Table of Contents
Mycovirus Transmission Methods: Overcoming Barriers in Fungal Virology
Last Updated: July 29, 2025
Author Bio: This research summary is based on the doctoral thesis of Wajeeha Shamsi, a researcher from the Atta-ur-Rahman School of Applied Biosciences at the National University of Sciences & Technology (NUST), Islamabad. Her work focuses on the identification and characterization of novel mycoviruses from environmental sources.
Introduction
To truly understand the impact of a virus on its host, scientists need a control group—an uninfected version of the same organism. In the world of mycology, this means creating “virus-free isogenic lines” of a fungus. This is easier said than done, as mycoviruses are notoriously difficult to manipulate. They don’t typically exist outside the cell, making conventional infection and curing protocols ineffective. A significant portion of Wajeeha Shamsi’s 2020 thesis was dedicated to this very challenge. This article explores the various mycovirus transmission methods and curing techniques she employed, revealing the scientific hurdles and the ultimate breakthrough that made her research possible.
The First Challenge: Curing the Fungus of its Virus
The initial goal was to create a virus-free version of the Alternaria alternata strain (4a) that was found to be infected with the novel AaBbV1 mycovirus. Several established methods for curing mycoviruses were attempted, each with its own rationale.
Attempt 1: Cycloheximide Treatment
It is noted that the synthesis of dsRNAs in fungi is inhibited by cycloheximide. This drug is a protein translation inhibitor that blocks translation by immobilizing ribosomes. The theory is that by interfering with the host’s protein production, you can also disrupt the machinery the virus needs to replicate.
Different concentrations of cycloheximide were added to the PDA growth media. While the treatment significantly inhibited the fungus’s growth rate, it was ultimately unsuccessful. Even at a high concentration of 150 mM, the drug failed to eliminate the mycovirus, which was still detectable via electrophoretic mobility.
Attempt 2: Single Spore and Hyphal Tipping Methods
If chemical methods fail, mechanical ones are the next logical step. These mycovirus transmission methods work on the principle that the virus might not be evenly distributed throughout the entire fungal colony.
- Single Spore Isolation: Spores from the infected fungus were collected and serially diluted to isolate individual spores. The hope is that some spores, as a consequence of asexual reproduction, might be naturally virus-free.
- Hyphal Tipping: Mycoviruses often spread to the growing tips of the fungal hyphae. This technique is based on the idea that the youngest mycelia at the very tip might not have been reached by the virus yet. Hyphal tips were carefully isolated under a microscope and re-cultured.
Unfortunately, both of these methods also proved unsuccessful. No observable difference was found in the resulting fungal colonies, and the virus infection was persistent. This suggested that the virus might have a high fitness within its host or be distributed ubiquitously throughout the cytosol, making it impossible to isolate a virus-free section.
The Second Challenge: Transmitting the Virus to a New Host
Since all attempts to cure the original fungus failed, the research strategy pivoted. The new approach was to transmit the virus into a known, virus-free strain of A. alternata. This would create the necessary isogenic lines (one infected, one not) to study the virus’s impact.
Attempt 1: Horizontal Transmission via Paired Culture
The most natural of the mycovirus transmission methods is horizontal transmission. This occurs when the hyphae of two different fungal strains fuse (anastomosis), allowing for the exchange of cytoplasmic material, including resident mycoviruses.
A paired culture was set up, with the virus-infected Pakistani strain (4a) and a virus-free Japanese strain (Ally-12) grown side-by-side on a PDA plate. However, even after 15 days of contact, the AaBbV1 virus failed to transmit to the virus-free strain.
The reason for this failure was heterogenic incompatibility. Just like an organ transplant can be rejected, fungal cells can recognize and reject genetically different strains. Because the two strains had different geographical origins, they had an allelic difference at their vegetative incompatibility (vic) loci, which prevented them from fusing and sharing their cytoplasm. Even using zinc chloride, a chemical known to sometimes facilitate fusion between incompatible strains, failed to work.
The Breakthrough: Protoplast Fusion
With natural transmission blocked and chemical curing ineffective, the research turned to a powerful, albeit complex, laboratory technique: protoplast fusion. This is one of the most effective mycovirus transmission methods for bypassing natural biological barriers.
The Protoplast Fusion Process:
- Cell Wall Removal: The tough outer cell walls of both the virus-infected and the virus-free fungal strains are digested using enzymes. This leaves behind the naked cell, known as a protoplast.
- Fusion: The protoplasts from the two strains are mixed in the presence of a chemical like PEG, which encourages their membranes to fuse together.
- Cytoplasmic Mixing: When the protoplasts fuse, their cytoplasmic contents mix. This allows the mycovirus from the infected strain to enter the cytoplasm of the previously virus-free strain.
- Regeneration: The fused cells are placed on a regeneration medium, where they regrow their cell walls and develop into a new hybrid fungal colony.
This approach was a resounding success. Out of 20 subcultures obtained from the protoplast fusion procedure, two were found to be AaBbV1-positive, carrying the same dsRNA profile as the original Pakistani strain. Crucially, these new virus-infected strains were genetically identical to the virus-free Japanese strain, finally creating the isogenic lines needed for the study.
Conclusion
The journey to study the impact of the AaBbV1 mycovirus highlights the immense practical challenges in fungal virology. Standard curing and transmission techniques can often fail due to the intricate biology of the host and virus.
This research demonstrates that while methods like cycloheximide treatment and hyphal tipping have their place, protoplast fusion stands out as a critical and powerful tool. As one of the most reliable mycovirus transmission methods, it allows researchers to bypass natural barriers like vegetative incompatibility, making it an indispensable technique for both fundamental research and the future development of mycoviruses as biocontrol agents.
Source & Citations
- Thesis Title: Molecular and Biological Characterization of Mycovirus Identified from Environmental Fungal Isolates
- Researcher: Wajeeha Shamsi
- Guide (Supervisor): Dr. Muhammad Faraz Bhatti
- University: Atta-ur-Rahman School of Applied Biosciences, National University of Sciences & Technology (NUST), Islamabad, Pakistan
- Year of Compilation: 2020
- Excerpt Page Numbers: 102, 103, 104, 105, 106, 107, 108, 110, 111, 117
Disclaimer: Some sentences have been lightly edited for SEO and readability. For the full, original research, please refer to the complete thesis PDF linked in the section above.
Join the Discussion!
What other advanced laboratory techniques do you think could revolutionize the study of mycoviruses in the future? Share your ideas in the comments section!
Discover more from Professor Of Zoology
Subscribe to get the latest posts sent to your email.
