Sampling,extraction and identification of plant parasitic nematodes PPN's

SAMPLING,
EXTRACTION &
IDENTIFICATION OF
PPNS

CONTENT
■ Introduction
■ Sampling
■ Extraction
■ Identification
■ Conclusion

Introduction
■ Nematodes are found virtually in every environment and can be parasitic or free-living
in the soil or plants.
■ Plant parasitic nematodes are known to reduce agricultural production by
approximately 11% globally (Agrios, 2005), which reduces production by a million
tonnes every year.
■ This calls for need to invest on sampling, extraction and identification and control of
nematodes so as to avoid the crops loss.

Sampling
■ Sampling for nematodes is advisable once symptoms which indicate possible or likely
nematode infestation have been identified.
■ Once collected the samples are taken to the laboratory for analysis to determine what
nematode is or are present and possibly quantify them.

Sampling tools
■ When sampling you need the appropriate tools.
■ These includes: a spade, hand trowel, soil auger (corer), knive (cutting roots), scissors,
polyethene sample bags, tags, maker pen (for labelling), pencil and note book for data
taking.

Nematode distribution■ Nematodes like most soil borne pathogens are not uniformly distributed in the field.
■ Nematodes have a patchy distribution with areas of high densities mixed among areas of very
low densities.
■ The species density vary in different areas.
■ The distribution of nematodes may be related to soil type e.g. high in sandy areas and lower in
heavier soil.
■ Edaphic factors, cropping history also influence nematode distribution.

Sample taking
■ When sampling a field with no plants grown in it the area is subdivide into portions for
sampling.
■ The most representative samples are obtained by taking a number of cores from a
given area, compositing the core into a single sample, mixing the soil thoroughly and
taking a sufficient subsample for analysis.
■ The pattern in which cores are selected is dependent on the cropping system being
sampled with perennial crops such as orchards and vineyards should be sampled in the
feeder-root zones.

Sample number
■ Taking enough samples ensure a proper representation of the field.
■ The more the sub-samples/cores combined for each sample the more accurate the
assessment will be.
■ From an area of 0.5 to 1 hectare the minimum number of core samples should be 10 to
a maximum of 50.
■ These should then be mixed to form one composite sample.

Sampling pattern
■ Sampling can be carried out at random or systematically.
■ Theoretically, random sampling (if really carried out in a random fashion!) is best as it
excludes bias; by sampling in a haphazard and crisscross way samples can be
considered independent observations.
Random sampling method

■ Systematic sampling is a more structured way to remove samples as it takes into
consideration the nature of the field and nematode distribution.
Randoms sampling b-d show systematic sampling of nematodes

Time of sampling
■ Sampling vary depending with season and crop.
■ Sampling before planting or after a cropping season is known as diagnostic sampling.
■ Normally nematode numbers are high during growing season and reduce during dry season.
■ This shows that its ideally best to sample in the middle of the season or at the end for diagnostic
purpose.
■ Perennial crop stand is better sampled during active period.

Taking root samples
■ Roots should be combined with soil to help preserve the roots.
■ About 25-100 grams of roots per sample or less incase of finer roots e.g. those of rice,
wheat.
■ Dead plant roots should be avoided.
■ Plant is lifted from the soil using a spade or trowel taking care to avoid roots from
breaking and then tapping the soil you can wash with water then using a knife remove
roots.

Care of sample
■ Samples should be placed in strong plastic bags.
■ Label them systematically with a permanent marker or with a label & pencil.
■ Key information on label include: crop, sample date, farmer, location e.g. GPS, reference no for
experimental trial and previous crop if any.
■ Because nematodes are sensitive to sunlight samples should be placed in cool box as soon as they
are sampled.

