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Abstract Localised fruit thinning strategies must be investigated to improve precision crop load management in narrow-canopy, multileader apple trees. This study aimed to determine the effects of within-leader and tree total crop load on leaders’ and trunk’s growth, fruit set, yield, and fruit quality in ‘Ruby Matilda’ apples (marketed as Pink Lady®) over three years. Different crop loads were imposed on two leaders (primary and secondary) of bi-axis trees. Leader and trunk relative growth rate, return fruit set, yield, and fruit quality parameters at harvest were measured. High within-leader crop loads led to a significant increase in yield and reductions in trunk growth, return fruit set, and deterioration of fruit quality parameters except for flesh firmness and starch index. Similar trends were observed in whole-tree relationships. High crop load in secondary leaders had moderate negative effects on trunk growth, yield, and fruit mass of primary leaders; it only marginally affected their return fruit set and had no significant effect (p > 0.05) on their fruit quality. A crop load of 6.8 fruit no. cm−2 of leader cross-sectional area was estimated to achieve a relatively consistent return fruit set within the same leader. At a whole-tree level, a similar crop load (6.9 fruit no. cm−2 of trunk cross-sectional area) produced a consistent return fruit set despite its higher variability. These crop loads produced high yields (120 and 111 t ha−1, respectively) and good quality fruit. Using individual leaders as management units is recommended to simplify operations and reduce variability.

In a bushfire burning of plant material generates volatile phenolics that may be absorbed by berries and leaves of grapes in nearby vineyards. In grapes these phenolics form glycoconjugates and the undesirable sensory attributes of smoke-exposed grapes only develop post-fermentation in the wine making process, when the free phenolics are released. To reduce the financial losses from producing smoke-tainted wines, phenolic glycosides associated with smoke-taint in grapes are currently monitored in analytical laboratories. Here, we performed LC-MS analysis on extracts of diverse grape varieties of various geographic sites and with varying levels of smoke taint exposure. Discovery of smoke-taint biomarkers, based on untargeted analysis led to the identification of additional phenolic compounds that could contribute significantly to the smoke-tainted flavour of wine. A total of 73 differentially expressed metabolites were detected and LCMSMS analysis resulted in the structural assignment of 15 compounds, including guaiacol and syringol derivatives and novel glycoconguate intermediaries.

Key Clinical Message Endometriosis may contribute to Mullerian adenosarcoma development but makes diagnosis challenging given similar symptoms. Survival benefit has not been definitively shown for chemotherapy, hormonal therapy, or radiotherapy, consolidating surgery as the mainstay treatment. Local excision may be a treatment option for patients with confined tumors wishing to preserve their fertility.

Abstract The combination of Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is the standard of care for hormone receptor-positive (HR + ), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (MBC). Currently, there are no robust biomarkers that can predict response to CDK4/6i, and it is not clear which patients benefit from this therapy. Since MBC patients with liver metastases have a poorer prognosis, developing predictive biomarkers that could identify patients likely to respond to CDK4/6i is clinically important. Here we show the ability of imaging texture biomarkers before and a few cycles after CDK4/6i therapy, to predict early response and overall survival (OS) on 73 MBC patients with known liver metastases who received palbociclib plus ET from two sites. The delta radiomic model was associated with OS in validation set (HR: 2.4; 95% CI, 1.06–5.6; P = 0.035; C-index = 0.77). Compared to RECIST response, delta radiomic features predicted response with area under the curve (AUC) = 0.72, 95% confidence interval (CI) 0.67–0.88. Our study revealed that radiomics features can predict a lack of response earlier than standard anatomic/RECIST 1.1 assessment and warrants further study and clinical validation.

