Viruses and Viroids in Cannabis and Hemp
Credits: Thanks to Joseph Ramahi PhD for his insights and efforts during the writing of this article. Joseph Ramahi currently runs a biotechnology company and tissue culture lab (Ploidy LLC) that specialises in cannabis in the Bay Area of California.
Definition of Terminology (scientific mumbo jumbo) Used
Vector: A “vector” can be defined as an organism that can acquire and subsequently transmit a pathogen (mostly viruses but also fungi and bacteria) to plants.
Chlorosis: In botany, chlorosis is a condition in which leaves produce insufficient chlorophyll. As chlorophyll is responsible for the green colour of leaves, chlorotic leaves are pale, yellow, or yellow-white.
Necrosis: When a living organism’s cells or tissues die or degenerate, the condition is called necrosis. In a plant, necrosis causes leaves, stems and other parts to darken and wilt.
Necrotic lesions or spots: This term describes tissue death in a localised section of a leaf. Necrotic lesions or spots usually begin with yellowing in a small area of leaf which then degenerates further into dry, dead, brown plant tissue. Necrotic lesions/spots can occur on one or more leaves at the same time. Additionally, these lesions can multiply and spread causing die off of entire leaves or sections of a plant.
Hypersensitive reaction: The hypersensitive reaction (HR) is a mechanism, used by plants, to prevent the spread of infection by microbial pathogens. The HR is characterized by the rapid death (necrosis) of cells in the local region surrounding an infection. The HR serves to restrict the growth and spread of pathogens to other parts of the plant.
Ribonucleic acid (RNA): RNA is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and DNA are nucleic acids, and, along with lipids, proteins and carbohydrates, constitute the four major macromolecules essential for all known forms of life.
Nucleic acid: Nucleic acids are the biopolymers, or small biomolecules, essential to all known forms of life. The term nucleic acid is the overall name for DNA and RNA. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base.
Capsid: A capsid is the protein shell of a virus. It consists of several oligomeric structural subunits made of protein called protomers. The capsid encloses the genetic material of the virus.
Virus Indexing or Virus Indexed: A virus-indexed variety/plant/cultivar is one that has been tested and deemed free of the viruses it was tested for up to the point of testing. Virus indexing ensures that farmers/growers can access plant stock that is free of the viroids and viruses that stock has been tested for.
Viruses and Viroids in Cannabis and Hemp: Overview
Annual crop losses due to plant diseases are estimated worldwide at $60 billion. Although losses caused by plant viruses alone are difficult to estimate, viruses are considered to be the second greatest contributor to yield loss – the foremost being fungi  – with one expert estimating that more than 40% of total crop losses can be attributed to several virus infections of horticultural crops grown in India.  Additionally, the symptoms induced by plant viruses lead to reduced crop quality and yield. The extent of these crop losses is demonstrated by the following three examples. Cacao swollen shoot virus (Bowers et al. 2001) is estimated to cause an annual loss of 50,000 tons of cocoa beans in Africa with an estimated value of $28 million dollars. In southeast Asia, infection of rice with Rice tungro virus leads to an estimated annual economic loss of $1.5 billion dollars annually (Hull 2002). Tomato spotted wilt virus infects a wide variety of plants including tomato, peanuts, and tobacco (Sherwood et al. 2003), and the estimated annual losses due to infection by this virus worldwide are estimated to be $1 billion dollars (Hull 2002).
The end result of virus infection is a reduction in plant growth, lower yield, inferior product quality, and economic loss to individuals who work in the plant industry. Most of the symptoms induced by viruses can also occur due to adverse environmental conditions or diseases caused by other plant pathogens. Because of this, correct diagnosis of viral diseases normally requires laboratory tests.
As I write this in early 2020, cannabis viroids and viruses have become a significant problem in North American cannabis and hemp production with one estimate suggesting that more than 50% of Californian cannabis plants being grown in commercial facilities may be infected with one or more viroids and/or viruses. Economic losses due to this situation should not be underestimated.
Recent reports on cannabis viruses and viroids have shown the occurrence of cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), Lettuce Chlorosis Virus (LCV), and the viroid hop latent viroid (HLVd). In addition, acryptic virus, cannabis cryptic virus (CCV) has been isolated in cannabis.  To add to this list, recently tobacco ringspot virus (TRV) was identified in cannabis by one Californian based lab, although this has not yet been widely reported.
Further, ‘Beet Curley Top Virus’ has been discovered in hemp being grown in Western Colorado and “Witches Broom” disease, has been found in Iranian hemp production. One report also suggesting that Witches Broom disease has recently been identified in Californian hemp and cannabis production.