Extraction
■ The easiest way to isolate nematodes from their host material is by submerging the
sample in water and select the nematodes under a microscope.
■ For that reason most of the extraction methods are indirect, making use of a number
of properties to separate nematodes from the surrounding medium:
 Weight and rate of settling
 Size and shape
 Mobility

Extraction methods
■ An ideal extraction method would make it possible to extract all stages
of all nematode species at 100% efficiency, irrespective of temperature
and soil type, and at low costs (labour, equipment, water) (McSorley
1987).
■ Nematode extraction methods depend on the media nematodes are
present and stage of nematodes adult/juvenile
Soil sample Root/foliar sample
Sedentary
nematodes
Migratory
nematodes
Sedentary
nematodes
Migratory
nematodes
Extraction tray X X
Sieving X X
Root/leaf
maceration
X X
Incubation X X

Extraction tray method
This method is also known as the modified Baermann technique, the pie-pan
method or whitehead tray method.
Advantages
■ Specialist equipment not required.
■ Its easy to adapt to basic circumstances using
locally available material.
■ Extract wide variety of mobile nematodes
■ It’s a simple technique.
Disadvantages
■ Large & slow moving nematodes not
extracted well.
■ Extraction can dirty thus difficulty in counting
■ Proportion of extracted nematode may vary
with temp.
■ Maximum recovery takes 3-4 days.

Soil/plant sample samples extraction
■ Sieve the soil removing stones/debris and break soil lumps.
■ Mix soil thoroughly and weigh 100g.
■ Place tissue paper in plastic sieve ensuring the base of sieve is full
covered by the tissue.

oil/plant sample samples extraction
■ Place soil sample on the tissue in the sieve.
■ Add water to the extraction plate till the soil in sieve is wet.
■ Leave undisturbed for 48 hours while adding water if soil dry out.
■ After extraction period drain excess water from sieve into the
extraction.
■ Remove sieve and dispose plant tissue /soil.

Soil/plant sample samples extraction
■ Pour the water from plate into a beaker and wash the plate
with wash bottle to rinse it then label the beaker.
■ For counting purpose reduce the excess water by: pouring off
gently, siphoning excess, or by passing through a very small
aperture sieve e.g.. 20-30µm.
■ The collected nematode sample is collected into a tube and
preserved for later counting.

Decanting and sieving: Cobb’s method
■ Cobb’s method is used for the
extraction of active nematodes from
soil and sediments.
Principle
■ The method makes use of
differences in size, shape, and
sedimentation rate between
nematodes and soil particles, and of
nematode mobility

Cobb’s method
■ The sample is stirred in a water-filled beaker, detaching the nematodes from the soil particles.
■ After heavy particles have settled, the nematode suspension is poured off (decanted) and sieved.
■ Nematodes remain on the sieves while tiny soil particles pass through.
■ Sieving is carried out with a series of sieves of decreasing mesh size, so nematodes of different size
are collected separately.
■ Soil particles in the suspension stay behind on the subsequent sieves, so the 45μm sieve at the
bottom is not easily clogged and samples up to 100 g can be extracted.
■ The debris collected from the sieves is placed on nematode filters.
■ Nematodes move through the filters into the water and can, after a certain extraction time, be
poured off as a clear suspension

Extraction of cysts using Baunacke
method
■ The Baunacke method (or white
bowl method) is used for the
extraction of cysts from dried soil
(max. 50 g) and sometimes for
isolating cysts from dried debris.
Principle
The method makes use of floating
properties of dried cysts and difference
in size, shape, and colour between cysts
and other fractions in the sample.

Procedure
■ Dry a (sub)sample of up to 50 g. Put it on a 175 μm sieve and wash it with water, forcing
fine particles through the sieve.
■ Wash the debris off the sieve into a white bowl.
■ The cysts float along the edge of the bowl (a lamp giving top light is useful); they can
be picked with a painting brush and transferred to a moist filter paper in a Petri dish or
onto a watch glass.
■ The sediment should be stirred, because cysts may be trapped under the settled soil
particles.
■ Please note: time to pick cysts is limited, because especially filled ones sink rapidly.

Fenwick method
■ Fenwick can is used for cyst
nematode extraction from dried
soils.
■ The method uses the principle of
floating properties of dried cyst and
of difference in size between and
other fraction of the sample.

