Lesson 8: Diagnosing Plant Viral Infections

Diagnosing plant viral infections is a crucial step in managing plant health and preventing the spread of viruses within crops. Plant virus diagnosis involves observing symptoms, using specialized diagnostic tools, and confirming the presence of the virus in plant tissues.

1. Visual Symptom Observation

The first step in diagnosing a plant viral infection is to observe and document the symptoms. Although symptoms alone can sometimes suggest a viral infection, they are not always definitive, as other factors (e.g., nutrient deficiencies, environmental stress, fungal or bacterial infections) can cause similar symptoms.

Common Viral Symptoms to Look For:

• Mosaic or Mottling Patterns: Light and dark patches on leaves.
• Yellowing and Chlorosis: Discoloration, often along leaf veins or in patches.
• Necrosis: Dead spots on leaves, stems, or fruits.
• Distorted Growth: Stunting, leaf curling, and deformation of leaves or stems.
• Abnormalities in Flowers and Fruits: Discolored or misshapen flowers and fruits.
• Wilting or Dieback: In some cases, viral infection can cause wilting or death of plant parts.

2. Sampling Plant Tissue

Once symptoms are observed, samples need to be collected for further diagnostic testing. Proper sampling is essential for accurate results.

• Leaf Samples: Collect symptomatic leaves, ensuring they are not dried or damaged. Young, infected leaves often contain higher virus concentrations.
• Other Plant Parts: In some cases, stems, flowers, or fruits may be sampled if symptoms are visible in those areas.
• Handling and Storage: Samples should be handled with gloves, placed in plastic bags, and kept cool until testing. Avoid exposing samples to high temperatures or prolonged sunlight, as this may degrade viral RNA or DNA.

3. Laboratory Diagnostic Techniques

There are several laboratory techniques available for detecting and identifying plant viruses:

a. Serological Tests

Serological tests use antibodies specific to plant viruses to detect the presence of viral antigens in plant tissues. Common serological methods include:

• Enzyme-Linked Immunosorbent Assay (ELISA): This is the most widely used test for plant viruses due to its simplicity, cost-effectiveness, and reliability.
  • Procedure: Plant sap is extracted and placed in wells coated with antibodies specific to the virus. If the virus is present, it binds to the antibody, resulting in a color change.
  • Advantages: ELISA is rapid, highly sensitive, and can detect multiple viruses simultaneously.
• Immunostrip Tests: Lateral flow devices (similar to pregnancy tests) provide on-site testing.
  • Procedure: A sample (leaf sap) is placed on a test strip that reacts with antibodies for specific viruses, providing a quick visual result.
  • Advantages: Ideal for field use, as it provides immediate results without specialized equipment.

b. Molecular Diagnostic Techniques

Molecular methods target the nucleic acids (RNA or DNA) of the virus, providing highly accurate identification.

• Polymerase Chain Reaction (PCR): PCR and its variants (RT-PCR for RNA viruses) are used to amplify specific viral DNA or RNA sequences, enabling detection of even small amounts of virus.
  • Procedure: Viral nucleic acids are extracted from plant tissue, then amplified using virus-specific primers. Results are visualized on a gel or through fluorescence.
  • Advantages: Highly sensitive and specific; can detect very low concentrations of virus.
• Quantitative PCR (qPCR): Allows for real-time detection and quantification of viral load.
• Loop-Mediated Isothermal Amplification (LAMP): A newer technique that amplifies viral DNA or RNA at a constant temperature, allowing for rapid diagnosis in the field without the need for thermal cycling.
  • Advantages: Quick, reliable, and does not require sophisticated equipment.

c. Next-Generation Sequencing (NGS)

NGS allows for comprehensive detection of all viruses present in a plant sample by sequencing the entire genome or transcriptome.

• Procedure: Plant RNA or DNA is extracted and sequenced, and bioinformatics tools are used to identify viral sequences.
• Advantages: Detects both known and novel viruses and can identify mixed infections. NGS is increasingly used in viral surveillance and epidemiology studies.

d. Electron Microscopy

Electron microscopy allows for the visualization of virus particles and can help confirm infection by detecting virions.

• Procedure: Plant tissue is prepared and examined under an electron microscope to identify virus particles based on their characteristic shapes (e.g., rod-shaped, spherical).
• Advantages: Useful for identifying virus morphology, especially for novel or unknown viruses, but requires specialized equipment and expertise.

4. Confirmation and Cross-Verification

For a robust diagnosis, it is best to use multiple methods to confirm the presence of the virus. For example, an ELISA test could be followed up with RT-PCR to confirm the virus type, especially if more than one virus is suspected or if visual symptoms are ambiguous.

5. Diagnostic Considerations for Specific Viruses

Each plant virus has unique traits that may guide diagnostic choices:

• RNA Viruses: RT-PCR is commonly used for RNA viruses like Tobacco mosaic virus (TMV) and Potato virus Y (PVY).
• DNA Viruses: PCR and LAMP are ideal for DNA viruses like Tomato yellow leaf curl virus (TYLCV).
• Mixed Infections: NGS is particularly useful when multiple viruses are suspected, as it can identify all viral sequences in the sample.
• Insect-Transmitted Viruses: Vector testing (e.g., testing aphids or thrips) may be useful for detecting viruses like Barley yellow dwarf virus (BYDV) and Tospoviruses.

6. Field Diagnostics and Early Detection

In-field diagnostics are especially valuable for rapid detection and management, reducing the time lag between infection identification and control measures. Immunostrip tests and portable LAMP kits are increasingly used for early detection in the field.

7. Interpretation and Management Recommendations

Once the viral infection is confirmed, specific management recommendations can be made:

• Remove and Destroy Infected Plants: To prevent further spread, infected plants should be removed and destroyed.
• Vector Control: If the virus is vector-borne, vector control measures (e.g., insecticide applications, netting) should be implemented.
• Use of Resistant Varieties: Whenever available, planting virus-resistant cultivars can prevent future outbreaks.
• Sanitary Practices: Cleaning tools, using virus-free seeds, and practicing crop rotation can help manage viral spread.

Summary Table of Diagnostic Methods