Nematodes, year. Therefore, In order to obtain

Nematodes, currently, are considered as one of the most numerous Metazoa on our planet. They can be either free-living or plant-parasitic or animal parasites. Although they occur in almost every habitat, they are essentially aquatic animals. Soil structure, soil pH, and other factors can affect nematodes by different ways {Decraemer, 2013 #12}(Decraemer & Hunt, 2013). Different groups of nematodes have adapted to different habitats through the evolution over time.

Up to now, approximately 4100 nematode species have been described as plant-parasites over the world (Decraemer & Hunt, 2013). In Belgium, the nematofauna has been relatively well studied. However, a lot of new species descriptions are being updated year by year. Therefore, In order to obtain a more comprehensive overview of the nematode diversity, it is necessary to investigate nematodes from various habitats.

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This thesis focuses on the investigation of plant-parasitic nematodes from neglected biotopes and this will provide a more detailed description of plant-parasitic nematode biodiversity in general and in Belgium in particular. The combination of molecular and morphological data in classification will contribute the knowledge to understand the controversial taxonomical problems as well as phylogenetic relationships.

1.1.  Nematodes in general

Nematodes are pseudocoelomate, unsegmented worm-like animals, commonly described as filiform or thread-like, a characteristic reflected by the taxon name nema (Greek, nema= thread) and its nominative plural nemata (Decraemer & Hunt, 2013).

The historical nematology was marked with the oldest reference from China in 2500 B.C. with the description of symptoms and treatment of the relatively large intestinal roundworm Ascaris or Huei Ch’ung (Maggenti, 1981). Due to their small size and atypical symptom, the reports of plant-parasitic nematodes were rarely found in ancient references. It is suggested that the first awareness of plant-parasitic nematodes were known in antiquity (235 B.C.) since the ancient Chinese symbol resembles in shape an adult female soybean cyst nematode that was used to describe itself (Noel, 1992). Needham (1742) provided the first description of wheat seed plant-parasitic nematodes. Recently, nematodes are generally regarded as a separate phylum named Nematoda (De Ley & Blaxter, 2002). De Ley and Blaxter (2002) presented the systematic scheme that distinguished three subgroups (Enoplia, Dorylaimia and Chromadoria) among nematodes, although their relationship to each other was not clear. A more recent molecular phylogenetic framework appoints 12 clades within the Nematoda (Holterman et al., 2006).

The phylum Nematoda consists of about 27 000 described species (Hugot et al., 2001). The prediction of nematode number can up to a hundred million, but more accurate numbers can be about 100 000 species (Coomans, 2000) to ten million (Lambshead, 2004). To date, the number of described plant-parasitic nematodes over the world is estimated to be approximately 4100 species (Decraemer & Hunt, 2013).

According to De Ley and Blaxter (2002) and more recent studies, plant-parasitic nematodes groups probably constitute several separate origins of parasitism (Quist et al., 2015; Sánchez-Monge et al., 2017). Plant-parasitism has evolved several times independently from fungivorous ancestors and they are located at four different clades, the more basal clades 1 (Trichodoridae), clade 2 (Longidoridae), clade 10 (Tylenchomorpha: Aphelenchoididae), and the more advanced clade 12 (Tylenchomorpha) (Holterman et al., 2006). Although the taxon Tylenchomorpha includes both non-plant-parasitic and plant-parasitic nematode groups, herein this thesis use the term “plant-parasitic nematodes” to indicate all the nematodes belong to the taxa Trichodoridae, Longidoridae and Tylenchomorpha.

1.2.  Taxonomy of nematodes

To assess biodiversity, to understand species distribution and to understand community structures and ecosystem functions, taxonomy of nematodes is really important. Hugot (2002) emphasized the importance of correct identification and taxonomy as a science. There are many species concepts, ranging from typological concept to biological and phylogenetic concepts. All of these concepts have their own limitations, the popular biological species concept, for example, is restricted to sexual and outcrossing populations, but it can’t be applied with parthenogenetic organisms (Subbotin & Moens, 2006). The search for a perfect concept has led to a distinction between theoretical species 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 also quite diverse (Hodda et al., 2009).

