Episode 134: Cannabis Viruses and Viroids with Dr. Tassa Saldi

Welcome to the Cannabis Cultivation and Science Podcast. I'm your host, Tad Hussey of KISS Organics. This is the podcast where we discuss the cutting edge of growing from a science based perspective and draw in top experts from around the industry to share their wisdom and knowledge. Now, before getting into the podcast, I wanted to let listeners know of a couple of things. First off, we just released a new bloom booster that we've been formulating and testing for the past two years. It's now available on our website and it is the cleanest organic bloom booster on the market. I'm really excited about it. And secondly, I want to let folks know that we have some new content on YouTube, including recordings of some recent talks I've given this year at conferences around the world, with one highlighting some of the content from six years of these podcasts. My guest this week is Dr. Tassa Saldi. Dr. Saldi is a molecular biologist with more than 25 years of research experience in RNA biology, infectious disease and molecular pathogen detection. Dr. Saldi graduated summa cum laude with a degree in molecular biology. She completed her graduate and postdoctoral studies at the University of Colorado where she studied RNA structure, small RNAs and RNA processing. She was awarded a prestigious fellowship from the American Cancer society and was one of 18 doctoral fellows nationwide invited to present her research at the ASPEN cancer conference. Dr. Saldi has authored more than 20 peer reviewed articles in top tier journals. As the Chief Science Officer and co founder of Tumi Genomics, Dr. Saldi continues to use her expertise to provide the cannabis industry with reliable, accurate diagnostic tools and pathogen mitigation guidance. Now on to the show. Hi Tassa, thanks for coming on the show today.

Yes, absolutely. Thanks so much for having me. This is an exciting opportunity for sure.

Yeah. Can you tell me a little bit about yourself and Tumi as well?

Yes, absolutely. So I'm Tassa. I'm the Chief Science Officer over at Tumi Genomics. As far as my background is concerned, I am a molecular biologist. I did all of my education, both my undergrad and graduate and postdoc work at the University of Colorado, either in Boulder or Denver. And pretty much all of my expertise is around rna, which is a molecule that can carry information very similar to DNA. It also has some other interesting functions and consequently it's also the molecule that represents hoplite and thyroid. That's all it is. And so I did a lot of work on understanding how RNA is processed, how that like a structure or the shape that it forms impacts how it's treated in the cell and and kind of just investigations into RNA processing in general. So as far as Tumi Genomics, our company was founded a little bit over four years ago. And so basically we were a group of postdocs and some entrepreneurs, and we just decided, you know, we're done with academia. We'd like to do something with what we know how to do that actually will help people and create products that will change something in the world. So we decided to found Tumi Genomics. And the beginning of our company was kind of crazy. We pivoted a bunch. We did cancer genomics. We ended up making Covid diagnostics for a while and worked in the COVID diagnostic lab at CU Boulder. And kind of underneath that, we. We had some connections with some growers in cannabis, and we had one grower contact us in California, and she was having issues with Hoplite and viroid, and she was having a hard time finding tests and labs that could test for it that she. That was reliable, that she would believe in. And so she asked us if we could help her out. And we're like, sure. You know, we're molecular biologists. We can definitely put something like that together. And we kind of just grew from there through word of mouth. And I think, you know, once we started getting into the cannabis industry, we realized that this was absolutely the place that we wanted to be, because we founded our company with the mission of bringing high quality, rigorous science to underserved industries. And when we realized how much the industry needed more research, more education, more outreach, it was. It was like, this is exactly why we started a company in the first place. And so, yeah, we've been working in cannabis for three years or something like that, and it's a great industry to be in. There's so many opportunities and so much to learn, which is the fun part of science. So.

Well, I. So I did a podcast with Dr. Jack Mun of Three Rivers Biotech talking a bit about HLVD and biroids and pathogens. So folks can go back and. And listen to that as well. But I want to kind of COVID some of the basics again in this one and sort of hear your. Your experience and take on things too. So let's talk initially. What is it important from a grower perspective to really understand the difference between a virus and a viroid?

I think certain aspects of understanding the difference between those two are important. Probably the two most important aspects for people to understand are that viroids lack any type of outside protection. So most, most organisms or life has like a genome that's the Information that tells that life how to be whatever it's supposed to be. Right. And then on the outside of it is like a fat layer or protein layer that keeps that information safe. So when you try to kill a pathogen, a lot of the things that you're going to treat it with are targeting that outside layer. Like soap dissolves fat, and so that'll make it, that'll make it fall apart. Like quaternary ammonia, though, like clean grow. Those kinds of things are, you know, targeting proteins because hoplite and vibrate lacks all of those things. There are a limited number of disinfectants that you can use that are effective, which is why you have, you know, basically bleach or veercon and there isn't a ton of other disinfectants. The other really important thing to understand is that viroids lack what I would say, unique biology. So if you get a bacterial infection, you can take an antibiotic, right. Because the antibiotic is targeting something specific about bacteria that's different than you, or an antifungal or even an antiviral. But because, um, viroids don't have that unique biology where you can, you can make an anti. Except for the sequence of the actual genome, it's much more difficult to come up with treatments. Right. So if you, if you have an antiviral or an antifungal, that's not going to have any impact on hop latent viroid. And so it's very unique because of, of those two factors.

