Brief Summary of Minutes of Annual Meeting

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.

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.  The group set priorities for collaborative research and grants for the coming year. Details on this project are:

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 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.

Book Chapter:

Schardl, Creamer, Cook, Afkami (and others) wrote a book chapter for Mycota:

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

Journal Articles:

Das, S., Gardner, D.R., Neyaz, M., Charleston, A.B., III, Cook, D., Creamer, R. 2023. Silencing of the transmembrane transporter (swnT) gene of the fungus Slafractonia leguminicola results in a reduction of mycotoxin transport. J. Fungi, 9, 370. https://doi.org/10.3390/ jof9030370