Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (2): 215-224 Research Article Kastamonu Univ., Journal of Forestry Faculty Doi: 10.17475/kastorman.349543

Survey for the detection of Bursaphelenchus -vector in the western part of Turkey

Mehmet DAYI*, Süleyman AKBULUT

Düzce University, Faculty of Forestry, Düzce, TURKEY *Corresponding author: [email protected]

Received Date: 06.11.2017 Accepted Date: 31.08.2018 Abstract Aim of the study: This study aimed to identify the insect vectors of these Bursaphelenchus species in conifer forests of the western Turkey. Area of study: Sampling was performed in conifer forests of the western part of Turkey. Material and Methods: Two different methods were used, trap trees and pheromone traps with specific lure combinations, to catch . Main results: A total number of 11,076 insects in the first year (2012), and 226,205 insects in the second year (2013) surveys were captured. The most represented insect order was Coleoptera and the most common insect species found in all sampling areas was Orthotomicus erosus. After morphological and molecular studies, B. mucronatus was found to be associated with insect bulk samples of Ips sexdentatus, while B. sexdentati was found to be associated with individual and insect bulk samples of O. erosus, Acanthocinus aedilis, and Arhopalus rusticus. Keywords: Wilt Disease, Bursaphelenchus species, conifer forests, insect vectors, insect traps. Türkiye`nin batı bölgelerinde Bursaphelenchus türlerinin taşıyıcı böceklerinin tespitine yönelik arazi taramaları

Özet Çalışmanın amacı: Bu çalışmada, Türkiye’nin batı bölgelerindeki Bursaphelenchus türlerinin taşıyıcı böceklerinin tespit edilmesi amaçlanmıştır. Çalışma alanı:Türkiye`nin batı bölgesinde iğne yapraklı ormanlarda çalışma yürütülmüştür. Materyal ve Yöntem: Böcekleri yakalamak için tuzak ağaçları ve özel koku karışımlarından oluşan feromon tuzakları olmak üzere iki farklı yöntem kullanılmıştır. Temel Sonuçlar: Çalışmanın ilk yılında (2012) toplamda 11,076 böcek yakalanırken, ikinci yılda (2013) ise 226,205 böcek yakalanmıştır. Tüm örnekleme sahalarında en fazla yakalanan böcek takımı Coleoptera olurken, en fazla yakalanan böcek türü ise Orthotomicus erosus olmuştur. Nematodların morfolojik ve moleküler açıdan teşhis çalışmalarından sonra, B. mucronatus’un Ips sexdentatus ile, B. sexdentati’nin ise O. erosus, Acanthocinus aedilis ve Arhopalus rusticus böcekleriyle ilişkili olduğu tespit edilmiştir Anahtar kelimeler: Çam Kuruma Hastalığı, Bursaphelenchus türleri, iğne yapraklı ormanlar, vektör böcekler, böcek tuzakları.

Introduction (Yano, 1913) further spreading to China and The pinewood (PWN), Korea (Yang, 2004; Yi., Byun, Park, Yang Bursaphelenchus xylophilus ((Steiner and and Chang, 1989) and to Europe (Abelleira, Buhrer, 1934) Nickle, 1970) is the causal Picoaga, Mansilla, Aguin, 2011; Fonseca et agent of pine wilt disease (PWD), which al., 2012; Mota et al., 1999; Vicente, Espada, causes severe ecological and economical Vieira, Mota, 2012). These new reports damages in conifer forests in East Asia and increased the concerns of a new southern Europe (Mota and Vieira, 2008). introduction/spread of the PWN into other The insect vectors belonging to the uninfected pine forests in Europe. Due to the Dejean 1821 carry this presence of susceptible pine species (e.g. nematode from symptomatic to new and Pinus brutia Ten, Pinus nigra Arnold and P. healthy pine trees (Akbulut and Stamps, sylvestris L.), the insect vector (Monochamus 2012). The disease was first reported from galloprovincialis) and geographic position Japan in the beginning of the 20th century and Mediterranean climate, the possible

