Annual/Termination Reports:

[12/17/2020] [07/11/2022] [08/01/2023]

Report Information

Participants

Daniel Cook – USDA/ARS Poisonous Plant Lab, Logan, UT Daniel.cook@usda.ars.gov.us;

Barbara Keith – Dept Land Resources and Environmental Sciences (LRES), Montana State University, bkeith@montana.edu;

Christopher Schardl – University of Kentucky, chris.schardl@uky.edu;

Tracy Sterling – Dept. LRES, Montana State University, tracy.sterling@montana.edu;

Rebecca Creamer – Dept EPPWS, New Mexico State University, creamer@nmsu.edu;

Sumanjari Das –Biology, NMSU, sdas@nmsu.edu;

Marwa Neyaz – Plant and Environmental Science, NMSU, marwane@nmsu.edu;

Kevin Welch, USDA/ARS Poisonous Plant Lab, Logan, UT, Kevin.welch@usda.gov;

Stephen Lee, USDA/ARS Poisonous Plant Lab, Logan, UT, Stephen.lee@usda.gov;

Ben Green, USDA/ARS Poisonous Plant Lab, Logan, UT, Ben.green@usda.gov;

Ram Nadathur – Molecular Biology, New Mexico State University, januj88@nmsu.edu;

Christopher Davies –Assoc. AES Director, Utah State University, chris.davies@usu.edu;

Jason Turner – Extension Animal Science, NMSU, jturner@nmsu.edu

Brief Summary of Minutes

Chris Davies, as project administrator, briefly discussed the Multistate project system. He explained that this project began in October 2020 and will continue for 5 years. Rebecca Creamer led introductions of all participants.

Marwa Neyaz, a Plant and Environmental Science PhD student working with Rebecca Creamer, presented her planned research project on Localization of the fungus Chaetothyriales spp. (Ascomycota) within its host Ipomoea carnea (Convolvulaceae).

The current study involves a seed transmitted fungus belongs to the order Chaetothyriales that live within morning glory plants Ipomoea carnea (family Convolvulaceae) and produces swainsonine. The sign of Chaetothyriales sp. on Ipomoea carnea appears as rich white mycelial growth clearly observed with the naked eyes only on the leaves adaxial surface of a fungal-infected plant. Since fungal growth is not present on surfaces of stems or petioles, but only on leaves adaxial surface, and since mycelia is not penetrating leaf epidermis neither found in cross sections, this study aims to answer the question: how does the fungus move within the morning glory plants? Here, we illustrate 2 hypotheses: Hypothesis 1. within the seed, fungus most likely to be found within endosperm, between tissue types associated with the surface of tissues, for example between leaf primordial. Hypothesis 2. The fungus is most likely extending as the plant extend upward, but, contained around leaf meristematic cells during the stem and petiole formation, and once leaf formation occurs, the fungus will exit through the adaxial leaf surface and grows as the leaf grows. This could possibly explain the absence of mycelia in cross sections and on the abaxial surface of leaves. To accept or reject these hypotheses we aim to use microbiology and molecular biology techniques including aseptic separation of embryo from seed coat and perform separate extraction followed by PCR and gel electrophoresis to investigate fungal presence within the seed. Microscopy techniques will be also used including thin sectioning, and Fluorescent in Situ Hybridization FISH and observations under the Scanning electron microscopy and Confocal Laser Scanning microscope.

Chris Schardl discussed a newly funded 5-year (beginning 2021) NSF grant proposal Dimensions US-China:  Phylogenetic breadth, genetic diversity and functions of seed-transmitted fungal endophytes, in which Rebecca Creamer and Daniel Cook are participants. He explained aspects of several seed-transmitted endophytes and the main objectives of the proposal.

Sumanjari Das, a Biology PhD student working with Rebecca Creamer, presented her research Analysis of the mycotoxins (slaframine and swainsonine) level and expression pattern of SWN gene cluster at different time points in Slafractonia leguminicola. The fungal pathogen Slafractonia leguminicola, the causal agent for black patch disease in red clover plants produces two important mycotoxins: slaframine and swainsonine. The indolizidine alkaloid swainsonine is a deadly mycotoxin to livestock causing locoism while slaframine causes slobbers syndrome. Genome sequence analyses revealed all the swainsonine-producing fungi, including S. leguminicola share orthologous gene clusters, “SWN,” which include 7 genes: a multifunctional swnK gene (NRPS-PKS) with domains for the initial steps of swainsonine biosynthesis, swnN and swnR (reductase genes), and swnH1 and swnH2 (nonheme iron dioxygenase gene). This study aimed to investigate the mRNA levels of all the genes of SWN clusters and level of toxin production in S. leguminicola at different time points. cDNAs from total mRNA were isolated from the mycelia at 5 time points post-inoculation and expression pattern were analyzed using RT-qPCR. swnK and and swnK2 (paralog 2) showed higher expression of mRNA in initial days while swn R, H1 and H2 showed increased expression in the later days. The total level of swainsonine and slaframine production from this fungus at these 4 time points were also examined. using liquid chromatography–mass spectrometry. Concentration of both the toxins increased with time, highest concentration detected on day 7. Knowledge on how the age of the mycelia affects toxin production by this fungus is an important step toward developing swainsonine management.

Tracy Sterling and Barbara Keith presented Locoweed research MSU update, endophyte/plant interactions.  The role of a vertically-transmitted fungal endophyte on various locoweed (Astragalus mollissimus var. mollissimus, Astragalus mollissimus var. thompsonae and Oxytropis sericea) plant growth parameters was evaluated in the common garden established in 2011 and located at the Montana Ag Experiment Station’s Post Farm near Bozeman MT. Growth parameters included evaluation of plant survival over winter, gas exchange of carbon assimilation and transpiration, flower and seed numbers to determine fecundity, and seed germination rates of those collected.  The summer of 2020 was the final year for the common garden and no additional gas exchange or fecundity data were collected this year.

The initial garden was established from plants grown from seeds collected in New Mexico in which the endophyte was mechanically removed from the seed to produce endophyte-free plants (E-).  Because vertically-transmitted endophytes are present in the seed during seed maturation, a legacy study to evaluate possible endophyte-induced transgenerational effects in E- plants was initiated in the garden by establishing O. sericea E+ and E- seedlings from seeds collected from 20, 1-year-old common garden plants in 2014 (10 E+ and 10 E-); from these, five seedlings from each were established in Fall 2015. To establish a second generation of plants free from the fungal endophyte, seeds from two plants from 5 E- and 5 E+ families were collected and established in the garden during spring 2017.

Preliminary conclusions to this multi-year-long study indicate survival for any of the species established in the common garden was not influenced by the endophyte, although there is a species survival difference with approximately fifty percent of O. sericea plants surviving three years across all generations, regardless of endophyte status and no A. mollissimus plants surviving beyond two years.  There is not an endophyte effect in plant photosynthesis or stomatal conductance in either of the locoweed species, however, there is a year effect for transpiration with O. sericea E+ plants from the NM seeds and the 1^st generation released from the endophyte legacy plants showing a higher rate of transpiration, but only for one of the six years analyzed. O. sericea NM E+ plants trend toward an increased number of seed pods per reproductive stem over NM E- plants, however, the number of seed pods per stem did not differ among the two generations of E- plants released from the fungal endophyte compared to the corresponding E+ for these generations. The presence (E+) or absence (E-) of the fungal endophyte did not influence germination of seeds collected from any of the generations over time.

