HpLVd, information, Vendors, Testing and Reviews

Copy and paste from dark heart, I thought this would be a good intro..

Hop latent viroid (HpLVd), the causal agent of dudding, is a current and important topic in the cannabis industry. Given the negative impacts to quality and yield, the ease of transmission, and the difficulty in identifying early infections, the disease will likely be an industry-wide problem for years to come. From a scientific perspective it is a very interesting pathogen, with characteristics that make it very well suited to take advantage of the standard practices in this industry.
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Part I: the Pathogen
Your plants are under vegetative lights and look good. Mostly. Well, maybe some have a less dense canopy, leaflets are overlapping, internodes are short, and branching angles are a bit off. It’s really subtle, though, so maybe you are imagining it. The schedule is to flip to flowering lights next week, so you don’t think much of it. A few weeks into flower you notice that a significant number of your plants have small, loose buds developing, or no flowers at all. You look at nutrient levels, light levels, and dial in as many parameters as possible. The worst-off plants have almost no flowers, and the small buds also have fewer trichomes. When harvest occurs the overall yield is way down, and the quality is lacking. This is a common scenario with hop latent viroid (HpLVd) infection, a very small pathogen causing big issues in cultivation.

First, a little introduction to the pathogen. HpLVd is one of a group of pathogens collectively known as viroids, of which there are 27 known species.[1] They infect higher order plants, with 25 that infect dicots and two that infect monocots.[2] They are all circular single-stranded RNA molecules that range in size from about 245 to 465 nucleotides in length.[1] HpLVd is on the smaller side at 256 nucleotides; the full sequence is at the end of this post.[3] To put that in perspective, the genome of SARS-CoV-2 is 29,903 nucleotides and the genome of cannabis is about 818 Megabase pairs.[4,5] Viroids differ from viruses in that they do not code for proteins and do not have a capsid structure. Even the simplest viruses have coding sequences to make proteins of their own for basic functions. They also have coat proteins forming a shell around their genomes to protect their genetic material against degradation when moving between hosts. Viroids rely entirely on the cells they take over for necessary processes: replication and movement. Their RNA genome has a lot of secondary structure, which allows for shapes that interact with the cellular proteins and enzymes. Viroids are also known to cause small RNA production within host cells, further disrupting normal processes.[1,6] By highjacking and disrupting cellular functions, they cause the symptoms we do not want.

If they have no proteins, no capsid structure, and rely on their host to do everything, how do viroids infect new plants? Through us, of course. Okay, we have not been around for the entire history of viroids, but once humans arrived on the scene and started developing agriculture life became easier for them. Viroids spread through seeds, clonal propagation, and sap transmission.[1,7,8] They are generally not transmitted by insects, though one has been shown to be effectively transmitted by aphids. Collectively, this means our actions and sanitation practices in the greenhouse directly impact the spread of HpLVd.

So why do we call it hop latent viroid? In virology, the first researcher who describes the disease and proves the causal organism gets naming rights. Plant virology naming conventions include the plant host it is first described in and symptoms associated with the disease it causes. HpLVd was originally described from symptomatic hop fields in 1988, Germany.[3] In hops, very few cultivars are symptomatic when infected with HpLVd and only show symptoms under certain environmental conditions.[9] This is pathogen latency, so the pathogen description, a viroid that is latent in hops, is shortened to hop latent viroid.
 
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A) HpLVd infected plant showing leaflets overlapping compared to B) uninfected leaves with normal leaflet spread. C) HpLVd infected plant with very large branch angles, which lead to poor canopy structure and broken branches compared to D) uninfected branching angles. Both of these symptoms can be seen in younger plants.
 
