• Extract leaf sap of the plants to be tested and inoculate it on the leaves of an indicator plant.

•  Wash the excessive sap from the inoculated leaves and keep the test plants under observation in the glass house.

•  Observe symptoms on inoculated and newly developed leaves 5 to 6 days after inoculation.

•  Indicator plants produce visible symptoms, like necrotic lesions, spots, venial chlorosis/necrosis, vein banding, vein clearing and even systemic mosaic.

• Symptoms developed on the inoculated or newly developed leaves indicate the presence of virus.

• This is another method for virus detection.

•  Graft a succulent stem or a bud (scion) of plant to be tested through wedge/bud/inarching graft method on a healthy plant (stock).

•  In graft, tissue of diseased and healthy plant comes in contact and union takes place. If the virus is transmitted then the young growing leaves/branch of the grafted indicator plant (stock) will show visible viral symptoms.

• The insects like aphids, whiteflies, thrips etc. are known to be vector for transmission of viral diseases.
• These healthy insects are allowed to feed on diseased plant.
• After feeding these insects should be released on healthy test plants for feeding.
• Kill the insects with insecticide after specified inoculation time. Protect inoculated plants from outside infection by putting them in insect proof glasshouse/ nethouse or cages.
• Test plants developing symptoms after 5 to 10 days or later are indicative of presence of viruses.

• In this test, a thin section from the nodal region of a stem of the test plant is treated with Rhodamin B or 1 per cent of the Phloroglucinol-HCl for 1 minute and is examined under simple microscope. The orange /red colour of the primary phloem where accumulation of lignin has occurred indicates the presence of potato leaf roll virus (PLRV).

• Varieties of serological/immunological techniques are available for viral diagnosis. In potato seed production, techniques like chloroplast agglutination, enzyme-linked immuno-sorbent assay and immuno electron microscopy are used.

• It involves the antigen antibody interaction in liquid phase. The virus antigen occurs in association with chloroplast and when antibody homologous to virus is added to the plant sap containing virus, antibody binds with virus particles which results in agglutination that can be seen readily with naked eye. However, this technique is less sensitive and less reliable.

• It employs catching of virus particles from virus extract and anti serum coated electron microscope grid. The presence of antibody on the grid promotes binding of the homologous virus particles but it prevents non-specific binding of other host proteins. As a result, a sort of immunopurification of virus on the grid, if the trapped virus particles are again made to react with homologous antiserum, antibodies bind to virus particles which results in the formation of clumps of virus particles due to cross-linking and are readily seen under electron microscope.

Enzyme-linked immunosorbent assay (ELISA) is a very sensitive serological test that determines the presence of an antigen (e.g. a plant virus) by means of an enzymatic reaction. It is based on the fact that yglobulin can be linked to an enzyme without losing its immunological properties and enzyme activity. Usually y-globulin is first bound to the solid phase (polystyrene plate or even nitrocellulose membrane) on which the sample is 'trapped', i.e. by an antibody linked with an enzyme. It is suitable for testing a large number of samples at a time without requiring large quantities of expensive antisera and chemicals. When enzyme is linked to the antibody (primary antibody) for ELISA, the test is termed as “Direct ELISA” but if it is linked to another molecule, which detects the primary antibody, the test is called “Indirect ELISA”. Commonly used ELISA methods are: double antibody sandwich ELISA (das- ELISA); direct antigen coating ELISA (dac-ELISA); and indirect or triple antibody ELISA.