Fenwick method
■ The course material is retained on the sieve, heavy particles passing through sink to
the bottom of the can and fine and light particles lick cyst keep afloat.
■ When the can overflows the floating cyst are carried off over the overflow collar and
drop on the sieve with a pore size smaller than the cyst diameter.

ugar floatation technique
Material
■ Five 8’’ diameter sieve -20,100,200,325 and 500-mesh/inch.
■ Two beakers -250 and 1000ml .
■ High speed stirrer .
■ Sugar flocculent solution .
■ Three nozzle fogger and two stainless pan or plastic bucket of at least
one gallon capacity.

Procedure
■ Pour sample to a pan add equal amount of water and sample and mix.
■ Pour surface liquid in pan a through the 20-mesh sieve held over pan B until
the soil begins to flow onto the sieve. Still holding the 20-mesh sieve over pan
B wash the soil through the sieve with the fogger nozzle.
■ Discard soil retained on the 20-mesh and in pan A. pour the matrial in pan B
through 100-mesh sieve held over panA until soil begins to flow onto the
sieve. Still holding the100-mesh sieve over pan B wash the soil through sieve
with the fogger nozzle.
■ Backwash the material retained on the 100-mesh sieve into a 1000 ml beaker.
Pour the material in pan A through 200-mesh sieve, held over pan B until soil
begin to flow onto the sieve. Still holding the 200-mesh sieve over pan B wash
the material though the sieve with the fogger nozzle.

Cont..
■ Backwash the material retained on the 200-mesh sieve into 1000ml beaker. Pour it
through 500-mesh sieve held over the sink until soil begins to flow onto the sieve.
Backwash the material retained on the 500-mesh sieve into a 1000ml beaker. Add
water to the 1000ml beaker containing the sieve material raising the volume of liquid
and residue to 300ml or 400ml.
■ Add an equal volume of sugar flocculent solution to the content of the 1000ml beaker
and stir for a minute at high speed with high speed stirrer. Allow the suspension to
settle for a minute.
■ Wash sugar off the residue on the 500-mesh sieve with the fogger nozzle. Backwash
the residue on 500-mesh sieve into a 250ml beaker.
■ Keep the final volume in the beaker to an absolute minimum 50ml maximum.
■ Label the beaker and examine the sample.

Advantages
■ Nematodes recovered quickly
making timely identification
possible.
Disadvantage
■ The technique needs some
expensive equipment's.
■ Sugar if left on the sample too long
may distort nematode making
identification difficult.

Floatation and sieve method
■ The technique for the extraction of motile nematodes from soil was introduced by
cobb(1988) and is mainly used forTylenchid species.
■ The soil is washed in water decanted and nematode are collected on sieve of different
aperture followed by cleaning the suspension with Baermann funnel/Oosternbrink
dish.
■ The method makes use of difference in size, shape and sedimentation rate between
nematodes and soil particles and of nematode motility.

Materials
Materials
■ Bucket of about 10 L;
■ Stirring rod;
■ 3 × 50 μm aperture sieves;
■ 500 mL glass beaker;
■ Watch glass (d = 6 cm);
■ Baermann funnel/Ostenbrink dish 100 mL
glass beaker.

Procedure
■ Add up to 200 mL of soil to a 10 L bucket and add approx. 5 L of water.
■ Stir the soil suspension vigorously for 10 sec.
■ Allow the soil to settle for 45 sec.
■ Pour the supernatant through a bank of 3 sieves of 50 μm aperture.
■ Wash the debris collected on the sieves in a clean glass beaker.
■ Carefully pour the suspension from the beaker with the help of a watch glass
onto the cotton-wool milk filter supported by a plastic sieve in the Baermann
funnel/Oostenbrink dish.
■ After 24 h, collect the nematodes from the Baermann funnel or Oostenbrink
dish in a glass beaker.

Advantage
■ Simple and rapid;
■ High extraction efficiency;
■ All nematode genera are recovered;
■ No elaborate apparatus needed.
Disadvantage
■ Maximum of 200 mL soil;
■ Not suitable for clay soil because
light particles remain in suspension
leading to a dirty final suspension.

