28 Jan 2021

Methods to Detect Viruses in Explants

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Virus Overview

The vegetatively propagated plants suffer from multiple virus or virus-like diseases that deteriorate the health status of the plant. You won’t know if the plant is suffering from any viral disease until the viral load increases and symptoms are observed. This problem is extensively observed in perennial woody plants. It’s a major challenge to grow plants free from viral diseases. However, growing healthy plants is only possible by the identification, development, and propagation of virus-free clones.

Before you proceed with the cloning of the plants, it is essential to make sure that your stock plants do not carry any viral diseases. If the stock plant carries any virus that went undetected, then it will affect your entire culture. This will result in loss of money and efforts, both at a large scale. So, it’s essential to perform tests on the source of explants for the presence of any viral strain.

This article discusses three main groups of detection methods that are used to detect the viruses in your source plants. This includes bioassays, molecular, and serological techniques.

Biological Methods to Detect The Virus Presence

Plant viruses are identified by using biological assays that include woody indexing and herbaceous indexing. These methods help to study the health status of the plants to be inoculated under in vitro conditions. With the plant to be inoculated, a plant sensitive to pathogen attacks is used for screening and comparison purposes.

Woody Indexing 

In this method, the indicator plants are kept in a greenhouse for proper growth. When they reach the height of 25-30 cm and diameter of 3-4 mm, chip budding is performed. In this technique, chips of bark are used from the source of inoculum and grafted on to each indicator plant (these are cut back before graft-inoculation). It is essential to prune the top graft to allow the growth and development of young shoots. These young shoots are more likely to show symptoms than the old shoots. Further, maintain the grafted plants in the greenhouse and check for the development or progress of any symptoms for 1-8 months.

Herbaceous Indexing

In this method, fresh leaves or freeze-dried leaves of the source of the explant (being tested for viral presence) are ground in a buffer or water (used for some virus and host species, not all). The buffer facilitates viral transmission by neutralizing the inhibitory effects of polyphenolic compounds on virus infectivity.

The sap collected from the grinding of the leaves is rubbed directly on the leaves of young herbaceous host plants, that were previously dusted with carborundum or diatomite. Then, maintain the indicator herbaceous plants in the greenhouse to monitor the symptoms weekly or more frequently. The symptoms that can be observed after 10-30 days of inoculation include local necrotic lesions on inoculated leaves, mosaics, systemic necrosis, and other symptoms affecting the top shoot or certain leaves.

Serological Methods to Detect The Virus Presence

The specific interactions between the antigen and similar antibodies are used to routinely monitor the sanitary status of the plant materials. The serological methods play a vital role in the diagnosis, detection, and identification of viruses in plants. These methods provide adequate information on the identity of the viruses.

Three famous serological techniques include enzymes linked immunosorbent assay (ELISA), dot immunoblotting assay (DBIA), and tissue blot immunobinding assay (TBIA). The most popular and extensively used technique of all these three is enzymes linked immunosorbent assay (ELISA). It is beneficial over other techniques because it shortens the time needed to get results, simplify detection, and allow the testing of large numbers of samples.

Shop PPM Here!

Molecular Methods to Detect The Virus Presence

This technique detects the presence of viral nucleic acid in the plant materials to be inoculated. Among several molecular techniques used for the detection of the virus, the most popular one is polymerase chain reaction (PCR). Before initiating PCR, a reverse transcription step is performed, as most of the viruses have RNA as the genome. PCR is an effective and sensitive tool that is used to detect viruses in woody plants, where the genetic diversity of the viruses infecting the plants is very high.

The points that should be kept in mind while performing PCR is the designing of primers and templates to be used for the identification purpose. The other modern techniques used over PCR include real-time quantitative PCR (qPCR), multiplex qPCR, and next-generation sequencing (NGS) technologies. The only limitation of the molecular methods is the high cost of the techniques. However, for broad screening of viruses, in the vegetatively propagated plant materials, these are best to use in combination with other techniques.

References

1. Varveri, C., Maliogka, V. I., & Kapari-Isaia, T. (2015). Principles for Supplying Virus-Tested Material. Control of Plant Virus Diseases - Vegetatively-Propagated Crops, 1–32. DOI:10.1016/bs.aivir.2014.10.004
2. Legrand, P. (2015). Biological assays for plant viruses and other graft-transmissible pathogens diagnoses: a review. EPPO Bulletin, 45(2), 240–251. doi:10.1111/epp.12222.
3. Abd El-Aziz MH. Three modern serological methods to detect plant viruses. J Plant Sci Phytopathol. 2019; 3: 101-106.