Tag Archives: pathogenic

By Bernie Lorenz, PhD

The journal Frontiers in Plant Science recently shared an important article from researchers at Simon Fraser University in British Columbia, highlighting the “Pathogens and Molds Affecting Production and Quality of Cannabis Sativa.”

As a chemist focused on the science of preventing and mitigating mold in greenhouse and indoor cannabis grow facilities, this piece was fascinating to me. Like many others, it details and explains prevalent mold like Penicillium, Cladosporium and Aspergillus – things I see in grows every day.

But wait, there’s more fungi

The research and resulting article also brought up another type of fungi – endophytic mold. Endophytic mold usually lives symbiotically with plants, or is at least beneficial for both plant and fungi.

But not always.

In the past, the industry has believed that damaging mold spores were found on the outside of the flower. When moved, that flower would release the spores and send them flying – often creating massive cross-contamination issues for indoor grows.

“While cannabis is an incredibly powerful plant in terms of its medicinal properties, it is unfortunately highly susceptible to many pest and pathogens,” says Hope Jones, PhD, CEO, Adivina & ECS. “And it is this susceptibility that is so challenging to many inexperienced or undisciplined grow operations.”

Now, however, we know that there’s another culprit to add to the list: the inner parts of the plant can also be a source of endophytic cross contamination and mold.

Since it grows inside of the plant, this fungus creates high spore counts that can cross contaminate from outside, into the flower.

Treating mold in a facility

Here’s the good news:

This seemingly bad news – that there’s a new fungus to worry about, and it is inside the flower – may actually help cannabis grows struggling with mold, and those who are following the proper protocols already.

A petri dish of mold growth from tested cannabis Photo credit: Steep Hill

Effective mitigation protocols can include things like treating HVAC systems, controlling humidity, using products like chlorine dioxide to treat irrigation lines, enforcing protective clothing and shoe covers for employees, reducing the amount of in-and-out for employees around grow rooms.

These are important upstream and environmentally-focused integrated pest management (IPM) programs that will usually keep facilities clean and relatively mold-free.

But if these programs are in place, and there’s still an issue, Endophytic fungi may be to blame.

If you are having ongoing mold issues but have ruled out cross-contamination and a facility without proper protocol, look to the mother plant.

“Small mistakes in agricultural practices are amplified with cannabis,” Dr. Jones continues. “And today’s propagation practices of traditional cloning add to this vulnerability. Cannabis is an annual plant and by keeping mothers in a perpetual state of vegetative growth for years, and taking repetitive cuttings produces clones in a highly stressed state. This stressed state diminishes genetic potential and weakens a plant’s ability to fight disease and pests.”

Testing for and addressing endophytic fungi

If these concerns are ringing a bell, remember, there is also a way to test for Endophytic mold.

Checking cuttings from suspected mother plants over a period of time is the best way to see if the Endophytic mold is present.

A section of the mother plant cutting is placed into a solution (for example, as outlined by the article, a very concentrated hypochlorite followed by 70% Ethanol) that will kill all of the microorganisms that are present on the surface of the plant tissues.

A large tissue culture facility run in the Sacramento area that produces millions of nut and fruit trees clones a year.

From there, an unadulterated dissection of the internal tissues can be extracted and cultured for quantification and identification of endophytic fungi.

“Tissue culture offers a form of genetic rebooting returning the plant to its natural genetic potential and thereby strengthening its natural ability to defend against environment assault,” says Dr. Jones. “It also allows the breeder to conduct pathogenic disease testing which provides the entire industry with a higher level of scientific certainty and analysis.”

If you find this mold inside of the mother plant, your facility’s mold problem could be a systemic issue, not an environmental one.

If you do find that Endophytic mold is causing issues, of course, you may have to destroy the mother plant.

This should not mean the end of a strain. Tissue culture on a cutting is an option that can eliminate the unwanted fungi and save the genetics. Using those genetics to regrow a mother will start fresh and avoid the intrinsic mold that was plaguing the strain prior.

Growing knowledge

The practice of checking mother plants for Endophytic mold is not yet commonplace in cannabis, but the hemp business is leading the way.

They’re testing to create very clean plants, so you don’t have issues during cultivation.

Major growers in the U.S. could save millions in lost harvests with mold mitigation. If your current IPM program isn’t doing the trick, you may want to follow in hemp’s footsteps and look inside the plant.

October 21, 2019

3 Essential Components of Microbial Safety Testing

By Heather Ebling

1 Comment

Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who are at risk of developing harmful and potentially fatal infections.