Sodium hypochlorite (NaOCl) extaction
technique
■ This technique is very commonly used to extract cyst of Heterodera and Globodera and
also for dissolving egg masses of Meloidogyne speciesare.
■ Care should be taken to minimize the exposure of the NaOCl.
■ Even with normal pre-caution only about 20% of the eggs extracted with NaOCl
produce infective juveniles.

Procedure
■ Collect and cut 6-10 week old infected roots into 1-2 cm segments.
■ Shake roots segments in 200ml of 0.5-0.1% NaOCl sol for 8-10 min
(for cyst extaction) and 1-4 min (for egg extraction).
■ Pass NaOCl sol through a 200 mesh (75 μm) sieve, nested over a
500-mesh sieve to collect freed eggs/cysts quickly place the 500-
mesh sieve with eggs under a stream of cold water to remove
residual NaOCl (rinse for several minutes).
■ Rinse remaning roots with water to remove additional egg/cysts
and collect them by sieving.

cont..
■ To maximize precision in experiment the eggs should be placed on 500-mesh nylon
hatching sieves in 1-2cm deep chlorine-free water.
■ Tap water is allowed to sit in the laboratory for 2-4 days before use is practically
chlorine-free due to evaporation.
■ The hatching juveniles are then collected and used as inoculum.

Introduction
Nematodes are identified by the different
morphological characteristic each one poses.
Some of these features include body shape,
length of the nematode, nature of stylet,
position of the vulva or spicule, esophagus, etc.

Oesophagus
■ Nematode oesophagus vary within the different nematode genera's;
■ esophagus is the area between the stylet and the start of the intestine
■ These types include;
■ Dorylaimoid
■ Tylenchoid
■ Criconematoid

Stylets
■ Length of the stylet and the relative length of its conus are useful, as are the size and
shape of the stylet knobs.Types of stylets:
■ Odontostylet- is a hollow needle-like structure evolved out of single sub ventral tooth
and passes through a guiding ring. Eg nematode of family dorylaimidae
■ onchiostyle- derived from a dorsal tooth supported by an extension of the dorsal
stoma lining.Usually curved, and never hollow. Eg nemaodes of family triplochidae
■ Stomatostylet- thought to have risen from various fused part of stoma. Divided into
an anterior tip or conus and posterior support or shaft. Eg nematode of family
aphelenchidae.

Sex and sexual dimorphism
■ Presence/absence of males and of sexual dimorphism are used as differentiating
features. Where males are absent, the female spermatheca is reduced and empty.
■ Sexual dimorphism in body shape is a good character of Heteroderidae and
Rotylenchulinae and in the anterior region (cephalic region, stylet and oesophagus) is a
characteristic of the Radopholinae as against the Pratylenchinae.

Shape
■ The different shapes of nematodes include:
 Lemon- eg heterodera
 Kidney- eg Rotylenchulus
 Pear shaped- eg tylenchorhynchus
 Ribbed/ringded- eg Criconematid
 Spherical/gourd shape- eg Meloidogyne
 C-shaped- scutellonema
 Fusiform/swollen- eg achlysiella and nacobbus
 Vermiform- eg Pratylenchus and Helicotylenchus

Cuticle
■ Cuticle thickness, striation, annulation (both transverse and longitudinal), punctation and
ornamentation, and cuticular modification (ridges, spines, scales, alae) serve as useful
characters as do the lateral field and their incisures.
■ Thickening of the cuticle at the tail tip is a good character for Trophurus and Paratrophurus.
Longitudinal ridges characterize Mulkorhynchus while the presence of five or six lateral
incisures are used to diagnose Quinisulcius and the Merliniinae, respectively.
■ Body pores are used as taxonomic characters inTrichodoridae and the excretory pore
position is used as a taxonomic character.
■ Bursal ribs on the tail are diagnostic for the Aphelenchidae while the presence of
hypoptygma around the cloacal aperture differentiates the Merliniinae from the
Telotylenchinae.

intestine, prerectum, rectum and anus.
■ The number of cells in a cross-section of intestine and the length of the prerectum are
useful in the taxonomy of the Dorylaimida.
■ The anus in members of the Aphelenchida is a large, posteriorly directed crescentic slit
compared with the small and pore-like structure in members of theTylenchida.
■ Rectum length in respect to anal body width is also used as a character.
■ A post-anal intestinal sac differentiates between Bitylenchus and Tylenchorhynchus
and its length is useful in differentiating species.