Are there any other viroids? I'm not aware of any. Are there any other viroids in cannabis that are a major economic importance?

Yeah, not right now. If you look in hops, which is very similar biology genetically to cannabis, in the same family, there are other viroids that can be very dangerous. There's hop stunt viroid, crackling bark, apple fruit, crinkle viroid. None of those have been characterized as of yet in cannabis. But because cannabis is now becoming legal and its presence is spreading all across, obviously all across the United States and all across the world, the possibility of some of these other viroids kind of jumping ship over into cannabis continues to increase. And so I suspect we will find other viroids in the near future, but as of yet we haven't found any.

Okay, and what are some best practices in general in regards to sort of all pathogens, you know, viruses, viroids, bacteria, fungi, all of these things that can damage our plants?

Yes, absolutely. So, so best practices, I think the first and most important thing that all growers, whether you're Growing a plant in your basement or, you know, your huge cultivation facility is to think about pathogens in a proactive way. It is infinitely easier to prevent them from entering your facility than it is to mitigate them once they have spread through your facility. So how do you prevent that? So we have kind of a dorky acronym or whatever at Tumi Genomics called stop, which kind of outlines the four big points of pathogen prevention. So the S stands for sterilization. You always need to be thinking about sterilizing your tools, sterilizing your pots. You want to make sure your water is sterilized if you're getting it from, you know, a well source or something else where there could be fungus in it surfaces. Sterilizing your hands as you move from plant to plant. Just making sure you have clean, clean practices. Right? The T is for testing. It's really important that you test. The single most common way anything is going to get into your facility is on plant tissue. All right? So if you're taking in clones or seeds, those things come with unwanted hitchhikers, right? And the only way to make sure that they don't gain access to your precious mother stock is to test them before you put them in next to the rest of your moms. So, and then preventative testing, just to make sure that nothing is brewing in your facility. The O is for organization. You want to make sure that you're documenting your testing. You have like a correct and organized schedule that you're thinking about the flow of traffic. Obviously you don't want people to start, you know, in a. In a high pathogen pressure area and then go to the mop room and start cutting or whatever, because you're just going to end up pulling those pathogens in. And then the P is for protecting your border waters. So that's obviously testing before you bring new stuff in. But also thinking about your water system, your growing medium can. You can bring pathogens in like that. You can bring it in on the air. And I mean, it can even. They can even come in on your friends. So you make sure that people are, you know, using gloves and covering their clothes because you want to keep all of that stuff out. So. So you have the control as to what organisms are allowed to grow on your plants. Basically.

Since we're talking about best practices, this question came up recently in regards to a new facility starting out that needs to get genetics. Now, obviously, clones can bring in hop, latent viroid or other things. And if we want to start clean and stay clean, what would be the best Practices, and I'll throw out a scenario for. And then you can tell me how you might approach it. So our thought was initially to grow a mother plant for two to three months, test that mother plant, test the roots of that mother plant for hoplite and viroid, and then if that's clean, then we could pull clones off of that. And then when those get a little larger, test those and feel fairly confident that the plant is clean. What would you do? Well, first off, how does that sound as just sort of a good protocol for moving something into your facility?

Yes. So, absolutely. So if you're so. So if the scenario is that you already have some plants and you're bringing in a set of new genetics, the first, most important step is to define a quarantine area, because you need to. To create a space with that has, like, unknown infection status. And that space has its own set of tools. And everybody knows that these plants could be dirty. And so they're incredibly careful. You don't. You don't use those tools anywhere else in the facility. You don't, like, work on those plants and then go to the other plants. Those plants, when you bring them in, there's a couple important things. First, I would definitely test, like you said right away, the root system for hop latent. I would also think about testing for Fusarium and Pythium. Those are two incredibly damaging root pathogens. And in some ways, especially Fusarium can be more damaging than hop latent because if it gets out of control, you can lose like an entire table in a week because it just spreads really quickly and it's lethal. Also observe. Right. Sometimes you'll. You'll have them there, you let them grow a little bit, and things like fungus gnats and whatnot fly out. And. And it's important to. To watch them and make sure that you don't see anything concerning. I would wait three to four weeks on those plants and then test them again. And if you get two negative tests, then you can feel pretty confident that those plants are not infected. That being said, once they go into the rotation with your regular mother plants, there should be some type of preventative testing that you're. You're just continually doing on your mother stock. And then it should go into that rotation of testing, because even if you're not bringing in these pathogens, especially the fungal ones, can get into your facility a lot of different ways. Um, so it's good to always be double checking and making sure that mom's doc is healthy. Yeah.

