Plant parasitic nematodes economically important Genera.

1. ECONOMICALLY IMPORTANT GENERA OF PLANT
PARASITIC NEMATODES
1

2. OUTLINE
 Root knot nematode
 Lesion nematode
 Cyst nematode
 Citrus nematode
 Barrowing nematode
 Aphelenchoides
 Management of PPNs
 Diagnostic tests for nematodes
2

3. Root-knot Nematode, Meloidogyne spp.
Systematic Position:-
Order - Tylenchida
Sub order - Tylenchina
Super family - Tylenchoidea
Family - Heteroderidae
Sub family - Meloidogyninae
Genus - Meloidogyne
Species -
i) Meloidogyne incognita
ii) Meloidogyne javanica
iii) Meloidogyne arenaria
iv) Meloidogyne hapla
3

4. Distribution and host range
Host range
 Cow pea
 Common bean (Phaseolus vulgaris)
 Cowpea (Vigna unguiculata)
 Tomato ( Solanum lycopersicon)
 Others; okra, cucumber, melon, carrot, gourds,
lettuce and peppers.
4
+
-
Geographical Distribution of Root-knot nematodes
in Africa (red marked)

5. Symptoms
 Affected plants are normally stunted and
eventually wilt and die.
 The most characteristic symptom is formation of
root galls (knots) and these can be seen with the
naked eye.
 Affected roots rot
5
Root-knot nematodes (Meloidogyne incognita
/M. javanica) Roots of severely attacked (left)
and healthy plant (right)..

6. Economic importance
 Disease complexes with other pathogens, such as Fusarium wilt,
Rhizoctonia solani and Thielaviopsis basicola, have been reported
(Manzanilla-López and Starr, 2009).
 The quarantine status of some species causes indirect costs in addition
to direct loss. For example, M. chitwoodi and M. fallax are increasingly
regulated as they can be spread through seed potatoes.
 yield losses of up to 87% have been reported (Lilley et al., 2011;)
 Losses of potatoes due to Meloidogyne species, mainly M.
incognita, are estimated at 25% or more.
6

7. Diagnostics
 Sexually dimorphic. Mature female sedentary, swollen, globular or pear-shaped, cuticle
thin, white, annulated. Non cyst forming.
 Tail absent, anus and vulva terminal surrounded by characteristic pattern of striae on the
cuticle (perineal pattern).
 Excretory pore anterior to median bulb and near to stylet knobs, head skeleton
hexaradiate.
 Stylet <25 m long, with well developed basal knobs; procorpus cylindrical followed by
spherical metacorpus with well developed musculature and cuticular valve plates;
 procorpus and metacorpus not amalgamated.
 Oesophageal glands overlapping intestine ventrally. Genital tracts paired, elongated,
anteriorly directed and much convoluted.
 Eggs laid in an external gelatinous matrix. The eggs are usually unembryonated and not
retained in the female body.
 The first moult occurs within the egg, the second-stage juveniles hatching and being the
infective stage.
7

8. Diagnosis
 Female: Sedentary, swollen, globular or pear-shaped, cuticle thin, white, annulated, tail
absent, anus and vulva terminal surrounded by characteristic pattern of striae on the cuticle
(perineal pattern), excretory pore anterior to median bulb near to stylet knobs, head skeleton
hexaradiate.
 Stylet <25μm long, well developed basal knobs: procorpus cylindrical followed by spherical
metacorpus with well developed musculature and cuticular valve plates, procorpus and
metacorpus not amalgamated.
 Oesophageal glands overlap intestine ventrally
 Genital tracts paired, elongated and convoluted.
 Eggs laid in gelatinous matrix, usually unembryonated and not retained in the female body.
 First moult within the egg, second-stage juveniles hatch and are infective.
 Male vermiform, migratory, stylet well developed, <33 m; cephalic framework well developed,
hexaradiate; short bluntly rounded tail; no bursa, usually one, but sometimes two testes;
paired slender spicules, simple gubernaculum. Intersexes or sex reversal may occur, particularly
in response to nutrient stress.
 j2 vermiform, infective, migratory; head skeleton hexaradiate, stylet slender, knobbed, stylet
<23 m long. Hyaline region of tail less than half the tail length. Heat relaxed form straight to
arcuate.
 The 3rd and 4th stage juveniles occur within the root and are swollen, sedentary, with a blunt
terminus and no stylet. They develop within the cuticle of 2nd stage juveniles, the tail spike of
which is retained.
8

9. Root-lesion Nematode, Pratylenchus spp.
 Systematic Position:-
 Order - Tylenchida
 Sub order - Tylenchina
 Super family - Tylenchoidea
 Family - Pratylenchidae
 Sub family - Pratylenchinae
 Genus - Pratylenchus
 Species - i) coffeae - Citrus, Banana & coffee
 ii) zeae - Maize
 iii) thornei – Pulses
9

10. Distribution and host range
Host range
 Root lesion nematode has a wide host
range, including hosts like apple, cherry,
conifers, roses, tomato, potato, corn, onion
and sugarbeets, and ornamentals such as
Narcissus.
 More than 164 hosts for P. penetrans have
been recorded.
 Some hosts that are susceptible, wheat, oat,
field pea, faba bean, and chickpea
 Some are moderately susceptible, barley
and canola.
10
P. brachyurus, P. coffeae and P. zeae are widely
distributed in tropical areas; P. penetrans mainly in
cooler regions of the tropics; P. goodeyi on banana
in Crete and the Canary Islands and in the cooler,
montane areas of Cameroon, Ethiopia, Kenya,
Tanzania, Uganda and Burundi.