215 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (2): 215-224 Dayı and Akbulut Kastamonu Univ., Journal of Forestry Faculty introduction of the PWN is a constant threat Pinus brutia and P. nigra. The average to conifer forests of Turkey (Akbulut et al., altitudes were 500 m and 1200 m for P. 2006). In Turkey, the survey for the presence brutia and P. nigra stands, respectively. All of the PWN in conifer forests started in 2003 study locations were established by using the (Akbulut et al., 2006). As a result of these same GPS (Global Positioning System) surveys, several Bursaphelenchus Fuchs, coordinates of previously known areas for 1937 species have been found, namely B. the presence of Bursaphelenchus species anamurius, Akbulut, Braasch, Baysal, (Akbulut et al., 2006; Akbulut et al., 2007; Brandstetter, Burgermeister, 2007, B. 2008a; Dayi et al., 2014). andrasyyi Dayi, Calin, Akbulut, Gu, For the 2012 survey, trap trees were Schroder, Vieira, Braasch, 2014, B. prepared before the flight period of possible hellenicus Skarmoutsos, Braasch, insect vector species (as early as at the Michalopoulou, 1998, B. mucronatus beginning of March). For each location, four Mamiya, Enda, 1979, B. pinophilus Brzeski, trap trees (20-30 m length and 12-15 cm Baujard, 1997, B. sexdentati Ruhm, 1960 and diam.) were selected from healthy trees: P. B. vallesianus Schonfeld, Polonski, brutia in the regions of İzmir, Manisa and Burgermeister, 2004 (Akbulut et al., 2006, Muğla; Abies cilicica Ant. and Kotschy Akbulut, Braasch, Baysal, Brandstetter, Carriére, and P. brutia in the region of Burgermeister, 2007; Akbulut, Elekçioğlu, Burdur, and P. nigra in the regions of Isparta Keten, 2008a; Dayi et al., 2014). So far, B. and Denizli, representing the forest type of xylophilus has not been detected in Turkey. the study areas. A total of 24 trap trees were In nature, the spread of Bursaphelenchus used in this study. For each location, trees species is totally dependent on its insect were felled while retaining their branches to vectors. The association established between prevent water loss. Trap trees were kept in Bursaphelenchus species and its insect vector the field to attract insect species from March can be more or less specific (Ryss, Vieira, to September and checked periodically for Mota, Kulinich, 2005). For example, species insect activities, such as oviposition marks, belonging to the Xylophilus group are mainly larval feeding (presence of frass), or insect associated with Monochamus species emergence. Several log samples (50-60 cm in (Cerambycidae), while species belonging to length) (about 5 logs of each tree) were taken other different morphological groups, such as from trap trees showing any insect activities the sexdentati, egersi or eremus can be and sent to the lab via cargo from March to vectored mainly by Scolytid species September. The logs were kept under (Curculionidae: Scolytinae) (Braasch, constant conditions (25-28oC, 65-75 % RH, Burgermeister, Gu, 2009; Ryss et al. 2005). and a 12L / 12D photoperiod) until In Turkey, the vector species of emergence of adult insects in the lab. Before Bursaphelenchus from different transferring each log into its own PVC morphological groups of Bursaphelenchus container (50-65 cm in length, 15-30 cm in species has not been studied. There are only diameter and the top part with a wire screen), few reports related to insect vectors of both ends of each log were waxed with hot Bursaphelenchus (Akbulut et al., 2010; paraffin to reduce desiccation. Emerged Giblin-Davis et al., 2005). Therefore, the insects were collected daily and identified to main goal of this study was to determine the species level. Then, were potential insect vectors of Bursaphelenchus extracted using a Baermann funnel or by species previously recorded in the western crushing (or dissecting) the insects in Petri parts of Turkey. dishes containing water for 24 h. Extracted nematodes were checked under a stereo- Material and Methods microscope (Olympus SZX-12), nematodes Insect surveys were transferred into a 2 ml tube containing Insect sampling studies were carried out DESS solution (Yoder et. al. 2006), and kept in the pine forests of Muğla, İzmir, Burdur, at room temperature for morphological and Isparta, Denizli and Manisa. Studied forest molecular studies. areas consisted of two main pine species,

