First report of Pratylenchus vulnus associated with apple in Tunisia

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VOLUME 50 , ISSUE 4 (December 2018) > List of articles

First report of Pratylenchus vulnus associated with apple in Tunisia

Noura Chihani-Hammas / Lobna Hajji-Hedfi * / Hajer Regaieg / Asma Larayedh / Ahmed Badiss / Yu Qing / Horrigue-Raouani Najet

Keywords : Pratylenchus, Morphometry, Molecular identification, Apple, Tunisia

Citation Information : Journal of Nematology. VOLUME 50 , ISSUE 4 , ISSN (Online) 2640-396X, DOI: 10.21307/jofnem-2018-056, December 2018 © 2018.© The Society of Nematologists

License : (PUBLISHER)

Published Online: 03-December-2018

ARTICLE

ABSTRACT

The root-lesion nematode of the genus Pratylenchus Filipjev (1936) has a worldwide distribution and cause severe production constraints on numerous important crops. In 2013-14, during a survey of the apple nurseries and orchards in center of Tunisia (Kairouan, Zaghouan, Monastir and Kasserine), 70 different roots and soil samples were collected. The populations of root-lesion nematode were identified on the basis of their morphological and morphometric characters, and by molecular methods. Microscopic observation of females and males demonstrated the occurrence of Pratylenchusd vulnus on apple trees. The ribosomal DNA D2-D3 expansion segments of the 28S rRNA and of the Pratylenchus populations were PCR amplified and sequenced. The sequences were compared with those of Pratylenchus species in the GenBank database with high similarity (99%). This comparison reconfirmed the morphological identifications. Phylogenetic studies placed those populations with P. vulnus. This is the first report of P. vulnus infecting apple in Tunisia.

Graphical ABSTRACT

Along with dates and citrus, which represent the main fruit tree commodities produced and exported in Tunisia, apples have an important place in the fruit sector. Pome and stone fruit trees can be infected by plant parasitic nematodes which are a serious problem influencing the growth and production of trees in all major fruit producing area (Askary et al., 2012). The root-lesion nematodes, Pratylenchus genus, are common endoparasites of plants worldwide. These nematodes cause severe production constraints and have great economic impact on numerous important crops (Castillo and Vovlas, 2007). They are migratory parasites, cause local lesions, and affect root growth by penetrating and feeding on young roots of host plant (Pinochet et al., 1996).

Within the genus Pratylenchus, 12 species have been reported as potential pathogens on apple (Castillo and Vovlas, 2007). Moreover, root-lesion nematodes, particularly Pratylenchus vulnus, is a major pest attacking pome and stone fruit crops in warm Mediterranean environments (Mckenry, 1989; Pinochet et al., 1992; Nyczepir and Becker, 1998; Askary et al., 2012).

The purpose of this study is to identity morphologically and by molecular tools the population of plant parasitic nematode (Pratylenchus spp.) in apple nurseries and orchards in Tunisia.

Materials and methods

Nematodes collection and extraction

Nematode surveys were conducted during 2013-14 in three different sites (stoolbeds, nurseries, and orchards) located in central Tunisia (Kairouan, Zaghouan, Kasserine, Jendouba, and Monastir). Samples were collected with a shovel from the upper 50 cm of soil and roots of four to five holes. Nematodes were extracted from 500 g of soil and from 1 g of roots by a modified sugar centrifugal-flotation method (De Grisse, 1969). Single females of root-lesion nematodes were transferred to carrot discs at 25°C (Castillo et al., 1995). After incubation of carrot cultures for up 8 wk, the carrot discs were cut into pieces in petri dishes containing sterile water. Then, the petri plates were incubated for 24 hr and collected by sterile Pasteur pipettes. Finally, purified nematodes extracted from carrot discs were used for further morphological and molecular analysis.

Morphological identification

Nematodes were preliminarily identified by morphological features. For that, five females and five males of each population of Pratylenchus were killed by gentle heat, fixed in buffered formalin (Seinhorst, 1959), and processed to pure glycerin. Specimens were examined using a Nikon Eclipse 80i compound microscope with Normarski differential interference contrast at powers up to 1,000× magnification. Measurements were made on glycerin infiltrated specimens with Nikon Digital Sight DS-FI2 K10410 camera and expressed in µm. The used measurement’s abbreviations were defined as Siddiqi (2000). Camera observations were performed as defined by De Man (1880). Nematodes were identified to the species level using Castillo and Vovlas (2007) diagnostic keys.

The scanning electron microscopy (SEM) of Pratylenchus males and females were assessed as described by Abolafi (2015).

