Fungal and mycotoxin contaminants in cannabis and hemp flowers: implications for consumer health and directions for further research.

Medicinal and recreational uses of Cannabis sativa, commonly known as cannabis or hemp, has increased following its legalization in certain regions of the world. Cannabis and hemp plants interact with a community of microbes (i.e., the phytobiome), which can influence various aspects of the host plant. The fungal composition of the C. sativa phytobiome (i.e., mycobiome) currently consists of over 100 species of fungi, which includes phytopathogens, epiphytes, and endophytes, This mycobiome has often been understudied in research aimed at evaluating the safety of cannabis products for humans. Medical research has historically focused instead on substance use and medicinal uses of the plant. Because several components of the mycobiome are reported to produce toxic secondary metabolites (i.e., mycotoxins) that can potentially affect the health of humans and animals and initiate opportunistic infections in immunocompromised patients, there is a need to determine the potential health risks that these contaminants could pose for consumers. This review discusses the mycobiome of cannabis and hemp flowers with a focus on plant-infecting and toxigenic fungi that are most commonly found and are of potential concern (e.g., Aspergillus, Penicillium, Fusarium, and Mucor spp.). We review current regulations for molds and mycotoxins worldwide and review assessment methods including culture-based assays, liquid chromatography, immuno-based technologies, and emerging technologies for these contaminants. We also discuss approaches to reduce fungal contaminants on cannabis and hemp and identify future research needs for contaminant detection, data dissemination, and management approaches. These approaches are designed to yield safer products for all consumers.

Review of nematode interactions with hemp (Cannabis sativa).

The many decades during which the cultivation of Cannabis sativa (hemp) was strongly restricted by law resulted in little research on potential pathogenic nematodes of this increasingly important crop. The primary literature was searched for hemp-nematode papers, resulting in citations from 1890 through 2021. Reports were grouped into two categories: (i) nematodes as phytoparasites of hemp, and (ii) hemp and hemp products and extracts for managing nematode pests. Those genera with the most citations as phytoparasites were Meloidogyne (root-knot nematodes, 20 papers), Pratylenchus (lesion nematodes, 7) and Ditylenchus (stem nematodes, 7). Several Meloidogyne spp. were shown to reproduce on hemp and some field damage has been reported. Experiments with Heterodera humuli (hop cyst nematode) were contradictory. Twenty-three papers have been published on the effects of hemp and hemp products on plant-parasitic, animal-parasitic and microbivorous species. The effects of hemp tissue soil incorporation were studied in five papers; laboratory or glasshouse experiments with aqueous or ethanol extracts of hemp leaves accounted for most of the remainder. Many of these treatments had promising results but no evidence was found of large-scale implementation. The primary literature was also searched for chemistry of C. sativa roots. The most abundant chemicals were classified as phytosterols and triterpenoids. Cannabinoid concentration was frequently reported due to the interest in medicinal C. sativa. Literature on the impact of root-associated chemicals on plant parasitic nematodes was also searched; in cases where there were no reports, impacts on free-living or animal parasitic nematodes were discussed.

Endophytic Beauveria bassiana increases galling of 'Rutgers' tomato roots with Meloidogyne incognita.

Beauveria bassiana is endophytic in many plant species and has been shown to protect host plants against insect pests and plant pathogens. However, less is known about its activity against plant-parasitic nematodes. In vitro and plant assays were conducted to determine the effect of B. bassiana 11-98 (Bb) on Meloidogyne incognita (root-knot nematode; RKN). Beauveria bassiana was confirmed as an endophyte in 'Rutgers' tomato and colonization patterns of Bb in 'Rutgers' (highly susceptible to RKN) were compared with those in 'Mountain Spring' (less susceptible to RKN). In greenhouse tests with 'Rutgers' at 30 and 60 days after treatment (DAT) with RKN and Bb, there were few differences in plant growth variables among treatments in repeated trials. However, RKN root galling and egg count/root system were enhanced in plants treated with Bb at 60 DAT. In an in vitro assay with egg masses from greenhouse tests, the percentages of hatched eggs, and mobile and immobile nematodes did not differ significantly for RKN and RKN+Bb treatments. The presence of viable Bb from roots was confirmed by collecting egg suspensions from root galls and plating them on selective medium. Colonies of Bb were verified on agar medium, but no parasitism of RKN eggs was observed. Research is needed to investigate factors responsible for increased galling by RKN in the presence of endophytic Bb in 'Rutgers' tomato.

