Research Article |
Corresponding author: Olivera Topalovic ( olivera.topalovic.best@gmail.com ) Academic editor: Sergei Subbotin
© 2017 Olivera Topalovic, John F. Moore, Toon Janssen, Wim Bert, Gerrit Karssen.
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:
Topalović O, Moore JF, Janssen T, Bert W, Karssen G (2017) An early record of Meloidogyne fallax from Ireland. ZooKeys 643: 33-52. https://doi.org/10.3897/zookeys.643.11266
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Root-knot nematodes, Meloidogyne spp., cause huge economic losses worldwide. Currently, three Meloidogyne spp. are present on the quarantine A2 list of EPPO, M. chitwoodi, M. fallax and M. enterolobii. As a quarantine organism, M. fallax has been detected in England and Northern Ireland on sport turf in 2011, and in England on leek in 2013. However, its presence in Ireland has probably been overlooked since 1965, when Mr. John F. Moore and Dr. Mary T. Franklin had detected a new Meloidogyne species for that time. While the relevant data was recorded and a preliminary manuscript describing the species was prepared but never submitted for publication, and together with the original slides, pictures and drawings, it was restudied recently. We compared the population of Irish Meloidogyne sp. to other similar Meloidogyne spp. Careful observation and comparison shows that it belongs to M. fallax. The characters found to be common for Irish Meloidogyne sp. and M. fallax are female stylet length (14.6 μm) with oval to rounded basal knobs, oval shaped perineal pattern with moderately high dorsal arch, slender stylet in males (18.5 μm) with set off and rounded basal knobs, slightly set off male head with one post-labial annule and incomplete transverse incisures, and second-stage juveniles with large and rounded stylet basal knobs, and a gradually tapering tail (46.9 μm) with a broadly rounded tip and a clearly delimitated smooth hyaline part sometimes marked by constrictions (12.9 μm). The host test and gall formation also correspond to M. fallax. The identification could not be additionally supported by molecular analysis, as we were unable to extract DNA from the old permanent slides. Nevertheless, our study reveals that the Meloidogyne species detected in Ireland in 1965 belongs to M. fallax.
Root-knot nematode, Cork, morphology, morphometrics, host, characters
Nematodes belonging to Meloidogyne spp. are among the most dangerous plant-parasitic nematodes worldwide and cause huge economic losses (
The main goal of our study was to compare the available original material of the population of Meloidogyne sp. detected in 1965 in Ireland to the type material of other similar Meloidogyne spp. Additionally, we tried to extract the DNA from the permanent slides originating from 1965 and 1966.
In 1995, the Dutch NPPO received the original permanent slides of 23 whole females, 18 males, 27 second-stage juveniles and 6 female perineal patterns, including pictures, measurements and an unpublished manuscript. In 2005, all slides were re-mounted in glycerol.
Morphological observations of glycerine-embedded permanent slides of Irish Meloidogyne sp. were done using a compound light microscope (Zeiss Axio Imager 2). Pictures were obtained using a Leica DFC 450 digital camera. A compound light microscope (DM 2500, LEICA) equipped with differential interference contrast (DIC) was used for making drawings. Drawings and pictures were subsequently edited using GNU Image Manipulation Program (http://gimp.org). Permanent slides of the Irish Meloidogyne population were compared to type material (slides & living type populations) and reference populations, of similar Meloidogyne spp. (Table
Populations of Meloidogyne spp. used for comparison to the original slides of an unknown Irish species.
