Clean stock: A Beginner’s Guide

by Josh Schneider

Zenleaf Labs

The legalization of cannabis in many states and the legalization of Hemp federally has substantially expanded the cultivation of this important crop.   However with increased movement of plants, the risk of diseases that can cause problems also increases.  There is much online to cause confusion and concern about how best to deal with these increased risks, but much of that information is wrong.  What I have compiled here is the distilled information from my 20 years in the horticulture and young plant business and can serve as a guide to growers looking to improve the health and vigor of their crops and preserve and protect the varieties that are important to the future of the industry.   The key to keeping your plants clean and green is sanitation, insect and human control.  Take a walk with me through the process and learn how we have successfully reduced the risk of virus diseases in challenging crops in ornamental horticulture.

What is a virus, and what does it do?

A virus is a snippet of DNA or RNA that is inanimate, but uses the cells of other plants or animals to replicate itself. In principle, a virus infects a cell and forces the cell to produce countless more copies of the virus. These viral copies can then infect other cells, and the process is repeated time and time again. Hijacking the cells and re-programming them to only focus on the production of more replicate virus copies in many cases has severe consequences for the cells, and in the end for the entire organism. Virus diseases are in many cases potentially deadly (e.g. Ebola, HIV in humans), but in most cases sublethal, severely decrease productivity, viability and health. In some cases, a virus infection can remain hardly visible for long periods of time (“latent”), only to explode into visible symptoms – and crop decimation – when the conditions are right (plant stress or environmental conditions are optimal).

Virus issues with clonal crops

In many cases Cannabis has been grown extensively from seed. Given the dioecious (male and female flowers on different plants) characteristics of this crop, this can result in a substantial percentage of (largely useless) male plants that increases both the cost of production and inefficiency. Furthermore, seed-raised plants can display a range of genetic variability; while this is acceptable for small-scale production, large-scale production of medical or recreational cannabis require reliability and predictability of crop characteristics and active ingredient content and so clonal production is more reliable and consistent.

With adoption of clonal cutting production with this crop, all-female clones with precise yield and active ingredient content are possible, making large-scale commercial production of this crop much, much more predictable and thus profitable.

Nevertheless, clonal production of any crop also poses inherent – and considerable – risks. In clonal production, all plants are derived from a single plant and are all genetically identical – hence also their resistance – and susceptibility – to plant diseases. Furthermore, if the plant that is used for propagation is infected by plant disease – ie virus, fungi or bacteria – the probability of all clonal progeny of that plant being likewise infected is extremely high.

Virus diseases in Cannabis production

In today’s cannabis industry, clonal material is readily traded and swapped between production sites. Given how widespread certain virus diseases are, and how all clonal progeny from an infected plant are likewise infected, this scenario is a potential catastrophe in the making. Furthermore, given the often clandestine nature of cannabis production, less research has been conducted to ascertain which other virus diseases could affect this crop.  Extensive experience with other crops, would suggest 10-15 virus diseases can potentially affect cannabis production.

We are only beginning to learn enough about the susceptibility of Cannabis to the vast range of diseases that can affect Cannabis.  Several serious virus and viroid diseases are among these believed to be of concern to cannabis growers. Among those are: hop latent viroid (HpLVd,) hemp streak virus, cannabis cryptic virus (CanCV), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV) and arabis mosaic virus (ArMV). Of these, cucumber mosaic, arabis mosaic and tobacco mosaic have very broad host ranges, are easily transmitted and could potentially decimate a cannabis crop. Transmission can be mechanical (TMV), by insects such as aphids (CMV) or nematodes (ArMV). Once a production crop or mother-stock range is infected, it can prove extremely difficult to eliminate, and the risk of re-infection is great. Hence, subsequent crops are at extreme risk without proper management and hygiene measures.

Not only virus disease – other pathogens cause problems too

Xanthomonas and Agrobacterium are both common, widespread and extremely damaging bacterial diseases that can affect and potentially devastate cannabis production. A clean-stock program for clonal cannabis production, coupled with stringent hygiene measures in production, are the only measures available to prevent such infection and their spread within a crop.

Fusarium, Sclerotinia, Phoma, Alternaria, Colletotrichum and many other fungal diseases can affect a cannabis crop; whilst these cannot be entirely excluded from a crop cycle with clean stock measures, starting from clean clonal material substantially reduces susceptibility and the risk of infection.

How virus diseases are spread

Plant viruses have developed multiple methods of transmission throughout history. Many are transmitted by insects that feed on the plants, ingesting the virus-infected sap and then moving on to other plants and infecting them as well. Aphids, whitefly, thrips and leafhoppers are common and widespread “vectors” of virus disease; insects from outside can easily infect an otherwise clean production range.

