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, seedborne pathogens may cause disease or death of plants resulting in crop loss.

Objectives
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 disease to new regions and to issue Phytosanitary certificate. Testing to make accurate decisions regarding the appropriate use of seed treatment . Testing for seed certification schemes. Testing for storage quality Testing for resistance of cultivars.

An ideal seed health testing method should have

Methodology required for seed health testing critiria . must fulfill

Specificity : that is the method should the ability to

distinguish 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 to enable test to be performed by simple technicians and not require 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 foreign material. 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 also be detected by this method. Disadvantages: The viability of the spores/ inoculums may not be determined by this method. moreover, the method does not 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 found associated with seeds are carefully examined and identified based on habit characters . Habit character of fungi can only be learnt by with practice . The blotter method is widely used for detecting fungi which are able to produce mycelial growth and fruiting structures under the incubation conditions available in the test. All kinds of 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 two

lamps) 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

Procedure
The seeds are plated on well water soaked filter papers, and incubated usually for 7 days at 22 C0 2C0 under 12 hours alternating cycles of light and darkness. After incubation, fungi developed on each seed are examined under different magnification of a

stereomicroscope and identified .

The identification of fungi is based on the way they grow

on 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 20 0 C for 24 and back to 20 0 2 0 C for further 3-5 days 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 obstruct

observations in standard blotter test. To overcome this, 2, 4-D is used to make the identification of mycoflora easy as it retards seed germination.
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 while weighing 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 colonies are produced . The number can be 3,4 and 5 or even higher depending 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 a compound microscope. Take notes on how the colonies appear when observed with the naked eye from both sides , e.g. color ,colony size , type of mycelium (fluffy, cottony or pressed).

6- Recording infection: Record the counts of the investigated fungi from each dish in 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 growing saprophytic or pathogenic fungi. The agar plate method is expensive and labour consuming and therefore this method only be used when fungi are not easily recorded by blotter method .

 Some of the seed borne fungi are capable of attacking seeds, making them ungerminable resulting in rotting of seeds and in producing symptoms on young seedlings or even killing the affected seedlings. These effects can be seen if seed are sown on suitable substrate and seedling grown under environmental conditions which support expressing of such effects. Some of the common examples of such fungal pathogens causing seedling diseases are species of Alternaria, Ascochyta, Biploris, Fusarium, Colletrotrichum, Macrophominia and Pyricularia.
Seed is sown in autoclaved soil , sand , gravel and similar

materials.

Cont. Infected seeds , under optimal conditions of temperature, light and humidity may develop symptoms comparable with those produced under field conditions. The seedling symptom test ,for certain types of pathogen , will provide information pertaining to field performance of 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.

Serological tests:
This test is generally applied for detection of viral

pathogens.

Identification of suitable method for detecting Incidence of Bipolaris sorokiniana on barley seeds
Method osmotic potato-sucrose-agar (PSA) Reis selective medium Bipolaris sorokiniana incidence 16.50 b 92.75 a 88.50 a

cv.(%)
LSD(%)

7.06
11.87
NADIA et al , 2001

The PSA and RSA has given good results about disease incidence in barley seeds compared to osmotic method. We can use PSA or RSM media for testing.

Identification of suitable method for detecting Incidence of Dreschlera teres on barley seeds
Method
osmotic potato-sucrose-agar (PSA) Reis selective medium cv.(%) LSD(%)

Dreschlera avenae incidence

18.oo a 12.00 b 15.25 a 14.11 5.43
NADIA et al , 2001

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

Testing efficiency of blotter test to detect pathogen Sclerotinia sclerotiorum in soyabean

Naturally infected Artificially inoculated

Days of incubation (Blotter test)

Luciane Henneberg, 2011

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 osmotic potato-sucrose-agar (PSA) Reis selective medium cv.(%) LSD(%) Dreschlera teres incidence 38.oo a 43.75 a 42.00 a 10.60 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 naturally infected commercial cotton (Gossypium hirsutum) seed lots by three different methods
Seed lot and cotton cultivar Fabrika Seed washing Semi selective media 4 (0.20%) Growing on test

Negative Negative Negative

0 0 0
0

Ita 90 A Makina

0 1 (0.05%)
0

Ita 90 B (treated Negative with unknown fungicide) ITA 90 C Positive*** Makina Positive***

21 (1.05%) 20 (1.00%)

No data due to short of seed 31 (1.55%)

Yeshwant et al., 2005

Identification of best media for xanthomonas axonopodis pv malvacearum

Recovery of Xanthomonas axonopodis pv malvacearum on difco nutrient agar (left) and on semi selective agar medium (right) three days after isolation. Yeshwant et al., 2005

Incidence of Bipolaris sorokiniana on wheat seeds detected by three methods of testing

Method

osmotic

Bipolaris sorokiniana incidence 17.50a

potato-sucrose-agar (PSA) 23.75a Reis selective medium cv.(%) 22.75a 17.71

LSD(%)

9.64
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 .