Species | Material | Number | Male | Female | J2 |
---|---|---|---|---|---|
M. chitwoodi | Type slides | (WT2076-WT2079) | 2 paratypes | 4 PP paraytpes | 26 paratypes |
Reference live material | E7149 | 31 | / | 31 | |
M. fallax | Type slides | WT3127-WT3130 | 2 paratypes | 2 PP paratypes | 5 paratypes |
Type live material | E6147 | 30 | / | 30 | |
M. minor | Type slides | WT3371-WT3374 | 2 paratypes | 2 PP paratypes | 5 paratypes |
Type live material | F714-4 | 27 | / | 30 | |
M. hapla | Reference live material | C3093 | / | / | 23 |
M. incognita | Reference live material | Rgi-23/42 | 30 | / | 30 |
The original manuscript from 1966 describes in detail the conducted host range test of the Irish Meloidogyne sp.: Infested soil from the original site was placed together with a potential host plant species (seed/plant transplant) in 4-inch earthenware pots which were maintained in a glasshouse. The plant species used for the host range test are listed in Table
As only permanent slides of the Irish Meloidogyne sp. originating from 1966 were available, we attempted to extract the DNA from fixed nematodes based on
For amplification of a 120 bp region of COX1 gene, the forward primer, JB3 (5’-TTTTTTGGG CATCCTGAGGTTTAT-3’) (
Morphological characters used for comparison in this study were selected according to
Body shape and perineal pattern
Females of Irish Meloidogyne sp. show oval to pyriform shape.
The perineal pattern of females of Irish Meloidogyne population was used for comparison according to
Stylet
The stylet is slender with dorsally curved shaft. Stylet knobs are large, oval to rounded, slightly backwardly sloping, which corresponds to the original description of M. fallax (
Differences in the stylet knob shape in females of compared Meloidogyne spp.
M & F + our observations | M. fallax (t.l.m. + o. d.) | M. chitwoodi (r.l.m. + o. d.) | M. minor (t.l.m. + o. d.) | M. hapla (o. d.) | M. incognita (o. d.) |
---|---|---|---|---|---|
Large, rounded | Large, rounded | Small, irregular | Large, ovoid | Small, rounded | Large, broadly elongate |
Second-stage juveniles
The stylet knobs shape, tail shape and hyaline tail terminus are used for morphological observations of second-stage juveniles according to
Stylet knob shape
Mr. Moore and Dr. Franklin described a slender stylet with rounded basal knobs. In specimens where it was possible to see, we observed large, rounded, set-off basal knobs that are characteristic for M. fallax (see Table
Comparison of the most important morphological characters in second stage juveniles of the studied Meloidogyne spp.
Irish Meloidogyne sp. (M. & F. + our observ.) | M. fallax (type material + o. d.) | M. chitwoodi (type slides/r. l. m. + o. d.) | M. minor (type material + o. d.) | M. hapla (r. l. m. + o. d.) | M. incognita (r. l. m. + o. d.) | |
---|---|---|---|---|---|---|
Stylet knob shape | Large, rounded | Prominent, rounded, set off | Small, irregular, sloping backwardly | Ovoid, slightly backwardly sloping | Small, rounded | Rounded, set off to transversely elongated, may indent anteriorly |
Tail shape | Rounded to broadly rounded, gradually tapering until hyaline part | Gradually tapering until hyaline terminus, bluntly rounded tip | Conical, narrowly rounded tip | Gradually tapering until finely pointed tail tip, rectum weakly inflated | Short, narrow, difficult to delimitate it from hyaline region | Slightly tapering to subacute terminus |
Hyaline tail terminus | Clear, rounded delimitation to the anterior, broadly rounded at the tip, sometimes with constrictions | Clearly delimitated, smooth hyaline part ending in a broadly rounded tip, faint constrictions | Short, clear rounded delimitation at the anterior end, narrowly rounded tip | Long, pointed terminus, rounded delimitation at the anterior region | Short, often irregularly shaped, delimitation at the anterior region difficult to observe | Pointed tip, clear delimitation at the anterior region |
Tail shape and hyaline tail terminus
Mr. Moore and Dr. Franklin observed a rounded tail with a clear hyaline tail terminus which is occasionally “swollen”. Based on our observations (Table
The comparison of tail and hyaline tail terminus shape in second-stage juveniles, lateral position. A an unknown Irish species B type material of M. fallax C type material of M. chitwoodi D type material of M. minor E reference material of M. hapla; F reference material of M. incognita. Scale bar = 20 µm.