Female reproductive system
■ Reproductive systems can be didelphic, monodelphic, pseudomonodelphic (when one
branch is secondarily reduced), prodelphic, opisthodelphic, monoprodelphic and mono-
opisthodelphic.
■ The length of the reproductive branch as a percentage of the body length is important in
some cases, but in others it is highly variable since it depends on the degree of
development of the ovary.
■ The shape and location of the vulva is a good character, as is the presence/absence of
lateral vulval membranes and epiptygma.
■ The shape and size of the spermatheca and its position in respect to the genital branch
(axial or offset) are good diagnostic characters.
■ When a branch is reduced to a sac, its length and the presence/absence of a reduced ovary
serve as good characters, e.g. in Pratylenchus.The length of the mature ovary and the
arrangement of oocytes are also important, e.g. in Aphelenchoides.

Male reproductive system
■ The size and shape of the spicules, gubernaculum and spermatozoa are useful
characters.
■ There is a distinct difference in the shapes of the spicula and gubernacula of the
Tylenchorhynchinae versus the Merliniinae and the gubernacula of the Pratylenchinae
versus Radopholinae.The shape of spermatozoa differs in species of Radopholus.
■ Genital papillae and ventromedian supplements are important characters of
trichodorid and dorylaimid nematodes.
■ Copulatory muscles were used by Siddiqi (1974) to differentiate between Trichodorus
and Paratrichodorus and the presence of hypoptygma was used by Siddiqi (1970) to
differentiate Merlinius from Tylenchorhynchus.

Tail
■ Tail length and shape are used in taxonomy but they show considerable variation.
■ The shape of the tail tip, although a useful character in many groups, was shown to be
variable in some species of Pratylenchus (Taylor and Jenkins, 1957).
■ Nevertheless, a pattern of tail tip shape emerges when a large number of specimens are
studied. For example, Pratylenchus panamaensis, P. coffeae and P. loosi have characteristic tail
tip shapes.
■ The tail is consistently elongated in theTylenchidae, hooked in the Halenchidae and short and
rounded in females of the Hoplolaiminae and males of the Heteroderidae and the
Meloidogynidae.

Meloidogyne-Root-knot nematode
 Body swollen.
 Body cuticle soft, no cyst
stage.
 Female neck usually short;
vulva and anus terminal;
perineal pattern present.
 Species- Javanica, hapla,
ingognita and arenaria

Trichodorus- stubby root nematode
■ Body slender, vermiform
■ Oesophagus dorylaimoid.
■ Body cigar-shaped; curved onchiostyle and
excretory pore present, pre-rectum absent
and protractors of spicule capsuliform and
not attached to body wall.
■ Vaginal length about half of the
corresponding body diameter lateral body
pore advulvar; sclerotization and
constrictor muscles distinct; male habitus a
j-shape; spicule protractor muscle capsule
strong.
■ Species – Trichodorus obtusus

Nanidorus-
excretory pore present, pre-rectum absent and
protractors of spicule capsuliform and not
attached to body wall.
■ Vagina shorter that half of the corresponding
body diameter with inconspicuous sclerotization
and constrictor muscle; male habitus nearly
straight spicule protractor muscle capsule weak
cuticle usually inflating strongly upon fixation
■ No lateral body pore or caudal pore in female;
vulva opening transverse slit in ventral view;
male absent or rare.

Paratrichodorus-stubby root
nematode
excretory pore present, pre-rectum absent
and protractors of spicule capsuliform and not
attached to body wall.
■ Vagina shorter that half of the corresponding
body diameter with inconspicuous
sclerotization and constrictor muscle; male
habitus nearly straight spicule protractor
muscle capsule weak cuticle usually inflating
strongly upon fixation
■ lateral body pore present caudal pore present
vulval opening a longitudinal slit in ventral
view male present.

