It’s been said that “big things have small beginnings.”
Take, for example, hop latent viroid (HLVd). This small semi-circular RNA, totaling just 256 bases [1], is responsible for billions of dollars in damages to cannabis grows. HLVd doesn’t code for any proteins. It doesn’t even have a capsid to protect its genome. Yet, it has still managed to become the biggest scourge on the cannabis industry since Reefer Madness.
What does HLVd look like?
HLVd is not known to cause any obvious signs of infection, such as leaf streaking, curling, or chlorosis. In fact, researchers first identified HLVd in cannabis while investigating the cause of “dudding”, a colloquial term used to describe plants that do not perform according to expectations. [2]
During the vegetative stage, HLVd-infected plants grow shorter with smaller leaves and tighter node spacing. Flowering plants infected with HLVd have smaller, looser buds with much fewer trichomes. One client estimated that cannabis plants infected with HLVd had half the cannabinoid content of healthy plants and overall yield was reduced 30%.
With mounting pressures in an increasingly competitive space, cannabis growers simply cannot afford loss of production at that or any level.
Where does HLVd come from?
HLVd is most commonly spread via mechanical infection from equipment. Cultivators who prune an infected plant, and then turn around and use the same shears/scissors to work on its neighbor have just spread the infection. That is why cultivators should always sterilize their equipment with a bleach solution before starting work on a new plant. Ethanol and isopropanol do not destroy viroids.
Because HLVd is a systemic infection found in nearly every tissue of the plant, cuttings that are taken from an infected mother will also carry the viroid. This will result in a garden full of infected clones, that “dud out” or fail to reach their full potential. It can be hard to identify infected mothers because symptoms of HLVd are not always obvious in the vegetative stage, especially if the infection occurs later in the plant’s development, since stunted growth will not be as apparent.
The problem of spreading HLVd through cuttings becomes compounded when nurseries are infected. Now instead of one cultivator filling one garden with infected clones, one nursery is infecting multiple facilities. We have worked with clients who traced their outbreak to a suspicious nursery clone.
How does HLVd harm the cannabis plant?
The precise pathology of viroids is still in debate. There are a few papers suggesting these viroids may induce RNA silencing in plants. Others suggest that viroids activate specific kinases that may interfere with other metabolic processes in the plant.
Our team at Medicinal Genomics is working on another theory grounded in the quantitative polymerase chain reaction (qPCR) data we see when screening plants for HLVd infection. In most infected plants, the value for the virus is 500-1000 times higher than the cannabis genomic DNA.
PCR amplifies a segment of target DNA or RNA to produce millions of copies – sometimes from just a single copy. This process is carried out over a series of heating cycles, each of which will double the amount of DNA or RNA present. qPCR assays include a fluorescent probe that will light up each time a new copy of the target sequence is made, which is read by the qPCR machine. The cycle at which the intensity of that fluorescent signal reaches the set threshold, it is called the Ct.
This high concentration of viroid RNA is a result of rolling circle amplification (RCA) and RNA ligase in the host plant. When RNA or DNA molecules are circular, they can be replicated like a wheel that becomes an infinite template.
These hypotheses are not mutually exclusive, and all are worthy of further investigation.
How can cultivators protect their operations from HLVd?
There are currently no viable treatment options available for HLVd infected plants. Meristem tissue culture can be used to rescue infected plants, but the process can take up to nine months and doesn’t always result in viroid-free plants. The success rate of tissue culture remediation is often cultivar-specific. Cultivators will surely put in the time and effort to save valuable heirloom cultivars, but many others will scrap infected plants and start a new phenohunt to replace them.
Prevention is surely the best way to protect your operation. There are a number of HLVd testing kits available that cultivators can use to confirm suspected infection or identify asymptomatic plants. Screening mother plants regularly, especially before taking a round of cuttings, would help to ensure only clean plants make it into production. Cultivators should also use extreme caution when introducing new genetics from another facility. New clones should be quarantined and screened for HLVd and other pathogens before they are allowed to share a space with existing plants.
What should cultivators look for in an HLVd test kit?
Not all HLVd testing kits are equal, and when a cultivator is facing the possibility of culling valuable mother plants, they want to make sure the decision is grounded on good data.
Choose quantitative over qualitative
Consider whether the kit provides a simple yes/no answer or if it can actually measure the viroid load in the plant. While the former seems like a convenient and unambiguous option, we believe quantitative methods are the way to go.
Given that we are still learning about this pathogen, quantitative methods can help the industry better understand how the disease progresses and whether treatment options can effectively lower the viral load. Could there be an acceptable level of viroid infection that does not lead to reduced yield and potency? Are there treatment options that will reduce the viroid load to an acceptable level? Does the success of these treatments depend on how early the infection is identified? How quickly does the viroid level build up in the plant? We will never know the answers to these questions unless we collect quantitative data.
Use an internal control
Simple yes/no kits are usually a single-plex reaction that uses loop-mediated isothermal amplification (LAMP). These kits amplify only one target (HLVd). If the target RNA is present, the result is positive. However, without using a second, internal control for each sample, the operator can’t truly trust the result. A negative result on the single-plex test could be the result of a botched sample prep or it could be a true negative from a clean plant. Are you willing to bet your farm on that coin toss?
Quantitative tests, such as qPCR, don’t have this problem. All the qPCR tests we designed at Medicinal Genomics target cannabis DNA as an internal control. If the internal control is missing, we know that there was an issue in the process upstream. Without internal controls, you run the risk of lots of false negatives.
Avoid kits that require opening tubes after amplification
Opening a tube with amplified DNA or RNA risks contaminating your facility with a PCR product that could cause false positives. LAMP’s amplification produces 10 times more DNA than qPCR, making it 10 times more prone to contamination. This means you should never open up a LAMP amplification tube in your facility. For this reason, we never design qPCR or LAMP assays that require the user to open the tube after amplification.
Moving forward
HLVd is a problem that is not likely to go away any time soon. Estimates claim that as much as 30% of plants tested in California are infected with the viroid. It’s very likely that many cultivators may have infected plants that they don’t even know about. To get a better understanding of the problem, cultivators should screen their plants with quantitative tests, and if they find infected plants, share the data with the cannabis community.
Furthermore, complete elimination of the viroid may not be realistic or even desirable. After all, there is a slight chance that there is a potential beneficial symbiosis for certain genotypes similar to how heterozygous sickle-cell humans are resistant to malaria.
References
[1] Puchta H, Ramm K, Sänger HL. The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Res. 1988;16(10):4197-4216. [journal impact factor = 16.97; times cited = 65][2] Warren JG, Mercado J, Grace D. Occurrence of hop latent viroid causing disease in Cannabis sativa in California. Plant Disease. 2019;103:2699. [journal impact factor = 4.438; times cited = 6]