11. Symptoms:-
 Late emergence of seedlings
 less germination and stunted growth with necrotic lesions on the
root surface which are initially small coalesce at the later stage
and cause death of the rootlets. Root system is reduced.
11
Patches of stunted wheat plants infected with root
lesion nematodes Pratylenchus spp
Severely abbreviated root system of olive tree

12. Economic importance
 In potatoes it has been shown that Pratylenchus penetrans has a relationship
with Verticillium dahliae in causing Potato Early Dying Syndrome.
 Potato Early Dying causes premature vine death, severe yield losses, scabby
appearance with sunken lesions, and dark, wart-like bumps that turn purple
on tubers in storage.
 The fungus is not shown to be transmitted by the nematode, however it has
an increased ability to infect because of the mechanical damage caused by
root lesion nematodes.
 Also documented. is that varieties ordinarily resistant to Verticillium
apparently becomes infected by it after previous infection by Pratylenchus.
12

13. Diagnostics
 Small nematodes (less than 1mm long) dying slightly curved ventrally on application of
gentle heat.
 No marked sexual dimorphism in form of anterior region.
 Head region low, flattened, usually appearing as a flat, black cap under the
stereomicroscope.
 Lip region divided into 2, 3 or 4 annules and continuous with the body contour; strongly
cuticularized.
 Stylet 20μm or less in length (i.e. about twice, or slightly more, of the head width),
moderately cuticularized and with rounded or anteriorly concave knobs.
 Oesophagus equally developed in both sexes, median bulb well developed; oesophageal
gland lobes overlapping the intestine ventrally.
 Female: vulva well posterior, usually at 70-80% of body length; genital system with a single
anteriorly directed tract and a variable post-vulval section which may show some
differentiation, but is never functional (mono-prodelphic); spermatheca oval or round and
usually filled with sperm in bisexual species; tail sub-cylindrical or more or less conoid with
a broad to narrowly rounded or truncate terminus which may be smooth or annulated.
 Male: tail short, dorsally convex-conoid; bursa extending to tail tip; spicules slender,
arcuate
13

14. Cyst Nematode, Heterodera spp& Globodera spp
Systematic Position:-
 Order - Tylenchida
 Sub order - Tylenchina
 Super family - Tylenchoidea
 Family - Heteroderidae
 Sub family - Heteroderinae
 Genus –
i) Heterodera
ii) Globodera
14
Heterodera
Globodera

15. Distribution of cysts nematodes
 Globodera
15
Heterodera

16. Host range
PCN
 Potato (Solanum spp.), tomato (Lycopersicon
spp.), egg plant (Solanum melongena) and some
solanaceous weeds.
 Although the preferred host is potato, PCN can
also infest tomatoes and other solanaceous
plants, including the nightshade weed.
 Thus populations of nematode can build up in the
soil as long as solanaceous crops are grown.
SCN
 Soybeans
 Phaseolus beans
16

17. Symptoms
Heterodera
 The diseased plants show yellowing of
leaves, stunted growth, reduced tillering.
Ear heads if formed are very small known as
‘Molya’ disease’
Globodera
 Typical symptoms of heavy infestation are
stunted plants with unhealthy foliage
 premature yellowing, poor development of
root system, reduction in size and number of
tubers.
 Such plants exhibit temporary wilting during
hotter part of the day
17
Chlorosis and stunted growth (
left) caused by Heterodera on
rice
Patchy growth and
chlorosis of lower
leaves of wheat caused
by Heterodera spp

18. Economic importance
PCN
 The pest can survive in a soil that has not be
planted for up to 20 years
 Takes a longer time before its detected up to
20 years
 Its easily dispersed by transport of infested
soil, in soil adhering to root tubers , farm
machinery, and implements,
SCN
 populations increases rapidly once
introduced and can complete 6-7
generations per season
 Any mechanism that spreads infested soils
can be a means of dispersal including wind,
water, and migratory birds
 Reported yield losses on soybean varies from
10-70% in japan
 Its invasive in its native range and outside
native range
 Difficult to identify/detect as a commodity
contaminant
 Difficult to identify/detect in the field
 Difficult /costly to control
18

19. Morphometrics
HETERODERA
 Body swollen
 Body cuticle of female transformed
into a leathery brown cyst.
 Cyst lemon-shaped with vulval cone.
GLOBODERA
 Body swollen
 Body cuticle of female transformed
into a leathery brown cyst.
 Cyst round to oval, no vulval cone
19

20. Citrus Nematode, Tylenchulus semipenetrans
 Systematic Position:-
 Order - Tylenchida
 Sub order - Tylenchina
 Super family - Criconematoidea
 Family - Tylenchulidae
 Sub family - Tylenchulinae
 Genus - Tylenchulus
 Species - semipenetrans
20

21. DISTRIBUTION AND HOST RANGE
Host range
 Preferred host; citrus species,
 related species, as Poncirus
trifoliata and its hybrids, can be
parasitized.
 Citrus nematodes also infect non-
rutaceous woody plants such as
grape, olive, and persimmon.
 Among grape species, Vitis
berlandieri, V. riparia, V. rupestris
and V. vinifera are infected by T.
semipenetrans
21

22. Symptoms:-
 The diseased trees show reduction in growth and vigor with yellowing of leaves.
Such trees show gradual dieback symptoms starting from the uppermost portion.
 Roots of infected trees appear larger in diameter and darker than the healthy trees
mainly due to adherence of soil particles to the gelationous matrix excreted by the
adult females
 Cortex of highy infested feeder roots decays and gets sloughed off easily
22

23. Economic importance
 These nematodes are considered as major plant-parasitic nematode because they
can cause 10-30% losses reported on citrus trees.
 They also parasitize other hosts such as olive, grape, persimmon and lilac
MORPHOMETRICS
 Mature female with long pointed tail.
 Mature female: Excretory pore near vulva, anus absent, posterior body exterior to
root.
23

24. Burrowing Nematode, Radopholus similis
Systematic Position:-
 Order - Tylenchida
 Sub order - Tylenchina
 Super family - Tylenchoidea
 Family - Pratylenchidae
 Sub family - Pratylenchinae
 Genus - Radopholus
 Species - similus
Parasitism: - Endoparasitic on roots of Banana and citrus.
24

25. Host range Distribution
Host range
 important pest of bananas and
citrus
 can be found on coconut, avocado,
coffee, sugarcane, other grasses,
and ornamentals
Distribution
 The majority of species have been described from
the Australasian region.
 However, R. similis is now found world-wide in
tropical regions and occurs virtually everywhere that
banana is grown.
 The citrus race, formerly known as R. similis
citrophilus, is recorded from Florida.
 Radopholus citri was described attacking citrus in
Java and may represent a threat to other citrus
growing areas if introduced
25