The term ?-taxonomy was first given by Turrill (1935) who differentiated between ?-taxonomy (traditional taxonomy) and ?-taxonomy (perfected taxonomy). ?-taxonomy was mostly based on morphology and ?-taxonomy was built upon a wider range of information from morphology, physiology, ecology, genetics, and relationships. 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 still mostly in view of morphology, morphometry, and geography. For taxonomical work, the light microscopic observations provide the very first bases for good morphological description. The addition of SEM and interference contrast photographs are really useful for identification (Coomans, 2000). However, in term of morphology, nematodes are highly diverse within the phylum Nematoda, but they seem to be relatively conserved when comparing within species level in some species (De Ley, 2006; Sánchez-Monge et al., 2017). 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 to the combination of both phenotypic and molecular methods is becoming more prevalent in nematology (Powers et al., 1997; Powers, 2004). Moreover, the phylogenetic species concept is widely accepted recently (Adams, 1998, 2002). Owing to the development of molecular approach, the terms ‘cryptic’ and ‘sibling’ species have been introduced to describe speciation without significant morphological differences. According to Bickford et al. (2006), ‘cryptic species’ are two or more particular species that are wrongly arranged (and hidden) under a single species name. ‘Sibling’ species has being used for sets of closely related cryptic species which are difficult to distinguish using conventional morphological characters, while ‘cryptic’ species is preferable to use because it does not reflect species relationships. There were many examples of cryptic species in plant-parasitic group and a few strategies, predominantly based on molecular information, have been produced to identify ‘cryptic’ species in recent years (Palomares-Rius et al., 2014). It is reasonable that cryptic species must be numerous in the Nematoda and molecular techniques may be the only practical approach to detect them (Powers, 2004). The best genomic regions that are suitable to identify cryptic species 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 totally replace the morphological approach by molecular approach in nematode identification. Rather we need to integrate morphological and molecular information as much as possible (Coomans, 2000).

1.2.1.     Molecular markers

The gene substitution rates, number of genes studied and type of molecular markers can influence species delimitation (Rittmeyer & Austin, 2012; Miralles & Vences, 2013). Various molecular techniques have been created that are fit for distinguishing and measuring nematodes at the species level and below. Techniques such as protein-based analysis, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analyses are very useful for nematode identification. However, amplification and sequencing of diagnostic regions of nematode DNA have been becoming the most reliable source of new information for enhancing our comprehension of evolutionary and genetic relationships (Hajibabaei et al., 2007; Meldal et al., 2007).

Especially, well-studied mitochondrial DNA and ribosomal coding genes are extremely useful for identification. rRNA genes such as the small subunit 18S, ITS, D2-D3 expansion of 28S fragments evolve relatively slowly (Blaxter, 2001; Subbotin & Moens, 2006). These genes are present in identical multi-copies, which makes them relatively easy to amplify as well as useful for phylogenetic studies of plant-parasitic, animal parasitic and free-living nematodes, or event to analyze the relations between orders within the Phylum Nematoda (Blaxter, 2001). Recently, the small subunit 18S, ITS, D2-D3 expansion of 28S fragments are being used extensively as the standard molecular marker throughout plant-parasitic nematode group (Blaxter et al., 1998; Subbotin et al., 1999; Subbotin et al., 2003; Subbotin & Moens, 2006; Subbotin et al., 2007; Holterman et al., 2009; Janssen et al., 2017). 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). The mitochondrial Cytochrome Oxidase I gene (COI) is being used as the standard barcode for almost all animal groups (Hebert et al., 2003) and they has been exploring for several nematode groups including marine taxa (Derycke et al., 2005; Derycke et al., 2010) and several plant-parasitic genus (Bickford et al., 2006; Palomares-Rius et al., 2014). For example, COI gene was successfully used in identifying Bursaphelenchus spp. (Ye et al., 2007; Kanzaki & Giblin-Davis, 2012). This gene seems to be very useful for identification of the genus Meloidogyne since even very closely-related esterase phenotypes can be reliably identified using mitochondrial haplotypes (Janssen, 2017). Identification of Pratylenchus spp. was also well-supported by COI gene (Troccoli et al., 2016; Janssen, 2017). Sánchez-Monge et al. (2017) used mtCOI successfully to diagnose the four main plant-parasitic Aphelenchoides species. According to Kolombia et al. (2017), the most suitable marker for barcoding and identifying Scutellonema spp. is the COI gene. With the increasing presence of nematode COI sequences in GenBank, sequence comparison of unknown species with the published sequences encourages fast identification of most plant-parasitic nematode 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). These molecular markers are highly efficient for the identification of different plant-parasitic groups due to the availability of several conserved primers that can amplify DNA from many taxa and this is also facilitated by the presence of phylogenetic informative sites (Blaxter et al., 1998; Subbotin et al., 2007).

