1. LITERATURE STUDY
1.1. State of the art of the research topic
Nematodes 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 & 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.
This thesis focuses on the investigation of plant-parasitic nematodes from neglected biotopes that provide a more detailed description of plant-parasitic nematode biodiversity in Belgium. The combination of molecular and morphological data in classification will contribute the knowledge to understand the controversial taxonomical problems as well as phylogenetic relationships.
1.2. 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 history of nematodes 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. Currently, nematodes are generally regarded as a separate phylum that is Nematoda or Nemata (De Ley & Blaxter, 2002). De Ley and Blaxter (2002) presented the systematic scheme that is based on the higher classification proposed and reflect new taxa proposals with three basal clades. Nonetheless, recent molecular phylogenetic analyses seemed to be more precise with 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 by approximately 4100 species (Decraemer & Hunt, 2013).
According to recent studies, plant-parasitic nematodes groups probably constitute several separate origins of parasitism (Quist et al., 2015; Sánchez-Monge et al., 2017). Strikingly, nowadays plant-parasitism has evolved several times independently from fungivorous ancestors and plant-parasitic taxa located in the basic clade 1 (Trichodoridae), clade 2 (Longidoridae) and in the more advanced clade 12 by Tylenchomorpha (Holterman et al., 2006).
1.3. 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 important of correct identification and taxonomy as a science. There are many species concepts that range widely from typological concept to biological and phylogenetic concepts. All of these concepts have its own limitations, for example the popular biological species concept is restricted to sexual and outcrossing populations, but 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 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 mostly based on morphology and ?-taxonomy was built upon a wider range of information from morphology, physiology, ecology, genetics and relationships. Later on, the presence of different taxonomy interpretations divided taxonomy into two or three main components. Mayr (1969) had given three definitions of taxonomy: ?-taxonomy includes the characterization and naming of species, ?-taxonomy aims to arrange species into a natural system, and ?-taxonomy consists of various biological and evolutionary aspects.
In nematology, taxonomy was generally limited to ?-taxonomy: the description of taxa, and mainly of species and genera. Recently, ?-taxonomy is still mostly in view of morphology, morphometry and geography. The light microscopic observations provided very first bases for good morphological description. The supports of SEM and interference contrast photographs, in addition, are really useful for identification (Coomans, 2000). However, the Nematoda seems to be highly conserved in morphological aspect which compromises the ease and reliability of species identification. Hence, morphological characters itself are insufficient to resolve all the monophyletic relationships, that 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 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 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).
1.3.1. 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 underneath. 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 supporting very well 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).
Well-studied mitochondrial DNA and ribosomal coding genes are 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). These genes are multicopy, which makes them relatively easy to amplify as well as using for phylogenetic studies among groups of plant-parasitic, animal parasitic and free-living nematodes, or between orders within the Phylum Nematoda (Blaxter, 2001). Recently, the D2-D3 expansion and the 18S segments are being used extensively as the standard molecular marker throughout plant-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 for identification of different plant-parasitic groups due to the availability of several conserved primers that can amplify DNA from many taxa and it is also facilitated by the presence of phylogenetic informative sites (Blaxter et al., 1998; Subbotin et al., 2007). In addition, sequence comparison of these genes from unknown species with published sequences in GenBank 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).
1.3.2. 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). Furthermore, the selection of loci can be critical: some, for example, the genes of animal mtDNA evolve quickly and are only useful for intraspecific analysis (Lazarova et al., 2006). Blouin (2002) showed that nematode mtDNA sequences have faster substitution accumulation than in ITS sequences and also have different mutation rates within mitochondrial genes. Moreover, these same loci can suffer from convergent evolution when compared across divergent taxa. As of late, Bik et al. (2013) discovered a large number of duplicate rRNA genes among nematode taxa. The potential presence of multiple and divergent consensus sequences in each species has critical implications for sequence-based approaches to biodiversity.
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. The inconsistency may appear in clustering of aligned DNA, a serious disadvantage to the definition and interpretation of Molecular Operational Taxonomic Units (MOTUs) (Blaxter, 2004).
Regardless the present level of data accumulated, DNA sequences alone are not adequate to describe a species, but their unique reproducibility helps to avoid duplicate descriptions (Tautz et al., 2002). 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. It is feasible to obtain both molecular and morphological data when analyzing plant-parasitic nematodes diversity at the sites close to the Nematology Unit’s laboratory. As a result, it will provide more substantial information about Belgian nematofauna.
1.4. Diversity of plant-parasitic nematodes 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. According to Bert et al. (2002), 6 out of 119 species were removed from the list of Coomans because they are synonym with another species in that list, 16 species were synonymised and presented with the correct nomenclature and 27 species were added. 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. 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 that, 10 species belong to Trichodoridae, 14 species belong to Longidoridae and 183 species belong to Tylenchomorpha (Steel et al., 2014).
Aside from the list provided by 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 the list of plant-parasitic nematodes in Belgium as his study on the evolution of the gall-forming plant-parasitic nematodes and their relationships with hosts. Damme et al. (2013) added the first report of the root-knot nematode Meloidogyne artiellia in Belgium. Qing et al. (2015) described the new species Abursanema quadrilineatum for the Infraorder Tylenchomorpha with the detailed descriptions of ultrastructural, phylogenetic and rRNA secondary structural analyses. Consoli et al. (2017) described Paratrophurus bursifer for the first time in Belgium.
2. RESEARCH PROPOSAL
o Objectives of research:
The research aims to identify plant-parasitic nematodes from Ugent botanical garden with the combination of morphological and molecular analyses that help to widen the Belgian nematofauna knowledge.
o Describe the methodology of research:
Soil and root samples will be collected randomly by a core that is 25cm in length and 5cm width throughout the Ugent botanical garden following the method of (Been & Schomaker, 2013).
Nematodes from soil and root samples will be extracted using the decanting and modified Baermann tray method (Whitehead & Hemming, 1965). Swollen nematodes will be dissected from root tissues under a stereomicroscope using a scalpel (Hartman & Sasser, 1985).
Fixing and mounting
For molecular work, nematodes can be stored for a long time by picking directly nematodes into DESS solution (Yoder et al., 2006).
For morphological work, permanent slides will be made by heat-killed nematodes with the methods of fixing by TAF and ethanol-glycerin dehydration (Seinhorst, 1959).
Pictures, drawings and measurements can be obtained through the Olympus light microscope with the supports of drawing tube and digital camera.
For scanning electron microscopy (SEM), formalin-fixed nematodes were transferred to a drop of 4% formalin. The nematodes were dehydrated by passing them through a gradual ethanol 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 electron microscope (Eisenback, 1986).
The sequences of the rRNA genes such as D2-D3 and ITS as well as mtDNA genes will be used. DNA sequences will be analyzed using the BLAST homology search program. The phylogenetic trees will be created by different methods and combined with morphology to identify to species level exactly.