26. Symptoms:-
 Infection causes toppling/Falling forward
disease of banana, yellows disease of pepper
and spreading decline of citrus.
 These diseases are the result of burrowing
nematode infection destroying root tissue,
leaving plants with little to no support or
ability to take up water and translocate
nutrients.
26
Toppling over of bananas caused by
Radopholus simili
Deformed banana root system
with swollen roots
Dieback of citrus

27. Economic importance
 The nematode causes economic problems throughout the world, most notably
in warmer regions, including South America, the Caribbean, Africa, Asia and
the Pacific.
 The lack of new land free from the burrowing nematode in banana-producing
countries has prevented the exclusion of the nematode from new banana
plantations and has caused the persistence of nematode problems, which
result in crop losses ranging 30-80% (Gowen et al. 2005).
 In Florida, the citrus race of the burrowing nematode causes spreading
decline symptoms only in the deep and coarse sandy soil of the Ridge in
central Florida, where yield losses range 40-80% (Duncan 2005).
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28. Diagnostics
 Small nematodes (less than 1mm long) dying more or less straight or slightly curved
ventrally on application of gentle heat.
 Marked sexual dimorphism in form of anterior region: female head region low, rounded,
continuous or slightly offset.
 Male cephalic sclerotization, stylet and oesophagus reduced; female cephalic sclerotization
strong, stylet and oesophagus well developed.
 Median bulb in female oesophagus well developed and oesophageal glands overlapping the
intestine mostly dorsally.
 Female: vulva median, with two functional and equally developed genital tracts
(amphididelphic);
 spermathecae rounded and with sperm in bisexual species; tail elongate, conoid (about
60μm long in R. similis).
 Male: tail elongate, conoid, ventrally arcuate; bursa not reaching to tail tip in R. similis and
most other species; spicules slender, arcuate.
28

29. Aphelenchoides
29
Phylum: Nematoda
Rank: Genus
Order: Tylenchida
Subclass: Diplogasterida
Genus: Aphelenchoides
Family: Aphelenchoididae

30. Distribution and Host range
 DISTRIBUTION
HOST RANGE
 Rice is the most important
host worldwide
 The nematode species can also
feed on numerous other mono-
and dicotyledonous plants (i.e.
Hockland, 2004).
30

31. Symptoms
 The disease is seed borne, and
infected plants are reduced in size.
 Symptoms of A. besseyi include the
production of chlorotic tips on new
leaves, which may subsequently
necrotize.
 The flag leaf enclosing the panicle
may also be distorted, leading to a
reduction in the number and size
of grains produced.
31
Symptoms of Aphelenchoides besseyi on rice.
(Photograph courtesy of Donald Groth, Louisiana
State University AgCenter, Bugwood.org.)

32. Economic importance
 It makes a significant contribution to the estimated $US16 billion worth of
damage caused by nematodes to rice crops (Lilley et al., 2011).
 Management by avoiding overhead irrigation. This is often difficult to
accomplish when costly overhead irrigation systems are already in place for
large sections of greenhouse space, and in outdoor nurseries where rain can
spread foliar nematodes, even in the absence of overhead irrigation.
32

33. Morphometric
 Body slender, vermiform.
 Oesophagus tylenchoid.
 Vulva situated much more posterior one genital tract.
 Dorsal Oesophageal gland secretes into Oesophageal lumen just anterior to valve
of median bulb; median bulb large distinct rounded-rectangular
33

34. MANAGEMENT OF PPNs
The general [principles of management
i) Most plant parasitic nematodes have a wide host range.
(ii) Dispersal of plant-parasitic nematodes is usually passive but may be active or aided by
vectors.
(iii) The principal dispersal agents of plant parasitic nematodes are water, man, wind and
arthropods.
(iv) Soil, water and plant residues are the main reservoirs of plant parasitic nematodes
(v) Knowledge of the factors that affect nematodes is invaluable in their management.
(vi) Control of nematodes induced diseases is usually directed at inhibition of the nematodes
themselves.
(vii) Control strategies should be more preventive rather than curative and aimed at
preventing build-up of high population densities.
(viii) Sustainable management of plant parasitic nematodes requires that all viable strategies
be combined into integrated pest management packages
34

35. MANAGEMENT OF PLANT PARASITIC NEMATODES
35

36. INTRODUCTION
The reduction of nematode population levels can be obtained by the following
methods:
(i) Killing the nematodes by starvation.
(ii) Directly killing the nematodes by a chemical or any other technique applied
before the crop is sown or planted.
(iii) Using chemicals that prevent the nematodes from feeding.
Sustainable management of plant parasitic nematodes can be achieved through
an integration of different tactics that fall into five broad strategies: -
(i) Preventing introduction and spread of nematodes
(ii) Cultural practices, particularly cropping systems, fallowing, resistant
cultivars and organic amendments.
(iii) Physical agents especially heat
(iv) Chemicals (nematicides)
(v) Biological control
36

37. PREVENTING INTRODUCTION AND SPREAD OF NEMATODE
 Exclusion is the first control to consider in nematode management
 Prevention of nematode spread can be considered at different levels; the farm, the nation and the world.
At farm level,
A judicious choice of the propagation material must be the basis for each crop.
 For this reason the multiplication sites (nurseries) should be established on unsuspected or disinfested
land.
 If the production of nematode free plants appears to be impossible, one must apply methods that
disinfest the soil such as thermotherapy and chemotherapy.
One of the ways nematodes are disseminated from one field to another is the soil adhering to farm
machinery..
 Cleaning the farm machinery would prevent the fields from infection.
Irrigation is another way of nematode dissemination.
 Nematodes are spread by water in both the field and in greenhouses.
 Sedimentation of the nematodes in a basin or a reservoir can reduce their presence in the water and
limit their spread.
 Filtration of irrigation water is also gaining popularity as a nematode management strategy especially
in capital-intensive production systems.
37

38. PREVENTING INTRODUCTION AND SPREAD OF NEMATODES
At nation level,
 To protect the individual farmer and to limit the nematode presence to a restricted
area, different countries restrict circulation of contaminated plant material.
 Legal measures aiming at the reduction of the infections of dangerous plant parasitic
nematodes are frequent.
At the international level,
 important phytosanitary problems are governed by quarantine regulations.
 Quarantine refers to regulatory actions aimed at preventing or retarding the
introduction, establishment and spread of dangerous pests.
 Quarantine pests are those of potential national economic importance that are not yet
present in the country or endangered region or that are present but not widely
distributed.
 Consignments must be free of quarantine pests (Zero tolerance).
38