Part Two: Identification and Management
HpLVd in cannabis is mainly known to cause bud quality and yield loss. However, it can cause other issues. In vertically integrated facilities, propagation can take a big hit. Cuttings from infected moms do not develop roots as readily as cuttings from healthy moms. Infected plants will also be more symptomatic when co-infected with other pathogens. HpLVd can also cause brittle stems in some strains, so lateral branches break more easily, leading to poor plant structure. We use the modifiers “may” and “can also” because viroids are known to be highly variable in causing symptoms, both due to environmental conditions and crop genetics.[8] Even though your strain is okay with infection now, later in the year it may respond differently to infection. Or strain A is asymptomatic, but strains B and C are heavily impacted.
Basic sanitation can greatly reduce the spread of HpLVd in the greenhouse. These measures include cleaning pruning shears between plants or plant lots, physically separating plants by lot/strain, and sanitizing greenhouse spaces between production cycles. All workers should wash their hands before entering a greenhouse or bay, and wash hands between bays. Any surface that has plant sap on it can be the source of infection for a clean plant. This also includes tubs of water that hold cuttings and any cloning machinery. Sanitize cutting surfaces between moms when taking cuts, including hands. During vegetative growth and training, make sure everyone who is touching and pruning the plants follow sanitation protocols. A little extra time spent on training and cleaning can make a big difference in the product.
Greenhouse sanitation keeps HpLVd from spreading within an operation, but how do you keep it out? Purchase plants from reputable sources and test new plants. For operations that buy most or all of their production plants as clones, make sure you purchase from a nursery that has robust sanitation practices and follow integrated pest management (IPM). You will get cleaner plants with fewer pathogen and pest problems. HpLVd can be asymptomatic in small plants, so testing is the only way to know if a plant is healthy.
If you do your own propagation and introduce new plants on a regular basis, then you should consider having a dedicated quarantine space for those plants. It should be physically separated from your other plants to allow a few weeks for testing and observation of other pests and pathogens. Test new plants for HpLVd when they arrive, then again at two and four weeks. Any infected plants with a low viroid titer will have detectable quantities by week four. Mother plants should be tested regularly. If you are experiencing dudding, then your moms are likely infected. For initial screening, test a couple plants of each strain. Once you determine where the problem is, test more extensively.
Here at Dark Heart, we test all the plants in the elite mom block monthly, and a percentage of production moms every week. When an outbreak occurs, we test the impacted strains more rigorously. All infected plants are destroyed unless we need to save the strain. In this case we put it through meristem tissue culture. Unlike nodal tissue culture, excising meristem domes can successfully exclude viruses and viroids from plants, a technique that has been in use for viroid exclusion for more than 30 years.[10] Of course, patience is necessary with this technique because it takes about a year to have greenhouse acclimatized plants produced this way.
Why did we say when an outbreak occurs? Because HpLVd is everywhere, and whenever you bring in new genetics there is a risk of those plants being infected and spreading HpLVd within the facility. Staying on top of industry trends means plant material is moving between facilities, with high-demand strains being propagated and distributed fast. The pace of this industry coupled with the fact that a new infection may not be detectable for six weeks means that complete eradication of HpLVd is not likely any time soon.
 
Part III: Research
There is not a lot of published research about HpLVd in cannabis. One of Dark Heart’s goals is to do some research to answer some fundamental questions about the disease. Some of these questions are: is HpLVd present in pollen? If it is present in pollen, can that cause seed infections? What is the seed transmission rate, and does it differ between the female or male being the infection source? How many plants can be infected from contaminated shears? How large of an impact can HpLVd have on yield and quality? How long is HpLVd infectious outside the plant? Are there other pathways of infection that are important?

DHN now has dedicated research space at its Half Moon Bay facility. We are currently working on a contaminated pruning shear experiment in the greenhouse, with three replicates of ten plants that were sequentially pruned after pruning a HpLVd infected plant. We are screening the plants weekly for viroid infection, and so far, it looks like shears can transmit HpLVd out to the eighth plant. More importantly, infected plants may not have detectable levels of the viroid until week seven. We will take the experiment through harvest and compare yield and quality metrics with infection dates. We will post an update when those results are finalized.

We also have a dedicated plant breeder, Dr. Kay Watt. Our lab is working with her on the breeding aspects of HpLVd, including pollen infection and transmission, and seedling infection rates by parentage. She will be guiding us through pollen collection and seed production in a parallel but separate space. We must be extremely careful both with pollen production in general and particularly with HpLVd infected plants.

