Seed health testing-A lecture note for L-4, S-1 students of BAU by K. M. Golam Dastogeer Bangladesh Agricultural University, Mymensingh, Bangladesh

1. STUDY OF SEED HEALTH TESTING
Seed health
Seed health concerns the overall condition of seeds. It includes pathogenic infection of seed, insect
infestation, morphological and physiological disorder, inert matter etc.
Seed health standard
Seed health standard is the maximum acceptable limit of the presence of a given pathogen in a given
seed lot. This is also referred to as "Pathogen tolerance level "or "Seed standard" for pathogen.
Field health standard
Field health standard is the maximum acceptable limit of a given seed borne disease present in a given
seed crop field. This is also referred as "Disease Tolerance level" or "Seed Standard" for disease of
seed crop.
Seed health testing
Seed health testing is a procedure by which can be determined whether tile seed is healthy or diseased
or it is a procedure by which the presence of absence of seed borne pathogen(s) in a seed lot can be
determined.
Seed borne pathogen
The establishment of a pathogen in, on and with the seed implies that the pathogen is seed borne.
Objectives of Seed health testing
1. Seed health testing is necessary for the improvement of seed stock in certification scheme.
2. It is necessary to satisfy quarantine requirement of a country.
3. It is done to know the planting value of a given seed lot in order to forecast the field
emergence and predict the health f the mature crop.
4. It is necessary to know the storage quality or feeding value of a seed lot.
5. It is necessary for checking the advisability of treatment.
6. It is done to know the efficacy of seed treating chemicals.
Methods of seed health testing
l. Dry inspection method
2. Microscopic examination of suspension obtained by

2. a. Washing test method
b. Whole embryo count method
3. Incubation tests
a. Blotter method
b. Agar plate method
c. Deep freezing blotter method
d. Water agar plate method
e. Test tube agar method
4. Seedling symptom test
- Hiltner's bricks stone method
- Sand methods
- Standard soil method
- Test tube agar method
5. Growing on test
6. Serological tests
7. Indicator plant test
8. Electron microscopy
Basic requirements or considerations
Routine methods for seed health testing must fulfill the following demands of efficiency and economy:
1. A test must give reliable information pertaining to field performance and quarantine requirements.
2. The results must be reproducible within the statistical limit
3. The time, labor and equipment for carrying through a test must be kept within economic limits.
4. The tests requiring incubation must be able to give the result quickly.
Equipment and chemicals required for seed health testing in the Laboratory
1. A hand lens.
2. A steriobinocular microscope.
3. A compound microscope.
4. An oven
5. A Refrigerator

3. 6. An autoclave
7. A centrifuge
8. Plastic/glass petridishes and/or earthen dishes
9. Filter papers or blotters/newsprints
10. Small equipments like forceps, glassware like-glass slides, cover slips, beakers, flasks, test
tube etc.
11. Chemicals and medium like-agar, dextrose, Nutrient agar, mounting 'fluid (lactophenol /Cotton
blue ctc,) chemicals for pretreatment like sodium hypochlorite (Clorox) and Hgc12 (mercuric
chloride) solution.
12. Potatoes, cheesecloth, pencils, markers ctc ,
13. Sampling equipments like treys, seed triers, dividers etc.
Seed lot
A lot is a specified quantity of seed physically identifiable in respect of which an International or
National Analysis Certificate may be issued.
Seed sampling
Primary sample
A primary sample is a small portion taken from one point of the seed lot.
Composite sample
The composite sample is formed by combining and mixing all the primary samples taken from the lot.
Submitted sample
A submitted sample is a sample which is submitted to the testing laboratory for analyses of the quality
of a seed lot
Working sample
The working sample is a sub-sample taken from the submitted sample in the laboratory, on which the
quality analyses will be done.
Dry inspection method
It is a very simple and preliminary method for testing the seed health.