OBJECTIVES/GOALS: We will describe the community-driven development and impact of the new Community Research Capacity-Building grants from the Alliance for Research in Chicagoland Communities, Northwestern University. Communities expressed that to enter equitably into partnerships with academics they need support to build their own community research capacity. METHODS/STUDY POPULATION: ARCC Seed Grants, since 2008, included Partnership Development and Research Pilots, which are both jointly submitted by a community-academic partnership. The new Community Grants are submitted only by community partners and don’t require an academic partner. These grants, $3,000 over 6 months, support the development or strengthening of organizational or community-level research capacity. This may include assessing community capacity to lead and/or collaborate on research; building research capacity of community organizations (staff, leadership, residents), developing community infrastructure (e.g. research principles; staff research responsibilities; process for assessing/ tracking researcher inquiries; template memorandum of understanding) or community research priorities, etc. RESULTS/ANTICIPATED RESULTS: Eight ARCC Community Research Capacity-Building Seed Grants have been awarded so far as a part of three cycles of applications over 2022-23 (2 in 2022, 6 in 2023). During this time period, data has been collected during the application process, in final reports, and in informal group and individual discussions. Information about the profile of grantees (community representation, health focus, etc.), the initial impact of grants, and feedback from grantees about the positive and challenging aspects of the grants will be shared. Grantees have informally shared that the awards have helped to address concerns that many low-income communities of color have their voices are not adequately included in research and other decision-making. The poster will be co-presented by a community grant recipient. DISCUSSION/SIGNIFICANCE: To ensure that research partnerships are community-driven & equitable, it is necessary to invest in community research capacity-building. More evaluation is needed to understand the grants impact, as well as other approaches to community research capacity and leadership development. Poster will be co-presented by a community grant recipient.

Phytohormones that trigger or repress flower meristem development in apple buds are thought to be locally emitted from adjacent plant tissues, including leaves and fruitlets. The presence of fruitlets is known to inhibit adjacent buds from forming flowers and thus fruits. The resulting absence of fruitlets the following season restores flower-promoting signalling to the new buds. The cycle can lead to a biennial bearing behaviour of alternating crop loads in a branch or tree. The hormonal stimuli that elicit flowering is typically referred to as the floral induction (FI) phase in bud meristem development. To determine the metabolic pathways activated in FI, young trees of the cultivar ‘Ruby Matilda’ were subjected to zonal crop load treatments imposed to two leaders of bi-axis trees in the 2020/2021 season. Buds were collected over the expected FI phase, which is within 60 DAFB. Metabolomics profiling was undertaken to determine the differentially expressed pathways and key signalling molecules associated with FI in the leader and at tree level. Pronounced metabolic differences were observed in trees and leaders with high return bloom with significant increases in compounds belonging to the cytokinin, abscisic acid (ABA), phenylpropanoid and flavanol chemical classes. The presence of cytokinins, namely adenosine, inosine and related derivatives, as well as ABA phytohormones, provides further insight into the chemical intervention opportunities for future crop load management strategies via plant growth regulators.

Safflower (Carthamus tinctorius L.) is an ancient oilseed crop of interest due to its diversity of end-use industrial and food products. Proteomic and metabolomic profiling of its organs during seed development, which can provide further insights on seed quality attributes to assist in variety and product development, has not yet been undertaken. In this study, an integrated proteome and metabolic analysis have shown a high complexity of lipophilic proteins and metabolites differentially expressed across organs and tissues during seed development and petal wilting. We demonstrated that these approaches successfully discriminated safflower reproductive organs and developmental stages with the identification of 2179 unique compounds and 3043 peptides matching 724 unique proteins. A comparison between cotyledon and husk tissues revealed the complementarity of using both technologies, with husks mostly featuring metabolites (99%), while cotyledons predominantly yielded peptides (90%). This provided a more complete picture of mechanisms discriminating the seed envelope from what it protected. Furthermore, we showed distinct molecular signatures of petal wilting and colour transition, seed growth, and maturation. We revealed the molecular makeup shift occurring during petal colour transition and wilting, as well as the importance of benzenoids, phenylpropanoids, flavonoids, and pigments. Finally, our study emphasizes that the biochemical mechanisms implicated in the growing and maturing of safflower seeds are complex and far-reaching, as evidenced by AraCyc, PaintOmics, and MetaboAnalyst mapping capabilities. This study provides a new resource for functional knowledge of safflower seed and potentially further enables the precision development of novel products and safflower varieties with biotechnology and molecular farming applications.

Abstract The ergot alkaloid ergotamine is produced by Claviceps purpurea, a parasitic fungus that commonly infects crops and pastures of high agricultural and economic importance. In humans and livestock, symptoms of ergotism include necrosis and gangrene, high blood pressure, heart rate, thermoregulatory dysfunction and hallucinations. However, ergotamine is also used in pharmaceutical applications to treat migraines and stop post-partum hemorrhage. To define its effects, metabolomic profiling of the brain was undertaken to determine pathways perturbed by ergotamine treatment. Metabolomic profiling identified the brainstem and cerebral cortex as regions with greatest variation. In the brainstem, dysregulation of the neurotransmitter epinephrine, and the psychoactive compound 2-arachidonylglycerol was identified. In the cerebral cortex, energy related metabolites isobutyryl-L-carnitine and S-3-oxodecanoyl cysteamine were affected and concentrations of adenylosuccinate, a metabolite associated with mental retardation, were higher. This study demonstrates, for the first time, key metabolomic pathways involved in the behavioural and physiological dysfunction of ergot alkaloid intoxicated animals.

