COLLECTION AND STORAGE OF SAMPLES AFFECTED BY PLANT PARASITIC NEMATODES

• PRACTICAL AND THEORETICAL CONSIDERATIONS

When making a sampling plan for estimating nematode numbers, biomass and species composition, a
number of practical and theoretical considerations must be observed. The design of the sampling plan
will partly depend on the purpose of the study, the required accuracy , the time frame, as well as the
costs involved. Besides, when making the plan, knowledge about the variation in space and time of
nematode populations, their biology, and the influence abiotic factors has to be considered, to arrive at
a correct choice regarding timing and depth of sampling, tools, number of samples and the sampling
pattern.

• THE SAMPLING PROCESS

1. Tools
Sampling can be carried out using a variety of tools, ranging from an ordinary spade to special
soil samplers or augers (fig.2). The type of sampling device depends on the purpose of sampling.
One sample consists of several cores. Since the level of accuracy increases with the nu::ber of
cores, it makes sense to take out only a small amount of soil per core, thus limiting total sample
size. However, the diameter of the soil sampler or auger should not be too small, since too much
friction harms the nematodes. A 17 mm auger is generally used. Il vulnerable nematode genera
of the Trichodoridae and Longidoridae families are to be sampled, it is recommended to use a
sampling,tube with a very wide diameter, e.g. a preserving can with both lids removed. The soil
sampler must be inserted at a straight angle and be given a quarter or half turn before
retracting, so that the soil will stick in the tube. Equipment always needs to be clean to avoid
contamination from a previously sampled plot and to prevent a ‘second degree error’ (wrongly
conclude that nematode species an attached to a dirty tool is present in the plot). Apart from
the described sampling tools, sketch paper and a pencil are used to record other details as the
condition of the crop, the weather, date, locality, host crop, extent of damage and collector etc.

2. Transport and Storage

To avoid dehydration, samples are preferably collected and stored in plastic bags. Use a
permanent waterproof marker for writing on the label or bag, to avoid discoloration and
bleaching. Avoid exposure of samples to high temperatures; a cooler box is preferred. The
samples nmust be handled with care because nematodes may die from shock and friction.
The samples must be processed as soon as possible, because storage may cause changes in
nematode numbers and species composition. Data on storage effects mostly refer to plant
parasites; increases and decreases in numbers have been observed. Bacteriovorous
Rhabditidae multiplies at temperatures as low as 4°C. For a general guideline Samples are
stored at 4°C. Keep storage time as short as possible. Samples can be stored up to half a
year in wet without considerable effects on nematode numbers.

• PRE-PROCESSING OF THE SAMPLES

a. Measuring: volume or weight?

The size of a sample can be described in terms of volume or weight, both having
advantages and disadvantages. In research for advisory purpose or phytosanitary
 certification, sample size is generally given as volume (100 or 200 ml soil), while in
other studies sample size is / usually based on weight (100 or 200 g). Sampling and
mixing destroy the natural structure of the soil, altering the degree of soil
compaction. For that reason it is difficult to find the amount of soil that would
correspond with a particular volume of soil under undisturbed field conditions.
Using weight to express sample size has the disadvantage that large differences in
the amount of soil per unit of weight occur, due to variations of soil moisture
content. This problem, however, can be solved by making a simple measurement of
dry matter content.

B. Taking sub-samples; Mixing:

Achieve sufficient accuracy, relatively large sample size is often needed. Usually, these
cannot be processed completely, making it necessary to take sub-samples (Note: this
reprešents an extra source of variance!). Before taking a representative sub sample, the
sample needs to be homogenized by mixing. If homogenization is carried out
thoroughly, analysis of one sub-sample is sufficient (Carbonell & Angulo 1979). Mixing
the sample can be done mechanically or manually. The sample should be mixed with
care as each treatment causes mortality of nematodes. Mixing by hånd-causes less
damage to the nematodes and is therefore preferred to mechanical mixing. Manual
mixing is a useful method for most samples, except for heavy clay soil. Disadvantages of
manual mixing are its labor intensity and difficulty to standardize. A procedure suitable
for soil or other fine materials (e.g. seed) is to pass the sample through a coarse sieve
(mesh size of 0.5-1 cm) to remove stones, roots,. Etc. Mix the sample on a small sheet (+
I x1 m) by lifting one sheet corner making the soil roll óver to the opposite corner. Do
the same with all comers and repeat a fixed number (generally six) of times. Take a sub-
sample of the desired volume or weight, using small scoops from different parts of the
sample. It is possible to obtain a representative sub-sample by taking small scoops from
different parts of the sample without previous mixing. This be preferred when
vulnerable nematodes are expected, e.g. Trichodorus. Mechanical mixing is generally
not used for taking sub-samples, but for homogenizing or making a suspension. For
plant material, a blender can be used. Prior to mixing, water is added to the sample until
it is just submerged. For mixing heavy clay or large soil samples, sodium
hexametaphospate or other appropriate chemical is added to the sample (a dose of 1%
of the soil weight) to break bonds between (clay) particles. Sodium hexametaphospate
is not harmful to mbst of the Tylenchid nematodes, but its effect on other nematodes is
not known.

Exercise no 4. PREPARATION OF TEMPORARY AND PERMANENT

SLIDES OF PLANT PARASITIC NEMATODES

MATERIAL:
Bamboo needle, watch-glass, dissecting microscope, slides, cover slip, fixative, spirit-
lamp.
 PROCEDURE:
A. Handling Nematodes: Sharpen the bamboo needles or a tooth brush bristle to a very
fine point and mount in a needle holder. Bring nematode suspension undèr a dissecting
microscope. Lift a nematode from the bottom with a pick and bring it to the surface of
water. Pass the pick under the specimen and it will be held on the pick by surface
tension.
B. Killing the Nematodes: Collect the nematodes in a small drop of water in a watch
glass. Hold it with forceps over a small flame moving it about 5-6 seconds. Watch the
nematode carefully from time to time to avoid overheating.
C. Temporary Mounts: Lift few nemat from watch glass and transfer to a drop of water
on a clean slide. The drop should contain just enough fluid to spread completely to the
edges of the cover slip but not more. Avoid crushing by the use of glass fibres. Cover the
drop with a cover glass and sealed the edges with melted paraffin, vaseline or nail polish
to prevent rapid evaporation.
D. Permanent Mounts: (Golden, 1971).
1. Bring nematodes to a watch glass with 2-3 ml of H20.
2. Incubate the specimens at 43 C for about 12 minutes to relax them,
3. After 12 min pour in warm (43 C) fixative to fill the watch glass almost to the top. The
fixative contains 30 ml formalin, 350 ml distilled water, 8 ml Glycerine.

4. Remove watch glass from oven, cover and maintain at room temperature for 24 hrs
or longer. Add 2 drops of saturated aqueous picric acid. This stain the stylet which other
wise becomes almost invisible in the final preparation.
5. Return the covered watch glass at 43 C and keep it at this termperature for 3 weeks
or longer. Every 2-3 days add warm fixture as needed to offset evaporation.
6. After 3 weeks or longer permit the fixative solution to evaporate completely. Now
add a few drops of pure, warm (43 C) glycerine.
7. . Mount in anhydrous glycerine or stored in sealed vials of glycerine.

