Molecular characterization of the Pratylenchus vulnus populations on cereals in Turkey

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Journal of Nematology

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Molecular characterization of the Pratylenchus vulnus populations on cereals in Turkey

Mehmet Sait Karaca / Elif Yavuzaslanoglu * / Gul Imriz / Ozlem Ates Sonmezoglu *

Keywords : Barley, D2-D3 expansion region of 28S rRNA, Detection, Real-time PCR, Sequencing, Walnut root lesion nematode, Wheat

Citation Information : Journal of Nematology. Volume 52, Pages 1-4, DOI: https://doi.org/10.21307/jofnem-2020-084

License : (CC-BY-4.0)

Received Date : 18-September-2020 / Published Online: 25-August-2020

ARTICLE

ABSTRACT

Pratylenchus vulnus (walnut root lesion nematode) is one of the most damaging root lesion nematode species worldwide. In this study, 17 populations of P. vulnus obtained from wheat and barley cultivated fields in 2016 to 2017 in Turkey (Karaman and Konya provinces) were identified using real-time PCR and melting curve analysis. Samples provided a single peak at 87.3˚C with real-time PCR. D2 to D3 expansion segments of the 28S rRNA of one population from Cihanbeyli district in Konya province was sequenced and recorded in GenBank (Accession number: MT320536.1). Alignments of the population was identical 98.66% to the populations of P. vulnus available in GenBank (Accs. nos: LT985479.1 and LT965052.1) and 98.65% (Accs. nos: KY424305.1 and KY424304.1).

Graphical ABSTRACT

Root lesion nematodes of the genus Pratylenchus (Filipjev, 1936) are migratory endoparasitic nematodes and the third most damaging nematode species in the world after root knot and cyst nematodes (Castillo and Vovlas, 2007). Currently, according to Janssen et al. (2017), there are 101 species of root lesion nematodes reported worldwide. The most economical important species are P. penetrans, P. thornei, P. neglectus, P. zeae, P. coffeae, and P. vulnus (Jones et al., 2013).

Pratylenchus vulnus infects the cortex of the plant roots and causes necrosis of roots, reduced root system, yellowed leaves, whole plant dwarfing, and plant dead (CABI, 2019). It reproduces on a wide range of plant species from strawberry to walnut on many field crops and forest trees (Pinochet et al., 1992).

The genus Pratylenchus spp. has high intra specific variation. Additionally, low number of diagnostic features depending on the reproductive strategy of the species requires molecular identification (Castillo and Vovlas, 2007). Molecular techniques as RAPD-PCR and sequencing of D2 to D3 expansion segments of the 28S rRNA was used for the identification of P. vulnus on different plant species (Subbotin et al., 2008; Bakooie et al., 2012; Lopez-Nicora et al., 2012). Moreover, real-time PCR provides sensitive identification of the species with species-specific primers using 1/128 of the DNA of one nematode (Huang and Yan, 2017).

Pratylenchus vulnus (Allen and Jensen, 1951) (walnut root lesion nematode) has been reported in China, India, Iran, Israel, Japan, Korea, Kyrgyzstan, Pakistan, Sri Lanka, Cameron, Egypt, Kenya, Reunion, Canada, USA, Cuba, Argentina, Brazil, Uruguay, Belgium, Bulgaria, Denmark, Finland, Russia, France, Germany, Greece, Italy, the Netherlands, Norway, Poland, Slovenia, Spain, UK, Australia, and New Zealand (CABI, 2019).

In Turkey, P. vulnus has been reported infected pepper (Capsicum annum) and rose (Rosa sp.) in Istanbul province, ornamental plants in Izmir province, olive in Samsun and sesame (Sesamum indicum) in Antalya and Mersin provinces (Saltukoglu, 1974; Borazanci, 1977; Kepenekci, 2001, 2002). It is firstly recorded on cereals in Turkey by Yavuzaslanoglu et al. (2020) using species-specific PCR technique. However, positive control specimen was not available in the study. Real-time PCR with positive control was performed for the 17 P. vulnus populations in the current study. In addition, one population was sequenced and recorded in GenBank.