4. Procedure
The dry seed samples were examined for impurities such as
a. Inert matter: It includes plant debris, spotted, unfilled & chaffy grains, sclerotia, galls, smut balls,
insects etc. It should also be incubated either on blotters or, agar media and examined, after a
standard period of infection.
b. Symptoms: Such as (Discoloration, staining, necrosis, malformation and similar indications of
infections, including fruiting bodies of fungi, resting hyphae on the seed-surface, spore or bacterial
masses on the seed as well as mechanical damage.
Seed samples were examined first by naked eye and the observed under a stereobinocular microscope
for confirmation of the above, impurities in, proper way. The inert matters need to be incubated for the
detection of the pathogens associated with these:
Advantages/Usefulness of dry inspection
1. Dry inspection method provides quick information.
2. It does not require much equipment.
3. It helps to take first hand measure
Disadvantages/Limitations of dry inspection
1. Only those diseases giving external, symptoms and signs can be detected by this method.
2. Information pertaining to viability of the seeds cannot be obtained.
3. It is not more reliable because badly infected seeds may look healthy.
The following samples were Supplied letr dry inspection
l. Discolored and spotted rice grains: the spikelets are dark brownish in color, upper surface showing
black velvety appearancxe with brown spots. Some spots are spindle or eye shaped.
a. Associated pathogens:
Bipolaris oryzae
Pyricularia oryzae
Curvularia oryzae

5. Curvularia lunata
Cladosporium oryzae
Nigrospora oryzae
b. Brick pieces
2. Black point of wheat: The embryonic point of wheat seed become black and the brains are also
shriveled.
The main causal organism associated:
Bipolaris sorokiniana
Other organisms may associate as saprophytes:
Alternaria tenuis
Curvularia spp.
Fusarium spp.
3. Discolored and shriveled light weight jute seeds: Due to attack of several pathogens, the seeds
are discolored and shriveled.
Causal organisms:
Macrophomina phaseolina
Botryodiplodia theohromae
Colletotrichum corchori
4. Discolored and shriveled seeds of mustard: Due to attack of seed-borne pathogens, the seeds
become discolored and shriveled.
Causal organisms:
Alternaria brassicae
Alternaria brassicicola
Aspergillus spp.
Penicillium spp.
Phoma lingam
5. Purple stained soybean seeds:
Purple stain of soybean: The diseased seeds became purple in color in one side and other side is
normal.
Causal organism associated: Cercospora kikuchi
6. Discolored and shriveled okra seeds: Dark brown in color rather than normal healthy seed and

6. most of the seeds arc shriveled.
Causal organisms may associate:
Colletotrichum dematium
Macrophomina pharseolina
Aspergillus spp.
Penicillium spp.
Fusarium spp.
7. Discolored and shriveled seeds of country bean
The diseased seeds become discolored and shriveled. Causal organisms may associate:
Colletotrichum lindemuthianum
Uromyces phaseoli
Cercospora spp.
Aspergillus spp.
Alternaria spp.
Fusarium spp.
Xanthomonus campestris pv. phaseoli
8. Discolored and shriveled seeds of mungbean:
Causal organisms may associate:
Colletotrichum dematium Mungbean golden mosaic virus
Cercospora canescens Mungbean mosaic virus
Macrophomina pharseolina Mungbean mosaic virus
Botryodiplodia theohromae
Fusurium spp.
Phoma spp.
9. Discolored seeds of white gourd
Pathogens may associate:
Colletotrichum spp.
Aspergillus spp.
Alternaria spp.
10. Discoloured seeds of sweet gourd
Pathogens may associate:

