assignment submitted to Dr. Sundar man shrestha sir

2. Presenter
Hem Raj Pant
frenndlyhem01234@gmail.com
Msc. Ag
Department of Plant Pathology
AFU- Agriculture and Forestry University
Rampur, chitwan
www.afu.edu.np
A Presentation on
CLASSIFICATION, CHARACTERIZATION AND
IDENTIFICATION OF DITYLENCHUS DIPSACI

3. INTRODUCTION
 Nematodes are triploblastic, bilaterally symmetrical, unsegmented,
pseudocoelomate, vermiform and colourless animals
The stem and bulb nematode Ditylenchus dipsaci (Kuhn,1857) is an
endoparasitic migratory nematode
 The nematode occurs worldwide but is particularly prevalent and
destructive in areas with temperate climate
Ditylenchus dipsaci is a plant pathogenic nematode that primarily
infects onion and garlic (Aftalion and Cohn, 1990).
 It was first recorded in Netherlands in 1883
 It can attack 400 plant species belonging to more than 40 families

4. Kingdom: Animalia
Phylum: Nematoda
Class: Secerenentea
Order: Tylenchida
Sub order: Tylenchina
Superfamily: Tylenchoidea
Family: Anguinidae
Subfamily: Anguininae
Genus: Ditylenchus
Species: dipsaci
CLASSIFICATION

5.  Females are 1000-1300 µm long and 35-40 µm wide. Males are
smaller than female
 They have 10-12 µm long and bottle shaped esophagus with a
distinct basal bulb which dose not overlap the intestine.
 The nerve ring is located at mid isthmus
Amphid aperature pore like
Stylet is always with basal knob
 Females have only one ovary and vulva is generally 75-80%
 Males have adenal bursa
MORPHOLOGICAL CHARACTERISTICS

6. Contd….
Ditylenchus can be distinguished as follows
 Nematode have lip region without obvious annules
 Lateral fields marked by four to six incisures
 Deirids very small but usually visible
 Phasmids rarely visible even from a dorso-ventral view
 Tails elongate-conoid to acute or subacute terminus
 Bursa enveloping one-fourth to three folds of tail
 Ovary single, outstretched, rarely with one or two flexures gonad cells
in one or two lines
 Rudimentary postuterine branch present

7. Figure: Structure of Male(left) and Female(right) Ditylenchus dipsaci

8.  Ditylenchus is one of the destructive plant parasitic nematode
which found worldwidely but is particularly prevalent and
destructive in temperate climatic areas.
 It can attack 400 plant species belonging to more than 40
families.
 Major cultivated host plants include bulbuous species such as
Garlic, Onion, Leek, Hyacinth, Tulip and non-bulbous ones
such as alfaalfa, broad bean, carnation, clover, corn, rye,
sugarbeet, strawberry
HOST RANGE AND DISTRIBUTION

9.  When infection occurs in seedlings, they become dwarfed, twisted and
abnormally white and also develop swollen areas where the epidermis
sometimes splits
 Most infected seedlings die within three weeks of planting and the
remainder usually die later
 Young plants show stunting, light yellow spots, swellings (“spikkles”) on
the stem, shorter and curled leaves, and swollen base
 Older infected bulbs show swelling (bloat) of scales
 Onion have a frosted appearance caused by the dissolution of cells that
results from nematode feeding
SYMPTOMS

10.  As the season advances the bulbs become soft and when cut
open show browning of the scales in concentric circles and give
off a foul odor
 The stem, neck, and individual scales of the bulb become
softened, loose, and pale gray
 Conversely, D. dipsaci does not induce deformation of leaves or
swelling in Garlic, but does cause leaf yellowing and death
Contd….

11. Figure 1:Onion leaves showing curling
symptom Figure 2:Bulbs showing
abnormal and corky tissue
Figure 3:Browning of scales in
concentric circle in cross section
Figure 4:Nematode infected field showing
leaf yellowing and early dieback
Figure 5: Difference between healthy
and nematode infected garlic plant

12.  Their lifecycle occurs in five stages where the first molt occur in the egg
and second and third molt occur in the soil producing preadult or infective
juvenile.
 The preadult/fourth stage juvenile can withstand adverse environmental
condition of freezing and of extreme dry for longer period in fragment of
plant tissue, in seeds or in soils.
 The fourth stage juvenile can withstand dessication for a long time,
sometimes 20 years or more (Barker and Lucas, 1984).
 During favourable temperature and moisture the preadult juvenile become
active, enter the host and pass through fourth molt and become males and
females.
Life Cycle and Disease development

13.  When seedlings are infected, fourth stage juvenile starts feeding intercellularly
on the parenchymatous cells of cortex
 Infected areas enlarge and seedlings are deformed
 The swelling of epidermis often breaks leading to fungal and bacterial entry.
Infected area disintegrates and finally seedlings die
 Fourth stage juvenile enter young plant through stomata or penetrate directly
and change to male and female nematodes
 Female start feeding intercellularly on nearby parenchyma cells and slight
hypertrophy and hyperplassia develop around nematode.
Contd….