Our observations disagree with those of Mr. Moore and Dr. Franklin regarding the hemizonid position; it is located at the same position of the excretory pore rather than 1-2 annules above the excretory pore as they described. The position of the hemizonid at the same level of the excretory pore is characteristic for second-stage juveniles of M. fallax (
Males
Stylet knob and head shape are considered the most important characters for male identification according to
Stylet knob shape
As presented in Table
The comparison of anterior region in males of populations of observed Meloidogyne species. A an unknown Irish species (ventral position) B type material of M. fallax (lateral position); C: type material of M. chitwoodi (ventral position) D reference material of M. hapla (lateral position) E reference material of M. incognita (lateral position) F type material of M. minor (lateral position). Scale bar = 20 µm.
M & F + our observations | M. fallax (type material + o. d.) | M. chitwoodi (type slides/r. l. m. + o. d.) | M. minor (type material + o. d.) | M. hapla (r. l. m. + o. d.) | M. incognita (r. l. m. + o. d.) | |
---|---|---|---|---|---|---|
Stylet knob shape | Large, rounded, set off from the shaft | Large, rounded, set off from the shaft | Smaller, oval to irregularly shaped, backwardly sloping | Larger, ovoid, slightly backwardly sloping | Small, rounded, slightly backwardly sloping | Oval, angle between the shaft and knobs is more than 90° |
Head shape | Labial disc elevated, head slightly set off with a post- labial annule, sometimes with an incomplete transverse incisure, as seen from the lateral view | Labial disc rounded and elevated, head slightly set off, one post-labial annule often with an incomplete transverse incisure | Labial disc not elevated, head not set off, no transverse incisures subdividing a single post-labial annule | Labial disc elevated, head not set off, one post-labial annule often with 1-2 transverse incisures | Labial disc elevated, head swollen, no transverse incisures on a post-labial annule | Labial disc not elevated, head slightly set off, incomplete transverse incisure on a post-labial annule |
Head shape
Mr. Moore and Dr. Franklin described three annules in lateral view of the head. The first one is deeply pinched off and succeeded by two other faintly seen annules. Our observations resemble the male head shape of type and reference material of M. fallax. It is described as a slightly set off with a single post-labial annule usually subdivided with a transverse incisure (
Males
The stylet length and stylet knob width, the most relevant morphometrical characters of males, were measured for populations of all observed species. Table
Morphometrical analysis of most important characters in females, males and second-stage juveniles {mean ± SD (range), all measurements in µm}.
M. incognita (r. l. m.) | / | 20.2±2.1 (18.0–25.0) | 4.1±0.8 (3.0–6.0) | 379.2±20.0 (340.0–435.0) | 55.0±2.9 (48.0–61.0) | 12.2±1.7 (9.0–15.0) | |
M. hapla (r. l. m.) | / | / | / | 364.2±31.3 (300–410) | 41.1±6.8 (31.0–50.0) | 8.8±1.2 (6.5–11.0) | |
M. minor | type slides | / | 18.7±0.7 (17.0–20.0) | 4.0±0.3 (3.0–4.5) | 369.0±32.5 (280–410) | 52.8±4.4 (46.0–62.0) | 16.9±1.6 (14.0–20.0) |