Staining nematodes within root tissue:

Wash roots thoroughly in tap water. Bring the tinted lactophenol (phenol crystal or liquid 20 g lactic acid
20 ml, Glycerine 40 ml, distilled water 20 ml) to a boil + 5 ml 1% sol of acid fuchsin) in a well ventilated
place and boil roots in staining solutions for 1 min. Wash out excess stain with tap water and blot off the
excess water. Place in clear lactophenol solution (without stain) for two days until the roots are
translucent. The nematodes retain dye while the plant tissues de-stained.

Exercise 5
PERMANENT MOUNT
(1) Bring nematodes to a watch glass with 2-3ml of water. Incubate the
specimens at 43°C for about 12 minutes to relax them.
(2) After 12 minutes pour in warm (43°C) fixative to fill the watch glass almost
to the top. The fixative contains 30ml formalin, 350ml distilled water, 8ml
glycerin.
(3) Remove watch glass from oven, cover and maintain at room temperature for
24 hours. Add 2 drops of saturated aqueous picric acid. This stains the stylet
which otherwise becomes almost invisible in the final preparation.
(4) Return the covered watch glass at 43°C and keep it at room temperature for
3 weeks. Every 2-3 days add warm fixture as needed to offset evaporation.
(5) After 3 weeks permit the fixative solution to evaporate completely. Now add
a few drops of pure, warm glycerin. Mount in anhydrous glycerin or stored in
sealed vials of glycerin.

Counting the Plant Parasitic Nematode

Extracted nematodes can be viewed and counted using a dissecting or a compound
microscope; access to both in ideal. Good quality illumination is essential.
A magnification of about 40X is usually suitable but a compound microscope can
also be used, which is useful for nematodes that are in poor condition or hard to
identify.
Material
3) Counting dishes 4) Dissecting microscope 5) Pipettes 6) Pipettes tips

Procedure:
1) Extract nematodes from a known weight of plant tissue or volume of soil.
2) Concentrate the extracted suspension to a precise known volume in a measuring
cylinder or graduated tube.
3) Shake the suspension immediately before removing aliquots. Use a wide mouth
pipette tips to remove aliquots, to prevent blockage by debris. Pipette tips can be
cut if they are too narrow.
4) Carefully pipette aliquots into the counting dish, avoiding splashing. If only a few
nematodes are present, count them in the total suspension volume.
5) If nematode density is high, count the nematode from an aliquot. Dilution of the
suspension may be necessary to aid-counting, for example doubling the volume.
6) Count all the nematodes in the counting dish in a systemic way following the
gridlines on the dish.
Sometimes nematodes may float on the surface, but adding a tiny spot of liquid
soap overcomes this.
7) Use a tally counter.
8) Return the counted aliquot to the suspension after counting.
9) Repeat using 2-3 aliquots per sample and then calculated the mean for the
combined aliquot score before calculating the total nematode number per sample.
10) The mean number of nematodes calculated from the aliquots should be
multiplied by the total volume of the suspension to calculate the total number of
nematodes present in the suspension.
 AS PLANTPARASITES
NEMATODES

JulieStantonand GrahamStirling
Contents

8.1 Introductton ............127

8.2 GeneraLstructure oJ nematodes........ ...... 127
8.3 Life cgcLe . . . . . . . . . . . . . .1. .3 O
8.4 Feeding tgpes.. ..........130
8.5 Sgmptomsof nematode damage .............131
8.6 PopuLattondgnamics and threshoLdleuets .............133
8.7 Nematode surutuoL1........,.. ...... 134
8.8 Eco1o9A.............. . . . . . .1 3 4
8.9 ControLoJnematodediseases............. ... 135
R e s i s t a n c e/ t o l e r a n c e . . . . . . . . . . . . . . . . . . . . . 1. 3 5
C r o pr o t a t i o n . . . . . . . . . . . . . . . . . . . .1 3 6
Chemicalcontrol ...............136
BtoLogtcal control ,.,,..,....,...138
O r g a n i ca m e n d m e n t s. . . . . . . . . . . . . . . . . . . . . .1. 3 8
PhgsicalcontroL ...,,....,...,.. 139
Interactions tuith ottrcr organisms ..... 139
8.7O CLassificatton oJptant parasttlc nematodes.............. .............140
8.11 ntrttrcrreading .... 142

8.1 lntroduction
Nematodes are the most abundant group of multicellular animals on earth in
terms of numbers of individuals. There are at least as many nematode species as
insect species. In a square metre of moderately fertile soil to 30 cm depth, there
are about 50 million nematodes. Nathan Cobb, father of nematology in Australia,
provided a good mental picture of the importance and diversity of nematodes
'if
when he stated that all matter in the universe except nematodes were swept
away, our world would still be dimly recognisable-we would find its mountains,
hills, valleys, rivers, lakes and oceans represented by a film of nematodes'. About
half of all nematode species are marine nematodes, 25o/oare free-living, soil-
inhabiting nematodes, I57o are animal and human parasites and lO%oare plant
parasites. Today, even with modern technologr, 5-loo/o of crop production is lost
to nematodes in developed countries.

8.2 General structure of nematodes

Nematodes are generally worm-shaped, being cylindrical but tapering at both
ends. However, adult females of some species swell to become kidney-shaped,
lemon-shaped or spherical. Some plant parasites may be up to 3 cm long but
most nematodes found in soil are O.5 to 1 mm long.
Regardless of their feeding habits, all nematodes have the same basic
structure. Just behind the mouth is a stoma (a chamber lined with cuticle)
through which the nematode ingests food. Nematodes have evolved a wide variety
T28 Julie Stanton and Graham Strlino

of feeding structures, each adapted to a particular method of feeding. Species

that feed on bacteria have a large cylindrical or funnel-shaped stoma through
which the nematode sucks its food. Some have lip extensions to tear dead plant
tissues apart. Carnivorous species have teeth for holding or piercing their victims
which may include other nematodes. Most plant-parasitic nematodes have a
hollow spear, called a stylet, with which the nematode pierces cell walls to suck
cytoplasmic contents from plant cells. Stylet shapes and sizes are useful
taxonomic characters as they vary markedly in different species.
Behind the stoma is the oesophagus (sometimes spelt esophagus) (Fig. 8.1).
This is a muscular, glandular tube with a lumen which is usuaily triradiate in
cross section. The lumen is also lined with cuticle. Radial muscles attached to
the lumen contract, pulling the walls apart, creating a suction. The oesophagus
of many nematodes is subdivided into four main regions (Fig. 8.2). The
procorpus connects the stoma to the metacorpus or median bulb. In plant
parasites, the median bulb contains a valvular apparatus which creates suction
and allows the nematode to feed via the style. The isthmus joins the metacorpus
to the basal bulb. The basal bulb is glandular and is sometimes expanded into a
flap which overlaps the intestines.

Figure 8.1 Structure of nematode oesophagus.(A) Rhabdittda (bacterialfeeder).

(B) plant parasitic form of Dorylaimida. (C) TAlenchrda(plant parasite).

