Detection surveys for quarantine pests are one of the most effective preventive measures given by the Regulation (EU) 2016/2031 of the European Parliament of the Council on protective measures against the introduction of non-native plant pathogens. The pine wood nematode
Currently, the information regarding the distribution of species within the genus
Pheromone-baited black cross traps (Ecconex/Galloprotect Pack) were established as a part of the workshops focused on general surveys for forest quarantine species, including the pine wood nematode and its insect vectors. Two traps were hanged in the crowns of most abundant pine trees (approximately 20 m high), one in 2017 in the area of Banja Luka (BiH) on
Nematodes were extracted by dissection and maceration of insects in sterile water followed by three hours incubation at room temperature. The presence or absence of nematodes was checked under the stereomicroscope (IPPC, 2016). The extracted nematode “dauer larvae” were fixed in 4 % formalin and transferred to pure glycerin according to De Grisse (1969) or prepared for molecular analysis. Where a large number of nematodes was available, a subset of living nematodes was transferred onto the plates containing the sporulating form of the fungus
Morphological and morphometric analyses were based on the main diagnostic features for the genus
For molecular identification, DNA from single nematode specimens (“dauer larvae”) was extracted and processed as follows: nematodes were homogenised in 50 μL of nematode lysis buffer (10mM Tris-HCl, pH 8.8; 1mM EDTA; 1 % Triton X-100 (v/v); 100 μg ml−1 proteinase K) in a 1.5 mL Eppendorf tube using a micropestle. Samples were incubated at 55 °C for 1 h and subsequently at 95 °C for 10 min. The resulting DNA extracts were used as a template for each PCR reaction. The identification of individual nematodes was performed using two different
The ITS1-5.8S-ITS2 PCR reaction was performed in a total volume of 50 μl containing 1x PCR buffer (75mM Tris-HCl pH 9.0; 50mM KCl; 20mM (NH4)2SO4, 4mM MgCl2, 400μM dNTPs, 600nM primers, 2 U of DNA polymerase (Biotools, Madrid, Spain) and 10 μl of undiluted template DNA, in a GenePro thermal cycler (Bioer Technology Co., LTD) using an initial denaturation step for 5 min at 94 °C, followed by 40 reaction cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 48 °C and extension for 2 min at 72 °C, with a final 10min extension at 72 °C. Six μl of the PCR product were digested for at least 2 hours at 37 °C, using 1x restriction buffer and 5 U of enzyme (
Amplification of the D2–D3 region was performed in a total volume of 25 μl using 1x PCRBIO Taq Mix (PCR Biosystems, London, UK), 400nM primers and 2 μl of undiluted template DNA, in a GenePro thermal cycler (Bioer Technology Co., LTD) using an initial denaturation step for 1 min at 95 °C, followed by 5 reaction cycles of denaturation for 15 s at 95 °C, annealing for 15 s at 45 °C and extension for 30 s at 72 °C and 35 cycles of 15 s at 95 °C, 15 s at 54 °C and 30 s at 72 °C.
