a) Methods used in sampling, extraction of motile stages and cysts, b) Different groups of plant parasitic nematodes found in soil samples, c) Calculate the ratio of eggs and cysts in soil samples as well as number of nematodes in a 100ml of soil, d) Effects of plant parasitic nematodes on a susceptible and resistant variety of fodder radish.

1.
Lab Work Protocol
Faculty of Organic Agricultural Sciences
Ecological Plant Protection
Submitted To
PD Dr. Johannes Hallmann
Julius Kühn-Institut, Bundesforschungsinstitut für Kulturpflanzen
Institut für Epidemiologie und Pathogendiagnostik
Submitted By
Gyanendra Dhakal
P20 PLANT NEMATOLOGY
Matriculation Number -21170080
7th November 13

2.
Table of Contents
Introduction
1
Methods and Materials form sampling
3
Sampling side and process
3
Methods of Extraction
3
Experiment 1
4
Experiment 2
6
Experiment 3
9
Experiment 4
11
Experiment 5
13
Summary
14
References
15

3.
1. Introduction
Nematodes belong to phylum Nematode-are non-segmented, elongated roundworms that are
colourless, without appendages, and usually microscopic. There are non-beneficial and
beneficial nematodes. Non-beneficial nematodes are also called “plant parasitic nematodes”
and cause damage to crops and other types of plants.
Beneficial nematodes attack soil borne insect pests, yet are not harmful to humans, animals,
plants, or earthworms, and can therefore be used as biological control organisms [2].
Beneficial nematodes that cause disease within an insect are referred to as
“entomopathogenic” and have the ability to kill insects. They have great distribution and live
almost in every corner of the world with an incredible diversity. By the mid 1980s more than
15,000 species 22,00 genera of nematode had been described [4].
Their number is increasing rapidly day by day. Most plant parasitic nematodes cause huge
problem to agricultural sector as well as human health. So nematodes are very important to
get knowledge on ecological potential in the sustainability of organic agriculture. Plant
parasitic nematodes have evolved into two groups the ones possessing stylet with knobs i.e.
the Tylenchida and the ones possessing stylet without knobs i.e. the Dorylamida and
Triplonchida.
Plant parasitic nematodes are economically important and cause severe economic loss to
farmers. Around 77 billion dollars of worldwide damages is lost annually due plant parasitic
nematodes [11]. So that farmers should have measured for the presence of nematodes to
adopt necessary control and preventive measures,
To control nematode and prevent economic loss in their farm farmers has to sample the soil
frequently for lab analysis. Representative sampling increases the accuracy of predication of
parasitic nematodes. After sampling extraction must be done to isolate nematode from soil
and plant materials to identify the nematodes present in soil and plant materials.
1

4.
The main objective of this lab protocol is to present the
a) Methods used in sampling, extraction of motile stages and cysts,
b) Different groups of plant parasitic nematodes found in soil samples,
c) Calculate the ratio of eggs and cysts in soil samples as well as number of nematodes
in a 100ml of soil,
d) Effects of plant parasitic nematodes on a susceptible and resistant variety of fodder
radish.
2

5.
2. Methods and Materias for sampling
2.1 Sampling Site
The sampling site bases in Neu-Eichenberg, 220-250 meters above sea level. It is clay-silt
soil on loess, which contains around 13-15% clay, 78-83% silt and 3-6% sand.
2.2 Sampling Process
Sampling is done from different site to increase the accuracy of predicting the presence of
parasitic nematodes. The sampling was done in a random manner. Sampling is done with the
assumption that whole farm cannot be analysed and the distribution of nematodes is also no
even on the farm. In this experiments sample were taken with probes which are hollow metal
devices inserted into the soil to obtain soil cores. Different soil cores were observed at
various depths and the soil sample was collected into plastic bag and labelled.
2.3 Methods of extraction used in experiments: Baermann Funnel method
This is a method used to extract nematodes and further analysis. Principally in the Baermann
Funnel method nematodes swim from plant materials or soil sample into water. The materials
essential to set up Baermann funnel apparatus are funnel with a rubber tube, cheesecloths and
screw clip [1].
The funnel is filled with water and the rubber tube attached to the funnel is clipped to prevent
the water from leaking. After that plant material sample or soil sample is placed in the funnel
so that it is just submerged by the water. The apparatus is allowed to stand for 72 hours after
which the nematodes swim from the sample into the water where the water is prevented from
flowing out by the clip. This water contains the nematodes from the sample and can be placed
on slides for further analysis.
3