Response of bitter and sweet Chenopodium quinoa varieties to cucumber mosaic virus: Transcriptome and small RNASeq perspective.

Saponins are secondary metabolites with antiviral properties. Low saponin (sweet) varieties of quinoa (Chenopodium quinoa) have been developed because seeds high in saponins taste bitter. The aim of this study was to elucidate the role of saponin in resistance of quinoa to Cucumber mosaic virus (CMV). Differential gene expression was studied in time-series study of CMV infection. High-throughput transcriptome sequence data were obtained from 36 samples (3 varieties × +/- CMV × 1 or 4 days after inoculation × 3 replicates). Translation, lipid, nitrogen, amino acid metabolism, and mono- and sesquiterpenoid biosynthesis genes were upregulated in CMV infections. In 'Red Head' (bitter), CMV-induced systemic symptoms were concurrent with downregulation of a key saponin biosynthesis gene, TSARL1, four days after inoculation. In local lesion responses (sweet and semi-sweet), TSARL1 levels remained up-regulated. Known microRNAs (miRNA) (81) from 11 miR families and 876 predicted novel miRNAs were identified. Differentially expressed miRNA and short interfering RNA clusters (24nt) induced by CMV infection are predicted to target genomic and intergenic regions enriched in repetitive elements. This is the first report of integrated RNASeq and sRNASeq data in quinoa-virus interactions and provides comprehensive understanding of involved genes, non-coding regions, and biological pathways in virus resistance.

Sciadopitys verticillata Resin: Volatile Components and Impact on Plant Pathogenic and Foodborne Bacteria.

Sciadopitys verticillata (Sv) produces a white, sticky, latex-like resin with antimicrobial properties. The aims of this research were to evaluate the effects of this resin (Sv resin) on bacterial populations and to determine the impact of its primary volatile components on bioactivity. The impact of sample treatment on chemical composition of Sv resin was analyzed using Fourier transform infrared spectroscopy (FTIR) coupled with principal component analysis. The presence and concentration of volatiles in lyophilized resin were determined using gas chromatography/mass spectrometry (GC/MS). Changes in bacterial population counts due to treatment with resin or its primary volatile components were monitored. Autoclaving of the samples did not affect the FTIR spectra of Sv resin; however, lyophilization altered spectra, mainly in the CH and C=O regions. Three primary bioactive compounds that constituted >90% of volatiles (1R-α-pinene, tricyclene, and ß-pinene) were identified in Sv resin. Autoclaved resin impacted bacterial growth. The resin was stimulatory for some plant and foodborne pathogens (Pseudomonas fluorescens, P. syringae, and Xanthomonas perforans) and antimicrobial for others (Escherichia coli, Bacillus cereus, Agrobacterium tumefaciens, and Erwinia amylovora). Treatment with either 1R-α-pinene or ß-pinene reduced B. cereus population growth less than did autoclaved resin. The complex resin likely contains additional antimicrobial compounds that act synergistically to inhibit bacterial growth.

Development and characterization of simple sequence repeats for Bipolaris sorokiniana and cross transferability to related species.

