Seed Health Testing in Field Crops Seeds...........Aaaaaaa

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Seed Health Testing Methods



Definition of seed health

In the context of this course, seed health refers to the

presence or absence of disease-causing organisms such

as fungi, nematodes, bacteria, viruses and insects, and to

the status of seeds in a seed lot.

Seed is the basic unit of crop production and therefore,

food production. When seeds are used for sowing, seed-

borne pathogens may cause disease or death of plants

resulting in crop loss.



Seed health testing may carried out basically to control seed

born diseases for the fallowing reasons : Testing for evaluation of planting value in the field .

Testing to assess the prevalence of seed borne infection.

Testing for quarantine purpose to avoid the spread of diseaseto new regions and to issue Phytosanitary certificate.

Testing to make accurate decisions regarding the appropriateuse of seed treatment .

Testing for seed certification schemes. Testing for storage quality

Testing for resistance of cultivars.

Objectives



An ideal seed health testing method should have

Methodology required for seed health testing must fulfill

critiria .

Specificity : that is the method should the ability todistinguish the target pathogen fro all organisms likely to

occur on seeds. Sensitivity : means that the method must be sensitive enough

to detect organism even in the low incidence in seed stocks.

Simplicity : the methodology should be simple so as to

minimize the number of stages to reduce room for error and toenable test to be performed by simple technicians and notrequire high qualified staff.

Cost effectiveness: that the methodology should be cheap.



Dry seed inspection

This is conducted in the Seed Inspection Area to

ensure that seeds are clean and free of foreignmaterial. Initial examinations are done and if live

storage insects are found, seeds are fumigated

immediately. Some significant risks that can be

eliminated through dry seed inspection are: weed seed contaminants

insect pests

seed discoloration



Macro testing After dry seed inspection, the bulk of the seed lot is

submitted to the laboratory for:Macro testing - to detect and identify the infection cause.The various symptoms are:

Seed rot necrosis

Shrunken seeds Mottling, wrinkling and

cracking of seeds

Fructification

Sclerotisation Seed discoloration



Washing test This method is for detecting fungi causing smut

and bunt in graminaceous hosts.Materials required:

Centrifuge(table model)

Compound microscope Test tube

Slides and cover slips

Mechanical shaker

Haemocytometer



Procedure of washing test

Seed is taken in a test tube with 10 ml of water and

shaken for 10 minutes on a mechanical shaker. The suspension is examined as such or the suspended

spores are concentrated by centrifuging at 3000 rpm for

15-20 mts. The supernatant is discarded and the spores are again

suspended 2 ml of lacto phenol .

This suspension is then examined under microscope for

the presence of spores, conidia and other fructifications Haemocytometer is used for semi-quantitative

estimation of spores present on the suspension obtained

from seed washing .



Advantages:

This method is good for quick detection of externally seed

borne mycoflora .Oospores and chlamydospores can alsobe detected by this method.

Disadvantages:The viability of the spores/ inoculums may not be

determined by this method. moreover, the method doesnot help to detect internally seed-borne pathogens.



Incubation methods Blotter and Agar plate methods are two widely used

incubation methods to study seed mycoflora.

Blotter method

Standard Blotter method:

This method was developed by Doyer in 1966. fungi foundassociated with seeds are carefully examined and identified

based on “habit characters” . Habit character of fungi canonly be learnt by with practice .

The blotter method is widely used for detecting fungi whichare able to produce mycelial growth and fruiting structuresunder the incubation conditions available in the test. All kindsof cereals , vegetables ,legumes, ornamentals and forest seeds

are tested by this method.



Facilities and equipment Incubation room Compound microscope(magnification up to X 400)

Stereomicroscope( magnification at least up to x 50 fitted with twolamps) Petri dishes made of clear plastic or pyrex/corning glass (9 cm

diameter) Filter paper (9 cm diameter ) with high water holding

capacity(saturated 3 layers of blotter must hold approximately 40 cc water).

