Developing Prediction Models Using Near-Infrared Spectroscopy to Quantify Cannabinoid Content in Cannabis Sativa.

Cannabis is commercially cultivated for both therapeutic and recreational purposes in a growing number of jurisdictions. The main cannabinoids of interest are cannabidiol (CBD) and delta-9 tetrahydrocannabidiol (THC), which have applications in different therapeutic treatments. The rapid, nondestructive determination of cannabinoid levels has been achieved using near-infrared (NIR) spectroscopy coupled to high-quality compound reference data provided by liquid chromatography. However, most of the literature describes prediction models for the decarboxylated cannabinoids, e.g., THC and CBD, rather than naturally occurring analogues, tetrahydrocannabidiolic acid (THCA) and cannabidiolic acid (CBDA). The accurate prediction of these acidic cannabinoids has important implications for quality control for cultivators, manufacturers and regulatory bodies. Using high-quality liquid chromatography-mass spectroscopy (LCMS) data and NIR spectra data, we developed statistical models including principal component analysis (PCA) for data quality control, partial least squares regression (PLS-R) models to predict cannabinoid concentrations for 14 different cannabinoids and partial least squares discriminant analysis (PLS-DA) models to characterise cannabis samples into high-CBDA, high-THCA and even-ratio classes. This analysis employed two spectrometers, a scientific grade benchtop instrument (Bruker MPA II-Multi-Purpose FT-NIR Analyzer) and a handheld instrument (VIAVI MicroNIR Onsite-W). While the models from the benchtop instrument were generally more robust (99.4-100% accuracy prediction), the handheld device also performed well (83.1-100% accuracy prediction) with the added benefits of portability and speed. In addition, two cannabis inflorescence preparation methods were evaluated: finely ground and coarsely ground. The models generated from coarsely ground cannabis provided comparable predictions to that of the finely ground but represent significant timesaving in terms of sample preparation. This study demonstrates that a portable NIR handheld device paired with LCMS quantitative data can provide accurate cannabinoid predictions and potentially be of use for the rapid, high-throughput, nondestructive screening of cannabis material.

Quantitation and Distribution of Epichloë-Derived Alkaloids in Perennial Ryegrass Tissues.

Perennial ryegrass (Lolium perenne L.), an economically important pasture and turf grass, is commonly infected with asexual Epichloë species endophytes. Endophytes provide enhanced bioprotection by producing alkaloids, and research often focusses on the negative impact on grazing animals. However, alkaloid distribution throughout the plant and their role in biocontrol of insect pests and diseases are less well understood. Additionally, intermediate compounds have not been investigated for their impacts on animal welfare and biological control in pasture-based scenarios. Here, a single liquid chromatography-mass spectrometry (LC-MS) method was used to measure seven alkaloids in different perennial ryegrass tissues infected with SE or NEA12 endophytes. High alkaloid recoveries and a clear plant matrix effect emphasize the importance of using matrix-matched standards for accurate quantitation. The method is sensitive, detecting alkaloids at low concentrations (nanogram levels), which is important for endophyte strains that produce compounds detrimental to livestock. Concentrations were generally highest in seeds, but distribution differed in the shoots/roots: peramine, terpendole E, terpendole C and lolitrem B were higher in shoots, whilst ergovaline, paxilline and epoxy-janthitrem I were more evenly distributed throughout the two tissues. Knowledge of alkaloid distribution may allow for concentrations to be predicted in roots based on concentrations in the shoots, thereby assisting future determinations of resistance to insects, especially subterranean root-feeding pests.

The Known Antimammalian and Insecticidal Alkaloids Are Not Responsible for the Antifungal Activity of Epichloë Endophytes.

Asexual Epichloë sp. endophytes in association with pasture grasses produce agronomically important alkaloids (e.g., lolitrem B, epoxy-janthitrems, ergovaline, peramine, and lolines) that exhibit toxicity to grazing mammals and/or insect pests. Novel strains are primarily characterised for the presence of these compounds to ensure they are beneficial in an agronomical setting. Previous work identified endophyte strains that exhibit enhanced antifungal activity, which have the potential to improve pasture and turf quality as well as animal welfare through phytopathogen disease control. The contribution of endophyte-derived alkaloids to improving pasture and turf grass disease resistance has not been closely examined. To assess antifungal bioactivity, nine Epichloë related compounds, namely peramine hemisulfate, n-formylloline-d3, n-acetylloline hydrochloride, lolitrem B, janthitrem A, paxilline, terpendole E, terpendole C, and ergovaline, and four Claviceps purpurea ergot alkaloids, namely ergotamine, ergocornine, ergocryptine, and ergotaminine, were tested at concentrations higher than observed in planta in glasshouse and field settings using in vitro agar well diffusion assays against three common pasture and turf phytopathogens, namely Ceratobasidium sp., Drechslera sp., and Fusarium sp. Visual characterisation of bioactivity using pathogen growth area, mycelial density, and direction of growth indicated no inhibition of pathogen growth. This was confirmed by statistical analysis. The compounds responsible for antifungal bioactivity of Epichloë endophytes hence remain unknown and require further investigation.