6.
Experiment 1
Identification of Nematodes with and without stylets from soil samples from Neu
Eichenberg
Materials and Methods
Nematode suspension from Neu Eichenberg soil sample was analysed for the identification
nematodes with and without stylet.1ml of the nematode suspension was provided to twelve
students to observe under the microscope. Each of the individuals counted nematode
with stylets and without stylates separately.
For the statistical analysis Microsoft excel software was used.
Results
The mean number of the nematodes without stylet was found to be higher than that of the
nematodes with stylet (Fig 1). However this result is not significantly different using SPSS to
do a t Test ∞ = 0.05. The variance of the nematodes with and without stylet was 389.5207
and 450.75 respectively. Similarly standard deviation of the nematodes of the nematodes with
and without stylets was 19.73 and 21.23 respectively.
Table 1: Number of nematodes with and without stylets in 12 samples
Sample
1
2
3
4
5
6
7
8
9
10
11
Total
Mean
With stylet
21
68
85
39
29
65
26
32
27
39
37
468
42.54
4
Without stylet
79
32
15
61
71
35
74
68
78
41
37
591
53.72

7.
Fig 1: Mean and Percentage of Nematode with stylet and without stylet
Discussion
There was not significant different between nematode with stylet and without stylet, which
mean that there is equal distribution of stylet bearing nematode and not bearing nematode in
Neu EIchenberg soil.
Similarly variance and standard deviations in both types of nematodes were high, that mean
individual data were further away from the mean of the group. This could be error in
counting nematode by different inexperience group of researcher. In addition unequal
homogenisation of the suspension possibly alter the results.
5

8.
Experiment 2
A biotest to investigate the effect of different of M. incognita on susceptible and resistant
Fodder varieties
Fodder radish is an edible root vegetable, which belong to Brassicaceae family. Fodder radish
is a very quick growing root vegetable used as feed for livestock. Fodder radish is also
though to contain chemicals that help supress nematodes e.g. cyst nematodes which attack
potatoes.
Meloidogyne incognita is an important plant parasitic nematode classified in parasitology as a
root-knot nematode, as it prefers to attack the root of its host plant. When M. incognita
attacks the roots of plants, it sets up a feeding location, where it deforms the normal root cells
and establishes giant cells. The roots become gnarled or nodulated, forming galls, hence the
term "root-knot" nematode.
M. incognita has been found to be able to move along shallower temperature gradients
(0.001C/cm) than any other known organism, [9] an example of thermotaxis. The response is
complicated and thought to allow the nematodes to move toward an appropriate level in soil,
[3] while they search for chemical cues that can guide them to specific roots.
They parasitize the roots, entering at the second juvenile stage. Crop yield is reduced and the
parasitism of the nematode alters the physiology of the infected plants. More than eighty
nominal species have been described so far, about ten species are agricultural pests, four are
major pests and distributed worldwide in agricultural areas [5].
Materials
1. Nematodes
Juvenile nematodes of M. Incognita were obtained from the Julius Küln Institute.
There were about 978 J2/ml of inoculums.
2. Fodder radish
Seven days old fodder radish was inoculated with 2 different concentrations of
M.incognita for 8 days. There were 2 varieties of fodder radish used one is susceptible
variety called Silentina and another resistant variety called Contra.
Method:
The original suspension of M.incognita was diluted to 2 different concentrations: 500
J2/plant and 2000 J2/plant. Subsequently each fodder radish variety was inoculated with the
different suspension concentrations. After 8th day inoculated treatments were pulled out and
examined by using microscope.
Procedure during biotest of fodder radish root
1. Wash and cut off stem removing all soil particle attached to roots,
6