One interesting anecdote we have heard several times is the following scenario: a facility that has cleaned up their garden and has no positive tests for months finds one or two positive plants. No new plants have been introduced, and greenhouse sanitation practices are followed. Where is the viroid infection coming from? Our theory is infected bud. We have tested dried bud samples from dispensaries, and some have tested positive for the viroid. We then used positive dried bud to mechanically inoculate cannabis plants and was able to cause infection. Then we handled positive dried bud and touched healthy cannabis plants, and this also caused infection in the plants. These results suggest that a person can handle infected bud then infect plants in the garden. This is another reason to require hand washing before entering grow spaces, and not allow smoke breaks during work hours.
 
Part III: Research
There is not a lot of published research about HpLVd in cannabis. One of Dark Heart’s goals is to do some research to answer some fundamental questions about the disease. Some of these questions are: is HpLVd present in pollen? If it is present in pollen, can that cause seed infections? What is the seed transmission rate, and does it differ between the female or male being the infection source? How many plants can be infected from contaminated shears? How large of an impact can HpLVd have on yield and quality? How long is HpLVd infectious outside the plant? Are there other pathways of infection that are important?
DHN now has dedicated research space at its Half Moon Bay facility. We are currently working on a contaminated pruning shear experiment in the greenhouse, with three replicates of ten plants that were sequentially pruned after pruning a HpLVd infected plant. We are screening the plants weekly for viroid infection, and so far, it looks like shears can transmit HpLVd out to the eighth plant. More importantly, infected plants may not have detectable levels of the viroid until week seven. We will take the experiment through harvest and compare yield and quality metrics with infection dates. We will post an update when those results are finalized.
We also have a dedicated plant breeder, Dr. Kay Watt. Our lab is working with her on the breeding aspects of HpLVd, including pollen infection and transmission, and seedling infection rates by parentage. She will be guiding us through pollen collection and seed production in a parallel but separate space. We must be extremely careful both with pollen production in general and particularly with HpLVd infected plants.
One interesting anecdote we have heard several times is the following scenario: a facility that has cleaned up their garden and has no positive tests for months finds one or two positive plants. No new plants have been introduced, and greenhouse sanitation practices are followed. Where is the viroid infection coming from? Our theory is infected bud. We have tested dried bud samples from dispensaries, and some have tested positive for the viroid. We then used positive dried bud to mechanically inoculate cannabis plants and was able to cause infection. Then we handled positive dried bud and touched healthy cannabis plants, and this also caused infection in the plants. These results suggest that a person can handle infected bud then infect plants in the garden. This is another reason to require hand washing before entering grow spaces, and not allow smoke breaks during work hours.
Taken together, we hope to publish results from all the experiments in a peer-reviewed journal later this year. None of this research is very enthralling but is important for better understanding the epidemiology and impacts of HpLVd in cannabis cultivation. Have ideas for other HpLVd experiments? Send us an email!

Common Questions

Is HpLVd seed transmitted?
Yes. It is not always present in each seed from an infected mom but seems to be transmitted to about 5% of the seedlings. More research is needed for seed transmission rates.
Is HpLVd transmitted through irrigation water?
In general, no. HpLVd is a naked RNA molecule and doesn’t last long outside a living host. There are lots of RNA degrading enzymes in the environment, and UV light quickly degrades it.
Is HpLVd in the air?
Again, no. It doesn’t aerosolize and infect new plants through air flow, again because it is so easily broken down.
How long is HpLVd infectious on surfaces?
Since HpLVd is a naked RNA molecule outside of plant hosts it is degraded quickly, and HpLVd on surfaces, such as benches and pots, is likely noninfectious within two hours.
Can plants rubbing together spread it?
Yes, this can happen, especially when there is vigorous movement that causes bruising or if plants rub across the same surface. It won’t be the cause of a big outbreak in the facility but will cause a cluster of infected plants.
How often should I test plants?
For mother plants of strains that you have had for a while, test all moms before starting to propagate. Cull all positive plants, and repeat testing every two weeks until no additional positive plants are found. When purchasing new plants, test when they arrive, again at two weeks, and once more at four weeks. For large production facilities that buy clones, test approximately 5% of shipments upon arrival and expand testing if HpLVd is detected.
If I find it in my garden, what should I do?
This is situation dependent. If it is in production plants, take extra care to not transmit it further. Terminating the plants will decrease yield more effectively than HpLVd infection, so your goal is to mitigate the impact. Increase sanitation practices and minimize plant handling. Work with infected plants after the rest of the garden. If it is found in mother plants, destroy those immediately and test all other mothers of the infected strain. There is not way to cure greenhouse plants of infection, so cull all infected plants to save the healthy plants.
 