Nutrients from the oesophagus are metabolised and absorbed by the intestine
into compounds required by the nematode. The intestine also stores reserve food
and excretes wastes combining the functions of both the intestine and liver of
mammals. Intestinal cells of adequately fed nematodes contain large numbers of
proteinaceous and lipid globules as well as glycogen and other storage products.
Without access to a suitable host, plant-parasitic nematodes starve, utilising
intestinal reserves so that the intestine becomes clear.
In most nematode species, the sexes are separate with females being generally
larger than males. Adults of the two sexes are easily distinguished
morphologically (Fig. 8.2A and D). The female gonad has one or two elongated
tubes which may be longer than the nematode's body and therefore they are
coiled or reflexed. At the distal end of the gonad is the ovary where cells divide
forming oocytes. The oocyles enlarge and move to the uterus via the oviduct. In
some species, there is a specialised pouch between the oviduct and uterus. This
is the spermatheca which stores sperm. The eggshell is formed around the
fertilised oocyte in the uterus. The egg is laid from the uterus via the vagina. This
leads to the vulva which is an opening on the ventral wall of the nematode.
 8. Nematodes as plant parasttes r29

C,o.
iiililr*€E

A,o-ggs-
B,c: loum

Figure 8.2 Morphology of the stunt nematode (Tglenchorttgnchusspp.). (A) female;

(B) anterior region of female (lateral view). (C) face view of female.
(D) posterior region of male (lateral view). (From O'Brien and Stirling,
1991.)

The testis of the male reproductive system (Fig. 8.2D) is similar to the ovary,
having a zorle of cell division, which produces sperrn, and a growth zone, where
the sperm cells enlarge. Sperm accumulates in the enlarged seminal vesicle and
is conducted to the exterior via the vas deferens which is a glandular and
muscular tube. The exterior opening of the reproductive system, the cloaca, is
shared with that of the alimentary canal. The male gonad also possesses two
hook-like structures, the spicules, which originate inside the cloaca. They form a
passageway for sperrn during mating when they are inserted into the vagina of
the female. In some species, the male has lateral cuticular flaps, the caudal alae
or bursa, in the tail region which they use to grasp the female during copulation.
Many types of reproduction have evolved. In most species the sexes are
separate but some species reproduce without males. These may be
hermaphroditic with a gonad that can produce spenn or eggs at different times.
Where only females are known, they lay eggs that develop parthenogenetically.
Parthenogenesis (development of an individual from an egg without fertilisation
by a sperm) is the most common form of reproduction in MeLoidoggne(root-knot
nematode). Depending on the species, the mechanism may be either mitotic or
meiotic. In meiotic parthenogenesis, the second nuclear division does not occur
so that the normal somatic chromosome number is restored and embryogenesis
can proceed. In mitotic parthenogenesis, the mature oocyte undergoes a single
mitotic division, forming a diploid egg. The somatic chromosome number is
consewed and embryogenesis can proceed.
130 Jutie Stanton and Graham Sttrltng

The most obvious component of the nervous system of nematodes is the nerve
ring which encircles the oesophagus just behind the median bulb. This connects
several hundred cells and some sense organs. Longitudinal nerve trunks run
from the nerve ring to the head and to the rest of the body. The nematode's
nervous system is quite sophisticated for an animal of its size. It allows the
nematode to move in any direction, to detect touch and changies in temperature,
to use chemical gradients to find members of the opposite sex and sources of
food, to feed by probing urith the stylet or by activating the stoma, to move
components of the oesophagus to swallow and digest food and to defecate, to
mate, lay eggs and to produce and secrete various compounds from their glands.
The nematode's sense organs are found near the head and around the vulva
and cloaca. A ring of papillae encircles tJre opening of the stoma. TWo pouches in
the head, the amphids, are connected to slits or pores on the lips through which
secretions are released. The amphids combine nerve endings and gland cells. In
hookworms, these are used to secrete anticoagulants into the host's blood.
Amphids of both plant- and animal-parasitic nematodes contain the eruzyrne,
acetylcholinesterase, which is important in nerve physiology. This enzyme is the
target of several nematicides and is similar to that in humans and other animals.

8.3 Life cycle

I)rpically, there are six stages within a nematode's life cycle: an egg, four juvenile
stages and the adult stage (Fig. 8.3). Each juvenile stage and the adult are
separated by a moulting phase. The first stage occurs in the egg. For most plant-
parasitic nematodes, the first moult also occurs in the egg so that it is the
second-stage juvenile which hatches.

^'"'r^oi""
j u v e n i l e4
1
I embry6eenesis
m o u l t3
I
\
''.^iuu"ri,",
)
i u v e n i l e3
\ /
tou"\,rvenire *'/ noultt
2

Figure 8.3 Generalised life cycle of nematodes. (From O'Brien and Stirling, 1991.)

8.4 Feeding types

Plant-parasitic nematodes feed either ecto- or endoparasitically (Fig. 8.4).
Ectoparasites feed from the outside of the root, inserting the stylet only or the
head and neck. Examples of ectoparasites are Xiphinema (dagger nematodes),
Longidorus (needle nematodes), Trichodorus (stubby-root nematodes),
HelicotgLenchus (spiral nematodes), Crtconemoides (ring nematodes),
Paratglenchus (pin nematodes) and TgLenchorhgncltus (stunt nematodes). In
contrast, endoparasites enter root or shoot tissue and feed within the plant.
They can be sedentary, establishing specialised feeding sites and remaining
there until they die. Examples of sedentary endoparasites include Heterodera and
GLobodera (cyst nematodes), MeLotdoggne (root-knot nematode), RotUlenchultts
reniJormis (reniform nematodes) and Tglenchulus semipenetrans (citrus
nematodes). Alternatively, they may be migratory, moving through plant tissue
Baermann Funnel / oestenbrink dish
This method for extraction of motile nematodes was introduced by
baermann in 1917 using an funnel in its………..
 Nematode extraction 475

original version, the sample was wrapped in a tissue cloth

and almost fully incubated in water resulting in very low
nematode recovery. Modified versions use a wire basket
plus filter to spread the sample over a larger area. In addi-
tion, the sample is only immersed half-way into the water.
Oostenbrink (1954) replaced the funnel by a dish. Since
then, several modifications have been published such as by
Whitehead & Hemming (1965), Rodrıguez-Kabana (1981)
and others.