9.
2. Transfer of root into glass vial and addition of 1% chlorine solution for 15 minutes,
3. Again wash chlorine with tap water and stain root with acid Fuchsin solution,
Preparation of Acid Fuchsin solution
Stock solution: 3.5g acid Fuchsin, 250ml acetic acid and 750ml water
Working solution: 1:20 dilution (50ml stock/1050ml water)
4.
Boil of root into microwave and wash of root again,
5.
Place roots between 2 microscope slides,
6.
Observation on Microscope with different magnification.
Results
The Contra variety had fewer numbers of nematodes in the root compared to Silentina
however there was no significant different between them which is well illustrated by table 2.
But there was a higher variation in the means of the different treatment variety in response to
the nematodes Fig. 2.
Table 2: Number of nematodes in root of fodder radish variety per different suspension
concentration
Fodder Radish variety
Silentina
Contra
Concentration
500J2
2000J2
500J2
2000J2
Number of nematodes found in roots
72.8
57
15.4
35.4
Fig 2: Effect of nematode concentration on susceptible and resistant fodder variety
7

10.
Table3: ANOVA for the Biotest
Source
Sum of Squares d.f.
Mean Square
Between 9425.35
3
3141.783
Within
26619.20
16
1663.7
Total
36044.55
F
19
1.89
Where
H1: There is no significant difference between nematodes numbers in all treatments.
H2: At least one mean is different
F critical 0.05= 3.16
From Table 3, F calculated <F critical, there is not adequate support to reject H1 that the
means are the same. Therefore the mean number of nematodes in all 4 varieties was the same
regardless nematode concentration used for infection.
Discussion
From the above result mean number of nematodes was found to be higher in the susceptible
Silentina variety in both concentrations compared to resistance Contra variety. But there
were not significant differences within all the treatments. Principally higher numbers of
juveniles are expected in treatments with higher juvenile concentration. However a higher
juvenile concentration leads to a higher infestation rate, which increases competition this
ultimately, reduces the nematode production.
Similarly higher variability in the means indicates that the individual nematode numbers were
further away from the mean. This can be recognized to counting errors of the nematodes.
Conclusion
M.incognita still attacks resistant fodder radish varieties, as the nematode will first attack the
fodder variety before reproduction. After that further damage can be done because of
susceptibility. This means that a resistant variety can still be subjected to fungi attacks due to
the holes made by the nematodes.
The objective of this experiment was not to ascertain the effect of M.incognita on
development on resistant and susceptible varieties. To know the effect of M. inognita on
development of susceptible and resistant fodder varieties further experiment should be done
8

11.
Experiment 3
Identification of number of eggs and juvennile in a single cyst.
Method
Fifty cysts of Heterodera schachtii were put in 30 ml of water with the objective of
identification of number of eggs and juvennile in a single cyst by using microscopes.
Results
Table 4: Numbers and ratio of Eggs and Juvennile
Eggs
157
60
83
66
43
149
134
66
110
63
116
134
Juvenile Ratio
299
0.525
303
0.198
198
0.419
296
0.223
263
0.163
323
0.461
197
0.680
236
0.279
197
0.558
222
0.283
241
0.481
376
0.356
9

12.
Fig. 4 Number of Eggs and Nematodes
Discussion
Above table number 4 shows that practically all the egg and juvenile ratios are below 0.5,
which suggest majority of the cysts were old.
From this information we can suggest farmers to make necessary modification to deprive the
juveniles suitable conditon to servive furthermore. There could be different options including
the use of trap crops, solarization, resistant varieties to reduce the risk pf serious infection and
damage by nematodes.
10

