testing on seeds by any disease, insect, nematodes occur or not

1. PRASATH S
2018611806

2.  Seed health refers to the presence or absence of
disease-causing agents such as fungi,
nematodes, bacteria, viruses and insects, and to
the status of seeds in a seedlot.
 These may be carried externally or internally
and caused severe damage in crop raised from
them.

3.  Seed Health Testing is a Science of
determining the presence or absence of
disease-causing agents/insects.
 To determine the health status of a seed
sample, and by inference seed lots.

4.  To determine the health status of a seed lot
which in turn establishes the sanitary condition
of the seed in commerce.
 To obtain objective proof of whether the seed
lot meets the requisite certification standards or
not.
 To obtain objective proof of whether the lot
meets requisite quarantine requirements.

6.  This simplest kind of pathological test for seed.
 with or without stereomicroscope.
 Inspected in dry state for the presence of
impurities such as ergots or other sclerotia.

7.  Infected seeds have a characteristic black
powdery mass generally along with the suture.
 Severe infection – most of the endosperm
together with pericarp and aleurone layer gets
destroyed giving seeds a boat like appearance

8.  Seeds are opened with the help of the blade.
 Whole seeds are transformed into black
powdery mass
 The infected seeds have a characteristic black
powdery mass when crushed with finger nails
or forcep.

9. Alternate procedure :
 Paddy seeds are soaked in 0.2% solution of
sodium hydroxide for 24 hours at 18 -25°C .
 Swollen seeds are visually examined for shiny
jet black colour.

10.  Soak seeds for 24hours at 20°C in Sodium
hydroxide and Trypan Blue.
 Separate the embryos by passing the soaked
material along with warm water (50-60°C)
 Use mesh sieve for separation of embryo
 Embryos are washed in wire basket and
dehydrated in 95% alcohol for 2min and later
transferred to a mixture of lactophenol and
water.

11.  Further separation of embryos can be made.
 Embryos are placed in lactophenol and boiled.
 Embryos are examined in microscope.
 Infected fungus with loose smut fungus is
present as hyphal strands.
 Golden brown thread like mycelium which is
septate with non uniform thickness and
swellings are classified as infected embryos.

12.  Fungi which are able to produce mycelial
growth and fruiting structures under the
incubation conditions available in the test.
 All kinds of seeds, cereals, vegetables,
legumes, ornamentals and forest seeds are test
by this method.

13.  Seeds are placed on moistened blotters, filter papers at
least 20mm apart.
 Usually 400 seeds/samples were taken.
 25 seeds placed in each Petri dish.
 Incubate the dishes at 20-22°C for 7 days in
alternating cycles of 12 hours darkness and 12
hours light.
 Examine the individual using stereomicroscope or
compound microscope.

14.  After pretreatment seeds are spaced on the
surface of 2% malt extract sterilised agar.
 After plating dishes are incubated at 20-25°C
for 5-8 days.

15.  Seeds are immersed in water with a wetting
agent and shaked vigorously to remove fungal
spores, hyphae etc.
 Excess liquid is removed by filtration or
centrifugation and the extracted material is
examined.

16.  Seeds are immersed in water, or other liquid to
make fruiting bodies, symptoms more easily
viable or to encourage the liberation of spores.
 After imbibition, the seeds are examined with a
stereoscopic microscope

18.  The ‘growing on’ bioassay of a working seed
sample involves the sowing of test seeds into
seedlings under conditions optimal for the disease
development in glass house or closed
environmental chambers.
 ‘Growing on’ test has been successfully used for a
large number of Xanthomonas sp. and
pseudomononas sp.

19.  Working seed sample is sterilized with (2.6%)
sodiumhypochlorite for 15 min, and rinsed with
sterile water.
 The seed sample is incubated for 18-24 h in sterile
water.
 The water suspension is inoculated by infiltration
into the primary leaf node of 10 day old bean
seedlings.
 The appearance of lesions followed systemic
necrosis is positive reaction.