Materials
• Knife, pair of scissors or blender;
• Cotton-wool milk filter or equivalent (e.g. cheesecloth,
filter paper, paper towel);
• Funnel made of glass with a piece of soft polyethylene
tube attached to the stem and closed with a spring or
screw clip (Fig. 1). Recommended slope of funnel is
approx. 30°. For the Oostenbrink dish method plastic or
stainless steel dishes (pie pan) are used (Fig. 2);
• Stand to hold the funnel;
• Support, such as plastic sieve or wire basket of large
enough aperture to allow nematode passage (i.e.
250 lm); Fig. 1 Modified Baermann funnel for extracting nematodes from plant
• 20 or 25 lm aperture sieve; material or soil (Photo: JKI, Germany).
• 100 mL glass beaker;
• Dissecting, inverse or compound microscope;
• Counting slide;
• Collect nematodes after 24–72 h by opening the spring or
• Microscope slide.
screw clip on the funnel stem or by collecting the nema-
todes of the dish in a glass beaker;
Procedure
• Let the nematodes settle in the glass beaker and
• Peel and chop plant tissue such as wood chips (for prepa-
remove the supernatant, or pass suspension in the bea-
ration of wood samples for Bursaphelenchus xylophilus
ker over a 20 or 25 lm sieve to reduce the volume of
see Appendix 3 of PM 9/1), leaves, roots, bulbs or tubers
water;
in 1 cm pieces or macerate in a blender for up to 1 min
• Examine nematodes within a counting slide at 25–409
depending on source of plant material; seeds remain
magnification using a dissecting or inverse microscope or
intact or can be split longitudinally to facilitate nematode
transfer nematodes with a handling needle to a micro-
removal (e.g. Aphelenchoides besseyi/rice, Hoshino &
scope slide for inspection at higher magnification in a
Togashi, 1999);
compound microscope.
• Place plant material on the cotton-wool milk filter placed
within a support (sieve);
Advantages
• Submerge support with sample gently in the water of the
•Simple and inexpensive;
funnel/dish;
•Small amount of water;
• Nematodes leave the plant tissue, pass through the cot-
•Final suspension is clean;
ton-wool milk filter and sink to the bottom of the funnel
•Good recovery of motile nematodes from small samples.
stem or dish, respectively;

Fig. 2 Oostenbrink dish. Left: Set-up showing

plastic dish, supporting sieve made of 250 µm
polyamide gauze and cotton-wool milk filter
(Photo: JKI, Germany). Right: Set-up
consisting of plastic dish, plastic basket and
cotton-wool milk filter for extracting
Bursaphelenchus xylophilus from wood chips
(Photo: Vladimir Gaar, Diag. Lab. Prague,
Czech Rep.).

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 471–495

476 Diagnostics

Disadvantages • Dissecting, inverse or compound microscope;

• Only suited for samples up to 50 g; • Counting slide;
• Lack of aeration in the water reduces nematode move- • Microscope slide.
ment;
• Rapid bacterial growth for some plant materials (e.g. Procedure
bulbs), especially after maceration; • Wash roots free from adhering soil and cut into short
• Poor recovery of relatively immotile nematodes (e.g. pieces (1–5 cm);
Xiphinema, Hemicycliophora, Criconemoides); • Approximately 5 g of roots are moistened and placed in a
• Poor recovery from large samples. polythene bag, that is closed and incubated for 3–4 days
at room temperature (20°C); during this period, most of
Remarks the nematodes will leave the root tissue;
Alternatively, funnels made of plastic or stainless steel and/ • Wash the roots and the inside of the bag with a small
or using silicone tubes can be used. However, regarding amount of water and collect the water containing the
the latter, diffusion of oxygen into water is lower than for nematodes in a glass beaker
polyethylene (Stoller, 1957) which could slowly lead to • Pass the nematode suspension in the beaker over a 20 or
asphyxiation. Depending on the plant tissue, most (50– 25 lm sieve to reduce the volume of water;
80%) of the motile nematodes present will be recovered • Examine nematodes within a counting slide at 25–409
within 24 h; however, samples can be left on the funnel magnification using a dissecting or inverse microscope or
for up to 72 h or for wood chips even up to 14 days to transfer nematodes with a handling needle to a micro-
increase recovery rate. For longer extraction periods regular scope slide for inspection at higher magnification in a
tapping and adding of fresh water increases nematode compound microscope.
motility and therefore recovery rate. Similar results can be
achieved by using a solution of 0.15% H2O2 instead of
Advantages
water, or by placing the Baermann funnel/Oostenbrink dish
in a mistifier (see 2.4). In general, extraction can be per-
• Simple and cheap;
formed at room temperature (20°C). The diameter of the
• Minimum labor and equipment.
funnel or dish should be chosen so that sample layer will
Disadvantages
not be more than 1–2 mm. For larger sample sizes use
aliquots or divide the sample over several funnels/dishes.
• Little efficacy;
The filter paper should retain remaining soil particles and
• Long time period until results are available.
plant debris but allow easy passage of nematodes at the
Remarks
same time. Milk filters made of cotton wool or fleece (e.g.
Efficacy of extraction can be improved by adding 1–3%
H€oschele GmbH, Remshalden, Germany; http://www.hoesc-
H2O2 for oxygen supply (Tarjan, 1967, 1972). Oxygen can
hele-nonwoven.de/) are commonly used. Usually, one or
also be supplied by adding air through a small pump as
two layers work well. However, users should be aware that
used in fish aquaria. In modified form, wood chips can be
nematode passage can vary highly depending on filter
directly emerged in water for extraction of nematodes such
material and thickness of filter. If in doubt, efficacy tests
as Bursaphelenchus spp. (Fig. 3). A comprehensive discus-
should be performed.
sion on this technique can be found in Ayoub (1980).

2.3 Root incubation

2.4 Mistifier technique
This method for the extraction of motile nematode stages
This technique was originally described by Seinhorst
is especially recommended for Nacobbus aberrans and
(1950) and is used to extract motile nematodes. It consists
Meloidogyne spp., but can also be used for other endo-
in principle of a Baermann funnel or Oostenbrink dish
parasitic nematodes, such as migrating endoparasites
placed in a mist or fog of water to avoid oxygen depletion.
(Radopholus spp., Bursaphelenchus spp.). The method was
The mist is produced by spray nozzles over the plant mate-
introduced by Young (1954) and later modified by Moun-
rial or by nozzles spraying upwards so that the droplets fall
tain & Patrick (1959), who used glass jars instead of plastic
in a soft curve onto the plant material. Sap and toxic
bags.
decomposition products of the plant material are washed
off with the funnel overflows allowing extraction times of
Materials
possibly up to 6 weeks (see remarks below).
• Pair of scissors or knife;
• Balance (scale);
Materials
• Polythene bag;
• 100 mL glass beaker; • Knife, pair of scissors or blender;
• 20 or 25 lm aperture sieve; • Mistifier spray apparatus (Figs 4 and 5);