sunsetdaydreamer said:
In general, no. HpLVd is a naked RNA molecule and doesn’t last long outside a living host. There are lots of RNA degrading enzymes in the environment, and UV light quickly degrades it.
Click to expand...
I believe this is one of the reasons infected plants can still do well outside so long as its not a full blown infection. A mineral rich soil along with the UVA/UVB rays seem to keep plants asymptomatic. Anecdotal but this is what I've observed.
 
Tangerine_ said:
I believe this is one of the reasons infected plants can still do well outside so long as its not a full blown infection. A mineral rich soil along with the UVA/UVB rays seem to keep plants asymptomatic. Anecdotal but this is what I've observed.
Click to expand...
I didnt realize branch angles were also affected by it. I grow 100% outdoor and I noticed with my last run, which was all clones, several plants had wider branch angles, like the pics a few posts above, and I was having to support the fuck out of them as they got heavier.

edit: I had an MB15 from pink box in with my last run, and it was acquired around the same time frame as other people who have tested the cut and got a positive diagnosis.
 
First Report of ‘Candidatus Phytoplasma trifolii’ Associated with a Witches’ Broom Disease in Cannabis sativa in Nevada, U.S.A. | Plant Disease (apsnet.org)

In September 2018, symptoms including leaf curling, mottling, chlorosis, witches’ broom, stunting, and node shortening were detected in Cannabis sativa L. plants at two growing sites in Central and Southern Nevada, U.S.A., respectively. Incidence of the disease varied between 5 and 20% at the growing site in Central Nevada, and 30% of plants were affected at the growing site in Southern Nevada. Symptomatic leaves showed “green islands” that were constrained by the veins on the upper leaf surface, and interveinal discoloration on the lower surface, forming a pale “pustule-like” appearance. One infected plant with 80% of leaves exhibiting symptoms was sampled from the Central Nevada site. One infected plant with 50% of leaves exhibiting symptoms was sampled from the Southern Nevada site. Two nonsymptomatic plants were provided by a local grower in California. Total DNA was extracted from the petioles of the most symptomatic leaves. DNA extracted from nonsymptomatic plants was used as the negative control. A diagnostic TaqMan real-time PCR test was conducted using the universal primer pair JH-F/JH-R (Hodgetts et al. 2009) targeting the 23S rRNA gene. Positive amplification signals were generated only from the symptomatic plants indicating the presence of Phytoplasma species. To identify the species, the 16S ribosomal RNA gene was amplified with the universal primer pairs P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), P1/Tint (Smart et al. 1996), followed by nested PCR primer pair R16F2/R2 (Lee et al. 1993). The anticipated amplicons (1.8, 1.6, and 1.2 kb, respectively) were gel purified and subjected to Sanger sequencing (UC Davis DNA Sequencing Facility). The sequence data were deposited in GenBank (accession no. MK377248). BLASTn results suggested a 99% sequence similarity with ‘Candidatus Phytoplasma trifolii’ (accession no. KF178706.1), a member of 16SrVI group associated with alfalfa witches’ broom disease. iPhyClassifier analysis (Zhao et al. 2013) was conducted to generate a restriction fragment length polymorphism profile using the amplified 16S rRNA gene sequence, revealing the most similarity (98.8%) to ‘C. P. trifolii’. Phytoplasmas of elm yellows group (16SrV) (Zhao et al. 2007) and aster yellows group (16SrI) (Raj et al. 2008), both associated with witches’ broom disease on Cannabis sp., have been reported in China and India, respectively. To our knowledge, this is the first report of 16SrVI group ‘Ca. P. trifolii’ infecting C. sativa in the United States. The identified pathogen may pose a significant threat to the production of C. sativa in the United States.
 
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