Nematodes to now, approximately 4100 nematode species have

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,
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 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.

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,

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,

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).

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 that range widely from typological concept to biological and
phylogenetic concepts. All of these concepts have its own limitations,
the popular biological species concept, for example, 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).

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).

1.2.2.     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). 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).


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.

1.3.  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
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.


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).

and mounting

For molecular work,
nematodes can be stored for a long time by picking directly nematodes into DESS
solution (Yoder et al.,

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).

Morphological observations

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).

DNA analyses

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.