13.
Experiment 4
Counting nematodes with stylet and without stylets
Plant parasitic nematodes obtain food by sucking juices from them. Feeding is accomplished
through a hollow, needlelike mouthpart called a stylet. They use stylet to puncture plant cells,
withdraw sap or food and parasitize the plant through secretion of protein and metabolites
[6].
Non stylet possessing nematodes are not plant parasitic e.g. Fungi feeding nematodes also
possess stylets, so the structure is not typical for only plant parasitic nematodes. Principally
all the stylet possessing nematodes are not plant parasitic however it can be useful for the
farmer to know the level of stylet possessing nematodes to give an idea of the health state of
the soil.
The aim of this experiment is to study the numbers of stylet possessing and non-stylet
possessing nematodes in 12 different soil samples.
Results
Table 5 Number of stylet and non stylet nematodes in different soil of 100ml
Sample
Conventional field 0-30cm
Conventional field 30-60cm
Buck Wheat
Legumes
Bush
Hebenhausen 0-10cm
Prof Finck’s garden
Clover
Corn
Private garden 0-8cm
Grassland
Garden compost
Golf course
With stylet
Without stylet Ratio
8200
9500
0.863
1700
800
2.125
4100
600
6.833
1600
5700
0.280
2300
4500
0.511
7500
1300
5.769
2700
16800
0.160
7500
2200
3.409
2400
5700
0.421
9100
22600
0.402
3700
7600
0.486
5200
19300
0.269
1900
1700
1.117
11

14.
Fig. 5 Number of nematodes in different 100 ml soil samples
Discussion
Greater number of nematodes without styple was observed thgan nematodes with stylets. Soil
sample collected from Buck Wheat field had the greatest ratio of nematodes with stylet and
nematode without stylet (6.833), followed by Hebenhausen 0-10cm (5.769), Clover field
(3.409) and Conventional field 30-60cm (2.125) subsequently. Considering the result from
table 5 it’s hard to conclude which factors are associated to the high stylet possessing
nematodes. So that futher investigation on previously grown crops, type of soil is essFrom the
result it is difficult to conclude what lead to the high stylet possessing nematodes. Further
essenital.
Where as soil sample from Prof Finckh had the lowest ratio (0.160) of nematodes with stylet
and nematodes without stylet, followed by the garden compost soil (0.269). There was not
any information on type of soil from Prof. Finckh, it will be difficult to determine which
characterstics of the soil influenced the low ratio of stylet and non-stylet neamtode. The
garden compost soil can be imagined to have a high level of nematode diversity which
reduces the level of plant parasitic nematodes.The supressive effect of compost on plant
parasitic nematodes was established by [8] and [10] as positive and potential to reduce plant
parasitic nematodes. Similarly Baby and Manibhushanrao also concluded that organic
ammendment have influence on parasitic nematode reduction through antagonistic activity
among soil microflora as the organic ammendment breaks down.
12

15.
Experiment 5
Isolation of nematode and classification based on Nematology key
Nematodes extracted from soil samples collected from diverse environment were identified
using a Nematology key and a microscope. Identification was based on main characterstics
like stylets, bulb, number of pharynx and other characteristics to further identify the
nematodes.
Most of the nematodes were ectoparasites. There were also some Paratylenchus sp. found
and they are important to farmers as well agriculturist to control the infestation casued by
neamtodes. These genera share a migratory habit and don’t enter into host root tissue but feed
externally by inserting their mouth stylets into root cell from the root surface. Ectoparasites
tend to congregate in the soil rhizosphere (soil on and around roots) where they browse along
roots and feed for a while, before moving on to a new feeding site [7].
Table 6: Nematodes found in soil samples
Ectoparasites
Migratory semiendoparasites
Migratory
endoparasites
Sedentary semi
endoparasites
Paratylenchus
Aphelenchoides
Helicotylenchus
Rotylenchus
Tylenchorynchus
Pratylenchus
Macrospothonia
13
Sedentary
endoparasites

