Research Article |
Corresponding author: Francesca De Luca ( francesca.deluca@ipsp.cnr.it ) Academic editor: Sergei Subbotin
© 2019 Elena Fanelli, Alessio Vovlas, Simona Santoro, Alberto Troccoli, Giuseppe Lucarelli, Nicola Trisciuzzi, Francesca De Luca.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Fanelli E, Vovlas A, Santoro S, Troccoli A, Lucarelli G, Trisciuzzi N, De Luca F (2019) Integrative diagnosis, biological observations, and histopathology of the fig cyst nematode Heterodera fici Kirjanova (1954) associated with Ficus carica L. in southern Italy. ZooKeys 824: 1-19. https://doi.org/10.3897/zookeys.824.26820
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Morpho-biological notes and histopathology, based on LM and SEM observations, of the fig cyst nematode Heterodera fici isolated from Ficus carica roots, collected in home and public gardens of Apulia region, southern Italy, are described and illustrated. Seventy-five localities throughout the Apulia region were sampled and one-quarter of the sampled localities had fig roots infested with H. fici, with population densities ranging from 44 to 180 cysts/100 ml of soil. All attempts to detect H. fici on ornamental Ficus spp. as well as on imported bonsai in Italy were unsuccessful. Morphometric characters of the Italian population conform to those of the type and re-description populations reported for H. fici. Molecular analysis using ITS, D2–D3 expansion domains of the 28S rRNA, and the partial 18S rRNA sequences of H. fici newly obtained in this study matched well with the corresponding sequences of H. fici present in the GenBank database. Phylogenetic trees confirmed and supported the grouping of H. fici in the Humuli group. Heterodera fici completes its embryogenic development in 14–16 days at 25 °C. Post-invasion development and maturity in the roots of F. carica seedlings is completed in 64–68 days at 25–28 °C with juveniles and adults showing different parasitic habits, being endoparasitic and semi-endoparasitic respectively. The establishment of permanent feeding sites that consist of the formation of large syncytia causes anatomical modification of vascular elements and general disorder in the root stelar structures. Syncytia structures associated with mature females showed different degrees of vacuolisation, numbers of syncytial cells, and contained nuclei and nucleoli which were constantly hypertrophied.
cyst-forming nematode, embryogenesis, histopathology, identification, phylogeny, SEM morphology
The fig cyst nematode Heterodera fici Kirjanova, 1954 was described from roots of rubber plants, Ficus elastica Roxb. ex Hornem from China by
Preliminary investigations indicated that H. fici was rather widespread in southern Italy and in particular in the Apulia region. The main objectives of the present research were to: (i) obtain additional information on distribution, morphology, and molecular details of H. fici from F. carica, (ii) establish the phylogenetic relationships among Heterodera species closely related to H. fici; (iii) obtain additional biological information (embryogenic and post-invasion development); and (iv) to provide morpho-biological details on the host-parasite relationships of this nematode species in fig-nematode-feeding sites and describe the F. carica host responses to the nematode parasitism.
In the framework of the project “Urban Phytonematology” and on the basis of occasional records of H. fici on edible figs (Ficus carica), an extensive survey, including more than 75 root and soil samples, was conducted in commercial orchards in several localities of the Apulia region and in private and public gardens of Bari city, southern Italy, in late May 2016. Twenty-five bonsai Ficus spp. from several import/export nurseries working with Asiatic Ficus plants, still in their original pots, were included in the present survey. The nematode population selected and used for the present study was collected at Bari University Campus (40°06'72"N, 16°52'54"E).
For diagnosis and identification, cysts and eggs were collected directly from infested roots, whereas second-stage juveniles, mature cysts, and males were extracted from soil by the flotation method (
Terminal cone structures of mature cysts were prepared for scanning electron microscopy (SEM) observations. Specimens fixed in formaldehyde (4% solution) were dehydrated in an ethanol gradient, critical-point dried, sputter-coated with gold, and observed according to procedures described by
Individual cysts were crushed with a sterile micro-spatula under a stereo-microscope and the second stage juveniles (J2) were collected. Genomic DNA was extracted from fifteen J2s as described by
PCR products of the ITS containing region, the 18S rRNA gene and the D2-D3 expansion domains from three individual nematodes were purified using the protocol given by the manufacturer (High Pure PCR elution kit, Roche, Germany). Purified DNA fragments were cloned and sequenced, in both directions, at MWG-Eurofin in Germany.
Ten μl of the ITS containing amplicons of H. fici from southern Italy were digested with the following restriction enzymes: Alu I (Roche), Hae III (Roche), Pst I (Roche) and Rsa I (Roche) (5 U of enzyme for each digestion) at 37 °C overnight. The digested DNA fragments were loaded onto 2.5% agarose gel and visualized by gel red staining gel. All gel images were stored digitally.