Air space volatile compounds from 1^st generation E- released from the endophyte and corresponding E+ plants were collected for a second year from the field plants when plants were beginning to flower. Amount and type of volatiles being released from E+ plants and E- plants did not differ, however, several terpenes were slightly and consistently elevated in E- plants compared to E+ plants. Work is continuing to identify these terpenes.

Several new studies were performed during the summer of 2020. Total nitrogen and carbon content of leaf tissue from E+ and E- 1^st generation legacy progeny was analyzed and was not affected by the fungal endophyte. The number of pollinator visits were recorded and the presence (E+) or absence (E-) of the fungal endophyte did not influence the number of pollinator visits. Root analysis was initiated by removing the surviving plant with as much of the root system as possible. No nodules were found on any of the roots. On-going analysis of the root system includes measuring the number of roots and leaf shoots initiating from the root crown, diameter of the thickest part of the crown, crown dry weight and nitrogen content of the crown.

The common garden study thus far has shown there is no apparent cost or benefit of the fungal endophyte on plant success for field-grown +/- E plants.

Kevin Welch presented his research on Water Hemlock Toxicity.  Water hemlock are plants in the genus Cicuta in the Apiaceae family (formerly Umbelliferae) comprised of four different species. Water hemlock plants are found in wet areas including small stream beds, riverbanks or marshy areas. The toxic components in water hemlock are C₁₇ polyacetylenes, with cicutoxin being the most studied. The proposed mechanism of action of these toxins is from their action as noncompetitive gamma aminobutyric acid (GABA_A) receptor antagonists in the central nervous system. Water hemlock is toxic to all species of livestock and to humans as well. The tubers are reported to be the most toxic plant part, with recent reports of death losses from cattle eating green seeds. Whereas the stems and leaves are thought to be relatively nontoxic, as there is considerable evidence in the field that animals graze the plants during the vegetative stage without any adverse effects. However, there are reports that the very early vegetative plants can be toxic. Additionally, there is some question as to how much variation in toxicity there is between plant populations from different geographical locations.

The variation in cicutoxin and total polyacetylene compounds in water hemlock populations across the great basin area of North America was determined showing minimal differences in risk of poisoning animals at the different locations. We confirmed that cicutoxin is most abundant in the tubers. Even though green seeds have slightly higher cicutoxin concentrations compared to leaves and stems, it is still approximately ten-fold less than that in the tubers. Additionally, we compared the concentration of these toxins in the various plant parts over the growing season. There is more cicutoxin in the leaves and stems of early vegetative plants than that found in mature plants, however, the tubers contain by far the most cicutoxin throughout the entire growing season.

Experiments were performed to evaluate the toxicity of the above ground parts of water hemlock plants. In mice, a water slurry of green seeds was found to be toxic. However, the dose of above ground parts required to poison a goat is so high that it is not likely a risk. The results of our studies suggest that there is little variation in the toxic risk of water hemlock plants across the western USA. The results also suggest that while the above ground parts of water hemlock do contain toxic components, the tubers are the plant part most likely to be a risk to poison animals. Recent research has also demonstrated that cicutoxin can be detected in the rumen contents of animals poisoned by water hemlock, which could be valuable for diagnostic purposes.

Daniel Cook presented his research on Lupinus sulphureus chemotypes and genetic relationships among those chemotypes.  Lupines (Lupinus spp.) are a common plant legume species found on western U.S. rangelands. Lupinus spp. may contain quinolizidine and/or piperidine alkaloids that can be toxic and/or teratogenic to grazing livestock. Alkaloid profiles may vary between and within a species. The objectives of this study were to (1) further explore the characteristic alkaloid profiles of Lupinus sulphureus using field collections and (2) explore the phylogenetic relationship of the different populations and chemotypes of L. sulphureus using the amplified fragment length polymorphism method of DNA fingerprinting, thus providing possible explanations to the phenomena of multiple chemotypes within a species. A total of 49 accessions of L. sulphureus were classified into seven chemotypes. The DNA profiles showed that one L. sulphureus chemotype, chemotype A, is genetically divergent from the other chemotypes of L. sulphureus, suggesting that it represents an unresolved lupine taxon, possibly a new lupine species. Additionally, the different chemotypes of L. sulphureus represented different genetic groups, as shown by Bayesian cluster analysis and principle component analysis.

Stephen Lee presented on Evaluation of earwax, hair, oral fluid, and nasal mucus as noninvasive specimens to determine livestock exposure to poisonous plants. The livestock industry in the western United States loses over $500 million annually from death losses and abortions due to poisonous plants (Holechek, 2002).  This may be underestimated because poisonous plant-induced death losses often go undiagnosed due to a lack of appropriate or available biological specimens for analysis.  Recommendations have been made to assist in collection and preparation of tissue specimens and gut contents for diagnosis of plant poisonings (Stegelmeier et al., 2009).  However, earwax, hair and other noninvasive specimens have been largely neglected as potential specimens for determining livestock consumption of poisonous plants.  Earwax, hair, oral fluid and nasal mucus from livestock in controlled dosing studies and livestock grazing lupine-infested ranges were analyzed for toxic/teratogenic lupine alkaloids by high-performance liquid chromatography-high resolution mass spectrometry (HPLC–HRMS).  Quinolizidine alkaloids including anagyrine were detected in the earwax of cattle grazing lupine-infested rangelands.  In addition, quinolizidine alkaloids including anagyrine were also detected in the earwax, hair, oral fluid and nasal mucus from cattle in controlled dosing studies.  In subsequent studies, larkspur alkaloids (norditerpenoid alkaloids) were detected in the earwax, oral fluid and nasal mucus in from cattle in larkspur dosing studies.  These noninvasive specimens may prove to be valuable tools in the assessment of livestock exposed to toxic and teratogenic lupines.

Ram Nadathur, a Molecular Biology PhD student working with Rebecca Creamer, presented his research on Regulation of swainsonine production and water stress in Alternaria. Locoweed consumption by animals has posed a toxin challenge in the arid western USA for more than 100 years. Locoweeds contain swainsonine, a mycotoxin which causes a neurodegenerative syndrome in livestock called locoism. The fungal genus Alternaria contains several species that adversely affect crop production and produce mycotoxins that are harmful to plants and animals. The fungus Alternaria oxytropis is prevalent in western North America and China in arctic and alpine regions and produces the toxin swainsonine, the consumption of which causes locoism in cattle. Genome analyses of the biosynthesis pathway of swainsonine in endophyte-infected locoweeds revealed a consensus region of orthologous gene clusters containing a multifunctional swnK gene. The ß-ketoacyl synthase identified as the swn-KS region in swainsonine-producing species, has been characterized. To determine the relationship between the swnK-KS region and the production of swainsonine, six fungal species primarily from the US and China were assessed. Q-PCR analysis revealed that the significant swainsonine-producing fungi Metarhizium anisopliae, Alternaria oxytropis (glabra isolate) and the clover pathogen Slafractonia leguminicola had higher swn-KS gene expression compared to Alternaria bornmuelleri, after 14 days in culture. However, only for Alternaria oxytropis (strictus isolate) was the production of swainsonine correlated to the gene expression of the swn-KS. These findings identified increased gene expression of swn-KS in species previously identified as high swainsonine-producers. These results question whether swn-KS gene expression can be used as a predictive factor for increased swainsonine production. Detailed understanding of other factors influencing swainsonine levels in fungi such as media composition and time in culture will improve methods to predict toxicity in locoweed populations. Further, the role of swainsonine production in fungal colonization and infection of the host plant remains unknown. Stress response has been identified as an underlying factor affecting pathogenesis. Particularly, the role of transcriptional stress response regulators has been studied in Alternaria alternata. I examined the role of two stress response modulators YAP-1 and SSK-1 previously identified in Alternaria alternata among several species of swainsonine-producing fungi. The expression of SSK-1 and YAP-1 in the pathogenic Alternaria alternata was significantly upregulated while the expression of YAP-1 in Alternaria bornmuellerii was downregulated. Although stress modulators have been known to largely be constitutively active, this suggests that pathogens and non-pathogens exist at varying stress levels during the growth phase in culture. Investigating the role of these two transcriptional regulators over a time course conclusively provided evidence of increased expression in all Alternaria species over 3, 7, and 10 days, respectively, although YAP-1 levels saw a downturn in Alternaria bornmuellerii.