1.2.2.     Some limitations of molecular markers

Because of the diminishing cost and expanded accessibility of sequence instruments, the number of published sequences on open databases has grown exponentially over the last 10 years (Muir et al., 2016). Regardless of many advantages of molecular data, they can violate the assumptions of phylogenetic analysis. For instance, the sequence evolving rates in different taxa can be very different perplexing their utilization in phylogenetic inference (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 for primer design (Blouin et al., 1998; Blouin, 2000). Blouin (2002) showed that nematode mtDNA sequences have faster substitution accumulation than in ITS sequences and also have different mutation rates within mitochondrial genes. Although the high mutational rates in mtDNA make them very useful for low-level phylogenetic applications, failure to correct for this severe substitution bias can potentially lead to phylogenetic error (Subbotin et al., 2013). Several flaws of COI gene have been reported such as the high diversity in nucleotide composition, the symbiont effects (i.e.Wolbachia), the anomalous properties (e.g recombination, insertion, multipartitioning) and particularly the Nuclear Mitochondrial Sequences (NUMTS), which are copies of mtDNA sequences into nuclear chromosomes (Sánchez-Monge et al., 2017). For rRNA genes, Bik et al. (2013) discovered a large number of duplicate rRNA genes among nematode taxa. The potential presence of multiple and divergent sequences in each species has critical implications for sequence-based approaches to biodiversity. Moreover, homoplasy or convergent evolution, that occur frequently at all taxonomic levels, has important implications for phylogenetic and evolutionary studies. In phylogenetic analyses, parallel or convergent genotypic changes, if common, may affect the estimations of phylogenetic relationships (Wood et al., 2005).

According to Janssen et al. (2017), a substantial part of the sequence data on GenBank can be incorrect, with faults ranging from sequence errors due to misassembled, mislabelled, unlabelled or misidentified sequences.

Similar to morphological approach, creating phylogenetic trees from DNA sequences has its own limitations that may affect the final conclusions. Alignment of sequences using computer algorithms may present predispositions, particularly when they are adjusted by eye (Abebe et al., 2011). Different methods of alignment may be a cause for discrepancies between aligned sequences from the same sequence. For example, the inconsistency may appear in clustering of aligned DNA, this is a serious disadvantage to the definition and interpretation of Molecular Operational Taxonomic Units (MOTUs) (Blaxter, 2004).It is not an easy work to find an ideal gene for taxonomic identification as well as phylogenetic inference in all nematode groups. Furthermore, choosing a DNA locus that provides a species-specific designation is still an open issue (Porazinska et al., 2009).

Consequently, in order to avoid 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. Regardless the present level of data accumulated, DNA sequences alone are not adequate to describe a species, not only because of their limitations but the work of taxonomists is also to provide knowledge of the organism, not just a few possibly unique nucleotides. DNA barcoding will provide meaningful information only if scientists can place it within the context of rich morphological, physiological, and behavioral knowledge. In any case, every barcode must be linked with a known, described specimen that is stored somewhere (Ebach & Holdrege, 2005a; b).

In this study, the plant-parasitic nematode diversity will be studied relatively close to the Nematology Research Unit’s laboratory and this will make it especially feasible to obtain both molecular and morphological data when analyzing. As a result, this will provide more substantial information about Belgian nematofauna.

1.3.  Nematofauna in Belgium

Belgium has a long “nematological tradition” with the relatively well-studied nematofauna. Coomans (1989) reviewed the Belgian nematofauna with the exclusion of the animal-parasitic nematodes. Based on new data and the data from Bert and Geraert (2000) and Coosemans (2002), Bert et al. (2003) had given an updated checklist of the Tylenchomorpha from Belgium, with the addition of 42 species, of which 11 were new records for the Belgium nematofauna. Five nominal species were removed because of synonymy and the list of 161 species was presented nomenclaturally. More recently, Steel et al. (2014) provided a Belgian nematode list of 418 species, 127 of them are new compared to the lists of Coomans (1989) and Bert et al. (2003). In this list, 10 species belong to Trichodoridae, 14 species belong to Longidoridae and 183 species belong to Tylenchomorpha (Steel et al., 2014).