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 471–495

478 Diagnostics

nozzles are generally used at 4–6 L h 1 under pressure of Remarks

about 2.8 kg cm 2. In case of fog, spray nozzles are best In case of potato peels, starch grains can complicate the
placed at the sides spraying into the interior of the appara- examination of the suspension from the 45 lm sieve. If that
tus. Fog spray runs constantly. When left for too long happens, addition of water can ease evaluation. For better
(>4 weeks), bacterial cultures can flourish and make the visualization, nematodes within root tissue can be stained
nematode suspension collected at the bottom of the funnel prior to extraction with Phloxine B (0.15 g L 1 water), acid
unclear and can even clog the tubing and filter. Complete fuchsin solution (875 mL lactic acid, 63 mL glycerol,
replacement of the water in the funnel every 3–4 days can 62 mL water, 0.1 g acid fuchsin) or 12.5% McCormick
improve extraction efficacy. Schilling red food color (Manzanilla-Lopez et al., 2002;
Thies et al., 2002; Hooper et al., 2005); however, applica-
bility of this method is questioned by some nematologists.
2.5 Maceration and filtration
Instead of mechanical maceration, enzymatic maceration
This method is suitable for the extraction of motile and can also be used (see 2.7). For Meloidogyne chitwoodi
immotile stages of Hirschmanniella spp., Radopholus extraction from potato tubers, mechanical and enzymatic
similis, Nacobbus aberrans and other endoparasitic nema- maceration yielded similar nematode numbers.
todes from roots, tubers and other plant tissues. Further
details on this method are given by Coolen et al. (1971)
2.6 Maceration and centrifugal flotation
and Seinhorst (1988).
This method is well suited to recover both motile and
Materials immotile nematode stages from plant tissues. In the first
• Pair of scissors or potato knife; step, nematodes are liberated from plant tissue by macera-
• Domestic blender (e.g. Waring blender); tion in a blender. They are then separated from the macer-
• 250, 150 and 45 lm aperture sieves; ated plant tissue by centrifugal flotation using a solution of
• 100 mL glass beaker; specific weight higher than the nematodes. Further details
• Dissecting, inverse or compound microscope; on this method are given by Coolen & D’Herde (1972), Co-
• Counting slide; olen (1979) and Greco & D’Abbaddo (1990).
• Microscope slide.
Materials
Procedure • Pair of scissors or knife;
• Cut and macerate roots and/or tuber peels (0.5 mm thick) • Blender (e.g. Waring blender, household blender);
in a blender at about 12 000 rev min 1 for 30 s; • 1200 lm aperture sieve;
• Pass the resulting suspension through a set of sieves with • Centrifuge plus centrifuge tubes (size ranging from 100
the 250 nested on top of the 150 and 45 lm aperture to 1000 mL);
sieves; • Kaolin;
• Debris collected on the 250 and 150 lm sieve is • MgSO4 solution with a density of 1.15–1.18 (or similar
generally discarded unless there is specific interest for extraction fluid, see 3.5);
swollen female stages such as those of Meloidogyne or • 20 or 25 lm aperture sieve;
Nacobbus; • 100 mL glass beaker;
• Transfer nematodes on the 45 lm sieve into a glass bea- • Dissecting, inverse or compound microscope;
ker; • Counting slide;
• Examine nematodes within a counting slide at 25–409 • Microscope slide.
magnification using a dissecting or inverse microscope or
transfer nematodes with a handling needle to a micro- Procedure
scope slide for inspection at higher magnification in a • Wash plant tissue and cut into pieces about 1 cm long.
compound microscope. Mix the sample carefully if only part of it is used for
nematode extraction;
Advantages • Macerate plant tissue in a blender at about 12 000 rev
• Examination within few minutes possible; min 1 for 30 s;
• Small amount of water. • Pour the resulting suspension through a 1200 lm sieve
and collect in a beaker;
Disadvantages • Wash the plant tissue on the sieve carefully with water to
• Nematodes might get damaged in blender; collect all nematodes;
• Maceration of plant tissue may release toxic or sticky • Centrifuge the collected washing water containing the
(e.g. banana roots) compounds; nematodes with 1% kaolin powder (approx. 1 tablespoon,
• Low efficacy. depending on size of tubes) at approx. 1800 g for 4 min;

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 471–495

 Nematode extraction 481

ald, 1989). As nematodes appear to move towards cold

(Robinson & Heald, 1989) a heating source (e.g. light
bulbs or similar) installed atop of the Baermann funnel/
Oostenbrink dish (see Nielsen, 1947/48; Sohlenius, 1976)
can speed up extraction time and improve overall efficacy;
however, this method is not commonly used in nematology.
Additional information on the different parameters affecting
nematode extraction by the Baermann funnel technique is
given by Viglierchio & Schmitt (1983a). See also remarks
made in 2.2.

3.2 Flotation and sieving

Fig. 6 Oostenbrink dishes for extracting nematodes from soil. Left:

This technique for the extraction of motile nematodes from
Oostenbrink dish with 15 cm inner diameter for samples up to 100 mL soil was introduced by Cobb (1918) and is mainly used
soil, Right: Oostenbrink dish with 24 cm inner diameter for samples up for Tylenchid species. In principle, soil is washed in water,
to 250 mL soil (Photo: JKI, Germany). decanted and nematodes are collected on sieves of differ-
ent aperture followed by cleaning the suspension with
• Let the nematodes settle in the glass beaker and remove Baermann funnel/Oostenbrink dish. The method makes use
the supernatant, or pass suspension over a 20 or 25 lm of differences in size, shape and sedimentation rate
sieve to reduce the volume of water; between nematodes and soil particles, and of nematode
• Examine nematodes within a counting slide at 25–409 motility.
magnification using a dissecting or inverse microscope or
transfer nematodes with a handling needle to a micro- Materials
scope slide for inspection at higher magnification in a • Bucket of about 10 L;
compound microscope. • Stirring rod;
• 3 9 50 lm aperture sieves;
Advantages • 500 mL glass beaker;
•Simple and inexpensive; • Watch glass (d = 6 cm);
•Small amount of water; • Baermann funnel/Ostenbrink dish (see 3.1.);
•Final suspension is clean; • 100 mL glass beaker;
•Good recovery of motile nematodes from small samples. • Dissecting, inverse or compound microscope;
• Counting slide;
Disadvantages • Microscope slide.
• Only suited for small samples up to 250 mL;
• Lack of aeration in the soil reduces nematode movement; Procedure
• Poor recovery of relatively immotile nematodes (e.g. • Add up to 200 mL of soil to a 10 L bucket and add
Xiphinema, Hemicycliophora, Criconemoides); approx. 5 L of water;
• Poor recovery from large samples. • Stir the soil suspension vigorously for 10 s;
• Allow the soil to settle for 45 s;
Remarks • Pour the supernatant through a bank of 3 sieves of 50 lm
Alternatively, funnels made of plastic or stainless steel and/ aperture;
or using silicone tubes can be used. However, regarding • Wash the debris collected on the sieves in a clean glass
the latter, diffusion of oxygen into water is less than for beaker;
polyethylene (Stoller, 1957) which could slowly lead to • Carefully pour the suspension from the beaker with the
asphyxiation. Depending on soil type and nematode spe- help of a watch glass onto the cotton-wool milk filter
cies, 50–80% of the motile nematodes present will be supported by a plastic sieve in the Baermann funnel/Oos-
recovered within 24 h (e.g. Verschoor & De Goede, 2000); tenbrink dish; if necessary add more water until the bot-
however, samples can be left on the funnel for up to 72 h. tom of the filter is just covered;
If so, regular tapping and adding of the water increases • After 24 h, collect the nematodes from the Baermann
nematode motility. Choose diameter of funnel or tray so funnel or Oostenbrink dish in a glass beaker;
that sample layer will not be more than 2–3 mm to achieve • Examine nematodes within a counting slide at 25–409
best recovery. For larger sample sizes use aliquots or magnification using a dissecting or inverse microscope or
divide the sample on several funnels/dishes. Covering the transfer nematodes with a handling needle to a micro-
soil sample to prevent evaporation can accelerate and scope slide for inspection at higher magnification in a
increase efficacy of nematode extraction (Robinson & He- compound microscope.

