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     LITERATURE STUDY 1.1. State of the art of theresearch topic Nematodes are considered as one of the most numerousMetazoa on our planet. They can be either free-living or plant-parasitic or animalparasites. Although they occur in almost every habitat, they are essentiallyaquatic animals. Soil structure, soil pH and other factors can affect nematodesby different ways (Decraemer & Hunt,2013).

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Different groups of nematodes have adapted to different habitats through theevolution over time. Up to now, approximately 4100 nematode species have beendescribed as plant-parasites over the world (Decraemer & Hunt,2013). InBelgium, the nematofauna has been relatively well studied. However, a lot ofnew species descriptions are being updated year by year. Therefore, In order toobtain a more comprehensive overview of the nematode diversity, it is necessaryto investigate nematodes from various habitats.This thesis focuses on the investigation of plant-parasiticnematodes from neglected biotopes that provide a more detailed description ofplant-parasitic nematode biodiversity in Belgium.

The combination of molecularand morphological data in classification will contribute the knowledge tounderstand the controversial taxonomical problems as well as phylogeneticrelationships.1.2. Nematodes in generalNematodes are pseudocoelomate, unsegmented worm-likeanimals, commonly described as filiform or thread-like, a characteristicreflected by the taxon name nema(Greek, nema= thread) and itsnominative plural nemata (Decraemer & Hunt,2013).The history of nematodes was marked with the oldestreference from China in 2500 B.C. with the description of symptoms andtreatment of the relatively large intestinal roundworm Ascaris or Huei Ch’ung (Maggenti, 1981).

Due to their small size and atypical symptom, thereports of plant-parasitic nematodes were rarely found in ancient references.It is suggested that the first awareness of plant-parasitic nematodes wereknown in antiquity (235 B.C.) since the ancient Chinese symbol resembles inshape an adult female soybean cyst nematode that was used to describe itself (Noel, 1992). Needham (1742) provided the first description of wheat seed plant-parasiticnematodes.

Currently, nematodes are generally regarded as a separate phylumthat is Nematoda or Nemata (De Ley & Blaxter,2002). De Ley and Blaxter (2002) presented the systematic scheme that is based on thehigher classification proposed and reflect new taxa proposals with three basalclades. Nonetheless, recent molecular phylogenetic analyses seemed to be moreprecise with 12 clades within the Nematoda (Holterman et al., 2006).The phylum Nematoda consists of about 27 000 describedspecies (Hugot et al., 2001).

The prediction of nematode number can up to a hundredmillion, but more accurate numbers can be about 100 000 species (Coomans, 2000) to ten million (Lambshead, 2004). To date, the number of describedplant-parasitic nematodes over the world is estimated by approximately 4100species (Decraemer & Hunt,2013).According to recent studies, plant-parasitic nematodesgroups probably constitute several separate origins of parasitism (Quist et al., 2015; Sánchez-Mongeet al.

, 2017).Strikingly, nowadays plant-parasitism has evolved several times independentlyfrom fungivorous ancestors and plant-parasitic taxa located in the basic clade1 (Trichodoridae), clade 2 (Longidoridae) and in the more advanced clade 12 byTylenchomorpha (Holterman et al., 2006).1.3. Taxonomy of nematodes To assess biodiversity, to understand speciesdistribution and to understand community structures and ecosystem functions,taxonomy of nematodes is really important. Hugot (2002) emphasized the important of correct identification andtaxonomy as a science.

There are many species concepts that range widely from typological concept to biological andphylogenetic concepts. All of these concepts have its own limitations,for example the popular biological species concept is restricted to sexual andoutcrossing populations, but can’t be applied with parthenogenetic organisms (Subbotin & Moens,2006). Thesearch for a perfect concept has led to a distinction between theoreticalspecies concepts and more operational species identification methods (Mayden, 1997; Adams,2002; Van Regenmortel, 2010). Furthermore,the concepts of diversity or the methods to measure diversity are quite diverse(Hodda et al., 2009). The term ?-taxonomy was first given by (Turrill, 1935) who differentiated between ?-taxonomy (traditionaltaxonomy) and ?-taxonomy (perfected taxonomy). ?-taxonomy mostly based onmorphology and ?-taxonomy was built upon a wider range of information frommorphology, physiology, ecology, genetics and relationships. Later on, thepresence of different taxonomy interpretations divided taxonomy into two orthree main components.

Mayr (1969) had given three definitions of taxonomy: ?-taxonomy includesthe characterization and naming of species, ?-taxonomy aims to arrange speciesinto a natural system, and ?-taxonomy consists of various biological andevolutionary aspects. In nematology, taxonomy was generally limited to ?-taxonomy:the description of taxa, and mainly of species and genera. Recently, ?-taxonomyis still mostly in view of morphology, morphometry and geography. The light microscopic observations provided very firstbases for good morphological description. The supports of SEM and interferencecontrast photographs, in addition, are really useful for identification (Coomans, 2000). However, the Nematoda seems to be highly conserved inmorphological aspect which compromises the ease and reliability of speciesidentification. Hence, morphological characters itself areinsufficient to resolve all the monophyletic relationships, that has resultedin controversial problems in nematode classification. Currently, the shift from using purely phenotypic to thecombination of both phenotypic and molecular methods is becoming more prevalentin nematology (Powers et al.

