Heterodera fici Kirjanova, 1954

Fanelli, Elena, Vovlas, Alessio, Santoro, Simona, Troccoli, Alberto, Giuseppe Lucarelli,, Trisciuzzi, Nicola & Luca, Francesca De, 2019, Integrative diagnosis, biological observations, and histopathology of the fig cyst nematode Heteroderafici Kirjanova (1954) associated with Ficuscarica L. in southern Italy, ZooKeys 824, pp. 1-19 : 1

Heterodera fici Kirjanova, 1954 Figures 1, 2, 3, 4

Nematode population.

The fig cyst nematode H. fici was recovered in our survey in established commercial fig orchards (more than 30 years old) as well as in private and public gardens. High infection rates were observed ranging from 44 to 180 cycts/100 ml of soil, 12-36 cysts per g of roots; 1.2-1.6 eggs - J2 /ml of soil, thus suggesting that the nematode might be causing damage. Furthermore, H. fici was detected in one-quarter of the 75 localities sampled throughout the Apulia region. All sampling attempts to detect H. fici from ornamental Ficus spp. as well as from imported bonsai in Italy were unsuccessful.

Description.

Measurements. See Table 1. Nematode diagnosis. Detailed morphometric observations of the Italian population based on second-stage juveniles, male body length and characteristic of tail, stylet length, adult female and cyst shape, and vulval cone features (Figs 1-4, Table 1) agree very well with most of the original morphometric data and the redescription.

Observing the morphology (Figs 1-4), as well the metric data of the Italian population (Table 1), together with molecular comparison, we conclude that our H. fici belongs to the Humuli group and is distinguished from similar species by a combination of morphological and morphometric characteristics; it differs from all other members of the Humuli group ( H. humuli Filipjev, 1934, H. ripae Subbotin, Sturhan, Rumpenhorst & Moens, 2003, H. vallicola Eroshenko, Subbotin & Kazachenko, 2001, and H. litoralis Wouts & Sturhan, 1996 by ambifenestrate rather than bifenestrate cysts and a longer vulval slit (43-48 vs. <40 μm), and by the prominent nipples on the male tail tip, which are regularly annulated and obtusely rounded in all other members of the group.

Females. Body basically lemon-shaped. Neck elongate, protruding vulval cone prominent. Cyst cuticle with zigzag pattern. Vulval cone well developed. Egg sac present, but few eggs deposited. Cuticular striae, extending to vulval slit are present at fenestral area (Fig. 2H).

Cysts. Body light to dark brown, basically lemon-shaped, neck and vulval cone distinct. Neck protruding, curved laterally. Cuticle thin, without sub-crystalline layer. External wall pattern at mid-body with interlocking ridges, forming zigzag pattern. Terminus of vulval cone with strongly developed zigzag ridges surrounding vulval slit and fenestra. Fenestra ambi-fenestrate, vulval slit equal in length to bridge. Few but distinct bullae are present. Anus distinct, on a depressed area surrounded by continuous cuticle edge/margin (Figs 2 F–J, 3F).

Males. Body slender, vermiform, slight ventral curvature. Cuticular annulation prominent. Lateral field areolated, with four incisures. Labial region slightly offset, hemispherical, with three or four annuli. Labial framework heavily sclerotised. Tail short, obtusely rounded, four prominent nipples on tail tip. Spicules arcuate, tapering distally. Gubernaculum slightly curved ventrally (Figs 2D, 3G).

J2 (Second stagejuveniles). Body vermiform; tapering at both extremities, more marked posteriorly. Cuticular annulation prominent. Lateral field with four incisures. Labial region slightly offset, rounded, with three or four annuli. Labial framework moderately sclerotised. Stylet well developed, basal knobs rounded, directed slightly anteriorly. Median bulb ovoid. Pharyngeal lobe usually distinct, with three large nuclei and overlapping anterior part of intestine. Tail long, tapering, terminus rounded. Anus distinct. Phasmid openings small but distinct, 11-14 μm, posterior to anus and anterior to middle of tail. Hyaline tail region half of tail length (Fig. 2B, C).

Embryogenesis. Mean dimensions of single-celled and embryonated eggs were 40 × 98 µm (Fig. 2A). The first cleavage was equatorial and the two-blastomeres stage appeared after 14-18 hours. The second and third divisions were also transverse and the four-cell stage phase was obtained after three days. Forty-eight hours later, rapid cell division resulted in the formation of multicell eggs. The gastrula stage was observed during days 9-12. First and second-stage juveniles appeared in 11-13 and 14-16 days, respectively, and were coiled three or four times within the eggshell. The embryogenic development was basically the same as in H. schachtii Schmidt, 1871 and H. mediterranea ( Mulvey and Golden 1983, Vovlas and Inserra 1983).