t. l. m. | / | 17.0±0.0 | 4.0±0.0 | 347.8±17.4 (331.5–372.3) | 49.0±3.3 (45.5–53.0) | 13.8±1.9 (11.5–16.5) | |
M. chitwoodi | r. l. m. | / | 18.3±0.7 (17.0–19.0)) | 3.75±0.3 (3.0–4.0) | 371.9±15.9 (330–400) | 44.0±2.6 (40.0–49.0) | 12.2±1.0 (10.0–14.0) |
Type slides | / | 18.0±0.0 | 3.75±0.3 (3.5–4.0) | 371.5±10.5 (350–385) | 39.9±2.3 (36.0–43.5) | 9.6±0.8 (8.0–11.0) | |
M. fallax | t. l. m. | / | 19.4±0.7 (18.0–21.0) | 4.6±0.4 (3.5–5.0) | 384.3±22.3 (330–420) | 47.9±2.6 (41.0–54.0) | 13.4±1.3 (10.5–15.0) |
Type slides | / | 18.7±0.3 (18.5–19.0) | 4.2±0.3 (4.0–4.5) | 347±7.5 (340–360) | 45.4±2.2 (43–49) | 13.1±0.8 (12.0–14.0) | |
Irish Meloidogyne sp. (our observ.) | 14.6±0.5 (14.0–15.0) | 18.5±1.1 (17.0–20.0) | 3.9±0.5 (3.0–4.5) | 358.6±27.6 (280–410) | 42.0±3.7 (33–50) | 11.3±1.8 (8.5–15.5) | |
Irish Meloidogyne sp. (M & F) | 18.0±2.4 (15.4–24.6) | 19.5±1.5 (17.0–24.6) | / | 406.1±16.1 (361.5–432.0) | 46.9±2.5 (43.0–52.3) | 12.9±1.8 (9.2–15.4) | |
Character | Female stylet length | Male stylet length | Male stylet knob width | J2 body length | J2 tail length | J2 hyaline tail length |
Second-stage juveniles
The body length, tail length and hyaline tail length are considered the most reliable for morphometrical observation of second-stage juveniles. The body length range in our observations of Irish slides (280–410 µm) is narrower than observed by Mr. Moore and Dr. Franklin (361.5–432 µm) (Table
Females
Although the female stylet length measured by Mr. Moore and Dr. Franklin is included in our study, it is considered unreliable as the length was measured from the anterior body end and not from the stylet tip. Therefore, the stylet length of the Irish Meloidogyne sp. was compared to the one originally described for species used for comparison in this study. Based on our measurements (Table
Host test
The host-range test for Irish Meloidogyne sp. included both weeds and cultural plants belonging to mono- and dicots. The Table
Comparison of female stylet length between Irish population and different Meloidogyne spp. {mean ± SD (range), all measurements in µm}
Species (females) | Unknown Irish sp. (Moore & Franklin) | Unknown Irish sp. (our observations) | M. fallax (original description) | M. chitwoodi (orig. descr.) | M. hapla (orig. descr.) | M. minor (orig. descr.) |
Stylet length | 18.0±2.4 (15.4–24.6) | 14.6±0.5 (14.0–15.0) | 14.5±0.4 (13.9–15.2) | 11.9±0.3 (11.2–12.5) | 13.0±/ (12.0–14.0) | 14.2±1.1 (12.6–15.2) |
Molecular analysis
The DNA extraction from glycerine-embedded nematodes in old slides was unsuccessful with both DNA extraction methods, as PCR product was not obtained. Contrastingly, the targeted region of COX1 gene was successfully amplified from all three fresh individuals of M. fallax. The primers used in this study have been designed to specifically amplify a short region of COX1 gene of M. fallax and M. chitwoodi, the two closely related species.
In the annual reports of Plant Sciences and Crop Husbandry Division from 1965 and 1966, a Meloidogyne species attacking tomato was recorded and briefly described by Mr. John F. Moore and Dr. Mary T. Franklin. Its host range was found to be very wide, including both dicots and monocots (Table
The host test conducted in 1966 for a population of Irish Meloidogyne sp.