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For the 2013 survey, three different found are classified here as non- pheromone-baited traps were used at each Bursaphelenchus only. location; radiator (Witasek-Germany), The same nematodes were then used for Scandinavian (VIT-Turkey) and multi-funnel single-nematode genomic DNA extraction (SMC-Turkey). For each location three using the Purelink Genomic DNA kit pheromone traps (radiator, Scandinavian and following the manufacturer’s instructions multi-funnel) were placed in open areas close (Life technologies–Invitrogen). For to location where Bursaphelenchus species amplification of the ITS1/ITS2 region of the were reported in previous studies mentioned rDNA the procedure used by Cermak et al., in the introduction section. A total of 18 (2013) was followed. For amplification of pheromone traps were used. Each trap was the ITS1/ITS2 region of the rDNA the placed 100 m away from each other, and a forward primer 5’- combination of three pheromone lures was CGTAACAAGGTAGCTGTAG-3’ (Ferris, used: α-pinene (20 mg), ipsdienol (95 mg) Ferris and Faghihi, 1993) and reverse primer and 2-methyl-3-buten-2-ol (MBO) (1500 mg) ’ ’ and kept from March to September in the 5 -TTTCACTCGCCGTTACTAAGG-3 field. The traps were checked weekly, all (Vrain, 1993) were used. Each PCR reaction insects were collected and sent to the lab in contained 1X Taq buffer, 2.5 mM MgCl2, 0.6 small cups via cargo. Each pheromone lure mM of each primer, 0.2 mM dNTPs, 2 units was periodically changed within a 20 day- Taq DNA Polymerase (Fermentas, interval with a new one. Collected insects ThermoScientific), and 5 μl of template were identified to species level (when DNA. The PCR reaction consisted of a possible) then dissected or crushed for denaturation step at 94°C for 4 minutes, nematode extraction as described above. followed by 35 cycles at 94°C for 30 Due to the great number of collected insects seconds, annealing at 50°C for 30 seconds, in some samples (mainly for bark beetles), and 72°C for 1 minute, and a final extension insects from the same trap were separated step at 72°C for 10 minutes. The amplified and processed for nematode extraction using PCR products were analyzed by bulk samples of approximately 100 electrophoresis in a 1.5 % agarose gel. individuals. Insects from other families (big- Posteriorly, the remaining PCR products sized insects belonging to the families such were purified using a combination of two as Cerambycidae or Buprestidae) were enzymes: Exonuclease I (0.5 μl) and Fast AP separated and processed individually. (Alkaline phosphatase) (1 μl) (Fermentas, ThermoScientific), and sent for sequencing Morphological identification and (Macrogen). The obtained sequences were molecular analysis of nematodes proofread manually using CLC Workbench Morphological and molecular studies to v. 7 software (CLC Bio, Qiagen). The ITS identify nematodes species isolated from rDNA region corresponding sequences were insects were done at University of Evora compared with a set of reference sequences (Portugal) in support of a fellowship from different species of Bursaphelenchus supported by COST (European Cooperation selected from GenBank (NCBI). The tree in Science and Technology). topology was obtained with the neighbour- Isolated nematodes were observed under a joining (NJ) analysis with 1000 bootstrap light microscope (Olympus BX50). For replications using CLUSTAL X. specimens belonging to the genus The phylogenetic tree was visualized and Bursaphelenchus, nematodes isolated from annotated using the program FigTree individual insects or bulks of insects were (http://tree.bio.ed.ac.uk/software/fig-tree/). identified according to Braasch et al., (2009) ITS-RFLP profiles presented for species of and Ryss et al., (2005) and and measured the sexdentati group were calculated from the using the software Cell^D (Imaging software ITS1/2 sequences available at GenBank for life sciences microscopy, Olympus). As (NCBI), using the software EnzymeX 3 Bursaphelenchus species were the main (www.mekentosj.com/science/enzymex). focus of this project, other nematode species

