The use of medical cannabis (MC) has increased tremendously in the last decade (Ruchlemer et al. 2015). One of the main reasons for the rising popularity and interest in MC are the therapeutic qualities of this plant (Ben Amar 2006; Cascio et al. 2017; Russo 2011; Sirikantaramas and Taura 2017). With the increase in use, both for recreational and therapeutic means, reports are starting to accumulate concerning the threat of microbial presence in MC inflorescences and the harmful potential to cannabis consumers, especially in immunocompromised patients (Gargani et al. 2011; Hamadeh et al. 1988; McPartland and McKernan 2017; Ruchlemer et al. 2015). The extent of MC inflorescence infections by fungal CFUs was determined in our initial survey of commercial farms, indicating that without sterilization treatments, levels of CFUs were extremely high, above that permitted by the IMCA in all tested sites, disregarding the sample condition; dried or un-dried, and with or without visible disease symptoms (Fig. 1). Since it is not feasible to cultivate commercial MC under a sterile environment there is an acute need for postharvest MC inflorescence sterilization before usage (Hazekamp 2016).

There have been many reports regarding the efficacy of different non-thermal treatments for food and herb sterilization, especially the utilization of gamma irradiation (Guerreiro et al. 2016; Jeong et al. 2015; Sádecká 2007). Alternatively, e-beam (beta irradiation) and cold plasma, are effective for food sterilization and are safe for human consumption (Jeong et al. 2015; Misra et al. 2019; Moreno et al. 2007; Van Impe et al. 2018). Even though these methods have been applied or suggested for decontaminating MC inflorescences to safeguard its use by immunocompromised patients, there is a lack of knowledge in regards to their efficacy in eliminating deleterious microorganisms.

In this research, we examined the effect of gamma irradiation, e-beam and cold plasma treatments, on the reduction of CFU contamination in artificially inoculated and naturally infected MC inflorescences and trimmed leaves (Table 2). Gamma irradiation was very effective in reducing the CFU levels by approximately 6-log-fold CFU/g inflorescences, at a minimal dosage of 7.5 KGy. Similarly, in the Netherlands, MC is sterilized using ⁶⁰Co gamma irradiation at a dosage of 10 KGy, which is well below the authorized dosage of 30 KGy, permitted by the FDA for irradiation of aromatic herbs and spices (Sádecká 2007). Likewise, in a recent report, ⁶⁰Co gamma irradiation was used to sterilize cherry tomatoes with a radiation dosage of 5.7 KGy that reduced CFU levels from 2.2 log CFU/g to nearly zero, an inactivation efficacy of 99.8% (Guerreiro et al. 2016). In spite of its effectiveness, gamma irradiation remains an expensive sterilization method requiring the usage of radioactive isotopes, specialized equipment and facilities. In contrast, e-beam does not require the use of radioactive isotopes and as such, is considerably more environmentally friendly (Leonhardt 1990). Moreover, in this research we found that e-beam treatments were very effective in eliminating CFUs from infected MC inflorescences applied at low temperatures, below 27 °C, that do not detrimentally affect the active ingredients of MC. In fact, this method is so effective that at a radiation dosage of 10.26 KGy, CFU levels were reduced from 6 log CFU/g inflorescence to 0 (Fig. 2). A similar reduction in CFUs was observed with artificially inoculated inflorescences (Fig. 3).

