Tag Archives: fungus

Soapbox

Clean Grow Still Failing? Check for Endophytic Mold

By Bernie Lorenz, PhD
No Comments

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.

Hope Jones, PhD, CEO of Adivina & ECS

“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.

Product Safety Hazards: Looking Beyond Food Safety in Cannabis

By Radojka Barycki
No Comments

I think that we need to start changing the terminology around the hazards associated with cannabis from food safety hazards to product safety hazards. These hazards have not only been associated with harmful effects for those that ingest cannabis infused products, but also for those that consume the cannabis products in other ways such as inhalation (vaping or smoking). So, when we refer to these hazards as food safety hazards, the immediate thought is edibles, which misleads cultivators, manufacturers and consumers to have a false sense of security around the safety of products that are consumed in other ways.

Food processing and sanitation
By standardizing and documenting safety procedures, manufacturers mitigate the risk of cannabis-specific concerns

There are several product safety hazards that have been associated with cannabis. These hazards can become a public health problem if not controlled as they could harm the consumer, regardless of the method of consumption.

Let’s take a look at the different types of hazards associated cannabis:

Biological Hazards refer to those microorganisms that can cause illness to the consumer of a product that contain them. They are not visible to the naked eye and are very dangerous when their metabolic by-products (toxins) are ingested or their spores are inhaled. The symptoms for illnesses caused by these microorganisms will vary. Consumers may experience gastrointestinal discomfort (vomiting, diarrhea), headaches, fever and other symptoms. The ingestion of these pathogens, allergens or their by-products may lead to death, if the illness is not treated on time or if the consumer of the product is immunocompromised. In addition, the inhalation of mold spores when smoking cannabis products, can lead to lung disease and death. Some of the biological hazards associated with cannabis are: Salmonella sp., E. coli, Clostridium botulinum, Aspergillus sp. and Penicillium sp.

Chemical Hazards refer to those chemicals that can be present in the plant or finished product due to human applications (pesticides), operational processes (extraction solvents and cleaning chemicals), soil properties (heavy metals), environmental contamination (radiological chemicals) or as a result of occurring naturally (mycotoxins and allergens). Consuming high concentrations of cleaning chemicals in a product can lead to a wide range of symptoms from mild rash, burning sensation in the oral-respiratory system, gastrointestinal discomfort or death. In addition, long term exposure to chemicals such as pesticides, heavy metals, radiological contaminants and mycotoxins may lead to the development of cancers.

Physical Hazards refer to those foreign materials that may be present in the plant or finished product. Foreign materials such as rocks, plastics or metals can cause harm to the consumer by chipping teeth or laceration of the mouth membranes (lips, inner cheeks, tong, esophagus, etc.) In the worst-case scenario, physical hazards may lead to choking, which can cause death due to asphyxiation.

These hazards can be prevented, eliminated or reduced to an acceptable level when foundational programs (Good Agricultural/Cultivation Practices, Good Manufacturing Practices, Allergen Management Program, Pest Control, etc.) are combined with a Food [Product] Safety Plan. These lead to a Food [Product] Safety Management System that is designed to keep consumers safe, regardless of the method of consumption.

Microbiology 101 Part One

By Kathy Knutson, Ph.D.
No Comments

I have been studying microorganisms for over 35 years, and the elusive critters still fascinate me! Here in Microbiology 101, I write about the foundation of knowledge on which all microbiologists build. You may have a general interest in microbiology or have concerns in your operation. By understanding microbiology, you understand the diversity of microorganisms, their source, control of microorganisms and their importance.

Part 1

The term microbiology covers every living being we cannot see with the naked eye. The smallest microbe is a virus. Next in size are the bacteria, then yeast and mold cells, and the largest microbes are the protozoans. The tiny structure of a virus may be important in the plant pathology of cannabis, but will not grow in concentrates or infused products. A virus is not living, until it storms the gate of a living cell and overtakes the functions within the cell. Viruses are the number one cause of foodborne illness, with the number one virus called Norovirus. Think stomach flu. Think illness on cruise ships. Viruses are a food service problem and can be prevented by requiring employees to report sickness, have good personal hygiene including good hand washing, and, as appropriate, wear gloves. Following Good Manufacturing Practices (GMPs) is critical in preventing the transfer of viruses to a product where the consumer can be infected.

The petri dishes show sterilization effects of negative air ionization on a chamber aerosolized with Salmonella enteritidis. The left sample is untreated; the right, treated. Photo courtesy of USDA ARS & Ken Hammond

The largest microbial cell is the protozoan. They are of concern in natural water sources, but like viruses, will not grow in cannabis products. Control water quality through GMPs, and you control protozoans. Viruses and protozoans will not be further discussed here. Bacteria, yeast and mold are the focus of further discussion. As a food microbiologist, my typical application of this information is in the manufacturing of food. Because Microbiology 101 is a general article on microbiology, you can apply the information to growing, harvesting, drying, manufacture of infused products and dispensing.