Ergot alkaloid mycotoxins: physiological effects, metabolism and distribution of the residual toxin in mice.

The complex ergot alkaloids, ergovaline and ergotamine, cause dysregulation of physiological functions, characterised by vasoconstriction as well as thermoregulatory and cardiovascular effects in grazing livestock. To assess the effect of the mycotoxins, blood pressure and heart rate of male mice were measured, and metabolite profiling undertaken to determine relative abundances of both ergotamine and its metabolic products in body and brain tissue. Ergotamine showed similar cardiovascular effects to ergovaline, causing elevations in blood pressure and reduced heart rate. Bradycardia was preserved at low-levels of ergovaline despite no changes in blood pressure. Ergotamine was identified in kidney, liver and brainstem but not in other regions of the brain, which indicates region-specific effects of the toxin. The structural configuration of two biotransformation products of ergotamine were determined and identified in the liver and kidney, but not the brain. Thus, the dysregulation in respiratory, thermoregulatory, cardiac and vasomotor function, evoked by ergot alkaloids in animals observed in various studies, could be partially explained by dysfunction in the autonomic nervous system, located in the brainstem.

Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry.

Lolitrem B is the most potent indole-diterpene mycotoxin produced by Epichloë festucae var. lolii (termed LpTG-1), with severe intoxication cases reported in livestock. To date, there are no in vivo metabolism studies conducted for the mycotoxin. A mouse model assay established for assessing toxicity of indole-diterpenes was used to investigate metabolic products of lolitrem B. Mice were administered lolitrem B at 0.5 and 2.0 mg/kg body weight (b.wt) intraperitoneally before body and brain tissues were collected at 6 h and 24 h post-treatment. Samples were cryoground and subjected to a biphasic or monophasic extraction. The aqueous and lipophilic phases were analysed using liquid chromatography high-resolution mass spectrometry (LC-HRMS); data analysis was performed with Compound Discoverer™ software. A total of 10 novel phase I metabolic products were identified in the lipophilic phase and their distribution in the liver, kidney and various brain regions are described. The biotransformation products of lolitrem B were found to be present in low levels in the brain. Based on structure-activity postulations, six of these may contribute towards the protracted tremors exhibited by lolitrem B-exposed animals.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography-mass spectrometry (LC-MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC-MS (for peramine). This work provides the first highly sensitive quantitative LC-MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes.

Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I-IV in Epichloë endophytes is proposed.

NMR Based Metabolomic Analysis of Health Promoting Phytochemicals in Lentils.

Lentils are a high-protein plant food and a valuable source of human nutrition, particularly in the Indian subcontinent. However, beyond sustenance, there is evidence that the consumption of lentils (and legumes in general) is associated with decreased risk of diseases, such as diabetes and cardiovascular disease. Lentils contain health-promoting phytochemicals, such as trigonelline and various polyphenolics. Fourteen lentil genotypes were grown at three locations to explore the variation in phytochemical composition in hulls and cotyledons. Significant differences were measured between genotypes and environments, with some genotypes more affected by environment than others. However, there was a strong genetic effect which indicated that future breeding programs could breed for lentils that product more of these health-promoting phytochemicals.

Tremorgenic Mycotoxins: Structure Diversity and Biological Activity.

Indole-diterpenes are an important class of chemical compounds which can be unique to different fungal species. The highly complex lolitrem compounds are confined to Epichloë species, whilst penitrem production is confined to Penicillium spp. and Aspergillus spp. These fungal species are often present in association with pasture grasses, and the indole-diterpenes produced may cause toxicity in grazing animals. In this review, we highlight the unique structural variations of indole-diterpenes that are characterised into subgroups, including paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines. A detailed description of the unique biological activities has been documented where even structurally related compounds have displayed unique biological activities. Indole-diterpene production has been reported in two classes of ascomycete fungi, namely Eurotiomycetes (e.g., Aspergillus and Penicillium) and Sordariomycetes (e.g., Claviceps and Epichloë). These compounds all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from tryptophan. Structure diversity is generated from the enzymatic conversion of different sites on the basic indole-diterpene structure. This review highlights the wide-ranging biological versatility presented by the indole-diterpene group of compounds and their role in an agricultural and pharmaceutical setting.

Elevated CO2 and virus infection impacts wheat and aphid metabolism.