Material and methods

Nematode populations

A total of 17 nematode populations obtained from wheat and barley cultivated fields in Konya and Karaman provinces in Central Anatolian Plateau in Turkey on April in 2016 and 2017 were investigated. Two populations were from Center (lat: 37.256700, lon: 33.402159, barley soil) and Ayrancı (lat: 37. 461495, lon: 33. 899667, barley soil) districts in Karaman province and 15 populations were from Çumra (lat: 37. 652138, lon: 32. 810197, wheat soil), Güneysınır (lat: 37. 267285, lon: 32. 703158, wheat plant), Bozkır (lat: 37.215825, lon: 32. 566730, wheat plant), Yalıhöyük (lat: 37.334460, lon: 32.094510, wheat plant), Beyşehir (lat: 37. 708799, lon: 31. 711387, barley plant), Yunak (lat: 38.813822, lon: 31.753218, wheat plant), Kulu (lat: 39.083393, lon: 32.985203, wheat plant; lat: 39.065963, lon: 33.054807, wheat plant; lat: 39.039809, lon: 33.040935, wheat soil; lat: 38.965648, lon: 33.008739, barley plant), Cihanbeyli (lat: 38.627013, lon: 32.920812, wheat plant; lat: 38.468969, lon: 32.833310, wheat plant), Karatay (lat: 37.877460, lon: 32.914944, wheat plant; lat: 37.978114, lon: 32.710529, barley plant), and Kadınhanı (lat: 38.573150, lon: 32.276917, wheat plant) districts in Konya province.

Nematodes were previously identified with PCR fragments at 287 bp using species-specific D3b-R/Pvul-F primer for P. vulnus (Yavuzaslanoglu et al., 2020).

DNA extraction

Total genomic DNA was extracted from five individual nematodes in 30 µl extraction buffer as described by Yavuzaslanoglu et al. (2018). The sample was placed at −20°C for 1 hr and then incubated at 65°C for 1 hr. The proteinase was deactivated at 95°C for 10 min. DNA template was re-suspended in 20 µl TE (10 mM Tris-HCl, 1 mM EDTA, pH: 8.0) (Al-Banna et al., 1997). Prepared DNA suspension was preserved at −20°C until use.

Real-time PCR

Real-time PCR experiment was set up with DNAs of 17 nematode populations. Negative amplification control (NAC) included distilled water and positive amplification control (PAC) included P. vulnus DNA. Samples were processed using Roche 480 real-time PCR. Study was carried out using Clear Detections nematode species-specific real-time PCR diagnostic kit (Product code: RT-N-D-2006, Wageningen, The Netherlands).

The PCR mixture including the nematode specific primer set was vortexed for 2 sec and transferred 15 µL into each well. A 5 µL of each DNA sample were added into their designated well. Real-Time PCR was run including the following steps; initial template denaturation for 3 min at 95°C, 35 cycles of amplification were DNA denaturation for 10 sec at 95°C, annealing for 60 sec at 63°C, and extension for 30 sec at 72°C. DNA melting curve analysis of the amplicon was performed by increasing the temperature from 72 to 95°C at 0.2 to 0.5°C/sec (ramp). The fluorescent signal was measured using the FAM or SYBR/FAM channel, after every cycle and after every temperature increment of the PCR melting curve.

Sequence alignment

Sequence alignments of one of the P. vulnus population from Cihanbeyli district in Konya province (sample number: 184) were determined.

Nematode DNAs was purified using ExoSAP-ITTM PCR Product Cleanup Reagent (Thermo Fisher Scientific, USA) prior to sequence alignment.

Purified DNA samples were sequenced using ABI3730XL Sanger sequencing device and BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA) in Macrogen laboratory in the Netherlands.

The sequences were deposited into the GenBank database and compared with those of the other P. vulnus populations available at the GenBank sequence database using the BLAST homology search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Results

The 17 P. vulnus populations provided single melting peak at 87.3°C indicating that a single amplicon was detected from real-time PCR (Fig. 1).

Figure 1:

Melting curve of specific amplicons for Pratylenchus vulnus with melting temperature at 87.3°C. (green bands indicate positive control, blue bands indicate negative control, and red bands indicate nematode samples).

10.21307_jofnem-2020-084-f001.jpg

The sequences of D2 to D3 expansion region of 28S rDNA for the one population of P. vulnus (sample no: 184) from Cihanbeyli district of Konya province was examined and deposited in the GenBank database with the accession number of MT320536.1 (https://www.ncbi.nlm.nih.gov/nuccore/1829742534?log$=activity).

Data blast revealed a sequence similarity of 98.66% with P. vulnus samples deposited in GenBank (e.g., Accession Nos: LT985479.1 and LT965052.1) and 98.65% (KY424305.1 and KY424304.1).