7. Colletotrichum spp.
Aspergillus spp.
Alternaria spp
Blotter method/test
The blotter test is a combination of the in vitro and the in vivo principles of investigation. In this method,
the seeds are sown in petridishes other suitable containers on moistened absorbent blotting paper,
usually three layers to provide enough moisture for duration of the test.
Materials required
1. Plastic petridishes 2. Blotting paper 3. Seeds
4. Sterilized water 5. Forceps 6. Stereoscopic microscope
7. Compound microscope 8. Cotton 9. Spirit
10. Mounting fluid, slide and cover slip etc.
Procedures
1. Required number of plastic petridishes was taken.
2. The plates/petridishes were then rinsed with methylated spirit and were dried.
3. The petridishes were left for a while to allow the spirit to be given off.
4. Required numbers of blotting papers were soaked in sterilized water and three sterilized
blotting papers were then placed on each petridish.
5. Then 400 seeds were taken randomly from a working sample.
Surface sterilization of seed lot: Then the seed lot or working sample were sterilized with 0.001%
HgCI2 solution or 10% Clorox solution for detection of internal seed-borne pathogens. For this, the seed
lot was soaked into the 10% Clorox solution in a petridish with repeated shaking for about one minute
and then the chemical solution were drained out from the petridish carefully. The seed lot was washed
with sterilized distilled water for 3 times to remove the chemical from the seed.
6. 5 or 10 or 25 seeds (with and without surface sterilized) were placed on the wet blotting papers kept
at the bottom per petridish or pyrex glass petridish or earthen dish (depending on the size of the seeds)
maintaining equal distance among the seeds.
7. Then the petridishes were kept into an incubation chamber at 20"C ± 2"C in 1 2/ 12 hours alternating

8. cycles under ultraviolet (UV) light and darkness for 7 days.
Observation
After a week, incubated petridishes containing seedlings and non-gerininated seeds were carefully
observed under stereobinocular microscope. The identification and the frequency of different category
of fungi associated with the seeds were performed by observing the color, growth habit and
morphological features. The data were recorded as shown in the following table
Table: Blotter method of Seed Health Testing
Nature of %Germ % Pre- % Post No. of Pathogen(s)
disinfection ination emergence emergence colonies associated
death death
Without surface
sterilized
With surface sterilized
The percentage of healthy seed in the sample tested=
% Germination- (% pre-emergence death + % post emergence death)
Remarks:
From the above results, it was observed that --------% healthy seed was present in the seed lot. So the
health status of the seed sample is good/poor. Therefore, the tested seed sample can be/ can not be
suggested for sowing at farmer’s level.
Advantages/Usefulness of blotter test
1. Pathogens can be detected quickly by observing their growth characters.
2. It is economic
3. Pathogenic potentials of the associated fungi can be determined by observing the signs and
symptoms produced by them on the germinating seeds and seedlings.
4. It can be applied for detecting wide range of fungal pathogens from all different kinds of seeds.
5. Results obtained by it, is more reliable because it is the combination of in vitro and in vivo.
6. Blotter method is widely used while agar plate method is impracticable.
Disadvantages/Limitations of Blotter method
1. Examination may be hampered due to the fast growth of certain Fungi over the slow growing
ones.

9. 2. Pathogenic bacteria can not be detected.
3. It is time consuming.
4. Pathogenicity cannot be detected.
5. Symptoms may not be detected.
Earthen dish newsprint method
This method is, in fact, the modification of f3lc3ttcr microflora of seed health testing (in this method
earthen dish and old newspaper are used in stead of petridish and filter papers, respectively.
Procedures:
1. The earthen dish was washed and the newspaper was then placed in tile centre of the dish.
2. Then 5 or 10 or 25 seeds (depending on the sire of the seeds) were placed on the wet newsprint
papers kept the bottom earthen dish maintaining equal distance among the seeds.
3. Then the dishes were covered with thin polythene sheet and were incubated in the incubation
chamber for at least seven days (depending on the crop species) .
4. Finally the data on the prevalence of seed borne pathogens, per cent infected seed, per cent pre
and post emergence death and per cent germination were recorded.
Agar plate method
This method is the modification of blotter incubation method. In this method, nutrient agar, malt extract
agar or potato dextrose agar (PDA) are used in place of blotting papers. But generally PDA is used for
the growth of fungal pathogens and Nutrient agar is used for bacteria.
Materials required
1. Seeds sample 2. Medium: PDA, NA etc. 3. Stereobinocular microscope
4. Compound microscope 5. Petridish 6. Needle
7. Forceps 8. Sterile water 9. Cotton
10. Spirit 11. Marker 12. Beaker
13. Mounting fluid, Slide and cover glass etc.
Procedure