INTRODUCTION: The aphid Rhopalosiphum padi L. is a vector of Barley yellow dwarf virus (BYDV) in wheat and other economically important cereal crops. Increased atmospheric CO2 has been shown to alter plant growth and metabolism, enhancing BYDV disease in wheat. However, the biochemical influences on aphid metabolism are not known. OBJECTIVES: This work aims to determine whether altered host-plant quality, influenced by virus infection and elevated CO2, impacts aphid weight and metabolism. METHODS: Untargeted 1H NMR metabolomics coupled with multivariate statistics were employed to profile the metabolism of R. padi reared on virus-infected and non-infected (sham-inoculated) wheat grown under ambient CO2 (aCO2, 400 µmol mol-1) and future, predicted elevated CO2 (eCO2, 650 µmol mol-1) concentrations. Un-colonised wheat was also profiled to observe changes to host-plant quality (i.e., amino acids and sugars). RESULTS: The direct impacts of virus or eCO2 were compared. Virus presence increased aphid weight under aCO2 but decreased weight under eCO2; whilst eCO2 increased non-viruliferous (sham) aphid weight but decreased viruliferous aphid weight. Discriminatory metabolites due to eCO2 were succinate and sucrose (in sham wheat), glucose, choline and betaine (in infected wheat), and threonine, lactate, alanine, GABA, glutamine, glutamate and asparagine (in aphids), irrespective of virus presence. Discriminatory metabolites due to virus presence were alanine, GABA, succinate and betaine (in wheat) and threonine and lactate (in aphids), irrespective of CO2 treatment. CONCLUSION: This study confirms that virus and eCO2 alter host-plant quality, and these differences are reflected by aphid weight and metabolism.

Insect-plant-pathogen interactions as shaped by future climate: effects on biology, distribution, and implications for agriculture.

Carbon dioxide (CO2 ) is the main anthropogenic gas which has drastically increased since the industrial revolution, and current concentrations are projected to double by the end of this century. As a consequence, elevated CO2 is expected to alter the earths' climate, increase global temperatures and change weather patterns. This is likely to have both direct and indirect impacts on plants, insect pests, plant pathogens and their distribution, and is therefore problematic for the security of future food production. This review summarizes the latest findings and highlights current knowledge gaps regarding the influence of climate change on insect, plant and pathogen interactions with an emphasis on agriculture and food production. Direct effects of climate change, including increased CO2 concentration, temperature, patterns of rainfall and severe weather events that impact insects (namely vectors of plant pathogens) are discussed. Elevated CO2 and temperature, together with plant pathogen infection, can considerably change plant biochemistry and therefore plant defense responses. This can have substantial consequences on insect fecundity, feeding rates, survival, population size, and dispersal. Generally, changes in host plant quality due to elevated CO2 (e.g., carbon to nitrogen ratios in C3 plants) negatively affect insect pests. However, compensatory feeding, increased population size and distribution have also been reported for some agricultural insect pests. This underlines the importance of additional research on more targeted, individual insect-plant scenarios at specific locations to fully understand the impact of a changing climate on insect-plant-pathogen interactions.

The effect of elevated CO2 and virus infection on the primary metabolism of wheat.

Atmospheric CO2 concentrations are predicted to double by the end of this century. Although the effects of CO2 fertilisation in crop systems have been well studied, little is known about the specific interactions among plants, pests and pathogens under a changing climate. This growth chamber study focuses on the interactions among Barley yellow dwarf virus (BYDV), its aphid vector (Rhopalosiphum padi) and wheat (Triticum aestivum L. cv. Yitpi) under ambient (aCO2; 400µmolmol-1) or elevated (eCO2; 650µmolmol-1) CO2 concentrations. eCO2 increased the tiller number and biomass of uninoculated plants and advanced the yellowing symptoms of infected plants. Total foliar C content (percentage of the total DW) increased with eCO2 and with sham inoculation (exposed to early herbivory), whereas total N content decreased with eCO2. Liquid chromatography-mass spectrometry approaches were used to quantify the products of primary plant metabolism. eCO2 significantly increased sugars (fructose, mannitol and trehalose), irrespective of disease status, whereas virus infection significantly increased the amino acids essential to aphid diet (histidine, lysine, phenylalanine and tryptophan) irrespective of CO2 concentration. Citric acid was reduced by both eCO2 and virus infection. Both the potential positive and negative biochemical impacts on wheat, aphid and BYDV interactions are discussed.

The effect of elevated temperature on Barley yellow dwarf virus-PAV in wheat.

Barley yellow dwarf virus-PAV (BYDV-PAV) is associated with yellow dwarf disease, one of the most economically important diseases of cereals worldwide. In this study, the impact of current and future predicted temperatures for the Wimmera wheat growing district in Victoria, Australia on the titre of BYDV-PAV in wheat was investigated. Ten-day old wheat (Triticum aestivum, cv. Yitpi) seedlings were inoculated with BYDV-PAV and grown at ambient (5.0-16.1°C, night-day) or elevated (10.0-21.1°C, night-day) temperature treatments, simulating the current Wimmera average and future daily temperature cycles, respectively, during the wheat-growing season. Whole above-ground plant samples were collected from each temperature treatment at 0 (day of inoculation), 3, 6, 9, 12, 15, 18, 21 and 24 days after inoculation and the titre of BYDV-PAV was measured in each sample using a specific one-step multiplex normalised reverse transcription quantitative PCR (RT-qPCR) assay. Physical measurements, including plant height, dry weight and tiller number, were also taken at each sampling point. The titre of BYDV-PAV was significantly greater in plants grown in the elevated temperature treatment than in plants grown in the ambient treatment on days 6, 9 and 12. Plants grown at elevated temperature were significantly bigger and symptoms associated with BYDV-PAV were visible earlier than in plants grown at ambient temperature. These results may have important implications for the epidemiology of yellow dwarf disease under future climates in Australia.