• a. Materials required: ELISA micro-titre plates; Multichannel volume adjustable pipettes (50-1200 μl); Plastic wares and glass wares; Minifuge; pH meter; Magnetic stirrer; ELISA reader; Wash bottles.
• b. Reagents: Purified y-globulin (IgG); alkaline phosphataselabelled y-globulin (IgG EC); p-nitrophenyl phosphate (pNPP).
• c. Sampling of leaves
  • During tuber indexing, plants from eye plugs attain testing stage (six leaf stage) after six weeks of planting.
  • Field testing should be completed well before they touch each other.
  • Collect the leaf samples from the lower, middle and top portion of the plant. The reason for taking sample from different parts is to avoid chances of escape of viruses in testing as the location and concentration of some viruses vary at different position of leaves
• d. Protocol for Double Antibody Sandwich ELISA (DASELISA)
  • The test is performed in micro-titre/ELISA plates. To each well add 100/200 μl of purified-globulin (diluted with coating buffer) and incubate in a refrigerator (4 °C) overnight* or 37 °C for 2-3 h.
  • Remove y-globulin solution and wash the plates with washing buffer 3-4 times, by incubating 4-5 min at every time.
  • Add 100/200 μl of plant extract to each well and incubate in a refrigerator, overnight or 37°C for 2-3 h.
  • Remove plant extracts and wash plates as described earlier.
  • Add 100/200 μl of enzyme conjugate to each well.
  • Incubate 3h at 37°C or overnight in refrigerator (4°C).
  • Remove conjugate and wash plates carefully as above.
  • To each well add 100/200 μl of enzyme substrate solution (@ 0.6 mg** of pNPP per ml of substrate buffer). Enzyme substrate should be used fresh.
  • Incubate at room temperature for 30 to 40 min.
  • Read the plate by using a ELISA reader at 405 nm.
  • However, within one experiment conditions should be the same.

    *Incubation with enzyme conjugate can even be done in a refrigerator for overnight.

    ** IMPORTANT: This is not a safe chemical-avoid skin contact. The simplest procedure to wash the plates is to strike out the contents above the sink and washing them carefully and thoroughly with a gentle flow of tap water, followed by rinsing with distilled water containing 0.05% Tween-20 or with PBST. The tap water should be clean.

• e. Troubleshooting

  During ELISA test, number of problems is encountered due to faulty procedure. Though the ELISA technique is very easy and simple yet it needs precision during conducting the test.

  • Plates: Depending on the manufacturing qualities of the plates, signals of varying levels may be obtained with microtitre plates. An important criterion for the assessment of the suitability of a plate for potato virus enzyme immunoassay is a low "background" signal (405nm < 0.1) for the virus-negative (healthy) samples.

  • Border row effect: Covering of plates tightly with parafilm/covers and placing them in a moist chamber during incubation will help in reducing the border-row-effect.

  • Washing the plate: For washing low number of plates squeezing bottle is enough, if the number of plate is more we can go for ELISA plate washer. Manual washing should be done carefully, empty the wells by quickly turning over the plate to prevent overflow into other wells and tap the plate onto absorbent tissue/towel before filling next time with washing buffer. Repeat washing 4-5 times.

  • Drying of test wells between steps: Do not let ELISA plate wells empty for a long time. Either keep the wells filled with washing buffer or place it upside down on a wet towel. During prolonged work on plates, a partial covering of the plate can prevent loss of activity and irregular results.

   

Molecular diagnostic techniques target the genomic nucleic acid of virus or viriod. The nucleic acid is detected either through nucleic acid spot hybridization (NASH) or through the detection of amplified DNA by polymerase chain reaction (PCR). Majority of potato viruses posses RNA genome therefore, it is first converted to complimentary DNA  (cDNA) which is then amplified through PCR and this process is called as reverse transcriptase polymerase chain reaction (RT-PCR).

• Nucleic acid spot hybridization (NASH)

  In this technique, a labeled complimentary nucleic acid probe is made to hybridize with the target nucleic acid. It is routinely used to detect potato spindle tuber viriod (PSTVd). DNA/RNA extracted from sample is spotted on nitrocellulose membrane, which is made to hybridize with labeled nucleic acid probe. Depending on the label it is autoradiograph (in case of radio labeled probe) or chemiluminiscence or a colour reaction is done for detection.

• Polymerase Chain Reaction

  It is employed to amplify the specific DNA or RNA after obtaining complimentary DNA. The amplified DNA or cDNA by PCR is detected through gel electrophoresis after staining with ethidium bromide.