Pratylenchus –lesion nematodes
■ BODY SLENDER,VERMIFORM.
■ OESOPHAGUS tylenchoid.
■ Vulva situated much more posterior one
genital tract.
■ Dorsal Oesophageal gland opening just
posterior to stylet knob, median bulb
smaller, more oval.
■ Tail blunt, finely or broadly rounded or
pointed, rarely spicate or mucronate;
stylet and stylet knobs well developed
cephalic framework strong.
■ Species- Pratylenchus coffeae

Ditylenchus- potato rot nematode
■ Vulva situated much more posterior
one genital tract.
■ Dorsal Oesophageal gland opening
just posterior to stylet knob, median
bulb smaller, more oval.
■ Tail tip elongated-conoid to nearly
filiform stylet and stylet knobs small,
weakly developed; cephalic framework
weak.
■ Species –ditylenchus dipsaci

Hemicycliophora-sheath nematode
■ OESOPHAGUS criconematoid.
one genital tract.
■ Stylet knobs sloping backwards;
vulva forms a conspicuous
discontinuity in body contour
medium to large nematode.
■ Species-

Paratylenchus-pin nematode
one genital tract.
■ Stylet knobs sloping backwards,
annuli small, inconspicuous; mostly
small slender nematodes (swollen
forms rarely found)
■ Species –Pratylenchus pratensis

Hemicriconemoides-false sheath
nematode
one genital tract.
■ Stylet cup shaped, annuli conspicuous
broad; robust cigar-shaped nematode.
■ Female body covered with fourth
stage juvenile cuticle which form a
closely fitting sheath over whole body,
but more loose post-vulvaly.
■ species-

Criconema, ogma, Criconemoides,
discocriconemella-ring nematode
one genital tract.
■ Stylet cup shaped, annuli conspicuous
broad; robust cigar-shaped nematode.
■ female body not covered with a
sheath, annuli, smooth or ornamental.
■ Species –Criconemoides morgensis

Heterodera- soybean cyst nematodes
■ Body swollen
■ Body cuticle of female transformed
into a leathery brown cyst.
■ Cyst lemon-shaped with vulval cone.
■ Species – heterodera exigua

Globodera- potato cyst nematode
■ Body swollen
■ Body cuticle of female transformed
into a leathery brown cyst.
■ Cyst round to oval, no vulval cone.
■ Species- globodera rostonchinsis

Helicotylenchus-spiral nematodes
■ Vulva usually situated medially, one or two
genital tracts,
■ Tail usually shorter than two times the anal
body width.
■ Stylet knobs anteriorly indented, flattened or
sloping backwards.
■ Phasmids punctiform, pre or post-anal.
■ Oesophageal overlap over intestine ventral or
dorsal Oesophageal usually more than one
quarter of stylet length behind knobs.
■ Species -

Scutellonema- false spiral nematode
■ Vulva usually situated medially, one or
two genital tracts,
■ Tail usually shorter than two times the
anal body width.
■ Stylet knobs anteriorly indented,
flattened or sloping backwards.
■ Phasmids greatly enlarged to a
scutellum, pre or post anal.
■ Species – scutellonema bradys

Xiphinema- dagger nematode
■ OESOPHAGUS DORYLAIMOID.
■ ODONTOSTYLET LONG, SLENDER,
WITH FLANGES,GUIDING-RING
NEAR BASE OF ODONTOSTYLE,
USUALLY DOUBLE AMPHID
OPENING SLIT.
■ Species – Xiphinema americana

Longidorus-needle nematode
■ ODONTOSTYLET LONG WITH OR
WITHOUT SMALL FLANGES,
GUIDING-RING USUALLY CLOSE
BEHIND LIP REGION, AMPHID
OPENING PORE-LIKE
■ Species- longidorus elongatus

PARALONGIDOROUS- needle
nematode
■ ODONTOSTYLE LONGWITHOUT
FLANGES,GUIDING-RING JUST
POSTERIORTO LIP REGION,
AMPHID OPENINGA LONG OR
SHORT SLIT, NEVER PORE-LIKE
■ Species – paralongidorous sali