The detection of beneficial microbes living within perennial ryegrass seed causing no apparent defects is challenging, even with the most sensitive and conventional methods, such as DNA genotyping. Using a near-infrared hyperspectral imaging system (NIR-HSI), we were able to discriminate not only the presence of the commercial NEA12 fungal endophyte strain but perennial ryegrass cultivars of diverse seed age and batch. A total of 288 wavebands were extracted for individual seeds from hyperspectral images. The optimal pre-processing methods investigated yielded the best partial least squares discriminant analysis (PLS-DA) classification model to discriminate NEA12 and without endophyte (WE) perennial ryegrass seed with a classification accuracy of 89%. Effective wavelength (EW) selection based on GA-PLS-DA resulted in the selection of 75 wavebands yielding 88.3% discrimination accuracy using PLS-DA. For cultivar identification, the artificial neural network discriminant analysis (ANN-DA) was the best-performing classification model, resulting in >90% classification accuracy for Trojan, Alto, Rohan, Governor and Bronsyn. EW selection using GA-PLS-DA resulted in 87 wavebands, and the PLS-DA model performed the best, with no extensive compromise in performance, resulting in >89.1% accuracy. The study demonstrates the use of NIR-HSI reflectance data to discriminate, for the first time, an associated beneficial fungal endophyte and five cultivars of perennial ryegrass seed, irrespective of seed age and batch. Furthermore, the negligible effects on the classification errors using EW selection improve the capability and deployment of optimized methods for real-time analysis, such as the use of low-cost multispectral sensors for single seed analysis and automated seed sorting devices.

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.

Abstract The neuroactive mycotoxin lolitrem B causes a neurological syndrome in grazing livestock resulting in hyperexcitability, muscle tremors, ataxia and, in severe cases, clonic seizures and death. To define the effects of the major toxin lolitrem B in the brain, a functional metabolomic study was undertaken in which motor coordination and tremor were quantified and metabolomic profiling undertaken to determine relative abundance of both toxin and key neurotransmitters in various brain regions in male mice. Marked differences were observed in the duration of tremor and coordination between lolitrem B pathway members, with some showing protracted effects and others none at all. Lolitrem B was identified in liver, kidney, cerebral cortex and thalamus but not in brainstem or cerebellum which were hypothesised previously to be the primary site of action. Metabolomic profiling showed significant variation in specific neurotransmitter and amino acid profiles over time. This study demonstrates accumulation of lolitrem B in the brain, with non-detectable levels of toxin in the brainstem and cerebellum, inducing alterations in metabolites such as tyrosine, suggesting a dynamic catecholaminergic response over time. Temporal characterisation of key pathways in the pathophysiological response of lolitrem B in the brain were also identified.

Aim: Describe naturalistic clinical course over 14 weeks in a mixed adolescent and a young-adult patient group diagnosed with developmental delays and catatonia, when the frequency of maintenance electroconvulsive therapy (M-ECT) was reduced secondary to 2020 COVID-19 pandemic restrictions.Methods: Participants were diagnosed with catatonia, and were receiving care in a specialized clinic. They (n = 9), F = 5, and M = 4, ranged in age from 16 to 21 years; ECT frequency was reduced at end of March 2020 due to institutional restrictions. Two parents/caregivers elected to discontinue ECT due to concern for COVID-19 transmission. Majority (n = 8) were developmentally delayed with some degree of intellectual disability (ID). Observable symptoms were rated on a three point scale during virtual visits.Results: All cases experienced clinically significant decline. Worsening of motor symptoms (agitation, aggression, slowness, repetitive self-injury, stereotypies, speech deficits) emerged within the first 3 weeks, persisted over the 14 week observation period and were more frequent than neurovegetative symptoms (appetite, incontinence, sleep). Four participants deteriorated requiring rehospitalization, and 2 among these 4 needed a gastrostomy feeding tube.Conclusion: Moderate and severe symptoms became apparent in all 9 cases during the observation period; medication adjustments were ineffective; resuming M-ECT at each participant's baseline schedule, usually by week 7, resulted in progressive improvement in some cases but the improvement was insufficient to prevent re-hospitalization in 4 cases. In summary, rapid deterioration was noted when M-ECT was acutely reduced in the setting of COVID-19 related restrictions.