Understanding Viroids and Viruses
Since Tobacco mosaic virus (TMV) was first recognized over a century ago, more than 1000 plant viruses have been found (King et al., 2011; Scholthof, 2000).
Viruses are submicroscopic particles made up of coat proteins called capsids and nucleic acid.
Plant viruses have no specific mechanism for entering the host cell. Cell walls and cuticles are difficult obstacles for them. Plant viruses depend, therefore, on injuries through mechanical means (e.g. cuts made to plant tissue while cloning or pruning etc) or on transmission via invertebrates (insects, nematodes, etc.) which act as vectors for disease. The invertebrate transmitter does in some cases also act as an intermediate host. This means that some plant viruses are able to multiply within invertebrate tissue. In some cases, viruses can also be spread via seed or pollen.
Viruses are perfect parasites. It has been known for decades that once a virus gets inside a cell, it hijacks the cellular processes to produce virally encoded protein that will replicate the virus’s genetic material. Viral mechanisms are capable of translocating proteins and genetic material from the cell and assembling them into new virus particles. It all may start with an insect bite or through mechanical wounding during cloning or pruning. A virus only has to reach a single cell to initiate infection. However, viruses cannot do anything by themselves; they need to hijack the infected cell’s mechanisms to produce copies of themselves. Eventually, progeny viruses are released to neighbouring cells and this cycle is repeated. Soon the virus is able to reach the vascular system in the plant (akin to the circulatory system in humans) and can spread long distances from the initial spot of infection, infecting everything from roots to young leaves.
In susceptible plants, virus infection can cause profound reorganization of host cells by altering the structure and integrity of intracellular membranes and organelles. A common symptom of virus infection is chlorosis, often expressed as yellow mosaic symptoms on the leaves.
Viroids differ somewhat in their makeup to viruses. Viroids are the smallest infectious pathogens known. A viroid (an infectious ‘RNA’ – Ribonucleic acid – molecule) is similar to a virus but not quite the same thing. It’s smaller than a virus and has no capsid, but, as with viruses, can reproduce only within a host cell. Viroids do not, however, manufacture any proteins. They produce only a single, specific RNA molecule. Intracellular movement of viroids is believed to be facilitated by host proteins. Viroids move cell to cell via the plasmodesmata, the natural openings connecting plant cells, and enter into the vascular system and spread systemically with plant sap.
The classic signs of viroid plant diseases, such as yellowing and curled leaves, are believed to be caused by the viroids paring their own RNA with that of the afflicted plant’s messenger RNA, interfering with proper translation.
In addition to symptomatic infections, viroids also cause latent infections where there is no visual evidence of infection in the host; however, transfer to a susceptible host can result in devastating disease.
Viroids typically spread via seeds or pollen or mechanical wounding through practices such as cloning. It is also possible that viroids may be spread via invertebrate (insect) vectors.
One particular viroid of major concern at the moment, in the US, is the viroid hop latent viroid disease (HLVd) – colloquially termed “Dudding” – which is spread via pollen and mechanical practices such as cloning. Viroids and viruses tend to be very host specific. However, hop plants are from the family Cannabaceae which is shared by the genus cannabis. Therefore, cannabis and hops are close cousins and it is likely that many viroids and viruses that infect hops also are capable of infecting cannabis.
In 2017 HLVd symptoms were noticed on multiple cultivars of Cannabis plants grown in California, including stunting, malformation (morphological changes), chlorosis of leaves, brittle stems, and reduction in yields and potency. To confirm the presence of HLVd in symptomatic plants, RNA was extracted from the original five symptomatic (showing symptoms of disease) and five asymptomatic (not showing symptoms of disease) plants, and HLVd-specific reverse transcription PCR (RT-PCR) was performed using the outward primers HLVdF and HLVdR. All five symptomatic plants tested positive for the presence of HLVd, and all the asymptomatic plants tested negative for the viroid.
What’s notable here is that the scientists behind isolating HLVd in Californian grown cannabis identified symptoms of HLVd/Dudding in approximately 35% of the cannabis plants being grown at one facility. This indicates that HLVd is potentially rampant and poses a serious threat to all cannabis producers.