, 1997; Powers, 2004). Moreover, the phylogenetic species concept is widelyaccepted recently (Adams, 1998, 2002). Owing to the development of molecular approach, theterms ‘cryptic’ and ‘sibling’ species have been introduced to describespeciation without significant morphological differences.

According to Bickford et al. (2006), ‘cryptic species’ are two or more particular speciesthat are wrongly arranged (and hidden) under a single species name. ‘Sibling’species has being used for sets of closely related cryptic species which aredifficult to distinguish using conventional morphological characters, while’cryptic’ species is preferable to use because it does not reflect speciesrelationships. There were many examples of cryptic species in plant-parasiticgroup and a few strategies, predominantly based on molecular information, havebeen produced to identify ‘cryptic’ species in recent years (Palomares-Rius et al., 2014).

It is reasonable that cryptic species must benumerous in the Nematoda and molecular techniques may be the only practicalapproach to detect them (Powers, 2004). The best genomic regions that are suitable toidentify cryptic species only should be evaluated on a case-by-case basis (Wu et al., 2007; Gutiérrez-Gutiérrez et al., 2010; Cantalapiedra-Navarrete et al., 2013).Nonetheless, it would be a big mistake if we totallyreplace the morphological approach by molecular approach in nematodeidentification. Rather we need to integrate morphological and molecularinformation as much as possible (Coomans, 2000).1.

3.1.    Molecular markers The gene substitution rates, number of genes studied and typeof molecular markers can influence species delimitation (Rittmeyer & Austin,2012; Miralles & Vences, 2013). Various molecular techniques have been created that arefit for distinguishing and measuring nematodes at the species level andunderneath. Techniques such as protein-based analysis, polymerase chain reaction(PCR), quantitative polymerase chain reaction (qPCR), restriction fragmentlength polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysesare supporting very well for nematode identification. However, amplification andsequencing of diagnostic regions of nematode DNA have been becoming the mostreliable source of new information for enhancing our comprehension of evolutionaryand genetic relationships (Hajibabaei et al., 2007; Meldal et al.

, 2007).Well-studied mitochondrial DNA and ribosomal coding genesare extremely useful for identification. rRNA genes, such as the small subunit,ITS, D2-D3 expansion segment of 18S and 28S fragments evolve relatively slowly (Blaxter, 2001; Subbotin& Moens, 2006).

Thesegenes are multicopy, which makes them relatively easy to amplify as well asusing for phylogenetic studies among groups of plant-parasitic, animalparasitic and free-living nematodes, or between orders within the PhylumNematoda (Blaxter, 2001). Recently, the D2-D3 expansion and the 18S segments arebeing used extensively as the standard molecular marker throughoutplant-parasitic nematode group. Conversely, mtDNA genes evolve more quickly,making them helpful for intraspecific and population genetic studies (Plantard et al., 2008) or intra-genus and intra-family studies (Blaxter, 2001). These molecular markers are highly efficient foridentification of different plant-parasitic groups due to the availability ofseveral conserved primers that can amplify DNA from many taxa and it is alsofacilitated by the presence of phylogenetic informative sites (Blaxter et al., 1998; Subbotin et al.

, 2007). Inaddition, sequence comparison of these genes from unknown species withpublished sequences in GenBank encourages fast identification of most plant-parasiticnematode species (Thiery & Mugniery,1996; Orui, 1997; Szalanski et al.,1997; Ferris et al., 1999; Subbotin et al., 1999; Subbotin et al., 2000; Eroshenko et al., 2001; Subbotin et al.

, 2001; He et al., 2005).1.3.2.     Some limitations of molecular markersBecause ofthe diminishing cost and expanded accessibility of sequence instruments, thenumber of published sequences on open databases has grown exponentially overthe last 10 years (Muir et al.

, 2016). Regardless of many advantages of molecular data, theycan violate the assumptions of phylogenetic analysis. For instance, the sequence evolving rates in differenttaxa can be very different perplexing their utilization in phylogeneticinference (Britten, 1986; Mallatt et al., 2010). Particular highlights for mtDNA genes in nematodes are,for example, high mutational rates, rich A +T content, inordinate saturation,biased substitution patterns and poorly conserved or non-evident regions forprimer design (Blouin et al., 1998; Blouin, 2000). Furthermore, the selection of loci can be critical:some, for example, the genes of animal mtDNA evolve quickly and are only usefulfor intraspecific analysis (Lazarova et al., 2006).

Blouin (2002) showed that nematode mtDNA sequences have fastersubstitution accumulation than in ITS sequences and also have differentmutation rates within mitochondrial genes. Moreover, these same loci can sufferfrom convergent evolution when compared across divergent taxa. As of late, Bik et al. (2013) discovered a large number of duplicate rRNA genes amongnematode taxa. The potential presence of multiple and divergent consensussequences in each species has critical implications for sequence-basedapproaches to biodiversity.According toJanssen et al. (2017), a substantial part of the sequence data on Genbank canbe incorrect, with faults ranging from sequence errors due to misassembled,mislabelled, unlabelled or misidentified sequences.Similar to morphological approach, creating phylogenetictrees from DNA sequences has its own limitations that may affect the finalconclusions.