Simple sequence repeats (SSR) markers were developed from a small insert genomic library for Bipolaris sorokiniana, a mitosporic fungal pathogen that causes spot blotch and root rot in switchgrass. About 59% of sequenced clones (n = 384) harbored SSR motifs. After eliminating redundant sequences, 196 SSR loci were identified, of which 84.7% were dinucleotide repeats and 9.7% and 5.6% were tri- and tetra-nucleotide repeats, respectively. Primer pairs were designed for 105 loci and 85 successfully amplified loci. Sixteen polymorphic loci were characterized with 15 B. sorokiniana isolates obtained from infected switchgrass plant materials collected from five states in USA. These loci successfully cross-amplified isolates from at least one related species, including Bipolaris oryzae, Bipolaris spicifera and Bipolaris victoriae, that causes leaf spot on switchgrass. Haploid gene diversity per locus across all isolates studied varied 0.633-0.861. Principal component analysis of SSR data clustered isolates according to their respective species. These SSR markers will be a valuable tool for genetic variability and population studies of B. sorokiniana and related species that are pathogenic on switchgrass and other host plants. In addition, these markers are potential diagnostic tools for species in the genus Bipolaris.

Endophyte isolate and host grass effects on Chaetocnema pulicaria (Coleoptera: Chrysomelidae) feeding.

Endophytic fungi belonging to the genus Neotyphodium, confer resistance to infected host grasses against insect pests. The effect of host species, and endophtye species and strain, on feeding and survival of the corn flea beetle, Chaetocnema pulicaria Melsheimer (Coleoptera: Chrysomelidae) was investigated. The grass-endophyte associations included natural and artificially derived associations producing varying arrays of common endophyte-related alkaloids or alkaloid groups, peramine, lolitrem B, ergovaline, and the lolines. Preference and nonpreference tests showed that C. pulicaria feeding and survival were reduced by infection of tall fescue with the wild-type strain of N. coenophialum, the likely mechanism being antixenosis rather than antibiosis. In the preference tests, endophyte and host species effects were observed. Of the 10 different Neotyphodium strains tested in artificially derived tall fescue associations, eight strongly deterred feeding by C. pulicaria, whereas the remaining two strains had little or no effect on feeding. Infection of tall fescue with another fungal symbiont, p-endophyte, had no effect. Perennial ryegrass, Lolium perenne L., infected with six strains of endophyte, was moderately resistant to C. pulicaria compared with endophyte-free grass, but four additional strains were relatively inactive. Six Neotyphodium-meadow fescue, Festuca pratensis Huds., associations, including the wild-type N. uncinatum-meadow fescue combination, were resistant, whereas three associations were not effective. Loline alkaloids seemed to play a role in antixenosis to C. pulicaria. Effects not attributable to the lolines or any other of the alkaloids examined also were observed. This phenomenon also has been reported in tests with other insects, and indicates the presence of additional insect-active factors.

Role of essential oils in control of Rhizoctonia damping-off in tomato with bioactive Monarda herbage.

Plants in the genus Monarda produce complex essential oils that contain antifungal compounds. The objectives of this research were to identify selections of monarda that reduce Rhizoctonia damping-off of tomato, and to determine relationships between essential oil composition of 13 monarda herbages (dried and ground leaves) and disease suppression. Herbages were grouped into five chemotypes, based on essential oil composition and effective concentrations for reducing growth by 50% for Rhizoctonia solani. Replicated and repeated disease control assays were conducted with monarda herbages in greenhouse medium, with or without Rhizoctonia. Percent survival, seedling height, and stem diameter were evaluated at 8 weeks. Survival, seedling height, and stem diameter in herbage-only treatments were not different from the control (no-herbage, no-pathogen) for most herbage treatments. In the pathogen control (no-herbage + Rhizoctonia), seedling survival was 10% that of the control. In pathogen-infested media, seedling survival ranged from 65 to 80% for treatments with thymol chemotypes and 55 to 65% for carvacrol chemotypes. Effective control of Rhizoctonia damping-off was correlated with phenolic monoterpenes; herbages classified as carvacrol chemotypes effectively protected tomato seedlings from Rhizoctonia damping-off disease without phytotoxicity. This study provides evidence that monarda herbages have potential as growing media amendments for control of Rhizoctonia damping-off disease.

Beauveria bassiana: endophytic colonization and plant disease control.

Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.

Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae.

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.