39. PREVENTING INTRODUCTION AND SPREAD OF NEMATODES
 Exclusion of plant material is confined to plants known to be hosts of pests or of biological
races or strains of pests with extremely high risk for the importing country, and originating
from countries where the pest is known to occur.
 Soil accompanying plant material is undoubtedly a serious quarantine risk.
 It is, therefore, prohibited by most quarantine regulations.
Spot Treatment
 Spot treatment entails marking out spots of high nematode density and treatment of the
same to reduce spread of the nematodes.
 The strategy is based on the fact that nematodes are hardly evenly distributed in a field.
 Treatment of the spots may be achieved through application of high amounts of organic
substrates or chemical nematicides.
39

40. CULTURAL PRACTICES
Crop rotation
 PPNs are obligate parasites: they need a host plant for both their development and
multiplication.
 Each species of phytonematodes has a range of hosts, which may be wide, but does not
include all crop plants.
 Nematodes numbers increase on favorable and decline on unfavorable hosts.
 In crop rotation susceptible crops are rotated with immune or resistant crops.
 The susceptible crop is usually the most profitable and the rotation crops less profitable.
 A rotation should be planned so that the nematode population is at its lowest level when
the principal or the most profitable and most susceptible crop is planted.
 After a tomato crop is harvested, RKNs population in the soil is usually high hence the
second tomato crop would be severely damaged.
 If the nematode species present is not M. hapla or race 1 of M. arenaria, peanuts can
follow a tomato crop without the risk of damage.
 While the peanuts are growing, the nematodes cannot reproduce. Instead, many of the
juveniles in the soil die or become non-infective because of starvation and the attacks of
predators, fungi and other natural enemies.
 Examples of poor host crops that are commonly used in rotations to suppress RKNs include
maize, onions, wheat, rhodes grass, and asparagus
40

41. CULTURAL PRACTICES
Early planting:
 PPNs are mainly harmful in the early stages of the crop, when the root system is not well
developed.
 In the temperate region growers can sow or plant in cool periods, so the crop gets started
before the nematodes are active.
 By the time the soil warms and juveniles of cyst nematodes (G. rostochiensis, G. pallida
and H. schachtii) invade growing roots, plants have already accumulated reserves and can
withstand attack.
 Continued early plantings, however, may select strains of plant parasitic nematodes
adapted to the system.
 Although tropical conditions are generally unfavourable for the cyst nematodes, high
altitude zones experience temperate-like climate and are therefore prone to cyst
nematode colonization.
41

42. CULTURAL PRACTICES
Desiccation:
 Nematodes in an active state are very sensitive to desiccation.
 When transferred directly from water to an environment with low relative humidity or
high osmotic pressure, they are killed in a few minutes.
 On the other hand slow drying of nematodes usually induces anhydrobiosis, increasing
their resistance to adverse environmental conditions including dryness and toxic
substances.
 In arid and semiarid areas, 80% mortality can be achieved by rapid desiccation of the soil
over a short period of time.
 In such areas, ploughing at intervals of 2 – 4 weeks during the dry season can reduce
populations of root-knot nematodes substantially.
 This exposes eggs and juveniles in roots and deeper layers of the soil to rapid desiccation
and may be sufficient to significantly increase the yield of a subsequent crop.
 Intermittent irrigation during the period of desiccation improves nematode control, owing
to the increased susceptibility of reactivated nematodes to desiccation.
42

43. CULTURAL PRACTICES
Flooding:
 Flooding of the soil cuts off its oxygen concentration to practically zero within one or two days.
 Carbon dioxide begins to increase, following flooding, due to reduction by anaerobic bacteria.
 Other chemical changes in flooded soil are: denitrification, accumulation of ammonia, reduction
of iron, manganese and sulphates and production of organic acids, methane and hydrogen
sulphide.
 Flooding has been practiced as an economical method of nematode control in bananas grown on
peaty clay soil in Surinam.
 In that country, banana fields become heavily infested by Radopholus similis after 4-5 years.
 The banana plants are then destroyed and the fields are flooded for 4 – 5 months and then
replanted with hot-water-treated rhizomes.
 Soil disinfestation by flooding is mainly caused by an indirect effect due to an increase of toxic
substances produced by anaerobic microbiological activity in the soil, rather than by the direct
effect of lack of oxygen.
 Flooding is restricted in adoption because it is only viable in the valley bottoms and in regions
that are endowed with enormous water resources.
 It may also be used to explain the general decline in nematode numbers after excessively wet
seasons.
43

44. CULTURAL PRACTICES
Soil amendments:
 Many substances can be added to soil to increase organic matter: manure from domestic animals,
sewage sludge from municipal waste disposal facilities, crop residues after harvest, and cover crops.
products obtained after the processing of agricultural products
 Organic soil amendments are known to improve the structure and water holding capacity of the soil.
 Plants developing in a substrate rich in organic matter usually grow more vigorously and thus tolerate
damage by harmful organisms including nematodes.
 Breakdown of organic matter releases compounds that may be toxic to nematodes. In particular,
decomposing residues of plant tissues release simple organic acids such as acetic, propionic and butyric
acids.
 These may remain for several weeks in concentrations sufficient to kill some PPNs with little or no
effect on free-living species.
 Organic substrates stimulate build-up of indigenous microorganisms some of which are antagonistic to
phytonematodes. For instance, addition of chitin to the soil, derived from crustaceans, stimulates the
growth of actinomycetes, some of which are antagonistic to plant parasitic nematodes.
 It also stimulates increase of fungi with enzymes capable of digesting chitin. Many of these fungi
penetrate cyst nematodes to attack chitinous walls of eggs. During degradation of organic matter by
microorganisms, toxic metabolites that kill nematodes are released. 44

