The cannabis industry has been rapidly expanding in recent years, driven by changing legal landscapes and growing consumer demand. However, this burgeoning sector is not immune to the pervasive threat of viruses that can devastate cannabis production.
These microscopic adversaries can infect cannabis plants, leading to reduced yields, compromised quality, and significant economic losses for growers.
In this article, we will explore current molecular tools available to cannabis labs looking to test for viruses in plant material, and important factors to consider when building out a diagnostic lab for cannabis plant health testing.
Unfortunately viroids do not have a protein coat, they are naked circular RNA molecules
Plant viruses
Plant viruses consist of genetic material (RNA or DNA) enclosed in a protein coat, often with additional outer layers, and they lack the cellular machinery found in host cells, relying on infected plant hosts for replication.
Plant viroids are tiny, circular RNA molecules lacking a protein coat, consisting solely of a short, single-stranded RNA with a characteristic secondary structure that allows them to infect plants and cause diseases.
Hop Latent Viroid (HLVd) is the most prevalent viroid on cannabis and is a major concern to growers due to its association with flower dudding and reduced yields.
Diagnostic products
Molecular tools are essential for detecting plant viruses. In many cases it is impossible to diagnose a virus infection in a plant without molecular confirmation, the main reason for this is that many viral symptoms (visible changes to the plant) can be confused with abiotic issues such as nutrient, water, and environmental stress.
Antibody-based detection assays: These tests usually take the form of a lateral test strip or an ELISA plate assay. These types of tests are able to detect the presence of the virus when the virus protein interacts with a complementary antibody in the test triggering a chemical reaction that results in a visual band on a lateral flow test or an absorbance (colour intensity) change in an ELISA based test.
These tests are relatively accurate, require a simple sample prep procedure (grinding sample in a buffer), and are usually cheaper per sample than nucleic acid-based assays. Unfortunately, viroids do not have a protein coat, they are naked circular RNA molecules, thus the only methods that can be used for viroid detection are nucleic acids based test kits.
Nucleic acid-based detection assays: Nucleic acid-based detection assays come in a variety of forms including: isothermal lamp-based assays, qPCR assays, traditional PCR, and RT-PCR microarrays. Each method has benefits and drawbacks, in general, nucleic acid-based assays require more sophisticated equipment to operate, thus incurring high capital costs. Equipment includes: Real-time PCR machines, microarray plate readers, thermocyclers, and isothermal blocks with the associated reader.
All of these assays first convert RNA to DNA with an enzyme called reverse transcriptase (as most plant viruses and all viroids are RNA based).
The DNA is then amplified with polymerase enzymes and special fluorescent dye interactions occur resulting in a signal that indicates the replication of the virus or viroid of interest. In general qPCR and RT-PCR microarrays have higher sensitivity than isothermal LAMP-based assays, however sensitivity is dependent on many factors discussed below.
It has been reported that HLVd is more likely to be detected in root tissue
Factors that can impact the accuracy of your plant virus diagnostic results
Viral titres: Viral titres refers to the amount of viral particles that are present in the sample you are testing. If a virus infection recently occurred in a plant it can take time for the number of virus particles in the plant to reach a detectable level. This is one of the reasons it is important to have a regular testing program for your grow operation.
Nucleic acid extraction methodology: (How are you extracting the genomic material of the virus out of your sample?): One of the most critical and often overlooked steps in nucleic acid-based detection assays is the methodology used to extract the nucleic acids (RNA/DNA) from the living plant tissue. RNA is a very unstable molecule and it is essential to extract it with methods that result in high quality RNA. The best extraction methods rely on liquid-phase organic solvent extraction such as CTAB (Cationic detergent cetyltrimethylammonium bromide). This method requires a significant amount of time and technical expertise. The next best method are silica-based column extraction kits; these kits contain necessary reagents, spin tubes and simple protocol to extract quality RNA from your plant sample and ensure the best chance of detecting a virus, though they can be more expensive per sample. The cheapest but lowest quality nucleic acids can be extracted with a crude extraction kit, which usually comes in the form of a simple lysis buffer. This extraction method can work well with high titre viruses, however, it is more likely to result in false negative calls, as nucleic acid recovery can be more variable.
Storage and maintenance of extracted nucleic acids or plant tissues: RNA and DNA samples extracted and eluted in nuclease-free water should be kept on ice and frozen at -20°C (week - months) or -80°C (years). If retesting samples make sure to prevent a large number of freeze/thaw cycles, this can degrade the DNA /RNA and diminish positive results over time. Crude lysis samples usually do not fare well and it is often best to prepare them quickly and add them to the PCR reaction immediately, rather than storing the lysed sample in the freezer and attempting a retest.
Sampling location/tissue type on plant to detect pathogen of interest: Some viruses and viroids replicate more readily in specific host tissue. It has been reported that HLVd is more likely to be detected in root tissue. In many viruses young leaf tissue is best to test as this is where the most active cell division is occurring and thus more viral replication is occurring in these active cells.
Physical Stability of the virus being tested: Some plant viruses are stable for years on a naked surface, while others cannot exist outside of their associated insect vector or plant host. If your virus of interest is less stable it may be more important to use a high quality extraction method for detection upstream of your kit.
Genetic variability of the virus or viroid in question: Some viruses are very diverse, detection kits can only work for the sequence they were designed for. If the virus is in a different group within a species the test may not work. It is important to understand the genetic variability of the virus you are testing for and to review technical documents from manufacturers to ensure the test can pick up all members of a specific virus species.