And knowing that Hoplitent can come in on seeds. Now, what's the best practice there in terms of testing and protocols for making sure that you're getting clean? Clean seed genetics, yes.

Yeah. So it. It depends on how many seeds you're buying and kind of how that works into the logistics of your facility. Hop latent viroid is in an incredibly high concentration in the flower. And so what we found is if a seed comes from an infected mother, even if the inside of that seed doesn't necessarily have hop latent, there's a really high concentration of it on the outside. So you can answer the question, did this seed come from an infected mother by just testing a few of the seeds from the batch that you've just purchased. Um, obviously the best way is to ask the seed producer to. To give you some data showing that they tested the mom before they went and put it into a breeding program. I would still go ahead and test it again. When you get it to your facility, if you only have 10 seeds or something like that, and you pop those, you can let that plant grow up into, like, the four leaf stage, and you can actually find it in that, the embryonic leaf pretty early on. So you can test them at that point and ditch the ones that test positive if any of them do.

But you can actually test seed, then you're saying fairly reliably.

Absolutely.

Good efficacy.

Yes, it's very good. And so the one important concept to remember, so hoplite and vibrate is in a very high concentration on the outside of the seed, like the coat, but it's also high concentration on the inside layer. But that doesn't necessarily mean every single one of those seeds will give rise to a positive plant. So when we did these experiments, we, if it was taken from a positive mother, we saw something like 90% of those seeds. 90 to 95% of those seeds tested very high positive. But when we grew the plants from those seeds, we got closer to 20 to 50%, depending on the cultivar. So not every single one of those seeds is going to become a positive plant. But if you don't want to risk it, then you can test the seeds themselves and say, okay, this all came from a positive mother. I don't even want to plant them in my facility.

So. Well, a couple questions. So a seed manufacturer could actually send you some seeds then, like five or ten seeds from a batch and feel fairly. If they all test negative, feel fairly confident in the seeds that they're sending out the door? It sounds like, is what you're saying.

Yes. Yeah. So what they would need to do is segregate the seeds so that each seed batch was actually representing a single mother. So that then, you know, we would expect most of the seeds, excuse me, taken from that positive mother. To test positive. Yeah. And of course, you know, if, If I were growing seeds, I would definitely be testing my mom's before I fertilized them. And, and the. The dads, too, because it can come through to a lesser extent in the pollen and just make sure that everything is clean. And that, that would be a question I would ask too, if I were purchasing seeds. Did you test the parents and of these plants before you fertilized it?

On the testing side of things, because testing is so expensive, I'm just trying to think of how we can do this most efficiently. Is it better to test the mother's roots or is it better to test the progeny, like the seeds from that mother? If you had to choose, for example.

Yeah, So I, I would definitely. If. If you're. If it. The econ. Economics is the thing, I would definitely test the mother. Because if. Yeah, if. Because then you'll know that it came from a positive mother, and then you don't need to. To test the seeds.

And how many tests do I need to do on that mother plant to feel reliably confident, you know, with a certain confidence interval that the plant is actually clean? Because I realize you send in one test, it could come back negative. That, that doesn't mean 100% that that plant is clean.

Yeah, absolutely. Yeah. So we generally tell people to wait about three weeks in between testing and to get at least two negative tests in the lifetime of the mother before you either use it in a breeding program or cut clones from it. So I would test that mother probably right when it was a freshly rooted clone, and then test it again before you decide to use it in, in production or to cut clones or. Or fertilize it.

Okay, now, you mentioned that not all of the plants that tested positive for. Of the viroid would express it or have a significant viroid load in the actual plant itself as it grew up? I think you said 20 to 50% was the number range. So, that being said, can you talk a little bit about viroid load and what that really means in relation to the media that we're growing in and the plant itself?

Yes, absolutely. So viroid load formally means how much viroid was detected in the tissue that you sent in to sample. And if the test that you have has an internal control that the test that you're getting, then that can even be normalized to the total amount of tissue. So that is a proxy for how infected the plant really is. Is it just a mild infection or is it like on fire with hot latent thyroid? Um, so in the research that we've been working on with state house cultivation, actually we found that there is a correlation between a higher viroid load and manifestation of symptoms. It's not a hundred percent. You can definitely find plants that have an incredibly high viroid load that don't appear to be terribly affected and plants that have like a medium load. But still you get a pretty significant loss in yield. But in general, if that viroid load is high, you're going to see a more affected plant and you're increasing your risk of spreading it. Because even if that plant doesn't look terribly sick, if there's a ton of viroid on it, then your chances of that plant spreading it throughout your facility is much higher.

