One of the most destructive pathogens plaguing commercial cannabis cultivation is also one of the least well-known among EU producers.
Fig.1: Example of the effects of HpLVd on cannabis plants. The image shows reduced trichome density and malformed apical flowers. Source: Dark Heart Nursery.
The Discovery of HpLVd in Cannabis
Hop Latent Viroid is an infectious plant pathogen caused by single-stranded circular infectious RNA molecules (Fig.2). First identified in hops in Spain and Germany in the late 1980s it was found to affect the production of secondary metabolites including acids, resins and essential oils that were critical for beer brewing impacting both the yield and overall quality of infected plants. However, these were considered extreme cases of the viroid which mostly presented as asymptomatic leading researchers in Germany to use the term “latent” in their nomenclature.
Fig.2. Some of the main similarities and differences between viruses and viroids. In terms of their general size, viroids are also smaller than any known virus. Source:neetlab.com.
Hops (Humulus lupulus) are in the same family of flowering plants as cannabis, Cannabacae. Unfortunately in cannabis, symptoms display greater severity and are colloquially known as “dudding.” These include growth stunting, loss of vigour, yield and potency reduction and overall changes in morphology.
Its presence in the crop has only recently been identified by two distinct groups of scientists in the US investigating this “Putative Cannabis Infectious Agent,” the name for the symptoms of HpLVd prior to its identification in commercial cannabis crops.
One group, affiliated with Dark Heart Nursery in California and led by Dr. Jeremy Warren began work in 2017. They performed next-generation RNA sequencing on a collection of symptomatic and asymptomatic plants and concluded Hop Latent Viroid was the offending pathogen. These findings were confirmed in 2019 by a second research group, Phylos Bioscience, a cannabis genetic testing facility based in Oregon.
Between 2018 and 2021 Dark Heart Nursery conducted over 200,000 tissue tests on samples of crops from over 100 Californian cannabis facilities. They found 30% of the tests from over 90% of facilities were testing positive for HpLVd leading them to conclude a potential crop loss of billions of dollars for the industry. Dr. Bryce Falk, Professor Emeritus in the University of California, Davis’ Plant Pathology Department concluded “Hop Latent Viroid is perhaps the greatest threat to the legal cannabis industry in the U.S.”
Symptoms of HLVd
Symptoms of HpLVd are varied and can be hard to spot since some symptoms can resemble those of other pathologies or those caused by inadequate growing conditions. HpLVd is more successively spotted in cases where the performance of a genetic has been well demonstrated and is consistent enough to become predictable. In this case, if a subset of plants from a genetic are underperforming under homogenous environments with automated controls, the viroid is more likely to be spotted. This was key in the early identification of HpLVd since scientists at Dark Heart were approached by Glass House Farms who provided suspicious plant material for identification when they found puzzling inconsistencies in the performance of their otherwise highly predictable commercial genetic.
Fig.3. One of the symptoms of HpLVd is an overall impact on the morphology of infected plants. Here the viroid has affected lateral branching culminating in a "Christmas tree" shaped plant. Source: Dark Heart Nursery
Fig.4. A clear comparison of the effects of the viroid on plant development. Infected plants are stunted and display reduced vigour. Source: Dark Heart Nursery
Its tendency to lay dormant also affects its ability to be diagnosed since infected plants can be asymptomatic for a period of time until exposure to secondary stressors such as heat, drought or nutrient imbalances can cause the viroid to proliferate and symptoms to appear.
In general, symptoms include:
Reduced vigour (Fig.3)
Stunted growth (Fig.3)
Reduced rooting
Abnormal branching - excessive lateral branch structure (Fig.4)
Brittle stems
Reduced trichome production (Fig.1)
Reduced cannabinoid production (Pinnacle Analytics, a cannabis and hemp testing laboratory in Oregan claim they have sampled infected material showing <50% of the content of healthy plants and in some cases as low as 6%).
Reduced flower mass and overall yields
Malformation and discolouration of leaves
Overall reduction in expected quality and yield
How Does HpLVd Spread?
The primary method of spread of HpLVd is mechanical means through infected tools and equipment typically used during cultivation tasks such as cutting of clones and pruning and defoliation. The viroid can continue to be spread through a facility by clonal propagation of infected mother stock.
Testing and Diagnosing HpLVd
The most accurate way to detect the viroid is through genetic testing using RT-PCR assays. Labs will typically test the same plant multiple times before determining if the virus is present or not. Unfortunately at the time of writing in Europe, there are currently no third-party cannabis licensed labs offering this testing service. This is likely to change in the near future as demand for the service increases and laboratories commission their own protocols.