See following image of a “Dudded” plant next to a healthy plant of the same cultivar…
ABOUT HOP LATENT VIROID (HLVd)
Hop latent viroid (HLVd) was first characterized in Humulus lupulus (hop) plants (Putcha et al. 1988). The initial research suggested it was a minor pathogen in hops; however, recent research has shown that infection with HLVd can have a significant impact on yields and secondary metabolite production (Adams et al. 1991, Barabra et al. 1990, Matousek 1994). These yield and metabolite impacts seem to be even more pronounced in cannabis. If plants are showing symptoms of HLVd infection, there are a few actions to consider. Always remove infected plants from the growing area to prevent spread. HLVd, like many viroids, has been shown to be primarily mechanically transmitted, so strong nursery sanitation protocols are necessary when pruning and processing plants. The secondary means of HLVd transmission are not yet fully understood. However, other viroids in the Cocadviroid genus have been shown to be pollen and seed transmissible. This suggests that HLVd may also be transmitted in a similar manner, but further study is required. Insect transmission of viroids is also still being studied. Cannabis plants can be carriers for HLVd without showing any outward symptoms of the disease. I.e. latent hop viroid is so named because the viroid can be ‘latent’, which by definition means “(of a quality or state) existing but not yet developed or manifest; hidden or concealed.” This is important to understand because while your plants may not be showing symptoms of LHVd they might be infected with the disease.
How Viroids and Viruses Impact on Plant Health and Yields
Although plants do not possess an equivalent to the animal adaptive immune system, they deploy a number of protein- and RNA-mediated defense mechanisms. Additionally, plants are able to defend themselves against viruses and viroids through launching hormonal (phytohormone) responses where, for example, signal molecules such as salicylic acid (a phytohormone) and Jasmonic Acid (a phytohormone) trigger a defense mechanism in the plant to help fight viruses. These defense responses are termed as systemic acquired resistance (SAS).
SAS sets of a cascade of plant defense mechanisms in order to fight the virus. For example, a hypersensitive reaction (HR), which causes rapid cell death and the formation of visible necrotic lesions on leaves, can occur as the plant attempts to limit cell to cell transfer of the virus. Necrotic responses associated with HR are generally thought to play a role in restricting virus movement. However, HR is not always efficient at restricting viruses and cells outside of the cell death zone of local necrotic lesions can harbor infectious virus. In some plants, induction of HR is either weak or delayed and does not prevent the systemic spread of viruses. Instead, this can result in runaway HR leading to systemic lethal necrosis.
The Chloroplast and Viruses
The chloroplasts of a plant are the machinery of photosynthesis. Chloroplasts work to convert the light energy of the sun into sugars that can be used by cells. The entire process is called photosynthesis and it all depends on the little green chlorophyll molecules in each chloroplast. When the energy from the sun hits a chloroplast and the chlorophyll molecules, light energy is converted into the chemical energy found in compounds such as ATP (Adenosine triphosphate) and NADPH (nicotinamide adenine dinucleotide phosphate). This energy is then used by the plant to power growth.
However, chloroplasts are also central in the deployment of plant defense responses with salicylic acid (SA), Jasmonic Acid (JA), and reactive oxygen species being produced in the chloroplast.
As a result, a common symptom of virus infection is chlorosis, often expressed as yellow mosaic symptoms on the leaves. Chlorotic symptoms have been linked to virus-induced changes in the number or size of chloroplasts, or with structural alterations. In addition, biotic stress including viral infection has been reported to cause global repression of plant photosynthetic genes. Specific interactions between viral and chloroplast proteins can also interfere with the normal functioning of the chloroplast resulting in reduced photosynthesis, and the rate of photosynthesis is directly related to the rate of plant growth. 
In simple terms, where rates of photosynthesis are reduced by virus infection, so too is the rate of plant growth and yields.
The Growth-Defense Tradeoff and Phytohormones
Growth–defense tradeoffs are thought to occur in plants due to resource restrictions, which demand prioritization towards either growth or defense, depending on external and internal factors. The ‘growth–defense tradeoff’ phenomenon was first observed in forestry studies of plant–insect interactions, and is based on the assumption that plants possess a limited pool of resources that can be invested either in growth or in defense. 
Therefore, when a plant is forced to defend itself against a virus, mounting the defense response requires energy resources, which are diverted at the expense of growth and development. While many of the molecular mechanisms underlying growth and defense tradeoffs remain to be fully understood, phytohormone crosstalk has emerged as a major player in regulating tradeoffs needed to achieve a balance. As a result, Induction of defense hormones can also result in reduced plant growth. So, for example, the activation of salicylic acid (SA) dependent defense responses has been linked to the dwarfing phenotypes observed in many plant – virus interactions. 
What this basically means is that where a plant is forced to defend itself against viruses and/or viroids it diverts energy away from growth towards defense, resulting in lower than optimal growth rates and yields.
How are Viroids and Viruses Spread?
In general, where agricultural crops are concerned, there is a serious scientific concern about the transmission of plant viroids and viruses sexually through seed and asexually through plant propagules (clones and tissue culture).
One of the most significant problems that cannabis growers face, where the spread of viroids and viruses is concerned, is that both viroids and viruses are readily passed from plant to plant via mechanical wounding. What this means is that practices such as cloning become central in the spreading of viroids and viruses from plant to plant, grow site to grow site. In the case of the latter, this is because select phenotypes of different cultivars are often passed grower to grower via clone sales.