45. CULTURAL PRACTICES
Antagonistic Plants
 Several plant species have been characterized as being antagonistic to plant parasitic
nematodes.
 The most intensively studied include marigolds, asparagus, sunnhemp, mustard and
neem.
 The antagonism results mainly from release of root exudates that have nematicidal
properties.
 Nematode-antagonistic plants have shown high potential in nematode management when
used in sequential or multiple cropping systems.
 It has been shown that marigolds and sunnhemp are more effective than fallow in root-
knot nematode suppression.
 Over 50% decline in juvenile numbers has been reported in fields left under marigolds for
three months.
 Wide-scale usage of the plants is, however, restricted because most of the plants have
little or no market value.
 When used as companion crops, most of them exhibit a strong weed effect resulting in
reduced yields of the principal crop. 45

46. PHYSICAL METHODS
Control by heating
 Nematodes are very susceptible to heat.
 Few nematodes resist temperatures higher than 600C for a duration of 30 minutes.
 Soil disinfestation by means of vapour has been practiced in intensive crop production systems
for almost a century. The steam moves as water vapour to the point where it is needed and
then condenses on soil particles cooler than itself.
 Sheet steaming is widely used because of its low capital and labour cost. In this system, steam
is blown under a plastic sheet 0.25 mm thick, anchored at the edges by sand bags or chains and
left for at least eight hours to penetrate into the soil.
 The drier the soil is, initially, the more condensed water it can absorb and the deeper the heat
can penetrate.
 Steam penetration is slower in loam than in sandy soils, therefore one is advised to cultivate
the soil before steaming.
 Most plant parasitic organisms are destroyed at a temperature of 600C for 30 minutes.
 Higher temperatures induce chemical changes in the soil, which may have adverse effects on
subsequent growth.
 The release of an excessively large amount of manganese may cause toxicity in crops,
especially in soils with low pH.
 Plant growth may also be affected by an increase in levels of nitrite and ammonium nitrogen
after steam sterilization.
 Moreover, at high temperatures most of the soil microflora is destroyed, creating a biological
vacuum that may support the rapid development of residue and re-contaminating organisms.
46

47. PHYSICAL METHODS
Soil Solarization
 The field is tilled to fineness and irrigated in mid-summer and while soil moisture is still at
or just below field capacity, a clear thin polyethylene sheet is then spread over the soil and
its edges buried.
 The sheet is left undisturbed for periods ranging from two to nine weeks depending upon
several factors including the level of solar radiation.
 The polyethylene sheet is removed at the end of the solarization period and the field is
available for normal use.
 Irrigation can be done simply by flooding or by sprinklers or underground drip irrigation
systems.
The choice of the mulching material is important.
 The material should have a high transparency to permit short-wave solar radiation to reach
the soil, but it should be impermeable to long-wave radiation (greenhouse effect) and to
water vapour and gases leaving the ground.
 Transparent mulching has been found to be more effective than black, since it transmits most
of the incident radiation to the soil, whereas the black polyethylene tends to heat up.
47

48. Soil Solarization
 The heating of soil under polyethylene is generally less near the edges of the plot.
 Thus it is preferable to treat larger plots with continuous sealed mulch.
 The optimum period of solarization depends on factors such as the quantity of solar
radiation available locally, the soil characteristics, the type of mulching materials, the
thermal sensitivity of the target organism and the available time in the cropping system
for solarization.
The principles of solarization include:-
(i) Accumulation of heat in polyethylene mulched soil by transmission of short wave solar
radiation and prevention of loss of long wave radiation from the soil.
(ii) High soil moisture improves the thermal conductivity of the soil.
(iii) Greater thermal sensitivity of hydrated than of desiccated organisms.
(iv) Shift of the biological equilibrium in favour of the natural enemies of plant pathogens.
(v) Production of toxic gases and release of toxic mineral ions.
(vi) Prolonged exposure to high temperature kills or weakens the pathogen, rendering them
more vulnerable to their natural enemies.
(vii) Water vapour condensing under the mulch reduces heat loss and may concentrate solar
radiation.
(viii) Control of weeds that serve as alternative hosts of nematodes and other pathogens
48

49. Soil Solarization
 Soil Solarization can be combined with other methods of control.
 It has been observed that control of nematodes with combinations of solarization and
either 1, 3-dichloropropene, ethylene dibromide, ethoprophos or formaldehyde is better
than with any of these treatments alone.
 Covering of soil with clear polythene is a mandatory step after application of Metham
sodium to reduce escape of gases from the soil.
 It should be noted that the polythene is too expensive for use by most small-scale
farmers in East and southern Africa.
 The method has high potential in treatment of small areas such as nurseries where
disease free planting materials are produced.
49

50. Disinfestation by heating with electricity
 Under intensive horticultural production in greenhouses, soil may be heated using buried
electrical resistances.
 Observations have demonstrated that when the soil temperature is kept at 500C for one
hour, root-knot nematodes are effectively controlled.
 The costs are reduced when the heating takes place in the warm season.
 Although the method is restricted in adoption due to the cost of energy, it has practical
value in nurseries producing high value planting materials especially for export.
 Such high value produce include chrysanthemum flowers, carnations and roses.
50

51. Field burning
 The most primitive way of heating soil is by burning stubble over it.
 However, experiments have showed that burning of dry leaf litter, 10 cm thick, killed
root-knot nematodes to a depth of only 9 cm.
 In some cases, burning of straw and stubble after harvest may be effective in
protecting the next crop from severe attack especially by foliar nematodes.
 Burning of crop resides is, however, disastrous because it denies the soil much-needed
organic matter.
 It also poses environmental hazards, coupled with the risk of fire spreading to non-
target areas.
 Addition of organic substrates into the soil helps to establish biological control against
nematodes and also improves plant growth as a result of enhanced nutrients and water
holding capacity.
51

52. Disinfestation of planting material using heat
 Hot water treatment has been successfully used in the control plant parasitic
nematodes.
 The treatment is useful as a preventive control measure in propagating material (seeds,
rootstocks, corms, tubers, rhizomes) that might harbour endoparasitic nematodes.
 The chances of success in hot water treatment increases as the difference between
thermal sensitivity of the host and nematodes increases, with the latter being more
sensitive.
 The smaller the difference is, the more accurately the temperature and time of the
treatment must be controlled.
 Both plants and nematodes are less susceptible to high temperatures when they are at
dormancy or dehydrated.
 To improve the efficiency of hot water treatments it is necessary to displace trapped air
by pre soaking the propagules in water, since the air acts as an insulator.
 Pre soaking for about two hours before hot water treatment is a common practice to
control Aphelenchoides besseyi in rice seed.
 Hot water treatment of rhizomes of bananas to control Radopholus similis is improved
by peeling all necrotic tissue from the corms before treatment.
52