Trays(30 X 60 cm) for holding petri dishes Beaker Forceps

Glass slides and cover slips Face masks Distilled water Alcohol lamp Dissecting needle



ProcedureThe seeds are plated on well water soaked filter papers,

and incubated usually for 7 days at 22 C0

±

2C0

under 12hours alternating cycles of light and darkness.

After incubation, fungi developed on each seed are

examined under different magnification of astereomicroscope and identified .

The identification of fungi is based on the way they growon seeds "habit characters” and on the morphological

characters of fruiting bodies , spores/conidia observed

under a compound microscope.



Deep freeze method

Material required :

All the material and methods required are the same as standard blotter test except deep freezer which is an additional requirement.

After plating the seeds as mentioned in standard blotter test , the

Petri dishes are incubated first at 20 0 C for 48 h, and again brought

back to –

200

C for 24 and back to 200 ±

20

C for further 3-5days under alternate 12 h light and dark periods.

Advantage: fungi like Fusarium and Septoria in cereals, Phoma in

sugar beet and Pyrenophora graminea and P. teres in barley can be detected by this method.

Disadvantage : Overgrowth of saprophytes, example Aspergillus,

Cladosporium and Penicillum is very common on seeds frozen for

24 h and is extremely disturbing during examination of seeds



2, 4-D Method

During incubation seeds germinate and obstructobservations in standard blotter test. To

overcome this, 2, 4-D is used to make the

identification of mycoflora easy as it retards seedgermination.

Instead of water the blotters are soaked in 0.2%solution of 2,4-D. Incubation and other condition

is similar to the standard blotter test



Agar plate method In the agar plate method, generally surface-

disinfected seeds are plated on an agar medium. plated seeds are usually incubated for 5-7 days at 22

– 250 C under 12 h alternating cycles of light and

darkness.At the end of incubation period, fungi growing out

from seeds on the medium are examined and

identified. Identification is based on colony characters and

morphology of sporulating structure under a

compound microscope .



1- Preparation of media and solution

Potato Dextrose Agar medium ( 1 liter)

Peeled potato 200 g

Dextrose 20g

Agar 20g

Distilled water 1 liter

Streptomycin sulfate 1 mg

Since streptomycin sulfate is toxic ,were gloves whileweighing and pouring it into the molten agar medium.

2. Pouring the medium in the plates : Pour the medium in the sterile Petri dishes, approximately

15 ml per dish.

Let the dishes solidify completely before plating seeds.



3- Plating seeds on agar:

4- Incubation of plates:

5- Examination of fungal colonies:

In the agar method more than one type of fungal coloniesare produced . The number can be 3,4 and 5 or even higherdepending on the level of infection present in the seeds.

The identification of the different colonies should be done visually and then under stereomicroscope and followed by an examination of the fruiting structures under acompound microscope.

Take notes on how the colonies appear when observed withthe naked eye from both sides , e.g. color ,colony size , typeof mycelium (fluffy, cottony or pressed).



6- Recording infection:

Record the counts of the investigated fungi from each dishin the working sheet and finally in the seed health report.

Advantages :

The agar plate method provides an efficient tool for quick

identification of specific seed infections.Disadvantages:

The slow growing fungi may be masked by fast growingsaprophytic or pathogenic fungi.

The agar plate method is expensive and labour consumingand therefore this method only be used when fungi arenot easily recorded by blotter method .



Some of the seed borne fungi are capable of attackingseeds, making them ungerminable resulting in rotting

of seeds and in producing symptoms on young seedlingsor even killing the affected seedlings. These effects canbe seen if seed are sown on suitable substrate andseedling grown under environmental conditions whichsupport expressing of such effects. Some of the commonexamples of such fungal pathogens causing seedlingdiseases are species of Alternaria, Ascochyta, Biploris,

Fusarium, Colletrotrichum, Macrophominia andPyricularia.