It is not possible to have sterile products. Even the canning process of high temperature for an extended time allows the survival of resistant bacterial spores. Astronauts take dehydrated food into space, and soldiers receive MREs; both still contain microbes. Sterility is never the goal. So, what is normal? Even with the highest standards, it is normal to have microbes in your products. Your goal is to eliminate illness-causing microorganisms, i.e. pathogens. Along the way, you will decrease spoilage microbes too, making a product with higher quality.

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

Yeast and mold were discussed on CIJ in a previous article, Total Yeast & Mold Count: What Cultivators & Business Owners Need to Know. Fuzzy mold seen on the top of food left in the refrigerator too long is a quality issue, not a safety issue. Mold growth is a problem on damaged cannabis plants or cuttings and may produce mycotoxin, a toxic chemical hazard. Following Good Agricultural Practices (GAPs) will control mold growth. Once the plant is properly dried, mold will not grow and produce toxin. Proper growing, handling and drying prevents mycotoxins. Like mold, growth of yeast is a quality issue, not a safety issue. As yeast grow, they produce acid, alcohol and carbon dioxide gas. While these fermentation products are unwanted, they are not injurious. I am aware that some states require cannabis-infused products to be alcohol-free, but that is not a safety issue discussed here.

What are the sources of microorganisms?

People. Employees who harvest cannabis may transfer microorganisms to the plant. Later, employees may be the source of microbes at the steps of trimming, drying, transfer or portioning, extract processing, infused product manufacture and packaging.

Ingredients, Supplies and Materials. Anything you purchase may be a source of microorganisms. Procure quality merchandise. Remember the saying, “you get what you pay for.”

Environment. Starting with the outdoors, microbes come from wind, soil, pests, bird droppings and water. When plants are harvested outdoors or indoors, microbes come from the tools and bins. Maintain clean growing and harvesting tools in good working condition to minimize contamination with microbes. For any processing, microbes come from air currents, use of water, and all surfaces in the processing environment from dripping overhead pipes to floor drains and everything in between.

In Part 2 I will continue to discuss the diversity of microorganisms, and future articles will cover Hazard Analysis and Critical Control Points (HACCP) and food safety in more detail. What concerns do you have at each step of operations? Are you confident in your employees and their handling of the product? As each state works to ensure public health, cannabis-infused products will receive the same, if not more, scrutiny as non-cannabis food and beverages. With an understanding and control of pathogens, you can focus on providing your customers with your highest quality product.

Total Yeast & Mold Count: What Cultivators & Business Owners Need to Know

By Parastoo Yaghmaee, PhD
2 Comments

Editor’s note: This article should serve as a foundation of knowledge for yeast and mold in cannabis. Beginning in January 2018, we will publish a series of articles focused entirely on yeast and mold, discussing topics such as TYMC testing, preventing yeast and mold in cultivation and treatment methods to reduce yeast and mold.


Cannabis stakeholders, including cultivators, extractors, brokers, distributors and consumers, have been active in the shadows for decades. With the legalization of recreational adult use in several states, and more on the way, safety of the distributed product is one of the main concerns for regulators and the public. Currently, Colorado1, Nevada and Canada2 require total yeast and mold count (TYMC) compliance testing to evaluate whether or not cannabis is safe for human consumption. As the cannabis industry matures, it is likely that TYMC or other stringent testing for yeast and mold will be adopted in the increasingly regulated medical and recreational markets.

The goal of this article is to provide general information on yeast and mold, and to explain why TYMC is an important indicator in determining cannabis safety.

Yeast & Mold

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

Yeast and mold are members of the fungi family. Fungus, widespread in nature, can be found in the air, water, soil, vegetation and in decaying matter. The types of fungus found in different geographic regions vary based upon humidity, soil and other environmental conditions. In general, fungi can grow in a wide range of pH environments and temperatures, and can survive in harsh conditions that bacteria cannot. They are not able to produce their own food like plants, and survive by breaking down material from their surroundings into nutrients. Mold cannot thrive in an environment with limited oxygen, while yeast is able to grow with or without oxygen. Most molds, if grown for a long enough period, can be detected visually, while yeast growth is usually detected by off-flavor and fermentation.

Due to their versatility, it is rare to find a place or surface that is naturally free of fungi or their spores. Damp conditions, poor air quality and darker areas are inviting environments for yeast and mold growth.

Cannabis plants are grown in both indoor and outdoor conditions. Plants grown outdoors are exposed to wider ranges and larger populations of fungal species compared to indoor plants. However, factors such as improper watering, the type of soil and fertilizer and poor air circulation can all increase the chance of mold growth in indoor environments. Moreover, secondary contamination is a prevalent risk from human handling during harvest and trimming for both indoor and outdoor-grown cannabis. If humidity and temperature levels of drying and curing rooms are not carefully controlled, the final product could also easily develop fungi or their growth by-product.

 What is TYMC?