Molecular identification

DNA extraction

DNA was extracted from several specimens from each sample. Each nematodes specimen was transferred to an Eppendorf tube containing 30 µL 10× PCR buffer (100 mMTris-HCl, pH 9.0 at 25°C, 500 mMKCl, 15 mM MgCl2), 10 µL Proteinase K (1 mg/mL), 50 µL distilled water. Specimens were crushed for 3 min with an ultrasonic homogenizer. The tubes were incubated at 68°C for 2 hr, then at 100°C for 15 min and stored at −20°C.

DNA amplification and cloning

The forward 18S-F (5′-TTGGATAACTGTGGTTTAACTAG-3′) and the reverse 18S-R (5′-ATTTCACCTCTCACGCAACA-3′) primers and the forward D2a (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and the reverse D3b (5′-TCGGAAGGAACCAGCTACTA-3′) were used for amplification and sequencing of the partial 18S r RNA genes and the D2-D3 expansion segments of the 28S r RNA, respectively. The amplification condition were: 95°C for 3 min, followed by 40 cycles of 30 sec at 95°C, 45 sec at 60°C and 2 min at 72°C, with final extension of 10 min at 72°C. All PCR reactions were performed in 25 µl volumes including 3 µl DNA, 2.5 µl 10× PCR buffer, 1.25 µl of 2.5 mM dNTPs, 0.4 µl from each primers and 0.25 µl Titanium Taq.

The PCR products were separated by electrophoresis (110 V, 45 min) in 2.0% agarose gels in TAE buffer with 2.5 µl DNA Ladder. The gels were stained with Ethidium bromide, visualized, and photographed under UV-light (Bio-rad DX, USA). All reactions were repeated twice for clear and stable banding patterns. The presence or absence of DNA fragments was scored as 1 or 0, respectively, in the binary matrix. Simple matching coefficients (SM) (Digby and Kempton, 1987).

Sequence alignment and phylogenetic analysis

The 18S and 28S fragments were sequenced in-house with an ABI Prism 377 sequencer (Perkin Elmer) in both directions and unambiguous consensus sequences obtained. The sequences were deposited into the GenBank database. DNA sequences were aligned by Clustal W (http://workbench.sdsc.edu, Bioinformatics and Computational Biology group, Department of Bioengineering, UC San Diego, CA). The sequences were compared with those of the other Pratylenchus species available at the GenBank sequence database using the BLAST homology search program. The model of base substitution was evaluated using MODELTEST (Posada and Crandall, 1998). The Pratylenchus sequences were aligned using CLUSTALW implemented in the MEGA package (Kumar et al., 2008). Clade reliability was examined though a nonparametric bootstrap with 1,000 replicated samples. The phylogenetic tree was constructed by neighbor-joining method with MEGA package v.7 (Kumar et al., 2016).

Results and discussion

Morphological identification showed the presence of P. vulnus in root and soil samples of apple trees located in the four regions visited and their abundance compared to other plant parasitic nematode varied between 58.24% in orchard in the region of Kasserine and 91.45% in nursery located in the region of Kairouan. All the qualitative and quantitative characters including number of lip annuli, spermatheca (presence and shape), post-vulval uterine sac, and tail shape of Tunisian populations of P. vulnus agreed with the original descriptions (Figs 13; Table 1). However, a few characters fell outside the range of the original descriptions. The measurements reported in the original description of Gao et al. (1999): 0.50 to 0.81 mm for female and 0.43 to 0.61 mm for male were similar to ours Pratylenchus specimens.

Table 1

Morphometrics of Pratylenchus vulnus from apples stoolbeds, nurseries (males and females), and orchards from four Tunisian regions (Kasserin, Kairouan, Monastir, and Zaghouan, Tunisia).

10.21307_jofnem-2018-056-tbl1.jpg

The qualitative morphological characteristics of the Pratylenchus populations used in this study followed the original species descriptions, but some morphometric discrepancies were found (Figs 2,3). Such morphometric variations in Pratylenchus confirmed the presence of different morphotypes used in this study. This intraspecific variability could be due different reproductive strategy of a population, isolates variation, environment conditions, and geographical localization (Pourjam et al., 1997; Troccoli et al., 2016).

Phylogenetic relationships within Pratylenchus species based on the 18S and 28S sequences were generated by MEGA and minimum evolution (Figs 4,5). The 18S and D2/D3 expansion fragments of the 28S rDNA idendity ranged from 98 to 99% identity with P. vulnus worldwide. The biogeography history of P. vulnus could be clarified by understanding the phylogenetic relationships among several species of nematodes. The 18S phylogenetic constructed tree showed that our P. vulnus is closely similar to two clades: P. thornei, clade included a group of P. crenatus, P. pratensis species. The phyologenetic tree based on 28S gene revealed the strong relationship of P. vulnus with P. neglectus, P. parazae, P. thornei, and P. mediterraneus.