A BLAST (Basic Local Alignment Search Tool) search at NCBI (National Center for Biotechnology Information) was performed in order to confirm nematode origins and species (
The embryogenic development of H. fici was studied in Petri dishes, using single-celled eggs obtained (deposited) from newly formed cysts, washed in distilled water, placed in 2% water agar, and maintained in an incubator at 26 ± 2 °C. Microscopic observations and micrographs were taken at six-hour intervals during the first week and daily for the second week.
The duration of post-invasion development was determined on fig (F. carica) seedlings transplanted in pots containing 250 ml of pasteurised sand, and inoculated, five days later, by using 1250 juveniles per pot. Inoculated young plants were maintained in a glasshouse at 26–28 °C. Invasion and nematode development was studied by stereoscope observations in acid fuchsin stained roots at seven-day intervals (Fig.
Host studies were made in a glasshouse at 26–28 °C using common Mediterranean fruit trees as possible hosts (almond, apple, orange, edible and wild fig, apricot, grapevine, loquat, walnut, and pistachio) and transplanted on naturally infested soil with estimated initial population 1500 juveniles + eggs per pot and exposed to the nematode for a three-month period. Plants were removed from pots, the roots washed free of adhering soil, and the nematode populations were recorded.
The histological changes induced by H. fici were studied in nematode-infected fig roots. Infected and healthy root segments were fixed for 48 hours in formalin-acetic acid-ethanol (FAA) solution, dehydrated in tertiary butyl alcohol, and embedded in 56–58 °C melting-point paraffin. Embedded tissues were sectioned transversely and longitudinally in 10–12 µm thick with a rotary microtome, stained with safranin and fast-green, and mounted in dammar xylene for microscopic examinations (
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.
Measurements. See Table
Morphometrics of an Italian population of Heterodera fici isolated from roots and soil around roots; specimens mounted in water agar temporary slides (Measurements are in µm).
Character* | Females | Cysts | Males | juveniles |
---|---|---|---|---|
n | 15 | 16 | 12 | 20 |
Body length (excl. neck) (L) | 355–638 | 420–680 | 750–880 | 330–416 |
Max. body width (W) | 230–422 | 280–550 | 25–26 | 17–20 |
L/W | 1.2–1.5 | 1.1–1.5 | ||
Neck length | 75–126 | – | ||
Fenestral length | – | 46–72 | ||
Fenestral width | – | 28–46 | ||
Vulva slit | – | 35–44 | ||
Bullae | – | Present, small | ||
Stylet length | 23–28 | – | 27–31 | 20–22 |
Dorsal gland orifice (DGO) | 4–5 | – | 5–6 | 4–5 |
Anterior end to centre of median bulb | 60–65 | – | 80–106 | 64–76 |
Excretory pore from anterior end | 132–138 | – | ||
Head to end of pharyngeal gland lobe | 200–275 | 136–156 | ||
Tail length | 7.5–8.2 | 39–52 | ||
Hyaline tail portion | – | 18–24 | ||
No of lines in lateral fields | 4 | 4 | ||
Spicule length | 26–32 | – | ||
Gubernaculum | 7–8 | – | ||
a | 30–38 | 17–22 | ||
b | 2.5–4.5 | 2.4–3.2 | ||
c | 102–128 | 8–9 |
Observing the morphology (Figs
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.
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
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
LM micrographs of Heterodera fici from Italy. A Embryonated egg with evident second stage juvenile stylet B Second stage juvenile anterior end C Second stage juvenile tail D Male tail with the characteristic tail tip E Females on Ficus carica roots F whole body of newly formed cyst G Females and cysts H-J Vulval cone structures, with clear illustration of vulval slit (in H), fenestral area (in I) and bullae (in J). Scale bars: 20 µm (A-D, H-J); 200 µm (F); 500 µm (E, G).
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.
Embryogenesis. Mean dimensions of single-celled and embryonated eggs were 40 × 98 µm (Fig.
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.
Post invasion development of Heterodera fici on Ficus carica roots. A Second stage juvenile within cortical layer, oriented in parallel position to the root axis B Newly formed cyst with gelatinous egg sac C Male inside 4th stage cuticle D Different sized syncytia (S) induced by female (the larger one), and by male. Abbreviations: j = juvenile; m = male; c = cyst; hn = hypertrophied nuclei. Scale bars: 50 µm (A, C); 200 µm (B); 100 µm (D).
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.
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 (
Phylogenetic trees using the ML method are given in Figures
Our study reports the occurrence of H. fici in commercial fig trees in southern Italy showing no symptoms of retarded growth or yellowing of leaves related to nematode presence. The morphology (Fig.
ITS-RFLP patterns of H. fici from South Italy showed no heterogeneity or differences to the published RFLP patterns (
In conclusion, our data confirm the occurrence of H. fici in two regions of southern Italy, Apulia and Basilicata, and on commercial fig orchards, approximately thirty years old, suggesting that this nematode despite its narrow host range is widespread all over the world and that it deserves attention.
Authors acknowledge Nicola Vovlas for critical review of and suggestions to the manuscript before submission.