14.  Spicules may develop on leaves and leaves remain short and thicken
at the base
 As the bulbs enlarge, the nematodes migrate downward from the
leaves either intercellularly or the surface of leaves and enter again at
the outer sheaths of stem or neck
 They enter into scales of bulb and infected scales appear as mealy or
discoloured ring
 The spread of the infection within a bulb continues in the field and in
storage until, usually, the entire bulb become affected
Contd….

15.  Females present in infected bulbs lay eggs and continue their
life cycle
 A female can lay about 200-500 eggs in her lifespan.
However, if conditions are unfavorable, the nematodes can
halt their lifecycle
 Some juveniles can survive on or in plant tissue by
entering cryptobiosis forming nematode wool and survive for
3–5 years in this stage
Contd….

17. The identification of D. dipsaci should preferably be based on morphological methods.
Molecular methods developed for identifying these species can be used for low infestation
levels or when only juveniles are present.
Molecular methods can be applied to damaged and atypical adults, and all life stages,
including the juvenile stages, for which morphological identification to species is not
possible.
Preparation of specimens
Temporary preparations for quick identification or study of features best seen in unfixed
specimens are prepared as follows (Kleynhans, 1997):
Live specimens are transferred to a small drop of water on a glass slide.
The slide is briefly heated over a spirit flame, checking frequently for nematode
movement. Heating should be stopped as soon as the specimens stop twitching.
IDENTIFICATION

18. A coverslip is applied and sealed around the edge with nail varnish. When the varnish has
dried, the slide with specimens is ready for study. For light microscopy, live nematodes are
extracted from soil or plant material, killed by gentle heat (65–70 °C), fixed in FAA (35%
distilled water, 10% of 40% formalin, 5% glacial acetic acid, 50% of 95% alcohol)
(Andrássy, 1984), transferred into glycerol (Hooper et al., 2005) and mounted in anhydrous
glycerine between coverslip slides as described by Seinhorst (1959).
For light microscopy identification work, magnification of 500× to 1000× (oil immersion
lens) in combination with differential interference contrast microscopy is recommended.
Morphological diagnostic characters
Keys for diagnosis for Ditylenchus species can be found in Viscardi and Brzeski (1993)
and Brzeski (1998). A key to distinguish Ditylenchus spp. from other tylenchid and
aphelenchid genera is presented in Table below.
Contd….

19. 1
Outlet of dorsal pharynx gland near base of stylet; median bulb roundish, ovoid or absent Tylenchida – 2
Outlet of dorsal pharynx gland in median bulb; median bulb a prominent feature, usually oblong Aphelenchida
2
Anterior part of oesophagus (procorpus) and median bulb not united into single unit; stylet never
exceptionally long
3
Procorpus gradually widened and fused with median bulb; stylet very long, its base often located in anterior
part of median bulb
Other genera
3
Adult female vermiform 4
Adult female saccate or pyriform sessile parasite on roots Other genera
4
Valvular median bulb 5
Median bulb without valve1 Other genera
5
Pharynx glands contained within basal bulb, not overlapping or slightly overlapping intestine; cephalic
framework rarely conspicuous; stylet weak to moderately strong
6
Pharynx glands lobe-like, overlapping intestine; cephalic framework strong; stylet massive Other genera
6
Single prodelphic ovary; vulva posterior 7
Ovaries two, amphidelphic; vulva slightly post-equatorial Other genera
7
Female not swollen; crustaformeria in female in form of quadricollumella with four rows of four cells each;
bursa in males enveloping one-third or more of tail
Ditylenchus
Female swollen; crustaformeria with more than 20 cells Other genera

20.  Strict quarantine regulations to prevent the entry of infected propagative materials
like bulb
 Use of certified plant propagative materials in uninfested soil is the best preventive
and economic method
 Hot water treatment of bulbs and tubers for 3-4 hours at 44-45ºC
 Crop rotation for at least 2-3 years with non host crops like Spinach, Carrots,
Beets, Crucifers and lettuce
 Wide row spacing and replacement of overhead irrigation with drip irrigation are
some of the cultural practices to prevent nematode build-up in the soil
 Fumigation of onion seeds with methyl bromide
Nematophagous fungi such as Verticillium balanoides may be suitable for
development as biological control agents for D. dipsaci (Hay and Bateson, 1997)
Management