Morphometric informations and phylogenetic analysis clarified the unambiguous species identification and complemented to reveal the first report of P. vulnus infecting apple in Tunisia. The morphometry was reported not robust and must be complemented by molecular tools in P. penetrans group (Handoo et al., 2008; Janssen et al., 2017).

Figure 1

Photo micrographs of Pratylenchus vulnus specimens: females (B) and males (A), (C): lateral field at mid – body; (D): spermatheca; (E): pharyngeal region; (F) and (H): female tails; (G): vulval region; (I): male tail (Scale bars: A, B = 100 mm; C–I = 20 mm).

10.21307_jofnem-2018-056-f001.jpg
Figure 2

SEM micrograph of P. vulnus male morphology from Tunisia (A, B, C, D): anterior region, (E, F) : tail, (G) : lateral fields; sp : spicules, lig : 4 lateral lines ; anx : 3 labial annuli).

10.21307_jofnem-2018-056-f002.jpg
Figure 3

SEM micrograph of P. vulnus female morphology from Tunisia (A; B; C): anterior region, (D): lateral lines at vulva region, (E): vulva, (F, G): tail, (H): lateral fields).

10.21307_jofnem-2018-056-f003.jpg
Figure 4

Phylogenetic relationships between Pratylenchus vulnus identified in Tunisia and other Pratylenchus already deposited in GenBank. The Pratylenchus sequences aligned in MEGA with CLUSTALW and the phylogenetic tree made by MEGA version 7.0 based on the Neighbor-Joining method as inferred from partial 18S rRNA gene.

10.21307_jofnem-2018-056-f004.jpg
Figure 5

Phylogenetic relationships between Pratylenchus vulnus identified in Tunisia and other Pratylenchus already deposited in GenBank. The Pratylenchus sequences aligned in MEGA with CLUSTALW and the phylogenetic tree made by MEGA version 7.0 based on the Neighbor-Joining method as inferred from D2-D3 expansion segments of the 28S rRNA.

10.21307_jofnem-2018-056-f005.jpg

Acknowledgements

Compliance with ethical standards: This research does not contain any conflicts of interest, nor research involving humans or animals. Lobna Hajji-Hedfi and Noura Chihani-Hammas contributed equally to this work.