Discussion

The real-time PCR was used to estimate the accuracy and sensitivity of molecular identification of P. vulnus and provided functional comparison. Qiu et al. (2007) showed specific detection of P. vulnus obtained from California orchards using real-time PCR assay and species-specific primers designed from ITS sequences of rDNA. Yan et al. (2012, 2013) and Huang and Yan (2017) reported melting curve analysis for P. thornei, P. neglectus, and P. scribneri, similarly to our observations, single peaks were produced at 88.4, 83.8, and 81.5°C temperatures, respectively.

In many research works, D3 expansion region of 26S rDNA has been using for evaluation and identification among species and genera of nematodes (Huang and Yan, 2017) as well as 28S rRNA (Subbotin et al., 2008; Lopez-Nicora et al., 2012; Janssen et al., 2017).

High similarity rate between the sequence alignments of P. vulnus populations from the current study and other geographical areas of the world reported on D2 to D3 expansion regions of 28S rDNA (Al-Banna et al., 1997; Subbotin et al., 2008; Lopez-Nicora et al., 2012; Chihani-Hammas et al., 2018) confirmed the specificity of the molecular identifications.

Molecular identification is specific and reliable in distinguishing nematode species (Nguyen et al., 2017). Nematode species difficult to distinguish morphologically from other species can be successfully identified using molecular characterization.

Detection of P. vulnus on wheat and barley as economically important commodities in Turkey is valuable in order to organize future studies on damage potential and control measurements.

Acknowledgements

This study was supported by Karamanoglu Mehmetbey University Scientific Research Projects Commission, Karaman, Turkey, Project No: 17-YL-17.

References


  1. Al-Banna, L. , Williamson, V. M. and Gardner, S. L. 1997. Phylogenetic analysis of nematodes of the genus Pratylenchus using nuclear 26S rDNA. Molecular Phylogenetics and Evolution 7:94–102.
  2. Allen, M. W. and Jensen, H. J. 1951. Pratylenchus vulnus, new species (Nematada: Prarylenchinae), a parasite of trees and vines in California. Proceedings of the Helminthological Society of Washington 18:4750.
  3. Bakooie, M. , Pourjam, E. and Javaran, M. J. 2012. Investigation of Iranian Pratylenchusvulnus populations by morphological and molecular market (RAPD-PCR). Journal of Agricultural Technology 8:219–231.
  4. Borazanci, N. 1977. Studies on identification and damage potential of plant parasitic nematodes on ornamental plants grown in greenhouses in Izmir province and around. master’s thesis, University of Aegean, Izmir, 180s.
  5. CABI 2019. Invasive species compendium, detailed coverage of invasive species threatening livelihoods and the environment worldwide, Pratylenchusvulnus (walnut root lesion nematode). available at: https://www.cabi.org/isc/datasheet/43904.
  6. Castillo, P. and Vovlas, N. 2007. Pratylenchus (Nematoda: Pratylenchidae). Diagnosis, biology, pathogenicity and management. Nematology monographs & perspectives, Vol. 6 Brill, Leiden.
  7. Chihani-Hammas, N. , Regaieg, H. , Larayedh, A. , Badiss, A. , Qing, Y. and Horrique-Raouani, N. 2018. First report of Pratylenchus vulnus associated with apple in Tunisia. Journal of Nematology 50:579–586.
  8. Filipjev, N. 1936. On the classification of the Tylenchinae. Proceedings of the Helminthological Society of Washington 3:80–82.