As a result, regulatory bodies in the United States and Canada mandate testing cannabis products for certain microbes. The two most popular methods for microbial safety testing in the cannabis industry are culture-based testing and quantitative polymerase chain reaction (qPCR).

When considering patient safety, labs should choose a method that provides an accurate account of what is living on the sample and can specifically target the most harmful microbes, regardless of the matrix.

1. The Method’s Results Must Accurately Reflect the Microbial Population on the Sample

The main objective of any microbial safety test is to give the operator an indication of the microbial population present on the sample.

Figure 1: MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

Culture-based methods measure contamination by observing how many organisms grow in a given medium. However, not all microbial organisms grow at the same rate. In some cases, certain organisms will out-compete others and as a result, the population in a post-culture environment is radically different than what was on the original sample.

One study analyzed fifteen medicinal cannabis samples using two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). Figure 1 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.

The Live Dead Problem

Figure 2: The enzyme is instantaneously inactivated when lysis buffer is added

One of the common objections to using qPCR for microbial safety testing is the fact that the method does not distinguish between live and dead DNA. PCR primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. Critics claim that this can lead to false positives because DNA from non-viable organisms can inflate results. This is often called the Live-Dead problem. However, scientists have developed multiple solutions to this problem. Most recently, Medicinal Genomics developed the Grim Reefer Free DNA Removal Kit, which eliminates free DNA contained in a sample by simply adding an enzyme and buffer and incubating for 10 minutes. The enzyme is instantaneously inactivated when lysis buffer is added, which prevents the Grim Reefer Enzyme from eliminating DNA when the viable cells are lysed (see Figure 2).

2. Method Must Be Able to Detect Specific Harmful Species

Toxic Aspergillus spp., which is responsible for at least one confirmed death of a cannabis patient, grows poorly in culture mediums and is severely underreported by current culture-based platforms. And even when Aspergillus does grow in culture, there is a certain non-pathogenic Aspergillus species that look remarkably similar to their pathogenic cousins, making it difficult to speciate using visual identification alone.

Conversely, qPCR assays, such as the PathoSEEK, are designed to target DNA sequences that are unique to pathogenic Aspergillus species, and they can be run using standard qPCR instruments such as the Agilent AriaMx. The primers are so specific that a single DNA base difference in the sequence can determine whether binding occurs. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.

Additionally, Medicinal Genomics has developed a multiplex assay that can detect the four pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus) in a single reaction.

3. The Method Must Work on Multiple Matrices

Figure 4: The team also placed TSB without any E. coli onto a petrifilm to serve as a control.

Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, The Medicinal Genomics team spiked a known amount of live E. coli into three different environments: tryptic soy broth (TSB), hemp oil and hard candy. The team then homogenized the samples, pipetted amounts from each onto 3M™ Petrifilm E. coli / Coliform Count (EC) Plates, and incubated for 96 hours. The team also placed TSB without any E. coli onto a petrifilm to serve as a control. Figures 3 and 4 show the results in 24-hour intervals.

Table 1: DNA was spiked into various MIPs

This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.

Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast, the qPCR signal from the Agilent AriaMx was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.

Table 3: SenSATIVAx DNA extraction can successfully lyse the cells of the microbes

Table 2: Different numbers of DNA copies spiked into chocolate

This is not an issue with DNA-based methods, so long as the DNA extraction method has been validated on these matrices. For example, the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table 1, and at different numbers of DNA copies into chocolate (Table 2). The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and the PathoSEEK detection assay can successfully detect that range of DNA. Table 3 demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a variety of organisms spiked onto cannabis flower samples.

February 5, 2018

Top 4 Food Safety Hazards for the Cannabis Industry

By Steven Burton

18 Comments

As many US States and Canadian provinces approach legalization of cannabis, the question of regulatory oversight has become a pressing issue. While public awareness is mainly focused on issues like age restrictions and impaired driving, there is another practical question to consider: should cannabis be treated as a drug or a food product when it comes to safety? In the US, FDA governs both food and drugs, but in Canada, drugs are regulated by Health Canada while food products are regulated under the CFIA.There are many food safety hazards associated with cannabis production and distribution that could put the public at risk, but are not yet adequately controlled

Of course, there are common issues like dosage and potency that pharmaceutical companies typically worry about as the industry is moving to classifying its products in terms of percentage of chemical composition (THC, CBD, etc. in a strain), much as we categorize alcohol products by the percentage of alcohol. However, with the exception of topical creams and ointments, many cannabis products are actually food products. Even the herb itself can be brewed into teas, added to baked goods or made into cannabis-infused butters, oils, capsules and tinctures.