References


  1. Abolafi, J.. 2015. A low-cost technique to manufacture a container to process meiofauna for scanning electron microscopy. Microscopy Research and Technique 78: 771-776.
    [CROSSREF]
  2. Askary, T. H., Banday, S. A., Iqbal, U., Khan, A. A., Mir, M. M. and Waliullah, M. I. S.. 2012. Plant parasitic nematode diversity in pome, stone and nut fruits, in Lichtfouse, E.. (Eds), Agroecology and strategies for climate change: sustainable agriculture reviews 8, Springer, Dordrecht.
  3. Castillo, M., Lam, Y. W. M., Dooley, M. A., Stahl, E. and Smith, P. C.. 1995. Disposition and covalent binding of Ibuprofen and its acyl glucuronide in the elderly. Clinical Pharmacology Therapeutics 57: 636-644.
    [CROSSREF]
  4. Castillo, P. and Vovlas, N.. 2007. Pratylenchus (Nematoda: Pratylenchidae): diagnosis, biology, pathogenicity and management, in Hunt, D. J. and Perry, R. N.. (Eds), Nematology monographs and perspectives 6, Brill, Leiden, p. 529.
  5. De Grisse, A. T.. 1969. Redescription ou modification de quelques techniques utilisées dans l’étude des nématodes phytoparasitaires. Meded. Rijksfac. Landb Wet. Gent 34: 351-359.
  6. De Man, J. G.. 1880. Die einheimischen, frei in der reinenErde und im süssen Wasser lebenden Nematoden. Tijdschr. Nederl. Dierk. Vereen 5: 1-104.
  7. Digby, P. and Kempton, R.. 1987), Multivariate analysis of ecological communities, Chapman and Hall, London.
  8. Doucet, M. E. and Lax, P.. 1997. Caracterización de una población y un aislado de Pratylenchus vulnus Allen et Jensen, 1951 (Nematoda: Tylenchida) provenientes de la Provincia de Córdoba, Argentina. Nematologia Mediterranea 25: 287-298.
  9. Filipjev, I. N.. 1936. On the classification of the Tylenchinae. Proceedings of the Helminthological Society of Washington, 3: 80-82.
  10. Gao, X., Cheng, H. and Fang, C.. 1999. New diagnostic characters for Pratylenchus vulnus. Nematologia Mediterranea 27: 9-13.
  11. Handoo, Z. A., Carta, L. K. and Skantar, A. M.. 2008. Taxonomy, morphology and phylogenetics of coffee-associated root-lesion nematodes, pratylenchus spp, in Souza, R. M.. (Ed.), Plant-parasitic nematodes of coffee, Springer, Dordrecht 29-50.
  12. 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, available at: http://dx.doi.org/10.1016/j.ympev
    [CROSSREF] [URL]
  13. Kumar, S., Nei, M., Dudley, J. and Tamura, K.. 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9: 299-306.
    [CROSSREF]
  14. Kumar, S., Stecher, G. and Tamura, K.. 2016. Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.
    [CROSSREF]
  15. Mckenry, M. V.. 1989. Damage and development of several nematode species in a plum orchard. Applied Agricultural Research 4: 10-14.
  16. Nyczepir, A. P. and Becker, J. O.. 1998. Fruit and citrus trees, in Barker, K. R., Pederson, G. A. and Windham, G. L.. (Eds), Plant and nematode interactions, American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, WI, 637-684.
  17. Pinochet, J., Fernandez, C., Alaniz, E. and Felipe, A.. 1996. Damage by a lesion nematode, Pratylenchus vulnus to Prunus rootstocks. Plant Disease 80: 754-757.
    [CROSSREF]
  18. 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 stocks in Spain. Journal of Nematology 24: 693-698.
  19. Posada, D. and Crandall, K. A.. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817-818.
    [CROSSREF]
  20. Pourjam, E., Kheiri, A. and Geraert, E.. 1997. The genus Pratylenchus Filipjev, 1936 (Tylenchida: Pratylenchidae) from north of Iran. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, University Gent, 62/3a, 741-756.
  21. Seinhorst, J. W.. 1959. Two new species of Pratylenchus. Nematologica 4: 83-86.
    [CROSSREF]
  22. Siddiqi, M. R.. 2000. Tylenchida Parasites of Plants and Insects, 2nd ed., CABI Publishing, Wallingford.
  23. Troccoli, A., Subbotin, S. A., Chitambar, J. J., Janssen, T., Waeyenberge, L., et al 2016. Characterization of amphimictic and parthenogenetic populations of Pratylenchus bolivianus Corbett, 1983 (Nematoda: Pratylenchidae) and their phylogenetic relationships with closely related species. Nematology 18: 651-678.
    [CROSSREF]
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FIGURES & TABLES

Figure 1

Photo micrographs of Pratylenchus vulnus specimens: females (B) and males (A), (C): lateral field at mid – body; (D): spermatheca; (E): pharyngeal region; (F) and (H): female tails; (G): vulval region; (I): male tail (Scale bars: A, B = 100 mm; C–I = 20 mm).

Full Size   |   Slide (.pptx)

Figure 2

SEM micrograph of P. vulnus male morphology from Tunisia (A, B, C, D): anterior region, (E, F) : tail, (G) : lateral fields; sp : spicules, lig : 4 lateral lines ; anx : 3 labial annuli).

Full Size   |   Slide (.pptx)

Figure 3

SEM micrograph of P. vulnus female morphology from Tunisia (A; B; C): anterior region, (D): lateral lines at vulva region, (E): vulva, (F, G): tail, (H): lateral fields).

Full Size   |   Slide (.pptx)

Figure 4

Phylogenetic relationships between Pratylenchus vulnus identified in Tunisia and other Pratylenchus already deposited in GenBank. The Pratylenchus sequences aligned in MEGA with CLUSTALW and the phylogenetic tree made by MEGA version 7.0 based on the Neighbor-Joining method as inferred from partial 18S rRNA gene.

Full Size   |   Slide (.pptx)

Figure 5

Phylogenetic relationships between Pratylenchus vulnus identified in Tunisia and other Pratylenchus already deposited in GenBank. The Pratylenchus sequences aligned in MEGA with CLUSTALW and the phylogenetic tree made by MEGA version 7.0 based on the Neighbor-Joining method as inferred from D2-D3 expansion segments of the 28S rRNA.