What this really means is that where growers access clones through other parties there is a chance that viroids and/or viruses may be present in these clones. It is my belief (and the belief of others) that clones have become the main mode of transmission of viroids and viruses through the North American grow scene. This presents as a serious biosecurity issue because all that it takes is for one infected plant to be introduced into a facility and for that plant to then become the source (patient zero) of further infection to other cultivars being grown at that facility. The end result is that viroids and viruses are spread cultivar to cultivar and then cuts from these cultivars may find their way into other grow facilities through clone distribution to other outside parties.
Another significant issue also presents in cannabis seeds because seeds are also a potential mode of transmission of viroids and viruses to grow sites. For example, in the case of latent hop viroid disease (LHVd), while research on cannabis seed transmission of HLVd is at this point non-existent, one study found that about 8% of hop seed is infected with HLVd when hop seed derives from infected crops. Based on this it is more probable than not that cannabis seed can also be infected with HLVd (among others).
What this means is that when you purchase seeds there is a chance that these seeds will become carriers for the initial infection of a crop.
Since plants and plant pathogens (viruses, fungi and bacteria) cannot travel by themselves, the majority of plant viruses that cause disease in agricultural crops rely on invertebrate vectors for transmission and survival. The largest class of plant virus-transmitting vectors are insects but other vectors include mites, nematodes and chytrid fungi. The most documented plant viral insect vectors are aphids, thrips, leafhoppers, planthoppers and whiteflies. However, many worm mites (Eriophyidae), false spider mites (Tenuipalpidae) and spider mites (Tetranychidae) are known as vectors of virus diseases infecting cereals, fruit trees, pulse crops and coffee plants.
Virtually all flowering plants are exposed to attack by pathogenic fungi. Necrotrophic fungal pathogens kill host tissue and absorb nutrients from the dead host cells, while biotrophic and hemibiotrophic fungal plant pathogens colonize the living host, and some commonly develop a specialized organ for uptake of sugars and other nutrients. To promote infection and disease development, fungal pathogens secrete or inject effector proteins that interfere with the host immunity and other physiological processes. Numerous studies have reported the occurrences of horizontal transfer of small RNAs between some pathogenic fungi and the roots of plants. It is through this mechanism that some fungi are able to vector viroids and viruses to plants. One study notes thirty soil borne viruses or virus-like agents are transmitted by five species of fungal vectors. For example, it has been shown that Rhizoctonia solani can act as a vector for cucumber mosaic virus (CMV) to plants. Additionally, infected Fusarium graminearum can efficiently transmit hop stunt viroid disease (HSVd) to a plant.
The Problem: The Cannabis AIDS Crisis
The economic significance, due to yield losses, of what is occurring now in North America surrounding viroid and virus infection of cannabis crops cannot be underestimated.
Reports of significant yield reductions from various grow sites are all too common. Often at these grow sites at least some plants show symptoms of viruses and/or viroids (e.g. chlorosis, necrotic lesions, malformation, reduced vigour, reduced yields and/or reduced medicinal oil production). When plants from these sites are then tested for viroids and viruses, one or more viroids and/or viruses is present.
One of the major problems is that in the commercial production of cannabis, clones over seed are seen as desirable in order to ensure the best phenotypes are in use and crop consistency/predictability. However, select cuts of clones are passed grow site to grow site. As a result, along the way some of these clones may become infected with viroids and/or viruses. This means that viroids and viruses are spread grow site to grow site, and once an infected cut has entered what may otherwise be a “clean” grow site, this cut becomes the mode of disease transmission (patient zero) to other cultivars at the grow site. These cultivars may then be passed onto to others (as clones) and the cycle of infection continues and snowballs. It is through this means that viroids and viruses have been spread across North America and today we face a growing crisis. Not to put too fine a point on things, this is the cannabis industry’s AIDS crisis where viral diseases are not being spread through blood to blood transfer (as with humans and AIDS) but through sap to sap transfer between infected and non-infected cultivars.