53. Control by irradiation
 Gamma-rays have multiple effects which may include sterilization, delayed gonadal
growth, delayed egg-hatching and morphological abnormalities.
 The movements of the nematodes are also known to become sluggish.
 The irradiation effects depend on the developmental stage of the nematode and also on
both the irradiation doses and the nematodes species.
 Eggs may develop normally after a UV-treatment but the juveniles hatching from
irradiated eggs exhibit reduced growth and reproduction is usually inhibited. #
 Increasing doses of UV cause an increase in mortality of the second stage juveniles
 The use of UV-, X- and gamma rays for soil and substrate disinfestations appears to be
impractical because the soil and substrate acts as a buffer and absorbs most of the
liberated energy.
 The irradiation of nematode infected plants is also not feasible because plants are more
susceptible than nematodes. Ionizing rays can, however, be applied in disinfestation of
nutrient solutions used in hydroponic-type systems.
 A small UV-unit can treat about 2500-liter water per hour.
 It can be recommended for adoption in high-technology flower production systems.53

54. Resistance
 Resistance describes the effects of host genes that restrict or prevent nematode multiplication in a
host species.
 Tolerance of damage is independent of resistance and relates to the ability of a host genotype to
withstand or recover from the damaging effects of nematode attack and yield well.
 Resistance induced by the nematode depends on the nematode biology.
 Ectoparasites generally have a necrotrophic relationship with the plant.
 The injected saliva liquefies the cytoplasm that accumulates around the stylet tip and is rapidly
ingested, usually killing the host cell.
 The nematode then moves to a new cell and repeats the process.
 Such behaviour limits the possibilities for effective induced resistance.
 Migratory endoparasites may have a necrotrophic type of feeding; in some species, however, the
feeding is partly biotrophic as it involves the induction of favourable changes in cells adjacent to
the feeding site.
 Resistant cultivars are available to species within this group, especially the stem and leaf parasites.
 Sedentary endoparasites are obligate biotrophs.
 Nematodes within this group are the most damaging to plants and resistance to them is widespread.
54

55. Tolerance
 Cultivars of a crop are regarded as differing in their tolerance if the decrease in their growth
and / or yield due to damage by nematodes differs significantly when they are grown in
uniformly infested soil.
 Where cultivars of different yield potentials are being compared, the proportional effect on
yield must be compared in adjacent, uniformly uninfested and infested conditions.
 Resistance and tolerance are independent attributes of plants but resistance may confer
tolerance especially if it decreases the incidence of nematode attack or parasitism.
 Equally, mechanisms of tolerance exist that are independent of resistance.
 Tolerance is probably widespread and important in wild plants but is likely to be lost during
crop breeding. Various mechanisms of tolerance have been suggested.
 They include: differences in numbers of roots, compensatory root growth, delayed
senescence and enhanced water uptake.
 Tolerance is independent of resistance and it appears to be largely nonspecific.
 The use of tolerance is a valuable strategy for many crops or situations where alternative
control measures are not available.
 However, tolerance of cyst and RKNs generally needs to be combined with a degree of
resistance, otherwise populations will be increased to densities where even tolerant cultivars
are damaged.
 Susceptible and tolerant genotypes may, however, be useful in rotations following resistant
genotypes as a means of reducing the rate of selection of virulence.
55

56. BIOLOGICAL CONTROL
 Many natural enemies attack plant parasitic nematodes in the soil and reduce their
populations.
 They include bacteria, rickettsia, fungi, protozoa, tardigrades, tubellarians, nematodes,
enchytraeids, mites, and insects.
 It is important to determine the nature and extent of such attacks on nematode
multiplication in order to establish whether these enemies can be exploited to reduce
damage and increase crop yield.
 There are two types of biological control: induced, where the biological control agent has
been applied by man, and natural, where the agents have increased to suppress nematode
multiplication without being specifically introduced.
 The term suppression is used to indicate a reduction in the numbers of nematodes not
simply a reduction in disease symptoms; it may be general or specific if only one or two
organisms are involved.
 Biological control should not be considered as a replacement for the chemical control.
 Growers that require rapid kills when their fields are found to be heavily infested have
few alternatives to chemical treatment.
 Biological control is slow acting and cannot fulfil this requirement. It, therefore, fits very
well in an integrated nematode management system.
56

57. Natural enemies as biological control agents
Pasteuria penetrans
 Pasteuria penetrans is an obligate parasite of some
plant parasitic nematodes.
 Nematodes become infected in soils when they get
in contact with the endospores, which adhere to
their cuticle.
 For instance, infected second -stage Meloidogyne
juveniles enter roots and begin feeding before the
spores germinate.
 A germ tube penetrates the cuticle and gives rise to
a vegetative microcolony that fragments and
proliferates throughout the nematode body cavity.
 Eventually females become filled with spores and
egg production is prevented.
 Although P. penetrans appears to have considerable
potential as a biological control agent, its
commercial exploitation is prevented by lack of
methods for large-scale production.
57
RKNs juvenile encumbered with
spores of P.penetrans

58. Nematode trapping fungi
 A commercial strain of Arthrobotrys
irregularis has been produced on rye grain
and marketed as Royal 350.
 It is recommended that the fungus be
applied at 1.4 t/ha at least 1 month before
planting and incorporated into the soil.
 Reduced root galling caused by Meloidogyne
spp. and increased tomato yield has been
reported
 Arthrobotrys robusta, sold under the
trademark Royal 300, is used for the control
of nematodes damaging mushrooms
(Ditylenchus myceliophagus).
 It is introduced into the compost on rye
grain at a rate of 1% with the mushroom
spawn.
 In one trial, Royal 300 increased yield by
20% and reduced the final populations by
40%..
58
nematodes trapped by the fungus (B & C) and
a nematode trapped in adhesive mycelial
columnar of Monacrosporium sp (D)

