Table of Contents
Screening for Mycoviruses: A Guide to Discovery in Fungal Isolates
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
The discovery of a new virus is like finding a viral needle in a biological haystack. For scientists studying mycoviruses—viruses that infect fungi—the process is a meticulous hunt that combines classic microbiology with cutting-edge molecular techniques. The successful screening for mycoviruses not only expands our fundamental knowledge of virology but also identifies potential candidates for biocontrol agents that could revolutionize agriculture. Drawing from the detailed methodology in Wajeeha Shamsi’s 2020 thesis, this article outlines the step-by-step scientific process used to find and identify novel mycoviruses. We’ll follow the journey from collecting environmental samples to the final genomic sequencing that brings a new virus to light.
Step 1: The Hunt Begins – Sample Collection & Fungal Isolation
The first step in screening for mycoviruses is to gather a diverse pool of potential fungal hosts. The search isn’t confined to a sterile lab; it begins out in the environment. In this research, samples of soil, water, and infected fruits and vegetables at the post-harvest stage were collected from various locations.
Isolating Fungal Spores:
- From Soil and Water: A small amount of a soil sample was dissolved in sterile water and left overnight to create a spore-rich solution. This solution, along with the water samples, was serially diluted and spread on Potato Dextrose Agar (PDA) plates, a common medium for fungal growth.
- From Fruits and Vegetables: Infected portions of produce were surface-sterilized to remove contaminants and then placed directly onto PDA plates.
After several days of incubation, various fungal species appear on the plates. From these, individual colonies are selected and re-cultured on fresh plates to obtain pure fungal isolates—the essential starting material for the screening process. In the study, a total of 120 different fungal isolates were obtained from the environmental samples.
Step 2: The Key Viral Indicator – Finding Double-Stranded RNA (dsRNA)
Many mycovirus families have genomes made of double-stranded RNA (dsRNA), which is uncommon in healthy fungal cells. Therefore, detecting the presence of dsRNA is a primary and highly effective method for the initial screening for mycoviruses.
The process begins with total nucleic acid extraction, where all DNA and RNA are pulled from the fungal mycelia. A key method described involves grinding the fungal tissue in liquid nitrogen and using an extraction buffer to isolate the nucleic acids.
Confirming the dsRNA Nature:
Once the total nucleic acid is extracted, it must be treated to eliminate everything except the target dsRNA.
- DNase I Treatment: This enzyme is used to specifically degrade all contaminating DNA in the sample.
- S1 Nuclease Treatment: This enzyme digests single-stranded nucleic acids (like messenger RNA and single-stranded DNA), leaving only the stable dsRNA behind.
After these treatments, if any nucleic acid remains, it is highly likely to be the dsRNA genome of a mycovirus. In the research, this screening process revealed that 8.3% of the 120 isolates were positive for mycoviral infection.
Step 3: Visualizing the Evidence with Agarose Gel Electrophoresis
How do scientists actually “see” the dsRNA? The answer lies in a fundamental laboratory technique called agarose gel electrophoresis.
The extracted dsRNA samples are loaded into a gel made of agarose. When an electric current is applied, the negatively charged RNA molecules move through the gel toward the positive electrode. Smaller molecules move faster and farther than larger ones. This separates the dsRNA segments by size. The gel is stained with a fluorescent dye (like ethidium bromide) that binds to the RNA, causing it to glow under UV light.
The result is a pattern of distinct bands, with each band representing a dsRNA segment of a specific size. This visualization not only confirms the presence of a mycovirus but can also give clues about its type. For example, the screening revealed different profiles:
- Monosegmented: A single dsRNA band.
- Tetrasegmented: Four distinct dsRNA bands.
- Multisegmented: Multiple bands of varying sizes.
Step 4: Identifying the Host – Molecular Characterization of the Fungus
Finding a virus is only half the story; you also need to know exactly what it’s infecting. To confirm the species of the fungal isolates that tested positive for dsRNA, a molecular identification technique was used. This method focuses on amplifying the Internal Transcribed Spacer (ITS) region of the fungal ribosomal RNA gene.
The ITS region is considered the universal DNA barcode for fungi because it is highly conserved within a species but varies between different species. By amplifying this region using Polymerase Chain Reaction (PCR) and sequencing it, researchers can compare the sequence to a public database (like NCBI) to get a precise species identification. This step confirmed the hosts for the discovered mycoviruses as species like A. alternata, F. oxysporum, and P. oxalicum.
Step 5: Unveiling the Viral Genome with Next-Generation Sequencing (NGS)
Once a mycovirus is detected and its host is identified, the final step is to determine its complete genetic sequence. Next-Generation Sequencing (NGS) is a powerful method that facilitates the sequencing of millions of nucleic acid molecules at once.
The purified dsRNA from an infected isolate is sent for NGS analysis. The process involves constructing a cDNA library from the RNA and then using a high-throughput platform to read the sequence. The resulting data consists of millions of short sequence reads, which are then assembled by computer algorithms into longer “contigs.”
This step in the screening for mycoviruses provides the complete genome sequence, allowing researchers to identify the virus’s open reading frames (genes), predict its proteins, and perform phylogenetic analysis to determine its relationship to other known viruses, ultimately confirming if it is a new species.
Conclusion
The screening for mycoviruses is a systematic and multi-faceted process that bridges field biology with advanced molecular genetics. From collecting diverse fungal isolates in the environment to the precise identification of viral dsRNA and the ultimate sequencing of the viral genome, each step is critical to discovery. This methodical approach, as detailed in the research, is essential for expanding our knowledge of the viral world and for identifying novel mycoviruses that may one day serve as powerful biocontrol agents in our quest for sustainable agriculture.
Source & Citations
- Thesis Title: Molecular and Biological Characterization of Mycovirus Identified from Environmental Fungal Isolates
- Researcher: Wajeeha Shamsi
- Guide (Supervisor): Dr. Muhammad Faraz Bhatti
- https://asab.nust.edu.pkUniversity: Atta-ur-Rahman School of Applied Biosciences, National University of Sciences & Technology (NUST), Islamabad, Pakistan
- Year of Compilation: 2020
- Excerpt Page Numbers: 38, 39, 40, 42, 45, 46, 68, 73, 74, 75, 78
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.
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