So when you say spreading it too, my understanding, and please do correct me if I, if I say this wrong, is that it's, it's not being, it's not being airborne spread. It's really through the same water, like sitting in the same water source or sitting in the same media or in actual like damage or like really rough touching of the plant tissue itself. Like if you brush against a plant and then brush against another plant, you probably didn't spread hop latent. Is that an accurate assumption or is it more contagious than I realized?

Yeah, no, that is correct. In order to spread hop latent viroid, there has to be SAP transfer. So basically breakage from the sick plant, that SAP has to get into a wound, even a micro wound on the, the healthy plant in order to transmit it. The water transmission happens in the root zone because you have cells that are kind of slow sloughing off of the root continuously and those, those are releasing biroid into the water. There's also a potential for insect spread. I know they're working on those experiments at the Colorado State here in Fort Collins, and I do think that they're finding some positive results for insect spread as well. But just touching a plant or having plants near each other, that one's sick and one isn't, isn't. Isn't necessarily sufficient to spread it.

Great. So this next question is one that comes from Steve Cantwell, a friend of mine at Greenlife Productions. And he brought up a good point around this question in the sense that as we do more testing or as we make hop latent into a bigger issue. We're potentially opening ourselves up as an industry to greater regulation and greater testing, which puts an undue burden upon growers and, and frankly raises the cost of everything across the board. Now obviously think testing is important, but are there ways that we can effectively manage this, I guess without testing? And, and his hypothesis was that if we are cycling our plants rapidly and reducing viroid load by growing healthy plants and, and cloning off of healthy plants, even if there was some viroid load in the plant itself, could we in theory reduce that viroid load cycle after cycle to where it's a non factor of any economic importance or virtually, you know, disappears? I guess. What are your thoughts on that?

Yeah, so it depends a little bit on the vibrate load of the plant that you begin with. So if you have a plant that has an incredibly low level infection or an early infection, I mean, what's really happening is that the infection enters at one spot and then it. Viroids can't move on their own, right? They don't have like tails or, or whatever. So it moves in the SAP with the photo assimilates and that's kind of a slow process. So theoretically you could have a viroid slowly spreading through the plant and if you clone from that plant and take an area where there isn't a high level of viroid and then you like continually repeat that process, you can probably get that viroid down to undetectable levels. And I've have had people tell me that they've done that process and gotten plants that didn't have anything and they've had it for a year and it's still testing negative. I've also had people that tried to do that and then they thought they were, everything was great and six months later it started to test positive again. The one thing to understand about viroids is they hijack the metabolism of the plant. And so if the plant is growing very vigorously and it's incredibly healthy, it's definitely going to be better at fighting off fungal pathogens, bacterial pathogens with its innate immune system. But a really healthy, vigorously growing plant isn't necessarily going to be better at fighting off hot blatant thyroid. It might be better at not manifesting those symptoms quite as dramatically. But the faster the plant grows, the more cells are replicating and, and, and metabolizing the viroid is hijacking that process and also replicating. What I think, what I, what I try to convince people to do when I talk to them about this issue is that you can reduce the amount of testing and still be effective if you change the way your mother stock is organized a little bit. So in general ag, usually what they have are nuclear stock, like mothers that are of heightened phytosanitary protocols. And there's only a few plants that represent each strain, and that's the core of your genetic library. And those plants should be tested all of the time for anything that could economically destroy your facility. But it doesn't have to be thousands of mother plants. Right. If you have two or three plants per strain and you have, I don't know, 10 strains or whatever, it's not a huge number of plants. From those plants, you can clone productive production mothers. And at that point you can start testing less or you can spot test, but you don't have to be quite as rigorous with those. And then the rigor can go down and down and down as it moves through the production facility. It's just important to keep that center of nuclear mother stock as clean as possible. And like that, you reduce the number of tests. But it's incredibly effective at keeping pathogens from moving down the production line.

Yeah, I see some challenges with that in cannabis. I love that idea. I just worry in the sense that, well, we, we try to turn our mother stock over because it gets woody. So every cycle we're usually taking a new mother for the next cycle. You can maybe go two cycles, but beyond that, it gets really challenging to keep cloning off the same plant. But, but I think your, your concept still, still works with this idea of keeping some being hyper vigilant about a select number of plants.

Yeah.

Kind of going downstream.

Exactly. So the concept would be I wouldn't recommend keeping mother plants more than a couple months, but when you go to clone to replace that, that mother, you don't clone all of your production plants, all your production mothers, and then randomly pick a few that will go back and represent your new like special nuclear mother. You'll clone a few plants and they stay in that special area and those will be. And from those, you select the one that will replace that mother. So it's like, it's, it's like a small cycle for your nuclear stock, and then a bigger cycle comes out for your production mothers and they go basically cloned into all veg plants at that point. So you continually refresh them, but you keep it internal into that special group of moms, if that makes sense. Yeah.