  9. Huang, D. and Yan, G. 2017. Specific detection of the root lesion nematode Pratylenchus scribneri using conventional and real time PCR. Plant Disease 101:359–365.
  10. Janssen, T. , Karssen, G. , Orlando, V. , Subbotin, S. A. and Bert, W. 2017. Molecular characterization and species delimiting of plant parasitic nematodes of the genus Pratylenchus from the penetrans group (Nematoda: Pratylenchidae). Molecular Phylogenetics and Evolution 117:30–48.
  11. Jones, J. T. , Haegeman, A. , Danchin, E. G. , Gaur, H. S. , Helder, J. , Jones, M. G. , Kikuchi, T. , Manzanilla-López, R. , Palomares-Rius, J. E. , Wesemael, W. M. and Perry, R. N. 2013. Top 10 plant parasitic nematodes in molecular plant pathology. Molecular Plant Pathology 14:946–961.
  12. Kepenekci, I. 2001. Preliminary list of Tylenchida Nematoda associated with olive in The Black Sea and The Mediterranean Regions of Turkey. Nematologia Mediterranea 29:145–147.
  13. Kepenekci, I. 2002. Plant parasitic nematodes species of Tylenchida Nematoda associated with sesame Sesamum indicum L growing areas in the Mediterranean Region of Turkey. Turkish Journal of Agriculture and Forestry 26:323–330.
  14. Lopez-Nicora, H. D. , Mekete, T. , Taylor, N. J. and Niblack, T. L. 2012. First report of lesion nematode (Pratylenchus vulnus) on Boxwood in Ohio. Disease Notes 96:1385.
  15. Nguyen, T. D. , Le, T. , Nguyen, H. T. , Nguyen, T. , Liebanas, G. and Trinh, Q. P. 2017. Morphological and molecular characteristics of Pratylenchus haiduongensis sp. n., a new species of root-lesion nematodes associated with carrot in Vietnam. Journal of Nematology 49:276–285.
  16. Pinochet, J. , Verdejo, S. , Soler, A. and Canals, J. 1992. Host range of a population of Pratylenchus vulnus in commercial fruit, nut, citrus, and grape rootstocks in Spain. Supplement to Journal of Nematology 24:693–698.
  17. Qiu, J. , Westerdahl, B. B. and Williamson, V. M. 2007. Detection and quantification of root-lesion nematode Pratylenchus vulnus using real time PCR. Journal of Nematology 39:95–96.
  18. Saltukoglu, M. E. 1974. A taxonomical and morphological study of Tylenchida (Nematoda) from the Istanbul Area (Turkey). PhD thesis, Ghent: Ghent University.
  19. Subbotin, S. A. , Ragsdale, E. J. , Mullens, T. , Roberts, P. A. , Mundo-Ocampo, M. and Baldwin, J. G. 2008. A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): evidence from 18S and D2–D3 expansion segments of 28S ribosomal RNA genes and morphological characters. Molecular Phylogenetics and Evolution 48:491–505.
  20. Yan, G. , Smiley, R. W. and Okubara, P. 2012. Detection and Quantification of Pratylenchus thornei in DNA extracted from soil using real time PCR. Phytopathology 102:14–22.
  21. Yan, G. , Smiley, R. W. , Okubara, P. A. , Skantar, A. M. and Reardon, C. L. 2013. Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease 97:757–764.
  22. Yavuzaslanoglu, E. , Ates Sönmezoglu, O. , Genc, N. , Akar, Z. and Terzi, B. 2018. Molecular characterization of Ditylenchus dipsaci on onion in Turkey. European Journal of Plant Pathology 151:195–200.
  23. Yavuzaslanoglu, E. , Karaca, M. S. , Ates Sonmezoglu, O. , Ocal, A. , Elekcioglu, H. I. and Aydogdu, M. 2020. Occurrence and abundance of cereal nematodes in Konya and Karaman Provinces in Turkey. Turkish Journal of Entomology 44:223–236.
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FIGURES & TABLES