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 471–495

484 Diagnostics

Advantages Material (Fig. 11 illustrates equipment used in centrifugal

• Efficient; flotation)
• Easy to standardize; • Centrifuge plus centrifuge tubes (size ranging from 100
• Good for large samples up to 1000 mL. to 1000 mL);
• Kaolin;
Disadvantages • Stirrer or Vibro mixer;
• Expensive equipment; • MgSO4 solution with a density of 1.15–1.18 (or similar
• High use of water. extraction fluid, see Table 5);
• 20 or 25 lm aperture sieve;
Remarks • 100 mL glass beaker;
The apparatus is generally used for samples of about • Dissecting, inverse or compound microscope;
250 mL soil; however, samples up to 1000 mL are possi- • Counting slide;
ble. Maximum sample size is determined by the fact that • Microscope slide.
the soil must be washed into the funnel before water
level 2 is reached. For small nematodes (e.g. Paratylen- Procedure
chus), the water current speed can be reduced, for larger • Fill 1000 mL centrifuge tube with up to 250 mL soil;
nematodes (e.g. Xiphinema, Longidorus) it can be • Add about 400 mL water plus a table spoon of kaolin;
increased to 1500–2000 mL min 1. Also for larger nema- kaolin forms a visible white layer that separates the sedi-
todes three nested sieves of 160–200 lm are recom- ment and nematodes from the supernatant (water and
mended. If nematode suspension needs to be examined light organic material);
the same day, cleaning can be done by centrifugal flota- • Stir suspension thoroughly with stirrer or Vibro mixer to
tion instead of Baermann funnel/Oostenbrink dish. For form a homogenous suspension;
detailed information on overall extraction efficacy and • Centrifuge tubes for approx. 4 min at 1800 g; time and
parameters influencing extraction efficacy see Verschoor g-force is not that critical, as long as a stable pellet is
& De Goede (2000). achieved; time lengths of 2–5 min and g-forces of 700–
A similar apparatus was built by Seinhorst: the ‘Seinhorst 2900 g can be used;
elutriator for free-living nematodes’. This device is used in • Gently pour off the supernatant and discard;
some laboratories and also gives excellent results. • Re-suspend the pellet in about 400 mL of a MgSO4 solution
(or similar extraction fluid) with a density of 1.15–1.18;
Modified Oostenbrink elutriator. The modified Oostenbrink • Centrifuge tubes again at 1800 g for 4 min;
elutriator made by MEKU (www.meku-pollaehne.de) pro- • Gently pour the supernatant containing the nematodes
cesses soil samples of 100–500 mL (Fig. 10). The soil sam- over a 20 or 25 lm sieve;
ple is washed with a jet nozzle at 600 mL min 1 through a • Rinse the sieve immediately with water to remove the
1 mm aperture sieve (4 mm for Xiphinema, Longidorus) MgSO4 solution;
into the apparatus. At the same time, the undercurrent water • Nematodes are transferred from the sieve in a glass beaker;
stream is set at 1000 mL min 1 and shortly later reduced • Examine nematodes within a counting slide at 25–409
to 600 mL min 1 (1500–2000 mL min 1 for large nema- magnification using a dissecting or inverse microscope or
todes). After 10–15 min, when the water has almost transfer nematodes with a handling needle to a micro-
reached the top of the extraction chamber, the side-outlet is scope slide for inspection at higher magnification in a
opened manually to pass the nematode suspension on four compound microscope.
nested 50 lm sieves.
Advantages
•Sluggish and immotile nematodes are extracted;
3.5 Centrifugal flotation
•Relatively clean nematode suspension;
This method is used in other areas of biology and was •Small amount of water;
adapted for the extraction of nematodes by Caveness & •Nematodes are available for examination within 15 min.
Jensen (1955). It is the only method that allows extraction
of motile and immotile nematodes from soil. Nematode Disadvantages
specimens are brought into a suspension with a specific • Extraction fluid can damage nematodes or alter their
gravity greater than their own, which is about 1.08, so they shape, which hampers identification; it can also influence
will float and heavier soil particles will sink. Centrifugation vitality (important when nematodes are needed for infec-
is used to speed up the separation of the sinking fraction tion studies or cultures);
and floating fraction. It is also used to clean extracts • Recovery of dorylaimid and triplonchid nematodes may
obtained by sieving or elutriation. The size of the sample be poor (e.g. Xiphinema, Longidorus, Trichodorus,
that can be processed is limited by the size of the centri- Paratrichodorus);
fuge tubes. Automated versions are available. • Expensive equipment.

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 471–495

486 Diagnostics

Methods for wet soil rely on upward water current that

keeps the cysts afloat in the suspension or flotation of cysts
in a solution with a higher density then their own. These
methods extract young full cysts as well as half depleted or
empty cysts.
Following extraction, the remainder of the sample left on
the sieve (the ‘float’) often contains high amounts of
organic matter. Cysts need to be hand-picked using a pair
of forceps, paint brush or a more robust hair (e.g. pig hair
in a holder). A great part of the organic matter can also be
removed with the help of 96% ethanol (Seinhorst, 1975) or
acetone (den Ouden, 1954; Oostenbrink, 1960). Prior to this
separation, the float needs to be dried. The float is then
Fig 12 Automated zonal centrifugation. The lower carousel feeds the placed in a conical flask and the solvent is added and thor-
samples to the centrifuge (1 L beaker) and the upper carousel receives oughly mixed with the float (Turner, 1998). Cysts will float
the nematode suspensions (150-mL beaker). The bowl of the zonal to the top while organic matter will sink to the bottom. The
centrifuge is kept inside a cupper cage for safety reasons. The sample, cysts are then carefully decanted into a filter paper-lined
kaolin and MgSO4 are fed to the centrifuge bowl through tubes (ILVO, funnel. Alternatively, centrifugal flotation using a solute
Merelbeke, Belgium).
with a specific density of 1.22–1.25 can also be used to
clean cysts from organic debris.
For comparison of methods and extraction efficacy see
D’Errico & Brzeski (1975), M€ uller (1980), Clayden et al.
4. Extraction of cysts from soil
(1985), Winfield et al. (1987), Riggs et al. (1997), Tenente
Cysts differ from other nematode stages in size, shape and et al. (2007) and Bellvert et al. (2008a,b).
weight. Therefore, special methods have been developed EPPO recommends the following methods for cyst
for extracting cysts from dry (Baunacke method, paper strip extraction in their respective Diagnostic Protocols
method, Fenwick can, Schuiling centrifuge) and wet or dry (Table 6).
soil (Seinhorst elutriator, centrifugal flotation, Wye washer).
Efficacy of cyst extraction generally decreases with increas-
4.1 Baunacke method and/or paper strip method
ing organic content. None of those methods provide satis-
factory separation of cysts from organic debris and soils Baunacke (1922) introduced the principle of drying soil for
with high content of peat. In those cases, cysts need to be the collection of Globodera and Heterodera cysts from field
handpicked. soil. The method makes use of the fact that dried cysts float
Cyst extraction from dried soil is based on the fact that in water. They can then be decanted and collected on
those cysts contain air bubbles and therefore float on sieves. The method was improved by Buhr (1954) using a
water. The soil must be completely dry. Therefore, keep paper strip to collect the cysts.
the sample in a porous (paper) bag at room temperature or
in a drying chamber at about 35°C. In case live specimens Materials
are required, note that while Globodera can resist drying, • 200 or 250 lm aperture sieve for collecting the cysts;
vitality of the cyst content of Heterodera spp. rapidly • Plastic beaker or bowl;
decreases during drying, especially when dried at tempera- • Stirring rod;
ture higher than 25°C and air humidity lower than 40%. • Paper strip (e.g. made from filter paper) in case of paper
Gentle air circulation accelerates the drying process. strip method;
Depending on the conditions, the soil will dry within 2– • Detergent;
4 weeks. Unfortunately, young, full cysts do not float very • Pair of forceps or fine painting brush;
well and can be lost; hence the total population can be • Dissecting microscope.
underestimated.

