201,129 79,24

H.mothi 1087 291,246,145, 1087 430,316, 350,246,195, 117,110,109,69 212,129 191,81,24

H.oryzicola 1010 326,295,203, 1010 320,276,195,131,8 358,354,194, *Fragments obtained in results of virtual sequence digestion using Webcutter2 (http://rna.lundberg.gu.se/cutter2/). Bold font-verified by PCR-RFLP study; normal font-not PCR-RFLP verified.

Figures

FIGURE 1. Heterodera fengi n. sp. A: Cysts; B: Anterior region of white female; C: Vulval cone; D: Pharyngeal region of J 2; E: Anterior region of J 2; F: Lateral field and tail of J 2; G: Pharyngeal region male; H: Anterior region of male; I: Lateral field and tail of male.

FIGURE 2. Cysts and females of Heterodera fengi n. sp. under light microscrope. A: Female (arrow) parasitizing roots of bamboo; B: Female with large egg sac; C: Eggs in female; D: Lip region of female; E: Stylet knobs and DGO of female; F-G: Median bulb of females; H: Entire cysts; I: Cuticle pattern of cyst in vulval region; J-L: En face view of vulval cone; K-M: Same as J and L, respectively, deeper level, with underbridge. (Scale bar: A, H = 100 μ m; B = 200 μ m; C = 100 μ m; D-G, I-M = 10 μ m.).

FIGURE 3. Males of Heterodera fengi n. sp. under light microscope. A: Entire body; B: Pharyngeal region; C: Lateral lines in the middle body; D: Lateral lines in the posterior body. E – F: Tail. (Scale bar: A = 100 μ m; B – F = 20 μ m.).

FIGURE 4. J 2 s of Heterodera fengi n. sp. under light microscope. A: Entire body; B: Pharyngeal region; C-D: Anterior region; E: Lateral lines; F: Tail. (Scale bar: A = 100 μ m; B, F = 20 μ m; C-E = 10 μ m.).

FIGURE 7. RFLP-ITS-rRNA pattern of Heterodera fengi n. sp. Lanes: M = 100 bp DNA ladder (NEB); 1 = ITS-rRNA amplification product; 2 = Alu I; 3 = Ava I; 4 = Bsh 1236 I (Bstu I); 5 = BsuRI (Hae III); 6 = Cfo I (Hha I); 7 = Mva I; 8 = Rsa I (Afa I).