59. Endoparasitic fungi of vermiform nematodes
 The endoparasitic fungi produce small spores that contain too little energy to initiate
colonization in soil.
 Hence spores remain dormant until they adhere to a passing nematode, after which they
germinate, penetrate the cuticle and colonize the host.
 Nematoctonus concurrens and N. haptocladus belong to this group and are quite effective
against nematodes.
Parasites of nematode eggs
 Eggs are more susceptible to infection before second stage juveniles have developed, and
if young females are parasitized their fecundity is decreased.
 Therefore these fungi are more effective if they are able to parasitize females and egg
masses soon after emergence on roots.
 Fungal parasites of eggs appear to be facultative parasites that can be grown readily in
vitro, and their survival in soil is probably not dependent on the presence of nematodes.
 Most infection occurs when the females and cysts are on the roots and not when they are
dispersed in soil. 59

60. Parasites of nematode eggs
 Assuming that the most convenient time for applying these fungi is before planting, they
must survive several weeks in soil in sufficient numbers to control the new generation of
nematodes.
 Application of an energy source with the inoculum‟ is therefore essential.
 Egg parasites do not kill active juveniles. Hence, in heavily infested field, crop damage
may not be decreased after treatment, particularly when the nematode has only one
generation per season.
 A considerable range of fungi have been reported to colonize eggs of cyst- and root- knot
nematodes.
 The best documented are: Paecilomyces lilacinus, Dactylella oviparasitica and Pochonia
chlamydosporia.
 Applications of Paecilomyces lilacinus on cereal grains readily colonize soil, and a single
treatment may be sufficient to establish the fungus.
 Rates of 0.4 t/ha seem to reduce damage caused by Meloidogyne. Dactylella
oviparasitica and Pochonia chlamydosporia parasitize eggs of root knot and cyst
nematodes and have been associated with the natural control of M. incognita on a wide
range of crops.
 It is easy to grow these fungi in vitro. They also colonize the rhizosphere of plants
without causing damage.
60

61. CHEMICAL CONTROL
Chemicals have been used to control nematodes for a long time.
 The use of nematicides is recommended when:
(i) The nematode population requires an immediate intervention
(ii) Other methods fail to reduce nematode populations to acceptable levels.
(iii) The crop protection should be at maximum eg on export materials.
(iv) Besides nematodes, other crop parasites are to be controlled.
Types of nematicides
 Nematicides fall into two groups:
 FUMIGANTS
 NON FUMIGANTS
61

62. CHEMICAL CONTROL
1. Fumigants
 volatile liquids that vaporize and dissolve in the soil solution.
 Their high vapour pressure distributes the gas in all directions through soil pores.
 Most of the fumigants are phytotoxic and directly kill nematodes and their eggs.
 Two types can be distinguished:
(i) Halagenated aliphatic hydrocarbons:
methyl bromide, ethylene dibromide (EDB) 1,3-dichloropropene mixtures and chloropicrine.
(i.i) Methyl isothiocyanate precursor compounds:
Metham sodium, dazomet, and methyl isothiocyanate mixtures: solutions in xylol or mixed
with, 1,3-dichloropropane mixtures (Vorlex).
.
62

63. CHEMICAL CONTROL
2. Nonfumigants nematicides
Often known as nematostats, as they do not kill nematodes directly but do affect their
behaviour. Most of these nematicides have a systemic action.
Different formulations exist and these nematicides can be applied at sowing or planting and
also later. Two chemical groups can be considered:
(i.) Organophosphates: fenamiphos, ethoprophos, thionazin, fensulphotion.
(ii) Carbamaaldicarb, oxamyl, carbofuran, methoxyl.
It should be emphasized that chemicals are potentially harzardous to the environment, man
and other non-target organisms.
Judicious application is strongly recommended with the aim of reducing the risks associated
with them and also the pesticide residues in agricultural produce.
Chemical application can only be economical in production of high value crops because they
are expensive
63

64. 64
MOLECULAR TECHNIQUES IN DETECTION
AND DIAGNOSIS OF PPNS

65. DNA EXTRACTION
 The first step in molecular diagnostic procedures is the preparation of the
template DNA.
 Several protocols for the extraction of nucleic acids from nematodes are available
 Some of these allow the isolation of microgram quantities of pure genomic DNA
DNA EXTRACTION INVOLVES TWO MAIN STEPS
I. mechanical destruction of nematode body and tissues in a tube using ultrasonic
homogenizer or other tools, or repeatedly freezing samples in liquid nitrogen; and
II. chemical lyses with proteinase K in a buffer for 1 h or several hours with subsequent brief
inactivation of this enzyme at high temperature.
 Various chemical treatments are also applied to remove cell components and purify the DNA.
 Phenol or phenol with chloroform extractions is often employed to remove proteins
 Ethanol is then used to precipitate and concentrate the DNA.
 Effective DNA extraction can be achieved by using commercial kits developed by different
companies.
 These approaches rely on DNA binding to silica in the presence of a high concentration of
chaotropic salt.
65

66. PCR-BASED METHODS
 This enzymatic reaction allows in vitro amplification of target DNA fragments by up to a billion
fold from complex DNA samples within a test tube.
 Any nucleic acid sequence can be detected by PCR amplification.
 The method requires a DNA template containing the region to be amplified, two oligonucleotide
primers flanking this target region, DNA polymerase and deoxyribonucleotide triphosphates
(dNTPs) mixed in buffer containing magnesium ions (MgCl2). The PCR is performed in tubes with
final volumes of 20–100L.
I. template denaturation at 95°C for 3-4 min
II. primer annealing at 55-60°C for 1-2 min
III. extension at 72°C for 1- 2 min.
 The PCR is carried out for 30-40 cycles in a thermocycler with programmed heating and cooling.
 Finally, PCR products are separated electrophoretically according to their size on agarose or
polyacrylamide gels and visualized by ethidium bromide under ultraviolet (UV) light or after silver
staining.
 Once identified, nematode target DNA generated by PCR amplification can be characterized
further by various analyses: restriction fragment length polymorphism (RFLP), single strand
conformation polymorphism (SSCP) or sequencing.
66