And just a reminder to folks to make sure you're sanitizing your tools when you're taking any, when you're when you're wounding a plant in any way. And I believe virkon was found. Dr. Zemir Punja did some research on this and found virkon to be the most effective sanitary tool for hoplitent. More so than bleach, is that correct?

So, so Vir Con 2% was found to be effective in the research against potato spindle tuber viroid, which comes from the same viroid family as hop latent. So we would expect it to be just as effective. 10% bleach has been used in the literature since 1969 or something. So that is generally been shown to be effective against every viroid in every plant species since the beginning of the time that they've been doing these experiments. So I would one of those two, either 2% Veercon and do be careful with that because 1% was shown not to be effective or that 10% bleach. Both of those seem to work really quite well.

Wonderful. On, let's see, on that note, I lost my, I lost my train of thought with that because I was thinking about sterilization, but I wanted to go back to this mother stock idea because we were talking about mother stock. So we're, we're taking our clones, we're making sure those are clean, we're putting them out and then hopefully going from there. Ah, my question was, have, have we discovered, has science figured out what triggers this latent viroid to express itself? Do we know what environmental factors are really key in causing expression?

Yeah, so what we've found, there's a couple factors. Genetics of the cultivar is an important factor. We found plants that were incredibly susceptible, mildly susceptible, all the way up to plants that were tolerant, which means that they had a ton of viroid and they just didn't care. It didn't seem to have a problem. So genetics is a really important factor in whether it gets expressed. Stress is another thing that tends to bring out the symptoms. If your plants dry out and then on accident and you water them, the ones that don't seem to recover very well, they tend to be the ones that are infected. You know, heat, stress, all those different types of stress can also bring it out. And then the level of viroid increases dramatically in the flower. So during the flower stage. So the stage of growth is also going to be something where that's going to bring out those symptoms more because the viroid level is going up at that point.

Are you seeing. I, I realize there's different levels of viroid in the leaf tissue versus the roots, but is There any difference in, let's say younger tissue versus older tissue? I would assume older tissue would have high viroid loads. So, like, would it, would it be best practice for the clones that we're keeping in theory to take those off the top of the plant versus lower down or older growth?

Right. So when a vibrate enters the plant, its movement is dictated by something called source to sink, which basically means it moves from where it was introduced to parts of the plant that need energy. Right. So it's obviously going to move towards new growth because new growth needs energy. And it has to move to the root ball because the root ball doesn't photosynthesize. So it's a huge sink on the plant. So some of this is a little bit of a guessing game because you don't really know when the plant was infected and you don't know which tissue represented a sink at the time the plant was infected. When we did this in our study, we did find higher viroid load in the older growth, and that's probably because when the plant was infected, that growth was a sink tissue, and so it moved there, and then it's been replicating in there for a while. But it's a weird guessing game because it really depends on where the plant was initially infected and what tissues were sources and what tissues were sinks at that time. So I don't have a really good answer for you, I'm afraid, because it just kind of depends on how that plant was infected and the timing.

No, that's great. That's fine. I just wanted to know if there was something new that I hadn't heard about that. So that's wonderful. What areas of the country are you seeing hop latent the most or other pathogens in general? Just out of curiosity?

Yes. Yeah, absolutely. So, so that's a. It's a difficult question because so obviously there's a lot of cultivation facilities on the west coast, and so a lot of people are testing. And so you have a lot of data from, you know, California, Colorado, Oregon, showing that there's a lot of hop latent. We have customers all across the United States and every single state in which we were able to have enough data to make the call. We've detected hop latent viroid. So I wouldn't say at this point that it's more specific in any part of the country. It's just kind of everywhere. We've also detected it in Canada, Thailand, South Africa, all across Europe. So it's, it's just kind of a global pathogen at this point. So it doesn't matter where you're getting your stuff. It's. It's something everybody is dealing with at this point.

Is it higher in any particular type of media or style of growing, like outdoor versus indoor? Are you seeing it more in chemical grows versus organics, anything like that at this point in time, or is it sort of agnostic? And everywhere It's.

I would definitely say it's agnostic because it's. It's an intercellular pathogen. It. It cares a lot less about, like, the media or the, the environmental conditions as opposed to, like, big funguses or whatever that can be impacted by a lot of those things. I would say it's. I. We don't see a huge difference. We. We definitely get less data from outdoor grows than we do from indoor grows, but it's. It's affecting all plants at this point, no matter how you're growing them.

And so the only real method for cleaning a plant right now that I'm aware of is, is like effectively tissue culture.

Yes. Yeah.

So how effective is tissue culture? Because, you know, it's something that we've been talking about for years. We see it in other industries. It seems more challenging in cannabis for some reason. So what real world. What's real world? Like, are you seeing a lot of success there, or is it proving to be more challenging than people realize?