Ben Green presented his research on Plant compounds which poison grazing livestock. Toxic plants poison grazing livestock causing large economic losses. The relative composition, chirality, and concentration of toxins in these plants affects their toxic potential. For example, differences in Delphinium (larkspur) spp. toxicity are due plant norditerpene alkaloid composition (chemotype) and the biogeographical distribution of plant chemotypes with some chemotypes significantly more toxic to grazing animals than others. There are also animal factors which influence poisonings. Experiments have demonstrated that sex, age, and cattle breed affect responses to larkspur. For example, Angus heifers are more susceptible to larkspur intoxication than are Angus steers or bulls. Yearling Angus steers are more susceptible to intoxication than are two-year old animals. Piperidine alkaloids like coniine from Conium maculatum are toxic and teratogenic to livestock. These differences in composition, chirality, and concentration of bioactive compounds in poisonous plants impacts the responses of livestock. The management of livestock must consider both plants and the animal grazing them.

 

There was a brief discussion as to suggested and possible collaborative projects for the upcoming year. Topics of interest include:

How does endophyte infection status influence plant competition. Are endophyte-infected plants more competitive than non-infected plants? Can plant interference be studied using pot experiments? Suggestion was to plant different proportions of E+ and E- plants together in the same pots and determine how that effects the overall competitiveness of the plants.

Do weather conditions have an impact on seedling emergence? Locoweeds emerge in a cyclic pattern based on moisture.

There is interest in working on irrigated forages and improved pastures. Jason Turner and Rebecca Creamer are interested in studying sorghum, sudangrass, and sorghum X sudangrass and dhurrin content in hay. There are questions about the breakdown products and exactly which products are the causal agents for toxicity to horses, and concerns about how treatment of the hay impacts the toxicity to horses.

Is the group still interested in submitting to Western SARE Professional Development grant? This is a good way to add impacts to our research and promotes ranchers and researchers working together. This would be particularly important in New Mexico because we have so many new extension agents that don’t have a strong grounding in ranching and the problems with poisonous plants.

Which extension range specialists should we invite to the group? – Eric Thacker, Utah and Casey Spackman, New Mexico will be invited to join.

Discussion on the 2021 meeting – The meeting will be hybrid (in person/zoom) to be held in October 2021 in Logan, UT.

Accomplishments

<p>The entire group met, discussed the current status of poisonous plants. A subset of the group worked together on cooperative research.&nbsp; The group set priorities for collaborative research and grants for the coming year. Because this project did not begin until October 2020, few accomplishments have been noted in a month.</p><br /> <p>Chris Schardl is the PI, Rebecca Creamer a Co-PI, and Daniel Cook is a collaborator on a 5-year NSF grant that begins in January 2021. This is a major accomplishment and will provide for abundant collaborative work on seed-borne fungal endophytes.</p>

Publications

<p>Nothing to report at this time.</p>

Impact Statements

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Report Information

Participants

Daniel Cook – USDA/ARS Poisonous Plant Lab, Logan, UT Daniel.cook@usda.ars.gov.us
Barbara Keith – Land Resources and Environmental Sciences (LRES), Montana State University, bkeith@montana.edu
Christopher Schardl – University of Kentucky, chris.schardl@uky.edu
Tracy Sterling – Dept. LRES, Montana State University, tracy.sterling@montana.edu
Rebecca Creamer – Dept EPPWS, New Mexico State University, creamer@nmsu.edu
Sumanjari Das –Biology, NMSU, sdas@nmsu.edu
Marwa Neyaz – Plant and Environmental Science, NMSU, marwane@nmsu.edu
Kevin Welch - USDA/ARS Poisonous Plant Lab, Logan, UT, Kevin.welch@usda.gov
Stephen Lee - USDA/ARS Poisonous Plant Lab, Logan, UT, Stephen.lee@usda.gov
Clint Stonecipher - USDA/ARS PPL, Logan, UT, clint.stonecipher@usda.gov
Christopher Davies –Assoc. AES Director, Utah State University, chris.davies@usu.edu
Jason Turner – Extension Animal Science, NMSU, jturner@nmsu.edu
James Strickland – Clemson University, jrstric@clemson.edu
David Weaver – Montana State University, Weaver@montana.edu
Rachel Sneed – Dept Plant Pathology, University of Kentucky, Rachel.sneed@uky.edu
Sarah Ward – Montana State University, sarahward1@earthlink.net
Jackson Strand – Montana State University, Jackson.strand@student.montana.edu
Roseann Wallander – Montana State University, rtw@montana.edu

Brief Summary of Minutes

Welcomed to Montana State University by Dr. Sreekala Bajwa, Dean of College of Agriculture, Associate director or MAES, and VP of Agriculture

Chris Davies, as project administrator, briefly discussed the Multistate project system. He explained that this project began in October 2020 and will continue for 5 years.

Rebecca Creamer led introductions of all participants.

 

Tracy Sterling presented talk on Locoweed common garden and seedling recruitment update 2022, by Keith, Sterling, and Ward.

The role of a vertically-transmitted fungal endophyte on various locoweed (Astragalus mollissimus var. mollissimus, Astragalus mollissimus var. thompsonae and Oxytropis sericea) plant growth parameters was evaluated in the common garden established in 2011 and located at the Montana Ag Experiment Station’s Post Farm near Bozeman MT to examine the influence of the endophyte on locoweed plant success when grown at the edge or beyond its native range. Growth parameters included evaluation of plant survival over winter, gas exchange of carbon assimilation and transpiration, flower and seed numbers to determine fecundity, and seed germination rates of those collected. Plants were sacrificed during summer 2020 to analyze roots. Seed bank recruitment at the site began in 2021 and the 1000 new seedlings in collaboration with USDA-ARS partners, each plant is being evaluated for SWA content with random pairs of plants with (E+) and without (E-) the endophyte sampled for mRNA to evaluate differential gene expression.

The initial garden was established from plants grown from seeds collected in New Mexico in which the endophyte was mechanically removed from the seed to produce endophyte-free plants.  Because vertically-transmitted endophytes are present in the seed during seed maturation, a legacy study to evaluate possible endophyte-induced maternal effects in E- plants was initiated in the garden by establishing O. sericea E+ and E- seedlings from seeds collected from 20, 1-year-old common garden plants in 2014 (10 E+ and 10 E-); from these, five seedlings from each were established in Fall 2015. To establish a second generation of plants free from the fungal endophyte, seeds from two plants from 5 E- and 5 E+ families were collected and established in the garden during spring 2017. 

