The following points highlight the top four techniques for detection of seed borne viruses. The techniques are: 1. Dry Examination 2. Biological Methods 3. Biochemical Method 4. Serological Methods.
Technique # 1. Dry Examination:
A close inspection of the see may give an indication of the presence of virus in certain seed.
Phatak (1974) found that in 13 genera no less than 16 seed borne viruses may leave a clue of their presence in the seed. e.g.:
1. Seeds of Allium cepa shows reduced in size due to onion yellow dwarf virus.
2. Seeds of Cucumis melo are poorly filled and deformed due to squash mosaic virus.
3. Seeds of Lupinus luteus are large in size due to Cucumber mosaic virus.
Dry examination of any seed sample is followed by Biological methods, Biochemical method. Serological methods and Biophysical methods.
Technique # 2. Biological Methods:
This method requires expensive green house facilities.
(a) Grow-on Test:
This test is also performed for detection of bean mosaic virus (BCMV) in bean seedlings. The major advantage of this method is the possible development of symptoms in the plants. In case of BCMV the seed borne infection is expressed on seedlings developed from infected seed up to third-trifoliate stage.
Procedure:
1. Count the seed and treat with a pinch of commonly used fungicides.
2. Fill the pots with soil and water until saturated.
3. Place the seeds on the top of wet soil and cover the soil. Do not water additionally.
4. Place the seeded pots under vector-free and optimal growth conditions.
5. Examine and record the symptoms 14 and 20 days after seeding.
6. If possible, confirm the virus infection by electron microscopy.
7. Follow the test by indicator plant or serology test.
8. The data is recorded.
The mosaic symptoms of the disease appear on developing young seedling. The systemic infection is expressed up to third trifoliate stage. This test is good and gives quick results of prevalence of virus in seed by developing disease symptoms on seedlings.
This test is usually done for seed transmitted viruses for routine testing but the test should be followed by indicator plant or serology test. The test is greatly influenced environmental conditions especially light and temperatures and requires vector-free conditions to avoid virus infection other than from the seed.
(b) Infectivity Test:
The test is most commonly performed for detection of seed borne viruses. Species of family Leguminosae and Cucurbitaceae are most commonly used indicator plants which react with large number of viruses.
Procedure:
1. Sow or transplant the seeds or seedlings of indicator plants for grow on test. Transplanting of indicator plants is done the same day of seeding of test plant. They are kept under vector-free and optimal growth conditions.
2. Prepare the inoculum by grinding the test plant (both young and old leaves) or seeds in water or a suitable buffer. (0.05 M phosphate buffer, pH 7). If the test sample is green material, the proportion of plant tissue/diluents should be approximately 1/3 (W/V). If it is seed (overnight soaked), the suspension should be like a thin paste.
3. Dust the indicator plant uniformly with carborundum or celite powder (300-600 mesh) so that a hardly visible layer of powder on the leaves is provided.
4. Apply the inoculum with the help of cotton swap by gentle on-way strokes. Inoculate the entire surface of the leaf but do not rub on the same spot. Include control with diluents only.
5. Rinse the inoculated leaves with tap water.
6. Keep the plants under vector-free and optimal growth conditions.
7. Examine for local and systemic symptoms after about one and two weeks, and after two or three weeks, respectively.
Technique # 3. Biochemical Method:
Phatak (1974) demonstrated viral inclusion by staining infected tissue with 1% aqueous phloxine solution. This method is based on the fact that viral infections cause biochemical and Physiological changes in host tissues.
Technique # 4. Serological Methods:
Serological tests are based on in vitro reactions between antigens and antibodies, here the antibodies unite only with corresponding antigen (virus- protein) and the union is detected as precipitation, agglutination etc. depending on the test employed.
The blood serum containing proteinaccous antibodies which are specific against a particular virus is termed as antiserum and is obtained by repeatedly injecting laboratory animals (rabbit, mouse, sheep, goat, etc.) with pure virus preparation.
(a) Gel Diffusion Techniques:
Macromolecules of viral protein antigens and corresponding antibodies can react with in agar substrate which is permeable to their diffusion.
1. Single Diffusion Test:
In this test the antiserum is earlier mixed with the agar before it solidifies. The antigen (virus) has to diffuse and react with antibodies present throughout the agar.
2. Double Diffusion Test:
In the test both reactants have to diffuse through agar from separate depots, Phatak (1974) employed this method for detection of Tobacco Mosaic virus (TMV) in tomato seed.
(b) Agglutination Technique:
This technique is based on reaction between molecular antigen such as viruses and corresponding antibodies which can be enhanced visibly by making use of carrier particles i.e., chloroplast (present along with virus particles in extract from infected leaf), Synthetic polystyrene latex, ion-exchange resins etc.
For testing, the carrier particles are mixed with test extract from seed or seedlings. These particles clump (agglutinate) if the serological reaction is possible.
1. Chloroplast Test:
Paper disc are pre-soaked with the virus antiserum. When extracts of germinated seed are placed in drops over the disc, clumping of chloroplasts indicate presence of virus.
2. Latex Test:
Phatak (1974) used latex agglutination test for detecting BCMV in bean and bean yellow mosaic virus in broad bean. He found this technique as one of the most promising for routine work. Biologically immune carrier particles e.g. synthetic polystyrene latex balls can be used to eliminate non specificity of reaction frequently met with in agglutination systems using organic or inorganic carrier particles.
Technique # 5. Biophysical Method:
The presence of virus in seed can be detected directly by Electron microscopy or indirectly by radiography of seed with X-rays.
(а) Electron Microscopy:
An electron microscope can be used in seed health testing for viruses. Gold (1954) observed particles of Barley stripe mosaic virus in individual seed of barley. It is difficult to identify the virus specifically, since several viruses belong to the same morphological class. This difficult can be eliminated by using Immuno Sorbent Election Microscopy (ISEM), A combination of serology and electron microscopy.
(b) Contact Radiography with “Soft” X-rays:
On the basis of decreased opacity to X -ray penetration it was possible to distinguish barley seeds infected with BSMV and runner bean (Phaseolus multiflorus) seeds inflected with Runner bean mosaic viruses.