Yeah. So it is possible to remove hoplite and viroid with tissue culture. The type of tissue culture that you have to do to remove it is not a beginner's procedure. It's. It's hard and it's not a hundred percent. And it definitely has differences in cultivar. The success rate is different depending on which strain you use. So we've definitely seen companies that were able to do it, and the plants have been cleaned for more than a year and it was successful. And then it just depends on the expertise of the company that's doing it, whether or not, you know, it's going to be successful. It helps to submit plants for tissue culture that have a lower fibroid load. If not, I think one of the first steps that they would take is to try to bring that load down before they do the tissue culture process. And it takes a long time because you have to keep that plant for a while. And if they're doing it right, they're continually testing and making sure that it was successful before they give it back to you. And that process can take like six months, sometimes even a year, just depending on, you know, how high the viroid load was in the beginning and differences between cultivars. So it definitely works. If you love a strain and you don't want to lose it and it's willing, you're willing to give it the time and the money to clean it, you know, you can do it like that. It's just be aware it's a lengthy process and it's not. I wouldn't recommend someone just go out and get a tissue culture hood and try to do it, do it yourself because it's pretty hard.

And are there. So I, I don't know if I'm just going to ask you and then you, if you choose not to answer it, you can choose not to answer it. But these companies that you worked with, that you saw good success with, are you comfortable naming them to at least point people in a direction for going down that path if they wish to?

Yes. If you guys send me an email, I will happy, I'll give you the, the names. How about that? Um, okay, so just send us a message. We do, we don't, we don't get a kickback. It's not like a monetary thing. I just know there are some companies that do really good science and they're very rigorous. And so if you guys want to know, feel free to send me a message and, and we're happy to give you some recommendations on that.

Okay, fair enough. Okay, well let's, let's kind of switch direction here.

Sure.

So TUMI has developed a on site, so like infield sort of HLVD testing platform. Is that correct or why don't you talk about it? Because it's your, it's your product. Let's talk a little about how it might work and what the, the benefits are for growers that are interested.

Yes, absolutely. So you're correct. We have created an on site testing platform and our goal with that was to provide growers with similar sensitivity and accuracy as an in lab pcr, but kind of give them the power so they can run the test themselves. They can see the results, you know, like after an hour or two and, and make decisions that will help maintain the efficiency of their facility. So the test itself is, it's a version of isothermal amplification. But we've made a lot of changes to sort of the base technology because what we wanted to provide, the cannabis facility, was not just an on site test that works, but something that really pushes the limits of what's possible with that type of technology. So we've been able to incorporate, I think probably one of the most critical things is an internal control. So what that means is if you run the test and there was a mistake made or the tissue wasn't any good, or maybe the reagents weren't good, or whatever, the test will fail. So you won't get a false negative and have that false sense of security. You'll know that that needs to be rerun. If it, if it's negative, you'll get the. You'll get a green, the test will glow green, and if it's positive, it'll glow somewhere between a yellow to a red, kind of depending on viroid load. The other ways that we've changed the chemistry have allowed us to bring that sensitivity to an incredibly high level. So we're able to detect four viroid particles per microliter, which comes in more than 95, 96% of all of the tests that we find with QPCR in lab. High complexity would also be detected by this technology. So it gives you the confidence that if you get a negative result with that level of sensitivity and the fact that you can tell the difference between a failed test and a negative test, that you can feel pretty confident that that plant is actually negative, or at least the tissue that you detected is or collected is negative. The other thing that we tried really hard on was to make this easy. Because growers aren't molecular biologists. I don't expect you to know how to use a pipette or know what the difference is between 10 microliters and 100. So we've taken all the complexity out. It's very easy to use. There's no pipettes, there's no scientific knowledge. The sample prep is, is all integrated into the tube. So you put some root in the tube, you close the lid, you shake it up just like our in lab tests, and then it's ready to be run in the test. So it's not hard at all to use. And it comes with a device that calls the results for you. So there's no ambiguity there. Whether it's positive, negative, or, oh, maybe it kind of changed color or it's glowing a little bit. The device will tell you the result. And it comes with a soft software that will give you a dashboard. So not only will you get the results, they'll be organized digitally and it'll track positivity for you over time. You can sort it by room, by strain, by user, by facility. And you have something to, you know, if you, if you need to send reports off, you have something that you can print out to show them how the facility is Doing with their pathogen prevention program. You can also print the reports out and give them to customers if they're buying your clones or your seeds to show them that you've tested. And it has all of the results in there so that you can give your customers that extra confidence. So, yeah, we've tried our very best to build a system that's accurate, sensitive, easy to use, and provides the type of convenience that I think growers need in order for it to be something that they can help them economically and they can utilize in their facility.

That's great. You answered a lot of the questions I was going to ask you about.

Okay.

So that was wonderful. So I don't have to be a scientist to use the product. I could just a little bit of training.

Yep.

Then I. And I could do it. That's great. How long does it take for me to, say, take a sample of root and run the whole. Run through the whole process and get a. Get a result?