Statistical analyses was conducted with the MSU Statistical Consulting Center. The main effects of taxon, generation, endophyte presence, and their interactions were analyzed using R (R Core Team 2021) Zone of each E-/+ pair, year planted, plant age (# of winters), and year sampled were treated as fixed effects where appropriate. Response variables involving counts were fit to a generalized linear mixed model with a Poisson response distribution. In initial models, we tested for an interaction between the two varieties of the A. mollissimus species and the endophyte +/- status. If there was no E x spp interaction, main effects of endophyte and main effects of species, comparing within genera were evaluated after adjusting for the species, and the random effects from the nested and repeated measurements design. There were no other fixed effects included due to aliasing of the species with the other variables, but the nesting and random effects structure accounted for the pair identifier matching the endophyte +/- plants within the 9 zones in the common garden.

For the O. sericea, we tested for an interaction between the generations and the endophyte +/- status. If there was no E x generation interaction, main effects of endophyte and main effects of species were evaluated after adjusting for the generation and the random effects from the nested and repeated measurements design. Similar to the A. mollissimus analysis, there were no other fixed effects included due to the aliasing of the generation with the other variables, but the nesting and random effects structure accounted for the pair identifier matching the endophyte +/- plants within the 9 zones in the common garden.

For continuous measurements such as diameter, weight, and the various Licor measurements a linear mixed effects model was used with the same fixed and random effects structure (where appropriate).

For time-to-germination analysis and survival analysis for the mixed effects model, a Gompertz discrete proportional hazards model was used to compare the different germination or surival curves first on the interaction between generation and endophyte +/- status and then on the main effects of endophyte +/- status and the main effect of generation after adjusting for the fixed effects of year of the seed collection for germination analysis and year planted for the survival analysis after adjusting for the nesting and repeated measurements structure.

Conclusions to this multi-year-long study indicate survival for any of the species established in the common garden was not influenced by the endophyte, although there is a species survival difference with approximately fifty percent of O. sericea plants surviving three years across all generations, regardless of endophyte status and no A. mollissimus plants surviving beyond two years.  There is not an endophyte effect in plant photosynthesis or stomatal conductance in either of the locoweed species, however, there is a year effect for transpiration with O. sericea E+ plants from the NM seeds and the 1^st generation released from the endophyte legacy plants showing a higher rate of transpiration, but only for one of the six years analyzed.

The common garden study provides the first comprehensive study demonstrating the endopyte/locoweed complex is a commensal relationship and there is no apparent cost or benefit of the fungal endophyte on plant success for field-grown +/- E plants. Results are being written up for submission to American Journal of Botany Fall 2022.

 

Jackson Strand, graduate student working with David Weaver, Montana State University, presented his research Using Organic Volatiles to Understand the Impact of Alternaria oxytropis on Locoweed Physiology: E+ / E- by Strand, Sterling, and Weaver.

Locoweeds are Astragalus and Oxytropis species that contain the toxic alkaloid swainsonine. The fungal endophyte Undifilum oxytropis is found in locoweed species and is responsible for the synthesis of swainsonine. Previous research has shown little noticeable difference in host plant fitness between endophyte positive and negative plants. Volatile Organic Compounds (VOCs) are involved in a wide array of biological and ecological functions, playing a crucial role in plants interacting with biotic and abiotic factors. Our objective was to collect VOCs produced by both endophyte positive and negative Oxytropis locoweed plants and compare the volatile composition and quantities between the two sample groups. We found that plants containing the endophyte exhibited decreased levels of β-ocimene, a key pollinator signal, and humulene. In addition, we found that endophyte positive plants produced increased levels of β-pinene, an important volatile in insect herbivore defense. Our results suggest a possible physiological change brought about by endophyte presence, the possible ecological implications of endophyte presence, and the need for further research.

 

Rachel Sneed, graduate student in Plant Pathology working with Dr. Chris Schardl at University of Kentucky presented her research on Assessing Epichloe endophyte frequency distributions and how genetic diversity influences functional and phylogenetic diversity.

Seed-transmissible fungal endophytes are best known for their roles as defensive mutualists. Historically the discovery of fungal endophytes was driven by investigations of plant toxicity to livestock, followed by extensive study of their diverse alkaloid profiles (chemotypes) for protection against insects and nematodes. These hereditary symbionts have diverse ecological functions, and considerable genetic and chemotypic diversity even within plant and endophyte species. Population analyses on genomic scales are underway to help us comprehend endophyte contributions to host species functional diversity as well as species diversity of their associated communities. We are investigating the diversity of the woodland grass Brachyelytrum erectum, its endophyte Epichloë brachyelytri, and the ecological relevance of that diversity. I first aim to screen populations from 20 locations across Kentucky using multiplex PCR testing for presence of various housekeeping genes, pyrrolopyrazine alkaloids, loline alkaloids, ergot alkaloids, indole-diterpenes, and mating types. We will also compare these results to a RNAseq screening to observe any transcriptomic changes due to endophyte presence or absence. 

 

Chris Schardl presented at talk CRISPR in the tall fescue endophyte.

The common forage and pasture grass, tall fescue (Lolium arundinaceum = Schedonorus arundinaceus) commonly harbors a seed-transmitted fungal symbiont (endophyte), Epichloe coenophiala (Neotyphodium coenophialum). The most common strains of E. coenophiala produce ergot alkaloids, which can cause episodes of toxicosis to livestock grazing the grass. Removing the endophyte from tall fescue seed stock, though technical feasible, is generally undesirable because stands of endophyte-free plants can be much less fit in the field than the parental lines containing E. coenophiala. Natural endophyte strains lacking ergot alkaloid genes have been identified from wild populations of other Lolium species, and some are deployed in U.S. cultivars, but in such cases there is concern about relative compatibility of the grass and endophyte genotypes and whether the same range of fitness enhancements are realized by the plant. In the past we have developed transgenic methods to modify E. coenophiala, but regulation and public acceptance are a concern. Most recently we used CRISPR technology and a procedure that resulted in no net introduction of exogenous DNA to simultaneously eliminate all ergot alkaloid biosynthesis genes from the endophyte. The resulting non-toxic and non-transgenic endophyte should be suitable for pasture and field cultivars of tall fescue.

 

Kevin Welch presented the talk, Mixture toxicology: Multiple plant toxins.

Poisonous plants can adversely impact livestock in numerous ways, including the acute poisoning of animals leading to their death. Often times as researchers, we oversimplify our evaluation of the effects of poisonous plants by focusing on a single plant, or even a single constituent from that plant. However, when livestock graze in a rangeland they have the potential to consume numerous poisonous plants. The consumption of multiple poisonous plants at the same time can lead to mixture effects, in that two or more toxins may have an additive or even synergistic effect on the animal. In this talk, I presented data demonstrating that in some plants more than one constituent needs to be quantitated to fully understand the risk of animals being poisoned from that plant. Also, I presented data demonstrating that co-exposure to multiple poisonous plants can have an additive effect on the animal. This may help explain why some animals appear to be more sensitive to some poisonous plants in a range setting compared to data obtained in controlled studies.

 

Clint Stonecipher presented a talk on Death Camas.