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Results 2013, with a total of 226,205 in comparison Insect captures to 11,076 individuals in 2012. In both survey In the 2012 survey, a total of 11,076 years, the most common scolytid species was insects were captured, and the most O. erosus, while the most common represented order was Coleoptera with most cerambycid species was Acanthocinus aedilis of the specimens belonging to the family Linnaeus and Arhopalus rusticus Linnaeus in Curculionidae followed by the family 2012 and 2013, respectively. Interestingly, Cerambycidae and only one specimen was M. galloprovincialis Olivier was only from the order Isoptera. captured, during the survey of 2013. The total number of insects was distributed among thirteen families of the Insect-nematode association and order Coleoptera, with most of the specimens identification belonging to the family Curculionidae From the total number of insects (twelve species), followed by the family collected, nematodes were only found in 11 Cerambycidae with five species. Insects individual insects and 73 insect bulk samples belonging to the families of Cerambycidae (100 individuals per bulk) from all sampling and Curculionidae were captured in all locations. Nematodes were found to be sampling areas - 170 and 10,875 specimens, associated with Curculionidae and respectively. Orthotomicus erosus Wollaston Cerambycidae species only. The majority of (Curculionidae) was the most common nematode species extracted from insect species found, followed by Ips sexdentatus samples were free-living nematodes, herein Boerner (Curculionidae). classified as non-Bursaphelenchus species. In the 2013 survey, a total of 226,205 insects Nematode carrying insect species were A. were captured. Almost all of the collected rusticus, O. erosus, Tomicus piniperda specimens were distributed within the family Linnaeus, I. sexdentatus, Spondylis Curculionidae (225,950 individuals). The buprestoides Linnaeus and A. aedilis. most common insect species found in all All Bursaphelenchus specimens were areas was O. erosus followed by I. identified based on the type of spicule and sexdentatus with few specimens of M. assigned to one of the morphological group galloprovincialis also captured. of species established for this genus, namely Even though the number of families the sexdentati- and xylophilus-group (Table collected was higher in 2012, the number of 1) and measured. insects captured increased dramatically in

Table 1. Bursaphelenchus species associated with insects captured in six different locations in the western part of Turkey. The group Number of Number of Number Number of Nematode species Location Insect total juveniles/dauer of females of males nematodes individuals larvae The Bursaphelenchus Orthotomicus1 xylophilus Burdur 3 (2) 0 3 0 sp. erosus group The Bursaphelenchus xylophilus Burdur O. erosus1 4 (1) 0 4 0 sp. group The Bursaphelenchus Arhopalus sexdentati Denizli 4 (1) 1 3 0 sexdentati rusticus2 group The Bursaphelenchus Aconthocinus sexdentati Izmir 3 (2) 1 2 0 sexdentati aedilis2 group The Bursaphelenchus sexdentati Izmir O. erosus1 16 (2) 15 1 0 sexdentati group The Bursaphelenchus Ips xylophilus Isparta 1 (1) 0 1 0 mucronatus sexdentatus1 group

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Note. The number of nematodes found in individual insect species is presented between parentheses. In the Insect column, the asterisk 1 represents the method “crashing”, and the asterisk 2 represents the method “dissecting” for nematode extraction.

Specimens belonging to the sexdentati- Only 3 individual and 6 bulk insect group presented stout and curved spicules samples were found to carry with prominent rostrum and condylus, the Bursaphelenchus specimens (Table 1). The presence of seven caudal papillae, and number of Bursaphelenchus specimens females with a small vulval flap. The extracted was very limited, varying from 1 to specimens belonging to the xylophilus-group 16 individuals (Table 1). presented the typical narrow and long Bursaphelenchus individuals extracted spicules, with flattened capitulum, and from all insect samples were female or male presence of cucullus, and seven caudal stages, with no juveniles or dauer larvae papillae. found within these nematode-insect associations (Figure 1).

