Brief Summary of Minutes of Annual Meeting

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.

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.

Chris Schardl is the PI, Rebecca Creamer a Co-PI, and Daniel Cook 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 (Schardl and Sneed) were directly related to that grant and three talks (Cook, Neyaz, and Das) 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.

Schardl, Creamer, Cook (and others) wrote a book chapter for Mycota on fungal endophytes of plants.

Schardl, Creamer, Cook (and others) wrote a book chapter for Mycota on fungal endophytes of plants.

Other joint publications by W2193 members/attendees published since the last report are listed below:

Neyaz, M., Das, S., Cook, D., Creamer, R. 2022. Phylogenetic comparison of swainsonine biosynthetic gene clusters among fungi.  Journal of Fungi 8:359.

Neyaz, M., Gardner, D. R., Creamer, R., Cook, D. 2022. Localization of the swainsonine-producing Chaetothyriales symbiont in the seed and shoot apical meristem in its host Ipomoea carnea. Microorganisms 10:545.

Noor, A.I., Nava, A., Neyaz, M., Cooke, P. Creamer, R. Cook, D. 2021. Ectopic growth of the Chaetothyriales fungal symbiont on Ipomoea carnea. Botany 99 (10):619-627.

Creamer, R., Hille, D.B., Neyaz, M., Nusayr, T., Schardl, C.L., Cook, D. 2021. Genetic relationship in the toxin producing fungal endophyte, Alternaria oxytropis, using polyketide synthase and non-ribosomal peptide synthase genes. Journal of Fungi 7(7), 538. https://doi.org/10.3390/jof7070538