Family | Genus + species | Result |
---|---|---|
Chenopodiaceae | Beta vulgaris L. *S | + |
Chenopodium album L. *Pl | + | |
Compositae | Matricaria matricarioides (Less.) Porter *Pl | + |
Senecio jacobaea L. *Pl | + | |
Sonchus sp. *Pl | + | |
Lactuca sativa L.* S | + | |
Caryophyllaceae | Stellaria media L. *Pl | + |
Cerastium sp. *Pl | + | |
Polygonaceae | Polygonum aviculare L. *Pl | + |
Rumex sp. *Pl | + | |
Graminaceae | Hordeum vulgare L. *S | + |
Triticum aestivum L. *S | + | |
Lolium multiflorum (Lam.) *S | + | |
Cruciferae | Capsella bursa-pastoris L. *Pl | + |
Brassica oleracea var. capitata *S | + | |
Brassica napus var. napobrassica *S | + | |
Euphorbiaceae | Mercuria lisannua L. *Pl | + |
Urticaceae | Urticadioica L. *Pl | + |
Labiatae | Lamium purpureum L. *Pl | + |
Umbelliferae | Daucus carota L. *S | + |
Fabaceae | Vicia faba L. *S | + |
Plantaginaceae | Plantago major L. *Pl | + |
Rosacae | Fragaria vesca L. *Pl | + |
Potentilla erecta L. *Pl | + | |
Solanaceae | Solanum tuberosum L. (potato tuber) | + |
Ranunculaceae | Ranunculus repens L. *Pl | + |
Ranunculus acris L. *Pl | + | |
Geraniaceae | Erodium moschatum L. *Pl | + |
Amaranthaceae | Spinacea oleraceaL. *S | + |
Alliaceae | Allium cepa L. *S | + |
Papaveraceae | Fumaria officinalis L. *Pl | – |
Our observations show that the perineal pattern of Irish females greatly corresponds to the one originally described for M. fallax (
Males and second-stage juveniles appeared to have much more informative morphological and morphometrical characters for comparison with other similar species. Mr. Moore and Dr. Franklin described the male head with 3 annules where the first one is deeply pinched off and succeeded by two faintly seen annules. Contradictory to this, we observed one post-labial annule which is interrupted with 1-2 incomplete transverse incisures visible from the lateral view on dorsal and ventral sides. We also found a slightly set-off head region with a slightly elevated labial region as was originally described for males of M. fallax (
Our careful observations show that the stylet length of males in Irish slides matches the one measured in paratypes of M. fallax and M. minor. Additionally, the stylet knob shape in Irish males, being rounded and set off from the shaft as originally described for M. fallax, mismatches ovoid and slightly backwardly sloping knobs characteristic for M. minor (
The stylet length of second-stage juveniles was excluded from the basic comparison (Table
It should also be pointed out that in Irish second-stage juveniles, a gradually tapering tail with bluntly rounded tip and a clearly delimitated hyaline part with broadly rounded tip and often constrictions resemble the tail and tail hyaline shape characteristic for M. fallax (
The host test of Irish Meloidogyne sp. conducted in 1966 showed a wide host range which included both dicots and monocots. Although
An additional molecular support for our data is lacking as we were unable to extract DNA from the 50-year-old slides with both protocols used. It was confirmed by PCR amplification of COX1 gene, showing products only for fresh M. fallax specimens. The COX1 gene was chosen for analysis as it has been previously proven as a good marker for distinguishing closely related Meloidogyne species (
In this study we showed a historical record of M. fallax in Ireland. It is not known which way it was introduced to the unheated glasshouse in the county Cork, either by infected tomato seedlings or by infested soil. Although
To conclude with, observations of the original material of a population of Meloidogyne sp. from Ireland and its comparison to other similar Meloidogyne spp. indicate that it belongs to M. fallax.
The authors are sincerely grateful to ir. Evelyn van Heese for the technical support; Alcides Sánchez-Monge for the help with GNU Image Manipulation Program; the National Plant Protection Organization in Wageningen, the Netherlands and a special research fund of Gent University 01N02312, Belgium.