Diagnosing the viroid can be difficult since it requires multiple samples to be taken from plants as the latent nature of the viroid means it can be present in some plant tissue and not others resulting in a high chance of false negatives.
Preventing HpLVd
As is the case for preventing any risk of plant pathogenic infection, prevention is key. This includes the maintenance of strict hygiene and sanitisation procedures and measures including, foot baths, PPE and gowning, equipment and tool disinfecting, updated SOPs that are rigorously followed and employee workflows. Frequent testing of mother stock is also recommended to identify any potential infection as quickly as possible.
Since the primary transmission is mechanical means cleaning and sanitising tools between plants should be done with a diluted 10% bleach solution or virucide. Simply using isopropyl alcohol is not sufficient. As well as this, quarantine procedures and destruction of plants known to be infected will help keep any possible infection at bay.
One of the best ways to prevent introducing HpLVd into a facility is by sourcing genetic stock from tissue culture. Meristem tissue culture combined with heat and cold therapy is the most successful way to eliminate the presence of the viroid and increase a company's chances of importing clean genetic stock into their facility. Tissue culture can also be used to clean a genetic and eradicate the virus from a prized cultivar. However, this is a slow and costly procedure typically taking between 6-9 months and is not currently available in Europe.
Aside from primary mechanical means of transmission secondary means of transmission from other viroids in the same genus (Cocadviroid) can be in seeds and pollen with insect transmission also providing an additional means of spread. In hops, the transmission of the viroid through seeds is 10% and it is thought to be even higher in cannabis.
Summary
Given the very recent discovery of this mysterious pathogen in cannabis, much work remains to be done to improve our understanding of all its modes of transmissibility and help develop techniques and procedures to prevent its continual and potentially economically disastrous spread in the commercial cannabis industry.
Current understanding of the means of mechanical transmission and the value of procuring genetics from tissue culture, facilities will give themselves a great start by ensuring employee compliance with strict hygiene and sanitisation procedures and by regularly testing their mother stock to identify infection as early as possible.
If you are interested in learning more about how your facility can manage and mitigate the risks associated with HpLVd and other plant pathogens contact Big Leaf Consultants for a discussion on some of our tailored services. We have a great network of tissue culture providers that import genetics into Europe and ensure your facility gets the best start to commercial production.
References:
Bektaş, A., Hardwick, K.M., Waterman, K. and Kristof, J., 2019. Occurrence of hop latent viroid in Cannabis sativa with symptoms of cannabis stunting disease in California. Plant Disease, 103(10), p.2699.
Faggioli, F., Durán-Vila, N., Tsagris, M. and Pallás, V., 2017. Geographical Distribution of Viroids in Europe. In Viroids and Satellites (pp. 473-484). Academic Press.
Pallas, V., Navarro, A. and Flores, R., 1987. Isolation of a viroid-like RNA from hop different from hop stunt viroid. Journal of General Virology, 68(12), pp.3201-3205.
Puchta, H., Ramm, K. and Sänger, H.L., 1988. The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic acids research, 16(10), pp.4197-4216.
Warren, J.G., Mercado, J. and Grace, D., 2019. Occurrence of hop latent viroid causing disease in Cannabis sativa in California. Plant Disease, 103(10), pp.2699-2699.
Punja, Z.K., 2021. Emerging diseases of Cannabis sativa and sustainable management. Pest management science, 77(9), pp.3857-3870.
"Dark Heart Nursery Identified New Pathogen, Creates Patent-Pending Process for Testing and Eliminating Cannabis “Dudding.” March 7th 2019. Business Wire. Available at: https://www.businesswire.com/news/home/20190307005678/en/Dark-Heart-Nursery-Identifies-New-Pathogen-Creates-Patent-Pending-Process-for-Testing-and-Eliminating-Cannabis-%E2%80%9CDudding%E2%80%9D
"Dark Heart Nursery Research Finds 90% of California Facilities Test Positive for Hop Latent Viroid." September 15th 2021.Cannabis Business Times. Available at: https://www.cannabisbusinesstimes.com/article/cannabis-hop-latent-viroid-infections-dark-heart-nursery-crop-loss/
"Hop Latent Viroid." Dark Heart Nursery. Available at: https://darkheartnursery.com/hop-latent-viroid/
"How Tackling Hop Latent Viroid Can Boost your Bottom Line." August 19th 2021. Greenhouse Grower. Available at:
The scientific reason for “dud” Plants — Hop Latent Viroid (HpLVd) Discovery in Cannabis." November 6th 2019. Phylos Biosciences. Available at:
"What is Wrong with my Cannabis?"Pinnacle Analytics. Available at:
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