20. By culturing the samples on the particular
media.
Presence of bacteria can be observed on the
media.

22. By growing seeds
 The seeds are examined and abnormal seeds are
separated.
 Both normal looking and abnormal looking seeds
are grown separately and the seedings are observed
for the characteristic symptom of the virus disease.
 Plants are kept free of insect vectors such as mites
and white fly.

23. Direct seed Test
 Seed are examined, normal and abnormal seeds
are separated and handled separately.
 Seeds are soaked in aqueous medium and then
triturated.
 The slurries produced are then applied to indicator
or test plants.
 The slurries containing virus produce symptoms
typical for a given virus on the indicator plant.

24. Double diffusion test
 Seeds to be tested are soaked in tapwater.
 Seed or part (embryo) of it is triturated
 Triturate is transferred to a well cut in a diffusion
media(agar gel).
 Antiserum specific for a suspect virus in a seed is
placed in separate well.

25.  In time the virus particles(antigen) and antibody
molecules diffuse towards one another.
 Since diffusion is in two direction it is called
Double diffusion
 The preciptate appears as a white band.

26. Radial Diffusion Test
 Procedure consists of charging the wells with the
seed or seedling preparations being tested for the
presence of virus.
 When virus molecules are present , they diffuse
radially from the well surface.
 A ring or halo results around the charged well.

27. Latex Flocculation test
 Specifically Prepared seed extracts are placed in a
pipette
 Specified quantity of tagged latex is added
 Pipette is then oscillated for about 15 minutes
 Observed under microscopes
 When Virus is present in the sample the latex
suspension becomes flocculated or aggregated.

28. ELISA Test
 In this procedure , antiserum specific for a given
virus is used to coat the polystyrene plate.
 Antibody molecules become absorbed.
 Then seed sample is added to the plate.
 It is followed by adding enzyme labelled specific
antibody to the plate.

29.  The enzyme alkaline phosphates is conjugated to
the antibody molecules specific for the virus under
examination.
 Finally enzyme substrate is added to the plate.
 Hydrolyzed substrate is determined by measuring
the extinction spectrophotometrically or by visual
observation.

30. SSEM(Serologically specific electron microscopy)
 Ground seeds or homogenised preparation is
applied to the electron microscope grid containing
an antiserum specific for virus in question.
 Samples from infected seed lots reveal the
presence of virus particle characteristics for the
suspect virus.

31. Radio Immuno Assay (RIA)
 Antiserum is added to the radio labelled antigens
and allowed to incubate in a centrifuge tube.
 After incubation , ammonium sulphate is added for
precipitation of antigen antibody complex.
 After centrifugation the supernatent is discarded
and the radio activity of the pallet is measured.
 The pallets showing radioactivity are counted as
infected.
 PCR is used inorder to detect the presence of
Viruses

33. Detection of presence of Insects
 Examined under magnification or stereoscopic
microscope
 Result is mentioned as number of insects per
weight of the sample

34. Detection of external insect injury
 Working sample is Examined under
magnification(10x) or stereoscopic microscope.
 The absence of insects does not however guarantee
that the seed lot is free from insect infestation, i.e
internal injury to the seeds or internal infestation.

35. Alkali or Glycerine Method
 Seeds are boiled in 10% NaOH solution for
10min(depending upon the kind of seeds to make
them transluscent)
 Alternatively the seeds can also be made
translucent by lactophenol solution.
 Washed with water
 Examined under magnification.
 seeds with visible internal infestation are
separated, cut opened to confirm the presence of
insect or its stage and counted.

37. Ear cockle of wheat (Anguina tritici)
 Working sample is visually examined for the presence
of ear cockle galls. (hard, small, dark purplish- black
colour structures)
 Galls are separated and soaked in water for 30 minutes
and are cut in water in petridish for observing the
release of nematode larvae, galls releasing nematode are
counted and result is reported in percentage.

38. Root knot Nematode of Sweet Potato (Meloidogyne
incognita)
 Entire submitted sample is examined for visible
symptoms of nematode infestation.
 Nematode infested tubers look ugly and disfigured.
 When tubers are cut across the knot , small glistening
round pin head shaped bodies are seen and such tubers
are separated and the results are reported.