Death camas (Zigadenus paniculatus) is a bulbous perennial forb that is toxic to cattle and sheep. Large losses occasionally occur in sheep and the problem occurs early in the spring. Grazing studies have been conducted to determine if animals that are hungry, when turned out to graze rangelands, will consume more death camas than animals that are satiated. Studies have been conducted in three locations in Utah and Idaho. One hungry animal was poisoned when grazing death camas in the early vegetative stage of plant growth and another hungry animal was poisoned when grazing death camas during the flowering stage of plant growth. None of the satiated animals were poisoned from death camas. Death camas is not a preferred forage by sheep but under some conditions, sheep will become poisoned from consuming death camas.

 

Stephen Lee presented the talk Noninvasive specimens to determine livestock exposure to poisonous plants.

The livestock industry in the western United States loses over $500 million annually from death losses and abortions due to poisonous plants (Holechek, 2002).  This may be underestimated because poisonous plant-induced death losses often go undiagnosed due to a lack of appropriate or available biological specimens for analysis.  Recommendations have been made to assist in collection and preparation of tissue specimens and gut contents for diagnosis of plant poisonings (Stegelmeier et al., 2009).  However, earwax, hair, oral fluid and nasal mucus have been largely neglected as potential specimens for determining livestock consumption of poisonous plants. These specimens in controlled poisonous plant dosing studies and livestock grazing poisonous plant infested ranges were analyzed for toxic/teratogenic alkaloids by high-performance liquid chromatography-high resolution mass spectrometry (HPLC–HRMS).  These noninvasive specimens may prove to be valuable tools in the assessment of livestock exposed to toxic and teratogenic plants.

 

Sumanjari Das, recent PhD graduate that worked with Rebecca Creamer, presented her research Characterization of a transmembrane transporter gene, the temporal expression pattern of the swn genes and toxin secretion levels, and swK paralogues in Slafractonia leguminicola

The plant pathogenic fungus Slafractonia leguminicola (reclassified Rhizoctonia leguminicola), causes black patch disease in red clover plants (Trifolium pratense L.) and other legumes. This plant pathogen produces two secondary metabolites: slaframine and swainsonine, that are toxic to livestock grazing on clover hay or pasture infested with the fungus. Swainsonine toxicosis causes locoism while slaframine causes slobbers syndrome. Genome sequence analyses revealed all the swainsonine-producing fungi, including S. leguminicola share orthologous gene clusters, “SWN,” which include 7 genes: a multifunctional swnK gene (NRPS-PKS) with domains for the initial steps of swainsonine biosynthesis, swnN and swnR (reductase genes), and swnH1 and swnH2 (nonheme iron dioxygenase gene). In addition to these 7 genes, two paralogs of swnK, swnK1 (paralog1) and swnk2 (paralog2) are found in S. leguminicola. All the genes in the SWN gene clusters are predicted to be involved in the swainsonine biosynthesis pathway. The overall goal of this research was to characterize a transmembrane transporter gene and study the temporal expression pattern of all the SWN genes and toxin secretion levels in S. leguminicola. The objective of the first part of the research is to investigate the role of a putative transmembrane transporter (swnT) in mycotoxin transport in S. leguminicola. swnT was silenced by RNA interference (RNA-i) using the silencing vector Psilent1 which includes inverted repeat transgenes of the swnT gene. This resulted in significant reduction in the swnT transcript levels compared to the controls. Silencing caused decline in active efflux of toxins from the mycelia to the media as shown by LC-MS analysis. SwnT transformants showed higher concentrations of both toxins in the mycelia compared to that in the media. SwnT transformants also exhibited visibly distinct phenotypes with much thicker and shorter mycelia compared to the wild type. These transformants were also unable to infect in detached leaves, unlike the controls, suggesting that swnT function might have role in pathogenesis in addition to toxin transportation. This research demonstrates the importance of this transporter to secretion of mycotoxins for this phytopathogenic fungus. For the second project, the study aimed to investigate the mRNA levels of all the genes of SWN clusters and level of toxin production in S. leguminicola at different time points. cDNAs from total mRNA were isolated from the S. leguminicola mycelia grown in the culture plates as well as from leaves inoculated with the fungal mycelia at different time points and expression pattern of the SWN genes were analyzed using RT-qPCR. The total level of swainsonine and slaframine production from this fungus at different time points were also examined using liquid chromatography–mass spectrometry. This research will help to develop a better foundation for the future study of the swainsonine and slaframine biosynthesis pathway and characterization of the associated catalytic enzyme genes in S. leguminicola. Knowledge on how the age of the mycelia affects toxin production by this fungus is an important step toward developing swainsonine management.

 

Daniel Cook presented a talk, Phylogenetic patterns of swainsonine presence in morning glories.

Endosymbionts play important roles in the life cycles of many macro-organisms. The indolizidine alkaloid swainsonine is produced by heritable fungi that occurs in diverse plant families, such as locoweeds (Fabaceae) and morning glories (Convolvulaceae) plus two species of Malvaceae. Swainsonine is known for its toxic effects on livestock following the ingestion of locoweeds and the potential for pharmaceutical applications. We sampled and tested herbarium seed samples (n = 983) from 244 morning glory species for the presence of swainsonine and built a phylogeny based on available internal transcribed spacer (ITS) sequences of the sampled species. We show that swainsonine occurs only in a single morning glory clade and host species are established on multiple continents. Our results further indicate that this symbiosis developed ∼5 mya and that swainsonine-positive species have larger seeds than their uninfected conspecifics.

 

Marwa Neyaz, a former  PhD student that worked with Rebecca Creamer, presented her research on Characterization of swainsonine producing fungi and localization in their host.

A diverse group of fungi including plant symbionts and pathogens of plants, insects, and mammals produce swainsonine; an indolizidine alkaloid and the toxic principle in several plant species worldwide and causes severe toxicosis in livestock grazing these plants. The goal of this research was to understand the association of fungi producing swainsonine to gain insights into their evolution and ecology. This was investigated by characterizing the SWN orthologous gene cluster, which is shared among these fungi and necessary for swainsonine synthesis, and localizing and identifying these fungi using microscopy and molecular biology techniques. Characterization of the SWN gene cluster suggests that in some fungal orders the SWN cluster was gained once from a common ancestor while in other orders it was likely gained several times from one or more common ancestors. Other patterns including the high conservation of swnK and swnH2 genes, genes rearrangements and inversions, and absence of genes provide evidence of a complex evolutionary history of this cluster. Localization of the Chaetothyriales fungus within Ipomoea carnea revealed presence of mycelia in the hilum, sclereids, hypocotyl, shoot apical meristem, and adaxial surface of immature folded leaves. Moreover, no cellular damage was observed due to fungal colonization, and the mycelia formed close association with the peltate glandular trichomes. These results provide explanation for how this symbiont and others may persist and transmit overtime. Continued studies on morphological, molecular, and ecological characteristics of this important taxa of fungi will likely advance knowledge of their evolution and understanding the association between swainsonine production, fungal morphology, and endophytic ecology.

 

Discussion of W2193 Project

There was a brief discussion as to how to best improve the group and its meetings. Primary suggestions were to bring new members in and increase the diversity of the group. Perhaps we should ask members to participate from Oregon or Carl Yeoman (Animal Biosciences) Montana State University. A personal request may be the most successful and a request for them to showcase their research might particularly bring folks for a trial visit.

The group much preferred the change in meeting dates from fall to summer.

Discussion on the 2023 meeting – The meeting will be hybrid (in person/zoom) to be held in late May or early June 2023 in Logan, UT.