Yeah. So. So to collect the root, we've timed ourselves. When you get pretty good at it, you can do it in about 30 seconds. It's we, we ask that people take four pieces of root about an inch long, cut it in half, put it in a tube. And so that takes. It depends on the root system, obviously. Thirty seconds, maybe per plant. Then you shake it up and you move 40 microliters of liquid using this, the specialized transfer stick that we provide into the reaction. So you can undo an entire plate of. Of that in maybe 20 minutes, and then it sits in the heat block for 90 minutes. But that's downtime for you. You hit start, you can walk away, you can go to lunch, you can do whatever else you need. And it beeps and turns itself off when it's done. Then the analysis bit, you can do an entire plate takes maybe a minute at the most. So it, it kind of depends on how many people you have collecting samples. But hands on time for an entire plate, Maybe two hours. Two hours.

Okay. When you say an entire plate, that means you're running multiple samples at the same time. Is that.

Yes, what you're referring to? Yes. Yeah. So the plates hold 48 samples at a time, and that's only limited by the number of heat blocks you have. So if you have four heat blocks, then you could do four times as many simultaneously. So by far the most time consuming part of this is actually just sample collection. And that's would be the same if you were doing an in lab test. That's, that's the part that takes the Most time, but running the test itself is minimal time commitment once you have those samples collected.

So could I run say six Tests at a time though, and save the other 42 to run another cycle?

Yes, absolutely. You can run one test all the way up to, you know, multiple plates at once. It just depends on what you need.

Okay. Because people are going to. These are. I'm trying to ask all the questions I think people might ask after listening to podcast and I, I know people are going to want to know, what does this cost? So we should probably talk about that just to get it out of the way for folks that are interested. I'm looking at the pricing here, but do you want to. Do you want to tackle that and kind of COVID that or do you want me to just.

Absolutely, yeah.

Okay.

So. So there's a one time cost for the device itself that's around 4800 and that's only paid once. And that comes with the device, the heat block, all of the trays and everything to sort of help with the organization. The reactions themselves range between $10 up to $18 and per reaction. And that just depends on bulk at that point, how many reactions you want to buy at once. And Obviously the, the 18 is the high price, but it comes down pretty quickly if you buy, you know, 100 reactions or 200 reactions and it keeps going down like that. Yeah, that's. That's the cost there. The. The device hopefully will be detecting multiple pathogens soon. So then there'll be multiple kits that you could purchase.

So it looks like the, like you said, the starter kit. 4775 US for the starter kit, and that comes with the device collection tube rack and two reaction tube racks and a dry bath and WI FI adapter are the components. And then the testing kit, it looks like the minimum size is $864, which works out to $18 a kit for 48 kits. Which you said you don't have to use all at once.

Correct.

And then you mentioned bulk options. My question there is like, how long could I store this stuff and how am I storing these reaction kits if I wanted to, like, stock up to save money, for example?

Yes. Yeah. So they. So you can store them for six months if you pop them in the fridge. They're more stable in the fridge than they would be on the counter if you have some freezer space. So just the reactions themselves are very little. So you can take the reactions out of the box and put them in the freezer. Then it's almost indefinite. You can keep them for up to a year and they're fine in the freezer. So it depends on what kind of storage options you have. But if you have like a little mini fridge or a mini freezer, you can stock up quite a few.

When you say indefinite, what's my range between a year and indefinite of probably having kits that are still good?

Yeah. Okay, so that's a great question. So I wouldn't recommend keeping them for more than a year. But the accelerated stability studies that we did showed that even if you keep them for a year in the fridge, we didn't see any decrease in sensitivity. So I wouldn't, I would try to use them up within the year. But the data we have doesn't suggest that they would be bad. If you use them after 13 months.

Is the issue sensitivity or is the issue potential kit failure where just you get a, a failed message back?

Like if you keep it for too long, you mean?

Yeah. Would the kit just automatically fail? I'm thinking of a COVID test, for example.

Yes. Yeah. So it's. So it's both. So the way that you do these accelerated stability studies is you kind of heat them up to a higher temperature and then you push the test to its limit of detection and ask how long can it sit and still capture, you know, near the sensitivity that, that it has when, when it's new. So you can also increase the number of fails if some components of the test aren't working as well. So the first thing you would see is sort of a decrease in sensitivity and then you might see a higher percentage of fails also if you push it past the expiration date.

Okay. And you have, it looks like publicly available data on this that people can really dive into that are interested in learning more. I want to just touch on a couple things here. I'm looking at a graph or that or a table. Sorry. That shows the percent detection success based on HLVD concentration in the sample. Can you talk a little bit about, about what that, what that graph or table is showing?