Background: The SARS-CoV-2 virus continues to spread rapidly across the globe afflicting many with Coronavirus Disease 2019 (COVID-19). As the infection rates rise, a growing number of SARS-CoV-2 positive individuals have been reported to complain of olfactory disturbances at an alarming rate. Postviral olfactory dysfunction (PVOD) is a well-known phenomenon that may explain the olfactory dysfunction reported by SARS-CoV-2 infected individuals. Methods: A scoping literature review was performed to identify studies that investigated the mechanisms of postviral olfactory dysfunction. Studies demonstrating pathophysiological, histological, immunochemical, and epidemiological outcomes of PVOD were included. Results: Fourteen studies were included in addition to one international news article. Three studies reported destruction of the olfactory epithelium following intranasal inoculation of various viral strains in mice. Three studies isolated pathogenic, anosmia inciting viruses (Parainfluenza virus, Human Coronavirus, Rhinovirus) through nucleic acid amplification. Eleven studies demonstrated female predilection in patients with PVOD and COVID-19 associated olfactory dysfunction, of which the majority were over 50 years old. Conclusions: PVOD and COVID-19 associated olfactory dysfunction demonstrates considerable similarities in epidemiological trends and disease sequela of other viruses to suggest identical pathophysiological mechanisms. Further studies such as intranasal inoculation and histological biopsies are needed to support our hypothesis.

Near-infrared (800–2500 nm; NIR) spectroscopy coupled to hyperspectral imaging (NIR-HSI) has greatly enhanced its capability and thus widened its application and use across various industries. This non-destructive technique that is sensitive to both physical and chemical attributes of virtually any material can be used for both qualitative and quantitative analyses. This review describes the advancement of NIR to NIR-HSI in agricultural applications with a focus on seed quality features for agronomically important seeds. NIR-HSI seed phenotyping, describing sample sizes used for building high-accuracy calibration and prediction models for full or selected wavelengths of the NIR region, is explored. The molecular interpretation of absorbance bands in the NIR region is difficult; hence, this review offers important NIR absorbance band assignments that have been reported in literature. Opportunities for NIR-HSI seed phenotyping in forage grass seed are described and a step-by-step data-acquisition and analysis pipeline for the determination of seed quality in perennial ryegrass seeds is also presented.

Epichloë endophytes, fungal endosymbionts of Pooidae grasses, are commonly utilized in forage and turf industries because they produce beneficial metabolites that enhance resistance against environmental stressors such as insect feeding and disease caused by phytopathogen infection. In pastoral agriculture, phytopathogenic diseases impact both pasture quality and animal production. Recently, bioactive endophyte strains have been reported to secrete compounds that significantly inhibit the growth of phytopathogenic fungi in vitro. A screen of previously described Epichloë-produced antifeedant and toxic alkaloids determined that the antifungal bioactivity observed is not due to the production of these known metabolites, and so there is a need for methods to identify new bioactive metabolites. The process described here is applicable more generally for the identification of antifungals in new endophytes. This study aims to characterize the fungicidal potential of novel, ‘animal friendly’ Epichloë endophyte strains NEA12 and NEA23 that exhibit strong antifungal activity using an in vitro assay. Bioassay-guided fractionation, followed by metabolite analysis, identified 61 metabolites that, either singly or in combination, are responsible for the observed bioactivity. Analysis of the perennial ryegrass-endophyte symbiota confirmed that NEA12 and NEA23 produce the prospective antifungal metabolites in symbiotic association and thus are candidates for compounds that promote disease resistance in planta. The “known unknown” suite of antifungal metabolites identified in this study are potential biomarkers for the selection of strains that enhance pasture and turf production through better disease control.

Current methods for measuring the abundance of proteogenic amino acids in plants require derivatisation, extended run times, very sensitive pH adjustments of the protein hydrolysates, and the use of buffers in the chromatographic phases. Here, we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of amino acids that requires only three steps: hydrolysis, neutralisation, and sample dilution with a borate buffer solution for pH and retention time stability. The method shows excellent repeatability (repeated consecutive injections) and reproducibility (repeated hydrolysis) in the amino acid content, peak area, and retention time for all the standard amino acids. The chromatographic run time is 20 min with a reproducibility and repeatability of