Figure 1. Light microscope observations of Bursaphelenchus specimens found in association with insects captured during the surveys of 2012 and 2013 in the western part of Turkey. A: anterior region of a specimen belonging to the genus Bursaphelenchus; B: female vulva region of B. sexdentati; C: male tail of B. sexdentati; D: male tail of B. mucronatus. Scale bars: 10 μm.

B. sexdentati was collected from regions of Denizli and İzmir that were individual insects of A. rusticus and A. identified as B. sexdentati, and 920 bp for a aedilis species (Cerambycidae), while the single nematode used for Isparta region and remaining Bursaphelenchus specimens identified as B. mucronatus (Table 2). The identified were collected from insect bulk nematodes belonging to B. sexdentati samples of O. erosus and I. sexdentatus clustered together with other isolates (Curculionidae) (Table 1). previously identified has belonging to the For each sample containing South European type. The ITS rDNA Bursaphelenchus specimens, a molecular sequences obtained for B. sexdentati characterization was performed based on the nematodes collected from the areas of ITS rDNA region using single nematode Denizli and Izmir also displayed several specimens. Amplification of the ITS1/2 point mutations in both ITS1 and ITS2 region (including a partial region of both 18S regions, including a point nucleotide and 28S rRNA genes) was obtained for five substitution at the 759 bp in the ITS2 region individuals out of four samples containing for two of the isolates, which led to the loss Bursaphelenchus specimens. Amplification of the MspI recognition site (Table 2), of this region resulted in a PCR product of generating a different ITS-RFLP pattern for 981 bp for nematodes collected from the this enzyme, in comparison to the established

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ITS-RFLP pattern for this species (Burgermeister et al., 2009).

Table 2. ITS-RFLP pattern of Bursaphelenchus species found in different forest areas of western part of Turkey. The ITS-RFLP pattern was generated using the sequencing results obtained for the ITS/ITS2 rDNA region. PCR Bursaphelenchus product Restriction fragments (bp) sp. (bp) RsaI HaeIII MspI HinfI AluI B. sexdentati 545 981 584 981 466 968 Izmir 414 279 279 13 22 118 211 25 B. sexdentati Denizli and 981 545 863 981 466 968 Izmir 414 118 279 13 22 211 25 B. mucronatus 920 487 620 354 408 275 Isparta 411 300 303 323 245 22 263 120 86 49 25

The isolate belonging to the xylophilus-group number of insects captured between both was identified as B. mucronatus showing type of traps herein used. The number of similar pattern of the ITS-RFLP established insects captured with pheromone-based traps for this species. was 20 times higher than those of trap trees. We were unable to successfully amplify The wide range dispersal of the pheromones the respective rDNA region of the nematodes in these types of traps compared to the trap collected from the Burdur area, which belong trees may explain this high difference. In to the xylophilus group of species based on addition, this high number of captures may the male spicules morphology (Ryss et al., also be related to the specific combinations 2005). of pheromones, such as α-pinene, ipsdienol and 2-methyl-3-buten-2-ol. Pheromone lures Discussion were renewed periodically, which may have In this study, insect vectors of previously increased the number of insects captured by reported Bursaphelenchus species were the pheromone traps compared to the trap investigated in six different locations of trees. Dodds, Darrel, Daterman, (2000) western forest sites of Turkey. The majority discussed that trap trees have a finite of the insect species collected mainly belong capacity for trapping insects due to their to the families of Curculionidae and limited area. On the other hand, in the Cerambycidae. The most common species pheromone traps, captured insects were are O. erosus and I. sexdentatus in all study removed periodically and the traps were locations. ready to capture new insects. However, the There is a huge difference in terms of the richness of insect species/families was higher