Seed is sown in autoclaved soil , sand , gravel and similar

materials.



Cont….

Infected seeds , under optimal conditions of temperature, light and humidity may develop symptomscomparable with those produced under field conditions.The seedling symptom test ,for certain types of pathogen , will provide information pertaining to field performanceof the seed lot.

Seedling symptom test can be conducted in the laboratory ,growth chambers and in glass houses using a variety of substrates.



Plant injection test In this method seeds to be tested are soaked in sterile

water for few hours. Then the leachate is injected into a young healthy

seedling and

the plant is then observed for development of disease

symptoms.



Bacteriophage technique : This test is performed by adding phage viruses to

seeds grown on agar media . If bacteria is present a clean plaque area appears

on the agar due to attack on the bacteria by virus.

S l i l



Serological tests:

This test is generally applied for detection of viral

pathogens.



Identification of suitable method for detecting

Incidence of Bipolaris sorokiniana on barley seedsMethod Bipolaris sorokiniana

incidence

osmotic 16.50 b

potato-sucrose-agar (PSA) 92.75 a

Reis selective medium 88.50 a

cv.(%) 7.06

LSD(%) 11.87

NADIA et al , 2001

The PSA and RSA has given good results about disease incidence inbarley seeds compared to osmotic method. We can use PSA or RSMmedia for testing.



Identification of suitable method for detecting

Incidence of Dreschlera teres on barley seeds

Method Dreschlera avenae incidence

osmotic 18.oo a

potato-sucrose-agar (PSA) 12.00 b


cv.(%) 14.11

LSD(%) 5.43

NADIA et al , 2001

The osmotic and RSA media has given good results about diseaseincidence in oats seeds compared to PSA method. We can useosmotic or RSM media for testing.



Testing efficiency of blotter test to detect pathogen

Sclerotinia sclerotiorum in soyabean

Days of incubation (Blotter test)

Luciane Henneberg, 2011

Naturally infected Artificially inoculated

For the detection of white mold in soybean the methods: Blotter Test are recommended .



Identification of suitable method for detecting Incidence

of Dreschlera teres on oat seeds

Method Dreschlera teres incidence

osmotic 38.oo a

potato-sucrose-agar (PSA) 43.75 a


cv.(%) 10.60

LSD(%) 11.66

NADIA et al , 2001

All the methods are on par. Three methods has given almost same readings so we can

go for any of three methods .

Detection of Xanthomonas axonopodis pv malvacearum (Xam) in six



Detection of Xanthomonas axonopodis pv. malvacearum (Xam) in sixnaturally infected commercial cotton (Gossypium hirsutum) seed lotsby three different methods

Seed lot andcotton cultivar

Seed washing Semi selectivemedia

Growing on test

Fabrika Negative 4 (0.20%) 0

Ita 90 A Negative 0 0

Makina Negative 1 (0.05%) 0

Ita 90 B (treated

with unknown

fungicide)

Negative 0 0

ITA 90 C Positive*** 21 (1.05%) No data due to short

of seedMakina Positive*** 20 (1.00%) 31 (1.55%)

Yeshwant et al., 2005

d f f b d f h



Identification of best media for xanthomonas

axonopodis pv malvacearum

Recovery of Xanthomonas axonopodis pv malvacearum on difco nutrient agar (left) and onsemi selective agar medium (right) three days after isolation.

Yeshwant et al., 2005

d f l k h d



Incidence of Bipolaris sorokiniana on wheat seeds

detected by three methods of testing

Method Bipolar is sorokiniana

incidence

osmotic 17.50a

potato-sucrose-agar (PSA) 23.75a

Reis selective medium 22.75a

cv.(%) 17.71

LSD(%) 9.64NADIA et al , 2001

All the methods are on par. Three methods has given almost same readings so we can

go for any of three methods .

Seed Health Testing in Field Crops Seeds...........Aaaaaaa

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