Accomplishments

<p>The entire group met, discussed the current status of poisonous plants. A subset of the group worked together on cooperative research. The group set priorities for collaborative research and grants for the coming year.</p><br /> <p>&nbsp;</p><br /> <p><strong>Chris Schardl </strong>is the PI, <strong>Rebecca Creamer</strong> a Co-PI, and <strong>Daniel Cook</strong> is a collaborator on a 5-year NSF grant that began in January 2021. This is a major accomplishment and will provided abundant collaborative work among the three investigators on seed-borne fungal endophytes. Two talks at this meeting (<strong>Schardl</strong> and <strong>Sneed</strong>) were directly related to that grant and three talks (<strong>Cook</strong>, <strong>Neyaz</strong>, and <strong>Das</strong>) included aspects from the grant. Since the grant has not held in in person meetings, this WERA 2193 meeting served as a chance to make progress presentations.</p><br /> <p>&nbsp;</p><br /> <p><strong>Schardl, Creamer, Cook</strong> (and others) wrote a book chapter for Mycota on fungal endophytes of plants.</p>

Publications

<p><strong>Schardl, Creamer, Cook</strong> (and others) wrote a book chapter for Mycota on fungal endophytes of plants.</p><br /> <p>&nbsp;</p><br /> <p><strong>Other joint publications by W2193 members/attendees published since the last report are listed below:</strong></p><br /> <p><strong>Neyaz</strong>, M., <strong>Das</strong>, S., <strong>Cook</strong>, D., <strong>Creamer,</strong> R. 2022. Phylogenetic comparison of swainsonine biosynthetic gene clusters among fungi.&nbsp; Journal of Fungi 8:359.</p><br /> <p><strong>Neyaz</strong>, M., Gardner, D. R., <strong>Creamer</strong>, R., <strong>Cook</strong>, D. 2022. Localization of the swainsonine-producing Chaetothyriales symbiont in the seed and shoot apical meristem in its host <em>Ipomoea carnea</em>. Microorganisms 10:545.</p><br /> <p>Noor, A.I., Nava, A., <strong>Neyaz,</strong> M., Cooke, P. <strong>Creamer</strong>, R. <strong>Cook</strong>, D. 2021. Ectopic growth of the Chaetothyriales fungal symbiont on <em>Ipomoea carnea</em>. Botany 99 (10):619-627.</p><br /> <p><strong>Creamer,</strong> R., Hille, D.B., <strong>Neyaz,</strong> M., Nusayr, T., <strong>Schardl,</strong> C.L., <strong>Cook,</strong> D. 2021. Genetic relationship in the toxin producing fungal endophyte, <em>Alternaria oxytropis</em>, using polyketide synthase and non-ribosomal peptide synthase genes. Journal of Fungi 7(7), 538. <a href="https://doi.org/10.3390/jof7070538">https://doi.org/10.3390/jof7070538</a></p><br /> <p>&nbsp;</p>

Impact Statements

1. The W2193 Multistate group has made major contributions towards our understanding of fungal endophytes of plants.

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Report Information

Participants

Daniel Cook – USDA/ARS Poisonous Plant Lab, Logan, UT Daniel.cook@usda.ars.gov.us
Christopher Schardl – University of Kentucky, chris.schardl@uky.edu
Rebecca Creamer – Dept EPPWS, New Mexico State University, creamer@nmsu.edu
Kevin Welch - USDA/ARS Poisonous Plant Lab, Logan, UT, Kevin.welch@usda.gov
Stephen Lee - USDA/ARS Poisonous Plant Lab, Logan, UT, Stephen.lee@usda.gov
Clint Stonecipher - USDA/ARS PPL, Logan, UT, clint.stonecipher@usda.gov
Dale Gardner – USDA/ARS PPL, Logan, UT, dale.gardner@usda.gov
Ben Green – USDA/ARS PPL, Logan, UT, ben,green@usda.gov
Eric Thacker – Utah State University, Cooperative Extension, eric.thacker@usu.edu
Casey Spackman – New Mexico State University, Extension Animal and Range Science, spackman@nmsu.edu
Christopher Davies –Assoc. AES Director, Utah State University, chris.davies@usu.edu
Rachel Sneed – Dept Plant Pathology, University of Kentucky, Rachel.sneed@uky.edu
Michelle Afkhami – University of Miami, michelle.afkhami@gmail.com
Chris Searcy – University of Miami, christopher.searcy82@gmail.com
Jack Kocher – University of Kentucky, jack.kocher@uky.edu
Andrew Tapia – University of Kentucky, andrew.tapia@uky.edu
Neil Moore – University of Kentucky, neil@cs.uky.edu

Brief Summary of Minutes

Daniel Cook welcomed everyone to the UADA, ARS Poisonous Plant Laboratory and explained who works there and what they do. The remainder of the meeting consisted of presentations by the participants and a field trip to look at the poisonous plants found around Logan, Utah.

 

Chris Schardl presented Facile and versatile CRISPR-based modification of alkaloid profiles of Epichloe coenophiala, the endophyte of tall fescue.

The common endophyte of the popular forage and pasture grass tall fescue (Lolium arundinaceum = Schedonorus arundinaceus) is the fungus Epichloë coenophiala, which produces up to four different classes of alkaloids that protect the grass from invertebrate herbivores: aminopyrrolizidines, ergot alkaloids, indole-diterpenes and pyrrolopyrazines. Two such alkaloid classes, the ergot alkaloids and indole-diterpenes, can also be toxic to livestock. Strains that lack the genes for these anti-mammalian toxins can be incorporated into breeding lines for tall fescue cultivar development. We adapted CRISPR technology to E. coenophiala and related fungi to precisely delete genes and clusters of genes for alkaloid biosynthesis, for example by deleting the entirety of two ergot alkaloid biosynthesis (EAS) gene clusters. Our approach involves transient co-transformation of single-guide RNAs, Cas9 protein and the selectable hygromycin B-resistance gene hph. Some of the targeted deletion mutants obtained in this approach lose hph and are non-transgenic. In addition to deleting EAS genes, we used this method to delete key genes for biosynthesis of indole-diterpenes (idtS), aminopyrrolizidines (lolC) and pyrrolopyrazines (ppzA). Because the role of idtS has not been established previously, Stephen Lee and Daniel Cook verified that its CRISPR-deletion mutants lack indole-diterpenes. The precise modification of E. coenophiala alkaloid profiles continues with the objective of generating a panel of mutants to allow for well-controlled studies of the effects of specific alkaloids on invertebrate herbivores as well as mammals.

 

Michelle Afkhami presented Big impact of tiny players: Chemical defense and drought tolerance from fungal endophytes affects plant species ranges and population persistence 15 years later.

Microbes are ubiquitous and can play key roles in the ecology and evolution of plants and animals with which they interact. In this talk, I show how phyllosphere fungal endophytes can ameliorate abiotic stress for their host plants, leading to profound effects on a species’ range limits and long term population dynamics of plants. First, combining field surveys of 92 populations, 10 common garden experiments throughout the host plant range, species distribution models and greenhouse experiments, I show that mutualistic fungal endophytes ameliorate drought stress and broaden the geographic range of their native grass host Bromus laevipes by thousands of square kilometres (~ 20% larger) into drier habitats. Second, I share research illustrating that these endophytes generate defensive chemicals that reduce herbivore damage and increase plant performance across the range, but that these chemical defenses do not underlie range divergence of their hosts. Finally, by resurveying the 92 B. laevipes populations ~15 years later, I demonstrate that populations with these endophytes were more likely to persist over time than populations without this fungal mutualist. Together, these results illustrate the importance of fungal endophyte mutualisms in host plant ecology over large spatial and temporal scales.