Figure 1:

Melting curve of specific amplicons for Pratylenchus vulnus with melting temperature at 87.3°C. (green bands indicate positive control, blue bands indicate negative control, and red bands indicate nematode samples).

Full Size   |   Slide (.pptx)

REFERENCES

  1. Al-Banna, L. , Williamson, V. M. and Gardner, S. L. 1997. Phylogenetic analysis of nematodes of the genus Pratylenchus using nuclear 26S rDNA. Molecular Phylogenetics and Evolution 7:94–102.
  2. Allen, M. W. and Jensen, H. J. 1951. Pratylenchus vulnus, new species (Nematada: Prarylenchinae), a parasite of trees and vines in California. Proceedings of the Helminthological Society of Washington 18:4750.
  3. Bakooie, M. , Pourjam, E. and Javaran, M. J. 2012. Investigation of Iranian Pratylenchusvulnus populations by morphological and molecular market (RAPD-PCR). Journal of Agricultural Technology 8:219–231.
  4. Borazanci, N. 1977. Studies on identification and damage potential of plant parasitic nematodes on ornamental plants grown in greenhouses in Izmir province and around. master’s thesis, University of Aegean, Izmir, 180s.
  5. CABI 2019. Invasive species compendium, detailed coverage of invasive species threatening livelihoods and the environment worldwide, Pratylenchusvulnus (walnut root lesion nematode). available at: https://www.cabi.org/isc/datasheet/43904.
  6. Castillo, P. and Vovlas, N. 2007. Pratylenchus (Nematoda: Pratylenchidae). Diagnosis, biology, pathogenicity and management. Nematology monographs & perspectives, Vol. 6 Brill, Leiden.
  7. Chihani-Hammas, N. , Regaieg, H. , Larayedh, A. , Badiss, A. , Qing, Y. and Horrique-Raouani, N. 2018. First report of Pratylenchus vulnus associated with apple in Tunisia. Journal of Nematology 50:579–586.
  8. Filipjev, N. 1936. On the classification of the Tylenchinae. Proceedings of the Helminthological Society of Washington 3:80–82.
  9. Huang, D. and Yan, G. 2017. Specific detection of the root lesion nematode Pratylenchus scribneri using conventional and real time PCR. Plant Disease 101:359–365.
  10. Janssen, T. , Karssen, G. , Orlando, V. , Subbotin, S. A. and Bert, W. 2017. Molecular characterization and species delimiting of plant parasitic nematodes of the genus Pratylenchus from the penetrans group (Nematoda: Pratylenchidae). Molecular Phylogenetics and Evolution 117:30–48.
  11. Jones, J. T. , Haegeman, A. , Danchin, E. G. , Gaur, H. S. , Helder, J. , Jones, M. G. , Kikuchi, T. , Manzanilla-López, R. , Palomares-Rius, J. E. , Wesemael, W. M. and Perry, R. N. 2013. Top 10 plant parasitic nematodes in molecular plant pathology. Molecular Plant Pathology 14:946–961.
  12. Kepenekci, I. 2001. Preliminary list of Tylenchida Nematoda associated with olive in The Black Sea and The Mediterranean Regions of Turkey. Nematologia Mediterranea 29:145–147.
  13. Kepenekci, I. 2002. Plant parasitic nematodes species of Tylenchida Nematoda associated with sesame Sesamum indicum L growing areas in the Mediterranean Region of Turkey. Turkish Journal of Agriculture and Forestry 26:323–330.
  14. Lopez-Nicora, H. D. , Mekete, T. , Taylor, N. J. and Niblack, T. L. 2012. First report of lesion nematode (Pratylenchus vulnus) on Boxwood in Ohio. Disease Notes 96:1385.
  15. Nguyen, T. D. , Le, T. , Nguyen, H. T. , Nguyen, T. , Liebanas, G. and Trinh, Q. P. 2017. Morphological and molecular characteristics of Pratylenchus haiduongensis sp. n., a new species of root-lesion nematodes associated with carrot in Vietnam. Journal of Nematology 49:276–285.
  16. Pinochet, J. , Verdejo, S. , Soler, A. and Canals, J. 1992. Host range of a population of Pratylenchus vulnus in commercial fruit, nut, citrus, and grape rootstocks in Spain. Supplement to Journal of Nematology 24:693–698.
  17. Qiu, J. , Westerdahl, B. B. and Williamson, V. M. 2007. Detection and quantification of root-lesion nematode Pratylenchus vulnus using real time PCR. Journal of Nematology 39:95–96.
  18. Saltukoglu, M. E. 1974. A taxonomical and morphological study of Tylenchida (Nematoda) from the Istanbul Area (Turkey). PhD thesis, Ghent: Ghent University.
  19. Subbotin, S. A. , Ragsdale, E. J. , Mullens, T. , Roberts, P. A. , Mundo-Ocampo, M. and Baldwin, J. G. 2008. A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): evidence from 18S and D2–D3 expansion segments of 28S ribosomal RNA genes and morphological characters. Molecular Phylogenetics and Evolution 48:491–505.
  20. Yan, G. , Smiley, R. W. and Okubara, P. 2012. Detection and Quantification of Pratylenchus thornei in DNA extracted from soil using real time PCR. Phytopathology 102:14–22.
  21. Yan, G. , Smiley, R. W. , Okubara, P. A. , Skantar, A. M. and Reardon, C. L. 2013. Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease 97:757–764.
  22. Yavuzaslanoglu, E. , Ates Sönmezoglu, O. , Genc, N. , Akar, Z. and Terzi, B. 2018. Molecular characterization of Ditylenchus dipsaci on onion in Turkey. European Journal of Plant Pathology 151:195–200.
  23. Yavuzaslanoglu, E. , Karaca, M. S. , Ates Sonmezoglu, O. , Ocal, A. , Elekcioglu, H. I. and Aydogdu, M. 2020. Occurrence and abundance of cereal nematodes in Konya and Karaman Provinces in Turkey. Turkish Journal of Entomology 44:223–236.

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