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Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (2): 215-224 Dayı and Akbulut Kastamonu Univ., Journal of Forestry Faculty for trap trees than pheromone traps. sampling areas. Thus, the insects may have Regarding to trap trees, different carried few or no nematodes. inhabiting insects were collected, whereas in Similar results were also observed during pheromone traps only insects attracted to the the survey of B. xylophilus. Since 2002, over specific pheromone lures were collected 3000 wood samples around Turkey were which could explain the reduced richness of taken from dead or dying conifer species, and species/families. The differences in the only 5 % of the total samples was contained number of insect species/families among the Bursaphelenchus species with limited studied locations can be also related to the number of individuals (Akbulut et al., 2006; stand types and tree species distributed Akbulut et al., 2007; Akbulut et al., 2008a; among the different locations. In Burdur Akbulut et al., 2013). All reported area, three different conifer species (Abies Bursaphelenchus species are believed to be cilicica, Cedrus libani and P. brutia) were native to Turkish conifer forests, and no identified in the stand, while in the other extensive wilting symptoms caused by these locations only one pine species was Bursaphelenchus species have been observed represented, either P. brutia or P. nigra. so far. Sarikaya and Avci (2011) reported that O. Nematodes belonging to the xylophilus erosus, I. sexdentatus and T. minor were group were found in Burdur and Isparta found as the most common species regions, and associated with bulk samples of associated with P. brutia and P. nigra forests O. erosus and I. sexdentatus, respectively. in the western part of Turkey, such as Nematodes belonging to the sexdentati group Antalya, Burdur, Denizli, Isparta, Muğla and were found in Denizli and İzmir regions and Afyonkarahisar. In the current study, isolated from individual insects belonging to similarly, O. erosus and I. sexdentatus were the species A. rusticus, A. aedilis, as well as the most common species, followed by T. bulk samples of O. erosus, respectively piniperda. (Table 1). Interestingly, only adult stages of The species of the genus Bursaphelenchus Bursaphelenchus were isolated from these are often found in association with different insects. This pattern of nematode stage-insect insect vector species (Penas et al., 2006; association is not usual, as the most common Ryss et al., 2005). In Turkey a total of seven Bursaphelenchus stage found with other Bursaphelenchus species have been insect species is called as “dauer larvae” identified associated with conifer trees, (Ryss et al., 2005). The occurrence of adult namely B. anamurius, B. andrassyi, B. stages (and in such reduced number of hellenicus, B. mucronatus, B. pinophilus, B. individuals) has been previously reported for sexdentati and B. vallesianus, (Akbulut et al., other Bursaphelenchus species, for example 2006, Akbulut et al., 2007; Akbulut et al., B. hellenicus in association with Hylurgus 2008a; Akbulut et al., 2008b; Akbulut et al., ligniperda, and B. leoni in association with a 2013, Dayi et al., 2014). However, no insect Pityogenes sp. (Penas et al., 2006). species have been reported as vectors of During this study, 39 individuals of M. these Bursaphelenchus species in Turkey galloprovincialis, the vector of B. xylophilus except the report of M. galloprovincialis as in Portugal (Sousa et al., 2001), were also the vector of B. mucronatus (Akbulut et al., collected, but none of them carried 2010). From the total of 237,281 insect nematodes. M. galloprovincialis is known as specimens captured during surveys, only a a vector of B. xylophilus (Sousa et al., 2001) very small percentage of insects (11 and B. mucronatus (Ryss et al., 2005). The individual and 73 bulk insect samples) were number of captured individuals was very low found to be associated with nematodes. compared to other insects. Since it has not In the particular case of Bursaphelenchus, been considered to be a for pine forests, only 3 individual and 6 bulk insect samples there is no study related to population density showed the presence of Bursaphelenchus of M. galloprovincialis in the field. In with limited number of individuals. This may sampling areas, previous studies on be caused by low population density or low cerambycid and curculionid species focused distribution frequency of these nematodes in on distribution of these species in conifer

