diversity, characterization, diagnostics and phylogeny of Plant-parasitic nematodes from neglected biotopes in belgium
Tien Nguyen Huu
Student number: 01608112
Academic year: 2016 – 2018
Indicate the state of the art.
1.1. Nematodes in general
Currently, nematodes are considered as one of the most numerous Metazoa on our planet. They can be either free-living, plant-parasitic nematodes or animal parasites. Different groups of nematodes have adapted to almost every habitat through the evolution over time. To date, the phylum Nematoda consists of about 27000 described species. However, the prediction of the real nematode number can up to a hundred million. Plant-parasitic nematodes are one of the most important pests due to their widespread distribution, host range and potential damage on various host plant. Currently, the number of described plant-parasitic nematodes is around 4100 species over the world (Perry & Moens, 2013).
1.2. Nematofauna in Belgium
Belgium has a long “nematological tradition” with the relatively well-studied nematofauna. Recently, Steel et al. (2014) provided a updated list of Belgian nematofauna with 418 species, 127 of them are new compared to the lists of Coomans (1989) and Bert et al. (2003). According to Sewell (1970), Moens and Hendrickx (1990), Subbotin et al. (2000), Damme et al. (2013), Maria et al. (2015), Qing et al. (2015), Troccoli et al. (2016), Consoli et al. (2017), Janssen (2017), and Slos et al. (2017), the list of 1 free-living and 14 plant-parasitic nematode species should be added to Belgian nematofauna comparing with Steel et al. (2014).
It is easyy to recognize that the new species descriptions for Belgian nematofauna list is being updated year by year. Certainly, it is predictable that there still exist a lot of unexplored species in the world generally and in Belgium particularly. In addition, the previous data of plant-parasitic nematodes in Belgium were mainly from ‘classic’ biotopes with a strong dominance of agricultural and horticultural ecosystems (Bert et al., 2003; Steel et al., 2014). Therefore, in order to obtain a more comprehensive overview of the Belgian nematode diversity, it is necessary to investigate nematodes from various habitats including agricultural, horticultural, coastal line and forest ecosystems.
1.3. Taxonomy of nematodes
To assess biodiversity, to understand species distribution and to understand community structures and ecosystem functions, the taxonomy of nematodes is really important. In the past, nematode taxonomy was generally limited to ?-taxonomy: the description of taxa, and mainly of species and genera. By that time, taxonomy was mostly in view of morphology. In term of morphology, although nematodes are highly diverse within the phylum Nematoda, but the intra-generic variations are very few in some genera. Hence, morphological characters alone are insufficient to resolve all the relationships, and this has resulted in controversial problems in nematode classification.
Currently, the shift from using purely phenotypic methods to using molecular approach is becoming more prevalent in nematology. Various molecular techniques such as protein-based analysis, PCR, q-PCR, RFLP and RAPD analyses were used successfully for nematode identification. However, amplification and sequencing of diagnostic regions of nematode DNA have become the most reliable source for enhancing our comprehension of evolutionary and genetic relationships. Regardless of many advantages of molecular approach, they have the own limitations ranging from the quality of sequences to the methods to analyze the evolution and relationships.
Consequently, in order to avoid the misidentifications and the appearance of mislabeled sequences on GenBank as well as other limitations of the molecular approach, the combination of DNA sequences and morphological characteristics is desperately needed. This study focuses on the investigation of plant-parasitic nematodes from all the neglected biotopes and this will provide a more detailed description of plant-parasitic nematode biodiversity in general and in Belgium particularly. The combination of molecular and morphological data in classification will contribute the knowledge to understand the controversial taxonomical problems as well as the phylogenetic relationships.
Describe the objectives of the research.
Describe the envisaged research and the research hypothesis, why it is important to the field, what impact it could have, whether and how it is specifically unconventional and challenging.
(1) To resolve the controversial taxonomical problems and obtain more comprehensive overview of nematode phylogenetic relationships by using integrate approach.
(2) To update the biology and taxonomy for poorly studied plant-parasitic nematodes
(3) To provide useful DNA barcodes for nematode identification in the future.
Describe the methodology of your research.