As more people gain access to and ingest cannabis products, it’s only a matter of time before food safety becomes a primary concern for producers and regulators. So when it comes to food safety, what do growers, manufacturers and distributors need to consider? The fact is, it’s not that different from other food products. There are many food safety hazards associated with cannabis production and distribution that could put the public at risk, but are not yet adequately controlled. Continue reading below for the top four safety hazards for the cannabis industry and learn how to receive free HACCP plans to help control these hazards.

Aflatoxins on Cannabis Bud

Just like any other agricultural product, improper growing conditions, handling and storage can result in mold growth, which produce aflatoxins that can cause liver cancer and other serious health problems. During storage, the danger is humidity; humidity must be monitored in storage rooms twice a day and the meter must be calibrated every month. During transportation, it is important to monitor and record temperatures in trucks. Trucks should also be cleaned weekly or as required. Products received at a cannabis facilities should be tested upon receiving and contaminated products must always be rejected, segregated and disposed of safely.

Petri dish containing the fungus Aspergillus flavus. It produces carcinogenic aflatoxins, which can contaminate certain foods and cause aspergillosis, an invasive fungal disease.
Photo courtesy of USDA ARS & Peggy Greb.

Chemical Residues on Cannabis Plants

Chemical residues can be introduced at several points during the production and storage process. During growing, every facility should follow instructions for applying fertilizers and pesticides to crops. This includes waiting for a sufficient amount of time before harvesting. When fertilizer is being applied, signs must be posted. After cannabis products have been harvested, chemical controls must be in place. All chemicals should be labelled and kept in contained chemical storage when not in use to prevent contamination. Only food-grade chemicals (e.g. cleaners, sanitizers) should be used during curing, drying, trimming and storage.

Without a comprehensive food safety program, problems will inevitably arise.There is also a risk of excessive concentration of chemicals in the washing tank. As such, chemical concentrations must be monitored for. In general, water (obviously essential for the growing process) also carries risks of pathogenic bacteria like staphylococcus aureus or salmonella. For this reason, city water (which is closely controlled in most municipalities) should be used with an annual report and review. Facilities that use well water must test frequently and water samples must be tested every three months regardless.

Pathogenic Contamination from Pest Infestations

Insects, rodents and other pests spread disease. In order to prevent infestations, a pest control program must be implemented, with traps checked monthly by a qualified contractor and verified by a designated employee. It is also necessary to have a building procedure (particularly during drying), which includes a monthly inspection, with no holes or gaps allowed. No product should leave the facility uncovered to prevent fecal matter and other hazards from coming into contact with the product. Contamination can also occur during storage on pallets, so pallets must be inspected for punctures in packaging material.

Furthermore, even the best controlled facility can fall victim to the shortcomings of their suppliers. Procedures must be in place to ensure that suppliers are complying with pest and building control procedures, among others. Certifications should be acquired and tracked upon renewal.

Pathogenic Contamination Due to Improper Employee Handling

Employee training is key for any food facility. When employees are handling products, the risk of cross-contamination is highest. Facilities must have GMP and personnel hygiene policies in place, with training conducted upon hiring and refreshed monthly. Employees must be encouraged to stay home when sick and instructed to wear proper attire (gloves, hair nets, etc.), while glass, jewelry and outside food must not be allowed inside the facility. Tools used during harvesting and other stages may also carry microorganisms if standard cleaning procedures are not in place and implemented correctly by employees.

As the cannabis industry grows, and regulatory bodies like the FDA and CFIA look to protect public safety, we expect that more attention will be paid to other food safety issues like packaging safety (of inks and labels), allergen control and others. In the production of extracts, for example, non-food safe solvents could be used or extracts can be mixed with ingredients that have expiration dates, like coconut oil. There is one area in which the cannabis industry may lead the way, however. More and more often, risks of food terrorism, fraud and intentional adulteration are gripping the food industry as the global food chain becomes increasingly complex. It’s safe to say that security at cannabis facilities is probably unparalleled.

All of this shows that cannabis products, especially edibles (and that includes capsules and tinctures), should be treated the same as other food products simply because they have the same kinds of hazards. Without a comprehensive food safety program (that includes a plan, procedures, training, monitoring and verification), problems will inevitably arise.