67. PCR METHODS
1. PCR-RFLP (RESTRICTION FRAGMENT LENGTH
POLYMORPHISM.
 The PCR product obtained from different species or
populations can be digested by a restriction enzyme and the
resulting fragment is separated by electrophoresis.
 Sequence differences in PCR products will lead to different
electrophoretic profiles.
 Comparison of restriction patterns derived from amplified
ITS regions has been used to distinguish species and
populations of Aphelenchoides (Ibrahim et al., 1994),
Bursaphelenchus (Hoyer et al., 1998), Ditylenchus (Ibrahim
et al., 1994), Nacobbus (Reid et al., 2003), Pratylenchus
(Waeyenberge et al., 2000), Radopholus (Fallas et al., 1996),
and root knot nematodes (Schmitz et al., 1998)
 Comparison of RFLP profiles from newly obtained samples
with those from known species provide a quick tool for
nematode identification.
 PCR-RFLPs are especially suited to identify nematodes of
monospecific probes; this strategy does not allow mixed
species populations to be identified.
67

68. 2. PCR-SSCP (SINGLE STRAND CONFORMATION POLYMORPHISM)
 This technique has been applied successfully for rapid identification of cyst-forming
nematodes and root knot nematodes from cultures and field samples (Clapp et al., 2000).
 The distinguishing patterns obtained with PCRSSCP
are sequence dependent and utilize minor nucleotide differences across several hundred bases
of sequences.
 It is a simple procedure where denatured, single-stranded PCR amplicons are separated
electrophoretically in a non denaturing polyacrylamide gel.
 The length, position and extent of selfcomplementary base pairs affect the conformation
taken up by the molecules and thus their electrophoretic mobility.
 This effect is enhanced by minor length polymorphisms and increasing amounts of
sequence variation.
 SSCP patterns are highly reproducible between gels and generate two markers from each
DNA sequence present.
 The band patterns are compared with those obtained from controls or from pattern
databases 68

69. 3. SEQUENCING
 Direct sequencing of PCR products or sequencing of cloned PCR fragments
provides full characterization of amplified target DNA.
 The sequences of the ITS regions, fragments of 18S and 28S of rRNA genes,
have been examined for a wide range of plant parasitic nematodes (Subbotin et
al., 2001b; Floyd et al., 2002; Reid et al., 2003).
 The comparison of newly obtained sequences from samples with those
published or deposited in the GenBank is a most reliable approach for
molecular identification.
 Increasing numbers of deposited nematode rDNA sequences as well as
decreasing costs for sequence analyses will allow wider application of this still
rather expensive procedure for routine nematode diagnostics in the future.
69

70. 4. PCR WITH SPECIES-SPECIFIC PRIMERS (MULTIPLEX PCR)
 This allows the detection of one or several species in a
nematode mixture by a single PCR test, thus decreasing
diagnostic time and costs.
 Species-specific primers are designed based on the broad
knowledge of sequence divergence of the target DNA
region in many populations of the same species and in
closely related species.
 This knowledge allows the detection of populations with
small differences in sequences, and avoids the
amplification of an identical specific fragment in other
species.
 The principle of this method is the alignment of the
sequences from target and non-target organisms and the
selection of primer mismatches to non-target organisms,
but it shows sufficient homology for efficient priming and
amplification of the target organism.
 This tool has been developed for identification of cyst-
forming and root knot nematodes and Pratylenchus
(Uehara et al., 1998), Xiphinema (Wang et al., 2003) and
Ditylenchus (Esquibet et al., 2003). .
70
Amplification product of PCR with species-specific
primer Finc/Rinc for Meloidogyne incognita. I,
Meloidogyne incognita; J, M. javanica; A, M. arenaria; M,
M. mayaguensis; H, M. hapla; C, M. chitwoodi; F,
M. fallax; W, no template DNA control; S, size marker
(Source: Zijlstra et al., 2000).

71. 5. REVERSE DOT-BLOT HYBRIDIZATION
 This technique involves the use of PCR
for simultaneous amplification and
labelling of target DNA to generate
digoxigenin-dUTP-labelled amplicons
which are hybridized to specific
immobilized oligonucleotide probes on a
membrane.
 This approach can be used for
simultaneous identification of many
different nematodes from a single
sample.
 Uehara et al. (1999) have demonstrated
that this technology can be used for the
identification of Pratylenchus species 71
Reverse dot-blot hybridization with immobilized specific
oligonucleotides. The Pratylenchus species
listed on the left were used for each hybridization (Source:
Uehara et al., 1999).

72. 6. RAPD-PCR (RANDOM AMPLIFIED POLYMORPHIC DNA-
PCR) In contrast to the above-mentioned classical PCR method, the random amplified polymorphic
DNA PCR (RAPD-PCR) or PCR with arbitrary primer (AP-PCR) does not require any information
on the primer design.
 This technology uses a single random primer about ten nucleotides long, approximately 50% GC
rich and lacking any internal inverted repeats.
 By lowering the annealing temperature during the amplification cycle, the primer anneals at
random in the genome, allowing the synthesis of highly polymorphic amplification products.
 RAPD-PCR distinguishes nematode species, subspecies and races and is used for root knot
nematodes (Cenis, 1993; Blok et al., 1997) and cyst-forming nematodes (Thiery et al., 1997)
 Specific sequences for certain species or races, called SCARs (sequence characterized amplified
regions), can be derived from RAPD fragments and further used to design species specific primers.
72
Fig. 4. RAPD patterns of 26
populations of the
Heterodera avenae
complex. Primers: A, A-16;
B, A-18.

73. 7. AMPLIFIED FRAGMENT LENGTH POLYMORPHISM
(AFLP)
 The technique is based on the selective amplification of genomic restriction
fragments and involves three steps:
(i) digestion of DNA with two restriction enzymes and ligation of specific
adapters to the restriction fragments
(ii) PCR amplification of a subset of the restriction/adapter fragments under
stringent conditions
(iii) gel electrophoresis analysis of the amplified restriction fragments.
 The AFLP technique has several advantages over RAPD in that it produces
results that are very reproducible and it has higher resolutions generating many
more amplified fragments.
 AFLP fingerprinting has been applied successfully for the evaluation of inter-
and intraspecific genetic variation of cyst-forming nematodes (Marche et al.,
2001) and root knot nematodes (Semblat et al., 1998). 73

74. END…..
THANKS
74