10. 1. Required numbers of petridishes were taken.
2. The petridishes were then sterilized with methylated spirit and were dried.
3. The petridishes were left for while to allow the spirit to be given off
4. 400 seeds were taken randomly from a working sample.
5. Seeds were surface sterilized with 10% Clorox solution or 0.001% HgCI2 for 1 minute.
6. Then 5 or 10 or 25 seeds (depending on the sized) with and without surface sterilized were
placed on PDA containing petridish with the help of forceps.
7. Then the petridishes were incubated at 20i2"C for an incubation period of from 5 to 7 days
with 12 hours darkness alternating with 12 hours NUV light.
Observation
After incubation, the incubated petridishes containing seedling and non-germinated seeds were
carefully observed macroscopically by naked eyes. The identification and the frequency of different
category of fungi associated with seeds were performed by observing the color, growth habit and
morphological features. The whole mounts were also prepared and observed under a compound
microscope for the identification of different fungal species associated with the seeds. The data were
recorded as shown in the following table.
Table: Agar plate method of Seed Health Testing
Nature of %Germination % Pre- % Post No. of Pathogen(s)
disinfection emergence emergence colonies associated
death death
Without surface
sterilized

11. With surface sterilized
The percentage of healthy seed in the sample tested=
% Germination- (% pre-emergence death + % post emergence death)
Remarks
From the above results, it was observed that ---------------% healthy seed was present in the seed lot.
So the health status of the seed sample is good/pour. Therefore, the tested seed sample can be/ can
not be suggested for sowing at farmer
Advantages/Usefulness of Agar plate method
1. This method is useful only for selective pathogens. .
2. Fruiting structures of fungi can develop quickly on agar.
3. The fungi that not identified by blotter method, can easily be identified by agar plate method.
4. Pathogenic bacteria can be detected by this method.
5. Agar plate method provides adequate conditions for the mycelial growth, sporulation and
symptoms on seedlings and seeds than blotter method.
Disadvantages
1. The method is costly, laborious and time consuming.
2. Slow-growing fungi can be suppressed by the rapidly growing ones.
3. Pathogenic potentials of the associated organisms cannot be determined as no disease
symptoms are produced oil the poorly germinated seeds and seedling on agar.
4. The analysts need experience to perform the test.
Growing-on test
Certain categories of seed-borne pathogens require a longer period of incubation to be detected than is
provided by the usual incubation procedures such as the blotter method, tile agar plate method and the
seedling symptom tests. This test is highly applicable for bacterial and viral pathogens. Also systemic
inflections of fungi may be detected in growing on .test.
Conditions:
1. The seeds are to be sown in sterilized soil in suitable containers and 'subjec.ted to suitable
conditions of temperature (21-30°C) and humidity.
2. The containers (Earthen pot; polythene bags, plastic tray etc.) must be kept in a glasshouse or
controlled environment cabinet (insect proof) protected against contamination.

12. Procedure
1. The pots or trays were tilled with sterilized soil/sand/saw dusts.
2. Then 25 or 100 seeds were sown in each pot/tray depending on the size of 'pot or tray.
3. The pots or trays were then placed in greenhouse/glasshouse at 21- 23% temperature for 2 to
3 weeks to allow the development of symptoms in plants. -
Observation
5. The seedlings/plants were examined for disease symptoms produced.
6. The number of infected seedlings was counted and expressed in percentage.
Advantages
1. It is of great importance in quarantine control as exemplified in the import inspection of seed
samples for breeding purposes.
2. The seed harvested from the plants grown under this test can be retested for health in the
laboratory before they are released for final surveillance under field conditions.
3. It is only method for detection of fungi, bacteria and viruses where other methods of seed
health testing are not available.
Limitations
1. The importance of growing-on tests is underestimated and the procedure is little used in
routine testing because it does require considerable space, greenhouse facilities or
environmental controlled rooms and also more time than laboratory procedures.
2. Growing-on test must be adopted when certain organisms are not detectable by available
laboratory procedures.
3. It does not provide reliable information because many seed transmitted pathogens may be
carried in a symptom less manner or may only produce vague symptoms.