After ExoSAP purification, PCR products of both regions were submitted for direct Sanger sequencing to the Centre of Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany (Olomouc, Czech Republic) using the corresponding forward and reverse primers. Resulting sequence data were analysed using Geneious Bioinformatics software platform (Biomatters). The online bioinformatic tool RestrictionMapper (
The D2–D3 sequences obtained from nematodes collected in BiH and Georgia, as well as 10 sequences downloaded from the GenBank database (
As mentioned above, the main goal of our study was to detect potential insect vectors of pine wood nematode in BiH and Georgia, and therefore our analyses focused primarilly on the genus
Morphometric comparison between the populations of
Dauers Georgian population | ♀♀ Georgian population | ♂♂ Georgian population | Dauers BiH population | Dauers Mamiya & Enda 1979 | ♀♀ Mamiya & Enda 1979 | ♂♂ Mamiya & Enda 1979 | |
---|---|---|---|---|---|---|---|
n | 5 | 25 | 12 | 5 | 30 | 40 | 35 |
L | 597.5±19.8 (570–620) | 866.4± 77.0 (738–1018) | 675.3 ± 19.7 (652.5–723.0) | 589 ± 23.7 (558–620) | 590 (500–650) | 870 (700–980) | 790 (640–970) |
a | 43.2±2.3 (39.5–45.6) | 40.9 ± 2.6 (35.0–45.7) | 42.6 ± 3.9 (35.8–50.4) | 41.7 ± 3.6 (35.8–45.0) | 39.8 (35.1–45.8) | 41.8 (36.5–45.9) | 44.0 (38.8–51.1) |
b | 9.5* | 11.0 ± 1.2 (8.0–13.3) | 9.1 ± 0.4 (8.4–9.9) | 9.3* | 8.9 (7.9–9.6) | 12.6 (9.6–15.6) | 11.4 (9.0–14.7) |
c | 20.7±1.40 (18.3–21.7) | 27.1 ± 2.8 (23.3–30.1) | 20.0 ± 1.1 (18.6–22.1) | 19.4 ± 1.3 (18.0–21.1) | 21.2 (19.7–24.4) | 26.2 (19.6–30.4) | 29.1 (25.7–35.6) |
c′ | 3.6±0.3 (3.3–4.0) | 2.9 ± 0.2 (2.5–3.3) | 2.4 ± 0.1 (2.1–2.5) | 3.5 ± 0.2 (3.0–3.7) | |||
V [%] | - | 74.1 ± 2.0 (67.0–76.9) | - | - | 75 (73–77) | ||
Stylet length | 8.5* | 13.3 ± 1.2 (10.2–15.1) | 13.6 ± 0.6 (12.8–14.6) | 8.8* | 15.8 (14–16) | 15.0 (14–16) | |
Excretory pore position1) | 54.9±4.5 (49–58.1) | 83.7 ± 3.6 (73.6–89.5) | 76.7 ± 4.2 (70.5–84.9) | 63,3±7,2 (54.0–74.4) | |||
Pharynx length | 59.6* | 77.9 ± 4.2 (72.3–91.4) | 74.9 ± 3.0 (69.2–80.8) | 58.9* | |||
MB2) | 59.9±4.9 (54.7–67.9) | 66.6 ± 3.7 (62.6–78.3) | 64.9± 2.5 (61.5–69.5) | 58.0 ± 5.7 (50.7–67.0) | |||
Hemizonid position1) | - | 95.8± 5.2 (76.0–101.8) | 91.0 ± 2.5 (86.9–94.9) | - | |||
Anal/cloacal body diameter | 8.1±0.2 (7.9–8.4) | 11.1± 0.7 (10.1–12.2) | 14.0 ± 0.7 (12.7–14.8) | 8.2 ± 0.3 (7.8–8.5) | |||
Tail length | 29.1±2.8 (27.0–33.9) | 32.0± 2.2 (26.0–36.9) | 33.9 ± 1.9 (30.4–36.9) | 28.3 ± 2.55 (27.0–33.9) | |||
Post-uterine sac length | - | 156.5 ± 17.9 (125.0–195.6) | - | - | |||
Spicule length (curved median line) | - | - | 31.2 ± 1.5 (28.5–33.6) | - | 26.0 (23–29) |
Distance from anterior end;
Distance from anterior end to base valves of median bulb,
- n=1
For both BiH and Georgian populations found during the survey, a molecular characterization was performed using the ITS1-5.8S-ITS2 and D2–D3 region of 28S of the ribosomal DNA generated from single dauer larvae specimens.