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Kastamonu Uni., Orman Fakültesi Dergisi, 2018, 18 (2): 215-224 Dayı and Akbulut Kastamonu Univ., Journal of Forestry Faculty forests and little is known as their abundance of pine wilt disease on Pinus pinaster in (Özdikmen, 2007; 2008, Sarıkaya and Avcı Northwestern Spain. Plant Disease, 95, 2011). Several other insect species such as A. 776. griseus, Ips spp., S. buprestoides and A. Akbulut, S., Vieira, P., Ryss, A., Yuksel, B., rusticus, were previously reported as Keten, A., Mota, M., Valadas, V. (2006). potential carriers of B. xylophilus in the Preliminary survey of the pinewood USA, although as a non-specific/casual nematode in Turkey. EPPO Bulletin, 36, association (Linit, 1988; Ryss et al., 2005). 538-542. Both A. rusticus and S. buprestiodes have a Akbulut, S., Braasch, H., Baysal, ̇I., wide distribution in Turkish pine forests Brandstetter, M., Burgermeister, W. (Çanakçıoğlu and Mol 1998), including the (2007). Description of Bursaphelenchus areas of Burdur, Denizli, İzmir, Muğla and anamurius sp. n. (Nematoda: Isparta sampled during the current study. Parasitaphelenchidae) from Pinus brutia In this study, Bursaphelenchus-vector in Turkey. Nematology, 9, 859-867. associations show differences when Akbulut, S., Elekçioğlu, İH., Keten, A. compared to the literature. For instance, a (2008a). First record of Bursaphelenchus Bursaphelenchus species of the sexdentati vallesianus Braasch, Schönfeld, Polomski group, B. sexdentati, was found in and Burgermeister in Turkey. Turkish association with a cerambycid insect, A. Journal of Agriculture and Forestry, 32, aedilis. Moreover, a Bursaphelenchus 273-279. species of the xylophilus group, B. Akbulut, S., Vieira, P., Ryss, A., Valadas, V., mucronatus, was found to be associated with Keten, A., Mota, M. (2008b). a curculinid insect, I. sexdentatus. These Bursaphelenchus Fuchs, 1937 (Nematoda: results suggest that vector specificity in Parasitaphelenchidae) species associated Bursaphelenchus species may vary depend with Pinus species in northern Turkey. on insect species in trees. Due to the critical Helminthology, 45, 89-95. geographical location, suitable climatic Akbulut, S., Yuksel, B., Cebeci, HH., Baysal, conditions, presence of vectors and highly I., Serin, M., Erdem, M. (2010). succeptible host trees for B. xylophilus, these Investigation of the pinewood nematode, surveys not only emphasize our lack of Bursaphelenchus xylophilus (Nematoda: knowledge related to the potential vectors of Parasitaphelenchidae) and other Bursaphelenchus species, but enable Bursaphelenchus species with their vector recognition of priority areas where PWN insects in pine forests of Turkey and their might succcesfully establish. pathogenicities. Project Report, The Scientific and Technological Council of Acknowledgements Turkey, Project No:107O088, Ankara, pp. This project was partly supported by 113. Düzce University Research Fund Project Akbulut, S., Stamps, T. (2012). Insect Number 2011.02.02.076. Mehmet Dayi was vectors of the pinewood nematode: a supported as a visiting scholar by COST review of the biology and of (European Cooperation in Science and Monochamus species. Forest Pathology, Technology) action “FP1002 Pathway 42, 89-99. Evaluation and Pest Risk Management in Akbulut, S., Braasch, H., Cebeci, HH. Transport (PERMIT) with a Short-Term (2013). First report of Bursaphelenchus Scientific Mission (STSM) for research of hellenicus Skartmoutsos, Braasch, his PhD dissertation at University of Évora, Michalopolou (Nematoda: Portugal. ) from Turkey. Forest Pathology, 43, 402-406. References Braasch, H., Burgermeister, W., Gu, J. Abelleira, A., Picoaga, A., Mansilla, JP., (2009). Revised intra-generic grouping of Aguin, O. (2011). Detection of Bursaphelenchus Fuchs, 1937 (Nematoda: Bursaphelenchus xylophilus, causal agent Aphelenchoididae). Journal of Nematode

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