I have heard some advising that because of the potential for clones to be infected with viroids and/or viruses growers should cultivate from seed. However, this advice misses the point that 1) seed too can carry viruses and/or viroids and 2) any one batch of seeds can contain multiple phenotypes with very different morphological traits (plant height, biomass, yield potential) and chemical profiles (potency, terpenes). That is, the whole point of pheno hunting from seed is to isolate that one special cut that surpasses all others within any strain group. This is how certain cuts have become the standard. So, for example, where talking of the Blue Dream cannabis strain, it is the Santa Cruz cut that made the Blue Dream name synonymous with top shelf big yielding cannabis. This particular cut has heavy sativa leanings (think Haze) and having purchased Blue Dream seed myself on several occasions I have never been able to find a similar phenotype of the same quality and yield potential. The same can be said for numerous other strains where select cuts achieved through the ‘pheno’ hunting efforts of others stand out above all others. It is these phenos’ that are highly sort after by growers, and these phenos’ are only available as cuts. Therefore, in a highly competitive market where potency, bag appeal and yields are of primary importance it is impractical advice to be telling commercial grow operators that in order to minimize the risk of viroids and viruses they should purchase overpriced, variable quality seed which in all instances provide multiple phenos of varying quality, growth characteristics and yield. That’s not to even mention the fact that in grow operations where several thousand plants may be being grown the price of purchasing seeds at $80.00 plus for 10 seeds would equate to tens of thousands of dollars every crop cycle. In other words, advising commercial grow operators to produce entirely from seed is counterproductive to the commercial realities of modern largescale commercial cannabis production.
It is important to note that viroids and viruses are spread by infected tools used in cloning or defoliation. Pruning multiple plants with the same tool is much like the situation with Aids and sharing needles. That is, sap to sap transfer occurs when tools are used on an infected plant and then used on uninfected plants. For this reason, tool sterilization between cutting one plant before moving to another is of the utmost importance. Attached is a paper that looks at different viral sterilising agents such as bleach and Virkon S. [Download]
This study found that the most effective disinfecting agents to combat several viroids and viruses were 10% Clorox regular bleach, and 2% Virkon S. Both of these chemicals are cheap and readily available. It’s important to use them with sufficient contact time with tools sitting in a sterilizing solution for above 1 minute to effectively stop transmission of viroids and viruses from plant to plant during manual wounding processes (e.g. pruning and cloning).
Seeds provide an efficient means in disseminating plant virus and viroid diseases. The success of modern cannabis agriculture depends on pathogen free seed with high yielding character and in turn disease management. There is a serious scientific concern about the transmission of plant viruses sexually through seed. This is something growers need to be aware of because many growers are under the misconception that if they grow from seed their plants will be viroid and virus free. Nothing could be further from the truth! While seed arguably presents as somewhat safer than accessing clones through third parties, numerous viroids and viruses are shown to be transmitted in seed that is derived from infected plants.
The Cannabis Industry and a Lack of Best Practice Agricultural Standards
For a very long time now, commercial horticulture/floriculture has embraced best practice biosecurity measures along with virus indexing systems where elite stock is stored in liquid nitrogen or refrigerated and heavily tested for disease before being distributed to growers. No such system (that I know of) exists in the cannabis industry. This needs to change in order to ensure the viability of what is now just another legal agricultural crop.
However, there is almost a collective sense of denial surrounding viroids and viruses in the cannabis industry. For example, the latent infection of HLVd is very concerning and yet many growers seem to refuse to acknowledge how big of a threat HLVd is to future production. The reaction that we often see is very concerning for the future of the industry, as a whole. This said, there is a small number of companies/entities that are making these viral/viroid concerns paramount, and I believe at some point in the not too distant future they will be introducing the idea of “virus indexing systems.”
The “Virus Indexing System” and its Importance in Future Commercial Cannabis Production
Virus indexing is the testing of plants for the presence or absence of viruses. A virus-indexed variety is one that has been tested and deemed free of viruses. These disease-free varieties are then stored cryogenically before being regenerated via tissue culture and circulated amongst commercial crop growers. Through this means commercial crop growers are able to access disease free stock.
The agricultural sector has long maintained a virus index system for numerous crop types. For example, about a decade ago there was a hop stunt viroid (HSVd) outbreak in the Pacific Northwest. The hop industry, the USDA, hop growers and universities all worked together to get hops into the National Clean Plant Network (http://nationalcleanplantnetwork.org/) and rescue the industry from cataclysmic crop/yield losses. Cannabis is just another crop with issues; fortunately, we don’t have to reinvent the wheel when it comes to what makes a “clean” plant. The standard was set with numerous other crops years ago. Cannabis cultivators/companies/entities simply need to adopt standards that have long been used in agriculture.
Lab Testing Methods for Viroids and Viruses
There are two key methods employed to test for viruses. These methods are based on serological testing and molecular methods. While there are numerous testing methods/standards within these groups, for reasons of expediency, and so as not to get bogged down in mind numbing jargon, I will cover a few commonly used examples – one from serological methods and two from molecular methods.
