Brief Summary of Minutes of Annual Meeting

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.

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. Because this project did not begin until October 2020, few accomplishments have been noted in a month.

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.

Nothing to report at this time.