Alignment of sequences using computer algorithms may presentpredispositions, particularly when they are adjusted by eye (Abebe et al., 2011). Different methods of alignment may be a cause fordiscrepancies between aligned sequences from the same sequence. Theinconsistency may appear in clustering of aligned DNA, a serious disadvantageto the definition and interpretation of Molecular Operational Taxonomic Units(MOTUs) (Blaxter, 2004).

 Regardless the present level of dataaccumulated, DNA sequences alone are not adequate to describe a species, buttheir unique reproducibility helps to avoid duplicate descriptions (Tautz et al., 2002). It is not an easy work to find an ideal gene for taxonomicidentification as well as phylogenetic inference in all nematode groups.Furthermore, choosing a DNA locus that provides a species-specific designationis still an open issue (Porazinska et al., 2009). Consequently,in order to avoid misidentifications and the appearance of mislabeled sequenceson Genbank as well as other limitations of the molecular approach, thecombination of DNA sequences and morphological characteristics is desperatelyneeded. It is feasible to obtain both molecular and morphological data whenanalyzing plant-parasitic nematodes diversity at the sites close to theNematology Unit’s laboratory.

As a result, it will provide more substantial informationabout Belgian nematofauna.1.4.  Diversity of plant-parasitic nematodes in BelgiumBelgium has a long “nematological tradition” with therelatively well-studied nematofauna.

Coomans (1989) reviewed the Belgian nematofauna with the exclusion ofthe animal-parasitic nematodes. According to Bert et al. (2002),6 out of 119 species were removed from the list of Coomans because they aresynonym with another species in that list, 16 species were synonymised andpresented with the correct nomenclature and 27 species were added. Basedon new data and the data from Bert and Geraert (2000) and Coosemans (2002), Bert et al. (2003) hadgiven an updated checklist of the Tylenchomorpha from Belgium, with theaddition of 42 species.

More recently, Steel et al. (2014) provided a Belgian nematode list of 418 species, 127 ofthem are new compared to the lists of (Coomans, 1989) and (Bert et al., 2003). In that, 10 species belong to Trichodoridae, 14 speciesbelong to Longidoridae and 183 species belong to Tylenchomorpha (Steel et al., 2014). Aside from the list providedby Steel et al.(2014), Sewell (1970) reported Paratylenchus projectus Jenkins, 1956 for Belgium. According to (Subbotin et al.

,2004) the species Anguina agrostis should be added to thelist of plant-parasitic nematodes in Belgium as his study on the evolution ofthe gall-forming plant-parasitic nematodes and their relationships with hosts. Damme et al. (2013) added the first report of theroot-knot nematode Meloidogyne artiellia inBelgium.Qing et al. (2015) described the new species Abursanema quadrilineatum for the Infraorder Tylenchomorpha withthe detailed descriptions of ultrastructural, phylogenetic and rRNA secondarystructural analyses. Consoli et al. (2017) described Paratrophurus bursifer for the first time in Belgium.2.

     RESEARCH PROPOSAL o Objectivesof research:The research aimsto identify plant-parasitic nematodes from Ugent botanical garden with thecombination of morphological and molecular analyses that help to widen theBelgian nematofauna knowledge.o Describe themethodology of research: SamplingSoil and root sampleswill be collected randomly by a core that is 25cm in length and 5cm widththroughout the Ugent botanical garden following the method of (Been & Schomaker, 2013).ExtractionNematodes from soiland root samples will be extracted using the decanting and modified Baermanntray method (Whitehead & Hemming, 1965). Swollen nematodes will be dissectedfrom root tissues under a stereomicroscope using a scalpel (Hartman & Sasser, 1985).Fixingand mountingFor molecular work,nematodes can be stored for a long time by picking directly nematodes into DESSsolution (Yoder et al.

,2006).For morphologicalwork, permanent slides will be made by heat-killed nematodes with the methodsof fixing by TAF and ethanol-glycerin dehydration (Seinhorst, 1959). Morphological observationsPictures, drawingsand measurements can be obtained through the Olympus light microscope with thesupports of drawing tube and digital camera.For scanning electronmicroscopy (SEM), formalin-fixed nematodes were transferred to a drop of 4%formalin. The nematodes were dehydrated by passing them through a gradualethanol concentration gradient 25 (overnight), 50, 75, 95 (3 h each) and 100%(overnight) at 25 ± C, and then were critical point dried with liquid CO2 ,mounted on SEM stubs, coated with gold, and studied using a scanning electronmicroscope (Eisenback, 1986).

DNA analyses The sequences of therRNA genes such as D2-D3 and ITS as well as mtDNA genes will be used. DNAsequences will be analyzed using the BLAST homology search program. The phylogenetictrees will be created by different methods and combined with morphology toidentify to species level exactly.

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