Be as detailed as necessary for a clear understanding of what you propose. Describe the different envisaged steps in your research, including intermediate goals. Indicate how you will handle unforeseen circumstances, intermediate results and risks. Show where the proposed methodology is according to the state of the art and where it is novel. Enclose risks that might endanger reaching project objectives and the contingency plans to be put in place should risk occur.
Sampling and extraction
Soil and root samples will be collected randomly by an auger (5x30cm) in three main ecosystem types including agricultural, horticultural, coastal line and forest ecosystems throughout Belgium. The information of sampling sites will be collected at the same time. Each sample (approximately 1kg) will be put in a separate plastic bag and brought to the lab to extract nematodes.
Samples can be kept at 4oC if the step of extraction can not be processed immediately. For extraction of nematodes, 100ml of soil (or pieces of roots) will be spread on the tissue that is supported by a 140 mm diameter wide-mesh plastic screen and put on another plastic tray. Water will be added until it just touch the soil and make the soil become wet enough. The screen will be removed after 24 hours to collect nematodes. Swollen nematodes will be dissected from root tissues under a stereomicroscope using a scalpel.
Preparation for PCR
For molecular work, temporary slides of nematode will be made and digital light microscope pictures will be taken as a morphological voucher. After that, nematode will be cut into pieces and put in an Eppendorf tube with 20µ of WLB and will be frozen for at least 10 min at ?20°C. 1?l proteinase K (1.2 mg ml?1) will be added before incubation in a PCR machine for 1 h at 65°C and 10 min at 95°C and centrifugation in 1 minute at maximum speed. Then, the sample can be stored for a long time at ?20°C before running PCR.
Fxing and mounting
For morphological work, permanent slides will be made by heat-killed nematodes after the steps of fixation by 4% PFA in PBS (+1% Glycerin) and ethanol-glycerin dehydration (Viaene et al., 2016).
Measurements and drawings will be prepared manually by an Olympus BX51 DIC Microscope with the supports of drawing tube and digital camera. Illustrations will be obtained using Illustrator ® CS 3 software based on light microscopic drawings. For scanning electron microscopy (Coosemans), specimens will be processed and viewed following the procedure of (Eisenback, 1986).
For each PCR reaction: 23 µl of Mastermix (17µl Water; 2.5 µl 10x buffer; 2 µl MgCL2; 2.5 µl Coralload; 0.5µl dNTP (10mM); 0.5µl Primer 1; 0.5µl Primer 2; 0.06 µl Toptaq) and 2µl DNA template will be used. The protocol for PCR reaction will be adapted for different primers (D2-D3, ITS, COI…, depending on the genus). The amplicon will be visualized by gel electrophoresis to check the result of PCR reaction quickly.
Successful PCR reactions will be purified and sequenced commercially by Macrogen Inc. (Europe). Consensus sequences will be assembled using GENEIOUS R11. All contigs will be used for a BLAST search on GenBank to check for closely related species.
MEGA 7 will be used for the alignment, selecting the best model and creating the phylogenetic trees. Poorly aligned regions can be removed from the alignments using Gblock. The phylogenetic trees can also be created by other methods such as MrBayes to analyze the phylogeny as well as the relationship. The data will be combined with morphological data to resolve the controversial taxonomical problems.
Provide a work plan, i.e. the different work packages and a detailed timetable.
Describe the different work packages (WP) the proposed research work will be divided in. Indicate for each WP the time that it is expected to take. You might use a table or another type of scheme to clarify the work plan.
1. Work plan
2. Time table
(for 4 ecosystem types)
Preparation for PCR
Note: Q1: January->March; Q2: April->June; Q3: July->September; Q4: October->December
Enumerate the bibliographical references that are relevant for your research proposal.
Bert, W., Coomans, A., Claerbout, F., Geraert, E. & Borgonie, G. 2003. Tylenchomorpha (Nematoda: Tylenchida) in Belgium, an updated list. Nematology 5, 435-440.
Consoli, E., Akanwari, J. & Subbotin, S. A. 2017. Morphological and molecular characterisation of Paratrophurus bursifer (Loof, 1960) Siddiqi, 1971 (Nematoda: Tylenchida) from Belgium. Russian Journal of Nematology 25, 17 – 22.