While insect control is an important factor in risk mitigation, one of the major causes of virus transmission is the human element. Sanitation protocols – no matter how stringent – are only reliable if followed to the letter, and humans – especially those in cannabis production –  often struggle with adherence to protocol.  Bringing new clones into a nominally-clean program without pathogen testing can easily contaminate a clean program, so strict quarantine processes are important for risk reduction.  In house clone propagation is another risk factor. The plant sap on knives and scissors used to propagate cuttings, prune plants or harvest flowers can likewise be extremely efficient in transmitting diseases from plant to plant and production range to production range. Indeed, some virus and viroid diseases are easily transmitted simply by touch – ie plants rubbing against each other, and/or workers and their clothing rubbing against or touching the plants. In fact, tobacco mosaic virus (TMV) is so aggressive and stable that it is often present on the clothes and skin of anyone who smokes cigarettes or lives with a smoker thus providing many opportunities for infection.

Testing and Indexing for Virus Diseases

Pathogen Indexing is a combination of sequential tests and processes used to ascertain – to a high degree of certainty  – the presence or absence of pathogens in the plant vascular system.  There are multiple established methods for identifying virus, bacterial and fungal infections. The so-called ELISA-method (ie “enzyme-linked immunosorbent assay”) is a commonly-used indexing method in commercial plant crops; it is based on antisera developed for that specific plant virus. This method allows for screening large volumes of plant material effectively and efficiently though regular randomized or specific testing.   While commercial antisera are available, they are often difficult to source and testing protocols should be done by seasoned laboratory technicians to ensure clonal material is correctly screened and test results are accurately interpreted.

PCR or “polymerase chain reaction” is another method for detecting the presence of virus.  This highly sensitive and specific  method is predominantly used to find previously unidentified viruses for which no antisera exists.

Keeping clones clean – the Laboratory Solution

The sterile, insect- and pathogen-free laboratory conditions in plant tissue culture, so-called in-vitro methods, are the safest and most reliable method for keeping clean, pathogen-indexed clonal material.  Stringent tracking and maintenance systems for all in-vitro clones, and re-indexing at regular, clearly defined intervals are necessary to guarantee cleanliness and long-term integrity of the clonal material. Clean material from the lab can be rooted and brought from the lab into the greenhouses and established as mother plants on a predetermined schedule to supply healthy and vigorous young plants for growers.

Utilizing tissue-culture methods to establish and maintain clean, disease-free clonal material is a highly-specialized, but widespread method in commercial agricultural and horticultural crops.  This process has nothing to do with genetic engineering / GMO. Clean clones from tissue culture can easily be adapted to organic and “Clean Green” cultivation systems.

Tissue culture is a proven method to preserve and protect clean, pathogen-indexed material under sterile conditions for both breeding and production.

Eliminating virus diseases – A Substantial Challenge

Many virus diseases can be eliminated from clonal plant material, but this requires a great deal of expertise, and cannot be conducted under standard greenhouse/growing room conditions but must be done in a qualified pathogen elimination lab using tissue culture techniques, ie initiation invitro and cultivation of clonal plant material under sterile conditions in the laboratory. Should a clone test positive or become infected, certain proprietary techniques and treatments can be used to eliminate virus infections.  However, these techniques are not always successful for all plant virus and viroid diseases, and can sometimes take a year or more – and multiple treatments – to complete.  Even then plants must be weaned from tissue culture and grown out and stressed to ascertain if the pathogen was elimimated.

NEXT TO GODLINESS: How to Keep Your Crop Clean

Starting clean in every grow cycle

In greenhouse / grow-room production of clonal crops, it is imperative that clean starter material of defined integrity is utilized for each and every crop cycle. Furthermore, the cultivation environment and all implements and tools used during the cultivation cycle must be disinfected before the new crop is brought in.

Hygiene procedures in production

In order to prevent re-infection of otherwise clean clonal material during the crop cycle, stringent hygiene measures should be implemented.  Greenhouse hygiene procedure implementation and staff training processes and evaluations as well as risk analysis for each pathogen are keys to maintaining a clean stock environment.  Gloves, lab coats and tool sterilization protocols can be very effective at reducing or eliminating the risk of infection and transmission.  But the real key is to train staff to use the same system every time.  Consistency will help keep infections from getting into your stock range, but also can help stop the transmission among stock plants should infection occur.  Whether tools are sterilized after every plant, every 5 plants, every 5 feet, or every 10 minutes is less important than adherence to the system consistently.   The optimal pathogen killing sterilizer is a 10% bleach solution.  Other products like alcohol or lysol can help but 10% bleach kills all known pathogens.   Some growers place tubs of bleach down the sides of tables labeled for dirty and clean tools.  Already used clippers are dropped into one tub and another tool is taken from the clean tub.  Each worker can carry with them a small bottle of 10% bleach spray solution that they use to clean their gloved hands when they change tools.   A firm, almost religous adherence to a sanitation protol will eliminate many of the worst problems before they can even become problems.

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