Asexual species of the genus Epichloë (Clavicipitaceae, Ascomycota) form endosymbiotic associations with Pooidae grasses. This association is important both ecologically and to the pasture and turf industries, as the endophytic fungi confer a multitude of benefits to their host plant that improve competitive ability and performance such as growth promotion, abiotic stress tolerance, pest deterrence and increased host disease resistance. Biotic stress tolerance conferred by the production of bioprotective metabolites has a critical role in an industry context. While the known antimammalian and insecticidal toxins are well characterized due to their impact on livestock welfare, antimicrobial metabolites are less studied. Both pasture and turf grasses are challenged by many phytopathogenic diseases that result in significant economic losses and impact livestock health. Further investigations of Epichloë endophytes as natural biocontrol agents can be conducted on strains that are safe for animals. With the additional benefits of possessing host disease resistance, these strains would increase their commercial importance. Field reports have indicated that pasture grasses associated with Epichloë endophytes are superior in resisting fungal pathogens. However, only a few antifungal compounds have been identified and chemically characterized, and these from sexual (pathogenic) Epichloë species, rather than those utilized to enhance performance in turf and pasture industries. This review provides insight into the various strategies reported in identifying antifungal activity from Epichloë endophytes and, where described, the associated antifungal metabolites responsible for the activity.

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.

Volatile phenols (VPs) derived from smoke-exposed grapes are known to confer a smoky flavor to wine. Current methods for determination of VPs in grape berries either involve complex sample purification/derivatization steps or employ two analytical platforms for free and bound VP fractions. We report here a simple gas chromatography-tandem mass spectrometry (GC-MS/MS) method for quantification of both free and bound VPs in grapes, based on optimized (1) GC-MS/MS parameters, (2) an analyte extraction procedure, and (3) phenol glycoside hydrolysis conditions. Requiring neither sample cleanup nor a derivatization step, this method is sensitive (LOD ≤ 1 ng/g berries) and reproducible (RSD < 12% for repeated analyses) and is expected to significantly reduce the sample turnover time for smoke taint detection in vineyards.

Asexual Epichloë spp. fungal endophytes have been extensively studied for their functional secondary metabolite production. Historically, research mostly focused on understanding toxicity of endophyte-derived compounds on grazing livestock. However, endophyte-derived compounds also provide protection against invertebrate pests, disease, and other environmental stresses, which is important for ensuring yield and persistence of pastures. A preliminary screen of 30 strains using an in vitro dual culture bioassay identified 18 endophyte strains with antifungal activity. The novel strains NEA12, NEA21, and NEA23 were selected for further investigation as they are also known to produce alkaloids associated with protection against insect pests. Antifungal activity of selected endophyte strains was confirmed against three grass pathogens, Ceratobasidium sp., Dreschlera sp., and Fusarium sp., using independent isolates in an in vitro bioassay. NEA21 and NEA23 showed potent activity against Ceratobasidium sp. and NEA12 showed moderate inhibition against all three pathogens. Crude extracts from liquid cultures of NEA12 and NEA23 also inhibited growth of the phytopathogens Ceratobasidium sp. and Fusarium sp. and provided evidence that the compounds of interest are stable, constitutively expressed, and secreted. Comparative analysis of the in vitro and in planta metabolome of NEA12 and NEA23 using LCMS profile data revealed individual metabolites unique to each strain that are present in vitro and in planta. These compounds are the best candidates for the differential bioactivity observed for each strain. Novel endophyte strains show promise for endophyte-mediated control of phytopathogens impacting Lolium spp. pasture production and animal welfare.

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.

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.

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.

The most potent of the indole diterpenes, lolitrem B, is found in perennial ryegrass (Lolium perenne L.) infected with the endophyte Epichloë festucae var. lolii (also termed LpTG-1). Ingestion causes a neurological syndrome in grazing livestock called ryegrass staggers disease. To enable the rapid development of new forage varieties, the toxicity of lolitrem B and its biosynthetic intermediates needs to be established. However, most of these indole diterpenes are not commercially available; thus, isolation of these compounds is paramount. A concentrated endophyte-infected perennial ryegrass seed extract was subjected to silica flash chromatography followed by preparative HPLC and purification by crystallization resulting in lolitrem B and the intermediate compounds lolitrem E, paspaline and terpendole B. The four-step isolation and purification method resulted in a 25% yield of lolitrem B. After isolation, lolitrem B readily degraded to its biosynthetic intermediate, lolitriol. We also found that lolitrem B can readily degrade depending on the solvent and storage conditions. The facile method which takes into consideration the associated instability of lolitrem B, led to the purification of indole diterpenes in quantities sufficient for use as analytical standards for identification in pastures, and/or for toxicity testing in pasture development programs.