Coomans, A. Overzicht van de vrijlevende nematofauna van België (Nematoda). Proceedings of the symposium “Invertebraten van België”, 1989 Brussels, Belgium. Royal Belgian Institute of Natural Sciences, 43-56.
Coosemans, J. Nematoden als indicators voor bodemclassificatie. In: Peeters, M. & Van Goethem, J. L., eds. Proceedings of the symposium “Status and trends of the Belgian fauna with a particular emphasis on alien species”, 2002 Brussels, Belgium Bulletin van het Koninklijk Belgisch Instituut voor Natuurwetenschappen Biologie, 51-62.
Damme, N., Waeyenberge, L., Viaene, N., Van Hoenselaar, T. & Karssen, G. 2013. First report of the root-knot nematode Meloidogyne artiellia in Belgium. Plant Disease 97, 152.
Eisenback, J. D. 1986. A comparison of techniques useful for preparing nematodes for scanning electron microscopy. J. Nematol 18, 479-87.
Janssen, T. 2017. Diversity, phylogeny, characterization and diagnostics of root-knot and lesion nematodes. PhD, Ghent University.
Maria, M., Fang, Y., He, J., Gu, J. & Li, H. 2015. Bursaphelenchus parantoniae n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from Belgium. Nematology 17, 1141-1152.
Moens, M. & Hendrickx, G. 1990. Nematode infection by recirculating nutrient solutions in gullies. Meded. Fac. Landbouwwet. Rijksuniv. (Gent) 55, 739-743.
Perry, R. & Moens, M. 2013. Plant nematology, CABI, pp.
Qing, X., Slos, D., Claeys, M. & Bert, W. 2015. Ultrastructural, phylogenetic and rRNA secondary structural analysis of a new nematode (Nematoda: Tylenchomorpha) from mushroom with recovery of intestinal crystals. Zoologischer Anzeiger 269, 13–25.
Sewell, R. 1970. Plant parasitic nematodes from Canada and abroad 1969. Can. Pl. Dis. Surv 50, 102-103.
Slos, D., Walter, S., Lewis, S., Wim, B. & Mark, B. 2017. Caenorhabditis monodelphis sp. n.: defining the stem morphology and genomics of the genus Caenorhabditis. BMC Zoology 2, 4.
Steel, H., Coomans, A., Decraemer, W., Moens, T. & Bert, W. 2014. “Nematodes from Terrestrial, Freshwater and Brackish Water Habitats in Belgium: An Updated List with Special Emphasis on Compost Nematodes.” Zootaxa 3756, 2208-2218.
Subbotin, S. A., Waeyenberge, L. & Moens, M. 2000. Identification of cyst forming nematodes of genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP. Nematology 2, 153-164.
Troccoli, A., Subbotin, S. A., Chitambar, J. J., Janssen, T., Waeyenberge, L., Stanley, J. D., Duncan, L. W., Agudelo, P., Uribe, G. E. M., Franco, J. & Inserra, R. N. 2016. Characterisation of amphimictic and parthenogenetic populations of Pratylenchus bolivianus Corbett, 1983 (Nematoda: Pratylenchidae) and their phylogenetic relationships with closely related species. Nematology 18, 651-678.
Viaene, N., Smol, N. & Wim, B. 2016. General techniques in nematology, Academia Press.
Indicate below whether you think the results of the proposed research will be suitable to be communicated to a non?expert audience and how you would undertake such communication.
FWO encourages its fellows to disseminate the results of their research widely, and valorize them where possible.
The proposed research will provide new insights and comprehensive data of plant-parasitic nematodes in Belgium. This should significantly contribute to the taxonomy and systematics of plant-parasitic nematodes that can be used as good reference for nematologists, students or people from other fields who are interesting in nematology.
On the other hand, the nematode biodiversity and biogeography studies provide the useful information for farmers and government in control and management of nematodes.
To popularize widely the useful results of this study, all the data can be printed as a thesis and the important information can be published as posters or articles.
Please provide full bibliographic details of your five main publications and update all your scientific publications through the E-portal. (Optional)
List all authors, title of publication and journal name (without abbreviations) with volume, page and year. Mention impact factor of the journal and whether the publication was peer reviewed or not.
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