Serological methods of testing for viroids and viruses
Serological detection systems use specific antibody developed in animals in response to antigens (Torrance, 1998). Viruses can be detected if viral antigens are used to develop antibodies. In fact, these kinds of techniques have been used for the routine diagnostic tool. Many serological methods have been reported including enzyme-linked immunosorvent assay (ELISA).
Identifying an unknown viral pathogen requires using a transmission electron microscope because they are so small, much smaller than bacteria. But, if a virus is known and can be isolated, an immunological reaction can be induced in an animal such as mice or rabbits. Using, then, antibodies from such a reaction can be used to detect the presence of the virus or viroid in infected tissue or liquid. This is the basic principle of the Enzyme-Linked-ImmunoSorbant-Assay, commonly referred to as the ELISA test, a serological test. ELISA is used in medicine to detect viruses such as HIV in people and it is used in agriculture to detect potato viruses such as PVX, PVY and PLRV. It is the ‘state of the art’ for seed certification. State seed Certification Agencies and Associations such as the one in Nebraska use ELISA to detect viruses in seed tuber lots in the ‘winter test’ conducted in Florida and other warmer climates. ELISA is quick and can detect viruses even in the absence of any plant symptoms of disease.
ELISA plates (having 96 wells) are available for each specific virus. Each well contains the virus-specific anti-body bound to its sides. Sap or liquid extracted from tissue or cells is added to the well. In order to minimize possible over-reactions or unwanted reactions, and to titer the virus, the liquid may be diluted several times with a buffer. If the virus is present in the test liquid, it will bind to its anti-body. Wells are rinsed to remove the liquid and its contents that did not bind and therefor not the targeted virus. More of the same anti-body bound to the well is added. This second set of anti-bodies also has an enzyme attached to it which will react with a pigment. These anti-bodies attach to the viruses held by the bound anti-body. After this second reaction, any unattached anti-body is rinsed away. And now, the pigment substrate is added. If the substrate attaches to the enzyme because it is present, it will develop or change color. A color change means the targeted virus is present in the sap or tissue extract and if no change occurs than the virus is absent.
Shortcomings of ELISA
Large amount of sample for ELISA is needed for capturing antigen of interest from the sample compared to sample requiring for molecular methods and it takes about 2 days for diagnosis (Lievens et al., 2005; Luminex, 2010). Since ELISA is antibody-antigen based assay, availability of antibody properly responding against the target agent is regarded as very important factor. ELISA often offers misdiagnosis due to false positive which is mainly resulted from non-specific reactions or cross-reactivity with certain factors in samples (Kfir and Genthe, 1993). Antibody used in ELISA can respond to many strains with an obvious different symptom because of lack of specificity. Therefore, strains of virus very related cannot be differentiated correctly by ELISA (Boonham et al., 2014). Although ELISA sensitivity was increased by adding some additives in extraction buffer (Fegla and Kawanna, 2013), ELISA is generally less sensitive when compared to molecular methods. Because of these reasons, although ELISAs have been widely used for diagnostic purpose up to date, the use of ELISA in terms of diagnosis seems to be gradually decreased. It is thought that alternative tools to be employed in coming age will be introduced in to a diagnostic market or more researches will be continued to overcome ELISA’s shortcomings.
Molecular methods of testing for viroids and viruses
Molecular methods can be applied for diagnosis of many viral diseases when genetic information of viruses is available. The most commonly used molecular method today is PCR (polymerase chain reaction).
PCR is currently the basis of all diagnostic methods, used with other detection methods (Lopez et al., 2008). PCR is a technique that enables the specific amplification and hence detection of target DNA sequences from complex mixtures of nucleic acid. A combination of short, specific primers and thermostable DNA polymerases are used to amplify the target sequence, through repeated cycles of denaturation, reannealing, and DNA synthesis at high temperatures, allowing an exponential increase in the amount of the DNA of interest. By addition of a reverse-transcription (RT) step, PCR can also be applied to cDNA generated from RNA templates. Its extreme sensitivity and high specificity make it an unparalleled technique for the detection and characterization of rare messages, including viral infections that are difficult to detect and diagnose by serology (e.g. ELISA) or electron microscopy. This high sensitivity facilitates detection of virus sequences during the early stages of infection of plants and in soil and vector samples.
Loop-mediated isothermal amplification (LAMP)
Apart from PCR, within the last decade, the development of a technique called “loop-mediated isothermal amplification” or LAMP has facilitated the development of hundreds of simple assays for plant disease diagnostics. There are now more than 200 LAMP publications per year, of which 20% identify plant disease pathogens. Among them, LAMP assays are available for pathogen detection of 50 plant viruses, 20 bacterial plant diseases, 7 fungal plant diseases and several phytoplasmas.