Table 6 Methods recommended for the extraction of nematode cysts from dry and wet soil as listed in the corresponding EPPO Diagnostic Protocols

Dried soil only Wet or dried soil

Baunacke method, Fenwick Schuiling Seinhorst Centrifugal

paper strip method can centrifuge elutriator flotation Wye washer

Globodera rostochiensis/G. pallida (PM 7/40) X X X X X X

Heterodera glycines (PM 7/89) X X X X X

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 471–495

 Nematode extraction 487

4.2 Fenwick can

A widely used apparatus for extracting cysts that was origi-

nally described by Fenwick (1940). The method is suitable
for dried soil samples up to 250 mL. Extraction is based on
the floating properties of dried cysts (containing air) and
the difference in size between other fractions of the sample.
Most of the cysts in the soil sample will be collected this
way. The soil at the base of the Fenwick can is elutriated
by means of water flowing rapidly through a long glass or
metal tube which is inserted deep into the can. The water
flow stirs the sediment and releases any trapped cysts. The
cysts move upward, into the collar and end up on the
250 lm aperture sieve. An automated version (carousel) is
available.

Materials
• Fenwick can (Fig. 14);
• 1 mm aperture top sieve;
• 200 or 250 lm aperture sieve (d = 20 cm) for collecting
the cysts;
• 840 lm aperture sieve (facultative);
• Filter paper;
• Dissecting microscope.
Fig. 13 Baunacke method (courtesy Van Bezooijen, 2006) performed
with a 180 µm sieve (for Globodera spp. and Heterodera glycines a
Procedure
200 or 250 µm sieve is recommended).
• Clean the can with water, close the outlet at the bottom
Procedure and fill the can to the rim with water;
• Pass the dried soil with a jet of water through a 200 or • Place a 200 or 250 lm aperture sieve under the outlet of
250 lm sieve to eliminate soil and small organic particles the overflow collar;
(Fig. 13); • Wash the air-dried soil sample through the top sieve
• Transfer the debris remaining on the 200 or 250 lm sieve (1 mm) into the can with a strong jet of water;
into a plastic beaker or bowl; • Leave the water running for approx. 5 min. until water
• Stir the suspension thoroughly; overflow is clean;
• Let the suspension settle for 30 s to some min; depending • Heavy soil particles will sink to the bottom of the can,
on the soil type, the water is cleared and the liquid will cysts and light root debris will overflow;
only contain the floating organic debris and cysts; • Collect overflow including cysts on the 200 or 250 lm
• Add a drop of detergent that causes the cysts to move to sieve beneath the outlet of the collar;
the edge; • Rinse the funnel and collar very well to gather all cysts
• Pick the cysts by hand under a dissecting microscope on the 200 or 250 lm sieve;
using forceps or a fine painting brush. • Remove stopper at bottom of can to remove the remain-
der of the sample, rinse the can and the 1 mm sieve
Advantages before the next sample is processed;
• Simple, quick and cheap; • Transfer the material collected on the 200 or 250 lm
• Little use of water. sieve on a filter paper;
• Count cysts and process them for identification using a
Disadvantages dissecting microscope.
• Sample size is limited to about 100 mL soil;
• Results depend highly on individual person. Advantages
• One person can process a large quantity of samples;
Remarks • Easy to construct.
Alternatively, use a paper strip around the beaker and raise
the water level so cysts can adhere to it (Buhr, 1954). Care- Disadvantages
fully remove the paper strip with the attached cysts from the • Soil samples must be dried beforehand;
beaker. Sieves with smaller aperture up to 100 lm should • Large amount of water.
be used for catching smaller cysts (e.g. Heterodera carotae).

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 471–495

826 1 5 . P L A N T D I S E A S E S C AU S E D B Y N E M ATO D E S

STUBBY-ROOT NEMATODES: PARATRICHODORUS AND TRICHODORUS

863

SEED-GALL NEMATODES: ANGUINA

865

FOLIAR NEMATODES: APHELENCHOIDES

867

PINE WILT AND PALM RED RING DISEASES: BURSAPHELENCHUS

870

INTRODUCTION microorganisms and microscopic plants and animals.

Numerous species of nematodes attack and parasitize

N
ematodes belong to the kingdom Animalia. Nema- humans and animals, in which they cause various dis-
todes are wormlike in appearance but quite eases. Several hundred species, however, are known to
distinct taxonomically from the true worms. feed on living plants, obtaining their food with spears
Most of the several thousand species of nematodes live or stylets (Fig. 15-1) and causing a variety of plant dis-
freely in fresh or salt waters or in the soil, and feed on eases worldwide. The annual worldwide losses caused

FIGURE 15-1 (A) Typical plant parasitic nematode. (B) Close-up of the head of a plant parasitic nematode showing
the spear or stylet. Scale bars: 10 mm. [From McClure and von Mende (1987), Phytopathology 77, 1463–1469.]
 C H A R AC T E R I S T I C S O F P L A N T PAT H O G E N I C N E M ATO D E S 827

by nematodes on the life-sustaining crops, which include CHARACTERISTICS OF PLANT

all grains and legumes, banana, cassava, coconut, PATHOGENIC NEMATODES
potato, sugar beet, sugarcane, sweet potato, and yam,
are estimated to be about 11%; Losses for most other Morphology
economically important crops (vegetables, fruits, and
nonedible field crops) are about 14%, for a total of over Plant-parasitic nematodes are small, 300 to 1,000
$80 billion annually. micrometers, with some up to 4 millimeters long, by
15–35 micrometers wide (Figs. 15-2 and 15-3). Their
small diameter makes them invisible to the naked eye,

Head Lateral View

Head–Face View Lips Mouth

Stylet
Stylet tip Mouth Lip region
Muscles
Esophagus
Median bulbs Stylet
of esophagus Lips

Cuticle
Salivary glands

Stylet knobs

Nerve ring

Intestine

Ovary

Salivary Nerve
gland ducts Testis
Cuticle

Esophagus
Hypodermal
cord
Egg
Body cavity
Body muscle Spermatheca

Uterus
Cuticular
annulations Vulva

Sperm Anus

Phasmid
Cross Section of Nematode
Spicule

FIGURE 15-2 Morphology and main characteristics of typical male and female plant parasitic nematodes.
828 1 5 . P L A N T D I S E A S E S C AU S E D B Y N E M ATO D E S

22
Longidorus
2 Helicotylenchus
Dolichodorus
3 21
Belonolaimus Rotylenchulus
4 Criconema 20
Anguina
5
Xiphinema Tylenchulus
19
6 Hoplolaimus
7 Rotylenchus
8 Hemicycliophora
9 Ditylenchus Meloidogyne 18
10 Aphelenchoides
11 Tylenchorhynchus
12 Trichodorus
13 Radopholus
14 Pratylenchus Heterodera 17
15 Criconemoides
16 Paratylenchus