Trophurus
■ Vulva usually situated medially, one
or two genital tracts,
■ Posterior tract reduced to uterine
sac, gubernaculum non-protruding

Radopholus –burrowing nematode
two genital tracts,
■ Tail usually longer than two times the
anal body width.
■ Oesophageal overlap over intestine
long and on dorsal side
■ Species –Radopholus similis

Dolichodorus- awl nematode
■ Tail longer than two times the anal
body width.
■ Vaginal sclerotization symmetrical
lateral field with three lines

Neodolichodorous
■ Tail longer than two times the anal
body width.
■ Vaginal sclerotization asymmetrical,
lateral field with four lines

Histotylenchus-
genital tracts,
■ Tail longer than two times the anal body
width.
■ Vaginal sclerotization absent
■ Oesophageal overlap over intestine short
but distinct.
■ Stylet strong with asymmetrical cone,
lateral field with four line, gubernaculum
usually not curved

Tylenchorhynchus – stunt nematode
genital tracts,
width.
■ Vaginal sclerotization absent
■ Oesophageal overlap over intestine short
but distinct.
■ Stylet weak to moderate strong with
asymmetrical cone, lateral field with three
to five lines.

Geocenamus
genital tracts,
width.
■ Vaginal sclerotization absent.
indistinct or absent.
■ Lateral fields with six lines.
■ Tail conical, terminal cuticle not
conspicuously thickened.

Tylenchorhynchus –stunt nematode
genital tracts,
width.
■ Lateral fields with five lines
■ Tail conical, terminal cuticle not
conspicuously thickened.

Paratrophurus
genital tracts,
width.
■ Lateral fields with five lines
■ Tail conical, terminal cuticle conspicuously
thickened, gubernaculum protruding.

Hoplolaimus- lance nematode
two genital tracts,
■ Tail usually shorter than two times the
anal body width.
■ Stylet knobs large; tulip shaped;
phasmid greatly enlarged, one
anterior, the other posterior to vulva.
■ Species- Hoplolaimus tylenchiformis

Rotylenchus –spiral nematode
■ Vulva usually situated medially, one or two genital
tracts,
■ Tail usually shorter than two times the anal body
width.
■ Stylet knobs anteriorly indented, flattened or
sloping backwards.
■ Oesophageal overlap over intestine mostly dorsal
Oesophageal opening usually less than one quarter
of stylet length behind stylet knobs.
■ Species- Rotylenchus robustus
MaleFemale protruding
from root tissue

Rotylenchulus- rainyform nematode
■ BODY SLENDER, VERMIFORM.
■ Vulva usually situated medially, one or two genital
tracts,
■ Tail usually shorter than two times the anal body
width.
■ Stylet knobs anteriorly indented, flattened or sloping
backwards.
■ Immature female habitus a c shape or spiral; dorsal
Oesophageal gland outlet one half to twice stylet
length behind knobs, Oesophageal overlap intestine
mostly laterally, vulva near or well behind midbody.

Aphelenchoides-leaf and bud
nematode
one genital tract.
■ Dorsal Oesophageal gland secretes
into Oesophageal lumen just
anterior to valve of median bulb;
median bulb large distinct rounded-
rectangular.
■ Species –Aphelenchoides besseyi

Tylenchulus- citrus nematode
■ Mature female not kidney shaped,
with long pointed tail.
■ Mature female: Excretory pore near
vulva, anus absent, posterior body
exterior to root.
■ Species – Tylenchulus semipenetrans

Meloinema
■ Body swollen.
■ Body cuticle soft, no cyst stage.
■ Female neck long; vulva and anus subterminal; perineal pattern absent.

Amplimerlinius
■ Vulva usually situated medially, one or two genital tracts,
■ Tail longer than two times the anal body width.
■ Oesophageal overlap over intestine indistinct or absent.
■ Lateral fields with six lines
■ Tail cylindrical, terminal cuticle conspicuously thickened.

Sampling,extraction and identification of plant parasitic nematodes PPN's

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Sampling,extraction and identification of plant parasitic nematodes PPN's