16.
Summary
Plant parasitic nematodes are microscopic and mainly ectoparasitic and endoparasitic. To
able to identify their presence on field, must be sampled using probes. After that Baermann
Funnel method is use where nematodes swim from plant materials or soil sample into water.
With the same solution, different individual have very different numbers. To get a more
desirable number, firstly the solution must be mix well so that the aliquots are similar with
each other. During counting the speed should be improved as much as possible since the
nematode are alive and mobile. If time allows, count all the 15 columns of the counting slides
since the distribution of nematodes on the slide may not be random.
From the identification result, different types of nematodes appear more frequently, such as
Pratylenchus and Tylenchorhynchus. It can be assumed these are the majority group in
German soil. They are all ectoparasitic nematodes and all linked with essential crop diseases,
which shows the importance of controlling research of nematodes. From the result of Neu
Eichenberg soil, the allocation of nematodes also correlates with depth. And in the arable
land, which we use to culture, it tends to have more diversity of nematodes, which strengthen
the importance of nematode control.
The standard deviation of the with/without stylet counting result is high so the result is kind
of non-reliable. But in general, we can still see that in the arable land more plant-parasitic
nematodes are found with also a higher amount. In the shallow Neu Eichenberg soil we have
much more plant-parasitic nematodes. This may due to the contribution of plant residues.
The cyst content has a huge difference among different nematode species as well as within
same species. Different species have different life strategy. Some are colonizer and some are
persister. So the colonizers produce more offspring to make dominant distribution while the
persister use most nutrients to maintain their own life. The difference within H. schachtii may
be caused by the living condition. For a good living condition, the nematodes tend to use the
extra energy to propagate but in a severe condition they will try to survive first.
While the Siletina seedling is susceptible and the contra seedling is resistant, the result
doesn’t show the big difference we expected. This may due to the short time we apply the
nematode solution or the contamination from the sand or seedling. But we can still find if we
apply more amount of solution, we got more difference. As we know the bio control result is
not as obvious as the others such as pesticide. So this is always the challenge in organic
farming. This also tells us the resistant seedling is just relative, it’s hard to find a plant that
can totally resist the nematode parasitism. Then we don’t have a simple answer to use plant
agent to control the nematode diseases.
14

17.
References
1. Baermann, G. (1917). Eine einfache Methode zur Auffindung von Ankylostomum
(nematoden) Larven in Erdproben. Geneesk. Tijdschr. Ned-Indië 57: 131-137.
2. Denno, R.F., D.S. Gruner, and I. Kaplan. 2008. Potential for Entomopathogenic
Nematodes in Biological Control: A Meta-Analytical Synthesis and Insights from
Trophic Cascade Theory. Journal of Nematology 40(2): 61-72.
3. Dusenbery, D.B. Biological Cybernetics, 60:431-437 (1989). A simple animal can use
a complex stimulus pattern to find a location.
4. http://entomology.ifas.ufl.edu/creatures/nematode/soil_nematode.htm, 2012.10.31
5. Karssen, G.The plant-parasitic nematode genus Meloidogyne Göldi, 1892
(Tylenchida) in Europe.- Koninklijke Brill NV, Leiden, The Netherlands, (2002),157
p.
6. Lambert, K. and Bekal., S 2002. Introduction to Plant-Parasitic Nematodes. The Plant
Health Instructor. DOI: 10.1094/PHI-I-2002-1218-01
7. McKenry, M.V. and P. A. Roberts. 1985. Phytonematology Study Guide. University
of California, Division of Agriculture and Natural Resources.
8. Oka, Y., and Yermiyahu, U. 2002. Suppressive effects of composts against the rootknot nematode Meloidogyne javanica on tomato. Nematology 4:891–898.
9. Pline, Diez, and Dusenbery, J. Nematology, 20:605-608 (1988). Extremely sensitive
thermotaxis of the nematode Meloidogyne incognita.
10. Renco, M and Kovacic, P.2012 Response of Plant Parasitic and Free Living Soil
Nematodes to CompostedAnimal Manure Soil Amendments. Journal of Nematology
44(4):329–336. 2012. The Society of Nematologists .
11. Sasser, J.N. and D.W. Freckman. 1987. A world perspective on nematology: the role
of the society. Pages 7-14 in: Vistas on Nematology, J.A. Veech and D.W. Dickson,
eds. Society of Nematologists, Inc. Hyattsville, MD.
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