0 250 m 500 m 750 m 1000 m 1250 m 1500 m 1750 m 2000 m 2250 m 2500 m 2750 m 3000 m

FIGURE 15-3 Morphology and related sizes of some of the most important plant parasitic nematodes.

but they can be observed easily under the microscope. The reproductive systems of nematodes are well
Nematodes are, in general, eel shaped and round in developed. Females have one or two ovaries, followed
cross section, with smooth, unsegmented bodies, by an oviduct and uterus terminating in a vulva. The
without legs or other appendages. The females of some male reproductive structure is similar to that of the
species, however, become swollen at maturity and have female, but there is a testis, seminal vesicle, and a ter-
pear-shaped or spheroid bodies (Fig. 15-3). minus in a common opening with the intestine. A pair
of protrusible, copulatory spicules are also present in the
male. Reproduction in plant parasitic nematodes is
Anatomy through eggs and may be sexual or parthenogenetic.
Many species lack males.
The nematode body (Fig. 15-2) is more or less trans-
parent. It is covered by a colorless cuticle, which is
usually marked by striations or other markings. The Life Cycles
cuticle molts when a nematode goes through the suc-
cessive juvenile stages. The cuticle is produced by the The life histories of most plant parasitic nematodes are,
hypodermis, which consists of living cells and extends in general, quite similar (Fig. 15-4). Eggs hatch into
into the body cavity as four chords separating four juveniles, whose appearance and structure are usually
bands of longitudinal muscles. The muscles enable the similar to those of the adult nematodes. Juveniles grow
nematode to move. in size, and each juvenile stage is terminated by a molt.
The body cavity contains a fluid through which cir- All nematodes have four juvenile stages, with the first
culation and respiration take place. The digestive system molt usually occurring in the egg (Fig. 15-4B). After the
is a hollow tube extending from the mouth through the final molt the nematodes differentiate into males and
esophagus, intestine, rectum, and anus. Lips, usually six females. The female can then produce fertile eggs either
in number, surround the mouth. Most plant parasitic after mating with a male or, in the absence of males,
nematodes have a hollow stylet or spear (Fig. 15-1B), parthenogenetically.
but a few have a solid modified spear. The spear is used A life cycle from egg to egg may be completed within
to puncture holes in plant cells and through which to 2 to 4 weeks under optimum environmental, especially
withdraw nutrients from the cells. temperature, conditions but will take longer in cooler
 C H A R AC T E R I S T I C S O F P L A N T PAT H O G E N I C N E M ATO D E S 829

B
A

E F
FIGURE 15-4 Stages in a life cycle and the infection process of plant parasitic nematodes. (A) Nematode eggs.
(B). Nematode eggs and hatching second-stage juvenile. (C) Typical plant parasitic nematode ready to infect plant.
(D) Juvenile and adult ectoparasitic ring nematodes feeding on root. (E) Aphelenchus nematodes present inside plant
cells. (F) Radopholus nematodes feeding inside plant root. [Photographs courtesy of (A, C, E, and F) University of
Florida, (B) U. Zunke, (D) S. W. Westcott III.]
830 1 5 . P L A N T D I S E A S E S C AU S E D B Y N E M ATO D E S

temperatures. In some species of nematodes the first or own power. Further spread takes place on contact of
second juvenile stages cannot infect plants and depend infected plant parts with adjacent healthy plants.
on the energy stored in the egg for their metabolic func- Two genera of the family Aphelenchoididae, namely
tions. When the infective stages are produced, however, Aphelenchoides (bud and leaf nematodes) and Bur-
they must feed on a susceptible host (Figs. 15-4D–15- saphelenchus (the pine wilt and red-ring nematodes),
4F) or starve to death. An absence of suitable hosts may seldom, if ever, enter the soil. They survive instead in the
result in the death of all individuals of certain nematode tissues of the plants they infect and, for the latter, in its
species within a few months, but in other species the insect vectors.
juvenile stages may dry up and remain quiescent or the
eggs may remain dormant in the soil for years.
Classification

Ecology and Spread All plant parasitic nematodes (Fig. 15-3) belong to the
phylum Nematoda. Most of the important parasitic
Almost all plant pathogenic nematodes live part of their genera belong to the order Tylenchida, but a few belong
lives in the soil. Many live freely in the soil, feeding to the order Dorylaimida.
superficially on roots and underground stems, and in all,
even in the specialized sedentary parasites, the eggs, the Phylum: Nematoda
preparasitic juvenile stages, and the males are found in Order: Tylenchida
the soil for all or part of their lives. Soil temperature, Suborder: Tylenchina
moisture, and aeration affect survival and movement of Superfamily: Tylenchoidea
nematodes in the soil. Nematodes occur in greatest Family: Anguinidae
abundance in the top 15 to 30 centimeters of soil. The Genus: Anguina, wheat or seed-gall
distribution of nematodes in cultivated soil is usually nematode
irregular and is greatest in or around the roots of sus- Ditylenchus, stem or bulb nematode of
ceptible plants, which they follow sometimes to consid- alfalfa, onion, narcissus, etc.
erable depths (30–150 centimeters or more). The greater Family: Belonolaimidae
concentration of nematodes in the region of host plant Genus: Belonolaimus, sting nematode
roots is due primarily to their more rapid reproduction of cereals, legumes, cucurbits, etc.
on the food supply available and also to attraction of Tylenchorhynchus, stunt nematode of
nematodes by substances released into the rhizosphere. tobacco, corn, cotton, etc.
To these must be added the so-called hatching factor Family: Pratylenchidae
effect of substances originating from the root that Genus: Pratylenchus, lesion nematode
diffuse into the surrounding soil, markedly stimulating of almost all crop plants and trees
the hatching of eggs of certain species. Most nematode Radopholus, burrowing nematode of
eggs, however, hatch freely in water in the absence of banana, citrus, coffee, sugarcane, etc.
any special stimulus. Nacobbus, false root-knot nematode
Nematodes spread through the soil slowly under their Family: Hoplolaimidae
own power. The overall distance traveled by a nematode Genus: Hoplolaimus, lance nematode
probably does not exceed a few meters per season. of corn, sugarcane, cotton, alfalfa,
Nematodes move faster in the soil when the pores are etc.
lined with a thin film of water (a few micrometers thick) Rotylenchus, spiral nematode of
than when the soil is waterlogged. In addition to their various plants
own movement, however, nematodes can be spread Heliocotylenchus, spiral nematode of
easily by anything that moves and can carry particles of various plants
soil. Farm equipment, irrigation, flood or drainage Rotylenchulus, reniform nematode of
water, animal feet, birds, and dust storms spread nema- cotton, papaya, tea, tomato, etc.
todes in local areas, whereas over long distances nema- Scutellonema, dry rot nematode of yam,
todes are spread primarily with farm produce and cassava, etc.
nursery plants. A few nematodes that attack the above- Family: Heteroderidae
ground parts of plants not only spread through the soil Genus: Globodera, round cyst nema-
as described earlier, but they are also splashed to the tode of potato
plants by falling rain or overhead watering. Some Heterodera, cyst nematode of tobacco,
species ascend wet plant stem or leaf surfaces on their soybean, sugar beets, cereals