Yes. You know what I'm referencing, so. Yes, absolutely. So the way that you determine the sensitivity of one of these molecular tests is that you, you create a gradient of viroid amount from a whole bunch to all the way down to basically nothing. And then you do a bunch of tests in each of the different levels and you need to show that you have 95% success of detection in order to find that range where the test is, is works and then the range where it kind of falls out. So. And that's that's exactly the way it's done if you're trying to do a human diagnostic or a diagnostic in any other industry where these types of things are standard and required. So that's what we did. We ran 20 or 30 tests at multiple different levels of thyroid and we pushed it until we hit the point where we got that 95% success. But we push it a little more and we went down, I think it was 80, then 50, and then we lost it or something like that. So that's how you kind of determine that, that, that special zone where, where you can detect down to that level with 95% confidence. Um, and so for us it was four viroid particles per microliter when we went down to 2 and 1, you can still find it now. And then it's just not 95% confident or 95% success or better.

And then you did other tests looking at different organisms. I'm looking at the list here. It's quite extensive. Yeah. Notably Pythium, botrytis, Rhizoctonia fusarium, all of these different, these different organisms and cross reactivity. And then you have a variety of percentages. Can you explain what I'm looking at to me here in terms of what these percentages really mean?

Yes, absolutely. Yeah. So for this test we did the, the cross reactivity and interference both in that table that you're looking at, which is a, a bioinformatic analysis and then we did a wet lab analysis. So for the bioinformatic analysis, you take the sequences that are used in the test to find hop latent viroid. And the idea is you want those sequences to find hop latent biroid, but you don't want them to find any other pathogens that might be present or the cannabis genome itself. Right. So you look through the genome of each of these different organisms and you make sure that the sequences you're using to find hop latent wouldn't also find those organisms. So those percentages are the highest percent overlap between the sequences we're using to find hop latent and all of those other organisms that we've looked at. And so the bar there is 80%. That's the bar set by the FDA. If you go below 80%, then it's not bioinformatically predicted to cross react. The other thing we did was a wet lab analysis. So that's where we just actually took that organism and we put it in a tube with the test and said, okay, do we a find it? If we don't add hop latent viroid and B, does it interfere with the detection of hoplite and viroid? When hoplite and viroid is really low, but there's like tons of Fusarium or pythium and both of those are very important. So we, we didn't find any interference or cross reactivity in our wet lab tests. Tests either.

Great. So if I had a plant with Fusarium or media with Fusarium and I pulled a root out of there, I should still get an accurate test result. Or trichoderma, pythium. You list a whole bunch of different pathogens in there, so. Okay, great, you answered that question. Last question I had was interfering substance studies that you did. So looking at growing medium.

Yes.

Can you touch on that?

Yes, absolutely. So to me, this is one of the most important analyses because growers use a lot of different inputs. And I think the big question is I just sprayed sulfur on my plants or I got some cocoa in the tube or I, you know, I use soil, is it going to interfere with this test? And so we set out to ask that of, of a ton of different substances that people might use, from IPM substances to different growing mediums, to different types of sterilization and identified the ones where used at an incredibly high concentration do interfere. There were a few. A lot of them didn't interfere. The ones that did interfere, we did follow up studies to see what level the interference would begin and then we can formulate recommendations. For example, soil tends to interfere. Its oil has a lot of PCR inhibitors. So if you have large amounts of soil on the roots, just for soil specifically, we recommend people, you know, wipe that soil off or, or clean it in some water before putting it in the test. But it's important that you do all of those studies so growers know, oh, I just used this particular product. Can I still do this or do I have to wait? And so we tried really hard to think of everything that might be in the root zone and then make sure that it wouldn't interfere. Or if it did, we provide advice on, on when to test the plant so they're not testing it and getting a bunch of fails and having to do it again.

Awesome. And I realize ultimately this product is not going to be for everyone. You know, it's more specific to people growing on a pretty large scale to make it worth it to do this level of testing. But I hope that folks that tuned in, even home growers, learned a little bit more about top latent viroid and pathogens in general and can take that back into their grow grow system and and make improvements as well. So I really thank you for your time and sharing your knowledge. I learned a bunch today and I will put links to the information that we referenced as well as anything else that you'd like into the podcast page itself. But I just want to say thank you for your time today.

Yes, absolutely. Thank you so much for having me on the show. It was really fun. So and I'm I'm glad to share the knowledge with the industry. Foreign.

That was Dr. Tassa Saldi and you are listening to the Cannabis Cultivation and Science Podcast. Don't forget to check out the podcast page at www.kisorganics.com just click on the Learn tab. Then podcast. And if you want access to podcasts as soon as they are recorded along with additional content and video, we do have a Patreon account. We also do book clubs on there hangouts and have a private Discord channel for members. The KISS Organics website also offers a variety of soil amendments, lighting fixtures, beneficial insects and the highest quality commercial soils on the market. We carefully research all our products we offer and manufacture to make sure they are science based and meet our standard of quality. Thanks for listening.

Sam.