The amplification and sequencing of ITS1-5.8S-ITS2 region yielded an 884 bp sequence (corresponding to the entire PCR product sequence after primer trimming) for both BiH and Georgian populations (OK500292 and OK500293, respectively) sharing 99.7 % identity to each other: BiH sequence included one ambiguous base (A/G=R) at position 161 and G at 642 (which prevented amplicon cutting with
The ITS-RFLP pattern was calculated virtually for the ITS sequences generated for both populations collected in BiH and Georgia. The virtual restriction profile generated for four enzymes were similar for both populations (Table 2) and showed the same reference ITS-RFLP pattern established for the
In case of D2–D3 region amplification and sequencing resulted in a sequence of 743 bp (corresponding to the entire PCR product sequence after primer trimming) for the population collected in BiH (OK523378) and partial 720 bp for the population collected in Georgia (OK523379). Both sequences shared 100 % identity to each other and also to the population of
Virtual ITS-RFLP patterns of
BiH population, OK523378 | Georgian population, OK523379 | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
ITS-PCR product | |||||||||||
Fragment sizes (~bp) | 884* | 623 | 355 | 411 | 678 | 623 | 355 | 411 | 678 | ||
196 | 304 | 232 | 246 | 196 | 304 | 232 | 246 | ||||
105 | 265 | 120 | 105 | 265 | 120 | ||||||
87 | 87 | ||||||||||
49 | 49 | ||||||||||
25 | 25 |
The amplification and sequencing of ITS1-5.8S-ITS2 region yielded an 884 bp sequence (corresponding to the entire PCR product sequence after primer trimming) for both BiH and Georgia populations (OK523378 and OK523379, respectively)
The genus
The diversity of the genus
List of
No. | status | Species | Insect vector | Associated plant/note | Group* | Reference |
---|---|---|---|---|---|---|
Hofmanni group | Kakulia & Shalibashvili, 1976a | |||||
Kakulia, Shalibashvili, Gorgadze, 1983 | ||||||
Xylophilus group | Kurashvili |
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Eggersi group | Kakulia & Maglakelidze, 1973 | |||||
Kurashvili |
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Eidemani group | Kakulia, 1971 | |||||
Georgia |
Kakulia & Devdariani, 1975** Kurashvili |
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Eremus group | Kurashvili |
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Insufficient description: not considered for Grouping | Kakulia & Devdariani, 1975** Mikaia |
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Hunt, 1993 ( |
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Eidemani group | Kurashvili |
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Kakulia, Shalibashvili, Gorgadze, 1983 | ||||||
Sexdentati group | Kakulia & Devdariani, 1975** | |||||
Kurashvili |
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- | Xylophilus group | Kakulia & Devdariani, 1975** | ||||
Kurashvili |
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Insufficient description: not considered for grouping | Devdariani |
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Kakulia, 1989 | ||||||
Hunt, 1993 ( |
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Ryss |
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Six-incisure ungrouped species | Kakulia & Devdariani, 1975** | |||||
Kurashvili |
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Sexdentati group | Kurashvili |
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Xylophilus group | This article | |||||
Not mentioned in the text. | Piniperdae group | Kakulia & Shalibashvili, 1976b | ||||
Piniperdae group | Kakulia, Devdariani, 1965 | |||||
Kakulia, 1967 | ||||||
Kurashvili |
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Kakulia, Shalibashvili, Gorgadze, 1983 | ||||||
Insufficient description: not considered for grouping | Devdariani, 1973* | |||||
Mikaia |
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Hunt, 1993 ( |
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Ryss |
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Hofmanni group | Kurashvili |
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Insufficient description: not considered for grouping | Kakulia, 1989 | |||||
Sexdentati gropu | Kakulia & Devdariani, 1975** | |||||
Kurashvili |
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Kakulia, Shalibashvili, Gorgadze, 1983 | ||||||
Sexdentati group | Devdariani, 1974 | |||||
Not mentioned in the text. | Insufficient description: not considered for grouping | Kakulia, 1989 | ||||
Hunt, 1993 ( |
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Ryss |
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Insufficient description: not considered for Grouping | Kakulia |
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Kurashvili |
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Kakulia, 1989 | ||||||
Hunt, 1993 ( |
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Ryss |
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Hofmanni/Abietinus group | Kakulia & Devdariani, 1967 | |||||
Kakulia & Devdariani, 1975** | ||||||
Kurashvili |
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Ryss |
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- | - | - | Kakulia, 1989 | |||
Hunt, 1993 | ||||||
Ryss |
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- | - | - | Kakulia, 1989 | |||
Hunt, 1993 | ||||||
Ryss |
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- | - | - | Kakulia, 1989 | |||
Hunt, 1993 | ||||||
Ryss |
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- | - | - | Devdariani, 1975** | |||
Kurashvili |
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This article (Nomina Nuda) | ||||||
Transfer to other genus | - | - | Kakulia, 1989 | |||
Meyl, 1961 | Goodey, 1960 | Ryss |
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Transfer to other genus | - | - | Kakulia & Devdariani, 1965 | |||
Kakulia, 1989 | ||||||
Ryss |
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Braasch, 2009 | ||||||
Transfer to other genus | - | - | Kakulia, 1967 | |||
Kakulia, 1989 | ||||||
Ryss |
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Braasch, 2009 |
Legend:
= grouping is based on Braasch
original publication not available to the authors
Six species were associated with longhorn beetle vectors (Cerambycidae):
Due to the association of