LAMP is a technique involving the use of four to six primers (two inner primers, two outer primers, and two loop primers) and the strand displacement activity of Bacillus subtilis-derived (Bst) DNA polymerase. The end result of strand displacement and loop formation and synthesis is the single-temperature amplification of a highly specific fragment from a DNA template at a much greater titre than that obtained with PCR. With LAMP, there are several methods to determine a positive reaction.
The LAMP assay has been recently applied for the rapid detection of several viruses in animal, such as Canine parvovirus (Cho et al., 2006). In addition, it has been used to determine sex of asparagus, genetically modified organisms (GMOs), and Phytoplasmas (Lee et al., 2009; Shiobara et al., 2011; Tomlinson, 2010). The RT-LAMP has been developed for simple monitoring of RNA viruses including PVY and PLRV (Ju, 2011; Nie, 2005).
Conclusion re Testing Methods
PCR based diagnostics have been adapted as a diagnostic system comparable with ELISA even becoming the predominant method in viroid and virus testing. There are a couple of reasons for this change. Firstly, PCR is standardized. Secondly, PCR-based assay has better sensitivity over ELISA and is faster. Isothermal nucleic acid amplification methods including LAMP are under development for virus detection because it is faster and has greater sensitivity over conventional PCR. Thus, LAMP is emerging as a desirable method over others (at least for now). 
Another testing method that needs to be covered is RNA Sequencing.
This is the method that was used to identify Hop Latent Viroid in Cannabis. How RNA sequencing works is you take RNA samples from sick plants, make a cDNA (complimentary DNA) library, and use sequencing machines (like Illumina, for example) to sequence all of the present RNA in the sample.
From this sequencing you get to read all the RNA transcripts in the samples, and go looking for virus/viroid sequences.
Usually you remove the known Cannabis sequences (which weren’t really available until recently) and look for any viral/viroid sequences left over. This is a very expensive way to go hunting for unknown viruses/viroids in a sample when you don’t know what to look for with PCR. It can cost a few thousand dollars to run a handful of RNA sequencing samples for comparison.
Eradicating Viroids and Viruses from Infected Plants
Unfortunately, there is no easy out from the Cannabis Aids Crisis. Unlike most fungal and bacterial diseases, which can effectively be controlled by chemical pesticides, control of virus diseases has focused on producing virus-free propagation materials. This really comes down to what is termed meristem culture along with heat and/or cold and/or chemotherapy and/or cryo-therapy treatments during the meristem culture process.
However, virus eradication is a slow and expensive process and is not guaranteed to work, as some organisms such as latent viruses and viroids are notoriously difficult, if not impossible, to.eradicate from infected plants. Adding to the challenges, cannabis is a relatively new commercial crop and there is limited research as to what viruses are important and cause negative effects on crop performance. Finally, removal of pathogenic and non-pathogenic viruses may have unexpected results on phenotypes such as cannabinoid profiles and growth habits.
To compound problems, viruses and viroids are notoriously hard to test for because some tissue from the plant may test positive for viruses while other tissue from the same plant may test negative. In other words, separate parts of the same plant can test positive and negative for a virus or viroid at the same time. So even negative tests can be misleading.
Because of this, growers need to remove tissue for virus testing from areas of the plant that are most likely to contain the virus.
Meristem Culture Overview
During meristem culture, key plant cells are excised from the apical shoot tip. These cells are then grown in a special medium, producing a new plant or plants.
In. the past, scientists have believed that meristem culture offered a way of guaranteeing virus free plant cells from which a new (virus free) plant could be grown. However, in more recent times it has been discovered that certain viroids and viruses cannot be eliminated through meristem culture alone. This has led to meristem culture being used in conjunction with other treatments such as heat (meristem tissue is subjected to a given duration of heat treatment), cold (meristem tissue is subjected to a given duration of cold treatment), cryo-treatment (tissue is rapidly frozen in liquid nitrogen for a short time and then unfrozen for in vitro growing) and chemotherapy (meristem tissue is placed in a medium which contains an antiviral agent such as Ribavirin) to eliminate viroids and viruses. The best method of treatment really comes down to what viroids or viruses are being treated and where multiple viroids and/or viruses are present one or more treatments may be required to eliminate disease.
The Problem Is
At this point in time very little viroid and virus lab analysis is being conducted by growers because testing is; 1) expensive with a full screen for viruses and viroids costing between $1000 and $1,500 per test; 2) should a plant test positive for one or more viroids or viruses cleaning the plant through the use of meristem tissue culture and other treatments is also prohibitively expensive (approx. $2000 per plant); 3) a lack of awareness about the impacts that viruses and viroids can have on yields is preventing growers from seeing the value in testing and cleaning plants of viruses and; 5) the technology involved in testing and removing viroids and viruses from cannabis is beyond the technical abilities of many/most grow operators.