Comparing with Steel et al. (2014) the list of 1 free-living and 14 plant-parasitic nematode species should be added. For free-living nematodes, Slos et al. (2017) have given the first description of Caenorhabditis monodelphis in Belgium. For plant-parasitic nematodes, Sewell (1970) reported the appearance of Paratylenchus projectus Jenkins, 1956 in the soil samples from Belgium. Moens and Hendrickx (1990) reported the presence of second-stage juveniles of Meloidogyne incognita (Kofoid , 1919) Chitwood, 1949 in the nutrient solution of hydroponic-like systems that was used for growing ornamental pot plants in Belgium. According to Subbotin et al. (2000), Heterodera iri Mathews, 1971 should be added to Belgian nematofauna. The species Anguina agrostis (Steinbuch, 1799) Filipjev, 1936 was first recorded from seed galls of Agrostis capillaris in Kasterlee, Belgium by Subbotin et al. (2004). Subbotin et al. (2006) gave the first information of Meloidodera alni Turkina & Chizhov, 1986 in Belgium. Heterodera fici Kirjanova, 1954 should also be added to Belgian nematode list (Subbotin et al., 2010). Damme et al. (2013) provided the first report of the root-knot nematode Meloidogyne artiellia Franklin, 1961 in Belgium. The new species Bursaphelenchus parantoniae was found in packaging wood from Belgium by (Maria et al., 2015). Qing et al. (2015) described the new species Abursanema quadrilineatum from a mushroom in Ghent. Pratylenchus bolivianus Corbett, 1983 is present in Belgium according to Troccoli et al. (2016). Consoli et al. (2017) described Paratrophurus bursifer for the first time in Belgium. Janssen (2017) reported the presence of Pratylenchus brzeskii Karssen, 2000, P. convallariae Seinhorst, 1959, and P. goodeyi Sher and Allen, 1953 for the first time in Belgium.

2.     RESEARCH PROPOSAL

o Objectives of research:

(1)   To characterize and identify plant-parasitic nematodes using the combination of morphological and molecular analyses to increase the knowledge of the diversity of plan-parasitic nematodes, to solve taxonomic problems and to provide useful DNA barcodes.

(2)   To increase the knowledge of the Belgian nematofauna.

o Describe the methodology of research:

Sampling and Extraction

Soil and root samples will be collected randomly by an auger throughout the UGent botanical garden and other relevant places. Each sample will be put in a separate plastic bag and brought to Nematology Research Unit’s laboratory to extract nematodes.

Nematodes from soil and root samples will be extracted using modified Baermann tray method (Whitehead & Hemming, 1965). Swollen nematodes will be dissected from root tissues under a stereomicroscope using a scalpel (Hartman & Sasser, 1985).

Preparation for PCR, fixing and mounting

For molecular work, temporary slides will be made and digital light microscope pictures will be taken as a morphological voucher before the preparation of nematode DNA template for running PCR. After that, each single nematode will be cut into two or three pecies and put in an Eppendorf tube with 20µ of worm lysis buffer (50mM KCl;10mM Tris pH 8.3; 2.5mM MgCl2; 0.45% NP 40 (Tergitol Sigma); 0.45% Tween 20) 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 long time at ?20°C before running PCR (Khun et al., 2015).

For morphological work, permanent slides will be made by heat-killed nematodes with the fixation by 4% PFA in PBS (+1% Glycerin) before ethanol-glycerin dehydration (Viaene et al., 2016).

Morphological observations

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 (Adobe Systems) base on light microscopic drawings. For scanning electron microscopy (SEM), specimens will be processed and viewed following the procedure of (Eisenback, 1986).

DNA analyses

Successful PCR reactions will be purified and sequenced commercially by Macrogen Inc. (Europe) in forward and reverse direction. Consensus sequences will be assembled using GENEIOUS R11. All contigs will be used for a BLAST search on the NCBI website (http://www.ncbi.nlm.nih.gov) to check for close related species (Slos et al., 2017).

MEGA 7 will be used for alignment, selecting best model and creating phylogenetic trees. Poorly aligned regions can be removed from the alignments using Gblock sever (http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks)

The phylogenetic trees can also be created by different methods as Mrbayes and the results will be combined with morphology to identify to species level exactly.