 

Rachel Sneed, graduate student in Plant Pathology working with Dr. Chris Schardl at University of Kentucky presented her research on Assessing frequency distributions and genetic and chemotypic diversity of the endophyte Epichloe brachyelytri and effects on its host grass Brachyelytrum erectum.

Seed-transmissible epichloid fungal endophytes are best known for their roles as defensive mutualists in cool-season grasses. Historically, the discovery of fungal endophytes was driven by investigations of plant toxicity to livestock, followed by extensive study of their alkaloids and protection against insects and nematodes. Epichloae can produce four classes of alkaloids: ergot alkaloids, lolines (saturated aminopyrrolizidines), indole–diterpenes, and peramine. It is increasingly evident that these hereditary symbionts have much more diverse chemical profiles both in individual populations and between them. To this end, differences in chemotypic profiles of these symbionts may translate to different evolutionary and environmental advantages across plant species, as well as influences on phenotypic measurements. In an ecological sense, the chemotypic diversity within the species may reflect frequency-dependent selection for the alkaloids, which can be metabolically expensive to produce. To date, there has yet to be extensive study on the alkaloid profiles of Epichloë brachyelytri in the cool-grass host Brachyelytrum erectum. This project aims to initiate the required genomic population analyses to comprehend endophyte frequency and chemotypic diversity among and between populations throughout the state of Kentucky. 

In a previous survey of B. erectum in the Kentucky Palisades, 50% of the plants had fungal endophytes, with two endophyte genotypes that differed in alkaloid profiles. In the investigation, 21 populations of B. erectum have been sampled and the genetic and chemotypic diversity analyzed by high-throughput tiller extraction and multiplex PCR. Biosynthesis genes for ergot alkaloids (e.g., chanoclavine and ergovaline), aminopyrrolizidines (e.g, lolines), indole-diterpenes (e.g., lolitrems) and pyrrolopyrazines (e.g., peramine), as well as genes for mating type and phylogenetic barcodes, are interrogated to indicate endophyte presence and diversity. Morphological measures for all samples were also assessed, and six populations were sampled for transcriptome analysis. The results of these studies will expand the understanding of how endophyte frequency distributions and genetic diversity can influence functional and phylogenetic diversity.

 

Jack Kocher, undergraduate student working with Dr. Chris Schardl at University of Kentucky presented his research on Preliminary introduction to fungal culturing for genotype confirmation.

 

Chris Searcy presented Soil microbiomes underlie population persistence of an endangered plant species.

Microbiomes can dramatically alter individual plant performance, yet how these effects influence higher order processes is not well resolved. In particular, little is known about how microbiome effects on individual plants alter plant population dynamics, a question critical to imperiled species conservation. Here, we integrate bioassays, multidecadal demographic data, and integral projection modeling to determine the soil microbiome’s role in plant population dynamics. Simulations indicated that the presence of soil microbiomes boosted population growth rates (λ) of the endangered Hypericum cumulicola by 13%, a difference between population growth versus decline in 76% of patches. The greatest benefit (47% increase in λ) occurred in low nutrient, high elevation habitats, suggesting that the soil microbiome may be responsible for expanding H. cumulicola’s distribution to include these stressful habitats. Our results demonstrate that soil microbiomes can significantly affect plant population growth and persistence, and support the incorporation of soil microbiomes into conservation planning. 

 

Andrew Tapia, graduate student at University of Kentucky working with Dr. Neil Moore presented Computing genetic similarity matrices among plants by using RNA-seq data.

Although RNA-seq data are commonly used for assessing functional diversity by measuring gene expression, this presentation describes an algorithm that uses RNA-seq data for measuring genetic diversity. The algorithm begins with a subset of genes from transcriptomes assembled from RNA-seq reads for two or more individual organisms (“samples”). The algorithm uses nucleotide BLAST to identify likely homologous genes for each pair of samples and constructs an undirected graph to find those genes with homologs in all samples. The algorithm computes a similarity value for each pair of samples using the statistics for BLAST hits between genes identified as having homologs in all samples. This approach was tested with sixteen tall fescue samples, and the similarity matrix computed showed the pattern expected. Pairs of samples with identical genotypes have very high similarity while pairs with different genotypes have lower similarity. Though the results of this initial test are reassuring, we plan further tests to verify the soundness of the approach and intend to investigate the mathematical properties of the similarity computed. 

 

Rebecca Creamer presented Ecology of swainsonine-producing fungi.

Swainsonine-producing fungi have diverse ecological roles including, plant pathogen, plant commensal, plant mutualist, entomopathogen, saprophyte, dermatophyte, and plant epiphyte. Swainsonine is not a pathogenicity factor for any of the fungi. The ecological role of the fungi appears to change with host quality and nutrient availability.

 

Neil Moore presented Computing orthologies for endophytes of Lolium species of grasses.

Discussion of various computer programs that were used including OrthoMCL which gives an output of ortholog groups, which contain in paralogs and out paralogs and co-orthologs. Another program, COCO-CL, uses the output from OrthoMCL to compute split scores giving the likelihood of speciation and duplication events.

 

Chris Schardl, Neil Moore, and Joanna Cholewo presented CURatio: A tool for assessing and visualizing phylogenetic tree relationships in comprehensive phylogenomic analysis.

As whole genomes are sequenced and annotated the tools for subsequent phylogenomic analyses are limited. For example, most tools require that each genome has an ortholog of each gene used in the analysis. This would preclude incorporation of "accessory" genes, defined as showing presence/absence polymorphism, which is a common feature of secondary metabolite biosynthesis genes among others. We developed a method to compare phylogenetic gene trees with presumed or inferred species trees whether or not the gene trees include representative orthologs in all of the genomes being compared. The method, called "constrained to unconstrained ratio" (CURatio), compares the gene tree length from phylogenetic inference using a species (or other) tree as a constraint to the length of the tree obtained from unconstrained inference from the same sequence alignment. The results, applied to sequenced and annotated genomes from the fungal family Clavicipitaceae, give CURatio scores close to 1.0 for most gene trees, but significantly higher ratios for many of the alkaloid biosynthesis genes, including those for the potent anti-mammalian ergot alkaloids.

 

Dale Gardner presented Salvia reflexa.

A case in which 500 range beef cows were fed alfalfa hay unknowingly contaminated with Salvia reflexa (lance-leaf sage) was investigated.  Over 165 died, many within 72 hours of feeding.  Clinical signs were observed in less than 24 hours and included feed refusal, depression, lethargy followed by sternal then lateral recumbency, bellowing, apparent pain and discomfort then death.  Some cows that did not die early showed aberrant behavior, aggression, apparent blindness, exhaustion and eventually death.  Necropsies by the attending Veterinarian revealed dark mottled livers.  Grossly, affected cattle had swollen livers with prominent lobular red discoloration.  The toxicity of the hay was experimentally determined using pen studies with cattle, followed by a mouse bioassay guided chemical extraction and fractionation process to identify hepatotoxic compounds. The hepatoxic compounds were identified as salviarin, salvianduline D, rhyacophiline and 7-hydroxyrhyacophiline.  All compounds were found to induce severe acute hepatic necrosis within 24 to 48 hours after a single oral dosage (260 - 280 mg/kg) in mice. The identified diterpenes are known to be found among different Salvia species which led to finding dried plant parts of S. reflexa within bales of weedy hay and subsequently a population of S. reflexa was found along the field edges and irrigation ditch banks of the alfalfa hay field.  It was concluded that S. reflexa contamination in the hay was responsible for the catastrophic death losses.