It is important to note that there are cheaper methods of conducting virus tests like Agdia virus test dipsticks here https://orders.agdia.com/pathogen-tests/immunostrip-tests. Additionally, enzyme-linked immunosorbent assays (ELISAs) are about $10.00 a test, and single PCR assays are available for anywhere from $50.00 to $100.00. The problem is that indexing multiple viroids and viruses becomes expensive.
What is the Meristem in Plants? And Why is it Used to Create Virus Free Plants?
The meristem is a region of plant tissue, found chiefly at the growing tips of roots and shoots and in the cambium, consisting of actively dividing cells forming new tissue. Meristematic cells give rise to various organs of a plant and are responsible for growth.
Typically, in meristem culture, the apical shoot tip meristem, also known as the growing tip, is excised from the plant and grown on a Murashige and Skoog (MS) medium. Cells at the shoot apical meristem summit serve as stem cells to the surrounding peripheral region, where they proliferate rapidly and are incorporated into differentiating leaf or flower primordia. In short, in meristem culture, tissue labs excise the stem cells of the plant from the growing tip and grow these cells to produce new plantlets.
Meristematic cells, have in the past, been considered as virus free because:
1). Meristematic cells have a higher multiplication rate than viral cell multiplication rates.
2). Viruses generally move in plant body through vascular bundles. There is the absence of vascular bundles in meristematic regions.
3). Meristematic cells have a high auxin concentration and cytokinin concentration and low pH value in which viruses can not survive.
4). Meristematic cells have a high metabolic activity.
5). Meristematic cells have an RNA silencing mechanism. According to some findings, it has been hypothesized that the defense systems in meristematic regions treat viruses as foreign particles thus preventing the viruses into the meristematic regions.
To view meristem culture in action visit this youtube link https://www.youtube.com/watch?v=cD9CFtpLL2s
Multiple Viruses and/or Viroids Exacerbate Yield Losses
There has been a lot of attention paid lately to Latent Hop Viroid disease (LHVd), AKA ‘Dudding’, in North American cannabis production. Based on this, firstly, let me stress that while growers focus solely on LHVd they may be losing site of a much broader problem. This being that a single viroid or virus is often not a limiting factor in producing plants such as cannabis; however, in many cases, what may instead be occurring is a complex infection with LHVd and other viroids and/or viruses. That is, loss of plant vigour and yields may be case of two or more viroids and/or viruses infecting the crop at the same time.
Other than this, if growers are fixated on a single virus or viroid they may miss the next viroid or virus that surfaces as a threat to North American cannabis production.
Preventing the Spread of Viruses and Viroids in Cannabis Production
What I will do now is outline a tissue culture and cannabis production facility I am currently designing for a large legal rec and medicinal cannabis producer in Colorado. In doing this, I will place up conceptual rough designs of a tissue culture lab and a clone and mother area that incorporates level 1 biosecurity (the highest level of biosecurity for this operaqtion) to ensure viruses and viroids are not introduced and spread through the grow operation. Additionally, I’ll include written material that acts as a brief/outline to the client who has commissioned me to conceptualise, design and implement the biosecure tissue culture lab and clone and mother area of the grow operation. Through approaching things this way, the following material should offer insights into what it takes to build a biosecure grow facility, where the introduction and/or spread of disease is significantly reduced when compared to non-biosecure grow operations.
I should add that this is part of a much larger project where there will also be biosecurity in light dep and ‘supp’ (supplemental lighting) green houses where vegetative and flowering plants will be produced. The aim though is to introduce disease free material into the vegetative and flowering greenhouses to ensure that plants are able to complete their lifecycle free of disease.
The brief to the client is as follows.
Biosecurity Measures for Preventing Viroids and Viruses (introduction and spread)
The key to preventing the spread of viroids and viruses into and through cannabis production facilities is biosecurity.
Biosecurity is a set of practices used to prevent, minimize and manage the transmission of diseases and pests including their introduction, spread and release. Implementing and enhancing biosecurity measures within a place of production will help to prevent the transmission/spread of viroids and viruses in crops.
Where preventing viroid and/or virus transmission in grow operations (whether indoor or greenhouse) is concerned, among other things, biosecurity comes down to preventing insect vectors entering the growing environment, identifying and quickly eliminating or reducing insect vectors should they enter the growing environment, and thoroughly sterilizing equipment that is used in manual wounding (e.g. cloning and defoliation) and other growing practices. Additionally, biosecurity requires establishing practices that keep potential threats isolated from the crop production area. For this reason, grow facilities should be designed and built to incorporate layered biosecurity zones with level 1 being the highest level of biosecurity and level 4 being the lowest.
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