Full Size   |   Slide (.pptx)

REFERENCES

  1. Abolafi, J.. 2015. A low-cost technique to manufacture a container to process meiofauna for scanning electron microscopy. Microscopy Research and Technique 78: 771-776.
    [CROSSREF]
  2. Askary, T. H., Banday, S. A., Iqbal, U., Khan, A. A., Mir, M. M. and Waliullah, M. I. S.. 2012. Plant parasitic nematode diversity in pome, stone and nut fruits, in Lichtfouse, E.. (Eds), Agroecology and strategies for climate change: sustainable agriculture reviews 8, Springer, Dordrecht.
  3. Castillo, M., Lam, Y. W. M., Dooley, M. A., Stahl, E. and Smith, P. C.. 1995. Disposition and covalent binding of Ibuprofen and its acyl glucuronide in the elderly. Clinical Pharmacology Therapeutics 57: 636-644.
    [CROSSREF]
  4. Castillo, P. and Vovlas, N.. 2007. Pratylenchus (Nematoda: Pratylenchidae): diagnosis, biology, pathogenicity and management, in Hunt, D. J. and Perry, R. N.. (Eds), Nematology monographs and perspectives 6, Brill, Leiden, p. 529.
  5. De Grisse, A. T.. 1969. Redescription ou modification de quelques techniques utilisées dans l’étude des nématodes phytoparasitaires. Meded. Rijksfac. Landb Wet. Gent 34: 351-359.
  6. De Man, J. G.. 1880. Die einheimischen, frei in der reinenErde und im süssen Wasser lebenden Nematoden. Tijdschr. Nederl. Dierk. Vereen 5: 1-104.
  7. Digby, P. and Kempton, R.. 1987), Multivariate analysis of ecological communities, Chapman and Hall, London.
  8. Doucet, M. E. and Lax, P.. 1997. Caracterización de una población y un aislado de Pratylenchus vulnus Allen et Jensen, 1951 (Nematoda: Tylenchida) provenientes de la Provincia de Córdoba, Argentina. Nematologia Mediterranea 25: 287-298.
  9. Filipjev, I. N.. 1936. On the classification of the Tylenchinae. Proceedings of the Helminthological Society of Washington, 3: 80-82.
  10. Gao, X., Cheng, H. and Fang, C.. 1999. New diagnostic characters for Pratylenchus vulnus. Nematologia Mediterranea 27: 9-13.
  11. Handoo, Z. A., Carta, L. K. and Skantar, A. M.. 2008. Taxonomy, morphology and phylogenetics of coffee-associated root-lesion nematodes, pratylenchus spp, in Souza, R. M.. (Ed.), Plant-parasitic nematodes of coffee, Springer, Dordrecht 29-50.
  12. 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, available at: http://dx.doi.org/10.1016/j.ympev
    [CROSSREF] [URL]
  13. Kumar, S., Nei, M., Dudley, J. and Tamura, K.. 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9: 299-306.
    [CROSSREF]
  14. Kumar, S., Stecher, G. and Tamura, K.. 2016. Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.
    [CROSSREF]
  15. Mckenry, M. V.. 1989. Damage and development of several nematode species in a plum orchard. Applied Agricultural Research 4: 10-14.
  16. Nyczepir, A. P. and Becker, J. O.. 1998. Fruit and citrus trees, in Barker, K. R., Pederson, G. A. and Windham, G. L.. (Eds), Plant and nematode interactions, American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, WI, 637-684.
  17. Pinochet, J., Fernandez, C., Alaniz, E. and Felipe, A.. 1996. Damage by a lesion nematode, Pratylenchus vulnus to Prunus rootstocks. Plant Disease 80: 754-757.
    [CROSSREF]
  18. 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 stocks in Spain. Journal of Nematology 24: 693-698.
  19. Posada, D. and Crandall, K. A.. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817-818.
    [CROSSREF]
  20. Pourjam, E., Kheiri, A. and Geraert, E.. 1997. The genus Pratylenchus Filipjev, 1936 (Tylenchida: Pratylenchidae) from north of Iran. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, University Gent, 62/3a, 741-756.
  21. Seinhorst, J. W.. 1959. Two new species of Pratylenchus. Nematologica 4: 83-86.
    [CROSSREF]
  22. Siddiqi, M. R.. 2000. Tylenchida Parasites of Plants and Insects, 2nd ed., CABI Publishing, Wallingford.
  23. Troccoli, A., Subbotin, S. A., Chitambar, J. J., Janssen, T., Waeyenberge, L., et al 2016. Characterization of amphimictic and parthenogenetic populations of Pratylenchus bolivianus Corbett, 1983 (Nematoda: Pratylenchidae) and their phylogenetic relationships with closely related species. Nematology 18: 651-678.
    [CROSSREF]

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