ED values are an important and useful tool as they indicate the efficacy of treatments and also allow for comparison between different sterilization systems; in general lower ED values represent a higher acute toxicity (Lorke 1983). ED values are extremely relevant in this study since they indicate a measurable value for the efficacy of the treatments. The ED values calculated for e-beam irradiation (Table 1) were also very promising; e.g. ED₁₀, the radiation dosage required to reduce 10% CFU populations of uninoculated and artificially inoculated inflorescences were calculated as 0.6 KGy and 1 KGy, respectively. In comparison, the ED₁₀ value for inactivation of enteric viruses e.g. poliovirus Type 1 on cantaloupe surfaces using e-beam was 4.76 KGy (Shurong et al. 2006). Similarly, e-beam treatment of red pepper powder at a dosage of 3 KGy, reduced CFU levels of total yeasts and molds from 6.62 to 3.71 log CFU/g (Kyung et al. 2019). In our research, we found that a dosage of 12.4 KGy resulted in 90% reduction in CFU levels in artificially inoculated B. cinerea MC inflorescence (Table 1). Although 12.4 KGy is considered a rather high radiation dosage, it is important to note that in this experiment the initial CFU levels were very high (more than 7.7 ± 0.11 log CFU/g). Accordingly, initial CFU levels in commercial MC were constantly much lower, measuring below 5 ± 0.25 CFU/g. This indicates that irradiation values of 8.1 KGy, resulting in a reduction of 90% of uninoculated MC inflorescences are very reasonable considering that the irradiation dosages are much lower than the maximum limit of 30 KGy, according to that authorized by the FDA for irradiation of dry herbs (Sádecká 2007).

Cold plasma treatment of MC inflorescences was also found to be effective for elimination of fungal propagules in this study. After 10 min of plasma exposure, CFU levels in uninoculated MC inflorescences were reduced by approximately 3 log-fold CFU/g. It is important to note that initial recorded infection levels in these experiments were approximately 3 log CFU/g indicating that given a higher infection level, an exposure to 10 min plasma treatment may have resulted in an even greater CFU reduction (Fig. 7). In an additional experiment (data not shown) a cold plasma treatment of 8 min vacuum time followed by 12 min of plasma exposure resulted in CFU reduction of approximately 6 log CFU/g of uninoculated MC inflorescences. Accordingly, in artificially inoculated B. cinerea MC inflorescences the results were improved with a reduction of approximately 4 log CFU/g, after 10 min of plasma exposure (Fig. 8). Similarly, cold plasma treatments conducted in Shaare Zedek Medical Center Israel, resulted in complete sterilization of MC inflorescences (Ruchlemer et al. 2015). In that research, autoclaving and ethylene gas sterilization were found to be as effective for MC inflorescence sterilization, although the two latter methods resulted in a greater reduction of Δ⁹-THC, which is one of the major phytocannabinoids in MC. Cold plasma was also found useful in decontamination of wheat seeds, resulting in complete inactivation of Fusarium nivale-artificially inoculated seeds after as little as 90 s treatment (Zahoranová et al. 2016). An increase in CFU counts after 5 min of plasma exposure compared to that after 2.5 min was recorded in our experiments, which is still unclear (Figs. 7 and 8) and will require further research. In various studies, cold plasma was reported to degrade both mycotoxins and pesticides in in vitro experiments (Ten Bosch et al. 2017; Misra et al. 2011; Sarangapani et al. 2016). Mycotoxins such as aflatoxins, zearalenone and fumonisins are secondary metabolites produced by certain fungi such as Aspergillus and Fusarium spp. that are ubiquitous in MC inflorescences and are known to cause health risks to humans and mammals (McPartland et al. 2017; Misra et al. 2019). Thus, cold plasma treatments may be even more beneficial by not only reducing CFU counts but also by reducing levels of mycotoxins (Ten Bosch et al. 2017; Misra et al. 2019).

Another important aspect when dealing with MC are the active compounds, comprised mainly of different phytocannabinoids and terpenoids. These compounds, especially phytocannabinoids, are responsible for MC therapeutic effects and currently more than 100 different phytocannabinoids have been identified (Cascio et al. 2017; Russo 2011). In recent years, there is a growing understanding that some of the MC therapeutic affects are a result of synergism among the bioactive compounds in certain MC lines and cultivars (Russo 2011). Thus, it is crucial to decontaminate MC inflorescences using a method that causes the least damage to the profiles of these active compounds.