 

Ben Green presented Can clays bind plant toxins in the rumen?

Poisonous plants cause losses through death, reduced production efficiency and compromised harvesting of forages. For example, toxic larkspurs (Delphinium Spp.) cause up to 10% herd mortality on an annual basis. Recent work at the Poisonous Plant Research Laboratory suggests that a clay-based mineral supplement decreases larkspur poisoning in cattle. This suggests that clay in mineral mixes can reduce poisonings in cattle. Recent rumen culture (RC) experiments identified bentonite as a larkspur and lupine toxin binder which sequesters plant toxins in the gut for elimination by feces.

 

Kevin Welch presented the talk, Pine needle abortion in cattle.

In my presentation, I reviewed the work done by the PPRL over the past 30 years on pine needle induced abortions in cattle. I covered the common scenarios of when these abortions are likely to occur, the outcome to the cow and the calf, the abortifacient compounds in the needles, and the current management recommendations.

 

Daniel Cook presented DNA barcoding as a diagnostic tool for poisonous plant research.

DNA metabarcoding is a powerful tool that allows for simultaneous identification of many taxa within a complex biological sample.  It has been successfully used to estimate diet composition of animals as well as estimate composition of complex natural environmental mixtures.  We will present results showing its utility as a diagnostic tool in poisonous plant research through evaluating rumen contents and contaminated feed mixtures. 

 

Clint Stonecipher presented a talk Tall larkspur (Delphinium spp.) geographical and season variation in alkaloids and grazing native vs. naïve cattle to potentially reduce animal losses.

Tall larkspurs (Delphinium spp.) are native plants that grow on mountain rangelands of western North America and have a long history of poisoning grazing cattle. The toxicity of larkspur has been attributed to the norditerpenoid alkaloids, which are divided into two main structural groups: the highly toxic (N-methylsuccinimido) anthranoyllycoctonine type (MSAL type) and the less toxic 7,8-methylenedioxylycoctonine type (MDL type). These two classes act together to form a toxic mixture that poisons cattle. Ranchers that graze cattle in rangelands with large populations of toxic larkspur often have yearly herd mortalities up to 10%. Toxic alkaloid mixtures and the concentrations of alkaloids change based upon growing season, plant growth stage, geographic location, and larkspur species. We collected larkspur plants from locations in Montana, Idaho, and Wyoming throughout the growing season over multiple years to look at geographical and seasonal variations in larkspur alkaloids in leaves, stems, and floral parts. There were differences in alkaloid concentrations in two locations in northwest Wyoming in the leaf and floral parts. At one location, the alkaloid concentrations remained high in the floral parts late into September. The rancher reported losses of animals to larkspur in late September, which is not typical. Understanding alkaloid concentrations in larkspur throughout the season and across geographic locations can help livestock producers reduce the risk associated with poisoning. The proper selection of replacement animals for grazing on larkspur containing rangelands is important. For example, ranchers often describe that the greatest larkspur losses occur with replacement animals, and once the initial losses are over, larkspur poisoning is much less of a problem. We compared animals from a herd grazed on larkspur-containing pastures (larkspur-native) to cattle from a herd that have never been exposed to larkspur (larkspur-naïve). We hypothesized that larkspur-native animals would consume less larkspur than larkspur-naïve cattle. The experiment was conducted in eastern Idaho on a mountain rangeland containing tall larkspur. Six native and six naïve Angus steers grazed in July 2022 when larkspur was in the flowering stage. Two animals from the same treatment group were randomly assigned to one of six pastures. Daily bite counts were used to determine animal diet composition. There was a tendency for naïve cattle to consume more larkspur (3 ± 0.4 % of diet; P = 0.056) than native cattle (1 ± 0.2 %) over the course of the grazing study. However, more of the native cattle showed signs of intoxication. The study will be repeated a second year to determine if naïve cattle consistently consume more larkspur than native cattle, and to determine if there is a difference in native or naive cattle becoming intoxicated.

 

Eric Thacker presented the talk Rangeland Issues in Utah.

Talk focused on many issues with extended discussion of sagebrush rangeland restoration as an example of ongoing efforts.

 

Casey Spackman presented the talk Rangeland Ecology and Management.

Managing vegetation in the arid and semi-arid regions of New Mexico is the primary focus for New Mexico Ranchers and Agencies, especially concerning poisonous plants. New Mexico State University Cooperative Extension is proactively assisting land managers to monitor and map vegetation across the state to better manage forage resources and avoid poisonous plant. Over the past three years we have documented eight large cases of animal deaths. The suspect plants have been locoweed, pale dock, broom snakeweed, pigweed, kochia, and alfombrilla. Overall, monitoring, responding to producer challenges, and poisonous plant education is and will continue to be a focus for New Mexico State University and land managers.

Accomplishments

<p>The entire group met, discussed the current status of poisonous plants. A subset of the group worked together on cooperative research project which is funded by NSF.&nbsp; The group set priorities for collaborative research and grants for the coming year. Details on this project are:</p><br /> <p><strong>Chris Schardl </strong>is the PI, <strong>Rebecca Creamer</strong> a Co-PI, and <strong>Daniel Cook</strong> is a collaborator on a 5-year NSF grant that began in January 2021. This is a major accomplishment and has provided abundant collaborative work among the three investigators on seed-borne fungal endophytes. Eight talks at this meeting were directly related to that grant or included aspects from the grant. Since the grant has not held in in person meetings, this WERA 2193 meeting served as a chance to make progress presentations.</p>

Publications

<p><strong>Book Chapter:</strong></p><br /> <p><strong>Schardl, Creamer, Cook, Afkami</strong> (and others) wrote a book chapter for Mycota:</p><br /> <p>Schardl, C.L., Afkhami, M.E., Gundel, P.E., Iannone, L.J., Young, C.A., Creamer, R., Cook, D.A., Berry, D. 2023. Diversity of seed endophytes: Causes and implications. In: Scott, B., Mesarich, C. (eds) Plant Relationships. The Mycota, vol 5. Springer, Cham. https://doi.org/10.1007 /978-3-031-16503-0_5</p><br /> <p>&nbsp;</p><br /> <p><strong>Journal Articles:</strong></p><br /> <p>Das, S., Gardner, D.R., Neyaz, M., Charleston, A.B., III, Cook, D., Creamer, R. 2023. Silencing of the transmembrane transporter (<em>swnT</em>) gene of the fungus <em>Slafractonia leguminicola </em>results in a reduction of mycotoxin transport. <em>J. Fungi</em>, <em>9</em>, 370. https://doi.org/10.3390/ jof9030370</p><br /> <p>&nbsp;</p>

Impact Statements

1. The primary long-term outcome for the project is increased comprehension of poisonous plants. This information will support progress in managing poisonous plants by developings tools to minimize the losses associated with toxic plants. Overall, this work can benefit society by improving understanding of poisonous plants, which in turn may ultimately protect the food supply by aiding ranchers whose livestock suffer from them.

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