TYMC, or total yeast and mold count, is the number of colony forming units present per gram of product (CFU/g). A colony forming unit is the scientific means of counting and reporting the population of live bacteria or yeast and mold in a product. To determine the count, the cannabis sample is plated on a petri dish which is then incubated at a specific temperature for three to five days. During this time, the yeast and mold present will grow and reproduce. Each colony, which represents an individual or a group of yeast and mold, produces one spot on the petri dish. Each spot is considered one colony forming unit.

Why is TYMC Measured?

TYMC is an indicator of the overall cleanliness of the product’s life cycle: growing environment, processing conditions, material handling and storage facilities. Mold by itself is not considered “bad,” but having a high mold count, as measured by TYMC, is alarming and could be detrimental to both consumers and cultivators. 

Aspergillus species niger
Photo: Carlos de Paz, Flickr

The vast majority of mold and yeast present in the environment are indeed harmless, and even useful to humans. Some fungi are used commercially in production of fermented food, industrial alcohol, biodegradation of waste material and the production of antibiotics and enzymes, such as penicillin and proteases. However, certain fungi cause food spoilage and the production of mycotoxin, a fungal growth by-product that is toxic to humans and animals. Humans absorb mycotoxins through inhalation, skin contact and ingestion. Unfortunately, mycotoxins are very stable and withstand both freezing and cooking temperatures. One way to reduce mycotoxin levels in a product is to have a low TYMC.

Aspergillus flavus on culture.
Photo: Iqbal Osman, Flickr

Yeast and mold have been found to be prevalent in cannabis in both current and previous case studies. In a 2017 UC Davis study, 20 marijuana samples obtained from Northern California dispensaries were found to contain several yeast and mold species, including Cryptococcus, Mucor, Aspergillus fumigatus, Aspergillus niger, and Aspergillus flavus.3 The same results were reported in 1983, when marijuana samples collected from 14 cannabis smokers were analyzed. All of the above mold species in the 2017 study were present in 13 out of 14 marijuana samples.4

Aspergillus species niger, flavus, and fumigatus are known for aflatoxin production, a type of dangerous mycotoxin that can be lethal.5 Once a patient smokes and/or ingests cannabis with mold, the toxins and/or spores can thrive inside the lungs and body.6, 7 There are documented fatalities and complications in immunocompromised patients smoking cannabis with mold, including patients with HIV and other autoimmune diseases, as well as the elderly.8, 9, 10, 11

For this reason, regulations exist to limit the allowable TYMC counts for purposes of protecting consumer safety. At the time of writing this article, the acceptable limit for TYMC in cannabis plant material in Colorado, Nevada and Canada is ≤10,000 CFU/g. Washington state requires a mycotoxin test.12 California is looking into testing for specific Aspergillus species as a part of their requirement. As the cannabis industry continues to grow and advance, it is likely that additional states will adopt some form of TYMC testing into their regulatory testing requirements.

References:

  1. https://www.colorado.gov/pacific/sites/default/files/Complete%20Retail%20Marijuana%20Rules%20as%20of%20April%2014%202017.pdf
  2. http://laws-lois.justice.gc.ca/eng/acts/f-27/
  3. https://www.ucdmc.ucdavis.edu/publish/news/newsroom/11791
  4. Kagen SL, Kurup VP, Sohnle PG, Fink JN. 1983. Marijuana smoking and fungal sensitization. Journal of Allergy & Clinical Immunology. 71(4): 389-393.
  5. Centre for Disease control and prevention. 2004 Outbreak of Aflatoxin Poisoning – Eastern and central provinces, Kenya, Jan – July 2004. Morbidity and mortality weekly report.. Sep 3, 2004: 53(34): 790-793
  6. Cescon DW, Page AV, Richardson S, Moore MJ, Boerner S, Gold WL. 2008. Invasive pulmonary Aspergillosis associated with marijuana use in a man with colorectal cancer. Diagnosis in Oncology. 26(13): 2214-2215.
  7. Szyper-Kravits M, Lang R, Manor Y, Lahav M. 2001 Early invasive pulmonary aspergillosis in a leukemia patient linked to aspergillus contaminated marijuana smoking. Leukemia Lymphoma 42(6): 1433 – 1437.
  8. Verweii PE, Kerremans JJ, Voss A, F.G. Meis M. 2000. Fungal contamination of Tobacco and Marijuana. JAMA 2000 284(22): 2875.
  9. Ruchlemer R, Amit-Kohn M, Raveh D, Hanus L. 2015. Inhaled medicinal cannabis and the immunocompromised patient. Support Care Cancer. 23(3):819-822.
  10. McPartland JM, Pruitt PL. 1997. Medical Marijuana and its use by the immunocompromised. Alternative Therapies in Health and Medicine. 3 (3): 39-45.
  11. Hamadeh R, Ardehali A, Locksley RM, York MK. 1983. Fatal aspergillosis associated with smoking contaminated marijuana, in a marrow transplant recipient. Chest. 94(2): 432-433.
  12. http://apps.leg.wa.gov/wac/default.aspx?cite=314-55-102