Post-invasion development. Post root-invasion development of H. fici on Ficus carica roots was completed in about 64-68 days at 26-28 °C. Invasion and nematode development stages were recorded by stereoscope observations on acid fuchsin-stained roots at seven-day intervals (Fig. 4). The maximum developmental phase observed was that of young cysts containing more than 25 eggs. The first period (24-26 days after inoculation) was utilized by the parasitic specimens for exploration, penetration, and selection of the feeding site (Fig. 4A). The second stage juveniles stop penetration and start to feed on the initial feeding cells by using a sedentary endoparasitic feeding position. Observations at 50-56 days after inoculation revealed that the 3rd moult was completed and that sexual differentiation occurred. The swollen posterior portion of sexually mature nematodes (piriform females and 4th stage vermiform males still inside the cuticle) protrudes from the root. Fourth-stage females and males were observed one week later (Fig. 4C). Lemon-shaped mature females were observed eight weeks after invasion. The post infection period was concluded about 8 weeks later. No new cysts were observed.

Histopathology. Heterodera fici establishes permanent, fully-developed feeding sites on F. carica roots and reaches maturity in 64-68 days. Histological examination of sectioned healthy and nematode-infected (Fig. 4) fig roots showed that infection by H. fici can cause cellular alterations in the cortex, endodermis, pericycle, and vascular parenchyma tissues of fig roots. Observations of cross-sections of H. fici infected roots indicated that the nematode can induce the formation of both cortical and endodermal syncytia. In many cases, the nematode female only penetrated one to three layers of the cortical root cells without reaching the stele. In these roots, the nematode established a permanent feeding site in a cortical cell that was fused with adjacent cells forming the syncytium. Structurally, vascular syncytia varied in shape and expansion when induced by females or males. Those induced by females were larger and with more cytoplasmic contents compared to those induced by males. All syncytial cells were hypertrophic with dense cytoplasm and large nuclei. In some cases, nematode females penetrated into the cortex with the anterior elongated body portion where they induced a permanent feeding site and continued to remain in a semi-endoparasitic position, probably because of the nature of root tissues (woody host).

Host-suitability test. The results presented in this paper as well as in the host-suitability test confirmed that H. fici has a narrow host range limited to Ficus species and for this reason it is not considered a major pest, as are other species of Heterodera .

Molecular characterization. The sequenced ITS, D2-D3 expansion domains of the 28S rRNA gene, and the 18S rRNA gene are 1035 bp, 780 bp, and 1627 bp long, respectively. BLAST search at NCBI revealed that the ITS, D2-D3 expansion domains of the 28S rRNA and the partial 18S rRNA sequences of H. fici from Italy, newly obtained in this study, matched well with the corresponding sequences of H. fici present in the database. The RFLP patterns of Italian H. fici were identical to those from Portugal, Greece, and another population from Potenza province, Basilicata, Italy ( Madani et al. 2004) (Fig. 5). In particular, the ITS region of the Italian population of H. fici showed 99% similarity (1027/1029) with the corresponding region of H. fici deposited in GenBank, differing from 1 to 4 nucleotides; the next closest Heterodera species were H. humuli (coverage: 897/962), H. vallicola (coverage: 899/975), H. ripae (coverage: 894/972), and H. litoralis (coverage: 884/962) with 92-93% similarities. Heterodera species belonging to Schachtii group, instead, showed about 82% similarity with the Italian population of H. fici . The D2-D3 expansion domains of the Italian H. fici , obtained for the first time in this study, showed 95% similarity with the corresponding region of H. latipons Franklin, 1969 and H. avenae Wollenweber, 1924, while with H. glycines Ichinohe, 1952 and H. schachtii the similarity was 94%. The 18S rRNA gene of H. fici showed 99% similarity with the corresponding region of H. avenae , H. hordecalis Andersson, 1975, H. schachtii , and H. glycines , while its similarity with H. elachista Ohshima, 1974 was 98%.

Phylogenetic trees using the ML method are given in Figures 6-8. ITS, D2-D3, and 18S trees confirmed the separation of the genus Heterodera into different groups ( Subbotin et al. 2010, De Luca et al. 2013). In particular, the Italian population of H. fici grouped within the Humuli group in all phylogenetic trees, closely related to the Schachtii group.